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(American Journal of Botany. 2001;88:1517-1522.)
© 2001 Botanical Society of America, Inc.

Systematics, Phytogeography, and Evolution

Molecular systematics of the Asplenium nidus complex from Mt. Halimun National Park, Indonesia: evidence for reproductive isolation among three sympatric rbcL sequence types1

Yoko Yatabe2, Shigeo Masuyama3, Dedy Darnaedi4 and Noriaki Murakami2,5

2Department of Botany, Graduate School of Science, Kyoto University, Kitashirakawa-Oiwake-cho, Kyoto 606-8502, Japan 3Faculty of Contemporary Culture, Tokyo Woman's Christian University, Zempukuji, Suginami-ku, Tokyo 112-8551, Japan 4Botanical Gardens, Bogor, Jl. Ir. H. Juanda 13, Bogor 16122, Indonesia

Received for publication July 25, 2000. Accepted for publication February 15, 2001.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 
Asplenium nidus is an epiphytic fern with large simple leaves. Because A. nidus lacks the good taxonomic characters available for species recognition, multiple cryptic species may exist within A. nidus. In Mt. Halimun National Park, West Java, Indonesia, three rbcL sequence types of A. nidus were recorded. All plants regardless of rbcL sequence were 2n = 144. Crossing experiments among these rbcL types were conducted, and it was observed that the molecularly distinct types were reproductively isolated because hybrids failed to form between at least two pairs of rbcL types. These results suggest that these rbcL types are cryptic species because they are reproductively isolated but they are difficult to distinguish morphologically. Thus, the characters of DNA sequences information are useful in the discovery of cryptic species in ferns.

Key Words: Asplenium nidus complex • molecular systematics • rbcL • reproductive isolation


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 
Ferns appear to have fewer taxonomically informative morphological characters than seed plants because they lack flowers, which provide valuable characters for analyzing evolutionary relationships. In systematic research on the ferns, cytotaxonomical and enzyme electrophoretic data have revealed cryptic species in many lineages such as Adiantum, Botrichium, Pityrogramma, and so on (see Paris, Wagner, and Wagner, 1989 ). Recently, it has become easier to collect DNA nucleotide sequence data from wild plants using polymerase chain reaction (PCR) and direct sequencing techniques. The characters of DNA sequences information might be also useful in discovering cryptic species in ferns. Once the evolutionary relationships are inferred from DNA sequence data, we can test the hypothesis that the members of a single species contain multiple cryptic species.

Asplenium sect. Thamnopteris, a group of epiphytic ferns with simple leaves, lacks good qualitative taxonomic characters for species recognition. Holttum (1974) monographed 15 species of sect. Thamnopteris using gross morphological characters. However, species delimitation is unclear, and the naturalness of these as species of this taxon is questionable because most of the characters that he adopted as keys to the species are quantitative characters such as frond width and frond apex shape. In cases such as this in which morphology fails to yield clear hypotheses, the characters of DNA sequences can provide alternative useful information for species recognition.

Asplenium nidus is one of the species assigned to Asplenium sect. Thamnopteris and it can be found throughout the Old World tropics. A large degree of rbcL variation has been found in plants identified as A. nidus sensu Holttum (1974) from various localities (Murakami et al., 1999b, c ). Because the evolutionary rates of rbcL were reported to be relatively slow (Chase et al., 1993 ), we made various comparisons among the plants of A. nidus with various rbcL types in order to clarify whether this rbcL variation was intraspecific or interspecific (Murakami et al., 1999b, c ). In the sympatric populations of Mt. Halimun National Park, located in West Java, Indonesia, Murakami et al. (1999b) found three rbcL types of A. nidus with different habitat preferences. We called those type A, type B, and type C, respectively. Type A and type B were distributed sympatrically even on a reduced scale at 1000–1250 m altitude, but their habitats were differentiated by their relative position on tree trunks. Type A grow on the deeply shaded lower section of tree trunks, whereas type B grow on higher sections, where they are half shaded. Type C are distributed almost parapatrically at 1170–1700 m altitude and never grow with type B and seldom with type A. Type C grow on either higher and lower sections of the trunks. Although these three types have been treated as conspecific because they are undistinguishable by their gross morphology, the former study provided indirect evidence that they are not intraspecific variations but biologically separate entities. However, it was not clear whether these three types were reproductively isolated.

To clarify this hypothesis, we conducted artificial crossing experiments among these rbcL types and examined their crossability. We also examined the somatic chromosome number of these three types because differences in ploidy would reduce the fecundity of hybrid plants and function as barriers to gene flow.

Furthermore, we determined the rbcL sequences of A. nidus plants from several other localities, added these sequences to the data set of Murakami et al. (1999b) , and conducted phylogenetic analyses.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 LITERATURE CITED
 
rbcL sequencing
For phylogenetic analyses, fresh green leaves of Asplenium sect. thamnopteris were collected from the sites listed in Table 1. The vouchers were kept in the herbaria of the Graduate School of Science, Kyoto University (KYO).


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  		Table 1. Taxa and vouchers for the plant materials sequenced

 
Total DNA was extracted using a 2 x CTAB (hexadecyl trimethyl ammonium bromide) solution according to Doyle and Doyle (1987) .

For phylogenetic analyses, PCR amplification of rbcL fragments followed Murakami et al. (1999c) . The PCR products were purified using a GENE CLEAN III kit (BIO101, Vista, California, USA) after electrophoresis in 1.0% agarose gel and then used as templates for direct sequencing. Sequencing reactions were prepared using a Big Dye terminator cycle sequencing kit (Perkin Elmer Applied Biosystems, Foster, California, USA). The reaction mixtures were analyzed on an Applied Biosystems Model 377 automated sequencer (Perkin Elmer Applied Biosystems). Sequences were aligned using Sequence Navigator Software (Perkin Elmer Applied Biosystems).

For the crossing experiments and cytological observations, ~500 bp (base pair) rbcL sequences were examined to identify the three types.

Phylogenetic analyses
The rbcL sequence data matrix contained data from 32 accessions. Their database accession numbers are listed in Table 1. Phylogenetic analysis was performed by the Maximum Parsimony method using PAUP (Phylogenetic Analysis Using Parsimony) version 3.1.1 (Swofford, 1993 ). A branch and bound search was conducted to find the most parsimonious trees. Asplenium griffithianum was used as an outgroup because its relevance as an outgroup was shown by Murakami et al. (1999a) . A bootstrap analysis with 10 000 replications was performed in order to estimate the reliability for various clades.

Cytological observation
We observed somatic chromosomes from root tips of the three rbcL types by pretreating with 0.002 mol/L 8-hydroxyquinoline solution for 4–6 h at ~20°C. After fixation in 45% acetic acid at 4°C for 20 min, the root tips were hydrolyzed in 60°C 1 mol/L HCl for 60 sec. They were then stained with 2% aceto-orcein for 1 h squashed and observed using a light microscope (BH2, OLYMPUS, Tokyo, Japan).

Allozyme analyses
Fresh leaves were ground in 1.0 mL of cold extraction buffer containing 0.1 mmol/L Tris-HCl, 1 mmol/L EDTA (4NA), 10 mmol/L KCl, 10 mmol/L MgCl2, 0.4% 2-mercaptoethanol, and 10% polyvinyl-pyrrolidone with the pH adjusted to 7.5. Enzymes were resolved on 6% polyacrylamide gels following the procedures of Shiraishi (1988) . We examined leucine aminopeptidase (LAP; EC 3.4.11.1), also following Shiraishi (1988) .

Crossing
Fresh green leaves with mature sori from eight individuals of Asplenium nidus were collected from Mt. Halimun National Park on December 1998. Four or five leaves were collected from each individual, and one was used for DNA extraction and allozyme analyses. Another was kept in KYO as a voucher specimen, and the others were used for collecting spores.

We examined the rbcL and LAP allozymes of the sporophytes. These sporophytes were shown to be appropriate parents for crossing experiments with specific genetic markers (Fig. 1). Spores of each sporophyte were sown on inorganic nutrient media in petri dishes 9 cm in diameter and cultivated in a chamber following the methods of Watano and Masuyama (1991) . Forty days after spore sowing, we obtained gametophytes with archgonia, which functioned as females, though gametophytes were not always found with antheridia. If necessary, we resowed the spores around the mature gametophytes. The spores were induced to germinate and to produce antheridia, probably owing to antheridogen, which was secreted by mature gametophytes. We then obtained gametophytes with antheridia, which functioned as males.



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  		Fig. 1. Electrophoretic band patterns of the allozyme (LAP) of each sporophyte used for the crossing experiments. A, B, and C and the numbers after these letters indicate the rbcL sequence types and the sample number of the sporophytes, respectively

 
In order to examine the possibility of cross-fertilization between type A and type B, 20 gametophytes with archegonia of type A were transferred into the dish where many gametophytes with antheridia of type B were growing and were flooded with water (Fig. 2, II). The same number of gametophytes of type A were also transferred into the dish of type A as a control (Fig. 2, I). These procedures were carried out reciprocally (Fig. 2, III, IV) and left overnight. We placed these 80 gametophytes on new media and dried for 1 h in a germ-free chamber (Clean Bench, Sanyo, Osaka, Japan) in order to prevent fertilization after the treatments. After 1 mo, we started to count the number of juvenile sporophytes growing on the gametophytes. After 3 mo, we examined LAP allozymes of the sporophytes. The allozyme polymorphisms were used to identify hybrid plants based on their heterozygous patterns combining those of their parents.



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  		Fig. 2. The method of crossing experiments between types A and B. A detailed explanation is given in MATERIALS AND METHODS

 
In order to examine the possibility of cross-fertilization between types A and C, we conducted the crossings in the same way as between types A and B.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 LITERATURE CITED
 
Molecular phylogenetic analyses
We determined 1194 bp nucleotide sequences of rbcL. Based on the characters of the rbcL sequence, the two equally most parsimonious trees (length = 156 steps; consistency index = 0.782; retention index = 0.860) were obtained. Their strict consensus tree with the bootstrap percentages is shown in Fig. 3. The general topology of the tree is same as that of Murakami et al. (1999b) . Type A is closely related to A. nidus from the southern peninsular part of Thailand as well as Yunnan, China and central Vietnam. The plant samples from the Malay Peninsula and the southern peninsular part of Thailand made a clade with type B and A. australasicum sensu Holttum (1974) from Australia and New Caledonia.



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  		Fig. 3. The strict consensus tree of the two most parsimonious trees (length = 156 steps; consistency index = 0.782; retention index = 0.860) based on the rbcL sequence data of Asplenium sect. Thamnopteris (see Table 1 ). The numbers above and below the branches are bootstrap percentages and the number of nucleotide substitutions (ACCTRAN optimization), respectively. Halimun A, Halimun B, and Halimun C indicate type A, type B, and type C in the text, respectively. An asterisk indicates nucleotide sequences of rbcL were analyzed for the first time in this study. 1 indicates species identified as A. setoi; 2 indicates species identified as A. australasicum; 3 indicates species identified as A. antiquum

 
Cytological observation
All plants surveyed were tetraploid 2n = 144 (Fig. 4).



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  		Fig. 4. Somatic metaphase chromosomes (2n = 144) of the three rbcL types of Asplenium nidus in Mt. Halimun National Park. (a) type A; (b) type B; (c) type C. Bar = 10 µm

 
Crossings between types A and B
We repeated the crossing experiments eight times, from which five data sets were obtained. No sporophytes were obtained even in one or both of the controls in three tests. The frequencies of occurrence of the juvenile sporophytes and hybrids between types are given in Fig. 5. The frequencies of juvenile sporophytes growing on gametophytes in the controls ranged from 50 to 95%. Though these frequencies in the controls were always significantly higher than those in the treatments when Fisher's exact probability tests were performed, we obtained juvenile sporophytes even in the treatments. None of the sporophytes obtained in crossings between types A and B were hybrid plants based on their LAP allozymes.



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  		Fig. 5. The frequencies of occurrence of juvenile sporophytes in crossing experiments between types A and B. The labels indicate the pairs of the parental sporophytes used for each crossing experiment. The numbers above bars are the number of hybrid plants. ** indicates that the number of the sporophytes obtained in I (or IV) were significantly higher than that in II (or III) (P < 0.01)

 
Crossings between types A and C
After eight trials, only three data sets with successful controls were obtained. The frequencies of juvenile sporophytes growing on gametophytes in the controls were always higher than those in the treatments, and these frequencies were significantly higher in one data set (Fig. 6). None of the obtained sporophytes were hybrid plants based on their LAP allozymes.



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  		Fig. 6. The frequencies of occurrence of juvenile sporophytes in crossing experiments between types A and C. The labels indicate the pairs of the parental sporophytes used for each crossing experiment. The numbers above bars are the number of hybrid plants. * and ** indicate that the number of the sporophytes obtained in the control were significantly higher than that in the treatment (*P < 0.05, **P < 0.01)

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
LITERATURE CITED
 
Murakami et al. (1999b) found three rbcL sequence types among plants from Mt. Halimun National Park, which were all identified as A. nidus according to Holttum's (1974) classification. All of these types were tetraploid at 2n = 144, a number that is consistent with other chromosome counts from various localities of the world (Bir, 1960 ; Abraham, Ninan, and Mathew, 1962 ; Kawakami, 1970 ; Koul, 1970; Tsai and Shieh, 1983 ). No hybrids between the rbcL sequence types were obtained in our crossing experiments. If an artificial or natural hybrid is sterile, it will provide evidence consistent with the hypothesis (Masuyama and Watano, 1994 ). However, not all species necessarily form interspecific hybrids even in ferns, as Schneller (1981) reported prezygotic isolation between two Athyrium species and Dryopteris filix-mas. Although various methods have been proposed for obtaining artificial hybrids in homosporous ferns (Lovis, 1968 ), none of these studies considered the frequency of hybrid formation. Focusing on the frequency of hybrid formation, we conducted quantitative crossing experiments with controls. The crossings were conducted between types A and B and between types A and C.

In the controls of crossings between types A and B, the frequencies of occurrence of juvenile sporophytes ranged from 50 to 95% (Fig. 5, I, IV). These successful results of the controls suggest that an abundance of gametophytes with antheridia, which functioned as males, existed in dishes flooded with water, and the gametophytes transferred into the dishes, which functioned as females, bore mature archegonia. Nevertheless, judging from LAP allozyme patterns, no hybrids between types A and B were obtained in the treatments (Fig. 5, II, III). It is hypothesized that the formation of sporophytes in the treatment resulted from the transferred gametophytes mating among themselves. The gametophytes functioning as females sometimes bear not only archegonia but also antheridia because the gametophytes of homosporous ferns are hermaphroditic and defectively dichogamous. However, the frequencies of occurrence of juvenile sporophytes in the controls were always significantly higher than those in the treatments. Therefore, it is assumed that the gametophytes that functioned as males supplied numerous spermatozoa. The absence of hybrids between types A and B in the treatments suggest that types A and B may be cryptic species.

In the crossings between types A and C, no hybrids were obtained, either. The frequencies of occurrence of juvenile sporophytes in the control were always higher than those in the treatments and significantly higher in one of three data sets. Therefore, types A and C may also represent cryptic species.

The two pairs of rbcL types that we used for the crossing experiments coexist, sometimes side by side, in Mt. Halimun National Park. Types A and B grew together even on the same tree trunk. Types A and C grew together only rarely, but they did in several sites at ~1200 m altitude. Nevertheless, no natural hybrids with irregularly shaped spores have been found in Mt. Halimun National Park. In the case of types A and B, since they are ecologically differentiated, the premating isolation mechanisms should function as barriers to gene flow to some extent. The habitat preference of the gametophytes may also be differentiated between type A and B. However, we could not find any natural hybrids even on the middle section of the tree trunks where these two types are adjacent to one another. It is evident that the postmating isolation observed in our crossing experiments functions as a barrier to gene flow between the two rbcL types to keep their biological identities separate. This postmating isolation may be prezygotic isolation or zygotic sterility.

Thus, our results suggest that these three rbcL types are cryptic species, though the crossability between types B and C has not yet been examined.

In Asplenium sect. Thamnopteris, the morphology does not provide good qualititative taxonomic characters for species recognition. However, DNA sequence data provide characters for separating apparently cryptic species. When we combine the DNA sequence data and crossing experiments, we are able to recognize these species more effectively. Coyne and Orr (1989) reported that the genetic distance of allozymes are well correlated with the degree of speciation in Drosophila. The DNA sequence data and the degree of speciation are also expected to correlate in Asplenium sect. Thamnopteris. In order to delimit the range of the species and revise the taxonomy of Asplenium sect. Thamnopteris, we are planning to examine the crossability of many other pairs of rbcL types in Asplenium sect. Thamnopteris.

Moreover, we examined intramorphospecific variations of rbcL sequences also in other distantly related fern groups such as Hymenasplenium obliquissimum (Aspleniaceae) (Murakami et al., 1998 ), H. cheilosorum (Murakami, Yokoyama, and Iwatsuki, 1998 ), Stegnogramma pozoi (Thelypteridaceae) (Yatabe, Takamiya, and Murakami, 1998 ), Osmunda cinnamomea, Os. claytonia, Os. regalis (Osmundaceae) (Yatabe, Takamiya, and Murakami, 1999 ), and Cheiropleuria bicuspis (Cheiropleuriaceae) (Kato et al., 2001). In each species, we found a large amount of rbcL sequence variation. We estimated the substitution rates of rbcL of Osmundaceae, and our estimation suggested that one nucleotide substitution between two rbcL sequences occurs only once in 5 million years on average (Yatabe, Nishida, and Murakami, 1999 ). Therefore, the large amount of rbcL sequence variation in these species may suggest that they consist of several different reproductively isolated criptic species, just as Asplenium nidus does. We are also planning to apply the same strategy used for A. nidus to these species in order to determine species status and to test the hypothesis that rbcL sequence data are generally useful for characterizing reproductively isolated groups within morphologically consistent assemblages.


   FOOTNOTES
 
1 The authors thank Mr. Bayu Adjie, Ms. Julisasi Tri Hadiah, Ms. Esti Endah Ariyanti, Bogor Botanical Gardens, and the staff members of Indonesian Institute of Science and Mt. Halimun National Park for their kind assistance in our collection of plant materials; Prof. K. Iwatsuki, The University of The Air, for his valuable advice and generous support; and Mr. K. Hirai (University of Tokyo) for their cultivation of our plant materials in their greenhouse. This study was supported in part by Grants-in-Aid Number 11440246 from the Japan Society for the Promotion of Science. Back

5 Author for reprint requests. Back


   LITERATURE CITED
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 
Abraham A. C. A. Ninan P. M. Mathew 1962 Studies on the cytology and phylogeny of the ptelydophytes. VII. Observations on one hundred species of south Indian ferns. Journal of the Indian Botanical Society 41: 339-421

Bir S. S. 1960 Cytological observations on the East Himalayan members of Asplenium Linn. Current Science 29: 445-447

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Doyle J. J. J. L. Doyle 1987 A rapid DNA isolation procedure for small quantities of fresh leaf tissue. Phytochemistry 19: 11-15

Holttum R. E. 1974 Asplenium Linn., sect. Thamnopteris Presl. Gardens' Bulletin Singapore 27: 143-154

Kato M. Y. Yatabe N. Sahashi N. Murakami 2001 Taxonomic studies of Cheiropleuria (Dipteridaceae). Blumea, in press

Kawakami S. 1970 Karyological studies on Aspleniaceae. II. Chromosomes of seven species in Aspleniaceae. Botanical Magazine Tokyo 83: 74-81

Koul A. K. 1970 Supernumerary cell divisions following meiosis in the spider plant. Genetica 41: 305-310[CrossRef][ISI]

Lovis D. 1968 Fern hybridists and fern hybridising. II. Fern hybridising at the university of Leeds. British Fern Gazette 10: 13-20

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Murakami N. S. Nogami M. Watanabe K. Iwatsuki 1999a Phylogeny of Aspleniaceae inferred from rbcL nucleotide sequences. American Fern Journal 89: 232-243[CrossRef]

———, Y. Yatabe H. Iwasaki D. Darnaede K. Iwatsuki 1999b Molecular {alpha}-taxonomy of a morphologically simple fern Asplenium nidus complex from Mt. Halimun National Park, Indonesia. In M. Kato [ed.], The biology of biodiversity, 53–66. Springer-Verlag, Tokyo, Japan

———, J. Yokoyama X. Cheng H. Iwasaki R. Imaichi K. Iwatsuki 1998 Molecular {alpha}-taxonomy of Hymenasplenium obliquissimum complex (Aspleniaceae) based on rbcL sequence comparisons. Plant Species Biology 13: 51-56

———, ———, and K. Iwatsuki 1998 Hymenasplenium inthanonense (Aspleniaceae), a new fern species from Doi Inthanon, and its phylogenetic status. Thai Forest Bulletin 26: 40-52

———, ———, Y. Yatabe H. Iwasaki S. Serizawa 1999c Molecular taxonomic study and revision of the three Japanese species of Asplenium sect. Thamnopteris. Journal of Plant Research 112: 15-25[CrossRef][ISI]

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Shiraishi S. 1988 Inheritance of isozyme variations in Japanese black pine, Pinus thunbergii Parl. Silvae Genetica 37: 93-100[ISI]

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Tsai J. L. W. C. Shieh 1983 A cytotaxonomic survey of the pteridophytes in Taiwan. Journal of Science 20: 137-158

Watano Y. S. Masuyama 1991 Inbreeding in natural populations of the annual, polyploid fern Ceratopteris thalictroides (L.) Brongn. Systematic Botany 16: 705-714[CrossRef][ISI]

Yatabe Y. H. Nishida N. Murakami 1999 Phylogeny of Osmundaceae inferred from rbcL nucleotide sequence and comparison to the fossil evidences. Journal of Plant Research 112: 397-404[CrossRef][ISI]

———, M. Takamiya N. Murakami 1998 Variation in the rbcL sequence of Stegnogramma pozoi subsp. mollisima (Thelypteridaceae) in Japan. Journal of Plant Research 111: 557-564[CrossRef][ISI]




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