Computational protocol: Sporadic nesting reveals long distance colonisation in the philopatric loggerhead sea turtle (Caretta caretta)

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Protocol publication

[…] Loggerhead turtle sporadic nesting events are very rare and thus their sampling is necessarily opportunistic (Table , Fig. ). When a nesting event occurred, we collected the basic biometric data of the nest and at least one sample per clutch from a dead hatchling and/or embryo found after emergence. When possible, several hatchlings were sampled in order to test for multiple paternities. The collection of samples was conducted in strict accordance with Spanish and European regulations. The Ethics Comitttee of Animal Experimentation of the University of Barcelona stated that the analysed procedures fit the essential ethical rules and the legislation in force according to the article RD2013 of B.O.E from 8th of February 2013. This sort of studies is excluded from the area of application of the legislation, and therefore, the corresponding authorization of this Ethics Comitee is not needed. Furthermore, although the study species is listed in CITES, transportation of samples within the European Union does not require CITES permits. Muscle or skin samples of 120 hatchlings and one female were collected from 18 clutches (Table ) and stored in 95% ethanol. DNA was extracted using the QIAamp extraction kit (QIAGEN®) following the manufacturer’s instructions.Individual assignments of all samples to either Atlantic or Mediterranean nesting beaches were done using a combination of a fragment of the mtDNA control region and seven microsatellites,. As a first step, we amplified a long (~800 bp) fragment of the mitochondrial DNA (mtDNA) control region of one sample per clutch, using the primers LCM15382 and H950 as it has been proven to be much more informative than the shorter fragment (~380 bp) of the same region used in previous studies,. We used the same PCR conditions as in previous studies (e.g.). Sequences were aligned using BioEdit v7.1.11 and compared to known loggerhead haplotypes found in the database maintained by the Archie Carr Centre for Sea Turtle Research ( Published haplotype frequencies on nesting populations,,,– were compared with our samples and the origin of the nesting female was directly determined if it had an exclusive haplotype. Additionally, we genotyped seven microsatellites of all the samples using primers previously used for this species: Cm84, Cc117, Cm72 and Ei8; Cc141 and Cc7; and a modified version of Ccar176 using the same protocols described in the literature. The combination of the seven microsatellites was checked against the baseline of Atlantic and Mediterranean individuals used in a previous study to perform individual assignments using the program STRUCTURE v 2.3.4. This baseline comprised a total of 112 individuals of Mediterranean origin and 56 individuals of Atlantic origin (Supplementary Dataset S1). A previous study indicated that the best number of clusters of this baseline was K = 2 and that Atlantic and Mediterranean samples were highly differentiated (FST = 0.029, P < 0.001). Five samples of this baseline were genotyped again for the present study in order to check for changes in allele sizing and thus all microsatellite data was compatible after a correction for allele size changes (data not shown). An input file was prepared including both the baseline and the sporadic nests genotypes. All the samples of the baseline were labelled as belonging either to the Mediterranean (1) or to the Atlantic (2) while all sporadic nests samples were labelled as belonging to an additional group (3). Only samples from the baseline were used as locprior and thus no prior was assumed for the sporadic nests samples. STRUCTURE was run under the assumption of no admixture, considering the differentiation between the Atlantic and Mediterranean samples of the baseline, performing 1,000,000 repetitions after a 100,000 burn-in period and assuming K = 2. Each assignation was replicated 10 times and the results were averaged using CLUMPP v1.2. Hence, a probability of being either Atlantic or Mediterranean was ascribed to each sample. Samples with assignment values higher than 0.8 to one of the two groups were assumed to be reliable as previous studies recommended.Multiple paternity was tested for all clutches sampled for more than two hatchlings, using the microsatellite data and the program GERUD v 2.0. This software allows the reconstruction of parental genotypes from half-sib progeny with unknown parents and infers multiple paternity by identifying more than four different parental alleles from a clucth. Additionally, we generated the genotypes of all possible mothers and fathers of our offspring and we made individual assignments of up to the 100 most probable parents using the same methodology described above. Furthermore, we calculated the Lynch & Ritland relatedness index between all hatchling pairs using GenAlex v 6.5. Values were multiplied by two as indicated in the program to allow the index to vary between −1 and 1. Relatedness values among all sample pairs were used to create a heatmap using ‘ggplots2’. […]

Pipeline specifications

Software tools BioEdit, CLUMPP, GenAlEx
Application Population genetic analysis