First Application of the SINE (Short Interspersed Repetitive Element) Method to Infer Phylogenetic Relationships in Reptiles: An Example from the Turtle Superfamily Testudinoidea

Takeshi Sasaki*,{dagger}, Kazuhiko Takahashi{dagger}, Masato Nikaido*, Seiko Miura{dagger}, Yuichirou Yasukawa{ddagger} and Norihiro Okada*,{dagger}

* Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, Yokohama, Japan
{dagger} Department of Cell Biology, National Institute for Basic Biology, Myodaiji, Okazaki, Japan
{ddagger} Tropical Biosphere Research Center, University of the Ryukyus, Nishihara, Okinawa, Japan

Correspondence: E-mail: nokada{at}bio.titech.ac.jp.


    Abstract
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 Literature Cited
 
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.

Key Words: tortoise • turtle • phylogeny • SINE • retroposon


    Introduction
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 Literature Cited
 
Bataguridae, which consists of approximately 60 extant species in 25 genera (Ernst, Altenburg, and Barbour 2000; Yasukawa, Hirayama, and Hikida 2001; Dalton 2003), is the largest family of turtles (order Testudines). Its species are distributed broadly across southern Europe, northern Africa, tropic temperate Asia, Central America, and northern South America. This family belongs to the suborder Cryptodira (hidden-necked turtles), which comprises 11 families. These families are grouped into four superfamilies, namely, Chelydroidea, Trionychoidea, Chelonioidea, and Testudinoidea (Mlynarski 1969; Gaffney and Meylan 1988; Ernst, Altenburg, and Barbour 2000). Bataguridae, together with Testudinidae and Emydidae, belongs to the Testudinoidea (Gaffney and Meylan, 1988). Formerly, Testudinoidea included only the two families Testudinidae and Emydidae, with the former group being terrestrial and the latter semi-aquatic. Also, Bataguridae was treated as a subfamily of Emydidae, but evidence from later morphological analyses suggested that this group is more closely related to Testudinidae than to Emydidae (McDowell 1964; Hirayama 1984). Subsequently, Gaffney and Meylan (1988) assigned Bataguridae to one of the three testudinoid families, leaving both the testudinoid intrarelationship and the batagurid monophyly unresolved. Thus, the phylogenetic position of the Bataguridae has been highly controversial, although most authors recognized the family.

Studies on phylogenetic relationships among members of Bataguridae have thus far been conducted from two viewpoints, namely morphology (McDowell 1964; Bramble 1974; Hirayama 1984; Gaffney and Meylan 1988; Yasukawa, Hirayama, and Hikida 2001) and molecular systematics (Sites et al. 1984; Wu, Zhou, and Yang 1999; McCord et al. 2000; Honda et al. 2002). McDowell (1964) defined the following four subgroups within Bataguridae primarily on the basis of cranial characteristics: the "Hardella complex" (Hardella, Morenia, and Geoclemys), the "Batagur complex" (Batagur, Callagur, Chinemys, Hieremys, Kachuga, Malayemys, and Ocadia), the "Orlitia complex" (Orlitia and Siebenrockiella), and the "Geoemyda complex" (Cuora, Cyclemys, Geoemyda, Heosemys, Mauremys, Melanochelys, Notochelys, Pyxidea, Sacalia, and Rhinoclemmys). Later, however, a morphological analysis suggested that the Hardella complex constitutes a polyphyletic group (Hirayama 1984). This study also suggested that Chinemys and Malayemys are sister genera within the Batagur complex. Gaffney and Meylan (1988) proposed the two subfamilies Batagurinae and Geoemydinae in Bataguridae, the former consisting of species of the Hardella, Batagur, and Orlitia complexes, and the latter corresponding to the Geoemyda complex. However, recent molecular phylogenetic analyses have suggested that a few genera of the subfamily Batagurinae are rather closely related to species in Geoemydinae (McCord et al. 2000; Honda, Yasukawa, and Ota 2002; Honda et al. 2002). McCord et al. (2000) suggested a close relationship between Siebenrockiella (Orlitia complex) and Melanochelys (Geoemyda complex) as well as between Chinemys (Batagur complex) and Mauremys (Geoemyda complex), based on a preliminary analysis of mitochondrial cytochrome b gene sequences. The latter relationship was further supported by an analysis of partial sequences of the mitochondrial 12S and 16S rRNA genes (Honda, Yasukawa, and Ota 2002; Honda et al. 2002). Thus, as with the phylogenetic position of the family Bataguridae, the proposed relationships among its members have also been a subject of vigorous discussion.

Over nearly a decade, the SINE (short interspersed element) method (Cook and Tristem 1997; Miyamoto 1999; Shedlock, Milinkovitch, and Okada 2000; Shedlock and Okada 2000; Rokas and Holland 2000; Graur and Li 2000; Nei and Kumar 2000) has been employed in a number of phylogenetic analyses (e.g., Murata et al. 1993; Shimamura et al. 1997; Takahashi et al. 2001; Nikaido et al. 2001a) as a powerful tool for identifying monophyletic groups. SINEs are retroposons that propagate in the genome by retroposition (Singer 1982; Weiner, Deininger, and Efstratiadis 1986; Okada 1991a, 1991b). Integration of a SINE sequence at a site in the genome is irreversible, and its target site is chosen almost at random (Okada et al. 1997). The latter characteristic of SINE retroposition indicates that we can actually exclude the possibility of parallel and independent insertion of such a sequence in distant phylogenetic lineages. Thus, when a SINE sequence is found at an orthologous locus in two or more lineages, it can be regarded as evidence for synapomorphy.

A SINE family sometimes consists of multiple subgroups that each contain different combinations of diagnostic nucleotides (e.g., Ohshima et al. 1993; Kido et al. 1994; Shimamura et al. 1999; Nikaido et al. 2001b; Nishihara, Terai, and Okada 2002; Takahashi and Okada 2002). Different SINE subgroups often show different relative frequencies of retroposition during the course of evolution. Therefore, when using the SINE method it is necessary to choose a certain subgroup (within the lineage to be tested) that appears to be specifically active (Kido et al. 1991; Shimamura et al. 1997). Otherwise, a large portion of the loci that are analyzed may appear to be phylogenetically uninformative because they might contain a significant number of SINE sequences that became inserted prior to the divergence of the lineage.

The tortoise polIII/SINE was reported to exist uniquely in the genomes of hidden-necked turtles (Endoh and Okada 1986; Endoh, Nagahashi, and Okada 1990; Ohshima et al. 1996). In the present study, we isolated tortoise polIII/SINEs from representative species of eight families of cryptodiran turtles, and subjected them to a comprehensive analysis via the SINE method to reveal their subgroup structure. On the basis of this information, we tested the applicability of the SINE method to deduce the phylogeny of these turtles, focusing especially on Bataguridae and related families within Testudinoidea. This study constitutes the first application of the SINE method to infer reptilian phylogeny.


    Materials and Methods
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 Literature Cited
 
DNA Extraction
A total of 23 species from eight families of cryptodiran turtles were analyzed (table 1). Total genomic DNA from each specimen was isolated by extraction with phenol-chloroform as described by Blin and Stafford (1976). Extracted DNAs were stored at 4°C in TE buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA) until use.


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Table 1 Species Analyzed in This Study.

 
Construction of Genomic Libraries, and Isolation and Sequencing of Clones Containing SINEs
To construct genomic libraries to isolate tortoise polIII/SINEs, genomic DNAs from nine representative species from eight families (table 1) were digested separately with HindIII. Each digest was fractionated by size via sucrose density gradient (10%–40%, w/v) centrifugation. DNA fragments obtained from the fraction corresponding to 2–4 kb were ligated into pUC18.

Genomic library screening was performed for two different purposes, namely to characterize the tortoise polIII/SINEs sequences themselves and to perform a phylogenetic analysis of Testudinoidea using the SINE method. For the former purpose, we screened genomic libraries of nine species from eight families (table 1) using the oligonucleotide probe TEF1 (5'-GGGAGGGATAGCTCAGTGGT-3'; Ohshima et al. 1996) corresponding to positions 1–20 of the Cry I consensus (cons) sequence (fig. 1). TEF1 is compatible among all the tortoise polIII/SINE subgroups. For the Testudinoidea phylogenetic analysis, we designed the probe Cry1tC (5'-GGGGCAAAAATTGGTCCTGC-3'), which corresponds to positions 86–105 of the CryIC cons sequence (fig. 1), to selectively isolate the SINE type IC from the genomic libraries of Kachuga smithii and Mauremys mutica kami (table 1). Each genomic library was screened with the above probes that were labeled with [{gamma}-32P] dATP. Hybridization was performed at 42°C overnight in a solution of 6x SSC, 1% SDS, 2x Denhardt's solution, and 100 µg/ml herring sperm DNA in a final volume of 50 ml. Washing was performed at 35°C for 20 min in a solution of 2x SSC and 1% SDS. The inserts of positive clones that appeared to contain these SINE sequences were sequenced by the dideoxy chain termination method (Sanger, Nicklen, and Coulson 1977). Purified plasmid DNAs were sequenced using the BigDye terminator cycle sequencing kit (Applied Biosystems) performed with 25 cycles consisting of denaturation at 96°C for 30 s, annealing at 50°C for 15 s, and extension at 60°C for 1 min in a total volume of 10 µl. The reaction contained 4 µl BigDye terminator premix, 50 ng sequencing primer, and 2 µl template DNA. Sequences were determined with an automated sequencer (3100 Applied Biosystems). The nucleotide sequence data have been deposited in GenBank (AB125391AB125574).



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FIG. 1. Comparison of consensus sequences of tortoise polIII/SINE subgroups and types. The horizontal line is the border between the sequences identified as the two subgroups Cry I and Cry II. The consensus (cons) sequence of each subgroup is indicated in boldface type. Diagnostic nucleotides and deletions specific to the Cry II subgroup are highlighted. Dots denote nucleotides identical to the Cry I cons sequence. Dashes indicate gaps inserted to improve the alignment. The regions showing a type-specific insertion/deletion are boxed with a dotted line. When two or three nucleotides were present predominantly at a certain position in the consensus sequence, the nucleotides were represented by the ambiguity code (IUB single-letter code)

 
Amplification of SINEs by Polymerase Chain Reaction (PCR)
To complement the above sequence data for the characterization of subgroups, the tortoise polIII/SINEs were directly amplified by PCR from the genomic DNAs of six representative species from five families of hidden-necked turtles (table 1). In these reactions, two different kinds of reverse primers were used to specifically amplify the respective sequences of the Cry I and Cry II subgroups, in combination with TEF1 as the common forward primer. For the reverse primers, we used Cry1R (5'-GATATACCAATCTCCTAGAA-3'), which corresponds to positions 170–189 of the Cry I cons sequence (fig. 1), and Cry2R (5'-AATCATAGAATATCAGGGT-3'), which corresponds to positions 164–182 of the Cry II cons sequence. Each reaction contained 200 ng genomic DNA, 100 ng of each primer, 0.2 mM dNTPs, 5µl ExTaq buffer (Takara, Japan), and 1 U of Takara ExTaq DNA polymerase in a final volume of 50 µl. Polymerase chain reaction was performed over 30 cycles consisting of denaturation at 94°C for 1 min, annealing at 50°C for 45 s, and extension at 72°C for 45 s. The PCR products were subcloned into pGEM-T (Promega). Sequences of the clones were determined by the dideoxy chain termination method as described above.

Alignment of the Tortoise PolIII/SINE Sequences
A total of 382 tortoise polIII/SINEs sequences (for representative sequences, see table 1 of the Supplementary Material online) obtained by screening genomic libraries and by PCR amplification were initially aligned using the ATGC version 3.00 software (Genetyx Corporation). The alignment was inspected by eye using the Genetix version 6.1.1 software.

Amplification of Orthologous Loci by PCR to Test the Presence/Absence of SINE Sequences
To examine the presence or absence of a SINE unit at orthologous loci in various species, we designed and synthesized a pair of primers that flanked the unit based on the sequences obtained from the isolated clones. Polymerase chain reaction amplification was carried out in 50 µl containing 200 ng genomic DNA, 100 ng of each primer, 0.2 mM dNTPs, 5µl ExTaq buffer (Takara, Japan), and 1 U of TaKaRa ExTaq DNA polymerase. Polymerase chain reaction was performed over 30 cycles of denaturation at 94°C for 1 min, annealing at 50°–55°C for 1 min, and extension at 72°C for 1 min. The PCR products were electrophoresed in a 1.5% gel of Agarose L03 (Takara, Japan). DNA bands were stained with ethidium bromide and visualized under UV irradiation. When necessary, PCR products were ligated into pGEM-T (Promega) and used as a template for sequencing (see table 2 of the Supplementary Material online). The nucleotide sequence data have been deposited in GenBank (AB125703AB125739).


    Results
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 Literature Cited
 
Identification of Subgroups and Types of the Tortoise PolIII/SINEs
A total of 382 tortoise PolIII/SINE sequences were isolated, of which 190 were screened from genomic libraries and 192 were amplified by PCR. Analysis of diagnostic nucleotides in the 190 full-length SINE sequences revealed that this SINE family comprised two subgroups that were designated Cry I and Cry II (see table 1 of the Supplementary Material online). Interestingly, the diagnostic nucleotides that distinguished these subgroups were located only in their 3' regions (fig. 1). To estimate relative frequency of each subgroup in the genomes of the turtle lineages we investigated, we compared the number of subgroups that were isolated randomly from the genomic libraries of representative species (table 2). Cry I was the only subgroup found in representative species of Trionychidae (Florida softshell turtle, Apalone ferox) and Carettochelyidae (pig-nose turtle, Carettochelys insculpta), whereas only the Cry II subgroup was observed in the representative species of Cheloniidae (green turtle, Chelonia mydas). Both subgroups were found in the remaining five turtle lineages, although their relative frequencies varied.


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Table 2 Number of Isolated SINEs in Each Subgroup for Each Taxon.

 
A more detailed comparison of the 382 sequences revealed that the Cry I and Cry II subgroups were further divided into four (IA–ID) and five (IIA–IIE) types, respectively, based on the characteristics of the insertion/deletion located in the middle part of each SINE sequence (fig. 1 and table 1 of the Supplementary Material online). The relative frequencies of these types in each SINE subgroup varied among the turtle lineages we investigated (tables 3 and 4; fig. 2). In the Cry I subgroup, sequences from each species of Trionychidae, Carettochelyidae, Chelydridae, and Emydidae belonged to a single type of SINE (IA, ID, IA, and IB, respectively). The species from Kinosternidae, Testudinidae, and Bataguridae shared the same three types of Cry I (IA, IB, and IC). The frequencies of the types of SINEs in the Cry II subgroup also varied. Type IIB predominated in species from Chelydridae and Cheloniidae, and was found only in these species. The Kinosternidae species displayed three types (IIA, IID, and IIE) of the Cry II group. Type IIA was the major member of Emydidae, Testudinidae, and Bataguridae, although IIC was also found in smaller numbers.


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Table 3 Number of Isolated SINEs of Each Type in the Cry I Subgroup.

 

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Table 4 Number of Isolated SINEs of Each Type in the Cry II Subgroup.

 


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FIG. 2. Comparison of the composition of the tortoise polIII/SINE subgroups/types elucidated in the present study. See tables 2 and 3 for the actual numbers of subgroups/types identified. The scale bar indicates percentage. The phylogenetic tree shown to the left is based on a consensus of those proposed in Gaffney and Meylan (1988) and Shaffer, Meylan, and McKnight (1997). The assumed emergence time of the tortoise polIII/SINE (Ohshima et al. 1996) is indicated on the tree

 
The relative frequencies of the SINE subgroups and types varied widely among the different lineages of Cryptodira (fig. 2). This is considered to be due to drastic differences in the relative retropositional activities among the lineages. Nevertheless, an obvious trend in the composition of the subgroups/types was recognized. Indeed, the species from Bataguridae, Testudinidae, and Emydidae, and to a lesser extent Kinosternidae, showed similarity with respect to the composition of the types in each subgroup, as described above for IB and IIA.

Phylogenetic Analysis of SINE Insertions in Testudinoidea
Based on the frequencies of the subgroups/types presented above, it was reasonable to assume that tortoise polIII/SINE types IB and IC exhibited higher rates of retroposition during the evolution of Bataguridae and its closely related families compared to other types. Hence, focusing on types IB and IC during the analysis increased the chance of obtaining loci that were informative with regard to elucidating the phylogenetic relationships among the turtle species within these groups. Thus, we investigated loci into which either type IB or IC had inserted. Such loci were either screened with the probe Cry1tC (specific to a type IC sequence) or isolated incidentally during genomic library screening with the general probe TEF1 (see Materials and Methods for details).

In the case of the BCr06 locus, the longer products that were expected when a SINE sequence existed in the locus were amplified from the genomes of 14 species that represented Testudinidae and Bataguridae (fig. 3A). In contrast, two species belonging to Emydidae yielded shorter products that were expected if the locus did not contain a SINE unit. These results on the presence/absence of SINEs at this locus (table 5) were further confirmed by sequencing the products amplified from the genomes of representative species (see table 2 of the Supplementary Material online). Similar results were obtained for the BCr01 locus (fig. 3B), although no product was obtained from Melanochelys trijuga, most likely because there was a mutation in the primer binding site. These results suggest that Bataguridae species are more closely related to those of Testudinidae than to those of Emydidae, assuming that the SINE inserted into these loci in a common ancestor of the relevant Testudinidae and Bataguridae species after it branched from the ancestor leading to the relevant Emydidae species.



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FIG. 3. Electrophoretic analysis of PCR products from 11 loci. Filled and open arrowheads indicate the expected positions of bands that contain or lack a SINE unit, respectively, at each locus. Size markers ({phi}X174 HincII-digest) are indicated in the leftmost lane (M) of each gel

 

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Table 5 Presence or Absence of a SINE Unit at Individual Loci.

 
The BKs11 locus suggested that Kachuga smithii, Callagur borneoensis, Malayemys subtrijuga, and Siebenrockiella crassicollis constitute a monophyletic group within Batagurinae, based on our observation that these four species alone yielded longer products containing a SINE unit (fig. 3C).

Data for the BKs36 locus revealed a close relationship among Kachuga smithii, Callagur borneoensis, and Malayemys subtrijuga, as only these species exhibited longer PCR products (fig. 3D). A similar result was obtained for the BKs52 locus (fig. 3E), although Trachemys scripta elegans, for which the BKs11 locus showed only a distant relationship with the above three species, did not yield a product.

Polymerase chain reaction amplification of the BCr61 locus (fig. 3F) yielded longer products for both Chinemys reevesii and Mauremys mutica kami, indicating a close relationship between these species. No PCR product was obtained for Cyclemys sp.

The BMm105 locus results showed a pattern similar to that of BCr61 (fig. 3G). C. reevesii displayed a product that was longer than expected for the incorporation of a single SINE unit. The sequence revealed one SINE unit at this locus as well as an additional 325-bp sequence unrelated to the tortoise polIII/SINE (see table 2 of the Supplementary Material online). Kachuga smithii and Sacalia bealei did not yield a PCR product from this locus; however, K. smithii is not closely related to either C. reevesii or M. mutica kami, based on the relationships suggested by the loci BKs11, BKs36, and BKs52.

Among the species examined, insertions of SINEs into the BKs31 locus appeared to have occurred specifically in Kachuga smithii, because this species was the only one to yield a long PCR product (fig. 3H). Similarly, the BCr04 locus showed a SINE insertion specific to Chinemys reevesii (fig. 3I). The results obtained for loci BMm74 and BMm85 (fig. 3J and 3K, respectively) suggested that the insertion of SINE units into these loci occurred independently and subsequent to the divergence of Mauremys mutica kami from all the other species investigated. This result was supported by data for the BCr61 and BMm105 loci. With regard to the BMm74 locus, the products obtained for K. smithii, C. borneoensis, and M. subtrijuga (fig. 3J, lanes 1, 2, and 3) were slightly shorter than the products for other species, suggesting the absence of a SINE unit at this locus. Sequencing of these three products confirmed a unique 46-bp deletion in the flanking region (see table 2 of the Supplementary Material online). This observation is consistent with the monophyly of these three genera, as suggested by the BKs36 and BKs52 loci, indicating that the above deletion occurred in a common ancestor.

A phylogenetic tree for Bataguridae and other related families was constructed on the basis of the above results obtained from a total of 11 loci (fig. 4).



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FIG. 4. Phylogenetic relationships among the 16 species of Testudinoidea. Arrowheads denote the insertion of tortoise polIII/SINEs into loci that were investigated in this study. White and black arrowheads indicate type IB and IC insertions, respectively. A–C on the nodes indicate the clades that were identified in the present study

 

    Discussion
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 Literature Cited
 
Two Subgroups of the Tortoise PolIII/SINEs
Four sequences of the tortoise polIII/SINE (TE3, TE5, TE6, and TE9) were previously reported for Chinemys reevesii (Endoh, Nagahashi, and Okada 1990). TE5, TE6, and TE9 were placed in the Cry I subgroup, whereas TE3 was included in the Cry II subgroup, according to the subgroup definitions for the tortoise polIII/SINE. Because TE3 was the only sequence of the Cry II subgroup isolated in that study, it was not recognized as an independent subgroup. In a later study (Ohshima et al. 1996), several clones of the tortoise polIII/SINE were further isolated from the genomic libraries of Florida softshell turtle, Apalone ferox, but all of these sequences were members of the Cry I subgroup. These data are consistent with our current observation that all 18 sequences isolated from the above species belong to Cry I (table 2 and fig. 2). Thus, because we used sufficient numbers of sequences and turtle taxa in our analysis, we were able to establish the Cry II subgroup of the tortoise polIII/SINE for the first time.

Tail Region Similarities Between the Cry I/II Subgroups and PsCR1 Long Interspersed Element (LINE)
The diagnostic nucleotides that differentiate the two tortoise polIII/SINE subgroups identified in the present study were found only in their 3' regions (fig. 1). In fact, the sequence of this region is homologous to that of the 3'-tail of PsCR1 LINE (long interspersed element) in turtles (Ohshima et al. 1996; Kajikawa, Ohshima, and Okada 1997). The LINEs encode a reverse transcriptase that facilitates their own retroposition, whereas the SINEs do not encode any protein. This fact, together with the discovery of the similarity between the 3'-tails of SINEs and LINEs (Ohshima et al. 1996; Okada et al. 1997), led to the hypothesis that SINE retroposition is dependent on the retropositional enzymatic machinery of LINEs, and that during retroposition of the SINE, its 3'-tail is recognized by a reverse transcriptase encoded by its partner LINE (Ohshima et al. 1996; Okada et al. 1997; Weiner 2000). This hypothesis was recently verified by experiments showing that the 3'-tail of eel UnaSINE1 is actually recognized by the reverse transcriptase encoded by eel UnaL2 LINE (Kajikawa and Okada 2002).

Of the two subgroups (Cry I and Cry II) identified in the present study, the consensus sequence of the 3'-tail region of Cry II SINEs is 95% identical to that of PsCR1 LINE, as suggested by Ohshima et al. (1996) for the TE3 sequence (fig. 5). Thus, the Cry II SINE subgroup appears to exploit the retropositional machinery of the PsCR1 LINE, the characterization of which was previously reported (Ohshima et al. 1996; Kajikawa, Ohshima, and Okada 1997). In contrast, the 3'-tail region of the consensus sequence of Cry I SINEs shows only 75% identity with that of the PsCR1 LINE. Although this 75% identity constitutes a significant level of homology, the 3'-tail sequence of a SINE is stringently recognized by the reverse transcriptase of its partner LINE (Okada et al. 1997). Thus, the fact that the Cry I SINE subgroup exists may indicate the existence of an as yet unreported PsCR1 LINE subgroup whose 3'-tail sequence is highly similar to that of the Cry I SINEs. This putative PsCR1 LINE subgroup may be directly responsible for retroposition of the Cry I subgroup.



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FIG. 5. Comparison of the 3'-tail sequences between PsCR1 LINE and the tortoise polIII/SINE Cry II subgroup. Structures of PsCR1 LINE and the Cry II subgroup are shown schematically. Homologous regions are indicated by rectangles shaded with slanted lines. Alignment of the tail sequences is shown at the bottom. The accession number for the PsCR1 LINE sequence is AB005891

 
Phylogenetic Analysis of Bataguridae and Its Related Families
The analysis of loci BCr01 and BCr06 (fig. 4) via the SINE method revealed close relationships between Bataguridae and Testudinidae, and suggested that these families constitute an outgroup of Emydidae. These results are consistent with earlier morphological studies (McDowell 1964; Hirayama 1984; Gaffney and Meylan 1988), as well as with a molecular study involving mitochondrial cytochrome b and 12S rRNA genes (Shaffer, Meylan, and McKnight 1997).

We also identified three new clades within Bataguridae (fig. 4). SINE insertions at loci BKs36 and BKs52 as well as a 46-bp deletion in BMm74, indicated that three species (Kachuga smithii, Callagur borneoensis, and Malayemys subtrijuga) out of the four we investigated from the Batagur complex (McDowell 1964) form a monophyletic group (clade A). In addition, Chinemys reevesii, another species of the Batagur complex that we investigated, was not included in this clade, although morphological studies suggested it is a sister species of Malayemys subtrijuga (Hirayama 1984; Gaffney and Meylan 1988).

The results obtained from the BKs11 locus suggested monophyly of the clade B (fig. 4), which was also supported by our preliminary analysis of locus BKs85 (unpublished data). This finding is inconsistent with an earlier phylogenetic analysis (McCord et al. 2000) based on the sequence of the mitochondrial cytochrome b gene, which suggested the close relationship between Siebenrockiella and Melanochelys, because the former genus was included in the clade B, but the latter was not a member of the clade in the present study.

The close relationship between Chinemys reevesii and Mauremys mutica kami, as suggested by loci BCr61 and BMm105 (clade C in fig. 4), is in agreement with a preliminary study based on the sequence of the mitochondrial 12S and 16S rRNA genes (Honda, Yasukawa, and Ota 2002; Honda et al. 2002). Sequence analysis of the cytochrome b gene also suggested that these species are closely related (McCord et al. 2000). However, this relationship is in conflict with the accepted taxonomy because Chinemys reevesii belongs to Batagurinae whereas Mauremys mutica kami is included in Geoemydinae (Gaffney and Meylan 1988; Ernst, Altenburg, and Barbour 2000; Yasukawa, Hirayama, and Hikida 2001). Accordingly, the monophyly of the Batagur complex (Callagur, Chinemys, Kachuga, and Malayemys, in terms of the specimens examined in the present study), as suggested by McDowell 1964, is also inconsistent with the present result. Chinemys reevesii shares several morphological traits, such as the broad and triangular fissura ethmoidalis and small foramen palatinum posterius, with other members of Batagurinae (Hirayama 1984; Gaffney and Meylan 1988). This conflict between the molecular systematics and the taxonomy may be due in part to extensive convergence in the morphological evolution of the Bataguridae (see Hirayama [1984] and Yasukawa, Hirayama, and Hikida [2001] for further details), which is largely affected by environmental selective forces, as suggested by Honda et al. (2002).

Applicability of the SINE Method for Inferring Turtle Phylogeny
The present study is the first application of the SINE method to reptilian phylogeny, specifically to that among families and genera of cryptodiran turtles. The phylogenetic tree that includes polIII/SINE insertions of the tortoise Cry I subgroup members (fig. 4) indicates that the individual types within this subgroup show different tendencies with respect to relative retropositional frequencies along the nodes. Indeed, retropositional insertion of the IB type SINE was evident only during the earliest evolutionary period of the turtles we investigated, whereas the retroposition of type IC was detected only during the later period, as shown in the phylogenetic tree. Such differences in relative retropositional frequencies within each evolutionary period or lineage is advantageous for phylogenetic analyses using the SINE method because the choice of the type(s) of the SINEs allows the analysis to focus on a specific relationship of interest. The above information regarding the choice of the types to be investigated will be useful in future detailed phylogenetic analyses within Bataguridae itself, as well as among Bataguridae and its related turtle families of Testudinoidea. In a similar manner, the present SINE methodology may be applied to other families of turtles, such as Chelydridae and Cheloniidae, although other types of tortoise polIII/SINEs (type IIB for these families, for example) must be used for such analyses.

The SINE method may be useful for resolving relationships not only among families or genera but also among species that diverged more recently. The loci BKs31, BCr04, BMm74, and BMm85 (table 5 and fig. 4) contain Cry I insertions within a single taxon. By including additional species and genera, more detailed analyses of SINE insertions at such loci would be informative for the above purpose (see Kido et al. [1991] and Murata et al. [1993] for examples of such analyses). The present study provides the indispensable groundwork for future phylogenetic analyses of cryptodiran turtles using the SINE method.


    Acknowledgements
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 Literature Cited
 
This work was supported by a grant-in-aid to N.O. from the Ministry of Education, Culture, Sports, Science and Technology of Japan.


    Footnotes
 
Pierre Capy, Associate Editor Back


    Literature Cited
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 Literature Cited
 

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Accepted for publication November 26, 2003.





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