CONFIRMING THE IDENTITY OF NEWLY RECORDED NYMPHAEA RUBRA ROXB . EX ANDREWS DISCERNING FROM NYMPHAEA PUBESCENS WILLD . USING MORPHOMETRICS AND MOLECULAR SEQUENCE ANALYSES

A multivariate statistical analysis was carried out to evaluate the morphological variation between Nymphaea pubescens Willd., and a deep purplish red flowered Nymphaea that occur in Sri Lanka. The plant resembles N. rubra Roxb. ex Andrews, a species that had been sometimes circumscribed as a variety under N. pubescens Willd. DNA sequences data of matK and psbA-trnH regions were used to obtain further support. Morphological data were scored from collected samples and analyzed using PAST software. Extracted DNA were amplified for matK and psbA-trnH gene regions. Obtained sequences were matched with the related accessions deposited in the GenBank. Multivariate analysis supported the recognition of deep purplish red flowered Nymphaea as a different species from N. pubescens, and was identified as N. rubra based on literature. GenBank accessions for the matK region of N. rubra showed 99% similarity while it gave only a 96% similarity for N. pubescens with query coverage of 97% and 96% respectively, corroborating with the morphological analysis. Comparison of the sequence divergence between N. pubescens and N. rubra sequences indicated a 95% similarity for matK gene region while 92% similarity for psbA-trnH gene region. The sequences generated during the present study would provide additional reference sequences for the two taxa. Introduction The genus Nymphaea L. (Nymphaeaceae Salisb.) or Water-lilies comprise of about 40-50 species and is widespread in tropical and temperate regions covering vast extents of natural waterbodies. All are aquatics with perennial or annual rhizomes (Jaime et al., 2000). Species of Nymphaea show a high morphological plasticity where the size of leaves and flowers are thought to be strongly dependent on hydrological and edaphic conditions (Polina and Alexy, 2007). They grow in open waters of large swamps, lakes, ponds, shallow ditches, and also in marshes. The species of Nymphaea may be either dayblooming or night-blooming. The flowers are showy and born solitarily, containing numerous petals, stamens, and many carpels. The genus Nymphaea is a taxonomically difficult group; many species are believed to have numerous subspecies, chromosomal races & forms of hybrids and of artificial origin (Polina and Alexy, 2007). The plants are very popular as ornamental aquatics in the landscape industry. Postgraduate Institute of Science, University of Peradeniya, Sri Lanka Corresponding author. Email: deepthiyakandawala@gmail.com Department of Horticulture & Landscape Gardening, Faculty of Agriculture & Plantation Management, Wayamba University of Sri Lanka.


Introduction
The genus Nymphaea L. (Nymphaeaceae Salisb.) or Water-lilies comprise of about 40-50 species and is widespread in tropical and temperate regions covering vast extents of natural waterbodies.All are aquatics with perennial or annual rhizomes (Jaime et al., 2000).Species of Nymphaea show a high morphological plasticity where the size of leaves and flowers are thought to be strongly dependent on hydrological and edaphic conditions (Polina and Alexy, 2007).They grow in open waters of large swamps, lakes, ponds, shallow ditches, and also in marshes.The species of Nymphaea may be either day-blooming or night-blooming.The flowers are showy and born solitarily, containing numerous petals, stamens, and many carpels.The genus Nymphaea is a taxonomically difficult group; many species are believed to have numerous subspecies, chromosomal races & forms of hybrids and of artificial origin (Polina and Alexy, 2007).The plants are very popular as ornamental aquatics in the landscape industry.
Among the diverse members of the family, N. nouchali Burm.f., N. rubra Roxb.ex Andrews, N. pubescens Willd, and N. alba L. are some of the most widely spread species in Asia (La-ongsri et al, 2009).Nymphaea rubra is common throughout the temperate and tropical Asia, such as in Bangladesh, India, Taiwan and Thailand, especially in shallow lakes and ponds.The species have somewhat big flowers compared to many other Nymphaea species, and it prefers to grow in nonacidic water above 15ºC (Hossain et al., 2007).According to the Revised Handbook to the Flora of Ceylon, the family Nymphaeaceae is represented in Sri Lanka only by the genus Nymphaea, with two species, N. nouchali Burm.f., and N. pubescens Willd.(Dassanayake, 1996).Other than these native species, during field visits, a deep purplish red flowered Nymphaea species with a morphological resemblance to N. pubescens was also encountered in natural water bodies in the dry lowland of the country.Many morphological features that are described under the N. pubescens (Dassanayake, 1996) overlap with this Nymphaea species.The plant has been referred to as N. pubescens variety rubra by de Vlas and de Vlas-de Jong in 2008.According to The Revised Handbook to the Flora of Ceylon (Dassanayake, 1996) the flower petals of N. pubescens are white, purplish pink or red, where inner petals are smaller.Leaf upper surface is glossy dark green and dark purplish green, velvety on leaf lower surface with very prominent veins.The petiole is red-brown, while the pedicel bears short prickles.However the presence of short prickles on the pedicels is a mis-conception as N. pubescens never poses prickles but Nelumbo nucifera, a species belonging to the family Nelumbonaceae, instead.The filament colour is described as yellowish white becoming deeper yellow distally, or pale purplish pink to crimson.Although the above description is accommodating many characters of N. pubescens, the description seems to include some characters of those of the deep purplish red flower species of Nymphaea as well.Characters such as red-brown petiole, dark purplish green lower surface with velvety appearance and highly prominent veins are more towards the plants with deep purplish red flowered Nymphaea rather than N. pubescens.On the other hand, according to literature, this deep purplish red flowered Nymphaea species share morphological similarities with N. rubra, a species that had not been recognized as occurring in the island during the revision of the Flora.According to Conard (1905), N. rubra possess deep purplish red coloured flowers with cinnabar red stamens, and the reddish leaves becoming greenish with the age, and rarely producing fruits or seeds.Mitra and Subramanyam (1982), questioned the treatment of N. rubra as a true species at par with other sexually reproducing species because of its failure to set fruits/seeds in nature.According to Gupta (1980), N. rubra has two cytotypes, one which is highly fertile and another nearly sterile.Further, La-ongsri et al. (2009), describes N. rubra as, leaf dark reddish above and below, nine pairs of prominent and angular veins below, petiole green or reddish-brown, and a deep purplish red flower bearing orange or cinnabar-red stamens, becoming brownish with age.Nymphaea pubescens and N. rubra are two closely related taxa (Jeremy et al., 2010).
Hence, a multivariate statistical analysis was carried out to evaluate the morphological variation between the ambiguous taxa and described N. pubescens, and further DNA sequences data of matK and psbA-trnH regions were used in verification of the identity between N. pubescens and the deep purplish red flowered Nymphaea species occurring in Sri Lanka.The matK is one of the rapidly evolving coding region in the plastid genome, while Chloroplast noncoding intergenic psbA-trnH spacer has recently become a popular tool in plant molecular phylogenetic studies at low taxonomic levels (Biswal et al., 2012).

Sample collection
Live plant material of the two Nymphaea species, including populations with both white and pink flowered N. pubescens, and deep purplish red flowered Nymphaea species, were collected from 50 different locations covering all the three major climatic zones of the island.The map showing the field localities are given in Fig. 1.From each locality, a minimal of five specimens were collected.All the collected populations were treated separately with a different acronym; DPRN (Deep Purplish Red flowered Nymphaea species), NPW (N.pubescens White) and NPR (N.pubescens Pink), for easy references.The collected specimens were examined in detail in the laboratory for different morphological characters.

Morphological studies
Vegetative characters such as leaf shape, length, petiole diameter and reproductive characters such as flower size, petal and sepal length, number of stamens were studied either with the naked eye, under a dissecting microscope or under a stereo microscope (Leica, 10446322, 2X WD).Five individuals from each population were studied in detail where the measurement was averaged.The mean value for up to three measurements of each character was recorded for each specimen.Special attention was paid to characters with distinct variations.Colour of the lower and upper surfaces of the leaf and petal, stamens, stigmatic segments, and petiole were determined using the Royal Horticulture Society Colour Chart (RHS Colour Chart 2001).Nineteen quantitative and 21 qualitative characters were coded for 50 representatives of N. pubescens including both white and pink flowered populations and deep purplish red flowered Nymphaea species.All qualitative characters were expressed quantitatively by giving a coding value, to avoid misrepresenting the possible range of variation (Stevens, 1991).

Morphometric analyses
The multivariate statistical analysis was carried out using the PAST -Paleontological Statistics program version 2.17 (Hammer et al., 2001).Cluster analysis, Principal Component Analysis (PCA) and Principal Coordination Analysis (PCoA) were performed.The cluster solution was selected from the best suitable similarity measure method and the algorithm; Gower similarity measure and 'Paired group' option (UPGMA), which produced the highest Co-phenetic correlation value of 0.933 over the other similarity distance methods and algorithms.Similarity Percentage Analysis (SIMPER) was performed to obtain overall average dissimilarity levels of the groups.Other than the SIMPER, PCA loadings were also used to rank characters regarding their contribution for the separation of clusters, and thereby to find the best vegetative characters to differentiate between the two species.
Consensus for resulted sequences of forward and reverse primers was compiled using Bioedit version 7.1.11and edited visually.Sequences deposited in the GenBank, for the matK gene region for the two taxa by other literatures were extracted using a BLAST (Basic Local Alignment Search Tool).ClustalW multiple sequence alignment was also used for Sequences alignment and comparison other than the BLAST.

Results and Discussion
The list of characters that were studied in detail together with their character states is given in the Table 1.The UPGMA dendrogram (co-phenetic correlation coefficient = 0.933) resolved two discrete clusters (denoted as cluster A and B), which separated respectively at approximately 0.35 distance units.The OTUs within each cluster grouped together closely, with none of them exceeding a distance of more than 0.7 units within any given cluster (Fig. 2).The scatter plot that resulted from the PCoA is given in Fig. 3 (Transformation component, C = 2).The first four (principal) eigenvalues recovered from the PCoA (1.3985, 0.2106, 0.1268, and 0.0878) accounted for 71.17% of the total variance (54.53%, 8.27%, 4.95%, and 3.43% respectively).A plot of the first and second coordinates (which provided the greatest separation of OTUs) returned a result similar to that obtained by the Cluster Analysis.Here the PCoA also resolved two discrete clusters, with each corresponding exactly to one of the clusters indicated by the UPGMA dendrogram.According to the results of PCA loading and SIMPER analysis, the number of stamens, leaf length, leaf width, number of petals and number of stigmatic segments are the highly contributed quantitative characters while lamina colour (both upper and lower), leaf venation pattern, petiole colour and stamen colour are the highly contributed qualitative characters for the separation.The character variation of the highly contributing six quantitative characters (number of stamens, leaf width, leaf length, number of stigmatic segments, number of petals and leaf size) between the two major groups; identified in analysis is given in Fig. 4 as box plots.
The morphometric analysis identifies two main phenetic groups A and B that corresponds to the deep purplish red flowered Nymphaea congregated in the cluster A while the cluster B corresponds to N. pubescens.Cluster B further branched at a distance of 0.2, where one group encompassed only pink flowered N. pubescens while the larger group consisted of both the white and pink flowered N. pubescens.Similarly the scatter plot obtained by PCoA clearly supports to the clustering of the populations into two major phenetic groups as A and B as recognized by the cluster analysis with non-overlapping distribution and the overlapped scattering of the members in group B. The detailed study of the characters of the members of the deep purplish red flowered Nymphaea and the character comparison with literature, Conard (1905) and La-ongsri et al. (2009), confirmed the identity of the group as N. rubra Roxb.ex Andrews, a species that has not been recorded before as occurring in the island.

DNA sequence analysis
The obtained sequence length of the matK and psbA-trnH gene regions were between 916-917 bp and 541-555 bp for deep purplish red flowered Nymphaea and N. pubescens respectively.The similarity percentage comparison of the obtained sequences of both N. pubescens (white and pink), with deep purplish red flowered Nymphaea with alignment scores obtained from BLAST search are given in Table 2.There were no sequence data deposited in the GenBank for both taxa for the psbA-trnH gene region.
Comparison of the obtained sequences with the GenBank (N.rubra -Acc.No. HQ592335.1)(Jeremy et al., 2010) gave a 99% similarity for the deep purplish red flowered Nymphaea for the matK gene region with N. rubra while it gave a 96% similarity for N. pubescens (Acc.No. FJ597753.1)(Jeremy et al., 2010).Comparison of the BLAST sequence divergence between N. pubescens and deep purplish red flowered Nymphaea for the sequences that were obtained in the study indicated only a 95% similarity for matK gene region existed between the two, while only 92% similarity for psbA-trnH gene region was indicated.Further, the comparison of both white and pink flowered N. pubescens sequences with the GenBank gave a 99% similarity match with N. pubescens (Acc.No. FJ597753.1).BLAST search results, while indicating that the deep purplish red flowered Nymphaea is a different taxa from the native N. pubescens, further confirms its identity as N. rubra.Comparison of the sequences using ClustalW, mismatches accounted for 6 point mutations and 17 insertion/ deletion (INDELS) events (single base pair) observed in the alignment for the two sequences of matK for the two species, N. rubra and white N. pubescens obtained in the present study while 4 point mutations and 3 INDELS (4, 5, and 17 bp length) were encountered for psbA-trnH sequence alignment.When compare between pink and white N. pubescens, there were only 2 gaps observed for matK and 5 gaps observed for psbA-trnH sequence.INDELS occurred in both matK and psbA-trnH gene regions, where the most informative was in the psbA-trnH region.
The results of both multivariate statistical analyses and the molecular sequences comparison have supported the recognition of the deep purplish red Nymphaea as a different species from N. pubescens.The detailed comparison of morphological characters has identified the species as N. rubra Roxb.ex Andrews while the molecular sequence comparison has further confirmed its identity.
According to the literature N. rubra could be easily separated from N. pubescens from the close examination of floral and leaf characters.Average dissimilarity value for the separation of N. rubra (DPRN) from white flowered N. pubescens (NPW) and pink flowered N. pubescens (NPP) is 15.31.According to the results, characters such as number of stamens, leaf length and width, and number of petals are good quantitative characters for delimitation of these two species while lamina colour (both upper and lower), leaf venation pattern, petiole and stamen colour are the highly contributed qualitative characters.Even though the cluster encompassing N. pubescens (Cluster B) initially separates a few individuals with pink flowers in a separate cluster, the remaining group includes both individuals with pink and white flowers once again separating the pink flowered into a sub-cluster.The overall dissimilarity value between the members of the two pink and white flowered groups within N. pubescens is 10.14 according to the SIMPER analysis while the gap was less than 0.2 in distance units (Fig. 2).Further in both BLAST and ClustalW sequence alignments for both pink and white flowered N. pubescens, the two sequences for both gene regions, showed a very high sequence identities with zero or very few mismatches, implying that they are just two color variations of the same species.A comparison of the characters between the two species is given in Table 3 and Fig. 5, and an identification key for the Sri Lankan Nymphaea is given below.
1. Leaves pubescent beneath with many short hairs, margin sharply dentatemucronate; stamens without a tongue-shaped appendage beyond the anther or appendage very short 2 -Leaves glabrous, margin entire to dentate with blunt teeth; stamens with a tongue-shaped appendage beyond the anther to 5 mm long N. nouchali 2. Leaf abaxial surface brown, venation pattern less prominent, petiole light green; flowers white, yellowish white or pink; stamens short, yellow; stigmatic surface yellow N. pubescens -Leaf abaxial surface dark purple, venation pattern very prominent, petiole reddish; flowers deep purplish red; stamen long, cinnabar red; stigmatic surface crimson red N. rubra All Water-lily species occur together in large water bodies in both dry and wet zones of the country.However, in many instances N. rubra occurs towards the center of the deep waters in isolation.The study has resulted in adding a new member to the genus Nymphaea in Sri Lanka enriching the islands biodiversity.Nymphaea is a taxonomically difficult group with many natural and man-made hybrids occurring in the nature and therefore, morphological features alone are not sufficient in confirming the identity.The present study provides additional reference sequences for both N. pubescens (Pink and white flowered groups) and N. rubra as well as a new gene region, psbA-trnH for reference.

Fig. 5 .
Fig. 5. Deep purplish red flowered Nymphaea species identified as N. rubra during the present study (A).Flowers of N. pubescens, pink (B) and white (C).Leaf upper surfaces of N. pubescens (left) and N. rubra (right) (D) Cinnabar red colour stamens of N. rubra (E) and yellow colour stamens of N. pubescens (F).