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Identity and limits of Limbella tricostata (Musci: Amblystegiaceae) Christy, John A. 1985

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IDENTITY AND LIMITS OF LIMBELLA TRICOSTATA (MUSCI: AMBLYSTEGIACEAEl by JOHN A. CHRISTY B.S., U n i v e r s i t y Of Oregon, 1978 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in • THE FACULTY OF GRADUATE STUDIES Department Of Botany We accept t h i s t h e s i s as conforming to the r e q u i r e d standard THE UNIVERSITY OF BRITISH COLUMBIA August 1985 © John A. C h r i s t y , 1985 In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t of the requirements f o r an advanced degree at the U n i v e r s i t y 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 r e e l y a v a i l a b l e f o r r e f e r e n c e and study. I f u r t h e r agree that p e r m i s s i o n fo r e x t e n s i v e copying of t h i s t h e s i s f o r s c h o l a r l y purposes may be granted by the Head of my Department or by h i s or her r e p r e s e n t a t i v e s . I t i s understood that copying or p u b l i c a t i o n of t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be allowed without my w r i t t e n p e r m i s s i o n . Department of Botany 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: August 1985 i i A b s t r a c t I n v e s t i g a t i o n of morphology, karyotype, isozymes, c u l t i v a t i o n , geography and h a b i t a t confirmed d i f f e r e n c e s between L i m b e l l a t r i c o s t a t a ( S u l l . ) CM. and Sciaromium f r y e i W i l l i a m s . S i g n i f i c a n t d i f f e r e n c e s were observed e x p e r i m e n t a l l y i n branch bud frequency, l e a f i n s e r t i o n angle, l e a f a r e o l a t i o n , chromosome le n g t h , m o b i l i t y of superoxide dismutase and shoot o r i e n t a t i o n . Sc iaromium f r y e i i s c o n s i d e r e d a subspecies of L i m b e l l a  t r i c o s t a t a . Chromosome numbers of both subspecies i s n=11. L.  t r i c o s t a t a ssp. t r i c o s t a t a has a Bryum type s p o r e l i n g and a hypnaceous peristome, and i s thought to have d e r i v e d from L.  pachyloma (Mont.) CM. or i t s antecedent a f t e r l o n g - d i s t a n c e d i s p e r s a l from South. America to Hawaii no l a t e r than the mid-T e r t i a r y . " L^ t r i c o s t a t a ssp. f r y e i i s thought to have d e r i v e d from C_ t r i c o s t a t a ssp. t r i c o s t a t a a f t e r l o n g - d i s t a n c e d i s p e r s a l from Hawaii to North America i n the mid- to l a t e T e r t i a r y . i i i T able of Contents A b s t r a c t i i L i s t of T a b l e s ix L i s t of F i g u r e s x Acknowledgement . x i i i Chapter I INTRODUCTION 2 A. SCOPE, PURPOSE AND OBJECTIVES OF STUDY 2 B. STUDY MATERIAL 2 C. TAXONOMIC HISTORY 3 1.1 Hawaiian P l a n t s 3 1.2 Oregon P l a n t s 3 D. PROBLEMS IN NEED OF INVESTIGATION 4 1. Taxonomic P o s i t i o n of Sciaromium f r y e i Based on L i m i t e d Data 4 2. D i s p a r i t y of Hawaiian and North American H a b i t a t s 4 3. D i s t r i b u t i o n i n North America 5 E. QUESTIONS TO BE ANSWERED 6 F. METHODS USED TO ANSWER QUESTIONS 6 1 . 3 Methods Used 6 1.4 R a t i o n a l e f o r Methods Used ° 6 G. BIOSYSTEMATICS OF BRYOPHYTES 7 Chapter II HABITAT 10 A. INTRODUCTION 10 B . HAW A11 AN ISLAND HABI TATS .' 10 2.1 C l i m a t e of Hawaiian H a b i t a t s 10 2.2 S o i l s of Hawaiian H a b i t a t s 13 2.3 Hydrology of Hawaiian H a b i t a t s 14 2.4 V e g e t a t i o n of Hawaiian H a b i t a t s 15 C. NORTH AMERICAN HABITATS 19 2.5 C l i m a t e of North American H a b i t a t s 19 2.6 S o i l s of North American H a b i t a t s 21 1. Sutton Lake S o i l s 21 2. Barview S o i l s 22 2.7 Hydrology of North American H a b i t a t s 23 1. Sutton Lake Hydrology 23 2. Barview Hydrology 23 2.8 V e g e t a t i o n of North American H a b i t a t s 24 i v 1. Sutton Lake V e g e t a t i o n 24 2. Barview Vege t a t i o n 29 D. DISCUSSION 31 2.9 Comparison of Climates 31 2.10 Comparison of S o i l s 32 2.11 Comparison of Hydrology 32 2.12 Comparison of Ve g e t a t i o n 33 E. CONCLUSION AND SUMMARY 33 Chapter I I I MORPHOLOGICAL ANALYSIS 35 A. INTRODUCTION 35 B. NEED FOR QUANTIFICATION OF DISCRETE MORPHOLOGICAL CHARACTERS 35 C. MORPHOLOGICAL SIMILARITY OF HAWAIIAN AND NORTH AMERICAN LIMBELLA 36 D. SIGNIFICANCE OF DIFFERENT BRANCHING PATTERNS IN LIMBELLA .36 3.1 C o n t r o l of Branching 37 E. METHODS 38 3.2 Q u a n t i f i c a t i o n of Branching P a t t e r n s 38 3.3 Phenetic A n a l y s i s 39 F. RESULTS 40 3.4 Q u a n t i f i c a t i o n , of Branching P a t t e r n s 40 3.5 Phenetic A n a l y s i s 43 G. DISCUSSION 46 H. SUMMARY AND CONCLUSIONS 46 Chapter IV KARYOTYPE ANALYSIS 50 A. INTRODUCTION 50 4.1 U t i l i t y of Karyotype A n a l y s i s 50 4.2 C y t o l o g i c a l Reports f o r L i m b e l l a Taxa 52 B. MATERIALS AND METHODS 52 4.3 Chromosome Squashes and Documentation 52 4.4 V a r i a t i o n s i n Methodology D i c t a t e d by Study M a t e r i a l 56 C. RESULTS 58 4.5 Karyotype A n a l y s i s 58 D. DISCUSSION 67 V 4.6 S i g n i f i c a n c e of N=11 Chromosome Number 67 4.7 S i g n i f i c a n c e of Chromosome Length Data 67 4.8 S i m i l a r i t i e s Between the Karyotypes 68 4.9 D i f f e r e n c e s Between the Karyotypes 68 E. CONCLUSIONS AND SUMMARY 69 Chapter V ISOZYME BANDING PATTERNS 70 A. INTRODUCTION 70 5.1 U t i l i t y of E l e c t r o p h o r e s i s 70 5.2 E l e c t r o p h o r e t i c Procedure 71 5.3 E l e c t r o p h o r e s i s of Bryophytes 71 B. MATERIALS AND METHODS 7 3 5.4 E l e c t r o p h o r e s i s 73 5.5 Enzyme Assays 75 C. RESULTS 76 5.6 I n c o n s i s t e n t S t a i n i n g 76 5.7 The Enzymes 77 D. DISCUSSION 80 A. CONCLUSION AND SUMMARY 81 Chapter VI PHENOLIC CHROMATOGRAPHY 83 A . INTRODUCTI ON 8 3 B. MATERIALS AND METHODS 84 C. RESULTS 85 D. DISCUSSION ...88 E. CONCLUSION AMD SUMMARY 89 Chapter VII EXPERIMENTAL CULTIVATION 91 A. INTRODUCTION 91 B. UTILITY OF MOSS CULTIVATION 91 C. CULTIVATION OF LIMBELLA 93 7.1 Need f o r L i v e M a t e r i a l 93 7.2 Need f o r C u l t i v a t i o n S t u d i e s of L i m b e l l a 93 1. • Whole Pla n t C u l t i v a t i o n '...93 2. Spore C u l t i v a t i o n 94 3. Fragment Regeneration ....94 D. MATERIALS AND METHODS 94 7.3 Whole Plant C u l t i v a t i o n 95 1. C u l t i v a t i o n of Hawaiian P l a n t s 95 2. C u l t i v a t i o n of North American P l a n t s 96 7.4 Spore C u l t i v a t i o n ; 96 7.5 Fragment Regeneration 97 1. L i v i n g M a t e r i a l 97 2. D r i e d M a t e r i a l 99 7.6 R e c i p r o c a l C u l t i v a t i o n 99 7.7 Shoot O r i e n t a t i o n Experiment 101 E. RESULTS 101 7.8 Whole Plant C u l t i v a t i o n 101 1. Hawaiian P l a n t s 101 2. North American P l a n t s 103 7.9 Spore C u l t u r e s , 103 7.10 Fragment Regeneration 105 1. L i v i n g M a t e r i a l 105 2. D r i e d M a t e r i a l 111 7.11 R e c i p r o c a l C u l t i v a t i o n 111 1. Hawaiian P l a n t s i n R e c i p r o c a l C u l t i v a t i o n ...112 2. North American P l a n t s i n R e c i p r o c a l C u l t i v a t i o n 112 7.12 Shoot O r i e n t a t i o n Experiment 112 F. DISCUSSION 11-3 7.13 Are Mo r p h o l o g i c a l D i f f e r e n c e s Between the Taxa St a b l e ? 113 7.14 Are V e g e t a t i v e Fragments V i a b l e Diaspores i n Limb e l l a ? 119 G. SUMMARY AND CONCLUSIONS .- 119 Chapter VIII TAXONOMIC TREATMENT 121 A. INTRODUCTION 121 B. TAXONOMIC TREATMENT 121 C. COLLECTIONS OF TAXONOMIC AND HISTORICAL SIGNIFICANCE .140 8.1 E a r l i e s t C o l l e c t i o n of L i m b e l l a t r i c o s t a t a 140 8.2 Type C o l l e c t i o n of Neckera t r i c o s t a t a S u l l 141 8.3 F i r s t C o l l e c t i o n of L. t r i c o s t a t a Sporophytes ....141 D. RATIONALE FOR TAXONOMIC DISPOSITION OF SUBSPECIES 142 E. CONCLUSION AND SUMMARY 143 Chapter IX PHYTOGEOGRAPHY 144 A. INTRODUCTION 144 B PRESENT D STRIBUTION 9.1 D i s t r i b u t i o n of L i m b e l l a t r i c o s t a t a ssp. t r i c o s t a t a 1 44 9.2 D i s t r i b u t i o n of L i m b e l l a t r i c o s t a t a ssp. f r y e i ...150 C. ORIGINS OF DISTRIBUTIONS 157 9.3 The O r i g i n Of Hawaiian P o p u l a t i o n s 157 1. R e l a t i o n of L. t r i c o s t a t a to South American L i m b e l l a 157 2. Did L i m b e l l a D i s p e r s e from South America to Hawaii? 158 9.4 The O r i g i n of North American P o p u l a t i o n s 162 1. Are Hawaiian and North American L i m b e l l a C l o s e l y Related? 162 2. Was L i m b e l l a Capable of Long-distance D i s p e r s a l from Hawaii to North America? .....162 D. DISCUSSION 165 9.5 Evidence Supporting Long-distance D i s p e r s a l in L i m b e l l a 165 1. Are L i m b e l l a Spores Capable of Long-distance D i s p e r s a l ? 168 2. Could L i m b e l l a Have Immigrated Into New Ha b i t a t s S u c c e s s f u l l y ? 169 9.6 A l t e r n a t i v e Hypotheses Concerning D i s p e r s a l to North America 172 1. Was D i s p e r s a l P r e g l a c i a l ? 172 2. Is North American L i m b e l l a Paleoendemic? ....174 3. D i d L i m b e l l a D i s p e r s e from North America to Hawaii? 174 4. Are Hawaiian and North American L i m b e l l a V i c a r i a d s ? 175 5. Was North American L i m b e l l a Introduced by Man? 1 76 E. CONCLUSION AND SUMMARY 176 Chapter X SUMMARY AND CONCLUSIONS 178 A. REVIEW OF EXPERIMENTAL EVIDENCE 178 1. Mo r p h o l o g i c a l D i f f e r e n c e s 178 2. Karyotype D i f f e r e n c e s 178 3. Isozyme D i f f e r e n c e s 178 4. C u l t i v a t i o n D i f f e r e n c e s 179 B. ANSWERS TO QUESTIONS POSED IN CHAPTER 1 179 10.1 Are Hawaiian and North American L i m b e l l a C o n s p e c i f i c ? 179 10.2 What E x p l a i n s the Geographical D i s t r i b u t i o n of the Taxa? 180 C. CONCLUSION ...180 LITERATURE CITED 182 APPENDIX A - SEQUENCE OF SSP. TRICOSTATA COLLECTIONS, AS REPRESENTED IN HERBARIA 199 APPENDIX B - PLANT SPECIES LIST, LIMBELLA HABITAT AT SUTTON LAKE , LANE COUNTY , OREGON . 200 APPENDIX C - PLANT AND ANIMAL REMAINS IN FILTRATE FROM WASHINGS OF SSP. FRYEI SPECIMENS 201 APPENDIX D - MORPHOLOGICAL CHARACTERS MEASURED IN SSP. TRICOSTATA AND SSP. FRYEI. 202 APPENDIX E - BRANCH BUD FREQUENCY DATA MATRIX. 1-20 = SSP. TRICOSTATA, 21-37 = SSP. FRYEI 203 APPENDIX F - DISCRIMINANT AND MAHALANOBIS DISTANCE ANALYSIS OF BRANCH BUD FREQUENCY DATA 204 APPENDIX G - PHENETIC DATA MATRIX. 1-12 = SSP. TRICOSTATA, 13-24 = SSP. FRYEI 205 APPENDIX H - DISCRIMINANT AND MAHALANOBIS DISTANCE ANALYSIS OF PHENETIC DATA 206 APPENDIX I - CORRELATION MATRIX OF PHENETIC DATA 207 APPENDIX J - ABSOLUTE AND RELATIVE CHROMOSOME LENGTHS. 1-154 = SSP. TRICOSTATA, 155-275 = SSP. FRYEI 208 APPENDIX K - DISCRIMINANT AND MAHALANOBIS DISTANCE ANALYSIS OF ABSOLUTE CHROMOSOME LENGTHS 209 APPENDIX L - CORRELATION MATRIX OF ABSOLUTE CHROMOSOME LENGTHS 210 APPENDIX M - RECIPES AND SOURCES FOR ELECTROPHORETIC STARCH GELS AND STAINS * 211 APPENDIX N - NORTH AMERICAN HABITATS SEARCHED FOR LIMBELLA. 215 ix L i s t of Tables 1. Diameters and ages of Pyrus fusca and S a l i x hookeriana i n L i m b e l l a h a b i t a t at Sutton Lake, Lane County, Oregon. 27 2. L i v i n g m a t e r i a l of L i m b e l l a used i n r e g e n e r a t i o n s t u d i e s 98 3. D r i e d m a t e r i a l used in re g e n e r a t i o n study 100 4. Developmental stages of s p o r e l i n g c u l t u r e s 104 5. Developmental stages of s i n g l e l e a f r e g e n e r a t i o n . ...107 6. Developmental stages of shoot t i p rege n e r a t i o n 108 7. Developmental stages of 1-8 cm shoot segment regeneration 109 8. R e l a t i v e s t a b i l i t y of morphological c h a r a c t e r s i n c u l t i v a t e d bryophytes, as reported i n l i t e r a t u r e . ...115 9. P h y s i c a l parameters i n f l u e n c i n g e x p r e s s i o n of morphological c h a r a c t e r s , as repo r t e d i n l i t e r a t u r e . 117 X L i s t of F i g u r e s 1. Temperature and p r e c i p i t a t i o n r e c o r d s . f o r three Hawaiian s t a t i o n s 12 2. Ssp. t r i c o s t a t a and h a b i t a t , Kawaikoi Stream t r i b u t a r y at A l a k a i T r a i l c r o s s i n g , Na P a l i - K o n a F o r e s t Reserve, Kauai , Hawai i 18 3. Temperature and p r e c i p i t a t i o n r e c o r d s f o r two c o a s t a l Oregon s t a t i o n s . A. - North Bend (m o d i f i e d from Loy 1976). B. - Newport (modified from Cooper 1958) 20 4. Ssp. f r y e i h a b i t a t , Sutton Lake, Lane County, Oregon. 25 5. Ssp. f r y e i on decaying l i t t e r and l i v i n g t r e e r o o t s , Sutton Lake. Scale = 1 dm 26 6. Branch bud f r e q u e n c i e s i n ssp. t r i c o s t a t a and ssp. f r y e i , per l e a f segment number 42 7. S c a t t e r p l o t of measurements of 28 morphological c h a r a c t e r s . T r i a n g l e = ssp. t r i c o s t a t a , c l o s e d c i r c l e = ssp. f r y e i 44 8. S c a t t e r p l o t of measurements of 7 morphological c h a r a c t e r s , a f t e r s e l e c t i o n by d i s c r i m i n a n t a n a l y s i s . T r i a n g l e = ssp. t r i c o s t a t a , c l o s e d c i r c l e = ssp. f r y e i . 45 9. Leaf i n s e r t i o n angles p l o t t e d a g a i n s t longest basal l e a f c e l l l e n g t h s . T r i a n g l e = ssp. t r i c o s t a t a , c l o s e d c i r c l e = ssp. f r y e i 47 10. Leaf i n s e r t i o n angles p l o t t e d a g a i n s t stem diameters. T r i a n g l e = ssp. t r i c o s t a t a , c l o s e d c i r c l e = ssp. f r y e i . 48 11. Sources of l i v i n g p l a n t s of ssp. t r i c o s t a t a used f o r l a b o r a t o r y experiments 54 12. Chromosome f i g u r e s of ssp. t r i c o s t a t a ( C h r i s t y 5368 ). 59 13. Chromosome f i g u r e s of ssp. f r y e i ( C h r i s t y 5505 ). ...60 14. Ssp. t r i c o s t a t a chromosome alignments. 1-10 - Kauai, C h r i s t y 5368. 11-14 - Kauai, C h r i s t y 5337 62 15. Ssp. f r y e i chromosome alignments. 1-6 - Oregon, C h r i s t y x i 5499. 7-11 - Oregon, C h r i s t y 5505 63 16. Absolute chromosome lengths, p l o t t e d a g a i n s t r e l a t i v e l e ngths, a l l chromosomes measured 64 17. Average a b s o l u t e chromosome lengths p l o t t e d a g a i n s t average r e l a t i v e l e n g t h s . T r i a n g l e = ssp. t r i c o s t a t a . Closed c i r c l e = ssp. f r y e i 65 18. Graph of average r e l a t i v e l e n g t h s of chromosome complements of ssp. t r i c o s t a t a and ssp. f r y e i 66 19. Zymograms of ssp. t r i c o s t a t a and ssp. f r y e i . Dark zones = enzymes s t a i n e d f o r ; white zones = SOD a c t i v i t y . A l l g e l s run i n Ridgway b u f f e r system 78 20. Phenolic spot p a t t e r n s on two-dimensional chromatograms. 86 21. Phenolic spot p a t t e r n s of s e q u e n t i a l l y - s p o t t e d f r a c t i o n s on one-dimensional chromatograms 87 22. Means of a t t a c h i n g shoot segments to f i l t e r paper, for shoot o r i e n t a t i o n experiment 102 23. Growth r a t e s of s p o r e l i n g c u l t u r e s , by d e n s i t y c l a s s . A. - 2 s p o r e l i n g s cm" 2. B. - 1 s p o r e l i n g cm - 2. C. - 43 s p o r e l i n g s cm" 2. D. - 27 s p o r e l i n g s cm" 2 106 24. Shoot o r i e n t a t i o n experiment. Ssp. t r i c o s t a t a on l e f t , ssp. f r y e i on r i g h t 114 25. Ssp. t r i c o s t a t a , dendroid p l a n t s . Note monopodial branching ( C h r i s t y 5196 ) 129 26. Ssp. t r i c o s t a t a , t r a i l i n g a q u a t i c p l a n t s . Branches spread to show branching p a t t e r n ( C h r i s t y 5245 ). ..130 27. Ssp. t r i c o s t a t a , upper branch l e a v e s . (Fosberg 10233 ). 131 28. Ssp. t r i c o s t a t a . A. - upper l e a f c e l l s . B. - b a s a l l e a f c e l l s (both Fosberg 10233 ) 132 29. Ssp. f r y e i , dendroid p l a n t s . Note monopodial branching ( C h r i s t y 5334 ) 136 30. Ssp. f r y e i , t r a i l i n g p l a n t s ( C h r i s t y 5338 ) 1 37 31. Ssp. f r y e i , upper branch leaves ( Frye s.n.) 138 32. Ssp. f r y e i . A. - upper l e a f c e l l s . B. - b a s a l l e a f c e l l s (both C h r i s t y 5338 ) 139 x i i 33. Surface and e l e v a t i o n a l d i s t r i b u t i o n of ssp. t r i c o s t a t a on Hawaii, Hawaii 145 34. Surface and e l e v a t i o n a l d i s t r i b u t i o n of ssp. t r i c o s t a t a on Kauai, Hawaii 146 35. Surface and e l e v a t i o n a l d i s t r i b u t i o n of ssp. t r i c o s t a t a on Maui, Hawaii 147 36. Surface and e l e v a t i o n a l d i s t r i b u t i o n of ssp. t r i c o s t a t a on Molokai, Hawaii 148 37. Surface and e l e v a t i o n a l d i s t r i b u t i o n of ssp. t r i c o s t a t a on Oahu, Hawaii 149 38. S i t e of e x i s t i n g ssp. f r y e i p o p u l a t i o n at Sutton Lake, Lane County, Oregon 151 39. T.C. Frye's map and note d e s c r i b i n g the type l o c a l i t y of Sciaromium f r y e i W i l l i a m s 153 40. Type l o c a l i t y of Sc iaromium f r y e i W i l l i a m s , Barview, Coos County, Oregon 154 41. S i t e s of h a b i t a t s searched f o r ssp. f r y e i . SL = Sutton Lake. B = Barview 156 42. World d i s t r i b u t i o n of L i m b e l l a 159 Acknowledgement I extend warm and h e a r t f e l t thanks to my s u p e r v i s o r , W i l f r e d B. S c h o f i e l d , f o r the moral and l o g i s t i c a l support and ex t e n s i v e b r y o l o g i c a l - b i b l i o g r a p h i c a l e x p e r t i s e given so f r e e l y throughout my time at UBC. I am a l s o indebted to many members of the UBC Botany Department, without whose a s s i s t a n c e and encouragement t h i s t h e s i s c o u l d not have been completed. S p e c i a l thanks are due to K.M. Cole, W.J. Hunter, T.T. Mcintosh, K.W. N i c h o l l s , K. R i t l a n d , J.R. Spence, N.L. Vogt and R.E. de Wreede, and to my committee members F.R. Ganders and B.A. Bohm. Thanks are a l s o due to W.J. Hoe f o r a s s i s t a n c e i n Hawaii. 1 TO David Dwight Baldwin (1831-1912), whose d i s c o v e r y l a y unknown for 105 years and Stephen Fowler C h r i s t y (1910-1982), whose love of enigmata l e d me t h i t h e r . 2 I. INTRODUCTION A. SCOPE, PURPOSE AND OBJECTIVES OF STUDY Th i s i n v e s t i g a t i o n was c a r r i e d out to r e s o l v e taxonomic and phytogeographical q u e s t i o n s r e g a r d i n g the taxonomic l i m i t s of the Hawaiian moss Sc iaromium t r i c o s t a t u m ( S u l l . ) M i t t . and the i d e n t i t y of Sc iaromium f r y e i W i l l i a m s of c o a s t a l northwestern North America (Oregon). Seven areas of i n v e s t i g a t i o n were used to determine i d e n t i t i e s and l i m i t s of the taxa and to provide a phyt o g e o g r a p h i c a l e x p l a n a t i o n of t h e i r present d i s t r i b u t i o n . B. STUDY MATERIAL L i m b e l l a i s a pleurocarpous moss genus in the f a m i l y Amblystegiaceae. L i m b e l l a t r i c o s t a t a ssp. t r i c o s t a t a i s aqu a t i c to semi-aquatic i n mountain streams and i s common on the f i v e major Hawaiian i s l a n d s . P l a n t s are t r a i l i n g or dendroid, up to 0.5 m long ( F i g s . 25,26). L i m b e l l a t r i c o s t a t a ssp. f r y e i grows i n a dense shrub-carr i n c o a s t a l Oregon. P l a n t s are dendroid to o c c a s i o n a l l y t r a i l i n g ( F i g s . 29,30). 3 C. TAXONOMIC HISTORY 1.1 Hawai ian P l a n t s L i m b e l l a t r i c o s t a t a ( S u l l . ) C M . was d e s c r i b e d as Neckera  t r i c o s t a t a S u l l . ( S u l l i v a n t 1854) and has been known r e c e n t l y as Sc iaromium t r i c o s t a t u m ( S u l l . ) M i t t . U n t i l 1971 i t was c o n s i d e r e d endemic to the Hawaiian I s l a n d s . Both male and female p l a n t s were known but sporophytes had never been r e p o r t e d i n the l i t e r a t u r e , even though they had been c o l l e c t e d as e a r l y as 1875 ( C h r i s t y 1980). Seven synonyms were c r e a t e d by v a r i o u s authors between 1889-1912, and over the years the taxon had been as s i g n e d to 5 d i f f e r e n t genera i n 3 d i f f e r e n t f a m i l i e s . 1.2 Oregon P l a n t s L i m b e l l a t r i c o s t a t a ssp. f r y e i (Williams) C h r i s t y comb. nov. was d e s c r i b e d as Sc iaromium f r y e i W i l l i a m s from a s i n g l e c o l l e c t i o n (Williams 1933). A second p o p u l a t i o n was d i s c o v e r e d i n 1978 ( C h r i s t y 1980). Lawton (1971) reduced Sc iaromium f r y e i to a synonym of S_^  t r i c o s t a t u m . 4 D. PROBLEMS IN NEED OF INVESTIGATION 1. Taxonomic P o s i t ion of Sc iaromium f r y e i Based on  L i m i t e d Data Lawton's (1971) d e c i s i o n to submerge Sc iaromium f r y e i as a synonym of S^ t r i c o s t a t u m was based s o l e l y on morphological grounds. U n f o r t u n a t e l y , she d i d not i n d i c a t e the number of specimens upon which she had based her o p i n i o n . At that time, WTU would have had only 2 specimens of S_^  t r i c o s t a t u m ( Neal s.n., 3.xi.l933; S v i h l a 2309 ) and 2 i s o t y p e s of S_^  f r y e i • Lawton would have had to borrow a d d i t i o n a l m a t e r i a l or base her d e c i s i o n on a l i m i t e d number of specimens. Although Hawaiian and Oregon p l a n t s are s u p e r f i c i a l l y s i m i l a r — even m i c r o s c o p i c a l l y -- apparent d i f f e r e n c e s i n s i z e , growth form, branching p a t t e r n and h a b i t a t c a l l e d f o r a r e - e v a l u a t i o n of t h e i r taxonomic r e l a t i o n s h i p . 2. D i s p a r i t y of Hawaiian and North American H a b i t a t s Habitat c o n d i t i o n s f o r L i m b e l l a i n Hawaii and Oregon are i n most -respects s t r i k i n g l y d i s s i m i l a r . Hawaiian p l a n t s u s u a l l y grow submerged or emergent on rocks i n streambeds. In c o n t r a s t , p l a n t s of the e x i s t i n g Oregon p o p u l a t i o n grow on wet peat, bark and r o t t e n wood, and no rocks are present i n the h a b i t a t . Accompanying v e g e t a t i o n of the two areas i s completely d i s s i m i l a r . The Hawaiian p l a n t s are most common between 1200-5 2130 m e l e v a t i o n , whereas Oregon p l a n t s grow at sea l e v e l . Although i t i s not uncommon f o r p l a n t s at the edge of t h e i r range to grow on s u b s t r a t a d i f f e r e n t from those occupied i n the center of t h e i r range, the d i s p a r i t y i n Sc iaromium t r i c o s t a t u m and £L f r y e i h a b i t a t s , c o u p l e d with other apparent d i f f e r e n c e s , r a i s e d the q u e s t i o n of whether they were r e a l l y c o n s p e c i f i c as Lawton had proposed. 3. D i s t r i b u t i o n i n North America Lawton (1971) proposed that the Oregon p o p u l a t i o n of Sc iaromium f r y e i had been i n t r o d u c e d by man from Hawaii. Discovery of a second p o p u l a t i o n i n 1978, i n an undisturbed h a b i t a t 80 km from the type l o c a l i t y , r a i s e d c o n s i d e r a b l e doubt that the p l a n t s had been i n t r o d u c e d . I t was a l s o u n l i k e l y that Sc iaromium t r i c o s t a t u m would be used for hobby or commercial purposes, the usual means by which a l i e n p l a n t s are introduced i n t o new g e o g r a p h i c a l a r e a s . I t seemed more l i k e l y that S.  f r y e i was a r a r e element of the n a t i v e moss f l o r a of the P a c i f i c Northwest. I n v e s t i g a t i o n of the d i s t r i b u t i o n a l anomaly c o u l d provide answers concerning the o r i g i n and s t a t u s of S_^  f r y e i , e s p e c i a l l y because the s p e c i e s was so p o o r l y known and had never been s t u d i e d c r i t i c a l l y . 6 E. QUESTIONS TO BE ANSWERED The problems o u t l i n e d above r a i s e d two s p e c i f i c q u e s t i o n s to be answered by the r e s e a r c h : 1. Were Sc iaromium t r i c o s t a t u m and S^ f r y e i c o n s p e c i f i c ? 2. What ex p l a i n e d t h e i r g e o g r a p h i c a l d i s t r i b u t i o n ? F. METHODS USED TO ANSWER QUESTIONS 1.3 Methods Used In order to answer the above q u e s t i o n s , the taxa were compared i n s i x areas of i n v e s t i g a t i o n : (1) h a b i t a t (2)' morphology ( i n c l u d i n g scanning e l e c t r o n microscopy and m u l t i v a r i a t e a n a l y s i s ) (3) karyotype morphology (4) isozyme banding p a t t e r n s (5) ph e n o l i c chromatography and (6) experimental c u l t i v a t i o n . Evidence from these areas of i n v e s t i g a t i o n was s y n t h e s i z e d to formulate taxonomic and phytogeographical treatments to answer the above q u e s t i o n s . 1.4 R a t i o n a l e f o r Methods Used Because W i l l i a m s ' (1933) d e s c r i p t i o n of Sc iaromium f r y e i seemed t o be based more on ge o g r a p h i c a l than on morphological d i f f e r e n c e s , and because Lawton's (1971) combination of S.  f r y e i and S_^  t r i c o s t a t u m seemed l e s s c o n v i n c i n g upon r e -7 examination of the problem, i t was d e s i r a b l e to apply, i n t h i s study, methods in a d d i t i o n to the t r a d i t i o n a l morphological approach, i n order to o b t a i n a d d i t i o n a l i n f o r m a t i o n with which to make taxonomic d e c i s i o n s . Although the morphological approach probably w i l l remain the cornerstone of bryotaxonomy, other taxonomic methods have begun to be a p p l i e d to bryophytes d u r i n g the l a s t 20 y e a r s . These methods i n c l u d e b i o c h e m i s t r y , c u l t i v a t i o n , c y t o l o g y , g e n e t i c s , numerical taxonomy and experimental p h y s i o l o g y , methods c o l l e c t i v e l y c a l l e d " b i o s y s t e m a t i c s " , which in botany were f i r s t a p p l i e d to v a s c u l a r p l a n t s y s t e m a t i c s . G. BIOSYSTEMATICS OF BRYOPHYTES Bi o s y s t e m a t i c s i s g e n e r a l l y d e f i n e d as an "experimental, b i o l o g i c a l l y based" approach to systematics (Vickery 1984), s y n t h e s i z i n g data from one or more of the methods l i s t e d above in c o n t r a s t to t r a d i t i o n a l taxonomy based s o l e l y on morphology — to c l a s s i f y "a group of organisms [and e l u c i d a t e ] the e v o l u t i o n a r y processes l e a d i n g to i t s d i f f e r e n t i a t i o n " (Longton 1982). Anderson's (1964) review of moss b i o s y s t e m a t i c s was l i m i t e d to cytology.. Other reviews (Anderson 1963; S t o t l e r 1976; Smith 1978; Koponen 1978; Szweykowski 1978, 1984; Longton 1982; Wyatt and Stoneburner 1984) i n c l u d e d a wider range of b i o s y s t e m a t i c methods in bryotaxonomy, and s p e c u l a t e d on the reasons why b i o s y s t e m a t i c s t u d i e s of bryophytes have lagged behind those of v a s c u l a r p l a n t s . 8 Longton (1982) a s t u t e l y observed that " v a r i o u s b i o s y s t e m a t i c techniques have a l l too o f t e n been a p p l i e d piecemeal to d i f f e r e n t bryophyte groups: our s t u d i e s have become i n c r e a s i n g l y b i o l o g i c a l but not yet s u f f i c i e n t l y s y s t e m a t i c . . . bryophyte systematics c o u l d most e f f e c t i v e l y be advanced by the a p p l i c a t i o n of a comprehensive approach [us i n g an a r r a y of b i o s y s t e m a t i c techniques s i m u l t a n e o u s l y ] to s e l e c t e d groups." Although the volume of p u b l i s h e d r e s e a r c h a p p l y i n g one or two b i o s y s t e m a t i c methods to a group of bryophytes i s now r e l a t i v e l y l a r g e , I know of only two s t u d i e s — both i n v o l v i n g l i v e r w o r t s -- that have used more than three such methods to solve s p e c i f i c taxonomic problems. Based on evidence from isozyme (Krzakowa and Szweykowski 1977; Krzakowa 1978, 1981; Szweykowski et a l . 1981; Z i e l i n s k i et a l . 1981), p h e n o l i c (Szweykowski and Krzakowa 1977), c y t o l o g i c a l (Mendelak 1981; Newton 1981) and s e r o l o g i c a l i n v e s t i g a t i o n s (Szweykowski et a l . 1981; Z i e l i n s k i et a l . 1981), r e l a t i o n s h i p s between c e n t r a l European P e l l i a taxa have been c l a r i f i e d (Szweykowski 1984). In a d d i t i o n to morphological data, Zehr (1980) used evidence from t e r p e n o i d , p h e n o l i c , isozyme, c y t o l o g i c a l and c u l t i v a t i o n i n v e s t i g a t i o n s to r e d e f i n e three s e c t i o n s i n the genus Scapania. T h i s i n v e s t i g a t i o n of L i m b e l l a t r i c o s t a t a , using s i x b i o s y s t e m a t i c methods, was a s i m i l a r attempt to r e d e f i n e the l i m i t s of the taxon. Based on the c o n t r i b u t i o n s of b i o s y s t e m a t i c s to v a s c u l a r p l a n t taxonomy, i t i s c l e a r that b i o s y s t e m a t i c s t u d i e s can provide a d d i t i o n a l i n f o r m a t i o n which can a l t e r or augment, but 9 not n e c e s s a r i l y supplant, systematic i n t e r p r e t a t i o n s based on t r a d i t i o n a l morphological a n a l y s i s . 10 I I . HABITAT A. INTRODUCTION T h i s chapter d e s c r i b e s the p h y s i c a l and f l o r i s t i c c h a r a c t e r i s t i c s of the Hawaiian and North American h a b i t a t s of L i m b e l l a . A n a l y s i s of these c h a r a c t e r i s t i c s emphasizes unexpected p h y s i c a l s i m i l a r i t i e s between the two l o c a l i t i e s and supports the assumption that ssp. f r y e i o r i g i n a t e d from long-d i s t a n c e d i s p e r s e d d i a s p o r e s of ssp. t r i c o s t a t a . B. HAWAIIAN ISLAND HABITATS 2.1 Climate of Hawai ian H a b i t a t s The Hawaiian I s l a n d s are renowned f o r t h e i r s u b t r o p i c a l c l i m a t e , i n f l u e n c e d by the P a c i f i c Ocean and the i s l a n d s ' l o c a t i o n at 20° north l a t i t u d e , both of which warm the r e g i o n a l a i r masses to produce m i l d temperatures year-round. However, d i v e r s e topography causes wide v a r i a t i o n i n temperature and p r e c i p i t a t i o n at any given p l a c e on the i s l a n d s . L i m b e l l a occurs most commonly in mountains between 1200-2130 m e l e v a t i o n , where p r e c i p i t a t i o n i s g r e a t e s t and temperatures c o o l e r than i n the lowlands. Annual temperature and p r e c i p i t a t i o n r e c o rds f o r three montane s t a t i o n s on three i s l a n d s — a l l near L i m b e l l a 11 l o c a l i t i e s -- are shown i n F i g . 1. In the mountains where L i m b e l l a i s most common, temperatures r a r e l y exceed 24°C or f a l l below 10°C. Data from these s i t e s show a r e d u c t i o n i n p r e c i p i t a t i o n from A p r i l to October, which c o i n c i d e s with the trade winds t y p i c a l of high-pressure a n t i c y c l o n i c weather systems. At t h i s time most r a i n f a l l i s o r o g r a p h i c , r e s t r i c t e d to higher mountain s l o p e s . Between November and March the montane s i t e s show an i n c r e a s e i n p r e c i p i t a t i o n , marked by the advent of c y c l o n i c storms of low-pressure weather systems. At t h i s time the r a i n f a l l i s widely d i s t r i b u t e d over the i s l a n d s , not r e s t r i c t e d to mountainous areas (Mink 1962; Blumenstock and P r i c e 1972; C a r l q u i s t 1980). In most windward mountains, where r a i n from both weather systems occurs, the annual r a i n f a l l i s more evenly d i s t r i b u t e d but s t i l l v a r i a b l e throughout the year ( F i g . 1C). Mt. Wa i a l e a l e , the h i g h e s t p o i n t on Kauai, r e c e i v e s over 12 m p r e c i p i t a t i o n a n n u a l l y , r e p o r t e d l y the r a i n i e s t s i t e known on e a r t h (Blumenstock and P r i c e 1972). At e l e v a t i o n s above 2100 m, annual p r e c i p i t a t i o n decreases to as l i t t l e as 380 mm (Blumenstock and P r i c e 1972). Maximum r a i n f a l l occurs between 610-1520 m, f r e q u e n t l y a s s o c i a t e d with c l o u d development beneath a trade wind i n v e r s i o n l a y e r forming between 1500-2100 m (Blumenstock and P r i c e 1972; C a r l q u i s t 1980; Whiteaker 1983). Mountain peaks are almost always shrouded i n c l o u d s . In t h i s zone, fog d r i p from condensation on v e g e t a t i o n c o n t r i b u t e s a p p r e c i a b l y to t o t a l p r e c i p i t a t i o n (Blumenstock and Pr i c e . 1 9 7 2 ) . During my v i s i t s to h a b i t a t s of L i m b e l l a on Maui and Kauai 12 F i g u r e 1 - Temperature and p r e c i p i t a t i o n records f o r three Hawaiian s t a t i o n s . A. - Makawao, Maui, e l e v . 640 m. B. - Kanalohuluhulu [Kokee], Kaui, e l e v . 1097 m. C. - Mountain View, Hawaii, e l e v . 466 m. A l l graphs m o d i f i e d from Armstrong 1983. 1 3 (June 1983, May 1984), water temperatures were 15-22°C and a i r temperatures 18-24°C. 2.2 S o i l s of Hawaiian H a b i t a t s The Hawaiian I s l a n d s are of igneous o r i g i n , having formed i n sequence over a "hot spot" near the c e n t e r of the n o r t h w e s t e r l y - s h i f t i n g P a c i f i c t e c t o n i c p l a t e . Most Hawaiian s o i l s are r e s i d u a l , having developed from the u n d e r l y i n g b a s a l t i c parent m a t e r i a l . The s o i l s are d i v e r s e , because the v a r y i n g age of d i f f e r e n t i s l a n d s determines the amount of weathering and s o i l formation which has o c c u r r e d at any one l o c a l i t y . Of the p r i n c i p a l e x i s t i n g i s l a n d s , Kauai, c o n s i d e r e d to to'be of P l i o c e n e age (5.7 m i l l i o n y e a r s ) , i s the o l d e s t and most i n t e n s e l y weathered, with the best-developed s o i l s . Hawaii i s the youngest i s l a n d , formed d u r i n g the P l e i s t o c e n e (450,000 years ago), and has the l e a s t - d e v e l o p e d s o i l s (Macdonald et a l . 1983). In the mountains, L i m b e l l a u s u a l l y grows on wet rocks, but o c c a s i o n a l l y i t i s found on s o i l i n i n t e r m i t t e n t stream beds or on peat i n boggy f o r e s t s . A h i s t o s o l s e r i e s i n Kauai's A l a k a i Swamp, a w e l l - c o l l e c t e d l o c a l i t y of L i m b e l l a , was d e s c r i b e d by Foote et a l . (1972) as mucky peat u n d e r l a i n by c l a y and b a s a l t , "extremely a c i d " (probably pH 4.5-5.0) and very p o o r l y d r a i n e d , o c c u r r i n g on mountaintops, r i d g e s and steep s l o p e s . A l a t o s o l a s s o c i a t i o n near O l i n d a , another w e l l - c o l l e c t e d l o c a l i t y f o r L i m b e l l a on Maui, was d e s c r i b e d by Foote et a l . (1972) as a 14 deep, w e l l - d r a i n e d s o i l of moderately f i n e t e x t u r e on s l o p i n g to very steep ground. Sherman (1972) d e s c r i b e d s o i l s of t h i s l a t o s o l group as a c i d (pH 5.0), d e r i v e d from v o l c a n i c ash. De Wreede (1967) d e s c r i b e d montane l a t o s o l s with a pH of 4.5-5.5. 2.3 Hydrology of Hawaiian H a b i t a t s L i m b e l l a u s u a l l y occurs i n p e r e n n i a l and i n t e r m i t t e n t streams, and u s u a l l y has been c o n s i d e r e d a q u a t i c . Most herbarium specimens were d e s c r i b e d as e i t h e r a q u a t i c or emergent i n p e r e n n i a l streams or exposed i n dry beds of i n t e r m i t t e n t streams. For a q u a t i c p l a n t s , c o l l e c t o r s d e s c r i b e d stream flows as s w i f t , slow or non-flowing, and water temperatures as c o l d to warm. The volume of water i n streams i s extremely v a r i a b l e . In ge n e r a l , the y o u t h f u l s o i l s are h i g h l y permeable and p e r e n n i a l streams are r a r e i n many p a r t s of the i s l a n d s (Stearns 1966). Surface r u n o f f d u r i n g moderate r a i n f a l l may be low, but may incr e a s e r a p i d l y d u r i n g heavy r a i n s , i r r e g u l a r l y r a i s i n g stream l e v e l s and sometimes causing f l a s h f l o o d i n g (Mink 1962; Stearns 1966; Macdonald et a l . 1983). The g r e a t e s t volume changes occur i n f i r s t - and second-order streams (sensu Horton 1945) that are f r e q u e n t l y i n t e r m i t t e n t , p e r i o d i c a l l y soaking L i m b e l l a p o p u l a t i o n s that otherwise e x i s t as t e r r e s t r i a l , non-aquatic p l a n t s . On rocks i n p e r e n n i a l streams, L i m b e l l a forms mats which a b r u p t l y cease 10-15 cm above water l i n e ( F i g . 2). I was unable t o o b t a i n pH measurements from these streams, but Maciolek (1967) recorded pH 6.0-7.0 i n small l a k e s at 2040 m, 1 5 and running streams at t h i s e l e v a t i o n presumably have a s i m i l a r pH. 2.4 V e g e t a t i o n of Hawai ian H a b i t a t s L i m b e l l a h a b i t a t s are g e o g r a p h i c a l l y , t o p o g r a p h i c a l l y , c l i m a t o l o g i c a l l y and e d a p h i c a l l y d i v e r s e . L i m b e l l a i s known from Hawaii, Kauai, Maui, Molokai and Oahu, from e l e v a t i o n s of 90 to over 2130 m (Chapter 9) and from h a b i t a t s r e c e i v i n g 760-12,000 mm p r e c i p i t a t i o n . As a r u l e , only recent c o l l e c t o r s (Appendix A) have pr o v i d e d adequate d e s c r i p t i o n s of v e g e t a t i o n a s s o c i a t e d with L i m b e l l a . However, v e g e t a t i o n of the h a b i t a t s can be d e s c r i b e d i n a general way because most c o l l e c t i o n s are from s i t e s at 1200-2130 m e l e v a t i o n , where the v e g e t a t i o n i s f a i r l y uniform. Based on my o b s e r v a t i o n s from Maui and Kauai, and data from recent herbarium c o l l e c t i o n s , the most t y p i c a l h a b i t a t of L i m b e l l a i s one of s e v e r a l M e t r o s i d e r o s polymorpha Gaud. f o r e s t communities d e s c r i b e d i n part by K r a j i n a (1963) and Whiteaker (1983). Common a s s o c i a t e s , depending on g r a z i n g d i s t u r b a n c e and e l e v a t i o n , are A c a c i a koa Gray, S a d l e r i a cyatheoides K a u l f . , S t y p h e l i a tameiameia (Cham.) F. M u e l l . , Coprosma, Cheirodendron, D r y o p t e r i s , and Athyrium. One l o w - e l e v a t i o n s i t e on Molokai was d e s c r i b e d as "mesic Eugenia cumini-Schinus f o r e s t " ( Bishop s.n., 22.i.1967, BISH). T h i s i s of i n t e r e s t because E^ cumini (L.) Druce and Schinus are weedy, int r o d u c e d s p e c i e s , t y p i c a l of low e l e v a t i o n s where n a t i v e p l a n t s have been 1 6 re p l a c e d almost completely by an a l i e n f l o r a ( v i d . Forbes 1911). At s e v e r a l p l a c e s up to 2000 m, e x o t i c t r e e s such as Cryptomeria j a p o n i c a D. Don and Euc a l y p t u s are the dominant spec i e s . L i m b e l l a most commonly grows on rocks i n streambeds and along the banks. I t occurs o c c a s i o n a l l y on t r e e s , t r e e r o o t s , s o i l and wood. P l a n t s a l s o have been c o l l e c t e d from man-made i r r i g a t i o n d i t c h e s and wooden flumes. On Maui I observed s e v e r a l p o p u l a t i o n s i n i n t e r m i t t e n t streams near the headwaters of Waikamoi Stream, NW slope of Ha l e a k a l a . In one, L i m b e l l a was r e s t r i c t e d to sheets of bedrock, the p l a n t s o c c u r r i n g (a) i n the shade of overhanging f e r n s or shrubs a l o n g the bank (b) on the lowermost exposures of bedrock, where r e c e d i n g waters accumulate and (c) around small pools of water remaining i n bedrock d e p r e s s i o n s . In another i n t e r m i t t e n t stream among dense understory v e g e t a t i o n , the p l a n t s grew on damp s o i l and no rocks were present i n the streambed. There was no evidence of pooled water. In both cases, M e t r o s i d e r o s canopy cover was 75-80 %, fern and shrub cover 20-85 %-. In p e r e n n i a l , t h i r d - o r d e r streams on Kauai (e.g., Kawaikoi and Mohihi Streams, Na Pali-Kona For e s t Reserve), L i m b e l l a i s abundant, i n p l a c e s c o v e r i n g a l l submerged rocks and choking tens of meters of stream channel with u n d u l a t i n g shoots up to 0.5 m long ( F i g . 2 ) . Here, i n streambeds 4-20 m wide, most of the L i m b e l l a i s a q u a t i c and i t i s o f t e n mixed with filamentous green a l g a e . M e t r o s i d e r o s canopy cover ranges from 0-30 %. P e r e n n i a l , f i r s t - o r d e r streams on Kauai (e.g., near foot 1 8 F i g u r e 2 - Ssp. t r i c o s t a t a and h a b i t a t , Kawaikoi Stream t r i b u t a r y at A l a k a i T r a i l c r o s s i n g , Na Pali-Kona F o r e s t Reserve, Kauai, Hawaii. 19 Pihea T r a i l and near gauging s t a t i o n at foot of Koaie-Waialae T r a i l , Na Pali-Kona F o r e s t Reserve) o f t e n occupy narrow g u l l i e s d ensely shaded by overhanging c l i f f s or v e g e t a t i o n . L i m b e l l a i s emergent, on moist rocks i n spray zones along the streambank, or a q u a t i c on submerged rocks i n the stream. Biomass i s never as great as i n h i g h e r - o r d e r streams. C. NORTH AMERICAN HABITATS 2.5 Climate of North American H a b i t a t s The c l i m a t e of c o a s t a l Oregon i s both oceanic and Mediterranean, i n f l u e n c e d by the P a c i f i c Ocean, which moderates summer and winter temperatures. P r e c i p i t a t i o n i s markedly seas o n a l , most o c c u r r i n g between October and A p r i l , caused by the presence of low-pressure weather systems o f f s h o r e , which form extended and o c c a s i o n a l l y v i o l e n t c y c l o n i c storms. High-p r e s s u r e weather systems i n the summer maintain c l e a r s k i e s , and p r e c i p i t a t i o n i s n e g l i g i b l e i n J u l y and August. Annual temperature and p r e c i p i t a t i o n records f o r North Bend (11 km northeast of the e x t i n c t p o p u l a t i o n of ssp. f r y e i at Barview) and Newport (70 km n o r t h of the e x i s t i n g p o p u l a t i o n of ssp. f r y e i at Sutton Lake) are shown i n F i g . 3. Summer drought occurs on the coast, but temperatures r a r e l y exceed 20°C and c o l d fogs are frequent. In winter, temperatures seldom f a l l below 5°C and s n o w f a l l i s r a r e . Cooper (1958) repo r t e d only 47 c l e a r days i n one year, the remainder with fog on 58 days, r a i n 20 F i g u r e 3 - Temperature and p r e c i p i t a t i o n records f o r two c o a s t a l Oregon s t a t i o n s . A. - North Bend (modified from Loy 1976). B. - Newport ( m o d i f i e d from Cooper 1958). 21 on 68, c l o u d and fog on 78 and c l o u d on 115. Both Cooper (1958) and Loy (1976) observed l i t t l e c l i m a t i c v a r i a t i o n at c o a s t a l s t a t i o n s of d i f f e r e n t l a t i t u d e s , the only general t r e n d being a gradual i n c r e a s e i n p r e c i p i t a t i o n and c o o l i n g of temperatures with i n c r e a s i n g l a t i t u d e . In c o n t r a s t , an abrupt change i n c o n d i t i o n s occurs only a few km i n l a n d : both maximum and minimum temperatures become more extreme, p r e c i p i t a t i o n i n c r e a s e s , and the c o o l i n g summer fogs are absent. During v i s i t s to Sutton Lake i n June 1983 and May 1984, water temperatures were 14-19°C and a i r temperatures 14-16°C. 2.6 S o i l s of North American H a b i t a t s 1. Sutton Lake S o i l s Sutton Lake was formed when p o s t - g l a c i a l sand dune advances blocked a stream which d r a i n e d the Coast Range to the • east (Cooper 1958). The p o p u l a t i o n of L i m b e l l a developed on an a d j o i n i n g 1 m-deep bed of f i n e medium- to light-brown peat, the t h i c k n e s s of which was determined by probing with a 1 m range p o l e . The peat i s densely f i b r o u s at a depth of c. 5 dm, becoming l e s s f i b r o u s above and below. The f i b r o u s m a t e r i a l i s composed of sedge and root remains. A black, mucky humic l a y e r occupies the top 1-5 cm. D r i e d peat samples analyzed f o r pH f o l l o w i n g the method of L a v k u l i c h (1981), gave average readings (5 g peat: 30 ml d i s t i l l e d water) of 4.9 at the s u r f a c e humic l a y e r , 4.8 at 10 cm depth and 4.7 at 20 cm depth. Sand, g r a v e l 22 and rocks are absent. The peat and ancient dune d e p o s i t s are u n d e r l a i n by marine s i l t s t o n e and sandstone of Eocene age. 2. Barview S o i l s The Barview popul a t i o n o c c u r r e d on a marine t e r r a c e of l a t e P l e i s t o c e n e o r i g i n ( A l l e n and Baldwin 1944). The t e r r a c e i s one of many that occur along the P a c i f i c coast, formed in part by e u s t a t i c sea l e v e l changes d u r i n g g l a c i a l maxima and minima durin g the l a t e P l i o c e n e and P l e i s t o c e n e ( A l l e n and Baldwin 1944; Baldwin 1981). The t e r r a c e s are composed of ancient beach and o f f s h o r e d e p o s i t s , and are mostly sand with some c l a y , g r a v e l , peat l a y e r s and woody remains, u n d e r l a i n by marine sandstone of P l i o c e n e age (Griggs 1945). The sandy s o i l s of these t e r r a c e s are u s u a l l y c l a s s i f i e d as podsols or spodosols. T y p i c a l l y , an iron-cemented hardpan develops at depths of 30-76 cm (Jenny et a l . 1969), impeding drainage and forming v e r n a l or p e r e n n i a l p o o l s . B l a c k l o c k s o i l s , d e s c r i b e d from a s i m i l a r marine t e r r a c e 60 km S of Barview, are o l i g o t r o p h i c with a pH of 5.0 ( S o i l Conservation S e r v i c e 1970). In Frye's i s o t y p e (UBC) I found remains of a t r i c h o p t e r a n ( c a d d i s f l y ) l a r v a l case c o n s t r u c t e d e n t i r e l y of sand, s u p p o r t i n g evidence f o r l o c a t i o n of the type l o c a l i t y on the sandy marine t e r r a c e at Barview. 23 2.7 Hydrology of North American H a b i t a t s 1. Sutton Lake Hydrology At Sutton Lake the water l e v e l i s 1-15 cm below the peat s u r f a c e , s a t u r a t i n g the peat and forming small pools i n depressions even in dry summer months. S c a t t e r e d occurrence of the a q u a t i c or hygrophytic mosses F o n t i n a l i s and F i s s i d e n s on branches and t r e e boles i n the v i c i n i t y of f l o o d e d d e p r e s s i o n s , as w e l l as the presence of small b i v a l v e s , s n a i l s and t r i c h o p t e r a n l a r v a l cases i n d i c a t e abundant p e r e n n i a l water. Probably the e n t i r e community i s i r r i g a t e d by subsurface p e r c o l a t i o n from the a d j o i n i n g l a k e . Peak winter runoff presumably causes a seasonal r i s e i n lake l e v e l , but I have never seen the shrub-carr f l o o d e d , except f o r enlargement of pools i n s c a t t e r e d d e p r e s s i o n s , even in midwinter. 2. Barview Hydrology It may have been impeded drainage caused by a subsurface i r o n hardpan that c r e a t e d the "more or l e s s wet p a s t u r e " seen by Frye at Barview i n August 1922 (Williams 1933). T h i s assumption i s supported by the f a c t t h at the p a s t u r e was s t i l l wet i n August 1922, the d r i e s t p a r t of the "Mediterranean" summer t y p i c a l of c o a s t a l Oregon. Remains o f . t h e t r i c h o p t e r a n l a r v a l case i s f u r t h e r evidence that the h a b i t a t was wet f o r at l e a s t part of the year ( C h r i s t y 1980). 24 2.8 V e g e t a t i o n of North American H a b i t a t s 1. Sutton Lake V e g e t a t i o n The Sutton Lake p o p u l a t i o n occurs i n a shrub-carr dominated by mature S a l i x hookeriana B a r r a t t and Pyrus fusca Raf., that form a dense canopy 4-6 m high ( F i g . 4).- P i c e a s i t c h e n s i s (Bong.) C a r r . , Pinus c o n t o r t a Dougl. and Pseudotsuga menziesi i (Mirb.) Franco are s c a t t e r e d and p e r i p h e r a l i n the c a r r . The herb l a y e r i s dominated by Carex obnupta B a i l e y and s c a t t e r e d Lysichiturn americanum Hulten & S t . John. A complete species l i s t i s given i n Appendix B. To date, no h a b i t a t of t h i s kind on the P a c i f i c Coast has been d e s c r i b e d f o r m a l l y by p l a n t e c o l o g i s t s (e.g., F r a n k l i n and Dyrness 1973). Canopy cover of S a l i x and Pyrus i s 60-90 %, i n c i d e n t l i g h t on sunny days v a r y i n g at ground l e v e l from t w i l i g h t c o n d i t i o n s to dappled s u n l i g h t . Carex coverage i s 70-98 %, except f o r l a r g e r , densely-shaded pool areas where cover approaches 0 %. The Pyrus and S a l i x are commonly multiple-stemmed, the stems f r e q u e n t l y l e a n i n g or decumbent. Increment cores from 14 t r e e s (Table 1) gave a mean age of 37.5 years at 125 mm DBH ( Pyrus ) and 40 years at 111 mm DBH ( S a l i x ) . The t r e e s are covered with a dense growth of e p i p h y t i c bryophytes and Polypodium g l y c y r r h i z a D.C. Eat-. Based on o b s e r v a t i o n s at other s i t e s with s i m i l a r v e g e t a t i o n ( v i d . Appendix N), t h i s Pyrus / S a l i x / Carex a s s o c i a t i o n appears to be a s e r a i but l o n g - l i v e d community 26 Table 1 - Diameters and ages of Pyrus fusca and S a l i x  hookeriana i n L i m b e l l a h a b i t a t at Sutton Lake, Lane County, Oregon. PYRUS FUSCA SALIX HOOKERIANA Diameter (mm) Age (yr) Diameter (mm) Age (yr) 110 35 110 * 120 40 115 * 145 45 38 * 130 35 50 * 80 30 120 40 160 40 210 * 130 * 131 * x=125 x=37.5 X=110.6 x=40 * Data l a c k i n g ; increment cores not countable i n f i e l d . 28 dependent upon p e r e n n i a l l y waterlogged s o i l s , and p o s s i b l y in the case of S a l i x hookeriana, p r o x i m i t y to to marine s a l t spray. I n d i v i d u a l t r e e s appear to d i e a f t e r about 40 yea r s , c r e a t i n g gaps in the canopy that are f i l l e d by young Pyrus, S a l i x , and Spi raea, the Spi raea e v e n t u a l l y dying a f t e r having been overtopped by the t a l l e r v e g e t a t i o n . I observed no Pyrus, S a l i x or Spiraea r e p r o d u c t i o n under a mature canopy. The community i s t h e r e f o r e probably maintained by gap phase s u c c e s s i o n and the formerly common c o a s t a l f i r e s . In the shrub-carr, L i m b e l l a i s s c a t t e r e d but frequent, forming small but dense stands or e x t e n s i v e , dense sods at the bases of l i v i n g Pyrus and S a l i x , on decumbent l i v i n g stems, on dead l i t t e r f a l l and r o t t i n g stumps, and on l e a f y muck and peat at the edge of pools ( F i g . 5). Ra r e l y , p l a n t s c l i m b to 0.5 m on stems of shrubs and t r e e s . Although p l a n t s were observed submerged to depths of 10 cm i n pools and at the edge of Sutton Lake, and p l a n t s sometimes o c c u r r e d on shrub and t r e e stems below, or next t o, the aquatic or hygrophytic mosses F o n t i n a l i s and F i s s i d e n s , most p l a n t s occur on s l i g h t l y e l e v a t e d s i t e s : hummocks of peat, bases of shrub stems or f a l l e n but e l e v a t e d branches. In a l l cases the p l a n t s are wet and most occur at the lowest l e v e l of a e r i a l v e g e t a t i o n and l i t t e r f a l l . In c o n t r a s t to t r i c o s t a t a , which has strong r h i z o i d a l c onnections to the substratum, ssp. f r y e i p l a n t s are e a s i l y d i s l o d g e d . Sods of L i m b e l l a are e f f e c t i v e seedbeds f o r v a s c u l a r p l a n t s , as observed f o r other moss taxa (e.g. Holcombe 1976). S e e d l i n g s of Carex, Lycopus, Pyrus and Spiraea a l l sprouted from 29 sods of ssp. f r y e i d u r i n g growth chamber c u l t i v a t i o n (Chapter 7). 2. Barview V e q e t a t i o n Frye's Barview h a b i t a t i s known only as "more or l e s s wet pasture l a n d " (Williams 1933). Having so l i t t l e i n f o r m a t i o n to work with, and not f i n d i n g any pasture i n Barview, I chose to search f o r v e g e t a t i o n approximating that at Sutton Lake. There i s no Pyrus/ S a l i x c a r r i n Barview. At the time of my l a s t v i s i t (June 1983), a small stand of S a l i x hookeriana e x i s t e d i n the back yard of a house, but no L i m b e l l a grew there (S.D. Sundberg, p e r s . comm. 1980). E l v a Lawton, who searched u n s u c c e s s f u l l y f o r ssp. f r y e i at the type l o c a l i t y i n 1956 (Chapter 9), doubted that the h a b i t a t was s u i t a b l e (pers. comm. 1980): "Because the [Hawaiian] h a b i t a t had been d e s c r i b e d as of t e n i n streams with w a t e r f a l l s e t c . I thought that t h i s was an e n t i r e l y d i f f e r e n t h a b i t a t and not s u i t a b l e f o r the moss, and that i t might p o s s i b l y have been introduced and then d i e d out." C a r e f u l comparison of extraneous p l a n t l i t t e r i n my and Frye's c o l l e c t i o n s of L i m b e l l a y i e l d e d some important c l u e s concerning h a b i t a t s . The Sutton Lake c o l l e c t i o n s were washed i n tubs of water, the p l a n t s removed, and the wash water f i l t e r e d through Whatman f i l t e r paper with a Buchner funnel and vacuum f l a s k ( V o e g t l i n 1982). The Frye c o l l e c t i o n s were not washed, but l i t t e r from a l l i s o t y p e packets was removed and combined. The l i t t e r thus obtained from both l o c a l i t i e s was then examined 30 and s o r t e d under a d i s s e c t i n g microscope. Aside from numerous arthropods, the m a t e r i a l from Sutton Lake contained l i t t e r from a l l the dominant v a s c u l a r p l a n t s of the h a b i t a t , g i v i n g a good p r o f i l e of the v e g e t a t i o n type. L i t t e r from the Frye c o l l e c t i o n , though scanty, y i e l d e d a few c l u e s which support h i s "wet p a s t u r e " d e s c r i p t i o n (Appendix C). Based on t h i s evidence, the two h a b i t a t s are s t r i k i n g l y d i s s i m i l a r , the only s i m i l a r i t y being the presence of L i m b e l l a , water and T r i c h o p t e r a . Presence of grass culms, Rubus p a r v i f l o r u s Nutt. seeds and the absence of any of the p l a n t l i t t e r found at Sutton Lake, i n d i c a t e that ssp. f r y e i can occur i n at l e a s t two very d i f f e r e n t h a b i t a t s , one a c l o s e d , dense shrub-carr and the other an open "pasture". N e v e r t h e l e s s , i t i s p o s s i b l e that the Barview s i t e once may have been a shrub-carr which had been c l e a r e d p r i o r to F r y e ' s 1922 v i s i t . Although L i m b e l l a appears to have very s p e c i f i c h a b i t a t requirements, t y p i c a l of d i s j u n c t i v e s p e c i es ( S c h o f i e l d and Crum 1972), i t may be a b l e to s u r v i v e anthropogenic h a b i t a t a l t e r a t i o n which would be l e t h a l to v a s c u l a r p l a n t s ( S c h o f i e l d 1969). C u l t i v a t i o n experiments (Chapter 7) showed L i m b e l l a to be hardy and p o s s i b l y able to s u r v i v e the r a d i c a l change i n i n s o l a t i o n , temperature and humidity a s s o c i a t e d with sudden o v e r s t o r y removal, as long as an adequate water supply remained. 31 D. DISCUSSION Comparison of the above-mentioned p h y s i c a l and b i o t i c parameters of h a b i t a t s of L i m b e l l a i n Hawaii and Oregon r e v e a l s some s t r i k i n g s i m i l a r i t i e s . 2.9 Comparison of Climates The Hawaiian s t a t i o n s show 2 p r e c i p i t a t i o n minima i n s t e a d of 1 as shown f o r Oregon, but the seasonal d i s t r i b u t i o n i s remarkably s i m i l a r , both r e g i o n s showing a major r e d u c t i o n i n summer p r e c i p i t a t i o n . The higher t o t a l p r e c i p i t a t i o n at the Hawaiian s t a t i o n s i s probably of small consequence to L i m b e l l a because both Hawaiian and Oregon h a b i t a t s are well-watered and most remain wet throughout the year, e s s e n t i a l l y independent of amount of p r e c i p i t a t i o n , as long as the water s u p p l i e s are maintained. The Hawaiian h a b i t a t s are u s u a l l y shrouded i n cl o u d , and r a i n occurs almost d a i l y . The Oregon s t a t i o n s are cloudy f o r 10.5 months per year and r a i n y f o r 8 months per year. The Hawaiian and Oregon s t a t i o n s both show s i m i l a r seasonal temperature d i s t r i b u t i o n s , the curve of the Oregon s t a t i o n s being s l i g h t l y more pronounced because of g r e a t e r seasonal f l u c t u a t i o n a s s o c i a t e d with higher l a t i t u d e . Average annual Hawaiian temperatures range from 10-25°C, Oregon from 5-20°C. The seasonal range of both l o c a l i t i e s i s i d e n t i c a l (15°C), the Hawaiian s t a t i o n s being on average 5°C warmer than those i n Oregon. 32 2.10 Comparison of S o i l s There i s no Sutton Lake c o u n t e r p a r t f o r the Hawaiian l a t o s o l s and igneous rocks s e r v i n g as L i m b e l l a substratum. However, the organic Hawaiian h i s t o s o l s may be c h e m i c a l l y and s t r u c t u r a l l y s i m i l a r to the Sutton Lake peat, the A l a k a i Swamp h i s t o s o l s being c. pH 5.0 and the Sutton Lake peat pH 4.9. The Ol i n d a l a t o s o l s have a PH of 5.0, the Barview spodosols c. pH 5.0 ( B l a c k l o c k s o i l s ) . The only s i g n i f i c a n t s i m i l a r i t y may be low pH. 2.11 Comparison of Hydrology L i m b e l l a t r i c o s t a t a ssp. t r i c o s t a t a occurs i n well-watered h a b i t a t s , e i t h e r submerged i n p e r e n n i a l streams or p e r i o d i c a l l y inundated i n i n t e r m i t t e n t streams. In a d d i t i o n to p e r i o d i c wetting by f l u c t u a t i n g water l e v e l s , p l a n t s i n i n t e r m i t t e n t streams r e c e i v e moisture from the frequent r a i n f a l l , fog d r i p and high humidity a s s o c i a t e d with the c l o u d f o r e s t . f r y e i , though s u b j e c t e d to dry summers, i s hydrated by the p e r e n n i a l l y waterlogged peat substratum and a s s o c i a t e d high humidity. In both l o c a l i t i e s , water i n some form i s p e r e n n i a l l y a v a i l a b l e and p l a n t s are hydrated throughout the year. 33 2.12 Comparison of V e q e t a t i o n Although there i s no f l o r i s t i c c o r r e l a t i o n between the Hawaiian and Oregon v e g e t a t i o n , the physiognomy of the more d e n s e l y - f o r e s t e d Hawaiian h a b i t a t s of L i m b e l l a i s s i m i l a r to the shrub-carr of Sutton Lake. I saw Hawaiian h a b i t a t s where i n c i d e n t l i g h t was l i m i t e d by dense t r e e canopy or v e r t i c a l w a l l s of f i r s t - o r d e r stream g u l l i e s . In a d d i t i o n , dense low-growing canopies and narrow stream g u l l i e s reduce a i r c i r c u l a t i o n , m a i n t a i n i n g high r e l a t i v e humidity that favors L i m b e l l a . Dense growths of e p i p h y t i c v e g e t a t i o n , evidence of h i g h humidity, f u r t h e r emphasizes the physiognomic s i m i l a r i t i e s of the two h a b i t a t s . V e g e t a t i v e s i m i l a r i t y , when such e x i s t s , i s t h e r e f o r e s t r u c t u r a l , not f l o r i s t i c , i n f l u e n c i n g l i g h t l e v e l and humidity. The open r i p a r i a n h a b i t a t s of L i m b e l l a i n Hawaii have no counterpart at the Oregon s i t e s , although Barview may have been s i m i l a r . E. CONCLUSION AND SUMMARY Despite the vast geographic d i f f e r e n c e s between the two h a b i t a t s , p h y s i c a l c o n d i t i o n s at 1200-2130 m i n Hawaii are n e a r l y the same as those at sea l e v e l on the north P a c i f i c coast of North America. The h a b i t a t s are i n f l u e n c e d by s i m i l a r l o c a l c l i m a t e s , m i c r o c l i m a t e s , s o i l pH and hydrology. The v e g e t a t i o n of Hawaii and c o a s t a l Oregon are f l o r i s t i c a l l y d i s p a r a t e , but sometimes physi o g n o m i c a l l y s i m i l a r . ' Given these s i m i l a r i t i e s , i t i s not i n c o n c e i v a b l e that 34 l o n g - d i s t a n c e d i s p e r s e d L i m b e l l a d i a s p o r e s c o u l d s u r v i v e i n e i t h e r l o c a l i t y . L_j_ t r i c o s t a t a ssp. t r i c o s t a t a , though growing i n " t r o p i c a l " Hawaii, can be c o n s i d e r e d a temperate or semi-temperate element of the Hawaiian moss f l o r a , i l l u s t r a t i n g the "quasi-temperate c o n d i t i o n s on mountain massifs at lower l a t i t u d e s " a l l u d e d to by Moore (1972). 35 I I I . MORPHOLOGICAL ANALYSIS A. INTRODUCTION If d i s c r e t e m o r p h o l o g i c a l d i f f e r e n c e s e x i s t between specimens of ssp. t r i c o s t a t a from Hawaii and Oregon, i t should be p o s s i b l e to i d e n t i f y them through c a r e f u l morphological examination. T h i s chapter d e s c r i b e s the p u t a t i v e morphological d i f f e r e n c e s between Hawaiian and Oregon specimens, how the d i f f e r e n c e s were measured q u a n t i t a t i v e l y , and which c h a r a c t e r s proved to be d i s t i n c t i v e . B. NEED FOR QUANTIFICATION OF DISCRETE MORPHOLOGICAL CHARACTERS Horton (1984) reviewed q u a n t i t a t i v e a p p l i c a t i o n s i n bryotaxonomy and o u t l i n e d t h e i r u s e f u l n e s s i n supplementing more t r a d i t i o n a l m o rphological d e s c r i p t i o n s . Although obvious morphological d i f f e r e n c e s such as presence or absence of a ch a r a c t e r can be d e s c r i b e d and i l l u s t r a t e d without ambiguity, l e s s obvious c h a r a c t e r d i f f e r e n c e s must be q u a n t i f i a b l e , c o n s i s t e n t , and u s u a l l y c o r r e l a t e d with other c h a r a c t e r or h a b i t a t d i f f e r e n c e s i f they are to have meaningful taxonomic use. 36 C. MORPHOLOGICAL SIMILARITY OF HAWAIIAN AND NORTH AMERI CAN  LIMBELLA When d e s i g n a t i n g L i m b e l l a f r y e i as a synonym of L. t r i c o s t a t a , Lawton (1971) i n d i c a t e d that no s i g n i f i c a n t m o r phological d i f f e r e n c e s e x i s t e d between specimens from Hawaii and North America. T h i s o p i n i o n was r e i t e r a t e d by C h r i s t y (1980). Cursory comparison of specimens, i n c l u d i n g m i c r o s c o p i c l e a f f e a t u r e s , r e v e a l s l i t t l e d i f f e r e n c e other than s i z e of p l a n t s : Hawaiian p l a n t s develop shoots and branches up to 3-5 times longer than those from Oregon. Leaf l e n g t h , a r e o l a t i o n and s e r r a t i o n appear to be i d e n t i c a l , given the polymorphism inherent i n most a q u a t i c mosses ( V i t t and Glime 1984). In a d d i t i o n , p l a n t s from both areas are e i t h e r dendroid or t r a i l i n g . To determine o b j e c t i v e l y i f these d i f f e r e n c e s were s i g n i f i c a n t , i t was necessary to measure the d i f f e r e n t c h a r a c t e r s and analyze them s t a t i s t i c a l l y . D. SIGNIFICANCE OF DIFFERENT BRANCHING PATTERNS IN LIMBELLA Close i n s p e c t i o n of Hawaiian and Oregon specimens i n d i c a t e d that t h e i r branching p a t t e r n s d i f f e r e d . T h i s d i f f e r e n c e was d i f f i c u l t to d e s c r i b e s a t i s f a c t o r i l y using standard branching p a t t e r n terminology. Hawaiian p l a n t s e x h i b i t branching at f a i r l y r e g u l a r i n t e r v a l s up to 1 cm d i s t a n t , which g i v e s the p l a n t s a pinnate or frondose h a b i t , e s p e c i a l l y i n aq u a t i c forms. However, the branches are n e i t h e r d i s t i c h o u s nor complanate but a r i s e symmetrically around the stem. In c o n t r a s t , i n Oregon 37 p l a n t s the branches a r i s e in c l o s e l y - s p a c e d groups, g i v i n g the p l a n t s a p e n i c i l l a t e , b r u s h - l i k e appearance. The p l a n t s are t h e r e f o r e n e a r l y always dendroid. 3.1 C o n t r o l of Branching Q u a n t i f i c a t i o n of branching p a t t e r n d i f f e r e n c e s must i n c l u d e both branches and branch buds. Although branch development from l a t e r a l buds appears to be i n h i b i t e d by a p i c a l dominance (Hebant 1977; C o l b e r t 1979; Bopp 1984) and s t i m u l a t e d by shoot d e c a p i t a t i o n (Bopp 1984), branch p o s i t i o n can be determined only by bud l o c a t i o n . Presumably, bud l o c a t i o n must be g e n e t i c a l l y c o n t r o l l e d , otherwise d i f f e r e n t taxa growing i n the same h a b i t a t s would e x h i b i t the same branching p a t t e r n s . Q u a n t i f i c a t i o n of branching p a t t e r n , t h e r e f o r e , must r e l y on bud l o c a t i o n and I chose to q u a n t i f y t h i s by c a l c u l a t i n g branch bud frequency per merophyte number. Each merophyte ("leaf segment" auct.) c o n s i s t s o f : (1) one l e a f (2) a subtending stem segment composed of c o r t i c a l and u n d e r l y i n g parenchymatous c e l l s and (3) at r e g u l a r i n t e r v a l s a branch bud l o c a t e d d i r e c t l y below the l e a f i n s e r t i o n (von Schoenau 1911; Lorch 1931; Schuster 1966; Kawai 1977). Branches that a r i s e subsequently from these buds are t h e r e f o r e hot, s t r i c t l y speaking, " a x i l l a r y , as s t a t e d erroneously by some authors. A l e a f y shoot i s thus composed of many adjacent merophytes and i s the product of s u c c e s s i v e d i v i s i o n s by the a p i c a l c e l l d u r i n g shoot e l o n g a t i o n . As such, merophyte 38 f e a t u r e s are d i f f e r e n t i a t e d at the time of shoot development and are t h e r e f o r e primary developmental t i s s u e s . Although r h i z o i d s a l s o a r i s e in c l u s t e r s from each merophyte, at the same s i t e as the branch bud or surrounding i t i f both are present, r h i z o i d s develop s e c o n d a r i l y from c o r t i c a l c e l l s and t h e r e f o r e are not i n i t i a t e d d i r e c t l y by the a p i c a l c e l l d u r i n g shoot e l o n g a t i o n . E. METHODS 3.2 Q u a n t i f i c a t i o n of Branching P a t t e r n s Bud f r e q u e n c i e s were determined by: (1) counting each merophyte, f o l l o w i n g the s p i r a l p a t t e r n from shoot base to shoot apex, u n t i l a bud or branch was encountered (2) r e c o r d i n g that merophyte number (3) resuming the count at zero with the next merophyte, f o l l o w i n g the s p i r a l p a t t e r n u n t i l the next bud or branch was encountered (4) r e c o r d i n g that merophyte number (5) resuming the count at zero with the next merophyte, e t c . , u n t i l the shoot apex was reached and the counting terminated. Twenty shoots (1134 merophytes) of ssp. t r i c o s t a t a and 17 shoots (689 merophytes) of ssp. f r y e i were counted. The numbers of buds or branches o c c u r r i n g at each merophyte number were t o t a l e d , the percent occurrence at each merophyte number (=frequency) c a l c u l a t e d , and the f r e q u e n c i e s p l o t t e d a g a i n s t the corr e s p o n d i n g merophyte numbers. The data set (Appendix E) was a n a l y z e d by computer to determine s i m i l a r i t y or d i s s i m i l a r i t y , u s i n g stepwise d i s c r i m i n a n t a n a l y s i s and Mahalanobis d i s t a n c e 39 (D 2) MIDAS programs. D i s c r i m i n a n t a n a l y s i s o r d i n a t e s c h a r a c t e r values i n axes of decreasing v a r i a n c e . U s u a l l y the f i r s t two or three axes c o n t a i n most of the v a r i a n c e i n the data s e t , thereby reducing and s i m p l i f y i n g i t . S c a t t e r p l o t s of the axes c o n t a i n i n g the g r e a t e s t v a r i a n c e thus should r e v e a l the g r e a t e s t d i f f e r e n c e s between the specimens sampled. Mahalanobis d i s t a n c e represents a g e n e r a l i z e d phenetic d i s t a n c e that measures d i f f e r e n c e s between p a i r s of c h a r a c t e r value means showing the g r e a t e s t v a r i a n c e . Distance i s measured as a f u n c t i o n of o v e r l a p between p a i r s of values and i n so doing the d i f f e r e n c e s between specimens are maximized. The method i s most u s e f u l at lower taxonomic ranks where d i f f e r e n c e s are more d i f f i c u l t to d e t e c t (Sneath and Sokal 1973; Dunn and E v e r i t t 1982). 3.3 Phenetic A n a l y s i s Twelve herbarium specimens of ssp. t r i c o s t a t a and 12 of ssp. f r y e i , i n c l u d i n g types, were score d f o r 28 morphological c h a r a c t e r s (Appendix D) and the scores (Appendix G) analyzed by computer, using stepwise d i s c r i m i n a n t a n a l y s i s and Mahalanobis d i s t a n c e (D 2) MIDAS programs. The 28 c h a r a c t e r s were chosen because they d e c r i b e d the s a l i e n t m o rphological f e a t u r e s of the gametophytes i n enough d e t a i l to d e t e c t s i g n i f i c a n t d i f f e r e n c e s between the taxa, should any e x i s t . Only gametophytes were scored because sporophytes of ssp. f r y e i are unknown. Because new shoots of ssp. t r i c o s t a t a up to c. 6 cm long can have very t h i c k stems and few branch l e a v e s , only shoots longer than 8 cm 40 were scored. Leaves and stem s e c t i o n s were measured 1.0-1.5 cm below t i p s of main shoots or l a r g e s t branches with i n t a c t a p i c e s . Leaf a p i c a l angle and marginal t o o t h angle were measured f o l l o w i n g the method of Wagner (1951). Measured values f o r each specimen represented means of 5-10 measurements of each c h a r a c t e r being scored. F. RESULTS 3.4 Q u a n t i f i c a t i o n of Branching P a t t e r n s Bud counts of the two taxa r e v e a l e d d i f f e r e n c e s i n bud f r e q u e n c i e s ( F i g . 6). In Hawaiian p l a n t s , buds were most frequent every f o u r t h merophyte (26.4%), with l e s s e r peaks every f i r s t , s i x t h , n i n t h and e l e v e n t h merophyte. A few buds were separated by up to 20 merophytes. In c o n t r a s t , buds in Oregon p l a n t s were most frequent (30.0%) every f i r s t merophyte, with l e s s e r peaks every f o u r t h , s i x t h and n i n t h merophyte. A few buds were separated by no more than 13 merophytes. In a very few cases, i n d i v i d u a l p l a n t s e x h i b i t e d a t y p i c a l bud f r e q u e n c i e s , but p o p u l a t i o n s as a whole appeared to be uniform when a l l bud f r e q u e n c i e s were averaged. Stepwise d i s c r i m i n a n t a n a l y s i s of bud f r e q u e n c i e s of the 37 p l a n t s measured (Appendix F) i n d i c a t e d that d i f f e r e n c e s between the Hawaiian and Oregon p l a n t s were s t a t i s t i c a l l y s i g n i f i c a n t , i n descending order of s i g n i f i c a n c e , at merophytes 1 (p < 0.0001), 8 (p < 0.001), 3 (p < 0.05) and 5 (p < 0.05). Based on 41 these 4 c h a r a c t e r s , the Mahalanobis d i s t a n c e between ssp. t r i c o s t a t a and ssp. f r y e i was 11.807 and was s t a t i s t i c a l l y h i g h l y s i g n i f i c a n t (p < 0.0001). 42 F i g u r e 6 - Branch bud f r e q u e n c i e s i n s s p . t r i c o s t a t a and s s p . f r y e i , per l e a f segment number. Leaf Segment Number 43 3 . 5 Phenetic A n a l y s i s Stepwise d i s c r i m i n a n t a n a l y s i s of data f o r the 28 m o r p h o l o g i c a l c h a r a c t e r s (Appendix H) i n d i c a t e d that 5 c h a r a c t e r s e x h i b i t e d 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 f e r e n c e s between the Hawaiian and Oregon p l a n t s . The f i v e c h a r a c t e r s were, in descending order of s i g n i f i c a n c e , l e a f i n s e r t i o n angle (p < 0.0001), stem diameter (p < 0.005), branch length (p < 0.01), c o s t a t h i c k n e s s at i n s e r t i o n (p < 0.05) and basal l e a f c e l l l e n g t h r w i d t h r a t i o (p < 0.05). Based on these 5 c h a r a c t e r s , the Mahalanobis d i s t a n c e between ssp. t r i c o s t a t a and ssp. f r y e i was 40.086 and was s t a t i s t i c a l l y h i g h l y s i g n i f i c a n t (p < 0.0001). The 23 remaining c h a r a c t e r s were not i d e n t i f i e d as being s t a t i s t i c a l l y s i g n i f i c a n t . However, examination of the c o r r e l a t i o n matrix (Appendix I) showed ba s a l l e a f c e l l lengths to be h i g h l y c o r r e l a t e d with b a s a l l e a f c e l l length:width r a t i o s . Because d i s c r i m i n a n t a n a l y s i s tends to s e l e c t only one of two or more c o r r e l a t e d c h a r a c t e r s as being s t a t i s t i c a l l y s i g n i f i c a n t , i t f o l l o w s that the other c o r r e l a t e d c h a r a c t e r s may be of d i s c r i m i n a n t value a l s o . Longest and s h o r t e s t basal l e a f c e l l l e ngths were t h e r e f o r e c o n s i d e r e d d i s t i n c t i v e c h a r a c t e r s i n a d d i t i o n t o the f i v e l i s t e d above. A s c a t t e r p l o t u s i n g a l l 28 c h a r a c t e r s r e v e a l e d l i t t l e d i s t i n c t i o n between the taxa ( F i g . 7). A s c a t t e r p l o t using the 7 c h a r a c t e r s i d e n t i f i e d by d i s c r i m i n a n t a n a l y s i s showed c l e a r d i s t i n c t i o n between the taxa ( F i g . 8). 44 F i g u r e 7 - S c a t t e r p l o t of measurements of 28 mo r p h o l o g i c a l c h a r a c t e r s . T r i a n g l e = ssp. t r i c o s t a t a , c l o s e d c i r c l e = ssp. f r y e i . <SCATTER VAR=29.30> SCATTER PLOT-N= 24 OUT OF 24 2 9 . V 2 9 V S . 3 0 . V 3 0 V29 6 1 4 . 9 8 •» A A A A A • 3 8 . 5 3 5 5 4 . 6 5 7 7 0 . 7 7 9 V30 3 0 . 4 7 4 4 G . 5 9 S S 2 . 7 1 8 7 8 . 8 4 0 45 F i g u r e 8 - S c a t t e r p l o t of measurements of 7 morphological c h a r a c t e r s , a f t e r s e l e c t i o n by d i s c r i m i n a n t a n a l y s i s . T r i a n g l e = ssp. t r i c o s t a t a , c l o s e d c i r c l e = ssp. f r y e i . ^SCATTER VAR=B,9> SCATTER PLOT N= 24 OUT OF 24 V8 6 0 1 . 9 3 8 . V 8 V S . 9 . V 9 5 5 3 . 3 1 4 5 6 . 0 7 - 3 6 . 4 6 3 - 2 4 . 4 6 7 - 1 2 . 4 7 2 - . 4 7 6 4 7 1 1 . 5 1 9 V9 - 3 0 . 4 6 5 - 1 8 . 4 7 0 - 6 . 4 7 4 2 5 . 5 2 1 2 1 7 . 5 1 7 46 G. DISCUSSION The c o n s i s t e n t and 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 f e r e n c e s i n bud f r e q u e n c i e s of Hawaiian and Oregon L i m b e l l a , based on mean bud counts, confirmed the e x i s t e n c e of q u a n t i f i a b l e d i f f e r e n c e s i n branching p a t t e r n which i s probably g e n e t i c a l l y f i x e d . Of the 7 c h a r a c t e r s i d e n t i f i e d by d i s c r i m i n a n t a n a l y s i s as being s t a t i s t i c a l l y s i g n i f i c a n t , 3 (branch l e n g t h , l e a f i n s e r t i o n angle, l o n g est b a s a l l e a f c e l l length) were used i n the dichotomous key presented i n Chapter 8. The remaining c h a r a c t e r s were c o n s i d e r e d too time-consuming to measure or s u p e r f l u o u s f o r convenient d i s c r i m i n a t i o n between the taxa. Leaf i n s e r t i o n angle and l ongest b a s a l l e a f c e l l l e n g t h were c o n v e n i e n t l y measurable, and when p l o t t e d ( F i g . 9) they d i s t i n g u i s h e d the taxa more e f f e c t i v e l y than l e a f i n s e r t i o n angle p l o t t e d a g a i n s t stem diameter ( F i g . 10). C u l t i v a t i o n experiments (Chapter 7) showed these two c h a r a c t e r s to be s t a b l e under a v a r i e t y of environmental c o n d i t i o n s and presumably more s t r o n g l y c o n t r o l l e d by g e n e t i c than by environmental f a c t o r s , c o r r o b o r a t i n g t h e i r importance as taxonomic c h a r a c t e r s . H. SUMMARY AND CONCLUSIONS Measurement of branch bud frequency i n ssp. t r i c o s t a t a and ssp. f r y e i r e v e a l e d s i g n i f i c a n t d i f f e r e n c e s i n bud d i s t r i b u t i o n which i s r e f l e c t e d by the d i s t r i b u t i o n of branches. Buds of ssp. t r i c o s t a t a most o f t e n occur every' f o u r t h merophyte, whereas buds of ssp. f r y e i most o f t e n occur every f i r s t 47 F i g u r e 9 - L e a f i n s e r t i o n a n g l e s p l o t t e d a g a i n s t l o n g e s t b a s a l l e a f c e l l l e n g t h s . T r i a n g l e = s sp . t r i c o s t a t a , c l o s e d c i r c l e = s s p . f r y e i . <SCATTER VAR =1,2> SCATTER PLOT N= 24 OUT OF 24 1.V1 V S . 2 . V 2 VI 6 0 . 0 0 0 t* • • • • 5 0 . 0 0 0 00 c 03 4 G . 6 S 7 a o u 0) 0) c 4-1 n) 4o. ooo A A A A AA A 2S.OOO 3 6 . 5 5 6 4 8 . 1 1 1 5 9 . 6 6 7 7 1 . 2 2 2 V2 3 0 . 7 7 8 4 2 . 3 3 3 5 3 . 8 8 9 6 5 . 4 4 4 . 7 7 . 0 0 0 Longest basal leaf cell (pin) 48 F i g u r e 10 - Leaf i n s e r t i o n angles p l o t t e d a g a i n s t stem diameters. T r i a n g l e = ssp. t r i c o s t a t a , c l o s e d c i r c l e = ssp. f r y e i . <SCATTER> SCATTER PLOT N= 24 OUT OF 24 t . V I VS . 2 . V 2 V 1 SO.OOO CD iH 60 (3 4 6 . 6 6 7 c o u, ts M-l 4 0 . OOO CO t-1 A A A A 1 6 5 . 0 0 2 6 1 . 6 7 3 5 8 . 3 3 4 5 5 . 0 0 5 5 1 . 6 7 V2 2 1 3 . 3 3 3 1 0 . 0 0 4 0 6 . 6 7 5 0 3 . 3 3 6 0 0 . 0 0 Stem diameter (pm) 49 merophyte. If and when such buds produce branches, the branches of ssp. t r i c o s t a t a are t h e r e f o r e by n e c e s s i t y more widely spaced, and those of ssp.- f r y e i more crowded. Such d i f f e r e n c e s i n branching are c l e a r l y e x h i b i t e d by many specimens. Average bud f r e q u e n c i e s observed i n ssp. t r i c o s t a t a and ssp. f r y e i suggest that bud formation by the a p i c a l c e l l d uring shoot e l o n g a t i o n i s g e n e t i c a l l y f i x e d . M u l t i v a r i a t e a n a l y s i s of phenetic measurements i n d i c a t e d that two gametophytic c h a r a c t e r s , l e a f i n s e r t i o n and b a s a l l e a f c e l l l e n g t h , were s i g n i f i c a n t l y d i f f e r e n t i n ssp. t r i c o s t a t a and ssp. f r y e i . Other c h a r a c t e r s measured were o v e r l a p p i n g or congruent, showing that ssp. t r i c o s t a t a was l a r g e r i n most morphological f e a t u r e s . The d i s t i n c t d i f f e r e n c e s i n l e a f i n s e r t i o n angle and b a s a l c e l l l e n g t h were confirmed by c u l t i v a t i o n experiments and support the use of these two c h a r a c t e r s to segregate the taxa. 50 IV. KARYOTYPE ANALYSIS A. INTRODUCTION Among m o r p h o l o g i c a l l y s i m i l a r taxa, chromosome numbers are o f t e n the same and t h e r e f o r e are of l i t t l e taxonomic use i n d i s t i n g u i s h i n g the taxa, although they may be u s e f u l i n c o n f i r m i n g s i m i l a r i t y based on morphology. In these cases, karyotype a n a l y s i s i s e s s e n t i a l to provide c r i t i c a l taxonomic i n f o r m a t i o n . T h i s chapter reviews a p p l i c a t i o n s of karyotype a n a l y s i s i n bryophytes and r e p o r t s the r e s u l t s of such an a n a l y s i s i n L i m b e l l a t r i c o s t a t a ssp. t r i c o s t a t a and L.  t r i c o s t a t a ssp. f r y e i . 4.1 U t i l i t y of Karyotype A n a l y s i s Ramsay (1984) pr o v i d e d an e x c e l l e n t review of the present s t a t e of moss c y t o l o g y . Chromosome numbers, though sometimes an important taxonomic or p h y l o g e n e t i c c o n t r i b u t i o n , by themselves are o f t e n of l i m i t e d u s e f u l n e s s because many d i v e r s e taxa have the same number, while others e x h i b i t i n t r a s p e c i f i c v a r i a t i o n (Koponen 1978). Buck (1981) d e c r i e d "the a l l - t o o - o f t e n 'count-and-run' r e p o r t s of chromosome numbers c l u t t e r i n g the l i t e r a t u r e , " such r e p o r t s p r o v i d i n g l i t t l e a d d i t i o n a l i n f o r m a t i o n and t h e r e f o r e being of l i m i t e d u s e f u l n e s s . C l e a r l y there i s a need for more d e t a i l e d c y t o l o g i c a l a n a l yses which can 51 provide more i n f o r m a t i o n than j u s t chromosome numbers. T h i s i s why karyotype a n a l y s i s , though h a r d l y new (Lewitsky 1931), has r e c e i v e d c o n s i d e r a b l e a t t e n t i o n i n recent b r y o l o g i c a l reviews, e s p e c i a l l y as a means of d i s c r i m i n a t i n g between c l o s e l y - r e l a t e d taxa with s i m i l a r karyotypes (Koponen 1978; Newton 1979; Inoue and Yamashita 1980; Ramsay 1982, 1984). Karyotype a n a l y s i s i s an attempt to d e s c r i b e karyotype morphology i n such a way as to make comparisons with other karyotypes p o s s i b l e . Analyses have i n c l u d e d an a r r a y of techniques ranging from photographs of chromosome squashes, i n t e r p r e t i v e drawings made from such photographs, ideograms and alignments showing chromosomes arranged by t h e i r l e n g t h s , to karyotype formulas which d e s c r i b e chromosome shape, l e n g t h , and heterochromatic s t a i n i n g a c t i v i t y (Inoue and Iwatsuki 1976; Newton 1979; Ramsay 1982). D e r i v a t i o n of such formulas i s dependent on the c o r r e c t i d e n t i f i c a t i o n and placement of the centromere. Lacking centromere data, some workers (Iverson 1963; Mendelak 1981) have r e l i e d on a b s o l u t e and r e l a t i v e chromosome lengths ( r e l a t i v e l e n g t h = absolute l e n g t h : t o t a l l e n g t h of complement, expressed as %) to d e t e c t d i f f e r e n c e s between c l o s e l y - r e l a t e d taxa. 52 4.2 C y t o l o g i c a l Reports f o r L i m b e l l a Taxa To my knowledge no taxa of L i m b e l l a have been i n v e s t i g a t e d c y t o l o g i c a l l y . Ramsay (1984) c i t e d the p a u c i t y of c y t o l o g i c a l i n f o r m a t i o n f o r both Gondwanalandic and Hawaiian moss f l o r a s . Under Sc iaromium M i t t . , only S^ l e s c u r i i ( S u l l . ) B r o t h . has been i n v e s t i g a t e d c y t o l o g i c a l l y , Crum and Anderson (1981) r e p o r t i n g a chromosome number of n=11, based on m e i o t i c counts ( F r i t s c h 1982). Sc iaromium l e s c u r i i i s not a L i m b e l l a and should be assigned to P l a t y l o m e l l a Andrews. B. MATERIALS AND METHODS 4.3 Chromosome Squashes and Documentation Squash p r e p a r a t i o n s of L^ t r i c o s t a t a were made from c. 150 d i f f e r e n t p l a n t s , using gametophytic shoot a p i c e s - (4 d i f f e r e n t Kauai p o p u l a t i o n s , 1 Oregon p o p u l a t i o n ; F i g . 11). Attempts to obt a i n chromosome f i g u r e s from Maui s p o r e l i n g s were u n s u c c e s s f u l . M a t e r i a l was obtained from f i e l d - c o l l e c t e d p l a n t s which had been maintained i n a growth chamber 4-5 months (Chapter 7). Although numerous shoot a p i c e s and a few immature sporophytes of ssp. t r i c o s t a t a were f i x e d i n the f i e l d , t h i s m a t e r i a l d i d not s t a i n p r o p e r l y and c o u l d not be used i n the study. I t was necessary to use somatic t i s s u e s because ssp. f r y e i sporophytes are unknown, and m i t o t i c chromosomes are e a s i e r to see and document because they are l e s s " s t i c k y " than 53 m e i o t i c chromosomes (Lowry 1948; Newton 1979). S t e e l ' s (1978) procedures were f o l l o w e d with some m o d i f i c a t i o n s : (1) 1-cm shoot t i p s were c o l l e c t e d from l i v i n g m a t e r i a l i n the growth chamber, p l a c e d with a few p-dichlorobenzene c r y s t a l s between sheets of moist f i l t e r paper i n a p e t r i p l a t e , and s t o r e d at 18°C f o r 4 hr. The m a t e r i a l was 54 F i g u r e 11 - Sources of l i v i n g p l a n t s of s s p . t r i c o s t a t a used for l a b o r a t o r y exper iment s . Na P a l i - K o n a F o r e s t R e s e r v e , K a u a i . M o d i f i e d from USGS Haena, Hawai i 7 .5 ' t o p o g r a p h i c map. A N 250 m 55 then (2) f i x e d i n Carnoy f i x a t i v e (3 p a r t s 100% e t h a n o l : 1 pa r t g l a c i a l a c e t i c a c i d ) f o r 3-8 hr at 5°C (3) r i n s e d 3 times i n d i s t i l l e d water (4) immersed i n pe c t i n a s e (Sigma P-5146, used f u l l - s t r e n g t h from the b o t t l e ) f o r 2 hr at 5°C (5) r i n s e d 3 times in d i s t i l l e d water (6) hydrolyzed i n 5N HCL at 18°C f o r 1 hr (7) r i n s e d 3 times i n d i s t i l l e d water (8) s t a i n e d i n aceto -i r o n - h a e m a t o x y l i n - c h l o r a l hydrate (Whitman's s t a i n ) f o r 2 hr at 18°C (9) r i n s e d 3 times i n d i s t i l l e d water and (10) d i s s e c t e d and squashed i n Hoyer's s o l u t i o n ( a f t e r Przywara and Kuta 1983). S l i d e s were heated on a hot microscope lamp p r i o r to squashing (Beeks 1955) and tapped r a t h e r v i g o r o u s l y with the end of an unsharpened p e n c i l to separate and spread the c e l l s . S l i d e s were then p l a c e d face down on absorbent paper and squashes were made by a p p l y i n g pressure with a p e n c i l e r a s er p l a c e d d i r e c t l y over the t i s s u e s . Cover s l i p s were ringed immediately with n a i l p o l i s h . D r i e d voucher specimens from the c u l t i v a t e d c o l l e c t i o n s , and s l i d e s showing chromosome f i g u r e s are d e p o s i t e d at UBC. Photographs of metaphase chromosome squashes were made under o i l immersion at 1000X with a L e i t z - compound microscope. Sketches of the f i g u r e s were made at the time they were photographed. I n t e r p r e t i v e drawings of the f i g u r e s were made by t r a c i n g the photographs and i n k i n g with i n d i a ink. Alignments of chromosomes were made by ar r a n g i n g c u t - o u t s from the i n t e r p r e t i v e drawings i n descending order from l e f t to r i g h t , a c c o r d i n g to t h e i r a b s olute l e n g t h s . S c a t t e r p l o t s and graphs were generated using the MIDAS s c a t t e r program. Absolute 56 lengths (Appendix J) were analyzed by computer using stepwise d i s c r i m i n a n t a n a l y s i s and Mahalanobis (D 2) MIDAS programs. S t e e l (1978) r e p o r t e d that the p r e f i x a t i o n treatment with p-dichlorobenzene r e t a r d e d m i t o s i s and improved chromosome s e p a r a t i o n . P e c t i n a s e and h y d r o l y s i s at room temperature with 5N HCL were e s s e n t i a l f o r s o f t e n i n g and s e p a r a t i n g t i s s u e s of L i m b e l l a , and d i d not appear to i n t e r f e r e with s t a i n i n g . Use of Hoyer's s o l u t i o n as a squashing medium simultaneously (a) provided an a d d i t i o n a l t i s s u e - s o f t e n i n g agent and (b) produced, once ringed w i t h n a i l p o l i s h , a semi-permanent s l i d e without any f u r t h e r treatment. The a c e t o - i r o n - h a e m a t o x y l i n - c h l o r a l hydrate s t a i n e d chromosomes and n u c l e i a dark p u r p l i s h - b l a c k . E i g h t months a f t e r s t a i n i n g , my p r e p a r a t i o n s showed no s i g n of f a d i n g . 4.4 V a r i a t i o n s i n Methodology D i c t a t e d by Study M a t e r i a l Methods f o r the p r e p a r a t i o n of bryophyte chromosome squashes, though f o l l o w i n g the same general schedule, vary widely i n c e r t a i n procedures. T h i s v a r i a t i o n , i n p a r t r e f l e c t i n g the p e r s o n a l p r e f e r e n c e s of r e s e a r c h e r s , i s a l s o determined by the nature of the p l a n t m a t e r i a l at hand. The most u s e f u l methods f o r squashing t i s s u e s of a given taxon can be i d e n t i f i e d o n l y by t r i a l and e r r o r . As a r e s u l t , recent reviews of methodology (e.g., Smith 1978) are not as u n i v e r s a l l y a p p l i c a b l e to a l l mosses as they f i r s t appear to be. One d i f f i c u l t y encountered i n v o l v e d c o n f l i c t i n g methodologies f o r storage of f i x e d m a t e r i a l i n e i t h e r f i x a t i v e 57 or e t h a n o l . Because Carnoy f i x a t i v e d e t e r i o r a t e s r a p i d l y unless r e f r i g e r a t e d , some r e s e a r c h e r s recommend storage of f i x e d m a t e r i a l i n 70% ethanol i f r e f r i g e r a t i o n i s u n a v a i l a b l e under f i e l d c o n d i t i o n s . T h i s was done, only to f i n d 4-5 months l a t e r that the m a t e r i a l s t a i n e d very p o o r l y . I t i s probable that the m a t e r i a l should never have been t r a n s f e r r e d to ethanol (Newcomer 1953; H.P. Ramsay, per s . comm. 1984), although McAdam (1982) observed no negative e f f e c t on s t a i n i n g a f t e r long storage in 70% e t h a n o l . There are numerous c o n f l i c t i n g methods i n v o l v i n g t i s s u e s o f t e n i n g and c e l l s e p a r a t i o n p r i o r to squashing. Many workers (e.g., Bowers 1969; Smith 1978; Przywara and Kuta 1983) recommended h y d r o l y z i n g f i x e d m a t e r i a l i n 1-2N HCL f o r 6-30 minutes at 18°C. S t i l l other r e s e a r c h e r s (e.g. Lowry 1948) used heat as the s o f t e n i n g agent, while Ramsay (1969) and Inoue and Iwatsuki (1976) used heat and 45% a c e t i c a c i d , and McAdam (1982) found that f i x a t i v e alone was s u f f i c i e n t . H y d r o l y z i n g L i m b e l l a with 1N HCL at 60°C (20-30 min) and 2N HCL at 18°C had l i t t l e e f f e c t on s o f t e n i n g t i s s u e s . However, S t e e l ' s (1978) methods, u s i n g p e c t i n a s e and h y d r o l y s i s with 5N HCL at 18°C, worked extremely w e l l on L i m b e l l a and s a t i s f a c t o r y spreading and f l a t t e n i n g were achieved. HCL c o n c e n t r a t i o n s up to 6N have been used to macerate t i s s u e s ( F r i t s c h 1983), emphasizing the f a c t that d i f f e r e n t taxa need d i f f e r e n t treatments. 58 C. RESULTS 4.5 Karyotype A n a l y s i s The chromosome number of both ssp. t r i c o s t a t a and ssp. f r y e i i s n =11 . McAdam (1982) and others noted how d i v i d i n g somatic c e l l s can vary from 0-40 i n any given squash p r e p a r a t i o n . C e l l d i v i s i o n s i n L i m b e l l a , never abundant, were most common in embryonic leaves from the a p i c a l c e l l r egion of shoot t i p s . Many chromosomes were " s t i c k y , " f a i l i n g to separate from one another i n a squash and thus p r e c l u d i n g a c c c u r a t e counting and measuring. Consequently, among approximately 150 p r e p a r a t i o n s , I observed and photographed 25 c e l l s (14 of ssp. t r i c o s t a t a , 11 of ssp. f r y e i ; F i g s . 12 & 13) which showed good s e p a r a t i o n in one plane. The chromosomes were extremely small (longest 3.5 Mm, s h o r t e s t 0.7 urn), making accurate counts d i f f i c u l t i n many p r e p a r a t i o n s . Although the chromosome f i g u r e s of L i m b e l l a e x h i b i t c o n s t r i c t i o n s and bends which resemble centromeres, these f e a t u r e s may not be centromeres at a l l but regions of d i f f e r e n t i a l heterochromatic s t a i n i n g (McAdam 1982). I was t h e r e f o r e r e l u c t a n t to i n d e n t i f y centromeres on these chromosomes, as placement of them in many cases would have amounted to guesswork. Non-placement of centromeres precluded i d e n t i f i c a t i o n of karyotype formulas. T h e r e f o r e , karyotypes of ssp. t r i c o s t a t a and ssp. f r y e i were compared on the b a s i s of absolute and 59 60 F i g u r e 13 - Chromosome f i g u r e s of ssp. f r y e i ( C h r i 5505 ) . 61 r e l a t i v e lengths of i n d i v i d u a l chromosomes. F i g s . 14 and 15 show chromosomes of a l l 25 m i t o t i c p l a t e s a l i g n e d from l e f t to r i g h t , a c c o r d i n g to t h e i r a b s o l u t e lengths ( v i d . Ramsay 1982). Absolute and r e l a t i v e chromosome lengths of the two taxa were almost completely congruent ( F i g . 16); the few non-congruent records merely r e f l e c t the l a r g e r ssp. t r i c o s t a t a data set (22 a d d i t i o n a l chromosome l e n g t h s ) . However, when the r e l a t i v e l e ngths of each chromosome in the data set were averaged and graphed ( F i g . 18), d i f f e r e n c e s between the two taxa were n o t i c e a b l e . Chromosomes 1-3 were longer i n ssp. f r y e i than i n t r i c o s t a t a , and chromosomes 4-11 were a l l s h o r t e r i n ssp. f r y e i than in t r i c o s t a t a . A l s o , F i g . 17 shows that average chromosome le n g t h s of ssp. f r y e i e x h i b i t e d a broader range of s i z e c l a s s e s than d i d those of t r i c o s t a t a . Stepwise d i s c r i m i n a n t a n a l y s i s of the absolute l e n g t h s of the 275 chromosomes measured (Appendix K) i n d i c a t e d t h a t d i f f e r e n c e s between the Hawaiian and Oregon p l a n t s were s t a t i s t i c a l l y s i g n i f i c a n t , i n descending order of s i g n i f i c a n c e , a t chromosomes 8 (p < 0.005) and 1 (p < 0.05). Based on these two c h a r a c t e r s , the Mahalanobis d i s t a n c e between LT and LF was 3.2912 and s t a t i s t i c a l l y s i g n i f i c a n t (p < 0.005). Chromosomes 6,7, and 9 were h i g h l y c o r r e l a t e d with chromosome 8, and chromosome 2 was c o r r e l a t e d with chromosome 1 (Appendix L ) . Despite these d i f f e r e n c e s , the average a b s o l u t e length of each chromosome i n each complement was i d e n t i c a l (1.6 urn), and the t o t a l average a b s o l u t e complement l e n g t h was a l s o i d e n t i c a l (17.5 am). F i g u r e 14 - Ssp. t r i c o s t a t a chromosome alignments. 1-10 -Kauai, C h r i s t y 5368. 11-14 - Kauai, C h r i s t y 5337. O l ! t l t l t " ))))>>ttm > m t t t u » t )t>»Slftl»*» Ulflftl AM* 9 liliutii'ft* 1 ) ) ) U t l l l M 63 F i g u r e 15 - Ssp. f r y e i chromosome alignments. 1-6 -Oregon, C h r i s t y 5499. 7-11 - Oregon, C h r i s t y 5505. 1 ) ) ) ) ! • ! « • • • 2 )l»)ftllftt* 5 ) » l > 1 l t l t # . In 7 )jj»|*»ftttl 9 J I ) I I } A | 4 « | 10 J > > l » t > W > t 11 UttitlM** 64 F i g u r e 16 - Absolute chromosome lengths p l o t t e d a g a i n s t r e l a t i v e l e n g t h s , a l l chromosomes measured. Square = measurements i d e n t i c a l to both t a x a . T r i a n g l e = measurements of a d d i t i o n a l s sp. t r i c o s t a t a chromosomes measured. <SCATTER V A R - 1 , 2 > SCATTER PLOT N « 121 OUT OF 275 1.AL V S . 2 . R L » L 3 . 0 0 0 0 •U 1 . 9 7 7 8 60 c CD o CO <! 1 . 4 6 6 7 • I I I I I I I A| • I A I I 11 I I I A A I A I AH A| I I | | | • I I I I • I I A I • I 1 2 . 3 7 7 1 5 . 1 1 2 RL 1 1 . 0 0 9 1 3 . 7 4 4 1 6 . 4 8 0 R e l a t i v e l e n g t h 65 F i g u r e 17 - Average a b s o l u t e a g a i n s t average r e l a t i v e l e n g t h s . C l o s e d c i r c l e = chromosome le n g t h s p l o t t e d T r i a n g l e = ssp. t r i c o s t a t a . ssp. f r y e i . <SCATTER V A R - 1 . 2 > SCATTER PLOT N » 11 OUT OF 11 1.AL VS . 2 .RLORA AL 2 . 8 0 O 0 + 2 . 5 8 8 9 1 cm c <u 4-1 3 r H O CO & (0 <u 00 CO > . 9 0 0 0 0 •A A A A • • A* • A 5 . 3 3 0 0 7 . 6 9 8 9 6 . 5 1 4 4 8 . 8 8 3 3 ' 0 . 0 6 B 1 2 . 4 3 7 1 4 . 8 0 6 RL0RA 1 1 - 2 5 2 1 3 . 6 2 1 1 5 . 9 9 0 Average r e l a t i v e length F i g u r e 18 - Graph of average r e l a t i v e lengths of chromosome complements of ssp. t r i c o s t a t a and ssp. f r y e i . Chromosome Number 67 D. DISCUSSION 4.6 S i g n i f i c a n c e of N=11 Chromosome Number The f o l l o w i n g represent the f i r s t chromosome number r e p o r t s in the genus L i m b e l l a . Given the morphological s i m i l a r i t y of the two karyotypes, the numbers themselves are of l i t t l e taxonomic use i n t h i s i n v e s t i g a t i o n , other than showing that both taxa have the same number. The number n=11 i s the most common chromosome number so f a r r e p o r t e d f o r pleurocarpous mosses (c. 36%), suggested to have a r i s e n by aneuploidy a f t e r d o u b l i n g of the base number x=6 (Smith 1978; Crosby 1980; Ramsay 1984). Based on the "remarkable s i m i l a r i t y i n the n=11 karyotype" e x h i b i t e d by the D i p l o l e p i d e a e , McAdam (1982) p o s t u l a t e d that i n that group the primary base number i s x=11, l e s s e r numbers having a r i s e n from chromosome f u s i o n s and " p o s s i b l y e l i m i n a t i o n of small chromosomes and fragments." I t remains to be seen whether t h i s concept i s t e n a b l e . 4.7 S i g n i f i c a n c e of Chromosome Length Data Although the sample s i z e i s s m a l l , and the absence of karyotype formulas makes i t d i f f i c u l t to compare the two complements o b j e c t i v e l y , s e v e r a l c o n c l u s i o n s can be drawn from the data on chromosome l e n g t h . S e v e r a l workers (Iverson 1963; Inoue and Iwatsuki 1976; Inoue and Yamashita 1980; Mendelak 1981; Ramsay 1982, 1984) have emphasized the u t i l i t y of a b s o l u t e 68 and r e l a t i v e chromosome lengths as taxonomic c h a r a c t e r s . Absolute chromosome lengths r e p o r t e d l y can be a f f e c t e d by treatment before f i x a t i o n , stage of d i v i s i o n and squash methods (Newton 1979; Ramsay 1984). Use of r e l a t i v e lengths minimizes these v a r i a t i o n s and p r o v i d e s a d i s c r i m i n a t i v e morphological parameter. 4.8 S i m i l a r i t i e s Between the Karyotypes F i g u r e s 14 and 15 show obvious s u p e r f i c i a l s i m i l a r i t i e s i n the chromosome lengths of the two karyotypes. T h i s s i m i l a r i t y i s supported.by the i d e n t i c a l values of average chromosome l e n g t h and t o t a l average complement leng t h , and F i g u r e 16. Such s i m i l a r i t y suggests that the two taxa are c l o s e l y r e l a t e d , but t h i s cannot be assumed, given the f a c t that most D i p l o l e p i d e a e with chromosome numbers n=11 e x h i b i t c l o s e m o r p h o l o g i c a l s i m i l a r i t i e s (McAdam 1982). 4.9 D i f f e r e n c e s Between the Karyotypes Fig u r e 18 r e v e a l e d d i f f e r e n c e s i n the r e l a t i v e l e n g t h s of i n d i v i d u a l chromosomes. Using s c a t t e r diagrams, Mendelak (1981) observed d i f f e r e n c e s i n l e n g t h among the l a r g e s t and s m a l l e s t chromosomes of P e l l i a megaspora and P^ e n d i v i i f o l i a and concluded that these d i f f e r e n c e s , i n c o n c e r t with other c h a r a c t e r s , were s u f f i c i e n t to d i s t i n g u i s h the two taxa. Based i n part on d i f f e r e n t chromosome len g t h s , Inoue and Yamashita 69 (1980) concluded that Rhodobryum roseum and R_^  o n t a r i e n s e are d i s t i n c t but c l o s e l y r e l a t e d , because they "show c o n s i d e r a b l e s i m i l a r i t y i n the chromosomal alignment p a t t e r n . " The same approach was used to analyze the karyotypes of L i m b e l l a . E. CONCLUSIONS AND SUMMARY Although small sample s i z e and lack of karyotype formulae compromise the karyotype data of L i m b e l l a to some extent, i t i s c l e a r that the karyotypes of ssp. t r i c o s t a t a and ssp. f r y e i are very s i m i l a r but e x h i b i t demonstrable d i f f e r e n c e s i n absolute and r e l a t i v e lengths of some chromosomes. Both taxa have the chromosome number n=11, the most common number so f a r repor t e d f o r pleurocarpous mosses. Therefore, there i s no evidence that secondary p o l y p l o i d y has occurred s i n c e the taxa became g e o g r a p h i c a l l y i s o l a t e d , as observed among m o r p h o l o g i c a l l y s i m i l a r " s p e c i e s p a i r s " in Mnium (Lowry 1948; Koponen and N i l s s o n 1978). The karyotypes of ssp. t r i c o s t a t a and ssp. f r y e i i n d i c a t e a c l o s e r e l a t i o n s h i p but show enough d i f f e r e n c e i n a b s o l u t e and r e l a t i v e lengths to j u s t i f y using t h i s d i f f e r e n c e as an a d d i t i o n a l taxonomic c h a r a c t e r to d i s t i n g u i s h the ta x a . 70 V. ISOZYME BANDING PATTERNS A. INTRODUCTION 5.1 U t i l i t y of E l e c t r o p h o r e s i s Isozymes have proved u s e f u l i n the study of p l a n t g e n e t i c s , b i o c h e m i s t r y , p h y s i o l o g y , p h y l o g e n e t i c s and s y s t e m a t i c s . Band p a t t e r n s produced by isozymes dur i n g e l e c t r o p h o r e t i c s e p a r a t i o n and enzyme s t a i n i n g may be i n t e r p r e t e d as e x p r e s s i o n s of s t r u c t u r a l genes and may be used as a t o o l to compare genotypes of i n d i v i d u a l p l a n t s . Bands are i n t e r p r e t e d as expressions of a l l e l e s at gene l o c i coding f o r enzymes, and have been analyzed q u a n t i t a t i v e l y to c a l c u l a t e a l l e l e f r e q u e n c i e s , locus polymorphisms, h e t e r o z y g o s i t y and genetic d i s t a n c e s w i t h i n and between s p e c i e s ( G o t t l i e b 1971, 1981, 1984). The data are used to determine genetic i d e n t i t i e s , g e n e t i c d i v e r s i t y i n p o p u l a t i o n s and phy l o g e n e t i c comparisons between taxa ( G o t t l i e b 1981). The method i s immensely u s e f u l i n r e v e a l i n g both g e n e t i c d i f f e r e n c e s between m o r p h o l o g i c a l l y and c y t o l o g i c a l l y s i m i l a r taxa, and genetic s i m i l a r i t y between m o r p h o l o g i c a l l y and c y t o l o g i c a l l y d i v e r s e taxa ( G o t t l i e b 1984). 71 5.2 E l e c t r o p h o r e t i c Procedure Isozymes are detected by means of g e l e l e c t r o p h o r e s i s , i n which p r o t e i n s i n a crude t i s s u e e x t r a c t are placed on a g e l ( s t a r c h or p o l y a c r y l a m i d e ) , s u b j e c t e d to e l e c t r i c c u r r e n t , and separated on the b a s i s of t h e i r e l e c t r o s t a t i c charge, s i z e and c o n f i g u r a t i o n . The g e l s are then assayed f o r s p e c i f i c enzymes by immersion i n s o l u t i o n s , one f o r each enzyme sought, c o n t a i n i n g . a c o f a c t o r and s u b s t r a t e f o r the r e a c t i o n that the enzyme c a t a l y s e s , and a s t a i n s p e c i f i c to the product of the r e a c t i o n ( S h i e l d s et a l . 1983; V a l l e j o s 1983). In t h i s way the enzymes can be dete c t e d by the s t a i n e d bands which appear at the s i t e s of a c t i v i t y . The i n t e r p r e t a t i o n of zymograms of h a p l o i d t i s s u e i s simpler than that of d i p l o i d t i s s u e because only one a l l e l e can be expressed per l o c u s . Each band u s u a l l y r e p r e s e n t s one l o c u s , although two l o c i may produce two superimposed bands. 5.3 E l e c t r o p h o r e s i s of Bryophytes I n v e s t i g a t i o n of g e n e t i c v a r i a b i l i t y i n bryophtes, u s i n g e l e c t r o p h o r e s i s , was reviewed by Cummins and Wyatt (1981) and de V r i e s et a l . (1983). T h e i r work s u b s t a n t i a t e d other workers' c o n c l u s i o n s that g e n e t i c polymorphism i n the bryophyte p o p u l a t i o n s i n v e s t i g a t e d i s as high as that r e p o r t e d f o r v a s c u l a r p l a n t s , d i s p e l l i n g the l o n g - h e l d b e l i e f that bryophytes "are a g e n e t i c a l l y d e p l e t e d group with l i m i t e d e v o l u t i o n a r y p o t e n t i a l . " They p o i n t e d out that the same had been found to be 72 true f o r other "phylogenetic r e l i c t s " such as the clubmoss Lycopodium lucidulum and horseshoe crab Limulus polyphemus. Both monomorphic and polymorphic l o c i have been observed i n mosses and h e p a t i c s . Polymorphism index values, g e n e t i c i d e n t i t i e s and g e n e t i c d i s t a n c e s have been c a l c u l a t e d f o r bryophyte p o p u l a t i o n s as they have been for phanerogams ( v i d . Gliddon 1980 and review by de V r i e s et a l . 1983). E l e c t r o p h o r e s i s was p o t e n t i a l l y u s e f u l i n i d e n t i f y i n g genotypic d i f f e r e n c e s between Hawaiian and North American p o p u l a t i o n s of L i m b e l l a . P o p u l a t i o n s of ssp. t r i c o s t a t a were expected to e x h i b i t g e n e t i c polymorphism s i m i l a r to that observed by other workers who i n v e s t i g a t e d s e x u a l l y - r e p r o d u c i n g bryophyte p o p u l a t i o n s . P o p u l a t i o n s of ssp. f r y e i were expected to be g e n e t i c a l l y monomorphic because the l a c k of sporophytes and a c t i v e v e g e t a t i v e propagation s t r o n g l y suggested that the p o p u l a t i o n i s c l o n a l . However, i t was a l s o expected that the banding p a t t e r n of ssp. • f r y e i would be only s l i g h t l y d i v e r g e n t — i f at a l l — from one of the ssp. t r i c o s t a t a p a t t e r n s , based on the assumption that ssp. f r y e i was r e c e n t l y d e r i v e d from L. t r i c o s t a t a . In reviewing comparisons between p r o g e n i t o r and d e r i v a t i v e s p e c i e s , G o t t l i e b (1981, 1984) emphasized that g e n e t i c d i f f e r e n c e s between such s p e c i e s are very s m a l l , dependent upon the amount of time elapsed s i n c e the d i f f e r e n t i a t i o n of the two taxa. I f the d e r i v a t i v e i s of recent o r i g i n , "most i f not a l l the a l l e l e s of the d e r i v a t i v e should s t i l l be present i n the parent" ( G o t t l i e b 1984). 73 B. MATERIALS AND METHODS 5.4 E l e c t r o p h o r e s i s Isozyme banding p a t t e r n s of L i m b e l l a were obtained by means of h o r i z o n t a l s t a r c h g e l e l e c t r o p h o r e s i s , f o l l o w i n g the methods of Helenurm (1983) and Layton and Ganders (1984). L i v i n g p l a n t s were procured from f i e l d - c o l l e c t e d m a t e r i a l which had been maintained i n a growth chamber f o r 1-4 months. I n i t i a l l y , f i v e 1 cm shoot t i p s were ground with a g l a s s g r i n d i n g rod i n 2 drops of d i s t i l l e d water (Szweykowski et a l . 1981) on a c h i l l e d spot p l a t e , the crude e x t r a c t s then absorbed onto wicks and the wicks i n s e r t e d i n the g e l s . F a i n t or no s t a i n i n g i n the subsequent enzyme assays i n d i c a t e d that l a r g e r q u a n t i t i e s of e x t r a c t were needed, with more thorough g r i n d i n g of the m a t e r i a l . Whole p l a n t s of 0.2-3.0 g wet weight (excess water b l o t t e d o f f ) were then ground with crushed g l a s s t u b i n g and 2 drops of d i s t i l l e d water i n a c h i l l e d mortar. The r e s u l t i n g paste was t r a n s f e r r e d to c h i l l e d 1.5 ml polypropylene micro t e s t tubes (Bio Rad 223-9501) and c e n t r i f u g e d f o r 15 min at 20,000 g. The supernatant was absorbed on wicks and s t a i n i n g i n t e n s i t y was much improved. The higher p r o p o r t i o n of stems in ssp. f r y e i made g r i n d i n g l a b o r i o u s and time-consuming. The task was s i m p l i f i e d by immersing p l a n t s of the above weights i n l i q u i d n i t r o g e n and g r i n d i n g the f r o z e n t i s s u e s to a powder i n a c h i l l e d mortar (Fahselt 1980). A few drops of d i s t i l l e d water were then added to the thawing powder to make a s l u r r y which c o u l d be 74 t r a n s f e r r e d to the micro t e s t tubes f o r c e n t r i f u g a t i o n . F r e e z i n g d i d not appear to a l t e r the s t a i n i n g i n t e n s i t y of bands. E x t r a c t s of L i m b e l l a were e l e c t r o p h o r e s e d s i multaneously on two g e l s , one with morpholine c i t r a t e b u f f e r and one with Ridgway b u f f e r ( r e c i p e s and sources i n Apendix M). Gels contained 12.5 % w/v s t a r c h ( E l e c t r o s t a r c h l o t 392), and 10 % w/v sucrose was added to improve enzyme r e s o l u t i o n and ease of h andling (Layton and Ganders 1984). Eight e l e c t r o p h o r e t i c runs were performed, using e x t r a c t s from: (1) 53 i n d i v i d u a l Hawaiian p l a n t s from 4 d i f f e r e n t Kauai p o p u l a t i o n s and 1 Maui p o p u l a t i o n , and 49 Oregon p l a n t s from 1 p o p u l a t i o n ( F i g . 11) (2) mature c u l t i v a t e d p l a n t s (3) small p l a n t s grown from spores ( " s p o r e l i n g s " , sensu Nehira 1984) and (4) r e c i p r o c a l l y c u l t i v a t e d m a t e r i a l . To d e t e c t banding v a r i a t i o n w i t h i n the Hawaiian or North American p o p u l a t i o n s , on some g e l s up to 14 i n d i v i d u a l s from one taxon were run together as a group, and 4 i n d i v i d u a l s from the other taxon run next to these f o r comparison of e l e c t r o p h o r e t i c m o b i l i t i e s (e.g., F i g . 19 A). On other g e l s , because uneven heat d i s s i p a t i o n from the charged g e l can d i s t o r t bands and give the appearance of d i f f e r e n t m o b i l i t y ("bowing", sensu O e l s h l e g e l and Stahmann 1973), p a i r s of i n d i v i d u a l s of one taxon were a l t e r n a t e d with p a i r s of i n d i v i d u a l s of the other, g i v i n g a more dependable comparison of t h e i r r e s p e c t i v e band m o b i l i t y (e.g., F i g . 19 B). 75 5.5 Enzyme Assays, Gels were s t a i n e d f o r 12 enzymes: diaphorase (DIA), e s t e r a s e (EST), glutamic dehydrogenase (GLDH), glucose-6-phosphate dehydrogenase (G6PDH), hexoseaminidase (HA), i s o c i t r i c dehydrogenase (IDH), l e u c i n e aminopeptidase (LAP), malic dehydrogenase (MDH), phosphoglucose isomerase (PGI), phosphoglucomutase (PGM), 6-phosphogluconate dehydrogenase (6PGDH) and superoxide dismutase (SOD). Recipes and sources f o r these s t a i n s are given i n Appendix M. A f t e r i n i t i a l t r i a l s , 5 enzyme assays (MDH, PGI, PGM, 6PGDH, SOD) were s e l e c t e d to use i n a l l subsequent runs, because they c o n s i s t e n t l y showed the most intense and b e s t - r e s o l v e d s t a i n i n g . In c o n t r a s t to dark-s t a i n i n g bands t y p i c a l of most enzymes, SOD appears as achromatic regions ("negative s t a i n i n g " auct.) on g e l s s t a i n e d with t e t r a z o l i u m dyes, because i t i n h i b i t s formation of blue formazan which gi v e s g e l s s t a i n e d with t e t r a z o l i u m dyes t h e i r c h a r a c t e r i s t i c b l u i s h - p u r p l e c o l o r (Beauchamp and F r i d o v i c h 1971). The negative s t a i n i n g of SOD i s enhanced when g e l s are exposed to l i g h t . Because the s t a i n s f o r MDH, PGI, PGM and 6PGDH a l l contained t e t r a z o l i u m s a l t s , a l l g e l s showed achromatic SOD bands as w e l l as s t a i n e d bands of the other enzymes being sought, thus being by d e f a u l t a l a b o r - s a v i n g way of t e s t i n g f o r two enzymes with a s i n g l e assay. 76 C. RESULTS 5. 6 Inconsistent- S t a i n i n g Banding was observed in a l l enzyme systems t e s t e d . However, poor band r e s o l u t i o n r e s u l t e d from t e c h n i c a l d i f f i c u l t i e s , and produced somewhat ambiguous r e s u l t s f o r 4 of the 5 enzymes t e s t e d . The primary d i f f i c u l t y encountered was o b t a i n i n g bands of c o n s i s t e n t s t a i n i n g i n t e n s i t y . I t was apparent from t r i a l runs that a minimum of 0.2-0.5 g of p l a n t m a t e r i a l was necessary to y i e l d enough e x t r a c t to show d i s t i n c t d a r k - s t a i n i n g bands which were s c o r e a b l e . However, even with 3.0 g of p l a n t m a t e r i a l , s t a i n i n g was i n c o n s i s t e n t with MDH, 6PGDH, PGI and PGM and i t was d i f f i c u l t to determine i f an apparent absence of a band i n one i n d i v i d u a l , while present in o t h e r s , i n d i c a t e d the e x i s t e n c e of a n u l l a l l e l e -- and t h e r e f o r e polymorphism — or simply the e x i s t e n c e of l e s s enzyme in the e x t r a c t . I t was o f t e n p o s s i b l e to demonstrate a s e r i e s of i n t e r g r a d i n g s t a i n i n g i n t e n s i t i e s among a group of i n d i v i d u a l s , ranging from no or very f a i n t bands to c l e a r l y -s t a i n e d bands. Because of t h i s handicap I c o u l d not c o n f i r m polymorphism in the p o p u l a t i o n s s t u d i e d . Because a l l e x t r a c t i o n and e l e c t r o p h o r e t i c procedures were c a r r i e d out at or below 4°C, i t i s u n l i k e l y that heat d e n a t u r a t i o n caused poor s t a i n i n g . Although the v i g o r of ssp. t r i c o s t a t a s l o w l y d e c l i n e d i n the growth chamber durin g the course of the study, ssp. f r y e i t h r i v e d , yet the l a t t e r a l s o 77 showed v a r i a b l e s t a i n i n g . The e r r o r may have been my s e l e c t i o n of e x t r a c t i o n or g e l b u f f e r s , s i n c e enzymes can be o x i d i z e d or denatured by b u f f e r s of u n s u i t a b l e pH ranges ( O e l s h l e g e l and Stahmann 1973). C a r e f u l s e l e c t i o n of optimal b u f f e r systems and enzyme assays f o r L i m b e l l a would have been a time-consuming matter of t r i a l and e r r o r which I judged to be beyond the l i m i t s of t h i s study. 5.7 The Enzymes Poor band r e s o l u t i o n of MDH, PGI, PGM and 6PGDH precluded r e l i a b l e s c o r i n g i n these enzyme systems. In some, banding appeared to be monomorphic and i d e n t i c a l i n both taxa, while i n others c o n f i g u r a t i o n s seemed to d i f f e r between the taxa ( F i g s . 19 A-D). Band m o b i l i t i e s u s u a l l y d i f f e r e d between the taxa. One to 3 l o c i were u s u a l l y e v i d e n t . SOD bands were the b e s t - r e s o l v e d among the 5 enzyme systems t e s t e d , and can be d e s c r i b e d i n some d e t a i l . Both L.  t r i c o s t a t a ssp. t r i c o s t a t a and ssp. f r y e i d i s p l a y e d t h e i r own i n v a r i a n t , monomorphic SOD p a t t e r n c o n s i s t i n g of 3 zones of a c t i v i t y , 2 m i g r a t i n g a n o d a l l y and 1 c a t h o d a l l y ( F i g . 19 E ) . .One a n o d a l l y - m i g r a t i n g zone showed f a s t e r m o b i l i t y and sometimes segregated i n t o 2 f r a c t i o n s . The other a n o d a l l y - m i g r a t i n g zone showed slower m o b i l i t y and showed e i t h e r much smearing between i t and the o r i g i n ( " t a i l i n g " sensu O e l s h l e g e l and Stahmann 1973) or segregation i n t o 3-4 f r a c t i o n s (sensu Baur and Schorr 1969). The c a t h o d a l l y - m i g r a t i n g zone d i d not appear to segregate i n t o 78 F i g u r e 19 - Zymograms of ssp. t r i c o s t a t a and ssp. f r y e i . Dark zones = enzymes s t a i n e d f o r ; white zones = SOD a c t i v i t y . A l l g e l s run i n Ridgway b u f f e r system. A. - MDH; 14 Hawaiian p l a n t s ( L ) , 4 Oregon p l a n t s (R). B. PGI; Hawaiian p l a n t s (with f a s t e r SOD m o b i l i t y ) a l t e r n a t e d with Oregon p l a n t s . C. - PGM; 14 Oregon p l a n t s (L) , 4 Hawaiian p l a n t s (R). D. - 6PGDH; 14 Hawaiian p l a n t s ( L ) , 4 Oregon p l a n t s (R). E. - SOD (dark bands are MDH); Hawaiian p l a n t s (with f a s t e r SOD m o b i l i t y ) a l t e r n a t e d with Oregon p l a n t s . 1. R e c i p r o c a l l y - c u l t i v a t e d Oregon m a t e r i a l . 2. R e c i p r o c a l l y -c u l t i v a t e d Hawaiian m a t e r i a l . 3. S p o r e l i n g s from Maui. 4. S p o r e l i n g s from Kauai. H o r i z o n t a l l i n e = o r i g i n . Note c a t h o d a l l y - m i g r a t i n g bands. 79 80 f r a c t i o n s . Cathodal banding a c t i v i t y was not s t a i n e d f o r in a l l e l e c t r o p h o r e t i c runs and i s mentioned here f o r documentation only. F r a c t i o n s were o f t e n more c l e a r l y segregated i n ssp. t r i c o s t a t a run on g e l s with morpholine c i t r a t e b u f f e r . Because i t was d i f f i c u l t to d i f f e r e n t i a t e f r a c t i o n s c o n s i s t e n t l y w i t h i n a zone of a c t i v i t y , they were grouped as a s i n g l e zone of enzymatic a c t i v i t y r a ther than each as a separate band. Szweykowski et a l . (1981) used the same approach when s c o r i n g i n d i s t i n c t peroxidase bands in P e l l i a . F o l l o w i n g t h i s i n t e p r e t a t i o n , two SOD l o c i were apparent. There was an obvious d i f f e r e n c e i n SOD m o b i l i t y between the two taxa: ssp. t r i c o s t a t a showed bands of c o n s i s t e n t l y g r e a t e r m o b i l i t y and u s u a l l y more intense negative s t a i n i n g . P o p ulations of ssp. t r i c o s t a t a from both Kauai (mature p l a n t s ) and Maui ( s p o r e l i n g s ) showed i d e n t i c a l band p a t t e r n s . R e c i p r o c a l l y c u l t i v a t e d m a t e r i a l (Chapter 7) of both taxa showed band p a t t e r n s i d e n t i c a l to those of t h e i r parent s t o c k s : the p a t t e r n s were not i n f l u e n c e d by* d i f f e r e n t environmental regimes. D. DISCUSSION L i m b e l l a t r i c o s t a t a ssp. t r i c o s t a t a and ssp. f r y e i have never been analyzed p r e v i o u s l y f o r isozymes, and as f a r as i s known ssp. t r i c o s t a t a i s the f i r s t Hawaiian bryophyte to be analyzed. Because s t a i n i n g of 4 of the 5 enzyme systems t e s t e d was i n c o n s i s t e n t , l i t t l e can be s a i d about genetic v a r i a b i l i t y of the two taxa. SOD banding, however, was c l e a r l y r e s o l v e d and 81 c o n s i s t e n t l y monomorphic w i t h i n the taxa but showed a d i f f e r e n c e in band m o b i l i t y between the taxa. SOD i s u s u a l l y monomorphic in p l a n t and animal t i s s u e s and only r a r e l y has been r e p o r t e d to be polymorphic (Baur and Schorr 1969; El-Kassaby 1980; Yeh and O'Malley 1980; Yamazaki 1981). Because SOD i s u s u a l l y monomorphic in p l a n t p o p u l a t i o n s , and was monomorphic i n 5 d i f f e r e n t L i m b e l l a p o p u l a t i o n s from 2 d i f f e r e n t i s l a n d s , i t i s probable that a l l ssp. t r i c o s t a t a p o p u l a t i o n s e x h i b i t an SOD m o b i l i t y c o n s i s t e n t l y d i f f e r e n t from that of the ssp. f r y e i p o p u l a t i o n . C o n s i s t e n t d i f f e r e n c e i n band m o b i l i t y i s a u s e f u l taxonomic c h a r a c t e r , and was used by Krzakowa and Szweykowski (1977) and Szweykowski et a l . (1981) as evidence f o r r e t e n t i o n of the h e p a t i c s p e c i e s P e l l i a b o r e a l i s Lorbeer. They supported t h e i r c o n c l u s i o n with a d d i t i o n a l b i o c h e m i c a l and c y t o l o g i c a l evidence (Szweykowski and Krzakowa 1977). The same approach was f o l l o w e d i n t h i s study of L i m b e l l a . The r e c i p r o c a l l y c u l t i v a t e d L i m b e l l a showed bands i d e n t i c a l to t h e i r parent s t o c k s , not to those of the other taxon with which they grew, i n d i c a t i n g t h at environmentally-caused v a r i a t i o n i n banding p a t t e r n s c i t e d by G o t t l i e b (1981) was not d e t e c t e d . A. CONCLUSION AND SUMMARY Banding p a t t e r n s were obtained f o r a l l enzyme systems t e s t e d , but r e s u l t s were ambiguous f o r 4 of 5 enzymes. The SOD enzyme system e x h i b i t e d the best r e s o l u t i o n , and appeared to be monomorphic w i t h i n each taxon but showed c o n s i s t e n t d i f f e r e n c e s 82 i n m o b i l i t y between the t a x a . In s s p . t r i c o s t a t a , the d i f f e r e n c e in SOD m o b i l i t y was i d e n t i c a l in 5 p o p u l a t i o n s from two d i f f e r e n t i s l a n d s , and i d e n t i c a l in both s p o r e l i n g s and mature p l a n t s . The c o n s i s t e n t d i f f e r e n c e s in m o b i l i t y i n d i c a t e s a f i x e d g e n e t i c d i f f e r e n c e between s s p . t r i c o s t a t a and s s p . f r y e i which was used as an a d d i t i o n a l c h a r a c t e r to d i s t i n g u i s h the two t a x a . 83 VI. PHENOLIC CHROMATOGRAPHY A. INTRODUCTION L i t e r a t u r e documenting the occurrence of non - f l a v o n o i d p h e n o l i c s i n bryophytes i s s c a r c e . Cinnamic a c i d d e r i v a t i v e s were repo r t e d by McClure and M i l l e r (1967), Koponen and N i l s s o n (1978), Mues and Zin s m e i s t e r (1978) and Zehr (1980). C a f f e i c a c i d d i g l u c o s i d e e s t e r s were re p o r t e d by Martensson and N i l s s o n (1974). In c o n t r a s t , l i t e r a t u r e documenting the occurrence of f l a v o n o i d s i n bryophytes i s abundant and has been reviewed r e c e n t l y by Markham et a l . (1978), Markham and Porter (1978), S u i r e and Asakawa (1979, 1981) and Huneck (1984). Most l i t e r a t u r e on the subj e c t i s i n the form of surveys, s c r e e n i n g taxa f o r compounds and/ or e l u c i d a t i n g chemical s t r u c t u r e s . R e l a t i v e l y few papers have expl o r e d taxonomic and ph y l o g e n e t i c i m p l i c a t i o n s of f l a v o n o i d occurrences in bryophytes, and most of these have concerned only Hepaticae ( v i d . Markham et a l . 1978; Markham and P o r t e r (1978), Koponen and N i l s s o n 1978; Campbell et a l . 1979; Markham 1980). Markham et a l . (1978) emphasized the value of f l a v o n o i d s as p o t e n t i a l taxonomic c h a r a c t e r s i n p l a n t s , c i t i n g t h e i r u b i q u i t y , s t a b i l i t y ( o b t a i n a b l e from m a t e r i a l up to 85 years o l d ) , i n t e r s p e c i f i c d i f f e r e n c e s , and r e l a t i v e r e s i s t a n c e to environmental m o d i f i c a t i o n . McClure and M i l l e r (1967) performed the f i r s t major 84 f l a v o n o i d survey in mosses. Working with samples as small as 0.1 g dry weight, they screened 70 s p e c i e s and de t e c t e d f l a v o n o i d s i n 34 of these. S i g n i f i c a n t l y , they found that f l a v o n o i d s were more common i n acrocarpous than pleurocarpous mosses, the l a t t e r e i t h e r l a c k i n g the pigments e n t i r e l y or having them i n very small q u a n t i t i e s (70% occurrence i n ac r o c a r p s , 20% i n pl e u r o c a r p s f i d . S u i r e and Asakawa 1979). T h e i r o b s e r v a t i o n s were v e r i f i e d by l a t e r workers (Vandekerkhove 1977; Aeon et a l . 1983). One of the taxa screened by McClure and M i l l e r (1967) was L i m b e l l a t r i c o s t a t a ssp. t r i c o s t a t a (as Sc iaromium ), which they found to be devoid of f l a v o n o i d s . Because that study was 18 years o l d , and because i t was p o s s i b l e that they may not have used enough p l a n t m a t e r i a l to detect f l a v o n o i d s i n L i m b e l l a , I thought i t worthwhile to attempt to v e r i f y or r e f u t e t h e i r r e s u l t s . I f f l a v o n o i d d i f f e r e n c e s c o u l d be d e t e c t e d between ssp. t r i c o s t a t a and ssp. f r y e i , t h i s would pro v i d e an a d d i t i o n a l taxonomic c h a r a c t e r with which to d i s t i n g u i s h the taxa. B. MATERIALS AND METHODS D r i e d p l a n t s were t r e a t e d f o l l o w i n g the methods of G o r n a l l and Bohm (1980). Two samples of ssp. t r i c o s t a t a were t e s t e d ( C h r i s t y 5209, 37.3 g; C h r i s t y 5245, 69.6 g) and one of ssp. f r y e i ( C h r i s t y 5334, 46.0 g ) . One-gram q u a n t i t i e s of these samples were a l s o t e s t e d s e p a r a t e l y . Voucher specimens were d e p o s i t e d at UBC. 85 P h e n o l i c s were e x t r a c t e d from whole L i m b e l l a p l a n t s i n hot 100% methanol. The n-butanol e x t r a c t s from the 1 g samples were run on two-dimensional chromatograms using aqueous- and benzene-based s o l v e n t s . P h e n o l i c s were det e c t e d by s p r a y i n g with 0.1% b-aminoethyl d i p h e n y l b o r i n a t e ( A l d r i c h Chemical Co.) and viewed under long-wave u l t r a - v i o l e t l i g h t . The n-butanol e x t r a c t s from the l a r g e r samples were loaded onto a Sephadex LH-20 column and e l u t e d with a s e r i e s of aqueous methanol s o l v e n t s of i n c r e a s i n g methanol c o n c e n t r a t i o n . F r a c t i o n s were c o l l e c t e d from the column, evaporated to dryness and spotted s e q u e n t i a l l y on a chromatographic p l a t e . P l a t e s were run i n the benzene so l v e n t and p h e n o l i c s v i s u a l i z e d as above. I n d i v i d u a l f r a c t i o n s with p h e n o l i c a c t i v i t y were then streaked on a p l a t e and run in s o l v e n t as above. Bands were scraped from the p l a t e s , the phenols e l u t e d with methanol, d r i e d down, and spotted again on a p l a t e to detect a c t i v i t y . Procedures were terminated a f t e r t h i s s t e p . C. RESULTS Both the two-dimensional chromatograms ( F i g . 20) and the s e q u e n t i a l l y - s p o t t e d f r a c t i o n s ( F i g . 21) e x h i b i t e d p h e n o l i c a c t i v i t y . Blue, y e l l o w and orange spots were v i s i b l e under UV a f t e r s p r a y i n g but were c o l o r l e s s i n v i s i b l e l i g h t . F a i n t UV-absorbing bands v i s i b l e on s t r e a k chromatograms disappeared when e l u t e d and run again by themselves. No p u r p l i s h UV-absorbing spots t y p i c a l of f l a v o n o i d s were detected, 86 F i g u r e 20 - Phe n o l i c spot p a t t e r n s on two-dimensional chromatograms. A. - ssp. t r i c o s t a t a (Kauai, C h r i s t y 5247 ). B. - ssp. f r y e i (Oregon, C h r i s t y 5336 ). L i g h t shading = orange, dark shading = blue. Aqueous-based solvent • 87 F i g u r e 21 - Phenolic spot p a t t e r n s of s e q u e n t i a l l y - s p o t t e d f r a c t i o n s on one-dimensional chromatograms. A. - ssp. t r i c o s t a t a (Maui, C h r i s t y 5209 ). B. - ssp. t r i c o s t a t a (Kauai, C h r i s t y 5245 ). C. - ssp. f r y e i (Oregon, C h r i s t y 5334 ). Unshaded = yellow, l i g h t shading = orange, dark shading = b l u e , s t r i p e d shading = y e l l o w - b l u e . A > i—I o XI w CO 000«@o c 88 nor were any such bands seen duri n g the column s e p a r a t i o n . D. DISCUSSION The spots seen on chromatograms of ssp. t r i c o s t a t a and ssp. f r y e i most l i k e l y i n d i c a t e d the presence .of p h e n o l i c s other than f l a v o n o i d s . However, because s p e c t r a and s t r u c t u r e s of the compounds were not obtained, t h e i r i d e n t i t y remains unknown. My chromatographic r e s u l t s , using up to 7 times more p l a n t m a t e r i a l than used by McClure and M i l l e r (1967), s u b s t a n t i a t e t h e i r c o n c l u s i o n that t r i c o s t a t a c o n t a i n s no f l a v o n o i d s . L i m b e l l a t r i c o s t a t a ssp. f r y e i has never before been analyzed f o r these compounds. McClure and M i l l e r ' s o b s e r v a t i o n that l a r g e r q u a n t i t i e s of p l a n t m a t e r i a l y i e l d e d no more f l a v o n o i d i n f o r m a t i o n than was a v a i l a b l e i n 0.3 g q u a n t i t i e s i s r e l i a b l e at l e a s t f o r L^ t r i c o s t a t a . They a l s o were c o r r e c t i n r e p o r t i n g 2 b l u e - f l u o r e s c i n g compounds in L.  t r i c o s t a t a ssp. t r i c o s t a t a . P u t a t i v e i d e n t i t i e s of v a r i o u s l y - c o l o r e d chromatographic spots de t e c t e d i n bryophtes have been re p o r t e d i n the l i t e r a t u r e as f o l l o w s : (a) blue = c a f f e i c a c i d d i g l u c o s i d e e s t e r s (Martensson and N i l s s o n 1974) (b) blue or blue-green = cinnamic a c i d d e r i v a t i v e s (Koponen and N i l s s o n 1978; Mues and Zinsmeister 1978; Zehr 1980) and (c) orange and yellow = c a r o t e n o i d s (Bendz et a l . 1968; Karunen and I h a n t o l a 1977; Zehr 1980). With the exception of Martensson and N i l s s o n (1974) and Karunen and I h a n t o l a (1977), none of these i n v e s t i g a t o r s reported the 89 s t r u c t u r e s of the compounds i n q u e s t i o n , and i t appears that t h e i r c o n c l u s i o n s are e n t i r e l y s p e c u l a t i v e . McClure and M i l l e r (1967) r e p o r t e d that n o n - f l a v o n o i d p h e n o l i c s were of l i t t l e taxonomic value because "they are r e a d i l y a l t e r e d by the environment and show l e s s v a r i a t i o n from s p e c i e s to s p e c i e s than do the f l a v o n o i d s . " However, i f non-f l a v o n o i d p h e n o l i c s were envir o n m e n t a l l y a l t e r a b l e , i n t e r s p e c i f i c v a r i a t i o n would tend to be g r e a t e r than that i n d i c a t e d by McClure and M i l l e r . Zehr (1980) found too much no n - f l a v o n o i d p h e n o l i c v a r i a t i o n between s p e c i e s , r e n d e r i n g them of l i t t l e taxonomic use "unless coupled with i d e n t i f i c a t i o n of these compounds." The chromatographic p a t t e r n s of n o n - f l a v o n o i d p h e n o l i c s in ssp. f r y e i d i f f e r e d from those of ssp. t r i c o s t a t a , but because the're was as much v a r i a t i o n w i t h i n ssp. t r i c o s t a t a as between i t and ssp. f r y e i , I c o u l d not d i s c e r n a p a t t e r n of any apparent taxonomic s i g n i f i c a n c e among the p l a n t s t e s t e d . T h e r e f o r e , chromatographic p a t t e r n s were not used as a c h a r a c t e r to d i s t i n g u i s h the two taxa. E. CONCLUSION AMD SUMMARY A n a l y s i s of up to 70 g of ssp. t r i c o s t a t a and ssp. f r y e i demonstrated the presence of s e v e r a l u n i d e n t i f i e d p h e n o l i c compounds. L_^  t r i c o s t a t a ssp. f r y e i had never before been screened f o r phenols. I t i s u n l i k e l y t h a t f l a v o n o i d s were pre s e n t , s u b s t a n t i a t i n g the f i n d i n g s of McClure and M i l l e r (1967). The chromatographic p a t t e r n s of phenols v a r i e d between the samples t e s t e d , r e n d e r i n g them of l i t t l e taxonomic use. 91 VII. EXPERIMENTAL CULTIVATION A. INTRODUCTION C u l t i v a t i o n of mosses i n "common garden" experiments can (1) h e l p determine what f a c t o r s c o n t r o l morphological p l a s t i c i t y of gametophytes and (2) i d e n t i f y which morphological f e a t u r e s remain s t a b l e under d i f f e r e n t environmental c o n d i t i o n s . S t a b l e morphological features can be u s e f u l taxonomic c h a r a c t e r s . T h i s chapter d e s c r i b e s c u l t i v a t i o n experiments i n L i m b e l l a and reviews the taxonomic and phytogeographic i m p l i c a t i o n s of the r e s u l t s . B. UTILITY OF MOSS CULTIVATION The importance of c u l t i v a t o n of mosses and the c o n t r i b u t i o n s and methods of numerous i n v e s t i g a t o r s using t h i s technique have been reviewed r e c e n t l y (Smith 1978; Longton 1982; Zander 1982; Wyatt and Stoneburner 1984; Frahm and Nordhorn-R i c h t e r 1984). C u l t i v a t i o n p r o v i d e s stocks of l i v i n g m a t e r i a l f o r experiments r e q u i r i n g l i v e p l a n t s (e.g., c y t o l o g y ) and i s a l s o a u s e f u l t o o l which may be used to compare p l a n t s grown under c o n t r o l l e d c o n d i t i o n s . In theory, c u l t i v a t i o n of mosses under uniform c o n d i t i o n s f o r taxonomic purposes should enable the i n v e s t i g a t o r to i d e n t i f y which morphological c h a r a c t e r s are g e n e t i c a l l y 92 c o n t r o l l e d and which c h a r a c t e r s are e n v i r o n m e n t a l l y a l t e r e d . G e n e t i c a l l y - c o n t r o l l e d c h a r a c t e r s should remain s t a b l e under a v a r i e t y of c u l t i v a t i o n regimes and should a l s o be s t a b l e i n w i l d p o p u l a t i o n s , whereas e n v i r o n m e n t a l l y - a l t e r e d c h a r a c t e r s should be m o d i f i e d by d i f f e r e n t c u l t i v a t i o n regimes and d i f f e r e n t h a b i t a t c o n d i t i o n s i n the f i e l d . In p r a c t i c e , c u l t i v a t i o n of mosses i n v o l v e s both t e c h n i c a l and i n t e r p r e t i v e d i f f i c u l t i e s . C u l t i v a t e d p l a n t s o f t e n develop appearances q u i t e d i f f e r e n t from those observed i n the f i e l d . P l a n t s may become e t i o l a t e d and e x h i b i t reduced l e a v e s , wider internodes, reduced branching and r h i z o i d a l development. Gametangial production may be suppressed. Under c e r t a i n c o n d i t i o n s d i f f e r e n t taxa may become i n d i s t i n g u i s h a b l e phenocopies (sensu Zander 1982). When working with m o r p h o l o g i c a l l y s i m i l a r taxa, or p u t a t i v e synonymous taxa, one cannot depend s o l e l y on i n f o r m a t i o n d e r i v e d from c u l t i v a t i o n s t u d i e s to d i s c e r n s i m i l a r i t i e s or d i f f e r e n c e s between them. C u l t i v a t i o n methods d i f f e r widely and i n g e n e r a l pleurocarpous mosses are more d i f f i c u l t to maintain than are a c r o c a r p s . Zander (1982) asked s e v e r a l p e n e t r a t i n g q u e s t i o n s concerning i n t e r p r e t a t i o n of i n f o r m a t i o n from c u l t i v a t i o n s t u d i e s and by doing so i d e n t i f i e d the l i m i t a t i o n s of such experiments. Despite these l i m i t a t i o n s , c u l t i v a t i o n can be a powerful taxonomic t o o l , as shown by Smith (1978) and Wyatt and Stoneburner (1984). 9 3 C. CULTIVATION OF LIMBELLA 7.1 Need f o r L i v e M a t e r i a l Aside from a need to maintain l i v i n g c u l t u r e s of L i m b e l l a f o r comparative s t u d i e s under c o n t r o l l e d c o n d i t i o n s , l i v e m a t e r i a l was a l s o needed for e l e c t r o p h o r e s i s and karyotype a n a l y s i s . Because the l a t t e r procedures were time-consuming and needed to be repeated over a p e r i o d of time i n order to accumulate s u f f i c i e n t data, i t was e s s e n t i a l that l i v i n g p l a n t s be maintained f o r the d u r a t i o n of the experiments. 7.2 Need f o r C u l t i v a t i o n S t u d i e s of L i m b e l l a -1. Whole Plant C u l t i v a t i o n Because phenetic a n a l y s i s i d e n t i f i e d two morphological c h a r a c t e r s that appeared to d i s t i n g u i s h ssp. t r i c o s t a t a from ssp. f r y e i , s t a b i l i t y of these d i f f e r e n c e s needed to be assessed b e f o r e they c o u l d be used as taxonomic c h a r a c t e r s to segregate the taxa. C u l t i v a t i o n of ssp. t r i c o s t a t a and ssp. f r y e i under a v a r i e t y of c u l t u r e c o n d i t i o n s , i n c l u d i n g r e c i p r o c a l c u l t i v a t i o n , p rovided a p o t e n t i a l l y u s e f u l t o o l to o b t a i n the needed evidence. 94 2. Spore C u l t i v a t i o n In order to t e s t the v i a b i l i t y of ssp. t r i c o s t a t a spores as p o s s i b l e v e c t o r s of l o n g - d i s t a n c e d i s p e r s a l , i t was necessary to c u l t u r e them. Furthermore, the s p o r e l i n g s (sensu Nehira 1984) c o u l d be used l a t e r i n e l e c t r o p h o r e s i s . 3. Fragment Regeneration Because sporophytes or s p e c i a l i z e d v e g e t a t i v e propagula such as gemmae were unknown i n ssp. f r y e i , and because i n s p i t e of t h i s handicap i t had s u c c e s s f u l l y c o l o n i z e d a r e l a t i v e l y e x t e n s i v e area, i t was necessary to t e s t the v i a b i l i t y of v e g e t a t i v e fragments — branches, shoot t i p s and s i n g l e leaves — as propagula. The r e g e n e r a t i v e c a p a c i t y of such fragments of ssp. t r i c o s t a t a a l s o needed to be assessed to determine whether v e g e t a t i v e fragments were as e f f e c t i v e propagules as spores appeared to be. D. MATERIALS AND METHODS C u l t u r e s were maintained f o r nine months, except f o r spore c u l t u r e s which were maintained f o r 13 months. Voucher specimens of a l l c u l t i v a t e d m a t e r i a l except s p o r e l i n g s were d e p o s i t e d at UBC. 95 7.3 Whole P l a n t C u l t i v a t i o n L i v i n g m a t e r i a l of ssp. t r i c o s t a t a was c o l l e c t e d from f i r s t - and second-order streams on Kauai i n May 1984 ( F i g . 11). Wet p l a n t s were se a l e d i n " z i p l o c k " p l a s t i c bags and r e f r i g e r a t e d as soon as p o s s i b l e . L i v i n g ssp. f r y e i was c o l l e c t e d i n the same manner at Sutton Lake in May 1984. The p l a n t s were pl a c e d i n a Conviron E15 growth chamber under f l u o r e s c e n t l i g h t i n g of 600-800 E i n s t m~2, 16 hr l i g h t : 8 hr dark at 18°C and 100% RH, c o n d i t i o n s approximating those in the f i e l d . 1. C u l t i v a t i o n of Hawaiian P l a n t s Six c o l l e c t i o n s of ssp. t r i c o s t a t a were p l a c e d i n s i x 12x12x25 cm p e r f o r a t e d p l a s t i c t r a y s , one c o l l e c t i o n per t r a y . A l l t r a y s were immmersed i n d e c h l o r i n a t e d water in a 20x50x60 cm p l e x i g l a s tank. The water was c i r c u l a t e d with an MGW Lauda T-1 pump and a e r a t e d with an aquarium stone. P l a n t s were immersed to the approximate depth of those in the f i e l d , e n a b l i n g emergent shoots to protrude above the s u r f a c e of the water. The water was changed biweekly. A f t e r f i v e months, a l g a l contamination — e s p e c i a l l y Cyanophyta — n e c e s s i t a t e d frequent c l e a n i n g of the tank and p l a n t s . In an e f f o r t to reduce a l g a l contamination, I r e p l a c e d the d e c h l o r i n a t e d water with c h l o r i n a t e d tap water, which slowed but d i d not e l i m i n a t e a l g a l p r o l i f e r a t i o n . 96 2. C u l t i v a t i o n of North American P l a n t s Seven c o l l e c t i o n s of ssp. f r y e i were p l a c e d i n 8x20x20 cm t r a n s p a r e n t p l a s t i c boxes with t i g h t - f i t t i n g l i d s . In an attempt to r e p l i c a t e f i e l d c o n d i t i o n s , the bottoms of the boxes were f i l l e d with 1 cm of d e c h l o r i n a t e d water, and the p l a n t s were misted with d e c h l o r i n a t e d water 3-5 times weekly. Water in the boxes was changed every 3-5 weeks. 7.4 Spore C u l t i v a t i o n Because sporophytes of ssp. f r y e i are unknown, only ssp. t r i c o s t a t a spores c o u l d be c u l t u r e d . Sporangia of ssp. t r i c o s t a t a c o l l e c t e d on Kauai and Maui were d r i e d at 38°C f o r 48 hr and s t o r e d at 18°C f o r 8 months. Spores were e x t r a c t e d f o l l o w i n g the methods of Schelpe (1952) and Hatcher (1965) and p l a c e d i n twenty 35x10 mm p e t r i d i s h e s f i l l e d with a u t o c l a v e d h a l f - s t r e n g t h Knopf's s o l u t i o n (Nehira 1966). The s p o r e l i n g s were grown i n a Psychrotherm R-27 incubator under c o n t r o l l e d c o n d i t i o n s i d e n t i c a l to those i n the growth chamber. The n u t r i e n t s o l u t i o n was changed every two weeks. Because mixing and a u t o c l a v i n g the n u t r i e n t s o l u t i o n i n v o l v e d c o n s i d e r a b l e e f f o r t , s p o r e l i n g growth was t e s t e d i n d e c h l o r i n a t e d water, and because no d i f f e r e n c e s i n subsequent growth were observed, a l l n u t r i e n t s o l u t i o n was r e p l a c e d with d e c h l o r i n a t e d water i n week 19 of the study. S p o r e l i n g s were grown under d i f f e r e n t a e r a t i o n regimes. In most d i s h e s , s p o r e l i n g s were grown in s t i l l (standing) water. 97 Some d i s h e s were submerged e x p e r i m e n t a l l y i n a transparent p l a s t i c box f i l l e d with d e c h l o r i n a t e d water a e r a t e d with an aquarium stone, while others were submerged and su b j e c t e d to c u r r e n t and a e r a t i o n with mature ssp. t r i c o s t a t a p l a n t s . A f t e r 41 weeks a l l s p o r e l i n g s were t r a n s f e r r e d to f i l t e r paper moistened with autoclaved d i s t i l l e d water, and grown i n p e t r i d i s h e s . Average s p o r e l i n g d e n s i t i e s in the p e t r i d i s h e s were ob t a i n e d by counting subsets of 1 cm 2. 7.5 Fragment Regeneration 1. L i v i n g M a t e r i a l Fragments of ssp. t r i c o s t a t a and ssp. f r y e i f r e q u e n t l y became detached from whole p l a n t s d u r i n g h a n d l i n g . The fragments c o n s i s t e d of (1) s i n g l e leaves (2) a p i c a l shoot t i p s 2-3 mm long and (3) shoot segments and branches 1-7 cm long (Table 2). To a s c e r t a i n t h e i r v i a b i l i t y as v e g e t a t i v e d i a s p o r e s , fragments were grown, segregated by type, on 4-5 sheets of moist f i l t e r paper (Gemmell 1953; M i l l e r and Ambrose 1976; S e l k i r k 1980, 1981) i n 100x15 mm p e t r i d i s h e s , with 2-5 r e p l i c a t e d i s h e s per fragment type. F i l t e r paper was moistened with a u t o c l a v e d d e c h l o r i n a t e d water which was r e p l e n i s h e d every two weeks. 98 Table 2 - L i v i n g m a t e r i a l of L i m b e l l a used i n r e g e n e r a t i o n s t u d i e s . ssp. t r i c o s t a t a spp. f r y e i 1 . s i n g l e leaves 50 50 2. shoot t i p s 20 20 3. shoot segments, 1-8 cm 120 80 99 2. D r i e d M a t e r i a l In an e f f o r t to regenerate d r i e d herbarium m a t e r i a l (Zander 1979a, 1979b), e n t i r e shoots or branches of ssp. t r i c o s t a t a and ssp. f r y e i were placed on f i l t e r paper and t r e a t e d as d e s c r i b e d above. Ages and i d e n t i t i e s of the taxa t r e a t e d , and d r y i n g procedures employed ( i f known) are given in Table 3. Only the most recent c o l l e c t i o n s a v a i l a b l e were used for the experiment. In a c a s u a l experiment to determine how d r y i n g temperature and d u r a t i o n of d r y i n g a f f e c t r e g e n e r a t i o n , f r e s h m a t e r i a l of ssp. f r y e i was d r i e d at (1) 40°C f o r 24 hr and (2) 20°C f o r 24 hr and 25 fragments from each treatment were p l a c e d on moist f i l t e r paper as d e s c r i b e d above. No r e p l i c a t e s were prepared. 7.6 R e c i p r o c a l C u l t i v a t i o n P o r t i o n s of c o l l e c t i o n s of ssp. t r i c o s t a t a and ssp. f r y e i were grown together e x p e r i m e n t a l l l y i n the same c o n t a i n e r s f o r 10 weeks. Some p l a n t s of ssp. t r i c o s t a t a were p l a c e d alone, without immersion, i n four 8x12x20 cm p l a s t i c boxes f i l l e d with 1 cm of d e c h l o r i n a t e d water, r e p l i c a t i n g the c u l t i v a t i o n c o n d i t i o n s of ssp. f r y e i • P o r t i o n s of two c o l l e c t i o n s of ssp. f r y e i were atta c h e d with rubber bands to a r o c k - f i l l e d bag of nylon window s c r e e n i n g , immersed in the tank c o n t a i n i n g ssp. t r i c o s t a t a , and p l a c e d 15 cm from the outflow of the c i r c u l a t i n g pump to ensure growth under c o n d i t i o n s of continuous c u r r e n t . 100 Table 3 - D r i e d m a t e r i a l used i n r e g e n e r a t i o n study. H it H Taxon Date Drying Time L i m b e l l a t r i c o s t a t a ssp. t r i c o s t a t a 1983 48 hr @ 38 deg C ssp. f r y e i 1983 " " 1922 ? L. capense 1984 ? L. pachyloma 1976 ? " 1981 ? Platylome-lla l e s c u r i i 1980 ? 101 7.7 Shoot O r i e n t a t i o n Experiment New shoots grown from L i m b e l l a fragments on f i l t e r paper i n p e t r i d i s h e s e x h i b i t e d two d i f f e r e n t o r i e n t a t i o n s , each p e c u l i a r to e i t h e r ssp. t r i c o s t a t a or ssp. f r y e i . To ensure that these movements were geniune growth responses and not a r t i f a c t s caused by s h i f t i n g of the m a t e r i a l on the f i l t e r paper, 1 cm shoot segments were s t r i p p e d of e x i s t i n g branches and f i r m l y attached to f i l t e r paper with monofilament f i s h i n g l i n e ( F i g . 22). Twelve r e p l i c a t e d i s h e s were prepared i n t h i s manner, c o n t a i n i n g 120 shoot segments, 60 of each taxon. The dishes were maintained under c o n d i t i o n s i d e n t i c a l to those of the spore c u l t u r e s . E. RESULTS 7.8 Whole P l a n t C u l t i v a t i o n 1. Hawai ian P l a n t s C u l t u r e s of both ssp. t r i c o s t a t a and ssp. f r y e i s u r v i v e d a growth chamber m a l f u n c t i o n at which time the temperature rose to 34°C f o r 12-18 hr. Glime and V i t t (1984) r e p o r t e d that high temperatures are o f t e n l e t h a l to a q u a t i c mosses. L_^  t r i c o s t a t a ssp. t r i c o s t a t a s u r v i v e d the h i g h temperature but began a slow d e c l i n e i n v i g o r a f t e r 4 months which c o i n c i d e d with o c c a s i o n a l l y severe a l g a l c ontamination. Most o r i g i n a l Hawaiian 1 02 F i g u r e 22 - Means of a t t a c h i n g shoot segments to f i l t e r paper , f o r shoot o r i e n t a t i o n exper iment . A . s e c t i o n a l v iew: 1 - shoot segment. 2 - monofi lament l i n e , knot ted at ends . 3 - f i l t e r p a p e r . 4 - p l a s t i c d i s c . 5 - h o l e s bored i n p l a s t i c d i s c w i t h hot n e e d l e . B. S u r f a c e v i ew . 6. - l i n e s c o r e d w i t h p e n c i l . 103 growth turned black or d i e d . No f u n g i were observed. However, new branches continued to be produced throughout the nine-month c u l t u r e p e r i o d . I t was unclear whether the d e c l i n e i n v i g o r was caused by the high temperature, n u t r i e n t d e f i c i e n c y or a l g a l contamination. Leaf i n s e r t i o n angle and b a s a l l e a f c e l l l e n g t h remained the same as i n t y p i c a l ssp. t r i c o s t a t a . 2. North American P l a n t s The Oregon p l a n t s produced v i g o r o u s v e r t i c a l shoots up to 6 cm long, which developed l a t e r a l branches a f t e r 2-3 months. A f t e r 7 months, p o r t i o n s of the c u l t u r e s yellowed and d i e d , accompanied by p r o l i f e r a t i o n of f u n g a l hyphae. Yellowing p o s s i b l y r e s u l t e d from n u t r i e n t d e f i c i e n c y (Frahm and Nordhorn-R i c h t e r 1984). Leaf i n s e r t i o n angle and b a s a l l e a f c e l l l e n g t h remained the same as i n t y p i c a l ssp. f r y e i . 7.9 Spore C u l t u r e s Developmental stages and growth r a t e s of the ssp. t r i c o s t a t a s p o r e l i n g s are shown i n Table 4. S p o r e l i n g s were of the Bryum type, t y p i c a l of other Amblystegiaceae s p o r e l i n g s i n v e s t i g a t e d (Nehira 1984). Throughout the c u l t u r e p e r i o d the s p o r e l i n g s resembled the f l a g e l l i f o r m ' branches o c c a s i o n a l l y produced by mature p l a n t s i n the f i e l d , e x h i b i t i n g very small (0.4-0.8x0.1-0.3 mm), widely-spaced and o f t e n wide-spreading to squarrose leaves with no or very reduced l i m b i d i a and sparse 104 Table 4 - Developmental stages of s p o r e l i n g c u l t u r e s . Week A c t i v i t y Observed 1 germination (a few d i d not germinate u n t i l week 9) 4 branched protonemal wefts 1 1 shoot buds; shoots developing with r h i z o i d s 13 shoots elongated 16 l i m b i d i a v i s i b l e , u n i s t r a t o s e ; a r e o l a t i o n and d e n t i t i o n as i n mature p l a n t s 19 secondary r h i z o i d i n i t i a t i o n at merophytes; some monopodial branching of shoots 26 strong secondary r h i z o i d a l attachment to substratum 30 shoots arched, some a e r i a l 1 05 c h l o r o p h y l l . The e t i o l a t e d appearance c o u l d have been caused by oxygen, carbon d i o x i d e or n u t r i e n t d e f i c i e n c y (Glime and V i t t 1984). F i g u r e 23 shows reduced shoot e l o n g a t i o n r a t e s i n di s h e s with higher s p o r e l i n g d e n s i t i e s . P l a n t s i n crowded d i s h e s developed m o r p h o l o g i c a l l y at approximately the same time as d i d those i n uncrowded d i s h e s , but because shoot e l o n g a t i o n was slower, the p l a n t s were of smaller s t a t u r e . A e r a t i o n of s p o r e l i n g s appeared to improve growth r a t e s and p l a n t s t a t u r e . In g e n e r a l , s p o r e l i n g s produced l a r g e r shoot t i p s and l a r g e r leaves w i t h i n two weeks of a e r a t i o n , f e a t u r e s which were suppressed w i t h i n three weeks a f t e r being returned to s t i l l water. Three weeks a f t e r having been t r a n s f e r r e d to moist f i l t e r paper, a l l s p o r e l i n g s developed l a r g e r l e a v e s , l a r g e r shoot t i p s , more c h l o r o p h y l l , more l a t e r a l branches and more a e r i a l shoots. 7.10 Fragment Regeneration 1. L i v i n g M a t e r i a l Developmental stages of the three r e g e n e r a t i o n treatments are shown i n Tables 5-7. A l g a l and i n v e r t e b r a t e ( r o t i f e r and o r i b a t i d mite) contamination oc c u r r e d i n some shoot segment c u l t u r e s . Fungi were not observed. Leaf i n s e r t i o n angle and ba s a l l e a f c e l l length remained s t a b l e i n a l l treatments. A. S i n g l e Leaf Regeneration. S i n g l e leaves 107 Table 5 - Developmental stages of s i n g l e l e a f r e g e n e r a t i o n . Week A c t i v i t y Observed ssp. t r i c o s t a t a 2 most p e l l u c i d or brown, dead 7 a few r h i z o i d s produced from base of c o s t a and l i m b i d i a 10 a few shoots produced from base of c o s t a and l i m b i d i a on a b a x i a l s i d e of l e a f 14 no c h l o r o p h y l l l e f t i n any leaves 17 secondary r h i z o i d s produced by the few shoots developed 19 shoots to 3 mm ssp. f r y e i 2 some r h i z o i d s produced from base of c o s t a and l i m b i d i a 7 many leaves s t i l l green; l i t t l e a c t i v i t y seen 10 a l l leaves brown, dead 108 Table 6 - Developmental stages of shoot t i p r e g e n e r a t i o n . Week A c t i v i t y Observed 2 r h i z o i d p r o d u c t i o n p r o l i f i c 5 a c t i v e shoot development and continued r h i z o i d p r o d u c t i o n 9 shoots to 6 mm, many m u l t i p l e shoots from each fragment 13 secondary r h i z o i d i n i t i a t i o n at merophytes 109 Table 7 - Developmental stages of 1-8 cm shoot segment regenerat i o n . Week A c t i v i t y Observed ssp. t r i c o s t a t a 3 r h i z o i d i n i t i a t i o n at merophytes and at p o i n t s - o f breakage; a p i c a l growth continued; new l a t e r a l shoots 6 new shoots 10-12 mm long, p l a g i o t r o p i s m and e r e c t - s p r e a d i n g l e a f i n s e r t i o n evident 9 new shoots to 15 mm long; r h i z o i d s numerous, some a e r i a l 13 shoots e t i o l a t e d - e l o n g a t e 19 shoots > 30 mm ssp f r y e i 2 new branching; r h i z o i d i n i t i a t i o n at merophytes 3 shoots to 5 mm 4 shoots to 7 mm; leaves e n l a r g i n g ; o r t h o t r o p i s m and spreading l e a f i n s e r t i o n evident 7 shoots to 10 mm 1 1 0 regenerated p o o r l y and most died w i t h i n 2-10 weeks. Only a few developed r h i z o i d s and produced shoots from the a d a x i a l base of the c o s t a . Development was slow and sparse when compared to the other regeneration treatments. B. Shoot T i p Regeneration. Shoot t i p s 2-3 mm long i n c l u d e d the a p i c a l c e l l , the surrounding embryonic leaves and the uppermost mature leaves of a shoot, and were common i n L i m b e l l a fragment l i t t e r . The t i p s e x h i b i t e d vigorous regeneration i n c u l t u r e , producing r h i z o i d s p r o l i f i c a l l y by week 2, numerous new shoots by week 5 and secondary r h i z o i d s from merophytes by week 13. The shoot t i p s regenerated much more r a p i d l y and p r o l i f i c a l l y than d i d s i n g l e l e a v e s . C. Shoot Segment Regeneration. L i v e shoot segments of both taxa e x h i b i t e d v i g o r o u s r h i z o i d and shoot p r o d u c t i o n . E n t i r e shoot segments and segments s t r i p p e d of branches both produced new brances and r h i z o i d s by week 3. R h i z o i d a l a t t a c h e n t s were numerous. Of the three r e g e n e r a t i o n treatments, l i v e shoots developed new growth most r a p i d l y and p r o l i f i c a l l y . 111 2. D r i e d M a t e r i a l None of the d r i e d m a t e r i a l t e s t e d e x h i b i t e d r e g e n e r a t i o n a f t e r 15 weeks of c u l t u r e . Most leaves u s u a l l y turned brown and fungal decomoposers appeared by week 1, although a few l e a f t i p s remained green u n t i l week 4. In the c a s u a l experiment t e s t i n g the e f f e c t of d r y i n g temperature and d u r a t i o n of d r y i n g on r e g e n e r a t i o n , 4% of the m a t e r i a l d r i e d at 40°C produced new growth, whereas 44% of the m a t e r i a l d r i e d at 20°C produced new growth. The m a t e r i a l d r i e d at 40°C had brown leaves and fungal decomposers by week 1. F a i l u r e of d r i e d m a t e r i a l to regenerate appears to be symptomatic of most a q u a t i c mosses, which i n d r y i n g undergo ex t e n s i v e and i r r e p a r a b l e c e l l u l a r d i s r u p t i o n (Glime and V i t t 1984). In these experiments, fungi never invaded s u c c e s s f u l l y r e g e n e r a t i n g c u l t u r e s . I t appears that fungi invade c u l t u r e s as decomposers of dead or dying p l a n t m a t e r i a l , and do not " d e s t r o y . . . p o t e n t i a l l y v i a b l e fragments" as r e p o r t e d by M i l l e r and Ambrose (1976). 7.11 R e c i p r o c a l C u l t i v a t i o n 112 1. Hawai ian P l a n t s i n R e c i p r o c a l C u l t i v a t i o n A f t e r 10 weeks, ssp. t r i c o s t a t a p l a n t s grown under ssp. f r y e i c u l t u r e c o n d i t i o n s produced numerous t e r e t e , o r t h o t r o p i c to p l a g i o t r o p i c shoots to 3 cm long. Leaf i n s e r t i o n angle and b a s a l l e a f c e l l l engths remained the same as i n t y p i c a l ssp. t r i c o s t a t a . Growth r a t e s were slow. 2. North American P l a n t s i n Rec i p r o c a l C u l t i v a t ion A f t e r 10-weeks' exposure to immersion and c u r r e n t , new growth produced by ssp. f r y e i was l i m i t e d to e l o n g a t i o n (to 6 cm) of e x i s t i n g shoots. Production of new shoots and l a t e r a l branches was minimal. I n i t i a l l y , immersion caused shoots to assume the appearance of f l a g e l l i f o r m branches d e s c r i b e d i n Chapter 8. T h i s form was produced f o r a 5 mm segment of shoot, a f t e r which normal l e a f morphology resumed. Except f o r leaves of the f l a g e l l i f o r m segments, l e a f i n s e r t i o n angle and b a s a l l e a f c e l l l engths remained the same as i n t y p i c a l ssp. f r y e i . In a few cases the leaves became squarrose. 7.12 Shoot O r i e n t a t i o n Experiment O r i e n t a t i o n s e x h i b i t e d by the shoot segments secured with monofilament l i n e were the same as those of unsecured segments, ssp. t r i c o s t a t a produced p l a g i o t r o p i c shoots ranging from h o r i z o n t a l to c. 60° o r i e n t a t i o n , whereas ssp. f r y e i e x h i b i t e d i n a l l cases o r t h o t r o p i c shoots of n e a r l y v e r t i c a l o r i e n t a t i o n 1 13 ( F i g . 24). Leaf i n s e r t i o n angle and basal l e a f c e l l l e n g t h remained s t a b l e . F. DISCUSSION 7.13 Are M o r p h o l o g i c a l D i f f e r e n c e s Between the Taxa Stable? R e s u l t s of a l l c u l t i v a t i o n methods except the spore c u l t u r e s (which i n c l u d e d only ssp. t r i c o s t a t a ) provide strong evidence that two key morphological c h a r a c t e r s i d e n t i f i e d i n Chapter 3 — angle of l e a f i n s e r t i o n and b a s a l l e a f c e l l l e n g t h are s t a b l e under v a r i o u s environmental c o n d i t i o n s and can be used r e l i a b l y to d i s t i n g u i s h the taxa. Other morphological c h a r a c t e r s are t h e r e f o r e subject to some form of environmental m o d i f i c a t i o n . S t a b i l i t y of the two c h a r a c t e r s i m p l i e s that they are g e n e t i c a l l y f i x e d . Based i n part on Wyatt and Stoneburner (1984), Table 8 summarizes l i t e r a t u r e r e p o r t s of the r e l a t i v e s t a b i l i t y of morphological c h a r a c t e r s i n mosses under c u l t i v a t i o n . Table 9 summarizes the p h y s i c a l parameters thought to i n f l u e n c e expression of morphological c h a r a c t e r s . Because the same ch a r a c t e r may by r e p o r t e d as both s t a b l e and unstable by d i f f e r e n t authors, i t i s c l e a r that r u l e s r e g a r d i n g s t a b i l i t y of a given c h a r a c t e r do not apply to a l l taxa. The two s t a b l e c h a r a c t e r s observed in ssp. t r i c o s t a t a and ssp. f r y e i may not be s t a b l e i n other taxa. I t appears that l e a f i n s e r t i o n angle has never before been measured for i t s s t a b i l t y i n comparative 1 14 F i g u r e 24 - Shoot o r i e n t a t i o n e x p e r i m e n t . S.sp. t r i c o s t a t a on l e f t , s s p . f r y e i on r i g h t . Table 8 - R e l a t i v e s t a b i l i t y of morphological c h a r a c t e r s in c u l t i v a t e d bryophytes, as reported i n l i t e r a t u r e . Character S t a b l e Unstable branching p a t t e r n 7 b u l b i l morphology 5 gametophyte morphology - 11 l e a f a l a r c e l l s 6,8 apex 7,12 12 " a u r i c l e s 6 c e l l l e n g t h - 6, 12 " c e l l shape 4,12 " c e l l w a l l t h i c k n e s s - ' 12 ?! c e l l width - 9,12 c h l o r o p l a s t s i z e - 12 " number - 12 c o s t a anatomy - 12 le n g t h 8 7 width 4 6 decurrency 4,13 10 d e n t i t i o n 4,8,11 1,13 le n g t h - 7,9 margin r o l l i n g - 12 morphology 7 o i l bodies 13 12 p a p i l l a number 12 1 16 " " p o s i t i o n 12 s i z e - 12 " p l i c a t i o n - 12 shape 11,12 6,8,12 s i z e - 6,8,12 " spa c i n g - 12 width - 9 p h y l l o t a x y - 12 propagula abundance - 12,13 protonemal morphololgy 7 -ps e u d o s t e r e i d s - 2 r h i z o i d abundance - 12 seta p a p i l l a e '11 sporophyte morphology 11 stem l e n g t h - 7,12 References: (1) Agnew 1958 (2) F l o r s c h u t z - d e Waard & W o r r e l l -Schets 1980 (3) Forman 1964 (4) Koponen 1967 (5) Lewis & Smith 1977 (6) Lodge 1960 (7) Longton 1981 (8) Sonessen 1966 (9) S t e e l 1978 (10) Wigh 1972 (11) Wigh 1975 (12) Zales 1973 (13) Zehr 1980. 117 Table 9 - P h y s i c a l parameters i n f l u e n c i n g e x p r e s s i o n of morphological c h a r a c t e r s , as reported i n l i t e r a t u r e . Character Humidity, Water L i g h t Temperature l e a f apex 2 - -" awn le n g t h 3 3 -c e l l l e n g t h 5 " c e l l s i z e 7 w a l l t h i c k n e s s 2,7 -" c h l o r o p l a s t number 2 -s i z e 2 - -" c o s t a length 4 4 -" " t h i c k n e s s 7 - -d e n t i t i o n 2 -l e n g t h 2 -" margin r o l l i n g 7 -shape 3,4 3,4 3 s i z e - - 3 spacing 7 - -II p h y l l o t a x y 4 4 p l a n t morphology 1 1 r h i z o i d abundance 2 stem c e n t r a l s t r a n d 7 l e n g t h 7 118 Changes i n humidity caused g r e a t e r m o r p h o l o g i c a l v a r i a b i l i t y than changes i n l i g h t ( 6 ) . References: (1) Buch 1928 (2) Davy de V i r v i l l e 1927 (3) Forman 1964 (4) Lodge 1960 (5) Seppelt & S e l k i r k 1984 (6) Wigh 1975 (7) Zastrow 1934. 119 c u l t i v a t i o n s t u d i e s . 7.14 Are V e g e t a t i v e Fragments V i a b l e Diaspores i n L i m b e l l a ? Based on the experimental evidence, v e g e t a t i v e fragments of L i m b e l l a are e x c e l l e n t d i a s p o r e s as long as they remain moist or are d r i e d below 20°C and remain dry f o r short p e r i o d s o n l y . Fragments d r i e d at higher temperatures or f o r longer p e r i o d s do not regenerate. Shoot fragments, r o u t i n e l y broken from mature p l a n t s in the f i e l d , e x h i b i t e d the most r a p i d and p r o l i f i c r e g e n e r a t i o n . Shoot t i p s , most commonly broken o f f a q u a t i c p l a n t s by stream c u r r e n t s , e x h i b i t e d r a p i d r e g e n e r a t i o n and are e x c e l l e n t propagules f o r t h e i r small s i z e . S i n g l e leaves seem to be the l e a s t e f f e c t i v e propagules, i n c o n t r a s t to the o b s e r v a t i o n s of Gemmell (1953), Longton and Greene (1979) and S e l k i r k (1980). G. SUMMARY AND CONCLUSIONS C u l t i v a t i o n of ssp. t r i c o s t a t a and ssp. f r y e i f o r nine months pro v i d e d (1) l i v e m a t e r i a l f o r e l e c t r o p h o r e s i s and karyotype a n a l y s i s (2) a means to determine the v i a b i l i t y of spores and v e g e t a t i v e fragments as d i a s p o r e s and (3) a means to determine whether m o r p h o l o g i c a l d i f f e r e n c e s between the two taxa are e n v i r o n m e n t a l l y or g e n e t i c a l l y a l t e r e d . Spores were c l e a r l y v i a b l e and produced a s p o r e l i n g of the Bryum type. L i v i n g v e g e t a t i v e fragments were a l s o shown to be v i a b l e d i a s p o r e s , t h e i r e f f i c i e n c y d i r e c t l y p r o p o r t i o n a l to the 120 s i z e of the fragment. V e g e t a t i v e fragments d r i e d longer than a few days or months d i d not regenerate. Two morphological c h a r a c t e r s which d i s t i n g u i s h the taxa, angle of l e a f i n s e r t i o n and b a s a l l e a f c e l l l e n g t h , were s t a b l e under a v a r i e t y of c u l t i v a t i o n regimes, i n c l u d i n g r e c i p r o c a l c u l t i v a t i o n , and appeared to be g e n e t i c a l l y c o n t r o l l e d . Other morphological c h a r a c t e r s v a r i e d with enviroment. In c u l t i v a t i o n , ssp. t r i c o s t a t a produced shoot growth of mostly p l a g i o t r o p i c o r i e n t a t i o n , whereas ssp. f r y e i produced c o n s i s t e n t l y o r t h o t r o p i c shoots. These d i f f e r e n c e s are u s e f u l taxonomic c h a r a c t e r s which support r e c o g n i t i o n of ssp. f r y e i as a d i s t i n c t taxon. 121 VI11. TAXONOMIC TREATMENT A. INTRODUCTION Based on the forego i n g experimental evidence, t h i s chapter documents the taxonomic d i s t i n c t i o n between t r i c o s t a t a ssp. t r i c o s t a t a and ssp. f r y e i . A generic d e s c r i p t i o n , a key to subspecies, c i t a t i o n of synonymy and type specimens, s p e c i e s d e s c r i p t i o n s , b i b l i o g r a p h i e s and p e r t i n e n t h i s t o r i c a l notes are presented. C i t a t i o n of synonyms, type specimens and specimens examined f o l l o w s Frahm and G r a d s t e i n (1984). D e s c r i p t i o n s f o l l o w the terminology of Flowers (1973) and I r e l a n d (1982). F i g u r e s were prepared with a Z e i s s drawing tube. B. TAXONOMIC TREATMENT Li m b e l l a (CM.) CM., F l o r a 82:466. 1896. Hypnum s e c t . L i m b e l l a CM., Forschungsr. " G a z e l l e " Bot. 4:37. 1889. Sciaromium ( M i t t . ) M i t t . , J . L i n n . Soc. Bot. 12:571. 1869, non Leskea s e c t . Sc iaromium M i t t . , J . L i n n . Soc. Bot. 8:7. 1864. [= Echinodium J u r . ] . PLANTS pleurocarpous, shoots dendroid or t r a i l i n g , to 0.5 m long. LEAVES smooth, arranged s p i r a l l y on stem, homomallous or 1 22 s t r a i g h t , to 3.5 mm long, concave, k e e l e d , u n i s t r a t o s e , sometimes with b i s t r a t o s e regions i n lamina or at i n s e r t i o n ; c o s t a s t r o n g , to 120 u.m t h i c k at base, of 4-6 l a y e r s of s t e r e i d s , p e r c u r r e n t or ex c u r r e n t , sometimes forked; margins limbate, l i m b i d i a s t r o n g , to 75 /xm t h i c k at base, of 2-4 l a y e r s of s t e r e i d s , u s u a l l y d i s a p p e a r i n g j u s t below acumen, u s u a l l y submarginal, sometimes forked; upper c e l l s quadrate to rhomboidal; b a s a l c e l l s mostly oblong. Note: L i m b e l l a CM. i s the c o r r e c t generic name (Buck i n ms., 1983). Sc iaromium, f i r s t d e f i n e d by M i t t e n ( 1864) as a s e c t i o n of Leskea, accomodated only Echinodium J u r . and predates that genus by two y e a r s . Echinodium should be conserved or submerged as a synonym of Sc iaromium. When M i t t e n (1869) e l e v a t e d Sc iaromium to generic rank, the s p e c i e s he d e s c r i b e d (sensu L i m b e l l a , non Echinodium ) must be as s i g n e d to L i m b e l l a , the next a v a i l a b l e name. KEY TO SUBSPECIES 1a. P l a n t s robust, mature shoots 7-20(50) cm long, lower branches 3-10 cm long; l e a v e s e r e c t - s p r e a d i n g 35-45°, b a s a l l e a f c e l l s to (32)60-77 Mm long; inner p e r i c h a e t i a l b r a c t s u s u a l l y e c o s t a t e ; on rock, r a r e l y on s o i l or wood, aq u a t i c or emergent i n p e r e n n i a l or i n t e r m i t t e n t streambeds; Hawaiian I s l a n d s L_j_ t r i c o s t a t a ssp. t r i c o s t a t a 1 23 1b. P l a n t s s l e n d e r , mature shoots 4-8(13) cm long, lower branches 1.5-4 cm long; leaves spreading 50-60°, b a s a l l e a f c e l l s to 25-50(60) m long; inner p e r i c h a e t i a l b r a c t s c o s t a t e ; on wood, peat, or bark, r a r e l y a q u a t i c , i n dense swampy shrub-carr; c o a s t a l northwestern North America (Oregon) t r i c o s t a t a ssp. f r y e i LIMBELLA TRICOSTATA ( S u l l . ) CM., F l o r a 82:466. 1896. Neckera t r i c o s t a t a S u l l . , Proc. Am. Acad. A r t s S c i . 3:81. 1854. Hypnum t r i c o s t a t u m ( S u l l . ) S u l l . , U.S. E x p l . Exped. Wilkes Musci 13. 1859. Sciaromium t r i c o s t a t u m ( S u l l . ) M i t t . i n Seem., F l . V i t . 400. 1873. Hypnodendron t r i c o s t a t u m ( S u l l . ) Jaeg. i n Jaeg. & Sauerb., Ber. That. S t . G a l l . Naturw. Ges. 1877-78:360. 1879. Lectotype nov.: " U.S. Ex. Ex. Wilkes 1838-42," (FH l e c t o , BM i s o l e c t o , NY i s o l e c t o ) . [ Un i t e d S t a t e s  E x p l o r i n g E x p e d i t i o n 12 ( ? ) , W20 ( ? ) , 1840-1841; "Hab. f o r e s t at eas t e r n base of Mauna Kea, Hawaii, Sandwich I s l a n d s ( S u l l i v a n t 1854)]. Note: The specimen at FH was chosen as the l e c t o t y p e because i t i s c l e a r l y the same specimen as that d e p i c t e d by S u l l i v a n t (1859) and i s from S u l l i v a n t ' s p e r s o n a l herbarium. 1 24 Hypnum su b t r i c o s t a t u m CM., Forschungsr. " G a z e l l e " Bot. 4: 37. 1889. nom. nud.  Sc iaromium subtr icostatum (CM.) Par., Ind. B r y o l . 1156. 1898. nom. nud. Type: not seen. May have been destroyed with the M i i l l e r herbarium (Schultze-Motel 1977). No l e c t o t y p e was chosen because no m a t e r i a l assigned to t h i s name was seen. L i m b e l l a i n t r a l i m b a t a Card., Annuair. Cons. J a r d . Bot. Geneve 15-16: 176. 1912. Hypnodendron intralimbatum (Card.) Broth., Nat. P f l . ed. 2, 11: 531. 1925. Type: not seen. May be at PC. Se v e r a l loan"requests sent to that i n s t i t u t i o n were not acknowledged. No m a t e r i a l assigned to t h i s name was seen. L i m b e l l a leptolomacea CM., F l o r a 82: 467. 1896. Sciaromium leptolomaceum (CM.) Par., Ind. B r y o l . 1155. 1898. Hypnodendron leptolomaceum (CM.) Broth., Nat. P f l . 1 (3) : 1170. 1909. Lectotype nov.: "Insulae Hawaiicae [Ka u a i ? ] , H i l l e b r a n d [s.n., no d a t e ] , Hb. C. M u l l . , Herb. Emile B e s c h e r e l l e : 1900." (BM). Li m b e l l a l i m b a t u l a CM., F l o r a 82: 467. 1896. 125 Sc iaromium 1 imbatulum (CM.) Par., Ind. B r y o l . 1155. 1898. Hypnodendron 1imbatulum (CM.) Broth., Nat. P f l . 1(3): 1170. 1909. Type: not seen. May have been destroyed with the M i i l l e r herbarium (S c h u l t z e - M o t e l 1977). No l e c t o t y p e was chosen because no m a t e r i a l assigned to t h i s name was seen. Sc iaromium f l a g e l l a r e Broth. i n Lev., B u l l . Bot. Soc. I t a l . 1904: 23. 1904. nom. nud. Sc iaromium f l e x i c a u l e Broth. i n Lev., B u l l . Bot. Soc. I t a l . 1904: 23. 1904. nom. nud. Sc iaromium p o r o t r i c h o i d e s Broth. i n Lev., B u l l . Bot. Soc. I t a l . 1904: 23. 1904. nom. nud. PLANTS yellow-green to dark green or b l a c k i s h , t r a i l i n g or sometimes d e n d r o i d , when submerged sometimes forming mats up to 5 m diameter, p r o l i f e r a t i n g by monopodial branching. STEMS 7-20(50) cm l o n g , round to t r a n s v e r s e l y e l l i p t i c i n s e c t i o n , (240)300-650(1200) urn diameter, 14-30(48) c e l l s wide, with small c e n t r a l s t r a n d ; c o r t i c a l l a y e r 20-40 txm wide, composed of 3-6 l a y e r s of s t e r e i d s ; r h i z o i d s dark r e d d i s h brown, i n dense c l u s t e r s at base of monopodial branches; branches + d i s t a n t , 3-10 cm long, u s u a l l y a r i s i n g from every f o u r t h merophyte. LEAVES 1 26 somewhat c o n t o r t e d when dry; Upper branch leaves ovate-oblong to o v a t e - l a n c e o l a t e , sometimes homomallous, e r e c t - s p r e a d i n g 30-45°, acuminate or c u s p i d a t e , concave, keeled, 2.2-3.5x0.5-1.4 mm; margins plane, u n i s t r a t o s e , 1(2) c e l l s wide, s e r r u l a t e or s e r r a t e i n upper t h i r d with 1-(2-) c e l l e d t e e t h 7-20 Mm long d e s c r i b i n g angle of 60-75°, u s u a l l y s e r r u l a t e to base by p r o j e c t i n g c e l l ends; c o s t a s t r o n g , p e r c u r r e n t to s h o r t l y e x c u r r e n t , depressed ovate i n s e c t i o n , 68-120x28-48 Mm at base, of 5-6 l a y e r s of s t e r e i d s ; l i m b i d i a submarginal, d i s a p p e a r i n g j u s t , below acumen, t r a n s v e r s e l y e l l i p t i c i n s e c t i o n , 30-50(75)x21-35 urn, of (2)4 l a y e r s of s t e r e i d s ; l e a f a p i c e s , e x c l u d i n g c u s p i d a t e t i p s , 30-50(80)°; upper c e l l s subquadrate to rhomboidal, 5-36x3-18 Mm; b a s a l c e l l s mostly oblong, 7-77x3-18 Mm, w a l l s at i n s e r t i o n i n c r a s s a t e , s p a r i n g l y p i t t e d , o f t e n y ellow. F l a g e l l i f o r m branches o c c a s i o n a l l y produced, the leaves reduced t o 0.4-0.8x0;1-0.3 mm, wide-spreading to squarrose, l i m b i d i a absent or po o r l y developed; Lower stem leaves brownish, e l o n g a t e - t r i a n g u l a r to d e l t o i d , p l i c a t e , acuminate-subulate, spreading 10-40°, 3-3.5x1.4-1.8 mm; margins u n i s t r a t o s e , 1(2) c e l l s wide, forming a u r i c l e s 2-10 c e l l s wide, s e r r a t e or s e r r u l a t e to base with t e e t h to 7 urn long, d e s c r i b i n g angle of 80°; c o s t a p e r c u r r e n t to long-excurrent, depressed obovate i n s e c t i o n , 165-225(300)x40~75 Mm at base, of 6 l a y e r s of s t e r e i d s ; l i m b i d i a submarginal, absent i n lowermost l e a v e s , d i s a p p e a r i n g w e l l below acumen, t r a n s v e r s e l y e l l i p t i c i n s e c t i o n , 40-75(100)x30-35 um at base, of 2(4) l a y e r s of s t e r e i d s ; l e a f a p i c e s 10°; upper c e l l s mostly oblong, 14-60x3-10 ixm; b a s a l c e l l 1 27 oblong-vermicular, 30-94x3-14 jum, w a l l s at i n s e r t i o n i n c r a s s a t e , p i t t e d , o f t e n yellow or brown. D i o i c o u s . PERICHAETTA 2-2.5 mm long, on upper p o r t i o n of stem or branches; b r a c t s 18-25, c o s t a weak or absent, l i m b i d i a absent; inner b r a c t s e l o n g a t e - t r i a n g u l a r , acuminate to subulate, squarrose, 1-2x0.8-1 mm, margins e n t i r e or s e r r u l a t e at apex; c e l l s vermiform above, oblong at base, 32-1 05(1 50)x3-1 8 (xm, w a l l s mostly i n c r a s s a t e , p i t t e d , o f t e n yellow-brown at i n s e r t i o n ; archegonia 45-55; paraphyses of 15-30 c e l l s , t r a n s v e r s e w a l l s i n c r a s s a t e d i s t a l l y , h y a l i n e or pale yellow except fo r 1-5(10) brownish b a s a l c e l l s , a p i c a l c e l l s acute. PERIGONIA 1-1.5 mm long, on upper p o r t i o n of stem or branches; b r a c t s 12-20, c o s t a weak or absent, l i m b i d i a absent; inner b r a c t s broadly ovate-acuminate, concave, 0.8-1x0.5 mm, margins e n t i r e or s e r r u l a t e at apex; c e l l s vermiform above, oblong at base, 18-80x5-14 Mm, w a l l s mostly i n c r a s s a t e , p i t t e d , o f t e n yellow-brown at i n s e r t i o n . A n t h e r i d i a 2-16. SETA 1.5-3.5 cm long, smooth, s t r a i g h t or f l e x u o s e , o f t e n tortuose i n a q u a t i c p l a n t s , 310-360 Mm diameter, with c e n t r a l s t r a n d ; c o r t i c a l l a y e r 35-45 Mm wide, composed of 3-5 l a y e r s of s t e r e i d s . SPORANGIUM h o r i z o n t a l to cernuous, o b l o n g - c y l i n d r i c , asymmetric, sometimes arc u a t e , 1.5-3 mm long, u s u a l l y shrunken below mouth when dry; annulus of 2-3 rows of c e l l s , deciduous; operculum c o n i c -a p i c u l a t e to r o s t r a t e , 0.5-1.5 mm long; stomata s u p e r f i c i a l ; peristome t e e t h 16, yellow-brown, l a n c e o l a t e , sometimes c r i b o s e at base, c r o s s - s t r i o l a t e below, p a p i l l o s e above, t r a b e c u l a t e at back; endostome h y a l i n e or pale yellow, with high b a s a l 128 membrane, a p p e n d i c u l a t e . SPORES minutely p a p i l l o s e , 14-18 um, green. CALYPTRA 4-5 Mm, naked, c y l i n d r i c , c u c u l l a t e , e a r l y -deciduous. CHROMOSOME NUMBER : n=11. ILLUSTRATIONS: FIGS. 25-28. Bartram 1933: F i g . 96 a-e. Brotherus 1909: F i g . 824 a-e; 1924: F i g . 385 a-e. C h r i s t y 1980: F i g s . 1-6. S u l l i v a n t 1859: P i . 9B, F i g s . 1-13. BIBLIOGRAPHY: Bartram 1933: 132; 1942: 330. Brotherus i n L e v i e r 1904: 23; 1909: 1170; 1924: 438; 1925: 531; 1927: 23, 37. Cardot 1912: 176. C h r i s t y 1980. Crosby 1965: 460. Degener et a l . 1973: 6. Hoe 1967: 111, 121; 1974: 5f 1979: 65, 78, 291, 305, 328. Hprmann 1965: 552. Jaeger and Sauerbeck 1879: 360. McClure and M i l l e r 1967: 120. M i l l e r 1954: 70. M i l l e r et a l . 1978: 252. M i t t e n 1873: 400. Mul l e r 1889: 37; 1896: 466; 1897: 119. P a r i s 1898: 1155; 1904: 375; 1905: 238, 240. Schu l t z e - M o t e l 1963: 102. Smith 1967: 241. S u l l i v a n t 1854: 81; 1859: 13; 1862: 46. Wijk et a l . 1962: 533; 1964: 168, 304, 460; 1967: 372; 1969: 761, 893. SPECIMENS EXAMINED: BISH (49), BM (13), CAS (3), COLO (5), FH (25), H-BR (16), 129 F i g u r e 25 - Ssp. t r i c o s t a t a , d endroid p l a n t s . Note monopodial branching ( C h r i s t y 5196 ). F i g u r e 26 - Ssp. t r i c o s t a t a , t r a i l i n g a q u a t i c p l a n t s . Branches spread t o show branching p a t t e r n ( C h r i s t y 5245 F i g u r e 27 - Ssp. t r i c o s t a t a , upper branch l e a v e s . Fosberg 10233 ). 1 32 F i g u r e 28 - Ssp. t r i c o s t a t a . A. - upper l e a f c e l l s . B, - b a s a l l e a f c e l l s (both Fosberg 10233 ). B 1 33 M (1), MO (5), NY (30), S (12), UBC (9), UC (5), US (6), WTU (3), YU (1). Note: 32 specimens c o l l e c t e d f o r t h i s r e s e a r c h , i n c l u d i n g 22 with sporophytes, w i l l be d i s t r i b u t e d to a l l h e r b a r i a shown above. LIMBELLA TRICOSTATA ssp. FRYEI ( W i l l i a m s ) C h r i s t y , comb. et s t a t . nov. Sc iaromium f r y e i W i l l i a m s , B r y o l . 35: 52. 1933. Lectotype nov.: "Cape Arago, Oregon," Frye s.n., 8 . v i i i . l 9 2 2 (NY l e c t o , UBC i s o l e c t o , WTU i s o l e c t o ) ["Oregon, Coos County, Cape Arago, 1-1.5 m i l e s northeast of C h a r l e s t o n on on east s i d e of highway. On ground i n more or l e s s wet p a s t u r e " (UBC, WTU)]. Note: The specimen at NY was chosen as the l e c t o t y p e because i t i s presumably the same specimen upon which Wi l l i a m s (1933) based h i s d e s c r i p t i o n and i l l u s t r a t i o n . PLANTS yellow-green to dark green, dendroid or sometimes t r a i l i n g , forming sods to 1m diameter, p r o l i f e r a t i n g by monopodial branching. STEMS 4-13 cm l o n g , round to t r a n s v e r s e l y e l l i p t i c i n s e c t i o n , 165-480 am diameter, 13-24 c e l l s wide, with sma l l c e n t r a l s t r a n d ; c o r t i c a l l a y e r 25-75 um wide, composed of 3-7 l a y e r s of s t e r e i d s ; r h i z o i d s dark reddish-brown, i n dense c l u s t e r s at base of monopodial branches; branches crowded, 1.5-4 cm long, u s u a l l y a r i s i n g i n c l u s t e r s from adjacent merophytes. 134 LEAVES somewhat c o n t o r t e d when dry; Upper branch leaves ovate-oblong to o v a t e - l a n c e o l a t e , spreading 50-60°, acuminate or c u s p i d a t e , concave, keeled, 1.5-3.8x0.1-1 mm; margins plane, u n i s r a t o s e , 1(2) c e l l s wide, s e r r a t e or s e r r u l a t e i n upper t h i r d with t e e t h 14-25 Mm long d e s c r i b i n g angle of 60-80°, u s u a l l y s e r r u l a t e to base by p r o j e c t i n g c e l l ends; c o s t a strong, p e r c u r r e n t to s h o r t l y e x c u r r e n t , depressed obovate i n s e c t i o n , 52-75(100)x35-48 Mm at base, of 5-6 l a y e r s of s t e r e i d s . Leaf a p i c e s , e x c l u d i n g cuspidate t i p , 15-60°; l i m b i d i a submarginal, d i s a p p e a r i n g j u s t below acumen, t r a n s v e r s e l y e l l i p t i c in s e c t i o n , 30-55(70)x(8)20-32 Mm at base, of 3(4) l a y e r s of s t e r e i d s . Upper c e l l s subquadrate to rhomboidal, 3-32x3-14 Min; b a s a l c e l l mostly oblong, 7-50(60)x3-14 Mm, w a l l s at i n s e r t i o n i n c r a s s a t e , s p a r i n g l y p i t t e d , o f t e n yellow. F l a g e l l i f o r m branches o c c a s i o n a l l y produced, the leaves reduced to 0.4-0.8x0.1-0.3 mm, wide-spreading to squarrose, l i m b i d i a absent or p o o r l y developed; Lower stem leaves e l o n g a t e - t r i a n g u l a r to d e l t o i d , p l i c a t e , acuminate-subulate, spreading 40-60°, 2.5-3.5x1 mm; margins u n i s t r a t o s e , 1(2) c e l l s wide, forming a u r i c l e s 2-3(6) c e l l s wide, s e r r a t e or s e r r u l a t e to base with t e e t h to 25 Mm long, d e s c r i b i n g angle of 45-80°; c o s t a p e r c u r r e n t to s h o r t l y e x c u r r e n t , depressed obovate i n s e c t i o n , 115-130x40-48 Mm at base, of 5-6 l a y e r s of s t e r e i d s ; l i m b i d i a submarginal, d i s a p p e a r i n g w e l l below acumen, t r a n s v e r s e l y e l l i p t i c i n s e c t i o n , 60-75x25-28 nm at base, of 2-4 l a y e r s of s t e r e i d s ; l e a f a p i c e s 10-35°; upper c e l l s quadrate to oblong, 7-36x5-18 Mm; b a s a l c e l l s mostly oblong, 7-40x5-14 Mm, w a l l s at i n s e r t i o n 1 35 i n c r a s s a t e , s p a r i n g l y p i t t e d , o f t e n yellow or brown. D i o i c o u s . PERICHAETIA 1.5-2.5 mm long, on upper p o r t i o n of stems or branches. B r a c t s 14-30, c o s t a t e , l i m b i d i a absent; inner b r a c t s e l o n g a t e - t r i a n g u l a r , acuminate to . sub u l a t e , squarrose, 1.5-2.5x0.8-1 mm, s e r r a t e or s e r r u l a t e to base with t e e t h to 18 Mm; c e l l s o blong-vermicular, 22-112x3-22 Mm, w a l l s mostly i n c r a s s a t e , o f t e n yellow-brown at i n s e r t i o n , not p i t t e d ; archegonia 30-55. PERIGONIA unknown. SPOROPHYTES unknown. CHROMOSOME NUMBER : n=11. ILLUSTRATIONS: •FIGS 29-32. Grout 1934: P i . 80, F i g s . 1-8. Kawai 1968: F i g I I , 1. Lawton 1971: P i . 158, F i g s . 11-17. W i l l i a m s 1933: F i g s 1-8. BIBLIOGRAPHY: Andrews 1945:102. C h r i s t y 1980. Crum & Steere 1958: 41. Grout 1934: 266. Hoe 1979: 65. I r e l a n d 1971: 328. Kawai 1968: 130, 147. Lawton 1971: 287. Wijk et a l . 1967: 372. Wi l l i a m s 1933. SPECIMENS EXAMINED: BISH (5), NY (6), UBC (7), US .(5), WTU (7). Note: 11 specimens c o l l e c t e d f o r t h i s r e s e a r c h w i l l be d i s t r i b u t e d to the h e r b a r i a shown above and on page 127. 136 '137 F i g u r e 30 - Ssp. f r y e i , t r a i l i n g p l a n t s ( C h r i s t y 5338 ). 138 139 F i g u r e 32 - Ssp. f r y e i . A. - upper l e a f c e l l s . B, b a s a l l e a f c e l l s (both C h r i s t y 5338 ). 1 40 C. COLLECTIONS OF TAXONOMIC AND HISTORICAL SIGNIFICANCE Appendix A o u t l i n e s the h i s t o r i c a l sequence of ssp. t r i c o s t a t a c o l l e c t i o n s , which l a r g e l y p a r a l l e l s the h i s t o r y of Hawaiian bryology. Hoe (1979) d e s c r i b e d the important c o l l e c t i o n s of most of these workers, and provided b i o g r a p h i c a l data f o r some. 8.1 E a r l i e s t C o l l e c t i o n of L i m b e l l a t r i c o s t a t a L. t r i c o s t a t a appears to have been c o l l e c t e d f i r s t by James Macrae i n 1825 (St. John 1978; Hoe 1979), 29 years before i t was d e c r i b e d by S u l l i v a n t (1854). Although Macrae's c o l l e c t i o n s were r e p o r t e d l y sent to Hooker and De C a n d o l l e , among others (Mann 1866), i t i s s u r p r i s i n g that L^ t r i c o s t a t a was not d e s c r i b e d f i r s t by European b r y o l o g i s t s . Macrae c o l l e c t e d ssp. t r i c o s t a t a on Oahu ("Woahoo; Ins. Sandwich. Macrae. Maio, 1825"; BM [Hb. Hooker], NY [Hb. M i t t e n ] ) and Hawaii ("Ins. Owhyhee, ad montem Kaah [Mauna Kea]. Macrae. -Junio, 1825"; BM [Hb. Hooker], NY [Hb. M i t t e n ] ) . Macrae's ssp. t r i c o s t a t a specimens at BM and NY were probably named by M i t t e n , who c i t e d them f o r t h e ' f i r s t time i n Seeman's F l o r a  V i t i e n s i s (Mitten 1873). Traces of glue on the sheets i n the Hooker herbarium at BM match — i n reverse — the p l a n t s i n the M i t t e n herbarium at NY, i n d i c a t i n g that M i t t e n obtained the specimens from Hooker, presumably a f t e r L_j_ t r i c o s t a t a had been d e s c r i b e d by S u l l i v a n t i n 1854. 141 8.2 Type C o l l e c t i o n of Neckera t r i c o s t a t a S u l l . The type specimen of ssp. t r i c o s t a t a was c o l l e c t e d i n 1840 or 1841, presumably by e i t h e r W i l l i a m Dunlop Brackenridge or C h a r l e s P i c k e r i n g , c o l l e c t o r s f o r the U n i t e d States E x p l o r i n g E x p e d i t i o n ("Wilkes E x p e d i t i o n " ) . Three type specimens e x i s t , d e p o s i t e d at FH ( l e c t o , herb. S u l l i v a n t ) , BM ( i s o l e c t o , herb. Hooker) and NY ( i s o l e c t o , herb. S m a l l ) . A l l bear handwritten l a b e l s s t a t i n g : "U.S. Ex. Ex. Wilkes 1838-1842," the only l a b e l data given f o r any of the E x p e d i t i o n ' s specimens (Sayre 1975). S u l l i v a n t (1854) s u p p l i e d a d d i t i o n a l data: "Hab. f o r e s t at the e a s t e r n base of Mauna Kea, Hawaii, Sandwich I s l a n d s . " The specimens at FH and BM are numbered "124," the NY specimen "W203." 8.3 F i r s t C o l l e c t i o n of L. t r i c o s t a t a Sporophytes Although a l l r e l e v a n t l i t e r a t u r e up u n t i l Hoe (1979) s t a t e d that sporophytes were unknown f o r ssp. t r i c o s t a t a , sporophytes were c o l l e c t e d by David Dwight Baldwin as e a r l y as 1875 ( C h r i s t y 1980). During the i n t e r v e n i n g 105 years, four specimens with sporophytes had been d i s t r i b u t e d : Baldwin 3 ("Insulae Sandwich, i n s . Maui o c c i d . , ad rupes i n f a u c i b u s humidis, 4500 p., 1875." [NY]), Baldwin 8 ("Maui, 1875-1900+." [BISH]), and Baldwin 206 and 207 ("In i n s u l a . " [NY]). The specimens at NY bear the l a b e l "Plantae Hawaiienses. D u p l i c a t e from herb D.C. Eaton, purchased from J.K. Small, 1920.", and were probably sent to Eaton by Baldwin ( M i l l e r 1956). The specimen at BISH was 1 42 obtained from Baldwin's granddaughter i n 1960. Some sporophytes of the three specimens at NY were mounted on mica s l i d e s , but i t i s not known by whom, and sporophytes were never rep o r t e d i n the l i t e r a t u r e u n t i l 1980. D. RATIONALE FOR TAXONOMIC DISPOSITION OF SUBSPECIES It i s w e l l known that no f i x e d d e f i n i t i o n e x i s t s f o r the term " s p e c i e s " (Wagner 1984), and i n v e s t i g a t o r s must d e f i n e species a c c o r d i n g to c r i t e r i a s p e c i f i c to the group being s t u d i e d (Ganders and Nagata 1984). The q u e s t i o n of s p e c i e s d e f i n i t i o n i n bryophytes has been reviewed r e p e a t e d l y without r e s o l u t i o n (Steere 1947; Anderson 1963; Crundwell 1970; Greene 1976; Richards 1978). However, there i s general agreement that bryophyte s p e c i e s must be d e l i m i t e d by a s u i t e of c o n s i s t e n t l y -expressed c h a r a c t e r s which, h o p e f u l l y , i n c l u d e data from s e v e r a l b i o s y s t e m a t i c sources (Szweykowski 1978). My d e c i s i o n to recognize Sc iaromium f r y e i at the s u b s p e c i f i c rank, i n s t e a d of s p e c i f i c rank or as a synonym of L. t r i c o s t a t a , was based on two reasons: 1. S i m i l a r i t i e s of the taxa are greater than t h e i r  d i f f e r e n c e s . Sciaromium f r y e i c o n s i s t e n t l y e x h i b i t s c h a r a c t e r s which are d i s t i n c t from L i m b e l l a t r i c o s t a t a , r e g a r d l e s s of geography. However, the m a j o r i t y of c h a r a c t e r s expressed by S_^  f r y e i are shared by 143 L. t r i c o s t a t a , i n d i c a t i n g that the two taxa are c l o s e l y r e l a t e d . 2. Cohesion w i t h i n the genus. A genus should be c o n s t r u c t e d i n a manner most c l e a r l y e x p r e s s i n g the r e l a t i o n s h i p s between i t s c o n s t i t u e n t s p e c i e s . Because Sc iaromium f r y e i and L i m b e l l a t r i c o s t a t a are c l e a r l y more s i m i l a r to one another than e i t h e r i s to any other member of the genus, and because they d i f f e r i n so few c h a r a c t e r s , t h e i r r e l a t i o n s h i p i s best expressed by s u b s p e c i f i c rank as d e f i n e d by R i c h a r d s (1978). Both s p e c i f i c rank and f u l l synonymy would obscure the r e l a t i o n s h i p between them. E. CONCLUSION AND SUMMARY Character d i f f e r e n c e s between Sc iaromium f r y e i and L i m b e l l a  t r i c o s t a t a are not great enough to warrant s p e c i f i c rank f o r S.  f r y e i . Sc iaromium f r y e i i s r e c o g n i z e d as a subspecies of L.  t r i c o s t a t a , thus best e x p r e s s i n g the r e l a t i o n s h i p between the two taxa. Ample type m a t e r i a l e x i s t s for both taxa, but types could not be found f o r three synonyms of ssp. t r i c o s t a t a . A r e l a t i v e l y l a r g e body of l i t e r a t u r e e x i s t s f o r ssp. t r i c o s t a t a , but ssp. f r y e i i s p o o r l y documented. 1 44 IX. PHYTOGEOGRAPHY A. INTRODUCTION The Northern Hemisphere d i s t r i b u t i o n of L i m b e l l a i s c h a r a c t e r i z e d by broad t r a n s o c e a n i c d i s j u n c t i o n s . I n t e r p r e t a t i o n of these d i s j u n c t i o n s r e q u i r e s a n a l y s i s of s e v e r a l p o s s i b l e d i s p e r s a l mechanisms and r e l a t e d h i s t o r i c a l events. T h i s chapter d e s c r i b e s the .modern d i s t r i b u t i o n s of L i m b e l l a t r i c o s t a t a ssp. t r i c o s t a t a and L_;_ t r i c o s t a t a ssp. f r y e i i n some d e t a i l , f o l l o w e d by o b s e r v a t i o n s on l o n g - d i s t a n c e d i s p e r s a l , i t s a l t e r n a t i v e s , and p u t a t i v e migratory paths. These o b s e r v a t i o n s are used to e x p l a i n both the means and approximate age of L i m b e l l a d i s p e r s a l . B. PRESENT DISTRIBUTION 9.1 D i s t r i b u t i o n of L i m b e l l a t r i c o s t a t a ssp. t r i c o s t a t a L i m b e l l a t r i c o s t a t a ssp. t r i c o s t a t a i s known from only the Hawaiian i s l a n d s of Hawaii, Kauai, Maui, Molokai and Oahu ( F i g s . 33-37; Hoe 1974). Although r a r e l y c o l l e c t e d as low as 90 m, (Hawaii: Bishop & Herbst s.n., 1 1 . v i i i . 1 9 6 6 , BISH; Molokai: Hoe  2216.0, BISH), most p l a n t s occur between 1200-2130 m. Common and o f t e n abundant where i t occurs, ssp. t r i c o s t a t a i s known from about 110 d i f f e r e n t herbarium c o l l e c t i o n s , many of these d i s t r i b u t e d widely as d u p l i c a t e specimens. Were i t not -44.5 1 46 F i g u r e 34 - Sur face , and e l e v a t i o n a l d i s t r i b u t i o n of s s p . t r i c o s t a t a on K a u a i , H a w a i i . A A i 1 A 10 km N Contour interval 300 m 4200 3600 3000 • 'a g 2400 • U «j 1800 • A A' Degrees Longitude 147 F i g u r e 35 - S u r f a c e and e l e v a t i o n a l d i s t r i b u t i o n of s sp . t r i c o s t a t a on M a u i , H a w a i i . A Contour interval 300 m 4200 -i 3600 1 B Degrees Longitude 1 4 8 F i g u r e 36 - Sur f a c e and e l e v a t i o n a l d i s t r i b u t i o n of ssp. t r i c o s t a t a on Mo l o k a i , Hawaii. MOLOKAI i . A 10 km N Contour interval 300 tn 4200 -I 3600 \ 3000 g 2400 • T-t U a « 1800 • A A' Degrees Longitude ,149 F i g u r e 37 - S u r f a c e and e l e v a t i o n a l d i s t r i b u t i o n of s s p . t r i c o s t a t a on Oahu, H a w a i i . 10 km N Contour interval 300 m 4200 l 3600 3000 2400 i 1800 A1 Degrees Longitude 1 50 for the p l a n t s ' l a r g e s i z e and Hawaii's renown as a c e n t e r of endemism, the l a t t e r having a t t r a c t e d many i n d u s t r i o u s b o t a n i s t s over the years (Appendix A), the taxon might not have been as w e l l c o l l e c t e d as i t has been. C e r t a i n l o c a l i t i e s provide r e l a t i v e l y easy access to the n a t i v e M e t r o s i d e r o s r a i n f o r e s t -- the most common h a b i t a t f o r L i m b e l l a — and many d i f f e r e n t b o t a n i s t s have c o l l e c t e d r e p e a t e d l y i n the same areas (e.g. O l i n d a P i p e l i n e T r a i l on Maui and A l a k a i T r a i l on Kauai; v i d . St. John 1946). In the l a s t 15 years, c o l l e c t i o n s by Hoe i n Maui's Kipahulu V a l l e y (Hoe 1967) and Herbst and Hoe at P a l i k u (Haleakala C r a t e r , Maui) have shown L i m b e l l a to occur i n areas never before e x p l o r e d b r y o l o g i c a l l y , and no doubt the moss occurs i n other unexplored regions of the i s l a n d s . 9.2 D i s t r i b u t i o n of L i m b e l l a t r i c o s t a t a ssp. f r y e i The only known l i v i n g p o p u l a t i o n of L i m b e l l a t r i c o s t a t a ssp. f r y e i occurs 9 m above sea l e v e l at Sutton Lake, Lane County, Oregon, 8.9 km north of F l o r e n c e and 3.5 km from the P a c i f i c Ocean ( F i g . 38; Township 17S, Range 12W, Sees. 26 and 35; 44°3'N, 124°6'W). I a c c i d e n t l y d i s c o v e r e d the p o p u l a t i o n i n A p r i l 1978 while s e a r c h i n g with S.D. Sundberg f o r c o a s t a l Sphagnum mires ( C h r i s t y 1980). W i l l i a m s (1933) c i t e d the type l o c a l i t y as "Cape Arago, Oregon", i d e n t i c a l to the l a b e l data of the l e c t o t y p e (NY). Labels of the i s o l e c t o t y p e s (UBC, WTU) are s l i g h t l y more i n f o r m a t i v e : "Coos County, Cape 1.5 1 F i g u r e 38 - S i t e of e x i s t i n g ssp. f r y e i p o p u l a t i o n at Sutton Lake, Lane County, Oregon. H a b i t a t d e p i c t e d by wetland symbols i M o d i f i e d from USGS Heceta Head, Oregon 7.5' topographic map. 250 m 1 52 Arago, 1-1.5 miles northeast of Cha r l e s t o n on east s i d e of highway." With the i s o l e c t o t y p e at WTU i s a map and note i n Frye's handwriting (Lawton, p e r s . comm. 1984) showing the general l o c a l i t y ( F i g . 39). Frye's map pl a c e s the type l o c a l i t y i n the present community of Barview, along an approximately 1-km segment of Cape Arago Highway, with F o s s i l P o int as the midpoint of the segment ( F i g . 40; Township 25S, Range 14W, Sec. 36, Township 26S, Range 14W, Sec. 1; 43°21'N, 124°18'W). The area i s 15 m above sea l e v e l and 2 km from the P a c i f i c Ocean, d i r e c t l y f a c i n g the o u t l e t of Coos Bay. Frye's 1950 note i n d i c a t e d t h a t the type l o c a l i t y was "now the back yard of a house," suggesting that he v i s i t e d the s i t e in 1950. I t i s uncl e a r from the note whether the L i m b e l l a p o p u l a t i o n s t i l l e x i s t e d i n 1950, or whether i t was destroyed by c o n s t r u c t i o n of the house. E l v a Lawton (p e r s . comm. 1984) co u l d not r e c a l l with c e r t a i n t y whether Frye a c t u a l l y returned to Barview i n 1950, and i f so, whether he found the ssp. f r y e i p o p u l a t i o n s t i l l e x t a n t . Dr. Lawton (pers. comm. 1980) gave me a d d i t i o n a l d e t a i l s r e g a r d i n g the Barview l o c a l i t y : In 1956 bef o r e we went on a c o l l e c t i n g t r i p to Oregon Dr. Frye t o l d me a l i t t l e more about the p l a c e where he c o l l e c t e d the moss. He s a i d there was a grove of t r e e s (a dozen or more, I think douglas f i r , ) beyond and on the o p p o s i t e s i d e of the road from where he got the moss. I am sure I found the p l a c e , the t r e e s were l a r g e r than he had d e s c r i b e d as i t was some years l a t e r . Some houses had been b u i l t i n the area but there was p l e n t y of u n d i s t u r b e d ground and a small stream. I hunted a l l over the area without f i n d i n g the moss. 153 F i g u r e 39 - T.C. Frye's map and note d e s c r i b i n g the type l o c a l i t y of Sc iaromium f r y e i W i l l i a m s . 1.54 F i g u r e 40 - Type l o c a l i t y of Sc iaromium f r y e i W i l l i a m s , Barview, Coos County, Oregon. Type l o c a l i t y was on east s i d e of highway, between'c. 1 km north to 1 km south of F o s s i l P o i n t . M o d i f i e d from USGS C h a r l e s t o n , Oregon 7.5' topographic map. 250 m 155 The Barview p o p u l a t i o n remains l o s t . My own searches i n 1980 (with S.D. Sundberg) and 1983 were u n s u c c e s s f u l . I t was unclear which Douglas f i r grove Lawton had r e f e r r e d to i n her l e t t e r , as s e v e r a l such stands o c c u r r e d along the west s i d e of the highway i n that v i c i n i t y . Most of the area was covered with housing or commercial developments, and the streams in the area had been impounded. Because three b r y o l o g i c a l l y - t r a i n e d i n d i v i d u a l s were unable to r e l o c a t e the p l a n t s , there i s a good chance that the Barview p o p u l a t i o n i s e x t i n c t ( C h r i s t y 1980). In 1983-1984 I searched 43 s i t e s (almost a l l S a l i x  hookeriana - Pyrus fusca - Carex obnupta sh r u b - c a r r s ) along 1600 km of c o a s t l i n e from southwestern B r i t i s h Columbia to northern C a l i f o r n i a <Fig. 41; Appendix N) without f i n d i n g any a d d i t i o n a l L i m b e l l a p o p u l a t i o n s , c o n f i r m i n g the r a r i t y of ssp. f r y e i . However, given the f a c t that two p o p u l a t i o n s have been documented -- 80 km d i s t a n t from one another — i t i s l i k e l y that other p o p u l a t i o n s e x i s t somewhere in the region and w i l l be d i s c o v e r e d e v e n t u a l l y . Few b o t a n i s t s e x p l o r e these dense, miry c a r r s , and any f u t u r e search f o r L i m b e l l a should c o n c e n t r a t e on such h a b i t a t s . The "dense, t a l l shrub communities" o c c u r r i n g on the many i s l a n d s at the mouth of the Columbia River (e.g., Columbia W h i t e - T a i l N a t i o n a l W i l d l i f e Refuge, v i d . F r a n k l i n and Dyrness 1973) may c o n t a i n promising s i t e s f o r i n v e s t i g a t i o n . 156 157 C. ORIGINS OF DISTRIBUTIONS 9.3 The O r i g i n Of Hawaiian P o p u l a t i o n s 1. R e l a t i o n of L. t r i c o s t a t a to South American L i m b e l l a The c l o s e s t r e l a t i v e of L i m b e l l a t r i c o s t a t a appears to be L. pachyloma (Mont.) CM., the nearest p o p u l a t i o n being i n the northern Andes Mountains near Bogota, Colombia, 9000 km ESE of Hawaii and 15° c l o s e r to the equator. Based on s i m i l a r i t i e s between the two taxa, L^ t r i c o s t a t a i s c o n s i d e r e d here to be d e r i v e d from South American L_j_ pachyloma. The two s p e c i e s e x h i b i t s i m i l a r a r e o l a t i o n , l i m b i d i a , sporophyte c h a r a c t e r s , h a b i t a t and c l i m a t i c preferences. 1 L^ pachyloma i s a polymorphic Southern Hemisphere s p e c i e s with no fewer than 16 h e t e r o t y p i c a l synonyms. Although the p l a n t s are h i g h l y v a r i a b l e , shoots are r a r e l y longer than 12 cm, the leaves always e n t i r e , and branching mostly simple. In c o n t r a s t , L^ t r i c o s t a t a ssp. t r i c o s t a t a can exceed 0.5 m i n l e n g t h , the leaves s h a r p l y s e r r a t e and branching i n a q u a t i c forms i s o f t e n subpinnate. The marked d i f f e r e n t i a t i o n i n t r i c o s t a t a , e s p e c i a l l y i t s l a r g e r s i z e when compared to i t s South American c o u n t e r p a r t , p a r a l l e l s the m o r p h o l o g i c a l d i f f e r e n t i a t i o n — from p u t a t i v e c o n t i n e n t a l p r o g e n i t o r s -- a s s o c i a t e d with the a d a p t i v e r a d i a t i o n of Hawaii's n a t i v e v a s c u l a r f l o r a ( C a r l q u i s t 1980). 1 58 2. Did L i m b e l l a D i s p e r s e from South America to Hawaii? In order to understand the phytogeographic r e l a t i o n s h i p of L. t r i c o s t a t a and L_;_ pachyloma, the d i s t r i b u t i o n of L. pachyloma must be examined. L_;_ pachyloma i s d i s c o n t i n u o u s l y c i r c u m s u b a n t a r c t i c , o c c u r r i n g on the major s u b a n t a r c t i c i s l a n d s and extending northward at middle and upper e l e v a t i o n s along the Andean c o r d i l l e r a and the c o a s t a l mountains of SE B r a z i l ( F i g . 42). L i m b e l l a capense (Dix.) comb. nov., p o s s i b l y a subspecies of pachyloma, occurs between 1220-1820 m i n South A f r i c a ' s Cape Pr o v i n c e . The genus i s d e c i d e d l y cool-temperate and r e s t r i c t e d to oceanic c l i m a t e s . E x h i b i t i n g i t s broadest d i s t r i b u t i o n i n the Southern Hemisphere, the genus p o s s i b l y o r i g i n a t e d as p a r t of the a n c i e n t Antarcto-Cretaceous (Gondwanlandic) f l o r a (Moore 1972). The s u b a n t a r c t i c i s l a n d s , now populated by L^ pachyloma, were ohce completely or almost completely g l a c i a t e d d u r i n g the P l e i s t o c e n e (Moore 1972; van Zanten and P6cs 1981). B a r r i n g the undocumented e x i s t e n c e of g l a c i a l r e f u g i a on these i s l a n d s , the modern d i s t r i b u t i o n of L_^  pachyloma can be a t t r i b u t e d only to p o s t g l a c i a l l o n g - d i s t a n c e d i s p e r s a l of spores over at 1500-6000 km of open ocean, presumably from p o p u l a t i o n s i n u n g l a c i a t e d p o r t i o n s of South America or South A f r i c a . Sporophytes of L_;_ pachyloma are rare i n herbarium specimens, but do e x i s t (e.g., Moreau s.n., 3 . i i i . 1 9 4 1 , (BA)) Based on my f i e l d and herbarium experience with L^ t r i c o s t a t a ssp. t r i c o s t a t a , L i m b e l l a sporophytes are not i n f r e q u e n t but are almost u n i v e r s a l l y overlooked by c o l l e c t o r s who do not take F i g u r e 42 - World d i s t r i b u t i o n of L i m b e l l a . 160 time to s c r u t i n i z e i n d i v i d u a l p o p u l a t i o n s ( v i d . Glime and V i t t 1984). The r a r i t y of pachyloma sporophytes i s t h e r e f o r e a p o s s i b l e a r t i f a c t of c o l l e c t i n g , and L i m b e l l a may be w e l l -equipped fo r l o n g - d i s t a n c e d i s p e r s a l . If L pachyloma c o u l d repopulate the s u b a n t a r c t i c i s l a n d s from a p u t a t i v e stock 1500-6000 km d i s t a n t and at lower l a t i t u d e , i t f o l l o w s that L^ t r i c o s t a t a ssp. t r i c o s t a t a c o u l d have been d e r i v e d from l o n g - d i s t a n c e d i s p e r s a l of L_;_ pachyloma (or i t s p r o g e n i t o r ) d i a s p o r e s from South America. Approximately 9000 km and 15° l a t i t u d e separate the two c l o s e s t d i s t r i b u t i o n a l areas (Colombia and Hawaii). Given the extent to which L.  t r i c o s t a t a ssp. t r i c o s t a t a has d i v e r g e d from pachyloma, and the amount of time needed f o r such divergence to occur, i t i s p o s s i b l e that d i s p e r s a l o c c u r r e d as e a r l y as the m i d - T e r t i a r y to one of the a n c i e n t volcanoes of the Leeward Hawaiian I s l a n d s , which have s i n c e mostly eroded to sea l e v e l . Kure, the o l d e s t of the e x i s t i n g i s l a n d s , i s of m i d - T e r t i a r y age. Many more an c i e n t i s l a n d s , now e x i s t i n g as only seamounts or guyots, are l o c a t e d northwest of Kure. If these a n c i e n t i s l a n d s supported antecedents of the Hawaiian b i o t a , McKenna (1983) noted that the Hawaiian f l o r a would have had c o n s i d e r a b l y more time to evolve, and the r a t e s of e v o l u t i o n and a d a p t i v e r a d i a t i o n would have been c o n s i d e r a b l y slower than has been assumed by most recent i n v e s t i g a t o r s . I t was on the Leeward I s l a n d s (or the more an c i e n t submerged i s l a n d s ) that Hawaii's n a t i v e a l p i n e f l o r a i s thought to have evolved ( S k o t t s b e r g , v i d . Stone 1967). Although some phytogeographers proposed e a r l y land b r i d g e s 161 between North America or Malaysia and A u s t r a l a s i a ( v i d . Gemmell 1955), most agree that the n a t i v e Hawaiian f l o r a d e r i v e d e x c l u s i v e l y from a n c e s t r a l immigrants reaching the a r c h i p e l a g o v i a l o n g - d i s t a n c e d i s p e r s a l from c o n t i n e n t a l areas. The importance of l o n g - d i s t a n c e d i s p e r s a l i n the o r i g i n of the Hawaiian f l o r a was f i r s t noted by H i l l e b r a n d (1888), based on Hawaii's permanently i s o l a t e d geographical p o s i t i o n and i t s igneous o r i g i n . The South American-Hawaiian d i s t r i b u t i o n i s n e a r l y p a r a l l e l e d by the A n t a r c t i c moss T o r t e l l a f r a g i l i s v a r . t o r t e l l o i d e s (S.W. Greene) Zand. & Hoe (Zander and Hoe 1979). Hawaiian Cyrtopus setosus (Hedw.) Hook. may have l i k e w i s e o r i g i n a t e d from southern South America, although i t c o u l d a l s o have d i s p e r s e d from New Zealand ( S c h o f i e l d and Crum 1972). Andreaea a c u t i f o l i a Hook. f . & W i l s . , known from southern South America, A u s t r a l i a , New Zealand and s e v e r a l s u b a n t a r c t i c i s l a n d s ( V i t t 1980), a l s o occurs i n Hawaii ( V i t t and Hoe 1980). Unless as yet undiscovered at high e l e v a t i o n s on i n t e r v e n i n g land masses (e.g. New Guinea or the northern Andes), or once o c c u r r i n g there but s i n c e e x t i r p a t e d , Andreaea a c u t i f o l i a spores had to t r a v e l a minimum of 12,000 km to reach Hawaii. As i n L_j_ pachyloma, the r a r i t y of U t r i c o s t a t a ssp. t r i c o s t a t a sporophytes ( S u l l i v a n t 1854; Bartram 1933; M i l l e r 1954; Crosby 1965; Hoe 1974) appears to be an a r t i f a c t of c o l l e c t i n g . Based on my f i e l d experience i n Hawaii, sporophytes are r a t h e r common and t h i s no doubt has enabled the s p e c i e s to c o l o n i z e a l l the major i s l a n d s having s u i t a b l e h a b i t a t s . Once 162 e s t a b l i s h e d from spores, L^ t r i c o s t a t a ssp. t r i c o s t a t a d isseminates r e a d i l y by v e g e t a t i v e fragmentation (Chapter 7), enab l i n g i t to populate any reach of a given stream. 9.4 The O r i g i n of North American P o p u l a t i o n s 1. Are Hawai ian and North American L i m b e l l a C l o s e l y  Related? A l l the data presented here c l e a r l y i n d i c a t e that ssp. t r i c o s t a t a and ssp. f r y e i are c l o s e l y r e l a t e d , ssp. f r y e i presumably having d e r i v e d from ssp. t r i c o s t a t a a f t e r l o n g -d i s t a n c e d i s p e r s a l from Hawaii. M o r p h o l o g i c a l f e a t u r e s (Chapter 3) are so s i m i l a r between the two taxa — f e a t u r e s not e x h i b i t e d by other members of the genus — that Lawton (1971) c o n s i d e r e d them to be c o n s p e c i f i c . 2. Was L i m b e l l a Capable of Long-distance D i s p e r s a l from  Hawaii to North America? In the Hawaiian I s l a n d s , L^ t r i c o s t a t a ssp. t r i c o s t a t a i s r e l a t i v e l y widespread, having s a t u r a t e d most a v a i l a b l e h a b i t a t s . In North America, however, the p o p u l a t i o n s of ssp. f r y e i a re very s m a l l , h i g h l y l o c a l i z e d and u n i s e x u a l . Male p l a n t s and sporophytes are unknown ( C h r i s t y 1980). These f e a t u r e s are c h a r a c t e r i s t i c of d i s j u n c t bryophyte p o p u l a t i o n s thought to have 163 o r i g i n a t e d from l o n g - d i s t a n c e d i s p e r s a l ( S c h o f i e l d and Crum 1972). Long-distance d i s p e r s a l i s the simplest and most l o g i c a l e x p l a n a t i o n f o r the occurrence of L i m b e l l a i n North America. Given the extent to which ssp. f r y e i has d i v e r g e d from ssp. t r i c o s t a t a , and the amount of time needed f o r such divergence to occur, d i s p e r s a l to North America c o u l d have occurred as e a r l y as the l a t e T e r t i a r y . The two p o p u l a t i o n s are separated by 4600 km of open ocean and 25° of l a t i t u d e . Biogeographers have concluded that a m a j o r i t y of the antecedents of the Hawaiian b i o t a were of Indo-Malaysian o r i g i n . Some of these founder s p e c i e s are thought to have reached Hawaii v i a the Northern Hemisphere j e t stream i l l u s t r a t e d by C a r l q u i s t (1980: 87). I t f o l l o w s that some elements of the Hawaiian montane b i o t a a l s o may have reached the c o o l and r a i n y c o a s t of the P a c i f i c Northwest v i a t h i s j e t stream, as s e v e r a l bryophyte d i s t r i b u t i o n s appear to p a r a l l e l the j e t stream t r a c k . The l i v e r w o r t L e p i d o z i a s a n d v i c e n s i s Lindenb. was once thought endemic to Hawaii but was l a t e r r e p o r t e d from the SE Alaska-NW B r i t i s h Columbia a r c h i p e l a g o (Evans 1914; S c h o f i e l d 1968). M i l l e r (1967) r e p o r t e d Herberta hawai i e n s i s M i l l e r (of q u e s t i o n a b l e i d e n t i t y f i d . S c h o f i e l d 1968) from the Queen C h a r l o t t e I s l a n d s , the only l o c a l i t y known o u t s i d e Hawaii. Frahm (1980, 1984) r e p o r t e d Campylopus aureus Bosch & Lac. from Hawaii, C a l i f o r n i a and Oregon — the Oregon p o p u l a t i o n o c c u r r i n g on s t a b i l i z e d sand dunes 3 km SSW of Sutton Lake — and concluded that t h i s d i s t r i b u t i o n "probably r e f l e c t s r e l a t i v e l y recent i n t r o d u c t i o n s " from i t s c e n t e r of d i s t r i b u t i o n i n Indo-164 M a l a y s i a . Claopodium whippleanum ( S u l l . ) Ren. & Card., Scapania o r n i t h o p o d i o i d e s (With.) Pears., P l e u r o z i a purpurea Lindb., Anastrepta o r c a d e n s i s (Hook.) S c h i f f n . and Andreaea  r u p e s t r i s Hedw., although r e s p e c t i v e l y showing i n c r e a s i n g l y wider d i s t r i b u t i o n s i n the northern hemisphere, a l l e x h i b i t a H a w a i i a n - P a c i f i c Northwest d i s j u n c t i o n ( M i l l e r 1956; S c h o f i e l d and Crum 1972; V i t t and Hoe 1980). The s h o r t e s t of these d i s j u n c t i o n s i s 3000 km of open ocean. M i l l e r (1956) s t a t e d that Hawaiian Anastrepta o r c a d e n s i s and L e p i d o z i a s a n d v i c e n s i s " a r r i v e d i n Hawaii from North America...perhaps d e r i v e d from d i s s e m i n u l e s c a r r i e d from Alaska by such m i g r a t i n g b i r d s as the golden p l o v e r . " U n f o r t u n a t e l y , M i l l e r d i d not present a r a t i o n a l e f o r h i s statement, and i t seems e q u a l l y p l a u s i b l e that these taxa c o u l d have d i s p e r s e d to North America from Hawaii, based on the p a r a l l e l d i s t r i b u t i o n of the other Hawaiian-North American d i s j u n c t s whose North American p o p u l a t i o n s are small and h i g h l y l o c a l i z e d . Because L i m b e l l a sporophytes are not inf r e q u e n t but only u n d e r c o l l e c t e d - n e a r l y every p o p u l a t i o n I examined had sporophytes, i n both i n t e r m i t t e n t and p e r e n n i a l streams - ssp. t r i c o s t a t a appears to be p e r f e c t l y capable of l o n g - d i s t a n c e d i s p e r s a l by means of spores. Only female p l a n t s have been found i n the two known North American p o p u l a t i o n s of ssp. f r y e i . Sporophytes and gemmae are unknown, and d i s p e r s a l i s ap p a r e n t l y l i m i t e d to v e g e t a t i v e fragmentation. C u l t i v a t i o n experiments (Chapter 7) showed that such fragments are e f f i c i e n t agents of v e g e t a t i v e propagation. 165 At Sutton Lake, detached branches or branch segments can be seen l y i n g on the substratum near the parent p l a n t s . Signs of b i r d s , beaver, Douglas s q u i r r e l s and mice were seen i n the shrub-carr and raccoons and mink probably a l s o occur t h e r e . Together with wind and water, these animals may be e f f e c t i v e v e c t o r s of L i m b e l l a d i s p e r s a l . By v e g e t a t i v e means alone, ssp. f r y e i has c o l o n i z e d 4-5 ha at Sutton Lake. I t i s unknown how e x t e n s i v e the Barview p o p u l a t i o n was. D. DISCUSSION 9.5 Evidence Supporting Long-distance D i s p e r s a l i n L i m b e l l a Most bryogeographers agree that only l o n g - d i s t a n c e d i s p e r s a l can account f o r the d i s t r i b u t i o n s of c e r t a i n s p e c i e s . In cases i n v o l v i n g the vast d i s t a n c e s p r e v i o u s l y d i s c u s s e d , the presumed d i s p e r s a l agents of wind and man seem to be the only l o g i c a l v e c t o r s . Van Zanten and Pocs (1981) argue a g a i n s t zoochory and hydrochory i n bryophytes, noted by Cain (1944) to occur i n v a s c u l a r p l a n t s , because no r e c o r d of t h i s kind of t r a n s p o r t e x i s t s f o r B r y o p s i d a . These authors a l s o argue that p l a t e t e c t o n i c s has been overemphasized as a major f a c t o r i n g e n e r a t i n g d i s c o n t i n u o u s d i s t r i b u t i o n s , contending that i t may account f o r g e n e r i c s i m i l a r i t i e s between d i s j u n c t f l o r a s , but c e r t a i n l y not s pecies d i s j u n c t i o n s . As s t a t e d i n Chapter 5 and by van Zanten and P6cs, s e v e r a l l i n e s of evidence now suggest that bryophytes are not g e n e t i c a l l y d e p l e t e d or " e v o l u t i o n a r y 166 f a i l u r e s " (Crum 1972). If p l a t e t e c t o n i c s was r e s p o n s i b l e f o r broad d i s j u n c t i o n s , d i f f e r e n t i a t i o n between the p o p u l a t i o n s most probably would have o c c u r r e d d u r i n g the long p e r i o d s of time a v a i l a b l e . I f t h i s were t r u e , why are so many d i s j u n c t bryophytes c o n s p e c i f i c or very c l o s e l y r e l a t e d ? A more recent d i s p e r s a l i s the most f e a s i b l e e x p l a n a t i o n . Wind d i s p e r s a l of spores remains the simplest and most l i k e l y e x p l a n a t i o n f o r l o n g - d i s t a n c e d i s p e r s a l of mosses. Given the c o n s t r a i n t s r e q u i r e d by (1) s u c c e s s f u l i n j e c t i o n of d i a s p o r e s i n t o the atmosphere (2) s u r v i v a l of t r a n s p o r t hazards (e.g. u l t r a - v i o l e t r a d i a t i o n , d e s i c c a t i o n and f r e e z i n g ) and (3) a r r i v a l i n a s u i t a b l e and unoccupied h a b i t a t , s u c c e s s f u l l o n g -d i s t a n c e d i s p e r s a l must by d e f i n i t i o n be a chance and extremely rare occurrence (van Zanten and P6cs 1981). A d d i t i o n a l c o n s t r a i n t s are the improbable mixing of a i r masses of d i f f e r e n t c l i m a t i c zones and the d i s p a r a t e hemispheric c i r c u l a t i o n p a t t e r n s at the equator (van Zanten and P6cs• 1981). Ne v e r t h e l e s s , the same authors review evidence that spores may be c a r r i e d " a g a i n s t and a c r o s s the d i r e c t i o n of the p r e v a i l i n g w e s terly a i r streams," thereby c r o s s i n g both c l i m a t i c zones and the equator. Gemmell's (1955) map "showing the number of moss spe c i e s which the Hawaiian a r c h i p e l a g o has i n common with" South America (24 s p e c i e s ) and A u s t r a l a s i a (47 s p e c i e s ) g r a p h i c a l l y i l l u s t r a t e s the c a p a c i t y of d i a s p o r e s to c r o s s c l i m a t i c and e q u a t o r i a l r e g i o n s . Despite the c o n s t r a i n t s of l o n g - d i s t a n c e wind d i s p e r s a l , i t i s the only l o g i c a l way the modern Hawaiian b r y o f l o r a 167 o r i g i n a t e d . Fosberg (1948, 1951) estimated that the n a t i v e Hawaiian v a s c u l a r f l o r a , c o n s i s t i n g of 1897 s p e c i e s and v a r i e t i e s , 92.4% of which are endemic (96% endemic f i d . Stone 1967), had d e r i v e d from 407 o r i g i n a l founder s p e c i e s -- "an average of one s u c c e s s f u l a r r i v a l and establishment every 20,000 to 30,000 ye a r s , " based on the 5-10 m i l l i o n - y e a r age of the an c i e n t i s l a n d s to the northwest. "Countless others may have come, f l o u r i s h e d , then become e x t i n c t l e a v i n g no t r a c e . " Fosberg (1951) estimated the American ( i n c l u d i n g South American) element i n the founder f l o r a to be about 16.2% (18.4% f i d . Hoe 1979). The n a t i v e Hawaiian moss f l o r a was estimated by Hoe (1979) to c o n s i s t of 233 s p e c i e s , 48.1% of which are endemic. F o l l o w i n g Fosberg's (1948) a n a l y s i s , Hoe (Hoe 1979; Zander and Hoe 1979) estimated that the moss f l o r a had d e r i v e d from 225 o r i g i n a l founder s p e c i e s , 7.1% of which were of American ( i n c l u d i n g South American) o r i g i n (Hoe 1979; V i t t and Hoe 1980). Using Hoe's f i g u r e s and the same time frame as Fosberg, Hawaii's modern moss f l o r a may have d e r i v e d from "an average of one s u c c e s s f u l a r r i v a l and establishment" every 25,000-35,000 yea r s . Such an immense time span c o u l d account f o r the marked morphological d i f f e r e n c e s between South American L_j_ pachyloma and Hawaiian t r i c o s t a t a , i f l o n g - d i s t a n c e d i s p e r s a l to one of the a n c i e n t Leeward I s l a n d s was e f f e c t i v e . By the same reasoning, the few m o r p h o l o g i c a l d i f f e r e n c e s between Hawaiian ssp. t r i c o s t a t a and North American ssp. f r y e i can be e x p l a i n e d most e a s i l y by a recent, p o s t g l a c i a l d i s p e r s a l , a f t e r which time the p l a n t s have had l i t t l e time to d i f f e r e n t i a t e . Fosberg 1 68 (1951) l i k e w i s e argued that m o r p h o l o g i c a l s i m i l a r i t i e s between c e r t a i n Hawaiian v a s c u l a r p l a n t s and t h e i r p u t a t i v e North American p r o g e n i t o r s evidenced a r e l a t i v e l y recent d i s p e r s a l . 1. Are L i m b e l l a Spores Capable of Long-distance  Di s p e r s a l ? Van Zanten and Pocs 1981) d e s c r i b e d the r e l a t i v e c a p a b i l i t i e s ( " f i t n e s s " ) f o r l o n g - d i s t a n c e d i s p e r s a l of bryophyte s p e c i e s producing "green" spores and "brown" spores. They c o n s i d e r e d green spores to have begun germination i n  sporangio, the green c o l o r i n d i c a t i n g c h l o r o p l a s t development which was the f i r s t stage of germination before rupture of the spore coat and protonematal e l o n g a t i o n . In c o n t r a s t , brown spores i n i t i a t e d c h l o r o p l a s t development only a f t e r r e l e a s e from the sporangium. Comparative study c o r r e l a t e d green spores with hygrophytic s p e c i e s , e s p e c i a l l y h e p a t i c s and pleurocarpous mosses, and brown spores with more x e r o p h y t i c , d r o u g h t - t o l e r a n t s p e c i e s . Experiments l e d van Zanten and Pocs to conclude that green spores were d r o u g h t - i n t o l e r a n t , because they germinated r a p i d l y (1-3 days) and were v i a b l e f o r only 6 days to 1 year. Brown spores were d r o u g h t - t o l e r a n t , slower to germinate (4-210 days) and v i a b l e up to 48 years. By i n f e r e n c e , green spores appeared to be best s u i t e d f o r r a p i d , l o c a l d i s s e m i n a t i o n and a p p a r e n t l y were unable to s u r v i v e the r i g o r s of l o n g - d i s t a n c e wind d i s p e r s a l , whereas brown spores a p p a r e n t l y c o u l d t o l e r a t e such hazards. They f u r t h e r p o i n t e d out that s m a l l e r spores (8-169 12 jum diameter) were more r e a d i l y borne a l o f t and t r a v e l f a r t h e r than l a r g e r spores (10-20 /um) , and that spore s i z e was c o r r e l a t e d with those species of wider d i s t r i b u t i o n s , and that the s p e c i e s of wider d i s t r i b u t i o n s produced spores more r e s i s t a n t to d e s i c c a t i o n and f r e e z i n g . These o b s e r v a t i o n s are of i n t e r e s t i n r e l a t i o n to L i m b e l l a . At time of l i b e r a t i o n , spores of both L_^_ pachyloma and L.  t r i c o s t a t a ssp. t r i c o s t a t a are green and average 14-18 diameter. Spores of ssp. t r i c o s t a t a f r e q u e n t l y lodge on the peristome and i n i t i a t e protonematal growth. These c h a r a c t e r i s t i c s t y p i f y those d e s c r i b e d by van Zanten and Pocs f o r hygrophytic p l e u r o c a r p s of r a p i d , l o c a l d i s p e r s i v e c a p a b i l i t y that are i l l - s u i t e d to l o n g - d i s t a n c e d i s p e r s a l . Yet L i m b e l l a has f e a t u r e s that make i t c l e a r l y capable of long-d i s t a n c e d i s p e r s a l . C u l t i v a t i o n of ssp. t r i c o s t a t a spores (Chapter 7), which had been d r i e d at 38°C fo r 48 hours and s t o r e d at 20°C fo r 8 months, showed e x c e l l e n t germination and protonematal development w i t h i n 7 days, i n d i c a t i n g a c o n s i d e r a b l e degree of drought t o l e r a n c e . Van Zanten and Pocs' o b s e r v a t i o n s t h e r e f o r e cannot be a p p l i e d to L i m b e l l a . 2. Could L i m b e l l a Have Immigrated Into New H a b i t a t s  S u c c e s s f u l l y ? In order f o r l o n g - d i s t a n c e d i s p e r s a l to be e f f e c t e d , immigrant d i a s p o r e s , having s u r v i v e d t r a n s p o r t , must s u c c e s s f u l l y invade a s u i t a b l e h a b i t a t unoccupied by the 170 e x i s t i n g v e g e t a t i o n . T h i s assumption i s based on the g e n e r a l l y -accepted o b s e r v a t i o n that an immigrant p l a n t cannot compete with the b e t t e r - a d a p t e d n a t i v e f l o r a , and most o f t e n occurs i n d i s t u r b e d s i t e s ' where com p e t i t i o n i s minimal. The s a x i c o l o u s aquatic or r i p a r i a n h a b i t a t of L i m b e l l a , h a r d l y appearing s u i t e d for immigrant or weedy s p e c i e s , may be one of reduced competition because of c o n t i n u a l e c e s i s caused by s h i f t i n g of rocks and a b r a s i o n by water and d r i f t m a t e r i a l d u r i n g f l o o d i n g (Conboy and Glime 1971). The a q u a t i c , s a x i c o l o u s h a b i t a t i n Hawaiian mountain streams may have been unoccupied at the time L i m b e l l a d i a s p o r e s a r r i v e d , and even now many streams e x h i b i t sparse bryophyte cover, l e a v i n g s u b s t r a t a unoccupied and a v a i l a b l e f o r new d i a s p o r e s . The Oregon h a b i t a t s a l s o may have "been unoccupied by the n a t i v e f l o r a at the time of L i m b e l l a d i a s p o r e i n t r o d u c t i o n from Hawaii. In shrub-carrs n e a r l y i d e n t i c a l to t h a t at Sutton Lake (e.g., D e v i l s Lake State Park, L i n c o l n C i t y , Oregon), much of the peaty s u r f a c e i s bare, and the r e s t i s covered by a d i s c o n t i n u o u s mat of K i n d b e r g i a praelonga (Hedw.) Ochyra and Climacium dendroides (Hedw.) Web. & Mohr. The Sutton Lake c a r r a l s o has c o n s i d e r a b l e exposures of bare peat, and i t would appear that an immigrant might encounter l i t t l e c o m p e t i t i o n from indigenous s p e c i e s . The occurrence of ssp. f r y e i on t e r r e s t r i a l peat or sand, i n s t e a d of submerged or emergent rocks as i s the usual substratum of L^ pachyloma and L^ t r i c o s t a t a ssp. t r i c o s t a t a , may have been a r e s u l t of c l i m a t i c and g e o l o g i c chance. Within 171 the narrow s t r i p of land along the immediate c o a s t l i n e , the c l i m a t i c regime a p p a r e n t l y best s u i t e d to L^ _ f r y e i , rocky streams are r e s t r i c t e d to headland areas. These streams probably were a l r e a d y occupied by a well-developed a q u a t i c bryophyte f l o r a composed of common h o l a r c t i c s p e c i e s found in the region today, p r e c l u d i n g p e n e t r a t i o n by immigrant spores. The only other l o t i c h a b i t a t s a v a i l a b l e i n the narrow c o a s t a l s t r i p would have been r i v e r s and streams c r o s s i n g the dune sheets. These streams are t y p i c a l l y low-gradient, meandering, with sandy bottoms and banks and v i r t u a l l y no rocks, b e a r i n g no resemblance to the Hawaiian h a b i t a t s . The r i v e r s , a l s o low-g r a d i e n t and meandering, are s u b j e c t to d i u r n a l changes in volume and s a l i n i t y caused by t i d a l i n f l u e n c e s , tidewater extending up to 20 km or more i n l a n d . As s t a t e d i n Chapter 2, c l i m a t e a b r u p t l y becomes more extreme j u s t a few km i n l a n d , a f u r t h e r b a r r i e r to immigration. Schuster (1979) and S c h o f i e l d (1984) emphasized that spores from c l o s e d f o r e s t , r i p a r i a n or t o p o g r a p h i c a l l y p r o t e c t e d h a b i t a t s were l e s s l i k e l y to become a i r b o r n e because of impeded wind c i r c u l a t i o n . Van Zanten and P6cs (1981), however, s t a t e d that d i a s p o r e s of s p e c i e s i n s h e l t e r e d s i t e s c o u l d d i s p e r s e j u s t as e f f e c t i v e l y as those of s p e c i e s i n exposed h a b i t a t s , as long as they c o u l d s u r v i v e the hazards of t r a n s p o r t and, upon a r r i v a l , p enetrate the n a t i v e v e g e t a t i o n . The world d i s p e r s a l p a t t e r n of L i m b e l l a g i v e s ample c r e d i b i l i t y to van Zanten and P6cs' a s s e r t i o n . 172 9.6 A l t e r n a t i v e Hypotheses Concerning D i s p e r s a l to North America 1. Was Di s p e r s a l Preglac i a l ? An absence of divergence among p o s t - P l e i s t o c e n e p o p u l a t i o n s of pachyloma on the s u b a n t a r c t i c i s l a n d s suggests that not enough time has elapsed f o r divergence to have occurred t h e r e . Because ssp. f r y e i has diverged s l i g h t l y from ssp. t r i c o s t a t a , Hawaiian p l a n t s p o s s i b l y d i s p e r s e d to North America p r i o r to P l e i s t o c e n e g l a c i a t i o n s , perhaps as e a r l y as the l a t e T e r t i a r y . The Oregon Coast Range was u p l i f t e d d u r i n g the P l i o c e n e ( F r a n k l i n and Dyrness 1973) but the modern c o a s t l i n e d i d not form u n t i l a f t e r c e s s a t i o n of P l e i s t o c e n e sea l e v e l f l u c t u a t i o n s a s s o c i a t e d with g l a c i a t i o n (Baldwin 1981). Today's L i m b e l l a h a b i t a t s would have been inundated d u r i n g the P l e i s t o c e n e , l e a v i n g two p o s s i b i l i t i e s f o r the estimated a r r i v a l time of Hawaiian spores: (1) the founder p o p u l a t i o n was e s t a b l i s h e d f u r t h e r i n l a n d , before P l i o c e n e Coast Range orogeny, when the western Cascade Range formed the c o a s t l i n e d u r i n g the l a t e Miocene (2) the founder p o p u l a t i o n was e s t a b l i s h e d during the P l i o - P l e i s t o c e n e on what are today higher Coast Range e l e v a t i o n s , such as the ol d e r P l i o c e n e marine t e r r a c e s , and along with other c o a s t a l p l a n t s remained at sea l e v e l d u r i n g orogeny. Although bryophytes are not represented i n the r e g i o n a l f o s s i l f l o r a of t h i s p e r i o d , a c o o l i n g and d r y i n g trend appears to have p r e v a i l e d . C o n i f e r s and x e r o p h y t i c Quercus a s s o c i a t i o n s 1 73 r e p l a c e d the temperate broad-leaf Mixed Mesophytic f o r e s t of the l a t e T e r t i a r y and e x t i n c t i o n s were numerous ( D e t l i n g 1968; Graham 1972). C l i m a t i c c o n d i t i o n s at that time would not seem f a v o r a b l e f o r L i m b e l l a , but i t must be remembered that the i n l a n d c l i m a t e — sometimes only a few k i l o m e t e r s i n l a n d bears l i t t l e resemblance to t h a t along the immmediate c o a s t , where c o n d i t i o n s may have been more f a v o r a b l e . M i g r a t i o n from the a n c i e n t western Cascade s h o r e l i n e to the present s h o r e l i n e would have n e c e s s i t a t e d a stepwise v e g e t a t i v e d i s p e r s a l of at l e a s t 100 km, u n l e s s e a r l y p o p u l a t i o n s produced sporophytes. Schuster (1984) p o s t u l a t e d t h a t c l i m a t i c d e t e r i o r a t i o n may suppress gametangium p r o d u c t i o n , forming u n i s e x u a l or s t e r i l e p o p u l a t i o n s that reproduce s o l e l y by v e g e t a t i v e means, c h a r a c t e r i s t i c s e x h i b i t e d by the ssp. f r y e i p o p u l a t i o n at Sutton Lake. He thought that such c l o n a l p o p u l a t i o n s , "presumably l a c k [ i n g ] a b i l i t y to e v o l v e , " would e v e n t u a l l y become e x t i n c t , but he noted that many taxa p e r s i s t under suboptimal c o n d i t i o n s as "depauperate p o p u l a t i o n s " which "may b a r e l y 'hang on' r e g i o n a l l y , unable to s i g n i f i c a n t l y expand [ t h e i r ] range." Although ssp. f r y e i does appear to be b a r e l y "hanging on" because so few p o p u l a t i o n s have been documented, the Sutton Lake p o p u l a t i o n h a r d l y seems depauperate. If Schuster's theory of c l i m a t i c suppression of gametangium formation i s c o r r e c t , the occurrence of a p p a r e n t l y healthy archegonia i n modern p o p u l a t i o n s of ssp. f r y e i would suggest that the c l i m a t e was l e s s f a v o r a b l e f o r a n t h e r i d i a l p r o d u c t i o n . Environmental c o n t r o l of sex d i f f e r e n t i a t i o n i n gametangia seems 174 very u n l i k e l y because the e a r l y developmental stages of a n t h e r i d i a and archegonia are thought to be i d e n t i c a l (Sharma 1971). Based on numerous p o l l e n p r o f i l e s , many from c o a s t a l l o c a l i t i e s , Hansen (1947) concluded that "The p o s t g l a c i a l c l i m a t e of the north P a c i f i c coast has probably been e s s e n t i a l l y marine, with l i t t l e v a r i a t i o n , even dur i n g the dry p e r i o d that developed f u r t h e r i n l a n d . While c l i m a t i c f l u c t u a t i o n s undoubtedly d i d occur, they were of i n s u f f i c i e n t magnitude to cause a systematic response by the v e g e t a t i o n . " 2 . I_s North Amer ican L i m b e l l a Paleoendemic? Paleoendemic s t a t u s of ssp. f r y e i would imply a n c i e n t d e r i v a t i o n , systematic i s o l a t i o n , and i t s present d i s t r i b u t i o n would have to be c o n s i d e r e d r e l i c t u a l . I t s morphological s i m i l a r i t y t o L^ t r i c o s t a t a , i n d i c a t i n g c l o s e r e l a t i o n s h i p and r e l a t i v e l y recent d e r i v a t i o n , p r e c l u d e s the systematic i s o l a t i o n r e q u i r e d f o r paleoendemism. 3. D i d L i m b e l l a D i s p e r s e from North America to Hawaii? Did ssp. t r i c o s t a t a o r i g i n a t e from ssp. f r y e i by long-d i s t a n c e d i s p e r s a l to Hawaii from North America? T h i s d i s t r i b u t i o n p a t t e r n i s p a r a l l e l l e d by the Madiinae ( C a r l q u i s t 1980). The p o s s i b i l i t y of such an o r i g i n cannot be r u l e d out, but the present d i s p a r a t e a r e a l d i s t r i b u t i o n of the two taxa 175 argues a g a i n s t i t . I t seems h i g h l y u n l i k e l y that the p r o g e n i t o r p o p u l a t i o n ( ssp. f r y e i ) would be so r e s t r i c t e d , and that of the d e r i v a t i v e so ( r e l a t i v e l y ) e x t e n s i v e . If t h i s were t r u e , ssp. f r y e i would appear to be r e l i c t u a l , having been more widespread i n the past, and p o s s i b l y s e x u a l l y r e p r o d u c t i v e . The e x i s t i n g ssp. f r y e i p o p u l a t i o n e x h i b i t s a l l the c h a r a c t e r i s t i c s of an immigrant p o p u l a t i o n r e c e n t l y e s t a b l i s h e d by l o n g - d i s t a n c e d i s p e r s a l , and the lack of sporophytes would seem to preclude any d i s p e r s a l to Hawaii, at l e a s t by means of spores. 4. Are Hawaiian and North American L i m b e l l a V i c a r i a d s ? V i c a r i a n c e i m p l i e s that both ssp. t r i c o s t a t a and ssp. f r y e i d e r i v e d from a common a n c e s t r a l p o p u l a t i o n , not from one another, and that they may have been sympatric i n the p a s t . Assuming that they arose from southern hemisphere L i m b e l l a stock, they may have been sympatric somewhere on the mainland between the P a c i f i c Northwest and the Andean c o r d i l l e r a . The p o s s i b i l i t y of v i c a r i a n c e cannot be r u l e d out, but the s i m i l a r i t y of the two taxa and d i s s i m i l a r i t y between them and L. pachyloma would seem to r e q u i r e e i t h e r (1) a common ance s t o r , now e x t i n c t , e x h i b i t i n g the fe a t u r e s shared by ssp. t r i c o s t a t a and ssp. f r y e i or (2) a p o l y t o p i c o r i g i n of ssp. t r i c o s t a t a and f r y e i , a p o s s i b i l i t y which s t r e t c h e s the imagination and i s c o n s i d e r e d by van Zanten and P6cs (1981) to be " n e g l i g i b l e . " 176 5. Was North American L i m b e l l a Introduced by Man? Anthropochory i s invoked too f r e q u e n t l y as a c a u s a t i v e agent to e x p l a i n troublesome d i s t r i b u t i o n s of p l a n t s . Lawton (1971) s p e c u l a t e d that L_j_ t r i c o s t a t a had been introduced from Hawaii. Frahm's (1980, 1984) o p i n i o n s concerning the occurrence of Campylopus aureus in Hawaii and North America were more circumspect i n t h i s regard. I t seems extremely u n l i k e l y that L.  t r i c o s t a t a would have been brought to North America e i t h e r i n t e n t i o n a l l y or u n i n t e n t i o n a l l y i n connection with any commercial or hobby e n t e r p r i s e , the usual means by which a l i e n p l a n t s are introduced i n t o new g e o g r a p h i c a l areas. E. CONCLUSION AND SUMMARY S i m i l a r i t i e s between ssp. t r i c o s t a t a and ssp. f r y e i , and d i s s i m i l a r i t y between : .them and other members of the genus, i n d i c a t e t h at they are c l o s e l y r e l a t e d . F i e l d and herbarium evidence showed that L i m b e l l a sporophytes were not i n f r e q u e n t but only u n d e r c o l l e c t e d . Fecundity was confirmed by _in v i t r o c u l t i v a t i o n of ssp. t r i c o s t a t a spores, which germinated r a p i d l y and produced p r o l i f i c protonemata, buds and s p o r e l i n g s . C u l t i v a t i o n of v e g e t a t i v e fragments showed them to be e x c e l l e n t propagative d i a s p o r e s . L i m b e l l a i s t h e r e f o r e amply endowed with d i s p e r s i v e mechanisms. P o s t g l a c i a l r e c o l o n i z a t i o n of g l a c i a t e d c i r c u m s u b a n t a r c t i c i s l a n d s i n d i c a t e s that pachyloma i s capable of l o n g - d i s t a n c e 1 77 d i s p e r s a l . Occurrence of ssp. t r i c o s t a t a on a l l p r i n c i p a l Hawaiian i s l a n d s i n d i c a t e s that i t has been able to c o l o n i z e a l l i s l a n d s by s h o r t - d i s t a n c e d i s p e r s a l . It has a l s o been ab l e to occupy most s u i t a b l e h a b i t a t s w i t h i n i t s p h y s i o l o g i c a l t o l e r a n c e s . Furthermore, i t i s widely acknowledged that the n a t i v e Hawaiian f l o r a d e r i v e d e n t i r e l y from l o n g - d i s t a n c e d i s p e r s a l of mainland p r o g e n i t o r s , the d i s p e r s a l o c c u r r i n g as long ago as the m i d - T e r t i a r y . The l i m i t e d d i s t r i b u t i o n and u n i s e x u a l c o n d i t i o n of ssp. f r y e i i s t y p i c a l of d i s j u n c t p o p u l a t i o n s most l i k e l y d e r i v e d from l o n g - d i s t a n c e d i s p e r s a l . Based on the f o r e g o i n g evidence, i t appears that ssp. t r i c o s t a t a d e r i v e d from Southern Hemisphere L i m b e l l a v i a l o n g -d i s t a n c e d i s p e r s a l , and that ssp. f r y e i d e r i v e d from ssp. t r i c o s t a t a by l o n g - d i s t a n c e d i s p e r s a l . I n t r o d u c t i o n of Southern Hemisphere L i m b e l l a to Hawaii c o u l d have occu r r e d as e a r l y as the m i d - T e r t i a r y , and i n t r o d u c t i o n of ssp. t r i c o s t a t a spores to North America c o u l d have occurred i n the l a t e T e r t i a r y . L.  t r i c o s t a t a ssp. f r y e i i s t h e r e f o r e of more recent o r i g i n and only s l i g h t l y d i f f e r e n t i a t e d from ssp. t r i c o s t a t a . 178 X. SUMMARY AND CONCLUSIONS A. REVIEW OF EXPERIMENTAL EVIDENCE Pre v i o u s chapters have d e s c r i b e d experimental evidence which shows Hawaiian and North American L i m b e l l a p l a n t s to be very s i m i l a r , but with the f o l l o w i n g d i f f e r e n c e s : 1. M o r p h o l o g i c a l D i f f e r e n c e s 1. Branching p a t t e r n (bud fre q u e n c y ) . 2. Leaf i n s e r t i o n angle. 3. Stem diameter. 4. Branch l e n g t h . 5. Costa t h i c k n e s s at i n s e r t i o n . 6. Basal l e a f c e l l l e n g t h . 2. Karyotype D i f ferences 1 . Chromosome length'. 3. Isozyme D i f f e r e n c e s 1. M o b i l i t y of superoxide dismutase. 1 79 4. C u l t i v a t i o n D i f f e r e n c e s 1. Geotropism. 2. R e c i p r o c a l c u l t i v a t i o n experiments confirmed that observed d i f f e r e n c e s i n (1) bud frequency (2) l e a f i n s e r t i o n angle (3) b a s a l c e l l l e a f l e n g t h and (4) superoxide dismutase m o b i l i t y were s t a b l e , and thus presumably g e n e t i c a l l y c o n t r o l l e d . B. ANSWERS TO QUESTIONS POSED IN CHAPTER J_ 10.1 Are Hawaiian and North American L i m b e l l a C o n s p e c i f i c ? Most of the evidence i n d i c a t e s that Hawaiian and Oregon L i m b e l l a are c o n s p e c i f i c , c o n f i r m i n g Lawton's (1971) viewpoint. However, enough d i f f e r e n c e s e x i s t between Hawaiian and North American p l a n t s — as o u t l i n e d above -- to support r e c o g n i t i o n of Sc iaromium f r y e i as a subspecies of L^ t r i c o s t a t a . The d i f f e r e n c e s are not great enough to warrant s p e c i f i c rank. In r e c o g n i z i n g Sc iaromium f r y e i as a subspecies, both s i m i l a r i t i e s and d i f f e r e n c e s between the taxa are accomodated i n a manner most i n d i c a t i v e of i n f r a s p e c i f i c and i n t e r s p e c i f i c r e l a t i o n s h i p s w i t h i n the genus L i m b e l l a . 180 10.2 What E x p l a i n s the Geographical D i s t r i b u t i o n of the Taxa? In Chapter 9, a l l phytogeographical p o s s i b i l i t i e s e x p l a i n i n g d i s t r i b u t i o n were d i s c u s s e d . Although routes and dates of m i g r a t i o n probably never w i l l be known with c e r t a i n t y , g e o g r a p h i c a l , g e o l o g i c a l and experimental evidence suggests that L i m b e l l a reached Hawaii and North America v i a lo n g - d i s t a n c e d i s p e r s a l , presumably by spores. The p u t a t i v e route of d i s p e r s a l would have been from South America to Hawaii, and from Hawaii to North America. A South American-North American-Hawaiian d i s p e r s a l route cannot be r e f u t e d with c e r t a i n t y , but modern d i s t r i b u t i o n s and r e p r o d u c t i v e c o n d i t i o n in L i m b e l l a , and s i m i l a r d i s t r i b u t i o n s i n c e r t a i n other bryophyte taxa argue a g a i n s t i t . Hawaiian and Oregon p l a n t s are more s i m i l a r to one another than e i t h e r i s to any other member of the genus, supp o r t i n g the Hawaiian-North American l i n k a g e . In order to account f o r the d i f f e r e n t degrees of s p e c i a t i o n that have occurred, d i s p e r s a l to Hawaii c o u l d have happened as e a r l y as the m i d - T e r t i a r y , and d i s p e r s a l to North America i n the l a t e T e r t i a r y . The Oregon p o p u l a t i o n s would have remained small because d i s p e r s a l was l i m i t e d to v e g e t a t i v e fragmentation. C. CONCLUSION T h i s i n v e s t i g a t i o n was the f i r s t c r i t i c a l study of ssp. t r i c o s t a t a and ssp. f r y e i and rev e a l e d c o n s i d e r a b l y more in f o r m a t i o n about the taxa than was a v a i l a b l e i n the l i t e r a t u r e . Lawton's (1971) c o n c l u s i o n that the two taxa were c o n s p e c i f i c 181 was confirmed, but enough d i f f e r e n c e s were found to j u s t i f y s u b s p e c i f i c s t a t u s . 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Poznan, Poland. 164 pp. 199 APPENDIX A - SEQUENCE OF SSP. TRICOSTATA COLLECTIONS, AS REPRESENTED IN HERBARIA. Date C o l l e c t o r I s l a n d 1825 J . Macrae Hawaii, Oahu 1 840-1841 U. S. E x p l o r i n g E x p e d i t i o n 1 Hawai i 1851- 1871 w. H i l l e b r a n d Kauai 1875- 1900 D. D. Baldwin Maui 1876 V. Knudsen Kauai 1886 F. L. C l a r k e Kauai 1895 A. A. H e l l e r Kauai 1910 J . F. Rock Maui 1915- 1919 C. N. Forbes Maui, Molokai 1919 J . A. Kusche Kauai 1 922 c . Skottsberg Hawaii, Kaui 1 927-X 0. Degener Hawaii, Kaui, Maui, Oahu 1 930 U n i v e r s i t y of Hawaii-Bishop Museum E x p e d i t i o n 2 Kauai 1930 E. B. Bartram Kauai, Maui 1 931 E. C h r i s t o p h e r s o n & B. Hume Oahu 1 932-1937 F. R. Fosberg Hawaii, Kauai, Molokai, Oahu 1933 M. C. Neal Maui, Oahu 1 934 R. D. S v i h l a Oahu 1 937 G. E. Olson Maui 1 938 Hawaiian Bog Survey 3 Kauai 1 948 F. F a g e r l i n d & R. Skotts b e r g Molokai 1 949 P. R. Needham Kauai, Maui 1 952 H. A. M i l l e r Kauai, Oahu 1 963 F. K. Sparrow Kauai 1 964 H. 0. W h i t t i e r Maui 1 964-1965 M. R. Crosby Kauai, Maui, Molokai 1 965-X W. J . Hoe Kauai, Maui, Molokai, Oahu 1 966-1973 L. E. Bishop Hawaii, Molokai 1 973-1977 D. R. Herbst Kauai, Maui 1 975 K. Nagata Molokai 1 977 F. R. Warshauer Hawai i 1978 C. W. Smith Maui 1983- 1 984 J . A. C h r i s t y Kauai, Maui 1. W. D. Brackenridge, C. P i c k e r i n g . 2. H. St. John, E. Y. Hosaka, E. Hume, R. Inafuku, J . C. Lindsay Masuhara, D. D. M i t c h e l l , W. Wong. 3. L. M. Cr a n w e l l , 0. H. S e l l i n g , C. Sk o t t s b e r g . 200 APPENDIX B - PLANT SPECIES LIST, LIMBELLA HABITAT AT SUTTON LAKE, LANE COUNTY, OREGON. BRYOPHYTA Hepaticae F r u l l a n i a t a m a r i s c i ssp. n i s q u a l l e n s i s ( S u l l . ) Hatt. Lophocolea h e t e r o p h y l l a (Schrad.) Dum. Metzgeria conjugata Lindb. P e l l i a neesiana (Gott.) Limpr. P o r e l l a n a v i c u l a r i s (Lehm. & Lindenb.) Lin d b . Radula b o l a n d e r i G o t t . R i c c a r d i a chamedryfolia (With.) G r o l l e R. m u l t i f i d a (L.) S. Gray Muse i A n t i t r i c h i a c u r t i p e n d u l a (Hedw.) B r i d . Dicranum sp. Drepanocladus sp. F i s s i d e n s sp. F o n t i n a l i s sp. Hooker i a lucens (Hedw.) Sm. Isothec ium s t o l o n i ferum B r i d . K i ndbergia praelonga (Hedw.) Ochyra L e u c o l e p i s m e n z i e s i i (Hook.) Steere ex Koch L i m b e l l a t r i c o s t a t a ssp. f r y e i ( Williams ) C h r i s t y Neckera d o u g l a s i i Hook. Orthotrichum l y e l l i i Hook. & T a y l . Rhizomnium glabrescens (Kindb.) T. Kop. VASCULAR PLANTS Carex obnupta B a i l e y G a u l t h e r i a s h a l l o n Pursh Ledum glandulosum (Pip e r ) H i t c h c . L y s i c h i t u m americanum Hulten & S t . John Picea s i t c h e n s i s (Bong.) C a r r . Pinus- c o n t o r t a Dougl. Polypodium g l y c y r r h i z a D.C. Eat. Pyrus fusca Raf. S a l i x hookeriana B a r r a t t S p i r a e a d o u g l a s i i Hook. Vaccinium p a r v i f o l i u m Smith 201 APPENDIX C - PLANT AND ANIMAL REMAINS IN FILTRATE FROM WASHINGS OF SSP. FRYEI SPECIMENS. SUTTON LAKE Pla n t Remains bud s c a l e s ( s p e c i e s u n i d e n t i f i e d ) Carex obnupta l e a f fragments and seeds L i m b e l l a branches and leaves Pinus c o n t o r t a needles Picea s i t c h e n s i s needles Pseudotsuga m e n z i e s i i needles Pyrus fusca flower p e d i c e l s S a l i x hookeriana l e a v e s , bud s c a l e s and seeds Sp i r a e a d o u g l a s i i f o l l i c l e s Animal Remains b e e t l e s and b e e t l e l a r v a e b i v a l v e s (<4 mm diameter) i n s e c t f r a s s isopod (sow bug) mouse droppings o r i b a t i d mites s n a i l s (<3 mm diameter, 2 spec i e s ) s p i d e r s c a d d i s f l y l a r v a l cases (composed of p l a n t m a t e r i a l , some of L i m b e l l a leaves BARVIEW Plan t Remains grass culms ( s p e c i e s u n i d e n t i f i e d ) F r u l l a n i a fragment f o l i o s e l i c h e n fragment leaves ( s p e c i e s u n i d e n t i f i e d ) Rubus p a r v i f l o r u s seeds (11) Animal Remains c a d d i s f l y l a r v a l case (composed of sand g r a i n s ; fragmented) APPENDIX D - MORPHOLOGICAL CHARACTERS MEASURED IN SSP. TRICOSTATA AND SSP. FRYEI. V a r i a b l e number Character measured 1 . shoot l e n g t h (cm) 2. branch " , longest (cm) 3. l e a f i n s e r t i o n angle (deg) 4. " lengt h , s h o r t e s t (mm) 5. " longest 6. " width, at widest part (mm) 7. " a p i c a l angle, e x c l u d i n g mucro (deg) 8. " tooth l e n g t h , longest (um) 9. upper l e a f c e l l l e n g t h , s h o r t e s t (um) 10. " " " , longest 11. " " " width, s h o r t e s t 12. " " " , longest 13. " " " length:width 14. basal " " le n g t h , s h o r t e s t (um) 15. " " " , longest 16. " " width, s h o r t e s t 17. " « " » f longest 18. " " length:width 19. c o s t a width at i n s e r t i o n (um) 20 l i m b i d i a " 21. number of c e l l l a y e r s of c o s t a 22. c o s t a t h i c k n e s s (um) 23. number of c e l l l a y e r s of l i m b i d i a 24. l i m b i d i a t h i c k n e s s (um) 25. stem diameter, longest dimension (um) 26. number of c e l l s i n stem diameter 27. width of stem c o r t i c a l l a y e r (um) 28. number of c e l l s i n stem c o r t i c a l l a y e r APPENDIX E " BRANCH BUD FREQUENCY DATA MATRIX. 1-20 = SSP. TRICOSTATA, 21-37 = SSP. FRYEI. 1 17, 3 . 1 , 6 . 3 , 2 9 . 6 , 1 . 9 , 1 7 , 6 . 3 , 1 . 9 , 1 0 . 7 , . 6 , 1 . 3 . 3 . 8 , . 6 . 0 , 0 , 0 , 0 , 0 . 0 , 0 , 1 2 1 6 . 2 , 1 . 4 , 0 , 2 7 , 0 , 9 . 5 . 2 . 7 , 0 , 2 3 , 5 . 4 . 0 , 9 . 5 , 1 . 4 . 1 . 4 , 1 . 4 , 1 . 4 , 0 , 0 , 0 , 0 , 1 3 2 2 . 7 , 5 . 5 , 9 . 1 , 2 8 . 2 , 0 , 1 0 . 9 , 7 . 3 , 5 . 5 , 6 . 4 , 1 . 8 , 1 . 8 , . 9 , 0 . 0 . 0 , 0 , 0 , 0 . 0 , 0 , 1 4 1 8 . 2 , 4 . 6 , 0 , 3 1 . 9 , 0 , 3 6 . 4 , 9 . 1 , 0 , 0 , 0 , 0 , 0 , 0 , 0 , 0 , 0 , 0 , 0 . 0 , 0 , 1 5 3 0 . 3 , 3 . 0 , 1 8 . 2 , 0 , 2 1 . 2 , 6 . 1 , 3 , 1 2 . 1 , 3 , 3 , 0 , 0 , 0 , 0 , 0 , 0 , 0 , 0 , 0 , 1 6 1 5 . 6 , 3 . 9 , 2 . 6 , 3 1 . 2 , 1 . 3 , 1 9 . 5 , 1 4 . 3 , 1 . 3 , 6 . 5 , 1 . 3 , 2 . 6 , 0 , 0 , 0 , 0 , 0 , 0 , 0 , 0 , 0 , 1 7 2 8 . 8 . 1 . 3 , 1 . 3 , 1 8 . 8 , 0 , 1 5 , 6 . 3 , 7 . 5 , 1 1 . 3 . 1 . 3 , 6 . 3 , 2 . 5 , 0 . 0 . 0 , 0 . 0 , 0 . 0 . 0 , 1 8 2 3 . 1 . 3 . 1 , 3 . 1 , 2 0 , 0 , 2 4 . 6 , 1 2 . 3 , 1 . 6 , 6 . 2 , 3 . 1 , 0 , 0 , 1 . 6 , 0 , 0 , 1 . 6 , 0 , 0 , 0 , 0 , 1 9 1 6 . 3 . 0 , 0 , 3 0 . 6 , 4 . 1 . 2 8 . 6 . 6 . 1 , 2 . 4 . 1 , 2 , 6 . 1 . 0 , 0 , 0 , 0 , 0 , 0 , 0 , 0 , 0 , 1 10 1 7 . 1 , 9 . 8 , 0 , 2 4 . 4 , 0 , 2 9 . 3 , 7 . 3 , 0 , 1 2 . 2 , 0 , 0 , 0 , 0 , 0 , 0 , 0 , 0 , 0 , 0 , 0 , 1 11 6 . 4 , 6 . 4 , 0 , 3 4 , 0 , 1 2 . 8 , 3 4 , 0 , 6 . 4 , 0 . 0 , 0 , 0 , 0 . 0 , 0 , 0 , 0 , 0 . 0 , 1 12 2 2 . 9 , 2 . 1 , 0 , 1 6 . 7 , 0 , 1 0 . 4 , 0 , 0 , 1 0 . 4 , 0 . 1 2 . 5 . 6 . 3 , 4 . 2 , 6 . 3 , 2 . 1 , 0 , 4 . 2 , 0 , 2 . 1 , 0 . 1 13 1 5 . 5 , 0 , 0 , 2 9 . 3 . 0 , 1 0 . 4 , 1 5 . 6 , 0 . 1 0 . 4 , 1 . 7 , 8 . 6 , 3 . 5 , 1 . 7 , 0 , 0 , 3 . 5 , 0 , 0 , 0 . 0 , 1 14 1 6 . 1 , 1 . 8 , 0 . 3 3 . 9 , 0 , 1 9 . 6 , 1 7 . 9 , 0 , 7 . 1 , 0 , 0 , 1 . 8 , 1 . 8 , 0 . 0 , 0 , 0 , 0 , 0 , 0 , 1 15 2 2 . 6 , 3 . 2 , 0 , 1 9 . 4 , 0 , 1 6 . 1 , 6 . 5 , 3 . 2 , 9 . 7 , 6 . 5 , 9 . 7 , 3 . 2 , 0 . 0 , 0 , 0 , 0 , 0 , 0 , 0 , 1 16 1 0 , 0 , 1 0 , 1 0 , 0 , 0 , 1 0 , 0 , 4 0 , 0 , 2 0 , 0 , 0 , 0 . 0 , 0 , 0 . 0 , 0 , 0 , 1 17 7 , 4 . 7 , 0 , 3 0 . 2 , 2 . 3 , 4 . 7 , 1 1 . 6 , 0 , 1 1 . 6 , 4 . 7 , 4 . 7 , 9 . 3 , 2 . 3 , 2 . 3 , 2 . 3 , 0 , 0 . 2 . 3 , 0 , 0 , 1 18 9 . 3 , 4 . 7 , 0 , 2 7 . 9 , 0 , 7 , 1 6 . 3 , 2 . 3 . 1 6 . 3 , 0 , 9 . 3 , 4 . 7 , 2 . 3 , 0 , 0 . 0 , 0 , 0 , 0 , 0 . 1 19 1 3 , 0 . 0 , 2 6 . 1 , 0 , 8 . 7 , 2 1 . 8 , 0 . 8 . 7 , 0 . 2 1 . 8 , 0 . 0 , 0 . 0 , 0 , 0 , 0 , 0 , 0 , 1 20 1 0 . 8 , 1 . 6 , 1 . 6 . 2 0 , 0 , 1 5 . 4 , 9 . 2 , 0 , 2 1 . 5 , 3 . 1 , 6 . 2 , 6 . 2 , 1 . 6 , 0 , 0 , 1 . 6 , 1 . 6 , 0 , 0 , 0 , 1 21 2 1 . 9 , 8 . 8 , 3 . 8 , 2 1 , 1 . 9 , 2 4 . 8 , 4 . 8 , 1 , 8 . 8 , 1 . 9 , 1 , 1 , 0 , 0 , 0 , 0 , 0 , 0 , 0 , 0 . 2 22 3 5 . 3 , 5 . 9 , 5 . 9 , 1 5 . 7 , 2 , 2 9 . 4 , 0 , 0 , 3 . 9 , 2 , 0 , 0 , 0 , 0 , 0 , 0 , 0 , 0 , 0 , 0 , 2 23 2 7 . 5 , 1 7 . 5 , 1 7 . 5 , 1 2 . 5 , 0 , 2 2 . 5 , 2 . 5 , 0 , 0 , 0 , 0 . 0 , 0 , 0 , 0 , 0 , 0 . 0 , 0 , 0 . 2 24 2 5 , 5 . 8 . 9 . 6 , 1 5 . 4 , 0 , 2 1 . 2 , 9 . 6 , 0 , 9 . 6 , 3 . 9 , 0 , 0 , 0 , 0 , 0 , 0 , 0 , 0 . 0 , 0 , 2 25 4 4 , 8 . 8 , 5 . 9 , 5 . 9 , 0 , 2 6 . 5 . 2 . 9 , 0 , 0 , 0 , 5 . 9 , 0 , 0 . 0 . 0 , 0 , 0 , 0 . 0 , 0 , 2 26 2 9 . 4 , 1 1 . 8 , 0 , 2 9 . 4 , 0 , 1 7 . 7 , 0 , 0 , 5 . 9 , 0 , 0 , 5 . 9 , 0 , 0 , 0 , 0 , 0 . 0 , 0 , 0 . 2 27 3 7 . 5 , 6 . 3 , 6 . 3 , 1 8 . 8 , 0 , 3 1 . 3 , 0 , 0 , 0 , 0 , 0 , 0 , 0 , 0 , 0 , 0 , 0 , 0 , 0 , 0 , 2 28 2 9 . 4 , 5 . 9 , 5 . 9 , 1 7 . 7 , 0 , 4 1 . 2 , 0 , 0 , 0 , 0 . 0 , 0 , 0 , 0 , 0 . 0 . 0 , 0 , 0 , 0 , 2 29 3 3 . 3 , 6 . 7 , 6 . 7 , 1 3 . 3 , 6 . 7 , 3 3 . 3 , 0 , 0 , 0 , 0 , 0 , 0 , 0 , 0 , 0 , 0 . 0 , 0 . 0 , 0 , 2 30 2 0 , 0 , 0 , 3 0 , 1 0 , 3 0 , 0 , 0 , 1 0 , 0 , 0 , 0 , 0 . 0 , 0 , 0 , 0 , 0 . 0 . 0 , 2 31 2 1 . 4 , 0 , 0 , 2 1 . 4 , 0 , 5 7 . 1 . 0 , 0 , 0 , 0 , 0 , 0 , 0 , 0 , 0 , 0 , 0 , 0 , 0 , 0 , 2 32 3 5 . 7 , 0 , 7 . 1 , 1 4 . 3 , 0 , 4 2 . 9 , 0 , 0 , 0 , 0 , 0 , 0 , 0 , 0 , 0 , 0 , 0 , 0 , 0 , 0 , 2 33 2 3 . 7 , 1 5 . 3 , 1 1 . 9 , 1 8 . 6 , 0 , 1 0 . 2 , 6 . 8 , 0 . 1 1 . 9 , 0 . 0 . 0 . 1 . 7 , 0 , 0 , 0 , 0 , 0 , 0 . 0 , 2 34 4 2 . 6 , 6 . 4 , 1 0 . 6 , 1 4 . 9 , 0 , 1 4 . 9 , 2 . 1 , 2 . 1 , 4 . 3 , 2 . 1 , 0 , 0 , 0 , 0 , 0 , 0 , 0 , 0 . 0 , 0 , 2 35 2 8 . 3 , 1 3 . 3 , 1 6 . 7 , 1 3 . 3 , 1 . 7 , 1 6 . 7 , 3 . 3 , 1 . 7 , 5 , 0 , 0 , 0 , 0 . 0 , 0 , 0 , 0 , 0 . 0 , 0 . 2 36 2 8 , 1 2 , 6 , 2 0 , 0 , 2 0 , 8 , 0 , 6 , 0 , 0 , 0 , 0 . 0 , 0 . 0 , 0 , 0 , 0 , 0 , 2 37 3 5 . 2 , 8 , 9 . 1 , 1 3 . 6 , 0 , 1 5 . 9 , 4 . 6 , 2 . 3 . 9 . 1 , 2 . 3 , 0 , 0 , 0 , 0 , 0 , 0 , 0 , 0 , 0 , 0 , 2 APPENDIX F - DISCRIMINANT AND MAHALANOBIS DISTANCE ANALYSIS BRANCH BUD FREQUENCY DATA. <SEPARATE OPTIONS=DISTANCE VAR=1-20 CASES=1-37 STRAT=V21> SEPARATION OF STRATA CASES=CASE#:1 -37 DISCRIMINANT FUNCTIONS (INCLUDE 5 . V 5 ) STEP= 4 VARIABLE F -STAT SIGNIF (1) (2) CONSTANT - 3 . 6 2 9 6 - 1 6 . 9 4 2 1 . V1 3 8 . 6 1 4 . 0 0 0 0 .43618 .94163 3 . V 3 9 . 2 2 3 9 .0047 .11955 .5988 1 5 . V5 5 . 5 6 7 4 .0246 .51792 1 . 3064 8 . V 8 16 .156 . 0003 - . 4 1 2 5 1 - 2 . 0 7 2 9 N (N= 37 OUT OF 37) 20 17 NOT INCLUDED F -STAT SIGNIF 2 . V 2 3 . 1 4 5 5 . 0 8 6 0 4 . V 4 .37539 .5445 6 . V6 2 . 7 4 0 8 .1079 7. V7 .24276 .6257 9 . V 9 .94346 .3389 10. V10 .38514 .5394 11. V11 2 . 6 9 5 7 .1107 12. V12 .76989 - 1 .7833 13. V13 1.9544 . 1 7 2 0 14. V14 2 . 3 8 9 6 .1323 15. V15 1.4418 .2389 16. V16 1.4854 .2321 17. V17 2 . 2 1 6 5 .1466 18. V18 . 6 0 6 9 3 - 2 .9384 19. V19 2 . 2 9 4 8 .1399 2 0 . V20 - O . 1 .0000 MAHALANOBIS DISTANCES BETWEEN STRATA V21 D-SOUARE F -STAT SIGNIF (1) (2 ) 11 .807 2 4 . 7 9 9 .OOOO STEP FORWARD SEPARATION . F -STAT SIGNIF 0 INCLUDE NO VARIABLES 1 INCLUDE 1 . V 1 3 5 . 5 4 2 .0000 2 INCLUDE 8 . V8 14.321 .0006 3 INCLUDE 3 . V3 7 . 2 3 3 5 .0111 4 INCLUDE 5 . V 5 5 . 5 6 7 4 .0246 <FINISH> 1 5 , 5 . 5 , 4 0 , 3 , 3 , . 8 , 5 0 , 14, 1 0 , 2 5 , 5 , 1 0 , 2 . 3 , 2 5 , 7 7 , 5 , 1 0 , 6 . 8 , 1 0 0 , 4 0 , 5 , 4 0 , 4 , 2 5 , 4 2 5 , 1 8 , 3 0 , 3 1 0 . 6 . 4 5 . 3 . 3 . 2 , . 8 , 4 0 , 1 4 . 7 , 3 2 , 7 , 1 4 , 1 . 9 , 2 2 , 7 0 , 3 . 8 , 8 . 4 , 9 0 , 6 0 , 5 , 4 4 , 4 , 3 2 , 3 8 5 , 1 8 , 2 0 , 3 1 2 , 3 , 3 0 , 3 , 3 . 2 , 1 , 4 5 , 2 1 , 8 , 2 0 , 4 , 12, 1 . 8 , 1 4 , 5 6 , 4 , 1 0 , 5 , 1 0 0 , 5 0 . 6 , 4 0 , 4 , 3 0 , 4 5 0 . 2 4 , 3 0 , 4 1 7 , 4 , 4 0 . 2 . 5 , 2 . 8 , . 6 . 2 0 . 7 , 7 . 1 8 , 3 . 7 , 2 . 5 . 7 . 3 2 . 3 . 7 , 3 . 9 . 7 5 , 3 0 . 5 . 3 6 , 4 , 2 5 , 2 6 0 . 1 8 , 4 5 , 4 1 8 . 5 , 6 . 4 5 , 2 . 5 , 2 . 8 , . 8 , 4 0 , 1 4 , 1 0 , 2 8 , 3 , 1 2 , 2 . 5 , 1 4 , 6 7 , 5 , 7 , 6 . 8 , 7 5 , 4 5 . 5 , 4 0 , 4 , 3 0 , 3 0 0 , 1 6 , 2 0 , 3 1 4 . 5 , 6 . 5 , 3 0 , 3 , 3 . 2 , 1 . 1 , 3 0 , 1 0 , 7 , 3 6 , 3 , 1 2 , 2 . 9 , 2 2 , 6 0 , 3 . 1 2 , 5 . 5 , 1 2 0 , 7 5 , 5 , 4 5 , 4 . 3 0 , 6 0 0 . 1 5 , 3 0 . 1 5 . 9 . 5 . 4 0 . 3 . 3 , . 8 , 3 0 , 1 4 , 1 0 , 3 6 , 5 , 1 0 , 3 . 1 , 1 8 , 7 4 , 3 , 8 , 8 . 4 , 9 5 , 5 5 , 6 , 4 8 , 4 , 3 5 , 5 0 0 , 2 5 , 3 0 . 3 . 5 1 1 , 3 , 4 0 , 2 . 5 , 2 . 8 , . 8 , 5 0 , 1 4 , 1 0 , 2 2 , 5 , 1 0 , 2 . 1 . 1 8 , 6 0 , 5 , 1 0 , 5 . 2 , 9 0 . 4 5 , 6 , 4 0 , 4 . 2 8 . 3 3 0 , 1 9 , 2 0 , 3 7 , 4 , 4 5 , 2 , 2 . 5 , . 5 , 6 8 , 1 4 , 7 , 2 5 , 5 , 1 0 , 2 . 1 , 14, 4 5 , 5 , 1 0 , 3 . 9 , 6 8 , 5 5 , 5 , 3 0 , 4 , 2 5 , 2 4 0 , 1 4 , 2 5 , 3 1 5 , 6 . 5 , 3 0 , 2 . 5 , 3 , 1 , 5 5 , 1 4 , 8 , 2 2 , 5 , 1 4 , 1 . 6 , 1 8 , 5 6 , 5 , 8 , 5 . 7 , 1 1 0 , 4 5 , 5 , 4 4 , 3 , 2 5 , 4 5 0 , 2 4 , 3 0 , 4 2 0 , 5 , 4 0 , 3 , 3 . 5 , 1 . 4 , 8 0 , 1 8 , 7 , 2 5 , 5 , 18, 1 . 4 , 1 8 , 6 6 , 5 , 1 8 , 3 . 7 , 8 5 , 4 5 , 5 , 2 8 , 2 , 2 1 , 4 1 5 , 2 0 , 2 0 , 3 1 3 , 3 , 3 5 , 2 . 2 , 2 , 2 , . 6 , 3 0 , 1 4 , 5 , 1 8 , 5 , 9 , 1 . 6 , 1 0 , 4 0 , 3 , 7 , 5 , 9 0 , 4 5 . 6 , 4 5 , 4 , 3 0 , 3 0 0 , 1 8 , 3 0 , 4 5.5 , 3 . 5 , 5 0 , 3 , 3 . 8 , . 6 , 1 5 , 1 8 , 1 0 , 3 2 , 5 , 1 0 , 2 . 8 , 1 4 , 6 0 , 5 . 1 4 , 3 . 9 , 9 0 . 5 0 , 6 , 4 8 , 4 , 3 0 , 4 0 0 , 2 0 . 2 5 , 4 5 , 1 . 5 , 5 0 , 2 . 8 , 3 . 5 , . 8 , 2 5 , 1 8 , 7 , 3 0 , 5 , 1 0 , 2 . 5 , 8 , 4 0 , 5 , 8 , 3 . 7 , 9 0 , 4 5 , 6 , 4 8 , 4 , 3 2 , 2 2 0 , 1 8 , 3 0 , 3 6 , 3 . 5 0 , 3 , 3 , 1 , 2 5 , 1 6 , 7 , 2 2 , 5 , 1 2 , 1 . 7 , 1 0 , 4 0 , 3 , 1 4 , 2 . 9 , 1 O 0 . 7 O , 5 , 3 5 , 4 , 3 0 . 5 0 0 , 2 4 , 3 0 , 3 9 . 5 , 4 , 6 0 , 1 . 9 , 2 . 5 , . 5 , 3 0 , 1 5 , 7 , 1 8 , 5 , 10, 1 . 7 , 7 , 2 7 , 3 , 1 0 , 2 . 6 , 7 5 , 5 0 , 5 , 3 8 , 4 , 1 9 , 4 0 0 , 2 2 , 2 5 , 4 6 , 4 , 6 0 , 1 . 8 , 2 . 8 , . 8 , 5 5 , 2 5 , 7 , 1 8 , 5 , 1 0 , 1 . 7 , 7 , 3 6 , 7 , 1 0 , 2 . 5 , 7 5 , 5 0 , 5 , 4 4 , 4 , 2 6 , 3 3 0 , 1 9 , 3 0 , 3 6 , 3 , 6 0 , 3 , 3 , . 8 , 6 0 , 2 2 , 7 , 1 8 , 5 , 1 0 , 1 . 7 , 8 , 4 0 , 3 , 1 0 , 3 . 7 , 7 5 , 5 5 , 6 , 4 0 , 4 , 3 0 , 4 2 0 . 2 3 , 3 0 , 4 5 , 2 , 5 0 , 2 , 2 . 2 , . 6 , 4 0 , 1 8 , 7 , 2 5 , 3 , 1 0 , 2 . 3 , 8 , 3 0 , 3 , 1 0 , 2 . 9 , 7 5 , 5 5 . 6 , 3 8 , 4 , 2 7 , 3 0 0 , 1 8 , 3 0 , 4 4.5 . 2.5 , 6 0 , 2 . 6 , 2.8, . 8 , 6 0 , 2 3 , 7 , 2 2 , 5 , 1 0 , 1 . 9 , 8 , 3 6 , 5 , 1 0 , 2 . 9 , 9 0 , 5 5 , 6 , 4 6 , 4 , 8 , 2 8 5 , 1 6 , 3 0 , 3 4 , 1 . 5 , 5 0 , 2 . 6 , 3 , . 6 , 4 0 , 1 4 , 7 , 2 2 , 5 , 14,1 . 5 , 7 , 3 2 , 5 , 8 , 3 , 8 3 , 4 5 , 6 . 4 3 , 4 . 2 8 , 2 7 0 , 2 0 , 3 0 , 3 1 0 , 2 . 5 , 6 0 , 1 . 5 , 2 , . 4 , 6 0 , 1 4 , 7 , 1 8 , 5 , 1 4 , 1 . 3 , 1 0 , 2 5 , 5 , 1 0 , 2 . 3 , 6 0 , 3 0 , 5 , 3 3 , 3 , 2 0 , 165, 1 3 , 3 0 , 3 1 2 . 5 , 2 , 5 0 , 2 . 5 , 3 , . 9 . 4 8 , 2 5 , 3 , 2 5 . 7 , 14, 1 . 3 , 8 . 5 2 , 3 , 1 0 , 4 . 6 , 5 2 , 4 8 , 5 , 4 0 , 4 , 3 0 , 3 3 0 , 2 0 , 3 0 , 4 8 , 2 , 55,2,2.5, .6 ,55, 1 4 , 7 , 1 8 , 5 , 10, 1 . 7 , 9 , 3 0 , 5 , 9 , 2 . 8 , 7 5 , 4 5 , 6 , 3 8 , 3 , 1 4 , 3 1 5 , 2 2 , 4 0 , 4 206 APPENDIX H - DISCRIMINANT AND MAHALANOBIS DISTANCE ANALYSIS OF PHENETIC DATA. DISCRIMINANT FUNCTIONS (INCLUDE 18.V18) STEP= 5 VARIABLE F -STAT SIGNIF (1) (2) CONSTANT - 1 2 6 . 9 4 - 2 2 6 . 6 5 2 . V2 7 . 3 5 3 3 .0143 - 7 . 5 6 2 8 - 1 1 .357 3 . V3 4 9 . 9 1 6 . 0 0 0 0 3 . 6 6 7 5 5 . 1 5 1 8 18 . V18 4 . 4 4 8 2 .0492 - 2 . 9 5 0 3 - 6 . 3 9 2 3 22 . V22 1 0 . 0 8 5 .0052 2 . 7 4 6 2 3 . 7 5 3 9 25 . V25 7 . 8 7 5 2 .0117 .15289 .20870 N (N= 24 OUT OF 24) 12 12 NOT INCLUDED F -STAT SIGNIF 1 . . V1 .52577 - 2 . 9 4 3 0 4. . V4 .32311 - 1 .8595 5. . V5 .63873 - 1 .8035 6 . V6 .29234 - 2 .9575 7 . V7 .94644 . 3443 8 .V8 .34626 - 3 .9854 9 . V9 .42696 .5222 10 . V10 3 . 2 2 2 3 .0904 11 . V1 1 .17066 .6847 12 . V12 3 . 2 0 6 4 .0912 13, . V13 .38342 - 3 .9846 14 , . V14 . 3 7 3 6 9 .5491 15, . V15 .16351 - 1 .8998 16 . V16 .44891 .5119 17. . V17 .25393 - 1 .8753 19, . V19 1 .4643 .2428 20 . . V20 .88095 - 1 .7702 21 , . V21 .30836 . 5859 23 . V23 .98508 . 3349 24 . V24 2 . 0 3 2 1 .1721 26 . V26 .94402 . 3449 27 . V27 .50394 . 4874 28 . V28 .45391 .5095 MAHALANOBIS DISTANCES BETWEEN STRATA V29 D.-SOUARE F - STAT SIGNIF (1 ) (2 ) 4 0 . 0 8 6 39 .357 . 0 0 0 0 STEP FORWARD SEPARATION F -STAT SIGNIF O INCLUDE NO VARIABLES 1 INCLUDE 3 . V 3 . 5 4 . 4 9 8 . 0 0 0 0 2 INCLUDE 2 . V 2 8 . 8 5 5 6 .0072 3 INCLUDE 2 5 . V 2 5 1 0 . 1 5 5 .0046 4 INCLUDE 2 2 . V 2 2 5 . 3 9 0 3 .0315 5 INCLUDE 18 .V18 4 . 4 4 8 2 .0492 207 APPENDIX I_ - CORRELATION MATRIX OF PHENETIC DATA. PO Kl M M M 10 IO to U M to IO C  Ul a Ul  (0 a tn Ul u < < < < < < < < < < < < < < < < < < < < IO IO M 10 10 M M u t» tn Ul 09 -4 01 Ul it u M -* O (0 ts tn Ul < M IO Ul co Ul Ul • o u u tn tn CD tn to o o o -CO O — — Ul IO u M & CO o o o o M tn 91 • o o o o < — t —. fa b Ul to b to 01 o 09 o u fa fa cn <n o 01 CD o cn o ro CO CO .& -J .Ul faO CO. to a 01 — IO cn — Ul GO fa o —• cn 09 o — Ul — Ul —• Ul • -J O M O u to cn -* u ui -fa O fa Ul -» KJ O -J u O O U) to -»J u o o o to — — w — O O O w to — O to < — • o CJ O U 09 O — CJ o U o o to u> o O -Nl o 1 1 I o CJ Ul u !* fa U) o CO OB Ul O fa o fa Ul cn u fa to *J to (0 Ul -4 -* m CJ fa fa m < w ro O b to cn to to o o O • Ul Ul Ul Ul _^ o o CJ -fa O to — •o fa fa o CD —• — CO CD O ~4 to o < 03 fO — — to O CO < to tow — O — tocJfaO to- -4 fa 01 O — CO O M CO fa — U O u O cn tn co —• o < M i -* fo to o O o O o u • tn U) tn M o Ul o & o o •o tn a> 03 o o < IO i IO -u b IO IO u b £k - IO o CO to o tn CO o o CO O II CN «* ID I -I • to X i- • o i-1 z w u .J [14 ta o • m cu! o in! 2 cn O K II CJ ID r» W > i t—< ID EH ID < w « < EH p < EH < in o W CJ EH t-H D tJ EH O to m ft < to i 10 M n z w a. < t 3 , 16 . 48 2 2 . 3 , 12.64 3 1 . 7 . 9 .34 4 1 . 7 . 9 34 5 1 6 , 8 . 79 6 1 5 8.24 7 1 5 8 . 24 8 1 5 8.24 9 1 3 7 . 14 IO 1 3 7.14 1 1 0 .8 4 .40 2 5 15.06 13 2 O 12 .05 14 1 7 10. 24 15 1 7 10.24 16 1 7 10. 24 17 1 5 9.04 18 1 3 7.83 19 1 3 7.83 20 1 3 7 .83 21 0 7 4 . 23 22 O 9 5 . 42 23 2 5 12.76 24 2 3 11.74 25 2 2 11 .23 26 2 0 10. 20 27 2 0 10. 20 28 2 O 10. 20 29 1 7 8.67 30 1 3 6 . 6 3 31 1 2 6 . 12 32 1 2 6.12 33 1 2 6. 12 34 2 8 15 . 39 35 2 5 13.74 36 1 7 9.34 37 1 7. 9.34 38 1 6, 8 . 79 39 1 6 8. 79 40 1 6 8 . 79 41 1 4 7 .69 42 1 3 7 . 14 43 1 1. 6.04 44 O 9| 4 .95 45 2 2. 12 .43 46 2 2. 12 .43 47 2 2. 12.43 48 2 0 , 11.3 49 1 7. 9.61 50 1 5, 8 . 4 8 51 1 3 , 7.35 52 1 3. 7.35 53 1 3 . 7.35 54 1 2. 6 . 7 8 55 0 8 , 4.52 56 2 1 . 12 . 28 57 2 0 . 11 .70 58 1 8. 10. 53 59 1 7. 9.94 60 1 6, 9 . 37 61 1 . 5 . 8 . 77 62 1 5, 8.77 63 1 . 4 . 8 . 19 64 1 4 , 8 . 19 65 1 2, 7.02 66 O 9- 5.26 67 2 . 5 . 15.82 68 1 B. 1 1 . 39 69 1 . 5 . . 9 . 4 9 70 1 . 5 . 9 .49 71 1 4. 8.86 72 1 3 . 8 .23 73 1 . 3 . 8 . 23 74 1 3. 8 .23 75 1 . 3 . 8 .23 76 1 1 . 6.96 77 0 8, 5.06 78 1 7. 11.33 79 1 7, 11.33 80 1 7 , 1 1 . 33 81 1 5. 10.0 82 1 3 . 8 . 67 83 1 3. 8.67 84 1 3 . 8 .67 85 1 2. 8 . 0 86 1 2. 8 . 0 87 1 1, 7 . 33 88 1 0 . 6 .67 89 2 5. 13 .02 90 2 3. 1 1 .98 91 2 0 . 10.42 92 2 0 , 10.42 93 1 9 . 9 . 38 94 1 7. 8 . 8 5 95 1 7. 8 . 8 5 96 1 4. 7 .29 97 1 3. 6.77 98 1 3. 6.77 99 1 2, 6 . 2 5 100 2 0 . 12.9 101 2 0 . 12.9 102 1 7. 10.97 103 1 5. 9.68 104 1 3 , 8 .39 105 1 3. 8 . 39 106 1 3. 8 . 39 107 1 3. 8 . 39 108 1 1, 7. 10 109 1 1. 7 . 10 1 10 0 9. 5.81 1 1 1 2 5. 14 .88 1 12 2 • 1 . 12.5 1 13 2 . 1 . 12.5 1 14 1 9. 11.31 115 1 6, 9.52 1 16 1 • 3 , 7.74 1 17 1 • 3 . 7.74 1 18 1 • 2, 7. 14 1 19 1 2, 7 . 14 120 0 • 9 , 5.36 121 0 •7, 4.17 122 2 • 3, 12.5 123 1 . 9 , 10.33 124 1 8, 9.78 125 1 7. 9 24 126 1 7, 9.24 127 1 6, 8 . 7 0 128 1 5, 8. 15 129 1 5, 8 . 15 130 1 6, 8 . 7 0 131 1 5, 8. 15 132 1 3 , 7 .07 133 2 7, 13.5 134 2 7, 13.5 135 2 1 , 10.5 136 1 8 , 9 . 0 137 1 7, 8 . 5 138 1 7. 8 . 5 139 1 5, 7.5 140 1 5, 7 . 5 141 1 5, 7.5 142 1 5. 7.5 143 1 3 , 6 . 5 144 2 3, 13.86 145 1 8. 10.84 146 1 7, 10.24 147 1 7. 10.24 148 1 6. 9 .64 149 1 5, 9.04 150 1 5, 9.04 151 1 3 , 7 .83 152 1 3, 7 .83 153 1 0 , 6.02 154 0 9, 5.42 155 2 7, 15.98 156 2. 7. 15.98 157 1 . 7, 10.06 158 1 . 5, 8.88 159 1 5, B.BB 160 1 4. 8.28 161 1 . 3. 7.69 162 1 . 3. 7.69 163 1 . 2, 7. 1 164 0 . 8. 4.73 165 0 . 8, 4 . 73 166 3 . 5. 15 70 167 2 . 7 . 12.11 168 2 . 7 , 12.11 169 2 . 2 , 9 . 87 170 2 . 0 . 8.97 171 2 . 0 , 8 . 97 172 1 . 7. 7 .62 173 1 . 6. 7 . 18 174 | 5. 6 . 73 175 1 3. 5 . 83 176 1 1 , 4 .93 177 3 3, 15 . 14 178 2 7, 12 . 39 179 2 6. 1 1 .93 180 2 1 . 9.63 181 2 0 . 9.17 182 1 8 . 8 . 26 183 1 7 . 7 .80 184 1 6. 7.34 185 1 4 , 6.42 186 1 3. 5 . 96 187 1 3. 5.96 188 3 0. 18 . 29 189 2 6, 15.85 190 1 7. 10. 37 191 1 5. 9 . 15 192 1 3. 7 .93 193 1 2. 7 . 32 194 1 2. 7 .32 195 1 1. 6.71 196 1 0 . 6. 10 197 0 9. 5 . 49 198 0 9, 5 .49 199 3 0. 15.54 200 2 7. 13 . 99 201 2 2. 1 1 .40 202 2 0. 10. 36 203 1 8. 9 .33 204 1 6. 8 . 29 205 1 4 , 7.25 206 1 . 3 . 6 .74 207 1 . 3 . 6.74 208 1 . 2 . 6 . 22 209 0 8, 4 . 15 2 IO 2 2. 14 .38 211 1 7, 11.11 212 1 . 7 . 11.11 213 1 . 4 . 9 . 15 214 1 . 3 . B .50 215 1 . 3 . 8 .50 216 1 . 2 , 7 . 84 217 1 . 2 , 7.84 218 1 . 2 , 7.84 219 1 . 1 . 7 . 19 220 1 . 0 . 6 .54 22 1 2 7 , 14 .44 222 2 5 . 13 . 37 223 2 5. 13 . 37 224 1 7 . 9.09 225 1 7 . 9 . 0 9 226 1 7. 9.09 227 1 6. 8.57 228 1 3. 6 .95 229 1 2, 6.42 230 0 9, 4.81 231 0 9 , 4.81 232 2 3, 19 .00 233 : i 7, 14 .05 234 i 2 , 9.92 235 1 1 . 9.09 236 1 0, 8 . 26 237 0 9 , 7 .44 238 0 B, 6.61 239 0 8, 6.61 240 o 8, 6.61 241 0 8. 6.61 242 0 7, 5. 79 243 2 8. 16 . 28 244 2 0 , 1 1 .63 245 2 0 . 1 1 .63 246 1 8. 10.47 247 1 7, 9.88 248 1 3. 7 . 56 249 1 3 . 7 .56 250 1 2, 6 . 98 251 1 1 . 6 . 40 252 1 1 , 6 . 4 0 253 O 9 , 5 . 2 3 254 2 3. 14.20 255 2 0 . 12 . 35 256 1 7 . 10. 49 257 i 5, 9 . 26 258 1 4. 8 .64 259 1 3, 8 .02 260 1 3. 8 .02 261 1 3 . 8 .02 262 1 2. 7.41 263.. 1 2, 7.41 264 1 o, 6 . 17 265 2 8. 16.97 266 2 2. 13.33 267 2 0 , 12 . 12 268 1 7, 10.30 269 1 7 , 10.30 270 1 3, 7 .88 271 1 2. 7.27 272 1 0 . 6.06 273 0 9. 5 . 4 5 274 0 9. 5.45 275 0 8. 4 .85 •2:03 APPENDIX K - DISCRIMINANT AND MAHALANOBIS DISTANCE ANALYSIS OF ABSOLUTE CHROMOSOME LENGTHS. <SEPARATE OPTIONS=DISTANCE VAR=1-11 CASES=1-25 STRAT=V12> SEPARATION OF STRATA CASES=CASE#:1 -25 DISCRIMINANT FUNCTIONS (INCLUDE 8 . V 8 ) VARIABLE F -STAT SIGNIF CONSTANT 1 . V1 8 . V 8 1 5 . 6 7 5 10.224 (N= 25 OUT OF 25) NOT INCLUDED F -STAT . V2 . V3 . V4 . V5 . V6 . V7 . V9 1 0 . V 1 0 1 1 . V1 1 .69056 1 .2742 1 . 2663 . 7 2 7 3 0 .26019 .80111 .23459 .64168 .99985 - 1 .0007 .0042 SIGNIF .7953 .2717 .2732 . 4034 .6153 . 3809 .6332 .8025 . 3287 (1) - 3 3 . 4 3 6 7 . 7 6 1 9 3 5 . 7 3 6 14 STEP= 2 (2) - 3 4 . 3 0 9 13.454 2 5 . 0 4 4 1 1 MAHALANOBIS DISTANCES BETWEEN STRATA D-SOUARE F -STAT SIGNIF V12 (1) (2 ) 3 . 2 9 1 2 9 . 6 9 6 3 . 0 0 1 0 STEP FORWARD SEPARATION F -STAT SIGNIF 0 INCLUDE NO VARIABLES 1 INCLUDE 1.V1 6 . 5 4 3 9 .0176 2 INCLUDE 8 . V 8 10.224 .0042 <FINISH> 240 APPENDIX L - CORRELATION MATRIX OF ABSOLUTE CHROMOSOME LENGTHS co I v o CO a  -1 CD CJ1 CO IO < V10 < CO < GO < 1^ < 05 < UI < < CO < ro < < > 73 > 00 r--4. CO CO ui UI UI ro _* UI ui -j & _» UI ro O cn 01 ro ro CO & CO CO cn CO CO UI o cn < ro IO ro —^  ro ui ui UI ui cn CO CO cn UI O cn cn CO Ol ro ro & ui CO CO CO cn UI ro O cn o CO < CO CO •o. CO UI cn 03 0  O -* •o. O ro cn cn ui O O ~j CO •u CO ro ui O -j -J CO ui CO ro CD O < .u .u & 01 cn '-J 0  CO o O CD cn CO ui CO o ~j o  _h cn -o cn o  o cn co co co O o < UI UI ui ui cn 09 03 b O CO cn ui 09 o •~i CO o  IO CO cn o ui UI cn cn 03 o o o o o o o o o ro ui ro CO O Ul O O coCO 01 o o O ui ro O A O O 73 a XI 73 m 73 r m > r-- i > i i -t O m Z < 2 > > 73 -i II ;a — < cn cn • cn Ul cn ~j CO O CO CO 03 ro O UI -1 ' cn O CO o o O CO -j -J o cn Ul 03 O ro ~4 o cn -I ro o CO CO CO o < 00 03 • — cn CO b — O o o ro o ro 03 a < co CO UI cn o CO CO o ro 03 o O Ul o < -o ^i o O O o o o -J o < o o 21 1 APPENDIX M - RECIPES AND SOURCES FOR ELECTROPHORETIC STARCH GELS AND STAINS. STARCH GEL RECIPES MORPHOLINE CITRATE One %el Two g e l s e l e c t r o d e b u f f e r pH 6.1 18 ml 30 ml d i s t i l l e d water 332 ml 570 ml s t a r c h 43.75 g 75 g sucrose 35 g 60 g Source: m o d i f i e d from C l a y t o n & T r e t i a k 1972 RIDGWAY concentrated g e l b u f f e r pH 8.5 35 ml 60 ml d i s t i l l e d water 315 ml 540 ml s t a r c h 43.75 g 75 g sucrose 35 g 60 g Source: m o d i f i e d from Ridgway et a l . 1970 STAIN RECIPES DIAPHORASE (DIA) 0.2 M t r i s HC1 b u f f e r pH 8.0 60 ml 2,6 d i c h l o r o p h e n o l i n d o p h e n o l 1 mg NADH 2.5 mg MTT 1 ml Source: Yeh & O'Malley 1980 ESTERASE (EST) a-naphthyl a c e t a t e } d i s s o l v e d b-naphthyl a c e t a t e } f a s t blue RR s a l t 0.2 M phosphate b u f f e r pH 6.4 D i s s o l v e f a s t blue RR i n small to remove i n p u r i t i e s . Source: m o d i f i e d from G o t t l i e b i n 10 ml acetone 50 mg 50 mg 100 mg 100 ml amount of b u f f e r and f i l t e r 1973 GLUTAMIC DEHYDROGENASE (GLDH) 212 0.2 M t r i s HC1 b u f f e r pH 8.0 60 ml L-glutamic a c i d 2 g NAD 1 ml NBT 1 ml PMS 1 ml Source: m o d i f i e d from Shaw & Prasad 1970 and Hartmann et a l . 1973 GLUCOSE-6-PHOSPHATE DEHYDROGENASE (G6PDH) 0.2 M t r i s HC1 b u f f e r .40 ml d i s t i l l e d water 20 ml 0.5 M MgC12 1 ml glucose-6-phosphate 200 mg NADP 1 ml NBT 1.5 ml PMS 1 ml Source: m o d i f i e d from Sing & Brewer 1969 and Shaw & Prasad 1970 HEXOSEAMINIDASE (HA) 0.05 M phosphate b u f f e r pH 4.0 50 ml d i s t i l l e d water 50 ml 4-methylumbelli f e r y l - N - a c e t y l -b-D-glucose aminide 100 mg Observe under long-wave UV l i g h t . Source: m o d i f i e d from S i c i l i a n o & Shaw 1976 ISOCITRIC DEHYDROGENASE (IDH) 0.2 M t r i s HC1 b u f f e r pH 8.0 40 ml d i s t i l l e d water 20 ml D L - i s o c i t r i c a c i d 250 mg 0.5 M MgC12 1 ml NADP 1 ml NBT 1 ml PMS 1 ml Source: m o d i f i e d from A l l e n d o r f et a l . 1977 LEUCINE AMINOPEPTIDASE (LAP) 0.5 M t r i s maleate b u f f e r pH 6.5 40 ml d i s t i l l e d water 20 ml 1-leucyl-B-naphthylamide 30 mg n,n dimethyl formamide 5 ml black K s a l t 30 mg 213 D i s s o l v e 1-leucyl-B-naphthylamide i n n,n dimethyl formamide. Incubate 45 min, then add black K s a l t . Source: Roose & G o t t l i e b 1976 MALIC DEHYDROGENASE (MDH) 0.2 M t r i s HC1 b u f f e r pH 8.0 1 M Na L malate pH 7.0 NAD MTT PMS Source: m o d i f i e d from S i c i l i a n o & Shaw 1976 PHOSPHOGLUCOSE ISOMERASE (PGI) 0.2 M t r i s HC1 b u f f e r pH 8.0 40 ml d i s t i l l e d water 20 ml 0.5 M MgC12 1 ml glucose-6-phosphate dehydrogenase 2 ml 0.018 M fructose-6-phosphate 2.5 ml NADP 1 ml MTT 1 ml PMS 1 ml Source: m o d i f i e d from G o t t l i e b 1973 PHOSPHOGLUCOMUTASE (PGM) 0.2 M t r i s HC1 b u f f e r pH 8.0 40 ml d i s t i l l e d water 20 ml 0.5 M MgC12 1 ml glucose-6-phosphate dehydrogenase 2 ml 0.00017 M K2 a-D-glucose-1-phosphate 1.5 ml Na2 a-D-glucose-1-phosphate 100 mg NADP 1 ml MTT 1.5 ml PMS 1 ml Source: m o d i f i e d from Roose & G o t t l i e b 1976 6-PHOSPHOGLUCONATE DEHYDROGENASE (6PGDH) 0.2 M t r i s HC1 b u f f e r pH 8.0 40 ml d i s t i l l e d water 20 ml 6-phosphogluconic a c i d 20 mg 0.5 M MgC12 1 ml NADP 1 ml 100 ml 10 ml 5 ml 4 ml 2 ml MTT PMS ml ml Source: m o d i f i e d from A l l e n d o r f et a l . 1977 215 APPENDIX N - NORTH AMERICAN HABITATS SEARCHED FOR LIMBELLA. L o c a l i t y Topographic Map BRITISH COLUMBIA Vancouver, St a n l e y Park, Beaver Lake Vancouver, U n i v e r s i t y of B r i t i s h Columbia Endowment Lands Ladner Marsh P i t t River Meadows Burnaby Lake Park WASHINGTON Grays Harbor Co. Ocean C i t y State Park Twin Harbors S t a t e Park Grayland, Ewart S t . P a c i f i c Co. Grayland Beach State Park O y s t e r v i l l e I s l a n d Lake Cranberry Marsh, 32nd Place Cranberry Marsh, 55th S t r e e t OREGON C l a t s o p Co. C u l l a b y Lake County Park Tillamook Co. Manzanita Manhattan Beach-Lake L y t l e Rockaway Old-Growth F o r e s t Preserve Chamberlain Lake T i e r r a d e l Mar Sears Lake Neskowin L i n c o l n Co. D e v i l s Lake State Park East D e v i l s Lake State Park South Beach S t a t e Park Lane Co. Sutton Lake Woahink Bog Douglas Co. Winchester Bay Coos Co. B l u e b i l l Lake Second Creek F o u r t h Creek R e s e r v o i r B a s t e n d o r f f Naval S t a t i o n Bog B a s t e n d o r f f Beach Bandon Curry Co. G a r r i s o n Lake CALIFORNIA Del Norte Co. North Vancouver 92 G/6 L u l u I s l a n d 92 G/3 « II Port Coquitlam 92 G/7 New Westminster 92 G/2 Copal i s Beach 7.5' Grayland 7.5' O y s t e r v i l l e 7.5' Ocean Park 7.5' Cape Disappointment 7.5 Gearhart 7.5' Nehalem 15' H II H H Tillamook 15' Hebo 15' Cape Foulweather 15' Euchre Mountain 15' Yaquina 15' Heceta Head 15' S i l t c o o s Lake 15' Reedsport 15' Empire 7.5' II H C h a r l e s t o n 7.5' H II II II Bandon 15' L a n g l o i s 15' 216 Yontocket Lake E a r l Dead Lake Crescen t C i t y South Humboldt C o . Freshwater Lagoon Dry Lagoon S ta te Park B i g Lagoon Mendocino C o . Inglenook Fen Lake Cleone Smith R i v e r 7 .5 ' Crescen t C i t y 7 .5 ' It TI S i s t e r Rocks 7 .5 ' O r i c k 7 .5 ' Rodgers Peak 7 .5 ' Inglenook 7 .5 ' F o r t Bragg 7 .5 ' PUBLICATIONS 1. I l l u c t r c t i o n s i n : Honon, C. 1975. Guide t o the P l a n t s of the Wallowa  Mount Bins of Nor the as t c m Oregon. S p e c i a l P u b l i c a t i o n s , Museum of N a t u r a l H i s t o r y , U n i v e r s i t y Of Oregon. 411 pp. 2 . C h r i s t y , J . A . 1979. Report on n Survey of Sphagnum-cent c l n l n g Wetlands  of the Oregon Const. Oregon N a t u r a l Area P r e s e r v e s A d v i s o r y Committee, E t c t e Lend Board, Solera. 92 pp. 3. . 1980. A d d i t i o n s t o the noes f l o r a o f Oregon. The B r y o l o g i s t 83: 355-358. 4. _.- 1980. r e d i s c o v e r y of £cicromiurn t r i c o c t c t u r o ( S u l l . ) K i t t . ("Limbello t r i c o E f t . t a ( S u l l . ) B c r t r . ) i n North America. The E r y o l o R i s t 83-521-523. 5. . _, J . H. L y f o r d L D. H. t'egner. 1982. C h e c k l i s t o f Orcg on Docses. The B r y o l o g i o t B5: 22-36. 6. _ • 1983. B i b l i o g r c p h y o f Oregon cocoes. S y c c i s 16: 43-52. 7. Pvuching, A . E . & J . A . C h r i c t y . 1984. B r u c h i a f l e x u o s a new t o western tforth America. The B r y o l o g i c t 87: 68-69. 8. C h r i s t y , J . A . 1984. New Sphagnum species from P a c i f i c Northwest. B u l l e t i n of the N a t i v e P l a n t S o c i e t y of Oregon 17(7): 11. 9. F r e n k e l , R. E., W. H. K o i r £, J . A . C h r i s t y . 1985. V e g e t a t i o n of Torrey Lake M i r e , c e n t r a l Cascade Range, Oregon. (submitted to Madrono). 10. C h r i s t y , J . A., F. D. Bowers £> M. J . Dibben. 1985. F i v e mosses new t o Wisconsin. (submitted to The B r y o l o g i s t ) . 11. ( , & 1985. A t l a s of Wi s c o n s i n b r y o p h y t e s : e d d i t i o n s . (submitted to Milwaukee P u b l i c Museum P r e s s ) . CBANT AWARDS 1983 N o r t h w e s t S c i e n t i f i c A s s o c i a t i o n R e s e a r c h Committee. S u p p o r t f o r h a b i t a t i n v e n t o r y o f L i m b e l l a t r i c o s t a t a ( M u s c i ) , W a s h i n g t o n , Oregon, n o r t h e r n C a l i f o r n i a . 1980 Mazamas R e s e a r c h Committee. S u p p o r t f o r s u r v e y of Sphapnim-cont a i n i n g w e t l a n d s o f C a s c a d e Range, O r e g o n . 1978 U n i v e r s i t y o f Oregon U n d e r g r a d u a t e R e s e a r c h Committee. S u p p o r t f o r purvey of Sphajmum-cont a i n i n g w e t l a n d s of Oregon c o a s t . 

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