Molecular Molecular SystematicsSystematics and and DNA DNA BarcodingBarcoding ofof

Orchids: A Cursory PerusalOrchids: A Cursory Perusal

Painting by: Ms. Hemalata Pradhan Renanthera imschootiana

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2 MOLECULAR SYSTEMATICS AND DNA BARCODING OF ORCHIDS:

A CURSORY PERUSAL

The family Orchidaceae is one of the highly evolved and largest families of angiosperms with approximately 25,000-35,000 species belonging to 750 to 850 genera distributed worldwide (Chase 2005, Hossain 2011). This highly advanced family of monocots is represented by mostly herbaceous plants which are epiphytes, lithophytic, saprophytes or terrestrial; possess distinct floral morphology, pollination mechanisms and minute seeds; and are associated with unique fungal partners (mycorrhizae), (Kumar et al. 2007). The characteristic features by which orchids can be distinguished from other plants are: bilateral symmetry/zygomorphic flowers, presence of labellum or lip (highly modified petal), fused stamens and carpels forming specialized structure called gymnostemium or column, inferior ovary which is generally twisted to 180° (resupinate flowers) and extremely minute seeds (Chowdhery 1998). Orchids are distributed worldwide mainly in the wet tropics. They have not been reported from the polar region and the driest of deserts (Chase 2005). The higher-level classification of Orchidaceae has traditionally been based on the construction of gymnostemium or column. The column because of its details is unique to the family (Chase 2005). The family Orchidaceae contains several megagenera with 1000+ sp. e.g., Bulbophyllum, Epidendrum, Pleurothallis and Dendrobium (Whitten et al. 2007). The reasons for such explosive speciation, possibly because of ecological adaptations, physiological/ morphological innovations, or accelerated rates of morphological/ molecular change, are still not properly understood (Whitten et al. 2007). However, orchid species are generally difficult to identify as these are classified primarily on the basis of floral morphology that changes with pollinator preferences (Cameron 2004). Therefore, for better circumscription and inferring relationships among the difficult to classify Orchidaceae genera, molecular systematic studies have been undertaken, by different groups, utilizing DNA regions from the mitochondrial, plastid and nuclear genomes. However, before perusing the molecular systematics of orchids, a brief appraisal of the morphology based classification of the family is necessary.

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2.1 CLASSIFICATION BASED ON MORPHOLOGICAL CLADISTICS

The father of taxonomy, Carl von Linnaeus was the first to publish binomials

of 69 orchid species belonging to 7 genera in the first edition (1753) of Species Plantarum and 85 species of 10 genera in the second edition (1762-63). Although, Linnaeus was the first to publish orchid binomials, the systematics of Orchidaceae began with the classification by Olof Swartz (1805, cited in Pridgeon 2009). Swartz provided a key to 25 orchid genera and divided them on the basis of number of anthers, one or two. This formed the basis for sub-familial classification. He further segregated the monandrous orchids into three groups depending on the position of the anther (cited in Pridgeon 2009). Louis Claude Richard (1817) stressed the need for taking into consideration the structure of pollinia and the column as well in characterizing orchids (cited in Pridgeon 2009). Based on Swartz’s classification, John Lindley (1830-1840), the father of orchid taxonomy, classified 3000 species of orchids that belonged to 394 genera distributed in seven tribes on the basis of the structure and position of the anther (cited in Pridgeon 2009). Lindley was the first to divide the family into tribes and recognized seven tribes (Lindley 1830-1840). However, Bentham (1881) recognized only five tribes and delineated 27 subtribes under these tribes in Genera Plantarum. Pfitzer (1887) criticized the classification of Bentham and laid more emphasis on vegetative characters, such as, vernation, number of pseudobulb internodes, and growth habit than the conventional column features for classifying orchids (cited in Pridgeon 2009). Pfitzer (1887) recognized 32 tribes, with a number of subtribes. Schlechter (1926) again classified Orchidaceae based on Pfitzer’s (1887) classification. In this classification, only four tribes were recognized and the remaining tribes and subtribes of Pfitzer’s classification were treated as subtribes (Schlechter 1926). The Schlechter’s system of classification was not in accordance with the new rules of nomenclature proposed at that time, therefore, Dressler and Dodson (1960) supplemented and changed slightly to bring it into compliance with the International Code of Botanical Nomenclature. Dressler and

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Dodson (1960) recognized forty subtribes in two subfamilies- Cypripedioideae (including Apostasia and Neuwiedia) and Orchidoideae. Garay (1972) based on additional characters relating to floral vascularization, embryology, and seeds divided the Orchidaceae into five subfamilies, which were Apostasioideae, Cypripedioideae, Orchidoideae, Neottioideae and Epidendroideae. In 1974, Dressler revised the classification earlier proposed by him along with Dodson (1960), by adding the sub-family Apostasioideae, as proposed by Garay (1972). Thus, the three sub-families recognized by Dressler (1974) were Apostasioideae, Cypripedioideae and Orchidoideae. Later Dressler (1981) once again revised the classification of the family Orchidaceae by recognising six sub-families. The three new sub-families added to his earlier classification (Dressler’s 1974) were Spiranthoideae, Epidendroideae and Vandoideae taking into consideration additional features, such as, subsidiary cell development and column structure. The latest and most accepted classification of the Orchidaceae that was based mainly on the anther morphology was proposed by Dressler (1993). According to this classification, the family comprises 850 genera and 20,000 species. These were arranged in 70 sub-tribes, 22 tribes and five sub-families (Dressler 1993). In Dressler’s system of classification an evolutionary progression on the basis of morphological characters, especially the number of anthers, was considered. The five sub-families recognised were: Apostasioideae, Cypripedioideae, Orchidoideae, Spiranthoideae and Epidendroideae, the last being the largest. The progression was envisaged to be from two or three anthers in the Apostasioid orchids (Apostasia and Neuwiedia) through two in the Cypripedioids (Cyprepedium, Medipedium, Paphiopedilum, Phragmipedium, and Selenipedium) to one in the monandrous orchids (Epidendroideae, Orchidoideae and Spirnthoideae; Dressler 1993). The major conclusion of Dressler’s classification was that Monandrae was monophyletic. However, the lack of complete androecial/gynoecial fusion in Apostasioideae indicated that this was sister of the rest, followed by Cypripedioideae in which though there is a complete fusion of androecium and gynoecium, diandrous condition is maintained. The other families though exclusively monandrous were not grouped together but were considered as sister clades (Dressler 1993). The major

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drawback of this classification was the narrow treatment of Neottioid orchids, which lack well-developed pollinia. These were kept as a tribe within sub-family Epidendroideae (Dressler 1993, Chase 2005). In addition to none-too-satisfactory circumscription of the Neottioids, the other major group of orchids that was problematic was Vanilloids, the columns of which are like those of the Epidendroids, but vegetatively they are highly divergent from all other orchids (Cameron and Dickison 1998, Stern and Judd 2000, Chase 2005). The classification of Orchidaceae based on floral morphology could never be accurate as orchid flowers are supposed to be highly susceptible to morphological plasticity due to pollinator selection pressure, yet floral characters above all others were used traditionally to classify the family (Dressler and Dodson 1960, Dressler 1974, 1981, 1993). With the increasing availability of DNA data and molecular phylogenetic studies, slightly different relationships among the different sub-families, tribes and sub-tribes have been revealed. The molecular studies have utilized genes from both nuclear and cytoplasmic genomes for phylogenetic circumscription of Orchidacecae at various taxonomic levels i.e., whole family, sub-family, tribe and sub-tribe. The pioneering two DNA based studies of Chase et al. (1994) and Cameron et al. (1999) had pointed out different patterns of relationships within Orchidaceae as compared to the morphological cladistics of Dressler (1993), discussed in detail in later section. Subsequent to these two initial reports, numerous DNA based phylogenetic analyses have been published, at the level of family (Freudenstein et al. 2004, Cameron 2004), sub-families (Neyland and Urbatsch 1995, 1996, Cox et al. 1997, Freudenstein and Chase 2001), tribes (Douzery et al. 1999, Kores et al. 2001, Goldman et al. 2001) and sub-tribes (Yukawa et al. 1996, Cozzolino et al. 1996, Pridgeon et al. 2001, van den Berg et al. 2005, Clements 2003). These studies had utilized many loci from the plastid genome viz., atpB, rbcL, matK, psaB, trnL-F; one, nad1 intron from the mitochondrial genome, and 26S rDNA and ITS from the nuclear genome. In terms of general patterns of relationships among orchids (described later), these studies have resulted in remarkably similar conclusions, regardless of the genome on which these were based. Based on these relationship patterns, as revealed by different DNA

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regions, a new classification of Orchidaceae was proposed by Chase et al. (2003). The new classification based on both morphological and molecular data divided the family Orchidaceae into five subfamilies – Apostasioideae, Vanilloideae, Cypripedioideae, Epidendroideae and Orchidoideae. In this classification, the members of the Neottioideae (Garay 1972), were distributed in Epidendroideae and Orchidoideae. The fifth family named as Vanilloideae was created by assembling some members, which were earlier included in Epidendroideae and which have distinct morphological characters, such as, reticulate venation and crustose seeds (Chase et al. 2003). The other salient features of this classification are:

1. Vanilloideae are sister to all other sub-families except Apostasioideae,

which means that the reduction to a single anther occurred at least twice. 2. Most of the Spiranthoid orchids are embedded in the Orchidoids and hence

are treated as a tribe in Orchidoideae. 3. The members of tribe Tropidieae earlier included in Spiranthoideae were

merged in Epidendroideae. The former, which was earlier segregated on the basis of terminal position of the anther, was put within Orchidiodeae, as this character was not considered reliable.

4. Except Epidendreae, Vandeae and Cymbidieae other tribes and sub-tribes within Epidendroideae could not be resolved.

Though the classification by Chase et al. (2003) correctly identifies sub-

familial relationships, many sub-tribes were left without any tribal affiliation. This concern was addressed in many subsequent studies that were carried out for the circumscription at sub-familial, tribal and sub-tribal levels utilizing different DNA regions. The examples of the use of different DNA regions in molecular systematics of Orchidaceae at various taxonomic levels are described and discussed below.

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2.2 DNA REGIONS/GENES USED FOR PHYLOGENETIC ANALYSIS AT VARIOUS TAXONOMIC LEVELS

2.2.1 Family Level

The nucleotide sequences have been considered to be of significant importance in phylogenetic studies as they could be rapidly produced and easily assessed for homology (Palmer et al. 1988). The first two pioneering studies on orchids by Chase et al. (1994) and Cameron et al. (1999), had used rbcL gene from the plastid genome to evaluate the monophyly of the family Orchidaceae. The rbcL gene (~ 1430 bp) encodes the large subunit of ribulose-1, 5-bisphosphate carboxylase/oxygenase (RUBISCO), a vital enzyme for photosynthesis (Soltis and Soltis 1998). The nucleotide variations in rbcL were analysed in 33 species by Chase et al. (1994) and 171 species by Cameron et al. (1999). The genera studied by Chase et al. (1994) were spread across the whole family and represented all sub-families. Out of 171 species analyzed by Cameron et al. (1999), 158 belonged to about 149 genera of Orchidaceae and 13 were out-group species. The orchid species analyzed represented all five sub-families and nearly all tribes and sub-tribes (Cameron et al. 1999). The data of both groups supported monophyletic nature of Orchidaceae with five major monophyletic clades. With a few exceptions, these major clades corresponded to the accepted sub-families defined by Dressler (1993). Besides leading to some important inferences, these studies revealed patterns of sub-familial relationships within Orchidaceae, which were different from those deduced through morphological cladistics. It was confirmed that the Apostasioids shared a unique genetic relationship with the rest of the orchids, thus making their treatment as a separate family unnecessary, as was suggested by Lindley (1830-1840). The monophyly of Apostasioideae with Apostasia and Nuewidia as two genera was established. As the representatives of the monandrous orchids did not co-seggregate as a clade their monophyletic nature was not established. Spiranthoideae were embedded in Orchidoideae, thus making the latter unnatural. The Vanilloid orchids were an unexpected major clade, thus justifying their treatment as a distinct subfamily (Chase et al. 1994, Cameron et al. 1999). Thus, these studies established the use of rbcL gene for

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phylogenetic reconstruction. In the data set provided by Cameron et al. (1999), rbcL was of greatest utility in inferring relationships at lower taxonomic levels despite its moderately conserved nature (Palmer et al. 1988). The inability of rbcL in obtaining monophyly in some clades was attributed to superficial sampling of the genera as in Epidendroideae, in which sampling and bootstrap support was lower than the values elsewhere in the family (Cameron et al. 1999). Thus, the final conclusion was that rbcL alone is not likely to provide robust estimates of phylogeny and therefore, more data from other gene regions along with complete sampling would be required to obtain complete phylogeny of Orchidaceae (Cameron et al. 1999). Neyland and Urbatsch (1996) sampled the family by comparing DNA nucleotide sequences of the plastid locus ndhF from 34 orchid species representing all five sub-families. Out of 34, twenty-nine represented ten tribes and 20 subtribes of the subfamily Epidendroideae, whereas only five were from other subfamilies. Along with, two out-group species were also included. The gene ndhF codes for a subunit of the type 1, multi sub-unit NADH: plastoquinone oxidoreductase (NADH dehydrogenase), an important respiratory enzyme complex (Shinozaki et al. 1986). The phylogenetic analysis revealed the family was monophyletic with five sub-families. However, the results of Neyland and Urbatsch (1996) and those of Chase et al. (1994) were considered as tentative because of limited sampling, and the importance of extended sampling for accurate phylogenetic reconstruction was again emphasized (Freudenstein et al. 2004). A few more studies using DNA sequences from the nuclear and mitochondrial genomes of Orchidaceae were published in the subsequent years. These include a study by Cameron and Chase (2000), which was based on the comparison of nuclear ribosomal 18S DNA among 26 genera. Based on this study, same five monophyletic sub-familial clades that were earlier found in the rbcL tree (Chase et al. 1994, Cameron et al. 1999) were recovered (Cameron and Chase 2000). This investigation based on a nuclear gene helped in assigning a few of the achlorophyllous orchid genera to their correct taxonomic positions, which could not be achieved based on the study of genes from the plastids. However, the rate of sequence mutation in 18S was too low to be of use in addressing relationship at levels lower than sub-family or tribe (Cameron and Chase 2000). Therefore, sequences from the mitochondrial genome were also tested to address higher

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level relationships within the Orchidaceae by Freudenstein and Chase (2001). The nad1b-c intron was sequenced from 100 orchid species representing all subfamilies of Orchidaceae and Hypoxis as outgroup taxon (Freudenstein and Chase 2001). The data provided evidence for the monophyly of at least four sub-families. However, the level of sequence variation found in this gene region was again not sufficient for addressing relationships below the rank of sub-family or tribe (Freudenstein and Chase 2001).

The inability of nuclear and mitochondrial genes in elucidating the

phylogenetic relationships below sub-family or tribal level made the researchers to switch back to plastid genes. The advantages of using plastid genes include unilateral inheritance, numerous copies per cell, ease of amplification and sequencing, as well as absence in animals and fungi (Palmer et al. 1988, Clegg and Zurawski 1992, Olmstead and Palmer 1994). The last one is especially significant for studies of Orchidaceae since they typically live in symbiotic association with fungal partners. An expanded plastid phylogeny for the family was reported by Freudenstein et al. (2004). In this study, along with rbcL, matK sequences were also generated for 173 representative species belonging to all five sub-families along with out-group species. The matK, a 1,500 bp long chloroplast gene is located within the trnK intron. It encodes a maturase-like protein that is involved in group II intron splicing (Ems et al. 1995). With the inclusion of matK sequences, the number of parsimony informative sites increased and hence, better resolution was obtained. The monophyly of each of the recognized subfamilies (Apostasioideae, Vanilloideae, Cypripedioideae, Orchidoideae and Epidendroideae) was supported at the 98% bootstrap percentage (BP) level or above in the combined analysis. This was a significant increase for some groups over the rbcL results [Orchidoideae was 78% and Epidendroideae was 65% with rbcL alone, Cameron et al. 1999] (Freudenstein et al. 2004). Branch support for many clades increased significantly when compared with an earlier analysis (Cameron et al. 1999), indicating that matK added substantially to the signal present in rbcL. Similarly, Cameron (2004) used psaB along with rbcL gene sequences for studying intra-familial relationships within the Orchidaceae. The psaB gene is one of five that code for a total of two large (psaA and psaB) and three smaller subunits (psaC, psaI,

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and psaJ) that make up protein P700 of photosystem I (Cameron 2004). The psaB gene was found to contain a similar amount of phylogenetic signal as rbcL as a low bootstrap support was obtained. However, the combined analysis of psaB +rbcL with strict consensus tree and good bootstrap values, presented strong support for each of the five subfamilies but failed to resolve the positions of Cypripedioideae and Vanilloideae (Cameron, 2004). Thus, the phylogenetic relationships of Orchidaceae at family level were best inferred by matK+rbcL sequences generated and analyzed by Freudenstein et al. (2004).

Table 1: A summary of the molecular phylogenetic studies at Family level in the

family Orchidaceae

DNA Region(s) Number of Species

Inference Reference

rbcL rbcL ndhF 18S (nuclear) nad1b-c intron (mitochondrial) rbcL+matK rbcL+psaB

33

171 34 26

100

173

173

Monophyletic with 5 sub-families Monophyletic with 5 sub-families Effective resolution at the subtribal level within Epidendroideae, but intertribal relationships are unresolved Low variations below sub-family or tribe level Monophyly of 4 sub-families was obtained Resolution increased with inclusion of matK sequences Resolution was same as with rbcL alone

Chase et al. (1994) Cameron et al. (1999) Neyland & Urbatsch (1996) Cameron & Chase (2000) Freudenstein & Chase (2001) Freudenstein et al. (2004) Cameron (2004)

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2.2.2 Sub-family Level

The sub-family Cypripediodeae contains five genera (Paphiopedilum, Phragmipedium, Cypripedium, Selenipedium and Mexipedium) with 150-170 species (Cox et al. 1997). Cox et al. (1997) tried to infer molecular phylogenetic relationships among these genera by comparing nuclear ribosomal internal transcribed spacer (nrITS) nucleotide sequences of 100 slipper orchid species. The ITS region of 18S-26S Nuclear DNA (nrDNA) contains two spacers, ITS1 and ITS2 (each <300 bp) with 5.8S rRNA gene in between (Baldwin et al. 1995). The parsimony analysis based on comparison of the ITS sequences revealed that the five genera of Cyprepediodeae were monophyletic, as was circumscribed earlier (Cox et al. 1997). Though ITS weakly supported the tree topology, it was found to be a reliable indicator of phylogenetic relationships only at higher levels within and among Cypripedioideae genera. The lack of support for some groups e.g., Paphiopedilum was due to the problems with DNA alignment. For this reason, use of ITS sequences for phylogenetic analyses at the sub-family level or above was not considered problem free in orchids (Cox et al. 1997). The sub-family Vanilloideae consists of 15 genera and about 180 species, belonging to two tribes Pogonieae and Vanilleae (Cameron 2009). Though, most of the molecular phylogenetic studies on Orchidaceae had been carried out on plastidial genes or inter-genic spacers, including the study where analysis of plastid genes could determine the sister taxon of Vanilla (Cameron and Molina 2006), this approach could not be used across Vanilloideae, as 40% of the genera within it are achlorophyllous mycoheterotrophs (Cameron 2009). Thus, it became necessary to evaluate non-plastid gene sequences from nucleus and mitochondria. Cameron (2009) studied phylogenetic relationships among Vanilloideae by comparing the nucleotide sequences of the nuclear 18S, 5.8S and 26S rDNA and two mitochondrial regions, atpA gene and nad1b-c intron, either individually or in combination. The nuclear and mitochondrial parsimony jack-knife trees showed similarities with each other and also with the previously published trees based solely on plastid data. Sequences for the achlorophyllous genera, Lecanorchis and Galeola, were obtained for the first time and their correct placements in the phylogenetic trees were established. The ITS sequence divergence was found to be high among the Vanilloid orchids. However, in

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Vanilliodeae members, the ITS region was hypervariable and was thus difficult to align. Interestingly, in this group, even the 5.8S gene was found to be variable with relatively large amount of phylogenetic information contained in it (Cameron 2009). The Epidendroideae is one of the largest sub-families of Orchidaceae with highest number of genera and species among all sub-families of Orchidaceae. The Epidendroideae is further divided into two clades or subgroups known as the higher and lower Epidendroids, each of which is further divided into a number of tribes and sub-tribes (Dressler 1993). The first molecular-based phylogenetic study of the sub-family Epidendroideae was conducted by Neyland and Urbatsch (1996) in which 3′ end of the chloroplast gene ndhF was used for analysis of 34 orchid genera and a lilioid monocot, Clivia miniata (Amaryllidaceae), the last being the outgroup species. The phylogenetic trees, constructed using both parsimony and maximum likelihood methods, revealed that the subfamily Epidendroideae is monophyletic, with Listera (Neottieae) as sister (Neyland and Urbatsch 1996). Although, in their investigation, sub-tribal relationships were resolved easily and had strong branch support, inter-tribal relationships were poorly resolved. This lack of resolution among tribes was attributed to rapid species radiations that overlapped with anatomical, physiological, and anatomical adaptations which initiated large-scale epiphytism in the ancestral Epidendroideae (Neyland and Urbatsch 1996). Another phylogenetic study based on the sequence of an intron within nad1, a mitochondrial gene, revealed a clade composed of Laeliinae, Pleurothallidinae, Bletia and Calypsoeae, all of which would qualify as Epidendreae (Freudenstein and Chase 2001). Both these regions, ndhF and nad1 intron, however, were still not sufficient to resolve many of the polytomies (i.e., evolutionary relationships cannot be resolved completely) within the sub-family. The higher Epidendroids are partly monophyletic (e.g., tribes Dendrobieae, Podochileae, and Malaxideae) and partly polyphyletic [e.g., tribes Arethuseae and Epidendreae] (Cameron et al. 1999). van den Berg et al. (2005) assessed phylogenetic relationships within the Epidendroid orchids with emphasis on tribes Epidendreae and Arethuseae using combined multiple DNA sequences from nrITS and four plastid regions viz., trnL intron, trnL-F spacer, matK (gene and spacers) and rbcL. The levels of variation varied markedly among the different DNA regions. The two ITS spacers, ITS1 and ITS2, had the greatest number of variable sites with the fastest rate of change. The

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spacers had almost double the number of variable sites that were present within the conserved 5.8S RNA region. The variations in all plastid regions were lower than that found in nrITS. Among the plastid regions, matK had maximum variations followed by non-coding trnL-F region, while rbcL had least variable sites. The circumscription of tribes Epidendreae and Arethuseae and relationships with Epidendroideae became clearer when multiple coding and non-coding regions of DNA were used rather than using single gene regions (van den Berg et al. 2005). Table 2: A summary of the molecular phylogenetic studies at Sub-family level in the

family Orchidaceae. Sub-family

DNA Region

studied Number

of Species studied

Inference Reference

Cypripediodeae Vanilliodeae Epidendroideae Higher Epidendroids

nrITS (Nuclear) 18S, 5.8S and 26S rDNA genes; mitochondrial atpA gene and nad1b-c intron ndhF ITS, trnL intron, trnL-F spacer, matK and rbcL

100

35 species belonging to 13 genera

34 species + 2 outgroups ~79 genera

Monophyletic with 5 genera. ITS was not useful in inferring sub-family and above relationships Monophyletic, Positions of achlorophyllus genera were established. ITS was hypervariable with high amount of variations even in 5.8S region. Monophyletic ITS was most variable. Among plastid genes, matK showed highest variation followed by trnL-F and rbcL

Cox et al. (1997) Cameron (2009) Neyland & Urbatsch (1996) van den Berg et al. (2005)

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2.2.3 Tribe Level The tribe Arethuseae (Epidendroideae) comprises 30 genera (Dressler 1993). The circumscription of this tribe has changed since it was first described by Lindley in 1840 (Goldman et al. 2001). The tribe is divided into four sub-tribes- Arethusinae, Bletiinae, Sobrallinae and Thuninae (Dressler, 1993). Goldman et al. (2001) was assessed the monophyly of this tribe by analyzing matK and rbcL sequences from its 24 representative genera and 46 other orchidaceous genera across the family. The parsimony analysis for both matK and rbcL individually and in combination, indicated that the tribe was polyphyletic and spread throughout Epidendroideae (Goldman et al. 2001). The number of variable positions in rbcL was much lower than in matK and hence, the former yielded less resolution. However, conclusions arrived at the phylogenetic relationships of tribe Arethuseae by Goldman et al. (2001) were not considered final because of the inadequate sampling of taxa and hence required further closer evaluation (van den Berg et al. 2005). Therefore, van den Berg et al. (2005) recircumscribed the tribe Arethuseae using combined analysis of multiple DNA regions (ITS spacer, trnL gene, trnL-F spacer, matK and rbcL genes). The spacers ITS1 and ITS2 were the most variable and among the plastid markers, matK showed maximum variable sites, while rbcL contained the least number of variable sites. The combined analysis of markers showed that this tribe has a distinct circumscription in relation to Dressler’s system of classification (1993) and it should include only Arethusinae (as in Dressler, 1993) and Coelogyninae (previously part of tribe Coelogyneae in Dressler, 1993). The analysis advocated the inclusion of Calopogon and Arundina in Arethusinae instead of Bletiinae, whereas Bletilla (formerly Bletiinae), Dilochia (formerly Arundininae, subtribe unplaced) and Glomera (formerly Glomerinae, Epidendreae) were included in Coelogyninae. Their results also suggested that Bletiinae, the main sub-tribe in terms of number of genera, should not be included in Arethuseae. Rather, they suggested that the type genus Bletia and a few of the genera earlier placed in this sub-tribe, are parts of the tribe Epidendreae (van den Berg et al. 2005). Furthermore, the analysis suggested that the genera earlier placed in Bletiinae by Dressler (1993) need to be placed with Collabiinae and Phaiinae (collectively Collabieae). Moreover, these two subtribes did not appear to be

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related to Arethuseae. Thus, with larger sampling of Epidendroideae, the polyphyletic nature of Arethuseae became more evident, and the genera previously placed in Arethuseae were assigned more precisely to other tribes and sub-tribes (van den Berg et al. 2005).

The tribe Vandeae (Epidendroideae) is divided into three sub-tribes

(Aeridinae, Aerangidinae and Angraecinae) on the basis of floral morphology and biogeography (Carlsward et al. 2006). The Vandeae tribe consists of horticulturally important genera with species having monopodial growth, leafless and epiphytic habit. To study the evolution of monopodial leaflessness, Carlsward et al. (2006) used molecular and structural evidence to generate phylogenetic hypotheses for Vandeae. Molecular relationships of various sub-tribes within Vandeae were assessed by the analyses of the data from nucleotide sequences of nrITS, trnL-F, and matK. Maximum parsimony analyses of these three DNA regions supported only two sub-tribes within the monopodial Vandeae. The two sub-tribes recognized were the original Aeridinae and Angraecinae, with the third sub-tribe Aerangidinae merged in the latter. Individually, Aerangidinae and Angraecinae were found to be polyphyletic, but together they formed a well-supported monophyletic group in all molecular analyses (Carlsward et al. 2006). This molecular phylogenetic analysis, based on three DNA regions from both nuclear and plastid genomes, supported the monophyly of the tribe Vandeae. The overall analysis indicated that leaflessness had arisen six to seven times within Vandeae (Carlsward et al. 2006).

The sub-family Orchidoideae (previously recognized as sub-family

Spiranthoideae) comprises two sub-clades with two tribes in each (Dressler, 1993). The first clade includes tribes Orchideae and Diseae, while the tribes Cranichideae and Diurideae make up the second clade (Dressler, 1993). In the first molecular phylogenetic study of tribe Diseae (Orchidoideae), nuclear ribosomal ITS sequences were compared for 30 Diseae genera along with 20 Orchideae, and four Cranichideae and Diurideae taxa as outgroups (Douzery et al. 1999). The tribe was further divided into five sub-tribes - Disinae (two genera), Coryciinae (five genera), Satyriinae (two genera), and the monogeneric sub-tribes Brownleeinae (seven species) and

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Huttonaeinae (five species). ITS sequences revealed that the tribe Diseae is paraphyletic which was in conjunction with the conclusion drawn on the basis of pervious morphological studies (Dressler 1993, Douzery et al. 1999). The analysis of ITS trees suggested the (i) removal of Disperis from Coryciinae, and (2) inclusion of all basitonic orchids in Diseae rather than distributing between Diseae and Orchideae, thus making Diseae paraphyletic. The genus Disperis, which was leading to paraphyly of tribe, could not be assigned correctly because of limited sampling therefore, need of further study with more extensive sampling was emphasized (Douzery et al. 1999).

As delimited by Dressler (1993), the tribe Diurideae, belonging to the second

clade of sub-family Orchidoideae, includes ten sub-tribes with 43 genera containing 900 species. The ten sub-tribes are Acianthinae, Caladeniinae, Cryptostylidinae, Diuridinae, Drakaeinae, Prasophyllinae, Pterostylidinae, Rhizanthellinae, Thelymitrinae and Chloraeinae (Dressler 1993). A molecular phylogenetic analysis, based on the comparison of DNA sequence data from plastid matK and trnL-F regions, indicated that the tribe Diurideae was non-monophyletic (Kores et al. 2001). However, if Chloraeinae and Pterostylidinae were excluded from Diurideae, the remaining sub-tribes formed a monophyletic group that was sister to a ‘‘spiranthid’’ clade. The tree topology obtained with matK was found to be similar to the one with matK+trnL-F. From the perspective of Diurideae, the trnL-F region did provide additional support for certain clades when combined with matK (Kores et al. 2001).

Salazar et al. (2003) carried out a phylogenetic assessment of the tribe

Cranichideae based on nucleotide sequences of plastid (rbcL, trnL-trnF and matK-trnK) and nuclear ribosomal (nrITS) DNA. The analysis with rbcL, trnL-trnF and matK-trnK did not provide good evidence for the monophyly of the Cranichideae. However, the combined analysis of all plastid genes supported monophyly of the tribe Cranichideae but with less bootstrap support (Bootstrap percentage (BP) - 69). The ITS analysis also supported monophyly of the tribe with BP< 50 (Salazar et al. 2003). In this study, the tribe Cranichideae was found to be monophyletic with moderately low bootstrap support (BP 6, BP 71) and Chloraeinae and a Pterostylis-Megastylis clade were found to be strongly supported sister clades to Cranichideae (Salazar et al. 2003).

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Table 3: A summary of the molecular phylogenetic studies at Tribe level in the family Orchidaceae.

Tribe

DNA Region

studied Number of

Taxa studied Inference Reference

Arethuseae Arethuseae Vandeae Diseae Diurideae Cranichidinae

matK+rbcL ITS spacer, trnL gene, trnL-F spacer, matK and rbcL genes ndhF nrITS, trnL-F, and matK ITS matK and trnL-F rbcL, trnL-trnF, matK-trnK) and nrITS

24Arethuseae genera+46 genera from other orchid ~79 genera 193 species 30 Diseae genera +20 Orchideae and 4 taxa from Cranichideae and Diurideae 95 genera 42 species belonging to 35 genera

Polyphyletic with 4 sub-tribes across all Epidendroideae Polyphyletic but genera were placed more accurately in tribes or sub-tribes Monophyletic with two sub-tribes, Aeridinae and a combined Angraecinae + Aerangidinae. Tribe was Paraphyletic Tribe appeared to be monophyletic if Chloraeinae and Pterostylidinae were excluded Combined analysis of four plastid genes increased the resolution and monophyly

Goldman et al. (2001) van den Berg et al. (2005) Carlsward et al. (2006) Douzery et al. (1999) Kores et al. (2001) Salazar et al. (2003)

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2.2.4 Sub-tribe Level

The molecular phylogenetics of sub-tribes Aeridinae and Angraecinae belonging to the tribe Vandeae, have been carried out by Hidayat et al. (2005) and Micheneau et al. (2008), respectively. The DNA sequences from matK and ITS were examined from 75 species representing 62 genera of sub-tribe Aeridinae to deduce relationships (Hidayat et al. 2005). The combined data set comprising sequences from both ITS and matK revealed much more robust topologies than those generated from the individual data sets. The monophyly of the tribe Aeridinae was established and 14 sub-clades reflecting phylogenetic relationships were recognized (Hidayat et al. 2005). These results were consistent with the previous classification of the sub-tribe by Dressler (1993). On the other hand the second sub-tribe Angraecinae was examined using DNA sequences from plastid genome only. The four plastid DNA regions used for inferring relationships within the sub-tribe were trnL intron, trnL-F intergenic spacer, matK gene and rps16 intron (Micheneau et al. 2008). Parsimony and Bayesian analyses provided identical sets of relationships within the sub-tribe. The large genus Angraecum did not form an exclusive clade. Bonniera was shown to be embedded in part of Angraecum. The phylogenetic analyses confirmed polyphyly of the large genus Angraecum as well as the unnaturalness of several of its sections (Micheneau et al. 2008). The phylogenetic relationships of sub-tribes Cranichidinae and Prescottiinae from the tribe Cranchideae were studied by Salazar et al. (2009), while Álvarez-Molina and Cameron (2009) assessed phylogenetic relationssips among the members of only the latter sub-tribe. Salazar et al. (2009) evaluated nucleotide sequences from both nuclear ribosomal (ITS) and plastid DNA (rbcL, matK-trnK and trnL-trnF) with cladistic parsimony and Bayesian inference for 45 species belonging to 14 genera of Cranichidinae and Prescottiinae (including suitable out-groups). Their analysis revealed that the sub-tribes Spiranthinae and Cranichidinae were paraphyletic with paraphyletic Presscottia embedded in it. Moreover, the evidence for monophyly of Prescottiinae was not found (Salazar et al. 2009). The nucleotide sequences from ITS,

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trnL-F, rbcL, atpB, and psaB regions were used by Álvarez-Molina and Cameron (2009) for inferring relationships of Prescottinae with its close allies i.e., Cranichidinae and Spiranthinae. The results were in conjunction with those of Salazar et al. (2009) and the sub-tribe was found to be non-monophyletic.

Yukawa et al. (1993) evaluated inter-generic relationships in the sub-tribe

(Podochileae) at molecular level by using plastid DNA restriction sites in 15 species belonging to 12 sections of the sub-tribe. The results indicated that the sub-tribe Dendrobiineae comprised three major clades. The first being represented by Epigeneium, while the second an Asian clade contained Callista, Dendrobium, Pedilonum, Distichophyllum, Formosae, Phalaenanthe and Aporum and the third, an Australian clade, comprised genera Rhizobium, Latouria, Spatulata, Grastidium, Cadetia, Diplocaulobium and Flickingeria. The Asian clade was reported to be an unresolved polytomy. Dendrobium appeared to be polyphyletic relative to Cadetia, Diplocaulobium and Flickingeria. However, Dendrobiineae was monophyletic, if Pseuderia was excluded from it. Similar results with a slight difference in tree topology were obtained when rbcL sequences and plastid restriction sites were used in combination (Yukawa et al. 1996). Clements (2003) who compared sequences of the nrITS from 23 species along with seven outgroup species, also arrived at the conclusion that the sub-tribe Dendrobiinae is made up of three major clades as was earlier concluded by Yukawa et al. (1993). Thus, Clements (2003) concluded that Dendrobiinae is polyphyletic, with the sections Pedilonum and Rhopalanthe being non-monophyletic (Clements 2003, reviewed in Adams, 2011). The sub-tribe Laeliinae (Epidendreae) comprises Neotropical orchids with 1500 species belonging to 50 genera (Dressler 1993), making the sub-tribe third largest in the family after Pleurothallidinae and Oncidiinae. The first broad phylogenetic analysis within Laeliinae was performed using ITS data for 295 taxa by van den Berg et al. (2000). They found little resolution and support along the spine of the tree, but relationships were clear enough to show that some groups were polyphyletic, which led to the transfer of many species from Laelia to Sophronitis

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(van den Berg et al. 2000). Further analysis of Laelline was carried out by van den Berg et al. (2009) using six DNA data sets: plastid trnL intron, trnL-F spacer, matK gene and trnK introns upstream and dowstream from matK, and nuclear ITS rDNA. The most variable datas et was ITS (as a whole), followed by the trnK introns. The trnL-F (intron+ exon+spacer) and matK gene had similar levels of variation. But in terms of informativeness (in terms of Retention Index), matK and trnL-F (intron+exon+spacer) performed similarly and slightly better than ITS. Based on combined molecular analysis from nuclear and plastid data sets, smaller alliances were formed within Laellinae which could be used for further detailed studies to explain morphological evolution and diversification patterns within the sub-tribe (van den Berg et al. 2009). The molecular phylogenetic studies based on plastid genes viz., ndhF and rbcL (Neyland and Urbatsch 1996 and Cameron 1999) contributed only modestly to understanding the phylogeny of sub-tribe Orchidinae (Bateman et al. 2003). nrITS of members of Orchidinae was first sequenced by Cozzolino et al. (1996). Five species of Orchis were resolved using ITS1 into two groups and subsequently into three groups by Pridgeon et al. (1997). The three groups recognized by latter were (i) O. coriophora, O. morio and O. laxiflora, (ii) O. simia and (iii) O. purpurea. The broader molecular phylogenetic analysis of the sub-tribe Orchidinae and species of Haberiinae were carried out by Bateman et al. (2003) using ITS sequences from 190 terrestrial orchid species. The results based on parsimony analysis indicated 12 well-resolved clades within the Orchidinae. However, relationships were less clearly resolved among these 12 clades. The monophyletic nature of the sub-tribe was weakly supported. It appeared as paraphyletic under maximum parsimony. The species-rich genus Habenaria was found to be highly polyphyletic. The relationships were also not clear within Habenariinae (Bateman et al. 2003). The sub-tribe Pleurothallidinae (Epidendreae) comprises an estimated 4000 Neotropical species distributed among 30 genera (Pridgeon et al. 2001). The phylogenetic status of the sub-tribe was evaluated using the nuclear ribosomal DNA

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internal transcribed spacers (ITS1 and ITS2) and 5.8S gene from 185 species and plastid sequences from matK, trnL intron, and trnL-F intergenic spacer. The latter were analyzed in only a sub-set of representative taxa to improve the overall assessments (Pridgeon et al. 2001). The results from nuclear and plastid sequences were highly congruent and the combined analysis of 58 representative taxa revealed the monophyletic nature of the sub-tribe that contained nine major clades encompassing all genera which were monophyletic. However, all the data sets pointed towards the polyphyly of Pleurothallis and its constituent subgenera (Pridgeon et al. 2001). The taxonomic position of sub-tribe Chloraeinae and the four genera (Bipinnula, Chloraea, Gavilea and Geoblasta) included in it was controversial. The placement of Chloraeinae in tribe Diurideae or Cranichideae by (Dressler 1993, Burns-Balogh and Funk 1986) was not supported (Pridgeon et al. 2001). A few molecular phylogenetic studies which dealt with taxonomic placement of the sub-tribe had agreed to polyphyly of the tribe Diurideae. These investigations also led to the suggestions of exclusion of the members of Chloraeinae and their placement in Chloraeinae under the tribe Cranichideae (Kores et al. 2001, Clements et al. 2002, Salazar et al. 2009). However, as these studies had not included representatives of all four genera of the sub-tribe Chloraeinae, the monophyly of the sub-tribe still needed to be tested. Therefore, broader molecular phylogenetic analysis was carried out by Chemisquy and Morrone (2010) in which three chloroplast markers, matK-trnK intron, atpB-rbcL spacer and rpoC1 gene, were used to assess (a) monophyly of the sub-tribe, (b) the phylogenetic relationships among Bipinnula, Chloraea, Gavilea and Geoblasta and (c) the monophyly of Chloraea and Gavilea. In all the analyses, Bipinnula, Chloraea, Gavilea and Geoblasta were grouped in a clade with high support, where Bipinnula, Geoblasta and Gavilea were nested inside Chloraea. Consequently, Chloraea was concluded to be paraphyletic, whereas Gavilea turned out to be monophyletic with high values of support. The other species of tribe Cranichideae appeared as sister groups of the Chloraeinae. Therefore, the placement of Chloraeinae in tribe Cranichideae was once again supported (Chemisquy and Morrone 2010).

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Table 4: A summary of the molecular phylogenetic studies at Sub-tribe level in the family Orchidaceae.

Sub-tribe

DNA Region

studied Number of

Species studied

Inference Reference

Aeridinae Angracinae Cranichidinae & Prescottiinae Prescottiinae Dendrobiinae Dendrobiinae Laeliinae Laeliinae

matK and ITS trnL intron, trnL-F intergenic spacer, matK and rps16 intron rbcL, matK-trnK, trnL-trnF and nrITS ITS, trnL-F, rbcL, atpB, and psaB regions Plastid DNA restriction sites Plastid DNA restriction sites+ rbcL nrITS nrITS Plastid trnL, trnL-F spacer, matK and trnK upstream and downstream from matK and nrITS

75 48

45 species belonging to 14 taxa 100 species, 8 genera 15 species belonging to 12 sections 15

23

295

125

Monophyletic with 14 sub-clades Polyphyletic with many unnatural sections. Both sub-tribes were Paraphyletic Paraphyletic Monophyletic, with Pseuderia excluded Monophyletic with three major clades Polyphyletic Some groups within the sub-tribe were polyphyletic Generic alliances were studied. matK and trnL-F trees had better resolution than nrITS

Hidayat et al. (2005) Micheneau et al. (2008) Salazar et al. (2009) Molina and Cameron (2009) Yukawa et al. (1993) Yukawa et al. (1996) Clements (2003) van den Berg et al. (2000) van den Berg et al. (2009)

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Sub-tribe

DNA Region studied

Number of Species studied

Inference Reference

Orchidinae Orchidinae and species of Haberiinae Pleurothallidinae Chloraeinae

ITS1 ITS ITS, matK, trnL intron, and trnL-F intergenic spacer matK–trnK intron, atpB–rbcL spacer and the rpoC1

5

180 species, ~27 genera

185

18 species belonging to four genera

Three groups were recognized Sub-tribe Orchidinae was paraphyletic and genus Habenaria was highly polyphyletic Monophyletic containing nine major clades Placement of Chloraeinae in tribe Cranichideae was supported

Cozzolino et al. (1996) Bateman et al. (2003) Pridgeon et al. (2001) Chemisquy and Morrone (2010)

2.2.5 Generic Level

The orchid genera are divided into various sections based on the morphological affinities (Dressler 1993). The relationships among different sections of a few genera have been analysed at molecular level using various regions from plastid and/or nuclear genome. The phylogenetic relationships analyzed for various genera are summarized below.

The phylogenetic relationships among the species of the genus Aerides

(Epidendroideae, Vandeae, Aeridinae) from Southeast Asia were evaluated by comparing sequences of one nuclear (nrITS) and two plastid (matK, trnL-trnL-F) regions of 48 taxa (21 Aerides species, 25 other Aeridinae and 2 outgroups) by Koycan et al. (2008). The combined analysis of all the data sets revealed that the

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genus Aerides is monophyletic and consisted of three well-supported sub-clades which were in accordance with previous sectional delimitations based on floral characters (Koycan et al. 2008).

The genus Calopogon (Epidendroideae, Arethuseae, Arethusinae) is a small

North American genus with five species (Goldman et al. 2004). Although, the genus has been circumscribed, the taxonomic position of species was not correct (Goldman et al. 2004). Almost all species in the genus were mistaken for or placed as varieties of other species (Goldman et al. 2004). The phylogenetic relationships among different species of Calopogon genus were studied at molecular level by using sequences of nrITS, amplified fragment length polymorphisms (AFLPs), chloroplast DNA restriction fragments, and chromosome counts (Goldman et al. 2004). The ITS analysis revealed that none of the taxa within the genus was coherent (discrete) groups with high bootstrap support, implying that the recognized taxa could not be strongly circumscribed. Calopogon multiflorus-C. pallidus and C. oklahomensis-C. barbatus were recognized as two sister species pairs based on the ITS analysis. However, this analysis could not unravel the relationships of C. barbatus, C. oklahomensis, and C. tuberosus. According to ITS analysis, C. oklahomensis was sister to C. barbatus whereas, on the basis of plastid restriction fragment analysis, C. oklahomensis was sister to C. tuberosus. Using combined analysis of all three techniques, all taxa appeared to be discrete groups. Thus, pointing towards the limitation of ITS data which alone was not sufficient for evaluating infra-generic relationships within Calopogon (Goldman et al. 2004).

The genus Coelogyne of sub-tribe Coelogyninae (tribe Coelogyneae, sub-

family Epidendroideae) with a total of around 550 species (Gravendeel et al. 2001) is one of the biggest genera in the sub-tribe. The monophyly of Coelogyne, sectional relationships and relations to allied genera in the sub-tribe Coelogyninae were evaluated using RFLP analyses from 11 plastid regions and sequences from matK and ITS of 42 taxa (28 Coelogyne species and 14 representatives of other genera) and three outgroups from Bletiinae and Thuniinae (Gravendeel et al. 2001). The separate

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analysis of three different regions resulted in weakly supported clades but a combined analysis identified three main clades in Coelogyninae with Coelogyninae being monophyletic. However, Coelogyne was polyphyletic as the species were spread into at least two well supported clades (Gravendeel et al. 2001).

The genus Cypripedium (Cyprepedioideae, Cypripedieae, Cypripediineae)

comprises approximately 50 species of terrestrial orchids (Li et al. 2011b). The molecular phylogenetic study of the genus Cypripedium utilizing nrITS and five chloroplast regions (trnH-psbA, atpI-atpH, trnS-trnfM, trnL-F spacer and trnL intron) of 56 species, including outgroups, confirmed that the genus is monophyletic with ten well supported clades. The eight monophyletic clades were Subtropica, Obtusipetala, Trigonopedia, Sinopedilum, Bifolia, Flabelinervia, Arietinum, and Cypripedium, whereas the remaining two clades (Irapeana and Retinervi) were paraphyletic (Li et al. 2011b).

The twelve Taiwan species of Dendrobium were classified and the genetic

relationships were inferred using ITS sequences by Tsai et al. (2004). The genetic distance was calculated using Kimura-2-parameter method and among the 12 Dendrobium species, the range of genetic distances was from 0.06 to 0.28. Each Dendrobium species could be easily identified based on the ITS sequence as inferred by genetic distance and Neighbour Joining methods.

Maxillaria, with 580 species is one of the largest genera (megagenus) of

Neotropical orchids. The circumscription and relationships within Maxillaria (sub-tribe-Maxillariinae, tribe-Cymbidieae) have long been regarded as artificial (Dressler 1993) till the phylogenetic relationships for the sub-tribe Maxillariinae with an emphasis on Maxillaria, using parsimony analyses of individual and combined DNA sequence data were inferred by Whitten et al. (2007). The DNA sequences from nrITS and the plastid regions, matK, trnK intron, rpoC1 and atpB-rbcL intergenic spacer, of 354 species were analyzed. The ITS sequences contained maximum variation/ number of parsimony informative sites, the most resolution and the greatest number of clades with boot strap support >70%.

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Table 5: A summary of molecular phylogenetic studies at genus level of the family Orchidaceae.

Genus

DNA Region

studied Number of

Species studied

Inference Reference

Aerides Calopogon Coelogyne Cypripedium Dendrobium Maxillaria Ophrys

nrITS, matK, trnL–trnL–F ITS, AFLPs and chloroplast DNA restriction fragments RFLPs from 11 plastid regions and matK and ITS sequences ITS, trnH-psbA, atpI-atpH, trnS-trnfM, trnL-F spacer, and trnL intron ITS nrITS DNA, the plastid matK gene and flanking trnK intron, rpoC1 gene, atpB-rbcL intergenic spacer nrITS, trnL-trnF

48 5 42 56 12

354

32 species and 6 outgroups

Monophyletic with three sub-clades Combined analysis showed the species form discrete groups. ITS alone was not able to resolve the relationships Coelogyne was polyphyletic with species in two clades Monophyletic with ten well supported clades ITS identified 12 species using genetic distance and NJ methods Polyphyletic with well supported clades Monophyletic with two clade

Koycan et al. (2008) Goldman et al. (2004) Gravendeel et al. (2001) Li et al. (2011b) Tsai et al. (2004) Whitten et al. (2007) Soliva et al. 2001

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This was followed by matK-trnK sequences which though containing more parsimony informative sites than nrITS, revealed 50% fewer clades with boot strap support of >70%. The least variable region was rpoC1. The combined analysis led to the conclusion that the genus Maxillaria is polyphyletic with well supported clades (Whitten et al. 2007).

The sexually deceptive orchid genus Ophrys has been placed in the sub-family

Orchidoideae, tribe Orchideae, sub-tribe Orchidinae (Dressler, 1993) based on morphological characters. Based on the phylogenetic studies by Cameron et al. (1999), who had used rbcL sequences, the genus was regarded as monophyletic. However, the taxonomy of this genus changed with variable number of species reported from time to time (Soliva et al. 2001), thus making it difficult to classify into a natural system. The reason could be hybridization among the con-generic species which was found to be common in Ophrys (Delforge 1994, cited in Soliva et al. 2001). Soliva et al. (2001) analysed the major lineages of Ophrys by comparing nrITS and non-coding chloroplast trnL-trnF sequences. The individual analysis of nuclear and chloroplast DNA sequences resulted in poorly resolved phylogenetic trees, whereas a combined analysis of both revealed better resolved phylogenetic relationships. The genus was found to be monophyletic with two well resolved groups that were not in congruence with traditional groups of Euophrys and Pseudophrys. Few taxa with similar sequences but distinct floral morphology were also observed (Soliva et al. 2001).

2.3 INFERENCES AND LIMITATIONS OF MOLECULAR

PHYLOGENY

The above described and discussed phylogenetic studies amply exemplify the utility of several coding and non-conding regions from both nuclear and cytoplamic genomes for deriving evolutionary relationships among Orchidaceae taxa. The chloroplast genes were found to be useful for deriving phylogenetic relationships at the sub-family and above level, as in one of the first few studies, Cameron et al.

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(1999) had shown family Orchidaceae to be monophyletic with five sub-families on the basis of rbcL sequences. Later, the combination of matK+rbcL provided maximum number of variable sites thus resulting in inferring monophyly of five sub-families of Orchidaceae (Freudenstein et al. 2004). Below sub-family level, nrITS was found to be better suited for deriving relationships among tribes and sub-tribes of different sub-families (Cameron 2009, van den Berg et al. 2005). However, above the tribe or sub-tribe level, the ITS region was found to be unsuitable/problematic (Cox et al. 1997). For deriving relationships among various sections of a genus, combinations to ITS, AFPL and RFLP markers yielded better results (Goldman et al. 2004, Gravendeel et al. 2001).

Molecular systematics, being a cladistic approach, is based on the assumption

that classification must correspond to phylogenetic descent and all valid taxa must be monophyletic (Soltis and Soltis 1998). Molecular phylogenies can be affected by numerous problems, including long-branch attraction and saturation (Philippe et al. 2011). In most of the studies dicussed above, the phylogenetic inferences or monophyly of taxonomic group(s) could not be achieved because of insufficient sampling of taxa (Chase et al. 1994, Neyland and Urbatsch 1996). Consequently, strikingly different results were obtained by applying different models to the same data set. To be used in phylogenetic studies, markers must exhibit sufficient variability to link species and groups of species by possessing shared (synapomorphic) substitutions (Chase et al. 2005). For this the use of whole gene is desirable. Conversely, for species specific markers, unique substitutions or autapomorphies are preferred, though these are generally not useful in assessing phylogenetic relationships of species and other taxa (Chase et al. 2005). The uniqueness/variation in several markers commonly used in phylogenetic studies could be utilized for identifying species using an innovative technique called DNA barcoding (Hebert et al. 2003a, Kress et al. 2005, Chase et al. 2007). The subsequent section provides a brief introduction of this technique, justifications for its need and summarizes the progress made so far in DNA barcoding of orchids.

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2.4 DNA BARCODING OF ORCHIDACEAE

DNA barcoding is a novel technique projected for rapid and reliable identification of an unknown biological sample to the level of species using short sequence(s) of DNA (Hebert et al. 2003a). The technique could be used for rapid identification and detection of species and relies on DNA sequence variations that provide a unique recognition tag to a species (Hebert et al. 2003a). The short sequences of DNA that can be from standardized and agreed upon locus/loci of either nuclear or cytoplasmic genome or both are called DNA barcodes. The differences in their nucleotide sequence provide a unique molecular identity to species. The advantage of DNA barcodes over the current taxonomic methods of identification is that a species can be identified even if a small amount of its tissue/DNA is available. Moreover, the amplification and direct PCR sequencing of short barcode sequences can be carried out across taxonomically diverse species using universal primers. The foremost advantage of DNA barcoding over molecular phylogeny is that it does not require cloning and sequencing of complete gene, thus making it less laborious and time consuming. Essentially, DNA barcoding is an additional taxonomic tool with high potential of reviving modern taxonomy (Schindel and Miller 2005).

Orchidaceae, one of the largest families of angiosperms comprises some of te

species that are difficult to identify and classify correctly even if available in flowering state (Dressler, 1993, van den Berg et al. 2000, Gravendeel et al. 2001, Cameron 2004). For example, Dactylorhiza contains different species complexes and aggregates e.g. D. maculata and D. incarnata aggregates. The main taxonomic problem is to elucidate the relationships of species in these complexes (Shupinov et al. 2004). Moreover, the family contains several megagenera (1000+sp.) viz. Bulbophyllum Thouars, Epidendrum L., Pleurothallis R. Br. and Dendrobium Sw. in which explosive speciation has taken place due to adaptive radiations (Whitten et al. 2007). The genus Holcoglossum consists of both long-evolved and recently radiated species that adds to difficulty in the identification of species (Xiang et al. 2011). In plants, events like hybridization play an important role in speciation (Soltis and Soltis 2009). Orchid

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species being promiscous in nature hybridize easily and due to hybridization a number of hybrids come in existence, as in genus Ophrys that has a number of hybrid species (Soliva et al. 2001). Due to the large number of hybrids it is difficult to classify the taxa into natural system (Soliva et al. 2001). The closely related species of Paphiopedilum form hybrids in nature as well as artifically. It is difficult to identify the hybrids from their parents especially in vegetative parts (Sun et al. 2011). The presence of cryptic species as in Serapias (Pellegrino et al. 2005) and sister species, like Anacamptis morio and A. longicornu (Zitari et al. 2011), also make the morphological calssification more difficult. For identification of these difficult to classify taxa, a rapid species identification technique like DNA barcoding is required (Schindel and Miller 2005).

The species of Orchidaceae are valued for cut flower production and as potted

plants [e.g. Dendrobiums, Paphiopedilums, Cypripediums, Phalaenopsis etc.] (www.orchidsasia.com). They are known for their longer lasting and enchantingly beautiful flowers which fetch a very high price in the international market (www.orchidsasia.com). Orchids since time immemorial have also been used for curing various diseases and ailments (Jalal et al. 2008). India being home to the rich repository of medicinal herbs has a long history of utilizing orchid species in traditional folk medicine such as ayurveda, siddha and unani (Jalal et al. 2008). Many orchidaceous species have also been used in traditional system of medicine for curing various ailments like tuberculosis, paralysis, stomach disorders, chest pain, arthritis, syphilis, jaundice, cholera, acidity, eczema, tumour, piles, boils, inflammations, menstrual disorder, spermatorrhea, leucoderma, diahorrhea, muscular pain, blood dysentery, hepatitis, dyspepsia, bone fractures, rheumatism, asthma, malaria, earache, sexually transmitted diseases, wounds and sores (Bulpitt et al. 2007, Hossain 2011). The therapeutic properties of different orchids are: aphrodisiac, rejuvenator, tonic, antibacterial, antioxidant and immunomodulating (Bulpitt et al. 2007). Chyavanprash, a popular Indian traditional polyherbal formulation that is widely used as tonic, rejuvenator, anabolic, immunomodulator and memory enhancer, consists of eight herbal components popularly known as ‘Ashtavarga’ in ‘‘Ayurveda’’ (Singh and Duggal 2009). Out of the eight components of ashtavarga, four are orchids. These are

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Riddhi (Habenaria intermedia), Vriddhi (Habenaria edgeworthii), Jivaka (Malaxis muscifera) and Rishabhaka (Malaxis acuminata) (Singh and Duggal 2009). Flickingeria macraei, known as jeevanti in ‘‘Ayurveda’’ is used as astringent to the bowels, aphrodisiac and in asthma and bronchitis (Hossain 2011). Other commonly used orchid drugs in the Ayurvedic system are salep (Orchis latifolia [accepted name is Dactylorhiza incarnata] and Eulophia latifolia), jivanti (Dendrobium alpestre), shwethuli and rasna (Acampe praemorsa and Vanda tessellata) (Hossain 2011). In ‘Sushruta samhita’ it is mentioned that the underground tubers of Orchis latifolia is used in the drug ‘munjatak’which pacifies cough (Hossain 2011). The leaves of Vanda roxburghii are prescribed in the ancient Sanskrit literature for external application in rheumatism, ear infections, fractures and diseases of nervous system (Hossain 2011).

Due to their ornamental and therapeutic properties the natural populations of

orchids have been over exploited in the past, thus rendering these species threatened and endangered. Therefore, all orchid species are listed in the Convention on International Trade of Endangered Species of Fauna and Flora (CITES) and thus their trade from the wild is banned (http://www.cites.org; Sun et al. 2011). In the absence of effective identification methods, collection, trade and export of these endangered orchid species especially in vegetative form can not be checked. The techniques of DNA barcoding could provide a potent method for checking these illicit practices by offering a fool proof method for their detection in any form, thus could indirectly help in their conservation. However, very few reports are available on DNA barcoding of orchids. The first study that sampled 1,036 Orchidaceae species from Mesoamerican biodiversity hotspot of southern Africa, was carried out by Lahaye et al. (2008). They tested eight potential barcode loci and demonstrated that matK could identify almost all species. Lahaye et al. (2008) sampled 86 species of flowering plants along with 1,036 orchid species in two different data sets. They observed that in the orchid data set, matK was amplified in all the sampled species, ndhJ and ycf5 did not amplify efficiently, the alignment of trnH-psbA was difficult as it required addition of several gaps, and presence of rps19 gene insertion in trnH-psbA and a less variable rbcL gene region. The barcodes exhibiting the lowest intra-specific divergence were rpoC1,

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acc/matK and matK. The highest inter-specific divergence was obtained in trnH-psbA, followed by matK. The matK data set was further expanded for orchids by mining the DNA sequences already available in GenBank. The coalescence analysis from UPGMA tree using 1,566 DNA sequences revealed 212 genetic clusters, of which 86 matched with previously recognized species and 25 with partially matched taxonomic species (Lahaye et al. 2008). The species identification success with matK alone was >90%. Their analysis pointed out that the 5′ end of matK exon was easy to amplify and align, and hence proposed matK as the preferred universal DNA barcode for flowering plants. However, the matK region was not found to be suitable in discriminating species of Australian orchid genus Cladenia (Farrington et al. 2009). Farrington et al. (2009) found matK to be less informative at the subgeneric level and therefore, was not suitable for discriminating species of Cladenia. On the other hand, they found trnL intron to be more useful for discrimination of Cladenia species (Farrington et al. 2009). In another investigation, the applicability of DNA barcoding technique in authentication of medicinal orchid species by the identification of 17 medicinally important Dendrobium species based on trnH-psbA spacer, one of the candidate DNA barcode (Yao et al. 2009). The intra-specific variation among the Dendrobium species studied ranged from 0% to 0.1%, while inter-specific divergence ranged from 0.3% to 2.3%. The sequence divergence of Dendrobium species from the outgroup species, Bulbophyllum odoratissimum ranged from 2.0% to 3.1%, with an average of 2.5%. The results of this investigation demonstrated that the trnH-psbA intergenic spacer region could be used as a barcode to distinguish various Dendrobium species and to differentiate Dendrobium species from other adulterating species (Yao et al. 2009). Asahina et al. (2010) too investigated the species discriminating abilities of matK and rbcL barcode regions for five species of Dendrobium. The five medicinal species investigated were Dendrobium fimbriatum, D. moniliforme, D. nobile, D. pulchellum and D. tosaense. The phylogenetic analysis carried out using whole gene sequences of matK and rbcL provided 100% species discrimination success only with matK. The rbcL tree showed less discrimination power which was attributed to low variation in rbcL gene sequences (Asahina et al. 2010). These results based on only five Dendrobium species indicated that matK could provide 100% species

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identification in this genus. However, in another investigation, matK could resolve only 76.92% species among 36 species of Dendrobium analyzed (Singh et al. 2012), while ITS in the same study yielded 100% resolution. As opposed to the observation of Yao et al. (2009), in this study, trnH-psbA spacer could not be tested for the discrimination of Dendrobium species because of the failure in obtainining its bi-directional sequences (Singh et al. 2012). The best multi-locus combination among the loci from chloroplast genome was matK + rpoB + rpoC1. This three-locus barcode could resolve 92.31% (48 out of 52) species (Singh et al. 2012). Earlier resported success of matk+rbcL combination in discriminating all five species of Dendrobium (Asahina et al. 2010) was ascribed to low sample size (Singh et al. 2012). The genus Holcoglossum (Aeridinae) with a complex taxonomy contains both long-evolved and recently radiated species, thus becoming an exceptional case to test DNA barcodes for Orchidaceae. The potential of DNA barcoding was tested on 12 species of this genus (Xiang et al. 2011). The DNA regions tested from a subset of proposed barcode loci were rbcL, matK, atpF-atpH, psbK-psbI, trnH-psbA from the plastid genome and nuclear ITS. The amplification success was 100% for all loci in 52 samples except for matK in which amplification success rate was 92.3%. The sequencing success rate was 100% for rbcL and trnH-psbA, 84% in ITS and ~75% in matK. The sequencing of atpF-atpH and psbK-psbI loci was not successful because of the presence of mononucleotide repeats. The highest variability was found in matK and ITS regions. Although none of the six candidate barcode loci could individually distinguish all 12 species of Holcoglossum, matK could resolved eight of the 12 species and thus, had the highest species discriminatory ability. Moreover, the combined matK+ITS sequences afforded better species discrimination than matK alone. This study concluded that matK was the best region to identify species of Holcoglossum but for identification of all species, other DNA regions would be required (Xiang et al. 2011). Parveen et al. (2012, part of the present thesis) tested the potential of five barcode loci in discriminating eight endangered Paphiopedilum species from India. In the analysis, nrITS although, had 4.4% average inter-specific divergence value, afforded only 50% species resolution. On the other hand, matK with 0.9% average inter-specific divergence value yielded 100% species resolution. The species identification

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capability of these sequences was further confirmed as each of the matK sequences was found to be unique for the species when a BLAST analysis of these sequences was carried out on NCBI. DNA barcodes of the three hybrids also reflected their parentage. The matK locus emerged as the signature sequence for the identification of closely related endangered species of Indian Paphiopedilums and also in elucidating the parentage of their inter-specific hybrids (Parveen et al. 2012).

Beside these few reports which are committed entirely to DNA barcoding of

orchid species, there are several other investigations in which floristic sampling of flowering plants also included few orchids. The CBOL Plant Working Group (2009) tested seven candidate barcode loci in 907 samples from 550 species belonging to different families of plants. Out of these 550 species, ten were orchids. The pooled data analysis revealed the matK+rbcL as core barcode region for all land plants with 72% species discrimination power. The validation of ITS2 as a novel barcode for medicinal plant species was reported by Chen et al. (2010). They had analyzed the discrimination ability of ITS2 in more than 6,600 plant samples belonging to 4,800 species from 753 distinct genera. The thirty medicinal orchid species included in this investigation were mainly from Dendrobium genus along with one species each of Bletilla and Bulbophyllum. They had demonstrated that the rate of successful identification with the ITS2 was 92.7% at the species level (Chen et al. 2010). Following this, Yao et al. (2010) downloaded 50,790 plant and 12,221 animal ITS2 sequences from GenBank and reported ITS2 as universal barcode for both animals and plant species. These downloaded sequences from GenBank included 1,237 Orchidaceae species belonging to 17 genera. The investigated orchid genera were Maxillaria, Oncidium, Dendrobium, Disa, Ophrys, Paphiopedilum, Phalaenopsis, Masdevallia, Gomesa, Satyrium, Dendrochilum, Cyrtochilum, Telipogon, Dichaea, Diuris, Scaphyglottis and Cymbidium. The percent species resolution obtained by analysing ITS2 sequences for different orchid genera is given in Table 6. The maximum species resolution (100%) was found among 33 species of Scaphyglottis followed by 98.3% species resolved in Satyrium and 91.9% in Dendrobium (Table 6).

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Table 6: Success rates of ITS2 for species identification in Orchidaceae genera (from Yao et al. 2010)

Genus No. of species No. of accessions % Species Resolution Maxillaria 227 482 62.9 Oncidium 139 215 65.1 Dendrobium 121 160 91.9 Disa 120 143 79.7 Ophrys 100 260 22.7 Paphiopedilum 85 192 76.6 Phalaenopsis 56 232 65.9 Masdevallia 48 49 79.6 Gomesa 46 55 49.1 Satyrium 42 59 98.3 Dendrochilum 42 52 71.2 Cyrtochilum 41 75 69.3 Telipogon 38 46 76.1 Dichaea 36 66 81.8 Diuris 33 61 31.1 Scaphyglottis 33 40 100 Cymbidium 30 58 74.1 Total 1237 2245