Fitness dynamics within a poplar hybrid zone: II. Impact of exotic sex on native poplars in an urban jungle

Trees bearing novel or exotic gene components are poised to contribute to the bioeconomy for a variety of purposes such as bioenergy production, phytoremediation, and carbon sequestration within the forestry sector, but sustainable release of trees with novel traits in large‐scale plantations requires the quantification of risks posed to native tree populations. Over the last century, exotic hybrid poplars produced through artificial crosses were planted throughout eastern Canada as ornamentals or windbreaks and these exotics provide a proxy by which to examine the fitness of exotic poplar traits within the natural environment to assess risk of exotic gene escape, establishment, and spread into native gene pools. We assessed postzygotic fitness traits of native and exotic poplars within a naturally regenerated stand in eastern Canada (Quebec City, QC). Pure natives (P. balsamifera and P. deltoides spp. deltoides), native hybrids (P. deltoides × P. balsamifera), and exotic hybrids (trees bearing Populus nigra and P. maximowiczii genetic components) were screened for reproductive biomass, yield, seed germination, and fungal disease susceptibility. Exotic hybrids expressed fitness traits intermediate to pure species and were not significantly different from native hybrids. They formed fully viable seed and backcrossed predominantly with P. balsamifera. These data show that exotic hybrids were not unfit and were capable of establishing and competing within the native stand. Future research will seek to examine the impact of exotic gene regions on associated biotic communities to fully quantify the risk exotic poplars pose to native poplar forests.     

Hybridization rapidly reduces fitness of a native trout in the wild

Human-mediated hybridization is a leading cause of biodiversity loss worldwide. How hybridization affects fitness and what level of hybridization is permissible pose difficult conservation questions with little empirical information to guide policy and management decisions. This is particularly true for salmonids, where widespread introgression among non-native and native taxa has often created hybrid swarms over extensive geographical areas resulting in genomic extinction. Here, we used parentage analysis with multilocus microsatellite markers to measure how varying levels of genetic introgression with non-native rainbow trout (Oncorhynchus mykiss) affect reproductive success (number of offspring per adult) of native westslope cutthroat trout (Oncorhynchus clarkii lewisi) in the wild. Small amounts of hybridization markedly reduced fitness of male and female trout, with reproductive success sharply declining by approximately 50 per cent, with only 20 per cent admixture. Despite apparent fitness costs, our data suggest that hybridization may spread due to relatively high reproductive success of first-generation hybrids and high reproductive success of a few males with high levels of admixture. This outbreeding depression suggests that even low levels of admixture may have negative effects on fitness in the wild and that policies protecting hybridized populations may need reconsideration.     

Incipient ecological speciation between successional varieties of a dominant tree involves intrinsic postzygotic isolating barriers

Whereas disruptive selection imposed by heterogeneous environments can lead to the evolution of extrinsic isolating barriers between diverging populations, the evolution of intrinsic postzygotic barriers through divergent selection is less certain. Long‐lived species such as trees may be especially slow to evolve intrinsic isolating barriers. We examined postpollination reproductive isolating barriers below the species boundary, in an ephemeral hybrid zone between two successional varieties of the landscape‐dominant Hawaiian tree, Metrosideros polymorpha, on volcanically active Hawai'i Island. These archipelago‐wide sympatric varieties show the weakest neutral genetic divergence of any taxon pair on Hawai'i Island but significant morphological and ecological differentiation consistent with adaptation to new and old lava flows. Cross‐fertility between varieties was high and included heterosis of F₁ hybrids at the seed germination stage, consistent with a substantial genetic load apparent within varieties through low self‐fertility and a lack of self‐pollen discrimination. However, a partial, but significant, barrier was observed in the form of reduced female and male fertility of hybrids, especially backcross hybrids, consistent with the accumulation of genetic incompatibilities between varieties. These results suggest that partial intrinsic postzygotic barriers can arise through disruptive selection acting on large, hybridizing populations of a long‐lived species.     

Effects of assortative mate choice on the genomic and morphological structure of a hybrid zone between two bird subspecies

Phenotypic differentiation plays an important role in the formation and maintenance of reproductive barriers. In some cases, variation in a few key aspects of phenotype can promote and maintain divergence; hence, the identification of these traits and their associations with patterns of genomic divergence is crucial for understanding the patterns and processes of population differentiation. We studied hybridization between the alba and personata subspecies of the white wagtail (Motacilla alba), and quantified divergence and introgression of multiple morphological traits and 19,437 SNP loci on a 3,000 km transect. Our goal was to identify traits that may contribute to reproductive barriers and to assess how variation in these traits corresponds to patterns of genome‐wide divergence. Variation in only one trait—head plumage patterning—was consistent with reproductive isolation. Transitions in head plumage were steep and occurred over otherwise morphologically and genetically homogeneous populations, whereas cline centres for other traits and genomic ancestry were displaced over 100 km from the head cline. Field observational data show that social pairs mated assortatively by head plumage, suggesting that these phenotypes are maintained by divergent mating preferences. In contrast, variation in all other traits and genetic markers could be explained by neutral diffusion, although weak ecological selection cannot be ruled out. Our results emphasize that assortative mating may maintain phenotypic differences independent of other processes shaping genome‐wide variation, consistent with other recent findings that raise questions about the relative importance of mate choice, ecological selection and selectively neutral processes for divergent evolution.     

A restricted hybrid zone between native and introduced red fox (Vulpes vulpes) populations suggests reproductive barriers and competitive exclusion

Introduced species can threaten native taxa in multiple ways, including competition and hybridization, which can reduce fitness, alter ecological niches or swamp native genomes. Encroachment and hybridization by introduced species also provide opportunities to study the dynamics of invasiveness and hybridization during early stages following contact. We used 33 microsatellites, 51 single nucleotide polymorphisms and a mtDNA marker to characterize the extent and spatial pattern of encroachment and hybridization between a native, endemic subspecies of red fox (Vulpes vulpes patwin) and an introduced red fox population composed of highly admixed, phylogenetically divergent stock, resulting from a century of domestication. Both nuclear and mtDNA markers indicated that hybridization was primarily restricted to a narrow zone where the two populations came into contact. Although a few introgressed genotypes were detected in the interior of the native range, we found no immigrant foxes or F(1) or F(2) hybrids there, suggesting native foxes excluded introduced individuals. We speculate that the observed interbreeding at the periphery was facilitated by low densities. In total, 98% of mtDNA haplotypes in the native range were native and 96% of the nuclear ancestry was estimated to be native. Although the introduced range had expanded fivefold over the past four decades, native and non-native haplotypes from museum samples collected in and near the native range three decades earlier showed a similar geographic distribution as today, suggesting that the native range and hybrid zone were relatively stable. We hypothesize that the monogamous mating system of red foxes and other wild canids may enhance their resistance to hybridization because of greater fitness consequences associated with mate discrimination.     

Quantified reproductive isolation in Heliconius butterflies: Implications for introgression and hybrid speciation

Heliconius butterflies have become a model for the study of speciation with gene flow. For adaptive introgression to take place, there must be incomplete barriers to gene exchange that allow interspecific hybridization and multiple generations of backcrossing. The recent publication of estimates of individual components of reproductive isolation between several species of butterflies in the Heliconius melpomene–H. cydno clade allowed us to calculate total reproductive isolation estimates for these species. According to these estimates, the butterflies are not as promiscuous as has been implied. Differences between species are maintained by intrinsic mechanisms, while reproductive isolation of geographical races within species is mainly due to allopatry. We discuss the implications of this strong isolation for basic aspects of the hybrid speciation with introgression hypothesis.     

Spatio-temporal dynamics of the Allonemobius fasciatus- A. socius mosaic hybrid zone: a 14-year perspective

Long-term studies of hybrid zones can provide valuable insight into a number of questions that have long attracted the attention of evolutionists. These questions range from the stability and fate of hybrid zones to the relative fitness of hybrids. In this paper we report the results of a 14-year survey of the Allonemobius fasciatus-Allonemobius socius hybrid zone. Populations were collected intensively in 1986 and 1987 and then more sporadically through the end of the 1980s and throughout the 1990s. By documenting changes in the genetic composition of populations near and within the zone during this period of time we assessed: the strength of the reproductive isolation between the two species; the relative growth rates (which can be considered a surrogate of relative fitness) of genotype classes corresponding to hybrids and to pure species individuals; and, the power of single-year and multi-year measurements of relative growth rates to predict changes in the genetic composition of mixed populations through time. In brief, we found very large year-to-year variation in the relative growth rates of pure species and hybrid individuals. This variation may reflect the fact that both species are at the edge of their range and perhaps at the limits of their ability to deal with environmental perturbations. As a consequence of the variation, even multi-year estimates of relative growth rates often provided imprecise predictions regarding the future genotypic composition of mixed populations. Despite our limited ability to predict the dynamics of individual populations, some trends are apparent. A. socius, the southern species, has clearly increased in frequency along a transect through the Appalachian Mountains, indicating that the zone is moving north in this region. In contrast, the zone appeared to be more stable along the East Coast transect. Within mixed populations, character-index profiles are often bimodal and stable through time, indicating relatively strong reproductive isolation between the two species that is not being reinforced, nor is it breaking down.     

Dispersal and selection mediate hybridization between a native and invasive species

Hybridization between native and non-native species has serious biological consequences, but our understanding of how dispersal and selection interact to influence invasive hybridization is limited. Here, we document the spread of genetic introgression between a native (Oncorhynchus clarkii) and invasive (Oncorhynchus mykiss) trout, and identify the mechanisms influencing genetic admixture. In two populations inhabiting contrasting environments, non-native admixture increased rapidly from 1984 to 2007 and was driven by surprisingly consistent processes. Individual admixture was related to two phenotypic traits associated with fitness: size at spawning and age of juvenile emigration. Fish with higher non-native admixture were larger and tended to emigrate at a younger age―relationships that are expected to confer fitness advantages to hybrid individuals. However, strong selection against non-native admixture was evident across streams and cohorts (mean selection coefficient against genotypes with non-native alleles (s) = 0.60; s.e. = 0.10). Nevertheless, hybridization was promoted in both streams by the continuous immigration of individuals with high levels of non-native admixture from other hybrid source populations. Thus, antagonistic relationships between dispersal and selection are mediating invasive hybridization between these fish, emphasizing that data on dispersal and natural selection are needed to fully understand the dynamics of introgression between native and non-native species.     

Structure of a mosaic hybrid zone between the field crickets Gryllus firmus and G. pennsylvanicus

Hybrid zones provide insight into the nature of species boundaries and the evolution of barriers to gene exchange. Characterizing multiple regions within hybrid zones is essential for understanding both their history and current dynamics. Here, we describe a previously uncharacterized region of a well‐studied hybrid zone between two species of field crickets, Gryllus pennsylvanicus and G. firmus. We use a combination of mitochondrial DNA sequencing, morphological data, and modeling of environmental variables to identify the ecological factors structuring the hybrid zone and define patterns of hybridization and introgression. We find an association between species distribution and natural habitat; Gryllus pennsylvanicus occupies natural habitat along forest edges and natural clearings, whereas G. firmus occupies more disturbed areas in agricultural and suburban environments. Hybridization and introgression occur across patch boundaries; there is evidence of substantial admixture both in morphological characters and mtDNA, over a broad geographic area. Nonetheless, the distribution of morphological types is bimodal. Given that F₁ hybrids are viable and fertile in the lab, this suggests that strong pre‐zygotic barriers are operating in this portion of the hybrid zone.     

Limited gene exchange between two sister species of leaf beetles within a hybrid zone in the Alps

Analysing genomic variation within and between sister species is a first step towards understanding species boundaries. We focused on two sister species of cold‐resistant leaf beetles, Gonioctena quinquepunctata and G. intermedia, whose ranges overlap in the Alps. A previous study of DNA sequence variation had revealed multiple instances of mitochondrial genome introgression in this region, suggesting recent hybridization between the two species. To evaluate the extent of gene exchange resulting from these hybridization events, we sampled individuals of both species inside and outside the hybrid zone and analysed genomic variation among them using RAD‐seq markers. Individual levels of introgression in the nuclear genome were estimated first by defining species‐specific SNPs (displaying a fixed difference between species) a priori and second by using model‐based methods. Both types of analyses indicated little gene exchange, if any, between species at the level of the nuclear genome. Whereas the first method suggested slightly more gene flow, we argue that it has likely overestimated introgression in the phylogeographic context of this study. We conclude that strong intrinsic barriers prevent genetic exchange at the level of the nuclear genome between the two species. The apparent discrepancy observed between introgression occurring in the nuclear and mitochondrial genomes could be explained by selection acting in favour of the latter. Also, these results have consequences for the phylogeographic study of each species, since we can assume that genetic diversity in the overlapping portion of their ranges is not the product of introgression.     

A hybrid zone dominated by fertile F1s: maintenance of species barriers in Rhododendron

Isolating barriers between interbreeding sympatric or parapatric interfertile species are maintained by processes that occur within their hybrid zones. Although the effects of intrinsic selection on hybrid fitness are well known, less is understood about extrinsic fitness variation. At Tiryal Dag, northeast Turkey, Rhododendron x sochadzeae (R. ponticum x caucasicum) forms large populations in which neither segregation nor backcrossing occur, in habitats intermediate between those of its parents. Using single-copy species-specific random amplified polymorphic DNA and inter simple sequence repeat markers, it was determined that most or all R. x sochadzeae plants are F1s, and that there are many separate genets present. Hand pollination and germination experiments showed that R. x sochadzeae plants can produce viable seed of F2s or backcrosses in either direction. Furthermore, adult backcrosses have been observed in habitats atypical for R. x sochadzeae. From this, all non-F1 hybrid derivatives appear to be eliminated in the hybrid zone at Tiryal Dag as a result of postgermination selection. This absence of post-F1 hybrid derivatives apparently prevents introgression. This type of hybrid population is here termed an F1-dominated hybrid zone (F1DZ), and also occurs in Encelia. The observed dominance of F1s within a narrow habitat range is best explained by habitat-mediated superiority of F1s over all other genotype classes. Therefore, habitat-mediated selection against the second hybrid generation might be preventing interspecific gene flow in R. x sochadzeae. F1DZ formation is postulated to require the formation of F1s in quantity, habitat-mediated superiority in F1s, and highly specific habitat conditions.     

Selection and dispersal in a multispecies oak hybrid zone

The four western North American red oak species (Quercus wislizeni, Q. parvula, Q. agrifolia, and Q. kelloggii) are known to produce hybrid products in all interspecific combinations. However, it is unknown whether hybrids are transitory resulting from interspecific gene flow or whether they are maintained through extrinsic selection. Here, we examine cryptic hybrid structure in Q. wislizeni through a broad region including contact and isolation from three other western North American red oaks using amplified fragment length polymorphism molecular markers. All four species were simultaneously detected in the genetic background of individuals morphologically assigned to Q. wislizeni, although the contribution of Q. kelloggii was minor. In some cases, introgression was detected well outside the region of sympatry with one of the parental species. Molecular structure at the individual level indicated this was due to long-distance pollen dispersal and not to local extinction of parental species. Species admixture proportions were correlated with climatic variables and greater proportions of Q. agrifolia and Q. parvula were present in the genetic background of Q. wislizeni in sites with cooler and more humid summers, corresponding with habitat preferences of the parental species. Partial Mantel tests indicated that climate was more important than distance from pollen source in this association. Despite high levels of introgression, species integrity was maintained in some populations in close proximity to the other species, providing further support to environmental selection in determining population genetic structure. Thus, the contribution of species mixtures to population genetic structure varies across the landscape according to availability of pollen, but more importantly to varying environmental selection pressures that produce a complex pattern of hybrid and pure gene pools.     

Morphological and molecular evidence for hybridization and introgression in a willow (Salix) hybrid zone

Hybrid zones provide biologists with the opportunity to examine genetic and ecological interactions between differentiated populations. Accurate identification of hybrid genealogies is considered a necessary prerequisite to understanding observed patterns of hybridization-related phenomena. We analysed molecular and morphological data from individuals in a hybrid zone between two species of willows (Salix sericea Marshall and S. eriocephala Michaux) and report the use of randomly amplified polymorphic DNA (RAPD), chloroplast DNA (cpDNA), and ribosomal DNA (rDNA) markers, as well as vegetative morphology and foliar chemistry data to identify individuals in terms of hybrid genealogy and to infer the direction and extent of backcrossing and introgression within the hybrid zone. A novel version of a maximum likelihood estimate approach (developed for this study) was used to calculate hybrid index scores from RAPD marker data; this method produced results similar to those obtained using traditional arithmetic methods. Distribution of rDNA, cpDNA, and chemistry data were examined within the graphical context of RAPD-based hybrid index score histograms and principal component analyses (PCA) on RAPD and morphology data. Seven of the 21 plants classified as S. eriocephala in the field were possible introgressants. Another plant presented an unequivocal example of backcrossed S. sericea chemistry and RAPD markers. Inter- and intraspecific chloroplast diversity found within the hybrid zone suggests both historic introgression (perhaps in a glacial refugium), and contemporary hybridization. Patterns of inheritance and expression within the hybrid zone suggest that morphological characters are often not expressed in a simple additive fashion, and problems associated with both morphological and molecular data are considered.     

Absence of conspecific pollen advantage in the dynamics of an Ipomopsis (Polemoniaceae) hybrid zone

The frequency of hybrid formation in angiosperms depends on how often heterospecific pollen is transferred to the stigma and on the success of that heterospecific pollen at fertilizing ovules. Even if heterospecific pollen is capable of effecting fertilization it may perform poorly when conspecific pollen is also available on the stigma. We applied pollen mixtures to stigmas to determine how pollen interactions affect siring success and the frequency of hybrid formation between two species of Ipomopsis (Polemoniaceae) in Colorado. Plants of both parental species and natural hybrids were pollinated with I. aggregata and I. tenuituba pollen in ratios of 100:0, 80:20, 50:50, 20:80, and 0:100 by mass. Plants were homozygous for different alleles at an isozyme marker, allowing us to distinguish the type of pollen parent for 2166 viable seeds from 273 fruits. In contrast to studies of many other hybridizing taxa, there was no evidence of an advantage to conspecific pollen, nor did composition of the stigmatic pollen load affect seed set. Instead, the frequency of seeds sired by a given species was proportional to its representation in the pollen load. In this hybrid zone, both the frequency of first-generation hybrid formation and the relative male fitness of the two parental species should be predictable from the rates of pollen transfer to stigmas.     

Cline coupling and uncoupling in a stickleback hybrid zone

Strong ecological selection on a genetic locus can maintain allele frequency differences between populations in different environments, even in the face of hybridization. When alleles at divergent loci come into tight linkage disequilibrium, selection acts on them as a unit and can significantly reduce gene flow. For populations interbreeding across a hybrid zone, linkage disequilibria between loci can force clines to share the same slopes and centers. However, strong ecological selection on a locus can also pull its cline away from the others, reducing linkage disequilibrium and weakening the barrier to gene flow. We looked for this “cline uncoupling” effect in a hybrid zone between stream resident and anadromous sticklebacks at two genes known to be under divergent natural selection (Eda and ATP1a1) and five morphological traits that repeatedly evolve in freshwater stickleback. These clines were all steep and located together at the top of the estuary, such that we found no evidence for cline uncoupling. However, we did not observe the stepped shape normally associated with steep concordant clines. It thus remains possible that these clines cluster together because their individual selection regimes are identical, but this would be very surprising given their diverse roles in osmoregulation, body armor, and swimming performance.     

Introgression between invasive and native blue mussels (genus Mytilus) in the central California hybrid zone

The ecological and genetic factors determining the extent of introgression between species in secondary contact zones remain poorly understood. Here, we investigate the relative importance of isolating barriers and the demographic expansion of invasive Mytilus galloprovincialis on the magnitude and the direction of introgression with the native Mytilus trossulus in a hybrid zone in central California. We use double‐digest restriction‐site‐associated DNA sequencing (ddRADseq) to genotype 1337 randomly selected single nucleotide polymorphisms and accurately distinguish early and advanced generation hybrids for the first time in the central California Mytilus spp. hybrid zone. Weak levels of introgression were observed in both directions but were slightly more prevalent from the native M. trossulus into the invasive M. galloprovincialis. Few early and advanced backcrossed individuals were observed across the hybrid zone confirming the presence of strong barriers to interbreeding. Heterogeneous patterns of admixture across the zone of contact were consistent with the colonization history of M. galloprovincialis with more extensive introgression in northern localities furthest away from the putative site of introduction in southern California. These observations reinforce the importance of dynamic spatial and demographic expansions in determining patterns of introgression between close congeners, even in those with high dispersal potential and well‐developed reproductive barriers. Our results suggest that the threat posed by invasive M. galloprovincialis is more ecological than genetic as it has displaced, and continues to displace the native M. trossulus from much of central and southern California.     

Fitness consequences of potential assortative mating inside and outside a hybrid zone in Chorthippus parallelus (Orthoptera: Acrididae): implications for reinforcement and sexual selection theory

We have examined the fitness consequences of random and potentially non-random matings within two populations taken from inside, and two from outside a hybrid zone in Chorthippus parallelus. When given the opportunity to mate non-randomly, females from all populations laid egg pods more quickly than females obliged to mate at random. A range of fitness parameters measured on the offspring did not show increased fitness following potential non-random mating for any population. However, in non-hybrid populations, the sons of non-randomly mated females had about twice the mating success of the sons of those females forced to mate at random, suggesting the existence of heritable variation for male reproductive success. Hybrid dysfunction did not occur amongst the offspring of randomly mated hybrid females, demonstrating that the lack of dysfunction within these populations is not due to the evolution of assortative mating within them.     

Introgression patterns in the mosaic hybrid zone between Mytilus edulis and M. galloprovincialis

Hybrid zones are fascinating systems to investigate the structure of genetic barriers. Marine hybrid zones deserve more investigation because of the generally high dispersion potential of planktonic larvae which allows migration on scales unrivalled by terrestrial species. Here we analyse the genetic structure of the mosaic hybrid zone between the marine mussels Mytilus edulis and M. galloprovincialis, using three length-polymorphic PCR loci as neutral and diagnostic markers on 32 samples along the Atlantic coast of Europe. Instead of a single genetic gradient from M. galloprovincialis on the Iberian Peninsula to M. edulis populations in the North Sea, three successive transitions were observed in France. From South to North, the frequency of alleles typical of M. galloprovincialis first decreases in the southern Bay of Biscay, remains low in Charente, then increases in South Brittany, remains high in most of Brittany, and finally decreases again in South Normandy. The two enclosed patches observed in the midst of the mosaic hybrid zone in Charente and Brittany, although predominantly M. edulis-like and M. galloprovincialis-like, respectively, are genetically original in two respects. First, considering only the various alleles typical of one species, the patches show differentiated frequencies compared to the reference external populations. Second, each patch is partly introgressed by alleles of the other species. When introgression is taken into account, linkage disequilibria appear close to their maximum possible values, indicating a strong genetic barrier within all transition zones. Some pre- or postzygotic isolation mechanisms (habitat specialization, spawning asynchrony, assortative fertilization and hybrid depression) have been documented in previous studies, although their relative importance remains to be evaluated. We also provided evidence for a recent migratory 'short-cut' connecting M. edulis-like populations of the Charente patch to an external M. edulis population in Normandy and thought to reflect artificial transfer of spat for aquaculture.     

The establishment of a hybrid zone between red and sika deer (genus Cervus)

Japanese sika deer ( Cervus nippon nippon ) were introduced to Scotland around 80 years (20 generations) ago. The sika phenotype is expanding its range and hybridizing extensively with native red deer ( Cervus elaphus ) leading to the establishment of a hybrid zone. This zone is currently moving and cannot be considered to be at equilibrium. Cervid genotypes and mitochondrial haplotypes were mapped across the sika phenotype range, using diagnostic protein isozymes, microsatellite nuclear DNA markers and RFLPs in mtDNA. These were analysed to estimate heterozygote deficits and nuclear linkage disequilibria and cytonuclear disequilibria in relation to gene frequencies and time since contact. Introgression was found in both taxa and strong linkage disequilibria and heterozygote deficits characterize the populations longest exposed to hybridization. Populations further from the introduction site, where hybridization is facilitated by the dispersal of sika-like stags, show low values for linkage disequilibria and heterozygote deficit. The observed patterns in genotype are explained in terms of assortative mating and a selective advantage of the sika genotype. The genetic integrity of the Scottish mainland red deer is shown to be at risk from the invasion of sika.     

Examination of prezygotic and postzygotic isolating barriers in tropical Ulva (Ulvophyceae,Chlorophyta): evidence for ongoing speciation

Phylogenetic clades based on DNA sequences such as the chloroplast rbcL gene and the nuclear ITS region are frequently used to delimit algal species. However, these molecular markers cannot accurately delimit boundaries among some Ulva species. Although Ulva reticulata and Ulva ohnoi occasionally bloom in tropical to warm‐temperate regions and are clearly distinguishable by their reticulate or plain blade morphology, they have few or no sequence divergences in these molecular markers and form a monophyletic clade. In this study, to clarify the speciation and species delimitation in the U. reticulata‐ohnoi complex clade, reproductive relationships among several sexual strains from the Philippines and Japan including offspring that originated from the type specimen of U. ohnoi were examined by culturing and hybridization in addition to the ITS‐based analysis. As a result, both prezygotic and postzygotic reproductive isolation were revealed to occur between genetically perforated U. reticulata and imperforate U. ohnoi. They were also separated on the basis of sequence analysis of the ITS region. That strongly supports that the two taxa are independent biological species. Although no prezygotic barrier among the Philippine and Japanese strains of U. reticulata was observed, unexpectedly zoospores produced by hybrid sporophytes in some of their combinations mostly failed to develop, indicating partial formation of a postzygotic barrier despite a 0.2% divergence in the ITS sequence. These findings suggest speciation is still ongoing in U. reticulata.