Genetic load and viability of a future restored northern white rhino population.

As biodiversity loss outpaces recovery, conservationists are increasingly turning to novel tools for preventing extinction, including cloning and in vitro gametogenesis of biobanked cells. However, restoration of populations can be hindered by low genetic diversity and deleterious genetic load. The persistence of the northern white rhino (Ceratotherium simum cottoni) now depends on the cryopreserved cells of 12 individuals. These banked genomes have higher genetic diversity than southern white rhinos (C. s. simum), a sister subspecies that successfully recovered from a severe bottleneck, but the potential impact of genetic load is unknown. We estimated how demographic history has shaped genome-wide genetic load in nine northern and 13 southern white rhinos. The bottleneck left southern white rhinos with more fixed and homozygous deleterious alleles and longer runs of homozygosity, whereas northern white rhinos retained more deleterious alleles masked in heterozygosity. To gauge the impact of genetic load on the fitness of a northern white rhino population restored from biobanked cells, we simulated recovery using fitness of southern white rhinos as a benchmark for a viable population. Unlike traditional restoration, cell-derived founders can be reintroduced in subsequent generations to boost lost genetic diversity and relieve inbreeding. In simulations with repeated reintroduction of founders into a restored population, the fitness cost of genetic load remained lower than that borne by southern white rhinos. Without reintroductions, rapid growth of the restored population (>20-30% per generation) would be needed to maintain comparable fitness. Our results suggest that inbreeding depression from genetic load is not necessarily a barrier to recovery of the northern white rhino and demonstrate how restoration from biobanked cells relieves some constraints of conventional restoration from a limited founder pool. Established conservation methods that protect healthy populations will remain paramount, but emerging technologies hold promise to bolster these tools to combat the extinction crisis.

Temperature-dependent gene regulatory divergence underlies local adaptation with gene flow in the Atlantic silverside.

Gene regulatory divergence is thought to play an important role in adaptation, yet its extent and underlying mechanisms remain largely elusive for local adaptation with gene flow. Local adaptation is widespread in marine species despite generally high connectivity and is often associated with tightly linked genomic architectures, such as chromosomal inversions. To investigate gene regulatory evolution under gene flow and the role of inversions associated with local adaptation to a steep thermal gradient, we generated RNA-seq data from Atlantic silversides (Menidia menidia) from two locally adapted populations and their F1 hybrids, reared under two temperatures. We found substantial divergence in gene expression and thermal plasticity between populations, with up to 31% of genes being differentially expressed. Reduced thermal plasticity, temperature-dependent gene misexpression and the disruption of co-expression networks in hybrids point towards a role of regulatory incompatibilities in local adaptation, particularly under colder temperatures. Chromosomal inversions show an accumulation of regulatory incompatibilities but are not consistently enriched for differentially expressed genes. Together, these results suggest that gene regulation can diverge substantially among populations despite gene flow, partly due to the accumulation of temperature-dependent regulatory incompatibilities within inversions.

Implications of methodologies for integrating empirical kinships into ex situ population management using PMx: A case study of Baer's Pochard (Aythya baeri) in North America.

The application of molecular tools to population management can improve the long-term genetic viability of ex situ populations. In this study, we aimed to understand the implications of integrating empirical kinships into the genetic management of an ex situ population of the endangered waterfowl, Baer's pochard (Aythya baeri), in North America. Single nucleotide polymorphism data were generated for 141 Baer's pochard using double digest restriction site-associated DNA sequencing and empirical kinships were derived and integrated into the population management software PMx. Analyses suggested 37.7% of pairwise relationships previously assumed to be unrelated were first, second, or third-order relatives. We determined that most genetic summary statistics were impacted through the calculation of the population's mean kinship, which increased from MK¯=0.0772 to MK¯=0.2074 after empirical kinships were integrated into our analyses. Our results also revealed the importance of understanding how molecular kinships derived from a particular estimator are scaled, if the scale differs significantly from pedigree-based kinships. We describe the theory behind the genetic metrics impacted and provide general guidance on incorporating empirical kinships into ex situ population management as well as provide suggestions for sampling strategies to minimize the biases inherent in merging two types of kinship estimators.

The contribution of historical processes to contemporary extinction risk in placental mammals.

Species persistence can be influenced by the amount, type, and distribution of diversity across the genome, suggesting a potential relationship between historical demography and resilience. In this study, we surveyed genetic variation across single genomes of 240 mammals that compose the Zoonomia alignment to evaluate how historical effective population size (Ne) affects heterozygosity and deleterious genetic load and how these factors may contribute to extinction risk. We find that species with smaller historical Ne carry a proportionally larger burden of deleterious alleles owing to long-term accumulation and fixation of genetic load and have a higher risk of extinction. This suggests that historical demography can inform contemporary resilience. Models that included genomic data were predictive of species' conservation status, suggesting that, in the absence of adequate census or ecological data, genomic information may provide an initial risk assessment.

Evolutionary constraint and innovation across hundreds of placental mammals.

Zoonomia is the largest comparative genomics resource for mammals produced to date. By aligning genomes for 240 species, we identify bases that, when mutated, are likely to affect fitness and alter disease risk. At least 332 million bases (~10.7%) in the human genome are unusually conserved across species (evolutionarily constrained) relative to neutrally evolving repeats, and 4552 ultraconserved elements are nearly perfectly conserved. Of 101 million significantly constrained single bases, 80% are outside protein-coding exons and half have no functional annotations in the Encyclopedia of DNA Elements (ENCODE) resource. Changes in genes and regulatory elements are associated with exceptional mammalian traits, such as hibernation, that could inform therapeutic development. Earth's vast and imperiled biodiversity offers distinctive power for identifying genetic variants that affect genome function and organismal phenotypes.

Genomes of endangered great hammerhead and shortfin mako sharks reveal historic population declines and high levels of inbreeding in great hammerhead.

Despite increasing threats of extinction to Elasmobranchii (sharks and rays), whole genome-based conservation insights are lacking. Here, we present chromosome-level genome assemblies for the Critically Endangered great hammerhead (Sphyrna mokarran) and the Endangered shortfin mako (Isurus oxyrinchus) sharks, with genetic diversity and historical demographic comparisons to other shark species. The great hammerhead exhibited low genetic variation, with 8.7% of the 2.77 Gbp genome in runs of homozygosity (ROH) > 1 Mbp and 74.4% in ROH >100 kbp. The 4.98 Gbp shortfin mako genome had considerably greater diversity and <1% in ROH > 1 Mbp. Both these sharks experienced precipitous declines in effective population size (Ne) over the last 250 thousand years. While shortfin mako exhibited a large historical Ne that may have enabled the retention of higher genetic variation, the genomic data suggest a possibly more concerning picture for the great hammerhead, and a need for evaluation with additional individuals.

A Chromosome-Length Reference Genome for the Endangered Pacific Pocket Mouse Reveals Recent Inbreeding in a Historically Large Population.

High-quality reference genomes are fundamental tools for understanding population history, and can provide estimates of genetic and demographic parameters relevant to the conservation of biodiversity. The federally endangered Pacific pocket mouse (PPM), which persists in three small, isolated populations in southern California, is a promising model for studying how demographic history shapes genetic diversity, and how diversity in turn may influence extinction risk. To facilitate these studies in PPM, we combined PacBio HiFi long reads with Omni-C and Hi-C data to generate a de novo genome assembly, and annotated the genome using RNAseq. The assembly comprised 28 chromosome-length scaffolds (N50 = 72.6 MB) and the complete mitochondrial genome, and included a long heterochromatic region on chromosome 18 not represented in the previously available short-read assembly. Heterozygosity was highly variable across the genome of the reference individual, with 18% of windows falling in runs of homozygosity (ROH) >1 MB, and nearly 9% in tracts spanning >5 MB. Yet outside of ROH, heterozygosity was relatively high (0.0027), and historical Ne estimates were large. These patterns of genetic variation suggest recent inbreeding in a formerly large population. Currently the most contiguous assembly for a heteromyid rodent, this reference genome provides insight into the past and recent demographic history of the population, and will be a critical tool for management and future studies of outbreeding depression, inbreeding depression, and genetic load.

Comparative linkage mapping uncovers recombination suppression across massive chromosomal inversions associated with local adaptation in Atlantic silversides.

The role of recombination in genome evolution has long been studied in theory, but until recently empirical investigations had been limited to a small number of model species. Here, we compare the recombination landscape and genome collinearity between two populations of the Atlantic silverside (Menidia menidia), a small fish distributed across the steep latitudinal climate gradient of the North American Atlantic coast. We constructed separate linkage maps for locally adapted populations from New York and Georgia and their interpopulation laboratory cross. First, we used one of the linkage maps to improve the current silverside genome assembly by anchoring three large unplaced scaffolds to two chromosomes. Second, we estimated sex-specific recombination rates, finding 2.3-fold higher recombination rates in females than males-one of the most extreme examples of heterochiasmy in a fish. While recombination occurs relatively evenly across female chromosomes, it is restricted to only the terminal ends of male chromosomes. Furthermore, comparisons of female linkage maps revealed suppressed recombination along several massive chromosomal inversions spanning nearly 16% of the genome. These inversions segregate between locally adapted populations and coincide near perfectly with blocks of highly elevated genomic differentiation between wild populations. Finally, we discerned significantly higher recombination rates across chromosomes in the northern population compared to the southern. In addition to providing valuable resources for ongoing evolutionary and comparative genomic studies, our findings represent a striking example of structural variation that impacts recombination between adaptively divergent populations, providing empirical support for theorized genomic mechanisms facilitating adaptation despite gene flow.

A beginner's guide to low-coverage whole genome sequencing for population genomics.

Low-coverage whole genome sequencing (lcWGS) has emerged as a powerful and cost-effective approach for population genomic studies in both model and nonmodel species. However, with read depths too low to confidently call individual genotypes, lcWGS requires specialized analysis tools that explicitly account for genotype uncertainty. A growing number of such tools have become available, but it can be difficult to get an overview of what types of analyses can be performed reliably with lcWGS data, and how the distribution of sequencing effort between the number of samples analysed and per-sample sequencing depths affects inference accuracy. In this introductory guide to lcWGS, we first illustrate how the per-sample cost for lcWGS is now comparable to RAD-seq and Pool-seq in many systems. We then provide an overview of software packages that explicitly account for genotype uncertainty in different types of population genomic inference. Next, we use both simulated and empirical data to assess the accuracy of allele frequency, genetic diversity, and linkage disequilibrium estimation, detection of population structure, and selection scans under different sequencing strategies. Our results show that spreading a given amount of sequencing effort across more samples with lower depth per sample consistently improves the accuracy of most types of inference, with a few notable exceptions. Finally, we assess the potential for using imputation to bolster inference from lcWGS data in nonmodel species, and discuss current limitations and future perspectives for lcWGS-based population genomics research. With this overview, we hope to make lcWGS more approachable and stimulate its broader adoption.

Chromosome-Level Assembly of the Atlantic Silverside Genome Reveals Extreme Levels of Sequence Diversity and Structural Genetic Variation.

The levels and distribution of standing genetic variation in a genome can provide a wealth of insights about the adaptive potential, demographic history, and genome structure of a population or species. As structural variants are increasingly associated with traits important for adaptation and speciation, investigating both sequence and structural variation is essential for wholly tapping this potential. Using a combination of shotgun sequencing, 10x Genomics linked reads and proximity-ligation data (Chicago and Hi-C), we produced and annotated a chromosome-level genome assembly for the Atlantic silverside (Menidia menidia)-an established ecological model for studying the phenotypic effects of natural and artificial selection-and examined patterns of genomic variation across two individuals sampled from different populations with divergent local adaptations. Levels of diversity varied substantially across each chromosome, consistently being highly elevated near the ends (presumably near telomeric regions) and dipping to near zero around putative centromeres. Overall, our estimate of the genome-wide average heterozygosity in the Atlantic silverside is among the highest reported for a fish, or any vertebrate (1.32-1.76% depending on inference method and sample). Furthermore, we also found extreme levels of structural variation, affecting ∼23% of the total genome sequence, including multiple large inversions (> 1 Mb and up to 12.6 Mb) associated with previously identified haploblocks showing strong differentiation between locally adapted populations. These extreme levels of standing genetic variation are likely associated with large effective population sizes and may help explain the remarkable adaptive divergence among populations of the Atlantic silverside.

Footprints of local adaptation span hundreds of linked genes in the Atlantic silverside genome.

The study of local adaptation in the presence of ongoing gene flow is the study of natural selection in action, revealing the functional genetic diversity most relevant to contemporary pressures. In addition to individual genes, genome-wide architecture can itself evolve to enable adaptation. Distributed across a steep thermal gradient along the east coast of North America, Atlantic silversides (Menidia menidia) exhibit an extraordinary degree of local adaptation in a suite of traits, and the capacity for rapid adaptation from standing genetic variation, but we know little about the patterns of genomic variation across the species range that enable this remarkable adaptability. Here, we use low-coverage, whole-transcriptome sequencing of Atlantic silversides sampled along an environmental cline to show marked signatures of divergent selection across a gradient of neutral differentiation. Atlantic silversides sampled across 1371 km of the southern section of its distribution have very low genome-wide differentiation (median F ST = 0.006 across 1.9 million variants), consistent with historical connectivity and observations of recent migrants. Yet almost 14,000 single nucleotide polymorphisms (SNPs) are nearly fixed (F ST > 0.95) for alternate alleles. Highly differentiated SNPs cluster into four tight linkage disequilibrium (LD) blocks that span hundreds of genes and several megabases. Variants in these LD blocks are disproportionately nonsynonymous and concentrated in genes enriched for multiple functions related to known adaptations in silversides, including variation in lipid storage, metabolic rate, and spawning behavior. Elevated levels of absolute divergence and demographic modeling suggest selection maintaining divergence across these blocks under gene flow. These findings represent an extreme case of heterogeneity in levels of differentiation across the genome, and highlight how gene flow shapes genomic architecture in continuous populations. Locally adapted alleles may be common features of populations distributed along environmental gradients, and will likely be key to conserving variation to enable future responses to environmental change.

Contrasting genomic shifts underlie parallel phenotypic evolution in response to fishing.

Humans cause widespread evolutionary change in nature, but we still know little about the genomic basis of rapid adaptation in the Anthropocene. We tracked genomic changes across all protein-coding genes in experimental fish populations that evolved pronounced shifts in growth rates due to size-selective harvest over only four generations. Comparisons of replicate lines show parallel allele frequency shifts that recapitulate responses to size-selection gradients in the wild across hundreds of unlinked variants concentrated in growth-related genes. However, a supercluster of genes also rose rapidly in frequency and dominated the evolutionary dynamic in one replicate line but not in others. Parallel phenotypic changes thus masked highly divergent genomic responses to selection, illustrating how contingent rapid adaptation can be in the face of strong human-induced selection.

Population genetic structure of a common host predicts the spread of white-nose syndrome, an emerging infectious disease in bats.

Landscape complexity influences patterns of animal dispersal, which in turn may affect both gene flow and the spread of pathogens. White-nose syndrome (WNS) is an introduced fungal disease that has spread rapidly throughout eastern North America, causing massive mortality in bat populations. We tested for a relationship between the population genetic structure of the most common host, the little brown myotis (Myotis lucifugus), and the geographic spread of WNS to date by evaluating logistic regression models of WNS risk among hibernating colonies in eastern North America. We hypothesized that risk of WNS to susceptible host colonies should increase with both geographic proximity and genetic similarity, reflecting historical connectivity, to infected colonies. Consistent with this hypothesis, inclusion of genetic distance between infected and susceptible colonies significantly improved models of disease spread, capturing heterogeneity in the spatial expansion of WNS despite low levels of genetic differentiation among eastern populations. Expanding our genetic analysis to the continental range of little brown myotis reveals strongly contrasting patterns of population structure between eastern and western North America. Genetic structure increases markedly moving westward into the northern Great Plains, beyond the current distribution of WNS. In western North America, genetic differentiation of geographically proximate populations often exceeds levels observed across the entire eastern region, suggesting infrequent and/or locally restricted dispersal, and thus relatively limited opportunities for pathogen introduction in western North America. Taken together, our analyses suggest a possibly slower future rate of spread of the WNS pathogen, at least as mediated by little brown myotis.

Risk factors associated with mortality from white-nose syndrome among hibernating bat colonies.

White-nose syndrome (WNS) is a disease responsible for unprecedented mortality in hibernating bats. First observed in a New York cave in 2006, mortality associated with WNS rapidly appeared in hibernacula across the northeastern United States. We used yearly presence-absence data on WNS-related mortality among hibernating bat colonies in the Northeast to determine factors influencing its spread. We evaluated hazard models to test hypotheses about the association between the timing of mortality and colony-level covariates, such as distance from the first WNS-affected site, colony size, species diversity, species composition and type of hibernaculum (cave or mine). Distance to origin and colony size had the greatest effects on WNS hazard over the range of observations; the type of hibernaculum and species composition had weaker effects. The distance effect showed a temporal decrease in magnitude, consistent with the pattern of an expanding epizootic. Large, cave-dwelling bat colonies with high proportions of Myotis lucifugus or other species that seek humid microclimates tended to experience early mortality. Our results suggest that the timing of mortality from WNS is largely dependent on colony location, and large colonies tend to be first in an area to experience high mortality associated with WNS.

Transmission efficiency of two flea species (Oropsylla tuberculata cynomuris and Oropsylla hirsuta) involved in plague epizootics among prairie dogs.

Plague, caused by Yersinia pestis, is an exotic disease in North America circulating predominantly in wild populations of rodents and their fleas. Black-tailed prairie dogs (Cynomys ludovicianus) are highly susceptible to infection, often experiencing mortality of nearly all individuals in a town as a result of plague. The fleas of black-tailed prairie dogs are Oropsylla tuberculata cynomuris and Oropsylla hirsuta. We tested the efficiency of O. tuberculata cynomuris to transmit Y. pestis daily from 24 to 96 h postinfection and compared it to previously collected data for O. hirsuta. We found that O. tuberculata cynomuris has over threefold greater transmission efficiency (0.18 infected fleas transmit Y. pestis at 24 h postinfection) than O. hirsuta (0.05 fleas transmit). Using a simple model of flea-borne transmission, we combine these laboratory measurements with field data on monthly flea loads to compare the seasonal vectorial capacity of these two flea species. Coinciding with seasonal patterns of flea abundance, we find a peak in potential for flea-borne transmission in March, during high O. tuberculata cynomuris abundance, and in September-October when O. hirsuta is common. Our findings may be useful in determining the timing of insecticidal dusting to slow plague transmission in black-tailed prairie dogs.

Early-phase transmission of Yersinia pestis by cat fleas (Ctenocephalides felis) and their potential role as vectors in a plague-endemic region of Uganda.

In recent decades, the majority of human plague cases (caused by Yersinia pestis) have been reported from Africa. In northwest Uganda, which has had recent plague outbreaks, cat fleas (Ctenocephalides felis) have been reported as the most common fleas in the home environment, which is suspected to be a major exposure site for human plague in this country. In the past, C. felis has been viewed as only a nuisance-biting insect because limited laboratory studies suggested it is incapable of transmitting Y. pestis or is an inefficient vector. Our laboratory study shows that C. felis is a competent vector of plague bacteria, but that efficiency is low compared with another flea species collected in the same area: the oriental rat flea, Xenopsylla cheopis. On the other hand, despite its low vector efficiency, C. felis is the most common flea in human habitations in a plague-endemic region of Uganda (Arua and Nebbi Districts), and occasionally infests potential rodent reservoirs of Y. pestis such as the roof rat (Rattus rattus) or the Nile rat (Arvicanthis niloticus). Plague control programs in this region should remain focused on reducing rat flea populations, although our findings imply that cat fleas should not be ignored by these programs as they could play a significant role as secondary vectors.

Oropsylla hirsuta (Siphonaptera: Ceratophyllidae) can support plague epizootics in black-tailed prairie dogs (Cynomys ludovicianus) by early-phase transmission of Yersinia pestis.

Plague, caused by the bacterium Yersinia pestis, often leads to rapid decimation of black-tailed prairie dog colonies. Flea-borne transmission of Y. pestis has been thought to occur primarily via blocked fleas, and therefore studies of vector efficiency have focused on the period when blockage is expected to occur (> or =5 days post-infection [p.i.]). Oropsylla hirsuta, a prairie dog flea, rarely blocks and transmission is inefficient > or =5 days p.i.; thus, this flea has been considered incapable of explaining rapid dissemination of Y. pestis among prairie dogs. By infecting wild-caught fleas with Y. pestis and exposing naïve mice to groups of fleas at 24, 48, 72, and 96 h p.i., we examined the early-phase (1-4 days p.i.) efficiency of O. hirsuta to transmit Y. pestis to hosts and showed that O. hirsuta is a considerably more efficient vector at this largely overlooked stage (5.19% of fleas transmit Y. pestis at 24 h p.i.) than at later stages. Using a model of vectorial capacity, we suggest that this level of transmission can support plague at an enzootic level in a population when flea loads are within the average observed for black-tailed prairie dogs in nature. Shared burrows and sociality of prairie dogs could lead to accumulation of fleas when host population is reduced as a result of the disease, enabling epizootic spread of plague among prairie dogs.

The potential role of swift foxes (Vulpes velox) and their fleas in plague outbreaks in prairie dogs.

Swift foxes (Vulpes velox) have been proposed as potential carriers of fleas infected with the bacterium Yersinia pestis between areas of epizootics in black-tailed prairie dogs (Cynomys ludovicianus). We examined antibody prevalence rates of a population of swift foxes in Colorado, USA, and used polymerase chain reaction (PCR) assays to examine their flea biota for evidence of Y. pestis. Fifteen of 61 (24%) captured foxes were seropositive, and antibody prevalence was spatially correlated with epizootic plague activity in prairie dog colonies in the year of, and previous to, the study. Foxes commonly harbored the flea Pulex simulans, though none of the fleas was positive for Y. pestis.

Early-phase transmission of Yersinia pestis by unblocked Xenopsylla cheopis (Siphonaptera: Pulicidae) is as efficient as transmission by blocked fleas.

For almost a century, the oriental rat flea, Xenopsylla cheopis (Rothschild) (Siphonaptera: Pulicidae), was thought to be the most efficient vector of the plague bacterium Yersinia pestis (Yersin). Approximately 2 wk after consuming an infectious bloodmeal, a blockage often forms in the flea's proventriculus, which forces the flea to increase its biting frequency and consequently increases the likelihood of transmission. However, if fleas remain blocked and continue to feed, they usually die within 5 d of blocking, resulting in a short infectious window. Despite observations of X. cheopis transmitting Y. pestis shortly after pathogen acquisition, early-phase transmission (e.g., transmission 1-4 d postinfection [ p.i.]) by unblocked fleas was viewed as anomalous and thought to occur only by mass action. We used an artificial feeding system to infect colony-reared X. cheopis with a fully virulent strain of Y. pestis, and we evaluated transmission efficiency 1- 4 d p.i. We demonstrate 1) that a single infected and unblocked X. cheopis can infect a susceptible host as early as 1 d p.i., 2) the number of fleas per host required for unblocked fleas to drive a plague epizootic by early-phase transmission is within the flea infestation range observed in nature, and 3) early-phase transmission by unblocked fleas in the current study was at least as efficient as transmission by blocked fleas in a previously published study using the same colony of fleas and same bacterial strain. Furthermore, transmission efficiency seemed to remain constant until block formation, resulting in an infectious period considerably longer than previously thought.