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Background  The Nymphaeales (waterlilly and relatives) lineage has diverged as the second branch of basal angiosperms and comprises of two families: Cabombaceae and Nymphaceae. The classification of Nymphaeales and phylogeny within the flowering plants are quite intriguing as several systems (Thorne system, Dahlgren system, Cronquist system, Takhtajan system and APG III system (Angiosperm Phylogeny Group III system) have attempted to redefine the Nymphaeales taxonomy. There have been also fossil records consisting especially of seeds, pollen, stems, leaves and flowers as early as the lower Cretaceous. Here we present an  in silico  study of the order Nymphaeales taking maturaseK ( matK ) and internal transcribed spacer (ITS2) as biomarkers for phylogeny reconstruction (using character-based methods and Bayesian approach) and identification of motifs for DNA barcoding.    Results  The Maximum Likelihood (ML) and Bayesian approach yielded congruent fully resolved and well-supported trees using a concatenated (ITS2+ matK) supermatrix aligned dataset. The taxon sampling corroborates the monophyly of Cabombaceae.  Nuphar  emerges as a monophyletic clade in the family Nymphaeaceae while there are slight discrepancies in the monophyletic nature of the genera  Nymphaea  owing to  Victoria-Euryale  and  Ondinea  grouping in the same node of Nymphaeaceae. ITS2 secondary structures alignment corroborate the primary sequence analysis. Hydatellaceae emerged as a sister clade to Nymphaeaceae and had a basal lineage amongst the water lilly clades. Species from  Cycas  and  Ginkgo  were taken as outgroups and were rooted in the overall tree topology from various methods.    Conclusions   MatK  genes are fast evolving highly variant regions of plant chloroplast DNA that can serve as potential biomarkers for DNA barcoding and also in generating primers for angiosperms with identification of unique motif regions. We have reported unique genus specific motif regions in the Order Nymphaeles from  matK  dataset which can be further validated for barcoding and designing of PCR primers. Our analysis using a novel approach of sequence-structure alignment and phylogenetic reconstruction using molecular morphometrics congrue with the current placement of Hydatellaceae within the early-divergent angiosperm order Nymphaeales. The results underscore the fact that more diverse genera, if not fully resolved to be monophyletic, should be represented by all major lineages.

bmc bioinformatics

Phylogenetic reconstruction in the Order Nymphaeales: ITS2 secondary structure analysis and in silico testing of maturase k (matK) as a potential marker for DNA bar coding