Using de novo transcriptomes to decipher the relationships in cutthroat trout subspecies (Oncorhynchus clarkii).

For almost 200 years, the taxonomy of cutthroat trout (Oncorhynchus clarkii), a salmonid native to Western North America, has been in flux as ichthyologists and fisheries biologists have tried to describe the diversity within these fishes. Starting in the 1950s, Robert Behnke reexamined the cutthroat trout and identified 14 subspecies based on morphological traits, Pleistocene events, and modern geographic ranges. His designations became instrumental in recognizing and preserving the remaining diversity of cutthroat trout. Over time, molecular techniques (i.e. karyotypes, allozymes, mitochondrial DNA, SNPs, and microsatellite arrays) have largely reinforced Behnke's phylogenies, but have also revealed that some relationships are consistently weakly supported. To further resolve these relationships, we generated de novo transcriptomes for nine cutthroat subspecies, as well as a Bear River Bonneville form and two Colorado River lineages (blue and green). We present phylogenies of these subspecies generated from multiple sets of orthologous genes extracted from our transcriptomes. We confirm many of the relationships identified in previous morphological and molecular studies, as well as discuss the importance of significant differences apparent in our phylogenies from these studies within a geological perspective. Specific findings include three distinct clades: (1) Bear River Bonneville form and Yellowstone cutthroat trout; (2) Bonneville cutthroat trout (n = 2); and (3) Greenback and Rio Grande cutthroat trout. We also identify potential gene transfer between Bonneville cutthroat trout and a population of Colorado River green lineage cutthroat trout. Using these findings, it appears that additional groups warrant species-level consideration if other recent species elevations are retained.

Heterochronic shift in gene expression leads to ontogenetic morphological divergence between two closely related polyploid species.

Heterochrony-alteration to the rate or timing of development-is an important mechanism of trait differentiation associated with speciation. Heterochrony may explain the morphological divergence between two polyploid species, June sucker (Chasmistes liorus) and Utah sucker (Catostomus ardens). The larvae of both species have terminal mouths; however, as adults, June sucker and Utah sucker develop subterminal and ventral mouths, respectively. We document a difference in the timing of shape development and a corresponding change in the timing of gene expression, suggesting the distinctive mouth morphology in June suckers may result from paedomorphosis. Specifically, adult June suckers exhibit an intermediate mouth morphology between the larval (terminal) and ancestral (ventral) states. Endemic and sympatric Chasmistes/Catostomus pairs in two other lakes also are morphologically divergent, but genetically similar. These species pairs could have resulted from the differential expression of genes and corresponding divergence in trait development. Paedomorphosis may lead to adaptive diversification in Catostomids.

Phylogenetic relationships of three rockfish: Sebastesmelanops, Sebastesciliatus and Sebastesvariabilis (Scorpaeniformes, Scorpaenidae) based on complete mitochondrial genome sequences.

We characterise the complete mitochondrial genomes (mitogenomes) of Black rockfish (Sebastesmelanops Girard, 1856; n = 1), Dark rockfish (Sebastesciliatus Tilesius, 1813; n = 2) and Dusky rockfish (Sebastesvariabilis Pallas, 1814; n = 2). The lengths of the mitogenomes are 16,405 bp for S.melanops, 16,400 bp for both S.ciliatus and 16,400 and 16,401 bp for S.variabilis. We examine these species' phylogenetic relationships using 35 previously published rockfish mitogenomes, representing 27 species. We find that S.melanops is sister to a clade consisting of S.rubrivinctus, S.nigrocinctus, S.umbrosus and S.oculatus, whereas S.ciliatus and S.variabilis are sister to a clade consisting of S.norvegicus, S.viviparus, S.mentella and S.fasciatus. We were unable to separate S.ciliatus and S.variabilis using their complete mitogenomes.

Complete mitochondrial genomes of two rockfish: Sebastes maliger and Sebastes norvegicus (Scorpaenidae, Scorpaeniformes).

We report the complete mitochondrial genomes of two rockfish: Sebastes maliger and Sebastes norvegicus. The mitogenomes consist of 13 protein-coding regions, 22 tRNAs, two rRNAs, and one control region. Sebastes mitogenome control regions are highly variable due to the presence of repeat sequences. The mitogenomes for S. maliger and S. norvegicus are 16,403 and 16,401 bp, respectively. Using these two mitogenomes and 25 additional Sebastes mitogenomes from GenBank, we examine the phylogenetic relationships in Sebastes. Sebastes maliger is sister to a clade including S. rubrivinctus, S. nigrocinctus, S. umbrosus, and S. oculatus, while S. norvegicus is sister to S. fasciatus.

Complete mitochondrial genomes of June sucker and Utah sucker (Chasmistes liorus and Catostomus ardens).

The relationship between June sucker (Chasmistes liorus, Jordan, 1878) and Utah sucker (Catostomus ardens, Jordan & Gilbert, 1881) has been a matter of controversy since the mid 1900s. Chasmistes liorus is endemic to Utah Lake, UT and has a subterminal mouth adapted for pelagic feeding. Catostomus ardens is widely distributed throughout the Bonneville Basin and Upper Snake River Basin and has a ventral mouth adapted for benthic feeding. Chasmistes has been recognized as a separate ancient genus. Despite being morphologically distinct, no study has successfully identified residual genetic markers that separate these species. Of these studies, several have used a subset of mitochondrial genes, but no study has analyzed the complete mitochondrial genomes (mitogenomes) of these suckers (Pisces: Catostomidae). To further explore the genetic relationships between these species, we report the complete mitogenomes of Chasmistes liorus and Catostomus ardens. DNA was sequenced using an Illumina HiSeq 2500 system and mitogenomes were assembled and annotated using Geneious v. 2021.2 and MitoAnnotator, respectively. The mitogenomes of Chasmistes liorus and Catostomus ardens are both 16,623 bp and are ∼0.072% divergent. We examine the phylogenetic relationship between Chasmistes liorus and Catostomus ardens using 33 mitogenomes, representing 16 species, from Catostomidae. Our data suggest that Chasmistes liorus is sister to Catostomus ardens. Additional samples from multiple localities and/or cohorts of these species will allow us to better resolve the complicated phylogenetic relationships between these species.

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

BACKGROUND: Phylogeographic 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. RESULTS: Our 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. CONCLUSIONS: This 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.

Pluvial Drainage Patterns and Holocene Desiccation Influenced the Genetic Architecture of Relict Dace, Relictus solitarius (Teleostei: Cyprinidae).

Changing drainage patterns have played a significant role in the evolution of western North American aquatic taxa. Relict dace, Relictus solitarius, is a Great Basin endemic cyprinid with a native range that is restricted to four valleys in eastern Nevada. Relictus solitarius now occupies spring systems that are the remnants of Pleistocene-era pluvial lakes, although it may have occurred in the area for much longer. Here we use mitochondrial DNA sequence data to assess range-wide genetic diversity of R. solitarius, and to estimate divergence times to determine whether pluvial drainages played an important role in shaping intraspecific genetic diversity. Genetic diversification within R. solitarius began during the early to mid-Pleistocene, separating populations within two sets of valleys (Butte/Ruby and Goshute/Steptoe). Additional diversification in each of the two sets of valleys occurred more recently, in the mid- to late-Pleistocene. Holocene desiccation has further isolated populations, and each population sampled contains unique mtDNA haplotypes. Pluvial drainage patterns did contribute to the genetic structure observed within R. solitarius, but most of the intraspecific diversification does not appear to be associated with the Last Glacial Maximum. Holocene desiccation has also contributed to the observed genetic structure. The relict dace populations we sampled are all unique, and we recommend that future management efforts should strive to preserve as much of the genetic diversity as possible.

Investigating the effects of Pleistocene events on genetic divergence within Richardsonius balteatus, a widely distributed western North American minnow.

BACKGROUND: Biogeographers seek to understand the influences of global climate shifts and geologic changes to the landscape on the ecology and evolution of organisms. Across both longer and shorter timeframes, the western North American landscape has experienced dynamic transformations related to various geologic processes and climatic oscillations, including events as recently as the Last Glacial Maximum (LGM; ~20 Ka) that have impacted the evolution of the North American biota. Redside shiner is a cyprinid species that is widely distributed throughout western North America. The species' native range includes several well-documented Pleistocene refugia. Here we use mitochondrial DNA sequence data to assess phylogeography, and to test two biogeographic hypotheses regarding post-glacial colonization by redside shiner: 1) Redside shiner entered the Bonneville Basin at the time of the Bonneville Flood (Late Pleistocene; 14.5 Ka), and 2) redside shiner colonized British Columbia post-glacially from a single refugium in the Upper Columbia River drainage. RESULTS: Genetic diversification in redside shiner began in the mid to late Pleistocene, but was not associated with LGM. Different clades of redside shiner were distributed in multiple glacial age refugia, and each clade retains a signature of population expansion, with clades having secondary contact in some areas. CONCLUSIONS: Divergence times between redside shiner populations in the Bonneville Basin and the Upper Snake/Columbia River drainage precedes the Bonneville Flood, thus it is unlikely that redside shiner invaded the Bonneville Basin during this flooding event. All but one British Columbia population of redside shiner are associated with the Upper Columbia River drainage with the lone exception being a population near the coast, suggesting that the province as a whole was colonized from multiple refugia, but the inland British Columbia redside shiner populations are affiliated with a refugium in the Upper Columbia River drainage.

Influence of introgression and geological processes on phylogenetic relationships of Western North American mountain suckers (Pantosteus, Catostomidae).

Intense geological activity caused major topographic changes in Western North America over the past 15 million years. Major rivers here are composites of different ancient rivers, resulting in isolation and mixing episodes between river basins over time. This history influenced the diversification of most of the aquatic fauna. The genus Pantosteus is one of several clades centered in this tectonically active region. The eight recognized Pantosteus species are widespread and common across southwestern Canada, western USA and into northern Mexico. They are typically found in medium gradient, middle-elevation reaches of rivers over rocky substrates. This study (1) compares molecular data with morphological and paleontological data for proposed species of Pantosteus, (2) tests hypotheses of their monophyly, (3) uses these data for phylogenetic inferences of sister-group relationships, and (4) estimates timing of divergence events of identified lineages. Using 8055 base pairs from mitochondrial DNA protein coding genes, Pantosteus and Catostomus are reciprocally monophyletic, in contrast with morphological data. The only exception to a monophyletic Pantosteus is P. columbianus whose mtDNA is closely aligned with C. tahoensis because of introgression. Within Pantosteus, several species have deep genetic divergences among allopatric sister lineages, several of which are diagnosed and elevated to species, bringing the total diversity in the group to 11 species. Conflicting molecular and morphological data may be resolved when patterns of divergence are shown to be correlated with sympatry and evidence of introgression.

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

BACKGROUND: Salmonids 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. RESULTS: Thirty 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. CONCLUSIONS: Genomic 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.

Phylogenetic relationships of the western North American cyprinid genus Richardsonius, with an overview of phylogeographic structure.

Diversification of many North American taxa, including freshwater fishes, has been heavily influenced by the effects of complex geological and climatic events throughout the Cenozoic that have significantly altered the landscape. Here, we employ an array of phylogenetic analyses using a multiple gene tree approach to address several questions regarding the phylogenetic relationships of the North American cyprinid genus Richardsonius and two other closely related genera, Clinostomus and Iotichthys. We also use divergence time estimates generated using fossil calibrations to qualitatively assess the phylogeographic implications of evolution within the group. Mitochondrial and nuclear DNA sequences show a sister relationship between Iotichthys and Richardsonius, with Clinostomus being sister to an Iotichthys-Richardsonius clade, hence the currently recognized sister relationship between Clinostomus and Richardsonius is not supported. These genera appear to be monophyletic lineages, and sister species within genera appear to be reciprocally monophyletic. The two species within the genus Richardsonius both exhibit phylogeographic structure that is worthy of further investigation. Divergence time estimates between genera and species are Miocene or Pliocene in age, and divergence between phylogroups within species occurred in the late Pliocene to Pleistocene. These splits coincide with documented geological and climatic events.

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.