Review and Assessment of Walleye Genetics and Stocking in Alberta

Transcription

1 Review and Assessment of Walleye Genetics and Stocking in Alberta CONSERVATION REPORT SERIES

2 The Alberta Conservation Association is a Delegated Administrative Organization under Alberta s Wildlife Act. CONSERVATION REPORT SERIES 25% Post Consumer Fibre When separated, both the binding and paper in this document are recyclable

3 Review and Assessment of Walleye Genetics and Stocking in Alberta By Fiona D. Johnston and Andrew J. Paul G8 Legacy Chair in Wildlife Ecology University of Calgary Calgary, AB T2N 1N4

4 Report Series Co editors PETER K.M AKU GARRY J. SCRIMGEOUR Alberta Conservation Association Alberta Conservation Twin Atria Building P.O. Box #111, Avenue Baker Centre Postal Outlet Edmonton, AB, T6B 2X3 Edmonton, AB, T5J 4M9 Conservation Report Series Types: Data, Technical ISBN printed: ISBN online: Publication Number: T/112 Disclaimer: This document is an independent report prepared by the Alberta Conservation Association. The authors are solely responsible for the interpretations of data and statements made within this report. Reproduction and Availability: This report and its contents may be reproduced in whole, or in part, provided that this title page is included with such reproduction and/or appropriate acknowledgements are provided to the authors and sponsors of this project. Suggested citation: Johnston, F.D. and Paul A.J Review and assessment of walleye genetics and stocking in Alberta. Technical report (T ) produced by Alberta Conservation Association, Edmonton, Alberta, Canada. 91 pp + App. Cover photo credit: David Fairless Digital copies of conservation reports can be obtained from: Alberta Conservation Association P.O. Box Baker Centre Postal Outlet Edmonton, AB, T5J 4M9 Toll Free: Tel: (780) Fax: (780) info@ab conservation.com Website: conservation.com i

5 EXECUTIVE SUMMARY Walleye (Sander vitreus) are native to Alberta occurring in large rivers and lakes throughout the province. However, overexploitation has resulted in the decline of many populations. Furthermore, walleye abundance in the province is limited by availability of large lakes that provide suitable walleye habitat and because Alberta falls near the northern limits of the species range. To address declining walleye populations and further extend their distribution, walleye have been stocked into 118 different waterbodies in the province. This report examines effects stocking may have on genetic diversity of walleye in Alberta. Four hazards are associated with loss in genetic diversity from any stocking program: extinction, loss of within population genetic variability, loss of among population genetic variability and domestication selection. Each step in the hatchery processes (i.e., broodstock selection, broodstock collection, mating, rearing or release) is associated with a genetic hazard. The vulnerability of a population to a particular hazard depends on the product of its risk (probability an event that produces the hazard occurs) and the consequence of the hazard. Assessing vulnerability can identify key weaknesses in the hatchery process and areas for improvement which provide the greatest benefit to maintenance of genetic diversity. In a survey of agencies responsible for walleye stocking in Canada and the United States, maintenance of genetic diversity is an important management concern. However, most agencies have only unofficial policies to prevent movement of walleye across major watershed boundaries. Few jurisdictions have comprehensive guidelines to minimise negative influences of stocking on genetic diversity from various components of the hatchery process. Several states are currently undergoing studies to examine existing and historical genetic diversity. With this information, management agencies intend to further identify: populations that represent distinct genetic groups; the vulnerability of these groups to alteration from a stocking program; and, how diversity can best be maintained. ii

6 Walleye stocking in the province of Alberta has occurred since The spatial pattern of stocking has tended to move walleye genes from northern lakes and watersheds to southern ones. The extent to which these genes have introgressed into southern populations is largely unknown. The only genetic work on walleye completed in Alberta suggests three important patterns. First, river and lake population of walleye are genetically distinct. Second, genetic diversity of walleye within the province may be substantial and unique to different drainage basins. Third, stocked walleye may have introgressed into both recipient lake populations and connected riverine populations. These patterns need to be confirmed using additional genetic techniques and populations. We believe the most appropriate way of proceeding with genetic analyses intended for management purposes in the province requires two initial steps. First, define evolutionary significant units (ESUs) based on a defined protocol (examples are provided in the text). The ESUs should be based on a combination of genetic, ecological and geographic information that designates populations with respect to: a) reproductive isolation and b) evolutionary lineage. Second, goals of a stocking program must be evaluated with respect to the relative importance of stock performance (e.g., survival, growth and reproductive success of stocked progeny) versus maintenance of genetic diversity, although these are not necessarily mutually exclusive goals. For example, jurisdictions that have no native walleye will likely place little emphasis on maintenance of genetic diversity and most efforts into performance. In contrast, jurisdictions with native populations should place more emphasis in walleye stocking on maintenance of diversity rather than short term performance. iii

7 ACKNOWLEDGEMENTS We thank the Alberta Conservation Association for funding this project, especially Paul Hvenegaard and Garry Scrimgeour for their input, support and review of the project. We thank Jim Wagner (Alberta Sustainable Resource Development; ASRD), Hugh Norris (ASRD), and Michael Sullivan (ASRD) for their participation in the planning stages and helpful suggestions. In particular, we thank Jim Wagner for providing us with a compilation of walleye stocking records for the province. We would like to thank all people contacted across Canada and the United States for their responses to inquiries about walleye stocking programs within respective jurisdictions; their feedback was very informative and appreciated. We would also like to thank Dr. Brian Sloss (United States Geological Survey, University of Wisconsin Stevens Point); Dr. Carol Stepien (University of Toledo); Dr. Rex Strange (University of Toledo); and Dr. Chris Wilson (Ontario Ministry of Natural Resources) for all their education and input regarding genetic techniques and population genetics. Finally, we thank Carly Greenway for developing the maps on walleye distributions and stocking locations. iv

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9 TABLE OF CONTENTS EXECUTIVE SUMMARY...ii ACKNOWLEDGEMENTS...iv TABLE OF CONTENTS...v LIST OF FIGURES...vi LIST OF TABLES... viii LIST OF APPENDICES...ix 1.0 INTRODUCTION Introduction and intent Walleye distribution GENETIC CONSIDERATIONS AND STOCKING Genetic processes Genetic hazards Genetic hazards in relation to the hatchery process Risk assessment and prioritization GENETIC CONSIDERATIONS BY OTHER JURISDICTIONS METHODS FOR ASSESSING AND MANAGING GENETIC DIVERSITY Molecular genetic techniques Evolutionary significant units GENETIC DIVERSITY OF WALLEYE IN ALBERTA Methods for assessing walleye stocking and distribution in Alberta Walleye and sauger distribution Walleye stocking history Stocking sources Walleye genetics in Alberta Sampling strategy CONCLUSIONS AND RECOMMENDATIONS Conclusions Recommendations LITERATURE CITED APPENDIX...92 v

10 LIST OF FIGURES Figure 1. The native distribution of walleye in North America... 2 Figure 2. The native distribution of sauger in North America... 3 Figure 3. Size of walleye stocking programs in North America in relation to their native distribution... 3 Figure 4. An illustration of random genetic drift Figure 5. The frequency of allele A1 in a population of 20 individuals after 100 generations Figure 6. Figure 7. Figure 8. Figure 9. The probability of maintaining heterozygotes in relation to the size of the population when under the influence of random genetic drift... 8 Change in allele frequency due to directional selection for dominant allele A The effect of gene flow on the frequency of an allele A1 in a recipient population Determining vulnerability of a population to genetic change from the hatchery activity of selecting dissimilar broodstock using a risk assessment framework Figure 10. A four step process to determine evolutionary significant units (ESUs) Figure 11. Figure 13 Distribution patterns of five major walleye mtdna haplotypes in North America in relation to their native distribution The distribution of stocking locations in the major drainage basins of the province of Alberta Figure 14 The distribution of stocking events over time in the province of Alberta Figure 15. Figure 16 The distribution of donor populations used for stocking walleye in Alberta Distribution of recipient populations in relation to the donor strain they were stocked Figure 17. The distribution of information on walleye genetics in Alberta Figure 18. The distribution of lakes in Alberta for which aging information is available vi

11 Figure 19. Figure 20 The distribution of rivers in Alberta for which aging information is available Distribution of waterbodies for which aging data is available for walleye prior to any stocking event vii

12 LIST OF TABLES Table 1. Table 2. Table 3. The time scale at which the five genetic processes generally work at, and their impacts on within and among population diversity Genetic hazards associated with the cultivation and introduction of artificially propagated fish to wild populations, and guidelines to reduce the risk of these hazards occurring Summary of information obtained from management personnel and documents regarding genetic considerations for walleye stocking programs from various jurisdictions in Canada and the United States viii

13 LIST OF APPENDICES Appendix 1. The history of stocking in Alberta Appendix 2. Annotated bibliography of genetic references with emphasis on walleye ix

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15 1.0 INTRODUCTION 1.1 Introduction and intent Walleye (Sander vitreus) is a species native to Alberta, occurring in 64 large rivers and approximately 177 lakes throughout the province (Berry 1995). However, due to their popularity as a sportfish, high levels of fishing pressure have resulted in the decline of many of these populations (Post et al. 2002). Walleye abundance in the province is further limited by the availability of large lakes that provide suitable walleye habitat and because Alberta falls near the northern limits of the species range (Berry 1992). To address declining walleye populations, the walleye stocking program was established to maintain self reproducing populations, re establish lost walleye populations and create new populations where suitable (Berry 1992). In order to achieve these goals and conserve native walleye populations in the province, information on the genetic diversity of these populations must be considered. It is the intent of this study to examine the following questions: 1. What are the genetic concerns when designing a stocking program, and what guidelines should be used? 2. What concerns do other jurisdictions consider when designing a stocking program and how do they address these concerns? 3. What is the history of walleye stocking in Alberta? 4. What do we know about genetic diversity of walleye in the province, and how would we assess diversity? 1.2 Walleye distribution Walleye are distributed throughout much of North America (Figure 1). This popular sportfish is native to many provinces and states, with a natural range extending from Québec south to northern Alabama, west to Alberta and as far northwest as Great Slave Lake (Billington and Maceina 1997, Scott and Crossman 1998). This distribution overlaps with the native distribution of sauger (Sander canadensis), a closely related species (Figure 2) (Billington 1996). In addition, walleye have been stocked into 1

16 numerous waterbodies both within and outside of their native range (Figure 3) (Billington 1996, Billington and Maceina 1997, Scott and Crossman 1998, Perrin et al. 2003). However, in recent years debate has arisen over the appropriateness of stocking as a management technique (Busack and Currens 1995, Incerpi 1996, Waples 1999). This is because concerns have been raised with regard to the effects of fish introductions on the genetic diversity of native populations (Evans and Willox 1991, Phillip 1991, Ferguson et al. 1995, Miller and Kapuscinski 2003, Page et al. 2005). Figure 1. The native distribution of walleye in North America (extracted from Billington 1996). 2

17 Figure 2. The native distribution of sauger in North America (extracted from Billington 1996). Figure 3. Size of walleye stocking programs in North America in relation to their native distribution (extracted from Fenton et al. 1996). It should be noted that this information may have changed in the last decade; however, it does provide some indication of the distribution of stocking effort. The native range of walleye is indicated by the black outline. 3

18 2.0 GENETIC CONSIDERATIONS AND STOCKING Genetic diversity is thought to reflect a population s resilience to adapt to environmental change (Busack and Currens 1995, Ferguson et al. 1995, Laikre 1999). Genetic variation is assumed to be the product of past selective pressures, non selective genetic processes, and ecological events. The genetic variation observed within populations can reflect the unique history and selection environment that the population has experienced (Philipp et al. 1993). Therefore, for long term management it is felt that genetic diversity both within and among populations should be conserved (Ryman 1991, Shaklee and Currens 2003). The hatchery process (i.e., collection, mating, rearing and release) may result in changes in genetic diversity (Waples 1991, Busack and Currens 1995, Laikre et al. 1999, Epifano et al. 2001, Miller and Kapuscinski 2003, Page et al. 2005). As a result genetic structure should be considered when designing a stocking program. However, in order to understand what genetic concerns are related to a stocking program, one must first understand what genetic diversity is and how it changes. 2.1 Genetic processes Genetic information of individuals is contained within molecules of deoxyribonucleic acid 1 (DNA) (Kapuscinski and Jacobson 1987). Strands of nuclear DNA become structured to form chromosomes. Each strand of DNA contains sequences of nucleotides that pair together to form base pairs. These sequences of nucleotides form genes (Laikre 1999). Genes contain an individual s genetic code and are the templates for the production of different proteins (Busack and Currens 1995). The position of a gene on the DNA is called a locus (Kapuscinski and Jacobson 1987). However differences in the sequence of nucleotides may produce variants of the same gene, called alleles (Laikre 1999). Fish generally have two copies of an allele, one from each parent, although mitochondrial DNA is inherited solely from the mother (Billington 2003). The combination of alleles may be homozygous (identical) or heterozygous (different), and this combination is the genotype of that individual (Busack and Currens 1995). The presence of different genotypes (i.e., the occurrence of different 1 Bolded words indicate important definitions or processes that are repeated throughout the document. 4

19 alleles) within a population represent within population genetic diversity, and differences in occurrence of alleles and their frequency within a population produce among population variability (Laikre 1999). The physical expression of the genotype is the phenotype, and reflects both the individuals genotype and the environment they experience. Most of the phenotypic traits that control a fish s performance or fitness (e.g. growth rates) are controlled by multiple loci on the chromosomes and can be strongly influenced by the environment (Hallerman 2003a). These phenotypes controlled by multiple loci are called quantitative traits. Information about differences in genetic structure of individuals or populations may be utilized to identify distinct genetic groups. The frequency of different alleles in a population, and hence the genetic diversity of the population is determined by the processes of: mutation, random genetic drift, gene flow (migration), selection, and non random mating. These five processes change allele frequency and/or the number of different alleles in a population Mutation Mutation is the only process that results in the production of new alleles (Hallerman and Epifano 2003), and occurs in a number of different ways that will not be discussed here. However, because mutation introduces new alleles it functions to increase genetic diversity. Mutation occurs at a very slow rate (i.e., 10 3 to 10 6 mutations per gene per generation depending on species, gene and the environment) (Laikre 1999, Hallerman and Epifano 2003). Therefore, mutation is unlikely to be a significant process when considering the effects of stocking on genetic diversity of fish populations due to the long time frame involved (Laikre 1999) Random genetic drift The second process that may result in a change in genetic diversity is random genetic drift. Genetic drift is a random, and hence, non selective process that has a more pronounced effect on small populations (Hallerman 2003b). Genetic drift occurs when there is a change in allele frequency within a population by chance. Genetic diversity is reduced if drift results in the frequency of an allele reaching zero and therefore the loss of the allele from the population. For example, if there was a catastrophic event that 5

20 killed a large percentage of the population, such as disease or overharvest, the genetic diversity of the remaining population is determined by the individuals that survived (Figure 4). As a result, within population genetic diversity declines due to random genetic drift, and the smaller the population of surviving individuals the lower the likelihood that the genetic diversity observed in the original population will be maintained in the new smaller population. The susceptibility of populations to genetic drift should be considered when managing systems where populations are near collapse. The founder effect is a special case of genetic drift (Hallerman 2003b). This is when a small number of individuals colonize a new area, for example the introduction of fish to previously uninhabited waterbodies, or when stocked fish are produced from a limited population or reared from a limited number of individuals. The genetic diversity of the founding population will often be low simply because the number of individuals available to contribute unique alleles is low. Future populations that descend from the original founder population will exhibit constrained genetic diversity as a result. Therefore, the effects of random genetic drift on both the donor and recipient populations should be considered when stocking. Population individuals removed a Population individuals removed b Frequency Frequency Allele Type Allele Type Figure 4. An illustration of random genetic drift. Panel (a) demonstrates the frequency distribution of different alleles in a population of 100, and that same population after 10 individuals have been randomly removed. Panel (b) demonstrates the frequency distribution of different alleles in a population of 50, and that same population after 10 individuals have been randomly removed. The dashed line in Panel (b) indicates the right part of the allele distribution is lost by chance from a small population of 50, a portion that was not lost in the larger population of

21 It may come as some surprise that, in the absence of other processes, genetic drift always results in loss of genetic diversity and fixation of alleles (Figure 5). This seems to counter the Hardy Weinburg law, which states the genotype frequency on average for diploids with a frequency p for allele A1 and 1 p (q) for allele A2 is: p 2 for A1A1; 2pq for A1A2; and, q 2 for A2A2. However, solely through chance, alleles will be lost through random mating when offspring consist of one homozygous combination (the allele of this homozygote is said to be fixed). The probability of extinction for an allele depends on its initial frequency (extinction probability = 1 initial frequency, fixation probability = initial frequency; Gillespie 2004). Once this occurs, the other allele is lost from the population and genetic diversity reduced. While genetic drift is an inevitable process for all populations of finite size, it is a process that is highly dependent on populations themselves. Allele A 1 frequency Generation Figure 5. The frequency of allele A1 in a population of 20 individuals after 100 generations. The initial frequency of allele A1 is 20% (frequency of allele A2 = 80%) and the plot shows six separate simulations. After 100 simulations, allele A1 was lost (i.e. fixation of A2) in 80% of the simulations. The rate of genetic drift is not constant among populations and depends on several factors of which population size is the most important. Since heterozygotes have a copy of both alleles, their presence in a population reflects genetic diversity. The 7

22 probability (Ht) of any individual being heterozygous within a diploid population at some generation t can be determined as 1 H t = H 0 (1 ) 2N t where H0 is the probability of being heterozygous at generation 0, N is population size and we assume alleles are drawn at random from the population for each generation (i.e., random mating; Gillespie 2004). Quite clearly, population size plays an important role in determining whether heterozygotes persist in a population (Figure 6). Probability of maintaining heterozygotes Population size Figure 6. The probability of maintaining heterozygotes in relation to the size of the population when under the influence of random genetic drift. However, this only considers the ideal case where alleles are drawn at random from the population. Several factors can affect this assumption (e.g., unequal sex ratios, inbreeding, variable family sizes) (Laikre 1999, Hallerman 2003b). We can correct for violations to random mating by adjusting true population size (N) in the above equation to effective population size (Ne). Where Ne simply represent the population as if it were an ideal population where alleles are chosen at random. For uneven sex ratios, Wright (1933) developed the correction for N necessary to produce the ideal randomly mating population Ne as 8

23 N e N f N m = 4 N + N f m where Nf and Nm are numbers of females and males, respectively. However, the sex ratio correction is often trivial when placed against uncertainty in the original population estimate (Gillespie 2004). The effective population size is, therefore, smaller than the population census size (Laikre 1999). The inverse of effective population size is proportionally related to the rate of inbreeding in the population, and gives a measure for the rate of loss of genetic heterogeneity (Ryman and Laikre 1991). In order to determine the effects of stocking on a population, the effective population size of both the donor (hatchery) and recipient populations must be considered to account for random genetic drift (Ryman and Laikre 1991) Selection Selection is the third process that results in a change to genetic diversity. In most cases selection is thought to act on deleterious phenotypes and resulting in loss of these alleles from the population. Or from the other perspective, selection results in fixation of advantageous alleles. This is directional selection (Hallerman 2003c). Directional selection is a destabilizing form of selection because it drives alleles towards fixation. Conversely, stabilizing selection drives allele frequencies towards an intermediate equilibrium value. Stabilizing selection can occur if there is frequency dependent selection, or if heterozygous individuals are more fit than homozygotes. Selection can influence allele frequencies in complex ways, particularly if there is linkage between genes. Ultimately, however, selection favours traits that increase an individual s fitness (survival and reproduction) (Hallerman 2003c), and usually results in a decrease in genetic diversity (Figure 7). There are many types of selection, such as natural selection, artificial selection and sexual selection. Sexual selection may limit the reproductive success of some individuals with unattractive traits. Fish culturing procedures may also have both intentional and unintentional selective effects on the donor populations genetic diversity. These artificial selection pressures and effects will be outlined in the following chapter. 9

24 Individual populations can also experience unique environments that impose unique selective pressures and result in the population becoming locally adapted to the environment it experiences (Hallerman 2003d). Local adaptation can function to increase genetic diversity between populations. However, local adaptation can also constrain the fitness of an individual if that individual is forced to survive in an environment that differs from the one it is locally adapted to. 1 Allele A 1 Frequency selection 0.4 selection Generations Figure 7. Change in allele frequency due to directional selection for dominant allele A1. The selection coefficient of 0.4 represents a relatively high selective pressure and 0.1 a moderate selective pressure Gene flow The fourth process that may alter genetic diversity is gene flow. Gene flow occurs when organisms migrate between discrete reproductive units and contribute to the local gene pool (Gharrett and Zhivotovsky 2003). This could mean movement between waterbodies or, within larger waterbodies where discrete reproductive subpopulations may exist, or simply a fish straying from its natal spawning grounds to one nearby (Laikre 1999). When migration occurs, the genetic material from one population is mixed with that of another population. For example, the introduction of cultured fish may result in introgression, the incorporation of the broodstock s genes into the gene 10

25 pool of a wild population (Billington 2003). If these two populations differ in their genetic composition, the result is a change in genetic diversity of the recipient population. This usually results in an increase of within population variability, at least initially, but a decrease in among population variability. If a large amount of gene flow occurs, then the populations that were once genetically distinct will become homogenized and essentially act as one randomly mating population with an equilibrium allele frequency that is the mean of the frequency of the donor and recipient populations (Figure 8a) (Ferguson et al. 1995, Gharrett and Zhivotovsky 2003). A migration rate of 10%, meaning 10% of the spawning population are immigrants, is sufficient to produce population homogenization (Ferguson et al. 1995). New alleles are also introduced to the population by gene flow. The frequency of this new allele in the recipient population changes in the same manner as alleles that were previously present, however in this case the initial allele frequency of the recipient population is zero (Figure 8b). Allele A 1 Frequency % Migration Rate 1% Migration Rate a Allele A 1 Frequency Initial frequency 10% Initial frequency 0% b Generations Generations Figure 8. The effect of gene flow on the frequency of an allele A1 in a recipient population. Panel (a) indicates what happens when gene flow is relatively fast (10% migration rate) and when gene flow is slow (1 % migration rate). Initial frequencies of allele A1 were 10% in the recipient population and 70% in the donor population. Panel (b) demonstrates how the frequency of allele A1 increases even when allele A1 is new to the population. 11

26 The process of gene flow occurs at a much quicker time scale than mutation or genetic drift, depending on connectivity of populations and migration rate. For example, shifts in allelic frequency may occur after a single introduction to a recipient population (Gharrett and Zhivotovsky 2003). However, the restriction of gene flow, either through geographic or reproductive isolation, may result in genetic divergence in populations over time as they adapt to their local environments. The longer these populations are isolated the more likely they are to be genetically different due to drift and local adaptation. The differences in local adaptation of these populations may result in outbreeding depression. Outbreeding depression occurs when the hybrids of donor and recipient populations are selected against and have decreased fitness (Hallerman 2003d). Outbreeding depression helps maintain genetic divergence between donor and recipient populations; however, it can result in a decreased genetic diversity of the recipient population through the loss of gametes to low fitness offspring, potentially resulting in extinction for extreme cases (Hallerman 2003d). The lack of local adaptations in introduced fish, both genetically and ecologically, may limit the success of the stocking program (Utter 2003) Non random mating and inbreeding Non random mating or small population sizes of genetically similar individuals can result in the loss of genetic heterozygosity and genetic diversity within a population. Inbreeding is the mating of related individuals and is an extreme form of non random mating. Inbreeding depression occurs when fitness decreases due to the expression of a deleterious homozygous recessive trait, resulting in extinction for extreme cases (Hallerman 2003e). However, even the non random mating of unrelated individuals may reduce heterozygosity, and decrease genetic diversity. For example fish that spawn early during the spawning run will mate with other early run spawners. If this is a genetically linked trait, then their offspring may spawn in the early part of the spawning run increasing the probability of related individuals mating. The effects of non random mating should be considered in gamete collection and captive breeding. 12

27 2.1.6 Summary The five processes outlined above have the potential to alter the genetic diversity both within and among populations. However, these processes do not work in isolation; it is the balance between processes that increase and those that decrease diversity, and their respective rates that produce the genetic distribution of a species (Table 1). Understanding these processes, and how they influence genetic diversity, is important to the development of effective stocking programs. Stocking has the potential to artificially induce all genetic processes (excluding mutation). Assuming that genetic diversity reflects the ability of populations to adapt to environmental change (Busack and Currens 1995, Ferguson et al. 1995, Laikre 1999), recognition and mitigation of genetic processes may help minimize adverse consequences of fish introductions. The following sections will examine how different stages of the hatchery process may alter genetic diversity through the genetic processes discussed. Table 1. The time scale at which the five genetic processes generally work and their impacts on within and among population diversity. Genetic Process Mutation Time Scale Slow (10-3 to 10-6 mutations per gene per generation) Impact on Within Population Genetic Diversity Increases Diversity Impact on Among Population Genetic Diversity Increases Diversity Random Genetic Drift Moderate to Slow (depends on size of population) Decreases Diversity May Increase or Decrease Diversity Selection Fast to Slow (depends on selective pressure) Can occur in one generation in hatchery Decreases Diversity May Increase or Decrease Diversity Gene Flow (Migration) Fast to Moderate (depends of rate) Can occur in one generation May Increase Diversity Decreases Diversity Non-random Mating and Inbreeding Moderate to Slow (depends on size of population) Decreases Diversity May Increase or Decrease Diversity 13

28 2.2 Genetic hazards Hatchery cultivation and introduction may change genetic diversity in a population through five processes: mutation, random genetic drift, selection, gene flow and nonrandom mating. Four major genetic hazards (i.e. potential adverse consequences) are associated with changes in genetic variability that result from the hatchery process. These are: extinction, loss of within population variability, loss of among population variability, and domestication selection (Busack and Currens 1995) Extinction Although ultimately a demographic process, extinction is the most severe genetic hazard which results in the complete loss of genetic information from a population, and could include the loss of rare alleles that are specific to a population (Busack and Currens 1995, Miller and Kapuscinski 2003) Loss of within population variation Loss of genetic diversity within a population means: a) the number of alleles decreases; b) the frequency an allele decreases; c) or adaptive allele combinations are lost (Busack and Currens 1995). Loss of within population variability often results from random genetic drift in small populations or inbreeding (Busack and Currens 1995, Miller and Kapuscinski 2003). Decreased genetic variation may lead to reduced fitness or inbreeding either of which increases the possibility of extinction (Miller and Kapuscinski 2003) Loss of among population variation Loss of distinct population units and their genetic uniqueness results in the loss of among population variation. The occasional occurrence of gene flow (i.e., low migration rates) to isolated populations may counter losses from genetic drift without overwhelming the local adaptation of populations (Miller and Kapuscinski 2003). However, excessive levels of gene flow may swamp local populations (as might occur during a successful stocking event), resulting in homogenization of populations and 14

29 loss of their genetic distinctiveness (Miller and Kapuscinski 2003). In addition, excessive gene flow may overwhelm a population s adaptive genetic diversity and outbreeding depression may occur, leading to further loss of within population variation. (Busack and Currens 1995, Miller and Kapuscinski 2003) Domestication selection Domestication selection may either be intentional or unintentional (Miller and Kapuscinski 2003). Domestication changes allele frequency in domestic populations relative to wild populations through artificial selection, a process that is often nonrandom and whose rate depends on selected traits and pressure imposed by the artificial environment (Busack and Currens 1995). For example, intentional selection would result if broodstock are chosen based on spawning timing or faster growth rates (Miller and Kapuscinski 2003). Less clear are unintentional selective forces encountered during broodstock collection, mating, rearing and release (Miller and Kapuscinski 2003). For example, collecting broodstock only during part of the spawning run or selective pressures on individual behaviour from artificial feeding or rearing conditions are examples of unintentional domestication (Miller and Kapuscinski 2003). It is important to consider the effects of both intentional and unintentional domestication selection on overall population variability. 2.3 Genetic hazards in relation to the hatchery process There are a number of stages involved in the hatchery process which may result in a population being at risk to one or more of the four genetic hazards outlined (Miller and Kapuscinski 2003). Examples of studies where stocking has resulted in the loss of genetic diversity are cited in Skaala et al. (1990), Hindar et al. (1991), Laikre (1999), and Epifano et al. (2001) (also see Appendix B for examples). The hazards associated with each stage of the cultivation process are summarized in Table 2, along with the mechanisms behind the potential hazard and guidelines to avoid or mitigate these hazards. It is important for the reader to consider that hatchery and wild populations are essentially subpopulations with individual effective population sizes and associated hazards (see Miller and Kapuscinski 2003 for details). However, more importantly, the relative survival and reproduction of two subpopulations will determine the impacts of 15

30 hatchery introductions on genetic diversity of recipient populations (Epifano et al. 2001, Miller and Kapuscinski 2003). 16

31 Table 2. Genetic hazards associated with the cultivation and introduction of artificially propagated fish to wild populations, and guidelines to reduce the risk of these hazards occurring. The hatchery processes discussed include: broodstock selection, broodstock collection, broodstock mating, progeny rearing, and progeny release. Information on hazards and guidelines was extracted primarily from Miller and Kapuscinski (2003), with additional information from Waples (1991), Busack and Currens (1995), Laikre et al. (1999), Epifano et al. (2001), Page et al. (2005). Hatchery Process Hatchery Activity Mechanism for Loss of Genetic Diversity and Associated Genetic Hazards (bulleted) Guidelines Broodstock Selection Dissimilar donor population used, based on genetic lineage, life history or ecology 1) Outbreeding depression through gene flow from a dissimilar donor population can decrease the population s adaptive ability, resulting in decreased reproductive success. This could lead to: Extinction Loss of Within Population Variation Loss of Among Population Variation First option: choose broodstock from target population Second option: choose broodstock that are as similar as possible in genetic lineage, life history, and ecology as the target population 2) Gene flow from a dissimilar donor population may cause the loss of a population s genetic distinctness through genetic introgression. Although genetic diversity within the population could either increase or decrease due to gene flow from introductions, populations will likely become homogenized genetically. This could lead to: This involves identifying populations or spawning stocks and their interactions Acceptable levels of introgression should be based on amount of genetic differentiation between populations Change of Within Population Variation Loss of Among Population Variation 3) Even without gene flow, dissimilarities in 17

32 Hatchery Process Hatchery Activity Mechanism for Loss of Genetic Diversity and Associated Genetic Hazards (bulleted) Guidelines life history traits between donor and recipient stocks may lead to unsuccessful stocking attempts. In addition, there may be negative repercussions to these populations due to broodstock removal, increased competition for resources, and mortality due to changes in population density. This could lead to: Extinction Loss of Within Population Variation Loss of Among Population Variation Broodstock Collection Excessive removal of broodstock from a depleted population 1) If excessive numbers of broodstock are removed from a system, there may be insufficient numbers in the donor population to maintain a viable population. This could lead to: Avoid excessive removal of adults from donor stock: < 50% provided this is still above the minimum sample size required (~ 50 spawners) Extinction Loss of Among Population Variation 2) If there is excessive removal of broodstock, and there is reduced reproductive success of hatchery progeny, or a catastrophic collapse of the hatchery population, this could lead to: Extinction An exceptional case is when a population is facing collapse (e.g. the wild population has very low or no reproductive success). In this case, a captive broodstock program could be considered where all adults are removed from the system to avoid population subdivision. However, this leads to an increased risk of domestication selection and catastrophic loss. 18

33 Hatchery Process Hatchery Activity Mechanism for Loss of Genetic Diversity and Associated Genetic Hazards (bulleted) Guidelines Loss of Among Population Variation 3) If only a small population is left in the wild as a result of excessive broodstock removal, genetic drift in the donor population will increase. This could lead to: Extinction Loss of Within Population Variation Changes in Among Population Variation 4) Excessive removal of broodstock may lead to increased inbreeding in the wild population. This could lead to: Extinction Loss of Within Population Variation Loss of Among Population Variation Broodstock Collection Collection of too few broodstock 1) If too few broodstock are collected, then fish may not be representative of the donor population s genetic diversity. As a result, rare alleles may be lost or amplified due to genetic drift (i.e., hatchery subpopulation and donor subpopulation will likely differ genetically). This will result in a change in allele frequencies when their progeny are reintroduced to the system, or if they are introduced as founders of a new population. This could lead to: 1) Collect an appropriate number of broodstock: A minimum sample of 50 spawners with an equal sex ratio is likely to minimize the loss of rare alleles. A minimum of 50 founders should be used for (re)introductions. 2) Maintain captive broodstock for only one generation and avoid use of hatchery 19

34 Hatchery Process Hatchery Activity Mechanism for Loss of Genetic Diversity and Associated Genetic Hazards (bulleted) Guidelines Domestication Selection (unintentional) Change of Within Population Variation Change of Among Population Variation 2) If too few broodstock are collected and the hatchery subpopulation is small, there may be a risk of increased random genetic drift. This could lead to: returns in broodstock collection: a) This avoids the enhancement of a few families genes over generations (assuming high survival of hatchery fish). b) This also potentially avoids substantial domestication selection from the hatchery environment that would occur over many generations. Loss of Within Population Variation Loss of Among Population Variation 3) Too few broodstock could lead to increased inbreeding in the hatchery population. This could lead to: 3) It is very important to monitor hatchery success. If it is low, reassess the stocking program. Loss of Within Population Variation Loss of Among Population Variation Broodstock Collection Source of broodstock (wild vs. hatchery) in long term stocking programs If hatchery fish have low survival and poor reproductive success in the wild, the continued collection of wild broodstock may decrease the donor population size (genetic mining ). This could lead to: 1) It is very important to monitor hatchery success. If it is low, reassess the stocking program. Extinction Loss of Within Population Variation 2) Establish a ratio of wild vs. hatchery returns for broodstock collection. The effective population size of both the hatchery and wild populations and their 20

35 Hatchery Process Broodstock Collection Hatchery Activity Broodstock collection procedure Mechanism for Loss of Genetic Diversity and Associated Genetic Hazards (bulleted) Broodstock collected in a non representative manner (e.g., run timing, habitat preferences, age or size structure, sex ratio, or fecundity) may alter the genetic variability of progeny produced, assuming these traits are heritable. Such collection may result in shifts in the expression of life history characteristics. This could lead to: Guidelines relative survival and reproductive success must be considered. Collect a representative sample for all life history traits that may affect fitness. Use a stratified random sampling design to collect representative proportions of broodstock over the duration of spawning season and over the course of the day. Domestication Selection (unintentional) Extinction Change of Within Population Variation Change of Among Population Variation This requires information about spawning timing and duration for the donor population, and variation in life history traits within this period (e.g. do older fish spawn later in the run?). Broodstock Mating Sex ratio used and variation in family size Mating one male with many females imposes non random mating, as does pooling sperm from multiple males if males sire an unequal proportion of progeny through differences in sperm potency. This leads to a decline in the hatchery effective population size (e.g. one male may be responsible for a large portion of progeny in the next generation). This could lead to: Loss of Within Population Variation Maximize the effective population size: 1) One male by one female if sex ratio even 2) If sex ratio uneven (likely to result from random daily collection of broodstock), divide the less numerous sex among the more numerous sex 3) If males are likely infertile, mix sperm from 2 males (as one is likely fertile) and fertilize eggs from one female. 4) Ideally with time and resources a 21

36 Hatchery Process Broodstock Mating Hatchery Activity Mate choice Mechanism for Loss of Genetic Diversity and Associated Genetic Hazards (bulleted) 1) It is unlikely that fish culture operations could ever anticipate all the complexities that occur during mate choice in a natural system. This could lead to: Domestication Selection (unintentional) Guidelines factorial mating system, or some variant on this, would be set up. In this case, gametes are equally divided and all individuals of the opposite sex are crossed. This is especially important for very small populations. However, the risk of inbreeding from the mating of relatives increases, which may have deleterious effects Use a random mating design for traits such as size to maintain variation. It is likely this will not mimic what occurs in the wild, but does help prevent the loss of diversity. 2) Non random mating from selective breeding programs (e.g. large fish together) may lead to changes in genetic diversity. This could lead to: Domestication Selection (intentional) Loss of Within Population Variation Progeny Rearing Release from natural selection In a hatchery environment, it is impossible to mimic all natural conditions the fish experience while rearing. This may result in the loss of adaptive traits or survival of individuals that would not have survived in 1) Minimize time in the hatchery 2) Attempt to simulate natural conditions. For example, densities of eggs and fry, feeding methods, flow regimes, light, temperature, substrate, etc. should be as 22

37 Hatchery Process Hatchery Activity Mechanism for Loss of Genetic Diversity and Associated Genetic Hazards (bulleted) Guidelines the wild. This could lead to: Domestication Selection (unintentional) similar as possible. Unfortunately, this is often not cost effective or realistic. Progeny Rearing Hatchery selection (intentional or unintentional) In the hatchery environment, not only are fish released from natural selection, but there may also be selection pressures for very different traits. This may simply be the product of cultivation, or intentional if there is selection by cultivators for a specific trait (e.g. faster growth). This could lead to: Domestication Selection (unintentional) Domestication Selection (intentional) Loss of Within Population Variation 1) Minimize time in the hatchery 2) Attempt to simulate natural conditions. 3) Rear all fish under similar conditions 4) Progeny from different mate pairings should be mixed as soon as possible to distribute them uniformly throughout hatchery operation. 5) Avoid intentional selection Progeny Release Number released 1) The recipient system has a limited carrying capacity which can be exceeded by the introduction of too many fish. This in turn leads to decreased growth and survival from density dependent competition. This could lead to: Estimate population size considering stocked and wild individuals, and compare this value to estimated carrying capacity of the system. Loss of Within Population Variation Collect an appropriate number of broodstock to represent the wild population s genetic diversity. 2) The goal of most hatchery programs is to have higher survival of hatchery progeny 23

38 Hatchery Process Hatchery Activity Mechanism for Loss of Genetic Diversity and Associated Genetic Hazards (bulleted) Guidelines when compared with wild recipient populations. If these fish are not genetically similar to the wild population, wild populations may be overwhelmed by the influx of hatchery recruits. Resulting gene flow may result in outbreeding depression through loss of adaptive characteristics. This could lead to: Attempts should be made not to overwhelm the wild population with large numbers of genetically dissimilar fish. Extinction Loss of Within Population Variation Loss of Among Population Variation 3) Even if no outbreeding depression occurs, the broodstock population may numerically overwhelm the natural population and result in the population s genetic composition being more similar to the hatchery population. This could lead to: Domestication Selection (unintentional) Extinction Change of Within Population Variation Change of Among Population Variation Progeny Release Fish health upon release Introduction of disease to wild populations by cultured fish can lead to high levels of mortality in the wild population. This could lead to: Cull fish with symptoms of disease in hatchery. 24

39 Hatchery Process Hatchery Activity Mechanism for Loss of Genetic Diversity and Associated Genetic Hazards (bulleted) Guidelines Extinction Loss of Within Population Variation Progeny Release Life stage released Fish that are maintained too long in the hatchery may experience artificial selection and release from natural selection. As a result the fish that survive in a hatchery may not be representative of those that would survive in the wild. However, fish must also be in a suitable condition to survive release. If there is poor survival upon release, genetic information is lost. This could lead to: Release the earliest lifestage possible that balances maximal adult return rates and minimal time in the hatchery: This minimizes exposure to hatchery selection and domestication, and also exposes fish to natural selective processes Domestication Selection (unintentional) Loss of Within Population Variation Progeny Release Location/Method of release 1) If fish are released into an area they would not normally occur in, or if they are released in disproportionately high densities then their survival may be low, and may also affect the survival of wild fish. This could lead to: 1) Release into habitat in which they would normally occur 2) Release over a broad area rather than in a concentrated spot Extinction Loss of Within Population Variation 2) Fish released in areas that are not in the vicinity of spawning habitat may not find suitable areas in which to reproduce once they sexually mature. This could lead to: 25

40 Hatchery Process Hatchery Activity Mechanism for Loss of Genetic Diversity and Associated Genetic Hazards (bulleted) Guidelines Extinction Loss of Within Population Variation 26

41 2.4 Risk assessment and prioritization The use of risk assessment techniques to evaluate or prioritize stocking programs is discussed in the literature with respect to both genetic and ecological threats (Busack and Currens 1995; Currens and Busack 1995; Pearsons and Hopley 1999; Ham and Pearsons 2000; Shaklee and Currens 2003). We do not intend to repeat this work but rather develop a common terminology and understanding of genetic risk assessment that should help clarify subsequent discussions. Genetic risk assessment is the process of understanding a population s vulnerability to genetic change (Shaklee and Currens 2003), with vulnerability being the product of risk and loss for a given activity (Figure 9). The probability that a particular hatchery activity leads to an adverse event is risk (Figure 9). Risk depends on proximate and ultimate control mechanisms. Proximate controls prevent the occurrence of an adverse event; selection of broodstock with similar genetic lineage as that of the recipient population is a proximate measure to prevent outbreeding depression. Whether proximate measures are effective or not depends on their reliability. For instance, how reliable is our ability to determine similar genetic lineages. Ultimate controls can be viewed as the backstop that safeguards systems when failure in proximate controls occur; and in this sense, ultimate controls provide resilience in the system to adverse events. Currens and Busack (1995) identify two sources of resilience in maintaining genetic diversity with respect to stocking: a) presence of genetic reserves and b) the ability of managers to alter or stop a stocking strategy (i.e., reactive management). The combination of proximate and ultimate controls determines risk. For instance, suppose we have a 10% probability of selecting broodstock with similar genetics to the recipient population (i.e., 10% reliability) and the recipient population is connected to a large intermixing metapopulation that we expect to provide an 80% chance of buffering outbreeding depression (i.e., 80% resilience), then the risk in occurrence of outbreeding depression and loss of within population variation is (1 0.1) X (1 0.8) = 18%. Quite clearly, in this example, system resilience is very important in reducing risk, as failure in proximate controls is high (90%). 27

42 Failure in both proximate and ultimate controls leads to occurrence of an adverse event, and realization of a hazard (Figure 9). Loss is the consequence of a hazard. This is perhaps most clearly illustrated when extinction is the hazard. If failure occurs and extinction is the resulting hazard, then loss in diversity for the extinct genotype is obviously 100%. Vulnerability takes into account the combination of risk and loss allowing managers to prioritize efforts. Take, for instance, the following hypothetical (and biologically improbable) problem. In the previous example risk of outbreeding depression from stocking was 18%, assume loss in genetic diversity through outbreeding depression is 50%. However, without stocking the population has a 25% chance of going extinct as bycatch in a commercial fishery. What should a manager do assuming maintenance of genetic diversity is their primary goal? Vulnerability to loss in genetic diversity from stocking is 0.18 X 0.50 = 9%; vulnerability to loss in diversity from not stocking is 0.25 X 1 = 25%. As stocking has a lower vulnerability, this would be the prudent management choice; however, one would certainly need to ask whether outbreeding depression were the only genetic process of importance (e.g., genetic drift) given effective population size is likely very low owing to the commercial fishery. Source of Hazard Proximate Control (reliability) Ultimate Control (resilience) Hazard Selection of dissimilar donor population Choose broodstock of similar genetic lineage 1) Large genetic reserve with recipient population s genotype 2) Reactive management Loss of withinpopulation genetic variation through outbreeding depression Risk Loss Vulnerability Risk X Loss = Vulnerability to loss of genetic variability by selecting broodstock from dissimilar donor populations Figure 9. Determining vulnerability of a population to genetic change from the hatchery activity of selecting dissimilar broodstock (Table 2) using a risk assessment framework (adapted and modified from Currens and Busack 1995). 28

43 Genetic hazards associated with the hatchery process (outlined in Table 2) do not infer anything about the risk of a hazard occurring; nor, the genetic loss associated with each hazard. Together (risk X loss) determines the vulnerability of a population to loss in genetic diversity (Currens and Busack 1995). The hatchery process produces an unnatural selective environment where broodstock are almost always going to be a subsample of the population (Ryman and Laikre 1991, Waples 1999, Miller and Kapuscinski 2003). Genetic risks associated with stocking can be minimised (Page et al. 2005); however, there will always be tradeoffs associated with hatchery processes, and these must be balanced appropriately (Waples 1999). Therefore, a method for assessing risk and prioritizing genetic conservation efforts helps managers in establishing the goals and methods behind a stocking program. 3.0 GENETIC CONSIDERATIONS BY OTHER JURISDICTIONS The role of stocking in fisheries management and the impacts of this process on genetic diversity is a matter or much debate (Philipp et al. 1993, Busack and Currens 1995, Campton 1995, Incerpi 1996, Waples 1999, Brannon et al. 2004, Mobrand et al. 2005). The degree to which genetic variation should be considered when stocking and the method of assessment of genetic diversity are not clearly defined and often depend on the goals of the stocking program (Epifano et al. 2001). The changes in genetic diversity from the hatchery process are not inherently bad or good (Table 2), and concerns associated with any changes are related to the goals of the management plan (Epifano et al. 2001). If the goal is to conserve genetic diversity of wild populations then changes in genetic variability are obviously of concern. However, if the goal is to produce a high quality fishery (regardless of genetic diversity) then some genetic hazards will be of little concern to managers. To determine what others consider in their walleye stocking programs in relation to genetic diversity, jurisdictions in both Canada and the United States were contacted. Information on official or unofficial policies and genetic studies were requested. A total of five provinces and one territory were contacted with responses received from all. A total of 26 states were contacted and responses were received from 22 of them (85% success). The four for which no response was received may be due to inappropriate 29

44 contact information. In addition, some of the states that responded did not supply information applicable to this project. In total, 98 people were contacted regarding this project, including both management personnel and geneticists. Contact information was obtained primarily from the Walleye Technical Committee membership list and from the internet or publications. The responses received from the various jurisdictions are summarized in Table 3. Table 3. Summary of information obtained from management personnel and documents regarding genetic considerations for walleye stocking programs from various jurisdictions in Canada and the United States. Country Canada Canada Canada Province or State British Columbia Manitoba Northwest Territories Summary of Considerations There is currently no walleye stocking program in B.C. (pers. comm. Baccante, N., B.C. Ministry of the Environment, Lands & Parks). Walleye are at the extreme of their range and only occur in the Peace and Liard River systems, although they may have invaded some southern parts of B.C. from Washington (Baccante and Down 2004). There is currently no provincial policy regarding genetic considerations for stocking walleye in Manitoba (pers. comm. Smith, T., Government of Manitoba) There is no walleye stocking program in the Northwest Territories (pers. comm. Low, G., Fisheries and Oceans Canada). A hatchery program for stocking was attempted in the late 1990ʹs. However, hatchery production was unsuccessful and so the project was discontinued. There are a number of viable walleye fisheries in the territory. 30

45 Country Canada Canada Province or State Ontario Québec Summary of Considerations Ontario has a very comprehensive stocking plan for all species including walleye. This is outlined in their ʺGuidelines for Stocking Fish in Inland Waters of Ontarioʺ (MNR 2002). These guidelines include general information on stocking goals, founding/broodstock collection, spawning and mating, rearing, waterbody suitability, fish health and disease, appropriateness of genetic stock, size and age of stock, stocking rates, and marking and assessment of stocking. In addition, specific guidelines for walleye are included with regards to: egg collection; habitat suitability; fish community structure in stocked lakes; stocking rate; stocking frequency and timing; temperature at stocking; locations of transfer; release sites; and stocking techniques. Some recommendations on watershed transfer are also discussed in Kerr et al. (1996). Other information on walleye management and stocking can be found in Kerr et al. 1994, Kerr et al. (1997), Fluri (1998), Kerr and Grant (2000), Lester et al. (2000), and Kerr et al. (2004). With regards to walleye genetic diversity, there was a study carried out by Ihssen and Martin (1995) that looked at genetic diversity in the province of Ontario. There is currently no stocking program in Québec for walleye (pers. comm. Nadeau, D., Ministère des ressources naturelles, de la faune). In the near future, they will produce walleye to re seed some natural lakes. For donor population they will choose: 1) the remnant population, 2) if this is not possible, a nearby population or a lake population with similar ecological and geological characteristics will be selected. Québec essentially follows the guidelines in Kerr et al. (1996) from the Ontario Ministry of Natural Resources. Consideration of shoaling vs. river spawning stock is made depending on lake habitat. There has also been an unofficial policy to separate the St. Lawrence and Hudson Bay drainages to protect genetic differences between the two. 31

46 Country Canada United States United States United States Province or State Saskatchewan Alabama Arkansas Connecticut Summary of Considerations There is currently no provincial policy regarding genetic considerations for stocking walleye in Saskatchewan (pers. comm. Murphy, K., Saskatchewan Environment). The province follows the ʺNational Code for Fish Introductions and Transfersʺ (DFO 2003). However a genetic study by Billington and Strange (1994) was carried out to determine the genetic similarity between a recipient and donor population in order to make an informed decision with regard to supplemental stocking of this lake. Work has also been done to examine the amount of hybridization occurring between walleye and sauger in Lake Diefenbaker (Billington et al. 2005). No information was received from management personnel in this state. However, a study by Billington and Maceina (1997) indicates that the walleye in the Mobile River basin are genetically distinct from walleye in the Tennessee River. Historically, walleye were stocked into the state of Arkansas from at least 8 states and 2 federal hatcheries (pers. comm. Perrin, C., Arkansas Game & Fish Commission). Since 1988 southern populations (Ouichita River drainages) and northern populations are stocked separately with no movement of fish between these basins (White River Drainage and Greers Ferry Lake), as they are thought to be genetically distinct (pers. comm. Stein, J., Arkansas Game & Fish Commission). However, there is no set protocol for these two spawning projects in relation to genetics. In addition, the Black River strain of walleye in Arkansas and Missouri is thought to be genetically distinct and efforts are being made to conserver them (pers. comm. Perrin, C., Arkansas Game & Fish Commission). They are currently undergoing a state wide genetics survey (pers. comm. Perrin, C., Arkansas Game & Fish Commission; and pers. comm. Stein, J., Arkansas Game & Fish Commission). Walleye are not considered native to this state; therefore, there is little concern about genetic integrity (pers. comm. Leonard, J., State of Connecticut). Fish were initially introduced into systems in 1993, and there no natural reproduction of these populations. Stock is received from Minnesota hatcheries. 32

47 Country United States United States Province or State Illinois Indiana Summary of Considerations In Illinois, the unofficial policy is to not mix stock across major drainage basins (pers. comm. Philipp, D., Illinois Natural History Survey). Impoundments are stocked with walleye with little concern for genetic integrity as they are introduced populations with no natural reproduction (pers. comm. Clodfelter, K., Illinois Department of Natural Resources). In addition, these systems are distant from naturally reproducing river populations. If river populations are stocked, they are stocked with fish from broodstock from that river (pers. comm. Clodfelter, K., Illinois Department of Natural Resources). Illinois has worked in collaboration with Wisconsin and Minnesota to do genetic analyses of a number of fish species in the Upper Midwest (Fields et al. 1997). In Indiana, the state wide stocking policy for all species regarding genetics is to consider genetic effects stocked fish may have on recipient populations. This includes avoiding the introduction of similar genera into a system (e.g. walleye/sauger), and avoiding the introduction of stocks from other watersheds. Whenever possible, fish from the same watershed are used. Fish from riverine systems are stocked into rivers when possible, as are lake fish be stocked into lakes. It is recommended that genetic information should be collected from frequently stocked fish for future management purposes (pers. comm. Schoenung, B., Indiana Department of Natural Resources). For the last 20 to 25 years broodstock have been collected from within the state (pers. comm. Sickles, D., Indiana Department of Natural Resources). 33

48 Country United States United States United States Province or State Iowa Kansas Kentucky Summary of Considerations Historically, little concern was given to genetics when stocking walleye in Iowa (pers. comm. McWilliams, D., Iowa Department of Natural Resources). Today broodstock is obtained from within the state (pers. comm. Christianson, J., Iowa Department of Natural Resources). Broodstock are given Visual Implant tags in order to reduce their repeated use (pers. comm. Gelwicks, G., Iowa Department of Natural Resources). In many areas there is very little natural recruitment and as a result stocking success is more of a concern than genetic integrity (pers. comm. Gelwicks, G., Iowa Department of Natural Resources). There were two studies done in the state that looked at genetic impacts of stocking and stocking success (Paragamian 1988, Paragamian and Kingery 1992). Iowa also restricts the in state movement between watersheds of walleye (Wingate 1991). Walleye are not considered native to this state (Fields and Phillips 2000); therefore there is little concern about genetic integrity and stocking (pers. comm. Steven, J., Kansas Department of Wildlife and Parks). Some impoundments do have self sustaining populations (pers. comm. Steven, J., Kansas Department of Wildlife and Parks). A study by Fields and Phillips (2000), found no distinguishable pattern of genetic divergence in seven walleye populations except one population. It was hypothesized that these seven populations are derived from similar stocks originally, or that gene flow has homogenized any differences that might have existed. Walleye have been stocked from the northern states (Lake Erie strain likely from New York) into reservoirs in Kentucky (pers. comm. Dreves, D., Kentucky Department of Fish and Wildlife). River populations are native. It was assumed that wild and stocked populations interbred, but it has since been determined that there is a ʺpureʺ native strain in the Rockcastle River. As a result, efforts are being made to restore this stock by hatchery propagation of Rockcastle River strain. 34

49 Country United States United States United States Province or State Michigan Minnesota Missouri Summary of Considerations I was unable to obtain information on walleye stocking policies and procedures from management personnel. However, Colby et al. (1994) and Dexter and O Neal (2004) provide information on management and stocking policies. Genetic studies have been carried out on walleye on the Great Lakes (Todd and Hass 1995, Todd and Hass 1993, Todd 1990). Michigan also restricts the in state movement walleye between watersheds of (Wingate 1991). Minnesota established genetic conservation units based on conservation management units reported by Fields et al. (1997). These units are guides to fish transfers for introduction and supplementation ensuring stocked fish are of similar genetics to recipient populations. As a result, Minnesotaʹs stocking guidelines suggest no mixing of stocks between major drainage basins (with some exceptions), and prioritization of stocking locations due to limitations in suitable stock availability (Wingate 1991, Beck et al. 1996). In 2000, law allowed for transfer of native species without a permit by the private aquaculture industry, but required a permit for the transfer of non native species and strains (Hirsch 2000). Applications for permits are now reviewed with consideration of maintaining areas critical to genetic integrity (Hirsch 2000). A genetic study on Minnesota walleye (McInerny et al. 1991) identified two genetically similar groups. However, these groups may have been influenced by previous indiscriminate stocking. In Missouri, a committee has currently been assigned to develop a statewide walleye plan (pers. comm. Banek, T., Missouri Department of Conservation). The plan will include considerations for conserving the genetic integrity of the distinct Black River strain. Any stocking in this system is done with Black River strain. Walleye in the rest of the state are not genetically distinct. At present, all broodstock are collected from within the state, but the state did receive stock from Nebraska historically. 35

50 Country United States United States United States United States Province or State Montana Nebraska New York North Dakota Summary of Considerations Walleye are not considered native to this state; therefore there is little concern about genetic integrity and stocking (pers. comm. Leathe, S., Montana Fish, Wildlife and Parks; and pers. comm. McDonald, K., Montana Fish, Wildlife and Parks). The biggest genetic concern with walleye is that it will hybridize with the native sauger populations which are a species of special concern in Montana (pers. comm. Steve Leathe, Montana Fish, Wildlife and Parks). Broodstock is now tested for hybridization prior to culturing (pers. comm. McDonald, K., Montana Fish, Wildlife and Parks). Walleye are no longer stocked into rivers to avoid hybridization with sauger (pers. comm. Leathe, S., Montana Fish, Wildlife and Parks). Work by Szalanski et al. (1999), suggests there are no unique strains of walleye in Nebraska, although two different haplotypes were observed. As a result, walleye are collected from traditional locations to stock within the state (pers. comm. Bauer, D., Nebraska Game & Parks Commission). New York restricts the in state movement walleye between watersheds of (Wingate 1991). In New York (NY), they assess stocking applications by examining the undesirable impacts and potential risks to the genetic integrity of walleye in the receiving watershed (New York State DEC 1998). They protect resources where possible by limiting permits and using the following guidelines for permit approval: 1) require the same strain of walleye to be stocked if the recipient waterbody directly enters a major wild walleye source population (i.e., Lake Erie, Lake Oneida, Lake Champlain, St. Lawrence River); 2) require NY strain walleye to be stocked if fish may migrate into other important wild populations (i.e., Delaware River, Susquehanna River, Silver Lake, Canadarago Lake, Chautauqua Lake); 3) do not require NY strain for private waters that are relatively isolated. The state uses Oneida Lake stock as broodstock (pers. comm. Keeler, S., New York State, Department of Environmental Conservation). In North Dakota, no information was obtained on the stocking policy related to walleye genetics. However, in Lake Sakakawea they are careful to avoid the use of walleye x sauger hybrids as broodstock (pers comm. Hendrickson, J., North Dakota Game and Fish Department; Ward and Berry 1995). 36

51 Country United States United States United States Province or State Ohio Oklahoma Pennsylvania Summary of Considerations Walleye are stocked into impoundments in Ohio. These fish are predominantly stocked with Lake Erie strain fish into the Lake Erie watershed. However, some are transferred into impoundments in the Ohio River Basin (pers. comm. Navarro, J., Ohio Department of Natural Resources). Populations in the impoundments are not natural and most are not self sustaining so there is little genetic concern (pers. comm. Hale, S., Ohio Department of Natural Resources; and pers. comm. Navarro, J., Ohio Department of Natural Resources). However, it is thought the Lake Erie strain and Ohio River strain are distinct (pers. comm. Navarro, J., Ohio Department of Natural Resources). Work has been done looking at genetic diversity in the Ohio River (White and Schell 1995, White et al. 2005). The biggest genetic concern in Ohio appears to be interbreeding of walleye with stocked saugeye. As a result, saugeye are no longer stocked into the Lake Erie Watershed in order to avoid escapement into Lake Erie (pers. comm. Hale, S., Ohio Department of Natural Resources). No response was received from personnel in this state. However, Gilliland and Boxrucker (1995) provide species specific guidelines for stocking reservoirs in Oklahoma. Genetic considerations do not appear to be important in the ranking of reservoirs to be stocked. In Pennsylvania, very little information was provided regarding walleye stocking in the state, as the person contacted deals with Lake Erie which is not stocked. 37

52 Country United States United States United States Province or State South Dakota Tennessee Texas Summary of Considerations Unofficially, in South Dakota only Missouri River walleye are stocked in Missouri River system (pers. comm. Lott, J., Barnes, M., Unkenholz, D., and Lucchesi, D., South Dakota Game, Fish and Parks). Current work is going on to determine if there are or were significant genetic differences between spawning stocks for each of the Missouri tributaries. Stocks in eastern lakes were distinct from Missouri river strain (Waltner 1988), but this may no longer be the case due to stocking of Missouri River strains. Walleye in the state are likely genetically distinct from other states, especially the Great Lakes or more distant places. However, this has not been tested. Stocks may be similar to North Dakota, Nebraska or Minnesota walleye from stock transfer. Spawning techniques are likely to have been adequate to maintain diversity. There is some question of genetic importance due to highly altered aquatic systems and interstate transfers. Comparisons of Missouri River walleye from South Dakota, with North Dakota and Montana walleye indicated that these fish were not different. Missouri River may be a ʺpureʺ strain as they have not been stocked with anything other than Missouri River strain. There is natural hybridization of walleye and sauger in the state. No response received. There are no genetic considerations for walleye stocking because Texas is outside the native range of walleye. However, they have tried to use genetics to influence growth (pers. comm. Munger, C., Texas Parks and Wildlife). Texas reservoirs were originally stocked from Spirit Lake, Iowa and then were not stocked for 30 years. Walleye were then stocked from Colorado and they had better growth traits. Some work has been done on identifying origin and amount of introgression but with no definitive results. 38

53 Country United States United States United States Province or State Vermont Virginia West Virginia Summary of Considerations Vermont does consider genetics in their stocking program of Lake Champlain (pers. comm. MacKenzie, C., Vermont Fish and Wildlife Department). Broodstock are collected from rivers which have been found to be genetically distinct (Hawley et al. 1991). Therefore, priority is placed on stocking fingerlings in areas where adults were captured (Vermont Department of Fish and Wildlife 1998). In addition, the attempt is made to collect at least 30 pairs from any river broodstock source (pers. comm. MacKenzie, C., Vermont Fish and Wildlife Department). No further genetic monitoring or assessment programs are planned at this time. No response received No response received 39

54 Country United States United States Province or State Wisconsin Wyoming Summary of Considerations One of the goals of the walleye management plan in Wisconsin is to maintain the genetic integrity of naturally reproducing populations (Hewett and Simonson 1998). Genetics policy was developed based on work by Fields et al. (1997) that Wisconsin participated in, in collaboration with Minnesota and Illinois (pers. comm. Staggs, M., Wisconsin Department of Natural Resources). An extensive review of the genetics of walleye and other species was carried out in the Midwest in early 1990ʹs (Fields et al. 1997). Results from this work suggest that there are five primary genetic stocks in Wisconsin (Wisconsin DNR 2005). Genetic boundaries are based on allozymes and mtdna (pers. comm. Jennings, M., Wisconsin Department of Natural Resources). In general, self sustaining populations are not stocked; and, when stocking other populations broodstock are collected from within major watershed boundaries (pers. comm. Martin Jennings, Wisconsin DNR 1999, Wisconsin DNR 2005). Wisconsin also restricts the in state movement walleye between watersheds of (Wingate 1991). Work is currently going on to assess what constitutes genetic integrity and how one should approach their management actions in an effort to preserve it (pers. comm. Sloss, B., USGS, University of Wisconsin Stevens Point). Work is also currently underway to review genetic considerations during the culturing process with draft guidelines expected at the end of August 2005 (pers. comm. Jennings, M., Wisconsin Department of Natural Resources). Wisconsin does not stock sauger in waters with naturally reproducing walleye populations, and does not stock saugeye at all (Wisconsin DNR 1999). Walleye are not native to Wyoming. However, sauger are native. Therefore, genetic considerations relate to hybridization with sauger rather than maintaining genetic integrity of walleye (pers. comm. Whaley, R., Wyoming Game and Fish Department; and pers. comm. Sharon, S., Wyoming Game and Fish Department). Walleye are no longer cultured within Wyoming. Stock is received from other states. 40

55 It would seem that the unofficial policy in most states and provinces, where walleye are native and there is an active stocking plan, is to minimize the transfer of fish between major drainage basins in order to conserve genetic integrity. In introduced populations, there is often little concern about genetic diversity, except in relation to connected waterbodies, as walleye are not native to the waterbody. Detection of hybridization between sauger and walleye is also a common use for genetic information in many states, as is assessing stocking success using genetic markers. However, states and provinces such as Ontario, Wisconsin, and Minnesota have more comprehensive stocking guidelines to minimize negative influences on genetic diversity from different components of the hatchery process. Some information on fish introduction policies is available in Leach and Lewis (1991) and Wingate (1991). Wingate (1991) identifies five states that restrict in state movement of walleye to reduce gene pool contamination. Many states are also currently undergoing studies to examine genetic diversity and conservation. With this information, management bodies may begin to identify areas that represent distinct genetic groups and the risk of these groups to alteration from the hatchery process. 41

56 4.0 METHODS FOR ASSESSING AND MANAGING GENETIC DIVERSITY In order to manage and conserve genetic diversity there must be an understanding of the distribution of genetic diversity. This involves not only a genetic inventory of stocks, but also an assessment of the inter relationship among stocks (Shaklee and Currens 2003). Molecular markers may be used to identify stocks. Statistical approaches for assessing diversity are discussed by Shaklee and Currens (2003). However, this information should not be used in isolation from information on the biological factors, environmental factors, and zoogeographic factors that influence the systems of interest (Shaklee and Currens 2003). Genetic and biological information should be used together to identify and prioritize areas where conservation efforts should be focused. 4.1 Molecular genetic techniques There are a number of molecular genetic markers that are used for determining genetic variability and identifying genetic stocks. These techniques have developed and will continue to develop as genetic technology advances. Genetic markers are scored for the number of alleles (polymorphisms) at a single locus, as there can be many variants on a single gene, and their frequency in a population (Hallerman et al. 2003). This information is then used to estimate genetic variation (Hallerman et al. 2003). Genetic markers must be selectively neutral, having no effect on the fitness of an individual (Kapuscinski and Jacobson 1987). This way, their presence and divergence is due to random genetic drift and not selective processes (Kapuscinski and Jacobson 1987, Brown and Epifano 2003). As a result, it may be assumed that the amount of genetic divergence is proportional to the time since separation (Brown and Epifano 2003). Both proteins and DNA, which includes mitochondrial and nuclear DNA, may be used as genetic markers. These markers may then be used to identify genetic stocks, to measure changes in genetic variation, to locate appropriate broodstock, to assess performance of stocked fish, to determine the genetic impacts of introduced fish on wild populations, as well as many other applications (Ferguson et al. 1995). However, since populations often differ in the frequency of alleles in a population rather than in the alleles present, bootstrapping techniques, such as outlined in Roff and Bentzen 42

57 (1989), are often used to determine genetic differentiation statistically (Ferguson and Danzmann 1998) Allozymes Allozymes are enzymatic proteins coded for at a single locus on the nuclear DNA, and are inherited in a predictable fashion (May 2003). If heterozygotes are codominant, meaning both alleles are expressed, they may produce unique signatures that can be identified in addition to identification of homozygotes. Like most molecular marker techniques, electrophoresis is used to identify the various alleles (May 2003). Allozymes are technically simple, quick to process and relatively cost effective (Ferguson et al. 1995, Shaklee and Currens 2003). However, the tissue required for these analyses (i.e. eye, liver, and muscle tissue) often require fatal sampling techniques which may be undesirable (Shaklee and Currens 2003). In addition, tissue must be stored at low temperatures (< 20ºC, ideally < 70ºC) as quickly as possible as the enzymes are very temperature labile (May 2003). Allozymes are not extremely polymorphic at a given loci limiting their ability to detect genetic variation (Ferguson et al. 1995). Allozymes have been found to be suitably polymorphic at some loci for walleye (Clayton et al. 1974, Todd and Hass 1993). However, allozymes sometimes lack the variability required for a population level study (Stepien 1995) Mitochondrial DNA (mtdna) Mitochondrial DNA (mtdna) is a circular strand of DNA from the mitochondria that is inherited clonally (without recombination) from the mother and is essentially haploid (single stranded) (Ferguson and Danzmann 1998, Shaklee and Currens 2003). This haploid genotype is called a haplotype (Billington 2003). The mtdna is generally digested with restriction endonucleases that recognize specific sequences in the mtdna and cleave it at these points (Shaklee and Currens 2003). The result is a number of fragments of varying length, called restriction fragment length polymorphisms (RFLP), which may be detected using electrophoresis (Billington 2003). In addition, polymerase chain reaction (PCR) methods may be used to amplify small sequences of mtdna, a technique which requires very small tissue samples (Shaklee and Currens 2003). Techniques exist for utilizing the whole molecule of mtdna or just 43

58 regions of the molecule that are amplified with PCR. One of the most common regions analyzed is the control region or D loop. This is chosen because it is the most variable portion of the molecule and is assumed to be selectively neutral (Stepien 1995). Mitochondrial DNA is a good genetic marker for a number of reasons. As a result of its maternal mode of inheritance, the effective population size drops to one quarter that of nuclear DNA (Ferguson and Danzmann 1998, Shaklee and Currens 2003). Therefore, mtdna is particularly sensitive to reductions in genetic variation through the process of genetic drift and low levels of gene flow (i.e., it takes four times the amount of gene flow to counteract divergence due to drift) (Ferguson and Danzmann 1998, Shaklee and Currens 2003). This makes mtdna a good population specific marker (Billington and Hebert 1991, Ferguson et al. 1995). In addition, mtdna does not recombine during replication and as a result the history of past isolations is preserved and it is slower to respond than nuclear DNA after gene flow is re established (Billington and Hebert 1991, Shaklee and Currens 2003). Mitochondrial DNA experiences a much higher mutation rate than nuclear DNA (5 to 10 times greater) which may increase genetic differences (Stepien 1995, Billington 2003). Finally, mtdna may be extracted using non lethal sampling and may even be isolated from preserved material (Billington 2003). However, a disadvantage of mtdna is that one is unable to detect the contribution of males to the population due to maternal inheritance (Ferguson and Danzmann 1998). In addition, in extreme genetic bottlenecks there is more likely to be fixation of a haplotype which makes it ineffective as a genetic marker, and its susceptibility to genetic drift may lead to spurious conclusions (Ferguson et al. 1995) Nuclear DNA There are numerous nuclear genetic markers such as allozymes, randomly amplified polymorphic DNA (RAPDs), amplified fragment length polymorphisms (AFLPs), satellite DNA, and variable number tandem repeats (VNTRs) which includes both minisatellite and microsatellite DNA (Ferguson and Danzmann 1998). The most common of these are allozymes (discussed above), RAPDs, and VNTRs. 44

59 RAPD Randomly amplified polymorphic DNA (RAPD) is produced from a small amount of nuclear DNA. PCR is used to amplify random fragments of the DNA from throughout the genome and of varying length (tens to thousands of base pairs) (Brown and Epifano 2003). A mutation in a DNA sequence will prevent the fragment from being amplified and its presence/absence provides the genetic marker (Brown and Epifano 2003). The technique is simple, inexpensive and requires no prior information on the genetic sequence that is needed to develop other types of genetic markers, such as mtdna and microsatellites (Brown and Epifano 2003). In addition, non lethal sampling is required to collect the required tissue samples (e.g., fin clips) (Thomas et al. 1999). However, the RAPD technique has been criticized for its lack of repeatability among laboratories as it is very sensitive to slight changes in amplification conditions (Ferguson et al. 1995, Ferguson and Danzmann 1998, Brown and Epifano 2003) Microsatellites Microsatellites are variable number tandem repeat (VNTR) sequences in the nuclear DNA. VNTRs are classified by their size, with satellite DNA being the largest (several thousand base pairs), minisatellites are of moderate size (9 to 100 base pairs), and microsatellites are the smallest (1 to 6 base pairs in length) (Ferguson et al. 1995). All of these VNTR sequences are repeated numerous times, as much as 100 times in the case of microsatellites (Ferguson et al. 1995). Unlike allozymes and mtdna which generally change due to changes in sequencing, VNTR involve different numbers of the repeated sequence making them highly variable with numerous rare alleles (Ferguson et al. 1995, Ruzzante 1998, Nielsen et al. 1999). As a result, microsatellites are a good genetic marker as they have variability in both heterozygosity (allele frequency) and allele number (Shaklee and Currens 2003), which may make them better suited to detecting loss in genetic variation (Ferguson and Danzmann 1998, Ruzzante 1998, Nielsen et al. 1999). Microsatellites have the ability to assess genetic structure at much smaller scales (Estoup et al. 1998). In addition, microsatellite DNA may be sampled non lethally since very little DNA is needed for the technique (Miller and Kapuscinski 1996, Shaklee and Currens 2003); a trait that makes them more suitable when using archived samples. Often archived material is degraded and only DNA fragments of less than 400 base 45

60 pairs are recovered; therefore, small microsatellites are desirable (Nielsen et al. 1999). However, for many species suitable genetic markers have not yet been established (Shaklee and Currens 2003), a process which may be time intensive and costly. The process involves the identification of tandem repeats that are suitably polymorphic and then the development of enzymes and genetic primers that allow the microsatellite to be amplified for analysis. This technique also requires large sample sizes when there is a lot of polymorphism at a locus in order to be reliable (Ferguson et al. 1995, White et al. 2005). In addition, microsatellites may be highly variable making their interpretation extremely complicated (pers. comm. Dr. Neil Billington). However, advances in statistics specific to population genetics have made the analysis and interpretation of highly polymorphic data more accessible (Falush et al. 2003, Wilson & Rannala 2003, Schweiger et al. 2004). Most measures of genetic diversity use markers that occur at a single or few loci. These are generally thought to be selectively neutral, and therefore represent the genetic diversity of the population(s) (Hallerman 2003a). However, these neutral markers may not necessarily have a straightforward relationship with the quantitative traits that influence an individual s fitness (Hard 1995, Hallerman 2003a, Shaklee and Currens 2003). Yet, the complex interactions of multiple loci and the environment make it difficult and expensive to measure genotypic diversity of quantitative traits (Busack and Currens 1995, Moran 2002). Therefore, it is important not only to consider the measured genetic diversity of populations, but also to consider ecological, life history and zoogeographic similarities of the donor and recipient populations prior to an introduction (Crandell 2000, Miller and Kapuscinski 2003, Shaklee and Currens 2003, Youngson et al. 2003). 4.2 Evolutionary significant units A fundamental step in managing biodiversity requires identifying populations with unique evolutionary histories (Moritz 1994). At a taxonomic scale, identifying unique evolutionary histories is straightforward when contrasting species defined under a traditional biological approach. For instance, walleye possess a unique evolutionary history compared to bull trout (Salvelinus confluentus). However, principles of conservation biology applied only to species defined under a biological definition (e.g., 46

61 individuals that breed and produce viable offspring) will result in the loss of different evolutionary lineages (e.g., phylogeny), and hence, genetic diversity. In response, the concept of an evolutionary significant unit (ESU) was developed to capture and conserve this genetic diversity. Precise definition of an ESU is difficult; however, the concept is simple: an ESU is a population having significant phylogenetic divergence from other populations (Moritz 1994). Moritz (1994) further quantified this definition by adding the caveat that divergence should be defined by mtdna, and then corroborated with nuclear DNA. Under the United States Endangered Species Act, an ESU must meet two criteria: a) the population must be reproductively isolated from other populations; and, b) it represents an important evolutionary legacy (Waples 1995). Placing clear guidelines on the meaning of important evolutionary legacy is difficult, however Management units In addition to an ESU, it is important to distinguish populations at a smaller scale in which demographic rates (e.g., births and deaths) are meaningful and management decisions often made (Youngson et al. 2003). Moritz (1994) termed this population scale the management unit (MU). The MU can be defined as a population of interbreeding individuals that have distinct allelic frequencies. Essentially, the MU differs from the ESU by not meeting the second criteria of the ESA definition for an ESU; that is, the population, in of itself, does not represent an important evolutionary legacy. Therefore, the MU does not require that the population represent a unique phylogeny. The ESU and MU fall on a hierarchical with an ESU always consisting of one or more MUs. Because MUs represent the scale at which management decisions are typically made, they have an obvious importance to maintenance of genetic integrity (preservation of individual MUs conserves the ESU). 47

62 4.2.2 Defining ESUs Bernatchez (1995) identified a hierarchical step wise approach to defining ESUs using genetic, phenotypic, ecological and geographic information (Figure 10). As part of the process, MUs are established (step 3); however, it is the ranking of these MUs (step 4) that is intended to establish important evolutionary lineages, and hence the ESU. The approach incorporates diverse information into the process and is thereby cognisant of important ecotypes that may lack detectable genetic differentiation from other populations. Despite a growing reliance on molecular phylogenies to distinguish ESUs, integrative approaches (that use information from molecular, ecological and geographic sources) have been advocated more recently (Crandall et al. 2000). The described fourstep approach (Figure 10) has several benefits in that a rating system (e.g., Dizon et al. 1992) that ranks the support for the ESU designation can be built into the process. In this sense, the definition of an ESU is not dichotomous but rather continuous (Crandall et al. 2000). For example, some group designations may strongly support the ESU criteria, while others less so. Furthermore, designation and ranks can be updated with additional information whether it be molecular or ecological. In defining ESUs using the approach, decisions are made regarding appropriate hierarchical levels at which ESUs will be distinguished from MUs (Waples 1995). This decision making process may be influenced within the logistic reality of what is feasible to conserve (Dodson et al. 1998). However, Dodson et al. (1998) identify that the incorporation of socioeconomic constraints should occur after designation of ESUs. That is, a clear set of biological principles and assumptions should be documented to describe how ESUs are established. This information is then used in conjunction with socio economic issues to determine how conservation efforts may or may not proceed among the identified ESUs (Dodson et al. 1998). 48

63 Step 1: Segregate species into groups based on phylogeny. Stizostedion vitreum A B C D Genetic techniques (e.g., mtdna) provide phylogeny. Phylogeny is used for initial segregation into groups. A B C D Phylogeny Step 2: Further divide groups based on phenetics (i.e., similarities and not evolutionary relationships). A A B C D B CD Genetic techniques (e.g., microsatellite DNA) could distinguish different allelic frequencies between groups B and C/D. OR Phenotypic (e.g., morphological differences) could distinguish between groups B and C/D. Step 3: Segregate groups from step 2 into discrete populations (using molecular, ecological or geographic information). A A B CD B C D For example, population C and D may be distinguished given their geographic isolation from each other (i.e., allopatric). Step 4: Rank the discriminated populations of step 3 for their adaptive uniqueness and priority for protection and conservation. A* ESU 1 B C D* ESU 2 ESU 3 For example, populations A and D may be ranked as high priority populations as: A shares a more distant ancestry to populations B/C/D (e.g., A may have originated from a different glacial refugia); and, D might occupy a unique ecological habitat (e.g., riverine species). This results in the designation of three ESUs. In this case, ESU 2 consists of two populations or management units (MUs). Figure 10. A four step process to determine evolutionary significant units (ESUs), adapted from Bernatchez (1995). 49

64 5.0 GENETIC DIVERSITY OF WALLEYE IN ALBERTA Given the large native range of walleye in North America (Figure 1), it is not surprising that a number of walleye groups are recognised as being genetically distinct (Billington 1996). In the parts of North America that experienced the Pleistocene glaciations, walleye are thought to have recolonised from three glacial refugia, the Atlantic, Mississippian and Missourian (Figure 11) (Billington 1996). Areas of the Great Lakes region were recolonised from a combination of these refugia. However, it is thought that western Canada, including Alberta, was recolonised predominantly from the Missourian refugium, with some influence from the Mississippian refugium (Billington 1996). This occurred at the end of the Pleistocene approximately 10,000 to 15,000 years ago (Ferguson et al. 1995), which is a relatively short time span evolutionarily speaking. As a result, little genetic differentiation is expected through mutation (Ferguson et al. 1995). Any differentiation observed is likely due to selection or genetic drift in cases where effective population size is small (Ferguson et al. 1995). Low levels of gene flow, on the other hand, will counter this process of differentiation. 50

65 Figure 11. Distribution patterns of five major walleye mtdna haplotypes in North America in relation to their native distribution (extracted from Billington 1996). Haplotype A represents walleye that originated from the Atlantic refugium after the last glaciation event. Haplotype B originated from the Mississippian refugium. Haplotype C originated from the Missourian refugium. Haplotype D is a mixture of fish from both the Atlantic and Mississippian refugia. Finally, Haplotype E represents a unique strain native to the south eastern USA designated as the Mobile drainage basin haplotype. 51

66 5.1 Methods for assessing walleye stocking and distribution in Alberta Walleye stocking records Stocking records for walleye in the province of Alberta were obtained from two sources. First, two compiled tables of walleye stocking locations, dates, number stocked, size class stocked, strain, etc. were obtained from Alberta Sustainable Resource Development (ASRD; Wagner, J., Alberta Sustainable Resource Development, Edmonton). The two tables were coalesced and any discrepancies between tables clarified (pers. comm. Wagner, J., ASRD, Edmonton). Second, walleye stocking records were independently gathered from data maintained in the Fish Culture Information System (FCIS; ASRD, Edmonton). Results from the FCIS queries were compared to the coalesced table to assess its completeness. Stocking locations (latitude and longitude, North American Datum 1983) were then digitised from 1: or 1: National Topographic Service maps and added to the coalesced table. The resulting table is provided in Appendix A. The original tables provided by ASRD contained information on the survival and reproductive success of stocked walleye cohorts. However, these data were not collected in a consistent and documented manner so their utility is questionable (pers. comm. Wagner, J., ASRD, Edmonton). Therefore, we omitted including this information within Appendix A. A quantitative assessment of the success of walleye stocking in Alberta is currently in progress (pers. comm. Sullivan, M., ASRD, Edmonton). The assessment proposes to utilise three indices for stocking success: 1) survival of stocked fish; 2) reproductive success of stocked fish; and, 3) reproductive success of first generation progeny sired from stocked fish. The indices provide an assessment of stocking goals whether they be establishing new populations, sustaining existing populations or enhancing depleted populations Walleye/sauger distribution and archived samples The distribution of walleye within Alberta lakes was determined from data maintained in the Fisheries Management Information System (FMIS; ASRD, Edmonton). FMIS was queried for all lakes in which walleye were known to be present. Localities were then 52

67 separated as to whether they were stocked or unstocked with walleye based on information from the previous table (i.e., Appendix A). However, unstocked localities are not necessarily native populations as they can include locations that were not stocked but which stocked walleye invaded. The distribution of walleye and sauger in Alberta rivers was determined by querying FMIS for all sample locations in which either fish have been collected. Therefore, unlike the lakes which have only one record of presence, a single river can have multiple sample locations at which walleye or sauger were collected. The presence of archived walleye samples for use in genetic analysis was determined using FMIS. Fin clips are used for aging walleye and are archived in ASRD offices following their determination of age (pers. comm. Norris, H. ASRD, Edmonton). Therefore, we assumed presence of aged walleye from a particular location indicates archived fin material is available. We determined when fin material was collected to assess whether archived samples existed from stocked lakes both before and after stocking. Furthermore, to increase the possibility of having archived samples prior to stocking, we also assessed whether aged fish were older than the earliest stocking event within a lake. That is, fish could not have originated from hatchery stocks as they predated the earliest stocking event. Our methods for determining the distribution of fish and archived samples are contingent on the completeness of the FMIS data. FMIS does not provide a complete record of all fish sampled and aged within the province. Therefore, our distribution maps should be viewed as providing minimum numbers Maps Compiled data on walleye and sauger for the province are displayed graphically on Alberta base maps showing provincial boundaries, cities, lakes, rivers, watersheds and elevation. Digital maps for provincial boundaries, cities, lakes, rivers and elevation were obtained from ArcCanada (ESRI Canada Ltd.). The watershed boundaries are from the Prairie Farm Rehabilitation Administration (Agriculture and Agri Food Canada). All map projections are Lambert Conformal Conic. 53

68 5.2 Walleye and sauger distribution Berry (1995) reports walleye occur throughout Alberta in 64 large rivers and approximately 177 lakes. Information we derived from FMIS indicate walleye presence in 230 lakes across all major drainage basins within the province (Figure 12). Walleye are present primarily in Alberta drainages to the Arctic Ocean and Hudson Bay. The presence of walleye in the Milk River Basin (part of the Missourian drainage) results from stocking of Milk River Ridge Reservoir which drains predominantly back into the South Saskatchewan River Basin through a network of canals. However, a portion of water from Milk River Ridge Reservoir does drain east into Tyrell Lake, Etzikom Coulee or Verdigris Coulee. Etzikom Coulee drains into Pakowki Lake which forms an internal drainage with no surface connection to the Milk River. Walleye from Milk River Ridge Reservoir have moved into Tyrell Lake but saline conditions in downstream coulees and the rarity of surface connections to the Milk River make it highly unlikely for walleye to reach the Milk River (pers. comm. Clayton, T., ASRD, Lethbridge). 54

69 Liard Hay Slave a b Athabasca Peace Churchill Walleye Lakes North Saskatchewan South Saskatchewan Milk Walleye Rivers Legend Stocked Unstocked c Sauger Presence Figure 12.. The distribution of walleye and sauger in the province of Alberta. Panel (a) illustrates the distribution of lakes in which walleye were found and includes stocked and unstocked lakes. Panel (b) illustrates the distribution of walleye in rivers, two of which were stocked. Panel (c) illustrates the distribution of sauger in the rivers of the province. The major drainage basins of Alberta are named in panel (a) and shown by the dark outlines. 55

70 Walleye are present in rivers from all major drainage basins in the province excepting the Liard and Milk river basins. Walleye are present in lakes within the Liard Drainage and their absence in rivers may be an artefact of sampling. Sauger are present within the Milk, Oldman, Bow, South Saskatchewan, Red Deer and North Saskatchewan rivers. Walleye have been stocked in only two rivers within the province, Owl River and Driedmeat Creek (Figure 12). The Owl River is a tributary to Lac la Biche; and, Driedmeat Creek is a tributary to Driedmeat Reservoir (a reservoir formed by damming the Battle River). However, the stocking record for rivers, which are spawning tributaries to lakes, may be incomplete; in that, the recipient lake may be listed as the stocked waterbody rather than the tributary stream which actually received the hatchery fish. For example, the stocking record for Lac la Biche lists walleye fry released into the lake in 1995 (Appendix A); whereas, the majority of these fish were reported to have been introduced to the Owl River rather than the lake specifically (pers. comm. T. Boag, Applied Aquatic Research Ltd., Calgary). The distribution of walleye and sauger overlap in southern and central Alberta (Figure 12). Both species are present in larger rivers of the South and North Saskatchewan drainage basins. However, sauger in the Milk River system are geographically isolated from walleye. Sympatric populations of sauger and walleye provide an opportunity for these species to hybridise, which has been known to occur (Billington et al. 1988, Todd 1990, Nelson and Paetz 1992, Billington et al. 2005). Sauger do not occur in lakes within Alberta (Nelson and Paetz 1992) minimising hybridisation that occurs in lentic systems. 5.3 Walleye stocking history Walleye have been stocked historically throughout the province, and in most drainage basins with the exception of the Slave Drainage (Figure 13). The first record of walleye stocking in the province was in 1926, when walleye were stocked into Sylvan and Buffalo lakes. Stocking continued to be prominent in years that followed. However, stocking reached a peak in the late 1980 s and early 1990 s to help counter effects of severe overharvest in several Alberta lakes (Nelson and Paetz 1992; Joynt and Sullivan 2003; Post et al. 2003). Forty three percent of all stocking events in the province occurred between 1985 and 1995 (Figure 14) (Appendix A). For example, maximum stocking effort occurred in 1991 when 28 lakes were stocked with a total of almost 40 56

71 million fry and fingerlings. Almost 238 million fry and fingerlings were stocked between 1990 and The period of peak stocking effort coincides with operation of the Walleye Enhancement Program. A program established in 1981 with the intent of increasing walleye production in order to meet increasing demands of recreational and commercial fisheries (Berry 1992). Liard Slave Hay Peace Athabasca Churchill North Saskatchewan South Saskatchewan Stocked Locations Milk Figure 13 The distribution of stocking locations in the major drainage basins of the province of Alberta. 57

72 Stocking effort is focused predominantly in the southern and central parts of the province (Figure 13). The goals of Alberta s stocking program are to: a) re establish lakes where walleye have been extirpated; b) enhance depleted stocks; or, c) introduce new populations in the province with the goal of producing self sustaining populations (Berry 1992). As a result, the distribution of effort is focused in areas that receive high angling pressure or may provide accessible angling opportunities if populations were introduced. Qualitative information from regional biologists suggests the success of walleye stocking has been low, with around 30% of lakes stocked producing catchable size fish and less than 10% producing self sustaining populations (pers. comm. Wagner, J., ASRD, Edmonton). 58

73 a Stocking History b c d e f Figure 14 The distribution of stocking events over time in the province of Alberta. Information was pooled into 20 or, in the more recent past, 10 year time periods. Multiple stocking events to a single lake within an indicated time period are shown only as a single point. 59

74 5.4 Stocking sources In order to stock all desired waterbodies in the province, eggs were collected from 6 donor lakes during the period from 1982 to 1991 (Berry 1992) (Figure 15). However, Bistcho Lake, Lesser Slave Lake, and Primrose Lake were the primary sources of gametes (Appendix A). Prior to the 1980 s, no information was available for the donor populations used for walleye stocking in the province, other than the Lac La Ronge strain that was used in the early 1960 s. Lake Diefenbaker stock was only used after It should be noted there is a high proportion of walleye X sauger hybrids in Lake Diefenbaker, although in the area of broodstock collection the walleye do not appear to be hybrids (Billington et al. 2005). 60

75 Bistcho Lake Source Strains Winefred Lake Lac La Ronge, Sask. Lesser Slave Lake Primrose Lake Diefenbaker Lake, Sask. Figure 15. The distribution of donor populations used for stocking walleye in Alberta. Donor lakes from Alberta also appear as stocked lakes because hatcheryraised juveniles were stocked back into donor systems to compensate for removal of gametes. With so few donor populations utilised in Alberta, these sources have been distributed to recipient populations across the province (Figure 16). In general, recipient lakes tend to be at much lower latitudes than donor populations. Bistcho Lake fish have been stocked into seven different drainage basins throughout the province, Lesser Slave Lake fish have been stocked into five basins, and Primrose fish have been stocked into six different basins. Although, as stated previously, Bistcho and Primrose fish stocked into the Milk River drainage are unlikely able to make it into the Milk River (pers. comm. Clayton, T., ASRD, Lethbridge). As a result of interbasin walleye transfers, it is possible there has been homogenization of genetic diversity (loss of among population 61

76 variability) across the province. The probability of this occurring depends on stocked walleye having survived to reproduce and interbreed with wild strain fish in recipient waterbodies. Unstocked lakes may also have been influenced by stocking if fish with donor genetic characteristics have migrated into other waterbodies. 62

77 a b c Bistcho Lesser Slave Lake d Primrose Strains Unknown e f g Winefred Diefenbaker Lac La Ronge Figure 16 Distribution of recipient populations in relation to the donor strain they were stocked. 63

78 5.5 Walleye genetics in Alberta Little is known about the genetic diversity of walleye in Alberta (see Appendix B, Location Alberta). However, Thomas et al. (1999) examined the genetic variation of walleye populations in central Alberta. Nuclear DNA extracted from pelvic fin ray clips was used to examine genetic variation within and among populations in the region. Genotypes were compared with those of donor lake populations in stocked systems and similarity was examined to determine survival of stocked fish and whether introgression had occurred with wild stock (Figure 17). RAPD analysis of DNA with PCR was used in this study, a technique criticized by some for its lack of repeatability among laboratories (Ferguson et al. 1995, Ferguson and Danzmann 1998, Brown and Epifano 2003). However, only fragments that consistently occurred in replicate samples from the same fish were used in the study by Thomas et al. (1999). Results from the study by Thomas et al. (1999) suggest that there has been survival of stocked walleye in central Alberta lakes which has resulted in greater genetic diversity within populations when compared to unstocked populations. Thomas et al. (1999) found genetically unique groups within the region. River populations appear to be distinct from lake populations and, as a result, likely have a high fidelity to spawning areas. However, there is evidence of genetic mixing among river populations as well as introgression into river populations of genetic material from fish stocked into lakes. This suggests fish are straying out of lakes or reservoirs that they were stocked. There is also evidence of local adaptation. For example, Pine Lake which was stocked with the same genetic source as Sylvan Lake is genetically distinct, whereas Sylvan Lake is not distinct from the donor Lac La Ronge strain. The results from the study by Thomas et al. (1999) suggest substantial genetic variation within central Alberta, even though evolutionarily speaking Alberta has only been deglaciated for a short period of time. Assuming walleye colonized Alberta from a single Missourian refugium (Billington 1996), the study by Thomas et al. (1999) suggests processes of selection and genetic drift have caused divergence of central Alberta s walleye populations. Little additional information is available for the province; although, mitochondrial results from Billington et al. (1992) for Bistcho Lake, 64

79 Primrose Lake and Lesser Slave Lake also suggest genetic differences may exist among watersheds. Lakes with Genetic Data Legend Wild Group Source Lake Mixed Group Figure 17. The distribution of information on walleye genetics in Alberta (information from Thomas et al. 1999). Grouping of samples was done by age. Fish the same age as past stocking events are included in the mixed group as they could be either stocked or wild. Fish that were not the same age as stocked walleye were included in the wild group (this group could be influenced by previous stocking events). Source lake fish represent samples of fish from donor stocks. 65

80 5.6 Sampling strategy In order to assess the impact of stocking on genetic diversity in the province, one must first understand natural diversity of walleye without the influence of stocking. This involves a genetic inventory of lakes within the province that represent natural diversity. Sampling should incorporate the entire geographic range of the species within both the province and native range. For example, sampling unstocked populations in all watersheds of Alberta (Figure 12 a and b) and comparing this to work completed in other jurisdictions throughout North America. However, in addition to genetic analyses, information on the physical environment, geologic history and geographic isolation should be considered in order to appropriately sample potentially distinct population units or ESUs (Shaklee and Currens 2003). Methods for identifying ESUs are discussed in Section 4.2. Genetic samples, such as fin clips or scales, exist for a number of systems in Alberta, as they were historically collected for aging purposes and have since been archived (pers. comm. Sullivan, M., ASRD, Edmonton). Figures 18 and 19, respectively, illustrate the lakes and rivers for which aging information is available. Although information is only as complete as FMIS, is does (at least) provide a starting point. Caution is also encouraged when selecting unstocked waterbodies. Unstocked systems may have been influenced by migrants from nearby stocked waterbodies. For example, the Tyrell Lake walleye population was founded from the stocked population in Milk River Ridge Reservoir (pers. comm. Council, T., ACA, Lethbridge). Information from regional biologists could help elucidate which systems are sufficiently isolated from stocked waterbodies to allow assessment of natural genetic diversity. 66

81 a Aged Lakes b All Aged Lakes Sample Size > 20 c d e f Figure 18. The distribution of lakes in Alberta for which aging information is available. They represent lakes for which fin clips or scales have been collected, archived and provide genetic samples. Panel (a) illustrates the distribution of all lakes for which age information is available. Panel (b) demonstrates those lakes for which the sample size is greater than 20, a minimum number required for genetic analysis. The additional panels (c f) represent the distribution of samples by 10 year time increments. 67

82 a Aged Rivers b All Aged Rivers Sample Size > 20 c d e Figure 19. The distribution of rivers in Alberta for which aging information is available. They represent rivers for which fin clips or scales have been collected, archived and provide genetic samples. Panel (a) illustrates the distribution of all lakes for which age information is available. Panel (b) demonstrates those lakes for which the sample size is greater than 20, a minimum number required for genetic analysis. The additional panels (c f) represent the distribution of samples by 10 year time increments. 68