A Phylogenetic Analysis Using Recombination Activating Gene 1

Euglena: 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. 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). 34 Euglena: 2013 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 35 Euglena: 2013 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

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