A framework for documenting homoploid hybrid speciation
This strategy was initially applied to the documentation of the homoploid hybrid origin of Ostryopsis intermedia (Wang et al., 2021; also see commentary by Owens, 2021). Ostryopsis is a member of the birch family (Betulaceae) and includes three species of shrubs –O. davidiana , O. intermedia , and O. nobilis – all of which are endemic to China. The three species are mostly allopatric, although O. intermedia and O. nobilis occasionally co-occur in Southwestern China. As implied by its name, O. intermedia is intermediate phenotypically relative to the other two members of the genus, and an earlier analysis of admixture based on 20 microsatellites and 11 nuclear loci supported a hybrid origin approximately 0.5-1.2 million years ago (mya), associated with Quaternary climate oscillations (Liu et al., 2014).
In their more recent and comprehensive investigation of the system, Wang et al. (2021) demonstrated that O. intermedia was reproductive isolated from its parental species (criterion 1 of Schumer et al. 2014), more robustly validated the hybrid origin of O. intermedia(criterion 2), and directly linked the origin of reproductive barriers to hybridization (criterion 3). While we primarily focus on the latter component of the study, the findings of the first two components provide useful background information. Briefly, Wang et al. (2021) found that the parental species were primarily isolated by two premating barriers (differential habitat adaptation and flowering time), whereas the hybrid was isolated from one parent (O. davidiana ) by habitat (mainly soil iron concentration) and the other parent (O. nobilis ) by flowering time. There also was a partial crossability barrier between the hybrid and parents, with total cumulative isolation exceeding 0.87 in all comparisons, even without considering ecogeographic isolation.
Next, Wang et al. (2021) used whole genome sequencing data and standard phylogenomic and admixture analyses (e.g., PhyloNet and ABBA-BABA tests) to more robustly document the hybrid origin of O. intermedia , as well as the timing of admixture. Consistent with the previous study, the two parental species were found to have diverged ~9.8 mya, with their hybrid originating approximately 1.8 mya. While this analysis was largely confirmatory of earlier work, such phylogenomic studies are useful because they can detect previously unsuspected cases of admixture (e.g., Green et al., 2010, and refine (Nevado et al., 2020; Owens et al., 2023) or even reject (Zalmat et al., 2021; Goulet-Scott et al., 2021) earlier hypotheses of homoploid hybrid speciation.
To show that hybridization contributed to the evolution of reproductive barriers isolating O. intermedia from its parental species, Wang et al. (2021) employed the following strategy (Figure 1). First, they searched for candidate genes underlying the key habitat (soil iron concentrations) and flowering time barriers. For tolerance to high soil iron concentrations, a combined population genomic data set of O. intermedia and O. nobilis was compared against O. davidiana , whereas for flowering time, population genomic data ofO. intermedia and O. davidiana was compared againstO. nobilis . Candidate isolation genes were expected to be under positive selection as indicated by Hudson–Kreitman–Aguade´ tests (Hudson et al., 1987), outliers with respect to divergence (in the top 2.5% of number of fixed non-synonymous mutation sites), and have functional annotations consistent with the putative control of variation in soil tolerance or flowering time. Three candidate genes (ZIP5, FRO4, and 018656 ) satisfied these criteria for the comparison ofO. intermedia + O. nobilis to O. davidiana ; all were annotated as being involved in iron reduction. Similarly, for the comparison of O. intermedia + O. davidiana to O. nobilis, three candidate genes (LHY , PIE1 , andFPA ) were under positive selection and had annotations consistent with their involvement in the control of flowering.
Second, Wang et al. (2021) analyzed the ancestry of O. intermediaalleles for each of these six genes. As expected, alleles forZIP5, FRO4, and 018656 were mainly derived from O. nobilis , whereas those for LHY , PIE1 , and FPA were mainly derived from O. davidiana . However, there were several instances in which a few genotypes of O. intermedia were heterozygous for alleles from both parental species, possibly due to post-speciation introgression.
Lastly, Wang et al. (2021) validated the function of alleles for three of the candidate premating barrier genes (FRO4 , ZIP5 , andLHY ) using a combination of gene expression analyses, enzymatic activity assays (for FRO4 and ZIP5 ), and functional complementation of mutations in Arabidopsis thaliana. In transgenic Arabidopsis plants, alleles of FRO4 andZIP5 from O. intermedia or O. nobilis outperformed those from O. davidiana under high iron concentrations, with opposite effects under low concentrations, strongly supporting their involvement in adaptation to different soil iron concentrations. Likewise, transgenic plants with LHY alleles from O. intermediaor O. davidiana flowered earlier than those with O. nobilis alleles, further confirming their contribution to the flowering time barrier isolating O. intermedia from O. nobilis .