Bot. Bull. Acad. Sin. (2003) 44: 223-228

Hao et al. — Phylogenetics of Bauhinia subgen. Phanera

Phylogenetics of Bauhinia subgenus Phanera (Leguminosae: Caesalpinioideae) based on ITS sequences of nuclear ribosomal DNA

Gang Hao, Dian-Xiang Zhang*, Ming-Yong Zhang, Li-Xiu Guo, and Shi-Jin Li

South China Institute of Botany, The Chinese Academy of Sciences, Guangzhou 510650, P. R. China

(Received October 2, 2002; Accepted April 11, 2003)

Abstract. The pantropical legume genus Bauhinia is currently subdivided into four subgenera. Subgenus Phanera, with ca. 150 species, is the largest. A parsimony analysis was conducted on ITS sequence data from 38 species, with representatives from all four subgenera, and species sampled from nine of 11 sections of subgen. Phanera, and including two Cercis species as the outgroup. The well-resolved strict consensus tree suggests that subgen. Phanera and the monotypic subgen. Barklya together form a monophyletic group. In the Phanera-Barklya clade, the African (sect. Tylosema), American (sect. Caulotretus), and Australasian (subgen. Barklya, subgen. Phanera sect. Lysiphyllum) species are the first (paraphyletic) lineages, while the Asian taxa (except species of sect. Lysiphyllum) form a monophyletic group. The ITS tree also revealed that sects. Phanera and Lasiobema are not monophyletic. Monophyletic groups supported by the phylogenetic analysis include ser. Fulvae and ser. Corymbosae of sect. Phanera, sect. Tubicalyx, and the Asian-Australasian sect. Lysiphyllum.

Keywords: Bauhinia; Caesalpinioideae; ITS; Leguminosae; Phylogenetic analysis; subgen. Phanera.

Introduction

Bauhinia L., a pantropical genus of about 300 species (Wunderlin et al., 1987) of trees, shrubs or lianas with bifoliolate, bilobed or simple entire leaves, has always been controversial in its taxonomy, including whether or how it could be divided into small genera.

According to Wunderlin et al. (1981, 1987), the ancestor of the genus gave rise to two main phyletic lines, one to the mostly arborescent or shrubby subgenera Bauhinia, Elayuna and Barklya, the second to subgenus Phanera, which mostly comprises lianas. A morphological cladistic analysis of the genus by Zhang (1995) and a survey of the rpL2 chloroplast intron for 78 species by Lai et al. (1997), however, both yielded results not fully compatible with the conclusions of Wunderlin et al.

Subgenus Phanera, comprising about 150 species, is the largest subgenus in Bauhinia. Pantropical in distribution, but with most of its species occurring in continental Asia and the Malesia region, the subgenus is subdivided into 11 sections (Wunderlin et al., 1987). Several of these sections are further subdivided into subsections or series.

In their recent phylogenetic analysis of the subfamily Caesalpinioideae based on chloroplast trnL intron sequences, Bruneau et al. (2001) suggested that the sub

genus Phanera, as well as the whole genus Bauhinia, might not be monophyletic.

Although taxonomic and floristic studies on the genus Bauhinia in general, and on subgen. Phanera in particular, have helped improve our knowledge of the group (Chen, 1988; Larsen and Larsen, 1973, 1983, 1991, 1996; Larsen et al., 1980, 1984; Zhang, 1993), systematic studies on the genus are still hampered by its large number of species, prevalence of morphological convergence, and difficulties in obtaining material. In this study, we newly sequenced nuclear ITS regions from representative species of Bauhina, with the aim of assessing the monophyly of subgen. Phanera as currently defined, and of clarifying the intra-subgeneric relationships within the group.

Materials and Methods

Taxon Sampling

A total of 38 species were included in this molecular study (Table 1). For the ingroup we sampled 40 accessions representing 32 species of subgen. Phanera, as well as two species of subgen. Bauhinia, one species of the monotypic subgen. Barklya, and one species of subgen. Elayuna. The ingroup taxa chosen covered all four subgenera recognized by Wunderlin et al. (1987). The 32 species of subgen. Phanera represented nine of the 11 sections (Wunderlin et al., 1987). Most of the species chosen were from the two larger sections that are apparently polymorphic, viz., sect. Lasiobema and sect. Phanera. For some sections, e.g., sects. Palmatifolia, Tubicalyx, and

*Corresponding author. Tel: 86-20-8523-2043; Fax: 86-20-8523- 2831; E-mail: dx-zhang@scib.ac.cn

Botanical Bulletin of Academia Sinica, Vol. 44, 2003

Hao et al. — Phylogenetics of Bauhinia subgen. Phanera

Tylosema, only one or two species were represented. However, these sections are readily identifiable morphologically and by leaf venation patterns (Wunderlin et al., 1987; Zhang, 1994) and have been consistently recognized as natural groups. The three species of sect. Lysiphyllum included in this study represented its two subsections. Recent molecular studies (Bruneau et al., 2001) confirmed that Cercis, another genus of tribe Cercideae (Wunderlin et al., 1987), has close affinity with Bauhinia. We thus chose two species of Cercis (C. chinensis and C. glabra) as the outgroup taxa in this study.

Genomic DNA Extraction, PCR Amplification, and Sequencing

Total DNA was extracted from fresh or silica-dried leaves, following a 2X CTAB method modified from Doyle and Doyle (1987). The entire ITS region was applied using universal primers "ITS 5" and "ITS 4" (White et al., 1990). PCR products were purified using QIAquick Gel Extraction Kit (QIAGEN). Automated sequencing was conducted using dye terminator on an ABI 377 DNA Sequencer, using at least two of the four primers N18L18 (Wen and Zimmer, 1996), ITS2, ITS3, and ITS4 (White et al., 1990).

Phylogenetic Analysis

The combined sequences of ITS1, 5.8S and ITS2 were initially aligned using Clustal X (Thomson et al., 1997), followed by minor adjustment by eye. Potentially informative and unambiguously assessed indels were scored as separate characters regardless of their length, and added to the data matrix as additional binary or multistate characters (25 such characters in total). Maximum parsimony analysis was performed using PAUP* (Swofford, 1998) on a Macintosh computer. The analysis used heuristic searches with random addition and TBR branch swapping. Clade robustness was evaluated by bootstrap analysis using 1000 replicates of heuristic searches, with simple addition sequence and TBR branch swapping. Sequence divergence values were calculated by the Kimura two-parameter method (Kimura, 1980).

Results

Variation in the ITS Sequences

The length of the entire ITS region in Bauhinia species studied ranges from 594 to 626 bp; the 5.8S region is 163 or 164 bp long while the ITS1 and ITS2 regions range between 212 - 247 and 203- 248 bp, respectively. The consensus sequence for the 42 sampled accessions is 689 bp in length. The Kimura two-parameter distance among the ingroup species is estimated as 0.19% to 40.19% (data matrix not shown). If the multiple accessions of B. championii, B. glauca, and B. corymbosa are ignored respectively, the lowest divergence is between B. didyma and B. glauca (both of subgen. Phanera); the highest value is between B. touranensis (of subgen. Phanera) and B. variegata (of subgen. Bauhinia).

Phylogenetic Analysis

The parsimony analysis generated nine trees of 1,282 steps, with a consistency index (CI) of 0.48, and a retention index (RI) of 0.65. The strict consensus tree is shown in Figure 1. The monophyly of sampled Bauhinia species has maximum support (100%). Two species representing subgen. Bauhinia (B. variegata and B. jenninsii), and the one species representing subgen. Elayuna (B. reticulata), form a clade with weak support (62%), which is the sister to the clade containing subgen. Phanera and Barklya. For the latter clade, the Africa species, B. fassoglensis of subgen. Phanera sect. Tylosema, is the first to branch out, followed by the American species, B. glabra (representing subgen. Phanera sect. Caulotretus) and the Australasian B. syringifolia (the sole species of subgen. Barklya). The remainder of subgen. Phanera forms a clade. The above branching patterns receive relatively high bootstrap support (Figure 1). A significant feature in the latter clade is that sect. Phanera, the biggest section of subgen. Phanera, is highly polyphyletic, including at least five distantly related lineages, although their relationships with other sections are always relatively weakly supported. Sect. Lasiobema is also revealed as a heterogeneous assembly of three lineages with the present sampling, with its members scattered throughout the strict consensus tree (Figure 1).

Discussion

Monophyly of Subgen. Phanera

As the ITS tree (Figure 1) shows, the genus Bauhinia is divided into two branches. The first branch only includes subgen. Bauhinia and subgen. Elayuna, not Barklya as Wunderlin et al. (1981, 1987) otherwise proposed. Subgen. Phanera is thus parapyletic since subgen. Barklya (B. syringifolia) is nested within it. The ITS analysis also revealed that in the subgen. Phanera - Barklya clade, the African (sect. Tylosema), American (sect. Caulotretus), and Australasian (subgen. Barklya, subgen. Phanera sect. Lysiphyllum) species are the first to branch out, while the Asian taxa form a monophyletic group. Unfortunately, the monotypic New Guinean section Austrocercis and the tropical American section Schnella were not sampled in the present analysis, and should be included to test the monophyly of the sections.

Taxonomic Implications and Character Evolution

The monophyletic groups supported by the present phylogenetic analysis based on ITS sequences, e.g., sect. Phanera ser. Fulvae, ser. Corymbosae, sect. Tubicalyx, and sect. Lysiphyllum, are species groups with very similar flower morphology (Wunderlin et al., 1987), pollen types (Larsen, 1975; Larsen and Larsen, 1983; Ferguson and Pearce, 1986; Schmitz, 1973, 1977) and leaf venation patterns (Zhang, 1994).

Wunderlin et al. (1987) conjectured that the ancestral stock of Cercideae consisted of moderate-sized trees with bifoliolate compound leaves as found in the extant

Botanical Bulletin of Academia Sinica, Vol. 44, 2003

Figure 1. Strict consensus tree of the nine most parsimonious trees from the entire ITS sequences of Bauhinia species. Tree length = 1282 steps, CI = 0.48, RI = 0.65. Numbers above lines represent bootstrap values in 1000 replicates. Supraspecific classification follows Wunderlin et al. (1987). Grey areas show taxa from subgen. Phanera. Series of sect. Phanera are indicated. B. corymbosa 1 = B. corymbosa var. corymbosa; B. corymbosa 2 = B. corymbosa var. longispes; B. glauca 1 = B. glauca ssp. glauca; B. glauca 2 = B. glauca ssp. hupehana.

Detarieae (Caesalpinioideae). The arborescent habit—found in all species of subgen. Bauhinia, Elayuna, and Barklya, and some species of subgen. Phanera—is presumably the plesiomorphic state in the genus Bauhinia. In the Phanera-Barklya branches in the strict consensus tree (Figure 1), the first clades to branch out, viz., representatives of sects. Tylosema and Caulotretus, as most of the species in the subgenus, are lianas, while the arborescent taxa, e.g., B. syringifolia (subgen. Barklya), B. carronii-B. gilva-B. winitii (sect. Lysiphyllum), B. roxburghiana (sect. Semla), and B. bohniana (sect. Pseudobauhinia), do not form a monophyletic group. A reversal to the arborescent habit has very likely occurred independently in these taxa.

The relationships between bifoliate, bilobed, and unifoliate leaves in Bauhinia have been a fascination, and several studies had been performed on the topic (Watari, 1934; Pijl, 1951; Cusset, 1966; Owens, 2000). The so-called "split

ter hypothesis" (e.g., Cusset, 1966, hypothesizing that the ancestral leaf form in Cercideae is unifoliate, from which the bilobed and the bifoliolate leaves are derived) and the "fusion hypothesis" (e.g., Pijl, 1951, suggesting that the bifoliolate leaf is the ancestral state in the tribe, from which the bilobed and unifoliate leaves are derived) have been proposed. The species with bifoliate leaves (e.g., B. winitii, B. carcinophylla) and unifoliate leaves (e.g., B. syringifolia, B. strychnifolia, B. strychnoidea, B. paucinervata) are scattered on the cladogram among species with bilobed leaves. Our study did not lend credence to either the "splitter hypothesis" or the "fusion hypothesis." Our only conclusion is that both bifoliate and unifoliate leaves have arisen many times in Bauhinia.

The number of fertile stamens is another important character in Bauhinia classification. Ten fertile stamens are found in all other genera of Cercideae, in subgen. Elayuna, Barklya, and in most sections of subgen.

Hao et al. — Phylogenetics of Bauhinia subgen. Phanera

Bauhinia. Three fertile stamens are found in most species of subgen. Phanera while 10 fertile stamens are found in sects. Lysiphyllum and Caulotretus, although species with two fertile stamens have been found in sects. Tylosema, Lasiobema (Zhang, 1993) and Phanera (Wunderlin et al., 1987). Judged from the topology of the ITS tree, the 10 fertile stamens very likely represent a plesiomorphic state in the subgenus while the three fertile stamens represent a derived one. The state of two fertile stamens may have independent origins in sects. Tylosema, Phanera, and Lasiobema.

Most of the sections in subgen. Phanera as presently circumscribed (Wunderlin et al., 1987) have to be redefined if only monophyletic groups are to be recognized. If Barklya is accepted as a subgenus, the subgeneric status of Caulotretus and Tylosema are also warranted. Sects. Phanera and Lasiobema may each need redefinition; sect. Phanera ser. Loxocalyx is best included in sect. Tubicalyx, a result similar to that of a previous cladistic analysis of morphological data in the genus Bauhina (Zhang, 1995); ser. Corymbosae, with unique leaf venation patterns (Zhang, 1994) and pollen morphology (Larsen, 1975; Zou et al., unpublished data), should be promoted to sectional level. Wunderlin et al. (1987) asserted that a close relationship exists between ser. Fulvae and sect. Palmatifolia and further suggested that the latter was derived from the former. Our study revealed these two groups to be sister groups. If the sectional status of Pseudobauhinia is preserved, a sectional status is also warranted for ser. Yunnanentes (with a sole species B. yunanensis). Semla is proved to be derived from within a paraphyletic Lasiobema. The monophyletic status of sect. Lysiphyllum, however, is strongly supported by this study.

Acknowledgements. This project was supported by grants from the Chinese Academy of Sciences Innovation Project (SCIB Director's Fund, Grants KSCX-2-1-06B, kscxz-sw-101A, and Fund for Teams of Young Scientists in Pursuit of Creative Research), and by grants for Students and Scholars returned from Overseas. We are indebted to Drs. Chawalit Niyomdham, Ruth Kiew, Shirley A. Owens, Benoit Jonckheere, Anne Bruneau, Peter Weston, M. H. Chapin, Lawrence Chow, Ruijiang Wang, Jacqueline Soule, Yunfei Deng, and several others for supplying silica dried leaf samples, and to one anonymous reviewer for critical comments and suggestions on the manuscript.

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