International Journal of Marine Science, 2017, Vol.7, No.33, 316-343
327
The methods of capture and data collection of the individuals as well as description of the variables are described
in
Of all the variables reported by these authors, we used the sexual class, the sexual
maturity stage and the straight carapace length (SCL) divided in five SCL classes of 10 cm modified fro
et al. (1998a). Individuals with SCL≥80 cm that were not directly examined by the authors and lacked secondary
sexual characteristics according to the fishermen, were classified as adult females according to
(1999). All procedures practiced on the turtles were made according to the national regulations established by the
Cuban Ministry of Fishery Industry. This institution provide us with the permissions to access to the fishery area
and take the samples for the genetics, reproduction and morphometry studies.
3.2 Collection and processing of samples for genetic analysis
Biopsies of skeletal muscle or skin were taken from each individual and preserved in 96% ethanol saturated with
EDTA and stored at room temperature. DNA extraction and purification were performed with FastDNA kit (MP
Biomedicals). The mtDNA control region (ca. 800 bp) was amplified using the primers LTEi9
(GGGAATAATCAAAAGAGAAGG-3') and H950 (GTCTCGGATTTAGGGGTTT-3') from Abreu-Grobois et al.
(2006). When DNA degradation hindered the PCR, we amplified the first half (ca. 400 bp) with LTEi9 and the
primer REi397 (CAGAGGCCAGAATAGATCAG-3'), designed in this research; and the second half (ca. 500 bp)
with Cont1a (ATTATTCTCAACCATGAATATCG-3') (Díaz-Fernández et al., 1999) and H950. The PCR was
developed in 25 μL of reaction volume for the 740 bp segment according to the PCR conditions of Velez-Zuazo et
al. (2008), and Díaz-Fernández et al. (1999) for the second half. For the first half the same cycles were used but
with an annealing temperature of 55°C for 45 seconds. The amplified products were purified using NucleoSpin
Extract II columns (Macherey-Nagel) and sequenced in both directions with fluorochrome-labeled ddNTP
(BidDye Terminator 3.1 Cycle Sequencing Kit, Applied Biosystems) in an ABI3100 genetic analyzer.
Sequences of 384 or 740 bp were aligned using Clustal X (Thompson et al., 1997) and matched against published
WC hawksbill mtDNA haplotypes (Bass et al., 1996; Bass, 1999; Díaz-Fernández et al., 1999; Bowen et al., 2007;
Velez-Zuazo et al., 2008; Leroux et al., 2012; Carreras et al., 2013). The haplotype (
h
) and nucleotide (π) diversity
indices were calculated using the software Arlequin version 3.11 (Excoffier et al., 2005). For the genetic
characterization of the JR fishing aggregation we compared the haplotype composition of non-adult individuals,
adult males, and adult females annually and to the previous decade; and in turn these treatments distributed in the
SCL classes through an X
2
test of pseudoprobability with 1000 Monte Carlo simulations (Roff and Bentzen, 1989),
through CHIRXC software (Zaykin and Pudovkin, 1993).
3.3 Mixed stock analysis
The ―mixstock‖ package in R version 2.15.1 (R Core Team, 2012) was used to compute a many to-many Bayesian
MSA (Bolker et al., 2007) with rookery size (annual number of nesting females) as a constraint, for both 384 and
740 bp sequence lengths. The Markov chain Monte Carlo (MCMC) method was used, implemented by
WINBUGS version 1.4 (Sturtz et al., 2005) executing 20000 iterations and a 10000 burning command. The
Gelman-Rubin diagnostic was used to determine convergence in the MSA posterior distributions (Gelman and
Rubin, 1992).
3.3.1 Aggregations
Genetic data from published feeding/breeding aggregations of the WC with neritic life-stages were used for the
MSAs (Figure 5). The aggregations were segregated in two maturation categories: non-adult or adult individuals,
declared as a ―non-adult‖ or ―adult aggregation‖ respectively. In each maturation category, the haplotype
frequencies were separated by decade of sampling,
i.e.
non-adult (or adult) aggregations from the 1990s or 2000s
(Supplementary Material 3A; Supplementary Material 3B). For the MSAs of non-adult aggregations the Nr of the
decade previous to their sampling was used considering that (Supplementary Material 2): 1) in WC the individuals
of
E. imbricata
have higher growth rates than in other regions (Moncada and Nodarse, 1994; van Dam and Diez,
1998a; Hawkes et al., 2014); 2) non-adult individuals of the present study had SCL classes similar to the adult
ones (SCL classes II and III according to Lee-González et al. (2015)) and; 3) no demographic data exists for the
rookeries during the 1970s. For the MSA of adult aggregations the Nr was selected from the same decade of
