Parallel evolution in an island archipelago revealed by genomic sequencing of Hipposideros leaf-nosed bats.

Body size is a key morphological attribute, often used to delimit species boundaries among closely related taxa. But body size can evolve in parallel, reaching similar final states despite independent evolutionary and geographic origins, leading to faulty assumptions of evolutionary history. Here we document parallel evolution in body size in the widely distributed leaf-nosed bat genus Hipposideros, which has misled both taxonomic and evolutionary inference. We sequenced reduced representation genomic loci and measured external morphological characters from three closely related species from the Solomon Islands archipelago, delimited by body size. Species tree reconstruction confirms the paraphyly of two morphologically designated species. The non-sister relationship between large-bodied H. dinops lineages found on different islands indicates large-bodied ecomorphs have evolved independently at least twice in the history of this radiation. A lack of evidence for gene flow between sympatric, closely related taxa suggests the rapid evolution of strong reproductive isolating barriers between morphologically distinct populations. Our results position Solomon Islands Hipposideros as a novel vertebrate system for studying the repeatability of parallel evolution under natural conditions. We conclude by offering testable hypotheses for how geography and ecology could be mediating the repeated evolution of large-bodied Hipposideros lineages in the Solomon Islands.

An elevational shift facilitated the Mesoamerican diversification of Azure-hooded Jays (Cyanolyca cucullata) during the Great American Biotic Interchange.

The Great American Biotic Interchange (GABI) was a key biogeographic event in the history of the Americas. The rising of the Panamanian land bridge ended the isolation of South America and ushered in a period of dispersal, mass extinction, and new community assemblages, which sparked competition, adaptation, and speciation. Diversification across many bird groups, and the elevational zonation of others, ties back to events triggered by the GABI. But the exact timing of these events is still being revealed, with recent studies suggesting a much earlier time window for faunal exchange, perhaps as early as 20 million years ago (Mya). Using a time-calibrated phylogenetic tree, we show that the jay genus Cyanolyca is emblematic of bird dispersal trends, with an early, pre-land bridge dispersal from Mesoamerica to South America 6.3-7.3 Mya, followed by a back-colonization of C. cucullata to Mesoamerica 2.3-4.8 Mya, likely after the land bridge was complete. As Cyanolyca species came into contact in Mesoamerica, they avoided competition due to a prior shift to lower elevation in the ancestor of C. cucullata. This shift allowed C. cucullata to integrate itself into the Mesoamerican highland avifauna, which our time-calibrated phylogeny suggests was already populated by higher-elevation, congeneric dwarf-jays (C. argentigula, C. pumilo, C. mirabilis, and C. nanus). The outcome of these events and fortuitous elevational zonation was that C. cucullata could continue colonizing new highland areas farther north during the Pleistocene. Resultingly, four C. cucullata lineages became isolated in allopatric, highland regions from Panama to Mexico, diverging in genetics, morphology, plumage, and vocalizations. At least two of these lineages are best described as species (C. mitrata and C. cucullata). Continued study will further document the influence of the GABI and help clarify how dispersal and vicariance shaped modern-day species assemblages in the Americas.

De novo assembly of a chromosome-level reference genome for the California Scrub-Jay, Aphelocoma californica.

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.

A highly contiguous reference genome for the Steller's jay (Cyanocitta stelleri).

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.

Mitonuclear discordance results from incomplete lineage sorting, with no detectable evidence for gene flow, in a rapid radiation of Todiramphus kingfishers.

Many organisms possess multiple discrete genomes (i.e. nuclear and organellar), which are inherited separately and may have unique and even conflicting evolutionary histories. Phylogenetic reconstructions from these discrete genomes can yield different patterns of relatedness, a phenomenon known as cytonuclear discordance. In many animals, mitonuclear discordance (i.e. discordant evolutionary histories between the nuclear and mitochondrial genomes) has been widely documented, but its causes are often considered idiosyncratic and inscrutable. We show that a case of mitonuclear discordance in Todiramphus kingfishers can be explained by extensive genome-wide incomplete lineage sorting (ILS), likely a result of the explosive diversification history of this genus. For these kingfishers, quartet frequencies reveal that the nuclear genome is dominated by discordant topologies, with none of the internal branches in our consensus nuclear tree recovered in >50% of genome-wide gene trees. Meanwhile, a lack of inter-species shared ancestry, non-significant pairwise tests for gene flow, and little evidence for meaningful migration edges between species, leads to the conclusion that gene flow cannot explain the mitonuclear discordance we observe. This lack of evidence for gene flow combined with evidence for extensive genome-wide gene tree discordance, a hallmark of ILS, leads us to conclude that the mitonuclear discordance we observe likely results from ILS, specifically deep coalescence of the mitochondrial genome. Based on this case study, we hypothesize that similar demographic histories in other 'great speciator' taxa across the Indo-Pacific likely predispose these groups to high levels of ILS and high likelihoods of mitonuclear discordance.

A complete species-level phylogeny of the Erythrura parrotfinches (Aves: Estrildidae).

Identifying species boundaries and phylogenetic relationships among groups of closely related species provides a necessary framework for understanding how biodiversity evolves in natural systems. Here we present a complete phylogeny of the avian genus Erythrura (family Estrildidae) commonly known as parrotfinches, which includes species threatened by habitat loss and the pet trade. Using both mitogenome and reduced-representation genome-wide nuclear DNA sequence data, we reconstructed the evolutionary history of the group by sampling all 12 recognized species, four of which had not previously been studied in a phylogenetic context. We included intra-species geographic sampling that allowed us to comment on species limits in some taxa. We recovered the Gouldian Finch (Chloebia gouldiae) of Australia which has often been placed in the monotypic genus Chloebia, as being sister to a clade comprising all Erythrura species. In addition, we recovered a well-supported clade comprising eight species distributed throughout the Pacific Island eco-region, whereas those species occurring in continental southeast Asia, the Greater Sundas, and the Philippines, were recovered as earlier branching lineages. Of note was the early branching of the Fiji-endemic E. kleinschmidti which corroborates its unique phenotype. We also found a deep phylogenetic split (8.59% corrected, 7.89% uncorrected divergence in the mitochondrial gene ND2) between the Java and Philippine populations of E. hyperythra, indicating unrecognized species-level diversity within this taxon. In contrast, genome-wide nuclear data suggested that the New Guinea endemic species E. papuana is embedded within the widespread species E. trichroa in all phylogenetic reconstructions, corroborating previously published mitochondrial data that suggested a similar pattern. By generating a phylogenetic hypothesis for the relationships among all species of Erythrura parrotfinches, we provide a framework for better understanding the extant diversity and evolutionary history of this group.

Temporal stability of the hybrid zone between Calocitta magpie-jays revealed through comparison of museum specimens and iNaturalist photos.

Hybrid zones are natural experiments for the study of avian evolution. Hybrid zones can be dynamic, moving as species adjust to new climates and habitats, with unknown implications for species and speciation. There are relatively few studies that have comparable modern and historic sampling to assess change in hybrid zone location and width over time, and those studies have generally found mixed results, with many hybrid zones showing change over time, but others showing stability. The white-throated magpie-jay (Calocitta formosa) and black-throated magpie-jay (Calocitta colliei) occur along the western coast of Mexico and Central America. The two species differ markedly in throat color and tail length, and prior observation suggests a narrow hybrid zone in southern Jalisco where individuals have mixed throat color. This study aims to assess the existence and temporal stability of this putative hybrid zone by comparing throat color between georeferenced historical museum specimens and modern photos from iNaturalist with precise locality information. Our results confirm the existence of a narrow hybrid zone in Jalisco, with modern throat scores gradually increasing from the parental ends of the cline toward the cline center in a sigmoidal curve characteristic of hybrid zones. Our temporal comparison suggests that the hybrid zone has not shifted its position between historical (pre-1973) and modern (post-2005) time periods-a surprising result given the grand scale of habitat change to the western Mexican lowlands during this time. An anomalous pocket of white-throated individuals in the northern range of the black-throated magpie-jay hints at the possibility of prehistorical long-distance introduction. Future genomic data will help disentangle the evolutionary history of these lineages and better characterize how secondary contact is affecting both the DNA and the phenotype of these species.

Hybrid zone or hybrid lineage: a genomic reevaluation of Sibley's classic species conundrum in Pipilo towhees.

Hybrid zones can be studied by modeling clines of trait variation (e.g., morphology, genetics) over a linear transect. Yet, hybrid zones can also be spatially complex, can shift over time, and can even lead to the formation of hybrid lineages with the right combination of dispersal and vicariance. We reassessed Sibley's (1950) gradient between Collared Towhee (Pipilo ocai) and Spotted Towhee (Pipilo maculatus) in Central Mexico to test whether it conformed to a typical tension-zone cline model. By comparing historical and modern data, we found that cline centers for genetic and phenotypic traits have not shifted over the course of 70 years. This equilibrium suggests that secondary contact between these species, which originally diverged over 2 million years ago, likely dates to the Pleistocene. Given the amount of mtDNA divergence, parental ends of the cline have very low autosomal nuclear differentiation (FST = 0.12). Dramatic and coincident cline shifts in mtDNA and throat color suggest the possibility of sexual selection as a factor in differential introgression, while a contrasting cline shift in green back color hints at a role for natural selection. Supporting the idea of a continuum between clinal variation and hybrid lineage formation, the towhee gradient can be analyzed as one population under isolation-by-distance, as a two-population cline, and as three lineages experiencing divergence with gene flow. In the middle of the gradient, a hybrid lineage has become partly isolated, likely due to forested habitat shrinking and fragmenting as it moved upslope after the last glacial maximum and a stark environmental transition. This towhee system offers a window into the potential outcomes of hybridization across a dynamic landscape including the creation of novel genomic and phenotypic combinations and incipient hybrid lineages.

Combining Species Delimitation, Species Trees, and Tests for Gene Flow Clarifies Complex Speciation in Scrub-Jays.

Complex speciation, involving rapid divergence and multiple bouts of post-divergence gene flow, can obfuscate phylogenetic relationships and species limits. In North America, cases of complex speciation are common, due at least in part to the cyclical Pleistocene glacial history of the continent. Scrub-Jays in the genus Aphelocoma provide a useful case study in complex speciation because their range throughout North America is structured by phylogeographic barriers with multiple cases of secondary contact between divergent lineages. Here, we show that a comprehensive approach to genomic reconstruction of evolutionary history, i.e., synthesizing results from species delimitation, species tree reconstruction, demographic model testing, and tests for gene flow, is capable of clarifying evolutionary history despite complex speciation. We find concordant evidence across all statistical approaches for the distinctiveness of an endemic southern Mexico lineage (A. w. sumichrasti), culminating in support for the species status of this lineage under any commonly applied species concept. We also find novel genomic evidence for the species status of a Texas endemic lineage A. w. texana, for which equivocal species delimitation results were clarified by demographic modeling and spatially explicit models of gene flow. Finally, we find that complex signatures of both ancient and modern gene flow between the non-sister California Scrub-Jay (Aphelocoma californica) and Woodhouse's Scrub-Jay (Aphelocoma woodhouseii) result in discordant gene trees throughout the species' genomes despite clear support for their overall isolation and species status. In sum, we find that a multi-faceted approach to genomic analysis can increase our understanding of complex speciation histories, even in well-studied groups. Given the emerging recognition that complex speciation is relatively commonplace, the comprehensive framework that we demonstrate for interrogation of species limits and evolutionary history using genomic data can provide a necessary roadmap for disentangling the impacts of gene flow and incomplete lineage sorting (ILS) to better understand the systematics of other groups with similarly complex evolutionary histories. [Gene flow; genomics; introgression; phylogenomics; population genetics; species delimitation; species trees; systematics.].

snpfiltr: An R package for interactive and reproducible SNP filtering.

Here, I describe the R package snpfiltr and demonstrate its functionality as the backbone of a customizable, reproducible single nucleotide polymorphism (SNP) filtering pipeline implemented exclusively via the widely adopted R programming language. SNPfiltR extends existing SNP filtering functionalities by automating the visualization of key parameters such as sequencing depth, quality, and missing data proportion, allowing users to visually optimize and implement filtering thresholds within a single, cohesive work session. All SNPfiltR functions require vcfr objects as input, which can be easily generated by reading a SNP data set stored in standard variant call format (vcf) into an R working environment using the function read.vcfR() from the R package vcfr. Performance and accuracy benchmarking reveal that for moderately sized SNP data sets (up to 50 M genotypes, plus associated quality information), SNPfiltR performs filtering with comparable accuracy and efficiency to current state of the art command-line-based programs. These results indicate that for most reduced-representation genomic data sets, SNPfiltR is an ideal choice for investigating, visualizing, and filtering SNPs as part of a user friendly bioinformatic pipeline. The snpfiltr package can be downloaded from CRAN with the command install.packages("snpfiltr"), and the current development version is available from GitHub at: (github.com/DevonDeRaad/SNPfiltR). Thorough documentation for SNPfiltR, including multiple comprehensive vignettes detailing realistic use-cases, is available at the website: devonderaad.github.io/SNPfiltR/.

Population connectivity across a highly fragmented distribution: Phylogeography of the Chalcophaps doves.

Chalcophaps is a morphologically conserved genus of ground-walking doves distributed from India to mainland China, south to Australia, and across the western Pacific to Vanuatu. Here, we reconstruct the evolutionary history of this genus using DNA sequence data from two nuclear genes and one mitochondrial gene, sampled from throughout the geographic range of Chalcophaps. We find support for three major evolutionary lineages in our phylogenetic reconstruction, each corresponding to the three currently recognized Chalcophaps species. Despite this general concordance, we identify discordant mitochondrial and nuclear ancestries in the subspecies C. longirostris timorensis, raising further questions about the evolutionary history of this Timor endemic population. Within each of the three species, we find evidence for isolation by distance or hierarchical population structure, indicating an important role for geography in the diversification of this genus. Despite being distributed broadly across a highly fragmented geographic region known as a hotspot for avian diversification, the Chalcophaps doves show modest levels of phenotypic and genetic diversity, a pattern potentially explained by strong population connectivity owing to high overwater dispersal capability.

Genome-environment association methods comparison supports omnigenic adaptation to ecological niche in malaria vector mosquitoes.

The concept of a fundamental ecological niche is central to questions of geographic distribution, population demography, species conservation, and evolutionary potential. However, robust inference of genomic regions associated with evolutionary adaptation to particular environmental conditions remains difficult due to the myriad of potential confounding processes that can generate heterogeneous patterns of variation across the genome. Here, we interrogate the potential role of genome environment association (GEA) testing as an initial step in building an understanding of the genetic basis of ecological niche. We leverage publicly available genomic data from the Anopheles gambiae 1000 Genomes (Ag1000g) Consortium to test the ability of multiple analytically unique GEA methods to handle confounding patterns of genetic variation, control false positive rates, and discern associations with broadly relevant climate variables from random allele frequency patterns throughout the genome. We found evidence supporting the ability of commonly implemented GEA methods to account for confounding patterns of spatial and genetic variation, and control false positive rates. However, we fail to find evidence supporting the ability of GEA tests to reject signals of adaptation to randomly simulated environmental variables, indicating that discerning between true signals of genome environment adaptation and genome environment correlations resulting from alternative evolutionary processes, remains challenging. Because signals of environmental adaptation are so diffuse and confounded throughout the genome, we argue that genomic adaptation to ecological niche is likely best understood under an omnigenic model wherein highly interconnected, genome-wide gene regulatory networks shape genomic adaptation to key environmental conditions.

Geographic potential of the world's largest hornet, Vespa mandarinia Smith (Hymenoptera: Vespidae), worldwide and particularly in North America.

The Asian giant hornet (AGH, Vespa mandarinia) is the world's largest hornet, occurring naturally in the Indomalayan region, where it is a voracious predator of pollinating insects including honey bees. In September 2019, a nest of Asian giant hornets was detected outside of Vancouver, British Columbia; multiple individuals were detected in British Columbia and Washington state in 2020; and another nest was found and eradicated in Washington state in November 2020, indicating that the AGH may have successfully wintered in North America. Because hornets tend to spread rapidly and become pests, reliable estimates of the potential invasive range of V. mandarinia in North America are needed to assess likely human and economic impacts, and to guide future eradication attempts. Here, we assess climatic suitability for AGH in North America, and suggest that, without control, this species could establish populations across the Pacific Northwest and much of eastern North America. Predicted suitable areas for AGH in North America overlap broadly with areas where honey production is highest, as well as with species-rich areas for native bumble bees and stingless bees of the genus Melipona in Mexico, highlighting the economic and environmental necessity of controlling this nascent invasion.