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Planarians (Platyhelminthes, Tricladida, Terricola) from the Family Rhynchodemidae, Microplana Species

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Planarians (Platyhelminthes, Tricladida, Terricola) from the Family Rhynchodemidae, Microplana Species Contributions to Zoology, 67 (4) 267-276 (1998) SPB Academic Publishing bv, Amsterdam Terrestrial planarians (Platyhelminthes, Tricladida, Terricola) from the Iberian first peninsula: records of the family Rhynchodemidae, with the description of a new Microplana species ¹ Eduardo Mateos Gonzalo Giribet¹,² & Salvador Carranza³ 'Departament de Universitat de Biologia Animal, Barcelona, Avinguda Diagonal 645, 08071 Barcelona, 2 Spain; Present address: American Department ofInvertebrates, Museum of Natural History, Central Park West 79th New New 3 at Street, York, York 10024-5192, USA; Departament de Genetica, Universitat de 08071 Barcelona, Avinguda Diagonal 645, Barcelona, Spain (corresponding author: S. Carranza, [email protected]) Keywords: Microplana, Rhynchodemidae, Terricola, new species, ITS-1, molecular markers, Iberian peninsula Abstract have been reported (see Minelli, 1974; 1977; Ball & Reynoldson, 1981). A review of the American The first records of terrestrial planarians belonging to the species of the family can be found in Ball & family Rhynchodemidae are reported for the Iberian Peninsula. Sluys (1990). A new endemic species from the Spanish Pyrenees, Microplana Data from terrestrial of the Iberian The planarians nana sp. nov., is described. characteristic features of this Peninsula small size in adult are and to our the species are: i) (8-10 mm) individuals, ii) very scarce, knowledge long conical penis papilla and iii) absence of seminal vesicle, introduced bipaliid Bipalium kewense Moseley, bursa copulatrix, genito-intestinal duct, and 1878 is well-developed the only species reported so far (Filella- bulb. the penial Moreover, widespread common land European Subirà, In 1983). contrast, the rhynchodemid Mi- planarian Microplana terrestris (Müller, 1774) is reported for croplana terrestris F. is the time from (O. Müller, 1774) widely first the Iberian Peninsula. The two species, M. distributed nana and M. throughout Europe, and has been sp. nov. terrestris, are described by means of found in Menorca external morphology using histological sections, and have been (Balearic Islands) (Minelli, characterized by the ITS-1 molecular marker. The study of 1974; 1977), while Microplana pyrenaica (Von molecular markers such as ITS-1 is proposed as a powerful Graff, 1899) has been described from the French technique for identification at the species level in terrestrial Pyrenees. planarians. The systematics of the land planarians, as that of the entire is difficult Tricladida, very because Introduction they usually lack clear external diagnostic char- acters. Thus, an exhaustive microscopical analy- Terrestrial planarians (Tricladida: con- sis of the internal with Terricola) anatomy emphasis on the stitute a understood poorly group of free-living copulatory apparatus is always needed for identi- flatworms somewhat hun- containing over eight fication at the species level. Although almost all dred species of flat or animals, 4-600 cylindrical of the land planarians are hermaphroditic, the mm in Current three male length. taxonomy recognizes and female are not systems necessarily pres- families: Bipaliidae, Geoplanidae, and ent at same Rhyncho- the stage of development in a given demidae. The several and Rhynchodemidae comprise individual, isolated descriptions of proteran- small with species an elongated and more or less drous and individuals well proterogynous may be cylindrical body, with a rounded or truncated an- one of the of causes the present taxonomic con- end terior and a of situated fusion single pair eyes an- (Ball & Sluys, 1990). Moreover, immature This is and teriorly. family cosmopolitan, widely organisms lack the copulatory apparatus, thus distributed throughout Europe, where two sub- preventing unequivocal identification. families and (Rhynchodeminae Microplaninae) based on Lately, the generally accepted hy- 268 E. Mateos, G. Giribet & S. Carranza - Rhynchodemidae from the Iberianpeninsula in- accessible tool pothesis that phenotypic similarities between region is proposed as an easy and and identification dividuals of the same species are a reflection of a for species differentiation even within the correspondingly similar genotype, some mole- genus Microplana. cular markers have been developed as species- specific markers or diagnostic characters (see DeSalle & These molecular Material and methods e.g., Birstein, 1996). markers have proved to be invaluable when deal- identi- Terrestrial planarians were collected from beneath stones lying ing with zoological groups where species in moist areas in two different localities of the northeasternpart fication is a challenge for systematists (Carranza, ofthe Iberian Peninsula (Fig. 1). Specimens were taken to the differ- 1997). In describing relationships among laboratory, reared in small capsules at 4°C, and recorded with a ent molecular systematists also em- organisms, VHS video-recorder connected to a stereo-microscope. In addi- involves di- studied ploy the comparative method, which tion, some specimens of these two populations were at the level, and their nucleic acids extracted in order rect or indirect information on nucleic acid and histological to inter- and intra-specific length variability in the ITS- related analyze protein sequences of species. 1 locus. Ribosomal DNA (rDNA) plays a pivotal role in For histological studies, specimens were fixed in Stein- the synthesis machinery of all prokaryotic protein mann’s fluid (1 part distilled water, 1 part concentrated nitric cells. The rDNA and eukaryotic eukaryotic array acid, 1 part 5% sodium chloride in saturated mercuric chloride) minutes transferred 70% typically consists of several hundred tandemly for four and to ethanol. Fixed speci- mens were processed to paraffin blocks, and 8 mm thin sagittal repeated copies of the transcription unit, sepa- sections were obtained. Sections were stained in haematoxylin/ nontranscribed The rated by spacers (NTS). tran- eosin. scription unit codes for the 18S, 5.8S and 28S The ITS-1 locus was amplified for two individuals of and internal transcribed genes and for external Microplana terrestris (“Font Groga” population) and for two and of nana nov. Alberes” spacers (ETS, ITS-1 ITS-2) (for a review, see individuals Microplana sp. (“Les pop- ulation). High-molecular weight genomic DNA from single Long & Dawid, 1980). The multiple copies of this individuals was purified according to a modification of the be identical within a cluster appear to nearly guanidine isothiocyanate method initially described for RNA given organism (Hillis & Dixon, 1991; Lane et by Chirgwin et al. (1979), ITS-1 was amplified using primers This conservation of the ah, 1985). sequence pre- 9F (5’-GTAGGTGAACCTGCGGAAGG-3’) and 1TSR (5’- sumably reflects functional constraints on the TGCGTTCAAATTGTCAAT-3’), which were designed to molecules, which are required for optimal trans- lational This of efficiency. process homogeniza- tion is called concerted evolution (Dover, 1982; Hillis & Dixon, 1991). The ITS-I (internal tran- scribed spacer) is located between the nucleait and the of ribosomal I8S 5.8S genes. Sequences the ITS-I segment in invertebrates have been re- ported for the coastal North American tiger beetle Cicindela dorsalis (Vogler & DeSalle, 1994), several dipteran species (Schldttlerer et ah, 1994; Miller et ah, 1996; Tang et ah, 1996), and the freshwater turbellarians of the Dugesia gonoce- phala species complex (Carranza, 1997). In the present work we report the finding of two species of terrestrial planarians of the family Fig. I. Map ofthe Iberian Peninsula with sampling localities of Rhynchodemidae in the northeastern part of the terrestrial planarians. Capital letters refer to the “Coll de Iberian Peninsula, one species being new to sci- Banyuls” site at Les Alberes (A) and “Font Groga” and “La ence. These are the first records for this in family Rierada” sites at Massis de Collserola (B). C refers to litera- the Iberian Peninsula. Amplification of the ITS-1 ture-cited specimens of M. terrestris from Menorca (Balearic Islands). Contributions to Zoology, 67 (4) - 1998 269 match the 3’ end ofthe 18S rDNA and the 5’ of the 5.8S gene rDNA carried out in gene, respectively. Amplification was a 50 volume reaction: 80 of 0.6 units of pi ng genomic DNA, DynaZyme™ polymerase, 200 pM of dNTP’s and 0.5 mM of each used for the PCR reaction. The PCR primer were program consisted of first a denaturingstep of 3 min at 94°C; and 30 sec at 94°C, 30 sec at 50°C, 30 sec at 72°C, 35 cycles. An aliquot of the reaction was 1.5% analyzed by electrophoresis on a agarose gel and visualized with ethidium bromide. Results 2. Fig. Microplana nana sp. nov,: sagittal reconstruction of the copulatory apparatus, anterior part ofthe specimen towards the Seventeen specimens of cylindrical terrestrial pla- right, dorsal surface to the top and ventral surface to the bot- narians assigned to the family Rhynchodemidae tom. Long conical penis papilla (P) with the anterior tip folded have = = been collected from two localities in the G V D = upwards. gonopore, female genital duct, vasa =oviducts northeastern part of the Iberian Peninsula. Two deferentia, O (scale bar 400 pm). to the species, assigned genus Microplana, have been identified through the study of histological file is'available from the anonymous ftp site: porthos.bio.ub.es/ sections of the specimens. it in pub/incoming; can be easily visualized a Macintosh or in a PC computer. section Systematic Habitat. - Specimens were found under stones in a shady
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