Molecular Phylogenetics and Evolution xxx (2012) xxx–xxx
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Molecular Phylogenetics and Evolution
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Marine turtle mitogenome phylogenetics and evolution
Sebastián Duchene a, Amy Frey a, Alonzo Alfaro-Núñez b, Peter H. Dutton a, M. Thomas P. Gilbert b, a, Phillip A. Morin ⇑ a Protected Resources Division, Southwest Fisheries Science Center, National Marine Fisheries Service, NOAA, 9801 La Jolla Shores Dr., La Jolla, CA 92037, USA b Centre for GeoGenetics, Natural History Museum of Denmark, University of Copenhagen, Øster Voldgade 5-7, 1350 Copenhagen, Denmark article info abstract
Article history: The sea turtles are a group of cretaceous origin containing seven recognized living species: leatherback, Received 7 March 2012 hawksbill, Kemp’s ridley, olive ridley, loggerhead, green, and flatback. The leatherback is the single mem- Revised 11 June 2012 ber of the Dermochelidae family, whereas all other sea turtles belong in Cheloniidae. Analyses of partial Accepted 13 June 2012 mitochondrial sequences and some nuclear markers have revealed phylogenetic inconsistencies within Available online xxxx Cheloniidae, especially regarding the placement of the flatback. Population genetic studies based on D- Loop sequences have shown considerable structuring in species with broad geographic distributions, Keywords: shedding light on complex migration patterns and possible geographic or climatic events as driving forces Sea turtle of sea-turtle distribution. We have sequenced complete mitogenomes for all sea-turtle species, including Molecular clock Mitogenome samples from their geographic range extremes, and performed phylogenetic analyses to assess sea-turtle Molecular adaptive evolution evolution with a large molecular dataset. We found variation in the length of the ATP8 gene and a highly Mitochondrial phylogenetics variable site in ND4 near a proton translocation channel in the resulting protein. Complete mitogenomes show strong support and resolution for phylogenetic relationships among all sea turtles, and reveal phy- logeographic patterns within globally-distributed species. Although there was clear concordance between phylogenies and geographic origin of samples in most taxa, we found evidence of more recent dispersal events in the loggerhead and olive ridley turtles, suggesting more recent migrations (<1 Myr) in these species. Overall, our results demonstrate the complexity of sea-turtle diversity, and indicate the need for further research in phylogeography and molecular evolution. Published by Elsevier Inc.
1. Introduction South Africa, Southern Argentina and Chile (Hirth et al., 1997). Ge- netic studies based on the mitochondrial D-Loop of C. mydas (Enca- The sea turtles comprise seven extant species grouped into two lada et al., 1996), D. coriacea (Dutton et al., 1999), and L. olivacea families: Dermochelidae, with the leatherback (Dermochelys coria- (Bowen et al., 1991; Karl and Bowen, 1999) suggest differentiation cea) as the single extant species, and Cheloniidae, with six species: of Indo-Pacific and Atlantic groups. This implies that South and hawksbill, Kemp’s ridley, olive ridley, loggerhead, green, and flat- Central America and the Isthmus of Panama represents a stronger back turtles (Eretmochelys imbricata, Lepidochelys kempii, L. oliva- geographic barrier to gene flow than do colder waters in the south- cea, Caretta caretta, Chelonia mydas, and Natator depressus, ern tip of Africa (Avise et al., 1992; Dutton et al., 1999), at least in respectively). Their phylogenetic placement has been somewhat these three species. debated, with different molecular data sets supporting different Recent advances in DNA sequencing technologies have made groupings within Cheloniidae. The placement of N. depressus has more molecular markers available for turtle phylogenetics. Previ- been particularly problematic, with different data supporting it ous studies have used as many as 14 nuclear markers across se- as the sister taxon either to a clade comprising the genera Eretm- lected turtle lineages (including freshwater and terrestrial ochelys, Caretta, and Lepidochelys (Dutton et al., 1996; Iverson turtles; Barley et al., 2010), and five nuclear and two mitochondrial et al., 2007), or to Chelonia only (Naro-Maciel et al., 2008). markers in marine turtles (Naro-Maciel et al., 2008). However, in Most sea turtles (except L. kempii and N. depressus) have a pan- terms of mitochondrial phylogenetics, only cytochrome b (Cytb) tropical distribution across a wide latitudinal range from Canada to (Bowen et al., 1993), D-Loop, ND4 (Dutton et al., 1996) and 12S and 16S (Naro-Maciel et al., 2008) regions have been used, produc- ing highly supported trees for contrasting topologies (see Naro- Corresponding author. ⇑ Maciel et al., 2008). E-mail addresses: [email protected] (S. Duchene), amy.frey@noaa. In other vertebrate groups, complete mitogenomes have gov (A. Frey), [email protected] (A. Alfaro-Núñez), [email protected] (P.H. Dutton), [email protected] (M. Thomas P. Gilbert), phillip.morin@noaa. demonstrated an increase in phylogenetic performance in terms gov (P.A. Morin). of branch support and divergence-time estimation relative to
1055-7903/$ - see front matter Published by Elsevier Inc. http://dx.doi.org/10.1016/j.ympev.2012.06.010
Please cite this article in press as: Duchene, S., et al. Marine turtle mitogenome phylogenetics and evolution. Mol. Phylogenet. Evol. (2012), http:// dx.doi.org/10.1016/j.ympev.2012.06.010 2 S. Duchene et al. / Molecular Phylogenetics and Evolution xxx (2012) xxx–xxx individual mitochondrial regions, and even nuclear markers (for olivacea (Tandon et al., 2006), E. imbricata (Tandon et al., 2006), examples in other vertebrates see Duchene et al., 2011; Okajima and C. mydas (Okajima and Kumazawa, 2010). The geographic and Kumazawa, 2010; Wang and Yang, 2011). provennance of GenBank sequences were not publically available, Although phylogenetic analyses using nuclear markers have so the D-Loop was compared to a stock assessment database to as- made important contributions in uncovering evolutionary relation- sign the most likely geographic region for these data. Table 1 lists ships in many taxa, branch support is sometimes low (Duchene all samples including outgroups, geographic origin, GenBank acces- et al., 2011). In contrast, complete mitogenomes often provide sion numbers, and bibliographic reference. highly supported trees and precise date estimates, often congruent with nuclear data, allowing for well-supported hypotheses for the 2.2. Sequencing true evolutionary histories of species. However, incomplete lineage sorting, hybridization, and past gene flow can obscure evolutionary The complete mtDNA genomes of a green turtle from Tortugero, relationships, and in some taxa the addition of independent lines of Costa Rica (haplotype Cmydas T CR); and a leatherback (haplotype evidence, such as nuclear markers or morphology is crucial to ob- D coriacea O CR) and Olive ridley (haplotype L olivacea O CR) from tain reliable phylogenetic resolution. In some cases, mitochondrial Ostional, Costa Rica were generated through Roche (454) FLX trees can be poorly supported even when using complete mitoge- sequencing of PCR amplicons. The mtDNA genome was first PCR- nomes (Talavera and Vila, 2011); in sea turtles, however, this has amplified in two long overlapping 2 kb and 15 kb fragments. Sub- not been the case (Drosopoulou et al., 2012; Shamblin et al., 2012). sequently the PCR products were purified, fragmented through In addition to phylogenetic relationships and divergence-time nebulization, converted into MID-tagged sequencing libraries and estimation, evolutionary reconstructions based on the entire sequenced as a partial fraction of an LR70 GS-FLX (Roche) run. mitogenome can benefit from genome characterization, identifica- The generated sequences were assembled into the complete tion of rates of evolution, and characterization of how these rates mitogenome using the previous green (Chelonia mydas; Genbank vary along particular genomic regions. Although some non-coding ID AB012104), hawksbill (E. imbricata; Genbank ID DQ533485) regions of the mitogenome are often assumed to evolve neutrally, and Olive ridley (L. olivacea; Genbank ID DQ486893) mitogenomes it is important to highlight the molecule’s crucial role in cellular as reference sequences. respiration. Therefore, the finding that some sites may be under Genomic DNA libraries for the rest of the samples were pre- positive selection and play an important role in environmental pared and given individual indexing sequences for multiplexing adaptation in other animals (Foote et al., 2011; Garvin et al., prior to pooling, library enrichment and sequencing as described 2011) must be taken into account in phylogenetic reconstructions in Hancock-Hanser et al. (submitted for publication). Sample li- and inferences of evolutionary processes. braries were pooled prior to capture array enrichment, and sample We address several important topics concerning sea turtle evo- libraries for all species were enriched using sequence baits from lution with large amounts of new data. The first is the phylogenetic the published mitochondrial genome of Chelonia mydas (Okajima relationships among species and the distinction of several groups, and Kumazawa, 2010). The pooled, enriched library was sequenced including the placement of N. depressus in relation to Chelonia and on the Illumina Genome Analyzer II (Illumina Inc., La Jolla, CA). the concordant phylogeographic patterns of some globally distrib- uted species. Secondly, the timing of sea turtle speciation events is key in understanding the timescale of turtle evolution and its rela- 2.3. Sequence assembly and mitogenome annotation tion to origins of geographic barriers such as the establishment of the Isthmus of Panama and changes in temperature of southern Contigs for the 24 mitogenomes were assembled using refer- ocean currents around Africa, as has been previously suggested ence sea turtle mitogenomes for L. olivacea (Tandon et al., 2006), for turtles (Dutton et al., 1999, 1996; Encalada et al., 1996; Naro- C. mydas (Okajima and Kumazawa, 2010) and E. imbricata (Tandon Maciel et al., 2008) and other marine organisms (Rosen, 1988). et al., 2006) found in GenBank (see Table 1) using Geneious v 4.7 Lastly, particular genomic features have been found in a wide vari- (Drummond et al., 2009). All mitogenomes including GenBank ref- ety of taxa, and have not been thoroughly investigated in sea tur- erences were then aligned using ClustalW v 2 (Larkin et al., 2007). tles, such as an extra base pair not translated in ND3 in birds and Gene identification and annotation were performed by import- terrestrial turtles (Mindell et al., 1998), as well as variation in ing GenBank sequence annotations into the newly assembled selective constraints across the mitogenome. mitogenomes, followed by a complete inspection of individual We have sequenced complete mitogenomes for a set of samples of gene coverage and reading-frame matching in each of the new all extant sea-turtle species, and collected sequences available from mitogenomes. GenBank to produce a large mitogenome phylogeny of these taxa. Samples from across geographic ranges have been included for 2.4. Phylogenetic models and mitogenome characterization several species to compile the genetic diversity and elaborate on intra-specific phylogeographic patterns and diversification events. Complete mitogenomes for terrestrial turtles to be used as out- Different molecular clock and phylogenetic frameworks were groups, Chelydra serpentina and Macrochelys temminckii (Nie and tested, and provide a basis for further mitogenomic studies in these Yan, 2006), were downloaded from GenBank and aligned with all taxa in the form of secondary calibrations (for a discussion on sec- sea turtles (including GenBank sequences) using Clustal W v2, pro- ondary calibrations see: Ho and Phillips, 2009; Ho et al., 2008). Fur- ducing a 17056 bp alignment. thermore, we explore particular characteristics of the mitogenome Individual genes and non-coding regions were extracted from and scan for codon sites under different selective constraints from the alignment (producing 39 partitions) according to the imported a structural and phylogenetic perspective. GenBank annotations. Reading frames were visually inspected and base frequencies and proportions of variable sites were estimated 2. Methods using the APE package v2.8 (Paradis et al., 2004). In order to avoid possible frameshift due to gene overlap (between 3 and 10 bp) 2.1. Sampling and geographic coverage such as in ATP8 and ATP6, extracted regions were concatenated after verifying their reading frames, producing a final alignment A total of 24 sea turtle samples from known localities were se- of 17094 bp. Although this procedure artificially increased the quenced and combined with additional GenBank sequences for L. alignment length by 38 bp, due to overlapping sites, it is effective
Please cite this article in press as: Duchene, S., et al. Marine turtle mitogenome phylogenetics and evolution. Mol. Phylogenet. Evol. (2012), http:// dx.doi.org/10.1016/j.ympev.2012.06.010 S. Duchene et al. / Molecular Phylogenetics and Evolution xxx (2012) xxx–xxx 3
Table 1 List of all sequences used, geographic origin, Sample ID, haplotype name, sequence length, sequence accession number (ACCN), and bibliographic reference. Locations marked with represent the inferred geographic origin of the sample using a turtle D-Loop stock assignment database. à Species Geographic origin Sample ID Haplotype name (Fig. 2 Sequence Reference ACCN key) length Lepidochelys North Pacific, Pelagic (NE Pacific ) SWFSC- L olivacea HI (A) 16719 This study à olivacea 55352 Lepidochelys Caribbean (International Waters) SWFSC- L olivacea Car (B) 16719 This study olivacea 78920 Lepidochelys Ostional, Costa Rica CGG-01 L olivacea OCR (C) 16718 This study olivacea Lepidochelys Indo-Pacific , See L olivacea Ind1 (D) 16808 Tandon et al. (2006) AM258984 à olivacea Reference Lepidochelys Indo-Pacific See L olivacea Ind2 (E) 16808 Tandon et al. (2006) DQ486893 à olivacea Reference Eretmochelys Kamehame, Hawaii, USA SWFSC- E imbricata HI (F) 16474 This study imbricata 5787 Eretmochelys Singapore SWFSC- E imbricata S (G) 16489 This study imbricata 61392 Eretmochelys Tortuguero, Costa Rica SWFSC- E imbricata T CR (H) 16472 This study imbricata 72489 Eretmochelys Pacific/Indian ocean See E imbricata Pa In (I) 16478 Tandon et al. (2006) DQ533485 à imbricata Reference Eretmochelys Pacific/Indian ocean See E imbricata Pa In (I) 16478 Tandon et al. (2006) NC_012398 à imbricata Reference Chelonia mydas Yap, Federated States of Micronesia SWFSC- C mydas Mi Mal (J) 16435 This study 71270 Chelonia mydas Archipielago de Revillagigedo, Mexico SWFSC- C mydas RMX (K) 16435 This study 13768 Chelonia mydas French Frigate Shoals, Hawaii, USA SWFSC- C mydas HI (L) 16435 This study 8855 Chelonia mydas Galapagos Islands, Ecuador SWFSC- C mydas Ec (M) 16435 This study 54903 Chelonia mydas Karpaz, Cyprus SWFSC- C mydas Cyp (N) 16440 This study 9277 Chelonia mydas Sipadan, Malaysia SWFSC- C mydas Mi Mal (J) 16435 This study 28666 Chelonia mydas Tortuguero, Costa Rica CGG-01 C mydas T CR (O) 16495 This study Chelonia mydas Atlantic ocean See C mydas ATL (P) 16497 Okajima and Kumazawa NC_000886 à Reference (1999) Chelonia mydas Atlantic ocean See C mydas ATL (P) 16497 Okajima and Kumazawa AB012104 à Reference (1999) Dermochelys Guerrero (nesting site), Mexico SWFSC- D coriacea GMX (Q) 16680 This study coriacea 5718 Dermochelys Maputaland (nesting site), South Africa SWFSC- D coriacea USVI MSA (R) 16681 This study coriacea 9790 Dermochelys St. Croix (nesting site), US Virgin SWFSC- D coriacea USVI MSA (R) 16676 This study coriacea Islands, USA 88903 Dermochelys Ostional, Costa Rica CGG-03 D coriacea O CR (S) 16680 This study coriacea Lepidochelys kempii South Padre Island (nesting site), Texas, SWFSC- L kempii SPI1 (T) 16715 This study USA 68090 Lepidochelys kempii South Padre Island (nesting site), Texas, SWFSC- L kempii SPI2 (P) 16715 This study USA 68091 Caretta caretta Hawaii (Pelagic), USA SWFSC- C caretta HI Pe (U) 16411 This study 46603 Caretta caretta Coastal waters, Florida, USA SWFSC- C caretta FL1 (V) 16454 This study 69599 Caretta caretta Coastal waters, Florida, USA SWFSC- C caretta FL2 (W) 16337 This study 69611 Caretta caretta Peruvian coast, Peru SWFSC- C caretta HI Pe (U) 16411 This study 87410 Natator depressus Australia SWFSC- N depressus Au (X) 16281 This study 21684 Chelydra serpentina Unknown See Chelydra serpentina 16631 Nie and Yan (2006) EF122793 Reference Macrochelys Unknown See Macrochelys temminckii 16569 Nie and Yan (2006) NC_009260 temminckii Reference
in assuring correct model assignment in subsequent phylogenetic used to test models of DNA sequence evolution for the complete analyses. mitogenome, each individual region, and the three codon positions. Base frequencies, numbers of haplotypes, and proportions of The best-fitting models were selected and used for all phylogenetic variable sites and nucleotide diversities were estimated for all analyses according to the Bayesian Information Criterion (BIC) alignments using the APE v2.8 and PEGAS 0.3-3 (Paradis, 2010) (sample size of 100) since it has been shown to perform better than packages, then the Phangorn package v1.4-0 (Schliep, 2011) was other similar methods (Luo et al., 2010).
Please cite this article in press as: Duchene, S., et al. Marine turtle mitogenome phylogenetics and evolution. Mol. Phylogenet. Evol. (2012), http:// dx.doi.org/10.1016/j.ympev.2012.06.010 4 S. Duchene et al. / Molecular Phylogenetics and Evolution xxx (2012) xxx–xxx
2.5. Phylogenetic analyses Development Core Team, 2006) (available from the authors upon request), and each shuffled alignment was analyzed under the par- Maximum likelihood and neighbor-Joining trees for the com- titioned framework (that with most parameters). This procedure plete mitogenome were obtained in Garli v2.0 (Zwickl, 2006) and assigns sites randomly to any of the five partitions, making the the APE package v2.8 (Paradis et al., 2004), respectively. In both specified substitution model and partitioning scheme inappropri- cases the GTR+G model was used (as selected with the BIC), and ate, which should lead to lower marginal likelihood. If this was 100 bootstrap replicates were performed to assess statistical sup- not the case one could infer that higher likelihoods are due to an port of taxonomic groups, and the maximum likelihood analysis increase in the number of parameters and not better overall fit of was repeated 10 times to ensure convergence. the model. In order to determine the most reliable divergence times and evolutionary rates, several Bayesian molecular-clock frameworks 2.6. Phylogenetic resolution of mitochondrial regions as implemented in BEAST v1.6.2 (Drummond and Rambaut, 2007) were compared using Bayes Factors (Kass and Raftery, Phylogenetic resolution was assessed for individual genes and 1995) of the harmonic mean of the marginal log-likelihoods as pro- the complete mitogenome to estimate the amount of phylogenetic duced in Tracer v1.5 (Rambaut and Drummond, 2005). In all anal- structure and diversity detected by the different mitochondrial re- yses the following fossil-derived maximum and minimum gions. This procedure entailed estimating the majority consensus divergence times were used as calibrations, as suggested by Bowen tree of 100 neighbor-Joining bootstrap replicates for every individ- et al. (1993); Dermochelidae–Cheloniidae 100–150 Ma (Weems, ual region as well as the complete mitogenome, to construct gene 1988; Zangerl, 1980), Cheloniidae 50–75 Ma (Ernst and Barbour, trees (and a mitogenomic tree) with polytomies in clades with 1989; Weems, 1988), Caretta–Lepidochelys 12–20 Ma (Carr Jr and bootstrap values below 50%. In every case the substitution model Marchand, 1942; Zangerl, 1980) and Lepidochelys 4.5–5 Ma (Dodd was chosen according to the BIC before tree estimation. We then Jr. and Morgan, 1992; Hendrickson, 1980). All calibrations were de- used the Penny and Hendy (1985) method to estimate the topolog- fined using truncated uniform prior distributions since there was ical distance between every pair of trees, thereby quantifying the no a priori information on the probability functions for any of the differences among all topologies. This method was performed fossil data. using a custom script in R (available from the authors upon The Dermochelidae–Cheloniidae divergence is of special impor- request). tance given that this date is inconsistent (59.76 Myr younger than fossil date) with fossil-derived calibrations using a bootstrap meth- 2.7. Detection of possible sites under selective constraints od across a turtle tree, including terrestrial taxa, and spanning back to 210 Ma (Near et al., 2005). However, more recent discussions An alignment of all coding genes was used to determine suggest that calibrations are not necessarily consistent across whether selective constraints were uniform throughout the mitog- broad arrays of taxa due to rate heterogeneity in long timeframes enome and whether any particular sites showed evidence of posi- (Parham and Irmis, 2008) (but see Near et al., 2008). The molecu- tive selection. The first step was to test codon models; M0, M1a, lar-clock method we used accounts for rate heterogeneity and M2a, M3, M7 and M8, as implemented in PAML v4.4. (Yang, encompasses sea turtles only (time span of 100–150 Myr); there- 2007) (for a mitogenome example see Garvin et al., 2011). The con- fore, we consider this calibration and the fossil-derived estimate catenated alignment of coding genes and the complete mitoge- (Weems, 1988; Zangerl, 1980) appropriate. nomic ML topology were used while allowing PAML to estimate A total of three molecular-clock analyses were conducted with branch lengths, transition/transversion ratio (dN/dS), and x values the following settings: across sites (x < 1 suggests purifying selection, x = 1 suggests neu- trality, and x > 1 suggests positive selection). Nested models were (1) Single substitution model (as selected by the BIC) and strict then compared (M0 vs. M1a, M1a vs. M2a, M0 vs. M3, and M7 vs. clock for the complete mitogenome (no partitioning). M8) using a likelihood-ratio test (LRT), and codon sites under posi- (2) Single substitution model (as selected by the BIC) and tive selection were determined based on the best-fitting models. relaxed log-normal clock (no partitioning). Since the PAML approach focuses on estimation of dN/dS ratios (3) Five partitions corresponding to the combined 12S and 16S per site and does not identify significant changes in protein struc- regions, the three codon positions for all coding genes and ture and function, we conducted an additional statistical analysis the tRNA regions (as individual partitions), and the com- based on the magnitude of changes in amino-acid properties due bined D-Loop and Stem-Loop (a common feature of reptile to non-synonymous mutations. This method was employed as mitogenomes; Jung et al., 2006). Each partition was assigned implemented in the TreeSaap v 3.2 program (Woolley et al., a separate substitution model and relaxed log-normal clock 2003), which estimates a score (Z) and statistical significance of to incorporate rate heterogeneity across the mitogenome, as amino-acid changes for 20 chemical properties including polarity, this has been shown to increase performance of Bayesian structure tendencies, bulkiness, isoelectric point, and others. phylogenetic analyses (Ho and Lanfear, 2010). 2.8. Mapping of variable sites in proteins BEAST analyses described above were run in the University of Oslo Bioportal web-based service (Kumar et al., 2009) with the fol- In order to find the location of potential positively-selected sites lowing settings: Chain length of 150,000,000, sampling every 600, in the proteins coded by each gene, we constructed 3D models and additional second runs to ensure convergence and ESS values using homology modeling. A high resolution (3 Å) crystallographic of at least 1000 for all parameters. structure for the membrane domain of the respiratory Complex I Since the harmonic mean of the marginal likelihood has been for Escherichia coli (Efremov and Sazanov, 2011) (PDB ID: 3RKO) criticized for favoring parameter-richer models (Beerli and Pal- was used as a template, where domains NuoA, NuoJ, NuoK, NuoN, czewski, 2010; Lartillot and Philippe, 2006), we employed a NuoM, and NuoL are homologous to ND3, ND4L, ND6, ND2, ND4, cross-validation procedure to assess reliability of Bayes-factor and ND5, respectively. Protein models were obtained in the comparisons. The nucleotide sites for the complete mitogenome SWISS-MODEL (Schwede et al., 2003) tool in the ExPASy Bioinfor- alignment were shuffled (sampled without replacement) three matics resource portal (Gasteiger et al., 2003), and viewed using times using custom R code (Ihaka and Gentleman, 1996; R the Swiss PDB Viewer v 4.0.1 (Guex and Peitsch, 1997) software.
Please cite this article in press as: Duchene, S., et al. Marine turtle mitogenome phylogenetics and evolution. Mol. Phylogenet. Evol. (2012), http:// dx.doi.org/10.1016/j.ympev.2012.06.010 S. Duchene et al. / Molecular Phylogenetics and Evolution xxx (2012) xxx–xxx 5
Superimposition of protein template and models was performed (bootstrap and posterior probabilities) (Fig. 1). This topology sup- using the ‘‘Magic Fit’’ function in the Swiss PDB viewer, and trans- ported major relationships found in previous studies based on membrane domains were identified according to Efremov and combined nuclear and mitochondrial data (Naro-Maciel et al., Sazanov (2011). 2008), but it was inconsistent with phylogenetic reconstructions using mitochondrial D-Loop and ND4L (Dutton et al., 1996), Cytb 3. Results (Bowen et al., 1993), and morphology (Zangerl, 1980). All nodes in Fig. 1 within and between species had bootstrap and poster- 3.1. Phylogenetic analyses ior-probability supports of 100% and 1.00, respectively, except within D. coriacea (within node VI), where the intra-specific haplo- Contig assembly for the 24 mitogenomes produced in this study type relationships had a low support of 47% and 0.44 (Fig. 1). yielded complete mitogenome lengths between 16281 and One finding of particular importance was high support for N. 16719 bp (Table 1). The complete mitogenome alignment of the depressus as the sister taxon to C. mydas. Previous studies based 32 sequences (24 from this study plus eight GenBank sequences) on mitochondrial 12S and 16S, and nuclear markers BDNF, Cmos, revealed a total of five shared haplotypes within the species C. R35, Rag1, and Rag 1 (Naro-Maciel et al., 2008) have supported this caretta, E. imbricata, C. mydas, and D. coriacea (see Table 1 for relationship, whereas D-Loop, ND4, and tRNA data have placed this mitogenomic haplotype naming in this study). This revealed a pro- species as the sister taxon to the clade containing Eretmochelys, portion of 0.83 (25/30) unique sea turtle haplotypes (number of Lepidochelys and Caretta (Dutton et al., 1999). unique haplotypes/total number of samples), and nucleotide diver- There was a common phylogeographic pattern for three of five sities (mean proportion of variable sites in pairwise comparison/ globally distributed species (E. imbricata, C. mydas and D. coriacea). alignment length) of 0.0078 (variance = 0.0033) for C. caretta, Phylogenetic groupings show two clades consisting of haplotypes 0.014 (variance = 0.005) for L. olivacea, 0.0002 (variance = 0.0002), from the geographic range extremes: The Atlantic and Indian, for L. Kempii, 0.011 (variance = 0.004) for E. imbricata, 0.006 (vari- and Pacific ocean regions (see color coding in Figs. 1 and 2), as sug- ance = 0.0017) for C. mydas, and 0.00036 (variance = 0.00016) for gested by previous studies (Bowen et al., 1998, 1994; Bowen and D. coriacea. Karl, 2007; Dutton et al., 1999; Encalada et al., 1996). In contrast, Model testing for the complete mitogenome showed a prefer- C. caretta did not display phylogenetic concordance with current ence for GTR+G as the best substitution model. The proportion of geographic distributions, given the high support in node XII variable sites was different among regions, the D-Loop having the (Fig. 1 and Table 2) for the Pacific haplotype (C caretta HI Pe) being highest variability, and the Stem-loop having the lowest (see nested within two Atlantic samples (C caretta FL1 and C caretta Fig. S1 for gene positions). Base frequencies were not homoge- FL2), with a median TMRCA of 2.37 Million Years Before Present neous among regions; the G content was particularly variable (Ma) (1.24–3.89 Highest Posterior Density (HPD)), as shown in (Table S1). Table 2. Within L. olivacea, the major split was approximately Maximum likelihood and all Bayesian phylogenetic analyses 2.7 Ma (2.40–3.36 HPD) between Indian Ocean samples and all revealed the same topology with comparable support values others, with samples from the Pacific clustering with high support
Fig. 1. Chronogram for complete mitogenomic analysis with haplotype key for Fig. 2. Branch support is shown for Posterior probability/Bootstrap support (maximum likelihood) only for branches where these values were below 0.99 and 95, respectively. Roman numbering corresponds to nodes listed in Table 2. Tip label colors represent haplotype geographic distribution as shown in Fig. 2.
Please cite this article in press as: Duchene, S., et al. Marine turtle mitogenome phylogenetics and evolution. Mol. Phylogenet. Evol. (2012), http:// dx.doi.org/10.1016/j.ympev.2012.06.010 6 S. Duchene et al. / Molecular Phylogenetics and Evolution xxx (2012) xxx–xxx
Fig. 2. Map with key for mitogenomic haplotypes in Fig. 1 and shaded with corresponding colors from Fig. 1 for the two main geographic regions: Atlantic and Indian, and Pacific oceans. Haplotypes marked with represent the inferred geographic origin of the sample using a turtle D-Loop stock assignment database. Ã
Table 2 Calibrations used in molecular clock analyses and posterior TMRCA estimates.
Taxonomic group Node in Fig. 1 Prior (MYBP) Median (MYBP) 95% HPD
Lepidochelys olivacea (haplotypes L_olivacea_O_CR, L_olivacea_HI, and L_olivacea_Car)I ÃTree prior 0.23 0.077–0.50 Chelonia mydas II ÃTree prior 3.09 1.76–4.87 Chelonia + Natator III ÃTree prior 36.43 21.92–52.51 Chelonidae IV 50.00–75.00 55.68 50.00–67.44 Dermochelidae + Chelonidae V 100.00–150.00 102.63 100.00–111.58 Dermochelys coriacea VI ÃTree prior 0.17 0.06–0.35 Eretmochelys imbricata VII ÃTree prior 5.63 3.44–8.85 Lepidochelys kempii VIII ÃTree prior 0.066 0.0082–0.17 Lepidochelys olivacea IX ÃTree prior 2.71 2.40–3.36 Lepidochelys X 4.50–5.00 4.84 4.56–5.00 Caretta caretta XI ÃTree prior 4.09 2.38–6.43 Caretta caretta (haplotypes C caretta HI PE and C caretta FL1) XII ÃTree prior 2.37 1.24–3.89 Caretta + Lepidochelys XIII 12.00–20.00 18.62 15.50–20.00
Table 3 Mean rate and coefficient of variation for partitions in Bayesian phylogenetic analyses.
3 Partition Mean rate (10À mutations/site MY) Coefficient of variation  Median 95% HPD Median 95% HPD 12S + 16S 0.88 0.70–0.99 0.85 0.49–1.58 First codon sites 1.71 1.44–1.97 0.88 0.64–1.18 Second codon sites 1.19 1.00–1.36 1.59 1.02–2.71 Third codon sites 1.86 1.56–2.13 0.67 0.46–0.93 D-Loop + Stem Loop 3.24 2.67–3.81 1.11 0.83–1.53
(node I in Table 2 and Fig. 1) with a sample from the Caribbean Molecular-clock analyses also permitted estimation of mean (Fig. 1), a pattern that has also been described based on shorter rates (substitutions/site/Myr) and a measure of clock-like behavior mitochondrial sequences (Bowen et al., 1998; Shanker et al., 2004). (coefficient of variation) of mitogenomic partitions (Table 3). The Of the three molecular clocks tested, the five-partition model fastest rate was found in the non-coding D-Loop + Stem-Loop re- 3 was selected according to a log Bayes Factors of 375.11 compared gions at 3.24 10À (2.67–3.81 HPD), and the slowest in the À  3 to the relaxed-non-partitioned model (second best performing; non-coding 12S + 16S at 0.88 10À (0.7–0.99 HPD). Since the cod-  Table S2). Reliability of the Bayes Factors clock comparison was ing regions were partitioned by codon positions, our estimates confirmed by the 3 randomized-site analyses, where the log-likeli- indicate that mean rate of third positions is the fastest 3 hoods were lowest among relaxed-clock analyses (log Bayes Fac- (1.86 10À (1.56–2.13 HPD)) and second positions is the slowest  3 tors between 483.69 and 476.72). (1.19 10À (1.00–1.36 HPD)), as expected from previous studies À À  Using the five-partitioned clock model, we estimated the time to on mitochondrial rates in reptiles (Jiang et al., 2007) and other ver- the most recent common ancestor (TMRCA) for all nodes in Table 2 tebrates (Nabholz et al., 2009). and Fig. 1. Median TMRCAS for species were between 0.066 Ma Previous estimates of mitochondrial substitution rates in mar- 3 (0.0082–0.17 HPD) for L. kempii, to 5.63 (3.44–8.85 HPD) for E. ine turtles range from 4 10À using Cytb (Bowen et al., 1993), 3  3 imbricata, whereas that for all marine turtles (families Cheloniidae to 1.2 10À (Encalada et al., 1996) and 6.7 10À Mutations/   and Dermochelyidae) was 102.63 (100.00–111.58 HPD). Site/Myr for D-Loop (Dutton et al., 1999). Although our methods
Please cite this article in press as: Duchene, S., et al. Marine turtle mitogenome phylogenetics and evolution. Mol. Phylogenet. Evol. (2012), http:// dx.doi.org/10.1016/j.ympev.2012.06.010 S. Duchene et al. / Molecular Phylogenetics and Evolution xxx (2012) xxx–xxx 7 employ more mitochondrial data (complete mitogenomes vs. sin- low RMS value of 0.7 Å. In both cases, codon site 169 was within gle genes) and different clock models (relaxed vs. strict), our rate an alpha helix (Fig. 3a). Fig. 3b and c show the predicted (ND4) estimates are comparable to those in previous reports (except for and template (NuoM) structures, the approximate path of the pro- the 12S + 16S regions), but exhibit a narrower range than those ton translocation channel (Efremov and Sazanov, 2011), and the based on smaller individual segments. location of site 169 (according to colors in Fig. S4). Although this When comparing phylogenetic resolution of the different mito- site is spatially close to the channel, the predicted structure is chondrial genes we observed that information content in terms of not considered a precise assessment of the effect of mutations at topologies is not uniformly distributed throughout the mitoge- this site. nome (a dendrogam of the distances among all topologies is shown in Fig. S2). Given each topology was estimated with a different dataset (partition) we could not determine whether any of the dif- 4. Discussion ferences were significant according to topology tests such as Shi- modaira and Hasegawa (1999), however we could establish Our results support previously suggested relationships among information content for partitions relative to the complete mitog- sea turtle species, notably the placing of N. depressus as the sister enome. The D-Loop, ND2, and 16S produced the most similar trees taxon to Chelonia (Naro-Maciel et al., 2008), rather than to the to the mitogenome tree. Other genes, such as ATP8, COX2, 12S, and clade comprising Eretmochelys, Lepidochelys, and Caretta (Dutton ND4 produced very different topologies and lower resolution. et al., 1999). This result is relevant in supporting phylogenetic rela- tionships within the family Cheloniidae, particularly the exclusion 3.2. Mitogenome features and possible selective constraints of N. depressus from the subfamily Carettini. Our exploration of the phylogenetic resolution of different Gene annotations as applied from published turtle mitoge- mitochondrial regions (Fig. S2) potentially explains the contrasting nomes in GenBank revealed several important features of the mar- phylogenetic relationships inferred from other mitochondrial ine-turtle mitogenome. As previously reported in birds and some markers in previous studies (Dutton et al., 1999; Naro-Maciel turtles (Mindell et al., 1998), there was an extra nucleotide causing et al., 2008). The congruence of the mitogenomic and nuclear– a frame shift in ND3 at nucleotide site 175 in most samples mitochondrial topology from this study and Naro-Maciel et al. (Table S3). However, the most striking finding was a change in (2008), respectively, demonstrates the importance of large the length of ATP8 caused by a point mutation in all D. coriacea, amounts of molecular data in resolving the turtle evolutionary four C. mydas (from the Caribbean, Cyprus, and Atlantic ocean), tree. Although mitochondrial markers are linked (Rand, 2001) two E. imbricata (from the Pacific–Indian region), and two L. oliva- and represent a single gene tree, using single regions or small por- cea (Indo-Pacific). This mutation eliminates a stop codon and in- tions of the mitogenome can produce poorly supported trees and creases the range of the gene until the next stop codon in the sometimes apparently conflicting estimates of that gene tree ATP8 reading frame (site 186 in Fig. S3, common to all sequences), (Duchene et al., 2011). Therefore, complete mitogenomes as used implying an additional 21 bp in ATP8 in the overlapping region be- here should provide a valuable source of data for phylogenetic tween ATP8 and ATP6 (as shown in Fig. S3 and Table S3). To date, no reconstructions with more reliable phylogenetic structure. high-resolution crystallographic structures are available for pro- We found some differences between our TMRCA estimates and teins coded by these genes, so it is not possible to show how this those of other studies, notably Naro-Maciel et al. (2008), who esti- mutation would affect structure and function. mated the Pacific and Atlantic C. mydas divergence time at 7 Ma PAML model comparisons provided evidence for possible sites (1.92–13.47 HPD), whereas our estimate was 3.09 (1.76–4.87). This subject to adaptive evolution. The Likelihood Ratio Test (LRT) for difference in the estimates of the TMRCA could reflect different lin- model comparisons M0 (same x for all sites) vs. M1a (x varies eages that may have been sampled in both studies. Nevertheless, among sites with two discrete categories x0 < 1 and x1 = 1), and given that calibrations were very similar, any differences and in- M0 vs. M3 (x varies among sites with three discrete categories crease in accuracy are likely due to the addition of molecular data of estimated x, x0, x1, x2) demonstrated better performance for (Duchene et al., 2011). Although an increase in accuracy (narrower models M1 and M3, respectively, suggesting heterogeneity in error bars) does not necessarily imply higher precision (reliability selective constraints across the coding regions. Positive selection in the estimation of the true divergence time), complete mitoge- was detected in one out of the two LRT model comparisons that ac- nomes have been shown provide more reliable divergence times counted for x > 1: M1a vs. M2a, and M7 vs. M8. Model M2a (x var- than single mitochondrial markers (Duchene et al., 2011; Ho and ies among sites with three categories of x: x0 <1, x1 = 1, x2 > 1) Lanfear, 2010). was not statistically distinguishable from M1a, but in the case of Although our sampling consisted of 10 or fewer samples per M7 (x has a beta distribution for x < 1) vs M8 (x has a beta distri- species, the wide geographic coverage permitted phylogeographic bution like M7, and an additional class for x > 1), model M8 per- inferences across all of the globally-distributed species. There formed better (Table S4). was clear differentiation between Indo-Pacific and Atlantic popula- The only site in model M8 with a significant x value suggestive tions in three out of five of the species with cosmopolitan distribu- of positive selection was found in codon site 169 of the ND4 gene tions (all but N. depressus and L. kempii), as observed by other (nucleotides 505–507) (Fig. S4), with a probability of positive authors (Dutton et al., 1999; Naro-Maciel et al., 2008), pointing selection (x > 1) of 0.97 and an x estimate of 1.480 ± 0.127. Anal- to the Americas as a barrier for migration in some sea turtles (E. ysis of changes in amino-acid properties in TreeSaap revealed that imbricata, C. mydas, and D. coriacea, but see below). D. coriacea dis- this codon site had 20 amino-acid properties with significant Z played this distinction with poor branch support, likely due to its scores (P < 0.001), the highest of any codon site (Fig. S5). more recent range expansion and little genetic differentiation, as SWISS-MODEL was used to obtain a structural prediction for the suggested by Dutton et al. (1999) (median TMRCA of 0.17 Ma, com- ND4 subunit. The structure obtained using the E. coli NuoM tem- pared to a median of 3.09 Ma in Chelonia). 11 plate had a mean Z-score of 6.75, an E-value of 2.48 10À , C. caretta samples were more divergent within the Atlantic than À Â and 65% sequence identity. Although the Z-score and E-value ob- between the Atlantic and Pacific, and in L. olivacea the only Atlantic tained suggest a poor structural prediction (Bowie et al., 1991), sample was nested within those from the Pacific. It is likely that superimposition of the template and model showed remarkable the current distribution of these two species was not shaped by similarities in the alpha helices and beta sheets, supported by a the same geographic barriers as the three other cosmopolitan
Please cite this article in press as: Duchene, S., et al. Marine turtle mitogenome phylogenetics and evolution. Mol. Phylogenet. Evol. (2012), http:// dx.doi.org/10.1016/j.ympev.2012.06.010 8 S. Duchene et al. / Molecular Phylogenetics and Evolution xxx (2012) xxx–xxx
(a) (b) (c)
Fig. 3. (a) Superimposition of structures. White corresponds to complete superimposition. Red and blue are respectively, turtle and E. coli residues only. (b and c) Turtle and Escherichia coli protein structures. Helixes are colored from blue to red, representing N-C terminal direction. In all proteins the red arrow points to amino-acid site 169. species. The phylogentic relationships between haplotypes of these et al., 2001) inferred for birds and widely used in other vertebrates, species is evidence of a possible connection between Atlantic and are an overestimate for the turtle mitochondrial clock by as much Pacific groups, possibly across the southern tip of Africa after the as an order of magnitude. This supports previous claims of a slower closing of the Panama isthmus. These contrasting patterns indicate molecular clock for the Testunidates (Avise et al., 1992). Molecular that the biogeographic history of marine turtles has been shaped clocks that use these canonical rates therefore tend to underesti- by different events; and barriers to geneflow are not the same mate divergence times (Ho et al., 2008). for all species. Changes in climatic conditions over the past 1 Ma Moreover, studies in cetaceans have found similar mitogenomic 3 have been particularly influential for geographic genetic patterns rates, ranging from to 2.6 10À in baleen whales (Jackson et al., 3  in C. caretta (Bowen et al., 1994; Encalada et al., 1998), and proba- 2009) to 3.9 10À in killer whales (Duchene et al., 2011; Morin  bly L. olivacea (Bowen et al., 1998; Shanker et al., 2004). Geographic et al., 2010). Summing these estimates from independent lines of genetic variation is much older in the other species (except for D. evidence may indicate that long-lived organisms with relatively coriacea, as discussed above). For example, divergence dates as large sizes may have more slowly evolving mitochondrial genomes old as 5.63 Ma (3.44–8.85 HPD) are needed to explain geographic (Jackson et al., 2009). genetic variation in E. imbricata. Complete mitochondrial genomes reveal several new features Timing of speciation and diversification events narrows hypoth- of sea turtle evolution. Most importantly, phylogeography of sea eses of possible geologic events driving evolution of some sea-tur- turtles follows a clear geographic differentiation for some species, tle species. Notably, the divergence between Pacific and Atlantic yet migration between ocean basins is likely for L. olivacea and C. clades of Chelonia mydas and E. imbricata occurred after the cooling caretta. Although more data is likely to yield similar phylogenetic of southern ocean waters in the mid to late Miocene (between 17 patterns to those we report (Karl et al., 2012), future research and 6 Ma) (Rogl, 1998), and possibly coincided with the closing of can uncover past migration patterns that may have shaped the the Panama isthmus (between 5 and 2.5 Ma) (Farrell et al., 1995). geographic genetic distribution of these species with nuclear Our evidence also supports L. olivacea and D. coriacea diverging markers. after the closing of the Panama isthmus in the Pliocene. Events such as these could have shaped current geographic distributions Acknowledgments by restricting gene flow between Atlantic and Pacific populations. Although some mitogenomic features may be the result of Samples archived in the US National Marine Fisheries Service adaptive evolution, most of the adaptive variation, as detected by (NMFS) Marine Turtle Molecular Research Sample Collection at the software used, refers to fixed differences among populations the Southwest Fisheries Science Center were collected under the or species. Therefore, the possibility that this variation is the result respective National authorizations and CITES permit conditions. of random fixed differences from stochastic processes cannot be We thank Erin LaCasella, Amanda Bowman, Gabriela Serra-Valente ruled out. Interestingly, the vast majority of coding-sites in the for laboratory assistance. For providing samples we thank the mitogenome have very low variability, suggesting strong selective Hawaiian Islands National Wildlife Refuge, US Fish and Wildlife constraints in a large proportion of the molecule and low overall Service, NMFS Pacific Islands Fisheries Science Center, NMFS-Paci- variation. fic Islands Regional Office Fisheries Observer Program, Galapagos Our data support previous rate estimates of single mitochon- National Park, Charles Darwin Research Foundation, SEMARNAT, drial regions (Bowen et al., 1993; Dutton et al., 1999; Encalada MINAE, US National Park Service, Padre Island National Seashore, et al., 1996), and although the rate is not constant throughout Sandy Point National Wildlife Refuge, US Virgin Islands Depart- 3 3 the mitogenome, it ranges between 0.7 10À and 3.81 10À   ment of Planning and Natural Resources, Natal Parks Board, George substitutions/site/Myr (inferred from HPD extremes of 12S–16S Balazs, Ana Barragan, Brian Bowen, Arturo Ceron, Jennifer Cruce, and D-Loop). These estimates indicate that canonical substitution Cheong Hoong Diong, Sheryan Epperly, Nancy Fitzsimmons, Bren- rates for mitochondrial regions used to calibrate clocks, such as dan Godley, Stacy Hargrove, Emma Harrison, George Hughes, Push- the standard 0.01 (Shields and Wilson, 1987) and 0.075 (Randi pa Palaniappan, Nelly de la Paz, Rotney Piedra, Laura Sarti, Donna
Please cite this article in press as: Duchene, S., et al. Marine turtle mitogenome phylogenetics and evolution. Mol. Phylogenet. Evol. (2012), http:// dx.doi.org/10.1016/j.ympev.2012.06.010 S. Duchene et al. / Molecular Phylogenetics and Evolution xxx (2012) xxx–xxx 9
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