J Mol Evol (1992) 34:324-330

Journal of MolecularEvolution (~) Springer-VerlagNew York Inc. 1992

Planarian Mitochondria I. Heterogeneity of Cytochrome c Oxidase Subunit I Gene Sequences in the Freshwater Planarian,

Dugesiajaponica Yoshitaka Bessho, Takeshi Ohama, and Syozo Osawa Laboratoryof MolecularGenetics, Department of Biology,School of Science, NagoyaUniversity, Chikusa-ku, Nagoya464-01, Japan

Summary. We have detected sequence heterogeneity in the cytochrome c oxidase subunit I (COI) gene of a freshwater planarian, Dugesia japonica, collected in one locality. A part of the COI gene was amplified via the polymerase chain reaction (PCR) using template DNA prepared from a mixture of 500 individuals or from each of 18 individuals. Analyses of DNA sequences by standard strategies for cloning and sequencing or by direct sequencing clearly show that (1) considerable sequence heterogeneity exists in DNA prepared from the mixed individuals, (2) 11 individuals have almost identical sequences (type A), and (3) 7 individuals have sequences different from one another (Seq-D 1 to SeqD7; collectively called type D). Each of the Seq-D l D7 sequences except for Seq-D5 shows some heterogeneity even in a single individual (heteroplasmy). A possible cause of the sequence heterogeneities is discussed. Key words: Planarian -- Dugesiajaponica -- Mitochondria -- Cytochrome c oxidase subunit I gene -- Heterogeneity -- Neoblast -- Asexual reproduction -- Heteroplasmy Introduction Sequence heterogeneity of mitochondrial DNA (mtDNA) from sexually reproducing animals has been observed when the population size is large and the mutation rate is high (Thomas et al. 1990; for review, see Takahata 1985), or when male-mediated transmission of mtDNA occurs from one populaOffprint requests to: Y. Bessho

tion to another as reported in Drosophila (Kondo et al. 1990)and in Mytilus (Hoeh et al. 1991). Introgression also increases the extent of variation in a population (e.g., Satta and Takahata 1990). In this study, we analyzed a part of the cytochrome c oxidase subunit I (COI) gene of the freshwater planarian, Dugesiajaponica. This species is a free-living flatworm common in far eastern Asia. Planarians are one of the early emerged multicellular animals as estimated by a comparison of 5S rRNA sequences (Ohama et al. 1983). The reproductive mode of planarians is unique. It is believed that D. japonica reproduces asexually several times a year by lateral cleavage of its body, and in winter some D. japonica reproduce sexually by producing zygotes (sexual-asexual reproduction). However, extensive studies by Tamura et al. (1979) have shown that races having a mixoploid or triploid karyotype are probably asexual, whereas those with a diploid karyotype would be sexual-asexual. In the population used in this study, individuals carrying two karyotypes have been found, mixoploid (2X and 3X) and diploid (2X), respectively (Tamura et al. 1979). The COI sequences were different among and within 7 individuals, whereas those from the other 11 individuals showed little sequence heterogeneity. We suggest that these sequence heterogeneities have resulted mainly from asexual reproduction of the mixoploid individuals in the planarian population. Materials and Methods Animal Material Dugesiajaponica used in this studywere collected at the limitedpoint of a streamat the foot of Mr. Fujiwara,

325 pr-a

C O I gene

pr-a2

I

1.5kbp pr.-b pr-b2

ix-a,?.

,

I

ilOObPl

450bp

Mie Prefecture, central Japan. These planarians were starved for at least 2 days before preparation of DNA.

Preparation of DNA. We tried to isolate planarian mitochondria according to Yonekawa et al. (1981) without success, because of existence of a large amount of melanin. Subsequently, the method described below was adopted for selective amplification of mtDNA. In this paper, the DNA so amplified was considered to be of mitochondrial origin. 1) About 500 individuals (ca. 5 g by wet weight) were digested in a reaction mixture containing 50 mM Tris-HC1 (pH 8.0), 10 mM EDTA, 0.6% sodium dodecyl sulfate (SDS), proteinase K (200 ~g/ml) for 12 h at 50°C, and the solution was treated once with a phenol-chloroform mixture. After precipitation of nucleic acids with ethanol, crude DNA was centrifuged in a CsC1 solution containing 200 ug/ml ethidium bromide for 48 h at 42,000 rpm (Beckman type 45 rotor). Chromosomal DNA and mtDNA, which formed a broad single band, were not separated from each other. This DNA fraction was used as a template for the polymerase chain reaction (PCR) (Saiki et al. 1985) to amplify a part of the COI gene using a pair of primers, pr-a and pr-b (see below). 2) Crude DNA was prepared independently from 18 individual planarians as follows: each individual was rinsed with distilled water several times and digested as described above without purification of DNA in CsC1 solution. The ethanol-precipitated DNA was dissolved in 20/A of 10 mM Tris-HC1 (pH 8.0). One microliter of this DNA solution was used as template for PCR using a pair of adapted primers, pr-a2 and pr-b2 (see below). Construction of Probesfor PCR. A computer search was performed on the published sequences of the COI genes (Bonitz et al. 1980; Anderson et al. 1981, 1982; Bibb et al. 1981; Hensgens et al. 1984; Clary and Wolstenholme 1985; Roe et al. 1985; Pritchard et al. 1986; Jacobs et al. 1988; Garey and Wolstenholme 1989). From these references we found several highly conserved amino acid sequences. Two regions having the least ambiguity were selected for PCR primer sites. The approximate location of the sequenced region in a COI gene and the sequences of two primers, pr-a and pr-b, are shown in Fig. 1. Two adapted primers, pr-a2 and pr-b2, were prepared considering the sequences obtained from the amplified DNA products using pr-a and pr-b as primers. These primers enabled direct sequencing via PCR using a single planarian as a DNA source. PCR. PCR using pr-a and pr-b primers was performed with the use of a DNA Thermal Cycler (Perkin-Elmer Cetus). Each amplification cycle consisted of DNA denaturation at 94°C for 1 min (except for the first cycle for 3 min), primer annealing at 45°C for 2 min, and extension at 72°C for 3 min. This cycle was repeated 40 times. The amplifications from 18 planarian individuals were performed using adapted primers, pr-a2 and pr-b2, under the conditions as above, except for the annealing temperature (50°C) and

Fig. 1. Schematic representation of an amplified region of the COI gene by PCR. The sequences of four primers are as follows: pr-a, 5'-TGGTTTTTTGTGCATCCTGAGGTTTA-3' (6634); pr-b, 5'-AGAAAGAACGTAATGAAAATGAGCAAC-3' (7023); pra2, 5 ' - A G C T G C A G T T T T G G T T T T T T G G A CATCCTGAGGT-3' (6631); pr-b2, 5'-ATGAGCAACAACATAATAAGTATCATG-3' (7005). The designated site number in parentheses of the 3' base is according to the numbering system for human mtDNA (Anderson et al. 1981). Direct sequencing was performed using the pr-a2 primer.

the number of cycles (35 cycles). Amplified DNA products were then subjected to preparation of the template for direct sequencing or used as a DNA source for cloning of a part of the COI gene.

Cloning and Sequencing or Direct Sequencing. Amplified DNA fragments using pr-a and pr-b had approximately the expected size, about 450 bp, so that they were subjected to cloning. Three clones were sequenced by the conventional chain-termination method. Each amplified DNA fragment from 18 planarian individuals, using primers pr-a2 and pr-b2, was analyzed by a direct sequencing method using Taq DNA polymerase and 32p-labeled pr-a2 primer according to the method of Innis et al. (1988) with slight modifications in the ratio ofdNTP to ddNTP. These were dGTP/ ddGTP (1:5), dATP/ddATP (1:34), dTTP/ddTTP (1:50), and dCTP/ddCTP (1:32). We determined at least 240 bp from each sample. In addition to direct sequencing of 11 individuals, we analyzed four clones derived from the independent PCR products of three planarian individuals. Most of the heterogeneous sites (see below) were confirmed by direct sequencing with other suitable primers (not shown).

Results The DNA sources for PCR, strategies for sequencing, a n d t h e t y p e s o f s e q u e n c e s o b t a i n e d a r e s u m m a r i z e d i n T a b l e 1.

1) Sequences o f D N A Prepared f r o m a M i x e d Population o f Planarians The amplified DNA product, of which template DNA had been prepared from about 500 individual planarians, was subjected to direct sequencing. The primary purpose of this experiment was to ascertain the homogeneity of planarian mtDNA for further studies on the genomic structure. Unexpectedly, however, the sequence ladders showed that a numb e r o f s i t e s h a d m o r e t h a n o n e b a n d at t h e s a m e horizontal position, suggesting sequence heterogeneity. It became apparent that two sequences, type A and type D, were the main contributors to the o b s e r v e d h e t e r o g e n e i t y (see s e c t i o n 2). W e f u r t h e r obtained three clones from the PCR product of the DNA mixture described above and sequenced them. Three different types of DNA sequences were obt a i n e d , i.e., t y p e A , t y p e B, a n d t y p e C (Fig. 2). T h e s e

326 Table 1.

Source of DNA and classification of sequences

DNA source Mixture of 500 individuals

Individual 1 Individuals 2-10 Individual 11 Individual 12 Individual 13

Individual Individual Individual Individual Individual

14 15 16 17 18

Table 2. Pairwise nucleotide and amino acid differences among the COI genes for seven individuals (DI-D7) of Dugesiajaponica

Strategy Actual for seType of sequencinga sequenceb quence Cln Cln Cln Direct Cln Direct Direct Direct Cln Direct Cln Cln Direct Direct Direct Direct Direct

A B C A A A A D E D E E D D D D D

Seq-A 1 Seq-B Seq-C Seq-A1 Seq-A1 Seq-A 1 Seq-A2 Seq-D1 Seq-E 1 Seq-D2 Seq-E2 Seq-E3 Seq-D3 Seq-D4 Seq-D5 Seq-D6 Seq-D7

D1 D2 D3 D4 D5 D6 D7

D1

D2

D3

D4

D5

D6

D7

-3 4 4 4 3 5

0 -3 2 3 2 4

0 0 -4 4 3 4

0 0 0 -3 3 4

0 0 0 0 -2 5

0 0 0 0 0 -4

0 0 0 0 0 0 --

For each pair of sequences (240 bp), the number of nucleotide differences (rounded) is given below the diagonal and the number of amino acid differences above the diagonal. Y (T and C) and T (Y/T), Y/C, Y/Y, W (T and A)/T, W/G, W/W, K (T and G)/ T, K/A, and K / K have been counted as 0.5 and Y/W as 0.25

Table 3. Pairwise nucleotide and amino acid differences among types A-E of the Dugesia japonica COI gene A

Cln: cloning and sequencing; Direct: direct sequencing without cloning b As determined by sequence similarity, Seq-A1 and Seq-A2 are collectively grouped as type A. Similar groupings are done for Seq-D1 to Seq-D7 (type D) and Seq-E1 to Seq-E3 (type E)

B

C

D

E

a

three types differed greatly from one another, indicating the presence of different types of COI sequences. One of the major sequences, type D, was not included in these clones. Types B and C would be minor components, for these sequences did not appreciably contribute to the sequence heterogeneity observed on the direct sequencing ladders.

2) Sequence Heterogeneity among Planarian Individuals (Interindividual Heterogeneity) Two adapted primers, pr-a2 and pr-b2, enabled the amplification of a part of the COI gene even from a single planarian body. DNA was prepared independently from 18 individuals, and the amplified products were subjected to direct sequencing. Sequences of at least 240 bp long were determined for each sample. The sequences from 11 individuals were identical with type A with one exception. Position 150 of one of them (named Seq-A2) was Y (T and C) instead of C as in the typical type A (named Seq-A1) (see autoradiogram in Fig. 3). The sequences from seven individuals were similar but significantly different from one another (Seq-D1-D7 in Table 2; interindividual heterogeneity). We designated these type D, collectively. In all, nine heterogeneous sites were found among them. Type D differed considerably from type A, type B, and type C (Table 3). Most of the heterogeneity among different types existed in the silent (neutral) sites ofcodons, i.e., the predicted

A B C D E

--

3

2

1

13 28 23 28

-34 30 31

5 -29 30

4 3 -23

5

8 6 5 --

For each pair of sequences (240 bp), the number of nucleotide differences (rounded) is given below the diagonal and the number of amino acid differences above the diagonal. Y (T and C) and T (Y/T), Y/C, Y/Y, W (T and A)/T, W/G, W/W, K (T and G)/ T, K/A, and K/K have been counted as 0.5, Y/W as 0.25, and gaps in type B tentatively as 1.0, respectively

amino acid sequences were nearly identical among all the detected sequences, indicating that the heterogeneities were the results of neutral mutations (cf. Kimura 1983). The average nucleotide sequence diversity zr as defined by Nei (1987) is 0.02% in 11 type A sequences, 1.4% in 7 type D sequences, and 5.0% in 18 sequences, respectively.

3) Presence of More than One Type of COI Gene Sequence within a Single Individual (Heteroplasmy) Each of the type D sequences (Seq-D1-D4, D6, and D7) from a single planarian individual revealed more than one band at the same horizontal position for several sites on the sequence ladder. This indicates the presence of at least two distinct sequences in a single individual (heteroplasmy). Heterogeneous sites were shown in Fig. 2 using abbreviations, Y (T and C), W (T and A), and K (T and G). Representative autoradiograms of such heterogeneous sites are shown in Fig. 3. Besides these typical heterogeneous sites, there were several more sites having a faint band appearing together with the main band on the ladder. These ambiguous heterogeneous sites

327 (a)

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..... .....

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................. .................

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2. Sequence heterogeneity in a part of the COI gene of planarian mitochondria. Abbreviations, Y: T and C, W: T and A, and K: T and G. In Seq-B, - designates a gap. a Sequences of cloned PCR products prepared from a mixed population of planaria, b Sequences of PCR products prepared from individual planaria by direct sequencing. A1 x 10 denotes that 10 independent individuals carry the same sequence, Seq-A 1. c Sequences of cloned PCR product prepared from individual planaria. See also Table 1. Fig.

were n o t t a k e n i n t o a c c o u n t i n the s e q u e n c e s o f Fig. 2. O n the other h a n d , n o s u c h h e t e r o g e n e i t y w a s d e t e c t e d i n 11 t y p e A s e q u e n c e s , e x c e p t for o n e site in S e q - A 2 .

T o o b t a i n a d d i t i o n a l e v i d e n c e for h e t e r o p l a s m y , i n d e p e n d e n t c l o n e s were o b t a i n e d ; o n e c l o n e f r o m the a m p l i f i e d S e q - A 1 D N A source, o n e f r o m the S e q - D 1 D N A source, and t w o f r o m the S e q - D 2 D N A

328 panel G A

- ] T C

Seq - A1

•p a n e l

(109-132) G A TC

D1

GA

T C

D3

GA

TC

D5

-

II

(142-159)

G A T C

G A T C

A1

A2

G

A

T

C

D1

Fig. 3. Examples of heterogeneous sites among individuals (interindividual) or within single individuals (intraindividual). SeqA1, Seq-A2, Seq-D1, Seq-D3, and Seq-D5 were obtained by direct sequencing of PCR products, each using a single planarian as the source of template DNA. Four interindividual heterogeneous sites are shown. Position 120 is C, Y, K, and G in SeqA1, Seq-D1, Seq-D3, and Seq-D5, respectively; position 123 is T in Seq-A1 while it is C in others; position 132 is C in Seq-A1

while it is T in others (in panel I); position 150 is A, Y, and T in Seq-A1, Seq-A2, and Seq-D1, respectively (in panel II). Intraindividual heterogeneous sites were also detected in the autoradiograms (each sequence ladder was obtained by direct sequencing of DNA from a single planaria); position 120 (in panel I) of Seq-D1 is T and C (designated as Y), position 120 of SeqD2 is T and G (K), and position 150 (in panel II) of Seq-A1 is Y and C (Y).

source. These three DNA sources were derived from each of three individuals as described in section 2. A clone from the Seq-A1 DNA source showed a sequence identical to Seq-A 1 (individual 1 in Table 1). The sequences of three clones, one from Seq-D 1 (individual 12) and two from Seq-D2 DNA (individual 13) sources, shared some similarities but were considerably different not only from type D, but also from all other sequences, so that these were collectively designated type E and individually as Seq-E 1 (from the Seq-Dl DNA source), Seq-E2, and SeqE3 (from the Seq-D2 DNA source), respectively. Only a single base difference was found between SeqE2 and Seq-E3 (see Table 1). Again, the differences between type E and type D resided mainly in the codon silent positions with, however, conservative amino acid substitutions at four or five sites. As type E was not detected by direct sequencing, this was considered to be a minor component like the type B and type C found in DNA from the mixed population (see section 1). The reason for failure to clone type D is not known. Perhaps type E has structure for more efficient ligation of the DNA fragment to a vector plasmid as compared with type D.

DNA, depending on the period after the integrations. At present it is not possible to rule out this possibility, but the following findings go against this. (1) Nucleotide substitutions observed between the sequences are almost exclusively synonymous. It would be hard to believe that mutations did not take place on the amino acid-replacement sites on the integrated genes, unless the genes were functionally active after the integrations. A further argument would be that the integrations could be recent events, so that substitutions would be found only at synonymous sites. Even if such events took place, the presence of heterogeneity by itself implies that the COI genes had at least similar heterogeneity before the integrations, because synonymous (neutral) mutation is a characteristic of functional genes (Kimura 1983). (2) The sequences from a group of individuals (individuals 12-18 in Table 1) show inter- as well as i n t r a i n d i v i d u a l heterogeneities, whereas those from another group (individuals 111) do not. It is unlikely that mitochondrial genes in some individuals were integrated into the nuclear genome and others were not. A more reasonable explanation would be that heterogeneity exists in the mtDNA of an asexual race (2X-3X mixoploidy) as suggested in the Introduction, whereas individuals of a sexual-asexual race (2X) reveal no such heterogeneity as described below. Asexual reproduction would be the most effective cause for mtDNA heterogeneity in this organism. Undifferentiated free parenchymal cells, neoblasts, that have scattered throughout the body are moving around in the body constantly. The number of neoblasts has been reported to be 104-105 per organism in Dugesia lugubris (Lender and Gabriel 1960; Brondsted and Brondsted 1961). Many of the neo-

Discussion The sequence heterogeneities of the COI gene revealed b y PCR in this study would most probably represent those that exist in mtDNA, because this gene has been known exclusively from mitochondria. One might argue that, if some mitochondrial genes had been integrated into the nuclear genome in the past, many substitutions could accumulate between integrated copies and their original mt-

329 blasts are believed to migrate to the prospective cleavage surface and multiply, thus participating in the f o r m a t i o n o f a new b o d y u p o n asexual reproduction (Dubois 1948; M c W h i n n i e and Gleason 1957; Lender and Gabriel 1960, 1965). During sexual reproduction, only limited n u m b e r s o f mitochondria are introduced into progeny via female gametes because o f a bottleneck effect, whereas this effect m a y be negligibly small during asexual reproduction. Thus, introduction into progeny o f mitochondrial mutations via neoblasts w o u l d be m o r e efficient than introduction through gametes, resulting in m o r e m t D N A sequence heterogeneity in asexual races than in sexual races. N o t e that m i t o c h o n dria in sexual-asexual individuals (a sexual line) are also subjected to the bottleneck effect in the sexual phase, so that no appreciable heterogeneity w o u l d result. Indeed, the planarian population used here consists o f m i x o p l o i d individuals (probably an asexual line) and diploid individuals (probably a sexualasexual line) ( T a m u r a et al. 1979). Thus, it is reasonable to assume that 11 individuals with identical type A sequences might be sexual-asexual, whereas the others with heterogeneous t y p e - D sequences might be asexual. As both population size and substitution rate o f the m t D N A are u n k n o w n at present, it is not certain whether the extent o f the detected heteroplasmy and interindividual heterogeneity are theoretically reasonable or not. Indeed, the n u m b e r o f neoblasts in an individual ( ~ 10 ~) estimated for D. lugubris is not enough to produce the observed heteroplasmy, assuming that substitution rate o f the m t D N A is 10-s/ site/year. One explanation might be that the actual n u m b e r o f neoblasts a n d / o r the substitution rate o f the m t D N A in the asexual race o l D . japonica could be m u c h greater, so that heteroplasmy would have occurred. Then, interindividual heterogeneity could have been p r o d u c e d if the population size o f the asexual race is large enough. Type B, type C, and type E are assumed to be m i n o r c o m p o n e n t s for the reasons discussed above. The origins o f these types are not known. The possibility that these types are c o n t a m i n a n t s from other organisms is not very likely, because the genetic code in these types is a characteristic o f type A or type D, i.e., A U A codes for isoleucine, A A A for asparagine, A G R for serine, and U G A for tryptophan (see Bessho et al. 1992). It has been suggested that u p o n asexual reproduction, certain organs ofplanarians are rebuilt from dedifferentiated cells derived f r o m the original organs, in addition to the neoblasts (Teshirogi and Ishida 1987). I f such is the case, then one might speculate that organ-specific m t D N A would be m a i n t a i n e d in asexual lines. Type B, type C, and type E could be such m t D N A species, because these

sequences differ considerably from the m a j o r type A or type D sequences. Alternatively, the heterogeneities o f type D discussed a b o v e might also somehow be connected with such cell lineages. The coexistence in one locality o f sexual and asexual races o f the same species m a y be explained by either one o f the following ways. (1) These two races had been geographically isolated in the past and intermingled relatively recently. (2) Or, these two lineages were separated long ago from their c o m m o n ancestor and have shared the same locality as if they were two independent species. W h a t e v e r the explanation m a y be, the coexistence o f sexual and asexual individuals is not confined to the present case, as exemplified by a population o f D. japonica in Form o s a ( T a m u r a et al. 1987).

Acknowledgments. We are grateful to Drs. N. Saitou and N. Takahata for helpful comments on the manuscript. This work was supported by Research Aid from the Inoue Foundation for Science, Inamofi Science Foundation, and grants from the Ministry of Education, Science and Culture, Japan.

References Anderson S, Bankier AT, Barrell BG, de Bruijn MHL, Coulson AR, Drouin J, Eperon IC, Nierlich DP, Roe BA, Sanger F, Schreier PH, Smith ALH, Staden R, Young IG (1981) Sequence and organization of the human mitochondrial genome. Nature 290:457-464 Anderson S, Bruijn MHL, Coulson AR, Eperon IC, Sanger F, Young IG (1982) The complete sequence of bovine mitocbondrial DNA: conserved features of the mammalian mitochondrial genome. J Mol Biol 156:683-717 Bessho Y, Ohama T, Osawa S (1992) Planarian mitochondria II. The unique genetic code as deduced from cytochrome c oxidase subunit I gene sequences. J Mol Evol 34:000-000 Bibb MJ, Van Etten RA, Wright CT, Walberg MW, Clayton DA (1981) Sequence and gene organization of mouse mitochondrial DNA. Cell 26:167-180 Bonitz SG, Coruzzi G, Thalenfeld BE, Tzagoloff A, Macino G (1980) Assembly of the mitochondrial membrane system: structure and nucleotide sequence of the gene coding for subunit I of yeast cytochrome oxidase. J Biol Chem 255:1192711941 Brondsted A, Brondsted HV (1961) Number ofneoblasts in the intact body of Euplanaria torva and Dendrocoelum lacteum. J Embryol Exp Morphol 9:167-172 Clary DO, Wolstenholme DR (1985) The mitochondrial DNA molecule of Drosophila yakuba: nucleotide sequence, gene organization, and genetic code. J Mol Evol 22:252-271 Dubois F (1948) Sur les conditions de la migration des cellules de r6g6n6ration chez les planaires d'eau douce. CR Soc Biol 142:533-535 GareyJR, WolstenholmeDR (1989) Platyhelminthmitochondrial DNA: evidence for early evolutionary origin of tRNAs~rAGNthat contains a dihydrouridine arm replacement loop, and serine-specifying AGA and AGG codons. J Mol Evol 28:374-387 Hensgens LAM, BrakenhoffJ, de Vries BF, Sloof P, Tromp MC, van Boom JH, Benne R (1984) The sequence of the gene for cytoehrome c oxidase subunit I, a frameshift containing gene for cytochrome c oxidase subunit II and seven unas-

330 signed reading frames in Trypanosoma brucei mitochondrial maxi-circle DNA. Nucleic Acids Res 12:7327-7344 Hoeh WR, Blakley KH, Brown WM (1991) Heteroplasmy suggests limited biparental inheritance of Mytilus mitochondrial DNA. Science 251:1488-1490 Innis MA, Myambo KB, Gelfand DH, Brow MAD (1988) DNA sequencing with Thermus aquaticus DNA polymerase and direct sequencing of polymerase chain reaction-amplified DNA. Proc Natl Acad Sci USA 85:9436-9440 Jacobs HT, Elliot DJ, Math VB, Farquharson A (1988) Nucleofide sequence and gene organization of sea urchin mitochondrial DNA. J Mol Biol 202:185-217 Kimura M (1983) The neutral theory of molecular evolution. Cambridge University Press, London, pp 34-50 Kondo R, Satta Y, Matsuura ET, lshiwa H, Takahata N, Chigusa SI (1990) Incomplete maternal transmission of mitochondrial DNA in Drosophila. Genetics 126:657-663 LenderT, GabrielA (1960) l~tudehistochimiquedesn6oblastes de Dugesia lugubris (Turbellari6 triclade) avant et pendant la r6g6n6ration. Bull Soc Zool Fr 85:100-110 Lender T, Gabriel A (1965) Les n6oblastes marqu6s par Furldine triti6e migrent 6t edifient le blast,me de r6g6n6ration des Planaires d'eau douce. CR Acad Sci Paris 260:4095-4097 McWhinnie MA, Gleason MM (1957) Histological changes in regenerating pieces ofDugesia dorotocephalatreated with colchicine. Biol Bull 112:371-376 Nei M (1987) Molecular evolutionary genetics. ColumbiaUniversity Press, New York, pp 254-259 Ohama T, Kumazaki T, Hori H, Osawa S, Takai M (1984) Evolution of multieellular animals as deduced from 5 S rRNA sequences: a possible early emergence of the Mesozoa. Nucleic Acids Res 12:5101-5108 Pritchard AE, Seilhamer J, Cummings DJ (1986) Paramecium mitochondrial DNA sequences and RNA transcripts for cytochrome oxidase subunit I, U R F 1, and three ORFs adjacent to the replication origin. Gene 44:243-253 Roe BA, Ma DP, Wilson RK, Wong JFH (1985) The complete

nucleotide sequence of the Xenopus laevis mitochondrial genome. J Biol Chem 260:9759-9774 Saiki RK, Scharf S, Faloona F, Mullis KB, Horn GT, Erlich HA, A r n h e i m N (1985) Enzymatic amplification of /3-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Science 230:1350-1354 Satta Y, Takahata N (1990) Evolution of Drosophila mitochondrial DNA and the history of the melanogastersubgroup. Proc Natl Acad Sci USA 87:9558-9652 Takahata N (1985) Population genetics of extranuclear genomes: a model and review. In: Ohta T, Aoki K (eds) Population genetics and molecular evolution. Japan Scientific Societies Press, Tokyo, pp 195-212 Tamura Y, Yamayosi T, Oki I (1979) Karyological and taxonomic studies of Dugesiajaponica Ichikawa et Kawakatsu II. Chromosomes of Dugesia japonica japonica collected from eighteen localities in Japan. Proc Jpn Soc Syst Zool 17:1-14 Tamura S, Oki I, Kawakatsu M, Lue K-Y, Takai M, Hori H, Muto A, Osawa S (1987) A new series of studies on the freshwater and land planarians from Taiwan V. Chromosomes of Dugesia japonica. Bull Fuji Women's Coll no 25, ser II:55-65 TeshirogiW, IshidaS (1987) Neoblastsandformationoftissues and organs. In: Teshirogi W (ed) Biology ofplanarians--foundation, application and experiment. Kyoritsu, Tokyo, pp 68-83 Thomas WK, P~i~iboS, Villablanca FX, Wilson AC (1990) Spatial and temporal continuity of kangaroo rat populations shown by sequencing mitochondrial DNA from museum species. J Mol Evol 31:101-112 Yonekawa H, Moriwaki K, Gotoh M, Hayashi J, Watanabe J, Miyashita N, Petras M (1981) Evolutionary relationships among five subspecies ofMus musculus based on restriction enzyme cleavage patterns of mitochondrial DNA. Gene 98: 801-816 Received March 27, 1991/Revised November 25, 1991