Contributions to Zoology, 67 (4) 267-276 (1998)

SPB Academic Publishing bv, Amsterdam

Terrestrial (Platyhelminthes, Tricladida, Terricola) from the Iberian first peninsula: records of the family Rhynchodemidae, with the

description of a new species

¹ Eduardo Mateos Gonzalo Giribet¹,² & Salvador Carranza³

'Departament de Universitat de Biologia , 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- (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- somewhat hun- containing over eight fication at the species level. Although almost all dred species of flat or , 4-600 cylindrical of the land planarians are hermaphroditic, the mm in Current three male length. recognizes and female are not systems necessarily pres- families: Bipaliidae, , 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 ) 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- 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 area near a running water course, on a Family Rhynchodemidae Von 1896 Graff, holm oak (¡Quercus ilex), and in riverside wood-

Subfamily Microplaninae Pantin, 1953 lands.

Genus Microplana Vejdovsky, 1890

Etymology. - The specific epithet nana refers to

the short body length of the four adult specimens

nana nov. 2-8) Microplana sp. (Figs. found: 4 mm for sectioned and specimens up to

10 mm for fully stretched living specimens.

Type material. - Holotype, sagittal sections of 1 specimen mounted on 3 slides stained with hematoxylin-eosin, BMNH Diagnosis. - Very small adult individuals (4 mm catalogue number 1998.2.9.1; Les Alberes (Girona, Spain), in fixed specimens and 10 mm in stretched “Coll de Banyuls”, February 7th, 1997 (E. Mateos & S. fully

Carranza leg.). Paratype, sagittal sections of 1 specimen mount- living specimens); long conical penis papilla; tes-

2 slides stained with labeled BMNH situated ed on hematoxylin-eosin, tes ventrally from behind the ovaries to catalogue number 1998.2.9.2, same locality and date, 1997 (E. the pharyngeal region; absence of the following Mateos & S. Carranza leg.). The type material has been structures: seminal vesicle, bursa copulatrix, gen- deposited at the Natural History Museum, London. ito-intestinal duct, and well-developed penis bulb.

Type locality. - “Coll de Banyuls” at Les Alberes (Rabos

- d’Emporda, Girona, Spain), coordinates UTM 31TEH0400. Description. Fully stretched living specimens

are about 8-10 mm long and 0.5 mm wide, while

Material examined. - Four the fixed x specimens, type locality, February two specimens measured up to 4 0.7

7th, 1997 (E. Mateos & S. Carranza leg.): two specimens for mm. The mouth is set at 2.5 mm and the genital histology, two specimens for nucleic acids extraction. aperture at 3 mm behind the anterior tip of the

body. The small ocelli are located at the anterior Video-recording. - A 90 seconds video of one specimen of end; the dorsal surface is greyish with darker Microplana nana was recorded during locomotion, with details all over the on the movement of the head. spots surface; anterior end darker; This video was digitalized and saved as in sole white. mnana.mov quick time format. The mnana.mov creeping 270 E. Mateos, G. Giribet & S. Carranza - Rhynchodemidae from the Iberian peninsula

of conical the anterior Figs. 3-8. Microplana nana sp. nov.: 3, sagittal section illustrating the presence a long penis papilla with tip

the duct and the Note ofseminal folded upwards (P), gonopore (G), the very narrow female genital (V), pharynx (H). the absence

vesicle and bursa copulatrix. Anteriorpart of the specimen towards the right, dorsal surface to the top and ventral surface to the bottom

the (scale bar 400 pm, BMNH catalogue number 1998.2.9.2); 4, sagittal section illustrating one of two oviduct openings (O). Anterior

ofthe towards the dorsal surface the and ventral the bottom 400 BMNH part specimen right, to top surface to (scale bar pm, catalogue

number ofthe the of the 1998.2.9.1);5, sagittal section illustrating one two vasa deferentia(D) receiving openings some testes on way Contributions to Zoology, 67 (4) - 1998 271

and musculature. - The Epidermis epidermis of Male reproductive system (Fig. 2). - Each of the the sole is about creeping 10 pm thick, its cilia approximately 10 pairs of testes (Fig. 5) is oval to

are and about 3 The about 100 in diameter. densely packed pm long. circular, pm The testes are

of the lateral and the dorsal wall epidermis body situated ventrally, dorsal to the nerve cords, ex- is about 15 thick and its cells pm are packaged tending from behind the ovaries to the pharyngeal

with rhabdites that have the same as the height region. The testes discharge medially and of epidemial cells. Nuclei the epidermal cells are ventrally into the vasa deferentia (Fig. 5). between 5-7 in diameter and basal to central pm The vasa deferentia are two visible ducts situ- in the cells. ated ventrally, lateral and slightly dorsal to each In the ventral part of the body, interior to the nerve cord, receiving on the way to the penis subepidermal circular and longitudinal muscle papilla the openings of some testes (Fig. 5). Be- and layers, separated from these by a layer of fore entering the penis papilla, the vasa deferentia

parenchymal tissue, there is a well-developed enlarge to form two independent false seminal layer of longitudinal parenchymal muscles about vesicles (Fig. 6), hereafter the vasa deferentia 15 mm thick. This parenchymal muscle layer oc- narrow again and at the base of the penis papilla curs only ventrally (Fig. 5). they fuse to form the ejaculatory duct, the latter

Nervous system. - The central nervous system lined with a nucleated Some epithelium. expan- consists of two large ventral nerve cords con- sions can be observed along the ejaculatory duct nected by several commissures lying between the due to the accumulation of spermatozoa (Fig. 3). parenchymal muscle layers and the vasa deferen- The penis papilla is a well-developed, long, tia. These nerve cords fuse at the anterior part of conical structure consisting of loose tissue sur- the body. rounded by circular and longitudinal muscle lay- Alimentary system. - The anterior ramus of the ers. The penial bulb is weakly muscular. The intestine runs forwards to within about 0.1 mm of is situated gonopore ventrally and centrally to the the anterior tip of the animal, giving off several atrium (Fig. 3). pairs of branched lateral diverticula. Both poste- Female reproductive stystem (Fig. 2). - The two rior rami extend to almost the hind end, each giv- ovaries are about 125 oval-shaped, pm high by ing off several diverticula. The diverticula are lie 100 pm, and ventrally above the brain at about lined with large columnar cells each about 100 0.3 mm from the anterior end of the animal (Fig. pm long. 7). The oviducts run posteriorly just above the In the holotype, the pharynx is about 0.5 mm nerve cords to the level of the narrow female and long 0.35 mm in diameter. It is externally genital duct (Fig. 3), opening independently into lined ciliated by a epithelium 4 pm thick, the cilia rise the latter (Fig. 4). The oviducts give to nu- 2 being pm long. This epithelium covers a longi- merous short, dorsal branches that are in con- tudinal muscle of about 2 fol- layer pm thick, tact with the vitellaria. The vitellaria are well lowed 4 thick of by a pm layer circular muscle, a developed and extend from the level of the ova- 100 layer pm thick of parenchymal tissue with ries towards the posterior end of the animal and elements, and a about 12 thick glandular layer pm fill much of the between the intestinal of circular and longitudinal muscle fibers. This is space branches. The cells of the vitellaria are large, bounded internally by a non-ciliated epithelium 6 vacuolated and with The thick. glandular cytoplasm. pm

to the penis papilla the of and the (T), well-developed layer longitudinalparenchymal muscles (M), pharynx (H). Anteriorpart of the

to the dorsal surface the ventral surface the bar 400 BMNH specimen bottom, to right and to left (scale pm, catalogue number

section 1998.2.9.1); 6, sagittal illustrating one of the two enlargements of the vasa deferentia (D) at the base of the penis papilla;

Anterior of the towards the dorsal surface the and pharynx (H). part specimen right, to top ventral surface to the bottom (scale bar 200

BMNH number section ofthe ovaries above the brain pm, catalogue 1998.2,9.2);7, sagittal illustrating one (A) (B). Anteriorpart ofthe

to the dorsal the left and ventral surface the bar 200 BMNH specimen top, surface to to right (scale pm, catalogue number 1998.2.9.1);

8, sagittal section one ofthe two oviducts Anterior of the to the dorsal to and illustrating (I). part specimen right, surface the top ventral

surface to the bottom bar 400 BMNH number (scale pm, catalogue 1998.2.9.1), 111 E. Mateos, G. Giribet & S. Carranza - Rhynchodemidae from the Iberian peninsula

oviducts are ciliated internally and have an exter- based on the similarities of the different struc-

of Minelli nal diameter of about 18 pm. tures the copulatory apparatus, (1977)

The female duct is about 25 with M. genital (Fig. 8) pm synonymized M. styriacus terrestris.

in diameter and 300 pm long and is lined with a

5 thick non-ciliated The pm epithelium. genital terrestris duct is surrounded by few muscle fibers and Microplana (O.F. Müller, 1774) (Figs. 9-13) many glands.

Material examined. - “Font Groga” at Massis de Collserola Remarks. - The previously known European spe- (Sant Cugat del Valles, Barcelona, Spain), coordinates UTM cies of Microplana differ from M. nana in the 31TDF2687: 1 specimen June 5th, 1996 (E. Mateos leg.); 3 traits. M. Graff, following pyrenaica (Von 1899): specimens June 11th, 1996 (E. Mateos leg.); 2 specimens size of bigger (50 mm); presence genito-intestinal February 12th, 1997 (E. Mateos leg.); 2 specimens February

1997 Mateos 4 1997 duct and seminal vesicle; smaller penis papilla. 13th, (F. leg.); specimens February 19th,

(E, Mateos leg.). Two specimens used for nucleic acids M. scharffi (Von Graff, 1899): bigger size (30 extraction and 3 specimens for histology. The slides containing semi- mm); presence of genito-intestinal duct and the histological sections have been deposited at the Natural nal vesicle. M. giustii Minelli, 1977: testes placed History Museum, London (U.K.) under BMNH catalogue between the pharynx and the genital atrium; pres- numbers 1998.2.9,3-5. ence of genito-intestinal duct. M. henrici Bendl,

of folds in the - 1908: presence genital atrium; Habitat. Specimens were found under stones in

of habitus intermittent presence genito-intestinal duct; striped a shady area near an running water of the animal. M. oak-ash woodland holm terrestris (O.F. Muller, 1774): course, on an containing bigger size (up to 26 mm); presence of bursa oak (Quercus ilex), pedunculate oak ( Quercus copulatrix, genito-intestinal duct and seminal robur), ash (Fraxinus excelsior), elm (Ulmus sp.), vesicle; penial bulb well developed. M. humicola water willow (Salix sp.), and white poplar ( Po-

1890: of seminal Some introduced such Vejdovsky, presence vesicle; pulus alba). tree species as smaller penial papilla; the female genital duct plane (Platanus sp.), black poplar (Populus ni- runs upwards to the dorsal part of the body and gra), acacia (Acacia sp.), and sycamore (Acer not backwards as in M. nana. M. mahnerti Mi- pseudoplatanus) occur as well. The predominant

seminal vesicle nelli, 1977: presence of a large species of the undergrowth are horsetails (Equi- and bursa copulatrix. setum telmateia) and soft shield ferns (Polysti-

Marcus & chum Microplana perereca Marcus, 1959, seritiferum).

M. is similar to nana in the general structure of

the - We copulatory apparatus. Nevertheless, the bigger Description. will only detail some relevant size diameter of features and M. perereca (the sectioned of this well-known species. Fully animal was 13 mm long and 1.5 mm in diameter), stretched living specimens are between 10 and 26 the of the the fixed animals measured x presence a well-developed penial bulb, mm long; up to 15 absence of 1 two independent enlargements (false mm. The body is cylindrical, becoming nar- seminal vesicles) in the vasa deferentia before rower towards the anterior end. The two tiny

fuse the base they at of the penis papilla, and its ocelli, placed in unpigmented areas, are situated particular geographical distribution (Sao Miguel, near the anterior tip. The dorsal surface is reddish

that M. and M. to brown with black all the Azores) suggests nana perereca granules over surface; are different species. anterior end darker; creeping sole white. There

Microplana styriacus (Freisling, 1935) differs are several testes situated ventrally from behind from M. in the nana presence of a copulatory the ovaries to the pharyngeal region. The penis bursa and and a very large highly musculated bulb and the conical penis papilla are well devel- bulb. This penial species was described on a sin- oped (Fig. 9) and a large seminal vesicle is

from Austria gle specimen that was destroyed present (Fig. 9). The two vasa deferentia, with

the Second World War. Later during on, and notable expansions due to sperm accumulation, Contributions 67 - to Zoology, (4) 1998 273

Figs. 9-13. Microplana terreslris (from Font Groga): 9, sagittal

section illustrating the long conical penis papilla (P), the

seminal vesicle and (S) the gonopore (G). Anterior part of the

specimen towards the right, dorsal surface to the top and

ventral surface to the bottom bar 400 BMNH (scale pm,

catalogue number 1998.2.9.3); 10, sagittal section illustrating the enlarged vasa deferentia (D), the oviduct opening (O), the

bursa copulatrix (C) and the penis bulb (E). Anterior part of the

specimen towards the right, dorsal surface to the top and

ventral surface to the bottom bar 400 BMNH (scale pm,

catalogue number 1998.2.9.3); 11, sagittal section illustrating

the female genital duct (V) and the bursa copulatrix (C).

Anterior part ofthe body towards the right, dorsal surface to the

top and ventral surface to the bottom. Note that this section is

folded at the bottom the bar 400 BMNH of specimen (scale pm,

catalogue number 1998.2.9.5); 12, sagittal section illustrating the ciliated bursa and the two copulatrix (C) genito-intestinal 'ducts (N). Anterior part ofthe body to the bottom, dorsal surface to the right and ventral surface to the left (scalebar 200 BMNH pm, catalogue number 1998.2.9.4); 13, sagittal section illustrating the genito-

intestinal duct (N) and a detail of the ciliated in the intestine-bursa epithelium (Z) copulatrix contact zone. Anterior part of the

towards the specimen top, dorsal surface to the left and ventral surface to the bar 100 BMNH right (scale pm, catalogue number 1998.2.9.5). 274 E. Mateos, G. Giribet & S. Carranza - Rhynchodemidae from the Iberian peninsula

open independently in the seminal vesicle (Fig.

from the the 10). The oviducts run ovaries to

level of the female genital duct where they open

independently (Fig. 10). The female genital duct

is connected with the bursa copulatrix (Fig. 11).

is with The bursa lined a ciliated, non-glandular,

epithelium (Fig. 12) and is connected with the

intestine by one (Fig. 13) or two (Fig. 12) genito-

intestinal ducts.

Remarks. - The sectioned specimens clearly

match the description of the neotype of Micro- plana terrestris designed by Minelli (1977). Fig. 14. Agarose gel electrophoresis showing the four ITS-1

PCR products: Microplana nana specimen 1 (Les Alberes)

(lane 1); M. nana specimen 2 (Les Alberes) (lane 2); M. ter-

restris specimen 1 (Font Groga) (lane 3); M. terrestris speci- ITS-I Amplification men 2 (Font Groga) (lane 4); negative control (lane 5); 100 bp

DNA size marker (lane 6). The amplification of the ITS-1 locus from both

individuals of Microplana terrestris yielded a restris group may consist of more than a single

product band size of about 900 bp, while it sized species (Minelli, 1977). about both of 650 bp for individuals of Microplana The description Microplana nana as a new

the nana (Fig. 14). Such a product band size differ- species is based on microscopical analysis of

ence clearly indicates that M. terrestris and M. the internal anatomy as well as on the small size

attained adult individuals. This is the first nana are differentiated from a molecular point of by spe-

view and, therefore, differences in the length of cies of terrestrial planarian described for the Ibe-

the ITS-1 be used rian Peninsula and be considered as an en- region may as a species-level may

molecular marker in land planarians. demic species of the Mediterranean slope of the

Spanish Pyrenees. Further collection expeditions

should test its presumed endemic status.

General discussion Identification at the species level of terrestrial

planarians belonging to the Rhynchodemidae is

Data task and be done of on terrestrial planarians in Europe have not an easy must by means

of the full been summarized by Minelli (1977), according,to histological study anatomy, emphasiz-

whom the is the the genus Microplana represented by a ing copulatory apparatus. Furthermore,

total of 7 species. From these species, only Mi- rhynchodemids are difficult to sort out by tradi-

terrestris croplana is widely distributed through- tional histological studies, mainly because appar-

review in Ball differences between out Europe (see Minelli, 1977; & ent morphological taxa may

and North America be small. almost Reynoldson, 1981) (Hyman, very Very often, they are impos-

The North American sible unless the material is well 1939; Ogren, 1984; 1991). to identify very representatives most probably have been intro- fixed. Other difficulties at species level identifi-

duced from cation the existence of and Europe (Ball & Sluys, 1990). M. are proterandrous pro-

terrestris has also been recorded in Madeira (De terogynous individuals, and the lack of sexual

Beauchamp, 1957; Marcus & Marcus, 1959). organs in immature animals (Jones et ah, 1990).

Thus, the finding of specimens of M. terrestris All together this led to the currently poorly under-

stood of the evidenced in the Iberian Peninsula is not surprising due to systematics group, as by their widespread distribution in the Mediterra- the numerous synonymized species within the

& nean area. Nevertheless, the identification of our Rhynchodemidae (see Minelli, 1977; Ball

specimens is not unequivocal, since the M. ter- Sluys, 1990). Contributions to 67 - 1998 Zoology, (4) 275

We of believe that examination should have different species-level species a ITS-1 sequence markers molecular could be in the due to the differentiation helpful study genetic processes that of these organisms that are difficult to occur to identify subsequently reproductive isolation by

by external or internal morphology and that can speciation.

only be reliably identified by using characters of

mature individuals (i.e., the genital apparatus).

The of molecular usage markers has already Acknowledgements be useful in proved to very analyzing the fresh-

water of the planarians The authors would like to thank Dugesia gonocephala J. Baguna and M. Riutort for

their species complex (Carranza, 1997). It can also be help. We are indebted to G. Marfany for discussions and

for English correction of the manuscript and to Asuncion successfully applied to terrestrial planarians, as Munoz from the I.C.F., Barcelona for the video digitalization. reported in this work on the basis of difference in This work was carried out at the laboratory of J. Baguna, and the size of the ITS-1 PCR product between M. was supported by a CIRIT grant GRQ93-1044 (to J.B.). G. terrestris and M. ITS nana. amplification can also Giribet was supported by an FPI fellowship from the MEG

be used for as well as for identification We also the juvenile, (Spain). appreciate comments and corrections of

two of live specimens, since DNA can be extracted anonymous referees that clearly improved this manuscript.

and PCR from small tissue amplified very sam-

ples removed from either alive or 100% ethanol-

fixed PCR References organisms. Thus, once the product pat-

tern for each species would be defined, identifica-

tion Ball, I.R. & T.B. 1981. British at the species level would be a much easier Reynoldson, planarians: 1- 141 (Cambridge University task than studies Press, Cambridge). histological are nowadays, so Ball, I.R. & R. Sluys, 1990. Turbellaria: Tricladida: Terri- that species (including immature, proterandrous cola. In: D. Dindal (ed.), Soil biology guide: 136-153 and proterogynous individuals) could be unam- (John Wiley & Sons, New York). identified. biguously Beauchamp, P. de, 1957. Planaires terrestres. Mission du

As commented C.N.R.S. in the introduction, there are a Madere 1957 (2). Bull. Soc. zool. Fr., 82: 357-

360. two levels of information that we can obtain Carranza, S., 1997. Taxonomia molecular DNA la se- when molecules are used in taxonomy: one quenciacio del DNA ribosomic 18S. Aplicacio a 1’origen i is the size of the molecule and the other is the dels filogenia Platihelmints. PhD thesis, Universitat de DNA itself. In our the ITS-1 sequence example, Barcelona, Barcelona, Spain. PCR of as- Chirgwin, product two individuals, previously J.M., A.E. Przybyla, R.J. MacDonald & W.J.

M. terrestris Rutter, 1979. Isolation signed to by means ofmorphological of biologically active ribonucleic acid from of the 250 sources enriched in ribonuclease. Biochemistry, analysis copulatory apparatus, is bp 18: 5294-5299. longer than the ITS-1 PCR product of the two DeSalle, R. & V.J. Birstein, 1996. PCR identification of individuals described as M. This dif- nana. large black caviar. Nature, 381: 197-198. ference in size of the ITS-1 between fragment Dover, G.A., 1982. Molecular drive: a cohesive model of individuals of M. and M. species terrestris nana strongly evolution. Nature, 299: 111-117.

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