Molecular and morphological evidence for two new species of terrestrial planarians of the genus Microplana (Platyhelminthes; Turbellaria; Tricladida; Terricola) from Europe Author Jones, Hugh D. 6 Off Hayfield Road, Birch Vale, High Peak, SK 22 1 DG, UK. (Scientific Associate, The Natural History Museum, London; Honorary Lecturer, University of Manchester) E-mail: flatworm @ btopenworld. com Department of Zoology, Natural History Museum, Cromwell Road, London, SW 7 5 BD, UK. E-mails: B. Webster @ nhm. ac. uk; T. Littlewood @ nhm. ac. uk Bridge House, Kirby Wiske, Thirsk, YO 7 4 ER, UK. E-mail: jill @ ntas. demon. co. uk Corresponding author Author Webster, Bonnie L. ebster@nhm.ac.uk Author Littlewood, D. Timothy J. ebster@nhm.ac.uk Author Mcdonald, Jillian C. text Zootaxa 2008 2008-11-28 1945 1 1 38 https://biotaxa.org/Zootaxa/article/view/zootaxa.1945.1.1 journal article 10.11646/zootaxa.1945.1.1 1175­5334 5231281 Microplana kwiskea n. sp. Material examined: Holotype K9. Syntypes K1a to K8 (see above). All collected a few days prior to, and fixed on, 25 October 2004 . Type locality: Bridge House , Kirby Wiske , Thirsk , Yorkshire , United Kingdom (Ordnance Survey Grid Reference: SE 375849) . Etymology: The specific epithet “ kwiskea ” is an abbreviation of Kirby Wiske, the type locality. Description: Live animals ( Fig. 1b , upper) are up to 2.5 cm long and about 2 mm in diameter at the widest point when extended. When contracted they are about 6 mm long and 3 mm wide ( Fig. 1c , upper). They are cylindrical and taper towards the anterior and posterior ends both of which are blunt. They are a uniform mid-brown (khaki) colour, even ventrally. There is a creeping sole mid-ventrally in the form a narrow ridge. Observations on creeping animals through transparent tubes showed little muscular distortion of the sole suggesting that locomotion is primarily by means of the cilia of the sole. Two small eyes, visible as black spots, are present at the anterior end. Otherwise there are no markings or orifices visible on live specimens. Preserved specimens are up to 18 mm long, the anterior end particularly being contracted and curled ventrally ( Fig. 16a & b ). The mouth (pharyngeal aperture) is about 8 mm from the anterior end (44% of body length) and the gonopore is about 12 mm from the anterior end (67%), but these must be regarded as approximate due to contraction. The gonopore in the remaining preserved specimens ( Fig. 16a & b ) and in the sectioned specimens is raised and prominent with a slightly protruded penis, presumably from muscular contraction at fixation. FIGURE 17. Microplana kwiskea n. sp. a. Specimen K2, NHML, 2006.6.28.31. Section through the pharynx. Anterior to the left. b-e. Specimen K8, NHML, 2006.6.28.37. b. Section through an eye. c. Section through an ovary near the anterior end. d. Transverse section, anterior to the pharynx. e. Enlarged view of the ventral portion of (d) FIGURE 18. Microplana kwiskea n. sp. Holotype, specimen K9, NHML, 2006.6.28.38. Longitudinal sections through the copulatory apparatus. Anterior is to the left and the sections are progressively from approximately the midline towards the right side. a. Through the tip of the penis and also the junction of the ovo-vitelline ducts, vagina and genitointestinal canal. b. Through the base of the penis showing the entry of the sperm ducts to the ejaculatory duct. c. Through the opening of the genito-intestinal duct to the intestine. FIGURE 19. Microplana kwiskea n. sp. Specimen K1a, NHML, 2006.6.28.30. Horizontal longitudinal sections through the copulatory apparatus. Anterior to the right. a–d are progressively more ventral. a. The penis and genito-intestinal canal. b. The penis and, to its right, the opening of the genito-intestinal canal into a digestive diverticulum. c. The conjunction of the genito-intestinal canal and the ovo-vitelline ducts. d. The vagina and penis. e. Micrograph in polarised light of the section in (b) showing the circular muscle of the penis and the highly refractive granules (white) present in the digestive epithelium. There are no granules in the genito-intestinal canal (cf. M. terrestris and M. scharffi ). FIGURE 20. Microplana kwiskea n. sp. Specimen K2, NHML, 2006.6.28.31. Longitudinal sections at an oblique angle. Anterior to the left. a–e show the course of the genito-intestinal canal from its origin at the posterior of the vagina (a) to its opening into the digestive diverticulum (e). c. shows the opening of the sperm ducts into the ejaculatory duct. f. Micrograph in polarised light of the section in (e) showing the circular muscle of the penis and the highly refractive granules (white) present in the digestive epithelium. There are no granules in the genito-intestinal canal (cf. M. terrestris and M. scharffi ). FIGURE 21. Microplana kwiskea . Manchester (1985) specimens (HDJ collection). a. Photograph of a live (partly autodigested) specimen. Anterior to the right. 2 mm graph paper. b & c. Sections through the copulatory apparatus. Anterior to the left. FIGURE 22. Microplana kwiskea . Shirdley Hill (1992) specimens (HDJ collection). a. Live specimen, anterior to the right, on 2 mm graph paper, thus the worm was about 2.2 cm long and 2 mm wide. b & c. Sections through the copulatory apparatus of a further specimen. Anterior to the left. The general anatomy is typically rhynchodemid. The two eyes of K8 have a diameter about 35 µm and length about 50 µm ( Fig. 17b ). The gut is triclad and there is a cylindrical pharynx. The pharynx of K2 is about 1.5 mm long and 0.5 mm in diameter ( Fig. 17a ). The wall of the pharynx is about 200 µm thick. The outer epithelium of the pharynx is about 2.5 µm thick and densely ciliated, the cilia being about 2.5 µm long. Inside this is a loose layer of radial and longitudinal muscle about 160 µm thick with an inner dense layer of circular and longitudinal muscle about 40 µm thick. The inner wall is not ciliated. The creeping sole ( Fig. 17d & e ) is distinct, narrow ( 0.5 mm wide, about one third of the total width) and raised as a mid-ventral ridge ( 0.25 mm high). The epidermis of the creeping sole is about 20 µm thick, has few, narrow, rhabdites and is densely ciliated with cilia about 5 µm long. Dorsal and lateral epithelium is about 30 µm thick with copious broad rhabdites but apparently devoid of cilia. Sub-epidermal muscle is indiscernable. Rhabdites are formed sub-epidermally, just inside the epidermis, about 50 µm from the outer surface. Inside the rhabdite-forming zone is a layer of parenchymal longitudinal muscle, about 50 µm thick dorsally and laterally, thickening ventrally over the creeping sole to about 100 µm . There are further longitudinal muscle bundles dorsal to the ventral nerve cords ( Fig. 17d & e ). The paired ventral nerve cords are joined by occasional transverse commissures ( Fig. 17d & e ). The epithelium of the intestinal diverticula is between 50 µm and 100 µm thick and contains numerous very small refractive granules. The paired ovaries of K8 are 500 µm from the anterior end ( Fig. 17c ). The paired ovo-vitelline ducts run posteriorly dorso-lateral to the ventral nerve cords. Testes lie ventrally between the intestinal diverticula. They are oval, about 100 µm by 70 µm . It is not possible to discern the sperm ducts in transverse sections of the anterior of the body, but posterior to the pharynx they run dorso-lateral to each ventral nerve cord in the vicinity of the ovo-vitelline ducts. The two sperm ducts broaden to about 30 µm in diameter just before they reach the penis to form sperm storage organs which are reflexed forwards ( Fig. 18 ). Presumably these straighten on penis eversion. Each then narrows to about 10 µm external diameter, 5 µm internal diameter, enters the muscular base of the penis and the two ducts discharge separately but close together into the ejaculatory duct. The penis is long, about 1 mm , and the distal half is free in the antrum ( Figs 16c & 18 ). For most of its length it is roughly parallel-sided, about 200 µm in diameter. The ejaculatory duct is broad, about 100 µm in diameter and has numerous internal short projections or folds. At its distal end (the last 250 µm ) the penis narrows to a blunt point and the ejaculatory duct in the distal 100 µm is narrow, about 10 µm in diameter, and has a smooth internal wall. The free portion of the penis has a dense outer layer of circular muscle fibres about 15 µm thick. Transverse sections of the penis show it to consist mostly of dense circular muscle fibres with a thin layer of longitudinal muscle near the outer epithelium ( Figs 16d & 19 ). The vagina is narrow and short, 320 µm long. The posterior end of the vagina receives the openings of the two ovo-vitelline ducts ventrally and the genito-intestinal duct dorsally. The genito-intestinal duct is clearly defined, strongly ciliated internally and has an internal diameter of 10 µm and external diameter of 30 µm for its entire length. It runs dorsally from the vagina, forwards and to the right to open into the right posterior intestinal diverticulum at about the level of the penis. The refractive granules of the digestive epithelium do not enter the genito-intestinal duct ( Figs 19e & 20f ). 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. Specimen from Manchester . Collected 20 October 1985 , Ecology Study Area, campus of the University of Manchester (Grid Reference: SJ846968). This specimen ( Fig. 21 ) partially autodigested before it was fixed. It is not quite fully mature in that the penis is not very large, but the ejaculatory duct extends for most of its length and has internal folds or projections, similar to the above. The genito-intestinal duct is well-defined, narrow and runs directly to a digestive diverticulum on one side. Its identity was uncertain until compared with the above specimens from Kirby Wiske and we are confident that it is a specimen of M. kwiskea . Specimens from Shirdley Hill, Lancashire . Collected October 1992 at the site of population studies of Australoplana sanguinea (sensu Jones 1981) reported by Jones et al. (1998 and 2001) (Grid reference: SD360129). Two specimens have been sectioned (in addition to undoubted M. terrestris from the same site). Both have the same penis structure and direct genito-intestinal duct typical of M. kwiskea ( Fig. 22 ) and again we are confident these are of that species. Discussion on Microplana kwiskea . The presence of a single pair of eyes and of a genito-intestinal connection means that these specimens are undoubtedly of the genus Microplana . There was little doubt about M. kwiskea being different from M. scharffi , the shape, size and colour being sufficient to distinguish the species. The different anatomy of the copulatory apparatus simply confirms this distinction. However, the size and shape of M. kwiskea is broadly similar to that of M. terrestris , though the colour is different, hence our original uncertainty. The molecular results are unequivocal, the specimens are of a different species to M. terrestris . The anatomy of the male ducts, penis and of the genito-intestinal duct are quite different in the two species. The expanded ejaculatory duct with internal projections (seminal vesicle of some authors) runs most of the length of the penis in M. kwiskea , but in M. terrestris it occupies only the basal half of the penis. In M. kwiskea , the genito-intestinal duct is of constant diameter for its entire length and it runs noticeably forwards to open into the intestine to one side (the right) of the penis. In M. terrestris the genito-intestinal duct broadens to a sinus-like cavity before narrowing to its opening or openings to the intestine which, though forwards of its origin, are somewhat dorsal and not as far forwards as in M. kwiskea . The pharyngeal opening (mouth) and gonopore are further apart in M. kwiskea than in M. terrestris . Apart from their different colour, probably the best discriminating feature between the two species when alive is the ventral creeping sole. This is best seen when the animals are viewed from the ventral side when placed in a transparent container. In M. terrestris the sole is wide, almost half the ventral width, hardly raised and almost white. In M. kwiskea the sole is narrow, ridged and virtually the same colour as the rest of the body.