Bergia_Montenegroetal2015

Zip file containing the full size, masked and concatenate alignments used for the phylogenetic reconstructions presented in Montenegro et al. (2015)

Sample information for specimens analyzed to determine prevalence of palytoxins in commercially available zoanthids.

<p>*closest match from publically available sequences (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0018235#pone-0018235-g002" target="_blank">Fig. 2</a>).</p><p>**also matched single COI sequence for <i>Zoanthus sociatus</i> (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0018235#pone-0018235-g002" target="_blank">Fig. 2</a>).</p><p><u>Weakly-Toxic</u> - defined as possessing a hemolytic component that was inhibited with an anti-PLTX antibody but too low in concentration to confirm as a PLTX by additional chemical means (methods described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0018235#pone.0018235-Deeds1" target="_blank">[8]</a>).</p><p><u>Non-Toxic</u> – defined as no detectable toxic activity using a hemolysis neutralization assay and no HPLC peak at 263 nm consistent with a PLTX standard.</p

Masses (<i>m/z</i>) used to determine palytoxin type (Top) and corresponding palytoxin detection in samples (Bottom).

<p>(Top) Calculated <i>m/z</i> values for palytoxin, 42-hydroxy-palytoxin, and deoxy-palytoxin, and their various derivatives, used to determine palytoxin type in aquarium store zoanthids using high resolution mass spectrometry.</p><p>(Bottom) Palytoxin type in highly toxic samples determined from information above. [+++] Major toxin: all ions present [except (M+3H)³⁺ - see text]. [+] Minor toxin: select ions also present as dominant ion in isotopic cluster [mainly (M+2H-H₂0)²⁺ and (M+2H+K)³⁺]. [−] No listed ions detected as dominant ion in isotopic cluster.</p

Toxin analysis of zoanthid samples collected from aquarium stores.

<p>High resolution mass spectrometric (left column) and high performance liquid chromatographic (right column) analysis of [A] palytoxin standard, from <i>P. tuberculosa</i>, purchased from Wako Pure Chemicals, Tokyo, Japan, [B] sample 305.11.2 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0018235#pone-0018235-g002" target="_blank">Figure 2C</a>), purchased from aquarium store #1, which contained approx. 500 µg palytoxin/g wet zoanthid and [C] sample 306.37.3 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0018235#pone-0018235-g002" target="_blank">Figure 2D</a>), purchased from aquarium store #2, which contained approx. 3500 µg deoxy-palytoxin/g wet zoanthid. For all 3, the inset graph in the right column is the UV spectra for the HPLC peak at 12.4 minutes. All samples contained a 327 Da. fragment and UV maxima at 233 and 263 nm, which are characteristic of palytoxins.</p

Species identification of zoanthid samples collected from aquarium stores.

<p>Maximum-likelihood (ML) phylogenies of zoanthids showing both the 16S [A] and COI [B] data sets. Support above branches: ML bootstraps/Neighbor-joining bootstraps/Bayesian posterior probabilities. [C-K] Representative specimens for <i>Palythoa</i> spp. (both toxic and non-toxic/weakly-toxic) and <i>Zoanthus</i> spp. (all non- or weakly-toxic) collected from local aquarium stores including: [C] sample 305.11.2, [D] sample 306.37.3, [E] sample 306.39.2, [F] samples 306.39.3 [open arrows] and sample 306.39.4 [closed arrows], [G] sample 305.13.1, [H] sample 306.39.1, [I] sample 305.11.6, [J] sample 305.11.5, and [K] sample 305.11.3. [Red Box] Visually and genetically consistent with <i>Palythoa</i> spp. and containing high concentrations of palytoxins (500–3500 µg/g wet zoanthid). [Green Box] Visually and genetically consistent with <i>Palythoa</i> spp. but non- or weakly-toxic, and [Blue Box] visually and genetically consistent with <i>Zoanthus</i> spp., and non- or weakly-toxic. For [C-K], bar represents 1 cm.</p

Zoanthid colony responsible for a severe respiratory reaction, collected from a home aquarium in 2008.

<p>[Referred to as VAZOA in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0018235#pone-0018235-g002" target="_blank">Figure 2A</a> and Virginia zoanthid in text]. Sample was found to contain approx. 600 µg palytoxin/g wet zoanthid. Bar represents 1 cm.</p

Relationship between <i>Desmophyllum dianthus</i> and principal taxa from Scleractinia families based on mitochondrial COI.

<p>Phylogenetic relationships among <i>D. dianthus</i> and representative species of the family Caryophylliidae. R, C and B indicate “robust”, “complex” and “basal” groups, respectively. The phylogenetic relationships were inferred by BI, MP and ML criteria (numbers show the Bayesian posterior probability and bootstrap supports given at branches, respectively). Stars indicate other well-supported clades (pp≥95; bootstrap >70).</p

Measures of DNA polymorphism and neutrality tests (Tajima's D and Fu's Fs tests) for nuclear and mitochondrial DNA marker sequences of <i>Desmophyllum dianthus</i> specimens.

<p>S = segregating (polymorphic) sites; η = total number of substitutions; <b>Hd</b> = haplotype diversity; <b>p</b> = nucleotide diversity; D = Tajima's D value; Fs = Fu's Fs value; p = p value.</p

Haplotypes network.

<p>Parsimony network of internal transcribed spacer (ITS) ribosomal DNA sequence haplotypes of <i>Desmophyllum dianthus</i> belonging to Mediterranean Sea populations (from this study) and South Pacific Ocean populations (in blue; from Miller <i>et al. </i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050215#pone.0050215-Miller1" target="_blank">[35]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050215#pone.0050215-Miller2" target="_blank">[36]</a>). Sizes of the circles are proportional to the number of samples presenting such haplotype. Numbers indicate the variable positions. A) Network based on depth (white = shallow <600 m; light green = medium 600–1000 m; dark green = deep >1000 m). B) Network based on sampling area (red = Ionian Sea; orange = Adriatic Sea; yellow = Strait of Sicily).</p

Lengths of the PCR products and the respective alignments of <i>Desmophyllum dianthus</i> and the main scleractinian family taxa (for more details see Fig. 1 and Table S2), along with the best-fit models selected by AIC in Modeltest 3.7.

*<p>Considering gaps as a fifth state of characters.</p