﻿Genital anatomy, jaw and radula of Guladentiasubtussulcata (Helicoidea, Cepolidae), endemic to western Cuba

﻿Abstract This study provides the first data on the genital anatomy, jaw and radula of Guladentiasubtussulcata (L. Pfeiffer, 1863). The auxiliary copulatory organ of this species is very peculiar, similar to that of Jeanneretia L. Pfeiffer, 1877, and different from that of other cepolids. It consists of an elongate, pedunculate mucus gland inserted apically on a muscular papilla and an atrial sac, all covered by a sheath. A sheath-like accessory gland is inserted at the base of the atrial sac. Another similarity with Jeanneretia is the presence of a fertilization pouch-spermatheca complex with a single exposed spermatheca. Like Jeanneretia, G.subtussulcata has an oxygnath, highly arched jaw with slight striae over the entire surface and a broad, well-developed median projection. The radula has triangular and monocuspid central and lateral teeth (the central teeth are smaller than the rest). The marginal teeth are multicuspid with the mesocone and ectocones smaller than the endocones. The similar structures of the auxiliary copulatory organ (without dart sac) and spermatheca (simple) strongly suggest that G.subtussulcata and Jeanneretia spp. are closely related. As such, it remains to be decided whether Guladentia Clench & Aguayo, 1951 and Jeanneretia should continue to be treated as separate genera.


Introduction
Guladentia Clench & Aguayo, 1951, is a Cuban endemic terrestrial snail genus of the family Cepolidae. It was originally described as subgenus of Jeanneretia L. Pfeiffer, 1877 (Clench and Aguayo 1951) with Helix subtussulcata L. Pfeiffer, 1863 as type species and comprising three other species, viz. Cepolis torrei Clench & Aguayo, 1933, Jeanneretia (Guladentia) modica Clench & Aguayo, 1951 and J. (G.) gundlachi Clench & Aguayo, 1951. While Guladentia was explicitly maintained as a subgenus by Ortea (1999, 2009) and implicitly by Fontenla et al. (2013), it was considered as a separate genus by Zilch (1960), Schileyko (2004) and Hernández et al. (2017), though without providing a rationale. As such, Guladentia differs conchologically from Jeanneretia by (1) possessing a shell with a well-defined "gular" fold, which is an invagination of the shell on the base of the body whorl, curved and parallel with the whorl about midway between the whorl periphery and the columella, and by (2) having a long and curved tooth on the base of the shell within the aperture. Thus, according to Clench and Aguayo (1951) the shell structure of Guladentia would resemble that of the genus Cepolis Montfort, 1810 ( Fig. 1).
Species of Guladentia occur in mountainous limestone areas in the central-western region of the Sierra de los Órganos, Cuba. They are quite rare (Clench and Aguayo 1951), yet, G. subtussulcata is the species with the widest distribution and that is the least difficult to collect. It is also the largest species in the genus (shell height: 16-31 mm; shell diameter: 21-31 mm). Hernández et al. (2020) emphasized that the genital anatomy of a majority of Cepolidae taxa, including the genus Guladentia, is still unknown. The present contribution, therefore, aims to provide the very first data on the genital anatomy, jaw, and radula of the type species of this genus, viz. G. subtussulcata, and on the basis of these new data it also briefly reflects on the (sub)generic status of Guladentia.

Materials and methods
Nine live specimens of G. subtussulcata were collected from three localities in Viñales, Pinar del Río Province, Cuba. The material examined is deposited in the Malacological Collection of the Institute of Ecology and Systematics (CZACC; Colección Zoológica de la Academia de Ciencias de Cuba). Specimens were drowned for 12 h in an airtight jar filled with water, after which they were removed from their shells and fixed in 70% ethanol. Specimens were dissected using a Carl Zeiss Stemi 2000 stereomicroscope. The reproductive tracts were photographed with a Nikon 5100 camera. Radula and jaw were extracted manually, cleaned by soaking in 10% KOH solution for about 6 h followed by rinsing in ethanol. They were mounted for scanning electron microscopy with a Thermo Fisher Quanta 200 scanning electron microscope.
The terminology of the reproductive apparatus follows Baur (2010) and Hernández et al. (2020). The length of seven genital structures was measured using scale paper (error 1 mm): flagellum, penis + distal epiphallus, proximal epiphallus, bursa-copulatrix duct, auxiliary copulatory organ, and length of the spermoviduct.

Jaw and radular morphology
The jaw is oxygnath (Fig. 3A), solid, high arched, almost smooth except for slight striae all over the surface, and with a wide, well-developed, median projection. The radula has a monocuspid, pointed and triangular central tooth, which is smaller than the other teeth and shorter than the base of the tooth (Fig. 3B). Lateral teeth monocuspid, pointed, triangular and as long as their base (Fig. 3B). Between the lateral and marginal teeth, there are transitional teeth with ectocones. Marginal teeth multicuspid with the mesocone and ectocones smaller than the endocones (Fig. 3C).

Discussion
The reproductive system of Guladentia subtussulcata is similar to that of other cepolid species by, amongst other features, the presence of an auxiliary copulatory organ. Yet, while in other cepolids this auxiliary copulatory organ contains a dart sac (on top of the muscular papilla), there is no dart sac in G. subtussulcata (Fig. 2B) and Jeanneretia ssp. (Hernández et al. 2021). Instead, these latter two taxa have a non-calcareous 'dart' (Fig. 2C), which is fused to a muscular papilla that opens directly into the atrial sac. More generally, the auxiliary copulatory organ of G. subtussulcata is similar in size and structure to that of Jeanneretia spp. as described by Hernández et al. (2021). In this sense it represents a unique feature within Cepolidae. Also, the simple spermatheca of G. subtussulcata is a unique feature within Cepolidae that links this species with Jeanneretia spp.
Conversely, G. subtussulcata and Jeanneretia ssp. differ markedly by their accessory glands: G. subtussulcata has a single sheath-like accessory gland (Fig. 2B), a unique feature within Cepolidae, whereas Jeanneretia ssp. have a pair of tubular accessory glands. Furthermore, compared to the data provided for Jeanneretia spp. by Hernández et al. (2021), G. subtussulcata has a much longer spermoviduct, but a much shorter flagellum, than Jeanneretia spp.
The jaw and radula of G. subtussulcata are extremely arched (Fig. 3A) like in Jeanneretia ssp. (Hernández et al. 2021), though the radular teeth of G. subtussulcata are a little more pointed than in Jeanneretia spp. In general, the radular morphology of G. subtussulcata complies with that of other cepolid genera, except Polymita Beck, 1937 A jaw B central and lateral teeth, central teeth marked with a black arrow C transitional and marginal teeth, the latter with their ectocones (e), mesocones (m), and endocones (ed). (Moreno 1950), in which the radula is arranged in a "v" shape with all teeth equal in size (Hernández et al. 2021).
The structure of the auxiliary copulatory organ (without dart sac) and the spermatheca (simple) strongly suggests that G. subtussulcata and Jeanneretia spp. are closely related. Hence, the question arises whether Guladentia should be regarded as a separate genus or as a subgenus of Jeanneretia. The conchological differences between G. subtussulcata and Jeanneretia spp. may indicate a separation at the genus level, with the gular shell fold (Fig. 1A) and the unique accessory gland structure (Fig. 2B) of Guladentia as the defining features of the genus. However, the genital anatomy of more Guladentia species should be studied and combined with DNA sequence data to infer the phylogenetic relationships of Guladentia and Jeanneretia. These additional data are not only necessary to verify whether these two taxa are monophyletic, but also to explore whether the conchological similarities between Guladentia and Cepolis suggested by Clench and Aguayo (1951) are synapomorphic or homoplasic character states.