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New insights into character evolution, hybridization and diversity of Indian Nymphaea (Nymphaeaceae): evidence from molecular and morphological data a

a

b

Jeremy Dkhar , Suman Kumaria , Satyawada Rama Rao & Pramod Tandon

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a

Plant Biotechnology Laboratory, Centre for Advanced Studies in Botany, North-Eastern Hill University, Shillong, 793 022, India b

Department of Biotechnology and Bioinformatics, North-Eastern Hill University, Shillong, 793 022, India c

Stress Physiology and Molecular Biology Laboratory, School of Life Sciences, Jawaharlal Nehru University, New Delhi, 110067, India Version of record first published: 03 Apr 2013.

To cite this article: Jeremy Dkhar , Suman Kumaria , Satyawada Rama Rao & Pramod Tandon (2013): New insights into character evolution, hybridization and diversity of Indian Nymphaea (Nymphaeaceae): evidence from molecular and morphological data, Systematics and Biodiversity, 11:1, 77-86 To link to this article: http://dx.doi.org/10.1080/14772000.2013.773949

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Systematics and Biodiversity (2013), 11(1): 77–86

Research Article New insights into character evolution, hybridization and diversity of Indian Nymphaea (Nymphaeaceae): evidence from molecular and morphological data

JEREMY DKHAR1, SUMAN KUMARIA1, SATYAWADA RAMA RAO2 & PRAMOD TANDON1 1

Plant Biotechnology Laboratory, Centre for Advanced Studies in Botany, North-Eastern Hill University, Shillong 793 022, India Department of Biotechnology and Bioinformatics, North-Eastern Hill University, Shillong 793 022, India

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Downloaded by [Jeremy Dkhar] at 07:50 03 April 2013

(Received 2 January 2013; revised 31 January 2013; accepted 4 February 2013) A comprehensive reassessment of Indian Nymphaea (Nymphaeaceae) based on morphology, RAPD and nucleotide sequence data of the ITS, trnK intron, matK and rbcL gene was conducted. Although considerable morphological variations have been reported, pollen colour and rhizome shape are two characters which have not been mentioned in previous studies. The transformation from yellow to white coloured pollen may have evolved independently and is probably associated with a strong selective pressure acted upon by the animal pollinators on white pollen of N. pubescens and N. rubra. Rhizome shape could easily discriminate among subgenera; the difference in shape (triangular, globular and cylindrical) probably highlights the success in their distribution. Our studies using RAPD revealed high genetic variability among individuals of N. caerulea. This may be attributed to the breeding system followed, which could be an outcrossing species. But the lack of genetic diversity in N. tetragona is probably due to founder events, whereby a solitary founder individual from China could have resulted in the establishment of this single population of ∼25–30 individuals with no detectable variations. Molecular cloning of the ITS region of N. rubra, necessitated by the presence of additional signals in the sequencing chromatogram, confirmed the origin of this plant taxon through hybridization involving N. lotus and N. pubescens as the parental species. Key words: founder events, hybrid, molecular cloning, Nymphaea, pollen colour, rhizome shape

Introduction Understanding the diversity and phylogeny of a plant species or genus is of great significance, primarily because of its connection to many branches of biological sciences. The presence or absence of genetic diversity is important both in determining the opportunity to improve the population through selection (Barrett & Schluter, 2008) and in devising appropriate steps for conservation (Rossetto et al., 1995; Lee et al., 2011). A well-corroborated phylogeny would provide a means for evaluating character evolution (Les et al., 1999; Borsch et al., 2008), molecular evolution (Grimm & Denk, 2007), global changes (Edwards et al., 2007) and historical biogeography (Löhne et al., 2008). Due to significant interest from the context of angiosperm origin, the genus Nymphaea L. has garnered great attention from scientists worldwide, but only a collaborative efCorrespondence to: Jeremy Dkhar. Present address: Stress Physiology and Molecular Biology Laboratory, School of Life Sciences, Jawaharlal Nehru University, New Delhi 110067, India. E-mail: [email protected] ISSN 1477-2000 print / 1478-0933 online C 2013 The Natural History Museum http://dx.doi.org/10.1080/14772000.2013.773949

fort can lead to a comprehensive understanding on species distribution, ecology and conservation status of the genus (Borsch et al., 2011); however, its cosmopolitan nature has hindered the efforts of several authors. Ten taxa (N. alba, N. alba var. rubra, N. caerulea, N. candida, N. × marliacea, N. micrantha, N. nouchali, N. pubescens, N. rubra and N. tetragona) have been reported from India (Mitra, 1990). However, the traditional method of classification was considered perplexing because of inappropriate nomenclature (Cook, 1996). Such an observation was also reported by Sanjappa et al. (2005), who noticed some lapses regarding Nymphaea species that appeared in the database on plants of South India, as described by Ganeshaiah & Uma Shaanker (2005). Notwithstanding all remarks, a recent report indicated an increase in the number of Nymphaea species available in India from 10 to 16 (Ansari & Jeeja, 2009). To validate such observations, a molecular-based phylogenetics and taxonomic revision of four Indian representatives of the genus Nymphaea was conducted (Dkhar et al., 2010). Because of limited taxon sampling and sole reliance upon molecular characters in our previous studies (Dkhar et al.,


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2010, 2012), evidences from morphology as well as DNAbased markers were deemed necessary in order to have a complete understanding of the Indian water lily species. Considerable variations of morphological characters have been reported among species of the genus Nymphaea (Borsch et al., 2007 and references therein). Such knowledge was used to evaluate character evolution in the different lineages of Nymphaea (Borsch et al., 2008), but not all species were represented, particularly those that are native to India. A review of the literature on genetic diversity in the genus Nymphaea involving marker-based systems revealed only four studies: (i) genetic variation among populations of N. odorata based on ISSRs (Woods et al., 2005); (ii) AFLP-based genetic variability among populations of Eurasian Nymphaea species such as N. alba, N. candida and N. tetragona (Volkova et al., 2010); (iii) ISSR-based species identification and differentiation of cultivars and natural populations of Nymphaea species found in Thailand (Chaveerach et al., 2011); and (iv) genetic diversity among and within populations of thermal Nymphaea species utilizing ISSR markers (Poczai et al., 2011). The results are contradictory with the three earlier reports indicating high polymorphism within and among populations, while the latter study showed low genetic diversity at both intra- and inter-populations. In line with these investigations, the present work endeavours to present a comprehensive study on seven Indian representatives of the genus Nymphaea based on morphology, RAPD and nucleotide sequence data of the ITS region, chloroplast trnK intron, matK and rbcL gene, and the attempt to provide additional information on morphological character evolution [since reconstructing phylogenetic trees requires a complete or near-complete representation of all representative taxa, approximately 50 species in the case of Nymphaea, phylogenetic estimation in this study (7 species, 14–15%) is solely used for providing new insights on character evolution in the genus Nymphaea], infers genetic variability among and within populations of Indian Nymphaea, and provides evidence for hybrid origin of N. rubra, commonly known as Indian red water lily.

Materials and methods Plant material and taxon sampling Plants of the genus Nymphaea were surveyed and collected from the states of Meghalaya and Assam (NorthEast India). Out of ten taxa reported from India, seven Nymphaea species viz. N. alba var. rubra, N. caerulea, N. × marliacea, N. nouchali, N. pubescens, N. rubra and N. tetragona were found (Appendix S1, distribution map, see supplementary material, which is available on the Supplementary tab of the article’s Taylor & Francis Online page at http://dx.doi.10.1080/14772000.2013.773949). Information on the place of collection, voucher specimens and the number of individuals used for morphological observations,

RAPD, and sequence data generated for ITS, trnK intron, matK and rbcL gene of all Nymphaea species investigated are provided in Table 1. Nuphar advena was chosen as outgroup because of its emergence as the first branch of family Nymphaeaceae (Les et al., 1999).

Morphological analysis Prior studies have indicated considerable morphological variation among Nymphaea species (Borsch et al., 2007, and references therein). In this regard, additional information was gathered on 36 morphological features comprising 27 qualitative and 9 quantitative characters for all seven Nymphaea species investigated. Qualitative characters encompassing both vegetative and reproductive features were evaluated from 417 and 184 samples, respectively. Quantitative characters (both vegetative and reproductive features) were counted and measured manually, employing callipers for seeds size (length and width) and light microscope for pollen diameter. Species means for 9 quantitative characters were calculated and the differences among the averages were evaluated with one-way ANOVA implementing the Scheffé method. If differences among means were statistically significant (P = 0.01), multiple range test (MRT) were performed to identify group of species means for each character. All tests were performed using STATGRAPHICS Centurion XVI Version 16.0.09.

DNA extraction Total genomic DNA was extracted from leaves (fresh or frozen) of each species by following the method of Doyle & Doyle (1987) with the addition of the saturated phenol extraction step prior to ethanol precipitation.

RAPD analysis For RAPD analysis, six primers with clear and reproducible bands were chosen from 30 initially screened primers for the amplification reactions of all individuals included in the present study. Standard PCR reaction conditions were employed. RAPD profiles were scored as 1 for presence and 0 for absence of a band for each individual. The percentage of polymorphism for the six RAPD primers was calculated manually. In addition to these measures, gene flow among sympatric species was also evaluated using POPGENE 1.31 (Yeh et al., 1999).

Amplification and sequencing of the ITS region, trnK intron, matK and rbcL gene PCR was used to amplify the ITS region, trnK intron (including the entire matK gene) and rbcL gene in N. caerulea, N. × marliacea and N. nouchali JD 07 following the protocol of White et al. (1990), Johnson & Soltis (1995)

Character evolution, hybridization and diversity of Indian Nymphaea

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Table 1. Nymphaea L. species investigated with their respective specimen voucher, place of collection and sampling numbers. For sequence data (ITS, trnK, matK, rbcL), numerical figures represent number of sequences generated. Number of samples Species

Specimen vouchera

Place of collection

Leaves

Flowers

RAPD

ITS

trnK

matK

rbcL

JD 03

Shillong, East Khasi Hills District, Meghalaya Smit, East Khasi Hills District, Meghalaya Mukhla, Jaintia Hills District, Meghalaya Shillong, East Khasi Hills District, Meghalaya Mukhla, Jaintia Hills District, Meghalaya Guwahati, Kamrup District, Assam Paikan, Goalpara District, Assam Paikan, Goalpara District, Assam Guwahati, Kamrup District, Assam Paikan, Goalpara District, Assam Nongpoh, Ri-Bhoi District, Meghalaya Chamata, Kamrup District, Assam Nongkrem, East Khasi Hills District, Meghalaya

54

16

5

2

1

1

1

57

16

5

2

1

1

1

26

8

5

2

1

1

1

62

25

5

3

1

1

1

31

10

5

2

1

1

1

12

11

2

2

1

1

1

19

11

5

2

1

1

1

15

19

5

2

1

1

1

6

1

1

1

1

1

1

35

19

5

2

1

1

1

21

20

5

2

1

1

1

40

20

5

2

1

1

1

45

9

5

2

1

1

1


N. alba var. rubra Lönnroth N. alba var. rubra Lönnroth N. alba var. rubra Lönnroth N. caerulea Savigny

JD 12 JD 13 JD 04

N. × marliacea Latour-Marliac N. nouchali Burm.f.

JD 02

N. nouchali Burm.f.b

JD 06

N. nouchali Burm.f.c

JD 07

N. pubescens Willd.

JD 09

N. pubescens Willd.

JD 08

N. rubra Roxb. ex Andrews N. rubra Roxb. ex Andrews N. tetragona Georgi

JD 10

aSpecimen vouchers deposited cwhite-coloured flowers.

JD 05

JD 11 JD 01

at the Herbarium, Department of Botany, North-Eastern Hill University, Shillong, India; bblue-coloured flowers;

and Fay et al. (1998), respectively. Amplified PCR products were purified using QIAQuick gel extraction kit (QIAGEN, Germany) and sequenced at Bangalore Genei, India and Axygen Scientific Pvt. Ltd, India. Sequencing of the different amplified regions was followed as mentioned in Dkhar et al. (2010, 2011b). Sequence data of the ITS region, trnK intron, matK and rbcL gene of N. alba var. rubra, N. nouchali JD 02, N. nouchali JD 06, N. pubescens, N. rubra and N. tetragona were taken from our previous studies (Dkhar et al., 2010, 2011b, 2012). Appendix S2 shows GenBank accession numbers of the submitted sequences (see supplementary material, which is available on the Supplementary tab of the article’s Taylor & Francis Online page at http://dx.doi.10.1080/14772000.2013.773949).

Cloning of the ITS region of N. rubra Due to the presence of additional signals in the sequencing chromatogram, amplified PCR product from the ITS region of N. rubra was cloned into plasmids using CloneJETTM PCR Cloning Kit (Fermentas, Canada). Primers pJET1.2 F and pJET1.2 R supplied along with the kit were used to amplify the cloned ITS region. Thirty clones were screened by sequencing the entire ITS region utilizing the above-

mentioned primers. Sequencing signals of each individual allele of the ITS region isolated through molecular cloning techniques were evaluated for singular peaks in their respective chromatogram and compared with chromatogram of direct sequencing. Nucleotide sequence data are available at GenBank database under accession numbers GU199454–GU199472 (complete and partial ITS alleles sequences). A maximum parsimony-based method using Phylip 3.69 was conducted to evaluate the genetic relatedness between the DNA sequences of the individual clones and the putative parental species.

Phylogenetic tree and mapping of morphological characters The phylogenetic tree used for reconstructing character evolution in seven Indian representatives of the genus Nymphaea is based on maximum parsimony analysis of the combined molecular datasets using NONA ver 1.6 (Goloboff, 1999). Clustal X (Thompson et al., 1997) aligned nucleotide sequence data matrix were incorporated into WinClada ver. 1.0000 (Nixon, 2002) and submitted into NONA using ‘spawn’. Prior to submission into WinClada, Clustal X generated multiple sequence

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alignments were further examined and verified manually. A heuristic search was conducted employing the multiple TBR + TBR strategy, with 1000 replications and 999 random seeds. Multiple sequence alignment data matrix used in phylogenetic tree reconstruction has been submitted to TreeBase and can be accessed at http://purl.org/phylo/treebase/phylows/study/TB2:S13951.

Results


Morphological analysis showed considerable variation Considerable morphological variation was observed among the investigated species of the genus Nymphaea (Appendix S3, details on qualitative characters evaluated, see supplementary material, which is available on the Supplementary tab of the article’s Taylor & Francis Online page at http://dx.doi.10.1080/14772000.2013.773949). Of significant interest are two sympatric races of N. nouchali viz., N. nouchali JD 06 and N. nouchali JD 07, resembling each other in all aspects, but showed flower colour polymorphism with blue- and white-coloured flowers respectively. Furthermore, pollen of N. pubescens and N. rubra were observed to be white in colour, in contrast to yellowcoloured pollen of the remaining species. In N. pubescens, a member of subg. Lotos, the white-petaled flowers exhibited a tinge of pink at the apex, resembling the petal colour of N. rubra. Rhizome shape, a character to our knowledge not mentioned in earlier studies, could discriminate among subgenera. Members of subg. Brachyceras had triangular-shaped rhizomes; globular rhizomes were observed in subg. Lotos, whereas subg. Nymphaea possessed rhizomes that were cylindrical in shape. For N. advena, selected as outgroup, morphological characters were compiled from available reports (Lippok et al., 2000; Padgett, 2007). Based on ANOVA and MRT, three quantitative characters, namely petal number, seed length and seed width, yielded non-overlapping homogeneous groups. Therefore, straightforward character state codes were assigned, five for petal number and four each for seed length and width. The remaining characters showed overlapping homogeneous groups (Appendix S4, the quantitative characters and their character state codes, see supplementary material, which is available on the Supplementary tab of the article’s Taylor & Francis Online page at http://dx.doi.10.1080/14772000.2013.773949). Data matrices of both qualitative and quantitative characters included in the study are summarized in Appendix S5 (see supplementary material, which is available on the Supplementary tab of the article’s Taylor & Francis Online page at http://dx.doi.10.1080/14772000.2013.773949).

RAPD analysis revealed variable percentage of genetic variation within and among populations The six RAPD primers selected for analysis of genetic diversity produced 156 scorable bands in 58 individuals of all species investigated (Appendix S6, data on polymorphism of 6 RAPD primers, see supplementary material, which is available on the Supplementary tab of the article’s Taylor & Francis Online page at http://dx.doi.10.1080/14772000.2013.773949). Three populations of N. alba var. rubra were identified with no variation detected among or within populations. Nymphaea caerulea showed the highest percentage of polymorphism (48.89%) among five randomly selected individuals (intrapopulation). Nymphaea × marliacea, N. rubra and N. tetragona did not show any variation. The three specimens of N. nouchali recorded 68.09% polymorphism. Nymphaea pubescens showed 16.67% variation. Shannon’s information index and Nei’s gene diversity calculated at different levels by POPGENE software yielded similar results. At the taxonomic level, N. nouchali recorded the highest, followed by N. caerulea. At the community level, Ward’s Lake, where N. alba var. rubra and N. caerulea were found growing, recorded the highest diversity followed by that at Paikan, where N. nouchali and N. pubescens existed. Estimation of gene flow (Nm) among sympatric species indicated the probability of gene flow at Ward’s Lake (Nm = 0.0737) and Paikan (0.0236), as shown in Table 2.

Molecular cloning of the ITS region of N. rubra Cloning and sequencing of the amplified ITS region of N. rubra resulted in the isolation of distinct ITS alleles (Fig. 1). Among the allelic variants isolated, Nyrclo24 and two others (Nyrclo6 and Nyrclo26) showed exact sequence matches to N. pubescens, confirming it to be one of the parental parents. Sequence data of allele Nyrclo4 was identical to sequences of both N. lotus and N. petersiana (Fig. 1), suggesting that one of them may represent the other parental species. Representation of these allelic DNA sequences in a phylogenetic framework using maximum parsimony methods demonstrated that some alleles (Nyrclo6, Nyrclo24 and Nyrclo26) grouped together with N. pubescens (clade 3) while some diverged into separate clades (clades 1 and 2), an outcome brought about through genetic recombination (Appendix S7, see supplementary material, which is available on the Supplementary tab of the article’s Taylor & Francis Online page at http://dx.doi.10.1080/14772000.2013.773949). It may be mentioned here that ITS alleles corresponding to the nucleotide sequences of N. lotus or N. petersiana were not obtained.

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Table 2. Estimation of gene flow (Nm) among sympatric species of different communities using POPGENE. Community (species) Ward’s Lake (N. alba var. rubra and N. caerulea) Mukhla (N. alba var. rubra and N. × marliacea) Paikan (N. pubescens and N. nouchali JD 06/JD 07) aGene

Samples

Hta

Hsb

Gstc

Nm

10

0.2431 ± 0.0547

0.0312 ± 0.0050

0.8715

0.0737

10

0.1186 ± 0.0456

0.0000 ± 0.0000

1.0000

0.0000

15

0.1524 ± 0.0425

0.0069 ± 0.0007

0.9550

0.0236

diversity in the total population; bgene diversity within subpopulation; cgene differentiation relative to the total population.


Phylogenetic relationship and mapping of morphological characters The present study was initiated to make use of the morphological variation observed in Nymphaea to complement molecular data for understanding the evolutionary history of seven Indian representatives of the genus Nymphaea,

encompassing three subgenera i.e. subg. Brachyceras, Lotos and Nymphaea. Members of subg. Anecphya and subg. Hydrocallis are restricted in distribution, and are not available in India. Nevertheless, subg. Anecphya is known to be closely related to subg. Brachyceras, while subg. Hydrocallis is a sister group of subg. Lotos (Löhne et al., 2007; Borsch et al., 2007, 2008). Phylogenetic analysis of

Fig. 1. Two distinct ITS alleles of Nymphaea rubra isolated through molecular cloning techniques. DNA sequence of allele Nyrclo24 (dark-shaded) is identical to that of N. pubescens whereas allele Nyrclo4 (light-shaded) showed nucleotide sequence corresponding to that of N. lotus. Numbers represented at the ends above each chromatogram refer to the nucleotide positions; ∗ specify exact match. Letters with numerical figures within parentheses following GenBank accession numbers indicate the ITS allele number.


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Fig. 2. Character evolution in seven Indian representatives of the genus Nymphaea. Unambiguous morphological transformation of rhizome shape (1) and pollen colour (19) mapped on the tree from analysis of combined molecular datasets. Black circles indicate nonhomoplasious changes (synapomorphies or autapomorphies). Numbers on top of branches represent character numbers whereas those below branches indicate character state transformation.

combined molecular datasets (ITS, trnK, matK, rbcL) placed subg. Nymphaea as the first branch, with subg. Lotos and subg. Brachyceras emerging as sister clades. Within subg. Brachyceras, N. nouchali JD 02 emerged as a separate clade with respect to N. nouchali JD 06 and N. nouchali JD 07, suggesting that this plant may represent a different taxon. Optimization revealed 18 unambiguously reconstructed phenotypic characters (both qualitative and quantitative). Two of these characters, rhizome shape and pollen colour, are depicted in Fig. 2.

Discussion Lack of genetic diversity in N. tetragona is probably due to founder events To our knowledge, the first study to utilize dominant markers such as ISSR for analysing genetic variation in the genus Nymphaea was reported by Woods et al. (2005).

They evaluated patterns of genetic variability in N. odorata and found it to be highly variable, probably influenced by breeding system. The breeding system greatly affects the diversity maintained in a particular population. Selfing tends to produce individuals with low genetic variation (Maki & Horie, 1999). However, Hamrick & Godt (1989) had earlier reported that the difference in genetic diversity between selfing and outcrossing species at the species level is not statistically significant. In the present study, high genetic variability recorded for five individuals of N. caerulea (48.89%) may be attributed to the breeding system followed, suggesting it to be an outcrossing species, or probably through gene flow from a sympatric species, N. alba var. rubra (Appendix S6, see supplementary material, which is available on the Supplementary tab of the article’s Taylor & Francis Online page at http://dx.doi.10.1080/14772000.2013.773949). Gene flow among these species was indicated with Nm = 0.0737 (Table 2). Although gene flow was significantly low as compared with a recent report of Poczai et al. (2011), genetic differentiation (Gst = 0.8715) was comparatively


Character evolution, hybridization and diversity of Indian Nymphaea higher (Table 2). Gene flow among sympatric species has been reported in Dubautia (Lawton-Rauh et al., 2007) and Crepis (Whitton et al., 2008). The latter assumption, however, seemed unlikely because of lack of seed setting in N. alba var. rubra, an Indian plant confirmed to have originated through hybridization (Dkhar et al., 2011b). Since these two taxa belong to distantly related genera, it is unlikely that hybridization would have occurred between them, nor has there been any previous report on their hybridization. RAPD failed to detect genetic variation among individuals of N. tetragona, a critically rare and endangered plant of India found only in one location (Appendix 6, see supplementary material, which is available on the Supplementary tab of the article’s Taylor & Francis Online page at http://dx.doi.10.1080/14772000.2013.773949). This evidence is in line with an earlier report based on an AFLP marker that showed relatively lower genetic variability in N. tetragona as compared with other Nymphaea species (Volkova et al., 2010). Because of its restricted distribution and morphological similarities shared with N. tetragona from China, it is believed that the plant taxon found in India probably migrated from China (Dkhar et al., 2011a) and colonized in one location of the state of Meghalaya, North-East India. The dynamics of genetic variation maintained in a population after colonization are affected by a number of factors viz. genetic bottlenecks, founder event, gene flow, effective size of founding populations and associated mating patterns within these populations (Jacquemyn et al. 2009, and references therein). So, which phenomenon could possibly explain the lack of genetic variation in N. tetragona? It is expected that cross pollination may have contributed to high within-population genetic variability in N. caerulea; the lack of/low intra-population genetic diversity in N. tetragona is probably due to founder events. A single founder individual could have resulted in the establishment of this population with no detectable variable individuals (∼25−30). This loss of genetic diversity would have increased inbreeding, resulting in reduction of population viability and evolutionary potential of the plant population (Uller & Leimu, 2011). However, this can be overcome by introducing larger numbers of individuals and multiple introduction events (Uller & Leimu, 2011). Therefore, conservation or multiplication of N. tetragona can be targeted at the probable translocation to other habitats through assisted colonization.

Molecular cloning and sequencing of ITS region confirmed hybrid origin of N. rubra To identify alleles unique to the parental species, molecular cloning techniques offer great potential because individual copies of biparentally inherited nuclear sequences (such

83

as ITS) can be isolated from the putative hybrid (Moody & Les, 2002; Les et al., 2004). In the present study, subsequent cloning and sequencing of the amplified ITS region of N. rubra resulted in the isolation of distinct ITS alleles (Fig. 1). The ITS sequences of N. lotus and N. petersiana were similar, differing by only one point mutation and an insertion (data not shown). Additional signals were not depicted at these positions, thus ruling out the possibility of N. petersiana as one of the putative parental species. Among the isolated allelic variants, three showed exact sequence matches to N. pubescens, confirming it to be one of the parental parents, while others had DNA sequences not exclusive of either N. pubescens or N. lotus. The latter observation suggested that genetic recombination between the two parental ITS alleles had occurred. Although ITS alleles exclusive to the nucleotide sequences of N. lotus were not obtained, the recombinant allelic DNA sequences confirmed that hybridization between N. lotus and N. pubescens had occurred. The occurrences of ITS alleles with sequences not exclusive of either of the parental species were detected in the hybrid Nymphaea ‘William Phillips’ (Les et al., 2004) and N. alba var. rubra (Dkhar et al., 2011b). Representation of these allelic DNA sequences in a phylogenetic framework using the maximum parsimony method demonstrated that some alleles grouped together with N. pubescens while some diverged into separate clades, an outcome brought about through genetic recombination (Appendix S7, see supplementary material, which is available on the Supplementary tab of the article’s Taylor & Francis Online page at http://dx.doi.10.1080/14772000.2013.773949). Comparing polymorphic nucleotide sites of all chloroplast markers that differentiate the two parental species indicated exact sequence matches between N. pubescens and the suspected hybrid, confirming N. pubescens as the maternal parent (data not shown). Molecular data were further substantiated by morphological features shared between N. rubra and N. pubescens such as shape of carpellary appendages, pollen colour, night-blooming of flowers, etc. Interestingly, the white petaled flowers of N. pubescens exhibited a tinge of pink at the apex, resembling the petal colour of N. rubra. Observations from previous cytological (Gupta, 1980) and reproductive (Mitra & Subramanyam, 1982) studies also support the hybrid origin of N. rubra. In his cytological investigation of Indian Nymphaea species, Gupta (1980) identified two cytotypes, cytotype I and II, for N. rubra with ploidy level 6× (hexaploid) and 8× (octoploid) respectively. Interestingly, pollen fertility is significantly reduced in cytotype II (19.3%), as compared with cytotype I (98.3%), resulting in cytotype II individuals lacking seed setting. Cytotype I individuals produced seeds and may represent plant materials studied by Venu et al. (2003), collected from South India. Mitra & Subramanyam (1982) also failed to observe seeds in N. rubra. As a result of its failure to set fruit/seed, they questioned the treatment of N. rubra as a true species, rather suggested as an apomict. These

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evidences strongly point to the origin of N. rubra through hybridization, or more appropriately an allopolyploidization event involving N. pubescens and N. lotus followed by chromosome duplication thereby explaining the difference in the ploidy level of this plant species.


The evolution of pollen colour and rhizome shape in the genus Nymphaea A brief but comprehensive description of the available knowledge of morphological characters in the order Nymphaeales, including the five subgenera of Nymphaea, was provided by Borsch et al. (2008). Although considerable morphological variation was reported, pollen colour and rhizome shape were two characters not mentioned in their (or earlier) studies (Borsch et al., 2008 did include rhizome in their study, but their character state definition is different from the present investigation). As flower colour is important in attracting different pollinators, pollen colour has also been reported to act as floral guides directing potential pollinators to their rewards (Ushimaru et al., 2007 and references therein). Due to their ecological significance, it is worth mentioning that the genus Nymphaea exhibited variation in pollen colour; in particular, members of subg. Lotos possessed white-coloured pollen while the remaining species showed yellow-coloured pollen (Fig. 2). Pollen colour polymorphism has been described in a number of species such as Lythrum, Erythronium, Linum, Campanula and Nigella (Jorgensen & Andersson 2005 and references therein). More elaborate, pollen grains of Nigella degenii and its close relatives show variable colouration and are mostly yellow or white, although violet/dark violet pollen are also reported (Jorgensen & Andersson, 2005). Jorgensen & Andersson (2005) suggested that the dark violet pollen of Nigella degenii is not likely a derived character but has evolved ‘as a result of direct selection on pollen colour or as a correlated genetic response to selection on other characters’. It is interesting to note that night-blooming species of subg. Lotos and Hydrocallis are pollinated by beetles (Ervik & Knudsen, 2003; Hirthe & Porembski, 2003) whereas day-blooming species are associated with pollination by several different pollinators (Wiersema, 1988). In the present investigation, optimization of pollen colour character was unambiguous, suggesting that it may have evolved independently at the base of subg. Lotos. This transformation may be associated with a strong selective pressure acted upon by the animal pollinators, but not due to selection on other characters viz. petal colour as some diurnal and nocturnal species shared the same characteristics (e.g. N. pubescens and N. tetragona have white-coloured petals). The evolutionary significance of the difference in rhizome shape probably highlights the success in the distribution of aquatic plant population, and may also play an

important role in their adaptation to seasonal drought, a condition exhibited by most tropical water lilies but not temperate subg. Nymphaea (Wiersema, 1987). Members of subg. Brachyceras had triangular-shaped rhizomes; globular rhizomes were observed in subg. Lotos, whereas subg. Nymphaea possessed rhizomes that were cylindrical in shape (Fig. 2). Rhizomes, in general, have two roles: as reproductive and storage organs (Kunii, 1993); a combination of both sexual and asexual modes of reproduction would enhance their dispersal (locally as well as distantly). Nymphaea alba var. rubra occurs as a densely populated plant species; attributed due to its rhizome shape as well as the direction in which these rhizome spread (horizontal), albeit lack of seed setting. In N. tetragona, dispersion is solely through seeds; the short and erect tuberous rhizome, although cylindrical in shape, function mainly as storage organ. But the lack of seed setting in N. rubra suggests that distribution of this plant taxon is primarily through human dispersal, as was accounted for in N. lotus (Wiersema, 1988) which belonged to the same subgenus. This proposition is supported by the specific choice of habitat adopted by N. rubra which is restricted to permanent ponds, in contrast to N. pubescens which is found in temporary habitats like seasonally inundated fields, rice swamps, shallow ditches along roadsides and railway tracks, etc. (Mitra & Subramanyam, 1982).

Acknowledgements The authors acknowledge two anonymous reviewers for their valuable comments made on an earlier version of the manuscript. The research work provided in this paper forms part of the PhD thesis submitted by JD to North-Eastern Hill University, Shillong, India, under the supervision of PT and SK. This work is partially supported by a project sanctioned to PT and SK by the Department of Biotechnology, Ministry of Science and Technology, India (file no. BT/PR-7055/BCE/08/437/2006). JD is thankful to University Grants Commission for awarding him the Rajiv Gandhi National Fellowship and to all his teachers, family members, friends and well-wishers.

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Associate Editor: Nadia Bystriakova