Page 8 - Colomba-et-al-2011-Erctella-mazzullii-complex-Zootaxa-3134

pendent MCMC runs were conducted and the log and tree files were combined using LogCombiner (v 1.4.8). The
results were examined with Tracer (v 1.5) to confirm stationary distribution and adequate effective sample sizes
(i.e. ESS>200) that had been obtained for all parameters, indicating that the sampled generations were uncorrelated
and the posterior distribution of the parameter was long and accurate. TreeAnnotator (v 1.4.8) was then used to
summarize a best supported tree and annotate the tree with the mean age and posterior probabilities of the nodes
under investigation. FigTree (v 1.3.1) was used to display the estimated tree with node ages and 95% confidence
intervals. Programs BEAST, BEAUTi, LogCombiner, Tracer, TreeAnnotator and FigTree were downloaded from
http://beast.bio.edu.ac.uk.

Results

Molecular data

DNA sequences and variability. The mitochondrial 16S rDNA sequence alignment showed nine different haplo-
types, defined by 23 polymorphic (segregating) sites (S = 23) including a total number of 26 mutations (Eta = 26).
Haplotypes (h) revealed to be restricted to unique geographical populations, in particular, one was sampled only in
the group from Cefalù, La Rocca; five from Monte Pellegrino and nearby mountains; and three from the sites
around Trapani. Haplotype diversity (Hd) was 0.892±0.002. Nucleotide diversity (π) was 0.043±0.004 and 0.045
when corrected according to Jukes and Cantor (πJC). The average number of nucleotide differences (k) was 8.968.

The mitochondrial 12S rDNA sequence alignment showed ten different haplotypes, defined by 37 polymorphic
(segregating) sites (S = 37) including a total number of 39 mutations (Eta = 39). In this case, haplotypes (h) also
revealed to be restricted to unique geographical populations, in particular, two haplotypes were from Cefalù, La
Rocca; five from Monte Pellegrino and nearby mountains; and three from the sites around Trapani. Haplotype
diversity (Hd) was 0.914±0.02. Nucleotide diversity (π) was 0.082±0.006 and 0.091 when corrected according to
Jukes and Cantor (πJC). The average number of nucleotide differences (k) was 15.591.

The ITS-2 sequence alignment showed six different genotypes, defined by 27 polymorphic (segregating) sites
(S = 27) including a total number of 29 mutations (Eta = 29). Genotypes revealed to be restricted to unique geo-
graphical populations, in particular, three were sampled only in the group from Monte Pellegrino and nearby moun-
tains; two from the sites around Trapani; and one from Cefalù. Genotype diversity was 0.845±0.026. Nucleotide
diversity (π) was 0.018±0.002 and 0.019 when corrected according to Jukes and Cantor (πJC). The average number
of nucleotide differences (k) was 9.856.

Phylogenetic structure and species delineation. All (NJ, MP, ML and BI) phylogenetic analyses yielded
highly congruent tree topologies that agreed on branching patterns and sister-taxon relationships. Analysis of 16S
rDNA, 12S rDNA and multigene combined datasets showed three phylogenetic groups with strong branch support,
thus confirming the occurrence of three taxa. The first phylogroup (mazzullii) included only the specimens col-
lected on Monte Pellegrino and nearby mountains, the second phylogroup (cephalaeditana) the specimens from
Cefalù, La Rocca and the third phylogroup (insolida) the specimens from Trapani province; mazzullii and cepha-
laeditana showed sister-taxon relations. The analysis of the ITS-2 dataset showed phylogenetic trees differing in a
few topological details; the BI consensus tree evidenced the occurrence of the three taxa but with a different sister-
group relationship; whereas NJ, ML and MP trees didn’t distinguish among the three taxa placing all specimens of
the mazzulli group within the same cluster supported by a strong BP value (86%). Since all reconstructions pro-
duced almost identical results not changing any of the interpretations, only the phylogenetic ML and BI consensus
trees obtained by the concatenate analysis are displayed as an example in figures 8a, b.

For mtDNA sequences the maximum divergence within each taxon was 1.3% (16S rDNA) and 1.6% (12S
rDNA); divergence among groups ranged from 6% to 10% (16S rDNA) and from 7% to 17% (12S rDNA). As far
as ITS-2 sequences are concerned, when comparing the taxa two at a time, genetic differences ranged from 0.5% to
2.4% in the mazzullii group. Estimates of evolutionary rates of mtDNA and nDNA fragments were 0.48% (both
16S rDNA and 12S rDNA) and 0.50% (ITS-2) per million years.

8 · Zootaxa 3134 © 2011 Magnolia Press COLOMBA ET AL.