Contributions to Zoology, 85 (2) – 2016Fabio Scarpa; Piero Cossu; Tiziana Lai; Daria Sanna; Marco Curini-Galletti; Marco Casu: Meiofaunal cryptic species challenge species delimitation: the case of the Monocelis lineata (Platyhelminthes: Proseriata) species complex
Appendix

To refer to this article use this url: http://ctoz.nl/vol85/nr02/a01

Appendix 2

In the following descriptions, Pore Index (a/c: b/c: c) is based on the following distances: a = mouth - vagina; b = vagina - male pore; c = male pore - female pore, with c = 1. Karyotype formula is represented as follows: fundamental number (NF) (according to Matthey, 1949); relative length and centromeric index of each chromosome; chromosome nomenclature within parentheses (m = metacentric; sm = submetacentric; st = subtelocentric; t = acrocentric).

Molecular diagnostic characters were checked on a subset composed by specimens belonging to the two new species, M. lineata complex sensu stricto, and M. fusca, in which molecular pure characters have been underlined (see Tables 1, 2). The state of the pure characters is present only across all members of a single clade (see Jörger and Schrödl, 2013).

This published work and the nomenclatural acts it contains have been registered in Zoobank, the online registration system for the ICZN. The ZooBank LSIDs (Life Science Identifiers) can be resolved and the associated information viewed through any standard web browser by appending the LSID to the prefix http://zoobank.org/. The LSID for this publication is: urn:lsid:zoobank.org:pub:F5F588A8-EE88-4FAE-B05D-99B8F9439F5E. The electronic edition of this work was published in a journal with an ISSN, and has been archived.

Types have been deposited at the Swedish Museum of Natural History (SMNH) (Stockholm, Sweden) and in the collections of the Zoological Museum (CZM), University of Sassary (Italy).

Phylum Platyhelminthes Minot, 1876

Order Proseriata Meixner, 1938

Family Monocelididae Hofsten, 1907

Subfamily Monocelidinae Midelburg, 1908

Genus Monocelis Ehrenberg, 1831

Monocelis algicola Curini-Galletti and Casu nov. sp.

urn:lsid:zoobank.org:act:D81936DB-350B-4042-8373- EC8B9EF7C0AF

(Fig. 5A-D()

FIG2

Fig. 5A-D: Monocelis algicola nov. sp.: sagittal reconstruction of the genital area (A); general organisation of a live specimens (B); sagittal section of copulatory organ (C); spermatogonial metaphase plate; arrows point to heterobrachial Chromosome II (D). E-H: Monocelis exquisita nov. sp.: sagittal reconstruction of the genital area (E); general organisation of a live specimens (F); sagittal section of copulatory organ (G); spermatogonial metaphase plate; arrows point to Chromosome III (H). I-M: Monocelis lineata s.s.: sagittal reconstruction of the genital area (I); sagittal section of copulatory organ (H); spermatogonial metaphase plate (M), from specimens from Helsingor, Denmark (HEo). Scale bar : C, G, L = 10 μm; D, H, M = 5 μm. Abbreviations used in figures: b: bursa, co: copulatory organ, e: eye, fd: female duct, fg: female glands, fp: female pore, gl: gut lumen, ml: muscular lining, mp: male pore, ov: ovary, pg: prostatic glands, ph: pharynx, pp: penis papilla, pt: prostatic tissue, rh: rhabdoid gland, sp: sphincter, st: statocist, t: testis, v: vagina, vi: vitellaria, vs: seminal vesicle.

Holotype. Italy: Sardinia, Cala Rossa (Lat. 41.027595N; Long. 8.892886E), 2-3 m deep on algae (05.15.2002): one specimen sagittally sectioned (SMNH-Type-8770).

Paratypes: same data as holotype, 32 specimens sagittally sectioned (CZM 614-645).

Other material: 15 specimens studied karyologically (in non-permanent mounts); five specimens sequenced.

Etymology: the specific epithet refers to the habitat of the species (from latin alga: seaweed; colere: to dwell).

Description. A very slender Monocelis, slightly tapering anteriorly. Living specimens very active; extremely adhesive due to the presence of numerous adhesive glands in the tail. The fixed holotype is 2.8 mm long: living, extended specimens up to 3 mm long. Many specimens show faint, irregular brownish lines in the cephalic area. With a large, pigmented eye-shield, just in front of the statocist. Epithelium with insunk nuclei, ciliated (cilia about 4 μm long) apart from the dorsal side of the caudal tip. With two types of rhabdoid glands: a) large, up to 30 μm long, numerous dorsally and caudally, with fine, filamentous content, appearing dot-like in transverse sections; b) smaller (up to 15 μm), more globular glands, with much thicker content (about 0.5 μm in diameter), particularly abundant caudally. With well developed ventral longitudinal musculature; rest of subepidermal musculature poorly developed. With a short, tubular pharynx, about 1/9th total body length, almost at mid-body. The pharynx is ciliated (cilia 3-4 μm long) a part from distal tip, where pharyngeal glands discharge. Musculature layers inverted with respects to body musculature, poorly developed a part from the layers surrounding the inner lumen. Oesophagous 1/4th to 1/5th the total length of the pharynx.

Male genital system. With about 30 testes, irregularly arranged in front of the pharynx. The copulatory organ is spherical (48.8 ± 1.03 μm wide, 47.18 ± 0.93 μm high; 30 measurements) (Fig. 5C(). It consists of a seminal vesicle, surrounded by a thin muscular layer, and provided with a short penis papilla. Thin (up to 2 μm), proximal musculature mostly longitudinal. Penis papilla 7.31 ± 2.01 μm long, surrounded by a thin muscular coating. The proximal basis of the papilla is lined by stronger, circular muscles. The penis papilla is lined internally by a thick layer of prostatic tissue, whose cell nuclei are mostly located outside the copulatory organ. Prostatic secretion dot-like. Male atrium small; opening to the outside though the male pore.

Female genital system. Ovaria just in front of pharynx. Vitellaria run from the level of the first testes to in front of bursa. Oviducts join behind pharynx to form an unciliated female duct. Just in front of the copulatory organ, the female duct is connected ventrally to a short, unciliated vaginal duct (10-15 μm long), surrounded by a strong sphincter, and opening to the outside through a vagina. Immediately above this duct, a very short bursal duct, also surrounded by a strong muscular sphincter, leads to a large bursa, up to 100 μm across, consisting mostly of resorbing vacuoles, many of which abutting the gut, with sperm in different stages of degeneration. Distally to this vaginal-bursa complex, the female duct is lined with an irregular, high, at least partly resorbiens epithelium, runs over the copulatory bulb and opens to the outside just posterior to the male pore through a female pore, surrounded by female glands (Fig. 5A().

Karyotype. Chromosome number n=3. With a distinctly heterobrachial chromosome pair. Chromosome I and II nearly even in size; Chromosome III about 83% the size of Chromosome I. Karyotype formula: FN=5; Chromosome I: 35.69 ± 0.56; 42.19 ± 0.8 (m); Chromosome II: 34.88 ± 0.62; 9.26 ± 0.78 (t); Chromosome III: 29.46 ± 0.43; 44.15 ± 0.61 (m) (Fig. 5D().

Diagnosis. slender Monocelis species with a large, pigmented eye-shield. With large rhabdoids with thin fibrillar content, and smaller rhabdoids with coarser content. With about 30 testes. Spherical copulatory organ (about 48 μm across), with a thin muscular coating, and a poorly developed penis papilla, with a thick layer of prostatic tissue. Prostatic secretion finely granular. With a large, mostly resorbiens bursa, and a vagina close to male pore; female duct at least partly resorbiens distally. Karyotype with n=3, and Chromosome II acrocentric. Pore Index: 6.7: 0.9: 1.

Molecular diagnosis: see Table 1.

Monocelis exquisita Curini-Galletti and Casu nov. sp.

urn:lsid:zoobank.org:act:26F6E96F-1D4C-4275-87E4- 895285647043

(Fig. 5E-H()

Holotype. Italy: Sardinia, Porto Puddu (Lat. 41.192306N; Long. 9.340800E), lagoon, about 30 cm deep in coarse silty sand (07.01.2002): one specimen sagittally sectioned (SMNH-Type-8769).

Paratypes: same data as holotype, 29 specimens sagittally sectioned (CZM 646-674).

Other material: 15 specimens from type locality studied karyologically (in non-permanent mounts); five specimens sequenced from type locality. Porto Puddu, mouth of creek close to the lagoon (see map in Fig. S3) (Lat. 41,983; Long. 9,317), about 20 cm coarse silty sand (07.01.2002; 05.28.2014), numerous specimens studied alive, three specimens studied karyologically.

Etymology: the specific epithet reflects the extensive search for populations of the species outside the type locality (lat. exquisitus: searched for, sought out).

Description. A slender, active Monocelis. The fixed holotype is 2.5 mm long: living specimens up to 3 mm long. Some specimens show faint, irregular brown lines in the cephalic area. With a pigmented eye-shield in front of statocist, irregular in shape; in a few specimens split into two closely lying spots. Epithelium with insunk nuclei, ciliated (cilia about 3-3.5 μm long) apart from the dorsal side of the caudal tip. With large rhabdoid glands, particularly numerous dorsally and caudally, in some areas appearing as a continuous layer parallel to the surface, with coarse-grained, thick (> 1 μm in diameter) fibrillar content, intensely stained with hematoxylin (Fig. 5C(). A few, scattered, subepithelial glands, with fine-grained, dot-like eosinophilous content are present dorsally. Posterior end with adhesive glands. Pharynx at the beginning of the posterior half of body, comparatively long (between 1/7th and 1/8th of total body length). It is ciliated externally (cilia 2-2.5 μm long), and internally (cilia 3-4 μm long) in its distal half, a part from its tip, where pharyngeal glands discharge. With very strong inner circular musculature. Oesophagous about 1/4th the total length of pharynx.

Male genital system. With numerous (40-60) testes, irregularly arranged in front of the pharynx. The copulatory organ is nearly spherical (44.12 ± 0.96 μm wide, 39.02 ± 0.92 μm high; 30 measurements), backward oriented, with an angle of about 40° to the vertical axis (Fig. 5G(). It consists of a seminal vesicle, surrounded by a thick muscular layer, with a thin, pointed penis papilla, ranging 12.86 ± 0.43 μm in length (30 measurements). Proximally, the muscular coating, up to 3 μm thick, consists mostly of longitudinal muscles. Circular musculature more developed distally; penis papilla lined by a thick layer of outer circular muscles, with a thin layer of longitudinal, inner musculature. The distal part of the copulatory bulb is lined internally by a thin layer of glandular epithelium, whose cell bodies are placed mostly externally to the copulatory bulb. These glandular cells have a fibrillar content. Male atrium small.

Female genital system. Similar to the previous species in general arrangement. Ovaria just in front of pharynx. Vitellaria run from the level of the first testes to in front of bursa. The vaginal duct is short (about 15 μm long), unciliated and surrounded by a strong sphincter. Above it, a large vacuolar bursa (up to 150 μm in diameter), abutting the gut, is present. The female duct, lined by an irregular, high, at least partly resorbiens epithelium, opens to the outside just posterior to the male pore through a female pore, surrounded by female glands (Fig. 5E().

Karyotype. Chromosome number n=3. Karyotype with Chromosome I and II almost of the same length; Chromosome III markedly smaller, about 60 % the size of Chromosome I. Karyotype formula: FN=6; Chromosome I: 39.26 ± 0.51; 45.07 ± 0.7 (m); Chromosome II: 37.38 ± 0.46; 31.72 ± 1.6 (sm); Chromosome III: 23.35 ± 0.42; 29.43 ± 1.09 (sm) (Fig. 5H().

Diagnosis. Slender Monocelis species with pigmented eye-shield. With large rhabdoid glands with coarse fibrillar content, and smaller rhabdoids with dot-like, eosinophylous content. With 40-60 testes. With a nearly spherical, backward oriented copulatory organ (about 40 μm across), with a slender penis papilla about 13 μm long, internally coated by a thin layer of prostatic tissue. Prostatic secretion fibrillar. With a large, mostly resorbiens bursa and external vagina close to male pore. Karyotype with n=3, and Chromosome III markedly smaller than the other pairs. Pore index: 6.3: 1.1: 1.

Molecular diagnosis: see Table 2.

Species justification

In addition to diagnostic characters of sequences (see above), the two new species are uniquely characterised by Chromosome III with low value of centromeric index and markedly smaller than the other pairs, and the penis papilla with a very thin prostatic component (Monocelis exquisita nov. sp.); Chromosome II acrocentric, extremely thin layer of proximal musculatura of the copulatory bulb, and the penis papilla with a thick prostatic component (M. algicola nov. sp.) (Tables S1-S4). The two new species appear externally quite similar, as both are more slender than the rest of the species-group, and may present similar cephalic pigmentation. In addition to the remarkably different morphology of their copulatory bulbs, the two species differ for morphology of prostatic secretion (dot-like in M. algicola nov. sp.; fibrillar in M. exquisita nov. sp.), and content of the larger, dorsal, rhabdoid glands: thin, fibrillar in M. algicola nov. sp., thick, coarse-grained in M. exquisita nov. sp. It is worth mentioning that the morphology of both rhabdoid and prostate gland secretion found in M. exquisita is also found in HEo (close to type locality of Monocelis lineata, see above) (Fig. 5I() and is then possibly plesiomorphic.