Geneious R6 Serial Number: Where to Find It and Why You Need It

The loggerhead marine turtle, Caretta caretta, is a widely distributed and endangered species that is facing critical population decline, especially in Colombian Caribbean rookeries. Mitochondrial DNA sequence data are of great importance for the description, monitoring, and phylogenetic analyses of migratory turtle populations. In this study, the first full mitochondrial genome of a loggerhead turtle nesting in the Colombian Caribbean was sequenced and analyzed. This mitochondrial genome consists of 16 362 bp with a nucleotide composition of T: 25.7 %, C: 27 %, A: 35 % and G: 12 %. Sequence annotation of the assembled molecule revealed an organization and number of coding and functional units as reported for other vertebrate mitogenomes. This Colombian loggerhead turtle (Cc-AO-C) showed a novel D-Loop haplotype consisting of thirteen new variable sites, sharing 99.2 % sequence identity with the previously reported Caribbean loggerhead CC-A1 D-Loop haplotype. All 13 protein-coding genes in the Cc-AO-C mitogenome were compared and aligned with those from four other loggerhead turtles from different locations (Florida, Greece, Peru, and Hawaii). Eleven of these genes presented moderate genetic diversity levels, and genes COII and ND5 showed the highest diversity, with average numbers of pair-wise differences of 16.6 and 25, respectively. In addition, the first approach related to t-RNAs 2D and 3D structure analysis in this mitogenome was conducted, leading to observed unique features in two tRNAs (tRNATrp and tRNALeu). The marine turtle phylogeny was revisited with the newly generated data. The entire mitogenome provided phylogenetically informative data, as well as individual genes ND5, ND4, and 16S. In conclusion, this study highlights the importance of complete mitogenome data in revealing gene flow processes in natural loggerhead turtle populations, as well as in understanding the evolutionary history of marine turtles.

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The loggerhead turtle, Caretta caretta (Cc) is distributed around the oceans of the world in tropical and subtropical latitudes (Amorocho, 2003). Its main nesting locations have been reported in the coasts of the peninsula of Florida (FWC 2015), in the western Brazilian Atlantic Ocean, in the Eastern Mediterranean Sea, in the Omani Arabian Sea, in Madagascar, and in Japan (Dodd 1988, Lancheros & Hernández 2013, Hernández et al. 2017). Despite its wide global distribution, it is considered as an endangered species (IUCN 2016). Loggerhead populations are directly threatened by several anthropic activities including: fisheries bycatch, excessive fishing/hunting, and illegal trade of eggs and meat. In addition, Loggerhead turtle populations are affected by habitat deterioration, coastal development, pollution, pathogens and climate change (Eckert et al. 2000, Lancheros & Hernández, 2013, Machado & Bermejo, 2012). Loggerhead turtles reach their sexual maturity at around 20-30 years of age (Machado & Bermejo, 2012), which does not offset the rampant overall population decline of the species. The threat to Loggerhead turtles has been well documented the Colombian Caribbean (Amorocho, 2003, Ceballos-Fonseca, 2004), where the world's second highest number of catches per year (approximately 600 turtles) has been reported (Humber et al. 2014). This, despite existing national laws and international agreements to protect the species from anthropic threats (SWOT 2012, IUCN 2016).

The sequencing strategy for the entire mitogenome was based on PCR amplification of overlapping fragments of 800 - 2 500 bp in length. The overlap among fragments was of 50 - 200 bp to facilitate full sequence assembly. A total of 22 primer pairs were employed to sequence the mitogenome of the Colombian Caribbean loggerhead turtle (Table 1). Seventeen primer pairs were designed using the Overlapping Primer Sets Program (Whitehead Institute, Cambridge, USA) based on the mitochondrial genome sequence of another loggerhead sea turtle (Cc-MS-G, GenBank accession number NC_016923.1). The remaining five primer pairs were used as previously described by Drosopoulou et al. (2012).

Different analyses were performed to identify chimeras between the mitochondrial genome and nuclear paralog sequences. First, the mitochondrial DNA was assembled with the reference genome of the loggerhead turtle (GenBank accession number NC_016923.1). Then, the mitogenome was aligned with mitogenomes of other four loggerhead turtles, and a phylogenetic tree was built using the complete mitogenome sequences of all six sea turtle species.

Standard diversity indices, such as number of haplotypes (k), number of polymorphic sites (S), haplotype diversity (H), average number of differences between pairs of sequences (n), and nucleotide diversity (n) according to Nei (1987) were estimated for each one of the thirteen mitochondrial protein coding genes from ad hoc sequence alignments of the Cc-AO-C turtle sequence (accession number KP256531.1) with sequences of other four loggerhead mitogenomes. These loggerhead mitogenomes were downloaded from the NCBI database and consisted of the Cc-AO-F (Florida-USA) (accession number JX454983), Cc-MS-G (Greece) (accession number NC_016923), Cc-PO-P (Peru) (accession number JX454988), Cc-PO-H (Hawaii) (accession number JX454977). All genetic variation estimators were obtained with DNAsp v5.10 (Librado & Rozas, 2009). A similar approach was also applied to the D-Loop region of the Cc-AO-C and the afore mentioned four loggerhead mitogenomes and a set of 92 loggerhead D-Loop haplotype stretches of the Archi Carr Center for Sea Turtle Research at the University of Florida (accstr.ufl.edu).

Phylogenetic inferences were made for the superfamily Chelonioidae using data from individual genes and complete mitochondrial genomes. The inference was made with mitogenome sequence data from seven sea turtle species: C. mydas (Cm-AO) (accession number NC_000886.1), N. depressus (Nd-PO-A) (accession number NC_018550.1), E. imbricata (Ei-AO-C) (accession number KP2218061), C. caretta (Cc-MS-G) (accession number NC_016923), L. olivacea (Lo-PO-CR) (accession number NC_028634.1), L. kempi (Lk-AO-US) (accession number JX_454982.1), and the mitogenome described in this study. The mitochondrial genome of D. coriacea (Dc-AO-US) (accession number JX_454989.1) was used as an outgroup.

The complete mitogenome sequence (16 362 bp in length) of the loggerhead turtle individual Cc-AO-C was obtained and deposited in the GenBank under accession number KP256531.1. Upon analysis of this mitogenome sequence, we confirmed that the sampled turtle was indeed a member of the Caretta caretta species. Since hybrids between sea turtles have been frequently reported (Bowen & Karl, 2007; Drosopoulou et al. 2012; Duchene etal. 2012), it was necessary to ascertain that the captured mitogenome was indeed from the loggerhead species. In addition, attention was paid during primers design to avoid unintended amplification and sequencing of nuclear paralogs of some mitogenome genes. Moreover, obtained sequence reads were inspected for double peaks, as seen in diploid nuclear sequences, before mitogenome assembly.

The parts of the mitogenome that showed the highest average number of differences between pairs of sequences (n), and thus the greatest genetic variation (Table 3) and the lowest sequence identity values (Table 2) were the D-Loop and protein-coding genes COII and ND5. Compared to other mitochondrial functional units, the D-Loop has been reported as the stretch with the highest levels of genetic diversity among sea turtle populations (Abreu-Grobois et al, 2006; Novelletto et al. 2016) as a non-coding and likely neutrally evolving DNA stretch, the D-Loop is possibly one of the top informative mitogenome fragments to perform gene flow analyses in populations of the species C. caretta. Based on the current results, the genes COII and ND5 could be equally useful when employed for this type of analyses.

In current phylogenetic analysis, the use of data from complete mitogenomes is gaining ground. With full or partial mitogenome data, phylogenetic analyses become more robust and gain in phylogenetic resolution and greater precision compared to analysis based on data from individual markers (Duchene et al. 2011). The current results support previous relationships among sea turtle species, N. depressus as the sister taxon to Chelonia (Duchene etal. 2012, Naro-Maciel et al. 2008) as well as the clade comprising Erecmochelys, Lepidochelys and Caretta (Fig. 3) (Dutton et al. 1999, Duchene et al. 2012). This result is important when explaining phylogenetic relationships within the family Cheloniidae, particularly the exclusion of N. depressus from the subfamily Carettini (Dutton et al. 1999, Duchene et al. 2012, Naro-Maciel et al. 2008). Out of the total number of mitochondrial markers, from which data were obtained to solve ancestry-descent relations among sea turtles, the ND5 gene produced highly supported trees. This marker can generate phylogenetic trees with a support comparable to that of a complete mitochondrial genome, and it confirms the topology of the proposed phylogeny for these species. This study presents the use of mitochondrial genomes as an alternative to improve phylogenetic analysis to estimate the evolutionary relations among sea turtles.

The matK and rbcLa sequences were combined using SequenceMatrix software (Vaidya, Lohman & Meier 2011). The MrBayes plugin Version 2.0.9 in Geneious R6 was used to construct a Bayesian Inference (BI) cladogram for phylogenetic analysis. The barcoding gap and species partitioning were determined with the online tool Automatic Barcode Gap Discovery (ABGD: Puillandre et al. 2011) with the default settings. A pairwise summary, best match and best close match were determined with the program SpeciesIdentifier (Meier et al. 2006). A threshold value of 0.349 was calculated from the pairwise summary (Meier et al. 2006). A median joining network was constructed in Network 4.6.1.1 to depict the number of mutational steps separating haplotypes of the six Clivia species. 2ff7e9595c