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Differentiation of mitochondrial DNA polymorphisms in populations of five Japanese Abies species.

The variation of organelle DNA sometimes shows quite different relationships to those suggested by nuclear DNA because of the differences in their inheritance mode and effective number of genes in a population. Although intraspecific chloroplast DNA variation is generally very low due to its slow rate of evolution, enough variation has been detected in many plants for it to be used in population analysis (Soltis et al. 1992b). Also, several population studies have shown that cpDNA variation is geographically structured (Soltis et al. 1989, 1991, 1992a; Swell et al. 1996). In contrast to chloroplast DNA, plant mitochondrial DNA variation is characterized by many genome rearrangements that result from its rapid structural evolution (Sederoff 1987; Palmer 1992a). This feature of mtDNA structure can be useful for classifying genome types at lower taxonomic levels (Palmer 1992a). In Pinaceae, the inheritance mode of organelle DNAs are unique because cpDNA is paternally inherited but mtDNA is of maternal origin (Neale et al. 1989; Wagner et al. 1991; Mogensen 1996). Thus, the genetic diversity of cpDNA and mtDNA clearly differs in populations of Pinaceae species (Strauss et al. 1993; Dong and Wagner 1993). Mitochondrial DNA variation in Fagus crenata also showed a clear dissemination along the Japanese archipelago since the last glacial period (Tomaru et al. 1998).

Japanese Abies populations consist of five species that are distributed throughout the Japanese archipelago as follows: A. firma is found in the warm temperate zone; A, homolepis in the cool temperate zone; A. veitchii and A. mariesii in the upper mountain zone; and A. sachalinensis on Hokkaido Island (which is the most northern island in Japan). Using allozyme analysis, several genetic studies in Japanese Abies showed their genetic diversity and possible dissemination following the last glacial period (Nagasaka et al. 1997; Suyama et al. 1997). In A. mariesii, cp DNA variation also showed a similar trend of that of allozyme study (Tsumura et al. 1994). Suyama et al. (1996) also analyzed sequence differences of a cpDNA spacer region among the five species and fossil Abies, and Tsumura et al. (1995) have studied their phylogeny using PCR-RFLP analysis of cpDNA genes. Nevertheless, the phylogenetic relationships of these species are not still entirely clear. By clarifying their relationships and determining the genetic diversity between and within species, we may know the speciation and dissemination within species, which might be closely related the geographical and climatic changes in ancient times. Therefore, we have conducted additional population and phylogeny studies using a combination of mtDNA variation and cpDNA sequence analysis.

MATERIALS AND METHODS

Plant Materials

Fresh needles were collected from 1,009 trees representing 40 populations dispersed widely across the natural distribution of five Abies species [ILLUSTRATION FOR FIGURE 1 OMITTED]. Abies sachalinensis is found in Hokkaido Island between 10 m and 1600 m above sea level (Yamazaki 1995). Abies mariesii is distributed in the upper mountain regions at elevations between 1000 m and 1800 m in northern Honshu and 1800 m and 2900 m in central Honshu. Abies veitchii is found mostly in central Honshu at elevations between 1200 m and 2800 m, except for two isolated populations, the Miser population of Kii Peninsula and the Ishizuchi population of Shikoku Island. The distribution of A. homolepis ranges from Fukushima southward through central Honshu and Shikoku to Kyushu Island between elevations 1000 m and 1800 m above sea level. Abies firma has wide distribution from the northern part of Honshu to the southern part of Kyushu Island, and its favorable elevation is between 50 m and 1600 m. The Mitsumine population of A. firma is said to be a hybrid of A. firma and A. homolepis, called A. umbellata (Mayr 1890). The Ishizuchi population of A. veitchii is said to be a variety of A. veitchii and has the name A. veitchii var. sikokiana (Liu 1971). This population is isolated in Shikoku from the main range of the species [ILLUSTRATION FOR FIGURE 1 OMITTED]. Fresh needles from individuals representing the 40 populations were stored at -30 [degrees] C until DNA extraction.

DNA Manipulation

Total DNA was extracted from needles of each sample by the method of Tsumura et al. (1995). Five grams of each needle sample was frozen in liquid nitrogen and ground in an Iwatani grinder (IFM-150). The ground tissues were then added to 50 ml of ice-cold buffer I (50 mM Tris-HCl, pH 8.0, 5 mM EDTA, 350 mM sorbitol, 0.1% BSA, 0.1% [Beta]-mercaptoethanol, and 10% polyethylene glycol 6000). The samples were then homogenized in the Iwatani grinder for 30 sec. The homogenate was filtered through two layers of cheesecloth and one layer of miracloth and pelleted by centrifugation at 3000 rpm for 10 rain at 4 [degrees] C. The pellet was resuspended in 10 ml of ice-cold buffer II (50 mM Tris-HCl, pH 8.0, 5 mM EDTA, 350 mM sorbitol, 0.1% BSA, 0.1% mercaptoethanol, and 1% sodium sarkosyl) and incubated for 30 min at room temperature. Then 10 ml of 2 x CTAB buffer (100 mM Tris-HCl, pH 8.0, 20 mM EDTA, 1.4 mM NaCl, 2% Cetytrimethylammoniumbromide, 0.4% mercaptoethanol) was added and the mixture was incubated at 60 [degrees] C for 10 min. The mixture was then shaken with 20 ml of chloroformisoamyl alcohol (24:1), and the resulting aqueous and organic layers were separated by centrifugation at 4000 rpm for 10 min at room temperature. The aqueous layer was transferred to a new tube and the DNA was precipitated by adding two-thirds volume of cold isopropanol. The DNA was recovered with a disposable pipette, washed with 70% cold ethanol, and dissolved in TE (10 mM Tris-HCl, pH 8.0, 0.1 mM EDTA). To remove contaminating RNA, each sample was incubated with RNase (20[[micro]gram]/mL) for 60 min at 37 [degrees] C. This was followed by phenol extraction of the DNA and precipitation with ethanol.

Portions of the DNA preparations were each digested by one of 18 restriction endonucleases: BamHI, Bg/II, DraI, EcoRI, EcoRV, HaeIII, HindIII, HinfI, HhaI, KpnI, PstI, PvuII, StyI, SalI, SmaI, TaqI, XbaI, and XhoI. The resulting fragments were separated by electrophoresis at 15 V for 20 hr on 0.7% agarose gels in TAE buffer (40 mM Tris-HCl, 20 mM sodium acetate and 2 mM EDTA, pH 8.0). After electrophoresis, the DNA samples were transferred to a nylon membrane (Hybond-N, Amersham Co. Ltd.) using the Southern blot method.

In Southern hybridization analysis of the mitochondrial DNA, two gene probes were used: coxI and coxIII. These probes were prepared by PCR amplification after designing primers based on the coxI and coxIII sequences published by Grabau et al. (1989) and Kemmerer et al (1989), respectively. PCR amplification was carried out by the method of Tsumura et al. (1995) using the following primers: 5[prime]-CGATGGCTGTTCTCCACTAA-3[prime] and 5[prime]-ATCTGGATAATCTGGAATGC-3[prime] for coxI, and 5[prime]-ATGATTGAATCTCAGAGGCA-3[prime] and 3[prime]-TATACCTCCCCACCAATAGA-3[prime] for coxIII. The probe DNAs (0.1-0.2 [[micro]gram]) were labeled by the digoxigenin nonradioactive labeling method (Boehringer Mannheim Co. Ltd). The nylon membranes were hybridized with probes for 18 hr at 42 [degrees] C in hybridization buffer (50%(w/v) formamide, 5 x SSC, 0.5%(w/v) dry skim milk, 0.1%(w/v) N-lauroylsarcosine sodium salt, 0.02%(w/v) SDS, 50 mg/ml salmon sperm DNA). Membranes were washed twice in 2 x SSC, 0.1% SDS for 15 min at room temperature and then twice in 0.1 x SSC, 0.1% SDS for 15 min at 68 [degrees] C. Immunological detection of the hybridized fragment was conducted following the protocol of the manufacturer (Boehringer Mannheim Co. Ltd.).

We also sequenced the partial rbcL gene (size = 1331 bp) from the five Abies species. PCR primers for rbcL were designed in the coding region, based on the sequence of the Pinus thunbergii gene published by Wakasugi et al. (1994). The gene was amplified by PCR using the following primers: 5[prime]-CAGCAGCTAGTTCAGGACTC-3[prime] and 5[prime]-ATGTCACCAAAAACAGAGACT-3[prime]. Then the amplified fragments were purified using S-400 spin columns (Pharmacia Co. Ltd) to prepare the template DNA for sequencing. This was carried out using the dye-terminator method with an ABI DNA sequencer model 377 using the following primers: 5[prime]-CAGCAGCTAGTTCAGGACTC-3[prime], 5[prime]-ACAATGGCCTACTTCTTCAC-3[prime], 5[prime]-GGACATACGCAATGCTTTAG-3[prime], 5[prime]-CCCTGCTTATTCCAAAACTT-3[prime], 5[prime]-ACCCAATTTTGGTTTGATAG-3[prime], and 5[prime]-ATGTCACCAAAAACAGAGACT-3[prime].

In addition, to confirm the sequences of the two genes used as probes, the PCR products were directly sequenced by the dideoxy termination method using the ABI 377 DNA sequencer. A MPsrch homology test (Collins 1993-1995), based on the Smith-Waterman dynamic programming algorithm (Smith and Waterman 1981), was also performed to identify the sequences.

Statistical Analysis

Frequencies of the polymorphic phenotypes in each population were determined by analysis of the coxI-HindIII, coxI-HhaI, coxI-SmaI, and coxIII-HindIII combinations. In this case, we considered the two polymorphisms as independent loci with the phenotypes as alleles. Haplotypes were subsequently determined according to phenotypes of the two polymorphisms. The frequencies of haplotypes in each population were then estimated, considering the mitochondrial genome as one locus with the haplotypes as alleles. Haplotype diversity (He) was calculated in coxI and coxIII polymorphisms of each population using their frequencies (Nei 1978). Haplotype diversity statistics were also calculated (Nei 1973, 1978) using the haplotype frequencies. These statistics partition genetic diversity into subdivisions within and among species components. Total gene diversity (HT) consists of the genetic variation within (HS) and among (DST) species such that HT - HS + DST. The HS can be further subdivided into gene diversity within (HP) and among (DPS) populations within species such that HS = HP + DPS. GST and GPT, the coefficients of gene differentiation among species and among populations within species, respectively, were then calculated to determine how gene diversity was partitioned at each level using the equations GST = DST/HT and GPT = DPS/HT. We also calculated the value of HP/HT to determine gene diversity within populations within species and GPS (from DPS/HS) for gene diversity in each species. Using the FSTAT program (Goudet 1995), the fixation index [Theta] (Wright 1965) was also estimated, together with its standard deviation, by the method of Weir (1990). The statistical significance of 0 was evaluated by the permutation test.

A phenetic approach was used to interpret relationships among populations by UPGMA analysis (Sheath and Sokal 1973) based on unbiased genetic identities (Nei 1972, 1978) estimated by the frequency of restriction fragment phenotypes.

Aligned sequences were analyzed using the PAUP 3.1.1 software package (Swofford 1993). Parsimony analysis was carried out using the branch-and-bound algorithm. The rbcL sequence of P. thunbergii was used as an outgroup. Robustness of the resulting tree was tested by bootstrap analysis (Felsenstein 1985) of 1000 replicates using the heuristic algorithm in PAUP.

RESULTS

Mitochondrial DNA Polymorphisms in Abies

For the preliminary survey, we investigated 36 combinations of probes and restriction endonucleases using six individuals per species. We selected four combinations (coxI-HindIII, coxI-HhaI, coxI-SmaI, and coxIII-HindIII), which gave clear polymorphism from 12 combinations [ILLUSTRATION FOR FIGURE 2 OMITTED]. We found five, six, two, and seven variants of coxI-HindIII, coxI-HhaI, coxI-SmaI, and coxIII-HindIII polymorphisms, respectively, among the five Abies species. Polymorphism of coxI (He = 0.704) was greater than that of coxIII (He = 0.519). With these probe-enzyme combinations, we observed one to four fragments, but the number of informative fragments was one or two. We found 15 haplotypes (I-XV) using the four probe-enzyme combinations (Table 1).

Diversity within and between Populations

Haplotype frequencies strongly differ among A. firma, A. homolepis, and A. sachalinensis populations (Table 1). Abies mariesii populations were found to have no coxI and coxIII variations and a fixed haplotype (denoted v). Abies veitchii populations had four haplotypes, but most of them shared a single haplotype (VII).

Abies firma populations were found to have seven haplotypes altogether, but five populations of this species had fixed, single haplotypes: the Tsukuba, Todai-chiba, and Dando populations had haplotype I; the Komagamine population had haplotype VII; and the Osugidani population had haplotype XIII [ILLUSTRATION FOR FIGURE 3 OMITTED]. The other two populations, Ishizuchi and Kirishima, showed intrapopulation variations, with He values of 0.587 and 0.142, respectively. Three southern populations (Osugidani, Ishizuchi, and Kirishima) and the northernmost, marginal population (Komagamine) differed from the other three central populations [ILLUSTRATION FOR FIGURE 3 OMITTED]. The Komagamine population shared the same haplotype (VII) with populations in A. veitchii and A. sachalinensis. The northern populations of A. homolepis also have haplotype VII.

Populations of A. homolepis showed great polymorphism. All populations except the Fuji and Chausu populations showed intrapopulational variation. The derived He ranged from 0.100 in the Yatsugatake population to 0.667 in the Mikunitoge population, and their average He value was 0.314. In A. homolepis, the main haplotype was I, as was true for A. firma. All populations in Honshu were found to have this haplotype, and only the Ishizuchi population on Shikoku island did not have it. The haplotypes of Ishizuchi population was very similar with those of Ishizuchi population of A. firma.

Nemuro and Hidaka A. sachalinensis populations showed intrapopulation variation, with five and six detected haplotypes, respectively [ILLUSTRATION FOR FIGURE 3 OMITTED]. The main haplotype in A. sachalinensis was VII, the same as that of A. veitchii. No within-population variation was observed in three A. sachalinensis populations of western Hokkaido (Table 1).

The Mitsumine population of A. umbellata had two haplotypes, which were similar to the main haplotypes of A. firma and A. homolepis (Table 1).

Diversity within and between Species

The derived gene diversity coefficients of the five Abies species were HT = 0.715, HS = 0.317, and HP = 0.130 (Table 2). Therefore, the gene diversity coefficients between populations and species, as given by DPS and DST, were 0.187 and 0.398, respectively. The gene differentiation coefficients among populations (GPS), among populations within species (GPT), and among species (GST) were 0.590, 0.262, and 0.557, respectively. The gene diversity coefficients of each species (HS) varied and were 0.741, 0.604, 0.039, 0.000, and 0.292 for A. firma, A. homolepis, A. veitchii, A. mariesii, and A. [TABULAR DATA FOR TABLE 1 OMITTED] sachalinensis, respectively (Table 2). The [Theta]-values also varied from 0.022 to 0.883 and were highly significant.

Using four combinations of probe and enzyme, we detected 15 haplotypes altogether. The three major haplotypes were VII, V, and I, and their frequencies were 0.458, 0.236, and 0.170 respectively. Most A. veitchii and A. sachalinensis individuals tested had haplotype VII, all individuals of A. mariesii had haplotype V, and approximately half of A. firma and A. homolepis individuals had haplotype I. Abies firma and A. homolepis had nine haplotypes between them, five of which they shared. The proportions of shared haplotypes were detected in 83.3% of A. firma and 90.3% of A. homolepis samples, respectively [ILLUSTRATION FOR FIGURE 4 OMITTED]. Abies veitchii and A. sachalinensis also shared a haplotype (VII), which was found in 98.0% and 83.8% of cases respectively. This haplotype was also observed [TABULAR DATA FOR TABLE 2 OMITTED] in A. firma and A. homolepis but not at such high frequency (23.0% and 6.5%, respectively). The main difference detected between A. veitchii and A. sachalinensis was that three coxIII-related variations were found in two A. sachalinensis populations, which were not detected in any A. veitchii populations (Table 2). In contrast, A. mariesii populations were found to have a fixed haplotype (V) and to be very different from populations of the other four Abies species [ILLUSTRATION FOR FIGURE 4 OMITTED]. Only one individual, from the Nikko population of A. veitchii, shared this haplotype (Table 2).

Phenetic Analysis

All of the topologies constructed had six major branches, four of which contained A. firma populations [ILLUSTRATION FOR FIGURE 5 OMITTED]. Three of these four branches also contained all the A. homolepis populations. All A. veitchii populations shared pairwise genetic identity of nearly 1.0 because little variation within species was found in these populations. This branch also included three populations of A. sachalinensis and the Komaganine population of A. firma, which shared the same haplotype. The other two A. sachalinensis populations that had coxIII variations were also included in this branch. A. mariesii populations were located in a separate branch because they had a different haplotype with no variation, unlike the other species. According to this tree, section Momi (Farjon 1990), which including the two species, A. firma and A. homolepis showed most diversity than sections Amabilis and Balsamea, which showed little or no mtDNA variation. Although they are different species, Ishizuchi populations between A. firma and A. homolepis were very similar.

Sequences of rbcL in the Five Abies Species

The 1331-bp rbcL sequences in A. sachalinensis, A. veitchii and A. homolepis were completely concordant. The sequences of A. firma and the former three species differed by only one base substitution, but A. mariesii showed 13 base substitutions from that of A. sachalinensis. We constructed a strict consensus tree based on rbcL sequence data using the parsimony method, which showed that A. mariesii was quite different from the other four species [ILLUSTRATION FOR FIGURE 6 OMITTED]. Parsimony analysis resulted in two equally parsimonious trees, each with a length 52 steps, a consistency index of 0.981 and a retention index of 0.833. Bootstrap analysis was in agreement with the strict consensus tree and the values were very high.

Discussion

Mitochondrial DNA Polymorphism in Abies

Inheritance of mtDNA is expected to be primarily maternal in Pinaceae (Neale and Sederoff 1989; Sutton et al. 1991; Wagner et al. 1991; Marshall and Neale 1992), whereas cpDNA is paternally inherited (Neale et al. 1986, 1989; Szmidt et al. 1987; Stine et al. 1989). Ziegenhagen et al. (1995) reported that cpDNA of Abies alba also showed paternal inheritance. It appears that A. mariesii is also able to transmit paternally, since we found cpDNA variations in megagametophytes of individual trees (Tsumura et al. 1994). Strauss et al. (1993) found high levels of population differentiation in studies of mtDNA polymorphism in Pinus spp. For this analysis of Abies species, we have therefore used the same method as Strauss et al. (1993); Southern hybridization of total genomic DNA, using PCR-amplified coxI and coxIII genes from the mtDNA as probes. The partial DNA sequences of the PCR-amplified mitochondrial coxI and coxIII genes from A. homolepis are very similar to the corresponding gene sequences of many angiosperm species in the MPsrch homology test (data not shown). This result verifies that the PCR-amplified genes used as probes were genuinely from the mtDNA. The hybridization intensity obtained with each mitochondrial probe appeared to be much too strong for it to be detecting single-copy nuclear genes, and the restriction fragment analysis did not give diploid-like patterns, which would be expected for nuclear genes. In addition, there are no reports so far that mtDNA sequences are incorporated into cpDNA (Palmer 1992b). The mtDNA polymorphisms were observed with the combination of two genes and many restriction enzymes, indicating that they likely resulted from structural rearrangements, which is consistent with current knowledge of plant mitochondrial DNA (Sederoff 1987; Palmer 1992a, Dong and Wagner 1993, Strauss et al. 1993). These observations strongly support the contention that the polymorphisms we observed were derived from mtDNA and not from either nuclear DNA or cpDNA.

Mitochondrial DNA polymorphism was investigated using PCR-amplified coxI and coxIII genes as probes. Polymorphism of coxI was found to be greater than that of coxIII. The number of haplotypes detected were eight and seven, respectively, and the derived gene diversity coefficients were HT = 0.704, HS = 0.282, GST = 0.600 for coxI, and HT = 0.519, HS = 0.211, GST = 0.593 for coxIII. The difference in gene diversity between coxI and coxIII might be related to their locations in the mitochondrial genome, because mtDNA contains large inverted and direct-repeat sequences within the genome, and recombination may occur through these repeat sequences. The process of recombination may not be restricted to intermolecular events but may also occur between different circular DNA molecules that share sequence homology (Lonsdale and Grienenberger 1992).

Highly Different mtDNA Variations Exist within and between Populations

Gene diversity was estimated at each level, and the partitioning was found to be as follows: 55.7% was found to be between species (GST), 26.2% occurred between populations (GPT), and 18.2% occurred within populations (HP/HT). The gene diversities between and within populations were highly variable, especially in A. firma and A. homolepis.

Populations of A. mariesii and A. veitchii appear to have little or no mtDNA variation. Populations of A. firma are highly polymorphic, but only two populations of southern Japan, Ishizuchi and Kirishima, showed intrapopulation variation. The other five populations have fixed, single haplotypes. The Komagamine trees, which form a marginal population of this species, have a unique haplotype for A. firma that is the same as a major haplotype of A. veitchii and A. sachalinensis. This might be evidence that hybridization/introgression occurred between A. firma and A. veitchii/A, sachalinensis in ancient times. The Osugidani population of A. firma also has a fixed haplotype (XIII), which it shares with the Ishizuchi populations. The Ishizuchi population of A. firma also has very similar haplotypes with the Ishizuchi population of A. homolepis which are mostly unique to A. homolepis. This might be also one of example of mtDNA capture in sympatric region between two species (Rieseberg and Soltis 1991). Brunsfeld et al. (1992) and Whittemore and Shaal (1991) reported similar phenomena in Salix and oaks, respectively. The marginal and southern populations in A. firma have unique haplotypes. Abies homolepis populations have similar trends to those of A. firma. In particular, the Kyushu, Shikoku, and Kii Peninsula populations of A. firma and A. homolepis, which inhabit areas corresponding to the probable refugia of several species in the late glacial period (Tsukada 1983, 1988), have great genetic diversity in their mtDNA. Because it is difficult to identify fossil pollen to the species level using morphology alone, we cannot be certain of the distribution of each Abies species during the last glacial period. However, if we obtain suitable samples of fossil pollen from many different stages and sites, we may be able to determine the past distribution of the five Abies species using sequence analysis of cpDNA in the fossil pollen (Suyama et al. 1996). It is known, nevertheless, that A. firma and A. homolepis were growing in the lowlands of southwestern Japan during the last glacial period (Tsukada 1983, 1988). Extension from these southwestern populations as centers of origin to the present distribution would be consistent with the above results. This is because, after the late glacial period, these ancestral populations of A. firma and A. homolepis should have expanded their distributions northward and to higher altitudes, and they might well have been affected by genetic drift during the migration. Although no mtDNA variation was observed in A. mariesii, allozyme studies of this species have shown clear evidence of genetic drift during the expansion of its distribution northward. This is seen in a cline of genetic variation, which decreases with increasing latitude (Suyama et al. 1997). The other Abies species are also considered to have been affected by genetic drift during the expansion of their distribution. Similarly, Tomaru et al. (1997, 1998) have shown by allozyme and mtDNA analysis that mtDNA variation in Fagus crenata has strong geographic structure, which may reflect the species' distribution in the last glacial maximum and subsequent colonization. The mtDNA variation they described probably also reflects intraspecific phylogeography of the species.

Two eastern populations of A. sachalinensis showed within-population mtDNA variations. Previous allozyme analysis has detected a significant east-west geographical variation pattern among A. sachalinensis populations, and, in general, the western populations have lower genetic variability than the eastern populations (Nagasaka et al. 1997). These authors mentioned that the east-west variation pattern may be due to regional environmental variations of factors such as warmth index, mean annual temperature, and snowfall (Tatewaki 1958; Nakamura et al. 1986). Genetic variation of cone, seed, and seedling characters; resistance to snow damage; freezing resistance; and winter desiccation resistance were also well documented (Hatakeyama 1981; Okada 1983; Eiga 1984). These morphological and physiological characters were shown to be closely related to the environmental variations and isozyme results. As discussed by Okada (1983), there may have been refugia of this species in northeast Hokkaido during the last glacial age. If so, the western populations might have lost their mtDNA variation during their migration westward after the last glacial period.

Mitochondrial DNA is predominately inherited maternally, although there are known to be some exceptions in conifers (Neale et al. 1989, 1991). In Pinaceae species, the inheritance mode is thought to be maternal (Neale and Sederoff 1989; Sutton et al. 1991; Wagner et al. 1991; Marshall and Neale 1992), and this is also believed to be true for the Abies species we studied. The strong population differentiation in mtDNA for Abies is most likely due to the maternal inheritance of mitochondria, and restricted seed dispersal. Many genera belonging to Pinaceae, including Pinus, Pseudotsuga, Larix, Picea, and Abies show paternal inheritance of cpDNA (Szmidt et al. 1987; Neale et al. 1989; Neale and Sederoff 1989; Sutton et al. 1991; Wagner et al. 1991; Ziegenhagen et al. 1995). Although the mode of inheritance in mitochondria of Japanese Abies species has not been investigated so far, there are some reports that mtDNA of Pinus and Picea species (which also belong to the Pinaceae family), is inherited maternally. Allozyme variations have been reported in two Japanese Abies species: A. mariesii (Suyama et al. 1997) and A. sachalinensis (Nagasaka et al. 1997). According to the allozyme data (Gst values of A. mariesii and A. sachalinensis are 0.144 and 0.015, respectively), the number of migrants exchanged per generation ([N.sub.e]m) of the two species are 4.6 and 16.6 respectively. However, the derived GST values of mtDNA variation among the five Abies species we studied range from 0.859 for A. firma to 0.198 for A. sachalinensis. We can, therefore, calculate values of 0.08 to 2.03 for [N.sub.eo][m.sub.e] (Birky et al. 1983, 1989) for the mitochondrial genome under the following assumptions: migration-drift equilibrium; a large number of populations; and negligible mutation rate. This result indicates that the migration rate is much higher for nuclear genes than for mtDNA genes. The derived value of [N.sub.eo][m.sub.e] for the chloroplast genome in Japanese five Abies is much higher, 28.91 (GST = 0.017) (Tsumura, Suyama, and Yoshimura, unpubl. data) presumably because cpDNA in Abies is also inherited paternally, so the gene flow is considered to occur through pollen dispersal. Consequently, the different rates of gene flow through seed and pollen dispersal are reflected in the differentiation between populations. Therefore, these results are in agreement with the theoretical expectation that organellar DNA variation should generally exhibit greater spatial structure than nuclear variation (Birky et al. 1989; Petit et al. 1993).

Differentiation of Mitochondrial DNA Variation in the Five Japanese Abies Species and Comparison with Chloroplast DNA Phylogenies

Our phenetic tree based on the genetic similarity of mtDNA suggests that some species are polyphyletic, especially A. firma and A. homolepis, which diverge from the other three species and have much greater mtDNA variation. However, we explain these results as being consequences of convergent mtDNA structural evolution (Palmer 1992a; Strauss et al. 1993). Frequencies of mtDNA haplotypes in the five Abies species clearly show their genetic relationships [ILLUSTRATION FOR FIGURE 5 OMITTED]. The haplotypes of A. firma and A. homolepis are very closely related because most of their haplotypes are shared. Abies umbellata, which is thought to be a hybrid between A. firma and A. homolepis, has similar haplotypes to these two species. However, we cannot confirm whether this species is hybrid from this data because we did not find species-specific mtDNA variation. Abies veitchii and A. sachalinensis also share the same major haplotype. However, A. mariesii is quite different from the other four species. A phylogeny based on rbcL sequences also supports the evolutionary position of A. mariesii [ILLUSTRATION FOR FIGURE 6 OMITTED]. The sequence data of A. homolepis, A. veitchii, and A. sachalinensis are completely concordant. The only differences detected between the sequences of A. firma and these three species are one base substitutions of the 1331-bp rbcL. Thus, these four Abies species are genetically very closely related according to the rbcL sequences. Even in the sequence of spacer region between rrn5 and trnR in cpDNA, the differences between these four Abies species are only one nucleotide substitution and one insertion/deletion (Suyama et al. 1996). However, A. mariesii is also quite different from the other four Abies species in terms of rbcL sequence data, which showed 13 nucleotide position difference from the rbcL of A. homolepis [ILLUSTRATION FOR FIGURE 6 OMITTED]. based on mtDNA variation, the five Abies species can be divided roughly into three groups consisting of (1) A. firma and A. homolepis; (2) A. veitchii and A. sachalinensis; and (3) A. mariesii [ILLUSTRATION FOR FIGURE 5 OMITTED]. The cpDNA sequence data showed that A. firma and A. homolepis were more closely related to A. sachalinensis and A. veitchii than to each other (Suyama et al. 1996), but the differences among these four species were only one-base substitutions. Therefore, these differences were too small to be significant.

Mitochondrial DNA variation more clearly shows the species-level genetic relationships of the five Abies species studied here. According to current classifications of Abies based on morphological traits (Farjon 1990; Yamazaki 1995), both A. veitchii and A. sachalinensis are included in the section Balsamea: A. firma is in the Momi section, A. homolepis is in the Homolepides section (Yamazaki 1995) or in the Momi section (Farjon 1990), and A. mariesii is in the Amabilis section. This classification is consistent with our results, because the mtDNA analysis showed A. veitchii and A. sachalinensis to be closely related and A. firma to be very similar to A. homolepis. Yamazaki (1995) classified A. firma and A. homolepis into different sections, but like Farjon's (1990) study, our data suggest these two species belong in the same section. Thus, the mtDNA variation that we observed is not only useful for studying the difference between populations and for establishing criteria to use in gene conservation, but also for understanding the geographical dissemination of each species. We may also be able to determine the differences between species within genera more clearly using mtDNA variation than we can using cpDNA sequence or allozyme analysis.

ACKNOWLEDGMENTS

We thank N. Tomaru and T. Nakamura for insightful comments and discussions concerning earlier versions of this manuscript. We also thank K. Nagasaka and K. Ishida for sampling Hokkaido populations of Abies sachalinensis, N. Tomaru for sampling the Ikawa population of A. veitchii, T. Tange for helping sample the Todai-Chiba population of A. firma, K. Yoshimura for helping sample some of the A. veitchii and A. homolepis populations, H. Taguchi for helping sample several populations of A. veitchii and A. mariesii, and M. Koshiba and H. Taguchi for technical assistance. We also thank K. Yoshimura for designing coxI and coxIII mitochondrial gene primers. This study was supported by a Grant-in-Aid from the Science and Technology Agency of Japan (Encouragement of Basic Research) and by the Program for Promotion of Basic Research Activities for Innovative Biosciences.

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Author:Tsumura, Yoshihiko; Suyama, Yoshihisa
Publication:Evolution
Date:Aug 1, 1998
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