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810 V. FIORENTINO ET AL.

(Marmorana)], the central-southern Apennines sample areas was planned considering the patchy
[assigned to Marmorana (Ambigua)] and Sicily distribution of Marmorana in the field. This patchi-
[assigned to Marmorana (Murella)]. The Sicilian ness is determined by the rock-dwelling life style of
Marmorana have impressive conchological diversity, this group which is confined to limestone rock faces.
with globular to flat-shelled populations, which has For shell measurement, ten specimens were col-
led to the description of approximately sixty nominal lected randomly in each site, making 20 specimens
taxa, now reduced to five species (Manganelli et al., per area and a total of 570 specimens. Only shells
1995) or five species with 59 subspecies (Bank, 2007): with a reflected lip were used because this indicates
Marmorana globularis (Philippi, 1836), Marmorana cessation of growth and maturity of the snail. Several
nebrodensis (Pirajno, 1840), Marmorana scabriuscula shell features (Fig. 1A) were measured to the nearest
(Deshayes, 1832), Marmorana platychela (Menke, 0.01 mm using digital images (Adobe Photoshop,
1830) and Marmorana muralis (Müller, 1774). Unfor- version 7.0.1) of standard views of shell with a Nikon
tunately, due to punctiform distribution of shell Coolpix 4500 digital camera (Fiorentino et al., 2008).
morphs, loss of typical material, vaguely indicated For each site, five sexually developed specimens
type locality (frequently only ‘Sicily’) and no consis- (genitalia at similar stages of development), randomly
tent or controversial application of names to different selected among those analysed for shell features, were
morphs, it is impossible to unequivocally assign popu- dissected under a light microscope (Wild M5A) using
lations to a particular taxon and, with the present fine-pointed watchmaker’s tweezers. Twelve linear
state of the art, nomenclatural and taxonomic revi- variables (Fig. 1B) of isolated genitalia were measured
sion is difficult. using a millimetric lens under a light microscope
As Arbogast & Kenagy (2001) note, ‘the goal of (0.01 mm) (Fiorentino et al., 2008).
phylogeography (Avise et al., 1987) is to characterize
the phylogenetic deployment of genealogical lineages
across the geographical landscape’. Phylogeographic ANALYSIS OF MORPHOLOGICAL DATA
analyses have often shown cryptic divergent evolu- Variables were log-transformed to obtain linear rela-
tionary lineages that are not reproduced in the tionships. Discriminant function analysis (DFA) was
current taxonomy, as well as the existence of nominal performed considering all the shell variables mea-
species found to be poly- or paraphyletic. sured (Jennrich, 1977; Chiu et al., 2002; Alonso et al.,
The present study aimed: (1) to determine the 2006b) to discover which measurements contributed
phylogenetic relationships of Sicilian Marmorana spe- to discriminate groups defined a priori, in this case
cies; (2) to test for shared patterns among putative the populations sampled. A sequential chi-square test
Marmorana species by morphological (shell and was used to quantify the significance with which the
anatomy) and molecular analysis [mitochondrial discriminant functions significantly separated the
(mt)DNA genes]; (3) to investigate the evolution of groups. Structure and canonical coefficient tables
shell shape (globular versus keeled flat shell); and (4) were used to establish the contribution of each mea-
to investigate the taxonomic implications of the three surement to the first two discriminant functions.
character sets analysed (conchological, anatomical Two-way analysis of variance (ANOVA) was used to
and molecular). distinguish populations on the basis of the variables
Accordingly, we obtained a molecular phylogeny of contributing most to the DFA functions. The a poste-
Sicilian populations of the genus Marmorana by riori Tukey test (P < 0.05) was used to check group
means of sequences of two commonly used mtDNA significance.
genes, cytochrome oxidase subunit I (CO I) and the DFA was then performed considering all genital
large ribosomal subunit (16S rRNA). variables measured. The analysis was run with the
groups identified by ANOVA, in an endeavour to
check for differences in genitalia among recognized
shell groups. With this analysis we assessed which
MATERIAL AND METHODS
measurements contributed to discrimination of
SAMPLING FOR MORPHOLOGICAL ANALYSIS groups defined a priori, in this case the populations
A two-factor sampling design was used for morpho- sampled. A sequential chi-square test was used to
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logical analysis: area factor (6 km ) and site factor quantify the extent to which each discriminant func-
(100 m ) (for sampling design, see Fiorentino, Manga- tion significantly separated groups and structure and
2
nelli & Giusti, 2008). The site was a two-level random canonical coefficient tables were used to establish the
nested factor: two sites were randomly selected in contribution of each measurement to the first two
each of the 30 sampling areas (in five areas, two-site discriminant functions.
sampling was not possible for logistic reasons) (see Size is generally the first determinant of biometric
Appendix). The sampling design and number of variation (Cadima & Jolliffe, 1996). Techniques that

© 2008 The Linnean Society of London, Biological Journal of the Linnean Society, 2008, 94, 809–823

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