Ted M. Townsend

Resolving the phylogeny of lizards and snakes (Squamata) with extensive sampling of genes and species

Squamate reptiles (lizards and snakes) are one of the most diverse groups of terrestrial vertebrates. Recent molecular analyses have suggested a very different squamate phylogeny relative to morphological hypotheses, but many aspects remain uncertain from molecular data. Here, we analyse higher-level squamate phylogeny with a molecular dataset of unprecedented size, including 161 squamate species for up to 44 nuclear genes each (33 717 base pairs), using both concatenated and species-tree methods for the first time. Our results strongly resolve most squamate relationships and reveal some surprising results. In contrast to most other recent studies, we find that dibamids and gekkotans are together the sister group to all other squamates. Remarkably, we find that the distinctive scolecophidians (blind snakes) are paraphyletic with respect to other snakes, suggesting that snakes were primitively burrowers and subsequently re-invaded surface habitats. Finally, we find that some clades remain poorly supported, despite our extensive data. Our analyses show that weakly supported clades are associated with relatively short branches for which individual genes often show conflicting relationships. These latter results have important implications for all studies that attempt to resolve phylogenies with large-scale phylogenomic datasets.

Rapid development of multiple nuclear loci for phylogenetic analysis using genomic resources: An example from squamate reptiles

Recently, as genome-scale data have become available for more organisms, the development of phylogenetic markers from nuclear protein-coding loci (NPCL) has become more tractable. However, new methods are needed to efficiently sort the large number of genes from genomic databases into more limited sets appropriate for particular phylogenetic questions, while avoiding introns and paralogs. Here we describe a general methodology for identifying candidate single-copy NPCL from genomic databases. Our method uses information from reference genomes to identify genes with relatively large continuous protein-coding regions (i.e., 700bp). BLAST comparisons are used to help avoid genes with paralogous copies or close relatives (i.e., gene families) that might confound phylogenetic analyses. Exon boundary information is used to identify appropriately spaced potential priming sites. Using this method, we have developed over 25 novel NPCL, which span a variety of desirable evolutionary rates for phylogenetic analyses. Although targeted for higher-level phylogenetics of squamate reptiles, many of these loci appear to be useful across and within other vertebrate clades (e.g., amphibians), and some are relatively rapidly evolving and may be useful for closely-related species (e.g., within genera). This general method can be used whenever large-scale genomic data are available for an appropriate reference species (not necessarily within the focal clade). The method is also well suited for the development of intron regions for lower-level phylogenetic and phylogeographic studies. We provide an online database of alignments and suggested primers for approximately 85 NPCL that should be useful across vertebrates.

Combining Phylogenomics and Fossils in Higher-Level Squamate Reptile Phylogeny: Molecular Data Change the Placement of Fossil Taxa

Molecular data offer great potential to resolve the phylogeny of living taxa but can molecular data improve our understanding of relationships of fossil taxa? Simulations suggest that this is possible, but few empirical examples have demonstrated the ability of molecular data to change the placement of fossil taxa. We offer such an example here. We analyze the placement of snakes among squamate reptiles, combining published morphological data (363 characters) and new DNA sequence data (15,794 characters, 22 nuclear loci) for 45 living and 19 fossil taxa. We find several intriguing results. First, some fossil taxa undergo major changes in their phylogenetic position when molecular data are added. Second, most fossil taxa are placed with strong support in the expected clades by the combined data Bayesian analyses, despite each having >98% missing cells and despite recent suggestions that extensive missing data are problematic for Bayesian phylogenetics. Third, morphological data can change the placement of living taxa in combined analyses, even when there is an overwhelming majority of molecular characters. Finally, we find strong but apparently misleading signal in the morphological data, seemingly associated with a burrowing lifestyle in snakes, amphisbaenians, and dibamids. Overall, our results suggest promise for an integrated and comprehensive Tree of Life by combining molecular and morphological data for living and fossil taxa.

Branch Lengths, Support, and Congruence: Testing the Phylogenomic Approach with 20 Nuclear Loci in Snakes

Many authors have claimed that short branches in the Tree of Life will be very difficult to resolve with strong support, even with the large multilocus data sets now made possible by genomic resources. Short branches may be especially problematic because the underlying gene trees are expected to have discordant phylogenetic histories when the time between branching events is very short. Although there are many examples of short branches that are difficult to resolve, surprisingly, no empirical studies have systematically examined the relationships between branch lengths, branch support, and congruence among genes. Here, we examine these fundamental relationships quantitatively using a data set of 20 nuclear loci for 50 species of snakes (representing most traditionally recognized families). A combined maximum likelihood analysis of the 20 loci gives strong support for 69% of the nodes, but many remain weakly supported, with bootstrap values for 20% ranging from 21% to 66%. For the combined-data tree, we find significant correlations between the length of a branch, levels of bootstrap support, and the proportion of genes that are congruent with that branch in the separate analyses of each gene. We also find that strongly supported conflicts between gene trees over the resolution of individual branches are common (roughly 35% of clades), especially for shorter branches. Overall, our results support the hypothesis that short branches may be very difficult to confidently resolve, even with large, multilocus data sets. Nevertheless, our study provides strong support for many clades, including several that were controversial or poorly resolved in previous studies of snake phylogeny.

Phylogeny of iguanian lizards inferred from 29 nuclear loci, and a comparison of concatenated and species-tree approaches for an ancient, rapid radiation

Iguanian lizards form a diverse clade whose members have been the focus of many comparative studies of ecology, behavior, and evolution. Despite the importance of phylogeny to such studies, interrelationships among many iguanian clades remain uncertain. Within the Old World clade Acrodonta, Agamidae is sometimes found to be paraphyletic with respect to Chamaeleonidae, and recent molecular studies have produced conflicting results for many major clades. Within the largely New World clade Pleurodonta, relationships among the 12 currently recognized major subclades (mostly ranked as families) have been largely unresolved or poorly supported in previous studies. To clarify iguanian evolutionary history, we first infer phylogenies using concatenated maximum-likelihood (ML) and Bayesian analyses of DNA sequence data from 29 nuclear protein-coding genes for 47 iguanian and 29 outgroup taxa. We then estimate a relaxed-clock Bayesian chronogram for iguanians using BEAST. All three methods produce identical topologies. Within Acrodonta, we find strong support for monophyly of Agamidae with respect to Chamaeleonidae, and for almost all relationships within agamids. Within Pleurodonta, we find strong Bayesian support for almost all relationships, and strong ML support for some interfamilial relationships and for monophyly of almost all families (excepting Polychrotidae). Our phylogenetic results suggest a non-traditional biogeographic scenario in which pleurodonts originated in the Northern Hemisphere and subsequently spread southward into South America. The pleurodont portion of the tree is characterized by several very short, deep branches, raising the possibility of deep coalescences that may confound concatenated analyses. We therefore also use 27 of these genes to implement a coalescent-based species-tree approach for pleurodonts. Although this analysis strongly supports monophyly of the pleurodont families, interfamilial relationships are generally different from those in the concatenated tree, and support is uniformly poor. However, a species-tree analysis using only the seven most variable loci yields higher support and more congruence with the concatenated tree. This suggests that low support in the 27-gene species-tree analysis may be an artifact of the many loci that are uninformative for very short branches. This may be a general problem for the application of species-tree methods to rapid radiations, even with phylogenomic data sets. Finally, we correct the non-monophyly of Polychrotidae by recognizing the pleurodont genus Anolis (sensu lato) as a separate family (Dactyloidae), and we correct the non-monophyly of the agamid genus Physignathus by resurrection of the genus Istiurus for the former Physignathus lesueurii.

Integrated analyses resolve conflicts over squamate reptile phylogeny and reveal unexpected placements for fossil taxa.

Squamate reptiles (lizards and snakes) are a pivotal group whose relationships have become increasingly controversial. Squamates include >9000 species, making them the second largest group of terrestrial vertebrates. They are important medicinally and as model systems for ecological and evolutionary research. However, studies of squamate biology are hindered by uncertainty over their relationships, and some consider squamate phylogeny unresolved, given recent conflicts between molecular and morphological results. To resolve these conflicts, we expand existing morphological and molecular datasets for squamates (691 morphological characters and 46 genes, for 161 living and 49 fossil taxa, including a new set of 81 morphological characters and adding two genes from published studies) and perform integrated analyses. Our results resolve higher-level relationships as indicated by molecular analyses, and reveal hidden morphological support for the molecular hypothesis (but not vice-versa). Furthermore, we find that integrating molecular, morphological, and paleontological data leads to surprising placements for two major fossil clades (Mosasauria and Polyglyphanodontia). These results further demonstrate the importance of combining fossil and molecular information, and the potential problems of estimating the placement of fossil taxa from morphological data alone. Thus, our results caution against estimating fossil relationships without considering relevant molecular data, and against placing fossils into molecular trees (e.g. for dating analyses) without considering the possible impact of molecular data on their placement.

Evolutionary diversification of clades of squamate reptiles

We analysed the diversification of squamate reptiles (7488 species) based on a new molecular phylogeny, and compared the results to similar estimates for passerine birds (5712 species). The number of species in each of 36 squamate lineages showed no evidence of phylogenetic conservatism. Compared with a random speciation–extinction process with parameters estimated from the size distribution of clades, the alethinophidian snakes (2600 species) were larger than expected and 13 clades, each having fewer than 20 species, were smaller than expected, indicating rate heterogeneity. From a lineage‐through‐time plot, we estimated that a provisional rate of lineage extinction (0.66 per Myr) was 94% of the rate of lineage splitting (0.70 per Myr). Diversification in squamate lineages was independent of their stem age, but strongly related to the area of the region within which they occur. Tropical vs. temperate latitude exerted a marginally significant influence on species richness. In comparison with passerine birds, squamates share several clade features, including: (1) independence of species richness and age; (2) lack of phylogenetic signal with respect to clade size; (3) general absence of exceptionally large clades; (4) over‐representation of small clades; (5) influence of region size on clade size; and (6) similar rates of speciation and extinction. The evidence for both groups suggests that clade size has achieved long‐term equilibrium, suggesting negative feedback of species richness on the rate of diversification.

Estimating divergence dates and evaluating dating methods using phylogenomic and mitochondrial data in squamate reptiles

Recently, phylogenetics has expanded to routinely include estimation of clade ages in addition to their relationships. Various dating methods have been used, but their relative performance remains understudied. Here, we generate and assemble an extensive phylogenomic data set for squamate reptiles (lizards and snakes) and evaluate two widely used dating methods, penalized likelihood in r8s (r8s-PL) and Bayesian estimation with uncorrelated relaxed rates among lineages (BEAST). We obtained sequence data from 25 nuclear loci (∼500-1,000 bp per gene; 19,020bp total) for 64 squamate species and nine outgroup taxa, estimated the phylogeny, and estimated divergence dates using 14 fossil calibrations. We then evaluated how well each method approximated these dates using random subsets of the nuclear loci (2, 5, 10, 15, and 20; replicated 10 times each), and using ∼1 kb of the mitochondrial ND2 gene. We find that estimates from r8s-PL based on 2, 5, or 10 loci can differ considerably from those based on 25 loci (mean absolute value of differences between 2-locus and 25-locus estimates were 9.0 Myr). Estimates from BEAST are somewhat more consistent given limited sampling of loci (mean absolute value of differences between 2 and 25-locus estimates were 5.0 Myr). Most strikingly, age estimates using r8s-PL for ND2 were ∼68-82 Myr older (mean=73.1) than those using 25 nuclear loci with r8s-PL. These results show that dates from r8s-PL with a limited number of loci (and especially mitochondrial data) can differ considerably from estimates derived from a large number of nuclear loci, whereas estimates from BEAST derived from fewer nuclear loci or mitochondrial data alone can be surprisingly similar to those from many nuclear loci. However, estimates from BEAST using relatively few loci and mitochondrial data could still show substantial deviations from the full data set (>50 Myr), suggesting the benefits of sampling many nuclear loci. Finally, we found that confidence intervals on ages from BEAST were not significantly different when sampling 2 vs. 25 loci, suggesting that adding loci decreased errors but did not increase confidence in those estimates.

Large-scale phylogeny of chameleons suggests African origins and Eocene diversification

Oceanic dispersal has emerged as an important factor contributing to biogeographic patterns in numerous taxa. Chameleons are a clear example of this, as they are primarily found in Africa and Madagascar, but the age of the family is post-Gondwanan break-up. A Malagasy origin for the family has been suggested, yet this hypothesis has not been tested using modern biogeographic methods with a dated phylogeny. To examine competing hypotheses of African and Malagasy origins, we generated a dated phylogeny using between six and 13 genetic markers, for up to 174 taxa representing greater than 90 per cent of all named species. Using three different ancestral-state reconstruction methods (Bayesian and likelihood approaches), we show that the family most probably originated in Africa, with two separate oceanic dispersals to Madagascar during the Palaeocene and the Oligocene, when prevailing oceanic currents would have favoured eastward dispersal. Diversification of genus-level clades took place in the Eocene, and species-level diversification occurred primarily in the Oligocene. Plio-Pleistocene speciation is rare, resulting in a phylogeny dominated by palaeo-endemic species. We suggest that contraction and fragmentation of the Pan-African forest coupled to an increase in open habitats (savannah, grassland, heathland), since the Oligocene played a key role in diversification of this group through vicariance.

Eastward from Africa: palaeocurrent-mediated chameleon dispersal to the Seychelles islands

Madagascar and the Seychelles are Gondwanan remnants currently isolated in the Indian Ocean. In the Late Cretaceous, these islands were joined with India to form the Indigascar landmass, which itself then split into its three component parts around the start of the Tertiary. This history is reflected in the biota of the Seychelles, which appears to contain examples of both vicariance- and dispersal-mediated divergence from Malagasy or Indian sister taxa. One lineage for which this has been assumed but never thoroughly tested is the Seychellean tiger chameleon, a species assigned to the otherwise Madagascar-endemic genus Calumma. We present a multi-locus phylogenetic study of chameleons, and find that the Seychellean species is actually the sister taxon of a southern African clade and requires accomodation in its own genus as Archaius tigris. Divergence dating and biogeographic analyses indicate an origin by transoceanic dispersal from Africa to the Seychelles in the Eocene–Oligocene, providing, to our knowledge, the first such well-documented example and supporting novel palaeocurrent reconstructions.