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46 D. D 'EsPOSITO, E. D ATTOLO, F. B ADA LAMENTI , L. 0RSINI, G. PROCACCIN I

Ameno (NA), Stareso (Corsica), Marettimo (TP) and Malta) and were genotyped
with a total of 64 microsatellite loci. 13 loci were anonymous, putatively neutra!
(A-SSR) and widely employed to assess neutra! genetic variation (e.g. Arnaud-Haond
et al., 2005; Serra et al., 2010), while 51 loci were EST-linked (E-SSR) and developed
ad hoc from two existing P. ocean.ica EST-libraries (http://drzompo.uni-muenster.
de/). Population structure was estimated with an analysis of molecular variance
(AMOVA; Excoffier et al., 1992) using the Genalex 6 software (Peakall & Smouse,
2006). A global AMOVA was conducted separately for A-SSR and E-SSR markers
by partitioning the dataset in the 6 populations and 12 sub-populations (two depths
for each population). A local AMOVA was used to estimate the contribution of the
two microsatellites sets in the detection of variance between the two depths at each
population. To identify loci under selection we used the software Lositan, a selection
detection workbench based on the method fdist (Antao et al., 2008; Beaumont &
Nichols, 1996). lt discriminates outlier loci (loci that depart from neutral expectation
based on the expectation of the island model of populations) from strictly neutra!
ones. Loci under selection will show a higher or a lower Fst (fixation index) in
relation to their expected heterozygosity. In order to detect outlier loci potentially
selected along a depth gradient, Lositan was run for pairwise comparison (the two
different depths) from each population and employing only polymorphic loci.

Results - The genetic diversity assessed comparing the two classes of markers
shows that A-SSR are more variable than E-SSR in terms of polymorphic loci, mean
number of alleles per locus and mean heterozygosity. The number of polymorphic
loci ranged between 9 and 12 for the 13 A-SSR loci and between 8 and 14 for the
51 EST-SSR, (Tab. 1). The A-SSR loci showed a higher number of alleles per locus
(from 2.077 to 4.077) in comparison to the E-SSR (from 1.255 to 1.431) (Tab. 1).
Expected heterozygosity ranged from 0.293 to 0.485 in the A-SSR set and from 0.069
to 0.121 in the E-SSR loci (Tab. 1). GIN values (number of genotypes/number of
samples) were almost similar in the two microsatellites sets (from 0.47 to 1.0 for
the A-SSR and from 0.42 to l for the E-SSR) (Tab. 1). Eleven outlier loci related to
depth were identified over ali the pairwise comparisons performed. The number of
outlier loci was lower for the putatively neutra! marker set, A-SSR (4 outliers over 12
polymorphic loci) than for EST-derived loci, E-SSR (7 outliers over 16 polymorphic
loci). Among the E-SSR markers, three loci result strong candidates for selection
related to the depth because they emerge from the comparison of more than one
population. The global AMOVA shows higher variance between depths when using
the E-SSR markers (variance=28%), in respect to the A-SSR (variance=l8%). The
local analysis, shows different values of variance among different sets of markers
(Tab. 1). The use of ali EST-linked loci showed higher variance than A-SSR markers
in ali populations except Malta (Tab. 1).

Conclusions - The aim of this work was to identify loci under selection related
to two different depths and to analyze the neutra! and adaptive variation in 6
populations [12 (sub)populations] of P. oceanica. In addition, we estimated the
resolution power of the new (EST-linked) microsatellite set in detecting depth-related
molecular variation, in respect to the existing one (A-SSR). Our analysis revealed
that A-SSR markers have higher number of alleles, heterozygosity and genotype
discrimination power but the variance between the two depths is generall y higher
when EST-linked are utilized. This suggests that meadows growing at different
depths are somehow adapted to different environmental forces. Results also show
that of the eleven loci that seem to be under balancing selection, seven belong to the
EST-linked microsatellites, while four loci were identified from the putative neutral