Biologia 69/6: 771—779, 2014
Section Zoology
DOI: 10.2478/s11756-014-0372-x
The Cochlostoma (Holcopoma) westerlundi group in Italy
(Caenogastropoda: Cochlostomatidae)
Mariastella Colomba1, Fabio Liberto2, Armando Gregorini1, Walter Renda3,
Agatino Reitano4 & Ignazio Sparacio5
1
Università di Urbino “Carlo Bo”, Dip. Scienze Biomolecolari, via Maggetti 22 (loc. Sasso), 61029 Urbino (PU), Italy;
e-mail: [email protected]
2
Strada Provinciale Cefalù-Gibilmanna n◦ 93, 90015 Cefalù (PA), Italy
3
via Bologna 18/a, 87032 Amantea (CS), Italy
4
Via Gravina 77, 95030 Tremestieri Etneo (CT), Italy
5
Via E. Notarbartolo 54 int. 13, 90145 Palermo, Italy
Abstract: Currently, the Cochlostoma (Holcopoma) westerlundi (Paulucci, 1879) group includes three subspecies inhabiting
southern Italy up to southeastern Sicily. C. w. westerlundi (Paulucci, 1879) is limited to southern Calabria, C. w. yapigium
(Westerlund, 1885) is widespread across the Salento (the southeastern extremity of the Apulia region) and C. w. dionysii
(Paulucci, 1879) is endemic to the environs of Siracusa (SE Sicily). There is also a fourth taxon, C. paganum (Westerlund,
1885) described for Otranto (LE, Apulia), considered a synonym of C. w. yapigium. Up to now, the molecular genetics
of C. westerlundi s.l. have been unknown and the morphological data of several populations are still lacking. Hence,
the systematic classification of the group is tentative. Aiming at filling this gap, mtDNA (16S rDNA and COI ) partial
sequences were investigated and, in addition, the reproductive apparatus of C. w. westerlundi was described for the first
time. Molecular sequences and anatomical data were used to test the taxonomic and phylogenetic status of the examined
populations. Maximum Likelihood and Bayesian analysis revealed three clusters, strongly supported, corresponding to the
three taxa. For the first time, synonymy between paganum and yapigium was confirmed by molecular evidence. Genetic
distances between groups (DxyJC ) ranged from 2.6% to 5% (16S rDNA) and from 6.3% to 8.3% (COI ). Molecular and
morphological data led us to suggest elevating the three subspecies to the species rank.
Key words: Cochlostoma westerlundi group; 16S rDNA; COI ; molecular phylogeny; taxonomic revision
Introduction
In zoological systematics, morphologically similar individuals are often grouped under the same nominal taxon. This method of individual classification
(not always practicable in terrestrial gastropods) gave
birth, over the past nearly sixty years, to several
slightly different classifications of the Cochlostoma
westerlundi s.l. morpho-types. Currently, the C. westerlundi group comprises four nominal taxa, three of
which are considered subspecies: C. w. westerlundi
(Paulucci, 1879), inhabiting very restricted areas of
southern Calabria; C. w. yapigium (Westerlund, 1885),
which is widespread in southern Apulia; and C. w.
dionysii (Paulucci, 1879), endemic to southeastern
Sicily (Fig. 1). The fourth taxon, C. paganum (Westerlund, 1885) described for Otranto (LE, Apulia), is
considered a synonym of C. w. yapigium (Forcart 1965;
Pintér & Szigethy 1976; Bodon et al. 1995; Ferreri et al.
2005; Bank 2012). As far as concerns a brief overview of
the Cochlostoma Jan, 1830 system (at least that of the
Italian species), Sacchi (1954) accounted yapigium as
distinct from westerlundi while Alzona (1971) reported
c 2014 Institute of Zoology, Slovak Academy of Sciences
westerlundi, yapigium and dionysii as different species,
and paganum as a subspecies.
Comparative anatomical data on the shell and genitalia are extremely limited. In males of C. w. yapigium,
the penis is simple, gradually tapering at the end; in
females, the sperm receptacle is absent (personal data;
see also Ferreri et al. 2005). In C. w. dionysii, the penis
is more or less cylindrical and narrows abruptly at the
end (Reitano et al. 2009), whereas nothing is known
about the female reproductive system. Finally, the genital anatomy of C. w. westerlundi has never been studied. Cochlostoma westerlundi s.l. prefers limestone environments and possesses very low active dispersal ability
(due to limited vagility), which is likely to have drastically reduced the gene flow, thus suggesting strong
isolation and (presumably) divergence among populations.
Recently, the combination of genetic data and
morphological characters (i.e., shell sculpture, genital
anatomy) has proven useful in unraveling taxonomic
issues in other groups of terrestrial mollusks (Uit De
Weerd & Gittenberger 2004; Liew et al. 2009; Colomba
et al. 2011). We herein applied a similar approach to
Unauthenticated
Download Date | 6/19/17 12:37 AM
772
M.S. Colomba et al.
Fig. 1. Geographic distribution of taxa ascribed to the Cochlostoma westerlundi group. • – yapigium;
– westerlundi; – dionysii;
◦ – C. paganum; – new records for dionysii: Priolo, Palazzolo Acreide, Eloro and contrada Palombara (Siracusa province, SR, Sicily).
solve the systematics of C. westerlundi s.l. and provide
the first comprehensive molecular phylogenetic study
of the taxon group by means of partial sequences of
two mitochondrial genes [16S rRNA and cytochrome
oxidase c subunit I (COI )] analyzed by Bayesian Inference and Maximum Likelihood algorithms. Finally,
we suggest a paleogeographic scenario which might account for the current spatial distribution and evolutionary history of the organisms under study.
Material and methods
Specimens
Two hundred and fifty-eight (258) specimens were analyzed
for both morphological and biometrical key features of shells
and animals. Individuals were drowned and fixed in 70–
90% ethanol. The reproductive apparatus was extracted by
means of a scalpel, scissors and forceps. Photographs were
taken with a digital camera. The maximum height and maximum diameter of the shell along with some parts of genitalia were measured (in mm) using digital calipers. Illustrations of genitalia were sketched using a camera lucida.
Voucher specimens were deposited at the I. Sparacio collection (CS), F. Liberto collection (CL) and W. Renda collection (CR). Twenty-one additional specimens including six
C. w. yapigium, three C. paganum, six C. w. dionysii and
six C. w. westerlundi were employed for the molecular characterization based on multigenic sequence analysis.
Examined material
C. w. yapigium
Lecce, Nardò, masserie, 40◦ 08 14 N, 18◦ 01 16 E, 60 m,
19.III.2009, leg. Renda W., 7 specimens, (CL); Lecce, Nardò,
Porto Selvaggio, 09.XI.2009, leg. Ferreri D. & Renda W., 7
specimens, (CL); Lecce, Lizzanello, 08.XI.2009, leg. Ferreri
D., 5 specimens, (CL); Lecce, Bosco La Lizza, 08.XI.2009,
leg. Ferreri D., 7 specimens, (CL); Otranto, Valle dell’Idro,
08.XI.2009, leg. Ferreri D., 6 specimens, (CL); Gallipoli,
Pineta, IV.1980, leg. Pirozzi N., 5 specimens (CS); Lecce,
Acaya, 4.V.1993, 11 specimens (CS); Lecce, Bosco La Lizza,
08.XI.2009, leg. Ferreri D., 2 specimens employed for molecular analyses, labeled as CYBL; Lecce, Nardò, Portoselvaggio, 09.XI.2009, leg. Ferreri D. & Renda W., 2 specimens
employed for molecular analyses, labeled as CYNA; Lecce,
Unauthenticated
Download Date | 6/19/17 12:37 AM
Cochlostoma (Holcopoma) westerlundi in Italy
773
Table 1. Origin of dissected specimens and data on mtDNA partial sequences generated in the present study.
Taxon
Collection site
Region/Country
dionysii
westerlundi
yapigium
yapigium
yapigium
paganum = yapigium
dionysii
westerlundi
yapigium
yapigium
paganum = yapigium
yapigium
roseoli
Siracusa (SR)
Mt. Consolino (RC)
Lecce (LE)
Nardò (LE)
Bosco La Lizza (LE)
Otranto (LE)
Siracusa (SR)
Mt. Consolino (RC)
Lecce (LE)
Nardò (LE)
Otranto (LE)
Bosco La Lizza (LE)
Moraca valley
Sicily/Italy
Calabria/Italy
Apulia/Italy
Apulia/Italy
Apulia/Italy
Apulia/Italy
Sicily/Italy
Calabria/Italy
Apulia/Italy
Apulia/Italy
Apulia/Italy
Apulia/Italy
Montenegro
loc. CAT, 08.XI.2009, leg. Ferreri D., 2 specimens employed
for molecular analyses, labeled as CYLE;
C. paganum
Valle dell’Idro, Otranto, 08.XI.2009, leg. Ferreri D., 3 specimens employed for molecular analyses, labeled as CYOTR.
C. w. dionysii
Noto, Noto Antica, 20.VIII.1993, legit Sparacio I., 7 specimens (CS); idem, 24.IV.2005, legit Brancato A., 3 specimens (CL); idem, 26.VI.2005, leg. Brancato A., 13 specimens (CL); Melilli, R.N.I. “Grotta Palombara” 37◦ 06 22
N, 15◦ 11 45 E, 130 m., 16.V.2009, leg. Liberto F., 8 specimens, (CL); Priolo Gargallo, Cava Sorciaro, 37◦ 09 49 N,
15◦ 09 24 E, 95 m, 09.V.2010, leg. Liberto F., 7 specimens (CL); idem, leg. Sparacio I., 12 specimens, (CS);
idem, 28.XI.2010, leg. Sparacio I., 6 specimens (CS);
Melilli, Sorgente Belluzza, 37◦ 13 16 N, 15◦ 06 21 E,
104 m, 21.XI.2010, leg. Liberto F., 10 specimens (CL);
Melilli, Santa Caterina, 37◦ 12 06 N, 15◦ 06 12 E, 200 m,
21.XI.2010, leg. Liberto F., 7 specimens, (CL); Palazzolo Acreide, Pianetti, 37◦ 02 14 N, 14◦ 58 37 E, 500 m,
05.III.2011, leg. Sparacio I., 28 specimens (CS); idem, leg.
Sparacio I., 8 specimens (CL); Siracusa, Santa Lucia, 50 m,
VIII.2011, leg. Brancato A., 5 specimens (CL); Siracusa,
Belvedere, Passo Piano, 37◦ 06 00 N, 15◦ 12 04 E; 130 m,
VIII.2011, leg. Brancato A., 3 specimens (CL); Siracusa, nei
pressi dell’Orecchio di Dionisio, 37◦ 04 36 N, 15◦ 16 33 E,
30 m, VIII.2011, leg. Brancato A., 6 specimens (CL); Siracusa, pressi del Teatro antico, leg. Sparacio I., 10.IX.1982, 4
specimens (CS); idem, leg. Sparacio I., 28.XI.2010, 8 specimens (CS); Sortino, Necropoli di Pantalica, 37◦ 08 28 N,
15◦ 01 51 E, 250 m, XI.2009, leg. Reitano A., 10 specimens
(CL); Noto, Eloro, 18.VIII.1993, leg. Sparacio I., 2 specimens (CS); Siracusa, Spinagallo, 37◦ 00 11 N, 15◦ 10 50 E,
119 m, 01.IV.2012, leg. Reitano A., 15 specimens (CL); Siracusa, Belvedere Nord, 17.III.2009, leg. Renda W., 6 specimens employed for molecular analyses, labeled as CDSR.
C. w. westerlundi
Pazzano, Monte Stella, 23.X.2009, leg. Renda W., 5 specimens (CR); idem, 09.V.2010, leg. Renda W., 2 specimens
(CR); Stilo, Monte Consolino, 38◦ 28 37 N, 16◦ 27 53 E,
550 m, 03.VII.2008, leg. Renda W., 12 specimens (CR);
idem, 14.III.2009, leg. Renda W., 3 specimens (CR); idem,
14.III.2009, leg. Renda W., 5 specimens, (CL); idem,
14.III.2009, leg. Renda W., 6 specimens employed for molecular analyses, labeled as CW.
mtDNA sequence
Length (bp)
Isolate
GenBank ID
16S
16S
16S
16S
16S
16S
COI
COI
COI
COI
COI
COI
COI
436
447
438
438
438
438
645
654
647
647
647
647
638
CDSR
CW
CYLE
CYNA
CYBL
CYOTR
CDSR
CW
CYLE
CYNA
CYOTR
CYBL
HNHM 95594
JN030448
JN030449
JN030450
KC609730
KC609732
JN162680
JN030453
JN030452
JN030451
KC609731
JN162681
KC609733
KF812521
DNA extraction, amplification and sequencing
Dissected specimens originated from the following populations/collection sites (Table 1); C. w. yapigium: Lecce (LE),
Nardò, Portoselvaggio (LE), and Bosco La Lizza (LE), Apulia; C. paganum: Otranto (LE), Apulia; C. w. dionysii Siracusa (SR), Sicily; C. w. westerlundi: Monte Consolino, Stilo
(RC), Calabria. Samples were stored at –20 ◦C in test tubes.
The entire specimen was used for total DNA extraction (by
Wizard Genomic DNA Purification Kit, Promega). For each
population/collection site, para-voucher specimens – sensu
Groenenberg et al. (2011), i.e., different specimens than the
ones used for DNA analysis, but from the same sample or
population/collection site – were stored at the University of
Urbino.
COI amplicons (645–654 bp) were obtained by the
primers LCO 1490 (5’-GGTCAACAAATCATAAAGATA
TTGG-3’) and HCO 2198 (5’-TAAACTTCAGGGTGACC
AAAAAATCA-3’) according to Folmer et al. (1994); 16S
rDNA fragments (436–447 bp) were amplified by the universal forward primer (5’-CCCGCCTGTTTACCAAAAACAT3’) reported in Mitani et al. (2009) and the reverse
primer (5’-TATCTCAATCCAACATCGAGG-3’) (designed
herein). PCR reactions were conducted as follows: for 16S,
95 ◦C for 5 min; 95 ◦C for 1 min, 55 ◦C for 1 min, 72 ◦C for
1 min (35 cycles); 72 ◦C for 5 min; for COI, 95 ◦C for 5 min;
95 ◦C for 1 min, 42 ◦C for 1 min, 72 ◦C for 1 min (37 cycles);
72 ◦C for 10 min. To remove primers and unincorporated
nucleotides, amplification products were purified using the
Wizard SV gel and PCR Clean-up kit (Promega). Sequencing of the purified PCR products was carried out using automated DNA sequencers at Eurofins MWG Operon (Germany). Chromatograms of each amplified fragment were visually inspected for reading mistakes by the sequencer. For
each collection site, the generated sequences were deposited
in GenBank (Table 1).
Phylogenetic analyses
All sequences were visualized with BioEdit Sequence Alignment Editor 7 (Hall 1999), aligned with the ClustalW option included in this software and refined by eye. For both
datasets, the strict molecular clock hypothesis could not be
rejected (P < 0.99, tested by MEGA 5.0.3). Phylogenetic
analyses were conducted in BEAST 1.6.1 (Drummond &
Rambaut 2007) using the *BEAST implementation (Heled
& Drummond 2010). A series of initial runs was performed
to optimize priors and runtime parameter choices to obtain
effective sampling sizes (ESS) above 500 for all estimated
parameters. Parameter estimates were gained from combined log files. The best-fit evolution model of nucleotide
Unauthenticated
Download Date | 6/19/17 12:37 AM
774
M.S. Colomba et al.
Fig. 2. Maximum Likelihood consensus tree obtained by the combined analysis of 16S rDNA and COI datasets (substitution method:
HKY+G, G = 0.1627). All positions containing gaps and missing data were eliminated; the tree is drawn to scale, with branch lengths
measured in the number of substitutions per site. Numbers above branches represent bootstrap values (after 1000 replicates) and
Bayesian posterior probabilities (in parentheses). A timeline in million years before present for the C. westerlundi complex evolution
performed by MEGA is reported below the tree.
substitution resulted in HKY with empirical base composition; the Yule Process tree prior for mitochondrial data
with piecewise linear population size model was applied with
a UPGMA-generated tree as the starting point. Five single runs were combined with the LogCombiner 1.6.1 implemented in the software package BEAST. Trees from all
runs were combined to produce an ultrametric consensus
tree using TreeAnnotator 1.6.1. The first 2 × 106 trees were
discarded as burnin. Support for nodes is expressed as posterior probabilities.
Moreover, the Maximum Likelihood (ML) method was
performed by MEGA 5.0.3 (Tamura et al. 2011); according to the “Find Best DNA Models (ML)” option avail-
able in this software, HKY + G resulted the best-fit
substitution model. Bootstrap values were assessed after
1000 replicates. C. patulum (Draparnaud, 1801), C. elegans (Clessin, 1879), C. septemspirale (Razoumowsky, 1789)
and C. roseoli (A.J. Wagner, 1901) (see also Webster et
al. 2012) were used as out-groups to root phylogenetic
trees. In this regard, please note that the C. roseoli COI
sequence (uploaded by us, GenBank ID: KF812521, Table 1) is from the same population as the C. roseoli 16S
sequence in Webster et al. (2012). Both Bayesian and
Maximum Likelihood analyses were performed either with
single (16S or COI ) or combined (16S-COI ) molecular
datasets.
Unauthenticated
Download Date | 6/19/17 12:37 AM
Cochlostoma (Holcopoma) westerlundi in Italy
775
Fig. 3. Shells of Cochlostoma w. yapigium from Nardò, Portoselvaggio (LE, Apulia) (A); C. w. dionysii from Siracusa (SR, Sicily)
(B); C. w. westerlundi from Monte Consolino (Stilo, RC, Calabria) (C).
Results
Molecular data
Three COI haplotypes, restricted to single geographic
areas, Sicily, Calabria and Apulia, were recorded. The
same was done for the 16S rDNA sequences, including three haplotypes each of which was unique for
one of the three Italian regions. Both Bayesian and
ML analyses resulted in phylogenetic trees, the topologies of which were largely consistent, showing three
clades with a strong branch support. Figure 2 illustrates the ML consensus tree obtained by the combined analysis of 16S rDNA and COI datasets. Three
distinct clusters are clearly evident. The first one includes C. w. yapigium and C. paganum specimens
(CYLE1-2, CYNA1-2, CYBL1-2 e CYOTR1-3); the
second includes C. w. dionysii specimens (CDSR1-6)
and, finally, the third includes C. w. westerlundi specimens (CW1-6). It is interesting to note that, in the
first cluster, C. w. yapigium and C. paganum specimens are intermixed. In addition, by comparison of
the mtDNA nucleotide sequences, it was found that
16S amplicons of C. w. yapigium were identical to
those of C. paganum and the same was found for COI,
which, for the first time, confirmed by molecular evidence the synonymy between the two nominal taxa
Unauthenticated
Download Date | 6/19/17 12:37 AM
776
(hereafter not further distinguished and indicated as
“yapigium”).
Divergence among groups (DxyJC ) ranged from
2.6% to 5% (16S rDNA) and from 6.3% to 8.3% (COI ).
By entering a standard COI rate estimate of ca. 1% per
million years per site (see Donald et al. 2005) into the
“calibrating MolClock” option included in MEGA software, it was determined that the C. westerlundi group
shared an exclusive common ancestor up to 5.01 million years ago (Mya), when C. westerlundi s. str. diverged from the yapigium-dionysii ancestor. Under the
same model, it was further estimated that yapigium and
dionysii were separated by about 2.57 Mya. Finally, the
most recent common ancestor (TMRCA) of C. westerlundi group and C. roseoli, both belonging to the subgenus Holcopoma Kobelt et Moellendorf, 1899, might
be dated back to about 7.14 Mya.
Morphological data
In this study we describe, for the first time, the reproductive system of C. (H.) w. westerlundi specimens
from Calabria, although, because of its restricted distribution area and relatively small populations, it was
possible to examine only five individuals (one male and
four females).
Cochlostoma w. yapigium
Shell (Fig. 3A). Protoconch smooth, with no ribs.
Teleoconch conical, ribbed, with rounded, regularly increasing whorls and deep sutures. Base of the last whorl
well-rounded. Shell yellowish with small brown-reddish
spots arranged in interrupted bands. Ribs oblique, condensed and regularly spaced, slightly prominent only
at the end of the last whorl. Last whorl slightly rising towards the aperture. Aperture rounded more or
less oblique with a well-developed lip. Columellar lobe
well-developed and abruptly curved, covering umbilicus. Shell wider and more elongated in females. Males:
shell height 7.24–8.10 mm, diameter 3.90–4.00 mm; females: height 7.52–9.02 mm, diameter 4.19–4.43 mm.
Male genitalia (Fig. 4A). Simple penis, cylinderconic, gradually tapering to the end with a long, thin
apex and internal penial ductus.
Female genitalia (Fig. 4B). Sperm receptacle absent. Bursa copulatrix medium-sized with a short and
narrow duct originating near the proximal portion.
Cochlostoma w. dionysii
Shell (Fig. 3B). Protoconch smooth, with no ribs. Teleoconch conical, prominently ribbed with seven rounded
whorls and deep sutures. Base of the last whorl wide
and rounded. Ribs oblique, prominent, close, regular
in space, becoming thicker and slightly prominent only
at the end of the last whorl. Last whorl slightly rising towards the aperture. Aperture rounded with a
well-developed lip. Columellar lobe well-developed and
abruptly curved, concealing umbilicus. Shell wider and
more elongated in females. Males: shell height 8.40–
8.94 mm, diameter 4.40–4.70 mm; females: height 8.62–
9.65 mm, diameter 4.30–4.90 mm.
M.S. Colomba et al.
Male genitalia (Fig. 4C). Simple penis, more or
less cylindrical and narrowing abruptly at the end; thin
apex, shorter than in yapigium.
Female genitalia (Fig. 4D). Sperm receptacle absent. Bursa copulatrix medium-sized with a short
(shorter than in yapigium) and narrow duct originating near the proximal portion.
Cochlostoma w. westerlundi
Shell (Fig. 3C). Protoconch without any ribbing. Teleoconch conical, elongated, densely ribbed, with rounded
whorls and deep sutures. Base slightly angled and relatively narrower than in yapigium and dionysii. Shell
yellowish-brown with small reddish marks arranged in
three interrupted bands. Ribs on first whorls slightly
oblique, prominent, widely spaced, thin, becoming indistinct towards the aperture. Last whorls with five to
eight thin, parallel, spiral ribs. Last whorl slightly rising towards the aperture. Aperture rounded, with duplicated broadly-reflected peristome only at the basal
and columellar side, inner lip well-developed and separated from the outer lip by a furrow. Columellar lobe
well-developed and abruptly curved, covering the umbilicus. Distance between base of the last whorl and
the upper edge of the columellar lobe wider than in
yapigium and dionysii. Shell wider and more elongated
in females. Males: shell height 7.50–9.60 mm, diameter
2.50–3.85 mm; females: height 10.00–11.00 mm, diameter 4.00–5.00 mm.
Male genitalia (Fig. 4E). Penis simple, robust,
broad, rough on the inner edge; apex short and bent
backwards.
Female genitalia (Fig. 4F). Sperm receptacle absent. Bursa copulatrix wide with a long duct (twice as
long as in dionysii and yapigium).
Comparative notes
The main differences between yapigium and dionysii
relate to: (i) rib count (ribs are fewer and more widely
spaced in dionysii); (ii) size and morphology of the last
whorl of the shell (wider and more rounded in dionysii);
(iii) penial apex (less elongated in dionysii); (iv) length
of the bursa duct (slightly shorter in dionysii).
Compared to yapigium-dionysii, westerlundi is
characterized by (i) an angled shell base (rounded in
yapigium and dionysii); (ii) the first whorls showing
spaced ribs becoming thinner and more widely spaced
in the last whorls (regular in yapigium and dionysii);
(iii) the final whorl showing blurred lines at the opening (contrary to dionysii and yapigium where the final
whorl is ribbed) and some parallel, thin, spiral lines
(absent in yapigium and dionysii); (iv) final portion of
the last whorl slightly ascending (markedly ascending
in yapigium and dionysii); (v) the edge of the peristome
poorly developed, with the exception of the columellar
part (entirely well developed in dionysii and yapigium);
(vi) a big distance between the columella and peristome, much greater than in dionysii and yapigium; (vii)
a more robust penis, ending with a stocky apex (thin
apex in yapigium and dionysii); and finally, (viii) feUnauthenticated
Download Date | 6/19/17 12:37 AM
Cochlostoma (Holcopoma) westerlundi in Italy
777
Fig. 4. Genitalia of Cochlostoma w. yapigium male (A) and female (B) from Nardò, Portoselvaggio (LE, Apulia); C. w. dionysii male
(C) and female (D) from Siracusa (SR, Sicily); and C. w. westerlundi male (E) and female (F) from Monte Consolino (Stilo, RC,
Calabria).
males showing a long channel of the bursa copulatrix
(much longer than in yapigium and dionysii).
Discussion
The present study provides original data accumulated
by this research team during the last few years on
C. westerlundi s.l. from Italy, a very interesting and
morphologically conservative group ascribed to the subgenus Holcopoma showing very low active dispersal
ability and a patchy scattered geographic distribution.
Main results include: (i) characters from morphological structures (i.e., shell and genital anatomy) show
some differences among the three taxa (C. w. yapigium,
C. w. dionysii and C. w. westerlundi); and (ii) molecular phylogenetic analysis revealed three clusters exactly
corresponding to the three subspecies. Taking into account the phenotypic features, nucleotide divergences
and topology of the phylogenetic trees, we strongly suggest elevating these three subspecies to the higher rank.
Hence, in the remainder of this paper – in line with Alzona (1971) – these three taxa will be referred to as
species.
In many animal groups, including Gastropoda,
species-level identification based on molecular markers is still contentious due to two main factors: (i) genetic distances are often expressed in different units,
which makes them difficult to compare and (ii) there
Unauthenticated
Download Date | 6/19/17 12:37 AM
778
is no constant or universal molecular clock. There is
no simple, “routine” way of interpretation of genetic
distances while assessing species distinctness. In fact,
distance values are applicable only within a group of
closely related species, and we are not aware of such
data for the Cochlostomatidae. Nevertheless, within
the C. westerlundi group, nucleotide divergences are
in line with genetic distances reported as discriminating among a few Caenogastropoda species. For example, 16S rDNA distances ≥0.03% were reported for several Ovulidae (Schiaparelli et al. 2005); 1.3% between
Kelletia kelletii and K. lischkei and 1.5% between Penion chathamensis and P. sulcatus (Hayashi 2005); less
than 4–5% for Conus species (Duda et al. 2009); and
ranging from 0.30% to 12.40% between specimens of
congeneric neogastropod species (Zou et al. 2011). For
COI distances, values of at least 3% were reported for
Hydrobia (Wilke et al. 2000), ranging from 0.3% to
26.6% in Trochidae (Donald et al. 2005); ≥1.5% in Bythinella (Bichain et al. 2007); from 2.1% to 19.8% in
40 Neogastropoda species (Zou et al. 2011). Therefore,
genetic differences among the three taxa under study
(DxyJC, 2.6–5% for 16S rDNA; 6.3–8.3% for COI ) may
be considered as the result of a certain degree of isolation and divergence, i.e., the very low active dispersal
ability of these animals is likely to have drastically reduced the gene flow and given rise to three genetically
independent species. All these findings led us to suggest that the three clusters, supported by strong bootstrap values and very high posterior probability, represent three phylogenetic species which we refer to as
C. westerlundi, C. yapigium and C. dionysii. Regarding
the morphological characters, the shell phenotype and
genital architecture in C. yapigium and C. dionysii are
rather conservative. In fact, these two taxa inhabiting
geographically well separated areas (Apulia and Sicily)
have maintained quite homogeneous features. On the
contrary, C. westerlundi s. str. appears to be quite different. Such an evident differentiation might be justified
by a longer evolutionary history (it was the first one to
split out from the group) and geographical and ecological isolation of the taxon (small populations occurring
in restricted mountainous areas of southern Calabria).
A possible paleogeographic reconstruction of the
history of the C. westerlundi group dates back to the
very end of the Miocene, in the Messinian (7.2–5.3
million years ago), when a series of tectonic movements caused the closure of the connections between the
Mediterranean and the Atlantic Ocean. The Mediterranean became a huge lake and gradually began to dry
up; many lands emerged from the waters and new connections between areas previously isolated by the sea
were settled. Divergence times reported herein would
be in line with La Greca (1967), who hypothesized
the ancestor of the Cochlostoma (Holcopoma) as one
of the numerous groups of terrestrial mollusks which,
at the beginning of the Messinian period, expanded
from the Balkan peninsula to southern Italy up to Sicily
thanks to the partial desiccation of the Mediterranean
(see also Sacchi 1954; Thake 1985, Giusti et al.1995;
M.S. Colomba et al.
Colomba et al. 2010). According to our results, the
separation between C. roseoli and C. westerlundi s.l.
might be roughly dated back to about 7.14 million
years ago. Then, in our opinion, the banishing of a relatively small group of specimens in isolated mountainous areas – combined with the low active dispersion
capacity of these calciphilous organisms – reduced the
gene flow within the original population, thus originating C. westerlundi s. str. which separated from the rest
of the group about 5.01 Mya. During Pliocene, the reopening of the Strait of Gibraltar caused the Mediterranean Sea level to rise and the formerly associated territories became separated, creating overall conditions
of greater insularity. In the late Pliocene (about 2.57
Mya), these populations, already fragmented, diverged
into two separate taxa: C. yapigium and C. dionysii. Alternatively, Girod & Sacchi (1967) suggested a passive
introduction of C. dionysii by humans in Sicily since it
was known only for the archaeological area of Siracusa.
However, C. dionysii is fairly widespread throughout
the Iblei mountains, even in areas not interested by human settlements (see Fig. 1). Hence, in our opinion, it
is to be considered a native species.
As far as concerns the ecology of these organisms,
Cochlostoma westerlundi s.l. are rupicolous and calciphilous; C. yapigium show a marked preference for
sunny habitats and are widespread across the Salento.
C. dionysii prefer shady areas and often take refuge in
rocky cracks. Occasionally, they occur in other types
of environment; for example, we found several specimens in the bark of poplars (Populus sp.) and willows
(Salix sp.) along river banks. They are also widespread
on the eastern side of the Iblei Mts. (SE Sicily) from
the first outcrops of limestone a few meters above sea
level (Eloro Cape, SR) up to medium altitudes (670 m
a.s.l., Palazzolo Acreide, SR). C. westerlundi are usually found both in shady areas and sunny rocks. They
are extremely localized, with populations showing very
low numerical density, which makes this species vulnerable to extinction and, therefore, worthy of attention
and more protection.
In conclusion, we hope that the present findings
may (i) shed some light on several aspects, never
focused on before, of the origin and evolution of
Cochlostoma (Holcopoma) westerlundi s.l. and (ii) provide new data to support a taxonomic revision of the
group by elevating the three subspecies to the species
rank.
Acknowledgements
The authors wish to thank D. Ferreri (Lecce, Italy), A.
Brancato (Siracusa, Italy) and A. Corso (Siracusa, Italy)
for contributing to specimen collection in the field and S.
Giglio (Cefalù, Italy) for taking pictures.
References
Alzona C. 1971. Malacofauna Italica. Catalogo e bibliografia dei
molluschi viventi, terrestri e d’acqua dolce. Atti della Società
Unauthenticated
Download Date | 6/19/17 12:37 AM
Cochlostoma (Holcopoma) westerlundi in Italy
Italiana di Scienze Naturali e del Museo Civico di Storia Naturale di Milano 111: 1–433.
Bank R.A. 2012. Fauna Europaea: Gastropoda, Cochlostomidae.
Fauna Europaea version 1.1. http://www.faunaeur.org (accessed 06.07.2012)
Bichain J.-M., Gaubert P., Samadi S. & Boisselier-Dubayle
M.C. 2007. A gleam in the dark: Phylogenetic species delimitation in the confusing spring-snail genus Bythinella MoquinTandon, 1856 (Gastropoda: Rissooidea: Amnicolidae). Mol.
Phylogen. Evol. 45 (3): 927–941. DOI: 10.1016/j.ympev.
2007.07.018
Bodon M., Favilli L., Giannuzzi Savelli R., Giovine F., Giusti
F., Manganelli G., Melone G., Oliverio M., Sabelli B. &
Spada G. 1995. Gastropoda Prosobranchia, Heterobranchia
Heterostropha. In: Minelli A., Ruffo S. & La Posta S.
(eds), Checklist delle specie della fauna italiana, Fascicola14,
Edizioni Calderini, Bologna, 60 pp. http://www.comitato.
faunaitalia.it/docs/F14.DOC (accessed 10.07.2013)
Colomba M.S., Gregorini A., Liberto F., Reitano A., Giglio S. &
Sparacio I. 2010. Molecular analysis of Muticaria syracusana
and M. neuteboomi from Southeastern Sicily (Gastropoda,
Pulmonata, Clausiliidae). Biodivers. J. 1 (1-4): 7–14.
Colomba M.S., Gregorini A., Liberto F., Reitano A., Giglio S. &
Sparacio I. 2011. Monographic revision of the endemic Helix
mazzullii (De Cristofori & Jan, 1832) complex from Sicily and
re-introduction of the genus Erctella Monterosato, 1894 (Pulmonata, Stylommatophora, Helicidae). Zootaxa 3134: 1–42.
Donald K.M., Kennedy M. & Spencer G.S. 2005. Cladogenesis
as the result of long-distance rafting events in South Pacific
Topshells (Gastropoda, Trochidae). Evolution 59 (8): 1701–
1711. DOI: 10.1554/04-553.1
Drummond A.J. & Rambaut A. 2007. BEAST: Bayesian evolutionary analysis by sampling trees. BMC Evol. Biol. 7: 214–
257. DOI: 10.1186/1471-2148-7-214
Duda T.F.Jr, Kohn A.J. & Matheny A.M. 2009. Cryptic species
differentiated in Conus ebraeus, a widespread tropical marine
gastropod. Biol. Bull. 217 (3): 292–305. PMID: 20040753
Ferreri D., Bodon M. & Manganelli G. 2005. Molluschi terrestri
della provincia di Lecce. Thalassia Salentina 28: 31–130. DOI:
10.1285/i15910725v28p31
Folmer O., Black M., Hoeh W., Lutz R. & Vrijenhoek R. 1994.
DNA primers for amplification of mitochondrial cytochrome
c oxidase subunit I from diverse metazoan invertebrates. Mol.
Mar. Biol. Biotechnol. 3 (5): 294–299. PMID: 7881515
Forcart L. 1965. Rezente Land- und Süsswassermollusken der
süditalienischen Landschaften Apulien, Basilicata und Calabrien. Verh. Naturforsch. Gesell. Basel 76: 59–184.
Girod A. & Sacchi C.F. 1967. Considerazioni biogeografiche sulla
malacofauna pugliese. Atti della Società Italiana di Scienze
Naturali e del Museo Civico di Storia Naturale di Milano 106
(4): 258–274.
Giusti F., Manganelli G. & Schembri P.J. 1995. The non-marine
molluscs of the Maltese Islands. Monografie Museo Regionale
Scienze Naturali Torino 15: 1–607. ISBN: 9788886041249
Groenenberg D.S.J., Neubert E. & Gittemberg E. 2011. Reappraisal of the “Molecular phylogeny of Western Palaeartic
Helicidae s.l. (Gastropoda: Stylommatophora)”: When poor
science meets GenBank. Mol. Phylogen. Evol. 61(3): 914–
923. DOI: 10.1016/j.ympev.2011.08.024.
Hall T.A. 1999. BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT.
Nucleic Acids Symposium Series 41: 95–98.
779
Hayashi S. 2005. The molecular phylogeny of the Buccinidae
(Caenogastropoda: Neogastropoda) as inferred from the complete mitochondrial 16S rRNA gene sequences of selected representatives. Mollusc. Res. 25 (2): 85–98.
Heled J. & Drummond A.J. 2010. Bayesian inference of species
trees from multilocus data. Mol. Biol. Evol. 27 (3): 570–580.
DOI: 10.1093/molbev/msp274
La Greca M. 1967. Considerazioni sulla origine del popolamento
faunistico pugliese. Archivio Botanico e Biogeografico Italiano 43: 297–320.
Liew T.-S., Schilthuizen M. & Vermeulen J.J. 2009. Systematic revision of the genus Everettia Godwin-Austen, 1891
(Mollusca: Gastropoda: Dyakiidae) in Sabah, northern Borneo. Zool. J. Linn. Soc. London 157 (3): 515–550. DOI:
10.1111/j.1096-3642.2009.00526.x
Mitani T., Akane A., Tokiyasu T., Yoshimura S., Okii Y. &
Yoshida M. 2009. Identification of animal species using the
partial sequences in the mitochondrial 16S rDNA gene. Legal
Medicine 1 (Suppl 1): S449–450. DOI: 10.1016/j.legalmed.
2009.02.002
Pintér L. & Szigethy A.S. 1976. Schnecken aus Sizilien [Szicíliai
csigák]. Soosiana 4: 27–38.
Reitano A., Liberto F., Sparacio I. & Giglio S. 2009. I molluschi terrestri della R.N.I. “Grotta Palombara” (Melilli, Sicilia sud-orientale) (Gastropoda Architaenioglossa, Neotaenioglossa, Stylommatophora). Il Naturalista siciliano S. IV,
33 (1-2): 177–205.
Sacchi C.F. 1954. Note di malacologia terrestre pugliese. Boll.
Zool. 21(1): 51–76. DOI: 10.1080/11250005409439190
Schiaparelli S., Barucca M., Olmo E., Boyer M. & Canapa
A. 2005. Phylogenetic relationships within Ovulidae (Gastropoda: Cypraeoidea) based on molecular data from the
16S rRNA gene. Mar. Biol. 147 (2): 411–420. DOI:
10.1007/s00227-005-1566-0
Tamura K., Peterson D., Peterson N., Stecher G., Nei M. & Kumar S. 2011. MEGA5: molecular evolutionary genetics analysis using Maximum Likelihood, Evolutionary Distance, and
Maximum Parsimony methods. Mol. Biol. Evol. 28 (10):
2731–2739. DOI: 10.1093/molbev/msr121
Thake M.A. 1985. The biogeography of the Maltese islands, illustrated by the Clausiliidae. J. Biogeogr. 12 (3): 269–287.
Uit De Weerd D.R. & Gittenberger E. 2004. Re-evaluating
Carinigera: molecular data overturn the current classification within the Clausiliid subfamily Alopiinae (Gastropoda,
Pulmonata). J. Mollusc. Stud. 70 (4): 305–318. DOI:
10.1093/mollus/70.4.305
Webster N.B., Van Dooren T.J. & Schilthuizen M. 2012. Phylogenetic reconstruction and shell evolution of the Diplommatinidae (Gastropoda: Caenogastropoda). Mol. Phylogen.
Evol. 63 (3): 625–638. DOI: 10.1016/j.ympev.2012.02.004
Wilke T., Rolán E. & Davis G.M. 2000. The mudsnail genus
Hydrobia s.s. in the northern Atlantic and western Mediterranean: a phylogenetic hypothesis. Mar. Biol. 137 (5-6):
827–833. DOI: 10.1007/s002270000407
Zou S., Li Q., Kong L., Yu H. & Zheng X. 2011. Comparing
the usefulness of distance, monophyly and character-based
DNA barcoding methods in species identification: A case
study of Neogastropoda. PLoS One 6 (10): e26619. DOI:
10.1371/journal.pone.0026619.
Received August 18, 2013
Accepted February 10, 2014
Unauthenticated
Download Date | 6/19/17 12:37 AM