Euglena: 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.) 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 Chelydra serpentina. Analysis of the RAG-1 gene and pulmonary morphological characters suggests that P. megacephalum is a member of the superfamily Testudinoidea. 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 skull (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 turtle 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 birds, and other reptiles is essential in order to reptile lineage was Rhynchocephalia due to a determine the basal condition and early fossil in Permian rock that was incorrectly transformations of amniote 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 anapsid amniotes, 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 Trionychidae 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 Chelydridae 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 evolution (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