Biological and genetic aspects of crosses between species of the genus Meccus (Hemiptera: Reduviidae Triatominae)

The degree of reproductive isolation between Meccus phyllosomus and the remaining five species of the genus Meccus, as well as between Meccus bassolsae and Meccus pallidipennis, Meccus longipennis and Meccus picturatus, was examined. Fertility and the segregation of morphological characteristics were examined in two generations of hybrids from crosses between these species. The percentage of couples with offspring (fertile) was high in the vast majority of sets of crosses, with the exception of that between ♀M. phyllosomus and ♂Meccus mazzottii. In sets of crosses involving M. bassolsae specimens, no first-generation (F1) individuals were morphologically similar to M. bassolsae, but instead shared the morphology of the other parental species. A similar phenomenon was observed in most sets of crosses involving M. phyllosomus. These results indicated that different degrees of reproductive isolation exist among the species of Meccus involved in this study. The biological evidence obtained in this study does not support the proposal that M. bassolsae is a full species. It could indicate that, on the contrary, it should be considered a subspecies of a single polytypic species. The biological evidence does support the proposal that M. phyllosomus is a full species.     

Biological aspects of crosses between Triatoma recurva (Stål), 1868 (Hemiptera: Reduviidae: Triatominae) and other members of the Phyllosoma complex

The degree of reproductive isolation between Triatoma recurva (Stål) (Hemiptera: Reduviidae: Triatominae) and the six species of the genus Meccus plus T. mexicana (Herrich‐Schaeffer) (Hemiptera: Reduviidae) was examined. Fertility and the segregation of morphological characteristics were examined in two generations of hybrids from crosses between these species. The percentage of couples with offspring (fertile) was low in the vast majority of sets of crosses, with the exception of that between T. recurva female and M. phyllosomus male. In all studied sets of crosses, no first‐ (F1) or second‐ (F2) generation individuals were morphologically similar to T. recurva but instead shared the morphology of the other parental species. A similar phenomenon was observed in the three successful sets of backcrosses. These results indicated that different recorded levels of reproductive fitness among T. recurva and the species of Meccus involved in this study, plus T. mexicana, are present and that they were apparently influenced by differing mechanisms of isolation. The presence of some degree of reproductive compatibility between studied triatomines of distinct genera (Meccus spp. and Triatoma spp.) reinforces the need for generic revision of the tribe Triatomini.     

Ongoing speciation within the Anastrepha fraterculus cryptic species complex: the case of the Andean morphotype

The Anastrepha fraterculus (Wiedemann) (Diptera: Tephritidae) cryptic species complex is currently composed of seven taxonomically recognized morphotypes. Both, pre‐ and post‐zygotic isolation has been documented among four of these morphotypes, revealing that in fact they appear to be distinct biological entities. In order to progress in the full delimitation of species within the complex, we examined reproductive isolation between a Colombian population of the Andean morphotype and populations belonging to four other morphotypes spanning from Mexico to Argentina. Flies from the Andean morphotype exhibited strong pre‐zygotic mating isolation through temporal partitioning of mating activity. Post‐zygotic isolation was observed for crosses of males of all morphotypes and Andean morphotype females, yet most of the F1 hybrid ♂ × F1 hybrid ♀ self‐crosses showed normal levels of fertility, a finding suggesting a nuclear–cytoplasmic interaction according to previous studies. Overall, the Andean morphotype within the complex also appears to be a distinct biological entity. We discuss the implications of these findings for the understanding of speciation mechanisms in the Neotropical genus Anastrepha.     

Genetic architecture of the cryptic species complex of Acanthocyclops vernalis (Crustacea: Copepoda). II. Crossbreeding experiments, cytogenetics, and a model of chromosomal evolution

Collectively, populations of Acanthocyclops vernalis, a species complex of freshwater copepods, are remarkably similar as to morphology and DNA content, despite variability in chromosome number. Reproductive isolation had been reported among some populations, but with each new investigation the species boundaries and factors that may influence them appeared less clear. To clarify the pattern of biological species within this group of populations, we adopted a comprehensive approach and examined patterns of reproductive isolation in populations for which morphology, chromosome number, DNA content, and 18S rDNA sequences are known. In this study we established nine isofemale lines from four sites in Wisconsin and performed 266 crosses. Crosses within and among these lines were used to relate the degree of reproductive isolation to chromosome differences and to construct a model to explain the origin and maintenance of chromosome number variability. Different gametic and somatic chromosome numbers were observed among specimens within some isofemale lines. In a few cases, gametes with different haploid numbers were produced by a single female. Matings within isofemale lines always produced at least some reproductively successful replicate crosses (produced viable, fertile offspring). Crosses between lines from the same site showed reduced success relative to within-line crosses. Crosses between populations from distant sites showed limited genetic compatibility, producing viable, fertile F1 offspring but infertile F2 adults. One cross between lines with different chromosome numbers (one with 2n = 8 and one with 2n = 10) produced fertile viable offspring, which reproduced for at least 60 generations. These hybrids had either eight or nine chromosomes in the third generation of inbreeding, and eight chromosomes after 20 generations. These hybrids also had reduced nuclear DNA contents at the third generation, a level that persisted through the 20th generation. Successful backcrosses between some hybrids and their parental lines further demonstrated the potential for genetic compatibility among forms with different chromosome numbers. We propose a model in which alterations due to Robertsonian fusions, translocations, and/or loss of chromosomal fragments generate heritable variation, only some of which leads to reproductive isolation. Hence, some of the criteria traditionally used to recognize species boundaries in animals (morphology, DNA content, chromosome number) may not apply to this species complex.     

Distribution and infection of triatomines (Hemiptera: Reduviidae) by Trypanosoma cruzi in the state of Michoacán, Mexico

An entomological study of triatomine species was carried out to assess their prevalence in 10 localities of the state of Michoacán, Mexico. Entomological indices were calculated to estimate the risk for vector-borne transmission of Trypanosoma cruzi to the human population in this area. Four triatomine species (Triatoma barberi, Triatoma dimidiata, Meccus pallidipennis and Meccus longipennis) were collected from the study area. This is the first report of M. longipennis and T. dimidiata in Michoacán. M. pallidipennis was significantly (p < 0.05) more abundant than any of the other species collected in the study area. Infection indices were greater than 50% for each of the four collected triatomine species. Significantly more triatomines were collected from intradomiciliary areas than from peridomiciliary or sylvatic areas. Infestation, crowding and density indices were low, whereas colonisation indices were high in five localities. The current vectorial conditions in the study area require continuous entomological and serological surveillance to diminish the risk of T. cruzi transmission to human populations.     

Biology of three species of the Meccus phyllosomus complex (Hemiptera: Reduviidae: Triatominae) fed on blood of hens and rabbits

Aspects related to hatching, life time, number of blood meals to molt, mortality, feeding time and postfeed defecation delay for each instar of Meccus phyllosomus, M. mazzottii, and M. bassolsae, life-cycle were evaluated and compared in two cohorts of each of those three species, fed on hens or rabbits. No significant (p > 0.05) differences were recorded among cohorts fed on hens respect to cohorts fed on rabbits in M. phyllosomus and M. mazzottii and the average time of hatching was 21.5 days for cohorts fed on hens and 22.5 for cohorts fed on rabbits. Average egg-to-adult development times were no significant (p > 0.05) different between both cohorts of M. phyllosomus and M. mazzotti, independent of the blood meal source. The average span in days for each instar fed on hens was not significantly different to the average span for each instar fed on rabbits, when comparisons were made by species. The number of blood meals at each nymphal instar varied from 1 to 6 in both cohorts of each species. The mortality rates were higher on older nymphs, in both cohorts of M. phyllosomus and M. bassolsae, whereas they were higher on first instar nymphs on M. mazzottii. Mean feeding time was no significant (p > 0.05) different in triatomines fed on hens or fed on rabbits, when each species were compared separately. A similar number of nymphs of each cohort, completed the cycle. Defecation delay was no significant (p > 0.05) different when cohorts fed on hens and fed on rabbits were compared by species. Most of the studied parameters showed no significant (p > 0.05) differences among those cohorts fed on hens and for fed on rabbits, which could mean a high degree of association of those species with birds as much as mammals, under wild conditions, increasing their capacity to colonize human dwellings.     

Accelerated mitochondrial evolution and "Darwin's corollary": asymmetric viability of reciprocal F1 hybrids in Centrarchid fishes

Reciprocal crosses between species can yield hybrids with different viabilities. The high frequency of this asymmetric hybrid viability ("Darwin's corollary") places it alongside Haldane's rule and the "large-X effect" as a general feature of postmating reproductive isolation. Recent theory suggests that reciprocal cross asymmetries can arise from stochastic substitutions in uniparentally inherited loci such as mitochondrial genomes, although large systematic differences in mitochondrial substitution rates can also contribute to asymmetries. Although the magnitude of asymmetry will be relatively insensitive to unequal rates of mitochondrial evolution in diverging species, we show here that rate asymmetries can have a large effect on the direction of viability asymmetries. In reciprocal crosses between species, the maternal parent with faster mitochondrial evolution will tend to produce less viable F(1) hybrids owing to an increased probability of mito-nuclear incompatibilities. We test this prediction using data on reciprocal hybrid viability and molecular evolution rates from a clade of freshwater fishes, Centrarchidae. As predicted, species with accelerated mitochondrial evolution tend to be the worse maternal parent for F(1) hybrids, providing the first comparative evidence for a systematic basis to Darwin's corollary. This result is consistent with the hypothesis that mito-nuclear incompatibilities can play an important role in reproductive isolation. Such asymmetrical reproductive isolation may help explain the asymmetrical mitochondrial introgression observed between many hybridizing species. However, as with any comparative study, we cannot rule out the possibility that our results arise from a mutual correlation with a third variable such as body size.     

Genetic divergence and reproductive isolation in the Ochthebius (Calobius) complex (Coleoptera: Hydraenidae)

The reproductive isolation in hydrenid beetles of the Ochthebius complex was studied by analysing gene exchange in natural populations of O. quadricollis, Ochthebius sp. A and O. brevicollis steinbuehleri collected along the Mediterranean coasts. The ranges of these three species are largely allopatric, but sympatric areas were detected between contiguous taxa, ie, O. quadricollis and Ochthebius sp. A; Ochthebius sp. A and O. b. steinbuehleri. Three levels of reproductive isolation and genetic divergence were observed. One level involves extensive intraspecific genetic divergence within the biological species O. quadricollis, Ochthebius sp. A and O. brevicollis, associated with both physical barriers (eg, sea and sand stretches) and the low dispersal capacity of larvae and adults. The finding of transitional samples between the most differentiated population groups should indicate, however, that there is still some gene flow between the populations of the three taxa. Another level is found between Ochthebius sp. A and O. b. steinbuehleri, whose gene pools appear to be fairly distinct in spite of the fact that reproductive isolation is still incomplete: in their few syntopic sites, some F1 hybrids appeared indeed to have lower fitness, since backcrosses or recombinant genotypes were never observed. The final level in the evolution of reproductive isolation (full reproductive isolation) has been achieved by the species O. quadricollis and Ochthebius sp. A. No F1 or F(n) hybrids, nor backcrosses were found in their sympatric areas. The relative importance of ecological factors and evolutionary forces in the prevention of gene exchange between taxa are discussed.     

Evidence of prezygotic isolation,but not assortative mating,between locally adapted populations of Fundulus heteroclitus across a salinity gradient

Selection along environmental gradients can drive reproductive isolation and speciation. Among fishes, salinity is a major factor limiting species distributions, and despite its importance in generating species diversity, speciation events between marine and freshwater are rare. Here, we tested for mechanisms of reproductive isolation between locally adapted freshwater and brackish water-native populations of killifish, Fundulus heteroclitus, from either side of a hybrid zone along a salinity gradient. There was evidence for pre-zygotic endogenous reproductive isolation with reduced fertilization success between crosses of freshwater-native males and brackish water-native females. Exogenous pre-zygotic isolation was also present where females had highest fertilization in their native salinity. We used a replicated mass spawning design to test for mate choice in both brackish and fresh water. After genotyping 187 parents and 2523 offspring at 2347 SNPs across the genome, 85% of offspring were successfully assign to their parents. However, no reinforcing mate choice was observed. These results therefore demonstrate emerging, yet limited, reproductive isolation and incipient speciation across a marine to freshwater salinity gradient and suggest that both endogenous and exogenous mechanisms, but not assortative mating, contribute to divergence.     

POSTZYGOTIC ISOLATION OVER MULTIPLE GENERATIONS OF HYBRID DESCENDENTS IN A NATURAL HYBRID ZONE: HOW WELL DO SINGLE‐GENERATION ESTIMATES REFLECT REPRODUCTIVE ISOLATION?

Understanding speciation depends on an accurate assessment of the reproductive barriers separating newly diverged populations. In several taxonomic groups, prezygotic barriers, especially preferences for conspecific mates, are thought to play the dominant role in speciation. However, the importance of postzygotic barriers (i.e., low fitness of hybrid offspring) may be widely underestimated. In this study, we examined how well the widely used proxy of postzygotic isolation (reproductive output of F₁ hybrids) reflects the long‐term fitness consequences of hybridization between two closely related species of birds. Using 40 species‐specific single nucleotide polymorphism (SNP) markers, we genotyped a mixed population of collared and pied flycatchers (Ficedula albicollis and F. hypoleuca) to identify grand‐ and great grand‐offspring from interspecific crosses to derive an accurate, multigeneration estimate of postzygotic isolation. Two independent estimates of fitness show that hybridization results in 2.4% and 2.7% of the number of descendents typical of conspecific pairing. This postzygotic isolation was considerably stronger than estimates based on F₁ hybrids. Our results demonstrate that, in nature, combined selection against hybrids and backcrossed individuals may result in almost complete postzygotic isolation between two comparatively young species. If these findings are general, postzygotic barriers separating hybridizing populations may be much stronger than previously thought.     

Genetic incompatibilities in killifish and the role of environment

Measuring reproductive isolation across multiple generations and environments is a key endeavor in speciation research because it indicates which isolating barriers currently prevent introgression and the extent to which they are intrinsic versus environmentally dependent. Here, I present data from several crosses (parental crosses, F1s, F2s, back-crosses) between two species of killifish (Lucania goodei and L. parva) that have diverged along a salinity gradient (L. goodei--freshwater, L. parva--euryhaline). Offspring were raised under high and low salinity to test for (1) extrinsic isolation, (2) intrinsic isolation manifested through genetic incompatibilities, and (3) environmentally dependent genetic incompatibilities. I found evidence for both intrinsic and extrinsic isolation, but no evidence for environmentally dependent genetic incompatibilities. The presence of extrinsic and intrinsic isolation varied among fitness measures, and all forms of reproductive isolation were asymmetric. Early egg survival was independent of salinity, but demonstrated pronounced intrinsic isolation. Both extrinsic and intrinsic isolation existed for egg hatching and survival of fry to the eating stage. Unfortunately, the order in which extrinsic and intrinsic isolation arose is unresolved. Understanding the extent to which adaptation to salinity creates multiple forms of reproductive isolation is critical for understanding diversification in many fish taxa.     

Population differentiation in the beetleTribolium castaneum. I. Genetic architecture

We used joint-scaling analyses in conjunction with rearing temperature variation to investigate the contributions of additive, non-additive, and environmental effects to genetic divergence and incipient speciation among 12 populations of the red flour beetle, Tribolium castaneum, with small levels of pairwise nuclear genetic divergence (0.033 < Nei's D < 0.125). For 15 population pairs we created a full spectrum of line crosses (two parental, two reciprocal F1's, four F2's, and eight backcrosses), reared them at multiple temperatures, and analyzed the numbers and developmental defects of offspring. We assayed a total of 219,388 offspring from 5147 families. Failed crosses occurred predominately in F2's, giving evidence of F2 breakdown within this species. In all cases where a significant model could be fit to the data on offspring number, we observed at least one type of digenic epistasis. We also found maternal and cytoplasmic effects to be common components of divergence among T. castaneum populations. In some cases, the most complex model tested (additive, dominance, epistatic, maternal, and cytoplasmic effects) did not provide a significant fit to the data, suggesting that linkage or higher order epistasis is involved in differentiation between some populations. For the limb deformity data, we observed significant genotype-by-environment interaction in most crosses and pure parent crosses tended to have fewer deformities than hybrid crosses. Complexity of genetic architecture was not correlated with either geographic distance or genetic distance. Our results support the view that genetic incompatibilities responsible for postzygotic isolation, an important component of speciation, may be a natural but serendipitous consequence of nonadditive genetic effects and structured populations.     

Asymmetrical crossing barriers in angiosperms

Patterns of reproductive isolation between species may provide insight into the mechanisms and evolution of barriers to interspecific gene exchange. We used data from published interspecific hybridization experiments from 14 genera of angiosperms in order to test for the presence of asymmetrical barriers to gene exchange. Reproductive isolation was examined at three life-history stages: the ability of interspecific crosses to produce seeds, the viability of F1 hybrids, and the fertility of F1 hybrids. Statistically significant asymmetries in the strength of reproductive isolation between species were detected in all genera and at each of the three life-history stages. Asymmetries in seed production may be caused by a variety of mechanisms including differences in stigma/style lengths, self compatibility and differential fruit abortion. Asymmetries in post-zygotic isolation are probably caused by nuclear-cytoplasmic interactions. Asymmetrical reproductive isolation between plant taxa may have important implications for the dynamics of hybrid zones, the direction of genetic introgression and the probability of reinforcement.     

Testing the viviparity-driven-conflict hypothesis: parent-offspring conflict and the evolution of reproductive isolation in a poeciliid fish

The evolution of viviparity increases the potential for genomic conflicts between mothers and offspring over the level of maternal investment. The viviparity-driven-conflict hypothesis predicts that such conflicts will drive the evolution of asymmetrical reproductive isolation between populations with divergent mating systems. We tested this hypothesis using crosses between populations of a poeciliid fish that differ in their level of polyandry. Our results support the prediction of an asymmetry in the rate of spontaneous abortion in reciprocal crosses, with the highest rate occurring in crosses between females from a relatively monandrous population and males from a relatively polyandrous population. The patterns of offspring size were not consistent with the pattern predicted by the viviparity-driven-conflict hypothesis: crosses between a monandrous female and a polyandrous male did not produce larger offspring than the reciprocal cross. This discrepancy was due to the presence of an effect of the maternal population on offspring size: polyandrous females produced larger offspring than monandrous females. In addition, offspring size was positively correlated with maternal size in crosses involving a polyandrous male. We discuss these results in light of models for intra- and intergenomic epistasis and the rapid origin of asymmetric reproductive isolation in viviparous taxa.     

Patterns of Reproductive Isolation in Toads

Understanding the general features of speciation is an important goal in evolutionary biology, and despite significant progress, several unresolved questions remain. We analyzed an extensive comparative dataset consisting of more than 1900 crosses between 92 species of toads to infer patterns of reproductive isolation. This unique dataset provides an opportunity to examine the strength of reproductive isolation, the development and sex ratios of hybrid offspring, patterns of fertility and infertility, and polyploidization in hybrids all in the context of genetic divergence between parental species. We found that the strength of intrinsic postzygotic isolation increases with genetic divergence, but relatively high levels of divergence are necessary before reproductive isolation is complete in toads. Fertilization rates were not correlated to genetic divergence, but hatching success, the number of larvae produced, and the percentage of tadpoles reaching metamorphosis were all inversely related with genetic divergence. Hybrids between species with lower levels of divergence developed to metamorphosis, while hybrids with higher levels of divergence stopped developing in gastrula and larval stages. Sex ratios of hybrid offspring were biased towards males in 70% of crosses and biased towards females in 30% of crosses. Hybrid females from crosses between closely related species were completely fertile, while approximately half (53%) of hybrid males were sterile, with sterility predicted by genetic divergence. The degree of abnormal ploidy in hybrids was positively related to genetic divergence between parental species, but surprisingly, polyploidization had no effect on patterns of asymmetrical inviability. We discuss explanations for these patterns, including the role of Haldane''s rule in toads and anurans in general, and suggest mechanisms generating patterns of reproductive isolation in anurans.     

Reproductive isolation and phylogenetic divergence in Neurospora: comparing methods of species recognition in a model eukaryote

We critically examined methods for recognizing species in the model filamentous fungal genus Neurospora by comparing traditional biological species recognition (BSR) with more comprehensive applications of both BSR and phylogenetic species recognition (PSR). Comprehensive BSR was applied to a set of 73 individuals by performing extensive crossing experiments and delineating biological species based on patterns of reproductive success. Within what were originally considered two species, N. crassa and N. intermedia, we recognized four reproductively isolated biological species. In a concurrent study (Dettman et al. 2003), we used genealogical concordance of four independent nuclear loci to recognize phylogenetic species in Neurospora. Overall, the groups of individuals identified as species were similar whether recognized by reproductive success or by phylogenetic criteria, and increased genetic distance between parents was associated with decreased reproductive success of crosses, suggesting that PSR using genealogical concordance can be used to reliably recognize species in organisms that are not candidates for BSR. In one case, two phylogenetic species were recognized as a single biological species, indicating that significant phylogenetic divergence preceded the development of reproductive isolation. However, multiple biological species were never recognized as a single phylogenetic species. Each of the putative N. crassa x N. intermedia hybrids included in this study was confidently assigned to a single species, using both PSR and BSR. As such, no evidence for a history of hybridization in nature among Neurospora species was observed. By performing reciprocal mating tests, we found that mating type, parental role, and species identity of parental individuals could all influence the reproductive success of matings. We also observed sympatry-associated sexual dysfunction in interspecific crosses, which was consistent with the existence of reinforcement mechanisms.     

The presence of six cryptic species of the whitefly Bemisia tabaci complex in China as revealed by crossing experiments

Abstract Recent phylogenetic analysis using mitochondrial cytochrome oxidase I (mtCOI) sequences of Bemisia tabaci worldwide indicates that the whitefly comprises at least 24 morphologically indistinguishable but genetically distinct cryptic species. While evidence of reproductive isolation has been reported for some of the putative species, more extensive crossing experiments are required to clarify the systematics of this species complex. In this study, we established laboratory cultures for six putative species of B. tabaci collected in China. We conducted 22 inter‐species crosses among the six putative species. The data and those reported previously were collated, and the combined dataset covered all the 30 possible inter‐species crosses among the six putative species. Intra‐species controls always produced female and male progeny and the proportions of females in the first generation (F1) ranged from 56% to 70%. However, in inter‐species crosses female progeny were rarely produced, and the few F1 females produced in four of the 30 inter‐species crosses were either sterile or significantly weaker in viability. These results demonstrate a pattern of complete reproductive isolation among the six putative species and show that they are six cryptic species in the B. tabaci complex.     

Reproductive Success and Body Size in the Cricket Gryllus firmus

Male body size influences mate choice and sexual selection in many animal species. Here we investigate the role of male body size in the reproductive success of the field cricket Gryllus firmus. This species hybridizes with a close smaller relative, G. pennsylvanicus, and it is thought that this size difference may affect reproductive isolation between these species. We paired large and small G. firmus males with a single G. firmus female and genotyped the resulting offspring. Overall, larger males sired a greater proportion of offspring and in a majority of the crosses the larger male sired all of the offspring. For crosses in which both males sired offspring, there was no difference in the proportion of offspring sired by small and large males. Intrasexual competition, female choice, and differences in ejaculates between males could all influence the patterns we observe. We discuss the implications of our findings within the context of reproductive isolation between G. firmus and G. pennsylvanicus.     

Differences in offspring size predict the direction of isolation asymmetry between populations of a placental fish

Crosses between populations or species often display an asymmetry in the fitness of reciprocal F₁ hybrids. This pattern, referred to as isolation asymmetry or Darwin''s Corollary to Haldane''s Rule, has been observed in taxa from plants to vertebrates, yet we still know little about which factors determine its magnitude and direction. Here, we show that differences in offspring size predict the direction of isolation asymmetry observed in crosses between populations of a placental fish, Heterandria formosa. In crosses between populations with differences in offspring size, high rates of hybrid inviability occur only when the mother is from a population characterized by small offspring. Crosses between populations that display similarly sized offspring, whether large or small, do not result in high levels of hybrid inviability in either direction. We suggest this asymmetric pattern of reproductive isolation is due to a disruption of parent–offspring coadaptation that emerges from selection for differently sized offspring in different populations.     

Reproductive isolation of a new hybrid species, Senecio eboracensis Abbott & Lowe (Asteraceae)

The nature and extent of reproductive isolation was examined between a new self-compatible hybrid species Senecio eboracensis (2n=40) and its parents, self-incompatible S. squalidus (2n=20) and self-compatible S. vulgaris (2n=40). The triploid F(1) of S. eboracensis x S. squalidus exhibited very low seed set (x=0.63%), and F(2) and F(3) progeny were able to recover nominal levels of fertility (x=23.9 and 9.7%), while F(1) and F(2) offspring of S. eboracensis x S. vulgaris showed reduced seed set (x=63.8 and 58.8%). In both cases, evidence from previous work indicates that reduced fertility is associated with meiotic chromosome mispairing, and is a likely consequence of recombining both parental genomes within this new taxon. No hybrid offspring between S. eboracensis and S. squalidus were found in the wild, and only one such hybrid was recorded among 769 progeny produced by S. eboracensis surrounded by S. squalidus on an experimental plot. Natural crossing between S. eboracensis and S. vulgaris was recorded to be very low (between 0 and 1.46%) in the wild, but rose to 18.3% when individuals of S. eboracensis were surrounded by plants of S. vulgaris. It was concluded that strong breeding barriers exist between the new hybrid species and its two parents. Prezygotic isolation between S. eboracensis and S. vulgaris is likely to be largely due to both species reproducing by predominant self-fertilisation. However, differences recorded for germination, seedling survival, time of flowering and characters associated with pollinator attraction, plus significant clumping of juvenile and adult conspecifics in the wild, probably also contribute to reproductive isolation and ecological differentiation.