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

Abstract

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.

Figure 1

ML topology of Nymphaeales from the aligned concatenated super matrix dataset using PhyML 3.0. Phylogeny reconstruction of the Order Nymphaeales based on concatenated dataset of two different loci (ITS2+ matK) using a taxon set of 51 taxa (including three outgroup taxa, Cycas revoluta, Cycas siamensis and Ginkgo biloba) with aLRT values (best ML tree, majority rule, aLRT values similar to 100 bootstrap replicates)

Figure 2

Bayesian Phylogram (majority rule consensus tree) inferred from the aligned supermatrix dataset (ITS2 + matk). Nymphaeale phylogeny reconstruction using sequence evolution model using GTR with 10 million generations, sample frequency, 1000, burn-in: 10% discarded in MrBayes 3.2. The third family Hydatellaceae represented by the genus Trithuria sps formed a sister basal lineage to Cabombaceae and Nymphaeaceae and clustered with the outgroup (Cycas and Ginkgo).

Figure 3

Median-joining network graphs with uncorrected p distances inferred with Splitstree version 4.10 from the supermatrix (ITS2+ matK).

Figure 4

Maximum Parsimonious tree of Nymphaeales using molecular clock test of matK sequence. Molecular clock test performed by comparing the ML value for the given topology with and without the molecular clock constraints under Jukes-Cantor (1969) model (+G). Differences in evolutionary rates among sites modeled using a discrete Gamma (G) distribution. The null hypothesis of equal evolutionary rate throughout the tree was rejected at a 5% significance level (P < 1.20575200719741E-58). The analysis involved 64 nucleotide sequences. Evolutionary analyses were conducted in MEGA 5.

Figure 5

ITS2 Consensus secondary structures of Nymphaeales with color legend using RNAz and LocaRNA. Validation of conserved ITS2 secondary structures across the three Nymphaeale families (Cabombaceae, Nymphaeaceae and Hydatellaceae). The three families are represented by the genera A. Brasenia, B. Cabomba, C. Euryale, D. Nuphur, E. Nymphaea F. Victoria G. Trithuria. Standard nucleotide ambiguity codes are used.

Figure 6

Profile Neigbour Joining (PNJ) tree from primary sequence- secondary structure alignment of Nymphaeale ITS2 data using 4SALE and ProfDistS. Simple correction Jukes and Cantor formula (Jukes and Cantor, 1969) operated on sequence-structure alignments. Based on the GTR RNA sequence-structure specific substitution model evolutionary distances between sequence-structure pairs are estimated by maximum likelihood and are also extended on the profile level. The group Hydatellaceae clustered with Cabombaceae and emerged as a sister clade to Nymphaeaceae. Consensus bootstrap values with 100 replicates are shown next to branches and ProfDistS output tree file viewed in NJplot. Tree viewing Profiles are marked by "Pi" (profile generated by identity threshold), "Pb" (profiles generated by bootstrap threshold) and "Po" (old profile generated in a previous iteration).