Microplana robusta Vila-Farré and Sluys, 2011

Vila-Farré, Miquel, Sluys, Ronald, Mateos, Eduardo, Jones, Hugh D. & Romero, Rafael, 2011, Land planarians (Platyhelminthes: Tricladida: Geoplanidae) from the Iberian Peninsula: new records and description of two new species, with a discussion on ecology, Journal of Natural History 45 (15 - 16), pp. 869-891 : 876-881

Microplana robusta Vila-Farré and Sluys , sp. nov.

( Figures 1 , 5 , Table 1)

Material examined

Holotype. ZMA V.Pl. 6857.1, Parque Natural Fragas do Eume , province of A Coruña (North Western Spain), 3 July 2003, sagittal sections on 13 slides.

Other material. CRBA-3776a-j, Parque Natural Fragas do Eume, province of A Coruña (North Western Spain), 12 April 2006, sagittal sections on 10 slides.

Etymology

The specific epithet is derived from the Latin adjective robustus, meaning strong, firm, and alludes to the relatively large body size.

Diagnosis

Microplana robusta sp. nov. can be distinguished anatomically from its congeners by a muscular penis bulb provided with intermingled longitudinal and circular muscle fibres, short and cylindrical penis papilla, very short genito-intestinal duct that links the long and narrow female genital duct to the gut, and absence of a copulatory bursa.

Description

In elongated state, living sexually mature specimens up to 50 mm long, 2–3 mm wide ( Figure 5A ). Preserved holotype specimen 12.9 mm long, as determined from histological sections. Cylindrical body (flattened when resting, Figure 5B ) tapers anteriorly and posteriorly to form blunt points. Dorsal surface orange with a lighter anterior end and a darker posterior end in ZMA V.Pl. 6857.1. Creeping sole reaches anterior end. Two black eyes (eye cup diameter 59 µm in sections) located laterally at a short distance anterior to the brain. Numerous longitudinal muscle fibres present in ventral body region .

Short cylindrical pharynx about one-tenth of total body length (0.9 mm in sections) in holotype and placed in a central, almost horizontal position. Outer epithelium of pharynx ciliated and underlain by layer of longitudinal muscles, followed by layer of circular muscles. Thin layer of longitudinal muscle fibres under inner pharynx epithelium, followed by intermingled layers of circular and longitudinal muscle fibres and a layer of longitudinal fibres ( Figure 5C ). Mouth located approximately in middle of pharyngeal pouch, 5 mm from tip of body in holotype. Spherical structure, origin unknown, bounded by circular muscle fibres, in intestinal lumen of holotype.

Very thick subintestinal mesenchymal transverse muscle layer in holotype, dorsal to brain and ventral nerve cords, ventral to intestine and continuing into posterior end, where it becomes weaker.

More than eight testes situated on either side of body in specimen CRBA-3776aj. Small rounded, or oval-shaped, irregularly-sized follicles occupy approximately one-fifth of the dorsoventral diameter of body, arranged in ventral longitudinal rows, extending anteriorly from about root of pharynx to an unknown distance (because frontal region of CRBA-3776a-j removed for use in DNA analysis). Sperm present in vasa deferentia; testes not observed in holotype. Ovaries not discovered in specimens examined.

Strongly muscular, spherical penis bulb ( Figure 5D ) with intermingled longitudinal and circular muscle fibres, especially well-developed in specimen CRBA-3776. Narrow vasa deferentia run directly dorsally to the ventral nerve cords and open separately into ejaculatory duct, which opens at tip of penis papilla ( Figure 5E ). Ejaculatory duct lined with nucleated epithelium and surrounded by thick layer of circular muscle fibres stronger at base of duct. Short and conical penis papilla of holotype has relatively broad base and an oblique, dorsoventral orientation. Conical penis papilla larger in CRBA-3776a-j. Outer wall of penis papilla covered with thin, nucleated epithelium, underlain with thin layer of circular muscles, bounded by layer of longitudinal muscles. Genital atrium lined with nucleated epithelium underlain with subepithelial circular muscle layer, followed by layer of longitudinal muscle. Gonopore 1.7 mm from mouth.

Rather narrow female genital duct with distinct cilia, openings of oviducts at posterior end. Anterior end of female genital duct projects slightly into atrium in holotype. Duct lined with nucleated epithelium underlain with thick, subepithelial layer of circular muscles, followed by layer of longitudinal muscle fibres. Shell glands open into female genital duct at same level as oviducts. Very short genito-intestinal duct arises from distal end of female genital duct ( Figures 5D,E ) and immediately communicates with gut. Genito-intestinal duct lined with ciliated epithelium by layer of circular muscle.

Discussion

The absence of ovaries in the sectioned specimens suggests that they were not fully mature. Despite this we suspect that the gross morphology of the copulatory apparatus will not change substantially after maturity because the copulatory apparatus of the type specimen has all the typical elements of the copulatory apparatus of a member of the genus Microplana , including the genito-intestinal duct. This species can be distinguished from other native European land planarians by a combination of external features and anatomical characteristics of the genital apparatus. The dorsal colouration pattern of M. robusta resembles that of M. pyrenaica (Von Graff, 1893) , M. scharffi and M. monacensis . Some individuals of M. groga can probably also exhibit a similar colouration pattern. However, M. pyrenaica is provided with an enormous penis bulb with a large intra-bulbar cavity and an extremely reduced penis papilla. In contrast, the penis bulb of M. robusta is much smaller than in M. pyrenaica and an intra-bulbar cavity is absent. Furthermore, its penis papilla is well developed. In addition, the female genital duct of M. pyrenaica is wider and almost horizontal and its genito-intestinal duct arises from its central section, whereas in M. robusta this duct is very narrow and has an oblique ventro dorsal disposition. In M. robusta the genito-intestinal duct communicates with the posterior end of the female genital duct, whereas in M. pyrenaica the middle section of the female duct gives rise to the genito-intestinal duct (cf. Heinzel 1929).

Microplana scharffi is yellow or cream when starved ( McDonald and Jones 2007). However, it is an extremely long and thin planarian (up to 9 cm), in contrast to M. robusta , which is shorter and thicker. With respect to anatomical features, the type specimen of M. scharffi has a very short genito-intestinal duct and possesses a copulatory bursa. Although a very short genito-intestinal duct is also present in M. robusta , the latter does not have a copulatory bursa. Furthermore, in M. scharffi the vasa deferentia fuse half-way along the elongated penis papilla, whereas in M. robusta the ducts fuse within the penis bulb and the penis papilla is conical.

In M. monacensis a copulatory bursa is present ( Heinzel 1929), whereas such a structure is absent in M. robusta . In addition, the penis papilla of M. monacensis is elongated, but it is conical in M. robusta .

Although M. groga was described originally as being “reddish to brown” ( Jones et al. 2008) we have also collected cream-coloured specimens similar to M. robusta . However, M. groga is more slender than M. robusta . Regarding anatomical features, in M. groga the vasa deferentia open separately at the base of the broad ejaculatory duct, close to the base of the penis bulb. In contrast, in M. robusta the ducts open separately at the base of a narrow ejaculatory duct, close to the base of the short and conical penis papilla. In addition, in M. groga there is a short, horizontal female genital duct (= vagina) of the same length as the genito-intestinal canal, whereas in M. robusta the very long female genital duct is oblique and the genito-intestinal canal is extremely short.

Among the known European species of Microplana a genito-intestinal duct has also been reported for the following species: M. humicola Vejdovsky, 1889 ; M. pyrenaica ; M. giustii Minelli, 1976 ; M. henrici ( Bendl, 1908) ; M. attemsi (Bendl, 1909) ; M. peneckei (Meixner, 1921) ; M. scharffi ; M. monacensis ; M. terrestris ; M. nana ; M. kwiskea Jones et al., 2008 ; M. groga ; M. gadesensis Vila-Farré et al. 2008 . Comparisons with the species M. pyrenaica , M. scharffi , M. monacensis and M. groga have been presented above. Microplana humicola is a small species (4 mm) that presents a hyaline colouration (cf. Schneider 1935), instead of the pigmented dorsal colouration pattern of the much larger M. robusta . Microplana humicola has two testes on either side of the body and its penis papilla is elongated, in contrast to the abundant testes and short conical penis papilla of M. robusta . In M. robusta the genito-intestinal canal is also shorter. Microplana giustii is a black species with post-pharyngeal testes (cf. Minelli 1976), contrasting with the orange-cream colouration of M. robusta , with its prepharyngeal testes. Microplana henrici , M. attemsi and M. peneckei present atrial folds (cf. Minelli 1977a), whereas these are absent in M. robusta . In M. terrestris a bursa is present, whereas such a structure is absent in M. robusta . In M. kwiskea (cf. Jones et al. 2008) the very elongated penis papilla houses a wide ejaculatory duct; the genito-intestinal canal is long. In M. robusta , in contrast, the penis papilla is short and conical and the genito-intestinal canal is very short. Microplana gadesensis has an elongated penis papilla and a very long genito-intestinal duct (cf. Vila-Farré et al. 2008), contrasting with the short and conical penis papilla and the very short genitointestinal duct of M. robusta . While in M. nana the penis papilla is elongated and the penis bulb is narrow, in M. robusta the penis papilla is short and conical and the spherical penis bulb is wide. The genito-intestinal canal is longer in M. nana than in M. robusta .

A female copulatory apparatus provided with a genito-intestinal canal is found only in two continental African species of Microplana , M. harea ( Du Bois-Reymond Marcus, 1953) and M. termitophaga Jones et al., 1990 . In M. harea (1) the testes extend to the level of the copulatory apparatus, (2) the almost spherical penis bulb is provided with a distinctive seminal vesicle and a reduced penis papilla, (3) the common oviduct meets the female genital duct, with approximately the same length as the genito-intestinal duct, at its distal part; all of these features are different in M. robusta . With respect to external features, M. harea is dorsoventrally flattened and “the back is dark-gray with a fine, partially faded black middle stripe...” and a “light area lies between the small eyes...” ( Du Bois-Reymond Marcus 1953), contrasting with the cylindrical body and the uniform orange-cream dorsal surface that is devoid of stripes in M. robusta .

Microplana termitophaga is very similar to M. harea with only one important exception that is relevant for the present discussion: the genito-intestinal duct of M. termitophaga is extremely short. However, the differences between M. harea and M. robusta pointed out above are also valid for M. termitophaga .

Among the known species of Microplana from Madagascar and neighbouring islands a genito-intestinal duct has been reported for M. mediostriata (Geba, 1909) , M. trifuscolineata (Kaburaki, 1920) ( Mauritius) and M. tristriata (Geba, 1909) ( Comores) . Unfortunately, the copulatory apparatus of M. gebavoeltzkowi Ogren and Kawakatsu, 1988 ( Comores) has never been described. Although M. mediostriata , M. striata and M. trifuscolineata have a genito-intestinal duct the gross morphology of their copulatory apparatus is completely different from that of M. robusta , notably the presence of a penis sheath in the three first-mentioned species. Furthermore, in M. tristriata the meeting point of the oviducts is far removed from the genitointestinal canal, whereas in M. robusta the point of communication of the oviducts with the female genital duct coincides with the origin of the genito-intestinal canal. Microplana gebavoeltzkowi is dirty yellow with a broad grey-brown median band, whereas M. robusta is orange and without a median band.

Among the known Asian species of Microplana a genito-intestinal duct has only been reported for M. unilineata (Frieb, 1923) and M. uniductus (De Beauchamp, 1930) . Microplana unilineata presents a mid-dorsal stripe, which is absent in M. robusta . Regarding anatomical features, the penis bulb of M. unilineata houses a bulbar cavity and the penis papilla is relatively long; the genito-intestinal duct is also relatively long. In contrast, in M. robusta , there is no seminal vesicle and the penis papilla and genito-intestinal duct are short. In addition, M. uniductus presents an elongated penis bulb and a common oviduct, whereas in M. robusta the penis bulb is rounded and a common oviduct is absent.

Microplana teres ( Von Graff, 1899) , also from Asia, is described on the basis of an immature specimen, showing a conical, slender penis papilla. In contrast, in M. robusta the penis papilla is not slender. The external colouration pattern is also different in both species.

The Asian Microplana ruteocephala Kaburaki, 1922 is “uniformly light black with a slight touch of reddish tint, in front grading over into a red or yellow tone at the tip of the body. Extending over the surface is a fine black median stripe, which loses itself in front” ( Kaburaki 1922). In contrast, M. robusta is uniformly orange cream. As the description of M. ruteocephala is only based on external morphology we cannot compare its anatomy with that of M. robusta .

Four species of the genus Microplana have been recorded from North America, two of which ( M. scharffi and M. terrestris ) have been discussed above. The third species, M. rufocephala ( Hyman, 1954) is a long, black species provided with a long genito-intestinal duct, contrasting with the features of M. robusta . Microplana atrocyanea (Walton, 1912) , the fourth North American member of the genus, is a black planarian with an elongated penis papilla and a long genito-intestinal duct (cf. Hyman 1954) and therefore also differs from M. robusta .

Among the known Microplana species of South and Central America a genitointestinal duct has been reported for the following species: M. yaravi (Du Bois- Reymond Marcus, 1957), M. montoyai ( Fuhrmann, 1914) and M. cockerelli (Von Graff, 1989) . However, M. yaravi is up to 20 mm long, provided with two broad longitudinal stripes and a copulatory bursa (“uterus” in Du Bois-Reymond Marcus 1957).

Mature specimens of M. montoyai are up to 40 mm long, with black dorsal colouration, a white mid-dorsal stripe, a big bulbar cavity, and a short genito-intestinal duct that arises from the middle of the roof of the female genital duct (“vagina” in Fuhrmann 1914). In contrast, M. robusta has orange dorsal surface, while a middorsal stripe is absent. Regarding anatomical features, a bulbar cavity is absent in M. robusta and its genito-intestinal duct arises from the distal end of the genital duct.

Microplana cockerelli ( Von Graff, 1899) has a bluish-black colouration with both body ends sharply marked off from the rest of the body by a different, lighter colouration; it also has a pale mid-dorsal longitudinal stripe, two seminal vesicles, one of them a presumed “prostatic organ” ( Prudhoe 1949), and a relatively long genito-intestinal canal, all being features that are different from those in M. robusta .

Specimens from las Fragas do Eume, with the external morphology of M. robusta , have been studied from a molecular point of view by Mateos et al. (2009). One of the animals analysed in the present paper, CRBA-3776a-j, was part of that study and its sequence is available from GenBank (accession number, COI: FJ969962 , 18S Tipus II: FJ969990 ). In the study of Mateos et al. (2009) these specimens were grouped together as morphotype M3, which formed a molecular clade consisting solely of specimens from this locality. Although Mateos et al. (2009) included land planarians from several areas of the Iberian Peninsula, this species did not occur at other localities and, consequently, it is probably restricted to a few areas with special and appropriate ecological conditions.

Figures

Figure 1. Distributional records of terrestrial planarians from the Iberian Peninsula used for this study. Filled circles: localities with previously known species. Stars: localities with new species.

Figure 5. Microplana robusta. (A–D) Specimens from Fragas do Eume: (A), extended living animal during movement; (B) living animal, resting; (C) diagrammatic transverse section through the pharynx, showing the arrangement of the rows of longitudinal and circular muscles; (D) holotype ZMA V.Pl. 6857.1, sagittal section of the copulatory apparatus; anterior to the left. (E) Holotype ZMA V.Pl. 6857.1, sagittal reconstruction of the copulatory apparatus; anterior to the left.