Genetic Variation at Nuclear Loci Fails to Distinguish Two Morphologically Distinct Species of Aquilegia

Nordborg M (2010) Genetic Variation at Nuclear Loci Fails to Distinguish Two Morphologically Distinct Species of Aquilegia. PLoS ONE 5(1): e8655. doi:10.1371/journal.pone.0008655 Genetic Variation at Nuclear Loci Fails to Distinguish Two Morphologically Distinct Species of Aquilegia Elizabeth A. Cooper 0 Justen B. Whittall 0 Scott A. Hodges 0 Magnus Nordborg 0 Simon Joly, McGill University, Canada 0 1 Department of Molecular and Computational Biology, University of Southern California, Los Angeles, California, United States of America, 2 Biology Department, Santa Clara University , Santa Clara , California, United States of America, 3 Department of Ecology , Evolution , and Marine Biology, University of California Santa Barbara, Santa Barbara, California, United States of America, 4 Gregor Mendel Institute of Molecular Plant Biology , Vienna , Austria Aquilegia formosa and pubescens are two closely related species belonging to the columbine genus. Despite their morphological and ecological differences, previous studies have revealed a large degree of intercompatibility, as well as little sequence divergence between these two taxa [1,2]. We compared the inter- and intraspecific patterns of variation for 9 nuclear loci, and found that the two species were practically indistinguishable at the level of DNA sequence polymorphism, indicating either very recent speciation or continued gene flow. As a comparison, we also analyzed variation at two loci across 30 other Aquilegia taxa; this revealed slightly more differentiation among taxa, which seemed best explained by geographic distance. By contrast, we found no evidence for isolation by distance on a more local geographic scale. We conclude that the extremely low levels of genetic differentiation between A. formosa and A.pubescens at neutral loci will facilitate future genome-wide scans for speciation genes. - The genetic mechanisms underlying the process of speciation are of critical interest to evolutionary biologists. In order to unravel this process, it is necessary to both identify the genes responsible for existing reproductive barriers and to consider what demographic and selective forces have shaped these traits. In particular, many recent studies have focused on the role of gene flow during the speciation process [310], even though the more traditional (allopatric) view of speciation posits that genetic exchange must be rare in order for species to remain distinct [11]. These studies have shown that adaptive differences between species can be maintained even in the face of significant amounts of introgression, especially if only a few genes or genomic regions control the traits that lead to reproductive isolation [4]. Genome-wide analyses of many species have shown that levels of introgression can vary across the genome, with divergent selection playing an active role in preventing gene flow at the loci underlying adaptive traits, but not acting at other areas in the genome [3,4,12]. Incipient species will also show varying levels of differentiation across the genome, with the most differentiated regions also being the most likely to contain genes that restrict random mating [3,7]. These species can appear almost identical at many loci, even in the complete absence of genetic exchange. Whether or not gene flow is a factor, closely related taxa offer an excellent opportunity to study the genetic changes and processes that lead to reproductive isolation, since genome-wide scans should be able to pinpoint loci with higher levels of differentiation, and these loci are most likely to be under the influence of natural selection [3,1315]. While the identification of potential speciation genes will not definitively prove a particular speciation model, comparing the pattern of variation in these loci with the pattern of shared variation in neutral loci will provide much more insight into the question of whether or not two species have diverged in the face of gene flow [3,7]. Thus, it is especially important to identify pairs or groups of species that maintain high levels of shared polymorphism over much of their genomes. The columbine genus Aquilegia [Ranunculaceae] is an excellent example of a recent, rapid adaptive radiation [1], and thus should provide an opportunity to identify the genetic changes important for speciation. The genus is comprised of approximately 70 outcrossing species that occupy a wide variety of habitats in North America, Europe, and Asia [16] and that differ substantially in floral morphology [16,17]. Despite these differences, species are usually cross-compatible [18,19]. Two species, Aquilegia formosa and A. pubescens, have long been studied for the purpose of understanding the factors controlling reproductive isolation between them [2023]. A. formosa is found throughout mountainous regions of western North America while A. pubescens is restricted to the southern Sierra Nevada range [22]. The species exhibit distinct differences in floral characters that have been shown to influence pollinator preference, thereby restricting gene flow between them [22,23](Figure 1). Additionally, they prefer different habitats: A. formosa populations typically occur in moist areas with well-developed soils at lower elevations (below 3,000m), whereas A. pubescens populations are found in drier, poorly developed soils at higher elevations (3,0004,000m) [20,21,24]. However, the two species are highly interfertile, and form natural hybrid zones at mid elevations where the two habitats co-occur [22]. Molecular markers exhibit more introgression than morphological characters near these zones, suggesting that gene flow could be extensive between these species for neutral markers [22]. Previous studies have uncovered limited DNA sequence variation between A. formosa and A. pubescens in both chloroplast and nuclear sequences [1,2]. However, these previous studies showed either low sequence variation across a wide range of Aquilegia species [1] or few individuals were sampled [2] and therefore do not address the degree of genetic differentiation between these species. Other studies suggest that intraspecific sequence variation may be quite similar in A. formosa and A. pubescens and thus that they may be especially useful for identifying speciation genes. For instance, microsatellite loci have similar numbers of alleles and size ranges [25], and another study including over 850 AFLP markers polymorphic in a small sample of both species found only one marker that showed complete differentiation [17]. Because these previous studies did not assess variation at the DNA sequence level or use relatively large population samples, we sought to gain insight into the inter- and intraspecific patterns of genetic variation in these species by sequencing nine nuclear loci from a total of 80 individuals from several populations. As a comparison, we also assessed variation among all of the North American species in the genus (plus (...truncated)