Molecular Characterization and DNA Barcoding of Arid-Land Species of Family Fabaceae in Nigeria

Author: Oshingboye, Aramide Dolapo

Supervisor: Oluwatoyin Temitayo Ogundipe

As a direct consequence of the appreciable threats confronting drylands, many arid-land species are lost with meagre information and research on their diversity, species composition, characteristics and endemism. It has become necessary to understand, describe and document species relationships and characterize the diversity of dryland species so as to help provide baseline information for efficient conservation strategies and sustain the livelihood of over one billion indigent inhabitants. A total of 324 leaf samples representing 118 species collected randomly across the arid and semi-arid regions of Nigeria were used for both morphological and molecular studies. Morphological features were recorded and used to develop keys for identification and to reveal species relationships. Molecular characterization involved isolation of total genomic DNA according to a modified CTAB protocol, amplification of four selected gene regions and sequencing. Raw sequenced data were contigned, edited and aligned, to be used for both DNA Barcoding and Phylogenetic analysis. Red-listing of species were performed according to IUCN categories and criteria. Samples comprised 22 Mimosoideae, 38 Caesalpinioideae and 58 Papilionoideae species; based on habit, members are either trees, shrubs, climbers or herbs, which are somewhat distinct in each sub-family. Morphological features recorded were mostly compound leaves (paripinnate, imparipinnate, pinnate, bipinnate, trifoliate and pinnate-trifoliate in some Papilionoideae species); flowers are zygomorphic in both Caesalpinioideae and Papilionoideae while actinomorphic in Mimosoideae. Fourty-seven characters and a hundred and eighty-four character states were recorded and used to develop a bracketed key and an electronic multi access key using Lucid 3.3 programme. Pair-wise analysis based on unweighted algorithm depicted various relationships and clustering of species taxonomically. For molecular studies, total genomic DNA isolated were predominantly high molecular weight DNA of 10,000bp and of good quality with A260/280 ratio within 1.75 – 2.01. The barcoding performance of 10 regions comprising three (3) cpDNA regions (rbcL, matK, trnL-F), one (1) nuclear region (ITS), six (6) combinations of regions (matK+rbcL, ITS+matK, ITS+rbcL, ITS+matK+rbcL+trnL-F, matK+trnL and rbcL+trnL-F) revealed the Internal Transcribed Spacer (ITS), exhibited significantly the highest inter-specific discrimination higher than matK and trnL-F while rbcL was the least. Wilcoxon signed rank tests further confirmed that ITS region exhibited the highest intra and inter-specific divergence between congeneric species, whereas rbcL exhibited the lowest. ITS region again exhibited the highest barcoding gap of 2.00, ITS+matK, ITS+rbcL, ITS+matK+rbcL+trnL-F, barcoding gap of 1.00 while other regions revealed no barcoding gap. This implies ITS region can either be used singly or in combination with either matK or rbcL as a potential plant barcode marker. Phylogenetic analysis based on three data matrices (ITS, rbcL+matK+trnL-F and ITS+rbcL+matK+trnL-F) support the monophyly of the family Fabaceae but depict two different topologies. Parsimony results based on ITS sequence data could not clearly resolve relationships among species; it inferred a monophyletic Fabaceae with Papilionoideae nested within a paraphyletic Caesalpinioideae while Mimosoideae is polyphyletic. The parsimony analysis of combined chloroplast regions and all combined regions produced a monophyletic family tree with a monophyletic Papilionoideae and Mimosoideae nested within a paraphyletic Caesalpinioideae; but members of the tribe Detarieae were the basal-most clade instead of Cercidieae. However, Bayesian analysis presented members of Cercidieae as the basal-most clade. IUCN red-listing of species revealed one, two, three and six species in the “Critically Endangered”, “Endangered”, “Vulnerable” and “Least Concerned” categories respectively. This study has explored and characterized the diversity of Nigerian arid land legumes while contributing to the ongoing project of re-constructing the phylogeny of Fabaceae and towards the quest of achieving the United Nation Strategic goals for Biodiversity 2011 - 2020.