Morphological variation within the genera <i>Nemertinoides</i> and <i>Sterreria</i>.

<p>Light microscope photographs of live specimens in squeeze preparation. a) <i>Sterreria rubra</i> from Southern Portugal, b) <i>S. psammicola</i> from Southern Portugal, c) <i>S. martindalei</i> n.sp. from Waimanolo, Hawaii, d) <i>S. ylvae</i> n.sp, from Waimanolo, Hawaii, e) <i>S. variabilis</i> n.sp. from New Caledonia, f) <i>S. variabilis</i> n.sp. from Bermuda, g) <i>Nemertinoides elongatus</i> from Southern Portugal, h) <i>S. rubra</i> from Helgoland, North Sea, i) <i>N. glandulosum</i> n.sp. from Southern Portugal, j) <i>N. wolfgangi</i> n.sp. from Croatia.</p

Molecular Diagnostic character of all newly described species in the three genes used in this study.

<p>Numbers refer to positions in the respective alignments and in brackets to the position in the sequences in the type specimen (genbank accession number).</p><p>Molecular Diagnostic character of all newly described species in the three genes used in this study.</p

Results of the BP&P analyses for the tested species.

<p>Results given as nodal support for all <i>Nemertinoides</i> species (green in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107688#pone-0107688-g002" target="_blank">Fig 2</a>), mainly European <i>Sterreria</i> species (red in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107688#pone-0107688-g002" target="_blank">Fig 2</a>) and the extra-European <i>Sterreria</i> species (blue in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107688#pone-0107688-g002" target="_blank">Fig 2</a>). Support values are Bayesian posterior probabilities for the different analyses in the order G(1/100), G(1/1000) and old root age (G(1/100) and G(1/10000)). The dataset was split in order to avoid artefacts due to unresolved topologies in the gene trees and increase confidence in the input topologies. Only clades represented by more than two specimens were tested in order to increase confidence.</p

Diversity within the genus <i>Sterreria.</i>

<p>a–c: <i>Sterreria lundini</i> n.sp. a) Overview over whole animal. b) Anterior with statocyst with double statoliths and oval frontal gland secretions. c) Posterior with fsv and male pore (mp). d, e: <i>Sterreria psammicola</i>. d) Posterior with fsv male pore (mp) and adhesive area (ad). e) Overview over male mature animal with large epidermal glands distributed more or less evenly over the whole body and mco in the posterior. f) Anterior part of an animal with epidermal glands, statocyst and central frontal gland opening (fp). g–j: <i>Sterreria rubra</i>. g) Posterior with two mature eggs (e) and mco. h) Overview over the same male and female mature specimen with mature eggs (e) and oocytes (oo). i) Anterior with statocyst and rod-shaped frontal gland secretions. j) Detail of the epidermis in the anterior of an animal with cell borders clearly visible, giving the animal a “scaly” appearance. k: <i>Stererria papuensis</i> n.sp. Anterior part of the holotype. All photographs are taken of live specimens in squeeze preparation. a, b, c, f and k are photographs of the holotype and neotype specimens respectively. The scale bars indicate 100 µm for each photograph.</p

List of all individuals used in this study sorted by clade, with Zoobank Life Science Identifiers (LSID) where applicable, connecting collection code (used in the scratchpads database for Acoela and Nemertodermatida at http://acoela.myspecies.info/), genbank accession numbers per gene and the museum collection numbers for type material.

<p>N. abbreviates the genus <i>Nemertinoides</i>, S. the genus <i>Sterreria</i>, abbreviations with numbers indicate putative species per genus not formally described in this paper. Type material is deposited at the Swedish Museum of Natural History (SMNH) in Stockholm, Sweden.</p><p>List of all individuals used in this study sorted by clade, with Zoobank Life Science Identifiers (LSID) where applicable, connecting collection code (used in the scratchpads database for Acoela and Nemertodermatida at <a href="http://acoela.myspecies.info/" target="_blank">http://acoela.myspecies.info/</a>), genbank accession numbers per gene and the museum collection numbers for type material.</p

Summary of the results of the different species identification methods (per genetic marker) and the multi-locus species validation (BP&P).

<p>The number of specimens is given per gene in the order LSU, SSU and H3. The uncorrected percentage of pairwise distances was calculated on the same reduced dataset as was used for the calculation of the parsimony networks; the lowest average percentage of pairwise distance between any two clades within the datasets versus the averaged intraspecific pairwise distance is shown. The parsimony networks were calculated with a threshold of 98% for LSU and SSU and 90% for H3; numbers specify the steps between the two clades in case they were recovered in one network, an asterisk indicates that a given clade was recovered in more than one network. Gene trees were calculated using RAxML and MrBayes, support is given in this order; non-monophyletic clades are indicated with a hash in superscript (^#). The program BP&P relies on a true guide tree, which due to the low taxon sampling we could not provide. We therefore decided to test only species with at least three specimens, thus some of the clades in the dataset were not tested and will not be named in this paper (except for S. martindalei and S. papuensis, which were well supported and described without BP&P validation). The posterior probabilities of the three different BP&P analyses are shown in the table. Due to the high variability of S. variabilis clade in all analyses, the clusters within this clade were tested for all analyses with the H3 gene. However, as this part of the tree was not resolved in either phylogenetic analysis, so no BP&B validation could be performed. A dash indicates missing data, or not tested clades.</p><p>Summary of the results of the different species identification methods (per genetic marker) and the multi-locus species validation (BP&P).</p

Majority rule consensus tree (75%) of the LSU ML tree with collapsed terminals.

<p>The colours correspond to partitions for BP&P analyses, green indicates the <i>Nemertinoides</i> group, red the mainly European <i>Sterreria</i> subgroup and blue the extra-European <i>Sterreria</i>; the distant <i>S. martindalei</i> n.sp. and <i>S. papuensis</i> n.sp. are shown in orange, as they was not tested with BP&P (s. text). Bootstrap support and Bayesian posterior probabilities are projected from different ML and Bayesian analyses in the order LSU, SSU and H3 where topologies were congruent. Clades supported in at least two of the three gene trees, present as separate networks by statistical parsimony, represented by at least three specimens, and validated by multi locus Bayesian analysis (except <i>S. martindalei</i> n.sp. and <i>S. papuensis</i> n.sp., see text), are formally described and named in this paper. Clades represented by two or less specimens were considered too poorly known for formal description but represent hypothetical species shown here with abbreviations (e.g. N1, S2).</p

List of localities with geographic coordinates, the number of specimens per gene included in this study.

<p>Species or clades collected at a given locality are shown with type localities for a given species shown in bold. N abbreviate clades belonging to the genus <i>Nemertinoides</i> and S indicates those belonging to the genus <i>Sterreria</i>.</p><p>List of localities with geographic coordinates, the number of specimens per gene included in this study.</p

Diversity within the genus <i>Nemertinoides</i>.

<p>a–c: <i>Nemertinoides glandulosum</i> n.sp. a) Posterior with mco. b) Overview (anterior region missing) of worm with mouth (m). c) Anterior with statocyst with double statoliths and frontal organ. d, e: <i>Nemertinoides elongatus</i>. d) Detail of the male copulatory organ with false seminal vesicle. e) Overview of whole animal with position of the mco. f, g: <i>Nemertinoides wolfgangi</i> n.sp. f) Anterior and posterior of fully mature animal with oocytes and fsv in the posterior. Only one statoliths in statocyst visible as photo is taken slightly laterally. g) Overview over the same animal with oocutes still visible but no fsv. Photographs were taken of live specimens, b–g are photographs of the respective holotypes. The scale bars indicate 100 µm for each photograph.</p