4. Molecular a-taxonomy of Asplenium nidus
55
differences in 1,194 bp (base pairs) sequences of the gene, though morphologically
it is not very different from the other species of the sect. Thamnopteris. It was also
discovered that the so called ''A. australasieum" in Japan has a very different rbeL
sequence from A. australasieum sensu Holttum distributed in Australia and South
Pacific Islands. Based on these molecular data, we described the Japanese "A.
australasieum" as a new species, Asplenium setoi N. Murak. et Seriz. However, it
is sometimes difficult to recognize species as realistic biological units by examining allopatric populations. Comparisons are required among sympatric populations
of the plants with various rbeL types which mayor may not be able to be reproductively isolated depending on their genetic differences.
In the present work, we examined rbeL variations of sympatric populations in
Mt. Halimun National Park where a large amount of biodiversity can be expected.
We used as materials, the plants that were all identified as one species, A. nidus
sensu Holttum (1974). Mt. Halimun National Park is located in west Java, Indonesia, and has primarily well preserved tropical lowland and mountain forests (10001800 m altitude). Relatively large morphological variations of A. nidus especially
in leaf shape and size, soral morphology and position, and the existence or absence
of a keel on the undersurface of the rachis and petiole, were observed. We found a
very large amount of rbeL sequence variation in the sympatric populations. Furthermore, we compared the rbeL sequences of the plants from Mt. Halimun with
those from other paleotropical localities, including its neighboring areas on Java
Island such as Mt. Gede and Bogor, in order to clarify their phylogenetic position.
We also compared differences in morphological characters and ecological habitats
between different rbeL types in order to recognize natural biological units in sect.
Thamnopteris distributed in Mt. Halimun.
2 Materials and Methods
2.1 Plant Materials
Fresh green leaves of 20 plants of A. nidus representing a wide spectrum of morphological variation were collected in a natural primary mountain forest (10001800 malt.) near Chikaniki station of Mt. Halimun National Park. For comparison, we also collected plant materials from Mt. Gede (3 individuals) and Bogor
Botanical Garden (naturally growing plants, 1 individual), which are 50-60 km
apart from Mt. Halimun. Usually two leaves were collected from each individual,
and one was used for DNA extraction and the other as voucher specimen. When
only one leaf was available, a small portion was used for molecular analysis, and
the remainder was deposited as a voucher specimen. Voucher specimens were kept
in the herbaria of the Graduate School of Science, Kyoto University (KYO); the
Graduate School of Science, The University of Tokyo (TI); and Aichi Kyoiku University (AICH). Morphological comparison was made using these voucher specimens. Voucher information is shown in Table 1.
55
differences in 1,194 bp (base pairs) sequences of the gene, though morphologically
it is not very different from the other species of the sect. Thamnopteris. It was also
discovered that the so called ''A. australasieum" in Japan has a very different rbeL
sequence from A. australasieum sensu Holttum distributed in Australia and South
Pacific Islands. Based on these molecular data, we described the Japanese "A.
australasieum" as a new species, Asplenium setoi N. Murak. et Seriz. However, it
is sometimes difficult to recognize species as realistic biological units by examining allopatric populations. Comparisons are required among sympatric populations
of the plants with various rbeL types which mayor may not be able to be reproductively isolated depending on their genetic differences.
In the present work, we examined rbeL variations of sympatric populations in
Mt. Halimun National Park where a large amount of biodiversity can be expected.
We used as materials, the plants that were all identified as one species, A. nidus
sensu Holttum (1974). Mt. Halimun National Park is located in west Java, Indonesia, and has primarily well preserved tropical lowland and mountain forests (10001800 m altitude). Relatively large morphological variations of A. nidus especially
in leaf shape and size, soral morphology and position, and the existence or absence
of a keel on the undersurface of the rachis and petiole, were observed. We found a
very large amount of rbeL sequence variation in the sympatric populations. Furthermore, we compared the rbeL sequences of the plants from Mt. Halimun with
those from other paleotropical localities, including its neighboring areas on Java
Island such as Mt. Gede and Bogor, in order to clarify their phylogenetic position.
We also compared differences in morphological characters and ecological habitats
between different rbeL types in order to recognize natural biological units in sect.
Thamnopteris distributed in Mt. Halimun.
2 Materials and Methods
2.1 Plant Materials
Fresh green leaves of 20 plants of A. nidus representing a wide spectrum of morphological variation were collected in a natural primary mountain forest (10001800 malt.) near Chikaniki station of Mt. Halimun National Park. For comparison, we also collected plant materials from Mt. Gede (3 individuals) and Bogor
Botanical Garden (naturally growing plants, 1 individual), which are 50-60 km
apart from Mt. Halimun. Usually two leaves were collected from each individual,
and one was used for DNA extraction and the other as voucher specimen. When
only one leaf was available, a small portion was used for molecular analysis, and
the remainder was deposited as a voucher specimen. Voucher specimens were kept
in the herbaria of the Graduate School of Science, Kyoto University (KYO); the
Graduate School of Science, The University of Tokyo (TI); and Aichi Kyoiku University (AICH). Morphological comparison was made using these voucher specimens. Voucher information is shown in Table 1.