Kate L. Sanders

Molecular phylogeny and divergence dates for Australasian elapids and sea snakes (hydrophiinae): evidence from seven genes for rapid evolutionary radiations

One of the most prolific radiations of venomous snakes, the Australo‐Melanesian Hydrophiinae includes ∼100 species of Australasian terrestrial elapids plus all ∼60 species of viviparous sea snakes. Here, we estimate hydrophiine relationships based on a large data set comprising 5800 bp drawn from seven genes (mitochondrial: ND4, cytb, 12S, 16S; nuclear: rag1, cmos, myh). These data were analysed using parsimony, likelihood and Bayesian methods to better resolve hydrophiine phylogeny and provide a timescale for the terrestrial and marine radiations. Among oviparous forms, Cacophis, Furina and Demansia are basal to other Australian elapids (core oxyuranines). The Melanesian Toxicocalamus and Aspidomorphus group with Demansia, indicating multiple dispersal events between New Guinea and Australia. Oxyuranus and Pseudonaja form a robust clade. The small burrowing taxa form two separate clades, one consisting of Vermicella and Neelaps calanotus, and the other including Simoselaps, Brachyurophis and Neelaps bimaculatus. The viviparous terrestrial elapids form three separate groups: Acanthophis, the Rhinoplocephalus group and the Notechis–Hemiaspis group. True sea snakes (Hydrophiini) are robustly united with the Notechis–Hemiaspis group. Many of the retrieved groupings are consistent with previous molecular and morphological analyses, but the polyphyly of the viviparous and burrowing groups, and of Neelaps, are novel results. Bayesian relaxed clock analyses indicate very recent divergences: the ∼160 species of the core Australian radiation (including sea snakes) arose within the last 10 Myr, with most inter‐generic splits dating to between 10 and 6 Ma. The Hydrophis sea snake lineage is an exceptionally rapid radiation, with > 40 species evolving within the last 5 Myr.

Evaluating molecular clock calibrations using Bayesian analyses with soft and hard bounds

A limiting factor in many molecular dating studies is shortage of reliable calibrations. Current methods for choosing calibrations (e.g. cross-validation) treat them as either correct or incorrect, whereas calibrations probably lie on a continuum from highly accurate to very poor. Bayesian relaxed clock analysis permits inclusion of numerous candidate calibrations as priors: provided most calibrations are reliable, the model appropriate and the data informative, the accuracy of each calibration prior can be evaluated. If a calibration is accurate, then the analysis will support the prior so that the posterior estimate reflects the prior; if a calibration is poor, the posterior will be forced away from the prior. We use this approach to test two fossil dates recently proposed as standard calibrations within vertebrates. The proposed bird–crocodile calibration (approx. 247 Myr ago) appears to be accurate, but the proposed bird–lizard calibration (approx. 255 Myr ago) is substantially too recent.

Ecological diversification in a group of Indomalayan pitvipers (Trimeresurus): convergence in taxonomically important traits has implications for species identification.

We analyse molecular and phenotypic evolution in a group of taxonomically problematic Indomalayan pitvipers, the Trimeresurus sumatranus group. Mitochondrial DNA sequencing provides a well‐resolved phylogeny, with each species representing a distinct lineage. Multivariate morphological analysis reveals a high level of phenotypic differentiation, which is congruent between the sexes but does not reflect phylogenetic history. An adaptive explanation for the observed pattern of differentiation is supported by independent contrasts analysis, which shows significant correlations between current ecology and the characters that most account for the variation between taxa, including those that are presently used to identify the species. Reduced precipitation and altitude, and increased temperature, are correlated with higher numbers of scales on the head, body and tail. It is hypothesized that scale number plays an important role in heat and water exchange by influencing the area of exposed of interstitial skin, and that colour pattern variation reflects selection pressures involving camouflage and thermoregulation. Ecological convergence in traits used for classification is found to have important implications for species identification where taxa are distributed over varying environments.

Venom Down Under: Dynamic Evolution of Australian Elapid Snake Toxins

Despite the unparalleled diversity of venomous snakes in Australia, research has concentrated on a handful of medically significant species and even of these very few toxins have been fully sequenced. In this study, venom gland transcriptomes were sequenced from eleven species of small Australian elapid snakes, from eleven genera, spanning a broad phylogenetic range. The particularly large number of sequences obtained for three-finger toxin (3FTx) peptides allowed for robust reconstructions of their dynamic molecular evolutionary histories. We demonstrated that each species preferentially favoured different types of α-neurotoxic 3FTx, probably as a result of differing feeding ecologies. The three forms of α-neurotoxin [Type I (also known as (aka): short-chain), Type II (aka: long-chain) and Type III] not only adopted differential rates of evolution, but have also conserved a diversity of residues, presumably to potentiate prey-specific toxicity. Despite these differences, the different α-neurotoxin types were shown to accumulate mutations in similar regions of the protein, largely in the loops and structurally unimportant regions, highlighting the significant role of focal mutagenesis. We theorize that this phenomenon not only affects toxin potency or specificity, but also generates necessary variation for preventing/delaying prey animals from acquiring venom-resistance. This study also recovered the first full-length sequences for multimeric phospholipase A2 (PLA2) ‘taipoxin/paradoxin’ subunits from non-Oxyuranus species, confirming the early recruitment of this extremely potent neurotoxin complex to the venom arsenal of Australian elapid snakes. We also recovered the first natriuretic peptides from an elapid that lack the derived C-terminal tail and resemble the plesiotypic form (ancestral character state) found in viper venoms. This provides supporting evidence for a single early recruitment of natriuretic peptides into snake venoms. Novel forms of kunitz and waprin peptides were recovered, including dual domain kunitz-kunitz precursors and the first kunitz-waprin hybrid precursors from elapid snakes. The novel sequences recovered in this study reveal that the huge diversity of unstudied venomous Australian snakes are of considerable interest not only for the investigation of venom and whole organism evolution but also represent an untapped bioresource in the search for novel compounds for use in drug design and development.

Phylogeny and divergence times of filesnakes (Acrochordus): Inferences from morphology, fossils and three molecular loci

Acrochordus is a species-poor but highly distinctive aquatic snake genus currently distributed from India to the western edge of the Pacific. We provide the first phylogeny for the three extant species using Bayesian and parsimony analyses of one mitochondrial and two nuclear gene sequences. Acrochordus javanicus is strongly recovered as sister to A. arafurae+A. granulatus, counter to expectations from superficial ecology, external phenotype and former taxonomy. We review and revise key fossil calibrations for dating snake divergences. Bayesian relaxed-clock analysis of the two nuclear loci yields deep interspecific divergences among extant species that occurred during the Miocene approximately 16 and approximately 20Mya (million years ago), pre-dating at least two of the three other living marine snake lineages. New morphological data for A. arafurae, and our molecular timescale, provide support for the placement of fossil taxon A. dehmi within the Acrochordus crown group, as sister to A. javanicus among nominate species. Finally, Acrochordus phylogeny provides an improved basis for taxon selection and character polarization in higher snake phylogenetics. Our study highlights the three Acrochordus species as old and highly distinct lineages that comprise an important component of the threatened Indo-Australian biodiversity.

Arthropod molecular divergence times and the Cambrian origin of pentastomids

Pentastomida are parasites of tetrapods (especially reptiles) and have had long contentious relationships. Traditionally seen as a separate phylum with arthropod affinities, recent ultrastructural and molecular evidence unites them to branchiuran crustaceans (fish lice). However, the discovery of Cambrian pentastomids has been interpreted to refute the view that pentastomids are closely related to advanced crustaceans (of presumably recent origins). Bayesian phylogenetic and relaxed-clock analyses of molecular data (18S and 28S ribosomal RNA, elongation factor 1α and 2, RNA polymerase II subunit) help reconcile these apparently contradictory views: a pentastomid-branchiuran grouping is recovered, but this clade is the sister group to other Tetraconata, whereas all other crustaceans form a monophyletic group that is sister to hexapods. Crustacean monophyly is widely supported by morphology, but has rarely been retrieved in other molecular studies. Estimated molecular dates for pentastomid origins range from ∼490 to ∼520 million years ago, consistent with Cambrian fossils. The basal position of branchiurans within tetraconatans means pentastomids can be simultaneously related to branchiurans, and also have a deep fossil record. All Cambrian pentastomids are tiny, resembling the larvae of living pentastomids that infect intermediate hosts including fish; as definitive hosts (tetrapods) of modern pentastomids were absent in the Cambrian, it is possible that the small Cambrian forms were adults rather than larvae, and their entire life cycle was confined to small fish-like vertebrates that were then present.

Uncoupling ecological innovation and speciation in sea snakes (Elapidae, Hydrophiinae, Hydrophiini).

The viviparous sea snakes (Hydrophiini) are by far the most successful living marine reptiles, with ∼60 species that comprise a prominent component of shallow‐water marine ecosystems throughout the Indo‐West Pacific. Phylogenetically nested within the ∼100 species of terrestrial Australo‐Melanesian elapids (Hydrophiinae), molecular timescales suggest that the Hydrophiini are also very young, perhaps only ∼8–13 Myr old. Here, we use likelihood‐based analyses of combined phylogenetic and taxonomic data for Hydrophiinae to show that the initial invasion of marine habitats was not accompanied by elevated diversification rates. Rather, a dramatic three to six‐fold increase in diversification rates occurred at least 3–5 Myr after this transition, in a single nested clade: the Hydrophis group accounts for ∼80% of species richness in Hydrophiini and ∼35% of species richness in (terrestrial and marine) Hydrophiinae. Furthermore, other co‐distributed lineages of viviparous sea snakes (and marine Laticauda, Acrochordus and homalopsid snakes) are not especially species rich. Invasion of the oceans has not (by itself) accelerated diversification in Hydrophiini; novelties characterizing the Hydrophis group alone must have contributed to its evolutionary and ecological success.

Diversification rates and phenotypic evolution in venomous snakes (Elapidae)

The relationship between rates of diversification and of body size change (a common proxy for phenotypic evolution) was investigated across Elapidae, the largest radiation of highly venomous snakes. Time-calibrated phylogenetic trees for 175 species of elapids (more than 50% of known taxa) were constructed using seven mitochondrial and nuclear genes. Analyses using these trees revealed no evidence for a link between speciation rates and changes in body size. Two clades (Hydrophis, Micrurus) show anomalously high rates of diversification within Elapidae, yet exhibit rates of body size evolution almost identical to the general elapid ‘background’ rate. Although correlations between speciation rates and rates of body size change exist in certain groups (e.g. ray-finned fishes, passerine birds), the two processes appear to be uncoupled in elapid snakes. There is also no detectable shift in diversification dynamics associated with the colonization of Australasia, which is surprising given that elapids appear to be the first clade of venomous snakes to reach the continent.

Anonymous nuclear loci in non-model organisms

MOTIVATION When working with non-model organisms, few if any species-specific markers are available for phylogenetic, phylogeographic and population studies. Therefore, researchers often try to adapt markers developed in distantly related taxa, resulting in poor amplification and ascertainment bias in their target taxa. Markers can be developed de novo and anonymous nuclear loci (ANL) are proving to be a boon for researchers seeking large numbers of fast-evolving, independent loci. However, the development of ANL can be laboratory intensive and expensive. A workflow is described to identify suitable low-copy anonymous loci from high-throughput shotgun sequences, dramatically reducing the cost and time required to develop these markers and produce robust multilocus datasets. RESULTS By successively removing repetitive and evolutionary conserved sequences from low coverage shotgun libraries, we were able to isolate thousands of potential ANL. Empirical testing of loci developed from two reptile taxa confirmed that our methodology yields markers with comparable amplification rates and nucleotide diversities to ANLs developed using other methodologies. Our approach capitalizes on next-generation sequencing technologies to enable the development of phylogenetic, phylogeographic and population markers for taxa lacking suitable genomic resources.

Evidence for a Müllerian mimetic radiation in Asian pitvipers

Müllerian mimicry, in which toxic species gain mutual protection from shared warning signals, is poorly understood in vertebrates, reflecting a paucity of examples. Indirect evidence for mimicry is found if monophyletic species or clades show parallel geographic variation in warning patterns. Here, we evaluate a hypothesis of Müllerian mimicry for the pitvipers in Southeast Asia using a phylogeny derived from DNA sequences from four combined mitochondrial regions. Mantel matrix correlation tests show that conspicuous red colour pattern elements are significantly associated with sympatric and parapatric populations in four genera. To our knowledge, this represents the first evidence of a Müllerian mimetic radiation in vipers. The putative mimetic patterns are rarely found in females. This appears paradoxical in light of the Müllerian prediction of monomorphism, but may be explained by divergent selection pressures on the sexes, which have different behaviours. We suggest that biased predation on active males causes selection for protective warning coloration, whereas crypsis is favoured in relatively sedentary females.