Polymorphism in Mitochondrial Coding Regions of ...

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Polymorphism in Mitochondrial Coding Regions of Mediterranean Loggerhead Turtles: Evolutionary Relevance and Structural Effects Andrea Novelletto1,* Letizia Testa1 Federico Iacovelli1 Paola Blasi1 Luisa Garofalo2 Toni Mingozzi3 Mattia Falconi1 1 Department of Biology, University Tor Vergata, Rome, Italy; 2 Istituto Zooprofilattico Sperimentale delle Regioni Lazio e Toscana, Centro di Referenza Nazionale per la Medicina Forense Veterinaria, Rieti, Italy; 3Department of Biology, Ecology, and Earth Sciences, University of Calabria, Rende, Italy Accepted 7/26/2016; Electronically Published 9/15/2016 Online enhancements: supplementary tables.

ABSTRACT We sequenced coding portions (1.6 kb) of the mtDNA in 170 loggerhead (Caretta caretta) turtles sampled in the central Mediterranean. The sequences spanned the entire ND1 and ND3 genes, the tRNAGly and tRNAArg, plus the 30 and 50 termini of COXIII and ND4L genes, respectively. Based on our sequencing results and published complete mitogenomes, we constructed a maximum parsimony phylogeny of C. caretta matrilines that sheds new light on the evolutionary relationships within the collection of lineages found in the Mediterranean and so far recognized by Dloop haplotypes only. We show that the new variants are useful to understand the ancestry of extant haplotypes, to improve genetically based studies on the philopatry and migratory behavior of the species, and for conservation purposes. In order to better understand the biological significance of the observed variation, we addressed intraspecific nonsynonymous substitutions in the context of the three-dimensional modeled structures of ND1 and ND3. The positions of variant amino acids within the folded subunits are consistent with a coadaptation with the restructuring of membrane thickness, fluidity, and lipid composition, a wellknown response mechanism to thermal conditions. The pattern of amino acid substitutions departs from neutrality, suggesting local adaptation and/or polymorphism-based local selection.

*Corresponding author; e-mail: [email protected]. Physiological and Biochemical Zoology 89(6):473–486. 2016. q 2016 by The University of Chicago. All rights reserved. 1522-2152/2016/8906-5144$15.00. DOI: 10.1086/688679

Keywords: mtDNA phylogeny, ND1 and ND3 coding genes, synonymous and nonsynonymous substitutions, marine turtles, protein structure, thermal adaptation.

Introduction The loggerhead turtle (Caretta caretta) inhabits tropical and subtropical waters of the Atlantic, Pacific, and Indian Oceans (Márquez 1990), extending its range also to the temperate Mediterranean Sea, where it is the most common marine turtle, and it is found both as a temporary foraging visitor and as a regularly nesting species (Casale and Margaritoulis 2010). Here and elsewhere the species displays a natal homing behavior, by which adult females mate in open waters and land to lay eggs on the same or in proximity of the beaches where they were born. The expected consequence, even over many generations, is a strong among-colony structuring for maternally inherited portions of the genome (e.g., mitochondrial DNA [mtDNA]), depending on the composition of the founder group, genetic drift, and additional subsequent bottlenecks. Indeed, extensive work on this species in the Mediterranean (Bagda et al. 2012; Clusa et al. 2013) and other seas/oceans (Shamblin et al. 2012b) has shown an extreme spatial structuring of matrilines, each colony being the result of extreme founder effects and remaining largely demographically independent (Shamblin et al. 2014), with examples also at very short distances (Shamblin et al. 2015a). In this context, a low level of breakdown of strict homing behavior (e.g., by social facilitation [Bowen et al. 1992; FitzSimmons et al. 1997] or straying [Shamblin et al. 2015a]) is able to maintain a residual connectivity between rookeries. This has major implications for conservation purposes, as far as it impacts the chances of recovery for declining threatened nesting populations. Initial studies on the polymorphism of the mtDNA D-loop in C. caretta revealed affinities between the molecular types found in Mediterranean and western Atlantic rookeries, suggesting that Mediterranean populations became isolated from Atlantic populations at the beginning of the Holocene (Bowen et al. 1993; Encalada et al. 1996, 1998). The colonization of the Mediterranean Sea was considered to include most likely the transplantation of the D-loop sequence variant called haplotype CC-A2 from southern Florida/Mexico, followed by local diversification. Subsequent works also considered an Atlantic origin for haplotype CC-A3 (the founder haplotype in Turkish rookeries), though its origin from CC-A2 within the Mediterranean could not be excluded based on D-loop diversity alone (Carreras et al. 2007; Yilmaz et al. 2011). Furthermore, the finding of haplotypes CC-A20

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and CC-A31 led us to conclude that the mitochondrial diversity of loggerhead turtles in a single nesting location in Italy could not be appropriately accounted for by a singular foundation event followed by molecular radiation in situ (Garofalo et al. 2009). The breakdown of a strict natal homing behavior is now considered to occur more often than previously thought (Shamblin et al. 2014, 2015a), and the initial settlement on Mediterranean beaches has been pushed back in time (Clusa et al. 2013). An appropriate reconstruction of this process requires the decoupling of the frequencies of a given lineage (e.g., identified as a D-loop haplotype) from its antiquity, as determined by the sequential accumulation of derived DNA features (allele states). It is then important to study the composition of foraging stocks as far as these represent the reservoir of potential new colonizers. Thus, in light of the unique geography of the Mediterranean, that is, a closed basin with a single entry point, different histories of colonization events can be tested, provided a sufficient knowledge of the mtDNA phylogeny is available. However, information on the amount and type of mitochondrial coding variation outside the D-loop at a population scale is scant for marine turtles (for a review see Lee 2008; Naro-Maciel et al. 2010). The existence of Mediterranean populations for as much as 65,000 years (Clusa et al. 2013) implies that they resisted the intervening climatic fluctuations, an outcome proposed also for at least some of the other subtropical species currently found in the Mediterranean (Wilson and Eigenmann Veraguth 2010). As poikilothermic vertebrates, marine turtles are expected to have been strongly impacted by such fluctuations, in view of the temperature dependency of many aspects of their biology (phenology, migration, reproduction, and sex determination; Mazaris et al. 2004; Hawkes et al. 2007). These conditions potentially allow the rise in frequency of novel positively selected mutations, eventually in the form of adapted genomic regions (Stiebens et al. 2013). Gathering evidence for such phenomena requires, as a first step, the sifting of functionally relevant variants from the whole array of variants. In view of high gene density in vertebrate mitogenome, surveys of short polymorphic regions may provide a wealth of variants that potentially impact the corresponding gene products. Moreover, the strong long-term functional conservation of mitochondrially encoded proteins enables a reliable inference of their three-dimensional (3D) structure starting from those experimentally obtained in related species. This opens new possibilities in the analysis of the distribution of variants, much more informative than statistics derived from the simple amino acid string, as in other methods (Woolley et al. 2003). In fact, based on the 3D protein model and the known properties of amino acids, the consequences of each substitution can be evaluated in the appropriate protein context, which includes embedding in the lipid bilayer, exposure to solvent, interaction with other subunits in multiprotein complexes, and so on. Thus, a specific effect on the structure of the protein domains, eventually relevant for its proper functioning, can be tentatively ascribed to each amino acid change as inferred from nonsynonymous substitutions in the DNA (Somero 2010). This study was thus aimed at (1) identifying regions other than the D-loop, harboring common polymorphisms in the mitoge-

nome of loggerhead turtles sampled in the central Mediterranean; (2) reaching an improved resolution of intraspecific mitochondrial lineages currently falling within a limited number of recognizable D-loop haplotypes and searching for a phylogeographic signal to enable the testing of current models for the colonization of the Mediterranean; (3) investigating the effects of coding variants on the corresponding protein structure to evaluate their biological significance.

Material and Methods Sampling Strategy The central Mediterranean is a crossroad for foraging juveniles and adults born in Mediterranean nesting grounds (central and eastern Mediterranean), as well as for Atlantic individuals that frequent this sea only for foraging (Carreras et al. 2006; Casale et al. 2008; Garofalo et al. 2013). We assembled a sample of individuals that could represent the widest divergence of lineages. We then replicated a strategy that proved successful in molecular evolutionary studies of other species (see, e.g., Achilli et al. 2012; Perego et al. 2012; Lancioni et al. 2013; Doro et al. 2014) by combining two orthogonal criteria. The first is the inclusion in the case series of individuals carrying mitochondria with different D-loop haplotypes (indicative of diverging lineages), and the second is, given the same D-loop haplotype, to include individuals sampled at distant geographical locations from each other. While an ideal study should entail sampling in the populations of origin, it is to be expected that a larger sampling area is able to provide an assemblage of matrilines originated in a much larger gene pool, from populations with geographically diverse and ancient origins. Previous mixed-stock analyses have shown that, in Mediterranean stocks, variable proportions of individuals can be inferred to be of Atlantic origin. However, our sampling areas also included waters shown to receive only a little (!10%) contribution from Atlantic rookeries (Garofalo et al. 2013; Clusa et al. 2014). The 170 loggerhead turtles experimentally examined (table 1) included three individuals (dead hatchlings) sampled in the Calabrian nesting colony and 167 from foraging stocks. Biological samples were collected in the frame of the TARTACare project (DiBEST, University of Calabria) or provided by collaborators who sampled individuals stranded on north Adriatic, Thyrrenian, west Ionian, and Maltese coasts; all the above individuals represent a subset of series previously described (Garofalo et al. 2009, 2013). Some individuals were included in the study based on their mtDNA D-loop haplotype, to improve the representation of types uncommon in central Mediterranean stocks. In assembling our series we tried to keep the proportions of the common haplotypes as similar as possible to those of the pooled Mediterranean reproductive units (Clusa et al. 2013; Carreras et al. 2014). A detailed listing of all individuals, partitioned by haplotype and sampling location, is provided in table 1. The study complies with institutional, national, and international ethics guidelines concerning the use of animals in research and/or the sampling of endangered species. None of the procedures used in the study met the criteria to define them “experi-

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Table 1: List of the individuals originally examined by the D-loop haplotype Species

D-loop haplotype

N

Representative isolate

Sampling area

Accession no. (ND1)

Accession no. (ND3)

Caretta caretta C. caretta C. caretta C. caretta C. caretta C. caretta C. caretta C. caretta C. caretta C. caretta C. caretta C. caretta C. caretta C. caretta C. caretta C. caretta

CC-A1.1 CC-A2.1 CC-A2.1 CC-A2.1 CC-A2.1 CC-A2.8 CC-A2.9 CC-A20.1 CC-A26.1 CC-A28.1 CC-A3.1 CC-A31.1 CC-A32.1 CC-A55.1 CC-A6.1 CC-A66.1

1 132 1 1 1 2 4 2 4 1 15 1 2 1 1 1

C4-02 CAL12-34 NADR-6 CAL12-33 SLM08-3 MB8 CAL12-8 0714_0101 CAL12-5 W08-12 CAL12-29 0704_0101 CAL12-13 W08-13BIS RA15 C4-01

West Ionian West Ionian North Adriatic West Ionian Malta West Ionian West Ionian Calabrian nesting colony West Ionian West Ionian West Ionian Calabrian nesting colony West Ionian West Ionian North Adriatic West Ionian

KJ810617 KJ810618 KJ810630 KJ810616 KJ810631 KJ810619 KJ810620 KJ810621 KJ810622 KJ810623 KJ810624 KJ810625 KJ810626 KJ810627 KJ810628 KJ810629

KJ768857 KJ768858 KJ810614 KJ768856 KJ810615 KJ768859 KJ768860 KJ768861 KJ768862 KJ768863 KJ768864 KJ768865 KJ768866 KJ768867 KJ768868 KJ768869

ments” as defined in article 2 of the Council of European Communities Directive 86/609/EEC regarding the protection of animals used for experimental and other scientific purposes. For this reason approval by the institutional ethics committee (Comitato Etico Indipendente at Fondazione PTV, Tor Vergata University) was not required.

ND1 and ND3 amplicons were subjected to Sanger sequencing on both strands using the same primers, followed by electrophoretic run on an ABI3100 automatic sequencer (Perkin-Elmer/ Applied Biosystems) under the conditions for sequences longer than 450 bp. Pherograms were inspected with the Sequencher software (Gene Codes Corporation; http://www.genecodes.com/). Variants were called if confirmed on both orientations.

Identification of the Target DNA Regions and Methods We first performed a search of the most variable segments in the alignment of five complete mitogenomes of Caretta caretta (Drosopoulou et al. 2012; Duchene et al. 2012) by considering the index of nucleotide diversity computed on sliding windows of 1,000 bp in steps of 100 bp. This revealed two regions of enhanced diversity (in addition to the D-loop) between nucleotide positions g.2500 and g.4000 and between g.8000 and g.10000, respectively (fig. A1). We then designed primer pairs to amplify from within these regions in different turtle species (table S1; tables S1–S3 available online). The first amplicon spanned the entire NADH ubiquinone oxidoreductase subunit ND1, while the second spanned ND3, the tRNA-Gly and tRNA-Arg genes, plus the 30 and 50 termini of COXIII and ND4L, respectively (fig. A2). Genomic DNA preparation and D-loop sequencing were as described (Garofalo et al. 2013). PCR reactions were conducted in 25-mL volumes containing 1# PCR buffer, 2.5 mM MgCl2, 5 pmol of each primer, 0.32 mM dNTPs, and 1 U Taq polymerase in the presence of 5 mL of purified DNA. Thermal profiles comprised an initial denaturation at 947C for 1 min, followed by 30 cycles of 1 min denaturation at 947C, 1 min primer annealing (527C for ND3 and 547C for ND1), and 1 min DNA chain extension at 727C. This was followed by a final extension at 727C for 10 min. Purification was performed on 90 ng of amplified PCR product with the exosap protocol (http://www.bmr-genomics.it/). The

Data Analysis Revised sequences were aligned with Clustal (Thompson et al. 1997) as implemented in MEGA (Tamura et al. 2011) and trimmed to the shortest length (positions g.2796–g.3762, 967 bp for ND1 and g.9380–g.10040, 661 bp for ND3, respectively). As the ND1containing segment of Chelonia mydas and Eretmochelys imbricata included three additional nucleotides, a gap was generated in the multispecies alignment, bringing the total length to 970 bp. A nonredundant set of sequences was deposited in GenBank and received accession numbers reported in table 1. Sequences from the same individual were concatenated in a spreadsheet and analyzed jointly (1,631 bp). Affiliation of published sequences to D-loop haplotypes was performed by BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi), using reference sequences (table S2 in Shamblin et al. 2014). The nomenclature of D-loop haplotypes was according to the Archie Carr Center for Sea Turtle Research (http://accstr.ufl.edu /resources/mtdna-sequences/). In order to fully represent the species here examined in the context of the Cheloniidae subtree, we included in our analysis the orthologous sequences (table 2) obtained from complete mitogenomes of C. caretta (5), Lepidochelys kempii (2), Lepidochelys olivacea (3), E. imbricata (5), and C. mydas (9; Tandon et al. 2006;

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D-loop haplotype

Caretta caretta C. caretta C. caretta C. caretta C. caretta Lepidochelys kempii L. kempii Lepidochelys olivacea L. olivacea L. olivacea Chelonia mydas C. mydas C. mydas C. mydas C. mydas C. mydas C. mydas C. mydas C. mydas Eretmochelys imbricata E. imbricata E. imbricata E. imbricata E. imbricata

CC-A2.1 CC-A5.1

CC-A1.1

Drosopoulou et al. 2012; Duchene et al. 2012; Shamblin et al. 2012a). The sequence JX454984 (C. caretta) contained 28 ambiguous positions at the 30 end of the ND3 amplicon. We used FR694649 (C. caretta) as reference for position numbering in the resulting alignment. Conceptual translation of all sequences in the alignment was obtained with MEGA. Note that ND1 in C. mydas and E. imbricata contains three asparagine residues at positions p.254–p.256 instead of two. This generates a slippage in the numbering of amino acids beyond 256 in the other species. Thus, for example, ND1 T315A should read as T314A in C. caretta. An unrooted maximum parsimony (MP) tree was obtained with MEGA (Tamura et al. 2011). The “show ancestral states” option was used for mapping mutation events to tree branches for each variable site in the same order as in the alignment. A number of uncertainties were encountered, attributable to recurrent mutational events. Single nucleotide variant states shared by all species but one were considered derived in the latter. Variant states shared by all C. mydas sequences only (fig. 1, branch 22) could not be polarized. Variants identified only in some sequences within the same species (hereafter referred to as intraspecific variants) were considered recurrent if found in one or more of the sequences of other species. Variants for which alternative polarizations required two versus three independent events were given the most parsimonious assignment (e.g., g.9853 A/G, assigned to branches

Sampling area Greek nesting colony Atlantic Ocean Pacific Ocean Pacific Ocean Atlantic Ocean Atlantic Ocean Atlantic Ocean Pacific Ocean Pacific Ocean Pacific Ocean Hawaii Ecuador Pacific Ocean Malaysia Micronesia Atlantic Ocean Atlantic Ocean Cyprus Atlantic Ocean Atlantic Ocean Pacific Ocean Pacific Ocean Indian Ocean Indian Ocean

Accession no. FR694649 JX454983 JX454988 JX454977 JX454984 JX454981 JX454982 JX454979 JX454987 JX454991 JX454971 JX454978 JX454974 JX454976 JX454985 JX454990 JQ034420 JX454972 JQ026233 JX454986 JX454980 JX454970 DQ533485 NC_012398

6 and 15 instead of 6, 16, and 22 of fig. 1). A complete list of mutations assigned to each branch is provided in table S2, with ambiguous nucleotide and amino acid states highlighted. A Bayesian tree reconstruction was performed with BEAST (Drummond and Rambaut 2007), under a birth-death model for speciation and gamma-distributed rate heterogeneity plus invariant sites. After a number of test runs to optimize the priors, we used a run of 10 million Markov chain Monte Carlo steps, sampled every 5,000, with a 20% initial burn-in. The performance of the run was monitored by analyzing the traces of the sampled values and the effective sample size of each parameter. For each group of identical sequences a single representative was used. In the absence of any firm value for the substitution rate and given the possibility that either one or both of inter- and intraspecific variation are not neutral (see “Results”), we present branch lengths/ node ages in mutational instead of time units (fig. A3). An unrooted median joining network of C. caretta intraspecific ND1– ND3 concatenated sequences (fig. A4) was obtained with the program Network 5.0 (Bandelt et al. 1999). Nonsynonymous changes were mapped onto multiple alignments of the 40 most similar hits obtained with BLASTp and aligned with COBALT at http://blast.ncbi.nlm.nih.gov/Blast.cgi, using accession numbers YP_005296176. 1 and YP_005296183.1 as queries from C. caretta ND1 and ND3, respectively.

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Figure 1. Unrooted maximum parsimony tree based on all DNA variants observed in the sequences here considered. The length of intraspecific branches is proportional to the number of mutations. Sequences identical over the mtDNA portions here considered are boxed together (for the number of sequences identical to each isolate, see table 1). Nonsynonymous intraspecific mutations are reported on the left of their corresponding branches, in their polarized form, and underlined if recurrent elsewhere in the tree. As the numbering of amino acid positions is that of the multispecies alignment, asterisks indicate positions that do not match the original numbering. Each branch is numbered to locate the corresponding mutations in table S2, available online. Each sequence is identified by its representative isolate (table 1) or accession number (table 2). The provenance of the individuals included in the analysis is reported in parentheses (A p Atlantic Ocean; I p Indian Ocean; M p Mediterranean Sea; P p Pacific Ocean); this refers to the place of sampling of the particular individual and not to the alleged origin of the D-loop haplotype. n p individual was sampled in a nesting ground. The D-loop haplotype is reported for Caretta caretta individuals.

As a neutrality test for the number of intraspecific variants as compared to fixed differences, the McDonald-Kreitman test (McDonald and Kreitman 1991) was performed with the program DNAsp (Librado and Rozas 2009). The sequences JX454981, JX454987, JX454970, and JX454978 were used as representatives of L. kempii, L. olivacea, E. imbricata, and C. mydas, respectively, as outgroups. Only the coding sequences of ND1 and ND3 were considered, excluding the incomplete segments of COXIII and ND4L. While the above analyses assume a random sampling of the population, we notice that rare D-loop haplotypes were observed to be associated with successful reproduction; thus, their

inclusion in our series did not result in the enrichment in severely deleterious variants. Modeling Procedure The ancestral (as inferred from allele states reported in fig. 1 and table S2) C. caretta NADH ubiquinone oxidoreductase subunit ND1 and ND3 3D structures were obtained using the iTASSER threading server (Zhang 2008; Xu et al. 2011). The iTASSER server provides an online workbench for high-resolution modeling of protein structures and functions (http://zhanglab.ccmb.med.umich

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.edu/I-TASSER/). The sequences used for the modeling were the NCBI GenBank entries ID YP_005296176.1 (C. caretta) and AFP52821.1 (C. caretta) for ND1 and ND3, respectively. The ND1 and ND3 models were structurally superimposed on the corresponding NADH ubiquinone oxidoreductase complex (PDB IDs: 4HE8 for ND1 and 3RKO for ND3) through the Matchmaker tool of the UCSF Chimera program (Pettersen et al. 2004). The mutations were mapped on the corresponding structures and investigated taking into account the class of substitution (hydrophobic to hydrophobic, hydrophilic to hydrophobic, and vice versa). In case of uncertain class assignment, the surrounding structural environment was checked.

Results Polymorphism, Phylogeny, and Neutrality Tests Overall we found 21 variable positions in the concatenated sequences experimentally obtained from the 170 loggerhead turtles. All of our sequences differed from the reference FR694649 for an additional C/T transition at position g.9974. We successfully aligned the concatenated sequences from our individuals with the corresponding portions obtained from five, two, three, five, and nine complete mitogenomes from Caretta caretta, Lepidochelys kempii, Lepidochelys olivacea, Eretmochelys imbricata, and Chelonia mydas for the ND1 and ND3 segments, respectively. The resulting MP tree is shown in figure 1. Our analysis revealed novel complexity in the phyletic relationships of matrilines as defined by the arrangement of the variants along the mtDNA molecule. Three major clades (fig. 1, top) replicated those obtained on complete mitogenomes (Duchene et al. 2012). The most basal lineage (fig. 1, branch 9), characterized by seven mutations, was found in the single carrier of the D-loop haplotype CC-A1.1, which produced a sequence identical to JX454984 sampled in the Atlantic (table S2). A second deeply rooted clade (with 15 shared mutations) included two sequences sampled in the Pacific (fig. 1, branch 7). A sister clade (identified by seven mutations) grouped all our remaining sequences sampled in the Mediterranean plus JX454983 (sampled in the Atlantic and assigned to the D-loop haplotype CC-A5.1), that is, all sequences assigned to the group of D-loop haplotypes related to CC-A2 (haplogroup II sensu Shamblin et al. 2014). A single variation (g.9440A1T; COXIII V254V) distinguished carriers of haplotypes CC-A2.9, CC-A26.1, and CC-A66.1 from the remaining carriers of haplogroup II haplotypes (fig. 1, branch 5). Among carriers of the mtDNA D-loop haplotype CC-A2.1, three out of 135 (plus FR694649) could be distinguished by one variant each (fig. 1, branches 1–4). In all the remaining cases, individuals carrying the same D-loop haplotype displayed identical sequences. Though with low support (posterior p 0.62), the Bayesian tree displayed an alternative order of branching of the Pacific and Atlantic clades within C. caretta (fig. A3). Indeed, several intraspecific variants replicated fixed differences between species, likely due to common recurrent events that generated variants equal in

state. We resolved most recurrent events in the entire MP tree by visual inspection (a complete list of mutations by branch is provided in table S2). In particular, of the 37 mutational events observed within C. caretta (branches 1–9 in fig. 1), 20 appeared to recur also in one or more of the other branches. This problem was acute for the C. caretta Pacific clade (branch 7), in which three, one, one, and one recurred also in branches 11, 15, 16, and 22, respectively, and two had uncertain assignment. This may explain the difference in topology in the MP tree versus the Bayesian tree. On the other hand, in both the MP and Bayesian trees, the depth of the three major C. caretta clades was comparable only to E. imbricata and much greater than those of the other species here considered. Within the ND1 and ND3 complete sequences, a number of 21 and 11 substitutions were synonymous and nonsynonymous, respectively. Though not significantly (Fisher exact test, P p 0.142), the majority of nonsynonymous substitutions (7 vs. 4) were nonrecurrent, as contrasted to synonymous substitutions (7 vs. 14; table S3). As such, this discrepancy denotes that nonsynonymous changes display some degree of departure from the likely neutral pattern of synonymous changes. Nonsynonymous substitutions greatly contributed to the divergence of each of the major C. caretta clades, with 1/7, 3/15, 2/3, and 3/7 amino acid replacements in branches 6, 7, 8, and 9 (fig. 1), respectively. We then assayed the excess of nonsynonymous changes by the McDonald-Kreitman test (McDonald and Kreitman 1991) by comparing the set of C. caretta sequences to one representative of each of the other species as outgroup (table 3). The results were significant in three out of four tests (P p 0.039, 0.018, and 0.021 using L. kempii, L. olivacea, and C. mydas, respectively; Fisher exact test).

Polymorphic Variants in Their 3D Context The observation that three C. caretta groups of sequences, characterized by distinct geographic provenances, harbor many coding nonsynonymous substitutions raises questions on whether the latter were allowed to spread by the different conditions in the seas/oceans inhabited by the corresponding populations. As shown previously, the relevance of a structural analysis in the study of effects induced by sequence variations may provide a rational interpretation of experimental data (Falconi et al. 1998; Napoli et al. 2008; Di Marino et al. 2010). In order to gain insight into the possible biological significance of the observed variation in this species, we then specifically addressed intraspecific nonsynonymous substitutions in the context of the 3D modeled structures of ND1 and ND3. To structurally characterize the observed mutations, we first performed a modeling of the 3D structures of the NADH ubiquinone oxidoreductase subunits ND1 and ND3 (fig. 2). A group of mutations in ND1 (table 4), including Ile73Thr, Thr315Ala, and Gly318Ser, has been classified as an hydrophilic/ hydrophobic shift, since they are located in the tails of transmembrane a-helices, where membrane thickness adjustments

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Table 3: Partition of synonymous and nonsynonymous changes in ND1 and ND3 interspecific comparisons contrasted to polymorphism in Caretta caretta

Polymorphic in Caretta caretta Divergence with Lepidochelys kempii (JX454981) Divergence with Lepidochelys olivacea (JX454987) Divergence with Eretmochelys imbricate (JX454970) Divergence with Chelonia mydas (JX454978)

support these kinds of substitutions. These three substitutions cooccur in the branch leading to the cluster of sequences peculiar to the Atlantic (branch 9, fig. 1). Unexpectedly, the Ala234Thr substitution is nonconservative and located in the protein hydrophobic core. Indeed, this is the sole substitution observed between amino acid positions p.190 and p.244 in the alignment of the 15 most similar protein sequences. This unusual substitution may be stabilized by the establishment of a hydrogen bond with the side-chain carboxyl group of the very conserved (100%) residue Asp56, proving full compatibility with the protein surrounding environment. Conversely, the Thr3Ala substitution is also nonconservative, but it is located in the membrane and participates to intersubunit contacts, establishing hydrophobic interactions with surrounding residues belonging to the flanking subunit. The ND1 Thr3Ala and Ala234Thr substitutions co-occur in the branch leading to the cluster of sequences peculiar to the Pacific (branch 7, fig. 1). Finally, Ile16Val and Val262Met may be regarded as conservative, as far as they are internal to the membrane and consist of hydrophobic to hydrophobic substitutions. The first one is the sole substitution observed between amino acid positions p.4 and p.32 in the alignment of the 13 most similar protein sequences; the second one is recurrent, alters a poorly conserved position, and was found as a private variant in a carrier of the CC-A2.1 D-loop haplotype. In the case of the ND3 subunit (fig. 2B; table 4), a group of mutations including Met5Thr, Ile7Thr, and Thr28Met is interpreted as a hydrophilic/hydrophobic adaptation to the membrane thickness. Also in this case these substitutions are located in the tails of transmembrane a-helices, where a diverse membrane thickness may be easily accommodated by the observed mutations. All three positions are poorly conserved in the multispecies protein alignment. The remaining Val108Ile substitution is permitted, being conservative and internal to the membrane. This substitution resides in a very conserved portion of the protein. In conclusion, intraspecific amino acid substitutions were predominantly located at the membrane boundary in both ND1 and ND3, that is, two polypeptide chains that lay in close physical proximity within the transmembrane portion of complex I (Efremov and Sazanov 2011). In ND3 they occur in protein domains (i.e., the poorly conserved region from aa.1 to aa.10 and the highly conserved region from aa.98 to aa.116) opposite those affected by interspecific substitutions (the region from aa.11 to aa.93; ta-

Synonymous substitutions

Nonsynonymous substitutions

P (exact test)

21 70 75 77 92

11 13 12 18 16

.039 .018 .089 .021

ble S2). This pattern is replicated by two intraspecific substitutions in ND1 (I16V and A234T), which fall in conserved portions of

Figure 2. Cartoon diagram of ND1 (A) and ND3 (B) protein models. The a-helices are indicated by gray spirals, while random coil structure and turns are shown as a gray wire. The residues affected by intraspecific mutations are depicted using spacefill models. The dashed lines roughly indicate the boundaries of the internal mitochondrial membrane, evaluated by detecting the hydrophobic/hydrophilic edge in the entire protein NADH ubiquinone oxidoreductase complexes used as model templates (PDB ID: 4HE8 for ND1 and 3RKO for ND3). In B, Q p probable position of the ubiquinone cofactor (Efremov and Sazanov 2011).

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Table 4: Structural classification of the amino acid substitutions in NADH ubiquinone oxidoreductase subunits ND1 and ND3 Gene

Position

Ancestral codon

Mutated codon

Ancestral residue

Mutated residue

Structural classification

ND1 ND1 ND1 ND1 ND1 ND1 ND1 ND3 ND3 ND3

3 16 73 234 262 315 318 5 7 28

ACC ATC ATC GCC GTA ACC GGC ATA ATC ACA

GCC GTC ACC ACC ATA GCC AGC ACA ACC ATA

Thr Ile Ile Ala Val Thr Gly Met Ile Thr

Ala Val Thr Thr Met Ala Ser Thr Thr Met

Ca B A Cb B A A A A A

Note. For structural classification, A p located in a transmembrane a-helix tail, qualified as hydrophilic or hydrophobic adaptation due to membrane thickness alteration; B p conservative residue substitution (hydrophobic to hydrophobic, internal to membrane); C p nonconservative residue substitution, structural adaptation derived from concerted mutations. a This mutation is most likely necessary to establish hydrophobic interactions with surrounding residues belonging to the flanking subunit. b This mutation is justified by a hydrogen bond established with the side-chain carboxyl of the conserved residue Asp56.

the protein, invariant among the four species here considered and displaying amino acid changes only in more distantly related ones. Discussion We performed a survey of variation in ND1- and ND3-containing segments of the mitogenome in Mediterranean loggerhead turtles. While our sampling and analyses focused only on individuals sampled in the Mediterranean (i.e., a marginal habitat for Caretta caretta), we used, as a benchmark, information from interspecific comparisons obtained from complete mitogenome sequences of individuals of the genera Caretta, Chelonia, Eretmochelys, and Lepidochelys sampled worldwide (Tandon et al. 2006; Drosopoulou et al. 2012; Duchene et al. 2012). This revealed that our data conveyed relevant information to better understand the evolutionary novelty of some variants and the phylogeography of matrilines found in Mediterranean stocks and to interpret the patterns of diversity as the result of active selective forces. In this work we report on many protein-coding synonymous and nonsynonymous, as well as non-protein-coding, mitochondrial variants (aim 1), obtained by screening a number of individuals much larger than previous complete mitogenome sequencing studies (Tandon et al. 2006; Drosopoulou et al. 2012; Duchene et al. 2012). These variants revealed new nodes in the intraspecific phylogeny of the species. Though the D-loop is by far the most variable portion of the mitochondrial genome, its variation among individuals nesting in or visiting the Mediterranean is still the limiting factor in fully understanding the population structure. In fact, the assessment of the provenance of individuals based on D-loop haplotyping is often unsuccessful, due to the overwhelming prevalence of one haplotype (CC-A2 and its subtype CC-A2.1). Among green turtles, Tikochinski et al. (2012, p. 23), by typing a tandemly repeated region at the 30 end of the D-loop (mtSTR), revealed cryptic variation within haplotype CM-A13, raising hopes “for the understanding of the fine-scale structure, population history and migration pat-

terns.” In fact, the same mitochondrial region was able to reveal structuring among Brazilian rookeries (Shamblin et al. 2015b). Our results add new single-nucleotide variants that increase the power of mtDNA in recognizing Mediterranean subpopulations of the loggerhead turtle and that can be of broader relevance in studies on the philopatry and migratory behavior of the species. It is to be expected that in some nesting grounds, inside or outside the Mediterranean, larger numbers of individuals carry variants that have appeared as private in this study, allowing the analysis of association between particular amino acid changes and the temperature experienced by the eggs during incubation on the beach (see below). A further step forward will involve the enlargement of the number of individuals screened, with indepth analyses of additional sampling locations, including nesting populations and feeding aggregates. This is particularly urgent for areas where haplogroup I (Shamblin et al. 2014) haplotypes are prevalent. In fact, these haplotypes were undersampled in this study, since in the Mediterranean they are found only in foraging visitors from the Atlantic. Our results are poorly informative on the diversity within haplogroup I. Within C. caretta, our MP tree replicates the tree obtained with complete mitogenomes (Duchene et al. 2012), with three deep clades. While the order of branching of the two deepest (Atlantic and Pacific) clades was not supported in the Bayesian analysis, we obtained a robust resolution of lineages within the third (Atlantic/Mediterranean) clade (aim 2). First, four different variants (branches 1–4 in figs. 1, A4) are associated with the D-loop haplotype CC-A2.1 Second, a nonrecurrent transversion (g.9440 A 1T; COXIII V254V) clusters together not only all sequences affiliated with haplotype CC-A2.1 but also all representatives of haplotypes CC-A55.1, CC-A31.1, CC-A28.1, CC-A20.1, CCA2.8, CC-A6.1, CC-A32.1, CC-A3.1, and CC-A5.1. This variant is not found in sequences affiliated with haplotypes CC-A2.9, CCA26.1, and CC-A66.1 (which share the ancestral state g.9440A) and thus defines branch 5 (figs. 1, A4). The first two of these haplotypes also appear in the network of D-loop haplotypes

mtDNA in Mediterranean Sea Turtles (Shamblin et al. 2014) within haplogroup II, in a cluster that forms a reticulation between a node ancestral to all haplotypes of haplogroup II and CC-A2.1. If we consider the complete linkage of variants along the mtDNA molecule, this evidence and our novel sequence data point to at least the combination of haplotypes CC-A2.9 and CC-A26.1 with g.9440A as basal in the intraspecific phylogeny of haplogroup II. The above results are promising for a much more complete resolution of evolutionary matrilines within haplogroup II at large and haplotype CC-A2.1 in particular. In fact, we screened only !10% of the mitogenome, and more variants can be expected to occur in the yet unexplored portions. Similar improvements with the same strategy were obtained in the resolution of the C. mydas haplotype CM-A5 in the greater Caribbean (Shamblin et al. 2012a). Interestingly, in this study markers that were phylogenetically informative had no population-level variation and vice versa. In this respect, our study benefited from the inclusion of mtDNAs with uncommon D-loop haplotypes, which proved highly rewarding for the reconstruction of both phylogeny and population structure. Do the phyletic relationships between mtDNA lineages inform on the genetic types that took part in the establishment of Mediterranean subpopulations (aim 2)? First, haplotypes CC-A2.9 and CC-A26.1 are found mainly in Libyan rookeries (Saied et al. 2012; Clusa et al. 2013), whereas CC-A66.1 was not yet assigned to a rookery so far. Thus, the Libyan population turns out to harbor at least two basal lineages, but this fact, per se, does not qualify it as the oldest population, as the colonization time cannot be equated to the age of the molecules. Second, arguments based on haplotype frequencies led to the consideration of CC-A2.1 as the founder haplotype for the Mediterranean nesting populations (Clusa et al. 2013). Based on phylogeographic considerations, we favor the hypothesis that the g.9440A1T mutation (defining branch 5 in figs. 1, A4) did not occur in a population already reproducing in Mediterranean nesting grounds. In fact, the complete mtDNA sequence JX454983, obtained from an individual sampled in the northwest Atlantic affiliated with haplotype CC-A5.1 (Duchene et al. 2012), carried the derived state (T; fig. 1). Also haplotype CC-A3.1, for which an Atlantic origin is possible (Carreras et al. 2007; Yilmaz et al. 2011), carries this allele state. Additionally, we (Garofalo et al. 2009) and other authors (Clusa et al. 2014) converge in considering CC-A20.1 as a marker of a recent and independent contribution to a Mediterranean rookery from the Atlantic, as compared to the widespread CC-A2.1. Yet, mtDNAs affiliated with CC-A20.1 share with those affiliated with CC-A2.1 and CC-A3.1 the derived state at position g.9440 (T), again pointing to an extraMediterranean origin (where CC-A2.1 and CC-A3.1 are also present) for this mutation. The direct testing of individuals of attested provenance from Atlantic rookeries is imperative to further support this hypothesis. In summary, the hypothesis that a large proportion of the D-loop haplotypes here surveyed have an Atlantic ancestry is strongly favored over the alternative, that is, that they evolved independently within the Mediterranean. In our series there is a minimum of five different haplotypes with an Atlantic ancestry that have been so far described in Mediterranean nesting grounds. As most of our haplotypes were not

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sampled in nesting females, we cannot put an upper bound to this number. However, from a conservation perspective, shifting from the alleged view of Mediterranean colonization as a singular event to a scenario with multiple though rare occurrences indicates that recolonization of suitable and appropriately conserved beach habitats is possible. Within C. caretta we found an overall excess of intraspecific nonsynonymous substitutions in ND1 and ND3, a finding considered as a signal of positive Darwinian selection (McDonald and Kreitman 1991; Yang and Bielawski 2000). In particular, nonsynonymous changes appeared to affect mostly ND1 in the lineage currently distributed mostly in the Atlantic (branch 9), whereas both ND1 and ND3 were affected in lineages distributed in the Pacific and the Mediterranean. By observing that seven intraspecific nonsynonymous changes were never observed elsewhere in the much deeper phylogeny connecting five species in our tree, we conclude that at least a subset of nonsynonymous changes represent an evolutionary novelty, allowed to spread only within C. caretta. This opens the possibility that local adaptation and/or polymorphism-based local selection is operating in this species (Watt and Dean 2000; Somero 2010). We then performed a structural modeling to improve our understanding of the possible significance of the observed substitutions at the phenotypic level (Mitchell-Olds et al. 2007; Ballard and Melvin 2010). The computational analysis of polymorphic sequence variants transferred on protein 3D models gave us useful insights for the interpretation of data that cannot be inferred purely on the basis of sequence analysis and prompts for further experimental investigation (e.g., on the lipid composition and physicochemical properties of mitochondrial membranes in subpopulations with differentiated mtDNA gene pools). In summary, our modeling results indicate (aim 3) that all the observed intraspecific nonsynonymous mutations are located in regions of the ND1 and ND3 proteins that do not compromise the functionality of the structure. Moreover, most of these mutations are located in proximity of the membrane borders, in line with a mechanism involving the restructuring of membrane thickness, fluidity, and lipid composition (relative proportions of saturated/unsaturated long-chain fatty acids). This phenomenon is comprehensively known as homeoviscous adaptation (reviewed in Hazel 1995) and is widely documented in poikilothermic vertebrates (e.g., Loftus and Crawford 2013; Strobel et al. 2013). As far as mitochondria are concerned, these changes have profound consequences for protein conformational mobility, in particular within the multiprotein complexes (Hazel 1995; Somero 2010). In this case, the conservation of an appropriate efficiency of proton translocation through the inner membrane by complex I is essential. The modulation of membrane fluidity represents an acclimation response (Vigh et al. 2007), setting the initial conditions for other genetically determined changes to become adaptive. Thus, the fact that the amino acid substitutions here described are viable at the population level potentially permits an adaptation operating on shorter timescales than the interspecific ones and related to the resettlements of populations in seas with varying temperature regimes (Watt and Dean 2000). This is a situation repeatedly encountered by C. caretta subpopulations during the expansion

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of their reproductive range. For example, the eastern US coasts and both sides of the Florida Peninsula, which are characterized by a sharp temperature gradient (http://www.nodc.noaa.gov/dsdt /cwtg/satl.html), were colonized northward with the establishment of independent but similar clines (Encalada et al. 1998; Shamblin et al. 2011, 2012b, 2014). Also, in the Mediterranean, these turtles are exposed to relevant fluctuations of surface temperatures(http://www.esa.int/spaceinvideos/Videos/2012/07 /Mediterranean_sea-surface_temperature, http://www.ifremer.fr/), for example, when frequenting the cooler northern portion of this sea (Carreras et al. 2006, 2011). In addition, it is worth stressing that sea turtles are exposed to widely different environmental temperatures during their life cycle. In particular, marine turtles are more exposed to temperature changes in habitats with seasonality (as the Mediterranean) than in warm tropical waters, with internal temperature strictly following external temperature and causing marked physiological changes in circulation, oxygen uptake, metabolic processes, and enzyme activities (Hochscheid et al. 2004). The sand temperature during egg incubation is also of critical importance in determining the individual’s development (Ackerman 1997). Finally, it has been noted (Somero 2010) that abundant temperature-adaptive molecular variants may allow “keeping the pace” with climate change. Our results then suggest that at least some of the variants here described may represent (part of) an adaptive response, in agreement with the emerging view of philopatry as a mechanism capable of maintaining a high adaptive potential and facilitating the retention of genetic polymorphism (Stiebens et al. 2013). Also in this respect, the finding of additional variants in other mitochondrially encoded proteins may turn out to be informative to confirm/dismiss the hypothesis of ongoing adaptation in Mediterranean loggerhead turtles, with a better understanding of their biology and adaptive potential. In view of the diversity of conditions experienced by these animals (outlined above), it will be

extremely difficult to work out the particular life stage in which a specific selective pressure (if any) operates. D-loop haplotypes have been so far considered neutral markers, ideal to trace historical processes and connectivity. The accumulation of variants by sequential mutational events only and the complete linkage along the entire mtDNA molecule make the evolutionary trajectories of neutral and selected markers inextricably bound. Only functional and physiological evidence will enable the distinction of the two types. With this information at hand, it will be possible to clarify the relative weight of directional selective forces in processes such as the starlike radiation of haplogroup II and the success in colonizing different portions of the Mediterranean basin. As far as the Mediterranean offers a wide range of sand temperatures for the most sensitive part of the life cycle (i.e., egg incubation), the establishment of colonies from founder females in surrogate beaches, after a nesting site loss, is a viable opportunity.

Acknowledgments We are grateful to Salvatore Urso, Giulia Cambiè, and all the researchers and students who took part in the fieldwork of the TARTACare Project. We also would like to thank Dino Scaravelli (University of Bologna, Italy), Luca Mizzan and Nicola Novarini (Natural History Museum of Venice, Italy), Leyla Knittweis and Carmen Mifsud (Fisheries Control and Environment Protection Directorates, Malta), Claudia Eleni (Istituto Zooprofilattico Sperimentale Lazio e Toscana), and Giovanni Scillitani (University Aldo Moro, Bari). This work was partially supported by Italian Ministry of Education grant PRIN 2012JA4BTY_003 to A.N. The funding source had no involvement in study design; in the collection, analysis, and interpretation of data; in the writing of the report; and in the decision to submit the article for publication.

APPENDIX

Figure A1. Linearized mtDNA map showing diversity (as summarized by the index v(p)) among five complete Caretta caretta mitogenome sequences computed on sliding windows of 1,000 bp in steps of 100 bp.

Figure A2. Organization of Caretta caretta mtDNA and sequences addressed in this work. The enlarged segments show sequences usefully recovered from all specimens after trimming, numbered as in the reference sequence FR694649.

Figure A3. Bayesian tree obtained with BEAST on all DNA variants observed in the sequences here considered. For identical sequences (see fig. 1) only one representative is shown. Numbers adjacent to nodes indicate the posterior support. The X-axis is scaled in mutational units. Note that the program coerces the resolution of multifurcations, so the five sequences at the top appear separated by nodes that are indeed nonexistent. Individual labels, provenance, and haplotype affiliation as in figure 1.

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Figure A4. Unrooted median-joining network of the Caretta caretta concatenated sequences examined here. Node size is proportional to the number of isolates (for the number of sequences identical to each isolate, see table 1). Branch length is proportional to the number of mutations (scale at bottom right). Branches discussed in the text are numbered as in figure 1. Individual labels, provenance, and haplotype affiliation as in figure 1. A color version of this figure is available online.

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