Preprint volume - SIBM

Pre-print Volume - Oral presentations
Topic 1: BIODIVERSITY AND CONSERVATION SCIENCE: CONTRIBUTING TO MANAGEMENT
method. A fragment of 642 bp of the COI locus and a portion of the ITS1 region
variable in length from 430 to 587 positions, were amplified by PCR using the primers
LCO1490/ HCO2198 and ITS1L/58C respectively. The PCR protocol was as follows:
95°C 5'; 95°C 1', 45°C (COI) or 55°C (ITS1) 1', 72°C 2', 35 cycles; 72°C 5'. All PCR
reactions were performed in a total volume of 25 µl included 2.5 µl of 2 mM of each
dNTP (GE Healthcare), 2.5 µl of 10× load buffer-MgCl2 (Qiagen), 2.5 µl of 2 µM of
each primer (Invitrogen), 0.25 µl of 5 U/µl Taq DNA polymerase (Qiagen), 19 µl of
demineralized water and 1 µl of the DNA template. Each amplicon was purified using
the GFX PCR DNA and GEL band purification kit (GE Healthcare) and sequenced on
both strands. Haplotype diversity (h), nucleotide diversity (π), hierarchical analysis of
molecular variance (AMOVA), ΦST and FST fixation indices were computed using
Arlequin 3.11. A combined data set composed by COI and ITS1 concatenated
haplotypes were used to investigate the phylogenetic relationship between the two
populations. The analysis was performed using the Bayesian inference (BI), computed
with MrBayes 3.1.2 and the Markov Chain Monte Carlo simulations tree sampling
procedure. The analysis was run for seven millions generations, assuming the GTR +I
+Γ evolution model and with parameter values and trees calculated et every 100 th step.
The estimated log-likelihood scores were plotted against generation time, to assess
when the log-likelihood values reached the stationary. The log-likelihood scores had
been clearly plateaued after 10,000 generations, that is after 10,000 generations the
changes in trees topology and parameter values did not continue to improve the trees
likelihood scores. Therefore the first 100 trees (from the first 10,000 generations) were
excluded and the remaining trees were used to make a 50% majority rule consensus
and to estimate the Bayesian Posterior Probability (BPP), to give support for tree
nodes. Only BPP values equal or above 95% were considered significant.
Results - COI sequences comparison revealed 63 variable sites, and 49 of them were
parsimony informative. This polymorphism defined 9 distinct haplotypes among the 31
individuals, giving the sample an overall haplotypic diversity of 0.391 and a nucleotide
diversity of 0.002. ITS1 sequences showed 58 variable sites 31 of which were
parsimony informative. This polymorphism defined 17 distinct haplotypes among the
31 individuals, giving the sample an overall haplotypic diversity of 0.965 and a
nucleotide diversity of 0.048. AMOVA of the COI partial sequence data showed that
the majority of the genetic variation was explained by the within populations
component, with only a small fraction of no-significant variance explained by the
between populations component (ΦST=0.008; p=0.145). Similarly, AMOVA of the
ITS1 partial sequence data showed that most variability was found within populations,
with a lack of significant variability between populations (FST=0.009; p=0.327).
Phylogenetic estimate made for the combined data set (COI+ITS1), reveals clearly two
major clades, one consisting of Mediterranean specimens and one including both
Mediterranean and Atlantic specimens (Fig. 1). Therefore, while the Atlantic group
appears monophyletic, the Mediterranean group is polyphyletic. However, all the BPP
values are lower than the limit of 95%.
41 st S.I.B.M. CONGRESS Rapallo (GE), 7-11 June 2010
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