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ResultsResults

Painting by: Ms. Hemalata Pradhan Vanda coerulea

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4 RESULTS

The potential of four loci viz., rpoB, rpoC1, rbcL and matK from the chloroplast genome and ITS from the nuclear genome, as DNA barcodes was tested across 435 individuals, belonging to 104 species of the family Orchidaceae. In addition, 95 individuals belonging to 68 other families of vascular plants were also included to ascertain the universality of the primers used and to compare the species discrimination capability of the selected loci at higher taxonomic levels. The five candidate barcode loci were evaluated and compared individually for their amplification and sequencing success rates and their intra- and inter-specific divergence values were calculated using genetic distance method. The species resolution for each candidate locus was calculated based on genetic distances, phylogenetic tree method, and through BLAST analysis. The intra-specific variations were estimated for only those orchid species, which were represented by more than one individual. The inter-specific K2P distances and species resolution analysis was carried out at family and generic levels. The family level analysis was based on the distance matrix prepared for each locus using the consensus sequence of each orchid species showing no intra-specific divergence along with the sequences of all individuals of a species exhibiting intra-specific variations and the sequences of species belonging to families of land plants other than Orchidaceae (data set I). The data set II comprised the sequences of only the orchid species analyzed in data set I. Subsequently, species discrimination rates among the orchid species provided by various multi-locus combinations of the five candidate loci were also calculated on the basis of K2P distances. At the generic level, the inter-specific variations among the species of a genus were calculated using the distance matrix prepared by aligning the sequences of all the accessions belonging to different species of a genus (data set III). The K2P distances and tree building methods were used to discriminate the congeneric species of a genus.

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4.1 AMPLIFICATION AND SEQUENCING SUCCESS RATES

Out of 435 orchidaceous individuals tested for PCR amplification, variable numbers of amplicons were obtained for different loci viz., 381 for ITS, 374 for matK, 413 for rbcL, 424 for rpoB and 426 for rpoC1. Thus, the amplification success rates of ITS, matK, rbcL, rpoB and rpoC1 for the orchids were 87.58%, 85.97%, 94.94%, 97.47% and 97.93%, respectively. In species other than orchids, these loci were amplified in 95 accessions, each belonging to an individual species. The amplification success rates of ITS, matK, rbcL, rpoB and rpoC1 in these species were 71.57%, 67.36%, 96.84%, 97.89% and 97.89%, respectively. The overall PCR success rates for both orchid and non-orchid species were 84.73% (ITS), 82.64% (matK), 95.28% (rbcL), 97.54% (rpoB) and 97.92% (rpoC1). All the five loci tested yielded multiple bands of amplicons in few of the investigated species. The band having molecular weight nearest to that of the target locus was gel extracted and sequenced. Examination of the sequence quality and coverage indicated that the five candidate loci routinely generated high quality bidirectional sequences. The sequencing success rates of ITS, matK, rbcL, rpoB and rpoC1 in orchid species were 88.97%, 87.70%, 92.49%, 92.21% and 93.42%, respectively. The sequencing success rates in non-orchid species were 67%, 70.31%, 79.12%, 90.21% and 84.78% for ITS, matK, rbcL, rpoB and rpoC1, respectively. Overall sequencing rates were 85.52% (ITS), 88.12% (matK), 89.9% (rbcL), 91.68% (rpoB) and 91.71% (rpoC1). The total number of barcode sequences generated were 384 for ITS, 386 for matK, 454 for rbcL, 474 for rpoB and 476 for rpoC1 (Table 9). Table 9: Amplification and sequencing success rates for the five candidate loci from

191 plant species (530 accessions).

Locus Length of amplicons obtained (bp)

No. of amplicons obtained

Amplification success

No. of finished sequences generated

Sequencing success

ITS 650-750 449 84.73% 384 85.52% matK 700-827 438 82.64% 386 88.12% rbcL 600-660 505 95.28% 454 89.9% rpoB 320-390 517 97.54% 474 91.68% rpoC1 380-458 519 97. 92% 476 91.71%

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4.2 INTRA-SPECIFIC VARIATIONS IN ORCHID SPECIES

The intra-specific distances were calculated only for those 78 orchid species that were represented by more than one accession. Out of 104 species, three were represented by more than 10 individuals, 25 species by 6-10 individuals, 39 by 3-5 individuals, 11 by 2 individuals and remaining 26 by only one individual. All the five tested loci exhibited intra-specific variations with variable range. ITS sequences of the multiple accessions of Crepidium acuminatum, Oberonia recurva, Paphiopedilum villosum and Rhynchostylis retusa revealed intra-specific variations. The average intra-specific distances, based on ITS, amomg the individuals of C. acuminatum, O. recurva, R. retusa and P. villosum were 0.001, 0.002, 0.002 and 0.003, respectively. The matK, sequences of individuals of eight species viz., Cottonia peduncularis, Geodorum densiflorum, Nervilia crociformis, Otochilus sp., Paphiopedilum venustum, Porpax reticulata, Rhynchostylis retusa and Vanda testacea had intra-specific variations with a range of 0-0.004. The accessions of G. densiflorum and P. reticulata had intra-specific distances of 0-0.001, while those of C. peduncularis exhibited distances between 0-0.002 and among the accessions of N. crociformis; Otochilus sp. and V. testacea these varied from 0-0.003. The intra-specific distance exhibited by E. nuda and Rhynchosylis retusa for matK was 0-0.004. The average intra-specific divergence for matK sequences of P. venustum was 0.0007 with a range of 0-0.0014. The rbcL locus revealed 0-0.002 intra-specific variations among the individuals of Eulophia spectabilis and Nervilia crociformis. The individuals of Habenaria heyneana and Luisia zeylanica exhibited 0-0.003 intra-specific distance for rpoC1. Likewise Otochilus sp. exhibited 0-0.005 intra-specific variations among rpoC1 sequences. Four species showing intra-specific variations in rpoB were Eulophia spectabilis with the range of distances being 0-0.006, while this was 0-0.003 among multiple accessions of Liparis deflexa, Rhychostylis retusa and Satyrium nepalense (Table 10).

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Table 10: Intra-specific K2P distance for the five candidate loci from Orchidaceae species

Species Intra-specific K2P distances based on genetic distance method ITS matK rbcL rpoB rpoC1

Cottonia peduncilaris - 0-0.002 - - -

Crepididium acuminaum 0-0.003 - - - -

Eulophia spectabilis - 0-0.004 0-0.002 0-0.006 -

Geodorum densiflorum - 0-0.001 - - -

Habenaria heyneana - - - - 0-0.003

Liparis deflexa - - - 0-0.003 - Luisia zeylanica - - - - 0-0.003 Nervilia crociformis - 0-0.002 0-0.002 - -

Oberonia recurva 0-0.003 - - - -

Otochilus sp. - 0-0.003 - - 0-0.005 Paphiopedilum venustum - 0-0.0014 - - -

P. villosum 0-0.0016 - - - - Porpax reticulata - 0-0.001 - - -

Rhynchostylis retusa 0-0.003 0-0.004 - 0-0.003 -

Satyrium nepalense - - - 0-0.003 -

Vanda testacea - 0-0.003 - - -

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4.3 INTER-SPECIFIC K2P DISTANCES AND SPECIES DISCRIMINATION RATES FOR THE FIVE CANDIDATE

LOCI AT THE FAMILY LEVEL

The inter-specific K2P distances and species discrimination rates, obtained on the basis of genetic distance, phylogenetic tree and BLAST methods for each locus are individually described below under separate heads.

4.3.1 ITS

4.3.1.1 Inter-specific K2P Distances

The average inter-specific K2P distance, among 145 ITS sequences of 131 species (86 orchid and 45 species other than orchids), was 0.164 with a range of 0-0.408. Out of 131 species analyzed, 20 species exhibited zero distance estimates, of which 18 belonged to the Orchidaceae and two were non-orchid species (Fig. 13). Four species exhibiting intra-specific variations exhibited zero distances among their accessions. The maximum inter-specific K2P distance was exhibited by the pair Magnolia sp. and Cymbidium devonianum (Fig. 13). The aligned ITS sequences of only the orchid species (data set II) yielded an average K2P distance of 0.247 with the range being 0-0.498. Eight species exhibited zero distance estimates in the data set II. The highest inter-specific K2P distance of 0.498 was between Paphiopedilum venustum-Cymbidium devonianum and P. wardii-C. devonianum (Fig. 14).

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4.3.1.2 Species Resolution The species discrimination rates calculated on the basis of three methods are

individually described below.

4.3.1.2.1 DISTANCE BASED METHOD The species resolution was calculated by preparing a K2P distance matrix of

all the 131 species from their aligned ITS sequences. The ITS distance matrix revealed 18 species pairs that had zero distance estimates. As the formation of these species pairs involved 20 species, the % species resolution was 84.73% (Table 11). The species pairs formed were: Coelogyne trinervis-C. quadritriloba; Diplocentrum congestum-Cottonia peduncularis; Eria andamanica-Pinalia mysorensis; Schoenorchis micrantha-C. peduncularis; Vandopsis undulata-C. peduncularis; S. micrantha-Diplocentrum congestum; V. undulata-D. congestum; Peristylus plantagineus-Habenaria stocksii; Oberonia recurva-O. ensiformis; Paphiopedilum venustum-P. insigne; P. villosum-P. insigne; P. wardii-P. insigne; P. villosum-P. venustum; P. wardii-P. venustum; P. wardii-P. villosum; Platanthera edgeworthii-P. latilabris; Vandopsis undulata-S. micrantha and Kigelia pinnata-Annona squamosa. The distance matrix prepared with only 86 Orchidaceae species yielded five species pairs involving ten species showing zero distance estimates. The four species pairs were- Eria andamanica-Pinalia mysorensis; Paphiopedilum villosum-P. insigne; P. wardii-P. venustum and Platanthera edgeworthii-P. latilabris. The fifth pair was of synonymous species i.e., Pholidota imbricata and P. pallida. Thus, the species discrimination rate was 90.69% (Table 12).

4.3.1.2.2 PHYLOGENETIC TREE-BUILDING METHOD

The analyses of the aligned ITS sequences amplified from the 86 orchidaceae species and 45 species from other families of land plants revealed that out of 905 nucleotide sites compared, 713 were parsimony-informative sites (a site is

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parsimony-informative if it contains at least two types of nucleotides or amino acids, and at least two of them occur with a minimum frequency of two) and 71 were singleton sites (a singleton site contains at least two types of nucleotides or amino acids with, at the most, one occurring multiple times). MEGA identifies a site as a singleton site if at least three sequences contain unambiguous nucleotides (Tamura et al. 2007). The Neighbour joining tree was constructed with one thousand bootstraps replicates, revealed eight different clusters comprising 20 species, thus resulting in 84.73% species resolution. The species clusters formed were- (i) Coelogyne trinervis and C. quadritriloba (ii) Diplocentrum congestum, Cottonia peduncularis, Schoenorchis micrantha and Vandopsis undulata (iii) Eria andamanica and Pinalia mysorensis (iv) Peristylus plantagineus and Habenaria stocksii (v) Oberonia recurva and O. ensiformis (vi) Paphiopedilum venustum, P. insigne, P. villosum, P. wardii (vii) Platanthera edgeworthii and P. latilabris and (viii) Kigelia pinnata and Annona squamosa. The individuals of two species viz. Crepidium acuminatum and Rhynchosylis retusa that had intra-specific variations however, formed single cluster with all the accessions of each of these two clustering together. All the individuals of Paphiopedilum villosum clustered together along with other species viz., P. wardii, P. insigne and P. venustum. The individuals of Oberonia recurva clustered together along with O. ensiformis accessions, showing zero inter-specific variation. Thus, the individuals of all the four species with intra-specific variations clustered with their own species and intra-specific divergence had no effect on their clustering (Fig. 15). The species of four subfamilies of Orchidaceae (viz. Epidendroideae, Orchidoideae, Cyprepedioideae and Vanilloideae) formed different clusters. The orchids were unambiguously distinguishable from the monocot species included in the analysis. However, a few dicot species were clustered in the monocot cluster, thus, the monocots and dicots were not resolved as two clades (Fig. 15).

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Figure 15: Neighbour joining tree of 131 plant species based on ITS sequences. The species showing

zero inter-specific divergence are enclosed in a rectangle and the individuals of four species exhibiting intra-specific divergence are shown by numbers in paranthesis.

(tree continued)

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zero inter-specific divergence are enclosed in a rectangle and the individuals of four species exhibiting intra-specific divergence are shown by numbers in paranthesis

(tree continued)

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zero inter-specific divergence are enclosed in a rectangle and the individuals of four species exhibiting intra-specific divergence are shown by numbers in paranthesis.

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4.3.1.2.3 BLAST ANALYSIS

In the BLAST analysis of ITS sequences of 131 species, sequences of 120 matched correctly with the sequences of their own species or were unique. Only 11 species were not correctly identified. Thus, the species resolution based on ITS locus using BLAST method was 91.6% (Table 11). The species discrimination for only the orchid species based on BLAST analysis was 91.86% (Table 12). All the individuals of three orchid species exhibiting intra-specific variations matched correctly with their own species or appeared as unique sequences. However, one individual of Paphiopedilum villosum, the species that had exhibited intra-specific variation, showed 100% identity with other species of Paphiopedilum viz. P. insigne and P. gratixianum and the remaining three individuals matched with their own species. The BLAST analysis was also carried out to determine that the query or the amplified sequence is of the targeted locus. All the 131 ITS sequences generated in the present study were found to be of only the targeted locus, ITS (Appendix II). The sequences generated in the present study were only of plant species and not of contaminations of fungi (as the latter forms symbiotic relationship with orchids) or, the host (many orchids being epiphytic) was also confirmed by BLAST analysis (Appendix II).

4.3.2 matK 4.3.2.1 Inter-specific K2P Distances

The distance matrix of 172 matK sequences belonging to 138 species, including 94 orchids and 44 of the other families, revealed an average inter-specific K2P distance of 0.147 with a range being 0-0.429. Out of 138 species analyzed, 24 exhibited zero distance estimates with one or the other species. Of these, 22 belonged to the Orchidaceae (Fig. 16). The maximum inter-specific K2P distance was between Oryza sativa and Melillotus indica (Fig. 16). Eight orchid species, exhibiting intra-specific variations revealed zero distances amongst their accessions. The aligned matK sequences of 94 orchid species, had 0.066 average inter- specific K2P distance with a range of 0-0.18. Eighteen species exhibited zero distance estimates in the Orchidaceae data set (II). The highest inter-specific K2P distance (0.18) was between Pecteilis gigantea and Vanilla planifolia (Fig. 17).

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4.3.2.2 Species Resolution

4.3.2.2.1 DISTANCE BASED METHOD The matK distance matrix of 138 species revealed 26 species pairs involving

24 species with distance estimates of zero. Therefore, the species resolution by this method was 82.6% (Table 11). The species pairs formed were- Platanthera edgeworthii-P. latilabris, P. venustospicerianum-P. wardii, Paphiopedilum fairrieanum-P. pradhanii, P. villosum-P. insigne, P. wardii-P. venustum, P. venustoinsigne-P. venustospicerianum, P. venustoinsigne-P. venustum, P. venustoinsigne-P. wardii, P. venustospicerianum-P. venustum, Pleione praecox-P. maculata, Nervilia infundibulifolia-N. plicata, Coelogyne nitida-C. quadritriloba, Otochilus sp.-C. nitida, Otochilus sp.-C. quadritriloba, Liparis deflexa-L. nervosa, Crepidium acuminatum-L. nervosa, C. acuminatum-L. deflexa, P. venustoinsigne-P. insigne, P. venustospicerianum-P. insigne, P. venustum-P. insigne, P. wardii-P. insigne, P. villosum-P. venustum, P. wardii-P. insigne, P. venustoinsigne-P. villosum, P. venustospicerianum-P. villosum, Vandopsis undulata-Acampe praemorsa and Mimusops elengi-Achras sapota. The distance matrix of the 94 orchidaceous species (data set II) had 13 species pairs involving 18 species with zero distance estimates. The species discrimination rate was 80.85% (Table 12). Of these, 11 pairs were the same as initial 11 listed above, obtained by the analysis of data set I. The other two exclusive in the data set II were Otochilus sp.-Coelogyne fusescens and Nervilia gammieana-N. aragoana.


The analyses of the aligned matK sequences amplified from the 94 Orchidaceae species and 44 species from other families revealed that out of 867 nucleotide sites compared, 672 were variable sites of which 554 were parsimony-informative and 118 were singleton. The Neighbour joining tree constructed with thousand bootstraps replicates, revealed nine different clusters comprising 24 species,

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thus resulting in 82.6% speices resolution. The species clusters formed were (i) Acampe praemorsa and Vandopsis undulata, (ii) Crepidium acuminatum, Liparis nervosa and L. deflexa, (iii) Nervilia plicata and N. infundibulifolia, (iv) Coelogyne nitida, C. quadritriloba and Otochilus sp., (v) Pleione maculata and P. praecox, (vi) Platanthera edgeworthii and P. latilabris, (vii) Paphiopedilum pradhanii and P. fairrieanum, (viii) Paphiopedilum venustum, P. insigne, P. villosum, P. wardii, P. venustoinsigne and P. venustoinsigne and (ix) Mimusops elengiae and Achras sapota (Fig. 18). For matK locus, eight species had exhibited intra-specific variations. Out of these eight species, all accessions of each of the seven species formed sigle clusters. However, the accessions of Paphiopedilum venustum clustered along with other Paphiopedilum species with zero inter-specific variations. The NJ tree with matK sequences resulted in resolving the species of Orchidaceae into four sub-families. However, as with ITS, monocots other than orchids and dicots did not form two different clades (Fig. 18).

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Figure 18: Neighbour joining tree of 138 plant species based on matK sequences. The species showing

zero inter-specific divergence are enclosed in a rectangle and the individuals of nine species exhibiting intra-specific divergence are shown by numbers in the paranthesis.

(tree continued)

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Figure 18: Neighbour joining tree of 138 plant species based on matK sequences. The species showing

zero inter-specific divergence are enclosed in a rectangle and the individuals of nine species exhibiting intra-specific divergence are shown by numbers in the paranthesis.

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The BLAST analysis of matK sequences correctly identified 92.02% of the 138 analyzed species (Table 11). Out of 138 species, 127 were recovered as correct match or unique sequences, whereas 11 did not correctly match with their own. The species discrimination rate for data set II using BLAST was 91.48% (Table 12). The individuals of seven orchid species exhibiting intra-specific variations matched correctly with their own species. However, one individual of Otochilus sp., (SBB-0557), the species exhibiting intra-specific variations, showed 100% similarity with Coelogyne fuscescens. However, the remaining two individuals matched with their own species. The BLAST analysis also confirmed that all the amplified sequences were of only the matK locus.

4.3.3 rbcL


The distance matrix of 184 rbcL sequences obtained from 172 species, including 102 orchids, revealed average K2P distance of 0.071 with a range of 0-0.272. Out of 172 species analyzed, 45 species had zero distances with one or more species. All these 45 species with zero inter-specific K2P distances belonged to the family Orchidaceae. The other 70 species had inter-specific variations of variable range (Fig. 19). The individuals of the four species of Orchidaceae that exhibited intra-specific variations, showed zero distances amongst themselves. The maximum inter-specific K2P distance was between Clarkia unguiculata and Adiantum capillus-veneris (Fig. 19). The aligned rbcL sequences of only the orchid species (102) revealed an average inter-specific K2P distance of 0.023, with a range of 0-0.062. In this data set the number of species with zero distance estimates was also 45. The highest inter-specific K2P distance (0.062) was between Vanilla planifolia and Peristylus densus (Fig. 20).

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4.3.3.2.1 DISTANCE BASED METHOD

The rbcL distance matrix of 172 species had 45 species which could not be resolved because of their zero distances with other species and thus, resulted in a number of species pairs with distance estimates as zero. Consequently, 45 species remained unresolved. All these species belonged to the family Orchidaceae and also remained unresolved even in the distance matrix prepared with data set II of rbcL. Therefore, the species resolution based on rbcL sequences was 73.83% in the data set I and 55.58% in the data set II (Table 11 and 12). The species with zero distance estimates in both the data sets were: Coelogyne cristata, C. fusescens, C. longipes, C. nitida, C. quadritriloba, C. trinervis, Cottonia peduncularis, Diplocentrum congestum, Herminium lanceum, Liparis deflexa, L. nervosa, Luisia zeylanica, Crepidium acuminatum, Nervilia aragoana, N. infundibulifolia, N. gammieana, N. plicata, Platanthera edgeworthii, P. latilabris, Otochilus sp., Paphiopedilum druryi, P. fairrieanum, P. pradhanii, P. insigne, P. spicerianum, P. venustum, P. villosum, P. wardii, P. venustoinsigne, P. venustospicerianum, Pecteilis gigantea, Habenaria roxburghii, Phalaenopsis sp., Pholidota articulata, Pleione praecox, P. maculata, Pinalia mysorensis, P. spicata, Renanthera imschootiana, Rhynchostylis retusa, Schoenorchis micrantha, Acampe praemorsa, Eria andamanica, E. convallarioides, and Habenaria foliosa.


The analyses of the aligned rbcL sequences amplified from the 102 Orchidaceae species and 70 species from other families revealed that out of 667 nucleotide sites compared, 280 were variable sites of which 225 were parsimony-informative sites and 55 were singleton sites. The Neighbour joining tree with thousand bootstraps replicates revealed 13 different clusters comprising 45 unresolved species, thus enabling species resolution of 73.83%. The species clusters formed were-

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(i) Eria andamanica and Pinalia mysorensis, (ii) E. convallarioides and P. spicata, (iii) Crepidium acuminatum, Liparis nervosa and L. deflexa, (iv) Pleione praecox and P. maculata, (v) Coelogyne cristata, C. fuscescens, C. nitida C. quadritriloba, C. trinervis, C. longipes, Pholidota articulata and Otochilus sp., (vi) Habenaria roxburghii and Pecteilis gigantea, (vii) H. foliosa and Herminium lanceum, (viii) Platanthera edgeworthii and P. latilabris, (ix) Nervilia aragoana and N. gammieana, (x) Nervilia plicata and N. infundibulifolia, (xi) Paphiopedilum venustum, P. venustoinsigne and P. venustospicerianum, (xii) Paphiopedilum druryi, P. pradhanii, P. fairrieanum, P. insigne, P. spicerianum, P. villosum and P. wardii, (xiii) Acampe praemorsa, Cottonia peduncularis, Diplocentrum congestum, Luisia zeylanica, Phalaenopsis sp., Renanthera imschootiana, Rhynchostylis retusa and Schoenorchis micrantha (Fig. 21). All the accessions of four orchid species which had exhibited variations among their rbcL sequences, however, clustered with their own species (Fig. 21). The species belonging to four subfamilies of Orchidaceae did not cluster as separate clades. However, monocots, dicots, gymnosperms and pteridophyte formed well defined clades (Fig. 21).

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Figure 21: Neighbour joining tree of 172 plant species based on rbcL sequences. The species showing

zero inter-specific divergence are enclosed in a rectangle and the individuals of two species exhibiting intra-specific divergence are shown by numbers in the paranthesis.

(tree continued)

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Figure 21: Neighbour joining tree of 172 plant species based on rbcL sequences. The species showing

zero inter-specific divergence are enclosed in a rectangle and the individuals of two species exhibiting intra-specific divergence are shown by numbers in the paranthesis.

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The rbcL locus of investigated species on BLAST analysis afforded 66.86% species resolution as the sequences of 115 of the 172 analyzed species were recovered as correct match or unique sequences. The species discrimination rate based on this method in the data set II was 58.82%. The individuals of four orchid species exhibiting intra-specific variations matched correctly with their respective species. The BLAST analysis also confirmed that all the 172 sequences generated in the present study were from rbcL gene.

4.3.4 rpoB


The distance matrix for rpoB was prepared using 201 sequences belonging to 186 plants species. The inter-specific variations were calculated among 104 species of Orchidaceae alone or along with 82 non-orchid species. The average inter-specific K2P distance in the data set I was 0.106 with a range of 0-0.412. Out of 186 species analyzed, 56 exhibited zero distance estimates. Among the unresolved species (i.e., with zero inter-specific K2P distance), 50 belonged to the family Orchidaceae (Fig. 22). The accessions of four orchid species, exhibiting intra-specific variations, showed zero distances amongst the individuals of the same species. The maximum inter-specific K2P distance (0.412) was between Dendrocalamus sp.-Agathis robusta as well as Oryza sativa-Agathis robusta (Fig. 22). The aligned rpoB sequences of only the orchidaceous species showed an average inter-specific K2P distance of 0.04 (with a range of 0-0.103). In this data set (II) only 46 species had zero distance estimates, as opposed to 50 in the data set I having both orchid and non-orchid species. The highest inter-specific K2P distance (0.103) was between species pairs of Vanilla planifolia-Conchidium filiforme, Vanilla planifolia-Eria exilis, Vanilla planifolia- Nervilia aragoana, Vanilla planifolia-N. gammieana, Vanilla planifolia-N. plicata and Vanilla planifolia-N. infundibulifolia species pairs (Fig. 23).

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4.3.4.2.1 DISTANCE BASED METHOD The rpoB distance matrix of 186 species revealed 56 species with zero distance

estimates. Therefore, the species resolution based on this method was 69.89% (Table 11). The species wth zero distance estimates were- Acampe praemorsa, Vanda cristata, V. stangeana, V. testacea, V. tessellata, Aerides maculosa, Epipactis veratrifolia, Cleisocentron sp., Pinalia mysorensis, Conchidium braccatum, Eria andamanica, Coelogyne cristata, C. fusescens, C. longipes, C. trinervis, Cottonia peduncularis, Diplocentrum congestum, Pleione praecox, P. maculata, Liparis deflexa, L. nervosa, Pholidota articulata, P. pallida, Oberonia brunoniana, O. ensiformis, Nervilia aragoana, N. infundibulifolia, N. gammieana, N. plicata, Platanthera edgeworthii, P. latilabris, Paphiopedilum druryi, P. fairrieanum, P. hirsutissimum, P. pradhanii, P. insigne, P. spicerianum, P. venustum, P. villosum, P. wardii, P. venustoinsigne, P. venustospicerianum, Peristylus densus, P. plantagineus, Habenaria heyneana, H. intermedia, H. longicorniculata, H. panchganiensis, H. crinifera, H. foliosa, Achras sapota, Mimusops elengiae, Datura inoxia, D. metel, Lagerstroemis indica and L. speciosa. The analysis of rpoB distance matrix of the data set II yielded a species discrimination rate of 55.76%, as 46 species had zero distance estimates (Table 12). The four species viz., C. braccatum, H.foliosa, H. crinifera and P. druryi showing zero distance estimates in data set I got resolved in data set II and the remaining 46 unresoled species were common in both the data sets.


The analyses of the aligned rpoB sequences of the data set I revealed that out of 417 nucleotide sites compared, 257 were variable sites of which 208 were parsimony-informative and 48 were singleton. The Neighbour joining tree constructed with thousand bootstraps replicates resulted in seventeen different clusters comprising 56 species, thus resulting in 69.89% spcies resolution. The species clusters formed

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were: (i) Acampe praemorsa, Aerides maculosa, Cottonia peduncularis and Vanda cristata, (ii) Diplocentrum congestum, Vanda stangeana and Cleisocentron sp., (iii) Vanda testacea, V. tessellata and Epipactis veratrifolia, (iv) Eria andamanica, Pinalia mysorensis and Conchidium braccatum, (v) Pleione praecox, P. maculata, Pholidota articulata, P. pallida, Coelogyne cristata, C. fuscescens, C. trinervis and C. longipes, (vi) Oberonia brunoniana and O. ensiformis, (vii) Liparis nervosa and L. deflexa, (viii) Nervilia aragoana and N. gammieana, (ix) Nervilia plicata and N. infundibulifolia, (x) Paphiopedilum venustum, P. venustoinsigne and P. venustospicerianum, Paphiopedilum druryi, P. pradhanii, P. fairrieanum, P. hirsutissimum, P. insigne, P. spicerianum, P. villosum and P. wardii, (xi) Platanthera edgeworthii and P. latilabris, (xii) Habenaria heyneana, H. intermedia and H. foliosa, (xiii) Peristylus densus and P. plantagineus, (xiv) H. crinifera, H. panchganiensis, and H. longicorniculata, (xv) Achras sapota and Mimusops elengiae, (xvi) Datura inoxia and D. metel, (xvii) Lagerstroemia indica and L. speciosa (Fig. 24). The accessions of each of the four species that exhibited intra-specific variations for rpoB locus, accumulated in single clusters (Fig. 24). The monocots, dicots and gymnosperm and the species from the four subfamilies of Orchidaceae formed distinct clades (Fig. 24).

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Figure 24: Neighbour joining tree of 186 plant species based on rpoB sequences. The species showing

zero inter-specific divergence are enclosed in a rectangle and the individuals of four species exhibiting intra-specific divergence are shown by numbers in the paranthesis.

(tree continued)

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zero inter-specific divergence are enclosed in a rectangle and the individuals of four species exhibiting intra-specific divergence are shown by numbers in the paranthesis

(tree continued)

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zero inter-specific divergence are enclosed in a rectangle and the individuals of four species exhibiting intra-specific divergence are shown by numbers in the paranthesis.

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A species resolution of 70.43% was obtained on BLAST analysis of rpoB sequences of 186 investigated species (Table 11). Out of 186 analyzed species, 131 were recovered as correct match or unique sequence, whereas sequences of 55 species showed homology with those of other species. The species discrimination rate in data set II was 61.53% (Table 12). The individuals of four orchid species exhibiting intra-specific variations matched correctly only with their respective species. The BLAST analysis also confirmed that all the queried sequences were from the rpoB locus.

4.3.5 rpoC1


The distance matrix for rpoC1 was prepared using 189 sequences belonging to 100 orchid and 76 out-group species (data set I) yielded an average inter-specific K2P distance of 0.095 with a range of 0-0.373. Out of 176 species analyzed, 59 species exhibited zero distance estimates. From the 59 unresolved species (i.e., with zero inter-specific K2P distance), 53 belonged to the family Orchidaceae (Fig. 25). Three orchid species, exhibiting intra-specific variations showed zero distances amongst their accessions. The maximum inter-specific K2P distance (0.373) was between Nicotiana tabacum and Ephedra gerardiana (Fig. 25). The aligned rpoC1 sequences of only the orchid species revealed an average inter- specific K2P distance of 0.03 (range: 0-0.093), with 53 having zero distance estimates with one or the other species. The highest inter-specific K2P distance (0.093) was between species pair of Vanilla planifolia-Nervilia aragoana, Vanilla planifolia-N. gammieana, and Vanilla planifolia-N. plicata (Fig. 26).

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4.3.5.2.1 DISTANCE BASED METHOD The number of species with zero distance estimates in the K2P distance matrix

generated by aligning rpoC1 sequences of 176 species was 59. Thus, the species resolution using distance method was 66.47% (Table 11). The species wth zero distance estimates with one or the other other species were: Acampe praemorsa, Aerides maculosa, A. multiflora, Vanda cristata, V. stangeana, V. tessellata, Pinalia mysorensis, P. spicata, Crepidium acuminatum, C. resupinatum, Eria andamanica, E. coronaria, E. convallariodes, Coelogyne cristata, C. longipes, C. trinervis, Cottonia peduncularis, Diplocentrum congestum, Eulophia flava, E. spectabilis, Otochilus sp., Pleione praecox, Liparis deflexa, L. nervosa, Pholidota articulata, P. pallida, Oberonia mucronata, O. ensiformis, Nervilia aragoana, N. infundibulifolia, N. plicata, Platanthera edgeworthii, P. latilabris, Paphiopedilum druryi, P. fairrieanum, P. pradhanii, P. insigne, P. spicerianum, P. venustum, P. villosum, P. wardii, P. venustoinsigne, P. venustospicerianum, Porpax jerdoniana, Habenaria heyneana, H. intermedia, H. longicorniculata, H. panchganiensis, H. furcifera, H. foliosa, Renanthera imschootiana, Rhynchostylis retusa, Holcoglossum amesianum, Kigelia pinnata, Adiantum capillus-veneris, Datura inoxia, D. metel, Lagerstroemis indica and L. speciosa. The distance matrix of rpoC1 sequences of only the orchid species (100 in data set II) yielded 53 species with zero distance estimates, thus yielding a species discrimination rate of 47% (Table 12). The 53 orchid species with zero distance estimates in data set set II were same as those in data set I.


The analyses of the aligned rpoC1 sequences of 176 species revealed that out of 474 nucleotide sites compared, 252 were variable sites of which 202 were parsimony-informative and 50 were singleton. The Neighbour joining tree, constructed with thousand bootstraps replicates, had fifteen different clusters,

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comprising 59 unresolved species, and thus resulting in 66.47% species resolution. The species clusters formed were: (i) Pholidota articulata, P. pallida, Otochilus sp., Pinalia mysorensis, Eria andamanica, E. coronaria, Coelogyne cristata, C. longipes, C. trinervis, C. quadritriloba, C. nitida, Pleione praecox and Porpax jerdoniana, (ii) N. infundibulifolia, Eulophia flava and E. spectabilis, (iii) Oberonia mucronata and O. ensiformis, (iv) Crepidium acuminatum, C. resupinatum and Liparis deflexa, (v) Nervilia aragoana and N. plicata, (vi) E. convallarioides and Pinalia spicata (vii) Vanda cristata, V. stangeana, V. tessellata and Holcoglossum amesianum, (viii) Acampe praemorsa, Aerides maculosa, A. multiflora, Cottonia peduncularis, Diplocentrum congestum, Renanthera imschootiana and Rhynchostylis retusa, (ix) H. longicorniculata and H. furcifera, (x) H. intermedia and H. foliosa, (xi) Platanthera edgeworthii and P. latilabris, (xii) Paphiopedilum druryi, P. fairrieanum, P. pradhanii, P. insigne, P. spicerianum, P. venustum, P. villosum, P. wardii, P. venustoinsigne and P. venustospicerianum (xiii) Kigelia pinnata and Adiantum capillus-veneris, (xiv) Datura inoxia- D. metel and (xv) Lagerstroemis indica and L. speciosa (Fig. 27). The accessions of four orchid species, exhibiting intra-specific variations in their rpoB sequences, formed individual clusters (Fig. 27). The species belongigng to four subfamilies of Orchidaceae clustered separately as four clades. The monocots, dicots and gymnosperms formed distinct clusters with the exception of Adiantum capillus-veneris (a pteridophyte) found nestled among dicots and showing 100% similarity to Kigelia pinnata rpoC1 sequence (Fig. 27).

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Figure 27: Neighbour joining tree of 176 plant species based on rpoC1 sequences. The species

showing zero inter-specific divergence are enclosed in a rectangle and the individuals of three species exhibiting intra-specific divergence are shown by numbers in the paranthesis.

(tree continued)

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Figure 27: Neighbour joining tree of 176 plant species based on rpoC1 sequences. The species

showing zero inter-specific divergence are enclosed in a rectangle and the individuals of three species exhibiting intra-specific divergence are shown by numbers in the paranthesis.

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The BLAST analysis of rpoC1 locus of 176 species provided 61.93% species resolution, as out of 176 analyzed species, 109 were recovered as correct match or unique sequences, whereas 67 species could not be correctly identified. The species discrimination rate for orchidaceous species was 50% (Table 11 and 12). The sequences of individuals of the four orchid species exhibiting intra-specific variations matched correctly with the sequence of their respective species. The BLAST analysis also confirmed that all the 189 sequences generated in the present study were from rpoC1 gene region. Table 11: Average inter-specific K2P distance and per cent species resolution of tested

plant species (orchids and out-group species) using five candidate loci.

Locus No. of Species

Average Inter-specific K2P distance (Range)

Species Discrimination Rates Distance Based Method

Phylogenetic Tree

Method

BLAST Method

ITS 131 0.164 (0-0.408) 84.73% 84.73% 91.6%

matK 138 0.147 (0-0.429) 82.6% 82.6% 92.02%

rbcL 172 0.071 (0-0.272) 73.83% 73.83% 66.86%

rpoB 186 0.106 (0-0.412) 69.89% 69.89% 70.43%

rpoC1 176 0.095 (0-0.373) 66.47% 66.47% 61.93%

Table 12: Average inter-specific K2P distance and per cent species resolution of

Orchidaceae species using five candidate loci.

Locus No. of Species analyzed

Average Inter-specific K2P distance (Range)

Species Discrimination Rates Distance Based Method

Phylogenetic Tree Method

BLAST method

ITS 86 0.247 (0-0.498) 90.9% 90.9% 91.86%

matK 94 0.066 (0-0.18) 80.85% 80.85% 91.48%

rbcL 102 0.023 (0-0.062) 55.88% 55.88% 58.82%

rpoB 104 0.04 (0-0.103) 55.76% 55.76% 61.53%

rpoC1 100 0.03 (0-0.09) 47% 47% 50%

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4.4 INTER-SPECIFIC K2P DISTANCES AND SPECIES DISCRIMINATION RATES FOR THE FIVE CANDIDATE LOCI

AT THE GENERIC LEVEL

The inter-specific variations and species discrimination rates among the congeneric species were also calculated individually for all the five loci for twenty orchid genera, which were represented by more than one species. The species discrimination rates were calculated both using genetic distance and phylogenetic tree methods. For the latter, the Neighbour joining trees with thousand bootstrap replicates were constructed for all the five tested loci. The genus wise inter-specific variations recorded and percent species resolutions yielded are detailed below. For the sake of simplicity, the genera have been arranged alphabetically.

4.4.1 Aerides

Three Aerides species were analyzed viz., A. maculosa [one (1) individual (I)], A. multiflora (4I) and A. odorata (1I). The ITS, matK and rbcL could be compared only for two species as sequences of these loci from one species (A. odorata) were not obtained. The average inter-specific distances for ITS, matK, rbcL, rpoB and rpoC1 were 0.072, 0.07, 0.01, 0.006 and 0.002, respectively. ITS, matK and rbcL sequences distinguished both the species, rpoB sequences were unique to each of the three species studied. However, rpoC1 sequences of two species (A. maculosa and A. multiflora) had distance estimate of zero, thus failing to differentiate between these two species.

The NJ trees constructed using accessions of two species A. multiflora and A.

maculosa for ITS, matK and rbcL distinguished both the species correctly (Fig. 28a,b,c). Likewise, rpoB distinguished all the three species. However, with rpoC1 species discrimination was only 33.33%. In rpoC1 tree, the individuals of A. maculosa and A. multiflora clustered together (Fig. 1e). Out of 729 nucleotides of ITS analyzed, 45 were parsimony informative sites. Out of 794 nucleotide sites of matK sequences analyzed, 5 were parsimony informative. For rbcL sequences, out of 660 nucleotides analyzed, 6 were parsimony informative. The number of parsimony informative sites for rpoB and rpoC1 were 2 out of 363 and 1 out of 431 nucleotides, respectively.

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(a)

0720 Aerides multiflora 0730 Aerides multiflora 0729 Aerides multiflora 0731 Aerides multiflora 0441 Aerides maculosa 0442 Aerides maculosa

100

0.005 (b)

SBB-0442 Aerides maculosa SBB-0858 Aerides maculosa SBB-0859 Aerides maculosa SBB-0441 Aerides maculosa SBB-0731 Aerides multiflora SBB-0730 Aerides multiflora SBB-0720 Aerides multiflora SBB-0729 Aerides multiflora

99

0.001 (c)

SBB-0442 Aerides maculosa SBB-0859 Aerides maculosa SBB-0441 Aerides maculosa SBB-0858 Aerides maculosa SBB-0860 Aerides maculosa SBB-0730 Aerides multiflora SBB-0731 Aerides multiflora SBB-0720 Aerides multiflora SBB-0729 Aerides multiflora

99

0.001 Figure 28: NJ trees for (a) ITS (b) matK and (c) rbcL of Aerides species

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(d)

SBB-0441 Aerides maculosa SBB-0859 Aerides maculosa SBB-0858 Aerides maculosa SBB-0860 Aerides maculosa

SBB-0731 Aerides multiflora SBB-0730 Aerides multiflora SBB-0720 Aerides multiflora SBB-0729 Aerides multiflora

SBB-0733 Aerides odorata

64

19

0.0005 (e)

SBB-0441 Aerides maculosa SBB-0860 Aerides maculosa SBB-0859 Aerides maculosa SBB-0442 Aerides maculosa SBB-0730 Aerides multiflora SBB-0858 Aerides maculosa SBB-0729 Aerides multiflora SBB-0720 Aerides multiflora SBB-0732 Aerides odorata SBB-0733 Aerides odorata

67

0.0002 Figure 28: NJ trees for (d) rpoB and (e) rpoC1 loci of Aerides species. The species

showing zero inter-specific divergence are enclosed in the rectangle.

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4.4.2 Coelogyne

A total of 14 accessions belonging to six Coelogyne species were analyzed using the five candidate loci. The analyzed species were C. cristata, C. fuscescens, C. longipes, C. nitida, C. quadritriloba and C. trinervis with 6, 1, 1, 4, 1 and 1 individuals, respectively. The average inter-specific distances for ITS, matK, rbcL, rpoB and rpoC1 were 0.054, 0.005, 0.001, 0.002 and 0.003, respectively. The ITS and matK sequences could resolve all six species of Coelogyne, thus resulting in 100% species resolution. The remaining loci from the chloroplast genome had resulted in number of species pairs with distance estimates as zero. The species discrimination rates for rbcL, rpoB and rpoC1 were 16.6, 33.3 and 33.33%, respectively.

Of the 710 nucleotide sites of ITS sequences of six Coelogyne species

compared, 86 were variable sites. Of these 54 were parsimony informative. All the six species with variable number of accessions (1-6) formed single species clusters (Fig. 29a), thus resulting in 100% species resolution. Out of 779 nucleotides of matK compared, the number of variable sites was 10, of which three were parsimony informative. All the six species could be unambiguously distinguished with matK barcodes (Fig. 29b). Slightly different trees topologies were obtained with ITS sequences as compared to matK but 100% monophyly of the Coelogyne species was affirmed with both the barcode sequences (Fig. 29 a,b).

The remaining three loci showed species clusters with different number of

unresolved species (Fig. 29c, d, e). Thus, the resulting species resolutions for rbcL, rpoB and rpoC1 loci were 16.6, 33.3 and 33.33% respectively. The rbcL sequences showed only one variable site from 660 nucleotides analyzed and none was found to be parsimony informative. The numbers of parsimony informative sites were 1 and 3 for rpoB and rpoC1 loci, respectively.

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(a)

0648 Coelogyne cristata 0650 Coelogyne cristata 0649 Coelogyne cristata 0644 Coelogyne cristata 0645 Coelogyne cristata

0990 Coelogyne longipes 0637 Coelogyne nitida 0640 Coelogyne nitida 0639 Coelogyne nitida 0633 Coelogyne nitida 0638 Coelogyne nitida

0607 Coelogyne fuscescens 0612 Coelogyne fuscescens

0312 Coelogyne quadritriloba 0308 Coelogyne trinervis

99

62

99

100

54

100

0.005 (b)

0634 Coelogyne nitida 0640 Coelogyne nitida 0633 Coelogyne nitida 0637 Coelogyne nitida 0638 Coelogyne nitida 0639 Coelogyne nitida 0607 Coelogyne fuscescens

0312 Coelogyne quadritriloba 0650 Coelogyne cristata 0645 Coelogyne cristata 0649 Coelogyne cristata 0647 Coelogyne cristata 0644 Coelogyne cristata 0648 Coelogyne cristata 0990 Coelogyne longipes

0308 Coelogyne trinervis69

63

63

0.0005 Figure 29: NJ trees for (a) ITS and (b) matK loci of Coelogyne species.

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SBB-0650 Coelogyne cristata SBB-0612 Coelogyne fuscescens SBB-0990 Coelogyne longipes SBB-0637 Coelogyne nitida SBB-0649 Coelogyne cristata SBB-0633 Coelogyne nitida SBB-0648 Coelogyne cristata SBB-0645 Coelogyne cristata SBB-0640 Coelogyne nitida SBB-0607 Coelogyne fuscescens SBB-0308 Coelogyne trinervis SBB-0639 Coelogyne nitida SBB-0312 Coelogyne quadritriloba

0.0002 (d)

SBB-0633 Coelogyne nitida SBB-0634 Coelogyne nitida SBB-0637 Coelogyne nitida SBB-0638 Coelogyne nitida

SBB-0990 Coelogyne longipes SBB-0645 Coelogyne cristata SBB-0308 Coelogyne trinervis SBB-0648 Coelogyne cristata SBB-0607 Coelogyne fuscescens SBB-0649 Coelogyne cristata SBB-0650 Coelogyne cristata SBB-0647 Coelogyne cristata SBB-0644 Coelogyne cristata

SBB-0312 Coelogyne quadritriloba

63

0.0001 Figure 29: NJ trees for (c) rbcL and (d) rpoB loci of Coelogyne species. The species

forming clusters are highlighted in the rectangle.

(c)

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(e) SBB-0312 Coelogyne quadritriloba

SBB-0308 Coelogyne trinervis SBB-0634 Coelogyne nitida SBB-0633 Coelogyne nitida SBB-0648 Coelogyne cristata SBB-0650 Coelogyne cristata SBB-0649 Coelogyne cristata SBB-0639 Coelogyne nitida SBB-0644 Coelogyne cristata SBB-0645 Coelogyne cristata SBB-0638 Coelogyne nitida SBB-0990 Coelogyne longipes SBB-0637 Coelogyne nitida SBB-0640 Coelogyne nitida SBB-0647 Coelogyne cristata

SBB-0607 Coelogyne fuscescens SBB-0612 Coelogyne fuscescens87

0.0005 Figure 29: NJ tree for (e) rpoC1 locus of Coelogyne species. The species forming

clusters are highlighted in the rectangle.

4.4.3 Conchidium

A total of 14 individuals of two species, C. braccatum and C. filiforme, with the former being represented by 5 and and the latter by 9 individuals, were analyzed. The average inter-specific K2P distances for ITS, matK, rbcL, rpoB and rpoC1 were 0.395, 0.059, 0.028, 0.037 and 0.014, respectively. The highest inter-specific variation was observed in ITS sequences followed by matK. The two species, C. braccatum and C. filiforme were resolved by all the five tested loci individually.

The seventeen individuals of two Conchidium species formed two separate

clusters with all the five tested barcode loci (Fig. 30 a-e), thus all the tested loci could individually distinguish two species. The number of variable and parsimony informative sites were 205 (out of 775 total nucleotide sites), 45 (787 total sites), 16

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(656 total sites), 13 (368 total sites) and 6 (434 total sites) for ITS, matK, rbcL, rpoB and rpoC1 sequences, respectively.

(a)

0452 Conchidium filiforme 0741 Conchidium filiforme 0455 Conchidium filiforme 0740 Conchidium filiforme 0739 Conchidium filiforme 0738 Conchidium filiforme 0456 Conchidium filiforme 0742 Conchidium filiforme 0453 Conchidium filiforme 0744 Conchidium braccatum 0745 Conchidium braccatum 0746 Conchidium braccatum 0743 Conchidium braccatum 0747 Conchidium braccatum

100

0.05 (b)

0452 Conchidium filiforme 0742 Conchidium filiforme 0453 Conchidium filiforme 0455 Conchidium filiforme 0740 Conchidium filiforme 0741 Conchidium filiforme 0456 Conchidium filiforme 0739 Conchidium filiforme 0964 Conchidium filiforme 0738 Conchidium filiforme 0963 Conchidium filiforme 0744 Conchidium braccatum 0747 Conchidium braccatum 0743 Conchidium braccatum 0962 Conchidium braccatum 0745 Conchidium braccatum 0746 Conchidium braccatum

100

0.005 Figure 30: NJ trees for (a) ITS and (b) matK loci of Conchidium species.

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(c)

SBB-0453 Conchidium filiforme SBB-0742 Conchidium filiforme SBB-0452 Conchidium filiforme SBB-0456 Conchidium filiforme SBB-0740 Conchidium filiforme SBB-0741 Conchidium filiforme SBB-0455 Conchidium filiforme SBB-0739 Conchidium filiforme SBB-0745 Conchidium braccatum SBB-0747 Conchidium braccatum SBB-0744 Conchidium braccatum SBB-0743 Conchidium braccatum SBB-0746 Conchidium braccatum

100

0.002 (d)

SBB-0738 Conchidium filiforme SBB-0740 Conchidium filiforme SBB-0456 Conchidium filiforme SBB-0739 Conchidium filiforme SBB-0742 Conchidium filiforme SBB-0741 Conchidium filiforme SBB-0453 Conchidium filiforme SBB-0455 Conchidium filiforme SBB-0452 Conchidium filiforme SBB-0745 Conchidium braccatum SBB-0746 Conchidium braccatum SBB-0744 Conchidium braccatum SBB-0743 Conchidium braccatum SBB-0747 Conchidium braccatum

100

0.005 Figure 30: NJ trees for (c) rbcL and (d) rpoB loci of Conchidium species.

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(e) SBB-0738 Conchidium filiforme SBB-0740 Conchidium filiforme SBB-0452 Conchidium filiforme SBB-0453 Conchidium filiforme SBB-0455 Conchidium filiforme SBB-0739 Conchidium filiforme SBB-0741 Conchidium filiforme SBB-0742 Conchidium filiforme SBB-0456 Conchidium filiforme SBB-0746 Conchidium braccatum SBB-0745 Conchidium braccatum SBB-0962 Conchidium braccatum SBB-0744 Conchidium braccatum SBB-0743 Conchidium braccatum SBB-0747 Conchidium braccatum

97

0.001 Figure 30: NJ tree for (e) rpoC1 locus of Conchidium species.

4.4.4 Crepidium

The Crepidium species investigated were C. acuminatum (5I) and C. resupinatum (7I). The average inter-specific distances for ITS, matK, rbcL, rpoB and rpoC1 were 0.057, 0.008, 0.002, 0.003 and zero, respectively. The highest inter-specific variation was exhibited by ITS and discrimination of both the species was possible on the basis of variation in this locus. Likewise, all the loci from the chloroplast genome, except rpoC1, could differentiate the two species.

The thirteen accessions of Crepidium belonging to two species clustered on two different branches thus resulting in 100% species resolution in ITS, matK, rbcL and rpoB trees (Fig. 31 a-d). For ITS sequences, out of 40 variable sites, 38 were parsimony informative sites (708 total sites). One individual of C. resupinatum clustered singly from rest of the individuals exhibiting intra-specific variations in ITS sequences (Fig. 31a). The numbers of variable and parsimony informative sites were 6 of 762 total sites in matK and one out of 630 and 369 total sites in rbcL and rpoB. In contrast, rpoC1 sequences resulted in collective clustering of individuals of two

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species, with no parsimony informative site and hence could not discriminate the two species (Fig. 31e). (a)

0886 Crepidium acuminatum 0890 Crepidium acuminatum

0888 Crepidium acuminatum 0887 Crepidium acuminatum 0889 Crepidium acuminatum

0356 Crepidium resupinatum 0357 Crepidium resupinatum100

24

2615

0.005 0948 Crepidium resupinatum 0950 Crepidium resupinatum 0949 Crepidium resupinatum 0952 Crepidium resupinatum 0357 Crepidium resupinatum 0951 Crepidium resupinatum 0358 Crepidium resupinatum 0356 Crepidium resupinatum 0886 Crepidium acuminatum 0889 Crepidium acuminatum 0888 Crepidium acuminatum 0887 Crepidium acuminatum 0890 Crepidium acuminatum

99

0.001 SBB-0356 Crepidium resupinatum SBB-0951 Crepidium resupinatum SBB-0950 Crepidium resupinatum SBB-0949 Crepidium resupinatum SBB-0357 Crepidium resupinatum SBB-0948 Crepidium resupinatum SBB-0952 Crepidium resupinatum SBB-0888 Crepidium acuminatum SBB-0889 Crepidium acuminatum SBB-0886 Crepidium acuminatum SBB-0887 Crepidium acuminatum SBB-0890 Crepidium acuminatum

64

0.0002 Figure 31: NJ trees for (a) ITS (b) matK and (c) rbcL loci of Crepidium species.

(b)

(c)

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SBB-0887 Crepidium acuminatum SBB-0889 Crepidium acuminatum SBB-0886 Crepidium acuminatum SBB-0888 Crepidium acuminatum SBB-0890 Crepidium acuminatum SBB-0952 Crepidium resupinatum SBB-0357 Crepidium resupinatum SBB-0356 Crepidium resupinatum SBB-0951 Crepidium resupinatum

65

0.0002 SBB-0888 Crepidium acuminatum SBB-0951 Crepidium resupinatum SBB-0950 Crepidium resupinatum SBB-0356 Crepidium resupinatum SBB-0948 Crepidium resupinatum SBB-0886 Crepidium acuminatum SBB-0952 Crepidium resupinatum SBB-0357 Crepidium resupinatum SBB-0889 Crepidium acuminatum SBB-0890 Crepidium acuminatum SBB-0887 Crepidium acuminatum SBB-0949 Crepidium resupinatum

50

0

5

50

0

0

0

Figure 31: NJ trees for (d) rpoB and (e) rpoC1 loci of Crepidium species.

4.4.5 Cymbidium

Three Cymbidium species evaluated in the present investigation were C. aloifolium (5I), C. cochleare (2I) and C. devonianum (1I). The average inter-specific distance for ITS, matK, rbcL, rpoB and rpoC1 were 0.123, 0.02, 0.008, 0.01 and 0.003, respectively. All the five tested loci were able to differentiate the three species individually on the basis of K2P distances. Likewise, with NJ trees also, 100% species resolution was provided by all the five tested loci (Fig. 32 a-e). The ITS sequences with 693 nucleotides showed 100 variable sites of which only 9 were parsimony informative. The matK sequences had 780 nucleotides of which 25 were variable and 18 were parsimony informative sites. The number of parsimony informative sites for

(d)

(e)

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rbcL, rpoB and rpoC1 sequences were 7 (out of 629 nucleotide sites), 4 (out of 369 sites) and 3 (out of 436 sites), respectively. (a)

0326 Cymbidium aloifolium 0332 Cymbidium aloifolium

0579 Cymbidium cochleare 0558 Cymbidium devonianum

99

0.02

(b)

0579 Cymbidium cochleare 0608 Cymbidium cochleare

0558 Cymbidium devonianum 0326 Cymbidium aloifolium 0332 Cymbidium aloifolium100

99

0.002

(c) SBB-0579 Cymbidium cochleare SBB-0608 Cymbidium cochleare SBB-0558 Cymbidium devonianum SBB-0326 Cymbidium aloifolium SBB-0332 Cymbidium aloifolium100

69

0.001 SBB-0326 Cymbidium aloifolium SBB-0332 Cymbidium aloifolium

SBB-0558 Cymbidium devonianum SBB-0579 Cymbidium cochleare SBB-0608 Cymbidium cochleare95

66

0.001 Figure 32: NJ trees for (a) ITS (b) matK (c) rbcL and (d) rpoB loci of Cymbidium species.

(d)

��

��

SBB-0579 Cymbidium cochleare SBB-0608 Cymbidium cochleare SBB-0558 Cymbidium devonianum SBB-0326 Cymbidium aloifolium SBB-0332 Cymbidium aloifolium90

68

0.001 Figure 32: NJ tree for (e) rpoC1 locus of Cymbidium species.

4.4.6 Eria

The four species of Eria used in present study were E. andamanica, E. coronaria (1I each); E. convallarioides (2I) and E. exilis (4I). The average inter-specific distance for ITS, matK, rbcL, rpoB and rpoC1 were 0.168, 0.04, 0.0.4, 0.027, and 0.02, respectively. For matK, sequence of E. coronaria was not obtained. The sequences of all the five candidate loci were divergent enough to provide 100 % species resolution among these species by both K2P distance and phylogenetic tree methods (Fig. 33 a-e).

Out of 812 nucleotides in matK, 47 were variable and 41 were parsimony

informative sites. In case of ITS 711 bases were obtained and out of 200 variable sites, 150 were parsimony informative. The number of parsimony informative sites in rbcL, rpoB and rpoC1 sequences were 13, 15 and 13 out of 628, 370 and 433 total sites, respectively.

0582 Eria convallarioides 0627 Eria convallarioides

0296 Eria andamanica 0585 Eria coronaria

0807 Eria exilis 0808 Eria exilis 0809 Eria exilis 0810 Eria exilis

10099

100

0.02 Figure 33: NJ tree for (a) ITS locus of Eria species.

(e)

(a)

��

��

(b)

0807 Eria exilis 0808 Eria exilis 0810 Eria exilis 0809 Eria exilis 0296 Eria andamanica

0582 Eria convallarioides

100

0.005 (c)

SBB-0627 Eria convallarioides SBB-0582 Eria convallarioides

SBB-0296 Eria andamanica SBB-0585 Eria coronaria SBB-0810 Eria exilis SBB-0809 Eria exilis SBB-0807 Eria exilis SBB-0808 Eria exilis

9565

99

0.002


SBB-0585 Eria coronaria SBB-0296 Eria andamanica

SBB-0808 Eria exilis SBB-0809 Eria exilis SBB-0807 Eria exilis SBB-0810 Eria exilis

8760

100

0.005 Figure 33: NJ trees for (b) matK (c) rbcL and (d) rpoB loci of Eria species.

(d)

��

��


SBB-0585 Eria coronaria SBB-0296 Eria andamanica

SBB-0810 Eria exilis SBB-0807 Eria exilis SBB-0808 Eria exilis SBB-0809 Eria exilis

9975

100

0.002 Figure 33: NJ tree for (e) rpoC1 locus of Eria species.

4.4.7 Eulophia

The average inter-specific variations between two species, E. flava (2I) and E. spectabilis [Syn: E. nuda] (5I) were 0.008, 0.002 and 0.009 in matK, rbcL and rpoB loci. However, the distance estimate in rpoC1 between two species was zero. In ITS, sequences for E. spectabilis accessions were not obtained. The two species of Eulophia could be distinguished by matK as well as rpoB. In rbcL, the average inter-specific distance was 0.002 and the maximum intra-specific variation among E. spectabilis accessions was also 0.002. Therefore, rbcL could not discriminate the two species.

The accessions of E. spectabilis and E. flava formed two different clusters in phylogenetic trees prepared using individually the three tested loci from the chloroplast genome (Fig. 34 a-c). Whereas, in rpoC1 NJ tree, all individuals of two Eulophia species were segregated in one cluster (Fig. 34 d). Out of 815 total nucleotide sites in matK sequences, the variable sites were 9, of which 7 were parsimony informative. The individuals of E. nuda showed intra-specific variations therefore, one individual formed separate branch from rest of the individuals (Fig. 34a). The rbcL tree showed two accessions of E. spectabilis with intra-specific variations as separate clusters. The number of parsimony informative sites in rbcL and rpoB sequences were 2 (out of 646 total nucleotide sites) and 5 (368 total sites), respectively.

(e)

��

��

(a)

0955 Eulophia nuda 0957 Eulophia nuda 0954 Eulophia nuda

0956 Eulophia nuda 0879 Eulophia flava 0880 Eulophia flava99

65

0.001 SBB-0955 Eulophia spectabilis SBB-0958 Eulophia spectabilis

SBB-0956 Eulophia spectabilis SBB-0957 Eulophia spectabilis SBB-0954 Eulophia spectabilis

SBB-0879 Eulophia flava SBB-0880 Eulophia flava68

70

0.0002 SBB-0954 Eulophia spectabilis SBB-0957 Eulophia spectabilis SBB-0956 Eulophia spectabilis SBB-0955 Eulophia spectabilis SBB-0958 Eulophia spectabilis SBB-0879 Eulophia flava SBB-0880 Eulophia flava95

66

63

0.001 SBB-0880 Eulophia flava SBB-0957 Eulophia spectabilis SBB-0955 Eulophia spectabilis SBB-0956 Eulophia spectabilis SBB-0958 Eulophia spectabilis SBB-0879 Eulophia flava SBB-0954 Eulophia spectabilis

10

10

6

8

Figure 34: NJ trees for (a) matK (b) rbcL (c) rpoB and (d) rpoC1 loci of Eulophia species.

(b)

(c)

(d)

��

��

4.4.8 Goodyera

The two species, G. procera and G. repens with five individuals each revealed the average inter-specific distances for ITS, rbcL, rpoB and rpoC1 sequences as 0.111, 0.011, 0.017 and 0.012 respectively. The matK sequences for none of the five individuals of G. repens were obtained. The highest inter-specific divergence was in ITS, followed by rpoB, rpoC1 and rbcL. The two species could be distinguished by comparing any one of the four loci i.e., ITS, rbcL, rpoB and rpoC1.

Five accessions of each of the two species clustered on two separate branches

on NJ trees of all the four candidate loci (Fig. 35 a-d). The numbers of variable and parsimony informative sites were 69 out of 690 total sites for ITS locus. The number of parsimony informative sites for rbcL, rpoB and rpoC1 sequences were 7 (out of 633 total nucleotide sites), 6 (372 total sites) and 5 (442 total sites), respectively. (a)

0084 Goodyera procera 0089 Goodyera procera 0085 Goodyera procera 0090 Goodyera procera 0086 Goodyera procera 0931 Goodyera repens 0932 Goodyera repens 0930 Goodyera repens 0929 Goodyera repens 0933 Goodyera repens

100

0.01 Figure 35: NJ tree for (a) ITS locus of Goodyera species.

��

��

(b) SBB-0084 Goodyera procera SBB-0086 Goodyera procera SBB-0085 Goodyera procera SBB-0089 Goodyera procera SBB-0090 Goodyera procera SBB-0929 Goodyera repens SBB-0932 Goodyera repens SBB-0930 Goodyera repens SBB-0931 Goodyera repens SBB-0933 Goodyera repens

99

0.001 SBB-0085 Goodyera procera SBB-0086 Goodyera procera SBB-0084 Goodyera procera SBB-0089 Goodyera procera SBB-0090 Goodyera procera SBB-0929 Goodyera repens SBB-0932 Goodyera repens SBB-0933 Goodyera repens SBB-0930 Goodyera repens SBB-0931 Goodyera repens

100

0.002 SBB-0084 Goodyera procera SBB-0089 Goodyera procera SBB-0086 Goodyera procera SBB-0090 Goodyera procera SBB-0085 Goodyera procera SBB-0933 Goodyera repens SBB-0929 Goodyera repens SBB-0932 Goodyera repens SBB-0930 Goodyera repens SBB-0931 Goodyera repens

99

0.001 Figure 35: NJ trees for (b) rbcL (c) rpoB and (d) rpoC1 loci of Goodyera species.

(c)

(d)

��

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4.4.9 Habenaria

A total of 65 individuals belonging to 10 species of Habenaria were analyzed. The species studied were H. crinifera (4I), H. foliosa (6I), H. furcifera (8I), H. grandifloriformis (7I), H. heyneana (7I), H. intermedia (5I), H. logicorniculata (12I), H. panchganiensis (6I) H. roxburghii (5I) and H. stocksii (5I). The average inter-specific distances for ITS, rbcL, rpoB and rpoC1 were 0.175 (range: 0.01-0.268), 0.007 (range: 0.002-0.013), 0.014 (range: 0-0.03) and 0.008 (range: 0-0.017), respectively. The matK sequences were obtained for only six species viz., H. foliosa, H. grandifloriformis, H. heyneana, H. intermedia, H. roxburghii and H. panchganiensis. The average inter-specific K2P distance for matK locus was 0.039 with a range of 0.014-0.056. The distance matrix of rpoB and rpoC1 of ten species revealed 4 and 2 species pairs, respectively, that had zero distance estimates. For the ten species of Habenaria analyzed, the species discrimination rate for ITS and rbcL was 100% with both distance based and phylogenetic methods (Fig. 36a, c). However, based on rpoB and rpoC1 comparison, only five and six species, respectively, could be assigned correctly. Therefore, the species resolutions for rpoB and rpoC1 were 50% and 60%, respectively by both distance based and phylogenetic tree methods (Fig. 36d, e). All the six species for which matK sequences could be generated were correctly identified by this locus using both the methods (Fig. 36b).

In the ITS sequences, the total number of nucleotide sites analyzed were 714.

The numbers of variable sites were 307 of which 301 were parsimony informative. Out of 804 sites in matK from six species, 109 were variable and 47 were parsimony informative. Out of 633 nucleotide sites analyzed for rbcL, 15 were parsimony informative. The two species clusters formed with rpoB sequences were – (i) H. crinifera, H. panchganiensis and H. longicorniculata and (ii) H. heyneana, H. foliosa and H. intermedia. Out of 381 mucleotides of rpoB locus compared, 17 were parsimony informative for. The rpoC1 tree resulted in two clusters with two species in each viz., (i) H. furcifera and H. longicorniculata and (ii) H. foliosa and H. intermedia. Out of 442 sites of rpoC1 analyzed, 14 were parsimony informative.

��

��

0370 Habenaria longicorniculata 0765 Habenaria longicorniculata 0368 Habenaria longicorniculata 0764 Habenaria longicorniculata 0372 Habenaria longicorniculata 0373 Habenaria longicorniculata 0762 Habenaria longicorniculata 0371 Habenaria longicorniculata 0763 Habenaria longicorniculata

0751 Habenaria panchganiensis 0754 Habenaria panchganiensis 0753 Habenaria panchganiensis 0749 Habenaria panchganiensis 0750 Habenaria panchganiensis 0752 Habenaria panchganiensis

0767 Habenaria crinifera 0769 Habenaria crinifera 0766 Habenaria crinifera 0768 Habenaria crinifera 0983 Habenaria crinifera 0779 Habenaria roxburghii 0781 Habenaria roxburghii 0782 Habenaria roxburghii 0783 Habenaria roxburghii 0780 Habenaria roxburghii

0775 Habenaria furcifera 0778 Habenaria furcifera 0774 Habenaria furcifera 0773 Habenaria furcifera 0776 Habenaria furcifera 0777 Habenaria furcifera

0893 Habenaria intermedia 0894 Habenaria intermedia 0895 Habenaria intermedia 0891 Habenaria intermedia 0896 Habenaria intermedia

0828 Habenaria stocksii 0829 Habenaria stocksii 0832 Habenaria stocksii 0831 Habenaria stocksii 0830 Habenaria stocksii

0755 Habenaria grandifloriformis 0761 Habenaria grandifloriformis 0758 Habenaria grandifloriformis 0760 Habenaria grandifloriformis 0756 Habenaria grandifloriformis 0757 Habenaria grandifloriformis 0759 Habenaria grandifloriformis

0770 Habenaria foliosa 0981 Habenaria foliosa 0772 Habenaria foliosa 0784 Habenaria foliosa 0976 Habenaria foliosa 0982 Habenaria foliosa 0350 Habenaria heyneana 0353 Habenaria heyneana 0979 Habenaria heyneana 0980 Habenaria heyneana 0352 Habenaria heyneana 0351 Habenaria heyneana 0355 Habenaria heyneana

100

100

100

100

100

100

97

59

100

99

100

60

100

79 94

74

99

0.02 Figure 36: NJ tree for (a) ITS locus of ten Habenaria species.

(a)

��

��

(b)

0784 Habenaria foliosa 0982 Habenaria foliosa 0770 Habenaria foliosa 0981 Habenaria foliosa 0771 Habenaria foliosa 0976 Habenaria foliosa 0772 Habenaria foliosa 0350 Habenaria heyneana 0353 Habenaria heyneana 0354 Habenaria heyneana 0355 Habenaria heyneana 0351 Habenaria heyneana 0352 Habenaria heyneana 0759 Habenaria grandifloriformis 0757 Habenaria grandifloriformis 0760 Habenaria grandifloriformis 0755 Habenaria grandifloriformis 0758 Habenaria grandifloriformis 0761 Habenaria grandifloriformis

0783 Habenaria roxburghii 0894 Habenaria intermedia 0891 Habenaria intermedia 0896 Habenaria intermedia 0893 Habenaria intermedia 0895 Habenaria intermedia

0946 Habenaria panchganensis

100

99

46

4798

93

94

0.01 Figure 36: NJ tree for (b) matK locus of six Habenaria species.

��

��

SBB-0768 Habenaria crinifera SBB-0769 Habenaria crinifera SBB-0983 Habenaria crinifera SBB-0766 Habenaria crinifera

SBB-0750 Habenaria panchganiensis SBB-0752 Habenaria panchganiensis SBB-0753 Habenaria panchganiensis SBB-0754 Habenaria panchganiensis SBB-0749 Habenaria panchganiensis SBB-0751 Habenaria panchganiensis SBB-0369 Habenaria longicorniculata SBB-0371 Habenaria longicorniculata SBB-0763 Habenaria longicorniculata SBB-0368 Habenaria longicorniculata SBB-0765 Habenaria longicorniculata SBB-0372 Habenaria longicorniculata SBB-0373 Habenaria longicorniculata SBB-0762 Habenaria longicorniculata SBB-0764 Habenaria longicorniculata SBB-0780 Habenaria roxburghii SBB-0782 Habenaria roxburghii SBB-0779 Habenaria roxburghii SBB-0781 Habenaria roxburghii SBB-0783 Habenaria roxburghii

SBB-0776 Habenaria furcifera SBB-0940 Habenaria furcifera SBB-0775 Habenaria furcifera SBB-0774 Habenaria furcifera SBB-0773 Habenaria furcifera SBB-0777 Habenaria furcifera SBB-0778 Habenaria furcifera SBB-0941 Habenaria furcifera

SBB-0828 Habenaria stocksii SBB-0830 Habenaria stocksii SBB-0832 Habenaria stocksii SBB-0829 Habenaria stocksii SBB-0831 Habenaria stocksii

SBB-0756 Habenaria grandifloriformis SBB-0755 Habenaria grandifloriformis SBB-0757 Habenaria grandifloriformis SBB-0761 Habenaria grandifloriformis SBB-0759 Habenaria grandifloriformis SBB-0760 Habenaria grandifloriformis SBB-0351 Habenaria heyneana SBB-0979 Habenaria heyneana SBB-0353 Habenaria heyneana SBB-0980 Habenaria heyneana SBB-0355 Habenaria heyneana SBB-0350 Habenaria heyneana SBB-0354 Habenaria heyneana

SBB-0895 Habenaria intermedia SBB-0896 Habenaria intermedia SBB-0891 Habenaria intermedia SBB-0894 Habenaria intermedia SBB-0893 Habenaria intermedia

SBB-0770 Habenaria foliosa SBB-0784 Habenaria foliosa SBB-0771 Habenaria foliosa SBB-0981 Habenaria foliosa SBB-0976 Habenaria foliosa SBB-0982 Habenaria foliosa

99

96

62

62

65

64

6

43

39

27

46

5

27

28

39

0.001 Figure 36: NJ tree for (c) rbcL locus of ten Habenaria species.

(c)

��

��

SBB-0769 Habenaria crinifera SBB-0754 Habenaria panchganiensis SBB-0763 Habenaria longicorniculata SBB-0369 Habenaria longicorniculata SBB-0749 Habenaria panchganiensis SBB-0368 Habenaria longicorniculata SBB-0764 Habenaria longicorniculata SBB-0370 Habenaria longicorniculata SBB-0766 Habenaria crinifera SBB-0371 Habenaria longicorniculata SBB-0765 Habenaria longicorniculata SBB-0753 Habenaria panchganiensis SBB-0372 Habenaria longicorniculata SBB-0767 Habenaria crinifera SBB-0750 Habenaria panchganiensis SBB-0983 Habenaria crinifera SBB-0762 Habenaria longicorniculata SBB-0373 Habenaria longicorniculata SBB-0751 Habenaria panchganiensis

SBB-0783 Habenaria roxburghii SBB-0780 Habenaria roxburghii SBB-0782 Habenaria roxburghii

SBB-0778 Habenaria furcifera SBB-0940 Habenaria furcifera SBB-0773 Habenaria furcifera SBB-0775 Habenaria furcifera SBB-0776 Habenaria furcifera SBB-0777 Habenaria furcifera SBB-0774 Habenaria furcifera SBB-0941 Habenaria furcifera

SBB-0779 Habenaria roxburghii SBB-0781 Habenaria roxburghii

SBB-0979 Habenaria heyneana SBB-0350 Habenaria heyneana SBB-0895 Habenaria intermedia SBB-0982 Habenaria foliosa SBB-0891 Habenaria intermedia SBB-0355 Habenaria heyneana SBB-0893 Habenaria intermedia SBB-0981 Habenaria foliosa SBB-0351 Habenaria heyneana SBB-0772 Habenaria foliosa SBB-0354 Habenaria heyneana SBB-0353 Habenaria heyneana SBB-0770 Habenaria foliosa SBB-0894 Habenaria intermedia SBB-0784 Habenaria foliosa SBB-0980 Habenaria heyneana

SBB-0758 Habenaria grandifloriformis SBB-0760 Habenaria grandifloriformis SBB-0756 Habenaria grandifloriformis SBB-0757 Habenaria grandifloriformis SBB-0761 Habenaria grandifloriformis SBB-0755 Habenaria grandifloriformis SBB-0759 Habenaria grandifloriformis

SBB-0896 Habenaria intermedia SBB-0976 Habenaria foliosa


99

95

64

3

4

0

0

0

0

0

88

9

63

0.002 Figure 36: NJ tree for (d) rpoB locus of ten Habenaria species. The species forming


(d)

��

��

SBB-0779 Habenaria roxburghii SBB-0782 Habenaria roxburghii SBB-0780 Habenaria roxburghii SBB-0781 Habenaria roxburghii SBB-0783 Habenaria roxburghii

SBB-0940 Habenaria furcifera SBB-0369 Habenaria longicorniculata SBB-0774 Habenaria furcifera SBB-0942 Habenaria longicorniculata SBB-0775 Habenaria furcifera SBB-0764 Habenaria longicorniculata SBB-0765 Habenaria longicorniculata SBB-0368 Habenaria longicorniculata SBB-0776 Habenaria furcifera SBB-0941 Habenaria furcifera

SBB-0766 Habenaria crinifera SBB-0769 Habenaria crinifera SBB-0767 Habenaria crinifera SBB-0768 Habenaria crinifera

SBB-0777 Habenaria furcifera SBB-0773 Habenaria furcifera SBB-0823 Habenaria longicorniculata SBB-0373 Habenaria longicorniculata

SBB-0752 Habenaria panchganiensis SBB-0754 Habenaria panchganiensis SBB-0749 Habenaria panchganiensis SBB-0750 Habenaria panchganiensis SBB-0751 Habenaria panchganiensis SBB-0753 Habenaria panchganiensis

SBB-0778 Habenaria furcifera SBB-0370 Habenaria longicorniculata SBB-0371 Habenaria longicorniculata SBB-0763 Habenaria longicorniculata SBB-0762 Habenaria longicorniculata SBB-0372 Habenaria longicorniculata

SBB-0771 Habenaria foliosa SBB-0896 Habenaria intermedia SBB-0891 Habenaria intermedia SBB-0895 Habenaria intermedia SBB-0893 Habenaria intermedia SBB-0981 Habenaria foliosa SBB-0784 Habenaria foliosa SBB-0772 Habenaria foliosa

SBB-0759 Habenaria grandifloriformis SBB-0761 Habenaria grandifloriformis SBB-0758 Habenaria grandifloriformis SBB-0756 Habenaria grandifloriformis SBB-0760 Habenaria grandifloriformis SBB-0755 Habenaria grandifloriformis SBB-0757 Habenaria grandifloriformis


SBB-0770 Habenaria foliosa SBB-0894 Habenaria intermedia

SBB-0980 Habenaria heyneana SBB-0355 Habenaria heyneana SBB-0354 Habenaria heyneana SBB-0979 Habenaria heyneana SBB-0350 Habenaria heyneana

SBB-0351 Habenaria heyneana

95

186

88

87

64

6

5

0

0

0

63

64

89

0.001 Figure 36: NJ tree for (e) rpoC1 locus of ten Habenaria species. The species forming


(e)

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4.4.10 Liparis

The average inter-specific distances in ITS, matK, rbcL and rpoB loci of the three species of Liparis viz., L. nervosa (10I), L. prazeri (3I) and Liparis sp. (1I), analyzed were 0.172 (with a range of 0.051-0.268), 0.024 (0.007-0.036), 0.009 (0-0.014) and 0.013 (0.003-0.02), respectively. The rpoC1 sequences of L. deflexa and L. nervosa exhibited 0.007 K2P distance. All the three species of Liparis could be distinguished on the basis of sequence variations in any of the three loci i.e., ITS, matK, and rpoB. Likewise the available rpoC1 sequences of only two species viz., L. deflexa and L. nervosa, could distinguish between these two species (Fig. 37 e). The rbcL sequences of L. deflexa and L. nervosa exhibited zero divergence. Therefore, the species resolution was 33.33% with rbcL.

The species resolution rates for all the loci remained same with tree method as

with distance based method (Fig. 37 a,b,d,e). The bootstrap percentage was 86 for matK, whereas ITS showed 100% bootstrap support. The ITS sequences showed 142 variable and 35 parsimony informative sites out of 710 sites analyzed. Out of 772 matK sites, 26 were variable and only four were parsimony informative. The rbcL tree showed L. nervosa and L. deflexa species as unresolved, thus resulting in 33.33% species resolution (Fig. 37c). Out of 646 sites, only 2 were parsimony informative for rbcL. The number of parsimony informative sites for rpoB and rpoC1 sequences were 1 (369 total sites) and 3 (431 total sites), respectively.

��

��

0072 Liparis nervosa 0073 Liparis nervosa 0075 Liparis nervosa 0079 Liparis nervosa 0074 Liparis nervosa 0076 Liparis nervosa 0077 Liparis nervosa 0071 Liparis nervosa 0078 Liparis nervosa 0080 Liparis nervosa

0824 Liparis deflexa 0825 Liparis deflexa 0826 Liparis deflexa 0329 Liparis sp.

100

100

0.02

0074 Liparis nervosa 0079 Liparis nervosa 0077 Liparis nervosa 0072 Liparis nervosa 0073 Liparis nervosa 0076 Liparis nervosa 0071 Liparis nervosa 0075 Liparis nervosa 0080 Liparis nervosa

0825 Liparis deflexa 0824 Liparis deflexa 0826 Liparis deflexa

0329 Liparis sp.

30

31

87

0.005 Figure 37: NJ trees for (a) ITS and (b) matK loci of Liparis species.

(b)

(a)

��

��

SBB-0825 Liparis deflexa SBB-0072 Liparis nervosa SBB-0826 Liparis deflexa SBB-0073 Liparis nervosa SBB-0075 Liparis nervosa SBB-0071 Liparis nervosa SBB-0329 Liparis sp.

0.002 SBB-0824 Liparis deflexa SBB-0826 Liparis deflexa SBB-0825 Liparis deflexa

SBB-0073 Liparis nervosa SBB-0076 Liparis nervosa SBB-0071 Liparis nervosa SBB-0078 Liparis nervosa SBB-0080 Liparis nervosa SBB-0075 Liparis nervosa SBB-0077 Liparis nervosa SBB-0074 Liparis nervosa SBB-0079 Liparis nervosa SBB-0072 Liparis nervosa

SBB-0329 Liparis sp.

65

0.002 SBB-0073 Liparis nervosa SBB-0078 Liparis nervosa SBB-0076 Liparis nervosa SBB-0077 Liparis nervosa SBB-0075 Liparis nervosa SBB-0079 Liparis nervosa SBB-0080 Liparis nervosa SBB-0074 Liparis nervosa SBB-0824 Liparis deflexa SBB-0825 Liparis deflexa SBB-0826 Liparis deflexa

95

0.001 Figure 37: NJ trees for (c) rbcL (d) rpoB and (e) rpoC1 loci of Liparis species. The

species forming clusters are highlighted in the rectangle.

(c)

(e)

(d)

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4.4.11 Nervilia

A total of 17 individuals belonging to five species of Nervilia viz., N. aragoana (1I), N. crociformis (8I), N. infundibulifolia (1I), N. gammieana (4I) and N. plicata (3I), were analyzed. The average inter-specific distances in ITS, matK, rbcL, rpoB and rpoC1 were 0.214, 0.033, 0.014, 0.034 and 0.039, respectively. None of the tested loci from the chloroplast genome resolved all species as different number of species pairs showing zero distance estimates were formed in the matrices of these loci. The matK sequences could not distinguish N. aragoana and N. plicata, while in the distance matrix of rbcL and rpoC1, species pair were N. infundibulifolia-N. plicata and N. aragoana-N. gammieana. As with matK, two species were indistinguishable with rpoB. However, the species pair was N. infundibulifolia-N. plicata. In contrast, the nuclear genome locus ITS provided distinction in all the three species for which sequences of this locus could be generated.

Even in the analysis based on phylogenetic tree method, the ITS sequences

obtained for three species showed 100% species resolution with 100 BP (Fig. 38a). There were 174 variable and parsimony informative sites in ITS out of 684 nucleotide sites compared. The four Nervilia species for which matK sequences were available returned with 50% species resolution (Fig. 38b). The matK sequences had 42 variable sites among 737 nucleotides compared, with 38 being parsimony informative. The accessions of N. crociformis which exhibited intra-specific variations formed two sub-branches (Fig. 38b). The NJ trees of rbcL and rpoC1 loci formed two species clusters with four species unresolved, thus resulting in 20% species resolution. The two clusters consisted of two species each viz., N. aragoana- N. gammieana and N. plicata and N. infundibulifolia (Fig. 38 c, e). The number of parsimony informative sites in rbcL and rpoC1 were 19 and 32 among 628 and 438 nucleotides compared. In the NJ tree of rpoB two species viz., N. plicata and N. aragoana formed a single cluster (Fig. 38d). The species resolution was 60% for rpoB and the parsimony informative sites were 14 in 340 nucleotide long sequences analyzed.

��

��

0881 Nervilia gammieana 0883 Nervilia gammieana 0882 Nervilia gammieana 0884 Nervilia gammieana 0885 Nervilia gammieana

0839 Nervilia plicata 0837 Nervilia plicata 0838 Nervilia plicata

0447 Nervilia crociformis 0847 Nervilia crociformis 0849 Nervilia crociformis 0848 Nervilia crociformis 0448 Nervilia crociformis 0850 Nervilia crociformis

100

100

100

0.02

0882 Nervilia gammieana 0883 Nervilia gammieana 0885 Nervilia gammieana 0881 Nervilia gammieana 0884 Nervilia gammieana

0837 Nervilia plicata 0838 Nervilia plicata 0945 Nervilia aragona 0839 Nervilia plicata

0847 Nervilia crociformis 0447 Nervilia crociformis 0848 Nervilia crociformis 0850 Nervilia crociformis 0448 Nervilia crociformis

0849 Nervilia crociformis176

100

98

100

0.005 Figure 38: NJ trees for (a) ITS and (b) matK loci of Nervilia species. The species


(b)

(a)

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SBB-0946 Nervilia aragoana SBB-0885 Nervilia gammieana SBB-0883 Nervilia gammieana SBB-0882 Nervilia gammieana SBB-0881 Nervilia gammieana

SBB-0839 Nervilia plicata SBB-0945 Nervilia infundibulifolia SBB-0837 Nervilia plicata SBB-0838 Nervilia plicata SBB-0450 Nervilia crociformi SBB-0849 Nervilia crociformis SBB-0850 Nervilia crociformis SBB-0848 Nervilia crociformis SBB-0847 Nervilia crociformis

SBB-0447 Nervilia crociformis SBB-0448 Nervilia crociformis SBB-0449 Nervilia crociformis

66

12

6

100

87

95

0.002

SBB-0883 Nervilia gammieana SBB-0885 Nervilia gammieana SBB-0881 Nervilia gammieana SBB-0882 Nervilia gammieana

SBB-0946 Nervilia aragoana SBB-0945 Nervilia infundibuifolia SBB-0837 Nervilia plicata SBB-0838 Nervilia plicata SBB-0839 Nervilia plicata

SBB-0850 Nervilia crociformis SBB-0447 Nervilia crociformis SBB-0849 Nervilia crociformis SBB-0448 Nervilia crociformis SBB-0449 Nervilia crociformis SBB-0847 Nervilia crociformis SBB-0450 Nervilia crociformis SBB-0848 Nervilia crociformis

66

96

96

99

0.005 Figure 38: NJ trees for (c) rbcL and (d) rpoB loci of Nervilia species.The species


(c)

(d)

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SBB-0946 Nervilia aragoana SBB-0881 Nervilia gammieana SBB-0885 Nervilia gammieana SBB-0883 Nervilia gammieana SBB-0884 Nervilia gammieana SBB-0882 Nervilia gammieana

SBB-0945 Nervilia infundibulifolia SBB-0839 Nervilia plicata SBB-0837 Nervilia plicata SBB-0838 Nervilia plicata SBB-0449 Nervilia crociformis SBB-0849 Nervilia crociformis SBB-0448 Nervilia crociformis SBB-0447 Nervilia crociformis SBB-0847 Nervilia crociformis SBB-0450 Nervilia crociformis SBB-0848 Nervilia crociformis SBB-0850 Nervilia crociformis

100

99

100

0.005 Figure 38: NJ tree for (e) rpoC1 locus of Nervilia species. The species forming


4.4.12 Oberonia

Six species of Oberonia evaluated in the present investigation were O. brunoniana (5I), O. falconeri (5I), O. ensiformis (3I), O. mucronata (9I), O. pachyrachis (3I) and O. recurva (7I). The average inter-specific K2P distances for ITS, matK, rbcL, rpoB and rpoC1 were 0.066, 0.018, 0.005, 0.01 and 0.008, respectively. The ITS, matK and rbcL sequences were able to distinguish all six species. Whereas, rpoB and rpoC1 matrices had one species pair each with zero distances. These species pairs formed were O. brunoniana-O. ensiformis and O. ensiformis-O. mucronata with rpoB and rpoC1, respectively.

The NJ trees of ITS, matK and rbcL from six Oberonia species provided 100%

resolution (Fig. 39 a-c). The 709 base pairs long ITS sequences had 105 parsimony informative sites. The matK sequences, which had 816 nucleotides, contained 40

(e)

��

��

variable sites with 39 being parsimony informative. Of 653 nucleotides of rbcL compared, 9 were parsimony informative. The NJ trees of remaining two loci, rpoB and rpoC1 showed two species forming one cluster (Fig. 39 d,e). Thus, the species discrimination rate was 83.33% for both of these loci. The number of parsimony informative sites in rpoB was 9 out of 372 nucleotides analyzed and 11 of 442 nucleotides in rpoC1.

0233 Oberonia mucronata 0234 Oberonia mucronata 0195 Oberonia mucronata 0194 Oberonia mucronata 0232 Oberonia mucronata 0190 Oberonia mucronata 0191 Oberonia mucronata 0231 Oberonia mucronata 0230 Oberonia mucronata

0790 Oberonia falconeri 0794 Oberonia falconeri 0791 Oberonia falconeri 0792 Oberonia falconeri 0793 Oberonia falconeri

0263 Oberonia pachyrachis 0264 Oberonia pachyrachis 0265 Oberonia pachyrachis

0789 Oberonia brunoniana 0795 Oberonia brunoniana 0785 Oberonia brunoniana 0787 Oberonia brunoniana 0786 Oberonia brunoniana 0796 Oberonia ensiformis 0797 Oberonia ensiformis 0798 Oberonia ensiformis

0800 Oberonia recurva 0392 Oberonia recurva 0799 Oberonia recurva

100

90100

100

100

100

85

88

56

100

0.005 Figure 39: NJ tree for (a) ITS locus of Oberonia species.

(a)

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0195 Oberonia mucronata 0230 Oberonia mucronata 0231 Oberonia mucronata 0233 Oberonia mucronata 0190 Oberonia mucronata 0191 Oberonia mucronata 0194 Oberonia mucronata 0232 Oberonia mucronata

0791 Oberonia falconeri 0792 Oberonia falconeri 0790 Oberonia falconeri 0793 Oberonia falconeri 0794 Oberonia falconeri

0389 Oberonia recurva 0801 Oberonia recurva 0392 Oberonia recurva 0799 Oberonia recurva 0390 Oberonia recurva 0391 Oberonia recurva

0798 Oberonia ensiformis 0789 Oberonia brunoniana 0795 Oberonia brunoniana 0785 Oberonia brunoniana 0788 Oberonia brunoniana 0786 Oberonia brunoniana 0787 Oberonia brunoniana

0796 Oberonia ensiformis 0797 Oberonia ensiformis

0263 Oberonia pachyrachis 0264 Oberonia pachyrachis 0265 Oberonia pachyrachis

99

99

99

86

77

60

26

12

20

66

0.005 Figure 39: NJ tree for (b) matK locus of Oberonia species.

(b)

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SBB-0788 Oberonia brunoniana SBB-0789 Oberonia brunoniana SBB-0785 Oberonia brunoniana SBB-0786 Oberonia brunoniana SBB-0787 Oberonia brunoniana SBB-0795 Oberonia brunoniana

SBB-0797 Oberonia ensiformis SBB-0796 Oberonia ensiformis SBB-0798 Oberonia ensiformis

SBB-0234 Oberonia mucronata SBB-0191 Oberonia mucronata SBB-0190 Oberonia mucronata SBB-0195 Oberonia mucronata

SBB-0793 Oberonia falconeri SBB-0794 Oberonia falconeri SBB-0790 Oberonia falconeri SBB-0791 Oberonia falconeri SBB-0792 Oberonia falconeri

SBB-0233 Oberonia mucronata SBB-0230 Oberonia mucronata

SBB-0265 Oberonia pachyrachis SBB-0263 Oberonia pachyrachis SBB-0264 Oberonia pachyrachis

SBB-0194 Oberonia mucronata SBB-0231 Oberonia mucronata SBB-0232 Oberonia mucronata

SBB-0391 Oberonia recurva SBB-0390 Oberonia recurva SBB-0801 Oberonia recurva SBB-0389 Oberonia recurva SBB-0392 Oberonia recurva

96

85

87

64

15

64

0.001 Figure 39: NJ tree for (c) rbcL locus of Oberonia species.

(c)

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SBB-0389 Oberonia recurva SBB-0390 Oberonia recurva SBB-0800 Oberonia recurva SBB-0801 Oberonia recurva SBB-0799 Oberonia recurva SBB-0391 Oberonia recurva SBB-0392 Oberonia recurva

SBB-0232 Oberonia mucronata SBB-0191 Oberonia mucronata SBB-0797 Oberonia ensiformis SBB-0798 Oberonia ensiformis SBB-0231 Oberonia mucronata SBB-0230 Oberonia mucronata

SBB-0791 Oberonia falconeri SBB-0790 Oberonia falconeri SBB-0792 Oberonia falconeri SBB-0793 Oberonia falconeri SBB-0794 Oberonia falconeri

SBB-0234 Oberonia mucronata SBB-0195 Oberonia mucronata

SBB-0786 Oberonia brunoniana SBB-0789 Oberonia brunoniana SBB-0788 Oberonia brunoniana SBB-0787 Oberonia brunoniana SBB-0785 Oberonia brunoniana SBB-0795 Oberonia brunoniana

SBB-0796 Oberonia ensiformis SBB-0190 Oberonia mucronata SBB-0233 Oberonia mucronata SBB-0194 Oberonia mucronata

SBB-0265 Oberonia pachyrachis SBB-0263 Oberonia pachyrachis SBB-0264 Oberonia pachyrachis

96

87

87

66

0.001 Figure 39: NJ tree for (d) rpoB locus of Oberonia species. The species forming


(d)

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SBB-0233 Oberonia mucronata SBB-0234 Oberonia mucronata SBB-0190 Oberonia mucronata SBB-0232 Oberonia mucronata SBB-0194 Oberonia mucronata SBB-0230 Oberonia mucronata SBB-0191 Oberonia mucronata SBB-0195 Oberonia mucronata SBB-0231 Oberonia mucronata SBB-0263 Oberonia pachyrachis SBB-0265 Oberonia pachyrachis SBB-0264 Oberonia pachyrachis SBB-0792 Oberonia falconeri SBB-0794 Oberonia falconeri SBB-0793 Oberonia falconeri SBB-0790 Oberonia falconeri SBB-0791 Oberonia falconeri

SBB-0785 Oberonia brunoniana SBB-0787 Oberonia brunoniana SBB-0798 Oberonia ensiformis SBB-0796 Oberonia ensiformis SBB-0797 Oberonia ensiformis SBB-0788 Oberonia brunoniana SBB-0786 Oberonia brunoniana SBB-0795 Oberonia brunoniana

SBB-0389 Oberonia recurva SBB-0392 Oberonia recurva SBB-0391 Oberonia recurva SBB-0799 Oberonia recurva SBB-0390 Oberonia recurva SBB-0801 Oberonia recurva

95

64

63

11

3

1

1

86

64

0.002 Figure 39: NJ tree for (e) rpoC1 locus of Oberonia species. The species forming


(e)

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4.4.13 Paphiopedilum

Out of the nine species and three natural interspecific hybrids of this genus occurring in India, all but one could be analyzed. The species and their inter-specific hybrids were: Paphiopedilum druryi (3I), P. hirsutissimum (6I), P. fairrieanum (5I), P. insigne (5I), P. villosum (5I), P. venustum (5I), P. spicerianum (5I), P. wardii (1I), P. venustospicerianum (1I), P. venusto-insigne (1I) and P. pradhanii (5I). The rbcL, rpoB and rpoC1 loci exhibited very low average inter-specific K2P distance value of 0.001, with number of species pairs showing distance estimates of zero. On the other hand, ITS and matK loci had 0.044 and 0.009 K2P distances, respectively. The locus matK could resolve all, whereas two species pairs comprising P. insigne -P. villosum and P. venustum-P. wardii were obtained with ITS matrix, thus, resulting in 50% species resolution. The sequencing of inter-specific hybrids for ITS locus yielded poor quality sequences with double peaks. The species discrimination rates in the remaining loci from chloroplast genome were 12.5, 12.5 and 25% for rpoB, rpoC1 and rbcL, respectively.

In the NJ tree prepared with ITS, accessions belonging to four different species

clustered in two branches with those of P. insigne and P. villosum on one branch and the P. venustum individuals clustering with those of P. wardii (Fig. 40a). Consequently, the species resolution was 50%. The numbers of variable and parsimony informative sites were 63 and 62, respectively, among the 706 nucleotides compared. All the eight Paphiopedilum species got distributed on different branches in matK tree, thus resulting in 100% species resolution. The three natural hybrids clustered with one of the parent e.g., P. pradhanii accessions with P. fairreanum accessions and P. venusto-insigne and P. venusto-spicerianum clustered with P. venustum (Fig. 40b). The numbers of variable and parsimony informative sites in matK were 20 and 19, respectively, among 791 sites compared. The NJ trees of rbcL, rpoB and rpoC1 loci resulted in 25%, 12.5% and 12.5% species resolution (Fig. 40 c-e). The number of parsimony informative sites for rbcL, rpoB and rpoC1 sequences were 2 (637 total sites), 1 (371 total sites), and 2 (444 total sites), respectively.

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0671 Paphiopedilum insigne 0493 Paphiopedilum villosum 0496 Paphiopedilum insigne 0659 Paphiopedilum villosum 0660 Paphiopedilum villosum 0670 Paphiopedilum insigne 0672 Paphiopedilum insigne 0669 Paphiopedilum insigne 0658 Paphiopedilum villosum 0657 Paphiopedilum villosum

0655 Paphiopedilum druryi 0492 Paphiopedilum druryi 0656 Paphiopedilum druryi

0673 Paphiopedilum spicerianum 0675 Paphiopedilum spicerianum 0674 Paphiopedilum spicerianum 0497 Paphiopedilum spicerianum

0495 Paphiopedilum hirsutissimum 0668 Paphiopedilum hirsutissimum 0666 Paphiopedilum hirsutissimum 0995 Paphiopedilum hirsutissimum 0665 Paphiopedilum hirsutissimum 0667 Paphiopedilum hirsutissimum

0494 Paphiopedilum fairrieanum 0661 Paphiopedilum fairrieanum 0664 Paphiopedilum fairrieanum 0662 Paphiopedilum fairrieanum 0663 Paphiopedilum fairrieanum

0501 Paphiopedilum wardii 0652 Paphiopedilum venustum 0654 Paphiopedilum venustum 0491 Paphiopedilum venustum 0651 Paphiopedilum venustum 0653 Paphiopedilum venustum

99

99

99

99

99

43

48

48

99

0.005 Figure 40: NJ tree for (a) ITS locus of Paphiopedilum species. The species forming clusters are highlighted in the rectangle.

(a)

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0673 Paphiopedilum spicerianum 0674 Paphiopedilum spicerianum 0675 Paphiopedilum spicerianum 0497 Paphiopedilum spicerianum 0493 Paphiopedilum villosum 0657 Paphiopedilum villosum

0671 Paphiopedilum insigne 0496 Paphiopedilum insigne 0670 Paphiopedilum insigne 0672 Paphiopedilum insigne 0669 Paphiopedilum insigne

0663 Paphiopedilum fairrieanum 0677 Paphiopedilum pradhanii 0494 Paphiopedilum fairrieanum 0662 Paphiopedilum fairrieanum 0679 Paphiopedilum pradhanii 0661 Paphiopedilum fairrieanum 0680 Paphiopedilum pradhanii 0664 Paphiopedilum fairrieanum 0678 Paphiopedilum pradhanii

0655 Paphiopedilum druryi 0492 Paphiopedilum druryi 0656 Paphiopedilum druryi

0668 Paphiopedilum hirsutissimum 0995 Paphiopedilum hirsutissimum 0665 Paphiopedilum hirsutissimum 0666 Paphiopedilum hirsutissimum 0667 Paphiopedilum hirsutissimum 0501 Paphiopedilum wardii

0653 Paphiopedilum venustum 0491 Paphiopedilum venustum 0651 Paphiopedilum venustum 0499 Paphiopedilum venustospicerianum 0500 Paphiopedilum venustoinsigne 0652 Paphiopedilum venustum

66

77

95

86

65

63

27

7

88

63

12

96

0.001 Figure 40: NJ tree for (b) matK locus of Paphiopedilum species. The two hybrid

species forming clusters with their respective female parents are highlighted in the rectangle and oval.

(b)

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SBB-0666 Paphiopedilum hirsutissimum SBB-0667 Paphiopedilum hirsutissimum SBB-0665 Paphiopedilum hirsutissimum SBB-0495 Paphiopedilum hirsutissimum SBB-0668 Paphiopedilum hirsutissimum SBB-0995 Paphiopedilum hirsutissimum

SBB-0678 Paphiopedilum pradhanii SBB-0664 Paphiopedilum fairrieanum SBB-0660 Paphiopedilum villosum SBB-0674 Paphiopedilum spicerianum SBB-0670 Paphiopedilum insigne SBB-0669 Paphiopedilum insigne SBB-0671 Paphiopedilum insigne SBB-0656 Paphiopedilum druryi SBB-0679 Paphiopedilum pradhanii SBB-0497 Paphiopedilum spicerianum SBB-0673 Paphiopedilum spicerianum SBB-0492 Paphiopedilum druryi SBB-0498 Paphiopedilum pradhanii SBB-0659 Paphiopedilum villosum SBB-0501 Paphiopedilum wardii SBB-0493 Paphiopedilum villosum SBB-0680 Paphiopedilum pradhanii SBB-0676 Paphiopedilum spicerianum SBB-0662 Paphiopedilum fairrieanum SBB-0675 Paphiopedilum spicerianum SBB-0663 Paphiopedilum fairrieanum SBB-0658 Paphiopedilum villosum SBB-0655 Paphiopedilum druryi SBB-0494 Paphiopedilum fairrieanum SBB-0677 Paphiopedilum pradhanii SBB-0661 Paphiopedilum fairrieanum SBB-0672 Paphiopedilum insigne SBB-0657 Paphiopedilum villosum

SBB-0499 Paphiopedilum venusto-spicer... SBB-0653 Paphiopedilum venustum SBB-0491 Paphiopedilum venustum SBB-0651 Paphiopedilum venustum SBB-0500 Paphiopedilum venusto-insigne SBB-0652 Paphiopedilum venustum SBB-0654 Paphiopedilum venustum

64

63

0.0002 Figure 40: NJ tree for (c) rbcL locus of Paphiopedilum species. The species forming


(c)

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SBB-0669 Paphiopedilum insigne SBB-0673 Paphiopedilum spicerianum SBB-0677 Paphiopedilum pradhanii SBB-0675 Paphiopedilum spicerianum SBB-0497 Paphiopedilum spicerianum SBB-0659 Paphiopedilum villosum SBB-0676 Paphiopedilum spicerianum SBB-0656 Paphiopedilum druryi SBB-0660 Paphiopedilum villosum SBB-0679 Paphiopedilum pradhanii SBB-0491 Paphiopedilum venustum SBB-0654 Paphiopedilum venustum SBB-0501 Paphiopedilum wardii SBB-0655 Paphiopedilum druryi SBB-0664 Paphiopedilum fairrieanum SBB-0661 Paphiopedilum fairrieanum SBB-0652 Paphiopedilum venustum SBB-0658 Paphiopedilum villosum SBB-0499 Paphiopedilum venusto-spicer... SBB-0657 Paphiopedilum villosum SBB-0680 Paphiopedilum pradhanii SBB-0492 Paphiopedilum druryi SBB-0493 Paphiopedilum villosum SBB-0494 Paphiopedilum fairrieanum SBB-0663 Paphiopedilum fairrieanum SBB-0670 Paphiopedilum insigne SBB-0500 Paphiopedilum venusto-insigne SBB-0671 Paphiopedilum insigne SBB-0651 Paphiopedilum venustum SBB-0653 Paphiopedilum venustum SBB-0662 Paphiopedilum fairrieanum SBB-0496 Paphiopedilum insigne SBB-0672 Paphiopedilum insigne SBB-0498 Paphiopedilum pradhanii SBB-0674 Paphiopedilum spicerianum SBB-0678 Paphiopedilum pradhanii SBB-0665 Paphiopedilum hirsutissimum SBB-0667 Paphiopedilum hirsutissimum SBB-0666 Paphiopedilum hirsutissimum SBB-0495 Paphiopedilum hirsutissimum SBB-0668 Paphiopedilum hirsutissimum

64

0.0005 Figure 40: NJ tree for (d) rpoB locus of Paphiopedilum species. The species forming


(d)

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SBB-0492 Paphiopedilum druryi SBB-0494 Paphiopedilum fairrieanum SBB-0655 Paphiopedilum druryi SBB-0664 Paphiopedilum fairrieanum SBB-0661 Paphiopedilum fairrieanum SBB-0662 Paphiopedilum fairrieanum SBB-0656 Paphiopedilum druryi SBB-0663 Paphiopedilum fairrieanum

SBB-0672 Paphiopedilum insigne SBB-0676 Paphiopedilum spicerianum SBB-0670 Paphiopedilum insigne SBB-0660 Paphiopedilum villosum SBB-0659 Paphiopedilum villosum SBB-0669 Paphiopedilum insigne SBB-0671 Paphiopedilum insigne SBB-0678 Paphiopedilum pradhanii SBB-0493 Paphiopedilum villosum SBB-0491 Paphiopedilum venustum SBB-0658 Paphiopedilum villosum SBB-0654 Paphiopedilum venustum SBB-0675 Paphiopedilum spicerianum SBB-0498 Paphiopedilum pradhanii SBB-0497 Paphiopedilum spicerianum SBB-0674 Paphiopedilum spicerianum SBB-0501 Paphiopedilum wardii SBB-0499 Paphiopedilum venusto-spicer... SBB-0500 Paphiopedilum venusto-insigne SBB-0651 Paphiopedilum venustum SBB-0653 Paphiopedilum venustum SBB-0677 Paphiopedilum pradhanii SBB-0679 Paphiopedilum pradhanii SBB-0673 Paphiopedilum spicerianum SBB-0652 Paphiopedilum venustum SBB-0680 Paphiopedilum pradhanii

SBB-0495 Paphiopedilum hirsutissimum SBB-0666 Paphiopedilum hirsutissimum SBB-0668 Paphiopedilum hirsutissimum SBB-0665 Paphiopedilum hirsutissimum SBB-0667 Paphiopedilum hirsutissimum SBB-0995 Paphiopedilum hirsutissimum

65

64

0.0005 Figure 40: NJ tree for (e) rpoC1 locus of Paphiopedilum species. The species forming


(e)

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4.4.14 Peristylus

The average inter-specific distances in ITS, matK, rbcL, rpoB and rpoC1 sequences of three species of Peristylus viz., P. densus (10I), P. elisabethae (3I) and P. plantagineus (5I), were 0.138, 0.06, 0.012, 0.006, and 0.014, respectively. The sequences of rpoB locus from P. densus and P. plantagineus were alike, thus these species had zero K2P distance. Each of the remaining four loci distinguished all three species unambiguously.

The species resolutions based on tree method were not different from those

obtained by genetic distance method for each of the tested loci. Thus, NJ tree analyses based on ITS, matK, rbcL and rpoC1 individually resolved all the three Peristylus species (Fig. 41 a,b,c,e), while species discrimination rate with rpoB was 33.33% (Fig. 41d). The 733 nucleotide long ITS sequences contained 128 variable sites with 125 being parsimony informative. The matK sequences, for which 777 sites were compared, had 53 variable sites including 11 parsimony informative sites. The number of variable and parsimony informative sites for rbcL and rpoC1 sequences were 11 (out of 642) and 9 (out of 435), respectively. The numbers of variable and parsimony informative sites were 3 among the 371 nucleotides analyzed.

0377 Peristylus densus 0814 Peristylus densus 0375 Peristylus densus 0378 Peristylus densus 0817 Peristylus densus 0376 Peristylus densus 0818 Peristylus densus 0813 Peristylus densus 0379 Peristylus densus 0815 Peristylus densus 0816 Peristylus densus

0977 Peristylus plantagineus 0978 Peristylus plantagineus 0975 Peristylus plantagineus 0380 Peristylus plantagineus 0381 Peristylus plantagineus

0936 Peristylus elisabethae 0937 Peristylus elisabethae 0934 Peristylus elisabethae 0935 Peristylus elisabethae

100

100

100

0.02 Figure 41: NJ tree for (a) ITS locus of Peristylus species.

(a)

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SBB-0375 Peristylus densus SBB-0813 Peristylus densus SBB-0815 Peristylus densus SBB-0818 Peristylus densus SBB-0378 Peristylus densus SBB-0816 Peristylus densus SBB-0376 Peristylus densus SBB-0377 Peristylus densus SBB-0379 Peristylus densus SBB-0814 Peristylus densus

SBB-0975 Peristylus plantagineus SBB-0977 Peristylus plantagineus SBB-0978 Peristylus plantagineus SBB-0936 Peristylus elisabethae

99

86

0.01

SBB-0377 Peristylus densus SBB-0378 Peristylus densus SBB-0814 Peristylus densus SBB-0815 Peristylus densus SBB-0375 Peristylus densus SBB-0813 Peristylus densus SBB-0376 Peristylus densus SBB-0379 Peristylus densus SBB-0817 Peristylus densus

SBB-0977 Peristylus plantagineus SBB-0381 Peristylus plantagineus SBB-0975 Peristylus plantagineus SBB-0380 Peristylus plantagineus SBB-0978 Peristylus plantagineus

SBB-0934 Peristylus elisabethae SBB-0935 Peristylus elisabethae SBB-0936 Peristylus elisabethae SBB-0937 Peristylus elisabethae

99

63

98

0.002 Figure 41: NJ trees for (b) matK and (c) rbcL loci of Peristylus species.

(b)

(c)

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SBB-0818 Peristylus densus SBB-0381 Peristylus plantagineus SBB-0977 Peristylus plantagineus SBB-0376 Peristylus densus SBB-0815 Peristylus densus SBB-0379 Peristylus densus SBB-0813 Peristylus densus SBB-0817 Peristylus densus SBB-0380 Peristylus plantagineus SBB-0375 Peristylus densus SBB-0378 Peristylus densus SBB-0975 Peristylus plantagineus SBB-0377 Peristylus densus SBB-0814 Peristylus densus SBB-0978 Peristylus plantagineus SBB-0937 Peristylus elisabethae SBB-0934 Peristylus elisabethae SBB-0935 Peristylus elisabethae

88

0.0005 SBB-0376 Peristylus densus SBB-0815 Peristylus densus SBB-0814 Peristylus densus SBB-0377 Peristylus densus SBB-0816 Peristylus densus SBB-0378 Peristylus densus SBB-0379 Peristylus densus SBB-0375 Peristylus densus SBB-0813 Peristylus densus SBB-0817 Peristylus densus SBB-0818 Peristylus densus

SBB-0975 Peristylus plantagineus SBB-0978 Peristylus plantagineus SBB-0380 Peristylus plantagineus SBB-0381 Peristylus plantagineus SBB-0977 Peristylus plantagineus SBB-0936 Peristylus elisabethae SBB-0935 Peristylus elisabethae SBB-0934 Peristylus elisabethae SBB-0937 Peristylus elisabethae

99

88

65

0.002 Figure 41: NJ trees for (d) rpoB and (e) rpoC1 loci of Peristylus species. The species


(d)

(e)

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4.4.15 Pholidota

The three species of Pholidota viz., P. articulata (4I), P. imbricata (1I) and P. pallida (2I), investigated in the present study, could not be discriminated from each other on the basis of comparison of rpoB and rpoC1 sequences, as the inter-specific divergence was zero. Whereas, in matK and rbcL, the average inter-specific distances were 0.006 and 0.002, respectively. The species pair, P. imbricata and P. pallida showed zero divergence in both matK and rbcL matrices. The ITS sequences of P. imbricata and P. pallida were not obtained. These two on literature survey were found to be synonyms and hence the species discrimination rate for matK and rbcL was 100%.

The matK and rbcL barcodes exhibited 100% species resolution with NJ analysis

too (Fig. 42 a,b). As expected the accessions of P. pallida and P. imbricata clustered together (Fig. 42 a,b). The numbers of variable and parsimony informative sites in matK and rbcL were 7 out of 745 and 2 out of 630, respectively. In the trees prepared using rpoB and rpoC1 sequences all the individuals clustered on one branch (Fig. 42 c,d).

SBB-0257 Pholidota articulata SBB-0260 Pholidota articulata SBB-0261 Pholidota articulata SBB-0259 Pholidota articulata SBB-0974 Pholidota pallida SBB-0328 Pholidota imbricata SBB-0811 Pholidota pallida

100

0.001 SBB-0260 Pholidota articulata SBB-0261 Pholidota articulata SBB-0257 Pholidota articulata SBB-0259 Pholidota articulata SBB-0328 Pholidota imbricata SBB-0811 Pholidota pallida SBB-0974 Pholidota pallida

87

0.0005 Figure 42: NJ trees for (a) matK and (b) rbcL loci of Pholidota species.

(a)

(b)

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SBB-0811 Pholidota pallida SBB-0974 Pholidota pallida SBB-0261 Pholidota articulata SBB-0260 Pholidota articulata SBB-0328 Pholidota imbricata SBB-0257 Pholidota articulata SBB-0259 Pholidota articulata15

186

6

SBB-0259 Pholidota articulata SBB-0260 Pholidota articulata SBB-0257 Pholidota articulata SBB-0328 Pholidota imbricata SBB-0974 Pholidota pallida SBB-0261 Pholidota articulata SBB-0811 Pholidota pallida12

123

4

Figure 42: NJ trees for (c) rpoB and (d) rpoC1 loci of Pholidota species.

4.4.16 Pinalia

The average inter-specific K2P distances based on ITS, rbcL, rpoB and rpoC1 between the two investigated species, P. mysorensis (7I) and P. spicata (2I), were 0.087, 0.005, 0.011 and 0.012, respectively. The matK sequences for P. spicata individuals were not obtained. The sequence variation in other tested loci from these two species was divergent enough to distinguish both the species by both methods (Fig. 43 a-d).

The numbers of variable and parsimony informative sites were 56 out of 697

total sites in ITS, 3 out of 639 sites in rbcL, 4 out of 371 sites in rpoB and 5 out of 445 sites in rpoC1.

(c)

(d)

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0199 Pinalia mysorensis 0592 Pinalia mysorensis 0198 Pinalia mysorensis 0197 Pinalia mysorensis 0593 Pinalia mysorensis 0200 Pinalia mysorensis 0201 Pinalia mysorensis 0241 Pinalia spicata 0250 Pinalia spicata

100

0.01

SBB-0198 Pinalia mysorensis SBB-0199 Pinalia mysorensis SBB-0593 Pinalia mysorensis SBB-0197 Pinalia mysorensis SBB-0592 Pinalia mysorensis SBB-0201 Pinalia mysorensis SBB-0241 Pinalia spicata SBB-0250 Pinalia spicata

96

0.0005

SBB-0198 Pinalia mysorensis SBB-0593 Pinalia mysorensis SBB-0199 Pinalia mysorensis SBB-0200 Pinalia mysorensis SBB-0197 Pinalia mysorensis SBB-0592 Pinalia mysorensis SBB-0201 Pinalia mysorensis SBB-0241 Pinalia spicata SBB-0250 Pinalia spicata

98

0.001 Figure 43: NJ tree for (a) ITS, (b) rbcL and (c) rpoB loci of Pinalia species.

(b)

(a)

(c)

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SBB-0201 Pinalia mysorensis SBB-0592 Pinalia mysorensis SBB-0197 Pinalia mysorensis SBB-0199 Pinalia mysorensis SBB-0200 Pinalia mysorensis SBB-0593 Pinalia mysorensis SBB-0198 Pinalia mysorensis SBB-0241 Pinalia spicata SBB-0250 Pinalia spicata

99

0.001 Figure 43: NJ tree for (d) rpoC1 loci of Pinalia species

4.4.17 Platanthera

Two species P. edgeworthii (6I) and P. latilabris (2I), analyzed in the present study, exhibited zero inter-specific divergence in their ITS, rbcL, rpoB and rpoC1 loci. Though, the K2P distance between two species based on matK was 0.002, all the individuals of two species clustered together in NJ trees of five tested loci (Fig. 44 a-e). Thus, none of the tested loci provided a distinguishing barcode for these species. There was only one variable site among the 801 nucleotides of matK compared. However, this site was not parsimony informative. ITS sequences had one variable site, which was parsimony informative, among 726 nucleotide sites compared. In the remaining three loci, none of the site was variable.

0898 Platanthera latilabris 0901 Platanthera edgeworthii 0902 Platanthera edgeworthii 0714 Platanthera edgeworthii 0903 Platanthera edgeworthii 0904 Platanthera edgeworthii 0712 Platanthera edgeworthii 0899 Platanthera edgeworthii 0713 Platanthera edgeworthii 0897 Platanthera latilabris 0900 Platanthera edgeworthii

6

6

1

5

1

0

0

0

Figure 44: NJ tree for (a) ITS locus of Platanthera species.

(a)

(d)

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SBB-0900 Platanthera edgeworthii SBB-0904 Platanthera edgeworthii SBB-0901 Platanthera edgeworthii SBB-0902 Platanthera edgeworthii SBB-0898 Platanthera latilabris SBB-0899 Platanthera edgeworthii SBB-0897 Platanthera latilabris SBB-0903 Platanthera edgeworthii SBB-0712 Platanthera edgeworthii SBB-0713 Platanthera edgeworthii SBB-0714 Platanthera edgeworthii

8

8

1

2

8

0

0

1

SBB-0899 Platanthera edgeworthii SBB-0903 Platanthera edgeworthii SBB-0897 Platanthera latilabris SBB-0902 Platanthera edgeworthii SBB-0900 Platanthera edgeworthii SBB-0901 Platanthera edgeworthii SBB-0904 Platanthera edgeworthii SBB-0898 Platanthera latilabris

10

11

21

3

SBB-0902 Platanthera edgeworthii SBB-0897 Platanthera latilabris SBB-0904 Platanthera edgeworthii SBB-0898 Platanthera latilabris SBB-0899 Platanthera edgeworthii SBB-0900 Platanthera edgeworthii SBB-0901 Platanthera edgeworthii

113

153

SBB-0899 Platanthera edgeworthii SBB-0903 Platanthera edgeworthii SBB-0901 Platanthera edgeworthii SBB-0900 Platanthera edgeworthii SBB-0897 Platanthera latilabris SBB-0904 Platanthera edgeworthii SBB-0902 Platanthera edgeworthii SBB-0898 Platanthera latilabris10

94

2

4

Figure 44: NJ trees for (b) matK (c) rbcL (d) rpoB and (e) rpoC1 loci of Platanthera

species.

(b)

(c)

(d)

(e)

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4.4.18 Pleione

Two species, P. maculata and P. praecox (one individual each) showed average inter-specific distances of 0.006, 0.001, 0.003 and 0.002 in their ITS, matK, rpoB and rbcL sequences, respectively. Except rbcL, sequences of all other loci were variable enough to distinguish two species.

4.4.19 Porpax

The species used in the present study were P. jerdoniana (3I) and P. reticulata (8I). The average inter-specific K2P distances based on ITS, matK, rbcL, rpoB and rpoC1 were 0.137, 0.019, 0.011, 0.011 and 0.009, respectively. All the five candidate loci were able to distinguish two species (Fig. 45 a-e).

The ITS sequences were 700 nucleotides long and had 84 variable sites

including 81 parsimony informative sites. In matK, out of 770 sites, 15 were variable, of which 13 were parsimony informative. The accessions of P. reticulata exhibited intra-specific variations and thus, formed two sub-branches (Fig. 45b). The number of parsimony informative sites for rbcL, rpoB and rpoC1 sequences were 7 (657 total sites), 4 (384 total sites), and 4 (458 total sites), respectively.

0383 Porpax reticulata 0384 Porpax reticulata 0385 Porpax reticulata 0387 Porpax reticulata 0386 Porpax reticulata 0344 Porpax jerdoniana 0348 Porpax jerdoniana

100

0.01 Figure 45: NJ trees for (a) ITS of Porpax species.

(a)

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SBB-0383 Porpax reticulata SBB-0966 Porpax reticulata SBB-0960 Porpax reticulata SBB-0384 Porpax reticulata SBB-0386 Porpax reticulata

SBB-0387 Porpax reticulata SBB-0385 Porpax reticulata

SBB-0344 Porpax jerdoniana SBB-0348 Porpax jerdoniana100

38

65

0.002 SBB-0386 Porpax reticulata SBB-0387 Porpax reticulata SBB-0960 Porpax reticulata SBB-0972 Porpax reticulata SBB-0384 Porpax reticulata SBB-0966 Porpax reticulata SBB-0383 Porpax reticulata SBB-0385 Porpax reticulata SBB-0344 Porpax jerdoniana SBB-0347 Porpax jerdoniana SBB-0348 Porpax jerdoniana

99

0.001 SBB-0386 Porpax reticulata SBB-0966 Porpax reticulata SBB-0384 Porpax reticulata SBB-0387 Porpax reticulata SBB-0383 Porpax reticulata SBB-0385 Porpax reticulata SBB-0960 Porpax reticulata SBB-0972 Porpax reticulata SBB-0347 Porpax jerdoniana SBB-0344 Porpax jerdoniana SBB-0348 Porpax jerdoniana

97

0.001 Figure 45: NJ trees for (b) matK (c) rbcL and (d) rpoB loci of Porpax species.

(b)

(c)

(d)

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SBB-0385 Porpax reticulata SBB-0386 Porpax reticulata SBB-0384 Porpax reticulata SBB-0960 Porpax reticulata SBB-0383 Porpax reticulata SBB-0966 Porpax reticulata SBB-0972 Porpax reticulata SBB-0387 Porpax reticulata SBB-0344 Porpax jerdoniana SBB-0347 Porpax jerdoniana SBB-0348 Porpax jerdoniana

99

0.001 Figure 45: NJ tree for (e) rpoC1 locus of Porpax species.

4.4.20 Vanda

Four species of Vanda evaluated in the present investigation were V. cristata (1I), V. stangeana (1I), V. tessellata (5I) and V. testacea (4I). The average inter-specific distances based on ITS, matK, rbcL, rpoB and rpoC1 were 0.021, 0.006, 0.003, 0.003 and 0.001, respectively. One species pair, V. cristata and V. tessellata in rpoB matrix showed zero distance estimate. In rpoC1, three species viz. V. cristata, V. stangeana and V. tessellata showed zero inter-specific variation. The species discrimination success for Vanda species evaluated in the present investigation for ITS, matK as well as rbcL was 100% by distance as well as phylogenetic method (Fig. 46 a,b). The species discrimination rates for rpoB and rpoC1 were 50 and 25%, respectively.

Based on phylogenetic analysis of ITS sequences obtained from two species,

both were monophyletic with two separate branches (Fig. 46a). The numbers of variable and parsimony informative sites among 787 nucleotides of ITS were 10 and 3, respectively. For matK (706 nucleotides long), the number of variable sites were 13. None of these was parsimony informative. The NJ trees of remaining three loci formed species clusters (Fig. 46 c-e) and resulted in variable rate of species discrimination i.e., 33.33% for rbcL, 50% for rpoB and 25% for rpoC1. The number

(e)

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of parsimony informative sites in rbcL, rpoB and rpoC1 sequences were 1 (641 total sites), 1 (379 total sites), and 1 (440 total sites), respectively.

0718 Vanda testacea 0857 Vanda testacea 0715 Vanda testacea 0717 Vanda testacea 0999 Vanda stangeana

0.002 SBB-0102 Vanda tessellata SBB-0105 Vanda tessellata SBB-0103 Vanda tessellata SBB-0101 Vanda tessellata SBB-0104 Vanda tessellata

SBB-0857 Vanda testacea SBB-0715 Vanda testacea SBB-0718 Vanda testacea SBB-0716 Vanda testacea

SBB-0999 Vanda stangeana

65

88

0.0005 SBB-0101 Vanda tessellata SBB-0715 Vanda testacea SBB-0102 Vanda tessellata SBB-0857 Vanda testacea SBB-0103 Vanda tessellata SBB-0718 Vanda testacea SBB-0104 Vanda tessellata SBB-0105 Vanda tessellata SBB-0717 Vanda testacea SBB-0999 Vanda stangeana

0.0005 Figure 46: NJ trees for (a) ITS (b) matK and (c) rbcL loci of Vanda species. The

species forming clusters are highlighted in the rectangle.

(b)

(a)

(c)

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SBB-0718 Vanda testacea SBB-0857 Vanda testacea SBB-0717 Vanda testacea

SBB-0102 Vanda tessellata SBB-0105 Vanda tessellata SBB-0104 Vanda tessellata SBB-0103 Vanda tessellata SBB-0586 Vanda cristata SBB-0101 Vanda tessellata

SBB-0999 Vanda stangeana

64

0.0005

SBB-0999 Vanda stangeana SBB-0105 Vanda tessellata SBB-0101 Vanda tessellata SBB-0102 Vanda tessellata SBB-0104 Vanda tessellata SBB-0586 Vanda cristata SBB-0103 Vanda tessellata SBB-0715 Vanda testacea SBB-0718 Vanda testacea SBB-0717 Vanda testacea SBB-0857 Vanda testacea

64

0.0002 Figure 46: NJ trees for (d) rpoB and (e) rpoC1 loci of Vanda species. The species


(e)

(d)

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Table 13: Average K2P distances along with the number of species analyzed for each candidate barcode loci.

S.No. Genus No. of

species Average Inter-specific K2P Distances

(Range) ITS matK rbcL rpoB rpoC1

1. Aerides 3 0.072 0.007 0.01 0.006 0.002 (0-0.002)

2. Coelogyne 6 0.054 (0.022-0.07)

0.005 (0.003-0.01)

0.001 (0-0.002)

0.002 (0-0.006)

0.003 (0-0.008)

3. Conchidium 2 0.395 0.059 0.028 0.037 0.014 4. Crepidium 2 0.057 0.008 0.002 0.003 0

5. Cymbidium 3 0.123 (0.043-0.168)

0.02 (0.017-0.025)

0.008 (0.003-0.01)

0.01 (0.006-0.015)

0.003 (0.002-0.005)

6. Eria 4 0.168 (0.093-0.205)

0.04 (0.009-0.063)

0.016 (0.005-0.024)

0.027 (0.01-0.045)

0.02 (0.006-0.034)

7. Eulophia 2 - 0.008 0.002 0.009 0 8. Goodyera 2 0.111 - 0.011 0.017 0.012

9. Habenaria 10 0.175 (0.01-0.268)

0.039 (0.014-0.056)

0.007 (0.002-0.013)

0.014 (0-0.03)

0.008 (0-0.017)

10. Liparis 3 0.172 (0.051-0.24)

0.024 (0.007-0.036)

0.009 (0-0.014)

0.013 (0.003-0.02) 0.007

11. Nervilia 5 0.214 (0.114-0.271)

0.033 (0-0.058)

0.014 (0-0.027)

0.034 (0-0.059)

0.039 (0-0.07)

12. Oberonia 6 0.066 (0.041-0.088)

0.018 (0.001-0.04)

0.005 (0.002-0.008)

0.01 (0-0.02)

0.008 (0-0.017)

13. Paphiopedilum 8 0.031 (0-0.044)

0.009 (0.001-0.015) 0.001 0.001 0.001

14. Peristylus 3 0.138 (0.102-0.17)

0.06 (0.014-0.087)

0.012 (0.008-0.016)

0.006 (0-0.008)

0.014 (0.009-0.017)

15. Pholidota 3 - 0.006 (0-0.01)

0.002 (0-0.003) 0 0

16. Pinalia 2 0.087 - 0.005 0.011 0.012 17. Platanthera 2 0 0.003 0 0 0 18. Pleione 2 0.006 0.001 0 0.003 0.002 19. Porpax 2 0.137 0.019 0.011 0.011 0.009

20. Vanda 4 0.021 0.006 (0.003-0.01)

0.003 (0.002-0.005)

0.003 (0-0.016)

0.001 (0-0.002)

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Table 14: Species Discrimination rates for five tested loci in twenty orchid genera. S.No. Genus No. of species Species Discrimination Rate

ITS matK rbcL rpoB rpoC1 1. Aerides 3 100% 100% 100% 100% 33.33%

2. Coelogyne 6 100% 100% 16.66% 33.33% 33.33%

3. Conchidium 2 100% 100% 100% 100% 100%

4. Crepidium 2 100% 100% 100% 100% 0

5. Cymbidium 3 100% 100% 100% 100% 100%

6. Eria 4 100% 100% 100% 100% 100%

7. Eulophia 2 - 100% 100% 100% 0

8. Goodyera 2 100% - 100% 100% 100%

9. Habenaria 10 100% 100% 100% 50% 60%

10. Liparis 3 100% 100% 33.33% 100% 100%

11. Nervilia 5 100% 50% 20% 60% 20%

12. Oberonia 6 100% 100% 100% 66.66% 66.66%

13. Paphiopedilum 8 50% 100% 25% 12.5% 12.5%

14. Peristylus 3 100% 100% 100% 33.33% 100%

15. Pholidota 3 - 100% 100% 0 0

16. Pinalia 2 100% - 100% 100% 100%

17. Platanthera 2 0 100% 0 0 0

18. Pleione 2 100% 100% 0 100% 100%

19. Porpax 2 100% 100% 100% 100% 100%

20. Vanda 4 100% 100% 100% 50% 25%

4.5 MULTI-LOCUS COMBINATIONS

As none of the five tested loci yielded 100% species resolution for all the analyzed orchid species, various multi-locus combinations were also tested. The species identification rates or percent species resolution was calculated for two, three, four and five locus combinations besides single locus analysis. The analysis was

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carried out on those species for which the sequences were obtained for all the five loci and the percent species resolution was calculated using genetic distance method. Out of 104 species, the number of species analyzed for multi-locus combinations were 76. The maximum percent species resolution for single locus analysis with 76 species, was obtained with ITS (92.11%) followed by matK with 89.47% (Table 15 and Fig. 47). The two-locus combinations of ITS + matK, ITS + rbcL, ITS + rpoB, ITS + rpoC1, matK + rbcL, matK + rpoB, matK + rpoC1, rbcL+ rpoB, rbcL + rpoC1 and rpoB + rpoC1 resolved 94.74%, 93.21%, 92.11%, 92.11%, 90.79%, 89.47%, 92.11%, 71.05%, 65.79% and 72.37%, respectively. The three-locus combination of ITS+matK+rbcL resulted in further increase in species resolution. This three-locus combination discriminated 96.05% of the species. The remaining three-locus combinations exhibited species discrimination rates above 90% but less than 96.05% with the exception of rbcL+rpoB+rpoC1 showing only 77.63% success. The inclusion of one more locus to the above three locus combination did not result in any further increase in species resolution. The four- and five-locus combinations containing three loci viz., ITS, matK and rbcL in combination with either rpoB or rpoC1 or both resulted in a maximum of 96.05% species resolution. The remaining four-locus combinations yielded 93-94% species resolution values (Table 15).

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combinations.

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Table 15: Percent species resolution calculated using genetic distance method for 76 Orchidaceae species based on single as well as multi-locus combinations of different loci from chloroplast and one locus from nuclear genome.

Locus/ Combination No. of species successfully

discriminated % Species Resolution

ITS 70 92.11 matK 68 89.47 rbcL 41 53.95 rpoB 42 55.26 rpoC1 41 53.95 Two-Locus ITS+matK 72 94.74 ITS+rbcL 71 93.42 ITS+rpoB 70 92.11 ITS+rpoC1 70 92.11 matK+rbcL 69 90.79 matK+rpoB 68 89.47 matK+rpoC1 70 92.11 rbcL+rpoB 54 71.05 rbcL+rpoC1 50 65.79 rpoB+rpoC1 55 72.37 Three-Locus ITS+matK+rbcL 73 96.05 ITS+matK+rpoB 72 94.74 ITS+matK+rpoC1 72 94.74 matK+rbcL+rpoB 69 90.79 matK+rbcL+rpoC1 71 93.42 rbcL+rpoB+rpoC1 59 77.63 matK+rpoB+rpoC1 70 92.11 ITS+rpoB+rpoC1 70 92.11 ITS+rbcL+rpoB 71 93.42 ITS+rbcL+rpoC1 71 93.42 Four-Locus ITS+matK+rbcL+rpoB 73 96.05 ITS+matK+rbcL+rpoC1 73 96.05 ITS+matK+rpoB+rpoC1 72 94.74 ITS+rbcL+rpoB+rpoC1 71 93.42 matK+rbcL+rpoB+rpoC1 71 93.42 Five-Locus ITS+matK+rbcL+rpoB+rpoC1 73 96.05

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4.6 EXEMPLIFYING THE APPLICATION OF DNA BARCODING IN DETECTING MARKET SUBSTITUTIONS

The authenticity of three medicinal orchids sold as Jivak (1), Jivak-Rishbhak,

Rishbhak (1), Riddhi (1), Riddhi-Vriddhi (1), Vridhi (2). Botanically Jivak, Rishbhak, Riddhi and Vriddhi are Malaxis muscifera, M. acuminata, Habenaria intermedia and H. edgeworthii, respectively. The authenticity of the samples procured from the local markets was checked by comparing the sequences of ITS, matK and/or rbcL, generated from these samples, with the corresponding DNA barcodes of the species developed from the authentic wild plants by genetic distance method and also BLAST analysis. Only matk and rbcL sequences could be obtained from the sample procured from the market as Jivak. Both the sequences matched with those of M. acuminata (Rishbhak) [Fig. 48 a-d]. However, as these sequences from an authentic sample of M. muscifera were neither available in our data sets or on NCBI, authenticity of such a sample could be doubted only if a clear distinction between these sequences from the two species is established. ITS and rbcL sequences were generated from the sample sold as Jivak-Rishbhak. ITS sequence was found to be of a fungus, Aspergillus fumigates (Fig. 49a,b), whereas, rbcL sequence revealed a zero distance with M. acuminata (Fig. 50a,b). The sample purchased as Rishbhak yielded only matK and ITS sequences both of which matched with M. acuminata thus, proving it to be an authentic sample (Fig. 48a, 49a). The sequence for none of the loci could be obtained from the sample purchased as Riddhi. In contrast, sequences of all the three loci were obtained from the sample procured as Riddhi-Vriddhi. In the distance based method, none of these sequences matched with the corresponding sequences of either H. intermedia (Riddhi) or H. edgeworthii (Vriddhi) [Fig. 51 a,b,c]. On BLAST analysis, matK and rbcL sequences had 99% and 100% homology with those of another orchid, Thunia alba (Fig. 52 b,c). When the sequences from this orchid were also included in the distance matrix analysis, zero distances were revealed between the respective sequences of the sample and the corresponding sequences of Thunia alba (Fig. 53a). In contrast, on BLAST analysis, ITS sequence from this sample appeared to be unique as it had only 94% similarity with the orchid, Anacamptis laxiflora (Fig. 52 a). Moreover, Thunia alba did not appear among the first 1000 matches, though two ITS sequences from this plant were available in the GenBank. The alignment of these two

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sequences (downloaded) with that of the sample revealed only 60% similarity. Only rbcL sequences could be obtained from both the samples of Vriddhi. Both the rbcL sequences did not match with the authentic sequences of either H. intermedia (Riddhi) or H. edgeworthii (Vriddhi) [Fig. 51c]. Rather, BLAST analysis showed their 99% similarity with those of the orchid, Habranthus martinezii (Fig. 52d).

Figure 48: Distance matrices (a, b) and BLAST analysis (c, d) of matK and rbcL

sequences, respectively, for Jivak.

(a)

(b)

(c)

(d)

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Figure 49: Distance matrix (a) and BLAST analysis (b) of ITS sequences for Jivak-

Rishbhak

Figure 50: Distance matrix (a) and BLAST analysis (b) of rbcL sequences for Jivak-

Rishbhak.

(a)

(b)

(b)

(a)

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Figure 51: Distance matrices of ITS (a), matK (b) and rbcL (c) sequences for Riddhi-

Vriddhi

(a)

(b)

(c)

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Figure 52: BLAST analysis of ITS (a), matK (b) and rbcL (c) sequences of Riddhi-

Vriddhi and rbcL (d) sequences of Vriddhi.

(a)

(b)

(c)

(d)

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Figure 53: Distance matrices of matK (a) and rbcL (b) sequences of Riddhi-Vriddhi,

Vriddhi and Thunia alba.

(a)

(b)

full thesis in pdfshodhganga.inflibnet.ac.in/bitstream/10603/13622/9/09... · 2015. 12. 4. ·...

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