Unusual patterns of hybridization involving two alpine Salvia species: Absence of both F1 and backcrossed hybrids

Abstract

Natural hybridization plays an important role in speciation; however, we still know little about the mechanisms underlying the early stages of hybrid speciation. Hybrid zones are commonly dominated by F₁s, or backcrosses, which impedes further speciation. In the present study, morphological traits and double digest restriction-site associated DNA sequencing (ddRAD-seq) data have been used to confirm natural hybridization between Salvia flava and S. castanea, the first case of identification of natural hybridization using combined phenotypic and molecular evidence in the East Asian clade of Salvia. We further examined several reproductive barriers in both pre-zygotic and post-zygotic reproductive stages to clarify the causes and consequences of the hybridization pattern. Our results revealed that reproductive isolation between the two species was strong despite the occurrence of hybridization. Interestingly, we found that most of the hybrids were likely to be F₂s. This is a very unusual pattern of hybridization, and has rarely been reported before. The prevalence of geitonogamy within these self-compatible hybrids due to short distance foraging by pollinators might explain the origin of this unusual pattern. F₂s can self-breed and develop further, therefore, we might be witnessing the early stages of hybrid speciation. Our study provides a new case for understanding the diversification of plants on the Qinghai-Tibet Plateau.

Keywords: RAD-seq; Salvia; ethological isolation; hybridization; reproductive isolation barriers; speciation.

Figure 1

Geographical distribution of S. flava and S. castanea (A) and the habitat distribution of S. flava, S. castanea and hybrids in MY (B). The area of circle represents the number of individuals of the three taxa in the plot. The map image derived from National Platform for Common Geospatial Information Services (https://www.tianditu.gov.cn/), topographic map from Geospatial Data Cloud (http://www.gscloud.cn).

Figure 2

(A) Morphological differences between Salvia flava (A1–A2), S. castanea (A5–A6) and putative hybrid individuals (A3–A4), and (B) PCA plot of first two components based on data from the 16 quantitative morphological characters.

Figure 3

Population genetic analyses of S. flava, S. casatanea and the putative hybrids based on SNP variation. (A) PCA plot of the first two components for MY and HSH locations. The ellipses represent the 95% confidence interval. (B) The STRUCTURE plot with K = 2. (C) Genotype class assignment by Newhybrids based on diagnostic loci.

Figure 4

Proportion of flower visitors to S. flava, S. castanea and hybrid plants in a natural setting (A), and transition percentages of pollinators in the manual plots (B). Transition types were categorized as: conspecific (S. flava–S. flava, or S. castanea–S. castanea) and heterospecific (S. flava–S.castanea, or S. castanea–S. flava).

Figure 5

Fruit set (A) and seed number per fruits (B) from inter- and intra-specific pollinations of S. flava and S. castanea (± S.E.).