Understanding the anatomical and genetic basis of complex phenotypic traits has long been a challenge for biological research. Domestic dogs offer a compelling model as they demonstrate more phenotypic variation than any other vertebrate species. Dogs have been intensely selected for specific traits and abilities, directly or indirectly, over the past 15,000 years since their initial domestication from the gray wolf. Because olfaction plays a central role in critical tasks, such as the detection of drugs, diseases, and explosives, as well as human rescue, we compared relative olfactory capacity across dog breeds and assessed changes to the canine olfactory system to their direct ancestors, wolves, and coyotes. We conducted a cross-disciplinary survey of olfactory anatomy, olfactory receptor (OR) gene variation, and OR gene expression in domestic dogs. Through comparisons to their closest wild canid relatives, the gray wolf and coyote, we show that domestic dogs might have lost functional OR genes commensurate with a documented reduction in nasal morphology as an outcome of the domestication process prior to breed formation. Critically, within domestic dogs alone, we found no genetic or morphological profile shared among functional or genealogical breed groupings, such as scent hounds, that might indicate evidence of any human-directed selection for enhanced olfaction. Instead, our results suggest that superior scent detection dogs likely owe their success to advantageous behavioral traits and training rather than an "olfactory edge" provided by morphology or genes.

The distribution of fitness effects (DFE) describes the selection coefficients ( s ) of newly arising mutations and fundamentally influences population genetic processes. However, the extent and mechanisms of DFE variation have not been systematically investigated across species with divergent phylogenetic histories and ecological functions. Here, we inferred the DFE in natural populations of eleven animal (sub)species, including humans, mice, fin whales, vaquitas, wolves, collared flycatchers, pied flycatchers, halictid bees, Drosophila, and mosquitoes. We find that the DFE co-varies with phylogeny, where the expected mutation effects are more similar in closely related species ( P a g e l ' s λ = 0.84 , P = 0.01 ). Additionally, mammals have a higher proportion of strongly deleterious mutations (22% to 47% in mammals; 0.0% to 5.4% in insects and birds) and a lower proportion of weakly deleterious mutations than insects and birds. Population size is significantly negatively correlated with the expected impact of new deleterious mutations ( P G L S λ , P = 0.03 ), and the proportion of new beneficial mutations ( R adj 2 = 0.73 , P < 0.001 ). These findings align with Fisher's Geometric Model (FGM), which defines organismal complexity as the number of phenotypes under selection. Consistent with the FGM's predictions, we observe that mutations are more deleterious in complex organisms, while beneficial mutations occur more frequently in smaller populations to compensate for the drift load. Our study demonstrates strong phylogenetic constraints in the evolution of a fundamental population genetics parameter, and proposes that, through mechanisms of global epistasis, long-term population size and organismal complexity drive variation in the DFE across animals.

Similar to other insular birds around the world, the Galapagos rail (Laterallus spilonota Gould, 1841) exhibits reduced flight capacity following its colonization of the archipelago ~1.2 mya. Despite their short evolutionary history, rails have colonized seven different islands spanning the entire width of the archipelago. Galapagos rails were once common on islands with sufficiently high altitudes to support shrubs in humid habitats. After humans introduced goats, this habitat was severely reduced due to overgrazing. Habitat loss devastated some rail populations, with less than 50 individuals surviving, rendering the genetic diversity of Galapagos rail a pressing conservation concern. Additionally, one enigma is the reappearance of rails on the island of Pinta after they were considered extirpated. Our approach was to investigate the evolutionary history and geographic distribution of Galapagos rails as well as examine the genome-wide effects of historical population bottlenecks using 39 whole genomes across different island populations. We recovered an early divergence of rail ancestors leading to the isolated populations on Pinta and a second clade comprising the rest of the islands, historically forming a single landmass. Subsequently, the separation of the landmass ~900 kya may have led to the isolation of the Isabela population with more panmictic populations found on Santa Cruz and Santiago islands. We found that rails genomes contain long runs of homozygosity (>2 Mb) that could be related to the introduction of goats. Finally, our findings show that the modern eradication of goats was critical to avoiding episodes of inbreeding in most populations.

The Yuma myotis bat (Myotis yumanensis) is a small vespertilionid bat and one of 52 species of new world Myotis bats in the subgenus Pizonyx. While M. yumanensis populations currently appear relatively stable, it is one of 12 bat species known or suspected to be susceptible to white-nose syndrome, the fungal disease causing declines in bat populations across North America. Only two of these 12 species have genome resources available, which limits the ability of resource managers to use genomic techniques to track the responses of bat populations to white-nose syndrome generally. Here we present the first de novo genome assembly for Yuma myotis, generated as a part of the California Conservation Genomics Project. The M. yumanensis genome was generated using a combination of PacBio HiFi long reads and Omni-C chromatin-proximity sequencing technology. This high-quality genome is one of the most complete bat assemblies available, with a contig N50 of 28.03 Mb, scaffold N50 of 99.14 Mb, and BUSCO completeness score of 93.7%. The Yuma myotis genome provides a high-quality resource that will aid in comparative genomic and evolutionary studies, as well as inform conservation management related to white-nose syndrome.

Townsend's big-eared bat, Corynorhinus townsendii, is a cave- and mine-roosting species found largely in western North America. Considered a species of conservation concern throughout much of its range, protection efforts would greatly benefit from understanding patterns of population structure, genetic diversity, and local adaptation. To facilitate such research, we present the first de novo genome assembly of C. townsendii as part of the California Conservation Genomics Project (CCGP). Pacific Biosciences HiFi long reads and Omni-C chromatin-proximity sequencing technologies were used to produce a de novo genome assembly, consistent with the standard CCGP reference genome protocol. This assembly comprises 391 scaffolds spanning 2.1 Gb, represented by a scaffold N50 of 174.6 Mb, a contig N50 of 23.4 Mb, and a benchmarking universal single-copy ortholog (BUSCO) completeness score of 96.6%. This high-quality genome will be a key tool for informed conservation and management of this vulnerable species in California and across its range.

Biodiversity monitoring in conservation projects is essential to understand environmental health, complexity, and recovery. However, traditional field surveys can be expensive, time-consuming, biased toward visual detection, and/or only measure a limited set of taxa. Environmental DNA (eDNA) methods provide a new approach to biodiversity monitoring that has the potential to sample a taxonomically broader set of organisms with a similar effort, but many of these approaches are still in the early stages of development and testing. Here, we used multi-locus eDNA metabarcoding to understand how the removal of invasive red swamp crayfish using cypermethrin pesticide impacts local biodiversity of a desert oasis ecosystem, as well as to detect crayfish both directly and indirectly. We tracked crayfish DNA signatures, microbial DNA associated with crayfish, and biodiversity of plant, fungal, animal, and bacterial communities through time. We were unsuccessful in detecting crayfish directly in either control tanks or oases using targeted metabarcoding primers for invertebrates and eukaryotes, similar to previous studies which have shown variable levels of success in detecting crayfish from environmental samples. However, we were successful in discerning a suite of 90 crayfish-associated taxa to serve as candidate bioindicators of invasive presence using 16S and fungal ITS2 metabarcoding. Ranking these 90 taxa by their geographic distribution in eDNA surveys and by evidence of crayfish associations in the literature, we support nine taxa to be high ranking, and suggest they be prioritized in future biomonitoring. Biodiversity analyses from five metabarcode loci including plants, animals, and both prokaryotic and eukaryotic microbes showed that communities differed but that species richness remained relatively similar between oases through time. Our results reveal that, while there are limitations of eDNA approaches to detect crayfish and other invasive species, microbial bioindicators offer a largely untapped biomonitoring opportunity for invasive species management, adding a valuable resource to a conservation manager's toolkit.

Wildlife populations are becoming increasingly fragmented by anthropogenic development. Small and isolated populations often face an elevated risk of extinction, in part due to inbreeding depression. Here, we examine the genomic consequences of urbanization in a caracal (Caracal caracal) population that has become isolated in the Cape Peninsula region of the City of Cape Town, South Africa, and is thought to number ~50 individuals. We document low levels of migration into the population over the past ~75 years, with an estimated rate of 1.3 effective migrants per generation. As a consequence of this isolation and small population size, levels of inbreeding are elevated in the contemporary Cape Peninsula population (mean FROH = 0.20). Inbreeding primarily manifests as long runs of homozygosity >10 Mb, consistent with the effects of isolation due to the rapid recent growth of Cape Town. To explore how reduced migration and elevated inbreeding may impact future population dynamics, we parameterized an eco-evolutionary simulation model. We find that if migration rates do not change in the future, the population is expected to decline, though with a low projected risk of extinction. However, if migration rates decline or anthropogenic mortality rates increase, the potential risk of extinction is greatly elevated. To avert a population decline, we suggest that translocating migrants into the Cape Peninsula to initiate a genetic rescue may be warranted in the near future. Our analysis highlights the utility of genomic datasets coupled with computational simulation models for investigating the influence of gene flow on population viability.

Island ecosystems provide natural laboratories to assess the impacts of isolation on population persistence. However, most studies of persistence have focused on a single species, without comparisons to other organisms they interact with in the ecosystem. The case study of moose and gray wolves on Isle Royale allows for a direct contrast of genetic variation in isolated populations that have experienced dramatically differing population trajectories over the past decade. Whereas the Isle Royale wolf population recently declined nearly to extinction due to severe inbreeding depression, the moose population has thrived and continues to persist, despite having low genetic diversity and being isolated for ∼120 years. Here, we examine the patterns of genomic variation underlying the continued persistence of the Isle Royale moose population. We document high levels of inbreeding in the population, roughly as high as the wolf population at the time of its decline. However, inbreeding in the moose population manifests in the form of intermediate-length runs of homozygosity suggestive of historical inbreeding and purging, contrasting with the long runs of homozygosity observed in the smaller wolf population. Using simulations, we confirm that substantial purging has likely occurred in the moose population. However, we also document notable increases in genetic load, which could eventually threaten population viability over the long term. Overall, our results demonstrate a complex relationship between inbreeding, genetic diversity, and population viability that highlights the use of genomic datasets and computational simulation tools for understanding the factors enabling persistence in isolated populations.

We announce the assembly of the first de novo reference genome for the California Scrub-Jay (Aphelocoma californica). The genus Aphelocoma comprises four currently recognized species including many locally adapted populations across Mesoamerica and North America. Intensive study of Aphelocoma has revealed novel insights into the evolutionary mechanisms driving diversification in natural systems. Additional insights into the evolutionary history of this group will require continued development of high-quality, publicly available genomic resources. We extracted high molecular weight genomic DNA from a female California Scrub-Jay from northern California and generated PacBio HiFi long-read data and Omni-C chromatin conformation capture data. We used these data to generate a de novo partially phased diploid genome assembly, consisting of two pseudo-haplotypes, and scaffolded them using inferred physical proximity information from the Omni-C data. The more complete pseudo-haplotype assembly (arbitrarily designated "Haplotype 1") is 1.35 Gb in total length, highly contiguous (contig N50 = 11.53 Mb), and highly complete (BUSCO completeness score = 97%), with comparable scaffold sizes to chromosome-level avian reference genomes (scaffold N50 = 66.14 Mb). Our California Scrub-Jay assembly is highly syntenic with the New Caledonian Crow reference genome despite ~10 million years of divergence, highlighting the temporal stability of the avian genome. This high-quality reference genome represents a leap forward in publicly available genomic resources for Aphelocoma, and the family Corvidae more broadly. Future work using Aphelocoma as a model for understanding the evolutionary forces generating and maintaining biodiversity across phylogenetic scales can now benefit from a highly contiguous, in-group reference genome.

Ethiopian wolves, a canid species endemic to the Ethiopian Highlands, have been steadily declining in numbers for decades. Currently, out of 35 extant species, it is now one of the world's most endangered canids. Most conservation efforts have focused on preventing disease, monitoring movements and behavior, and assessing the geographic ranges of sub-populations. Here, we add an essential layer by determining the Ethiopian wolf's demographic and evolutionary history using high-coverage (∼40×) whole-genome sequencing from 10 Ethiopian wolves from the Bale Mountains. We observe exceptionally low diversity and enrichment of weakly deleterious variants in the Ethiopian wolves in comparison with two North American gray wolf populations and four dog breeds. These patterns are consequences of long-term small population size, rather than recent inbreeding. We infer the demographic history of the Ethiopian wolf and find it to be concordant with historic records and previous genetic analyses, suggesting Ethiopian wolves experienced a series of both ancient and recent bottlenecks, resulting in a census population size of fewer than 500 individuals and an estimated effective population size of approximately 100 individuals. Additionally, long-term small population size may have limited the accumulation of strongly deleterious recessive mutations. Finally, as the Ethiopian wolves have inhabited high-altitude areas for thousands of years, we searched for evidence of high-altitude adaptation, finding evidence of positive selection at a transcription factor in a hypoxia-response pathway [CREB-binding protein (CREBBP)]. Our findings are pertinent to continuing conservation efforts and understanding how demography influences the persistence of deleterious variation in small populations.

Effective population size estimates are critical information needed for evolutionary predictions and conservation decisions. This is particularly true for species with social factors that restrict access to breeding or experience repeated fluctuations in population size across generations. We investigated the genomic estimates of effective population size along with diversity, subdivision, and inbreeding from 162,109 minimally filtered and 81,595 statistically neutral and unlinked SNPs genotyped in 437 grey wolf samples from North America collected between 1986 and 2021. We found genetic structure across North America, represented by three distinct demographic histories of western, central, and eastern regions of the continent. Further, grey wolves in the northern Rocky Mountains have lower genomic diversity than wolves of the western Great Lakes and have declined over time. Effective population size estimates revealed the historical signatures of continental efforts of predator extermination, despite a quarter century of recovery efforts. We are the first to provide molecular estimates of effective population size across distinct grey wolf populations in North America, which ranged between Ne  ~ 275 and 3050 since early 1980s. We provide data that inform managers regarding the status and importance of effective population size estimates for grey wolf conservation, which are on average 5.2-9.3% of census estimates for this species. We show that while grey wolves fall above minimum effective population sizes needed to avoid extinction due to inbreeding depression in the short term, they are below sizes predicted to be necessary to avoid long-term risk of extinction.

Background The international Dog10K project aims to sequence and analyze several thousand canine genomes. Incorporating 20 × data from 1987 individuals, including 1611 dogs (321 breeds), 309 village dogs, 63 wolves, and four coyotes, we identify genomic variation across the canid family, setting the stage for detailed studies of domestication, behavior, morphology, disease susceptibility, and genome architecture and function. Results We report the analysis of > 48 M single-nucleotide, indel, and structural variants spanning the autosomes, X chromosome, and mitochondria. We discover more than 75% of variation for 239 sampled breeds. Allele sharing analysis indicates that 94.9% of breeds form monophyletic clusters and 25 major clades. German Shepherd Dogs and related breeds show the highest allele sharing with independent breeds from multiple clades. On average, each breed dog differs from the UU_Cfam_GSD_1.0 reference at 26,960 deletions and 14,034 insertions greater than 50 bp, with wolves having 14% more variants. Discovered variants include retrogene insertions from 926 parent genes. To aid functional prioritization, single-nucleotide variants were annotated with SnpEff and Zoonomia phyloP constraint scores. Constrained positions were negatively correlated with allele frequency. Finally, the utility of the Dog10K data as an imputation reference panel is assessed, generating high-confidence calls across varied genotyping platform densities including for breeds not included in the Dog10K collection. Conclusions We have developed a dense dataset of 1987 sequenced canids that reveals patterns of allele sharing, identifies likely functional variants, informs breed structure, and enables accurate imputation. Dog10K data are publicly available.

Aging, often considered a result of random cellular damage, can be accurately estimated using DNA methylation profiles, the foundation of pan-tissue epigenetic clocks. Here, we demonstrate the development of universal pan-mammalian clocks, using 11,754 methylation arrays from our Mammalian Methylation Consortium, which encompass 59 tissue types across 185 mammalian species. These predictive models estimate mammalian tissue age with high accuracy ( r > 0.96). Age deviations correlate with human mortality risk, mouse somatotropic axis mutations and caloric restriction. We identified specific cytosines with methylation levels that change with age across numerous species. These sites, highly enriched in polycomb repressive complex 2-binding locations, are near genes implicated in mammalian development, cancer, obesity and longevity. Our findings offer new evidence suggesting that aging is evolutionarily conserved and intertwined with developmental processes across all mammals.

Role-playing activities in the classroom promote students’ critical thinking, research, and communication skills. We present an activity where students debate a current controversy in conservation. In our case study, students debate the topic of wolf reintroduction in California. Each student is assigned a stakeholder role (e.g., rancher, environmental scientist, hunter, or politician) and a position (either pro or con). First, the whole class participates in a vote on the debate topic so as to register pre-debate sentiment. Then, in the first part of the activity (75 minutes or as homework), students prepare arguments with others representing their stakeholder group by reading the primary and secondary literature and answering guided questions. In the second part of the activity (75 minutes), students participate in a live debate divided into three sections: introductory arguments, questions from the jury, and concluding arguments. The whole class then votes again to decide the winner of the debate, leading to a discussion about which factors do and do not lead to changes in understanding and opinion. The interdisciplinary nature of this activity reinforces student knowledge on ecological networks, keystone species, and natural history, as well as introduces the importance of non-scientific stakeholders in conservation. While this case study focuses on the reintroduction of wolves in California, the activity can be adapted to the reintroduction of controversial species in other regions, or used as a framework for any debatable topic in conservation biology. Primary Image: The reintroduction of the gray wolf Canis lupus is a controversial topic in conservation biology and environmental policy.

The Steller’s jay is a familiar bird of western forests from Alaska south to Nicaragua. Here, we report a draft reference assembly for the species generated from PacBio HiFi long-read and Omni-C chromatin-proximity sequencing data as part of the California Conservation Genomics Project (CCGP). Sequenced reads were assembled into 352 scaffolds totaling 1.16 Gb in length. Assembly metrics indicate a highly contiguous and complete assembly with a contig N50 of 7.8 Mb, scaffold N50 of 25.8 Mb, and BUSCO completeness score of 97.2%. Repetitive elements span 16.6% of the genome including nearly 90% of the W chromosome. Compared with high-quality assemblies from other members of the family Corvidae, the Steller’s jay genome contains a larger proportion of repetitive elements than 4 crow species (Corvus), but a lower proportion of repetitive elements than the California scrub-jay (Aphelocoma californica). This reference genome will serve as an essential resource for future studies on speciation, local adaptation, phylogeography, and conservation genetics in this species of significant biological interest.

Twentieth century industrial whaling pushed several species to the brink of extinction, with fin whales being the most impacted. However, a small, resident population in the Gulf of California was not targeted by whaling. Here, we analyzed 50 whole-genomes from the Eastern North Pacific (ENP) and Gulf of California (GOC) fin whale populations to investigate their demographic history and the genomic effects of natural and human-induced bottlenecks. We show that the two populations diverged ~16,000 years ago, after which the ENP population expanded and then suffered a 99% reduction in effective size during the whaling period. In contrast, the GOC population remained small and isolated, receiving less than one migrant per generation. However, this low level of migration has been crucial for maintaining its viability. Our study exposes the severity of whaling, emphasizes the importance of migration, and demonstrates the use of genome-based analyses and simulations to inform conservation strategies.

The remarkable radiation of South American (SA) canids produced 10 extant species distributed across diverse habitats, including disparate forms such as the short-legged, hypercarnivorous bush dog and the long-legged, largely frugivorous maned wolf. Despite considerable research spanning nearly two centuries, many aspects of their evolutionary history remain unknown. Here, we analyzed 31 whole genomes encompassing all extant SA canid species to assess phylogenetic relationships, interspecific hybridization, historical demography, current genetic diversity, and the molecular bases of adaptations in the bush dog and maned wolf. We found that SA canids originated from a single ancestor that colonized South America 3.9 to 3.5Mya, followed by diversification east of the Andes and then a single colonization event and radiation of Lycalopex species west of the Andes. We detected extensive historical gene flow between recently diverged lineages and observed distinct patterns of genomic diversity and demographic history in SA canids, likely induced by past climatic cycles compounded by human-induced population declines. Genome-wide scans of selection showed that disparate limb proportions in the bush dog and maned wolf may derive from mutations in genes regulating chondrocyte proliferation and enlargement. Further, frugivory in the maned wolf may have been enabled by variants in genes associated with energy intake from short-chain fatty acids. In contrast, unique genetic variants detected in the bush dog may underlie interdigital webbing and dental adaptations for hypercarnivory. Our analyses shed light on the evolution of a unique carnivoran radiation and how it was shaped by South American topography and climate change.

Long-term studies of mountain lions (Puma concolor) in Southern California have documented persistent small population sizes and the lowest genetic variation of any mountain lion population, except for the Federally endangered mountain lion subspecies, the Florida panther (Puma concolor coryi). There is overwhelming molecular evidence supporting inbreeding and low genetic diversity in these Southern California populations but there is a lack of phenotypical evidence of inbreeding depression. The primary goal of this study was to assess male mountain lions, in Southern California, for teratospermia (>60% abnormal sperm production), one of the first signs of inbreeding depression in mountain lions that are associated with decreased reproduction and population decline. From December 2019 to December 2020, we surveyed mountain lions during live captures, after mortality events, and in images collected from camera traps in the following populations: Santa Monica Mountains, Santa Susana Mountains, Santa Ana Mountains, and the Eastern Peninsular Range. Mountain lions were sampled for known physical abnormalities associated with inbreeding depression such as teratospermia, cryptorchidism, and distal tail kinks. For teratospermia, we extracted testes from five males post-mortem to assess sperm morphology. Epididymal sperm evaluations revealed all males were teratospermic. Across all samples, on average, 93% of observed spermatozoa were abnormal. We physically examined 32 mountain lions (males and females) for distal tail kinks, and we observed one individual affected. We examined 15 male mountain lions for cryptorchidism, and we observed one unilaterally cryptorchid male and one male with testes that differed significantly in size, likely reflecting asynchronous migration of the testes during puberty. Further, we identified three other animals in camera-trap images that had distal tail kinks, for a total of four. In conclusion, from December 2019 to December 2020, we identified nine individuals exhibiting physical signs of inbreeding depression. These reproductive and physical signs of inbreeding depression in Southern California mountain lions increase the urgency of conservation efforts in the region.