Corresponding author: Thomas Inäbnit (
Academic editor: Martin Haase
The generic allocation of
La répartition générique de
Bouaziz-Yahiatene H, Inäbnit T, Medjdoub-Bensaad F, Colomba MS, Sparacio I, Gregorini A, Liberto F, Neubert E (2019) Revisited – the species of Tweeting vineyard snails, genus
The hitherto monotypic genus
The tribe originates from the Maghrebinian radiation centre (
The specimens for this study were collected by the authors of the study, particularly by the senior author. Missing sequences for
DNA was extracted from a piece of foot muscle tissue using Qiagen Blood and Tissue Kit (Qiagen cat nr. 69506) and the QIAcube extraction robot (Protocol 430, DNeasy Blood Tissue and Rodent tails Standard). Our phylogenetic hypotheses were reconstructed using five phylogenetic markers (mitochondrial COI (657 base pairs (bp)), 16S (374 bp) and nuclear 28S (528 bp), H3 (304 bp) and ITS2 (909 bp)), resulting in a length of 2772 bp (see Table
Taxa used in this study: family, species, locality, voucher, GenBank accession numbers for COI, 16S, H3, and 5.8S-ITS2-28S.
Species | Locality | Coordinates | Voucher | GenBank accession number | ||||||
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CO1 | 16S | H3 | 28S | 5.8S-ITS2 | Origin | |||||
(N) | (E) | |||||||||
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Hannover-Anderten, N side of Mittelland Canal/ Lower Saxony |
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MN_2551-Hel/MN_012 |
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Makouda, Tizi Ouzou, DZ |
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Beach between Agia Napa and Capo Greco, CY |
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Draâ-Ben Khedda/ Tizi Ouzou, DZ |
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Draâ-Ben Khedda/ Tizi Ouzou, DZ |
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Ighil Bourmi, DZ |
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This work | ||
Ighil Bourmi, DZ |
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This work | |||
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Tigzirt/ Tizi Ouzou, DZ |
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This work | ||
Draa Ben Kheda/ Tizi Ouzou, DZ |
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This work | |||
Djelfa, Algeria |
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MVHN-2013 | - |
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- | - |
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Marincola, Amantea, Calabria |
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This work | ||
Marincola, Amantea, Calabria |
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This work | |||
Palermo: Cefalú, Cocuzzola |
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Enna: Assoro, C. da Cernigliere |
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Enna: Assoro, C. da Cernigliere |
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Italy, Strada del Casone (Siena) |
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FGC 36599 |
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Italy, Strada del Casone (Siena) |
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FGC 36599 |
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Italy, Strada del Casone (Siena) |
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FGC 36599 |
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Draa Ben Kheda/ Tizi Ouzou, DZ |
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- | This work | ||
Ait Bouadou, Tizi Ouzou, DZ |
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- | This work | |||
Palermo: Cefalú, Mazzaforno |
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Palermo: Cefalú, Mazzaforno |
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Palermo: Cefalú, Mazzaforno |
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Trapani: San Vito lo Capo, Cala Mancina |
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Trapani: San Vito lo Capo, Cala Mancina |
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Trapani: San Vito lo Capo, Cala Mancina |
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Trapani: Custonaci, Monte Cofano |
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Trapani: Custonaci, Monte Cofano |
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Trapani: Custonaci, Monte Cofano |
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- |
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Trapani: Custonaci, Monte Cofano |
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Trapani: Custonaci, Monte Cofano |
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Trapani: Custonaci, Monte Cofano |
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- |
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Palermo: Cefalú, La Rocca |
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Palermo: Cefalú, La Rocca |
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Palermo: Cefalú, La Rocca |
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Palermo: Cefalú, La Rocca |
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Palermo: Monte Pellegrino |
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Palermo: Monte Pellegrino |
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Palermo: Monte Pellegrino |
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Palermo: Cinisi, Monte Pecoraro |
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Palermo: Cinisi, Monte Pecoraro |
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Palermo: Cinisi, Monte Pecoraro |
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Palermo: Sferracavallo |
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Palermo: Sferracavallo |
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Palermo: Sferracavallo |
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Palermo: Sferracavallo |
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Palermo: Sferracavallo |
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Palermo: |
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The PCR included the following admixture: 2 µL template, 12.5 µL GoTaq (Promega) polymerase, 8.5 µL of nuclease-free water and 1 µL of both forward and reverse primer (10 µmol) respectively. In cases where the PCR signal was judged too weak, the reaction was repeated using 3 µL template DNA, 3 µL of the previous PCR product and 5.5 µL of nuclease-free water. The amount of GoTaq and primers stayed the same. The PCR was conducted using the following protocols: For COI, the admixture was first heated up to 95 °C for 1 minute (min), followed by 30 cycles of 30 seconds (s) at 95 °C, 30s at 52 °C and 30s at 72 °C, finishing with 3 min at 72 °C. For 16S, the protocol started with 2:30 min at 90 °C, followed by ten cycles of 30s at 92 °C, 30s at 44 °C and 40s at 72 °C, followed again by 30s at 92 °C, 40s at 48 °C and 40s at 48 °C. The protocol for 28S started with 1 min at 96 °C, then went into 35 cycles of 30s at 94 °C, 30s at 50 °C and 1 min at 72 °C, finishing with 10 min at 72 °C. The ITS2 protocol started with 1 min at 96 °C, followed by 35 cycles of 30s at 94 °C, 30s at 44 °C and 1 min at 72 °C, ending with 10 min at 72 °C. For H3 the admixture was first heated up to 95 °C for 3 min, followed by 40 cycles of 45s at 94 °C, 45s at 50 °C and 2 min at 72 °C, finishing with 10 min at 72 °C. The protocols for COI and H3 could be used for both markers. The PCR products were sequenced at the LGC Genomics GmbH (Berlin, Germany) and at Eurofins Genomics (Ebersberg, Germany) using their respective standard protocol. In total, 48 helicid specimens were used, chiefly from the genera
Topologies were estimated using two different phylogenetic methods: Maximum Likelihood (
The Bayesian tree, which was used as a basis for the combined tree (Fig.
Combined RaxML and Bayesian tree of
The Bayesian and RaxML reconstructions yielded the same topology for all species involved and are shown in Fig.
All three genera treated here in the analysis split in monophyletic lineages, and the nodes on the generic level have high support values. The species
The five markers used in this study.
Marker | Primer Name | Primer sequence | Reference |
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COI | LCO1490 | 5’-GGTCAACAAATCATAAAGATATTGG-3’ |
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HCO2198 | 5’-TAAACTTCAGGGTGACCAAAAAATCA-3’ | ||
16S | 16s F | 5’-CGGCCGCCTGTTTATCAAAAACAT-3’ |
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16s R | 5’-GGAGCTCCGGTTTGAACTCAGATC-3’ | ||
28S | LSU-2 | 5’-GGGTTGTTTGGGAATGCAGC-3’ |
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LSU-4 | 5’-GTTAGACTCCTTGGTCCGTC-3’ | ||
5.8S-ITS2-28S | ITS2ModA | 5’-GCTTGCGGAGAATTAATGTGAA-3’ |
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ITS2ModB | 5’-GGTACCTTGTTCGCTATCGGA-3’ | ||
H3 | H3-F | 5’-ATGGCTCGTACCAAGCAGAC(ACG)GC-3’ |
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H3-R | 5’-ATATCCTT(AG GGCAT(AG) AT(AG)GTG-3’ |
Character matrix including the genera
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1 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 1 |
2 | 0 | 0 | 1 | 2 | 0 | 0/1 | 0 | 1 |
3 | 0 | 0 | 1 | 0 | 0 | 2 | 3 | 0/4 |
4 | 0 | 1 | 0 | 1 | 0 | 0 | 0 | NA |
5 | 0 | 0 | 1 | 0 | 0 | 0 | 0 | 0 |
6 | 0 | 0 | 0 | 1 | 0 | 0 | 0 | 1 |
7 | 0 | 1 | 1 | NA | 0 | 0 | 0 | NA |
8 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 0 |
9 | 0/2 | 2 | 2 | 1 | 1 | 1 | 1 | 2 |
10 | 1 | 2 | 1 | 0 | 0 | 2 | 1 | V |
11 | 1 | 2 | 2 | 2 | 0 | 0 | 0 | 0/1 |
In Table
Anticipating a north African origin of the syntype
Syntype
Italy: Foggia, Ordona,
Shell thick, medium sized if compared to other helicid species, with a depressed spire and a large last whorl occupying more than two thirds of the complete height of the shell; protoconch small, consisting of 1.5 smooth whorls; teleoconch consisting of approximately 4 whorls, separated by a deep, sometimes crenulated suture; basic colour of teleoconch greenish-brownish, often with longitudinal yellow streaks and a few scattered zig-zag markings; surface of teleoconch smooth, but also often covered by low longitudinal riblets; aperture almost perfectly rounded, enormously large, old specimens with an inconspicuous whitish lip; umbilicus always completely closed.
Genital organs: penis short, club-shaped, epiphallus short, of the same length as penis, mrp attaching in the distal third of epiphallus or even closer to penis; flagellum twice the length of the epiphallus; atrial and penial lumen with numerous crests, penial chamber lumen is wrinkled, pp2 a short broad papilla with a central perforation structured by thick annuli to almost completely reduced forming a septum; pp1 a blind papilla, in a central position inside the penial chamber, elongate, sometimes with a broadened tip; epiphallial pore in a lateral position; distal epiphallial lumen with six broad pilasters, the proximal lumen with elongated ridges.
Vagina short, stem of pedunculus thickened and short, diverticulum slightly longer than the vesicle stem + vesicle, longer than the flagellum; glandulae mucosae longer than the dart sac, with a thickened basal part and two subsequent ramifications, tubules thin and weak, less than 10 tubules per stem; atrium with a medium sized stimulator flap.
Syntype
South-eastern France including Corsica, Italy, Sicily, south-eastern Adriatic coast, Albania, western Greece; scattered found introduced on some Aegean Islands, and in Turkey, Muğla, Gökçebel (
The anatomy of the genital organs of
Algeria, Kabylie: Tiguemounine (Ouacif), 1100 m alt. coll. Bouaziz; Ighil Bourmi (Ait Bouaddou), 950 m alt.
Shell medium sized to large, thin, globose with a broad to relatively acute conical spire; protoconch whitish, large, with a diameter of up to 6 mm and 2.5 smooth whorls; basic shell colour olive yellowish with up to five separate brown spiral bands; teleoconch covered by a dense granulation, sometimes accompanied by very fine, deep spirals; teleoconch usually covered by irregularly arranged riblets of even ribs, usually stronger around the umbilical area; periostracum thick, often preserved on the shell in small patches; in eroded shells, ribs and riblets whitish; aperture large, elongate oval, slightly thickened forming a lip callus, with a parietal callus in fully adult specimens; aperture whitish inside, with the spiral bands shining through the thin shell; peristome sharp; umbilicus closed, periomphalum covered by a thickened calcareous layer.
Penis short, epiphallus reaching at least three times the length of penis, mrp attaching in the distal third of epiphallus; flagellum twice the length of the epiphallus; penial lumen smooth, pp2 a short broad papilla with a central perforation structured by thick annuli, pp1 a blind papilla, the epiphallial pore in a lateral position; distal epiphallial lumen with broad pilasters, the proximal lumen with elongated ridges.
Vagina short, stem of pedunculus thickened and short, diverticulum longer than the vesicle stem + vesicle, longer than the flagellum; glandulae mucosae longer than the dart sac, with a thickened basal part and two subsequent ramifications, tubules thin and weak, less than 10 tubules per stem; atrium dominated by a massive stimulator.
Syntypes figured (n = 4): H = 26 mm; D = 27.5 mm;
As far as known, this species is restricted to the Djudjura Mts., where it inhabits quite high altitudes. It also occurs in the northern promontory of this mountain ridge.
In the description of
Algeria: Tigzirt/ Tizi Ouzou/ Kabylie,
Shell thin, medium sized, with a relatively elevated spire and a large last whorl occupying more than half of the complete height of the shell; protoconch medium-sized, consisting of 1.5 smooth whorls; teleoconch with approximately four whorls, separated by a deep, crenulated suture; colour of teleoconch brownish, surface of teleoconch covered by low longitudinal riblets, which are more prominent below the suture, disappearing on the last whorl; aperture rounded, very large, with an inconspicuous whitish lip; umbilicus always completely closed.
penis elongate, club-shaped, epiphallus as long as penis, mrp attaching in the distal third of epiphallus or even closer to penis; flagellum twice the length of the epiphallus; penial lumen smooth; pp2 a broad acute conical papilla with a central perforation structured by thick annuli; pp1 a blind papilla with a slightly broadened apex, the epiphallial pore in a lateral position; atrial and penial lumen with numerous strong crests; distal epiphallial lumen with six broad pilasters, the proximal lumen with elongated ridges.
Vagina short, stem of pedunculus thickened and short, diverticulum extremely longer than the vesicle stem + vesicle, and almost three times longer than the flagellum; glandulae mucosae longer than the dart sac, with a thickened basal part and two subsequent ramifications, tubules thin and weak, less than 10 tubules per stem; atrium dominated by a massive stimulator flap.
(of lectotype): H = 26.3 mm; D = 27.0 mm;
the two genetically identified specimens originate from Eastern Algeria.
The description of the genital organs is based on the specimen
It is almost impossible to define differences in shell morphology between this new species and
This specimen was collected by Kobelt in the Gorge d’Isser; it lacks the malleation typical for
Pictures of living specimens of
The main results of this work consist of the allocation of
The data presented here suggest the need for a more careful investigation of the phylogenetic relationships among the populations of
Neubert and Bank (2006: 105) argue that the transformation of the papilla system represents a synapomophic character state for an “
We are very grateful to Harriet Wood and Ben Rowson, Cardiff, who supplied photographs of the type specimens of