Recombination was only detected in O. rufipogon, so the longest nonrecombining blocks were only utilized in the comparisons including O. rufipogon. Each comparison was run in M-mode with wide value cutoffs for all parameters to determine where posterior probability distributions ranged. After the initial run, three runs were conducted with different random number seeds and smaller cutoff values that were based on the distribution of parameter values from the first run.All runs had 100,000,000 MCMC steps after a burn-in of 100,000 steps. Each run had 10 chains with a mixing rate of five chain swaps per step. All three M-mode outputs were checked for convergence and L-mode runs were conducted on the tree files to test nested models. The maximum likelihood estimates were scaled into demographic values based on a mutation rate of 1 × 10−8 and a generation time of one year, as done with previous work, based on. All IMa runs were computed on the Condor cluster at Clemson University using primarily an extensive web-enabled system to simultaneously manage and monitor performances of each set of input priors. Use of a cluster allowed for more than 28 simultaneous runs, where priors could be checked and adjusted as needed.The goal of these phenotypic analyses was to elucidate genotype-phenotype relationships between California Oryza cultivar and weedy rice ecotypes. Thus, we determined the most influential phenotypic footprints of rapid divergence in domestic and wild-like traits of rice and its conspecific weed within the California floristic province. To characterize trait variability and by extension morphological relationships among domesticated and weedy rice ecotypesin infested fields, grow rack we quantified phenotypic diversity by first describing the variance partitioning of weedy populations and comparing the adaptive traits which characterized weedy rice to those that defined cultivars.
To more accurately characterize dimensionality in weedy or feral rice morphology, a subset of unique gourmet varieties were added to the medium-grain cultivars in the rice dataset for the phenotypic diversity analyses. Qualitative descriptors were transformed using the PRINQUAL procedure of SAS with the OPSCORE option for optimal scoring and MONOTONE option for monotonic preservation of order. Principal Components Analysis with maximum total variance was performed on the combined quantitative and transformed qualitative descriptors. The variables describing cultivars were reduced by eliminating any that did not vary by descriptive statistics and then using both random and a priori sampling to preserve group partitioning while identifying the eigenvectors which most clearly separated groups. UPGMA hierarchical clustering using the CLUSTER procedure of SAS was performed to confirm separation of clusters on PCs and to generate a dendrogram using average Euclidean distances.Average estimates of genetic differentiation between weedy rice in California rice fields are very low, ranging from 0 to 0.0026 . There are no significant differences in FST estimates for any of the 48 loci. The highest FST estimate was 0.077, betweenCRR1 and CRR4 at STS085. These low values indicate no population structure and no divergence of weedy rice in the fields sampled, which supports the appropriateness of a genetic diversity assessment for California weedy rice . Measures of genetic diversity for California weedy rice within each field as well as for weedy rice within all fields combined are also very low , consistent with a recent founder event, or strong population bottleneck.
These values are a full order of magnitude lower than what was calculated for strawhull and blackhull weedy rice ecotypes collected from the southern US. Due to the lack of population substructure and low genetic diversity, we placed all California weedy rice into one group for the remaining analyses. Values for average population differentiation estimates across all 48 loci indicate high divergence between California weedy rice and all other sampled groups. The lowest mean value is with O. rufipogon collected from Southeast Asia . Taking the median values across the 48 loci allows better understanding of the patterns across all loci. The lowest divergence was between California SHA weedy rice and BHA and SH; median ФST values indicated that for at least half of the loci tested divergence was an order of magnitude lower than the mean estimates . This indicates that the mean ФST is high due to divergence at a few loci, and that California weedy rice does share some similarity to weedy rice from the southern US at several loci. The most recent divergence of California weedy rice from other Oryzasis from California rice cultivars , which was estimated at about 118 generations ago . The other divergence estimates were over an order of magnitude older . Interestingly, both SH and BHA weedy rice from the southern US have very old divergence estimates: approximately 30,000 and 17,000 , respectively. These numbers are likely inflated compared to the reported origin of domesticated rice approximately 10,000 generations ago due to interactions among other closely related genotypes. This follows work examining model testing performance of IMa under several scenarios,which showed that divergence estimates inflate when gene flow from other populations is included in the model. A model of relative divergence times shows a shallow, recent coalescence of California weedy rice and California crop rice alleles, whereas SH and BHA southern US weedy rice and Chinese O. rufipogon show a much older divergence from California weedy rice .
Migration estimates between California weedy rice and all other groups were quite low, with higher estimates of migration into California weedy rice in all cases. Indeed, these data should not be interpreted as absolute numbers but instead as relative values. Any overestimation of the generation values could otherwise indicate that the divergence actually happened even more recently. The effective population size for California weedy rice is very small in all cases,supporting a recent founder event or bottleneck. California weedy rice has a straw-colored hull with long awns , whereas only 7%of SH weedy rice in the southern US has awns. Nevertheless, California weedy rice shares important weedy traits with those of southern US weedy rice including high seed shattering and a red-colored pericarp in addition to tall stature and high tillering habit.Principal components analysis reduced the set of observed variables for California weedy and cultivated rice by loading them on orthogonal lines of fit based on contributions to variance. No variation was observed amongst weedy and cultivated rice for leaf texture and angle, ligule shape, ligule color, ligule pubescence, auricle color, node color, or panicle secondary branching, so these traits were excluded from the analysis. When multiple traits represented the same metric, we chose the variable with the highest eigenvector value to represent each group or suite of highly correlated traits, although each group member or variable has an impact when describing the underlying mechanism responsible for phenotypic selection differences between the cultivar and weedy rice in California. Importantly, pericarp color clearly distinguishes weedy from cultivated rice in California , but is not highlighted in the dimension-reducing PCA because it was scored as a qualitative trait following International Rice Research Institute descriptor guidelines. The remaining phenotypic traits included vegetative growth habit characters, reproductive morphologies, and yield metrics related to grain morphology. Principal components analysis was conducted on these informative traits, excluding highly correlated variables . A second PCA was performed on a reduced dataset, which included the five traits with highest eigenvector values for each principal component in the initial analysis . Principal Components 1 , 2 , and 3 together account for 45.18% of the total cumulative variance in cultivated and weedy rice in the first PCA . Traits most greatly discriminating California weedy from cultivated rice include panicle type, leaf width ,flowering , awn color, and culm length ; lemma pubescence, texture of the panicle axis, length of the first leaf below the flag leaf, length/width ratio of grain, and width of flag leaf ; 100-grain weight of the field-collected mother plant, awn length of the field-collected and offspring plants, spikelet fertility , and grains per panicle described most of the variation along PC3. Becausewe were interested in the contribution of suites of traits describing each statistically significant orthogonal vector, vertical racks the 15 traits contributing most to variance along the first three PCs in PCA 1 were subjected to a second analysis. In PCA 2 of “key discriminating traits,” principal components 1 , 2 , 3 , and 4 account for 74.91% of the cumulative variance in the phenotype of California weedy rice . Since components or dimensions with an eigenvalue greater than one are statistically relevant to the result , we report four principal components for this second PCA.Traits in Californiaweedy rice plants that mimic both medium-grain and gourmet specialty cultivars include erect leaf angles, cleft ligule shape, culm strength , green node color, panicle exertion , and ligule pubescence .
Crop traits in weedy rice specific to co-occurring medium-grain cultivars include basal leaf sheath color and hull color . Crop traits grouping weedy rice with gourmet specialty varieties include intermediate tillering or spreading growth habit . These crop-like traits are presumably carried by the immediate progenitors of the weedy rice, the crop, and have not been lost by the crop. When shared between weedy and cultivated rice, some of these traits could reinforce weed persistence and adaptation to cultivation by visually disguising the weed, thus preventing detection.Wild-like traits in California weedy rice traits exhibit high variance and differentiate this nascent feral population from both medium-grain cultivated varieties M-104, M-202, M-204, and M-205 as well as gourmet varieties in several ways. Compared with medium-grain cultivars, California weedy rice has a purple pericarp, long fully developed awns, lower seed set but with more grains per panicle, open panicles with scabrous texture, more tillers and panicles, spreading growth habit, long culm with gold internodes and delayed and extended flowering period . The medium-grain cultivars in California have brown pericarp; short awns, , high seed set, less tillers and panicles than weedy rice, have compact panicles, are less than 100 cm tall, flower earlier than weedy rice, and have erect culms. Distinct clusters within the California weedy rice population can be resolved by a multivariate analysis of variance . UPGMA cluster analysis confirmed morphogroups based on the key partitioned traits identified through PCA . The number of clusters N was given as three for several clustering methods. More clusters could be resolved amongst the weedy rice, but an additional split did not offer more information when co-occurring rice cultivars were included .Weedy rice in California is a newly established and distinct group in the USA. Isolation with migration modeling suggests that California SHA weedy rice diverged from California rice cultivars approximately 118 generations ago. The relatively recent divergence, distinct morphology, and small genetic relatedness with other US weedy rice indicate that this unique population has evolved separately from a cultivated ancestor. The recent origin of California weedy rice suggests that the population has differentiated since the establishment of rice cultivation in California and is in the early stages of segregating weedy traits, such as more tillers, extended flowering, and pigmented pericarp. Across all loci, we find no haplotypes in California cultivated rice that are not present in other japonicas . Further, there are no additional shared polymorphisms between California weedy rice and other japonicas that are not shared between California weedy and California crop rice. California weedy rice either diverged from japonicas outside of the US and was brought in to California after becoming weedy or diverged from California japonica cultivated in California. The former argument that this weed was brought in is highly unlikely due to strict laws in rice seed import into California. Regardless, California weedy rice is distinct from the other US weedy rices and our coalescent IM estimates point to a recent de-domestication from the California japonica line. Indeed, while the generation values may be inflated , this does not necessarily mean that the estimate is greater than it should be. The relative values are indicative of the relatively recent divergence, which was our objective with this analysis. This result does not demonstrate definitively that time since divergence between California cultivated and weedy rice is different from that between BHA and California weedy rice; however, it is clear that California cultivated rice and California weedy rice have different origins, and more importantly, that the divergence of California weedy rice is more recently from cultivated rice in the same area than that from all other Oryza groups investigated.