Limited, asymmetric hybridization between coastal cutthroat trout and steelhead in a Northern California river.

Hybridization between coastal cutthroat trout (Oncorhynchus clarkii clarkii) and steelhead (O. mykiss) was assessed in the Smith River, California. Individuals were categorized as pure or as 1 of 10 hybrid classes using 30 "diagnostic" single-nucleotide polymorphisms positioned on 26 separate chromosomes. Most of the individuals examined (n = 876), were pure coastal cutthroat trout (n = 634) or pure steelhead (n = 213), and 29 individuals were identified as having hybrid ancestry. Among hybrids, first generation hybrids (n = 15) and coastal cutthroat trout backcrosses (n = 12) were the most common. No individuals were identified as backcrosses to SH, suggesting the presence of genetic or behavioral mechanisms constraining such backcrosses, or the growth and survival of their progeny. Mitochondrial DNA of 14 of 15 F1 hybrids was of steelhead origin, suggesting that hybridization was driven primarily by sneak-mating of male coastal cutthroat trout with female steelhead. Evaluation of classical phenotypic characters for coastal cutthroat trout and steelhead (i.e. jaw slash, maxillary length, and hyoid teeth) were not reliable by themselves for identification of either pure parental fish or hybrids. In contrast, analysis with geometric morphometrics revealed distinctive body shapes for pure coastal cutthroat trout and steelhead, and the combination of classical traits and geometric morphology was mostly accurate in distinguishing them. However, first generation hybrids and backcrosses overlapped completely with parental types, highlighting challenges in hybrid identification using phenotypic traits.

Using environmental DNA and occupancy modelling to estimate rangewide metapopulation dynamics.

We demonstrate the power of combining two emergent tools for resolving rangewide metapopulation dynamics. First, we employed environmental DNA (eDNA) surveys to efficiently generate multiseason rangewide site occupancy histories. Second, we developed a novel dynamic, spatial multiscale occupancy model to estimate metapopulation dynamics. The model incorporates spatial relationships, explicitly accounts for non-detection bias and allows direct evaluation of the drivers of extinction and colonization. We applied these tools to examine metapopulation dynamics of endangered tidewater goby, a species endemic to California estuarine habitats. We analysed rangewide eDNA data from 190 geographically isolated sites (813 total water samples) surveyed from 2 years (2016 and 2017). Rangewide estimates of the proportion of sites that were occupied varied little between 2016 (0.52) and 2017 (0.51). However, there was evidence of extinction and colonization dynamics. The probability of extinction of an occupied site (0.106) and probability of colonization of an unoccupied site (0.085) were nearly equal. Stability in site occupancy proportions combined with nearly equal rates of extinction and colonization suggests a dynamic equilibrium between the 2 years surveyed. Assessment of covariate effects revealed that colonization probability increased as the number of occupied neighbouring sites increased and as distance between occupied sites decreased. We show that eDNA surveys can rapidly provide a snapshot of a species distribution over a broad geographic range and, when these surveys are paired with occupancy modelling, can uncover metapopulation dynamics and their drivers.

Recent, small beginnings: genetic analysis suggests Catostomus rimiculus (Klamath smallscale sucker) in the Smith River, California, are introduced.

Identification of introduced species can be important to understanding ecological systems and meeting conservation and management goals, but the process can be surprisingly challenging. The Klamath smallscale sucker Catostomus rimiculus seems likely to be native to the Smith River because the drainage separates two basins believed to be within the fish's native range, the Rogue and Klamath rivers. Further, C. rimiculus is broadly distributed in the Smith River, and the indigenous Dee-ni' People of the Smith River have a unique word for sucker. Nonetheless, a historical survey of fishes that described C. rimiculus from the Rogue and Klamath rivers did not include C. rimiculus among the fishes of the Smith River. To determine whether the genetic structure of the Smith River C. rimiculus reflects expectations for a native sucker population, the authors of this study examined variation in microsatellite and mitochondrial genetic markers from the Smith River and surrounding drainages. The genetic analyses revealed a pattern consistent with extreme founder effects in Smith River C. rimiculus, as would be expected from a single introduction of six or fewer effective individuals. The sharing of a high-frequency haplotype between the Smith River and Klamath River that is not detected in the Rogue River suggests the Klamath River as the likely source for the introduction. The findings highlight that local-scale introductions can be easily overlooked because the newly established populations can appear to be parts of contiguous natural distributions.

Genomic islands of divergence infer a phenotypic landscape in Pacific lamprey.

High rates of dispersal can breakdown coadapted gene complexes. However, concentrated genomic architecture (i.e., genomic islands of divergence) can suppress recombination to allow evolution of local adaptations despite high gene flow. Pacific lamprey (Entosphenus tridentatus) is a highly dispersive anadromous fish. Observed trait diversity and evidence for genetic basis of traits suggests it may be locally adapted. We addressed whether concentrated genomic architecture could influence local adaptation for Pacific lamprey. Using two new whole genome assemblies and genotypes from 7,716 single nucleotide polymorphism (SNP) loci in 518 individuals from across the species range, we identified four genomic islands of divergence (on chromosomes 01, 02, 04, and 22). We determined robust phenotype-by-genotype relationships by testing multiple traits across geographic sites. These trait associations probably explain genomic divergence across the species' range. We genotyped a subset of 302 broadly distributed SNPs in 2,145 individuals for association testing for adult body size, sexual maturity, migration distance and timing, adult swimming ability, and larval growth. Body size traits were strongly associated with SNPs on chromosomes 02 and 04. Moderate associations also implicated SNPs on chromosome 01 as being associated with variation in female maturity. Finally, we used candidate SNPs to extrapolate a heterogeneous spatiotemporal distribution of these predicted phenotypes based on independent data sets of larval and adult collections. These maturity and body size results guide future elucidation of factors driving regional optimization of these traits for fitness. Pacific lamprey is culturally important and imperiled. This research addresses biological uncertainties that challenge restoration efforts.

Evidence for the genetic basis and epistatic interactions underlying ocean- and river-maturing ecotypes of Pacific Lamprey (Entosphenus tridentatus) returning to the Klamath River, California.

Surveys of genomic variation have improved our understanding of the relationship between fitness-related phenotypes and their underlying genetic basis. In some cases, single large-effect genes have been found to underlie important traits; however, complex traits are expected to be under polygenic control and elucidation of multiple gene interactions may be required to fully understand the genetic basis of the trait. In this study, we investigated the genetic basis of the ocean- and river-maturing ecotypes in anadromous Pacific lamprey (Entosphenus tridentatus). In Pacific lamprey, the ocean-maturing ecotype is distinguished by advanced maturity of females (e.g., large egg mass) at the onset of freshwater migration relative to immature females of the river-maturing ecotype. We examined a total of 219 adult Pacific lamprey that were collected at-entry to the Klamath River over a 12-month period. Each individual was genotyped at 308 SNPs representing known neutral and adaptive loci and measured at morphological traits, including egg mass as an indicator of ocean- and river-maturing ecotype for females. The two ecotypes did not exhibit genetic structure at 148 neutral loci, indicating that ecotypic diversity exists within a single population. In contrast, we identified the genetic basis of maturation ecotypes in Pacific lamprey as polygenic, involving two unlinked gene regions that have a complex epistatic relationship. Importantly, these gene regions appear to show stronger effects when considered in gene interaction models than if just considered additive, illustrating the importance of considering epistatic effects and gene networks when researching the genetic basis of complex traits in Pacific lamprey and other species.

Novel Concordance Between Geographic, Environmental, and Genetic Structure in the Ecological Generalist Prickly Sculpin (Cottus asper) in California.

Ecological generalists may contain a wealth of information concerning diversity, ecology, and geographic connectivity throughout their range. We explored these ideas in prickly sculpin (Cottus asper), a small generalist freshwater fish species where coastal forms have potentially undergone radiations into inland lacustrine and riverine environments. Using a 962bp cytochrome b mtDNA marker and 11 microsatellites, we estimated diversity, divergence times, gene flow, and structure among populations at 43 locations throughout California. We then incorporated genetic and GIS data into ecological niche models to assess ecological conditions within identified groups. Though not reciprocally monophyletic, unique mtDNA haplotypes, microsatellite clustering, and measures of isolation by distance (Coastal: r = 0.960, P < 0.001; Inland: r = 0.277, P = 0.148) suggest 2 novel taxonomic groups, Coastal and Inland (constrained to Great Central Valley). Divergence estimates of 41-191 kya combined with the regional biogeographic history suggest geographic barriers are absent between groups since divergence, but ecological niche modeling revealed significant environmental differences (t = 10.84, P < 0.001). Introgressed individuals were also discovered between groups in an ecologically and geographically intermediate region. Population structure was limited, predominately found in tributaries of the San Joaquin basin in the Inland group. Overall, C. asper exhibited substantial genetic diversity, despite its ecological generality, reflecting California's historically unique and complex hydrology. More broadly, this study illustrates variable environments within the range of a generalist species may mask genetic divergences and should not be overlooked in biodiversity assessments.

Temporal genetic analysis of the endangered tidewater goby: extinction-colonization dynamics or drift in isolation?

Extinction and colonization dynamics are critical to understanding the evolution and conservation of metapopulations. However, traditional field studies of extinction-colonization are potentially fraught with detection bias and have rarely been validated. Here, we provide a comparison of molecular and field-based approaches for assessment of the extinction-colonization dynamics of tidewater goby (Eucyclogobius newberryi) in northern California. Our analysis of temporal genetic variation across 14 northern California tidewater goby populations failed to recover genetic change expected with extinction-colonization cycles. Similarly, analysis of site occupancy data from field studies (94 sites) indicated that extinction and colonization are very infrequent for our study populations. Comparison of the approaches indicated field data were subject to imperfect detection, and falsely implied extinction-colonization cycles in several instances. For northern California populations of tidewater goby, we interpret the strong genetic differentiation between populations and high degree of within-site temporal stability as consistent with a model of drift in the absence of migration, at least over the past 20-30 years. Our findings show that tidewater goby exhibit different population structures across their geographic range (extinction-colonization dynamics in the south vs. drift in isolation in the north). For northern populations, natural dispersal is too infrequent to be considered a viable approach for recolonizing extirpated populations, suggesting that species recovery will likely depend on artificial translocation in this region. More broadly, this work illustrates that temporal genetic analysis can be used in combination with field data to strengthen inference of extinction-colonization dynamics or as a stand-alone tool when field data are lacking.

Complex phylogeography and historical hybridization between sister taxa of freshwater sculpin (Cottus).

Species ranges that span different geographic landscapes frequently contain cryptic species- or population-level structure. Identifying these possible diversification factors can often be accomplished under a comparative phylogeographic framework. However, comparisons suffer if previous studies are limited to a particular group or habitat type. In California, a complex landscape has led to several phylogeographic breaks, primarily in terrestrial species. However, two sister taxa of freshwater fish, riffle sculpin (Cottus gulosus) and Pit sculpin (Cottus pitensis), display ranges based on morphological identifications that do not coincide with these breaks. Using a comprehensive sampling and nuclear, mitochondrial and microsatellite markers, we hypothesized that proposed species ranges are erroneous based on potential hybridization/gene flow between species. Results identified a phylogeographic signature consistent with this hypothesis, with breaks at the Coast Range Mountains and Sacramento/San Joaquin River confluence. Coastal locations of C. gulosus represent a unique lineage, and 'true' C. gulosus were limited to the San Joaquin basin, both regions under strong anthropogenic influence and potential conservation targets. C. pitensis limits extended historically throughout the Sacramento/Pit River basin but currently are restricted to the Pit River. Interestingly, locations in the Sacramento River contained low levels of ancestral hybridization and gene flow from C. gulosus but now appear to be a distinct population. The remaining population structure was strongly correlated with Sierra Nevada presence (high) or absence (low). This study stresses the importance of testing phylogeographic breaks across multiple taxa/habitats before conservation decisions are made, but also the potential impact of different geographic landscapes on evolutionary diversification.

Life history and biogeographic diversification of an endemic western North American freshwater fish clade using a comparative species tree approach.

The west coast of North America contains a number of biogeographic freshwater provinces which reflect an ever-changing aquatic landscape. Clues to understanding this complex structure are often encapsulated genetically in the ichthyofauna, though frequently as unresolved evolutionary relationships and putative cryptic species. Advances in molecular phylogenetics through species tree analyses now allow for improved exploration of these relationships. Using a comprehensive approach, we analyzed two mitochondrial and nine nuclear loci for a group of endemic freshwater fish (sculpin-Cottus) known for a wide ranging distribution and complex species structure in this region. Species delimitation techniques identified three novel cryptic lineages, all well supported by phylogenetic analyses. Comparative phylogenetic analyses consistently found five distinct clades reflecting a number of unique biogeographic provinces. Some internal node relationships varied by species tree reconstruction method, and were associated with either Bayesian or maximum likelihood statistical approaches or between mitochondrial, nuclear, and combined datasets. Limited cases of mitochondrial capture were also evident, suggestive of putative ancestral hybridization between species. Biogeographic diversification was associated with four major regions and revealed historical faunal exchanges across regions. Mapping of an important life-history character (amphidromy) revealed two separate instances of trait evolution, a transition that has occurred repeatedly in Cottus. This study demonstrates the power of current phylogenetic methods, the need for a comprehensive phylogenetic approach, and the potential for sculpin to serve as an indicator of biogeographic history for native ichthyofauna in the region.

KNEMIDOKOPTES MITES AND THEIR EFFECTS ON THE GRIPPING POSITION OF THE FEET OF STELLER'S JAYS (CYANOCITTA STELLERI).

Steller's Jays (Cyanocitta stelleri) with swollen legs and feet resembling the signs of scaly leg have been commonly seen around Arcata, California, US. The clinical signs are thought to be caused by knemidokoptic mites, a group of parasites specialized on avian hosts. Between February 2019 and March 2020, we analyzed the long-term database of Steller's Jays collected by Humboldt State University for trends in the prevalence of signs of scaly leg, compared the gripping position in the feet of Steller's Jays with variable signs of this condition as an index of their ability to perch, identified the mites using a partial sequence of the cytochrome oxidase subunit I gene, and examined genetic distances between mites collected from different host species both sequenced in this study and from GenBank. Overall, 27% of jays recorded in the long-term database had shown signs of scaly leg. Jays with signs captured in this study had greater variability in and a reduced degree of contraction in the gripping position of their feet compared to jays without signs, suggesting that infestation may have an impact on the host's ability to perch. The cytochrome oxidase subunit I sequence (578 base pairs) from mites collected from Steller's Jays was compared to sequences from Knemidokoptes jamaicensis, Knemidokoptes derooi, and to unidentified Knemidokoptes spp. collected from different hosts. The mites from Steller's Jays were most closely related to Knemidokoptes jamaicensis but had a relatively high sequence divergence, 7.8%, supporting the possibility that the form infesting these jays may be an undescribed species.

Rampant drift in artificially fragmented populations of the endangered tidewater goby (Eucyclogobius newberryi).

Habitat fragmentation and its genetic consequences are a critically important issue in evaluating the evolutionary penalties of human habitat modification. Here, we examine the genetic structure and diversity in naturally subdivided and artificially fragmented populations of the endangered tidewater goby (Eucyclogobius newberryi), a small fish restricted to discrete coastal lagoons and estuaries in California, USA. We use five naturally fragmented coastal populations from a 300- km spatial scale as a standard to assess migration and drift relative to eight artificially fragmented bay populations from a 30- km spatial scale. Using nine microsatellite loci in 621 individuals, and a 522-base fragment of mitochondrial DNA control region from 103 individuals, we found striking differences in the relative influences of migration and drift on genetic variation at these two scales. Overall, the artificially fragmented populations exhibited a consistent pattern of higher genetic differentiation and significantly lower genetic diversity relative to the naturally fragmented populations. Thus, even in a species characterized by habitat isolation and subdivision, further artificial fragmentation appears to result in substantial population genetic consequences and may not be sustainable.

A complex phenotype in salmon controlled by a simple change in migratory timing.

Differentiation between ecotypes is usually presumed to be complex and polygenic. Seasonal patterns of life history in salmon are used to categorize them into ecotypes, which are often considered "distinct" animals. Using whole-genome sequencing and tribal fishery sampling of Chinook salmon, we show that a single, small genomic region is nearly perfectly associated with spawning migration timing but not with adiposity or sexual maturity, traits long perceived as central to salmon ecotypes. Distinct migration timing does not prevent interbreeding between ecotypes, which are the result of a simple, ancient polymorphism segregating within a diverse population. Our finding that a complex migratory phenotype results from a single gene region will facilitate conservation and restoration of this iconic fish.

A new microsporidian infecting the musculature of the endangered tidewater goby (Gobiidae).

A previously unrecognized microsporidian (Kabatana newberryi n. sp.) is described from the musculature of Eucyclogobius newberryi (Gobiidae) in Big Lagoon, Humboldt County, California. Spores are ovoid, ranging in size from 2.8 +/- 0.3 microm in total length and 1.9 +/- 0.4 microm in width (measurements of 30 spores made by calculation from micrograph). The polar filament has 9-10 coils in 1-2 rows. Development occurs in direct contact with host muscle cell cytoplasm, without xenoma or sporophorous vesicle. Phylogenetic analysis of the new species and of 35 other microsporidians known to infect fish using 1115 base pairs of aligned 16S rRNA gene indicate the new species is most closely related to Kabatana takedai. However, the new species differs by 11% sequence divergence from K. takedai. Divergence in morphology and genetic data allow for diagnosis from all other fish-infecting microsporidia and supports recognition of a new species of microsporidian, Kabatana newberryi n. sp., presently known only from a suspected specific host, the endangered tidewater goby Eucyclogobius newberryi.

Using occupancy modelling to compare environmental DNA to traditional field methods for regional-scale monitoring of an endangered aquatic species.

Environmental DNA (eDNA) monitoring approaches promise to greatly improve detection of rare, endangered and invasive species in comparison with traditional field approaches. Herein, eDNA approaches and traditional seining methods were applied at 29 research locations to compare method-specific estimates of detection and occupancy probabilities for endangered tidewater goby (Eucyclogobius newberryi). At each location, multiple paired seine hauls and water samples for eDNA analysis were taken, ranging from two to 23 samples per site, depending upon habitat size. Analysis using a multimethod occupancy modelling framework indicated that the probability of detection using eDNA was nearly double (0.74) the rate of detection for seining (0.39). The higher detection rates afforded by eDNA allowed determination of tidewater goby occupancy at two locations where they have not been previously detected and at one location considered to be locally extirpated. Additionally, eDNA concentration was positively related to tidewater goby catch per unit effort, suggesting eDNA could potentially be used as a proxy for local tidewater goby abundance. Compared to traditional field sampling, eDNA provided improved occupancy parameter estimates and can be applied to increase management efficiency across a broad spatial range and within a diversity of habitats.

Increased extinction potential of insular fish populations with reduced life history variation and low genetic diversity.

Theoretical work has shown that reduced phenotypic heterogeneity leads to population instability and can increase extinction potential, yet few examples exist of natural populations that illustrate how varying levels expressed diversity may influence population persistence, particularly during periods of stochastic environmental fluctuation. In this study, we assess levels of expressed variation and genetic diversity among demographically independent populations of tidewater goby (Eucyclogobius newberryi), show that reductions in both factors typically coincide, and describe how low levels of diversity contribute to the extinction risk of these isolated populations. We illustrate that, for this annual species, continuous reproduction is a safeguard against reproductive failure by any one population segment, as natural, stochastically driven salinity increases frequently result in high mortality among juvenile individuals. Several study populations deviated from the natural pattern of year-round reproduction typical for the species, rendering those with severely truncated reproductive periods vulnerable to extinction in the event of environmental fluctuation. In contrast, demographically diverse populations are more likely to persist through such periods through the continuous presence of adults with broader physiological tolerance to abrupt salinity changes. Notably, we found a significant correlation between genetic diversity and demographic variation in the study populations, which could be the result of population stressors that restrict both of these diversity measures simultaneously, or suggestive of a causative relationship between these population characteristics. These findings demonstrate the importance of biocomplexity at the population level, and assert that the maintenance of diversity contributes to population resilience and conservation of this endangered species.

Small founding number and low genetic diversity in an introduced species exhibiting limited invasion success (speckled dace, Rhinichthys osculus).

Molecular evaluations of successful invaders are common, however studies of introduced species that have had limited invasion success, or have died out completely, are rare. We studied an introduced population of speckled dace (Rhinichthys osculus) from northern California, USA that has rapidly increased in abundance but remained restricted to a 25-km stretch of river since its introduction in the mid-1980s. Field and laboratory analyses indicate that invasion success of speckled dace is constrained by the combined effects of multiple predators. The role of bottleneck effects associated with the introduction has not been studied. We assayed variation in seven microsatellite loci and one mitochondrial DNA gene in the introduced population and nine putative source populations to identify the source population and evaluate bottleneck effects. The Trinity River system was supported as the source owing to its genetic similarity and geographic proximity to the introduced population. Consistent with a bottleneck, the introduced population exhibited reduced allelic and haplotype richness in comparison to source populations. Estimates of the genetically effective number of individuals founding the introduced population using nuclear coalescent analyses and a mitochondrial simulation procedure were highly concordant in suggesting that the initial colonizing group was comprised of about 10 individuals. A bottleneck effect in an exotic species exhibiting limited invasion success has rarely been documented and thus introduction of speckled dace represents an important model system for future investigation. Establishing a relationship between genetic diversity and factors limiting invasion success in this system (e.g., predator avoidance) will help determine the extent to which genetic diversity loss has constrained invasion success in speckled dace.