Microplana groga, Jones & Webster & Littlewood & Mcdonald, 2008

Jones, Hugh D., Webster, Bonnie L., Littlewood, D. Timothy J. & Mcdonald, Jillian C., 2008, Molecular and morphological evidence for two new species of terrestrial planarians of the genus Microplana (Platyhelminthes; Turbellaria; Tricladida; Terricola) from Europe, Zootaxa 1945 (1), pp. 1-38 : 32-36

Microplana groga n. sp.

Material examined: Specimens labelled “ M. terrestris Font Groga ”, NHML 1998.2.9.3-5, deposited by Mateos et al. (1998). Three specimens, FG1 (3 slides), FG2 (3 slides), FG3 (4 slides).

Type locality: “Font Groga”, Massis de Collserola (Sant Cugat del Vallès, Barcelona, Spain).

Etymology: the specific epithet is after the type locality.

Holotype: Specimen FG 1, three slides FG1.1, FG1.2 and FG 1.3, labelled Font Groga, M. Terrestris , NHML 1998.2 .9.3. Co-types FG 2 (4 slides, NHML 1998.2.9.4) and FG 3 (4 slides, NHML 1998.2.9.5), similarly labelled.

Mateos et al. (1998) identified five specimens as M. terrestris . Three were sectioned and are the specimens deposited above. A further two were sacrificed for nucleic acid extraction and one of these is the source of the GenBank sequence AF178318 (Carranza, pers. comm.) which is identical to that of our specimens of M. kwiskea (K2, K8, K9) ( Fig. 2 ). The worms were described as “reddish to brown with black granules all over the surface: anterior end darker; creeping sole white” ( Mateos et al. 1998). Representative sections of these three specimens are shown in Figs 23–26 . The distance between the mouth (pharyngeal aperture) and the gonopore can be measured in all three specimens. In FG1 it is 1.4 mm, in FG2 it is 1.7 mm, in FG3 it is 1 mm. The pharynx is cylindrical, 1.1 mm long in FG2 and 0.9 mm in FG3 (it is reflexed in FG 1), and about 0.4 mm in diameter in all three specimens ( Fig. 23a–d ). The pharyngeal aperture is two-thirds along the pharyngeal cavity. The outer epithelium of the pharynx is densely ciliated, cilia about 2.5 µm long. The pharyngeal musculature consists of an outer thin, sub-epidermal layer of circular muscle about 10 µm thick, a loose layer of longitudinal and radial fibres about 130 µm thick and an inner, dense layer of mainly circular fibres, but with some longitudinal fibres, about 40 µm thick. The inner epithelium is not ciliated. Though there are no transverse sections of the whole body, it can be seen in longitudinal sections that there is no longitudinal muscle immediately sub-epidermal but that longitudinal muscle fibres are present 30 to 60 µm inside the epidemis ( Fig. 23e ). There are few longitudinal fibres dorsal to the ventral nerve cords. In all three specimens, the sperm ducts are exceptionally broadly expanded between the pharynx and the anterior end of the penis ( Fig. 23e ). The ducts narrow, internal diameter about 5 µm, enter the penis musculature where they are convoluted, and discharge separately into the base of the ejaculatory duct (which is labeled “seminal vesicle” by Mateos et al. 1998). The penis is muscular and free in the antrum for about three-quarters of its length. The penis musculature is quite dense with, from outer to inner: dense circular muscle about 15 µm thick: dense longitudinal muscle about 10 µm thick; more loose radial muscle about 100 µm thick; circular muscle about 10–20 µm thick around the ejaculatory duct. The ejaculatory duct is broad, about 70 µm in diameter, for most of the length of the penis but narrows at the distal end of the penis to about 10 µm. There are no internal projections or folds on the wall, though there are some possible secretory products. There is a short vagina which at its posterior end receives the two ovo-vitelline ducts ventrally and the genito-intestinal canal dorsally (Mateos et al. label this as “bursa copulatrix”). In FG1 and FG2 the single genito-intestinal canal opens into one of the digestive diverticula via a confused, plate-like opening, possibly with more than one opening ( Figs 24 , 25 ). In FG3 the genito-intestinal canal forks to become Y-shaped with each upper branch opening into a digestive diverticulum on the opposite side ( Fig. 26 ). These openings are more distinct than in FG1 and FG2.

Discussion of M. groga . The presence of a single pair of eyes and of a genito-intestinal connection means that these specimens also are of the genus Microplana . However, they are distinct from both M. terrestris and M. kwiskea . The penis of M. terrestris has a broad, spherical proximal ejaculatory duct with internal projections (“seminal vesicle” of many authors) and a narrow, distal, muscular penis papilla. The ejaculatory duct of M. groga lacks such a differentiated portion. The genito-intestinal canal of M. terrestris broadens to a sinuslike cavity before narrowing to its opening or openings to the intestine. That of FG1 and FG2 is narrow for its entire length. That of FG3 is narrow but forked. The penis of FG1, FG2 and FG3 are similar, so the significance of the forked genito-intestinal canal in FG3 is uncertain. Its openings into the intestine on either side are rather different from FG1 and FG2. The lack of a distinct layer of longitudinal muscle dorsal to the ventral nerve cord also distinguishes these specimens from M. terrestris and also from M. kwiskea .

The cox1 results ( Fig. 2 ) suggest these specimens are identical with M. kwiskea . However, Mateos et al. (1998) noted that their specimens had a white sole, but the sole of M. kwiskea is of the general body colour. The mouth and gonopore are much closer together in the Font Groga specimens (1–1.7 mm apart) than in M. kwiskea (4 mm apart). The internal surface of the ejaculatory duct appears smooth in the Font Groga specimens, unlike M. kwiskea in which there are internal folds or projections. The musculature of the penis of the Font Groga specimens (outer to inner circular, longitudinal, radial and circular fibres) is different from that of M. kwiskea (dense circular fibres with a longitudinal layer). In the Font Groga specimens the genito-intestinal canal is narrow for its entire length and in two of the three specimens the duct divides near its entry to the digestive diverticulum so that there are two or three openings into the diverticulum ( Figs 24 & 25 , and Fig. 12 of Mateos et al. 1998). FG3 has a forked, Y-shaped genito-intestinal canal ( Fig. 26 ). These openings are posterior to the penis and antrum and somewhat dorsal. The genito-intestinal canal of M. kwiskea is narrow for its entire length and opens into the intestine on one side at the level of the penis via a distinct papillate opening. These differences lead us to conclude on morphological evidence that these specimens are of a different species to M. kwiskea . That is despite the apparently unambiguous identity of the cox1 sequence from the specimen AF178318 . Thus they are a previously undescribed species, Microplana groga .

How can the identical cox1 sequences reported above ( Fig. 2 , GenBank AF178318 , K2, K8 and K9) be explained? There are two possibilities. Firstly, since different individuals were sectioned and sequenced by Mateos et al. (1998) there is the possibility that the sequenced individual ( AF178318 ) was in fact not of the same species as FG1, FG2 and FG3. Secondly, although less likely, it is possible that the small fragment of cox1 characterized is identical between M. kwiskea and M. groga . Although widely popular as a ‘barcoding’ gene, partial cox1 is not always capable of discriminating between species with absolute accuracy (e.g. Meyer and Paulay 2005). Also, the fragment of cox1 characterized by Mateos et al. (1998) and herein, is approximately half the size of the fragment (~650 bp) suggested by Hajibabaei et al. (2007) for DNA barcoding. Whilst it remains possible that the fragment of cox1 used in the current study fails to differentiate between what are clearly distinct species ( M. kwiskea and M. groga ), there is a need to sequence new specimens of M. groga as detailed in this study; namely to section part of the worms for species verification and sequence the remaining tissues. If the sequences remain identical, or insufficiently different to separate them in a phylogeny, the possibility that the fragment of cox1 is incapable of differentiating these taxa remains and additional sequence data will be required for further verification.

Figures

FIGURE 2. Phylogenetic analysis of cox1 genes showing results from maximum likelihood (ML) analysis. The topology of the tree was identical with maximum parsimony (MP). Nodal support indicated is from bootstrap resampling (ML/MP; n=100/1000 respectively; =50% only shown).

FIGURE 12. Microplana terrestris (Müller 1774). Specimens from NHMW, originating from the University of Graz. a. Longitudinal section through the copulatory apparatus of a specimen from County Galway, Ireland. b & c. Transverse sections through a specimen of unknown origin.

FIGURE 23. Microplana groga n. sp. a & b, the pharynx of specimen FG2. a is of the whole pharynx and (b) is a high magnification view of part of (a). c & d, similar low and high magnification views of the pharynx of specimen FG3. Anterior to the right. e. Composite diagram of the copulatory apparatus, based mostly on specimen FG1 (cf. Figure 24). Anterior to the left.

FIGURE 24. Microplana groga n. sp. Specimen FG1 of Mateos et al. (1998). NHML, 1998.2.9.3. Anterior to the left. a. Longitudinal section showing the expanded sperm duct, penis and vagina. b–e. Sections at intervals through the vagina and genito-intestinal canal. All to the same scale.

FIGURE 25. Microplana groga n. sp. Specimen FG2 of Mateos et al. (1998). NHML, 1998.2.9.4. Anterior to the right. a. Longitudinal section showing the penis and genito-intestinal canal. b–e. Sections at intervals through the genito-intestinal canal. The serial sections are incomplete. All to the same scale.

FIGURE 26. Microplana groga n. sp. Specimen FG3 of Mateos et al. (1998). NHML, 1998.2.9.5. Anterior to the right. a. Longitudinal section showing the expanded sperm duct, penis and vagina. b–g. Sections at intervals through the vagina and genito-intestinal canal. Note that the genito-intestinal canal forks and there are two openings to the intestine, one on either side. (b) shows the opening of the genito-intestinal canal on one side, and (g) on the other side. All to the same scale.