Zoosyst. Evol. 97 (1) 2021, 111-119 | DOI 10.3897/zse.97.60254 gee BERLIN A new hydrobiid species (Caenogastropoda, Truncatelloidea) from insular Greece Canella Radea!, Paraskevi Niki Lampri!*, Konstantinos Bakolitsas*, Aristeidis Parmakelis! 1 Section of Ecology and Systematics, Department of Biology, National and Kapodistrian University of Athens, 15784 Panepistimiopolis, Greece 2 High School, Agrinion, 3 Parodos Kolokotroni 11, 30133 Agrinion, Greece 3 Institute of Marine Biological Resources and Inland Waters, Hellenic Centre for Marine Research, 46.7 km of Athens — Sounio ave., 19013 Anavissos Attica, Greece http://zoobank.org/FE7CB458-945 9-409C-B254-DA0A1BA65B86 Corresponding author: Canella Radea (kradea@biol.uoa.gr) Academic editor: T. von Rintelen # Received 1 November 2020 Accepted 18 January 2021 # Published 5 February 2021 Abstract Daphniola dione sp. nov., a valvatiform hydrobiid gastropod from Western Greece, is described based on conchological, anatomical and molecular data. D. dione is distinguished from the other species of the Greek endemic genus Daphniola by a unique combination of shell and soft body character states and by a 7-13% COI sequence divergence when compared to congeneric species. The only population of D. dione inhabits a cave spring on Lefkada Island, Ionian Sea. Key Words Freshwater diversity, Lefkada Island, taxonomy, valvatiform Hydrobiidae Introduction The Mediterranean Basin numbers among the first 25 Global Biodiversity Hotspots due to its biological and ecological biodiversity and the plethora of threatened bi- ota (Myers et al. 2000). The region from the Iberian Pen- insula to the Balkans is characterized by the high diversi- ty and endemicity of freshwater flora and fauna (Cuttelod et al. 2008; De Figueroa et al. 2013; Smith et al. 2014) and the biota of Greek freshwater ecosystems are widely recognized as a major component of this diversity (Gloer and Maassen 2009; Gloer et al. 2010). Among freshwater molluscs, the family Hydrobiidae (hydrobiids) is one of the largest and the most diverse gastropod family throughout the Mediterranean region (Cuttelod et al. 2008). The valvatiform hydrobiids is a group of minute gastropods with depressed trochiform shells resembling those of the genus Valvata O. F. Miller, 1773 (Heterobranchia, Valvatoidea). The species of this group are highly endemic and phylogenetically not relat- ed (Radea 2018). In Europe, 37 valvatiform genera have been described so far. More than 60% of these genera inhabit the freshwater systems of the Balkan Peninsula (Radea 2018; Boeters et al. 2019; Delicado et al. 2019). The Mediterranean Basin, the Balkan, the Iberian and the Italian Peninsulas seem to be evolutionary centers of valvatiform hydrobiids throughout, especially of the sty- gophile and stygobiont taxa (e.g. Radoman 1983; Bodon et al. 2001; Arconada and Ramos 2001, 2002, 2006, 2007; Arconada et al. 2007; Callot-Girardi and Boeters 2012: Radea et al. 2016a; Quifionero-Salgado and Rolan 2017; Boeters et al. 2019). To date, nine valvatiform hydrobi- id genera live in lotic and lentic habitats of Greece, and five of them, namely Daphniola Radoman, 1973, Grae- coarganiella Falniowski & Szarowska, 2011, /simerope Radea & Parmakelis, 2013, Myrtoessa Radea, 2016 and Pseudoislamia Radoman, 1979, are endemic to Greece. Daphniola (type species Daphniola graeca Radoman, 1973), which is the only non-narrow range Greek endemic valvatiform genus, was the first extant endemic valvati- form hydrobiid described from Greece. Daphniola is dis- tributed in the eastern part of the central and southern main- Copyright Canella Radea et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which per- mits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. ra Canella Radea et al.: Hydrobiid from Lefkada Island 1:4,000,000 Figure 1. The distribution of the Greek endemic genus Daphniola. A. D. exigua and B. D. magdalenae, Larissa prefecture; C. D. di- one, Lefkada Island; D. D. hadei, Lakonia prefecture; E. Daphniola sp., Rhodos and Chios Islands; F. D. eptalophos Parnassos Mt.; G. D. louisi Attiki prefecture; (?) the record of D. exigua (as Horatia (Daphniola) exigua pangaea) in Kavala prefecture (Reischitz 1984) should be re-examined because it is based on empty shells identification. land and comprises five species: D. exigua (A. Schmidt, 1856) (as Horatia (Daphniola) exigua in Schitt 1980), D. louisi Falniowski & Szarowska, 2000, D. eptalophos Radea, 2011, D. hadei (Gittenberger, 1982) and D. mag- dalenae Falniowski, 2015. D. graeca Radoman, 1973 is accepted as synonym of D. exigua (MolluscaBase 2020). Another sequenced undescribed Daphniola species has been recorded in Chios and Rhodos Islands, Aegean Sea (Szarowska et al. 2014). Two Daphniola species, D. exi- gua and D. louisi, have been evaluated as Endangered and Critically Endangered in the IUCN Red List of Threat- ened Species, respectively IUCN 2020.2). Herein, we describe and analyze phylogenetically a new Daphniola species from Lefkada Island, Ionian Sea and delineate morphological and anatomical characters, which are evident and efficient to support the existence of the new species. Materials and methods Live specimens and empty shells of the new taxon were found on Lefkada Island, Ionian Sea (Fig. 1). All the ma- terial was collected by hand from a spring spouting in- zse.pensoft.net side a cave. Two specimens were stored at -20 °C and preserved in 100% ethanol to be used in subsequent mo- lecular analyses, whereas the remaining specimens were preserved unrelaxed in 70% ethanol for further morpho- logical and anatomical analysis. DNA extraction, amplification and sequencing Due to the small body size, the entire animals were used for genomic DNA isolation. DNA was extracted using the CTAB protocol as described in Parmakelis et al. (2003). Amplification of a fragment of approximate- ly 700 bp long of the mitochondrial cytochrome C oxi- dase subunit I (COI), was carried out using the universal primers LCO1490 5'-GGTCAACAAATCATAAAGA- TATTGG-3' and HCO2198 5'-TAAACTTCAGGGT- GACCAAAAAAT-3' (Folmer et al. 1994). Each PCR was performed in a 25 uL volume, where 2 uL of template DNA were mixed with 0.2 mM dNTPs, 3.5 mM MgCl2, 0.4 uM of each primer, and 0.5 unit of Taq Polymerase (Kappa). Thermocycling was performed in a BioRad My- Cycler Thermal Cycler. The PCR program comprised an initial denaturation at 95 °C for 3 min, followed by 42 Zoosyst. Evol. 97 (1) 2021, 111-119 cycles of 15 sec at 94 °C, 1 min at 42 °C, and 1.5 min at 72 °C. The cycling was ended with 10 min sequence ex- tension at 72 °C. PCR amplicons were purified using the NucleoSpin Gel and PCR Clean-up kit (Macherey-Nagel GmbH and Co KG Neumann-Neander, Germany). Both strands of the PCR product were sequenced. The primers in the sequencing reactions were the same as in the ampli- fication procedure. Out of the two specimens sequenced, only one generated a high-quality COI sequence. Sequence alignment and genetic data analysis Both strands of the sequenced fragment were combined in a single contig and edited with CodonCode Aligner v. 2.06. The generated sequence has been deposited in GenBank. To reconstruct the phylogenetic relationships of our specimen, to other published Daphniola species, Daphniola COI sequence data were obtained from Gen- Bank. Daphniola eptalophos, which was recorded and described for the first time in 2011 from Mt. Parnassos, was not included in the phylogenetic analysis due to the lack of COI sequence data available for the species (Ra- dea 2011, 2018). Phylogenetic relationships were inferred using Bayesian Inference (BI) framework. In order to se- lect a proper outgroup taxon several preliminary analyses involving other hydrobiid genera, were performed. Cor- bellaria celtiberica Girardi & Boeters, 2012 was a valvat- iform species leading to a well resolved phylogenetic tree, and thus was ultimately used as the outgroup species of our analyses. BI analysis was performed using MrBayes v.3.2.2 (Ronquist et al. 2012) following the selection of 113 the best-fit model of molecular evolution using Partition Finder v2.1.1 (Lanfear et al. 2012). The nucleotide substitution model suggested by Parti- tion Finder was the HK Y +G. No codon partition was im- plemented. For the BI analysis, the number of generations was set to 2 x 10°. The first 25% of trees were discarded as burn-in and the analysis was summarized on a 50% majority-rule tree. Support for the nodes was assessed by posterior probabilities. Following the completion of the phylogenetic analysis, a rogue taxa analysis was per- formed using the RogueNaRok webserver (Aberer et al. 2013). Estimates of evolutionary divergence between Species were estimated using both the p-distance and the HKY substitution model (Hasegawa et al. 1985) as im- plemented in SeaView v.4 (Gouy et al. 2010). The genetic distance estimates are presented 1n Table 2. Shell morphology and soft body anatomy Shell characters (shell height and width, aperture height and width) were taken from 10 specimens using the mi- crometer of a stereomicroscope Stemi 2000-C, Zeiss, Germany. Four ratios were generated from the raw data; these were SH/SW, AH/AW, SH/AH and SW/AW (see abbreviations at the end of this section). Before dissec- tion, the shells were removed by soaking in Perenyi solution (Clayden 1971). Shell and soft body features were photographed using a Canon EOS 1000D camera attached to the stereomicroscope. The new taxon was compared with specimens of D. exigua, D. eptalophos, D. graeca and D. louisi which have been collected from Table 1. Species, families, locality details, GenBank accession numbers and publication references for COI sequences used in the phylogenetic analysis of this study. The hydrobiid species Corbellaria celtiberica was used as outgroup. Species Family Country Region Sampling locality GenBank Reference accession number Daphniola exigua Thessaly Large spring at Agia Paraskevi, JF916467 Falniowski and Hydrobiidae | Greece Daphniola graeca Daphniola hadei Peloponnese Hydrobiidae | Greece Daphniola louisi Hydrobiidae | Greece Attiki Tembi Valley, N of Larisa ii Spring at Dhiaselo, W of Sparta, N Taigetos Mts. Spring at Agia Paraskevi in Szarowska 2011 Falniowski et al. 2007 Falniowski and Szarowska 2011 Falniowski et al. 2007 JF916479 EU047766 Tembi Valley Daphniola magdalenae Hydrobiidae | Greece Thessaly Melissotripa Cave KT825580 Falniowski and Sarbu 2015 Daphniola dione lonian Islands Daphniola sp. Hydrobiidae | Greece Lefkada island MW581160 Present study Szarowska et al.2014 Corbellaria celtiberica Hydrobiidae | Greece | Dodecanese Rhodes island KM887915 Islands Hydrobiidae | Spain Manubles River, Soria MH350207 Delicado et al. 2019 Table 2. Pairwise genetic distances between Daphniola species and undescribed specimens estimated using both the p-distance and the HKY substitution model (p-distance/HKY). Species 1 2 3 4 5 6 1. D. exigua JF916467 2. D. graeca EU047763 0.02/0.02 3. D. louisi EU047766 0.01/0.01 0.02/0.01 4. D. dione sp.n. MW581160 0.07/0.07 0.08/0.09 0.07/0.08 5. Daphniola sp. KM887915 0.09/0.09 O.T0/0.L1 0.10/0.10 0.09/0.08 6. D. magdalenae KT825580 0.14/0.15 0.14/0.16 0.14/0.14 O.1370:15 O12 7/005 7. Corbellaria celtiberica MH350207 0.18/0.21 0.18/0.21 0.18/0.20 0.17/0.20 Osl7/0.21 0.21/0.25 zse.pensoft.net 114 0.04 D. sp. KM887915 Canella Radea et al.: Hydrobiid from Lefkada Island D. exigua JF916467 0.94 D. graeca EU047763 D. louisi EU047766 D. dione sp. n. D. magdalenae KT825580 Corbellaria celtiberica MH350207 Figure 2. 50% majority-rule consensus tree of the BI analysis. Number on nodes denote the posterior probability values. Scale bar indicates number of substitutions per site. The valvatiform species Corbellaria celtiberica was used as outgroup. their respective loci typici. These specimens are included in the personal collection of C. Radea deposited in the Section of Ecology and Systematics, Department of Bi- ology, National and Kapodistrian University of Athens. Morphological terminology follows that of Hershler and Ponder (1998) and thus the character states may be differ- ent from those given by the authors which have described Daphniola species. Abbreviations used in the text, Tables and Figures are: Ag = albumen gland, AH = aperture height, AW = aperture width, Bc = bursa copulatrix, Bd = bursal duct, Cg = capsule gland, CV* (1+1/4n)*SD/Y = coefficient of variation corrected for sample size (Sokal and Rohlf 1995), GNHM = Goulandris Natural History Museum, Max = maximum, Min = minimum, n= number of specimens, NW = Number of whorls, NKUA = National & Kapodistrian University of Athens, Ov = Coiled oviduct, Pd = penial duct, Pl = penial lobe, rs] = distal seminal receptacle, rs2 = proximal seminal receptacle, SD = standard deviation, SH = shell height, SW = shell width, X = mean. Results Phylogenetic tree and evolutionary divergence The phylogenetic relationships of Daphniola species de- scribed so far are reflected in the tree shown in Fig. 2. In a preliminary phylogenetic analysis, the species Daphniola hadei was identified as a rogue taxon. Thus, it was removed from the analysis since it was signifi- cantly distorting the phylogenetic information tncluded in the dataset. To date, D. hadei is considered a valid zse.pensoft.net Daphniola species, and for reasons of comparison with the new species described herein, 1t was maintained in the morphological assessment of the species. The tree ob- tained after excluding D. hadei from the analysis is quite well resolved with the majority of nodal support being above 0.90. D. exigua and D. graeca appear to be very closely related, whereas D. /ouisi is firmly associated with the former group of species. D. dione sp.n. seems to be a separate lineage that is well separated from the exigua-graeca-louisi group as well as from D. magdale- nae. The latter is directly, albeit distantly, related to all the former. The most phylogenetically distant species of all Daphniola seems to be Daphniola sp. from Rhodes. The relationships reflected in the phylogenetic tree, are corroborated by the pairwise genetic distances of the se- quences (Table 2) as well. D. dione is separated from all the other Daphniola species by distances ranging from 7% (D. exigua) to 13% (D. magdalenae). Systematic description Family Hydrobiidae Stimpson, 1865 Daphniola dione sp. nov. http://zoobank.org/4F 1 D8C21-1EEF-4248-8299-012692C20D79 Figs 3, 4 Etymology. The specific name (in apposition) derives from Greek mythology: Dione, (A1i@vn in Greek), was the mother of the goddess Aphrodite according to the Greek poet Homer, author of Iliad and Odyssey. Zoosyst. Evol. 97 (1) 2021, 111-119 115 Figure 3. Shells of Daphniola dione sp. nov. A., E. Dorsal view, B., F., K. Ventral view; C., D., G., H., L. Lateral view; I., J. Shape of rectum in females and males respectively. Scale bar 1 mm. Diagnosis. Shell minute (maximum height 0.98 mm, maximum width 1.31 mm), valvatiform; soft body without any pigmentation; eyes small; penis long, nar- row, tapered, with wider wrinkled proximal portion, filamentous distal portion and an obtuse outgrowth on the left side forming an acute angle with the penis dis- tal portion; female genitalia with large pyriform bursa copulatrix, renal oviduct coiled in a equilateral triangle. Differentiated from D. exigua by its smaller size, wid- er and more open umbilicus, paler operculum, lack of pigmentation in soft body, pyriform-shaped bursa cop- ulatrix, wider bursal duct, triangle-shaped oviduct and obtuse rather than pointed outgrowth at the distal pe- nis portion. Differentiated from D. /ouisi by its smaller size, lack of pigmentation in soft body, large, pyriform and protruding bursa copulatrix, triangle-shaped ovi- duct, nearly centered penial duct, and more prominent penial outgrowth. Differentiated from D. eptalophos by its smaller size, paler operculum, lack of pigmentation in soft body, protruding bursa copulatrix, wider bursal duct, triangle-shaped oviduct, more pointed penial apex, outgrowth on distal rather than proximal penial portion and nearly centered penial duct. Differentiated from D. hadei by its larger size, wider and more open umbilicus, pyriform and protruding bursa copulatrix, more point- ed penial apex and more prominent penial outgrowth. Differentiated from D. magdalenae by its smaller size, wider and more open umbilicus, paler operculum, pro- truding bursa copulatrix, more pointed penial apex and presence of eyes. Type-locality. Cave spring on Lefkada Island, Ionian Sea, Greece, 38.722532°N, 20.682713°E, 240 m a.s.l., 15.V.2017, K. Bakolitsas leg. Type material. Holotype. Ethanol-fixed specimen, GNHM 39587. Paratypes (from the same lot). Two etha- nol-fixed specimens, GNHM 39588. Seven ethanol-fixed mature specimens dissected for anatomical study. Three mature and seven immature specimens are in the personal collection of C. Radea deposited in the Section of Ecolo- gy and Systematics, Department of Biology, NKUA. zse.pensoft.net 116 Table 3. Shell morphometry of Daphniola dione sp. nov. Mea- surements are in mm. For abbreviations see the section of Ma- terials and methods. n=10 SH SW AH AW SH/SW AH/AW SH/AH SW/AW Min 0.83 1.14 0.47 0.52 0.68 0.79 1.62 2.05 Max 0.98 1.31 0.53 0.61 0.80 1202 2.08 22d. x 02914 FZ, Ot Ori O75 0.90 1.78 2/13 SD 0.04 0.05 0.03 0.03 0.03 0.07 0.12 0.06 CV 0.05 0.05 0.06 0.06 0.05 0.08 0.07 0.03 Description. She// (Fig. 3A—L). Valvatiform with up to 3.25 whorls, thin, colorless, transparent and crystalline when fresh, finely striated; spire more or less depressed; whorls rounded, regularly growing with shallow sutures. Measurements are given in Table 3. Periostracum light cream-colored; aperture adhering to the last whorl, pro- socline, roundish to ovate; peristome continuous, slightly thickened at columellar margin, the outer margin simple; umbilicus open, deep, wide so that the first whorls can be seen through it (Fig. 3B, F). Operculum (Fig. 3F). Ovate, thin, pliable, corneous, paucispiral, yellowish darker at the nucleus, with weakly convex inner face without any peg, nucleus sub-central. Soft body pigmentation (Fig. 3A—L). Soft body total- ly unpigmented and visible under the transparent shell; snout longer than wide, parallel-sided with medium distal lobation; eye spots very small. Nervous system. Cerebral ganglia of the same size, white-colored; supraoesophageal and suboesophageal ganglia of the same size, smaller than cerebral ganglia, white-colored; supraoesophageal connective longer than suboesophageal connective. Ctenidium-Osphradium. Ctenidium with ca 8-12 lamellae. Osphradium of intermediate width, opposite approximate middle of ctenidium. Digestive system. Radula very fragile, not investigat- ed; stomach unpigmented. Style sac smaller than stom- ach, not protruding to the intestinal loop; rectum (Fig. 3], J) with V-shaped bend, 1n some specimens with irregular V-shaped bend. The V-shaped bend is narrower in males (Fig. 3J) than in females (Fig. 31). Faecal pellets are lon- gitudinally packed. Male reproductive system (Fig. 4A, B) Penis whitish, medium-sized, narrow, long, gradually tapering bearing an obtuse outgrowth on the left side forming an acute an- gle with the penis distal portion; distal portion filamen- tous and usually bent; proximal portion bent upon itself and wrinkled; base of penis of intermediate width, its at- tachment area behind the right eye; penial duct strongly undulating especially in the proximal penis portion, near outer edge positioned, penial opening terminal; prostate like an elongate bean. Female reproductive system (Fig. 4C). Albumen and capsule glands very small, total mean height 0.38 mm (three specimens); bursa copulatrix large-sized, pyriform, posteriorly positioned and fully protruding from the pos- terior end of the albumen gland with longitudinal orienta- tion relative to albumen gland; bursal duct anterior, wide zse.pensoft.net Canella Radea et al.: Hydrobiid from Lefkada Island Figure 4. A., B. Penis (dorsal aspect); C. Female genitalia. Ab- breviations are given in the section of Materials and Methods. Scale bar 0.5 mm. Table 4. Morphometry of Daphniola species. Measurements are in mm. For abbreviations see Materials and methods. Species NW SH SW AH AW D. dione sp. nov. 2.75-3.25 0.83-0.98 1.14-1.31 0.47-0.55 0.52-0.61 D.eptalophos =. 3..25-3.50 0.90-1.25 1.10-1.90 0.50-0.80 0.50-0.75 D. exigua 3.00-3.50 1.10-1.52 1.10-1.40 0.60-0.80 0.60-0.76 D. hadei 2.25-2.50 0.84-0.85 1.14-1.15 0.55-0.57 0.52-0.54 D. louisi 3.50 1.09-1.45 1.17-1.69 0.59-0.98 0.59-0.79 D. magdalenae 3.50-3.75 2.34 - 1.35 1.28 Table 5. Morphological and anatomical diagnostic characters of D. dione sp. nov. and its congeneric species. Species 123 4 5678910 11 12 13 14 15 DP atone’ sp. nova! -O* Or-Oe2 9 I 2 -S20)-O) “TOs dade 1 D. eptalophos Or. Orel sl? eel’ As Bel 0 D. exigua dee 26Oe1 Rd eO-2O8 1 Om On gIS Os" ah 20 D. hadei 1003100--- 1 201 - 1 «0 D. louisi Ome 1 eG Os 2 we BOO Bes Os Gh EO) D. magdalenae 130 2 00--- 1 21 0- 0 1 Umbilicus: wide, open (0); semi-open (1) Operculum: light yellowish darker on the nucleus (0); orange; darker on the nucleus (1); yellow; orange on the nucleus (2); pink reddish (3) Soft body pigmentation: no pigmentation (0); light pigmentation (1); pigmentation (2) Bursa copulatrix shape: globular (0); ovoid (1); pyriform (2) Bursa copulatrix size: medium (0); large (1) Bursa copulatrix position relative to posterior end of albumen gland: non-protrud- ing (0); protruding (1) Bursal duct width: narrow (0); medium wide (1); wide (2) Coiled oviduct shape: circular-ovoid (0); Ovoid (1); ellipsoid, transverse relative to albumen gland (2); triangular (3) Primary oviduct loop shape: V (0); U (1); circular (2) Penis pigmentation and shape: unpigmented with pointed apex (0); unpigmented with long, narrow filament (1); pigmented with pointed apex or short filament (2) Penial outgrowth shape: prominent; forming a right angle with the distal part of pe- nis (0); prominent; forming an acute angle with the distal part of penis (1); slightly prominent (2) Penial outgrowth position: distal penis portion (0); medial (1); basal penis portion (2) Penial duct: near center (0); near outer edge (1) Eyes: absent (0); present (1) Habitat: spring (0); cave spring (1) —: No data and longer than bursa length; coiled oviduct unpigment- ed, well-developed forming an equilateral triangle with a vertical, V-shaped primary loop; two seminal receptacles; distal seminal receptacle (rs,) very small, globular with- out duct; proximal seminal receptacle (rs,) larger than distal one and smaller than bursa copulatrix, pyriform, with very short duct and a pink pearly shine, lying over the renal oviduct and against bursa copulatrix; an egg Zoosyst. Evol. 97 (1) 2021, 111-119 capsule with a single egg was found inside the umbilicus of one specimen (Fig. 3K, L). Morphometric data along with conchological and ana- tomical characters of the nominal Daphniola species are provided in Tables 4 and 5, respectively. TAF Distribution and habitat. So far, the distribution of Daphniola dione sp. nov., is restricted to the type locality. The live specimens of the new species were found crawl- ing on the roots of woody plants reaching the bottom of the spring. The dichotomous key to species of the genus Daphniola 1 SOU, HOC O tally sUIMOIS GENIE BS. cae rons cee tca bees raaers ccteey see rey Pee eee tts