Advances in genomic studies are revealing that gene flow between species is more frequent than previously understood, although the ways in which hybridization can bias gene flow across species boundaries or the extent to which introgression might be adaptive remain unexplored in most systems. We report on an annotated chromosome-level genome assembly for the Gilbert's skink, Plestiodon gilberti, one of 18 clades of reptiles and amphibians selected for reference genome sequencing in the California Conservation Genomics Project. This assembly was produced using Pacific Biosciences HiFi long reads and Omni-C proximity ligation data. Although members of the Scincidae comprise nearly one-quarter of all lizard species (1785 described species), this de novo assembly represents one of only 10 skink species globally and the first North American skink with a reference genome. The assembly has a total length of ~ 1.57 Gb, a scaffold N50 length of ~ 231.32 Mb, read coverage of ~56X, and BUSCO completeness score of 97.2% based on the Tetrapoda ortholog database. Plestiodon gilberti is a member of the Plestiodon skiltonianus species complex, a group with many of the characteristics of ecological speciation but where ancient hybridization and biased introgression present challenges to retracing the initial patterns of lineage divergence. Combined with dense sampling of resequenced genomes in the California Conservation Genomics Project, including other members of the P. skiltonianus complex, this reference genome will enable future analyses of the links between divergent selection and the genes underlying speciation, as well as the potential for introgression to enable adaptation to new or changing environments.

The California poppy (Eschscholzia californica), a native wildflower of western North America and the state wildflower of California, is characterized by extensive ecological variation and adaptation to diverse climatic conditions. Its broad geographic range and adaptability make it a valuable model for studying how plants respond to changing environmental conditions. Here, we present an updated, near-chromosome-level genome assembly for E. californica, developed as part of the California Conservation Genomics Project. This assembly spans 0.401 Gb and represents an advancement over previous versions, with a scaffold N50 of 66.4 Mb, a contig N50 of 11.8 Mb, and BUSCO completeness of 99.2%, providing near-complete genomic coverage. The enhanced genome assembly described here facilitates precise whole-genome resequencing, providing insights into genetic diversity and gene flow between populations-key factors in understanding the adaptive mechanisms that will support the species' survival in the face of environmental challenges.

The flowering plant genus Aquilegia (columbine) is an important contributor to biodiversity and an example of both biotic and abiotic niche adaptation across much of the Northern Hemisphere, especially in California. Here we report a near-chromosome level draft genome assembly for A. eximia, a California endemic species. A. eximia is a serpentine-soil specialist and is very closely related to 2 columbine species also being studied for the California Conservation Genomics Project (CCGP), A. formosa (widespread) and A. pubescens (high alpine). Utilizing high throughput, long reads (PacBio) and chromatin capture (Omni-C), the A. eximia genome makes marked contiguity improvements compared to the existing reference genome for another North American columbine, A. coerulea "Goldsmith." The A. eximia genome will also be more useful for aligning whole genome resequencing data from California columbines than the genomes for more distantly related columbine species, the Asian A. oxysepala var. kansuensis and the European A. vulgaris. Notably, we found evidence that A. eximia, A. coerulea "Goldsmith," and A. vulgaris all share the same overall genome structure and differ from A. oxysepala var. kansuensis by the same reciprocal translocation. The A. eximia reference genome will be a valuable tool for identifying patterns of plant biodiversity across California for the CCGP, as well as for future population genomic and trait mapping studies.

Colusa grass, Neostapfia colusana, is a listed California endangered plant endemic to the vernal pools of California. Vernal pool habitat is highly degraded and threatened by further anthropological development, with only 10% of its historical range remaining. With only 42 confirmed extant populations, it is a major conservation concern to understand patterns of genomic diversity. Here we report the first complete genome assembly of Colusa grass. The assembly includes two haplotypes: haplotype one spans 2.13 Gb with contig N50 of 10.62 Mb, scaffold N50 of 112.31 Mb, and BUSCO completeness of 98.1%. Haplotype two spans 2.04 Gb with contig N50 of 10.05 Mb and scaffold N50 of 138.31 Mb, with a BUSCO completeness of 97.6%. This genome assembly will allow for in-depth analysis of genomic variation and gene flow in populations of this threatened grass and will be a major asset to studies supporting its conservation. This genome was assembled as part of the California Conservation Genomics Project, which contributes to a collection of resources and tools to support state-wide conservation efforts.

We report on an annotated chromosome-level genome assembly for the long-nosed leopard lizard, Gambelia wislizenii, as part of the California Conservation Genomics Project (CCGP). All 17 species of reptiles, including two turtles, seven lizards, and seven snakes targeted for reference genome sequencing by the CCGP are now complete and posted on NCBI, and this article is the third of seven CCGP lizard release papers to be published. It is also the first species of the family Crotaphytidae to have a released reference genome. Following the CCGP pipeline, the G. wislizenii genome was produced using Pacific Biosciences HiFi long reads and Omni-C proximity ligation data. The de novo assembly includes 69 scaffolds and has a total length of ~ 2.47 Gb, a scaffold N50 length of 380.1 Mb, and a BUSCO completeness score of 97.4% based on the tetrapod gene set. We improved the annotation of the genome using transcriptome sequencing (seven tissue types), identifying 23,279 genes, with BUSCO completeness of 98.9%. This reference genome, when combined with CCGP's on-going state-wide resequencing efforts for the three species of Gambelia in California, including the federally endangered blunt-nosed leopard lizard (Gambelia sila), and Cope's leopard lizard (Gambelia copei), will be a powerful tool enabling researchers to characterize hybridization dynamics between Gambelia species, document the remaining diversity within G. sila, and explore the genetic underpinnings of key traits that vary between the three Gambelia species, such as territoriality, sexual size dimorphism, presence versus absence of male breeding coloration, and skull morphologies.

Invasive species present one of the most challenging threats to native biodiversity, particularly when they hybridize with imperiled native taxa. In California, hybridization between the endangered California tiger salamander ("CTS," Ambystoma californiense) and the invasive barred tiger salamander ("BTS," Ambystoma mavortium) is one of the best understood examples of this management challenge. Reclusive life history and cryptic hybridization, often on private land, render eradication programs difficult or impossible. This study evaluates hydroperiod management as a tool to conserve and maintain native CTS populations threatened by hybridization. We adapt a recent, empirically informed Bayesian integral projection model (IPM) for CTS to incorporate new results that link genotype and ecology to fitness, and use this individual-based model to evaluate alternative management scenarios. We found overwhelming support for the importance of hydrology in both native and hybrid populations, where a 10-day increase in hydroperiod can increase population growth rate ( λ $$ \uplambda $$ ) 17% and triple the carrying-capacity (K). We assess hydroperiod management as a strategy to control and contain hybrid introgression, and suggest a three-pronged strategy. First, for native populations not at risk of hybridization, hydroperiod should be increased to >120 days to support robust populations. Second, within the geographic hybrid zone, hydroperiod should be reduced to limit hybrid populations, maintain vernal pool function, and improve the efficiency of adult hybrid removal. Finally, our models indicate that managers should combine hydroperiod management with rapid field-based genotyping and hybrid removal, focusing on ponds where hybrids are rare, typically at the leading edge of the hybrid swarm. Efforts should also prioritize high-intensity surveys and early removal as opposed to long-duration (10+ years), lower effort surveys. This study demonstrates the value of integrating demographic, genetic, and ecological information to evaluate strategies for endangered species management, and may serve as modeling framework for a wide variety of imperiled species.

The spread and impact of wildlife pathogens is often seasonal, and identifying the seasons of high impact is critical to biodiversity and public health management. Here, we report new evidence that winter host dormancy, a period generally neglected in terms of pathogen seasonal dynamics, promotes the spread of infection from the globally threatening amphibian fungal pathogen, Batrachochytrium dendrobatidis (Bd) . Bd surveillance of Yosemite toads ( Anaxyrus canorus ) during their first year of life showed that: (a) detectable Bd infections were nearly absent in the tadpole stage and immediately after metamorphosis, (b) Bd prevalence and intensity gradually increased during the first two months of post-metamorphic growth, and (c) Bd prevalence and intensity increased four-fold in toads during the first winter dormancy. High prevalence and intensity of Bd infections after winter dormancy was observed in yearling toads across two consecutive years and was much higher than that observed in aquatically breeding adults during the same timeframe. To our knowledge, this is the first evidence from wild amphibians that Bd proliferates during terrestrial winter dormancy. This discovery calls for a reconsideration of the seasons enabling the proliferation and persistence of Bd and identifies recently metamorphosed hosts as overwinter Bd reservoirs. More generally, the study underscores the importance of host dormancy in pathogen persistence and seasonal infection spread. ### Competing Interest Statement The authors have declared no competing interest. La Kretz Center for California Conservation Science Yosemite Conservancy

The winter ant, Prenolepis imparis, is one of the most common, widespread, and conspicuous ant species in North America. P. imparis is well adapted to cold climates, and consequently, is often noted as the only active ant species during colder months. This specialized life history makes P. imparis a useful model organism for exploring thermal physiology and understanding the potential impacts of a warming climate on insects. Phylogeographic studies have revealed deeply divergent lineages across North America, as well as a single collection of an apparent social parasite in California. In light of its distinctive cold adaptation and recently discovered geographic diversity, a better understanding of the underlying genetic patterns of the winter ant is valuable to future conservation efforts for this species. Here, we present a high-quality genome assembly of P. imparis from Santa Clara County, California. This genome assembly consists of 787 scaffolds spanning 327.3 Mb, with contig N50 of 901.9 kb, scaffold N50 of 18.7 Mb, and BUSCO completeness of 96.5%. This genome assembly provides an essential foundation for future studies of the winter ant and will be particularly useful for understanding the genetic basis of thermal adaptation, cold resistance, chemical ecology, and the resilience of organisms in response to a changing climate.

Salamanders of the genus Lyciasalamandra are represented by as many as 20 narrow-range endemic taxa inhabiting the Mediterranean coast of Turkey and a handful of Aegean Islands. Despite recent molecular phylogenetic studies, the genus is rife with uncertainty about the number of contained species and their phylogenetic relationships, both of which can interfere with needed conservation actions. To test species limits and infer interrelationships we generated as many as 113,176 RAD loci containing 229,427 single nucleotide polymorphisms (SNPs), for 110 specimens of Lyciasalamandra representing 19 of the 20 described taxa. Through a conservative species delimitation approach, we found support for eight species in the genus which broadly agree with currently described species-level diversity. We then use multiple coalescent-based species tree methods to resolve relationships in this relatively old, synchronous species radiation. We recommend synonymization of the largely over-split subspecific taxa, and the elevation of L. luschani finikensis to full species status as L. finikensis. Our hope is that this revised taxonomic framework provides a stable foundation for conservation management in these fragile, microendemic taxa.

The black surfperch, Embiotoca jacksoni , exhibits limited dispersal due to its lack of a pelagic larval stage and offers a unique model for studying local adaptation and potential cryptic speciation in marine species. This study employs medium-coverage whole genome resequencing to explore population structure, local adaptation, and genetic divergence across a latitudinal gradient from central California to Baja California, Mexico, including offshore islands. We identify strong genetic differentiation between five distinct groups: a coastal group and four island groups (Santa Catalina Island, San Clemente Island, Isla Guadalupe, and Isla San Jerónimo), from Principal Components Analysis (PCA), Fst estimation, ancestry (sNMF), and phylogenetic analyses. For coastal populations, genetic structure correlates with geographic distance, following isolation-by-distance (IBD) expectations. Further, we identify numerous outlier loci associated with adaptive traits, particularly genes linked to reproductive isolation, such as Spermine oxidase, Izumo sperm-egg fusion protein 1, and SPAG1, which are involved in fertilisation success. These loci suggest that reproductive barriers are contributing to divergence among genetic groups. The presence of signatures of both genetic drift and selective pressures, particularly in genes governing fertilisation, indicates that these distinct populations may represent incipient or cryptic species. Our findings highlight the role of low dispersal in fostering local adaptation and speciation in marine systems and underscore the potential for rapid evolutionary responses to environmental gradients. This research provides insights into the complex processes of speciation in marine vertebrates, particularly those with limited gene flow, and offers a baseline for monitoring genetic shifts in response to climate change.

Genetic rescue, or the translocation of individuals among populations to augment gene flow, can help ameliorate inbreeding depression and loss of adaptive potential in small and isolated populations. Genetic rescue is currently under consideration for an endangered butterfly in Canada, the Half-moon Hairstreak (Satyrium semiluna). A small, unique population persists in Waterton Lakes National Park, Alberta, isolated from other populations by more than 400 km. However, whether genetic rescue would actually be helpful has not been evaluated. Here, we generate the first chromosome-level genome assembly and whole-genome resequence data for the species. We find that the Alberta population maintains extremely low genetic diversity and is genetically very divergent from the nearest populations in British Columbia and Montana. Runs of homozygosity suggest this is due to a long history of inbreeding, and coalescent analyses show that the population has been small and isolated, yet stable, for up to 40k years. When a population like this maintains its viability despite inbreeding and low genetic diversity, it has likely undergone purging of deleterious recessive alleles and could be threatened by the reintroduction of such alleles via genetic rescue. Ecological niche modelling indicates that the Alberta population also exhibits environmental associations that are atypical of the species. Together, these evolutionary and ecological divergences suggest that population crosses may result in outbreeding depression. We therefore infer that genetic rescue has a relatively unique potential to be harmful rather than helpful for this population at present. However, because of its reduced adaptive potential, the Alberta population may still benefit from future genetic rescue as climate and habitat conditions change. Proactive experimental population crosses should therefore be completed to assess reproductive compatibility and progeny fitness.

The Gophersnake, Pituophis catenifer, is a habitat generalist that ranges throughout the western half of the United States and southward into México. Five of the six subspecies, P. catenifer affinis (Sonoran Gophersnake), P. catenifer annectens (San Diego Gophersnake), P. catenifer catenifer (Pacific Gophersnake), P. catenifer deserticola (Great Basin Gophersanke), and P. catenifer pumilus (Santa Cruz Island Gophersnake), occur in California and span virtually all the state's diverse terrestrial habitats. These subspecies are ecologically and morphologically distinct from one another, although existing genetic data indicate there is genetic admixture across some of their contact zones. Given that these subspecies occur in such different environments they will not all respond to climate change and anthropogenic stressors equally. Here, we report a new, chromosome-level assembly of P. catenifer as part of the California Conservation Genomics Project (CCGP). Consistent with the reference genome strategy of the CCGP, we used Pacific Biosciences HiFi long reads and Hi-C chromatin-proximity sequencing technology to produce a de novo assembled genome. The assembly comprises 426 scaffolds covering 1,804,944,895 bp, has a contig N50 of 37.5 Mb, a scaffold N50 of 161 Mb, and BUSCO completeness score of 95.3%. This genome will be a foundational resource for future studies on the conservation, adaptation, biogeography, and systematics of P. catenifer.

We describe a highly isolated population of hairstreak butterfly from Waterton Lakes National Park, Alberta, Canada, as a new species, Satyrium curiosolus sp. nov., previously recognized as Satyrium semiluna (Half-moon Hairstreak). We propose “Curiously Isolated Hairstreak” as the common name due to its disjunct and unusual distribution. Previous whole-genome analyses revealed S. curiosolus has extremely low genomic diversity and is highly divergent from the nearest S. semiluna populations in British Columbia and Montana, more than 400 km distant. Further analysis suggested prolonged inbreeding and isolation for up to ~40,000 years BP. Ecological niche modeling indicated that S. curiosolus occupies environmental conditions that are distinct from S. semiluna, suggesting niche divergence driven by long-term geographical and ecological separation. While host plant and ant associations have not been definitively resolved, they likely differ between S. curiosolus and S. semiluna. As part of this description, we provide whole-genome consensus sequences for each individual of the type series and identify 21,985 single nucleotide polymorphisms (SNPs) that are divergently fixed between S. curiosolus and S. semiluna, including 117 unlinked SNPs distributed across the genome as putative diagnostic markers. Previously listed as Endangered in Canada as the Waterton population of S. semiluna, S. curiosolus should retain this conservation status due to its extreme isolation, small population size, and flatlined genomic diversity. We propose species recognition as a testable hypothesis under the General Lineage Concept and recommend further research to explore the taxonomy, ecological relationships, and conservation of the greater species complex, including S. curiosolus, S. semiluna, and S. fuliginosa.

1. Climate change and infectious disease jointly impact species worldwide. In addition to causing conspicuous mortality events, these threats produce a range of non-lethal effects that are often overlooked, yet can affect individual survival and fecundity, and ultimately, population and species viability. 2.We develop an energetic framework that structures the study of non-lethal effects of climate change and infectious disease and their downstream demographic consequences. The framework identifies pathways by which climate change and infectious disease affect the acquisition, storage and mobilisation of energy required for organismal survival and reproduction. 3.The joint energetic effects of climate change and infectious disease, while often non-lethal, can reduce fitness by increasing energetic demands, exacerbating energetic trade-offs and accelerating physiological ageing. 4. Considering the energetic mechanisms underlying non-lethal effects can explain when and why recurrent and/or chronic events associated with climate change and infectious disease can be important limiting forces for populations and species.Read the free Plain Language Summary for this article on the Journal blog.

PremiseAzolla is a genus of floating ferns that has closely evolved with a vertically transmitted obligate cyanobacterium endosymbiont-Anabaena azollae-that fixes nitrogen. There are also other lesser-known Azolla symbionts whose role and mode of transmission are unknown.MethodsWe sequenced 112 Azolla specimens collected across the state of California and characterized their metagenomes to identify the common bacterial endosymbionts and assess their patterns of interaction.ResultsFour genera were found across all samples, establishing that multiple Azolla endosymbionts were consistently present. We found varying degrees of cophylogenetic signal across these taxa as well as varying degrees of isolation by distance and of pseudogenation, which demonstrates that multiple processes underlie how this endosymbiotic community is constituted. We also characterized the entire Azolla leaf pocket microbiome.ConclusionsThese results show that the Azolla symbiotic community is complex and features members at potentially different stages of symbiosis evolution, further supporting the utility of the Azolla microcosm as a system for studying the evolution of symbioses.

Few genomic resources currently exist for the American endemic family Cactaceae, a group of around 1850 species, which are world renowned for their amazing growth forms and succulent habits. These icons of arid landscapes across the Americas are threatened in many parts of their range, including in parts of California, and developing more comprehensive genomic data will aid efforts to better understand and preserve these plants. We sequenced and assembled the genome of the beavertail cactus, Opuntia basilaris, which is represented by three varieties in California, one of which is threatened, and another endangered. The genome assembly has a BUSCO complete score of 98.1%, and a total scaffold length of 980 Mb, with a scaffold N50 length of 83 Mb. The genome size of diploid O. basilaris is markedly smaller than other diploid members of Cactaceae that have been assembled to date. This is the first nuclear genome sequenced in subfamily Opuntioideae and the most complete nuclear genome for Cactaceae to date and will lay the foundation for future genomic work across the biologically and taxonomically complicated prickly pear cacti.

Azolla is a genus of freshwater ferns that is economically important as a nitrogen-fixing biofertilizer, biofuel, bioremediator, and for potential carbon sequestration, but also contains weedy invasive species. In California, only 2 species are currently recognized but the actual diversity may include up to 6 species, with the discrepancy being due to the difficulty in identifying taxa, hybridization, and the introduction of non-native species. Here, we report a new haplotype-resolved, chromosome-level assembly and annotation of Azolla caroliniana as part of the California Conservation Genomics Project (CCGP), using a combination of PacBio HiFi and Omni-C sequencing technologies. The assembly is 521 Mb in length, with a contig N50 of 1.6 Mb, and is scaffolded into 22 pseudochromosomes. A total of 21,848 protein-coding genes was predicted with a Benchmarking Universal Single-Copy Orthologs (BUSCO) completeness score of 89.88%. In combination with the previously published Azola filiculoides genome, this A. caroliniana genome will be a powerful tool for understanding the population genetics and taxonomy of one of the most cryptic, economically important, and poorly circumscribed fern taxa, and for facilitating land plant genomics more broadly.

Carpenter ants in the genus Camponotus are large, conspicuous ants that are abundant and ecologically influential in many terrestrial ecosystems. The bicolored carpenter ant, Camponotus vicinus Mayr, is distributed across a wide range of elevations and latitudes in western North America, where it is a prominent scavenger and predator. Here, we present a high-quality genome assembly of C. vicinus from a sample collected in Sonoma County, California, near the type locality of the species. This genome assembly consists of 38 scaffolds spanning 302.74 Mb, with contig N50 of 15.9 Mb, scaffold N50 of 19.9 Mb, and BUSCO completeness of 99.2%. This genome sequence will be a valuable resource for exploring the evolutionary ecology of C. vicinus and carpenter ants generally. It also provides an important tool for clarifying cryptic diversity within the C. vicinus species complex, a genetically diverse set of populations, some of which are quite localized and of conservation interest.

Established invasive species represent one of the most harmful and challenging threats to native biodiversity, necessitating methods for Early Detection and Rapid Response. Cryptic invasions are particularly challenging and often require expensive and time-consuming molecular surveys which limits their usefulness for management. We present a novel application of the Fluidigm SNP-Type Assay to identify rare non-native alleles that significantly reduces the cost and time to generate diagnostic results. We demonstrate the efficacy of this method using experimental Fluidigm pools (99% accuracy) and sequence data (96% accuracy). We apply our novel methodology to an endangered population of California tiger salamanders in Sonoma County where two individual non-native tiger salamander hybrids have previously been detected since 2008. We screened 5805 larvae in 387 sample-pools containing 15 larvae each. We did not detect any non-native hybrids in the population, a result that was verified with sequence data, though we strongly recommend additional years of sampling to confirm hybrid absence. Our success with a challenging, large-genome amphibian suggests this method may be applied to any system, and would be particularly useful when it is necessary for conservation practitioners to rapidly identify rare taxa or genes of interest.

We describe a highly contiguous and complete diploid genome assembly for the Chryxus Arctic, Oeneis chryxus (E. Doubleday, [1849]), a butterfly species complex spanning much of northern and western North America. One subspecies, the Ivallda Arctic (O. c. ivallda), is endemic to California's Sierra Nevada and of particular biogeographic interest and conservation concern. Extreme alpine habitats occupied by this subspecies include the summit of Mt. Whitney, California, representing the highest elevation butterfly population in North America. The assembly presented here consists of two haplotypes, 738.92 and 770.85 Mb in length, with contig N50 values of 10.49 and 10.13 Mb, scaffold N50 values of 25.35 and 25.69 Mb, scaffold L50 values of 13 and 14, and BUSCO completeness scores of 96.5 and 98.3%, respectively. More than 97% of the assembly is organized into 29 scaffolds, which likely represent whole chromosomes. This assembly is the first major genomic resource for Oeneis, providing a foundational reference for future genomic studies on the taxonomy, evolutionary history, and conservation of the genus. As part of the California Conservation Genomics Project, we will use this assembly in conjunction with short-read resequencing to resolve patterns of evolutionary differentiation, adaptive genomic variation, and gene flow among remaining O. c. ivallda populations. These data can and will be used to inform the subspecies' conservation as warming climatic conditions continue to lead to the loss and fragmentation of alpine habitats. We also provide genome assemblies for the O. chryxus mitochondrion and a Wolbachia endosymbiont.