Genetic erosion in an endangered desert fish during a megadrought despite long-term supportive breeding.

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.

El uso humano del agua, combinado con una megasequía reciente, ha reducido el flujo de los ríos y arroyos en el suroeste de los Estados Unidos y ha provocado la seca periódica de segmentos de ríos que antes eran perennes. Las reducciones en la escorrentía del deshielo y el aumento de la sequía amenazan colectivamente a especies obligatoriamente acuáticas de vida corta, incluyendo la amenazada carpa chamizal (Hybognathus amarus). Esta especie está sujeta a una dinámica poblacional de explosión y colapso, bajo la cual se espera que grandes fluctuaciones en la abundancia reduzcan las estimaciones del tamaño efectivo de la población y erosionen la diversidad genética con el tiempo. Las tasas de pérdida de la diversidad también se ven afectadas por la adición de peces procedentes de criaderos usados para suplementar la población silvestre. Utilizamos datos demográficos y genéticos de individuos silvestres y de criaderos para examinar la relación entre la diversidad genética y el tamaño efectivo de la población con la abundancia durante las últimas dos décadas. La diversidad genética fue baja a principios de los 2000, pero las métricas de diversidad y demografía estabilizaron después de que se inició el programa de criadero y mejoraron las condiciones ambientales. Sin embargo, a partir de 2017, la diversidad alélica disminuyó (d de Cohen = 1,34) y se mantuvo baja a pesar de la suplementación con individuos de criaderos y la breve recuperación de la población silvestre. A lo largo del tiempo, las estimativas de muestras individuales del tamaño efectivo de la población basados en el desequilibrio de ligamiento (LD Ne) estaban asociadas positivamente (r = 0,53) con la abundancia silvestre y la abundancia total, pero a medida que la proporción de desovadores originados en criaderos aumentó, el LD Ne disminuyó (r = -0,55). La megasequía limitó la abundancia de desovadores silvestres e impidió el reabastecimiento de las poblaciones en cautiverio con peces silvestres; por lo tanto, predecimos una población ribereña cada vez más dominada por individuos procedentes de criaderos y una pérdida acelerada de diversidad genética a pesar de la suplementación. Recomendamos un plan de gestión adaptativo y acelerado que integre la gestión del caudal del río y las operaciones de criaderos para frenar el ritmo de pérdida de diversidad genética exacerbada por la megasequía.

The complete mitochondrial genomes of two imperiled species endemic to the Southwestern United States: Peppered Chub (Macrhybopsis tetranema) and Gila Trout (Oncorhynchus gilae).

Macrhybopsis tetranema and Oncorhynchus gilae are fish species endemic to the Southwestern United States. We present the complete mitochondrial genomes for these species. Each genome consisted of 13 protein-coding genes, two ribosomal (rRNA) genes, 22 transfer RNA (tRNA) genes, and the control region (D-loop). Mitogenome lengths were 16,916 base pairs (bp) for M. tetranema, and 16,976 bp for O. gilae. The GC content was 41% for M. tetranema and 46% for O. gilae. The relationships of M. tetranema and O. gilae were consistent with previous phylogenetic analyses.

Limited evidence for extensive genetic differentiation between X and Y chromosomes in Hybognathus amarus (Cypriniformes: Leuciscidae).

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 FST, 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.

Transitioning from microsatellites to SNP-based microhaplotypes in genetic monitoring programmes: Lessons from paired data spanning 20 years.

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.

The complete mitochondrial genomes of three imperiled cyprinid fishes Bonytail (Gila elegans), Rio Grande Silvery Minnow (Hybognathus amarus) and Loach Minnow (Tiaroga cobitis).

Gila elegans, Hybognathus amarus, and Tiaroga cobitis (Family Cyprinidae, Order Cypriniformes) are endemic and endangered fishes in the southwestern United States. We present complete mitochondrial genomes for each species. Each mitochondrion consisted of 13 protein-coding genes, 2 ribosomal (rRNA) genes, 22 transfer RNA (tRNA) genes, and a single control region (D-loop), and gene order was consistent with other cyprinid fishes. Total genome lengths were 16,593 base pairs (bp) for G. elegans, 16,705 bp for H. amarus, and 16,802 for T. cobitis. The GC content in G. elegans and H. amarus was 44%, but higher in T. cobitis at 48%. Phylogenetic trees were generated to confirm relationships inferred via novel mitogenomes, and best-supported trees were consistent with previous research.

Variance in Reproductive Success is Driven by Environmental Factors, not Mating System, in Bonytails.

Studying the reproductive ecology of aggregate broadcast spawning fishes is difficult because it generally is not feasible to sample all potential parents and unambiguously assign their offspring. We used molecular-based parentage analysis to gain insights into the reproductive ecology of the endangered Bonytail, and to evaluate whether protected off-channel habitats could be used as an alternative to hatchery production. By genotyping adults and offspring stocked (n = 4130) into two experimental backwaters across three years, we determined that most adults (82-97%) contributed to progeny production across years and backwaters, with one exception. Both sexes mated multiply and the number of mates and family size were positively correlated. There was also a positive correlation between adult size and metrics of reproductive success. There were strong interactions between sample years and backwaters suggesting that environmental factors are the primary driver of variance in reproductive success. Knowledge of mating systems and sources of variance in reproductive success is important for management of endangered fish because high variance in reproductive success leads to substantial losses of genetic variation when few individuals reproduce successfully. Although variance in reproductive success was observed, most adults contributed to genetically diverse progeny in experimental backwaters. These results support the use of predator-free, but otherwise natural, backwaters as an effective conservation tool for reintroducing Bonytail to its native habitat.

Spatio-temporal variation in parasite communities maintains diversity at the major histocompatibility complex class IIß in the endangered Rio Grande silvery minnow.

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 IIß 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 IIß 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.

Biogeography of "Cyprinella lutrensis": intensive genetic sampling from the Pecos River 'melting pot' reveals a dynamic history and phylogenetic complexity.

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 emphasized the Pecos River due to its complex geological history and potential to harbor 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.

Comparative riverscape genetics reveals reservoirs of genetic diversity for conservation and restoration of Great Plains fishes.

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.

Genetic analysis of captive spawning strategies for the endangered Rio Grande Silvery Minnow.

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 (H e : F = 0.52, P = 0.67; H o : 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.

Genetic monitoring and complex population dynamics: insights from a 12-year study of the Rio Grande silvery minnow.

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 (N(e)) 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 N(e) 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.

Isolation and characterization of major histocompatibility class IIß genes in an endangered North American cyprinid fish, the Rio Grande silvery minnow (Hybognathus amarus).

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.

Genetic effective size, N(e), tracks density in a small freshwater cyprinid, Pecos bluntnose shiner (Notropis simus pecosensis).

Genetic monitoring tracks changes in measures of diversity including allelic richness, heterozygosity and genetic effective size over time, and has emerged as an important tool for understanding evolutionary consequences of population management. One proposed application of genetic monitoring has been to estimate abundance and its trajectory through time. Here, genetic monitoring was conducted across five consecutive year for the Pecos bluntnose shiner, a federally threatened minnow. Temporal changes in allele frequencies at seven microsatellite DNA loci were used to estimate variance effective size (N(eV)) across adjacent years in the time series. Likewise, effective size was computed using the linkage disequilibrium method (N(eD)) for each sample. Estimates of N(e) were then compared to estimates of adult fish density obtained from traditional demographic monitoring. For Pecos bluntnose shiner, density (catch-per-unit-effort), N(eV) and N(eD) were positively associated across this time series. Results for Pecos bluntnose shiner were compared to a related and ecologically similar species, the Rio Grande silvery minnow. In this species, density and N(eV) were negatively associated, which suggested decoupling of abundance and effective size trajectories. Conversely, density and N(eD) were positively associated. For Rio Grande silvery minnow, discrepancies among estimates of N(e) and their relationships with adult fish density could be related to effects of high variance in reproductive success in the wild and/or effects of supplementation of the wild population with captive-bred and reared fish. The efficacy of N(e) as a predictor of density and abundance may depend on intrinsic population dynamics of the species and how these dynamics are influenced by the landscape features, management protocols and other factors.

Life history and environmental variation interact to determine effective population to census size ratio.

Successful recovery and sustainability of threatened and exploited species depends in part on retention and maintenance of genetic diversity. Theory indicates that genetic diversity is lost at a rate inversely proportional to the genetically effective population size (N(e)), which is roughly equal to one-half the adult census size (N) in many organisms. However, N(e) has been reported to be up to five orders of magnitude lower than N in species with life histories that result in type III survivorship (high fecundity, but heavy mortality in early life stages, e.g. bony fishes), prompting speculation that low values of N(e) may be a general feature of such organisms despite sometimes vast abundances. Here, we compared N(e) and the ratio N(e)/N across three ecologically similar fish species from the arid southwestern United States, all with type III life histories but with differing expectations of egg and larval survivorship that correlate with the degree of human-imposed habitat fragmentation. Our study indicates that type III life history may be necessary, but this alone is insufficient to account for extraordinarily low values of N(e)/N. Rather, life history interacts with environmentally imposed mortality to determine the rate and magnitude of change in genetic diversity in these desert fish species.