An ecogeographic analysis of Oryza series Sativae in Asia and the Pacific

The non-cultivated speciesof the genus Oryza can provide a genetic arsenal of useful traits for improving the widely cultivated and consumed Asian rice (O. sativa). The diversity of these valuable plant resources must be well understood to ensure their effective in- and ex-situ conservation. In this thesis, we examined the ecogeographic variations within and between the three species of Oryza series Sativae in Asia and the Pacific. We looked at species differentiation from different spatial scales by analysing sympatric accession pairs of O. meridionalis and O. rufipogon and of O. nivara and O. rufipogon. We conducted phenotypic analyses in Chapter 2. The strong influence of ecology on species morphology was demonstrated in the ordination and cluster analyses results where O. meridionalis and O. nivara grouped together and were separated from O. rufipogon. We detected greater differentiation of O. nivara and O. rufipogon in South Asia and positive correlations between spatial and intraspecific (interpopulation) morphological distances in continental Asia. We found significant correlations between geoclimatic factors and certain character measurements within species and observed that seedling height, culm number and diameter, leaf size, and anther length exhibit contrasting responses for O. nivara and O. rufipogon. We confirmed significant morphological differences between the three species, between the South and Southeast Asian populations of O. nivara, and between the Australasian and the non-Australasian populations of O. rufipogon and provided botanical descriptions to delineate O. meridionalis, O. nivara and O. rufipogon morphologically. In Chapter 3, we genotyped the same set of accessions with 29 SSR markers and applied a variety of methods for genetic diversity analysis. Based on ordination and phylogenetic results, we verified that O. meridionalis is a genetically distinct species and that O. nivara and O. rufipogon overlap genetically across their geographic distribution. However, Bayesian clustering analysis recognized local-scale species separation of O. nivara and O. rufipogon implying stronger interspecific gene flow barriers in smaller spatial units. Concurrently, AMOVA indicated that the bulk (64%) of genetic variation in Asia Pacific series Sativae can be found among accessions and the lesser portions within accessions (26%) and among species (10%). We captured contrasting intraspecific variation patterns for O. nivara and O. rufipogon where the former exhibited low diversity, high population differentiation and isolation by distance mainly in South Asia while the latter displayed high diversity, low population differentiation and isolation by distance primarily in continental Southeast Asia. We established that altitude is correlated negatively to accession diversity and positively to local-scale species differentiation. Using Bayesian inference, we identified eight genetically distinct population groups: C1) Indian and Bangladeshi O. nivara; C2) Cambodian O. nivara; C3) Southeast Asian O. rufipogon; C4) O. meridionalis; C5) Nepalese O. nivara; C6) non-Cambodian Southeast Asian O. nivara; C7) Australasian O. rufipogon; and C8) South Asian O. rufipogon. Cluster analysis grouped the aromatic and japonica cultivar groups of O. sativa with O. rufipogon in South Asia and the indica and aus groups with O. nivara from Thailand and Cambodia, respectively. O. nivara from Nepal seemed genetically isolated from the other population groups. We also detected variation patterns that agreed with the results in Chapter 1 such as the South and Southeast Asian divisions of O. nivara, the divergence of Australasian populations from the rest of O. rufipogon and the greater differentiation of O. nivara and O. rufipogon in South Asia. In Chapter 4, we conducted artificial crossing experiments to 15 selected parental accessions of O. meridionalis, O. nivara, and O. rufipogon and assessed the extent of several post-pollination isolating mechanisms in Oryza series Sativae. We observed reproductive incompatibility within and between the inbreeding species O. meridionalis and O. nivara and high intraspecific crossability of the outcrossing O. rufipogon where viable and non-sterile F1 hybrids were produced only by combinations with a parental distance that ranged from 1062 to 3813 kilometers. Insular Southeast Asian and/or Australasian accessions of O. rufipogon were the most reproductively successful parents. O. rufipogon exhibited significant pre-zygotic species isolation (in terms of seed set) and reduced post-zygotic isolation, and seemed symmetrically compatible with O. nivara and asymmetrically compatible with O. meridionalis. We obtained few annual hybrids with relatively high fertilities from crosses between O. rufipogon and O. nivara and numerous perennial hybrids with low fertilities from crosses between O. rufipogon and O. meridionalis. Crossability estimates did not show significant correlations with geographic distance between parents. However, we discerned reduced seed set and F1 fertility in interspecific combinations with sympatric parents compared to crosses with non-sympatric parents, indicative of reinforced species isolation in sympatry. We evaluated the F1 offspring of different cross combinations and found a mixture of intermediate and parental character traits in interspecific hybrids. We discussed the taxonomic implications of the research results in Chapter 5 where we specifically dealt with the opposing views of lumping or splitting of O. nivara and O. rufipogon. We concluded that these two taxadeserve to be treated as separate species based on the following biosystematic evidence obtained from the thesis: 1) ecological distinction; 2) considerable prezygotic barriers; 3) opposing patterns of gene flow and genetic variation; 4) local-scale genetic divergence and 5) enhanced reproductive barriers under sympatric conditions. We identified ecogeography as a major driving force in the diversification of Oryza series Sativae in Asia and the Pacific and suggested that ecological speciation gave rise to O. nivara and O. rufipogon. We also presented recognizable geographic races within species. Ultimately in Chapter 6, we emphasized the importance of our study in several aspects of rice science and identified results that agreed with prior Oryza diversity studies. At the same time, we presented previously unreported morphological and genetic variation patterns that were established in this thesis. We discussed the possible applications of the research results to wild rice conservation, covering in situ strategies as well as gene bank practices. We also highlighted the potential role of O. nivara in Asian rice domestication where it could have either directly given rise to the indica cultivar group or hybridized/introgressed with migrated japonica cultivars in India, eventually leading to the development of indica.