Genetic relationships and gene flow between resident and migratory brook trout in the Salmon Trout River

Journal of Great Lakes Research 38 (2012) 152–158

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Genetic relationships and gene flow between resident and migratory brook trout in the Salmon Trout River Kim Scribner a, b,⁎, Casey Huckins c, 1, Edward Baker d, 2, Jeannette Kanefsky a a

Department of Fisheries and Wildlife, Michigan State University, East Lansing, MI 48824, USA Department of Zoology, Michigan State University, East Lansing, MI 48824, USA Department of Biology, Michigan Tech. University, Houghton, MI 49931, USA d Michigan Department of Natural Resources and Environment, Marquette Fish Hatchery, 488 Cherry Creek Rd., Marquette, MI 49855, USA b c

a r t i c l e

i n f o

Article history: Received 26 July 2011 Accepted 14 November 2011 Available online 22 December 2011 Communicated by Michael E. Sierszen Index words: Brook trout Coaster Salvelinus fontinalis Genetic differentiation Bayesian analyses Population assignment

a b s t r a c t Genetic differentiation among brook trout (Salvelinus fontinalis) of different life history forms and populations can result from reproductive isolation imposed by natural or anthropogenically derived barriers to gene flow, behavioral incompatibilities, or differential exposure to environmental cues. We used multilocus microsatellite genotypes and likelihood and Bayesian-based analyses to characterize the degree of genetic differentiation and evidence of introgression among stream resident brook trout above a natural barrier, and putative stream residents and adfluvial (coaster) brook trout from below the barrier in the Salmon Trout River (STR); the sole tributary along the southern shore of Lake Superior known to be inhabited by a viable remnant population of coaster brook trout. Two genetically differentiated populations were identified, generally associated with individuals inhabiting sections of the STR above and below the falls. No evidence of differentiation was found between a priori classified resident and coaster brook trout from below the falls. Gene flow from individuals above the falls was detected based on evidence of interbreeding between upper river individuals and coasters below the falls. We collected only a relatively small number of individuals that we a priori classified as being stream residents below the falls, and these individuals had a high probability of having ancestry originating from the population above the barrier, which suggests that the stream-resident life history may be exceptionally rare or absent in the lower Salmon Trout River. © 2011 International Association for Great Lakes Research. Published by Elsevier B.V. All rights reserved.

Introduction Brook trout (Salvelinus fontinalis) are a notable feature of the native aquatic community in the Upper Great Lakes (Newman et al., 2003; Schreiner et al., 2008; Scott and Crossman, 1973). Within the Lake Superior basin, some brook trout are stream residents, while others are lacustrine or lacustrine–adfluvial (Huckins et al., 2008). The lacustrine or lacustrine–adfluvial life history form of brook trout, referred to as a ‘coaster’ was historically widespread and abundant throughout Lake Superior's near-shore waters. Coasters were unique because of their Great Lake residency where they lived longer and grew to larger sizes than stream-resident brook trout. Coaster brook trout displaying the lacustrine–adfluvial life history undertook migrations from Lake Superior into tributaries for reproduction, feeding, or refuge and provided a popular sport fishery along the Lake

⁎ Corresponding author at: Department of Fisheries and Wildlife, Michigan State University, East Lansing, MI 48824, USA. Tel.: + 1 517 353 3288. E-mail addresses: [email protected] (K. Scribner), [email protected] (C. Huckins), [email protected] (E. Baker). 1 Tel.: + 1 906 487 2475. 2 Tel.: + 1 906 249 1611x309.

Superior shore, particularly near river mouths (MacCrimmon and Gots, 1980). Coaster abundance and distribution have been drastically reduced from historic levels probably due to the combined effects of overexploitation, habitat changes, and biotic interactions with exotic salmonines (Huckins et al., 2008; Newman et al., 2003; Schreiner et al., 2008). In recent years coasters have become a focus for restoration across the Lake Superior basin. Coaster restoration is a federal, state, tribal and international goal. Fish community objectives for Lake Superior and research priorities for the entire Great Lakes basin (Horns et al., 2003) outline the need for evaluation of the current population structure, habitat conditions, genetic profiles and potential impediments to restoration of coaster brook trout. In a recent status review of Upper Great Lakes brook trout conducted in response to the petition for listing under the Endangered Species Act (73 FR 14950), the U.S. Fish and Wildlife Service identified the need for genetic data to facilitate identification of conservation units and assist with conservation status assessments (USFWS, 2009a, 2009b). Restoration initiatives focused on coaster brook trout in Lake Superior will be dictated in part by information on current coaster population status and life history, both of which are limited by the paucity of direct observational data on the few remaining extant

0380-1330/$ – see front matter © 2011 International Association for Great Lakes Research. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.jglr.2011.11.009

K. Scribner et al. / Journal of Great Lakes Research 38 (2012) 152–158

populations (Schreiner et al., 2008). Data on life history variation within streams, stream fidelity of resident brook trout and on interactions between coaster and resident brook trout from the same drainage, and movements by coaster brook trout among drainages is currently lacking. Use of molecular genetic markers has begun to fill in some of the data gaps by providing indirect insight on past dispersal and ancestry of brook trout lineages (D'Amelio and Wilson, 2008; Theriault et al., 2007; Wilson et al., 2008) but much remains unknown. Data regarding the historical distribution of coaster brook trout along the southern shores of Lake Superior are incomplete (Huckins et al., 2008; Schreiner et al., 2008). Both lake- and stream-dwelling brook trout were historically widespread and abundant across the Upper Peninsula of Michigan. The lacustrine–adfluvial life history is characteristic of the coaster brook trout of the Salmon Trout River (STR), Marquette Co., MI. The STR in the Upper Peninsula of Michigan is the only river verified to be supporting a remnant and viable spawning population of coaster brook trout along the south shore of Lake Superior (Huckins and Baker, 2008). Brook trout have been observed leaving other Lake Superior tributaries but they have not been detected returning to the rivers to spawn, for example, the Gratiot River, Keweenaw County (Carlson, 2003), and the Hurricane River in the Pictured Rocks National Lakeshore (Kusnierz et al., 2009). Coaster brook trout are widely believed to provide a vehicle for dispersal (and gene flow) among tributary brook trout populations (e.g., D'Amelio and Wilson, 2008) and represent a potentially important source for recolonization of other rivers. The STR coaster population may also be important for future broodstock development and stocking efforts if the coaster life history is shown to have nonzero heritability (e.g., Theriault et al., 2007). A fundamental question pertaining to brook trout in the STR is the extent to which there is life history and genetic variation among individuals such that migratory coaster brook trout are behaviorally divergent and reproductively isolated from stream resident brook trout within the STR. The importance of maintaining the viability, integrity, and diversity of natural populations underlies the need for better information on population relationships and interactions among individuals from different life history forms and spatially segregated groups. Our objectives were to describe the degree of genetic differentiation between migratory and suspected resident brook trout in the STR and among spatially segregated brook trout within the STR isolated by a natural barrier, and to quantify levels of interbreeding among individuals from these different groups.

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Methods Study area The STR is a Lake Superior tributary located in northern Marquette County of Michigan's Upper Peninsula (Fig. 1). The STR drains a relatively undeveloped and predominantly forested watershed of 12,823 ha. There is a series of waterfalls referred to as the upper, middle, and lower falls on the river mainstem approximately 11 km upstream from Lake Superior. The falls are a barrier to upstream fish migration and therefore brook trout upstream from the falls were predicted to be reproductively isolated from brook trout below the falls. Fish below the falls have unrestricted access to Lake Superior. Field methods We sampled brook trout using backpack electrofishing during late July and early August, 2001–2003 from river reaches both upstream and downstream of the STR falls. In addition, during summer 2000–2003 we collected fish at two locations within the river with two-way traps that separately collected fish moving upstream and those moving downstream. The fish trap sites were near the river mouth approximately 150 m upstream from Lake Superior and approximately 3 km upstream from Lake Superior (Huckins and Baker, 2008). An additional electrofishing effort was undertaken in June, 2008 that focused on the goal of collecting additional suspected resident brook trout from the STR downstream of the falls. The majority of the wadeable river (over 6 km of river) downstream of the falls was sampled with a relatively continuous run using two backpack electrofishers simultaneously (Fig. 1). The additional June collection effort resulted in 119 brook trout 42–186 mm TL (mean ± SD: 127 ± 33 mm) and only 5 brook trout 205–270 mm TL (mean ± SD: 224 ± 27 mm). Each captured brook trout was measured for total length (TL, mm) and weight (g). We also collected the adipose fin from all captured brook trout for genetic analysis. Criteria for classifying brook trout in the STR We can identify an individual brook trout as a coaster if the fish is captured in Lake Superior or as it enters a tributary from the lake. However, morphological characteristics such as body size cannot reliably differentiate coaster brook trout captured in a stream

Fig. 1. Location of Salmon Trout River and details of river mainstem showing falls and reaches sampled with electrofishing gear. Samples were classified as upper and lower river groups. Lower river brook trout were further designated as migratory or resident on the basis of body size and date of collection (see text for details).

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from putative stream-resident brook trout. Our a priori hypothesis was that a small brook trout captured in the river could be a small coaster that had not yet migrated to Lake Superior or a streamresident that likely never will migrate. We devised criteria to differentiate coasters from putative stream residents in the Salmon Trout River based on previous research on the coasters of the Salmon Trout River (see also Robillard et al., 2011 for support of criteria). Previous research in the Salmon Trout has shown that for the first several years of life, coaster brook trout grow an average of approximately 100 mm TL annually and they tend to return to the river at approximately 300 mm TL, near the end of their third summer (Huckins and Baker, 2008). Although a major juvenile outmigration of brook trout from the river has not been reported, small groups of brook trout of similar length (210.7 ± 5.0 mm TL and 216.3 ± 14.9 mm TL) were detected moving downstream into Lake Superior (Huckins and Baker, 2008) during fall, 2001. Based on their lengths, these outmigrants were likely in the fall of their second year of life, which matches literature accounts of out-migrations in other populations (Naiman et al., 1987; Power, 1980; Ritchie and Black, 1988). Further, body condition of brook trout (estimated by relative weights) in the Salmon Trout population was found to be relatively greater at lengths over approximately 220 mm TL, which suggests that is the approximate size or age at which individuals significantly begin to utilize the lake habitat assuming resource availability is greater in the lake than in the river (Huckins and Baker, 2008). Similarly, Robillard et al. (2011) documented faster growth of known coaster brook trout captured in the open waters of Lake Superior when compared to putative stream resident brook trout captured in nearby tributaries to the lake during summer. Based on these results, we classified putative stream-resident brook trout as individuals of approximately 210 mm in length or longer that were captured in the river in the spring or early summer. If coaster brook trout in the Salmon Trout grow faster and move out into the lake in the fall at lengths ~ 210–220 mm TL, or if they happen to be that length at the age or time of year (i.e., fall) when they outmigrate, then individuals of that length or longer captured the following spring would likely be residents. They would likely be fish that were in their third year of life (Huckins and Baker, 2008). We predicted that if they were going to display the coaster life history, they would have already out-migrated. Adfluvial coaster migration from Lake Superior tends to begin near the end of July. Thus, the specific criteria we used to categorize putative stream resident brook trout (PR) were those individuals of ~ 200 mm TL that were collected in the river before the second week of July. Individuals b200 mm were also retained and genotyped but were classified simply as small brook trout. Finally, brook trout captured in the river mouth trap as they entered the river from Lake Superior were classified as definitive coaster brook trout. Laboratory genetic analyses Adipose fin tissue samples were dried and placed in individual sampling tubes. The DNA was extracted from all samples using QIAGEN DNeasy kits (QIAGEN, Valencia, California, USA). Samples were then quantified using a spectrophotometer, and diluted to working concentrations of 20 ng/μl. Nine microsatellite markers were employed including Sfo23, Sfo8, Sfo12, and Sfo18 (Angers et al., 1995), Ots1 (Banks et al., 1999), Sco19 (Taylor et al., 2001), and C24, D75, C28 (T. King, unpubl. data). Loci were amplified using polymerase chain reaction (PCR) in 10 μl or 25 μl reaction volumes including PCR Buffer (10 mM Tris–HCl at pH 8.3, 1.5 mM MgCl2,50 mM KCl, 100 μg/ml gelatin, 0.01% NP-40, and 0.01% Triton-X 100), 80 μM dNTPs, (Sfo18- Perkin Elmer 10 × PCR Buffer II (Roche, Indianapolis, Indiana)); Sco19-LGL buffer (10 mM Tris–HCl at pH 8.5, 1.5 mM MgCl2, 50 mM KCl, 100 μg/ml nuclease-

free BSA, 0.04% Tween 20), 10 pmol of forward and reverse fluorescently-labeled primers, 0.25 units of Taq polymerase, dimethyl sulfoxide (DMSO) and 40 or 100 ng of DNA. Two sets of three primer pairs were co-amplified: Sfo23, C24, D75 and Sfo8, C28 and Sfo12. Thermocycler conditions included a 2-min denaturation at 94 °C followed by 35–42 cycles of 1-min at 94 °C, 1-min at locus-specific annealing temperature, and 1-min at 72 °C with an additional 5min at 72 °C. A Perkin Elmer GeneAmp 9600 (Boston, Massachusetts) was used for Sfo18 and Sco19. Conditions for these included a 2-min denaturation at 94 °C followed by 15 cycles of 1-min at 94 °C, 35-s at locus-specific annealing temperature, and 10-s at 72 °C with an additional 20 cycles of 45-s at 94 °C, 35-s at locusspecific annealing temperature, and 10-s at 72 °C. Following electrophoresis on denaturing 6.5% polyacrylamide gels, PCR products were visualized using a LI-COR 4300 (Lincoln, Nebraska). 6% polyacrylamide gels were used for Sfo18 and Sco19; products were visualized on an FMBIO II laser scanner (Hitachi Software Engineering Co, Alameda, California). Genotypes were scored based on 20 base-pair standards and individual standards of known genotypes. All genotypes were scored by two experienced laboratory personnel. Statistical analyses Estimates of allele frequency and measures of genetic diversity (observed and expected heterozygosity, and allelic richness) were obtained using the program FSTAT (Goudet, 1995). Measures of deviation of population genotype frequencies from Hardy–Weinberg expectations were estimated using Fisher's exact tests (Guo and Thompson, 1992) implemented in program GENEPOP (Raymond and Rousset, 1995). P-values associated with Hardy–Weinberg exact tests were adjusted for multiple comparisons using sequential Bonferroni corrections (Rice, 1989). Relationships among coaster and PR individuals and smaller individuals of unknown background sampled within the lower STR and individuals sampled above the falls were assessed using pair-wise estimates of variance in allele frequency (Fst, Weir and Cockerham, 1984) using groupings defined a priori based on location (above vs below the falls) and based on phenotype (see criteria above). We also used program STRUCTURE (Falush et al., 2003; Pritchard et al., 2000) to ascertain whether there was evidence for significant substructuring within the stream without a priori group assignment, and whether phenotype or location correlated significantly with probability of assignment to different genetic clusters if present. Ten replicate runs were conducted using a 100,000 burn-in and 1,000,000 bootstrap replicates per run to estimate the likelihood of the data under each hypothesized number of genetic clusters (K = 1–3). Assessment of the number of significant genetic clusters was assessed using Evanno et al. (2005) and the ΔK statistic was assessed for K = 1 to 3. Analyses were also performed using the Bayesian Analysis of Population Structure (BAPS) program (Corander et al., 2004) version 4.0. Analyses were conducted as described above to assign posterior probabilities of genetic cluster affiliation to each individual. The use of multiple population assignment programs, each based on different analytical methods, to converge on a single population assignment helps to ensure that assignments are robust. Programs BAPS and STRUCTURE have been shown to reliably assign individuals to their populations of origin when populations are not well differentiated in allele frequency (as measured based on Fst; Latch et al., 2006). Additionally, we utilized the non-spatial genetic mixture analysis of BAPS to assign individuals to genetic clusters within the STR and an admixture analysis (Corander and Marttinen, 2006) to document evidence of, and statistical support for introgression (i.e., individuals of admixed ancestral origins with ancestral contributions from members of different genetic clusters).

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Results Variation within population segments of the Salmon Trout River Summary measures of genetic diversity were generally high across the sampling groups assigned on the basis of location and phenotype (Table 1). No locus deviated from Hardy–Weinberg expectations for samples from PR and migratory brook trout from the lower STR or from samples from the upper STR. Allele frequency estimates for 2 groups of migratory STR brook trout (captured in the weirs at the mouth and upstream) were indistinguishable (data not shown), and were combined for all analyses within and among drainages. Genetic relationships between migratory and putative resident brook trout (PR) from the Salmon Trout River Based on a priori assignment of individuals to migrant and PR groups in the lower river and to an above falls group, pair-wise estimates of variance in allele frequency (Fst) between lower STR PR and migrant forms with the upper STR brook trout were highly asymmetrical. Estimate of variance in allele frequency (Fst) between migrants from the lower STR and individuals above the falls was 0.093 (P b 0.05) while estimate of Fst between PR brook trout from the lower STR and upper STR was less than half this level (Fst = 0.043, P b 0.05). Allele frequency differences between migratory and PR individuals were not significant (Fst = 0.029; P > 0.05). Further investigations into relationships among STR brook trout from upper and lower segments of the river were conducted using the aspatial admixture models in BAPS and STRUCTURE. In analyses using both complementary approaches, no a priori information regarding group membership was used. Results of the Evanno et al. (2005) test revealed that the number of genetic clusters most consistent with the data was K = 2. One cluster was composed of individuals with high posterior probabilities of assignment (mean 0.94) which originated from the upper river above the falls. A second cluster was composed of all migratory, PR and unassigned (younger and smaller) individuals from the lower river (Fig. 2). Based on posterior probabilities of cluster assignment to ‘upper river’ and ‘lower river’ clusters, three of the 12 PR individuals captured below the falls were clearly from the upper river cluster based on posterior probabilities (mean posterior probability 0.96; Table 1; Fig. 2). Posterior probabilities of assignment to 2 clusters based on BAPS were likewise high (mean 0.99). The lower STR PR samples also contained a number of individuals that were identified as having significant evidence of admixture (either first or second generation introgression). PR and unassigned (young/small) individuals had high posterior probabilities of assignment to both clusters. Brook trout classified as PR (i.e., suspected residents) were comprised of individuals from genetically distinct lower and upper river clusters, including individuals with evidence for introgression. Thus, the genetic distinctions

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between PR and migratory lower river fish were due in part to the individuals assigned a priori as residents being a mixture of highly genetically differentiated fish from the lower and upper STR. BAPS analyses also revealed that several individuals sampled from the upper STR also possessed multi-locus genotypes consistent with introgression between lower and upper river individuals. Because the falls separating the lower and upper river sections of the STR are a complete barrier to natural upstream migration, one interpretation of these results is that lower STR fish have historically been transported upstream. Discussion Estimates of variance in the frequency of alleles at neutral genetic markers such as microsatellites or mitochondrial (mt)DNA are routinely used in ecological and conservation genetic studies (DeSalle and Amato, 2004; Sunnocks, 2000). However, genetics data must be interpreted in the context of the degree, recency, and causes of isolation (Fraser and Bernatchez, 2008). Degree of differences among groups of brook trout from the STR relates to the length of time in which above- and below-barrier populations have been physically separated and the strength of the barriers to gene flow. Above- and below-barrier subpopulations were presumably isolated with the fall of lake levels 8000 to 10,000 years bp (Underhill, 1986). Some gene flow has been observed directionally from above- to below stream sections based on the genotypes of below-barrier fish with above-barrier genotypes (Fig. 2). However, the levels of gene flow have been sufficiently low that levels of variance in allele frequency between above and below-barrier brook trout far exceed levels of variance among brook trout from below-barrier sections of other drainages or between migratory brook trout and individuals collected from instream waters below-barrier sections of the same stream. D'Amelio and Wilson (2008) also found that brook trout above and below barrier water falls were more highly differentiated than were brook trout from below a barrier. Similar phenomena have been described for other salmonid species (e.g., Pearse et al., 2009) where reproductive isolation imposed by elevational gradients (water falls) has been partially overcome by bi-directional gene flow of natural (downstream) and anthropogenic (upstream) origin. For STR brook trout we documented significant differences in microsatellite allele frequency between samples taken above a waterfall barrier and putative resident (PR) and migrant brook trout (Fst = 0.093 and 0.043, respectively) that were higher than pairwise comparisons between PR and migrant brook trout collected from the lower river (Fst = 0.026). Data interpreted based on interpopulation variance (Fst) using groups defined a priori suggest that there is a much higher rate of gene flow between migratory and PR brook trout than between above- and below-barrier brook trout from the Salmon Trout River or between brook trout from immediately adjacent drainages. Fish sampled and analyzed in each of the 3

Table 1 Summary measures of genetic diversity for wild brook trout sampled from above and below a falls on the Salmon Trout River, MI. Samples include coaster and resident life history forms from the lower river as well as smaller individuals that were not classified. Sampling groupa Measures of genetic diversity

Lower Salmon Trout River (coaster) (N = 68)

Lower Salmon Trout River (resident) (N = 12)

Lower Salmon Trout River (small) (N = 86)

Upper Salmon Trout River (N = 23)

Observed heterozygosity Expected heterozygosity Allelic richness Inbreeding coefficient Number (%) of transposed individuals Number (%) of admixed individuals

0.573 0.603 3.10 0.051 0 (0.0)

0.661 0.718 3.70 0.075 3 (25.0)

0.621 0.614 3.31 0.034 2 (2.3)

0.725 0.783 3.95 0.069 0 (0.0)

6 (8.8)

1 (8.3)

2 (2.3)

2 (8.6)

a

Group membership was determined based on size and location in the stream as described in the Methods section.

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Fig. 2. Posterior probabilities of origin to 2 genetic clusters (K = 2) based on output from program BAPS (Y-axis) for each individual from the Salmon Trout River. Individuals are grouped based on location as above the impassable barrier (upper river) and below the falls into migrant, resident, and small undetermined forms. Gray and clear portions of the individual bars correspond to posterior probabilities of different genetic clusters. Arrows across top of figure indicate fish with significant likelihood of admixture between upper river and lower river individuals. There is evidence for down-stream movements and gene flow but no evidence that migrant and resident groups collected below the falls represent distinct genetic clusters.

categories (migrant, putative resident, and small) included individuals collected over at least 3 years and in 2001 and 2002 fish were collected from each category supporting the conclusion that detected patterns were not driven by temporal variation in recruitment. Upstream fish were collected in only 1 year. Results of the genetic analysis support field observations in the STR that a true resident life history may be exceedingly rare or nonexistent in this river. Genetic relationships between coaster and resident phenotypes We found that most lower river (below-barrier) PR and migrant brook trout from the Salmon Trout River were part of the same genetic ‘cluster’ or statistically supported groupings of brook trout (Fig. 2). Of 80 coaster and PR brook trout analyzed from the lower river, 70 individuals had posterior probabilities indicating membership to one genetic cluster, three individuals had posterior probabilities associated with a second genetic cluster and 7 individuals were admixed. These proportions are consistent with random mating among cluster members in the lower river. Based on microsatellite data from 8 loci collected from migratory (coaster) brook trout in Lake Superior and brook trout in Ontario streams, D'Amelio and Wilson (2008) found that coaster brook trout were not a genetically cohesive group divergent from instream brook trout. Lake Superior coasters appear to be life history variants of brook trout. Lack of genetic variation between resident and adfluvial life history forms is wide spread in salmon (Northcote, 1997). Low levels of variance in allele frequency between instream (small and PR) and migratory brook trout in the Salmon Trout River are consistent with high levels of inter-breeding as has been found between migratory and resident populations of brook trout elsewhere (Theriault et al., 2007). Theriault et al. (2007) used genetic determination of parentage based on microsatellite genotyping of breeding adults and juveniles. There was no evidence for significant differences in allele frequency between putative resident and migratory brook trout from the river sampled. The authors found no evidence for assortative mating. Half of all male–female pairs reconstructed as parents of offspring (21 of 42) involved an anadromous and resident spawner (Theriault et al., 2007). The few putative resident and the numerically dominant migratory brook trout in the river belonged to the same breeding population. Matings between migratory and putative resident individuals were mediated by the instream males (possibly young or fish with above the barrier origins) breeding with migratory females. We have observed multiple instances of interactions between large migratory females on redds and multiple small and presumably male brook trout in the Salmon Trout River

(Huckins and Baker, unpublished data). Levels of introgression possibly occurring in the Salmon Trout and as described in Theriault et al. (2007) would effectively homogenize allele frequencies at neutral genetic markers that are bi-parentally inherited (e.g., microsatellite loci). However, markers that are maternally inherited might be more informative. Several additional papers have estimated the magnitude of genetic differentiation between resident and migratory brook trout either directly or indirectly. Results vary across studies. Rogers and Curry (2004) sampled brook trout from 12 locations throughout the Miramichi river in New Brunswick using 6 microsatellite loci. The authors used individual assignment tests without a priori assumptions of population association to determine the number of ‘genetic populations’ consistent with the genotypic data. Five genetic clusters were inferred from the data but cluster membership did not correspond with geographic proximity among sampling locales. The authors concluded that habitat selection and life history (anadromous vs resident) stage may underlie population structuring. Jones et al. (1997) used mitochondrial DNA and allozymes to compare sympatric resident and migratory brook trout for the same drainage and hatchery strains to their progenitor populations. The authors found that sympatric river resident and migratory brook trout from the same stream were genetically more similar to each other than either form was to brook trout from other drainages, suggesting that resident and migratory individuals are not reproductively isolated. Mitochondrial DNA data suggest that migratory and resident individuals are part of the same evolutionary lineage. Theriault et al. (2007) used genetic markers to reconstruct family pedigrees, used a mixed effects model to estimate the heritability of the residency/anadromy life history trait, and tested for genetic correlations between phenotypic traits and the life history strategy, where phenotypic correlations between body size, shape and life history were previously shown (Morinville and Rasmussen, 2003, 2006; Theriault and Dodson, 2003). Theriault et al. (2007) estimated significant heritabilities for life history tactic, body size and body depth (heritabilities ranging from 0.39 to 0.56). A significant genetic correlation was found between life history form and body size and anadromous fish were found to be genetically associated with larger body size at age. Perry et al. (2005) used both neutral genetic markers (microsatellites) and a quantitative genetic breeding design to determine genetic differences between anadromous and resident populations of brook trout collected from the Laval River drainage, Quebec. The study is important because the authors were able to estimate genetic differentiation among resident and anadromous brook trout by examining

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the relative effects of neutral genetic processes (drift, migration) versus selection by jointly using neutral genetic marks such as microsatellites, which are not under selection, and measures of phenotypic variation in fitness-related traits. The authors proposed that differentiation in adaptive traits may be most apparent for populations that have evolved to utilize different niches (Bernatchez, 2004). The idea behind the study was that high quantitative differentiation between life history forms relative to differentiation in neutral markers would provide compelling evidence for the importance of adaptation to local environmental conditions which could evolve rapidly depending on the relative costs and benefits to alternative forms (Hendry et al., 2004; Perry et al., 2005). This point has direct relevance to the evolution of migratory forms in Great Lakes habitats. The authors found evidence for significant variance in allele frequency between resident and anadromous samples confirming that they were genetically distinct. The estimate of variance between anadromous and resident forms (mean Fst = 0.153) was considerably higher than observed differences among anadromous populations (range 0.05–0.10; Castric and Bernatchez, 2003). Migratory tendencies vary among species, populations or groups occupying different habitats, sexes, individuals, and years, and numerous factors generate and maintain variation in migratory behavior (review in Hendry et al., 2004). The expression of an anadromous or resident life history is thought to be determined in part by consideration of the fitness consequences of alternate patterns (Gross, 1985). Fitness consequences can be cast in terms of the trade-offs between probabilities of current and future reproductive success owing to differential probabilities of survival, growth, and fecundity resulting from occupancy of different habitats (resident stream or lake/ocean). Expression of either life history form would be expected to likewise vary depending on environmental circumstances (Hendry et al., 2004; Quinn, 2005). For example, fish remaining in streams will have comparatively higher probabilities of survival but will on average grow at a slower rate than individuals which migrate to lacustrine (or ocean) habitats. Accordingly, age and size at sexual maturity are likely to differ between life history forms. Likewise, fecundity (of females) in terms of egg number and size will also vary. Sample sizes of PR brook trout below the barrier on the Salmon Trout River were small. Accordingly, there is considerable uncertainty about variance estimates when allele frequencies were estimated for PR individuals prior to analyses and without clarification of cluster (or admixture) status. Sample size is important and has been shown to affect DPS designations based on genetic data (review in Fallon, 2007). The exceptionally small number of brook trout we collected in June, 2008 in the Salmon Trout River (i.e., 126 total brook trout in over 6 km of river) and the fact that only 5 of them fit our a priori criteria for being putative residents (>200 mm TL), supports the hypothesis that fish below the falls that were members of the lower river genetic cluster may all be coasters and there are no stream resident fish below the lower falls. A lack of resident brook trout sympatric with mainstem populations of migrant brook trout has been confirmed in other larger rivers (e.g., Theriault et al., 2007). Conclusions Data from this study reveal that relative differences between coaster and instream lower river brook trout from the Salmon Trout River are small compared to levels of variation among geographically proximal streams (Scribner et al., 2005). Further, based on individualbased analyses, interpretations of genetic relationships between coaster and purported resident forms from the Salmon Trout River are confounded by evidence of gene flow between upper and lower Salmon Trout River groups and evidence of inter-breeding. If present, the rarity of the resident life history in the Salmon Trout River and the apparent association between the resident behavior and gene flow from a true resident population above a natural barrier highlight the

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complexity of brook trout life histories and the mechanisms driving this variation. Data reported herein and elsewhere in restricted regions of Lake Superior represent important first steps towards understanding the complexity of existing diversity in Great Lakes brook trout. A comprehensive and standardized genetic data set will be required if pressing management issues in the Great Lakes and adjoining areas are to be addressed. Acknowledgments Funding for this project was provided by the National Fish and Wildlife Federation, the Huron Mountain Wildlife Foundation, the Michigan Department of Natural Resources, Michigan Technological University, and Michigan State University through the Partnership for Ecosystem Research and Management (PERM) cooperative program between the Department of Fisheries and Wildlife at Michigan State University and the Michigan Department of Natural Resources. Kristi Filcek assisted with the laboratory analysis. 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