Trocheta ariescornuta n. sp. (Annelida, Hirudinida: Erpobdellidae) – a new cavernicolous leech from Motena Cave in Georgia

A new leech species Trocheta ariescornuta n. sp. is described and illustrated here based on two specimens found in Motena Cave in western Georgia, Caucasus. This species differs from the known congeners and other similar Erpobdellids of the region, including Georgian cavernicolous leech - Dina ratschaensis Kobaķhidze, 1958 in a complex of external and internal morphological characteristics: colour, annulation, position of genital pores, shape of the genital atrium with strong coiled cornua (similar to the horns of a ram). Cave inhabitant invertebrate community for Motena Cave is provided.


Introduction
The leech fauna of Georgia is not intensively researched and largely unknown. In Georgia, Kvavadze (2002) reported only 17 leech species, belonging to 3 families and 11 genera, including 7 species of Erpobdellidae. The Erpobdellid Dina ratschaensis Kobaķhidze, 1958 was described as stygobiotic leech from the Sakishore and Tsivtskala caves in the catchment area of the stream Sakishorestskali at Ratscharegion in western Georgia. First, this taxon was considered as a subspecies of D. absoloni Johansson, 1913(Kobaķhidze 1958. Both taxa would not be clearly separated in the older literature. Lukin (1976) considered D. absoloni as a representative of the leech fauna of the Soviet Union. But the figure and description of the reproductive system provided in Lukin (1976) belong to D. ratschaensis. However, both taxa are clearly different, in their reproductive system also. The taxonomical status of D. ratschaensis was clarified by Neubert & Nesemann 1995. At present, both leeches are considered as separate species (Neubert & Nesemann 1995;Nesemann & Neubert 1999). D. stschegolewi (Lukin & Epshtein, 1960) is an other Georgian Erpobdellid-species with a recent record (Khomenko et al. 2017). The genus Trocheta Dutrochet, 1817 is characterized by a heteronomous annulation with eight or more annuli per somite in combination with praeatrial loops of the vas deferens, extending to the ganglion in the somite before male gonopore. Until now 11 species of this genus were recorded in the area of Western Palaearctic: T. subviridis Dutrochet, 1817, T. cylindrica Örly, 1886, T. vignai (Minelli, 1978, T. pseudodina Nesemann, 1990, T. riparia Nesemann, 1993, T. africana, Nesemann & Neubert, 1994, T. falkneri Nesemann & Neubert, 1996 haskonis Grosser, 2000, T. tunisiana Ahmed, Cichocka, Tekaya, Gorzel &Harrath, 2013, T. taunensis Grosser 2015, Trocheta blanchardi Khomenko, Utevsky, Utevsky & Trontelj, 2020. Several species of the genus Trocheta were listed particularly for Georgia or in general for the Caucasus region in various papers. Stschegolew (1949) reported T. subviridis Dutrochet, 1817 from the river Dniester, the Crimean peninsula and the Caucasus. In the figure 77 in Stschegolew (1949), 15г is the position of the gonopores similar to T. blanchardi described from the Crimean peninsula (Khomenko et al. 2020). Lukin (1976) reported T. subviridis also from Caucasus and mentioned the region around Erewan (Armenia). Farther, Lukin (1976) listed T. bykowskii Gedroyć, 1913 for a doubtful very large area, from the Bretagne (France) to Afghanistan, including the Caucasus. T. bykowskii was described from the Eastern Carpathians (Gedroyć 1913). Later, Košel (2004) considered T. bykowskii to be a synonym of Trocheta cylindrica Örley, 1886. The taxonomical status of some populations of "T. bykowskii" is unclear, because the specimens are distinctly different from T. cylindrica. For this reason, the German populations were described as a separate species under the name Trocheta taunensis Grosser, 2015. Trocheta bykowskii is not a valid name, and the actual Trocheta-species is unclear, which listed under this name for the Caucasus region by Lukin (1976) and for Georgia by Kvavadze (2002). Nesemann (1991Nesemann ( , 1993a reported T. cylindrica Örley, 1886 from Georgia. But this author points out explicitly (Nesemann 1991), that T. cylindrica is known from Georgia under the name Trocheta subviridis f. danastrica Stschegolew, 1938. Nesemann & Neubert (1999 cited the description of T. subviridis f. danastrica Stschegolew, 1938 from Georgia. But this taxon (recent considered as distinct species T. danastrica) was described from the river Dniester near Odessa (Ukraine). Nesemann confused T. cylindrica with T. danastrica in his earlier publications. Stschegolew and Lukin did not make distinctions between T. subviridis and T. danastrica, respectively T. s. f. danastrica. All records of T. subviridis from the territory of the former Soviet Union refer to T. danastrica and perhaps also to some other species to be newly described (particularly from the eastern part, e.g. Sakhalin). The taxonomical status of T. danastrica was clarified by Grosser & Epshtein (2009).
There is no evidence, that the Trocheta-species, recorded from Georgia in the past, were collected in subterranean waters. In 2020, two specimens of Trocheta were found in Motena Cave, western Georgia, Caucasus. Perhaps Dina cf. ratschaensis Kobaķhidze 1958mentioned in Chertoprud et al. (2020 is this species. The two Trocheta-specimens differ from other known Trocheta-species in a complex of external and internal morphological characteristics. The most important feature is the shape of the cornua of the genital atrium. The cornua are large and wound up, similar to the horns of a ram. The sampled leech specimens are described as a new species -Trocheta ariescornuta n. sp. in this paper

Material and methods
Material: 2 specimens; Caucasus, Georgia, Motena Cave, 19.VII.2020, leg. Sh. Barjadze & Eter Maghradaze. The holotype is deposited in the collection of the Senckenberg Natural History Museum (Frankfurt a. Main in Germany). The paratype is classified in the collection of the Ilia State University, Institute of Zoology, Tbilisi, Georgia.
Methods: Leeches were collected by hand or with pincers from wet stalagmites outside the water (Fig. 1). The external morphology (the number and position of eyes, the annulation, coloration, papillation and the position of genital pores) was examined on two specimens. The characters of sexual organs of T. ariescornuta n. sp. (location, shape and extension of the genital atrium with the cornua, shape of the ovarian sacks and vasa deferentia), were checked on the single adult specimen (paratype), which shows well developed sexual organs with visible oocytes inside the ovisacs. The paired organs of the male and female reproductive systems were formed almost symmetrically. Therefore, the found shape of these organs is considered as representative of this new species.
Measurements were taken with a ruler (in the authors opinion the precision of such measurement is sufficient, because they anyway largely depend on the body contraction). Material was examined using a stereomicroscope (Novex RZT.PL), and photographs were taken with a microscope camera (Euromex, VC 3031C) and the camera Canon EOS 400D (with several macro lens). x 25 x 12 m) which are connected to each other with narro wholes. The cave ends with a small syphon lake, from which permanent subterranean water stream is flowing and meets the Abasha River as its left tributary. Air temperature close to cave end is 13.0°C. There are different speleothems and big stones in the caves (Tatashidze et al. 2009). Its plan and profile are given in Figs   Literature: Antić & Reip, 2020;Grego et al. 2020;Barjadze et al. 2015Barjadze et al. , 2019a.

Taxonomical part
Taxonomy (according to Tessler et al. 2018 andNeubert 1999 Diagnosis: Trocheta-like large leeches with a heteronomous annulation due to a high tendency to split the annuli, male and female genital pores are separated by four annuli; unicoloured without dark patterns, preserved specimens yellowish grey, living reddish brown; dorsal and ventral surface rough with numerous small papillae; the cornua of the genital atrium are large and wound up.

Description
Habitus: Large leeches with a body length more than 100 mm of preserved specimens. The body is elongated and cylindrical, the last third is slightly flattened. The dorsal surface of the posterior part of the body is convex. Lateral keels are absent. The caudal sucker is medium sized and clearely exceeds half of the maximum body diameter. The head region is clearly tapered off to the mouth opening. The dorsal part of the cranial sucker is only slightly elongated. The dorsal and ventral sides are covered with numerous small papillae, especially on the ventral surface, arranged irregularly (Fig. 3).
Size: Large leeches. Preserved adult specimens up to ca. 110 mm in the length and 13 mm in width. This information based on moderately contracted specimens. Living specimens were stretched and reached ca. 200 mm. Annulation: Leeches with a typical Trocheta-like annulation. The midbody somites are subdivided by clear furrows into annuli c1, c2, b2, a2, b5 and b6 heteronomously into c11, d23, d24. Annuli b2, a2 and b5 broadened. But a2 and b5 are subdivided by shallow furrows into b3 and b4 (= a2) and c9 and c10 (= b5). The furrow on b5 is a little clearer. Annulus b2 dosen't show a furrow or is only very slighthly subdivided.
The male genital pore is situated in the furrow b2/b3, the female in the furrow c10/c11. The genital pores are separated by four narrow annuli (Fig. 4). Colouration: Preserved specimens yellowish gray with a bright median stripe on the dorsal side. The colour on both sides of this stripe is slightly darker grey (Fig. 5). Living leeches are unicoloured reddish brown without any dark patterns (Fig. 1)   Figure 5. Trocheta ariescornuta n. sp., colouration, dorsal sureface, clitellum-region.
Eyes: The eyes of the studied preserved adult specimens are invisible. Maybe it is an individual feature of the two examined specimens. But it is also conceivable, that the eyes are reduced with increasing age, similar to Dina minuoculata Grosser, Moritz & Pešić, 2007 and Trocheta danastrica (Grosser et al. 2007, Grosser & Epshtein 2009 or visible eyes are generally absent as in other cavernicolous species. Sexual organs: The characteristics of the male and female sexual organs, which are important for species characterization and differentiation, were studied (Fig. 6, 7). Figure 6. Several species of the genus Trocheta, genital atrium. A -B Trocheta ariescornuta n. sp. paratype, A -atrium ventral, B -atrium lateral; C -D Fadejewobdella quinqueannulata, C -drawing of the atrium, ventral, by Lukin 1976 (slightly changed), D -preserved specimen from Kharkiv (Ukraine); E -Trocheta danastrica; F -Trocheta taunensis; a -atrium body; c -cornua; g -ganglion.
The male sexual organs are characterized by a small atrial body with long cornua. The cornua are strongly wound up in their entire length, reminescent of horns of a ram. The cornua are not extending to the ganglion of the previous somite, due to their strong winding (Fig. 6). The paired vasa deferentia form praeatrial loops reaching in cranial direction to the ganglion of the previous somite in front of the male gonopore and are extending to the seventh ganglion after the female genital pore, the section of testisacs starts here. After the third ganglion, the vasa deferentia are thickened and coiled (Fig. 7).
The female sexual organs are characterized by the paired ovarian sacks, which are lying dorsally to the vas deferens in their entire length. They are reaching to the third somite (ca. on annulus b2) after the female genital pore. Than, the ovisacs turn cranially and they are several times winding to the ganglion of the previous somite (Fig. 7). Holotype: The holotype was studied in its external morphology. The measuring shows a body length of 108 mm and a width of 13 mm. The width of the caudal sucker is 7 mm. The annuli a2 and b5 are clearly subdivided by shallow furrows into b3, b4 and c9, c10. A tendency to split the annulus b2 into c3 and c4 by a very slight furrow is visible. The genital pores are separated by four annuli (b3, b4, c9, c10). The preserved holotype specimen is unicoloured yellowish grey, dorsally with a slightly brighter median stripe. In addition, some important morphological features of this specimen are shown in Figs. 3 A-D, 4 A-B, 5, 8.

Etymology:
Trocheta ariescornuta n. sp. is named after the cornua of the genital atrium. These cornua remind of the strong wound up horns of a handsome ram in their shape. In Latin aries = ram in English; in Latin cornuta = horned in English.).

Differential diagnosis:
The distribution of T. ariescornuta n. sp. is unknown. This species is only found on the locus typicus. Some other Erpobdellids are reported from Georgia (listed here according to the valid nomenclature): Erpobdella octoculata (Linnaeus, 1758), E. verrucosa (Örley, 1886), E. testacea (Savigny, 1822), Dina lineata (O. F. Müller, 1774), D. ratschaensis Kobaķhidze, 1958, D. stschegolewi Lukin &Epshtein, 1960, andTrocheta spec. (named as T. bykowskii) by Kvavadze (2002), further T. danastrica (by Nesemann 1991Nesemann , 1993a. Besides, some leech species, living in neighboring regions and could be found in Georgia in the future or were also recorded from similar habitats (e.g. Croatobranchus mestrovi Kerovec, Kučinić & Jalžić, 1999, Erpobdella borisi Cichocka & Bielecki, 2015 or Trocheta haskonis Grosser, 2000). A confusion of these species with T. ariescornuta n. sp. would be conceivable, and differentiating features are highlighted in the following section. The new species differs from the stygobiotic leeches Dina ratschaensis, Dina absoloni and Croatobranchus mestrovi by coloration: of these species who are paler/whitish, while the new species is clearly darker. The Georgian cave leech D. ratschaensis reaching a maximum body length of 50 mm and is clearly smaller than T. ariescornuta n. sp. (100 mm). Erpobdella borisi, described from a cave in north-west Iran, is also not whitish, but the dorsal surface shows dark pattern and the annulation is Dina-like (Cichocka et al. 2015). The somites of D. ratschaensis, D. absoloni and E. borisi are quinqueannulate with the last annulus (b6) broadened. The same annulation is present in non stygobiotic Dina stschegolewi and D. orientalis Grosser, Nesemann & Pešić, 2011, latter species known from Iran, Turkey, Lebanon, Israel and Syria (Grosser et al. 2011). Both of these leeches differ further from T. ariescornuta n. sp. by the dark coloured body with yellow spots. C. mestrovi is unmistakably through the gills on the lateral sides. The mid-body somites of this species are homonomousely quinqueannulate. T. ariescornuta n. sp. has a heteronomous annulation with more than five annuli per somite (c1, c2, b2, a2, b5 and b6 heteronomously subdivided into c11, d23, d24; annuli b2, a2 and b5 broadened and only sligthly subdivided); see the point "annulation" in the part "description" of this paper.
Trocheta vignai (Minelli, 1978) is also a stygobiotic leech, known from Turkey. The surface is smooth, any papillae are lacking. The genital pores are separated by two annuli. The shape of the genital atrium is unknown. In contrast, T. ariescornuta n. sp. differs from all other species of the genus in this characteristic. Only the atrium of T. ariescornuta n. sp. has strong wound up cornua. In the other Trochetaspecies the cornua are more or less elongated, sometimes slightly curved (Fig. 6 E-F). In some species the ends of the cornua (slightly) coiled. Furthermore T. ariescornuta n. sp. differs from Trocheta danastrica Stschegolew 1938, distributed in Southeast Europe with an unknown Eastern border of the distribution area, and Trocheta blanchardi, described from the Crimean peninsula, Ukraine, in the position of the genital pores. The male gonopore is situated on anulus a2 in the shallow furrow b3/b4 and the female in the furrow c10/c11 on the same somite in T. blanchardi. T. danastrica has the same position of the male gonopore, but the female is typically slightly shifted caudal on annulus c11. The gonopores of T. ariescornuta n. sp. are separated by four narrow annuli. Trocheta haskonis, described from Germany (Grosser 2000) and recorded from Southeast Europe (Grosser 2013, Grosser et al. 2018 and Iran (Darabi-Darestani et al. 2016), is well characterized by accessory pores left and right beside the male gonopore. Male and female gonopore are separated by two to three annuli.
T. ariescornuta n. sp. and Fadejewobdella quinqueannulata (Lukin, 1929) are similar in the shape of their genital atrium (Fig. 6 A-D). But Fadejewobdella has a smooth surface without papillae and the somites of the mid-body are homonomousely quinqueannulate. The genital pores of this leech are separated by three annuli.
T. ariescornuta n. sp. differs from all here listed species in the simultaneous existence of two characteristics: a Trocheta-like heteronomous annulation combined with the strong wound up cornua of the genital atrium.

Variability:
The species only known by two specimens. The holotype and the paratype are very similar in their external morphology and differs lightly in size, annulation and expression of the papillae. The holotype shows a higher tendency to subdivide annulus b2 into c3 and c4 by a very slight furrow. The annulus b2 of the paratype is not, or only in some somites subdivided by an almost imperceptible furrow. The shallow furrows of a2 and b5 are more clearly visible in the holotype. The furrow on b5 is most pronounced. The papillae are more prominent in the paratype (Fig. 8). A conspicuous variability between the type material is not visible. Habitat: The species is only known from a cave with an air temperature of 13°C. The collected specimens lived on wet stalagmites outside the water. It's unknown, if the leeches also inhabit the water body as a semiaquatic species.
Distribution: Georgia (Motena Cave). At the moment, Trocheta ariescornuta n. sp. is only known by the here described two specimens from the type locality. Therefore, the distribution of the species is unclear. Furthermore is it currently unknown whether T. ariescornuta n. sp. is a stygobiotic leech, like D. ratschaensis, D. absoloni and C. mestrovi or can be also found in surface waters, similar to T. haskonis. The last species was described from flood plain areas of the river Elbe in Saxony-Anhalt, Germany (Grosser