The apomictic Ranunculus cassubicus complex became a model system for the origin of asexual seed reproduction when Paun, Stuessy and Hörandl (2006) inferred, from AFLP and microsatellite data on 448 individuals, a unique hybrid origin of the apomictic hexaploid R. carpaticola from the sexual autotetraploid R. cassubicifolius and diploid R. carpaticola, with unique alleles arising from genomic reorganization that might have triggered apomixis, and tentative ages linking the hybridization events to the last glacial period. The paper left its causal and temporal claims tentative, the possibility of unsampled parental contributions or later introgression remained open and was documented as such by the group's subsequent work, and whether hybridization plus polyploidy is a general pathway to apomixis became a further comparative question pursued across the wider R. auricomus complex. Experimental hybridization has since shown that hybridization alone can trigger spontaneous, dosage-dependent apospory within two generations, and transcriptomics has nominated candidate meiosis- and sporogenesis genes (ASY1, APC1, MSP1, XRI1), but the causal molecular changes and their stable inheritance remain unconfirmed. Genome-wide phylogenomics supports predominantly allopolyploid origins with substantial post-origin evolution, yet each genetic group still involves an unknown progenitor species, and the age of the asexual lineages remains without independent calibration. The problem - a causal, ancestral and temporal account of how these apomictic lineages originated and established - remains open.

Background

Apomixis in flowering plants is asexual reproduction via seeds: embryos develop without meiotic reduction and without fertilization of the egg, so offspring are clonal copies of the maternal genotype. In the gametophytic form called apospory, which occurs in the buttercup genus Ranunculus, a somatic cell of the ovule's nucellus (the aposporous initial cell) develops into an unreduced embryo sac, bypassing meiosis (apomeiosis); the unreduced egg cell then develops into an embryo without fertilization (parthenogenesis), while the endosperm, the nourishing tissue of the seed, usually still requires fertilization (pseudogamy). Apomixis therefore requires several concerted alterations of the sexual developmental program. Across angiosperms, natural apomicts are almost always polyploids (organisms with more than two complete chromosome sets) and frequently hybrids, and it has long been suspected that hybridization allows apomixis to escape hybrid sterility; an influential version of this idea holds that combining divergent parental genomes deregulates the timing and spatial patterning of reproductive-gene expression, which in turn unlocks the asexual pathway (this and related hypotheses are reviewed in the works cited below).

The Ranunculus auricomus complex (goldilocks buttercups, Ranunculaceae) comprises a handful of diploid and tetraploid sexual species and several hundred polyploid apomictic microspecies (agamospecies) distributed across Europe and adjacent Asia. It is a standard model for hybrid speciation via apomixis and for geographical parthenogenesis, the tendency of asexual organisms to occupy larger and more northern distribution areas than their sexual relatives (Hörandl 2006). Within the complex, the R. cassubicus group is distinguished by floral morphology and contains the sexual diploid R. carpaticola of central Slovakia, the sexual (auto)tetraploid R. cassubicifolius of the northern pre-Alps (an autopolyploid: a polyploid whose extra chromosome sets derive from the same species), and hexaploid apomictic populations morphologically referable to R. carpaticola.

Paun, Stuessy and Hörandl (2006) combined AFLP fingerprints (a dominant, multi-locus DNA-marker technique) with microsatellite genotypes (simple sequence repeats, SSRs - codominant markers whose stepwise mutations can be modelled) for 448 individuals, together with morphology, isozymes (variant forms of the same enzyme, distinguishable by protein electrophoresis), karyology (chromosome numbers and morphology) and distribution data. They inferred a unique hybrid origin of the apomictic hexaploids from the two sexual parents, R. cassubicifolius and diploid R. carpaticola. The hexaploids were genetically intermediate but carried unique alleles attributed to genomic reorganization in the allopolyploids (polyploids combining chromosome sets from different species), which the authors suggested might also have triggered apomixis. Distribution patterns and age estimates based on microsatellite mutation models suggested that the hybridization events may correlate with the last glacial period. Earlier population-genetic work had shown that the hexaploid populations behave as a limited number of widespread clones with residual sexuality (Paun et al. 2006, Molecular Ecology), and classical crossing work in the complex had established the genetic machinery of apospory as a heritable, dosage-sensitive trait.

The tentative conclusions of the 2006 paper, together with the subsequent literature, framed two decades of research on this complex around the following open questions: (1) the causal mechanistic link between hybridization/polyploidization and the developmental switch to apomictic reproduction is unresolved - which molecular changes (gene loss, silencing, epigenetic alteration, transposable-element activation) underlie the expression of apomixis in these hybrids is not established; (2) additional, unsampled parental contributions or subsequent introgression from other apomictic microspecies remain possible, since a minority of marker fragments unique to the hexaploids are shared with other apomicts, and more conserved-marker studies were needed to confirm or reject complex hybrid/introgression scenarios; (3) the age estimates for the asexual lineages are tentative and span orders of magnitude because they rest on uncertain microsatellite mutation rates and generation-time assumptions, so more precise molecular-clock calibrations or independent dating approaches are needed to test the correlation with the last glacial period; and (4) as a comparative background question beyond this complex, the generality of the inferred pathway - hybridization plus polyploidy followed by establishment via apomixis - across the wider R. auricomus complex and in other plant groups remained to be tested with dense, genome-wide and cytotypically informed sampling. Subsequent work made the system experimentally tractable: synthetic F1 and F2 hybrids between the sexual progenitor species now exist in cultivation, and reduced-representation genome sequencing, target capture of hundreds of nuclear genes, and a chromosome-scale reference genome of the progenitor species R. cassubicifolius have become available, so these questions are now addressable with tools far beyond the AFLP/SSR toolkit of 2006.

Problem Statement

The problem is to establish a causal, ancestral and temporal account of the origin of the apomictic Ranunculus cassubicus complex. For this complex, the account sought is: (1) Mechanism: identify the specific molecular changes that causally underlie the developmental switch to apomixis in the hybrids - which gene mutations or losses, gene silencing or misregulation, epigenetic alterations or transposable-element activations make aposporous initial cells replace the meiotic lineage, how these changes are coupled to parthenogenesis and functional pseudogamous endosperm formation, and how they are stably inherited across generations - and demonstrate their necessity or sufficiency by functional or segregation evidence, not merely by association with the hybrid or polyploid state. (2) Ancestry: resolve the full reticulate ancestry of the polyploid apomicts - determine whether the identified sexual progenitors completely account for apomict genomes or whether additional, unsampled parental contributions or subsequent introgression from other apomictic microspecies must be invoked, and, if necessary, identify the unknown progenitor lineages implicated. (3) Age: establish robust, independently calibrated age estimates for the asexual lineages that do not rest solely on uncertain microsatellite mutation rates and generation-time assumptions, and thereby test whether their origins correlate with the last glacial period as proposed. These three parts serve one overarching objective: a causal, ancestral and temporal account of how, when, from which ancestry, and by which molecular mechanism the asexual seed-reproducing lineages of this complex arose and established. The generality of the hybridization-plus-polyploidy pathway across the wider R. auricomus complex and in other plant groups is background context (see Background), not part of the account sought here.

References

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