Catharine E. Pook

Coevolution of diet and prey-specific venom activity supports the role of selection in snake venom evolution

The processes that drive the evolution of snake venom variability, particularly the role of diet, have been a topic of intense recent research interest. Here, we test whether extensive variation in venom composition in the medically important viper genus Echis is associated with shifts in diet. Examination of stomach and hindgut contents revealed extreme variation between the major clades of Echis in the proportion of arthropod prey consumed. The toxicity (median lethal dose, LD50) of representative Echis venoms to a natural scorpion prey species was found to be strongly associated with the degree of arthropod feeding. Mapping the results onto a novel Echis phylogeny generated from nuclear and mitochondrial sequence data revealed two independent instances of coevolution of venom toxicity and diet. Unlike venom LD50, the speed with which venoms incapacitated and killed scorpions was not associated with the degree of arthropod feeding. The prey-specific venom toxicity of arthropod-feeding Echis may thus be adaptive primarily by reducing venom expenditure. Overall, our results provide strong evidence that variation in snake venom composition results from adaptive evolution driven by natural selection for different diets, and underscores the need for a multi-faceted, integrative approach to the study of the causes of venom evolution.

A nesting of vipers: Phylogeny and historical biogeography of the Viperidae (Squamata: Serpentes)

Despite their medical interest, the phylogeny of the snake family Viperidae remains inadequately understood. Previous studies have generally focused either on the pitvipers (Crotalinae) or on the Old World vipers (Viperinae), but there has been no comprehensive molecular study of the Viperidae as a whole, leaving the affinities of key taxa unresolved. Here, we infer the phylogenetic relationships among the extant genera of the Viperidae from the sequences of four mitochondrial genes (cytochrome b, NADH subunit 4, 16S and 12S rRNA). The results confirm Azemiops as the sister group of the Crotalinae, whereas Causus is nested within the Viperinae, and thus not a basal viperid or viperine. Relationships among the major clades of Viperinae remain poorly resolved despite increased sequence information compared to previous studies. Bayesian molecular dating in conjunction with dispersal-vicariance analysis suggests an early Tertiary origin in Asia for the crown group Viperidae, and rejects suggestions of a relatively recent, early to mid-Tertiary origin of the Caenophidia.

When continents collide: Phylogeny, historical biogeography and systematics of the medically important viper genus Echis (Squamata: Serpentes: Viperidae)

We analyze the phylogeny of the medically important and taxonomically unresolved viper genus Echis using four mitochondrial gene fragments. The results show that the populations of the genus fall into four main clades: the Echis carinatus, E. coloratus, E. ocellatus and E. pyramidum groups. The E. pyramidum and E. coloratus groups are sister taxa but the interrelationships of this clade and the E. ocellatus and E. carinatus groups are unresolved. The initial divergence of the genus appears to coincide with the collision between Afro-Arabia and Eurasia, and that between the E. coloratus and E. pyramidum clades appears to be associated with the opening of the Red Sea. Later land connections between Africa and Arabia may have contributed to shaping the distribution of the E. pyramidum complex. The present distribution of E. carinatus may be the result of range expansion from southern India. Taxonomically, our results provide molecular evidence for the validity of Echis omanensis, E. khosatzkii, E. borkini and E. jogeri, for the presence of unsuspected genetic diversity within the E. pyramidum complex in eastern Africa, and for the conspecificity of E. carinatus and E. multisquamatus. The status of E. leucogaster remains to be confirmed.

Molecular clocks and geological dates: cytochrome b of Anolis extremus substantially contradicts dating of Barbados emergence

Even though molecular clocks vary in rate to some extent, they are widely used and very important in a range of evolutionary studies, not least in interpreting cause and colonization in phylogeography. Evolutionists may use island age and emergence to give the earliest possible date for colonization by a species and hence give the lower limit in a molecular clock calibration. The geology of the Lesser Antilles is well studied and Barbados, although composed of some ancient rocks, is thought to have emerged only about 1million years ago (Ma). The cytochrome b mitochondrial gene is the most widely used gene in vertebrate phylogeography, and generally evolves at a rate of 1–2% per million years (Myr) for poikilothermic vertebrates. Divergence measured across almost all of this gene in the endemic anole (Anolis extremus) reveals a mean patristic distance of approximately 8.3% between this clade and its sister, together with distinct divergence and phylogeographical structure within Barbados. The divergence time, estimated by a range of procedures using four calibration points, is not in the least compatible with the proposed geological time of emergence of Barbados. Hence, either the molecular clock rate does not apply to the Barbadian anole population, or the geological dating of the emergence of Barbados is erroneous. The compatibility of geological times and molecular divergence of this complex on Martinique, together with relative rates tests comparing the rates on Barbados and Martinique, do not suggest atypical clock rates. The question of whether Barbados emerged much earlier than is currently thought, or whether the molecular clock assumptions are inappropriate, remains open.

Targeting optimal introns for phylogenetic analyses in non‐model taxa: experimental results in Asian pitvipers

Nuclear introns are increasingly used as phylogenetic markers. Here, we present a multidisciplinary approach towards optimal locus selection and amplification using Asian pitvipers as an example of a non‐model taxon, and raise the profile of length variant heterozygotes (LVHs) in intron loci. Taxon‐specific primers were identified using a bioinformatic approach, and also designed from existing exon primed, intron crossing (EPIC) primer amplifications. Eleven further universal EPIC primer pairs were assayed using a range of PCR optimization strategies. Taxon‐specific primers yielded the most consistent amplifications, but assaying a large number of universal EPIC primers yielded another appropriate locus for phylogenetic purposes. Modified Taq DNA polymerases such as JumpStart™Taq either significantly improved the specificity and yield of EPIC PCR amplifications (of low copy number nuclear targets), or resulted in amplifications that were not significantly worse than those derived from a generic Taq DNA polymerase. Finally, LVHs were detected in all loci that were sequenced suggesting that they are relatively common in introns. This study provides an efficient and cost effective template for the successful identification of intron markers for molecular systematics which is universally applicable to other non‐model taxon groups.