FLUVIAL MOUNTAIN WHITEFISH ( williamsoni) IN THE UPPER FRASER RIVER: A MORPHOLOGICAL, BEHAVIOURAL, AND GENETIC COMPARISON OF FORAGING FORMS.

PETER M. TROFFE

B.Sc, University of British Columbia, 1994

A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE IN THE FACULTY OF GRADUATE STUDIES (Department of Zoology)

We accept the thesis as conforming to the required standard

THE UNIVERSITY OF BRITISH COLUMBIA April 2000

© Peter M. Troffe, 2000 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission.

Department of

The University of British Columbia Vancouver, Canada

DE-6 (2/88) Abstract:

Members of the family Coregonidae are notoriously plastic in their morphology and life histories, but in British Columbia there is little evidence of the kind of variation in trophic structures seen elsewhere in North America and Europe. There is, however, one exception — the mountain whitefish, Prosopium williamsoni. Museum, and field collections of fluvial mountain whitefish from the upper Peace, Columbia, and Fraser river systems commonly contain two sympatry phenotypes of fluvial mountain whitefish.

One form (the most common) is characterized by a short blunt snout while, the other

form has a long slightly upturned snout. I refer to this latter from as the 'pinocchio' form.

Individuals with the pinocchio nose are not confined to British Columbia but are also known from isolated populations in Utah, the upper Missouri system, and the Olympic

Peninsula in Washington State.

The phenotypes from the Upper Fraser system differ in morphological features

usually associated with trophic adaptations including gill raker counts and cranial

architecture. Furthermore, the forms exhibit different foraging behaviours in sympatry,

suggesting they occupy different foraging niches.

A mitochondrial DNA survey reveals that pinocchio and normal mountain

whitefish from Upper Fraser River tributaries have significantly different haplotype

frequency distributions. The nature of the haplotype variation suggests either

asymmetrical reproductive isolation — with normal males avoiding pinocchio females —

or strong selection against hybrid progeny. iii

Table of Contents

Abstract..... ii List of Figures iv List of Tables v Acknowledgements vi

1.0 CHAPTER I - Introduction 1

2.0 CHAPTER II - Nature of the variation 2.1 Methods 6 2.11 Characters 7 2.12 Assumptions and Size Adjustment 8 2.13 Distribution of residuals 9 2.14 Multivariate Analysis 9 2.15 Gill rakers 10 2.2 Results 11 2.21 Character residual distribution 11 2.22 Multivariate Analysis 11 2.23 Gill rakers 12

2.3 Morphometric survey (conclusions) 14

3.0 CHAPTER III - Genetic survey 3.1 Methods 15 3.11 DNA extraction 15 3.12 Mitochondrial DNA analysis 16 3.2 Results 19 3.3 Genetic survey (conclusions) 20

4.0 CHAPTER IV - Foraging observations 4.1 Methods 21 4.11 Statistical analysis 22 4.2 Results 24 4.3 Foraging observations (conclusions) 27

5.0 CHPATER V - General Discussion 28

Literature cited 57 Appendix 1 65 Appendix II 68 List of Figures:

Figure 1: Distribution of mountain whitefish in North America 38

Figure 2: Head profiles of normal and pinocchio mountain whitefish phenotypes 39

Figure 3: Upper Fraser River, including Prince George, BC 40

Figure 4: Head vs. standard length regression residuals, Swift River 41

Figure 5: Maxillary vs. standard length regression residuals, Swift River 42

Figure 6: Snout vs. standard length regression residuals, Swift River 43

Figure 7: Distribution of head vs standard length residuals, Swift River 44

Figure 8 Distribution of maxillary vs standard length residuals, Swift River 45

Figure 9: Distribution of snout vs standard length residuals, Swift River 46

Figure 10: Scatter plot of Swift River cranial character principal component analysis....47

Figure 11: Hierarchical cluster analysis of Swift River principal component values 48

Figure 12: Scatter plot of combined drainage principal component analysis 49

Figure 13: Hierarchical cluster analysis of combined drainage PCA 50

Figure 14: Gill raker counts from Swift River mountain whitefish phenotypes 51

Figure 15: Gill raker counts from four upper Fraser River tributaries 52

Figure 16: Mitochondrial DNA haplotype arranged by life history and collection sites...53

Figure 17: Mitochondrial DNA haplotypes arranged by phenotype 54

Figure 18: Foraging preferences of mountain whitefish phenotypes in Dome Creek 55

Figure 19: Foraging rates of mountain whitefish phenotypes in Dome Creek 56 V

List of Tables:

Table 1: Upper Fraser River mountain whitefish included in morphometric survey 34

Table 2: Upper Fraser River mountain whitefish included in gill raker survey 34

Table 3: Comparison of gill raker counts of mountain whitefish phenotypes 34

Table 4: ANOVA -gill raker counts among drainages and phenotypes 35

Table 5: Tissue samples used in mitochondrial DNA analysis 35

Table 6: Distribution of mountain whitefish mitochondrial DNA haplotypes 36

Table 7: Mitochondrial DNA haplotypes arranged by mountain whitefish phenotype.. ..36

Table 8: Foraging preferences of mountain whitefish phenotypes (Top site) 37

Table 9: Foraging preferences of mountain whitefish phenotypes (Bottom site) 37 vi

Acknowledgements:

I would like to thank all of my committee members for their patience over the last four years. My advisor Dr. J.D. McPhail sowed the seeds for this thesis and continued to be very generous with his time, and vast scientific experience. Dr. E.B. Taylor provided all aspects of assistance in the genetics lab. He was able to convince me that I should " put down the quill pen, and jump into the world of molecular markers".

Many thanks to Dr. Peter Mylecreest and Ian Kusabs for their help in the sometimes-trying field conditions. Peter provided this study with excellent photographs and he was instrumental in the collection of many mountain whitefish specimens. Dr.

CC. Lindsey was generous with his whitefish experiences during his luncheon rounds at the UBC ichthyology collections.

Additional tissue samples from outside the Fraser Basin were provided by James

Baxter, David O'Brien, and E.B. Taylor. The maps of mountain whitefish distribution in

North America and mountain whitefish head diagrams are after Diana McPhail's drawings. The Natural History Curators at the Royal B.C. Museum provided valuable support during the final writing. CHAPTER I

1.0 Introduction:

The study of phenotypic, genetic and behavioural variation is a fundamental

component of evolutionary biology. Although virtually any aspect of an organism's

phenotype can vary, morphological variation often is the most conspicuous and easily

assayed form of variation. In North Temperate freshwater , intraspecific variation in

morphology is especially noticeable and well documented. Individuals within a

population of the same size and sex normally vary in their body proportions and meristic

counts, but in some cases, the phenotypic variation is strikingly discontinuous. This latter

situation is the subject of this thesis.

Among freshwater fish, discontinuous variation in structures associated with

foraging are common (e.g. Robinson and Wilson 1996; Meyer 1989; Liem 1973) and

found in a wide range of phyletic groups distributed throughout both the northern and

southern hemispheres (Ruzzante at al. 1998; Taylor and McPhail 1999; Schluter 1996;

Northcote 1988). Most examples of discontinuous morphological variations involve

structures associated with foraging ecology such as gill rakers, mouth size, cranial

architecture and dentition (Galis and Drucker 1996; Skulason and Smith 1995; Taylor and

Bentzen 1993; Galat and Vicinich 1983). In some cases where discontinuous

morphological variation is observed there are clear genetic or inherited differences

between two or more sympatric forms (e.g. Skulason at al. 1993, 1989; Taylor and

Bentzen 1993; Ferguson and Mason 1981). In other cases, the variation is present in the

absence of detectable genetic differences and variations in morphology are thought to 2 represent examples of environmentally induced phenotypic plasticity (Meyer 1989; Liem

1973; Hindar and Jonsson 1992; Jonsson and Hindar 1982).

One group of fish in which the presence of different trophic morphs is especially common in are the whitefish (Coregonidae). Coregonids are a variable, Holarctic family

of freshwater and anadromous fish that are distributed primarily in glaciated areas.

Whitefish are notoriously variable in their morphology and life histories and, so far, have eluded the attempts of taxonomists to establish clear species boundaries within the family

(Svardson 1979; Lindsey 1981; Bernatchez at al.. 1999). In British Columbia there appear to be ten species of whitefish but, surprisingly, there is little evidence of the kind

of discontinuous variation in trophic structures seen elsewhere in North America and

Eurasia. There is, however, one noticeable exception — the mountain whitefish,

Prosopium williamsdni.

Prosopium differs from other genera of whitefish in that they are adapted primarily for life in rivers rather than lakes, and they reach their highest diversity in western North

America (Norden 1970; McCart 1970). Among the North American species of Prosopium the mountain whitefish has the widest geographic distribution and occupies a variety of aquatic habitats — large silty rivers, small clear headwater streams, and small to large lakes. Mountain whitefish are distributed along the eastslope of the Sierra in

California and Nevada, they are present in internal drainage systems in Utah, Idaho and

Oregon, and are widespread in the Columbia, Fraser, and Skeena systems as well as in rivers draining the eastslope of the Rocky Mountains in Wyoming, Montana and Alberta

(Fig. 1). They also occur in the Mackenzie system (Athabasca, Peace and Liard drainages) and extend down the mainstem Mackenzie River at least as far as its confluence with the 3

Bear River. Throughout this extensive geographic range the mountain whitefish is an interior species and only approaches the coast in a few large rivers that have cut through the coastal mountains (e.g., the Fraser, Skeena, and Stikine rivers). The species is absent from Vancouver Island and from rivers that rise in the coastal mountains. Again, however, there is an exception: mountain whitefish are abundant in the short coastal rivers that drain the west slope of the Olympic Peninsula (Fig. 1). The significance of the

Olympic populations, and the presence of isolated populations in arid areas like eastern

California and Utah, is that they establish that mountain whitefish likely survived glaciation in more than one glacial refugium (McPhail and Lindsey 1970, 1986). Thus,

British Columbia may have been colonized postglacially by more than one isolated or genetic form of mountain whitefish.

Relative to trout and salmon the life history of this species is not well known; however, it is clear that fluvial populations perform complex reproductive, overwintering, and feeding migrations (Davies and Thompson 1976; Northcote and Ennis 1994; McPhail and Troffe 1998). In rivers, the diet of both juveniles and adults consists mostly of the larvae and nymphs of aquatic insects. Unlike trout, mountain whitefish rarely take terrestrial insects and appear to feed mainly on drift and benthic materials (McHugh

1940).

Observations of several fluvial mountain whitefish specimens from the upper

Peace, Columbia, and Fraser systems housed in the University of British Columbia ichthyology museum collection suggests the presence of phenotypes that differ in cranial architecture, most noticeably snout length. One form (the most common) is characterized by a short, rather blunt snout. I refer to this form as the "normal" form (Fig.2). The 4

alternate form, occurs at approximately a 20% frequency in the upper Fraser River

system, and has a long, slightly upturned snout (Fig. 2). The elongation of the snout

becomes progressively prominent with increasing specimen size, and I refer to this long

snout morph as the "pinocchio" form. Individuals with the elongate snout are not

confined to British Columbia but are also known from isolated populations in Utah

(Stalnaker at al. 1974), the upper Missouri system in Wyoming (Evermann 1892), and the

Olympic Peninsula in Washington (P. Mongillo, Washington Dept. of Fish and Game,

pers. comm.).

The function of the elongate snout of some fluvial mountain whitefish populations

is unknown and it is unclear if this characteristic is an inducible, polymorphic feature or if

it has a heritable genetic foundation (e.g. Meyer 1989; Lindsey 1981). Stalnaker at al.

(1974) suggested the long rostrum of some mountain whitefish is a male secondary sexual

characteristic. This seems unlikely since, in museum collections, the trait occurs in adults

of both sexes, and my field collections, made throughout the spring and summer

contained long snouted individuals of both sexes, even during the fall breeding season.

Thus, there is no evidence that the elongate snout is sexually dimorphic or associated

with reproduction. Also, there are adults that are intermediate between the two forms. The

first question I address in this thesis is — is there more than one morphological form of

fluvial mountain whitefish in the upper Fraser Basin (i.e. is the variation in head

morphology discontinuous or, represented by more than one clear group)? Given a positive answer to this question, I then asked if there are genetic differences between the

forms using a survey of mitochondrial DNA restriction fragment length polymorphisms.

Finally, because head shape and gill raker architecture differences suggest the possibility 5 of trophic differences, I address the question is there a relationship between phenotype and foraging behaviour? 6

CHAPTER II

2.0 Nature of the Variation:

The first question posed in the introduction relates to the distribution of the morphological variation between and within fluvial mountain whitefish populations in upper Fraser River Basin, British Columbia. Specifically, is there more than one distinct group of mountain whitefish based on cranial characters commonly associated with trophic foraging efficiency, or is the morphological variation in these traits uniformly distributed? Furthermore, if morphological differences are revealed among or within populations are there any differences in gill raker number among phenotypes?

2.1 Methods

The data for my morphometric survey of mountain whitefish cranial characters came from 84 mountain whitefish specimens from three upper Fraser River tributaries.

Adult specimens of similar standard lengths were used in the morphometric survey to minimize any potential differences in allometry (Table 1).

The first two data sets are museum specimens; the Wright Creek, and Swift River specimens were collected during June, 1955 and June, 1956, respectively (BC 55-355,

BC 56-344) and are currently housed within the University of British Columbia ichthyology museum. Wright Creek is a moderately slow, small clear water creek that meets the Salmon River near its confluence with the Fraser River, approximately 15 kilometeres north of Prince George, BC (Fig. 3). The Swift River is a fast water tributary of the Cottonwood River which has its confluence with the Fraser River approximately

80 kilometeres south of Prince George, BC (Fig 3). The presence of juvenile and adult 7

specimens in the Wright Creek, and Swift River collections (0+, 1+, 2+ and older)

suggest that mountain whitefish were using these reaches as both foraging and rearing

grounds during the 1955 and 1956 summer seasons.

The third morphometric data set included in this survey of fluvial mountain whitefish cranial characters comes from mountain whitefish I collected in the autumn of

1995 at the confluence of the Nechako and Fraser rivers at Prince George, BC (Fig 3).

Adult specimens were collected with 25-metere seine net sets at the rivers' confluence

during seasonal reproductive congregations. The 25 specimens were individually labeled

and preserved in 10% buffered formaldehyde solution for five days before they were

fixed in 50% isopropanol for final storage.

2.11 Characters:

Four characters were measured on each specimen: head length, maxillary length,

snout length and standard length. Straight-line measurements were conducted with digital

calipers accurate to the nearest 0.01 mm following the procedures of Hubbs and Lagler

(1947). When necessary, finer measurements were made under the resolution of a

dissecting microscope at three times magnification.

An estimate of measurement accuracy was made by remeasuring 25 Swift River

specimens and comparing both the individual and total variation to original measurements in a one-way-ANOVA (Haas and McPhail 1991). Measurement error was

low, with the variance ratio of original to the recollected measurements being close to one

(0.98), suggesting that the measurement procedure is repeatable. 2.12 Assumptions and Size Adjustment:

To be statistically robust multivariate morphometric analysis usually requires the number of specimens to exceed the number of characters used in the multivariate analysis

(Reist 1985; Sharp at al. 1978). A total of 84 mountain whitefish form the three drainages were found to be suitable for the morphometric analysis (i.e. they were not twisted or warped during preservation). The morphometric measurements were collected in ASCII format and analyzed with Systat Version 8. Bivariate regressions of were made with LOGio transformed data (Zar 1984; Humphries at al. 1981; Bookstein at al. 1985).

As indicated in the literature (Holt 1960; Scott and Crossman 1973) I assumed that the sex ratio of mountain whitefish is equal, stable, and that the species is not sexually dimorphic. There are colour and lateral tubercle differences between the sexes when they are in spawning condition, but these differences are slight and do not involve any of the characters I measured (Scott and Crossman 1973; McAfee 1966).

Despite attempts to obtain specimens of equal size for comparison, specimen sizes varied both within and among populations and character measurements were correlated with specimen size and required size adjustment. The morphometric data sets were univariately adjusted for differences in allometry by regressing each of the three cranial characters against individual specimen standard length and collecting deviations from the regression line as residuals (Reist 1985). Thus, differences among character measurements are expressed as deviations (residuals) from the linear relationship describing the overall body size relationship for the character set. The residual variation results from two sources; character measurement error due to the observer, and biological effects representing the deviations of individual specimens from the overall size relationship (Haas and McPhail 1991). Since assessment of measurement error was low, it can be assumed that the resulting residual variation represents a biological effect. The regression based morphometric technique of size adjustment is thought to be superior when compared to other univariate methods (e.g. Ratios - division of character by standard length) as it avoids problems resulting from non-linear relationships between the size adjusted and the original characters (Reist 1985; Haas 1988).

2.13 Distribution of residuals:

Measurements of snout, maxillary and head length were taken on 38 Swift River specimens. The character measurements were regressed against individual specimen standard length as outlined in the aforementioned section outlining character size adjustment. The distribution of variance surrounding the regression line (residuals) of each bivariate plot was then represented in scatter plots and histograms to determine if character residuals are uniformly or asymmetrically distributed about the mean regression line.

2.14 Multivariate Analysis:

Two separate variance-covariance principal component analysis (PCA) procedures were performed on the size corrected character matrices to determine if the cranial characters are represented by more than one morphometric group (Winans 1984;

Reist 1985; Haas 1988). The first PCA examined the 38 Swift River specimens, while the second analysis involved a combined 84 specimens from all three drainages (Table 1). 10

After collecting the PCA loading scores, a hierarchical cluster analysis using

Systat V. 8 was performed on first principal component loading scores (principle component expressing the majority of explained variance) from the Swift River and

Combined drainage PCA. The cluster analysis was used to determine if any homogenous groups could be assigned based on the measured characters in a method similar to Taylor and Bentzen (1993). Hierarchical cluster analysis begins with each individual case in a separate cluster and then combines clusters sequentially using a least squares method, reducing the number of cluster groups until the variance is minimized.

2.15 Gill rakers:

Gill raker counts were made after dissecting the left primary gill arch of 55 adult mountain whitefish from four upper Fraser tributaries (Table 2). The specimens were sorted into morphological groups as suggested by the initial PCA and cluster analysis before the counts were made. All gill raker counts were made under a dissecting microscope (3x) and counts include all rudimentary rakers (McPhail 1992). To test for effects of locality and phenotype, gill raker counts were compared against each other with a two-way-ANOVA. Morphotype comparisons within a single drainage were compared univariately with a two-tailed student's t-test (assuming equal variance). 2.2 Results

2.21 Character residual distribution:

The distribution of the head character residuals about the mean regression line

suggests there are two separate groups of mountain whitefish within the Swift River collection. Each of the regressions (snout, maxillary and head length) exhibited high squared multiple R-values (Maxillary =0.809, Snout =0.816, Head =0.941) suggesting the regressions against standard length were robust. The distribution of each residual data set is asymmetrical when plotted against specimen standard length. In each case positive residual values cluster together in a group while the negative values were more widely distributed (Figs. 4, 5, 6). Furthermore, frequency plots describing the distribution of the residuals for each of the three adjusted characters about the mean regression line are represented by strikingly disrupted distributions, with the lowest frequency counts occurring near the mean regression line (Figs. 7, 8, 9).

2.22 Multivariate Analysis:

The total explained variance in the Swift River PCA was 99%, with 94.8%, and

4.5% variance accounted for by the first and second principal components, respectively.

This high level of explained variance suggests that inference made from this analysis is robust (Winans 1984). The component loading values were highest for snout length

(0.973) followed by head length (0.960) and maxillary length (0.957). A scatter plot of principal component Factor 1 against Factor 2 suggests the data are represented by two distinct clusters with most of the separation being attributed to differences along the first principal component axis (Fig. 10). Hierarchical cluster analysis performed on the factor 1 principal component loading values separates the data into two distinct clusters (Fig.

11). The division between the two clusters occurs at the first branch of the cluster tree

(Euclidean distance = 0.7).

Scatter plots of the PCA loading including the combined specimens from Wright

Creek, and the Swift and Nechako Rivers are similar to the single drainage Swift River

PCA. The total explained variance in the combined drainage PCA was 99%, with 94.8%) and 5% variance accounted for by the first and second components respectively. Two clusters are represented in the PCA, separated by differences along the first principal component similar to the single drainage PCA (Fig. 12). Hierarchical cluster analysis performed on the first principal component loading values divides the specimens into two distinct clades at the first branch of the cluster (Euclidean distance = 0.35) (Fig. 13).

2.23 Gill raker counts:

Frequency plots of gill raker counts of pinocchio and normal fish from the Swift

River specimens, and data from combined drainages, reveal that individuals with long snouts have on average more gill rakers than sympatric short nosed individuals. The differences in gill raker number between pinocchio and normal fish are represented by a disrupted distribution rather than merely describing differences along both tails of a single normal distribution (Figs. 14, 15) (Lindsey 1981,1988).

Although there is variation among drainages, there are consistent differences in gill raker number between pinocchio and normal mountain whitefish (Figs. 14, 15). Two- tailed t-tests show that, regardless of drainage, pinocchio mountain whitefish on average 13 have a significantly higher (22.4 ± 0.27) gill raker count than sympatric normal whitefish

(20.1 ±0.3) (Table 3).

Independent of drainage designation, analysis of variance indicates consistent differences in gill raker counts between pinocchio and normal fish (Table 4). There are also significant differences in the mean gill raker count between drainages independent of mountain whitefish phenotype (Table 4). 14

2.3 Morphometric survey (conclusions)

The variation in cranial morphology and gill raker count suggests the presence of two distinct mountain whitefish morphotypes in the upper Fraser River, B.C. The distribution of the residuals from regressions of maxillary, head and snout lengths against

individual specimen standard length also indicates that there are two morphotypes of mountain whitefish. In all characters examined, the magnitudes of residuals above and below the regression line, was higher than along the mean regression line where they would be expected if the residuals were distributed normally. Hierarchical cluster analysis of PCA loading scores from a single, and three combined drainages confirms the results examining the character regression residuals by assigning mountain whitefish to one of two constructed clades.

Although there is variation in mountain whitefish gill raker number between upper Fraser Basin tributaries, there are consistently significant differences between the

long snout 'pinocchio' and blunt snout 'normal' morphotypes of mountain whitefish. In all four drainages where gill raker number was surveyed pinocchios had, higher gill raker counts than their normal snouted sympatric counterparts. The significance of these differences in gill raker architecture will be discussed below.

The first question posed in my introduction to this thesis was — Is there evidence of discontinuous morphometric variation in fluvial mountain whitefish populations in the

Upper Fraser system? Clearly, the answer to this question is — Yes. The fact that different phenotype can be maintained in sympatry suggests there could also be genetic and behavioural differences among upper Fraser River mountain whitefish populations. 15

CHAPTER III

3.0 Genetic survey:

The second question posed in my introduction concerns the genetic relationship between the morphotypes outlined in the previous chapter. This question was addressed by survey of a hypervariable portion of the mitochondrial DNA genome of mountain whitefish in the upper Fraser River and adjacent drainages.

3.1 Methods

In 1995, mountain whitefish tissue samples were collected from six upper Fraser tributaries, the Fraser mainstem, two drainages in the Peace system, and the Duncan River in the Columbia system. The tissue samples were obtained from both adult and juvenile fish collected by pole seine, beach seine, trapping, electroshocking and angling (Table 5).

Juvenile fish were slit ventrally and stored whole in 95% EtOH. Adult fish were sorted according to morphology, their adipose fins clipped, and the tissues stored in 95% EtOH.

An RFLP (restriction fragment length polymorphism) analysis was performed on the tissues samples to assess mitochondrial DNA variability among mountain whitefish in the upper Fraser Basin.

3.11 DNA Extraction:

Mitochondrial DNA was extracted using a modified version of the

Chloroform/Phenol procedure. Liver, heart, and peduncle muscle were dissected from preserved juvenile specimens. Whole adipose fins from adult specimens and 20-50 mg of tissue from juvenile specimens were blotted dry, and macerated before extraction.

Weighed tissue samples were digested overnight in a buffered Pronase solution. The 16

digestion temperature was 37° C. Samples that were poorly digested after 8-10 hours were reincubated and, in some cases, additional aliquots of Pronase were added until the

samples appeared completely digested.

Aliquots of RNAse were added to the fully digested tissue samples, and the

samples were then incubated at 37° C for one to three hours. Equal volumes of phenol

and chloroform were added to the digested samples. They were then centrifuged and the

aqueous phase removed in preparation for final DNA precipitation.

Cold isopropanol was added to the aqueous extractions, they were then gently mixed and held at -20° C for 20 minutes to maximize the yield of DNA precipitate. The precipitated samples were centrifuged, the isopropanol aspirated, and the DNA pellets washed for 1-3 hours in ice cold 70% lab grade EtOH. After alcohol washing the samples were re-centrifuged, the EtOH aspirated, and DNA pellets resuspended in 75-150 ul of

TE buffer (pH 8.0) and stored at -20 0 C.

The DNA concentration of the extracts was determined with a spectrophotometer.

A volume of 3ul of the resuspended DNA precipitate solution was diluted in 247p.l of 0.5

x TBE buffer (pH 8.0). The diluted suspension was compared to a control standard in a

Pharmacia spectrophotometer and DNA concentrations were calculated and recorded.

3.12 Mitochondrial DNA analysis:

Dilution trials and reaction conditions were standardized and refined for the

Polemerase Chain Reaction (PCR) amplification of the combined Cytochrome b, and D- loop portion of the mountain whitefish mtDNA genome. These portions of the 17 mitochondrial genome were surveyed because they are considered highly polymorphic and are easily assayed (Hall and Nawrocki 1995).

To define a standardized reaction condition that yielded consistent, high quality chain replicated products, trial PCR reactions were conducted at a variety of initial concentrations of DNA and PCR reagents. The primers used in the PCR reaction are after

Bernatchez and Osinov (1995); and 0.4 units of Taq Polymerases was used per 25 pi PCR reaction. The final concentrations of the standard reaction consisted of:

- O.lpg/ul of raw DNA extract - 0.8mM dNTP nucleotide mix - 0.6mM of HN20, CGlu (Primers A,B) - 2.0 p/ul Taq polymerase (added last) - 2.0mM Magnesium Chloride

The DNA extract and the PCR reagents were combined and the reaction conducted in a Robocycler unit via the hot start method. The mtDNA loci were chain replicated for 30 cycles with each of the denaturing, annealing, and extension phases continuing for 1.5 minutes at 95, 55, and 72° C, respectively. After 30 amplification cycles the samples were held in a single extension phase for 5 minutes before final storage at 6°C.

PCR products were qualified by staining aliquots of product with 1.5% Ethidium

Bromide/ loading buffer solution. The stained products were electophoretically separated by running them between 60-75 volts for 1 to 1.5 hours on a 1% agarose gel loaded with a

lkilobase (Kb) molecular weight standard ladder. The separated gel was placed under ultra-violet light and the fluorescent profiles photographed on Polaroid film. 18

As recommended by the vendor, (New England Biolabs) aliquots of high quality

PCR products were digested in individual vials with one multi-hexameric (Sty /restriction endonucleases. The digested fragments were combined with loading buffer, loaded into, and electrophoretically resolved on, 1.5 % agarose gels immersed in TBE buffer at between 75 and 90 volts for 2.5 to 3.5 hours. Each gel was run with a 1Kb ladder as a molecular weight standard. The separated gels were stained for 20 minutes in an ethidium bromide solution, washed in 0.5x TBE buffer for a further 20 minutes and finally illuminated with UV light for Polaroid photography.

A random selection of five samples were re-amplified via PCR, and re-digested to determine if there is any variability in the RFLP procedure. There were no detectable differences in the RFLP trials. This indicates the procedure is repeatable.

Distinct endonuclease genotype (haplotype) patterns were identified and given composite codes. In an attempt to understand the distribution of mountain whitefish mtDNA haplotype frequencies in the upper Fraser system the haplotype patterns were grouped according to their collection location and life-history stage (Table 6).

Contingency tests were conducted using the MONTE algorithm in the REAP statistical package to determine if there was any association between fish morphology and haplotype frequency (McElroy at al. 1992). Using a priori knowledge the contingency tests were blocked into two groups: a universal sample containing all pinocchio and normal mountain whitefish tissue collections (Bowron, Willow, McGregor and Nechako rivers), and a localized comparison from the Nechako River where fish of both morphotypes were collected at the same time and place. 19

3.2 Results:

The RFLP survey (PCR products cut with Styl) revealed three haplotype patterns.

All of the mountain whitefish populations examined from the Peace (Chowade and Burnt rivers), Columbia (Duncan River), and upper Fraser rivers possessed some mix of these mtDNA haplotypes, however, the Burnt and McGregor rivers appear to contain only patterns A and C (Table 6). Haplotypes A and B were found throughout the entire sampling area (Table 6). The frequencies of these two common haplotypes (A and B) are relatively homogeneous throughout the upper Fraser and account for 97% of the mtDNA variation in upper Fraser mountain whitefish populations (Fig. 16).

In the Nechako system, contingency tests indicate that there is a difference between the mtDNA haplotype frequencies found in pinocchio and normal whitefish (X

= 8, p<0.005). Samples from the Nechako River show that normals are exclusively haplotype A, while pinocchios possess both patterns (A and B) at about equal frequency

(Fig. 17; Table 7). This difference in haplotype frequencies between pinocchio and normals also occurs in the combined samples from the Bowron, Willow and Nechako rivers (Fig. 17; Table 7). The haplotype distribution in juvenile fish is similar to the distribution in adults suggesting that, although the individuals are too small to be identified by their phenotype, both pinocchio and normal haplotype patterns are present in juvenile samples (Fig. 16). 20

3.3 Genetic survey (conclusions):

The mitochondrial RFLP evidence implies that mountain whitefish populations in

British Columbia are not a genetically homogeneous group. The haplotypes present in the upper Fraser system appear to be shared amongst all the tributaries but the relative frequency of the haplotypes differs among localities. This suggests that there are some genetic differences among upper Fraser mountain whitefish populations.

The haplotypes, and their frequencies in the pinocchio and normal morphotypes, are significantly different. Haplotypes A and B are equally frequent in pinocchios; while normal fish are almost exclusively haplotype A and never haplotype B. The sample size is small (14 pinocchio and 15 normals); nevertheless these haplotype differences imply that the pinocchio and normal populations belong to different reproductive groups. Thus, although it is not clear what mechanisms maintain these haplotype differences in sympatry, it is clear that there are genetic differences between the two morphotypes.

Consequently, the answer to the second question posed in the introduction — are there genetic differences between the morphotypes? — is yes. What this means is discussed in the final chapter. 21

CHAPTER IV

4.0 Foraging observations:

The third and final question posed in the introduction concerns the functional significance of different fluvial mountain whitefish morphotypes. This question was addressed through in situ field observations on the foraging behaviour of the two morphs in Dome Creek, B.C.

4.1 Methods

In late August 1996, two days of foraging observations were conducted on pinocchio and normal mountain whitefish in Dome Creek, a small tributary of the upper

Fraser River (Fig.3). Seasonal surveys of Dome Creek suggested that this tributary, like many in the region, is used as both a summer foraging area, and a reproductive site by both pinocchio and normal mountain whitefish. The hydrology of Dome Creek is highly variable, with variable flow regimes and low visibility for the majority of the ice-free

season. During select periods in early August thorough to September the water levels decrease and the water visibility improves allowing for safe snorkel observation.

Under-water snorkel observations focusing on mountain whitefish foraging behaviours were made at two sites (designated Top site and Bottom site). At both sites

adult pinocchios and normals were distinguishable under water at distances of up to 10

meters. Observations were conducted for 5 minute periods. Focal individuals alternated between pinocchio and normal whitefish. Approximate standard lengths of focal

were determined by comparing them to two mountain whitefish that had been angled,

measured and tagged two weeks earlier. The whitefish showed no obvious fright response 22 to the presence of a snorkeling observer and fish continued to forage until the observer approached closer than 2 meters. Even then, only sudden movements by the observer displaced the fish. The turbidity and flow regime of Dome Creek allowed for only two days of foraging data collection, on a total of 13 mountain whitefish; eight normal and five pinocchio adult whitefish. The low density of larger fish at the top and bottom observation pools did not allow for larger sample sizes.

The number, and type, of foraging attempts made by the focal in a five minute period were recorded with a soft pencil on a polyvinyl slate. Foraging attempts were categorized as either benthic or in the water column. Benthic foraging attempts occurred when fish foraged actively amongst, or on, the substrate. Water column attempts were recorded when fish were seen to make foraging movements directed at items suspended in the moving water column. The frequency of unique benthic foraging behaviours (e.g. rock-rolling and substrate burrowing) were recorded along with the two standard foraging categories. The success rate during the foraging trials is unknown but it was assumed that that not all foraging attempts were fruitful.

4.11 Statistical analysis

Foraging observations conducted on pinocchio and normal whitefish were standardized by observing individuals of both forms in alternate trials of equal duration.

The estimated size range of observed animals was similar for both forms and the number of observation periods on pinocchio and normal fish at each site was nearly equal (Tables

8, 9). Mann-Whitney U-tests were performed on the foraging data to determine if pinocchio and normal fish have different foraging rates for benthic and water column 23 prey. The number of benthic and water column foraging attempts were compared for each observation site individually, then the data were pooled to test for overall rate differences: 24

4.2 Results:

In situ snorkel observations conducted in Dome Creek during August 1996 confirmed that pinocchios and normals forage together at the same time but in different ways. At each observation site pinocchios foraged alongside normal fish, however, the morphotypes exhibited different foraging behaviours. Normal mountain whitefish foraged only on drift in the water column. Pinocchio forms also foraged on drifting prey suspended in the water column but, in addition, they foraged by burrowing amongst the gravel substrate with their snouts (Table 8, 9; Fig. 18).

Normal mountain whitefish oriented themselves on a plane horizontal to the substrate and about 30 cm above the bottom. They hold themselves in the current with tail movements and hold their pectoral fins against the body. Movement was limited, and most of the normal fish did not move more than a couple of meters during the five minute observation periods. All of the foraging attempts by normal mountain whitefish in both observation pools were made in the water column and directed at drift items the fish appeared to identify visually. Most foraging attempts involved fish moving to, and then striking at, items drifting no more than 30 cm from their holding position. After a strike attempt these fish returned to a horizontal holding posture facing into the current.

Pinocchios appear to be more benthically oriented than normal fish. They hold in the current about 20 cm above the substrate but, unlike normals, they did not remain in a single holding area during the observation trials. Instead pinocchios moved, stopped and moved again in a saltatorial pattern. While holding, and cruising over the substrate, pinocchios extend their pectoral fins away from their bodies and tilt their heads towards the substrate. 25

Approximately one-half of the foraging attempts made by pinocchios were directed at drifting items suspended in the water column while they held themselves into the current like normal fish (Tables 8, 9; Fig. 18). Unlike the normal fish, however, pinocchios also actively foraged in and amongst the substrate (Tables 8. 9; Fig 18). Thus, pinocchios appeared to forage in two modes: one involves holding a position and focusing on foraging for drift with occasional probes at the substrate; the other mode occurred during saltatorial movements and seemed to be oriented solely at the bottom.

Occasionally, while in a holding position, pinocchios would turn their eyes away from the oncoming current and, looking at the substrate, they would sink their snouts into the gravel and burrow through the gravel, or wedge their noses between interstices among rocks while thrusting with their caudal fins. Many pinocchios were seen "coughing-up" small gravel and sand after such benthic foraging episodes. When moving between holding positions pinocchios cruise over the substrate with their eyes oriented towards the bottom and slow to a stop before burrowing into the gravel. During several bottom foraging episodes, pinocchios used their elongated snouts to flip and roll rocks then struck quickly at the freshly exposed substrate.

Mann-Whitney U- tests comparing the number of benthic foraging attempts normal and pinocchio fish made at both observation sites are significantly different (both

U o.io(i) 4,3= 12; U o.io(i) 4,2 =8; U> 15 for both sites) (Tables 8, 9; Fig 18). There were significant differences (U o.os(i) 8,5 =32; U> 37) in the total number of water column foraging attempts made in the top pool, when compared to the bottom pool (Fig. 18).

Pinocchios directed more foraging attempts at the benthos in the top pool, and picked more drift in the bottom pool. This suggests that pinocchios can exhibit slightly different foraging behaviour in different habitats. Foraging rates of normal fish were similar at each observation site, and usually slightly elevated compared to their pinocchio counterparts (Fig. 19). 27

4.3 Foraging observations (conclusions):

Observations of foraging behavior under natural conditions in Dome Creek, combined with the morphometric differences suggest that pinocchio and normal mountain whitefish have different foraging ecologies while in sympatry.

The two forms of mountain whitefish did not differ in their location within the observation pools and did not appear to hold foraging territories. Normal fish made more foraging attempts than pinocchios, but the differences are slight and any differences between the morphs in foraging rates may be attributed to differences in foraging behaviours. The movements of the pinocchio fish were saltatorial, and directed at both the substrate and water column thereby affording less time for drift feeding than the more stationary and exclusively drift-feeding normal mountain whitefish morph. The elongate snout of the pinocchios was used to flip rocks, burrow into the substrate, and poke into the interstices between larger rocks. These benthic-oriented behaviours were not observed in the normal form of fluvial mountain whitefish. 28

CHAPTER V

5.0 General Discussion:

Coregonid fish were subjected to multiple bouts of range fragmentation, divergence, and range expansions during the Pleistocene glaciations (Lindsey 1981;

McPhail and Lindsey 1986; Bernatchez and Dodson 1991). Many species show unique local divergences, and there are several examples of multiple forms reported from the same lake (Bodaly 1979; Bird 1979). Most of these investigations have focused on the genus and little attention has been directed at variation in the genus

Prosopium. Members of Prosopium are distributed throughout Siberia and western North

America and the oldest known fossils (Miocene) are from deposits in southwest Idaho and northeast Oregon (Smith 1975). This suggests that the genus probably has its evolutionary roots in western North America rather than in Siberia (Norden 1970;

Vuorinen 1998). The mountain whitefish, Prosopium williamsoni, is considered by morphological and molecular analyses to be the least derived of the six known species within Prosopium (Norden 1970; Sajdak and Phillips 1997; Vuorinen 1998). Curiously, all recent morphological accounts of this species ignore the long snouted (pinocchio) form of mountain whitefish and treat the species as if it were homogenous throughout its wide western North American distribution. In contrast, older accounts (e.g. Evermann

1892; Jordan and Snyder 1909) recognized the long snouted morph and emphasized morphological variation within the species.

The first question posed in the introduction of this thesis concerns the nature of the morphological variation of fluvial mountain whitefish observed in upper Fraser River system -— are there discrete forms of mountain whitefish in the system, or is the variation 29 continuously distributed? The PCA conducted on size-adjusted Swift River and two other upper Fraser tributary specimens suggests that the presence of two groups of mountain whitefish based on head length, maxillary length, and snout length. Hierarchical cluster analysis of the first principal component loading scores identifies two phenotypes of upper Fraser mountain whitefish. The phenotypic differences vary between collection sites, but one form has consistently longer cranial features than the other group.

Whatever the cause, the consistent differences in head shape and mouth suggest differences in either microhabitat use or foraging behaviour in fluvial environments (e.g.;

Liem 1973; Bodaly 1979; Taylor and Bentzen 1993; Gailis and Metz 1998).

In fish, differences in gill raker number often reflect differences in trophic ecology, especially in coregonid species (Bodaly 1979; McPhail 1992; Gardener at al..

1988). My data indicate that pinocchios have a higher gill raker counts than normal whitefish. It is unclear whether these differences in gill raker counts are an effect of exposure to different micro-habitats early in life or if there are heritable gill raker differences between the pinocchio and normal forms; however, most coregonid taxonomists consider differences in gill raker numbers to be a reliable indicator of genetic differences among whitefish (Svardson 1957; Lindsey 1981). Thus, these differences in cranial features, in conjunction with the disrupted distribution in gill raker counts, suggest that the two forms of mountain whitefish in upper Fraser River tributaries differ in their trophic ecology.

The second question posed in the introduction concerns the genetic structuring of mountain whitefish in the upper Fraser system, specifically — are there genetic differences between the phenotypes? Again, the answer is yes. 30

The haplotype data compiled from mitochondria DNA restriction fragment length polymorphisms in upper Fraser River mountain whitefish indicate that the fish from different locations vary in haplotype frequencies. The presence of this diversity in the mitochondrial genome implies that these populations have been subjected to historical restrictions in gene flow that arose as a result of founder effects or population bottlenecks

(Bernatchez and Dodson 1990, 1991).

Interestingly, over ninty-seven percent of mountain whitefish mtDNA haplotype diversity in the upper Fraser is attributable to two haplotypes — A and B. The frequencies of these haplotypes appear to be fixed in the upper Fraser system, since they are independent of life-stage and location. Mitochondrial DNA haplotype frequency distributions also are significantly different in the two morphological forms — pinocchios and normals. Haplotypes A and B are equally represented in pinocchio fish, while normal fish were almost exclusively Haplotype A and never contained haplotype B. This difference in mtDNA haplotype frequencies in pinocchios and normals suggests the two forms are partially reproductively isolated even though they occur together in spawning congregations; Since mtDNA is passed mainly through the maternal line, crosses between normal males and pinocchio females should yield some normal fish with the B haplotype.

Thus, the absence of haplotype B in the normal form implies either asymmetrical reproductive isolation — with normal males avoiding pinocchio females (positively assorted mating) — or strong selection against hybrid progeny of normal male and pinocchio female matings.

Examples of reproductive isolation are common among closely related fish and amphibia taxa (Michalak and Rafinski 1999; Dgebuadze at al. 1999; Rocha-Olivares at 31 al. 1999). Most examples of reproductive isolation in fish occur in north temperate,

species poor, lacustrine environments and involve morphological features associated with foraging behaviours (Wimberger 1994). The genetic data suggests the pinocchio and normal mountain forms of whitefish represent a possible example of asymmetric reproductive isolation in a fluvial environment. Further research is needed before any more speculation surrounding mountain whitefish reproductive isolation is disscussed.

The third, and final question posed in the introduction concerns the function of the morphological differences observed in upper Fraser system mountain whitefish populations. Observations in Dome Creek clearly show that in sympatry the long-snouted pinocchio and blunt-snouted normal forms of mountain whitefish have different foraging behaviours. When foraging, normal mountain whitefish are less motile than pinocchios foraging exclusively on water-born drift. In contrast, pinocchios cruise the substrate in a

saltatory fashion, poking their elongated snouts into interstices and often rolling over rocks. Pinocchios direct their foraging attempts about equally to the benthos and the water column, but their over-all foraging rate is similar to the normal fish. The

differences between pinocchios and normals in body position and pectoral fin extension when holding in the current suggest that pinocchios are inducing downward forces on the

anterior end of their bodies. This benthically oriented body angle, as well as differences in head shape, may produce differences in near point focus. Thus, allowing pinocchios to

search the substrate for prey items while remaining in a position to forage on passing

drift. The morphological differences, especially in rostral length and gill raker

architecture, combined with the striking differences in their foraging tactics suggest that 32 these fluvial forms of mountain whitefish — pinocchios and normals — use overlapping, but slightly different foraging niches (Fausch at al. 1997; Skulason and Smith 1995).

The combined evidence from my morphometric, genetic and foraging behaviour investigations of mountain whitefish suggest that sympatric pairs of whitefish are not restricted to lacustrine environments. The mountain whitefish in the upper Fraser system are functionally dimorphic and, although there is molecular evidence supporting reproductive isolation between the morphs, it is unclear how this dichotomy is maintained. It is clear, however, that the observed differences in mountain white fish are not exclusive to the Fraser Basin. The two forms occur throughout a large portion of the species' range and the pinocchio form bears a striking resemblance to Coregonus oregonius, a species from the McKenzie River, Oregon proposed by D. S. Jordan and J.

Snyder in 1909.

While it is clear that there are two forms of mountain whitefish in upper Fraser

River tributaries, it is unclear where or how the pinocchio and normal forms originated.

The presence of both pinocchio and normal populations of mountain whitefish on both the Olympic Peninsula, Washington, and above the barrier at Shoshone Falls on the

Snake River, Idaho, suggest that the propensity to form this dichotomy probably predates the Wisconsinan glacial period. Thus, postglacial migrants into British Columbia may have carried with them the genetic capacity to produce the pinocchio form. The presence of interpopulation variation in the development of elongate snout implies that either a strong environmental influence or local adaptation is involved in the expression of the trait. Thus, the pinocchio and normal forms may have diverged in situ. The present study is only a beginning. Questions about the morphological features associated with the 33 pinocchio morphotype and the role of environment and genetics in their expression clearly require more research. Further life history, laboratory rearing experiments, foraging observations, and genetic studies may unravel these problems now that the presence of two forms has been documented. 34

Drainage Collection date NORMAL PINOCCHIO n SL±se (n) SL±se (n) Nechako Oct./95 165 ± 15 (19) 171 ± 19 (11) 25 Wright June/56 171 ± 35 (11) 173 ± 32 (10) 21 Swift June/55 176 ± 35 (25) 182 ± 23 (13) 38 n - 55 34 84 Table 1 Ranges of standard lengths (mm) and collection sizes of the upper Fraser mountain whitefish measured in the morphometric survey.

Drainage Collection date NORMAL, (n) PINOCCHIO, (n) total (n)

Bowron 1996 5 5 10 Wright 1956 6 6 12 Swift 1955 10 11 21 Nechako 1995 6 6 12 total - 28 27 55 Table 2 Sample sizes (n) of the upper Fraser mountain whitefish used in comparison of gill raker number in pinocchio and normal froms.

Drainage Gill raker count ± s.'e. Gill raker count ±s.e. p-value (NORMAL) (PINOCCHIO) Swift 20.8 + 0.4 22.5 ± 0.43 0.0047 Bowron 20.0 ± 0.6 23.0 ±0.61 0.005 Wright 18.8±0.5 21.3 ±0.5 0.027 Nechako 20.5 ± 0.5 22.6 ± 0.5 0.0078 mean 20.1 ±0.3 22.4 ± 0.27 Table 3 Comparison of pinocchio and normal mountain whitefish primary gill arch raker number from various upper Fraser tributaries. 35

source sum of degrees of mean F-ratio p-value squares freedom square Ecotype 69.26 1 69.26 37.44 <0.001 Drainage 21.48 3 7.16 3.87 0.015 Ecotype* 3.117 3 1.039 0.562 0.643 Drainage Error 85.1 46 1.85 Table 4 Result of analysis of variance comparing pinocchio and normal mountain whitefish gill raker numbers across upper Fraser tributaries.

Drainage Adults Adults Juveniles Total normal pinocchio McGregor - - 4 4 Willow 1 3 4 8 Salmon - - 6 6 Chilako - - 3 3 Bowron 2 3 3 8 Nechako 11 6 - 17 Fraser mainstem - - 12 12 Chowade (Peace) • - - 7 7 Burnt (Peace) - - 4 4 Duncan (Columbia) - - 2 2 Totals 14 12 45 71

Table 5 Mountain whitefish tissue sample sizes and collection sites included in the mtDNA analysis. 36

Composite Total upper Fraser upper Peace System Columbia Haplotype sample tributaries (adults), Fraser (Chowade, system (juveniles) mainstem Burnt) (Duncan) A 51 19 73% , 13 9 75% 6 85.7%, 3 1 50% 74.7% 76.4% 75% B 15 7 27% , 3 3 25% 1 14.3% ,0 1 50% 22.5% 17.6% 0% C 2 0 0% , 1 0 0% 0 0% , 1 0 0% 2.8% 6% 25% Total 68 26 , 17 12 7 , 4 2 Table 6 Distribution of D-loop/cyt. b RFLP haplotype patterns in adult and juvenile mountain whitefish populations in British Columbia.

Composite Pinocchios, Normal, Pinocchios, Normal, Haplotype (Nechako) (Nechako) (Total sample) (Total pattern sample)

A 3, 50% 11, 100% 6, 50% 14, 100% B 3, 50% 0 6, 50% 0 C 0 0 0 0 Totals 6 11 12 14 Table 7 Distribution of D-loop/cyt b RFLP haplotype patterns from pinocchio and normal mountain whitefish populations in the upper Fraser system. 37

Morphotype S.L± Sample observation Benthic Water column Total s.e. size trials foraging foraging attempts ±s.e (mm) attempts (±s.e) (±s.e) Normal 285± 4 10 0 13.6±2.7 13.6± 54 2.7 Pinocchio 273± 3 10 6.5+1.1 2.5±2 9.0±3 45 .1 Table 8 Standard length, sample sizes, number of observation trials and mean number of foraging attempts made by pinocchio and normal mountain whitefish in the top observation pool in Dome Creek.

Morphotype S.L+ Sample observation Benthic Water column Total s.e. size trials foraging foraging attempts +s.e (mm) attempts (+s.e) (+s.e) Normal 250± 4 14 0±0 12.2412.2 12.2 53 +2.2 Pinocchio 215+ 2 12 3.6±1.5 9.511.1 10.1 45 11.2 Table 9 Standard length, sample sizes, number of observation trials and mean number of foraging attempts made by pinocchio and normal mountain whitefish in the bottom pool observation pool in Dome Creek. 38

Figure 1 — Distribution of mountain whitefish, Prosopium williamsoni, in North

America. 39

Figure 2 — Head profiles of normal and pinocchio phenotypes of mountain whitefish,

Prosopium williamsoni.

NORMAL PINOCCHIO 40

Figure 3 —Upper Fraser River Basin including Prince George, B.C.

Upper Fraser River 41

Figure 4 — Distribution of residuals collected from a head vs. standard length regression of Swift River mountain whitefish.

HEAD LENGTH

o \— Z) OQ Cd \- co

Q _i <

Q CO LU CL -0.04 h -0.05 2.0 2.1 2.2 2.3 2.4 2.5 STANDARD LENGTH 42

Figure 5 — Distribution of residuals collected from a maxillary vs. standard length regression of Swift River mountain whitefish.

MAXILLARY LENGTH

0.10

o 0.05h CQ

SCO o.ooh Q

< Q CO -0.05h UJ CrT

2.0 2.1 2.2 2.3 2.4 2.5 STANDARD LENGTH 43

Figure 6 — Distribution of residuals collected from a snout vs. standard length regression of Swift River mountain whitefish.

SNOUT LENGTH

0.10

o I- 0.05h GO

\— ^CO o.oof- Q < Q CO -0.05h LU

-0.10 2.0 2.1 2.2 2.3 2.4 2.5 STANDARD LENGTH 44

Figure 7 — Frequency distribution of residuals collected from a head vs. standard length regression of Swift River mountain whitefish.

HEAD LENGTH

16 H0.4

12 0.3 o "O o C 3- o 8 o O 0.2 => "D CD 0.1 S

0 0.0 -0.050 -0.025 0.000 0.025 0.050 RESIDUAL DISTRIBUTION gure 8 —Frequency distribution of residuals collected from a maxillary vs. standard length regression of Swift River mountain whitefish.

MAXILLARY LENGTH

15

0.3 10h

3 0.2 o o 5h CTJ 0.1 -

0 0.0 -0.10 -0.05 0.00 0.05 0.10 RESIDUAL DISTRIBUTION 46

Figure 9 — Frequency distribution of residuals collected from a snout vs. standard length regression of Swift River mountain whitefish.

SNOUT LENGTH

15

0.3 TJ —i O 10h "O o H0.2 2- o O "O o CD 0CJ 03 0.1 ^

o 0.0 -0.10 -0.05 0.00 0.05 0.10 RESIDUAL DISTRIBUTION 47

Figure 10 — Scatter plot of principal component analysis loading values for Swift River mountain whitefish cranial measurements.

Swift River

-2-1012 Factor 1 (94.8% Variance) 48

Figure 11 — Hierarchical cluster analysis of factor one principal component loading values of mountain whitefish cranial measurements from the Swift River.

Case 19 Case 36 Case 2 Case 37 Case 14 Case 16 Case 15 Case 1 Case 30 Case 11 Case 24 Case 5 Case 35 Case 22 ase 38 Sase 10 Case 9 Case 17 ase 31 Sase 33 Case 23 Case 21 Case 28 Case 4 Case 3 Case 32 Case 12 Case 8 Case 26 Case 18 Case 6 Case 20 Case 34 ase 13 ase 29 BCase 7 Case 25 Case 27 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 Euclidean Distance 49

Figure 12 — Scatter plot of principal component analysis loading values of mountain whitefish cranial measurements from three upper Fraser River tributaries.

Combined Drainages

CD o C ro i_ >ro

un CN o ' o CO LL

-2-10123 Factor 1 (94.9% Variance) 50

Figure 13 — Hierarchical cluster analysis of factor one principal component loading values of mountain whitefish cranial measurements from three upper Fraser River tributaries.

0.0 0.1 0.2 0.3 0.4 Euclidean Distance 51

Figure 14 — Frequency distribution of gill raker counts from Swift River mountain whitefish.

GILL RAKERS

8 0.4 TJ o 6 MORPHOTYPE J 0.3 "D —I O • NORMAL 4 O • PINOCCHIO 0.2 E "O O CD 2 0.1 -f 00 Q) 0 0.0 —% TJ tj —^ O 2 0.1 "O o 4 0.2 o O "O o CD 6 0.3 —\ DO CD 8 0.4 18 20 22 24 26 28 GILLRAKER NUMBER 52

Figure 15 — Frequency distribution of gill raker counts from mountain whitefish from four upper Fraser River tributaries.

GILL RAKERS

20 -0.3 o 15 O

10h -0.2 o "O CD 5 -0.1 rjj 0 -0.0 TJ O 5h -0.1 "O o oa- g 10h MORPHOTYPE -0.2 "O O CD iNORMAL —^ 15 u PINOCCHIO -0.3 LTJ Q) 20 15 20 25 30 GILLRAKER NUMBER 53

Figure 16 — Frequency distributions of composite mitochondrial DNA RFLP haplotypes from upper Fraser River mountain whitefish arranged by collection location and life-history stage.

• Haplotype A • Haplotype B • Haplotype C

90 -i

80

70 u e 60 s u" 50 (Ll be tt*, 40

30 OH 20

10 0 I Mainstem Fraser Tributaries Tributaries Total Samples (adults) (juveniles) 54

Figure 17 — Frequency distribution of upper Fraser River mountain whitefish composite mitochondrial RFLP haplotypes arranged by phenotypes.

• Haplotype A • Haplotype B

120 n

Pinocchio Normal Nechako Pinocchio Normal Nechako R. R. Combined Combined drainage draianges 55

Figure 18 — Foraging preferences of pinocchio and normal mountain whitefish from two observation sites in Dome Creek (Top site and Bottom site).

a) TOP SITE

BENTHIC WATER COLUMN

MORPHOTYPE MORPHOTYPE

b) BOTTOM SITE

BENTHIC WATER COLUMN

==• 20

• > & 111 t— 1 1 o z o - • $ £ o

MORPHOTYPE MORPHOTYPE 56

Figure 19 — Foraging rate of pinocchio and normal mountain whitefish from two observation sites in Dome Creek (Top and Bottom site).

TOP SITE

MORPHOTYPE

BOTTOM SITE

MORPHOTYPE Literature cited:

Bernatchez, L. and J.J. Dodson. 1990. Allopatric origin of sympatric populations of lake

whitefish (Coregonus clupeaformis) as revealed by mitochondrial-DNA restriction

analysis. Evolution 44: 1263-1271.

Bernatchez, L., and J.J. Dodson. 1991. Phylogeographic structure in mitochondrial DNA

of the lake whitefish (Coregonus clupeaformis) and its relation to Pleistocene

Glaciations. Evolution 45: 1016-1035.

Bernatchez, L., and A. Osinov. 1995. Genetic diversity of trout (Genus Salmo) from its

most eastern native range based on mitochondrial DNA and nuclear gene variation.

Molecular Ecology 4: 285-298.

Bernatchez, L., A. Chouinard, and G. Lu. 1999. Integrating molecular genetics and

ecology in studies of adaptive radiation: whitefish, Coregonus sp., as a case study.

Biological Journal of the Linnean Society of London. 68:173-194.

Bird, F.H. 1979. Pygmy whitefish, Prosopium coulteri, in three lakes of the Copper River

system in Alaska. J. Fish. Res. Board Can. 36: 468-470.

Bodaly, R.A. 1979. Morphological and ecological divergence within the lake whitefish

(Coregonus clupeaformis) species complex in the Yukon Territory. Fish. Res.

Board Can. Bull. 36:1214-1222.

Bookstein, F.L., B. Chernoff, R. Elder, J. Humphries, G. Smith, and R. Strauss. 1985.

Morphometries in evolutionary biology. The Academy of Natural Sciences of

Philadelphia. Special Publication 15. 58

Davies, R.W. and G.W. Thompson. 1976. Movements of the mountain whitefish

(Prosopium williamsoni) in the Sheep River watershed. Alberta. J. Fish. Res. Board

Can. 33:2395-2401.

Dgebuadze, Y.Y., M.V. Mina, S.S. Alesgev, and A.S. Golubtsov. 1999. Observation on

reproduction of the lake Tana barbs. J. Fish Biology. 54:417-423.

Evermann, B.W. 1892. A reconnaissance of the streams and lakes of Western Montana

and Northwestern Wyoming. Bull. U.S. Fish. Comm. XF3-60.

Fausch K.D., Nakano S., Kitano S. 1997. Experimentally induced foraging mode shift by

sympatric charr in a Japanese mountain stream. Behavioral Ecology 8: 414-420.

Ferguson, A., and F.M. Manson. 1981. Allozyme evidence for reproductively isolated

sympatric populations of brown trout, Salmo trutta, in Lough Melvin, Ireland. J.

Fish Biology 18: 629-42.

Galat, D.L., and N. Vucinich. 1983. Food partitioning between young of the year of two

sympatric tui chub morphs. Trans. Am. Fish. Soc. 112:486-497.

Galis, F. and E.G. Drucker. 1996. Pharyngeal biting mechanisms in Centrachids and

chiclids: Insights into key evolutionary innovation. J. Evol. Biology 9:641-670.

Galis, F. and J.A.J. Metez. 1998. Why are there so many cichlid species? Trends Ecol.

Evol. 13:1-2.

Gardner, A.S., A.F. Walker, and R.B. Greer. 1988 Morphometric analysis of two

ecologically distinct forms of Arctic charr, Salvelinus alpinus (L.), in Loch

Rannoch, Scotland. J. of Fish Biology 32:901-910.

Haas, G.R. 1988. The sytematics, zoogeography and evolution of Dolly Varden and bull

trout in British Columbia. M.Sc. thesis, Dept.of Zoology, U.B.C., Vancouver, B.C. 59

Haas, G.R., and J.D. McPhail. 1991. Systematics and distributions of Dolly Varden

(Salvelinus malmd) and Bull trout (Salvelinus confluentus) in North America. Can.

J. Fish. Aquat. Sci. 48:2191-2211.

Hall H.J., and L.W. Nawrocki. 1995. A rapid method of detecting mitochondrial DNA

variation in the brown trout, Salmo trutta. J. of Fish Biology 46:360-364.

Hindar, K., and B. Jonsson. 1992. Ecological polymorphism in Arctic charr. Biological

Journal of the Linnean Society 48:63-74.

Holt, R.D. 1960. Comparative morphometry of the Mountain Whitefish, Prosopium

williamsoni: Copeia, 1960:192-2001.

Hubbs, C.L., and K.F. Lagler. 1947. of the Great Lakes region. Univ. Michigan

Pess, Ann Arbor, Michigan.

Humphries, J.M., F.l. Bookstein, B. Chernoff, G.R. Smith, R.L. Elder, and S.G. Poss.

1981. Multivariate discrimination of shape in relation to size. Systematic Zoology

30:291-308.

Jonsson, B., and K. Hindar. 1982. Reproductive strategy of dwarf and normal Arctic charr

(Salvelinus alpinus) from Vangsvatnet Lake, Western Norway. Can. J. Fish. Aq.

Sci. 39:1404-1413.

Jordan, D.S., and J.O. Snyder. 1909. Description of a new whitefish (Coregonus

oregonius) from McKenzie river, Oregon. Proceedings of the United States

National Museum XXXVI: 425-430.

Lagler, Carl, F. 1956. Freshwater Fishery Biology. W.M.C. Brown Company, Dubuque,

Iowa. Liem, K.F. 1973. Evolutionary strategies and morphological innovations: Cichilid

pharyngeal jaws. Syst. Zoology 22:245-441.

Lindsey, CC. 1988. Factors controlling meristic variation, pp. 197-274. In D.J. Randall

and W.S. Hoar, (eds.) Fish Physiology, vol XIB. Academic Press, New York.

Lindsey, CC 1981. Stocks are chameleons: plasticity in gill rakers of coregonid fishes.

Can. J. Fish. Aquatic Sci. 38:1497-1505.

McAfee, W.R.I 966. Mountain whitefish. In L.A. Calhoun (ed.) Inland fisheries

management. California Department of Fish and Game, Bulletin: 299-303.

McCart, P. 1970. Evidence for the existence of sibling species of pygmy whitefish

(Prosopium coulteri) in three Alaskan lakes. In CC Lindsey and CS. Woods (ed.)

Biology of Coregonid Fishes, pp 81-98.

McElroy, M.D., E. Bermingham, and I. Kornfeild. 1992. REAP: an integrated

environment for manipulation and phylogenetic analysis of Restriction data. J of

Heredity 83: 157-158.

McHugh, J.L. 1940. Food of the Rocky Mountain Whitefish, Prosopium williamsoni

(Girard). J. Fish. Res. Board Canada. 5:131-137.

McPhail J.D. 1992. Ecology and evolution of sympatric sticklebacks (Gasterosteus):

evidence for a species-pair in Paxton Lake , Texada Island, British Columbia. Can.

J. Zoology 70: 361-369.

McPhail, J.D. and CC Lindsey. 1970. Freshwater fishes of northwestern Canada and

Alaska. Fish. Res. Board Can. Bull. 173. McPhail, J.D., and C.C. Lindsey. 1986. Zoogeography of the freshwater fishes of

Cascadia. In Hocutt and E.O. Wiley. John Wiley and Sons (ed.) The zoogeography

of North American freshwater fishes. 615-637.

McPhail, J.D., and P.M. Troffe. 1998. The mountain whitefish (Prosopium williamsoni):

A potential indicator species for the Fraser system. Technical report submitted to

the Fraser River Action Plan.

Meyer, A. 1989. Plasticity in morphology and performance in the trophically polymorphic

cichlid fish, Cichlasoma citrinellum. Dissertation Abstracts International, Part B:

The Sciences and Engineering 50: 229p.

Michalak, P. and J. Rafinski. 1999. Sexual isolation between two newt species (Triturus

valgaris and T. montandoni, Amphibia, Salamanridae). Biological Journal of the

Linnean Society 67:343-352

Norden C.R. 1970. Evolution and distribution of the genus Prosopium. In C.C. Lindsey

and C.S. Woods (ed.) Biology of CoregonidFishes. 81-98.

Northcote, T.G. and G.L. Ennis. 1994. Mountain Whitefish Biology and Habitat use in

Relation to Compensation and Improvement Possibilities. Reviews in Fisheries

Science 2(4): 347-371.

Northcote, T.G. 1988. Meristic variation in the New Zealand smelt, Retropinna

retropinna, (Richardson): New questions for an old problem. Verhandlungen.

Internationale Vereinigung fur theoretische und angewandte

Limnologie/Proceedings International 23(3) Available as: Congress in New Zealand

1987. Proceedings, pp 1797-1804. Reist, J.D. 1985. An empirical evaluation of several univariate methods that adjust for

size variation in morphometric data. Can. J. Zooi. 63: 1429-1439.

Robinson, B.W., and D.S. Wilson. 1996. Genetic variation and phenotypic plasticity in a

trophically polymorphic population of pumpkinseed sunfish (Lepomis gibbosus).

Evolutionary Ecology 10: 631-652.

Rocha-Olivares, A., R.H. Rosenbalt, and R.D. Vetter. Molecular evolution, systematics

and zoogeography of the rockfishes subgenus Sebastomus (Sebastes, scorpaenidae)

based on Mitochondrial Cytochrome b and control region sequences. Molecular

Phylogenetics and Evolution 11:441-458.

Ruzzante, D.E., S.J. Waide, V.E. Cussac, P.J. Macchi and M.F. Alonso. 1998. Trophic

polymorphism, habitat and diet segregation in Percichthys trucha (Pisces:

Percichthyidae) in the Andes. Biological Journal of the Linnean Society 65: 191-

214.

Sajdak, S.L., and R.B. Phillips. 1997. Phylogenetic relationships among Coregonus

species inferred from the DNA sequence of the first transcribed spacer (ITS1) of

ribosomal DNA. Can. J. Fish. Aquat. Sci. 54:1494-1503.

Schluter, D. 1996. Ecological speciation in postglacial fishes. Philosophical Transactions

of the Royal Society of London 351: 807-814.

Scott, W.B., and E.J. Crossman. 1973. Freshwater fishes of Canada. Fish. Res. Board

Canada. Bull. 184:966.

Sharp, J.C., K.W. Able and W.C. Leggett. 1978. Utility of meristic and morphometric

characters for identification of capelin (Mallotus vdlous) stocks in Canadian

Atlantic waters. J. Fish. Res. Board Can. 35: 124-130. 63

Skulason, S., D.L.G. Noakes, and S.S. Snorrason. 1989. Ontogeny of trophic morphology

in four sympatric morphs of Arctic charr, Salvelinus alpinus, in Thingvallavatn,

Iceland. Biological Journal of the Linnean Society 38:281-301.

Skulason, S., and T.B. Smith. 1995. Resourse polymorphisms in vertebrates. Trends Ecol.

Evol. 10:366-370.

Skulason, S., S.S. Snorrason, D. Ota, and D.L.G. Noakes. 1993. Genetically based

differences in foraging behaviour among sympatric morphs of Arctic charr (Piscces:

Salmonidae). Animal Behaviour 45:1179-1192.

Smith, G.R. 1975. Fish of the Pliocene Glenns Ferry formation, southwest Idaho.

University of Michigan Papers in Palaeontology 14:1-68.

Stalnaker, C.B., R.E. Gresswell, and R.E. Siefert. 1974. Early life history and feeding of

young mountain whitefish. U.S. Environmental Protection Agency, Ecological

Research Series, EPA-660/3-73-019.

Svardson, G. 1957. The coregonid problem, vi. The Palearctic species and their

intergrades. Rep. Inst. Freshwater Res. Drottningholm 38:261-356.

Svardson, G. 1979. Speciation of Scandinavian Coregonus. Rep. Inst. Freshwater Res.

Drottningholm 57:95p.

Taylor, E.B., and P. Bentzen. 1993. Evidence for multiple origins and sympatric

divergence of trophic ecotypes of smelt (Osmerus) in northeastern North America.

Evolution 47:813-822.

Taylor, E.B., and J.D. McPhail. 1999. Evolutionary history of an adaptive radiation in

species pairs of threespine sticklebacks (Gastewsteus): insights from mitochondrial

DNA. Biological Journal of the Linnean Society. 66: 271-291. Vuorinen, J.A., R.A Bodaly, J.D. Riest, and M. Lucynski. 1998. Phylogeny of five

Prosopium species with comparisons with other Coregonine fishes based on

isozyme electrophoresis. J. Fish Biology. 53(5).

Wimberger, P.H. 1994. Trophic polymorphisms, plasticity, and speciation in vertebrates.

Theory and Application of Fish Feeding ecology. University of South Carolina

Press, Columbus, South Carolina, pp. 19-44.

Winans, G.A. 1984. Multivariate morphometric variability in Pacific salmon: Technical

demonstration. Can. J. Fish. Aquat. Sci. 41:1150-1159.

Zar, J.H. 1984. Biostatistical Analysis, 2 ed. Prentice-Hall, Inc., Englewood Cliffs, New

Jersey. 65

Appendix I: Mountain whitefish, Prosopium williamsoni, mitochondrial RFLP haplotype patterns by drainage collection, (p) = pinocchio phenotype

Sty I Haplotype restriction fragments

Haplotype A 762 bp— Haplotype B 762bp — Haplotype C 762bp — 883 bp—

995 bp — 1096bp —

Drainage Sample # Stvl McGregor 1 19 A McGregor 2 21 A McGregor 3 23 C McGregor 4 24 A

Willow 1 wil 1 A Willow 3 wil 3 A Willow 4 (p) 25 B Willow 5 26 A Willow 6 (p) P(2) B Willow 7 B017 A Willow 8 B018 A Willow 9 B019 A

Salmon 1 9 B Salmon 2 10 A Salmon 3 11 A Salmon 6 90 A Salmon 7 91 A Salmon 8 92 B

Chilako 1 chil2 A Chilako 2 chil 3 B Chilako 3 chil4 B

Fr. Mainstem 1 33 B Fr. Mainstem 4 48 A Fr. Mainstem 5 59 A Fr. Mainstem 6 60 A Fr. Mainstem 7 65 A Fr. Mainstem 9 70 B Fr. Mainstem 10 71 A Fr. Mainstem 11 80 A Fr. Mainstem 12 85 A Fr. Mainstem 13 86 A Fr. Mainstem 14 95 A Fr. Mainstem 15 96 B

Nechako 1 (p) DI A Nechako 3 D3 A Nechako 4 D4 A Nechako 5 D5 B Nechako 6 D6 A Nechako 7 D7 A Nechako 8 (p) D8 A Nechako 9 D9 A Nechako 10 (p) D10 B Nechako 11 (p) Dll A Nechako 13 D13 A Nechako 14 D14 A Nechako 15 D15 A Nechako 16 D16 A Nechako 17 Nek 2 A Nechako 18 (p) Nek 5 B Nechako 19 Nek 9 A

Bowron 1 Bow 1 A Bowron 2 Bow 2 A Bowron 3 Bow 3 A Bowron 4 Bow 4 A Bowron 5 Bow 5 A Bowron 6 (p) Bw20 A Bowron 7 (p) Bw21 A Bowron 8 (p) Bw37 B

Duncan 1 D61 A Duncan 2 D62 B

Chowade 1 Chi A Chowade 2 Ch2 B Chowade 3 Ch3 A Chowade 4 Ch4 A Chowade 5 Ch5 A Chowade 6 Ch6 A Drainage Sample # Sty I Chowade 7 Ch7 A

Burnt 1 BI A Burnt 2 B2 A Burnt 3 B3 A Burnt 4 B4 C Appendix II:

Comment on Wright Creek:

The University of British Columbia fish museum contains mountain whitefish

samples from Wright Creek, a tributary to the Salmon River. The sample was collected in

June, 1955 and contains mountain whitefish of all size classes, including young-of-the- year and large adults of both the pinocchio and normal forms. This collection suggests that at one time mountain whitefish used Wright Creek during summer months, presumably as an adult foraging site and a juvenile rearing area. In 1995 repeated pole-

seining and back-pack electroshocker surveys in Wright Creek — from the source to its confluence with the Salmon River— uncovered no mountain whitefish. The absence of adults, and especially young-of-the-year and juveniles suggest that the creek no longer supports a mountain whitefish population.