Comparing the selective landscape of TLR7 and TLR8 across primates reveals unique sites under positive selection in Alouatta.

Among primates, susceptibility to yellow fever (YFV), a single-stranded (ss) RNA virus, ranges from complete resistance to high susceptibility. Howler monkeys (genus Alouatta) are the most susceptible to YFV. In order to identify Alouatta-specific genetic factors that may be responsible for their susceptibility, we collected skin samples from howler monkey museum specimens of the species A. caraya and A. guariba clamitans. We compared the rate of nonsynonymous to synonymous (dN/dS) changes of Toll-like receptor (TLR) 7 and TLR8, the two genes responsible for detecting all ssRNA viruses, across the Primate order. Overall, we found that the TLR7 gene is under stronger purifying selection in howler monkeys compared to other New World and Old World primates, but TLR8 is under the same selective pressure. When we evaluated dN/dS at each codon, we found six codons under positive selection in Alouatta TLR8 and two codons under positive selection in TLR7. The changes in TLR7 are unique to A. guariba clamitans and are found in functionally important regions likely to affect detection of ssRNA viruses by TLR7/TLR8, as well as downstream signaling. These amino acid differences in A. guariba clamitans may play a role in YFV susceptibility. These results have implications for identifying genetic factors affecting YFV susceptibility in primates.

A very short, functionally constrained sequence diagnoses cone snails in several Conasprella clades.

The traditional taxonomy of ca. 700 cone snails assigns all species to a single genus, Conus Linnaeus 1758. However, an increasing body of evidence suggests that some belong to a phylogenetically distinct clade that is sometimes referred to as Conasprella. Previous work (Kraus et al., 2011) showed that a short (259 bp) conserved intronic sequence (CIS) of the γ-glutamyl carboxylase gene (intron 9) can be used to delineate deep phylogenetic relationships among some groups of Conus. The work described here uses intron 9 (338 bp) to resolve problematic relationships among the conasprellans and to distinguish them from Conus proper. Synapomorphic mutations at just 39 sites can resolve several groups within Conasprella because the informative region of intron 9 is so well conserved that the phylogenetic signal is not obscured by homoplasies at conflicting sites. Intron 9 also unambiguously distinguishes Conasprella as a whole from Conus because the conserved regions that are so well conserved within each group are not alignable and clearly not homologous between them. This pattern suggests that expression of the γ-glutamyl carboxylase gene may have undergone a functionally significant change in Conus or Conasprella shortly after they diverged.

Against expectation: a short sequence with high signal elucidates cone snail phylogeny.

A short (259 nucleotide) conserved intronic sequence (CIS) is surprisingly informative for delineating deep phylogenetic relationships in cone snails. Conus species previously have been assigned to clades based on the evidence from mitochondrial 12S and 16S rRNA gene sequences (1129 bp). Despite their length, these genes lack the phylogenetic information necessary to resolve the relationships among the clades. Here we show that the relationships can be inferred from just 46 sites in the very short CIS sequence (a portion of "intron 9" of the γ-glutamyl carboxylase gene). This is counterintuitive because in short sequences sampling error (noise) often drowns out phylogenetic signal. The intron 9 CIS is rich in synapomorphies that define the divergence patterns among eight clades of worm- and fish-hunting Conus, and it contains almost no homoplasy. Parsimony, maximum likelihood and Bayesian analyses of the combined sequences (mt rRNA+CIS) confirm most of the relationships among 23 Conus sequences. This phylogeny implies that fish-hunting behavior evolved at least twice during the history of Conus-once among New World species and independently in the Indo-Pacific clades.

Phylogeny of the genus Turris: correlating molecular data with radular anatomy and shell morphology.

There are over 10,000 species of venomous marine molluscs, the vast majority of these, which are generally referred to as "turrids", are traditionally assigned to a single family, Turridae (Powell 1966). Here, we provide an initial molecular analysis of the type genus of the family, Turris Röding, 1798, thought to be among the most well characterized groups in the family. We show that the type genus is not monophyletic. We analyzed specimens conventionally assigned to 9 different Turris species using molecular markers, combined with the shell morphology and radular anatomy whenever feasible. The results suggest that species assigned to the genus Turris, provisionally assigned to two different subgenera are not monophyletic. Five previously described species belong to the subgenus Turris (s.s.) Röding 1798: Turris babylonia, (Linne, 1758), Turris grandis, (J. E. Gray, 1834), Turris dollyae, (Olivera, 1999), Turris normandavidsoni (Olivera, 1999) and Turris spectabilis (Reeve, 1843). With a change in species designation, Turris assyria (formerly T. babylonia1010) is added to a well-defined clade, which is in turn more closely related to Lophiotoma and Gemmula species than to the other five Turris species. We show that these five species conventionally assigned to Turris do not belong in the same subgenus, and form a clade provisionally designated as AnnulaturrisPowell, 1966: Turris annulata, (Reeve, 1843), Turris undosa, (Lamarck, 1816), Turris cristata, (Vera-Peláez, Vega-Luz, and Lozano-Francisco 2000) Turris cryptorrhaphe (G. B. Sowerby, 1825) and Turris nadaensis (Azuma, 1973). Implications of the molecular phylogenetic results and its correlation with radular morphology are discussed.

Phylogeography of ninespine sticklebacks (Pungitius pungitius) in North America: glacial refugia and the origins of adaptive traits.

The current geographical distribution of the ninespine stickleback (Pungitius pungitius) was shaped in large part by the glaciation events of the Pleistocene epoch (2.6 Mya-1 Kya). Previous efforts to elucidate the phylogeographical history of the ninespine stickleback in North America have focused on a limited set of morphological traits, some of which are likely subject to widespread convergent evolution, thereby potentially obscuring relationships among populations. In this study, we used genetic information from both mitochondrial DNA (mtDNA) sequences and nuclear microsatellite markers to determine the phylogenetic relationships among ninespine stickleback populations. We found that ninespine sticklebacks in North America probably dispersed from at least three glacial refugia-the Mississippi, Bering, and Atlantic refugia-not two as previously thought. However, by applying a molecular clock to our mtDNA data, we found that these three groups diverged long before the most recent glacial period. Our new phylogeny serves as a critical framework for examining the evolution of derived traits in this species, including adaptive phenotypes that evolved multiple times in different lineages. In particular, we inferred that loss of the pelvic (hind fin) skeleton probably evolved independently in populations descended from each of the three putative North American refugia.

Evolution of Conus peptide toxins: analysis of Conus californicus Reeve, 1844.

Conus species are characterized by their hyperdiverse toxins, encoded by a few gene superfamilies. Our phylogenies of the genus, based on mitochondrial genes, confirm previous results that C. californicus is highly divergent from all other species. Genetic and biochemical analysis of their venom peptides comprise the fifteen most abundant conopeptides and over 50 mature cDNA transcripts from the venom duct. Although C. californicus venom retains many of the general properties of other Conus species, they share only half of the toxin gene superfamilies found in other Conus species. Thus, in these two lineages, approximately half of the rapidly diversifying gene superfamilies originated after an early Tertiary split. Such results demonstrate that, unlike endogenously acting gene families, these genes are likely to be significantly more restricted in their phylogenetic distribution. In concordance with the evolutionary distance of C. californicus from other species, there are aspects of prey-capture behavior and prey preferences of this species that diverges significantly from all other Conus.

Defining a Clade by Morphological, Molecular and Toxinological Criteria: Distinctive Forms related to Conus praecellens A. Adams, 1854.

We carried out a definition of the species group to which Conus praecellens A. Adams 1854 belongs using a combination of comparative morphological data, molecular phylogeny based on standard genetic markers and toxinological markers. Prior to this work, Conus praecellens was generally postulated to belong to a clade of similarly high-spired, smaller Conusspecies such as Conus pagodus Kiener, 1845, Conus memiae (Habe & Kosuge, 1970) and Conus arcuatus Broderip & Sowerby, 1829. The molecular phylogeny and toxinological data demonstrate that these prior hypotheses are incorrect, and that instead, Conus praecellens is in a branch of Conus that includes Conus stupa (Kuroda, 1956), Conus stupella (Kuroda, 1956), Conus acutangulus Lamark, 1810 and surprisingly, some species that are morphologically strikingly different, Conus mitratus Sowerby, 1870 and Conus cylindraceus Broderip & Sowerby, 1830. A more careful analysis of the morphologically diverse forms assigned to Conus praecellens suggests that from the Philippine material alone, there are at least three additional undescribed species, Conus andremenezi, Conus miniexcelsus and Conus rizali. A reevaluation of protoconch/early teleoconch morphology also strongly suggests that Conus excelsus Sowerby III, 1908 is related to these species. Together, the different data suggest a clade including the 10 species above that we designate, the Turriconus (Shikama and Habe, 1968) (clade; there are additional distinctive forms within the clade that may be separable at the species level. The phylogenetic definition using the multidisciplinary approach described herein provides a framework for comprehensively investigating biodiverse lineages of animals, such as the cone snails.

Multiple genes elucidate the evolution of venomous snail-hunting Conus species.

The species-rich Cone snails (Conus sp.) are predatory, marine gastropods known for small venom peptides that are valuable for pharmacological research applications. Phylogenetic analyses with mitochondrial rRNA sequences have facilitated peptide discovery. However, these relatively conserved genes leave unresolved the closer relationships among many species. We sequenced 26 internal transcribed spacer 2 (ITS2) sequences from genomic ribosomal DNA to elucidate the evolutionary relationships among molluscivorous species and to piscivorous and vermivorous species. We show that ITS2 sequences are well conserved within species but are sufficiently variable among species to resolve recent divergences. Using Bayesian, maximum likelihood and log-determinant methods, we use the ITS sequences to resolve portions of the tree that could not be resolved using the more conventional mt rRNA sequences. When the ITS2 sequences are added to existing COI and to the more conserved rRNA sequences and then properly modeled, support throughout the tree is increased. This enables us to show finer relationships among the molluscivorous species that reveal three well-supported clades (Conus, Cylinder, and Darioconus) and renders the ITS2 sequences an essential component in advancing the discovery and pharmacological characterization of novel peptides from the venoms of these molluscs.

Characterization of a venom peptide from a crassispirid gastropod.

The crassispirids are a large branch of venomous marine gastropods whose venoms have not been investigated previously. We demonstrate that crassispirids comprise a major group of toxoglossate snails in a clade distinct from all turrids whose venoms have been analyzed. The isolation and biochemical definition of the first venom component from any crassispirid is described. Crassipeptide cce9a from Crassispira cerithina (Anton, 1838) was purified from crude venom by following biological activity elicited in young mice, lethargy and a lack of responsiveness to external stimuli. Using Edman sequencing and mass spectrometry, the purified peptide was shown to be 29 amino acid residues long, with the sequence: GSCGLPCHENRRCGWACYCDDGICKPLRV. The sequence assignment was verified through the analysis of a cDNA clone encoding the peptide. The peptide was chemically synthesized and folded; the synthetic peptide was biologically active and coelution with the native venom peptide was demonstrated. When injected into mice of various ages, the peptide elicited a striking shift in behavioral phenotype between 14 and 16 days, from lethargy to hyperactivity.

THE INDO-PACIFIC GEMMULA SPECIES IN THE SUBFAMILY TURRINAE: ASPECTS OF FIELD DISTRIBUTION, MOLECULAR PHYLOGENY, RADULAR ANATOMY AND FEEDING ECOLOGY.

The biology, feeding ecology and phylogenetic relationships of marine snails in the family Turridae remain poorly understood. Here we report our study on four deep-water species in the genus Gemmula, a major group in this family. The four species G. speciosa (Reeve 1843), G. sogodensis (Olivera 2005), G. kieneri (Doumet 1940) and G. diomedea (Powell 1964) were collected at five different sites in the Philippines, and their pattern of distribution in the sites, their feeding behaviour as well as their phylogenetic relationships with each other and with other members of the subfamily Turrinae were investigated. The radular morphology (of two Gemmula species) and potential prey (for one Gemmula species) were also examined. Actual feeding observations were also conducted for Gemmula speciosa and compared with two turrids from other genera.All four Gemmula species showed strikingly different patterns of distribution; each species was found to be relatively much more abundant at one site but not at the other sites. Molecular phylogenetic analysis based on 16S sequences correlated with previously reported 12S sequences and revealed that the four species all belong to a well-supported Gemmula clade within the subfamily Turrinae; and that this clade appeared more closely related to the clades Xenuroturris, Turris and Lophiotoma than to the other clades in the subfamily (i.e., Turridrupa, Unedogemmula and Polystira). Morphological analysis of the radula of both G. speciosa and G. sogodensis revealed that the radulae of the two species were similar but differed from the other turrids, Lophiotoma acuta and Unedogemmula bisaya, by the absence of central teeth, consistent with the separation of the Gemmula clade from the Lophiotoma and Unedogemmula clade.To identify the polychaete group that is targeted as prey by species of Gemmula, analysis of regurgitated food fragments was made; phylogenetic analysis of an mtCOI gene fragment that was PCR-amplified from the regurgitated tissue of one specimen (G. diomedea) indicated close affinity of the prey to the terebellid polychaete Amphitritides. Specimens of Gemmula speciosa, when challenged with the terebellid polychaete Loimia sp., were observed to attack the worm suggesting that Gemmula species feed on terebellid polychaetes. Lophiotoma acuta were also observed to feed on the same species of terebellid but were usually group-feeding in contrast to the solitary feeding of G. speciosa. Unedogemmula bisaya did not feed on the terebellid which also supports the separation of the Gemmula and Unedogemmula clade.Two lines of proof (i.e. the molecular phylogenetic analysis and the feeding challenge) supporting the toxin homology findings previously reported, provide consistent evidence that Gemmula is a distinct clade of worm-hunting Turrinae that feeds on Terebellidae.