Although turtles (order Testudines) constitute one of the major reptile groups, their phylogenetic relationships remain largely unresolved. Hence, we attempted to elucidate their phylogeny using the SINE (short interspersed repetitive element) method, in which the sharing of a SINE at orthologous loci is indicative of synapomorphy. First, a detailed characterization of the tortoise polIII/SINE was conducted using 10 species from eight families of hidden-necked turtles (suborder Cryptodira). Our analysis of 382 SINE sequences newly isolated in the present study revealed two subgroups, namely Cry I and Cry II, which were distinguishable according to diagnostic nucleotides in the 3' region. Furthermore, four (IA-ID) and five (IIA-IIE) different SINE types were identified within Cry I and Cry II subgroups, respectively, based on features of insertions/deletions located in the middle of the SINE sequences. The relative frequency of occurrence of the subgroups and the types of SINEs in this family were highly variable among different lineages of turtles, suggesting active differential retroposition in each lineage. Further application of the SINE method using the most retrotranspositionally active types, namely IB and IC, challenged the established phylogenetic relationships of Bataguridae and its related families. The data for 11 orthologous loci demonstrated a close relationship between Bataguridae and Testudinidae, as well as the presence of the three clades within Bataguridae. Although the SINE method has been used to infer the phylogenies of a number of vertebrate groups, it has never been applied to reptiles. The present study represents the first application of this method to a phylogenetic analysis of this class of vertebrates, and it provides detailed information on the SINE subgroups and types. This information may be applied to the phylogenetic resolution of relevant turtle lineages.

The family Geoemydidae is one of three in the superfamily Testudinoidea and is the most diversified family of extant turtle species. The phylogenetic relationships in this family and among related families have been vigorously investigated from both morphological and molecular viewpoints. The evolutionary history of Geoemydidae, however, remains controversial. Therefore, to elucidate the phylogenetic relationships of Geoemydidae and related species, we applied the SINE insertion method to investigate 49 informative SINE loci in 28 species. We detected four major evolutionary lineages (Testudinidae, Batagur group, Siebenrockiella group, and Geoemyda group) in the clade Testuguria (a clade of Geoemydidae + Testudinidae). All five specimens of Testudinidae form a monophyletic clade. The Batagur group comprises five batagurines. The Siebenrockiella group has one species, Siebenrockiella crassicollis. The Geoemyda group comprises 15 geoemydines (including three former batagurines, Mauremys reevesii, Mauremys sinensis, and Heosemys annandalii). Among these four groups, the SINE insertion patterns were inconsistent at four loci, suggesting that an ancestral species of Testuguria radiated and rapidly diverged into the four lineages during the initial stage of its evolution. Furthermore, within the Geoemyda group we identified three evolutionary lineages, namely Mauremys, Cuora, and Heosemys. The Heosemys lineage comprises Heosemys, Sacalia, Notochelys, and Melanochelys species, and its monophyly is a novel assemblage in Geoemydidae. Our SINE phylogenetic tree demonstrates extensive convergent morphological evolution between the Batagur group and the three species of the Geoemyda group, M. reevesii, M. sinensis, and H. annandalii.

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