Cloacal gaping in females.

<p>The graphs represent female Relative Cloacal Gaping (RCG) at t₀ and t_max in courtship water and aged tap water (negative control). RCG was measured as the cloacal width (cw) divided by the tail base width (tb). The width of the cloaca was measured orthogonal to the center of the cloacal length (cl). The tail base was measured at the hind limb initiation region. If the cloaca was closed, RCG was set to 0.00.</p

Late Cretaceous vicariance in Microhylidae and Natatanura.

<div><p>(<b>A</b>) Molecular timetree (TK method, all calibration points except G <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000074#pone.0000074-1" target="_blank">[12]</a>).</p> <p>Horizontal colored bars and lines at internal nodes (Standard deviation and 95% credibility interval, respectively) indicate vicariance events reconstructed by DIVA-analyses, and interpreted as follows: orange: Australia <–> Indo-Madagascar; yellow: Africa <–> South America; blue: Africa <–> Indo-Madagascar; purple: Madagascar <–> India (Seychelles); green: S. America-Antarctica <–> Indo-Madagascar (the intervening Kerguelen Plateau being involved).</p> <p>The latter splits in our timetree are interpreted as vicariance events between the Kerguelen plateau and Antarctica or Indo-Madagascar <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000074#pone.0000074-1" target="_blank">[12]</a>.</p> <p>The branches denoting the latest colonization of Eurasia, as reconstructed by DIVA, are indicated by an asterisk.</p> <p>Numbers at terminals correspond to taxon numbers in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000074#pone-0000074-g001" target="_blank">figure 1</a>. (<b>B</b>) Late Cretaceous Gondwana, with indication of corresponding geological break-ups.</p> <p>Abbreviations: AF = Africa, MA = Madagascar, IN = India, EU = Eurasia, SA = South America, AN = Antarctica, AU = Australia-New Guinea, KP = Kerguelen Plateau.</p></div

Structure of hymenochirin transcript 1 and comparison to other precursors and AMPs.

<p><b>A</b> Structure of hymenochirin transcript 1 (obtained from a breeding skin gland cDNA library of <i>Hymenochrius boettgeri</i> males) and of two AMP precursor proteins from <i>Xenopus laevis</i> and <i>Silurana tropicalis</i>. preproPGLa-Xl1: <i>X. laevis</i> PGLa precursor; preproCPF-St7: <i>S. tropicalis</i> CPF precursor. Region coloration distinguishes UTR (white), and sequences encoding signal peptide (dark green), spacer (light green) and antimicrobial peptides (blue). Dashed lines indicate known exon boundaries in <i>Xenopus</i> and <i>Silurana</i> AMP precursors. <b>B</b> Comparative alignment of the deduced amino acid sequence of the coding part of hymenochirin transcript 1 with two <i>X. laevis</i> and <i>S. tropicalis</i> AMP precursors and the previously published hymenochirins <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086339#pone.0086339-Mechkarska4" target="_blank">[27]</a>. Predicted signal peptides are printed in lower case, antimicrobial peptides are printed in bold. Amino acids shared between the hymenochirin precursor and at least one of the other precursors are indicated in light grey. Residues identical between all hymenochirin peptides but not present in preproPGLa-Xl1 or preproCPF-St7 are labeled in dark grey. Small arrowheads indicate putative cleavage sites.</p

Overview of hymenochirin transcripts: amino acid sequences, structure and encoded peptides.

<p><b>A</b> Deduced amino acid sequences of the hymenochirin transcripts. Previously published hymenochirins <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086339#pone.0086339-Mechkarska4" target="_blank">[27]</a> are marked in grey; predicted novel encoded hymenochirins are marked in black. Small arrowheads indicate putative cleavage sites for the hymenochirins. Names of encoded hymenochirins are indicated on the right. <b>B</b> Comparative schematic representation of repeat sequences in the transcripts. The number of cDNA sequences represented by each transcript is indicated between brackets. Each repeat is represented by one larger and one smaller block (repeat sections), corresponding to exons 2 and 3 in <i>S. tropicalis</i> and <i>X. laevis</i> AMP genes. The numbers in the blocks correspond to unique repeat sections as used in the phylogenetic analyses. Hymenochirins encoded by the corresponding transcripts are indicated on the right; previously published hymenochirins <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086339#pone.0086339-Mechkarska4" target="_blank">[27]</a> are labeled grey, the novel hymenochirins are labeled black.</p

Helical wheel projections of the four peptides that were synthesized and tested for antimicrobial activity.

<p><b>A</b> hymenochirin-6B; <b>B</b> hymenochirin-7B; <b>C</b> hymenochirin-10B; <b>D</b> hymenochirin-12B. Only the region of the peptide predicted to have an alpha-helical structure with a confidence level of 5 or more (PSIPRED v3.0 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086339#pone.0086339-Buchan1" target="_blank">[30]</a>) is shown in the projection. Amino acids are shaded according to the Combined Consensus Scale <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086339#pone.0086339-Tossi1" target="_blank">[31]</a> with hydrophobic residues in white, nearly neutral residues in light grey, polar residues in dark grey, charged residues in black with+or - sign indicating charge. Helical wheels are adapted from <a href="http://cti.itc.virginia.edu/~cmg/Demo/wheel/wheelApp.html" target="_blank">http://cti.itc.virginia.edu/~cmg/Demo/wheel/wheelApp.html</a>. Circle sizes indicate the relative distance from the N-terminal end of the peptide; smaller circles are further away.</p

Figure 6. Phylogenetic relationships among pipid AMPs (exon 2).

<p>Phylogenetic relationships as inferred by Bayesian analysis of a data set consisting of exon 2 of amphibian cck genes and AMP genes in <i>X. laevis</i> and <i>S. tropicalis</i>, aligned with the corresponding repeat sections of the hymenochirin precursor proteins. Repeat numbers correspond to those in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086339#pone-0086339-g002" target="_blank">Figure 2</a>. The depicted tree represents the Bayesian consensus phylogram rooted with cck genes. Branches are shown in bold if Bayesian posterior probability is above 0.95 and RAxML bootstrap is more than 75%.</p

Comparison of pairwise sequence similarities between AMPs of <i>H. boettgeri</i>, <i>S. tropicalis</i> and <i>X. laevis</i>.

<p>Box plots comparing the distribution of pairwise sequence similarities (in %) between the 14 hymenochirins of <i>H. boettgeri</i> and all known AMP peptides of <i>S. tropicalis</i> and <i>X. laevis</i> respectively. Boxes indicate median, and 25- and 75- percentiles, and whiskers indicate minimum and maximum values.</p

Alignment of all known and predicted hymenochirin peptides.

<p>Amino acids shared by more than 50% of the peptides are marked in grey. Brackets delineate a conserved central sequence motif.</p

Structural properties of the novel hymenochirin peptides predicted from the <i>H. boettgeri</i> transcripts.

<p>Average mass was calculated with <a href="http://www.peptidesynthetics.co.uk/tools" target="_blank">http://www.peptidesynthetics.co.uk/tools</a>. Hydrophobicity was calculated following the Combined Consensus Scale <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086339#pone.0086339-Tossi1" target="_blank">[31]</a> as implemented in HydroMCalc. The mean hydrophobic moment is the vectorial sum of all the hydrophobicity indices, divided by the number of residues. The mean hydrophobicity is the total hydrophobicity (sum of all residue hydrophobicity indices) divided by the number of residues. Helicity was calculated with PSIPRED v3.0 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086339#pone.0086339-Buchan1" target="_blank">[30]</a>.</p

Number of codon sites predicted to evolve under diversifying or purifying selection according to three different methods.

a<p>Using a Bayes factor of 50.</p>b<p>Using a 0.05 significance level.</p>c<p>calculated on the Bayesian consensus trees.</p>d<p>minimum and maximum number of codon sites calculated on 20 randomly selected trees from the Baysesian posterior tree set.</p