FBIP: One baobab species or two-morphological and molecular data to address this question

Study Extent

Measurements taken from herbarium specimens housed in various herbaria. Sequence data from material collected in South Africa, Venda, Namibia and Mozambique.

Sampling

Several approaches will be used to rigorously establish the presence of a second baobab species and to test the hypothesis that ‘poor-producer’ trees in Limpopo are triploids resulting from hybridization between the two forms or species of baobab.

Method steps

1. 1. Testing the hypothesis that there are two species of Adansonia in Africa: Sampling: Extensive sampling of baobab populations across southern Africa will be conducted, as well as samples from across the rest of Africa. In southern Africa, extensive sampling of Limpopo populations in Venda and the Kruger National Park has/will be done in collaboration with Dr. Venter and Mr. Rob Taylor respectively. Field trips to Namibia, Botswana, Zimbabwe, and Mozambique will be undertaken and/or local botanists will be contacted to assist with collection of samples. Given the difficulties in reaching locations further afield, we will collaborate with Dr. Jens Gebauer (Germany) and local African scientists to use previously collected specimens/collect samples for us. Field trips will be conducted in December 2013–March 2014 to collect fresh leaf tissue samples and voucher specimens, flowering where possible. Leaf tissue will be dried in silica gel for DNA analyses. Leaf tissue to be used for stomatal measurements will be frozen and pressed to ensure the integrity of the stomata. Voucher specimens will be deposited at J and relevant national herbaria. Necessary permits will be obtained prior to collection. Morphological measurements: Floral traits will be measured in the field where possible, or from voucher specimens collected for that purpose. Pollen grain diameter, androecium length and stalk diameter, maximum calyx diameter, and staminal corolla width will be measured and the number of free staminal filaments will be counted. In addition, stomatal counts and length and width measurements of stomata will be made under a compound microscope following Saltonstall et al. (2005). This will be completed by Oct 2014. Ploidy levels: Via chromosome counts and flow cytometry (details given below). To be completed by Oct 2014. DNA Sequence data and barcoding: A representative number of individuals of different ploidy will be sequenced and included in a phylogenetic analysis to establish the relationship of baobabs. Genbank data will be used for the other species of baobab from Madagascar (6 species) and Australia (1 species). The regions chosen will include those previously sequenced (nuclear ITS and plastid trnL-trnF regions) as well as additional regions useful for DNA barcoding (e.g. matK). Sequencing and analyses completed by Dec 2014. 2. Testing the hypothesis that poor-producers are triploid due to hybridization between diploid and tetraploid forms Sampling: Intensive sampling of previously well-studied populations in Venda has been done in collaboration with Dr. Venter in order to investigate ploidy and related features of producer versus poor-producer trees. Thirty samples from three villages and wild populations have been collected, and their productivity measured over the last 5 years. The type of A. kilima was included in the sampling effort. Ploidy Estimation: Seeds will be germinated and root tips of seedlings macerated and stained with propionic acid-ethanol-carmine using the method of Snow (1963). Chromosomes will be counted and photographed under 100x magnification using a compound microscope. Flow cytometry will be used to quantify the DNA content of cells and thereby confirm ploidy levels of the various samples via fluorescence of the stained nuclei of leaf tissue. This aspect of the study will be completed by Oct 2013. AFLP/Microsatellite data: Amplified fragment length polymorphisms (AFLPs) and microsatellites will be used to examine hybridization patterns between ‘producers’ and ‘non-producers’ and between ploidies. Amplified fragment length polymorphism (AFLP) profiles will be generated using a modified protocol6 and microsatellite primers will be developed using available protocols7 to obtain additional markers for species identification. These data will be visualized using an ABI Genetic Analyzer. Combined, these markers provide a comprehensive picture of hybridization - AFLPs cover a large portion of a non-model organism’s genome, whereas microsatellites provide detailed descriptions of parental contributions to putative hybrid progeny given their co-dominant nature. These data will be analysed using appropriate software. Microsatellite markers will be collected in collaboration with Dr. Jens Gebauer (Germany) who has successfully developed markers for baobabs. To be completed by Oct 2013. The species level investigation study will be offered initially as an MSc (2014–15), supervised by Prof. G. Cron, Dr. K. Glennon & Prof. E. Witkowski at the University of the Witwatersrand. The ploidy level study of poor producers will be done by MSc student Mr. Ronie Tivakudze from June to Nov. 2013 in collaboration with Dr. Venter, supervised by the same team at Wits. In both projects, Prof. Witkowski will be responsible for population level analyses, statistical analyses and supervision, Prof. Cron for supervising and assisting with morphological analyses and DNA sequencing, Dr. Glennon for supervision of determination of ploidy levels and AFLP/microsatellite data collection.

Boabab identified to species

1. Adansonia digitata
   common name: Baobab rank: species

Namibia (Caprivi Strip); South Africa (Venda) and Mozambique (Quelilea Island); Botswana; Kenya; Burkina Faso; South Somalia; Tanzania; Zimbabwe; Republic of Congo; Comoros Island; Togo; Hawaii; West Indies; Haitii; Kauai; Ivory Coast; Malawi; Ivory Coast; Oman; Zambia; Mauritius; Sri Lanka; Sierra Leone; Nigeria; Sudan; Senega