Evolution and Biodiversity of New Caledonian Diospyros

New Caledonia is one of the 34 biodiversity hotspots defined by Conservation International (Mittermeier et al., 2005), and the flora has a high level of endemism. Of the 500 species of Diospyros 32 grow in New Caledonia and all except one are endemic to the territory. Twenty-one native species were included in a previous investigation that showed four species form a clade with Australian sister species, indicating a Gondwana lineage; the others formed a clade with Asian taxa, indicating long distance dispersal. We hypothesize that speciation in New Caledonian Diospyros has been mainly due to ecological shifts (edaphic and climatic, shifts in flowering time, pollinators and dispersal mechanisms). This may have been accompanied by polyploidization and structural rearrangements of the chromosomes if any are observed in New Caledonian species. In the present proposal we plan to get the species boundaries for closely related species of Diospyros using AFLP, sampling a minimum of 10-20 individuals per species collected from five different localities. Clearly defined taxa (OTU, Operational Taxonomic Units) will be tested for their phylogenetic relationships as well as to detect hybridization events, which are suspected in some New Caledonian Diospyros, using DNA sequences of fast evolving plastid regions, nuclear rDNA and low- or single copy nuclear genes such as glutamine synthetase (GS), phytochrome C (PHYC) and malate synthetase (MS). A robust phylogenetic tree will provide an indirect record of the speciation events that have led to present day species. Ecological shifts in speciation will be investigated by making field observations on flowering time, pollinators, seed dispersal as well as edaphic and climatic factors. Chromosome counts will be made for the New Caledonian species to see if polyploidization (allo/autopolyploidy) has contributed to speciation in Diospyros. The impact of ecological speciation and adaptive radiation on structural rearrangements of the chromosomes, leading to reproductive isolation, will be investigated using FISH (using 5S and 45SrDNA). The time of divergence of the New Caledonian Diospyros will be estimated using sequences of the low copy nuclear gene PHYC, with other Diospyros species as well as genera Royena, Euclea of Ebenaceae and few species of Ericales (families Theaceae, Ericaceae) where fossil records are available as outgroups for calibration. The fossil record available for the Australian Diospyros will be used as a second calibration point to estimate the time of divergence of the basal-most node of the clade containing New Caledonian Diospyros together with the Australian species and thus verification of the type of endemism. We will investigate the patterns and processes of speciation and genetic diversity within and among these endemics, for evaluating biodiversity and compiling conservation strategies for the New Caledonian Diospyros.

Diospyros forms a large genus of woody flowering plants of the family Ebenaceae. Of 31 Diospyros species found in New Caledonia, 30 are endemic. Previous phylogenetic studies of the genus Diospyros based on sequences of plastid markers, showed the New Caledonian species to form three groups. Two of these groups contain only few species (2 to 5), and the majority of species (24) forms the third group. In our first study based on sequence data we observed the species of group 3 are closely related. Diospyros vieillardii is clearly shown to be sister to the rest of this group and individuals of only four species formed unique groups. The morphological species concept, is supported by our AFLP (Amplified Fragment Length Polymorphisms) results. However, with AFLP data we were not able to elucidate the phylogenetic relationships between these closely related species. Species delimitations inferred from next generation sequencing technique RAD (Restriction site associated DNA) are comparable to those obtained from AFLP data. Phylogenetic trees based on thousands of RAD-derived SNPs (single nucleotide polymorphism) are much better resolved than those based on Sanger sequencing of nuclear and plastid markers. Most of the 21 included species formed monophyletic groups in AFLP and RAD analyses. The observed phylogenetic relationships do not follow an ecological structure, pointing to a role of environmental heterogeneity of New Caledonia in shaping speciation events in this group. Functional annotations of genomic regions consistently exhibiting high differentiation between pairs of sister species occurring on different substrates (e.g. D. flavocarpa D. umbrosa, D. labillardierei D. trisulca) pointed to genes involved in binding and transporting compounds to/through the cell membrane. Species from group 3 revealed nearly 3-fold larger genome sizes compared to Diospyros species from other groups. Chromosome counts showed no indication of polyploidy in this group. Whole genome sequencing using next generation sequencing techniques showed that the species with larger genomes generally contain more copies of repeated elements such as LTR/gypsy elements, without a significant enrichment for a particular element type. Beside the repeated elements we were able to obtain plastid sequences and reads corresponding to the nuclear ribosomal DNA (nrDNA), from the low-coverage whole genome sequencing. These reads could be assembled to whole plastid genomes and nearly complete nrDNA regions. The obtained plastomes were compared to the plastid sequence of Camellia sinensis instructural organization and gene content. Dating analyses based on DNA sequence and RAD data showed that the crown group 3 is around seven million years old and the group with low statistical support in the RAD based analysis to be around four million years. Diospyros are woody plants with long generation time, thus not more than 500,000 generations passed since the most recent common ancestor of the latter Diospyros group. The low number of generations after the original long distance dispersal event, together with the rapid radiation across different habitats can explain the presence of the low genetic divergence in this group.