Charles W. Linkem

MITOCHONDRIAL INTROGRESSION AND INCOMPLETE LINEAGE SORTING THROUGH SPACE AND TIME: PHYLOGENETICS OF CROTAPHYTID LIZARDS

Abstract We investigate the roles of mitochondrial introgression and incomplete lineage sorting during the phylogenetic history of crotaphytid lizards. Our Bayesian phylogenetic estimate for Crotaphytidae is based on analysis of mitochondrial DNA sequence data for 408 individuals representing the 12 extant species of Crotaphytus and Gambelia. The mitochondrial phylogeny disagrees in several respects with a previously published morphological tree, as well as with conventional species designations, and we conclude that some of this disagreement stems from hybridization-mediated mitochondrial introgression, as well as from incomplete lineage sorting. Unidirectional introgression of Crotaphytus collaris (western collared lizard) mitochondria into C. reticulatus (reticulate collared lizard) populations in the Rio Grande Valley of Texas has resulted in the replacement of ancestral C. reticulatus mitochondria over approximately two-thirds of the total range of the species, a linear distance of ∼270 km. Introgression of C. collaris mitochondria into C. bicinctores (Great Basin collared lizard) populations in southwestern Arizona requires a more complex scenario because at least three temporally separated and superimposed introgression events appear to have occurred in this region. We propose an “introgression conveyor” model to explain this unique pattern of mitochondrial variation in this region. We show with ecological niche modeling that the predicted geographical ranges of C. collaris, C. bicinctores, and C. reticulatus during glacial maxima could have provided enhanced opportunities for past hybridization. Our analyses suggest that incomplete lineage sorting and/or introgression has further confounded the phylogenetic placements of additional species including C. nebrius, C. vestigium, C. insularis, C. grismeri, and perhaps G. copei. Despite many independent instances of interspecific hybridization among crotaphytid lizards, the species continue to maintain morphological and geographic cohesiveness throughout their ranges.

Phylogenomics of Phrynosomatid Lizards: Conflicting Signals from Sequence Capture versus Restriction Site Associated DNA Sequencing

Sequence capture and restriction site associated DNA sequencing (RADseq) are popular methods for obtaining large numbers of loci for phylogenetic analysis. These methods are typically used to collect data at different evolutionary timescales; sequence capture is primarily used for obtaining conserved loci, whereas RADseq is designed for discovering single nucleotide polymorphisms (SNPs) suitable for population genetic or phylogeographic analyses. Phylogenetic questions that span both “recent” and “deep” timescales could benefit from either type of data, but studies that directly compare the two approaches are lacking. We compared phylogenies estimated from sequence capture and double digest RADseq (ddRADseq) data for North American phrynosomatid lizards, a species-rich and diverse group containing nine genera that began diversifying approximately 55 Ma. Sequence capture resulted in 584 loci that provided a consistent and strong phylogeny using concatenation and species tree inference. However, the phylogeny estimated from the ddRADseq data was sensitive to the bioinformatics steps used for determining homology, detecting paralogs, and filtering missing data. The topological conflicts among the SNP trees were not restricted to any particular timescale, but instead were associated with short internal branches. Species tree analysis of the largest SNP assembly, which also included the most missing data, supported a topology that matched the sequence capture tree. This preferred phylogeny provides strong support for the paraphyly of the earless lizard genera Holbrookia and Cophosaurus, suggesting that the earless morphology either evolved twice or evolved once and was subsequently lost in Callisaurus.

Phylogeography and historical demography of Polypedates leucomystax in the islands of Indonesia and the Philippines: evidence for recent human-mediated range expansion?

Southeast Asias widespread species offer unique opportunities to explore the effects of geographical barriers to dispersal on patterns of vertebrate lineage diversification. We analyzed mitochondrial gene sequences (16S rDNA) from a geographically widespread sample of 266 Southeast Asian tree frogs, including 244 individuals of Polypedates leucomystax and its close relatives. Our expectation was that lineages on island archipelagos would exhibit more substantial geographic structure, corresponding to the geological history of terrestrial connectivity in this region, compared to the Asian mainland. Contrary to predictions, we found evidence of numerous highly divergent lineages from a limited area on the Asian mainland, but fewer lineages with shallower divergences throughout oceanic islands of the Philippines and Indonesia. Surprisingly and in numerous instances, lineages in the archipelagos span distinct biogeographical provinces. Phylogeographic analyses identified four major haplotype clades; summary statistics, mismatch distributions, and Bayesian coalescent inference of demography provide support for recent range expansion, population growth, and/or admixture in the Philippine and some Sulawesi populations. We speculate that the current range of P. leucomystax in Southeast Asia is much larger now than in the recent past. Conversion of forested areas to monoculture agriculture and transportation of agricultural products between islands may have facilitated unprecedented population and range expansion in P. leucomystax throughout thousands of islands in the Philippine and Indonesian archipelagos.

EVIDENCE FOR CLIMATE‐DRIVEN DIVERSIFICATION? A CAUTION FOR INTERPRETING ABC INFERENCES OF SIMULTANEOUS HISTORICAL EVENTS

Approximate Bayesian computation (ABC) is rapidly gaining popularity in population genetics. One example, msBayes, infers the distribution of divergence times among pairs of taxa, allowing phylogeographers to test hypotheses about historical causes of diversification in co‐distributed groups of organisms. Using msBayes, we infer the distribution of divergence times among 22 pairs of populations of vertebrates distributed across the Philippine Archipelago. Our objective was to test whether sea‐level oscillations during the Pleistocene caused diversification across the islands. To guide interpretation of our results, we perform a suite of simulation‐based power analyses. Our empirical results strongly support a recent simultaneous divergence event for all 22 taxon pairs, consistent with the prediction of the Pleistocene‐driven diversification hypothesis. However, our empirical estimates are sensitive to changes in prior distributions, and our simulations reveal low power of the method to detect random variation in divergence times and bias toward supporting clustered divergences. Our results demonstrate that analyses exploring power and prior sensitivity should accompany ABC model selection inferences. The problems we identify are potentially mitigable with uniform priors over divergence models (rather than classes of models) and more flexible prior distributions on demographic and divergence‐time parameters.

A hybrid phylogenetic-phylogenomic approach for species tree estimation in African Agama lizards with applications to biogeography, character evolution, and diversification

Africa is renowned for its biodiversity and endemicity, yet little is known about the factors shaping them across the continent. African Agama lizards (45 species) have a pan-continental distribution, making them an ideal model for investigating biogeography. Many species have evolved conspicuous sexually dimorphic traits, including extravagant breeding coloration in adult males, large adult male body sizes, and variability in social systems among colorful versus drab species. We present a comprehensive time-calibrated species tree for Agama, and their close relatives, using a hybrid phylogenetic-phylogenomic approach that combines traditional Sanger sequence data from five loci for 57 species (146 samples) with anchored phylogenomic data from 215 nuclear genes for 23 species. The Sanger data are analyzed using coalescent-based species tree inference using (*)BEAST, and the resulting posterior distribution of species trees is attenuated using the phylogenomic tree as a backbone constraint. The result is a time-calibrated species tree for Agama that includes 95% of all species, multiple samples for most species, strong support for the major clades, and strong support for most of the initial divergence events. Diversification within Agama began approximately 23 million years ago (Ma), and separate radiations in Southern, East, West, and Northern Africa have been diversifying for >10Myr. A suite of traits (morphological, coloration, and sociality) are tightly correlated and show a strong signal of high morphological disparity within clades, whereby the subsequent evolution of convergent phenotypes has accompanied diversification into new biogeographic areas.

Species boundaries and cryptic lineage diversity in a Philippine forest skink complex (Reptilia; Squamata; Scincidae: Lygosominae).

In the megadiverse conservation hotspot of the Philippines, biodiversity is not uniformly distributed throughout the archipelago, but hierarchically partitioned into islands and island groups that were conjoined during the mid- to late-Pleistocene. Few species groups are widely distributed throughout the archipelago, but some exceptions exist, such as the common scincid lizards of the Sphenomorphus jagori complex (including S. jagori, S. coxi, and S. abdictus). Using mtDNA haplotype data we test biogeographic and taxonomic predictions in these abundant, large-bodied, forest floor lizards and arrive at conclusions that differ significantly from both past, and current, appraisals of species diversity. In contrast to expectations based on existing taxonomy (three species, each with two subspecies), we find evidence of at least eleven highly divergent species lineages diagnosed by haplotypic variation. Each lineage corresponds to a biogeographically circumscribed distribution (i.e., isolated islands or geological components of islands), suggesting lineage cohesion and allopatric differentiation. Parametric bootstrapping tests reject taxonomic and biogeographic hypotheses and suggest a complex pattern of unpredicted relationships. Only one of the former species (S. jagori) appears as a monophyletic entity (including four allopatric, highly divergent lineages that we suspect may represent evolutionary species), and the remaining species are paraphyletic, necessitating a comprehensive future taxonomic revision. The pattern of biogeographic provincialism and hidden cryptic species diversity detected here leads us to suspect that even the most common, presumably well-studied, and widespread species complexes in the Philippines are in need of thorough analysis with modern genetic and phylogenetic techniques. Such studies of speciation genetics in these common, widely distributed groups may lead to a better understanding of the genetic underpinnings of biodiversity, allow for an enhanced appreciation of the evolutionary history of this model island archipelago, and enable more informed conservation planning in a global biodiversity hotspot.

Molecular systematics of the Philippine forest skinks (Squamata: Scincidae: Sphenomorphus): testing morphological hypotheses of interspecific relationships

Skinks of the genus Sphenomorphus are the most diverse clade of squamates in the Philippine Archipelago. Morphological examination of these species has defined six phenotypic groups that are commonly used in characterizations of taxonomic hypotheses. We used a molecular phylogeny based on four mitochondrial and two nuclear genes to assess the groups biogeographical history in the archipelago and examine the phylogenetic validity of the currently recognized Philippine species groups. We re‐examined traditional characters used to define species groups and used multivariate statistics to quantitatively evaluate group structure in morphometric space. Clustering analyses of phenotypic similarity indicate that some (but not all) members of previously defined species groups are phenotypically most similar to other members of the same group. However, when species group membership was mapped on our partitioned Bayesian phylogenetic hypothesis, only one species group corresponds to a clade; all other species group arrangements are strongly rejected by our phylogeny. Our results demonstrate that (1) previously recognized species group relationships were misled by phenotypic convergence; (2) Sphenomorphus is widely paraphyletic; and (3) multiple lineages have independently invaded the Philippines. Based on this new perspective on the phylogenetic relationships of Philippine Sphenomorphus, we revise the archipelagos diverse assemblage of species at the generic level, and resurrect and/or expand four previously recognized genera, and describe two new genera to accommodate the diversity of Philippine skinks of the Sphenomorphus group.

Philippine Bent-Toed Geckos of the Cyrtodactylus agusanensis Complex: Multilocus Phylogeny, Morphological Diversity, and Descriptions of Three New Species

Abstract We review the taxonomic status of Philippine bent-toed geckos previously referred to Cyrtodactylus agusanensis. We delineate four evolutionary lineages within the C. agusanensis complex from the southeastern islands of the archipelago and describe three of these lineages as new species. The new species and true C. agusanensis are identified by numerous, nonoverlapping morphological characters and by allopatric ranges on separate islands. Our morphology-based taxonomic conclusions are bolstered by biogeographic evidence and marked interspecific divergence between monophyletic groups defined by mitochondrial and nuclear DNA sequences. To compliment these descriptions and enable future taxonomic work on Philippine Cyrtodactylus, we rediagnose and redescribe C. agusanensis. Because the holotype of C. agusanensis was destroyed in World War II, we designate a neotype for this species and restrict its geographic range to north central Mindanao Island. Our phylogenetic estimate suggests that the C. agusanensis complex originated in Mindanao and spread progressively north, diversifying incrementally with colonization of successive islands in a south-to-north pattern of biogeographic expansion and allopatric speciation.

Detecting the Anomaly Zone in Species Trees and Evidence for a Misleading Signal in Higher-Level Skink Phylogeny (Squamata: Scincidae).

The anomaly zone, defined by the presence of gene tree topologies that are more probable than the true species tree, presents a major challenge to the accurate resolution of many parts of the Tree of Life. This discrepancy can result from consecutive rapid speciation events in the species tree. Similar to the problem of long-branch attraction, including more data via loci concatenation will only reinforce the support for the incorrect species tree. Empirical phylogenetic studies often employ coalescent-based species tree methods to avoid the anomaly zone, but to this point these studies have not had a method for providing any direct evidence that the species tree is actually in the anomaly zone. In this study, we use 16 species of lizards in the family Scincidae to investigate whether nodes that are difficult to resolve place the species tree within the anomaly zone. We analyze new phylogenomic data (429 loci), using both concatenation and coalescent-based species tree estimation, to locate conflicting topological signal. We then use the unifying principle of the anomaly zone, together with estimates of ancestral population sizes and species persistence times, to determine whether the observed phylogenetic conflict is a result of the anomaly zone. We identify at least three regions of the Scincidae phylogeny that provide demographic signatures consistent with the anomaly zone, and this new information helps reconcile the phylogenetic conflict in previously published studies on these lizards. The anomaly zone presents a real problem in phylogenetics, and our new framework for identifying anomalous relationships will help empiricists leverage their resources appropriately for investigating and overcoming this challenge.

Comparative Species Divergence across Eight Triplets of Spiny Lizards (Sceloporus) Using Genomic Sequence Data

Species divergence is typically thought to occur in the absence of gene flow, but many empirical studies are discovering that gene flow may be more pervasive during species formation. Although many examples of divergence with gene flow have been identified, few clades have been investigated in a comparative manner, and fewer have been studied using genome-wide sequence data. We contrast species divergence genetic histories across eight triplets of North American Sceloporus lizards using a maximum likelihood implementation of the isolation–migration (IM) model. Gene flow at the time of species divergence is modeled indirectly as variation in species divergence time across the genome or explicitly using a migration rate parameter. Likelihood ratio tests (LRTs) are used to test the null model of no gene flow at speciation against these two alternative gene flow models. We also use the Akaike information criterion to rank the models. Hundreds of loci are needed for the LRTs to have statistical power, and we use genome sequencing of reduced representation libraries to obtain DNA sequence alignments at many loci (between 340 and 3,478; mean = 1,678) for each triplet. We find that current species distributions are a poor predictor of whether a species pair diverged with gene flow. Interrogating the genome using the triplet method expedites the comparative study of species divergence history and the estimation of genetic parameters associated with speciation.