Molecular Identification Of Water Lily(Nymphaea)in Africa

Nymphaeales(Water lily and relatives)besides being highly preferred aquatic ornamental herbs have high cultural and economic values.Their position as basal angiosperm qualifies them as a model for studying the origin and the evolution of flowering plants.The importance of water lilies in the horticultural industry and phylogenetic studies emphasize the need for a fast and reliable identification method.Assessment of biodiversity at the species level is the most informative and desired,but requires correct identification.Morphology-based determination has limitations and therefore,the necessity for integrating molecular information.DNA barcoding has proved to be a useful identification tool to overcome this taxonomic challenge and facilitate the conservation of biodiversity in temperate regions.However,its utility in Africa aquatic habitats has rarely been considered.This study aims at investigating the efficiency of DNA barcoding in identifying water lily species in tropical Africa.Water lily species were collected across Africa continent for the specificity of a nuclear-encoded internal transcribed spacer(ITS),the chloroplast non-coding region trnT-trnF and the coding region rpl16.We used the similarity-based method(Blastn search)and phylogenetic method(Maximum Likelihood and Neighbor-Joining trees)to assess the effectiveness of the three barcode regions,both singly and in combination.To enhance the species discrimination,an integrative approach that combined sequence similarity method alongside a priori and a posteriori data obtained from morphology,literature,other molecular markers and geographical distribution of species was also used.This method was introduced to compensate for the high ambiguity incidences in trnT-trnF and rpl16 as well as correctly identify Nymphaea species in Africa lacking sequence data.For the single barcodes:ITS,trnT-trnF and rpl16 each identified seven,five,five species respectively.For the concatenated sequences seven species were determined from the unknown samples while the integrated approach identified ten species.The feasibility of the integrative approach used in this study was tested in identifying one of the two rare yellow-flowered water lily species in Africa without molecular data.The plastid trnT-trnF and rpl16 sequence data were less conclusive.These results are consistent with earlier studies inferring a close relationship of Nymphaea guineensis Schum.&Thonn.,a native of West Africa and Nymphaea minuta K.C.Landon,R.A.Edwards&Nozaic from Eastern Africa.Additionally,the transfer of Nymphaea petersiana Klotzsch.,from Nymphaea subgenus Brachyceras to Nymphaea subgenus Lotos is hereby confirmed.In addition to the three barcodes,concatenated sequences failed to resolve a close relationship between two similar varieties of Nymphaea nouchali Burm.f.,from Uganda.The occurrence of Nymphaea micrantha Guill.&Perr.,Nymphaea lotus L.,Nymphaea capensis Thunb.,Nymphaea caerulea Savigny over great geographical locations as well as Nymphaea heudelotii Planch.,Nymphaea nouchali Burm.f.,and Nymphaea sulphurea Gilg.,in restricted areas serve as the candidate for investigating both historical and contemporary factors influencing their distributional pattern.The use of an integrated approach increased the identification of the unknown samples by 1.43 times.Therefore,this study affirms the suitability of internal transcribed spacer together with an integrative approach to DNA barcoding as a powerful tool in taxonomy and biogeography with great use in identifying biogeographical patterns and resolving classifications issues at the rank of genus and species.This study also expands the global DNA barcode database considerably for the African Nymphaea.