R. Paul Evans

Across the great divide: genetic forensics reveals misidentification of endangered cutthroat trout populations

Accurate assessment of species identity is fundamental for conservation biology. Using molecular markers from the mitochondrial and nuclear genomes, we discovered that many putatively native populations of greenback cutthroat trout (Oncorhynchus clarkii stomias) comprised another subspecies of cutthroat trout, Colorado River cutthroat trout (Oncorhynchus clarkii pleuriticus). The error can be explained by the introduction of Colorado River cutthroat trout throughout the native range of greenback cutthroat trout in the late 19th and early 20th centuries by fish stocking activities. Our results suggest greenback cutthroat trout within its native range is at a higher risk of extinction than ever before despite conservation activities spanning more than two decades.

Phylogeographic and nested clade analysis of the stonefly Pteronarcys californica (Plecoptera:Pteronarcyidae) in the western USA

Abstract Long-distance dispersal by aquatic insects can be difficult to detect because direct measurement methods are expensive and inefficient. When dispersal results in gene flow, signs of that dispersal can be detected in the pattern of genetic variation within and between populations. Four hundred seventy-five base pairs of the mitochondrial gene, cytochrome b, were examined to investigate the pattern of genetic variation in populations of the stonefly Pteronarcys californica and to determine if long-distance dispersal could have contributed to this pattern. Sequences were obtained from 235 individuals from 31 different populations in the western United States. Sequences also were obtained for Pteronarcella badia, Pteronarcys dorsata, Pteronarcys princeps, Pteronarcys proteus, and Pteronarcys biloba. Phylogenies were constructed using all of the samples. Nested clade analysis on the P. californica sequence data was used to infer the processes that have generated the observed patterns of genetic variation. An eastern North American origin and 2 distinct genetic lineages of P. californica could be inferred from the analysis. Most of the current population structure in both lineages was explained by a pattern of restricted gene flow with isolation by distance (presumably the result of dispersal via connected streams and rivers), but our analyses also suggested that long-distance, overland dispersal has contributed to the observed pattern of genetic variation.

Inferring dispersal of aquatic invertebrates from genetic variation: a comparative study of an amphipod and mayfly in Great Basin springs

Abstract Whether active or passive, dispersal accompanied by gene flow shapes the genetic makeup of populations and ultimately the evolutionary divergence of species. Our objective was to determine if 2 very different aquatic invertebrates with overlapping distributions show similar dispersal histories in their phylogeographic patterns and genetic uniqueness. Two spring-dwelling invertebrates, Hyalella azteca and Callibaetis americanus, were collected from 6 adjacent closed basins in the Great Basin of western North America. Cytochrome c oxidase subunit I (COI) and the 28S ribosomal subunit were used as genetic markers in Hyalella, and COI with the 16S ribosomal subunit of the mitochondrial genome were examined in Callibaetis. Maximum parsimony (MP) and likelihood (ML) analyses, FST values, analysis of molecular variance (AMOVA), Mantel tests, and nested clade phylogeographical analysis (NCPA) were used to evaluate geographical associations. Hyalella azteca appears to have been in the adjacent basins much longer than has Callibaetis. FST values in H. azteca reached near fixation. Callibaetis americanus FST values were lower suggesting greater gene flow and, consequently, higher dispersal rates. Mantel tests did not detect significant isolation by distance for either species, but NCPA on smaller networks of closely related haplotypes found the genetic structure in C. americanus dominated by restricted gene flow with isolation by distance. Hyalella azteca was characterized more by gradual range expansion followed by fragmentation. These results suggest that these isolated freshwater communities are amalgams of species that entered at different times, with weak dispersers having greater constraints on movement and, thus, reflecting an older geographical story than do species with stronger dispersal capabilities.

Phylogenetic divergence in a desert fish: differentiation of speckled dace within the Bonneville, Lahontan, and upper Snake river basins

ABSTRACT. Historical events have had a great impact on the biogeography of fishes of western North America. We examined the genetic variation of the speckled dace (Rhinichthys osculus) to determine the effects on this species of extensive hydrological changes during the last 10 million years in the Bonneville and Lahontan basins of the Great Basin and the upper Snake River Basin. Eight hundred sixty-nine base pairs of the mitochondrial gene cytochrome b were sequenced from 97 individuals representing 22 populations within these 3 basins, as well as from 2 individuals of longnose dace (Rhinichthys cataractae) that served as outgroups. Additionally, 13 speckled dace sequences representing 3 Bonneville populations were used from GenBank. Phylogenetic relationships were reconstructed using maximum parsimony and maximum likelihood criteria. Analysis of molecular variance was used to determine population structure and to estimate the amount of gene flow across the community boundaries. Three distinct clades were reconstructed representing the Lahontan Basin, the northern Bonneville and upper Snake River basins, and the southern Bonneville Basin. Additionally, most of the population structuring was explained by variation among basins (65.33%) Speckled dace demonstrated high genetic variation. As hypothesized, the northern and southern Bonneville specimens formed separate clades; however, the southern Bonneville clade was basal to a sister clade formed by the northern Bonneville/upper Snake River and Lahontan clades. These relationships indicate that Pliocene connections between the Snake, Lahontan, and Bonneville drainages, rather than more recent Pleistocene connections, best explain population structuring in speckled dace.

Single nucleotide polymorphism discovery in cutthroat trout subspecies using genome reduction, barcoding, and 454 pyro-sequencing

BackgroundSalmonids are popular sport fishes, and as such have been subjected to widespread stocking throughout western North America. Historically, stocking was done with little regard for genetic variation among populations and has resulted in genetic mixing among species and subspecies in many areas, thus putting the genetic integrity of native salmonid populations at risk and creating a need to assess the genetic constitution of native salmonid populations. Cutthroat trout is a salmonid species with pronounced geographic structure (there are 10 extant subspecies) and a recent history of hybridization with introduced rainbow trout in many populations. Genetic admixture has also occurred among cutthroat trout subspecies in areas where introductions have brought two or more subspecies into contact. Consequently, management agencies have increased their efforts to evaluate the genetic composition of cutthroat trout populations to identify populations that remain uncompromised and manage them accordingly, but additional genetic markers are needed to do so effectively. Here we used genome reduction, MID-barcoding, and 454-pyrosequencing to discover single nucleotide polymorphisms that differentiate cutthroat trout subspecies and can be used as a rapid, cost-effective method to characterize the genetic composition of cutthroat trout populations.ResultsThirty cutthroat and six rainbow trout individuals were subjected to genome reduction and next-generation sequencing. A total of 1,499,670 reads averaging 379 base pairs in length were generated by 454-pyrosequencing, resulting in 569,060,077 total base pairs sequenced. A total of 43,558 putative SNPs were identified, and of those, 125 SNP primers were developed that successfully amplified 96 cutthroat trout and rainbow trout individuals. These SNP loci were able to differentiate most cutthroat trout subspecies using distance methods and Structure analyses.ConclusionsGenomic and bioinformatic protocols were successfully implemented to identify 125 nuclear SNPs that are capable of differentiating most subspecies of cutthroat trout from one another. The ability to use this suite of SNPs to identify individuals of unknown genetic background to subspecies can be a valuable tool for management agencies in their efforts to evaluate the genetic structure of cutthroat trout populations prior to constructing and implementing conservation plans.

Climate oscillations, glacial refugia, and dispersal ability: factors influencing the genetic structure of the least salmonfly, Pteronarcella badia (Plecoptera), in Western North America

BackgroundPhylogeographic studies of aquatic insects provide valuable insights into mechanisms that shape the genetic structure of communities, yet studies that include broad geographic areas are uncommon for this group. We conducted a broad scale phylogeographic analysis of the least salmonfly Pteronarcella badia (Plecoptera) across western North America. We tested hypotheses related to mode of dispersal and the influence of historic climate oscillations on population genetic structure. In order to generate a larger mitochondrial data set, we used 454 sequencing to reconstruct the complete mitochondrial genome in the early stages of the project.ResultsOur analysis revealed high levels of population structure with several deeply divergent clades present across the sample area. Evidence from five mitochondrial genes and one nuclear locus identified a potentially cryptic lineage in the Pacific Northwest. Gene flow estimates and geographic clade distributions suggest that overland flight during the winged adult stage is an important dispersal mechanism for this taxon. We found evidence of multiple glacial refugia across the species distribution and signs of secondary contact within and among major clades.ConclusionsThis study provides a basis for future studies of aquatic insect phylogeography at the inter-basin scale in western North America. Our findings add to an understanding of the role of historical climate isolations in shaping assemblages of aquatic insects in this region. We identified several geographic areas that may have historical importance for other aquatic organisms with similar distributions and dispersal strategies as P. badia. This work adds to the ever-growing list of studies that highlight the potential of next-generation DNA sequencing in a phylogenetic context to improve molecular data sets from understudied groups.

A description of Echinorhynchus baeri Kostylew, 1928 (Acanthocephala: Echinorhynchidae) from Salmo trutta in Turkey, with notes on synonymy, geographical origins, geological history, molecular profile, and X-ray microanalysis.

A population of Echinorhynchus baeri Kostylew, 1928 with 18–24 rows of 8–10 proboscis hooks each and long fusiform eggs measuring 95–110 × 18–22 μm collected from Salmo trutta (Salmonidae) in a branch of the Murat River in Turkey is described and specimens are designated as neotype. Specimens of two similar populations of E. baeri (E. baeri Kostylew, 1928 and E. sevani Dinnik, 1932) were previously described from Salmo ischchan in Lake Sevan, Armenia. Waters of Lake Sevan and the Murat River were previously joined during the Middle Miocene-Pliocene. The two populations from Lake Sevan and ours from Turkey had identical morphology and size eggs. The proboscis armature and eggs, among other features of our Turkish specimens, proved intermediate between E. baeri and E. sevani, thus eliminating the significance of the described differences between these two species and confirming their synonymy with priority to Echinorhynchus baeri (junior synonym: Echinorhynchus sevani Dinnik, 1932). Echinorhynchus baeri is apparently a highly variable species. The two descriptions from Lake Sevan did not include features or illustrations of females, except for references to trunk and egg size but the eggs were illustrated. Complete morphometric comparisons are made and females of the Turkish material are described for the first time. DNA sequencing (mitochondrial cytochrome oxidase subunit I gene; nuclear 18S rRNA gene) results from two available E. baeri individuals were equivocal. New features to the Acanthocephala include the presence of rootless uncalcified apical proboscis hooks studied with X-ray microanalysis.

Morphological and molecular descriptions of Moniliformis saudi sp. n. (Acanthocephala: Moniliformidae) from the desert hedgehog, Paraechinus aethiopicus (Ehrenberg) in Saudi Arabia, with a key to species and notes on histopathology.

A new acanthocepohalan species, Moniliformis saudi sp. n. is described from the desert hedgehog, Paraechinus aethiopicus (Ehrenberg), in central Saudi Arabia. Fourteen other valid species of Moniliformis Travassos, 1915 are recognised. The new species of Moniliformis is distinguished by having a small proboscis (315-520 µm long and 130-208 µm wide) with two apical pores, 14 rows of 8 hooks each and small hooks, thre largest being 25-31 µm long anteriorly. Distinguishing features are incorporated in a dichotomous key to the species of Moniliformis. The description is augmented by scanning electron microscopical (SEM) observation and DNA analysis of nuclear (18S rRNA) and mitochondrial (cytochrome oxidase subunit 1; cox1) gene sequences. Attached worms cause extensive damage to the immediate area of attachment in the host intestine. This includes tissue necrosis and blood loss due to damage to capillary beds. Worms also obstruct essential absorbing surfaces.

Evaluating the genetic status of a Great Basin endemic minnow: the relict dace ( Relictus solitarius )

The relict dace (Relictus solitarius) is an endemic minnow restricted to isolated spring systems within Butte, Goshute, Ruby and Steptoe valleys in the eastern Nevada portion of the Great Basin. It is the only native fish in these basins, but speckled dace (Rhinichthys osculus) have been introduced into some localities in Ruby Valley and, based on morphology, the two species are believed to have hybridized in those areas. We used DNA sequence data from one mitochondrial gene (cytochrome b) and one nuclear intron (S7) to determine if putative relict dace populations in Ruby Valley share mtDNA haplotypes with speckled dace, or show signs of hybridization. Results of our analyses show that 11 of 16 Ruby Valley populations sampled contain only speckled dace, and one speckled dace population exhibited signs of limited introgressive hybridization. The remaining five Ruby Valley populations that were sampled contain relict dace that remain genetically distinct from speckled dace. The integrity of the Ruby Valley relict dace populations should be actively protected, particularly in the area surrounding Ruby Lake where speckled dace have been widely introduced.

Genetic Characterization of Two Populations of Bonneville Cutthroat Trout in Great Basin National Park, USA

Abstract. The cutthroat trout (Oncorhynchus clarkii) is a popular sport fish that is native to the waters of western North America. Cutthroat trout comprises many subspecies, each of which has experienced range reduction resulting from anthropogenic activities. Hence, there is a general interest from management agencies to assess the genetic structure of managed populations of cutthroat trout subspecies to ensure that proper conservation plans are implemented. Herein, we utilize microsatellite data to characterize the genetic composition of 2 populations of Bonneville cutthroat trout in Great Basin National Park: Mill Creek and South Fork Big Wash. Mill Creek was used as a source population for reintroduction into South Fork Big Wash in the year 2000, and there is concern that South Fork Big Wash may have experienced a population bottleneck after, or during, the stocking effort. We found that both populations exhibit low genetic diversity, and that the source population, Mill Creek, exhibited mixed signals of having undergone a recent population bottleneck. Structure analysis revealed 4 distinct groups, but those groups did not segregate geographically, although a significant pairwise FST (0.06727, P < 0.00001) between Mill Creek and South Fork Big Wash populations suggests that some genetic differentiation has occurred.