A multi-disciplinary evaluation of the hybrid anemonefish Amphiprion leucokranos: behaviour shaping evolutionary outcomes of hybridization

Causes and consequences of natural hybridisation among coral reef butterflyfishes (Chaetodon: Chaetodontidae)

Hybrid zones between Townsend's Warblers (Dendroica townsendi) and Hermit Warblers (D. occidentalis) in the Pacific Northwest are narrow relative to estimated dispersal distances and appear to be moving, with Townsend's replacing Hermits. We examined whether the habitat-transition and parental-fitness asymmetry models can explain why these zones are narrow and moving by comparing habitat variables associated with warbler territories in the Washington Cascades hybrid zone. Habitat variables did not differ among phenotypes, suggesting that the habitat-transition model cannot explain the narrow and dynamic nature of this hybrid zone. Habitat characteristics of Hermit Warbler territories did not differ inside versus outside the hybrid zone, also suggesting that this zone is not associated with a region of habitat transition. The lack of difference in habitat use could be the result of comparing variables that are not important to pairing success. However, warblers tended to select territories on west-southwest aspects. South aspects in the southern Washington Cascades are dominated by Douglas fir (Pseudotsuga menziesii) and true fir, which is a habitat selected by female warblers when choosing among territories and males. The parental-fitness asymmetry model does not necessarily make predictions about habitat use within the hybrid zone but predicts the superiority of one parental species over the other. However, if significant overlap occurs in habitat use or niche (as in these warblers), then competition between parental species is likely to occur. To determine whether these species compete, we mapped 12 warbler territories and monitored an additional 94 territories throughout the breeding season and found that all males with neighbors compete for and hold exclusive territories. Thus, the pattern of habitat use and territoriality is consistent with the parental-fitness asymmetry model.

Hybrid zones between Townsend's Warblers (Dendroica townsendi) and Hermit Warblers (D. occidentalis) in the Pacific Northwest are narrow relative to estimated dispersal distances and appear to be moving, with Townsend's replacing Hermits. We examined whether the habitat-transition and parental-fitness asymmetry models can explain why these zones are narrow and moving by comparing habitat variables associated with warbler territories in the Washington Cascades hybrid zone. Habitat variables did not differ among phenotypes, suggesting that the habitat-transition model cannot explain the narrow and dynamic nature of this hybrid zone. Habitat characteristics of Hermit Warbler territories did not differ inside versus outside the hybrid zone, also suggesting that this zone is not associated with a region of habitat transition. The lack of difference in habitat use could be the result of comparing variables that are not important to pairing success. However, warblers tended to select territories on west-southwest aspects. South aspects in the southern Washington Cascades are dominated by Douglas fir (Pseudotsuga menziesii) and true fir, which is a habitat selected by female warblers when choosing among territories and males. The parental-fitness asymmetry model does not necessarily make predictions about habitat use within the hybrid zone but predicts the superiority of one parental species over the other. However, if significant overlap occurs in habitat use or niche (as in these warblers), then competition between parental species is likely to occur. To determine whether these species compete, we mapped 12 warbler territories and monitored an additional 94 territories throughout the breeding season and found that all males with neighbors compete for and hold exclusive territories. Thus, the pattern of habitat use and territoriality is consistent with the parental-fitness asymmetry model.

. 1 We asked three questions about the patterns of relative abundance of insect herbivores across host plant taxa at a palo verde hybrid zone. (1) What is the morphological structure of the hybrid zone and does this suggest a certain pattern of introgression? (2) How are putative parental seed defence mechanisms expressed in hybrid plants? (3) Do ovipositing females prefer host plant taxa on which their offspring have best survivorship? 2 Morphologically, hybrids were either intermediate or tended to resemble one parental species. Previous studies have suggested that unidirectional introgression results in loss of parental defence mechanisms against herbivores. Hybrid plants in general lacked seed coat resistance and early pod abscission which are known to act as plant defence mechanisms against bruchid beetles in the parental palo verde trees. 3 All other sources of bruchid mortality that we examined did not vary across parental and hybrid taxa, with the possible exception of egg parasitism which occurred at a lower frequency on one parental palo verde species. 4 Thus, survivorship of bruchid offspring should be greater on hybrid palo verdes. 5 Patterns of egg densities suggested that females may prefer hybrid hosts in some years but not others. An oviposition choice experiment conducted in the field, however, showed bruchids have no preference for hybrids over one of the parental species. 6 These results suggest that some insect herbivores may have higher densities on hybrid host plants because they are less resistant.

Hybrid zones provide interesting systems to study genetic and ecological interaction between different species. The correct identification of hybrids is necessary to understand the evolutionary process involved in hybridization. An oak species complex occurring in Mexico formed by two parental species, Quercus crassifolia H. & B. and Q. crassipes H. & B., and their putative hybrid species, Q. dysophylla, was analyzed with molecular markers (random amplified polymorphic DNA [RAPDs]) and morphological tools in seven hybrid zones (10 trees per taxa in each hybrid zone) and two pure sites for each parental species (20 trees per site). We tested whether geographic proximity of hybrid plants to the allopatric site of a parental species increases its morphological and genetic similarity with its parent. Seventeen morphological traits were measured in 8700 leaves from 290 trees. Total DNA of 250 individuals was analyzed with six diagnostic RAPD primers. Quercus crassifolia differed significantly from Q. crassipes in all the examined characters. Molecular markers and morphological characters were highly coincident and support the hypothesis of hybridization in this complex, although both species remain distinct in mixed stands. Clusters and a hybrid index (for molecular and morphological data) showed that individuals from the same parental species were more similar among themselves than to putative hybrids, indicating occasional hybridization with segregation in hybrid types or backcrossing to parents. Evidence does not indicate a unidirectional pattern of gene flow.

Hybrid zones are areas where gene flow between related species is currently occurring, so information on the compatibility between related species and their hybrids is essential for predicting the dynamics of such zones generated by introgressive hybridization. In this study, we quantified the compatibility among Magnolia stellata, M. salicifolia, and their hybrids in a hybrid zone using gene dispersal modeling. After determining the genealogical classes of adult trees in the hybrid zone, the paternity of 574 open-pollinated seeds from 37 known maternal trees was analyzed with microsatellite markers. A neighborhood-based Bayesian gene dispersal model developed by us for estimating compatibility was then applied to the paternity data. When M. stellata or M. salicifolia were mothers, interspecific mating to produce F1 hybrids yielded significant incompatibility, but backcrossing with F1 hybrids did not. Furthermore, when F1 hybrids became mothers, no significant incompatibility resulted from backcrossing to parental species or intra-F1 mating to produce F2 hybrids. The estimated proportion of F1 hybrids in the outcrossed seeds (1.7%) in the hybrid zone was much lower than that in the adult trees (14.0%). While it is difficult to obtain F1 hybrids, their low incompatibility makes it easy to produce advanced generation hybrids, once they have been successfully obtained. Although the production of F1 seeds is rare, heterosis and/or weak selection pressure in an empty niche between the parental species' niches may have contributed to the increased proportion of adult F1 hybrids in the hybrid zone.

Adaptations to different environments between closely related species can be important drivers of reproductive isolation during speciation due to either habitat isolation between species or reduced fitness in hybrids that possess suboptimal adaptations. Hybrid zones are useful natural arenas to explore how ecologically divergent species compete for habitat in sympatry and how possible differences in their habitat use may contribute to the speciation process. We investigated habitat selection by sister species of tree squirrels, the Douglas squirrel and the red squirrel, that have evolved in different forest types in allopatry and hybridize in a transitional forest. We first used genome-wide SNP data and admixture analyses to classify individuals into parental or hybrid classes. Next, we estimated home ranges with radio telemetry data and then used a novel ground-based lidar system to measure forest canopy structure of squirrel home ranges, midden sites, and marginally used forest habitat. We found hybrids consisting of multiple hybrid classes were intermixed with both parental species in the same forest with varying canopy structure complexity. On average, Douglas squirrels utilized forests with slightly greater structural complexity than either red squirrels or hybrids, while marginally used forests were the least structurally complex. Interestingly, hybrid squirrels were not relegated to marginal habitat and were successful in mating among each other and with both parental species. As such, our study suggests that prezygotic-ecological isolation and postzygotic-hybrid infertility, and postzygotic ecological inviability of hybrids are not strong barriers in the speciation process between Douglas squirrels and red squirrels. Closely related species that become geographically divided often encounter different environments and thus evolve different adaptations. Sometimes, these species meet again and produce hybrids. Hybrids often have lower fitness due to their inferior adaptations. We studied a pair of closely related squirrels that evolved in different environments and meet and produce hybrids in a secondary contact zone. Despite these species evolving in allopatry in very different forest types, they do not select different forest characteristics while in sympatry in the hybrid zone. Furthermore, hybrids do not show major differences in types of habitats that they choose and are capable of defending territories and reproducing among each other and with both parental species. In summary, prezygotic and postzygotic isolating mechanisms associated with habitat selection do not appear to have an important role in promoting species divergence.

Hybridization is an important evolutionary process, with ecological and behavioural factors influencing gene exchange between hybrids and parent species. Patterns of hybridization in anemonefishes may result from living in highly specialized habitats and breeding status regulated by size-based hierarchal social groups. Here, morphological, ecological and genetic analyses in Kimbe Bay, Papua New Guinea, examine the hybrid status of Amphiprion leucokranos, a nominal species and presumed hybrid between Amphiprion sandaracinos and Amphiprion chrysopterus. We test the hypothesis that habitat use and relative size differences of the parent species and hybrids determine the patterns of gene exchange. There is strong evidence that A.leucokranos is a hybrid of smaller A.sandaracinos and larger A.chrysopterus, where A.chrysopterus is exclusively the mother to each hybrid, based on mtDNA cytochrome b and multiple nDNA microsatellite loci. Overlap in habitat, depth and host anemone use was found, with hybrids intermediate to parents and cohabitation in over 25% of anemones sampled. Hybrids, intermediate in body size, colour and pattern, were classified 55% of the time as morphologically first-generation hybrids relative to parents, whereas 45% of hybrids were more A.sandaracinos-like, suggesting backcrossing. Unidirectional introgression of A.chrysopterus mtDNA into A.sandaracinos via hybrid backcrosses was found, with larger female hybrids and small male A.sandaracinos mating. Potential nDNA introgression was also evident through distinct intermediate hybrid genotypes penetrating both parent species. Findings support the hypothesis that anemonefish hierarchical behaviour, habitat use and species-specific size differences determine how hybrids form and the evolutionary consequences of hybridization.

Quercus cerris L. and Q. suber L. are sympatric for a small portion of their distribution area, where they occasionally hybridize. The hybrid's fitness is low in the wild and there is no significant record of backcrossing; due to occasional reproduction and to the long life span, however, rare individuals of hybrid origin occur scattered in southeastern Europe, even outside of the current range of one or other of the parent species, and can be assigned to the corresponding nothospecies. Several names have been published for this taxon, and others have been variously misapplied to it for more than two centuries, resulting in a true nomenclatural jumble. A thorough examination of the types and protologues of all relevant names leads to the conclusion that Q. × crenata Lam., a name often overlooked, is the correct name for this nothospecies; Q. × hispanica Lam., the name adopted by most authors, is a name of unresolved application, which definitely refers to a different hybrid; all other competing names are junior heterotypic synonyms.

Natural hybridization is a crucial evolutionary process and a long‐standing topic of study in evolutionary biology. Hybrid zones, where two congeneric species interact, can provide insight into the process of natural hybridization, especially with respect to how taxon diversity is maintained. In this study, we used double digest restriction‐site associated DNA sequencing technology (ddRAD‐seq) to examine genetic structure and estimate introgression in four hybrid zones of Ligularia tongolensis and Ligularia cymbulifera. Our analysis demonstrated that parental species were highly differentiated, whereas pairwise FST between parents and their hybrids was low, indicating that sympatric sites can form hybrid zones. As most F1 hybrid individuals were observed within these zones, our finding also implied the presence of substantial barriers to interbreeding. Furthermore, some individuals that possessed the typical morphology of the parental species belonged to the F1 generation. Genomic clines analysis revealed that a large fraction of single nucleotide polymorphisms (SNPs) deviated from a model of neutral introgression in the four hybrid zones, and most SNPs exhibited selection favoring the L. cymbulifera genotype. Bidirectional but asymmetric introgression was revealed as evident in the four hybrid zones. Habitat differences between the four hybrid zones may affect isolation barriers between both species. Taken together, these findings suggest that where incomplete reproductive barriers allow natural hybridization, the introgression between species generates rich genetic recombination that contributes to the fast adaptation and diversification of the widespread Ligularia in the Hengduan Mountains Region (HMR).

Two sinica varieties, Tainung 67(TNG 67) and Taiken 1(TK1), and a wild rice, O. nivara (Acc.104705) were used as the recurrent parents and non-recurrent parent, respectively, to investigate the genetics and breeding behaviors for the fertility, panicle number, grain number of per panicle and 1000-grain weight of the interspecific hybrid progenies of Oryza species. The F1, F2, BC1F1, BC1F2, BC2F1, BC2F2, BC3F1 and BC3F2 populations of two cross combinations were simultaneously planted at the experimental farm of Taiwan Agricultural Research Institute in the first crop of 1996. The following results were obtained. The F1 cross compatibility of two crosses, TNG67×O. nivara and TK1×O. nivara, was 51% and 58% respectively. These results might suggest that no serious reproductive barrier between two scinca varieties and the wild rice. Heterosis was found in both number of panicle per plant and 1000-grain weight for the hybrids. The grain number per panicle was similar to the wild parent. From observing the distribution patterns of F2 population, it was found that there were many plants leaned toward the wild parent, the variation was height and showed transgressive segregation in all of these characters. The results put F2 populations at a disadvantage in selection. In the BC1F1 generation, backcrosses were carried out in both cross combinations based on plant type (similar to cultivated varieties). The selected hybrid plant was ratooned to obtain F2 seeds. The variation in yield components for various backcrosses were then observed. Results indicated that the plants with superior characters would be observed in duplication in various backcross populations. For example, the mean of fertility and 1000-grain weight of BC3F2 and BC3F2 in TK1×O. nivara cross combination were better than the recurrent parent(TK1). Furthermore, as increasing in the number of backcross the variation in the characters became small. This indicated that plants with homogeneous genetic background could be obtained from the populations in a s hort time spectrum. According to the results found, that the wild rice O. nivara could be a germplasm potential for improving cultivated rice yield components, i.e., spikelet fertility, panicle number per plant, grain number of per panicle and 1000-grain weight, by recurrent backcross method should be hypothesized.

Refining the ecological role of stingrays in coral reef ecosystems

Hybrid zones, where two divergent taxa meet and interbreed, offer unique opportunities to investigate how climate contributes to reproductive isolation between closely related taxa and how these taxa may respond to climatic changes. Red‐naped (Sphyrapicus nuchalis) and Red‐breasted (Sphyrapicus ruber) sapsuckers (Aves: Picidae) hybridize along a narrow contact zone that stretches from northern California to British Columbia. The hybrid zone between these species has been studied extensively for more than 100 years and represents an excellent system for investigations of the evolution of reproductive isolation. Shifts in the proportions of phenotypes at hybrid localities since 1910 that were inferred using specimens from museum collections were confirmed using species distribution models. We predicted the historical, current, and future distributions of parental and hybrid sapsuckers using Random Forests models to quantify how climate change is affecting hybrid zone movement in the Pacific Northwest. We found observed distribution shifts of parental sapsuckers were likely the result of climate change over the past 100 years, with these shifts predicted to continue for both sapsuckers over the next 80 years. We found Red‐breasted Sapsuckers are predicted to continue to expand, while Red‐naped Sapsuckers are predicted to contract substantially under future climate scenarios. As a result of the predicted changes, the amount of overlap in the distribution of these sapsuckers may decrease. Using hybrid phenotypes, we found the climate niche occupied by the hybrid zone is predicted to disappear under future conditions. The disappearance of this climate niche where the two parental species come into contact and hybridize may lead to a substantial reduction in genetic introgression. Understanding the impacts of global climate change on hybrid zones may help us to better understand how speciation has been shaped by climate in the past, as well as how evolution may respond to climate change in the future.

Hybridization and introgression are evolutionarily significant phenomena breaking down species boundaries. “Hybrid zones” (regions of species overlap and hybridization) enable quantification of hybridization frequency and examination of mechanisms driving and maintaining gene flow. The hybrid anemonefish Amphiprion leucokranos is found where parent species (A. chrysopterus; A. sandaracinos) distributions overlap. Here, we examine geographic variation in hybridization and introgression, and potential impacts on parent species integrity through assessing relative abundance, social group composition, and genetic structure (mtDNA cytochrome b, 21 microsatellite loci) of taxa at three hybrid zone locations: Kimbe Bay (KB) and Kavieng (KA), Papua New Guinea; the Solomon Islands (SO). Relative abundances of and size disparities between parent species apparently drive hybridization frequency, introgression patterns, and genetic composition of taxa. Conspecific groups are most common in KB (65%) where parent species are similarly abundant. Conversely, mixed species groups dominate SO (82%), where A. chrysopterus is more abundant. Hybrids most commonly cohabit with A. sandaracinos in KB (17%), but with A. chrysopterus in KA (22%) and SO (50%). Genetic differentiation (nDNA) analyses indicate that parent species remain distinct, despite ongoing hybridization and hybrids are genetically similar to A. sandaracinos—resulting from persistent backcrossing with this smallest species. This study shows that hybridization outcomes may depend on the social and ecological context in which taxa hybridize, where relative abundance and disparate size of parent species explain the frequency and patterns of hybridization and introgression in the A. leucokranos hybrid zone, reflecting size‐based dominance behaviors of anemonefish social groups.

The hybrid anemonefish, Amphiprion leucokranos, is known to be the product of ongoing, introgressive hybridization between parent taxa Amphiprion sandaracinos and Amphiprion chrysopterus. Hybridization is an important evolutionary phenomenon contributing to biodiversity within marine systems, where hybrid zones provide ideal systems in which to study hybridization events. Here, a suite of 42 Amphiprion microsatellite markers (including development of 8 novel markers) were cross-amplified in individuals from parent taxa and hybrid populations to facilitate investigation into the relatedness of hybridizing species across the A.leucokranos hybrid zone. Analysis revealed 15, 20 and 24 highly polymorphic loci (PIC > 0.5) in the two parent species and hybrid, respectively, for use in population genetic and parentage studies, with 305 unique alleles found overall (ranging from 1 to 13 alleles per locus) and 7 alleles per locus on average. Observed and expected heterozygosities ranged from 0.000 to 1.000 and 0.000 to 0.978, respectively. Significant deviations from Hardy-Weinberg equilibrium were found in eight loci, possibly due to relatedness among samples or the presence of null alleles. Use of the suite of markers tested here will provide valuable insights into the contemporary population structure and introgression among species and hybrids within the Amphiprion leucokranos hybrid zone, as well as inform future ecological and evolutionary studies of anemonefishes more broadly.