Evidence for interspecific hybridization between exotic ‘Dam manel’ (Nymphaea × erangae) and native ‘Nil manel’ (Nymphaea nouchali Burm. f.) in Sri Lanka

Biological invasions are considered a serious threat to the biodiversity, and second only to habitat loss, but predicted to soon become the key cause of environmental degradation globally. In addition to competing with natives in natural habitats, another serious threat possessed by Invasive Alien Species (IAS) is their ability to hybridize with natives. The exotic violet flowered Nymphaea × erangae has been introduced to the country for ornamental purposes where it has got naturalized. Now it is recognized as a silent invader in wetlands of the country. The revealing of Nymphaea populations with intermediate characters, both of the native N. nouchali and Nymphaea × erangae in the wetlands of the island raised the question of the occurrence of natural hybridization. The present study was carried out to investigate the event of natural hybridization between the native and the exotic using morphological data. Data collected from putative hybrids and pure populations of the two parents were subjected to multivariate statistical analyses. The results confirmed the identity of the populations with intermediate characters as hybrids between the native N. nouchali and Nymphaea × erangae, highlighting the importance of conserving the natural populations of the native, as hybridization with the exotic pose a threat to its genetic purity.


INTRODUCTION
Global biodiversity is under serious threat due to either direct or indirect human activities. Apart from habitat destruction, human-involved transport of species beyond the native range of organisms continues to increase, with threats posed by biological invasions to native biodiversity and ecosystem functioning are also rising (Levine, 2008 andWu et al., 2015). Biological invasions are considered as serious threats to the biodiversity and presently graded second only to habitat loss (Gurevitch and Padilla, 2004;Didham et al., 2005;Tiebre et al., 2007). It is predicted that biological invasions may shortly surpass habitat loss as the key cause of environmental degradation globally (Chapin et al., 2000). This has raised the attention towards Invasive Alien Species (IAS) in the past decades (Gurevitch and Padilla, 2004;Didham et al., 2005;Tiebre et al., 2007). Apart from competing with natives in natural habitats affecting the ecological balance, another serious threat possess by IAS is hybridizing with natives (Yakandawala and Yakandawala, 2011;Wu et al., 2015). According to Bleeker et al. (2007), interspecific hybridization between more abundant IAS and rare natives can threaten native populations through outbreeding depression and/or through high rates of gene flow swamping native populations. This phenomenon is currently identified as a major threat that could lead to the extinction of the native flora (Levin et al., 1996;Wolf et al., 2001;D'Antonio and Meyerson, 2002).
Water-lilies or Nymphaeas have long been admired for its large and attractive flowers around the world. Since the end of the 19 th century intensive breeding between selected Nymphaea species with desired characters have produced a large number of hybrids for the ornamental plant industry (Nierbauer et al., 2014).
The Sri Lankan violet flowered Nymphaea (Violet water lily or 'Dam manel') is a plant that has been introduced to the country for ornamental purposes, and later got naturalized. The identity of the plant led to many controversial arguments recently, especially due to its mis-identification as the native Nymphaea nouchali Burm. f. ('Nil manel' or Blue water lily) and thereby using its image to depict the national flower of the country. Recent studies have cleared the ambiguity of the identity and also scientifically named this exotic hybrid Nymphaea species as Nymphaea × erangae (Yakandawala et al., in press). This exotic violet flowered Nymphaea is a hybrid between N. micrantha Guill. & Perr. and N. caerulea Savigny (Yakandawala et al., in press). The plant has been spreading in natural water bodies in the recent years and Yakandawala and Yakandawala (2011) recognize the plant as a silent invader in the lowlands of the country. A preliminary study has identified Nymphaea populations with intermediate characters, both of the native N. nouchali and Nymphaea × erangae in the wetlands of the island (Yakandawala and Yakandawala, 2011) raising the question of the occurrence of natural hybridization between the two, the exotic and the native (Figure1). Therefore, the present study was conducted with the aim of investigating the event of natural hybridization between the native N. nouchali and Nymphaea × erangae using morphological data. Confirmation of such events between exotics and the natives are important in terms of biodiversity conservation.

Sample collection and coding of characters
Field work was carried out covering all three major climatic zones (Wet, Intermediate and Dry zones) of the island from 2006 to 2015. Live plant material was collected from putative hybrid populations mixed with either both parents or either parent. Pure populations of the two parents were also sampled to provide morphological standards for the analyses. At each site, at least 4-5 plants were selected to represent the range of gross morphological variations observed within the population. The locations of the sample collection sites are given in Figure 2. Individuals were collected from 143 populations that included 104 pure populations of N. nouchali [the samples included both varieties of N. nouchlai, blue flowered (N. nouchali var. nouchali) and, pink or white flowered (N. nouchali var. versicolor (Sims) Guruge and Yakandawala) Guruge et al., in press]; 28 pure populations of Nymphaea × erangae and 11 tentative hybrid populations. Individuals of each population or Operational Taxonomic Unit (OTUs) were denoted by an acronym, N. nouchali (NN), Nymphaea × erangae (VN) and putative hybrid (HN) for easy reference. Morphological data were studied in detail at the Plant Systematics laboratory, Department of Botany, University of Peradeniya, Sri Lanka. Data were obtained from randomly selected individuals from each population and qualitative and quantitative characters were examined in the laboratory, either by naked eye or under a dissecting or stereomicroscope (Leica, 10446322, 2X WD). Colour of the flower, leaf abaxial and adaxial surfaces and petiole was determined using the Royal Horticultural Society Colour Chart (RHS Colour Chart 2001). A standard suite of morphological characters complied during previous studies were adopted giving special attention to the characters with distinct variations. The list of characters together with the coded character states employed in the morphometric analyses is given in the Table 1.

Data analysis
Morphological data were subjected to Principal Coordinate Analysis (PCoA) and Hierarchical Cluster Analysis (CA) using the statistical software PAST (ver. 2.15) (Hammer et al., 2001). The cluster solution was selected from the best suitable algorithm where Gower distance was used to calculate the similarity measures with the 'paired group' Unweighted Pair Group Method with Arithmetic Mean (UPGMA) option and the Single Linkage algorithm with the highest Cophenetic correlation value. The ordination analysis was performed with Gower distance (transformation exponent C=2) to generate a distance matrix to use in the PCoA. Following the results of the above analyses, each major, consistently recovered cluster was identified.

RESULTS
The UPGMA dendrogram (cophenetic correlation coefficient = 0.9726) that resulted from the Cluster Analysis resolved two main clusters encompassing the parents, Cluster A (N. nouchali) and Cluster B (Nymphaea × erangae) (Figure 3) separating at a distance of 0.44. Cluster A further divided at a distance of 0.26 separating three OTUs of putative hybrids. Another two putative hybrid OTUs clustered within Cluster A. On the other hand, the OTUs corresponding to hybrid taxa that are clustered in Cluster B, progressively separates from the main cluster, showing a very little distance between each separation and is mixed with the Nymphaea × erangae, the violet flowered parent OTUs.
The first four (principal) eigen values recovered from the PCoA (5.0423, 0.41488, 0.30782 and 0.11717) accounted for 76.3073% of the total variance (65.412%, 5.382%, 3.9933%, and 1.52%, respectively). A plot of the first and second coordinates (which provided the greatest separation of OTUs) resulted in a separation similar to that obtained by the Cluster Analysis. Here, the PCoA also resolved two discrete clusters (Figure 4), with each corresponding exactly to one of the phenetic group clusters indicated by the UPGMA dendrogram while the OTUs corresponding to the tentative hybrids overlapped with the main cluster. Results of both analyses support the separation of the two parents and the inclusion of the putative hybrids within the parent clusters showing the resemblance of the Nymphaea populations with intermediate characters, to parental populations. This provides evidence for the hybrid origin of the Nymphaea populations with intermediate characters as a result of natural hybridization between the native N. nouchali and the exotic violet flowered Nymphaea × erangae.
According to the Principle Component Analysis (PCA) PC 1 loading the separation of the main groups were supported mainly by quantitative characters such as the number of stamens (0.9829), stigmatic segments (0.08871), and petals (0.08291) and, flower circumference (0.0709) and leaf size (0.05713) ( Figure 5). The PCA loadings of the highly contributed characters are given in Table 2.

DISCUSSION
Confirming the identity of the populations with intermediate characters as hybrids between the native N. nouchali and the violet flowered exotic Nymphaea × erangae leads to many consequences regarding the 'genetic purity' and the conservation of the native water lily. The geographic ranges of the two species overlap and in some localities they are sympatric. The study identified hybrid populations distributed in wet zone as well as intermediate zone of the country. Of the three recorded Nymphaea species in the country (Dassanayake1996 and Guruge et al., 2016), only N. nouchali is a day bloomer while others, N. pubescens Willd. and N. rubra Roxb. ex Andrews are night bloomers. Since the exotic Nymphaea × erangae is also a day bloomer the natural hybridization between the two plants is favored in the natural environment. The natural hybridization in Nymphaea is a common phenomenon (Huxel et al., 1999) and a large number of artificial hybrids have been developed as ornamentals. Nierbauer et al. (2014), in a similar study addresses threats posed by whiteflowered Nymphaea hybrids that have escaped from cultivation in Germany. These white flowered Nymphaea hybrids now grow in the same habitats as the native water lily, N. alba in Germany and the worse situation is that the native cannot be identified from the hybrids by the morphology alone and therefore pose a threat for the conservation of the native, which is considered endangered. The hybrid populations identified during the study were mostly concentrated in the lowland wet zone, and the reason could be that the water in these wetlands does not dry off completely, while the hybrid populations encountered during the study period in the dry zone have disappeared. The long dry spells in the lowland dry zone during certain years could contribute for this. Further, these dry spells also affect the pure populations of the native in the lowland dry zone.
Nymphaea nouchali, even though secured an important position during the past in cultural and social activities as well as in legendary history in Sri Lanka, the situation has gradually changed with the introduction of the exotic violet flowered water lily and its spread in local water bodies. Even though there are no exact records of the introduction of this violet flowered Nymphaea, Nymphaea × erangae, this could have happened well over half a century as at the time of the declaration of the national flower of Sri Lanka in 1986 the plant has been abundant in nature and embedded within the hearts of the people of the country. At present, both the position and status of the native is acquired by its imposter, Nymphaea × erangae ('Dam manel') due to the erroneous recognition of this species as N. nouchali ('Nil manel').
The native N. nouchali is listed under vulnerable category during the recent Red-listing (MOE, 2012). Considering the threat through hybridization, population sizes and the vulnerability of the habitats they occur, it is imperative to pay attention on the conservation of this native water lily in its natural environment.
The study also highlights the negative aspects of the introduction of exotic ornamental plants in to the ornamental plant industry, especially aquatics where once escaped from the controlled environment, they could possess detrimental damage to the local biodiversity. Transport of plants as ornamentals across the globe is increasing and this contributes for the introduction of aliens to new destinations. According to the prevailing statistics for the ornamental aquatic plant industry, of the 389 species that are currently on trade in Sri Lanka, 62% is occupied by exotic plants (Yakandawala et al., 2013). Most of these plants have their related counterparts in Sri Lanka occurring in the natural environment. This could lead to similar situation reported in this paper; where once escaped from the controlled environment, these exotics get naturalized, leading to hybridization with the natives. According to research, carried out by Elton (1958) and Donlan et al. (2003) IAS possess an intense ecological threat over island flora.

CONCLUSION
The study has confirmed the occurrence of natural hybridization between the native N. nouchali and the violet flowered exotic Nymphaea × erangae in the wetlands of Sri Lanka. As Nymphaea × erangae has invaded the natural wetlands and the habitats of N. nouchali silently, the natural hybridization would pose a threat to the 'genetic purity' of the native species.