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Molecular systematics of genus Bulbophyllum (Orchidaceae) in Peninsular Malaysia based on combined nuclear and plastid DNA sequences
Biochemical Systematics and Ecology 65 (2016) 40e48
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Biochemical Systematics and Ecology journal homepage: www.elsevier.com/locate/biochemsyseco
Molecular systematics of genus Bulbophyllum (Orchidaceae) in Peninsular Malaysia based on combined nuclear and plastid DNA sequences Shahla Hosseini a, *, Kourosh Dadkhah b, Rusea Go c a
Department of Biological Science and Biotechnology, University of Kurdistan, P.O. Box 416, Sanandaj, Iran Department of Statistics, Faculty of Science, University of Kurdistan, P.O. Box 416, Sanandaj, Iran c Department of Biology, University Putra Malaysia, 43400 UPM Serdang, Selangor Darul Ehsan, Malaysia b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 21 July 2015 Received in revised form 4 January 2016 Accepted 31 January 2016 Available online xxx
Phylogenetic relationships were inferred for representative Bulbophyllum species of 13 sections from subtribe Bulbophyllinae (Epidendroideae, Orchidaceae) in Peninsular Malaysia. The combined data matrix consists of sequences from ITS nuclear gene region and trnL-F, matK, and rbcL plastid gene regions with 3114 characters. Molecular data were analysed using parsimony and Bayesian inference. The results show that several recognized sections are monophyletic. Section Hirtula with paraphyletic status must split up and section Desmosanthes contain misplaced elements. Furthermore, generic status of Cirrhopetalum and Epicrianthes cannot be supported, because they are deeply embedded within the genus Bulbophyllum. Section Desmosanthes is recognized as the closest group to section Cirrhopetalum; therefore, they can be merged in some aspects. © 2016 Elsevier Ltd. All rights reserved.
Keywords: Bulbophyllum Monophyly Orchidaceae Peninsular Malaysia Phylogenetic
1. Introduction Bulbophyllum Thouars is the largest genus of the pantropical subtribe Bulbophyllinae and comprises approximately 2032 species (Frodin, 2004). Sieder (2007) reported 184 species with 48 endemic species from Malaysia. It is mostly an epiphytic species found in different habitats, ranging from (sub) tropical dry forests to wet montane cloud forests and most of them are adapted to fly pollination (Nishida et al., 2004; Teixeira et al., 2004). They are distributed in the most northern parts (Perlis) to the most southern parts (Johor) in Peninsular Malaysia and their geography can range from lowland to highland areas like Cameroon and Genting Highlands. In recent years, several molecular phylogenetic studies of orchids have been published (Van Den Berg et al., 2005; Carlsward et al., 2006) placing genus Bulbophyllum as sister to the genus Dendrobium within tribe Dendrobieae in the higher Epidendroids clade of subfamily Epidendroideae. Orchids of the genus Bulbophyllum are one of the important plants in Malaysia in terms of their abundance, but identification of Bulbophyllum at species level still remains a problem for practising taxonomists. Uncertainty in taxonomic status of the Bulbophyllum is a major problem which requires molecular taxonomic revision. Attempts to split up this gargantuan genus into smaller, more manageable units have generally faltered due to the fact that most of the main groups differ only in rather loose combinations of characters. In every section, there were species that do not
* Corresponding author. E-mail addresses: [email protected] (S. Hosseini), [email protected] (K. Dadkhah), [email protected] (R. Go). http://dx.doi.org/10.1016/j.bse.2016.01.003 0305-1978/© 2016 Elsevier Ltd. All rights reserved.
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possess all the diagnostic characters of that section, and yet seem to belong in it; conversely, species that seemingly belong to a certain group based on its diagnostic characters sometimes show other characters that contradict the affinity (Schuiteman et al., 2010). Such genera as Cirrhopetalum have been split off, but based on molecular studies of matK sequences analysis (Gravendeel et al., 2004; Hosseini et al., 2012), generic status of Cirrhopetalum failed. In this study, analysis of some Bulbophyllum species with putative out groups was performed on 4 DNA regions: plastid trnL-F spacer, matK gene, rbcL gene, and the ITS data. In this paper, the main objectives were to clarify the internal topology within Bulbophyllum spp., to test the monophyly of studied taxa, and to explicate the generic status of sections Cirrhopetalum and Epicrianthes based on reconstructed multi-gene based phylogeny. 2. Materials and methods 2.1. Taxon sampling Sampling of species required for this study was one of the greatest challenges faced. It was not possible to cover every part of the forested area in Peninsular Malaysia during this study period. Nevertheless, areas of earlier collection site were visited for possible sampling. However, not all the previous collection sites are forested areas. At least 50% of the recorded collection sites are now palm oil plantation area. Therefore, it was not possible to obtain some of the species required for this study. Some of the collected samples were without flower, so the correct name of these species could not be declared. In this case, blooming season needs to be waited for or the plants should be placed in green house. However, some species in the new conditions could not survive and some of them never flower even after three years in the green house. Species representing 13 sections of Peninsular Malaysian Bulbophyllum, as described by Seidenfaden and Wood (1992), were sampled (Table 1). For some sections, it was not possible to identify all the accessions to species or to apply a valid name based on only vegetative characters, so they appeared as B. sp1- B. sp9. Living samples were collected in Peninsular Malaysia in the wild or taken from plants in the green house of University Putra Malaysia. Herbarium dried samples from the herbarium in Biology Department of University Putra Malaysia were also used for some species. Voucher specimens for all accessions were deposited in herbarium of Biology Department, University Putra Malaysia (UPM). Three taxa (Dendrobium rosellum, Dendrobium pahangensis and Dendrochilum pallideflavens) were selected as the out group. 2.2. DNA extraction, amplification, and sequencing DNA was extracted from the herbarium or fresh material using Wizard® Genomic DNA Purification Kit (Promega). Polymerase chain reaction (PCR) amplification was performed using Bioline Biolasetaq (5 U/ul), Bioline 10 biolase buffer, dNTPs (10 mM) following the manufacturer's protocol and 2e8 ng template DNA for a 50 ml reaction mixture. The Primers 26SE and 17SE of Sun et al. (1994) were used for the amplification of the nrITS regions with the following PCR program: an initial 2 min premelt at 94 C and 34 cycles of 1 min denaturation at 94 C, 1 min annealing at 50 C and 1 min extension at 72 C followed by a final extension for 7 min. For sequencing, the primers ITS4 and ITS5 (White et al., 1990) were used. The cpDNA trnLetrnF intron was amplified using forward primer designed by Fischer et al. (2007) and universal reverse primer designed by Taberlet et al. (1991). The rbcL and matK chloroplast region were amplified using the designed primers pair by David Erickson and Ki-Joong Kim, respectively, from the Consortium for the Barcode of Life (CBOL, 2009). These primers were amplified partial sequences of rbcL and matK with length of 500e600 bp and 1000 bp, respectively. Amplification of cpDNA was carried out with an initial 3 min premelt at 94 C and 30 cycles of 30 s denaturation at 94 C, 30 s annealing at 55 C and 1 min 40 s extension at 72 C followed by a final extension for 7 min at 72 C. DNA was purified from gel slice or PCR product by Wizard® SV Gel and PCR clean-up system (Promega) following the manufacturer's protocol. All DNA sequences produced for this study were submitted to GenBank. For several species, it was not possible to amplify all the molecular markers. 2.3. DNA sequence alignment Multiple sequence alignments were performed using Clustal W (Thompson et al., 1994) with default settings by MEGA 4 and BioEdit softwares. Sequences from forward and reverse primers were assembled and compared. Verified sequences were visually inspected and manually adjusted using these softwares. The primer part was trimmed from the first of each sequence, because the character base sequence should not include primer. Then, the end of the sequences was matched together and the unmatched sequence was trimmed. In the trnLe trnF matrix, an unalignable 64 bp part of the spacer region had to be excluded from the phylogenetic analyses for all the species analyzed. 2.4. Phylogenetic analysis Maximum parsimony (MP) analyses were undertaken for the individual markers (nrITS, trnLeF, matK, and rbcL), for the combined cpDNA dataset and for all the molecular markers combined using PAUP* 4.0b10 (Swofford, 2002). To find islands of equally most parsimonious trees, maximum parsimony analyses were run using a heuristic search strategy of branch-
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Table 1 List of studied taxa. Species Section No.
Taxon
Location
Specimen Voucher
Genbank accession number
1 2
Hirtula Hirtula
B. dayanum Rchb. f. B. hirtulum Ridl.
Gunung Jerai, Malaysia Fraser s Hill, Malaysia
JF428000 JF305780 JF428055 JF428119 JF428030 JF305797 JF428091 JF428126
3
Hirtula
B. hirtulum Ridl.
4
Hirtula
B. limbatum Lindl.
B0054
JF428036 JF305814 JF428081 JF428122
5 6
Hirtula Altisceptrum
B. dayanum Rchb. f. B. farinulentum J.J. Sm.
Gunung Belumut, Malaysia Gunung Belumut, Malaysia Gunung Jerai, Malaysia Gunung Jerai, Malaysia
B0014 FAN. FH314 SH.K-100
JF428018 JF305799 JF428067 JF428120 JF428022 JF305803 JF428069 e
7 8
Cirrhopetalum B. flabellum veneris (J. Koenig) Seidenf. & Ormerod ex Aver. Cirrhopetalum B. purpurascens Teijsm. & Binn
SH.K-101 FAN. FH200 RG 1945 B0027
JF428010 JF305795 JF428084 e
9
Cirrhopetalum B. vaginatum (Lindl.) Rchb. f.
JF428005 JF305785 JF428057 e
10 11
Cirrhopetalum B. corolliferum J. J. Sm. Cirrhopetalum B. acuminatum (Ridl.) Ridl.
FAN. FH503 B0026 RG 2291
JF428009 JF305789 JF428061 e JF428021 JF305802 JF428073 JF428117
12
Cirrhopetalum B. auratum (Lindl.) Rchb. f.
B0060
JF428040 JF305817 JF428089 e
13 14
Cirrhopetalum B. gracillimum (Rolfe) Rolfe Cirrhopetalum B. sp1
B0053 SH.K-102
JF428037 JF305813 JF428080 JF428125 JF428017 JF305798 JF428066 JF428114
15
Cirrhopetalum B. sp2
SH.K-103
JF428024 JF305805 JF428087 e
16
Cirrhopetalum B. dentiferum Ridl.
B0048
JF428025 JF305806 JF428072 e
17 18 19
Cirrhopetalum B. sp3 Cirrhopetalum B. biflorum Teijsm. & Binn. Aphanobulbon B. flavescens (Blume) Lindl.
Gua Musang, Kelantan, Malaysia Gunung Panti, Malaysia Malaysia Fraser s Hill, Malaysia
JF428027 JF305808 JF428074 e e e EF195919 JF427998 JF305778 JF428053 JF428102
20
Aphanobulbon B. mutabile (Blume) Lindl.
Fraser s Hill, Malaysia
21
Aphanobulbon B. linearifolium King & Pantl.
Fraser s Hill, Malaysia
22 23
Aphanobulbon B. odoratum (Blume) Lindl. Aphanobulbon B. apodum Hook. f.
24 25 26
Aphanobulbon B. armeniacum J. J. Sm. Aphanobulbon B. caudatisepalum Ames & C. Schweinf. Aphanobulbon B. sp4
Pahang, Malaysia Cameron Highlands, Malaysia Fraser s Hill, Malaysia Fraser s Hill, Malaysia Fraser s Hill, Malaysia
27
Desmosanthes B. concinnum Hook. f.
SH.K-104 e FAN. FH062 FAN. FH105 FAN. FH258 B0056 FAN. FH276 SH.K-105 SH.K-106 FAN. FH419 RG 2207
28
Desmosanthes B. sulcatum (Blume) Lindl.
JF427995 JF305775 JF428076 JF428106
29
Desmosanthes B. angustifolium (Blume) Lindl.
FAN. FH304 RG 2313
30 31 32 33
Desmosanthes Desmosanthes Desmosanthes Desmosanthes
34
Desmosanthes B. sp5
35 36
Desmosanthes B. sp6 Sestochilus B. macranthum Lindl.
37 38
Sestochilus Sestochilus
B. inunctum J. J. Sm. B. lobbii Lindl.
39
Sestochilus
B. uniflorum (Blume) Hassk.
40 41
Sestochilus Sestochilus
B. patens King ex Hook. f. B. pileatum Lindl.
42
Sestochilus
B. lasianthum Lindl.
B. B. B. B.
medusae (Lindl.) Rchb. f. bakhuizenii van Steenis planibulbe (Ridl.) Ridl. obtusum (Blume) Lindl.
Cameron Highlands, Malaysia Cameron Highlands, Malaysia Melacca, Malaysia Penang, Malaysia Gunung Belumut, Malaysia Cameron Highlands, Malaysia Gunung Panti, Malaysia Genting Highland, Malaysia Gunung Belumut, ̕ Malaysia
Genting Highland, Ulu Kali, Malaysia Gunung Jerai, Malaysia Genting Highland, Ulu Kali, Malaysia Pahang, Malaysia Gunung Jerai, Malaysia Gunung Jerai, Malaysia Fraser s Hill, Malaysia ̕ Highland, Ulu Genting Kali, Malaysia ̕ Panti, Malaysia Gunung Cameron Highlands, ̕ Malaysia Gunung Jerai, Malaysia Cameron Highlands, Malaysia Genting Highland, ̕ Malaysia ̕ Jerai, Malaysia Gunung ̕ Belumut, Gunung Malaysia Fraser s Hill, Malaysia
rbcL
matK
trnL-F
ITS
JF428031 JF305810 JF428090 JF428127
JF427996 JF305776 JF428052 e
JF427997 JF305777 JF428077 JF428101 JF427999 JF305779 JF428054 JF428103 JF428034 JF305793 JF428083 JF428113 JF428039 e JF428079 JF428121 JF428015 JF305794 JF428064 JF428112 JF428008 e JF428060 JF428108 JF428014 e JF428065 e JF428006 JF305790 JF428058 e
JF427993 JF305773 JF428051 e e JF428068 JF428062 e
e e e e
B0052 SH.K-107 SH.K-108 FAN. FH172 B0010
JF428038 JF428019 JF428011 JF427994
JF305812 JF305800 JF305791 JF305774
e
JF305786 e
B0057 FAN. FH153 SH.K-109 FAN. FH426 FAN. FH107 B005 RG 2281
JF428033 JF305816 JF428085 e JF427988 JF305768 JF428047 JF428097
RG 1922
e
JF427989 JF305769 JF428048 JF428110 JF427991 JF305771 JF428050 JF428099 JF427990 JF305770 JF428049 JF428098 JF427992 JF305772 JF428075 JF428100 JF428007 JF305787 JF428059 JF428105 JF428026 JF305818 JF428086 JF428116
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Table 1 (continued ) Species Section No.
Taxon
Location
Specimen Voucher
Genbank accession number
43 44
Sestochilus Sestochilus
B. singaporeanum Schltr. B. sp7
B0050 SH.K-110
JF428032 JF305811 JF428078 e JF428020 JF305807 e JF428115
45
Sestochilus
B. membranifolium Hook. f.
e
e
e
e
EF195935
46 47 48 49 50 51 52 53
Sestochilus Careyana Careyana Epicrianthes Epicrianthes Epicrianthes Polyblepharon Polyblepharon
B. B. B. B. B. B. B. B.
e B0029 SH.K-111 B0018 B0031 B0015 B0024 B0045
e JF428012 JF428002 JF428004 JF428013 JF428001 JF428028 JF428029
e JF305796 JF305782 JF305784 JF305792 JF305781 JF305788 JF305809
e JF428063 JF428056 e e e e e
EF195924 JF428111 e JF428109 e e JF428107 e
54
Monilibulbus
B. ovalifolium (Blume) Lindl.
RG 2167
JF428003 JF305783 JF428092 JF428104
55
Monilibulbus
B. stormii J. J. Sm.
B0058
JF428042 e
56 57
Ephippium Leptopus
B. restrepia (Ridl.) Ridl. B. tenuifolium (Blume) Lindl.
B0055 B0061
JF428035 JF305815 JF428082 JF428123 JF428043 JF305820 JF428094 e
58
Globiceps
B. coniferum Ridl.
RG1757
JF428041 JF305819 JF428088 e
59 60 61 62
? ? Aporum Distichorchis
B. sp8 B. sp9 D. rosellum Ridl. D. pahangensis Carr.
Johor, Malaysia Gunung Belumut, Malaysia Cameron Highlands, Malaysia Peninsular Malaysia Gunung Jerai, Malaysia Gunung Jerai, Malaysia Fraser s Hill, Malaysia Penang, Malaysia Gunung Jerai, Malaysia Fraser s Hill, Malaysia Cameron Highlands, Malaysia Cameron Highlands, Malaysia Cameron Highlands, Malaysia Johor, Malaysia Cameron Highlands, Malaysia Cameron Highlands, Malaysia Penang, Malaysia Penang, Malaysia Fraser s Hill, Malaysia Fraser s Hill, Malaysia
JF428016 JF428023 JF428046 JF428045
63
Dendrochilum D. pallideflavens Blume.
SH.K-112 SH.K-113 D001 FAN. FH180 OYS 041
dearei (Hort.) Rchb. f. lilacinum Ridl. sichyobulbon Parish & Rchb. f. cheiropetalum Ridl. haniffii Carr. mobilifilum Carr. membranaceum Teijsm. & Binn. membranaceum Teijsm. & Binn.
Genting Highland, Ulu Kali, Malaysia
rbcL
matK
JF305801 JF305804 JF305822 JF305823
trnL-F
ITS
JF428093 JF428124
JF428070 JF428071 JF428095 e
e JF428118 JF428129 e
JF428044 JF305821 JF428096 JF428128
̕
swapping by tree bisection-reconnection (TBR) step-wise addition with 1000 random-addition replicates and holding 10 ̕ trees at each step. Levels of support were estimated with 1000 bootstrap replicates (BP), using the TBR algorithm of branch swapping for 10 random-addition replicates per bootstrap replicate. Bootstrap percentages were interpreted as weak (50e74%), moderate (75e84%) or high (85e100%). Congruence between nrITS and the combined cpDNA data sets were tested using the incongruence length difference (ILD) test (Farris et al., 1995) as implemented by the partition homogeneity test in PAUP* for 100 replicates (heuristic search, simple addition, and TBR branching swapping), each saving a maximum of 1000 most parsimonious trees per replicate. Each pH test was performed on 100 replicates. Moreover, bootstrap trees generated from each gene region were then manually compared for congruency, as described by Whitten et al. (2000). Data were combined where there were no conflicting, well-supported clades (BP greater than 74%) between gene regions. If probability values (P-value) greater than 0.5 were used to identify data sets, it means they are not significantly different from each other and therefore the regions could be combined. Bayesian inference (BI) was generated for the individual markers, for the combined cpDNA dataset and for all molecular ̕ markers combined using Mr Bayes 3.2.1 (Huelsenbeck and Ronquist, 2001) using default settings. The best fitting model of ̕ sequence evolution (GTR þ I þ G) was chosen based on the result of a hierarchical likelihood ratio test conducted with Model test 3.7 (Posada and Crandall, 2001). Four Markov chains were run simultaneously for 1,000,000 generations and every 10th generation was sampled. After 250,000 generations, a stable probability was reached. All non-significant generations (p < 0.5) were discarded for the consensus tree.
3. Results 3.1. Analysis of chloroplast sequence data Combined data analysis provided better insight of section separation rather than single matrix analyses. For example, rbcL data yielded high level of conserved nucleotide with low level of substitution that made a weak arrangement of clades with low support. Sequences analysis of matK and trnL-F was much better than rbcL, because of the higher percentage of sequence variation. However, polyphyletic clades were still being observed specially in trnL-F data analysis. Therefore, analysis on a combination of data is the way to solve this kind of problems.
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The pH test between plastid data sets gave a value of 0.63 indicating that the null hypothesis that they were sampled from the same phylogeny, could not be rejected and consequently they could be combined. The combined chloroplast matrix included 56 in-group species and three out group species. The combined alignment of all the chloroplast markers consists of 2436 positions and contained 421 phylogenetic informative substitutions. Average percentage sequence divergence (uncorrected p distance) within Bulbophyllum spp. was 6.4%. MP analyses yielded >10,000 most parsimonious trees (MPTs) with a length of 1265 steps, a consistency index (CI) of 0.65 and a retention index (RI) of 0.69. The strict consensus and the BI trees (data not shown) were highly congruent. In both analyses, Peninsular Malaysian Bulbophyllum were strongly supported (BP99/PP100) as monophyletic. 3.2. Analysis of nuclear sequence data The aligned sequences consisted of 678 nucleotide sites, with 81 characters which are constant among all the taxa, 603 sites were variable and 86 variable characters are parsimony-uninformative, and 517 sites were parsimony informative. Average percentage sequence divergence (uncorrected p distance) within Bulbophyllum spp. was 9.8%. MP analyses yielded >10,000 MPTs with a length of 1261 steps, CI of 0.61 and RI of 0.68. The strict consensus tree and the BI trees were highly congruent (data not shown) with the cpDNA trees and internal nodes received similar statistical support. The monophyly of studied species was supported by BP100/PP95. The major incongruences between combined cpDNA and nrITS trees were related to situation of sections Sestochilus and Aphanobulbon. These sections showed polyphyletic status inside the lineage, but the problem was roughly solved in analysis of nuclear ITS sequences. Moreover, B. gracillimum (Rolfe) Rolfe was separated from other species of the section Cirrhopetalum. 3.3. Combined molecular data analysis Probability values from the partition homogeneity tests between nrITS and cpDNA data sets (P-value ¼ 0.01) showed significant heterogeneity. However, given the oversensitivity of this statistical test seen in other studies (Graham et al., 2000), it seems likely that the partition homogeneity test would be overly sensitive comparing nuclear and chloroplast data sets. The much better resolved tree of the combined analysis as compared to the separate analysis of the data sets supports the assumptions that the partition homogeneity test of this study sometimes reveal unreliable results especially for large data sets (Norup et al., 2006). It is more likely that these incongruencies are due to technical issues, such as phylogenetic signal and homoplasy (Wendel and Doyle, 1998). Therefore, the data sets were combined to give a more robust phylogeny analysis of the genus Bulbophyllum than would otherwise be attainable with individual data sets (Soltis et al., 1998). The total alignment consisted of 3114 characters with 662 parsimony informative characters and result of Bayesian analysis with 3 106 generation Markov chain in a similar topology that was, however, more fully resolved and better supported than the combined cpDNA and nrITS trees separately (Fig. 1). 4. Discussion Based on the combined data analysis, the most significant result regarding the phylogenetic relationships of Bulbophyllum is the demonstration of monophyly of the genus. All the sections with the exception of section Hirtula as described by Seidenfaden and Wood (1992), strongly supported the monophyletic groups in the combined molecular data analysis. Unique morphological synapomorphies characterizing clades are scarce, but supporting combinations of characters are abundant. All species of section Sestochilus (clade A; PP100) are large plants with distinct pseudobulb (Fig. 2B) and rhizome of all species have been covered by sheaths (Fig. 2A). They have one non-resupinate (Fig. 2E) flower with the exception of B. singaporeanum Schltr and B. lasianthum Lindl which have many flowers on racemose inflorescence and glabrous petals are more than half as long as the sepals. Seidenfaden and Wood (1992) used the above-mentioned features to characterize section Sestochilus. Section Monilibulbus comprises B. ovalifolium (Blume) Lindl and B. stormii J. J. Sm These species are tiny, single flowered and they have contiguous bilaterally flattened pseudobulb. Most of the species in this section have been observed with papilloselip (Fig. 2F). Bulbophyllum stormii is one of the exceptions. Nevertheless, they are in the same group with high level of support. It seems pseudobulb shape has a stronger effect to place B. stormii and B. ovalifolium in the same section. Vermeulen and Lumb (1988) also recognized the section by pseudobulb character. Section Polyblepharon (clade A; PP100) is characterized by small plants, single flower carry with short pedicel. In all the molecular data sets and combined data analysis, this section was sister to section Epicrianthes (PP98). Section Epicrianthes is recognizable by thick leaf, single flower on the short pedicel, lip with elaborate construction (cover with small vesicles and hairs) (Fig. 2F) and diverging petals (Fig. 2D), the last character is a unique synapomorphy character for this group. Garay and Kittredge (1985) proposed a generic status for section Epicrianthes, but this analysis placed section Epicrianthes inside the genus Bulbophyllum. Majority of the species in section Aphanobulbon (clade B) are small to medium-size plants with very small or sometimes undetectable pseudobulbs (Fig. 2B) as unique character and multi-flowered raceme inflorescences. Most of the species were recognized by a hairy lip, except of B. linearifolium King & Pantl and B. mutabile (Blume) Lindl. Vermeulen (1991) used majority of above characters to recognize Aphanobulbon.
Fig. 1. Consensus tree resulting from BI analysis of the combined (nrITS and cpDNA) data, posterior probability values are indicated above the nodes. Section names shaded refer to paraphyletic sections whereas names in bold were found to be monophyletic. Nodes for the recognized sub-clades are marked with black dot.
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Fig. 2. Reconstruction of selected morphological key features mapped on the simplified combined (nrITS and cpDNA) phylogeny.
Vermeulen (1991) characterized section Desmosanthes (clade C; PP100) by small plants, distinct pseudobulb, inflorescence with two or more flowers, rachis very short and flowers arranged on subumbellate inflorescence with very tiny flowers except for B. medusae (Lindl.) Rchb. f. Virtually, all the characters have homoplasious status, but with a combination of them, distinct section was formed. However, trnL-F data analysis showed polyphyletic status, but in combined data analysis, situation of all species was recovered. In all molecular results Desmosanthes was sister to section Cirrhopetalum with strong support (PP100). Based on the results, B. medusae belong to Cirrhopetalum with 100% support and this is in conflict with earlier description by Seidenfaden and Wood (1992). Unique synapomorphy character of Cirrhopetalum (Fig. 2C) and angled pseudobulb can be observed in B. medusae as well. Leaf shape and scape of B. medusae covered by several sheaths are similar with B. vaginatum
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(Lindl.) Rchb. f, beside edges of dorsal sepal and petals are not fringed and base of the lateral sepals are free unlike Cirrhopetalum. Molecular results showed that the length of lateral sepal and pseudobulb shape have sufficient power to detect species of section Cirrhopetalum. Cirrhopetalum with more than 80 species around the world is a section which has always been considered as a separate genus. Umbellate inflorescence, longer length of lateral sepals than dorsal sepal (Fig. 2C), fringed edges of petals and dorsal sepal and angled pseudobulb, predominantly characterize section Cirrhopetalum. All the molecular data analysis showed common ancestor between Bulbophyllum and Cirrhopetalum and there was no evidence to accept the generic status of Cirrhopetalum. Combined molecular data sets showed that Cirrhopetalum is also deeply nested between Bulbophyllum spp. As was earlier mentioned, Cirrhopetalum was sister group to Desmosanthes, even B. medusae was placed inside Cirrhopetalum. The same position was proposed by Holttum (1964). Nevertheless, Seidenfaden and Wood (1992) removed the species from section Cirrhopetalum and they were placed inside the monotypic section Desmosanthes, because of its non-fringed petals and dorsal sepal. In this analysis, it was clear that B. medusa is nested within Cirrhopetalum. Sample developing can help to improve the status of section Desmosanthes and it may be proposed to merge some species of sections Cirrhopetalum and Desmosanthes. Three unknown species (B. sp6, B. sp8, and B. sp9) were placed between Desmosanthes and Cirrhopetalum. Morphological evidence of leaf and pseudobulb (thick leaf with blunt tip and angled pseudobulb) was highly similar to section Cirrhopetalum even though B. sp8 and B. sp9 were placed in the same clade with B. dentiferum Ridl. Unfortunately, inflorescence characters were not available, so these species can be included in section Cirrhopetalum or Desmosanthes. Seidenfaden and Wood (1992) proposed to place B. lilacinum Ridl and B. sichyobulbon Parish & Rchb. f. in section Careyana (clade C, PP100). Meanwhile, Holttum (1964) assigned B. lilacinum in section 12 along with a few species of section Sestochilus. In combined molecular analysis, these species appeared as sister to section Cirrhopetalum (PP96). Morphologically, combination of characters, such as large and angled pseudobulb, thick and fleshy leaf, adherent lateral sepals at the lower margins and longer length of lateral sepal than dorsal sepal make close affiliation between sections Careyana and Cirrhopetalum. Molecular data have confirmed the status of B. lilacinum and B. sichyobulbon in an independent section. Section Hirtula showed paraphyletic status in both individual and combined molecular data analysis. Hirtula consists of three species, namely B. hirtulum Ridl, B. dayanum Rchb. f, and B. limbatum Lindl (Seidenfaden and Wood, 1992). Holttum (1964) described B. hirtulum in section 9 and B. hespidum (synonym of B. dayanum) in section 12 along with B. limbatum. For B. hirtulum and B. limbatum, Ridley (1924) proposed Hirtula as sectional name. There are some common characters showed by species in section Hirtula, such as inflorescence with more than one flowers, equal length of sepals, and ciliate petals. Combined molecular tree and separate marker analysis showed B. hirtulum and B. limbatum in the same clade with strong support (clade B, PP100) independent of B. dayanum (clade C). So, it is proposed to split down section Hirtula; B. hirtulum and B. limbatum in section Hirtula. Bulbophyllum dayanum must be detached. In conclusion, considerable evidence has been made in delimiting natural sections and the relationships between species could be clearly determined. Nevertheless, the addition of a new molecular dataset with enhanced number of species will allow significant progress in clarifying the sectional delimitation in Bulbophyllum. Acknowledgements The authors would like to knowledge the following institutions: Singapore Herbarium (SING), Kepong Herbarium (KEP) in FRIM, and University Malaya Herbarium (KLU) for their hospitality and kindness providing facilities to work in their herbarium. This research work is made possible through Research University Grant scheme no. RUGS 03-01-07- 0035RU and RUGS 05-04-08-0556RU made available by University Putra Malaysia. References Carlsward, B.S., Whitten, W.M., Williams, N.H., Bytebier, B., 2006. Molecular phylogenetics of Vandeae (Orchidaceae) and the evolution of leaflessness. Am. J. Bot. 93, 770e786. CBOL, P.W.G., 2009. A DNA barcode for land plants. Proc. Natl. Acad. Sci. U. S. A. 106, 12794e12797. €, M., Kluge, A.G., Bult, C., 1995. Constructing a significance test for incongruence. Syst. Biol. 44, 570e572. Farris, J.S., K€ allersjo Fischer, G.A., Gravendeel, B., Sieder, A., Andriantiana, J., Heiselmayer, P., Cribb, P.J., Smidt, E. d. C., Samuel, R., Kiehn, M., 2007. Evolution of resupination in Malagasy species of bulbophyllum (Orchidaceae). Mol. Phylogenetics Evol. 45, 358e376. Frodin, D.G., 2004. History and concepts of big plant genera. Taxon 53, 753e776. Garay, L.A., Kittredge, W., 1985. Notes From the Ames Orchid Herbarium. Harvard University, Cambridge, Massachusetts, USA. Graham, S.W., Reeves, P.A., Burns, A.C.E., Olmstead, R.G., 2000. Microstructural changes in noncoding chloroplast DNA: interpretation, evolution, and utility of indels and inversions in basal angiosperm phylogenetic inference. Int. J. Plant Sci. 161, S83eS96. Gravendeel, B.S.A., Slik, F.J.W., Schuiteman, A., 2004. Epiphytism and pollinator specialization: drivers for orchid diversity? Philosophical Trans. R. Soc. Lond. B 359, 1523e1535. Holttum, R.E., 1964. A Revised Flora of Malaya. Government Printing Office, Singapore. Hosseini, S.G.R., Dadkhah, K., Ainuddin Nuruddin, A., 2012. Studies on maturase K sequences and systematic classification of Bulbophyllum in Peninsular Malaysia. Pak. J. Bot. 44, 2047e2054. Huelsenbeck, J.P., Ronquist, F., 2001. MRBAYES: Bayesian inference of phylogenetic trees. Bioinformatics 17, 754e755. Nishida, R., Tan, K.H., Wee, S.L., Hee, A.K.W., Toong, Y.C., 2004. Phenylpropanoids in the fragrance of the fruit fly orchid, Bulbophyllum cheiri, and their relationship to the pollinator, Bactrocera papayae. Biochem. Syst. Ecol. 32, 245e252. Norup, M.V., Dransfield, J., Chase, M.W., Barfod, A.S., Fernando, E.S., Baker, W.J., 2006. Homoplasious character combinations and generic delimitation: a case study from the indo-pacific arecoid palms (Arecaceae: Areceae). Am. J. Bot. 93, 1065e1080. Posada, D., Crandall, K.A., 2001. Selecting the best-fit model of nucleotide substitution. Syst. Biol. 50, 580e601.
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Ridley, H.N., 1924. Flora of the Malaya Peninsula. Reeves, London. Schuiteman, A., Vermeulen, J.J., de Vogel, E.F., 2010. Genus Bulbophyllum. Flora Malesiana: Orchids of New Guinea. National Herbarium of the Netherlands, Leiden. Seidenfaden, G., Wood, J.J., 1992. The Orchids of Peninsular Malaysia and Singapore. Olsen & Olsen, Fredensborg. Sieder, A., Rainer, H., Kiehn, M., 2007. CITES Checklist for Bulbophyllum and Allied Taxa (Orchidaceae)in Botanical Garden of the University of Vienna. Available from: http://www.cites.org/common/com/NC/tax_ref/Bulbophyllum.pdf. Soltis, D.E., Soltis, P.S., 1998. Choosing an Approach and an Appropriate Gene for Phylogenetic Analysis. Kluwer Academic, Boston. Sun, Y., Skinnder, D.Z., Liang, G.H., Hulbert, S.H., 1994. Phylogenetic analysis of Sorghum and related taxa using internal transcribed spacers of nuclear ribosomal DNA. Theor. Appl. Genet. 89, 26e32. Swofford, D.L., 2002. PAUP*. Phylogenetic Analysis Using Parsimony (* and Other Methods). Version 4.0b10. Sinauer Associates, Sunderland, Massachussets, USA. Taberlet, P., Gielly, L., Pautou, G., Bouvet, J., 1991. Universal primers for amplification of three non-coding regions of chloroplast DNA. Plant Mol. Biol. 17, 1105e1109. Teixeira, S.P., Borba, E.L., Semir, J., 2004. Lip Anatomy and its Implications for the pollination Mechanisms of bulbophyllum species (Orchidaceae). Ann. Bot. 93, 499e505. Thompson, J.D., Gibson, T.J., Higgins, D.G., 1994. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position specific gap penalties and weight matrix choice. Nucleic Acids Res. 22, 4673e4680. Van Den Berg, C., Goldman, D.H., Freudenstein, J.V., Pridgeon, A.M., Cameron, K.M., Chase, M.W., 2005. An overview of the phylogenetic relationships within Epidendroideae inferred from multiple DNA regions and recircumscription of Epidendreae and Arethuseae (Orchidaceae). Am. J. Bot. 92, 613e624. Vermeulen, J.J., Lamb, A., 1988. Six new species of bulbophyllum Sect. Monilibulbus (Orchidaceae). Gardens' Bull. 41 (pt. 2), 29e41. Vermeulen, J.J.A., 1991. Bulbophyllum. Bentham - Moxon Trust, Royal Botanic Gardens, Kew and Toihaan Publishing Company, Kota Kinabalu. Wendel, J.F., Doyle, J.J., 1998. Phylogenetic incongruence: window into genome history and molecular evolution. In: Soltis, D., Soltis, P., Doyle, J. (Eds.), Molecular Systematics of Plants II: DNA Sequencing, 265e296 (Kluwer, Boston, Massachusets, USA). White, T.J., Bruns, T., Lee, S., Taylor, J., 1990. In: Innis, M.A., Gelfand, D.H., Sninsky, J.J., White, T.J. (Eds.), Amplification and Direct Sequencing of Fungal Ribosomal RNA Genes for Phylogenetics. Academic Press, San Diego, California, USA. Whitten, W.M., Williams, N.H., Chase, M.W., 2000. Subtribal and generic relationships of Maxillarieae (Orchidaceae) with emphasis on Stanhopeinae: combined molecular evidence. Am. J. Bot. 87, 1842e1856.
Contents lists available at ScienceDirect
Biochemical Systematics and Ecology journal homepage: www.elsevier.com/locate/biochemsyseco
Molecular systematics of genus Bulbophyllum (Orchidaceae) in Peninsular Malaysia based on combined nuclear and plastid DNA sequences Shahla Hosseini a, *, Kourosh Dadkhah b, Rusea Go c a
Department of Biological Science and Biotechnology, University of Kurdistan, P.O. Box 416, Sanandaj, Iran Department of Statistics, Faculty of Science, University of Kurdistan, P.O. Box 416, Sanandaj, Iran c Department of Biology, University Putra Malaysia, 43400 UPM Serdang, Selangor Darul Ehsan, Malaysia b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 21 July 2015 Received in revised form 4 January 2016 Accepted 31 January 2016 Available online xxx
Phylogenetic relationships were inferred for representative Bulbophyllum species of 13 sections from subtribe Bulbophyllinae (Epidendroideae, Orchidaceae) in Peninsular Malaysia. The combined data matrix consists of sequences from ITS nuclear gene region and trnL-F, matK, and rbcL plastid gene regions with 3114 characters. Molecular data were analysed using parsimony and Bayesian inference. The results show that several recognized sections are monophyletic. Section Hirtula with paraphyletic status must split up and section Desmosanthes contain misplaced elements. Furthermore, generic status of Cirrhopetalum and Epicrianthes cannot be supported, because they are deeply embedded within the genus Bulbophyllum. Section Desmosanthes is recognized as the closest group to section Cirrhopetalum; therefore, they can be merged in some aspects. © 2016 Elsevier Ltd. All rights reserved.
Keywords: Bulbophyllum Monophyly Orchidaceae Peninsular Malaysia Phylogenetic
1. Introduction Bulbophyllum Thouars is the largest genus of the pantropical subtribe Bulbophyllinae and comprises approximately 2032 species (Frodin, 2004). Sieder (2007) reported 184 species with 48 endemic species from Malaysia. It is mostly an epiphytic species found in different habitats, ranging from (sub) tropical dry forests to wet montane cloud forests and most of them are adapted to fly pollination (Nishida et al., 2004; Teixeira et al., 2004). They are distributed in the most northern parts (Perlis) to the most southern parts (Johor) in Peninsular Malaysia and their geography can range from lowland to highland areas like Cameroon and Genting Highlands. In recent years, several molecular phylogenetic studies of orchids have been published (Van Den Berg et al., 2005; Carlsward et al., 2006) placing genus Bulbophyllum as sister to the genus Dendrobium within tribe Dendrobieae in the higher Epidendroids clade of subfamily Epidendroideae. Orchids of the genus Bulbophyllum are one of the important plants in Malaysia in terms of their abundance, but identification of Bulbophyllum at species level still remains a problem for practising taxonomists. Uncertainty in taxonomic status of the Bulbophyllum is a major problem which requires molecular taxonomic revision. Attempts to split up this gargantuan genus into smaller, more manageable units have generally faltered due to the fact that most of the main groups differ only in rather loose combinations of characters. In every section, there were species that do not
* Corresponding author. E-mail addresses: [email protected] (S. Hosseini), [email protected] (K. Dadkhah), [email protected] (R. Go). http://dx.doi.org/10.1016/j.bse.2016.01.003 0305-1978/© 2016 Elsevier Ltd. All rights reserved.
S. Hosseini et al. / Biochemical Systematics and Ecology 65 (2016) 40e48
41
possess all the diagnostic characters of that section, and yet seem to belong in it; conversely, species that seemingly belong to a certain group based on its diagnostic characters sometimes show other characters that contradict the affinity (Schuiteman et al., 2010). Such genera as Cirrhopetalum have been split off, but based on molecular studies of matK sequences analysis (Gravendeel et al., 2004; Hosseini et al., 2012), generic status of Cirrhopetalum failed. In this study, analysis of some Bulbophyllum species with putative out groups was performed on 4 DNA regions: plastid trnL-F spacer, matK gene, rbcL gene, and the ITS data. In this paper, the main objectives were to clarify the internal topology within Bulbophyllum spp., to test the monophyly of studied taxa, and to explicate the generic status of sections Cirrhopetalum and Epicrianthes based on reconstructed multi-gene based phylogeny. 2. Materials and methods 2.1. Taxon sampling Sampling of species required for this study was one of the greatest challenges faced. It was not possible to cover every part of the forested area in Peninsular Malaysia during this study period. Nevertheless, areas of earlier collection site were visited for possible sampling. However, not all the previous collection sites are forested areas. At least 50% of the recorded collection sites are now palm oil plantation area. Therefore, it was not possible to obtain some of the species required for this study. Some of the collected samples were without flower, so the correct name of these species could not be declared. In this case, blooming season needs to be waited for or the plants should be placed in green house. However, some species in the new conditions could not survive and some of them never flower even after three years in the green house. Species representing 13 sections of Peninsular Malaysian Bulbophyllum, as described by Seidenfaden and Wood (1992), were sampled (Table 1). For some sections, it was not possible to identify all the accessions to species or to apply a valid name based on only vegetative characters, so they appeared as B. sp1- B. sp9. Living samples were collected in Peninsular Malaysia in the wild or taken from plants in the green house of University Putra Malaysia. Herbarium dried samples from the herbarium in Biology Department of University Putra Malaysia were also used for some species. Voucher specimens for all accessions were deposited in herbarium of Biology Department, University Putra Malaysia (UPM). Three taxa (Dendrobium rosellum, Dendrobium pahangensis and Dendrochilum pallideflavens) were selected as the out group. 2.2. DNA extraction, amplification, and sequencing DNA was extracted from the herbarium or fresh material using Wizard® Genomic DNA Purification Kit (Promega). Polymerase chain reaction (PCR) amplification was performed using Bioline Biolasetaq (5 U/ul), Bioline 10 biolase buffer, dNTPs (10 mM) following the manufacturer's protocol and 2e8 ng template DNA for a 50 ml reaction mixture. The Primers 26SE and 17SE of Sun et al. (1994) were used for the amplification of the nrITS regions with the following PCR program: an initial 2 min premelt at 94 C and 34 cycles of 1 min denaturation at 94 C, 1 min annealing at 50 C and 1 min extension at 72 C followed by a final extension for 7 min. For sequencing, the primers ITS4 and ITS5 (White et al., 1990) were used. The cpDNA trnLetrnF intron was amplified using forward primer designed by Fischer et al. (2007) and universal reverse primer designed by Taberlet et al. (1991). The rbcL and matK chloroplast region were amplified using the designed primers pair by David Erickson and Ki-Joong Kim, respectively, from the Consortium for the Barcode of Life (CBOL, 2009). These primers were amplified partial sequences of rbcL and matK with length of 500e600 bp and 1000 bp, respectively. Amplification of cpDNA was carried out with an initial 3 min premelt at 94 C and 30 cycles of 30 s denaturation at 94 C, 30 s annealing at 55 C and 1 min 40 s extension at 72 C followed by a final extension for 7 min at 72 C. DNA was purified from gel slice or PCR product by Wizard® SV Gel and PCR clean-up system (Promega) following the manufacturer's protocol. All DNA sequences produced for this study were submitted to GenBank. For several species, it was not possible to amplify all the molecular markers. 2.3. DNA sequence alignment Multiple sequence alignments were performed using Clustal W (Thompson et al., 1994) with default settings by MEGA 4 and BioEdit softwares. Sequences from forward and reverse primers were assembled and compared. Verified sequences were visually inspected and manually adjusted using these softwares. The primer part was trimmed from the first of each sequence, because the character base sequence should not include primer. Then, the end of the sequences was matched together and the unmatched sequence was trimmed. In the trnLe trnF matrix, an unalignable 64 bp part of the spacer region had to be excluded from the phylogenetic analyses for all the species analyzed. 2.4. Phylogenetic analysis Maximum parsimony (MP) analyses were undertaken for the individual markers (nrITS, trnLeF, matK, and rbcL), for the combined cpDNA dataset and for all the molecular markers combined using PAUP* 4.0b10 (Swofford, 2002). To find islands of equally most parsimonious trees, maximum parsimony analyses were run using a heuristic search strategy of branch-
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S. Hosseini et al. / Biochemical Systematics and Ecology 65 (2016) 40e48
Table 1 List of studied taxa. Species Section No.
Taxon
Location
Specimen Voucher
Genbank accession number
1 2
Hirtula Hirtula
B. dayanum Rchb. f. B. hirtulum Ridl.
Gunung Jerai, Malaysia Fraser s Hill, Malaysia
JF428000 JF305780 JF428055 JF428119 JF428030 JF305797 JF428091 JF428126
3
Hirtula
B. hirtulum Ridl.
4
Hirtula
B. limbatum Lindl.
B0054
JF428036 JF305814 JF428081 JF428122
5 6
Hirtula Altisceptrum
B. dayanum Rchb. f. B. farinulentum J.J. Sm.
Gunung Belumut, Malaysia Gunung Belumut, Malaysia Gunung Jerai, Malaysia Gunung Jerai, Malaysia
B0014 FAN. FH314 SH.K-100
JF428018 JF305799 JF428067 JF428120 JF428022 JF305803 JF428069 e
7 8
Cirrhopetalum B. flabellum veneris (J. Koenig) Seidenf. & Ormerod ex Aver. Cirrhopetalum B. purpurascens Teijsm. & Binn
SH.K-101 FAN. FH200 RG 1945 B0027
JF428010 JF305795 JF428084 e
9
Cirrhopetalum B. vaginatum (Lindl.) Rchb. f.
JF428005 JF305785 JF428057 e
10 11
Cirrhopetalum B. corolliferum J. J. Sm. Cirrhopetalum B. acuminatum (Ridl.) Ridl.
FAN. FH503 B0026 RG 2291
JF428009 JF305789 JF428061 e JF428021 JF305802 JF428073 JF428117
12
Cirrhopetalum B. auratum (Lindl.) Rchb. f.
B0060
JF428040 JF305817 JF428089 e
13 14
Cirrhopetalum B. gracillimum (Rolfe) Rolfe Cirrhopetalum B. sp1
B0053 SH.K-102
JF428037 JF305813 JF428080 JF428125 JF428017 JF305798 JF428066 JF428114
15
Cirrhopetalum B. sp2
SH.K-103
JF428024 JF305805 JF428087 e
16
Cirrhopetalum B. dentiferum Ridl.
B0048
JF428025 JF305806 JF428072 e
17 18 19
Cirrhopetalum B. sp3 Cirrhopetalum B. biflorum Teijsm. & Binn. Aphanobulbon B. flavescens (Blume) Lindl.
Gua Musang, Kelantan, Malaysia Gunung Panti, Malaysia Malaysia Fraser s Hill, Malaysia
JF428027 JF305808 JF428074 e e e EF195919 JF427998 JF305778 JF428053 JF428102
20
Aphanobulbon B. mutabile (Blume) Lindl.
Fraser s Hill, Malaysia
21
Aphanobulbon B. linearifolium King & Pantl.
Fraser s Hill, Malaysia
22 23
Aphanobulbon B. odoratum (Blume) Lindl. Aphanobulbon B. apodum Hook. f.
24 25 26
Aphanobulbon B. armeniacum J. J. Sm. Aphanobulbon B. caudatisepalum Ames & C. Schweinf. Aphanobulbon B. sp4
Pahang, Malaysia Cameron Highlands, Malaysia Fraser s Hill, Malaysia Fraser s Hill, Malaysia Fraser s Hill, Malaysia
27
Desmosanthes B. concinnum Hook. f.
SH.K-104 e FAN. FH062 FAN. FH105 FAN. FH258 B0056 FAN. FH276 SH.K-105 SH.K-106 FAN. FH419 RG 2207
28
Desmosanthes B. sulcatum (Blume) Lindl.
JF427995 JF305775 JF428076 JF428106
29
Desmosanthes B. angustifolium (Blume) Lindl.
FAN. FH304 RG 2313
30 31 32 33
Desmosanthes Desmosanthes Desmosanthes Desmosanthes
34
Desmosanthes B. sp5
35 36
Desmosanthes B. sp6 Sestochilus B. macranthum Lindl.
37 38
Sestochilus Sestochilus
B. inunctum J. J. Sm. B. lobbii Lindl.
39
Sestochilus
B. uniflorum (Blume) Hassk.
40 41
Sestochilus Sestochilus
B. patens King ex Hook. f. B. pileatum Lindl.
42
Sestochilus
B. lasianthum Lindl.
B. B. B. B.
medusae (Lindl.) Rchb. f. bakhuizenii van Steenis planibulbe (Ridl.) Ridl. obtusum (Blume) Lindl.
Cameron Highlands, Malaysia Cameron Highlands, Malaysia Melacca, Malaysia Penang, Malaysia Gunung Belumut, Malaysia Cameron Highlands, Malaysia Gunung Panti, Malaysia Genting Highland, Malaysia Gunung Belumut, ̕ Malaysia
Genting Highland, Ulu Kali, Malaysia Gunung Jerai, Malaysia Genting Highland, Ulu Kali, Malaysia Pahang, Malaysia Gunung Jerai, Malaysia Gunung Jerai, Malaysia Fraser s Hill, Malaysia ̕ Highland, Ulu Genting Kali, Malaysia ̕ Panti, Malaysia Gunung Cameron Highlands, ̕ Malaysia Gunung Jerai, Malaysia Cameron Highlands, Malaysia Genting Highland, ̕ Malaysia ̕ Jerai, Malaysia Gunung ̕ Belumut, Gunung Malaysia Fraser s Hill, Malaysia
rbcL
matK
trnL-F
ITS
JF428031 JF305810 JF428090 JF428127
JF427996 JF305776 JF428052 e
JF427997 JF305777 JF428077 JF428101 JF427999 JF305779 JF428054 JF428103 JF428034 JF305793 JF428083 JF428113 JF428039 e JF428079 JF428121 JF428015 JF305794 JF428064 JF428112 JF428008 e JF428060 JF428108 JF428014 e JF428065 e JF428006 JF305790 JF428058 e
JF427993 JF305773 JF428051 e e JF428068 JF428062 e
e e e e
B0052 SH.K-107 SH.K-108 FAN. FH172 B0010
JF428038 JF428019 JF428011 JF427994
JF305812 JF305800 JF305791 JF305774
e
JF305786 e
B0057 FAN. FH153 SH.K-109 FAN. FH426 FAN. FH107 B005 RG 2281
JF428033 JF305816 JF428085 e JF427988 JF305768 JF428047 JF428097
RG 1922
e
JF427989 JF305769 JF428048 JF428110 JF427991 JF305771 JF428050 JF428099 JF427990 JF305770 JF428049 JF428098 JF427992 JF305772 JF428075 JF428100 JF428007 JF305787 JF428059 JF428105 JF428026 JF305818 JF428086 JF428116
S. Hosseini et al. / Biochemical Systematics and Ecology 65 (2016) 40e48
43
Table 1 (continued ) Species Section No.
Taxon
Location
Specimen Voucher
Genbank accession number
43 44
Sestochilus Sestochilus
B. singaporeanum Schltr. B. sp7
B0050 SH.K-110
JF428032 JF305811 JF428078 e JF428020 JF305807 e JF428115
45
Sestochilus
B. membranifolium Hook. f.
e
e
e
e
EF195935
46 47 48 49 50 51 52 53
Sestochilus Careyana Careyana Epicrianthes Epicrianthes Epicrianthes Polyblepharon Polyblepharon
B. B. B. B. B. B. B. B.
e B0029 SH.K-111 B0018 B0031 B0015 B0024 B0045
e JF428012 JF428002 JF428004 JF428013 JF428001 JF428028 JF428029
e JF305796 JF305782 JF305784 JF305792 JF305781 JF305788 JF305809
e JF428063 JF428056 e e e e e
EF195924 JF428111 e JF428109 e e JF428107 e
54
Monilibulbus
B. ovalifolium (Blume) Lindl.
RG 2167
JF428003 JF305783 JF428092 JF428104
55
Monilibulbus
B. stormii J. J. Sm.
B0058
JF428042 e
56 57
Ephippium Leptopus
B. restrepia (Ridl.) Ridl. B. tenuifolium (Blume) Lindl.
B0055 B0061
JF428035 JF305815 JF428082 JF428123 JF428043 JF305820 JF428094 e
58
Globiceps
B. coniferum Ridl.
RG1757
JF428041 JF305819 JF428088 e
59 60 61 62
? ? Aporum Distichorchis
B. sp8 B. sp9 D. rosellum Ridl. D. pahangensis Carr.
Johor, Malaysia Gunung Belumut, Malaysia Cameron Highlands, Malaysia Peninsular Malaysia Gunung Jerai, Malaysia Gunung Jerai, Malaysia Fraser s Hill, Malaysia Penang, Malaysia Gunung Jerai, Malaysia Fraser s Hill, Malaysia Cameron Highlands, Malaysia Cameron Highlands, Malaysia Cameron Highlands, Malaysia Johor, Malaysia Cameron Highlands, Malaysia Cameron Highlands, Malaysia Penang, Malaysia Penang, Malaysia Fraser s Hill, Malaysia Fraser s Hill, Malaysia
JF428016 JF428023 JF428046 JF428045
63
Dendrochilum D. pallideflavens Blume.
SH.K-112 SH.K-113 D001 FAN. FH180 OYS 041
dearei (Hort.) Rchb. f. lilacinum Ridl. sichyobulbon Parish & Rchb. f. cheiropetalum Ridl. haniffii Carr. mobilifilum Carr. membranaceum Teijsm. & Binn. membranaceum Teijsm. & Binn.
Genting Highland, Ulu Kali, Malaysia
rbcL
matK
JF305801 JF305804 JF305822 JF305823
trnL-F
ITS
JF428093 JF428124
JF428070 JF428071 JF428095 e
e JF428118 JF428129 e
JF428044 JF305821 JF428096 JF428128
̕
swapping by tree bisection-reconnection (TBR) step-wise addition with 1000 random-addition replicates and holding 10 ̕ trees at each step. Levels of support were estimated with 1000 bootstrap replicates (BP), using the TBR algorithm of branch swapping for 10 random-addition replicates per bootstrap replicate. Bootstrap percentages were interpreted as weak (50e74%), moderate (75e84%) or high (85e100%). Congruence between nrITS and the combined cpDNA data sets were tested using the incongruence length difference (ILD) test (Farris et al., 1995) as implemented by the partition homogeneity test in PAUP* for 100 replicates (heuristic search, simple addition, and TBR branching swapping), each saving a maximum of 1000 most parsimonious trees per replicate. Each pH test was performed on 100 replicates. Moreover, bootstrap trees generated from each gene region were then manually compared for congruency, as described by Whitten et al. (2000). Data were combined where there were no conflicting, well-supported clades (BP greater than 74%) between gene regions. If probability values (P-value) greater than 0.5 were used to identify data sets, it means they are not significantly different from each other and therefore the regions could be combined. Bayesian inference (BI) was generated for the individual markers, for the combined cpDNA dataset and for all molecular ̕ markers combined using Mr Bayes 3.2.1 (Huelsenbeck and Ronquist, 2001) using default settings. The best fitting model of ̕ sequence evolution (GTR þ I þ G) was chosen based on the result of a hierarchical likelihood ratio test conducted with Model test 3.7 (Posada and Crandall, 2001). Four Markov chains were run simultaneously for 1,000,000 generations and every 10th generation was sampled. After 250,000 generations, a stable probability was reached. All non-significant generations (p < 0.5) were discarded for the consensus tree.
3. Results 3.1. Analysis of chloroplast sequence data Combined data analysis provided better insight of section separation rather than single matrix analyses. For example, rbcL data yielded high level of conserved nucleotide with low level of substitution that made a weak arrangement of clades with low support. Sequences analysis of matK and trnL-F was much better than rbcL, because of the higher percentage of sequence variation. However, polyphyletic clades were still being observed specially in trnL-F data analysis. Therefore, analysis on a combination of data is the way to solve this kind of problems.
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The pH test between plastid data sets gave a value of 0.63 indicating that the null hypothesis that they were sampled from the same phylogeny, could not be rejected and consequently they could be combined. The combined chloroplast matrix included 56 in-group species and three out group species. The combined alignment of all the chloroplast markers consists of 2436 positions and contained 421 phylogenetic informative substitutions. Average percentage sequence divergence (uncorrected p distance) within Bulbophyllum spp. was 6.4%. MP analyses yielded >10,000 most parsimonious trees (MPTs) with a length of 1265 steps, a consistency index (CI) of 0.65 and a retention index (RI) of 0.69. The strict consensus and the BI trees (data not shown) were highly congruent. In both analyses, Peninsular Malaysian Bulbophyllum were strongly supported (BP99/PP100) as monophyletic. 3.2. Analysis of nuclear sequence data The aligned sequences consisted of 678 nucleotide sites, with 81 characters which are constant among all the taxa, 603 sites were variable and 86 variable characters are parsimony-uninformative, and 517 sites were parsimony informative. Average percentage sequence divergence (uncorrected p distance) within Bulbophyllum spp. was 9.8%. MP analyses yielded >10,000 MPTs with a length of 1261 steps, CI of 0.61 and RI of 0.68. The strict consensus tree and the BI trees were highly congruent (data not shown) with the cpDNA trees and internal nodes received similar statistical support. The monophyly of studied species was supported by BP100/PP95. The major incongruences between combined cpDNA and nrITS trees were related to situation of sections Sestochilus and Aphanobulbon. These sections showed polyphyletic status inside the lineage, but the problem was roughly solved in analysis of nuclear ITS sequences. Moreover, B. gracillimum (Rolfe) Rolfe was separated from other species of the section Cirrhopetalum. 3.3. Combined molecular data analysis Probability values from the partition homogeneity tests between nrITS and cpDNA data sets (P-value ¼ 0.01) showed significant heterogeneity. However, given the oversensitivity of this statistical test seen in other studies (Graham et al., 2000), it seems likely that the partition homogeneity test would be overly sensitive comparing nuclear and chloroplast data sets. The much better resolved tree of the combined analysis as compared to the separate analysis of the data sets supports the assumptions that the partition homogeneity test of this study sometimes reveal unreliable results especially for large data sets (Norup et al., 2006). It is more likely that these incongruencies are due to technical issues, such as phylogenetic signal and homoplasy (Wendel and Doyle, 1998). Therefore, the data sets were combined to give a more robust phylogeny analysis of the genus Bulbophyllum than would otherwise be attainable with individual data sets (Soltis et al., 1998). The total alignment consisted of 3114 characters with 662 parsimony informative characters and result of Bayesian analysis with 3 106 generation Markov chain in a similar topology that was, however, more fully resolved and better supported than the combined cpDNA and nrITS trees separately (Fig. 1). 4. Discussion Based on the combined data analysis, the most significant result regarding the phylogenetic relationships of Bulbophyllum is the demonstration of monophyly of the genus. All the sections with the exception of section Hirtula as described by Seidenfaden and Wood (1992), strongly supported the monophyletic groups in the combined molecular data analysis. Unique morphological synapomorphies characterizing clades are scarce, but supporting combinations of characters are abundant. All species of section Sestochilus (clade A; PP100) are large plants with distinct pseudobulb (Fig. 2B) and rhizome of all species have been covered by sheaths (Fig. 2A). They have one non-resupinate (Fig. 2E) flower with the exception of B. singaporeanum Schltr and B. lasianthum Lindl which have many flowers on racemose inflorescence and glabrous petals are more than half as long as the sepals. Seidenfaden and Wood (1992) used the above-mentioned features to characterize section Sestochilus. Section Monilibulbus comprises B. ovalifolium (Blume) Lindl and B. stormii J. J. Sm These species are tiny, single flowered and they have contiguous bilaterally flattened pseudobulb. Most of the species in this section have been observed with papilloselip (Fig. 2F). Bulbophyllum stormii is one of the exceptions. Nevertheless, they are in the same group with high level of support. It seems pseudobulb shape has a stronger effect to place B. stormii and B. ovalifolium in the same section. Vermeulen and Lumb (1988) also recognized the section by pseudobulb character. Section Polyblepharon (clade A; PP100) is characterized by small plants, single flower carry with short pedicel. In all the molecular data sets and combined data analysis, this section was sister to section Epicrianthes (PP98). Section Epicrianthes is recognizable by thick leaf, single flower on the short pedicel, lip with elaborate construction (cover with small vesicles and hairs) (Fig. 2F) and diverging petals (Fig. 2D), the last character is a unique synapomorphy character for this group. Garay and Kittredge (1985) proposed a generic status for section Epicrianthes, but this analysis placed section Epicrianthes inside the genus Bulbophyllum. Majority of the species in section Aphanobulbon (clade B) are small to medium-size plants with very small or sometimes undetectable pseudobulbs (Fig. 2B) as unique character and multi-flowered raceme inflorescences. Most of the species were recognized by a hairy lip, except of B. linearifolium King & Pantl and B. mutabile (Blume) Lindl. Vermeulen (1991) used majority of above characters to recognize Aphanobulbon.
Fig. 1. Consensus tree resulting from BI analysis of the combined (nrITS and cpDNA) data, posterior probability values are indicated above the nodes. Section names shaded refer to paraphyletic sections whereas names in bold were found to be monophyletic. Nodes for the recognized sub-clades are marked with black dot.
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Fig. 2. Reconstruction of selected morphological key features mapped on the simplified combined (nrITS and cpDNA) phylogeny.
Vermeulen (1991) characterized section Desmosanthes (clade C; PP100) by small plants, distinct pseudobulb, inflorescence with two or more flowers, rachis very short and flowers arranged on subumbellate inflorescence with very tiny flowers except for B. medusae (Lindl.) Rchb. f. Virtually, all the characters have homoplasious status, but with a combination of them, distinct section was formed. However, trnL-F data analysis showed polyphyletic status, but in combined data analysis, situation of all species was recovered. In all molecular results Desmosanthes was sister to section Cirrhopetalum with strong support (PP100). Based on the results, B. medusae belong to Cirrhopetalum with 100% support and this is in conflict with earlier description by Seidenfaden and Wood (1992). Unique synapomorphy character of Cirrhopetalum (Fig. 2C) and angled pseudobulb can be observed in B. medusae as well. Leaf shape and scape of B. medusae covered by several sheaths are similar with B. vaginatum
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(Lindl.) Rchb. f, beside edges of dorsal sepal and petals are not fringed and base of the lateral sepals are free unlike Cirrhopetalum. Molecular results showed that the length of lateral sepal and pseudobulb shape have sufficient power to detect species of section Cirrhopetalum. Cirrhopetalum with more than 80 species around the world is a section which has always been considered as a separate genus. Umbellate inflorescence, longer length of lateral sepals than dorsal sepal (Fig. 2C), fringed edges of petals and dorsal sepal and angled pseudobulb, predominantly characterize section Cirrhopetalum. All the molecular data analysis showed common ancestor between Bulbophyllum and Cirrhopetalum and there was no evidence to accept the generic status of Cirrhopetalum. Combined molecular data sets showed that Cirrhopetalum is also deeply nested between Bulbophyllum spp. As was earlier mentioned, Cirrhopetalum was sister group to Desmosanthes, even B. medusae was placed inside Cirrhopetalum. The same position was proposed by Holttum (1964). Nevertheless, Seidenfaden and Wood (1992) removed the species from section Cirrhopetalum and they were placed inside the monotypic section Desmosanthes, because of its non-fringed petals and dorsal sepal. In this analysis, it was clear that B. medusa is nested within Cirrhopetalum. Sample developing can help to improve the status of section Desmosanthes and it may be proposed to merge some species of sections Cirrhopetalum and Desmosanthes. Three unknown species (B. sp6, B. sp8, and B. sp9) were placed between Desmosanthes and Cirrhopetalum. Morphological evidence of leaf and pseudobulb (thick leaf with blunt tip and angled pseudobulb) was highly similar to section Cirrhopetalum even though B. sp8 and B. sp9 were placed in the same clade with B. dentiferum Ridl. Unfortunately, inflorescence characters were not available, so these species can be included in section Cirrhopetalum or Desmosanthes. Seidenfaden and Wood (1992) proposed to place B. lilacinum Ridl and B. sichyobulbon Parish & Rchb. f. in section Careyana (clade C, PP100). Meanwhile, Holttum (1964) assigned B. lilacinum in section 12 along with a few species of section Sestochilus. In combined molecular analysis, these species appeared as sister to section Cirrhopetalum (PP96). Morphologically, combination of characters, such as large and angled pseudobulb, thick and fleshy leaf, adherent lateral sepals at the lower margins and longer length of lateral sepal than dorsal sepal make close affiliation between sections Careyana and Cirrhopetalum. Molecular data have confirmed the status of B. lilacinum and B. sichyobulbon in an independent section. Section Hirtula showed paraphyletic status in both individual and combined molecular data analysis. Hirtula consists of three species, namely B. hirtulum Ridl, B. dayanum Rchb. f, and B. limbatum Lindl (Seidenfaden and Wood, 1992). Holttum (1964) described B. hirtulum in section 9 and B. hespidum (synonym of B. dayanum) in section 12 along with B. limbatum. For B. hirtulum and B. limbatum, Ridley (1924) proposed Hirtula as sectional name. There are some common characters showed by species in section Hirtula, such as inflorescence with more than one flowers, equal length of sepals, and ciliate petals. Combined molecular tree and separate marker analysis showed B. hirtulum and B. limbatum in the same clade with strong support (clade B, PP100) independent of B. dayanum (clade C). So, it is proposed to split down section Hirtula; B. hirtulum and B. limbatum in section Hirtula. Bulbophyllum dayanum must be detached. In conclusion, considerable evidence has been made in delimiting natural sections and the relationships between species could be clearly determined. 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