Molecular Phylogenetics and Evolution 69 (2013) 950–960

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Molecular Phylogenetics and Evolution

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Molecular systematics of (, ) from mainland based on plastid and nuclear sequences

Xiao-Guo Xiang a, André Schuiteman b, De-Zhu Li c, Wei-Chang Huang d, Shih-Wen Chung e, Jian-Wu Li f, ⇑ Hai-Lang Zhou a, Wei-Tao Jin a, Yang-Jun Lai a, Zhen-Yu Li a, Xiao-Hua Jin a, a State Key Laboratory of Systematic and Evolutionary Botany, Institute of Botany, Chinese Academy of Sciences, Beijing 10093, b Herbarium, Library, Art and Archives Directorate, Royal Botanical Gardens, Kew, Richmond, Surrey TW9 3AB, UK c Key Laboratory of Biodiversity and Biogeography, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming, 650204, China d Chenshan Shanghai Botanical Garden, Chenhua Road 3888, Songjiang, Shanghai 201602, China e Botanical Garden Division, Forestry Research Institute, 53 Nanhai Road, Taipei 10066, Taiwan f Xishuanbanna Tropical Botanical Garden, Chinese Academy of Sciences, Menglun Township, Mengla County, Yunnan 666303, China article info abstract

Article history: Dendrobium is one of the three largest genera and presents some of the most intricate taxonomic prob- Received 29 January 2013 lems in the family Orchidaceae. Based on five DNA markers and a broad sampling of Dendrobium and its Revised 5 June 2013 relatives from mainland Asia (109 species), our results indicate that mainland Asia Dendrobium is divided Accepted 17 June 2013 into eight clades (with two unplaced species) that form polytomies along the spine of the cladogram. Available online 27 June 2013 Both Dendrobium and Epigeneium are well supported as monophyletic, whereas sect. Dendrobium, sect. Densiflora, sect. Breviflores, sect. Holochrysa, are paraphyletic/polyphyletic. Many ignored phylogenetic Keywords: relationships, such as the one of major clades formed by D. jenkinsii and D. lindleyi (two members of sect. Dendrobium Densiflora), the Aphyllum group, the Devonianum group, the Catenatum group, the Crepidatum group, and Mainland Asia Molecular systematics the complex are well supported by both molecular and morphological evidence. Sect. Dendrobium Based on our data, we propose to broaden sect. Dendrobium to include sect. Stuposa, sect. Breviflores, and Sect. Densiflora sect. Holochrysa and to establish a new section to accommodate D. jenkinsii and D. lindleyi. Our results Sect. Holochrysa indicated that it is preferable to use a broad generic concept of Dendrobium and to pursue an improved infrageneric classification at sectional level, taking into account both morphology and current molecular findings. Ó 2013 Elsevier Inc. All rights reserved.

1. Introduction intergrading and overlapping morphological variation within and among species (Adams, 2011; Morris et al., 1996; Yukawa and The Dendrobium belongs to the tribe Dendrobieae Uehara, 1996). Since the establishment of Dendrobium by Swartz (Orchidaceae: ) and is one of the three largest gen- (Swartz, 1799), various generic delimitations and infrageneric sys- era in the family Orchidaceae with approximately 800–1500 spe- tems have been proposed based on morphological characters cies (Cribb and Govaerts, 2005; Wood, 2006; Zhu et al., 2009), (Adams, 2011; Brieger, 1981; Burke et al., 2008; Clements, 2003, mainly distributed in tropical Asia, Australasia, and Australia, with 2006; Clements and Jones, 2002; Kranzlin, 1910; Schlechter, a few species extending into the temperate Asian regions and New 1912; Schuiteman, 2011; Seidenfaden, 1985; Tang and Wang, Zealand. Dendrobium is characterized by lateral inflorescences aris- 1951; Wood, 2006; Yukawa et al., 2000). Lindley (1829) estab- ing from the upper part of the shoot, lateral sepals forming a men- lished the subtribe Dendrobiinae, initially recognizing four sections tum with the column foot, and four naked pollinia. An in Dendrobium (Lindley, 1830), and later expanding the number of autapomorphy of this genus is that the rostellum is hollow and sections to 10 (Lindley and Paxton, 1851). Schlechter (1912) pro- swollen and when touched released an opaque, glue-like liquid. posed an infrageneric system of Dendrobium with four subgenera The of Dendrobium is considered to present one of the and 41 sections, which was followed by most authors with a few most intricate problems in Orchidaceae, due to the morphological modifications recognizing a broad Dendrobium with a reasonably diversity, wide distribution range, large number of species, and the natural infrageneric system at section level (not at subgenus level). Brieger (1981) divided the Dendrobiinae into 28 genera, most of ⇑ Corresponding author. which correspond to Schlechter’s sections of Dendrobium. Recently, E-mail addresses: [email protected] (X.-G. Xiang), A.Schuiteman@ results of molecular analyses have shed new light on the taxonomy kew.org (A. Schuiteman), [email protected] (D.-Z. Li), [email protected] (W.-C. Huang), bifl[email protected] (S.-W. Chung), [email protected] (J.-W. Li), of Dendrobium. Most phylogenetic reconstructions indicate that [email protected] (X.-H. Jin). the subtribe Dendrobiinae can be subdivided into three main

1055-7903/$ - see front matter Ó 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.ympev.2013.06.009 X.-G. Xiang et al. / Molecular Phylogenetics and Evolution 69 (2013) 950–960 951 clades (Burke et al., 2008; Clements, 2003, 2006; Schuiteman, were amplified using a standard polymerase chain reaction 2011; Yukawa et al., 1993, 2000). At the same time, many morpho- (PCR). The sequencing reactions were performed using the ABI logical characters of the Dendrobinae appear to be homoplasious, Prism Bigdye Terminator Cycle Sequencing Kit (Applied Biosys- and several previously defined infrageneric taxa of Dendrobium tems, ABI). are not monophyletic (Adams, 2011; Brieger, 1981; Burke et al., 2008; Clements, 2003, 2006; Clements and Jones, 2002; Kranzlin, 2.3. Phylogenetic analyses 1910; Schlechter, 1912; Schuiteman, 2011; Seidenfaden, 1985; Tang and Wang, 1951; Wood, 2006; Yukawa et al., 2000). Some Sequences were aligned using the program Clustal X 1.83 divergent ideas about the taxonomy and classification of Dendrobi- (Thompson et al., 1997) and manually adjusted using BioEdit (Hall, um have been put forward. Based on analyses of nuclear ITS, Cle- 1999). The homogeneity between the nrDNA ITS data and the com- ments and Jones (2002) and Clements (2003, 2006) divided the bined plastid dataset (rbcL, matK, trnH-psbA spacer and trnL intron) broad Dendrobium s.l. into three subtribes with approximately 50 was tested using the incongruence length difference (ILD) test genera. Also based on ITS data, Schuiteman (2011) proposed a (Farris et al., 1995), implemented in PAUP v4.0b10 (Swofford, broader Dendrobium, and subsumed several small genera of the 2002). Following Cunningham (Cunningham, 1997), a significance subtribe Dendrobiinae into Dendrobium. Dressler (1993) and Wood level of P = 0.01 was adopted for this test. In the present study, (2006) presented a traditional, morphology-based consensus view the congruence between data sets was assessed by a comparing of Dendrobium and Dendrobiinae with five or six genera. the topologies and support values of the strict consensus trees of Although there are some agreements, for example, on the exclu- data partitions. This ‘‘hard incongruence’’ test was performed sion of Oxystophyllum and from the Dendrobiinae, such a directly by visually comparing the support and resolution of each tremendous divergence of taxonomic views on Dendrobium s.l. has of the clades in the separate analyses that had a higher bootstrap led to considerable taxonomic uncertainty and debates. As previ- percentage (BP) and posterior probability (PP) than BP > 75 and ous results of molecular systematics were largely based on sparse PP > 90 (Norup et al., 2006; Wiens, 1998). sampling across Dendrobium, or focused on a single section, and The phylogenetic analyses for each matrix were performed mainly utilized either a single DNA marker (especially ITS), or using the maximum parsimony (MP) and Bayesian inference (BI) two, some conclusions and results were weakly supported or even methods in PAUP v4.0b10 (Swofford, 2002) and MrBayes v3.0b4 without statistical support. Therefore, it is desirable to understand (Ronquist and Huelsenbeck, 2003), respectively. the phylogenetic relationships within Dendrobium s.l., as well as For the MP analyses, heuristic searches were conducted with the delimitation of the infrageneric taxa, the evolutionary history 1000 replicates of random addition, one tree held at each step dur- of morphological characters that characterize the infrageneric taxa, ing the stepwise addition, tree-bisection-reconnection (TBR) and the interrelationships among major clades within Dendrobium, branch swapping, MulTrees in effect, and steepest descent off. All to base the analyses on multiple DNA markers and a denser sam- of the characters were unordered and equally weighted, and the pling across Dendrobium. However, it is impossible to sample all gaps were coded as missing data. To evaluate the node support, of the species throughout the range of this large and widespread bootstrap analyses (Felsenstein, 1988) were performed using genus. In the present study, phylogenetic relationships were in- 1000 replicates, with 10 random taxon additions and heuristic ferred using five DNA markers (plastid rbcL, matK, trnH-psbA spacer search options. and trnL intron and nuclear ITS sequences), with sampling of Prior to the Bayesian analysis, the Akaike Information Criterion Dendrobium primarily from mainland Asia with the aims of (1) (AIC) implemented in ModelTest v3.7 (Posada and Crandall, 1998) understanding the delimitation of the infrageneric taxa of Dendro- was used to select the best-fit model of molecular evolution for bium from mainland Asia; (2) reconstructing the phylogenetic rela- each dataset. For the BI analyses, four chains of the Markov Chain tionships within sect. Dendrobium; (3) investigating the Monte Carlo (MCMC) chains were run, sampling one tree every interrelationships among major infrageneric taxa from mainland 1000 generations for 5,000,000 generations, starting with a ran- Asia; (4) reconsidering the taxonomy of Dendrobium. dom tree. Majority rule (>50%) consensus trees were constructed after removing the ‘‘burn-in period’’ samples (the first 25% of the 2. Materials and methods sampled trees).

2.1. Taxon sampling 2.4. Character reconstruction

The taxon sampling in this study included 192 accessions of 109 The evolution of five diagnostic morphological characters that Dendrobium species and of the traditionally recognized genera Epi- have been used to define infrageneric taxa of Dendrobium from geneium and Flickingeria species, in order to represent the geo- mainland Asia was reconstructed using a maximum likelihood ap- graphic and taxonomic diversity in mainland Asia. proach with the Markov k-state one-parameter (Mk1) model newportii, Oxystophyllumchangjiangense, Liparis kumokiri and implemented in Mesquite v2.74 (http://mesquiteproject.org/mes- Malaxis spicata were used as outgroups. The species and voucher quite/mesquite.html). The five reconstructed characters are: (1) specimens used in this study and the GenBank accession numbers distinct elongate rhizome present; (2) leaf sheathing at base; (3) are listed in Table A.1. bright yellow flowers; (4) stems and leaves hairy; (5) hairs on stems and leaves blackish. For the purpose of this analysis we fol- lowed the infrageneric system of Dendrobium as presented by 2.2. Molecular methods Wood (2006) and Zhu et al. (2009). Total genomic DNA from silica-dried material was extracted using a modified CTAB protocol from Doyle and Doyle (1987). For 3. Results this study, four plastid markers (the coding gene rbcL, matK, trnH-psbA spacer and trnL intron) and the internal transcribed 3.1. Sequences and alignment spacers from the nuclear ribosomal DNA (ITS) were used. The PCR and sequencing primers for rbcL, matK, trnH-psbA spacer, trnL In this study, 527 new sequences were obtained. Sequence intron and ITS are listed in Table A.2. The selected DNA regions lengths were as follows: 875 bp for ITS region, 1315 bp for rbcL, 952 X.-G. Xiang et al. / Molecular Phylogenetics and Evolution 69 (2013) 950–960

1993 bp for matK, 1416 bp for trnH-psbA spacer, and 1477 bp for groups. The Fimbriata subclade comprises eight sampled species. trnL intron. Of the combined four plastid regions 6202 bp, 25% The Core subclade includes 41 sampled species and is further sub- were variable (14% parsimony-informative) in the dataset. The divided into seven well supported groups: Group 1 (G1; PP = 100, combined alignment of nrITS and plastid regions comprised BS = 91), the Northern group with 16 species; G2 (PP = 7077 bp, 21% of which were parsimony-informative. Table 1 sum- 100, BS = 100), the Henryi group with 2 species; G3 (PP = 100, marizes the properties of each aligned data partition. BS = 100), the Falconeri group with one species; G4 (PP = 100, BS = 89), the Pendulum group with nine species; G5 (PP = 3.2. Incongruence between data partitions 100, BS = 100), the Devonianum group with two species; G6 (PP = 100, BS = 99), the Crepidatum group with two species; The partition homogeneity test for plastid DNA + ITS shows G7 (PP = 100, BS = 94), and the Aphyllum group with six species character incongruence (P = 0.01), whereas visual inspection indi- (Fig. 2). cates that there are very few ‘‘hard’’ conflicts between the nuclear Clade II (PP = 100, BS = 100) comprises two species from sect. vs. plastid trees (Bull et al., 1993; Mason-Gamer and Kellogg, 1996; Densiflora. Clade III includes 9 species from sect. Stachyobium Quicke et al., 2007). Such conditions have been interpreted by (PP = 100, BS = 100), with four strongly supported subclades. Clade Wendel and Doyle (1998) as a soft incongruence, which might dis- IV (PP = 100, BS = 100) consists of nine species from sect. Formosae. appear with additional data. Gatesy et al. (1999) demonstrated Clade V includes two species belonging to sect. Distichophyllae with that concatenating truly incongruent data sets could still increase weak support (PP = 53). Clade VI (PP = 100, BS = 100) includes eight resolution and branch support. Therefore, we combined the data- species, seven of which are from sect. Crumenata, and the remain- sets for simultaneous analyses. ing two are from sect. Strongyle and sect. Aporum, respectively. Clade VII includes two species from sect. Pedilonum and sect. Calca- rifera (PP = 100, BS = 100). Clade VIII consisting of 4 species from 3.3. Phylogenetic analyses sect. Densiflora, is moderately supported (PP = 98, BS = 51; Fig. 2), and sister to clades VI and VII. Clade IX (PP = 99, BS = 70) includes Phylogenetic relationships based on the ITS data had a better 4 species from sect. Grastidium. resolution than the four combined plastid DNA data (results not shown here). Based on the combined ITS and plastid DNA data, 3.4. Evolution of morphological characters our findings are consistent in the overall topology of the trees pro- duced with maximum parsimony (MP) and Bayesian inference (BI) The analyses of the evolution of morphological characters methods, except for a few of the collapsed nodes. BS values (Boot- indicate complex evolutionary patterns of these morphological strap percentages) were often lower than the PP values (Posterior characters (Fig. 3): (1) an elongate rhizome probably evolved Probability) from the Bayesian analysis. at least 3 times independently in Dendrobiinae: in Clade II, Epi- The BI topology from the combined dataset is the primary tree geneium, and Flickingeria; moreover, this character state also oc- used for discussion of phylogenetic relationships (Fig. 1; the MP curs in the Australasian sections Cadetia, Diplocaulobium and strict consensus tree is not shown). Our results indicate that the Microphytanthe, which were not included in our analysis; (2) subtribe Dendrobiinae can be divided into two well supported sis- the absence of a leaf sheath has probably evolved at least three ter clades (PP = 91, BS = 89), Epigeneium (recognized at genus rank times independently within the Asian clade alone, in Clade I, II, by Wood (2006) and Zhu et al. (209)) (PP = 100, BS = 100) and and VIII; it is also found in Flickingeria, Epigeneium and several Dendrobium (PP = 100, BS = 98), while the latter can be subdivided sections in the Australasian clade, e.g., sect. Latouria, sect. Dend- into a predominantly Australasian clade (PP = 100, BS = 93) and a rocoryne and sect. Rhizobium; (3) bright yellow flowers probably predominantly Asian clade (PP = 100, BS = 98). The genus Flickinge- evolved more than ten times independently; (4) hairs on stems ria is nested within the Australasian clade (PP = 88, BS = 62) and leaves at least twice; (5) blackish hairs on stems and leaves (Fig. 1). These findings agree with previous studies. Within the once or twice. Asian Dendrobium clade, eight major clades are supported; how- ever, these clades form a polytomy along the backbone of the Asian clade, and the interrelationships among them are therefore unre- 4. Discussion solved (Fig. 1). Our results indicate that several sections of the Asian clade in Zhu et al. (2009) and Wood (2006) are not 4.1. The infrageneric taxa of Dendrobium from mainland Asia monophyletic. Clade I comprises 59 species (PP = 95, BS = 51), most of which With more sampling and more DNA sequences, our results not belong to sect. Dendrobium. The remaining are from sect. Breviflo- only recover and strongly support three major clades in the Dend- res, sect. Holochrya, sect. Densiflora, and sect. Stuposa. Clade I is sub- robiinae as found in previous studies based on ITS/matK (Yukawa divided into two major subclades, a Core subclade (PP = 100, et al., 1993; Burke et al., 2008; Clements, 2003, 2006; Schuiteman, BS = 91) and what we informally call Fimbriata subclade 2011), but also revealed eight major clades forming a polytomy (PP = 100, BS = 64), along with several unplaced species/species along the spine of our cladogram of Dendrobium, which indicates

Table 1 The statistics from the analyses of the chloroplast and nuclear data sets from the parsimony analysis.

Information ITS rbcL matK trnH-psbA trnL intron plastid DNA Combined No. taxa 195 167 165 134 114 177 197 Aligned length 875 1315 1993 1417 1477 6202 7077 No. variable characters 600 175 512 202 628 1477 2077 No. parsimony-informative characters 503 84 322 107 370 877 1380 Tree length (steps) 3851 286 854 349 1087 2762 6834 Consistency index (CI) 0.292 0.689 0.696 0.645 0.735 0.640 0.430 Retention index (RI) 0.787 0.869 0.830 0.822 0.807 0.773 0.774 Model GTR + I + G GTR + I + G TVM + I + G TVM + I + G TVM + I + G TVM + I + G GTR + I + G X.-G. Xiang et al. / Molecular Phylogenetics and Evolution 69 (2013) 950–960 953

Fig. 1. Bayesian tree from the analysis of the combined ITS and four plastid DNA regions. The previous recognized sections from Zhu et al. (2009) and Wood (2006) are highlighted by the color of branches. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) 954 X.-G. Xiang et al. / Molecular Phylogenetics and Evolution 69 (2013) 950–960

Fig. 2. (A–C) Phylogenetic tree obtained by Bayesian analysis of the combination of ITS and plastid regions, showing the detailed relationships of the Australasian group and the outgroups (Part A), Clade II – VIII (Part B) and Clade I (Part C). Numbers above the branches indicate posterior probabilities (PP) and bootstrap percentages (BS). that Dendrobium radiated in mainland Asia. In addition, some new of species from the traditionally defined sect. Dendrobium as well clades were discovered. Our analyses show that the taxonomy of as several species from two other sections, sect. Breviflores and the infrageneric taxa of Asian Dendrobium is more complicated sect. Holochrya, and has strong support. Most branches within than previous results have indicated. On one hand, some sections the Core subclade are also strongly supported (Fig. 2). and genera, such as sect. Stachyobium, sect. Grastidium, Epigeneium Two of three sampled species of sect. Breviflores, including the and Dendrobium, are well supported as monophyletic (but with type of the section, are nested with species of sect. Dendrobium. sect. Grastidium very sparsely sampled). On the other hand, some The remaining one is nested with D. stuposum, the type species of currently defined infrageneric taxa of Dendrobium, such as sect. sect. Stuposa, forming a branch as the sister group of D. fimbriatum, Dendrobium, sect. Holochrysa, and sect. Densiflora, are paraphylet- a species of sect. Holochrya. Except for the unplaced D. capillipes, all ic/polyphyletic, and their taxonomic status needs to be reassessed of the sampled members of sect. Holochrya are nested within Clade and redefined. I and mainly with sect. Dendrobium and several other sections. Sect. Densiflora is divided into three groups, one (comprising D. chrys- otoxum only) is nested within Clade I with species of sect. Dendrobi- 4.1.1. Sect. Dendrobium and its alliance um and other sections, while the remaining two form two different As the type section of Dendrobium, the delimitation of sect. clades along the spine of the Dendrobium of mainland Asia. Dendrobium has nomenclatural implications for the entire infra- Based on these findings, we propose to subsume three sections, generic system. Wongsawad et al. (2005) stated that sect. Dendro- i.e., sect. Breviflores, sect. Holochrya, and sect. Stuposa, into sect. bium is not monophyletic, and Clements (2006) also argued that Dendrobium. One species of sect. Densiflora is also included in sect. the currently defined sect. Dendrobium is polyphyletic. Our results Dendrobium. Under this scenario, the delimitation of sect. Dendro- indicate that sect. Dendrobium is paraphyletic, and that some spe- bium is broadened to include at least one species without leaf cies from several other sections, such as sect. Breviflores, sect. Holo- sheaths (D. chrysotoxum). chrya, and sect. Densiflora, are nested in different subclades within Clade I. The sampled species of sect. Dendrobium (as traditionally circumscribed) form two groups (Figs. 1 and 2), one is the Core 4.1.2. Sect. Densiflora subclade of Clade I, and the other is formed by two species, D. Wongsawad et al. (2005) demonstrated that sect. Densiflora is hookerianum and D. ochreatum. The Core subclade largely consists not monophyletic, while Clements (2006) recognized part of sect. X.-G. Xiang et al. / Molecular Phylogenetics and Evolution 69 (2013) 950–960 955

Fig. 2 (continued)

Densiflora as a well-supported group within Dendrobiinae, which (Fig. 2). Our morphological studies indicate that these two clades he treated as the separate genus Callista. Our results indicate that are readily distinguished by their vegetative characters (Fig. 3). sect. Densiflora is probably polyphyletic. Apart from D. chrys- of Clade II, comprising D. lindleyi and D. jenkinsii, have a otoxum, which is included in sect. Dendrobium, the remaining sam- more or less elongate rhizome, depressed, short, clustered, multi- pled species of sect. Densiflora form two clades, i.e., Clade II and VIII noded and fleshy pseudobulbs, each pseudobulb with one terminal 956 X.-G. Xiang et al. / Molecular Phylogenetics and Evolution 69 (2013) 950–960

Fig. 2 (continued) X.-G. Xiang et al. / Molecular Phylogenetics and Evolution 69 (2013) 950–960 957

Fig. 3. Character evolution based on maximum likelihood method. The topology and species order is the same as in Figs. 1 and 2.

leaf, a thick-coriaceous leaf blade, slender and sparsely-flowered perianth. The distinct morphological differences suggest that inflorescences, and a lip which is much longer than the other the two clades (II and VIII) could be treated as separate sections. parts of the perianth. Plants of Clade VIII (which includes the However, considering that they form part of a polytomy in our type species of sect. Densiflora, D. densiflorum) have very short cladogram, we suggest that it is premature to make formal rhizomes; erect and more cane-like, fleshly stems with several proposals, especially given the great morphological similarity leaves in the upper part, robust and densely flowered inflores- between D. chrysotoxum (sect. Dendrobium) and D. densiflorum cences, and the lip similar in length to the other parts of the (sect. Densiflora). 958 X.-G. Xiang et al. / Molecular Phylogenetics and Evolution 69 (2013) 950–960

4.1.3. Sect. Formosae and sect. Distichophyllae 4.2. Systematics of redefined sect. Dendrobium Sathapattayanon (2008) showed that the traditionally circum- scribed sect. Formosae was subdivided into two groups along with Our current molecular systematics include approximately 70% two unplaced species (D. trigonopus and D. jerdonianum). Our re- of the species of the redefined sect. Dendrobium, and the phyloge- sults indicate that sect. Formosae from mainland Asia is well char- netic relationships are well resolved among most species. Wongsa- acterized by the black hairs on leaves and leaf sheaths (Fig. 3), very wad et al. (2005) suggested that sect. Dendrobium comprises at short inflorescence with few relatively large, predominantly white least seven clades. However, our results indicate that four of these flowers, and is strongly supported as monophyletic except for the clades, D1, D3, D5, and D7, are paraphyletic/polyphyletic. The cur- unplaced D. trigonopus. Wood (2006) included D. senile into sect. rent defined sect. Dendrobium can be subdivided into two major Formosae, and Schuiteman (2011) treated D. senile as an unplaced subclades, the Core subclade and the Fimbriata subclade, along species; our results suggest that D. senile belongs to sect. Dendrobi- with several unplaced species/species group. The Fimbriata subc- um (Fig. 2). The sister group relationships between sect. Formosae lade includes species from three different traditionally defined sec- and sect. Distichophyllae needs further study. We sampled only tions and is supported by few vegetative characters, such as fleshy two species of sect. Distichophyllae, of which one may not belong but hard stems more or less longitudinally ridged, leaves more or to this section (D. jerdonianum); this clade is weakly supported less leathery and at the upper part of stem, several inflorescences and needs more sampling (Fig. 2). arising from the top of older stems. This morphologically diverse group needs more study to understand the evolutionary patterns. The Core subclade mostly consists of species from the traditional 4.1.4. Sect. Stachyobium morphologically defined sect. Dendrobium, including D. monili- Sect. Stachyobium is strongly supported as monophyletic forme, which is the type of the genus. (Fig. 2). This section is mainly distributed in mountainous regions in mainland Asia and is well characterized by its tufted growth ha- bit, fleshy stems enclosed by the leaf sheaths, inflorescences arising 4.2.1. Phylogenetics of seven groups within the Core subclade from the current year’s and usually immature stems, peduncle and Within sect. Dendrobium, the Northern group is strongly sup- rachis almost filiform, more or less elongate with several to many ported. It is mainly distributed in subtropical/temperate mainland flowers. Our results indicate that sect. Stachyobium is subdivided Asia, while most tropical species are basal in Clade I (Fig. 2). In gen- into four strongly supported subclades, and that species of reduced eral, species distributed in subtropical and temperate mainland vegetative stature, such as D. microbulbon and D. peguanum, are sis- Asia can be subdivide into three groups: (1) some species, such ter to the remaining species. as D. denneanum and D. hancockii, are distributed northwards along the valleys of major rivers (such as the Mekong and the Salween); (2) some species, such as the Dendrobium moniliforme complex, the 4.1.5. Sect. Aporum, sect. Crumenata and sect. Strongyle D. scoriarum complex, and D. hercoglossum, are distributed along These three sections form the Clade VI, which is strongly sup- the northern geographical limit of Dendrobium; (3) some species, ported (Fig. 2). Although only eight species were sampled, our re- such as D. nobile, are widespread. This pattern suggests that Dendr- sults indicate that boundaries of these sections need to be obium may have spread from tropical to subtropical regions. reevaluated. Yukawa and Uehara (1996) stated that several ana- Although there are considerable differences in size and floral tomical characters of these three sections are associated with xer- color pattern among members of the Northern group (Fig. 2, omorphy. Schuiteman (2011) suggested that these three sections Fig. 3C), it is characterized by uniform stems usually not swollen; and sect. Bolbodium should be treated as one section, which is sup- leaves at upper part of stem; very short and few-flowered inflores- ported by the current results. cence arising from the leafless previous year’s stems; lip more or less hairy and with a callus. Dendrobium wangliangii is a very special and narrowly endemic 4.1.6. Sect. Pedilonum and sect. Calcarifera Chinese species, characterized by its 1–3 cm long and clustered Two species of these two sections form the Clade VII. It is inter- stems prostrate on the trunk, shedding leaves before the 4-month esting to note, as was found by previous workers, that this clade is dry season. Hu et al. (2008) considered it close to D. flexicaule; the sister group of the Clade VI with strong support (Fig. 2). Plants however, our results indicate that it is the sister species of D. devo- of these two clades differ significantly in their habitat: species of nianum, which is consistent with morphological characters, such as Clade VI mostly grow in relatively dry habitats, while those of the short inflorescence, hairy lip and the distinctive floral color Clade VII grown in more humid habitats. However, some species pattern. of clades VI and VII may co-occur in the same habitat, although Dendrobium henryi is the sister species of D. menglaense (most the hottest and driest habitats are mainly inhabited by species likely a synonym of D. chrysocrepis) with strong support from clade VI. Despite the differences in vegetative characters, (PP = 100, BS = 100), which is supported by several morphological these two clades share many inflorescence and floral characters: characters, such as very short and 1–2-flowered inflorescence aris- short lateral inflorescences arising from penultimate and older ing from the leaf-bearing stem, a golden-yellow and hairy lip canes; pedicel frequently inserted at a right angle to mentum; which is more or less concave. These two species are sister group mentum elongate and longer than column, lip usually entire and of the Northern group plus another two species, D. leptocadum expanded. and D. unicum, with strong support. is an isolated species in sect. Dendrobium by its unique vegetative characters, such multi-branched and pen- 4.1.7. Sect. Grastidium dent stems, moniliform internodes, and very narrow leaves. In our Sect. Grastidium is strongly supported as monophyletic and is analyses it is sister to the clade comprising the Northern group, the nested within the Australasian clade including Flickingeria (Dendr- Henryi group, and D. leptocadum, and D. unicum, with strong sup- obium sect. Crinifera), and other species from Australasia (Fig. 2). port (PP = 96, BS = 82). This section is poorly represented in mainland Asia, but highly spe- The Crepidatum group comprises two species and is strongly cies-rich in New Guinea. It is the largest section in the genus supported by morphological characters, such as fleshy stems Dendrobium. which are uniformly green and not swollen, many very short X.-G. Xiang et al. / Molecular Phylogenetics and Evolution 69 (2013) 950–960 959 inflorescences usually with two flowers arising along the old 4.4. The taxonomy of Dendrobium stems, pedicel and ovary very long, and densely hairy lip. The Pendulum group is a morphologically more or less uniform Using several markers and a denser species sampling, especially group within the Core subclade, characterized by their fleshy and in sect. Dendrobium from mainland Asia, our results are consistent yellow stems with nodes more or less swollen; papery leaves usu- with previous identification of the three major clades of the sub- ally at the upper part of the new stems, short inflorescences with tribe Dendrobiinae, and the widespread homoplasy of some diag- one to several arising from leafless stems, colorful flowers usually nostic morphological characters And our results are parallel with with pink tips. previously reported results for the Australasian clade of Dendrobi- The Aphyllum group is very widely distributed in tropical re- um (Adams, 2011) in the degree of resolution, finding of polyto- gions, from Sri Lanka to Australia, and is well supported by mor- mies, and well supported subclades. Therefore, the genus can phological characters, such as the terete, uniformly green stem, now be looked at as an entity. These data can in our opinion best which is usually not much swollen, many short and 1–2-flowered be interpreted in terms of a broad generic concept for Dendrobium inflorescences arising from nodes along the stem, and the distinc- with infrageneric sectional classification, as has been concluded for tive trumpet-shaped lip enclosing the column. the Australasian clade (Adams, 2011). It is not practical to use the phylograms to construct numerous genera, as such an attempt would lead to many small, equivocal genera with little or no mor- 4.2.2. Phylogenetics of the species complex phological support, leading to ongoing generic taxonomic instabil- Dendrobium catenatum (syn. D. officinale) is often treated as a ity. The broad concept for Dendrobium meets the key plant member of the D. moniliforme complex (Pearce and Cribb, 2002), taxonomic principles of stability, monophyly, predictability and but our analyses indicate that it is nested within the branch includ- use of morphological characters. ing D. flexicaule, D. scoriarum, and D. aduncum with strong support In summary, although only about 20% of the total Dendrobium (PP = 100, BS = 100). All species in this clade share many charac- species have so far been included in molecular studies (including ters, such as fleshy stems with dark color, uniform, not swollen this one), our sampling has enabled revision or refinement of pre- internodes, leaves and leaf sheaths usually with purple spots, short vious conclusions such as phylogenetic relationships of section and few-flowered inflorescences arising from old leafless stems. Dendrobium, sect. Densiflora and sect. Formosae. We have clarified Dendrobium moniliforme is one of the most widespread species the Asian sections and some subgroups, broadened sect. Dendrobi- of Dendrobium, ranging from Japan through China to India and Ne- um to include sect. Stuposa, sect. Breviflores and sect. Holochrysa, pal, mostly along the northern geographical limit of Dendrobium. and suggested a new section for D. jenkinsii and D. lindleyi. Further Dendrobium moniliforme complex, characterized by the uniformly work should focus on refinement and development of the sectional slender stem, slightly bifid leaves, inflorescences usually arising classification of the remaining majority of unstudied species. from upper part of old leafless stems, pale white bracts with a brownish zone, and more or less hairy lip, presents problems in species delimitation. Wood (2006) recognized only one species in Acknowledgments this complex; Jin et al. (2009) recognized D. moniliforme and D. wilsonii besides D. fanjingshanense; Zhu et al. (2009) recognized We appreciate the critical comments of two anonymous four species. All of the six named entities of this complex, i.e. D. reviewers. Funds were provided by a grant from the National Nat- fanjingshanense, D. henanense, D. huoshanense, D. moniliforme, D. ural Science Foundation of China (Grant No. 31107176) and the okinawense and D. xichouense were sampled in the current study. Knowledge Innovation Program of the Chinese Academy of Sci- Our results indicate that D. moniliforme is not a monophyletic spe- ences (Grant No. KSCX2-EW-Z-1). cies, with different accessions nesting in various positions with the five other species mentioned. This suggests that it would be logical Appendix A. Supplementary material to follow Wood (2006) and to recognize only one species in this complex. 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