Advanced Studies in Biology, Vol. 5, 2013, no. 7, 337 - 346 HIKARI Ltd, www.m-hikari.com http://dx.doi.org/10.12988/asb.2013.3315

Molecular Phylogeny of the Endangered Vietnamese

Paphiopedilum Species Based on the Internal Transcribed

Spacer of the Nuclear Ribosomal DNA

K. H. Trung1, T. D. Khanh2 , L. H. Ham2, T. D. Duong2 and N. T. Khoa2

1Genetic Engineering Lab, Agricultural Genetics Institute, Hanoi, Vietnam

2Molecular Biology Division, Agricultural Genetics Institute, Hanoi, Vietnam Corresponding author: [email protected]

Copyright © 2013 K. H. Trung et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

We have sequenced the internal transcribed spacer (ITS) region (including ITS1, 5.8S, and ITS2) of 16 species and two varieties of the slipper orchid genus Paphiopedilum in Vietnam. The identification of relationship between those species was also assessed using molecular tool. Most of slipper orchid species in Vietnam are now endangered or threatened to extinction due high demand of ornamental trade of these beautiful species. However, it is difficult to distinguish them without their flowers. Therefore, it is needed to develop molecular markers to accurately identify these species. Our present study is in the view to set the first steps in constructing a molecular database of slipper orchids for future assessment of inter- and intra-specific molecular diversity of Paphiopedilum species from Vietnam, to contribute to the conservation of these valuable orchids. The results showed that highly variable areas (that contain many indels) in ITS region are useful for phylogenetic analysis as the dataset containing these long-indels areas and gave better Jack-knife supports in resolving the relationships of Paphiopedilum species than the modified dataset did.

Keywords: Paphiopedilum, slipper orchids, Vietnam, internal transcribed spacer region

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1. Introduction

The genus Paphiopedilum, a genus first narrated by E.Pfitzer in 1886 [1] comprises approximately 80 species, and is the largest one in the sub-family Cypripedioideae, which is referred as slipper orchids. This genus has been distributed from India, to Eastern and Southeastern in Asia. In Vietnam, there are 18 species (four endemic) and seven varieties recorded [2]. For a long time, the slipper orchids have been attracted from collectors and florists due to their magnificent beauty and high commercial value. However, it has brought up concerns about the conservation of these slipper orchids. Most of them are now endangered due to over exploitation by orchid collectors, traders etc [2]. Many of slipper orchid species are not easy to separate without their flowers. This causes difficulties in identifying them in monitoring the orchid trade (many of slipper orchids are now protected under CITES) and in conservation activities. In Vietnam, traditionally, the botanists have identified and classified the relatedness of orchid species based on morphological and cytological characteristics. However, these methods are limited by environmental effects and diagnostic resolution. Molecular markers may be a feasible method to distinguish the related plants [3-4]. In order to build the molecular database for the conservation and molecular identification of slipper orchids, we have been investigating genetic diversity of this genus in Vietnam. The internal transcribed spacer (ITS) region of 18S-26S nuclear ribosomal DNA (including ITS1, 5.8S and ITS2) has been popularly used in plant systematic to reconstruct phylogenetic relationships at different taxonomic levels [5-9]. The ITS region is claimed to be useful for low-level phylogenetic analysis, such as infra-generic level, due to its relatively fast rate of evolution [10-11]. On the other hand, ITS is one of the most extensively used molecular markers for angiosperm phylogenetic inference and genetic relatedness and is a proven useful source of character for phylogenetic studies in may orchids [12-14, 4]. Specifically, ITS sequences have shown as molecular evidence to evaluate the phylogeny of several taxonomic groups, e.g. in the studies of the subfamily Cypripedioideae [15], of the sub-family Apostasioideae [16] and of the genus Phalaenopsis [17]. The aim of the present study was to investigate genetic variability and relationship among several Vietnamese slipper orchid species by sequencing the internal transcribed spacer (ITS) region.. We obtained the sequences of ITS region of Paphiopedilum species in Vietnam as initial steps in building molecular database of Vietnamese slipper orchids. We have observed in aligned sequences certain highly variable areas with many insertion-deletions (indels). Hence, we also accessed the effects of these highly variable areas in ITS region in analysing the relationships among the studied species.

2. Materials and Methods

Plant Materials

Sixteen species and two varieties of Paphiopedilum from Vietnam are included in the present study. Information about plant materials, including localities of collection, geographic distribution, and numbers of Genbank accession are presented in Table 1. Three

Molecular phylogeny of the endangered Vietnamese Paphiopedilum species 339

representatives of closest genera in the sub-family Cypripedioideae were used as outgroups for cladistic analysis. They are Cypripedium guttatum, Phragmipedium lindleyanum, and Mexipedium xerophyticum. Outgroups’ ITS sequences were downloaded from Genbank (Table 1).

Table 1: List of species used in analyses, locality information, geographic distribution and ITS sequence details.

Species Locality data Voucher Geographic Sequence Genbank distribution lengths Accession No. Paphiopedilum Khanh Hoa HD8 Thailand, Laos, 662 bp GU461596.1 appletonianum (Gower) Cambodia, southern Rolfe Vietnam, China (Hainan island) P. callosum (Rchb.f.) Lam Dong HD5 Thailand, Laos, 662 bp GU461597.1 Stein Cambodia, southern Vietnam P. concolor (Bateman) Lang Son TD10 Myanmar, southern 687 bp GU461599.1 Pfitzer China, Laos, Thailand, northern Vietnam P. delenatii Guillaum Khanh Hoa HD10 Southern Vietnam: 750 bp GU461600.1 Dak Lak, Khanh Hoa (endemic) P. dianthum Tang & Lao Cai HD18 Southern China, 656 bp GU461601.1 Wang northern Vietnam P. emersonii Koop. & Bac Kan HD2 Southern China, 705 bp GU993841.1 Cribb northern Vietnam (Tuyen Quang) P. gratrixianum Vinh Phuc TD55 Laos, northern 659 bp GU993842.1 (Masters) Rolfe (Tam Dao) Vietnam P. hangianum Perner & Ha Giang HD1 Southern China, 700 bp GU993843.1 Gruss northern Vietnam (Tuyen Quang) P. henryanum Braem Ha Giang HD15 Southern China, 698 bp GU993845.1 northern Vietnam (Ha Giang) P. hirsutissimum (Lindl. Lang Son HD7 India, Myanmar, 720 bp GU993847.1 ex Hook.) Stein southern China, Laos, northern Vietnam P. malipoense Chen & Son La HD13 Southern China, 675 bp GU993848.1 Tsi northern Vietnam P. malipoense var. jackii Tuyen Quang HD12 China (Yunnan), 698 bp HQ123428.1 (Hua) Aver. northern Vietnam (Tuyen Quang) P. malipoense var. Ha Giang HD4 China (Yunnan), 720 bp HQ123427.1 hiepii (Aver.) Cribb northern Vietnam (Tuyen Quang) P. micranthum Tang & Tuyen Quang HD9 Southern China, 732 bp GU993849.1 Wang northern Vietnam (Tuyen Quang)

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Table 1 (continued): List of species used in analyses, locality information, geographic distribution and ITS sequence details.

P. purpuratum (Lindl.) Lam Dong HD6 Southeastern China, 662 bp GU993850.1 Stein Vietnam P. tranlienianum Gruss Bac Kan HD11 Northern Vietnam: 659 bp GU461602.1 & Perner Tuyen Quang, Thai Nguyen, Cao Bang, Bac Can (endemic) P. vietnamense Gruss & Thai Nguyen LH Northern Vietnam: 674 bp GU461603.1 Perner Cao Bang, Tuyen Quang, Thai Nguyen P. villosum (Lindl.) Khanh Hoa HD19 India, Myanmar, 659 bp GU993851.1 Stein southern China, Thailand, Laos, Vietnam Cypripedium guttatum see Cox et al. USA, Russia, 730 bp Z78526.1 Cw. (1997) China, Japan, India, Korea, Nepal, Bhutan Phragmipedium see Cox et al. Brazil, French 727 bp Z78506.1 lindleyanum (Lindl.) (1997) Guiana, Guyana, Rolfe Suriname, Venezuela Mexipedium see Cox et al. Mexico 765 bp Z78515.1 xerophyticum (Soto (1997) Arenas, Salazar & Hágsater) Albert & Chase

Total DNA extraction, PCR and DNA sequencing

Total genomic DNA was extracted from the fresh leaves by using the CTAB (cetyltrimethylammonium bromide) extraction protocol method of Doyle and Doyle (1987) [18]. The approximate DNA yields were then determined using a spectrophotometer [19]. PCRs and DNA sequencing: nuclear ribosomal Internal Transcribed Spacers including the ITS1, 5.8S and ITS2 regions were amplified using primers IT1 (5'- AGTCGTAACAAGGTTTCC-3') and IT2 (5'-GTAAGTTTCTTCTCCTCC-3') designed by Tsai (2003) [17]. Amplification was conducted in a polymerase chain reaction (PCR) containing 2.5 units of the Taq-polymerase (Promega, Madison, WI) and 200 ng of the two primers. Amplification reactions were performed in two-step thermal cycles. The first step was carried out for 10 cycles in conditions with denaturation at 94°C for 30s, annealing at 63°C for 20s and extension at 72°C for 60s. The second step was carried out for 30 cycles in conditions with denaturation at 94°C for 30s, annealing at 57°C for 20s and extension at 72°C for 60s, and final extension at 72°C for 7min. Amplified double stranded DNA fragment (- 750bp) were purified using the "Wizard" DNA cleanup system (Promega, Madison, WI) and directly sequenced on an ABI 373A automated sequencer using standard dye-terminator chemistry following manufacturer's protocols (Applied Biosystems Inc.).

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Sequence analyses

Sequences obtained were corrected by using Codon Code Aligner version 3.5 (CodonCode Corp., Massachusetts). Sequences then were aligned using ClustalX version 2.0.12 [20] before being manually aligned in MEGA version 4.0 [21], especially for indels areas. Because we observed the certain segments of ITS sequences with long indels, we have examined the effects of including and excluding these long indels on the outcome of cladistic analysis. First, we used online version of Gblocks [22-23] for an alignment refinement to remove those long indels, under less stringent parameters. Both the original dataset (after alignment in Clustal X and manual alignment) and the modified dataset with long indels removed were analyzed under parsimony using “New Technology search” in TNT version 1.1 [24]. Trailing gaps were treated as “missing”, internal gaps were treated as “fifth character state”. Internal gaps are treated as fifth character state (as default setting in TNT) instead of being treated as “missing”, because indels may provide additional information for phylogenetic inference [25]. The Jack-knife analysis, with 36% cut-offs and 1000 replicates, was used to assess the support of each dataset to the most parsimonious trees.

3. Results and Discussion

Sequence alignment

After the default alignment in Clustal X and manual alignment, lengths of aligned sequences varied from 731 to 754 bp, inclusive of internal gaps. This result revealed containing 21 taxa and 754 characters in a dataset with external gaps (trailing gaps) coded as missing characters “?”. In this dataset, 286 characters are constant, 253 variable characters are parsimony- uninformative, and 215 characters are parsimony-informative. We observed in aligned sequences few highly variable areas containing many indels: an area of ca. 90 bp in length (from site 196 to site 286) and few smaller areas (with length ≤10 bp) scattered in the sequenced region. Notably within the long-indels area, there were four areas of around 5-10 bp could not be aligned unambiguously: they were at sites 208-287, sites 205-209, sites 224- 232, and sites 268-271 (Figure. 1). These highly variable areas indicated relatively fast rate of evolution. Thus, it is uncertain if these areas are useful to resolve the relationships among studied taxa, because they may contain misleading signals. Therefore, we have performed a re-finement alignment to remove these long indels from the original dataset using online Gblocks. The alignment in Gblocks resulted in a modified dataset containing 648 characters, of which 274 are constant, 225 are parsimony-uninformative, and 149 are parsimony- informative.

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Figure. 1: Aligned sequences of ITS region with long-indels area (in parentheses) in the ITS sequences of Paphiopedilum

Parsimony analysis

The parsimony analyses using New Technology search resulted in one of the most parsimonious tree from each data set (tree scores: 830 for tree produced by original dataset, 620 for tree produced by modified dataset). The topology of two trees generally agreed, except for the clade containing five taxa, P. hangianum, P. emersonii, P. delenatii, P. vietnamense, and P. micranthum (Figure. 2).

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Figure. 2: Most parsimonious trees produced from parsimony analysis based on original dataset (A) and modified dataset (B) with Jack-knife supports (%): values above branches are supports of the original dataset, values below branches are supports of the modified dataset.

Results from Jack-knife analyses showed that original dataset gave better supports than the modified dataset did. Especially, at the clade of five taxa above, the modified dataset has been poorly supported, with Jack-knife supports below 50%. This indicates that areas containing many indels which hold useful information to resolve relationships among Paphiopedilum taxa. The ITS region is a non-coding region, thus, it is not surprising to find highly variable areas there. These highly variable areas may not be useful to study the phylogenetic relationships of high-level taxa, but could be a good source to investigate phylogenetic relationships at lower levels, like intra-generic levels. Our observation agreed with several studies that claimed ITS region was useful in resolving relationships at low phylogenetic levels [10-11, 26]. To date, Sun et al. (2011) [12] applied rDNA-ITS (internal transcribed spacer) sequences to design species-specific SCAR (sequence characterized amplified regions) markers to distinguished P.micranthum, P.delenatii and their respective hybrids. Moreover, Yao et al. (2010) [27] based on the analysis of ITS2 sequences of 85 paphiopedilum species and 76.6% of paphiopedilum species could be resolved.

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Based on the alignment results, we propose that ITS region could be useful in study intra- specific taxa, i.e. varieties. However, we found that ITS sequences of P. malipoense and its two varieties, P. malipoense var. jackii and P. malipoense var. hiepii, were identical, except that the sequence of P. malipoense var. hiepii, at one end was 18 bp longer than those of P. malipoense and P. malipoense var. jackii because the sequencer managed to obtain a longer sequence for the former. Thus, these three taxa were always grouped together in the parsimony analysis, with Jack-knife supports at 100% at the clade containing them, in both datasets, but the relationships within the clade were not resolved. This finding supported the con-specificity of these varieties, but not their intra-specific differences at the molecular level. However, it is premature to give the conclusion about the potential use of ITS region for separating taxa below species level. Note that the results were only based on single-ton representatives. Series of samples for these varieties are needed to re-examine this ITS region and population analysis are suggested in order to confirm the taxonomy of this species. Our results were congruent with the previous study of Yuan et al. (2009) [28] whom reported the genetic relationship among 36 Dendrobium species in China including two different species based on sequences analysis of the rDNA ITS region.

4. Conclusion

The ITS region is the possible marker to evaluate the relationships of Vietnamese Paphiopedilum species. However, this paper has only established the first steps in constructing a molecular database of slipper orchids for future assessment of inter- and intra- specific molecular diversity of Paphiopedilum species from Vietnam. Our subsequent studies would extend to other molecular markers, and focus on population study in order to re- examine the systematics of below-species level taxa in Paphiopedilum, especially varieties of species occurring in Vietnam. Another focal point in our future studies is to find specific markers to fill the need of a molecular tool in identifying Vietnamese Paphiopedilum species and varieties that are difficult to be determined without their flowers.

Acknowledgements The first author thanks his colleagues in the Genetic Engineering Lab, Agricultural Genetics Institute of Vietnam for helping with the collection of the orchid samples. The study is a part of the project KC.04/06-10 funded by the National Key Program of Science and Technology Biotechnology, the office of National Science & Technology Research Program, Ministry of Science and Technology, Vietnam.

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Received: March 7, 2013