Molly R. Stephens

Migration‐related phenotypic divergence is associated with epigenetic modifications in rainbow trout

Migration is essential for the reproduction and survival of many animals, yet little is understood about its underlying molecular mechanisms. We used the salmonid Oncorhynchus mykiss to gain mechanistic insight into smoltification, which is a morphological, physiological and behavioural transition undertaken by juveniles in preparation for seaward migration. O. mykiss is experimentally tractable and displays intra‐ and interpopulation variation in migration propensity. Migratory individuals can produce nonmigratory progeny and vice versa, indicating a high degree of phenotypic plasticity. One potential way that phenotypic plasticity might be linked to variation in migration‐related life history tactics is through epigenetic regulation of gene expression. To explore this, we quantitatively measured genome‐scale DNA methylation in fin tissue using reduced representation bisulphite sequencing of F2 siblings produced from a cross between steelhead (migratory) and rainbow trout (nonmigratory) lines. We identified 57 differentially methylated regions (DMRs) between smolt and resident O. mykiss juveniles. DMRs were high in magnitude, with up to 62% differential methylation between life history types, and over half of the gene‐associated DMRs were in transcriptional regulatory regions. Many of the DMRs encode proteins with activity relevant to migration‐related transitions (e.g. circadian rhythm pathway, nervous system development, protein kinase activity). This study provides the first evidence of a relationship between epigenetic variation and life history divergence associated with migration‐related traits in any species.

Subspecies‐informative SNP assays for evaluating introgression between native golden trout and introduced rainbow trout

We characterize 20 single nucleotide polymorphism assays for evaluating hybridization between native golden trout subspecies (Oncorhynchus mykiss aguabonita and O. m. whitei) and introduced rainbow trout strains. These assays utilize the 5′‐nuclease reaction, facilitating high‐throughput genotyping of many individuals and making them useful in quantifying and monitoring introgression and potentially applicable to studies of other O. mykiss groups. Minor allele frequency differentials (δq) among native and introduced rainbow groups ranged from 0 to 1, with an average differential of 0.75 for both subspecies aguabonita and whitei relative to the hatchery rainbow trout strain.

Fishing for SNPs: A Targeted Locus Approach for Single Nucleotide Polymorphism Discovery in Rainbow Trout

Abstract The combination of whole-genome sequencing efforts and emerging high-throughput genotyping techniques has made single nucleotide polymorphisms (SNPs) a marker of choice for molecular genetic analyses in model organisms. This class of marker holds great promise for resolving questions of phylogeny, population structure, introgression, and adaptive genetic variation. Fifty-five polymerase chain reaction primer pairs were used to target variable regions of the rainbow trout Oncorhynchus mykiss genome, 48 of which were designed from information found in publicly available DNA sequence databases. Forty of these primer pairs yielded sequenceable products. These sequences were compared across 1−10 individual fish from each of the following representative subspecies and strains: Sacramento redband trout O. mykiss stonei, California golden trout O. mykiss aguabonita, Little Kern golden trout O. mykiss whitei, coastal rainbow trout O. mykiss irideus, and the Mount Shasta hatchery strain. A total of 208 SNP...

Transcriptional Response to Acute Thermal Exposure in Juvenile Chinook Salmon Determined by RNAseq

Thermal exposure is a serious and growing challenge facing fish species worldwide. Chinook salmon (Oncorhynchus tshawytscha) living in the southern portion of their native range are particularly likely to encounter warmer water due to a confluence of factors. River alterations have increased the likelihood that juveniles will be exposed to warm water temperatures during their freshwater life stage, which can negatively impact survival, growth, and development and pose a threat to dwindling salmon populations. To better understand how acute thermal exposure affects the biology of salmon, we performed a transcriptional analysis of gill tissue from Chinook salmon juveniles reared at 12° and exposed acutely to water temperatures ranging from ideal to potentially lethal (12° to 25°). Reverse-transcribed RNA libraries were sequenced on the Illumina HiSeq2000 platform and a de novo reference transcriptome was created. Differentially expressed transcripts were annotated using Blast2GO and relevant gene clusters were identified. In addition to a high degree of downregulation of a wide range of genes, we found upregulation of genes involved in protein folding/rescue, protein degradation, cell death, oxidative stress, metabolism, inflammation/immunity, transcription/translation, ion transport, cell cycle/growth, cell signaling, cellular trafficking, and structure/cytoskeleton. These results demonstrate the complex multi-modal cellular response to thermal stress in juvenile salmon.

Identifying Introgressive Hybridization in Native Populations of California Golden Trout Based on Molecular Markers

Abstract The California golden trout Oncorhynchus mykiss aguabonita is one of three subspecies within the rainbow trout–redband trout complex endemic to the Kern River basin and historically restricted to Golden Trout Creek and the South Fork Kern River. Past allozyme studies have indicated that native populations of California golden trout in the Golden Trout Creek drainage may have become introgressed with rainbow trout alleles through interaction with hybrids of rainbow trout and California golden trout stocked into nearby headwater lakes that are connected to tributaries in the drainage. We used six microsatellites and a minisatellite marker to estimate the genetic diversity and levels of introgression in approximately 700 California golden trout taken from 23 locations in Golden Trout Creek, its tributaries, and surrounding lakes. Indications of introgression were found in all but one of the sampled Golden Trout Creek drainage locations, the lowest average levels (0–8%) occurring in the lower reaches...

Six diagnostic single nucleotide polymorphism markers for detecting introgression between cutthroat and rainbow trouts

Ten primer pairs were screened to develop single nucleotide polymorphism (SNP) TaqMan assays that will distinguish California golden trout and some rainbow trouts (Oncorhynchus mykiss sspp., O. m. aguabonita) from the Paiute and Lahontan cutthroat trouts (Oncorhynchus clarkii seleniris, O. c. henshawi). From these 10 primer pairs, one mitochondrial and five nuclear fixed SNP differences were discovered and developed into TaqMan assays. These six assays will be useful for characterizing and monitoring hybridization between these groups. Additional Oncorhynchus clarkii sspp. and Oncorhynchus mykiss sspp. were assayed to determine if these assays are useful in closely related species.

Genetic structure of the tick Ornithodoros coriaceus (Acari: Argasidae) in California, Nevada, and Oregon.

Abstract The argasid tick Ornithodoros coriaceus (Koch) is the only confirmed vector of epizootic bovine abortion (EBA) in the United States. The disease and its tick vector have historically been reported in the foothills of the Sierra Nevada Mountains and coast ranges of California. In the past two decades, the range of EBA has apparently expanded into southern Oregon and northern Nevada. Possible explanations for this expansion include 1) increased recognition and reporting of EBA in these regions; 2) widespread movement of tick-infested and EBA-infected hosts with subsequent colonization of these regions by infected ticks; and 3) widespread movement of the EBA agent, independent of tick movements, into extant tick populations in these new regions. The current study was performed to evaluate these hypotheses by examining patterns of variability in a 420-bp segment of the 16S mitochondrial rDNA gene sequence among 210 O. coriaceus individuals from 14 sites in California, Oregon, and Nevada. Sixty-three unique haplotypes were identified in the ticks tested, with 84% of the sequence variation attributable to among-population variation and 16% to within-population variation. A majority of the haplotypes were unique to their particular collection site, whereas only four collection sites shared haplotypes. Overall, very little evidence of gene flow among tick populations was detected, making it unlikely that widespread tick movement had introduced O. coriaceus and the EBA agent into new regions.

Sequencing improves our ability to study threatened migratory species: Genetic population assignment in California's Central Valley Chinook salmon

Abstract Effective conservation and management of migratory species requires accurate identification of unique populations, even as they mix along their migratory corridors. While telemetry has historically been used to study migratory animal movement and habitat use patterns, genomic tools are emerging as a superior alternative in many ways, allowing large‐scale application at reduced costs. Here, we demonstrate the usefulness of genomic resources for identifying single‐nucleotide polymorphisms (SNPs) that allow fast and accurate identification of the imperiled Chinook salmon in the Great Central Valley of California. We show that 80 well‐chosen loci, drawn from a pool of over 11,500 SNPs developed from restriction site‐associated DNA sequencing, can accurately identify Chinook salmon runs and select populations within run. No other SNP panel for Central Valley Chinook salmon has been able to achieve the high accuracy of assignment we show here. This panel will greatly improve our ability to study and manage this ecologically, economically, and socially important species and demonstrates the great utility of using genomics to study migratory species.

Genetic characterization of California's Central Valley chinook salmon

This data set includes genotypes for 5000 chinook salmon individuals collected from throughout Californias Central Valley between 1998 and 2013. We genotyped these samples using a panel of 96 single nucleotide polymorphism (SNP) markers. This is the most comprehensive genetic characterization published to date, covering all of the California Central Valley Evolutionary Significant Units (ESUs) and including all major river drainages within each ESU (total of 17 rivers and 5 hatchery populations). These populations are the foci of considerable basic and applied scientific research given the ecological, economic, and cultural importance of salmonid species. Moreover, all Central Valley ESUs are listed as federally threatened, endangered, or species of concern. This data set improves our ability to study basic ecological questions about salmonid biology, including testing hypotheses about population structure, genetic diversity, introgression between ESUs, and levels of gene flow among populations. Addition...