Phylogeny of the Lady Fern Group, Tribe Physematieae (Dryopteridaceae), Based on Chloroplast rbcL Gene Sequences

Molecular Phylogenetics and Evolution Vol. 15, No. 3, June, pp. 403– 413, 2000 doi:10.1006/mpev.1999.0708, available online at http://www.idealibrary.com on

Phylogeny of the Lady Fern Group, Tribe Physematieae (Dryopteridaceae), Based on Chloroplast rbcL Gene Sequences Ryosuke Sano,* ,† Masayuki Takamiya,‡ Motomi Ito,† Siro Kurita,† and Mitsuyasu Hasebe* ,1 *National Institute for Basic Biology, 38 Nishigonaka, Myodaiji-cho, Okazaki 444-8585, Japan; †Department of Biology, Faculty of Science, Chiba University, 1-33 Yayoi-cho, Chiba 263-0022, Japan; and ‡Department of Environmental Science, Faculty of Science, Kumamoto University, Kumamoto 860-8555, Japan Received March 31, 1999; revised July 16, 1999

Nucleotide sequences of the chloroplast gene rbcL from 42 species of the fern tribe Physematieae (Dryopteridaceae) were analyzed to gain insights into the inter- and intrageneric relationships and the generic circumscriptions in the tribe. The phylogenetic relationships were inferred using the neighbor-joining and maximum-parsimony methods, and both methods produced largely congruent trees. These trees reveal that: (1) Athyrium, Cornopteris, Pseudocystopteris, and Anisocampium form a clade and Athyrium is polyphyletic; (2) Deparia sensu lato is monophyletic and Dictyodroma formosana is included in the Deparia clade; (3) Diplaziopsis forms a clade with Homalosorus, which is isolated from the other genera of the Physematieae; (4) Monomelangium is included in the monophyletic Diplazium clade; and (5) Rhachidosorus is not closely related to either Athyrium or Diplazium. © 2000 Academic Press

INTRODUCTION The lady fern group, Physematieae (⫽Athyrieae), is one of five tribes of the Dryopteridaceae sensu Kramer et al. (1990) and contains about 700 species distributed mainly in tropical temperate forests. The tribe is characterized by its monomorphic or nearly monomorphic leaves and vascular anatomy (Tryon and Tryon, 1982). Pichi Sermolli (1977) listed 24 genera in the Physematieae, while Ching (1978a,b) recognized 23 genera of Chinese species alone. On the other hand, Kramer and Kato (1990) divided the Physematieae into 12 genera and treated several previously proposed genera as synonyms of their 12 genera. The discrepancies depend on differences in the characters that each author considers significant. The differences in the three generic classifications are summarized in Fig. 1. Since information from phylogenetic inferences is scarce and there are no cladistic analyses of morphological or cyTo whom correspondence should be addressed. Fax: ⫹81-564-557546. E-mail: [email protected]. 1

totaxonomical characters, the phylogenetic relationships among the genera are not well understood, which makes the classification of the Physematieae confused. Two large genera, Athyrium and Diplazium, include more than 80% of the species of Physematieae (Kramer and Kato, 1990). The difference in the basic chromosome number of Athyrium (n ⫽ 40) and Diplazium (n ⫽ 41) is a useful diagnostic character (Tryon and Tryon, 1982), although there are some exceptions (reviewed in Kato, 1977). Several genera, whose phylogenetic relationships to Athyrium and Diplazium are ambiguous, complicate the intratribal classification of the Physematieae. The genus Cornopteris is distinguished from Athyrium and Diplazium by its exindusiate sori and leaf morphology, while its phylogenetic relationships to the two genera are controversial (Ching, 1945; Kato, 1977). Anisocampium was originally established based on its characteristic goniopteroid anastomosing vein, which is rare in Athyrium, whereas it has other morphological traits that are similar to Athyrium. Later, the circumscription of Anisocampium was expanded to include Athyrium sheareri, which is generally similar to Anisocampium, except that it has free veins (Iwatsuki, 1970; Kramer and Kato, 1990). The distinction between Anisocampium and Athyrium then became unclear. Ching (1964a) separates Pseudocystopteris from Athyrium based on its characteristic frond, sorus, and spore morphology, but other authors (Kato, 1977; Kramer and Kato, 1990) do not think that the differences between Athyrium and Pseudocystopteris are sufficient to divide the two genera. Rhachidosorus shares morphological characters with both Athyrium and Diplazium (Ching, 1964a). Recent authors (Kato, 1975a; Kramer and Kato, 1990) place the genus under Diplazium because it shares more morphological characters with Diplazium than with Athyrium (Kato 1975a). Dictyodroma and Diplaziopsis have similar anastomosing veins and are believed to be closely related to each other (Copeland, 1929; Ching, 1964a; Kato, 1977; Price, 1990), although their phylogenetic

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FIG. 1.

Comparison of three recent classifications of the Physematieae.

relationships to Diplazium are controversial (Copeland, 1929; Ching, 1964a,b; Kato 1975b; Kramer and Kato, 1990). Kato (1977) reclassified four previously proposed genera (Deparia, Lunathyrium, Dryoathyrium, and Athyriopsis) as sections in the genus Deparia. The monophyletic relationship of the four genera has not been analyzed by molecular phylogenetic analyses. Monomelangium pullingeri bears Asplenium-like linear sori, multicellular hairs on both stipes and rhizomes, and a densely echinate spore surface. These characters are unusual in Diplazium, while other morphological characters are similar to those of Diplazium. The separation of the genus from Diplazium is controversial (Ching, 1964a; Kato, 1973). Homalosorus pycnocarpos has been placed in either Athyrium (Mickel, 1979; Lellinger, 1985; Kramer and Kato, 1990) or Diplazium (Tryon and Tryon, 1982; Kato and Iwatsuki, 1983) owing to its distinctive combination of characters, such as simply pinnate fronds with entire pinnae, thin herbaceous texture, and linear sori. Cystopteris, Acystopteris, Gymnocarpium, and Hypodematium have been treated as members of other tribes or families by some authors. Cystopteris and

Acystopteris share characteristic orbicular sori and are thought to be closely related. They have been considered to be related to the Athyrium (e.g., Kato 1977), Dryoathyrium (Holttum, 1954), or the tribe Dryopterideae (Copeland, 1947). Similarly, Gymnocarpium and Hypodematium have been variously classified in the Thelypteridaceae (Copeland, 1947; Ching, 1940), Dryopteridoideae (Holttum, 1947, 1954), or Physematieae (Kramer and Kato, 1990). Recent molecular phylogenetic studies using nucleotide sequences of the gene encoding the large subunit of ribulose 1,5-bisphosphate carboxylase/oxygenase (rbcL) have successfully revealed the phylogenetic relationships of ferns at both generic and familial levels (Hasebe et al., 1994, 1995; Wolf et al., 1994). Some representative species of the Physematieae sensu Kramer and Kato (1990) were included in the previous studies and the polyphyletic relationships of the Physematieae were revealed (Hasebe et al., 1995). Some members of the Physematieae used in the study formed a monophyletic group with the Blechnaceae and Thelypteridaceae, as well as with the tribe Onocleeae of the same subfamily. Cystopteris and Gymnocarpium formed a clade and were not closely related to the other

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members of the Physematieae. These results suggest that further studies including problematic taxa are necessary to reveal the phylogenetic relationships of members of the Physematieae. In this paper, we obtained new rbcL nucleotide sequences from 32 taxa in the Physematieae and 4 taxa in other groups. Our analyses aim to address the following questions: (1) Is the generic circumscription of Athyrium, Cornopteris, Anisocampium, and Pseudocystopteris reasonable? (2) Is Deparia sensu Kato (1977) monophyletic? (3) Do Dictyodroma and Diplaziopsis form a clade? (4) Should Homalosorus and Monomelangium be included in Diplazium? (5) Is Rhachidosorus more closely related to Athyrium or Diplazium? MATERIALS AND METHODS The living materials used in this study are listed in Table 1. Total DNA extraction and sequencing generally followed Hasebe et al. (1994). Some extracted DNA samples were further purified by CsCl density gradient centrifugation (Sambrook et al., 1989) or with Qiagentip 20 (Qiagen GmbH, Hilden, Germany). Three overlapping fragments, which cover most of the rbcL gene, were amplified by the polymerase chain reaction. The primers for the amplification followed Hasebe et al. (1994) and Murakami et al. (1999). The amplified products were electrophoresed on 1% agarose gels and purified with GeneClean III (BIO 101 Inc., La Jolla, CA). The purified double-stranded DNA was sequenced in both directions using either an AutoCycleSequencing Kit (Amersham Pharmacia Biotech, Uppsala, Sweden) and an ALF autosequencer (Amersham Pharmacia Biotech) or a BigDye Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems, Foster City, CA) and an ABI PRISM 377 DNA sequencer (Applied Biosystems). The sequences used in this study were aligned manually. Phylogenetic analyses were performed by the neighbor-joining (NJ) (Saitou and Nei, 1987) and maximum-parsimony (MP) methods. In the NJ analyses, we used the NEIGHBOR program of PHYLIP version 3.572 (Felsenstein, 1995). Kimura’s two-parameter model of nucleotide substitutions (Kimura, 1980) was implemented with the DNADIST program in that package. Based on Hasebe et al. (1994), the transition/transversion ratio was fixed at 3.0. One-thousand bootstrap replications were conducted with the SEQBOOT program of the package. In the MP analyses, we used PAUP ver. 3.1.1 (Swofford, 1993). We assigned equal weight to each codon position and had the MULPARS and STEEPEST DESCENT options in effect in all analyses. The heuristic search option using 100 replications of random sequence addition with Nearest-Neighbor Interchange (NNI) branch swapping was employed to search for multiple islands of the most-parsimonious trees (Mad-

dison, 1991). We could not use the Tree Bisection– Reconnection (TBR) branch swapping at this step because of the long calculation time. The trees obtained were used as starting trees for the next heuristic search with Tree Bisection–Reconnection branch swapping. Bootstrap (Felsenstein, 1985) and decay analyses (Bremer, 1988; Donoghue et al., 1992) were used to obtain a measure of support for each branch. One-thousand bootstrap replications were carried out with simple sequence addition and NNI branch swapping. The decay indexes for representative branches were calculated with PAUP in conjunction with the program AutoDecay version 3.0 (provided by T. Eriksson, Stockholm University, Stockholm). In the decay analyses, the most-parsimonious trees were searched for under the reverse-constraint option of PAUP with 3 replications of random sequence addition using TBR branch swapping. The constraint searches were performed under the constraint option of PAUP with 3 replications of random sequence addition using TBR branch swapping. The g1 value (Hillis and Huelsenbeck, 1992) was calculated to evaluate the structure of the data. The expected numbers of synonymous and nonsynonymous nucleotide substitutions per site (Ks and Ka, respectively) were estimated with the method of Li et al. (1985) using the program LWL91 (Li, 1993). The average intergeneric Ks and Ka values were calculated between pairs of species in different genera of the Physematieae. Adiantum capillus-veneris, Hypolepis punctata, and Asplenium normale were selected as outgroup taxa, based on the previously published broad-scale analysis (Hasebe et al., 1995). A specimen of Dictyodroma formosana (voucher Sano 102) was examined cytologically. Observations of somatic chromosomes followed Yatabe et al. (1998). Root tips were pretreated with a 0.002 M 8-hydroxyquinoline solution for 4 h at about 20°C. After fixation in 45% acetic acid at 4°C for 20 min, the root tips were hydrolyzed in 60°C 1 M HCl for 20 s. Then they were stained with 2% aceto-orcein for 1 h and squashed. RESULTS The 1206-bp region between base pairs 73 and 1278, numbered from the initial methionine codon of Nicotiana tabacum (Shinozaki et al., 1986), was used for the phylogenetic analyses. The nucleotide sequences were aligned without any insertions or deletions. The data matrix for the 68 taxa contained 453 variable sites (37.6%), of which 342 were phylogenetically informative. The distribution of the lengths of 10,000 random trees was significantly skewed with a g1 value of ⫺0.47, indicating that a strong nonrandom structure

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TABLE 1 List of Taxa Used in This Study Family (subfamily, tribe) Species Dryopteridaceae, Athyrioideae, Onocleeae Matteuccia struthiopteris (L.) Todaro Onoclea orientalis (Hook.) Hook. Onoclea sensibilis L. var. interrupta Maxim. Oncoleopsis hintonii Ballard Dryopteridaceae, Athyrioideae, Physematieae Acystopteris japonica (Luerss.) Nakai Anisocampium sheareri (Bak.) Ching Athyriopsis conilii (Fr.et Sav.) Ching Athyriopsis minamitanii (Serizawa) Z. R. Wang Athyriopsis petersenii (Kunze) Ching Athyrium filix-femina (L.) Roth ex Mertens Athyrium niponicum (Mett.) Hance Athyrium vidalii (Miq.) Koidz. Athyrium yokoscense (Fr.et Sav.) Christ Cornopteris crenulatoserrulata (Makino) Nakai Cornopteris decurrenti-alata (Hook.) Nakai Cystopteris fragilis (L.) Bernh. Deparia bonincola (Nakai) M. Kato Deparia fenzliana (Luerss.) M. Kato Deparia prolifera (Kaulf.) Hook.et Grev. Dictyodroma formosana (Rosenst.) Ching Diplaziopsis cavaleriana (Christ.) C. Chr. Diplazium chinense (Bak.) C. Chr.

Locality

Reference

Sano 16 (CBM)

D43917 U62030 U05640 U62033

a b c b

Japan (Aichi) Japan (Aichi) Japan (Kagoshima) Japan (Miyazaki) New Zealand (Auckland) Unknown Japan (Tokyo) Japan (Chiba) Japan (Chiba) Japan (Fukushima) Japan (Nagasaki) USA (Utah) Japan (Tokyo) USA (Hawaii) USA (Hawaii) Taiwan (Wu-lai) Japan (Chiba) Japan (Kumamoto)

Sano Sano Sano Sano Sano

AB021725 D43892 D43901 AB021717 D43905 U05908 D43891 D43893 D43894 D43896 D43897 U05916 D43899 D43900 D43906 AB021723 D43909 AB021718

a a a a a d a a a e a d e a a a a a

AB021719

a

D43911 U05619 U05920 AB021720

a c d a

D43912

a

AB021721 D43915 D43903 AB021715 D43908 U05925 U05626 AB021722

a a a a a d c a

D43916

e

AB021716 D43907 D43904 AB021724

a a a a

AB021713 AB021714 D43910 AB021726 U05657

a a a a c

U30608 D43898 U05622 U30832

e a c e

U05648

c

Japan (Kagoshima)

Diplazium donianum (Mett.) Tard. Diplazium esculentum (Retz.) Sw. Diplazium lonchophyllum Kunze Diplazium sibiricum (Turcz. ex Kunze) Kurata var. sibiricum Diplazium squamigerum (Mett.) Matsum.

Japan (Kagoshima) Japan (Kagoshima) Unknown Japan (Akita)

Diplazium subserratum T. Moore Diplazium wichurae (Mett.) Diels Dryoathrium okuboanum (Makino) Ching Dryoathyrium unifurcatum (Bak.) Ching Dryoathyrium viridifrons (Makino) Ching Gymnocarpium dryopteris (L.) Newman Gymnocarpium oyamense (Baker) Ching Homalosorus pycnocarpos (Spreng.) Pic. Ser.

Indonesia (Jawa Is.) Japan (Shizuoka) China (Yun-nan) Japan (Gifu) Japan (Chiba) USA (Idaho) Japan (Saitama) USA, cultivated in Tokyo Japan (Saitama)

Pseudocystopteris atkinsonii (Bedd.) Ching Pseudocystopteris spinulosa (Maxim.) Ching Rhachidosorus mesosorus (Makino) Ching Woodsia manchuriensis Hook. Woodsia polystichoides D. C. Eaton Dryopteridaceae, Dryopteridoideae, Dryopterideae Arachniodes aristata (G. Forst.) Tindale Ataxipteris sinii (Ching) Holttum Dryopteris dickinsii (Fr. & Sav.) C. Chr. Polystichum tripteron (Kunze) Presl Dryopteridaceae, Dryopteridoideae, Rumohreae Rumohra adiantiformis (G. Forst.) Ching

Accession no.

Japan (Fukushima) China (Hubei) Japan (Fukushima) Mexico (Oaxaca)

Diplazium dilatatum Blume

Hypodematium crenatum (Forsk.) Kuhn subsp. fauriei (Kodama) K. Iwats. Lunathyrium pterorachis (Christ.) Kurata Lunathyrium pycnosorum (Christ.) Koidz. Lunathyrium otomasui Kurata Monomelangium pullingeri (Bak.) Tagawa

Voucher specimen

Japan (Kumamoto)

Japan Japan Japan Japan

(Tochigi) (Tochigi) (Kumamoto) (Okinawa)

Japan Japan Japan Japan Japan

(Kochi) (Saitama) (Tochigi) (Tochigi) (Fukushima)

Japan Japan Japan Japan

(Mie) (Kagoshima) (Kumamoto) (Mie)

Australia (Victoria)

42 41 32 31 10

(CBM) (CBM) (CBM) (CBM) (CBM)

Sano 38 (CBM) Sano 25 (CBM) Sano 22 (CBM) Sano 18 (CBM)

Sano 14 (CBM) Sano 15 (CBM) Sano 102 (CBM) Sano 11 (CBM) Ohta & Takamiya 739 (KUMA) Ohta & Takamiya 602 (KUMA) Sano 29 (CBM)

Ohta & Takamiya 695 (KUMA) Ohta & Takamiya 893 (KUMA) Sano 51 (CBM) Sano 13 (CBM) Sano 21 (CBM) Sano 47 (CBM) Sano 24 (CBM)

Sano 46 (CBM)

Sano 48 (CBM) Sano 26 (CBM) Sano 28 (CBM) Ohta & Takamiya 1439 (KUMA) Sano 101 (CBM) Sano 103 (CBM) Sano 44 (CBM) Sano 52 (CBM)

Sano 40 (CBM)

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MOLECULAR PHYLOGENY OF LADY FERN GROUP

TABLE 1—Continued Family (subfamily, tribe) Species Dryopteridaceae, Dryopteridoideae, Tectarieae Tectaria devexa (Kunze) Copel. Aspleniaceae Asplenium normale D. Don Blechnaceae Blechnum orientale L. Doodia maxima J. Sm. Sadleria pallida Hook. & Arm. Stenochlaena palustris (Burm.) Bedd. Woodwardia orientalis Sw. Davalliaceae Davallia mariesii T. Moore ex Baker Dennstaedtiaceae Hypolepis punctata (Thunb.) Mett. Lomariopsidaceae Elaphoglossum yoshinagae (Yatabe) Makino Oleandraceae Oleandra pistillaris (Sw.) C. Chr. Polypodiaceae Polypodium australe Fee Pteridaceae Adiantum capillus-veneris L. Thelypteridaceae Cyclosorus opulentus (Kaulf.) Nakaike Thelypteris acuminata (Houtt.) Morton Thelypteris beddomei (Baker) Ching Thelypteris palustris (Salisb.) Schott. var. pubescens (Laws.) Pernald

Locality

Voucher specimen

Accession no.

Reference

Japan (Okinawa)

D43918

e

Japan (Mie)

AB014701

f

Japan (Kagoshima) Unknown USA (Hawaii) Singapore (Singapore) Japan (Fukushima)

U05606 U05921 U05943 U05652 AB021727

c d d c a

Japan (Fukushima)

U05617

c

Japan (Nara)

U05628

c

Japan (Kagoshima)

U05623

c

Malay Peninsula

U05639

c

England (Cornwall)

U21140

g

Japan (Tokyo), cultivated

D14880

h

U05915 D43919 U05655 U05947

d a c d

Equador Japan (Chiba) Japan (Saga) USA

Sano 49 (CBM)

Sano 39 (CBM)

Note. CBM, Natural History Museum and Institute, Chiba; KUMA, Herbarium of Kumamoto University; a, present study; b, Gastony and Ungerer, 1997; c, Hasebe et al., 1994; d, Wolf et al., 1994; e, Hasebe et al., 1995; f, Murakami et al., 1999; g, Haufler and Ranker, 1995; h, Hasebe et al., 1993.

exists in the data matrix (Hillis and Huelsenbeck, 1992). The NJ tree obtained using Kimura’s two-parameter model (Kimura, 1980) is shown in Fig. 2. In the MP analyses, 2160 equally most-parsimonious trees of 1694 steps were found with 100 replications of NNI branch swapping. The 2160 trees were then used as starting trees for a further search with the TBR branch swapping option, and an additional 8712 trees of 1694 steps were found. The 10,872 most-parsimonious trees are likely to be on a single island, because 10,872 trees were found in the search with the TBR branch swapping using a randomly selected tree from the 10,872 trees as a starting tree. The most-parsimonious trees had a consistency index of 0.343 (0.292 excluding uninformative sites), a retention index of 0.540, and a rescaled consistency index of 0.185. The strict consensus of the 10,872 most-parsimonious trees is shown in Fig. 3. Although the trees obtained by the NJ and MP analyses are not completely congruent, branches with high bootstrap values in the NJ tree are also statistically supported in the MP analyses by both bootstrap values

and decay indexes. The branch indicated by an asterisk in the NJ tree (Fig. 2) is an exception. It is supported with a 91% bootstrap value in the NJ tree (Fig. 2) but with less than 50% in the MP tree (Fig. 3). The somatic chromosome number of Dictyodroma formosana was 2n ⫽ 80 (Fig. 4); thus, this species is a diploid based on n ⫽ 40. DISCUSSION Monophyly of Athyrium, Cornopteris, Pseudocystopteris, and Anisocampium Two species of Pseudocystopteris form a well-supported clade with the three species of Athyrium. Neither the NJ nor the MP trees support the monophyly of each genus, and the phylogenetic relationship of the five species is not well resolved. The average Ks and Ka values between Pseudocystopteris and the three species of Athyrium are 0.0097 ⫾ 0.0014 and 0.0015 ⫾ 0.0004, respectively. Both values are much smaller than the average intergeneric values in ferns (0.411 ⫾ 0.258 and 0.018 ⫾ 0.011, respectively; Hasebe et al., 1995).

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FIG. 2. Tree obtained using the neighbor-joining method. The horizontal branch lengths are proportional to the estimated number of nucleotide substitutions per site. Bootstrap values are indicated for the branches occurring in more than 50% of 1000 bootstrap replicates. The higher classification sensu Kramer and Green (1990) is shown on the right.

MOLECULAR PHYLOGENY OF LADY FERN GROUP

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FIG. 3. Strict consensus of the 10,872 equally most-parsimonious trees in the maximum-parsimony analyses with equal weighting. Bootstrap values are indicated above the branches occurring in more than 50% of 1000 bootstrap replicates, and decay indexes are shown in brackets. Number of nucleotide substitutions (ACCTRAN optimization) is designated below each branch. The higher classification sensu Kramer and Green (1990) is shown on the right.

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sheareri, but its other morphological characters are similar to those of other Athyrium species. A long creeping rhizome is also observed in Pseudocystopteris, suggesting that the morphology of the rhizome is plesiomorphic or results from parallel evolution in different lineages. A hybrid between A. niponicum and A. sheareri has been reported, while no hybrids between A. niponicum or A. sheareri and any other Athyrium species have been reported (Iwatsuki et al., 1995). Hybrids among Athyrium other than A. niponicum are often observed. These facts are concordant with the close alliance between A. sheareri and A. niponicum, as well as with the distant relationship between the two species and the other Athyrium. The separation of A. niponicum from other members of Athyrium should be confirmed with a wider sampling of species from both Anisocampium and Athyrium. FIG. 4. Somatic metaphase chromosomes of Dictyodroma formosana (2n ⫽ 80). Bar, 5 ␮m.

These results suggest the taxonomic inclusion of Pseudocystopteris in Athyrium (Kato, 1977; Kramer and Kato, 1990). Ching (1964a) mentioned that Pseudocystopteris was an intermediate taxon connecting Athyrium and Cystopteris and was more closely related to the latter genus, but the rbcL trees do not support his statement. Kato (1977) treated Pseudocystopteris as a synonym of Athyrium and divided Athyrium into two groups: the A. puncticaule group and the A. filix-femina group. Of the species used in this study, Pseudocystopteris atkinsonii (⫽Athyrium atkinsonii Bedd.) is included in the former group and the other species are in the latter group. Although Kato (1977) suggested that the former group has more primitive characters in Athyrium and is a basal-branched taxon, the rbcL trees do not positively support this hypothesis because P. atkinsonii does not occupy a basal position. Cornopteris is an Asian genus. It is defined by the absence of indusia and the horn-like appendages on the adaxial bases of the costae and costules, while other morphological characters are similar to those of Athyrium. Although Ching (1945) considered Cornopteris to be an exindusiate derivative of Diplazium, Kato (1977) stated that Cornopteris was closely related to Athyrium rather than to Diplazium, based on his more detailed observation of sorus morphology. Our rbcL trees show the monophyly of the two Cornopteris species used in this study, as well as that of Anisocampium, Athyrium, Cornopteris, and Pseudocystopteris. Athyrium niponicum forms a clade with Anisocampium sheareri instead of the other Athyrium species in both rbcL trees. A. sheareri has a long creeping rhizome, pinnate fronds with a chartaceous texture, and orbicular sori. The combination of these morphological characters is unusual in Athyrium. A. niponicum has a long creeping rhizome similar to that of A.

Monophyletic Relationship of Deparia sensu Kato (1977) Hooker and Greville established the genus Deparia in 1829 based on Deparia prolifera. Kato (1977, 1984) stressed the similarities of the articulate hairs, frond architecture, and frond segmentation of Deparia, Athyriopsis, Dryoathyrium, and Lunathyrium, although D. prolifera has peculiar cup-shaped sori that are not observed in any other species of Deparia sensu Kato (1977). Kato (1977) considered that the monotypic genus Deparia is congeneric with the other three genera and changed the latter three genera to sections under Deparia. The rbcL trees clearly show a monophyletic relationship of the four genera and support Kato’s (1977) classification of the four groups into a single genus. The average Ks values between the four genera range from 0.017 ⫾ 0.0069 (between Deparia and Athyriopsis) to 0.0647 ⫾ 0.0042 (between Dryoathyrium and Deparia), and the average Ka values range from 0.0029 ⫾ 0.0014 (between Deparia and Athyriopsis) to 0.0065 ⫾ 0.0015 (between Dryoathyrium and Deparia). Even the largest values are smaller than the average intergeneric values in ferns (0.411 ⫾ 0.258 and 0.018 ⫾ 0.011, respectively; Hasebe et al., 1995), suggesting that Kato’s (1977) treatment is adequate. D. prolifera forms a clade with other Deparia species, and the peculiar sori should be an autoapomorphic character in the D. prolifera lineage. The monophyletic relationship of the species selected from Deparia and Athyriopsis is supported with high bootstrap values in both NJ and MP trees, although the interspecific relationships of the genera are not well resolved. The morphological similarity of the nonwinged stipe bases and predominantly elongate sori with rather thin indusia of Deparia and Athyriopsis suggest their close relationship (Kato, 1984). Lunathyrium otomasui is included in the monophyletic Deparia–Athyriopsis clade, supporting Kato’s (1984) placement of L. otomasui under Athyriopsis.

MOLECULAR PHYLOGENY OF LADY FERN GROUP

The monophyletic relationship of the species of Dryoathyrium examined has weak support in the NJ tree and is not well resolved in the MP tree. The 1206 bases of the rbcL genes of D. okuboanum and D. viridifrons are identical. D. okuboanum is a triploid apogamous species (Kato, 1984) and likely originated from a hybrid between diploid sexual D. viridifrons and another apogamous species. Our result supports the hypothesis that D. viridifrons is a maternal parent of D. okuboanum. L. pterorachis occupies a basal position in Deparia sensu Kato (1977) in the MP tree and forms a monophyletic group with Dryoathyrium in the NJ tree. L. pycnosorum forms a sister group with the clade composed of Deparia, Athyriopsis, and L. otomasui in both trees. These results reveal the polyphyletic relationship of Lunathyrium. The diagnostic characters of Lunathyrium are also observed in some taxa outside the clade containing the species of Deparia sensu Kato (1977), such as Athyrium (Kato, 1977), suggesting that the genus Lunathyrium is likely defined by plesiomorphic characters. Ching (1964a) established Dictyodroma based on its pinnate or deeply pinnatifid fronds, reticulate venation, and the scale-like hairs on the stipe. It has been suggested that the genus is related to Diplaziopsis or the species in Diplazium with reticulate venation (Copeland, 1929; Ching, 1964a; Kato, 1975b, 1977; Price, 1990). The rbcL trees clearly show that Dictyodroma formosana is not closely related to either Diplaziopsis or Diplazium, but it is related to Deparia sensu Kato (1977), suggesting that the species should be classified under Deparia sensu Kato (1977). Although a close relationship between Dictyodroma and Deparia sensu Kato (1977) has not been proposed in any previous studies, some morphological characters support the rbcL trees. The characteristic groove of the frond axis and the special type of multicellular hairs on the stipe, both of which are important diagnostic characters of Deparia sensu Kato (1977), have also been observed in Dictyodroma (Kato, 1973). Kato (1973) assumed that the reticulate venation was a more important character for phylogenetic inference than the morphology of the fronds and the hairs, but the rbcL trees do not support his assumption. The spore morphology of Dictyodroma differs from that of Diplazium or Diplaziopsis (Tryon and Lugardon, 1991) and it is consistent with the rbcL trees. The chromosome number of D. formosana examined was 2n ⫽ 80 (Fig. 4), which concurs with the basic number in Deparia (n ⫽ 40) but is different from that of Diplazium and Diplaziopsis (n ⫽ 41). The chromosome number of D. formosana has been reported to be n ⫽ 41 (Hirabayashi, 1970; Kurita, 1972; Tsai and Shieh, 1978), but in the picture of the chromosomes (Kurita, 1972) it is possible to count n ⫽ 40. Therefore, we judge that the basic number of D. formosana is n ⫽ 40.

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Phylogenetic Relationships of Enigmatic Genera Pichi Sermolli (1973) established the monotypic genus Homalosorus, but other authors classified it under Athyrium (Mickel, 1979; Lellinger, 1985; Kramer and Kato, 1990) or Diplazium (Tryon and Tryon, 1982; Kato and Iwatsuki, 1983). A relationship between Homalosorus pycnocarpos and Diplaziopsis (Tryon and Tryon, 1973; Kato and Iwatsuki, 1983) has been suggested from their similar pinna shape, rachis-grooving, indusia, and spores, although they have different lamina apex, venation, and chromosome numbers (Kato and Darnaedi, 1988; Price, 1990). The rbcL trees support the sister relationship between H. pycnocarpos and Diplaziopsis cavaleriana with 100% bootstrap value. The trees also do not support a close relationship between Diplaziopsis and Diplazium. The constraint searches, assuming the monophyly of the Diplazium clade and the Diplaziopsis–Homalosorus clade, and that of the Athyrium–Anisocampium–Cornopteris– Pseudocystopteris clade and the Diplaziopsis–Homalosorus clade, resulted in trees with 10 and 8 steps additional to those of the most-parsimonious tree, respectively. This suggests that Diplaziopsis and Homalosorus should be treated as a separate genus from Athyrium and Diplazium. Ching (1964a) stressed the distinctness of the monotypic genus Monomelangium from other members of Diplazium, based on the presence of multicellular hairs on the stipes and rhizomes, which are rarely observed in other members of Diplazium. He pointed out the close relationship between Monomelangium and Athyriopsis, based on their similar multicellular hairs, but Kato (1973) observed that the multicellular hairs of Monomelangium lack a glandular cell at the tip of each hair and are different from those of Athyriopsis. He also found that Monomelangium has an adaxial groove on the frond axis similar to that in Diplazium but different from that in Athyriopsis. Therefore, Monomelangium pullingeri was classified under Diplazium (Kato, 1977). The rbcL trees support Kato’s (1977) treatment, and the gain of multicellular hairs likely occurred in the M. pullingeri lineage. The chromosome number of M. pullingeri is indicated as n ⫽ 41 (2n ⫽ 164; Takamiya and Ohta, unpublished data). This is not consistent with the basic number of Deparia (n ⫽ 40) but is with that of Diplazium (n ⫽ 41), suggesting the close relationship of M. pullingeri to Diplazium rather than to Deparia. Monomelangium pullingeri and the species of Diplazium used in this study form a clade, and three groups with high bootstrap values are recognized: (D. lonchophyllum and D. wichurae), (D. squamigerum and D. sibiricum), and (the other species of Diplazium plus M. pullingeri). This result is preliminary because we examined only a few of the more than 300 species of Diplazium. D. wichurae from eastern Asia and D. lon-

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chophyllum from central America form a clade in the rbcL trees, suggesting that more detailed studies including species from wider areas should be useful for biogeographic studies of Diplazium. Rhachidosorus mesosorus has been classified in Athyrium (Makino, 1899; Tagawa, 1936) or Diplazium (Koidzumi, 1924; Kato, 1977; Kramer and Kato, 1990), while Ching (1964a) established the genus Rhachidosorus. Based on our rbcL analyses, R. mesosorus does not cluster with either Athyrium or Diplazium but occupies a position isolated from the other taxa. The constraint searches, assuming the monophyly of the Diplazium clade and R. mesosorus, and that of the Athyrium–Anisocampium–Cornopteris–Pseudocystopteris clade and R. mesosorus, resulted in trees with three steps additional to those of the most-parsimonious trees in either search. Therefore, this study supports the taxonomic treatment separating Rhachidosorus from Athyrium and Diplazium. Phylogenetic Relationships of Other Genera and Families Cystopteris and Acystopteris form a clade in the rbcL trees. The two genera are sister to the clade of Gymnocarpium in the NJ tree but not in the MP tree. Acystopteris has a morphology of indusium similar to that of Cystopteris (Blasdell, 1963), which agrees with the rbcL trees. Gymnocarpium, Cystopteris, and Acystopteris have been suggested to be monophyletic according to their similar morphology of rhizome, trichome, and frond texture (Ching, 1940). Cystopteris has been considered to be closely related to Athyrium (reviewed in Kato, 1977). The constraint search, assuming the Acystopteris–Cystopteris–Gymnocarpium clade and the Athyrium–Anisocampium–Cornopteris– Pseudocystopteris clade monophyletic, resulted in trees with three steps more than those of the most-parsimonious trees, suggesting that the two clades do not form a clade. Two species of Woodsia form a clade with weak support of the bootstrap values and decay index, and the phylogenetic relationship to other genera is not resolved. In previous studies, the Blechnaceae and Thelypteridaceae are nestedly clustered with Dryopteridaceae, although phylogenetic relationships of these families and the genera of Dryopteridaceae are not supported with high bootstrap values (Hasebe et al., 1994, 1995; Wolf et al., 1994). In this study, we included more taxa of the Physematieae than in previous studies, but wellsupported phylogenetic relationships were not obtained. Other molecular data may be useful for deducing the relationships of the families and genera.

Kitaoka for living material of Dictyodroma, and to T. Nakaike, T. Iwata, M. Kato, and H. Ishikawa for help in our field collection of the plants. We also acknowledge T. Nishiyama for calculation of Ks and Ka, T. Nakaike for suggesting some references, and M. Kato for critical comments on the manuscript. This work was supported in part by the Ministry of Education, Science, Sports and Culture, Japan (08404055 to M.H.).

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