Subgeneric division of the genus Orcula Held 1837 with remarks on Romanian orculid data (Gastropoda, Pulmonata, Orculidae)

Abstract The genital anatomy of Orcula jetschini (Romania), Orcula zilchi (Bulgaria), and Orcula wagneri (Albania) is described. Based on anatomical features (morphology of the penial caecum) shell characters (sculpture and shape) and unpublished molecular data the genus Orcula is subdivided into three subgenera. Orcula zilchi was classified within the monotypic subgenus Orcula (Hausdorfia) subgen. n.; Orcula jetschini, Orcula wagneri, and Orcula schmidtii were classified to Orcula (Illyriobanatica) subgen. n. (type species: Pupa schmidtii) whereas the other Orcula species remain in the nominotypical subgenus. Orcula (Hausdorfia) is known from South-Eastern Bulgaria and North-Western Turkey, Orcula (Illyriobanatica) inhabits Western Romania, North-Western Greece, Albania, Macedonia, Kosovo, and Montenegro. The nine species of Orcula (Orcula) are known mainly from the Alps and the Western Carpathians (from Eastern France to Eastern Hungary and Slovakia). The occurrence of only one Orcula species namely Orcula jetschini is verified from Romania. Available information suggests that data on the Romanian occurrence of Orcula dolium and Orcula gularis were based on wrongly identified specimens. Sphyradium dobrogicum (=Orcula dobrogica) is considered as a synonym of Sphyradium doliolum.

within Orcula based on shell and genital characters. These divisions are further supported by unpublished molecular data (Harl et al. in prep.).
Furthermore, we discuss the orculid species reported from Romania. Two species, namely Pupa (Orcula) jetschini Kimakowicz 1883 and Sphyradium dobrogicum Grossu 1986 were originally described from Romania. Two other species (O. dolium and O. gularis) were also reported from Romania by Bielz (1863) and later by other authors. Distributions of the last two species (see Welter-Schultes 2012), unreliable data sources and inaccessible or lost voucher material make Romanian occurrence data questionable.
Photographs of several focal planes were made with a Wild Makroskop M420 and a Nikon DS Camera Control Unit DS-L2. The different layers were combined with Helicon Focus 4.75 Pro to obtain one completely focused image.
Shells were directly observed without coating under a low vacuum SEM (Miniscope TM-1000, Hitachi High-Technologies, Tokyo). Teleoconch sculpture was noted on the dorsal or dorsolateral area of the penultimate whorl.
Type species. Pupa dolium Draparnaud 1801, by subsequent designation Gray: 1847: 176. Diagnosis. Shell yellowish-greenish to dark brown; cylindrical to conical and elongated; 8-10 weakly convex whorls; sculpture of first 0.5-1.0 protoconch whorl usually smooth, but may be of fine spiral lines, which may be extremely weak; teleoconch axial sculpture variable, ranging from irregular growth lines to equally spaced, conspicuous radial structure; apertural barriers: one parietal and 1-3 columellar lamellae; palatal side of the aperture smooth or with strong tooth or thickening parallel to the apertural lip; parietal callus weak, subangularis sometimes present; palatalis plicae missing.
Penis cylindrical, penial caecum of variable length and shape; penial appendix absent; interior of penis, epiphallus and caecum with longitudinal folds; retractor muscle attaches to the penis-epiphallus junction on the opposite side of the penial caecum; diverticulum absent; distal part of vas deferens sometimes slightly swollen, entering epiphallus terminally; bursa copulatrix long, club-like.
Habitat. Orcula species occur in humid limestone areas, usually forests, or rocky boulder fields at high altitudes. Animals live under stones, leaf litter or decaying wood, or at the base of large rocks.
In general, species of Schileykula and Orculella usually inhabit dry limestone areas in the Mediterranean. The only exceptions known are the closely related Orculella bulgarica (Hesse) and Orculella aragonica (Westerlund) which both prefer very humid, marshy stream banks (Garrido et al. 2005, Arrébola et al. 2012.

Content
The penial caecum of Orcula (Orcula) restituta is very short compared to other Orcula (Orcula) species, but the shell is similar to that of Orcula (Orcula) gularis. Prior to Klemm (1967), restituta was considered a subspecies of gularis.
A third columellar lamella is rarely present, but can occur in a small percentage of individuals within a population. Brancsik (1888: 84) noted a third columellar fold in only one individual of thousands in each O. dolium titan (Brancsik 1888) and O. dolium dolium.
Distribution. Most species have limited distributions in the Alps (mainly Austria). O. dolium is widely distributed in Central Europe, in the Alps (eastern France, Switzerland, Southern Germany, Northern Italy, Austria, Slovenia, Northern Croatia, and Slovenia) and the Western Carpathians (Northern Hungary, Slovakia, Eastern Czech Republic). The Croatian records of O. dolium and O. gularis (Stossich 1880, 1899, Zimmermann, 1932 have not been verified by recent investigations. Our knowledge of the distribution of O. dolium is distorted due to misidentified material. Probably all reports of this species (living and fossil) from Spain (e.g. Llamas et al. 1995) refer to Orculella aragonica (see Arrébola et al. 2012). Italian (Toscana) records (Zanchetta et al. 2004(Zanchetta et al. , 2006 refer to a yet unknown Orculella species (see photo in Zanchetta et al. 2006). Damjanov and Likharev (1975) reported O. dolium from the Balkan Peninsula, South, Central and West Europe, the Crimea, Western Ukraine, Central Asia, Tunisia, Ethiopia and northen Iran. This distribution is much broader than that of O. dolium and probably refers to the distribution of the family Orculidae. Likharev and Rammelmejer (1952) and Sysoev and Schileyko (2009) speculated that O. dolium occurs in Ukraine. This supposition has been included in distribution maps (Welter-Schultes, 2012), but to date the taxon's occurrence in Ukraine has not been verified data (Balashov and Gural-Sverlova 2012). Soós (1943) mentioned that during careful collections around Munkács (Mukachevo, southwest Ukraine), Traxler was not able to find the species.
Sacco (1897) described Orcula dolium var. pliopedemontana from the middle Pliocene sediments at Ceresole d'Alba (Italy: ''Villafranchiano''). The description is unfortunately insufficient and the taxonomic position of this form is uncertain (Ferrero-Mortara et al. 1984, Pilsbry 1922). More recently, Ciangherotti et al (2007) made no mention of the species from the same sediment layers. Diagnosis. Shell usually with strong axial sculpture (irregular ribs), with two columellar lamellae, apex rather rounded, not attenuate. The penial caecum usually consists of two parts (''tubercles'') and its length is less than half that of the penis.

Orcula
Etymology. The name of this new subgenus refers to its distribution in the Illyrian and Banatic biogeographical regions. It is feminine.
Remarks. The reproductive anatomy of O. schmidtii transversalis (Westerlund 1894) was described by Hausdorf (1987) and Reischütz and Sattmann (1990). According to Hausdorf (1987), the penial caecum of O. schmidtii transversalis is short, but simple. The caecum appears double in the illustration provided by Reischütz and Sattmann (1990    Description of the genitalia. Two specimens were anatomically examined. Penis slim, with the retractor muscle attached at its distal end; penial caecum very small, vestigial, consisting of two "tubercles"; epiphallus very long and cylindrical; there is clear distinction between the vas deferens and the epiphallus; vas deferens long and relatively thick; a slender retractor muscle is attached near the proximal end. Vagina short and thick, but pedunculus relatively long; bursa copulatrix extremely long, with the distal end slightly expanded. In one specimen an elongated, simple spermatophore was found with the apical portion slightly thickened. Distribution. Orcula (I.) jetschini is known only from western part of Romania (Banat, Crişana and Western Transylvania). The Hungarian record (Pintér and Suara 2004) is apparently based on a flotsam specimen so its origin is suspect (Varga, 2009). Negrea (1966) reported the species from Moldova Nouă, which lies close the Serbian border and, therefore, it is expected to occur in Serbia. The Pleistocene distribution of Orcula dolium included the ''Požarevac Danube Area'' (Mitrović 2007), which is just on the other bank of the Danube River, but temporal and spatial sympatry of O. (I.) jetschini and O. (O.) dolium is not verified.
Ecology. The species inhabits deciduous forests. It is found most commonly between small stones and leaf litter on the forest floor or under hazelnut (Corylus) bushes. The species is known from non-limestone bedrock, such as the Zarand Mountains.
Conservation status. Least concern (LC) according to IUCN criteria (Fehér 2011). Remarks. All living specimens found were covered in mud, causing them to appear like tiny grains of soil. The ribbed shell is possibly an adaptation for camouflaging. The photographs herein are of cleaned shells. Description of the genitalia. One specimen was dissected. Penis cylindrical and slim, with a short, but thick penial caecum, the proximal portion broader than the short and slimmer distal portion; retractor muscle attaches at the penis-epiphallus transition; epiphallus more than twice as long as the penis and much thicker, its transition to the vas deferens is gradual, barely discernable; there is a slim retractor muscle attached to the proximal portion of the epiphallus; proxim\al portion of the vas deferens thicker than the distal part. Vagina and free pedunculus extremely short; bursa copulatrix almost twice as long as the combined length of the penisepiphallus complex.

Type species. Orcula zilchi Urbański 1960 (by monotypy).
Diagnosis. Shell with conical apex and strong axial sculpture (irregular axial growth lines), with three columellar lamellae (columellar, supracolumellar and one short lamellae above), palatalis reaches its maximum height on the dorsolateral side. Penial caecum very long with thickened base, canal connecting the proximal end of the epiphallus to the penial caecum.
Etymology. The new subgenus is named in honour of Dr Bernhard Hausdorf (University of Hamburg), who first noted the unusual shell characters of Orcula zilchi and questioned its generic status (Hausdorf 1996). It is feminine.
Distribution  Remarks. According to Schileyko (2012), the penial caecum of Orcula fuchsi is long and slender, with a thickened base, making it similar in morphology to O. zilchi. However, the characteristic canal connecting the proximal end of the epiphallus with the penial caecum of O. zilchi is lacking in O. fuchsi. The long caecum of this species is also illustrated by Gittenberger (1978), but its base is not conspicuously thickened. This may vary between populations or during an individual's life history. Description of the genitalia. Two specimens were dissected. Penis cylindrical, relatively long; retractor muscle short, attaches on the proximal portion; penial caecum very long, with a thickened base and a cylindrical distal portion; an additional canal (?) connects the proximal end of the epiphallus with the penial caecum; epiphallus long, with a thickened distal part; the separation between the vas deferens and epiphallus is distinct; vas deferens relatively thick. Vagina cylindrical and relatively short; bursa copulatrix extremely long with a pointed end.

Orcula
A developing egg covered with small calcareous crystals was found in the uterus of the figured specimen. In the other specimen, an elongated, simple bursa copulatrix was found with a slightly thickened apical part.
We were able to find O. zilchi only in deciduous forests. In Bulgaria (near Kondolovo village), living specimens were collected in an oriental beech (Fagus orientalis) forest in shady and moist microhabitats between the leaf litter and soil. These conditions were very similar to the Abant Gölü locality (Turkey). The other Turkish locality (between Bozüyük and İnegöl) was slightly different, with a deciduous forest at the base of a large limestone rock, on a slope covered with smaller stones and larger rocks.
The species is very rare wherever it has been encountered yet, especially in Turkey.  Remarks. Two of four living specimens had beetle (possibly drilid beetle) larvae in the body whorl.
The dissected specimens were collected about 23 km south-southwest of the type locality. The Strandzha Mountains (incl. the collecting site) belongs to the drainage of the Ropotamo River. It is reasonable to suppose that Urbański's population was "washed down" from somewhere in the Strandzha Mts. and settled a temporary subpopulation in the Ropotamo floodplain. This might be a reasonable explanation why A. Irikov could not find this species in the type locality.  Remarks. Sphyradium dobrogicum was described based on a single shell. The holotype could not be located in the collection of the Grigore Antipa National Museum of Natural History (Bucharest) during a recent search (2012). It could still be in Grossu's house (Oana Popa, pers. comm., 2011) but, at present, the holotype seems to be lost. Bank (2011) andWelter-Schultes (2012) assigned the species to Orcula without supporting evidence. S. dobrogicum has a domed apex, a ribbed shell and very weak lamellae (see original description and drawing), indicating that it may represent a dwarf specimen of S. doliolum. We visited the type locality of S. dobrogicum in 2011 but found only Sphyradium doliolum.

Genus
Based on available information we suggest using Sphyradium dobrogicum as a synonym of S. doliolum.

Orcula gularis and O. dolium in Romania
Orcula gularis: Bielz (1863Bielz ( , 1867 reported Pupa (Pupilla) gularis from Gușterița, which is presently part of Sibiu. Grossu (1987Grossu ( , 1993 cited this record in his account of Sphyradium gularis. Grossu (1986) discussed Bielz's specimens housed in the museum in Sibiu, but specimens were not located in the collection of Bielz in the NHMS. Although Bank (2011) reports the species from Romania, recent authors consider this record as erroneous (Falkner et al 2011) or simply ignore it (Welter-Schultes 2012). Indeed, the occurrence of O. gularis in Romania, more than 650 km from its main distribution area is very unlikely. However, to our knowledge, no one has searched for the species at the respective Romanian locality which was well-defined by Bielz. Nevertheless, despite intensive collecting efforts by competent malacologists over the last 150 years, O. gularis has not been encountered in Transylvania.

Discussion
In this paper we describe the genitalia of the Eastern European Orcula jetschini and O. zilchi for the first time. We also examine and describe the anatomy of O. wagneri from a locality that lies 200 km north of populations examined by Schileyko (2012). This additional information and data published by other authors (mainly Gittenberger 1983 and Schileyko 2012) allows us to review the taxonomic relationships of the entire genus.
The genus can be subdivided into three subgenera (Orcula, Illyriobanatica subgen. n. and Hausdorfia subgen. n.) based on the shell characters and the morphology and size of the penial caecum, which serves as the primary diagnostic character. Unpublished results of molecular phylogenetic analysis (Harl et al. in prep.) of most Orcula species and subspecies indicate the monophyly of these three groups. This subdivision is in good agreement with biogeographic information.
Three species included herein have some shell and anatomical characters which differ from characters used to the features mentioned in the diagnoses of certain subgenera: (1) the shell sculpture of many populations of Orcula (Illyriobanatica) wagneri is almost smooth, which is unusual in the subgenus. (2) The penial caecum of Orcula (Orcula) restituta is very short compared to other species assigned to the subgenus. (3) The penial caecum of O. (Illyriobanatica) schmidtii transversalis is short, but simple (Hausdorf 1987), not ''tuberculated'' as the other forms of the subgenus. The caecum of O. schmidtii transversalis seems to be double in the illustration of Reischütz and Sattmann (1990). Based