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Phylogenetic systematics and the evolutionary history of some intestinal flatworm parasites (Trematoda… O'Grady, Richard Terence 1987

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PHYLOGENETIC SYSTEMATICS AND THE EVOLUTIONARY HISTORY OF INTESTINAL FLATWORM PARASITES (TREMATODA: DIGENEA: PLAGIORCHI01DEA) OF ANURANS SOME by RICHARD TERENCE 0'GRADY B . S c , Univers i ty Of B r i t i s h Columbia, 1978 M . S c , M c G i l l U n i v e r s i t y , 1981 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES Department Of Zoology We accept th i s thes is as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA March 1987 © Richard Terence O'Grady, 1987 In presenting th i s thesis in p a r t i a l fu l f i lment of the requirements for an advanced degree at the Univers i ty of B r i t i s h Columbia, I agree that the L i b r a r y s h a l l make i t f ree ly a v a i l a b l e for reference and study. I further agree that permission for extensive copying of t h i s thes is for s cho lar ly purposes may be granted by the Head of my Department or by his or her representat ives . It i s understood that copying or p u b l i c a t i o n of th i s thesis for f i n a n c i a l gain s h a l l not be allowed without my written permission. Department of Zoology The U n i v e r s i t y of B r i t i s h Columbia 2075 Wesbrook Place Vancouver, Canada V6T 1W5 Date: March 24, 1987 i i Abstract H i s t o r i c a l s tructura l i sm is presented as a research program in evolutionary bio logy. It uses patterns of common ancestry as i n i t i a l hypotheses in explaining evolutionary h i s t o r y . Such patterns , represented by phylogenetic trees , or cladograms, are postulates of pers i s tent ances tra l t r a i t s . These t r a i t s are evidence of h i s t o r i c a l constra ints on evolutionary change. Patterns and processes consistent with a cladogram are considered to be consistent with an i n i t i a l hypothesis of h i s t o r i c a l cons tra in t . As an app l i ca t ion of h i s t o r i c a l s t ruc tura l i sm, a phylogenetic analys i s is presented for members of the digenean p l a g i o r c h i o i d genera Glypthelmins S ta f ford , 1905 and Haplometrana Lucker, 1931. The eight species studied are i n t e s t i n a l paras i tes of frogs and toads in North, C e n t r a l , and South America. In a Wagner parsimony analys i s of 21 morphological characters with both the PAUP and PHYSYS computer programs, a s ingle phylogenetic tree with a consistency index of 84.8% can be i n f e r r e d . This suggests strong h i s t o r i c a l constra int in the evolut ion of the characters examined. It i s postulated that the eight species form a monophyletic group (c lade) , cons i s t ing of two less inc lus ive c lades . Glypthelmins  hyloreus and G. pennsylvaniensis comprise one of these clades; G. robustus, G. shas ta i , H. i n t e s t i n a l i s , G. c a l i f o r n i e n s i s , G. qu ie ta , and G. f a c i o i comprise the other. G. robustus, found in Bufo marinus in Colombia, i s both the southernmost and the most plesiomorphic member of i t s c lade . Glypthelmins c a l i f o r n i e n s i s , G. qu ie ta , and G. f a c i o i form a c lade , and p a r a s i t i z e frogs in the Rana pipiens complex in Mexico, eastern North America, and Central America, re spec t ive ly . Glypthelmins  shastai and H. i n t e s t i n a l i s , the l a t t e r of which is the only member of i t s genus, form a western North American c lade , and p a r a s i t i z e Bufo boreas and Rana pre t io sa , r e spec t ive ly . The phylogenetic analys i s includes a redescr ipt ion of G. shas ta i , the synonymy of the genus Haplometrana with Glypthelmins, the redescr ipt ion of H. intest i n a l i s as G. i n t e s t i n a l i s , an emended diagnosis of the genus Glypthelmins, and the f i r s t account of the l i f e cyc le of G. c a l i f o r n i e n s i s . Three aspects of phylogenetic analys is are examined in d e t a i l . These are the coding of mult i s tate character trees , the use of paras i te data to infer host r e l a t i o n s h i p s , and the propert ies of the Consistency Index and the F - R a t i o . It is proposed that the Consistency Index be ca l cu la ted without non-homoplasious autapomorphic characters . For the present study, th i s modif icat ion gives a value of 76.9%. Using the phylogenetic tree as a general reference system of patterns of common ancestry, i t is in ferred from developmental studies that (1) there i s no c o n f l i c t between the phylogenetic re la t ionsh ips indicated by only l a r v a l or only adult characters , and that (2) the evolution of some of the characters involved cer ta in types of heterochrony. Paedomorphic heterochrony i s in ferred to have occurred in the evolut ion of the uterus in G. shas ta i , H. i n t e s t i n a l i s , G. c a l i f o r n i e n s i s , G. qu ie ta , and G. f a c i o i . Peramorphic heterochrony is in ferred i v to have occurred in the evolut ion of the penetration glands in G. f a c i o i , and of the hindbody in H. i n t e s t i n a l i s . The r e l a t i v e l y longer hindbody of H. i n t e s t i n a l i s was experimentally induced to show paedomcrphic development by r a i s i n g specimens of H. i n t e s t i n a l i s in Bufo boreas, which i s the host of G. shas ta i , i t s s i s t e r - s p e c i e s . By one year after i n f e c t i o n , the r e l a t i v e length of the hindbody is shorter , and is equal to that of the pr imi t ive state found in G. shas ta i . The phylogenetic re la t ionsh ips among the anuran hosts are re-analyzed. There i s 80% congruence between them and the postulated phylogenetic tree for the i r paras i t e s , suggesting strong h i s t o r i c a l assoc iat ion between the paras i te and host groups. This inference of coevolution is further supported by the concordance of the present geographical d i s t r i b u t i o n s of the paras i tes and the i r hosts with the h i s t o r i c a l geology of the areas in which they occur. This implies an h i s t o r i c a l assoc iat ion between the areas and the organisms. V Table of Contents Abstract i i L i s t of Tables v i i i L i s t of Figures ix Acknowledgements x i i i Chapter I INTRODUCTION 1 Chapter II HISTORICAL STRUCTURALISM AS A RESEARCH PROGRAM IN EVOLUTIONARY BIOLOGY 7 BIOLOGICAL SYSTEMS 8 END-ATTAINING ACTIVITY 10 HISTORICAL AND NONHISTORICAL APPROACHES IN BIOLOGY .17 NATURAL SELECTION 21 ADAPTATION 25 A SINGLE EXPLANATORY FRAMEWORK 28 1. INTRASPECIFIC INVESTIGATIONS 30 2. PHYLOGENETIC INVESTIGATIONS 31 3. LIFE HISTORY INVESTIGATIONS 33 4. COMMUNITY STRUCTURE INVESTIGATIONS 36 "WHY" QUESTIONS 37 SUMMARY 38 CONCLUSIONS 41 Chapter III PHYLOGENETIC ANALYSIS OF HAPLOMETRANA LUCKER, 1931 AND SPECIES OF GLYPTHELMINS STAFFORD, 1905 (DIGENEA: PLAGIORCHI01DEA) IN NORTH, CENTRAL, AND SOUTH AMERICA 42 INTRODUCTION 42 TAXA STUDIED 4 5 MATERIALS AND METHODS 47 SPECIMENS EXAMINED 47 1. MUSEUM SPECIMENS 47 2. FIELD COLLECTIONS 52 3. EXAMINATION OF SPECIMENS 53 THE INFERENCE OF PHYLOGENETIC RELATIONSHIPS 54 1 . HOMOLOGY 54 2. OUTGROUPS 57 3. MULTISTATE CHARACTERS 59 4. COMPUTER-ASSISTED PARSIMONY ANALYSIS 60 CHARACTER ANALYSIS 63 1. CHARACTERS EXCLUDED FROM ANALYSIS 84 RESULTS AND DISCUSSION 87 PHYLOGENETIC ANALYSIS 87 TAXONOMIC CONSIDERATIONS 91 Chapter IV USING THE PHYLOGENETIC TREE TO STUDY EVOLUTIONARY EVENTS .94 PHYLOGENETIC CONCORDANCE OF LARVAL AND ADULT CHARACTERS 95 INTRODUCTION 95 LIFE HISTORY DATA 100 1. PREVIOUS STUDIES , 101 2. NEW DATA 103 .ANALYSIS OF LARVAL CHARACTERS 107 ANALYSIS OF LIFE CYCLE EVOLUTION 108 HETEROCHRONIC DEVELOPMENT 109 INTRODUCTION 109 ANALYSIS 114 ALLOMETRY AS HETEROCHRONY 118 EXPERIMENTALLY-PRODUCED HETEROCHRONY IN HAPLOMETRANA INTESTINALIS 121 INTRODUCTION 121 ANALYSIS 121 DISCUSSION 124 COEVOLUTION AND BIOGEOGRAPHY OF PARASITES AND HOSTS ..126 INTRODUCTION 126 HOST AND DISTRIBUTION DATA 128 1 . PREVIOUS STUDIES , 128 2. NEW DATA 134 3. DISCUSSION 140 COEVOLUTION ANALYSIS 142 1. FAMILY LEVEL 1 42 2. SPECIES LEVEL 144 BIOGEOGRAPHIC ANALYSIS 152 SUMMARY 156 LITERATURE CITED 275 APPENDIX A - COMPUTER ANALYSES WITH PAUP AND PHYSYS 292 APPENDIX B - HOST AND DISTRIBUTION DATA FOR GLYPTHELMINS QUIETA 303 APPENDIX C - COLLECTION SITES OF ANURANS 306 APPENDIX D - REDESCRIPTION OF GLYPTHELMINS SHASTAI, SYNONYMIZATION OF HAPLOMETRANA WITH GLYPTHELMINS, AND REDESCRIPTION OF G. INTESTINALIS N. COMB 307 APPENDIX E - THE LIFE CYCLE OF GLYPTHELMINS CALIFORNIENSIS 313 v i i APPENDIX F - CODING MULTISTATE CHARACTERS 318 APPENDIX G - SOME PROPERTIES OF THE CONSISTENCY INDEX AND THE F-RATIO 327 v i i i L i s t of Tables I . SPECIES STUDIED . . . . 4 6 I I . CHARACTERS ANALYZED 85 I I I . CHARACTERS RENUMBERED FROM UNORDERED ANALYSIS 90 ix L i s t of Figures 1. End-Atta in ing A c t i v i t y in B i o l o g i c a l Systems .159 2. Ultimate and Proximate Causa l i ty 159 3. Types of Evolut ionary Explanations 161 4. H i s t o r i c a l Structural i sm in Phylogenetic Systematics 161 5. Using a Cladogram to Study the Evolut ion of L i f e His tory T r a i t s 163 6. The Appl i ca t ion of H i s t o r i c a l Structural i sm to the Study of Community Structure 165 7. Postulated Relat ionships among Glypthelmins, from Brooks (1977) 167 8. C o l l e c t i o n Si tes of Anurans in B r i t i s h Columbia 169 9. C o l l e c t i o n s S i tes of Anurans in C a l i f o r n i a 171 10. Tegumental Project ions 173 11. Penetration Glands in Adult Glypthelmins f a c i o i 173 12. Medial Glands in Haplometrana i n t e s t i n a l i s 175 13. Pharyngeal Glands in Glypthelmins quieta 175 14. Schematic Representation of the D i s t r i b u t i o n of the V i t e l l a r i a in Glypthelmins and Haplometrana 177 15. D i s t r i b u t i o n of V i t e l l a r i a in Glypthelmins quieta and G. f a c i o i 179 16. D i s t r i b u t i o n of V i t e l l a r i a in Glypthelmins c a l i f o r n i e n s i s 181 17. D i s t r i b u t i o n of V i t e l l a r i a in Glypthelmins shastai and Haplometrana i n t e s t i n a l i s 181 18. V i t e l l i n e Ducts in Haplometrana i n t e s t i n a l i s and Glypthelmins c a l i f o r n i e n s i s 183 19. Shape of the Excretory V e s i c l e 185 20. Postulated Phylogenetic Relat ionships among Glypthelmins and Haplometrana 187 X 21. Mul t i s ta t e Character Trees: One 189 22. Mul t i s ta te Character Trees: Two 191 23. Disrupt ive Grouping C r i t e r i a in Systematics 193 24. Congruence and Consistency in Tree Topologies 195 25. General ized L i f e Cycle of a Digenean Flatworm 197 26. Phylogenetic Analys i s of Larva l Characters of Species of Glypthelmins and Haplometrana 199 27. Phylogenetic Analys i s of L i f e Cycle Evolut ion in Species of Glypthelmins and Haplometrana 201 28. Categories of Heterochronic Change 203 29. Using a Cladogram to Di s t ingu i sh Paedomorphic, Peramorphic, and Symplesiomorphic Morphologies 205 30. Some Limits to the Use of a Cladogram to Detect Heterochrony 205 31. The Growth of the Hindbody in Haplometrana i n t e s t i n a l i s and Glypthelmins quieta 207 32. An Estimation of the Plesiomorphic State of the Hindbody for Haplometrana i n t e s t i n a l i s 209 33. Inference of Plesiomorphic Hosts for Species of Glypthelmins and Haplometrana: One 211 34. Experimentally-Produced Heterochronic Development of the Hindbody in Haplometrana i n t e s t i n a l i s 213 35. A l tered Growth of the Hindbody in Haplometrana  i n t e s t i n a l i s 215 36. D i s t r i b u t i o n in the Americas of the Digenean Species, G. hyloreus , G. pennsylvaniensis , G. f a c i o i , and G. robustus 217 37. D i s t r i b u t i o n in the Americas of the Digenean Species, Haplometrana i n t e s t i n a l i s and Glypthelmins shas ta i , and of the Anuran Species, Bufo boreas, Rana p r e t i o s a , R. cascadae, and R. muscosa 219 38. D i s t r i b u t i o n in the Americas of the Digenean Species, Glypthelmins quieta and G. c a l i f o r n i e n s i s , and of the Anuran Species, Rana aurora, R. b o y l i i , and the species of the R. pipiens Group 221 39. Inference of Plesiomorphic Hosts for Species of xi Glypthelmins and Haplometrana; Two 223 40. Coevolutionary Analys i s of Species of Glypthelmins and Rana in North and Centra l America 225 41. Summary Cladogram of Higher Level Relat ionships among Ranids in the Americas 227 42. Phylogenetic Relat ionships among Ranids in Western North America 229 43. Postulated Phylogenetic Relat ionships among Members of the Rana pipiens Group, from H i l l i s e_t a l . (1983) . . .231 44. Biogeographic and Phylogenetic Patterns for Species of Glypthelmins and Haplometrana in the Americas 233 45. D i s t r i b u t i o n of the Hosts of Glypthelmins c a l i f o r n i e n s i s 235 46. Morphology of Glypthelmins shas ta i : One 237 47. Morphology of Glypthelmins shas ta i : Two 239 48. Morphology of the Larva l Stages of Glypthelmins c a l i f o r n i e n s i s 241 49. Young Adult of Glypthelmins c a l i f o r n i e n s i s 243 50. Mul t i s ta te Transformation Series 245 51. Demonstration Tree for Mul t i s ta te Coding Methods . . . . 247 52. Addi t ive Binary Coding 249 53. Redundant Linear Coding 251 54. Nonredundant Linear Coding 253 55. Coding a B a s a l l y - B i f u r c a t i n g Mul t i s ta te Series 255 56. Incorporating a Mul t i s ta te Tree into a Character Matrix 257 57. Using Paras i te Data to Infer Host Relat ionships 259 58. Using Inclus ive ORing to Deal with the Occurrence of more than One Paras i te Taxon in a Host Group 261 59. The L imi t s of Inc lus ive ORing 263 60. The I n a b i l i t y of the Consistency Index to D i s t ingu i sh between Autapomorphies and Synapomorphies 265 x i i 61. C a l c u l a t i o n of the F-Rat io 267 62. The S e n s i t i v i t y of the F-Rat io to Factors Relevant to Unrooted, Rather than Rooted, Trees 269 63. Agreement between the F-Rat io and the Consistency Index 271 64. Disagreement between the F-Rat io and the Consistency Index 271 65. A Demonstration that the F-Rat io i s not Biased towards Para l l e l i sms or Reversals when Comparing Trees of Equal Length 273 x i i i Acknowledgements The moving finger • wri tes . I am grate fu l to NSERC, and to both the Faculty of Graduate Studies and the Department of •Zoology at the Univers i ty "of B r i t i s h Columbia for f i n a n c i a l support during the course of th i s research. I thank the Parks Board of B r i t i s h Columbia, the College of the Siskiyous (COS), and San Francisco State Univers i ty (SFSU) for t h e i r cooperation with some of the f i e l d c o l l e c t i o n s . In p a r t i c u l a r , I thank Leona Kegg and Dr. Ken Beatty, of COS, and Stacy G i l e s , of SFSU. I am grate fu l to Dr . J . Ralph L i c h t e n f e l s , U .S . National Museum, Helminthological C o l l e c t i o n , B e l t s v i l l e , Maryland, for his help with the loaning of specimens, to Dr. John C. Holmes, Dept. of Zoology, Univ. of A l b e r t a , for specimens of Glypthelmins  shas ta i , Haplometrana i n t e s t i n a l i s , and G. hyloreus, and to Dr. James J . S u l l i v a n , CDC, A t l a n t a , Georgia, for specimens of G. f a c i o i . Dr . Gordon G. Gibson, National Museum of Natural Sciences, Ottawa, Ontar io , helped me to track down Staf ford's specimens of G. qu ie ta . The fol lowing parts of th i s d i s s e r t a t i o n have been modified from papers that I have authored or co-authored: Chapter I I : O'Grady, 1986. Can. J . Zoo l . 64:1010-1020; O'Grady and Brooks, in Entropy, Information, and Evo lut ion , in press , MIT Press; Appendix F : O'Grady and Deets, Syst . Zool . in review; Appendix G: Brooks et §_1. , 1987. Syst . Zool . 35:571-581. I appreciate the help of my research committee members during the course of th i s work. They are: Drs . J . R . Maze, C V . Finnegan, H . E . Kasinsky, and J . D . McPhai l , of U . B . C . Jack Maze, in p a r t i c u l a r , kept me on my toes. I am also grate fu l to Prof . Mary H. P r i t c h a r d , Curator of the Harold W. Manter Laboratory, Un ivers i ty of Nebraska, for her interes t in my work and for her c r i t i c a l assessment of th i s d i s s e r t a t i o n . Most importantly, I thank Dr. Daniel R. Brooks, my major professor , for his help, encouragement, and guidance during the f ive eventful years that we have spent together. And, having w r i t , moves on. Here's to my parents , for the ir quiet love; to Cathy, for her strength at the beginning of a l l of t h i s ; to Ryan, my son, the l i t t l e king; and to Maggie, for helping me r e a l i z e that there i s nothing you can do that can't be done. During my time at U . B . C , I have been lucky enough to have had some pret ty t e r r i f i c people as f r i ends , col leagues, and even fellow frog-catchers . They include: Dave Glen , Gayle Brown, N e i l Benson, Helen Benson, Dawn McArthur, Tom Petersen, Pat Shaw, Greg Deets, George Benz, and Kate Shaw. The complete l i s t i s much longer, for I have benefited from so many. I am grate fu l to Maggie Hampong, Cheryl Macdonald, Greg Deets, and e spec ia l ly Kathy Lauriente for the ir help with the i l l u s t r a t i o n s . What else can I say? I t ' s been fun. 1 I . INTRODUCTION Despite the ir apparent complementarity, evolutionary theory and systematic theory have long been operating on d i f f erent conceptual bases. That i s , not only have they been interested in d i f f erent things - the former in processes, the l a t t e r in patterns - but they have each been assuming the operation of d i f f erent forces in the production of organic form. One reason for th is d i s p a r i t y i s that humans were categor iz ing organisms before there was any thought that those organisms might have evolved from one another. P la to , with h i s concept of natural kinds as immutable, d i s c r e t e , and eternal essences, wanted to "cut nature at her j o i n t s " . Others, such as Linnaeus, wanted to catalogue the ir god's handiwork. Regardless of the causative factor presumed, these e f for t s amounted to grouping organisms by phenetic s i m i l a r i t y , that i s , by o v e r a l l s i m i l a r i t y of form. It could have been l i t t l e e l se , given that there was no concept of genealogical r e l a t i o n s h i p . Systematic theory, then, has an ancestry of non-evolutionary thought. It i s important to note that the concept of homology introduced by Owen (1848) was based upon s i m i l a r i t y of form, and not upon evolut ionary r e l a t i o n s h i p . Homology d id not acquire i t s evolutionary connotation u n t i l workers such as Darwin (185S), Haeckel (1866), Lankester (1870) and Gegenbaur (1878). Once th i s connotation was recognized, systematists began seeing homologues as ind icators of k inship among organisms. Thus, the evolutionary re la t ionsh ips among organisms came to be in ferred from s t r u c t u r a l c r i t e r i a . 2 Evolut ionary theory, on the other hand, has operated predominantly with funct ional c r i t e r i a . I am r e f e r r i n g to Darwin's theory, and i t s d e r i v a t i v e , neo-Darwinism. For various h i s t o r i c a l reasons (see, e . g . , O'Grady, 1984), these consider the primary force o f . evo lu t ion to be natural se lec t ion act ing upon r e l a t i v e funct ional d i f ferences among organisms. Thus, while systematic theory considers s tructure before funct ion, current evolutionary theory considers function before s tructure . This d i s p a r i t y creates a c o n f l i c t of interest because i t i s systematic theory that reconstructs the genealogies whose o r i g i n evolutionary theory t r i e s to exp la in . For most of the twentieth century, the resul t has been a systematics that attempts to represent both genealogical r e la t ionsh ips (pattern) and degree of funct ional change (presumed process) at the same time, within the same evolutionary tree (see Mayr, 1981). This creates problems when the descendants of a common ancestor are s p l i t into two or more groups of equal taxonomic rank because of di f ferences in the ir funct ional proper t i e s . Such an operation can obscure more re la t ionsh ips than i t c l a r i f i e s because i t d i srupts the h i e r a r c h i c a l in ternes t ing of the pattern of inher i ted homologues. The apparent loss of pattern can d irec t evolut ionary explanations away from s t r u c t u r a l propert ies and towards funct ional a t t r i b u t e s . Phylogenetic systematics i s one attempt to base systematic theory on s t r u c t u r a l evolut ionary terms. Developed by Hennig (1950; 1966), i t has been ref ined extensively in the las t twenty years ( e . g . , Camin and Sokal , 1965; Kluge and F a r r i s , 1969; 3 F a r r i s , 1970, 1982, 1985; Maddison et a l . , 1984; Swofford and Maddison, in review). Procedures for i n f e r r i n g homology have been improved, and there are now a number of computer programs capable of analyzing data sets q u i c k l y , e f f i c i e n t l y , and in large volume. This d i s s e r t a t i o n i s offered as an app l i ca t ion of phylogenetic systematics to the study of evo lut ion . It begins with a prel iminary chapter (Chapter II) that attempts to bring the concerns of evolutionary theory and systematic theory together into an approach c a l l e d h i s t o r i c a l s t ruc tura l i sm. This is offered as an onto log ica l j u s t i f i c a t i o n of phylogenetic systematics. Chapter III presents the empir ica l core of the d i s s e r t a t i o n , a phylogenetic ana lys i s of eight species of digenean trematodes, a group of flatworms (Platyhelminthes) p a r a s i t i c in vertebrates and invertebrates . The species are members of two genera in the Superfamily P lag iorchio idea D o l l f u s , 1930: Glypthelmins S t a f f o r d , 1905 and Haplometrana Lucker, 1931. They are i n t e s t i n a l paras i tes of anurans in North, C e n t r a l , and South America. The analys is i s based upon 21 morphological characters . The animals examined consisted of type mater ia l , specimens from pr ivate c o l l e c t i o n s , and specimens from my f i e l d and laboratory s tudies . The study of type mater ia l and the recent a v a i l a b i l i t y of add i t i ona l specimens has allowed a redescr ipt ion of Glypthelmins  shas ta i . In a d d i t i o n , Haplometrana i n t e s t i n a l i s i s redescribed as G. i n t e s t i n a l i s n. comb., and the genus Haplometrana is synonymized with the genus Glypthelmins. F i e l d c o l l e c t i o n s have 4 e s t a b l i s h e d new l o c a l i t i e s f o r some of the p a r a s i t e s p e c i e s . Laboratory work has r e s u l t e d i n the f i r s t d e s c r i p t i o n of the l i f e c y c l e of G. c a l i f o r n i e n s i s and the c o l l e c t i o n of v a r i o u s developmental stages of G. q u i e t a and H. i n t e s t i n a l i s . The ph y l o g e n e t i c r e l a t i o n s h i p s of the taxa are i n f e r r e d with computer-assisted parsimony techniques, which c o n s t r u c t g e n e a l o g i c a l t r e e s by maximizing the number of p o s t u l a t e d homologous s e r i e s of c h a r a c t e r s . The p h y l o g e n e t i c t r e e , or cladogram, i s then used as a general r e f e r e n c e system with which to examine the occurrence of c e r t a i n e v o l u t i o n a r y events. T h i s a n a l y s i s i s presented i n Chapter IV. Chapter IV begins with a demonstration of the congruence of the t r e e s i n f e r r e d from e i t h e r the l a r v a l or the a d u l t c h a r a c t e r s of the worms. Included i n t h i s s e c t i o n i s a d i s c u s s i o n of why some e a r l i e r e v o l u t i o n a r y e x p l a n a t i o n s , drawing i m p l i c i t l y or e x p l i c i t l y from concepts of Haeckelian r e c a p i t u l a t i o n , d i d not expect t h i s congruence to e x i s t . T h i s d i s c u s s i o n leads i n t o an examination of the e v o l u t i o n of the l i f e c y c l e s i n the study group. Next, there i s an a n a l y s i s of the h e t e r o c h r o n i c development of c e r t a i n morphological p r o p e r t i e s of Glypthelmins and Haplometrana. T h i s a n a l y s i s uses F i n k ' s (1982) methodology f o r a n a l y z i n g heterochrony from a ph y l o g e n e t i c p e r s p e c t i v e . I t not only looks at three c h a r a c t e r s of the worms that are the p o s s i b l e r e s u l t of d i f f e r e n t types of heterochrony, but i t a l s o r e p o r t s on the experimentally-produced h e t e r o c h r o n i c development of the hindbody in one of the s p e c i e s , H. i n t e s t i n a l i s . 5 The remaining sections of Chapter IV assess the extent of host -paras i te coevolut ion, as well as the biogeographic and speciat ion patterns involved. For th i s study, I found i t necessary to re-analyze the data of some e a r l i e r phylogenetic studies on the re la t ionsh ips of the anuran hosts . These comparisons are further demonstrations of the uses to which an hypothesis of phylogenetic re la t ionsh ips can be put. The digenean trematodes are good organisms for such a m u l t i - l e v e l study because of the number of l eve l s of re la t ionsh ips that ex i s t . These are the resu l t of the paras i tes ' complex l i f e cyc le s , in d i f f erent environments, with an intimate assoc iat ion with d i f f erent types of hosts . F i n a l l y , th i s d i s s e r t a t i o n contains two appendices, F and G, which explore c e r t a i n methodological procedures in systematics. Appendix F evaluates the strengths and weaknesses of the methods that can be used to represent the shape of an evolutionary tree with a numerical code, so as to allow various forms of computer a n a l y s i s . Appl icat ions include mult i s tate character trees (an evolut ionary transformation ser ies of more than two homologous character states) and the use of paras i te phylogenies to infer host r e l a t i o n s h i p s . Appendix G examines the propert ies of two commonly-used opt imal i ty measures in systematics: the Consistency Index (Kluge and F a r r i s , 1969; F a r r i s et a l . , 1970) and the F-Rat io ( F a r r i s , 1972). These measures are used to assess the goodness-of-f i t of a tree to the data . Each has what I bel ieve are previous ly unnoticed propert ies that af fect i t s performance with c e r t a i n types of 6 character d i s t r i b u t i o n s . Some of these propert ies are shortcomings of the measures, and in an e f for t to avoid such problems, I describe the c a l c u l a t i o n of a modified Consistency I ndex. 7 I I . HISTORICAL STRUCTURALISM AS A RESEARCH PROGRAM IN EVOLUTIONARY BIOLOGY Any phys ica l en t i ty can be examined with or without considerat ion of the means by which i t came to e x i s t . An approach that takes e t io logy into account can be c a l l e d h i s t o r i c a l , meaning that events that occurred in the past are considered to be of use in explaining current proper t i e s . The a l t e r n a t i v e perspective can be c a l l e d n o n h i s t o r i c a l . 1 Some quest ions, such as "What i s i t made of?" and "What does i t do?" are n o n h i s t o r i c a l ; they are interested in the propert ies of the thing now. Other quest ions, such as "How did i t come to be?" are h i s t o r i c a l . S t i l l others , such as "What causes i t to be as i t is?" and "Why i s i t as i t is?" look for e i ther n o n h i s t o r i c a l or h i s t o r i c a l answers, depending upon the completeness of explanation des i red . (The use of "why" questions should be minimized because of the ambiguity of that adverb; see the sec t ion , "WHY" QUESTIONS.) Consider a window broken by a f a l l i n g rock. The nonh i s tor i ca l question i s "What breaks the window?". The corresponding h i s t o r i c a l questions can address many leve ls of past c a u s a l i t y , such as "How did the rock come to be moving?", "How did i t come to be a rock?", and "How 1 O'Grady (1984) referred to the nonh i s tor i ca l as " a h i s t o r i c a l " . As noted in O'Grady (1986), i t has since become necessary to d i s t i n g u i s h between those views that simply do not consider h i s t o r i c a l phenomena in biology ( n o n h i s t o r i c a l ) , and those that a c t i v e l y deny the ir importance ( a h i s t o r i c a l ) . Only the former is discussed here. 8 did gravi ty come to have such an ef fect upon rocks?" With respect to b i o l o g i c a l systems, I use the term "history" in a phylogenetic sense. That i s , I use i t to refer to the evolut ionary h i s tory of organisms. I do not use th i s term to refer to simple persistence through time, or to intragenerat ional change over time, such as that occurring during ontogeny. Ontogeny i s , of course, a product of evo lu t ion . Nevertheless, I consider i t separately here because, unl ike phylogenesis, i t need not necessar i ly be viewed as the product of such a process . H i s t o r i c a l and nonh i s tor i ca l perspectives can be examined with respect to invest igat ions of two basic organic propert ies : s tructure and funct ion . I suggest that many disagreements over the v a l i d i t y of b i o l o g i c a l explanations are the resul t of i n s u f f i c i e n t de l ineat ion of these perspect ives . In the fol lowing pages, I examine h i e r a r c h i c causa l i ty in phys ica l systems, and then use th i s framework to assess the explanatory strengths and weaknesses of s t r u c t u r a l i s t and f u n c t i o n a l i s t approaches. I hope to demonstrate the advantages to evolutionary biology of h i s t o r i c a l s t ruc tura l i sm. BIOLOGICAL SYSTEMS B i o l o g i c a l systems are s p e c i f i c a l l y configured phys ica l systems, and so they possess, minimally, the same phys ica l propert ies as an inanimate object . Cats , af ter a l l , f a l l from heights just as rocks do. But there are also propert ies unique 9 to b i o l o g i c a l systems. The two with which I am concerned here are the ir f u n c t i o n a l i t y and the ir evo lut ion . At any moment of an organism's existence, a p a r t i c u l a r organizat ion of i t s components must be maintained in the face of p o t e n t i a l l y d i srupt ive forces (heat, c o l d , energy flows, e t c . ) . L i v i n g systems must always be "doing" something. They cannot pass ively e x i s t , as do inanimate objects . Any phys ica l object has s tructure , and thus some degree of order. A l i v i n g system must have i t s s tructure organized in a manner that performs p a r t i c u l a r functions that resu l t in the continued existence of the organism. Organisms do not function in order to survive . Rather, i t i s t r i v i a l l y true that they w i l l not survive i f they do not funct ion . Organisms must a c t i v e l y e x i s t . If they d i e , the ir constituent elements w i l l s t i l l exist and s t i l l possess basic phys ica l propert ies - dead cats f a l l from heights just as l i v e cats do - but the organisms w i l l no longer have the organizat ional propert ies of l i v i n g things . The second property of organisms that is of concern here i s the ir evo lut ion . An ancestor-descendant continuum is establ ished through reproduction. As th i s continuum i s produced, whether i n t r a s p e c i f i c a l l y or t r a n s - s p e c i f i c a l l y , ancestra l propert ies become incorporated into the ontogeny of the descendants. Every new descendant, does not, so to speak, s tart from scratch . It inher i t s many of i t s propert ies from i t s ancestors, which inher i ted many of the i r s from the i r ancestors -and so on. This has two consequences. The f i r s t i s that a property that ar i ses for the f i r s t time in a species ( i . e . , 10 unique, and contingent upon previous ly unreal ized circumstances) may become a f ixed , inher i ted property in i t s descendants. Because of t h i s , an inves t igat ion of an organism's existence must deal with more than questions about ult imate o r i g i n s , that i s , about the o r i g i n of l i f e on ear th . It i s a lso necessary to ask questions about any subsequent evo lut ion , for a ser ies of events, spanning m i l l i o n s of years, i s responsible for the organism appearing as i t does now. A phylogeny is thus not a passive "trace" of organisms' existence through time, but a record of the r e l a t i v e times and l eve l s of genera l i ty at which the causes of various organic propert ies evolved. For example, a cat has three propert ies caused by three h i s t o r i c a l events during i t s evo lut ion: i t has inher i ted a nuclear membrane from the common ancestor of eukaryotes, i t has inher i ted an in terna l skeleton from the common ancestor of vertebrates , and i t has inher i ted fur from the common ancestor of mammals. Each of these propert ies or ig inated as a unique, contingent character of a p a r t i c u l a r species , and each became an inher i ted character through the subsequent evolution of descendent species . END-ATTAINING ACTIVITY The f u n c t i o n a l i t y and the evolut ion of b i o l o g i c a l systems are often considered together, in that i t i s asked whether the fact that organisms must function in a p a r t i c u l a r manner i f they are to survive has any causal connection with the means through 11 which they came to possess the s tructures that perform those funct ions . This introduces the subject of te leo logy , or goal -d i rec ted a c t i v i t y . It i s at th i s point that a d i s t i n c t i o n between h i s t o r i c a l and n o n h i s t o r i c a l explanations i s important. To a s s i s t in th i s d i s t i n c t i o n , discussed- in the next sect ion , i t i s h e l p f u l f i r s t to recognize three types of processes that a t t a i n end-states . F i r s t , natural e n t i t i e s or systems ( i . e . , not made by humans) may at ta in- end-states because of the operation of processes whose existence depends not on the ent i ty being a l i v e or purposeful ly designed, but on the propert ies of i t s const i tuent matter. Some of these propert ies , such as gravi ty or entropic decay, operate u n i v e r s a l l y . Others, such as radioact ive decay and react ion gradients , are more r e s t r i c t e d in the ir operat ion. A l l of th i s i s teleomatic a c t i v i t y (Mayr, 1974; Wicken, I98 la ,b) . The end-state , such as a rock coming to rest at the bottom of a c l i f f , can simply be said to resul t from the propert ies of the e n t i t y . There i s no "control" or "purpose" involved. I refer to th i s a c t i v i t y as end-resu l t ing . Second, there i s the f u n c t i o n a l i t y of l i v i n g systems that i s dependent upon the operation of inher i ted genetic and epigenetic fac tors . These factors determine the end-states of processes such as homeostasis (maintenance of a spec ie s - spec i f i c p h y s i o l o g i c a l s ta te ) , ontogeny (development into an adult of the spec ies ) , and reproduction (production of new members of the spec ies ) . This is teleonomic a c t i v i t y (P i t tendr igh , 1958; Mayr, 1961, 1974). The end-states are reached because of inher i t ed , 1 2 i n t e r n a l , c o n t r o l l i n g fac tors . It i s end-directed . The t h i r d category is that of purposeful behavior, in which cer ta in outcomes occur because events were d e l i b e r a t e l y brought about so as to produce them. This behavior requires some degree of cogn i t ion . It is most prevalent in humans, and ex i s t s to various extents in other animals. The end-state i s d e l i b e r a t e l y sought, and i s therefore a goal ( i . e . , one type of end-state) . O'Grady (1984) referred to th i s a c t i v i t y as goa l -d i rec ted ; i t i s perhaps better to c a l l i t goal-seeking (O'Grady, 1986), so as to make i t c l ear that such behavior is the resul t of a consciousness capable of some amount of premeditation and choice . I think that only th i s type of end-atta ining a c t i v i t y should be c a l l e d t e l e o l o g i c a l . My pos i t ion is a departure from e a r l i e r usage of the term. T r a d i t i o n a l l y (see, e . g . , Nagel, 1984; Wicken, 1985), teleology has been used as a general term for a l l a c t i v i t i e s that achieved an end - which was not d is t inguished from a goal . Thus, not only have the teleomatic and the teleonomic been considered to be types of te leology, but so, too, have b i o l o g i c a l function and organizat ion . From th i s p o s i t i o n , for example, Gray (1874) was correct in saying that Darwin had "wedded morphology to teleology" - simply because Darwin's theory deals with f u n c t i o n a l i t y . I suggest that such terminology inter feres with the search for , and the study of, d i f f erent end-atta ining processes in b i o l o g i c a l systems. With respect to A r i s t o t l e ' s concept of un iversa l c a u s a l i t y , which is in some ways responsible for much of the uncertainty over the status of t e l e o l o g i c a l explanations (see, e . g . , Nagel, 13 1984; J a k i , 1966), i t seems that the fol lowing observations apply. (1) A l l three types of end-atta ining a c t i v i t i e s have materia l causes (that in which change occurs) and e f f i c i e n t causes (that by which change occurs) . (2) Teleonomic and t e l e o l o g i c a l a c t i v i t y a l so have formal causes ( in terna l representat ions: that into which something changes). (3) Only t e l e o l o g i c a l a c t i v i t y a lso has f i n a l causes (that for the sake of which change occurs) . Of course, the nature of the in terna l representations in teleonomic and t e l e o l o g i c a l a c t i v i t y i s quite d i f f e r e n t . In the former, i t consists of inher i ted developmental fac tors ; in the l a t t e r i t i s a cogni t ive ac t . Nevertheless, in c o n t r a d i s t i n c t i o n to teleomatic a c t i v i t y , i t can be said that in both there i s something within the organism that has a representation of the end-state and is involved in the taking of steps that a t t a i n that s tate . While not a l l phys ica l e n t i t i e s show a l l three types of end-atta ining a c t i v i t y , those that do are a subset of those that do not (F ig . 1). A l l phys ica l e n t i t i e s show some type of teleomatic a c t i v i t y ; a subset of these also shows teleonomic a c t i v i t y ( b i o l o g i c a l systems); and a subset of these a lso shows t e l e o l o g i c a l a c t i v i t y (cognit ive b i o l o g i c a l systems). Thus, while the act of a rock f a l l i n g to earth is teleomatic (gravity) and the act of a k i t ten becoming a cat is teleonomic (ontogeny), the act of a k i t ten f a l l i n g to earth i s also teleomatic ( g r a v i t y ) . To complete the example, a human parachutist t e l e o l o g i c a l l y reaches for the r ipcord as his te leonomical ly-maintained body plummets te leomat ica l ly to the ground below. 1 4 If the et io logy of the e n t i t i e s involved in th i s internest ing i s to be addressed, i t i s necessary to introduce an h i s t o r i c a l perspect ive . It i s at th i s point that there i s a r i sk of introducing inappropriate te leology - i . e . , an inappropriate assumption of goal-seeking behavior - by incorporat ing the attainment of the end-state into the causal explanation for the ent i ty coming to ex i s t . Evolut ionary theory seeks to provide mechanistic explanations for the et io logy of organic s tructure and funct ion, and to keep such ideas d i s t i n c t from t e l e o l o g i c a l explanations for the existence of man-made objects . C r i t i c s of evo lut ion , such as Wil l iam Paley (1802), b lurred th i s d i s t i n c t i o n when they presented the i r arguments from design. Paley's te leology of the designer appealed to external , imposed c a u s a l i t y . H u l l (1973) has c a l l e d th i s Platonic te leo logy . Others wr i t er s , such as those orthogenet ic i s t s who approved of teleology (discussed l a t e r ) , appealed to i n t e r n a l , immanent c a u s a l i t y . H u l l (1973) has c a l l e d th i s A r i s t o t e l i a n te leo logy . I think that i t resu l t s from mistaking formal causation for f i n a l causat ion. That i s , the propert ies of the state into which something changes are seen as the reason for the change occurr ing . Later in th i s section I suggest that th i s l i n e of argument can be p a r t l y a t t r ibuted to an inappropriate analogy of evolut ion with ontogenetic development. Both Platonic and A r i s t o t e l i a n teleology assume that the existence of f u n c t i o n a l i t y in organisms i s i n d i c a t i v e , as i t i s in human works, of purpose. They assume that i t must have come to exist for some reason. 1 5 For evolutionary theory, the recognit ion of teleonomic a c t i v i t y provides a mechanistic n o n h i s t o r i c a l explanation for organic f u n c t i o n a l i t y , and thus deals with the "organic teleology" of design discussed by writers such as Russe l l (1916) and von Bertalanffy (1962). (See a lso P i t t e n d r i g h ' s l e t t e r to Mayr, in Mayr, 1974.) But th i s does not address the question of where those factors came from. What produced teleonomic systems in the f i r s t p lace , and what causes the evolut ion of one spec i e s - spec i f i c teleonomic system into another? Unless a mechanistic explanation can be found for these h i s t o r i c a l questions, teleonomy w i l l mean nothing more than "purposefully designed but current ly unattended organic a c t i v i t y " , l i k e a machine with servo-mechanisms. The so lut ion seems to l i e in seeing teleonomic a c t i v i t y as the resu l t of cer ta in types of teleomatic a c t i v i t y . With respect to the o r i g i n of teleonomic systems - the o r i g i n of l i f e on earth - th i s means that the l i v i n g emerged from the nonl iv ing through processes of s e l f - o r g a n i z a t i o n , such as those proposed by Eigen (1971) (see also Wicken, 1979). With respect to the transformation of one teleonomic system into another - the evolut ion of species - th i s means that evolut ion is caused by teleomatic changes in inher i ted teleonomic propert ies during reproduction and ontogeny. Genetic d r i f t (King and Jukes, 1969; Kimura and Ohta, 1971) and molecular dr ive (Dover, 1982a, b) are examples of such changes. More general ly , I am simply r e f e r r i n g to mutations, which are changes in the reproductive message from one generation to the next. The impact of th i s a c t i v i t y upon 16 the teleonomic system in which i t occurs may exceed that system's homeostatic c a p a b i l i t i e s enough to cause the death of the i n d i v i d u a l , or even the ext inc t ion of the species . If the system remains together, but nevertheless changes to a c e r t a i n degree, then speciat ion occurs. This view appears to be consistent with Paterson's (1980, 1981, 1982) argument that speciat ion is an inc identa l effect of breakdowns in species-maintaining systems. Brooks and Wiley (1986) have argued that th i s process i s the resu l t of entropic d i s s i p a t i o n of species-cohesive fac tor s . There is no immutable te los in the teleonomic a c t i v i t y of b i o l o g i c a l funct ion . There i s only an end-state, s u f f i c i e n t l y stable for generations of i n t r a s p e c i f i c reproduction, that eventual ly evolves into the end-state of a descendent species . The teleomatic processes responsible for th i s transformation a t t a i n the i r end-states because of the natural propert ies of the phys ica l e n t i t i e s involved. They do not do so for the sake of the organism in which they happen to occur. Teleomatic a c t i v i t y does not occur for anything. The teleomatic produces the teleonomic (or ig in of l i f e ) , whereupon the teleonomic sets the context of operation for , but does not d i r e c t or c o n t r o l , further teleomatic a c t i v i t y (evo lut ion) . Teleonomic a c t i v i t y i s an i n t r a s p e c i f i c phenomenon. I see no evidence for t r a n s - s p e c i f i c teleonomic a c t i v i t y , in which c o n t r o l l i n g factors d i rec t the attainment of an end-state that is e v o l u t i o n a r i l y beyond the maintenance of the ex i s t ing species . The t r a n s - s p e c i f i c process of evolut ion is therefore 17 not analogous to the i n t r a s p e c i f i c process of ontogeny, for only the l a t t e r i s d irec ted towards an end-state . Ontogeny, a teleonomic a c t i v i t y , operates with an in terna l representation of the end-state(s) to be a t ta ined . It cannot be sa id that evolut ion operates with a s i m i l a r , c o n t r o l l i n g representation of what is to be. Such an analogy between evolut ion and ontogeny has been drawn a number of times (e .g . Po lany i , 1968), and i t would seem to be responsible for many of the shortcomings of orthogenetic theory at the turn of the century. As with any end-atta ining a c t i v i t y , th i s analogy has also a t trac ted t e l e o l o g i c a l in terpreta t ions of evolut ionary change, such as Bergson's (1911) "creative evolution" and the "progressive evolution" of some neo-Lamarckians (see Bowler, 1983). HISTORICAL AND NONHISTORICAL APPROACHES IN BIOLOGY As products of evo lut ion , organisms are h i s t o r i c a l e n t i t i e s . They can, however, be examined from e i ther h i s t o r i c a l or n o n h i s t o r i c a l perspect ives . The in trus ion of t e l e o l o g i c a l explanations into what should be mechanistic explanations in biology is prevented by maintaining a d i s t i n c t i o n between these perspect ives . Even though the questions may be phrased in s imi lar ways, each is looking for a d i f f erent type of answer. Each is usual ly associated with p a r t i c u l a r b i o l o g i c a l d i s c i p l i n e s , and disagreements over what const i tutes a v a l i d explanation of a phenomenon may ar i se because h i s t o r i c a l and nonh i s tor i ca l perspectives are being mixed. In a d d i t i o n , some 18 d i s c i p l i n e s are more interested in s t r u c t u r a l propert i e s , while others are more interested in funct ional proper t i e s . These c r i t e r i a give four ways of looking at biology: nonh i s tor i ca l s t ruc tura l i sm, nonhi s tor i ca l funct ional i sm, h i s t o r i c a l s t ruc tura l i sm, and h i s t o r i c a l funct ional i sm. NONHISTORICAL STRCUTURALISM AND FUNCTIONAL!SM: Studies of th i s type take the existence of an organism for granted and ask questions such as "What is i t made' of?" and "What does i t do?". They are usual ly known as form-and-function s tudies . Anatomy, physiology, molecular bio logy, biochemistry, biomechanics, and medicine, for example, are nonh i s tor i ca l d i s c i p l i n e s . By th i s I mean not that one cannot do evolut ionary research from within these areas of study, but that one does not have to do such research in order to study these subjects . Embryology can also take a nonh i s tor i ca l perspect ive , for even though i t studies organic development through time, i t need not address the evolutionary o r i g i n of those ontogenies. Even much of population biology i s n o n h i s t o r i c a l , for the same, reason that i t does not address evolut ionary o r i g i n s . For example, K e t t l e w e l l ' s (1955, 1961, 1965) c l a s s i c studies on i n d u s t r i a l melanism in Biston b e t u l a r i a were concerned with changes in the r e l a t i v e frequencies of pigmentation patterns in a given system, and not with how the pigmentation propert ies themselves came to e x i s t . (But see Lambert et al., 1986, for comments on the v a l i d i t y of K e t t l e w e l l ' s conclus ions . ) By d i sassoc ia t ing themselves from the question of h i s t o r i c a l o r i g i n , n o n h i s t o r i c a l studies can take e i ther a 19 mechanistic or a t e l e o l o g i c a l approach without creat ing any problems. The questions "What i s i t made of?" and "What does i t do?" are mechanist ic . But i t may be possible to discover more about the propert ies of the organism by asking the t e l e o l o g i c a l "What i s th i s for?" . From a nonh i s tor i ca l perspect ive , "what for" questions are per fec t ly l eg i t imate . This i s the proper place of teleology in b io logy. But there is a pr i ce pa id , for i t must be acknowledged that such t e l e o l o g i c a l causation plays no role, in the o r i g i n of the proper t i e s . Nagel (1984) considered t e l e o l o g i c a l explanations in biology to be acceptable when they simply refer to funct ional a t t r i b u t e s . Even though such explanations contain phrases such as "in order to" and "the purpose of", they create no problems because they are not intended as an e t i o l o g i c a l explanation. I concur. An anatomist studies the c i r c u l a t o r y system of vertebrates , asks "What i s the heart for?", and answers "For pumping the blood." An endocrinologist studies nervous transmiss ion, asks "What i s acety lchol inesterase for?", and answers "For maintaining proper leve ls of ace ty l cho l ine ." This approach treats organisms as if_ they had been purposeful ly designed; i t looks for explanations for functions and structures in the same way that a mechanic would examine an o i l pump or the e l e c t r i c a l system of an engine. It treats the end-directed a c t i v i t y of teleonomic systems as i f i t were the goal-seeking a c t i v i t y of a t e l e o l o g i c a l system. The te leology that th i s introduces is not problematic because i t is divorced from mechanistic causal explanations for the existence of organisms and i s used as only 20 an explanatory analogy. This decoupling i s one reason why centuries of data on the propert ies of organisms can s t i l l be used today, even though most of them were c o l l e c t e d not only before evolutionary theory was developed, but under i m p l i c i t or e x p l i c i t assumptions of purposeful design. Biology today makes use of the f indings of Harvey, Vesa l ius , Owen, and Agassiz - and i t does not matter how those people thought l i v i n g systems came to be. HISTORICAL STRUCTURALISM: I apply th i s term to the view of evolution advocated here. In th i s view, function i s an ef fect of s tructure , and has no causal power of i t s own; evolut ionary change is s t r u c t u r a l change, from which funct ional change emerges; and any end-atta ining aspects of evolut ionary change comes from teleomatic processes operating wi th in , but not because of, a teleonomic context. In no sense of the word does evolution occur for something. "What for" questions are not appl icable in h i s t o r i c a l s t r u c t u r a l i s m . Instead, the questions asked are of the type, "How did i t come to be?", or , "Through what means d id i t come to have these propert ies?". HISTORICAL FUNCTIONAL!SM: This approach looks to funct ion , rather than to s t ruc ture , for the explanation of how things came to be. In doing so, i t looks for causal powers in what are , in fac t , e f f ec t s . Other authors have noted the shortcomings of such a viewpoint in morphological ( e . g . , Lauder, 1981, 1982; Smith, 1982) and behavioral studies (Jamieson, 1986). The  i n a b i l i t y to f ind mechanistic evolutionary causes in 21 f u n c t i o n a l i t y resu l t s in the extension of the t e l e o l o g i c a l  c a u s a l i t y of nonh i s tor i ca l studies into the h i s t o r i c a l realm. This extension is not v a l i d because such t e l e o l o g i c a l causa l i ty does not ac tua l ly e x i s t . If the extension i s made, the resu l t i s an h i s t o r i c a l teleology that asks a second type of "what for" quest ion. This type i s unacceptable. It asks "What d id th i s or ig inate for?", or "For what purpose d id th i s come to be?" H i s t o r i c a l funct ional ism does not necessar i ly recognize a process of evolut ion through descent with modi f i ca t ion . If i t does not, then i t s teleology comes from nothing- more than an argument from design: t h i s performs a funct ion , therefore , i t came to exis t so as to perform that funct ion. If h i s t o r i c a l funct ional ism does recognize evo lut ion , the question then becomes "What d id th i s evolve for?", and the h i s t o r i c a l te leology of the answers becomes an evolutionary teleology of the type "The heart evolved in order to c i r c u l a t e the blood." As discussed below, an over -re l iance on natural se lec t ion in evolut ionary explanations can produce a var iant of th i s evolutionary teleology that I have termed adaptat ional teleology (O'Grady, 1984, 1986). NATURAL SELECTION Although I have suggested that the transformational causes recognized by h i s t o r i c a l s t ructura l i sm are necessary and s u f f i c i e n t for some kind of b i o l o g i c a l evolut ion to occur, I am not suggesting that they are the only causes a f fec t ing the 22 appearance of the organisms that have ac tua l ly evolved on earth . F u n c t i o n a l i t y does play a ro le in c e r t a i n evolutionary phenomena. This is the basis of Darwin's theory, and of the der iva t ive theory, neo-Darwinism. 1 In addi t ion to being effected by the generative processes of h i s t o r i c a l s t r u c t u r a l i s t causes, b i o l o g i c a l systems can be affected by imposed e l iminat ive processes. Natural se lec t ion is an e l iminat ive force . It involves the surv iva l of a subset of organisms from a heterogeneous grouping according to the r e l a t i v e funct ional e f f i c i ency of the organisms in a p a r t i c u l a r environment. Those organisms that function best (at ta in and maintain the i r teleonomic end-state best) w i l l be in a pos i t ion to leave more o f f spr ing than those that p e r i s h . This view of natural se l ec t ion appears to agree with that of Brandon (1981), who i d e n t i f i e d three d i f f e r e n t i a l processes at work: (1) d i f f e r e n t i a l reproduction re su l t ing from (2) the d i f f e r e n t i a l s u r v i v a l of p o t e n t i a l progeni tors , such d i f f e r e n t i a l s u r v i v a l r e su l t ing from (3) d i f ferences in the  organisms' r e l a t i v e funct ional e f f i c i e n c i e s . Brandon considered a l l three factors to be necessary for natural s e l e c t i o n . This i s because subsets of one or two of the factors can occur in 1 For current purposes, th i s i s defined as those evolutionary explanations that look to natural se lec t ion as the primary cause of evo lu t ion , that i s , the explanation of f i r s t choice . Neo-Darwinism is thus not a labe l that should be appl ied to every view that recognizes natural se l ec t ion somewhere in i t s framework. The nature of a theory depends upon the way in which i t s components are s tructured , and not on a simple enumeration of the components contained. 23 other circumstances. For example, d i f f e r e n t i a l reproduction can a lso be caused by acc identa l events, unrelated to funct ional e f f i c i e n c y , that k i l l off potent ia l breeders. D i f f e r e n t i a l "surviva l" can also occur among inanimate objects , such as granite and sandstone deposits on a mountainside. In the face of eros ional forces , the granite w i l l maintain i t s form for a longer time than w i l l the sandstone. One cannot c a l l th i s process natural se lect ion and expect that term to re ta in i t s usefulness for b i o l o g i c a l s tudies , e spec ia l l y with respect to concepts of adaptation (discussed below). Lewontin (1980) gave three factors that he considered necessary and s u f f i c i e n t for evolut ion by natural s e l ec t ion : (1) v a r i a t i o n , (2) h e r i t a b i l i t y of that v a r i a t i o n , and (3) d i f f e r e n t i a l f i tness of the inher i tors of that v a r i a t i o n (I suggest that the t h i r d factor i s the net resu l t of Brandon's three aspects of natural s e l e c t i o n ) . I think that th i s t r i a d can be restated as: the processes that produce novelty (factor 1), the processes that perpetuate that novelty (factor 2), and the processes that e l iminate some of that novelty (factor 3) . Stated in th i s way, I suggest that factors 1 and 2 are s u f f i c i e n t not only for some kind of b i o l o g i c a l evolut ion to occur, but for i t s products to possess s u f f i c i e n t organizat ion to survive . This is because of the phylogenet ica l ly internested nature of the causes of b i o l o g i c a l organizat ion . A newly evolved organism does not develop from a state of disorder which must be somehow ordered by an imposed force such as natural s e l e c t i o n . Instead, the continuum of inheritance provides 24 h i s t o r i c a l l y accumulated proper t i e s , and thus sets the i n i t i a l  condit ions that resu l t in the production of descendent order from ancestra l order. Natural se lec t ion can thus be said to act as a proximate cause operating among b i o l o g i c a l systems, each of which i s a hierarchy of ultimate and proximate within-system causa l i ty ( F i g . 2) . Se lect ion may operate during the evolutionary h is tory of a l ineage, but because i t s among-system causa l i ty is e x t r i n s i c to any p a r t i c u l a r organism, i t cannot become incorporated into the h i s t o r i c a l causa l i ty of the l ineage. Invest igat ions of se lec t ion may therefore address the h i s tory of the evolut ion of an organism, but not the h i s t o r i c a l , transformational processes that caused the organism to e x i s t . I am not suggesting that any evolutionary b i o l o g i s t today would argue that a l l b i o l o g i c a l order is the resu l t of natural se l ec t ion operating upon t o t a l l y unordered v a r i a t i o n . Futuyma (1984), for example, considered such an unqual i f i ed concept to be "s impl i s t i c" . But there can be a tendency to lean towards modified versions of th i s p o s i t i o n . This tendency appears to be a r e l i c t of the controvers ies of the 1860s to the 1920s. There was at that time a s truggle , f i r s t for the acceptance of Darwinian evolutionary theory, and then for the primacy of neo-Darwinian, mutat ionis t , or thogenet i c i s t , or neo-Lamarckian explanations (see Bowler, 1983). One of the major disputes was whether evolut ion was in any way predetermined or d irected towards a goa l . Orthogenet ic i s t s and neo-Lamarckians had d i f f i c u l t y keeping the ir proposed mechanistic explanations free 25 from te leo logy . In a d d i t i o n , some c r i t i c s of evolut ion offered b la tant ly t e l e o l o g i c a l a l t e r n a t i v e explanations. The concept of purposeless change with which the Darwinians and neo-Darwinians attempted to introduce mechanistic causa l i ty advocated natural se lec t ion act ing upon undirected v a r i a t i o n . This means that v a r i a t i o n is not d irected towards the future needs of organisms. It can thus be said to be "random" with respect to those needs, but i t cannot be said to be random with respect to the sources that produced i t . Res tr ic ted v a r i a t i o n , persistence of ordered s tates , and morphological trends in evolut ion are not ind ica t ive of predetermination; they resul t from unique h i s t o r i c a l events. When change must take place within the i n i t i a l condit ions establ i shed by these past events, i t can be said to be past- determined. This is not the same as fu ture -d irec ted change that serves the needs of the organism. ADAPTATION Darwin's theory recognized a p a r t i c u l a r type of organic f u n c t i o n a l i t y , the r e l a t i v e e f f i c i ency of which allowed s u r v i v a l from the e l iminat ive forces of natural s e l e c t i o n . This f u n c t i o n a l i t y i s adaptat ion. (I do not address the app l i ca t ion of the term "adaptation" to refer to in tra -generat iona l adjustments to new condi t ions , other than to say that i t s use in such instances should be avoided.) Some workers ( e . g . , Bock, 1967, 1979, 1980), have taken the view that "On t h e o r e t i c a l grounds, a l l ex i s t ing features of animals are adaptive." (1967: 26 63). Others, such as Will iams (1966) and Gould and Vrba (1982), have argued that the designation of adaptation should be reserved for features that have been affected by natural s e l e c t i o n . I agree with Brandon (1981), van der Steen (1983), and van der Steen and Voorzanger (1984) that the t ight coupling of cause and effect in the l a t t e r approach is pre ferable . However, I do not accept Wil l iams' (1966: 9) argument that only adaptive characters have "functions", while any remaining characters have "effects". A l l funct ion , I suggest, i s an effect of s tructure , regardless of whether se lec t ion and adaptation are involved. Adaptive f u n c t i o n a l i t y is simply one type of organic f u n c t i o n a l i t y , and adaptive s t r u c t u r a l changes must occur within the context of organic s t r u c t u r a l changes. Adaptations are "useful" to the organism in that they have allowed i t s surv iva l from se lec t ion because of a r e l a t i v e super ior i ty of funct ion . But no matter how useful a character i s , i t has not necessar i ly become that way because of s e l ec t ion . It could have been produced by genetic and epigenetic assoc iat ions with other characters that have been se lected. (Brandon, 1981, termed these epiphenomenal t r a i t s , and included with them gene linkage and p l e i o t r o p i c e f f ec t s . ) A useful character could also have evolved as a monomorphic der ivat ive of an ances tra l cond i t ion , or i t could have been inher i t ed , unchanged, from an ancestor. Se lect ion can be c i t e d as a contr ibut ing cause of character usefulness only when there are grounds to bel ieve that there has been a process of d i f f e r e n t i a l s u r v i v a l from a polymorphic stage 27 because of r e l a t i v e funct ional d i f ferences in that character . Otherwise, there is the r i s k of t r i v i a l i z i n g the concept of adaptation by synonomizing i t with p r a c t i c a l l y any aspect of b i o l o g i c a l organizat ion . This would cause some of the perceived evidence for natural se lec t ion to come, in fac t , from the f a l l a c y of Aff irming the Consequent: from the statement "i f adaptation through s e l e c t i o n , then usefulness and f u n c t i o n a l i t y " , i t is concluded "usefulness and f u n c t i o n a l i t y , therefore adaptation through se l ec t ion ." Some authors appear to have adopted th i s l ine of argument. Bock (1967: 63), for example, stated "If [ a l l ex i s t ing features of animals] were not adaptive, then they would be el iminated by se l ec t ion and would disappear". Ayala (1976) and Simpson (1958) have presented s imi lar arguments that equate b i o l o g i c a l organizat ion and f u n c t i o n a l i t y with adaptat ion. Once the state of adaptation has been synonymized with b i o l o g i c a l organizat ion , the process of adaptation may then become synonymized with the process that produces that organizat ion , namely, evolut ion (see Eldredge, 1985: 107). Such a perspective confounds studies of causes and the i r e f f ec t s . It a lso ensures a specious supply of confirmations of the e f f i cacy of neo-Darwinian theory. When natural se lect ion is taken to be the primary cause of evo lu t ion , i t s e f f ec t , adaptat ion, becomes incorporated into explanations of why organisms, or parts of organisms, evolved. The resu l t i s a t e l e o l o g i c a l causa l i ty that is r e l i e d upon to explain the phenomena concerned. This teleology i s a form of h i s t o r i c a l funct ional ism, and i t is not d i f f i c u l t to f ind 28 examples of i t in the research l i t e r a t u r e (see O'Grady, 1984). Organisms are imbued with powers of foresight and a c t i o n . Questions such as "Why does th i s frog have green skin?" are given answers such as "I_n order to be better camouflaged" and "So that i t could not be seen by predators". I agree with Nagel (1984) that sentences of th i s type are acceptable for expla ining contemporary funct ional propert ies ( i . e . , from what I have termed a nonh i s tor i ca l perspec t ive ) . But I suggest that they are p o s i t i v e l y misleading when they are used in evolutionary explanations. One undesirable aspect of the re l iance on t e l e o l o g i c a l causa l i ty that h i s t o r i c a l funct ional ism produces i s that i t gives a fa lse sense of securi ty about how well an evolut ionary phenomenon has been explained. That i s , i f some kind of causal explanation seems to have been provided, then there w i l l be less incentive to study the matter fur ther . A SINGLE EXPLANATORY FRAMEWORK Natural se lec t ion and h i s t o r i c a l s t ructura l i sm can be brought together in a protocol that factors out the r e l a t i v e contr ibut ions of causes by considering the more general causes f i r s t , and then the less general causes only i f necessary. As an extreme example, one would not look to a se lec t ion l e v e l cause to explain why a cat walks upon the ground (se lect ion against those that disobeyed grav i ty and rose into the a i r ? ) . By extension, i t may not be j u s t i f i a b l e to immediately look to 29 se lec t ion when t ry ing to explain why cats have four l imbs, or a p lacenta , or r e t r a c t i l e claws. The internest ing of causes in h i s t o r i c a l l y - p r o d u c e d systems means that there i s a primacy of  act ion of some causes over others . It follows that explanations a t t r i b u t i n g phenomena to more general causes have l o g i c a l  pr imacy over those employing less general causes. The more general explanation should thus be the explanation of f i r s t  choice , or the i n i t i a l hypothesis• Its acceptance gives the least departure from the data and the greatest consistency with the causes known to be capable of producing the e f fects in quest ion. It is to be retained so long as a d d i t i o n a l data do not show i t to be inadequate. It must be noted that in the internested hierarchy of evolutionary c a u s a l i t y , the i n i t i a l hypothesis for any p a r t i c u l a r l e v e l appeals to the causal propert ies of the immediately lower l e v e l . Thus, for a hierarchy of n l e v e l s , there are n-1 i n i t i a l hypotheses. As w i l l be shown in the fol lowing four subsections, th i s internest ing of explanation resu l t s in a sequence of evolut ionary analyses, each of which begins with the resu l t s of an analys i s of the immediately lower l e v e l of c a u s a l i t y . 30 1. INTRASPECIFIC INVESTIGATIONS Suppose one wanted to provide an evolutionary explanation for a species of frog having s t r i c t l y green skin c o l o r a t i o n . B i o t i c or a b i o t i c se lec t ion may have acted as a proximate cause (C1) by e l iminat ing non-green frogs from a polymorphic state produced by a non-green immediate ancestor ( F i g . 3a). If so, the developmental a l t e r a t i o n that f i r s t produced the color change is the' ult imate cause (C2), and the complete explanation for green co lora t ion must refer to both the developmental and s e l e c t i o n a l causes. There are , however, at least three other means by which the species could come to be green and only green. (1) The non-green immediate ancestor may have produced only green descendants that were not subject to any se lect ion for color ( F i g . 3b), in which case only the ancestra l developmental a l t e r a t i o n would need to be c i t e d as a cause. (2) Se lect ion may have acted upon a polymorphic state produced by a green immediate ancestor (F ig . 3c) . (3) The current green species may simply be descended from a green immediate ancestor, and be unaffected by se lec t ion ( F i g . 3d). The las t two cases do not address the question of the o r i g i n of green c o l o r a t i o n , and so the answer must be sought further back in the phylogeny, in even more general l eve l s of causa l i ty (C3). However, for the species whose characters prompted the study, the cause of green co lora t ion has been determined. In a l l four cases above, the complete causal explanation for the condi t ion of the species includes a component of h i s t o r i c a l c a u s a l i t y . In the absence of data suggestive of 31 s e l e c t i o n , t h i s h i s t o r i c a l causa l i ty should be accepted as a s u f f i c i e n t explanation. H i s t o r i c a l causa l i ty is thus the explanation of f i r s t choice, while h i s t o r i c a l plus s e l e c t i o n a l causa l i ty i s the explanation of second choice . This r e l a t i o n s h i p among causes in an internested system d i f f e r s from the more t r a d i t i o n a l e i ther -or method of a n a l y s i s , which is derived from A r i s t o t l e ' s Law of the Excluded Middle . It i s not a question of h i s tory or s e l ec t i on , but one of whether se lec t ion has acted upon a system already affected by more ult imate causes. 2. PHYLOGENETIC INVESTIGATIONS At the next l eve l up, the inference of the evolutionary re la t ionsh ips of a group of organisms takes as i t s i n i t i a l hypothesis the supposition that s i m i l a r i t i e s among organisms are caused by descent from a common ancestor. These s i m i l a r i t i e s are homologues. The app l i ca t ion of Hennigian methodology (Hennig, 1966) then constructs a putat ive genealogy by internest ing the organic s i m i l a r i t i e s a t t r i b u t a b l e to shared derived homologues (synapomorphies) within those a t t r i b u t a b l e to shared pr imi t ive homologues (symplesiomorphies). This attempt to minimize the number of postulated instances of character evolut ion i s a parsimony argument, hence, the connection in the systematics l i t e r a t u r e between c l a d i s t i c s and parsimony ( e . g . , Sober, 1983; Fe l sens te in , 1982, 1984; F a r r i s , 1982, 1985). An analys i s of , say, four taxa ( F i g . 4a) begins with any basal 32 synapomorphies and autapomorphies (derived t r a i t s on terminal branches of the t r e e ) . Then, through the app l i ca t ion of Hennigian Argumentation (Hennig, 1966), the tree i s resolved with the addi t ion of other character data ( F i g . 4b). As each character i s added, i t s c a u s a l i t y i s , whenever poss ib le , a t t r ibuted to a s ingle h i s t o r i c a l o r i g i n . The resul t i s an internest ing of characters and the i r causes ( F i g . 4d). It is c r u c i a l to note that , as an a p p l i c a t i o n of h i s t o r i c a l s t ruc tura l i sm, phylogenetic systematics constructs genealogies from internested patterns of in ferred homologous t r a i t s regardless of the perceived funct ional importance or lack  thereof of those t r a i t s . The focus is on inher i ted t r a i t s as indicators of k insh ip , and i t does not matter to what use those t r a i t s may have been put. Thus, no character of an organism, no matter how useless or t r i v i a l i t may seem, is to be considered a p r i o r i to be uninformative of phylogenetic r e l a t i o n s h i p s . Conversely, the retention of a character through an evolutionary lineage is not to be immediately taken as ind ica t ive of i t s funct ional importance to the organisms. It may be important, but that should not be be concluded merely from i t s continued existence. H i s t o r i c a l funct ional ism can produce an argument of the form, "If i t i s here, i t must be doing something; and i f i t has been here for a long time, i t must be doing something indispensable". 33 3. LIFE HISTORY INVESTIGATIONS Because a phylogenetic tree postulates patterns of common descent, i t can be used to invest igate the evolut ion of propert ies other than those used to construct the t ree . L i f e h i s tory t r a i t s are one such type of property . The digenean trematodes are an in teres t ing group for such a study because of the numerous l i f e h i s tory components involved in the ir complex l i f e cycles in an invertebrate intermediate host and a vertebrate f i n a l host. The o r i g i n of such a condit ion can be inferred from a phylogenetic tree for the p a r a s i t i c flatworms. Figure 5a gives the resul t of such a study (Brooks et a l . , 1985a). This is a cladogram 1 for the p a r a s i t i c platyhelminths , a group that includes digenean and monogenean "trematodes", as well as the Cestoidea, or tapeworms. There are three types of l i f e cyc les : d i r e c t in an invertebrate host (DI), d i r e c t in a vertebrate host (DV), or i n d i r e c t (complex), involv ing both host groups (CB). These propert ies can be mapped onto the tree , and the ances tra l states can be in ferred by optimizing the nodal values so as to give the most parsimonious ( i . e . , most homologous) in terpre ta t ion of character evo lu t ion . The opt imizat ion procedures of both F a r r i s (1970) and of Swofford and Maddison ( in review) give the nodal values in Figure 5b. This pos i t s that there were three changes in an ances tra l d i r e c t 1 An hypothesis of phylogenetic r e l a t i o n s h i p s , constructed from inferred synapomorphic characters , and cons i s t ing of an internested set of monophyletic groups sensu Hennig (1966), or c lades . See Chapter I I I . 34 l i f e cycle in an invertebrate: the addi t ion of a vertebrate host in the Digenea, the addit ion of a vertebrate host in the common ancestor of the Cercomeromorphae (the four taxa on the r i g h t ) , and the addi t ion of an invertebrate host in the Cestoidea. This analys i s was f i r s t presented in Brooks (1982; see also Brooks and Wiley, 1984, and Brooks et a l . , 1985a). O'Grady (1985) discussed at length the means by which the cladogram could be used to infer the evolutionary events that were involved in the production of a complex l i f e cycle in both the Digenea and the Cestoidea. The re la t ionsh ips in the cladogram suggest that cer ta in characters in the adults of the Monogenea, Gyrocoty l idea , and Amphilinidea are homologous with those in the larvae of the Cestoidea. For example, a hook-bearing adhesive disc at the poster ior of the body, which is a synapomorphy for the Cercomeromorphae, i s present only in the early l a r v a l stages of the Cestoidea. The characters usual ly associated with tapeworms, such as s t r o b i l i z a t i o n into mult ip le body segments, and the formation of an anter ior holdfast organ, c a l l e d a scolex, are adult characters that seem to have been added onto the ances tra l ontogeny by terminal a d d i t i o n . Furthermore, whereas the other three members of the Cercomeromorphae develop in vertebrate hosts, the comparable ontogenetic stage in the tapeworms, the l a r v a , develops in an invertebrate , which is in ferred to be a more recent ly acquired host . The plesiomorphic host, the vertebrate , contains the apomorphic ontogenetic stage, the adu l t . This suggests an i n t e r c a l a t i o n , or a nonterminal  add i t ion , of an invertebrate host into the ancestral l i f e c y c l e . 35 The evolut ion of the Digenea appears to have involved just the opposite processes. The cladogram supports an inference of homology between cer ta in characters of the adult stages of digeneans and the ir s i s t e r group, the Aspidocoty lea . The sequence of digenean l a r v a l stages (miracidia - sporocyst redia - cercar ia ) associated with the complex l i f e cycle has no homologues in the Aspidocotylea . This suggests that they have been i n t e r c a l a t e d , by nonterminal a d d i t i o n , into the ancestral ontogeny. Conversely, the invertebrate host present in the D a l y e l l o i d e a , Temnocephalidea, and Aspidocotylea l i f e cycles holds the apparently more recent ly evolved digenean l a r v a l stages. Thus, i t i s the more recent ly acquired vertebrate host that holds the plesiomorphic, adult ontogenetic stage of the Digenea. This suggests a terminal addi t ion of a vertebrate host into the ancestra l l i f e c y c l e . This analys i s suggests that the complex l i f e cycles of tapeworms and digeneans arose through d i f f erent means. Tapeworms show terminal addi t ion of ontogenetic stages and nonterminal addi t ion of a host. Digeneans show nonterminal addi t ion of ontogenetic stages and terminal addi t ion of a host . The general ized term "complex l i f e cycles" would thus appear to be an instance of funct ional equivalence in the face of s t r u c t u r a l d i f f erences . 36 4. COMMUNITY STRUCTURE INVESTIGATIONS L o g i c a l primacy and explanations of f i r s t choice have also been examined in community s tructure analyses ( e . g . , ' Quinn and Dunham, 1983; Roughgarden, 1983; Simberloff , 1983; Strong, 1983). The question has been whether one hypothesis has primacy of cons iderat ion over another, or whether there i s such a m u l t i p l i c i t y of impinging causes of equal status that one hypothesis is as reasonable a s t a r t i n g point as any. Brooks (1985; see a lso Brooks and Wiley, 1986) suggested that neither of the hypotheses discussed so f a r , random d i spersa l (Roughgarden, 1983) or competition effects (Simberloff , 1983; Strong, 1983) i s a su i table i n i t i a l hypothesis for b i o l o g i c a l systems. I concur. The assumption of h i s t o r i c a l l y determined re la t ionsh ips appeals to the most general causes in community s tructure a n a l y s i s . Of course, th i s approach does not deny that factors such as co lon iza t ion and competition may have affected communities. It simply argues that the h i s t o r i c a l contr ibut ion must be factored out f i r s t ( F i g . 6) . Simberloff (1983) and Strong (1983) presented arguments that can be interpreted to support th i s approach. They noted the need to examine i n t r a s p e c i f i c , au teco log ica l , fac tors , such as v a g i l i t y , before deal ing with i n t e r s p e c i f i c , syneco log ica l , f ac tors , such as competit ion. Strong (1983: 639) came close to a t t r i b u t i n g th i s requirement to the h i s t o r i c a l nature of b i o l o g i c a l systems. It would seem that these goals are best met by formulating i n i t i a l hypotheses of h i s t o r i c a l causa l i ty ( e . g . , Brooks, 1979a, 1979b, 37 1980, 1981a, 1985; see also Brooks and M i t t e r , 1984; Mit ter and Brooks, 1983; Cressey et a l . , 1983; C o l l e t t e and Russo, 1985). "WHY" QUESTIONS The recognit ion of the d i f f erent types of b i o l o g i c a l questions discussed here is made a l l the more d i f f i c u l t when the adverb "why" is used. Questions are sometimes categorized according to the interrogat ive adverb they use. I suggest that there are no d i s t i n c t c lasses of "what" quest ions, "how" questions, or "why" questions in b io logy , but that i t i s the form of the sentence that is more important in determining what kind of question i s being asked. The adverb "why" is problematic because the same sentence can be used to ask questions that ant i c ipate h i s t o r i c a l , n o n h i s t o r i c a l , s e l e c t i o n a l , or even t e l e o l o g i c a l answers. For example, the quest ion, "Why do cats have fur?" can be answered with "epidermal der iva t ion" , "common inheritance from a mammalian ancestor", "common inheritance from a mammalian ancestor whose fur gave i t a r e l a t i v e funct ional advantage during a period of se lect ion", or "so that they can keep warm". In a d d i t i o n , in comparative b i o l o g i c a l studies a question such as "Why does i t have this?" i s often intended simply to mean "Is there a function for th is?" or "Is there a function associated with th i s being d i f f erent in th i s p a r t i c u l a r organism?". The ambiguity produced by using "why" questions as abbreviated forms of mechanistic causal questions can introduce 38 the teleology of h i s t o r i c a l funct ional i sm. Mayr (1961) avoided th i s ambiguity by noting that the "why" questions of evolutionary biology (= the h i s t o r i c a l perspect ive , discussed herein) should be taken to mean the mechanistic "how come", and not the f i n a l i s t i c "what for". I recommend the approach put forward in th i s d i s s e r t a t i o n because i t recognizes two types of "what for" questions - one of which is v a l i d , one of which is not - and because i t avoids the use of "why" questions a l together . SUMMARY The causes responsible for the existence of an organism are internested in l eve l s of genera l i ty . As spec i f i c conf igurat ions of phys ica l elements, organisms are subject to general , phys ica l causes, and then to p a r t i c u l a r , b i o l o g i c a l causes. Within th i s hierarchy are evolut ionary causes that were once contingent for a p a r t i c u l a r species , but have subsequently been incorporated into the h i s t o r i c a l c a u s a l i t y of i t s descendent species . This internest ing of proximate and ult imate causa l i ty i s best studied with a theory that appeals to s i m i l a r l y internested causes. The more general , h i s t o r i c a l causes have operative and thus l o g i c a l p r i o r i t y over the less general causes. The interact ions of the s tructures in b i o l o g i c a l h i erarch ies produce functions that contribute to the s u r v i v a l of the organism. Organisms must function i f they are to continue ex i s t ing as organisms, rather than as c o l l e c t i o n s of s tructured , 39 but inanimate, matter. Function i s an effect of s t ruc ture . There are three types of end-atta ining a c t i v i t y that can ex i s t in b i o l o g i c a l systems: teleomatic, or end-resu l t ing ; teleonomic, or end-directed; and t e l e o l o g i c a l , or goal-seeking. Organic f u n c t i o n a l i t y a t ta ins end-states, but these are not goals , and the f u n c t i o n a l i t y is not t e l e o l o g i c a l . An h i s t o r i c a l l ineage of organisms p e r s i s t s over generations of teleonomic a c t i v i t y u n t i l teleomatic d i srupt ions in species cohesion resu l t in the evolution of new species with der ived , but a l t e r e d , s t r u c t u r a l proper t i e s . Function may change a l so , but th i s i s not a necessary consequence. Teleomatic processes occur because of the phys ica l propert ies of the matter cons t i tu t ing the organism, and not because of any ef fects they might have on the teleonomic system in which they take place . At any point in th i s evolutionary process, natural se lec t ion can el iminate those organisms whose r e l a t i v e funct ional e f f i c i e n c y is inadequate for s u r v i v a l in the ir environment. Although i t can become part of the evolutionary h i s tory of a l ineage, se lec t ion acts as a proximate cause operating among organisms; i t i s not responsible for the wi th in -organism h i s t o r i c a l causa l i ty that produces changes in organic form. The r e s u l t i n g adaptation of the surviv ing organisms is an e f f ec t , not a cause, of the ir evo lu t ion . Only natura l se lec t ion produces adaptat ion. An adaptive character i s useful to the organism that possesses i t , in that i t has allowed s u r v i v a l of 40 s e l e c t i o n , but not a l l useful characters , no matter how indispens ib le to continued s u r v i v a l , are necessar i ly adaptations. Select ion and adaptation are not necessary for the existence of f u n c t i o n a l i t y , usefulness, organizat ion , or teleonomic a c t i v i t y . As products of evo lut ion , organisms are h i s t o r i c a l e n t i t i e s . They can, however, be studied from e i ther a nonh i s tor i ca l or h i s t o r i c a l perspect ive . The f i r s t i s interested in current propert ies and does not ask where they came from. The second is interested in how organisms came to be, and is thus evolut ionary . Nonhis tor ica l studies can be  s t r u c t u r a l i s t or f u n c t i o n a l i s t , and can ask mechanistic or t e l e o l o g i c a l quest ions. The l a t t e r are a form of "what for" quest ion, but the teleology creates no problems because i t comes from a machine analogy and i s not intended to be extended to h i s t o r i c a l explanations. H i s t o r i c a l s t ructura l i sm is the term given to the view advocated in th i s d i s s e r t a t i o n : evolutionary change i s s t r u c t u r a l change, from which funct ional change emerges. In no sense of the word does evolut ion occur for something; "what for" questions are not appl icable in h i s t o r i c a l s t ruc tura l i sm. H i s t o r i c a l funct ional ism i s the resu l t of attempting to use current functions to explain the o r i g i n s of the s tructures that make those functions poss ib le . This places ef fect before cause in the explanatory sequence, and produces an unacceptable te leo logy. In an evolutionary context, th i s can be termed evolutionary te leo logy, a var iant of which can be termed 41 adaptat ional te leo logy. This has i t s own type of "what for" quest ion: i t asks "What d id th i s evolve for?", and i t answers "In order to adapt the organism to x". Adaptational te leology is not the inev i tab le resu l t of se lect ion-adaptat ion explanations, for such explanations are mechanistic as long as they are r e s t r i c t e d to dealing with proximate among-system c a u s a l i t y . Adaptational teleology i s the resu l t of improperly extending se lect ion-adaptat ion explanations to the realm of h i s t o r i c a l within-system c a u s a l i t y . Neo-Darwinian approaches can encounter problems d i s t ingu i sh ing th i s .error because, on the one hand, natural se lec t ion acts upon the same funct ional propert ies with which t e l e o l o g i c a l nonh i s tor i ca l studies dea l , and, on the other hand, i t s explanatory framework does not include those causes responsible for the production and transformation of organic form. The recognit ion of these types of questions i s made a l l the more d i f f i c u l t by the ambiguity produced with the use of the adverb "why". CONCLUSIONS I have argued here that h i s t o r i c a l s tructura l i sm is a productive way of studying evo lu t ion . Its use seems to allow the examination of both pattern and process from within a s ingle explanatory framework. The fol lowing two chapters provide an app l i ca t ion of h i s t o r i c a l s t r u c t u r a l i s t methodology. The study begins with the construct ion of a phylogenetic t ree . 42 I I I . PHYLOGENETIC ANALYSIS OF HAPLOMETRANA LUCKER, 1931 AND SPECIES OF GLYPTHELMINS STAFFORD, 1905 (DIGENEA: PLAGIORCHIOIDEA) IN NORTH, CENTRAL, AND SOUTH AMERICA As discussed in the previous chapter, h i s t o r i c a l s t ructura l i sm in phylogenetic systematics postulates that h i s t o r i c a l processes produce a set of t r a i t s that covary with phylogeny. These are homologues. As each character is brought into a phylogenetic a n a l y s i s , the ex i s t ing tree acts as an hypothesis that predic t s that the new character w i l l show the same patterns of r e l a t i o n s h i p as do the previous ly-cons idered characters . The ex i s t ing tree can be supported, modified, or re jec ted . This chapter presents such a procedure. INTRODUCTION Glypthelmins S ta f ford , 1905 and Haplometrana Lucker, 1931 are members of the Superfamily P lag iorchio idea D o l l f u s , 1930. Approximately 19 species have at one time or another been placed in the genus Glypthelmins (see below). They are p a r a s i t i c in the i n t e s t i n e , rare ly the g a l l bladder, of amphibians in the New and Old World. The seven species of Glypthelmins studied in th i s d i s s er ta t i on are a l l p a r a s i t i c in the in tes t ine of anurans, p r i m a r i l y ranids , in North, C e n t r a l , and South America. The eighth species studied here, Haplometrana i n t e s t i n a l i s Lucker, 1931, is p a r a s i t i c in the in tes t ine of ranids in western North America and is the only member of that genus. 43 The f a m i l i a l and generic status of Glypthelmins has been revised a number of times since Staf ford (1905) erected the genus to" receive Pistomum quietum Sta f ford , 1900. On the basis of two characters , excretory v e s i c l e shape and c e r c a r i a l t a i l morphology, the genus has been placed in e i ther the P lag iorch i idae Luhe, 1901 (F i shtha l* and Kuntz, 1967; Mart in , 1969; Ulmer, 1970; S u l l i v a n , 1976), or the Macroderoididae McMullen, 1937 (Schell' , 1962a; Odening, 1964; S u l l i v a n and Byrd, 1970; S u l l i v a n , 1976). On the basis of four characters , excretory v e s i c l e shape, uterine extent, v i t e l l a r i a n extent, and presence of pharyngeal glands, there have been seven revis ions of the genus ( M i l l e r , 1930; Olsen, 1937; Cabal l ero , 1938; Cheng, 1959; Byrd and Maples, 1963; Nasir and Diaz , 1970; S u l l i v a n , 1976). Brooks' (1977) phylogenetic a n a l y s i s 1 of a number of genera of p l a g i o r c h i o i d trematodes (see the sec t ion , CHARACTER ANALYSIS) concluded that Glypthelmins and Haplometrana are each other's c losest r e l a t i v e s , that i s , they are s i s t e r taxa. In a study of species that have at one time or another been placed in the genus Glypthelmins, Brooks (1977) examined 11 morphological characters and presented a cladogram ( F i g . 7) containing four monophyletic l ineages . The f i r s t two lineages are the subject of th i s d i s s e r t a t i o n , and consis t of a l l the species in North America, and some of those in Centra l and South 1 The study by Brooks (1977) used Camin-Sokal parsimony to construct the tree and the common-equals-primitive c r i t e r i o n to po lar i ze characters . See the sec t ion , THE INFERENCE OF PHYLOGENETIC RELATIONSHIPS. 44 America. Lineage I cons is ts of G. hyloreus and G. pennsylvaniensi s. Lineage II consists of G. robustus, G. shas ta i , G. c a l i f o r n i e n s i s , G. quie ta , and G. f a c i o i . Members of the remaining two c lades 1 are found in South America, A f r i c a , A s i a , and the western P a c i f i c . Brooks (1977) postulated the paraphy le t i c 2 or p o l y p h y l e t i c 3 nature of a l l of the genera to which some of the species had at some point been assigned. These are: Glypthelmins S ta f ford , 1905; Choledocystus Pere ira and Cuocolo, 1941; Rauschie l la Baberb, 1951; and Repandum Byrd and Maples, 1963. Brooks' cladogram supported M i l l e r ' s (1930) transference of Margeana c a l i f o r n i e n s i s C o r t , 1919 to Glypthelmins, and N a s i r ' s (1966) transference of Reynoldstrema afr icana (Dol l fus , 1950) Cheng, 1959 to Glypthelmins. Brooks proposed that the ent i re monophyletic assemblage be referred to as Glypthelmins. This provided systematic support for N a s i r ' s (1966) proposal to subsume the genera Choledocystus, Reynoldstrema, and Repandum in Glypthelmins. The question asked in th i s part of the present study was whether the app l i ca t ion of more recent ly developed phylogenetic ana ly t i c methods to a larger set of character data for 1 A monophyletic group sensu Hennig (1966): containing an ancestor and a l l of i t s descendants (see also F a r r i s , 1974). 2 A group that includes a common ancestor and some, but not a l l , of i t s descendants ( F a r r i s , 1974). 3 A group in which the most recent common ancestor i s assigned to some other group, and not to the group i t s e l f ( F a r r i s , 1974). 45 Haplometrana and the seven species of Glypthelmins concerned would continue to support the ex i s t ing cladogram. The re -analys i s used morphological characters observed through the examination of type mater ia l , specimens from pr ivate c o l l e c t i o n s , specimens from f i e l d surveys, and specimens from laboratory s tudies . As an hypothesis of common ancestry and homologous character inheri tance , the r e s u l t i n g cladogram provided the basis of the further comparative studies reported in Chapter IV. TAXA STUDIED A l l of the members of the glypthelminth lineages I and II postulated by Brooks (1977) (F ig . 7) were examined (Table I ) . The genus Haplometrana was included in the analys i s because of (1) i t s postulat ion as the s i s t e r taxon to Glypthelmins by Brooks (1977), (2) i t s phenetic s i m i l a r i t y to species of Glypthelmins, and (3) i t s paras i t i sm of R. p r e t i o s a , a ranid c lose ly re la ted to the ranid hosts of the species of Glypthelmins in western North America (see Chapter IV, COEVOLUTION ANALYSIS). 46 Table I - SPECIES STUDIED Glypthelmins hyloreus Mart in , 1969 G. pennsylvaniensis Cheng, 1961 [=Choledocystus pennsylvaniensis (Cheng, 1961) Byrd and Maples, 1963] G. robustus Brooks, 1976 G. shastai Ingles , 1936 G. c a l i f o r n i e n s i s (Cort , 1919) M i l l e r , 1930 [=Margeana c a l i forn iens i s C o r t , 1919] G. quieta (Staf ford, 1900) S ta f ford , 1905 [=Distomum quietum S t a f f o r d , 1900] [=Glypthelmins subtropica Harwood, 1932] G. f a c i o i Brenes et a l . , 1959 Haplometrana i n t e s t i n a l i s Lucker, 1931 [=Haplometrana utahensis Olsen, 1937] 47 MATERIALS AND METHODS SPECIMENS EXAMINED 1. MUSEUM SPECIMENS USNM Helm. C o l l . refers to the U.S. National Museum, Helminthological C o l l e c t i o n , B e l t s v i l l e , Maryland. Glypthelmins hyloreus Mart in , 1969 Specimens: USNM Helm. C o l l . No. 70463: holotype; No. 70464: paratypes; add i t i ona l specimens from the c o l l e c t i o n of D.R. Brooks, Dept. of Zoology, Univ . of B r i t i s h Columbia (co l lec ted in Nebraska, see Brooks, 1976a) Type L o c a l i t y : Near C o r v a l l i s , Oregon Type Host: Hyla regi11a Baird and G i r a r d 48 Glypthelmins pennsylvaniensis Cheng, 1961 [=Choledocystus pennsylvaniensis (Cheng, 1961) Byrd and Maples, 1963] Specimens: USNM Helm. C o l l . No. 59515: holotype and paratype Type L o c a l i t y : Lake Warren, Pennsylvania Type Host: Hyla c r u c i f e r Weid Glypthelmins robustus Brooks, 1976 Specimens: USNM Helm. C o l l . No. 73482: holotype; No. 73483: paratype Type L o c a l i t y : 15 km west of Neiva, H u i l a , Colombia Type Host: Bufo marinus L . 49 Glypthelmins shastai Ingles , 1936 Specimens: USNM Helm. C o l l . No. 8925: holotype; a d d i t i o n a l specimens from the c o l l e c t i o n of J . C . Holmes, Dept. of Zoology, Univ. of Alberta (co l l ec ted in B r i t i s h Columbia and Alberta) Type L o c a l i t y : Glenburn, Shasta County, C a l i f o r n i a Type Host: Bufo boreas Baird and G i r a r d Glypthelmins c a l i f o r n i e n s i s (Cort , 1919) M i l l e r , 1930 [=Margeana c a l i f o r n i e n s i s Cor t , 1919] Specimens: USNM Helm. C o l l . No. 51701: syntypes; a d d i t i o n a l specimens from the c o l l e c t i o n of D.R. Brooks, Univ. of B r i t i s h Columbia ( co l l ec ted in Langley, B r i t i s h Columbia) Type L o c a l i t y : San Francisco area, C a l i f o r n i a Type Host: Rana aurora Baird and Girard 50 Glypthelmins f a c i o i Brenes et a l . , 1959 Specimens: USNM Helm. C o l l . No. 72275: deposited by Su l l i van (1976); add i t iona l specimens from the c o l l e c t i o n of J . J . S u l l i v a n , CDC, A t l a n t a , Georgia (co l lec ted in Costa Rica; see S u l l i v a n , 1976) Type L o c a l i t y : C o r i s , Cartago Province, Costa Rica Type Host: Rana pipiens Schreber Glypthelmins quieta (Staf ford, 1900) S ta f ford , 1905 [=Distomum quietum Staf ford , 1900] [=Glypthelmins subtropica Harwood, 1932] Specimens: USNM Helm. C o l l . No. 72268-72271: deposited by S u l l i v a n (1976); a d d i t i o n a l specimens from the c o l l e c t i o n of D.R. Brooks, Univ . of B r i t i s h Columbia (co l lected in Nebraska; see Brooks, 1976a) 51 Type L o c a l i t y : Eastern Canada; 1 Toronto area presumed Type Host: Rana virescens Garman (= R. p i p i e n s ) , R. catesbeiana Shaw, and Hyla p i c k e r i n g i i Kennicott (= H. c r u c i f e r Weid) Haplometrana i n t e s t i n a l i s Lucker, 1931 [=H. utahensis Olsen, 1937] Spec imens: USNM Helm. C o l l . No. 29903: holotype; No. 29904: paratypes; No. 9025: holotype of H. utahensis Olsen, 1937; No. 9026: paratype of H. utahensis; add i t i ona l specimens from the c o l l e c t i o n of J . C . Holmes, Univ. of Alberta ( co l l ec ted in Kelowna, B r i t i s h Columbia: o r i g i n a l l y i d e n t i f i e d as Glypthelmins sp . ; i d e n t i f i e d as H. i n t e s t i n a l i s by the present author) Type L o c a l i t y : B o t h e l l , King County, Washington State Type Host: Rana pret iosa Baird and G i r a r d 1 S ta f ford ' s (1900, 1905) reports spec i f i ed neither l o c a l i t y nor type specimens. I have recently discovered that there are f ive of S ta f ford ' s specimens deposited at the National Museum of Natural Sciences, Ottawa, Ontar io , Canada (Invertebrate Zoology D i v i s i o n , nos. NMCP 1900-1694 to 1900-1698). It may thus be poss ib le to designate a lectotype for G. q u i e t a . I thank Dr. Gordon G. Gibson, NMNS, for his help with th i s search. 52 2. FIELD COLLECTIONS During the spring and summer of 1983 to 1986, anurans were c o l l e c t e d in the fol lowing areas (see also Chapter IV, HOST AND DISTRIBUTION DATA): 1) Southern B r i t i s h Columbia, along an east-west transect remaining within 50 km of the Canada - U . S . border, from Vancouver Island to the B r i t i s h Columbia - Alber ta border (F ig . 8) . The species of anurans c o l l e c t e d , and the pert inent species of paras i tes found, were: (a) Rana aurora: G. c a l i f o r n i e n s i s ; (b) R. pret iosa: H. i n t e s t i n a l i s ; (c) Bufo boreas: no i n t e s t i n a l digeneans found; and (d) Hyla regi11a: no i n t e s t i n a l digeneans found. 2) S i sk iyou , Shasta, Modoc, Lassen, Plumas, and S i e r r a Counties in northern C a l i f o r n i a ( F i g . 9) . Co l l ec t ions in Shasta, Modoc, and Lassen Counties centered around the drainage basin of the P i t R iver . This r iver runs through Glenburn, the type l o c a l i t y of G. shastai in B. boreas. The species of anurans c o l l e c t e d were R. cascadae and B. boreas. No i n t e s t i n a l digeneans were found. 3) Southeast Nebraska, in the region of L i n c o l n . 4) Northwest Wyoming. The Nebraska c o l l e c t i o n s acquired specimens of G. quieta from 53 R. p ip iens , so as to obtain ear ly developmental stages of the paras i te for laboratory s tudies . Co l l ec t ions of R. aurora from Langley, B r i t i s h Columbia y ie lded specimens of G. c a l i f o r n i e n s i s for use in laboratory s tudies . Co l l ec t ions in Wyoming were d irected towards the i d e n t i f i c a t i o n of "Glypthelmins sp ." , reported by Turner (1958) from R. pret iosa at F i sh ing Bridge . 3. EXAMINATION OF SPECIMENS A l l of the specimens examined from publ ic and pr ivate c o l l e c t i o n s were whole mounts, stained with e i ther hematoxylins or acetocarmine. Worms obtained from dissect ions of anurans co l l ec t ed in the f i e l d were f lat tened under a c o v e r s l i p , f ixed for 24 hours in Alcoho l -Formal in-Acet ic A c i d , and then stored in 70% EtOH. They were stained with hematoxylins, acetocarmine, or Fast Green, then whole-mounted. Developmental stages obtained from laboratory infect ions were processed in the same manner. L i v i n g specimens were studied with v i t a l s tains (Nile Blue and Methylene Red). Unless stated otherwise, observations were taken from f ixed specimens. I l l u s t r a t i o n s from microscope work were done with the a id of a drawing tube. 54 THE INFERENCE OF PHYLOGENETIC RELATIONSHIPS Hennig (1950, 1966) developed the methodology of phylogenetic systematics (= c l a d i s t i c s ) as a formalized procedure for i n f e r r i n g phylogenetic re la t ionsh ips by grouping organisms together on the basis of the ir shared possession . of derived homologous characters . 1 Such hypotheses of common ancestry can serve as general reference systems for other studies in comparative b io logy . 1. HOMOLOGY Two characters (= a t t r i b u t e , or t r a i t ) are considered to be homologues (= "special homology": R u s s e l l , 1916; R i e d l , 1978; Wiley, 1981a) of one another i f one is e v o l u t i o n a r i l y der ived , through t r a n s - s p e c i f i c reproductive l i n k s , from the other. These two characters are re ferred to as a transformation s e r i e s . 1 As introduced by Owen (1848), the concept of homology had no evolut ionary connotations. Later workers, such as Darwin (1859), Haeckel (1866), Lankester (1870), and Gegenbaur (1878) used the term to refer to s t r u c t u r a l s i m i l a r i t i e s that were the resu l t of common ancestry. The c r i t e r i a of s t r u c t u r a l s i m i l a r i t y used today are general ly those del ineated by Remane (1956): (1) s i m i l a r i t y of p o s i t i o n , (2) degree of resemblance, and (3) cont inu i ty through intermediate species . To these, Hennig (1950, 1966) added the c r i t e r i o n of concordance between hypotheses of i n d i v i d u a l characters ' homologies and the phylogenetic re la t ionsh ips that such homology suggests. If two structures are in ferred to be homologues sensu Remane, and i f they are a l so in ferred to be shared, derived characters from a common ancestor, then the genealogical grouping of taxa supported by that shared character w i l l continue to be supported when other characters are examined. This c r i t e r i o n introduced the concept of evolut ion producing a covarying sui te of inher i ted characters . 55 The character that existed f i r s t i s termed the plesiomorphic ("near the form"), or ancestra l s tate , of the s e r i e s , and the character that evolved from i t i s termed the apomorphic ("away from the form"), or derived s tate , of the s e r i e s . In the same manner, three or more characters can be homologues of each other, with the added p o s s i b i l i t y that the character states in the ser ies can be derived from one another in a l i n e a r sequence, such as: A - - B - - C or in a branching sequence, termed a mult i s tate character tree , such as that given below (see Chapter IV and Appendix F for further d iscuss ion of mult i s tate character t rees ) : D I B A character found in two or more taxa is homologous in a l l of those taxa when the most recent common ancestor can also be in ferred to have had the character . I f , in such a case, the character can be considered to have or ig inated in that most recent common ancestor, i t i s termed a synapomorphy, a shared 56 derived t r a i t . If i t i s considered to have or ig inated in an e a r l i e r ancestor, i t i s termed a symplesiomorphy, a shared ancestra l t r a i t . If a character i s the derived state of an homologous s e r i e s , but i s present in only one of the taxa in the a n a l y s i s , i t i s termed an autapomorphy. C l a d i s t i c methodology attempts to reconstruct the phylogenetic re la t ionsh ips among organisms by grouping them on the basis of in ferred synapomorphic t r a i t s . This minimization of the number of postulated evolutionary events i s a parsimony argument ( e . g . , F a r r i s , 1982, 1985; Sober, 1983; Fe l sens te in , 1982, 1984) Plesiomorphy and apomorphy refer to the r e l a t i v e degree of evolutionary d e r i v a t i o n , and every species i s a composite of the d i f f erent homologue types. This i s because a new evolutionary character can or ig inate as an autapomorphy, af ter which, through the evolut ion of descendent species , i t can become f i r s t a synapomorphy and then a symplesiomorphy. For example, ha ir i s an autapomorphy of mammals when addressing the evolut ion of the Tetrapoda within the Vertebrata , a synapomorphy of mammals when addressing the evolution of the Mammalia within the Tetrapoda, and a symplesiomorphy of mammals when addressing the evolut ion of F e l i s within the Fe l idae . Characters that cannot be in ferred to be homologues on a cladogram are termed homoplasies (Lankester, 1870). These characters do not sa t i s fy Hennig's c r i t e r i o n of phylogenetic congruence with other characters . They are considered e i ther to have or ig inated independently of one another, that i s , from two d i f f erent pre -ex i s t ing characters (convergence), or to have 57 or ig inated from the same character at d i f f erent times or in d i f f erent taxa ( p a r a l l e l i s m ) . In the l a t t e r case, homoplasious characters may sa t i s fy Remane's (1956) c r i t e r i a of s i m i l a r i t y of p o s i t i o n , resemblance, and c o n t i n u i t y . As with homologues, homoplasies show s t r u c t u r a l s i m i l a r i t i e s . They are thus not equivalent to Owen's (1848) other type of comparative anatomical property, an analogue. This l a t t e r category refers to s i m i l a r i t y in function regardless of s i m i l a r i t y in form. Rather, homoplasies are s t r u c t u r a l l y s imi lar a t t r ibutes that are considered to be non-homologues only after a phylogenetic ana lys i s shows that they cannot be in ferred to be synapomorphic for a l l of the taxa in which they occur. 1 2. OUTGROUPS A number of protocols have been proposed for determining the r e l a t i v e plesiomorphy and apomorphy of character transformation ser ies ( e . g . , Stevens, 1980), a procedure termed p o l a r i z a t i o n . To date, they have been found e i ther to give incorrect estimates (such as the p r i n c i p l e of common-equals-p r i m i t i v e : e . g . , Estabrook, 1978; C r i s c i and Steussy, 1980), or 1 Homoplasious characters can be synapomorphic, i . e . , homologous, for less inc lus ive clades on the tree . This follows from the recognit ion that regardless of whether a character ar i se s as a p a r a l l e l i s m , convergence, or r e v e r s a l , i t i s capable of being inher i ted in descendent species . One consequence of th i s i s that i f a paraphylet ic group possesses, say, the "1" state in a 0-1-0 or 0-1-2 transformation ser ie s , then that state i s an homologous t r a i t of that group. 58 to be spec ia l cases of the more general method of "outgroup" comparisons (see Lundberg, 1972). This method i s based upon the assumption that a t r a i t found in at least one member of the study group and in a taxon outside the group (the outgroup - a close r e l a t i v e , preferably the s i s t e r taxon) i s ples iomorphic . Such a t r a i t is hypothesized to have been inher i t ed by the species that is now the ancestor of the study group. Because outgroups can themselves evolve ( i . e . , what i s taken to be a plesiomorphy is a c t u a l l y an apomorphy of the outgroup taxon), i t i s necessary to use more than one outgroup to determine the plesiomorphy of a character (see Maddison e_t a l . , 1984). One must e s tab l i sh the character state of the outgroup node, which i s the ances tra l state on the tree immediately below (= e v o l u t i o n a r i l y p r i o r to) the group of taxa being studied (= the ingroup, or study group). This i s done by examining at least two outgroup taxa that are not themselves monophyletic. For th i s reason, a cladogram w i l l , depending upon the characters used in the study, often be rooted at a composite  outgroup ( i . e . , at a node cons i s t ing of plesiomorphic character s tates , rather than at an ex i s t ing taxon). This follows from Hennig's (1966) argument that any organism is a composite of plesiomorphic and apomorphic t r a i t s . The necessity to po lar i ze characters by examining at least two non-monophyletic outgroup taxa continues to hold as an analys i s attempts to resolve re la t ionsh ips within the ingroup. This i s because a member of the ingroup can be an outgroup to the remaining ingroup members at a less inc lus ive l e v e l of 59 a n a l y s i s . Thus, the c r i t e r i a of Maddison et a l . (1984) are to be appl ied in the Funct ional Outgroup Analys is of Watrous and Wheeler (1981), which deals with ingroup character p o l a r i z a t i o n s . 3. MULTISTATE CHARACTERS If a character occurs in only two states in the taxa studied, i t i s termed binary , and i t s p o l a r i z a t i o n into plesiomorphic and apomorphic states w i l l concurrently e s tab l i sh i t s order of transformation. That i s , i f one state i s (» a n c e s t r a l , then the other state must be the derived state in a l inear transformation s e r i e s . This concurrence does not apply when a character has more than two states . These are c a l l e d mult i s tate characters . When one of the i r states i s po lar ized as the ances tra l s tate , there i s s t i l l ambiguity as to the subsequent transformations among the remaining s tates . . There has long been a tendency in systematics to order these states into an in tu i t ed transformation s er i e s . Much of t r a d i t i o n a l evolutionary taxonomy is based upon choosing an appropriate ly "key" character , and then postu lat ing the evolut ion of i t s states according to various funct ional or s t r u c t u r a l c r i t e r i a . 1 At the very l ea s t , th i s approach suffers simply because i t uses so few characters that a c t u a l l y test the phylogenetic 1 A commonly used c r i t e r i o n is a l i n e a r increase or decrease in s ize or number. 60 hypothesis . As discussed in the fol lowing subsection, i t i s poss ib le to analyze mult i s tate characters c l a d i s t i c a l l y , in a manner comparable to that for binary characters . 4. COMPUTER-ASSISTEP PARSIMONY ANALYSIS A number of algorithms have been developed for construct ing phylogenetic trees on the basis of in ferred synapomorphies. The Wagner method (Kluge and F a r r i s , 1969; F a r r i s , 1970) places no r e s t r i c t i o n s on the kinds of character changes that are postulated so as to produce a minimum length tree . The Camin and Sokal (1965) algorithm proh ib i t s character reversals (a return to a more p r i m i t i v e s ta te ) . The Dol lo algorithm ( F a r r i s , 1977) minimizes mult ip le or ig ins (paral le l i sms and convergences) of characters . Wagner parsimony is the most defensible method because i t makes the fewest a p r i o r i assumptions about evolut ionary events. The method does not deny the existence of reversals or mult ip le o r i g i n s : i t simply makes no i n i t i a l assumptions and then l e t s the d i s t r i b u t i o n of the characters on the tree indicate a p o s t e r i o r i postulat ions of such types of character change (see F a r r i s , 1985; Sober, 1983). This form of tree-wide, or g l o b a l , parsimony can produce trees that are shorter than those produced by Camin-Sokal or Dol lo parsimony c r i t e r i a . With respect to evolutionary "reversals", i t should be noted that s imi lar codes for various characters in a data matrix can refer to completely d i f f erent organic proper t i e s . For 61 example, in a two-state character , the plesiomorphic state is usual ly coded as "0", and the derived state i s coded as "1" ( i . e . , a "binary" character ) . But the "0" code can refer to (1) the p r i m i t i v e absence of a character whose derived state i s i t s presence, (2) the p r i m i t i v e presence of a character whose derived state i s i t s absence, or (3) the p r i m i t i v e morphology of a character whose derived state i s an a l tered morphology. If a computer program for phylogenetic analys i s p r o h i b i t s evolut ionary reversa l s , the only thing ac tua l ly being prohib i ted may be a "0 - 1 - 0" transformation in the coding. The two programs implementing Wagner parsimony that were used in th i s study are PAUP (version 2.4, 1985, developed by D . L . Swofford) and PHYSYS (1983 vers ion , developed by J . S . F a r r i s and M . F . Mickevich) . Both PAUP and PHYSYS contain algorithms that b u i l d trees by the step-wise addi t ion of taxa (often re ferred to in th i s context as OTUs: Operational Taxonomic Uni.ts) to a less inc lus ive tree containing a l l of the character data such that the number of postulated changes for a l l of the character states i s minimized. 1 Another approach is simply to contruct a l l poss ible trees for the taxa, and then pick the shortest one. This may rather ine legant ly e l iminate any problems concerning the appropriate manner of step-wise addi t ion of OTUs, but i t i_s guaranteed to f ind the optimal tree 1 Although the goal i s c lear enough, the manner in which an algorithm can and should accomplish th i s task is the subject of much discuss ion in the systematics l i t e r a t u r e (see, e . g . , Swofford and Maddison, in review; F ink , 1986). 62 according to whatever opt imal i ty c r i t e r i o n is being appl i ed . Such an operation is only p r a c t i c a l when a computer is ava i lab le to carry out the extensive c a l c u l a t i o n s (for nine taxa, there are 135,135 t r e e s ) . PAUP's a b i l i t y to do th i s for a l imi t ed number of taxa was u t i l i z e d in t h i s study. Parsimony procedures have been developed for mult i s tate characters ( F i t c h , 1971; Mickevich, 1982; Swofford and Maddison, in review). Mickevich's technique is termed Transformation Series A n a l y s i s , and is d i s t r i b u t e d with the PHYSYS computer program. The Swofford and Maddison technique, which is a form of F i t c h Optimization ( F i t c h , 1971), is the Unordered Analys is procedure of the PAUP computer program. A l l of the techniques attempt to f ind the most parsimonious arrangement of character states on a tree regardless of any d i r e c t i o n a l i t i e s of transformation implied by the coding of the character s tates . The main di f ferences l i e in the c r i t e r i a of homology, and the means by which ambiguity over the character state at an in terna l node is handled. I w i l l not invest igate these di f ferences here, other than to say that PAUP's Unordered Analys i s was used because I consider i t to make fewer a p r i o r i assumptions about character evo lu t ion . 63 CHARACTER ANALYSIS Twenty-one morphological characters were used in the analys i s of Haplometrana i n t e s t i n a l i s and the seven species of Glypthelmins. The l i s t i n g below indicates the inferred states of. each character , and the r e l a t i v e plesiomorphy postulated from outgroup comparisons. These comparisons u t i l i z e d the resu l t s of c l a d i s t i c analyses of digenean f a m i l i a l re la t ionsh ips (Brooks et a l . , 1985b), cer ta in p l a g i o r c h i o i d genera (Brooks, 1977), and spec i f i c re la t ionsh ips within Glypthelmins (Brooks, 1977). In addi t ion to Glypthelmins and Haplometrana, the p l a g i o r c h i o i d genera included in the analys i s by Brooks (1977) were: Brachycoelium (Dujardin, 1845) S t i l e s and H a s s a l l , 1898, Tremiorchis Mehra and Negi, .1926, Mesocoelium Odhner, 1911, Haplometroides Odhner, 1911, Ost io lo ides Odening, 1960, Xenopodistomum MacNae et §_1. , 1973, Metaplagiorchis Timofeeva, 1962, Laiogonimus Vercammen-Grandjean, 1960, Qpisthioglyphe Looss, 1899, and Astiotrema Looss, 1900. The c r i t e r i a of Maddison et a l . , (1984) for e l iminat ing ambiguity as to the plesiomorphic state were appl ied whenever poss ib le . The p o l a r i z a t i o n procedures appl ied here use the term "widespread character state" in the sense of Kluge and F a r r i s (1969) (see a l so F a r r i s , 1982). Although outgroup taxa from outside the study group were examined for each character , the p a r t i a l reso lut ion of the tree by some characters rendered these comparisons moot for other characters . This i s because the former gave s u f f i c i e n t ingroup reso lut ion to provide funct ional outgroups (sensu Watrous and Wheeler, 1981) that establ ished the 64 plesiomorphy of the l a t t e r characters at the l e v e l of general i ty  encompassed by the study group. In the l i s t i n g below (summarized in Table I I ) , plesiomorphic states are coded with a "0". The derived states of binary characters are coded with a "1", while those of mult i s tate characters are given l e t t e r codes. Such alphanumeric coding was done so as to indicate that the order of transformation in the mult is tate characters was in ferred a p o s t e r i o r i in the c l a d i s t i c a n a l y s i s . This coding does not af fect the manner in which mult i s tate characters are treated in the Unordered Analys is of PAUP. Missing data or l o g i c a l l y inappl i cab le character states are coded with a question mark (?) in Table I I . (1) Tegumental project ions (2 states) ( F i g . 10): (0) spines; (1) scales Character States: Although only M i l l e r (1930) ac tua l ly i l l u s t r a t e d scaled project ions on the tegument of G. qu ie ta , "sca le - l ike spines" have been noted on G. quieta by Rankin (1944), on G. c a l i f o r n i e n s i s by Cort (1919), and on G. f a c i o i by Su l l i van (1976). Spines are approximately 9 jum long, 4 .um wide, and do not overlap one another. Scales are approximately 18 jum long, 8 jum wide, and over lap. Scales appear to be expanded spines, since the centers of the bases of both are approximately 18 jum apart 65 from one another on the surface of the tegument. P o l a r i t y : Based upon an analys i s of digenean f a m i l i a l re la t ionsh ips (Brooks e_t a_l. , 1985b), spines are postulated to be symplesiomorphic for the l eve l of th i s study. (2) Proport ion of body bearing tegumental project ions (3 states) : (0) from anter ior end to pos ter ior f i f t h of body; (A) from anter ior end to l eve l of pharynx; (B) from anter ior end to middle of hindbody 1 Character States: The character states reported here agree for the most part with published accounts. Differences l i e with reports of project ions to the l e v e l of the testes in G. shastai (by Ingles , 1936), G. f a c i o i (by S u l l i v a n , 1976), and H. i n t e s t i n a l i s (by Olsen, 1937). As Cort (1919) noted for G. c a l i f o r n i e n s i s , the density of the project ions begins to th in poster ior to the testes , most l i k e l y as a resul t of the growth of the hindbody (see Chapter IV, HETEROCHRONIC DEVELOPMENT). Nevertheless, large specimens of H. i n t e s t i n a l i s and G. shas ta i , which 1 The region from the anter ior edge of the ventra l sucker to the poster ior end of the body. 66 are the largest species s tudied, bear project ions to the poster ior of the body. P o l a r i t y : Based upon an analys i s of digenean f a m i l i a l re la t ionsh ips (Brooks et a l . , 1985b), the plesiomorphic state of spination in the Digenea is postulated to be state (0). (3) Diameter of ora l sucker / diameter of ventra l sucker (2 s ta tes ) : (0) 1:0.40 - 1:0.75; (1) >1:0.75 Character States: Ranges and (means), a l l specimens: G. hyloreus, n = 10, 1:0.80 - 1:0.84 (1:0.82) G. pennsylvaniensis , n = 2, 1:0.75 - 1:0.78 (1:0.77) G. robustus, n = 2, 1:0.45 - 1:0.50 (1:0.48) G. shas ta i , n = 19, 1:0.58 - 1:0.74 (1:0.69) G. c a l i f o r n i e n s i s , n = 20, 1:0.52 - 1:0.67 (1:0.60) G. f a c i o i , n = 10, 1:0.58 - 1:0.65 (1:0.61) G. qu ie ta , n = 15, 1:0.50 - 1:0.68 (1:0.58) H. i n t e s t i n a l i s , n = 25, 1:0.47 - 1:0.68 (1:0.62) P o l a r i t y : State (0) i s es tabl i shed as the plesiomorphic state because of i t s widespread d i s t r i b u t i o n throughout Glypthelmins l ineages III and IV (sensu Brooks, 1977; see 67 F i g . 7) and other p l a g i o r c h i o i d genera p a r a s i t i c in anurans. (4) Penetration glands (2 states) ( F i g . 11): (0) present in c e r c a r i a , absent in adul t ; (1) present in c e r c a r i a , present in adult Character States and P o l a r i t y : Many digenean cercar iae possess penetration glands whose ducts empty near the oral, sucker. The glands usual ly degenerate when adulthood i s reached, although the ducts can sometimes s t i l l be seen in young adu l t s . Although cercar iae of G. f a c i o i have not been examined, the pos i t ion of the glands and the i r ducts in the adults suggests that these glands are penetration glands p e r s i s t i n g from the c e r c a r i a l stage. (5) Medial glands (2 states) (Figs 11, 12, 13 and 47) (0) at l eve l of prepharynx; (1) at l e v e l of pharynx and esophagus Character States: This character and character no. 6 comprise what have been previously referred to c o l l e c t i v e l y in Glypthelmins as the pharyngeal or the peripharyngeal 68 glands. They l i e on each side of the body in the region of the pharynx. Byrd and Maples*s (1963) caution against the use of these glands for systematic purposes was d irec ted against Cheng 1s ( 1 959) attempt to r e - e s t a b l i s h the genus Margeana Cort , 1919 for species lacking such glands. My work on the study group indicates that there is always at least one type of gland, with two types present in G. qu ie ta . The f i r s t are herein referred to as the medial glands (from Leigh , 1946, see below), the second as the pharyngeal glands. A l l of the species possess the more a n t e r i o r , and smal ler , medial glands. They were d i f f i c u l t to observe in H. i n t e s t i n a l i s , and the v i t a l s ta in ing of l i v e specimens was necessary. Only G. quieta has the l arger , more p o s t e r o - l a t e r a l pharyngeal glands. The two types of glands are located in d i f f erent areas, and, although they often overlap each other when both are present, each has ducts with d i f f erent o r i e n t a t i o n s . Those of the medial glands enter the prepharynx d o r s o - l a t e r a l l y , while those of the pharyngeal glands enter ventro-media l ly . Leigh (1946) f i r s t reported medial glands in G. qu ie ta , although he concluded that they degenerated as the animal matured. Rankin (1944) can also be considered to have observed them in G. qu ie ta , since he noted that the pharyngeal glands (sensu lato) were sometimes arranged in an anter ior group and a pos ter ior group on each side of the body. 69 Po lar i ty: Although p o l a r i t y i s establ ished by ingroup r e l a t i o n s h i p s , the r e l a t i v e plesiomorphy of state (0) i s not known. Accordingly , i t i s coded as a missing datum for the composite outgroup in the analys i s (a question mark in Table I I ) . (6) Pharyngeal glands (2 states) ( F i g . 13): (0) absent; (1) present Character States: As noted in the remarks for character (5), only G. quieta possesses these large and conspicuous glands. They l i e p o s t e r o - l a t e r a l to the pharynx, with ducts that enter the prepharynx ventro-medial ly . P o l a r i t y : This i s es tabl i shed by ingroup r e l a t i o n s h i p s . Pharyngeal glands are absent in the outgroup taxa. (7) Pos i t ion of ovary (2 s tates ) : (0) s i n i s t r a l : on l e f t side of body; (1) d e x t r a l : on r ight side Character States: Brooks (1976b) i l l u s t r a t e d the dextra l condi t ion in G. robustus. Examination of the type 70 specimens confirmed th i s character . It should be noted that amphitypy ( i . e . , in th i s case, a s i n s t r a l or dextra l ovary pos i t ion in d i f f erent specimens of the same species) i s not uncommon in digeneans, and that only the holotype and the paratype of G. robustus were ava i lab le for examination. Nevertheless, such amphitypy has not been reported for any of the species studied here. Polarit-y: The dextral pos i t ion also occurs in the South American species , Choledocystus hepaticus Lutz , 1928 (= G. hepaticus, l ineage III sensu Brooks, 1977) and Rauschie l la palmipedes Lutz , 1928 (= G. palmipedes, l ineage IV) (see S u l l i v a n , 1977a,b). Nevertheless, the widespread d i s t r i b u t i o n of the s i n i s t r a l condit ion among other glypthelminths and p l a g i o r c h i o i d s es tabl i shes the s i n i s t r a l state as plesiomorphic. Pos i t ion of Laurer ' s canal (2 states) (0) a r i s i n g between seminal receptacle and common v i t e l l i n e duct; (1) a r i s i n g d i s t a l to common v i t e l l i n e duct Character States; Brooks (1976b) i l l u s t r a t e d the d i s t a l locat ion of the canal in G. robustus. I have not ascertained the condit ion in Centra l and South American members of Choledocystus and Rausch ie l l a . 71 P o l a r i t y : This i s es tabl i shed by ingroup r e l a t i o n s h i p s . The r e l a t i v e plesiomorphy of state (0) i s not known, and i t i s coded as a missing datum for the composite outgroup in the analys i s (Table I I ) . Anter ior extent of anter ior v i t e l l i n e f i e l d (3 states) (Figs . 14, 15, 16, 17, 18) (0) anter ior v i t e l l i n e duct present, with anter ior v i t e l l i n e f i e l d extending to l e v e l of b i f u r c a t i o n ; (A) anter ior v i t e l l i n e duct present, with anter ior v i t e l l i n e f i e l d extending to l e v e l of pharynx; (B) anter ior v i t e l l i n e duct .absent , no anter ior v i t e l l i n e f i e l d Character States: The v i t e l l i n e system is herein considered to consist of f ive characters , presented here as characters (9) to (13). The v i t e l l i n e glands on each side of the body are composed of anter ior and pos ter ior f i e l d s , each of which empties into i t s own duct . These ducts jo in and form a s ingle duct that runs mediad towards the ootype region. The s ingle ducts from each side of the body unite into the common v i t e l l i n e duct . I define the anter ior and poster ior f i e l d s of v i t e l l a r i a with respect to the duct from which they a r i s e . Although the f i e l d s often overlap along the sides of the body, the anter ior f i e l d comprises the majority of the v i t e l l a r i a anter ior to the ootype region, 72 while the poster ior f i e l d comprises the majority of the v i t e l l a r i a poster ior to that region. Figure 14 presents a schematic diagram of the d i s t r i b u t i o n of the v i t e l l a r i a as a reference for characters (9) to (13). Figure 15c i s an i l l u s t r a t i o n of a specimen of G. quieta with reduced v i t e l l i n e development that c l e a r l y shows the anter ior and pos ter ior ducts and f i e l d s . Figures 15a and b show the usual condi t ion in G. qu ie ta . The v i t e l l i n e d i s t r i b u t i o n s for some of the other species are i l l u s t r a t e d i n : Figs 15d and e, for G. f a c i o i ; Figs 16a and b, for G. c a l i f o r n i e n s i s ; Figs 17a and b, for G. shasta i ; and Figs 17c and d, for H. i n t e s t i n a l i s . The absence of the anter ior v i t e l l i n e f i e l d s in H. i n t e s t i n a l i s is associated with the absence of the anter ior v i t e l l i n e ducts ( F i g . 18a). On the basis of anter ior v i t e l l i n e d i s t r i b u t i o n , Rankin (1944) synonymized G. c a l i f o r n i e n s i s with G. q u i e t a . S u l l i v a n (1976) concurred with th i s p o s i t i o n . I disagree. Only G. quieta possesses pharyngeal glands (sensu character 6), and i t s anter ior v i t e l l a r i a only occas ional ly extend to the l e v e l of the esophagus, whereas those of G. c a l i f o r n i e n s i s almost always do. Furthermore, there i s a d i f ference in the poster ior extent of the v i t e l l a r i a (character 11). P o l a r i t y : This is es tabl i shed State (0) i s widespread among p l a g i o r c h i o i d s . by ingroup r e l a t i o n s h i p s , other glypthelminths and 73 (10) Dorso-medial confluence of anter ior v i t e l l i n e f i e l d s (2 states) (Figs . 14, 15, 16) (0) absent; (1) present Character States: In a l l of the species s tudied, the v i t e l l a r i a l i e d o r s a l , l a t e r a l , and ventra l to the i n t e s t i n a l ceca ( F i g . 14). In some of the species , the v i t e l l a r i a from each side extend mediad and become confluent , or nearly so, with each other. The four v i t e l l i n e areas that may be involved are the dorsa l or ventra l parts of the anter ior or poster ior v i t e l l i n e f i e l d s . Dorsal confluence of what I herein term the anter ior f i e l d has been noted in G. qu ie ta , by Rankin (1944) (see Figs 15a,b), G. f a c i o i , by S u l l i v a n (1976) (see Figs I5d,e) , and G. c a l i f o r n i e n s i s , by Cort (1919) (see Figs 16a,b). Rankin (1944) synonymized G. subtropica Harwood, 1932 with G. quieta on the basis of th i s character . P o l a r i t y ; This i s es tabl i shed by ingroup r e l a t i o n s h i p s . State (0) i s widespread among other glypthelminths and p l a g i o r c h i o i d s . (11) Poster ior extent of poster ior v i t e l l i n e f i e l d (4 states) (F igs . 15, 16, 17, 18) 74 (0) to posterior t h i r d of hindbody (= mid-way between pos ter ior t e s t i s and poster ior end of body; (A) to pos ter ior end of body; (B) to within approximately one t e s t i s diameter pos ter ior to testes; (C) no further pos ter ior than testes Character States: As noted in character (9), th i s character i s another in which G. c a l i f o r n i e n s i s d i f f e r s from G. qu ie ta . The former has state C (Figs 16a,b), while the l a t t e r has state B (Figs 15a,b). However, there are two discrepancies . in the l i t e r a t u r e . F i r s t , in the o r i g i n a l descr ip t ion of G. c a l i f o r n i e n s i s , Cort (1919) presented an i l l u s t r a t i o n of a l i v i n g specimen in which the v i t e l l a r i a extend s l i g h t l y pos ter ior to the testes . But in both the diagnosis and the syntypes, the v i t e l l a r i a possess state C, as above. In my observations on l i v i n g specimens of G. c a l i f o r n i e n s i s , c o l l e c t e d from R. aurora in B r i t i s h Columbia, I found no di f ferences in the d i s t r i b u t i o n of the v i t e l l a r i a in l i v i n g and f ixed specimens. As shown in Figure 18b, th is r e s t r i c t e d d i s t r i b u t i o n is associated with the reduced development of the poster ior v i t e l l i n e ducts and t h e i r accompanying v i t e l l i n e f i e l d s . The other discrepancy l i e s with Sta f ford ' s (1900) o r i g i n a l descr ip t ion of G. quieta (= Pistomum quietum), in which the v i t e l l a r i a are i l l u s t r a t e d as extending to mid-way between the poster ior t e s t i s and the poster ior end of the body ( i . e . , state 0 here in ) . The diagnosis i s unclear on t h i s 75 po int : i t i s not spec i f i ed whether a l i v i n g or f ixed specimen is i l l u s t r a t e d , and no type specimens were deposited. (But see the sec t ion , SPECIMENS EXAMINED, for comments on Staf ford's specimens.) Subsequent studies on G. quieta have occas ional ly found the same state of v i t e l l i n e extent ( e . g . , Brooks, 1976a), but the usual condit ion is that of state B. Polar i t y : State (0) i s establ ished as the plesiomorphic state because of i t s widespread d i s t r i b u t i o n throughout Glypthelmins lineages III and IV (sensu Brooks, 1977) and other p l a g i o r c h i o i d genera p a r a s i t i c in anurans. (12) Dorso-medial confluence of poster ior v i t e l l i n e f i e l d s (2 states) (Figs 15, 17) (0) absent; (1) present Character States: The derived state of th i s character has been previous ly noted in G. f a c i o i , by S u l l i v a n (1976) (see Figs 15d,e) and H. i n t e s t i n a l i s , by Olsen (1937) (see Figs 17c,d). P o l a r i t y : This i s establ ished by ingroup r e l a t i o n s h i p s . State (0) occurs in most of the outgroup taxa. 76 (13) Ventro-medial confluence of both v i t e l l i n e f i e l d s (2 states) (Figs 14, 17) (0) absent; (1) present Character States; In the derived state of th i s character , the f i e l d s are confluent , or nearly so, v e n t r a l l y at the l eve l of the ootype, where the v i t e l l i n e ducts empty. Although th i s condi t ion has occas ional ly been reported in G. quieta (by Rankin, 1944) and G. c a l i f o r n i e n s i s (by Cort , 1919), i t occurs regular ly only in G. shas ta i . P o l a r i t y : This i s establ ished by ingroup r e l a t i o n s h i p s . State (0) occurs in the outgroup taxa. (14) L a t e r a l extent of uterine loops (2 states) (0) ex traceca l , overlapping i n t e s t i n a l ceca v e n t r a l l y ; (1) i n t e r c e c a l , l y i n g between i n t e s t i n a l ceca Character States: The ear ly uterine development of both states i s in terceca l (Sul l ivan and Byrd, 1970; Olsen, 1937; see a lso Appendix E , and the sect ion , HETEROCHRONIC DEVELOPMENT). Add i t i ona l l a t e r a l growth of the uterus produces the extracecal condi t ion (Sul l ivan and Byrd, 1970). 77 P o l a r i t y : State (0) i s establ ished as the plesiomorphic state because of i t s widespread d i s t r i b u t i o n throughout Glypthelmins l ineages III and IV (sensu Brooks, 1977) and other p l a g i o r c h i o i d genera p a r a s i t i c in anurans. (15) Anter ior extent of uterine loops (2 states) (0) throughout hindbody, with p r e t e s t i c u l a r loops present; (1) throughout hindbody, with p r e t e s t i c u l a r loops absent Character States: On the basis of characters (14) and (15), Byrd and Maples (1963) d i s t inguished among Glypthelmins (no extracecal loops, no p r e t e s t i c u l a r loops) , Repandum (no extracecal loops, p r e t e s t i c u l a r loops present) , and Choledocystus (extracecal loops present, p r e t e s t i c u l a r loops present) . P o l a r i t y : State (0) i s es tabl i shed as the plesiomorphic state because of i t s widespread d i s t r i b u t i o n throughout Glypthelmins l ineages III and IV (sensu Brooks, 1977) and other p l a g i o r c h i o i d genera p a r a s i t i c in anurans. It i s poss ible that future studies on G. robustus w i l l resu l t in i t s removal from the group of glypthelminths studied here, and i t s grouping with some of the other South American species of Glypthelmins previously placed in the genera Choledocystus and Rausch ie l l a . This p o s s i b l i t y ex i s t s for 78 two reasons. F i r s t , the holotype and paratype of G. robustus are not f u l l y mature specimens, and i t cannot be known for the moment whether the mature uterine condit ion exhib i t s extracecal and p r e t e s t i c u l a r loops. Second, the dextral ovary pos i t i on possessed by G. robustus a lso occurs in Choledocystus hepaticus (= G. hepaticus) and Rauschie l la palmipedis (= G. palmipedis) (see Character 7). (16) Relat ive pos i t ion of testes (3 states) (Figs 15, 16, 17, 35) (0) obl ique; (A) symmetrical; (B) tandem Character States: Symmetrical testes are l a t e r a l to one another (Figs 15a,b; 16a,b), oblique testes are diagonal to one another (Figs 15c,d; 17a,b), and tandem testes are arranged one poster ior to the other (Figs 35a,b). One of the characters by which Olsen (1937) considered H. utahensis to d i f f e r from H. i n t e s t i n a l i s was the oblique testes pos i t ion in the former and the tandem testes pos i t ion in the l a t t e r . In his synonymization of the two species , Waitz (1959) considered the two states to be the extremes of a continuum. In most of the specimens of H. i n t e s t i n a l i s examined in the present study, the testes are tandem. In some, they are obl ique . Within the study group, tandem testes occur only in H. intest i n a l i s. 79 P o l a r i t y : State (0) i s es tabl i shed as the plesiomorphic state because of i t s widespread d i s t r i b u t i o n throughout Glypthelmins l ineages III and IV (sensu Brooks, 1977) and other p l a g i o r c h i o i d genera p a r a s i t i c in anurans. (17) Seminal v e s i c l e (3 states) (0) i n t e r n a l , u n i p a r t i t e , c o i l e d ; (A) i n t e r n a l , u n i p a r t i t e , s t ra igh t ; (B) i n t e r n a l , b i p a r t i t e , s tra ight Character States; The character states reported here agree with published accounts. The b i p a r t i t e condit ion of G. shastai has not been reported before (see Appendix D) . P o l a r i t y : State (0) is es tabl i shed as the plesiomorphic state because of . i t s widespread d i s t r i b u t i o n throughout Glypthelmins l ineages III and IV (sensu Brooks, 1977) and other p l a g i o r c h i o i d genera p a r a s i t i c in anurans. (18) Length of c i r r u s sac / length of forebody 1 (2 states) (0) <0.5:1; (1) >0.5:1 1 The region from the anter ior edge of the ventra l sucker to the anter ior end of the body. 80 Character States: This character quant i f i e s the noticeably greater c i r r u s sac length in G. shastai and H. i n t e s t i n a l i s . In a l l of the species s tudied, the c i r r u s sac overlaps the ventra l sucker, and the gen i ta l pore i s immediately anter ior to the ventra l sucker. Longer c i r r u s sacs extend further pos ter iad . P o l a r i t y : This i s es tabl i shed by ingroup r e l a t i o n s h i p s . The plesiomorphic state i s widespread among glypthelminth l ineages III and IV (sensu Brooks, 1977) and other p lag iorch io ids p a r a s i t i c in anurans. (19) Mean egg length (3 states) (0) <30jLim; (A) 30-40 jam; (B) >40jjm Character States: Ranges and (means), in jjm, a l l specimens: G. hyloreus, 46-52 (50) G. pennsylvaniensis , 21-43 (34) G. robustus, 27-37 (31) G. shas ta i , 42-50 (45) G. c a l i f o r n i e n s i s , 42-50 (45) G. f a c i o i , 28-32 (29) G. qu ie ta , 42-50 (45) H. i n t e s t i n a l i s , 45-53 (50) Brooks (1976b) reported a range of 23-26 pm for the length 81 of the eggs in G. robustus. Brenes e_t a_l. (1959) reported a range of 33-47 jum for the length of the eggs in the ir specimens of G. f a c i o i (not examined). The values for G. fac i o i in the current study agree with those of S u l l i v a n (1976). P o l a r i t y : State (0) is es tabl i shed as the plesiomorphic state because of i t s widespread d i s t r i b u t i o n throughout Glypthelmins lineages III and IV (sensu Brooks, 1977) and other p l a g i o r c h i o i d genera p a r a s i t i c in anurans. ) C e r c a r i a l s ty l e t (2 states) (0) present; (1) absent Character States and P o l a r i t y : The x i p h i d i o c e r c a r i a l condit ion (presence of s ty l e t ) i s symplesiomorphic for the study group, being postulated by Brooks et a l . (1985b) as a synapomorphy for the Order P lag iorch i i formes . The gymnocephalous condit ion (no s ty l e t ) occurs in G. hyloreus and G. pennsylvaniensis . ) Shape of excretory ves i c l e (2 states) ( F i g . 19) (0) b i f u r c a t i n g anter ior to l e v e l of testes; ( l ) 82 b i f u r c a t i n g a t , or poster ior to , l e v e l of testes Character States: I consider these in ferred character states to be of some help in circumventing problems with t r a d i t i o n a l terminology for the shape of the excretory ves i c l e in digeneans. This terminology has referred to V, Y, or I shapes. Another t r a d i t i o n a l descr ip t ive term is that of a "tubular" v e s i c l e , p r i m a r i l y for reference to an I-shaped v e s i c l e . Manter (1969) noted the ambiguity of th i s term, since a l l three shapes of ves i c l e s are tubular in some sense of the word. On the basis of a Y or I-shaped v e s i c l e , c e r t a i n members of Glypthelmins have been assigned to the P lag iorch i idae (Y-shaped), while others have been assigned to the Macroderoididae (I-shaped) (see S u l l i v a n , 1976). Sche l l (1961) placed H. i n t e s t i n a l i s in the Macroderoididae on the basis of i t s I-shaped v e s i c l e . With the exception of H. i n t e s t i n a l i s , which was not included in the a n a l y s i s , and of G. shas ta i , for which there was at the time i n s u f f i c i e n t information, S u l l i v a n (1976) placed a l l of the species included in th i s present study into the genus Glypthelmins on the basis of the ir I-shaped excretory v e s i c l e . The fol lowing can be sa id of ve s i c l e shape in general . Entering the v e s i c l e postero-medial ly from each side of the body is an excretory tubule. The shape of the ves i c l e i s determined by (a) the r e l a t i v e width of the tubules in the area of th i s b i f u r c a t i o n , and (b) the point along the length of the v e s i c l e at which i t s body 83 b i f u r c a t e s . If the v e s i c l e b i furcates towards i t s base at the excretory pore, i t i s V-shaped. If i t b i furcates further anter iad , i t i s Y-shaped. If the b i furca t ion involves only the unexpanded tubules, i t i s I-shaped, with the tubules forming the thin arms of a Y. A l l of the species studied here have an I-shaped excretory ves i c l e in which the b i furca t ion occurs a t , or poster ior to , the l eve l of the testes (F ig . 19a) (reported here in G. shastai for the f i r s t time> see Appendix D). Although Brooks (1976b) reported that the excretory v e s i c l e of G. robustus i s I -shaped, th i s is not c l ear in the specimens examined ( a l l ava i l ab l e specimens). Accordingly , th is character i s coded as a missing datum for that species (Table I I ) . P o l a r i t y : Glypthelminths in lineages III and IV (sensu Brooks, 1977), in Centra l and South America, possess a Y-shaped v e s i c l e in which the b i furca t ion occurs anter ior to the l e v e l of the testes ( F i g . 19b) (see S u l l i v a n , I977a,b). This state i s widespread throughout p l a g i o r c h i o i d genera p a r a s i t i c in anurans. 84 1. CHARACTERS EXCLUDED FROM ANALYSIS Metraterm: The metraterm has been described as muscular in G. pennsylvaniensis , by Byrd and Maples (1963), and in G. shas ta i , by Ingles (1936). I observed the same condit ion in specimens of G. robustus, G. quieta (both stained with hematoxylin), G. c a l i f o r n i e n s i s , and H. i n t e s t i n a l i s (both stained with Fast Green). In a l l cases, l ong i tud ina l muscle f ibers run the length of the metraterm in the region of the surrounding gland c e l l s . The observation of the muscle f ibers proved to be highly dependent upon the preparation of the in d iv idua l worm, and was great ly f a c i l i t a t e d by the use of Fast Green. I d id not include th i s character in the analys i s because I was unable to examine a l l of the species in a comparable manner. Vas deferens; I observed that the two vasa e f f erent ia were joined into a vas deferens before they entered the c i r r u s sac in older adults of H. i n t e s t i n a l i s , as well as in some specimens of G. shastai and G. fac i o i (presence not re lated to body s i z e ) . The vasa e f f erent ia were observed to enter the c i r r u s sac separately in a l l ava i lab le specimens of G. hyloreus, G. pennsylvaniensis , and G. robustus, as well as in young adults of H. i n t e s t i n a l i s , G. c a l i f o r n i e n s i s , and G. quieta (although Rankin, 1944, i l l u s t r a t e d a vas deferens in a specimen of G. qu i e ta ) . Given that the presence of a vas deferens i s age-85 dependent in H. i n t e s t i n a l i s , the lack of s u f f i c i e n t specimens of known ages for a l l of the species studied necessitated the exclusion of th i s character from the a n a l y s i s . Table II presents the codings assigned to the 21 characters l i s t e d above. A "0" code in the l i s t i n g s below is an a r b i t r a r y coding value for the plesiomorphic state; i t does not necessar i ly indicate absence. Table II - CHARACTERS ANALYZED Character Number 1 1 1 1 1 1 1 1 1 1 2 2 1 2 3 4 5 6 7 8 9 0 1 2 3 4 5 6 7 8 9 0 1 0 0 0 0 7 0 0 7 0 0 0 0 0 0 0 0 0 0 0 0 0 composite outgroup 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 A A O B 1 1 G. hyloreus 0 0 1 0 0 0 0 0 0 0 B 0 0 0 0 0 0 0 A 1 1 G. pennsylvaniensis 0 A 0 0 0 0 1 1 0 0 A 0 0 1 1 0 0 0 A 0 7 G. robustus 0 0 0 0 1 0 0 0 0 0 0 0 1 1 1 0 B 1 B 0 1 G. shastai 0 0 0 0 1 0 0 0 B C 0 1 0 1 1 B B 1 B 0 1 H. i n t e s t i n a l i s 1 0 0 0 0 0 0 0 A 1 C 0 0 1 1 A B 0 B 0 1 G. c a l i f o r n i e n s i s 1 0 0 0 0 1 0 0 0 1 B 0 0 1 1 A B 0 B 0 1 G. quieta 1 B 0 1 0 0 0 0 0 1 B 1 0 1 1 0 B 0 A 0 1 G. f a c i o i A l l analyses were conducted on the Amdahl 5850 computer at the Univers i ty of B r i t i s h Columbia. I n i t i a l runs of PAUP used the 86 MULPARS step-wise addi t ion of OTUs algorithm with g lobal branch swapping (SWAP=GLOBAL). This algorithm examines a l l minimum-length trees that are obtained for a data set . Each of these trees i s , in turn , input to the branch-swapping procedure. In g lobal branch-swapping, the f i t of each OTU at each pos i t ion on the tree is examined. In l a t er computer runs, when ambiguities in character coding had been el iminated as much as poss ib le , the branch-and-bound (BANDB) algorithm was used. This approaches an exhaustive inspection of a l l poss ible topologies of a tree through a modif icat ion of the methodology of Hendy and Penny (1982). As a f i n a l a n a l y s i s , the ALLTREES algorithm was used. For small numbers of taxa, th i s examines every poss ible topology of a tree for minimum-length representat ions . In the Unordered Analys is of the mult i s tate characters with PAUP, a CSPOSS output was requested. This l i s t s the nodes for which there i s ambiguity of the character state after opt imizat ion . From the re su l t ing cladogram, the in ferred transformation ser ies of each mult i s tate character was represented with a numerical code. The coding method is herein referred to as Nonredundant Linear Coding, and i t s propert ies are examined in Appendix F . The recoded mult i s tate characters were then re inserted into the data matrix . This new matrix was then reanalyzed with both the ALLTREES algorithm of PAUP (Ordered Analysis' on a l l characters) and the WAGNER.S algorithm of PHYSYS. The l a t t e r performs step-wise addi t ion of OTUs with Wagner parsimony and subsequent g lobal branch-swapping. 87 RESULTS AND DISCUSSION PHYLOGENETIC ANALYSIS The cladogram for the eight species , rooted with a composite outgroup (ROOT=ANCESTOR), is presented in Figure 20. The postulated homologous ser ies for both binary and mult i s tate characters can be interpreted by reading the tree in conjunction with the character l i s t in the preceding sec t ion . With an Unordered Analys i s of the mult i s tate characters , nos. 2, 9, 11, 16, 17, and 19, the same tree i s obtained with MULPARS SWAP=GLOBAL, BANDB, and ALLTREES. The computer runs for ALLTREES are given in Appendix A. The goodness-of- f i t of a parsimony tree to the data from which i t i s in ferred is usual ly measured with the Consistency Index (CI) (Kluge and F a r r i s , 1.969; F a r r i s et a l . , 1970). This i s a r a t i o of the minimum number of evolutionary changes (character steps) required by the data, d iv ided by the number of changes postulated by the t ree . A perfect f i t of the tree to the data gives a CI of 1.0. The CI of the cladogram in Figure 20 is 0.848. The standard c a l c u l a t i o n of the CI does not d i s t i n g u i s h between autapomorphies and synapomorphies. It can be increased by the addi t ion of e i ther type of character to a tree . Since only the l a t t e r strengthen a phylogenetic hypothesis , the inc lus ion of autapomorphies in the c a l c u l a t i o n can produce a misleadingly high goodness-of-f i t value. For th i s 88 reason, i t i s h e r e i n suggested that non-homoplasious autapomorphies be excluded from the c a l c u l a t i o n of the CI. For the present study, t h i s procedure g i v e s a CI of 0.769. In a s s o c i a t i o n with t h i s proposed m o d i f i c a t i o n , the p r o p e r t i e s of the CI are examined i n Appendix G. Each m u l t i s t a t e t r a n s f o r m a t i o n s e r i e s was recoded (Nonredundant L i n e a r Coding, see Appendix F) f o r ordered a n a l y s i s with both PAUP and PHYSYS. T h i s r e c o d i n g assigned a numerical value to each s t a t e of the m u l t i s t a t e c h a r a c t e r such that the m u l t i s t a t e t r e e , which had been i n f e r r e d through parsimony c r i t e r i a , was unambiguously represented as a set of l i n e a r l y coded t r a n s f o r m a t i o n s e r i e s . In a d d i t i o n to p r o v i d i n g an example of the m u l t i s t a t e coding methods d i s c u s s e d i n Appendix F, t h i s procedure allowed an a n a l y s i s of the f u l l matrix with the WAGNER.S a l g o r i t h m of PHYSYS, which operates only on ordered data. M u l t i s t a t e c h a r a c t e r s 2, 9, 16, 11, and 17 have no a m b i g u i t i e s as to t h e i r nodal v a l u e s , and t h e i r recoding i s given i n F i g u r e 21. Character 19, the mean egg l e n g t h , shows ambiguity at some nodes ( F i g . 22). As noted i n the documentation f o r PAUP ( v e r s i o n 2.4, 1985), the r e s o l u t i o n of t h i s ambiguity f o r the purpose of the program's output i s completely a r b i t r a r y (thus, the importance of r e q u e s t i n g a CSPOSS l i s t i n g ) . In the Unordered ALLTREES a n a l y s i s given i n Appendix A, s t a t e 19(B) was chosen. These nodal assignments are o u t s i d e any parsimony c o n s i d e r a t i o n s . T h e i r a l t e r n a t i v e r e s o l u t i o n s do not a f f e c t the l e n g t h of the t r e e . 89 So as to provide a tentat ive reso lut ion for character 19, a d d i t i o n a l c r i t e r i a were considered. The character i s that of mean egg length, with state 0 <30jjm, state A = 30-40 jum, and state B >40jLim. With an assumption of l inear increase of egg length, state A was given precedence in the transformation s e r i e s . However, at the node uni t ing G. shas ta i , H. i n t e s t i n a l i s , G. c a l i f o r n i e n s i s , G. qu ie ta , and G. f a c i o i , state B was given precedence because there i s some uncertainty as to whether the mean egg length of G. fac i o i i s state A or B (see the sec t ion , CHARACTER ANALYSIS). The a p o s t e r i o r i recoding of the six mult i s tate characters produced f ive new character columns in the data matrix. The reassigned character numbers are given in Table I I I . When run through an Ordered ALLTREES analys i s in PAUP, as well as a WAGNER.S ana lys i s in PHYSYS, the new matrix produced the same cladogram as that in Figure 20 (see Appendix A ) . 90 Table III - CHARACTERS RENUMBERED FROM UNORDERED ANALYSIS Character No. in Character State Character No. in Unordered Analys i s Transformations Ordered Analys i s 1 1 0 - 1 2 0 - A d ) - B d ) 0 3 0 - 1 4 0 - 1 5 0 - 1 6 0 - 1 7 0 - 1 8 0 - 1 9 0 - A d ) - B d ) 0 10 0 - 1 11 0 - A d ) - B d ) -0 - C(2) 12 0 - 1 13 0 - 1 14 0 - 1 15 0 - 1 16 0 - A d ) , - B d ) , 0 17 0 - A d ) , - B d ) , 0 18 0 - 1 19 0 - A d ) -- 1 ... - B(2) 20 0 2 3 4 5 6 7 8 9 10 1 1 1 2 1 3 1 4 1 5 1 6 1 7 18 19 20 21 22 23 24 25 21 0 - 1 26 91 As a f i n a l a n a l y s i s , the expanded matrix was mapped onto the Brooks (1977) cladogram for Glypthelmins (see Appendix A ) . Thus, the hypothesis of re la t ionsh ips postulated by the e a r l i e r cladogram was compared to the present hypothesis by seeing how well a l l of the present character data supported i t . The e a r l i e r analys is used 11 characters , and d i f f e r e d in i t s placement of G. fac i o i and by i t s non- inc lus ion of H. i n t e s t i n a l i s . The mapping-on can be done by hand, but i t is more eas i ly accomplished, with the DIAGNOSE routine of PHYSYS. The new data f i t onto the e a r l i e r cladogram with an unmodified CI of 0.788. TAXONOMIC CONSIDERATIONS The higher l eve l monophyly of Brooks' (1977) analys is of North, C e n t r a l , and South American species of Glypthelmins i s supported by the resu l t s of the present study. The clade cons i s t ing of G. hyloreus and G. pennsylvaniensis corresponds to the genus Hylotrema, proposed by Su l l i van (1972). Brooks' analys i s also supported th i s correspondence. Despite i t s use by Sche l l (1985), Hylotrema is not a v a l i d genus. This i s because i t has been proposed in no place other than a d i s s er ta t i on ( A r t i c l e 9, Internat ional Code of Zoological Nomenclature, 3rd e d i t i o n ) . The resu l t s presented in Figure 20 postulate that Haplometrana is not the s i s t e r taxon of Glypthelmins, but i s in fact a member of one of the clades (lineage II sensu Brooks, 92 1977) within Glypthelmins. Thus, Glypthelmins w i l l not be monophyletic i f the monotypic genus Haplometrana i s excluded from i t . It i s therefore proposed that Haplometrana be synonymized with Glypthelmins as a junior subject ive synonym. Appendix D presents th i s synonymy in conjunction with a redescr ipt ion of H. i n t e s t i n a l i s and the necessary emendation of the generic diagnosis of Glypthelmins. Also in Appendix D is a redescr ipt ion of G. shasta i , made possible by the examination of the add i t i ona l specimens used in th i s study. Such c l a s s i f i c a t o r y changes are d irec ted towards maintaining consistency between c l a s s i f i c a t i o n s and the evolutionary trees whose re la t ionsh ips they are intended to communicate ( H u l l , 1964; Wiley, 1979, 1981b). This consistency can be los t when cer ta in characters are considered, a p r i o r i , to be more important than others as c r i t e r i a of group membership. See Figure 23a. The tree at the top i s a cladogram for f ive taxa with four characters , each of which occurs in e i ther a "U" or "X" s tate . The cladogram postulates the evolut ion of six character states ( i . e . , i t i s six character steps long) , inc luding the p a r a l l e l evolut ion of character 4 and the reversa l of character 1. In a departure from the c r i t e r i o n of grouping by monophyly, some of the characters might be considered more important because of t h e i r funct ional r o l e , as discussed in Chapter I I . Or, some might be considered more important as, say, generic or f a m i l i a l l e v e l characters . In such cases, trees such as those in Figures 23b-d would r e s u l t . Tree 23b re su l t s from putt ing 93 taxa A and B in the ir own group because they do not possess the derived state of character 3 that makes taxa C, D, and E look d i f f e r e n t . The tree i s seven character steps long, and is an example of grouping by shared p r i m i t i v e characters . Tree 23c resul t s from putt ing A, D, and E together because of the ir common possession of the derived state of character 4. It i s seven steps long, and is an example of grouping by convergent t r a i t s . Tree 23d resu l t s from excluding taxon E from the group because i t does not possess the derived state of character 1. It i s eight steps long, and is an example of f a i l i n g to d i s t i n g u i s h between plesiomorphic and apomorphic absence. A l l three types of character in terpre ta t ion above d i s t o r t phylogenetic re la t ionsh ips and in ter fere with the use of a phylogenetic tree as a general reference system in comparative bio logy. 94 IV. USING THE PHYLOGENETIC TREE TO STUDY EVOLUTIONARY EVENTS Once a cladogram has been constructed, i t can be used as a general reference system for further comparative b i o l o g i c a l s tudies . This chapter presents such studies of f ive types of events in ferred to have occurred during the evolut ion of the species of Glypthelmins and Haplometrana studied here. For the remainder of th i s d i s s e r t a t i o n , th i s group w i l l be re ferred to as the Glypthelmins c lade . Although the synonymization of H. intest i n a l i s as G. i n t e s t i n a l i s n. comb. i s supported by the analys i s in Chapter III (see Appendix D), the former name is retained here for c o n t i n u i t y . The f ive evolutionary events considered here are: (1) the phylogenetic concordance of l a r v a l and adult characters , (2) l i f e cyc le evo lut ion , (3) heterochronic character development, (4) coevolution of paras i tes and hosts, and (5) biogeographic and spec iat ion patterns . In add i t i on , heterochronic development i s studied fur ther , with a report on the experimentally-produced heterochronic development of the hindbody in H. i n t e s t i n a l i s . 95 PHYLOGENETIC CONCORDANCE OF LARVAL AND ADULT CHARACTERS INTRODUCTION Cladograms constructed from the characters of d i f f erent developmental stages can be compared in order to determine whether the d i f f erent character sets indicate the same phylogenetic r e l a t i o n s h i p s . This comparison i s important for two reasons. F i r s t , the ontogenetic stages (= l a r v a l + adult stages) of digeneans occur, to varying extents, in d i f f erent environments. At the very l ea s t , there are three such environments: the mollusc intermediate host , in which the m i r a c i d i a , sporocysts , rediae, and cercariae develop (see next sec t ion) ; the water, in which the cercar iae disseminate; and the vertebrate f i n a l host, in which the adult digeneans develop. If phylogenetic analyses of the d i f f erent developmental stages produce the same hypothesis of r e l a t i o n s h i p s , then there i s evidence of constra ints on character evolution that produce s i m i l a r l y covarying sets of characters regardless of environmental d i f ferences and presumed se lect ion pressures. Second, a p r i o r i assumptions of recap i tu la t ion can be used to promote or discount the usefulness of c e r t a i n characters in phylogenetic inference. By th i s I mean the fo l lowing . There are s ix basic types of sequence changes that can occur in an ances tra l developmental sequence (say, A - B - C - D ) . There can be an addi t ion of a stage that i s terminal (A-B-C-D-X) or 96 nonterminal (A-B-X-C-D) . There can be a de le t ion of a stage that i s terminal (A-B-C) or nonterminal (A-C-D) . And there can be a subs t i tu t ion of a stage that i s terminal (A-B-C-X) or nonterminal (A-B-X-D) . As discussed in O'Grady (1985b), a study of l i f e cyc le evolution in the p a r a s i t i c flatworms, so long as there is some sort of inher i ted temporal ser ies involved, a "developmental sequence" can refer to both ontogenetic stages and l i f e h i s tory stages. Thus, as discussed in Chapter II (A SINGLE EXPLANATORY FRAMEWORK)-, i t was postulated that the evolut ion of the Digenea involved a nonterminal addi t ion of ontogenetic stages and a terminal addit ion of a host. To the extent that evolut ionary transformations are added to and retained in developmental sequences, information about those evolut ionary events w i l l be retained in i n d i v i d u a l developmental sequences. Conversely, to the extent that the sequences are a l t e r e d , such information w i l l be l o s t . Haeckelian recap i tu la t i on (Haeckel, 1866) ex i s t s when there has been terminal addit ion to an ances tra l sequence (see F ink , 1982; Gould, 1977; Lovtrup, 1978); i t allows for the strongest inference of phylogenetic re la t ionsh ips from developmental sequence observations. Von Baerian r e c a p i t u l a t i o n (von Baer, 1828) ex i s t s when there has been terminal subs t i tu t ion to an ancestra l sequence (see F ink , 1982); i t allows for a weaker inference of phylogenetic re la t ionsh ips from developmental sequence observations. Nonterminal a l t e r a t i o n s to a developmental sequence w i l l d i srupt the p a r a l l e l between evolut ionary h i s tory and i n d i v i d u a l sequences, and thus v i o l a t e 97 both Haeckelian and von Baerian c r i t e r i a of r e c a p i t u l a t i o n . Using Haeckel's terminology, sequence characters that are recap i tu la t ions (sensu la to : the term w i l l henceforth be used here to refer to both Haeckelian and von Baerian r e c a p i t u l a t i o n ) , are pa l ingenet i c , while those that are not are cenogenet i c . Cenogenetic changes have come to be often a t t r i b u t e d to the e f fects of natural se lect ion ( e . g . , R u s s e l l , 1916). Some p a r a s i t o l o g i s t s have attempted to infer phylogenetic re la t ionsh ips s t r i c t l y from characters that are considered to be pa l ingenet i c . Chitwood and Chitwood (1974: 213), for example, considered cer ta in l a r v a l characters of nematodes to be cenogenetic adaptations associated with l a r v a l se lec t ion pressures, and thus not ind ica t ive of phylogenetic r e l a t i o n s h i p s . Goodchild (1943) presented a s imi lar argument for the cercar iae of phyllodistome digeneans. Cable (1974) considered the l a r v a l stages of digeneans to be recap i tu la t ions of ances tra l adult stages, and so based h i s phylogenetic inferences on the larvae . Conversely, Gibson (1981) considered the l a r v a l stages to be cenogenetic adaptations, and so based his phylogenetic inferences on the adul t s . There are two drawbacks to such a re l iance on recapi tu la tory phenomena: (1) the perception that recapi tu la tory developmental sequences are the only source of information on phylogenetic r e l a t i o n s h i p s , and (2) the app l i ca t ion of th i s perception in conjunction with some a p r i o r i assumption about which of the characters in an analys i s are recap i tu la tory . 98 Recapitulatory characters are not the only source of information for phylogenetic inference because sequence analys i s is not a primary source of such information. 1 The study on the p a r a s i t i c flatworms by Brooks e_t a_l. (1985a), discussed above, i s not only an example of other types of character data being used to draw phylogenetic inferences , i t a lso shows that a cladogram can be used to come to a p o s t e r i o r i decis ions about the types of sequence changes involved in the evolut ion of a c lade . Thus, an i n i t i a l assumption of r ecap i tu la t i on as the  primary c r i t e r i o n with which to infer phylogeny encounters the  same problems as does any evolut ionary in terpre ta t ion based upon a s ingle transformation ser i e s : namely, without a phylogenetic hypothesis corroborated by other characters , i t i s not poss ible to determine the p o l a r i t y or the order of transformation. Just as a phylogenetic hypothesis i s necessary to d i s t i n g u i s h p r i m i t i v e absence from secondary absence, so too is i t necessary to d i s t i n g u i s h whether, say, in a study of three taxa, the presence of the developmental sequence A-B-C in one of them and the presence of the sequence A-B-C-D in the other two is ind ica t ive that the l a t t e r two taxa are apomorphic. The c l a d i s t i c analys i s of the Digenea by Brooks e_t a l . (1985b) was, in part , a test of previous arguments that only l a r v a l or adult characters are of use in i n f e r r i n g the 1 Nelson (1978, 1985) and Nelson and Platn ick (1981) argued for the u t i l i t y of ontogenetic sequence data for p o l a r i z i n g characters . Brooks and Wiley (1985) and Kluge (1985) offered counter-arguments. 99 re la t ionsh ips of these worms (see Cable, 1974; Gibson, 1981). This question was e spec ia l ly in teres t ing because our e a r l i e r study (Brooks et a l . , 1985a) had hypothesized that the l a r v a l stages of digeneans are nonterminal addit ions to an ancestra l ontogeny. By r e c a p i t u l a t i o n i s t c r i t e r i a , these cenogenetic characters should be phylogenet ica l ly uninformative. Furthermore, by s e l e c t i o n i s t c r i t e r i a , they should instead corre la te with the d i f f erent environmental condit ions * experienced by the larvae , as compared to the a d u l t s . The study found that (1) l a r v a l characters alone resolved 74% of the tree , (2) adult characters alone resolved 76% of the tree , (3) there was no disagreement in phylogenetic inferences drawn from only l a r v a l or adult characters , and (4) eco log ica l characters resolved 26% of the tree . The concordance of l a r v a l and adult characters was taken as evidence that there are evolutionary constra ints capable of producing a s ing le , phylogenet ica l ly informative, covarying set of characters despite the nonterminal i n t e r c a l a t i o n - i . e . , the non-recapi tulatory modif icat ion - of ontogenetic stages, and despite development in d i f f erent environments. Such comparisons of phylogenetic hypotheses drawn from d i f f erent data sets involve the comparison of the branching patterns , or topologies , of two or more trees . The terms used in systematics for the degrees of s i m i l a r i t y of trees are congruence (Sokal and Sneath, 1963; F a r r i s , 1971; Mickevich, 1978) and consistency ( H u l l , 1964; Wiley, 1981b). When trees have the same topology, as do Figures 24a and b, they are said 100 to be completely congruent ( so l id arrows) with each other. When a tree i s ambiguous about the re la t ionsh ips involved, as i s the polytomy in Figure 24c, then a l l poss ible reso lut ions of that tree , such as Figures 24a, b, and d, are said to be consistent (dashed arrows) with i t . Complete congruence requires that trees have i d e n t i c a l topologies , consistency only requires that there are no disagreements among the trees . Any intermediate condit ion ( i . e . , only parts of trees are iden t i ca l ) i s referred to as p a r t l y congruent ( e . g . , Mickevich , 1978; Miyamoto, 1981; Crother e_t a_l. , 1986). Trees with mutually exclusive topologies , such as Figures 24b and d, are said to be incongruent with each other (= the incompatibl i ty c r i t e r i o n of Camin and Sokal , 1965; see F a r r i s , 1971). LIFE HISTORY DATA Figure 25 shows a general ized digenean l i f e c y c l e . It i s a complex l i f e c y c l e , involv ing one or more intermediate hosts in addit ion to the f i n a l , or d e f i n i t i v e host , in which the paras i te develops to maturity . Eggs pass into the water. The miracidium emerges from the egg and enters the f i r s t intermediate host, usual ly a gastropod mollusc, sometimes a polychaete. Within the host t i s sues , the miracidium metamorphoses into a r e l a t i v e l y undi f ferent ia ted stage c a l l e d a sporocyst . Asexual reproduction then occurs, as numerous rediae develop from germinal c e l l s in each sporocyst. This reproduction may be preceded by a second generation of sporocysts . There can also be a second generation 101 of rediae . Rediae are somewhat more d i f f e r e n t i a t e d than sporocysts , possessing a mouth, pharynx, and gut. Again, asexual reproduction occurs, as numerous cercar iae develop from germinal c e l l s in each r e d i a . A c e r c a r i a is the juveni le of the adult digenean, in that i t s body develops into the adult body. It possesses a complete d iges t ive and osmoregulatory system, and often has the gen i ta l primordia as w e l l . In a d d i t i o n , there i s usual ly a t a i l . In some groups, there i s a lso a s ty l e t in the o r a l sucker. The c e r c a r i a emerges from the s n a i l host and enters the body of the next host e i ther through penetration or ingest ion . If a two-host l i f e cycle is involved, the paras i te w i l l develop to maturity in th i s second host. If a three-host l i f e cycle i s involved, the paras i te develops into a metacercaria in the t i ssues of the second intermediate host, and then infects the f i n a l host when the second intermediate host i s eaten. 1. PREVIOUS STUDIES There are published reports of the l i f e h i s t o r i e s of the fol lowing species: G. hyloreus , by Martin (1969), G. pennsylvaniensi s, by S u l l i v a n and Byrd (1970), G. qu ie ta , by Rankin (1944) and Leigh (1946), and H. i n t e s t i n a l i s , by Olsen (1937). In a l l of these species , there i s a three-host l i f e cyc le in which the frog i s both second intermediate host and f i n a l host. Glypthelmins hyloreus develops in Lymnaea s tagnal i s L . ; the cercar iae possess a f i n f o l d , but no s t y l e t ; they enter 102 the nares of H. regi11a tadpoles, remain as unencysted metacercariae in the coelom, then enter the gut when the host metamorphoses. Glypthelmins pennsylvaniensis develops in Physa  gyrina Say; the cercariae possess a f i n f o l d , but no s t y l e t ; they penetrate the body of H. c r u c i f e r tadpoles and remain as unencysted metacercariae beneath the epidermis; the host becomes infected when i t ingests the sloughed epithel ium sometime after metamorphosis. Glypthelmins quieta develops in P. gyrina and P. integra Haldeman; the cercar iae possess a f i n f o l d and s t y l e t ; they penetrate and encyst in the epithel ium of R. pipiens adul t s , which become infected when they ingest the sloughed epi thel ium. Haplometrana i n t e s t i n a l i s has been reported to develop in P. utahensis (Clench), by Olsen (1937), in P. gyrina and P. ampullacea Gould, by Sche l l (1961), and in Lymnaea  s tagnal i s wasatchensis (Hemphil l) , L . bulimoides Lea, and Helisoma t r i v o l v i s (Say), by Current and Lang (1975). The cercar iae possess a f i n f o l d and s t y l e t ; they penetrate and encyst in the epithelium of R. pret iosa adul t s , which become infected when they ingest the sloughed epi thel ium. The cercar iae w i l l encyst i n , but not develop to maturity i n , adults of R. p i p i e n s , R. clamitans L a t r e i l l e , and R. areolata (see Olsen, 1937). 1 03 2. NEW DATA The l i f e cycles of three paras i te species were maintained in the laboratory: G. c a l i f o r n i e n s i s , G. qu ie ta , and H. i n t e s t i n a l i s . This i s the f i r s t report of the l i f e cycle of G. c a l i f o r n i e n s i s (see Appendix E ) . For each of the three species, the appropriate s n a i l and anuran host were maintained in the laboratory . Uninfected populations were establ ished for the fol lowing species of l o c a l l y - c o l l e c t e d sna i l s ( i d e n t i f i c a t i o n s based on Clarke , 1981): Physa gyrina Say, P. propinqua Tyron, P. l o r d i B a i r d , Stagnicola elodes (Say) [=Lymnaea p a l u s t r i s ( M u l l e r ) ] , and Pseudosucc inea columella (Say). This las t species is indigenous to eastern North America and has been introduced to the western part of the continent (Clarke, 1981). D i f f i c u l t i e s were encountered in rearing anurans from eggs to adul t s . Five species were involved: R. aurora, R. pre t io sa , R. cascadae, R. pipiens and B. boreas. Each suffered high morta l i ty as tadpoles and during the t r a n s i t i o n from tadpoles to a d u l t s . As a r e s u l t , only R. pret iosa was completely reared in the laboratory . For the remaining four species, young adults were c o l l e c t e d from areas where there had been no reports of the paras i te species involved. Surveys were conducted of these areas to support the conclusion of an absence of paras i t i sm. The areas were: R. aurora: Streams associated with Hicks and Moss Lakes, Sasquatch P r o v i n c i a l Park, B r i t i s h Columbia (approximately 20 km NE of Chi l l iwack) (c lear , fast moving water, no s n a i l s seen) 104 R. cascadae: Dead F a l l Lakes, S i sk iyou County, C a l i f o r n i a ( c l ear , g l a c i a l lakes at approximately 2200m e levat ion , no s n a i l s seen) R. p ip iens : Elk Point , Union County, South Dakota (flooded gravel p i t , no sna i l s seen) B. boreas; Meadows north of Kangaroo Lake, S i sk iyou County, C a l i f o r n i a (no R. pret iosa and no H.. i n t e s t i n a l i s found) The l i f e cycles were maintained as fo l lows. For G. qu ie ta , the procedures of Rankin (1944) and Leigh (1946) were used for reference: Physa gyrina and P. propinqua were fed overnight on eggs that had been teased out of mature worms and had been allowed to s i t in f i l t e r e d pond water at 15°C for at least a week. Approximately one month l a t e r , cercar iae began to emerge from the s n a i l . Adult R. pipiens were placed for two hours in f inger bowls containing shedding sna i l s in f i l t e r e d pond water. The frogs were then kept in a common tank at 1 5 ° C . For H. i n t e s t i n a l i s , Olsen's (1937) study was used for reference: P. gyrina and P. propinqua were fed overnight on H. intest i n a l i s eggs that had been prepared in the above manner. Approximately one month l a t e r , cercar iae began to emerge from the s n a i l s . Adult R. pre t iosa were exposed to them as above and then kept in a common tank. Attempts were made to e s tab l i sh infect ions of H. i n t e s t i n a l i s in the adults of R. cascadae, R. aurora, and B. boreas. Frogs were e i ther exposed to H. i n t e s t i n a l i s 105 cercar iae or were fed metacercariae encysted in the skin of R. p r e t i o s a . In add i t ion , the penetration behavior of cercar iae of H. i n t e s t i n a l i s was observed on pithed adults of R. cascadae, R. aurora, R. clamitans, R. catesbeiana, R. p ip i ens , and B. boreas. The penetration behavior of cercar iae of G. quieta was observed on pithed adults of R. cascadae and R. aurora. In an attempt to produce an i n f e c t i o n , l i v e adults of R. aurora were also exposed to cercar iae of G. qu ie ta . The m i r a c i d i a , daughter sporocysts , and cercar iae of G. c a l i f o r n i e n s i s were obtained. The r e s u l t s , presented in Appendix E , show that members of th i s species develop as x iph id iocercar iae in sporocysts in the physid s n a i l s , Physa  gyrina and P. propinqua. The cercar iae encyst in the skin of adults of R. aurora, and develop in the small in tes t ine of the frog when the host eats the shed s k i n . Cercariae of G. quieta and H. i n t e s t i n a l i s were a lso obtained from infect ions of P. gyrina and P. propinqua. Metacercariae of both species were obtained encysted in the skin of specimens of R. pipiens. and R. p r e t i o s a , respec t ive ly . Adults of G. quieta were obtained from R. pipiens at one, four, e ight , and twelve months af ter exposing adult frogs to c e r c a r i a e . Adults of H. i n t e s t i n a l i s were obtained at the same ages from R. pret iosa and R. cascadae af ter exposing adult frogs to c ercar iae . Attempts to infect adults of R. aurora with cercar iae and metacercariae of H. i n t e s t i n a l i s were unsuccessful . Adults of H. i n t e s t i n a l i s were obtained at one, four, and twelve months from B. boreas af ter feeding the adult toads metacercariae encysted in the skin 106 of R. p r e t i o s a . Changes in the r e l a t i v e body dimensions of these specimens are examined in the sect ion , HETEROCHRONIC DEVELOPMENT. The in fec t ion of B. boreas with H. i n t e s t i n a l i s proved to be d i f f i c u l t . Exposure to cercar iae was unsuccessful . The cercar iae los t the ir t a i l s , penetrated the s k i n , and ba l l ed up under the epithel ium within approximately 30 minutes. Not only is th i s penetration process twice as long as that shown by H. intest i n a l i s when penetrating ranids (see below), but the encysted metacercariae on the toads were not as deeply embedded in the epithel ium, and could be dislodged . f a i r l y e a s i l y . A l t e r n a t i v e l y , feeding the toads metacercariae encysted in the skin of R. pret iosa produced an in fec t ion on the t h i r d t r i a l . The in fec t ion of tadpoles of B. boreas was not attempted. Olsen (1937) reported that the cercar iae of H. i n t e s t i n a l i s were capable of encysting i n , but not developing to maturity i n , adults of R. areo la ta , R. p i p i e n s , and R. c lamitans. The present study observed c e r c a r i a l encystment in adults of R. p ip i ens , R. c lamitans, R. catesbeiana, and R. aurora. Of these, only R. aurora ind iv idua l s were subsequently studied for development of the worm in the gut. The resu l t s were negative. In a l l four ranid species , as with R. pret iosa and R. cascadae, cercar iae took approximately 15 minutes to penetrate the epi the l ium. In each case, the c e r c a r i a would stop swimming upon contact ing the frog . It would then crawl over the surface for a short d i s tance , penetrate the s k i n , lose i t s t a i l as i t entered, and b a l l up. The longer penetration time shown by specimens of 1 07 H. i n t e s t i n a l i s when exposed to B. boreas resu l t s from both extended crawling and penetration per iods . It was also observed that cercar iae of G. quieta could encyst in adults of R. p r e t i o s a , R. aurora, and R. cascadae. Penetration behavior and timing i s the same as that for H-. i n t e s t i n a l i s with the ranids noted above. Subsequent study of R. aurora ind iv idua l s found G. quieta to develop for one month, but no more, in the gut. ANALYSIS OF LARVAL CHARACTERS Figure 26 presents a cladogram for the f ive species of Glypthelmins or Haplometrana for which there are l a r v a l data . It is constructed from the fol lowing f ive c e r c a r i a l t r a i t s : (1) c e r c a r i a l s t y l e t , (2) dorso-ventral f i n f o l d , (3) tandem testes , (4) symmetrical testes , and (5) tegumental sca les . Observations for G. c a l i f o r n i e n s i s , G. quie ta , and H. i n t e s t i n a l i s were conducted on specimens reared in the laboratory during the course of th i s research. Observations for G. hyloreus and G. pennsylvaniensis were taken from the studies by Martin (1969) and S u l l i v a n and Byrd (1970), re spec t ive ly . The tree i s congruent with the cladogram in Figure 20 with respect to i t s support of the G. hyloreus + G. pennsylvaniensis c lade , and the G. c a l i f o r n i e n s i s + G. quieta c lade , and i t i s consistent with respect to the placement of H. i n t e s t i n a l i s (the dashed l i n e shows that species' placement in Figure 20. There i s thus no disagreement between phylogenetic 108 inferences drawn from the ava i lab le adult and l a r v a l data . This conclusion can be contrasted with the comments by Martin (1969) and by Su l l i van and Byrd (1970) that the cercar iae of G. hyloreus and G. pennsylvaniensis appear to be instances of l a r v a l divergence from the usual condit ion in Glypthelmins. In conjunction with l i f e h i s tory data, discussed in the next s ec t ion , the l a t t e r authors supported the in terpre ta t ion of th i s divergence as a phylogenet ica l ly uninformative cenogenetic property . The present study has shown that although G. hyloreus and G. pennsylvaniensis may, in cer ta in respects , be highly derived members of the c lade , th i s der ivat ion does not obscure t h e i r phylogenetic r e l a t i o n s h i p s . ANALYSIS OF LIFE CYCLE EVOLUTION Seven l i f e cyc le propert ies can be examined: (1) a physid s n a i l as at least one of the f i r s t intermediate host types, (2) a lymnaeid s n a i l as at least one of the f i r s t intermediate host types, (3) a lymnaeid s n a i l as the only f i r s t intermediate host type, (4) a hel isomid s n a i l as at least one of the f i r s t intermediate host types, (5) an anuran as both second intermediate host and f i n a l host , (6) development as an unencysted metacercaria in anuran tadpoles, and (7) development as an encysted metacercaria in anuran adul t s . The tree supported by these characters i s presented in Figure 27. Again, there i s no disagreement with the primary cladogram in Figure 20. And again, any l a r v a l divergence in 109 G. hyloreus and G. pennsylvaniensis does not obscure phylogenetic r e l a t i o n s h i p s . Physid sna i l s are concluded to be the plesiomorphic f i r s t intermediate host, with lymnaeids replac ing them in G. hyloreus, and augmenting them in H. i n t e s t i n a l i s . The l a t t e r species i s also postulated to have acquired the a b i l i t y to develop in hel isomid s n a i l s . Development as an unencysted metacercaria in anuran tadpoles is concluded to have ar isen as a synapomorphy for the G. hyloreus + G. pennsylvaniensis c lade , while development as an encysted metacercaria in anuran adults is concluded to have ar isen as a synapomorphy for the H. i n t e s t i n a l i s + G. c a l i f o r n i e n s i s + G. quieta c lade . HETEROCHRONIC DEVELOPMENT INTRODUCTION Heterochrony ex i s t s when there are di f ferences in the r e l a t i v e developmental timing of characters . It i s a comparative phenomenon: developmental timing and i t s e f fects must be d i f f eren t with respect to something, and that something can be the a n c e s t r a l , or ples iomorphic , s tate . Heterochrony in a descendant can resu l t in a derived condit ion that i s , with respect to some c r i t e r i o n of change, less developed or more developed than the plesiomorphic cond i t ion . If i t i s less developed, the heterochrony involved i s termed paedomorphosis 110 ( e . g . , Gould, 1977; Alberch et a l . , 1979). If i t is more developed, i t i s termed peramorphosis (Alberch et a_l. , 1979). Alberch et a l . (1979) gave three parameters of developmental change that could produce heterochrony. These are i l l u s t r a t e d in Figure 28, in which the degree of development (gamma) of the ances tra l state (A) and the derived state (D) is p lo t ted against the time period over which development occurs . The parameters of change are: the rate of development (_k) , the time at which development s tarts (alpha), and the time at which i t stops (beta). Paedomorphosis can be produced by (1) a slower developmental rate: neoteny; 1 (2) a la ter i n i t i a t i o n of development: postdisplacement ; and (3) an e a r l i e r cessat ion of development: progenesis . Peramorphosis can be produced by (1) a faster rate: acce lerat ion; (2) an e a r l i e r i n i t i a t i o n : predisplacement ; and (3) a la ter cessat ion: hypermorphosis. These are the six basic types of heterochrony that occur when the three parameters change one at a time. Although Alberch et a l . (1979) d id not discuss them, add i t i ona l types of heterochrony would resul t i f more than one parameter changed. For example, a slower rate in conjunction with an e a r l i e r cessat ion of development would produce paedomorphosis. It i s even possible for a process that i s usual ly associated with paedomorphosis, such as a slower ra te , to produce a peramorphic 1 In th i s usage, th i s term is d isassoc iated from p a r t i c u l a r reference to gonadal development. The terminology here treats a l l development equal ly , and thus does not give spec ia l weight to gonadal maturation. 111 resu l t through a s u f f i c i e n t l y ear ly i n i t i a t i o n or a s u f f i c i e n t l y delayed cessation of development, or both. It should also be noted that peramorphosis i s only poss ible when the apomorphic development i s capable of developing beyond the plesiomorphic s ta te . If there i s no such c a p a b i l i t y , acce lerat ion or predisplacement w i l l simply resu l t in the derived developmental t ra jec tory reaching the plesiomorphic f i n a l state r e l a t i v e l y sooner and then l e v e l l i n g off with respect to gamma. Thus, the six developmental changes in Figure 28 should be seen as the processes that w i l l produce the types of heterochrony described under condit ions of c e t e r i s par ibus . Heterochrony may or may not involve a l t e r a t i o n s in developmental sequences. When i t does, i t provides the processes by which some of the sequence changes discussed in the f i r s t section of th i s chapter can evolve. With respect to Figure 28, a l l three types of paedomorphosis involve a descendant not a t ta in ing the plesiomorphic state because of a terminal de le t ion to i t s developmental sequence, and a l l three types of peramorphosis involve a descendant evolving beyond the plesiomorphic state because of a terminal addi t ion to i t s developmental sequence. Peramorphosis therefore exhib i t s Haeckelian r e c a p i t u l a t i o n . It appears that the t h i r d type of terminal change, s u b s t i t u t i o n , i s not produced by heterochronic processes. As for nonterminal sequence changes, i t appears that heterochrony can be involved sometimes, but i f i t i s , i t w i l l not be one of the six types out l ined by Alberch et a_l. (1979). This i s par t ly because in a nonterminal change, the 1 12 plesiomorphic terminal state is s t i l l a t ta ined . There may, however, be a l t e r a t i o n s in the r e l a t i v e time at which the descendant a t ta ins that s ta te . For example, a nonterminal de le t ion (1-2-3-4 to 1-2-4) may resu l t in state 4 being at ta ined sooner. Conversely, a nonterminal addi t ion (1-2-X-3-4) or even subs t i tu t ion (1-2-X-4) may resu l t in state 4 being at ta ined l a t e r . F i n a l l y , there can be heterochrony that does not involve any terminal or nonterminal changes to a developmental sequence. In t h i s , the plesiomorphic sequence s t i l l occurs in the descendant, but i t simply takes place faster or slower ( i . e , changes in k that cannot be categorized as acce lerat ion or neoteny because there i s no change in development with respect to gamma). Fink (1982) demonstrated that a cladogram could be used to detect the types of heterochrony noted by Alberch et a l . (1979). He showed that a cladogram is needed to d i s t i n g u i s h between paedomorphic and symplesiomorphic morphologies (F igs . 29a and b) , and that an inspect ion of developmental sequences may be necessary to d i s t i n g u i s h between paedomorphosis and peramorphosis ( F i g . 29c). An example from F i n k ' s (1982) study w i l l help to c l a r i f y the methodology before examining heterochrony in Glypthelmins and Haplometrana. Figure 30 presents Figure 5, a contr ived example from Fink (1982)'. The cladogram in Figure 30a i s assumed to be supported by a number of characters . One of these is a poss ible instance of heterochrony because i t s less complex form (state 0) i s present not only in the outgroups and the most 113 plesiomorphic taxon of the study group, taxon A, but also in one of the more derived taxa, C. Taxon B, on the other hand, has the more complex form (state 1). The question asked in the analys i s i s whether character state 0 in taxon C is symplesiomorphic, paedomorphic, or peramorphic. Figure 30b gives one case in which th i s question could be answered. A and C could both have state 0, but the developmental rate of the character may be slower in C than in e i ther A or B, and the l a t er cessation of development in B may coincide with that of C. In such a case, the inference is that the common ancestor of B and C evolved character state 1 through hypermorphosis (cessation of development occurred l a t e r ) , subsequent to which C evolved state 0 through neoteny (slower rate of development). As noted by Fink (1982), there can also be instances in which a cladogram cannot help to detect heterochrony. An example is given in Figure 30c. The common ancestor of taxa B and C could have evolved character state 1 through hypermorphosis, subsequent to which C evolved state 0 through progenesis ( e a r l i e r cessation of development). Because the plesiomorphic and apomorphic developmental rates are the same, and because the time of cessation of development in A and C i s the same, the parsimony c r i t e r i o n of phylogenetic systematics would produce the spurious conclusion that the common occurrence of state 0 in A and C is symplesiomorphic. 114 ANALYSIS Of the 21 characters analyzed, three were examined for heterochrony because t h e i r apomorphic states exhibi t morphologies that are" conspicuously less developed or more developed than those of the plesiomorphic s tates . These three characters are not presented as an exhaustive c o l l e c t i o n of the poss ibly heterochronic characters in the data set , but simply as those that were chosen for examination. A fourth character , r e l a t i v e hindbody length, was also examined. The apomorphic states of the f i r s t three characters (refer to Chapter I I I , CHARACTERS ANALYZED, and the cladogram in F i g . 20) are: the absence of extracecal uterine loops (character 14, state 1); the absence of p r e t e s t i c u l a r uterine loops (character 15, state 1); and the persistence of penetration glands in the adult (character 4, state 1); The apomorphic state of the fourth character , r e l a t i v e hindbody length, that i s of interest here i s that of an e spec ia l l y long hindbody (HBL) in re la t ion to t o t a l body length (TBL) in H. i n t e s t i n a l i s . The mean values for HBL/TBL in each species , for a l l ava i lab le adult specimens, are as fol lows: G. hyloreus: 0.68; G. pennsylvaniensis: 0.71; G. robustus: 0.62; G. shasta i : 0.75; G. c a l i f o r n i e n s i s : 0.69; G. quie ta: 0.71; G. f a c i o i : 0.74; H. i n t e s t i n a l i s : 0.80. (For comments on s t a t i s t i c a l l y s i g n i f i c a n t d i f ferences , see the sec t ion , EXPERIMENTALLY-PRODUCED HETEROCHRONY IN HAPLOMETRANA INTESTINALIS.) Comparisons involv ing G. c a l i f o r n i e n s i s , G. quie ta , and 1 15 H. i n t e s t i n a l i s made use of observations on specimens reared in the laboratory during the course of th i s research (see the sec t ion , LIFE HISTORY DATA). Comparisons involv ing G. hyloreus and G. pennsylvaniensis made use of the data of Martin (1969) and of S u l l i v a n and Byrd (1970), re spec t ive ly . There are no developmental data for G. robustus, G. shasta i , and G. fac i o i . The absence of extracecal and p r e t e s t i c u l a r uterine loops: S u l l i v a n and Byrd (1970) observed that during the development of G. pennsylvaniensis , the uterus remains in terceca l and post-t e s t i c u l a r u n t i l i t has grown to the poster ior of the body. Subsequent la terad and anteriad growth produces the extracecal and p r e t e s t i c u l a r loops, r e spec t ive ly . In my observations of young specimens of H. i n t e s t i n a l i s and G. quie ta , I found that during both i t s growth to the poster ior of the body and i t s subsequent enlargement, the uterus remains in terceca l and post-t e s t i c u l a r . These observations for H. i n t e s t i n a l i s agree with those of Olsen (1937). The conclusion i s therefore that the absence of extracecal and p r e t e s t i c u l a r uterine loops in mature adults i s an instance of paedomorphosis. There are i n s u f f i c i e n t data to determine whether th i s is produced through a l t e r a t i o n s in the ra te , time of i n i t i a t i o n , or time of cessat ion of the developmental t r a j e c t o r i e s . The presence of penetration glands in the adul t : These glands, located in the forebody and emptying into the o r a l region, are found in many digenean cercar iae . They usual ly degenerate and 1 16 disappear in adulthood. They p e r s i s t , however, in the adults of G. fac i o i . Using the t r a d i t i o n a l usage of the term (see Gould, 1977), th i s property might be termed neotenic because i t i s a l a r v a l character appearing in an adult - comparable to the retent ion of g i l l s in sexually mature salamanders. However, according to the c r i t e r i a for heterochrony discussed here in , i t i s most l i k e l y peramorphic. The a v a i l a b i l i t y of only mature adult specimens of G. fac i o i forces a tentat ive conclus ion, but two observations can nevertheless be made. F i r s t , the persistence of the glands in the adult indicates that the operation of whatever homeostatic mechanisms maintain them has been extended into the adult stage. Second, i f the glands in the adults of G. fac i o i are compared to the glands in the cercar iae of the two s i s t e r species, G. c a l i f o r n i e n s i s and G. qu ie ta , they are seen to be l a r g e r . Therefore, not only are the glands maintained in adulthood, but they also continue to grow. This i s peramorphosis, presumably through hypermorphosis. Hindbody length: The development of a longer hindbody in H. i n t e s t i n a l i s was followed by examining specimens at the metacercaria l stage, and at 1, 4, 8, and 12 months after development in the gut of the f i n a l host, R. p r e t i o s a . Figure 31 presents the growth curve, p lo t ted as hindbody l eng th / to ta l body length (HBL/TBL) vs. time. Growth data are also given for specimens of G. quieta reared in the laboratory in R. p ip iens . This i s the only other species in the study group for which such data are a v a i l a b l e . Both species show the same growth pattern 1 1 7 up to four months after in fec t ion (MAI). Subsequent to that , the rate of development of G. quieta slows down, while that of H. i n t e s t i n a l i s continues at approximately the same rate as during the 1 MAI to 4 MAI per iod . By 12 MAI, specimens of H. i n t e s t i n a l i s have at ta ined a mean HBL/TBL value of 0.79 (n = 10; s = 0.03), while those of G. quieta have at ta ined a mean value of 0.71 (n = 10; s = 0.03). Figure 31 a lso includes the mean HBL/TBL values for mature adu l t s , of unknown ages, of the other species of Glypthelmins that are in the same clade as H. i n t e s t i n a l i s and G. qu ie ta . These values f a l l within the 0.62 to 0.75 range. The value for H. i n t e s t i n a l i s i s s i g n i f i c a n t l y d i f f erent at the 0.01 l eve l (difference of two means from independent samples) from that of the c losest species , G. shas ta i , which is 0.75 (n = 14; s= 0.03). The plesiomorphic value of the HBL/TBL r a t i o for H. i n t e s t i n a l i s ( i . e . , the value of the node uni t ing H. i n t e s t i n a l i s and G. shastai) can be estimated by set t ing the nodes on the cladogram to the median values of the ra t ios ( F i g . 32). This gives a value of 0.75. 1 The conclusion for H. i n t e s t i n a l i s i s that given the equal i ty of i t s ear ly HBL/TBL values to those of G. quieta ( i . e . , the time of i n i t i a t i o n of development i s the same), and given the equal i ty of the time at which f i n a l measurements were taken ( i . e . , the time of cessat ion of development is the same), 1 As noted in Chapter I I I , the ava i lab le specimens of G. robustus are not f u l l y mature; th i s species was therefore omitted from the c a l c u l a t i o n s . 1 18 a longer hindbody evolved through peramorphosis by acce lerat ion of the rate of development subsequent to 4 MAI. ALLOMETRY AS HETEROCHRONY Huxley (1932) introduced the concept of al lometry for reference to r e l a t i v e d i f f e r e n t i a l growth of body par t s . It is best considered as a phenomenological labe l that can be appl ied to cer ta in patterns of character change brought about by various developmental processes. As such, i f heterochrony involves di f ferences in the r e l a t i v e developmental timing of characters a f fec t ing body dimensions, then the resul t w i l l show an a l lometr ic growth pat tern . Huxley (1932) demonstrated how an a l lometr ic growth constant can be ca lcu la ted by double logarithmic p lots of the r e l a t i v e growth in any two body p a r t s . This allowed the q u a n t i f i c a t i o n of instances in which body features .were observed to become longer / shorter , wider / th inner , b igger / smal ler , e tc . than cer ta in other features . If the growth of the two parameters i s equal , then the a l lometr ic growth constant has a value of 1.0, and the condit ion i s referred to as i sometr ic . If the growth of body feature X is greater than that of body feature Y over the same period of time, then the constant w i l l be greater than 1.0, and the growth of X is referred to as p o s i t i v e l y a l lometr ic with respect to that of Y. If the converse holds , the growth of X is negatively a l l o m e t r i c . Studies of a l lometr ic growth of various body parts in 1 19 digeneans or monogeneans have been conducted by: (1) Thomas (1965), for Mesocoelium monodi D o l l f u s , 1929; (2) Rohde (1966), for Anchitrema sanguineum (Sonsino, 1894) Looss, 1899, Platynosomum fastosum Kossack, 1910, Zonorchis s p . , Mesocoelium s p . , Diaschi s torch i s m u l t i t e s t i c u l a r i s Rohde, 1962, Maxbraunium  baeri Rohde, 1964, Odeningotrema hypergeni ta l i s Rohde, 1962, Novotrema nyt i ceb i Rohde, 1962, Renschetrema malayi Rohde, 1964, Kaurma intermedia Rohde, 1963, Parorientodiscus magnus Rohde, 1962, Opisthorchis v i v e r i n i P o i r i e r , 1886, Polystomoides malayi Rohde, 1963, and P. renschi Rohde, 1965; (3) F i s c h t h a l and Kuntz (1967), for Pleuroqenoides t a y l o r i (Tubangui, 1928) Travassos, 1930; (4) S u l l i v a n (1977a), for Choledocystus hepaticus; and (5) F i s c h t h a l ( l978a,b) , for Apocreadium mexicanum Manter, 1937, Pseudocreadium l a m e l l i forme (Linton, 1907) Manter, 1946, Paracryptogonimus americanus Manter, 1940, M u l t i t e s t i s rotundus Sparks, 1954, Stenopera equi la ta Manter, 1933, Leurodera decora L i n t o n , 1910, and Prosorhynchus p a c i f i c u s Manter, 1940. In a l l of these species , the hindbody shows pos i t i ve a l lometr ic growth in r e l a t i o n to t o t a l body length. The observations on G. quieta and H. i n t e s t i n a l i s reported herein a lso indicate such pos i t i ve allornetry of the hindbody. Rohde (1966) supported the suggestion by Huxley (1932) and Needham (1964) that a l lometr ic re la t ionsh ips be used as taxonomic characters . Thus, the a l lometr i c growth constant would be seen as a taxon-spec i f ic character determined by taxon-s p e c i f i c growth patterns . F i s c h t h a l and Kuntz (1967) cautioned against the weighting of such a character over any other 120 characters of the taxa involved. I concur, and I a lso note that (1) a l lometr ic growth constants can change during the ontogeny of an organism, and (2) since al lometry i s not necessar i ly produced by a s ingle process, there is the danger, as with any character , of mistaking homoplasy for homology. Just as Fink (1982) demonstrated that heterochrony can be placed in a phylogenetic context, so too can a l lometry . Thus, with reference to Figure 31 and the phylogenetic analys is presented here in , the fol lowing can be sa id . The growth of the hindbody in H. i n t e s t i n a l i s and G. quieta is equally p o s i t i v e l y a l lometr ic with respect to t o t a l body length for the f i r s t four months of adult development. Subsequent to t h i s , the apomorphic character of H. intest i n a l i s i s expressed when, through peramorphosis of the plesiomorphic growth rate , the hindbody continues to increase in length such that i t s growth is p o s i t i v e l y a l lometr ic not only with respect to i t s t o t a l body length, as before, but now also with respect to the plesiomorphic value of the same growth parameter, represented in G. qu ie ta . 121 EXPERIMENTALLY-PRODUCED HETEROCHRONY IN HAPLOMETRANA INTESTINALIS INTRODUCTION As an hypothesis of common ancestry, a cladogram can be used as a general reference system not only for making inferences about evolutionary events, but also for experimental manipulations of character development. In t h i s context, the development of the hindbody in H. intest i n a l i s was invest igated. I postulated in the previous sect ion that the r e l a t i v e l y longer hindbody in th i s species evolved through peramorphosis of the plesiomorphic developmental ra te . In an attempt to see whether th i s autapomorphic t r a i t could be perturbed, the development of H. i n t e s t i n a l i s in anurans other than R. pret iosa was s tudied. ANALYSIS The choice of experimental hosts was based upon an optimizat ion of the Glypthelmins cladogram for hosts (F ig . 33). The eight paras i te species studied develop in ranid (R), h y l i d (H), or bufonid (B) anurans. Optimization of the nodal values of the tree indicates an ambiguity at the node un i t ing G. shastai and H. i n t e s t i n a l i s . The host of the common ancestor of these species i s in ferred to have been e i ther a bufonid or a r a n i d . Following the procedures in the sec t ion , LIFE HISTORY 1 22 DATA, attempts were made to infect adults of Bufo boreas, Rana  cascadae and R. aurora with cercar iae or metacercariae of H. i n t e s t i n a l i s . Bufo boreas was chosen because i t i s the host of G. shas ta i , which is postulated to be the s i s t e r species of H. i n t e s t i n a l i s . If B. boreas i s the apomorphic host, that i s , a newly acquired host colonized by G. shastai during that paras i t e ' s evo lut ion , then the development of H. i n t e s t i n a l i s in that host may somehow be abnormal. Conversely, i f B. boreas is the plesiomorphic host of the two species , then the development of H. i n t e s t i n a l i s in that host should proceed as i t does in R. pret iosa . Of the two ranid experimental hosts, R. cascadae is parapatr ic with R. pret iosa in eastern Washington and Oregon ( F i g . 37), and R. aurora i s parapatr ic with R. pret iosa in B r i t i s h Columbia ( F i g . 38). 1 C l a d i s t i c ana lys i s of ranid re la t ionsh ips (see F i g . 42a, and the sec t ion , COEVOLUTION ANALYSIS) postulates that R. aurora and R. cascadae are the c losest r e l a t i v e s of R. pret iosa . Of the three experimental host species , infect ions developed in R. cascadae and B. boreas. Specimens of H. i n t e s t i n a l i s were obtained from adults of each of these two host species at 1, 4, and 12 months after in fec t ion (MAI). Changes in the r a t i o of hindbody length to t o t a l body length 1 L i c h t (1969, 1974) reported sympatric populations of R. aurora and R. pret iosa in Langley, B r i t i s h Columbia. My recent c o l l e c t i o n s in the same area found only R. aurora . The only digenean gut paras i tes obtained from these frogs were G. c a l i f o r n i e n s i s and Megalodiscus microphagus. 1 23 (HBL/TBL) over time are given in Figure 34. This p l o t , which can be compared to that of the HBL/TBL growth curve of specimens of H. i n t e s t i n a l i s in R. pre t iosa ( F i g . 31), gives the values for H. i n t e s t i n a l i s developing in R. cascadae (upper dashed l i n e ) , the values for H. i n t e s t i n a l i s developing in B. boreas ( s o l i d l i n e ) , and the values for G. quieta developing in R. pipiens (lower dashed l i n e : from F i g . 31). Specimens of H. i n t e s t i n a l i s developing in R. cascadae follow the same growth curve as when development occurs in R. p r e t i o s a . Figure 35a is an i l l u s t r a t i o n of one of the specimens from R. pret iosa at 12 MAI. 1 In B. boreas, however, the rate of development slows af ter 4 MAI, such that the mean value of HBL/TBL at 12 MAI, 0.74 (n = 6 ; s = 0.02), is within the range of values present in the other species in the c lade . Figure 35b i s an i l l u s t r a t i o n of one of the specimens from B. boreas at 12 MAI. 2 Test ing for the di f ference between means from independent samples shows that there is no s i g n i f i c a n t d i f ference between the HBL/TBL value for H. i n t e s t i n a l i s from B. boreas at 12 MAI and that for mature adults of i t s postulated s i s t e r species , G. shastai (mean HBL/TBL = 0.75, n = 14; s = 0.03). Reca l l that the plesiomorphic HBL/TBL value for the node un i t ing G. shastai and H. i n t e s t i n a l i s can be estimated to be 1 2 Voucher specimens of H. i n t e s t i n a l i s at 12 MAI in R. pret iosa (HWML no. 23659) and B. boreas (HWML no. 23660) have been deposited at the Harold W. Manter Laboratory, Un ivers i ty of Nebraska State Museum, 529-W Nebraska H a l l , Un ivers i ty of Nebraska, L i n c o l n , NE 68588-0514. 124 0.75 ( F i g . 32). Thus, i t can be sa id that when H. i n t e s t i n a l i s develops in B. boreas i t exhib i t s not the apomorphic HBL/TBL value of approximately 0.80, but the plesiomorphic value of approximately 0.75. Because the a l tered growth parameter that brings th i s change about i s a slowing of the rate of development af ter 4 MAI, the heterochrony involved - with respect to the  development of H. i n t e s t i n a l i s in i t s natural host, R.  pret iosa - i s that of paedomorphosis through neoteny. Heterochrony can thus be studied from a phylogenetic perspective both as an evolutionary event and as an experimentally-produced a l t e r a t i o n in development. DISCUSSION There are at least three terms that might be appl ied to the a l t ered heterochronic growth of the hindbody in H. i n t e s t i n a l i s . F i r s t , although i t i s s t i l l p o s i t i v e l y a l lometr ic with respect to t o t a l body length, i t is not as p o s i t i v e l y a l lometr ic as i t i s in development in R. pret iosa or R. cascadae. Second, i t could c o r r e c t l y be termed "retarded growth", for th i s term is in fact a reference to heterochrony. T h i r d , and most relevant to digenean systematics, i t could be c i t e d as an instance of "host-induced v a r i a t i o n " . Such v a r i a t i o n has been reported for a number of digeneans developing in other than the ir usual host: e . g . , (1) Beaver (1937), for Echinostoma revolutum ( F r o e l i c h , 1802); (2) Boddeke (1960), for Prosthogonimus ovatus Rudolphi, 1803; (3) Grabda-Kazubska (1967), for Opisthoqlyphe ranae 1 25 F r o e l i c h , 1791; (4) Watertor (1967), for Te lorch i s bonnerensis Waitz, 1960; (5) Blankespoor (1974), for P lag iorch i s noblei Park, 1936; and (6) Palmieri (1977), for Posthodiplostomum  minimum (MacCallum, 1921). Studies such as these have concentrated on determining the characters of the worms that would vary under such developmental condi t ions , but only in so far as such v a r i a t i o n in ter fered with the correct i d e n t i f i c a t i o n of the species of p a r a s i t e . Thus, such characters were considered to be not as taxonomically useful as those that remained constant. T y p i c a l l y , the characters affected include body dimensions, v i t e l l i n e d i s t r i b u t i o n , r e l a t i v e sucker s i zes , and gonad l o c a t i o n . In the context of such s tudies , i t i s poss ible to say that the a l t e r a t i o n of hindbody growth in H. i n t e s t i n a l i s developing in B. boreas i s yet another example of host-induced v a r i a t i o n . (There are s t i l l two autapomorphies - tandem testes and the absenqe of an ter ior v i t e l l i n e f i e l d s - that allow i d e n t i f i c a t i o n of the spec ies . ) Nevertheless, th i s term and the aspects of comparative biology i t represents are of l i m i t e d use in studying the developmental propert ies of H. i n t e s t i n a l i s discussed here. This i s because host-induced v a r i a t i o n i s t r a d i t i o n a l l y considered with respect to the host, and not to the p a r a s i t e . Character v a r i a t i o n is seen as something that a host "induces" in a paras i t e , and a paras i t e ' s " p l a s t i c i t y " in the face of such induction i s seen as problematic for evolutionary b io logy . Phylogenet ica l ly determined l i m i t s to th i s v a r i a t i o n are recognized, but th i s recognit ion is not e x p l i c i t enough to avoid 126 the perception that such characters are not of much use in paras i te systematics. Without denying the importance of e s tab l i sh ing ranges of character v a r i a t i o n and the hosts in which i t can occur, and without denying the problems that highly var iab le characters create for paras i te taxonomy, the studies herein demonstrate that var iab le characters may be relevant to paras i te systematics and evolutionary bio logy. The reduced hindbody development in specimens of H. intest i n a l i s developing in B. boreas i s not just any v a r i a t i o n - i t is the plesiomorphic s tate . From within the context of an hypothesis of phylogenetic r e l a t i o n s h i p s , changes in characters can be invest igated . Some of these changes may be environmental ly- tr iggered, and since the environment of a paras i te i s usual ly another organism, that t r i g g e r i n g w i l l be what i s c a l l e d host-induced v a r i a t i o n . COEVOLUTION AND BIOGEOGRAPHY OF PARASITES AND HOSTS INTRODUCTION A concordance between the phylogenetic re la t ionsh ips of paras i tes and of the ir hosts has been recognized since the nineteenth century (von Iher ing , 1891). Hennig (1966) discussed th i s concordance and the resultant p o s s i b i l i t y of i n f e r r i n g host phylogenies from paras i te data. The continued discovery of covarying associat ions between paras i tes and the i r hosts has led 127 to the formulation of various "rules" of evo lut ion , of which the best known is probably Farenholz 's Rule: Paras i te Phylogeny Mirrors Host Phylogeny (for reviews, see I n g l i s , 1971; Brooks, 1979a, 1981a, 1985). Brooks (1981a) developed th i s concept of coevo lut ion 1 in terms of phylogenetic systematics and demonstrated that host -paras i te coevolution can ar i se through processes comparable to the evolut ion of homologous and homoplasious characters . Symplesiomorphic paras i t i sm occurs when a host l ineage speciates , i t s paras i tes do not, and a paraphylet ic d i s t r i b u t i o n of retained p r i m i t i v e paras i tes in derived hosts r e s u l t s . Synapomorphic and autapomorphic paras i t i sm occurs when hosts and paras i tes cospeciate , and the ir phylogenies do in fact "mirror" each other. Homoplasious parasi t i sm occurs when eco log ica l assoc iat ion resul t s in paras i tes co lon iz ing hosts other than those with which they have been e v o l u t i o n a r i l y assoc iated. This homoplasy can occur in a p a r a l l e l or a convergent manner. Analys i s of the biogeography and speciat ion patterns of organisms re lates the i r d i s t r i b u t i o n patterns to the i r postulated phylogenetic re la t ionsh ips and the geographical h i s tory of the areas in which the organisms occur. As with other l eve l s of phylogenetic a n a l y s i s , the intent i s to 1 This term is used here in the sense of Brooks (1979a), who appl ied i t to those instances of a common evolutionary h i s tory of l ineages of hosts and the i r paras i t e s . This resu l t s in congruent, or at least cons is tent , host and paras i te cladograms. The term coevolution has a lso been used to refer to r e c i p r o c a l adaptive responses between hosts and paras i tes during evolut ion ( e . g . , E h r l i c h and Raven, I 9 6 4) . 128 determine the contr ibut ion of h i s t o r i c a l processes to contemporary phenomena ( e .g . , Wiley, 1980). Any group of organisms can be analyzed with respect to i t s geographical d i s t r i b u t i o n ( e . g . , Nelson, 1974; Rosen, 1978). When a host-paras i te system is involved, there i s an a d d i t i o n a l l e v e l of re la t ionsh ips that can be examined, namely, paras i te re la t ionsh ips with respect to host re la t ionsh ips with respect to geographical re la t ionsh ips (see Brooks, 1985). HOST AND DISTRIBUTION DATA 1. PREVIOUS STUDIES The fol lowing are e i ther published reports in the l i t e r a t u r e or personal communications, as ind icated . New reports a r i s i n g from the current study are given in the next sec t ion . Figures refer to d i s t r i b u t i o n maps. G. hyloreus; ( F i g . 36) Type l o c a l i t y : near C o r v a l l i s , Oregon, from Hyla r e g i l l a Baird and G i r a r d Other reports : Nebraska, from Pseudacris t r i s e r i a t a Weid (by Brooks, 1976a) Colorado, from P. t r i s e r i a t a (by Ubelaker et a l . , 1967: reported as G. pennsylvaniensis , 1 29 i d e n t i f i e d as G. hyloreus by Brooks, 1976a) Spokane County, Washington State , from H- regi11a (by B. Lang, pers . comm.: Dept. of Biology, Eastern Washington State U n i v . , Cheney, Wash.) George Lake, A l b e r t a , from P. t r i s e r i a t a (by J . C . Holmes, Dept.of Zoology, Univ. of A l b e r t a , pers . comm.) 1 G. pennsylvaniensis: ( F i g . 36) Type l o c a l i t y : Lake Warren, Pennsylvania, from Hyla c r u c i f e r Weid 1 These specimens were o r i g i n a l l y i d e n t i f i e d as "G. quieta from Pseudacris n i g r i t a " by the c o l l e c t o r , Z. Hameed. Given the l o c a l i t y , in southwestern A l b e r t a , the host i s more l i k e l y to have been P. t r i s e r i a t a . Even though the worms are immature specimens, the i r possession of tegumental spines, rather than scales , indicates that they are not specimens of G. qu ie ta . A l s o , the peripharyngeal glands are of the medial type, rather than of the pharyngeal type. The testes are obl ique, rather than symmetrical, and the I-shaped excretory ves i c l e extends to the l eve l of the poster ior tes tes . Both of these are c h a r a c t e r i s t i c of G. pennsylvaniensis . The length of the eggs, however, i s c h a r a c t e r i s t i c of G. hyloreus, being 45 - 53 (mean of 49) jjm. The specimens are t en ta t ive ly i d e n t i f i e d as G. hyloreus. 1 30 Other reports: Clarke and Chatham Counties , Georgia , from H. c r u c i f e r and Pseudacris n i g r i t a (Le Conte) (by Byrd and Maples, 1963) Oconee County, Georgia , from H. c r u c i f e r . (by S u l l i v a n and Byrd, 1970) G. robustus: (F ig . 36) Type l o c a l i t y : 15 km west of Neiva, H u i l a , Colombia, from Bufo marinus L . Other reports : None G. shasta i : ( F i g . 37) Type l o c a l i t y : Glenburn, Shasta County, C a l i f o r n i a , from Bufo boreas Other reports : Gorge Creek, R . B . M i l l e r B i o l o g i c a l S ta t ion , 60 km SW of Calgary , A l b e r t a , from B. boreas (by J . C . Holmes, Dept. of Zoology, Univ. of A l b e r t a , pers . comm.) 131 Nelson, B r i t i s h Columbia, from B. boreas ( i b i d . ) G. c a l i f o r n i e n s i s : ( F i g . 38) Type l o c a l i t y : San Francisco area, C a l i f o r n i a , from Rana aurora Baird and G i r a r d Other reports : San Diego and Butte Counties, C a l i f o r n i a , from R. b o y l i i Baird (by Ingles, 1936) Marin and Sonoma Counties, C a l i f o r n i a , from R. boy l i i (by Lehmann, 1960) Cienaga de Lerma, and Lago de Xochimilco, Mexico, from R. montezumae B a i r d , and from R. pipiens Schreber (by Cabal lero Y . C . , 1942; Cabal lero Y C. and Sokoloff , 1934) Langley, B r i t i s h Columbia (approximately 65 km SE of Vancouver), from R. aurora (by R. Douthwaite and D.R. Brooks, pers . comm.) G. f a c i o i : ( F i g . Type l o c a l i t y : 36) C o r i s , Cartago Province, Costa R i c a , from Rana 1 32 pipiens (by Brenes e_t a l . , 1959) Other reports: T u r r i a l b a , Cartago Province, Costa Rica , from R. pipiens (by S u l l i v a n , 1976) G. quie ta: ( F i g . 38) Type l o c a l i t y : Eastern Canada (Toronto area presumed), 1 from Rana catesbeiana Shaw, R. virescens Garman (=R. p i p i e n s ) , and Hyla picker ingi i Kennicott (=H. c r u c i f e r Weid) Other reports: Over s i x t y , p r i m a r i l y throughout eastern North America, from members of the R. pipiens and R. catesbeiana groups (see the sec t ion , COEVOLUTION ANALYSIS). See Figure 38, in conjunction with the Index Catalogue of Medical and Veter inary Zoology, Oryx Press , Phoenix, Ar izona , U . S . A . 1 S ta f ford ' s (1900, 1905) reports spec i f i ed neither l o c a l i t y nor type specimens. However, as noted in the sect ion , SPECIMENS EXAMINED, there are specimens from Staf ford ' s c o l l e c t i o n deposited at the National Museum of Natural Sciences, Ottawa, Ontar io , Canada. 1 33 Haplometrana i n t e s t i n a l i s ; ( F i g . 37) Type l o c a l i t y : B o t h e l l , King County, Washington State, from Rana pret iosa Baird and G i r a r d Other reports: S p r i n g v i l l e , Utah, from R. pret iosa (Olsen, 1937) Idaho, from R. p r e t i o s a , R. pret iosa X R. s y l v a t i c a , and B. boreas (Waitz, 1961) Spokane County, Washington State; cercar iae from lymnaeid and hel isomid sna i l s (Current and Lang, 1975); adults from R. pret iosa (B. Lang, pers . comm.) Coleman, A l b e r t a , from R. pret iosa ( J . C . Holmes, Univ. of A l b e r t a , pers . comm.) Lake County and Flathead County, Montana, from R. p ipiens ( i b i d . ) Gorge Creek, A l b e r t a , from R. sy lva t i ca ( i b i d . ) P o s t i l l Lake, Kelowna, B r i t i s h Columbia, from R. pret iosa ( i b i d . ; o r i g i n a l l y i d e n t i f i e d as Glypthelmins sp; i d e n t i f i e d as H. i n t e s t i n a l i s by the present author) 1 34 2. NEW DATA From 1983 to 1986, surveys were conducted in southern B r i t i s h Columbia. The study area lay along an east-west transect remaining within 50km of the Canada - U .S . border, from Vancouver Island to the B r i t i s h Columbia - Alberta border ( F i g . 8: see Appendix C for f i e l d c o l l e c t i o n s i t e s ) . The fol lowing data on paras i t i sm by species of Glypthelmins and Haplometrana were obtained. G. hyloreus: Negative, in 76 Hyla regi11a examined G. c a l i f o r n i e n s i s : Langley, B r i t i s h Columbia, in area of L i t t l e Campbell River; from Rana aurora; prevalence = 62% (35 frogs examined) New L o c a l i t i e s : Bonsal l Creek, Duncan, Vancouver I s land , B . C . ; from R. aurora; prevalence = 42% (26 frogs examined) Remarks: 25 R. catesbeiana and 18 H. regi11a from the same areas in Langley were examined and found to be negative for in fec t ions by any species of Glypthelmins. 1 35 H. i n t e s t i n a l i s ; New L o c a l i t i e s : L i t t l e Muddy Pond, Manning Park, B . C . ; from Rana pre t iosa ; prevalence = 83% (15 frogs examined) Okanagan F a l l s , B . C . ; from R. pre t io sa ; ; prevalence = 80% (15 frogs examind) Wilgress Lake, B . C . ; from R. pre t io sa ; (1 frog examined) Champion Lakes, B . C . ; from R. pret iosa; prevalence = 74% (31 frogs examined) Creston, B . C . ; from R. pre t io sa ; prevalence = 50% (6 frogs examined) Loon Lake, B . C . ; from R. pre t io sa ; (1 frog examined) Remarks: (1) Other than Holmes' unpublished report of H. i n t e s t i n a l i s from R. pret iosa in Kelowna (see PREVIOUS STUDIES), these are the f i r s t reports of H. i n t e s t i n a l i s in B r i t i s h Columbia. (2) Although the d i s t r i b u t i o n of R. pre t iosa i s pr imar i ly throughout the Columbia and Snake River Plateaus, there are populations occurring further 136 north and further west (see F i g . 37). L ich t (1969, 1974) reported sympatric populations of R. pret iosa and R. aurora along the L i t t l e Campbell R iver , in Langley, B . C . . I have examined L i c h t ' s data and deposited specimens (Cowan Vertebrate Museum, Univers i ty of B r i t i s h Columbia; R. aurora; nos. 461, 1250, 1251, 1307; R. pre t io sa ; nos. 477, 478, 415), and I agree with the i d e n t i f i c a t i o n of the two species . During the course of the present study, from 1983 to 1986, 35 R. aurora were c o l l e c t e d at and around the areas surveyed by L i c h t . No R. pret iosa were observed, and no specimens of H. i n t e s t i n a l i s were recovered from R. aurora , as might happen i f there had been a host transfer when R. pret iosa were present. (Note: as reported in the sect ion , LIFE HISTORY DATA, attempts to infect R. aurora with H. i n t e s t i n a l i s in the laboratory were unsuccessful . ) (3) Two specimens of B. boreas were c o l l e c t e d from L i t t l e Muddy Pond, Manning Park, B . C . , but were found to be free of digenean paras i t e s . G. quie ta: Negative, no R. pipiens c o l l e c t e d in B r i t i s h Columbia Remarks: Three of the four l o c a l i t i e s for R. pipiens in B . C . reported by C a r l (1949) were surveyed: Osoyoos, Creston, and Loon Lake. No specimens were found. Two specimens of R. catesbeiana, another host of G. quie ta , 137 were c o l l e c t e d in Osoyoos, but were negative for digenean gut paras i t e s . Green (1978) reported a population of R. pipiens on Vancouver Island that was establ ished in the 1930s (see also Orchard, 1984), but th i s was not examined. From 1983 to 1986 surveys were conducted in northern C a l i f o r n i a . The study area covered S i sk iyou , Shasta, Modoc, Lassen, Plumas, and S i erra Counties ( F i g . 9). C o l l e c t i o n s in Shasta, Modoc, and Lassen Counties centered around the drainage basin of the P i t R i v e r . This r iver runs through Glenburn, Shasta County, the type l o c a l i t y of G. shastai in B. boreas. Co l l ec t i ons in S i e r r a County were d irec ted towards obtaining specimens of R. muscosa; while those in Siskiyou County were d irected towards obtaining specimens of R. cascadae. Both of these ranids have areas of sympatry with B. boreas ( F i g . 37), and are thus po tent ia l hosts of G. shas ta i . The fol lowing data on paras i t i sm by species of Glypthelmins and Haplometrana were obtained. (1) 82 specimens of R. cascadae were examined from areas in S iskiyou and Shasta Counties where th i s species i s sympatric with B. boreas. A l l were free of i n t e s t i n a l digenean paras i t e s . (2) No specimens of R. muscosa were c o l l e c t e d from the Gold Lakes area , S i erra County. 138 (3) No specimens of R. pret iosa were c o l l e c t e d from Medicine Lake, S i sk iyou County; Upper Mud Lake, Modoc County; and Blue Lake, Lassen County. This was unexpected, given the report by Hayes and Jennings (pers. comm.) that th i s species was c o l l e c t e d at these s i t es in 1980. R. pret iosa i s a slow, basking frog , and there i s l i t t l e r i sk of missing i t in a survey. 1 (4) 25 specimens of B. boreas were examined and found to be free of i n t e s t i n a l digenean paras i t e s . Four of these toads were c o l l e c t e d from Medicine Lake, S iskiyou County; two from L i t t l e Bear F l a t s , Shasta County; and 19 from Brown R d . , in Glenburn, Shasta County: the type l o c a l i t y of G. shastai in B. boreas. (5) Ingles (1933) reported that no digenean paras i tes had been c o l l e c t e d in C a l i f o r n i a from the introduced species , R. catesbeiana, despite i t s sympatry with R. aurora and R. boy l i i , and despite the presence of su i table s n a i l hosts. This led him to suggest that bu l l f rogs were incapable of acquir ing the digenean paras i tes of the indigenous frogs . Three years l a t e r , Ingles (1936) reported Meqalodiscus temperatus, a digenean paras i te of a number of western anurans, from R. catesbeiana in Butte 1 Marc. P. Hayes, Dept. of Biology, Univers i ty of Miami, Coral Gables, F l o r i d a ; Mark R. Jennings, U .S . F i s h and W i l d l i f e Serv ice , Los Banos, C a l i f . 139 County, C a l i f o r n i a . As w i l l be reported elsewhere, in my work in C a l i f o r n i a I recovered gorgoderid and haematoloechid digeneans from specimens of R. catesbeiana. From North American survey s i t e s other than those in B r i t i s h Columbia and C a l i f o r n i a , the fol lowing data on paras i t i sm by species of Glypthelmins and Haplometrana were obtained. G. qu ie ta : New L o c a l i t i e s : . White Earth River , Mountrai l County, North Dakota; from R. p ip iens; prevalence = 17% (6 frogs examined) H. i n t e s t i n a l i s : New L o c a l i t i e s : Pe l ican Creek, Wyoming; from R. pre t iosa; prevalence = 50% (2 frogs examined) 1 40 3. DISCUSSION The fol lowing can be concluded from the information given in the previous two sect ions . 1) Glypthelmins hyloreus p a r a s i t i z e s h y l i d frogs west and east of the Rockies, but not east of the Missouri River; G. , pennsylvaniensis p a r a s i t i z e s hy l ids east of the Missouri (F ig . 36). 2) The d i s t r i b u t i o n of G. shastai in B. boreas in North America is d i s j u n c t , occurring in northeast C a l i f o r n i a and southwest Alberta ( F i g . 37). Surveys of B. boreas in Idaho (Waitz, 1961), Utah (Frandsen and Grundmann, 1960), and southern B . C . (present study) have not found H. i n t e s t i n a l i s . 3) Glypthelmins c a l i f o r n i e n s i s p a r a s i t i z e s western ranid species along the west coast of North America, with add i t i ona l occurrence in a l l o p a t r i c members of the Rana pipiens group ( H i l l i s et a l . , 1983, see the sec t ion , COEVOLUTION ANALYSIS) in Mexico ( F i g . 38). 4) Glypthelmins quieta p a r a s i t i z e s members of the R. pipiens and R. catesbeiana groups (see the sec t ion , COEVOLUTION ANALYSIS) in eastern North America, with a d d i t i o n a l occurrence in some sympatric hy l ids and bufonids ( F i g . 38). Glypthelmins quieta can be considered to be a paras i te p r i m a r i l y of the R. pipiens group, rather than of the R. catesbeiana group. This is because 141 i t occurs in members of the R. p ipiens group where there are no members of the R. catesbeiana group, but i t does not occur in members of the R. catesbeiana group where there are no members of the R. pipiens group. It i s a l so of in teres t that the reports of G. quieta in Seat t l e , Washington (Rankin, 1944) and in Cuba (Odening, 1968) were both from R. catesbeiana, a species indigenous to southeast North America and frequently transplanted because of i t s commercial value. 1 5) Haplometrana i n t e s t i n a l i s p a r a s i t i z e s R. pret iosa in western North America, with occurrence in a sympatric bufonid, B. boreas, as well as in populations of R. pipiens and R. s y l v a t i c a ( F i g . 37). 6) Turner (1958) reported "Glypthelmins sp." from R. pre t iosa at F i sh ing Bridge , Wyoming. My c o l l e c t i o n of H. i n t e s t i n a l i s from th i s species of frog at Pe l ican Creek, which is within 10 miles of F i sh ing Bridge, suggests that Turner ac tua l ly c o l l e c t e d H. i n t e s t i n a l i s . 1 Hayes and Jennings ( in press) report that R. catesbeiana was introduced to western North America in C a l i f o r n i a in 1896. Green (1978) reported a population of R. catesbeiana introduced on Vancouver I s land , B r i t i s h Columbia, in the 1930s (see a l so Orchard, 1984). 1 42 COEVOLUTION ANALYSIS 1. FAMILY LEVEL The species of anurans p a r a s i t i z e d by the eight paras i te species studied here are members of the Hyl idae , Bufonidae, or Ranidae. These fami l ies were mapped onto the paras i te cladogram in Figure 33- for the heterochrony study, reported above. That study el iminated some of the ambiguity of the nodal values by demonstrating disrupted development of H. i n t e s t i n a l i s in B. boreas, the host of G. shas ta i . Disrupted development in a bufonid provides evidence that the plesiomorphic host for the G. shastai + H. i n t e s t i n a l i s clade was a r a n i d . With th i s inference, the nodal values on the cladogram are as in Figure 39a. These values imply the host re la t ionsh ips shown in Figure 39b. Because i t i s a trichotomy ( a l l three taxa united at a s ingle node), the t ree ' s topology would not change i f , as discussed in Chapter I I I , G. robustus were placed as the s i s t er taxon to the other seven species . The conclus ion, therefore , i s that a l l of the paras i te species except G. shastai show coevolution with the ir hosts at the family l e v e l . Glypthelmins  shastai appears to have colonized B. boreas. Comparison of the host tree in Figure 39b to f a m i l i a l l eve l c l a d i s t i c analyses of anurans shows that , with respect to the Hyl idae , Bufonidae, and Ranidae, those studies have also postulated a trichotomous r e l a t i o n s h i p . There have been three 143 such s tudies , using pr imar i ly morphological data . That of Kluge and F a r r i s (1969) presented a cladogram (their Figure 5) that , interpreted in the conventional manner, seems to place the hy l ids and bufonids in a separate c lade . However, the text and the ir Figure 4 make i t c l ear that a trichotomy is being postulated. The study by Lynch (1973) also placed the hy l ids and bufonids in a clade separate from the ranids . This was done on the basis of three characters: the absence of transverse processes on the coccyx, the presence of an accessory head on the adductor maqnus muscle, and the presence of an a x i l l a r y amplectic p o s i t i o n . These characters were interpreted in th i s manner because of the use of Camin-Sokal parsimony in the a n a l y s i s . By p r o h i b i t i n g character state reversa l s , th i s method of phylogenetic inference causes the postulat ion of an add i t iona l four instances of character state evolut ion for the f i r s t two characters (see Figure 3.7, Lynch, 1973), and i t e l iminates an equal- length postu lat ion of reversa l in the t h i r d character . The postulat ion of a trichotomous r e l a t i o n s h i p i s therefore better supported by the data . L a s t l y , in an analys i s performed with the WAGNER78 computer program developed by J . S . F a r r i s , Duellman and Trueb (1986: F i g . 17.3) postulated a trichotomous r e l a t i o n s h i p for the h y l i d s , bufonids, and ranids . My re -ana lys i s of the ir data with both PAUP (Unordered Analys i s on mult i s tate characters) and PHYSYS (WAG.S) d id not a l t e r the phylogenetic conclusions perta in ing to these three f a m i l i e s . 1 44 2. SPECIES LEVEL A study of paras i te-host coevolution at the species l e v e l was done for the ranid hosts . Figure 40 presents the Glypthelmins cladogram with the species of ranid hosts mapped on. These are: R. pret iosa (pr) , R. aurora (au), R. boy l i i (bo), R. montezumae (mo), R. pipiens ( p i ) , R. b l a i r i ( b l ) , R. sphenocephala (sp), R. p a l u s t r i s (pa), and R. palmipes (pp). With the exception of R. catesbeiana and R. clamitans (see the sec t ion , HOST AND DISTRIBUTION DATA), a l l of the hosts of G. quieta have been postulated to be members of the R. pipiens group ( H i l l i s e_t a l . , 1983), a clade containing at least 20 species endemic to areas in Mexico and eastern North America ( F i g . 38). With respect to the hosts of G. c a l i f o r n i e n s i s , R. pipiens i s put in inverted commas because given the l o c a l i t y of i t s c o l l e c t i o n by Cabal lero (1942) and Cabal lero and Sokoloff (1934), on the Mexican Plateau, i t was most l i k e l y not R. p ip i ens , but some other member of the group. The other host of G. c a l i f o r n i e n s i s in that area , R. montezumae, i s a lso a member of the R. pipiens group. The host of G. fac i o i is marked as R. palmipes, rather than as R. p ip i ens , as reported by Brenes et a l . (1959) and Su l l i van (1976). This change was made because the l o c a l i t y from which the host was c o l l e c t e d , Costa R i c a , indicates that i t was most l i k e l y R. palmipes, a member of the R. tarahumarae group (Webb, 1977; Case, 1978; H i l l i s e_t a l . , 1983). This group contains about four species endemic to areas of Centra l and South America. The remaining hosts on the cladogram, R. p r e t i o s a , 145 R. aurora, and R. boyl i i , are considered to be members of a clade of ranids endemic to western North America (Figs 37, 38). The monophyly of t h i s group, whose other members are R. cascadae, and R. muscosa, has been postulated by morphological (Chante l l , 1970), immunological (Wallace et a l . , 1973; Case, 1978), e lec trophoret ic (Case, 1978; Green, 1986a), and k a r y l o g i c a l (Green, 1986b) analyses. F a r r i s et a l . (1979, 1982) reanalysed the data of Case (1978) and Post and U z z e l l (1981) and concluded that the European species , R. temporaria, should a lso be included in th i s group. This conclusion has been disputed by Green (1986a), with e lec trophoret ic data, and by U z z e l l and Post (1986), with immunological data. A summary cladogram of the higher l e v e l re la t ionsh ips of ranids in the Americas i s given in Figure 41. The tree presents two species: R. temporar ia and R. sylvat i c a , and four clades of species: the R. boyl i i group, the R. tarahumarae group, the R. pipiens group, and the R. catesbeiana group (with respect to the monophyly of th i s l a s t group, endemic to eastern North America, see Case, 1978, and Lynch, 1965). The s i s t e r r e l a t i o n s h i p of the R. tarahumarae and R. pipiens groups has been postulated by Case (1978) and H i l l i s et a l . (1983), and that c lade ' s s i s t e r r e l a t i o n s h i p to the R. catesbeiana group has been postulated by Case (1978). The monophyly of the clade containing R. s y l v a t i c a , R. temporaria, and the R. b o y l i i group has been postulated by F a r r i s et a l . (1979) and Green (1986b). The monophyletic status of the ent i re assemblage is unclear . Savage (1973) considered a l l ranids in the Americas to be 1 46 descended from an ancestra l stock entering western North America v ia Beringia during the Eocene. Case (1978) considered the immunological data to support the conclusion that only the R. b o y l i i group arr ived in th i s manner, while the R. tarahumarae, R. p ip iens , and R. catesbeiana groups entered eastern North America v ia a- Laurasian land connection that existed u n t i l the mid-Eocene (McKenna, 1975). Regardless of the resolut ion of th i s quest ion, there appear to be two main lineages of ranids in the Americas: a north-west clade and a south-east c lade . The re la t ionsh ips of the species within the R. b o y l i i group have been c l a d i s t i c a l l y analyzed a number of times. The most recent studies are those with allozyme (Green, 1986a) and karyolog ica l (Green, 1986b) data . Green's analys i s of the e lec trophoret ic propert ies of the allozyme data followed the coding procedures of Mickevich and Mit ter (1981) and Buth (1984). Both studies used Camin-Sokal parsimony, in conjunction with a p r i o r i transformation ser ies for the mult i s tate characters . I have re-analyzed the data sets with the Wagner parsimony algorithms of PAUP, using Unordered Analys i s for the mult i s tate characters (see Appendix A for the data matr ices ) . Such treatment of these types of data i s supported by s imi lar studies on l e p t o d a c t y l i d frogs (Miyamoto, 1983, 1984, 1986) and xantus i id l i z a r d s (Crother et a_l. , 1986). These authors analyzed the mult i s tate characters with e i ther Swofford's Unordered Analys i s or Mickevich's (1982) Transformation Series Ana lys i s . 147 The trees I obtained are given in Figures 42a (karyolog ica l data) and 42b (e lectrophoret ic data) . Green (1986b) presented a s ingle tree for the karyo log ica l data, the topology of which is the same as that shown here. 1 Although no goodness-of- f i t s t a t i s t i c s for th i s tree were given, analys i s of Green's o r i g i n a l 13-character data set with WISS, the Camin-Sokal parsimony algorithm in PHYSYS, gives a consistency index (CI) of 0.94. 2 If Wagner parsimony is used and the author's a p r i o r i mult i s tate transformation series are maintained, analys i s with the BANDB algorithm of PAUP gives two trees with a CI of 0.94, one of which i s the tree published by Green. When, in conjunction with BANDB, Unordered Analys i s i s performed on the mult i s tate characters , the s ingle tree shown in Figure 42a i s obtained. It i s the same tree published by Green (1986b), and i t has a CI of 1.0, i n d i c a t i n g a perfect f i t to the data. In my re -analys i s of the e lec trophoret ic data of Green (1986a), I co l lapsed the populations of R. aurora and R. pret iosa used by Green to a s ingle OTU (Operational Taxonomic Unit) each. Analys is of the o r i g i n a l 47-character data set with the Camin-Sokal parsimony WISS algorithm of PHYSYS gives the 1 The tree has the same topology whether or not R. aurora is taken as the outgroup, as i t was by Green. If th i s species is not taken as the outgroup, then there w i l l be a composite outgroup, cons i s t ing of zero s tates , in the data matrix in Appendix A. 2 Unless otherwise noted, CI values presented here are ca lcu la ted as o r i g i n a l l y proposed by Kluge and F a r r i s (1969). Appendix G of th i s d i s s e r t a t i o n presents a modified C I , in which non-homoplasious autapomorphies are not included in the c a l c u l a t i o n s . 148 tree he publ ished, with a CI of 0.58. Analys is with the Wagner parsimony BANDB algorthm of PAUP while maintaining the a p r i o r i mul t i s ta te transformation ser ies gives the same tree again, with a CI of 0.61. Analys is with BANDB and Unordered Analys i s on the mult i s ta te characters gives eight trees with a CI of 0.80 (modified CI of 0.69; according to the c r i t e r i a in Appendix G) . None of these trees i s the same as the tree published by Green (1986a). Figure 42b gives the two topologies of the mult ip le trees , as well as the consensus t ree . The l a t t e r d i f f e r s from Green's resu l t s p r i m a r i l y in the paraphylet ic nature of the two species of "stream frogs": R. boy l i i and R. muscosa (see Zweife l , 1955; C h a n t e l l , 1970; Green, 1986a). The phylogenetic inferences drawn from the karyo log ica l and e lec trophoret ic data d i f f e r p r i m a r i l y in the placement of R. aurora. It i s e i ther placed as the s i s t e r species to the rest of the c lade , or i t is placed further up in the tree , grouped with R. cascadae and R. b o y l i i . The immunological and e lec trophoret ic studies by Case (1978), re-analyzed by F a r r i s e_t a l . (1979) a lso place R. aurora with R. boy l i i . The tree in ferred from the karyo log ica l data i s consistent with the morphological data that are ava i lab le (overlap of dorsal flange and ventra l crest on shaft of humerus, vastus prominence of i l i u m : C h a n t e l l , 1970; skin texture and c o l o r a t i o n : Dumas, 1966). It i s a lso consistent with the biogeographical study by Dumas (1966), in which a conclusion of v i c a r i a n t speciat ion associated with late-Cenozoic orogeny in western North America was reached. The species on the tree f a l l 1 49 into three groups previously recognized on the basis of phenetic s i m i l a r i t y . These are: the "wood frogs" (Dumas, 1966): R. s y l v a t i c a (one of the outgroups, see F i g . 41) and R. aurora; the "pond frogs" (Dumas, 1966): R. pret iosa and R. cascadae; and the "stream frogs" (Zweifel , 1955; C h a n t e l l , 1970; Green, I986a,b): R. b o y l i i and R. muscosa. Of these three groups, only the t h i r d i s postulated to be monophyletic. The f i r s t two groups are components of a l a r g e r , apparently paraphylet ic assemblage, the Pa learc t i c "brown frogs". Green (1986a,b) postulated that th i s assemblage i s the resu l t of a geographical ly and systemat ical ly widespread symplesiomorphic morphology. I consider the tree inferred from the k a r y l o g i c a l data to be the preferred hypothesis of the phylogenetic re la t ionsh ips of the species in the R. boy l i i group. This conclusion is based on the t ree ' s optimal CI value and on i t s support by other types of data . Its acceptance necessitates the conclusion that in the e lec trophoret ic and immunological data analyzed so f a r , there has been convergent evolut ion that resu l t s in the grouping of R. aurora with R. boy l i i . When the plesiomorphic states for the ranid hosts are optimized on the Glypthelmins cladogram ( F i g . 40; i . e . , host re la t ionsh ips in ferred from paras i te r e l a t i o n s h i p s ) , and the appl icable port ions of the host cladogram from Figures 41 and 42a (dashed l ines ) are superimposed, four conclusions can be drawn. The f i r s t i s that the plesiomorphic host of the G. c a l i forn iens i s + G. quieta + G. fac i o i clade was a member of 1 50 the R. pipiens group. The inference of a common "R. pipiens" node for a l l three of the paras i te species involved indicates coevolut ion of hosts and p a r a s i t e s . A coevolutionary pattern continues to be found when the host and paras i te phylogenies are examined at a lower l e v e l . Figure 43a presents the cladogram of H i l l i s et a l . (1983) for the R. pipiens group. The reported occurrences of paras i t i sm by G. c a l i f o r n i e n s i s (ca), G. quieta (qu), and G. fac i o i (fa) are noted. In a process comparable to generating a reduced area cladogram in biogeographic analys i s (see Rosen, 1978), Figure 43b shows the c l a d i s t i c re la t ionsh ips of the p a r a s i t i z e d members of the R. p ipiens c lade . The postulated s i s t e r group re la t ionsh ips are congruent with those of the paras i te cladogram (see F i g . 40). That i s , s i s t e r paras i te taxa are found in s i s t e r host taxa. The second conclusion of th i s analys i s is that G. c a l i f o r n i e n s i s subsequently colonized two members of the R. b o y l i i group: R. aurora and R. b o y l i i . The occurrence of G. c a l i f o r n i e n s i s in only R. aurora and R. boy l i i i s evidence of e i ther (a) the descent of these two species of frogs from a common ancestor co lonized by G. c a l i f o r n i e n s i s , or (b) G. c a l i f o r n i e n s i s co lon iz ing f i r s t one and then the other host species . Interpretat ion (a) is consistent with the host phylogeny in ferred from immunological and e lec trophoret ic data ( F i g . 42b), while in terpre ta t ion (b) is consistent with the host phylogeny in ferred from the karyo log ica l and morphological data ( F i g . 42a). Given the better support for the l a t t e r tree , the tentat ive conclusion is that the common paras i t i sm of 151 R. aurora and R. b o y l i i by G. c a l i f o r n i e n s i s i s a resul t of sequential or independent c o l o n i z a t i o n . In l i g h t of th i s conc lus ion , i t i s in teres t ing to note that these two species of frogs share a number of immunological characters (thus, the ir grouping in Figure 42b). It i s not unreasonable to expect hosts' immunological propert ies to play some role in the ir common s u s c e p t i b i l i t y to the same species of p a r a s i t e . The t h i r d conclusion that can be drawn from comparing the host and parasi te cladograms is that H. i n t e s t i n a l i s coevolved with R. pret iosa , another species in the R. boy l i i group. Because only one species of paras i te and one species of host are involved, th i s r e la t i onsh ip is minimally coevolut ionary. Nevertheless, H. i n t e s t i n a l i s can only be said to have colonized R. pret iosa in the sense that every coevolutionary assoc iat ion must begin with a species of paras i te co lon iz ing a species of host . The fourth conclusion i s that the paras i te data are not of assistance in determining whether the north-west clade and south-east clade of ranids in the Americas form a monophyletic group ( i . e . , the dotted l i n e in F i g . 40). This i s because, with only two converging nodal values, i t i s not poss ible to e l iminate ambiguity at the bottom node of the host tree . An appeal to outgroups cannot help because the remaining species of Glypthelmins analyzed are not paras i tes of ran ids . 1 52 BIOGEQGRAPHIC ANALYSIS As noted above, phylogenetic analys i s of species of Rana in the Americas postulates a south-east l ineage cons i s t ing of the R. tarahumarae, R. p ip iens , and R. catesbeiana groups, and a north-west l ineage cons i s t ing of R. s y l v a t i c a , R. temporaria, and the R. b o y l i i group. Rana palmipes has the most southern d i s t r i b u t i o n of a l l the ranids in the Americas. Its range extends from Centra l America to the northern areas of South America, 'where i t i s the only ranid on that cont inent . The hypothesis of a northern entry of the ranids into the Americas i s supported by the occurrence of ranid f o s s i l s in North America only af ter the mid-Miocene (Estes , 1970), and the i r occurrence in South America not u n t i l the Holocene (Baez and de G a s p a r i n i , 1979), despite the ir appearance in the Old World, p a r t i c u l a r l y in A f r i c a , in the Oligocene, (Estes , 1970; Savage, 1973). The grouping of the R. b o y l i i group with the European species , R. temporaria, by F a r r i s et a l . (1979, 1982) i s consistent with an hypothesis of northern entry. As discussed by Savage (1973), ranids may have entered South America only af ter the re -establishment of a land connection between North and South America in the Pl iocene, fol lowing the disappearance of such a connection in the Cretaceous ( e . g . , Coney, 1982; but see Carey, 1976, and H . G . Owen, 1976 for geophysical analyses that suggest that the two continents have never been apar t ) . Bufonids and hy l ids have been postulated to have a South America-Afr ica o r i g i n (Savage, 1973), with f o s s i l s of both fami l ies occurr ing in Paleocene deposits in South America 153 (Estes, 1970). The appearance in North America of h y l i d f o s s i l s in the Oligocene and bufonid f o s s i l s in the Miocene (Estes, 1970) would seem to be evidence against the ir entry into North America from South America (Savage, 1973) so long as there was no land connection between the continents for most of the T e r t i a r y . C l a d i s t i c analyses of bufonids and hy l ids for the ir re la t ionsh ips with Old World species would help to c l a r i f y th i s matter. The Glypthelmins cladogram can be mapped onto a map of the Americas ( F i g . 44) fol lowing the method of Brundin (1972; see Brooks, 1977). With respect to those species p a r a s i t i c in ranids and bufonids, there i s , as with the ranids themselves, a north-west l ineage , cons i s t ing of G. shastai and H. i n t e s t i n a l i s , and a south-east l ineage, cons i s t ing of G. fac i o i , G. c a l i forn iens i s, and G. qu ie ta . This pattern suggests v i c a r i a n t speciat ion in assoc iat ion with mid-Cenozoic orogeny and c l i m a c t i c changes in northern Mexico and western North America (see Axelrod, 1975, Rosen, 1978). The Centra l America / Mexico / eastern North America v icar iance shown by the s i s t e r species , G. fac i o i , G. c a l i f o r n i e n s i s , and G. qu ie ta , re spec t ive ly , i s a pattern found among the i r ranid hosts within the R. pipiens group ( H i l l i s et al., 1983), as well as among cer ta in p lants , f i shes , salamanders, snakes, b i r d s , and mammals (see Rosen, 1978) . The presence of the r e l a t i v e l y plesiomorphic paras i te species , G. robustus, in B. marinus in Colombia does not suggest a plesiomorphic assoc iat ion of Glypthelmins with bufonids. This 1 54 is because, as discussed in the f a m i l i a l l e v e l coevolution analys i s above, there i s an ambiguity in that part of the tree ( F i g . 39) as to whether the plesiomorphic host was a h y l i d or a bufonid. Another consequence of th i s ambiguity is that i t i s d i f f i c u l t to interpret the associat ion of G. hyloreus and G. pennsylvaniensis with h y l i d frogs in only North America. These two paras i te species are postulated to be not as c lose ly re lated to the North American species of Glypthelmins as is G. robustus. I f , as discussed in Chapter I I I , the examination of more mature specimens of G. robustus warrants that species' re locat ion on the cladogram to a pos i t ion below G. hyloreus and G. pennsyIvaniensis, th i s question would be p a r t i a l l y reso lved. In his analys i s of these species of Glypthelmins, Brooks (1977) considered that the placement of the G. hyloreus and G. pennsylvaniensis l ineage to the s i s t e r taxon pos i t ion below G. robustus supported an in terpre ta t ion of d i spersa l of a common glypthelminth ancestor before v i c a r i a n t spec iat ion events took place (Nelson, 1974). I concur. Given that the d i s t r i b u t i o n s of R. aurora and R. b o y l i i are d i s junct from that of R. montezumae ( F i g . 45), the postulated co lon iza t ion of the f i r s t two species by G. c a l i f o r n i e n s i s would have to (a) have occurred during a time when the d i s t r i b u t i o n s were not d i s j u n c t , or (b) involve the paras i t i sm of a c lo se ly re la ted host species whose contemporary d i s t r i b u t i o n l i e s between those of the other three host species. Rana tarahumarae and R. ch ir icahuens i s are two such species , being found in the S i e r r a Madre Occ identa l . The analys is by H i l l i s et a_l. ( 1 983) 1 55 places the f i r s t species as the s i s t e r taxon to the R. pipiens group, and the second species in the same clade as R. montezumae ( F i g . 43). These species have not been examined for paras i t i sm. The remaining lineage to be examined is the G. shastai + H. i n t e s t i n a l i s c lade , for which I have postulated a co lon iza t ion of B. boreas by G. shastai from a common ranid ancestor. While H. i n t e s t i n a l i s has been reported from R. pret iosa throughout the Columbia and Snake Rivers Plateaus, from southern B r i t i s h Columbia-Alberta to Utah, the d i s t r i b u t i o n of G. shastai in B. boreas i s strongly d i s junct (Fig 37). The species has been c o l l e c t e d in northeastern C a l i f o r n i a and southwestern A l b e r t a . Surveys of B. boreas in Idaho (Waitz, 1961), Utah (Frandsen and Grundmann, 1960), and southern B r i t i s h Columbia (present study) have not found G. shas ta i . A biogeographic pattern in th i s l ineage suggestive of v icar iance is not evident. An a l t e r n a t i v e explanation is that the divergence of G. shastai and H. i n t e s t i n a l i s from a common ancestra l paras i te of ranids took place within a sympatric assoc iat ion of the hosts , and may have involved an a l l o h o s p i t a l i c condit ion (parasites r e s t r i c t e d to d i f f erent host species: analagous to a l l opatry ) created through the co lon iza t ion by G. shastai of B. boreas. 156 SUMMARY The phylogenetic hypothesis of the re la t ionsh ips among Glypthelmins and Haplometrana that was produced in Chapter III serves in th i s chapter as a general reference system for the study of f ive evolutionary processes and one laboratory manipulation. With respect to the phylogenetic re la t ionsh ips that they support, the l a r v a l and adult characters studied are found to be consistent with one another. This suggests the existence of constra ints on character evolut ion that produce a s i m i l a r l y covarying set of characters regardless of environmental d i f ferences and assumed se lec t ion pressures. L i f e cyc le propert ies are mapped onto the cladogram, and are found to be consistent with the phylogenetic re la t ionsh ips supported by the l a r v a l and adult characters . The cladogram is used to study poss ible instances of heterochronic character development. It i s concluded that heterochrony has occurred in the evolut ion of three characters: (1) paedomorphosis, in the retent ion of in terceca l and post-t e s t i c u l a r uterine loops in G. shas ta i , H. i n t e s t i n a l i s , G. c a l i f o r n i e n s i s , G. qu ie ta , and G. fac i o i ; (2) hypermorphic peramorphosis, in the retent ion of penetration glands in adults of G. fac i o i ; and (3) accelerated peramorphosis, in the development of a longer hindbody in H. intest i n a l i s. A coevolutionary and biogeographic a n a l y s i s , which involves re-analyses of data from e a r l i e r studies on anuran 1 57 r e l a t i o n s h i p s , makes the fol lowing conclus ions . ( 1 ) There i s a north-west clade and a south-east clade in the phylogenetic tree for the glypthelminth species in North and Centra l America, as there i s for the ir ranid hosts . (2) Glypthelmins fac i o i , G. c a l i f o r n i e n s i s , and G. quieta have coevolved with host species of the Rana pipiens group in a manner that shows a Central America / Mexico / southeastern U.S . v icar iance pat tern , re spec t ive ly . This pattern is a lso shown by the ir ranid hosts . (3) Glypthelmins c a l i forniens i s subsequently colonized two members of the R. b o y l i i group in western North America: R. aurora and R. b o y l i i ; th i s common s u s c e p t i b i l i t y to paras i t i sm may be associated with the apparent convergence of some of the immunological propert ies of these two frog species . (4) Haplometrana i n t e s t i n a l i s coevolved with R. p r e t i o s a . (5) The common ancestor of H. i n t e s t i n a l i s and G. shastai was a paras i te of ranids , subsequent to which, G. shas ta i , in i t s spec ia t ion , colonized a bufonid, Bufo boreas. Experimental infect ions of adults of B. boreas with specimens of H. i n t e s t i n a l i s produced an a l t ered development of the hindbody of H. i n t e s t i n a l i s a f ter 12 months of development in the host. This a l t e r a t i o n is considered to be an instance of neotenic paedomorphosis, produced through a decrease in the rate of growth of the hindbody af ter 4 months of development. The a l t ered morphology is that of the plesiomorphic s tate , which i s exhibi ted by G. shas ta i , the postulated s i s t e r species of H. i n t e s t i n a l i s . Heterochrony can thus be studied from a phylogenetic perspective both as an evolutionary event and as an 1 58 experimentally-produced a l t e r a t i o n in development. 1 59 Figure 1 - End-Attaining A c t i v i t y in B i o l o g i c a l Systems Three types of a c t i v i t y are involved. They are internested, rather than mutually exc lus ive . A l l phys ica l systems show teleomatic a c t i v i t y ; a subset ( b i o l o g i c a l systems) a lso shows teleonomic a c t i v i t y ; a subset of these (cognit ive systems) also shows t e l e o l o g i c a l a c t i v i t y . (From O'Grady, 1986. Can. J . Zoo l . 64:1010.) Figure 2 - Ultimate and Proximate Causa l i ty Three organisms composed of a hierarchy of within-system ultimate and proximate c a u s a l i t y . These are subsequently d i f f e r e n t i a l l y el iminated by an among-system proximate cause: natural s e l e c t i o n . (From O'Grady, 1986. Can. J . Zool . 64:1010.) 160 t e l e o m a t i c : e n d - r e s u l t i n g t e l e o n o m i c : e n d - d i r e c t e d t e l e o l o g i c a l : g o a l - s e e k i n g Proximate 1 j Proximate Ultimate Proximate \ I Proximate | Ultimate" 2 161 Figure 3 - Types of Evolut ionary Explanations Four a l t e r n a t i v e evolutionary h i s t o r i e s for four skin colors of a species of frog: brown (Br) , yellow (Ye), red (Re),and green (Gr) . The causes involved are: natural se l ec t ion (C1), and inheritance at two l eve l s (C2, C3). (From O'Grady, 1986. Can. J . Zool . 64:1010.) Figure 4 - H i s t o r i c a l Structura l i sm in Phylogenetic Systematics Hennigian phylogenetic analys i s of four taxa. As the tree is resolved with the addi t ion of character data, the analys is incorporates as much internested h i s t o r i c a l caua l i ty as pos s ib l e . (From O'Grady, 1986. Can. J . Zoo l . 64:1010.) 162 0 d) A B 163 Figure 5 - Using a Cladogram to Study the Evolut ion of L i f e His tory T r a i t s Top f igure: a phylogenetic tree for the p a r a s i t i c platyhelminths (Brooks, 1982; Brooks et a l . , 1985a; O 'Grady, 1985). DI indicates a d i r e c t l i f e cycle in an invertebrate host, DV indicates a d i r e c t l i f e cyc le in a vertebrate host, CB indicates a complex l i f e cycle involv ing both host groups. Bottom f igure: the same tree , with arrows ind ica t ing the postulated changes in the type of l i f e c y c l e . . The changes are in ferred with the opt imizat ion procedures of F a r r i s (1970) and Swofford and Maddison (in review). 164 165 Figure 6 - The Appl i ca t ion of H i s t o r i c a l S tructura l i sm to the Study of Community Structure The pattern of inheritance suggested by a phylogenetic analys is can be used as the i n i t i a l hypothesis in explanations of eco log ica l t r a i t s . Departures from congruence between the postulated phylogeny and such t r a i t s can be postulated to be ind ica t ive of nonhi s tor ica l events, such as c o l o n i z a t i o n . (From O'Grady, 1986. Can. J . Zoo l . 64:1010.) PHYLOGENETIC ANALYSIS characters 1 2 3 4 5 A • - - - -m • • - - -C • • • -D * •» •• • E • • • • • / A B C D E A B C D E COMMUNITY STRUCTURE ANALYSIS ecology 1 2 3 A • - • B + - -C • D - -t -E • ON 167 Figure 7 - Postulated Relat ionships among Glypthelmins, from Brooks (1977) Species: a, hyloreus; b, pennsylvaniensis; c, robustus; d, fac i o i ; e, shasta i ; f, quieta; g, c a l i f o r n i e n s i s ; h, v i t e l l i n o p h i l u m ; i , incurvatum; j , 1inguatula; k, hepat ica; 1, palmipedes; m, proximus; n, repandum; o, t i n e r i ; p, a f r i c a n a ; q, diana; r , s t a f f o r d i ; s, rugocaudata. The lower f igure (reproduced from Brooks, 1977. Syst . Zool . 26:277) shows the biogeographic d i s t r i b u t i o n s of the four l ineages . 168 169 F i g u r e 8 - C o l l e c t i o n S i t e s of Anurans i n B r i t i s h Columbia See Appendix C f o r d e t a i l s . 171 Figure 9 - Co l l e c t i ons Si tes of Anurans in C a l i f o r n i a See Appendix C for d e t a i l s . S o l i d squares indicate c o l l e c t i o n s of R. cascadae; small s o l i d t r iang l e s indicate c o l l e c t i o n s of B. boreas; the large s o l i d t r iang le indicates the c o l l e c t i o n of B. boreas at Glenburn, the type l o c a l i t y of G. shasta i ; open diamonds indicate l o c a l i t i e s at which no anurans were found. The course of the P i t River i s indicated; i t flows from the north-east of the state towards Redding, Shasta County, jo in ing with the Sacramento and McCloud Rivers at Shasta Lake, north of Redding. 172 173 Figure 10 - Tegumental Project ions The two types of project ions that occur among the species s tudied, drawn from the ventra l surface of the forebody of a specimen of (a) H. i n t e s t i n a l i s , and (b) G. qu ie ta . (The anter ior of the worm is towards the top of the p la te . ) The project ions in (a) are termed spines, those in (b) are termed scales; (bar = 0.1 mm). Figure 11 - Penetration Glands in Adult Glypthelmins fac i o i A ventra l view of specimen no. 222, from the c o l l e c t i o n of J . J . S u l l i v a n , 1 showing penetration glands (pen. g l . ) and medial glands (med. g l . ) ; (bar = 0.2 mm). 1 Center for Disease C o n t r o l , A t l a n t a , Georgia. 174 175 Figure 12 - Medial Glands in Haplometrana i n t e s t i n a l i s A ventral view of a specimen from the c o l l e c t i o n of the author, showing medial glands (med. g l . & mgl) and ducts; (bars = 0.2 mm). Figure 13 - Pharyngeal Glands in Glypthelmins quieta A ventra l view of a specimen from the c o l l e c t i o n of D.R. Brooks, Univ. of B r i t i s h Columbia, showing pharyngeal glands (ph. g l . ) , medial glands (med. g l . ) , and the ir ducts; (bar 0.2 mm). 176 1 77 Figure 14 - Schematic Representation of the D i s t r i b u t i o n of the V i t e l l a r i a in Glypthelmins and Haplometrana Representations for some of the species s tudied , showing the d i f f erent regions in which medial confluence of the v i t e l l a r i a occurs . The view is antero -dorsa l , i . e . , with the anter ior of the animal at the bottom of the p l a t e . The dark cy l inders represent the i n t e s t i n a l ceca, the l i g h t areas represent the v i t e l l a r i a , and the s t ipp led l ine s represent the v i t e l l i n e ducts . The species i l l u s t r a t e d are: a, H. i n t e s t i n a l i s ; b, G. shasta i ; c , G. qu ie ta ; d, G. c a l i forn iens i s; e, G. fac i o i . 178 1 79 Figure 15 - D i s t r i b u t i o n of V i t e l l a r i a in Glypthelmins quieta and G. facioi-(a)-and (b): Ventral views of a specimen of G. quieta from the c o l l e c t i o n of D.R. Brooks, Univ. of B . C . The dark shaded areas represent (a) the d i s t r i b u t i o n of the v i t e l l a r i a dorsal to the gut and gonads, and (b) the d i s t r i b u t i o n of the v i t e l l a r i a ventra l to the gut and gonads. (c): A dorsal view of a specimen of G. quieta from the c o l l e c t i o n of D.R. Brooks, in which the reduced development of the v i t e l l a r i a allows the anter ior and poster ior f i e l d s to be seen c l e a r l y . (d) and (e): Ventra l views of G. f a c i o i specimen no. 200, from the c o l l e c t i o n of J . J . S u l l i v a n . The dark shaded areas represent (d) the d i s t r i b u t i o n of the v i t e l l a r i a dorsal to the gut and gonads, and (e) the d i s t r i b u t i o n of the v i t e l l a r i a ventra l to the gut and gonads; ( a l l bars = 1.0 mm). 180 181 Figure 16 - D i s t r i b u t i o n of V i t e l l a r i a in Glypthelmins c a l i f o r n i e n s i s Ventra l views of a specimen of G. c a l i f o r n i e n s i s from the c o l l e c t i o n of D.R. Brooks, Univ. of B . C . The dark shaded areas represent (a) the d i s t r i b u t i o n of the v i t e l l a r i a dorsa l to the gut and gonads, and (b) the d i s t r i b u t i o n of the v i t e l l a r i a ventra l to the gut and gonads; (bars = 1.0 mm). Figure 17 - D i s t r i b u t i o n of V i t e l l a r i a in Glypthelmins  shastai and Haplometrana i n t e s t i n a l i s (a) and (b): Ventra l views of G. shastai specimen no. UAPAR 720, from the c o l l e c t i o n of the Dept. of Zoology, Univ. of A l b e r t a . The dark shaded areas represent (a) the d i s t r i b u t i o n of the v i t e l l a r i a dorsal to the gut and gonads, and (b) the d i s t r i b u t i o n of the v i t e l l a r i a ventra l to the gut and gonads; (bars = 1.0 mm). (c) and (d): Ventra l views of a specimen of H. i n t e s t i n a l i s from the c o l l e c t i o n of the author. The dark shaded areas represent (c) the d i s t r i b u t i o n of the v i t e l l a r i a dorsa l to the gut and gonads, and (d) the d i s t r i b u t i o n of the v i t e l l a r i a ventra l to the gut and gonads; 0, ovary; T, t e s t i s ; (bars = 0.5 mm). 182 183 Figure 18 - V i t e l l i n e Ducts in Haplometrana i n t e s t i n a l i s and Glypthelmins c a l i f o r n i e n s i s (a) A ventra l view of a specimen of H. i n t e s t i n a l i s from the c o l l e c t i o n of the author, showing the s ingle v i t e l l i n e duct on each s ide; (bar = 0.2 mm). (b) A dorsal view of a specimen of G. c a l i f o r n i e n s i s from the c o l l e c t i o n of the author, showing the reduced development of the poster ior v i t e l l i n e duct (a s t er i sk ) ; (bar = 0.2 mm). T, t e s t i s ; SR, seminal receptacle; 0, ovary; L C , Laurer ' s canal ; MG, Mehl is ' gland; U, uterus; VD, v i t e l l i n e duct. 184 185 Figure 19 - Shape of the Excretory V e s i c l e (a) , I-shaped ves i c l e b i f u r c a t i n g a t , or pos ter ior to , the l e v e l of the testes; (b), Y-shaped ves i c l e b i f u r c a t i n g anter ior to the l e v e l of the tes tes . 186 a b 187 Figure 20 - Postulated Phylogenetic Relat ionships among Glypthelmins and Haplometrana The cladogram obtained from a phylogenetic ana lys i s of seven species of Glypthelmins and the s ingle species of Haplometrana p a r a s i t i c in the intes t ines of anurans in North, C e n t r a l , and South America. Twenty-one morphological characters are s tudied . The Consistency Index of the tree i s 0.848. When non-homoplasious autapomorphic characters are not included in the c a l c u l a t i o n (see Appendix G) , the Consistency Index i s 0.769. Character states are given in brackets , with binary characters having a numeric code, and mult i s tate characters having an alphabetic code. As ter i sks indicate characters for which there i s ambiguity as to the ir state at cer ta in nodes on the tree . See the sec t ion , CHARACTER ANALYSIS, as well as Table II and Appendix A. 188 189 F i g u r e 21 - M u l t i s t a t e Character Trees: One The m u l t i s t a t e t r a n s f o r m a t i o n s e r i e s for c h a r a c t e r s no. 2, 9, 11, 16, and 17, as p o s t u l a t e d by an Unordered A n a l y s i s with the PAUP computer program ( v e r s i o n 2.4, 1985), developed by D.L. Swofford. 190 Cl-0.75 191 Figure 22 - Mul t i s ta te Character Trees: Two L e f t : the mult i s tate transformation ser ies for character no. 19 (mean egg length) , as postulated by an Unordered Analys is with the PAUP computer program (version 2.4, 1985), developed by D . L . Swofford. Right: a poss ible reso lut ion of the ambiguous nodal values, based upon an assumption of a l inear increase in mean egg length. 192 193 Figure 23 - Disrupt ive Grouping C r i t e r i a in Systematics Figure (a) gives the most parsimonious cladogram that can be constructed with respect to the d i s t r i b u t i o n of four characters , in which the plesiomorphic states are coded as "U", and the apomorphic states are coded as "X". Character steps on the tree mark the appearance of the apomorphic state . This tree i s six character steps long. If one character is given precedence over the others when grouping the taxa, the resul t can be trees such as those in (b) to (d). These are incongruent (see Figure 24) with the tree in (a) . (b): If taxa A and B are grouped together because they both lack state X of character 3 (grouping by symplesiomorphy), the tree i s seven steps long. (c): If a l l of the taxa possessing state X of character 4 are grouped together (grouping by convergent t r a i t s ) , the tree is seven steps long, (d): If a l l of the taxa possessing state X of character 1 are grouped together ( f a i l i n g to recognize a r e v e r s a l ) , the tree is eight steps long. 194 C h a r a c t e r S t a t e s 4 X U U X X 3 U U X X X 2 U X X X X 1 X X X X U A B C D E E A B C D 195 Figure 24 - Congruence and Consistency in Tree Topologies Trees (a) and (b) are congruent with one another ( s o l i d arrows); each of these is consistent with tree (c) (dashed arrows); tree (d) i s a lso consistent with tree (c) , but i t i s incongruent with trees (a) and (b). 196 197 Figure 25 - Generalized L i f e Cycle of a Digenean Flatworm The double-headed arrows indicate instances of asexual reproduct ion. The sequences at lower l e f t indicate the permutations of the developmental sequence of miracidium (M), sporocyst (S), redia (R), and cercar iae (C) stages that have been reported in the l i t e r a t u r e . (Figure reproduced from O'Grady, 1985. C l a d i s t i c s 1:165.) See O'Grady (1985) and Brooks et a l . (1985b) for phylogenetic analyses that suggest that the sporocyst stage i s always present. 198 DEFINITIVE HOST ( VERT.) . adult / metacercaria egg ^ 2 n d INTERMEDIATE /K \ HOST ^ ' \ WATER (VERT. OR) ^ ^ ( INVERT ) 1 ^ ^ \ * c e r c a r i a miracidium ^ \ ^ \ ^ jxS-^C ^ r e d i a ^ sporocyst M < ^ S - » R - > C ^ \ 1 St INTERMEDIATE HOST ( MOLLUSC ) 199 Figure 26 - Phylogenetic Analys i s of Larva l Characters of Species of Glypthelmins and Haplometrana The species included are a l l of•those in the study group for which there are data: hy, G. hyloreus; pe, G. pennsylvaniensis; i n , H. i n t e s t i n a l i s ; ca , G. c a l i f o r n i e n s i s ; qu, G. q u i e t a . A l l of the character states are t r a i t s of the c e r c a r i a e , and are: 1, a s t y l e t ; 2, a dorso-ventral f i n f o l d ; 3, tandem testes; 4, symmetrical testes; 5, tegumental sca les . Character states placed on the v e r t i c a l l ine at the bottom of the tree are considered to be symplesiomorphic for the l e v e l of the study group. The dashed l ine shows the placement of H. i n t e s t i n a l i s in the primary cladogram in Figure 20. 200 1 201 Figure 27 - Phylogenetic Analys i s of L i f e Cycle Evolut ion in Species of Glypthelmins and Haplometrana The species included are a l l of those in the study group for which there are 'data: hy, G. hyloreus; pe, G. pennsylvaniensis; i n , H. i n t e s t i n a l i s ; ca , G. c a l i f o r n i e n s i s ; qu, G. qu ie ta . The character states are: 1, a physid s n a i l as at least one of the f i r s t intermediate host types; 2, a lymnaeid s n a i l as at least one of the f i r s t intermediate host types; 3, a lymnaeid s n a i l as the only f i r s t intermediate host type; 4, a hel isomid s n a i l as at least one of the f i r s t intermediate host types; 5, an anuran as both the second intermediate and the f i n a l host; 6, development as an unencysted metacercaria in anuran tadpoles; 7, development as an encysted metaceraria in anuran adul t s . Character states placed on the v e r t i c a l l i n e at the bottom of the tree are considered to be symplesiomorphic for the l e v e l of the study group. The dashed l i n e indicates the placement of G. c a l i f o r n i e n s i s in the primary cladogram in Figure 20. 202 203 Figure 28 - Categories of Heterochronic Change A l t e r a t i o n s in the rate of development (k), i t s time (t) of i n i t i a t i o n (alpha), or i t s time of cessation (beta) can produce changes in the morphology (gamma) of the descendant (D), as compared to that of i t s ancestor (A). There are s ix categories of change; three are paedomorphic, in that the descendant's morphology is less developed than that of the ancestor; three are peramorphic, in that the descendant's morphology is more developed than that of the ancestor. (From Alberch et a l . , 1979, and F ink , 1982) PAEDOMORPHOSIS a p • a P a 0 < • slower rate starts later stops sooner NEOTENY POSTDISPLACEMENT PROGENESIS O PERAMORPHOSIS a 0 a< • 0 a ——> $ faster rate starts sooner stops later ACCELERATION PREDISPL ACEMENT HYPERMORPHOSIS 205 Figure 29 - Using a Cladogram to Dis t ingui sh Paedomorph'ic, Peramorphic, and Symplesiomorphic Morphologies The trees show the developmental sequences of the taxa involved. In a l l three trees , taxa A and D exhib i t terminal state 2, even though D is more c lose ly re la ted to taxa B and C, which exhibi t terminal state 3. In tree (a), th i s i s due to symplesiomorphy (retained p r i m i t i v e t r a i t , state 3 i s a synapomorphy of B and C); in tree (b), i t is due to paedomorphosis (a less developed descendent morphology); and in tree (c) , i t i s due to peramorphosis (a more developed descendent morphology, the terminal state of which (state 2') i s s imi lar to the e a r l i e r state 2. Figure 30 - Some Limits to the Use of a Cladogram to Detect Heterochrony An example taken from Fink (1982; F i g . 5). The taxa in the cladogram in (a) possess e i ther state 0 or 1 of a character . If the growth patterns of the taxa are as in (b), i t can be concluded that the common ancestor of B and C evolved state 1 through hypermorphosis ( la ter cessation of development), subsequent to which C evolved state 0 through neoteny (slower rate of development). However, i f the growth patterns are as in (c) , in which taxon C has evolved state 0 through progenesis ( e a r l i e r cessat ion of development), then i t w i l l not be poss ible to detect such paedomorphosis. Instead, i t w i l l be wrongly concluded that state 0 in taxon C is symplesiomorphic (refer to Figure 28 for the parameters of the growth p l o t s ) . 206 207 Figure 31 - The Growth of the Hindbody in Haplometrana  i n t e s t i n a l i s and Glypthelmins quieta Values for the mean r a t i o (with ranges) of hindbody length to t o t a l body length (HBL/TBL) 1 in specimens of the two species as metacercariae (META) and at 1, 4, 8, and 12 months after in fec t ion (MAI) in Rana pret iosa , for H. i n t e s t i n a l i s ( i n ) , and in R. p ip i ens , for G. quieta (qu). The values for mature adults of other species of Glypthelmins, of unknown ages, are given at 12+ MAI (see the sec t ion , HETEROCHRONIC DEVELOPMENT, for comments on G. robustus) . 1 The hindbody is the distance from the anter ior edge of the ventra l sucker to the pos ter ior end of the body. 208 ce '5 nai/iaH 209 Figure 32 - An Estimation of the Plesiomorphic State of the Hindbody for Haplometrana i n t e s t i n a l i s The mean values for the r a t i o of hindbody length to t o t a l body length in the species studied are given across the top of the f igure : hy, G. hyloreus; pe, G. pennsylvaniensis; ro , G. robustus; sh, G. shasta i ; i n , H. intest i n a l i s ; ca , G. c a l i f o r n i e n s i s ; qu, G. quie ta; f a , G. fac i o i . These are mapped onto the cladogram of the species' r e l a t i o n s h i p s , from Figure 20 (see the sec t ion , HETEROCHRONIC DEVELOPMENT, for comments on G. robustus) . The plesiomorphic state of the r a t i o for H. i n t e s t i n a l i s can be estimated by se t t ing the nodal values on the tree to the median values of the r a t i o s . This y i e lds a value of 0.75 for the node un i t ing H. i n t e s t i n a l i s and G. shas ta i . The value of 0.80 in f u l l y mature specimens of H. i n t e s t i n a l i s is s i g n i f i c a n t l y d i f f erent from th i s value Tp<0.0l). 210 .68 hy .71 pe ro .75 sh .80 in .69 ca .71 qu .74 fa 21 1 Figure 33 - Inference of Plesiomorphic Hosts for Species of Glypthelmins and Haplometrana: One The paras i te species indicated are: hy, G. hyloreus; pe, G. pennsylvaniensis; ro , G. robustus; sh, G. shas ta i ; i n , H. i n t e s t i n a l i s ; ca , G. c a l i f o r n i e n s i s ; qu, G. quie ta; fa , G. fac i o i . The anurans host fami l ies indicated are: H, Hyl idae; B, Bufonidae; R, Ranidae. Optimization according to the c r i t e r i a of Swofford and Maddison (in review) y i e lds the indicated nodal values. 212 B B R ro sh 213 Figure 34 - Experimentally-Produced Heterochronic Development of the Hindbody in Haplometrana i n t e s t i n a l i s The r a t i o of hindbody length to t o t a l body length (HBL/TBL) i s p lo t ted against time, for specimens of H. i n t e s t i n a l i s (in) as metacercariae (META) and at 1, 4, 8, and 12 months after in fec t ion (MAI). The upper dashed l ine gives the mean values for specimens of H. i n t e s t i n a l i s developing in e i ther Rana  pret iosa or R. cascadae. The middle s o l i d l i n e gives the values for specimens of H. i n t e s t i n a l i s developing in Bufo boreas. The lower dashed l i n e , g iv ing the values for G. quieta developing in R. p ip i ens , is repeated from Figure 31, as are the values for mature adults of other species of Glypthelmins (see the sec t ion , HETEROCHRONIC DEVELOPMENT, for comments on G. robustus) . The HBL/TBL value at 12 MAI for specimens of H . 0 i n t e s t i n a l i s developing in B. boreas i s (1) s i g n i f i c a n t l y d i f f erent (p<0.01) from the same measure for specimens developing in R. cascadae and R. p r e t i o s a , and (2) not s i g n i f i c a n t l y d i f f eren t from the same measure for mature adu l t s , of unknown age, of G. shas ta i . MAI 215 Figure 35 - Al tered Growth of the Hindbody in Haplometrana i n t e s t i n a l i s (a) Ventral view of specimen of H. i n t e s t i n a l i s from Rana  p r e t i o s a , 12 months after i n f e c t i o n ; (b) ventra l view of specimen of H. i n t e s t i n a l i s from Bufo boreas, 12 months after i n f e c t i o n ; (bars = 1.0 mm) 216 217 Figure 36 - D i s t r i b u t i o n in the Americas of the Digenean Species, G. hyloreus, G. pennsylvaniensis , G. fac i o i , and G. robustus Symbols: dots, G. hyloreus; squares, G. pennsylvaniensis; t r i a n g l e , G. fac i o i ; diamond, G. robustus. 218 219 Figure 37 - D i s t r i b u t i o n in the Americas of the Digenean Species, Haplometrana i n t e s t i n a l i s and Glypthelmins shas ta i , and" of the Anuran Species, Bufo boreas, Rana p r e t i o s a , R. cascadae, and R. muscosa 221 Figure 38 - D i s t r i b u t i o n in the Americas of the Digenean Species, Glypthelmins quieta and G. c a l i f o r n i e n s i s , and of the Anuran Species , Rana aurora , R. b o y l i i , and the species of the R. pipiens Group See the sec t ion , COEVOLUTION ANALYSIS, for comments on the species composing the R. pipiens group. 223 Figure 39 - Inference of Plesiomorphic Hosts for Species of Glypthelmins and Haplometrana: Two (a) The species of paras i tes are: hy, G. hyloreus; pe, G. pennsylvaniensis; ro , G. robustus; sh, G. shasta i ; i n , H. i n t e s t i n a l i s ; ca , G. c a l i f o r n i e n s i s ; qu, G. qu ie ta ; f a , G. fac i o i . The host taxa are the anuran fami l i e s : H, Hyl idae; B, Bufonidae; R, Ranidae. The nodal optimizations are obtained with the Swofford and Maddison (in review) method. The bufonid /ranid ambiguity that , in Figure 33, is assigned to the node uni t ing G. shastai and H. i n t e s t i n a l i s i s t en ta t ive ly removed here. This i s done on the basis of the in fec t ion studies reported in the sec t ion , EXPERIMENTALLY-PRODUCED HETEROCHRONY IN HAPLOMETRANA INTESTINALIS. These suggest that G. shastai colonized a bufonid, Bufo boreas. Figure (b) gives the re la t ionsh ips among the host groups that are indicated by the i r d i s t r i b u t i o n in tree (a) . 224 225 Figure 40 - Coevolutionary Analys i s of Species of Glypthelmins and Rana in North and Central America The paras i te species are: i n , H. i n t e s t i n a l i s ; ca , G. c a l i f o r n i e n s i s ; qu, G. quie ta; f a , G. fac i o i . The host species are: p r , R. pre t io sa ; au, R. aurora; bo, R. b o y l i i ; mo, R. montezumae; p i , R. p ip iens ; b l , R. b l a i r i ; sp, R. sphenocephala; pa, R. p a l u s t r i s ; pp, R. palmipes. The notation of "pi" refers to other members of the R. pipiens group (see the sec t ion , COEVOLUTION ANALYSIS). The arrow indicates a postulated co lon iza t ion event. The s o l i d l ines indicate the phylogenetic re la t ionsh ips among the paras i te species (from F i g . 20). The dashed l ine s indicate the phylogenetic re la t ionsh ips among the host species (from F i g . 42a). 226 227 Figure 41 - Summary Cladogram of Higher Level Relat ionships among Ranids in the Americas Compiled from various sources (see the sec t ion , COEVOLUTION ANALYSIS). 228 229 Figure 42 - Phylogenetic Relat ionships among Ranids in Western North America The species are: au, R. aurora; pr , R. pret iosa; ca , R. cascadae; bo, R. boy l i i ; mu, R. muscosa. Figure Ca) gives the resu l t s of a re -ana lys i s of the karyolog ica l data of Green (1986b); Figure (b) gives the resu l t s of a re -ana lys i s of the allozyme data of Green (1986a). The l a t t e r i s a consensus tree , the two resolut ions of which are shown at bottom l e f t . Re-analyses used Wagner parsimony and . Unordered Analys is of mul t i s ta te characters . See Appendix A for the data matrices from which these trees were constructed. 2 3 0 231 Figure 43 - Postulated Phylogenetic Relat ionships among Members of the Rana pipiens Group, from H i l l i s et al_. (1983) The occurrence of G. fac i o i ( fa ) , G. c a l i f o r n i e n s i s (ca) , and G. quieta (qu) is noted on the ranid cladogram in (a). Figure Tb) shows the re la t ionsh ips among the paras i tes that are implied by the re la t ionsh ips among the ir hosts . This tree i s congruent with the cladogram for these species , in Figure 20. 1% 1X 1% 233 Figure 44 - Biogeographic and Phylogenetic Patterns for Species of Glypthelmins and Haplometrana in the Americas The cladogram for the species of Glypthelmins and Haplometrana, Figure 20, f i t t e d onto a contemporary map of the Americas according to the species' d i s t r i b u t i o n : hy, G. hyloreus; pe, G. pennsylvaniensis; ro , G. robustus; sh, G. shasta i ; in , H. i n t e s t i n a l i s ; ca , G. c a l i f o r n i e n s i s ; qu, G. quie ta; fa , G. f a c i o i . 234 235 Figure 45 - D i s t r i b u t i o n of the Hosts of Glypthelmins c a l i f o r n i e n s i s The paras i te species are: ro, G. robustus; sh, G. shasta i ; i n , H. i n t e s t i n a l i s ; ca , G. c a l i f o r n i e n s i s ; qu, G. quie ta; gf, G. f a c i o i . The c l a d i s t i c re la t ionsh ips are as in Figure 44. 236 237 Figure 46 - Morphology of Glypthelmins shas ta i : One (a) Ventral view of specimen no. UAPAR 692, Univ. of A l b e r t a ; (b) i l l u s t r a t i o n of the holotype of G. shas ta i , USNM Helm. C o l l . 1 no. 8925, reproduced from Ingles (1936); (bars = 1.0 mm) . 1 U .S . National Museum Helminthological C o l l e c t i o n , B e l t s v i l l e , Maryland. 238 239 Figure 47 - Morphology of Glypthelmins shas ta i : Two Specimen numbers are of those in the c o l l e c t i o n of the Dept. of Zoology, Univ. of A l b e r t a . (a) Ventral view of the anter ior of specimen no. 692, showing the medial glands (med. g l . ) and the i r ducts (bar = 0.5 mm); (b) c i r r u s sac of specimen no. 715 (pc, pros ta t i c c e l l s ; sv, seminal v e s i c l e ; ve, vasa e f f e r e n t i a ; ga, gen i ta l atrium; bar = 0.5 mm); (c) ventra l view of ootype region of specimen no. 7660 (MG, Mehl is ' gland; LC, Laurer ' s canal ; SR, seminal receptacle; 0, ovary; VD, v i t e l l i n e duct; bar 0.2 mm); (d) ventra l view of c i r r u s sac of specimen no. 715, showing metraterm (me) and associated gland c e l l s (gc), uterus (ut) , and ventra l sucker (vs) (bar = 0.5 mm); (e) ventra l view of specimen no. 690 (immature), showing I-shaped excretory v e s i c l e (bar = 0.5 mm). 240 241 Figure 48 - Morphology of the Larva l Stages of Glypthelmins c a l i f o r n i e n s i s (a) Ventra l view of a c e r c a r i a , with the excretory system i l l u s t r a t e d on one side only (bar = 0.1 mm); (b) s ty l e t (bar = 20 jum); (c) miracidium (bar = 20 jum); (d) ventra l view of a c e r c a r i a , showing the b i p a r t i t e excretory ves i c l e (EV) and the developing testes (T) , ovary (0), and c i r r u s sac (dorsal to the ventra l sucker) (bar = 0.1 mm); (e) daughter sporocyst, containing c e r c a r i a e , from a moribund s n a i l (bar = 0.2 mm). 242 243 Figure 49 - Young Adult of Glypthelmins c a l i f o r n i e n s i s A ventra l view of a specimen of G. c a l i f o r n i e n s i s recovered from an adult of Rana aurora 20 days af ter exposing the frog to cercar iae ; (bar = 0.2 mm). 244 245 Figure 50 - Mul t i s ta te Transformation Series Series (a) an.d.(b) are complex, (c) is l i n e a r , and (d) is basa l ly b i f u r c a t i n g . 246 247 F i g u r e 51 - Demonstration Tree f o r M u l t i s t a t e Coding Methods A c o n t r i v e d example; t r e e s (a) and (b) are e q u i v a l e n t 248 b) A B C D 249 Figure 52 - Addi t ive Binary Coding (a) The i n i t i a l tree; (b) the ABC matrix; (c) the reconstructed polytomy; (d) the reconstructed tree , with a l l nine new binary characters ; (e) the reconstructed tree , without the two hypothet ica l characters , i and i i . 2 5 0 a) b) 1 2 3 A 5 6 7 8 9 A R C D E F G i ii A 1 0 0 1 0 0 0 1 1 B 0 1 0 1 0 0 0 1 1 C 0 0 1 1 0 0 0 0 1 D 0 0 0 1 0 0 0 0 0 E 0 0 0 1 1 0 0 0 0 F 0 0 0 1 1 1 0 0 0 G 0 0 0 1 1 0 1 0 0 i 0 0 0 1 0 0 0 1 1 ii 0 0 0 1 0 0 0 0 1 d) A B F G e) A F 6^  251 Figure 53 - Redundant Linear Coding (a) The i n i t i a l tree; (b) the RLC matrix; (c) the reconstructed polytomy; (d) the reconstructed t ree . 252 1 2 3 4 5 A 3 2 1 0 0 B 2 3 1 0 0 C 1 1 2 0 0 D 0 0 0 0 0 E 0 0 0 1 1 F 0 0 0 2 1 G 0 0 0 1 2 0 A B C D F E ••5(0) ••4(0) --3(0) -•2(0) ••1(0) 253 Figure 54 - Nonredundant Linear Coding (a) The i n i t i a l tree; (b) the NLC matrix; (c) the reconstructed polytomy; (d) the reconstructed tree; (e) an a l t e r n a t i v e coding for the l e f t side of the tree . 254 255 Figure 55 - Coding a B a s a l l y - B i f u r c a t i n g Mul t i s ta te Series (a) The i n i t i a l ser ies ; (b) coding with the ABC method; (c) coding with the RLC and NLC methods; (d) " in terna l ly rooting" a s ingle l inear code. 256 a) B D b) 0 d) 1 2 3 4 5 A 1 1 1 0 0 B 0 1 1 0 0 C 0 0 1 0 0 D 0 0 1 1 0 E 0 0 1 1 1 1 2 A 2 0 B 1 0 C 0 0 D 0 1 E 0 2 A B * C D E 1 0 1 2 3 A ABC R L C NLC internal * rooting A E \ 1 / 5 B n A U 2 \ E D 2(1) + 2(0) + 1(0) A E 1 ( 0 ) \ / l (4) B D 1(1 ) \ / i ( 3 ) C f 1(2) 257 Figure 56 - Incorporating a Mul t i s ta te Tree into a Character Matrix (a) The cladogram, supported by seven binary characters , with the d i s t r i b u t i o n of a four-s tate mult is tate character ( i - i v ) ; (b) the character matrix for the cladogram; (c) the mult is tate tree , coded with the NLC method; (d) the NLC matrix , from which the appropriate hor izonta l codes are put into the character matrix in (b); (e) the cladogram constructed from a l l nine characters . 2 5 8 IV 1(2) \ i \ 2(1) 1 (1) 2(0) 1 (0) b) d) x A B C D 1 2 3 A 5 6 7:8 9 0 0 0 0 0 0 0!0 0 ( 1 1 0 0 0 0 0:1 0 (ii 1 0 1 1 0 0 OH Odi 1 0 1 0 1 1 0!2 0 (iii 1 0 1 0 1 0 111 1 (iv 1 2 I 0 0 i i 1 0 iii 2 0 iv 1 1 ii i i A B 259 Figure 57 - Using Parasi te Data to Infer Host Relat ionships Figures (a) to (d) show the method of c l u s t e r i n g hosts according to shared paras i te taxa, while (e) to ( i ) show the method of using a paras i te cladogram as a mult i s ta te character of the hosts . (a) A presence/absence matrix for paras i tes (upper case l e t t er s ) in hosts (roman numerals); (b) the re su l t ing host cladogram; (c) another presence/absence matrix; (d) the resu l t ing host cladogram; (e) a cladogram for the paras i te taxa in (c) , coded with the NLC method; (f) the NLC matrix for the paras i te cladogram; (g) the host matrix constructed by taking the appropriate hor izonta l code from the NLC matrix in (f) according to the information on paras i te presence given in (c); (h) the host cladogram constructed from the host matrix in (g); ( i ) the paras i te re la t ionsh ips implied by the host cladogram. 260 261 Figure 58 - Using Inclus ive ORing to Deal with the Occurrence of more than One Parasi te Taxon in a Host Group (a) The cladogram for paras i te taxa A - D , showing the ir occurrence in hosts I - IV; (b) the ABC matrix for the paras i te cladogram; (c) the expanded host matrix, constructed from the ABC matrix - host IV receives the hor izonta l codes of the two paras i te taxa, C and D; (d) the compressed host matrix, produced by the inc lus ive ORing of the two rows for host IV; (e) the host cladogram constructed from the matrix in (d); (f) the paras i te re la t ionsh ips implied by the host cladogram - these are congruent with those in the i n i t i a l paras i te cladogram in (a) . 262 a ) I A IL B ILL c F b) LY D ABC A B C D E F G 1 2 3 4 5 6 7 A B C D E F G 1 0 0 0 0 0 0 1 0 0 0 1 0 0 1 0 1 1 0 0 0 1 1 1 0 0 0 0 1 1 0 0 0 0 0 1 1 1 0 0 0 0 0 0 1 0 d) 1 2 3 4 5 6 7 I 1 0 0 0 0 0 1 n 0 1 0 0 0 1 1 in 0 0 1 0 1 1 1 _ LY 0 0 1 0 1 1 1 (C)l 1Y 0 0 0 1 1 1 1 (D). OR ing 1 2 3 4 5 6 7 I 1 0 0 0 0 0 1 II 0 1 0 0 0 1 1 ILL 0 0 1 0 1 1 1 HYOO 1 1 1 1 1 e) f) I H HI 12 I IL ILL 263 Figure 59 - The Limits of Inclusive ORing (a) The cladogram for paras i te taxa A - G, showing the ir occurrence in hosts I - VI ; (b) the compressed host matrix produced by the inc lus ive ORing of the expanded host matrix (not shown) constructed from the ABC matrix (not shown) for the paras i te cladogram in (a); (c) the host cladogram constructed from the compressed host matrix; (d) the paras i te re la t ionsh ips implied by the host cladogram - these are incongruent with those in the i n i t i a l paras i te cladogram in (a) . This i s because, unl ike the example i l l u s t r a t e d in Figure 58, the paras i te taxa that occur in more than one host group ( v i z . , paras i tes C, D, and G in host III) are not each other's s i s t er taxa. 264 a) b) I II in LY Y 21 1 2 3 4 5 6 7 8 9 10111213 1 0 0 0 0 0 00 0 0 0 0 1 0 1 0 0 0 0 0 0 0 0 0 1 1 0 0 1 1 0 0 1 1 1 1 1 1 1 0 0 0 0 1 0 0 0 1 1 11 1 0 0 0 0 0 1 0 1 1 1 1 1 1 0 0 0 0 0 0 1 1 1 1 1 1 1 c) I JJ 12 Y YL d) I H r i i i i 265 Figure 60 - The I n a b i l i t y of the Consistency Index to Distinguish-between Autapomorphies and Synapomorphies Four trees with a common d i s t r i b u t i o n of seven binary characters (1 - 7), and d i f f erent d i s t r i b u t i o n s of two other binary characters (8 - 9); the Consistency Index for each of the trees is the. same. (From Brooks e_t a l . 1987. Syst. Zoo l . 35:571.) 266 A B C D A B C D 267 Figure 61 - C a l c u l a t i o n of the F-Rat io A matrix of phenetic distances (c) i s ' constructed from the character matrix (a), then compared with a matrix of p a t r i s t i c distances (d) for a tree (b) in ferred from the data . The sum of the matrix of the di f ferences between the phenetic and p a t r i s t i c distance matrices is normalized by d i v i d i n g i t by the sum of the phenetic distance matrix. (From Brooks e_t a l . 1987. Syst . Zool . 35:571.) 268 (a) (b) 1 2 3 4 5 X 0 0 0 0 0 A 1 0 0 0 1 B 1 \ 0 0 0 C 1 1 t 1 0 D 1 1 1 1 1 X A B C D X A B C D 2 2 2 i I 2 5 3 3 1 X A B C X A B C D 2 2 2 A 4 2 5 © 3 1 (c) (d) 269 Figure 62 - The S e n s i t i v i t y of the F-Rat io to Factors Relevant to Unrooted, Rather than Rooted, Trees Figures (b) - (d) present d i f f erent d i s t r i b u t i o n s of character 4 on the tree in (a) . When character 4 i s placed as an autapomorphy (b), an in terna l synapomorphy (c) , and a basal synapomorphy (d), the F-Rat io only d is t inguishes between cases in which the character is on a terminal branch (b) and (d), and a nonterminal (c) branch of the tree . (From Brooks et a l . 1987. Syst . Zool . 35:571.) 270 P H E N E T I C PATRISTIC X A B C X A B X X A 2 A 2 B 3 1 B 3 (D C 2 2 1 C 2 2 1 X A B C X A B C X X A 2 A 2 B 3 1 B 3 (D C 3 3 2 C 3 3 2 (c) X A B C X A B C X X A 2 A 2 B U 2 B C 3 3 1 C 3 3 1 X A B C X A B C X X A 3 A 3 B i. 1 B C 3 2 1 C 3 2 1 271 Figure 63 - Agreement between the F-Rat io and the Consistency Index Tree (a) has the higher CI value and the lower F-Rat io (character reversals are marked with an X ) . (From Brooks e_t a l . 1987. Syst . Zoo l . 35:571.) Figure 64 - Disagreement between the F-Rat io and the Consistency Index A demonstration that the shortest tree inferred from a data set i s not necessar i ly the tree with the lowest F - R a t i o . The tree in (b) i s shorter , and thus has the higher CI, while the tree in (a) has the lower F - R a t i o . (From Brooks et a l . 1987. Syst . Zoo l . 35:571.) 2 7 2 273 Figure 65 - A Demonstration that the F-Rat io i s not Biased towards Para l l e l i sms or Reversals when Comparing Trees of Equal Length Three pa irs of trees are presented. Each pair i s of equal length. The trees that postulate para l l e l i sms are (a), (c ) , and (e); the trees that postulate reversals (marked with an X) are (b) , (d), and ( f ) . The trees with the lower F-Rat ios are (b), (c) =(d), and (e) . 274 275 LITERATURE CITED Alberch . P . S . , S . J . Gould, G . F . Oster , and D . B . Wake. 1979. Size and shape in ontogeny and phylogeny. P a l e o b i o l . 5:296-317. Axelrod, D . I . , 1975. Evolut ion and biogeography of Madrean-Tethyan s c l e r o p h y l l vegetat ion. Ann. Missouri Bot. Gard. 62:280-334. Ayala , F . J . 1976. Biology as an autonomous sc ience. 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Zool . 54:207-292. 292 APPENDIX A - COMPUTER ANALYSES WITH PAUP AND PHYSYS This appendix contains (1) ALLTREES analyses from PAUP (with both Ordered and Unordered Analys is of mul t i s ta te characters ) , (2) WAG.S analyses from PHYSYS, (3) the re su l t s of using the DIAGNOSE command of PHYSYS to assess how well the character data used in the present study f i t the cladogram of Brooks (1977), and (4) the data matrices from Green (1986a,b) that were reanalyzed in Chapter IV. A l l runs were conducted on the Amdahl 5850 computer at the Univers i ty of B r i t i s h Columbia, operating on the Michigan Terminal System. Parts of the printout have been edited for c l a r i t y . 2 9 3 $Log Output: R i c h a r d O'Grady. #1 th.mx.unord ZENO. Job#=6194. Host*G, 10:10:30 Sat Jan 17/87 1 5 6 65 7 8 g 92 94 96 98 (GLYPTHELMINS AND HAPLOMETRANA DATA !INITIAL RUN. WITH UNORDERED MULT I STATE CHARACTERS !UNORDERED ANALYSIS ON CHARACTERS 2.9.11,16.17. and 19 2.22 2.24 2.4 HARACTERS (SEE TEXT AND TABLE I I FOR CHARACTER STATES): 1) shape of tegumental p r o j e c t i o n s (2 s t a t e s ) 2) e x t e n t o f tegumental p r o j e c t i o n s (3 s t a t e s ) 3) o r a l s u c k e r diam. / v e n t r a l s u c k e r diam. (2 s t a t e s ) 4) p e n e t r a t i o n g l a n d s (2 s t a t e s ) 5) m e d i a l g l a n d s (2 s t a t e s ) 6) p h a r y n g e a l g l a n d s (2 s t a t e s ) 7) p o s i t i o n o f o v a r y (2 s t a t e s ) * ( 8 ) p o s i t i o n o f L a u r e r ' s c a n a l (2 s t a t e s ) 02 * ( 9 ) a n t e r i o r e x t e n t of a n t e r i o r v i t e l l i n e f i e l d (3 s t a t e s ) 04 * ( 1 0 ) d o r s a l c o n f l u e n c e o f a n t e r i o r v i t e l l i n e f i e l d (2 s t a t e s ) 06 * ( 1 1 ) p o s t e r i o r e x t e n t of p o s t e r i o r v i t e l l i n e f i e l d (4 s t a t e s ) OB * ( 1 2 ) d o r s a l c o n f l u e n c e of p o s t e r i o r v i t e l l i n e f i e l d (2 s t a t e s ) 1 * ( 1 3 ) v e n t r a l c o n f l u e n c e o f b o t h v i t e l l i n e f i e l d s (2 s t a t e s ) 12 * ( 1 4 ) l a t e r a l e x t e n t o f u t e r i n e loops (2 s t a t e s ) 14 * ( 1 5 ) a n t e r i o r e x t e n t of u t e r i n e loops (2 s t a t e s ) 16 * ( 1 6 ) p o s i t i o n of t e s t e s (3 s t a t e s ) 18 * ( l 7 ) s e m i n a l v e s i c l e (3 s t a t e s ) 2 * ( 1 8 ) c i r r u s s a c l e n g t h / f o r e b o d y l e n g t h (2 s t a t e s ) 19) egg l e n g t h (3 s t a t e s ) 20) c e r c a r l a l s t y l e t (2 s t a t e s ) 21) shape of e x c r e t o r y v e s i c l e (2 s t a t e s ) 2.5 * 3 param notu=9 nchar=21 outwldth=80 echo n o l i n k s s t a t r e p 3.3 o t u l a b = r 1 g h t ; 4.2 symbols 0-1 A-C: 5 d a t a (21A2.2X.A8) 6 0 0 0 0 7 0 0 7 0 0 0 0 0 0 0 0 0 0 0 0 0 7 O 0 1 O O O 0 0 0 O O O O 0 O A A 0 B 1 1 8 O 0 1 O 0 O O 0 0 O B O O O 0 0 0 0 A 1 1 9 0 A 0 0 0 0 1 1 0 0 A 0 0 1 1 0 0 0 A 0 7 10 0 0 0 0 1 0 0 0 0 0 0 0 1 1 1 0 B 1 B 0 1 11 0 0 0 0 1 0 0 0 8 0 0 1 0 1 1 B B 1 B 0 1 12 1 0 0 0 0 0 0 0 A 1 C 0 0 1 1 A B 0 B 0 1 13 1 0 0 0 0 1 0 0 0 1 B 0 0 1 1 A B 0 B 0 1 14 1 B 0 1 0 0 0 0 0 1 B 1 0 1 1 0 B 0 A 0 1 15 u n o r d e r e d 2 9 11 16 17 19 #$RUN PAUP ^ E x e c u t i o n b e g i n s ' P A U P P h y l o g e n e t i c A n a l y s i s U s i n g P a r s i m o n y I l l i n o i s N a t u r a l H i s t o r y Survey 10:11:05 JAN 17. 1987 E n t e r name of d a t a f i l e : th.mx.unord E n t e r name of o u t p u t f i l e : - unord GLYPTHELMINS AND HAPLOMETRANA DATA INITIAL RUN, WITH UNORDERED MULTISTATE CHARACTERS UNORDERED ANALYSIS ON CHARACTERS 2.9.11.16,17. and 19 C h a r a c t e r - s t a t e symbols r e a d . R e a d i n g d a t a m a t r i x . . . ou t g r o u p hy1oreus pennsy1 v a n i ens i s r o b u s t u s s h a s t a i i n t e s t i n a l i s c a l i f o r n i e n s i s q u i e t a f a c i o i 294 Data m a t r i x s t o r e d . U n o r d e r e d c h a r a c t e r s s e t . E n t e r i n g i n t e r a c t i v e mode . . . E n t e r command: g o / a l l t r e e s c s p o s s c h g l i s t t r e e o u t = 2; * * * * * * * * * * * * * * * * * * * * A n a l y s i s No. 1 * * * * * * * * * * * * * * * * * * * * O p t i o n s e t t i n g s : NOTU 9 NCHAR 21 U s e r - t r e e ( s ) NO HYPANC 1 ADDSEO N/A HOLD N/A SWAP N/A MULPARS N/A OPT FARRIS ROOT ANCESTOR Weights a p p l i e d NO OUTWIDTH 80 M i s s i n g d a t a code ? MAXTREE N/A The f o l l o w i n g c h a r a c t e r s a r e u n o r d e r e d : 2 9 11 16 17 19 E x h a u s t i v e s e a r c h o f a l l p o s s i b l e t o p o l o g i e s p e r f o r m e d . Computing l e n g t h s f o r a l l p o s s i b l e t r e e s . . . E x h a u s t i v e s e a r c h c o m p l e t e d . T o t a l number of t r e e s examined • 135135. 1 t r e e ( s ) f o u n d a t 33.000 s t e p s P o s s i b l e HTU c h a r a c t e r - s t a t e a s s i g n m e n t s C h a r a c t e r lode 1 2 3 4 5 6 7 8 9 1 0 1 1 1 2 1 3 1 4 1 5 1 6 1 7 1 8 1 9 2 0 2 1 10 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 A B 1 1 11 o 0 0 0 1 0 0 0 0 0 0 0 0 1 1 0 B 1 B 0 1 12 1 0 0 0 0 0 0 0 0 1 B 0 0 1 1 A B O B 0 1 13 1 0 0 0 0 0 0 0 0 1 B 0 0 1 1 0 B 0 A B 0 1 14 0 0 0 0 o 0 o 0 0 0 0 0 0 1 1 0 B 0 A B 0 1 15 o 0 0 0 0 0 0 0 0 0 0 0 0 1 1 0 0 0 A B 0 1 16 0 0 0 o 0 0 0 0 0 0 0 o 0 0 0 0 0 0 A 0 1 B S t a t i s t i c s f o r t r e e no. 1 L e n g t h * 33.COO OOO' I s r n s n q o j < s i s r n s n q o j < s i OOO' I 000' I OOO' I OOO' I H < f l l O J O B i < Cl 01 < 91 OOO' I s n i s n q o j <--}0»08J < - -- Sl - ei V a o o 000' I ei < — f i A o u a i s jsuoo Mouejq Buotv o i IUOJJ j a i o e j e u . 3 io(oe> 6 ********************** * e ^ e i n b 8 ************ ei*******»»* Z l * * * * * * * * * * s i s u a i u j o i i i e o L ************ * ********** i e u ( i s a i u t 9 * * * * * * * * * * * * * * * * * * * * * * • * 11 ********** * ieiseu.s s ********************** S l * * * * * * * * * * * s n i s n q o j f ******************************************** * 91 ********** AiAsuudd E ******************************************** * * 0 1 * * * * * * * * * * snajoiAu, z ******************************************** * * dnoj6j.no 1 * j o i s a o u e p e a e u B i s a p 6 u j s n p a i o o j 1 ou a a j i Bt?BO = x a p u i A o u a i s i s u o o S6Z 296 1 .coo 10 13 14 15 18 20 0 A 12 > c a l I f o r n O B 11 > i n t e s t l n 14 > 13 11 O B 14 > 13 B C 12 > c a l i f o r n O A 15 > r o b u s t u s O B 10 > p e n n s y l v 12 O 1 13 > f a c l o i O 1 11 > m t e s t i n 11 > s h a s t a i 16 > 15 16 > 15 16 O A 13 > 12 O B 11 > m t e s t i n O A 10 > h y l o r e u s 17 O B 15 > 14 O A 10 > h y l o r e u s 14 > 11 19 O B o u t g r o u p > 16 B A 13 > f a c i o i B A 15 > r o b u s t u s B A 10 > p e n n s y l v 1 .OOO 1 .000 0.750 0.500 1 .OOO 1 .OOO 1 .OOO 0.667 1 .OOO 1 .OOO 16 > 10 0.500 1 .OOO 297 21 O 1 o u t g r o u p > 16 1 .COO *** A n a l y s i s no. 1. c o m p l e t e d . E n t e r command: mts R e t u r n i n g t o MTS. Use SRESTART t o c o n t i n u e . #1 th.mx.ord 1 !GLYPTHELMINS AND HAPLOMETRANA DATA 2 !FINAL RUN. WITH MULTISTATE CHARACTERS ORDERED A POSTERIORI 4 • 5 "CHARACTERS (SEE TEXT AND TABLE I I I FOR CHARACTER STATES): 6 » ( D shape of tegumental p r o j e c t i o n s 7 * ( 2 ) e x t e n t of tegumental p r o j e c t i o n s ( 1 s t a x i s ) 7 . 2 • ( 3 ) e x t e n t of tegumental p r o j e c t i o n s (2nd a x i s ) 8 • ( 4 ) o r a l s u c k e r d i a m e t e r / v e n t r a l s u c k e r d i a m e t e r 9 • ( 5 ) p e n e t r a t i o n g l a n d s 10 * ( 6 ) m e d i a l g l a n d s 1 1 • ( 7 ) p h a r y n g e a l g l a n d s 12 • ( 8 ) p o s i t i o n o f o v a r y 13 * ( 9 ) p o s i t i o n o f L a u r e r ' s c a n a l 14 *( 10) a n t e r i o r e x t e n t o f a n t e r i o r v i t e l l i n e f i e l d ( 1 s t a x i s ) 14 . 2 *(11) a n t e r i o r e x t e n t of a n t e r i o r v i t e l l i n e f i e l d (2nd a x i s ) 15 •( 12) d o r s a l c o n f l u e n c e of a n t e r i o r v i t e l l i n e f i e l d 16 *( 131 p o s t e r i o r e x t e n t o f p o s t e r i o r v i t e l l i n e f i e l d ( 1 s t a x i s ) 16 2 *( 14] p o s t e r i o r e x t e n t o f p o s t e r i o r v i t e l l i n e f i e l d (2nd a x i s ) 17 *(15) d o r s a l c o n f l u e n c e of p o s t e r i o r v i t e l l i n e f i e l d 18 *(16) v e n t r a l c o n f l u e n c e of b o t h v i t e l l i n e f i e l d s 19 *( 17) l a t e r a l e x t e n t o f u t e r i n e l o o p s 20 *( 18) a n t e r i o r e x t e n t o f u t e r i n e l o o p s 21 *( 19) p o s i t i o n o f t e s t e s ( 1 s t a x i s ) 21 2 *(20) p o s i t i o n o f t e s t e s (2nd a x i s ) 22 * ( 2 i : s e m i n a l v e s i c l e ( 1 s t a x i s ) 22 2 *(22) s e m i n a l v e s i c l e (2nd a x i s ) 23 •(23) c i r r u s s a c l e n g t h / f o r e b o d y l e n g t h 24 •(24) egg l e n g t h 25 •(25) c e r c a r i a l s t y l e t 26 •(26) shape o f e x c r e t o r y v e s i c l e 26 2 * 27 param notu=9 nchar=26 outwidth=80 echo n o l i n k s s t a t r e p mlssing=9 28 o t u l a b = r l g h t ; 30 d a t a (26I2.2X.A8) 31 0 0 0 0 0 9 0 0 9 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 ou t g r o u p 32 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 1 0 0 2 1 1 h y l o r e u s 33 0 0 0 1 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 1 1 1 p e n n s y I v a n l e n s 1 34 0 1 O O O O O I 1 O 0 0 1 0 0 0 1 1 0 0 0 0 0 1 0 9 r o b u s t u s 35 0 O 0 0 0 1-0 O O O O O O O O 1 1 1 0 0 0 1 1 2 0 1 s h a s t a i 36 0 0 0 0 0 1 0 0 0 0 1 0 0 0 1 0 1 1 0 1 0 1 1 2 0 1 i n t e s t i n a l i s 37 1 0 0 0 0 0 0 0 0 1 0 1 0 2 0 0 1 1 1 0 0 1 0 2 0 1 c a l i f o r n i e n s i s 38 1 0 0 0 0 0 1 0 0 0 0 1 0 1 0 0 1 1 1 0 0 1 0 2 0 1 q u i e t a 39 1 0 1 0 1 0 0 0 0 0 0 1 0 1 1 0 1 1 0 0 0 1 0 1 0 1 f a c i o i * $ r e s t a r t E n t e r command: newdata E n t e r name of d a t a f i l e : t h.mx.ord E n t e r name of o u t p u t f i l e : 298 - o r d GLYPTHELMINS AND HAPLOMETRANA DATA FINAL RUN. WITH MULTISTATE CHARACTERS ORDERED A POSTERIORI Reading data m a t r i x . . . Data m a t r i x s t o r e d . E n t e r i n g I n t e r a c t i v e mode . . . Enter command: go/all t r e e s t r e e o u t * 2 ; • A n a l y s i s No. 1 * Option s e t t i n g s : NOTU 9 NCHAR 26 U s e r - t r e e ( s ) NO HYPANC 1 ADDSEO N/A HOLO N/A SWAP N/A MULPARS N/A OPT FARRIS ROOT ANCESTOR Weights a p p l i e d NO 0UTWIDTH 80 M i s s i n g d a t a code 9 MAXTREE N/A Exh a u s t i v e s e a r c h of a l l p o s s i b l e t o p o l o g i e s performed. Computing l e n g t h s f o r a l l p o s s i b l e t r e e s . . . Exh a u s t i v e s e a r c h completed. T o t a l number of t r e e s examined » 135135, 1 t r e e ( s ) found a t 33.000 steps S t a t i s t i c s f o r t r e e no. 1 Length « 33.OOO Co n s i s t e n c y index • 0.848 Tree no. 1 r o o t e d u s i n g d e s i g n a t e d a n c e s t o r 1 outgroup '10 •16 '15 '11 '14 '13 '12 2 hyloreus 3 p e n n s y l v a n i e n s i s 4 robustus 5 s h a s t a i 6 i n t e s t i n a l i s 7 ca11forn1 ens 1s 8 q u i e t a 9 f a c i e i *** A n a l y s i s no. 1. completed. 299 E n t e r command: end; PAUP e x e c u t i o n c o m p l e t e d . ^ E x e c u t i o n t e r m i n a t e d #1 th.mx.phy GLYPTHELMINS AND 026 009 (26F2.0.2X.2A4) 0 0 0 0 0 9 0 0 1 1 0 0 0 0 0 O 001 0 O 0 0 0 1 1 1 : PHYSYS b e g i n s HH HH HH HH HH HH HHHHHHHH HHHHHHHH 1 26 28 29 30 31 32 33 34 35 36 37 jf$RUN CLAD <f Execut i o n >PPPPPP >PPPPPPP >PP PP >PP PP >PPPPPPP >PPPPPP >PP >PP >PP > > COPYRIGHT 1983 BY d. > ALL >/data,th.mx.phy;xread >XREAD INPUT >GLYPTHELMINS AND HAPLOMETRANA DATA > >WAGNER INPUT HAPLOMETRANA DATA. MULTIS ORDERED WITH PAUP 9.0 9 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 outgr o u p 0 0 0 0 0 0 0 0 0 0 1 0 1 0 0 2 1 1 h y l o r e u s 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 1 1 1 pennsy1 van i ens 1s 1 0 0 0 1 0 0 0 1 1 0 0 0 0 0 1 0 9 r o b u s t u s 0 0 0 0 0 0 0 1 1 1 0 0 o 1 1 2 0 1 shast a 1 0 0 1 0 0 0 1 0 1 1 0 1 0 1 1 2 0 1 I n t e s t i n a l i s 0 1 0 1 0 2 0 0 1 1 1 0 0 1 0 2 0 1 c a l i f o r n i e n s i s 0 0 0 1 0 1 0 0 1 1 1 0 0 1 0 2 0 1 q u i e t a 0 0 0 1 0 1 1 0 1 1 0 0 0 1 0 1 0 1 f a c i o i YY YY YY YY YY YY HH HH HH HH HH HH HH HH YYY YY YY YY YY YY SSSSSS SSSSSSS SS SSSSS SSSSS SS ss SSSSSSS SSSSSS YY YY YY YY YY YY YYY YY YY YY YY YY SSSSSS SSSSSSS SS SSSSS SSSSS ss ss SSSSSSS SSSSSS MICKEVICH S. FARRIS & M. RIGHTS RESERVED 1 n p u t ; w a g . s . i n p u t . o u t p u t ; 1 f 1 1 ; t p / INPUT MULTIS ORDERED WITH PAUP > > >LFIT > >TREE >LENGTH >C-INDEX >F-RATI0 > >TPL0T > 1 TREES. LENGTH" 33.000 OUTPUT 33.000 84.848 9.524 OUTPUT >TREE 1 > 2 .OOO c a l i f o r n > I > 2 .OOO STEM0O12 > I I > 1 .OOO q u i e t a * + I > I > 3 .OOO STEM0013 > I I > 3 .OOO f a c i o i + + I > I > 1 .OOO STEM0014 300 > I I > 3.000 I n t e s t l n I I > I I I > 3.OOO STEM0011 I > I I > 1.000 s h a s t a i * I > I > 2.000 STEM0015 * 1 1 > 4.000 r o b u s t u s I > I > 3.OOO h y l o r e u s I > I I > 2.OOO STEM0010 > I I •> 1.000 p e n n s y l v • I > I > 33.OOO STEM0016 > I > 2.OOO o u t g r o u p > >$end ^ E x e c u t i o n t e r m i n a t e d #1 th.mx.br #1 t h 1 GLYPTHELMINS MATRIX FOR MAPPING ONTO BROOKS 1977 CLADOGRAM 2 026 008 001 9.0 3 (26F2.0.2X.2A4) 4 0 0 0 0 0 9 0 0 9 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 outgroup 5 0 0 0 1 0 0 0 0 0 0 0 0 1 0 0 0 0 0 1 0 1 O 0 2 1 1 h y l o r e u s 6 0 0 0 1 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 O O 0 0 1 1 1 pennsy1 van1ens 1s 7 0 1 0 0 0 0 0 1 1 0 0 0 0 0 0 0 1 1 0 0 O 0 0 1 0 9 ro b u s t u s 7.5 1 0 1 0 1 0 0 0 0 0 0 1 0 1 1 0 1 1 0 0 0 1 0 1 0 1 f a c i o i 8 0 0 0 0 0 1 0 0 0 0 0 0 1 0 0 1 1 1 0 0 O 1 1 2 0 1 s h a s t a i 10 1 0 O 0 0 0 O 0 0 1 0 1 O 2 0 0 1 1 1 O O 1 0 2 0 1 c a l i f o r n i e n s i s 1 1 1 0 0 0 0 0 1 0 0 0 0 1 0 1 0 0 1 1 1 0 0 1 0 2 0 1 q u i e t a . t r . b r 1 1977 BROOKS CLADOGRAM FOR GLYPTHELMINS 2 008 3 o u t g r o u p h y l o r e u s p e n n s y l v r o b u s t u s f a c i e i s h a s t a i c a l i f o r n q u i e t a 4 015 5 15 9 9 13 12 11 10 10 14 11 12 13 14 15 #$RUN CLAD:PHYSYS ^ E x e c u t i o n b e g i n s >PPPPPP HH HH YY YY SSSSSS YY YY SSSSSS >PPPPPPP HH HH YY YY sssssss YY YY sssssss >pp pp HH HH YY YY ss YY YY ss >pp pp HHHHHHHH YYY sssss YYY sssss >ppppppp HHHHHHHH YY sssss YY sssss >pppppp HH HH YY ss YY ss >pp HH HH YY ss YY ss >pp HH HH YY sssssss YY sssssss >pp HH HH YY SSSSSS YY SSSSSS > > COPYRIGHT 1983 BY d. S. FARRIS 8. M. F. MICKEVICH > ALL RIGHTS RESERVED >/data.th.mx.br;xread.matr i x ; d a t a . t h . t r . b r ; t r e a d . t r e e ; t p , t r e e ; > / d l a g . t r e e , m a t r i x , o u t p u t ; 1 f i t , o u t p u t / 301 >XREAD MATRIX INPUT >GLYPTHELMINS MATRIX FOR MAPPING ONTO BROOKS 1977 CLADOGRAM >TREAD >1977 BROOKS >TREE 1: > >TPLOT > >TREE 1 > c a 1 i f o r n > I > STEMOOIO > I >qu1eta++ > > STEM001 > >shasta1+ > > > > f a c i o i + + -> > > > r o b u s t u s -> > > >pennsy1v-> > > > h y l o r e u s -> > > >outgroup-> > >LFIT > >TREE >LENGTH >C-INDEX >F-RATIO >$end (CExecut i o n TREE INPUT CLADOGRAM FOR GLYPTHELMINS TREE I I I STEM0012---I I I I STEM0013---I I I I STEM0014 I — — I I I STEM0009 I STEM0015-I OUTPUT 33.OOO 78.788 12.644 t e r m i n a t e d 302 !allozyme data for the JL boyl j i group (Green, 1986a) Schars 4 5 6 17 19 22 25 31 32 41 43 deleted as uninformative param notu=11 nchar=47 otulab=right outwidth=80 echo statrep; data (4711,IX,A8) 00000000000000000000000000000000000000000000000 outgroup 10000133543220130400100100215200122035110004011 Ra.er 10000133442220020200100100005200123034110003011 Ra.me 00000133333220010201000100004200211034110003011 Ra.br 01000134333220010300100100004200211034210003011 Ra.t i 00200136333220010200000100004200211034110003011 Ra.du 10000135332321010100002000116200111034120101021 Rc.ca 00000143462321010000002001003000000201140000200 Rp.id 10000143440001010000002000123000122200140001200 Rp.or 10000113452320030000001000212200132003130202011 Rb.bo 10000142222220040000002000002200124024140003100 Rm.mu unordered 7-12 16 18 23 27-29 33-35 37-40 42 44-46 ! karyological data for the JB.. boyl i i group (Green, 1986b) param notu=5 nchar=l3 outwidth=80 echo statrep otulab=right; data (13I1,1X,A8) 0111001000000 Ra 0010110001002 Rc 1010000001101 Rp 2000100002011 Rb 2000100113001 Rm unordered 1 3 7 9 10 11 12 13 303 APPENDIX B ~ HOST AND DISTRIBUTION DATA FOR GLYPTHELMINS QUIETA (L i t era ture c i t a t i o n s are l i s t e d as they appear in the Index Catalogue of Medical and Veter inary Zoology, Oryx Press , Phoenix, Ar izona , U . S . A . ) BUFONIDAE Bufo americanus Holbrook Presque I s l e , Maine (Bouchard, 1951) B. microscaphus Cope Utah (Parry and Grundmann, 1965) B. woodhousi i Girard Nebraska (Brooks, 1976) Utah (Parry and Grundmann, 1965) HYLIDAE A c r i s crepitans Baird Iowa (Ulmer, 1970) Hyla cruc i fer Weid (= H. p i c k e r i n g i i Kennicott) Eastern Canada (Staf ford, 1905) Western Massachusetts (Rankin, 1945) Athens, Clarke C o . , Georgia (Byrd and Maples, 1963) Ann Arbor, Michigan (Najarian, 1955) Pseudacris n i g r i t a (LeConte) Athens, Clarke C o . , Georgia (Byrd and Maples, 1963) P. t r i s e r i a t a Weid Iowa (Ulmer, 1970) RANIDAE Rana b l a i r i Meacham et a l . Nebraska (Brooks, 1976) R. catesbeiana Shaw 304 Eastern Canada (Staf ford, 1905) l i e Perrot , Quebec (Rau et a l . , 1978) Gaspe Penninsula, Quebec~TRankin , 1944) Urbana, I l l i n o i s ( M i l l e r , 1930) I l l i n o i s (Leigh, 1937) Ohio (Odlaug, 1954) Ann Arbor, Michigan (Najarian, 1955) Indiana (Lank, 1971) Wisconsin (Williams and T a f t , 1980) Houston and H u n t s v i l l e , Texas (Harwood, 1932) East Texas ( H o l l i s , 1972) Cleveland C o . , Oklahoma (Trowbridge and Hefley, 1934) Oklahoma (Brooks, 1979) Arkansas (Rosen and Manis, 1976) Beaufort C o . , N. Caro l ina (Brandt, 1936) F l o r i d a (Manter, 1938) Louisiana (Bennett, 1938) Athens, Clarke C o . , Georgia (Parker, 1941) Amherst, Massachusetts (Rankin, 1944) North Carol ina (Rankin, 1944) V i r g i n i a (Campbell, 1968) Seat t le , Washington (Rankin, 1944) Iowa (Ulmer, 1970) Burke, Chatham, T a l i a f e r r o , Oconee, and Screven C o . , Georgia ( S u l l i v a n , 1976) Terrebonne and East Baton C o . , Louis iana ( S u l l i v a n , 1976) Oktibbeha C o . , M i s s s i s s i p p i ( S u l l i v a n , 1976) M i s s i s s i p p i (Brooks, 1979) Nye C o . , Nevada (Babero and G o l l i n g , 1974) Havana, Cuba (Odening (1968) R. clamitans L a t r e i l l e Western Massachusetts (Rankin, 1945) Urbana, I l l i n o i s ( M i l l e r , 1930) Ann Arbor, Michigan (Najarian, 1955) V i r g i n i a (Campbell, 1968) Presque I s l e , Maine (Bouchard, 1951) De Kalb and Oglethorpe C o . , Georgia ( S u l l i v a n , 1976) Warren C o . , New Jersey ( S u l l i v a n , 1976) Connecticut (Brooks, 1976) Louisiana (Brooks, 1979) Wisconsin (Williams and T a f t , 1980) l i e Perrot , Quebec (Rau et a l . , 1978) R. p a l u s t r i s Le Conte Presque I s l e , Maine (Bouchard, 1951) R. pipiens Schreber (= R_^  virescens Garman) Eastern Canada (Staf ford, 1905) Frank l in C o . , Ohio ( S u l l i v a n , 1976) Urbana, I l l i n o i s ( M i l l e r , 1930) 305 I l l i n o i s (Leigh, 1937) Iowa (Ulmer, 1970) Alamance C o . , N. Caro l ina ( S u l l i v a n , 1976) Frank l in C o . , Tennessee ( S u l l i v a n , 1976) Arkansas (Rosen and Manis, 1976) West V i r g i n i a (Brooks, 1979) Mountrai l C o . , N. Dakota (present study) Utah (Parry and Grundmann, 1965) R. s eptentr iona l i s Baird Presque I s l e , Maine (Bouchard, 1951) R. sphenocephala Cope Cleveland C o . , Oklahoma (Trowbridge and Hefley, 1934) Houston and H u n t s v i l l e , Texas (Harwood, 1932) R. u t r i c u l a r i a Harlan Louisiana (Brooks, 1979) M i s s i s s i p p i (Brooks, 1979) 306 APPENDIX C - COLLECTION SITES OF ANURANS Rana p ip iens : 9 km E . of Sprague, Neb. / White Earth R . , Mountrai l C o . , 85 km E of W i l l i s t o n , N. Dak. / Elk Point , Union C o . , S. Dak. R. aurora: Bonsal l C r k . , Duncan, Vancouver Is land, B . C . / L i t t l e Campbell R . , approximately 9 km E of White Rock, B . C . / Ponds at 232nd and 0 ave . , Langley, B . C . / Hicks and Moss L . , Sasquatch Park, 20 km NE of C h i l l i w a c k , B . C . R. pre t iosa : L i t t l e Muddy Pond, Manning Park, 65 km E of Hope, B . C . / Okanagan F a l l s Prov. Campground, Okanagan F a l l s , B . C . / . Wilgress L . , 15 km E of Greenwood, B . C . / Champion L . , Champion Lakes Park, 25 km NE of T r a i l , B . C . / Creston, B . C . / Loon L . , 25 km S of E lko , B . C . / Pel ican C r k . , Yellowstone National Park, Wyoming R. catesbeiana: L i t t l e Campbell R . , 9 km E of White Rock, B . C . 7 Ponds at 232nd and 0 ave . , Langley, B . C . / Osoyoos Lake, Osoyoos, B . C . / Parks C r k . , 25 km NW of Weed, S iskiyou C o . , C a l i f . / Thousand Springs, 10 km NW of Glenburn, Shasta C o . , C a l i f . / Glenburn, C a l i f . / McArthur, Shasta C o . , C a l i f R. cascadae: L i l y Pad L . , 16 km W of Cal lahan, S iskiyou C o . , C a l i f . / Kangaroo L . , 16 km W of Cal lahan, S iskiyou C o . , C a l i f . / Dead F a l l L . , 20 km W of Weed, Siskiyou C o . , C a l i f . / L i t t l e and Big Bear F l a t s , 30 km N of Burney, Shasta C o . , C a l i f . Bufo boreas: L i t t l e Muddy Pond, Manning Park, 65 km E of Hope, B . C . 7 Kings C r k . , Lassen Volcanic National Park, C a l i f . / Medicine Lake, 75 km N of Burney, Shasta C o . , C a l i f . / L i t t l e and' Big Bear F l a t s , 30 km N of Burney, C a l i f . / Brown R d . , Glenburn, Shasta C o . , C a l i f . Hyla regi11a; Ponds at 232nd and 0 ave . , Langley, B . C . / Pent icton, B . C . / C h r i s t i n a L . 25 km E of Grand Forks , B . C . 307 APPENDIX D - REDESCRIPTION OF GLYPTHELMINS SHASTAI, SYNONYMIZATION OF HAPLOMETRANA WITH GLYPTHELMINS, AND REDESCRIPTION OF G. INTESTINALIS N. COMB. REDESCRIPTION OF GLYPTHELMINS SHASTAI This species has not been reported since Ingles (1936) described i t from Bufo boreas c o l l e c t e d in the area of Glenburn, Shasta County, C a l i f o r n i a . In the present study, I examined 19 adults of B. boreas from the type l o c a l i t y during 1985-1986, but I d id not f ind any i n t e s t i n a l digenean paras i t e s . I obtained a d d i t i o n a l specimens of G. shastai through the courtesy of Dr . John C. Holmes, Department of Zoology, Univers i ty of A l b e r t a , Edmonton, Canada. These worms had been c o l l e c t e d from B. boreas in two l o c a l i t i e s in southwestern Canada during the 1960s: Gorge Creek, Alberta (approximately 60km southwest of Ca lgary) , and Nelson, B r i t i s h Columbia. 1 The examination of 24 specimens from th i s c o l l e c t i o n , as well as the re-examination of the holotype, has allowed a redescr ipt ion of the species . In addit ion to providing a larger set of observations and measurements, i t i s necessary to make some correct ions and augmentations to the o r i g i n a l d e s c r i p t i o n . A l l specimens examined were wholemounts stained with hematoxylins or acetocarmine and mounted in balsam. Glypthelmins shastai Ingles , 1936 (Figs 10, 17, 46, 47) DESCRIPTION ( F i g . 46a; measurements given as ranges and (means), based upon the holotype, and 24 f ixed specimens from the small in tes t ine of adults of Bufo boreas in southwestern Canada): P lag iorch io idea : body rounded at both ends; 1.62 - 5.87 (4.14) mm long; 0.60 - 1.15 (1.12) mm wide; tegument spinose ( F i g . 10); spines f la t tened , tapering with ends rounded, non-overlapping, extending to poster ior end of body. Oral sucker subterminal , 0.25 - 0.39 (0.32) mm long, 0.25 - 0.40 (0.32) mm wide. Ventra l sucker medial , in second f i f t h of body, 0.16 0.27 (0.22) mm long, 0.0.16 - 0.27 (0.22) mm wide. Prepharynx short , rece iv ing ducts of medial glands d o r s o - l a t e r a l l y ; medial glands at l e v e l of pharynx and esophagus ( F i g . 47a). Pharynx 0.14 - 0.26 (0.18) mm long, 0.14 - 0.27 (0.2l)mm wide. Esophagus b i f u r c a t i n g mid-way between pharynx and ventra l sucker; i n t e s t i n a l ceca extending to near pos ter ior end of body. Geni ta l pore medial , mid-way between ceca l b i f u r c a t i o n and ventra l sucker. C i r r u s sac 0.46 - 0.92 (0.68) mm long, more 1 C o l l e c t o r s : W.R. Turner, M. Aleks iuk , L Graham, and E . Huebner. 308 than one-half length of forebody, containing s t r a i g h t , b i p a r t i t e , in terna l seminal v e s i c l e ( F i g . 47b). Vas deferens present in some adul ts , absent in others, presence not corre la ted with s i ze . Testes pa i red , i n t e r c e c a l , obl ique, spher ica l to oval with smooth edges, in middle f i f t h of body; anter ior t e s t i s 0.21 - 0.45 (0.36) mm long, 0.34 - 0.41 (0.35) mm wide; poster ior t e s t i s 0.35 - 0.52 (0.42) mm long, 0.25 0.44 (0.35) mm wide. Ovary s i n i s t r a l , p r e t e s t i c u l a r , postacetabular, spher ica l to oval with smooth edges, 0.20 - 0.31 (0.26) mm long, 0.19 - 0.31 (0.25) mm wide; seminal receptacle s p h e r i c a l , immediately postero-medial to ovary; Laurer ' s canal o r i g i n a t i n g in ootype region between seminal receptacle and common v i t e l l i n e duct ( F i g . 47c); uterus extending posteriad to end of body, in numerous in terceca l and p o s t - t e s t i c u l a r transverse loops, f i l l i n g body poster iad to i n t e s t i n a l ceca; metraterm muscular, ventra l to c i r r u s sac, shorter than c i r r u s sac, surrounded by gland c e l l s (F ig . 47d). . V i t e l l a r i a f o l l i c u l a r , l y i n g d o r s a l , l a t e r a l , and ventra l to ceca, extending along sides of body from l e v e l of cecal b i f u r c a t i o n to poster ior t h i r d of hindbody, often ventro-medial ly confluent , or nearly so, at l e v e l of ootype region ( F i g . 17). Excretory bladder I-shaped, extending to just poster iad to l eve l of testes ( F i g . 46e). Eggs 41 - 50 (44) jum long. HOST: Bufo boreas Baird and G i r a r d . SITE OF INFECTION: Small i n t e s t i n e . TYPE LOCALITY: Glenburn, Shasta County, C a l i f o r n i a . OTHER LOCALITIES : Nelson, B r i t i s h Columbia; Gorge Creek, A l b e r t a . SPECIMENS : Holotype, U .S . Helm. C o l l . No. 8925; add i t i ona l specimens from the Dept. of Zoology, Univers i ty of A l b e r t a . Specimens from th i s c o l l e c t i o n have been deposited (HWML no. 23661) at the Harold W. Manter Laboratory, Univers i ty of Nebraska State Museum, 529-W Nebraska H a l l , Un ivers i ty of Nebraska, L i n c o l n , NE 68588-0514. Add i t i ona l specimens from th i s c o l l e c t i o n w i l l be deposited at the National Museum of Natural Sciences, Ottawa, Ontar io , Canada. REMARKS: The fol lowing characters were not included in the descr ip t ion by Ingles (1936) (see F i g . 46b): the b i p a r t i t e condit ion of the seminal v e s i c l e , the pos i t ion of the Laurer ' s cana l , and the shape of the excretory v e s i c l e . Through the examination of add i t iona l specimens, the v i t e l l a r i a are found to extend from the l eve l of the cecal b i furca t ion to the poster ior t h i r d of the hindbody; there is a lso frequently a ventro-medial confluence of the v i t e l l a r i a at the l e v e l of the ootype reg ion. The range and (mean) lengths for the eggs in the holotype are 41 - 46 (43) jam. The values for the specimens from southwestern Canada are 42 - 50 (45) jum. (Differences between the two sets of measurements are not s i g n i f i c a n t at the 0.05 l e v e l . ) Ingles (1936) reported a range of 31 - 32 jum for the length of the eggs in the holotype. The fol lowing two observations are a lso not in 309 accord w i t h ; those given in the o r i g i n a l d e s c r i p t i o n : the presence of small glands (herein termed medial glands, af ter Le igh , 1946) l a t e r a l to the pharynx and esophagus, and the locat ion of the metraterm ventra l to the c i r r u s sac (Ingles reported the locat ion to be d o r s a l , but a re-examination of the holotype shows i t to be v e n t r a l ) . Cheng (1959) c i t e d G. shastai as one of the species included in a proposed re-establishment of Margeana Cort , 1919. Byrd and Maples (1963) argued against the re-establishment of th i s genus. I concur. Rankin (1944) included G. shastai in a synonymization of a number of glyptheminths with G. qu ie ta . This opinion was based p r i m a r i l y upon the d i s t r i b u t i o n of the v i t e l l a r i a . Although a small number of specimens of G. quieta have v i t e l l a r i a with a ventro-medial confluence and an extension to the middle of the hindbody, these two characters are regular ly observed only in G. shas ta i . The two species a lso d i f f e r with respect to: (1) the presence in G. quieta of large pharyngeal glands in addit ion to the smaller , more a n t e r i o r , medial glands that are also present in G. shasta i ; (2) a scaled tegument in G. quie ta , and a spined tegument in G. shasta i ; (3) the presence in G. shastai of a c i r r u s sac that i s more than one-half the length of the forebody; and (4) symmetrical testes in G. qu ie ta , and oblique testes in G. shas ta i . Nasir and Diaz (1970) synonymized G. shastai with G. l inguatu la (Rudolphi, 1819) Travassos, 1924 [= Choledocystus 1. (Rudolphi, 1819) Byrd and Maples, 1963]. I do not accept th i s because (1) G. l inguatu la has a Y-shaped excretory v e s i c l e , b i f u r c a t i n g anter ior to the l e v e l of the testes , while that of G. shastai is I-shaped, b i furca t ing poster ior to the l e v e l of the testes , and (2) only G. l inguatu la has extracecal and p r e t e s t i c u l a r loops of the uterus. As noted by S u l l i v a n (1976), the holotype of G. shastai i s not a f u l l y mature specimen. This l imi t ed the conclusions that could be drawn from Ingles' (1936) d e s c r i p t i o n . 310 SYNONYMIZATION OF HAPLOMETRANA WITH GLYPTHELMINS AND REDESCRIPTION OF GLYPTHELMINS INTESTINALIS N. COMB. The resu l t s of the phylogenetic analys i s presented in Chapter III support the synonymization of Haplometrana with Glypthelmins, Haplometrana being the junior subject ive synonym. Haplometrana i n t e s t i n a l i s shares a number of t r a i t s with the species of Glypthelmins s tudied, in p a r t i c u l a r , with i t s postulated s i s t e r species , G. shas ta i . Its exclusion .from Glypthelmins would render that genus paraphyle t i c . Glypthelmins i n t e s t i n a l i s (Lucker, 1931) n. comb. (Figs 10, 12, 17, 18, 35) SYNONYMS: Haplometrana intest i n a l i s Lucker, 1931 H. utahensis Olsen, 1937 DESCRIPTION ( F i g . 35a; measurements given as ranges and (means), based upon the holotypes and paratypes of H. i n t e s t i n a l i s and H. utahensis , and 20 f ixed specimens of H. i n t e s t i n a l i s co l l e c t ed from R. pret iosa in southern B r i t i s h Columbia): P lag iorch io idea : body rounded at both ends; 2.70 5.28 (4.03) mm long, 0.55 - 0.67 (0.62) mm wide. Tegument spinose ( F i g . 10); spines f la t tened , tapering with ends rounded, in non-overlapping rows to poster ior end of body. Oral sucker subterminal , 0.27 - 0.35 (0.29) mm long, 0.25 - 0.38 (0.29) mm wide. Ventral sucker medial , in second f i f t h of body, 0.16 - 0.23 (0.18) mm long, 0.13 - 0.23 (0.18) mm wide. Prepharynx short , rece iv ing ducts of medial glands dorso-l a t e r a l l y ; medial glands at l e v e l of pharynx and esophagus ( F i g . 12). Pharynx 0.10 - 0.14 (0.12) mm long, 0.11 - 0.14 (0.12)mm wide. Esophagus b i f u r c a t i n g mid-way between pharynx and .ventral sucker; i n t e s t i n a l ceca extending to poster ior end of body. Geni ta l pore medial , immediately anter ior to ventra l sucker; c i r r u s sac 0.35 - 0.76 (0.45) mm long, more than one-half length of forebody, containing s t r a i g h t , b i p a r t i t e , i n t e r n a l seminal v e s i c l e . Vas deferens present in older adul t s , absent in younger a d u l t s . Testes pa i red , i n t e r c e c a l , spher ica l to ova l , with smooth edges, in middle f i f t h of body, usual ly tandem, occas ional ly obl ique; anter ior t e s t i s 0.23 - 0.32 (0.28) mm long, 0.19 - 0.26 (0.23) mm wide; poster ior t e s t i s 0.25 - 0.35 (0.29) mm long, 0.21 - 0.28 (0.24) mm wide. Ovary s i n i s t r a l , p r e t e s t i c u l a r , postacetabular, spher ica l to ova l , with smooth edges, 0.17 - 0.21 (0.19) mm long, 0.14 - 0.19 (0.17) mm wide; seminal receptacle s p h e r i c a l , immediately postero-medial to ovary; Laurer ' s canal o r i g i n a t i n g in ootype region between seminal receptacle and common v i t e l l i n e duct; uterus extending poster iad to end of body, in numerous in terceca l and post-t e s t i c u l a r transverse loops, f i l l i n g body poster iad to i n t e s t i n a l ceca; metraterm muscular, ventra l to c i r r u s sac, shorter than c i r r u s sac, surrounded by gland c e l l s . V i t e l l a r i a f o l l i c u l a r , l y ing d o r s a l , l a t e r a l , and ventra l to ceca, extending along sides of body from l e v e l of ovary to poster ior 31 1 t h i r d of hindbody, dorso-medially confluent poster ior to testes , emptying into s ingle v i t e l l i n e duct on each side (Figs 17, 18). Excretory bladder I-shaped, extending to just poster ior to l eve l of testes; flame c e l l pattern 2 (3 + 3 + 3)(3 + 3 + 3) = 36. Eggs 45-53 (50) jum long. HOST : Rana pret iosa (natural i n f e c t i o n s ) , R. cascadae ( laboratory 1 i n f e c t i o n s ) , Bufo boreas ( laboratory 2 and n a t u r a l 3 i n f e c t i o n s ) . SITE OF INFECTION: Small in tes t ine TYPE LOCALITY: B o t h e l l , King County, Washington State SPECIMENS: USNM Helm. C o l l . No. 29903, holotype; no. 29904, paratypes. Nos. 9025-9026, holotype and paratype, respec t ive ly , of H. utahensis . Add i t i ona l specimens c o l l e c t e d from R. pret iosa in southern B r i t i s h Columbia. REMARKS: This genus has been monotypic since Waitz (1959) synonymized H. utahensis Olsen, 1937 with H. i n t e s t i n a l i s Lucker, 1931. The character states c i t ed by Waitz as j u s t i f i c a t i o n for a synonymy are supported by the present redescr ip t ion . These are: the length/width r a t i o , the shape of the ventra l sucker, a b i p a r t i t e seminal v e s i c l e , tandem testes , and the pos i t ion of the vasa e f f e r e n t i a , vas deferens, and Laurer ' s cana l . Addi t iona l characters are reported herein that were observed from the examination of l i v i n g and f ixed specimens co l l ec t ed from R. pret iosa in southern B r i t i s h Columbia. These consist of: (a) medial glands at the l eve l of the pharynx and esophagus, with ducts entering the prepharynx d o r s o - l a t e r a l l y ; (b) the absence of a vas deferens in young adul t s ; and (c) the absence of the anter ior v i t e l l i n e f i e l d s and the i r associated ducts . 1 2 Present study. 3 Waitz (1959) . 312 EMENDED DIAGNOSIS OF GLYPTHELMINS In conjunction with the synonymization of Haplometrana and Glypthelmins, the generic diagnosis of Glypthelmins must be emended so as to' include species with tandem paired testes . The diagnosis includes the resul t s of the present study and those of Brooks (1977). Emendations are underl ined. Glypthelmins S ta f ford , 1905, emend. from Su l l i van (1976) SYNONYMS: Marqeana Cort , 1919 Haplometrana Lucker, 1931 Choledocystus Pereira and Cuoculo, 1941 Rauschie l la Babero, 1951 Reynoldstrema Cheng, 1959 Repandum Byrd and Maples, 1963 DIAGNOSIS: P lag iorch io idea: Body elongate, c y l i n d r i c a l to s u b c y l i n d r i c a l . Tegument spined or sca led . Oral sucker subterminal. Ventral sucker medial , in anter ior half of body. Pharynx well-developed. Esophagus present. Cecal b i furca t ion midway between pharynx and ventra l sucker; ceca long, reaching to poster ior quarter of body. Testes postacetabular, symmetrical, obl ique, o_r tandem in p o s i t i o n . C i r r u s pouch elongate, usual ly overlapping ventra l sucker. Ovary p r e t e s t i c u l a r , close to ventra l sucker, s i n i s t r a l or d e x t r a l ; seminal receptacle present; Laurer ' s canal present, o r i g i n a t i n g  in ootype region between seminal receptacle and common v i t e l l i n e  duct, or d i s t a l to common v i t e l l i n e duct. Uterus transversely c o i l e d , reaching to poster ior extremity of body; uterine c o i l s  in terceca l or extraceca l , pretest i c u l a r or not; metraterm  muscular or not, surrounded by numerous gland c e l l s . Geni ta l pore medial , between ventra l sucker and cecal b i f u r c a t i o n . V i t e l l a r i a f o l l i c u l a r , in l a t e r a l f i e l d s of body, ly ing d o r s a l , l a t e r a l and ventra l to ceca, may intrude mediad, dorsa l l y or  v e n t r a l l y . Longi tudinal extent of v i t e l l a r i a v a r i a b l e , ranging  from l eve l of pharynx to poster ior extremity of body. Excretory  ve s i c l e I-shaped or Y-shaped. P a r a s i t i c in in te s t ine , rare ly  g a l l bladder, of anurans. TYPE SPECIES: Glypthelmins quieta (Staf ford, 1900), S ta f ford , 1905. 313 APPENDIX E - THE LIFE CYCLE OF GLYPTHELMINS CALIFORNIENSIS From 1983 to 1986, I conducted surveys of the populations of sna i l s in the ponds and streams in Langley, B r i t i s h Columbia, where specimens of R. aurora infected with G. c a l i f o r n i e n s i s had been caught. Five species of sna i l s were examined ( i d e n t i f i c a t i o n s based ' on Clarke , 1981): Physa gyrina Say (70 specimens), P. propinqua Tyron (238 specimens), P. l o r d i Baird (24 specimens), Stagnicola elodes (Say) [= Lymnaea p a l u s t r i s (Muller)] (193 specimens) , and Pseudosuccinea columella" (Say) (46 specimens). This last species is indigenous to eastern North America and has been introduced to the western part of the continent (Clarke , 1981). The only cercar iae obtained from these f i e l d studies were echinostome cercar iae and furcocercous c e r c a r i a e . Accordingly , attempts were made to infect sna i l s with the eggs of G. c a l i f o r n i e n s i s in the laboratory . The resu l t s provided information on the l i f e c y c l e , m i r a c i d i a , daughter sporocysts , and cercariae of th i s species . MATERIALS AND METHODS Uninfected s n a i l populations were establ ished by r a i s i n g second generation stocks of Physa gyr ina , P. propinqua, Stagnicola elodes, and Pseudosucc inea columella in tanks of dechlorinated water, containing bo i led l e t tuce , stems of Elodea, and crushed s n a i l s h e l l s . Snai l s were fed overnight on eggs of G. c a l i f o r n i e n s i s that had been teased out of mature worms and had been allowed to s i t in a mixture of f i l t e r e d pond water and the host frog's feces for at least a week. In addi t ion to the four species of sna i l s reared in the laboratory , m i r a c i d i a l hatching behavior was also studied in specimens of the planorbid s n a i l , Gyraulus c ircumstr iatus Tryon, c o l l e c t e d from the same areas of water. The developmental stages obtained were examined as l i v i n g specimens, stained with Neutral Red or N i l e Blue v i t a l s ta ins , and immobilized with s l i g h t c o v e r s l i p pressure. M i r a c i d i a and sporocysts were placed in 0.3% s a l i n e , while cercar iae were placed in f i l t e r e d pond water. Drawings from l i g h t microscope examinations were made with the a id of a drawing tube. RESULTS EGG AND MIRACIDIUM ( F i g . 48c): Eggs range in length from 42 to 50 jum, with a mean length of 45 jam. They are l i g h t brown in c o l o r , operculated, and contain a f u l l y developed miracidium when e i ther ovipos i ted or removed from the d i s t a l part of the uterus . In a l l of the species of phys id , lymnaeid, and planorbid sna i l s examined, i t was found that af ter an overnight feeding of eggs, followed by the c o l l e c t i o n of the s n a i l s ' feces over the next 12 hours, mirac id ia had emerged from approximately 30% of the eggs in the feces. Eggs kept in f i l t e r e d pond water 314 for up to four months at 15°C s t i l l showed th i s hatching act i v i t y . Morphological examinations were done on mirac id ia obtained from the gut contents of specimens of P. propinqua one hour af ter feeding eggs to the s n a i l s . The mirac id ia range in length from 40 to 47 jum, with a mean of 44 jum. The surface is c i l i a t e d . An examination for epidermal plates was not conducted, but the p lates ' presence is suggested by the interupt ion of the c i l i a r y pattern in the anter ior and poster ior regions of the miracidium. At the anter ior end of the miracidium, which l i e s at the operculated end of the egg, there i s a l o n g i t u d i n a l l y s t r i a t e d a p i c a l p a p i l l a that can be protruded. Immediately poster ior to the p a p i l l a are two large , i r r e g u l a r l y rounded, penetration glands, the contents of which are granular . L a t e r a l to these glands are two longer glands. These are spindle-shaped and run from the anter ior region of the a p i c a l p a p i l l a to the poster ior region of the miracidium. The contents of these glands are r e l a t i v e l y more granular when the mirac id ia are within the eggs than when they are recovered from the intes t ine of the s n a i l , suggesting that the glandular secretions are associated with hatching. At the pos ter ior of the miracidium there are two large nucleated germinal c e l l s and a s ingle pair of flame c e l l s . SPOROCYSTS ( F i g . 48e): Development beyond the m i r a c i d i a l stage occurred only in P. gyrina and P. propinqua. The morta l i ty of infected sna i l s was very high, reaching approximately 90% within the f i r s t week of i n f e c t i o n . The sporocysts examined were obtained by crushing moribund sna i l s that were s t i l l shedding c e r c a r i a e . For th i s reason, observations on intramolluscan development are incomplete, in that i t was not poss ible to look for two sporocyst generations or the ir s i t e of development in the host. The sporocysts from which cercar iae emerge l i e loose in the d iges t ive gland of the s n a i l . They are elongate, ranging in length from 0.5 to 1.5 mm ( F i g . 48e), containing 4 to 10 cercar iae . There i s no b i r t h pore evident, and the cercar iae escape through ruptures in the body wall of the sporocyst . This wall gives the appearance of having two layers , the inner one of which is the t h i c k e r . The poor condit ion of the sporocysts precluded further observations. CERCARIA (Figs 48a,b ,d): Specimens of P. gyrina and P. propinqua began shedding cercariae at 50 days after i n f e c t i o n . No phototropism or p e r i o d i c i t y was observed. The cercar iae are act ive swimmers and are usual ly found near the surface of the water in the c o l l e c t i n g vesse l . 315 DESCRIPTION: Distomate x i p h i d i o c e r c a r i a . Body 0.47 - 0.51 (0.49) mm long, 0.26 - 0.32 (0.30) mm wide at ventra l sucker. T a i l 0.45 - 0.49 (0.48) mm long, w i t h extensive dorso-ventral f i n f o l d ; ventra l port ion of f i n f o l d running nearly ent ire length of t a i l , dorsal port ion running along poster ior t h i r d of t a i l . Narrow scales present on tegument to poster ior of body. Oral sucker subterminal , bearing s t y l e t . Stylet c lear and rounded at base, tapering to a point , with s l i g h t shouldering; 24 - 26 (25) jum long ( F i g . 48b). Ventral sucker medial , postequator i a l . Prepharynx present. Pharynx muscular, approximately one-half width of o r a l and ventra l suckers. Cecal b i furca t ion immediately anter ior to ventra l sucker; ceca terminating at poster ior end of body. Geni ta l primordia d i f f e r e n t i a t e d into testes , ovary, and c i r r u s sac ( F i g . 48d). Testes symmetrical, a n t e r o - l a t e r a l to anter ior chamber of excretory v e s i c l e . Penetration glands composed of at least f ive glands on each s ide , occupying l a t e r a l areas of body from poster ior border of pharynx to anter ior border of ventra l sucker, emptying at o r a l sucker near base of s t y l e t . Excretory bladder dumbbell-shaped, c o n t r a c t i l e . Flame c e l l pattern = 2[(3+3+3)(3+3+3)] = 36. When placed in a d ish of pond water with pithed adults of R. aurora, the cercar iae continue to swim about a c t i v e l y . When a c e r c a r i a contacts the frog , i t stops swimming, crawls over the surface of the skin for a short dis tance , and begins to penetrate the epi thel ium, los ing i t s t a i l in the process . Once under the epi thel ium, the c e r c a r i a b a l l s up and begins r o l l i n g ac t ions . The elapsed time from contact with the frog to b a l l i n g up beneath the epithel ium is approximately 15 minutes. Contact with the frog does not inev i tab ly lead to penetrat ion. Some cercariae crawl over the skin for up to ten minutes, and then swim away. Penetration behavior with respect to tadpoles of R. aurora was not examined. DEVELOPMENT IN RANA AURORA ( F i g . 49): Attempts to raise uninfected R. aurora to adulthood in the laboratory were unsuccessful . Frogs were therefore co l l ec t ed from areas where no paras i t i sm by G. c a l i f o r n i e n s i s had been encountered. These areas were the streams associated with Hicks and Moss Lakes, Sasquatch P r o v i n c i a l Park, B r i t i s h Columbia (approximately 20 km NE of C h i l l i w a c k ) . Frogs were e i ther kept separate after exposure to c e r c a r i a in the laboratory , or were fed port ions of the skin of another adult of R. aurora in which cercar iae had encysted. At the end of the twenty-day period during which these infec t ions were studied, both groups of frogs contained young adults of G. c a l i f o r n i e n s i s in the anter ior region of the small in tes t ine T F i g T 49). These worms are approximately 1 mm long. A l l of the major organ systems are developed. Medial glands are evident in the region of the prepharynx and pharynx. The uterus extends poster iad beyond the ends of the i n t e s t i n a l ceca in a s ing l e , in t erceca l loop. A lack of infected frogs precluded the study of development beyond twenty days after i n f e c t i o n . 316 DISCUSSION My incomplete study of the intramolluscan development stages of G. c a l i f o r n i e n s i s prevents a f u l l comparison to l i f e h i s t o r y studies of G. qu ie ta , by Leigh (1946), Rankin (1944), and Sche l l (1962a!, and of H. i n t e s t i n a l i s , by Sche l l (1961). In both of these species, the mother and daughter sporocysts possess two wal l s , the outer of which is derived from the basement membrane of the s n a i l in tes t ine ( S c h e l l , 1961, 1962a). As noted by Sche l l (1962b), although th i s surrounding layer of host o r i g i n , termed a pa le to t , occurs in the sporocyst generations of many p l a g i o r c h i o i d s (Biehringer, 1885; Leuckart, 1863, 1886-1901; Cort and O l i v i e r , 1943; Cort and Ameel, 1944; Cort et a_l. , 1952), i t does not always form from the same source. Sche l l (1962a) noted that the s i m i l a r i t i e s in the host response that produces the paletots in G. quieta and H. i n t e s t i n a l i s appear to resu l t from the depth to which the mirac id ia penetrate the wall of the host' i n t e s t i n e . On the basis of th i s character and s i m i l a r i t i e s in c e r c a r i a l and sporocyst morphology, Sche l l (1962a) considered G. quieta and H. i n t e s t i n a l i s to be more c lo se ly re lated to one another than they are to Te lorch i s bonnerensis Waitz, 1960, another p l a g i o r c h i o i d parasi te of amphibians that develops in physid sna i l s (Sche l l , 1962b). Furthermore, I infer from S c h e l l 1 s d iscuss ion that , based upon the data of Cheng (1961), he included G. pennsylvaniensis as a c lose r e l a t i v e of G. quieta and H. i n t e s t i n a l i s . S u l l i v a n and Byrd's (1970) work on G. pennsylvaniensis of fers l imi t ed evidence that supports th i s i n c l u s i o n . In l i g h t of these s tudies , and in l i g h t of the add i t iona l observation by Sche l l (1962a) that the mother sporocysts of H. i n t e s t i n a l i s develop in the wall of the stomach, cecum, and foregut of the s n a i l , while those of G. quieta develop in the wall of the midgut, i t i s unfortunate that my work on G. c a l i f o r n i e n s i s could not examine these same proper t i e s . Limited observations on the daughter sporocysts show that the ir s ize and shape are s imi lar to those of G. quieta (as reported by Le igh , 1946; Rankin, 1944; S c h e l l , 1962a), H. i n t e s t i n a l i s (as reported by S c h e l l , 1961), G. pennsylvaniensis (as reported by Cheng, 1961; Su l l i van and Byrd, 1970), and T. bonnerensis (as reported by S c h e l l , 1962b). A second layer i s apparent, but because of the poor condit ion of the sporocysts , i t could not be determined whether th i s was a paletot or the resul t of delamination of the inner l ayer . The cercar ia of G. c a l i f o r n i e n s i s infects the anuran host in the same manner as do the cercar iae of G. quieta and H. i n t e s t i n a l i s , in that they a l l penetrate and encyst in the skin of the adult frog, which infects i t s e l f upon eating the shed s k i n . The morphology of the c e r c a r i a of G. c a l i f o r n i e n s i s places i t in the Cercariae Ornatae group. From surveys of physid sna i l s in I l l i n o i s , M i l l e r (1936) reported Cercar ia  mesotyphla M i l l e r , 1935 as another member of t h i s group, and 317 concluded that i t was the c e r c a r i a of G. qu ie ta . The studies of Leigh (1946) and Rankin (1944) confirmed t h i s . In addi t ion to the characters described by M i l l e r (1936), Leigh (1946) also observed medial glands, pharyngeal glands, and recognizably d i f f e r e n t i a t e d gonads in the cercar iae of G. qu ie ta . I observed these same characters in the cercar iae of th i s species that I obtained during the present study (Chapter IV, LIFE HISTORY DATA). It was also observed that , although the di f ference is not as marked as in the adu l t s , the tegumental project ions are more sca le - l ike , than s p i n e - l i k e ( i . e . , more rectangular than taper ing) . The c e r c a r i a of G. c a l i f o r n i e n s i s d i f f e r s from that of G. quieta only by i t s lack of medial and pharyngeal glands. Although the former are present in the adults of both species , I could not observe them in the ava i lab le specimens of G. c a l i f o r n i e n s i s . A l l of the other characters observed in the c e r c a r i a of the two species are the same. These cercar iae d i f f e r from the cercar ia of H. i n t e s t i n a l i s with respect to (a) the ir symmetrical testes , rather than the oblique to tandem testes of the l a t t e r species (Olsen, 1937), and (b) a scaled, rather than spined, tegument. In my observations of the cercar iae of H. i n t e s t i n a l i s (Chapter IV, LIFE HISTORY DATA), I found that the t a i l fo ld i s not s t r i c t l y v e n t r a l , as described by Olsen (1937), but that i t a lso has a shorter , shallower, dorsal port ion that often l i e s against the surface of the t a i l . 318 APPENDIX F - CODING MULTISTATE CHARACTERS This is a technica l note with three p a r t s . The f i r s t demonstrates four mult i s tate character coding methods, the second compares the i r propert i e s , and the t h i r d examines some of the i r uses in phylogenetic s tudies . This las t part i s p r i m a r i l y concerned with the use of paras i te data to infer host r e l a t i o n s h i p s . A mult i s tate character i s herein considered to be any set of more than two organic or inorganic states that have, through some process, transformed from one into the other. The order of transformation of these states w i l l describe a mult i s tate t ree . This d e f i n i t i o n is broad enough to include phenomena such as organic character evo lut ion , changes in eco log ica l propert i e s , host -paras i te coevolut ion, and biogeographic events. Whatever type of transformation is involved, there are two aspects to deal ing with mult i s tate characters . The f i r s t i s that of determining the order of transformation. In evolut ionary s tudies , the transformations have not been observed, and so the ir order must be i n f e r r e d . This note i s not p r i m a r i l y concerned with these methods of inference. The use of parsimony analys i s and outgroup comparisons in such procedures has been addressed by Mickevich (1982), F i t c h (1971), Swofford's PAUP program, and by Swofford and Maddison (in review) (see Chapter I I I , THE INFERENCE OF PHYLOGENETIC RELATIONSHIPS). If one wants to construct a mult i s tate tree by other c r i t e r i a , such as excluded transformations, funct ional r e s t r i c t i o n s , e t c . , i t i s necessary to j u s t i f y that t ree ' s di f ferences from a tree constructed with parsimony techniques that place no r e s t r i c t i o n s on the order of character transformation. The second aspect of deal ing with mult i s tate characters , and that with which th i s note is concerned, i s that regardless of how the order of character state transformations in a tree i s determined, the t ree ' s shape must be unambiguously represented when using that tree in further phylogenetic s tudies . The goal i s always the same: to represent the mult i s tate tree so that the re la t ionsh ips among i t s states can be used to study the re la t ionsh ips among the e n t i t i e s (taxa, land masses, e t c . ) possessing those s tates . CODING MULTISTATE CHARACTERS The f i r s t method i s Addit ive Binary Coding (ABC), developed by F a r r i s ejt a l . ( 1970). I refer to the second method as Redundant Linear Coding (RLC), and to the t h i r d method as Nonredundant Linear Coding (NLC). The RLC method was explained to me by Mary Mickevich (pers. comm., 1983), and the NLC method by David Swofford (pers. comm., 1984). The fourth method, in t erna l root ing , has spec ia l i zed appl i ca t ions for coding 319 basa l ly dichotomous mult i s tate s er i e s . A mult i s tate tree can have various conf igurat ions . There may be observed states at only the terminal branches (F ig . 50a), as occurs when a paras i te phylogeny is used as a character of the hosts (see below). Or, a tree may have observed states at the nodes as well (F ig . 50b), as occurs with a transformation series for character evo lut ion , ontogenetic stages, or land mass break-up. The tree shape of th i s second type of mult i s tate character may be complex ( F i g . 50b), l inear ( F i g . 50c), or basa l ly dichotomous ( F i g . 50d). I w i l l demonstrate the coding procedures with the contrived tree in Figure 51a. This tree has unspecif ied nodal states on i t s l e f t s ide , and spec i f i ed nodal states on i t s r ight s ide . For each of the three main coding methods, th i s tree w i l l be coded, a data matrix w i l l be made, and the tree w i l l be reconstructed from the matrix in order to demonstrate the retent ion of the topo log ica l information. When examining these reconstruct ions i t should be noted that the trees in Figures 51a and 51b are equivalent: a state col lapses to the node beneath when that state i s not character ized by an autapomorphy. Addi t ive Binary Coding - Every node on the tree i s l a b e l l e d . In the example in Figure 52a, there are nine states to be connected: f ive terminal branches and four in terna l nodes. The tree i s represented in a matrix ( F i g . 52b) with nine columns of binary characters . The matrix values are determined by assigning a "1" code to every state ly ing along the minimum path between each state and the base of the tree . For example, the minimum path from A to D passes through i and i i , and so only these four states receive a "1" in the code entered h o r i z o n t a l l y in the matrix. When reconstruct ing a tree from the matrix, with no reference to the o r i g i n a l tree in Figure 52a, one f i r s t bui lds a polytomy for a l l nine states onto which the character d i s t r i b u t i o n s are mapped ( F i g . 52c). This produces the o r i g i n a l tree (F ig . 52d). Redundant Linear Coding - Each terminal branch on the tree becomes the terminal state in a l inear transformation series constructed as the minimum path from the base of the tree . Unspecif ied nodal values are not l a b e l l e d , and nonterminal branches become intermediate transformation s tates . Every transformation ser ies s tar t s as a "0" state at the bottom of the tree and changes at every nonterminal branch as i t passes up the tree to the terminal state ( F i g . 53a). The data matrix in the example ( F i g . 53b) has f ive mult i s tate characters , one for each terminal branch. The matrix values are entered h o r i z o n t a l l y , and are determined by ass igning the most derived state of every character passed through. For example, the minimum path from A to the base of the tree contains states 1, 2, and 3 of character 1, states 1 and 2 of character 2, state 1 of character 3, and state 0 of characters 4 and 5. The code for A is thus 32100. The tree constructed from the matrix begins as a polytomy (F ig . 53c), and f in i shes as the o r i g i n a l tree ( F i g . 53d). 320 Nonredundant Linear Coding - This method is s imi lar to the RLC method, except that not a l l of the transformation ser ies change states at every nonterminal branch they pass through. There are major and minor axes. In the tree in Figure 54a, for example, there i s a four state ser ies (character 1) running from the base of the tree to A. This i s the major a x i s . B is part of- a minor a x i s , and is assigned state 1 of character 2, rather than state 3 as i t was with the RLC method. It should be noted that the major axis i s a r b i t r a r i l y set , and need not even be the longest mult i s tate s e r i e s . For example, the l e f t side of the tree could be coded as in Figure 54e. There appear to be no reasons for pre ferr ing one major axis over another. The matrix in the example has f ive mult i s tate characters , one for each terminal branch ( F i g . 54b). As in the RLC method, the values are determined by assigning the most derived state of every character passed through. For example, the minimum path from A to the base of the tree contains state 3 of character 1, and state 0 of the other four characters . The code for A is thus 30000. Figure 54c shows the polytomous tree constructed from the matrix, and Figure 54d shows the o r i g i n a l t ree . Internal Root ing - This i s an a d d i t i o n a l coding method that can be used when a transformation ser ies contains no unspecif ied nodal values and consis ts of a s ingle basal b i f u r c a t i o n ( F i g . 55a). This tree could be coded with the ABC ( F i g . 55b), RLC ( F i g . 55c), or NLC ( F i g . 55c) methods (the l a t t e r two give the same matr ix ) . Or, i t can be assigned a s ingle l inear mult is tate character that s tarts at one terminus of the tree , runs through the base, and ends at the other terminus ( F i g . 55d). This ser ies is rooted i n t e r n a l l y by speci fy ing the basal state as the outgroup in the data matrix (C in th i s example). The diverging numerical values of the coding w i l l describe the basal b i f u r c a t i o n . COMPARISONS A l l of the coding methods described above are capable of unambiguously representing a mult i s tate tree . This i s not a t r i v i a l c a p a b i l i t y , for i t i s poss ib le to code a mult is tate tree so that i t s Consistency Index (Kluge and F a r r i s , 1969; F a r r i s et a l . , 1970) i s less than 1.0. This can occur when the propert ies of the character s tates , which may have already been u t i l i z e d in construct ing the mult i s tate tree , are allowed to influence the coding of the representation of the tree . Glen and Brooks (1985) give an example in which a basa l ly b i f u r c a t i n g mult is tate tree might be inadvertently coded with a l inear code because of a numerical progression in the character states . The ABC, RLC, and NLC methods can represent trees of various conf igurat ions . In i t s s p e c i a l i z e d a p p l i c a t i o n , the in terna l rooting method has the advantage of occupying a s ingle matrix column, compared with two columns in the RLC and NLC methods, and three or more columns in the ABC method. A disadvantage is that (1) i t produces a plesiomorphic state with other than the standard "0" code, which could be confusing in an otherwise standardly coded 321 matrix of other characters , and (2) one arm of the transformation ser ies w i l l have a code with values that decrease (2-1-0 in F i g . 55) even though no reversals are being postulated. This would, for example, preclude i t s use with a Camin-Sokal parsimony t r e e - b u i l d i n g a lgori thm. There are three things to note about the ABC method. (1) It assigns a s ingle character state change to each branch, (2) only the codes for the terminal states (A, B, C, F , and G in F i g . 52) are needed to reconstruct the topology of the tree ( F i g . 5e), and (3) the number of new binary characters created is the number of terminal states plus the number of nodes to be connected. Because of the t h i r d property, ABC matrices for a l l but the simplest trees can be quite large . Because of the second property , some of th i s s ize comes from what might be unnecessary codings of in terna l nodes, depending on whether or not the nodal states are observed ( e . g . , in F i g . 52, E is observed, while i and i i are not) . Two things to note about the RLC method are that (1) i t assigns more than one character state change to some nonterminal branches, and (2) the matrix is smaller than that produced with the ABC method: there are only as many new mult i s tate characters as there are terminal branches on the tree . The RLC method can be seen as a methodological precursor to the NLC method. However, i t s redundant coding can produce trees with areas of mis leadingly high synapomorphic support. That i s , in t erna l branches receive another instance of character support every time they are included in the minimum path between a terminal branch and the base of the tree . It i s thus the NLC method that appears to be pre ferab le . It avoids the unnecessary node l a b e l l i n g and the re su l t ing large matrix s ize of the ABC method, and i t avoids the redundant branch coding and the r e s u l t i n g u n j u s t i f i e d weighting of the RLC method. SOME USES OF MULTISTATE CODING METHODS I n t r i n s i c Characters - I refer here to an organism's propert ies that are manifested during i t s ontogenetic development, so as to make a d i s t i n c t i o n with i t s paras i t e s , which w i l l be discussed next. If such an i n t r i n s i c character i s d i s t r i b u t e d among the study taxa in a binary manner, then i t s order of transformation is establ ished when i t i s p o l a r i z e d . If i t occurs in a mult i s tate manner, then i t s order i s not es tabl i shed with the determination of the plesiomorphic s tate . A phylogenetic ana lys i s w i l l often contain both binary and mult i s tate characters . An invest igator may want to (1) see the ef fect of using a mult i s tate tree suggested by funct ional cons iderat ions , previous s tudies , e t c . ; or (2) he may want to produce a mul t i s ta te tree with parsimony techniques such as F a r r i s Optimizat ion ( F a r r i s , 1970), Transformation Series Analys is (Mickevich, 1982), and Unordered Character Analys i s (Swofford, PAUP, vers ion 2.4, 1985), and then incorporate that tree into the character matrix for the taxa s tudied. 322 Figure 56a shows a cladogram for f ive taxa produced by Wagner parsimony analys i s of seven binary characters . These are represented in the f i r s t seven columns of the data matrix in Figure 56b. There i s also a mult i s tate character ( i - iv) d i s t r i b u t e d among the taxa as shown in Figure 56a. Figure 56c shows a poss ible tree , determined by whatever means, for that character . The tree has been coded with the NLC method, which gives the mult i s tate matrix in Figure 56d. This information is then put into the character matrix in Figure 56b by using the mult i s tate matrix as a source from which the appropriate hor i zonta l code is taken, depending on which state a p a r t i c u l a r taxon possesses ( i . e . , the ent ire mult is tate matrix i s not necessar i ly used). Figure 56e is the cladogram constructed from a l l nine characters . With e i ther the ABC or NLC method, the codings for the mult i s tate tree w i l l have no greater weight in the cladogram than w i l l the codings for the other characters . Paras i tes as Characters of Their Hosts - There are a number of operations involved in assessing the degree of host -paras i te coevolut ion , and the reader should refer to Brooks (1979a, 1980, 1981a, and 1985) for a f u l l e r d i scuss ion . Appl ica t ions can be found in Brooks and Mit ter (1984), C o l l e t t e and Russo (1985), Cressey e_t a l . (1983), Glen and Brooks (1986), and Mit ter and Brooks (1983). One can compare phylogenetic trees constructed from the i n t r i n s i c organic characters of hosts and paras i te s , but th i s necessitates the designation of e i ther the host tree or the paras i te tree as the standard of comparison ( i . e . , as the more l i k e l y of the two to be c o r r e c t ) . A l t e r n a t i v e l y , based upon the aforementioned homologous and homoplasious nature of host -paras i te assoc ia t ions , one can treat the phylogenetic tree for the paras i tes l i k e a mult is tate character tree of the hosts . Hosts are assigned the mult i s tate code associated with the paras i te they harbor, and a host phylogeny is generated. This tree can then be compared to host phylogenies produced using other paras i te data ( i . e . , looking for cons i l i ence with other taxonomic groups of p a r a s i t e s ) , or i t can be compared to a phylogeny produced through an analys i s of i n t r i n s i c organic characters of the hosts themselves. In a l l of these approaches, which are complementary and not mutually exc lus ive , the goal is to d i s t i n g u i s h homologous from homoplasious paras i te as soc ia t ions , and to use the former for information on host r e l a t i o n s h i p s . In th i s note I w i l l demonstrate the representation of a paras i te phylogeny as a mul t i s ta te tree in l i g h t of my e a r l i e r comments on coding methods. Brooks (1981a) showed the use of the ABC method, and I f ee l that further exp l i ca t ion would be h e l p f u l . I w i l l a lso discuss coding procedures for the occurrence of more than one paras i t e taxon per host. I begin by contrast ing the mult i s tate approach with another method of i n f e r r i n g host re la t ionsh ips from paras i te data. This groups hosts on the basis of a common presence or absence of a 323 paras i te (see Glen and Brooks, 1986, for add i t i ona l d i scuss ion) . If o v e r a l l s i m i l a r i t y c r i t e r i a are used, the resu l t has the usual shortcomings of using phenetic analys i s in evolutionary studies (see Ernst and Erns t , 1980, and the reply of Brooks, 1981b). Provided that e i ther the presence or absence of the paras i te is taken to be plesiomorphic, one can instead c lus ter by spec ia l s i m i l a r i t y . As noted by Glen and Brooks (1986), th i s plesiomorphy can be set as simply an a p r i o r i assumption, or by reference to whether or not the outgroup of the hosts possesses the paras i te ( this would, of course, necessitate the e l iminat ion of ambiguity about the state of the outgroup node). While i t i s true that th i s t h i r d approach introduces an accountabi l i ty to evidence that i s missing from the other two, i t requires a previous phylogenetic analys is to have establ ished an outgroup for the hosts . It thus does not re ly exc lus ive ly on paras i te data to infer host r e l a t i o n s h i p s . Figures 57a~d show the l i m i t s of presence/absence a n a l y s i s . In Figure 57a, there has been retention of the ancestral paras i tes af ter each speciat ion event with the hosts . The presence of a paras i te taxon ( c a p i t a l l e t t er s ) in a host taxon (roman numerals) i s indicated with a dot. By treat ing each paras i te as an independent binary character , and by considering the presence of the paras i te to be apomorphic, the host phylogeny in Figure 57b i s produced. But i f there has been no such re tent ion , as in Figure 57c, then presence/absence analys i s w i l l produce an unresolved tree with nothing but autapomorphies. There are , of course, intermediate degrees of retent ion that would give p a r t i a l r e s o l u t i o n . A l t e r n a t i v e l y , evolutionary re la t ionsh ips can be taken into considerat ion by f i r s t doing a phylogenetic ana lys i s of the paras i t e s , and then t rea t ing that cladogram as a mult i s tate character tree of the hosts . Such a tree i s constructed with a source of information that i s not ava i lab le in standard mult i s tate analyses, namely, the c l a d i s t i c analys i s of the paras i tes themselves. The characters have characters , so to speak, that can be used to infer the i r r e l a t i o n s h i p s . Because of th i s property , a parsimony analys i s of the order of transformation of the mult i s tate characters can be done i n t r i n s i c a l l y , without having to refer to a tree topology determined by a preceding analys i s of binary characters at that  l e v e l . At the same time, i t must be noted that th i s add i t iona l source of information introduces an add i t i ona l source of e r r o r . Incorrect inference of host re la t ionsh ips can be made because (1) the paras i te cladogram is correct but there i s homoplasious d i s t r i b u t i o n of the paras i tes among the hosts, (2) the paras i te cladogram i s incorrect despite s t r i c t l y homologous d i s t r i b u t i o n of the paras i tes among the hosts , or (3) the paras i te cladogram is incorrect and there is homoplasious d i s t r i b u t i o n of the paras i tes among the hosts . Figures 57e-i i l l u s t r a t e the mult i s tate approach with paras i te data . Assume that a phylogenetic analys i s of the four 324 paras i te taxa in Figure 57c produces the cladogram in Figure 57e. This can be represented with the NLC matrix in Figure 57f. Using th i s matrix as the source of the hor i zonta l code for each host taxon, the host matrix in Figure 57g is produced. This matrix gives the host re la t ionsh ips in Figure 57h, for which the corresponding paras i te re la t ionsh ips are shown in Figure 57 i . These l a t t e r re la t ionsh ips are the same as those in Figure 57e. Thus, the information in the transformation ser ies i s retained in the tree that is constructed with that ser ies as i t s only source of information. Inc lus ive ORing - Presence/absence analys i s r e l i e s upon there being a hierarchy of mult ip le occurrences of a paras i te taxon in d i f f erent host groups to produce a resolved host tree ( F i g . 57a). Mul t i s ta te analys is is most straightforward when there are no mult iple occurrences of any sor t . This l a t t e r condi t ion , in which the host simultaneously possesses more than one state in the mult is tate tree , creates a coding s i t u a t i o n not usual ly encountered with standard mult i s tate data of i n t r i n s i c organic characters (although i t might ar i s e with s e r i a l homologues, for example). There i s current ly one method that has been appl ied in such a s i t u a t i o n . Known as inc lus ive ORing (see, e . g . , Copi , 1968, p. 216), i t has been used with the ABC method i m p l i c i t l y by Brooks (1981a: Figs 20-21) and Glen and Brooks (1986), and e x p l i c i t l y by Cressey et a l . (1983), and C o l l e t t e and Russo (1985). Consider Figure 58a, a paras i te phylogeny with the hosts (roman numerals) ind ica ted . Host IV has the s i s t e r paras i te taxa C and D. I use the ABC method for the tree ( F i g . 58b), for c l a r i t y . As Cressey et a_l. (1983) note, when i n c l u s i v e l y ORing more than one set of characters (the hor izonta l codes for paras i tes C and D in th i s example), a character state is sa id to be present in the union of the sets so long as i t is present in at least one of the sets . (In formal l o g i c , an inc lus ive OR statement uses the word "or" in i t s weak sense, as an AND/OR statement. This i s in c o n t r a d i s t i n c t i o n to exclusive ORing, in which something can be one state or another, but not both.) Thus, the two hor izonta l codes assigned to host IV in the host matrix in Figure 58c can be compressed into the s ingle code shown in the host matrix in Figure 58d. E i ther one or two occurrences of the "1" code in a column resul t in the union being set to "1". The compressed matrix produces the tree in Figure 58e, with the implied paras i te re la t ionsh ips in Figure 58f. Paras i te C is in ferred to have evolved in the same speciat ion event in which the common ancestor of hosts III and IV evolved, and then to have been retained by hosts III and IV ( i . e . , l i k e an ancestra l t r a i t ) when paras i te D evolved with host IV. (See Brooks, 1981a, pp. 240-242 for comments on i n f e r r i n g such ancestor-descendant r e l a t i o n s h i p s . ) There are , however, data conf igurat ions in which inc lus ive ORing w i l l d i s t o r t phylogenetic information and thus lead to incorrect inferences of host r e l a t i o n s h i p s . This occurs because 325 the most apomorphic code is always given precedence in determining the compressed code, no matter what the d i s t r i b u t i o n or number of the more plesiomorphic codes might be. The l o g i c a l basis for th i s dec is ion is that only the apomorphic codes are considered to represent rea l propert ies of organisms ("true" statements) that can be used as grouping c r i t e r i a . The plesiomorphic codes are considered to represent the absence of a property ("not" statements), which, c l e a r l y , cannot be used to group. Thus, with the ABC method, a " 1 " code takes precedence over any number of "0" codes. And, with the RLC and NLC methods, the most derived state of a mult i s tate ser ies takes precedence over a l l preceding s tates . This procedure does not create any problems when a paras i te i s shared by s i s t er host taxa, as in Figure 58, because i t produces a synapomorphy for that clade (character 5, paras i te C, in Figs 58e-f) . Problems a r i s e , however, when a host group possesses paras i tes that are phylogenet ica l ly d is tant from one another, as would occur when there has been symplesiomorphic retent ion of some paras i tes and host-switching of others . In such a case, inc lus ive ORing w i l l lose the kinship information contained in the d i s t r i b u t i o n s of the more plesiomorphic codes. I f , for example, the co lon iz ing paras i te taxa are more derived ( i . e . , a " 1 " code) than the coevolved paras i te taxa, then the coevolutionary pat tern , and thus the data for a correct inference of host phylogeny, w i l l be l o s t . This i s demonstrated in Figure 59a, in which paras i te taxa C, D, and G are present in host I I I . The "1" code that G, the more derived paras i te group, receives in the ABC matrix (not shown) overides the "0" codes that the more plesiomorphic paras i te taxa (C and D) rece ive . The compressed host matrix ( F i g . 59b) gives the phylogeny in Figure 59c, in which taxon III has been misplaced. (I say that i t i s misplaced because there are two characters , C and D, p lac ing i t between II and IV, and one character , G, p lac ing i t with V I . This occurs whether C and D are paraphylet ic or monophyletic.) The paras i te re la t ionsh ips implied by th i s tree ( F i g . 59d) are inconsistent with those in the i n i t i a l cladogram in Figure 59a. (For example, G is placed as the ancestor of C and D.) Restating th i s in a context broader than that of host -paras i te coevolut ion: the pattern of re la t ionsh ips among the group, "capi ta l l e t t ers" , which here was used as the only source of information on the re la t ionsh ips of the group, "roman numerals", i s not re tr ievable from the inferred roman numeral cladogram. The data have been d i s t o r t e d by the method with which they were handled. CONCLUSIONS The purpose of th i s study has been to expl icate the usage of some coding methods for mult i s tate characters , and to examine the ir u t i l i t y in cer ta in types of mult i s tate character ana lys i s . My concerns come p r i m a r i l y from the perspective of studying host -paras i te r e l a t i o n s h i p s . A major part of such studies i s 326 examining the host re la t ionsh ips that are indicated by a paras i te phylogeny. This necessitates the use of a coding method that does not d i s t o r t the paras i te tree (as can inc lus ive ORing) or bias the resu l t s (as can the RLC method). For these reasons, we f ind the ABC and NLC methods to be pre ferab le . The ABC method produces coding which i s perhaps a l i t t l e easier to follow (hence i t s use in Figures 58 and 59), while the NLC method produces a smaller and more e f f i c i e n t code. 327 APPENDIX G - SOME PROPERTIES OF THE CONSISTENCY INDEX AND THE F -RATIO CONSISTENCY INDEX The Consistency Index (CI) (Kluge and F a r r i s , 1969) i s ca l cu la ted as the sum of the ranges of a l l characters in the data set ( i . e . , the minimum tree length required to explain the data) d iv ided by the number of evolutionary changes, or steps, postulated on the tree ( i . e . , the tree length) . A value of 1.0 indicates a perfect f i t of the tree to the data. 1 A data set of four binary characters , say, would have a t o t a l range of four steps [(0 - 1) x 4] , If a tree in ferred from those data posited f ive steps, i t s CI_ would be 4/5 = 0.80. The CI_ does not take into account the d i s t r i b u t i o n of characters on a tree or in a data set . The range of a binary character , for example, i s counted as "one" in a data set whether the derived state i s present in one or many taxa, and i t s appearance on the tree i s counted as "one" whether i t i s shared or unique. Because of t h i s , synapomorphies are not d i s t inguished from autapomorphies. Figures 60a-c show three trees , constructed from three data sets with the same f i r s t seven binary characters , and with d i f f erent d i s t r i b u t i o n s of two other binary characters . A l l three trees have a CI of 0.75 (9/12), even though characters 8 and 9 are autapomorphic in 60a, i n t e r n a l l y synapomorphic in 60b, and basal ly synapomorphic in 60c. Consider two systematists working on the ABCD c lade , one of whom has constructed tree 60a, the other of whom has constructed tree 60c. A comparison of CI_ values alone w i l l not indicate that one person has found two autapomorphies, thus corroborat ing the monophyly of that taxon, but adding no support to the proposed genealogical groupings, while the other has found two synapomorphies corroborat ing the monophyly of the ent i re c lade . The CI_ can be increased by the addi t ion of autapomorphies. The tree in Figure 60d presents the f i r s t seven characters used in Figures 60a-c. Its CI_ is 0.70 (7/10). The addi t ion of characters 8 and 9 as autapomorphies in Figure 60a increases the CI to 0.75. The discovery of one add i t i ona l autapomorphy for each of the taxa would further increase the C_I to 0.81 (13/16). This apparent increase in opt imal i ty occurs although the degree of synapomorphic support for the postulated genealogy remains the same. The s e n s i t i v i t y of the CI to autapomorphies could be e l iminated by modifying the measure so as to not count any non-CI values are often reported as percentages. 328 homoplasious characters on terminal branches. Some systematists have begun to report both modified and unmodified CI values with the ir re su l t s ( e . g . , F ink , 1985). The phylogenetic trees presented in th i s thes is (Chapter III) are reported with both values . 1 So long as the number of steps of character evolut ion are equal , the CI_ w i l l not d i s t i n g u i s h between para l l e l i sms and reversa l s . Character 5 in Figure 60d, for example, i s explained as a p a r a l l e l i s m evolving in two steps: on the terminal branch of taxon A, and on the nonterminal branch bearing taxa C and D. It could a lso be explained as a reversa l in two steps: appearing at the base of the tree , and revers ing in taxon B. The CI thus of fers no way of choosing among equal- length trees with a l t ernat ive in terpretat ions of homoplasious character evo lut ion . Of course, i f a postulate of p a r a l l e l i s m requires more steps than one of r e v e r s a l , or v ice versa , the CI^  w i l l d i s t i n g u i s h the shorter t ree . For th i s reason, there w i l l not be equal CI^  values for postulates of reversa l and convergence in a homoplasious character in po lyphyle t i c taxa ( e . g . , i f in Figure 60d there were a character common to Taxa A and D). F-RATIO The F-Rat io i s derived from the ^ - s t a t i s t i c of F a r r i s (1972), and i s a comparison of Manhattan distance matrices constructed from the o r i g i n a l data set and an in ferred tree . Differences in F-Rat io values can be used to choose between trees of equal CI_. Figure 61 demonstrates the c a l c u l a t i o n . From the character matrix (F ig . 61a), a matrix of the phenetic distances between each pair of taxa i s constructed ( F i g . 61c). These values are the f ixed , observed distances between taxa. For any tree in ferred from the character matrix ( e . g . , 61b), a matrix of p a t r i s t i c distances between pa irs of taxa is constructed ( F i g . 61d). These p a t r i s t i c values are the v a r i a b l e , postulated distances between taxa. If the tree f i t s the data p e r f e c t l y , i t s postulated p a t r i s t i c distances w i l l be the same as the observed phenetic dis tances . If there i s homoplasy, the p a t r i s t i c distance w i l l exceed the phenetic di stance. 1 This modif icat ion of the CI requires a d i s t i n c t i o n between d i f f erent types of autapomorphic characters , that i s , characters that appear on the terminal branches of a tree . As homoplasies, they may appear one (a s ingle reversal) or more times (para l e l l i sms , convergences, and mult ip le reversals) on the ent ire t ree . As homologues, they appear only once. In order to give a truer measure of the support for a tree , a modified CI should e l iminate only the non-homoplasious autapomorphies. No homoplasy, be i t on terminal or nonterminal branches, should be e l iminated . 329 The sum of the d i f f e r e n c e matrix f o r the phenetic and p a t r i s t i c d i s t a n c e m a t r i c e s i s the _ f - s t a t i s t i c . T h i s i s normalized with d i v i s i o n by the sum of the phenetic d i s t a n c e matrix, g i v i n g the F - R a t i o . 1 For the example i n F i g u r e 61, homoplasy i n c h a r a c t e r 5 r e s u l t s i n an f_-sta t i s t i c of 2 (an e x t r a 2 steps i n the p a t r i s t i c d i s t a n c e between taxa A and D), and an F-Ratio of 7.14 [(2/28) x 1 00]. As with a CI_ l e s s than 1, an F-Ratio g r e a t e r than 0 i n d i c a t e s a l e s s - t h a n - p e r f e c t f i t of the t r e e to' the data. U n l i k e the CI_, the F-Ra t i o i s s e n s i t i v e to the d i s t r i b u t i o n of c h a r a c t e r s among taxa, but only under c e r t a i n c o n d i t i o n s . I t can d i s t i n g u i s h between e q u a l - l e n g t h p o s t u l a t e s of p a r a l l e l i s m and r e v e r s a l , as w e l l as between autapomorphies and i n t e r n a l synapomorphies. I t cannot, however, d i s t i n g u i s h between autapomorphies and b a s a l synapomorphies. T h i s i s because i n i t s p a i r w i s e comparison of taxa i t t r e a t s r e l a t i o n s h i p s as u n d i r e c t e d , r a t h e r than d i r e c t e d , t r e e s . I t thus d i s c r i m i n a t e s between c h a r a c t e r s on t e r m i n a l and nonterminal branches, r a t h e r than between synapomorphies and autapomorphies. Basal synapomorphies are t r e a t e d i n the same manner as autapomorphies i n p a t r i s t i c d i s t a n c e c a l c u l a t i o n s because both occur on a t e r m i n a l branch of the t r e e . These two types of c h a r a c t e r d i s t r i b u t i o n w i l l g ive d i f f e r e n t m a t r i c e s , but the f > s t a t i s t i c and the F-Ratio w i l l be the same. T h i s p r o p e r t y i s demonstrated i n F i g u r e 62. The F-Ratio of t r e e 62a i s 18.18 (2/11: e x t r a steps i n the AB d i s t a n c e ) . The val u e drops to 14.29 (2/14) when a f o u r t h c h a r a c t e r i s added as an autapomorphy ( F i g . 62b) (thus showing t h a t , as with the CI, the F-Ratio can be improved by the a d d i t i o n of such characters')"", and to 13.33 (2/15) when i t i s added as an i n t e r n a l synapomorphy ( F i g . 62c). When i t i s added as a basal synapomorphy ( F i g . 62d), however, the F- R a t i o i s once more 14.29 (2/14). The value f o r the i n t e r n a l synapomorphy i s lower not because of a change i n the amount of departure of the p a t r i s t i c d i s t a n c e from the phe n e t i c d i s t a n c e (the ^ - s t a t i s t i c s of a l l the t r e e s are 2), but because of an i n c r e a s e i n the denominator of the F-Ratio, that i s , the sum of the phen e t i c d i s t a n c e matrix. The F-Ratio cannot be used as the only c r i t e r i o n f o r choosing the p r e f e r e d t r e e , f o r the t r e e with the lowest F-Ratio i s not n e c e s s a r i l y the s h o r t e s t t r e e . T h i s has a l s o been noted by Swofford, i n the documentation f o r PAUP ( v e r s i o n 2.4, 1985), 1 The p h y l o g e n e t i c s computer program PHYSYS (developed by J.S. F a r r i s and M.F. Mickevich) g i v e s the normalized value m u l t i p l i e d by 100 as the F-Ratio, while the p h y l o g e n e t i c s program PAUP (developed by D. L. Swofford) simply g i v e s the normalized v a l u e . 330 and in Swofford and Maddison (in review). Compare the trees in Figures 63a and 63b. The f i r s t i s from Figure 61, but with character 5 interpreted as a reversa l rather than as a p a r a l l e l i s m . This adds a step to the tree and decreases i t s CI_ from 0.83 (5/6) to 0.71 (5/7) . The F-Rat io increases from 7.14 to 28.57 (8/28:. extra steps in the XB, XC, XD, and AD d i s tances ) . In Figure 63b, a d i f f erent tree for the same data has an even lower CJ_, of 0.56 (5/9) , and an even higher F - R a t i o , of 42.86 (12/28: extra steps in the XA, AC, BC, and CD, d i s tances ) . In th i s case, then, both measures indicate a poorer f i t to the data. But th i s agreement of C_I_ and F-Rat io assessments does not always ho ld . Consider the trees in Figures 64a and 64b. The f i r s t postulates a p a r a l l e l i s m in character 5, g iv ing a CI_ of 0.80 (8/10). This tree is longer than the tree in Figure 64b, which postulates a r e v e r s a l , g iv ing a CI of 0.89 (8/9). The F-Rat ios , however, rank the trees in the opposite order. The F-Ratio for 64a, the p a r a l l e l i s m , i s 5.61 (6/107: extra steps in the AB, AC, and BC d i s tances ) , while the value for 64b, the r e v e r s a l , i s 7.48 (8/107: extra steps in the XD, XE, XF, and XG d i s tances ) . Assessing trees on the basis of the ir F-Rat ios alone would resul t in choosing a longer tree . When choosing among equal- length shortest trees , the F -Ratio i s not biased towards p a r a l l e l i s m or reversa l in a homoplasious character . Returning to character 5 in Figure 60d, the F-Rat io for the p a r a l l e l i s m postulate i s 30.00 (12/40: extra steps in the AC, AD, BC, BD d is tances ) , while for the reversa l (character 5 appearing on the bottom branch, then being los t in taxon B) i t is 25.00 (10/40: extra steps in the XB, AC, BC, and BD d i s tances ) . The F-Rat io prefers the reversa l t ree . It i s poss ib le , however, to have trees with F-Rat ios that prefer para l l e l i sms over reversa l s , or that do not d i s t i n g u i s h between e i t h e r . In Figure 65a, a p a r a l l e l i s m in character 4 gives an F -Ratio of 18.18 (4/22: extra steps in the AC and BC distancesT, while a reversa l ( F i g . 65b) gives a better value, of 9.09 (2/22: extra steps in the XD d i s tance ) . In Figures 65c and 65d, taxon E has been added to the a n a l y s i s , and the F-Rat ios for p a r a l l e l i s m and reversa l in character 4 are equal , at 10.26 (4/39: extra steps in e i ther the AC and BC, or the XD and XE d i s tances ) . F i n a l l y , in Figures 65e and 65f, taxon F has been added, and the F-Rat io for a p a r a l l e l i s m ( F i g . 65e) i s 6.45 (4/62: extra steps in the AC and BC distances) while the reversa l tree ( F i g . 65f) gives a poorer value, of 9.68 (6/62: extra steps in the XD, XE, and XF d i s tances ) . The F-Rat io has th i s property because in the pairwise comparison of taxa, the more taxa there are on e i ther side of a homoplasious character , the more times w i l l that step appear in the p a t r i s t i c distance matrix , and the more w i l l i t contribute to departures from the ( phenetic distance matrix. As with the C I , the F-Rat io can be improved by the addi t ion of autapomorphies to an a n a l y s i s . From Figure 61, the tree in Figure 61b has an F-Rat io of 7.14 (2/28). Adding one autapomorphy to taxon B reduces i t to 6.25 (2/32); adding 331 another reduces i t to 5.56 (2/36). This w i l l a lso occur with the addi t ion of basal synapomorphies, since they are a lso on terminal branches of the undirected tree . In a d d i t i o n , the F -Ratio can be improved by adding taxa to a t ree . If a taxon with the characters of taxon C is added to the tree in Figure 61b, the F-Rat io decreases to 5.13 (2/39). If another taxon with the same characters i s added, the F-Rat io decreases further to 4.00 (2/50). Because of the measure's d i s t i n c t i o n between terminal and nonterminal characters , the same improved f i t can be produced by adding taxa i d e n t i c a l to the outgroup. This creates more pairwise comparisons to be made, and increases the sum of the phenetic distance matrix. 

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