Corresponding author: Marian A. Ramos (
Academic editor: E. Gittenberger
Several
The Mediterranean basin, and within it the Iberian Peninsula, has been identified as a biodiversity hotspot for animal species including those of hydrobiid gastropods (
Two subgenera are currently recognized within the
One of these 11 species is
Our paper provides a wide conchological and anatomical description of a new species of
The study area comprised the Departments of Alpes-Maritimes and Var in southeastern France and the provinces of Castellón and Valencia in eastern Spain. Specimens were collected from several sites in this area (see
Map of localities of
Anatomical observations and morphometric measurements were made on specimens relaxed with menthol crystals and fixed in ethanol following the procedures described in
Specimens were dissected under a Leica MZ 16 A stereomicroscope and photographed using a Nikon ds fi1 camera. All measurements were made using Nis-Elements V. 2.2. software. Anatomical illustrations were prepared from camera lucida drawings. Environmental scanning electron microscope (ESEM) images of shells were captured using a Philips Quanta 200 in low-vacuum mode, after removal of the periostracum by immersion in 5% sodium hypochlorite and then cleaning by ultrasonication. The radula and operculum were cleaned by immersion in KOH solution (10g/l) at room temperature. Both structures were then rinsed in distilled water and air-dried before mounting on stubs and coating with a thin (10–20 nm) gold layer in an Emitech K550X sputter coating unit followed by observation in high-vacuum mode.
Total DNA was isolated from the foot tissue of the snails using the ChargeSwitch gDNA Micro Tissue (Invitrogen, Paisley, UK) extraction kit. Partial COI sequences were amplified by polymerase chain reaction (PCR) using LCO1490 (
Species name, locality details, Genbank accession numbers and publication references for mtCOI sequences.
Species name | Locality | Genbank accession number | Reference |
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La Gitana spring, La Peza, Granada, Spain. |
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La Armada spring, Orce, Granada, Spain. |
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La Garganta stream, Nava de San Pedro, Jaén, Spain. |
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Spring in Ermita de las Santas, Granada, Spain. |
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Pilar del Mono spring, Dúrcal, Granada, Spain. |
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Don Pedro spring, Loja, Granada, Spain. |
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La Salud spring, Toscarejo, Jaén, Spain. |
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Thermal spring in Bagni di Lucca, Tuscany, Italy. |
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Artificial pond in Marmaris, Evvoia island, Greece. |
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Lac de Tunis, Tunisia. |
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Lake Garda, Brescia, Italy. |
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Surroundings of Grasse, France (Dupuy, 1851).
Shells of
Operculum and radula of
Anatomy of
A few specimens collected from Source d’Argens , Brue-Aurillac, Var, France after finding the type area and other localities in Alpes-Maritimes and Var practically destroyed by severe storms. A total of two females and four males have been examined for anatomical descriptions.
Source d’Argens, Brue-Aurillac, Var, France,
Shell yellowish or whitish with a body whorl occupying 2/3 shell length and a deep suture between whorls; protoconch microsculpture granulated; central radular tooth formula 7-C-7; style sac surrounded by a black pigmented intestine; elongate bursa copulatrix U-shaped; elongate seminal receptacle without duct; penis slender with a black patch of pigmentation and some folds in its middle region; nervous system brown pigmented with supraoesophageal connective about two times longer than suboesophageal.
Shellovate-conic with 4–4.75 spire whorls, height 2.5–3.5 mm (
Shell measurements (in mm) of
T-test<br/> ( |
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SL | 3.11<br/> (3.56–2.58) | 0.25 | 0.08 | 2.50<br/> (2.85–2.17) | 0.18 | 0.07 | p < 0.001 |
SW | 1.92<br/> (2.28–1.74) | 0.15 | 0.08 | 1.59<br/> (1.78–1.44) | 0.10 | 0.06 | p < 0.001 |
SL/SW | 1.62<br/> (1.76–1.45) | 0.10 | 0.06 | 1.57<br/> (1.73–1.46) | 0.07 | 0.04 | n.s. |
AH | 1.37<br/> (1.78–1.26) | 0.14 | 0.10 | 1.20<br/> (1.36–1.04) | 0.08 | 0.07 | p < 0.001 |
SL–LBW | 1.10<br/> (1.39–0.76) | 0.16 | 0.15 | 0.69<br/> (1.00–0.49) | 0.13 | 0.19 | p < 0.001 |
WBW | 1.76<br/> (1.89–1.59) | 0.09 | 0.05 | 1.42<br/> (1.57–1.29) | 0.07 | 0.05 | p < 0.001 |
AL | 1.39<br/> (1.72–1.28) | 0.12 | 0.08 | 1.26<br/> (1.41–1.11) | 0.09 | 0.07 | p < 0.01 |
AW | 1.01<br/> (1.33–0.85) | 0.14 | 0.14 | 0.89<br/> (1.11–0.78) | 0.08 | 0.09 | p < 0.01 |
WPW | 1.23<br/> (1.33–1.11) | 0.08 | 0.07 | 0.95<br/> (1.12–0.80) | 0.08 | 0.08 | p < 0.001 |
WAW | 0.19<br/> (0.32–0.15) | 0.06 | 0.32 | 0.28<br/> (0.37–0.19) | 0.04 | 0.14 | p < 0.001 |
NSW | 4.30<br/> (4.75–4.00) | 0.31 | 0.07 | 4.18<br/> (4.50–4.00) | 0.21 | 0.05 | n.s. |
SDN, Unbiased estimate for Standard Deviation, CV, Coefficient of Variation.
Operculum corneous, yellowish, thin, pliable, ellipsoidal, paucispiral with nucleus submarginal (
Operculum measurements (in mm) of
Mean (Max-Min) | SDN | CV | Mean (Max-Min) | SDN | CV | |
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OL | 1.10 (1.18–0.96) | 0.05 | 0.05 | 1.08 (1.18–0.96) | 0.08 | 0.08 |
OW | 0.79 (0.84–0.73) | 0.04 | 0.05 | 0.74 (0.84–0.66) | 0.06 | 0.08 |
OLWL | 0.48 (0.56–0.40) | 0.05 | 0.11 | 0.49 (0.63–0.36) | 0.08 | 0.17 |
OLWW | 0.34 (0.40–0.25) | 0.05 | 0.16 | 0.33 (0.38–0.29) | 0.03 | 0.09 |
NL | 0.47 (0.52–0.39) | 0.04 | 0.09 | 0.36 (0.44–0.26) | 0.06 | 0.17 |
NW | 0.36 (0.42–0.31) | 0.04 | 0.12 | 0.29 (0.45–0.21) | 0.06 | 0.22 |
SDN, Unbiased estimate for Standard Deviation, CV, Coefficient of Variation.
Radula intermediate length (20% total shell length) bearing some 50 rows of teeth (
Radula formulae and measurements (in mm) of three radulae of
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Central teeth | 7+C+7/1–1 | 5+C+5/1–1 |
Central teeth width | ~ 20 µm | ~ 15 µm |
Lateral teeth | 3–C–3 | 3–C–3 |
Inner marginal teeth | ≥ 18 cusps | ≥ 15 cusps |
Outer marginal teeth | ≥ 19 cusps | ≥ 19 cusps |
Radula length | ~ 700 µm | ~ 600 µm |
Radula width | ~ 90 µm | ~ 95 µm |
Number of rows | ~ 55 | ~ 50 |
Ctenidium, osphradium and digestive system measurements (in mm) of
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Ct L | 1.06<br/> (1.25–0.90) | 0.15 | 0.14 | 1.11<br/> (1.27–0.92) | 0.16 | 0.14 |
Os L | 0.32<br/> (0.43–0.20) | 0.09 | 0.27 | 0.33<br/> (0.45–0.24) | 0.07 | 0.22 |
Os W | 0.13<br/> (0.15–0.11) | 0.02 | 0.16 | 0.21<br/> (0.25–0.15) | 0.03 | 0.15 |
Ss L | 0.59<br/> (0.67–0.50) | 0.06 | 0.11 | 0.62<br/> (0.66–0.58) | 0.03 | 0.05 |
Ss W | 0.35<br/> (0.37–0.31) | 0.02 | 0.06 | 0.37<br/> (0.41–0.33) | 0.03 | 0.08 |
St L | 0.71<br/> (0.74–0.66) | 0.04 | 0.06 | 0.73<br/> (0.84–0.66) | 0.06 | 0.09 |
St W | 0.61<br/> (0.67–0.56) | 0.04 | 0.07 | 0.69<br/> (0.78–0.61) | 0.06 | 0.09 |
SDN, Unbiased estimate for Standard Deviation, CV, Coefficient of Variation.
Female genitalia with a slender pallial oviduct (
Female and male genitalia measurements (in mm) of two females and four males of
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Po L | 2.22<br/> (2.35–2.08) | 0.24 | 0.11 | 2.04<br/> (2.32–1.75) | 0.26 | 0.13 |
Po W | 0.58<br/> (0.59–0.56) | 0.03 | 0.04 | 0.45<br/> (0.49–0.41) | 0.04 | 0.10 |
Ag. L | 0.95<br/> (1.04–0.84) | 0.18 | 0.18 | 0.81<br/> (1.00–0.71) | 0.14 | 0.17 |
Cg. L | 1.01<br/> (1.11–0.91) | 0.18 | 0.17 | 1.00<br/> (1.11–0.93) | 0.09 | 0.09 |
SR1 L | 0.14<br/> (0.15–0.14) | 0.01 | 0.09 | 0.18<br/> (0.22–0.15) | 0.03 | 0.18 |
BC L | 1.24<br/> (1.32–1.15) | 0.15 | 0.12 | 1.37<br/> (1.56–0.95) | 0.30 | 0.22 |
BC W | 0.30<br/> (0.35–0.25) | 0.09 | 0.29 | 0.31<br/> (0.36–0.25) | 0.05 | 0.18 |
dBC L | 0.60<br/> (0.75–0.65) | 0.09 | 0.15 | 0.62<br/> (0.72–0.46) | 0.13 | 0.21 |
Pr L | 1.67<br/> (1.86–1.55) | 0.14 | 0.08 | 1.37<br/> (1.46–1.26) | 0.09 | 0.06 |
Pr W | 0.58<br/> (0.69–0.52) | 0.09 | 0.15 | 0.45<br/> (0.51–0.41) | 0.05 | 0.12 |
P L | 1.26<br/> (1.35–1.15) | 0.09 | 0.07 | 1.28<br/> (1.60–1.12) | 0.24 | 0.19 |
P W | 0.37<br/> (0.40–0.33) | 0.03 | 0.09 | 0.66<br/> (0.75–0.50) | 0.12 | 0.18 |
PL/Head length | 1.02<br/> (1.16–0.86) | 0.16 | 0.16 | 0.91<br/> (1.07–0.82) | 0.15 | 0.17 |
SDN, Unbiased estimate for Standard Deviation, CV, Coefficient of Variation.
Male genitalia bear a bean-shaped prostate gland about three times longer than wide (
Nervous system brown pigmented, consisting of disperse points of pigmentation; cerebral ganglia equal in size; supraoesophageal connective more than two times longer than suboesophageal (
Nervous system measurements (in mm) of
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LRCG | 0.22<br/> (0.24–0.20) | 0.02 | 0.10 | 0.21<br/> (0.23–0.18) | 0.02 | 0.10 |
LLCG | 0.22<br/> (0.25–0.18) | 0.03 | 0.15 | 0.22<br/> (0.24–0.19) | 0.02 | 0.10 |
LCC | 0.11<br/> (0.12–0.07) | 0.02 | 0.19 | 0.15<br/> (0.19–0.12) | 0.02 | 0.14 |
LRPG | 0.13<br/> (0.18–0.10) | 0.03 | 0.25 | 0.15<br/> (0.16–0.12) | 0.02 | 0.14 |
LLPG | 0.17<br/> (0.20–0.13) | 0.03 | 0.19 | 0.23<br/> (0.28–0.20) | 0.03 | 0.14 |
LsupG | 0.14<br/> (0.17–0.11) | 0.03 | 0.23 | 0.12<br/> (0.15–0.10) | 0.02 | 0.18 |
LsubG | 0.11<br/> (0.13–0.09) | 0.01 | 0.10 | 0.12<br/> (0.14–0.09) | 0.02 | 0.18 |
LPsupC | 0.18<br/> (0.24–0.11) | 0.05 | 0.30 | 0.29<br/> (0.39–0.21) | 0.07 | 0.26 |
LPsubC | 0.07<br/> (0.09–0.06) | 0.01 | 0.15 | 0.10<br/> (0.13–0.05) | 0.03 | 0.32 |
RPG | 0.40<br/> (0.49–0.38) | 0.05 | 0.13 | 0.51<br/> (0.58–0.46) | 0.05 | 0.10 |
SDN, Unbiased estimate for Standard Deviation, CV, Coefficient of Variation.
The only available information on the anatomy of this species in the literature corresponded to populations from Foux à Draguignan (figure 2, 4, 7, 9 in
Comparingshell sizes among the
Los Nogales spring, Benafer, Castellón, Spain,
Holotype MNCN 15.05/60026a (SEM preparation,
Shells of
Four males and four females from type locality were examined for anatomical study. In addition, some populations from provinces of Castellón and Valencia (Spain) were also found and studied, dissecting likewise two males and two females from each for their identification.
Los Nogales spring, Benafer, Castellón, Spain (type locality),
Shell, anatomical, operculum and radular measurements (
Dedicated to the malacologist and ecologist Torsten Hauffe, for his help and support during the stay of the first author in Germany.
Shell yellowish with body whorl occupying 2/3 shell length; umbilicus slightly visible; protoconch microsculpture grooved; central radular tooth formula 5-C-5; style sac protruding below non-pigmented intestine; elongate bursa copulatrix J-shaped; renal oviduct pigmented until seminal receptacle, which has a pigmented short duct; penis triangular with a wide base attached to central area of head; nervous system brown pigmented with supraoesophageal connective about three times longer than suboesophageal.
Shellovate-conic (
Operculum corneous, yellowish, thin, pliable, ellipsoidal, paucispiral, with nucleus submarginal (
Operculum and radula of
Radula with around 50 rows of teeth, medium in size (25% total shell length) (
Anatomy of
Bayesian 50% majority rule consensus tree inferred employing COI mitochondrial gene partial sequence. The numbers above branches represent Bayesian posterior probabilities. The numbers between brackets symbolize specimens with identical haplotypes. Scale bar: expected changed per site.
Female genitalia with a pallial oviduct about four times longer than wide (
Male genitalia bearing a bean-like prostate gland about three times longer than wide (
Nervous system brown pigmented, but ganglia darker than connectives and commissures; cerebral ganglia equal in size; supraoesophageal and suboesophageal ganglia similar in shape and size; supraoesophageal connective around three times longer than suboesophageal (
Some of the localities where this species was found were cited by
Compared to the other
The data set analysed included data for 11
Genetic divergence matrix for the species examined based on the COI gene sequence.
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1. |
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2. |
7.44 | - | |||||||||||
3. |
10.26 | 10.41 | - | ||||||||||
4. |
10.79 | 12.16 | 6.00 | - | |||||||||
5. |
8.13 | 9.42 | 6.38 | 6.69 | - | ||||||||
6. |
9.11 | 10.64 | 6.46 | 6.54 | 5.39 | - | |||||||
7. |
8.97 | 8.81 | 9.19 | 10.33 | 7.83 | 8.05 | - | ||||||
8. |
7.86 | 7.65 | 9.14 | 9.59 | 7.87 | 8.50 | 8.42 | - | |||||
9. |
8.73 | 8.74 | 9.65 | 11.15 | 8.81 | 9.03 | 7.67 | 5.46 | - | ||||
10. |
15.06 | 14.78 | 15.15 | 15.21 | 13.69 | 14.31 | 13.66 | 12.90 | 14.22 | - | |||
11. |
14.45 | 14.47 | 14.85 | 14.89 | 14.16 | 14.32 | 13.67 | 14.42 | 14.54 | 7.68 | - | ||
12. |
17.91 | 19.49 | 17.60 | 17.26 | 15.78 | 16.35 | 16.62 | 17.12 | 18.29 | 18.65 | 18.97 | - | |
13. |
16.67 | 17.44 | 17.55 | 16.67 | 14.99 | 16.82 | 17.58 | 16.09 | 17.05 | 16.90 | 18.14 | 17.38 | - |
Based on this wide morphological study and our molecular data, we were able to delimit both species and clearly rule out the hypothesis of the presence of
Through a phylogenetic approach based on partial sequence data for the COI gene provided in GenBank for other
Besides clarifying the taxonomic status of these two species and their phylogenetic relationship as sister species, our findings point to a greater diversity of
The authors thank Dr. M. Haase and Dr. E. Gittenberger for their constructive comments and suggestions. A.L. Tormo, M. Furio and A. Jorge from the MNCN assisted with the ESEM study and photomicrographs. Drawings were redone by I. Díaz Cortaberría. Dr. M.A. Alonso-Zarazaga provided advice on nomenclature. The English was reviewed by A. Burton. The manuscript was also improved by the comments and suggestions of Dr. R. Hershler. This work has been financed by the MICINN projects Fauna Ibérica IX (CGL2007-66786-C8-01) and FaIb X (CGL2010-22267-C07-01) and the support of a JAE Predoctoral fellowship (JAEPre047) to DD.