*JT Biohistory Research Hall, Osaka, Japan;
and
Institute of Zoology, Chinese Academy of Sciences, Beijing, China;
Department of Gynecology, Tôkyû General Hospital, Tokyo, Japan;
and
§Shibatsuji-Cho, Nara, Japan
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Abstract |
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Introduction |
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Materials and Methods |
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Direct sequencing was performed with an automated ABI PRISM 377 DNA sequencer using the dideoxy chain termination method (Sanger, Nicklen, and Coulson 1977
). The reaction mixture for cycle sequencing consisted of 6 µl of dRhodamine terminator cycle sequencing ready reaction with AmpliTaq DNA Polymerase, FS (Applied Biosystems, Foster City, Calif.), 0.10.3 pmol/µl of DNA, 2.4 µl (1 pmol/µl) of sequencing primer, and distilled water to a total volume of 15 µl. The cycle-sequencing conditions were 25 cycles at 96°C for 10 s, 50°C for 5 s, and 60°C for 4 min, followed by an indefinite hold at 4°C using a GeneAmp PCR system 9600 (Perkin Elmer). The DNA product was cleaned with Centri-Sep spin columns (Applied Biosystems) and vacuum-dried before applying. The nucleotide sequence data reported in this paper will appear in the DDBJ, EMBL, and GenBank nucleotide sequence databases with the accession numbers shown in table 2
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Phylogenetic Analysis
The ND5 and the 28S rDNA sequences were aligned and compared using the multiple-alignment program CLUSTAL W (Thompson, Higgins, and Gibson 1994
) and DNASIS, version 3.7 (Hitachi Software Engineering, Japan). The evolutionary distances (D) were computed by Kimuras (1980)
two-parameter method, and the phylogenetic trees were constructed by the neighbor-joining (NJ) method (Saitou and Nei 1987
) and the unweighted pair grouping method with arithmetic means (UPGMA). All of these processes were performed with the DNA sequence analysis package SINCA, version 3.0 (Fujitsu System Engineering, Japan). A maximum-parsimony (MP) tree was also constructed with PHYLIP, version 3.5 (Felsenstein 1993
). Bootstrap analysis was performed for all the trees (Felsenstein 1985
) based on 500 resamplings. The gene sequences of two Calosomina species were used as an outgroup.
Dating
For setting the timescale, a 0.01D unit corresponding to 3.6 Myr was used (Su et al. 1998
; Osawa et al. 1999;
revised by Su et al. [unpublished data]).
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Results and Discussion |
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The possibility of lineage sorting of ancestral polymorphism is now considered. If the ancestral species or population is assumed to contain both P and S (and the other) types, the current character distribution may be explained by random lineage sorting. For the reason mentioned above, there is at present no way to specify the ancestral form. Suppose that the P type is the ancestral form. The distribution range of the P type in lineage 1 is exclusively distributed in central China, where no other types have been discovered in reasonably extensive expeditions by a number of entomologists. The distribution range of lineage 1 does not overlap with that of the other types (see fig. 2 ). There is no evidence showing the presence of polymorphism in a single population. Of course, this does not exclude its absence in the past. The ancestral polymorphismrandom lineage sorting possibility may be theoretically pleasing, but it cannot be verified.
One might argue that the ND5 phylogeny would be brought about by introgression of the mitochondrial gene via hybridization. This possibility can be largely ruled out by the overall congruence of the 28S rDNA and the ND5 phylogenies. Furthermore, the distributional isolation of the three lineages, which would have occurred long time ago, is not consistent with the introgression hypothesis.
One plausible interpretation for the appearance of the same type in different lineages would be that parallel evolution took place for the P type in lineages 1 and 3 and for the S type in lineages 2 and 3. If the P type is the ancestral form, parallelism should be considered only for the S type. In addition, the intermingled occurrence of different types in lineages 2 and 3 suggests the morphological transformation from one type to another within the respective lineages. The situation resembles that of Ohomopterus (Su et al. 1996b
), in which taxonomically the "same species" or the members belonging to the same species group (=type) appear in more than two different places on the ND5 tree. Su et al. (1996b
) proposed that parallel evolution took place in different lineages, possibly through discontinuous morphological transformation called "type-switching."
Diversification Within the Respective Lineages
The branching of the three lineages mentioned above seems to have started within a short time, as judged from the ND5 tree. No ancestral lineage can be defined (see above). Within lineage 2, to which the northern and eastern Eurasian species belong, initiation of diversification into various species is much older than that in lineage 3, to which all of the Japanese species belong. Leptocarabus koreanus, L. truncaticollis, L. seishinensis, and L. semiopacus, respectively, form a well-defined cluster. The L. canaliculatus specimens from various localities are clustered together, and L. kurilensis from Hokkaido is included in this cluster, although L. canaliculatus and L. kurilensis are usually regarded as two distinct species by most morphologists. This suggests that L. kurilensis branched off from L. canaliculatus, with accompanying morphological changes. Thus, taxonomy based on morphology at the species level is consistent with the ND5 and 28S rDNA phylogeny in lineage 2, except that L. kurilensis was not separated from L. canaliculatus. In lineage 3 (fig. 5 ), two sublineages are recognized: the L. kyushuensis sublineage (sublineage 1), which is further divided into two clades containing, respectively, the habitants in Kyushu and those in Honshu (the Chugoku district), and sublineage 2, which consists of the habitants in Hokkaido, Honshu, Shikoku, and Kyushu. Within sublineage 2, four clades are recognized: clade a (L. procerulus miyakei in Kyushu), clade b (L. hiurai in Shikoku), clade c (L. arboreus in Hokkaido), and clade d (the habitants in Honshu [L. procerulus procerulus, L. kumagaii, and L. arboreus]). Thus, clustering is more or less linked to geographical distribution. Especially noteworthy are the inhabitants of Honshu (sublineage 2, clade d). As noted above, in clade d there occur three morphological species, L. procerulus (P type), L. kumagaii (P type), and L. arboreus (S type). These three species are clearly separable morphologically, and yet the evolutionary distances among them are either null or very small (less than 0.5% difference), such that they are unresolved on the ND5 phylogenetic tree. This would represent "evolution in action"; i.e., these (sub)species recently started their speciation, in which "type-switching" would be involved. Thus, besides intermingled occurrence of the species of different types in one sublineage, taxonomically the "same species" appear in different sublineages or clades. For example, L. procerulus appears in clade d (subspecies procerulus) and clade a (subspecies miyakei), and L. arboreus appears in clade c (subspecies arboreus and pararboreus) and clade d (many subspecies). The "species" mentioned above are most probably paraphyletic as judged from the ND5 phylogenetic tree; i.e., each "species" arose in parallel in the different pyhlogenetic lines with minor morphological differences and can be recognized as subspecies or local races. Although the 28S rDNA tree is mostly consistent with the ND5 tree, some discrepancies between them do exist, especially in lineage 3. In the 28S rDNA phylogeny, L. hiurai form a clade with L. kyushuensis (Sublineage 1), while in the ND5 phylogeny, L. hiurai clusters with the other members of sublineage 2. In the 28S rDNA tree, L. procerulus procerulus of Honshu forms a clade with L. procerulus miyakei of Kyushu, whereas in the ND5 tree, L. procerulus procerulus of Honshu clusters with the other Honshu members, L. kumagaii and L. arboreus. As was noted above, the maximum D among the Japanese species or races in the 28S rDNA tree is less than 0.005 (0.81%), in contrast to that in the ND5 gene (maximum D = 11.42%). Therefore, it is not possible to decide whether the apparent discrepancies between the ND5 and the 28S rDNA phylogenies are really meaningful or whether or not this situation can be explained by the introgression hypothesis.
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The origin of the Japanese Leptocarabus (lineage 3) is now considered. As noted above, the Japanese species started to diversify much later (ca. 1210 MYA) than the continental species (ca. 2825 MYA). We tentatively assume that the ancestor of the Japanese Leptocarabus inhabited the ancient Japanese area in the eastern periphery of the continent. Upon its split from the continent ca. 15 MYA, followed by the archipelago formation by an extensive submergence, the Japanese Leptocarabus ancestor was isolated on some island(s). Upon subsequent upheaval of the Japanese Islands (96 MYA), the ancestor spread all over Japan, resulting in various species (even different types) and subspecies upon isolation by various barriers such as straits, tectonic lines, rivers, mountains, etc. (For geohistory of the Japanese Islands, see Su et al. [1998
]). It has been generally believed that all the Japanese Leptocarabus immigrated from the Korean Peninsula via land-bridges in the glacial era (<2 MYA) (Ishikawa 1989
). Indeed, morphologies including genitalia of the S type Japanese Leptocarabus are very similar to those of L. seishinensis or L. semiopacus from the Korean Peninsula, and the P type Japanese Leptocarabus species resemble L. koreanus from Korea, or L. yokoae, L. marchilhaci, and Leptocarabus sp. from central China. In particular, L. kyushuensis from Japan is morphologically quite similar to L. yokoae. However, lineage 3 includes solely the Japanese species and none from the Korean Peninsula or China, suggesting that there exist no direct sister relationships between the Japanese and the Korean (and Chinese) species. Moreover, the emergence of the Japanese species is much older (1210 MYA) than previously thought (<2 MYA). Thus, the present phylogenetic analyses and dating would reject the Korean origin hypothesis, although it is possible, but not very likely, that such a Korean species, if they existed, became extinct or have not been discovered.
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Acknowledgements |
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Footnotes |
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1 Keywords: Leptocarabus ground beetles,
mitochondrial ND5 gene,
nuclear 28S rDNA,
phylogeny,
parallel evolution,
2 Address for correspondence and reprints: Zhi-Hui Su, JT Biohistory Research Hall, 1-1 Murasaki-Cho, Takatsuki, Osaka 569-1125, Japan. E-mail: su.zhihui{at}ims.brh.co.jp
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References |
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Clark, C. G., B. W. Tague, V. C. Ware, and S. A. Gerbi. 1984. Xenopus laevis 28S ribosomal RNA: a secondary structure model and its evolutionary and functional implications. Nucleic Acids Res. 12:61976220.[Abstract]
Felsenstein, J. 1985. Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39:783791.
. 1993. PHYLIP (phylogeny inference package). Version 3.5c. Distributed by the author, Department of Genetics, University of Washington, Seattle.
Imura, Y., and H. Kezuka. 1992. Geographical and individual variation of carabid beetles in the species of the subtribe Carabina (3), carabid beetles of the southern part of the Korean Peninsula. Illustrations of selected insects in the world. Series B (Coleoptera), No. 3. Mushi-Sha, Tokyo.
Imura, Y., C.-G. Kim, Z.-H. Su, and S. Osawa. 1998. An attempt at the higher classification of the Carabina (Coleoptera, Carabidae) based on morphology and molecular phylogeny, with special reference to Apotomopterus, Limnocarabus and Euleptocarabus. Elytra Tokyo 26:1735.
Imura, Y., and K. Mizusawa. 1996. The Carabus of the world. Mushi-Sha, Tokyo.
Ishikawa, R. 1972. Studies on Leptocarabus and its related subgenera of the genus Carabus L. (Coleoptera: Carabidae). Bull. Natl. Sci. Mus. Tokyo 15:1927.
. 1989. The Japanese Carabina: geographical distribution and speciation within an archipelago (Coleoptera: Carabidae). Nature and Culture No.1. The University Museum, The University of Tokyo.
. 1992. Taxonomic studies on Leptocarabus (Adelocarabus) arboreus (Lewis) (Coleoptera, Carabidae). TMU Bull. Nat. Hist. 1:140.
Kimura, M. 1980. A simple method for estimating evolutionary rate of base substitutions through comparative studies of nucleotide sequences. J. Mol. Evol. 16:111120.[ISI][Medline]
Kwon, Y. J., and S.-M. Lee. 1984. Classification of the subfamily Carabinae from Korea (Coleoptera: Carabidae). Insecta Koreana, Series 4, Editorial Committee of Insecta Koreana, Seoul, Korea.
Nakane, T. 1961. New or little known Coleoptera from Japan and its adjacent regions. XV. Fragm. Coleopt. 1:16.
. 1962. Insecta Japonica, Series 2, Part 3. Coleoptera: Carabidae (1). Hokuryukan, Tokyo.
Osawa, S., Z.-H. Su, C.-G. Kim, M. Okamoto, O. Tominaga, and Y. Imura. 1999. Evolution of the carabid ground beetles. Adv. Biophys. 36:65106.[ISI][Medline]
Saiki, R. K., D. H. Gelfand, S. Stoffl, S. T. Scharf, R. Higuchi, G. T. Horn, K. B. Mullis, and H. A. Erlich. 1988. Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239:487491.
Saitou, N., and M. Nei. 1987. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol. Biol. Evol. 4:406425.[Abstract]
Sanger, F., S. Nicklen, and A. R. Coulson. 1977. DNA sequencing with chain-terminating inhibitors. Proc. Natl. Acad. Sci. USA 74:54635468.
Su, Z.-H., T. Ohama, T. S. Okada, K. Nakamura, R. Ishikawa, and S. Osawa. 1996a. Phylogenetic relationships and evolution of the Japanese Carabinae ground beetles based on mitochondrial ND5 gene sequences. J. Mol. Evol. 42:124129.
Su, Z.-H., O. Tominaga, T. Ohama, E. Kajiwara, R. Ishikawa, T. S. Okada, K. Nakamura, and S. Osawa. 1996b. Parallel evolution in radiation of Ohomopterus ground beetles inferred from mitochondrial ND5 gene sequences. J. Mol. Evol. 43:662671.
Su, Z.-H., O. Tominaga, M. Okamoto, and S. Osawa. 1998. Origin and diversification of hindwingless Damaster ground beetles within the Japanese Islands as deduced from mitochondrial ND5 gene sequences (Coleoptera, Carabidae). Mol. Biol. Evol. 15:10261039.[Abstract]
Tautz, D., J. M. Hancock, D. A. Webb, C. Tautz, and G. A. Dover. 1988. Complete sequences of the rRNA gene of Drosophila melanogaster. Mol. Biol. Evol. 5:366376.[Abstract]
Thompson, J. D., D. G. Higgins, and T. J. Gibson. 1994. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, positions-specific gap penalties and weight matrix choice. Nucleic Acids Res. 22:46734680.[Abstract]