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Euglena: 2013

Using RAG-1 and Morphological Characters To Define Phylogenetic Relationships of : Separation of and , Designating Two Novel Infraorders Cryptodira and Trionychida (Tax. Nov.) Kaitryn Ronning, Emily Beliveau, Emily McCaffery, Cierra Omlor, and Ellie Rosenblum.

Department of Biology, Susquehanna University, Selinsgrove, PA 17870.

Abstract The Anapsida, and the turtles within the Anapsida, have proven difficult to classify. This study supports several phylogenetic relationships among turtles, including the separation of the suborders Cryptodira and Pleurodira, the designation of two infraorders (Cryptoda and Trionychida) within Cryptodira, as well as the placement of Platysternon megacephalum. Using the molecular data of the recombination-activating gene (RAG-1) we generated two cladograms using the Maximum Parsimony and Maximum Likelihood methods in MEGA 5.1. A consensus tree was then constructed using these cladograms and known morphological characters. We interpreted the analysis to mean that two suborders, Pleurodira and Cryptodira, exist. However, an additional classification of infraorders Cryptoda and Trionychida was supported. The results also show that P. megacephalum is not a sister taxon to serpentina. Analysis of the RAG-1 gene and pulmonary morphological characters suggests that P. megacephalum is a member of the superfamily .

Please cite this article as: Ronning, K., E. Beliveau, E. McCaffery, C. Omlor, and E. Rosenblum. 2013. Using RAG-1 and morphological characters to define phylogenetic relationships of turtles: separation of cryptodira and pleurodira, designating two novel infraorders cryptodira and trionychida (tax. nov.). Euglena. doi:/euglena. 1(1): 1-9.

Introduction Additionally, the numbers of similarities Anapsida is a class, which traditionally has between morphological and molecular characters been defined the lack of the zygomatic arch in are very high between groups (Rieppel 2000). the (Williston 1917). Clearly, because the Shaffer et al. (1997) conducted a absence of a character is not a character in itself phylogenetic study of twenty-three taxa, there have been difficulties classifying Anapsida. representing all extant families and subfamilies Even during the early stages of classification of turtles, using the molecular character there were many issues that arose surrounding cytochrome b. This study was conducted because Anapsida. Credner (cited in Bayley 1889) the position of the turtles in relation to mammals, initially hypothesized that the beginnings of , and other is essential in order to lineage was due to a determine the condition and early in rock that was incorrectly transformations of characters (Shaffer et identified. This identification error and other al. 1997). These turtle families have been difficulties in classification lead to many classified within two suborders, Pleurodira (side- problems in the understanding of reptiles, necked) and Cryptodira (hidden-neck), since the including Anapsida (Hill 2005, Williston 1917). work of Cope in 1871. The Cryptodira, Anapsida includes the order Testudines (turtles), specifically, are poorly understood because most the only living , which are of the data is based solely on a few monophyletic. The biggest conflict in morphological characters. Their analysis of two classifying turtles appears to be the wide array of mitochondrial genes and morphological data of morphological characters studied and a 115 characters of these twenty-three taxa was discontinuity between previous investigators. informative for some sections of a cladogram, but became uninformative in deeper levels. From

1 Euglena: 2013 their data set, they found strong support of a One defining characteristic of some turtle sister-group relationship between Chelydra taxa is the presence of skin covering the serpentina and Platysternon carapace. While the majority of turtles do not megacephalum. They also found evidence have any covering on the carapace, some have an against the monophyly of Trionychoidae obvious leathery covering (Bonin et al. 2006). (Shaffer et al. 1997). Based on the interpretations Another feature is the trochlear apparatus, a of their study, they made the suggestion that a unique jaw-closing system among turtle species rapid series of radiations must have occurred (Gaffney 1975). Turtles have a main adductor about 100 million years ago that then established tendon that lands over a trochlea that changes its the families within Cryptodira. At the time of direction of movement (Gaffney 1975). This the analysis, the lack of sequenced data then led tendon contains cartilage that is classified by them to believe the use of a starburst tree would Hanken and Hall (1993) as cartilago transiliens. be a useful alternative. However, they proposed This trochlear apparatus differs among turtle that if more sequences from nuclear genes were species in that some have a true joint of the obtained, it would lead to concrete support for processus trochlearis octum and the otic chamber the proposed rapid change in phylogeny (Shaffer while others do not true joint of the processus et al. 1997). trochlearis oticum and the otic chamber Krenz et al. (2005) studied the (Gaffney1975). recombination activating gene 1 (RAG-1) of Turtles are also differentiated by the way twenty-four turtle species, twenty-three of which that they retract their neck into their shell. Some were the same taxa used by Shaffer et al. (1997). species of turtles pull their neck back into their This was done in order to further investigate the shells in an s-shape motion giving the look that ambiguous evolutionary relationships among the neck is being retracted sideways. Other turtles. Morphological characters, mitochondrial species of turtles pull their necks directly back. DNA and nuclear DNA were analyzed in order They are called “hinge-necks” because the neck to compare these species. Their analysis suggests bends slightly when it is retracted into the shell, the are monophyletic and a sister looking similar to that of a hinge (Benton 2005 group to the family Cryptodira. Even though the and Williams 1950). morphological data suggests that the Yet another way to classify turtles is to and Platysternidae families have a sister compare pulmonary morphology. Certain turtle relationship, the RAG-1 sequence illustrates that species have a visible horizontal intrapulmonary Chelydra serpentina and Platysternon septation in the medial chambers, which creates megacephalum are not sister taxa and P. a dorsal and ventral lobe. Others do not have megacephalum does not belong in the this character (Lambertz et al. 2010). Chelydridae family. Instead, they suggest that One family in particular, Carettochelys, has morphological similarities could be due to been difficult to place within turtle cladograms parallel adaptive (Krenz et al. 2005). because it is very unique both morphologically Krenz et al. (2005) chose RAG-1 because and molecularly. For example, these are the only the sequences are not saturated which makes it a turtles that have thick porcine snout (Bonin et al. good sequence for analyzing phylogenetic 2006 and Ernst et al. 1989). However, molecular relationships. Notably, Krenz et al. (2005) studies have found this group to be uncertain reported that in the RAG-1 sequences there was (Shaffer et al. 1997 and Krenz et al. 2005). no saturation in the transitions or transversions The purpose of this paper is to confirm that whereas cytochrome b, which Shaffer et al. there are two larger clades of turtles, the (1997) used, the transitions were slightly Pleurodira and Cryptodira, and suggest that saturated. Another reason why RAG-1 is a good within the Cryptodira there are two infraorders, gene for comparison is because there is minimal the Cryptoda and Trionychida. Additionally, we bias in the nucleotide base composition. In further explore the relationships of Platysternon cytochrome b there are high levels of adenine megacephalum and the Carettochelyidae and and cytosine and low levels of guanine and Trionychidae families. thymine whereas in RAG-1 there is only slightly more adenine. Finally, the RAG-1 gene Materials and Methods sequences among the selected species of turtles Twenty-five taxa of turtles were examined are relatively similar which increases the chances in this study, all of which are listed with of determining the true evolutionary authority in Appendix A. Also in Appendix A is relationships (Krenz et al. 2005).

2 Euglena: 2013 the accession number for the RAG-1 gene, the the turtle taxa could be associated with other molecular basis of this study. Among the taxa amniotes. that were studied, twenty-three of these were In order to complete this phylogenetic also used in the studies of Shaffer et al. (1997) analysis, we first obtained the RAG-1 sequenced and Krenz et al (2005). These taxa were data from an NCBI BLAST search for each originally chosen because they represent all species. These sequences were then aligned recognized turtle families and subfamilies (Ernst using CLUSTAL W in MEGA 5.1, which was and Barbour, 1989). The other two turtle taxa also used to generate a maximum parsimony and examined in this study were chosen because they maximum likelihood (using the Tamura-Nei have similar genetic characters as the other model) phylogenetic tree. For both trees, twenty-two taxa. Three outgroup taxa were also bootstrapping was set to 1000 replications. included in the analysis; a chicken, crocodile, These trees were then compared to create a and cat, which are also included with authority consensus tree. Morphological characters were and appropriate accession number in Appendix also taken into consideration in the construction A. These three outgroups were selected so that of the consensus tree. These characters are listed in Table 1.

Table 1: The morphological characters that were used in this study to generate the consensus cladogram. Character State One State Two Trochlear apparatus True joint of the processus trochlearis No true joint of processus trochlearis oticum and the otic chamber oticum and the otic chamber Neck morphology Hidden neck (retracts neck straight Side-neck (retracts neck in s-like back) motion) Snout Thick and porcine Not thick and porcine Carapace Covered with layer of skin Not covered with layer of skin Intrapulmonary Septation in medial Present Absent chambers

Results higher than the corresponding values in Figure 1. Many sections remain constant in both Figures 1 and 2, including the organization of the Discussion Pleuridae suborder. Also, species generally Figure 3, a summary cladogram, remain within the same families. The two species demonstrates the major clades among the turtles that differ the most between Figures 1 and 2 are that are most strongly supported by both Platysternon megacephalum and Carettochelys morphological and molecular data. Figure 4 is a insculpta. However, the larger phylogenetic complete analysis of Figures 1 and 2 taking organization is contradictory. These include known morphological characters into differences in placement of the Pleurodira, consideration. These depict a clear separation Trionychidae, Carettochelys, and P. between the Pleurodira and the Cryptodira, megacephalum. where the Pleurodira are the most basal of the In Figure 1, the cladogram generated using turtles. Additionally, Figure 4 shows that the maximum parsimony, there is a relationship family Trionychidae is paraphyletic and a sister where the Trionychidae are the most basal group group to the family Carettochelyidae. and the Pleurodira are nested within the Figure 4 is based more strongly on Figure 2 Cryptodira, which has very low bootstrap values. than Figure 1 because of bootstrap (BP) values. C. insculpta branches off before Trionychidae In Figure 1 the BP value associated with the and P. megacephalum is a sister group to five early separation of the Trionychidae is thirty- other taxa within Cryptodira. five, which is extremely low compared to that of In Figure 2, the cladogram generated using the Figure 2, which has a BP value of ninety- the Tamura-Nei model maximum likelihood, nine. This indicates that Pleurodira branches off there are two strongly supported large clades, the first. These results confirm the results of Krenz suborders Pleurodira and Cryptodira. Within the et al. (2005), but differ from those of Shaffer et suborder Cryptodira, C. insculpta is a sister al. (1997). In their analysis using cytochrome b, group to the family Trionychidae and P. Shaffer et al. (1997) refute the monophyly of megacephalum is sister to three other taxa. The Trionychidae. However, in our study the high bootstrap values for these relationships are

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Trionychidae. However, in our study the high character used in our study is less saturated than bootstrap values in both Figures 1 and 2 strongly cytochrome b which Shaffer et al. (1997) used, support the monophyly of Tionychidae. supporting the accuracy of our conclusions. Additionally, RAG-1, which was the molecular

Figure 1: The Maximum Parsimony cladogram generated for the twenty-five turtle species (see Appendix A) examined using the RAG-1 gene. The cladogram was generated using MEGA 5.1. This cladogram illustrates three major nodes separating the twenty-five turtle taxa and displays the Trionychidae as the most basal group. This places members of the suborder Cryptodira amongst the most basal and most derived clades of turtles with the suborder Pleurodira nested within. The most questionable taxa are Carettochelys insculpta and Platysternon megacephalum.

Euglena: 2013

Figure 2: The Maximum Likelihood (using the Tamura-Nei model) for the twenty-eight species (see Appendix A) examined using the RAG-1 gene. The cladogram was generated using MEGA 5.1. This cladogram illustrates the suborder Pleurodira to be the most basal and the suborder Cryptodira to be the most derived. The placement of Carettochelys insculpta and Platysternon megacephalum are the most questionable.

Another important instance where BP values due to the low BP value of forty-four in Figure 1. were used in constructing Figure 4 was in Morphological data were examined in order determining the proper location of the outgroups. to further support the conclusions The separation of outgroups in Figure 2 was used reached in Figures 3 and 4.

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The separation of Pleurodira and Cryptodira is Trionychidae and the rest of the Cryptodira. supported by the morphology of the trochlear Most obviously, the carapace on all species apparatus and neck. Pleurodira do not have a true within Trionychidae is covered with a joint in the trochlear apparatus and are the “side- continuous leathery skin, which explains their necked” turtles, whereas Cryptodira have a true common name, the “soft-shelled” turtles (Bonin joint and are the “hinge-necked” turtles (Gaffney et al. 2006). Our results clearly support this 1975 and Benton 2005). It is because of these separation, as shown in Figure 6. Indeed, we clear differences in morphology and genetics that suggest that, for ease of discussion, the two a current hypothesis states that the most recent groups be considered infraorders, Cryptoda and common ancestor of all turtles occurred before Trionychida, which is how they have been this proposed split of Cryptodira and Pleurodira labeled in Figure 6. (Chiari et al. 2012). One major difference between Figures 1 and While the separation of suborders 2 was the location of Carettochelys insculpta Cryptodira and Pleurodira is widely accepted, (representing the Carettochelyidae family). the separation of the family Trionychidae from While in Figure 1 it is shown as the most basal within the Cryptodira suborder has recently of the turtles, it is shown as a sister to the emerged. For the last 200 years, Trionychidae Trionychidae family in Figure 2. In Figure 1 C. has been considered a part of Cryptodira because insculpta is placed with no bootstrap value of the synapomorphies which delineate the given, so it is assumed that the BP value is Pleurodira from the Cryptodira, two of which are negligibly low. Therefore, when constructing the mentioned above. However, with developments consensus tree we placed C. insculpta in a in molecular technologies, the separation of similar position to where it is located in Figure 2, Trionychidae from the rest of the Cryptodira is locating it within the Trionychida infraorder. becoming more strongly supported (Krenz et al. This is also supported by morphological 2005). similarities, the most notable of which is the Although there are many unifying characters presence of skin covering the carapace, a of the suborder Cryptodira, there are character shared by the entire Trionychida morphological differences between the infraorder.

Figure 3: The summary consensus tree we generated from Figures 1 and 2. This tree simplifies the turtles to suborder and infraorder where appropriate. The suborder classifications are in bold and the infraorder is in plain text. The outgroup species are italicized.

The taxon Platysternon megacephalum has phylogenetic relationships between turtle taxa. been particularly challenging to place within the This method has led scientists to believe that P. evolutionary history of turtles (Krenz et al. 2005, megacephalum and Chelydra serpentina were Shaffer et al. 1997). One of the main reasons for sister taxa in the family Chelydridae (Gaffney this difficulty is that, in the past, mainly cranial 1975, Gaffney and Meylan 1988, Shaffer et al. morphological has been used for determining 1997). However, these morphological

Euglena: 2013 similarities could be due to adaptive parallel involving the RAG-1 gene, including our evolution (Krenz et al. 2005). This sister investigation, have also questioned this sister relationship is now in question based on results relationship (Krenz et al. 2005). Instead, these from nuclear genes, the mitochondrial genome, results suggest that P. megacephalum is more as well as vertebrae and pulmonary morphology closely related to the superfamily Testudinoidea (Krenz et al. 2005 and Lambertz 2010). Studies (Lambertz et al., 2010).

Figure 4: The complete consensus tree we generated based on Figures 1 and 2, and previously published literature. This tree classifies the evolutionary relationship of the twenty-eight species and notes significant morphological characters. On the right, families are indicated by smaller font, infraorders by larger font, and suborders in bold. The morphological characters are represented by numbers 1-5. (1) side neck, no true joint of processus trochlearis oticum and the otic chamber, (2) hidden neck, true joint of processus trochlearis oticum and the otic chamber, (3) carapace covered with layer of skin, (4) thick and porcine snout, (5) intrapulmonary septation in medial chambers.

Euglena: 2013

Figures 1 and 2 suggest that Platysternon Johns Hopkins University Press, Maryland. megacephalum and Chelydra serpentina are not pp. 152 sister taxa. Both Krenz et al. (2005) and Chiari, Y., V. Cahais, N. Galtier, and F. Delsuc. Lambertz et al. (2010) support this conclusion. 2012. Phylogenomic analyses support the Figure 1 suggests a sister relationship between P. position of turtles as the sister group of birds megacephalum and Testudinoidea whereas and crocodiles (Archosauria). BMC Figure 2 suggests P. megacephalum to be within Biology. 10:1-14. the Testudinoidea superfamily. Thus, in Figure Ernst, C.H., Barbour, R.W. 1989. Turtles of the 4, we placed P. megacephalum within World. Smithsonian Institution Press, Testudinoidea. This was because even though Washington, DC. the bootstrap value in the Figure 2 is thirty-four Gaffney, E. S. 1975. A Phylogeny and at the node where P. megacephalum separates. Classification of The Higher Categories of Lambertz et al. (2010) suggest that P. Turtles. Bulletin of the American Museum megacephalum is within Testudinoidea due to of Natural History. 155:387-436. pulmonary morphology, specifically the Gaffney, E.S. and Meylan, P.A. 1988. The presence of visible horizontal intrapulmonary Phylogeny and Classification of the septation in the medial chambers. Other . Oxford University Press, New pulmonary synapomorphies between the York. 33:157-178. Testudinoidea (which contains the family Hanken, J. and B.K. Hall. 1993. The Skull, ) and P. megacephalum include fewer Volume 2: Patterns of Structural and chambers, the first lateral chamber is larger, and Systematic Diversity. The University of a greater stabilization from the post-pulmonary Chicago Press, Chicago. septum. C. serpentina does not share any of Hill. R. V. 2005. Integration of morphological these pulmonary traits (Lambertz et al. 2010 and data sets for phylogenetic analysis of Joyce et al. 2004). The nodes within the Amniota: The importance of integumentary Testudinoidea superfamily in Figure 1 have very characters and increased taxonomic low bootstrap values of fourteen and sixteen, sampling. Systematic Biology. 54: 530-547. which suggest these relationships are not strong. Joyce, W.G., J.F. Parham, and J.A. Gauthier. These low bootstrap values further influenced 2004. Developing a protocol for the our decision to place P. megacephalum in the conversion of rank-based taxon names to Testudinoidea. phylogenetically defined clade names, as The research conducted supports two large exemplified by turtles. The Paleontological suborders of turtles: Pleurodira and Society. 78:989-1013. Cryptodira. An additional classification was Krenz, J.G., J.P. Naylor, H.B. Shaffer, F.J. made among the Cryptodira suggesting that two Janzen. 2005. Molecular and infraorders are present: Cryptoda and evolution of turtles. Molecular Trionychida. Additionally, our results strongly Phylogenetics and Evolution. 37:178-191. suggest that P. megacephalum is not a sister Lambertz, M., W. Bohme, and S. F. Perry. 2010. taxon to C. serpentina. This relationship is The anatomy of the respiratory system in strongly supported through morphological and Platysternon megacephalum Gray, 1831 molecular data. While we feel our argument is (Testudines:Cryptodira) and related species, strong, further research encompassing multiple and its phylogenetic implications. genetic and morphological characters would Comparative Biochemistry and Physiology strengthen our position on the classification of Part A: Molecular & Integrative Physiology. turtles. 156:330-336. Rieppel 2000. Turtles as reptiles. Literature Cited Zoologica Scripta. 29:199-212. Bayley, W.S. 1889. Geology and Paleontology. Shaffer, H. B., P. Meylan, and M. L. McKnight. The American Naturalist. 23:148-155. 1997. Tests of turtle phylogeny: molecular, Benton, M. J. 2005. Paleontology. morphological, and paleontoloical Third Edition. Blackwell Publishing, approaches. Systematic Biology. 46:235- Malden, MA. 268. Bonin, F., B. Devaux, A. Duprè, and P.C.H., Williams, E. E. 1950. Variation and selection in Pretchard. 2006. Turtles of the World. The the cervical central articulation of living turtles. Bulletin of the American Museum of Natural History. 94: 537.

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Williston, S. W. 1917. The phylogeny and classification of reptiles. The Journal of Geology. 25:411-421.

Submitted 8 February 2013 Accepted 1 March 2013

Appendix A: The Latin Bionomial, accession number for RAG-1, and the authorities for all taxa studied including 25 ingroup species and 3 outgroup species. All taxa, except those denoted by an asterisk, were those used by Krenz et al. (2005) and Shaffer et al. (1997). Latin Binomial Accession Number (RAG-1) Authority reevesii AY687914.1 Gray,1831 triporcatus AY687909.1 Wiegmann, 1828 odoratus AY687911.1 Latreille, 1801 Dermochelys coriacea AY687908.1 Vandelli, 1761 pardalis AY687912.1 Miller, 1779 spinosa AY687913.1 Gray, 1830 pseudogeographica AY687916.1 Gray, 1831 marmorata AY687917.1 Baird & Girard, 1852 Carettochelys insculpta AY687904.1 Ramsay, 1886 punctata AY687902.1 Lacépède, 1788 senegalensis AY687903.1 Duméril & Bibron, 1835 spinifera AY687901.1 LeSueur, 1827 williamsi AY687923.1 Laurent, 1965 Pelomedusa subrufa AY687922.1 Lacépède, 1788 expansa AY687924.1 Schweigger, 1812 longicollis AY687921.1 Shaw, 1974 Fimbriata AY687918.1 Schneider, 1783 latisternum AY687920.1 Gray 1867 gibbus AY687919.1 Schweigger, 1812 Chelydra serpentina AY687906.1 Linnaeus, 1758 Platysternon megacephalum AY687905.1 Gray, 1831 Chelonia mydas AY687907.1 Linnaeus, 1758 Crocodylus porosus EU375509.1 Schneider, 1801 Gallus Gallus AF143730.1 Linnaeus, 1758 Felis catus AF203761.1 Linnaeus, 1758 temminckii* FJ234441.1 Troost & Harlan, 1835 Dermatemys mawii AY687910.1 Gray, 1847 sinensis* FJ230871.1 Wiegmann, 1835

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