Hyphaene – Material and methods

International scientific team

Previous studies of the genus Hyphaene (i.e. Beccari, 1924; Tuley, 1995) have failed to incorporate local scientific partners, which in turn strongly hindered the incorporation of critical local knowledge (locally-recognized varieties, morphological variability and distribution, ecology, ethnobotanical uses, etc.). We have established an international scientific network of Swiss, European and African partners that will allow us to tackle the main objectives of the project. The scientific network includes:

a) The Conservatory and Botanical Garden of Geneva (CJB), including the collaboration of Dr. Yamama Naciri, specialist on population genetics, Dr. Didier Roguet (Ethnobotanist) and Dr. Mathieu Perret, specialist on phylogenetics and phylogeography of Angiosperms, both at the Laboratory of Phylogeny and Molecular Genetics;

b) European partners, particularly Dr. John Dransfield and Dr. William Baker (Royal Botanic Gardens, Kew) and Dr. Frédérique Aberlenc (Head of the Laboratory of Palm Development and Evolution, French Institute for Research and Development, IRD -Montpellier);

c) The African-based network, critical for our project, relying on close partnerships with local institutions. Several African scientific partners have confirmed their interest in the project and have agreed to collaborate in shared field work. In West Africa: Prof. Patrick Ekpe (University of Accra, Ghana), Dr. Koffi Koudouvo (Faculty of Sciences, University of Lomé, Togo); Dr. Bienvenu Sambou (Institut des Sciences de l'Environnement de l'Université Cheikh Anta Diop de Dakar, Sénégal), Dr. Altiné Traoré (Institut des Eaux et Forêts, Sénégal); Dr. Georges Yameogo (University of Ouagadougou, Burkina Faso); Prof. Afiedegnon Akoegninou (Université d’Abomey-Calavi, Cotonou, Benin), Prof. Zoul-Kifouli Adeoti (Université d’Abomey-Calavi, Cotonou, Benin); Prof. Adama Bakayoko, Mr. Doudjo Ouattara (University Nangui Abrogua, Abidjan. Ivory Coast). In Central Africa: Dr. Thomas Couvreur (Institute of Research and Development - IRD, Yaoundé, Cameroon). In East Africa: Dr. Abdourahman Daher (Institut des sciences de la vie, Centre de Recherche de Djibouti) and Dr. Alain Borgel (Institute of Research and Development - IRD, Nairobi, Kenya).

Field work missions

Wild populations of Hyphaene are widely distributed in dry lowlands of tropical and subtropical Africa, Madagascar, the Red Sea and the coasts of the Gulf of Aqaba (Gulf of Eilat), with outliers in western India. We are planning to prioritize field expeditions to the countries reporting the largest numbers of taxa for the group (Appendix 1) and to countries hosting doubtful, poorly known species. These two expeditions (Kenya-Tanzania, Mozambique-South Africa) will be realized either through personal visits to these countries or by way of the local partnerships we have established. With respect to future field work in India, we will establish contact with local palm experts associated to the Central National Herbarium in Calcutta (Office of Botanical Survey of India), whereas in Sri Lanka preliminary contacts will be established with experts from the Royal Botanic Gardens in Peradeniya. The study of the single, relatively well understood Madagascar species (H. coriacea) will benefit from the current efforts undertaken by Rolf Eiserhardt and William Baker (Royal Botanic gardens, Kew), who have kindly agreed to share DNA samples with us. Additional material required from this species will be available to us from the Montgomery Botanical Center (Florida, USA).
The ebola outbreak that was currently affecting several West African countries will almost certainly preclude collections for the indefinite future in the affected regions. For many reasons, of course, we hope the epidemic will come under control as quickly as possible.  Realistically, however, this is going to require a major effort and will require time. We will thus put aside, for the time being, a planned mission to Liberia.  We have strong collaborations established with several non-affected neighboring countries, and we plan to work in this contingent on developments with the epidemic. We will be following closely announcements from the World Health Organization, and if any change is made in the original schedule for the project it will be discussed with the members of the Lombard Foundation.

Taxonomical and structural biology studies

The macro-mophological study of the species will be based on the analysis of herbarium specimens (including all existing types) deposited in international herbaria such as the Royal Botanic Gardens Kew (K), Herbarium of the University of Leiden (LE), Belgium National Herbarium (BR), Tropical Herbarium of the University of Florence (FT), the Natural History Museum of Paris (P) and the Tropical Research Institute in Lisbon (LISC) (Table 1). It is worth mentioning that Palm specimens, including Hyphaene, are in most cases not sent on loan because of their extreme size and bulk.  In the context of earlier work we have developed a precise protocol for analyzing herbarium and fresh material in other palm genera, which we successfully tested in the field in the frame of our floristic inventories of palms in Ghana, Ivory Coast and Senegal. In addition to existing herbarium specimens and new material resulting from our field collections, we may count as well on samples of H. compressa, H. coriacea and H. thebaica from living collections of the Fairchild Tropical Gardens and the Montgomery Botanical Center, both in Florida and hosting important collections propagated from wild source seed. Floral structures, until known poorly known in Hyphaene and potentially informative from a taxonomic point of view, will be investigated in most taxa using the anatomical techniques employed in the Laboratory of Micro-Morphology of the CJB. The flowers sampled in the field will be directly fixed in a FAA solution, whereas dried material (i.e. extracted from herbarium specimens) will be rehydrated in water and afterwards kept in 70% alcohol. For the macro-morphological analysis flowers will be dissected under a stereomicroscope (Kombistereo, Wild HEERBRUGG/ M3Z - Switzerland).  As part of anatomical investigations, flowers at late bud stage will be selected, then dehydrated and embedded in Kulzer’s Technovit 7100 (2-hydroxyethyl metacrylate [HEMA]) (Igersheim & Cichocki, 1996). The material will be cross and longitudinally sectioned at 5-7 microns using a rotary automatic microtome (Microm HM-355), stained with ruthenium red and toluidine blue, and permanently enclosed in Histomount. Observations and photographs will be carried out with an electronic microscope (NIKON Eclipse 80i) at the Laboratory of Micro-Morphology (CJB) and the Laboratory of Cytology and Vegetal Histology (University of Geneva); the permanent slides will be deposited at Laboratory of Micro-Morphology of the Conservatory and Botanical Garden of Geneva. Additionally, part of the material will be dehydrated, critical-point dried and sputter-coated with gold for Scanning Electron Microscopy (SEM) observations using the Zeiss DSM 940A – Orion 6.60 Imaging System at the Natural History Museum of Geneva or the Faculty of Sciences of the University of Geneva.

Table 1. Major repositories of type and non-type specimens in Hyphaene and main geographical regions represented.

Phylogenetics and population genetics within the genus

The phylogenetic position of Hyphaene in the subfamily Coryphoideae and relationships within this genus will be estimated based on analyzing DNA sequencing from the nuclear and plastid genomes. Silica-dryed plant material suitable for DNA extraction will be collected in the field. Several populations for each species of Hyphaene will be sampled. DNA markers that have been widely used in other studies on Coryphoideae will be used to maximize overlap with existing data. These include the low copy nuclear genes RPB2, PRK, CISP4, CISP5 and the plastid genes matK and trnD-T. Sequenced DNA regions will be aligned using standard bioinformatic tools and protocols. Aligned matrices will be analyzed independently for each marker and in combination following current procedures for multi-gene matrices reconstruction (Nylander et al. 2004). Phylogenetic inference will be performed using Maximum Likelihood and Bayesian Inference analyses. Age of Hyphaene and divergence time between lineages will be estimated using the software BEAST and the existing fossil record for Hyphaene (Pan et al., 2005) or for other Coryphoids.
The higher the differentiation, the smaller the optimal sample size per population is necessary to achieve a correct estimation of this differentiation. Pons & Petit (1995) have demonstrated that, when working with chloroplast markers, it is necessary to sample many populations rather than many individuals per population for an accurate measurement of genetic structure. This is because chloroplast markers show low levels of within-population variation, but high differentiation between populations. In contrast, some nuclear markers such as microsatellites are known to be more variable and to show lower genetic differentiation than chloroplast DNA. Therefore, intensive sampling within populations of Hyphaene is recommended in order to have accurate estimate of allele frequencies. Considering these contradictory trends, we will sample 15-20 individuals per locality and a minimum of 10 localities for the 7-8 species recognized in the genus.
We will use two types of markers to analyze the structure of species concerned: chloroplast spacers and nuclear SNP (Single Nuclear Polymorphism) markers. Different spacers will be used for the chloroplast markers (trnH-psbA, trnL-trnF, atpB-rbcL and trnD-trnY) because they already showed some interspecific divergence (trnL-trnF, atpB-rbcL and trnD-trnY) and even some intraspecific polymorphism (trnH-psbA). Genes such as matK, rpoB or rbcL seem to be too conserved among species. SNPs will be identified using NGS techniques and outsourced to the Institute for Genomic Diversity (www.igd.cornell.edu/page/GBS). At least 20 of the identified polymorphic SNPs will be used to characterize the species diversity and structuration within Hyphaene. All individuals will be genotyped on the Beckman-Coulter automated sequencer of the Phylogeny and Molecular Genetics Unit (CJB). The activities associated with the phylogenetic analysis and the population genetic studies will depend on the quality and quantity of the material sampled.

Phytogeographical and conservation studies

The distribution maps for the genus and all species recognized within Hyphaene will be produced with the R-notch software or Arc-view. Details of these applications are available and permanently updated at www.unil.ch/biomapper/Download/Chatelain-SysGeoPla-2002.pdf. Georeferencing points will be obtained from three main sources: 1) direct observation (GPS data) during fieldwork, 2) data obtained from collections deposited in international herbaria and 3) data from specialized online databases (i.e. Tropicos, GBIF). We have already contacted Dr. Anne Overgaard (Department of Bioscience, University of Aarhus), who has agreed to share with us a very complete database of the African palm collections deposited in herbaria worldwide assembled in the frame of her recent studies (Greve et al., 2011; Overgaard et al., 2013)
Species richness throughout the range of distribution of the genus will be calculated using the Zonation software for spatial conservation prioritization (Moilanen & Kujala, 2008). The conservation status assessment for all species will be evaluated with the model of the Generalized Regression Analysis and Spatial Prediction (GRASP), which optimizes the spatial predictions (Lehmann et al., 2002). The final assessment for each species will be obtained using the IUCN Red List version 3.1 (IUCN, 2001).

Ethnobotanical analysis

The ethnobotanical study (inventory of uses, common names) will be mostly qualitative and based on surveys and structured interviews to be held with local populations (handcraft makers, local artisans, market sellers, etc.) in the regions where Hyphaene species are found and utilized. Specific methods include the classical ethnobotanical approaches described by Bellamy (1993), Martin (1995) Alexiades (1996), Cotton (1996) and Cunningham (2001). Many of these methods were already employed in the frame of a Master’s study on the ethnobotany and structural biology of useful palms in Ivory Coast (da Giau, 2014), field work partially funded by the Lombard Foundation.

Dissemination of results

All the results associated with this project are planned to be disseminate in international publications, congresses and workshops, but efforts will also be made towards the distribution of critical results in publications or meetings of more local interest (including documents and workshops). The achievements associated with the more specific objectives are planned to be presented in at least 4 international and national publications, in different degrees aiming to promote new programs of conservation and domestication or the economically important species of Hyphaene. It is worth to mention that the dissemination of these results will be extremely important to highlight the main achievements issued from our research activities.