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Phylogenetics, biogeography, and evolutionary trends of the Phalaenopsis sumatrana complex inferred from nuclear and chloroplast DNA
Biochemical Systematics and Ecology 37 (2009) 633–639
Contents lists available at ScienceDirect
Biochemical Systematics and Ecology journal homepage: www.elsevier.com/locate/biochemsyseco
Phylogenetics, biogeography, and evolutionary trends of the Phalaenopsis sumatrana complex inferred from nuclear and chloroplast DNA Chi-Chu Tsai a, b, *, Sheng-Chung Huang c, Chang-Hung Chou d, ** a
Kaohsiung District Agricultural Improvement Station, Pingtung 908, Taiwan National Pingtung University of Science and Technology, Pingtung 912, Taiwan c National Plant Genetic Resources Center, Agricultural Research Institute, Taichung 413, Taiwan d Research Center for Biodiversity, China Medical University, Taichung 404, Taiwan b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 9 February 2009 Accepted 4 September 2009
Phylogenetic trees inferred from the internal transcribed spacers 1 and 2 (ITS1 þ ITS2) region from nuclear ribosomal DNA (nrDNA) and the intergenic spacer of atpB-rbcL from chloroplast DNA (cpDNA) were used to clarify the phylogenetics and evolutionary trends of the Phalaenopsis sumatrana complex. The P. sumatrana complex includes the two species P. sumatrana and Phalaenopsis corningiana as well as a problem species, Phalaenopsis zebrina, according to the concepts of Sweet (1980) and Christenson (2001). Based on the phylogenetic tree inferred from the ITS sequence, the accessions of P. sumatrana cannot be separated from those of P. corningiana. In contrast, the accessions of P. zebrina can be separated from those of both P. sumatrana and P. corningiana. However, analyses of the sequences of the atpB-rbcL spacer apparently cannot discriminate among these three species of the P. sumatrana complex. An inspection of the morphological characters of the plants of the P. sumatrana complex and the floral fragrances of P. zebrina can be used to separate it from both P. sumatrana and P. corningiana. Based on the molecular data and floral fragrances, P. zebrina perhaps should not be treated as a synonym of P. sumatrana. On the evolutionary trend of the P. sumatrana complex, P. zebrina was suggested to be the relative origin group of the P. sumatrana complex based on the phylogenetic tree and biogeography. Ó 2009 Elsevier Ltd. All rights reserved.
Keywords: Phalaenopsis sumatrana Species complex Phylogenetics Biogeography
1. Introduction The Phalaenopsis sumatrana complex includes P. sumatrana and Phalaenopsis corningiana plus one questionable species, Phalaenopsis zebrina. P. sumatrana is widely distributed in Sumatra, Java, Borneo, Mentawei Is, Malaya, Perak, Johore, and Thailand. P. corningiana and P. zebrina have restricted distributions in Borneo/Sarawak and Borneo/Palawan, respectively (Sweet, 1980; Masaaki, 2002). Based on morphology, the characters of these three species of this complex are not easily differentiated. P. zebrina was treated as a synonym of P. sumatrana by Sweet (1968). Fowlie (1982) attempted to raise the taxonomic position of plants in the P. sumatrana complex with a white ground color flower into a separate species, P. zebrina, distinct from plants of P. sumatrana with a yellowish ground color. Christenson (2001), however, disagreed with Fowlie’s separation because the type specimen of P. zebrina had a yellowish ground color. Therefore, P. zebrina is retained as a synonym
* Corresponding author at: Kaohsiung District Agricultural Improvement Station, Pingtung 908, Taiwan. Tel.: þ886 8 7746735; fax: þ886 8 7389106. ** Corresponding author. Tel./fax: þ886 4 22071500. E-mail addresses: [email protected] (C.-C. Tsai), [email protected] (C.-H. Chou). 0305-1978/$ – see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.bse.2009.09.004
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of P. sumatrana. Nevertheless, P. zebrina is still used to represent plants with narrower brown markings on a white or yellowish ground color in horticulture to the present (Masaaki, 2002). Furthermore, Sweet (1968), Fowlie (1969), and Christenson (2001) have the congruent concept of P. sumatrana and P. corningiana as being separate from each other and being treated as two separate species. In addition, confusion has also surrounded plants of P. corningiana and various darker-colored plants of P. sumatrana. Plants of P. corningiana can be separated from those of P. sumatrana based on their callus morphology and the marking pattern on their petals and sepals. The callus morphology of plants of P. corningiana is uniseriate, sulcate, bifid, and, at the base, continuous with a structure analogous to a posterior keel, forming a sunken pit. The callus of P. sumatrana, on the other hand, is biseriate, with the posterior callus being four lobulate at the apex and granular at the base and the anterior callus being longer, sulcate, and bifid. The petal markings of P. corningiana show longitudinal stripes toward the apex of the sepals and petals. In contrast, those of P. sumatrana always show transverse stripes (Sweet, 1968, 1980; Christenson, 2001). However, these different characteristics between P. corningiana and P. sumatrana are still obscured in some individual clones. For example, a solid red clone, P. sumatrana var. sanguinea, has been treated as a synonym of P. corningiana (Sweet, 1980). In addition, the length of the floral inflorescences of P. corningiana (much shorter than the leaves) is considered to be shorter than that in P. sumatrana (Sweet, 1968). Wallbrunn (1971), however, did not accept this concept according to inspections of several living plants of these two species. Furthermore, Christenson (2001) proposed that the floral fragrance is the best character for separating these two sister species. Plants of P. corningiana bear a wonderful scent of spicy candy. Those of P. sumatrana, on the other hand, bear a mildly acrid fragrance. Doubtlessly, species of the P. sumatrana complex, namely P. zebrina, P. sumatrana, and P. corningiana, are closely related. To evaluate the phylogenetics and evolutionary trends of this complex, sequences of the ITS from nrDNA and the atpB-rbcL spacer from chloroplast DNA were analyzed in this study. 2. Materials and methods 2.1. Plant materials Material from 14 accessions of the P. sumatrana complex were selected and used for this study (Table 1). The geographical distribution of each species of this complex is shown in Fig. 1. Furthermore, one species of the section Amboinenses, Phalaenopsis gigantea and one species of the section Fuscatae, Phalaenopsis fuscata, were used as outgroups of the P. sumatrana complex to clarify the evolutionary trend of this complex, since this complex (section Zebrinae) and the Amboinenses and Fuscatae sections (Tsai et al., 2006a) share a common ancestor. 2.2. DNA extraction Total DNA was extracted using the CTAB (cetyltrimethylammonium bromide) method (Doyle and Doyle, 1987). The approximate DNA yields were then determined using a spectrophotometer (Hitachi U-2001). 2.3. PCR amplification and electrophoresis The primer sets designed for amplifying the ITS from nrDNA and the atpB-rbcL spacer from chloroplast DNA (cpDNA) from Phalaenopsis plants as well as the PCR conditions are described in Tsai et al. (2006a) and Tsai et al. (2006b), respectively. These
Table 1 The taxon names, geographical distributions, source information, and GenBank accession numbers for the samples used in this study. Taxa and systematic classification
Distribution
P. P. P. P. P. P. P. P. P. P. P. P. P. P.
sumatrana sumatrana sumatrana sumatrana sumatrana sumatrana corningiana corningiana corningiana corningiana corningiana zebrina zebrina zebrina
Sumatra; Java; Borneo; Sumatra; Java; Borneo; Sumatra; Java; Borneo; Sumatra; Java; Borneo; Sumatra; Java; Borneo; Sumatra; Java; Borneo; Borneo, Sarawak Borneo, Sarawak Borneo, Sarawak Borneo, Sarawak Borneo, Sarawak Borneo, Palawan Borneo, Palawan Borneo, Palawan
a
Kaohsiung District Agricultural Improvement Station.
Mentawei Mentawei Mentawei Mentawei Mentawei Mentawei
Is.; Is.; Is.; Is.; Is.; Is.;
Malaya; Malaya; Malaya; Malaya; Malaya; Malaya;
Perak, Perak, Perak, Perak, Perak, Perak,
Johore; Johore; Johore; Johore; Johore; Johore;
Thailand Thailand Thailand Thailand Thailand Thailand
Sourcea
GenBank accession no. ITS
atpB-rbcL spacer
KDAIS KDAIS KDAIS KDAIS KDAIS KDAIS KDAIS KDAIS KDAIS KDAIS KDAIS KDAIS KDAIS KDAIS
AY390239 AY390240 AY390241 AY390242 AY390243 AY390244 AY390245 AY390246 AY390247 AY390248 AY390249 AY390250 AY390251 AY390252
AY389415 FJ460408 FJ460409 FJ460410 FJ460411 FJ460412 FJ460413 FJ460414 FJ460415 FJ460416 FJ460417 FJ460418 FJ460419 FJ460420
kc-32 kc-160 kc-161 kc-403 kc-404 kc-405 kc-330 kc-345 kc-346 kc-383 kc-384 kc-57 kc-257 kc-231
C.-C. Tsai et al. / Biochemical Systematics and Ecology 37 (2009) 633–639
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Fig. 1. The geographical distributions of P. sumatrana, P. corningiana, and P. zebrina.
PCR products were detected by agarose gel electrophoresis (1.0%, w/v in TBE), stained with 0.5 mg/mL of ethidium bromide, and photographed under UV light exposure. 2.4. DNA recovery and sequencing The PCR products of different DNA fragments from the plant material studied were recovered with Glassmilk (BIO 101, California) and sequenced directly by the dideoxy chain-termination method using an ABI377 automated sequencer with a BigdyeÔ Terminator Cycle Sequencing Ready Reaction Kit (PE Biosystems, California). Sequencing primers were the same as those used for the PCR. These reactions were performed as recommended by the manufacturers. 2.5. Data analyses The boundaries of the ITS regions (including ITS1, 5.8S rDNA, and ITS2) and those of the atpB-rbcL spacer in species of the P. sumatrana complex were determined by comparison to several published sequences in GenBank. The sequences were aligned using the program Clustal W Multiple alignment in BioEdit (Hall, 1999). Maximum parsimony (MP) analyses (Fitch, 1971), using code modified from the Close-Neighbor-Interchange (CNI) algorithm (Rzhetsky and Nei, 1992) as implemented in the DNAMP program, and a maximum likelihood (ML) method (Kishino and Hasegawa, 1989), as implemented in the DNAML program, were conducted using the PHYLIP package version 3.65 (Felsenstein, 2002). Bootstrapping (1000 replicates) was carried out to estimate the support for both the MP and ML topologies (Felsenstein, 1985; Hillis and Bull, 1993). The strict consensus parsimonious tree was then constructed using the PHYLIP package version 3.65. Both the MP and ML trees were drawn with the TREEVIEW program (Page, 1996). The aligned data matrix and tree files are available from the corresponding author ([email protected]). The number of haplotypes was counted with all the polymorphic sites, including indels (insertions/deletions), for all the accessions of the P. sumatrana complex studied. The haplotype diversity (h), nucleotide diversity (p) (Nei, 1987), and Tajima’s D (Tajima, 1989) test for the departure from neutrality on the total number of segregating sites were calculated using the DnaSP program version 2.0 (Rozas et al., 2003). 3. Results and discussion 3.1. Sequence characteristics The accession numbers of the 14 accessions of the P. sumatrana complex are shown in Table 1. The length and sequence of the 5.8S rDNA among the 14 accessions of the P. sumatrana complex were the same. We combined the ITS1 and ITS2 regions, and the resulting alignment contained 497 characters. There were 11 variable sites and five potentially parsimony-informative sites. Five haplotypes were detected among the 14 accessions examined, with a haplotype diversity of 0.5055 and
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a nucleotide diversity of 0.0039. Within species of P. sumatrana, two haplotypes were detected, with a haplotype diversity of 0.3333 and a nucleotide diversity of 0.0005. Only one haplotype was found among the five samples of P. corningiana examined. Within species of P. zebrina, three haplotypes were detected, with a haplotype diversity of 1.0000 and a nucleotide diversity of 0.0061 (Table 2). The sequence lengths of the atpB-rbcL spacer from the 14 accessions of the P. sumatrana complex were the same at 681 bp. The alignment of those sequences resulted in 682 characters. There were two variable sites and two potentially parsimonyinformative. Three haplotypes were detected among the 14 accessions examined, with a haplotype diversity of 0.7143 and a nucleotide diversity of 0.0015. Within the species of P. sumatrana, two haplotypes were detected, with a haplotype diversity of 0.3333 and a nucleotide diversity of 0.0005. Two haplotypes were found among the five samples of P. corningiana, with a haplotype diversity of 0.6000 and a nucleotide diversity of 0.0009. Only one haplotype was found among the five samples of P. zebrina examined (Table 2). 3.2. Phylogenetic reconstructions The phylogenetic tree inferred from the combined ITS1 and ITS2 nrDNA sequences from the 14 accessions of the P. sumatrana complex plus the outgroups, namely P. fuscata and P. gigantea, was reconstructed using the MP and ML methods. Based on the MP method, the analysis yielded 210 equally parsimonious trees with a length of 85 steps, a consistency index (CI) of 1.0, and a retention index (RI) of 1.0. The strict consensus MP tree is shown in Fig. 2. The bootstrap values greater than 50% are shown above the supported branches for MP tree. The ML tree is congruent with the MP strict consensus tree (data not shown). Based on both phylogenetic trees, the accessions of both P. sumatrana and P. corningiana formed a clade with 98% support by the bootstrap test and were separated from P. zebrina. In addition, the accessions of P. zebrina formed the basal group within the P. sumatrana complex. Using the atpB-rbcL spacer sequences, the phylogenetic tree of the 14 accessions of the P. sumatrana complex plus the two outgroups, P. fuscata and P. gigantea, was reconstructed following the MP and ML methods. Based on the MP method, the analysis yielded 230 equally parsimonious trees with a length of 15 steps, a consistency index (CI) of 1.0, and a retention index (RI) of 1.0. The strict consensus tree is shown in Fig. 3. The bootstrap values greater than 50% are shown below/above the supported branches for MP tree. The ML tree is congruent with the MP strict consensus tree (data not shown). Based on the phylogenetic tree, the species of P. sumatrana, P. corningiana, and P. zebrina cannot be separated from one another. Of all the accessions of the P. sumatrana complex, the accessions of P. zebrina were closer to two accessions of P. corningiana, namely P. corningiana-kc-330 and P. corningiana-kc-345, and were separated from the remaining accessions of both species of P. corningiana and P. sumatrana. In addition, one accession of P. sumatrana, namely P. sumatrana-kc-405, was closer to three accessions of P. corningiana, namely P. corningiana-kc-346, P. corningiana-kc-383, and P. corningiana-kc-384. The results indicate that the relationships of the species of the P. sumatrana complex could not be resolved based on the atpB-rbcL spacer phylogeny. Therefore, only the analysis of the ITS from nrDNA offered valuable information for identifying the relationships between the accessions of the P. sumatrana complex, namely the accessions of P. zebrina. Based on the ITS sequences, P. sumatrana and P. corningiana could not be separated from each other. This result does not support P. corningiana and P. sumatrana being treated as two separate species, as described by Sweet (1968), Fowlie (1969), and Christenson (2001). Although characteristics of the callus morphology and marking pattern on the petals could be identified between P. sumatrana and P. corningiana as described by Sweet (1980) and Christenson (2001), we found that these two characteristics of P. sumatrana and P. corningiana were not consistent based on the samples in this study. In our inspection, some accessions of P. corningiana showed a marking pattern with longitudinal stripes toward the apex of sepals and petals, but the callus morphology was similar to that of P. sumatrana, which was biseriate without forming a sunken pit at the base of the callus (data not shown). Furthermore, the differentiation between P. corningiana and P. sumatrana was also proposed based on the floral fragrance (Christenson, 2001). The accessions of P. zebrina in this complex could be separated from both P. sumatrana and P. corningiana based on the ITS sequence of nrDNA. In the analyses of the sequences of the atpB-rbcL spacer, neither species of this complex could be separated from the other. Therefore, the molecular data in this study only support P. zebrina being separated from both P. corningiana and P. sumatrana. This separation was supported by a lower FST value (0.0000) between P. sumatrana and P. corningiana than those between other pairs (0.6667 between P. zebrina and P. sumatrana; 0.6842 between P. zebrina and P. corningiana) based on the ITS sequences. Based on the atpB-rbcL spacer, the FST value (0.9091) between P. sumatrana
Table 2 The species names, sample size, number of haplotypes, haplotype diversity (h), nucleotide diversity (p) accompanied by neutrality test statistics for three species of the P. sumatrana complex based on the ITS sequences and atpB-rbcL spacer sequences (in parenthesis). Species
No. of samples
No. of haplotypes
Polymorphic sites
Parsimony-informative sites
h
P. P. P. P.
14 6 5 3
5 2 1 3
11 (2) 1 (1) 0 (1) 6 (0)
5 0 0 0
0.5055 0.3333 0.0000 1.0000
sumatrana complex sumatrana corningiana zebrina
(3) (2) (2) (1)
(2) (0) (1) (0)
p (0.7143) (0.3333) (0.6000) (0.0000)
0.0039 0.0005 0.0000 0.0061
Tajima’s D (0.0015) (0.0005) (0.0009) (0.0000)
1.01133 (1.55373) 0.93302 (0.93302) – (1.22474) – (–)
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Fig. 2. The MP tree of the 14 accessions from the P. sumatrana complex plus the outgroups, P. maculata and P. gigantea, obtained from sequence comparisons of the ITS region of nrDNA. The numbers above the internodes indicate the values of the bootstrap test from 1000 replicates. Bootstrap values greater than 50% are shown.
and P. zebrina was higher than that between the other pairs (Table 3). This result supports the treatment of P. zebrina described by Fowlie (1982) and Masaaki (2002). However, the floral ground color of P. zebrina is not a unique characteristic of this species as described by Fowlie (1982). Both white and yellowish ground color in the flower of P. zebrina can be found according to our inspection, as described by Masaaki (2002). Based on our inspection of the plants of the P. sumatrana complex for this study, flowers of P. zebrina have a special floral fragrance which smells like that of a cockroach. Therefore, it was easy to separate the plants of P. zebrina from the other two species of this complex based on floral fragrances. Furthermore, all the sepals and petals of these three species are retained after pollination (Christenson, 2001). We found that the red marking pattern of anthocyanin of the retained sepals and petals in both P. zebrina and P. sumatrana was partly visible after pollination, but that of P. corningiana was not. In conclusion, the plants of P. zebrina could be
Fig. 3. The MP tree of the 14 accessions from P. sumatrana complex plus the two outgroups, P. fuscata and P. gigantea, obtained from sequence comparisons of the atpB-rbcL spacer of chloroplast DNA. The numbers above the internodes indicate the values of the bootstrap test from 1000 replicates. Bootstrap values greater than 50% are shown.
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Table 3 The FST values among the species of the P. sumatrana complex based on the ITS and atpB-rbcL spacer sequences. ITS sequence
P. sumatrana P. corningiana P. zebrina
atpB-rbcL spacer
P. sumatrana
P. corningiana
P. sumatrana
P. corningiana
0.0000 0.6667
0.6842
0.6216 0.9091
0.5000
separated from both P. sumatrana and P. corningiana based on floral fragrances and molecular data. Therefore, it is not suitable to treat P. zebrina as a synonym of P. sumatrana as described by Sweet (1968, 1980) and Christenson (2001).
3.3. Biogeography and evolutionary trends Regarding the biogeography of the P. sumatrana complex, the distributions of these three species overlap in Borneo (Fig. 1). This allows a much greater chance for these three species to hybridize in their natural environment, making these three species difficult to discriminate. In fact, some naturally hybridized plants between species of P. sumatrana and P. corningiana have been collected (Christenson, 2001). According to the phylogenetic tree inferred from the ITS sequences, the evolutionary trend of the P. sumatrana complex was deduced. Since the accessions of P. zebrina were located as the basal group within the P. sumatrana complex, P. zebrina was suggested to be the relative origin group of the P. sumatrana complex. The suggestion is partly supported by the analysis of the atpB-rbcL spacer sequences. Based on the evolutionary trend derived from the molecular data, the dispersal pathway of the P. sumatrana complex was deduced and is shown in Fig. 4. According to this model, P. zebrina developed in Borneo and dispersed into Palawan, the Philippines. P. corningiana and P. sumatrana might have evolved from P. zebrina in Borneo. Since then, P. sumatrana dispersed into Sumatra, Malay Peninsula, and the Andaman Is. across land bridges in glacial times. It has been shown to be a widespread species. Land bridges among Sumatra, the Malay Peninsula, and Borneo could have formed, since Borneo, Sumatra, the Malay Peninsula, and Java make up the Sunda Shelf (van Oosterzee, 1997). However, specimens of P. sumatrana have not been found in Java to date. This result is in agreement with the biogeography of the genus Phalaenopsis, with Phalaenopsis species found in Java differing from those of other lands of the Sunda Shelf (Christenson, 2001; Tsai et al., 2003). Furthermore, Andaman Is. might have been interconnected to Sumatra in ancient times based on the evidence from both biogeography and the evolutionary trends of the P. sumatrana complex.
Fig. 4. The evolutionary trend of the P. sumatrana complex based on the phylogenetic tree.
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Acknowledgements This research was supported by funding from the National Science Council, Executive Yuan, Taiwan. We are grateful to Director F. H. Lin for his valuable comments and helpful discussion in the course of the study. References Christenson, E.A., 2001. Phalaenopsis. Timber Press, Portland, Oregon, 330 pp. Doyle, J.J., Doyle, J.L., 1987. A rapid DNA isolation procedure for small quantities of fresh leaf tissue. Phytochem. Bull. 19, 11–15. Felsenstein, J., 1985. Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39, 783–791. Felsenstein, J., 2002. PHYLIP (Phylogeny Inference Package) Version 3.65. Department of Genetics, University of Washington, Seattle, WA. Fitch, W.M., 1971. Towards defining the course of evolution: minimum change for a specific tree topology. Syst. Zool. 20, 406–416. Fowlie, J.A., 1969. A note on Phalaenopsis sumatrana and Phalaenopsis corningiana. Orchid Dig. 33, 301. Fowlie, J.A., 1982. Malaya revised, pt. 21: Phalaenopsis corningiana on saplings and streamside trees near waterfalls. Orchid Dig 46, 138–142. Hall, T.A., 1999. BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucl. Acids Symp. Ser. 41, 95–98. Hillis, D.M., Bull, J.J., 1993. An empirical test of bootstrapping as a method for assessing confidence in phylogenetic analysis. Syst. Biol. 42, 182–192. Kishino, H., Hasegawa, M., 1989. Evaluation of the maximum likelihood estimate of the evolutionary tree topologies from DNA sequence data, and the branching order in Hominoidea. J. Mol. Evol. 29, 170–179. Masaaki, C., 2002. Phalaenopsis Species. Chiba Masaaki Phalaenopsis Species Publishing Society, Saitama-ken, Japan. Nei, M., 1987. Molecular Evolutionary Genetics. Columbia University Press, New York. Page, R.D.M., 1996. TreeView: an application to display phylogenetic trees on personal computers. Comput. Appl. Biosci. 12, 357–358. Rozas, J., Sanchez-DelBarrio, J.C., Messequer, X., Rozas, R., 2003. DnaSp, DNA polymorphism analyses by the coalescent and other methods. Bioinformatics 19, 2496–2497. Rzhetsky, A., Nei, M., 1992. Statistical properties of the ordinary least-squares, generalized least-squares, and minimum-evolution methods of phylogenetic inference. J. Mol. Evol. 35, 367–375. Sweet, H.R., 1968. A revision of the genus Phalaenopsis-II. Am. Orchid Soc. Bull. 37, 1091. Sweet, H.R., 1980. The Genus Phalaenopsis. The Orchid Digest, Pomona, California. Tajima, F., 1989. Statistical method for testing the neutral mutation hypothesis by DNA polymorphism. Genetics 123, 585–595. Tsai, C.C., Huang, S.C., Chou, C.H., 2006a. Molecular phylogeny of Phalaenopsis Blume (Orchidaceae) based on the internal transcribed spacers 1 and 2 of nuclear ribosomal DNA. Plant Syst. Evol. 256, 1–16. Tsai, C.C., Huang, S.C., Huang, P.L., Chen, F.Y., Su, Y.T., Chou, C.H., 2006b. Molecular evidences of a natural hybrid origin of Phalaenopsis intermedia Lindl. J. Hort. Sci. Biotechnol. 81, 691–699. Tsai, C.C., Huang, S.C., Huang, P.L., Chou, C.H., 2003. Phylogeny of the genus Phalaenopsis (Orchidaceae) with emphasis on the subgenus Phalaenopsis based on the sequences of the internal transcribed spacers 1 and 2 of rDNA. J. Hort. Sci. Biotechnol. 78, 879–887. van Oosterzee, P., 1997. Where Worlds Collide: the Wallace Line. Cornell Univ. Press, Ithaca, NY. Wallbrunn, H.M., 1971. A second look at the ‘‘revision of the genus Phalaenopsis’’ Am. Orchid Soc. Bull. 40, 223–231.
Contents lists available at ScienceDirect
Biochemical Systematics and Ecology journal homepage: www.elsevier.com/locate/biochemsyseco
Phylogenetics, biogeography, and evolutionary trends of the Phalaenopsis sumatrana complex inferred from nuclear and chloroplast DNA Chi-Chu Tsai a, b, *, Sheng-Chung Huang c, Chang-Hung Chou d, ** a
Kaohsiung District Agricultural Improvement Station, Pingtung 908, Taiwan National Pingtung University of Science and Technology, Pingtung 912, Taiwan c National Plant Genetic Resources Center, Agricultural Research Institute, Taichung 413, Taiwan d Research Center for Biodiversity, China Medical University, Taichung 404, Taiwan b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 9 February 2009 Accepted 4 September 2009
Phylogenetic trees inferred from the internal transcribed spacers 1 and 2 (ITS1 þ ITS2) region from nuclear ribosomal DNA (nrDNA) and the intergenic spacer of atpB-rbcL from chloroplast DNA (cpDNA) were used to clarify the phylogenetics and evolutionary trends of the Phalaenopsis sumatrana complex. The P. sumatrana complex includes the two species P. sumatrana and Phalaenopsis corningiana as well as a problem species, Phalaenopsis zebrina, according to the concepts of Sweet (1980) and Christenson (2001). Based on the phylogenetic tree inferred from the ITS sequence, the accessions of P. sumatrana cannot be separated from those of P. corningiana. In contrast, the accessions of P. zebrina can be separated from those of both P. sumatrana and P. corningiana. However, analyses of the sequences of the atpB-rbcL spacer apparently cannot discriminate among these three species of the P. sumatrana complex. An inspection of the morphological characters of the plants of the P. sumatrana complex and the floral fragrances of P. zebrina can be used to separate it from both P. sumatrana and P. corningiana. Based on the molecular data and floral fragrances, P. zebrina perhaps should not be treated as a synonym of P. sumatrana. On the evolutionary trend of the P. sumatrana complex, P. zebrina was suggested to be the relative origin group of the P. sumatrana complex based on the phylogenetic tree and biogeography. Ó 2009 Elsevier Ltd. All rights reserved.
Keywords: Phalaenopsis sumatrana Species complex Phylogenetics Biogeography
1. Introduction The Phalaenopsis sumatrana complex includes P. sumatrana and Phalaenopsis corningiana plus one questionable species, Phalaenopsis zebrina. P. sumatrana is widely distributed in Sumatra, Java, Borneo, Mentawei Is, Malaya, Perak, Johore, and Thailand. P. corningiana and P. zebrina have restricted distributions in Borneo/Sarawak and Borneo/Palawan, respectively (Sweet, 1980; Masaaki, 2002). Based on morphology, the characters of these three species of this complex are not easily differentiated. P. zebrina was treated as a synonym of P. sumatrana by Sweet (1968). Fowlie (1982) attempted to raise the taxonomic position of plants in the P. sumatrana complex with a white ground color flower into a separate species, P. zebrina, distinct from plants of P. sumatrana with a yellowish ground color. Christenson (2001), however, disagreed with Fowlie’s separation because the type specimen of P. zebrina had a yellowish ground color. Therefore, P. zebrina is retained as a synonym
* Corresponding author at: Kaohsiung District Agricultural Improvement Station, Pingtung 908, Taiwan. Tel.: þ886 8 7746735; fax: þ886 8 7389106. ** Corresponding author. Tel./fax: þ886 4 22071500. E-mail addresses: [email protected] (C.-C. Tsai), [email protected] (C.-H. Chou). 0305-1978/$ – see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.bse.2009.09.004
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of P. sumatrana. Nevertheless, P. zebrina is still used to represent plants with narrower brown markings on a white or yellowish ground color in horticulture to the present (Masaaki, 2002). Furthermore, Sweet (1968), Fowlie (1969), and Christenson (2001) have the congruent concept of P. sumatrana and P. corningiana as being separate from each other and being treated as two separate species. In addition, confusion has also surrounded plants of P. corningiana and various darker-colored plants of P. sumatrana. Plants of P. corningiana can be separated from those of P. sumatrana based on their callus morphology and the marking pattern on their petals and sepals. The callus morphology of plants of P. corningiana is uniseriate, sulcate, bifid, and, at the base, continuous with a structure analogous to a posterior keel, forming a sunken pit. The callus of P. sumatrana, on the other hand, is biseriate, with the posterior callus being four lobulate at the apex and granular at the base and the anterior callus being longer, sulcate, and bifid. The petal markings of P. corningiana show longitudinal stripes toward the apex of the sepals and petals. In contrast, those of P. sumatrana always show transverse stripes (Sweet, 1968, 1980; Christenson, 2001). However, these different characteristics between P. corningiana and P. sumatrana are still obscured in some individual clones. For example, a solid red clone, P. sumatrana var. sanguinea, has been treated as a synonym of P. corningiana (Sweet, 1980). In addition, the length of the floral inflorescences of P. corningiana (much shorter than the leaves) is considered to be shorter than that in P. sumatrana (Sweet, 1968). Wallbrunn (1971), however, did not accept this concept according to inspections of several living plants of these two species. Furthermore, Christenson (2001) proposed that the floral fragrance is the best character for separating these two sister species. Plants of P. corningiana bear a wonderful scent of spicy candy. Those of P. sumatrana, on the other hand, bear a mildly acrid fragrance. Doubtlessly, species of the P. sumatrana complex, namely P. zebrina, P. sumatrana, and P. corningiana, are closely related. To evaluate the phylogenetics and evolutionary trends of this complex, sequences of the ITS from nrDNA and the atpB-rbcL spacer from chloroplast DNA were analyzed in this study. 2. Materials and methods 2.1. Plant materials Material from 14 accessions of the P. sumatrana complex were selected and used for this study (Table 1). The geographical distribution of each species of this complex is shown in Fig. 1. Furthermore, one species of the section Amboinenses, Phalaenopsis gigantea and one species of the section Fuscatae, Phalaenopsis fuscata, were used as outgroups of the P. sumatrana complex to clarify the evolutionary trend of this complex, since this complex (section Zebrinae) and the Amboinenses and Fuscatae sections (Tsai et al., 2006a) share a common ancestor. 2.2. DNA extraction Total DNA was extracted using the CTAB (cetyltrimethylammonium bromide) method (Doyle and Doyle, 1987). The approximate DNA yields were then determined using a spectrophotometer (Hitachi U-2001). 2.3. PCR amplification and electrophoresis The primer sets designed for amplifying the ITS from nrDNA and the atpB-rbcL spacer from chloroplast DNA (cpDNA) from Phalaenopsis plants as well as the PCR conditions are described in Tsai et al. (2006a) and Tsai et al. (2006b), respectively. These
Table 1 The taxon names, geographical distributions, source information, and GenBank accession numbers for the samples used in this study. Taxa and systematic classification
Distribution
P. P. P. P. P. P. P. P. P. P. P. P. P. P.
sumatrana sumatrana sumatrana sumatrana sumatrana sumatrana corningiana corningiana corningiana corningiana corningiana zebrina zebrina zebrina
Sumatra; Java; Borneo; Sumatra; Java; Borneo; Sumatra; Java; Borneo; Sumatra; Java; Borneo; Sumatra; Java; Borneo; Sumatra; Java; Borneo; Borneo, Sarawak Borneo, Sarawak Borneo, Sarawak Borneo, Sarawak Borneo, Sarawak Borneo, Palawan Borneo, Palawan Borneo, Palawan
a
Kaohsiung District Agricultural Improvement Station.
Mentawei Mentawei Mentawei Mentawei Mentawei Mentawei
Is.; Is.; Is.; Is.; Is.; Is.;
Malaya; Malaya; Malaya; Malaya; Malaya; Malaya;
Perak, Perak, Perak, Perak, Perak, Perak,
Johore; Johore; Johore; Johore; Johore; Johore;
Thailand Thailand Thailand Thailand Thailand Thailand
Sourcea
GenBank accession no. ITS
atpB-rbcL spacer
KDAIS KDAIS KDAIS KDAIS KDAIS KDAIS KDAIS KDAIS KDAIS KDAIS KDAIS KDAIS KDAIS KDAIS
AY390239 AY390240 AY390241 AY390242 AY390243 AY390244 AY390245 AY390246 AY390247 AY390248 AY390249 AY390250 AY390251 AY390252
AY389415 FJ460408 FJ460409 FJ460410 FJ460411 FJ460412 FJ460413 FJ460414 FJ460415 FJ460416 FJ460417 FJ460418 FJ460419 FJ460420
kc-32 kc-160 kc-161 kc-403 kc-404 kc-405 kc-330 kc-345 kc-346 kc-383 kc-384 kc-57 kc-257 kc-231
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Fig. 1. The geographical distributions of P. sumatrana, P. corningiana, and P. zebrina.
PCR products were detected by agarose gel electrophoresis (1.0%, w/v in TBE), stained with 0.5 mg/mL of ethidium bromide, and photographed under UV light exposure. 2.4. DNA recovery and sequencing The PCR products of different DNA fragments from the plant material studied were recovered with Glassmilk (BIO 101, California) and sequenced directly by the dideoxy chain-termination method using an ABI377 automated sequencer with a BigdyeÔ Terminator Cycle Sequencing Ready Reaction Kit (PE Biosystems, California). Sequencing primers were the same as those used for the PCR. These reactions were performed as recommended by the manufacturers. 2.5. Data analyses The boundaries of the ITS regions (including ITS1, 5.8S rDNA, and ITS2) and those of the atpB-rbcL spacer in species of the P. sumatrana complex were determined by comparison to several published sequences in GenBank. The sequences were aligned using the program Clustal W Multiple alignment in BioEdit (Hall, 1999). Maximum parsimony (MP) analyses (Fitch, 1971), using code modified from the Close-Neighbor-Interchange (CNI) algorithm (Rzhetsky and Nei, 1992) as implemented in the DNAMP program, and a maximum likelihood (ML) method (Kishino and Hasegawa, 1989), as implemented in the DNAML program, were conducted using the PHYLIP package version 3.65 (Felsenstein, 2002). Bootstrapping (1000 replicates) was carried out to estimate the support for both the MP and ML topologies (Felsenstein, 1985; Hillis and Bull, 1993). The strict consensus parsimonious tree was then constructed using the PHYLIP package version 3.65. Both the MP and ML trees were drawn with the TREEVIEW program (Page, 1996). The aligned data matrix and tree files are available from the corresponding author ([email protected]). The number of haplotypes was counted with all the polymorphic sites, including indels (insertions/deletions), for all the accessions of the P. sumatrana complex studied. The haplotype diversity (h), nucleotide diversity (p) (Nei, 1987), and Tajima’s D (Tajima, 1989) test for the departure from neutrality on the total number of segregating sites were calculated using the DnaSP program version 2.0 (Rozas et al., 2003). 3. Results and discussion 3.1. Sequence characteristics The accession numbers of the 14 accessions of the P. sumatrana complex are shown in Table 1. The length and sequence of the 5.8S rDNA among the 14 accessions of the P. sumatrana complex were the same. We combined the ITS1 and ITS2 regions, and the resulting alignment contained 497 characters. There were 11 variable sites and five potentially parsimony-informative sites. Five haplotypes were detected among the 14 accessions examined, with a haplotype diversity of 0.5055 and
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a nucleotide diversity of 0.0039. Within species of P. sumatrana, two haplotypes were detected, with a haplotype diversity of 0.3333 and a nucleotide diversity of 0.0005. Only one haplotype was found among the five samples of P. corningiana examined. Within species of P. zebrina, three haplotypes were detected, with a haplotype diversity of 1.0000 and a nucleotide diversity of 0.0061 (Table 2). The sequence lengths of the atpB-rbcL spacer from the 14 accessions of the P. sumatrana complex were the same at 681 bp. The alignment of those sequences resulted in 682 characters. There were two variable sites and two potentially parsimonyinformative. Three haplotypes were detected among the 14 accessions examined, with a haplotype diversity of 0.7143 and a nucleotide diversity of 0.0015. Within the species of P. sumatrana, two haplotypes were detected, with a haplotype diversity of 0.3333 and a nucleotide diversity of 0.0005. Two haplotypes were found among the five samples of P. corningiana, with a haplotype diversity of 0.6000 and a nucleotide diversity of 0.0009. Only one haplotype was found among the five samples of P. zebrina examined (Table 2). 3.2. Phylogenetic reconstructions The phylogenetic tree inferred from the combined ITS1 and ITS2 nrDNA sequences from the 14 accessions of the P. sumatrana complex plus the outgroups, namely P. fuscata and P. gigantea, was reconstructed using the MP and ML methods. Based on the MP method, the analysis yielded 210 equally parsimonious trees with a length of 85 steps, a consistency index (CI) of 1.0, and a retention index (RI) of 1.0. The strict consensus MP tree is shown in Fig. 2. The bootstrap values greater than 50% are shown above the supported branches for MP tree. The ML tree is congruent with the MP strict consensus tree (data not shown). Based on both phylogenetic trees, the accessions of both P. sumatrana and P. corningiana formed a clade with 98% support by the bootstrap test and were separated from P. zebrina. In addition, the accessions of P. zebrina formed the basal group within the P. sumatrana complex. Using the atpB-rbcL spacer sequences, the phylogenetic tree of the 14 accessions of the P. sumatrana complex plus the two outgroups, P. fuscata and P. gigantea, was reconstructed following the MP and ML methods. Based on the MP method, the analysis yielded 230 equally parsimonious trees with a length of 15 steps, a consistency index (CI) of 1.0, and a retention index (RI) of 1.0. The strict consensus tree is shown in Fig. 3. The bootstrap values greater than 50% are shown below/above the supported branches for MP tree. The ML tree is congruent with the MP strict consensus tree (data not shown). Based on the phylogenetic tree, the species of P. sumatrana, P. corningiana, and P. zebrina cannot be separated from one another. Of all the accessions of the P. sumatrana complex, the accessions of P. zebrina were closer to two accessions of P. corningiana, namely P. corningiana-kc-330 and P. corningiana-kc-345, and were separated from the remaining accessions of both species of P. corningiana and P. sumatrana. In addition, one accession of P. sumatrana, namely P. sumatrana-kc-405, was closer to three accessions of P. corningiana, namely P. corningiana-kc-346, P. corningiana-kc-383, and P. corningiana-kc-384. The results indicate that the relationships of the species of the P. sumatrana complex could not be resolved based on the atpB-rbcL spacer phylogeny. Therefore, only the analysis of the ITS from nrDNA offered valuable information for identifying the relationships between the accessions of the P. sumatrana complex, namely the accessions of P. zebrina. Based on the ITS sequences, P. sumatrana and P. corningiana could not be separated from each other. This result does not support P. corningiana and P. sumatrana being treated as two separate species, as described by Sweet (1968), Fowlie (1969), and Christenson (2001). Although characteristics of the callus morphology and marking pattern on the petals could be identified between P. sumatrana and P. corningiana as described by Sweet (1980) and Christenson (2001), we found that these two characteristics of P. sumatrana and P. corningiana were not consistent based on the samples in this study. In our inspection, some accessions of P. corningiana showed a marking pattern with longitudinal stripes toward the apex of sepals and petals, but the callus morphology was similar to that of P. sumatrana, which was biseriate without forming a sunken pit at the base of the callus (data not shown). Furthermore, the differentiation between P. corningiana and P. sumatrana was also proposed based on the floral fragrance (Christenson, 2001). The accessions of P. zebrina in this complex could be separated from both P. sumatrana and P. corningiana based on the ITS sequence of nrDNA. In the analyses of the sequences of the atpB-rbcL spacer, neither species of this complex could be separated from the other. Therefore, the molecular data in this study only support P. zebrina being separated from both P. corningiana and P. sumatrana. This separation was supported by a lower FST value (0.0000) between P. sumatrana and P. corningiana than those between other pairs (0.6667 between P. zebrina and P. sumatrana; 0.6842 between P. zebrina and P. corningiana) based on the ITS sequences. Based on the atpB-rbcL spacer, the FST value (0.9091) between P. sumatrana
Table 2 The species names, sample size, number of haplotypes, haplotype diversity (h), nucleotide diversity (p) accompanied by neutrality test statistics for three species of the P. sumatrana complex based on the ITS sequences and atpB-rbcL spacer sequences (in parenthesis). Species
No. of samples
No. of haplotypes
Polymorphic sites
Parsimony-informative sites
h
P. P. P. P.
14 6 5 3
5 2 1 3
11 (2) 1 (1) 0 (1) 6 (0)
5 0 0 0
0.5055 0.3333 0.0000 1.0000
sumatrana complex sumatrana corningiana zebrina
(3) (2) (2) (1)
(2) (0) (1) (0)
p (0.7143) (0.3333) (0.6000) (0.0000)
0.0039 0.0005 0.0000 0.0061
Tajima’s D (0.0015) (0.0005) (0.0009) (0.0000)
1.01133 (1.55373) 0.93302 (0.93302) – (1.22474) – (–)
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Fig. 2. The MP tree of the 14 accessions from the P. sumatrana complex plus the outgroups, P. maculata and P. gigantea, obtained from sequence comparisons of the ITS region of nrDNA. The numbers above the internodes indicate the values of the bootstrap test from 1000 replicates. Bootstrap values greater than 50% are shown.
and P. zebrina was higher than that between the other pairs (Table 3). This result supports the treatment of P. zebrina described by Fowlie (1982) and Masaaki (2002). However, the floral ground color of P. zebrina is not a unique characteristic of this species as described by Fowlie (1982). Both white and yellowish ground color in the flower of P. zebrina can be found according to our inspection, as described by Masaaki (2002). Based on our inspection of the plants of the P. sumatrana complex for this study, flowers of P. zebrina have a special floral fragrance which smells like that of a cockroach. Therefore, it was easy to separate the plants of P. zebrina from the other two species of this complex based on floral fragrances. Furthermore, all the sepals and petals of these three species are retained after pollination (Christenson, 2001). We found that the red marking pattern of anthocyanin of the retained sepals and petals in both P. zebrina and P. sumatrana was partly visible after pollination, but that of P. corningiana was not. In conclusion, the plants of P. zebrina could be
Fig. 3. The MP tree of the 14 accessions from P. sumatrana complex plus the two outgroups, P. fuscata and P. gigantea, obtained from sequence comparisons of the atpB-rbcL spacer of chloroplast DNA. The numbers above the internodes indicate the values of the bootstrap test from 1000 replicates. Bootstrap values greater than 50% are shown.
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Table 3 The FST values among the species of the P. sumatrana complex based on the ITS and atpB-rbcL spacer sequences. ITS sequence
P. sumatrana P. corningiana P. zebrina
atpB-rbcL spacer
P. sumatrana
P. corningiana
P. sumatrana
P. corningiana
0.0000 0.6667
0.6842
0.6216 0.9091
0.5000
separated from both P. sumatrana and P. corningiana based on floral fragrances and molecular data. Therefore, it is not suitable to treat P. zebrina as a synonym of P. sumatrana as described by Sweet (1968, 1980) and Christenson (2001).
3.3. Biogeography and evolutionary trends Regarding the biogeography of the P. sumatrana complex, the distributions of these three species overlap in Borneo (Fig. 1). This allows a much greater chance for these three species to hybridize in their natural environment, making these three species difficult to discriminate. In fact, some naturally hybridized plants between species of P. sumatrana and P. corningiana have been collected (Christenson, 2001). According to the phylogenetic tree inferred from the ITS sequences, the evolutionary trend of the P. sumatrana complex was deduced. Since the accessions of P. zebrina were located as the basal group within the P. sumatrana complex, P. zebrina was suggested to be the relative origin group of the P. sumatrana complex. The suggestion is partly supported by the analysis of the atpB-rbcL spacer sequences. Based on the evolutionary trend derived from the molecular data, the dispersal pathway of the P. sumatrana complex was deduced and is shown in Fig. 4. According to this model, P. zebrina developed in Borneo and dispersed into Palawan, the Philippines. P. corningiana and P. sumatrana might have evolved from P. zebrina in Borneo. Since then, P. sumatrana dispersed into Sumatra, Malay Peninsula, and the Andaman Is. across land bridges in glacial times. It has been shown to be a widespread species. Land bridges among Sumatra, the Malay Peninsula, and Borneo could have formed, since Borneo, Sumatra, the Malay Peninsula, and Java make up the Sunda Shelf (van Oosterzee, 1997). However, specimens of P. sumatrana have not been found in Java to date. This result is in agreement with the biogeography of the genus Phalaenopsis, with Phalaenopsis species found in Java differing from those of other lands of the Sunda Shelf (Christenson, 2001; Tsai et al., 2003). Furthermore, Andaman Is. might have been interconnected to Sumatra in ancient times based on the evidence from both biogeography and the evolutionary trends of the P. sumatrana complex.
Fig. 4. The evolutionary trend of the P. sumatrana complex based on the phylogenetic tree.
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