Multiple causation of phylogeographical pattern as revealed by nested clade analysis of the bamboo viper (Trimeresurus stejnegeri) within Taiwan

In order to assess the utility of nested clade analysis, both standard phylogenetic algorithms and nested clade analysis were performed on a geographically widespread survey of mitochondrial DNA haplotypes of the bamboo viper, Trimeresurus stejnegeri, within Taiwan. Gross tree topologies were congruent for all analyses and indicated the presence of two geographically overlapping clades within Taiwan. The smaller lineage was restricted to the north and east coasts, whereas the larger lineage occupied all but the northern range of the species within Taiwan including the Pacific offshore populations of Green and Orchid Islands. The phylogeographical pattern supports the existence of at least one colonization event from the continent since the initial isolation of Taiwan from the mainland in the Pliocene. However, determining the exact number of colonization events was not possible due to the simultaneous vicariant forces of hypothesized continental landbridge connections and the occurrence of dramatic in situ orogenesis throughout the Pleistocene. Nested clade analysis provided multiple temporal and spatial population historical inferences that are not possible with standard analyses and therefore should become widely applied to future phylogeographical studies.     

The use of amplified fragment length polymorphism in determining species trees at fine taxonomic levels: analysis of a medically important snake, Trimeresurus albolabris

There are a huge number of phylogenetic studies based on mitochondrial DNA (mtDNA); however, these may represent gene trees that may not be congruent with the species tree. A solution to this problem is to include additional, independent, loci from the nuclear genome. At fine taxonomic levels, i.e. between populations and closely related species, previously suggested nuclear markers such as intron sequence data may not be appropriate. In this study we investigate the use of amplified fragment length polymorphisms (AFLP) to aid determination of the species tree for 24 specimens of a medically important snake, Trimeresurus albolabris. This is of particular importance for many venomous snakes as venom often varies intraspecifically. Five different primer combinations produced 434 bands and were analysed by constructing a phylogenetic tree using neighbour joining and principal component analysis. Results were similar across all methods and found distinct groupings. The results were compared with mtDNA data and a reconciled tree was constructed in order to determine the species tree for T. albolabris. We found that T. albolabris (sensu lato) is not monophyletic. Specimens from the Indonesian islands (except West Java) form a distinct clade and we propose elevation to species level. A specimen from Nepal is also distinct and suggests that this population also deserves specific status. We suggest that AFLPs may prove a valuable aid in determining species trees as opposed to gene trees at fine taxonomic levels and this should facilitate the incorporation of molecular data into such activities as antivenom production and conservation management.     

Enhancing the catalytic repertoire of nucleic acids: a systematic study of linker length and rigidity

The incorporation of potentially catalytic groups in DNA is of interest for the in vitro selection of novel deoxyribozymes. A series of 10 C5-modified analogues of 2'-deoxyuridine triphosphate have been synthesised that possess side chains of differing flexibility and bearing a primary amino or imidazole functionality. For each series of nucleotide analogues differing degrees of flexibility of the C5 side chain was achieved through the use of alkynyl, alkenyl and alkyl moieties. The imidazole function was conjugated to these C5-amino-modified nucleotides using either imidazole 4-acetic acid or imidazole 4-acrylic acid (urocanic acid). The substrate properties of the nucleotides (fully replacing dTTP) with TAQ polymerase during PCR have been investigated in order to evaluate their potential applications for in vitro selection experiments. 5-(3-Aminopropynyl)dUTP and 5-(E-3-aminopropenyl)dUTP and their imidazole 4-acetic acid- and urocanic acid-modified conjugates were found to be substrates. In contrast, C5-amino-modified dUTPs with alkane or Z-alkene linkers and their corresponding conjugates were not substrates. The incorporation of these analogues during PCR has been confirmed by inhibition of restriction enzyme digestion using XBAI and by mass spectrometry of the PCR products.     

4-Hydroxycinnamoyl-CoA: an ionizable probe of the active site of the medium chain acyl-CoA dehydrogenase

4-OH-Cinnamoyl-CoA has been synthesized as a probe of the active site in the medium chain acyl-CoA dehydrogenase. The protonated form of the free ligand (lambda(max) = 336 nm) yields the corresponding phenolate (lambda(max) = 388 nm) with a pK of 8.9. 4-OH-Cinnamoyl-CoA binds tightly (K(d) = 47 nM, pH 6) to the pig kidney dehydrogenase with a prominent new band at 388 nm, suggesting ionization of the bound ligand. However, this spectrum reflects polarization, not deprotonation, of the neutral form of the ligand. Thus, the 388 nm band is abolished as the pH is raised (not lowered), and analogous spectral and pH behavior is observed with the nonionizable analogue 4-methoxycinnamoyl-CoA. Studies with wild type, E99G, and E376Q mutants of the human medium chain acyl-CoA dehydrogenase showed that these two active site carboxylates strongly suppress ionization of the 4-OH ligand. Binding to the double mutant E99G/E376Q gives an intense new band as the pH is raised (pK = 7.8), with an absorbance maximum at 498 nm resembling the natural 4-OH-cinnamoyl-thioester chromophore of the photoactive yellow protein. Raman difference spectroscopy in water and D(2)O, using the free ligand and wild-type and double-mutant enzyme.ligand complexes, confirms that the 4-OH group of the thioester is ionized only when bound to the double mutant. These data demonstrate the strong electrostatic coupling between ligand and enzyme, and the critical role Glu376 plays in modulating thioester polarization in the medium chain acyl-CoA dehydrogenase.     

A phylogeny of the trimeresurus group of pit vipers: new evidence from a mitochondrial gene tree

The Trimeresurus group is an important radiation of over 40 Asian pit viper species. Once considered congeneric, four genera are generally currently recognized (Trimeresurus sensu stricto, Ovophis, Protobothrops, and Tropidolaemus) but relationships within and between these are still unclear. This study, based on mitochondrial cytochrome b sequences, is the first to include a large number of species (21) and demonstrates that the current taxonomy does not adequately represent either the relationships or the genetic diversity present in the complex. Although many deeper nodes are not strongly supported, the following novel conclusions are all well supported: (1) the paraphyly of Trimeresurus sensu stricto, (2) the presence of several divergent clades within Trimeresurus sensu stricto, (3) the paraphyly of some widespread, medically significant, species, (4) the nonmonophyly of Ovophis, and (5) the monophyly of Protobothrops. Mapping of morphological characters onto the mitochondrial tree further supports the four groups proposed for Trimeresurus sensu stricto.     

The dynamics of natural selection and vicariance in the Dominican anole: patterns of within-island molecular and morphological divergence

The larger islands of the Lesser Antilles are ecologically and geologically complex and are inhabited by single, but morphologically variable, Anolis species. Although earlier work has indicated that a large part of the morphological variation in Anolis oculatus from Dominica can be attributed to selection, a history of recurrent volcanic activity over the last few million years suggests that vicariance may have also played a significant role. We report a study of variation in the cytochrome b gene of mitochondrial DNA across the island to address this issue. We uncovered a very high degree of polymorphism, with an overall gene diversity of 0.97 and a nucleotide diversity of 0.04. Sequences, on average, differ by 3.82% and the maximum pairwise divergence (corrected for multiple hits) is 9.29%. Most haplotypes are restricted to single localities (a pattern not changed by increasing the sample size). Phylogenetic analysis revealed the presence of two distinct lineages on the island with strong phylogeographic structure. One of these is geographically restricted to a relatively small part of the central Caribbean coast. Sublineages were also discernible within the other more widely distributed lineage, but resolution within and support for these sublineages was poor. The phylogeographic pattern is not congruent with generalized body shape and scalation, but is significantly correlated with color pattern. Even when correcting for this lineage effect with partial Matrix correspondence tests, the relationship between color pattern and vegetation is reaffirmed, suggesting that although both vicariance and selection have played a role in the morphological differentiation of this species, selection for current environmental conditions has been more important. We discuss the causes of the phylogeographic structure in light of the volcanic history of the island and highlight the exceptional instance of congruence between all morphological character systems and lineage boundaries, which occurs at the transition between the northern and southern Caribbean ecotypes.     

Sticky-end assembly of a designed peptide fiber provides insight into protein fibrillogenesis

Coiled-coil motifs provide simple systems for studying molecular self-assembly. We designed two 28-residue peptides to assemble into an extended coiled-coil fiber. Complementary interactions in the core and flanking ion-pairs were used to direct staggered heterodimers. These had "sticky-ends" to promote the formation of long fibers. For comparison, we also synthesized a permuted version of one peptide to associate with the other peptide and form canonical heterodimers with "blunt-ends" that could not associate longitudinally. The assembly of both pairs was monitored in solution using circular dichroism spectroscopy. In each case, mixing the peptides led to increased and concentration-dependent circular dichroism signals at 222 nm, consistent with the desired alpha-helical structures. For the designed fiber-producing peptide mixture, we also observed a linear dichroism effect during flow orientation, indicative of the presence of long fibrous structures. X-ray fiber diffraction of partially aligned samples gave patterns indicative of coiled-coil structure. Furthermore, we used electron microscopy to visualize fiber formation directly. Interestingly, the fibers observed were at least several hundred micrometers long and 20 times thicker than expected for the dimeric coiled-coil design. This additional thickness implied lateral association of the designed structures. We propose that complementary features present in repeating structures of the type we describe promote lateral assembly, and that a similar mechanism may underlie fibrillogenesis in certain natural systems.     

Social status and growth rates as determinants of site attachment in juvenile Atlantic salmon

This paper describes an experimental study of the effects of food supply, growth rates and social interactions on homing by juvenile Atlantic salmon Salmo salar in response to displacement. Groups of five fish were housed in a section of an artificial stream and given either rations allowing maximum growth (the rich condition) or 0·1 of this amount (the poor condition); daily specific growth rates were significantly higher in the rich condition. After a 6-day settlement period, the fish were captured, displaced downstream and their movements recorded over the next 3 h. Prior to displacement, the fish showed a high degree of site fidelity and high levels of aggression. Dominant fish and those with stronger site attachment grew faster prior to displacement, these effects being independent. Following displacement, 24% of all fish returned to their previously favoured site and stayed there, 23% returned home initially, but subsequently moved on, 5% settled in a new site and 49% failed to move. The distribution of responses was identical for the rich and poor conditions, but fish that homed were dominant and had grown faster during the pre-displacement period.