Waves of Colonization and Gene Flow in a Great Speciator.

Secondary contact between previously allopatric lineages offers a test of reproductive isolating mechanisms that may have accrued in isolation. Such instances of contact can produce stable hybrid zones-where reproductive isolation can further develop via reinforcement or phenotypic displacement-or result in the lineages merging. Ongoing secondary contact is most visible in continental systems, where steady input from parental taxa can occur readily. In oceanic island systems, however, secondary contact between closely related species of birds is relatively rare. When observed on sufficiently small islands, relative to population size, secondary contact likely represents a recent phenomenon. Here, we examine the dynamics of a group of birds whose apparent widespread hybridization influenced Ernst Mayr's foundational work on allopatric speciation: the whistlers of Fiji (Aves: Pachycephala). We demonstrate two clear instances of secondary contact within the Fijian archipelago, one resulting in a hybrid zone on a larger island, and the other resulting in a wholly admixed population on a smaller, adjacent island. We leveraged low genome-wide divergence in the hybrid zone to pinpoint a single genomic region associated with observed phenotypic differences. We use genomic data to present a new hypothesis that emphasizes rapid plumage evolution and post-divergence gene flow.

First Insights into Population Structure and Genetic Diversity Versus Host Specificity in Trypanorhynch Tapeworms Using Multiplexed Shotgun Genotyping.

Theory predicts relaxed host specificity and high host vagility should contribute to reduced genetic structure in parasites while strict host specificity and low host vagility should increase genetic structure. Though these predictions are intuitive, they have never been explicitly tested in a population genomic framework. Trypanorhynch tapeworms, which parasitize sharks and rays (elasmobranchs) as definitive hosts, are the only order of elasmobranch tapeworms that exhibit considerable variability in their definitive host specificity. This allows for unique combinations of host use and geographic range, making trypanorhynchs ideal candidates for studying how these traits influence population-level structure and genetic diversity. Multiplexed shotgun genotyping (MSG) data sets were generated to characterize component population structure and infrapopulation diversity for a representative of each trypanorhynch suborder: the ray-hosted Rhinoptericola megacantha (Trypanobatoida) and the shark-hosted Callitetrarhynchus gracilis (Trypanoselachoida). Adults of R. megacantha are more host-specific and less broadly distributed than adults of C. gracilis, allowing correlation between these factors and genetic structure. Replicate tapeworm specimens were sequenced from the same host individual, from multiple conspecific hosts within and across geographic regions, and from multiple definitive host species. For R. megacantha, population structure coincided with geography rather than host species. For C. gracilis, limited population structure was found, suggesting a potential link between degree of host specificity and structure. Conspecific trypanorhynchs from the same host individual were found to be as, or more, genetically divergent from one another as from conspecifics from different host individuals. For both species, high levels of homozygosity and positive FIS values were documented.

A few essential genetic loci distinguish Penstemon species with flowers adapted to pollination by bees or hummingbirds.

In the formation of species, adaptation by natural selection generates distinct combinations of traits that function well together. The maintenance of adaptive trait combinations in the face of gene flow depends on the strength and nature of selection acting on the underlying genetic loci. Floral pollination syndromes exemplify the evolution of trait combinations adaptive for particular pollinators. The North American wildflower genus Penstemon displays remarkable floral syndrome convergence, with at least 20 separate lineages that have evolved from ancestral bee pollination syndrome (wide blue-purple flowers that present a landing platform for bees and small amounts of nectar) to hummingbird pollination syndrome (bright red narrowly tubular flowers offering copious nectar). Related taxa that differ in floral syndrome offer an attractive opportunity to examine the genomic basis of complex trait divergence. In this study, we characterized genomic divergence among 229 individuals from a Penstemon species complex that includes both bee and hummingbird floral syndromes. Field plants are easily classified into species based on phenotypic differences and hybrids displaying intermediate floral syndromes are rare. Despite unambiguous phenotypic differences, genome-wide differentiation between species is minimal. Hummingbird-adapted populations are more genetically similar to nearby bee-adapted populations than to geographically distant hummingbird-adapted populations, in terms of genome-wide dXY. However, a small number of genetic loci are strongly differentiated between species. These approximately 20 "species-diagnostic loci," which appear to have nearly fixed differences between pollination syndromes, are sprinkled throughout the genome in high recombination regions. Several map closely to previously established floral trait quantitative trait loci (QTLs). The striking difference between the diagnostic loci and the genome as whole suggests strong selection to maintain distinct combinations of traits, but with sufficient gene flow to homogenize the genomic background. A surprisingly small number of alleles confer phenotypic differences that form the basis of species identity in this species complex.

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.

Diversification and historical demography of Rhampholeon spectrum in West-Central Africa.

Pygmy Chameleons of the genus Rhampholeon represent a moderately diverse, geographically circumscribed radiation, with most species (18 out of 19 extant taxa) limited to East Africa. The one exception is Rhampholeon spectrum, a species restricted to West-Central African rainforests. We set out to characterize the geographic basis of genetic variation in this disjunctly distributed Rhampholeon species using a combination of multilocus Sanger data and genomic sequences to explore population structure and range-wide phylogeographic patterns. We also employed demographic analyses and niche modeling to distinguish between alternate explanations to contextualize the impact of past geological and climatic events on the present-day distribution of intraspecific genetic variation. Phylogenetic analyses suggest that R. spectrum is a complex of five geographically delimited populations grouped into two major clades (montane vs. lowland). We found pronounced population structure suggesting that divergence and, potentially, speciation began between the late Miocene and the Pleistocene. Sea level changes during the Pleistocene climatic oscillations resulted in allopatric divergence associated with dispersal over an ocean channel barrier and colonization of Bioko Island. Demographic inferences and range stability mapping each support diversification models with secondary contact due to population contraction in lowland and montane refugia during the interglacial period. Allopatric divergence, congruent with isolation caused by geologic uplift of the East African rift system, the "descent into the Icehouse," and aridification of sub-Saharan Africa during the Eocene-Oligocene are identified as the key events explaining the population divergence between R. spectrum and its closely related sister clade from the Eastern Arc Mountains. Our results unveil cryptic genetic diversity in R. spectrum, suggesting the possibility of a species complex distributed across the Lower Guinean Forest and the Island of Bioko. We highlight the major element of species diversification that modelled today's diversity and distributions in most West-Central African vertebrates.

Rivers, not refugia, drove diversification in arboreal, sub-Saharan African snakes.

The relative roles of rivers versus refugia in shaping the high levels of species diversity in tropical rainforests have been widely debated for decades. Only recently has it become possible to take an integrative approach to test predictions derived from these hypotheses using genomic sequencing and paleo-species distribution modeling. Herein, we tested the predictions of the classic river, refuge, and river-refuge hypotheses on diversification in the arboreal sub-Saharan African snake genus Toxicodryas. We used dated phylogeographic inferences, population clustering analyses, demographic model selection, and paleo-distribution modeling to conduct a phylogenomic and historical demographic analysis of this genus. Our results revealed significant population genetic structure within both Toxicodryas species, corresponding geographically to river barriers and divergence times from the mid-Miocene to Pliocene. Our demographic analyses supported the interpretation that rivers are indications of strong barriers to gene flow among populations since their divergence. Additionally, we found no support for a major contraction of suitable habitat during the last glacial maximum, allowing us to reject both the refuge and river-refuge hypotheses in favor of the river-barrier hypothesis. Based on conservative interpretations of our species delimitation analyses with the Sanger and ddRAD data sets, two new cryptic species are identified from east-central Africa. This study highlights the complexity of diversification dynamics in the African tropics and the advantages of integrative approaches to studying speciation in tropical regions.

Transcriptomic Analysis of Skin Color in Anole Lizards.

Color and color pattern are critical for animal camouflage, reproduction, and defense. Few studies, however, have attempted to identify candidate genes for color and color pattern in squamate reptiles, a colorful group with over 10,000 species. We used comparative transcriptomic analyses between white, orange, and yellow skin in a color-polymorphic species of anole lizard to 1) identify candidate color and color-pattern genes in squamates and 2) assess if squamates share an underlying genetic basis for color and color pattern variation with other vertebrates. Squamates have three types of chromatophores that determine color pattern: guanine-filled iridophores, carotenoid- or pteridine-filled xanthophores/erythrophores, and melanin-filled melanophores. We identified 13 best candidate squamate color and color-pattern genes shared with other vertebrates: six genes linked to pigment synthesis pathways, and seven genes linked to chromatophore development and maintenance. In comparisons of expression profiles between pigment-rich and white skin, pigment-rich skin upregulated the pteridine pathway as well as xanthophore/erythrophore development and maintenance genes; in comparisons between orange and yellow skin, orange skin upregulated the pteridine and carotenoid pathways as well as melanophore maintenance genes. Our results corroborate the predictions that squamates can produce similar colors using distinct color-reflecting molecules, and that both color and color-pattern genes are likely conserved across vertebrates. Furthermore, this study provides a concise list of candidate genes for future functional verification, representing a first step in determining the genetic basis of color and color pattern in anoles.

Geography is more important than life history in the recent diversification of the tiger salamander complex.

The North American tiger salamander species complex, including its best-known species, the Mexican axolotl, has long been a source of biological fascination. The complex exhibits a wide range of variation in developmental life history strategies, including populations and individuals that undergo metamorphosis; those able to forego metamorphosis and retain a larval, aquatic lifestyle (i.e., paedomorphosis); and those that do both. The evolution of a paedomorphic life history state is thought to lead to increased population genetic differentiation and ultimately reproductive isolation and speciation, but the degree to which it has shaped population- and species-level divergence is poorly understood. Using a large multilocus dataset from hundreds of samples across North America, we identified genetic clusters across the geographic range of the tiger salamander complex. These clusters often contain a mixture of paedomorphic and metamorphic taxa, indicating that geographic isolation has played a larger role in lineage divergence than paedomorphosis in this system. This conclusion is bolstered by geography-informed analyses indicating no effect of life history strategy on population genetic differentiation and by model-based population genetic analyses demonstrating gene flow between adjacent metamorphic and paedomorphic populations. This fine-scale genetic perspective on life history variation establishes a framework for understanding how plasticity, local adaptation, and gene flow contribute to lineage divergence. Many members of the tiger salamander complex are endangered, and the Mexican axolotl is an important model system in regenerative and biomedical research. Our results chart a course for more informed use of these taxa in experimental, ecological, and conservation research.

Evidence of two deeply divergent co-existing mitochondrial genomes in the Tuatara reveals an extremely complex genomic organization.

Animal mitochondrial genomic polymorphism occurs as low-level mitochondrial heteroplasmy and deeply divergent co-existing molecules. The latter is rare, known only in bivalvian mollusks. Here we show two deeply divergent co-existing mt-genomes in a vertebrate through genomic sequencing of the Tuatara (Sphenodon punctatus), the sole-representative of an ancient reptilian Order. The two molecules, revealed using a combination of short-read and long-read sequencing technologies, differ by 10.4% nucleotide divergence. A single long-read covers an entire mt-molecule for both strands. Phylogenetic analyses suggest a 7-8 million-year divergence between genomes. Contrary to earlier reports, all 37 genes typical of animal mitochondria, with drastic gene rearrangements, are confirmed for both mt-genomes. Also unique to vertebrates, concerted evolution drives three near-identical putative Control Region non-coding blocks. Evidence of positive selection at sites linked to metabolically important transmembrane regions of encoded proteins suggests these two mt-genomes may confer an adaptive advantage for an unusually cold-tolerant reptile.

Phylogenomics Reveals Ancient Gene Tree Discordance in the Amphibian Tree of Life.

Molecular phylogenies have yielded strong support for many parts of the amphibian Tree of Life, but poor support for the resolution of deeper nodes, including relationships among families and orders. To clarify these relationships, we provide a phylogenomic perspective on amphibian relationships by developing a taxon-specific Anchored Hybrid Enrichment protocol targeting hundreds of conserved exons which are effective across the class. After obtaining data from 220 loci for 286 species (representing 94% of the families and 44% of the genera), we estimate a phylogeny for extant amphibians and identify gene tree-species tree conflict across the deepest branches of the amphibian phylogeny. We perform locus-by-locus genealogical interrogation of alternative topological hypotheses for amphibian monophyly, focusing on interordinal relationships. We find that phylogenetic signal deep in the amphibian phylogeny varies greatly across loci in a manner that is consistent with incomplete lineage sorting in the ancestral lineage of extant amphibians. Our results overwhelmingly support amphibian monophyly and a sister relationship between frogs and salamanders, consistent with the Batrachia hypothesis. Species tree analyses converge on a small set of topological hypotheses for the relationships among extant amphibian families. These results clarify several contentious portions of the amphibian Tree of Life, which in conjunction with a set of vetted fossil calibrations, support a surprisingly younger timescale for crown and ordinal amphibian diversification than previously reported. More broadly, our study provides insight into the sources, magnitudes, and heterogeneity of support across loci in phylogenomic data sets.[AIC; Amphibia; Batrachia; Phylogeny; gene tree-species tree discordance; genomics; information theory.].

Genomic Data Reveal Conserved Female Heterogamety in Giant Salamanders with Gigantic Nuclear Genomes.

Systems of genetic sex determination and the homology of sex chromosomes in different taxa vary greatly across vertebrates. Much progress remains to be made in understanding systems of genetic sex determination in non-model organisms, especially those with homomorphic sex chromosomes and/or large genomes. We used reduced representation genome sequencing to investigate genetic sex determination systems in the salamander family Cryptobranchidae (genera Cryptobranchus and Andrias), which typifies both of these inherent difficulties. We tested hypotheses of male- or female-heterogamety by sequencing hundreds of thousands of anonymous genomic regions in a panel of known-sex cryptobranchids and characterized patterns of presence/absence, inferred zygosity, and depth of coverage to identify sex-linked regions of these 56 gigabase genomes. Our results strongly support the hypothesis that all cryptobranchid species possess homologous systems of female heterogamety, despite maintenance of homomorphic sex chromosomes over nearly 60 million years. Additionally, we report a robust, non-invasive genetic assay for sex diagnosis in Cryptobranchus and Andrias which may have great utility for conservation efforts with these endangered salamanders. Co-amplification of these W-linked markers in both cryptobranchid genera provides evidence for long-term sex chromosome stasis in one of the most divergent salamander lineages. These findings inform hypotheses about the ancestral mode of sex determination in salamanders, but suggest that comparative data from other salamander families are needed. Our results further demonstrate that massive genomes are not necessarily a barrier to effective genome-wide sequencing and that the resulting data can be highly informative about sex determination systems in taxa with homomorphic sex chromosomes.

The influence of locus number and information content on species delimitation: an empirical test case in an endangered Mexican salamander.

Perhaps the most important recent advance in species delimitation has been the development of model-based approaches to objectively diagnose species diversity from genetic data. Additionally, the growing accessibility of next-generation sequence data sets provides powerful insights into genome-wide patterns of divergence during speciation. However, applying complex models to large data sets is time-consuming and computationally costly, requiring careful consideration of the influence of both individual and population sampling, as well as the number and informativeness of loci on species delimitation conclusions. Here, we investigated how locus number and information content affect species delimitation results for an endangered Mexican salamander species, Ambystoma ordinarium. We compared results for an eight-locus, 137-individual data set and an 89-locus, seven-individual data set. For both data sets, we used species discovery methods to define delimitation models and species validation methods to rigorously test these hypotheses. We also used integrated demographic model selection tools to choose among delimitation models, while accounting for gene flow. Our results indicate that while cryptic lineages may be delimited with relatively few loci, sampling larger numbers of loci may be required to ensure that enough informative loci are available to accurately identify and validate shallow-scale divergences. These analyses highlight the importance of striking a balance between dense sampling of loci and individuals, particularly in shallowly diverged lineages. They also suggest the presence of a currently unrecognized, endangered species in the western part of A. ordinarium's range.