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812 V. FIORENTINO ET AL.

stored in 95% ethanol for morphological examination treated as unknown and were estimated during the
and kept in the F. Giusti Collection (Dipartimento di run. The separate genes were treated as ‘unlinked’, so
Scienze Ambientali, Siena, Italy). that separate parameter estimates were obtained for
each gene. The default value of four Markov chains
was used (one cold and three heated) and each chain
DNA EXTRACTION, POLYMERASE CHAIN REACTION
was started from a random tree. The ‘temperature’
(PCR) AND SEQUENCING parameter was set at 0.2. The Markov Chain Monte
Total genomic DNA was extracted from foot muscle of Carlo length was 4 000 000 generations and trees
fresh specimens using C-TAB buffer (0.1 M Tris-HCl, were sampled every 100 generations.
pH 8.0, 1.4 M NaCl, 0.02 M ethylenediaminetetracetic Parsimony analysis was performed with PAUP*,
acid, 2% cetyl trimethylammonium bromide, 0.2% version 4.0 (Swofford, 2001) using a heuristic search
2-mercaptoethanol) and standard phenol–chloroform/ under equal weighting of all characters (1000 random
ethanol extraction (Hillis et al., 1996). stepwise addition and tree bisection-reconnection
A fragment of CO I was amplified by PCR using branch swapping). To evaluate clade support, non-
the specifically-designed primer pair (5′-CCTATTATA parametric bootstrap resampling was used with 1000
ATTGGGGGTTTTGG-3′) and (5′-GTATCGGCTCTAA replicate datasets and 100 random-addition sequ-
AATAAGC-3′). A fragment of the mitochondrial gene ences per dataset.
coding for the large ribosomal subunit (16S rRNA)
was also amplified, using the primer combination
LR-J-12887 [5′-CGATTTGAACTCAGATCA-3′ (Simon RESULTS
et al., 1994) and 5′-GTGCAAAGGTAGCATAATCA-3′
MORPHOLOGICAL ANALYSIS
(Gantenbein et al., 1999)].
PCR reactions were run in a total volume of 50 mL Regarding the DFA of shell, the first discriminant
under the following conditions: 95 °C for 20 s, 48 °C function accounted for most morphological variance
for 30 s and 72 °C for 30 s (repeated for 25-s cycles), (74%) and the second for only 12%. Considering the
plus a final extension step at 72 °C for 5 min. Ampli- canonical and structural coefficients making up the
fied products were examined on 1.5% agarose gels. first function, the highest loadings (Table 1) were for
DNA fragments were then excised and purified with a maximum diameter (MaxD) (1.42) and shell height
‘Nucleospin extract’ (Genenco) column kit, following (ShH) (-1.29) respectively. A sequential chi-square
the manufacturer’s instructions. test showed that the first function contributed to
Detecting the appropriate amplification products population discrimination to a very large extent
2
was straightforward for both genes because no sec- (c = 3506, d.f. = 319, P < 0.001) whereas the contribu-
2
ondary bands due to nonspecific priming occurred. tion of the second (c = 1981, d.f. = 280, P < 0.001) and
Approximately 30 mg of the purified products was subsequent functions was less significant. Indeed, the
then used as templates for direct sequencing of both first function discriminated scores from negative to
strands, using the same amplification primers and
a CEQ dye terminator cycle sequencing kit. DNA
sequences then underwent electrophoresis on a CEQ Table 1. Canonical coefficients of discriminant analysis of
8000XL (Beckman Coulter). All sequences generated shell measurements
in this study were deposited in GenBank (see
Appendix). Canonical variables
Variable First Second
GENETIC DATA ANALYSIS
AH -0.27 -0.06
For both mitochondrial regions, sequences were easily
AW 0.45 0.44
aligned unambiguously by eye using the computer MaxD 1.42 0.98
program BIOEDIT, version 7.0 (Hall, 1999).
MinD 0.21 0.10
Phylogenetic relationships were estimated by Baye- HMaxD -0.61 -0.19
sian inference (BI) and maximum parsimony (MP).
LWW -0.26 -0.15
Prior to Bayesian inference, we used MRMODELT- ShH -1.29 -0.56
EST, version 2.2 (Nylander, 2004) to determine the SpH 0.30 0.23
best model of nucleotide evolution for each dataset. MaxLWH -0.76 0.12
The selected models were then used in a partitioned a -0.07 -0.17
Bayesian analysis carried out with MRBAYES, b -0.11 0.12
version 3.1 (Huelsenbeck & Ronquist, 2001; Ronquist
& Huelsenbeck, 2003). Model parameter values were For abbreviations, see Fig. 1.

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

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