Re-creating ancient hybrid species' complex phenotypes from early-generation synthetic hybrids: three examples using wild sunflowers

Can the complex phenotypes that characterize naturally occurring hybrid species be re-created in early-generation artificial hybrids? We address this question with three homoploid hybrid species (Helianthus anomalus, Helianthus deserticola, Helianthus paradoxus) and their ancestral parents (Helianthus annuus, Helianthus petiolaris) that are phenotypically distinct and ecologically differentiated. These species, and two synthetic hybrid populations of the ancestral parents, were characterized for morphological, physiological, and life-history traits in greenhouse studies. Among the synthetic hybrids, discriminant analysis identified a few individuals with the multitrait phenotype of the natural hybrid species: 0.7%-1.1% were H. anomalus-like, 0.5%-13% were H. deserticola-like, and only 0.4% were H. paradoxus-like. These relative frequencies mirror previous findings that genetic correlations are favorable for generating the hybrid species' phenotypes, and they correspond well with phylogeographic evidence that demonstrates multiple natural origins of H. deserticola and H. anomalus but a single origin for H. paradoxus. Even though synthetic hybrids with hybrid species phenotypes are rare, their phenotypic correlation matrices share most of the same principal components (eigenvectors), setting the stage for predictable recovery of hybrid species' phenotypes from different hybrid populations. Our results demonstrate past hybridization could have generated hybrid species-like multitrait phenotypes suitable for persistence in their respective environments in just three generations after initial hybridization.     

The genomic organization of Ty3/gypsy-like retrotransposons in Helianthus (Asteraceae) homoploid hybrid species

The origin of new diploid, or homoploid, hybrid species is associated with rapid genomic restructuring in the hybrid neospecies. This mode of speciation has been best characterized in wild sunflower species in the genus Helianthus, where three homoploid hybrid species (H. anomalus, H. deserticola, and H. paradoxus) have independently arisen via ancient hybridization events between the same two parental species (H. annuus and H. petiolaris). Most previous work examining genomic restructuring in these sunflower hybrid species has focused on chromosomal rearrangements. However, the origin of all three homoploid hybrid sunflower species also is associated with massive proliferation events of Ty3/gypsy-like retrotransposons in the hybrid species' genomes. We compared the genomic organization of these elements in the parent species and two of the homoploid hybrid species using fluorescence in situ hybridization (FISH). We found a significant expansion of Ty3/gypsy-like retrotransposons confined to the pericentromeric regions of two hybrid sunflower species, H. deserticola and H. paradoxus. In contrast, we detected no significant increase in the frequency or extent of dispersed retrotransposon populations in the hybrid species within the resolution limits of our assay. We discuss the potential role that transposable element proliferation and localization plays in the evolution of homoploid hybrid species.     

Origin(s) of the diploid hybrid species Helianthus deserticola (Asteraceae)

Homoploid hybrid speciation has traditionally been considered a rare event, dependent on the establishment of both a novel, balanced genotype and reproductive isolating barriers between the new species and its progenitors. However, more recent studies have shown that synthetic hybrids converge toward the chromosomal structure of natural hybrids after only a few generations, suggesting that this phenomenon may be more frequent than previously assumed. Here, the possibility that the diploid hybrid species Helianthus deserticola arose from more than one hybrid speciation event was investigated using patterns of variation from cpDNA, 18 nuclear microsatellite loci, and population interfertility. Helianthus deserticola contains cpDNA haplotypes characteristic of both parental species, is polyphyletic with one parental species based on nine microsatellite loci, and has a high degree of interfertility among populations. The data are consistent with either a single origin followed by introgression with the parental species or multiple origins. Analysis of microsatellite variation places the origin of H. deserticola between 170?000 and 63?000 years before present, making it unlikely that anthropogenic disturbances influenced its origin. Finally, the hybrid species generally has lower levels of genetic diversity but higher levels of differentiation among populations than either parental species.     

Reconstructing the origin of Helianthus deserticola: survival and selection on the desert floor

The diploid hybrid species Helianthus deserticola inhabits the desert floor, an extreme environment relative to its parental species Helianthus annuus and Helianthus petiolaris. Adaptation to the desert floor may have occurred via selection acting on transgressive, or extreme, traits in early hybrids between the parental species. We explored this possibility through a field experiment in the hybrid species' native habitat using H. deserticola, H. annuus, H. petiolaris, and two populations of early-generation (BC(2)) hybrids between the parental species, which served as proxies for the ancestral genotype of the ancient hybrid species. Character expression was evaluated for each genotypic class. Helianthus deserticola was negatively transgressive for stem diameter, leaf area, and flowering date, and the latter two traits are likely to be advantageous in a desert environment. The BC(2) hybrids contained a range of variation that overlapped these transgressive trait means, and an analysis of phenotypic selection revealed that some of the selective pressures on leaf size and flowering date, but not stem diameter, would move the BC(2) population toward the H. deserticola phenotype. Thus, H. deserticola may have originated from habitat-mediated directional selection acting on hybrids between H. annuus and H. petiolaris in a desert environment.     

Selective sweeps in the homoploid hybrid species Helianthus deserticola: evolution in concert across populations and across origins

The evolution of different populations within a species in response to selective pressures can potentially happen in three different ways. It can occur in parallel, where similar changes occur independently in each population in response to selection; in concert, where the spread of an adaptive mutation across a species' range results in a single allele fixing in each population; or populations can diverge in response to local selective pressures. We explored these possibilities in populations of the homoploid hybrid species Helianthus deserticola relative to its parental species Helianthus annuus and Helianthus petiolaris using an analysis of variation in 96 expressed sequence tag-based microsatellites. A total of nine loci showed evidence consistent with recent selection at either the species or population level, although two of these genes were discarded because the apparent sweep did not occur relative to the parent from which the locus was derived. Between one and five loci showed a putative sweep across the entire species range with the same microsatellite allele fixed in each population. This pattern is consistent with evolution in concert despite geographical isolation and potential independent origins of the populations. Only one population of H. deserticola showed candidate sweeps that were unique compared to the rest of the species, and this population has also potentially experienced recent admixture with the parental species.     

Patterns of genetic variation suggest a single,ancient origin for the diploid hybrid species Helianthus paradoxus

Abstract.— Experimental and comparative evidence implies that homoploid hybrid speciation is a reproducible process, mediated in part by ecological selection. Here, molecular data from the chloroplast genome and 17 nuclear microsatellite loci were employed to determine whether a well-documented homoploid hybrid species, Helianthus paradoxus , has arisen multiple times. Helianthus paradoxus is ecologically divergent from its parental species, and has a disjunct geographic distribution consistent with multiple origins. The molecular data, however, strongly support a single hybrid origin. First, all sampled populations of H. paradoxus are fixed for a single chloroplast DNA (cpDNA) haplotype, whereas local populations of both parental species, H. annuus and H. petiolaris , have multiple cpDNA haplotypes. Second, H. paradoxus populations form a single, well-supported clade (99.8% bootstrap support) in a neighbor-joining tree based on microsatellite allele frequencies. The microsatellite data also tentatively place the origin of H. paradoxus between 75,000 years and 208,000 years before present, indicating that anthropogenic disturbance likely did not play a role in the formation of this species. Finally, the genetic structure of this species is not consistent with passive riparian dispersal, which has been suggested for other wetland plant species, but may be explained by dispersal mechanisms implicated for H. annuus , such as large migratory mammals.     

Extensive chromosomal repatterning and the evolution of sterility barriers in hybrid sunflower species

New species may arise via hybridization and without a change in ploidy. This process, termed homoploid hybrid speciation, is theoretically difficult because it requires the development of reproductive barriers in sympatry or parapatry. Theory suggests that isolation may arise through rapid karyotypic evolution and/or ecological divergence of hybrid neospecies. Here, we investigate the role of karyotypic change in homoploid hybrid speciation by generating detailed genetic linkage maps for three hybrid sunflower species, Helianthus anomalus, H. deserticola, and H. paradoxus, and comparing these maps to those previously generated for the parental species, H. annuus and H. petiolaris. We also conduct a quantitative trait locus (QTL) analysis of pollen fertility in a BC2 population between the parental species and assess levels of pollen and seed fertility in all cross-combinations of the hybrid and parental species. The three hybrid species are massively divergent from their parental species in karyotype; gene order differences were observed for between 9 and 11 linkage groups (of 17 total), depending on the comparison. About one-third of the karyoypic differences arose through the sorting of chromosomal rearrangements that differentiate the parental species, but the remainder appear to have arisen de novo (six breakages/six fusions in H. anomalus, four breakages/three fusions in H. deserticola, and five breakages/five fusions in H. paradoxus). QTL analyses indicate that the karyotypic differences contribute to reproductive isolation. Nine of 11 pollen viability QTL occur on rearranged chromosomes and all but one map close to a rearrangement breakpoint. Finally, pollen and seed fertility estimates for F1's between the hybrid and parental species fall below 11%, which is sufficient for evolutionary independence of the hybrid neospecies.     

Behavioural isolation may facilitate homoploid hybrid speciation in cichlid fish

Hybrid speciation is constrained by the homogenizing effects of gene flow from the parental species. In the absence of post‐mating isolation due to structural changes in the genome, or temporal or spatial premating isolation, another form of reproductive isolation would be needed for homoploid hybrid speciation to occur. Here, we investigate the potential of behavioural mate choice to generate assortative mating among hybrids and parental species. We made three‐first‐generation hybrid crosses between different species of African cichlid fish. In three‐way mate‐choice experiments, we allowed hybrid and nonhybrid females to mate with either hybrid or nonhybrid males. We found that hybrids generally mated nonrandomly and that hybridization can lead to the expression of new combinations of traits and preferences that behaviourally isolate hybrids from both parental species. Specifically, we find that the phenotypic distinctiveness of hybrids predicts the symmetry and extent of their reproductive isolation. Our data suggest that behavioural mate choice among hybrids may facilitate the establishment of isolated hybrid populations, even in proximity to one or both parental species.     

Evolution of a hybrid zone of two willow species (Salix L.) in the European Alps analyzed by RAD-seq and morphometrics

Natural hybridization of plants can result in many outcomes with several evolutionary consequences, such as hybrid speciation and introgression. Natural hybrid zones can arise in mountain systems as a result of fluctuating climate during the exchange of glacial and interglacial periods, where species retract and expand their territories, resulting in secondary contacts. Willows are a large genus of woody plants with an immense capability of interspecific crossing. In this study, the sympatric area of two diploid sister species, S. foetida and S. waldsteiniana in the eastern European Alps, was investigated to study the genomic structure of populations within and outside their contact zone and to analyze congruence of morphological phenotypes with genetic data. Eleven populations of the two species were sampled across the Alps and examined using phylogenetic network and population genetic structure analyses of RAD Seq data and morphometric analyses of leaves. The results showed that a homoploid hybrid zone between the two species was established within their sympatric area. Patterns of genetic admixture in homoploid hybrids indicated introgression with asymmetric backcrossing to not only one of the parental species but also one hybrid population forming a separate lineage. The lack of F1 hybrids indicated a long-term persistence of the hybrid populations. Insignificant isolation by distance suggests that gene flow can act over large geographical scales. Morphometric characteristics of hybrids supported the molecular data and clearly separated populations of the parental species, but showed intermediacy in the hybrid zone populations with a bias toward S. waldsteiniana. The homoploid hybrid zone might have been established via secondary contact hybridization, and its establishment was fostered by the low genetic divergence of parental species and a lack of strong intrinsic crossing barriers. Incomplete ecological separation and the ability of long-distance dispersal of willows could have contributed to the spatial expansion of the hybrid zone.     

PATTERNS OF MATING IN WILD SUNFLOWER HYBRID ZONES

Theory predicts that homoploid hybrid speciation will be facilitated by selfing, yet most well-documented hybrid species are outcrossers. One possible explanation for this puzzle is that conditions in hybrid populations may favor selfing, even in otherwise outcrossing species. For example, in self-incompatible plants, mixtures of self and interspecific pollen often induce selfing. Here, we examine patterns of mating in three hybrid zones and four “pure” populations of Helianthus annuus and H. petiolaris, wild, self-incompatible sunflower species that are thought to have parented three homoploid hybrid species. Fourteen to 16 maternal families from each pure population and 44–46 maternal families from each hybrid zone were analyzed for seven polymorphic isozyme loci. Maximum-likelihood (ML) methods were used to estimate multilocus outcrossing rates (T_m) and hybridization frequencies for each maternal family, each phenotypic group within each hybrid zone (annuus-like, hybrid, and petiolaris-like), and each population. As predicted for self-incompatible species, all four parental populations have outcrossing rate ML estimates of 1.0. Within the hybrid zones, outcrossing rates were lowest in the H. annuus–like fraction of the population (0.73, 0.72, and 0.74 in the three hybrid zones, respectively), largely intermediate in the H. petiolaris–like group (0.94, 0.90, and 0.94), and highest in the hybrid group (0.97, 0.93, and 0.97). Although outcrossing rates are lower in hybrid zones than in pure populations, it is unlikely that the observed decrease facilitates hybrid speciation because outcrossing rates in the critical hybrid fraction of the population do not differ significantly from 1.0. Dividing the outcrossed pollen pool into intraspecific and interspecific components revealed that maternal plants are largely fertilized by conspecific pollen, confirming an important role for pollen competition as a reproductive barrier. Highly sterile hybrid plants do not appear to discriminate between parental species pollen, but hybrids with higher fertility tend to be fertilized by pollen from the parental group they resemble genetically. Thus, gametic selection leads to substantial assortative mating in these hybrid zones.     

The role of parental and hybrid species in multiple introgression events: evidence of homoploid hybrid speciation in Centaurea (Cardueae,Asteraceae)

Molecular approaches have greatly increased the number of confirmed homoploid hybrids, which suggests that the frequency of this phenomenon was underestimated in the past because it was much more difficult to detect than allopolyploidy. Centaurea is a suitable model group for studying homoploid speciation, as hybridization events have been commonly reported for this genus. Based on this, here we study Centaurea × forsythiana, a naturally occurring homoploid hybrid between two Sardinian endemics, C. horrida and C. filiformis, using a molecular approach involving nuclear and plastid markers, to understand the underlying population dynamics between homoploid hybrids and their parents. Our results confirm that C. × forsythiana is a hybrid between the above‐mentioned species and define the roles of the parents. Plastid markers point towards C. horrida as the maternal progenitor, and nuclear markers reveal that the other parental species, C. filiformis, is itself an old, stabilized homoploid hybrid related to the C. paniculata complex from the Italian mainland. Homoploid hybrid speciation is discussed and C. × forsythiana and C. filiformis are compared with other similar examples. The study confirms the importance of introgression between parental species mediated by hybrids and its potential implications in conservation. Furthermore, it shows how hybridization studies become even more complex when the parents are themselves of probable hybrid origin. © 2014 The Linnean Society of London, Botanical Journal of the Linnean Society, 2014, 175 , 453–467.     

Natural selection for salt tolerance quantitative trait loci (QTLs) in wild sunflower hybrids: implications for the origin of Helianthus paradoxus,a diploid hybrid species

For a new diploid or homoploid hybrid species to become established, it must diverge ecologically from parental genotypes. Otherwise the hybrid neospecies will be overcome by gene flow or competition. We initiated a series of experiments designed to understand how the homoploid hybrid species, Helianthus paradoxus, was able to colonize salt marsh habitats, when both of its parental species (H. annuusxH. petiolaris) are salt sensitive. Here, we report on the results of a quantitative trait locus (QTL) analysis of mineral ion uptake traits and survivorship in 172 BC2 hybrids between H. annuus and H. petiolaris that were planted in H. paradoxus salt marsh habitat in New Mexico. A total of 14 QTLs were detected for mineral ion uptake traits and three for survivorship. Several mineral ion QTLs mapped to the same position as the survivorship QTLs, confirming previous studies, which indicated that salt tolerance in Helianthus is achieved through increased Ca uptake, coupled with greater exclusion of Na and related mineral ions. Of greater general significance was the observation that QTLs with effects in opposing directions were found for survivorship and for all mineral ion uptake traits with more than one detected QTL. This genetic architecture provides an ideal substrate for rapid ecological divergence in hybrid neospecies and offers a simple explanation for the colonization of salt marsh habitats by H. paradoxus. Finally, selection coefficients of +0.126, -0.084 and -0.094 for the three survivorship QTLs, respectively, are sufficiently large to account for establishment of new, homoploid hybrid species.     

Genomic alterations in the interspecific hybrid Helianthus annuus×Helianthus tuberosus

 The genome of a Helianthus annuus (2n=34) ×Helianthus tuberosus (2n=102) hybrid was studied at cytological, biochemical and molecular levels and compared to those of the parental species. Cytophotometric analyses showed that the hybrid has a 4C DNA content higher than expected and with a larger variability than in the parents. This high variability is probably not related to chromosome-number variations since the hybrid always had 2n=68 chromosomes. Moreover, hybrid interphase nuclei showed lower heterochromatin condensation than the parental ones. Thermal denaturation of genomic DNAs indicated that quantitative variation of some DNA families occurred in the hybrids compared to parents. Finally, molecular analyses of DNAs restricted with different enzymes, after Southern blotting and hybridization with HR probes, showed restriction patterns in the hybrid different from those observed in parents. These results indicate that interspecific hybridization between H. annuus and H. tuberosus may determine quantitative variation of some DNA families and differential DNA methylations that probably modify the nuclear structure. These phenomena are probable responses to a “genomic shock” following the interspecific cross. Received: 22 May 1998 / Accepted: 4 June 1998     

The ecological genetics of homoploid hybrid speciation

Our understanding of homoploid hybrid speciation has advanced substantially since this mechanism of species formation was codified 50 years ago. Early theory and research focused almost exclusively on the importance of chromosomal rearrangements, but it later became evident that natural selection, specifically ecological selection, might play a major role as well. In light of this recent shift, we present an evaluation of ecology's role in homoploid hybrid speciation, with an emphasis on the genetics underlying ecological components of the speciation process. We briefly review new theoretical developments related to the ecology of homoploid hybrid speciation; propose a set of explicit, testable questions that must be answered to verify the role of ecological selection in homoploid hybrid speciation; discuss published work with reference to these questions; and also report new data supporting the importance of ecological selection in the origin of the homoploid hybrid sunflower species Helianthus deserticola. Overall, theory and empirical evidence gathered to date suggest that ecological selection is a major factor promoting homoploid hybrid speciation, with the strongest evidence coming from genetic studies.     

Genome size and base composition variation in natural and experimental Narcissus (Amaryllidaceae) hybrids

Background and Aims

Although there is evidence that both allopolyploid and homoploid hybridization lead to rapid genomic changes, much less is known about hybrids from parents with different basic numbers without further chromosome doubling. Two natural hybrids, Narcissus × alentejanus (2n = 19) and N. × perezlarae (2n = 29), originated by one progenitor (N. cavanillesii, 2n = 28) and two others (N. serotinus, 2n = 10 and N. miniatus, 2n = 30, respectively) allow us to study how DNA content and composition varies in such hybrids.

Methods

Flow cytometry measurements with two staining techniques, PI and DAPI, were used to estimate 2C values and base composition (AT/GC ratio) in 390 samples from 54 wild populations of the two natural hybrids and their parental species. In addition, 20 synthetic F₁ hybrid individuals were also studied for comparison.

Key Results

Natural hybrids presented 2C values intermediate between those found in their parental species, although intra-population variance was very high in both hybrids, particularly for PI. Genome size estimated from DAPI was higher in synthetic hybrids than in hybrids from natural populations. In addition, differences for PI 2C values were detected between synthetic reciprocal crosses, attributable to maternal effects, as well as between natural hybrids and those synthetic F₁ hybrids in which N. cavanillesii acted as a mother.

Conclusions

Our results suggest that natural hybrid populations are composed of a mixture of markedly different hybrid genotypes produced either by structural chromosome changes, consistent with classic cytogenetic studies in Narcissus, or by transposon-mediated events.     

Homoploid hybridization of plants in the Hengduan mountains region

The Hengduan Mountains Region (HMR) is a major global biodiversity hotspot. Complex tectonic and historical climatic conditions created opportunities for natural interspecific hybridization. Likewise, anthropogenic disturbance potentially raises the frequency of hybridization. Among species studies to date, the frequency of homoploid hybridization appears in the HMR. Of nine taxa in which natural hybridization has been detected, three groups are involved in homoploid hybrid speciation, and species pairs from the remaining six genera suggest that continuous gene flow occurs in hybrid zones. Reproductive isolation may greatly affect the dynamic and architecture of hybrid zones in the HMR. Asymmetrical hybridization and introgression can primarily be attributed to both prezygotic and postzygotic barriers. The frequent observation of such asymmetry may imply that reproductive barrier contributes to maintaining species boundaries in the alpine region. Ecological isolations with environmental disturbance may promote breeding barriers between parental species and hybrids. Hybrid zones may be an important phase for homoploid hybrid speciation. Hybrid zones potentially provided abundant genetic resources for the diversification of the HMR flora. The ecological and molecular mechanisms of control and mediation for natural hybridization will help biologists to understand the formation of biodiversity in the HMR. More researches from ecological and molecular aspects were required in future studies.     

Habitat divergence between a homoploid hybrid sunflower species, Helianthus paradoxus (Asteraceae), and its progenitors

The diploid hybrid species Helianthus paradoxus is restricted to salt marshes with sodium concentrations that exceed those found in the habitats of its progenitors, H. annuus and H. petiolaris. The observed association with saline habitats has led to the hypothesis that H. paradoxus is more salt tolerant than its progenitors. This hypothesis was tested by growing all three species in three NaCl treatments (0 mmol/L, 100 mmol/L, and 200 mmol/L). Helianthus paradoxus treated with NaCl was found to be more than five times as fit, in terms of biomass and survivorship, than its progenitors. Selection for salt tolerance in early generation hybrids may have contributed to the formation of H. paradoxus because theory predicts that homoploid hybrid speciation is feasible even when selection favoring hybrid genotypes is much weaker. Additionally, we show that H. paradoxus is significantly different from its parental species for several traits that often distinguish salt-tolerant species and suggest a mechanistic basis for the elevated salt tolerance expressed by H. paradoxus.     

Microsatellite signature of ecological selection for salt tolerance in a wild sunflower hybrid species, Helianthus paradoxus

The hybrid sunflower species Helianthus paradoxus inhabits sporadic salt marshes in New Mexico and southwest Texas, USA, whereas its parental species, Helianthus annuus and Helianthus petiolaris, are salt sensitive. Previous studies identified three genomic regions - survivorship quantitative trait loci (QTLs) - that were under strong selection in experimental hybrids transplanted into the natural habitat of H. paradoxus. Here we ask whether these same genomic regions experienced significant selection during the origin and evolution of the natural hybrid, H. paradoxus. This was accomplished by comparing the variability of microsatellites linked to the three survivorship QTLs with those from genomic regions that were neutral in the experimental hybrids. As predicted if one or more selective sweeps had occurred in these regions, microsatellites linked to the survivorship QTLs exhibited a significant reduction in diversity in populations of the natural hybrid species. In contrast, no difference in diversity levels was observed between the two microsatellite classes in parental populations.