Species Persistence with Hybridization in Toad-Headed Lizards Driven by Divergent Selection and Low Recombination.

Speciation plays a central role in evolutionary studies, and particularly how reproductive isolation (RI) evolves. The origins and persistence of RI are distinct processes that require separate evaluations. Treating them separately clarifies the drivers of speciation and then it is possible to link the processes to understand large-scale patterns of diversity. Recent genomic studies have focused predominantly on how species or RI originate. However, we know little about how species persist in face of gene flow. Here, we evaluate a contact zone of two closely related toad-headed lizards (Phrynocephalus) using a chromosome-level genome assembly and population genomics. To some extent, recent asymmetric introgression from Phrynocephalus putjatai to P. vlangalii reduces their genomic differences. However, their highly divergent regions (HDRs) have heterogeneous distributions across the genomes. Functional gene annotation indicates that many genes within HDRs are involved in reproduction and RI. Compared with allopatric populations, contact areas exhibit recent divergent selection on the HDRs and a lower population recombination rate. Taken together, this implies that divergent selection and low genetic recombination help maintain RI. This study provides insights into the genomic mechanisms that drive RI and two species persistence in the face of gene flow during the late stage of speciation.

A combined approach of mitochondrial DNA and anchored nuclear phylogenomics sheds light on unrecognized diversity, phylogeny, and historical biogeography of the torrent frogs, genus Amolops (Anura: Ranidae).

The genus Amolops ("torrent frogs") is one of the most species-rich genera in Ranidae, with 59 recognized species. This genus currently includes six species groups diagnosed mainly by morphology. Several recent molecular studies indicated that the classification of species groups within Amolops remains controversial, and key nodes in the phylogeny have been inadequately resolved. In addition, the diversity of Amolops remains poorly understood, especially for those from incompletely sampled regions. Herein, we investigate species-level diversity within the genus Amolops throughout southern China and Southeast Asia, and infer evolutionary relationships among the species using mtDNA data (16S, COI, and ND2). Molecular analyses indicate nine unnamed species, mostly distributed in the Himalayas. We then utilized anchored hybrid enrichment to generate a dataset representing the major mitochondrial lineages to resolve phylogenetic relationships, biogeography, and pattern of species diversification. Our resulting phylogeny strongly supports the monophyly of four previously identified species groups (the A. ricketti, A. daiyunensis, A. hainanensis, and A. monticola groups), but paraphyly for the A. mantzorum and A. marmoratus groups, as previously defined. We erect one new species group, the A. viridimaculatus group, and recognize Dubois' (1992) subgenus Amo as the A. larutensis species group. Biogeographic analysis suggests that Amolops originated on the Indo-Burma/Thai-Malay Peninsula at the Eocene/Oligocene boundary, and dispersed outward, exemplifying a common pattern observed for the origin of Asian biodiversity. The early divergence within Amolops coincides with the Himalayan uplift and the lateral extrusion of Indochina at the Oligocene/Miocene boundary. Our results show that paleoclimatic and geomorphological events have profoundly influenced the patterns of lineage diversification within Amolops.

Hidden diversity within the depauperate genera of the snake tribe Lampropeltini (Serpentes, Colubridae).

Accurate representation of lineage diversity through complete taxon sampling is crucial to understanding the evolution of biodiversity, particularly when using molecular phylogenetics to estimate evolutionary relationships. In this interest, taxonomic diversity is often used as a proxy for lineage diversity even though the two concepts are not synonymous. We explore this within the snake tribe Lampropeltini which includes some of the most conspicuous and heavily studied snakes in North America. Both the taxonomy and hypothesized relationships within this tribe have been in flux. The number of species has increased from 23 to 51 over the last thirty years, predominately within three of the nine genera (Lampropeltis, Pantherophis, Pituophis). The remaining six depauperate genera (Arizona, Bogertophis, Cemophora, Pseudelaphe, Rhinocheilus, and Senticolis) have been poorly represented in phylogenetic studies. To estimate evolutionary relationships and determine if the dichotomy in depauperate and speciose genera within Lampropeltini is a function of taxon sampling or truly represents the lineage diversity, we estimated the phylogeny of this group using nuclear and mitochondrial loci in a concatenated and coalescent framework with the largest sampling of the six depauperate genera to date. In addition, we estimated the divergence dates among the genera to assess whether the instability of Lampropeltini phylogenetic relationships is due to an adaptive radiation. While some nodes still remain unresolved, the generic-level relationships we recovered agree with those of a recent next-generation study that used a much larger set of loci for fewer individuals. We also tested two putative species, Arizona pacata and Pseudelaphe phaescens, for the first time phylogenetically and find evidence that they are distinct lineages. Overall, we find that the taxonomic and genetic diversity are not correlated in Lampropeltini and that representing putative diversity in phylogenies will lead to a better estimate of evolutionary histories, especially in groups with complex radiations.

Historical mitochondrial genome introgression confounds species delimitation: evidence from phylogenetic inference in the Odorrana grahami species complex.

Species delimitation is essential to informing conservation policy and understanding ecological and evolutionary processes. Most of our recent gains in knowledge on animal diversity rely on morphological characteristics and mitochondrial (mt) DNA variation. Concordant results based on both have led to an unprecedented acceleration in the identification of new species and enriched the field of taxonomy. However, discordances are also found commonly between morphological and mtDNA evidence. This confounds species delimitation, especially when gene flow or mt genome introgression has occurred. Here, we illustrate how mt genome introgression among species of the Odorrana grahami complex confounds species delimitation using the combined evidence of morphological characters, mt variation, and thousands of nuclear single-nucleotide polymorphisms (SNPs) from genotyping-by-sequencing (GBS). Fifty-eight samples across the distribution of the O. grahami complex were included. The mtDNA matrilineal genealogy indicated 2 clades, with O. grahami and Odorrana junlianensis clustered together. In contrast, all nuclear evidence including gene trees, species trees, and genetic structure analyses based on GBS data support 3 species with distinct genetic clusters. These 3 distinct genetic clusters also correspond to distinct morphological characters. They affirm the distinct taxonomic entities of both O. grahami and O. junlianensis, as well as a third clade distinct from either. Which species the third clade belongs to remains unclear and will require further testing. The nuclear genomic loci contradict the COI evidence, with indications of rampant historical mt genome introgression among the species of the O. grahami complex. These discordant signals previously confused species delimitation efforts in this group. Based on these findings, we recommend the integration of independent data, especially nuclear genomic evidence, in species delimitation so as to be robust against the pitfalls of mt introgression.

Benchmarking ultra-high molecular weight DNA preservation methods for long-read and long-range sequencing.

BACKGROUND: Studies in vertebrate genomics require sampling from a broad range of tissue types, taxa, and localities. Recent advancements in long-read and long-range genome sequencing have made it possible to produce high-quality chromosome-level genome assemblies for almost any organism. However, adequate tissue preservation for the requisite ultra-high molecular weight DNA (uHMW DNA) remains a major challenge. Here we present a comparative study of preservation methods for field and laboratory tissue sampling, across vertebrate classes and different tissue types. RESULTS: We find that storage temperature was the strongest predictor of uHMW fragment lengths. While immediate flash-freezing remains the sample preservation gold standard, samples preserved in 95% EtOH or 20-25% DMSO-EDTA showed little fragment length degradation when stored at 4°C for 6 hours. Samples in 95% EtOH or 20-25% DMSO-EDTA kept at 4°C for 1 week after dissection still yielded adequate amounts of uHMW DNA for most applications. Tissue type was a significant predictor of total DNA yield but not fragment length. Preservation solution had a smaller but significant influence on both fragment length and DNA yield. CONCLUSION: We provide sample preservation guidelines that ensure sufficient DNA integrity and amount required for use with long-read and long-range sequencing technologies across vertebrates. Our best practices generated the uHMW DNA needed for the high-quality reference genomes for phase 1 of the Vertebrate Genomes Project, whose ultimate mission is to generate chromosome-level reference genome assemblies of all ∼70,000 extant vertebrate species.

A new species of the genus Megophrys Gunther, 1864 (Amphibia: Anura: Megophryidae) from Mount Wuyi, China.

A new species of horned toad, Megophrys ombrophila sp. nov., is described based on specimens found from Guadun village from Mount Wuyi in northwestern Fujian Province, China. The species is distinguished from other described Megophrys by morphology, bioacoustics, and molecular data of the 12S and 16S mitochondrial loci. The species is characterized by its rotund stature, head length approximately equal to head width, canthus rostralis well developed, tympanum large and distinct, vomerine teeth absent, margin of tongue smooth, not notched from behind, heels of the feet not meeting when femurs are held at 90° to the axis of the body and tibias are depressed against the femur, toes weakly webbed at base, dorsal skin mostly smooth with scattered granules and ridges, usually the presence of two discontinuous dorsolateral ridges, supratympanic fold distinct and well-developed, and with females ranging from 32.8-35 mm snout-to-vent length, and males ranging from 27.4-34.5 mm SVL. In its type locality, the species is sympatric with M. boettgeri and M. kuatunensis; and phylogenetically, the most closely related described species is M. obesa from southwest Guangdong province. As more cryptic species are described within the subfamily Megophryinae, the more apparent becomes the need for fine-scale molecular and phenotypic assessment to capture the many forms of this hyper-diverse group.