RICERCA ITALIANA     

Research program

Integrated approach to the identification of problematic taxa of the marine meiofauna: drafting of volumes of the series "Fauna d'Italia" and development and evaluation of methods of DNA-barcoding in Gastrotrichs, Proseriates and Rotifers

University Co-ordinator

Università degli Studi di SASSARI - ZOOLOGIA E GENETICA EVOLUZIONISTICA - ()

Research Unit Leader

Marco Curini Galletti

Description

The project aims to contribute to the identifications of Proseriata (Platyhelminthes) both in the grounds of traditional and DNA-based taxonomy, by means of the drafting of an issue of the series “Fauna d’Italia” and with the development and evaluation of a DNA-barcoding approach. The realisation of the project in the time at disposal will be possible thanks to the commitment of the tenured researchers, as well as to the presence of collaborators, part of whom has already been formed to the study of Proseriata during the project PRIN-2004. Most of the financial requests of this application is thus devoted to the financing of further scholarships which will enable them, as well as new graduate students, to continue with the research. The partecipation of these collaborators to the various steps of the research will allow not only to respect the timing of the project, but also to perform an important role of formation of new students, with a multidisciplinary approach, in the field of taxonomy.
1) drafting of an issue of the series “Fauna d’Italia” devoted to the Proseriata.
According to the standards of ”Fauna d’Italia”, the issue will be articulated as follows:
a) a general part, which will include chapters devoted to morphology and biology, phylogenetical relationships, ecology, techniques of collection, preparation and study, zoogeography and systematics of the taxon.The principal investigator of this O.U. has personal experiences in all the topics, at least partly synthetized in Curini Galletti, 2001. The task will be here to make the whole introductory section of interest both to a reader already experienced in the field of systematics and phylogeny of Platyhelminthes, and to readers with different cultural back-ground, who may face for the first time the study of Platyhelminthes.We feel that this aim is particularly important, as this may constitute a cultural inheritance for future generations of researchers.Particular care will thus be devoted to the description of specific techniques which can only be acquired with working experience on the group, such as techniques of collection and isolation from the sediment, observation of semisquashed living specimens, establishment of permanent mounts, as well as the possibility to obtain karyotypes by means of coloration with lactic-acetic orcein, which, at least in some instances, allows reliable identifications of members of complexes of sibling species. The general part will also include a chapter with the lists of the species present/common in the different geographic sectors of the Mediterranean, with indications, as an aid to determination, of their preferences for sediment type, bathymetrical distribution and ecological conditions.
b) a special part, where all the Proseriate species known for the italian coasts and the Mediterranean (as recommended by the Scientific Committee for the marine groups), will be presented. Members of this O.U. have already produced checklists of italian and Mediterranean Platyhelminthes (Curini Galletti & Casu, 2003; Curini Galletti, 2007) , which could be used as bases upon which the new distributional data gathered from sampling campaigns sponsorshipped by PRIN-2004 will be added, as well as numerous new species, presently under publication. The number of species which will be included in the issue is 221, belonging to the suborder Unguiphora (with the fam. Nematoplanidae) and Lithophora (with the fam. Archimonocelididae, Coelogynoporidae, Monocelididae and Otoplanidae). Species descriptions, as well as sections devoted to supra-specific taxa, will be written according to the editorial rules of the “Fauna d’Italia”. Similarly to the general part, in descriptions and figures a particular emphasis will be given to characteristics allowing identification of living and/or fixed organisms. Identification keys will also be produced, especially pictorial keys, which may result in an easier use by non-expert taxonomists. What follows below is a species’ card, exemplificative of the general lines of the issue, relative to a species whose representatives are widely distributed across the Mediterranean and particularly abundant, but whose identification may be problematical due to the lack of sclerotized structures and for its belonging to a complex of sibling species.
2) development and evaluation of a DNA-barcoding approach to the identification of Proseriata.
Given the lack of any a-priori knowledge, this section of the project will proceed according to the following steps:
i) tests will be performed in order to standardise the optimal conditions for the amplification of the mtDNA region corresponding to the cytochrome oxydase 1 (cox1). In particular, the pair of "universal" primers for cox1 known in literature (Folmer et al. 1994) will be used to test whether they may produce sequences of adeguate length (at least 450 bp). In cases of suboptimal results, specific primers for the group will be designed.
ii) the usefulness of a sequence as barcode is related to the possibility of its amplification, from a single pair of primers or a given set, in all the species of a group. This aspect will be tested by means of a broad taxonomic sample, essaying at least three species belonging to each of the five families constituting the order Proseriata.
iii) the efficacy of cox1 sequences to unambiguously assign unknown specimens to clusters of reference sequences deposited in a database will be tested determining its intraspecific variability in relation to interspecific variability, and extrapolating a treshold index for interspecific distinction according to the method described by Pons et al. (2006). In order to trial the actual resolutive taxonomic power of cox1 sequences, tests will be performed on particularly problematic cases, evidenced as results of the PRIN-2004 project:
- sibling species complex of Monocelis lineata, indistinguishable on morphological bases (cf Casu & Curini-Galletti, 2004). The study will be performed on 11 Atlantic populations and 8 Mediterranean populations – from whom the genomic DNA has already been extracted.
- sympatric sister species of the genus Archilina – a monophyletic group which includes eight species, restricted in range to the upper Adriatic (Curini-Galletti & Campus, submitted).
In case of failure of cox1 as a diagnostic tool in the discrimination of Proseriata, other molecular markers (SSU, LSU), on which the OU has already experiences of successful amplification, will be tested according to the same procedure described above.



EXEMPLIFICATIVE SPECIES’ CARD:

Pseudomonocelis ophiocephala (Schmidt, 1861)
Monocelis ophiocephala Schmidt, 1861: pag 24, figs 3-5.
Pseudomonocelis ophiocephala, Meixner 1943: pag 460-467, figs 2, 4. Ax 1959: pagg 79, 80, figs. 38, 39; Murina 1981 pag. 129, 130; Schockaert & Martens 1987 pp. 101-113, figs. 6-7; Curini-Galletti & Casu 2005 pag 7-13, fig 2 D-G; Casu & Curini-Galletti 2006.
Locus typicus: Corfu Island (Greece), beneath the Corfu castle.
Description
Colourless animals, slender and elongate, about 10 times as long as broad. Adult, living worms may reach about 4 mm in length. Fixed specimens are 2-2.5 mm long. Anterior end obtuse, provided with a pair of pigmented eye-spots in front of the statocyst. The eye pigment is diffused, and may cohalesce into a single, irregular median spot.The epidermis is of the insunk-type and ciliated all over the body, except at the caudal tip, where numerous adhesive papillae are present.Cilia range 2.5-4 µm in length, and are longer ventrally than dorsally.The narrowly elongate rhabdoids (10-15 µm long) are very dense at both the posterior and the anterior ends of the body.
The pharynx is long and tubiform. It is provided with a longitudinal muscle sheath at the epithelial side and with numerous circular muscles at the parenchymal side. The external and the luminal epithelia have insunk-nuclei and are ciliated; only the distalmost tip of the pharynx (where glands discharge) is devoid of cilia. A comparatively very long oesophagus extends into the proximal third to quarter of the pharynx.
Male genital organs. The number of testes is high, up to 80.The ovoid copulatory bulb (33-42 µm high) consists of a seminal vesicle, lined by a nucleated epithelium, which becomes glandular distally, with nuclei located outside the bulb.Its muscular wall is formed by thin layers of inner circular fibres, and outer longitudinal muscles.The distalmost portion of the bulb forms a muscular penis papilla, and is provided with a much stronger coating of circular fibres than the rest of the copulatory organ.The penis papilla protrudes into a narrow male antrum which is unciliated and opens to the outside through a pore, nearly halfway between vaginal and female pores.
Female genital organs.The dorso-lateral vitellaria stretch from behind the anterior end of the intestine till the level of the vaginal pores.The ovaries are situated ventrolaterally behind the pharynx (a diagnostic feature for the genus, appreciable in semi-squashed specimens, cf Fig. 2, A,B). The oocytes are in one row, with the oldest one more posteriorly placed.They are enveloped in a thin cellular tunica propria from which the oviducts emerge.The two very short oviducts fuse just posterior to the ovaries into the common female duct which, in front of the copulatory bulb, is overlied by a large bursa, lined by a vacuolar epithelium with intraepithelial nuclei, of the resorbiens type.The female duct is partly embedded in the bursal tissue, but remains clearly distinguishable from the latter.The bursa is connected posteriorly to a vaginal duct, which splits distally into two narrow canals above the female duct; it has thus two ventro-lateral pores close in front of the male pore, appreciable in semi-squashed mounts (Fig. 2 C).The vaginal duct is lined with an anucleated epithelium; the paired ducts are surrounded by moderately developed muscles, the unpaired horizontal canal by a layer of strong circular muscle fibers.The female duct opens to the exterior through the female pore, posterior to the male pore, and surrounded by numerous eosinophilous glands
Karyotype: with three chromosomes in its haploid set. Chromosome 1 is acrocentric, chromosome 2 metacentric, and chromosome 3 subtelocentric, with low index value (see Curini Galletti & Casu, 2005)
Distribution (Fig. 1 D): a Mediterranean endemic, absent from the westernmost sector. Widely distributed and often very common in the central basin. Since it belongs to a complex of sibling species, only localities where specimens could be unequivocally attributed to P. ophiocephala on karyological or molecular bases are listed below: France: Corsica, St. Fleurent; Calvi (not found since 1999); Ventilegne.Italy: Sard.: Dragunara (Capo Caccia); Porto Torres; Alghero; Bosa Marina; S. Antioco Is., La Maddalena. Lig.: Santa Margherita. Tusc: Livorno, Quercianella, Punta Ala, Porto Ferraio (Elba Is.). Lat.: Santa Marinella. Camp.: Ischia. Sic.: Trapani, Castellammare del Golfo. Apul.: Porto Cesareo, Acquatina.Croatia: Omis.Greece: Corfu, Glyfada (Piraeus).Israel: Shiqmona (Haifa). The other localities cited in literatura – Naples, S. Rossore (Schockaert and Martens, 1987); Bosporus (Ax, 1959), Sebastopol, Black Sea (Murina, 1981) could not be checked, and at least the latter may pertain to Pseudomonocelis caputanguis Casu & Curini Galletti, 2006 (see under the species’ chapter). Research at Banyuls sur Mer, where Ax (1959) found the species, were unsuccessful, and suitable habitat appears at present lacking, due to the enlargement of the harbour.
Habitat and ecological notes: exclusively found in well sorted, medium-coarse sand, in bays or small coves with moderate hydrodynamism, from lower midlittoral to about 30 cm. deep. Avoids brackish conditions. In its restricted habitat, may be among the dominant metazoan taxa, reaching very high densities (a mean of 14640 specimens per mq have been reported (Murina, 1981), but see above). Species of the genus are reported to feed on crustaceans (Murina, 1981), and are easily kept in cultures feeding on crushed specimens of Sphaeroma sp.–being apparently unable to prey upon living crustaceans. Albeit predation may be facilitated by the more limited escape reaction of the prey within the sediment, the species seems to act as a scavenger.
Remarks: the main discriminatory features of the P. ophiocephala species complex (post-pharyngeal ovaria, bifurcated vagina, lack of pigment in the parenchime) can be appreciated in semi-squashed living specimens, without the need of hystological sections (see fig. 2).In most of the Mediterranean, P. ophiocephala s.s. is the only strictly marine monocelidid with eyes-spot(s) occurring in clean, well sorted mid-littoral habitats. Care should be taken in the presence of fresh-water outlets, where specimens of the nearly identical, at low magnification, Monocelis lineata (Muller, 1774) complex frequently occur.Differences may easily be appreciated in semi-squashed specimens, which reveal the presence of prepharyngeal ovaria, round vacuolar bursa and single vaginal pore in the latter. Problems in identification may only arise where distribution of P. ophiocephala overlaps with its siblings’ ranges – P. caputserpentis Casu & Curini Galletti, 2006 in the Corsican-Sardinian complex and Elba Is., P. caputdraconis Casu & Curini Galletti, 2006 and P. caputanguis Casu & Curini Galletti, 2006 along western and eastern coasts of Greece respectively. Habitat and sediment choice however differ among siblings, as P. caputserpentis is exclusively found in reduced sediment beneath stranded Posidonia oceanica (L.) Delile leaves (the so-called ‘banquette’), and the other two species occur in mixed, silty sediments. For an univocal attribution to P. ophiocephala, the karyotype is the discriminating tool: in its three siblings, in fact, chromosome 1 is at the border between subtelo- and submetacentric, and chromosome 2 submetacentric.The preferred habitat of the species (typically consisting of small coves) is particularly subject to human induced disturbance across the Mediterranean. One of the known populations (Calvi) is extinct since 1999, due to the upgrading of the harbour (Casu & Curini Galletti, 2006) – the same fate has probably happened to the population of Banyuls sur Mer (see above). Populations in densely inhabited areas may thus be tagged as “Locally Threatened” according to IUCN (1994) categories.

Legends to figures.
Fig. 1: Pseudomonocelis ophiocephala (Schmidt, 1861). A: general organisation from a living animal; B: recostruction of the genital organs from serial sections; C: genital organs as seen in semi-squashed living specimens; D: distribution of the species in central Mediterranean (O: presumably extinct population). B and C redrawn from Schockaert & Martens (1987).
Fig. 2: Pseudomonocelis ophiocephala (Schmidt, 1861). A: semi-squashed living specimen; B: detail of the post-pharyngeal area; C: detail of the genital area. Scale bar: A, B: 100 µm; C: 50 µm.