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Phylogenetic relationships within Araucariaceae based on rbcL gene sequences¹

^a Makino Herbarium, Faculty of Science, Tokyo Metropolitan University, Tokyo 192–03, Japan; ^b Department of Biology, Faculty of Science, Chiba University, Chiba 246, Japan; and ^c Department de Botanique, Centre ORSTOM de Nouméa, BP A5 Nouméa, New Caledonia

	ABSTRACT

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Phylogenetic relationships were determined in the Araucariaceae, which are now found mainly in the Southern Hemisphere. This conifer family was well diversified and widely distributed in both hemispheres during the Mesozoic era. The sequence of 1322 bases of the rbcL gene of cpDNA was determined from 29 species of Araucariaceae, representing almost all the species of the family. Phylogenetic trees determined by the parsimony method indicate that Araucariaceae are well defined by rbcL sequences and also that the monophyly of Agathis or Araucaria is well supported by high bootstrap values. The topology of these trees revealed that Wollemia had derived prior to Agathis and Araucaria. The rbcL phylogeny agrees well with the present recognition of four sections within Araucaria: Araucaria, Bunya, Eutacta, and Intermedia. Morphological characteristics of the number of cotyledons, position of male cone, and cuticular micromorphologies were evaluated as being phylogenetically informative. Section Bunya was found to be derived rather than to be the oldest taxon. Infrageneric relationships of Agathis could not be well elucidated because there are few informative site changes in the rbcL gene, suggesting the more recent differentiation of the species as their fossil records indicate. The New Caledonian Araucaria and Agathis species each formed a monophyletic group with very low differentiation in rbcL sequences among them, indicating rapid adaptive radiation to new edaphic conditions, i.e., ultramafic soils, in the post-Eocene era.

Key Words: Agathis • Araucaria • Araucariaceae • Gondwana • rbcL • Wollemia

	INTRODUCTION

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From the biogeographical point of view, the Araucariaceae are one of the most interesting families with a primarily Southern Hemisphere distribution. They occur in South America, Australia, New Zealand, New Guinea, New Caledonia, and other South Pacific islands. The family consists of three well-defined genera, Araucaria de Jussieu (19 extant species sensu de Laubenfels [1972, 1988], Agathis Salisbury (13 species: Mabberley, 1987) and the monotypic genus Wollemia W. G. Jones, K. D. Hill and J. M. Allen. The first two genera have been recognized for more than a century; Wollemia was recently found in New South Wales, Australia, and described in 1995. All species of Araucaria and most of Agathis species are restricted to the Southern Hemisphere. Some species of Agathis occur north of the equator in Malaysia, but this is regarded as a result of migration from the Southern Hemisphere to the Northwest during the Plio-Pleistocene (Florin, 1963). Thus, the extant species of the family are assumed to be of southern origin.

The genera Araucaria and Agathis each have distinctive morphological characteristics of leaves and cone elements (bract scale, ovuliferous scale, and seed), described before Wollemia was known. Wollemia shows intermediate characteristics between Araucaria and Agathis (Jones, Hill, and Allen, 1995), and similar characteristics are known only in fossil taxa such as Podozamites from the Jurassic to Tertiary Periods.

Araucaria is the most diversified genus in the family, being disjunctively distributed throughout the Southern Hemisphere (Chile, Argentina, southern Brazil, New Caledonia, Norfolk Island, Australia, and New Guinea). Although this distribution from a southern Pacific-Antarctic link to South America is generally taken as the result of a vicarious event and/or long-distance dispersal (Pole, 1994; Macphail, 1997), araucarian fossils have been widely excavated not only from the Southern but also the Northern Hemisphere. Further, they had been a major component of the Mesozoic forests (Miller, 1977; Stockey, 1982; Stocky, Nishida, and Nishida, 1992; Hill, 1995). Therefore, Araucaria's present distribution might be considered as the relic type, although its extant species show a distribution pattern very similar to that of Nothofagus and other Gondwanan groups.

Fossils of Araucaria are among the oldest fossils of extant coniferous genera that have been found since the Triassic or Jurassic eras (Miller, 1977, 1988; Stockey, 1982). Before 1995, Araucaria had been infragenerically classified into four extant Sections: Araucaria (= Columbea), Bunya, Eutacta, and Intermedia (Wilde and Eames, 1952). In 1995, Ohsawa, Nishida, and Nishida (1995) added the new Section Yezonia to include the extinct species. This sectional classification was based on morphology: leaves, attachment of pollen cones and ovulate cones, cone-scale, vascular system cone-scale complex, type of seedling germination, and seedling. Fossil records and extant species within these sections are compared in Table 1. The oldest records of Araucaria fossils are those of the Sections Bunya and Eutacta; they were widespread across both hemispheres during the Jurassic (we are cautious in regard to the treatment of Triassic fossils as Hill [1995] recommended). In the other two extant sections, Intermedia and Araucaria, the distribution of both extant and extinct species is limited to the Southern Hemisphere (Australia, New Zealand, Tasmania, and Argentina), and their fossil records are relatively recent, starting with the late Cretaceous. Therefore, it is expected that Sections Bunya or Eutacta will be revealed to be the oldest in the genus Araucaria and will locate at the base of the phylogenetic tree of the extant species.

The highest concentration of specific diversity of Araucariaceae is in New Caledonia. In spite of its small area (19 000 km), New Caledonia possesses a very rich and distinctive flora (Jaffré, 1995). This is especially true in the case of the Araucariaceae: 13 of the 19 species of Araucaria and five of the 13 species of Agathis are endemic to this island and are well diversified in their morphological characteristics (e.g., de Laubenfels, 1972; Veillon, 1978, 1980). Thus in the phylogenetic study of the Araucariaceae, it is indispensable to include all or many New Caledonian species.

This study aims to construct a sequence database for the chloroplast encoded rbcL gene in order to reconstruct a molecular phylogeny for the family and to use this phylogeny to address the following questions: (1) Are the three genera of Araucariaceae supported? (2) What is the relationship among Wollemia, Araucaria, and Agathis? (3) Is the infrageneric classification of Araucaria supported? (4) Which section is located in the basal cluster within Araucaria? (5) What phylogenetic relationships are observed among species of Araucaria and Agathis in New Caledonia?

	MATERIALS AND METHODS

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Material
Plant material was collected from natural habitats or cultivated plantings, dried and preserved in silica gel. In total, 29 species were selected representing all species of Araucaria (19 species) sensu de Laubenfels (1972, 1988), and ten species of Agathis. The rbcL sequence of Wollemia nobilis was referenced from GenBank. Materials and collection/source data are listed in Table 2.

Amplification and sequencing of the rbcL gene of cpDNA
The double-stranded DNA of most of the rbcL gene of cpDNA, ~1350 bp, was amplified by 30 cycles of symmetric polymerase chain reaction (PCR) as shown by use of primers aF and cR of Hasebe et al. (1994) for rbcL. PCR cycle conditions in the first cycle consisted of 2 min at 94°C for denaturation, 1 min at 45°C for primer annealing, and 1 min at 72°C for primer extension. Denaturation time at 94°C was reduced to 1 min during the next 28 cycles. The time at 72°C was increased to 5 min in the last cycle. PCR products were purified by electrophoresis in 1.0% agarose gel using 1 x TAE buffer. The gel was stained with ethidium bromide and the DNA was eluted using Geneclean II (Bio 101, Vista, California, USA). Purified DNAs were sequenced in both directions by the standard methods of the Taq dye deoxy terminator cycle sequencing kit (Perkin Elmer, Foster City, California, USA) on an Applied Biosystems Model 373A automated sequencer (Applied Biosystems, Foster City, California, USA). Primers for this sequence determination were aF, cF, cR, bR, and cR of Hasebe et al. (1994) and originally designed primers ARGbF, ARGsF, and ARGsR. The sequences of these original primers are as follows: ARGbF, 5'-TACCCCTTAGACCTTTTTGAAGAAGGTTC; ARGsF, 5'-ACTGTAGTAGGTA AACTTGAAGGTGAACG; ARGsR, 5'-GAACCTTCCTCAAAAAGGTCTAAGGGGTA. Sequence data were aligned manually with the GENETYX program (The Software Development Co., Tokyo, Japan).

Outgroup
Before the phylogenetic analysis of the Araucariaceae, we examined the phylogenetic position of Araucariaceae among conifers in order to select the outgroup to Araucariaceae. Following Chase et al. (1993), we used the rbcL sequences of 20 coniferous taxa and Ginkgo (as an outgroup), which were registered in GenBank, along with several species of Araucariaceae. As a result, a cluster of Cupressaceae, Podocarpaceae, and Taxodiaceae formed the sister group to Araucariaceae, and we used the rbcL sequences of Juniperus conferta (GBANL12573: Cupressaceae), Taxodium distichum (GBANS75127: Taxodiaceae), and Podocarpus gracilior (GBANX58135: Podocarpaceae) to root phylogenetic trees of Araucariaceae.

Phylogenetic analysis
A database of 1322 bp of the rbcL gene was used for the phylogenetic analyses. For the present study, rbcL gene sequence (positions 28–1350) were phylogenetically analyzed using PAUP (Phylogenetic Analysis Using Parsimony) version 3.1.1 (Swofford, 1993). We performed the heuristic search under the equal weighting criteria using Tree Bisection Reconnection (TBR) branch-swapping algorithm with MULPARS on, Steepest Descent on, and 100 replicates of random taxon addition. Accelerated transformation (ACCTRAN) was used for optimization in the analyses. We used the bootstrap analysis (Felsenstein, 1985; Felsenstein and Kishino, 1993) of 1000 replicates by the heuristic search under unweighted criteria to assess the internal support for clades. Character state reconstructions were performed using MacClade, version 3.05 (Maddison and Maddison, 1992). All character changes were treated as unrooted, equally weighted, and were resolved using two ways of optimization: accelerated transformation (ACCTRAN) and delayed transformation (DELTRAN).

	RESULTS

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Sequences for 29 species were obtained and deposited in GenBank (Table 2). Pairwise distances between taxa are listed in Table 3. Values of pairwise percentage sequence divergence ranged from 0 to 1.7% among Araucaria species, from 0 to 0.8% among Agathis species, from 2.1 to 3.2% between Araucaria and Agathis species, from 1.3 to 2.0% between Wollemia and Araucaria species, from 1.6 to 2.0% between Wollemia and Agathis species, and from 7.4 to 9.0% between Araucariaceae and outgroup taxa. In particular, the values were very low among New Caledonian Araucaria and Agathis species, between 0 and 0.5% and between 0.1 and 0.2%, respectively (Table 3).

View larger version (35K): [in this window] [in a new window]   Fig. 1. Consensus tree of the 20 equally parsimonious ones for Araucariaceae based on cpDNA rbcL sequences, showing the clades that are supported in the strict consensus by solid lines and the clades that collapse by dotted ones. Length = 281 steps, CI = 0.766 (excluding uninformative data), RI = 0.925. A percentage of the 1000 bootstrap values (>50%) is given for each node. Distribution area for each species is indicated within parentheses. Sectional attribute for each species is indicated by a dashed line. Figure abbreviations: AU = Australia, FJ = Fiji, MA = Malaysian region, NC = New Caledonia, NF = Norfolk Island, NG = New Guinea, NZ = New Zealand, SA = South America, VA = Vanuatu.

Phylogeny within Araucaria
The phylogeny of Araucaria is relatively well resolved in the present phylogenetic analysis. The phylogenetic trees divided into two clades at first: one clade consists of all 15 species of Section Eutacta and another consists of the remaining sections, Araucaria, Bunya, and Intermedia. In the former clade, Araucaria cunninghamii (distributed in Australia and New Guinea) was derived first, and after that, A. heterophylla (from Norfolk Island). The 13 endemics of New Caledonia formed a monophyletic group. The homology of rbcL sequences among New Caledonian species is very high, between 99.5 and 100% (Table 3). The sequence in ten of the 13 endemics (Araucaria biramulata, A. columnaris, A. humboldtensis, A. laubenfelsii, A. luxurians, A. montana, A. nemorosa, A. schmidii, A. scopulorum, and A. subulata) was identical.

The second major clade in Araucaria further divided into two clades, one cluster consists of two South American species of Section Araucaria (A. araucana and A. angustifolia), and of New Guinean species of Section Intermedia (A. hunsteinii) and Australian species of Section Bunya (A. bidwilli).

Phylogeny within Agathis
Unlike Araucaria, the rbcL sequences resolved the relationships in Agathis poorly. In the strict consensus tree (Fig. 1), only two monophyletic groups were present: one of Agathis borneensis and A. palmerstonii (distributed in South East Asia) and another of A. obtusa (Vanuatu), A. robusta (Australia), and A. vitiensis (the Fiji Islands). The 50% majority rule consensus suggested a closer relationship between all four endemics to New Caledonia, and also a relationship between A. dammara (distributed in Malaysian region), A. obtusa, A. robusta, and A. vitiensis, but there is no support for these clusters in the data (bootstrap value <50%).

	DISCUSSION

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In this study, the rbcL sequence data set indicates that the Araucariaceae are a highly coherent taxon and that the monophyly of the genera Araucaria and Agathis is well supported.

Phylogenetic position of Wollemia
In the phylogenetic tree, the genus Wollemia is derived before the divergence of Araucaria and Agathis. Based on the topology, Wollemia can be interpreted as the earliest diverged genus in Araucariaceae. Wollemia shares morphologies among Araucaria and Agathis (Jones, Hill, and Allen, 1995). Morphological characteristics shared by both Wollemia and Araucaria (i.e., closely crowned sessile and amphistomatic leaves, aristate bract scales) or Wollemia and Agathis (i.e., fully fused bract and ovuliferous scale, winged seed) are interpreted as plesiomorphic characters in the family. To examine this theory, further knowledge is needed, especially on the comparative morphology of Wollemia and related fossil taxa, for understanding the relationships among Wollemia, Araucaria and Agathis.

Infrageneric relationships of Araucaria
The rbcL phylogeny agrees well with the present infrageneric classification system, which recognizes four sections of extant species: Araucaria, Bunya, Eutacta, and Intermedia. The classification is based on the combination of morphological characteristics, such as leaf shape, stomatal distribution and orientation, polar extension, position of male cone, number of vascular supplies to cone scale and their arrangement, dehiscence of cone scale, number of cotyledons, and germination manner (Wilde and Eames, 1952; Stockey, 1982; Stockey and Ko, 1986). Therefore, most of these morphological characteristics well reflect the phylogenetic relationships of the genus. In particular, the major branching of the rbcL phylogeny in Araucaria, which splits Section Eutacta from the other sections, corresponded to the number of cotyledons (four in Section Eutacta and two in other sections), position of male cone (terminal in Section Eutacta and axillary in other sections), stomatal orientation to long axis of leaf (mostly oblique or perpendicular in Section Eutacta and mostly parallel in other sections), and stomatal distribution (mostly two bands or groups in Section Eutacta and discontinuous rows in other sections). These morphological characteristics should be evaluated as being phylogenetically informative and will be pertinent to estimating the phylogenetic position of extinct species of Araucaria.

We further polarized the morphological characteristics discussed above (Figs. 2–6). Figure 2 shows the evolutionary trend in the number of cotyledons. Cotyledons of Araucaria vary in number as discussed above, but all species of Agathis and Wollemia have two (de Laubenfels, 1972, 1988; Whitmore, 1980; Jones, Hill, and Allen, 1995). ACCTRAN and DELTRAN without outgroups showed the same distribution and evolutionary trends of these characters. Parsimony evaluated the plesiomorphy as two both in Araucaria and Araucariaceae. This fact agrees well with palaeobotanical data since all araucarian fossil species from the Mesozoic have dicotyledonous embryos (Stockey, 1975, 1978, 1980a, b; Stockey, Nishida, and Nishida, 1992). An increase from two to four cotyledons occurred once at the level of the common ancestor of section Eutacta.

View larger version (44K): [in this window] [in a new window]   Fig. 2. Parsimonious replacement of characters of the number of cotyledons with ACCTRAN and DELTRAN optimization on the most parsimonious phylogenetic tree based on the rbcL gene sequences (Fig. 1 ). There is no difference in the character distribution between ACCTRAN and DELTRAN optimization.

View larger version (49K): [in this window] [in a new window]   Fig. 3. Parsimonious replacement of the position of the male cone with ACCTRAN and DELTRAN optimization on the most parsimonious phylogenetic tree based on the rbcL gene sequences (Fig. 1 ). There is no difference in the character distribution between ACCTRAN and DELTRAN optimization.

View larger version (40K): [in this window] [in a new window]   Fig. 4. Parsimonious replacement of stomatal orientation with ACCTRAN and DELTRAN optimization on the most parsimonious phylogenetic tree based on the rbcL gene sequences (Fig. 1 ). There is no difference in the character distribution between ACCTRAN and DELTRAN optimization.

View larger version (47K): [in this window] [in a new window]   Fig. 5. Parsimonious replacement of stomatal distribution with ACCTRAN and DELTRAN optimization on the most parsimonious phylogenetic tree based on the rbcL gene sequences (Fig. 1 ). There is no difference in the character distribution between ACCTRAN and DELTRAN optimization.

Figure 4 shows the evolutionary trend in stomatal orientation. Stomata of Agathis are obliquely and/or perpendicularly oriented to the long axis of the leaf (Stockey and Atkinson, 1993) and those of Wollemia have not been observed. ACCTRAN and DELTRAN evaluated the plesiomorphy as being oblique and/or perpendicular in Araucaria. Parallel orientation arose in the ancestor of sections Araucaria, Bunya, and Intermedia. It has been difficult to assess the placement of the leaves in any extant section because of bad preservation of leaf cuticles in Mesozoic fossil leaves (Stockey, 1994). Therefore, further details of micromorphology of fossil Araucaria and cuticle micromorphology in Wollemia are needed to determine the evolutionary trends in this character in the family.

Figure 5 shows the evolutionary trend of stomatal distribution. Stomata of Agathis occur in discontinuous rows (Stockey and Atkinson, 1993), and those of Wollemia are unknown. ACCTRAN and DELTRAN evaluated the plesiomorphy of the stomatal distribution as occurring in discontinuous rows in Araucaria. A shift from discontinuous rows to two bands appears to have occurred once in the ancestor of section Eutacta, with reversal among New Caledonian Araucaria.

Phylogenetic position of the Section Bunya in the genus Araucaria
Before we obtained the rbcL phylogeny of Araucariaceae, we expected that Araucaria bidwillii of Section Bunya would be placed at the bottom of the phylogenetic tree of Araucaria since Section Bunya is one of the oldest recorded sections from the Jurassic (Wieland, 1935; Calder, 1953; Stockey, 1975, 1978). However, the molecular data do not support the early divergence of Section Bunya, because A. bidwillii was found to be located in the terminal cluster together with A. hunsteinii.

Several palaeobotanical works have already pointed out the differences between Araucaria bidwillii and the fossil species assigned to Section Bunya in seed size and seedling anatomy. The seed size of A. bidwillii is large, 5–6 cm in length and 2.5–3.5 cm in width (Burrows, Boag, and Stockey, 1992) compared with 0.8–1.3 cm in length and 0.2–0.6 cm in width in A. mirabilis from Argentina (Stockey, 1975, 1978), 0.8 cm in length and 0.3 cm in width in A. brownii from the UK (Stockey, 1980a), 1.6 cm in length and 0.7 cm in width in A. sphaerocarpa from the UK (Stockey, 1980b). Seed size is also small in all other fossil species, such as A. nihongii (Stockey, Nishida, and Nishida, 1992) or A. nipponensis (Stockey, Nishida, and Nishida, 1994). Burrows, Boag, and Stockey (1992) indicated that seeds of extinct species were too small for cryptogeal (hypogeal) germination, and Stockey (1994) even suggested that hypogeal germination was a relatively recent phenomenon in the Araucariaceae. Thus it is suggested that epigeal germination is the general condition (plesiomorphy) within Section Bunya.

Stockey, Nishida, and Nishida (1990), Burrows, Boag, and Stockey (1992), and Burrows and Stockey (1994) showed that seedlings of Araucaria bidwillii develop concentric rings of vascular tissue between shoot apex and hypocotyl, but such a structure is not present in the fossil seedlings.

The present finding on the phylogenetic position of Araucaria bidwillii is consistent with these paleobotanical data. We conclude that the Mesozoic araucarians assigned to Section Bunya, which were distributed in both hemispheres, should be separately treated from the extant Bunya species. Hypogeal germination can be evaluated as an autapomorphy for A. bidwillii in the section. We should add further molecular data to enhance the statistical probability concerning the present position of A. bidwillii in the phylogenetic tree.

Evolution of Araucaria
The rbcL tree indicates that the genus Araucaria is divided into two clades. In the cluster of Sections Araucaria, Bunya, and Intermedia, extant species attributed to these sections are distributed in South America, Australia, or New Guinea. This distribution pattern linking South America and Australasia is often considered to be the result of the Mesozoic break up of Gondwanaland followed by continental drift (see Raven and Axelrod, 1972, 1974). Fossils of Sections Araucaria and Intermedia have also been found only from the Southern Hemisphere (Argentina + Australia and New Zealand, respectively; see Table 1), and fossils assigned to Section Bunya should be separately treated from the extant Bunya species as discussed above. Therefore, common ancestral taxa of Sections Araucaria and Intermedia, and possibly Section Bunya, were distributed in Gondwanaland during the Mesozoic and the early Tertiary, and their evolution into Sections Araucaria and Intermedia (and possibly Section Bunya) were completed before South America separated from Antarctica, during the Eocene at the latest. The oldest fossil records in these sections are late Cretaceous (Bose, 1975) and agree well with the fact that there was a continuous landmass from South America to Antarctica, Australia, New Zealand, and New Guinea 70–80 million years ago, in the late Cretaceous (Wilford and Brown, 1994). Therefore, the present finding indicating the monophyly of Sections Araucaria, Intermedia, and Bunya agrees with these fossil and geological facts.

The monophyly of Section Eutacta was strongly supported. Section Eutacta has been regarded as the oldest taxon together with Section Bunya based on their Jurassic to Cretaceous fossil records. In the present study, rbcL phylogeny suggests that Section Eutacta is older than the other sections. However, recent paleobotanical findings have indicated that some Mesozoic Northern Hemisphere species may have been inappropriately assigned to Section Eutacta.

Fossils of Araucaria are usually fragmentary, i.e., found as a part of the whole plant and have been discussed in their sectional attributes on the basis of partial fossils such as cones or leaves. Ohsawa, Nishida, and Nishida (1995) found a new specimen of Araucaria whose cone was attached to the vegetative organs that had been described as Yezonia vulgaris. They reconstructed the whole plant and proposed a new section, Yezonia, based on the combination of features quite different from other extant sections. The cone anatomy of this fossil coincided with that of Araucaria nihongii (Stockey, Nishida, and Nishida, 1992), which has cones externally similar to those of Section Eutacta. External and anatomical features of shoots of Yezonia closely resemble Brachyphyllum, which is sometimes associated with araucarian cone fossils assigned to Section Eutacta. Thus, the taxonomic positions of Mesozoic araucarian fossils that have been included in Section Eutacta are now doubtful after the finding of Yezonia. Reexamination of Mesozoic fossils assigned to Section Eutacta is needed.

Evolution of Agathis
Fossils of Agathis have been excavated from Tertiary sediments restricted to Australia and New Zealand (Cookson and Duigan, 1951; Florin, 1963; Hill and Bigwood, 1987). The oldest known fossil is a collection of leaves from the middle Cretaceous (Albian) in Australia (Cantrill, 1992), but no cones are known from the Mesozoic. Stockey (1994) stated that Agathis is known from the Cretaceous, and Hill (1995) gave its time of appearance as the beginning of the Tertiary. Despite the molecular evidence for the sister-group relationship between Agathis and Araucaria, our current knowledge of the fossil record only traces Agathis back to the Cretaceous/Tertiary boundary and suggests that the genus may always have been more restricted to the southern hemisphere.

Infrageneric relationships of Agathis were poorly resolved by the data, and the low sequence divergence suggests the recent diversification of extant agathian species. The bootstrap values are very low in each node of the tree, and we will not discuss the evolution of Agathis based on the present trees. We are now examining matK sequence data for the Araucariaceae in order to derive a better understanding of the evolution of Agathis as well as to solve some other problems uncovered in the present study.

New Caledonian species
New Caledonia possesses 13 endemics of Araucaria and five of Agathis, and their morphological characteristics and preferred habitats are well diversified (see de Laubenfels, 1972; CTFT, 1975; Veillon, 1978, 1980; Jaffré, 1995). Nevertheless, the very high or identical homology of their rbcL gene sequences was observed among the species in each genus (Table 3). High morphological diversity with the low sequence divergence, the low restriction site divergence, or low genetic differentiation is usually observed in endemics to the oceanic islands, by means of rapid adaptive radiation and speciation to new niches (e.g., Carr and Kyhos, 1986; Crawford, Stuessy, and Silva, 1987; Ito and Ono, 1990; Ito et al., 1990; Crawford et al., 1992, 1993; Soejima et al., 1994).

New Caledonia, one of the continental islands, originated from a part of Gondwanaland and was separated from the continent between the Triassic and the end of the Jurassic (Raven and Axelord, 1974; Paris, 1981). However, a large area of New Caledonia is covered with ultramafic soil derived from peridotite, and it is suggested that this area was formed at the end of the Eocene. Most of the species of Araucariaceae are distributed in this soil (12 of the 13 species in Araucaria and four of the five species in Agathis). Jaffré (1995) suggested that there was differentiation of new species of Araucaria in the post-Eocene, after the emplacement of peridotite. His discussion agrees with the findings of very low sequence divergence in the rbcL gene among endemics of Araucaria and Agathis. It is suggested that the adaptive radiation to new edaphic conditions, i.e., ultramafic soils, caused their rapid differentiation after the Eocene.

As a result, although New Caledonia is an old continental island that had been a part of Gondwanaland, the evolutionary trend of the Araucariaceae is rather like that of oceanic islands. Setoguchi et al. (1997) found a similar pattern in New Caledonian Nothofagus using an atpB-rbcL intergenic spacer sequence. Further case studies will be needed to examine the origin and speciation of Gondwanan floristic elements in New Caledonia.

	FOOTNOTES

 
¹ The authors thank Drs. G. J. Jordan and R. S. Hill(University of Tasmania), C. J. Quinn (The University of New South Wales), and G. Humphrey (The University of Sydney) for reviewing the manuscript; Mr. M. Bullet (Service de l'environnement à la Direction de l'Economie Rurale de la Province Sud, New Caledonia), Drs. M. Ono and M. Nakazawa (Tokyo Metropolitan University), S. Vodonaivalu (The University of the South Pacific, Fiji), A. Abrahim (Singapore Botanic Gardens), M. Higuchi and T. Kitayama (National Science Museum), M. Tsuda (Kyoto University), M. Ito (Chiba University), H. Nishida (Chuou University, Japan), K. Ueda (Kanazawa University, Japan) for their cooperation in collecting plant materials and/or fruitful discussion; and N. Tanaka for helping with the analyses. This study was partly supported by Grant in Aid from Ministry of Education, Science and Culture Japan to H. S. (number 08740670) and T. A.-O. (number 0730457), Sasagawa Scientific Grant from Japan Science Society to T. A.-O., Grant in Aid for overseas research from Ministry of Education, Science and Culture Japan number 05041093 (Chief: M. Tsuda, Kyoto University) and number 08041135 (Chief: M. Ito, Chiba University).

⁶ Current address: School of Natural Science, Faculty of Integrated Human Studies, Kyoto University, Yoshida, Nihonmatsu-machi, Kyoto 606–01, Japan (e-mail:seto{at}gaia.h.kyoto-u.ac.jp ).

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