Microplana scharffi (Graff 1896)

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 : 7-15

Microplana scharffi (Graff 1896)

Though the identity of this species amongst European species seems clear, examination of several specimens and the material of some authors, has shown that published descriptions and illustrations are in some ways unclear, deficient or inaccurate, even the fairly comprehensive description of Graff (1899). Descriptions and figures of the copulatory apparatus may be found in Graff (1896, 1899, as Rhynchodemus scharffi ), Percival (1925), Eastham (1933) and Adam and Leloup (1944) (all as R. britannicus ), Beauchamp (1934, as R. hovassei ), Battalgazi (1945, as R. decennii ), Minelli (1977) and Ball and Reynoldson (1981) (both as M. scharffi ). Minelli (1977) synonymised the above species as Microplana scharffi (Graff 1896) . Minelli (1977) also considered R. monacensis Heinzel 1929 to be a synonym of M. scharffi . It will be shown that this is not the case.

Rhynchodemus scharffi Graff 1896 . Holotype, NHMW, L154 /M254, Inventory number 2832: I, 9 slides, anterior TS; II, 2 slides, mid-body LS; III, 9 slides, posterior LS. This is of a single specimen found by R.F. Scharff in Dublin, Ireland and is undoubtedly the original specimen. It is in excellent condition. It was designated as holotype by Minelli (1977).

In general shape and proportion, the copulatory apparatus of this specimen is as figured by Graff (1896, Fig. 9 ; 1899, Text Fig. 53). The same diagram is used in both, but with slightly different labeling. However, after examination of the slides, the diagrams are in some respects misleading as follows.

1. The bulk of the penis ( Fig. 3 ) consists of dense layers of radial muscle, giving a stratified appearance in section. This is not conveyed by the Graff diagrams. The outer ends of these fibres turn towards the tip and become longitudinal (as figured by Graff, 1899, Taf. XLVII, Fig. 2 ).

2. The sperm ducts each widen (as in most terrestrial planarians) to form a sperm storage organ anterior to the penis, turn dorsally and enter the base of the penis separately as narrow ducts. They continue separately as even narrower ducts, internal diameter about 5µm, external diameter about 15µm. They are difficult to follow through the penis because of their narrowness and the dense musculature of the penis, but they can be traced. They join nearly half way along the penis to form a short common sperm duct of the same width ( Fig. 3 ). These ducts within the penis musculature are narrower and their junction is closer to the bulb of the ejaculatory duct than suggested by the Graff (1896, 1899) diagrams. The common sperm duct opens into the ejaculatory duct which has a broad proximal bulb and narrows towards the pointed penis tip. The internal wall of this bulb and duct is smooth, with no projections. There is a layer of circular muscle surrounding the ejaculatory duct, not very clear in the type specimen, but clearer in other specimens.

3. Graff (1896) labeled the penis with a chitinous tip though Graff (1899) corrected this in the diagram legend and the text. The tip is not chitinous.

4. The female copulatory apparatus is as illustrated by Graff, with the exception that it is possible to discern a narrow connection from the anterior of the bursa (labeled “uterus” by Graff) to the intestine on the right side ( Fig. 3 , a & d), not noted or figured by Graff. Thus there is a genito-intestinal connection. As Minelli (1977) commented, there is a pencil note (author unknown) on slide III.5 of the holotype series indicating a genito-intestinal connection. The canal leading to this bursa is strongly ciliated.

Rhynchodemus britannicus Percival 1925 . Percival’s material was mostly from Yorkshire, UK, the same county as specimens S1-8. It was described as a new species but Percival did not clearly distinguish between that and R. scharffi . Percival worked at the University of Leeds, UK, and then at the University of Canterbury, New Zealand. Neither can trace any of Percival’s material (pers. comms., Baker 2007; Johns 1983; Norris 1982). Percival (1925) figures the copulatory apparatus, in outline, of three specimens. The penis of specimens A and C is rooted dorsally and is quite long and in one case flexed, similar to S1, S2 and S4. However, the penis of specimen B is illustrated as short and conical and seems rather less typical. Unfortunately both Minelli (1977) and Ball and Reynoldson (1981) used this diagram as the basis for their diagrams, which are thus misleading. The sperm ducts of all three specimens are shown as converging at or near the base of the penis, which, from other specimens, may not be accurate. Percival considered the ciliated genito-intestinal duct (= bursal canal) to open directly into the gut. As we have shown, there is a genito-intestinal connection in the type specimen of M. scharffi via an expanded bursa.

Eastham (1933) figures the copulatory apparatus of a specimen identified as R. britannicus . His material is lost (pers. comm., Eastham 1977). He listed several differences between R. scharffi and R. britannicus but most can be discounted as resulting from different fixation or individual variation. The most significant is the genito-intestinal connection in R. britannicus but its apparent absence in R. scharffi . But as we have seen there is a genito-intestinal connection in R. scharffi . Thus we can be confident that the specimens of both Percival (1925) and Eastham (1933) were of M. scharffi (Graff 1896) .

Rhynchodemus britannicus , material of Adam and Leloup (1944) from Belgium. The material is extant, IRSNB, IG 13876 (5 slides, longitudinal sections of posterior half including copulatory apparatus, examined), IG 14582 (one specimen in alcohol, not examined). The diagram in Adam & Leloup (1944) is mostly accurate, the main discrepancy being that they indicate a direct connection from the genito-intestinal canal to the intestine (their Fig. 1 , c.g.i. and d.i. respectively). Examination of the slides confirms that there is a constriction between the bursa (labeled d.i. by Adam & Leloup) and the intestine ( Fig. 4 ), as in other specimens of M. scharffi . The penis also has dense, layered musculature and narrow sperm ducts joining within the penis just before entering the ejaculatory duct. We are confident that this specimen is of M. scharffi (Graff 1896) .

Rhynchodemus hovassei Beauchamp 1934 . The type specimen was from Turkey, but Beauchamp (1957) reports further specimens (as Microplana hovassei ) from Madeira. The slides of the specimens from both locations are extant and have been examined. Turkey: MNHNP, 1 slide, longitudinal sections of copulatory apparatus labeled “ Rhynchodemus hovassei de B, type, Balta Liman (illegible) 29.IV.32”, label on reverse: AO 29. As Beauchamp (1934, 1957) comments, the sections are poor and badly folded. The penis is as figured by Beauchamp (1934), quite well developed ( Fig. 5a ) and has the dense layered muscular core typical of M. scharffi , though is rather horizontal compared with other specimens. There is a well-defined duct running dorsally from the posterior of the vagina, similar to the bursal duct of other specimens, but the extent of the bursa and any connection to the gut is obscured by folding of the sections. However, enough is visible to be reasonably certain that this was a specimen of M. scharffi (Graff 1896) but probably not fully mature.

Microplana hovassei Madeira specimens ( Beauchamp 1957): MNHNP, 7 slides, 2 TS, 5 LS, variously labeled “Madé a”, labels on reverse AO30-36. Paper label on AO 33 reads “ Microplana hovassei (Beau. Coi.) Madére a, IV.57”. (“Coi” is a reference to M. Coiffait, the collector.) The sections are apparently of a single mature individual. As well as TS sections of this specimen, AO 31 also has a whole mount of an immature specimen 11 mm long. The paper label reads: “ Microplana hovassei (Beauchamp) Madére (Coiffait) , IV.57, Monte Gueymadas C. de d Rabaçal”. This seemingly refers to the whole mount which must be the “non sexue” specimen mentioned by Beauchamp (1957). The sectioned specimen is mature and, as Beauchamp comments, the structure of the penis leaves no doubt that this is of the same species as the specimen from Turkey ( Fig. 5b ). The bursal canal, bursa and genito-intestinal connection are as the holotype of M. scharffi and there is no doubt that these specimens are of that species.

Rhynchodemus decennii Batalgazzi 1945 . Minelli (1977) considered this to be a synonym of M. scharffi . We have been unable to trace the material of Batalgazzi. From the diagram of the copulatory apparatus in Batalgazzi (1945) there are some apparent differences from M. scharffi . The penis is shown as horizontal; the two sperm ducts are shown separately entering the bulb of the ejaculatory duct (labeled as Vesicula seminalis); it is not clear if there is any bursa in the genito-intestinal connection. Nevertheless it otherwise seems similar to the specimen from Turkey described by Beauchamp (1934) and we can be reasonably certain that this was a specimen of M. scharffi .

Thus we concur with Minelli (1977) in considering Rhynchodemus britannicus Percival 1925 , Rhynchodemus hovassei Beauchamp 1934 and Rhynchodemus decennii Batalgazzi 1945 as junior synonyms of Microplana scharffi (Graff 1896) .

Microplana scharffi (Graff 1896) , Specimens S1, S2, S4 and S5 (see above). Specimens S1, S2 and S4 each have a well-developed penis with the dense layered radial musculature typical of this species ( Figs. 6 , 7 & 8 ). The penis of both S1 and S2 has a dorsal origin and they are both flexed at the level of the bulb of the ejaculatory duct. The two sperm ducts are very narrow and join just before entering the bulb of the ejaculatory duct. The bulb and canal of the ejaculatory duct are surrounded by circular muscle within the body of the penis. The bursal canal opens into the bursa which has a narrow anterior connection to an intestinal diverticulum. Examination with polarised light reveals that the intestinal epithelium has many refractive granules (of unknown origin or significance, but possibly a product of prey digestion). These granules are also found in the epithelium of the bursa but only in the region near to the intestinal connection, not in the end near the bursal canal ( Figs 6c , 7c & 8d ). This suggests that digestive products enter the bursa from the intestinal connection.

Discussion of Microplana scharffi . Amongst the European land planarian species, M. scharffi is characterised when alive by its length and elongate, slender shape when extended, its extensibility and its colour, though colour is variable due to different prey ( McDonald & Jones 2007). Internally it is characterised by the unusual length of the penis which may have a dorsal origin and is free for most of its length, by its dense, layered penis musculature, the narrow sperm ducts within the penis which join before discharging into the ejaculatory duct, and by the nature of the genito-intestinal connection with a narrow duct leading to a broad bursa which may receive digestive products. Thus it is a relatively easily recognisable and distinctive species both when alive and when sectioned.

Rhynchodemus monacensis Heinzel 1929 . Minelli (1977) also considered this to be a synonym of M. scharffi . The specimen, from Monaco, exists (not destroyed as assumed by Minelli 1977), NHMW, L153/ M249, Inventory number 2849. I 1-2, 2 slides TS anterior end; II 1-2, 2 slides TS mid-body; III 1-3, LS posterior end. Sections cut at 5 µm. Heinzel (1929) states that the specimen was identified as R. scharffi by Bendl. All slides are labeled “ Bendl 1908 ” and labeled by him “ Rhynchodemus scharffi Graff ” with “ scharffi ” crossed out and replaced by “ monacensis ”, presumably by Heinzel. The copulatory apparatus ( Fig. 9 ) is as figured by Heinzel (1929, Taf. 10, Fig. 4 ). The specimen was only 5 mm long, fully mature, and the penis, bursa and genito-intestinal connection are very different from any specimen of M. scharffi (compare Fig. 9 with Figs 3–8 ). In view of these differences, it is surprising that either Bendl or Minelli (1977) should consider this to be conspecific with M. scharffi . Thus we consider the proposal of Minelli (1977) to be invalid and that Microplana monacensis ( Heinzel 1929) should be reinstated as a valid species. Beauchamp (1961) figures the copulatory apparatus of M. monacensis (his Fig. 59a) but the legend incorrectly states that it is of M. terrestris .

Figures

FIGURE 1. Microplana spp. Colour photographs of live specimens (stills from digital video). Scale line = 1 cm (the circular depressions on the background stone are 1 cm apart and about 2 mm in diameter): a. Microplana scharffi (about 35 mm long, anterior to the right). b. Microplana kwiskea n. sp. a khaki flatworm (upper, about 23 mm long, anterior to the right) and M. terrestris (lower, the dark grey specimen, about 20mm long, anterior to the bottom). c. The same specimens as (b) when contracted and lying on their dorsal side. The narrow end of both is anterior. The ventral creeping sole is visible in both. d. A “granular” specimen of M. terrestris (about 15 mm long, anterior to the right). All specimens from Kirby Wiske, Yorkshire.

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 3. Microplana scharffi (Graff 1896). Holotype (as Rhynchodemus scharffi), NHMW, L154/M254, Inventory number 2832. a. Diagram of the copulatory apparatus, based on the following micrographs. b–d. Micrographs of the copulatory apparatus at different planes through the serial sections. e. Micrograph at larger magnification of the penis showing the combined sperm duct entering the ejaculatory duct. Anterior to the left.

FIGURE 4. Microplana scharffi (Graff 1896). Specimen of Adam & Leloup (1944) from Belgium (as Rhynchodemus britannicus Percival 1925), IRSNB, IG 13876. a – c. Sections through the copulatory apparatus at different planes. Anterior to the left.

FIGURE 5. Microplana scharffi (Graff 1896). a. The holotype specimen of Rhynchodemus hovassei Beauchamp 1934 from Turkey, MNHNP, AO 29. b. Specimen from Madeira identified as Microplana hovassei by Beauchamp (1957), MNHNP, AO 30-36. Sections though the copulatory apparatus, both of merged adjacent images. Anterior to the left.

FIGURE 6. Microplana scharffi (Graff 1896). Specimen S1, NHML 2006.6.28.22. a. Section through the penis. Note the circular muscle around the ejaculatory duct. b. Section through the female part of the copulatory apparatus. c. Same section as (b) viewed with polarised light, showing the refractive granules (white) in the intestinal epithelium and in the bursal epithelium nearest to the entry to the intestine. Anterior to the left.

FIGURE 7. Microplana scharffi (Graff 1896). Specimen S2, NHML, 2006.6.28.23. a. Section through the penis. Note the circular muscle around the ejaculatory duct. b. Section through the female part of the copulatory apparatus. c. Same section as (b) viewed with polarised light, showing the refractive granules (white) in the intestinal epithelium and in the bursal epithelium nearest to the entry to the intestine. Anterior to the left.

FIGURE 8. Microplana scharffi (Graff 1896). Specimen S4. NHML, 2006.6.28.25. a & b. HLS sections through the penis showing the junction of the sperm ducts (a) and the single combined sperm duct (b). c & d. Sections showing the bursal canal and bursa (genito-intestinal duct) d. is in polarised light showing the refractive granules (white) of the intestinal epithelium and in the bursa.

FIGURE 9. Microplana monacensis (Heinzel 1929). Holotype. NHMW, L153/M249, Inventory number 2849. a. Section through the penis. b. Section through the bursa. Anterior to the left.