Phylogenies often contain both well-supported and poorly supported nodes. Determining how much additional data might be required to eventually recover most or all nodes with high support is an important pragmatic goal, and simulations have been used to examine this question. Most simulations have been based on few empirical loci, and suggest that well supported phylogenies can be determined with a very modest amount of data. Here we report the results of an empirical phylogenetic analysis of all 10 genera and 25 of 48 species of the new world pond turtles (family Emydidae) based on one mitochondrial (1070 base pairs) and seven nuclear loci (5961 base pairs), and a more biologically realistic simulation analysis incorporating variation among gene trees, aimed at determining how much more data might be necessary to recover weakly-supported nodes with strong support.

The western pond turtle (WPT), recently separated into two paripatrically distributed species (Emys pallida and Emys marmorata), is experiencing significant reductions in its range and population size. In addition to habitat loss, two potential causes of decline are female-biased road mortality and high juvenile mortality from non-native predatory bullfrogs (Rana catesbeiana). However, quantitative analyses of these threats have never been conducted for either species of WPT. We used a combination of historical museum samples and published and unpublished field studies shared with us through personal communications with WPT field researchers (B. Shaffer, P. Scott, R. Fisher, C. Brown, R. Dagit, L. Patterson, T. Engstrom, 2019, personal communications) to quantify the effect of roads and bullfrogs on WPT populations along the west coast of the United States. Both species of WPT shift toward increasingly male biased museum collections over the last century, a trend consistent with increasing, female-biased road mortality. Recent WPT population studies revealed that road density and proximity were significantly associated with increasingly male-biased sex ratios, further suggesting female-biased road mortality. The mean body size of museum collections of E. marmorata, but not E. pallida, has increased over the last 100 years, consistent with reduced recruitment and aging populations that could be driven by invasive predators. Contemporary WPT population sites that co-occur with bullfrogs had significantly greater average body sizes than population sites without bullfrogs, suggesting strong bullfrog predation on small WPT hatchlings and juveniles. Overall, our findings indicate that both species of WPT face demographic challenges which would have been difficult to document without the use of both historical data from natural history collections and contemporary demographic field data. Although correlational, our analyses suggest that female-biased road mortality and predation on small turtles by non-native bullfrogs are occurring, and that conservation strategies reducing both may be important for WPT recovery.

Arctostaphylos (Ericaceae) species, commonly known as manzanitas, are an invaluable fire-adapted chaparral clade in the California Floristic Province (CFP), a world biodiversity hotspot on the west coast of North America. This diverse woody genus includes many rare and/or endangered taxa, and the genus plays essential ecological roles in native ecosystems. Despite their importance in conservation management, and the many ecological and evolutionary studies that have focused on manzanitas, virtually no research has been conducted on the genomics of any manzanita species. Here, we report the first genome assembly of a manzanita species, the widespread Arctostaphylos glauca. Consistent with the genomics strategy of the California Conservation Genomics project, we used Pacific Biosciences HiFi long reads and Hi-C chromatin-proximity sequencing technology to produce a de novo assembled genome. The assembly comprises a total of 271 scaffolds spanning 547Mb, close to the genome size estimated by flow cytometry. This assembly, with a scaffold N50 of 31Mb and BUSCO complete score of 98.2%, will be used as a reference genome for understanding the genetic diversity and the basis of adaptations of both common and rare and endangered manzanita species.

Incorporating measures of taxonomic diversity into research and management plans has long been a tenet of conservation science. Increasingly, active conservation programs are turning toward multispecies landscape and regional conservation actions, and away from single species approaches. This is both a reflection of changing trends in conservation science and advances in foundational technologies, including genomics and geospatial science. Multispecies approaches may provide more fundamental insights into evolutionary processes and equip managers with a more holistic understanding of the landscapes under their jurisdiction. Central to this approach are data generation and analyses which embrace and reflect a broad range of taxonomic diversity. Here, we examine the family-level phylogenetic breadth of the California Conservation Genomics Project (CCGP) based on family-level phylogenetic diversity (PD), family-level phylogenetic distinctness, and family richness. We place this in the context of the diversity present in California and compare it to the 35-plus years of genetic research compiled in the CaliPopGen Database. We found that the family-level PD in the CCGP reflected that of California very well, slightly overrepresenting chordates and underrepresenting arthropods, and that 42% of CCGP PD represented new contributions to genetic data for the state. In one focused effort, the CCGP was able to achieve roughly half the family-level PD studied over the last several decades. To maximize studied PD, future work should focus on arthropods, a conclusion that likely reflects the overall lack of attention to this hyperdiverse clade.

The northwestern pond turtle, Actinemys marmorata, and its recently recognized sister species, the southwestern pond turtle, A. pallida, are the sole aquatic testudines occurring over most of western North America and the only living representatives of the genus Actinemys. Although it historically ranged from Washington state through central California, USA, populations of the northwestern pond turtle have been in decline for decades and the species is afforded state-level protection across its range; it is currently being considered for protection under the US Endangered Species Act. Here, we report a new, chromosome-level assembly of A. marmorata 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 198 scaffolds spanning 2,319,339,408 base pairs, has a contig N50 of 75 Mb, a scaffold N50 of 146Mb, and BUSCO complete score of 96.7%, making it the most complete testudine assembly of the 24 species from 13 families that are currently available. In combination with the A. pallida reference genome that is currently under construction through the CCGP, the A. marmorata genome will be a powerful tool for documenting landscape genomic diversity, the basis of adaptations to salt tolerance and thermal capacity, and hybridization dynamics between these recently diverged species.

The Virginia rail, Rallus limicola, is a member of the family Rallidae, which also includes many other species of secretive and poorly studied wetland birds. It is recognized as a single species throughout its broad distribution in North America where it is exploited as a game bird, often with generous harvest limits, despite a lack of systematic population surveys and evidence of declines in many areas due to wetland loss and degradation. To help advance understanding of the phylogeography, biology, and ecology of this elusive species, we report the first reference genome assembly for the Virginia rail, produced as part of the California Conservation Genomics Project (CCGP). We produced a de novo genome assembly using Pacific Biosciences HiFi long reads and Hi-C chromatin-proximity sequencing technology with an estimated sequencing error rate of 0.191%. The assembly consists of 1,102 scaffolds spanning 1.39 Gb, with a contig N50 of 11.0 Mb, scaffold N50 of 25.3 Mb, largest contig of 45 Mb, and largest scaffold of 128.4 Mb. It has a high BUSCO completeness score of 96.9% and represents the first genome assembly available for the genus Rallus. This genome assembly will help resolve questions about the complex evolutionary history of rails and evaluate the potential of rails for adaptive evolution in the face of growing threats from climate change and habitat loss and fragmentation. It will also provide a valuable resource for rail conservation efforts by quantifying Virginia rail vagility, population connectivity, and effective population sizes.

We describe the genome of the western painted turtle, Chrysemys picta bellii, one of the most widespread, abundant, and well-studied turtles. We place the genome into a comparative evolutionary context, and focus on genomic features associated with tooth loss, immune function, longevity, sex differentiation and determination, and the species' physiological capacities to withstand extreme anoxia and tissue freezing.

Delimiting the boundaries of species involved in radiations is critical to understanding the tempo and mode of lineage formation. Single locus gene trees may or may not reflect the underlying pattern of population divergence and lineage formation, yet they constitute the vast majority of the empirical data in species radiations. In this study we make use of an expressed sequence tag (EST) database to perform nuclear (nDNA) and mitochondrial (mtDNA) genealogical tests of species boundaries in Ambystoma ordinarium, a member of an adaptive radiation of metamorphic and paedomorphic salamanders (the Ambystoma tigrinum complex) that have diversified across terrestrial and aquatic environments. Gene tree comparisons demonstrate extensive nonmonophyly in the mtDNA genealogy of A. ordinarium, while seven of eight independent nuclear loci resolve the species as monophyletic or nearly so, and diagnose it as a well-resolved genealogical species. A differential introgression hypothesis is supported by the observation that western A. ordinarium localities contain mtDNA haplotypes that are identical or minimally diverged from haplotypes sampled from a nearby paedomorphic species, Ambystoma dumerilii, while most nDNA trees place these species in distant phylogenetic positions. These results provide a strong example of how historical introgression can lead to radical differences between gene trees and species histories, even among currently allopatric species with divergent life history adaptations and morphologies. They also demonstrate how EST-based nuclear resources can be used to more fully resolve the phylogenetic history of species radiations.

Cryptic species are a challenge for systematics, but their elucidation also may leave critical information gaps about the distribution, conservation status, and behavior of affected species. We use the leopard frogs of the eastern U.S. as a case study of this issue. We refined the known range of the recently described Rana kauffeldi, the Atlantic Coast Leopard Frog, relative to the region's two other leopard frog species, conducted assessments of conservation status, and improved methods for separating the three species using morphological field characters. We conducted over 2,000 call and visual surveys and took photographs of and tissue samples from hundreds of frogs. Genetic analysis supported a three-species taxonomy and provided determinations for 220 individual photographed frogs. Rana kauffeldi was confirmed in eight U.S. states, from North Carolina to southern Connecticut, hewing closely to the Atlantic Coastal Plain. It can be reliably differentiated in life from R. pipiens, and from R. sphenocephala 90% of the time, based on such characters as the femoral reticulum patterning, dorsal spot size and number, and presence of a snout spot. However, the only diagnostic character separating R. kauffeldi from R. sphenocephala remains the breeding call described in 2014. Based on our field study, museum specimens, and prior survey data, we suggest that R. kauffeldi has declined substantially in the northern part of its range, but is more secure in the core of its range. We also report, for the first time, apparent extirpations of R. pipiens from the southeastern portion of its range, previously overlooked because of confusion with R. kauffeldi. We conclude with a generalized ecological research agenda for cryptic species. For R. kauffeldi, needs include descriptions of earlier life stages, studies of niche partitioning with sympatric congeners and the potential for hybridization, and identification of conservation actions to prevent further declines.

Pricklebacks (Family Stichaeidae) are generally cold-temperate fishes most commonly found in the north Pacific. As part of the California Conservation Genomics Project (CCGP), we sequenced the genome of the Monkeyface Prickleback, Cebidichthys violaceus, to establish a genomic model for understanding phylogeographic patterns of marine organisms in California. These patterns, in turn, may inform the design of marine protected areas using dispersal models based on forthcoming population genomic data. The genome of C. violaceus is typical of many marine fishes at less than 1 Gb (genome size = 575.6 Mb), and our assembly is near-chromosome level (contig N50 = 1 Mb, scaffold N50 = 16.4 Mb, BUSCO completeness = 93.2%). Within the context of the CCGP, the genome will be used as a reference for future whole genome resequencing projects, enhancing our knowledge of the population structure of the species and more generally, the efficacy of marine protected areas as a primary conservation tool across California's marine ecosystems.

Sea turtles represent an ancient lineage of marine vertebrates that evolved from terrestrial ancestors over 100 Mya. The genomic basis of the unique physiological and ecological traits enabling these species to thrive in diverse marine habitats remains largely unknown. Additionally, many populations have drastically declined due to anthropogenic activities over the past two centuries, and their recovery is a high global conservation priority. We generated and analyzed high-quality reference genomes for the leatherback (Dermochelys coriacea) and green (Chelonia mydas) turtles, representing the two extant sea turtle families. These genomes are highly syntenic and homologous, but localized regions of noncollinearity were associated with higher copy numbers of immune, zinc-finger, and olfactory receptor (OR) genes in green turtles, with ORs related to waterborne odorants greatly expanded in green turtles. Our findings suggest that divergent evolution of these key gene families may underlie immunological and sensory adaptations assisting navigation, occupancy of neritic versus pelagic environments, and diet specialization. Reduced collinearity was especially prevalent in microchromosomes, with greater gene content, heterozygosity, and genetic distances between species, supporting their critical role in vertebrate evolutionary adaptation. Finally, diversity and demographic histories starkly contrasted between species, indicating that leatherback turtles have had a low yet stable effective population size, exhibit extremely low diversity compared with other reptiles, and harbor a higher genetic load compared with green turtles, reinforcing concern over their persistence under future climate scenarios. These genomes provide invaluable resources for advancing our understanding of evolution and conservation best practices in an imperiled vertebrate lineage.

Surfperches (Family Embiotocidae) are viviparous temperate reef fishes that brood their young. This life history trait translates into limited dispersal, strong population structure, and an unusually strong potential for local adaptation in a marine fish. As part of the California Conservation Genomics Project (CCGP), we sequenced the genome of the Black Surfperch, Embiotoca jacksoni, to establish a genomic model for understanding phylogeographic patterns of marine organisms in California. These patterns, in turn, may inform the design of marine protected areas using dispersal models based on genomic data. The genome of E. jacksoni is typical of marine fishes at less than 1Gb (genome size = 635 Mb), and our assembly is near-chromosome level (contig N50 = 6.5Mb, scaffold N50 = 15.5 Mb, BUSCO = 98.1%). Within the context of the CCGP, the genome will be used as a reference for future whole genome resequencing projects aimed at enhancing our knowledge of the population structure of the species, and efficacy of Marine Protected Areas across the state.

Keystone species are known to play a critical role in kelp forest health, including the well-known killer whales, sea otter, sea urchin, kelp trophic cascade in the Aleutian Islands, Alaska, USA. In California, a major player in the regulation of sea urchin abundance, and in turn, the health of kelp forests ecosystems, is a large wrasse, the California Sheephead, Semicossyphus pulcher. We present a reference genome for this ecologically important species that will serve as a key resource for future conservation research of California's inshore marine environment utilizing genomic tools to address changes in life-history traits, dispersal, range shifts, and ecological interactions among members of the kelp forest ecological assemblages. Our genome assembly of S. pulcher has a total length of 0.794 Gb, which is similar to many other marine fishes. The assembly is largely contiguous (N50 = 31.9 Mb) and nearly complete (BUSCO single-copy core gene content = 98.1%). Within the context of the California Conservation Genomics Project (CCGP), the genome of S. pulcher will be used as an important reference resource for ongoing whole genome resequencing efforts of the species.

The California Pipevine Swallowtail Butterfly, Battus philenor hirsuta, and its host plant, the California Pipevine or Dutchman's Pipe, Aristolochia californica Torr., are an important California endemic species pair. While this species pair is an ideal system to study co-evolution, genomic resources for both are lacking. Here, we report a new, chromosome-level assembly of B. philenor hirsuta as part of the California Conservation Genomics Project (CCGP). Following the sequencing and assembly 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. Our genome assembly, the first for any species in the genus, contains 109 scaffolds spanning 443 mega base (Mb) pairs, with a contig N50 of 14.6 Mb, a scaffold N50 of 15.2 Mb, and BUSCO complete score of 98.9%. In combination with the forthcoming A. californica reference genome, the B. philenor hirsuta genome will be a powerful tool for documenting landscape genomic diversity and plant-insect co-evolution in a rapidly changing California landscape.

Snakes in the family Colubridae include more than 2,000 currently recognized species, and comprise roughly 75% of the global snake species diversity on Earth. For such a spectacular radiation, colubrid snakes remain poorly understood ecologically and genetically. Two subfamilies, Colubrinae (788 species) and Dipsadinae (833 species), comprise the bulk of colubrid species richness. Dipsadines are a speciose and diverse group of snakes that largely inhabit Central and South America, with a handful of small-body-size genera that have invaded North America. Among them, the ring-necked snake, Diadophis punctatus, has an incredibly broad distribution with 14 subspecies. Given its continental distribution and high degree of variation in coloration, diet, feeding ecology, and behavior, the ring-necked snake is an excellent species for the study of genetic diversity and trait evolution. Within California, six subspecies form a continuously distributed "ring species" around the Central Valley, while a seventh, the regal ring-necked snake, Diadophis punctatus regalis is a disjunct outlier and Species of Special Concern in the state. Here, we report a new reference genome assembly for the San Diego ring-necked snake, D. p. similis, as part of the California Conservation Genomics Project. This assembly comprises a total of 444 scaffolds spanning 1,783 Mb and has a contig N50 of 8.0 Mb, scaffold N50 of 83 Mb, and BUSCO completeness score of 94.5%. This reference genome will be a valuable resource for studies of the taxonomy, conservation, and evolution of the ring-necked snake across its broad, continental distribution.

Sandblossoms, Linanthus parryae is a widespread annual plant species found in washes and sandy open habitats across the Mojave Desert and Eastern Sierra Nevada of California. Studies in this species have played a central role in evolutionary biology, serving as the first test cases of the shifting balance theory of evolution, models of isolation by distance, and metrics to describe the genetic structure of natural populations. Despite the importance of L. parryae in the development of landscape genetics and phylogeography, there are no genomic resources available for the species. Through the California Conservation Genomics Project, we assembled the first genome in the genus Linanthus. Using PacBio HiFi long reads and Hi-C chromatin conformation capture, we assembled 123 scaffolds spanning 1.51 Gb of the 1.96 Gb estimated genome, with a contig N50 of 18.7 Mb and a scaffold N50 of 124.8 Mb. This assembly, with a BUSCO completeness score of 88.7%, will allow us to revisit foundational ideas central to our understanding of how evolutionary forces operate in a geographic landscape. In addition, it will be a new resource to uncover adaptations to arid environments in the fragile desert habitat threatened by urban and solar farm development, climate change, and off-road vehicles.

Rattlesnakes play important roles in their ecosystems by regulating prey populations, are involved in complex coevolutionary dynamics with their prey, and exhibit a variety of unusual adaptations, including maternal care, heat-sensing pit organs, hinged fangs, and medically-significant venoms. The western rattlesnake (Crotalus oreganus) is one of the widest ranging rattlesnake species, with a distribution from British Columbia, where it is listed as threatened, to Baja California and east across the Great Basin to western Wyoming, Colorado and New Mexico. Here, we report a new reference genome assembly for one of six currently recognized subspecies, C. oreganus helleri, as part of the California Conservation Genomics Project (CCGP). Consistent with the reference genomic sequencing 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 a total of 698 scaffolds spanning 1,564,812,557 base pairs, has a contig N50 of 64.7 Mb, a scaffold N50 of 110.8 Mb, and BUSCO complete score of 90.5%. This reference genome will be valuable for studies on the genomic basis of venom evolution and variation within Crotalus, in resolving the taxonomy of C. oreganus and its relatives, and for the conservation and management of rattlesnakes in general.

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.

Hybrid zones represent valuable opportunities to observe evolution in systems that are unusually dynamic and where the potential for the origin of novelty and rapid adaptation co-occur with the potential for dysfunction. Recently initiated hybrid zones are particularly exciting evolutionary experiments because ongoing natural selection on novel genetic combinations can be studied in ecological time. Moreover, when hybrid zones involve native and introduced species, complex genetic patterns present important challenges for conservation policy. To assess variation of admixture dynamics, we scored a large panel of markers in five wild hybrid populations formed when Barred Tiger Salamanders were introduced into the range of California Tiger Salamanders.

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.