UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

Geographic variation in Novumbra hubbsi Schultz 1929 (Pisches, Umbridale) : External meristic characters,… Rosenfeld, Mark Jay 1983

Your browser doesn't seem to have a PDF viewer, please download the PDF to view this item.

Notice for Google Chrome users:
If you are having trouble viewing or searching the PDF with Google Chrome, please download it here instead.

Item Metadata

Download

Media
831-UBC_1983_A6_7 R67.pdf [ 8.11MB ]
Metadata
JSON: 831-1.0095677.json
JSON-LD: 831-1.0095677-ld.json
RDF/XML (Pretty): 831-1.0095677-rdf.xml
RDF/JSON: 831-1.0095677-rdf.json
Turtle: 831-1.0095677-turtle.txt
N-Triples: 831-1.0095677-rdf-ntriples.txt
Original Record: 831-1.0095677-source.json
Full Text
831-1.0095677-fulltext.txt
Citation
831-1.0095677.ris

Full Text

GEOGRAPHIC VARIATION IN NOVUMBRA HUBBSI SCHULTZ 1929 (PISCES: UMBRIDAE): EXTERNAL MERISTIC CHARACTERS, CHROMOSOMAL STATE AND NUCLEAR DNA CONTENT by MARK JAY ROSENFELD B . S c , U n i v e r s i t y o f Utah, 1976 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n THE FACULTY OF GRADUATE STUDIES (Department of Zoology) We accept t h i s t h e s i s as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA October 1983 ©Mark Jay Rosenfeld, 1983 In presenting t h i s thesis i n p a r t i a l f u l f i l m e n t of the requirements fo r an advanced degree at the University of B r i t i s h Columbia, I agree that the Library s h a l l make i t f r e e l y a v a i l a b l e for reference and study. I further agree that permission for extensive copying of t h i s 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 representatives. 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 for f i n a n c i a l gain s h a l l not be allowed without my written permission. Department of Zoology  The University of B r i t i s h Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 Date 14 October 1983 DE-6 (3/81) i i ABSTRACT Geographic V a r i a t i o n i n Novumbra hubbsi Schultz 1929 ( P i s c e s : Umbridae): External M e r i s t i c Characters, Chromosomal State and Nuclear DNA Content V a r i a t i o n i n external m e r i s t i c c h a r a c t e r s , chromosomal s t a t e and nuclear DNA content was examined across the range o f a r e l i c t f i s h , Novumbra hubbsi S c h u l t z , o c c u r r i n g w i t h i n the general area o f the Chehalis p e r i g l a c i a l refuge near the terminal moraine of the l a t e P l e i -stocene Vashon G l a c i e r i n western Washington, U.S.A. N^ . hubbsi shows considerable geographic v a r i a t i o n i n several external m e r i s t i c c h a r a c t e r s . The geographic d i s t r i b u t i o n s o f mean counts f o r l a t e r a l bars and anal f i n rays i n males as well as those f o r the l a t e r a l s c a l e row and scales above the l a t e r a l s c a l e row f o r males and females combined i n d i c a t e that the Montesano H i l l s , f o o t h i l l s o f the Olympic Mountains, are a b a r r i e r to gene flow. This b a r r i e r may have been present s i n c e at l e a s t the P l e i -stocene. L a t e r a l bar counts are lower and dorsal f i n ray counts higher i n males from predator-free h a b i t a t s . These character trends are con-sid e r e d important f o r mate a t t r a c t i o n and t e r r i t o r i a l defense. Reductions i n the numbers of pectoral and p e l v i c f i n rays are a l s o a s s o c i a t e d with predator-free h a b i t a t s . Lower numbers o f pectoral and p e l v i c f i n rays as w e l l as l a t e r a l bars and greater numbers o f dorsal f i n rays may cause f i s h to be more prone to predation. Character frequencies may change over time, as i n t e r p r e t e d from comparisons o f f i s h from the present 111 study with those caught i n 1947 and 1968. The elevated counts f o r l a t e r a l row scales and s c a l e rows before the dorsal f i n o r i g i n i n f i s h from the Wishkah River drainage may be due to a b i o t i c developmental f a c t o r s . As to chromosome number and morphology, hubbsi appears i n -v a r i a t e i n t e r s e x u a l l y and g e o g r a p h i c a l l y . Forty-eight chromosomes com-p r i s e the d i p l o i d number. The fundamental number i n chromosome comple-ments i s 80 chromosome arms, c o n t r a s t i n g with a published value of 74 arms. The r e v i s e d fundamental number i s based on an extensive s e r i e s o f measurements on 186 elongate chromosome s e t s . Nuclear DNA contents are reported f o r male and female N_. hubbsi from four populations. No i n t e r s e x u a l or geographical d i f f e r e n c e s appear present. The Feulgen-DNA determination f o r erythrocyte n u c l e i i s 1.68 pg/nucleus± 0.02(S.E.), about 80% of a published value. The d i f f e r e n c e between determinations may be due to the use of d i f f e r e n t c o n t r o l c e l l s or too high a reference DNA value by e a r l i e r workers. Since the Feulgen-DNA determination i s not very d i f f e r e n t from the f l u o r o m e t r i c ones i n t h i s study, as well as near a value determined f o r a c l o s e r e l a -t i v e , the present DNA estimate f o r N_. hubbsi i s considered accurate. The observed conservatism i n chromosomal s t a t e and DNA content may be a consequence of _N. hubbsi p e r s i s t i n g i n the same environment f o r several m i l l i o n y e a r s , p o s s i b l y since the Oligocene. i v TABLE OF CONTENTS ABSTRACT i i LIST OF TABLES v i LIST OF FIGURES x ACKNOWLEDGEMENTS x i i Int r o d u c t i o n 1 Ma t e r i a l s and Methods 11 Sampling l o c a l i t i e s 11 C l a s s i f i c a t i o n o f sampling l o c a l i t i e s 11 Physical and chemical determinations on the water 11 Methods o f c o l l e c t i o n 14 Morphomeristic determinations 14 An a l y s i s o f m e r i s t i c data 16 Chromosome methodology I 8 Feulgen-DNA determinations 2 0 Fluorometric-DNA technique 2 2 Results 2 3 Habitat c l a s s i f i c a t i o n 2 3 Presence o f f i s h e s other than N. hubbsi 2 3 P h y s i c a l and chemical determinations on the water 2 5 M e r i s t i c c h a r a c t e r i s t i c s 2 6 Sexual dimorphism o f dorsal and anal f i n rays 2 7 Dorsal f i n rays i n males 2 7 Dorsal f i n rays i n females 36 V Anal f i n rays i n males 3 6 Anal f i n rays i n females 42 Caudal f i n rays 42 Pectoral f i n rays 42 P e l v i c f i n rays 51 Scale rows before the dorsal f i n o r i g i n 51 La t e r a l row scales 51 Scales above the l a t e r a l s c a l e row 62 Scales below the l a t e r a l s c a l e row 62 La t e r a l bars i n males 62 Chromosome c h a r a c t e r i s t i c s 73 DNA q u a n t i f i c a t i o n 87 Discussion 91 The J u n c t i o n C i t y c o l l e c t i n g s i t e 91 M e r i s t i c v a r i a t i o n 92 Chromosomal s t a t e 99 Heteromorphy of chromosomes 15 and 16 100 Nuclear DNA 101 General d i s c u s s i o n 103 Summary and Conclusions 106 L i t e r a t u r e C i t e d 108 v i LIST OF TABLES TABLE PAGE 1 Map co-ordinates and s i t e d e s c r i p t i o n s f o r N. hubbsi c o l l e c t i n g s i t e s 12 2 C o r r e l a t i o n s between m e r i s t i c c h a r a c t e r values and standard l e n g t h f o r _N. hubbsi 17 3 Selected parameters of _N. hubbsi h a b i t a t s 24 4 Chi-square t e s t s on the d i s t r i b u t i o n s of dorsal and anal f i n ray s t a t e s compared between sexes by l o c a t i o n f o r _N. hubbsi 28 5 Sexual dimorphism i n the mean number of dorsal and anal f i n rays i n H. hubbsi evaluated w i t h t - t e s t s 29 6 Frequency d i s t r i b u t i o n s , l o g a r i t h m i c means and a r i t h m e t i c means f o r the number of dorsal f i n rays i n male _N. hubbsi 30 7 Frequency d i s t r i b u t i o n s , l o g a r i t h m i c means and a r i t h m e t i c means f o r the number of dorsal f i n rays i n female _N. hubbsi 31 8 Frequency d i s t r i b u t i o n s , l o g a r i t h m i c means and a r i t h m e t i c means f o r the number of anal f i n rays i n male _N. hubbsi 32 9 Frequency d i s t r i b u t i o n s , l o g a r i t h m i c means and a r i t h m e t i c means f o r the number of anal f i n rays i n female _N. hubbsi 33 10 S t a t i s t i c a l analyses using l o g a r i t h m i c means t e s t i n g f o r i n t e r p o p u l a t i o n a l d i f f e r e n c e s i n the number of dorsal f i n rays i n male N. hubbsi. 34 11 Scheffe's m u l t i p l e c o n t r a s t s t e s t s on the mean counts of dorsal and anal f i n rays f o r male N. hubbsi. 37 12 A n a l y s i s of variance i n the number of dorsal f i n rays i n female N. hubbsi 38 v i i TABLE (cont'd) PAGE 13 S t a t i s t i c a l analyses using l o g a r i t h m i c means t e s t i n g f o r i n t e r p o p u l a t i o n a l d i f f e r e n c e s i n the number of anal f i n rays i n male N. hubbsi. 40 14 S t a t i s t i c a l analyses using l o g a r i t h m i c means t e s t i n g f o r i n t e r p o p u l a t i o n a l d i f f e r e n c e s i n the number of anal f i n rays i n female N. hubbsi. 43 15 Frequency d i s t r i b u t i o n s , l o g a r i t h m i c means and a r i t h m e t i c means f o r the number of caudal f i n rays i n N. hubbsi 45 16 S t a t i s t i c a l analyses using l o g a r i t h m i c means t e s t i n g f o r i n t e r p o p u l a t i o n a l d i f f e r e n c e s i n the number of caudal f i n rays i n N. hubbsi 46 17 Frequency d i s t r i b u t i o n s , l o g a r i t h m i c means and a r i t h m e t i c means f o r the number of pectoral f i n rays i n N. hubbsi 48 18 S t a t i s t i c a l analyses using l o g a r i t h m i c means t e s t i n g f o r i n t e r p o p u l a t i o n a l d i f f e r e n c e s i n the number of pec t o r a l f i n rays i n N. hubbsi 49 19 Frequency d i s t r i b u t i o n s , l o g a r i t h m i c means and a r i t h m e t i c means f o r the number of p e l v i c f i n rays i n N. hubbsi 52 20 A n a l y s i s of variance and K r u s k a l l - W a l l i s t e s t s f o r the number of pevic f i n rays i n N. hubbsi 53 21 Frequency d i s t r i b u t i o n s , l o g a r i t h m i c means and a r i t h m e t i c means f o r the number of s c a l e rows before the dorsal f i n o r i g i n i n N. hubbsi 55 22 S t a t i s t i c a l analyses using l o g a r i t h m i c means t e s t i n g f o r i n t e r p o p u l a t i o n a l d i f f e r e n c e s i n the number of s c a l e rows before the dorsal f i n o r i g i n i i n N. hubbsi 56 23 Frequency d i s t r i b u t i o n s , l o g a r i t h m i c means and a r i t h m e t i c means f o r the number of l a t e r a l row sc a l e s i n N. hubbsi 58 i i x TABLE (cont'd) PAGE 24 S t a t i s t i c a l analyses using l o g a r i t h m i c means t e s t i n g f o r i n t e r p o p u l a t i o n a l d i f f e r e n c e s i n the number of l a t e r a l s c a l e s i n N. hubbsi 60 25 Frequency d i s t r i b u t i o n s , l o g a r i t h m i c means and a r i t h m e t i c means f o r the number of sca l e s above the l a t e r a l s c a l e row i n N. hubbsi 63 26 S t a t i s t i c a l analyses using l o g a r i t h m i c means t e s t i n g f o r i n t e r p o p u l a t i o n a l d i f f e r e n c e s i n the number of sca l e s above the l a t e r a l s c a l e row i n N. hubbsi 64 27 Frequency d i s t r i b u t i o n s , l o g a r i t h m i c means and geometric menas f o r the number of scales below the l a t e r a l s c a l e row i n N. hubbsi 66 28 S t a t i s t i c a l analyses using l o g a r i t h m i c means t e s t i n g f o r i n t e r p o p u l a t i o n a l d i f f e r e n c e s i n the number of scales below the l a t e r a l s c a l e row i n N. hubbsi 67 29 Frequency d i s t r i b u t i o n s , l o g a r i t h m i c means and a r i t h m e t i c means f o r the number of l a t e r a l bars i n male N. hubbsi 69 30 S t a t i s t i c a l analyses using l o g a r i t h m i c means t e s t i n g f o r i n t e r p o p u l a t i o n a l d i f f e r e n c e s i n the number of l a t e r a l bars i n male N. hubbsi 70 31 Summary of N. hubbsi examined f o r chromosomal s t a t e s 74 32 Frequency d i s t r i b u t i o n of d i p l o i d chromosome numbers obtained from t i s s u e s of N. hubbsi 76 33 Frequency d i s t r i b u t i o n of chromosome counts from the g i l l e p i t h e l i u m of N. hubbsi 79 34 Chromosome morphometries of N. hubbsi, based on 186 karyotypes obtained from 32 males and 30 females from several populations 80 35 Determinations of nuclear DNA contents f o r erythr o c y t e s of N. hubbsi 88 (cont'd) S t a t i s t i c a l analyses using the arc s i n square root transform o f r e l a t i v e Feulgen-DNA values f o r the n u c l e i o f N_. hubbsi erythrocytes X LIST OF FIGURES FIGURE PAGE I Map of the Olympic Peninsula and adjacent regions showing the range of N_. hubbsi 2 II Geographic v a r i a t i o n i n the number of dorsal f i n rays i n male j ^ . hubbsi 35 I I I Geographic v a r i a t i o n i n the number of dorsal f i n rays i n femal e N_. hubbsi 39 IV Geographic v a r i a t i o n i n the number of anal f i n rays i n nale N. hubbsi 41 V Geographic v a r i a t i o n i n the number of anal f i n rays i n female.NL hubbsi 44 VI Geographic v a r i a t i o n i n the number of caudal f i n rays i n N_. hubbsi 47 VII Geographic v a r i a t i o n i n the number of pectoral f i n rays i n _N. hubbsi 50 11X Geographic v a r i a t i o n i n the number of p e l v i c f i n rays i n N_.. hubbsi 54 IX Geographic v a r i a t i o n i n the number of s c a l e rows before the dorsal f i n o r i g i n i n N. hubbsi 57 X Geographic v a r i a t i o n i n the number of l a t e r a l row scales i n H. hubbsi 61 XI Geographic v a r i a t i o n i n the number of scales above the l a t e r a l s c a l e row i n J\L hubbsi 65 XII Geographic v a r i a t i o n i n the number of scales below the l a t e r a l s c a l e row i n H_. hubbsi 68 XIII Geographic v a r i a t i o n i n the number o f l a t e r a l bars i n male 'N_. hubbsi 71 XIV Male N_. hubbsi from four d i f f e r e n t regions 72 XV Metaphase chromosome spreads from N_. hubbsi 77 XVI Sorted karyotypes of N. hubbsi 82 x i FIGURE (Cont'd) PAGE XVII Idiogram of m i t o t i c metaphase chromosomes from hubbsi, based on the measurements of arm r a t i o and r e l a t i v e length 84 XIIX Metaphase chromosome spreads obtained from the g i l l e p i t h e l i u m o f N_. hubbsi captured at various l o c a t i o n s 85 XIX A m i t o t i c spread comprised of elongate chromosomes, obtained from a male N_. hubbsi c o l l e c t e d i n the Wishkah R. drainage 86 x i i ACKNOWLEDGEMENTS I am e s p e c i a l l y g r a t e f u l to Dr. N. J . Wilimovsky, my a d v i s o r , f o r his continual guidance, support and patience. Technical advice and c r i t i c a l a n a l y s i s o f t h i s paper were provided by the members o f my committee: Dr. N. R. L i l e y , Dr. J . D. McPhail, Dr. G. E. E. Scudder and Dr. A. G. Lewis. Several people have provided help of various kinds: Dr. J . D. Berger, U n i v e r s i t y o f B r i t i s h Columbia, f a c i l i t i e s f o r Feulgen-DNA densitometry; Dr. T. Gurney, U n i v e r s i t y o f Utah, f a c i l i t i e s f o r f l u o r o m e t r i c DNA determinations; Dr. F. J . D i l l , U n i v e r s i t y of B r i t i s h Columbia, use of a Zeiss photomicroscope; Dr. F. Hoppensteaedt, U n i v e r s i t y of Utah, advice on s t a t i s t i c s ; Mr. E. Ridges, U n i v e r s i t y o f Utah, access to the UNIVAC 1160 computer; Mr. G. McMines, Quinault Indian Nation, permission to c o l l e c t f i s h on Indian lands; and Mr. S. E. Campana, U n i v e r s i t y o f B r i t i s h Columbia, f i e l d a s s i s t a n c e . Ms. L. C. A. Rosenfeld provided e d i t o r i a l a s s i s t a n c e during the f i n a l stages of t h i s work. Last, but by no means l e a s t , I wish to express my a p p r e c i a t i o n to L. J . Romero f o r the typ i n g of t h i s manuscript. This i n v e s t i g a t i o n was supported by a National Sciences and Engineering Research Council grant to N. J . Wilimovsky. INTRODUCTION Watershed a l t e r a t i o n s r e s u l t i n g from g e o l o g i c a l and long-term c l i m a t i c processes have been important determinants f o r the d i s t r i b u t i o n s of freshwater f i s h e s (Myers, 1951; D a r l i n g t o n , 1957; Nelson and P l a t n i c k , 1981). One consequence of these drainage changes has been the r e s t r i c t i o n of some species t o small parts of the former range. For these r e l i c t (= e p i b i o t i c ) f i s h e s , recent populations are of p o t e n t i a l e v o l u t i o n a r y i n t e r e s t regarding the c o n t r i b u t i o n s of i s o l a t i o n and h a b i t a t i n determining t h e i r genetic d i f f e r e n t i a t i o n . Perhaps the most-e x t e n s i v e l y s t u d i e d r e l i c t species i n North America are the cyprinodonts t h a t survived the p o s t - P l e i s t o c e n e d e s s i c a t i o n of Death V a l l e y and contiguous regions (see Naiman and S o l t z , 1981). Considerable i n t e r e s t has a l s o been d i r e c t e d towards the a s s o c i a t i o n between r e l i c t f i s h e s and former P l e i s t o c e n e g l a c i a l r e f u g i a (e.g., Franz and Lee, 1976; Trautman, 1981). The Chehalis River drainage comprises a former pe r i g l a c i a l refuge near the terminal moraine of the l a t e P l e i s t o c e n e Vashon G l a c i e r i n western Washington, U.S.A. (Heusser, 1964). R e s t r i c t e d t o t h i s general area i s a f i s h , Novumbra hubbsi Schultz 1929a (E s o c o i d e i : Umbridae), that probably had a more widespread d i s t r i b u t i o n i n past g e o l o g i c a l times (McPhail, 1967; Cavender, 1969). _N. hubbsi belongs t o a s y s t e m a t i c a l l y -confusing f a m i l y d i s t r i b u t e d on three continents (Cavender, 1969). il* hubbsi occurs over a 12000 km 2 range ( F i g . I ) . Schultz (1929a,b) described t h i s f i s h as having a d i s j u n c t d i s t r i b u t i o n wholly w i t h i n the Chehalis River refuge, but other capture records have i n d i c a t e d a post-Figure I. Map of the Olympic Peninsula and adjacent regions showing the range o f N. hubbsi (shaded a r e a ) , based on data i n McPhail (1967), Meldrim (1968) and Hagen and Moodie (1979). The l o c a t i o n s of c o l l e c t i n g s i t e s are approximated by s t a r s ( * ) , with the exception o f the Copalis River s i t e , and a corresponding number as f o l l o w s : 1, Quinault River drainage (Taholah); 2, Copalis River drainage; 3, Conner Creek drainage; 4, coastal drainage (Oyhut); 5, Humptulips River drainage; 6, Wishkah River drainage; 7, Chehalis River drainage ( J u n c t i o n C i t y ) ; 8, Wynoochee River drainage; 9, Satsop River drainage; 10, Black River drainage; 11, Hanaford Creek drainage. The Copalis River c o l l e c t i o n was obtained from the U n i v e r s i t y o f Washington f i s h c o l l e c t i o n , from which l o c a t i o n a l data was not d e t a i l e d f o r these specimens. See Table 1 f o r map co-ordinates o f and d i r e c t i o n s to the c o l l e c t i n g s i t e s . kilometers 4 P l e i s t o c e n e d i s p e r s a l i n t o contiguous areas once covered by g l a c i a l i c e . See Heusser (1964) and McKee (1972) f o r d e t a i l s of the Vashon g l a c i a t i o n i n the regions c u r r e n t l y i n h a b i t e d by _N. hubbsi. This species was f i r s t noted i n the Deschutes R i v e r drainage by F i t z g e r a l d (1957). McPhail (1967) and Meldrim (1968) recorded t h i s f i s h i n Olympic coastal drainages as f a r north as the Queets R i v e r . I n t r o -ductions by man are probably responsible f o r the presence of _N. hubbsi even f u r t h e r north i n Lake Ozette (Wydoski and Whitney, 1979). Hagen et a l . (1979) c o l l e c t e d _N. hubbsi i n Puyallup and Skookum Creeks, the former system d r a i n i n g west i n t o southern Puget Sound and the l a t t e r d i s c h a r g i n g i n t o the same from the east near Shelton, Washington. C e r t a i n marshes and swamps contained w i t h i n the Chehalis River system sometimes overflow i n t o contiguous drainages during periods of heavy p r e c i p i t a t i o n a l l o w i n g f i s h t o invade regions outside the peri g l a c i a l refuge (Wydoski and Whitney, 1979). Skookum Creek o r i g i n a t e s i n the same area as the Cloquallum R i v e r , the l a t t e r system part of the Chehalis River drainage. Novumbra from Skookum Creek may have come from t h i s Chehalis R i v e r t r i b u t a r y . Puyallup Creek f i s h could have o r i g i n a t e d from Deschutes River populations through d i s p e r s a l across the Puget Lowlands. The r e s t r i c t i o n of _N. hubbsi t o l o t i c peaty waters has been noted several times (Schultz 1929a,b; McPhail, 1967; Meldrim, 1968; Hagen et a l . 1979) and such habitat appears to be t y p i c a l l y occupied by the other c l o s e r e l a t i v e s (Ostdiek and Nardone, 1959, f o r P a l l i a pectoral i s Bean; Lelek, 1980, f o r Umbra krameri (Walbaum); Peckham and Dineen, 1957, f o r JJ. 1 imi ( K i r t l a n d ) ; Hoese, 1963, f o r ]J_. pygmaea (DeKay)). Experimental observations have i n d i c a t e d that JJ. hubbsi p r e f e r s water w i t h minimal f l o w , l i t t l e or no d i s s o l v e d s a l t , and reduced l i g h t i n t e n s i t y (Meldrim, 5 1968), c h a r a c t e r i s t i c s of the habitat occupied i n nature. There i s a l s o f o s s i l evidence f o r the occupation of bogs, swamps or freshwater marshes by Novumbra during the Oligocene (Cavender, 1969). The habitat preference of_N. hubbsi i s l i k e l y a major determinant of i t s patchy d i s t r i b u t i o n . Since h a b i t a t s are d i s c o n t i n u o u s , s i g n i f i c a n t genetic d i f f e r e n t i a t i o n may have occurred between populations. The extent of t h i s d i f f e r e n t i a t i o n would depend on population s i z e , gene flow and time as well as natural s e l e c t i o n (Bush et a l . , 1977; Endler, 1977; Johnson and Mickevich, 1977). _N. hubbsi may not have been subjected t o c l i m a t i c heterogeneity s u f f i c i e n t f o r s i g n i f i c a n t i n t e r p o p u l a t i o n a l d i f f e r e n t i a t i o n . This f i s h i s found almost e x c l u s i v e l y w i t h i n a s p e c i f i c h a b i t a t , rather than a wide v a r i e t y of waters, over a l i m i t e d geographic area. The c l i m a t e does not g r e a t l y d i f f e r over the species range, everywhere c h a r a c t e r i z e d by high p r e c i p i t a t i o n during the c o o l e r months, r e l a t i v e l y dry summers, and m i l d r a r e l y - f r e e z i n g winters (Ruffner, 1980). Consequently, i n t e r r e g i o n a l c l i m a t i c f a c t o r s could have l i t t l e i n f l u e n c e upon i n t e r p o p u l a t i o n a l v a r i a t i o n . Some information i s a v a i l a b l e on i n t r a - and i n t e r p o p u l a t i o n a l v a r i a t i o n i n external morphology f o r _N. hubbsi. Schultz (1929a) enumerated external characters on specimens captured at the type l o c a l i t y , a peaty d i t c h between Elma and Satsop about 35 km upstream from the mouth of the Chehalis River ( F i g . I ) . _N. hubbsi showed a range of v a r i a t i o n as f o l l o w s : number of pectoral f i n rays, 18-23; p e l v i c f i n r a y s , 6-7; caudal f i n rays, 17-22; dorsal f i n r a y s , 12-15; anal f i n rays, 11-13; and l a t e r a l row s c a l e s , 52-58. Most specimens examined by Schultz (1929a) were j u v e n i l e s l e s s than 1.5 cm standard l e n g t h (personal o b s e r v a t i o n ) . Since counting e r r o r s are 6 more l i k e l y t o occur w i t h small specimens (Moyle and Cech, 1982), some characters may have been i n c o r r e c t l y evaluated. Schultz (1929a) reported dark l a t e r a l b a r r i n g on reproductive males but i n d i s t i n c t or f a i n t patterns on breeding females. Although t h i s was his only reference t o sexual dimorphism, other characters may have d i f f e r e d between sexes, e s p e c i a l l y i f these were somehow important t o sexual r o l e s during reproducti on. il« hubbsi does not possess a l a t e r a l l i n e but has pored scales i n the p o s i t i o n occupied by a t y p i c a l l a t e r a l l i n e ( S c h u l t z , 1929a). Meldrim (1968) looked at geographic v a r i a t i o n i n the number of these l a t e r a l row s c a l e s . _N. hubbsi from the Chehalis River drainage had 50-59 s c a l e s . Specimens from the Quinault R i v e r system, an Olympic coastal drainage ( F i g . 1), had l e s s e r numbers, 42-48, while f i s h from swamps i n the Humptulips River drainage had 43-58. These data may be i n d i c a t i v e of a c l i n e i n l a t e r a l s c a l e numbers i n c r e a s i n g from the P a c i f i c coast t o i n l a n d l o c a l i t i e s . Since s c a l e counts were most v a r i a b l e i n the Humptulips River c o l l e c t i o n , two Novumbra morphs may have been i n d i c a t e d w i t h t h e i r zone of i n t e r g r a d a t i o n w i t h i n the Humptulips River region. The number of specimens examined by Meldrim (1968) was fewer than 100 i n d i v i d u a l s . The f i s h were not deposited i n a museum c o l l e c t i o n , so the study material i s not r e a d i l y a v a i l a b l e f o r v e r i f i c a t i o n . Adults of both sexes as well as j u v e n i l e s were enumerated together, so s e x u a l l y -dimorphic or ontogenetic f a c t o r s were not considered. Since Meldrim (1968) d i d not define how he d i d his m e r i s t i c counts, v e r i f i c a t i o n of his r e s u l t s might be d i f f i c u l t even i f his c o l l e c t i o n s were a v a i l a b l e . Meldrim (1968) sta t e d that Chehalis River _N. hubbsi had barred l a t e r a l markings, while those from the Quinault River drainage possessed mottled •7 pa t t e r n s . This observation was n e i t h e r defined nor i l l u s t r a t e d . Although there e x i s t many i c h t h y o l o g i c a l s t u d i e s on geographic v a r i a t i o n i n external morphology, few i n v e s t i g a t o r s have examined chromosomal s t a t e s across a species range. Chromosomal s t a t e r e f e r s t o the o r g a n i z a t i o n of the chromosome complement. In t h i s study, the term i s r e s t r i c t e d to chromosome numbers and centromeric l o c a t i o n s determined by means of Giemsa or o r c e i n s t a i n i n g . Methods are a v a i l a b l e , e s p e c i a l l y w i t h chromosomes from higher vertebrates (Hsu, 1973; B l a x h a l l , 1975), f o r l o o k i n g at v a r i a t i o n i n intrachromosomal features such as n u c l e o l a r o r g a n i z i n g and heterochromatic regions as well as s p e c i f i c genes or DNA sequences. See Swanson et a l . (1981) f o r a d i s c u s s i o n on chromosomal analyses i n systematic b i o l o g y . The d i s j u n c t d i s t r i b u t i o n of _N. hubbsi could mean that t h i s f i s h occurs i n small breeding u n i t s , a c r i t e r i o n considered important by some i n v e s t i g a t o r s f o r the establishment of i n t e r p o p u l a t i o n a l chromosomal polymorphisms (Bush, 1975; Wilson et a l . , 1975). The cytogenetic study of salmonids by Thorgaard (1978) in v o l v e d k a r y o t y p i c analyses on animals from Alaska t o C a l i f o r n i a , perhaps the most comprehensive examination of i n t r a s p e c i f i c chromosomal d i v e r s i t y i n f i s h e s to date. Black and Howell (1978, 1979) have looked at geographic chromosomal v a r i a t i o n i n cy p r i n o d o n t i d s . The above researchers have shown that chromosomal s t a t e s can vary between populations of c o n s p e c i f i c s . Chromosomal v a r i a t i o n i n f i s h e s has u s u a l l y been associated w i t h geographic i s o l a t i o n (Gold et a l . , 1977; Black and Howell, 1978, B e r t o l l o et a l . , 1979, LeGrand and Cavender, 1980) although environmental f a c t o r s have a l s o been considered determinants of chromosomal s t a t e s (Nikolsky and V a s i l y e v , 1974; N i k o l s k y , 1976). •8 Most cytogenetic s t u d i e s on f i s h e s have i n v o l v e d few chromosome spreads from even fewer animals u s u a l l y captured at one l o c a t i o n (e.g., most references l i s t e d i n Gold et a l . , 1980), probably under the assumption that chromosomal s t a t e s are f i x e d w i t h i n species (White, 1978). The only published chromosomal a n a l y s i s on _N. hubbsi was done w i t h s i x m i t o t i c spreads from two specimens caught at an u n s p e c i f i e d l o c a l i t y (Beamish et a l . , 1971). Consequently, nobody has looked at i n t e r p o p u l a t i o n a l v a r i a t i o n i n chromosomal s t a t e s i n t h i s s p e c i e s . The modal d i p l o i d number f or _N. hubbsi was reported as 48 chromosomes or 24 chromosome p a i r s (Beamish et a l . , 1971), but t h i s should be considered as t e n t a t i v e f o r the species as a whole u n t i l i n d i v i d u a l s from several populations are examined. Beamish et a l . (1971) determined nuclear DNA values f or JN. hubbsi as well as other c l o s e r e l a t i v e s . These are presented here i n two groupings, w i t h DNA contents expressed as m u l t i p l e s of the determination f o r _N. hubbsi (with an absolute DNA content of 2.08 pg (picograms)/-di p i o i d nucleus): P a l l i a pectoral i s Bean and Esox spp. w i t h 1.08-1.31 times; and Umbra spp. w i t h 2.32 - 2.42 times. The ONA values f o r _ N . hubbsi, JJ. pectoral i s and Esox spp. were 40-56% of those determined f o r salmonids by Vendrely (1953), Knobloch et a l . (1957) and Hinegardner (1968), perhaps evidence f o r the hypothesized t e t r a p l o i d a l d e r i v a t i o n of the salmonid genome from that of a d i p l o i d salmoniform ancestor (Zenzes and V o i c u l e s c u , 1976). The accuracy of the esocoid DNA determinations by Beamish et a l . (1971) i s questionable. Only Feulgen-DNA methodology was used, although i t may have been b e t t e r t o c o r r e l a t e other DNA q u a n t i f i c a t i o n techniques wit h Feulgen-DNA cytophotometry because of d i f f i c u l t i e s i n the 9-s t a n d a r d i z a t i o n of the l a t t e r (McLeish and Sunderland, 1961; Miksche, 1971). A l s o the sample s i z e s were s m a l l . For example, only two _N. hubbsi from a s i n g l e u n s p e c i f i e d l o c a l i t y were examined. Although the DNA constancy p r i n c i p l e (Mirsky and and R i s , 1949) was l i k e l y assumed i n a s c e r t a i n i n g genomic contents, l i t e r a t u r e i s a v a i l a b l e showing that DNA values may d i f f e r between populations of c o n s p e c i f i c s . The l a r g e s t d i f f e r e n c e s i n n u c l e i c a c i d content are p l o i d a l ones and external morphology or s i z e may not be usable f o r d i s c e r n i n g between i n d i v i d u a l s w i t h d i f f e r e n t p l o i d a l s t a t e s (e.g., the races of c o b i t i d s s t u d i e d by Sezaki and Kobayashi, 1978). P l o i d a l d i f f e r e n c e s are r e a d i l y i d e n t i f i e d by counting chromosomes si n c e complements would cont a i n m u l t i p l e s of the haploid chromosome number. Sherwood and Patton (1982) found extensive i n t e r p o p u l a t i o n a l DNA v a r i a t i o n i n the rodent, Thomomys bottae (Eydoux and G e r v a i s ) , although specimens had s i m i l a r karyotypes. DNA contents were found t o d i f f e r by as much as 35%, i n d i c a t i n g that d i f f e r i n g n u c l e i c a c i d contents may not be always detectable by l o o k i n g at chromosome spreads. No f i s h e s other than some d i p l o i d - p o l y p l o i d forms (Sezaki et a l . , 1977) have been examined f o r geographic v a r i a t i o n i n DNA content. This study intends to examine geographic v a r i a t i o n in_N. hubbsi, addressing the f o l l o w i n g questions: (a) How does the external phenotype, s p e c i f i c a l l y m e r i s t i c c h a r a c t e r s , vary across the range? Is _N. hubbsi a polymorphic species i n the manner suggested by Meldrim's (1968) data? (b) Does chromosome morphology vary? (c) Do i n d i v i d u a l s from d i f f e r e n t populations have d i f f e r e n t amounts of DNA? 10 The r e s u l t s of the external m e r i s t i c , chromosomal, and nuclear DNA analyses w i l l be c o r r e l a t e d w i t h g e o l o g i c a l and e c o l o g i c a l information t o i n f e r zoogeographical and s e l e c t i o n a l h i s t o r i e s . As emphasized by Chernoff (1982), such hypotheses are a necessary p r e l i m i n a r y t o under-standing character v a r i a t i o n . Land use p r a c t i c e s have e l i m i n a t e d much Novumbra habitat (Wydosky and Whitney, 1979) and such a c t i v i t i e s have r e s u l t e d i n the e x t i n c t i o n of r e l i c t f i s h e s elsewhere ( P i s t e r , 1981). Consequently, base l i n e data on population v a r i a t i o n i s important information f o r fu t u r e p r o t e c t i o n and management of N. hubbsi. 11 MATERIALS AND METHODS Sampling l o c a l i t i e s _N. hubbsi were c o l l e c t e d from a major part of t h e i r range, w i t h i n the Chehalis River drainage as well as Olympic coastal ones. The sampling s i t e s are shown i n F i g . I and map co-ordinates of as well as d i r e c t i o n s t o each s i t e are given i n Table 1. C l a s s i f i c a t i o n of sampling l o c a l i t i e s Each c o l l e c t i n g s i t e was categorized according to i t s source of water. Water bodies kept f i l l e d only by p r e c i p i t a t i o n and groundwater p e r c o l a t i o n were termed close d (land l o c k e d ) , while those fed by streams were c l a s s i f i e d as open (drained). Surface areas were c a l c u l a t e d f o r a l l sampling s i t e s by pacing o f f s h o r e l i n e perimeters t o obtain circumferences from which areas were c a l c u l a t e d and/or by reference t o topographic maps. This measure was considered i n d i c a t i v e of h a b i t a t s i z e s i n c e swampy h a b i t a t s were mostly f l a t and shallow. Since _N. hubbsi i s u s u a l l y found i n water l e s s than 1.5 m deep (Meldrim, 1968), t h i s f i s h was considered t o have had a p o t e n t i a l presence anywhere w i t h i n a p a r t i c u l a r s i t e . P h ysical and chemical determinations on the water Physical and chemical determinations of the water were made upon each v i s i t t o a l l c o l l e c t i n g s i t e s . Temperature was a s c e r t a i n e d w i t h a standard centigrade thermometer, pH with Hydrion t e s t papers, d i s s o l v e d oxygen using the Winkler method, and carbon d i o x i d e by means of a Hach Table 1. Drainage l o c a t i o n s , map co-ordinates and s i t e d i r e c t i o n s f o r H. hubbsi c o l l e c t i n g s i t e s . The names f o r c o l l e c t i n g s i t e s are those used i n the t e x t . Drainages marked w i t h a s t e r i s k s (*) are t r i b u t a r i e s of the Chehalis R i v e r , the remaining ones discharge d i r e c t l y i n t o the P a c i f i c Ocean. Latit u d e s and longitudes were obtained from U. S. Geological Survey 15-minute Series topographic maps. Col 1 e c t i ng S i t e Taholah Copal i s Drainage Quinault R. Copalis R. Conner Creek Conner Creek Oyhut Humptul i ps Coastal Humptulips R. Latitude Longitude 47°29'50" 124°17'35" 47°11'05" 124°07'30" 47°15'00" 124°01'45" 47°03'48" 124°09'15" 47°01'45" 124 o09'10" 47°03'45" 124°01'15" D i r e c t i o n s t o c o l l e c t i n g s i t e 0.4 km west of Quinault Indian Nation headquarters at Taholah, swamp adjacent t o westernmost housing complex. Specimens from the Copalis R. drainage were c o l l e c t e d i n 1948 and are curated by the U n i v e r s i t y of Washington College of F i s h e r i e s . L o c a l i t y i n f o r m a t i o n i s apparently l o s t . The l i s t e d l a t i t u d e s and longitudes comprise the geographic area covered by the Copalis R. Approximately 6 km upstream from the mouth of Conner Creek, s i t e destroyed during the co n s t r u c t i o n of a housing p r o j e c t (Wydoski and Whitney, 1979). 1.1 km south of the entrance t o Ocean Shores State Park on highway to Oyhut, swamp on the east s i d e of the road where highway abruptly turns west. Burrows Road 1 km north of i t s i n t e r s e c t i o n w i t h State Route 9C, swamp on the east s i d e of the road. Table 1. (Continued) C o l l e c t i n g S i t e Drainage L a t i t u d e Longitude Wishkah Wishkah R. 47°00'11" 123°48'12" Junction C i t y Chehalis R. 46°58'43" 123°45'58" Wynoochee Wynoochee R.* 46°59'20" 123°39'04" Satsop Satsop R.* 47°06'40" 123°24'45" Black River Black R.* 46°52'30" 123°01'20" Hanaford Creek Hanaford Creek* 46°45'03" 122°55'08" D i r e c t i o n s t o c o l l e c t i n g s i t e 1.1 km north of the entrance t o Fern H i l l Cemetery on East Wishkah Road, where stream crosses under the highway, swamp on the east side of the road. 0.6 km southeast of the bridge across Northern E l l i o t Slough on the highway t o J u n c t i o n C i t y , marshy area on the west s i d e of the road. 2.1 km north of U. S. Route 410 on West Wynoochee Road, where stream crosses under the road, swamp contiguous t o the west s i d e of the hi g hw ay. 13 km northeast of Schaefer State Park and 2 km northeast of the Matthews homestead on Matlock-Shelton Road, flooded region along Dry Run Creek on the northwest s i d e of the highway. 2.2 km south of L i t t l e Rock on State Route 1M, marshy slough 40 m west of the highway. 5.6 km east of State Route 507 on Hanaford V a l l e y Road, approximately 100 m south of the highway, an oxbow pond contiguous t o Hanaford Creek. 14 k i t . D issolved s a l t concentration was a s c e r t a i n e d w i t h a Yellow Springs Instrument Model 33 c o n d u c t i v i t y meter. Methods of c o l l e c t i o n F i s h were captured w i t h a 3-m long 6.4-mm mesh beach seine as well as metal minnow t r a p s . The l a t t e r were u s u a l l y b a i t e d w i t h cat food and placed i n the water overnight. Both capture methods were used at a l l s i t e s ; however, most specimens were obtained w i t h traps s i n c e many s i t e s were c l u t t e r e d w i t h sunken debris that tended t o entangle and t e a r the s e i n e . A finely-meshed dipnet w i t h a 30 cm opening was used on at l e a s t one occasion at each l o c a t i o n . A l l _N. hubbsi were caught between November 1978 and December 1979. Specimens from Conner Creek and the Copalis R i v e r were borrowed from the f i s h c o l l e c t i o n at the College of F i s h e r i e s of the U n i v e r s i t y of Washington at S e a t t l e . Morphomeristic determinations Captured f i s h were f i x e d i n 10% borate-buffered f o r m a l i n f o r one week, then r i n s e d i n running tap water f o r 6 h, drained t o remove excess moisture, and placed i n 37.5% isopropanol p r e s e r v a t i v e . A p i l o t study on 10 animals had shown that weight-loss d i d not s t a b i l i z e u n t i l a f t e r 8 wks i n p r e s e r v a t i v e . Consequently, a l l samples were stored f o r at l e a s t 60 d p r i o r t o measurement of standard lengths t o minimize v a r i a t i o n due t o post mortem changes i n p r e s e r v a t i v e . F i s h used f o r morphomeristic determinations, other than Copalis and Conner Creek ones, were deposited i n the f i s h c o l l e c t i o n of the U n i v e r s i t y of B r i t i s h Columbia. With the exception of the l a t e r a l bar count on males, morphomeristic determinations were made according t o the methods of Hubbs and Lagler (1964). Measurements were obtained using a Helios d i a l c a l i p e r and taken 15 t o the nearest 0.1 mm; these were taken three times on the same specimen and averaged t o give a s i n g l e value. A l l m e r i s t i c counts were made under a d i s s e c t i n g microscope, with each count redone at l e a s t once t o assure accuracy. D e s c r i p t i o n s of the characters used f o l l o w . 1. Standard length (SL): the measurement from the t i p of the snout t o the end of the hypural p l a t e , the l a t t e r i d e n t i f i e d by passing l i g h t through the specimen. 2. L a t e r a l s c a l e row: the number of s c a l e s along a l i n e i n the p o s i t i o n t h a t would be occupied by a t y p i c a l l a t e r a l l i n e and recognized by pored s c a l e s . The count terminates at the end of the hypural p l a t e and begins at the upper o r i g i n of the opercular opening. 3. Scales above the l a t e r a l s c a l e row: the number of s c a l e rows above the l a t e r a l s c a l e row, from the o r i g i n of the dorsal f i n and counted down and back f o l l o w i n g the s c a l e row, the count terminated w i t h the s c a l e immediately before the l a t e r a l row s c a l e . 4. Scales below the l a t e r a l s c a l e row: the number of s c a l e rows below the l a t e r a l s c a l e row, from the o r i g i n of the anal f i n and counted up and forward f o l l o w i n g the s c a l e row, the count terminated w i t h the s c a l e immediately below the l a t e r a l row s c a l e . 5. Scale rows before the dorsal f i n o r i g i n : the number of s c a l e rows from the upper o r i g i n of the opercular opening t o the o r i g i n of the dorsal f i n . 6. Dorsal and anal f i n rays: the count i n c l u d i n g a l l branched rays plus one unbranched one, s i n c e only a s i n g l e unbranched ray reaches the outer margin of e i t h e r f i n ; the l a s t ray may be d i v i d e d through i t s base but i s s t i l l counted as only one. 7. Caudal f i n rays: the count of a l l p r i n c i p a l r a y s , the number of 16 branched rays plus two unbranched ones. 8. Pectoral and p e l v i c f i n rays: the count of a l l r a y s , i n c l u d i n g the smallest ones v i s i b l e only w i t h a d i s s e c t i n g microscope. 9. L a t e r a l bars on males: the count of the prominent dark l a t e r a l bars s t a r t i n g w i t h the f i r s t obvious one p o s t e r i o r t o the upper o r i g i n of the opercular opening, enumerated along the l a t e r a l row and ter m i n a t i n g w i t h the l a s t complete bar before the hypural p l a t e . A n a l y s i s of m e r i s t i c data Whether or not a character had been sexually-dimorphic was determined w i t h Chi-square t e s t s on m e r i s t i c character frequencies. I f sexes at even one l o c a l i t y d i f f e r e d at the p = 0.05 l e v e l , then the relevant parameter was f u r t h e r t e s t e d separately f o r each sex f o r a l l populations. Variances of the raw data were u s u a l l y heterogeneous as checked w i t h B a r t l e t t ' s t e s t , so base-10 l o g a r i t h m i c conversions were made p r i o r to parametric analyses. C o r r e l a t i o n analyses between m e r i s t i c characters and standard length suggested l i t t l e or no f u n c t i o n a l r e l a t i o n s h i p s (Table 2 ) , so each m e r i s t i c parameter was evaluated by i t s e l f and not i n r e l a t i o n t o l e n g t h . For characters determined t o have been sex u a l l y - d i m o r p h i c , mean counts f o r males and females w i t h i n each population were compared using t - t e s t s . Geographic comparisons were f i r s t made w i t h a n a l y s i s of variance (ANOVA). ANOVA i s robust enough t o operate well even w i t h considerable heterogeneity of variances and departures from normality (Zar, 1974). Although variances may not be equal, acceptance of ANOVA F-values i s j u s t i f i e d by concordance with the Welch and Brown-Forsythe t e s t s (Brown and Forsythe, 1974). The Welch t e s t as wel l as Brown-17 Table 2. C o r r e l a t i o n s between m e r i s t i c character s t a t e s and standard l e n g t h f o r N. hubbsi. C o l l e c t i o n s from each sampled l o c a l i t y were evaluated i n d i v i d u a l l y y i e l d i n g the l i s t e d range of c o r r e l a t i o n s . The m e r i s t i c characters used i n t h i s study d i d not appear t o be i n f l u e n c e d by standard l e n g t h , a measure of s i z e . M e r i s t i c character _N Dorsal f i n rays i n males 160 Dorsal f i n rays i n females 149 Anal f i n rays i n males 160 Anal f i n rays i n females 149 Caudal f i n rays 306 Pectoral f i n rays 248 P e l v i c f i n rays 319 Scale rows before dorsal f i n o r i g i n 307 La t e r a l row s c a l e s 307 Scales above l a t e r a l row 303 Scales below l a t e r a l row 208 L a t e r a l bars i n males 207 Range of C o r r e l a t i o n s -0.29 - 0.45 -0.33 - 0.48 -0.38 - 0.47 -0.39 - 0.43 -0.05 - 0.23 -0.39 - 0.36 -0.42 - 0.12 -0.01 - 0.40 0.01 - 0.30 -0.09 - 0.09 -0.07 - 0.10 -0.32 - 0.48 Overal1 Correl a t i o n 0.23 0.08 0.21 0.10 0.08 0.01 -0.09 0.08 0.03 0.03 -0.02 0.04 18 " Forsythe t e s t s are e s s e n t i a l l y ANOVA's without the assumption of equal variance. Each ANOVA i s presented w i t h the Levene t e s t f o r equal variance i n turn followed by the Welch and Brown-Forsythe t e s t s . If the F-value was s i g n i f i c a n t , then i t was expected that the l a t t e r two should be a l s o f o r the ANOVA F-value t o be accepted. C o r r e l a t i o n a n a l y s i s , the Levene t e s t f o r equal variance as well as the ANOVA, Welch t e s t and Brown-Forsythe t e s t were done on the U n i v e r s i t y of Utah Univac 1160 computer, u t i l i z i n g the BMDP-81 software package (Dixon et a l . , 1981). A p o s t e r i o r i comparisons were made wi t h the Student-Newman-Keul m u l t i p l e range t e s t f o r unequal sample s i z e s (SNK) and Scheffe's m u l t i p l e c o n t r a s t s t e s t (SMC) as o u t l i n e d by Sokal and Rohlf (1969). SMC i s not as powerful f o r pairwise comparisons as SNK (Zar, 1974); consequently, a p p l i c a t i o n of the former was r e s t r i c t e d t o s e l e c t e d cases f o r which i t was e s p e c i a l l y s u i t a b l e , m u l t i p l e c o n t r a s t s . Chromosome methodology M e i o t i c and m i t o t i c chromosomes were obtained using a m o d i f i c a t i o n of the Kligerman and Bloom (1977) technique. F i s h were i n t r a p e r i t o n e a l l y i n j e c t e d w i t h 20 g c o l c h i c i n e / g body weight. The c o l c h i c i n e was made up as a 0.04% s o l u t i o n i n Cortland's p h y s i o l o g i c a l s a l i n e (Wolf, 1963) co n t a i n i n g 0.25% calcium c h l o r i d e . This a d d i t i o n of calcium followed Subrahmanyam (1969), who used t h i s ion t o s t i m u l a t e c e l l d i v i s i o n and counter c o l c h i c i n e - i n d u c e d c o n t r a c t i o n of chromosomes. Injected f i s h were placed i n a we l l - a e r a t e d container at room temperature (20 - 22°C) f o r 6 h. Specimens were s a c r i f i c e d by t r i c a i n e methanosulfonate overdose and, as a p p r o p r i a t e , g i l l arches, f i n edges, kidney, i n t e s t i n a l t r a c t , and t e s t e s were removed. The e x t i r p a t e d t i s s u e was gently and q u i c k l y 19 <• washed with 0.4% hypotonic KC1 s o l u t i o n . Tissues were then placed i n a v i a l w i t h approximately 15 times t h e i r volume of 0.4% KC1 s o l u t i o n f o r 20 - 30 min at room temperature. A f t e r removal of as much hypotonic s o l u t i o n as p o s s i b l e , the sample was f i x e d i n the same v i a l . F i x a t i o n e n t a i l e d at l e a s t 3 changes of freshly-prepared 3:1 anhydrous methanol-a c e t i c a c i d at 4°C f o r at l e a s t 30 min each. Tissues were u s u a l l y processed onto s l i d e s immediately a f t e r t h i s f i x a t i o n , but they were sometimes stored i n the r e f r i g e r a t o r f o r up t o 72 h. A l l s l i d e s were cleansed w i t h Alconex, washed w i t h hot water, and r i n s e d thoroughly with d i s t i l l e d water. F i n i s h e d s l i d e s were stored i n methanol u n t i l needed. S l i d e s were wiped dry w i t h a Kimwipe and placed on a s l i d e warmer heated t o 40 - 45°C. A piece of t i s s u e was removed from the f i x a t i v e , touched t o a clean paper towel t o remove excess f i x a t i v e , and placed i n t o the well of a Boerner s l i d e . Four t o s i x drops of 50% a c e t i c a c i d were q u i c k l y added and the t i s s u e gently minced f o r approximately 1 min to form a c e l l s l u r r y . A non-heparinized microhematocrit tube ( S c i e n t i f i c Products, B4416-1), mounted t o a t u b e r c u l i n syringe f o r c o n t r o l l e d s u c t i o n , was f i l l e d w i t h c e l l suspension and e x p e l l e d onto a warmed s l i d e . The s l u r r y was allowed t o remain on the s l i d e f o r 15 - 20 sec and then withdrawn back i n t o the c a p i l l a r y tube. A c i r c l e of c e l l s would remain on the s l i d e . One tube f i l l e d w i t h c e l l suspension s l u r r y was s u f f i c i e n t f o r 3-4 c i r c l e s . S l i d e s were allowed to dry, placed i n a d u s t - f r e e s l i d e box, and s t o r e d i n a c o o l , dry l o c a t i o n f o r 72 h before s t a i n i n g . Aging s l i d e s f o r a few days seemed t o improve s t a i n i n g q u a l i t y . S l i d e s were s t a i n e d i n f r e s h 10% Giemsa (Gurr Improved R66) prepared i n M/15 Sorensen b u f f e r at pH 6.8. St a i n was always f i l t e r e d before 20 use. Stained s l i d e s were r i n s e d with d i s t i l l e d water, b l o t t e d w i t h bibulous paper, a i r d r i e d , and placed i n xylene f o r at l e a s t 15 min. S l i d e s were mounted i n Permount with a No. 1 c o v e r s l i p . F i n i s h e d s l i d e s were observed under a Zeiss Photomicroscope II equipped w i t h a 100 W mercury vapor l i g h t source. Photographs were taken through a 100X phase o b j e c t i v e and a green substage f i l t e r w i t h Kodak Technical Pan 2415 f i l m ASA 50. Film was developed i n Kodak D19 s o l u t i o n and p r i n t e d on Kodak paper using Dektol developer. A f t e r p r e l i m i n a r y s o r t i n g of those enlarged photographs i n which chromosomes were n e i t h e r e x c e s s i v e l y overlapping nor contracted, each chromosome was measured. Q u a n t i t a t i v e c h a r a c t e r i z a t i o n s i n v o l v e d measurements of the r e l a t i v e l e n g t h (expresssed as percent of t o t a l l e ngth of d i p l o i d complement) and the arm r a t i o (short arm/long arm X 100). A l l measurements were taken w i t h c a l i p e r s and a r u l e r graduated i n 1/60 i n c h u n i t s . The terminology of Levan et a l . (1964) was used f o r d i s c u s s i n g chromosome morphology. Feulgen-DNA determinations Nucleated erythrocytes were chosen since they are d i p l o i d , uniform i n s i z e and shape, and r e a d i l y a v a i l a b l e as s i n g l e c e l l s . Blood was obtained by s a c r i f i c i n g the f i s h and d r a i n i n g the heart w i t h a heparinized c a p i l l a r y tube ( S c i e n t i f i c Products, B4416-2). No more than 2 h before f i s h were t o be processed, blood was a l s o taken from a Rhode Island Red chicken through a wing vein using a heparinized Vacutainer tube and the r e s u l t a n t sample stored i n a r e f r i g e r a t o r u n t i l needed. Clean dry s l i d e s were d i v i d e d i n t o 2 s e c t i o n s w i t h a diamond p e n c i l . Blood was smeared t h i n l y onto the s l i d e s , one s e c t i o n r e c e i v i n g f i s h 21 er y t h r o c y t e s and the other chicken blood as a reference. The smears were a i r - d r i e d f o r 5 min and f i x e d according t o the method of Mizuno and MacGregor (1974). S l i d e s were placed f o r 15 min i n freshly-mixed 3:1 absolute e t h a n o l - a c e t i c a c i d at 4°C and then passed through two 5 min soaks i n 95% ethanol and a i r - d r i e d . S l i d e s were wetted with d i s t i l l e d water and hydrolyzed i n 5N hy d r o c h l o r i c a c i d at 20°C f o r 40 min, sin c e DNA l o s s should have been minimized under these c o n d i t i o n s (Deitch et a l . , 1968; Murgatroyd, 1968; Vahs, 1973). The method of Mizuno and MacGregor (1974) was followed f o r s t a i n i n g . A f t e r washing the s l i d e s i n three 5 min changes of d i s t i l l e d water, they were s t a i n e d i n S c h i f f ' s reagent at 23°C i n the dark f o r 90 min. The s l i d e s were then washed i n three changes of 0.5% sodium m e t a b i s u l f i t e f o r 2 min each, and then passed through 5 min soaks i n d i s t i l l e d water, 70% eth a n o l , 95% e t h a n o l , absolute e t h a n o l , and xylene. Smears were mounted i n Carl Zeiss immersion o i l (nD = 1.515) with a No. 1 c o v e r s l i p . Two-area cytophotometry of s t a i n e d n u c l e i was done w i t h a Zeiss PM-2 microspectrophotometer l i n k e d t o a p h o t o m u l t i p l i e r - a n a l o g d i g i t a l computer-microprocessor u n i t . Each determination f o r one nucleus was recorded as the mean of 32 automatic readings. A l l q u a n t i f i c a t i o n s were made using a 40X (n.a. = 0.75) water-immersion lens and a 100 W mercury vapor l i g h t source. Green i n c i d e n t l i g h t was used and, by b a r r i e r f i l t e r s , the emitted l i g h t measured at 546 nm. R e l a t i v e and absolute DNA values were determined on the basis of co-stained chicken blood as a reference. Twenty-five f i s h erythrocytes and 25 chicken blood c e l l s were evaluated f o r each s l i d e and two s l i d e s were evaluated per i n d i v i d u a l . 22 1 Fluorometric-DNA technique Erythrocyte density was determined on blood drawn from the caudal vein using a Thomas p i p e t . The blood was d i l u t e d w i t h Hayem c e l l counting s o l u t i o n and erythrocytes counted with a Neubauer-ruled hemocytometer. A d d i t i o n a l blood was obtained w i t h a heparinized micro-hematocrit tube ( S c i e n t i f i c Products, B4416-2) and i t s volume measured. The blood was d i l u t e d t o 1.0 ml w i t h 0.5% heparin i n 0.8% sodium c h l o r i d e and kept under r e f r i g e r a t i o n f o r no more than 1 h before subsequent processing. One-hundred and 200 1 known c e l l - d e n s i t y samples were assayed f o r DNA amount, using a f l u o r o m e t r i c q u a n t i f i c a t i o n of a c i d - i n s o l u b l e deoxypurine nucleotides based on the methods of Hinegardner (1971) and Foster and Gurney (1976). Samples were mixed w i t h 100 g of p u r i f i e d yeast RNA as a c a r r i e r and p r e c i p i t a t e d i n 5 ml of 10% t r i c h l o r o a c e t i c a c i d f o r 30 min at 0°C. The p r e c i p i t a t e was c o l l e c t e d by s u c t i o n on 2.4 cm Whatman GF/C glass f i b e r f i l t e r s . F i l t e r s were r i n s e d twice w i t h 1 M HC1 at 0°C, once w i t h 95% ethyl alcohol at room temperature, and a i r d r i e d . Each d r i e d f i l t e r was placed i n a v i a l c o n t a i n i n g 0.25 ml 2 M 3,5-diaminobenzoic a c i d d i h y d r o c h l o r i d e f o r 40 min at 60°C. The f i l t e r was then e x t r a c t e d f o r 10-15 min w i t h 3 - 5 ml of 1 M HC1 at room temperature. The fluorescence of the HC1 e x t r a c t was checked at 510 nm w i t h e x c i t a t i o n at 410 nm. The fluorescence values were c a l i b r a t e d against a c o n t r o l c o n s i s t i n g of a known amount of p u r i f i e d salmon sperm DNA (Sigma). 23 RESULTS Habitat c l a s s i f i c a t i o n Along the P a c i f i c coast of the Olympic P e n i n s u l a , _N. hubbsi were found i n cl o s e d systems, s t a b i l i z e d dune swamps or bogs (Table 3). Such h a b i t a t s o r i g i n a t e as s t a b i l i z e d beach sediments w i t h water l e v e l s c o n t r o l l e d by r a i n f a l l and groundwater p e r c o l a t i o n (Hutchinson, 1957). Closed sampling l o c a l i t i e s were r e l a t i v e l y s m a l l , the surface areas of both the Taholah and Oyhut s i t e s having been about 0.1 ha (hectares). Since closed h a b i t a t s do not interconnect by means of waterways, the t r a n s f e r of i n d i v i d u a l s between populations would appear r e s t r i c t e d t o episodes of f l o o d i n g . The remaining c o l l e c t i n g s i t e s were i n open systems, regions flooded by stream overflows (Table 3). These h a b i t a t s were l a r g e r than the clo s e d ones, 2.0 - 6.5 ha, as well as connected by streams t o other s i t e s s u i t a b l e f o r JJ. hubbsi. _N. hubbsi probably move more r e a d i l y between populations because of such i n t e r c o n n e c t i n g waterways and, i n f a c t , _N. hubbsi were i n f r e q u e n t l y caught i n the streams f l o w i n g i n t o or out of open systems. Closed systems occurred e x c l u s i v e l y i n the lowlands along the P a c i f i c coast while open ones were found throughout the sampling area. Presence of f i s h e s other than N. hubbsi Attempts were made to capture f i s h e s on at l e a s t f i v e occasions at the l o c a t i o n s l i s t e d i n Table 3. The Oyhut, Humptulips, Wishkah, 24 Table 3. Selected parameters of _N. hubbsi c o l l e c t i n g s i t e s . 'Location' i n d i c a t e s the c o l l e c t i n g s i t e s used i n t h i s study, shown i n F i g . I and described i n Table 1. Surface area i s considered r e p r e s e n t a t i v e of ha b i t a t s i z e , s i n c e h a b i t a t s were u s u a l l y shallow r e l a t i v e l y f l a t depressions up to 1.5 m deep. JN. hubbsi may occur anywhere w i t h i n such waters (Meldrim, 1968). 'Closed' drainages are h a b i t a t s maintained by p r e c i p i t a t i o n , groundwater p e r c o l a t i o n , and runoff . 'Open' drainages are sustained by streams. Fishes other than G. aculeatus co-occurred w i t h H. hubbsi only i n open drainage s i t u a t i o n s . Location Surface area (hectares) S i t e drai nage Fishes co-oc c u r r i n g w i t h N. hubbsi* Tahol ah 0.1 closed 6 Oyhut 0.1 closed 6 Humptul i p s 6.5 open 2, 3, 4, 6, 8 Wi shkah 5.0 open 2, 6, 8 Wynoochee 4.5 open 2, 3, 4, 6, 8 Black River 5.8 open 2, 3, 4, 6, 7, 8 Hanaford 2.0 open 5, 6, 8 Satsop 6.0 open 1, 2, 6, 8 Junction C i t y 4.5 open 6, 8 *1 = Lampetra sp.; 2 = Oncorhynchus k i s u t c h Walbaum; 3 = Sal mo gai rdneri Richardson; 4 = Rhinichthys osculus "(Gi r a r d ) ; 5 = Richardsonius  balteatus (Richardson); 6 = Gasterosteus aculeatus Linnaeus; 7 = Cottus  gulosus ( G i r a r d ) ; 8 = C_. asp'er Richardson. Wynoochee, and Satsop s i t e s were sampled once a month f o r 12 months. Only _N. hubbsi and Gasterosteus aculeatus Linnaeus were caught at closed s i t e s . The strong co-occurrence of these f i s h e s has been d e t a i l e d by McPhail (1967, 1969) as well as Hagen et a l . (1979). G. aculeatus males u s u a l l y had b l a c k , i n s t e a d of the usual red, spawning c o l o r s w i t h i n h a b i t a t s a l s o occupied by _N. hubbsi; McPhail (1969) and Hagen et a l . (1980) have presented explanations of t h i s spawning c o l o r polymorphism based on experimental and observational data. Several f i s h e s were captured i n open h a b i t a t s . Red G. aculeatus males were recorded at the Junction C i t y s i t e , c o n t r a s t i n g only black morphs elsewhere. Perhaps important f i s h e s t o _N. hubbsi were the salmonids, Onchorynchus k i s u t c h (Walbaum) and Sal mo g a i r d n e r i Richardson, as well as two c o t t i d s , Cottus gulosus (Girard) and C_. asper Richardson, s i n c e these species are p i s c i v o r o u s (Moodie and Reimchen, 1976). Although salmonids were not recorded from a l l l o c a l i t i e s these could have been present i f c o t t i d s were captured. Moodie and Reimchen (1976) have documented a strong co-occurrence of these t a x a . The n o n - p a r a s i t i c Lampetra sp. captured at the Satsop s i t e had a unique d e n t i t i o n f i t t i n g no a v a i l a b l e keys, probably an example of the endemism w i t h i n the Chehalis River system noted by McPhail (1967). The most-often encountered c y p r i n i d i n open systems was Rhinichthys osculus ( G i r a r d ) . Since R. osculus i s p r i m a r i l y i n s e c t i v o r o u s (Scott and Crossman, 1972), i t i s l i k e l y not a s i g n i f i c a n t predator on _N. hubbsi. Phy s i c a l and chemical determinations on the water Waters at a l l c o l l e c t i n g s i t e s but one c o n s i s t e n t l y had a c i d i c pH's, 4.5-6.5, and no d e t e c t a b l e d i s s o l v e d s a l t s . The Junction C i t y s i t e was 26 an exceptional h a b i t a t f o r _N. hubbsi, s i n c e monthly determinations from February - June 1979 y i e l d e d pH's between 7.2 and 7.4 as well as 2-4 ppt (parts per thousand) d i s s o l v e d s a l t s . The lowest pH's, 4.5 - 5.0, were recorded during the 23-29 J u l y 1979 f i e l d t r i p s t o the Oyhut and Satsop s i t e s , when r a i n f a l l had been approaching the y e a r l y minimum (Ruffner, 1980). Evaporation had reduced the Oyhut and Satsop c o l l e c t i n g s i t e s from l a r g e r continuous bodies of water t o s e r i e s of smaller pools. No oxygen was detected i n some of the l a t t e r at Satsop, i n contrast t o 4-7 parts per m i l l i o n elsewhere. Carbon d i o x i d e contents i n Oyhut and Satsop waters during t h i s period of time were as much as 5-6 ppt, l i k e l y c o n t r i b u t i n g t o the low pH's. Water temperatures everywhere had ranged from 4°C i n the winter t o 23°C during the summer, but some pools at the Satsop s i t e were 27-30°C on 23 Ju l y 1979. M e r i s t i c c h a r a c t e r i s t i c s _N. hubbsi from the Copalis River and Conner Creek drainages had been captured i n 1947 and 1968, r e s p e c t i v e l y . These specimens were borrowed from the U n i v e r s i t y of Washington c o l l e c t i o n . Their p r e s e r v a t i o n was such t h a t fewer m e r i s t i c characters were evaluated than on specimens caught by myself. Counts of the median and paired f i n rays were obtained. Scale counts, w i t h the exception of sca l e s above the l a t e r a l s c a l e row, were not done because too many f i s h were misshapen and/or missing s c a l e s . L a t e r a l bar counts on males were taken from some Copalis f i s h ; however, the remaining U n i v e r s i t y of Washington specimens were too faded or d i s c o l o r e d f o r accurate e v a l u a t i o n of t h i s c haracter. Small numbers of N. hubbsi were obtained from the Black R i v e r and Hanaford 27 Creek c o l l e c t i n g s i t e s , so m e r i s t i c data from these f i s h were not included i n the analyses. There was no strong c o r r e l a t i o n between m e r i s t i c character s t a t e s and standard l e n g t h , a measure of s i z e (Table 2). S i m i l a r l y , Chernoff (1982) found t h a t m e r i s t i c characters on a t h e r i n i d s were not c o r r e l a t e d w i t h mensural ones. Only adult _N. hubbsi were evaluated, ranging from 3.43 - 6.68 cm SL f o r females and 3.19 - 6.19 cm SL f o r males, so the absence of j u v e n i l e specimens could have been p a r t l y r esponsible f o r the lack of c o r r e l a t i o n . Since s i z e d i d not appear t o have i n f l u e n c e d m e r i s t i c character s t a t e s , the l a t t e r were analyzed by means of u n i v a r i a t e s t a t i s t i c a l t e s t s i n s t e a d of a n a l y s i s of covariance. Sexual dimorphism of dorsal and anal f i n rays The d i s t r i b u t i o n s of dorsal and anal f i n ray counts appeared sexually-dimorphic w i t h i n some populations (Table 4). When samples from any c o l l e c t i n g s i t e were sorted by sex, males u s u a l l y had higher counts than d i d females (Tables 5-8). Both sexes were found t o have the same modal dorsal f i n count, 13 rays , at a l l s i t e s but two. Tahol ah and Oyhut males had modal dorsal f i n counts of 14 rays (Table 6) contrasted by only 13 rays f o r females (Table 7). Wishkah and Humptulips males had modal counts of 11 anal f i n rays, i n c o n t r a s t t o males from elsewhere which had 12 (Table 8). A s i m i l a r d i s t r i b u t i o n of modal anal f i n ray counts was found i n females, although Tahol ah specimens had a mode of only 11 rays and those from Conner Creek were bimodal, w i t h 11 and 12 rays (Table 9). Dorsal f i n rays i n males (Tables 6, 10; Figure I I ) Mean dorsal f i n ray counts were separable i n t o two c a t e g o r i e s . The 2 8 Table 4. Chi-square analyses on the d i s t r i b u t i o n s of dorsal and anal f i n ray numbers compared between sexes by l o c a t i o n f o r N. hubbsi. Although several s c a l e and f i n ray characters were examined Tn t h i s study, only dorsal and anal f i n rays were found t o be sexually-dimorphic, (n.s. = not s i g n i f i c a n t ) . Dorsal f i n rays: Location Tahol ah Copali s Conner Creek Oyhut Humptuli ps Wi shkah Wynoochee Satsop Chi-square 2.799 1.714 3.776 12.228 0.222 42.243 3.418 4.159 d.f. 1 1 2 2 1 2 3 2 P. n.s. n.s. n.s. X 20.005(2)=10.597, r e j e c t n u l l n.s. X 20.001(2)=13.16, r e j e c t n u l l n.s. n.s. Anal f i n rays: Location Tahol ah Copal i s Conner Creek Oyhut Humptul i ps Wi shkah Wynoochee Satsop Chi-square 2.334 0.948 1.437 2.449 0.187 0.250 14.707 d.f. 1 1 2 1 1 1 3 n.s. n.s. n.s. n.s. n.s. n.s. X 20.005(3)=12.838, r e j e c t n u l l Table 5. Sexual dimorphism i n the mean number of dorsal and anal f i n rays i n J N . hubbsi evaluated with t - t e s t s . A l l comparisons between sexes are s i g n i f i c a n t at the p=0.001 l e v e l . Males had greater numbers of rays on average than d i d females. Dorsal f i n ray counts Anal f i n ray counts Location t - t e s t df s i g n i f i c a n c e t - t e s t df s i g n i f i c a n c e Tahol ah 18.38 17 t0.001(2)=4.02 32.37 17 t0.001(2)=3.97 Copal i s 32.59 38 t0.001(2)=3.57 10.68 38 t0.001(2)=3.57 Conner Creek 37.03 51 t0.001(2)=3.50 49.16 51 t0.001(2)=3.49 Oyhut 125.53 65 t0.001(2)=3.45 23.92 64 t0.001(2) = 3.45 Humptulips 29.83 32 t0.001(2)=3.62 21.79 32 t0.001(2)=3.62 Wishkah 75.73 81 t0.001(2)=3.41 24.33 80 t0.001(2) = 3.42 Wynoochee 12.47 38 t0.001(2)=3.57 33.93 38 t0.001(2)=3.57 Satsop 73.32 67 t0.001(2)=3.43 49.67 66 t0.001(2)=3.44 Table 6. Frequency d i s t r i b u t i o n s , l o g a r i t h m i c means and a r i t h m e t i c means f o r the number of dorsal f i n rays i n male_N. hubbsi. 'R' i s the ordering of l o g a r i t h m i c means according t o i n c r e a s i n g magnitude and i d e n t i f i e s the populations as used i n the Student-Newman-Keuls t e s t (R, Table 10b). Number of dorsal f i n rays Location 11 12 13 14 15 1 Logarithm!' c mean s.d. A r i t h m e t i c mean 95% c o n f i dence i n t e r v a l R_ Tahol ah 4 5 9 1.1318 0.0431 13.56 12.55 - 15.62 7 Copal i s 1 17 9 1 28 1.1253 0.0206 13.36 13.10 - 13.59 6 Conner Creek 2 13 11 26 1.1249 0.0214 13.35 13.06 - 13.60 5 Oyhut 1 12 15 5 33 1.1369 0.0244 13.76 13.43 - 13.98 8 Humptuli ps 3 11 2 16 1.1114 0.0200 12.94 12.61 - 13.24 1 Wishkah 7 32 9 48 1.1149 0.0200 13.04 12.86 - 13.20 4 Wynoochee 2 2 12 6 22 1.1130 0.0305 13.00 12.57 - 13.38 2 Satsop 4 24 5 33 1.1146 0.0184 13.03 12.82 13.21 3 Table 7. Frequency d i s t r i b u t i o n s , l o g a r i t h m i c means and a r i t h m e t i c means f o r the number of dorsal f i n rays i n female N. hubbsi. Number of dorsal f i n rays 95% Location 11 12 13 14 15 N Logarithmi c mean s.d. Arithmeti c mean confidence i n t erval Tahol ah 1 6 3 10 1.1201 0.0210 13.20 12.74 - 13.65 Copal i s 9 3 12 1.1220 0.0147 13.25 12.96 - 13.53 Conner Creek 1 1 18 6 1 27 1.1194 0.0247 13.19 12.87 - 13.46 Oyhut 3 20 11 34 1.1213 0.0201 13.24 13.01 - 13.44 Humptuli ps 3 15 18 1.1081 0.0136 12.83 12.72 - 13.03 Wishkah 5 28 1 34 1.1098 0.0143 12.88 12.73 - 13.02 Wynoochee 1 1 13 3 18 1.1198 0.0378 13.17 13.00 - 13.36 Satsop 3 21 11 35 1.1211 0.0198 13.23 13.01 - 13.45 Table 8. Frequency d i s t r i b u t i o n s , l o g a r i t h m i c means and a r i t h m e t i c means f o r the number of anal f i n rays i n male JN. hubbsi. 'R' i s the ordering of l o g a r i t h m i c means according t o i n c r e a s i n g magnitude and i d e n t i f i e s the populations as used i n the Student-Newman-Keuls t e s t (R, Table 13b). Number of anal f i n rays Location 11 11 12 13 _N Logarithmi c mean Tahol ah 1 2 6 9 1.0620 Copalis 2 8 18 28 1.0627 Conner Creek 11 13 2 26 1.0659 Oyhut 1 13 17 1 32 1.0624 Humptuli ps 1 10 5 16 1.0506 Wishkah 32 16 48 1.0540 Wynoochee 6 15 1 22 1.0705 Satsop 2 3 24 4 33 1.0752 Ar i t h m e t i c confidence s.d. mean i n t e r v a l R 0.0282 11.56 10.98 - 12.11 3 0.0246 11.57 10.98 - 11.42 5 0.0233 11.65 11.39 - 11.89 6 0.0235 11.56 11.32 - 11.77 4 0.0223 11.25 10.93 - 11.55 1 0.0181 11.33 11.19 - 11.46 2 0.0196 11.77 11.53 - 11.99 7 0.0257 11.91 11.64 - 12.14 8 C J O ro Table 9. Frequency d i s t r i b u t i o n s , l o g a r i t h m i c means and a r i t h m e t i c means f o r the number of anal f i n rays i n female _N. hubbsi. 'R' i s the ordering of l o g a r i t h m i c means according t o i n c r e a s i n g magnitude and i d e n t i f i e s the populations as used i n the Student-Newman-Keuls t e s t (R, Table 14b). Number of anal f i n rays Location 10 _11 _12 13 1 Logarithmic mean s.d. A r i t h m e t i c mean 95% confidence i n t e r v a l R_ Tahol ah 7 3 10 1.0527 0.0182 11.30 10.96 - 11.63 3 Copalis 1 8 3 12 1.0474 0.0225 11.17 10.79 - 11.53 1 Conner Creek 14 13 27 1.0596 0.0192 11.48 11.27 - 11.67 6 Oyhut 3 12 19 34 1.0589 0.0258 11.47 11.22 - 11.69 5 Humptuli ps 15 3 18 1.0477 0.0145 11.17 10.98 - 11.34 2 Wishkah 21 13 34 1.0558 0.0186 11.38 11.20 - U.54 4 Wynoochee 6 12 18 1.0666 0.0183 11.67 11.41 - 11.90 7 Satsop 1 9 24 1 35 1.0682 0.0218 11.71 11.50 - 11.90 8 33 34 Table 10. S t a t i s t i c a l analyses using l o g a r i t h m i c means t e s t i n g f o r i n t e r -p opulational d i f f e r e n c e s i n the number of dorsal f i n rays i n male N. hubbsi: (a) a n a l y s i s of variance and (b) Student-Newman-Keuls (SNK) f o r paTrwise comparisons. 'R' i n the SNK i d e n t i f i e s the population means i n Table 6; s i g n i f i c a n c e l e v e l s are denoted by s u p e r s c r i p t s t a r s ( 0.05=*; 0.01=**; 0.001=***). a. A n a l y s i s of variance f o r data i n Table 6 Source Sums Mean of V a r i a t i o n of Squares df Squares Total 0.1128 214 Groups 0.0163 7 0.0023 Error 0.0965 207 0.0005 F = 4.60 F 0.0005(1) 7,207 = 3.92; r e j e c t n u l l Levene's t e s t f o r equal variances = 2.94; p < 0.02 One-way a n a l y s i s of va r i a n c e , t e s t s t a t i s t i c s f o r within-group variances not assumed t o be equal: Welch = 4.60; p < 0.005 Brown-Forsythe = 4.65; p < 0.005 b. Student .-Newman-Keuls t e s t f o r data i n Table 6 R 1 2 3 4 5 6 7 X 1.1114 1.1130 1.1146 1.1149 1.1249 1.1253 1.1318 N 16 22 34 48 26 28 9 R X N 1 1.1114 16 -2 1.1130 22 0.0016 -3 1.1146 34 0.0032 0.0016 -4 1.1149 48 0.0035 0.0019 0.003 -5 1.1249 26 0.0135 0.0119 0.0103 0.0100 -6 1.1253 28 0.0139 0.0123 0.0107 0.0104 0.0004 -7 1.1318 9 0.0204 0.0188 0.0172 0.0169 0.0069 0.0065 -8 1.1369 33 0.5255 0.5239 0.5223 0.5220 0.0120 0.0116 0.0051 8 1.1369 33 1 5 r > co ^ To 2 w E 1 4 o Q 1 3 e 2 8 2 2 0 33 1 2 m o SI CO I I I I CO a o O a> <u O c c o o o in a a E I I I CO CD o o o c >> 5 a o m +~ CO CO Figure I I . Geographic v a r i a t i o n i n the number of dorsal f i n rays i n male_N. hubbsi. A r i t h m e t i c mean i s represented as the center of the cross-hatched c i r c l e , upper and lower 95% confidence i n t e r v a l s as the v e r t i c a l l i n e s . The number immediately above the upper confidence i n t e r v a l i s sample s i z e . 3 6 . Humptulips, Wishkah, Wynoochee, and Satsop c o l l e c t i o n s were s t a t i s t i c a l l y equal, with a combined mean of 13.02 rays. The Taholah, Oyhut, Copal i s and Conner Creek samples were a l s o i n d i s t i n g u i s h a b l e from each other, w i t h a combined mean of 13.51 rays. SMC i n d i c a t e d a s t a t i s t i c a l d i f f e r e n c e between these two groupings (Table 11). The more western assemblage (Taholah, Copal i s , Conner Creek and Oyhut) had a greater combined mean dorsal f i n ray count than d i d the eastern one (Humptulips, Wishkah, Wynoochee and Satsop). Dorsal f i n rays i n females (Tables 7, 12; Figure I I I ) ANOVA i n d i c a t e d no s i g n i f i c a n t d i f f e r e n c e s between populations regarding dorsal f i n ray counts i n females. Anal f i n rays i n males (Tables 8, 13; Figure IV) There were no s i g n i f i c a n t d i f f e r e n c e s between samples from s i t e s west of and i n c l u d i n g Wishkah. Wynoochee and Satsop means counts were s t a t i s t i c a l l y equal t o each other, but counts from both d i f f e r e d from the Wishkah mean at the p = 0.05 and p = 0.001 l e v e l s r e s p e c t i v e l y . The Humptulips sample mean d i f f e r e d from the Satsop one at the p = 0.01 l e v e l . SMC i n d i c a t e d a s i g n i f i c a n t d i f f e r e n c e between combined Wynoochee and Satsop mean counts compared against combined Wishkah and Humptulips ones, n o n - s i g n i f i c a n c e f o r Humptulips and Wishkah mean counts compared against those from other samples from west of the Montesano H i l l s (Table 11). The coastal populations appeared t o have been equal regarding mean anal f i n ray counts Average counts may have been l e s s at Humptulips and Wishkah, but w i t h i n - p o p u l a t i o n sample s i z e s were too s m a l l , p r o h i b i t i n g s t a t i s t i c a l v e r i f i c a t i o n of t h i s p o s s i b i l i t y . Mean counts s i g n i f i c a n t l y increased from 11.33 rays at Wishkah to 11.77 rays at at Wynoochee. 37 Table 11. Scheffe's m u l t i p l e c o n t r a s t s t e s t s on the mean dorsal and anal f i n ray counts i n males and anal f i n ray counts i n females f o r N. hubbsi. 'X' r e f e r s t o p o p u l a t i i o n mean, with each population i d e n t i f i e d "by a s u b s c r i p t number (R i n Table 6 f o r dorsal f i n rays i n males, Table 8 f o r anal f i n rays i n males, Table 9 f o r anal f i n rays i n females). See t e x t f o r an explanation of r e s u l t s . Contrast Dorsal ray means , male x5+x6+x7+x8 x1+x2+x3+x4 _ _ = 0.0162 Anal ray means, male X7+X8 XJ+X2+X3+X4+X5+X5 Standard e r r o r 0.0010 2 0.0132 0.0038 x 7 + x 8 Xj + X 2 2 2 = 0.0205 0.0045 Xj+X2 x 3+x 4x 4x 5x 6 2 4 = 0.0110 Anal ray means, female Xj+X^+Xs+Xy x3+x4+x6+x8 _ 4 = 0.0049 0.0042 0.0033 16.20 3.47 4.56 2.62 1.48 Conclus ion SO.0005(1) 7,206=5.24; p<0.0005; r e j e c t n u l l SO.05(1) 7,208=3.79; p>0.05, accept n u l l SO.01(1),7,208 = 4.37; p<0.0005; r e j e c t n u l l SO.05(1) 7,208=3.79; p>0.05; accept n u l l SO.05(1) 7,180-3.80; p>0.05,accept n u l l 38 Table 12. A n a l y s i s of variance on mean dorsal f i n ray counts i n female i i * hubbsi captured at several l o c a l i t i e s . The data i s presented i n Table 7. No d i f f e r e n c e s between population samples were i n d i c a t e d . Source Sums Mean of V a r i a t i o n of Squares df Squares Total 0.0880 187 Groups 0.0052 7 0.0007 Er r o r 0.0828 180 0.0005 F = 1.40 F 0.05(1), 7,180 = 2.06; accept n u l l Since no d i f f e r e n c e s were i n d i c a t e d , the data was not f u r t h e r analyzed w i t h a Student-Neuman-Keuls t e s t . 14 CO CO Q) — «j co E CO <D O w o 13 10 34 18 34 18 35 12 « o co I I I I CO a o O CD 0) k— O 0) c c o O D .C o a a E X co (0 CD o o o c >> 5 a o m ca CO Figure I I I . Geographic v a r i a t i o n i n the number of dorsal f i n rays i n female N. hubbsi. See F i g . II f o r an explanation of symbols. -^ o 40 Table 13. S t a t i s t i c a l analyses using l o g a r i t h m i c means t e s t i n g f o r i n t e r p o p u l a t i o n a l d i f f e r e n c e s i n the number of anal f i n rays i n male N. hubbsi: (a) a n a l y s i s of variance and (b) Student-Newman-Keuls (SNK) f o r pa i r w i s e comparisons. 'R' i n the SNK i d e n t i f i e s the population means i n Table 8; s i g n i f i c a n c e l e v e l s are denoted by s u p e r s c r i p t a s t e r i s k s ( 0.05=* ; 0.01=**; 0.001=***). a. A n a l y s i s of variance f o r data i n Table 8 Source Sums Mean of V a r i a t i o n of Squares df Squares Total 0.1178 213 Groups 0.0127 7 0.0018 Error 0.1051 206 0.0005 F = 3.60 F 0.0025(1) 7,206 = 3.29; r e j e c t n u l l Levene's t e s t f o r equal variances = 2.97; p < 0.02 One-way a n a l y s i s of va r i a n c e , t e s t s t a t i s t i c s f o r within-group variances not assumed t o be equal: Welch = 3.00; p < 0.005 Brown-Forsythe = 3.16; p < 0.005 b. Student-Newman-Keuls t e s t f o r data i n Table 8 R 1 2 3 4 5 6 7 8 X 1.0506 1.0540 1.0620 1.0624 1.0627 1.0659 1.0705 1.0752 N 16 48 9 32 28 26 22 33 R X N 1 1.0506 16 -2 1.0540 48 0.0034 -3 1.0620 9 0.0114 0.0080 -4 1.0624 32 0.0118 0.0084 0.0004 -5 1.0627 28 0.0121 0.0087 0.0007 0.0003 -6 1.0659 26 0.0153 0.0119 0.0039 0.0035 0.0032 -7 1.0705 22 0.0199 0.0165 0.0085 0.0081 0.0078 0.0046 -8 1.0752 33 0.Q&U 0.6M2 0.0132 0.0128 0.0125 0.0093 0.0047 ro - n X -"• — 1 £Z o> - J 3 rt> cu r+ i — i - '• < O • 3 O !7> - h ro O I/) c Q << -5 3 Cu c r - o O 3 " —J - J . o • < r T a h o l a h Anal rays (male) T T " 3 r-t-3 " ro 3 cr ro cu 3 Cu cu 3 CU cr ro ro C o p a l i s C o n n e r C r e e k O y h u t — Humptul ips W i s h k a h W y n o o c h e e — S a t s o p — v X "> /TV K l ^ l 0 /TV O ) /TV I o cu / / / 42* Anal f i n rays i n females (Tables 9, 14; Figure V) The only p a i r - w i s e comparisons i n which 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 were found included Satsop f i s h compared with Copalis specimens and Satsop f i s h w i t h Humptulips ones, the Satsop c o l l e c t i o n s having the l e s s e r mean i n both cases. SMC d i d not i n d i c a t e other intersample d i f f e r e n c e s (Table 11). Geographic trends i n character v a r i a t i o n were not obvious i n SNK, but those populations with l e s s e r mean counts i n an absolute sense occurred west of the Wynoochee c o l l e c t i n g s i t e . C o palis and Conner Creek specimens were captured 32 and 11 y e a r s , r e s p e c t i v e l y , before the other samples, which may have been a f a c t o r i n producing mean counts s i g n i f i c a n t l y d i f f e r e n t from that obtained from Satsop f i s h . Caudal f i n rays (Tables 15, 16; Figure VI) Caudal f i n ray means were s t a t i s t i c a l l y - i n d i s t i n g u i s h a b l e between Taholah, Oyhut, Humptulips, Wishkah, Wynoochee, and Satsop samples. Copalis and Conner Creek mean counts were greater than the Wynoochee and Satsop ones at the p = 0.05 l e v e l . No trends i n character v a r i a t i o n were suggested by the analyses except f o r d i f f e r e n c e s r e s u l t i n g from the time when Copalis and Conner Creek specimens were c o l l e c t e d . Pectoral f i n rays (Tables 17, 18; Figure VII) The SNK i n d i c a t e d t h a t nearest-neighbor sample means were not d i f f e r e n t from each other, but that there was a c l i n a l i ncrease i n mean ray counts from west t o east. Taholah, C o p a l i s , Conner Creek, and Oyhut mean counts appeared equal, t h e i r combined mean having been 19.63 pect o r a l f i n rays. The next equivalent group included Oyhut, Humptulips, Wishkah and Wynoochee specimens w i t h a combined mean of 20.20 rays. The 43 Table 14. S t a t i s t i c a l analyses using l o g a r i t h m i c means t e s t i n g f o r i n t e r -p opulational d i f f e r e n c e s i n the number of anal f i n rays i n female N. hubbsi: (a) a n a l y s i s of variance and (b) Student-Newman-Keuls (SNK) f o r p a i r w i s e comparisons. 'R' i n the SNK i d e n t i f i e s the population means i n Table 9; s i g n i f i c a n c e l e v e l s are denoted by s u p e r s c r i p t a s t e r i s k s ( 0.05=*). a. Analysis of variance f o r data i n Table 9 Source of V a r i a t i o n Total Groups E r r o r Sums of Squares 0.0856 0.0087 0.0769 df 187 7 180 Mean Squares 0.0012 0.0004 F = 3.00 F 0.01(1) 7,180 = 2.74; r e j e c t n u l l Levene's t e s t f o r equal variances = 1.55; p > 0.10 Since the variance appeared homodescacic, Welch and Brown-Forsythe t e s t s were not performed. b. Student-Newman-Keuls t e s t f o r data i n Table 9 R 1 2 3 4 5 6 7 8 X 1.0474 1.0477 1.0527 1.0558 1.0589 1.0596 1.0666 1.0682 n _ N M 12 18 10 34 34 27 18 35 K 1 X 1.0474 IN 12 _ 2 1.0477 18 0.0003 -3 1.0527 10 0.0053 0.0050 -4 1.0558 34 0.0084 0.0081 0.0031 -5 1.0589 34 0.0115 0.0112 0.0062 0.0031 -6 1.0596 27 0.0122 0.0119 0.0069 0.0038 0.0007 -7 1.0666 18 0.0192 0.0189 0.0139 0.0108 0.0077 0.0076 -8 1.0682 35 0.0208 0.02*05 0.0155 0.0124 0.0093 0.0086 0.0016 -12 g i 1 0 « -'12 O 27 34 I I I I </) _ C 0 CD o To a CD -C >. Tar Co O O CD C c o O 34 18 co a a E 3 I ca 18 CD CD JO o o o c >. 35 I I a o co ca 03 Figure V. Geographic v a r i a t i o n explanation of symbols. i n the number of anal f i n rays i n female N. hubbsi. See F i g . II f o r an Table 15. Frequency d i s t r i b u t i o n s , l o g a r i t h m i c means and a r i t h m e t i c means f o r the number of caudal f i n rays i n N. hubbsi. 'R' i s the ordering of l o g a r i t h m i c means according t o i n c r e a s i n g magnitude and i d e n t i f i e s the populations as used i n the Student-Newman-Keuls t e s t (R, Table 16b). Number of caudal f i n rays 95% Location i i XL 18 Ii _N Logarithm!' c mean s.d. Arithmeti c mean confidence i n terval R_ Tahol ah 2 17 19 1.2527 0.0078 17.89 17.74 -• 18.05 4 Copalis 1 2 49 3 55 1.2547 0.0101 17.98 17.86 -• 18.09 8 Conner Creek 4 47 2 53 1.2543 0.0082 17.96 17.86 -• 18.05 6 Oyhut 5 59 64 1.2533 0.0067 17.92 17.85 -• 17.99 5 Humptul i p s 2 31 1 34 1.2545 0.0078 17.97 17.86 -• 18.08 7 Wishkah 1 20 58 2 81 1.2491 0.0127 17.75 17.63 -• 17.86 3 Wynoochee 4 5 31 40 1.2471 0.0170 17.68 17.44 • • 17.88 1 Satsop 7 6 55 68 1.2478 0.0165 17.71 17.53 -• 17.86 2 46 Table 16. S t a t i s t i c a l analyses using l o g a r i t h m i c means t e s t i n g f o r i n t e r p o p u l a t i o n a l d i f f e r e n c e s i n the number of caudal f i n rays i n \L hubbsi: (a) a n a l y s i s of variance and (b) Student-Newman-Keuls t e s t (SNK) f o r p a i r w i s e comparisons. 'R' i n the SNK i d e n t i f i e s the population means i n Table 15; s i g n i f i c a n c e l e v e l s are denoted by s u p e r s c r i p t a s t e r i s k s ( 0.05=*). a. A n a l y s i s of variance f o r data i n Table 15 Source Sums Mean of V a r i a t i o n of Squares df Squares Total 0.0608 413 Groups 0.0037 7 0.005 Error 0.0571 406 0.0001 F = 5.00 F 0.0005(1) 7,406 = 3.83, r e j e c t n u l l Levene's t e s t f o r equal variances = 13.41; p < 0.001 One-way a n a l y s i s of v a r i a n c e , t e s t s t a t i s t i c s f o r within-group variances not assumed t o be equal: Welch = 3.62; p < 0.005 Brown-Forsythe = 3.15; p < 0.009 b. Student-Newman-Keuls t e s t f o r data i n Table 15 R 1 2 3 4 5 6 7 8 X 1.2471 1.2478 1.2491 1.2527 1.2533 1.2543 1.2545 1.2547 N 40 68 81 19 64 53 34 55 R X N 1 1.2471 40 -2 1.2478 68 0.0008 -3 1.2491 81 0.0020 0.0013 -4 1.2527 19 0.0056 0.0049 0.0036 -5 1.2533 64 0.0062 0.0055 0.0042 0.0006 -6 1.2543 53 0.0072 0.0065 0.0052 0.0016 0.0010 -7 1.2545 34 0.0074 0.0067 0.0054 0.0018 0.0012 0.0002 -8 1.2547 55 0.0$76 0.0069 0.0056 0.0020 0.0014 0.0004 0.0002 rt) -n X —*• T3 CO — ' c cu 3 rt> Ol rt- < O • 3 O -t! rt> o </> u s v< - J 3 CU cr-o o 3-in o < CU o 3 rt) 3 3 cr n> o CU Q. CU "5 CU * < c cr cr oo rt> Caudal rays N r 00 T a h o l a h — -(Jhw C o p a l i s — <J>5 C o n n e r C r e e k — O y h u t — $ 2 H u m p t u l i p s — W i s h k a h — <J>~! W y n o o c h e e — -<3>-o S a t s o p — -($)-£ •o CU 3 Table 17. Frequency d i s t r i b u t i o n s , l o g a r i t h m i c means and geometric means f o r the number of pectoral f i n rays i n N. hubbsi. 'R' i s the ordering of l o g a r i t h m i c means according t o i n c r e a s i n g magnitude and i d e n t i f i e s the populations as used i n the Student-Newman-Keuls t e s t (R, Table 18b). Number of pectoral f i n rays 95% Location 17 18 19 20 21 22 23 _N Logarithmic mean s.d. A r i t h m e t i c mean confidence i n t e r v a l R_ Tahol ah 5 9 5 19 1.2785 0.0152 19.00 18.67 - 19.31 1 Copal i s 5 15 20 9 49 1.2934 0.0201 19.67 19.39 - 19.91 3 Conner Creek 2 2 25 19 1 49 1.2854 0.0169 19.31 19.07 - 19.51 2 Oyhut 2 9 24 5 40 1.2964 0.0172 19.80 19.54 - 20.04 4 Humptulips 1 4 13 10 28 1.3038 0.0191 20.14 19.78 - 20.47 5 Wishkah l ' 22 30 53 1.3126 0.0149 20.55 20.35 - 20.74 7 Wynoochee 2 6 15 16 1 40 1.3049 0.0211 20.20 19.86 - 10.50 6 Satsop 1 3 9 50 4 1 68 1.3183 0.0176 20.82 20.61 - 21.02 8 CO 49 Table 18. S t a t i s t i c a l analyses using l o g a r i t h m i c means, t e s t i n g f o r i n t e r p o p u l a t i o n a l d i f f e r e n c e s i n the number of pectoral f i n rays i n NI. hubbsi: (a) a n a l y s i s of variance and (b) Student-Newman-Keuls (SNK) f o r pa i r w i s e comparisons. 'R' i n the SNK i d e n t i f i e s the population means i n Table 17; s i g n i f i c a n c e l e v e l s are denoted by s u p e r s c r i p t a s t e r i s k s ( 0.005=*; 0.01=**; 0.001=***). a. A n a l y s i s of variance f o r data i n Table 17 Source Sums Mean of V a r i a t i o n of Squares df Squares Total 0.5661 345 Groups 0.2299 7 0.0328 Error 0.3362 338 0.0010 F = 32 80 F 0.0005(1) 7,338 = 384; r e j e c t n u l l Levene's t e s t f o r equal variances = 2.93; p < 0.014 One-way a n a l y s i s of var i a n c e , t e s t s t a t i s t i c s f o r within-group variances not assumed t o be equal: Welch = 32.68; p < 0.001 Brown-Forsythe = 31.98; p < 0.001 b. Student-Newman-Keuls t e s t f o r data i n Table 17 R 1 2 3 4 5 6 7 x 1.2785 1.2854 1.2934 1.2964 1.3038 1.3049 1.3126 N 19 49 49 40 28 40 53 R X N 1 1.2785 19 -2 1.2854 49 0.0069 -3 1.2934 49 0.0149 0.0080 -4 1.2964 40 0.0179 0.0110 0.0030 -5 1.3038 28 0.02*53 0.0l84 0.0104 0.0074 -6 1.3049 40 0.0264 0.0195 0.0115 0.0085 0.0011 -7 1.3126 53 0.6541 oMIz 0.0f92 0.0162 0.0088 0.0077 -8 1.3183 68 0.0^98 0.65^9 oMh 0.02*19 0.0148 0.0134 0.0057 - n X — J . T J CO cr Cu - 3 3 rt> CU r+ < —'• »—i O 3 • o - h CD rt> CO O << CQ 3 ~ 3 c r Oi o T 3 — ' 3 " c n — i . • O < CU CU n -o co 3 C T rt> rt> n c+ O "5 Cu -tt 3 - 3 CU v < cn 3 " SZ CT c r cn es r T a h o l a h C o p a l i s — C o n n e r C r e e k — O y h u t Humptul ips — W i s h k a h — W y n o o c h e e — S a t s o p Pectoral rays •o T" ro o T CD <o / T N ft <£> — / T N •o ro 0 (TJ o -51;. l a s t group was made up of Humptulips, Wishkah, Wynoochee, and Satsop f i s h w i t h a combined mean of 20.52 rays. P e l v i c f i n rays (Tables 19, 20; Figure V I I I The ANOVA F - s t a t i s t i c could not be used due t o i t s lack of agreement with the Welch and Brown-Forsythe t e s t s . A non-parametric K r u s k a l l -W a l l i s t e s t showed no d i f f e r e n c e s i n mean counts. P e l v i c f i n rays were an almost i n v a r i a t e c h a r a c t e r , most f i s h having seven rays. The only v a r i a t i o n was f i n ray l o s s r e s u l t i n g i n s i x rays. Lesser f i n ray counts comprised a very small f r a c t i o n , 6% or l e s s , of those c o l l e c t i o n s i n which v a r i a t i o n was found. The Taholah sample was noteworthy s i n c e 3 out of the 19 f i s h (16%) had 6 p e l v i c f i n rays. Scale rows before the dorsal f i n o r i g i n (Tables 21, 22; Figure IX) With the exception of comparisons with the Wishkah sample, a l l c o l l e c t i o n s were s t a t i s t i c a l l y equal and had a combined mean of 21.38 s c a l e rows. Wishkah specimens averaged 23.82 s c a l e rows, s i g n i f i c a n t l y d i f f e r e n t at the p = 0.001 l e v e l from a l l other samples. On average, Wishkah f i s h had 2.02 s c a l e rows more than _N. hubbsi from other 1 o c a l i t i e s . L a t e r a l row sca l e s (Tables 23, 24; Figure X) Taholah, Oyhut, and Humptulips samples had s t a t i s t i c a l l y equal means, with a combined average of 50.80 s c a l e s . The SNK i n d i c a t e d t h a t s c a l e counts f o r these populations averaged l e s s than the means f o r Wynoochee and Satsop c o l l e c t i o n s , the l a t t e r s t a t i s t i c a l l y i n d i s t i n g u i s h a b l e from each other w i t h a combined mean of 52.21 s c a l e s . Non-significance between Taholah and Satsop sample means may have been Table 19. Frequency d i s t r i b u t i o n s , l o g a r i t h m i c means and a r i t h m e t i c means f o r the number of pelv f i n rays i n N. hubbsi. Number of p e l v i c f i n rays 95% Location 6. _7 _N Logarithmic mean s.d. A r i t h m e t i c mean con f i dence i nterval Tahol ah 3 16 19 0.8345 0.0252 6.84 6.64 - 7.03 Copal i s 2 55 57 0.8428 0.0126 6.96 6.91 - 7.02 Conner Creek 53 53 0.8451 0.0000 7.00 -Oyhut 1 63 64 0.8441 0.0087 6.98 6.94 - 7.02 Humptuli ps 1 33 34 0.8431 0.0117 6.97 6.90 - 7.03 Wishkah 82 82 0.8451 0.0000 7.00 -Wynoochee 1 39 40 0.8434 0.0109 6.97 6.92 - 7.03 Satsop 68 68 0.8451 0.0000 7.00 53 Table 20. A n a l y s i s of variance and K r u s k a l l - W a l l i s t e s t s on the number of p e l v i c f i n rays i n _N. hubbsi. The data f o r these t e s t s are presented i n Table 19. The Levene as well as the Brown-Forsythe and Welch t e s t s i n d i c a t e d t h a t the a n a l y s i s of variance F - s t a t i s t i c should not be used, so data was analyzed w i t h a non-parametric K r u s k a l l - W a l l i s t e s t . No d i f f e r e n c e s between populations were i n d i c a t e d by the l a t t e r t e s t . Source of V a r i a t i o n Sums of Squares df Mean Squares Total 0.0293 416 Groups 0.0021 7 0.0003 Er r o r 0.0272 409 0.0001 F = 3.00 F 0.0005(1), 7,409 = 2.96; r e j e c t n u l l Levene's t e s t f o r equal variances = 19.13; p < 0.001 One-way a n a l y s i s of v a r i a n c e , t e s t s t a t i s t i c s f o r within-group variances not assumed t o be equal: Welch=0.52; p < 0.70 Brown-Forsythe=2.22 p < 0.12 Note - Only those groups w i t h non-zero variance were used i n the computations of the Levene, Welsh and Brown-Forsythe Tests. K r u s k a l l - W a l l i s , H=8.58; X 2 0.05(7)=14.067, accept nul1 (V TI X - i . T3 tQ — 1 C CU -) 3 rt) cu 3 i—c CD to CD << O 3 to c r -5 O Cu — ' T J to zr o O) < cu o 3 T 3 < to c c r c r to CO (t> T a h o l a h — c C o n n e r C r e e k — Pelvic rays I 7 -«J CO C o p a l i s em u O y h u t — Humptul ips — 2 W i s h k a h 0 o W y n o o c h e e — S a t s o p — 0 c cu 3 Table 21. Frequency d i s t r i b u t i o n s , l o g a r i t h m i c means and a r i t h m e t i c means f o r the number of s c a l e rows before the dorsal f i n o r i g i n i n N. hubbsi. 'R' i s the ordering of l o g a r i t h m i c means according t o i n c r e a s i n g magnitude and i d e n t i f i e s the populations as used i n the Student-Newman-Keuls t e s t (R, Table 22b). Number of scal e rows before the dorsal f i n o r i g i n 95% Location 18 19 20 21 22 23 24 25 26 27 _N Logarithmic mean s.d. A r i t h m e t i c mean confidence i n t e r v a l R_ Taholah 6 5 4 2 2 19 1.3300 0.0222 21.42 20.85 - 21.91 3 Oyhut 1 4 25 14 14 4 1 1 64 1.3192 0.0264 20.89 20.54 - 21.17 1 Humptul i p s 5 4 7 11 6 1 34 1.3285 0.0288 21.35 20.82 - 21.80 2 Wi shkah 2 12 23 23 12 3 7 82 1.3763 0.0263 23.82 23.47 - 24.10 6 Wynoochee 1 4 4 11 8 6 6 40 1.3327 0.0329 21.57 21.00 - 22.04 4 Satsop 1 12 19 19 12 1 3 1 68 1.3360 0.0273 21.72 21.34 - 22.01 5 cn 56 Table 22. S t a t i s t i c a l analyses using l o g a r i t h m i c means, t e s t i n g f o r i n t e r - p o p u l a t i o n a l d i f f e r e n c e s i n the number of s c a l e rows before the dorsal f i n o r i g i n i n _N. hubbsi: (a) a n a l y s i s of variance and (b) Student-Newman-Keuls (SNK) f o r pairw i s e comparisons. 'R' i n the SNK i d e n t i f i e s the population means i n Table 21; s i g n i f i c a n c e l e v e l s are denoted by s u p e r s c r i p t a s t e r i s k s ( 0.0001=***). a. A n a l y s i s of variance f o r data i n Table 21 Source Sums Mean of V a r i a t i o n of Squares df Squares Total 0.3768 306 Groups 0.1447 5 0.0289 Error 0.2322 301 0.0008 F = 36.13 F 0.0005(1) 5,301 = 4.56, r e j e c t n u l l Levene's t e s t f o r equal variances = 0.90; p < 0.50 One-way a n a l y s i s of va r i a n c e , t e s t s t a t i s t i c s f o r within-group variances not assumed t o be equal: Welch = 39.88; p < 0.001 Brown-Forsythe = 36.28; p < 0.001 b. Student -Newman i - Keuls t e s t f o r data i n Table 21 R 1 2 3 4 5 x 1.3192 1.3285 1.3300 1.3327 1.3360 N 19 64 34 82 40 R X N 1 1.3192 19 -2 1.3285 64 0.0093 -3 1.3300 34 0.0108 0.0015 -4 1.3327 82 0.0135 0.0042 0.0027 -5 1.3360 40 0.0168 0.0075 0.0060 0.0033 -6 1.3763 68 0.65^1 0.8*75 0.5^3 0.8$3"6 Figure IX. Geographic v a r i a t i o n i n the number of s c a l e rows before the dorsal f i n o r i g i n i n N. hubbsi. See F i g . II f o r an explanation of symbols. Table 23. Frequency d i s t r i b u t i o n s , l o g a r i t h m i c means and a r i t h m e t i c means f o r the number of l a t e r a l row scales i n NL hubbsi. 'R' i s the ordering of l o g a r i t h m i c means according t o i n c r e a s i n g magnitude and i d e n t i f i e s the populations as used i n the Student-Newman-Keuls t e s t (R, Table 24b). Number of l a t e r a l row s c a l e s 61 62 63 _N 19 64 34 1 82 40 68 Location 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Tahol ah 2 1 7 2 3 2 2 Oyhut 1 1 4 12 11 17 4 6 2 2 3 1 Humptuli ps 1 2 7 7 8 1 2 4 2 Wi shkah 2 8 9 12 20 15 3 8 4 Wynoochee 2 2 5 5 6 16 1 1 1 1 Satsop 1 4 5 17 11 10 10 4 3 3 Table 23. Continued Location Logarithmic mean s.d. Taholah 1.7055 0.0172 Oyhut 1.7065 0.0191 Humptuli ps 1.7057 0.0177 Wishkah 1.7485 0.0163 Wynoochee 1.7159 0.0161 Satsop 1.7189 0.0112 Ar i t h m e t i c c o n f i dence mean i n t e r v a l R_ 50.79 49.81 - 51.72 1 50.92 50.31 - 51.44 3 50.82 50.07 - 51.50 2 56.56 55.58 - 56.50 6 52.02 51.39 - 52.60 4 52.38 52.01 - 52.69 5 cn vo 60 Table 24. S t a t i s t i c a l analyses using l o g a r i t h m i c means, t e s t i n g f o r i n t e r p o p u l a t i o n a l d i f f e r e n c e s i n the number of l a t e r a l s c a l e s i n JN. hubbsi: (a) a n a l y s i s of variance and (b) Student-Newman-Keuls (SNK) f o r pairwis e comparisons. 'R' i n the SNK i d e n t i f i e s the population means i n Table 23; s i g n i f i c a n c e l e v e l s are denoted by s u p e r s c r i p t a s t e r i s k s (0.05=*; 0.01=**; 0.001=***). a. A n a l y s i s of variance t a b l e f o r data i n Table 23 Source Sums Mean of V a r i a t i o n of Squares df Squares Total 0.1802 306 Groups 0.0893 5 0.0179 Error 0.0909 301 0.0003 F = 59.27 F 0.0005(1) 5,301 = 4.56; r e j e c t n u l l Levene's t e s t f o r equal variances = 0.35; p > 0.85 One-way a n a l y s i s of var i a n c e , t e s t s t a t i s t i c s f o r within-group variances not assumed t o be equal: Welch = 60.27; p < 0.001 Brown-Forsythe = 58.61; p < 0.001 b. Student-Newman-Keuls t e s t f o r data i n Table 23 R 1 2 3 4 5 6 X 1.7055 1.7057 1.7065 1.7159 1.7189 1.7485 n 19 34 64 68 40 82 R X n 1 1.7055 19 -2 1.7057 34 0.0002 -3 1.7065 64 0.0010 0.0008 -4 1.7159 68 0.0104 0.0102 0.§094 -5 1.7189 40 0.0t35 0.M32 0.0l2"4 0.0030 -6 1.7485 82 0.8130 oMh oMh 5 7 r 56 5 5 5 41 CO CO o CO « 5 3 CD +-> CO 52 51 50 19 4> 6 4 4> 3 4 62 4 0 0 6 8 Figure X. Geographic v a r i a t i o n i n the number of l a t e r a l row seal hubbsi. See F i g . II f o r an explanation of symbols. 62 due t o the small sample s i z e of the former c o l l e c t i o n s i n c e d i f f e r e n c e s between Oyhut and Satsop as well as Humptulips and Satsop were a r i t h m e t i c a l l y - l e s s but t e s t e d as s i g n i f i c a n t l y d i f f e r e n t . Means from the Taholah, Oyhut, Humptulips, Wynoochee and Wishkah samples were each s t a t i s t i c a l l y d i f f e r e n t from the Wishkah mean at the p = 0.001 l e v e l . The Wishkah mean was 5.28 and 3.87 sca l e s g r e a t e r , r e s p e c t i v e l y , than the above combined means. Scales above the l a t e r a l s c a l e row (Tables 25, 26; Figure XI) S t a t i s t i c a l l y - e q u a l Copalis and Conner Creek means were a r i t h m e t i c a l l y greater than other sample mans f o r c o l l e c t i o n s made west of the Wynoochee s i t e , although t h i s d i f f e r e n c e was not s t a t i s t i c a l l y s i g n i f i c a n t . The Taholah, Oyhut, Humptulips and Wishkah means were s t a t i s t i c a l l y equal. These f o u r , i n t u r n , averaged l e s s than Wynochee and Satsop counts. The combined mean count f o r Taholah, Oyhut, Humptulips and Wishkah f i s h was 9.40 sca l e s i n con t r a s t t o 9.81 s c a l e s f o r Wynoochee and Satsop f i s h . Scales below the l a t e r a l s c a l e row (Tables 27, 28; Figure XII) Nearest-neighbor sample means were not s t a t i s t i c a l l y d i f f e r e n t . However, means increased c l i n a l l y from Taholah and Oyhut, w i t h a combined mean of 9.25 s c a l e s , t o Wishkah, which averaged 9.71 s c a l e s . Mean counts from Wynoochee f i s h were s t a t i s t i c a l l y equal t o those from Wishkah_N. hubbsi; however, means from Satsop f i s h were l e s s than those from Wishkah ones but equivalent t o those from Wynoochee specimens. Lat e r a l bars i n males (Tables 29, 30; Figure X I I I , XIV) The Wynoochee and Satsop sample means were s i g n i f i c a n t l y d i f f e r e n t Table 25. Frequency d i s t r i b u t i o n s , l o g a r i t h m i c means and a r i t h m e t i c means f o r the number of sca l e s above the l a t e r a l scale row i n H. hubbsi. 'R' i s the ordering of l o g a r i t h m i c means according t o i n c r e a s i n g magnitude and i d e n t i f i e s the populations as used i n the Student-Newman-Keuls t e s t (R, Table 26b). Number of scales above l a t e r a l row 95% Logarithmi c Ar i t h m e t i c c o n f i dence Locat i on 9 10 11 N mean s.d. mean i nterval R Tahol ah 13 6 19 0.9686 0.0219 9.32 9.08 - 9.53 2 Copali s 17 21 38 0.9795 0.0231 9.55 9.37 - 9.71 5 Conner Creek 22 28 50 0.9798 0.0230 9.56 9.40 - 9.69 6 Oyhut 2 40 21 63 0.9678 0.0246 9.30 9.15 - 9.42 1 Humptulips 18 16 34 0.9757 0.0232 9.47 9.28 - 9.63 4 Wishkah 4 34 41 79 0.9754 0.0280 9.46 9.31 - 9.59 3 Wynoochee 6 34 40 0.9931 0.0164 9.85 9.72 - 9.96 8 Satsop 1 14 52 1 68 0.9898 0.0223 9.78 9.65 - 9.89 7 cn 64 Table 26. S t a t i s t i c a l analyses using l o g a r i t h m i c means, t e s t i n g f o r i n t e r p o p u l a t i o n a l d i f f e r e n c e s i n the number of sca l e s above the l a t e r a l s c a l e row i n _N. hubbsi; (b) a n a l y s i s of variance and (b) Student-Newman-Keuls (SNK) f o r p a i r w i s e comparisons. 'R' i n the SNK i d e n t i f i e s the population means i n Table 25; s i g n i f i c a n c e l e v e l s are denoted by s u p e r s c r i p t a s t e r i s k s ( 0.05=*; 0.01=**; 0.001=***). a. A n a l y s i s of variance f o r data i n Table 25 Source Sums Mean of V a r i a t i o n of Squares df Squares Total 0.2221 390 Groups 0.0269 7 0.0038 Erro r 0.1952 383 0.0005 F = 7.60 F 0.0005(1) 7,383 = 3.84; r e j e c t n u l l Levene's t e s t f o r equal variances = 9.56; p < 0.001 One-way a n a l y s i s of va r i a n c e , t e s t s t a t i s t i c s f o r within-group variances not assumed t o be equal Welch = 11.17; p < 0.001 Brown-Forsythe = 10.08; p < 0.001 b. Student ;-Newman-Keuls t e s t f o r data i n Table i 25 R 1 2 3 4 5 6 7 X 0.9678 0.9686 0.9754 0.9757 0.9795 0.9798 0.9898 n 63 19 79 34 38 50 68 R X n 1 0.9678 63 -2 0.9686 19 0.0008 -3 0.9754 79 0.0076 0.0068 -4 0.9757 34 0.0079 0.0071 0.0003 -5 0.9795 38 0.0117 0.0109 0.0041 0.0038 -6 0.9798 50 0.0120 0.0112 0.0044 0.0041 0.0003 -7 0.9898 68 oMn 0.8144 0.ol41 0.0103 0.0100 -8 0.9931 40 0.6245 0.8177 0.0174 0.o!36 0.6133 0.0033 8 0.9931 40 0) o o ©10 CO g 0} CO CD _ - 2 L O CD o U to ~ CO 19 JC CO 3B co a o O 50 CO CO CJ cu c c o o 34 6 3 7 9 Z3 SZ O co a a E D I I I I I I SZ CO JC co 40 CU cu SZ o o o c >> 68 I I a o co CO CO Figure XI. Geographic var iat ion in the number of scales above the la tera l scale row in _N. hubbsi. See F ig . II for an explanation of symbols. Table 27. Frequency d i s t r i b u t i o n s , l o g a r i t h m i c means and a r i t h m e t i c means f o r the number of s c a l e s below the l a t e r a l s c a l e row in_N. hubbsi. 'R' i s the ordering of l o g a r i t h m i c means according t o i n c r e a s i n g magnitude and i d e n t i f i e s the populations as used i n the Student-Newman-Keuls t e s t (R, Table 28b). Number of scales below l a t e r a l row 95% Locat i on 8 9 10 11 12 N Logarithmic mean s.d. A r i t h m e t i c mean co n f i dence i n t e r v a l R_ Taholah 3 13 3 19 0.9534 0.0285 9.00 7.83 - 10.31 1 Oyhut 2 42 19 2 65 0.9687 0.0270 9.32 8.21 - 10.53 3 Humptulips 1 14 10 2 27 0.9757 0.0318 9.48 8.13 - 10.99 4 Wi shkah 33 37 8 1 79 0.9861 0.0308 9.71 8.41 - 11.15 6 Wynoochee 22 17 1 40 0.9759 0.0251 9.48 8.42 - 10.63 5 Satsop 1 48 19 68 0.9663 0.0220 9.26 8.36 - 10.23 2 67 Table 28. S t a t i s t i c a l analyses using l o g a r i t h m i c means, t e s t i n g f o r i n t e r p o p u l a t i o n a l d i f f e r e n c e s i n the number of sca l e s below the l a t e r a l s c a l e row i n H. hubbsi: (a) a n a l y s i s of variance and (b) Student-Newman Keuls (SNK) f o r pairw i s e comparisons. 'R' i n the SNK i d e n t i f i e s the population means i n Table 29; s i g n i f i c a n c e l e v e l s are denoted by s u p e r s c r i p t a s t e r i s k s (0.05=*; 0.01=**; 0.001=***). a. A n a l y s i s of variance t a b l e f o r data i n Table 27 Source Sums Mean of V a r i a t i o n of Squares df Squares Total 0.2433 297 Groups 0.0256 5 0.0051 Error 0.2177 292 0.0007 F = 7.29 F 0.0005(1) 5,292 = 4.57; r e j e c t n u l l Levene's t e s t f o r equal variance = 5.62; p < 0.001 One-way a n a l y s i s of var i a n c e , t e s t s t a t i s t i c s f o r w i t h i n -group variances not assumed t o be equal: Welch = 6.91; p < 0.001 Brown-Forsythe = 6.75; p < 0.001 b. Student-Newman-Keuls t e s t f o r data i n Table 27 R 1 2 3 4 5 X 0.9534 0.9663 0.9687 0.9757 0.9759 n 19 68 65 27 40 R X n 1 0.9534 19 -2 0.9663 68 0.0129 -3 0.9687 65 0.0153 0.0024 -4 0.9757 27 0.0223 0.0094 0.0070 -5 0.9759 40 0.02*25 0.0096 0.0072 0.0002 -6 0.9861 79 oMb 0.8198 0.6174 0.0104 0.0102 10 o CO 9 CO Q) (0 _ O CD I CO (0 CO 79 19 e 6 5 40 6 8 o CO I I I I O a a E 3 X ca 10 CL) O o o c >< D. O in ca CO Figure X I I . Geographic v a r i a t i o n i n the number of scales below the l a t e r a l s c a l e row in _N- hubbsi See F i g . II f o r an explanation of symbols. Table 29. Frequency d i s t r i b u t i o n s , l o g a r i t h m i c means and a r i t h m e t i c means f o r the number of l a t e r a l bars i n male H. hubbsi. 'R' i s the ordering of l o g a r i t h m i c means according t o i n c r e a s i n g magnitude and i d e n t i f i e s the populations as used i n the Student-Newman Keuls t e s t (R, Table 13b). Number of l a t e r a l bars 95% Locati on 9_ 10 11 12 13 14 15 16 17 18 19 ' N Logarithmi c mean s .d. Ari t h m e t i c mean confidence i n t e r v a l R_ Tahol ah 4 2 3 3 12 1.0149 0.0520 10.41 9.59-11.17 1 Copali s 4 8 4 2 1 19 1.0906 0.0384 12.37 11.81-12.85 3 Oyhut 7 11 13 10 7 5 1 54 1.0876 0.0553 12.33 11.82-12.66 2 Humptuli ps 1 3 5 5 1 1 16 1.1224 0.0394 13.31 12.64-13.90 5 Wishkah 2 11 .12 12 17 2 2 1 59 1.1059 0.0508 12.84 12.37-13.16 4 Wynoochee 1 1 5 14 1 22 1.2193 0.0211 16.59 16.21-16.92 7 Satsop 1 2 13 10 7 8 2 1 44 1.1825 0.0420 15.29 14.78-15.68 6 70 Table 30. S t a t i s t i c a l analyses using l o g a r i t h m i c means t e s t i n g f o r i n t e r p o p u l a t i o n a l d i f f e r e n c e s i n the number of l a t e r a l bars i n male N. hubbsi; (a) a n a l y s i s of variance and (b) Student-Newman-Keuls (SNK) f o r pa i r w i s e comparisons. 'R' i n the SNK i d e n t i f i e s the population means i n Table 29; s i g n i f i c a n c e l e v e l s are denoted by s u p e r s c r i p t a s t e r i s k s ( 0.001=***). a. A n a l y s i s of variance f o r data i n Table 29 Source Sums Mean of V a r i a t i o n of Squares df Squares Total 1.0876 225 Groups 0.6676 6 0.1113 Er r o r 0.4200 219 0.0019 F = 58.58 F 0.0005(1) 6,219 = 4.19; r e j e c t n u l l Levene's t e s t f o r equal variances = 3.84; p < 0.003 One-way a n a l y s i s of var i a n c e , t e s t s t a t i s t i c s f o r within-group variances not assumed t o be equal: Welch = 52.82; p < 0.001 Brown-Forsythe = 42.00; p < 0.001 b. Student -Newman -Keuls t e s t f o r data i n Table 29 R 1 2 3 4 5 6 X 1.0149 1.0876 1.0906 1.1059 1.1224 1.1825 N 12 54 19 59 16 44 R X N 1 1.0149 12 -2 1.0876 54 0.0*27 -3 1.0906 19 0.8857 0.0030 -4 1.1059 59 *** 0.0910 0.0183 0.0153 -5 1.1224 16 o.Ws oMfo 0.85*8 0.0165 -6 1.1825 44 o.fele 0M%9 0.8319 0.6*66 0.8*01 -7 1.2193 22 0.f»7 o.!!54 0.8*>*.9 7/ 17 r 1 6 h 44 1 5 h 14 h 16 CO k-C3 JO « 1 3 » -k_ CD CO 12 19 0 54 59 1 1 ioH 12 9 »-J O o .c to t-I I I I CO a o O O CO .a 3 Q. E X CO .c co 5 CD .c o o o c >> 5 a o CO CO CO " Z b b s T ^ S P P ^ ^ T ' f " 3 " ' 3 * 1 0 " ! 1 " ^ n U m b 6 r ° f l a t 6 r a l b a r S i n m a l e IN. nuDbsi. See F i g . II f o r an explanation of symbols. I I I 0 1 2 centimeters Figure XIV. Male N. hubbsi from four d i f f e r e n t regions: (a) the Ouinault R. drainage, (b) the Wishkah R. drainage, (c) trie Satsop R. drainage and (d) the Wynoochee R. drainage. Note that the specimens from west of the Montesano H i l l s ( a, b) have l e s s e r numbers of l a t e r a l bars than those f i s h c o l l e c t e d - 3 east of the Montesano H i l l s ( c , d). The Quinault R. male was captured at a close d h a b i t a t while a l l 5 0 others were obtained from open ones. 73 from each other at the p = 0.001 l e v e l , w i t h average bar counts of 16.59 and 15.29 bars, r e s p e c t i v e l y . This was contrasted by s i g n i f i c a n t l y lower bar means from c o l l e c t i o n s made elsewhere. The Taholah sample mean t e s t e d as l e s s than a l l other mean bar counts at the p = 0.001 l e v e l . C o p a l i s , Oyhut and Humptulips means were not s t a t i s t i c a l l y d i f f e r e n t i a t e . The three c o l l e c t i o n s had a combined mean of 12.51 bars. The Wishkah mean, 12.84 bars, was s i g n i f i c a n t l y greater than means from C o p a l i s , Oyhut and Humptulips f i s h at the p = 0.001 l e v e l . A west t o east i n c r ease i n bar numbers occurred from coastal l o c a l i t i e s t o the Wishkah s i t e , t h i s having been 2.43 bars i n magnitude. An abrupt increase i n mean counts was found between the Wishkah and Wynoochee s i t e s , almost 4 bars i n magnitude, although the two c o l l e c t i n g s i t e s had been i n c l o s e r proximity than were the o t h e r s , 16 km compared t o an average of approximately 26 km. Chromosome c h a r a c t e r i s t i c s No obvious d i f f e r e n c e s were observed i n comparisons between males and females or between populations i n chromosome numbers or morphology. A summary by l o c a t i o n of _N. hubbsi examined i n the chromosomal a n a l y s i s i s presented i n Table 31. A t o t a l of 84 males and 77 females were examined f o r chromosome numbers but only 32 males and 30 females were used i n s o r t i n g and q u a n t i f y i n g m i t o t i c spreads. In these l a t t e r cases, 3 karyotypes per i n d i v i d u a l were evaluated, w i t h care having been taken t o use e q u a l l y as well as not too contracted chromosomes. Both males and females were examined from a l l c o l l e c t i n g s i t e s , w i t h two exceptions. Only males were c o l l e c t e d at Hanaford Creek and only one female from the Ju n c t i o n C i t y l o c a l i t y was karyotyped. 74 Table 31. The categories of N. hubbsi used i n the chromosome a n a l y s i s . "Number examined" r e f e r s t o tRbse f i s h from which karyotypes were obtained; "number q u a n t i f i e d " r e f e r s t o that subset of the "number examined" f o r which 3 sorted karyotypes were measured. Males Females Location Number exami ned Number q u a n t i f i e d Number exami ned Numbe r quanti f i e d Tahol ah 5 3 5 3 Oyhut 21 12 20 8 Humptuli ps 6 2 5 2 Wi shkah 20 4 17 6 Junction C i t y - - 1 1 Wynoochee 8 3 5 3 Satsop 20 4 20 5 Black River 2 2 4 2 Hanaford Creek 2 2 - -Total 84 32 77 30 75 The frequency d i s t r i b u t i o n of chromosome numbers i n g i l l e p i t h e l i a l c e l l s f rom _N. hubbsi c o l l e c t e d at d i f f e r e n t s i t e s i s presented i n Table 32. The modal d i p l o i d count was 48 chromosomes. Counts greater than 48 chromosomes were probably the r e s u l t s of m i t o t i c n o n d i s j u n c t i o n , and chromosome breakage or separation as well as chromosomes o r i g i n a t i n g from other m i t o t i c c e l l s ; hypomodal counts were l i k e l y a consequence of chromosome l o s s during sample p r e p a r a t i o n . C e n t r i c fusions and f i s s i o n s have been found responsible f o r an i n t e r t i s s u e chromosomal polymorphism i n the salmonid, S_. i r i d e u s Gibbons (Ohno et a l . , 1965). Most st u d i e s on f i s h chromosomes have i n v o l v e d only somatic chromosome spreads. The polymorphism i n ^. i r i d e u s i n d i c a t e s t h a t f u s i o n s and f i s s i o n s could be responsible f o r d i f f e r e n c e s i n chromosome number between somatic and gonadal c e l l s f o r other f i s h e s . Since i n t r a i n d i v i d u a l polymorphisms would be detectable as d i f f e r e n c e s i n chromosome number, somatic d i p l o i d counts should be compared w i t h m e i o t i c ones to determine whether somatic chromosome spreads are r e p r e s e n t a t i v e of gonadal ones. Metaphase II chromosome sets from t e s t e s c o n s i s t e n t l y y i e l d e d 24 chromosomes while metaphase I spreads had 48 ( F i g . 15). The frequency d i s t r i b u t i o n of m i t o t i c counts f o r c e l l s from f i n , g i l l , i n t e s t i n e and kidney are presented i n Table 33. Since the modal d i p l o i d number was 48 chromosomes f o r a l l t i s s u e s and t h i s number was the same as observed f o r Metaphase I t e s t i c u l a r c e l l s as well as two times that seen i n metaphase II c e l l s , _N. hubbsi was considered i n v a r i a t e regarding i n t r a i n d i v i d u a ! polymorphisms i n chromosome number. M i t o t i c c e l l s from d i f f e r e n t t i s s u e sources could have been interchanged f o r my a n a l y s i s , but only g i l l e p i t h e l i u m was used i n the remainder of t h i s a n a l y s i s . A summary of chromosome morphometries i s given i n Table 34. A 76 Table 32. Frequency d i s t r i b u t i o n of d i p l o i d chromosome numbers as obtained from c e l l s i n d i f f e r e n t t i s s u e s of _N. hubbsi. The r e s u l t s of f i n , i n t e s t i n e , and kidney were combined from the a n a l y s i s of 1 male and 1 female from each of the f o l l o w i n g l o c a t i o n s : Oyhut, Wynoochee and Satsop. The r e s u l t s f o r g i l l i n c l u d e those ta b u l a t e d i n Table 33. Number of chromosomes per m i t o t i c spread Tissue 40 41 42 43 44 45 46 47 48 49 50 F i n 1 2 0 1 0 1 0 5 29 0 0 G i l l 3 1 4 1 7 8 10 24 113 4 2 I n t e s t i ne 0 1 0 2 1 1 1 2 18 1 0 Kidney 0 1 0 2 3 1 3 3 28 0 1 Total 4 5 4 6 11 11 14 34 188 5 3 ~7~J Figure XV. Metaphase chromosome spreads from N. hubbsi: (a) second spermatogonia! d i v i s i o n showing 24 b i v a l e n t s , l b ) m i t o t i c complement from g i l l e p i t h e l i u m with 48 chromosomes and (c) m i t o t i c complement from kidney t i s s u e with 48 chromosomes. 10/Lim 79 Table 33. Frequency d i s t r i b u t i o n of chromosome counts from the g i l l e p i t h e l i u m of _N. hubbsi. Eleven spreads c o n t a i n i n g l e s s than 40 chromosomes are not presented i n t h i s t a b l e . Note the negative skew of the d i s t r i b u t i o n as well as the 48 chromosome mode i n a l l cases. Location Number of chromosomes per m i t o t i c spread 40 41 42 43 44 45 46 47 48 49 50 51 Satsop 1 0 0 1 2 2 1 7 20 1 0 1 Wynoochee 0 0 1 0 0 2 3 5 18 0 1 0 Wi shkah 0 1 0 0 2 1 3 8 39 2 1 0 Oyhut 1 0 0 0 1 0 2 0 9 0 0 1 Taholah 0 0 1 0 1 1 0 3 12 0 0 0 Black River 0 0 0 0 1 2 1 1 15 1 0 0 A l l s i t e s 1 0 2 1 7 8 10 24 113 4 2 2 80 Table 34. Morphometries of the N. hubbsi chromosome complement based on 186 chromosome spreads obtained Tro'm 32 males and 30 females. R e l a t i v e length Arm r a t i o P a i r (0/0 of d i p l o i d set) /short arm x 1 Q Q \ 1ong arm No. Mean S.D. Mean S.D. 1 6.8 0.4 59.5 10.4 2 6.0 0.4 60.3 6.3 3 5.5 0.4 44.6 9.6 4 5.2 0.3 44.0 8.7 5 5.1 0.3 53.0 8.5 6 4.8 0.3 66.2 7.6 7 4.6 0.3 56.3 5.9 8 4.4 0.3 48.8 8.6 9 4.3 0.2 53.8 7.6 10 4.1 0.2 63.8 9.3 11 3.9 0.3 55.1 9.9 12 3.7 0.4 40.6 12.8 13 3.3 0.3 41.9 10.2 14 3.1 0.3 42.4 10.6 15 4.6 0.5 14.4 12.7 16 4.1 0.5 12.8 12.0 17 3.8 0.3 0 0 18 3.6 0.2 0 0 19 3.4 0.2 0 0 20 3.3 0.2 0 0 21 3.2 0.2 0 0 22 3.1 0.2 0 0 23 2.8 0.3 0 0 24 2.7 0.2 0 0 81 sorted karyotype i s presented i n Figure XVI, followed by a diagrammatic ideogram i n Figure XVII. The d i p l o i d spread was s o r t a b l e i n t o three chromosomal groups. Chromosomes i n which mean arm r a t i o s were submedian or subterminal made up the f i r s t group, designated chromosome p a i r s 1-14. The second one c o n s i s t e d of terminal region chromosomes, p a i r s 15 and 16, while the t h i r d category contained terminal sensu s t r i c t o ones, p a i r s 17-24. Within each group the chromosomes were ranked by decreasing mean r e l a t i v e l e n g ths. Chromosomes 1 and 2 were u s u a l l y i d e n t i f i a b l e without s o r t i n g or measuring ( F i g s . XV and X V I I I ) . Chromosomes 15 and/or 16 had very short l e s s e r arms although the l a t t e r sometimes appeared absent i n m i t o t i c spreads from the same i n d i v i d u a l ( F i g . XVI). When l e s s e r arms were present, these chromosomes could a l s o be discerned without s o r t i n g or measuring. The remaining chromosomes required some q u a n t i f i c a t i o n f o r proper assignment. Terminal sensu s t r i c t o chromosomes were r e a d i l y i d e n t i f i e d as a group. Short or l e s s e r arms were never seen i n chromosome p a i r s 17-24, even when chromosome spreads were very elongate ( F i g . 19). For the most p a r t , a s s i g n a t i o n of chromosomes was not hard, si n c e chromosome arms had f e a t u r e s , euchromatic and heterochromatic r e g i o n s , that helped the p a i r i n g and s o r t i n g process. Measurements of chromosomes f u r t h e r aided i d e n t i f i c a t i o n . The number of chromosome arms i s r e f e r r e d t o as the fundamental number. This term o r i g i n a t e d w i t h the assumption that biarmed chromosomes arose from the f u s i o n of uniarmed ones (Matthey, 1953), but the r e a l i z a t i o n that other cytogenetic phenomena (e.g., i n v e r s i o n s , c e n t r i c t r a n s p o s i t i o n s ; see Jackson, 1975) may generate biarmed chromosomes has rendered the 'fundamental number' a d e s c r i p t i o n of Figure XVI. Sorted karyotypes of N. hubbsi: (a) from a male c o l l e c t e d a t Oyhut and (b) a female captured i n the Satsop River drainage. Both m i t o t i c complements were obtained from g i l l e p i t h e l i a l c e l l s . Note that chromosomes 15 and 16 do not have l e s s e r arms i n (a) but do have them i n ( b ) . The presence or absence of these l e s s e r arms does not represent as i n t e r s e x u a l o f i n t e r p o p u l a t i o n a l polymorphism. Rather, chromosome spreads from a s i n g l e i n d i v i d u a l can have e i t h e r s t a t e s . c -o _i o ro 4 -r — O cu 2 3 6 7 8 9 10 11 12 1 3 14 1 5 16 1 7 18 19 2 0 21 2 2 2 3 2 4 Chromosome Number Figure XVII. Idiogram of m i t o t i c metaphase chromosomes from N. hubbsi, based on the measurements o f arm r a t i o and r e l a t i v e l ength. This f i g u r e was derived from the q u a n t i f i c a t i o n of 186 chromosome spreads from 32 males and 30 females. O O %5 Figure XIIX. Metaphase chromosome spreads from the g i l l e p i t h e l i u m o f N. hubbsi captured at various l o c a t i o n s : (a) Quinault River male, (b) Oyhut female, (c) Humptulips River male, (d) Wishkah River female, (e) Hanaford Creek male and ( f ) Satsop River female (used to c o n s t r u c t the karyotype i n F i g . XVIb). Chromosomes 1 and 2 are r e l a t i v e l y easy to d i s c e r n i n unsorted chromosome spreads (e.g., those chromosomes denoted by arrows i n b, e and f ) . The chromosomes spreads i n c and d are enlarged more than the other ones. The spread obtained from the Wishkah River female (d) i s more contracted than i s the complement from the Humptulips River male ( c ) . Excessive c o n t r a c t i o n r e s u l t s i n an accompanying l o s s o f chromosomal d e t a i l s , with d i f f e r e n t chromosome pa i r s tending to be s i m i l a r i n the more contracted spreads. Such preparations were not used f o r chromosomal a n a l y s i s i n t h i s study. • 85a Figure XIX. A m i t o t i c spread comprised o f elongate chromosomes, obtained from a male N. hubbsi c o l l e c t e d i n the Wishkah R. drainage. Arrows point out some of those chromosomes appearing devoid of l e s s e r arms. These chromosomes are considered terminal sensu s t r i c t o using the terminology of levan et a l . (1964). Terminal sensu s t r i c t o chromosomes are assigned to p a i r s 17 - 24 i n sorted karyotypes ( F i g s . XVI and XV I I ) . 87 centromeric p o s i t i o n (White, 1978; Swanson et a l . , 1981). For _N. hubbsi, the fundamental number was ascer t a i n e d t o be 80 chromosomal arms per d i p l o i d karyotype. DNA q u a n t i f i c a t i o n Feulgen-DNA determinations were done on er y t h r o c y t e n u c l e i from f i v e male and f i v e female _N. hubbsi f o r each of four c o l l e c t i n g s i t e s (Table 35). A t - t e s t i n d i c a t e d no i n t e r s e x u a l d i f f e r e n c e s i n DNA values and an ANOVA revealed no i n t e r p o p u l a t i o n a l d i f f e r e n c e s (Table 36). Feulgen-DNA determinations were based on r a t i o s between the d e n s i t i e s of f i s h blood c e l l n u c l e i and those from chicken e r y t h r o c y t e s . Ratio data u s u a l l y f o l l o w s a binomial d i s t r i b u t i o n and should be transformed t o arc s i n e s f o r a near-normal d i s t r i b u t i o n (Zar, 1974). My data were converted t o arc s i n e s i n order to permit the above parametric analyses. The grand mean absolute Feulgen-DNA estimate f o r H. hubbsi was 1.68 pg/nucleus ± 0.02 (S.E.). Fluorometric assays r e s u l t e d i n absolute DNA estimates of 1.5 and 1.6 pg/nucleus (Table 35). These were 11 and 7% l e s s than the mean Feulgen-DNA estimate. Previous i n v e s t i g a t o r s (e.g., Miksche, 1971; Mizuno and Macgregor, 1974) have reported s i m i l a r d i s c r e p a n c i e s when comparing biochemical and microdensitometric DNA measurements. The Feulgen-DNA determination i s an assay on aldehydes of deoxyribose sugars (Sharma and Sharma, 1972) while the f l u o r o m e t r i c one i s a q u a n t i f i c a t i o n of purine n u c l e o t i d e s (Foster and Gurney, 1976). Differences between the Feulgen and f l u o r o m e t r i c r e s u l t s could have been because each of the two methods i n v o l v e d a d i f f e r e n t aspect of the DNA molecule. Erythrocytes could have been miscounted f o r the f l u o r o m e t r i c assay. Hinegardner 88 Table 35. Determinations of nuclear DNA contents i n the erythrocytes of H. hubbsi: (a) Feulgen-densitometry and (b) f l u o r o m e t r i c determinations f o r two females from the Satsop R. s i t e . Although only two f i s h were processed i n the f l u o r o m e t r i c assay, the r e s u l t a n t DNA estimates were considered c l o s e enough to the Feulgen ones t o a t t e s t t o the r e l i a b i l i t y of the l a t t e r . a. Feulgen-densitometry Rel a t i ve T Location _N DNA s.d. Oyhut males 5 0.665 0.070 females 5 0.672 0.057 combined 10 0.668 0.064 Wishkah males 5 0.668 0.058 females 5 0.674 0.066 combined 10 0.671 0.062 Wynoochee males 5 0.671 0.052 females 5 0.673 0.063 combined 10 0.672 0.058 Satsop males 5 0.673 0.042 females 5 0.670 0.051 combined 10 0.672 0.047 *DNA content r e l a t i v e t o chicken e r y t h r o c y t e s , which have 2.50 pg/nucleus ± 0.04 (S.E.). i . Grand mean f o r r e l a t i v e Feulgen-DNA = 0.671 + 0.017 (S.E.) i i . Mean absolute Feulgen-DNA estimate using the grand mean f o r r e l a t i v e Feulgen-DNA = 1.68 p g / d i p l o i d c e l l ± 0.02 (S.E.). b. Fluorometric determinations Specimen 1. 1150000 erythrocytes/mm 3 blood; 7.36 yg DNA f o r 4866000 eryth r o c y t e s i n sample Specimen 2. 985000 erythrocytes/mm 3 blood; 7.01 yg DNA f o r 3815000 erythrocytes i n sample Absolute DNA estimates = 1.5 and 1.6 pg/erythrocyte nucleus 89 Table 36. S t a t i s t i c a l analyses using the arc s i n square root transform of r e l a t i v e Feulgen-DNA values f o r the nuc l e i of N. hubbsi e r y t h r o c y t e s : (a) a n a l y s i s of variance t e s t i n g f o r d i f f e r e n c e s between populations and (b) t - t e s t comparing male and female nuclear DNA contents. Since no in t e r s e x u a l or i n t e r p o p u l a t i o n a l DNA d i f f e r e n c e s were found, a s i n g l e mean absolute DNA value, 1.68 peg/nucleus + 0.02 (S.E.) as derived i n Table 35, was considered r e p r e s e n t a t i v e of a l l _N. hubbsi. a. A n a l y s i s of variance f o r data i n Table 35a Source of V a r i a t i o n Sums of Squares df Mean Squares Total 180.23 39 Groups 4.58 3 1.53 Error 175.65 36 4.88 F = 0.31 F 0.05(1) 3,36 = 2.86; do not r e j e c t n u l l B a r t l e t t ' s t e s t f o r equal variance = 2.66 2 0.05,3 = 7.82; do not r e j e c t n u l l b. t - t e s t f o r data i n Table 35a t = 0.28 t 0.05,38 = 2.02; do not r e j e c t n u l l F-max t e s t f o r equal variance = 1.17 F 0.025,19,19 = 2.53; do not r e j e c t n u l l (1968) found t h a t counting e r r o r s with a hemocytometer were great enough to s i g n i f i c a n t l y a f f e c t DNA estimates. Since f l u o r o m e t r i c measurements were not extremely d i f f e r e n t from the Feulgen-densitometric ones, the former were considered to be an i n d i c a t o r of the accuracy of the l a t t e r . 91 DISCUSSION The Junction C i t y c o l l e c t i n g s i t e The Junction C i t y c o l l e c t i n g s i t e was near the mouth of the Chehalis Ri v e r . Only two _N. hubbsi were captured at Junction C i t y a f t e r 150 trap nights (one b a i t e d t r a p submerged overnight = one t r a p night) as well as s e i n i n g and d i p n e t t i n g one day a month f o r f i v e months. Several of the _G. aculeatus males caught during the March - May 1979 c o l l e c t i n g t r i p s had red spawning c o l o r s . The presence of red morphs as well as water w i t h an a l k a l i n e pH and 2-4 ppt d i s s o l v e d s a l t s suggested a connection w i t h e s t u a r i n e Grays Harbor, about one kilometer t o the west. Meldrim (1968) showed that _N. hubbsi has a behavioral aversion t o d i s s o l v e d s a l t , suggesting a p h y s i o l o g i c a l i n t o l e r a n c e t o i t . Small numbers of _N. hubbsi may occur at Junction C i t y because of s a l i n e c o n d i t i o n s . The Junction C i t y s i t e was considered t o be marginal habitat f o r _N. hubbsi and p o s s i b l y an outer l i m i t of d i s t r i b u t i o n . Conditions s i m i l a r t o those at Junction C i t y may be responsible f o r the absence of_N. hubbsi elsewhere. McPhail (1967) and Meldrim (1968) have suggested that s u i t a b l e Novumbra ha b i t a t s occurred along the southern shore of Grays Harbor immediately southwest of the Junction C i t y c o l l e c t i n g s i t e , but gave no reasons f o r the seeming absence of _N. hubbsi t h e r e . Caged i n d i v i d u a l s survived several months i n swamps or marshes w i t h i n the region (Meldrim, 1968), i n d i c a t i n g that c o n d i t i o n s could be at l e a s t b r i e f l y s u i t a b l e f o r JN. hubbsi. The topography of streams and excessive s a l i n i t i e s could be 92 -r e s p o n s i b l e f o r the absence of t h i s f i s h . Streams t r a v e r s e steep gradients from the W i l l a p a H i l l s almost t o the shores of Grays Harbor (U. S. Geological Survey map: Hoquiam (1962): 1:250000 s c a l e ) , r e s t r i c t i n g l o t i c waters p r e f e r r e d by _N. hubbsi c l o s e t o the Grays Harbor estuary. S a l i n e i n t r u s i o n s i n t o these marshes or swamps because of t h e i r proximity t o a marine h a b i t a t may p r o h i b i t long-term establishment of_N. hubbsi. The Junction C i t y c o l l e c t i n g s i t e was suggested t o contain few H. hubbsi f o r the same reason. M e r i s t i c v a r i a t i o n A b i o t i c developmental f a c t o r s can cause m e r i s t i c v a r i a t i o n (Barlow, 1961; Lindsey and Harrington, 1972). Consequently, whether or not characters are under s t r i c t genetic c o n t r o l cannot be determined without c o n t r o l l e d l a b o r a t o r y s t u d i e s . Since _N. hubbsi i n h a b i t s a s p e c i f i c h a b i t a t w i t h i n a r e s t r i c t e d geographic area having a s i m i l a r c l i m a t e throughout, i n t e r p o p u l a t i o n a l m e r i s t i c v a r i a t i o n could have been mostly g e n e t i c a l l y - b a s e d . Climate could have a f f e c t e d m e r i s t i c character f r e q u e n c i e s . The h a b i t a t s of _N. hubbsi are r e g u l a r l y subjected t o summer d e s s i c a t i o n (Ruffner, 1980). M o r t a l i t y i n _N. hubbsi was noted at Oyhut and Satsop i n J u l y 1979, when hab i t a t s i z e s had been reduced and water chemistry and temperatures p o s s i b l y most s t r e s s f u l t o f i s h . Several dead f i s h were found i n dried-up pools and the remaining bodies of water had the lowest pH's recorded during the study, as low as 4.5, w i t h no detectable oxygen i n some. Higher summer water temperatures, e s p e c i a l l y at the Satsop c o l l e c t i n g s i t e , may have been m e t a b o l i c a l l y s t r e s s f u l . I n d i v i d u a l s w i t h c e r t a i n character s t a t e s could have died during such periods of 93 environmental s t r e s s , due t o e i t h e r poorer f i t n e s s or chance. The patterns i n the geographic d i s t r i b u t i o n of mean counts f o r l a t e r a l bars and anal f i n rays i n males as well as sca l e s above the l a t e r a l s c a l e row f o r males and females combined may be a t t r i b u t a b l e t o h i s t o r i c a l events. Character means were d i s t r i b u t e d i n a manner i n d i c a t i n g a b a r r i e r t o gene flow between the Wishkah and Wynoochee Ri v e r s . L a t e r a l bar means increased c l i n a l l y from coastal t o i n l a n d s i t e s w i t h the l a r g e s t d i f f e r e n c e i n mean counts between the Wishkah and Wynoochee c o l l e c t i n g s i t e . The magnitude of t h i s increase was approximately two times greater than any other nearest-neighbor d i f f e r e n c e . Anal f i n ray counts i n males had a d i s t r i b u t i o n i n which Wynoochee and Satsop specimens had lower mean counts than f i s h from more western s i t e s . A d i f f e r e n t pattern was seen w i t h s c a l e s above the l a t e r a l s c a l e row. Wishkah specimens and those from more western s i t e s had lower mean s c a l e counts than d i d Wynoochee and Satsop ones. The former s e r i e s of c o l l e c t i o n s d i d not d i f f e r among themselves, but t h e i r mean counts were l e s s than the s t a t i s t i c a l l y - e q u a l Wynoochee and Satsop ones. The Montesano H i l l s ( F i g . 1) c o n s i s t of several h i l l s , 200 - 400 m i n height, o r i g i n a t i n g i n the north as f o o t h i l l s of the Olympic Mountains and making up the east and west s l o p e s , r e s p e c t i v e l y , of the Wishkah and Wynoochee drainages. The Montesano H i l l s end abr u p t l y i n the south as b l u f f s , 30 - 40 m i n height, along the f i n a l kilometers of the Chehalis River before the l a t t e r enters Grays Harbor (personal o b s e r v a t i o n ; U. S. Geological Survey map: Aberdeen (1962), 1:62500 s c a l e ) . Along t h i s lower s e c t i o n of the r i v e r , the low r e l i e f t e r r a i n s u i t a b l e f o r Novumbra ha b i t a t s was scarce due t o the b l u f f s and l i t t l e or no s u i t a b l e 94 ' updrainage h a b i t a t appeared present because of the steep slope of the Montesano H i l l s (U. S. Geological Survey map: Aberdeen (1962); 1:62500 s c a l e ) . Such topography would i n h i b i t or prevent d i s p e r s a l of _N. hubbsi through the region. This f i s h was not captured more than 5 km from the mouths of the Wishkah and Wynoochee R i v e r s , although c o l l e c t i o n s were made f u r t h e r up the drainages. Conditions w i t h i n the Chehalis River may a l s o r e s t r i c t movements of _N. hubbsi between the Wishkah and Wynoochee drainages. Meldrim (1968) has documented the aversion by _N. hubbsi t o s a l i n e c o n d i t i o n s . S a l i n e waters from Grays Harbor extend u p r i v e r past the Montesano H i l l s , p o s s i b l y making the lower part of the r i v e r i n t o l e r a b l e t o _N. hubbsi. Beverage and Swecker (1969) reported 10 ppt d i s s o l v e d s a l t s i n Chehalis River water samples from about 6 km west of Montesano, at the southeast corner of the Montesano H i l l s , as well as 2 ppt s a l t f o r water samples at Montesano i t s e l f . Beverage and Swecker (1969) a l s o found t h a t water flows i n the Chehalis River annually f l u c t u a t e d between 20 and 75 m /sec, consider a b l y more than the n e g l i g i b l e water movement i n t y p i c a l Novumbra h a b i t a t s (Meldrim, 1968). Large p i s c i v o r o u s f i s h e s occur i n the Chehalis R i v e r (Wydoski and Whitney, 1979), these p o s s i b l y preying upon _N. hubbsi i f and when i t enters the system. The separation of the range of JN. hubbsi i n t o two g e o g r a p h i c a l l y -i s o l a t e d regions ( i . e . , c o a s t a l and Grays Harbor drainages; and the i n l a n d Chehalis River subdrainage) may have been i n existence s i n c e at l e a s t the P l e i s t o c e n e . S u i t a b l e habitat f o r _N. hubbsi seems t o have been l e s s l i k e l y w i t h i n the Wishkah and Wynoochee drainages, s i n c e a southern lobe of the Olympic G l a c i e r extended i n t o the northern Montesano H i l l s (Heusser, 1964). Ice melt from t h i s g l a c i a l lobe l i k e l y r e s u l t e d i n 95 greater stream flows and could be a reason f o r present streams being s i t u a t e d i n deep, narrow ravines (U. S. Geological Survey map: Aberdeen (1962); 1:62500). The Puget Lobe of the Vashon G l a c i e r extended south of the Juan de Fuca S t r a i t s and formed a peri g l a c i a l lake drained by the Chehalis River (McPhail, 1967; McKee, 1972). The r e s u l t a n t l a r g e r i v e r flow was f u r t h e r increased by drainage from ice-blocked Cascade Mountain v a l l e y s , making Chehalis River discharge greater than the present flow i n the Columbia River (McKee, 1972). I f ice-covered Montesano H i l l s blocked the d i s p e r s a l of _N. hubbsi during the P l e i s t o c e n e , east-west movements could only have occurred by means of the Chehalis R i v e r . However, flow rates were probably too f a s t f o r _N. hubbsi t o use the r i v e r as a passageway. As an aside t o t h i s study, s t a r c h gel e l e c t r o p h o r e s i s was done w i t h muscle e x t r a c t s from ten _N. hubbsi, f i v e from the Wishkah c o l l e c t i n g s i t e and f i v e from the Wynoochee one. Three of the four assayed enzymes migrated the same distance on the gel regardless of the geographic o r i g i n of the sample. However, i s o c i t r a t e dehydrogenase was found t o d i f f e r . Wishkah Novumbra were f i x e d f o r a gene product t h a t migrated f u r t h e r anodally than d i d i s o c i t r a t e dehydrogenase obtained from Wynoochee specimens. Although the sample s i z e was s m a l l , the f i x e d d i f f e r e n c e s i n i s o c i t r a t e dehydrogenase may have been i n d i c a t i v e of the faunal b a r r i e r i n d i c a t e d by the m e r i s t i c character a n a l y s i s . The r e s u l t s of analyses on the other m e r i s t i c characters d i d not c o n t r a d i c t the Montesano H i l l s being a faunal b a r r i e r . Pectoral f i n ray counts increased from western t o eastern s i t e s , p o s s i b l y because of gene flow and the r e s t r i c t i o n of predator-free h a b i t a t s t o coastal regions. Reductions i n the number of p e l v i c f i n rays were a l s o most common i n 96 c l o s e d h a b i t a t s . Lower f i n ray numbers could make a f i s h l e s s able t o escape from predators. Closed s i t e s were small i n comparison t o the open ones, and movements i n t o or from the former were r e s t r i c t e d . E f f e c t i v e population s i z e s i n closed h a b i t a t s may be small enough f o r chance events t o have a s i g n i f i c a n t e f f e c t . Reductions i n the numbers of p e l v i c and pectoral f i n rays could have been the r e s u l t of genetic d r i f t as proposed f o r the Death V a l l e y cyprinodonts by M i l l e r (1948). Sexual s e l e c t i o n may be strong i n closed systems due t o the small h a b i t a t s i z e s and the lack of predators. Male_N. hubbsi are t e r r i t o r i a l during the breeding season (Hagen et a l . , 1972). Spawning t e r r i t o r i e s i n the small areas comprising closed s i t e s could be at a premium, so any features enhancing the p r o b a b i l i t y of successful a c q u i s i t i o n and defense of good spawning s i t e s as well as a t t r a c t i o n of mates might be s t r o n g l y favored ( W i l l i a m s , 1975). Even i f c e r t a i n sexual character s t a t e s made f i s h more v i s i b l e t o predators, s e l e c t i o n f o r these characters would not have t o be compromised with a n t i - p r e d a t o r adaptation because of the absence of p i s c i v o r e s . Both male co u r t s h i p and male-male aggressive i n t e r a c t i o n s i n v o l v e expansion and f l u t t e r i n g of the dorsal f i n (Hagen et a l . , 1972). The highest modes and means f o r the number of dorsal f i n rays i n males were found i n males from close d s i t e s . Greater numbers of dorsal f i n rays may increase the expanse of the dorsal f i n making the l a t t e r more apparent t o c o n s p e c i f i c s . The lowest counts i n l a t e r a l bars were found i n males caught at c l o s e d s i t e s . A l e s s e r number of bars may be favored f o r male-male competition and female a t t r a c t i o n . Lesser l a t e r a l bar counts may have 97-been a s s o c i a t e d w i t h broader bar width but the l a t t e r was not measured. Lesser bar numbers, as well as greater numbers of dorsal f i n r a y s , could make male _N. hubbsi more conspicuous t o predators. If t h i s were t r u e , f i s h from open systems would be expected t o have greater numbers of l a t e r a l bars and l e s s e r numbers of dorsal f i n rays. A c l i n e e x i s t e d f o r the number of l a t e r a l bars, i n c r e a s i n g from the P a c i f i c coast t o i n l a n d areas. The higher mean dorsal f i n ray counts were i n specimens from the P a c i f i c coastal region. The sexual s e l e c t i o n hypothesis may be t e s t a b l e by means of mate choice and preference t e s t s (see McPhail, 1969). Females o r i g i n a t i n g from anywhere w i t h i n the range would be expected t o p r e f e r e n t i a l l y o r i e n t a t e towards or mate w i t h _N. hubbsi males having lower numbers of l a t e r a l bars and higher dorsal f i n ray counts. If t e r r i t o r i a l defense were a f f e c t e d by b a r r i n g or dorsal f i n ray s t a t e s , male-male encounters during the breeding season could be examined. Males w i t h lower bar counts but higher dorsal f i n ray counts would be expected t o be b e t t e r able to hold onto a s i t e . No geographic patterns of v a r i a t i o n were i n f e r r e d from the a n a l y s i s on anal f i n ray counts i n females, although Satsop specimens d i d have more ray s , on average, than d i d Humptulips ones. In males, anal f i n ray counts d i d not have the same geographic pattern as counts on dorsal f i n rays. The d i s t r i b u t i o n of anal f i n ray means was b e t t e r associated w i t h a g e o l o g i c a l b a r r i e r r a t h e r than open or c l o s e d h a b i t a t s suggesting that anal f i n rays may not be under the same (sexual) s e l e c t i v e regime as dorsal f i n rays. No s t a t i s t i c a l d i f f e r e n c e s were discerned f o r the geographic d i s t r i b u t i o n dorsal f i n ray means i n females; however, the d i s t r i b u t i o n of mean counts numerically approximated the pattern 9 8 -s t a t i s t i c a l l y - a s c e r t a i n e d f o r males. This r e s u l t could be expected i f males and females shared some of the genes f o r determining t h i s character s t a t e , w i t h s e x - l i m i t e d a l l e l e s r e sponsible f o r the dimorphism. Although both sexes might have had a s i m i l a r pattern of v a r i a t i o n , males would have expressed i t more s t r o n g l y . The only s i g n i f i c a n t d i f f e r e n c e s found i n the a n a l y s i s on caudal f i n ray means were Copalis and Conner Creek ones t e s t i n g as greater than those from Wynoochee or Satsop. Anal f i n ray counts d i f f e r e d between the Satsop and Copalis samples. The l a t e r a l bar and pectoral f i n ray means f o r Copalis f i s h were the same as those obtained f o r recent c o l l e c t i o n s from other coastal drainages. No s t a t i s t i c a l d i f f e r e n c e s i n the number of anal f i n rays i n males were found i n comparisons between Taholah, C o p a l i s , Conner Creek, and Oyhut. Whether or not character s t a t e s i n Copalis River and Conner Creek specimens were r e p r e s e n t a t i v e of current populations r e q u i r e s updated c o l l e c t i o n s . C opalis _N. hubbsi had been caught i n 1947, Conner Creek ones i n 1968. The Conner Creek swamp has sin c e been drained f o r r e s i d e n t i a l development (Whitney and Wydowski, 1979; personal observation) and the Copalis l o c a t i o n was not l i s t e d i n the U n i v e r s i t y of Washington records. Temporal changes i n m e r i s t i c s t a t e s are documented f o r other f i s h e s (e.g., Lindsey, 1953; Chernoff, 1982), so characters i n Co p a l i s and Conner Creek f i s h may have changed from the past t o the present. The pattern of geographic v a r i a t i o n i n the number of l a t e r a l row sc a l e s suggested by data i n Meldrim (1968) was not found i n my a n a l y s i s . P o s s i b l e reasons f o r the high means f o r s c a l e rows before the dorsal f i n o r i g i n and l a t e r a l row s c a l e s at the Wishkah c o l l e c t i n g s i t e are not 99 •' ' obvious. Perhaps there are f a c t o r s t o which developing eggs of _N. hubbsi are h y p e r s e n s i t i v e . Mean counts f o r both characters were about 10% higher than averages from elsewhere. Lindsey and Harrington (1972) have demonstrated s i m i l a r v e r t e b r a l and, to a l e s s e r e x tent, p e l v i c f i n ray v a r i a t i o n produced by d i f f e r e n t developmental temperatures. My p h y s i c a l and chemical determinations of the water at Wishkah were not designed to d e t a i l the aquatic h a b i t a t during egg development. At the l e v e l of r e s o l u t i o n i n t h i s study, c o n d i t i o n s at the Wishkah s i t e d i d not appear d i f f e r e n t from those i n waters elsewhere. Excepting the Wishkah c o l l e c t i o n , there was no geographic v a r i a t i o n i n s c a l e rows before the dorsal f i n o r i g i n . If the mean l a t e r a l row s c a l e count f o r Wishkah f i s h i s removed from the a n a l y s i s , remaining samples have t h e i r means d i s t r i b u t e d i n a manner parsimonious w i t h the Montesano H i l l s being a b a r r i e r t o gene flow. Wynoochee and Satsop f i s h had equivalent means. Counts were s t a t i s t i c a l l y - g r e a t e r than the r e s u l t s obtained from c o l l e c t i o n s made west of the Montesano H i l l s . These l a t t e r c o l l e c t i o n s had s t a t i s t i c a l l y - e q u a l means. Chromosomal s t a t e No geographic v a r i a t i o n s i n chromosome number or morphology were found. My work agreed w i t h that by Beamish et a l . (1971) regarding the d i p l o i d number, 48 chromosomes i n both s t u d i e s . My fundamental number f o r _N. hubbsi d i f f e r e d from the one by Gold et a l . (1980), 74 arms i n t h e i r report c o n t r a s t i n g w i t h 80 i n mine. Colchicine-induced d i f f e r e n c e s i n chromosomal condensation may have been responsible f o r the discrepancy i n fundamental number. Thompson (1979) considered t h i s t o be the case f o r arm number d i f f e r e n c e s between 100 , h i s k a r y o l o g i e s of c i chl i d f i s h e s and those produced by Ohno and A t k i n (1966). Excessive exposure t o c o l c h i c i n e can cause the smaller and/or more c o n t r a c t i l e l e s s e r arms t o become too small f o r r e s o l u t i o n w i t h a l i g h t microscope (Smith, 1965). Elongate chromosome spreads would, t h e r e f o r e , be e s s e n t i a l components of any cytogenetic study, s i n c e these would be the m i t o t i c or meiot i c sets l e a s t a f f e c t e d by a n t i m i t o t i c s . Gold et a l . (1980) counted chromosome arms i n the s i n g l e contracted m i t o t i c spread for_N. hubbsi i n Beamish et a l . (1971) t o obtain the fundamental number. My determination was derived from the enumeration of 186 elongate chromosome s e t s . The newer fundamental arm number does not r e s o l v e questions on umbrid or esocoid i n t e r r e l a t i o n s h i p s , these f i s h e s remaining a confusing chromosomally-diverse group as discussed by Beamish et a l . (1971). Heteromorphy of chromosomes 15 and 16 Chromosomes 15 and 16 were anomalous, appearing uniarmed i n some m i t o t i c spreads but biarmed i n others. The uniarmed and biarmed chromosomal s t a t e s were found i n d i f f e r e n t chromosome spreads obtained from the same f i s h . If l e s s e r arms were hi g h l y contracted then they may not have been r e s o l v a b l e under the l i g h t microscope. The long arms of biarmed chromosomes 15 and 16 f e l l w i t h i n s i z e ranges equivalent t o the r e l a t i v e lengths of terminal chromosomes 15 and 16, suggesting t h a t i n v e r s i o n or c e n t r i c t r a n s p o s i t i o n were not re s p o n s i b l e f o r the polymorphism. The polymorphism could have been caused by o r i e n t a t i o n s of these l e s s e r arms. White (1973) has argued that t r u e terminal chromosomes do not e x i s t s i n c e a l l experimentally-induced ones are unstable. There 101 could be fundamental d i f f e r e n c e s between natural and induced terminal chromosomes, but White (1973) stat e d that l e s s e r heterochromatic arms were always present but not n e c e s s a r i l y r e s o l v a b l e w i t h a l i g h t microscope. Since small l e s s e r arms c o n s i s t c h i e f l y of p a r a c e n t r i c heterochromatin, h i g h l y r e p e t i t i v e DNA sequences s i m i l a r l y repeated on the proximal region of the major arm (Swanson et a l . , 1981), the seeming absence of l e s s e r arms i n chromosomes 15 and 16 could have been the r e s u l t of DNA-DNA h y b r i d i z a t i o n . John and Freeman (1975) have observed that l e s s e r arms on terminal-appearing chromosomes were sometimes f o l d e d back onto the major arm during meiosis and m i t o s i s . I f DNA sequences on the l e s s e r arm were a l i g n i n g themselves w i t h s i m i l a r ones on the major arm, t h i s would be v i s u a l l y - o b s e r v a b l e as bac k f o l d i n g . Nuclear DNA Previous q u a n t i f i c a t i o n of nuclear DNA contents f o r _N. hubbsi as well as the other esocoids (Beamish et a l . , (1971) may be too high. Beamish et a l . (1971) used human buccal e p i t h e l i u m as the reference t o ery t h r o c y t e s from _N. hubbsi. Buccal e p i t h l i u m c o n s i s t s mostly of k e r a t i n i z e d dead c e l l s (Bloom and Fawcett, 1968) not n e c e s s a r i l y having the same DNA content. The n u c l e i of buccal e p i t h e l i a l c e l l s are k a r y o r r h e c t i c or p y c n o t i c , both s t a t e s i n v o l v i n g changes i n and loss e s of DNA (Pharr, 1976; Frankel et a l . , 1970). Nucleic a c i d contents would not n e c e s s a r i l y be the same between c e l l s as well as l e s s than i n l i v i n g c e l l s . Although Beamish et a l . (1971) purportedly followed the methods of Ohno and A t k i n (1966) f o r DNA q u a n t i f i c a t i o n , the c o n t r o l f o r these l a t t e r workers was t h y r o i d e p i t h e l i u m , which c o n s i s t s of l i v i n g c e l l s (Bloom and Fawcett, 1968) f o r which DNA determinations would be 102 r e p r e s e n t a t i v e of the d i p l o i d genome s i z e . Beamish et a l . (1971) c a l c u l a t e d absolute amounts of DNA by a s s i g n i n g t o t h e i r e p i t h e l i a l c e l l standard a n u c l e i c a c i d content averaged from an u n s p e c i f i e d l i s t of p l a c e n t a l mammals. It i s my understanding t h a t such l i s t s are u s u a l l y incomplete compilations i n c l u d i n g values without regard t o technique or accuracy (e.g., Shapiro, 1976); such compendia are meant t o be b i b l i o g r a p h i e s not trustworthy data s e t s . The absolute DNA value assigned t o human buccal e p i t h e l i a l c e l l s was about 14% more than the average per c e l l content e s t a b l i s h e d f o r humans (Swanson et a l . , 1981). Using t h i s value would have made the DNA estimates i n Beamish et a l . (1971) too high. Nucleated chicken er y t h r o c y t e s were chosen as reference c e l l s i n my study. The q u a n t i f i c a t i o n of DNA i n chicken erythrocytes has been repeated by several i n v e s t i g a t o r s w i t h l i t t l e v a r i a t i o n i n the determinations (averaging 2.50 p g / c e l l +_ 0.04 (S.E.), n = 7: Davidson et a l . , 1950; Mirsky and R i s , 1951; L e s l i e , 1955; Vendrely and Vendrely, 1956; Rasch et a l . , 1971), so they were a w e l l - s u i t e d standard f o r DNA comparisons in_N. hubbsi. Using a f l u o r o m e t r i c technique, Vendrely and Vendrely (1953) and Knobloch et a l . (1958) e s t a b l i s h e d 1.7 pg DNA/diploid nucleus f o r an esocoid, _E. l u c i u s . The DNA values from my study were i n c l o s e agreement w i t h t h i s determination p o s s i b l y i n d i c a t i n g the DNA estimate f o r H. hubbsi as having been accurate. The n u c l e i c a c i d q u a n t i f i c a t i o n s of Beamish et a l . (1971) were o f f e r e d e a r l i e r as supporting evidence f o r p o l y p i o i d a l o r i g i n of the salmonid genome. Revisions i n absolute DNA values i f one accepts r e l a t i v e DNA amounts from Beamish et a l . (1971) obscure t h i s n o t i o n . 103 Using my absolute Feulgen-DNA estimate f o r _N. hubbsi, the D a l l i a - Novumbra-Esox group would have 26-37% of the nuclear DNA found i n the c e l l s of salmonids rather than approximately 50%. F l u c t u a t i n g environmental regimes may s e l e c t f o r d i f f e r e n t h e r i t a b l e DNA contents (Durrant, 1962), with more extreme or v a r i a b l e s i t u a t i o n s a s s o c i a t e d w i t h higher n u c l e i c a c i d amounts. For example, a c l i n e f o r nuclear DNA has been reported i n the gymnosperm, Picea s i t c h e n s i s , i n c r e a s i n g from northern C a l i f o r n i a t o the Kenai Peninsula of Alaska (Miksche, 1971). Perhaps no i n t e r p o p u l a t i o n a l DNA d i f f e r e n c e s were found in_N. hubbsi s i n c e t h i s f i s h does not encounter a s i g n i f i c a n t amount of environmental heterogeneity; i t occurs i n a very p a r t i c u l a r h a b i t a t w i t h i n a region having a s i m i l a r c l i m a t e throughout. General d i s c u s s i o n Although v a r i a t i o n i n some external m e r i s t i c characters was considerable between populations of _N. hubbsi, obvious d i f f e r e n c e s were not found i n chromosome morphology and number as well as nuclear DNA content. Congruence i n the patterns of morphological and chromosome v a r i a b i l i t y w i t h i n a species has been documented f o r i n s e c t s ( A t c h l e y , 1981). Among f i s h e s , a l l o p a t r i c morphotypes of the p o e c i l i i d , Gambusia  a f f i n i s , have been found w i t h d i f f e r e n t chromosome morphologies (Black and Howell, 1979), yet _N. hubbsi from e i t h e r s i d e of the Montesano H i l l s appeared a l i k e regarding nuclear parameters. S i m i l a r t o _N. hubbsi, cyprinodontids of the genus Cyprinodon have been c h a r a c t e r i z e d by a l a c k of obvious chromosomal d i f f e r e n c e s but considerable morphological d i f f e r e n t i a t i o n (Turner and L i u , 1977; Stevenson, 1981). N. hubbsi i s found w i t h i n a temperate r a i n f o r e s t , and the general 104 region i n which t h i s animal occurs has l i k e l y contained r a i n f o r e s t s i n c e the e a r l y Eocene (Wolfe, 1981). The John Day deposit i n Oregon from which f o s s i l Novumbra have been recovered had a r a i n f o r e s t o r i g i n (Cavender, 1969), suggesting t h a t t h i s f i s h has e x i s t e d w i t h i n the same environment s i n c e at l e a s t the Oligocene epoch. Conservatism i n chromosome morphology and DNA content may be a consequence of Novumbra having p e r s i s t e d i n the same environment f o r several m i l l i o n y ears. One outcome of t h i s may have been the e v o l u t i o n of an optimum karyotype and DNA content. With f i t n e s s regarding chromosomal s t a t e and DNA content maximized, f u r t h e r e v o l u t i o n a r y changes would occur predominately through means other than a l t e r a t i o n s i n genome s i z e and r e l a t i v e gene p o s i t i o n (Bickham and Baker, 1979). The primary amino a c i d sequence of a p r o t e i n i s determined by a s i n g l e gene and some d i f f e r e n c e s i n t h i s sequence may be detected by e l e c t r o p h o r e s i s . Genie, or e l e c t r o p h o r e t i c , characters in_N. hubbsi were only examined as an aside. The p i l o t e l e c t r o p h o r e t i c study suggested geographic v a r i a t i o n i n i s o c i t r a t e dehydrogenase, but f u r t h e r comments on genie characters should not be made u n t i l the completion of s u b s t a n t i a l e l e c t r o p h o r e t i c analyses. Morphological characters may vary due t o d i f f e r e n c e s i n t h e i r r e g u l a t o r y c o n t r o l (Bickham and Baker, 1979). External morphological d i f f e r e n c e s can be extensive between populations w i t h s i m i l a r karyotypes (Swanson et a l . , 1981) and would be an expected outcome of geographic i s o l a t i o n and s e l e c t i o n f a v o r i n g d i f f e r e n t morphological s t a t e s i n d i f f e r e n t places. External morphological d i f f e r e n t i a t i o n i n _N. hubbsi occurred as i n f e r r e d from my data, i n response t o the presence or absence of predators as well as a l l o p a t r y due t o the Montesano H i l l s . 105 Chromosomal d i f f e r e n c e s w i t h i n _N. hubbsi may e x i s t but t h e i r d e t e c t i o n could be beyond the r e s o l u t i o n of the Giemsa-staining technique. For example, p a r a c e n t r i c i n v e r s i o n s would have been undetectable no matter how l a r g e they were. Inadequate methodology, t h e r e f o r e , may have been p a r t l y r e s p o n s i b l e f o r the perceived chromosome conservatism. Intrachromosomal s t a i n i n g techniques may have been more s u i t a b l e f o r a n a l y z i n g chromosomal v a r i a t i o n . Such techniques f o r f i s h e s have had a slow development ( B l a x h a l l , 1975), u n l i k e the advances i n intrachromosomal r e s o l u t i o n achieved with chromosome spreads from higher vertebrates (Hsu, 1973). G-banding would have been most appropriate s i n c e almost every chromosome could be unambiguously i d e n t i f i e d and chromosomal rearrangements traced (Bickham and Baker, 1976). Attempts t o G-band f i s h chromosomes have been unsuccessful so f a r (Denton, 1973; Abe and Muramoto, 1974; Kligerman and Bloom, 1976; Thorgaard, 1978; Hafez, 1979; Gregory et a l . , 1980). Another way t o uncover chromosomal d i f f e r e n c e s i n _N. hubbsi may be t o examine m e i o t i c b i v a l e n t s i n hybrids between f i s h from d i f f e r e n t p o p u l a t i o n s ; the only m e i o t i c spreads examined i n my study were from wild-caught males f o r the purpose of e s t a b l i s h i n g haploid chromosome numbers. 106 SUMMARY AND CONCLUSIONS 1. N^ . hubbsi has considerable geographic v a r i a t i o n i n external m e r i s t i c characters caused by a l l o p a t r y , d i f f e r e n t i a l predation pressure, and sexual s e l e c t i o n . 2. The geographic d i s t r i b u t i o n o f mean counts f o r l a t e r a l bars and anal f i n rays i n males as well as sca l e s above the l a t e r a l s c a l e row f o r males and females combined i n d i c a t e t h a t the Montesano H i l l s , those f o o t h i l l s o f the Olympic Mountains separating the Wishkah River and Wynoochee River drainages, are a b a r r i e r to gene flow. The Montesano H i l l s may have been such a b a r r i e r s i n c e a t l e a s t the P l e i s t o c e n e , s i n c e a lobe of the Olympic G l a c i e r extended i n t o t h i s region during t h i s g e o l o g i c a l epoch. 3. The r e s u l t s regarding l a t e r a l bar and dorsal f i n ray numbers may be a consequence o f sexual s e l e c t i o n i n the absence o f predators. Lower numbers of l a t e r a l bars and higher dorsal f i n ray counts i n males were as s o c i a t e d with small predator-free h a b i t a t s . These character trends may be important f o r mate a t t r a c t i o n and male-male aggressive i n t e r -a c t i o n s , s i n c e dorsal f i n expansion and l a t e r a l d i s p l a y s are part o f the behavioral r e p e r t o i r e during breeding and t e r r r i t o r i a l defense. 4. The la c k o f predators and small h a b i t a t s i z e may e x p l a i n the tendency f o r pectoral and p e l v i c f i n ray numbers to be reduced i n small closed s i t e s . Genetic d r i f t could be i n c r e a s i n g the frequency of f i s h with l e s s e r numbers o f paired f i n rays. A l s o , the absence o f predators may r e l a x s e l e c t i o n f o r greater numbers of f i n rays i f the l a t t e r were important f o r swimming speed and, hence, escape. 107 4. The high counts on l a t e r a l row sca l e s and s c a l e rows before the dorsal f i n o r i g i n i n f i s h from the Wishkah River drainage may be due, i n p a r t , to a b i o t i c developmental f a c t o r s . Since mean counts are about 10% greater than those from elsewhere, f u r t h e r i n v e s t i g a t i o n s are necessary i n order to p r o v i s i o n a l l y e x p l a i n the r e s u l t s i n a more s a t i f a c t o r y manner. 5. N_. hubbsi appears to be i n v a r i a t e i n t e r s e x u a l l y and geogra-p h i c a l l y with respect to chromosome number and gross morphology. Like the r e s u l t s obtained by Beamish et a l . (1971), 48 chromosomes comprise the d i p l o i d number. The fundamental number determined i n t h i s study i s 80 chromosome arms, c o n t r a s t i n g with 74 arms reported i n Gold et a l . (1980). The present study e n t a i l e d measurements on 186 elongate chro-mosome sets instead o f the e v a l u a t i o n o f fewer contracted ones. 6. No d i f f e r e n c e s appear present i n t e r s e x u a l l y or ge o g r a p h i c a l l y regarding DNA content. The Feulgen-DNA estimate f o r erythrocyte n u c l e i i s about 1.68 pg/nucleus, about 80% of a p r e v i o u s l y - p u b l i s h e d value. This discrepancy may be a t t r i b u t e d to improper c o n t r o l c e l l s and absolute DNA reference values being used i n the previous work. Since the Feulgen determination i s not very d i f f e r e n t from my f l u o r o m e t r i c ones as well as cl o s e to values a s c e r t a i n e d f o r another esocoid, the DNA estimate f o r H. hubbsi i s considered accurate. 7. The observed conservatism i n chromosomal s t a t e and DNA content could be a consequence o f N. hubbsi having p e r s i s t e d i n a r a i n f o r e s t environment f o r several m i l l i o n y e a r s , perhaps s i n c e a t l e a s t the Oligocene. An optimum karyotype and DNA content may have evolved. 108 LITERATURE CITED Abe, S., and J . Muramoto. 1974. D i f f e r e n t i a l s t a i n i n g of chromosomes of two salmonid s p e c i e s , S a l v e l i n u s leucomaenis ( P a l l a s ) and S a l v e l i n u s  malma (Walbaum). Proc. Jpn. Acad. 50:507-511. A t c h l e y , W. R. 1981. Chromosomal e v o l u t i o n and morphometric v a r i a b i l i t y i n the thelytokous i n s e c t Warramaba v i r g a (Key). Pages 371-397 U}_ W. R. Atchley and D. S. Woodruff, eds. E v o l u t i o n and S p e c i a t i o n . Essays i n honor of M. J . D. White. Cambridge U n i v e r s i t y Press, Cambridge. Barlow, G. W. 1961. Causes and s i g n i f i c a n c e of morphological v a r i a t i o n i n f i s h e s . Syst. Zool. 10:105-117. Beamish, R. J . , M. J . M e r r i l e e s , and E. J . Crossman. 1971. Karyotypes and DNA values f o r members of the suborder Esocoidei (Osteichthyes: Salmoniformes). Chromosoma ( B e r l . ) 34:436-447. B e r t o l l o , L. A. C , C. S. Takahashi, and 0. M. Fi 1 ho. 1979. Karyotypic s t u d i e s of two a l l o p a t r i c populations of the genus Hoplias ( P i s c e s , E r y t h r i n i d a e ) . Rev. Bras. Genet. I I 1:17-37. Beverage, J . P., and M. N. Swecker. 1969. Estuarine s t u d i e s i n upper Grays Harbor, Washington. U. S. Geological Survey Water-Supply Paper 1873-B, 40 p. Bickham, J . W., and R. J . Baker. 1976. Chromosome homology and e v o l u t i o n of emydid t u r t l e s . Chromosoma ( B e r l . ) 54:201-219. Bickham, J . W., and R. J . Baker. 1979. C a n a l i z a t i o n model of chromosomal e v o l u t i o n . B u l l . Carnegie Mus. Nat. H i s t . No. 13:70-84. Black, A. and W. M. Howell. 1978. A d i s t i n c t i v e chromosomal race of the cyprinodontid f i s h , Fundulus notatus, from the Upper Tombigbee River system of Alabama and M i s s i s s i p p i . Copeia 1978:280-288. Black, D. A. and W. M. Howell. 1979. The North American m o s q u i t o f i s h , Gambusia a f f i n i s : a unique case i n sex chromosome e v o l u t i o n . Copeia 1979:509-513. B l a x h a l l , P. C. 1975. F i s h chromosome techniques - a review of s e l e c t e d l i t e r a t u r e . J . F i s h B i o l . 7:315-320. Brown, M. B. and A. B. Forsythe. 1974. Robust t e s t s f o r the e q u a l i t y of var i a n c e s . J . Amer. S t a t i s t . Assoc. 69:364-367. Bush, G. L. 1975. Modes of animal s p e c i a t i o n . Annu. Rev. E c o l . Syst. 6:339-364. Bloom, W. and D. W. Fawcett. 1968. A Textbook of H i s t o l o g y . 9th ed. Saunders, P h i l a d e l p h i a (Pa.). 109 Bush, G. L., S. M. Case, A. C. Wilson and J . L. Patton. 1977. Rapid s p e c i a t i o n and chromosomal e v o l u t i o n i n mammals. Proc. Nat. Acad. S c i . USA 74:3942-3946. Cavender, T. 1969. An Oligocene mudminnow (f a m i l y Umbridae) from Oregon with remarks on r e l a t i o n s h i p s w i t h i n the E s o c o i d e i . Occ. Pap. Mus. Zool. Univ. Mich., No. 660:1-33. Chernoff, B. 1982. Character v a r i a t i o n among populations and the a n a l y s i s of biogeography. Am. Zool. 22:425-439. D a r l i n g t o n , P. J . , J r . 1957. Zoogeography. The geographical d i s t r i b u t i o n of animals. Wiley, New York (N.Y.). Davidson, J . N., I. L e s l i e , R. M. S. S m e l l i e , and R. Y. Thomson. 1950. Chemical changes i n the developing chick embryo r e l a t e d t o the d e o x y r i b o n u c l e i c a c i d content of the nucleus. Biochem. J . 46, Proc. XI. D e i t c h , A. D., D. Wagner, and R. M. R i c h a r t . 1968. Conditions i n f l u e n c i n g the i n t e n s i t y of the Feulgen r e a c t i o n . J . Histochem. Cytochem. 16:371-379. Denton, T. E. 1973. F i s h Chromosome Methodology. Charles C. Thomas, S p r i n g f i e l d (111.). Dixon, W. J . , M. B. Brown, L. Engelman, J . W. Frane, M. A. H i l l , R. I. J e n n r i c h , and J . D. Toporek, eds. 1981. BMDP S t a t i s t i c a l Software 1981. U n i v e r s i t y of C a l i f o r n i a Press, Berkeley ( C a l i f . ) Durrant, A. 1962. The environmental i n d u c t i o n of h e r i t a b l e changes i n Li num. Heredity 17:27-61. Endl e r , J . A. 1977. Geographic V a r i a t i o n , S p e c i a t i o n and C l i n e s . Princeton U n i v e r s i t y Press, Princeton (N.J.). F i t z g e r a l d , J . W. 1957. Range extension f o r the western mudminnow, Novumbra S c h u l t z . Copeia 1957:248. F o s t e r , D. N. and T. Gurney, J r . 1976. Nuclear l o c a t i o n of mammalian DNA polymerase a c t i v i t i e s . J . B i o l . Chem. 251:7893-7898. Franz, R. and D. S. Lee. 1976. A r e l i c t population of the mottled s c u l p i n , Cottus b a i r d i , from the Maryland coastal p l a i n . Chesapeake S c i . 17:301-302. Gold, J . R., W. J . K a r e l , and M. R. Strand. 1980. Chromosome formulae of North American f i s h e s . Progressive F i s h - c u l t u r i s t 42:10-23. Gregory, P. A., P. N. Howard-Peebles, R. D. E l l e n d e r and B. J . M a r t i n . 1980. Analysis of a marine f i s h c e l l l i n e from a male sheepshead. J . Hered. 71:209-211. 110 Hafez, R. 1979. Analyse du caryotype de l a tanche (Tinea t i n e a L.) par 1'obtention des bandes C et G. Cymbium 3e s e r i e 7:15-26. Hagen, D. W., and G. E. E. Moodie. 1979. Polymorphism f o r breeding c o l o r s i n Gasterosteus aculeatus . I . Their genetics and geographic d i s t r i b u t i o n . E v o l u t i o n 33:641-648. Hagen, D. W., G. E. E. Moodie and P. F. Moodie. 1972. T e r r i t o r i a l i t y and c o u r t s h i p i n the Olympic mudminnow (Novumbra hubbsi). Can. J . Zool. 50:1111-1115. Hagen, D. W., G. E. E. Moodie and P. F. Moodie. 1980. Polymorphism f o r breeding c o l o r s i n Gasterosteus aculeatus. I I . Reproductive success as a r e s u l t of convergence f o r t h r e a t d i s p l a y . E v o l u t i o n 34:1050-1059. Heusser, C. J. 1964. Palynology o f four bog sections from the western Olympic Peninsula, Washington. Ecology 45:23-40. Hinegardner, R. T. 1968. E v o l u t i o n o f c e l l u l a r DNA content i n t e l e o s t f i s h e s . Am. Nat. 102:517-523. Hinegardner, R. T. 1971. An improved f l u o r o m e t r i c assay f o r DNA. Anal. Biochem. 39:197-201. Hoese, H. D. 1963. S a l t t olerance of the eastern mudminnow, Umbra  pygmaea. Copeia 1963:165-166. Hsu, T. C. 1973. Longitudinal d i f f e r e n t i a t i o n of chromosomes. Annu. Rev. Genet. 7:153-176. Hubbs, C. L., and K. F. Lagler. 1964. Fishes o f the Great Lakes Region. U n i v e r s i t y of.Michigan Press, Ann Arbor ( M i . ) . Hutchinson, G. E. 1957. A T r e a t i s e on Limnology. V o l . I . Wiley, New York (N. Y.). John, B., and M. Freeman. 1975. Causes and consequences of Robertsonian exchange. Chromosoma ( B e r l . ) 52:123-130. Johnson, M. S., and M. F. Mickevieh. 1977. V a r i a b i l i t y and e v o l u t i o n a r y rates o f c h a r a c t e r s . E v o l u t i o n 31:642-648. Kligerman, A. D., and S. E. Bloom. 1976. D i s t r i b u t i o n o f F-bodies, heterochromatin, and n u c l e o l a r organizers i n the genome of the c e n t r a l mudminnow, Umbra 1i m i . Cytogenet. C e l l Genet. 18:182-196. Kligerman, A. D., and S. E. Bloom. 1977. Rapid chromosome preparations from s o l i d t i s s u e s o f f i s h e s . J . F i s h . Res. Board. Can. 34:2166-2169. Knobloch, A., C. Vendrely, and R. Vendrely. 1957. The amount of desoxyribonucleic a c i d i n a s i n g l e t r o u t sperm. Biochim. Biophys. Acta 24:201-202. ' 111 LeGrande, W. H., and T. M. Cavender. 1980. The chromosome complement of the stonecat madtom, Noturus f l a v u s ( ?Sil u r i formes: I c t a l u r i d a e ) , with evidence f o r the existence of a p o s s i b l e chromosomal race. Copeia 1980:341-344. Lelek, A. 1980. Threatened freshwater f i s h e s of Europe. Council of Europe (Strasbourg), Nature and Environment S e r i e s , No. 18:269p. L e s l i e , I. 1955. The n u c l e i c a c i d content of t i s s u e s and c e l l s . Pages 1-50 ijn E. Chargaff and J . N. Davidson, eds. The Nucleic A c i d s . Vo l . 2. Academic Press, New York (N.Y.). Lindsey, C. C. 1953. V a r i a t i o n i n anal ray count of the redside shiner Richardsonius balteatus (Richardson). Can. J. Zool. 31:211-225. Lindsey, C. C , and R. W. H a r r i n g t o n , J r . 1972. Extreme v e r t e b r a l v a r i a t i o n induced by temperature i n a homozygous clone of the s e l f - f e r t i l i z i n g c y p r i n o d o n t i d f i s h Rivulus marmoratus. Can. J . Zool . 50:733-744. Matthey, R. 1953. Les chromosomes des Muridae. Revision c r i t i q u e et materiaux nouveaux pour s e r v i r a l ' h i s t o i r e de l ' e v o l u t i o n chromosomique chez ces Rongeurs. Rev. S u i s s e Zool. 50:225-283. McLeish, J . , and N. Sunderland. 1961. Measurement o f deoxyribosenucleic a c i d (DNA) i n higher plants by Feulgen photometry and chemical methods. Exp. C e l l Res. 24:527-540. McKee, B. 1972. Cascadia. The geologic e v o l u t i o n of the P a c i f i c Northwest. McGraw-Hill, New York (N.Y.). McPhail, J . D. 1967. D i s t r i b u t i o n of freshwater f i s h e s i n western Washington. Northwest S c i . 41:1-11. McPhail, J . D. 1969. Predation and the e v o l u t i o n of a s t i c k l e b a c k (Gasterosteus). J . F i s h . Res. Board Can. 26:3183-3208 Meldrim, J. W'. 1968. The e c o l o g i c a l zoogeography of the Olympic mudminnow (Novumbra hubbsi, S c h u l t z 1929). Ph.D. D i s s e r t a t i o n , U n i v e r s i t y of Washington, S e a t t l e . Miksche, J. P. 1971. I n t r a s p e c i f i c v a r i a t i o n of DNA per c e l l between Picea s i t c h e n s i s (Bong.) Carr. Provenances. Chromosoma ( B e r l . ) 32:343-352. M i l l e r , R. R. 1948. The cyprinodont f i s h e s of the Death V a l l e y system of eastern C a l i f o r n i a and southwestern Nevada. Misc. Publ. Mus. Zool. Univ. Mich. 68:1-155. Mirsky, A. E., and H. R i s . 1949. V a r i a b l e and constant components of chromosomes. Nature (London) 163:666-667. 112 M i r s k y , A. E. and H. R i s . 1951. The desoxyribonucleic a c i d content of animals and i t s e v o l u t i o n a r y s i g n i f i c a n c e . J . Gen. P h y s i o l . 34:451-462. Mizuno, S. and H. C. Macgregor. 1974. Chromosomes, DNA sequences, and e v o l u t i o n i n salamanders of the genus Plethodon. Chromosoma ( B e r l . ) 48:239-296. Moodie, G. E. E. and T. E. Reimchen. 1976. Phenetic v a r i a t i o n and habitat d i f f e r e n c e s i n Gasterosteus populations of the Queen C h a r l o t t e i s l a n d s . Syst. Zool. 25:49-61. Murgatroyd, L. B. 1968. A q u a n t i t a t i v e i n v e s t i g a t i o n i n t o the e f f e c t of f i x a t i o n temperature and a c i d s t r e n g t h upon the Feulgen r e a c t i o n . J . Roy. Microscop. Soc. 88:133-144. Myers, G. S. 1951. Fresh-water f i s h e s and East Indian zoogeography. Stanford I c h t h y o l . B u l l . 4:11-21. Naiman, R. J . and D. L. S o l t z , eds. 1981. Fishes i n North American Deserts. Wiley, New York (N.Y.). Nelson, G. J . and N. P l a t n i c k . 1981. Systematics and Biogeography: c l a d i s t i e s and v i c a r i a n c e . Columbia U n i v e r s i t y Press, New York (N.Y.). N i k o l s k y , G. 1976. The i n t e r r e l a t i o n between v a r i a b i l i t y of c h a r a c t e r s , e f f e c t i v e n e s s of energy u t i l i z a t i o n , and karyotype s t r u c t u r e i n f i s h e s . E v o l u t i o n 30:180-185. N i k o l s k y , G., and V. P. V a s i l y e v . 1973. On some r e g u l a r i t i e s i n the d i s t r i b u t i o n of chromosome numbers i n f i s h . J . I c h t y o l . (Eng. T r a n s l . Vopr. I k h t i o l . ) 13:1-19. Ohno, S. and N. B A t k i n . 1966. Comparative DNA values and chromosome complements of eight species of f i s h e s . Chromosoma ( B e r l . ) 18:456-466. Ohno, S., C. S t e n i u s , E. F a i s s t , and M. T. Zenzes. 1965. P o s t - z y g o t i c chromosomal rearrangements i n rainbow t r o u t (Salmo i r i d e u s , Gibbons). Cytogenetics 4:117-129. Ostdiek, J . L. and R. M. Nardone. 1959. Studies on the Alaskan b l a c k f i s h P a l l i a pectoral i s I. H a b i t a t , s i z e and stomach analyses. Am. M i d i . Nat. 61:218-229. Peckham, R. S. and C. F. Dineen. 1957. Ecology of the c e n t r a l mudminnow Umbia 1 imi ( K i r t l a n d ) . Am. M i d i . Nat. 58:222-231. Pharr, S. L. 1970. I d e n t i f i c a t i o n of benign and malignant c e l l s . Pages 996-1003 i n S. F r a n k e l , S. Reitman and A. C. Sonnerwirth, eds. Gradwohl's C l i n i c a l Laboratory Methods and Diagnosis. 7th Ed. V o l . I. C. V. Mosby, Saint Louis (Mo.). 113 P i s t e r , E. P. 1981. The conservation of desert f i s h e s . Pages 411-446 i n R. J . Naiman and D. L. S o l t z , eds. Fishes i n North American Deserts. Wiley, New York (N.Y.). Rasch, E. M., H. J . Barr and R. W. Rasch. 1971. The DNA content of sperm of Drosophila melanogaster. Chromosoma ( B e r l . ) 33:1-18. Ruffner, J . A. 1980. Climates of the S t a t e s , National Oceanic and Atmospheric A d m i n i s t r a t i o n n a r r a t i v e summaries, t a b l e s , and maps f o r each s t a t e w i t h overview of s t a t e c l i m a t o l o g i s t programs. 2nd ed. V o l . 2. Gale Research, D e t r o i t ( M i . ) . S c h u l t z , L. P. 1929a. D e s c r i p t i o n of a new type of mud-minnow from western Washington, with notes on r e l a t e d s p e c i e s . Univ. Wash. Publ. F i s h 2:73-81. S c h u l t z , L. P. 1929b. The discovery of a remarkable type of mud-minnow i n western Washington. J . Pan-Pac. Res. Inst. 4:12-16. S c o t t , W. B. and E. J . Crossman. 1973. Freshwater f i s h e s of Canada. B u l l . F i s h . Res. Board Can. 184: 966 pp. S e z a k i , K. and H. Kobayasi. 1978. Comparison of e r y t h r o c y t e s i z e between d i p l o i d and t e t r a p l o i d i n spinous l o a c h , C o b i t i s biwae. B u l l . Jpn. Soc. S c i . F i s h 44:851-854. Sezaki, K., H. Kobayasi, and M. Nakamura. 1977. Size of erythrocytes i n the d i p l o i d and t r i p i o i d specimens of Carassius auratus  1 a n g s d o r f i . Jpn. J . I c h t h y o l . 24:135-140. Shapiro, H. S. 1976. Deoxyribonucleic a c i d content per c e l l of various organisms. Pages 284-311 j_n_ G. E. Fasman, ed. Handbook of Biochemistry and Molecular B i o l o g y . 3rd ed. Nucleic a c i d s . V o l . I I . CRC Press, Cleveland (Oh.). Sharma, A. K. and A. Sharma. 1972. Chromosome Techniques. Theory and p r a c t i c e . U n i v e r s i t y Park Press, Baltimore (Md.) Sherwood, S. W. and J . L. Patton. 1982. Genome e v o l u t i o n i n pocket gophers (genus Thomomys). I I . V a r i a t i o n i n c e l l u l a r DNA content. Chromosoma ( B e r l . ) 85:163-179. Sokal, R. R. and F. J . Rohlf. 1969. Biometry. The p r i n c i p l e s and p r a c t i c e of s t a t i s t i c s i n b i o l o g i c a l research. W. H. Freeman, San Francisco (Ca.). Stevenson, M. M. 1981. Karyomorphology of several species of Cyprinodon. Copeia 1981: 494-498. Subrahmanyan, K. 1969. A k a r y o t y p i c study of the e s t u a r i n e f i s h Boleopthalmus boddareti ( P a l l a s ) w i t h calcium treatment. Curr. S c i . (Bangalore) 38:437-439. 114 Swanson, C. P., T. Merz and W. J . Young. 1981. Cytogenetics. The chromosome i n d i v i s i o n , i n h e r i t a n c e and e v o l u t i o n . 2nd ed. P r e n t i c e - H a l l , Englewood C l i f f s (N.J.). Thompson, K. W. 1979. Cytotaxonomy of 41 species of n e o t r o p i c a l C i c h l i d a e . Copeia 1979:679-691. Thorgaard, G. H. 1978. Chromosome rearrangements and sex chromosomes i n the rainbow t r o u t and sockeye salmon. Ph.D. d i s s e r t a t i o n , U n i v e r s i t y of Washington, S e a t t l e . Trautman, M. B. 1981. The Fishes of Ohio. Ohio State U n i v e r s i t y Press, Columbus (Oh.). Turner, B. J . and R. K. L i u . 1977. Extensive i n t e r s p e c i f i c genetic c o m p a t i b i l i t y i n the New World k i l l i f i s h genus Cyprinodon. Copeia 1977:259-269. Vahs, W. 1973. Die Bedeutung der Hydrolyse-Art i n der Feulgen-Cytophotometrie von Kernen mit u n t e r s c h i e d l i c h e n P l o i d i e g r a d e n . Histochemie 33:341-348. Vendrely, R. 1953. Arginine and deoxyribonucleic a c i d content of e r y t h r o c y t e n u c l e i and sperms of some species of f i s h e s . Nature (London) 172:30-31. Vendrely, R. and C. Vendrely. 1956. The r e s u l t s of cytophotometry i n the study of the deoxyribonucleic a c i d (DNA) content of the nucleus. I n t . Rev. C y t o l . 5:171-197. White, M. J . D. 1973. Animal Cytology and E v o l u t i o n . 3rd ed. Cambridge U n i v e r s i t y , London. White, M. J . D. 1978. Modes of S p e c i a t i o n . W. H. Freeman, San Francisco (Ca.). Wilson, A. C , G. L. Bush, S. M. Case and M.-C. King. 1975. S o c i a l s t r u c t u r i n g of mammalian populations and rate of chromosomal e v o l u t i o n . Proc. Nat. Acad. S c i . USA 72:5061-5065. Wydoski, R. S. and R. R. Whitney. 1979. Inland Fishes of Washington. U n i v e r s i t y of Washington Press, S e a t t l e (Wa.). W i l l i a m s , G. C. 1975. Sex and E v o l u t i o n . Princeton U n i v e r s i t y Press, Princeton (N.J.). Wolf, K. 1963. P h y s i o l o g i c a l s a l i n e s f o r fresh-water t e l e o s t s . Progressive F i s h - c u l t u r i s t 25:135-140. 115 Wolfe, J . A. 1981. A chronologic framework f o r Cenozoic megafossil f l o r a s of northwestern North America and i t s r e l a t i o n t o marine geochronology. Pages 39-47 j_n J . M. Armentrout, ed. P a c i f i c Northwest Cenozoic B i o s t r a t i g r a p h y . Geol. Soc. Am. Spec. Pap. No. 184. Zar, J . H. 1974. B i o s t a t i s t i c a l A n a l y s i s . P r e n t i c e - H a l l , Englewood C l i f f s , (N.J.). Zenzes, M. T., and I. V o i c u l e s c u . 1976. C-banding patterns i n Sal mo  t r u t t a , a species of t e t r a p l o i d o r i g i n . Genetica 45:531-536. 

Cite

Citation Scheme:

        

Citations by CSL (citeproc-js)

Usage Statistics

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            data-media="{[{embed.selectedMedia}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
https://iiif.library.ubc.ca/presentation/dsp.831.1-0095677/manifest

Comment

Related Items