Terrestrial planarians (Platyhelminthes, Tricladida, Terricola) from the Iberian Peninsula: new records and description of three new species

Accepted by W. Sterrer: 31 Jan. 2008; published: 2 Apr. 2008 1

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Terrestrial planarians (Platyhelminthes, Tricladida, Terricola) from the Iberian Peninsula: new records and description of three new species

MIQUEL VILA-FARRÉ1,2, EDUARDO MATEOS3, RONALD SLUYS4 & RAFAEL ROMERO1,5

1Departament de Genètica, Facultat de Biologia, Universitat de Barcelona2E-mail: [email protected] de Biologia Animal, Facultat de Biologia, Universitat de Barcelona; E-mail: [email protected] for Biodiversity and Ecosystem Dynamics & Zoological Museum, University of Amsterdam, P. O. Box 94766, 1090 GT Amsterdam, The Netherlands. E-mail: [email protected]: [email protected]

Abstract

Little is known about the taxonomy and distribution of terrestrial planarians on the Iberian Peninsula. Few studies havetried to investigate the local diversity of these animals, due to both their lack of economic interest and their low abun-dance. In this study we have made extensive searches and collections of terrestrial planarians from the Iberian Peninsula,thus gathering new information on their taxonomy and biogeography. The study includes the description of three newspecies of the genus Microplana, viz. Microplana aixandrei sp. nov., Microplana grazalemica sp. nov., and Microplanagadesensis sp. nov. We present distribution maps summarizing published and new records of land planarians. The presentwork substantially increases our knowledge on this group of animals in Spain and Portugal and at the same time also evi-dences the scarcity of data and studies on the biology of these organisms.

Key words: Platyhelminthes, Tricladida, Rhynchodemidae, Microplana, Rhynchodemus, Iberian Peninsula, Andalusia,Grazalema, biogeography, taxonomy, spermatophore

Introduction

Although a considerable amount of taxonomic and ecological knowledge on several European faunal groupsis currently available, there are still some autochthonous elements of Europe that remain virtually unknown.Such is the case for terrestrial planarians (Platyhelminthes, Tricladida, Terricola). Research efforts on somerelatively recently introduced terrestrial flatworm species has been considerable, due to their potential eco-nomic impact (Boag & Yeates, 2001; Murchie et al., 2003), but resources for the study of autochthonous ter-restrial planarians are virtually nonexistent. This apparent lack of interest of the scientific community resultsfrom different factors. First, autochthonous terrestrial flatworms have no economic impact. Second, these spe-cies are usually rare and hard to find, as is reflected in their taxonomic literature: 7 of the 19 known autochtho-nous European species are described on the basis of a single specimen. In addition, some of this material waslost, destroyed or is poorly preserved (Minelli, 1977). Finally, specimen identification requires an elaborateprocess that involves the production of histological sections, which have to be examined under a compoundmicroscope. As a result, the general knowledge on this group of invertebrates in Europe is rather poor.

The Iberian Peninsula presents a low number of records for terrestrial planarians, as compared to othergroups of animals in this area or to terrestrial planarians in some other European regions (Sluys, 1999). Fur-thermore, some of these planarian records correspond to unidentified species or to specimens with doubtfulidentity (H. Jones, personal communication).

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Simroth (1881) provided the two first records for Iberian terrestrial planarians, concerning two speciescollected in the area of Oporto, Portugal. Von Graff (1899) considered one of these two species to be M.pyrenaica (von Graff, 1899) (Jones, 1998). It was only after a long period of time that a third record wasreported for the Iberian Peninsula when Filella (1983) collected Bipalium kewense Moseley, 1878, an intro-duced species, from the area of Barcelona. This worker failed in finding new animals in later visits (E. Filella,personal communication). Mateos et al. (1998), described the new species M. nana Mateos et al., 1998 fromLes Alberes (Eastern Pyrenees) and reported the presence of another species, M. terrestris (Müller, 1774),from the area of Barcelona. More recently, Boix and Sala (2001), collected Rhynchodemus sylvaticus Leidy,1851 from the Estany d’Espolla (NE Spain) and Mateos et al. (2004) described the presence of two unidenti-fied terricolans from three localities in the Montnegre-Corredor (NE Spain).

FIGURE 1. Distributional records of autochthonous terrestrial planarians in the Iberian Peninsula. A. Iberian Peninsulaexcluding Catalonia. B. Catalonia. Numbers indicate the total number of localities in each area. Arrow points to the typelocality of the three new species.


Zootaxa 1739 © 2008 Magnolia Press · 3THREE NEW TERRICOLA FROM THE IBERIAN PENINSULA

In the present paper we describe three new species of the genus Microplana from Andalusia, in the mostsouthern part of Spain. Furthermore, we provide distribution maps and a table specifying 55 new localitieswhere terrestrial planarians were found. These data substantially increase our knowledge on the diversity ofthe Iberian land planarians but at the same time reveal the scarcity of studies on this group of animals.

Material and methods

Between 1998 and 2006 different areas from the Iberian Peninsula were sampled for terrestrial planarians(Fig. 1). Forested and potentially humid areas, usually close to rivers and streams, were selected in order tomaximize detection probability. Oak, beech, and gallery forests were the main prospected areas. Planarianswere collected with a fine paint brush from underneath stones and logs. Specimens were individually trans-ported to the laboratory in little plastic containers with humid soil. Live specimens were photographed. Forhistological studies specimens were killed (under field or laboratory conditions) using Steinmann’s fluid(Sluys, 1989a) and were subsequently fixed in 70% ethanol. Fixed specimens were cleared in clove oil andthen embedded in paraffin, cut at intervals of 5–7 µm, and mounted on gelatine-coated slides. The sectionswere stained in Mallory-Heidenhain (Sluys, 1989a) and mounted in DePeX. In addition to this routine over-sight stain, several series of sections were stained with the alcian blue-periodic acid-Schiff (AB-PAS) tech-nique to detect polysaccharides (Romeis, 1989). The identification of R. sylvaticus has not been based onhistological sections but on its special external morphology, typically showing two dark longitudinal stripes, amedial black circular spot, and two large eyes situated a little distant from the anterior tip (Ball & Reynoldson,1981). The material examined is deposited in the Centre de Recursos de Biodiversitat Animal, Facultat deBiologia, Universitat de Barcelona.

Abbreviations used in figuresbl, bulbar lumen; br, brain; dep, dorsal epidermis; e, eye; ed, ejaculatory duct; fgd, female genital duct;

gid, genito-intestinal duct; gp, gonopore; h, head; i, intestine; cm, circular muscles; lm, longitudinal muscles;mf, muscular fibres; od, oviduct; ov, ovary; pb, penis bulb; ph, pharynx; phc, pharyngeal cavity; pp, penispapilla; sg, shell glands; sp, sperm; sph, sphincter; spt, spermatophore; t, tail; te, testis; va, vacuolated paren-chymal area; vap, vacuolated epidermis; vd, vas deferens; vep, ventral epidermis; vnc, ventral nerve cord.

Systematic account

Order Tricladida Lang, 1884

Suborder Terricola Hallez, 1892

Family Rhynchodemidae von Graff, 1896

Subfamily Microplaninae Pantin, 1953

Genus Microplana Vedjovsky, 1890

Microplana aixandrei sp. nov.

Material. Holotype, CRBA435, Llano del Berral (lat. 36.75428, long. -5.45399; alt. approx. 657 m) in thecentral sector of the Sierra de Grazalema, Cádiz (Spain), 5 December 2004, sagittal sections on 1 slide.Paratypes: CRBA436, ibid., sagittal section on 1 slide; CRBA437, ibid., horizontal sections on 1 slide.


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Diagnosis. Microplana aixandrei sp. nov. can be distinguished from its congeners by its small size (up to10 mm long), cylindrical body tapering anteriorly to a blunt point, bluntly pointed tail, and hyaline body sur-face. Regarding anatomical features, the species differs from its congeners in the following features: presenceof two ventral testes on either side of the body; spherical penis bulb provided with a strong musculature and adistinct bulbar lumen; short and vertically oriented penis papilla; atrium divided in a cup-shaped cavity and atubular distal cavity; wide and obliquely orientated bursal canal with a sphincter at its proximal end; a copula-tory bursa without genito-intestinal connections; use of spermatophore in the transfer of sperm.

Ecology and distribution. The species is known only from the type locality. In contrast to other Iberianterrestrial planarians, M. aixandrei can be considered a relatively common species of the soil fauna at the typelocality. During mating the sperm is transferred to the copulatory bursa of the partner aggregated in a sper-matophore.

Etymology. The specific epithet is based on the nickname of Vila’s grandfather, Miquel Farré Servent,who lived in a house known as “casa l’Aixandre” in his hometown Salàs del Pallars.

Description. The living, sexually mature specimen measured 10 mm in length and about 0.5 mm inwidth, in elongated state (Fig. 2A). The cylindrical body tapers anteriorly to a blunt point; tail also bluntlypointed. The body surface is hyaline and therefore the species appears white in colour due to the content of theintestine. The anterior and posterior ends, where the intestine is absent, are transparent. The ventral surface ishyaline. The hyaline colouration and the tiny size of the preserved specimens prevented us to adequatelyobserve the creeping sole.

The two small eyes (eye cup diameter 11 µm in sections) are located at a short distance in front of thebrain (Fig. 2B). In living and preserved specimens the eyes are only clearly visible under observation througha dissecting microscope.

The anterior body region is filled with vacuolated parenchymal tissue (Fig. 2B, C) that is reduced betweenthe eyes. This region with vacuolated tissue extends backwards to the level of the testes. The adjacent ventralepidermis is also vacuolated and thick (about 25–30 µm in longitudinal sections while approximately 18 µmin the adjacent non-vacuolated ventral and dorsal epidermis); this vacuolated part of the epidermis (Fig. 2C)extends to almost the level of the ovaries.

The subepidermal longitudinal fibres of the body musculature are weak. In the ventral body region numer-ous longitudinal fibres are distributed in two parenchymal bands, which are especially strong over and underthe ventral nerve cords. The scarce dorsal longitudinal parenchymal fibres are very weak and apparently dis-continuous.

The cylindrical pharynx is about one-eighth of the body-length (0.3 mm) and is situated in the posteriorthird of the animal, in an almost horizontal position. The outer epithelium, which is ciliated only at the poste-rior part of the pharynx, is underlain by a layer of longitudinal muscles, followed by a layer of circular mus-cles (Fig. 3A), intermingled with some additional longitudinal fibres (not represented in Fig. 3A). Close to thedorsal insertion of the pharynx, the inner epithelium is underlain by a thin layer of circular muscles thatbecomes thicker at the posterior end of the pharynx. At the tip of the pharynx this circular layer narrows againbefore reaching the lumen. This circular layer is followed by a thick layer of longitudinal muscles. The mouthis situated at the posterior portion of the pharyngeal pocket, close to the hind wall of the pharyngeal pouch. Inspecimen CRBA435 the mouth is situated at 1.63 mm from the tip of the body and 0.15 mm from the gonop-ore.

There are two pairs of ventrally located, oblong testes follicles, occupying about one-fourth of the dorso-ventral diameter in the prepharyngeal part of the body. The testes are located in the posterior third of the ante-rior body region. In specimen CRBA435 there is an additional immature third testis situated on one side of theanimal.


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FIGURE 2. Microplana aixandrei. Holotype. CRBA435. A. Living animal. B. Sagittal section through the head. C. Sag-ittal section through front end. D–E. Sagittal sections of the copulatory apparatus; anterior to the left. F. Sagittal recon-struction of the copulatory apparatus; anterior to the left.



The strongly muscular, spherical penis bulb is covered with intermingled longitudinal and circular musclefibres and is provided with a well-developed bulbar lumen (Fig. 2D). This bulbar lumen tapers gradually toform an ejaculatory duct that opens at the tip of the penis papilla. The bulbar lumen and ejaculatory duct arelined with a nucleated epithelium that is underlain with a layer of circular muscle fibres. At the level of theposterior section of the pharyngeal pocket the thin vasa deferentia (diameter about 6 μm at the mid-level of thepharyngeal pocket) enlarge to form spermiducal vesicles, which narrow before entering the penis bulb. Afterhaving penetrated the penis bulb, the vasa deferentia increase in diameter, subsequently opening separatelyinto the dorsal part of the seminal vesicle.

The vertically oriented penis papilla is short and conical. The papilla is covered with a thin, nucleated epi-thelium that is underlain with a thick, subepithelial layer of circular muscle bound by a layer of longitudinalfibres.

The atrium consists of a dorsal cup-shaped cavity and a distal tubular part. The lining epithelium of theatrium is underlain with a subepithelial circular muscle layer, thickened at the posterior wall of the tubularpart, followed by a thin layer of longitudinal muscles.

The ovaries are situated immediately above the ventral nerve cords. They lie at about one-third of the dis-tance between the anterior end of the body and the root of the pharynx, occupying about one–fifth of thedorso-ventral diameter. The oviducts arise from the ventral side of the ovaries. In running backwards, theducts follow the course of the ventral nerve cords. Behind the gonopore the ducts turn dorsally and open sepa-rately into the bursal canal.

The copulatory bursa is an irregular sac-shaped structure, lined with tall vacuolated cells (Fig. 2E). Sev-eral muscle fibres traverse in dorso-ventral direction the parenchyma between the posterior wall of the copula-tory bursa and the adjacent intestinal branch (Fig. 2F). The copulatory bursa is not connected with the gut.

All three of the animals examined had in their copulatory bursa remnants of an irregular structure (37x21µm in specimen CRBA435) formed by a blue homogeneous substance partially enveloped by a thin, brownlayer, most likely of sclerotic nature (Fig. 2E). We have not found the origin of this substance in penial oratrial glands, but the colour and texture resemble the wall of a cocoon capsule. The location and nature of thisstructure suggest that it forms part of a spermatophore.

The wide bursal canal, which receives the opening of the shell glands at the same level as it receives theoviducts, is lined with nucleated cells. The distal section of the oviducts, just before communicating with thebursal canal, also receives the secretion of the shell glands. The bursal canal is surrounded by a subepitheliallayer of circular muscles and some scattered longitudinal muscles fibres. A sphincter, consisting of circularmuscle fibres, surrounds the proximal section of the bursal canal (Fig. 2F).

Discussion. Among the approximately 19 species of native land planarians known from Europe, three ofwhich are newly reported in the present paper, M. aixandrei stands apart from the other species by a combina-tion of external features and the anatomy of its genital apparatus.

A hyaline body colouration is also found in M humicola Vedjovsky, 1890. However, this species shows agreenish anterior end, in contrast to M. aixandrei. Regarding anatomical features, M. humicola has dorsal tes-tes and a genito-intestinal duct, while a copulatory bursa is absent (Schneider, 1935). In contrast, M. aixandreipresents ventral testes and a copulatory bursa, but lacks a genito-intestinal duct.

A copulatory bursa that is devoid of any connection with the intestine occurs also in M. mahnerti Minelli,1977, M. styriaca (Freisling, 1935), and M. grazalemica sp. nov. described below.

Microplana mahnerti shows about twenty testes on each side of the body and an elongated penis papilla.In contrast, M. aixandrei shows two testes on each side of the body and a short and conical penis papilla. In M.mahnerti the bursal canal runs from the wall of the atrium parallel to the body surface and the oviducts open atits distal section. A sphincter is absent (Minelli, 1977). In contrast, in M. aixandrei the bursal canal is aobliquely running structure that receives the oviducts at its central region, while a sphincter is present in theproximal section of the canal. With respect to external features, M. mahnerti shows a grey colouration and big



size (13 mm in preserved specimens), contrasting with the hyaline colouration and reduced size of M. aixan-drei (about 4 mm in preserved specimens).

In M. styriaca (Freisling, 1935) an expanded bulbar lumen is absent, while the penis bulb and papilla areelongated. In contrast, in M. aixandrei a distinct bulbar lumen is present, while the penis bulb is rounded andthe penis papilla is short and conical. In M. styriaca the oviducts form a short common oviduct before theyenter the bursal canal (Freisling, 1935), contrasting with the oviducts in M. aixandrei that open separately intothe bursal canal. Furthermore, M styriaca is dark-brown, whereas M. aixandrei is hyaline.

The new species M. grazalemica sp. nov. is not hyaline, contrasting with the hyaline colouration of M.aixandrei. With respect to anatomical features, M. grazalemica possesses about 15 testes on each side of thebody, in contrast to the two pairs present in M. aixandrei. The presence of a spermatophore in the copulatorybursa of the three specimens of M. aixandrei suggests that the animals are adult worms and not juveniles of M.grazalemica sp. nov. The use of spermatophores represents a unique feature of this species, not previouslyrecorded in any species of the Microplaninae. A vacuolated parenchymal tissue in the anterior part of the bodyis absent in M. grazalemica but present in M. aixandrei.

There are various explanations for the vacuolation of the epidermis and parenchyma observed in M. aix-andrei: vacuolation as an artefact of tissue fixation, processing and subsequent histological preparative treat-ment, apparent vacuolation due to the chromophobicity of the contents of the vacuoles, normally vacuolatedplanarian tissues, or vacuolation due to a disease process.

A heavy infection of gregarine parasites can result in pathological peri-intestinal histolytic vacuolation ofthe mesenchyme. In this condition the epidermis is not involved; rather gregarine gamonts and zygocysts arepresent in the parenchyma surrounding the gut trunk, branches and diverticula, and gamonts are generallypresent in the gut lumen and mesenchyme (L. Winsor, personal communication). No gregarines were presentin the specimens of M. aixandrei examined here, and both epidermis and parenchyma exhibit vacuolation.

Normally vacuolated and vesicular tissues in planarians are associated with the reproductive organs, suchas the phagocytic cells of sperm resorptive tissues in various bursae, vitelline follicles, ovarian tubae, andparovarian tissues (Cernosvitov, 1931; Sluys, 1989b; Winsor, 2006). The vaculoate epidermis and paren-chyma in M. aixandrei are located at the anterior tip and are not associated with reproductive structures. Nordo they exhibit the fine cytological characteristics of resorptive tissues.

In addition to the routine oversight stain, sections were stained with the alcian blue-periodic acid-Schiff(AB-PAS) technique to detect hexose-containing and sialic acid-containing mucosubstances, and alciano-philic carbohydrates with carboxylated and sulphate ester groups, which in the adhesive musculo-glandularorgan in the terricolan Pimea can be weakly basiphil to chromophobic (Winsor, 1991). The AB-PAS gavenegative results. It is, therefore, concluded that nothing is present in the vacuoles. The most likely explanationis that something has been lost during fixation or processing. Strongly acidic fixatives such as Heidenhain’sSUSA and Bouin’s dissolve or fail to stabilize certain secretory elements such as types of acidophil or“zymogen” granules (Drury & Wallington, 1980; Leal-Zanchet & Hauser, 1999). Nitric acid is a protein-coag-ulant fixative, a suitable concentration in a fixative for which is 0.5M (Baker, 1958). However the concentra-tion of nitric acid in Steinmann’s fixative is approximately 5M, making the reagent very strongly acidic. Whenadditional specimens of M. aixandrei are available they will be fixed in non-acid and formaldehyde-based fix-atives for further histochemical studies.

Microplana grazalemica sp. nov.

Material. Holotype, CRBA438, CRBA439, Llano del Berral (lat. 36.75428, long. -5.45399; alt. approx. 657m) in the central part of the Sierra de Grazalema, Cádiz (Spain), 5 December 2004, sagittal sections on 2slides.



Other material examined: CRBA440, CRBA441, CRBA442, Río Majaceite (lat. 36.77368, long. -5.486587; alt. approx. 278 m) in the western part of the Sierra de Grazalema, Cádiz, 6 December 2004, sagit-tal sections on 3 slides.

Diagnosis. With respect to external features, M. grazalemica sp. nov. can be distinguished from its conge-ners by its size (12 mm), cylindrical body tapering anteriorly to a blunt point, bluntly pointed tail, brown dor-sal surface with dark spots, and anterior end without conspicuous eyes. Anatomically the species ischaracterized by the presence of a large and rounded penis bulb, obliquely oriented conical penis papilla, acopulatory bursa not connected to the intestine and communicating with the atrium through a slightlyobliquely orientated bursal canal.

Ecology and distribution. The species is known only from two localities in the Sierra de Grazalema.Etymology. The specific epithet is based on the name of the mountain system from where the specimens

were collected, i.e. the Sierra de Grazalema in Southern Spain.Description. In elongated state the living, sexually mature specimens measured 10–12 mm in length, with

a width of about 1 mm (Fig. 3B). The preserved holotype specimen measured 3.45 x 0.68 mm. The cylindricalbody tapers anteriorly and posteriorly to form blunt points. The dorsal surface is brown with darker spots allover the surface; the anterior end is dark brown. The anterior end of the body is slightly invaginated. Thecreeping sole is less than one-third of the body width.

The two small eyes (eye cup diameter 13–16 μm in sections) are located at a short distance anterior to thebrain and are only clearly visible under observation through a dissecting microscope. In CRBA440-442 a thirdreduced eye is present close to the anterior body margin.

The subepidermical longitudinal fibres of the body musculature are weak. In the ventral body regionnumerous longitudinal fibres are especially present over and under the ventral nerve cords. The scarce dorsallongitudinal parenchymal fibres are very weak and apparently discontinuous.

In specimen CRBA438-439, the cylindrical pharynx is about one-eight of the total body length (0.3 mm)and placed in a posterior horizontal position, while in specimen CRBA440-442 the pharynx represents aboutone-twelfth of the body length (0.4 mm) and is situated slightly posterior to the central region. The outer epi-thelium is ciliated and underlain by a layer of longitudinal muscles followed by a layer of circular muscles.Underneath the inner pharynx epithelium lies an outer layer of circular muscles, followed by an inner layer oflongitudinal muscles fibres. The mouth is located in the middle of the pharyngeal pouch in specimenCRBA438-439 at 2.2 cm from the tip of the body, but in specimen CRBA440-442 it is situated in the posteriorportion of the pharyngeal pocket, somewhat anterior to the hind wall of the pharyngeal pouch at 2.7 cm fromthe tip of the body. In specimens CRBA438-439 and CRBA440-442 the gonopore is situated at 0.23 and 0.6mm from the mouth, respectively.

There are 13 to 15 testes situated on either side of the body (CRBA438-439 and CRBA440-442, respec-tively). The rounded or oval-shaped, irregularly sized follicles occupy approximately one-eight of the dorso-ventral diameter and are arranged in ventral longitudinal rows, extending anteriorly from about the root of thepharynx up to two-fifth of the distance between the root of the pharynx and the ovaries.

The vasa deferentia open separately into the bulbar lumen, which communicates with an ejaculatory ductthat opens at the tip of the penis papilla (Fig. 4A). The bulbar lumen and the ejaculatory duct are lined with anucleated epithelium that is underlain with a layer of circular muscle fibres.

The conical penis papilla has a relatively broad base. It has an oblique ventro-caudal orientation and inboth specimens the tip projects into the bursal canal. The outer wall of the penis papilla is covered with a thinepithelium, which is underlain with a layer of circular muscles at its distal section; on the middle and moreproximal parts of the papilla the circular muscle layer is considerably thicker and bounded by a layer of longi-tudinal muscles.

The genital atrium is lined with a nucleated epithelium that is underlain with a subepithelial circular mus-cle layer, followed by a longitudinal one.



The small ovaries are situated close to the ventral nerve cord, occupying about one-fifth of the dorso-ven-tral diameter. The ovaries are located at approximately one-third of the distance between the first testis and thebrain. Somewhat posterior to the gonopore, the oviducts turn towards the middle of the body and open sepa-rately into the bursal canal.

The copulatory bursa is rounded and slightly flattened; it is lined with tall, vacuolated cells, with thenuclei mainly in peripheral position (Fig. 4B). The bursa is connected with the atrium by means of a some-what obliquely running bursal canal (Fig. 5A, B). The lining epithelium of the canal bears long cilia and con-sists of nucleated cells surrounded by a subepithelial layer of circular muscle fibres. Shell glands could not bediscerned.

FIGURE 3. A. Diagrammatic transverse section through the pharynx of Microplana aixandrei showing the arrangementof the rows of longitudinal and circular muscles. B. Microplana grazalemica. Living animal. Scale bar 1mm.



Discussion. This species can be distinguished from the other European native land planarians by a combi-nation of external features and anatomical characteristics of the genital apparatus.

The dorsal colouration pattern of M. grazalemica resembles that of M. nana and M. gadesensis sp. nov,the latter described below. However, in both M. nana and M. gadesensis a copulatory bursa is absent.

There are three European species that are provided with a copulatory bursa and lack a genito-intestinalduct, viz. M. mahnerti, M. styriaca, M. aixandrei. In M. mahnerti the bursal canal runs parallel to the bodysurface and the oviducts open at its most distal end. In contrast, in M. grazalemica the bursal canal is anobliquely running structure with the opening of the oviducts at its central region. (Minelli, 1977). In M. styri-aca the penis bulb is very large and elongated, while an expanded bulbar lumen is absent, in contrast to therounded penis bulb from M. grazalemica that is provided with a distinct bulbar lumen. In M. styriaca the ovi-ducts form a short common oviduct that enters the bursal canal (Freisling, 1935), contrasting with the oviductsof M. grazalemica, which open separately into the bursal canal.

Differences between M. aixandrei and M. grazalemica were detailed above.

FIGURE 4. Microplana grazalemica. CRBA438-A439.A–B. Sagittal sections of the copulatory apparatus; anterior tothe left.



FIGURE 5. Microplana grazalemica. CRBA438-A439. A. Sagittal reconstruction of the copulatory apparatus; anteriorto the left. CRBA440-442. B. Sagittal reconstruction of the copulatory apparatus; anterior to the left.

Microplana gadesensis sp. nov.

Material: Holotype, CRBA443, CRBA444, Llano del Berral (lat. 36.75428, long. -5.45399; alt. aprox. 657m) in the central part of the Sierra de Grazalema, Cádiz (Spain), 5 December 2004, sagittal sections on 2slides. This specimen was accidentally broken in two pieces while it was being embedded in paraffin.Paratype: CRBA445, ibid., sagittal sections on 1 slide.

Diagnosis. Microplana gadesensis sp. nov. can be distinguished anatomically from its congeners by amuscular penis bulb provided with a very thick layer of circular muscle fibres, rounded bulbar lumen, largepenis papilla provided with a wide ejaculatory duct, genito-intestinal duct that links the female genital duct tothe gut, and absence of a copulatory bursa.



Ecology and distribution. The species is known only from the type locality.Etymology. The specific epithet is based on the name of the region where the specimens were collected,

Cádiz, named Gades in Latin, in southern Spain.Description. When the animals were fixed in the field and later prepared for sectioning, they were consid-

ered to be morphologically similar to M. grazalemica, and therefore no particular attention was paid to theirexternal features. It may be assumed that their external appearance did not differ much from that of M. graza-lemica. From the sections of the holotype it could be determined that the animal was about 10 mm long.

The two small eyes (eye cup diameter 16 µm in sections) are located at a short distance in front of thebrain (Fig. 6) and are only clearly visible under observation through a dissecting microscope.

The subepidermical longitudinal fibres of the body musculature weak. In the ventral body region numer-ous strong parenchymal longitudinal fibres are present especially dorsally and ventrally of the ventral nervecords. This ventral musculature becomes especially strong at the cephalic region, where it attaches at the epi-thelium of an invaginated area (Fig. 6). This longitudinal musculature partially encloses the eyes.

The short pharynx is about one-twelfth of the total body length (0.3 mm), its root being located slightlyposterior to the middle of the body. The outer epithelium is ciliated from the root to the tip of the pharynx. It isunderlain by a thin layer of longitudinal muscles followed by a thin layer of circular muscles. The inner epi-thelium of the pharynx is underlain with a very thick outer layer of circular muscles, followed by a longitudi-nal muscles layer, the latter in some areas intermingled with the circular layer. In specimen CRBA445 themouth is situated close to the posterior wall of the pharyngeal pouch at 2.5 cm from the tip of the body and 0.9mm from the gonopore.

The number of elongated testes varies from four (CRBA445) to six (CRBA443-444) on either side of thebody. The follicles occupy approximately one-fourth of the dorso-ventral diameter and are arranged in longi-tudinal rows, extending from some distance behind the ovaries to the root of the pharynx.

The penis bulb is provided with a rounded bulbar lumen, leading to a very wide ejaculatory duct that runscentrally through the penis papilla and gradually narrows before opening at the tip of the papilla (Fig. 7A).The bulbar lumen and the ejaculatory duct are lined with a nucleated epithelium. The bulbar lumen is sur-rounded by a thick layer of circular muscle fibres that extends on the proximal part of the penis papilla; thiscoat of circular muscle is highly developed on the ventral side of the seminal vesicle.

The large and elongated penis papilla is highly muscular; it is oriented parallel to the body surface. At thelevel of the penis bulb, the two vasa deferentia turn dorsally and open separately into the anterior portion ofthe bulbar lumen.

The penis papilla projects into the female genital atrium. The gonopore opens into the mid-ventral sectionof the atrium.

The small ovaries occupy one-fifth of the dorso-ventral diameter, lying on top of the ventral nerve cords.The ovaries are located at approximately one-sixth of the distance between the brain and the root of the phar-ynx. The oviducts arise from the ventral side of the ovaries. The rather narrow female genital duct is providedwith distinct cilia and receives the openings of the oviducts at its posterior end. The duct is lined with nucle-ated epithelium that is underlain with a thick, subepithelial layer of circular muscles followed by a layer oflongitudinal muscle fibres. The female genital duct of specimen CRBA445 receives the openings of the shellglands anteriorly to the openings of the oviducts. Secretion granules are present in the same area in the holo-type, but not the glands itself. A long genito-intestinal duct arises from the distal end of the female genitalduct (Fig. 7B) and immediately recurves to run approximately parallel to the dorsal wall of the atrium, subse-quently communicating with the gut. The genito-intestinal duct is lined with a ciliated epithelium and is sur-rounded by a layer of circular muscles (Figs. 8A, B).

The wall of the elongated atrium is lined with a thin epithelium, provided with a thin circular muscularlayer, surrounded by a thin longitudinal layer of muscles.



FIGURE 6. Microplana gadesensis. CRBA443-444. Sagittal section through the head.

Discussion. Microplana gadesensis can be distinguished from the other known native European land pla-narians species by the anatomy of its copulatory apparatus.

Regarding the external features, similar body colouration is found in M. nana and M. grazalemica. How-ever, M. nana and M. grazalemica lack a genito-intestinal duct and also the thick layer of circular musclefibres around the bulbar lumen.

Among the known European species of Microplana a genito-intestinal duct has been reported also for M.humicola, M. pyrenaica, M. howesi (Scharff, 1900), M. giustii Minelli, 1976, M. henrici (Bendl, 1908), M.attemsi (Bendl, 1909), M. peneckei (Meixner, 1921), M. scharffi (von Graff, 1899), M. monacensis (Heinzel,1929), and M. terrestris. However, M. humicola presents a hyaline colouration (Schneider, 1935), instead ofthe pigmented dorsal colouration pattern of M. gadesensis. With respect to anatomical features, M. humicolahas two testes on either side of the body, while its penis papilla is vertically oriented, in contrast to the 4-6 tes-tes and horizontally oriented penis papilla of M. gadesensis. M. pyrenaica and M. howesi are bigger species(up to 5 cm long) with a poorly developed penis papilla (Minelli, 1977), in contrast to the large and welldefined papilla of M. gadesensis. In M. giustii, the testes are present in a postpharyngeal position (Minelli,1976), contrasting with the prepharyngeal testes of M. gadesensis. M. henrici, M. attemsi, and M. peneckeishow atrial folds (Minelli, 1977), whereas atrial folds are absent in M. gadesensis. In M. scharffi the vasa def-erentia fuse to a common vas deferens at the base of the penis papilla (Ball & Reynoldson, 1981). In M. gade-sensis the vasa deferentia open into the bulbar lumen, far anterior to the root of the penis papilla. With respectto external features, M. scharffi is a very long (maximum 90 mm) animal with a pale colouration, whereas M.gadesensis is shorter and with darker colouration. In M. monacensis (Heinzel, 1929) and M. terrestris(Minelli, 1977) a bursa is present, whereas such a structure is absent in M. gadesensis.



FIGURE 7. Microplana gadesensis. CRBA443-444. A–B. Sagittal sections of the copulatory apparatus; anterior to theleft.

Biogeography and biodiversity

In the present study, we present 54 new locality records for 202 individuals (Fig. 1, Table 1) from the IberianPeninsula, with an undetermined number of species per record. The intrinsic complexity of the identificationof planarian specimens (see Introduction) presently prevents us to provide an extensive list of species col-lected at each locality. Since this is a work in process, further results will be presented in future publications.However, the external features of the specimens collected suggest that usually more than one species ispresent at a single locality.

The southern sector of the Iberian Peninsula has been sampled in three distinct areas, yielding 7 newlocalities and 43 specimens. We have focused our analysis on the region of the Sierra de Grazalema, whichhas the maximum average annual rainfall (more than 2000 mm) of the entire Iberian Peninsula, thus poten-tially forming a suitable area for terrestrial planarians. The material that has been analysed so far yielded three



TABLE 1. List of localities with new records of autochthonous terrestrial planarians in the Iberian Peninsula. The first

locality of the unidentified species belongs to Portugal while the rest belongs to Spain. Latitude and longitude are

expressed in decimal degrees (Datum European 1979). *: locality with populations of the three new species described in

the present paper (M. aixandrei, M. grazalemica, M. gadesensis).

Locality Province Latitude Longitude Species

Eo river Asturias 43.42404956 -7.13722351 Rynchodemus sylvaticus

Luarca Asturias 43.53122231 -6.54066729 Rynchodemus sylvaticus

Serra del Corredor, riera Can Rimbles Barcelona 41.594097 2.46444 Rynchodemus sylvaticus

Serra del Corredor, riera de Canyamars Barcelona 41.598317 2.44302 Rynchodemus sylvaticus

Montjuïc mountain, Vivers dels Tres Pins

Barcelona 41.36903802 2.16342328 Rynchodemus sylvaticus

Collsacabra, Puig de Rajols Girona 41.996119 2.488137 Rynchodemus sylvaticus

Sierra de Grazalema, Río Majaceite Cádiz 36.773681 -5.486587 Microplana grazalemica sp.nov

Sierra de Grazalema, Llano del Berral* Cádiz 36.75428 -5.45399 three newly described Microplana

Mata da Margaraça, Serra do Açor Coimbra 40.21681 -7.90961 unidentified

Sant Llorenç del Munt, Sot Teixoneres Barcelona 41.66168 1.99706 unidentified

Collserola, La Rierada Barcelona 41.437904 2.056765 unidentified

Collserola, Can Catà Barcelona 41.474183 2.147384 unidentified

Serra del Corredor, torrent de Canyamars

Barcelona 41.605197 2.45639 unidentified

Serra de l'Obac Barcelona 41.649761 1.97323 unidentified

Montnegre, Sot la Massaneda Barcelona 41.668975 2.602379 unidentified

Montnegre, Pantà la Brinxa Barcelona 41.678624 2.587049 unidentified

Montnegre, àrea Ramió, Sot de Can Masó


Montseny mountains, Tordera river shore


Montseny mountains, Tordera river shore


Montseny town Barcelona 41.758725 2.399094 unidentified

Montseny mountains, el Molar Barcelona 41.763575 2.365855 unidentified

Montseny mountains Barcelona 41.768404 2.467023 unidentified

Montseny mountains, Sot les Mines Barcelona 41.777115 2.353205 unidentified

Font del Vidre Barcelona 42.151601 1.93114 unidentified

Los Alcornocales Cádiz 36.091333 -5.6222 unidentified

Sierra de Grazalema, Llanos del Republicano

Cádiz 36.696744 -5.376621 unidentified

Sierra de Grazalema, salto del cabrero path

Cádiz 36.706868 -5.421502 unidentified

Sierra de Grazalema, Las Covezuelas Cádiz 36.711273 -5.3684 unidentified

La Miel Cádiz 36.11933041 -5.47959327 unidentified

Fragas do Eume A Coruña 43.417185 -8.063563 unidentified

Joanetes Girona 42.116900 2.400990 unidentified

Puigpardines Girona 42.142877 2.42516 unidentified



new species, suggesting that upon further research the species richness of this area might turn out to be higherthan in other sections of the Iberian Peninsula.

The presence of R. sylvaticus is reported for new areas of the northern sector of the Iberian Peninsula, viz.the Cantabric and the Mediterranean regions (Fig. 1), where other elements of the Centro-European fauna arealso usually present. It should be noted that one of the records corresponds to a plant nursery (Viver dels TresPins, Barcelona, NE Spain). However, the others correspond to natural areas.

General discussion

For practical purposes we have adopted in this paper the higher classification of the land planarians that is cur-rently available (Kawakatsu et al., 2003 and references therein). However, we are well aware of the fact thatthe higher classification of triclads in general and that of land planarians in particular, is in need of revision.Since a revised classifcation of the triclad flatworms is in preparation and will be published elsewhere and theprimary purpose of the present paper is the description of three new species, we have adopted the currentlyclassical higher taxonomy of the terrestrial planarians.

There are three genera of autochthonous European land planarians, viz. Rhynchodemus (subfamily Rhyn-codeminae), Geobenazzia and Microplana (subfamily Microplaninae). We have assigned the three new spe-cies to the genus Microplana on the basis of the taxonomic definition of this genus presented in Kawakatsu etal. 2003: “Microplaninae with an elongate, rounded body (preserved worms may have rounded anterior withgreatest diameter anterior to mouth); with two small eyes; male copulatory organ consists of well-developed

Fageda d'en Jordà Girona 42.149366 2.519729 unidentified

Ansó valley Huesca 42.886305 -0.805996 unidentified

Serra del Tallat, Montblanquet Lleida 41.481613 1.124143 unidentified

Montsec, Sant del Bosc Lleida 42.066583 0.901788 unidentified

Talarn Lleida 42.18945694 0.89633052 unidentified

Capdella, camí Filià Lleida 42.47181881 0.98914475 unidentified

Capdella Lleida 42.47638271 0.99366981 unidentified

Sierra Cebollera, Puente Ra Logroño 42.04626 -2.68545 unidentified

Monasterio de Valvanera Logroño 42.23156 -2.85017 unidentified

Isaba, Belabarte creek shore Navarra 42.877835 -0.862635 unidentified

Roncal valley, Belagua river shore Navarra 42.890385 -0.892624 unidentified

Anduña river shore Navarra 42.921576 -1.011301 unidentified

Irati forest Navarra 42.988866 -1.098518 unidentified

Irati forest Navarra 43.007606 -1.201696 unidentified

Road NA174, Artesiaga river shore Navarra 43.113056 -1.538889 unidentified

Tximista river shore Navarra 43.233825 -1.659663 unidentified

Etxalar, water channel margin Navarra 43.240646 -1.662193 unidentified

Road NA 4400 from Etxalar to Sare Navarra 43.242546 -1.62907 unidentified

Road NA 4000 from Lesaka to Endara's marsh

Navarra 43.269574 -1.749217 unidentified

Tail of Endara's marsh Navarra 43.273254 -1.775105 unidentified

Els Ports de Beceït Tarragona 40.707546 0.236715 unidentified

Ergoyen Vizcaya 43.32776899 2.81787795 unidentified



penis with muscular bulbus, bulbar cavity and elongate penis papilla projecting into short antrum; femaleorgan has one simple canal (vagina) entering common genital antrum; typically a genito- intestinal canal ispresent arising from vagina or behind the common ovovitelline duct; seminal bursa can be present with a shortconnection (bursasteles, bursal canal, or proximal duct) to female tract, with or without connections to intes-tine, or bursa is blind lacking connections to intestine; when bursa is absent a genito-intestinal connection isoften present; without adenodactyls, tentacles or suckers.”

FIGURE 8. Microplana gadesensis. CRBA443-444. A. Sagittal reconstruction of the copulatory apparatus; anterior tothe left. CRBA445. B. Sagittal reconstruction of the copulatory apparatus; anterior to the left.

A genito-intestinal duct is reported here only for M. gadesensis. The taxonomic characters of this speciesare in complete agreement with those mentioned in the definition of the genus Microplana (Kawakatsu et al.,2003). Microplana aixandrei and M. grazalemica possess a copulatory bursa (or seminal bursa) that is con-nected to the female tract but not to the intestine. Kawakatsu et al. (2003) consider M. nana and M. perereca



Marcus & Marcus, 1959 to be good species of the genus Microplana, albeit that these species lack a genito-intestinal duct. Therefore we assign M. aixandrei, M. grazalemica, and M. gadesensis to the genusMicroplana and not to Rhynchodemus. In Rhynchodemus a genito-intestinal duct and a well developed penispapilla are absent and the subepithelial longitudinal muscle fibres of the strong cortical musculature aregrouped into large, definite bundles, in contrast to species of Microplana, including the three described as newin the present paper.

In the present work we have not considered the synonymic treatment proposed in Minelli (1977) andKawakatsu et al. (2003). In this paper we follow the advice of H. Jones (personal communication), who hasexamined part of the original slides corresponding to the synonymized species and considers that some ofthem must maintain their original generic assignments.

Presence of a spermatophore is extremely rare in terrestrial flatworms. It has been described only for thegeoplanid Pimea monticola Winsor, 1991 and the Australian rhynchodemid Platydemus victoriae (Dendy,1898), a member of the subfamily Rhynchodeminae (Winsor, 1998). The spermatophore in P. monticola dif-fers in size (500 µm) and composition (multiple layers that completely envelope sperm) from the structureobserved in M. aixandrei. In P. victoriae the spermatophore presents a thin red single layer enveloping thesperm similar to the single layered structure observed in M. aixandrei, but probably of different nature.Although we did not observe sperm in the putative spermatophore of M. aixandrei the existence of a sper-matophore of sclerotic structure is reported also for the freshwater planarians (Sluys, 1989a). Similary, Sluysand Ball (1989) suggested the existence of a spermatophore in Vatapa tumidosa (Sluys & Ball 1989), a marineplanarian, on the basis of similar reasons (localization in the copulatory bursa and sclerotic nature), althoughsperm was not observed. The recurrent observation of this structure only in the copulatory bursa of M. aixan-drei reinforces our idea that it represents a spermatophore. This spermatophore in M. aixandrei represents thefirst report of such a structure in a member of the subfamily Microplaninae.

The amphiatlantic species R. sylvaticus is widely distributed in continental Europe and the British Isles,including an isolated record in northern Spain (Minelli, 1977; Boix & Sala, 2001). On the basis of our newfindings, R. sylvaticus is probably widely distributed in the northern sector of the Iberian Peninsula. Further-more, its special strategy of locomotion in which the animals leave a dotted slime-trail and thus use lessmucus (mainly composed of water) allows the species to live in relative dry areas (Pantin, 1950). Such areasare present in southern Spain and Portugal (with Mediterranean climatic characteristics), where the specieshas not yet been collected.

Sluys (1999) published several maps of the world summarizing the biogeographic data on species richnessand endemism of land planarians. Only few data have been added to the European fauna since then, thus thiswork still holds at the present time.

In his map of species richness, Sluys (1999, Fig. 1), the two grid cells corresponding to the northern sectorof the Iberian Peninsula and the Balearic Islands have two and three records, corresponding with at least fourdifferent species in total, while no record is provided for the southern sector. The new data presented in thispaper suggest that this low specific richness is due to a collector’s artefact.

In contrast to the few records available in the literature, our intensive sampling efforts show that land pla-narians are widely distributed across the Iberian Peninsula. The ecological conditions required for their pres-ence (Kawaguti, 1932; Ball & Reynoldson, 1981), especially high humidity, are present on the IberianPeninsula in forested areas and near shores of rivers. The extremely low density of the worms, their cryptichabitus, and their specific habitat requirements ask for a thorough sampling methodology (detailed searches ofcovered surfaces of stones and adjacent ground) and an intensive sampling effort for successful fieldwork.The results presented in this study show that future studies on terrestrial planarians most likely will yield othernew and interesting information on this group of animals on the Iberian Peninsula.



Acknowledgements

We are grateful to Edgar Vila Farré, Sergi Rodríguez, Oscar Gordo, Mar Sancho Prat, Salvador Carranza andCristina Cabrera for their help in the field and to Eduard Filella for his information on the current status ofBipalium kewense on the Iberian Peninsula. We are indebted to Hugh Jones and Manuel Irimia for their valu-able suggestions during the preparation of this paper and to Kay Eckelt and Michael Lang for their help in thetranslation of the German texts. We are grateful to Dr. L. Winsor and Prof. Dr. M. Kawakatsu for reviewingand commenting on the penultimate version of the manuscript. Dr. L. Winsor also kindly assisted with the his-tological interpretation of the vacuolated tissues in one of the species. MVF acknowledges financial supportfrom SYNTHESYS, a program of the European Commission under the Sixth Research and TechnologicalDevelopment Framework Programme “Structuring the European Research Area”, which enabled MVF towork at the Zoological Museum of the University of Amsterdam during March 2005 (grant number NL-TAF-179). This work is also supported by grant BFU2004-05015/BFI (to R.R.) from the “Ministerio de Uducacióny Ciencia” of Spain. Prof. Dr. M. Kawakatsu and Dr. T. Kifune are thanked for nomenclatural advice.

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Terrestrial planarians (Platyhelminthes, Tricladida, Terricola) from the Iberian Peninsula: new records and description of three new species