Dipartimento di Scienze della Natura e del Territorio – Sezione di Zoologia, Archeozoologia e Genetica, Centro di Eccellenza dell’Universit a di Sassari, Universit a di Sassari, Sassari Italy Biodiversity patterns in interstitial marine microturbellaria: a case study within the genus Parotoplana (Platyhelminthes: Rhabditophora) with the description of four new species MARCO CASU,FABIO SCARPA,VALENTINA DELOGU,PIERO COSSU,TIZIANA LAI ,DARIA SANNA and MARCO CURINI -GALLETTI Abstract Four new species of Otoplanidae (Platyhelminthes: Rhabditophora: Proseriata) with ranges restricted to the central Mediterranean are described. These species are characterized by the presence of a tubular copulatory stylet and by a bursa lacking an open connection to the atrium. Both characters are novel for the family Otoplanidae. The four species differ mainly in details of the morphology of the sclerotized structures of the copulatory organ. Generic attribution of the new species has been problematic. Morphological characters shared with either Parotoplanina or Parotoplanella were detected. Molecular data (based on 18S rRNA and 28S D1-D6 rRNA genes) evidenced that the new species constitute a monophyletic group falling within species of Parotoplana, with a sister/taxon relationship with Parotoplana spathifera. The genus Parotoplana, however, appears to be paraphy- letic, as Parotoplanella progermaria nests within Parotoplana species. The inadequate molecular sampling, combined with the lack of sequences from the type species of both Parotoplana and Parotoplanina, suggested a cautionary taxonomic approach, and the new species are therefore attributed to the earliest generic taxon available, Parotoplana. Key words: Microturbellarians – taxonomy – biodiversity – molecular analysis – phylogeny Introduction The recent register of the world’s marine species singled out the Rhabditophora as a taxon with a higher than average percentage of unknown species (Appeltans et al. 2012). This lack of knowl- edge is particularly acute for free-living marine interstitial groups (collectively known as microturbellarians), whose inventory is far from complete. Recent metagenetic evidence has revealed that microturbellarians may be particularly abundant in meiofaunal communities, although they are often neglected due to the extrac- tion techniques used in traditional ecological research (Fonseca et al. 2010). Given that most microturbellarians occupy top pred- ator roles in benthic ecosystems (Martens and Schockaert 1986), their prominence demonstrates that conventional diversity assess- ments may provide a distorted perspective of the relationships within the benthos. The discrepancy between the diversity and the ecological rele- vance of this group and its state of knowledge is obvious. The study of these minute interstitial organisms is, admittedly, prob- lematic, as external morphology provides no clues to taxonomy, and both living and sectioned specimens must be obtained for accurate observations (Cannon and Faubel 1988). The Proseriata (Rhabditophora: Neoophora) are among the largest taxa of marine microturbellarians; however, over 400 spe- cies known so far are thought to comprise only a fraction of their worldwide diversity (Curini-Galletti 2001; Appeltans et al. 2012). Furthermore, information on the actual geographic ranges of the Proseriata, and of microturbellarians in general, is particu- larly limited. This lack of information on distribution further complicates estimates of the global species richness of the taxon. Over the course of several sampling periods in the Mediterra- nean, we discovered specimens of Otoplanidae (Proseriata), belonging to four different species, that shared a hitherto unknown construction of the male sclerotized system, and dif- fered in subtle, yet constant, morphological details. The generic attribution of these new species was particularly problematic, either on morphological or on molecular grounds. These species are presented and discussed here. Materials and Methods Sampling and morphological study Samples were collected manually by scooping up the superficial layer of sediment. Extraction of the animals from the sediment was accomplished using MgCl 2 decantation (Martens 1984). Each specimen was first stud- ied alive by slight squeezing under the cover slip and then retrieved and treated for further analyses. For microscopical study, specimens were fixed in Bouin’s fluid, embedded in 60°C Paraplast, serial sagittal sections were obtained at 4 lm intervals, stained with Hansen’s haematoxylin and eosin-orange and mounted in Eukitt (see Beccari and Mazzi 1966 for details). The contribution to the description of the species by the various authors differs, and the authorship of the new species is limited to the authors who contributed to morphological description. Type material is stored in the collections of the Swedish Museum of Natural History (SMNH; Stockholm, Sweden). Additional voucher mate- rial is stored in the collection of the Zoological Museum of the Univer- sity of Sassari (Italy; CZM). Measurements of sclerotized structures of the male copulatory organ were taken on squeezed, karyological slides. We measured copulatory stylet, two companion spines and two lateral spines (see species descrip- tions). In species diagnoses, the ratio among sclerotized pieces is given as: (1) length of the copulatory stylet = 1; (2) length of companion spines/length of copulatory stylet; (3) length of lateral spines/length of copulatory stylet. For statistical analysis, the following parameters were measured (see spe- cies descriptions for explanation of the morphological terminology used): (1) length of copulatory stylet; (2) length of basal lobe/total length of copulatory stylet; (3) width-to-length ratio (W/L) of copulatory stylet; (4) length of companion spines; (5) length ratio between companion spines and copulatory stylet; (6) length of distal tip/total length of companion spines; (7) W/L of companion spines; Corresponding author: Marco Casu (marcasu@uniss.it) Contributing authors: Fabio Scarpa (fscarpa@uniss.it), Valentina Delogu (vdelogu@uniss.it), Piero Cossu (picossu@uniss.it), Tiziana Lai (laitiz@uniss.it), Daria Sanna (darsanna@uniss.it), Marco Curini-Galletti (curini@uniss.it) Accepted on 30 November 2013 © 2014 Blackwell Verlag GmbH J Zoolog Syst Evol Res doi: 10.1111/jzs.12058