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Large mitochondrial DNA fragments and nearly complete mitogenomes were obtained from nextera-tagmented reductively amplified DNA (nextRAD) data from the Rio Grande silvery minnow (Hybognathus amarus). We used these data to characterize variation across the mitogenome and to evaluate changes in mitochondrial diversity across a 20 year time series punctuated by marked species decline. We observed different evolutionary rates among protein-coding genes and inflated values of the ratio of non-synonymous change to non-synonymous sites to synonymous changes per synonymous site for several protein-coding genes, violating assumptions of selective neutrality. Analyses suggested that non-synonymous substitutions are effectively neutral, and our results were not consistent with genetic draft. Mitogenomes revealed large long-term effective population size for Rio Grande silvery minnow, and time-series data indicated recent reductions in mitochondrial diversity via genetic drift associated with population bottlenecks. The results presented in this study show that in some cases, nextRAD sequences contain informative mitochondrial DNA data. [ABSTRACT FROM AUTHOR]

Human water use combined with a recent megadrought have reduced river and stream flow through the southwest United States and led to periodic drying of formerly perennial river segments. Reductions in snowmelt runoff and increased extent of drying collectively threaten short‐lived, obligate aquatic species, including the endangered Rio Grande silvery minnow (Hybognathus amarus). This species is subject to boom‐and‐bust population dynamics, under which large fluctuations in abundance are expected to lower estimates of effective population size and erode genetic diversity over time. Rates of diversity loss are also affected by additions of hatchery‐origin fish used to supplement the wild population. We used demographic and genetic data from wild and hatchery individuals to examine the relationship of genetic diversity and effective population size to abundance over the last two decades. Genetic diversity was low during the early 2000s, but diversity and demographic metrics stabilized after the hatchery program was initiated and environmental conditions improved. Yet, from 2017 onward, allelic diversity declined (Cohen's d = 1.34) and remained low despite hatchery stocking and brief wild population recovery. Across the time series, single‐sample estimates of effective population size based on linkage disequilibrium (LD Ne) were positively associated (r = 0.53) with wild abundance and total abundance, but as the proportion of hatchery‐origin spawners increased, LD Ne declined (r = −0.55). Megadrought limited wild spawner abundance and precluded refreshment of hatchery brood stocks with wild fish; hence, we predict a riverine population increasingly dominated by hatchery‐origin individuals and accelerated loss of genetic diversity despite supplementation. We recommend an adaptive and accelerated management plan that integrates river flow management and hatchery operations to slow the pace of genetic diversity loss exacerbated by megadrought. [ABSTRACT FROM AUTHOR]

Sex determination systems and genetic sex differentiation across fishes are highly diverse but are unknown for most Cypriniformes, including Rio Grande silvery minnow (Hybognathus amarus). In this study, we aimed to detect and validate sex-linked markers to infer sex determination system and to demonstrate the utility of combining several methods for sex-linked marker detection in nonmodel organisms. To identify potential sex-linked markers, Nextera-tagmented reductively amplified DNA (nextRAD) libraries were generated from 66 females, 64 males, and 60 larvae of unknown sex. These data were combined with female and male de novo genomes from Nanopore long-read sequences. We identified five potential unique male nextRAD-tags and one potential unique male contig, suggesting an XY sex determination system. We also identified two single-nucleotide polymorphisms (SNPs) in the same contig with values of F ST, allele frequencies, and heterozygosity conforming with expectations of an XY system. Through PCR we validated the marker containing the sex-linked SNPs and a single nextRAD-tag sex-associated marker but it was not male specific. Instead, more copies of this locus in the male genome were suggested by enhanced amplification in males. Results are consistent with an XY system with low differentiation between sex-determining regions. Further research is needed to confirm the level of differentiation between the sex chromosomes. Nonetheless, this study highlighted the power of combining reduced representation and whole-genome sequencing for identifying sex-linked markers, especially when reduced representation sequencing does not include extensive variation between sexes, either because such variation is not present or not captured. [ABSTRACT FROM AUTHOR]

Climate change and a host of other human stressors on aquatic systems in the American Southwest are rapidly exceeding our ability to conserve native fish diversity. The most severe megadrought in over a millennium has compromised current management plans by exacerbating the impacts of altered hydrology, poor water quality, invasive species, and habitat fragmentation. Drought legacies are of particular concern, and existing conservation actions may not be sufficient to maintain resilient native fish populations, particularly if this drought persists. We draw examples from current literature and empirical research to support our contention that more resources and novel approaches to prioritizing environmental flows, improving ecological connectivity of populations, preventing and controlling invasive species, and establishing refuge populations are necessary to stave off extinctions. Moreover, shifts in socio‐political attitudes that better recognize environmental concerns must be integrated into water resource policy and management to achieve native fish conservation goals. [ABSTRACT FROM AUTHOR]

Many long‐term genetic monitoring programmes began before next‐generation sequencing became widely available. Older programmes can now transition to new marker systems usually consisting of 1000s of SNP loci, but there are still important questions about comparability, precision, and accuracy of key metrics estimated using SNPs. Ideally, transitioned programmes should capitalize on new information without sacrificing continuity of inference across the time series. We combined existing microsatellite‐based genetic monitoring information with SNP‐based microhaplotypes obtained from archived samples of Rio Grande silvery minnow (Hybognathus amarus) across a 20‐year time series to evaluate point estimates and trajectories of key genetic metrics. Demographic and genetic monitoring bracketed multiple collapses of the wild population and included cases where captive‐born repatriates comprised the majority of spawners in the wild. Even with smaller sample sizes, microhaplotypes yielded comparable and in some cases more precise estimates of variance genetic effective population size, multilocus heterozygosity and inbreeding compared to microsatellites because many more microhaplotype loci were available. Microhaplotypes also recorded shifts in allele frequencies associated with population bottlenecks. Trends in microhaplotype‐based inbreeding metrics were associated with the fraction of hatchery‐reared repatriates to the wild and should be incorporated into future genomic monitoring. Although differences in accuracy and precision of some metrics were observed between marker types, biological inferences and management recommendations were consistent. [ABSTRACT FROM AUTHOR]

Climate change will strongly impact aquatic ecosystems particularly in arid and semi-arid regions. Fish-parasite interactions will also be affected by predicted altered flow and temperature regimes, and other environmental stressors. Hence, identifying environmental and genetic factors associated with maintaining diversity at immune genes is critical for understanding species' adaptive capacity. Here, we combine genetic ( MHC class IIβ and microsatellites), parasitological and ecological data to explore the relationship between these factors in the remnant wild Rio Grande silvery minnow ( Hybognathus amarus) population, an endangered species found in the southwestern United States. Infections with multiple parasites on the gills were observed and there was spatio-temporal variation in parasite communities and patterns of infection among individuals. Despite its highly endangered status and chronically low genetic effective size, Rio Grande silvery minnow had high allelic diversity at MHC class IIβ with more alleles recognized at the presumptive DAB1 locus compared to the DAB3 locus. We identified significant associations between specific parasites and MHC alleles against a backdrop of generalist parasite prevalence. We also found that individuals with higher individual neutral heterozygosity and higher amino acid divergence between MHC alleles had lower parasite abundance and diversity. Taken together, these results suggest a role for fluctuating selection imposed by spatio-temporal variation in pathogen communities and divergent allele advantage in maintenance of high MHC polymorphism. Understanding the complex interaction of habitat, pathogens and immunity in protected species will require integrated experimental, genetic and field studies. [ABSTRACT FROM AUTHOR]

Thorough sampling is necessary to delineate lineage diversity for polytypic 'species' such as Cyprinella lutrensis. We conducted extensive mtDNA sampling (cytochrome b and ND4) from the Pecos River, Rio Grande, and South Canadian River, New Mexico. Our study focussed on the Pecos River due to its complex geological history and potential to harbour multiple lineages. We used geometric-morphometric, morphometric, and meristic analyses to test for phenotypic divergence and combined nucDNA with mtDNA to test for cytonuclear disequilibrium and combined our sequences with published data to conduct a phylogenetic re-assessment of the entire C. lutrensis clade. We detected five co-occurring mtDNA lineages in the Pecos River, but no evidence for cytonuclear disequilibrium or phenotypic divergence. Recognized species were interspersed amongst divergent lineages of ' C. lutrensis'. Allopatric divergence among drainages isolated in the Late Miocene and Pliocene apparently produced several recognized species and major divisions within ' C. lutrensis'. Pleistocene re-expansion and subsequent re-fragmentation of a centralized lineage founded younger, divergent lineages throughout the Rio Grande basin and Edwards Plateau. There is also evidence of recent introductions to the Rio Grande, Pecos and South Canadian Rivers. Nonetheless, deeply divergent lineages have coexisted since the Pleistocene. [ABSTRACT FROM AUTHOR]

We used comparative landscape genetics to examine the relative roles of historical events, intrinsic traits and landscape factors in determining the distribution of genetic diversity of river fishes across the North American Great Plains. Spatial patterns of diversity were overlaid on a patch-based graphical model and then compared within and among three species that co-occurred across five Great Plains watersheds. Species differing in reproductive strategy (benthic vs. pelagic-spawning) were hypothesized to have different patterns of genetic diversity, but the overriding factor shaping contemporary patterns of diversity was the signature of past climates and geological history. Allelic diversity was significantly higher at southern latitudes for Cyprinella lutrensis and Hybognathus placitus, consistent with northward expansion from southern Pleistocene refugia. Within the historical context, all species exhibited lowered occupancy and abundance in heavily fragmented and drier upstream reaches, particularly H. placitus; a pelagic-spawning species, suggesting rates of extirpation have outpaced losses of genetic diversity in this species. Within most tributary basins, genetically diverse populations of each species persisted. Hence, reconnecting genetically diverse populations with those characterized by reduced diversity (regardless of their position within the riverine network) would provide populations with greater genetic and demographic resilience. We discuss cases where cross-basin transfer may be appropriate to enhance genetic diversity and mitigate negative effects of climate change. Overall, striking similarities in genetic patterns and in response to fragmentation and dewatering suggest a common strategy for genetic resource management in this unique riverine fish assemblage. [ABSTRACT FROM AUTHOR]

Captive breeding and rearing are central elements in conservation, management, and recovery planning for many endangered species including Rio Grande Silvery Minnow, a North American freshwater cyprinid. Traditionally, the sole purpose of hatcheries was to produce as many fish as feasible for stocking and harvest. Production quotas are also an important consideration in hatchery programs for endangered species, but they must also maintain and maximize genetic diversity of fish produced through implementation of best breeding practices. Here, we assessed genetic outcomes and measures of productivity (number of eggs and larval viability) for three replicates of three mating designs that are used for this small, pelagic-spawning fish. These were 1) monogamous mating, 2) hormone-induced communal spawning, and 3) environmentally cued communal spawning. A total of 180 broodstock and 450 progeny were genotyped. Genetic diversity and egg productivity did not differ significantly among spawning designs (He: F = 0.52, P = 0.67; Ho: F = 0.12, P = 0.89; number of eggs: F = 3.59, P = 0.09), and there was evidence for variance in reproductive success among individuals in all three designs. Allelic richness declined from the broodstock to progeny generation in all breeding designs. There was no significant difference in the genetic effective size (regardless of the method used) among designs. Significantly more viable eggs were produced in environmentally cued communal spawn compared to the alternative strategies (F = 5.72, P = 0.04), but this strategy is the most difficult to implement. [ABSTRACT FROM AUTHOR]

The endangered Rio Grande silvery minnow persists as a remnant population in a highly fragmented and regulated arid-land river system. The species is subject to dramatic annual fluctuations in density. Since 2003, the wild population has been supplemented by hatchery-reared fish. We report on a 12-year (1999-2010) monitoring study of genetic diversity and effective population size (Ne) of wild and hatchery stocks. Our goals were to evaluate how genetic metrics responded to changes in wild fish density and whether they corresponded to the number and levels of diversity of hatchery-reared repatriates. Genetic diversity and all measures of Ne in the wild population did not correlate with wild fish density until hatchery supplementation began in earnest. Estimates of variance and inbreeding effective size were not correlated. Our results suggest source-sink dynamics where captive stocks form a genetically diverse source and the wild population behaves as a sink. Nevertheless, overall genetic diversity of silvery minnow has been maintained over the last decade, and we attribute this to a well-designed and executed propagation management plan. When multiple factors like environmental fluctuation and hatchery supplementation act simultaneously on a population, interpretation of genetic monitoring data may be equally complex and require considerable ecological data. [ABSTRACT FROM AUTHOR]

Abstract: The major histocompatibility complex (MHC) is a critical component of the adaptive immune response in vertebrates. Due to the role that MHC plays in immunity, absence of variation within these genes may cause species to be vulnerable to emerging diseases. The freshwater fish family Cyprinidae comprises the most diverse and species-rich group of freshwater fish in the world, but some are imperiled. Despite considerable species richness and the long evolutionary history of the family, there are very few reports of MHC sequences (apart from a few model species), and no sequences are reported from endemic North American cyprinids (subfamily Leuciscinae). Here we isolate and characterize the MH Class II beta genes from complementary DNA and genomic DNA of the non-model, endangered Rio Grande silvery minnow (Hybognathus amarus), a North American cyprinid. Phylogenetic reconstruction revealed two groups of divergent MH alleles that are paralogous to previously described loci found in deeply divergent cyprinid taxa including common carp, zebrafish, African large barb and bream. Both groups of alleles were under the influence of diversifying selection yet not all individuals had alleles belonging to both allelic groups. We concluded that the general organization and pattern of variation of MH class II genes in Rio Grande silvery minnow is similar to that identified in other cyprinid fishes studied to date, despite distant evolutionary relationships and evidence of a severe genetic bottleneck. [Copyright &y& Elsevier]

We used 991 bp of mtDNA (D-loop/ND4L/ND4) and eight microsatellite loci to examine population structure and genetic diversity of Catostomus plebeius from nine localities across the northern part of its range in New Mexico. Two phylogeographic regions (Mimbres and Rio Grande) were defined by phylogenetically distinct collections of mtDNA haplotypes. Mismatch distribution analysis and relatively deep coalescence of haplotypes within the upper Rio Grande suggested rapid population expansion of Rio Grande populations following putative colonization from the ancestral Cabeza de Vaca lake system during the Miocene. Mitochondrial haplotypes indicated that individuals from Palomas Creek (near present-day Elephant Butte Reservoir) shared most recent common ancestry with the Rio Grande phylogeographic region. Microsatellite diversity and allele identity supported the hypothesis of native status of C. plebeius from Sapillo Creek in the Gila River drainage. Analysis of molecular variance within and among samples from the upper Rio Grande suggested low levels of historic gene flow across that basin, with ΦST = 0.19 and FST = 0.14 for mtDNA and microsatellites, respectively. [ABSTRACT FROM AUTHOR]

Hatchery programs are critical for conservation and management of many imperiled fishes. Most traditional aquaculture programs negatively affect ecological performance, genetic, and phenotypic diversity of hatchery-origin fish compared with wild counterparts. Here, we synthesize outcomes of three conservation programs aimed at enhancing 'wildness'. Each program focuses on a different species: lake sturgeon, razorback sucker, and Rio Grande silvery minnow. These species differ in key life history traits including size and age at sexual maturity, reproductive and migratory behavior, and habitat requirements. Threats to persistence of the focal taxa, however, exemplify common pressures experienced by freshwater fishes worldwide. Conservation hatchery programs for lake sturgeon, razorback sucker, and Rio Grande silvery minnow capitalize on natural spawning in the wild followed by collections of wild-fertilized eggs/larvae for hatchery rearing. Individuals are repatriated to the wild after rearing to body sizes less susceptible to mortality. Protocols include collections of eggs or larvae across the entire spawning period and at appropriate geographic scales to maximize retention of genetic diversity and, to increase the likelihood of preserving variation for heritable life history traits. Using direct and indirect evidence we show that hatchery programs that allow individuals to fulfill parts of the life-cycle in their native habitats can be conducted without compromising genetic diversity. Adoption of similar strategies in other imperiled fishes would improve understanding of species life history, and provide an incentive to protect native habitats so they may eventually support self-sustaining populations. • Outcomes of conservation programs that capitalize on wild spawning are synthesized. • Wild produced eggs/larvae are used in hatchery activities for imperiled fishes. • Long-term genetic monitoring reveals retention of genetic diversity. • Larval growth and survival is higher in lake sturgeon streamside hatcheries. • Broader adoption of these strategies may increase success of conservation programs. [ABSTRACT FROM AUTHOR]

Rio Grande sucker (Pantosteus plebeius) is continuously distributed in parts of its range, but occurs in geographically isolated populations at the periphery. We used 10 microsatellites and DNA sequence data from the mitochondrial cytochrome b gene to characterize genetic diversity and reconstruct evolutionary relationships of five peripheral populations in the southwestern United States. An isolated population in Rio Bonito (Pecos River drainage) is most closely related to a central Rio Grande population in Alamosa Creek, and part of the monophyletic Rio Grande lineage. Another disjunct population, Bluewater Creek in the western Rio Grande drainage, has a Mimbres lineage mtDNA haplotype that likely originated via interbasin transfer. Both peripheral populations are important for conservation of the species as a whole. Three other isolated populations are closely related to mainstem ‘‘core’’ populations in the Mimbres and Gila rivers that colonized these intermittently connected tributaries. One peripheral population in Allie Canyon, Mimbres River, has persisted longer in isolation than Rocky Canyon or Trout Creek in the Gila system. In general, a process of range expansion during relatively cool and wet periods, followed by retreat to wetted refugia during warm and dry periods, leads to genetic differentiation of peripheral populations of Rio Grande sucker at local and across-basin scales. [ABSTRACT FROM AUTHOR]

In the late 1940s, Rio Grande Sucker Pantosteus plebeius was reported in tributaries of the Gila River in the Colorado Basin and presumed to be introduced because of its absence in mid-19th century surveys. We assayed genetic variation at ten microsatellite loci and two mtDNA genes (cyt b and ND4) to test the hypothesis of human-mediated introduction into the Gila within the last century. Phylogeographic analysis indicated that Gila River populations shared recent common ancestry with populations in the Mimbres River. Using approximate Bayesian computation (ABC), we rejected the hypothesis that Rio Grande Sucker was introduced to the upper Gila River within the last century. Rather, we hypothesize an older (∼4000 years before present) headwater capture event that facilitated transfer of fishes from the Mimbres to Sapillo Creek in the Gila Basin. From there, suckers dispersed to the San Francisco River and became established in upland stream habitats. Rio Grande Sucker exhibits low levels (<10%) of introgression with Desert Sucker but maintains species cohesion in the Gila Basin. We conclude that Rio Grande Sucker is native to the Gila Basin, and that these populations harbor unique diversity that could play an important role in species conservation. More generally, rigorous testing for human-mediated translocation is a difficult analytical problem, but a definitive means to do so is essential to fully understand the biogeography of freshwater fishes of the American Southwest. [ABSTRACT FROM AUTHOR]

Two endemic, "large river" fishes of the Colorado River basin of western North America, bonytail Gila elegans and razorback sucker Xyrauchen texanus, are among several critically endangered species in the system. Wild populations of bonytail are gone, and there are no self-sustaining populations of razorback sucker anywhere; reproduction occurs but recruitment does not. Both species have been under intensive management in the Lower Colorado River since the 1980s. Today, with the single exception of Lake Mead, remaining populations are composed entirely of repatriated individuals that depend on stocking for their continued existence. In 2003, a conceptual off-channel habitat (OCH) management plan for these and other large river fishes of the system was published by the late W.L. Minckley and colleagues. The cornerstone of the approach was to move away from hatchery-based fish production and instead use isolated OCHs that were free of predatory and competitive nonnative fishes, where populations of native species could live, grow, reproduce, and recruit. Populations of adult fish also would live in open waters of the system, and through active management, individuals would be exchanged with those in OCHs to maintain genetic integrity and diversity of both species. Progress in the last 2 decades toward implementing the plan includes creation of new OCHs, studies of population dynamics and genetics of "wild" and captive populations, development of appropriate metrics to assess status of OCH populations, and refinement of the OCH concept itself. Our goals in this paper are to review management of bonytail and razorback sucker in the Lower Colorado River, present examples of species dynamics in OCHs, offer data-driven refinements to the OCH concept, and explore practical aspects including challenges and constraints to implementation of the concept. We conclude that bonytail and razorback sucker never can meet quantitative criteria required by current recovery plans, but long-term conservation of these species can be achieved if an OCH concept of management is successfully implemented and maintained. [ABSTRACT FROM AUTHOR]

We report results from a 3-year field investigation to limit hybridization between native and nonnative suckers in 2 small tributaries of the Gunnison River basin, Delta County, Colorado. Upstream movement to spawning habitat was selectively impeded by picket weir in intermittent Cottonwood Creek. There, native suckers were identified based on morphological characters in the field and were transferred upstream of the weir, whereas nonnative fishes were excluded from upstream movement. Another stream, Potter Creek, was not impeded by weir. Genetic identification of adult fishes in both streams was consistent with morphological identification in the field. Larval genetic identification was consistent with probable spawning species in Potter Creek. However, in Cottonwood Creek, larval fish proportions differed significantly from likely spawners. Proportions of nonnative suckers were high in Cottonwood Creek because exclusion structures were compromised by flooding during the spawning season. Yet, instream control of spawning by nonnative fishes has potential for reducing the incidence of hybridization, but practical challenges remain for implementation in the field. [ABSTRACT FROM AUTHOR]

Biodiversity in stream networks is threatened globally by interactions between habitat fragmentation and altered hydrologic regimes. In the Great Plains of North America, stream networks are fragmented by >19 000 anthropogenic barriers, and flow regimes are altered by surface water retention and groundwater extraction. We documented the distribution of anthropogenic barriers and dry stream segments in five basins covering the central Great Plains to assess effects of broad-scale environmental change on stream fish community structure and distribution of reproductive guilds. We used an information-theoretic approach to rank competing models in which fragmentation, discharge magnitude, and percentage of time streams had zero flow (a measure of desiccation) were included to predict effects of environmental alterations on the distribution of fishes belonging to different reproductive guilds. Fragmentation caused by anthropogenic barriers was most common in the eastern Great Plains, but stream desiccation became more common to the west, where rivers are underlain by the depleted (i.e., extraction > recharge) High Plains Aquifer. Longitudinal gradients in fragmentation and desiccation contributed to spatial shifts in community structure from taxonomically and functionally diverse communities dominated by pelagic reproductive guilds where fragmentation and desiccation were least, to homogenized communities dominated by benthic guilds where fragmentation and desiccation were common. Modeling results revealed these shifts were primarily associated with decline of pelagic reproductive guilds, notably small-bodied pelagophilic and lithopelagophilic fishes that declined in association with decreased fragment length and increased number of days with zero flow. Graph theory combined with a barrier prioritization approach revealed specific fragments that could be reconnected to allow fishes within these guilds to colonize currently unoccupied fragments with the mitigation or removal of small dams (<10 m height). These findings are useful for natural resource managers charged with halting or reversing the prevailing pattern of declining fish diversity in the Great Plains. Our study represents one of the most comprehensive assessments of fish diversity responses to broad-scale environmental change in the Great Plains and provides a conservation strategy for addressing the simultaneous contributions of fragmentation and flow alteration to the global freshwater biodiversity crisis. [ABSTRACT FROM AUTHOR]