Page 5 - Colomba-et-al-2011-Erctella-mazzullii-complex-Zootaxa-3134

In a previous and preliminary study (Colomba et al. 2008), mainly based on anatomical data and molecular
analysis of partial mitochondrial 16S rRNA gene sequences, we suggested that Helix mazzullii (therein reported as
Cornu mazzullii) might indeed constitute a complex (i.e., the mazzullii group) structured in three discrete taxa
clearly defined by distribution, morphology and molecular evidence, corresponding to (i) the populations living in
Monte Pellegrino (Palermo) and nearby mountains (taxon mazzullii s. str.) (Figs. 2 and 5); (ii) the endemic popula-
tion of Cefalù, La Rocca (Palermo) (taxon cephalaeditana) (Figs. 3 and 6); and (iii) the populations living around
Trapani (taxon insolida) (Figs. 4 and 7). These findings highlighted a controversy about the taxonomy of the maz-
zullii group with two conflicting hypotheses, the one-species hypothesis versus the three-(sub)species hypothesis.

In order to be able to test the two hypotheses we present a multidisciplinary perspective on distribution, ecol-
ogy, shell and genital morphology, DNA and fossil record of these nominal taxa. In addition, we 1) better define the
still poorly known distribution areas and the eco-biological features of the mazzullii group from Sicily; 2) provide
detailed morphological descriptions of shells and genitalia; 3) perform a molecular analysis of this group which has
never before been investigated from this point of view; and 4) unravel the phylogenetic relationships of its pre-
sumed taxa. To this end, sampling was carried out throughout northwestern Sicily, including many localities never
reported before as collection sites for the mazzullii group. Our data were combined with paleontological records,
bibliographic references and records from museum collections (Appendix 1). Observations on the animals’ biology
were made directly in the field. For each taxon, morphological features were illustrated in detail.

Two mitochondrial DNA markers (16S rDNA and 12S rDNA) which are very informative for high resolution
analysis of evolutionary processes and the Internal Transcribed Spacer 2 (ITS-2), a relatively fast-evolving nuclear
non-coding element which can be used in phylogenetic reconstructions on the species and genus level, were
employed for molecular investigations. Time to the most recent common ancestor (TMRCA) for each lineage and
its closest relative was calculated based on 16S rDNA phylogeny. The results and their taxonomic implications are
discussed.

Material and methods

Twenty-six specimens (Table 1) were analysed for both morphological and biometrical key features of shells and
animals. Individuals were drowned in water and fixed in 75% ethanol. The reproductive apparatus was extracted by
means of scalpel, scissors and forceps. Photographs were taken with a Panasonic Lumix FZ 30 digital camera.
Maximum height and maximum diameter of the shell along with some parts of the genitalia were measured (in mil-
limeters) with digital calipers. Illustrations of genitalia were sketched using a camera lucida mounted on a Leica
MZ12.5 stereomicroscope, scanned and finished in GIMP (GNU Image Manipulation Program) 2.6. Voucher spec-
imens for the pictures are deposited in the Museo Civico di Storia Naturale di Genova, Italy (catalogue numbers:
MSNG55994–MSNG56005).

An additional thirty-one specimens of the mazzullii group and six (three per species) Cornu aspersum (O.F.
Müller, 1774) and Cantareus apertus (Born, 1778), chosen as confamilial outgroups, were used for molecular anal-
yses. Voucher specimens (catalogue numbers: CA1–CA3; CAP1–CAP3; CM1–CM5, CMS1–CMS5; COF1–
COF4; COL1–COL3; CU4–CU5; MP1–MP5; PEC1–PEC3; SV1–SV4) were stored in the Laboratory of Cytoge-
netics and Molecular Biology, University of Urbino (Table 2).

Place names are given following the Portale Cartografico Nazionale (PCN, http://www.pcn.minambiente.it/
PCN/). Each locality and/or collection site is named in the original language (Italian).

DNA extraction, amplification and sequencing. Samples were stored separately at -20°C in test tubes. Of
each individual, a piece of foot tissue was used for total DNA extraction (by Wizard Genomic DNA Purification
Kit, Promega). Fragments of 16S rDNA, 12S rDNA and ITS-2 sequences were amplified using three pairs of prim-
ers (Table 3) designed on alignments of several homologous sequences of Helicidae species downloaded from the
GenBank database. PCR cycles were as follows: for 16S rDNA (209–223 bp) and ITS-2 (536–541 bp) amplicons,
95°C for 5 min; 95°C for 1 min, 55°C for 1 min, 72°C for 1 min (30 cycles); 72°C for 5 min. For 12S rDNA (192–
196 bp) amplicons, 95°C for 5 min; 95°C for 1 min, 50°C for 1 min, 72°C for 1 min (35 cycles); 72°C for 5 min. To
remove primers and unincorporated nucleotides, the amplified products were purified with the Wizard SV gel and
PCR Clean-up kit (Promega).

FROM HELIX MAZZULLII COMPLEX TO ERCTELLA Zootaxa 3134 © 2011 Magnolia Press · 5