Research Article |
Corresponding author: Andrzej Falniowski ( andrzej.falniowski@uj.edu.pl ) Academic editor: Menno Schilthuizen
© 2015 Andrzej Falniowski, Serban Sarbu.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Falniowski A, Sarbu S (2015) Two new Truncatelloidea species from Melissotrypa Cave in Greece (Caenogastropoda). ZooKeys 530: 1-14. https://doi.org/10.3897/zookeys.530.6137
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In the small lake located in the cave Melissotrypa in Thessalia, Greece, truncatelloidean gastropods representing two species were found, new to science. One of them, represented by two specimens only, has been described based on the shell characters only; with its cytochrome oxidase sequence it has been assigned to the genus Iglica, and to the family Moitessieriidae, Iglica hellenica sp. n. For the other species, represented by 30 collected specimens, the shell, protoconch, radula, head, penis and female reproductive organs have been described; all the morphological characters and cytochrome oxidase sequences have confirmed its assignment to the genus Daphniola (Hydrobiidae: Sadlerianinae), Daphniola magdalenae Falniowski, sp. n.
Gastropoda , Hydrobiidae , Moitessieriidae , aquatic snails, morphology, cytochrome oxidase, taxonomy, troglobionts
In June 2014, in Melissotrypa Cave in Greece (39°52'38"N and 22°02'58"E), several specimens of Truncatelloidea gastropods were collected. This was the third visit by the second author to this cave, but the snails were found for the first time.
The cave is located in Melissotrypa Kefalovriso Elassona, north of Larissa, and is the largest known underground karstic form of karst system Kranias Elassona, drilled in marbles. The character of the cave is demonstrated by the remaining forms of dissolution and growth of the cave, the gypsum and detected hydrogen sulfide in the lakes of the cave. The cave covers an area 0.06 km2 and has a total length of mapped passageways about 2103.6 m. The elevation in the region of the inlet orifice is 299 m while the interior reaches a depth up -47.3 m i.e. absolute altitude 251.7 m. The depth of the precipitous entry is 14.6 m (http://7gym-laris.lar.sch.gr/perivalon/spilaia.htm).
Many specimens of gastropods were concentrated in just one area in the sulfuric lake, close to the shore in a depth of approximately 10 cm. In the vicinity of the lake there are no terrestrial animals, although there are microbial biofilms and organic matter. The aquatic fauna is highly interesting: the most abundant form is an amphipod Niphargus, which swims upside down, seemingly an adaptation to such water chemistry. The snails do not live everywhere, but only in one place on a limestone wall, at 5–10 cm beneath the water surface. There were hundreds of individuals gathered in a compact group. Maybe there are more such groups, but the water is deep and one cannot reach the walls except by means of a small boat, the lake being very narrow. In this cave, there is also another lake, at several hundred meters away from the former, in which the water has no sulfur, and which is sometimes dry. No snails have been found in it.
Only two specimens with a turriform shell were collected, and approximately 30 specimens with a valvatiform shell. The aim of the paper is to describe these two snails collected in Melissotrypa Cave.
The snails were collected by hand and placed directly in 95% ethanol. The ethanol was changed twice, and the material stored at -20 °C.
The shells were photographed with a CANON EOS 50D digital camera, attached to a NIKON SMZ18 stereoscope microscope with dark field. They were dissected using a NIKON SMZ18 stereoscope microscope with a NIKON drawing apparatus, and a NIKON DS-5 digital camera. Radulae and protoconchs were examined using a JEOL JSM-5410 scanning electron microscope, applying the techniques described by
DNA was extracted from foot tissue of two specimens. The tissue was hydrated in TE buffer (3 × 10 min); total genomic DNA was then extracted with the SHERLOCK extracting kit (A&A Biotechnology), and the final product was dissolved in 20 µl TE buffer. The PCR reaction was performed with the following primers: LCOI490 (5’-ggtcaacaaatcataaagatattgg-3’) (
The PCR conditions were as follows: initial denaturation step of 4 min at 94 °C, followed by 35 cycles of 1 min at 94 °C, 1 min at 55 °C 2 min at 72 °C, and a final extension of 4 min at 72 °C. The total volume of each PCR reaction mixture was 50 µl. To check the quality of the PCR products 10 µl of the PCR product was ran on 1% agarose gel. The PCR products were purified using Clean-Up columns (A&A Biotechnology) and were then amplified in both directions using BigDye Terminator v3.1 (Applied Biosystems), following the manufacturer’s protocol and with the primers described above. The sequencing reaction products were purified using ExTerminator Columns (A&A Biotechnology); DNA sequences then underwent electrophoresis on an ABI Prism sequencer.
The COI sequences were aligned by eye using BioEdit 5.0.0 (
Taxa used for phylogenetic analyses, with their GenBank Accession Numbers and references.
Species | COI GB# | References |
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Adrioinsulana conovula (Frauenfeld, 1863) | AF367628 |
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Agrafia wiktori Szarowska & Falniowski, 2011 | JF906762 |
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Alzoniella finalina Giusti & Bodon, 1984 | AF367650 |
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Anagastina zetavalis (Radoman, 1973) | EF070616 |
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Avenionia brevis (Draparnaud, 1805) | AF367638 |
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Belgrandiella kusceri (Wagner, 1914) | KT218520 |
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Bithynia tentaculata (Linnaeus, 1758) | AF367643 |
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Boleana umbilicata (Kuščer, 1932) | KT218521 |
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Bythinella austriaca (Frauenfeld, 1857) | FJ545132 |
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Bythiospeum sp. | AF367634 |
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Bythiospeum acutum (Geyer, 1904) | HM107120 | unpublished, from GenBank |
Bythiospeum francomontanum Bernasconi, 19730 | HM107131 | unpublished, from GenBank |
Bythiospeum hungaricum (Soós, 1927) | KP296923 | unpublished, from GenBank |
Bythiospeum husmanni (C.R. Boettger, 1963) | HM107134 | unpublished, from GenBank |
Bythiospeum pellucidum (v. Wiedersheim, 1973) | HM107124 | unpublished, from GenBank |
Bythiospeum suevicum (Geyer, 1905) | HM107118 | unpublished, from GenBank |
Dalmatinella fluviatilis Radoman, 1973 | KC344541 |
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Daphniola exigua (A. Schmidt, 1856) | EU047767 |
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Daphniola hadei (Gittenberger, 1982) | JF916477 |
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Daphniola graeca Radoman, 1973 | EF070618 |
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Daphniola louisi Falniowski & Szarowska, 2000 | EU047769 |
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Daphniola sp. | KM887915 |
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Daphniola magdalenae sp. n. | KT825578-80 | present study |
Dianella thiesseana (Kobelt, 1878) | AY676127 |
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Fissuria boui Boeters, 1981 | AF367654 |
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Graecoarganiella parnassiana Falniowski & Szarowska, 2011 | JN202348 |
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Graziana alpestris (Frauenfeld, 1863) | AF367641 |
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Grossuana codreanui (Grossu, 1946) | EF061919 |
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Hauffenia tellinii (Pollonera, 1898) | AF367640 |
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Heleobia dalmatica (Radoman, 1974) Horatia klecakiana Bourguignat, 1887 |
AF367631
KJ159128 |
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Hydrobia acuta (Draparnaud, 1805) | AF278808 |
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Iglica hellenica sp. n. | KT825581 | present study |
Islamia piristoma Bodon & Cianfanelli, 2001 | AF367639 |
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Lithoglyphus naticoides (C. Pfeiffer, 1828) | AF367642 |
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Marstoniopsis insubrica (Küster, 1853) | AY027813 |
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Moitessieria cf. puteana (Coutagne, 1883) | AF367635 |
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Montenegrospeum bogici (Pešić & Glöer, 2012) | KM875510 |
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Pseudamnicola lucensis (Issel, 1866) | AF367651 |
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Pyrgula annulata (Linnaeus, 1767) | AY341258 |
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Radomaniola callosa (Paulucci, 1881) | AF367649 |
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Rissoa labiosa (Montagu, 1803) | AY676128 |
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Sadleriana fluminensis (Küster, 1853) | AY273996 |
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Tanousia zrmanjae (Brusina, 1866) | Xx |
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Trichonia kephalovrissonia Radoman, 1973 | EF070619 |
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Ventrosia ventrosa (Montagu, 1803) | AF118335 |
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Ethanol-fixed specimen, Melissotrypa Cave, Thessalia, Greece, 39°52'38"N, 22°02'58"E, sulphidic lake, near the shore, June 2014, S. Sarbu coll., ZMUJ-M.2107.
One specimen destroyed for DNA extraction details as for holotype.
Shell relatively big, turriform, readily distinguished from geographically close and related species I. sidariensis, I. maasseni, I. wolfischeri and I. alpheus by its larger size and more convex whorls Iglica hellenica is readily distinguished from the geographically closest species Paladilhiopsis thessalica by its larger size and narrow aperture.
Shell (Fig.
The specific epithet (hellenica) is a Greek adjective meaning Greek.
Known from two specimens from the type locality only.
Ethanol-fixed specimens, Melissotrypa Cave, Thessalia, Greece, 39°52'38"N, 22°02'58"E, sulphidic lake, near the shore, June 2014, S. Sarbu coll., holotype: ZMUJ-M.2109; 20 paratypes: ZMUJ-M.2110-ZMUJ-M.2130.
Shell relatively big, valvatiform-trochiform; soft parts with no pigment, no eyes, penis with long and slender filament and big outgrowth on the left side. Readily distinguished from geographically and closely related D. exigua (= D. graeca) by its bigger size (2.5 vs. 1.5 mm), reddish operculum, broader base and longer and thinner filament of the penis. Differentiated from D. louisi (from Kessariani at Athens) by its larger size, higher spire, longer and thinner filament and more prominent outgrowth on the left side of the penis. Differs from D. hadei (from Gythion at Peloponnese) by its double size, higher spire and much more prominent outgrowth on the left side of the penis.
Shell (Fig.
shell heigth | shell heigth (mm) | spire heigth (mm) | body whorl width (mm) | aperture heigth (mm) | aperture width (mm) | whorl number |
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holotype | 2.51 | 0.38 | 1.99 | 1.37 | 1.34 | 3.5 |
mean | 2.335 | 0.363 | 1.895 | 1.346 | 1.280 | 3.70 |
sd | 0.1788 | 0.0503 | 0.1506 | 0.0797 | 0.0643 | 0.1083 |
minimum | 2.16 | 0.28 | 1.76 | 1.20 | 1.19 | 3.50 |
maximum | 2.68 | 0.43 | 2.21 | 1.44 | 1.39 | 3.75 |
Inner and outer sides of operculum smooth. Operculum pink (Fig.
Radula (Figs
Animal brownish, with no pigment, and no eyes (Fig.
Renal and pallial section of female reproductive organs of Daphniola magdalenae (bc – bursa copulatrix, cbc – duct of bursa copulatrix, ga – albuminoid gland, gn – nidamental gland, gp – gonoporus, ov – oviduct, ovl – loop of (renal) oviduct, rs1, rs2 – receptacula seminis, nomenclature after
Named in memory of Dr Magdalena Szarowska, a malacologist, wife and best friend of the first author.
Known from the type locality only.
The saturation test of
With one (since the other had to be destroyed for DNA extraction) available specimen of Iglica hellenica sp. n. it has not been possible to study its soft parts. However, nearly all the representatives of Bythiospeum, Paladilhiopsis, Iglica, etc. are known as empty shells only. The distinction between these genera remains unclear. The molecular tree, as well as the phylogeny presented by
The shells of Daphniola exigua are highly variable (
The molecular tree confirms relationships of both new species Iglica hellenica and Daphniola magdalenae. As it is based on one short fragment of mitochondrial DNA, it presents the phylogeny of this fragment, certainly not of the species/genera (e.g.,
The study was supported by a grant from the National Science Centre (2012/05/B/NZ8/00407).