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A Phylogenetic Analysis Using Recombination Activating Gene 1 (RAG-1) and an Examination of Four Distinguishing Characters to Determine the Placement of the Anapsids within the Amniota Clade. Austin Iovoli, Steven Cole, Erica Meader, Danielle Reber.
Department of Biology, Susquehanna University, Selinsgrove, PA 17870.
Abstract We investigated the taxa of the Amniota clade in order to determine the phylogentic placement of Anapsids among the Mammal, Squamata, Bird and Crocodilia clades. In addition, we examined the different relationships within the Anapsid clade. The Anapsids are unique because of several highly derived features. Using recombination activating gene 1 (RAG-1), a maximum likelihood tree and a neighbor-joining tree were constructed. These molecular trees were used to trace several morphological characters that were essential to defining the relationships between the Anapsids and other Amniota. The characters examined were the fusion of the ribs and vertebrae to form a carapace, the ability to temperature regulate the sex of offspring, the number of temporal fenestrae, and the presence of mesoplastra bones. The molecular and morphological trees generated indicated that the Anapsids are most closely related to the Crocodilia clade, followed closely by Birds, Squamata, and then Mammals. This suggests that the Anapsids are derived from within the Diapsids.
Please cite this article as Iovoli, A., S. Cole, E. Meader, and D. Reber. 2013. A phylogenetic analysis using recombination activating gene 1 (RAG-1) and an examination of four distinguishing characters to determine the placement of the Anapsids within the amniota clade. Euglena. doi:/euglena 1(1):34-42.
Introduction use the SRY gene to trigger male development The turtles are a unique group of amniotes (Sekido and Lovell-Badge 2008). In turtles and with a highly derived anatomy (Li et al. 2008). The crocodiles, however, a genetic trigger is not present. placement of this group within the phylogenetic tree Instead, turtles and crocodiles use temperature as a of amniotes has been a topic of discussion for a long determinant for sexual development (Chojnowski et time. Traditionally thought of as a sister clade to the al. 2012). diapsids, turtles are typically separated from other The presence of a carapace is not a character amniotic reptiles because of the temperature unique to turtles and tortoises. Invertebrates such as regulated determination of sex in their offspring and lobsters and crabs also have this derived feature. because of the ensnarement of the ribs and vertebrae What is unique about the structure in turtles is that within the carapace (Hedges 2012; Chojnowski et al. the carapace fuses with the ribs and vertebrae of the 2012). Turtles are most commonly characterized by organism (Gilbert et al. 2001). In embryonic turtles the lack of temporal fenestrae in their skulls, thus the and tortoises, a carapacial ridge begins developing on frequently used group term, anapsids (Modesto and the surface dorsal to the limb buds. This ridge Anderson 2004). Derived characters within the turtle eventually forms the edges of the carapace and clade have also lead to dispute over the phylogenetics directs the lateral formation of the ribs in the embryo within the group. The clade of turtles known as (Gilbert et al. 2001). Pleurodira, for example, is known for having an One of the most commonly analyzed additional set of dermal bones in the plastra known as features of the turtle is the skull. Within the amniotes, the mesoplastra bones (De La Fuente et al. 2001). most organisms have temporal openings in the skull Most vertebrate groups such as amphibians, called fenestrae. In mammals, only one temporal hole snakes, birds, and mammals use a mechanism called is found in the skull, and in remaining groups such as genetic sex determination to form the sex of offspring snakes, lizards, crocodiles and birds, two temporal (Chojnowski et al. 2012). For these organisms, a gene holes are present. The mammals, therefore, are or group of genes triggers a certain path of sexual referred to as synapsids while the other amniotes are development in an embryo. Mammals, for instance, considered diapsids (Meyer and Zardoya 2003).
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Turtles, on the other hand, lack temporal fenestrae Pleurodira clade, for instance, an additional set of giving them the name anapsids (Modesto and bones called the mesoplastra are present on the Anderson 2004). While this character was originally underside of the included turtles. Members of the thought of as being an ancestral feature, recent Chelonioidia and Testudinoidia clades, however, do analyses summarized by Hedges (2012) have not have these extra bones (De La Fuente et al. suggested that turtles actually derived from a diapsid 2001). lineage. The new placement of turtles within the The placement of turtles among the Amniota diapsids implies that the anapsid condition is actually has been up for debate for an extended period of a derived state (Meyer and Zardoya 2003). time. Different placements of turtles arise depending Finally, within the turtle group, variations in on the type of analysis performed: molecular or the plastron have led to various organized clades morphological (Hedges 2012). Analysis of characters within the family. The plastron, or ventral dermal such as sex determination, temporal fenestrae, rib bone of a turtle shell, is made up of different pieces fusion, and mesoplastra bones could lead to a better of dermal bone (Li et al. 2008). The arrangement of understanding of the turtle group and their relation to these bones varies from one clade to the other. In the other amniotes.
Table 1: This displays the species, group, authority, and accession number to the NCBI database for the recombination activating gene 1 (RAG-1). The NCBI accession number was used to find a DNA sequence for each species. The proposed clades show where each species belongs within the amniotes and Hoplophryne rogersi was used as an out-group to the amniotes. Species Group Authority, Year RAG 1 Myuchelys latisternum Anapsid Gray, 1867 AY687920 Chelodina longicollis Anapsid Shaw, 1794 AY687921 Chelus fimbriatus Anapsid Schneider, 1783 AY687918 Pelusios williamsi Anapsid Laurent, 1965 AY687923 Caretta Caretta Anapsid Linnaeus, 1758 FJ009032 Chelydra serpentina Anapsid Linnaeus, 1758 AY687906 Macrochelys temminckii Anapsid Troost, 1835 FJ230864 Lepidochelys olivacea Anapsid Eschscholtz, 1829 FJ039982 Mauremys reevesii Anapsid Gray, 1831 HQ442404 Heosemys spinosa Anapsid Gray, 1830 AY687913 Trachemys scripta Anapsid Thunberg, 1792 AY687915 Sacalia bealei Anapsid Gray, 1831 HQ442391 Alligator mississippiensis Crocodilia Daudin, 1802 AF143724 Gavialis gangeticus Crocodilia Gmelin, 1789 AF143725 Crocodylus siamensis Crocodilia Schneider, 1801 EU375508 Crocodylus porosus Crocodilia Schneider, 1801 EU375509 Harpactes diardii Birds Temminck, 1832 AY625243 Sylvia nana Birds Hemprich & Ehrenberg, 1833 AY057033 Lanius excubitor Birds Linnaeus, 1758 AY443293 Struthidea cinerea Birds Gould, 1837 AY443335 Eunectes murinus Squamata Linnaeus, 1758 HQ399517 Epicrates alvarezi Squamata Baez & Nader, 1964 HQ399522 Trachyboa boulengeri Squamata Peracca, 1910 EU402863 Phymaturus palluma Squamata Molina, 1782 JF806209 Calotes emma Squamata Gray, 1845 JF806189 Chalarodon madagascariensis Squamata Peters, 1854 FJ356745 Tonatia bidens Mammals Spix, 1823 AF203753 Noctilio albiventris Mammals Desmarest, 1818 AF447509 Melogale personata Mammals Saint-Hilaire, 1831 EF987988 Hoplophryne rogersi Amphibia Barbour & Loveridge, 1928 EF396089
Materials and Methods Birds, six species from the Squamata, and three The sample size consisted of twenty-nine species from the Mammals (Table 1). species (Table 1) from within the Amniota clade and Using the National Center for one Amphibian species, Hoplophryne rogersi, as an Biotechnology Information (NCBI) online database, out-group. An amphibian species was chosen as an sequences for the recombination activating gene 1 out-group because they are the closest related group (RAG-1) were found for each of the species to the Amniota. Within the Amniota, the study analyzed. These sequences for RAG-1 were then included twelve species from the Anapsids, four uploaded to Molecular Evolutionary Genetic species from the Crocodilia, four species from the Analysis (MEGA5) to generate phylogentic trees
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based on molecular data (Tamura et al. 2011). The each phylogenetic analysis, a bootstrap method of sequences were then aligned by ClustalW and 1000 bootstrap replications was used. The bootstrap trimmed to 1317 base pairs. This ensured the values generated through this aid in supporting the sequences were correctly lined up with each other accuracy of the results. and cut them all to the same length. Following this, A data matrix was constructed to show the the RAG-1 sequences were aligned again by progression of several morphological characters ClustalW to ensure they are appropriately paired. across the species sampled (Table 2). These Once the sequences were aligned, the species characters include the fusion of the ribs and vertebrae sampled were ready to be analyzed by statistical to form a carapace, the ability to temperature regulate estimation methods. the sex of offspring, the number of temporal With the aligned RAG-1 nucleic acid fenestrae, and the presence of mesoplastra bones. sequences for every sampled species, Maximum These characters were chosen in order to trace the Likelihood (Figure 1) and Neighbor-Joining (Figure evolutionary relationships between the Anapsids with 2) phylogentic trees were generated on MEGA5. We the rest of the Amniota clade. Using MEGA5, a constructed our Maximum Likelihood tree (Figure 1) consensus tree based on the Maximum Likelihood using the Tamura-Nei model. In Figure 2, a tree (ML) and Neighbor-Joining (NJ) trees was based on the distance between each pair of species constructed that tracks the different character states was generated. A Maximum Composite Likelihood of these distinguishing features. model was utilized for the Neighbor-Joining tree. In
Table 2: Comparison of four morphological characters for various species within the Amniota clade. The characters include: number of temporal fenestrae pairs (synapsid, diapsid, or anapsid); presence of fused ribs and vertebrae to form a carapace; presence of mesoplastra bones; and presence of temperature dependant sex determination. These characters were chosen to differentiate the clades within the amniotes.
Synapsid/Diapsid/Anapsid Fusion of Ribs and Vertebrae Presence of Temperature Dependent Species Skull Configuration to form Carapace Mesoplastra Bones Sex Determination Myuchelys latisternum Anapsid + + + Chelodina longicollis Anapsid + + + Chelus fimbriatus Anapsid + + + Pelusios williamsi Anapsid + + + Caretta Caretta Anapsid + - + Chelydra serpentina Anapsid + - + Macrochelys Anapsid + - + temminckii Lepidochelys olivacea Anapsid + - + Mauremys reevesii Anapsid + - + Heosemys spinosa Anapsid + - + Trachemys scripta Anapsid + - + Sacalia bealei Anapsid + - + Alligator Diapsid - - + mississippiensis Gavialis gangeticus Diapsid - - + Crocodylus siamensis Diapsid - - + Crocodylus porosus Diapsid - - + Harpactes diardii Diapsid - - - Sylvia nana Diapsid - - - Lanius excubitor Diapsid - - - Struthidea cinerea Diapsid - - - Eunectes murinus Diapsid - - - Epicrates alvarezi Diapsid - - - Trachyboa boulengeri Diapsid - - - Phymaturus palluma Diapsid - - - Calotes emma Diapsid - - - Chalarodon Diapsid - - - madagascariensis Tonatia bidens Synapsid - - - Noctilio albiventris Synapsid - - - Melogale personata Synapsid - - - Hoplophryne rogersi Synapsid - - -
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Results from morphological characters and fossil records Figure 1 is split into five major clades. (Hedges and Poling 1999). Contrary to this, our Additionally, the Anapsid clade is further divided proposed phylogeny (Figure 3) based on molecular into three clades, corresponding to the Pleurodira, and morphological evidence suggests the Anapsids Chelonioidea, and Testudinoidea. There are 29 are derived from the Archosauria. species listed in the ML tree. Figure 1 places the The Maximum Likelihood tree in Figure 1 Anapsids in a polytomy with the Squamata, places the Anapsids next to the Squamata, suggesting Crocodilia, and Birds. This is suggested by the low a sister relationship between the two. However, the support of the relationships. Within the Anapsid bootstrap values do not support this relationship and group, the divisions of the Pleurodira, Chelonioidea, our consensus tree (Figure 3) places the Squamata as and Testudinoidea clades are strongly supported. The an out-group to the Archosauria and Anapsids. In the Mammals and the Amphibian outgroup appear on the past, the Squamata and Anapsid sister clade outside tree, suggesting a distant relationship with the relationship has been widely accepted based Anapsid + Diapsid polytomy. primarily morphological characters (Hedges and In contrast, the NJ tree displayed in Figure 2 Poling 1999; Chiari et al. 2012). The morphological suggests that Anapsids are most closely related to the characters that Anapsids share with Squamata and Crocodilia and Birds. Figure 2 is separated into six other reptiles has suggested their positions are close different clades and displays several sister together within the Amniota (Chiari et al. 2012). relationships between them. The Crocodilia clade is Anapsids have traditionally been classified as either presented as a sister group to the Anapsids. This basal to all reptiles or as a part of the reptiles, making relationship has high support from the bootstrap their phylogenetic relationship within the Amniota value. Following this, the Birds are shown to be a difficult to interpret (Chiari et al. 2012; Hedges 2012; sister clade to the Crocodilia + Anapsid clade, backed Zardoya and Meyer 2001). The lack of temporal by moderate support. The Squamata then appear as a fenestration in the skull has placed Anapsids as a sister clade to the Archosauria + Anapsid clade. This sister group to reptiles in several studies (Chiari et al. is also backed by moderate support from the 2012; Hedges 2012). Additionally, Anapsids have bootstrap. Mammals and the Amphibian are again been described as having a “modified reptile body presented as distant relatives to the Anapsids. Figure plan” by Chiari et al. (2012), which indicates another 2 also has the Anapsid clades of Pleurodira, morphological character supporting a close Chelonioidea, and Testudinoidea emerge with strong relationship between the two. Our consensus support. phylogenetic tree (Figure 3), generated through molecular and morphological analysis, challenges the Discussion classic phylogeny of Anapsids and Squamata. Figure 3 shows that the Anapsids are highly derived Inferred Phylogeny Amniotes, but do not share a sister relationship with We interpret Figure 1 and Figure 2 the Squamata. Contrary to this, our consensus tree (especially Figure 2) to conclude that the Anapsids (Figure 3) suggests that the Anapsids share a sister are most closely related to crocodiles and birds, and relationship with Crocodilia. share a sister relationship with the Crocodilia. We The Neighbor Joining tree in Figure 2 places used the relationships indicated by the Neighbor- the Anapsids next to the Crocodilia, suggesting a Joining (Figure 2) tree to create a consensus tree close relationship with Archosauria (crocodiles and (Figure 3) that shows the morphological character birds grouped together). We used the relationships evolution of several derived features relevant to the suggested by Figure 2 to construct our consensus tree Amniota. Our interpretation was based on Figure 2 (Figure 3) because the relationships were strongly because the relationships had higher support from the supported by their bootstrap values. Although several MEGA5 bootstrap values. Several molecular studies morphological studies (Chiari et al. 2012; Hedges (Chiari et al. 2012; Hedges 2012; Iwabe et al. 2004; and Poling 1999) support Squamata and other reptiles Hedges and Poling 1999) favor Crocodilia and Birds as the closest relatives to Anapsids, many molecular as the sister group of the Anapsids, while other studies favor the Archosauria as the living sister studies (Zardoya and Meyer 2001; Hedges 2012) group of Anapsids (Zardoya and Meyer 2001; Iwabe have analyzed morphological data that support et al. 2004). The molecular studies that contrast the Squamata as the closest related relative. These two traditional phylogeny of Amniotes suggest that interpretations of the placement of the Anapsids have Anapsids are not derived from primitive reptiles continuously been at odds. Placing Squamata as a (Zardoya and Meyer 2001) or diapsid reptiles sister clade to the Anapsids has traditionally been a (Hedges and Poling 1999). The molecular data widely accepted phylogeny for years, due to evidence presented in the consensus tree (Figure 3) proposes
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Euglena: 2013 that Anapsids are highly derived rather than Another phylogenetic analysis of Amniotes (Iwabe et primitive. The lack of temporal fenestration in the al. 2004) examined DNA-coded protein sequences skull is presented as a character of Anapsids that has and obtained a tree topology supporting the been derived from diapsids such as Squamata and hypothesis that turtles are the sister group to Crocodilia. A particular study by Hedges and Poling Archosauria. Several emerging molecular discoveries (1999) analyzed sequences from nuclear proteins, (Zardoya and Meyer 2001; Hedges and Poling 1999; resulting in significant support (97% confidence) that Iwabe et al 2004) are consistent with the molecular suggests an Anapsid-Crocodilia sister relationship. analysis presented in Figure 3.
Figure 1: A phylogenetic tree generated through the Maximum Likelihood Method using MEGA5 (Tamura et al. 2011). This method provides a tree with the maximum likelihood of producing the correct relationships. This figure shows the phylogenetic relationship between various clades within the amniotes and further subdivides the Anapsid clade. The Anapsid, Squamata, Crocodilia, Bird, and Mammal clades are formed by the genetic data. This tree was based on recombination activating gene 1 genetic data. The sequences and accession numbers were obtained through NCBI. The tree was produced with 1000 bootstrap replications to support the accuracy of the relationships. Bootstrap values designated by a star (*) indicate values with less than 60% certainty. An amphibian, Hoplophryne rogersi, was used as an out-group to the Amniota clade.
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The phylogenetic consensus tree (Figure 3) derived Diapsids. This leaves the classical hypothesis generated from our molecular analyses was more placing Anapsids as a more basal group to be consistent with molecular studies (Hedges and Poling questioned. Our data presented by Figure 3 is most 1999; Iwabe et al. 2004) than morphological studies consistent with the molecular data placing Anapsids (Hedges 2012; Chiari et al. 2012). Over the past few as a sister clade to Crocodilia, suggesting they are decades, conflicting molecular evidence has emerged among the most highly derived Amniotes. that provides strong support that Anapsids are highly
Figure 2: A phylogenetic tree generated through the Neighbor-Joining method using MEGA5 (Tamura et al. 2011). This method produces a tree based on the distance between each pair of species. The phylogenetic relationship between the Anapsid, Crocodilia, Bird, Squamata, and Mammal clades is shown. The tree was generated using recombination activating gene 1 nucleic acid sequences from NCBI. To support the accuracy of the data, 1000 bootstrap replications were conducted. Bootstrap values designated by a star (*) indicate values with less than 60% certainty An amphibian, Hoplophryne rogersi, was used as an out-group to the Amniota clade.
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A(1) B(2)
D(1) C(1)
A(0) B(0) B(1) C(0) D(0)
Figure 3: A consensus phylogenetic tree based on the data from the Maximum Likelihood and Neighbor-Joining tree (Figure 1 and 2, respectively). This tree shows the morphological character evolution of several characters including: fused ribs and vertebrae forming a carapace (A), the number of temporal fenestrae pairs in the skull (B), temperature dependent sex determination (C), and presence of mesoplasta bones (D). For morphological characters A, C, and D, character state (0) indicates the absence of the character and character state (1) indicates its presence. For character B, character state (0) indicates a synapsid skull, character state (1) indicates a diapsid skull, and character state (2) indicates an anapsid skull. Detailed information for each character state is found in Table 2. These morphological characters separate the amniotes into several clades and further divide the Anapsids into the clades of Pleurodira, Chelonioidea, and Testudinoidea.
Character Evolution Nagashima et al. (2009) determined that initial The characters examined for each species Anapsid development conforms to the typical pattern are located in the character's taxon matrix, displayed in which all amniotes develop. However, Nagashima in Table 2. These characters were chosen based on et al. (2009) also showed in their study that during salient characters that classify the Anapsid clade and embryogenesis the Anapsids differ from other how those characters compare to those of the four amniotes in that rib growth is lateral, leading to other clades examined within the Amniotes: the Anapsid specific muscle attachments and carapace Crocodilia, Birds, Squamata, and Mammals. formation. This presence of fused vertebrae and ribs One character we observed was the fusion of forming a carapace as a protective shell is a highly ribs with vertebrae to form a carapace. The consensus derived character, distinguishing the Anapsids as a tree (Figure 3) indicates that the Anapsids are monophyletic clade within the Amniota. separated from all other Amniotes due to the fusion Another observed character was the number of ribs and vertebrae to form a carapace and plastron. of temporal fenestrae pairs among the Amniotes. The three conditions examined were anapsid, diapsid and
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Euglena: 2013 synapsid. Figure 3 shows that Mammals are Amniota groups and illuminates the highly derived separated from the other Amniotes with the presence nature of the Anapsids. of a synapsid skull. The Squamata, Birds and Crocodilia all displayed a diapsid skull, leaving the Anapsids as the only clade with an anapsid skull. The Literature Cited separation of Anapsids with their sister group, the Chiari, Y., V. Cahais, N. Galtier, and F. Delsuc. diapsid Crocodilia, supports the notion that the 2012. Phylogenomic analyses support the anapsid condition could be derived from diapsids or position of turtles as the sister group of birds synapsids through the loss of arches over time (Holt and crocodiles (Archosauria). BMC and Iudica 2013). Additionally, if Anapsids are truly Biology. 65(10): doi: 10.1186/1741-7007- relatives of Archosauria, it is possible that they lost 10-65. their skull fenestration secondarily, rather than never Chojnowski, J. L. and E. L. Braun. 2012. An having evolved it (Zardoya and Meyer 1998). Our unbiased approach to identify genes results confirm this and indicate that the Anapsids involved in development in a turtle with have lost their skull fenestration secondarily from the temperature-dependent sex determination. Diapsids. BMC Genomics. 308(13): doi: Temperature-dependent sex determination is 10.1186/1471-2164-13-308. a morphological character among the Amniota where De La Fuente, M., F. de Lapparent de Broin, and T. the gender of an offspring is dependent on the Manera de Bianco. 2001. The oldest and temperature of the surrounding environment during first nearly complete skeleton of a chelid, of embryonic development (Valenzuela 2008). This is a the Hydromedusa sub-group (Chelidae, derived character because it is only present in Pleurodira), from the Upper Cretaceous of Crocodilia and Anapsid clades. Figure 3 supports the Patagonia. Bulletin de la Societe Geologique notion that temperature dependent sex determination de France. 172(2): 237-244. is a derived character present in the Crocodilia and Gilbert, S. F., G. A. Loredo, A. Brukman and A. C. Anapsids. The presence of this character among Burke. 2001. Morphogenesis of the turtle Anapsids and Crocodilia is another indication that shell: the development of a novel structure supports they are closely related groups. in tetrapod evolution. Evolution and Figure 3 also exhibits variation within the Development. 3(2): 47-58. Anapsid clade. This is based on the presence of Hedges, S. B. 2012. Amniote phylogeny and the mesoplastra bones as an extra bone fusion, which position of turtles. BMC Biology. 64(10): functions in carapace support and development. This doi:10.1186/1741-7007-10-64. character is derived and aids in separating the Hedges, S. B., L. Poling. 1999. A Molecular Anapsids into three major groups: Testudinoidea, Phylogeny of Reptiles. Science. 283: 998- Chelonioidea, and Pleurodira. The Pleurodira are 1001. separated from Testudinoidea and Chelonioidea due Holt, J., C. Iudica. 2012. Diversity of Life. to the presence of mesoplastra bones within the
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Annual Review of Ecology, Evolution, and Methods. Molecular Biology and Evolution. Systematics. 34: 311-338. 28: 2731-2739. Modesto, S. P. and J. S. Anderson. 2004. The Valenzuela, N. 2008. Evolution of the gene network phylogenetic definition of Reptilia. underlying gonadogenesis in turtles with Systematic Biology. 53(5): 815-821. temperature-dependent and genotypic sex Nagashima, H., F. Sugahara, M. Takechi, R. determination. The Society for Integrated Ericsson, Y. Kawashima Ohya, Y. Narita, and Comparative Biology. 48(4): 476-485. and S. Kuratani. 2009. Evolution of the Warner, D.A. and R. Shine. 2008. The adaptive turtle body plan by the folding and creation significance of temperature-dependent sex of new muscle connections. Science. determination in a reptile. Nature. 451: 566- 325(5937): 193-196. 568. Sekido, R. and R. Lovell-Badge. 2008. Sex Zardoya, R. and A. Meyer. 1998. Complete determination and SRY: Down to a wink mitochondrial genome suggests diapsid and a nudge. Trends in Genetics. 25: 19-29. affinities of turtles. Proceedings of the Tamura, K., D. Peterson, N. Peterson, G. Stecher, M. National Academy of Sciences of the United Nei, and S. Kumar. 2011. MEGA5: States of America. 95: 14226-14231. Molecular Evolutionary Genetic Analysis Zardoya, R. and A. Meyer. 2001. The evolutionary using Maximum Likelihood, Evolutionary position of turtles revised. Distance, and Maximum Parsimony Naturwissenschaften. 88(5): 193-200.
Submitted 15 February 2013 Accepted 1 May 2013
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