Figure 1.
Morphological variation within the genera Nemertinoides and Sterreria.
Light microscope photographs of live specimens in squeeze preparation. a) Sterreria rubra from Southern Portugal, b) S. psammicola from Southern Portugal, c) S. martindalei n.sp. from Waimanolo, Hawaii, d) S. ylvae n.sp, from Waimanolo, Hawaii, e) S. variabilis n.sp. from New Caledonia, f) S. variabilis n.sp. from Bermuda, g) Nemertinoides elongatus from Southern Portugal, h) S. rubra from Helgoland, North Sea, i) N. glandulosum n.sp. from Southern Portugal, j) N. wolfgangi n.sp. from Croatia.
Table 1.
List of localities with geographic coordinates, the number of specimens per gene included in this study.
Table 2.
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.
Table 3.
Primers used in this study for sequencing of SSU, LSU and H3.
Figure 2.
Majority rule consensus tree (75%) of the LSU ML tree with collapsed terminals.
The colours correspond to partitions for BP&P analyses, green indicates the Nemertinoides group, red the mainly European Sterreria subgroup and blue the extra-European Sterreria; the distant S. martindalei n.sp. and S. papuensis 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 S. martindalei n.sp. and S. papuensis 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).
Figure 3.
Parsimony haplotype networks calculated with TCS.
a) LSU gene dataset, b) SSU gene dataset and c) Histone 3 gene dataset. The datasets were reduced and trimmed in order to reduce artefacts from missing data. The colours indicate the subgroups Nemertinoides (green), mainly European Sterreria (red) and extra-European Sterreria species (blue), and the not validated S. martindalei n.sp. and S. papuensis n.sp. in orange (see Fig 2). Some haplotypes are not connected to any other haplotype given the threshold and are represented by single boxes.
Table 4.
Summary of the results of the different species identification methods (per genetic marker) and the multi-locus species validation (BP&P).
Figure 4.
Results of the BP&P analyses for the tested species.
Results given as nodal support for all Nemertinoides species (green in Fig 2), mainly European Sterreria species (red in Fig 2) and the extra-European Sterreria species (blue in Fig 2). 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.
Figure 5.
World map showing the distribution of all named species of Nemertodermatida in this study, Europe is shown in an expanded view. Records for presence of Nemertinoides species are shown as squares, Sterreria species as dots with numbers corresponding to the species (see legend within Figure). Localities with records for “filiform” Nemertodermatida from the literature are marked as triangles and type localities of the previously described species are shown as stars.
Figure 6.
Diversity within the genus Nemertinoides.
a–c: Nemertinoides glandulosum 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: Nemertinoides elongatus. d) Detail of the male copulatory organ with false seminal vesicle. e) Overview of whole animal with position of the mco. f, g: Nemertinoides wolfgangi 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.
Table 5.
Molecular Diagnostic character of all newly described species in the three genes used in this study.
Figure 7.
Diversity within the genus Sterreria.
a–c: Sterreria lundini 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: Sterreria psammicola. 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: Sterreria rubra. 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: Stererria papuensis 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.
Figure 8.
Diversity within the genus Sterreria.
a: Sterreria martindalei n.sp. Overview over slightly damaged specimen with mature egg (e) and oocytes in the posterior. b, c: Sterreria ylvae n.sp. b) Anterior part with statocyst with double statoliths. c) Mid-section of the body with one mature egg (e) and few oocytes (oo). d, e: Sterreria monolithes n.sp. d) Overview over immature animal with only one statolith in the statocyst. e) Detail of the anterior of the same specimen with fanning out frontal gland opening and frontal gland secretions. The single statolith fills nearly the whole statocyst. f: Sterreria boucheti n.sp. Overview over complete, immature specimen. g, h: Sterreria variabilis n.sp. g) Overview over male mature specimens with club-shaped frontal gland secretions and food particles (f) in gut. h) Detail of male copulatory organ with fsv anterior of male pore (mp) and oocyte (oo). All photographs were taken of the live holotype specimens in squeeze preparation. The scale bars indicate 100 µm for each photograph.