The mountain whitefish ( williamsoni ): a potential indicator species for the Fraser System.

DOE FRAP 1998-16

Prepared for:

Environment Canada Environmental Conservation Branch Aquatic and Atmospheric Sciences Division 700-1200 West 73rd Avenue Vancouver, BC V6P 6H9

Prepared by:

J.D. McPhail and P.M. Troffe

Department of Zoology, University of , Vancouver, BC, V6T 1Z4

May 1998 DISCLAIMER

This report was funded by Environment Canada under the Action Plan through the Environmental Quality Technical Working Group. The views expressed herein are those of the authors and do not necessarily state or reflect the policies of Environment Canada

Any comments regarding this report should be forwarded to:

Aquatic and Atmospheric Sciences Division Environmental Conservation Branch Environment Canada 700-1200 West 73rd Avenue Vancouver, B.C. V6P 6H9

ii Acknowledgements

This project was funded by the Fraser River Action Plan. The patience and perseverance of Colin Gray and Erika Szenasy at Environment Canada were especially appreciated. Don Cadden and Ted Zimmerman (BC Ministry of Environment, Lands and Parks) provided collecting permits and local knowledge. The work would not have been completed without the enthusiasm and expertise of Peter Mylechreest.

iii Abstract

In summary, the results of this project indicate that the life history, habitat use, and movements of mountain whitefish in the Prince George area are similar to what has been found in other areas. In the Prince George area, there is evidence of age-related habitat shifts; complex migrations to spawning, over-wintering, and summer-foraging sites; genetically different foraging forms; and distinctive stocks.

Despite this complex life history and stock structure, mountain whitefish have a number of characteristics that suit them for the role of an indicator species in the Fraser Basin. They are widely distributed throughout the basin and are relatively long-lived (up to 15 years) so that there is time for individuals to accumulate contaminants and reach a body size sufficient for tissue sampling. Their diet (primarily nymphs and pupae of aquatic insects) is unlikely to be confounded by inputs of terrestrial or aerial contaminants, and the species shows remarkable fidelity to summer foraging sites. Thus, in spite of evidence from this, and other studies for complex migrations, individual appear to return to summer foraging sites that they used in previous years and, if this pattern of seasonal movement is consistent from year to year, these fish may provide a useful picture of contaminant loading at these foraging sites. Consequently, fish that forage in sites exposed to contaminants are likely to return those sites on an annual basis.

Stock structure in fall aggregations is still a problem. The available data suggest that these aggregations contain fish from a mixture of stocks and, potentially, a mixture of contaminant histories. Thus, if the practice of drawing monitoring samples from such aggregations is continued, some method of identifying the origin of individual fish is needed.

iv Résumé

En résumé, on peut dire que les résultats de ce projet montrent que le cycle vital, l’utilisation de l’habitat et les déplacements chez le ménomini de montagnes dans la région de Prince George sont semblables à ce qui a été observé dans d’autres régions. Dans la région de Prince George, on a observé les phénomènes suivants : changements d’habitat liés à l’âge; migrations complexes vers les sites de fraye, d’hivernage et d’alimentation estivale; existence de formes (ménominis « normaux »et « pinocchios »), qui sont fonction du mode d’alimentation, différentes génétiquement; et existence de stocks distincts. Malgré la complexité de son cycle vital et de la structure de ses stocks, le ménomini de montagnes possède diverses caractéristiques qui le rendent apte à remplir le rôle d’espèce indicatrice dans le bassin du Fraser. Les ménominis de montagnes sont présents partout dans le bassin du Fraser et vivent relativement longtemps (jusqu’à 15 ans) de sorte que les individus ont le temps d’accumuler des contaminants et d’atteindre une taille suffisante pour qu’on puisse échantillonner leurs tissus. Leur régime alimentaire (principalement des nymphes et des pupes d’insectes aquatiques) est tel qu’on peut être assez certain que les contaminants qu’ils ingèrent ne proviennent pas de sources terrestres ou atmosphériques; en outre, cette espèces est remarquablement fidèle à ses sites d’alimentation estivale. Ainsi, malgré l’existence de migrations complexes démontrée par cette étude et d’autres études, les poissons semblent revenir à leurs sites d’alimentation estivale d’une année à l’autre, et si ces déplacements saisonniers se répètent bel et bien d’année en année, ces poissons peuvent nous donner une bonne idée de la charge de contaminants à ces sites d’alimentation. Ainsi, les poissons qui s’alimentent dans des sites exposés à des contaminants reviendront probablement à ces sites chaque année. La question de la structure des rassemblements automnaux, en termes de stock, est encore problématique. Les données existantes laissent entendre que ces rassemblements renferment des poissons de divers stocks, et peut-être des poissons qui n’ont pas tous connus la même contamination. Ainsi, si l’on continue de prélever des échantillons de surveillance dans ces rassemblements, il faudra disposer d’une méthode permettant d’identifier l’origine des différents poissons.

v Table of Contents

Acknowledgements ...... iii

Abstract ...... iv

Résumé ...... v

Table of Contents ...... vi

List of Tables ...... viii

List of Figures ...... ix

1.0 INTRODUCTION...... 1

2.0 REVIEW OF MOUNTAIN WHITEFISH BIOLOGY ...... 2

2.1 Geographic distribution...... 2 2.2 Life history ...... 7

3.0 MOUNTAIN WHITEFISH IN THE UPPER FRASER SYSTEM...... 10

3.1 Life history ...... 10 3.1.1 Spawning...... 10 3.1.2 Emergence...... 10 3.1.3 Growth of young-of-the-year ...... 18 3.1.4 Diet of the young-of-the-year...... 19 3.1.5 Growth of juveniles and adults ...... 19 3.1.6 Habitat of juveniles and adults...... 21 3.1.7 Diet of juveniles and adults...... 24 3.2 Movements...... 26 3.2.1 Radio-tagging ...... 26 3.2.1.1 Autumn tags...... 26 3.2.1.2 Spring tags...... 27 3.2.2 Conventional tags ...... 32 3.2.1.1 Within season tag returns ...... 32 3.2.1.2 Between year tag returns...... 33 3.3 Laser ablation ...... 34 3.3.1 Evidence of stocks ...... 34 3.3.2 Evidence of movement...... 38 3.4 Evidence for two foraging forms of mountain whitefish...... 43

vi 3.4.1 Morphological analysis...... 43 3.4.1.1 Head shape...... 43 3.4.1.2 Behaviour ...... 46 3.4.2.3 Length-weight relationships...... 48 3.4.3 Molecular analysis...... 48 3.4.3.1 Pinocchios vs Normals ...... 50 3.5 Evidence for a mainstem stock...... 50 3.5.1 Molecular evidence ...... 50

4.0 CONCLUSIONS...... 51

5.0 LITERATURE CITED...... 55

APPENDIX ...... 58

A.1 DNA Extraction...... 59 A.2 Mitochondrial DNA analysis ...... 59

vii List of Tables

Table 1. Sites surveyed for newly-emerged fry in the upper Fraser system (1996)...... 12

Table 2. Substrate characteristics of sites used by newly-emerged mountain whitefish fry.18

Table 3. Distribution of scale “signatures” (number of first year circuli) on the scales of 32 adults collected from a fall aggregation in the mainstem Fraser River near the confluence of Naver Creek...... 20

Table 4. Mean length at age for selected BC populations of mountain whitefish...... 20

Table 5. Comparison of depth distribution of young-of-year and juvenile mountain whitefish...... 24

Table 6. Length, weight, and sex of whitefish radio-tagged in October, 1995 ...... 26

Table 7. Length, weight, and sex of whitefish radio-tagged in April, 1996...... 27

Table 8. Within season returns (conventional tags)...... 32

Table 9. Between year returns (conventional tags)...... 34

viii List of Figures

Figure 1. Geographic distribution of the mountain whitefish, Prosopium williamsoni...... 4

Figure 2. British Columbian distribution of the mountain whitefish...... 5

Figure 3. Fraser Basin distribution of the mountain whitefish...... 6

Figure 4. Mean length of newly-emerged fry in the mainstem Fraser and tributary rivers. 13

Figure 5. Temperature regimes in a "western" and "eastern" Fraser tributary...... 14

Figure 6. Growth of mountain whitefish fry in the mainstem Fraser and major tributaries.15

Figure 7. Mean first year scale circuli counts (scale signatures) in the mainstem Fraser and an "western" (Nechako) and "eastern" (McGregor) tributaries...... 16

Figure 8. Seasonal changes in habitat use by young-of-the-year mountain whitefish in the upper Fraser system...... 17

Figure 9. Diet of young-of-year mountain whitefish...... 22

Figure 10. Adult length-at-age relationships in "western" (Nechako) and "eastern" (McGregor) upper Fraser tributaries...... 23

Figure 11. Diet composition of juvenile and adult mountain whitefish...... 25

Figure 12. Movement of fish radio-tagged downstream of Bridge, Prince George, October 1995...... 29

Figure 13. Movement of fish radio-tagged at McMillan Park, Prince George, in October 1995...... 30

Figure 14. Movement of fish radio-tagged at McMillan Park, Prince George, in April 1996...... 31

Figure 15. Principal Component Analysis of element ratios obtained from adult mountain whitefish scales...... 36

Figure 16. Principal Component Analysis of element ratios obtained from young-of-the- year mountain whitefish scales...... 37

Figure 17. Change in the relative level of Mercury 202 over the width of an adult mountain whitefish scale...... 39

ix Figure 18. Change in the relative level of Magnesium 25 over the width of an adult mountain whitefish scale...... 40

Figure 19. Change in the relative level of Zinc 68 over the width of an adult mountain whitefish scale...... 41

Figure 20. Change in the relative level of Rubidium 85 over the width of an adult mountain whitefish scale...... 42

Figure 21. The two foraging forms of mountain whitefish (upper fish is a "normal" and lower fish is a "pinocchio")...... 44

Figure 22. Principal Component Analysis of head shape in the two forms of mountain whitefish...... 45

Figure 23. Frequency distributions of gill rakers in the "normal" and "pinocchio" forms of mountain whitefish...... 47

Figure 24. Differences in the foraging behaviour of the "normal" and "pinocchio" forms of mountain whitefish. Note the absence of bottom directed foraging in the "normal" form...... 49

Figure 25. Length-weight relationship in the "normal" and "pinocchio" forms of mountain whitefish...... 52

Figure 26. Haplotype frequencies in "normal" and "pinocchio" mountain whitefish...... 53

x 1.0 INTRODUCTION

Indicator species are used to monitor the health of ecosystems. An assumption underlying this practice is that some species, by virtue of their distribution and ecology, are convenient integrators of the anthropogenic stresses that effect the overall health of the ecosystem in which they live. In aquatic ecosystems, fish are often chosen as indicator species because of their large size and the relative ease of identification; however, not all fish are suited to this role and many factors influence the choice of an appropriate indicator species. For example, in a large drainage system like the Fraser Basin any potential indicator species should be distributed throughout the basin. Such a basin-wide distribution allows replication of samples from both pristine and contaminated sites. Also, since contaminant transport is downstream, if there are multiple contaminant sources, contaminant gradients can be assayed with the same species in different parts of the basin. Potentially, this could allow replicate tests of contaminant uptake models.

Ideally, indicator species should be sedentary and reflect the level of contaminant exposure at the sites where the species was sampled. Very few, if any, fish fulfill this criterion. In addition, a good indicator species' diet should be restricted to aquatic organisms. This will minimize exposure to contaminants of terrestrial origin. Also, a good indicator species should be easy to sample, should be abundant enough to sample without depleting local populations, should be large enough to provide adequate tissue samples, and not be a commercially or culturally important species. Finally, the biology of any potential indicator species must be well enough known that the distribution, behaviour, and trophic status of its different life-history stages do not confound the adult's role as an integrator of ecosystem health.

The mountain whitefish (Prosopium williamsoni) has been used as an indicator species in the Columbia system (Mah et al 1989; Dwernychuk et al 1993; Nener et al 1995) and in the Peace-Athabasca system (Muir and Pastershank 1996). Thus, the mountain whitefish is a promising candidate for the role of indicator species in the Fraser Basin. Unfortunately, little is known about the habitat use and seasonal movements of the various life-history

1 stages of this species in the Fraser system. Consequently, this project was initiated to provide information on the life history and biology of mountain whitefish in a critical part of the Fraser Basin --- the area above and below the pulp mills at Prince George.

2.0 REVIEW OF MOUNTAIN WHITEFISH BIOLOGY

Over most of its geographic range, the mountain whitefish is a riverine species. In British Columbia, however, there are three distinct life-history patterns: a lacustrine pattern where the life cycle is completed entirely within a lake, a riverine pattern where the life cycle is completed entirely within flowing water, and an adfluvial pattern where the life cycle involves migrations between lakes and rivers. This review deals only with the riverine life- history pattern, however, information on the other life-history patterns is available in an excellent review of the biology of mountain whitefish (Northcote and Ennis 1994). The following summary of this species' biology is based on the Northcote and Ennis review, supplemented where appropriate, with additional B.C. information that relates to their potential as an indicator species in the upper Fraser system. The purpose of this summary is to provide a background against which upper Fraser life-history and habitat data can be interpreted.

2.1 Geographic distribution

Mountain whitefish occur only in western North America (Figure 1). Here, they are widely distributed along both slopes of the Rocky Mountains from Idaho and Wyoming in the south, to the Mackenzie River in the north. On the east slope of the Rockies they rarely extend out onto the Great Plains; however, west of the Continental Divide, mountain whitefish occur in suitable environments throughout most of Idaho (including the Snake River above Shoshone Falls), Washington, Oregon, north-central California, and British Columbia. Along the southern edge of this range there are a number of isolated, relict populations. These isolated populations are confined to altitudes above 1400 m and imply that the species' southern distribution is restricted by temperature.

2 In B.C., mountain whitefish are an interior species that only reaches the coast where large rivers like the Fraser, Skeena, Nass, and Stikine have cut canyons through the Coast Mountains. In the northwestern part of the province mountain whitefish occur in the upper Liard and Stikine systems, but they are absent from the Taku, Alsek and Yukon drainages (Figure 2). In the Fraser Basin, mountain whitefish occur throughout the system from the estuary to the Rocky Mountains (Figure 3).

3 Figure 1. Geographic distribution of the mountain whitefish, Prosopium williamsoni.

4 Figure 2. British Columbian distribution of the mountain whitefish.

5 Figure 3. Fraser Basin distribution of the mountain whitefish.

6 2.2 Life history

Mountain whitefish in the late fall or early winter. Depending on altitude and latitude, spawning has been recorded at water temperatures ranging from 0 to 10°C; however, peak spawning activity usually occurs at temperatures below 6° C (Northcote and Ennis 1994). Most B.C. populations spawn in either October or November, but in northern B.C. (Liard system) spawning occurs earlier in late September and early October (O'Neil et al 1982); while in southern B.C. (the Castlegar-Trail area) spawning in the mainstem Columbia and lower Kootenay rivers peaks in January and continues into early February (Anonymous 1997). Spawning occurs at dusk or, perhaps, at night. Consequently, field observations on breeding behaviours are scarce. Egg surveys in the Columbia and lower Kootenay rivers suggest that spawning occurs in shallow water (usually <3m) over coarse substrates and just upstream of riffles or rapids (Anonymous 1997). A similar preference for spawning in shallow water over coarse gravel and in, or close to riffles, has been recorded from the Liard and Parsnip rivers (McLeod et al 1978; O'Neil et al 1982).

Apparently, there is no site preparation and the eggs are simply scattered over the substrate. Like most fish, egg number in mountain whitefish is a function of female body size, and large females produce disproportionately more eggs than small females. In B.C., fecundity ranges from about 4,000 to 17,000 eggs, and newly-fertilized eggs range in size from 3 to 4 mm. The eggs lodge in crannies among the cobbles and rocks on the bottom and incubate over winter. The fry emerge in the spring or early summer (late March to early June, depending on latitude and altitude). Newly-emerged fry are small and upon emergence may drift some distance downstream before moving into shallow, low velocity areas along the river margins (e.g., side-channels and backwaters).

2.3 Growth, diet, and maturity:

Although mountain whitefish are small (about 15 mm in length) when they begin feeding, their mouth size relative to body size is larger than at any other stage in their life cycle. At

7 this time they are day-active and feed on the smallest life-stages of aquatic insects. The diet of juveniles and adults is similar to that of the young (predominately aquatic insects) but the size of prey increases with the size of the fish. The young-of-the-year grow rapidly and reach about 60-100 mm by October (the end of their first growing season). By the end of their second growing season, populations in northern B.C. usually exceed 100 mm in length, while southern populations of the same age often exceed 200 mm. Rapid growth continues through their third growing season and then slows as some individuals reach sexual maturity. Typically, males mature about a year earlier than females. The youngest mature males are at the end of their third growing season, and most individuals of both sexes are mature by the age of six. The maximum recorded age is 23 years but in B.C. relatively few individuals live longer than 12 years.

2.4 Habitat use, movements, and migrations:

During their first summer, young-of-the-year mountain whitefish are strongly associated with shallow, low velocity, deposition areas. They remain in this habitat until early autumn when they gradually moved into faster and deeper water. This tendency to move into faster, deeper water as body size increases continues throughout life. Thus, the summer habitat of juveniles is deeper and faster than the summer habitat of young-of-the-year, and the summer habitat of adults is faster and deeper water than the summer habitat of juveniles. In summer, adult foraging sites are usually located immediately downstream of riffles where the fast water breaks into either a pool or a slightly deeper area, or in quiet water just downstream of sites where the current sweeps around large woody debris or some other obstruction.

In many populations, there are well marked seasonal shifts in habitat use by adults, young- of-the-year, and juveniles. In B.C., the nature of these seasonal shifts differs among populations. For example, in the (Peace system) young-of the-year move downstream in the fall from small tributaries into larger streams where they over-winter (McLeod et al 1978); while in the Columbia and the Sukunkwa rivers the fall movement of fry is upstream (Ash et al 1981; Stuart and Chislett 1979). In the McGregor and Parsnip

8 rivers, apparently there is a fall movement of juveniles from tributaries into the mainstems of these rivers, presumably, these also are over-wintering migrations. Similar movements of juveniles from tributaries to over-wintering sites in mainstems occur in the Liard system in B.C. (Stewart et al 1982), the Sheep River in Alberta (Davies and Thompson 1976), and the Clearwater River in Idaho (Pettit and Wallace 1975).

Occasionally mountain whitefish spawn in the same region as their summer foraging area (MacAfee 1966), but most populations migrate from summer foraging areas to spawning sites. Usually these pre-spawning migrations are downstream into the lower reaches of large tributaries or into mainstem rivers, but upstream spawning migrations also are known in the Columbia and Kootenay rivers (Anonymous 1997). In addition, there usually are well marked post-spawning migrations to over-wintering sites (Davies and Thompson 1976; Pettit and Wallace 1975). Tagging studies in Idaho (Pettit and Wallace 1975) indicate that over-wintering sites can be used by a mixture of fish from several different summer foraging areas. In the spring, shortly after ice-out, both juveniles and adults perform return migrations (usually upstream) from over-wintering sites to summer foraging sites. In the Sheep River, Alberta, there is an additional migration to a spring foraging site that is followed by a later migration to a summer feeding area (Davies and Thompson 1976). The tagging studies in Idaho (Pettit and Wallace 1975) indicate that individual fish return to the summer foraging sites where they were originally tagged. In the , tagging studies suggest strong in-season fidelity to specific foraging sites, as well as the presence of several sub-populations or stocks between Castlegar and the US border (Anonymous 1997).

2.5 Summary

This short review of mountain whitefish life history suggests two aspects of their biology that could compromise their role as an indicator species in the upper Fraser system. First, the literature clearly establishes that migrations and seasonal habitat shifts are normal for this species. This means that the mountain whitefish sampled at a site do not necessarily reflect the level of contaminant exposure at that site. Second, the limited tagging data

9 available for this species suggest the presence of multiple stocks, often within relatively restricted geographic areas, and also suggests that these stocks may mix at some times of the year and segregate at other times. Thus, individuals taken from summer foraging sites on different years may represent the same stock, while individuals taken from the same over-wintering site on the same day may represent different stocks.

3.0 MOUNTAIN WHITEFISH IN THE UPPER FRASER SYSTEM

3.1 Life history

3.1.1 Spawning

In the upper Fraser system, mountain whitefish spawn in the late autumn (October and November). Although spawning was not observed in the Prince George region, inferences can be made about the time and places of spawning based on the presence of adults with mature gonads and the distribution of newly-emerged fry. Thus, “running” ripe males with well developed spawning tubercles were collected in the lower Nechako, mainstem Fraser, Willow, and Bowron rivers in mid-October, 1995 (water temperatures ranged from 4-7ºC). Presumably, these fish were either spawning or close to spawning.

3.1.2 Emergence

Fry (15-20 mm in length) were collected in early spring (late April and early May) near the sites where ripe adults had been observed; however, the fry may have been displaced downstream from where the adults actually spawned. Yolk-sac fry remain in the substrate until they reach about 14 mm. They then emerge from the substrate and begin exogenous feeding (Thompson 1974). Table 1 presents the results of a fry survey conducted in May and early June of 1996. This table indicates that fry less than 20 mm in length are widely distributed throughout the upper Fraser and its tributaries. For example, the Willow River was surveyed throughout its length and fry were found from headwater streams near Barkerville to its confluence with the Fraser (Table 1). This suggests that spawning not only occurs in the mainstem Fraser and the lower reaches of major tributaries, but also

10 occurs at suitable sites throughout the length of tributary rivers. Because there is evidence in both Idaho (Pettit and Wallace 1975) and Alberta (Thompson and Davies 1976) of homing to spawning sites, the wide but scattered distribution of fry in large tributaries like the Willow River suggests a complex, within drainage, stock structure.

One unexpected observation made during the fry survey was the similarity in the size and stage of development of the fry in tributaries with different temperature profiles (Figure 4). Like most fish, incubation rates in mountain whitefish are temperature dependent (Rajagopal 1979) and, since the temperature regimes in "western" tributaries (e.g., the Nechako) are mediated by large lakes while tributaries like the McGregor River

11 Table 1. Sites surveyed for newly-emerged fry in the upper Fraser system (1996).

Drainage Date Substrate +/- Whitefish Fry Faser Mainsteam U/S of Dome Cr. May 14, 1996 mud on gravel + D/S of Dome Cr. May 14, 1996 base gravel (clean) + Penny May 15,1996 sand on gravel + Between Avrial/Olsson Cr. July 3, 1996 sand on gravel + Northwood bridge 2 May 19, 1996 silt/mud on gravel + Simon Fraser bridge 1 May 16, 1996 sand on gravel + Stone Cr. confluence May 6,1996 sand on gravel + Woodpecker May 6, 1996 sand on gravel + Tree nursery May 3, 1996 silt/mud on gravel + Huble May 20,1996 sand on gravel + Willow river Willow, Giscome bridge 1 May 4, 1996 sand on gravel + Willow, FSR bridge 2 May 12, 1996 sand on gravel + Wansa lake outlet May12, 1996 gravel (clean) - Wansa creek May 12, 1996 gravel (clean) - Eaglet lake outlet May 13, 1996 gravel (clean) - Thurday creek May 24, 1996 large rock and gravel - Georage creek May 24, 1996 large rock and gravel - Pitoney creek May 24, 1996 large rock and gravel - Piney Creek May 24, 1996 large rock and gravel - Narrow lake outlet May 24, 1996 sand on gravel - Willow, Stony lake creek confluence May 24, 1996 silt/mud on gravel + Big Valley creek May 25, 1996 silt/mud on gravel + Willow, Tregillus creek confluence May 25, 1996 sand on gravel + Tregillus creek May 25, 1996 sand on gravel + Jack-of-clubs lake outlet May 25, 1996 sand and fine gravel - Williams creek May 25, 1996 gravel (clean) - Creek out of Barkerville May 25, 1996 sand on gravel + Nechako,Wilkins park May 27, 1996 sand on gravel + Stuart river May 28, 1996 sand on gravel + Nechako, 15 u/s of Fraser confluence May 10, 1996 sand on gravel + Nechako, at Finnmore May 17, 1996 sand on gravel + Chilako, Dahl creek confluence May 17, 1996 gravel (clean) - Chilako, Upper Mud river Rd. June 2, 1996 silt/mud on gravel + Bobtail lake outlet May 31, 1996 gravel (clean) - Salmon river Salmon, Hwy 97 bridge May 5, 1997 sand on gravel + Wright creek May 20, 1996 gravel (clean) - McGregor river McGregor, Chrurch Rd. bridge May 8, 1996 sand on gravel + Seebach creek May 8, 1996 sand on gravel + Bowron river Bowron, Bowron Rd. FSR bridge May 4, 1996 silt/mud on gravel + Bowron, Beaver Rd. FSR bridge May 11, 1996 sand on gravel + Purden creek May 15, 1996 gravel (clean) - Bowron, Coalmine Rd. May 12, 1996 sand on gravel + Naver creek Naver, Hwy 97 crossing May 23, 1996 gravel (clean) - Naver, Naver FSR bridge May 23, 196 silt/mud gravel + Dome Creek Dome, u/s of rail bridge May 15, 1996 gravel (clear) - Dome, Broderick Rd. bridge June 12, 1996 sand on gravel + Slim Creek Slim, Hwy 16 crossing May 15, 1996 gravel (clear) -

12 Figure 4. Mean length of newly-emerged fry in the mainstem Fraser and tributary rivers.

13 Figure 5. Temperature regimes in a "western" and "eastern" Fraser tributary.

14 Figure 6. Growth of mountain whitefish fry in the mainstem Fraser and major tributaries.

15 Figure 7. Mean first year scale circuli counts (scale signatures) in the mainstem Fraser and an "western" (Nechako) and "eastern" (McGregor) tributaries.

16 Figure 8. Seasonal changes in habitat use by young-of-the-year mountain whitefish in the upper Fraser system.

17 are glacial, it was expected that McGregor fry would emerge several weeks later than Nechako fry. Instead, fry of about the same size and state of development were present in all the tributaries at about the same time (Table 1 and Figure 4). Apparently, emergence from the gravel and the onset of feeding is triggered by some stimulus other than temperature. One such stimulus, the onset of spring flooding, has been suggested as a primary hatching stimulus in Scandinavian whitefish (Nasje et al 1995). If spring flooding also triggers hatching in mountain whitefish, this may account for this similarity in size and state of development in different tributaries at the beginning of the growing season.

3.1.3 Growth of young-of-the-year As water temperatures begin to rise in spring, the fry in the main river and in different tributaries are exposed to different temperature regimes (Figure 5). These differences in temperature clearly influences the growth rate of the fry. In the warmer "western" tributaries (e.g., Nechako and Chilako rivers) fry grow more rapidly and reach a larger size by the end of the growing season than fry in the cooler tributaries (e.g., McGregor, and Bowron rivers; Figure 6). These differences in first year growth produce different circuli counts in the mainstem Fraser River and in different tributaries (Figure 7) and provide a scale "signature" that stays with the fish for life. During the 1996 fry survey measurements were made on the habitats occupied by young-of-the-year whitefish. The results are summarized in Table 2. Basically, they occupied shallow, quiet, inshore waters (a habitat they share with young-of-the-year chinook salmon). Although such sites are ephemeral and dependent on water levels, young-of-the-year mountain consistently occur at sites with this combination of habitat characteristics. For example, during the fry survey they were strongly associated (P<0.001) with deposition areas (29 of 30 sites with y-o-y mountain whitefish) even though many of these deposition areas are clearly temporary (i.e., only a thin layer of silt or sand over coarse gravel). This habitat preference confines the young-of-the-year to the edges of streams and rivers, and it is only late in their first growing season that they show a detectable shift into deeper, faster water (Figure 8).

18 Table 2. Substrate characteristics of sites used by newly-emerged mountain whitefish fry.

Sites with clean gravel Sites with silt or sand whitefish fry present 1 29 whitefish fry absent 17 1

3.1.4 Diet of the young-of-the-year

Young-of-the-year mountain whitefish feed on the smallest life-history stages of aquatic insects and crustaceans (Figure 9). The major items (percent frequency) in their diet are chironomids (57%), mayflies (23%), crustaceans (8%), and simuliids (6%). As they grow, the frequency of mayflies in their diet increases (P=0.005) and the frequency of chironomids decreases (P<0.05). These changes in diet may reflect growth related changes in foraging preferences but, equally, they could also reflect seasonal changes in either prey size or prey abundance.

3.1.5 Growth of juveniles and adults

Age in juvenile and adult mountain whitefish was estimated from scales. In whitefish this method is reliable for younger adults but for older fish scales tend to under-estimate age relative to otoliths (Barnes and Power 1984). Nonetheless, our length-at-age estimates suggest growth trajectories similar to other central and northern B.C. populations (Table 3). These data also suggest consistent differences in growth rate between the "western" tributaries (e.g., the Nechako and Chilako rivers) and glacier-fed tributaries like the McGregor River (Figure 10).

In the upper Fraser system in October, the youngest reproductive males were aged at 2+ and the youngest females at 3+. This is consistent with other studies that suggest most male mountain whitefish reach sexual maturity at the end of their third growing season

19 (Sigler 1951; Brown 1952), and that females typically mature later than males (Thompson and Davies 1976).

Differences in the growth rates of young-of-the-year mountain whitefish in different tributaries of the upper Fraser produced differences in the number and spacing of circuli in the first growing season. Scales from adult whitefish collected for tissue samples from a fall aggregation in the mainstem Fraser near the mouth of Naver Creek were examined for their first year scale "signatures". In this regard, Table 4 indicates that about half the fish at this site displayed the mainstem Fraser "signature", but other individuals clearly that spent their first growing seasons in different environments. This suggests that this fall aggregation is made up of fish from more than one source and, potentially, more than one history of contaminant exposure.

Table 3. Distribution of scale “signatures” (number of first year circuli) on the scales of 32 adults collected from a fall aggregation in the mainstem Fraser River near the confluence of Naver Creek.

Circuli number 5 6 7 8 9 10 11 12 13 14 number of fish 1 2 6 5 3 4 7 2 1 1 with count

Table 4. Mean length at age for selected BC populations of mountain whitefish.

River Age 0 1 2 3 4 5 6 7 8 9 10 Columbia1 138 210 270 304 341 355 370 375 373 401 432 Nechako 91 175 232 250 281 300 319 336 349 360 McGregor2 63 102 156 191 210 229 247 269 284 309 312 Peace3 93 172 220 249 273 295 323 342 365 369 Liard4 52 110 168 227 278 314 336 354 394 421 426 1 Anonymous 1997. 2 O’Neil et al 1990, plus original data. 3 Pattenden et al 1990. 4 McLeod et al 1978.

20 3.1.6 Habitat of juveniles and adults

Like young-of-the-year, juvenile (1+) mountain whitefish are found throughout the upper Fraser system (i.e., in major and minor tributaries and in the Fraser mainstem). The most obvious change in habitat use by juveniles relative to fry is a shift to deeper, faster water. During the fry survey, over 300 seine hauls in young-of-the-year habitat produced only 2 juveniles, whereas 30 seine hauls in deeper, faster water (Table 5) produced over 100 juveniles. Late summer underwater observations in clear streams indicate that juveniles are associated with areas adjacent to adult habitats but in shallower and slower water than the adults.

Adult habitat use is more difficult to describe than that of either fry or juveniles. Late summer underwater observations in clear streams indicate that adults concentrate where shallow riffles or rapids break into deeper scour pools or sweep around large woody debris. At such sites, aggregations of adult mountain whitefish maintain position in the current just off the bottom and forage on drifting insects. They are often associated with but, unlike trout, whitefish rarely make foraging movements that are directed towards the surface. In large turbid tributaries (e.g., the McGregor, Salmon, Bowron and Willow rivers), and in the mainstem Fraser, adults could not be observed, and neither seining nor electroshocking were efficient methods of collection. However, angling with worms in the types of habitats described for clear streams produced adequate samples of adults.

21 Figure 9. Diet of young-of-year mountain whitefish.

22 Figure 10. Adult length-at-age relationships in "western" (Nechako) and "eastern" (McGregor) upper Fraser tributaries.

23 Table 5. Comparison of depth distribution of young-of-year and juvenile mountain whitefish. Pole seine sites: shallow water (<0.5 m) Beach seine sites: deeper water (>0.5 m) number of number of number of number of number of number of hauls fry juveniles hauls fry juveniles >300 >2,000 2 32 0 106

3.1.7 Diet of juveniles and adults

The summer diets of juveniles (1+) and adults extend the size-related trends seen in the diets of the young-of-the-year (i.e., an increase in the proportion of mayflies in the diet and a decrease in the proportion of chironomids; Figure 11). The three major items (percent frequency) in the adult diet are mayflies (67%), chironomids (11%), and tricopterans (9%). Among adults (2+ or older), the largest fish appeared to contain more stoneflies than the smaller adults; however, the sample size is too small to establish statistical significance. A striking feature of the diet of all size classes of mountain whitefish is the relative unimportance of prey items of terrestrial origin -- winged insects and spiders appear sporadically in the diet of adults but rarely make up more than 0.5% of the stomach contents. This dominance of aquatic insects in the diet of riverine mountain whitefish clearly reflects their habit of foraging close to the substrate.

24 Figure 11. Diet composition of juvenile and adult mountain whitefish.

25 3.2 Movements

3.2.1 Radio-tagging

Radio-tags (Lotech Engineering, Model FSM-3) were applied in mid-October of 1995 and mid-April, 1996. These tags were the smallest available tags that had a life-span of at least a month. The autumn tags were used in an attempt to track spawning and over-wintering migrations. The purpose of the spring tags was to determine if fish from a known over- wintering site dispersed to more than one summer feeding site.

3.2.1.1 Autumn tags Eight tags were applied to fish collected in the Fraser mainstem at a site 3.5 km downstream of Simon Fraser Bridge (Prince George). Five fish were tagged on October 11, one on October 12, and two on October 14, 1995 (Table 6). An additional five fish were tagged on October 15 and 16 at McMillan Park on the Nechako River about 3 km upstream of its confluence with the Fraser River (Table 7). The pattern of movement of

Table 6. Length, weight, and sex of whitefish radio-tagged in October, 1995 Tag number Date Length Weight Sex Tagging site 150.595 11/10/95 257 178.8 m below Simon Fraser Bridge 150.596 14/10/95 236 136.5 f (?) “ 150.615 14/10/95 251 171.2 f “ 150.636 12/10/95 269 213.6 m “ 150.716 11/10/95 223 111.6 ? “ 150.737 11/10/95 246 171.9 m “ 150.756 11/10/95 245 156.6 f “ 150.996 11/10/95 246 153.8 m “ 150.005 15/10/95 240 140.4 f MacMillan Park 150.017 15/10/95 248 149 m “ 150.026 15/10/95 235 152.9 m “ 150.036 15/10/95 247 176.8 m “ 150.166 16/10/95 261 194.6 f “

26 fish from both sites was similar (Figures 12 and 13). In all cases, movement was downstream and proceeded as a staccato series of stops and starts. Typically, tagged fish held position for a few days then moved rapidly downstream for several kilometers, held position again for a few days and then started down again. They were tracked from October 11 until November 26, or until the tags could no longer be located. The minimum downstream movement was by two Nechako fish that moved 2 km on the first day. Contact with one of these tags (150.026) was lost on the third day. The other tag

Table 7. Length, weight, and sex of whitefish radio-tagged in April, 1996.

Tag number Date Length Weight Sex Tagging site 149.266 17/04/96 286 258.3 ? MacMillan Park 149.956 17/04/96 277 224.5 ? “ 149.996 17/04/96 295 252.4 ? “ 149.976 17/04/96 306 301.8 ? “ 149.966 17/04/96 273 227.6 ? “

(150.005) did not move over the next 32 days and probably represents either a dead fish or a lost tag. The maximum downstream movement was also by a Nechako fish (150.017). This moved 1 km downstream, held position for 8 days, suddenly shifted to the mainstem Fraser and moved downstream for a total movement of 24 km in one day, then held for 10 days, moved another 15 km downstream in a single day and held for 12 days after which contact was lost. Over the 32 day tracking period this fish moved a total 40 km. Since all of the autumn-tagged fish were adults in, or close to, reproductive condition, these downstream movements may be part of a spawning migration. Alternatively, the tags may have been marginal for the size of mountain whitefish available and, consequently, the downstream movement could be an artifact.

3.2.1.2 Spring tags Five whitefish were radio-tagged at the McMillan Park site in the spring of 1996 (April 17). According to local anglers, mountain whitefish aggregate at this site during the winter but begin to move out of the area shortly after ice-out. The spring tags were followed

27 from April 18 until May 11. Like the autumn movements, the spring movements proceeded as a staccato series of stops and starts (Figure 14). In this case, however, fish moved both upstream and downstream from the tagging site. Again, the maximum movement was 40 km before contact was lost. Over 25 days this fish (149.966) moved up the Nechako River and into a major tributary (the Chilako River). Over the same time period another fish (149.996) moved downstream into the mainstem Fraser (a total distance of 17 km). The movements (both upstream and downstream) of the spring-tagged fish indicate that mountain whitefish of the size tagged are able to carry the tags. Furthermore, the pattern of spring movements suggests dispersal of more than one stock from a common over-wintering site.

28 Figure 12. Movement of fish radio-tagged downstream of Simon Fraser Bridge, Prince George, October 1995.

29 Figure 13. Movement of fish radio-tagged at McMillan Park, Prince George, in October 1995.

30 Figure 14. Movement of fish radio-tagged at McMillan Park, Prince George, in April 1996.

31 3.2.2 Conventional tags

Over the course of two summers (1995 and 1996) conventional tags were applied in the mainstem Fraser, in major tributaries (e.g., the Salmon, Bowron, McGregor, and Willow rivers) and one minor tributary (Dome Creek). Dome Creek was chosen because a fall aggregation of whitefish near its confluence with the Fraser is regularly sampled for tissues as a control site (i.e., no major contaminant sources upstream). Thus, if Dome Creek fish over-winter at this site, some tags might be recovered during tissue sampling. Although the number of conventional tags applied was relatively small (123), the results of the tag returns, both within season and between years, are instructive.

3.2.1.1 Within season tag returns Tags were recovered within a season in the Willow and McGregor rivers and in Dome Creek. Sixteen out of 123 conventional tags were recovered --- all within meters of the site where they were applied (Table 8).

Table 8. Within season returns (conventional tags). Locality Tagging Recapture Elapsed time Movement date date (days) (m) McGregor 8/1/95 9/20/95 50 0 Willow 9/14/95 10/24/95 41 0 Dome 6/28/96 8/5/96 38 0 Dome 6/29/96 7/1/96 2 0 Dome 6/29/96 7/19/96 20 0 Dome 7/18/96 9/9/96 72 0 Dome 7/18/96 7/18/96 0.5 0 Dome 7/18/96 8/5/96 18 0 Dome 7/19/96 8/5/96 18 0 Dome 7/19/96 7/24/96 5 0 Dome 7/19/96 7/24/96 5 0 Dome 7/19/96 8/2/96 14 0 Dome 7/24/96 7/25/96 1 0 Dome 7/24/96 9/19/96 57 0

32 The time at large ranged from half a day to 57 days (the average was 24 days). This 13 percent tag return is exceptional for mountain whitefish, but the lack of significant within season movement is typical of this species. For example, in the Columbia and Kootenay rivers, 55 out of 1,887 tagged mountain whitefish (3%) were recaptured (Anonymous 1997). Ten of these recaptures were made within 45 days of the original capture, and 9 of the fish were recaptured at the site where they were marked.

The within season recaptures suggest that once a summer foraging site is established, mountain whitefish in the upper Fraser system show little movement during the summer growing season.

3.2.1.2 Between year tag returns Tags were recovered after a year at large in both the McGregor River and in Dome Creek (Table 9). The McGregor recapture in the summer of 1996 was fortuitous (i.e., no special effort was made to re-sample the river for tags); however, the Dome Creek recaptures in 1997 were deliberate. The single recapture in the McGregor River was at the site where the fish originally had been tagged in 1995. During the summer of 1996, 87 mountain whitefish were tagged at five sites on Dome Creek. At the end of July 1997 an attempt was made to re-sample these sites. Unfortunately, time was limited (2 days) and weather conditions were poor --- the creek was high and turbid, and only three of the 1996 sites were fishable. Nonetheless, 14 mountain whitefish of the size range tagged in 1996 were collected at the three tagging sites. Of these, four (28%) were tagged: three at one tagging site and one at another site. All of the recaptures were at the exact sites where the fish had been tagged in 1996. Given the low number of tagged fish, the tag returns after one year are startling; especially for Dome Creek where underwater observations and late season sampling indicated that whitefish had left the Dome Creek tagging areas by late September, 1996. It is not known whether these fish migrated to the mainstem Fraser or to the lower reaches of Dome Creek, but it is clear that on their return migration the next summer tagged individuals return to the exact foraging sites that they occupied the previous year. These results imply a remarkable year-to-year site fidelity in adult mountain whitefish.

33 Table 9. Between year returns (conventional tags).

Locality Tagging Recapture Elapsed time Movement date date (days) (m) McGregor 8/1/95 8/1/96 365 0 Dome 6/29/96 7/30/97 396 0 Dome 7/18/96 7/30/97 378 0 Dome 7/19/96 7/30/97 377 0 Dome 8/18/96 7/31/97 357 0

3.3 Laser ablation

Laser ablation inductively coupled plasma mass spectrometry (LAICPMS) is a technique that allows the assessment of the relative concentrations of various elements in micro- samples. In this study scales were used as samples since they are calcified and exhibit annular growth (i.e., new layers are laid down each growing season). The technique is still in its infancy and the results should be treated with some caution.

3.3.1 Evidence of stocks

During the summer of 1995, scales were collected from adult fish in four rivers --- the mainstem Fraser, the Nechako, the Willow and the McGregor rivers. Scales from five individuals from each river were subject to laser ablation of the scale focus (centre). The ratios of 16 elements were recorded and subjected to a Principal Components Analysis. The results are presented in Figure 15 and show a complete separation between the four rivers. Since the focus of a scale is laid down during the first growing season, this result implies that the five adult fish collected from each river spent their first growing season in the same elemental environment, but that these environments were different for the fish collected from different rivers. The simplest explanation for this observation is that each group of adults was spending the summer in the same river where they had spent their first growing season.

34 To test this hypothesis we sampled young-of-the-year from the same rivers and added a sixth river (the Chilako). Again, the results were subject to Principal Components Analysis and, again, good separation was obtained between most rivers (Figure 16); however, the Nechako and Fraser samples were not totally separable. Since the Nechako River is a major tributary of the Fraser River, and the Nechako sample was taken about 1 km above the confluence of the two rivers, this result is not unexpected. What was unexpected, is that the elemental ratios obtained in the first analysis could not be used to build a Discriminant Function Analysis to classify fish by river in the second analysis. Apparently, the LAICPMS machine requires re-calibration after each run and its performance changes with changing environmental conditions. Therefore, without external reference standards of known composition, the technique provides only relative elemental concentrations. Consequently, samples run at different times are not directly comparable and, at its present stage of development, the technique can not be used to classify fish of unknown origin.

Interestingly, a trial run of the technique by the Department of Fisheries and Oceans using scales from Skeena system salmon and steelhead achieved a similar result. The technique distinguished fish of the same brood year, but from geographically different sites, with a high degree of certainty. However, a discriminant function based on the results of analysis of one brood year did not successfully classify fish of the same origin from different brood years. Apparently, there is significant variation between sample years in the concentrations of some of the elements (e-mail message to B. Ward from Dr. R. E. McNichol, August 25, 1997).

Still, within the technical constraints of the system, the results from samples run at the same time argue that the mainstem Fraser and its major tributaries in the Prince George area contain sufficiently distinctive elemental profiles that the portion of the scale laid down in the first growing season can be used to distinguish fish from the mainstem and from the different tributaries.

35 Figure 15. Principal Component Analysis of element ratios obtained from adult mountain whitefish scales.

36 Figure 16. Principal Component Analysis of element ratios obtained from young-of- the-year mountain whitefish scales.

37 3.3.2 Evidence of movement

Scales from two adult fish collected from the mainstem Fraser in a fall aggregation located near the confluence of the Fraser River and Naver Creek were ablated at eight equally spaced sites from the focus (centre) to the edge of the scale. This was done to determine if there were interpretable changes in the elemental composition of the parts of the scale laid down in different years. Since the eight sites on each scale were sampled consecutively on the same run, the relative concentrations of elements in different parts of the scale are directly comparable. Both of the scales chosen for this analysis showed an abrupt change in the spacing of the circuli in the third or fourth year of life and then a return to the previous circuli spacing pattern. This apparent change in growth rate implies a change in habitat.

The changes in elemental concentration over the scales was not the same for all elements. Some heavy metals (e.g., mercury 202) steadily increased in concentration from the centre of the scale to the edge (Figure 17), other elements (e.g., magnesium 25) steadily decreased along the same axis (Figure 18), whereas the concentration of still other elements (e.g., zinc 68) was remarkably variable (Figure 19). However, a few elements (e.g., rubidium 85), showed a pattern of change in concentration consistent with the circuli pattern on the scale. For these elements there is a clear peak in elemental concentration in the third or fourth year of life followed by a return to the original concentration (Figure 20). One interpretation of this pattern is that the fish moved to a new environment, perhaps for spawning or over-wintering, and then returned to the original environment. If this interpretation is correct, it suggests long-term fidelity to a specific elemental environment with occasional short-term shifts to different environments.

38 Figure 17. Change in the relative level of Mercury 202 over the width of an adult mountain whitefish scale.

39 Figure 18. Change in the relative level of Magnesium 25 over the width of an adult mountain whitefish scale.

40 Figure 19. Change in the relative level of Zinc 68 over the width of an adult mountain whitefish scale.

41 Figure 20. Change in the relative level of Rubidium 85 over the width of an adult mountain whitefish scale.

42 3.4 Evidence for two foraging forms of mountain whitefish

3.4.1 Morphological analysis

Early in the project it was noticed that there is considerable variation in head shape and general appearance in riverine mountain whitefish. This variation can be categorized into two forms --- one is the "normal" form and the other is the "pinocchio" form (Figure 21). The most obvious difference between these forms is the elongate snout in the "pinocchio". Both pinocchios and normals occur in the upper Fraser mainstem and in all of its major tributaries. This dichotomy is not confined to the Fraser system and pinocchios also occur in the Peace and Columbia systems; however, not enough observations are available to determine if there are differences in the frequency of pinocchios in different systems.

3.4.1.1 Head shape Extreme examples of the "pinocchio" form are obvious and are usually large adults; however, it is often difficult to categorize smaller adults and juveniles (<120 mm). This suggests that there are differences in the growth patterns of the two forms. To examine this question we made a series of head measurements (Table 11) and subjected them to Principal Components Analysis. Figure 22 presents the results of this analysis for a single

43 Figure 21. The two foraging forms of mountain whitefish (upper fish is a "normal" and lower fish is a "pinocchio").

44 Figure 22. Principal Component Analysis of head shape in the two forms of mountain whitefish.

45 population (Swift River, a Willow River tributary). Although there are two clearly different head shapes in the Fraser mainstem and in most upper Fraser tributaries, there also is variation among tributaries and even some apparently intermediate individuals. Most of the ambiguity in categorization, however, occurs in fish below 200 mm in length. One possibility is that this dichotomy in head shape influences the foraging performance of the two forms which, in turn, might influence the rate of contaminant intake from food. We attempted to examine stomachs contents taken from paired samples of the two forms (i.e., pairs of fish of the same size taken at the same time and place). Unfortunately, the sample sizes are too small to detect differences against the background of diurnal, seasonal and site-to-site variation in stomach contents. In an attempt to indirectly examine the question of foraging differences between the two forms, we examined gill raker number (a trait associated with foraging in fish), and made underwater observations in Dome Creek on the foraging behaviour of the two forms. Because condition is often used to assess fish health, we also examined the length-weight relationships in the two forms.

Over most of their geographic range, gill rakers number in mountain whitefish ranges from 17 to 26. In this respect, the upper Fraser populations are normal --- the range was from 17 to 25 --- but there was a significant difference (P <0.05) between pinocchios and normals in gill raker number (Figure 23). This suggests that they are morphologically equipped to exploit somewhat different prey.

3.4.1.2 Behaviour In late August 1996, underwater observations were made on pinocchios and normals at two sites associated with a pool below an obstruction on Dome Creek. At both sites adults of the two forms could be identified underwater at distances up to 10 meters. To estimate the size of fish underwater and at a distance, two weeks before the observation period two fish were angled, measured, tagged, and released into the pool. During an observation period a focal fish was observed for five minutes, and the number and nature, of the foraging movements were recorded. Focal fish alternated between pinocchios and

46 Figure 23. Frequency distributions of gill rakers in the "normal" and "pinocchio" forms of mountain whitefish.

47 normals, and foraging movements were classified as either water column directed (feeding movements made in a plane horizontal to the bottom) or bottom directed (vertical or oblique downward movements that made contact with the substrate).

Typically, normal whitefish make foraging movements directed towards items drifting in the current. Normal oriented themselves horizontally about 30 cm off the substrate, and hold a position from which they strike at drifting prey. In contrast to normal mountain whitefish, pinocchios are more bottom oriented. They position themselves about 20 cm above the bottom and do not hold at a site like the normals. Instead, pinocchios cruise over the substrate in short start-stop bursts, often with their heads tilted towards the bottom. About half of the foraging movements of pinocchios were directed at the substrate, with the other half of their foraging movements directed at drifting prey. When substrate foraging, pinocchios wedge their snouts into interstices amongst the rocks and swim vigorously into the substrate. This behaviour often dislodges gravel and pinocchios were observed to turn stones and strike quickly at the newly exposed substrate. Normal whitefish did not show this substrate shifting behaviour (Figure 24). These observations suggest that the two forms of mountain whitefish differ in their foraging behaviour.

3.4.2.3 Length-weight relationships Comparison of the length-weight relationships in pinocchios and normals reveals a significant difference (P <0.05) between the two forms. Pinocchios either are lighter for a given length than normals or, alternatively, they are longer for a given weight than normals. This difference in the length-weight relationship is most apparent in fish over 200 mm in length (Figure 25).

3.4.3 Molecular analysis

To determine if there are genetically differentiated mountain whitefish stocks in the Prince George area, tissue samples from fish collected in the mainstem Fraser, and in different tributaries, were examined for mitochondrial DNA restriction fragment length

48 Figure 24. Differences in the foraging behaviour of the "normal" and "pinocchio" forms of mountain whitefish. Note the absence of bottom directed foraging in the "normal" form.

49 polymorphisms (details in Appendix 1). This technique identified six haplotypes in the Prince George area (Figure 26). Two of the haplotypes (A and B) were common and geographically widespread. They occurred both within, and outside of, the Fraser Basin. The other four haplotypes (C, D, E, and F) were rare and had more restricted distributions. Although the frequency of the two common haplotypes (A and B) varied from site to site, there was no evidence of significant differences in their frequency among sites.

3.4.3.1 Pinocchios vs Normals Interestingly, however, the "normal" and "pinocchio" forms of mountain whitefish differed dramatically in the frequency of the two common haplotypes. All normals (100%) are haplotype A, while both haplotypes A and B occurred with about equal frequency (50%) in "pinocchios". Thus, although both haplotypes occurred in "pinocchios", only “pinocchios” displayed haplotype B. This is clear evidence of a genetic difference between the "normal" and "pinocchio" forms of mountain whitefish. How this genetic difference is maintained is unknown, but one possibility is assortative mating. Since mitochondrial haplotypes are passed through the maternal lineage, "normal" males may rarely, if ever, mate with "pinocchio" females.

3.5 Evidence for a mainstem stock

3.5.1 Molecular evidence

The frequencies of the rarer haplotypes (C, D, E, and F) are generally too low to permit comparisons among sites. There is, however, one instructive comparison. It is clear that haplotype D is more common in the mainstem Fraser than it is in any of the tributaries and, if all of the tributary sites are pooled and compared with the mainstem samples, there is a significant (P<0.05) difference in the frequency of haplotype D between the tributaries and the main river. Thus, the RFLP analysis suggest the presence of a distinct mainstem stock.

50 4.0 CONCLUSIONS

The goal of this study was to determine if the biology and population structure of the mountain whitefish suit this species for the role of an indicator species in the upper Fraser system. To this end we examined its life history, biology, population structure, and movements.

1) The biology and life history of mountain whitefish in the upper Fraser are complex but essentially similar to those in other places where the species has been studied.

a) There is evidence of movements between the main river and its tributaries that include spawning migrations, movements to over-wintering sites, and movements to summer foraging sites.

b) The distribution of newly-emerged fry indicates reproduction in both the main river and in the major tributaries.

c) Laser ablation of scales from both adults and fry indicate discrete stocks in the main river and major tributaries.

d) Restriction Fragment Length Polymorphism (RFLP) analysis suggests the presence of a mainstem stock.

e) Morphological and behavioural observations indicate the presence of two foraging forms of mountain whitefish in the upper Fraser system.

f) Molecular analysis indicate the two foraging forms are genetically distinct.

g) Scale "signatures" suggest that fall aggregations near the confluence of the Fraser and various tributaries consist of fish of mixed origins.

51 Figure 25. Length-weight relationship in the "normal" and "pinocchio" forms of mountain whitefish.

52 Figure 26. Haplotype frequencies in "normal" and "pinocchio" mountain whitefish.

53 2) Despite the complexity of its life history and stock structure, among the available fish species, the mountain whitefish remains the most suitable candidate for an indicator species. However, some precautions should be taken when sampling this species for contaminant loads.

a) Adult mountain whitefish are difficult to sample in the mainstem Fraser, except in the fall when they aggregate near the mouths of tributary streams. Since these fall aggregations appear to consist of fish of mixed origins, care should be taken in pooling samples. Ideally, the population of origin of each individual should be determined, and only samples from individuals from the same stock should be pooled. At present there is no simple way of determining the origin of individuals but micro-satellite analyses show promise in other salmonid (Angers et al. 1995).

b) Condition (length-weight relationship) is often used as a measure of fish health. Since the two foraging forms of mountain whitefish differ in their length- weight relationship and, the two forms are genetically distinct, they should be analyzed separately.

54 5.0 LITERATURE CITED

Angers, B., L. Bernatchez, and L. Desgroseillers. 1995. Specific microsatellite loci for brook charr reveal strong population subdivision on a microgeographic scale. Journal of Fish Biology 47 (Suppl. A): 177-185.

Anonymous. 1997. Lower Columbia River mountain whitefish monitoring program, 1994- 1996 investigations. Draft report prepared for BC Hydro by R. L. and L. Environmental Services Ltd., Report No. 514D, 101 p.

Ash, G., W. Luedke, and B. Herbert. 1981. Fisheries inventory and impacts assessment in relation to the proposed Murphy Creek Project on the Columbia River, B.C. Report prepared for BC Hydro by R.L.&L. Environmental Services Ltd.

Barnes, M. a., and G. Power. 1984. A comparison of otolith and scale ages from western Labrador lake whitefish, clupeaformis. Environmental Biology of Fishes 10: 297-299.

Brown, C. J. D. 1952. Spawning habits and early development of the mountain whitefish, Prosopium williamsoni, in Montana. Copeia 1952: 109-113.

Dwernychuk, L. W., T. G. Boivin, and G. S. Bruce. 1993. Fraser and Thompson rivers dioxin/furan trend monitoring program 1992. Final Report. Hatfield Consultants, West Vancouver, B.C.

Mah, F. T. S., D. D. MacDonald, S. W. Sheehan, T. M. Tuominen, and D. Valiela. 1989. Dioxins and furans in sediment and fish from the vicinity of ten inland pulp mills in British Columbia. Inland Waters Directorate, Environment Canada, Vancouver, B.C.

McAfee, W. R. 1966. Mountain whitefish. In L. A. Calhoun [ed.]. Inland fisheries management. California Fish and Game, Bulletin

McLeod, C., J. O'Neil, and M. Psutka. 1978. McGregor River Diversion Project. Fisheries and benthic fauna. Vols. I III. Report prepared for BC Hydro and Power Authority by Renewable Resources Consulting Services Ltd. on behalf of Reid, Crowther and Partners Ltd.

Muir, D. C. G., and G. M. Pastershank. 1996. Environmental contaminants in fish: spatial and temporal trends of polychlorinated Dibenzo-p-Dioxins and Dibenzofurans in Athabasca, Peace, and Slave river drainages, 1992-94. Northern River Basins Study Technical Report No. 129

55 Naesje, T. B., B. Jonsson, and J. Skurdal. 1995. Spring flood: a primary cue for hatching of river spawning Coregoninae. Canadian Journal of Fisheries and Aquatic Sciences 52: 2190-2196.

O'Neil, J., C. McLeod, L. Noton, L. Hildebrand, and T. Clayton. 1982. Aquatic investigations of the , British Columbia and Northwest Territories, relative to proposed hydroelectric development at Site A. Report prepared for BC Hydro and Power Authority by R.L.&L. Environmental Services Ltd., Edmonton.

Pattenden, R., C. McLeod, G. Ash, and K. K. English. 1990. Site C hydroelectric development, pre-construction fisheries studies: fish movements and population status, 1989. Report prepared by R.L.&L. Environmental Services Ltd. and LGL Limited, for BCHydro, Environmental Resources Division, Vancouver.

Pettit, S. W., and R. L. Wallace. 1975. Age, growth, and movement of mountain whitefish, Prosopium williamsoni, (Girard), in the North Fork Clearwater River, Idaho. Transactions of the American Fisheries Society 104:68-76.

Rajagopal, P. K. 1979. The embryonic development and the thermal effects on the development of the mountain whitefish, Prosopium williamsoni (Girard). Journal of Fish Biology 15: 153-158.

Sigler, W. F. 1951. The life history and management of the mountain whitefish Prosopium williamsoni (Girard) in the Logan River, Utah. Agricultural Experimental Station, Utah State College, Bulletin 347, 21 p.

Stewart, R. J., R. E. McLenehan, J. D. Morgan, and W. R. Olmsted. 1982. Ecological studies of Arctic grayling (Thymallus arcticus), Dolly Varden char (Salvelinus malma), and mountain whitefish (Prosopium williamsoni) in the Liard drainage, B. C. Report prepared for Westcoast Transmission Company Ltd. and Foothills Pipe Lines (North B.C.) Ltd. by E.V.S. Consultants Ltd., Vancouver.

Stuart , K. M., and G. R. Chislett. 1979. Aspects of the life history of Arctic grayling in the Sukunka drainage. B.C. Ministry of Environment, Fish and Wildlife Branch, Prince George, B.C.

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.

Thompson, G. E. 1974. The ecology and life history of the mountain whitefish (Prosopium williamsoni Girard) in the Sheep River, Alberta. MSc thesis, University of Calgary, Calgary, Alberta.

56 Thompson, G. E., and R. W. Davies. 1976. Observations on the age, growth, reproduction, and feeding of mountain whitefish (Prosopium williamsoni) in Sheep River, Alberta. Transactions of the American Fisheries Society 105:208-219.

57 APPENDIX

Restriction Fragment Length Polymorphism (RFLP) analysis

58 A.1 DNA Extraction

Mitochondrial DNA was extracted using a modified version of the method of Taylor and Bentzen (1993). Liver, heart, and peduncle muscle were dissected from EtOH 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 digestion temperature was 37° C. After 8-10 hours poorly digested samples were re-incubated 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 quantitatively extracted in preparation for final DNA precipitation.

Cold isopropanol was added to the aqueous extractions, they were 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 re-suspended in 75-150 ml of TE buffer (pH 8.0) and cold stored.

The DNA concentration of the extracts was determined with Spectrophotometer analysis. A standardized volume of 3ml of the re-suspended DNA precipitate sample solutions were diluted in 247ml of 0.5M TE buffer (pH 8.0). The diluted suspension was compared to a control standard in a Promega spectrophotometer and DNA concentrations were calculated and recorded.

A.2 Mitochondrial DNA analysis

59 Dilution trials and reaction conditions were standardized and refined for the Polymerase Chain Reaction (PCR) amplification of the NAD5/6, and the combined Cytochrome b, D- loop loci of the mountain whitefish mtDNA genome.

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 extract and PCR reagents. The same reaction concentrations were used for the isolation and replication of both the NAD5/6, and d-loop/Cyt b loci. The standard PCR cocktail consisted of:

- 0.1mg/ml of raw DNA extract

- 0.8 mM dNTP nucleotide mix

- 0.6 mM of HN20, CGlu (Primers A,B)

- 2.0 m/ml TAQ polymerase (added last)

- 2.0 mM Magnesium Chloride

The DNA extract and the PCR reagents were combined and the reaction conducted via the hot start method in a Robocycler unit. 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 quantified by staining aliquots of product with 1.5% Ethidium Bromide/ loading buffer solution. The stained products were electophoreticlly separated on a 1% agarose gel loaded with a 1Kb molecular weight standard ladder at between 60- 75 volts for 1 to 1.5 hours. The separated gel was fluoresced under ultra-violet light and the fluorescent profiles photographed on Polaroid film.

As recommended by the vendor (New England Biolabs), aliquots of high quality PCR products were digested in individual vials with one multi-pentameric (Nci I), one multi-

60 hexameric (Sty I), one pentameric (Hinf I), and four quatrameric ( Dpn I, Hae III, Msp I, Rsa I ) restriction endonucleases .The digested fragments were stained with loading buffer, loaded into, and electrophoretically resolved, on 1.5 % agarose gels emersed in TE buffer at between 75 and 90 volts for 2.5 to 3.5 hours. Each gel was run with a 1kilobase ladder as a molecular weight standard. The separated gels were stained for 20 minutes in an ethidium bromide solution, washed in 0.5M TE buffer for a further 20 minutes and finally illuminated with UV light for Polaroid photography.

A random selection of 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 and 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 blocked according to their collection location and life-history stage.

Yates’ corrected Chi-squared contingency tests were conducted to determine if there was any association fish morphology and haplotype frequency. 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) regardless of time or space, and a localized comparison from the Nechako river where fish of both morphotypes were collected at the same time and space.

61