Haploweb analysis of ITS sequences of Leiopathes glaberrima (Cnidaria: Antipatharia)

This a reanalysis of the ITS sequences of Leiopathes glaberrima published in Ruiz-Ramos DV, Saunders M, Fisher CR, Baums IB (2015) Home bodies and wanderers: sympatric lineages of the deep-sea black coral Leiopathes glaberrima. PLoS ONE 10:e0138989 (https://doi.org/10.1371/journal.pone.0138989). I thank the authors for sharing with me the original chromatograms of their study.

The chromatograms were assembled into contigs in Sequencher then scrutinized to detect double peaks (using the procedure outlined in Fontaneto D, Flot J-F, Tang CQ (2015) Guidelines for DNA taxonomy, with a focus on the meiofauna. Marine Biodiversity 45:433–451; https://doi.org/10.1007/s12526-015-0319-7). Of the 43 individuals sequenced, 22 were homozygous (or had double peaks so small or noisy that it was impossible to infer their second haplotype with certainty, in which case they were considered as homozygous); two had a single double peak each (in which case phasing them was trivial); one had two double peaks (with strong differences in peak heights allowing direct phasing); and the remaining 18 were length-variant heterozygotes (characterized by many double peaks) that were phased using Champuru (Flot J-F (2007) Champuru 1.0: a computer software for unraveling mixtures of two DNA sequences of unequal lengths. Molecular Ecology Notes 7:974–977; https://doi.org/10.1111/j.1471-8286.2007.01857.x; available online at http://jfflot.mnhn.fr/champuru/). The two alleles of heterozygous individuals were named by appending -a and -b to the names of the corresponding individuals.

Sequences were aligned using MAFFT's E-INS-i mode (Katoh K, Rozewicki J, Yamada KD (2019) MAFFT online service: multiple sequence alignment, interactive sequence choice and visualization. Briefings in Bioinformatics 20:1160–1166; https://doi.org/10.1093/bib/bbx108; available online at https://mafft.cbrc.jp/alignment/server/) then the resulting FASTA alignment was turned into a haploweb (Flot J-F, Couloux A, Tillier S (2010) Haplowebs as a graphical tool for delimiting species: a revival of Doyle’s “field for recombination” approach and its application to the coral genus Pocillopora in Clipperton. BMC Evolutionary Biology 10:372; https://doi.org/10.1186/1471-2148-10-372) using HaplowebMaker (Spöri Y, Flot J (2020) HaplowebMaker and CoMa: two web tools to delimit species using haplowebs and conspecificity matrices. Methods in Ecology and Evolution;  https://doi.org/10.1111/2041-210X.13454; available online at https://eeg-ebe.github.io/HaplowebMaker/).

In the resulting haploweb all haplotypes are connected into a single field for recombination (FFR sensu Doyle JJ (1995) The irrelevance of allele tree topologies for species delimitation, and a non-topological alternative. Systematic Botany 20:574–588; https://doi.org/10.2307/2419811), thereby supporting the authors' original conclusion that all the specimens they sequenced were conspecific.

Included in this dataset are:

- a zipped folder containing the original chromatograms communicated by the authors (Leiopathes_ITS_chromatograms.zip);

- a Sequencher 4.1 project file containing the chromatograms assembled into contigs, cleaned and phased (Leiopathes_ITS_Sequencher.spf);

- a FASTA alignment of the phased sequences obtained (Leiopathes_ITS_MAFFT-E-INS-i.fasta);

- a HaplowebMaker project file (in JSON format) containing the results of the haploweb analysis (Leiopathes_ITS_JSON.HaplowebMaker);

- and a PDF printout of the final haploweb (Leiopathes_ITS_haploweb.pdf).