Springer Japan KK s. Osawa · Z.-R. Su . Y. Imura Molecular Phylogeny and Evolution of Carabid Ground Beetles With 119 Figures, Including 63 in Color , Springer SYOZO OSAWA, Sc.D. Professor Emeritus, Nagoya University Professor Emeritus, Hiroshima University 2-4-7-1003 Ushita-Asahi, Higashi-ku, Hiroshima 732-0067, Japan ZHI-HUI Su, Ph.D. JT Biohistory Research Hali 1-1 Murasaki-cho, Takatsuki, Osaka 569-1125, Japan YUKI IMuRA, M.D. 1249-8 Shinohara-cho, Kohoku-ku, Yokohama 222-0026, Japan Library of Congress Cataloging-in-Publication Data Osawa, Syozo, 1928- Molecular phylogeny and evolution of carabid ground beetles I S. Osawa, Z.-H. Su, Y.lmura. p. cm. Inc1udes bibliographical references and index. ISBN 4-431-00487-4 (alk. paper) 1. Ground beetles-Phylogeny. 2. Ground beetles-Evolution. I. Su, Z.-H. (Zhi-Hui), 1963- II. Imura, Y. (Yuki), 1954- III. Title. QL596.C2073 2003 595.76'2-dc22 ISBN 978-4-431-67969-1 ISBN 978-4-431-53965-0 (eBook) 2003059005 DOI 10.1007/978-4-431-53965-0 This book is based on DNA de tadoru osamushi no keito to shinka (Molecular Phylogeny and Evolution of the Carabid Ground Beetles of the World), written in Japanese by Syozo Osawa, Zhi-Hui Su, and Yllki Imura and published in 2002 by Tetsugakushobo (Philosophy Press), Tokyo, Japan. The publication of this book is supported in part by a Grant-in-Aid for Publication of Scientific Research Results No. 155306. ISBN 4-431-00487-4 Springer-Verlag Tokyo Berlin Heidelberg New York Printed on acid-free paper © Springer Japan 2004 OriginaHy published by Springer-Verlag Tokyo Berlin Heidelberg New York in 2004 This work is subject to copyright. AH rights are reserved, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustra­ tions, recitation, broadcasting, reproduction on microfilms or in other ways, and storage in data banks. The use of registered names, trademarks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protec­ tive laws and regulations and therefore free for general use. Typesetting, print ing, and binding: SNP Best-set Typesetter Ud., Hong Kong SPIN: 10905692 Dedicated to our friend, the former director of JT Biohistory Research Hall, Takatsuki, Osaka, Tokindo S. Okada without whose constant encouragement this work could not have been undertaken. Preface One of the authors, Syozo Osawa, still clearly remembers the afternoon when one of his colleagues, a young embryologist, succeeded in extracting RNA from the optic tissues of a newt. He was so impressed and showed the test tube containing the trans­ parent RNA solution to a well-known professor of morphological embryology. The professor said, "What, nucleic acid? I don't believe what I cannot see with my eyes." This took place about 50 years ago. DNA is indeed invisible to the naked eye but has become a powerful tool in the study of phylogeny, evolution, and taxonomy. Naturally, without morphology molecular phylogeny alone does not have much meaning. Morphology and molecular phylogeny are like the two wheels of a cart: both are necessary for the vehicle to run smoothly. Nowadays, there is no one who does not believe that DNA can be useful in deter­ mining such things as parent -child relationships or the identity of a criminal. For entomologists, regardless of whether they are professional or amateur, it is very enjoyable to look at well-arranged insect specimens in a box. A DNAsolution in a test tube is prosaic by comparison, yet it is this solution that provides a greater wealth of information to the researchers . It is true , however, that DNAanalysis has not yet been embraced by a large number of insect taxonomists. One reason for this is the assumption that molecular biology is difficult to understand. This may be true to some extent for traditional entomolo­ gists and amateur insect lovers, but a detailed knowledge of molecular biology is not necessary to study phylogeny,evolution, and taxonomy with the aid of DNA. What is required is a fundamental knowledge of the following kind. The first im­ portant point to keep in mind is that DNAto be used for phylogenetic study using "a molecular clock" (see below) is unrelated to phenotypes, i.e., morphological struc­ tures, various physiological functions, etc. Togive an example, if one has examples of three species of carabid ground beetle, a, b, and c, and finds the difference in the nucleotide (A, G, C, and T) sequence between a and b is 1% and that between c and a (or b) is 5%, these results may be explained in the following way. The percentage of difference in the nucleotide sequence of the species may indicate that c and alb descended from a common ancestor, after which a separated from b. If we assume that a period of 0,4 million years is required to produce a 1% differ­ ence in nucleotide sequences, then the separation of c and alb from a common ances­ tor took place 2 million years ago. Nucleotide sequence changes, the substitution of A by T for example, takes place at a more or less constant rate, which means that it can be regarded as a molecular (or DNA) clock. Construction of a phylogenetic tree can be successfully completed by applying this feature of DNA. To put it simply, the smaller the difference in the nucleotide sequence between species, the closer the phylogenetic relationship and vice versa. Of course, there are some complicated technical and theoretical problems associated with this approach, which will be considered in Chapter 4. As the changes occurring in DNA that are used as a reference for this molecular clock are neutral, i.e.,they are neither deleterious nor are they related to morphology and function, they do not cause any phenotypic changes. On the contrary, some non-neutral changes result in morphological and physiological alterations that are VII VIII Preface unrelated to the number of neutral changes. This is because changes leading to phenotypic alterations occur on sites different from the neutral site. Motoo Kimura (l986) stresses the fact that the "phenotype is conventional; molecu­ lar evolution is conservative:' Morphological character is a part of the phenotype that does not change relative to time, while a molecular clock ticks at a constant rate. Some claim that the use of the mitochondrial DNA molecular clock is equivalent to an examination based on morphology. This is incorrect, however, because it confuses the concept of phenotype and that of molecular clock. The purpose of molecular phylogeny is to show the order in which species or other taxa in a given group of organisms have diversified against a relative (or absolute) time axis. When molecular phylogenetic methods were not available, one could only construct a phylogenetic tree by means of cladistic analysis using morphological char­ acteristics. This allowed researchers to deduce phylogenetic relationships with some degree of accuracy, because the phenotype, even if it changes in a conventional way, reflects phylogeny to some extent. It is the case, however, that various researchers choose widely different morpho­ logical characteristics when undertaking cladistic analysis, which means that the phylogenetic trees they produce quite often disagree. The "character" or the "character condition" referred to by cladists does not necessarily have a genetic basis and does not therefore necessarily correspond to a "genetic character:' In most cases, it is not possible to know what genetic event(s) created a morpho­ logical change that is apparent to the naked eye as a character. It has been known since Mendel's time that one mutation sometimes leads to a change in a morpholog­ ical character, but sometimes one genetic change is responsible for multiple pheno­ typic alterations. There is no way to verify which of these is the case using only a traditional cladistic approach . This means that cladistic analysis can be thought of as having played an important role only until molecular phylogenetic analysis tech­ niques became available. About nine years have passed since we began to study the DNA phylogeny of the carabid ground beetle. In the initial stages of this study, we experienced much resis­ tance to the idea of using DNA analysis in the field of entomology, with its long tra­ dition dating from the time of Carl von Linne. For all of Linne's achievements, about 200 years have passed since he pioneered the field. In the twenty-first century, it is to be expected that new techniques will supplant the old. When we submitted a paper on our early findings to a journal in the field, one reviewer said, "I don't have faith in the molecular results, because they do not agree with findings based on morphology. Molecular phylogeny has value only when it sup­ ports the results of cladistic analysis:' Even today, a fraction of entomologists con­ tinue to view the results of our studies on molecular phylogeny with doubt. This reaction is not surprising, as it is always the case that new techniques are met with skepticism. Indeed, when we began molecular phylogenetic studies, only a few researchers around the world were engaged in this kind of study. The validity of molecular phylogeny has, however, gradually been acknowledged as a useful tool by a considerable number of young entomologists. In Japan, molecular phylogenetic studies are now being rapidly extended to cover many groups of insects, such as butterflies, moths, longhorn beetles, lucanid beetles, and dragonflies, in addition to the carabid ground beetles. Wehave studied the phylogeny and evolution of the carabid beetle as objectively as possible.