Haldane's rule (HR) is the most universal observation in speciation research on animals, wherein hybridization between species leads to lower viability or fertility in either males with XY sex chromosomes or females with ZW sex chromosomes. Since its inception, an animal-centric perspective has dominated work on HR. This study will test for HR and its genetic basis using five flowering plant species in the genus Silene. Three experiments involving between-species crosses, together with quantitative-genetic analyses, will allow documentation of (1) whether HR occurs in plants for either viability or fertility, (2) the extent to which the autosomes, X and Y chromosomes, cytoplasmic genetic elements, and their interactions are involved in HR, and (3) whether genes controlling male reproductive traits evolve relatively fast and contribute to reduced male fertility.
The results of this study will address fundamental aspects of how reproductive isolation leads to speciation and will therefore be of general interest to most biologists. By working with plants with relatively young sex chromosomes, the research will address the important question of the generality of HR and the timing of the reproductive incompatibilities that lead to HR. The work will allow the training of numerous undergraduate and graduate students in speciation research and evolutionary genetics.
Haldane’s rule is based on a phenomenon first pointed out by J. B. S. Haldane, concerning hybrid crosses between species of animals with sex chromosomes: hybrids of the heterogametic sex suffer in terms of either rarity and/or sterility. Hence, in animals with XY sex-chromosome systems (e.g., mammals) hybrid males are absent, rare and/or sterile, while in animals with ZW systems (e.g., birds) it is hybrid females that suffer. Haldane’s rule is the most universal observation in speciation in animals, and we tested whether it also applies to plants with sex chromosomes. In addition, we determined the underlying genetic basis of the rule, as well as investigating other reproductive isolating mechanisms. We used three species of flowering plants in the genus Silene. These species are closely related and crossable (in other words, hybrids can be formed), and they have separate males and females as determined by sex chromosomes (females have two X chromosomes, while males have one X and one Y chromosome [i.e., males are heterogametic]). Following the production of hybrids, we quantified whether hybrid males were significantly rare and whether less viable pollen was produced, relative to pure-species males, as predicted by Haldane’s rule. Our experiment revealed that hybrid males were both relatively rare and sterile, thereby extending the applicability of the rule to plants with sex chromosomes. We then examined the genetic basis of this hybrid breakdown, as a way of providing insight into the genetic mechanisms of this commonly observed isolating phenomenon. We chose two species from the above experiment, Silene diclinis and Silene latifolia, which showed strong conformity with Haldane’s rule. We produced a set of 15 different types of hybrid crosses, including, for example, crossing hybrid males and females to each other as well as back to pure-species individuals. This high level of crossability between these two plant species, which we termed the "Goldilocks zone," gave us the ability to dissect the underlying genetic architecture of their divergence. Our results differed somewhat from those seen in crosses between animal species. In animals, it is usually the lack of genes on the Y chromosome (which is often the smallest of the chromosomes) that leads to the rarity of hybrid males, but not their sterility, which has a different underlying mechanism. This lack-of-genes theory is called the "dominance" theory. In contrast, we found results consistent with dominance theory for the sterility of males, but not their rarity. Our findings suggest that the genetic architecture of Haldane’s rule in plants with separate sexes may differ from those commonly found in animals. The two species in this part of the study shared a common ancestor with an XY sex-chromosome system. Subsequently, one of the species, S. diclinis, evolved a XYY system, by splitting the ancestral Y chromosome into two. We found that this "neo" sex-chromosome system also contributed to the rarity of males, but that both Y chromosomes could be inherited in hybrid crosses and did not completely prevent the formation of hybrids. Haldane’s rule is an example of reproductive isolation between species that occurs after fertilization has led to the formation of a zygote (an embryo), and which is therefore referred to as "post-zygotic" reproductive isolation. We therefore also investigated pre-zygotic reproductive isolation between S. diclinis and S. latifolia, as a way of understanding why hybrids between these two species do not occur in the wild, in spite of the fact that they grow in the same place and flower at the same time. We found that the pollinators of these two species are different: S. diclinis flowers are open during the day and are visited by bumblebees, but S. latifolia flowers open at dusk and are visited by night-flying moths. This contributes to a lack of pollen being deposited on the flowers of females of the other ("wrong") species. Moreover, when pollen is deposited, the pollen of the relatively small-flowered S. diclinis cannot grow all the way down the long styles (the reproductive tract) of the relatively large flowers of S. latifolia: fertilization of the eggs is therefore prevented and hybrid seeds are not formed. Overall, we found that a variety of pre- and post-zygotic reproductive isolating mechanisms exist among three closely related dioecious Silene species. Taken together, this work contributes to our understanding of the maintenance of species boundaries. Two graduate students wrote PhD dissertations based primarily on this work. In addition, one component of two other PhD students’ research was also based on this work. Several undergraduate and high-school students participated, and one of the undergraduates won the Outstanding Honors Thesis award for her research as an REU (Research Experience for Undergraduates) student. Website postings, seminar talks, meeting talks, and publications in referred journals disseminated the research findings funded by this grant.
