Diagnostic single nucleotide polymorphisms for identifying westslope cutthroat trout (Oncorhynchus clarki lewisi), Yellowstone cutthroat trout (Oncorhynchus clarkii bouvieri) and rainbow trout (Oncorhynchus mykiss)

We describe 12 diagnostic single nucleotide polymorphism (SNP) assays for use in species identification among rainbow and cutthroat trout: five of these loci have alleles unique to rainbow trout (Oncorhynchus mykiss), three unique to westslope cutthroat trout (O. clarkii lewisi) and four unique to Yellowstone cutthroat trout (O. clarkii bouvieri). These diagnostic assays were identified using a total of 489 individuals from 26 populations and five fish hatchery strains.     

Hybrid zone structure and the potential role of selection in hybridizing populations of native westslope cutthroat trout (Oncorhynchus clarki lewisi) and introduced rainbow trout (O. mykiss)

Introgressive hybridization is a common feature of many zones of contact between divergent lineages of fishes. This is particularly common when taxa that are normally allopatric come into artificial (human-induced) secondary contact. We examined 18 native populations of westslope cutthroat trout (Oncorhynchus clarki lewisi, WCT) to determine the extent of introgressive hybridization with introduced rainbow trout (O. mykiss, RBT) and the genetic structure of hybridizing populations in the upper Kootenay River, southeastern British Columbia, Canada. Using four diagnostic nuclear loci we calculated a hybrid index, inbreeding coefficient, FIS, and the linkage disquilibrium correlation coefficient, Rij, for each locality to determine the distribution of genotypes in each population. We also categorized the 142 hybrid individuals found across localities into four hybrid classes based on their genotypes. The majority of localities (11/18) showed a unimodal distribution of genotypes skewed towards genotypes of WCT. Two localities, however (lower Gold Creek and Lodgepole Creek) showed a flat to bimodal distribution and one site (lower Bull River) showed a unimodal distribution skewed towards RBT genotypes. The majority of hybrid individuals were classified genotypically as WCT backcrosses (59%) and post-F1 individuals (24%). We found a skewed ratio of pure WCT to pure RBT (17:1) and only four F1 hybrids (3%), suggesting that the spread of RBT alleles may be facilitated by hybrids straying to neighbouring populations. We also tested for the action of selection in one population using cohort analyses, but found little evidence of differential selection between pure WCT and hybrid individuals. Pooled across age classes there were significant differences in genotypic frequencies among loci suggesting differential introgression. There was no asymmetry to the hybridization between rainbow trout and westslope cutthroat trout because both species' mitochondrial DNA haplotypes were observed at similar frequencies in the hybrids. Our analyses suggest that hybrid swarms are likely to form in the upper Kootenay River drainage and that certain native WCT populations in British Columbia are at risk of local genomic extinction.     

An analysis of spatial and environmental factors influencing hybridization between native westslope cutthroat trout (Oncorhynchus clarki lewisi) and introduced rainbow trout (O. mykiss) in the upper Kootenay River drainage, British Columbia

Westslope cutthroat trout (Oncorhynchus clarki lewisi, WCT) and introduced rainbow trout (O. mykiss, RBT) readily hybridize and introgression has occurred in many drainages across the historic native range of WCT. In British Columbia (Canada), the upper Kootenay River drainage is the heart of the westslope cutthroat trout (WCT, Oncorhynchus clarki lewisi) distribution in Canada this drainage harbours native WCT gene pools that are thought to be under threat from hybridization with introduced rainbow trout (RBT, O. mykiss). In this study, we assess the extent and distribution of WCT × RBT hybridization in the upper Kootenay River drainage. We used four diagnostic nuclear loci to determine the extent of hybridization in 981 fish collected from 23 sample localities across 12 different streams in the upper Kootenay River drainage. About 14% (142/981) of individuals were identified as hybrids (an individual with both RBT and WCT alleles), 3.4% (33/981) were identified as pure RBT, and the remaining individuals were identified as pure WCT. Although pure RBT were absent from the majority of locales (20/23), we found evidence of hybridization at 78% (18/23) of the localities and the percentage of heterospecific alleles (% I) ranged from 0.7% to 97.1%. Only 22% (5/23) of the localities showed no evidence of hybridization. Spatial analysis showed clustering among hybridized locations and decreasing hybridization with increasing distance from Koocanusa Reservoir, suggesting that the reservoir acts as a RBT source. We found no evidence that stream order, stream magnitude, or stream elevation influenced the extent of hybridization among localities. We compared our results to an analysis conducted in 1986, which indicated that hybridization is relatively recent in the upper Kootenay River drainage and that it is increasing in magnitude and distribution. In the absence of timely management intervention, the genetic integrity of WCT populations in the heart of their Canadian range may be lost. Our results indicate the dynamic nature of hybridization in fluvial systems and that for closely related taxa such as WCT and RBT, hybridization appears to be largely influenced by physical barriers to dispersal and contact between species.     

A molecular analysis of hybridization between native westslope cutthroat trout and introduced rainbow trout in southeastern British Columbia, Canada

Restriction site variation in the Ikaros gene intron was used to assess the incidence of westslope cutthroat trout ( Oncorhynchus clarki lewisi ), rainbow trout ( O. mykiss ) and interspecific hybrids at 11 localities among eight streams tributary to the upper Kootenay River system in south-eastern British Columbia, Canada. Out of 356 fish assayed by this technique, hybrids ( n =16) were found at seven of the 11 sites across five different streams. Rainbow trout ( n =6) were found at two of the 11 sites. Analysis of hybrids with a second genetic marker (heat shock 71 intron) indicated that most represented either backcrosses to both westslope cutthroat and rainbow trout, or post F₁ hybrids. Mitochondrial DNA analysis indicated that hybrid matings occur between male rainbow trout and female westslope cutthroat trout and vice versa. Comparison of present hybridization in five tributaries relative to an allozyme-based analysis in the mid-1980s, that documented hybrids in only a single tributary of seven that were common to the two studies, suggests that hybridization and introgression has increased in upper Kootenay River tributaries. The present analysis is a conservative estimate of genetic interaction between the species because introgression was not tested in the majority of samples. Identification of genetically pure westslope cutthroat trout populations, and why they might be resistant to introgression from rainbow trout, are crucial conservation priorities for this unique subspecies of cutthroat trout.     

Novel molecular markers differentiate Oncorhynchus mykiss (rainbow trout and steelhead) and the O. clarki (cutthroat trout) subspecies

A suite of 26 PCR‐based markers was developed that differentiates rainbow (Oncorhynchus mykiss) and coastal cutthroat trout (O. clarki clarki). The markers also differentiated rainbow from other cutthroat trout subspecies (O. clarki), and several of the markers differentiated between cutthroat trout subspecies. This system has numerous positive attributes, including: nonlethal sampling, high species‐specificity and products that are easily identified and scored using agarose gel electrophoresis. The methodology described for developing the markers can be applied to virtually any system in which numerous markers are desired for identifying or differentiating species or subspecies.     

Microsatellite analysis of genetic population structure in an endangered salmonid: the coastal cutthroat trout (Oncorhynchus clarki clarki)

The genetic population structure of coastal cutthroat trout ( Oncorhynchus clarki clarki ) in Washington state was investigated by analysis of variation in allele frequencies at six highly polymorphic microsatellite loci for 13 anadromous populations, along with one outgroup population from the Yellowstone subspecies ( O. clarki bouvieri) (mean heterozygosity = 67%; average number of alleles per locus = 24). Tests for genetic differentiation revealed highly significant differences in genotypic frequencies for pairwise comparisons between all populations within geographical regions and overall population subdivision was substantial ( F _ST = 0.121, R _ST = 0.093), with 44.6% and 55.4% of the among-population diversity being attributable to differences between streams ( F _SR = 0.054) and between regions ( F _RT = 0.067), respectively. Analysis of genetic distances and geographical distances did not support a simple model of isolation by distance for these populations. With one exception, neighbour-joining dendrograms from the Cavalli-Sforza and Edwards' chord distances and maximum likelihood algorithms clustered populations by physiogeographic region, although overall bootstrap support was relatively low (53%). Our results suggest that coastal cutthroat trout populations are ultimately structured genetically at the level of individual streams. It appears that the dynamic balance between gene flow and genetic drift in the subspecies favours a high degree of genetic differentiation and population subdivision with the simultaneous maintenance of high heterozygosity levels within local populations. Results are discussed in terms of coastal cutthroat trout ecology along with implications for the designation of evolutionarily significant units pursuant to the US Endangered Species Act of 1973 and analogous conservation units.     

Development and evaluation of 200 novel SNP assays for population genetic studies of westslope cutthroat trout and genetic identification of related taxa

DNA sequence data were collected and screened for single nucleotide polymorphisms (SNPs) in westslope cutthroat trout (Oncorhynchus clarki lewisi) and also for substitutions that could be used to genetically discriminate rainbow trout (O. mykiss) and cutthroat trout, as well as several cutthroat trout subspecies. In total, 260 expressed sequence tag‐derived loci were sequenced and allelic discrimination genotyping assays developed from 217 of the variable sites. Another 50 putative SNPs in westslope cutthroat trout were identified by restriction‐site‐associated DNA sequencing, and seven of these were developed into assays. Twelve O. mykiss SNP assays that were variable within westslope cutthroat trout and 12 previously published SNP assays were also included in downstream testing. A total of 241 assays were tested on six westslope cutthroat trout populations (N = 32 per population), as well as collections of four other cutthroat trout subspecies and a population of rainbow trout. All assays were evaluated for reliability and deviation from Hardy–Weinberg and linkage equilibria. Poorly performing and duplicate assays were removed from the data set, and the remaining 200 assays were used in tests of population differentiation. The remaining markers easily distinguished the various subspecies tested, as evidenced by mean G_ST of 0.74. A smaller subset of the markers (N = 86; average G_ST = 0.40) was useful for distinguishing the six populations of westslope cutthroat trout. This study increases by an order of magnitude the number of genetic markers available for the study of westslope cutthroat trout and closely related taxa and includes many markers in genes (developed from ESTs).     

Spatial partitioning and asymmetric hybridization among sympatric coastal steelhead trout (Oncorhynchus mykiss irideus), coastal cutthroat trout (O. clarki clarki) and interspecific hybrids

Hybridization between sympatric species provides unique opportunities to examine the contrast between mechanisms that promote hybridization and maintain species integrity. We surveyed hybridization between sympatric coastal steelhead (Oncorhynchus mykiss irideus) and coastal cutthroat trout (O. clarki clarki) from two streams in Washington State, Olsen Creek (256 individuals sampled) and Jansen Creek (431 individuals sampled), over a 3-year period. We applied 11 O. mykiss-specific nuclear markers, 11 O. c. clarki-specific nuclear markers and a mitochondrial DNA marker to assess spatial partitioning among species and hybrids and determine the directionality of hybridization. F1 and post-F1 hybrids, respectively, composed an average of 1.2% and 33.6% of the population sampled in Jansen Creek, and 5.9% and 30.4% of the population sampled in Olsen Creek. A modest level of habitat partitioning among species and hybrids was detected. Mitochondrial DNA analysis indicated that all F1 hybrids (15 from Olsen Creek and five from Jansen Creek) arose from matings between steelhead females and cutthroat males implicating a sneak spawning behaviour by cutthroat males. First-generation cutthroat backcrosses contained O. c. clarki mtDNA more often than expected suggesting natural selection against F1 hybrids. More hybrids were backcrossed toward cutthroat than steelhead and our results indicate recurrent hybridization within these creeks. Age analysis demonstrated that hybrids were between 1 and 4 years old. These results suggest that within sympatric salmonid hybrid zones, exogenous processes (environmentally dependent factors) help to maintain the distinction between parental types through reduced fitness of hybrids within parental environments while divergent natural selection promotes parental types through distinct adaptive advantages of parental phenotypes.     

Characterization of 13 microsatellites for Lahontan cutthroat trout (Oncorhynchus clarki henshawi) and cross-amplification in six other salmonids

Thirteen newly developed tri- and tetranucleotide repeat microsatellite markers were developed for Lahontan cutthroat trout (Oncorhynchus clarki henshawi), a threatened subspecies endemic to the Lahontan hydrographic basin in the western USA. These loci are highly polymorphic with five to 30 alleles per locus and observed heterozygosities ranging from 0.4 to 0.7. Cross-species amplification of these markers was most successful in the closely related rainbow trout, Oncorhynchus mykiss, with only three loci amplifying in brown trout, Salmo trutta. Nonoverlapping allelic distributions for many of these loci among the six salmonid species screened suggest these markers may be useful for hybrid determination.     

Diet overlap among size classes of Colorado River cutthroat trout (Oncorhynchus clarki pleuriticus) in a high-elevation mountain stream

We examined the taxonomic composition, abundance, and size of food items consumed by young-of-year, juvenile, and adult Colorado River cutthroat trout (Oncorhynchus clarki pleuriticus) in order to determine the degree of diet overlap occurring in a relatively unproductive, high-elevation, mountain stream. Overall, we identified 49 Families of insects representing nine Orders, and 4 other Classes of organisms in the diets of the trout sampled and saw no evidence of piscivory. Each size class of fish consumed significantly different taxa and significantly different sizes of food items. However, despite these differences, the proportional similarity index (PSI) indicated that there was considerable overlap in taxa and sizes of organisms consumed by the three size classes. The greatest overlap occurred between young-of-year and juveniles, and between juveniles and adults. Both the relatively high proportion of small items in the adult diet and the slow growth rate of adults in these streams indicate that food may be limiting for adults and that intraspecific competition between adults and smaller size classes may be high.     

Interaction for food and space between experimental populations of juvenile coho salmon (Oncorhynchus kisutch) and coastal cutthroat trout (Salmo clarki) in a laboratory stream

Populations of juvenile coho salmon and coastal cutthroat trout frequently cohabit small coastal streams in western North America. The pattern and mechanism of interactions for food and space between experimental populations of underyearlings of these two salmonids were examined at 3, 5 and 13 °C in a laboratory riffe/pool environment simulating winter and summer conditions. When tested separately at 13 °C, their habitat demands were similar and approximately 60–75% of either species occurred in pools. When tested together they segregated, with approximately 75% of coho in pools and up to 63% of cutthroat trout in riffles. In winter, at 3 °C, both species preferred pools and overhead cover, whether tested separately or together. At 5 °C, they partially segregated in a pattern similar to, but far less pronounced than, that in summer. Both species used similar forms of aggressive behaviour, although aggressive displays were more frequently used by coho, while nipping was more frequently used by cutthroat trout. Both salmonids were most aggressive when food was presented, irrespective of season, although coho responded with greater rapidity and intensity to feeding than did cutthroat trout. When tested together in summer, aggressiveness was high for both species, and agonistic interference by coho in pools and cutthroat trout in riffles appeared to largely account for their segregation. At 3 °C, aggression was low and both species weakly defended pools. At 5 °C, their aggression rose considerably and they partially segregated in a pattern resembling that at 13 °C. The mechanism of segregation between these two salmonids is clearly that of Nilsson's interactive type, which presumably functions to attenuate competition when streams are most likely to be resource limiting; typically, that is the late summer period of low flows and relatively high fish population densities.Based on a dissertation in partial fulfillment of the requirements for the degree of Doctor of Philosophy, Institute of Animal Resource Ecology, University of British Columbia, Vancouver, B.C.Ministry of Agriculture and Fisheries, Fisheries Research Division, P.O. Box 8324, Riccarton Christchurch, New Zealand     

Discovery and characterization of novel genetic markers for use in the management of Lahontan cutthroat trout (Oncorhynchus clarkii henshawi)

The Lahontan cutthroat trout (Oncorhynchus clarkii henshawi) is threatened by habitat destruction, over‐harvest and hybridization with nonnative trout. Currently, three Geographic Management Units (GMUs) are recognized within the taxon. Here, we describe a suite of 68 single‐nucleotide polymorphism (SNP) genetic markers for use in the study and management of Lahontan cutthroat trout and a closely related subspecies, the Paiute cutthroat trout (O. c. seleneris). These include markers variable within the two subspecies (n = 35), diagnostic for the two subspecies (n = 23) and diagnostic for Yellowstone cutthroat trout (O. c. bouvieri) and other closely related subspecies (n = 10). Sixty‐three markers were discovered by Sanger sequencing of 171 EST loci in an ascertainment panel including Lahontan cutthroat trout from four populations representing all GMUs. Five markers were identified in a secondary sequencing effort with a single population of Lahontan cutthroat trout. TaqMan assays were validated on six Lahontan cutthroat trout populations and a diverse panel of other trout. Over 90% of the markers variable in Lahontan cutthroat trout were polymorphic in at least two populations, and 66% were variable within all three GMUs. All Lahontan diagnostic markers were also fixed for the Lahontan allele in Paiute cutthroat trout. Most of the Yellowstone diagnostic markers can also be used for this purpose in other cutthroat trout subspecies. This is the first set of SNP markers to be developed for Lahontan cutthroat trout, and will be an important tool for conservation and management.     

Diet overlap among non‐native trout species and native cutthroat Trout (Oncorhynchus clarkii) in two U.S. ecoregions

The invasion of freshwater ecosystems by non‐native species can constitute a significant threat to native species and ecosystem health. Non‐native trouts have long been stocked in areas where native trouts occur and have negatively impacted native trouts through predation, competition, and hybridization. This study encompassed two seasons of sampling efforts across two ecoregions of the western United States: The Great Basin in summer 2016 and the Yellowstone River Basin in summer 2017. We found significant dietary overlaps among native and non‐native trouts within the Great Basin and Yellowstone River Basin ecoregions. Three orders of invertebrates (Ephemeroptera, Trichoptera, and Diptera) composed the majority of stomach contents and were responsible for driving the observed patterns. Great Basin trout had higher body conditions (k), and non‐native Great Basin trout had higher gut fullness values than Yellowstone River Basin trout, indicating a possible limitation of food in the Yellowstone River Basin. Native fishes were the least abundant and had the lowest body condition in each ecoregion. These findings may indicate a negative impact on native trouts by non‐native trouts. We recommend additional monitoring of native and non‐native trout diets, regular invertebrate surveys to identify the availability of diet items, and reconsidering stocking efforts that can result in overlap of non‐native fishes with native cutthroat trout.     

The contribution of newly established populations to the dynamics of range expansion in a one-dimensional fluvial-estuarine system: rainbow trout (Oncorhynchus mykiss) in Eastern Quebec

Aim Rainbow trout (Oncorhynchus mykiss, Walbaum 1792) is an exotic salmonid invading eastern Canada. First introduced for recreational fishing in Ontario, Quebec and the Maritime provinces, the species is now spreading in salmon rivers located in Eastern Quebec, where its stocking is strictly forbidden. Newly established populations have been found along the St Lawrence Estuary. To effectively mitigate the potential threat the invasion poses to native salmonids, we aimed to document the invasion’s origin and progress in the St Lawrence River and estuary. We first determined genetic origins among several potential wild and cultured source populations, found at the upstream and downstream extremities of the St Lawrence system. Thereafter, we studied the range expansion, predicting that the invasion process conforms to a one‐dimensional stepping‐stone dispersion model. Location Recently invaded salmon rivers that flow into the Estuary and Gulf of St Lawrence in Quebec, and watercourses supporting naturalized populations (Lake Ontario, Lake Memphremagog and Prince‐Edward‐Island rivers). Methods Rainbow trout from 10 potential source populations (wild and domestic strains) and 243 specimens captured in salmon rivers were genotyped at 10 microsatellite loci. Genetic origins of specimens and relationship between colonies were assessed using assignment analyses based on individuals and clusters. Results Invasion of rainbow trout in Eastern Quebec is directed downstream, driven by migrants from upstream naturalized populations, found in the Ganaraska River (Lake Ontario), and, to a lesser extent, in Lake Memphremagog. Populations from the Maritime provinces and domestic strains do not contribute to the colonisation process. A recently established population in Charlevoix (Eastern Quebec) supplies other downstream colonies. Main conclusions Rainbow trout is spreading from Lake Ontario downstream to Eastern Quebec using the St Lawrence River system as an invasion corridor. Range expansion in a downstream direction is driven by a more complex stepping‐stone dispersion model than predicted. Management options to protect native salmonids include reducing the effective size of the Charlevoix population, impeding reproduction in recently colonized rivers, halting the upstream migration of anadromous spawners, and curtailing stocking events inside the stocking area.     

Landscape topography and the distribution of Lahontan cutthroat trout (Oncorhynchus clarki henshawi) in a high desert stream

Lahontan cutthroat trout, Oncorhynchus clarki henshawi, are currently limited in their distribution to a patchwork of small isolated populations, the result of habitat degradation and natural variation in landscape and in-stream conditions. The objectives of this study were to determine if landscape topography influences trout distribution, and if water temperatures control this response. The work was carried out in a sub-basin of the Quinn River system, McDermitt Creek, which drains the sagebrush desert of southeastern Oregon and northern Nevada. Headwater tributaries of this creek consist of alternating canyon-confined and valley bounded reaches. Trout within these systems are challenged by low discharge and high temperatures during the summer, and anchor ice during the winter. Contiguous whole stream surveys were used to look at trout distribution during the summer of 2003 and spring and fall of 2004. Our results suggested that topography can affect trout distribution. Trout numbers were highest in areas with greater numbers of nick-points (the transition zones between less confined and more confined valley segments) and greater valley confinement. Additionally, in the downstream portion of our headwater reaches, more trout were found in nick-points than expected based on the availability of this habitat type. Our data suggest that hyporheic inputs may be high in such areas, thus providing trout with shelter from warm water in the summer, anchor ice in the winter, and shallow stream depths during all seasons. Spatial occurrence of these areas of refugia can be taken into consideration when planning land use activities and restoration efforts. Further research is required to confirm that topography can affect the distribution of Lahontan cutthroat trout in other systems, and to better understand the mechanisms behind these patterns.     

Population genetic structure of the endangered crayfish Austropotamobius pallipes in France based on microsatellite variation: biogeographical inferences and conservation implications

1. One important goal in conservation biology is to characterise evolutionary lineages within endangered species before management decisions are taken. Here, we assess population differentiation in the freshwater crayfish Austropotamobius pallipes, an endangered species endemic to western Europe and provide valuable information for the conservation of French populations. 2. Analysis of five microsatellite loci in 44 populations revealed very different within population levels of genetic diversity (0.000 < H₀ < 0.564). Two groups, corresponding to northern and southern French populations, showed a high degree of genetic differentiation in both allele frequencies and allele sizes. Comparison of these results with previous studies of A. pallipes strongly suggests that the divergence between northern and southern populations could have occurred during the last glaciation period of the Pleistocene from one Atlantic and one Mediterranean refuge. 3. Evidence for genetic admixture between these two lineages was revealed by correspondence analyses in southern populations, probably as the result of artificial translocations. 4. French populations appeared significantly differentiated among the different river drainages and were highly structured within rivers. The impact of population size, population bottlenecks and founder events on the population genetic differentiation are discussed. 5. Based on these results, we propose the designation of two evolutionarily significant units for A. pallipes in France. Our data also support the maintenance of separate demographic management strategies for crayfish inhabiting different river systems. However, genetic analyses will have to be combined with demographic and ecological data for sustainable conservation programmes.     

Mitochondrial DNA variation in rainbow trout (Oncorhynchus mykiss) across its native range: testing biogeographical hypotheses and their relevance to conservation

North-western North America has been repeatedly glaciated over most of the past two million years, with the most recent glaciation occurring between 60 000 and 10 000 years ago. Intraspecific genetic variation in many species has been shaped by where they survived glaciation and what postglacial recolonization routes were used. In this study, molecular techniques were used to investigate biogeographical, taxonomic and conservation issues in rainbow trout, Oncorhynchus mykiss. Mitochondrial DNA (mtDNA) variation was assessed using a restriction fragment length polymorphism (RFLP) analysis, focusing mainly on the previously understudied northern extent of the species' range. Two phylogenetically distinct mitochondrial lineages were found that differed from each other by up to 1.8% in sequence. Although the geographical distributions of the two clades overlap extensively, diversity and distributional analyses strongly suggest that trout survived glaciation in both coastal and inland refugia followed by postglacial gene flow and secondary contact. Postglacial dispersal into British Columbia most likely occurred from the Queen Charlotte Islands and the Columbia River. Although trout most likely also survived glaciation along the coast of Washington, Oregon and California, as well as near the Bering Strait, evidence suggests that dispersal into British Columbia from these areas was limited. Sequence analysis of mitochondrial haplotypes revealed higher diversity in California than in the northern part of the species' range, indicating an ancient presence of the species in the south. Phylogeographic divergence probably predates adaptive variation in the species as suggested by evidence for parallel evolution of life history types across the range of O. mykiss.     

Development and characterization of novel tetra‐, tri‐, and dinucleotide microsatellite markers in rainbow trout (Oncorhynchus mykiss)

We discuss the development and characterization of 40 polymorphic rainbow trout (Oncorhynchus mykiss) microsatellite loci. We used enriched libraries to isolate 14 dinucleotide, seven trinucleodide, eight compound di/tetranucleotide, and 11 tetranucleotide loci. These markers will be useful for selective breeding via marker‐assisted selection, population genetics studies, parentage analysis, and have already been used for genome mapping.     

Population structure in the highly fragmented range of Tor douronensis (Cyprinidae) in Sarawak, Malaysia revealed by microsatellite DNA markers

1. Understanding the structure of threatened populations, particularly those that exist in degraded or fragmented habitats is crucial for their effective management and conservation. Recently developed methods of individual-based analysis of genetic data provide an unprecedented opportunity to understand the relationships amongst fragmented populations.
2. In the present study, population structure of an important cyprinid species ( Tor douronensis ), which is indigenous to Sarawak, Malaysia, is investigated as part of an ongoing conservation effort to restore threatened wild populations of the species. The population structure inferred using data from seven autosomal microsatellite loci was generally consistent with geography and habitat fragmentation.
3. The results indicate that there are two well-defined clusters of T. douronensis in Sarawak, namely the 'northeastern' and the 'southwestern' clusters. In addition, a further subdivision was observed in each of the clusters distributed between river systems. Low levels of gene flow were also observed and migrants between habitat fragments were identified, possibly resulting from human-mediated translocations.
4. Implications of the findings for management and conservation of T. douronensis are discussed.     

Dynamic micro-geographic and temporal genetic diversity in vertebrates: the case of lake-spawning populations of brown trout (Salmo trutta)

Conservation of species should be based on knowledge of effective population sizes and understanding of how breeding tactics and selection of recruitment habitats lead to genetic structuring. In the stream‐spawning and genetically diverse brown trout, spawning and rearing areas may be restricted source habitats. Spatio–temporal genetic variability patterns were studied in brown trout occupying three lakes characterized by restricted stream habitat but high recruitment levels. This suggested non‐typical lake‐spawning, potentially representing additional spatio–temporal genetic variation in continuous habitats. Three years of sampling documented presence of young‐of‐the‐year cohorts in littoral lake areas with groundwater inflow, confirming lake‐spawning trout in all three lakes. Nine microsatellite markers assayed across 901 young‐of‐the‐year individuals indicated overall substantial genetic differentiation in space and time. Nested gene diversity analyses revealed highly significant (≤P = 0.002) differentiation on all hierarchical levels, represented by regional lakes (F_LT = 0.281), stream vs. lake habitat within regional lakes (F_HL = 0.045), sample site within habitats (F_SH = 0.010), and cohorts within sample sites (F_CS = 0.016). Genetic structuring was, however, different among lakes. It was more pronounced in a natural lake, which exhibited temporally stable structuring both between two lake‐spawning populations and between lake‐ and stream spawners. Hence, it is demonstrated that lake‐spawning brown trout form genetically distinct populations and may significantly contribute to genetic diversity. In another lake, differentiation was substantial between stream‐ and lake‐spawning populations but not within habitat. In the third lake, there was less apparent spatial or temporal genetic structuring. Calculation of effective population sizes suggested small spawning populations in general, both within streams and lakes, and indicates that the presence of lake‐spawning populations tended to reduce genetic drift in the total (meta‐) population of the lake.