A recent study of the magnitude of marine biodiversity downscaled the global number of species to the lower end of previous estimates (Appeltans et al., 2012). This result has been challenged on the basis that, in many taxa, the actual number of cryptic species may be underestimated (Mora et al., 2013; Adams et al., 2014). The lack of resolution of cryptic complexes, whose species may have limited geographic ranges and unique ecological requirements (Cox and Moore, 2005), may indeed hinder our chances of determining both the actual level of biodiversity and the extinction risks of the individual components of a cryptic complex.
Molecular techniques are the preferred tool for the resolution of cryptic complexes and, although they have been applied in a minority of instances (see Jörger and Schrödl, 2013 and references therein), nowadays their use is increasing following the implementation of new specific software for automated species delimitation (see Fontaneto et al., 2015). This is particularly true for obscure, minute marine meiofaunal organisms, for which even the description of morphologically distinct species still lags given the tremendous heterogeneity and species-richness of this group. For instance, a recent study of two nominal species of Nemertodermatida, which revealed the existence of 20 supported species (Meyer-Wachsmuth et al., 2014), is of particular interest as it points to the existence of hyper-cryptic complexes in morphologically simple, ‘worm-like’ taxa that lack clear diagnostic features.
Among meiofaunal organisms, the marine microturbellarians (Platyhelminthes) are a group whose contribution to marine biodiversity has yet to be fully assessed (Appeltans et al., 2012). They may be tremendously common and species-rich in littoral marine habitats (Martens and Schockaert, 1986), ranking second in abundance after Nematoda (Fonseca et al., 2010), as evidenced by environmental metagenomics (Fonseca et al., 2014). This group includes known examples of hyper-cryptic complexes; for instance, in the supposedly cosmopolitan Gyratrix hermaphroditus Ehrenberg, 1831, studies of karyotype and fine morphology have revealed the existence of seven cryptic species in a small portion of a tidal creek at Darwin (NT, Australia) (Curini-Galletti and Puccinelli, 1990). The paucity of diagnostic morphological characters is particularly felt in a taxon of marine microturbellaria, the Monocelididae Monocelidinae (Proseriata), where the copulatory organ of the simplex type (Litvaitis et al., 1996) provides very limited taxonomic information, and species detection and diagnoses increasingly rely on nucleotide-based information (Casu et al., 2009). The case of Monocelis lineata O.F. Müller, 1774 is exemplary as the nominal species has an unusually wide distribution for an interstitial microturbellarian, ranging from both coasts of the North Atlantic through the entire Mediterranean and the Black Sea, and a similarly unusually wide ecological tolerance, occurring in brackish to marine habitats on any type of substrate (Ax, 1956). In stark contrast, most Mediterranean species of Proseriata have ranges limited to single sectors of the basin, and either occur in brackish-water or fully marine habitats (Martens and Curini-Galletti, 1994; Delogu and Curini-Galletti, 2009; Casu et al., 2014). A previous study carried out by means of allozyme electrophoresis (Casu and Curini-Galletti, 2004) on 15 populations morphologically attributable to M. lineata, based on the general morphology of the reproductive structures, suggested the presence of a complex composed of at least six sibling species in Europe alone: five species from the Mediterranean [three (siblings A, B, and E) with a pigmented eye-shield from brackish areas, one unpigmented (sibling C) from sandy marine habitats, and one (sibling D) with a pigmented eye-shield from algae] and one (sibling F) from the Atlantic. However, the study ultimately had no impact on taxonomy due to the lack of more detailed information about markers other than the allozymatic patterns.
This study aimed to provide further insights into the species complex of M. lineata using a multifaceted approach. In fact, since the publication of the paper by Casu and Curini-Galletti (2004), there has been an emerging consensus that an integrative taxonomic approach (Padial et al., 2010; Schlick-Steiner et al., 2010 and references therein), which considers species boundaries from multiple complementary perspectives, is the best way to overcome the potential caveats of any species delimitation method (Dayrat, 2005; Will et al., 2005; Padial et al., 2010).
For the purposes of our study, we planned a workflow that established the six siblings, hereafter treated as OTUs – operational taxonomic units (sensu Sokal and Sneath, 1963; Sneath and Sokal, 1973, identified by Casu and Curini-Galletti (2004) as a taxonomic null hypothesis to be tested against different datasets. The new data sources included information on karyotype, morphology, reproductive biology and genetics (DNA gene sequences). For the genetic approach, we chose to use two nuclear ribosomal markers, the complete nuclear small subunit rDNA (18S) gene and the partial nuclear large subunit rDNA (28S) fragment (spanning the D1-D6 variable domains). These markers have been chosen because of their potential in depicting the genetic variability at inter-specific level, combined to their low level of intra-specific variation, that make them a powerful tool for molecular phylogeny and taxonomy (see Litvaitis et al., 1996; Littlewood et al., 2000; Curini et al., 2010; Casu et al., 2011a; 2014). Furthermore, 18S and 28S genes represent the most complete molecular dataset available for Proseriata in GenBank. Molecular datasets were analysed using automated species delimitation methods, two of which, GMYC (Pons et al., 2006), and PTP/bPTP (Zhang et al., 2013), are based on the PSC (phylogenetic species concept); one, ABGD (Puillandre et al., 2012) is based on genetic distances, and one, K/θ method (Birky et al., 2010) is based on the 4× rule criterion. Finally, we estimated the divergence time among taxa to obtain a better understanding of the evolutionary pathways (see Lemey and Posada, 2009).