Evolution of pre‐zygotic and post‐zygotic barriers to gene flow among three cryptic species within the Anastrepha fraterculus complex

Tropical tephritids are ideally suited for studies on population divergence and speciation because they include species groups undergoing rapid radiation, in which morphologically cryptic species and sister species are abundant. The fraterculus species group in the Neotropical genus Anastrepha is a case in point, as it is composed of a complex of up to seven A. fraterculus morphotypes proposed to be cryptic species. Here, we document pre‐ and post‐zygotic barriers to gene flow among adults of the Mexican A. fraterculus morphotype and three populations (Argentina, Brazil, and Peru) belonging to two separate morphotypes (Brazilian 1 and Peruvian). We unveiled three forms of pre‐zygotic reproductive isolation resulting in strong assortative mating. In field cages, free‐ranging male and female A. fraterculus displayed a strong tendency to form couples with members of the opposite sex belonging to their own morphotype, suggesting that male pheromone emission, courtship displays, or both intervene in shaping female choice before actual contact and coupling. In addition, males and females of the Peruvian morphotype became receptive and mated significantly later than adults of the Mexican and Brazilian 1 morphotypes. After contact, Mexican females exhibited greater mating discrimination than males when facing adults of the opposite sex belonging to either the Peruvian or the Brazilian 1 morphotype as evidenced by vigorous resistance to penetration once they had been forcefully mounted by heterotypic males. Forced copulations resulted in production of F1 hybrids that were either less viable (and partially fertile) than parental crosses or even sterile. Our results suggest that the Mexican morphotype is a distinct biological entity and that pre‐zygotic reproductive isolation through divergence in courtship or male‐produced pheromone and other mechanisms appear to evolve faster than post‐zygotic isolation in the fraterculus species group.     

Natural and sexual selection against immigrants maintains differentiation among micro‐allopatric populations

Local adaptation to divergent environmental conditions can promote population genetic differentiation even in the absence of geographic barriers and hence lead to speciation. But what mechanisms contribute to reproductive isolation among diverging populations? We tested for natural and sexual selection against immigrants in a fish species inhabiting (and adapting to) nonsulphidic surface habitats, sulphidic surface habitats and a sulphidic cave. Gene flow is strong among sample sites situated within the same habitat type, but low among divergent habitat types. Our results indicate that females of both sulphidic populations discriminate against immigrant males during mate choice. Furthermore, using reciprocal translocation experiments, we document natural selection against migrants between nonsulphidic and sulphidic habitats, whereas migrants between sulphidic cave and surface habitats did not exhibit increased mortality within the same time period. Consequently, both natural and sexual selection may contribute to isolation among parapatric populations, and selection against immigrants may be a powerful mechanism facilitating speciation among locally adapted populations even over very small spatial distances.     

Genetic structure of cherry fruit fly (Rhagoletis cingulata) populations across managed,unmanaged, and natural habitats

The cherry fruit fly (CFF), Rhagoletis cingulata Loew (Diptera: Tephritidae: Trypetini), is endemic to eastern North America and Mexico, where its primary native host is black cherry [Prunus serotina Ehrh. (Rosaceae)]. Cherry fruit fly is also a major economic pest of the fruit of cultivated sweet (Prunus avium L.) and tart (Prunus cerasus L.) cherries. Adult CFF that attack wild black cherry and introduced, domesticated cherries in commercial and abandoned orchards are active at different times of the summer, potentially generating allochronic isolation that could genetically differentiate native from sweet and tart CFF populations. Here, we test for host‐related genetic differences among CFF populations in Michigan attacking cherries in managed, unmanaged, and native habitats by scoring flies for 10 microsatellite loci. Little evidence for genetic differentiation was found across the three habitats or between the northern and southern Michigan CFF populations surveyed in the study. Local gene flow between native black cherry, commercial, and abandoned orchards may therefore be sufficient to overcome seasonal differences in adult CFF activity and prevent differentiation for microsatellites not directly associated with (tightly linked to) genes affecting eclosion time. The results do not support the existence of host‐associated races in CFF and imply that flies attacking native, managed, and unmanaged cherries should be considered to represent a single population for pest management purposes.     

The evolution of host preference in allopatric vs. parapatric populations of Timema cristinae walking-sticks

Divergent habitat preferences can contribute to speciation, as has been observed for host-plant preferences in phytophagous insects. Geographic variation in host preference can provide insight into the causes of preference evolution. For example, selection against maladaptive host-switching occurs only when multiple hosts are available in the local environment and can result in greater divergence in regions with multiple vs. a single host. Conversely, costs of finding a suitable host can select for preference even in populations using a single host. Some populations of Timema cristinae occur in regions with only one host-plant species present (in allopatry, surrounded by unsuitable hosts) whereas others occur in regions with two host-plant species adjacent to one another (in parapatry). Here, we use host choice and reciprocal-rearing experiments to document genetic divergence in host preference among 33 populations of T. cristinae. Populations feeding on Ceanothus exhibited a stronger preference for Ceanothus than did populations feeding on Adenostoma. Both allopatric and parapatric pairs of populations using the different hosts exhibited divergent host preferences, but the degree of divergence tended to be greater between allopatric pairs. Thus, gene flow between parapatric populations apparently constrains divergence. Host preferences led to levels of premating isolation between populations using alternate hosts that were comparable in magnitude to previously documented premating isolation caused by natural and sexual selection against migrants between hosts. Our findings demonstrate how gene flow and different forms of selection interact to determine the magnitude of reproductive isolation observed in nature.     

The evolution of reproductive isolation in spatially structured populations

Abstract.— Recent models of speciation have incorporated population structure and migration into the classic model of speciation in which reproductive isolation arises as a by-product of divergence. In this paper, we expanded these models to explore the joint effects of migration and population subdivision on speciation in a spatially explicit context. The results of our simulation support previous results concerning the influence of population subdivision on the accumulation of reproductive isolation. The simulation also shows that speciation in subdivided populations occurs most rapidly when subpopulations are not strictly allopatric. These results counter the widespread notion that speciation is most likely to occur in allopatric populations and suggest that there are useful insights to be gained by incorporating increasingly realistic types of population structure into models of speciation.     

Speciation of silverside Chirostoma attenuatum (Pisces: Atheriniformes) in Central Mexico

The high speciation rate of Chirostoma in Central Mexico has been associated with allopatric speciation events promoted by the emergence of vicariant barriers in freshwater habitats, as well as by sympatric ecological segregation, common in those species inhabiting lacustrine ecosystems. Through nuclear and mitochondrial markers, this study revealed a speciation process within Chirostoma attenuatum resulting in two evolutionary independent units that coincide with their morphological differentiation, indicating that Chirostoma attenuatum and Chirostoma zirahuen may be considered separate species. This process was the result of vicariance associated with geological dynamics of the region. Phylogeographic findings indicated two speciation stages: early allopatric isolation, during which the isolated populations accumulated unique adaptations, and secondary contact with low migration rate and the maintenance of the evolutionary trajectory. Historical demographic analysis indicated that the two well‐differentiated lineages underwent independent evolutionary histories in their respective lakes. Chirostoma zirahuen from Zirahuen and C. attenuatum from Patzcuaro represent unique and irreplaceable genetic diversity that must to be conserved.     

Patterns of reproductive isolation in a haplodiploid – strong post‐mating,prezygotic barriers among three forms of a social spider mite

In speciation research, much attention is paid to the evolution of reproductive barriers, preventing diverging groups from hybridizing back into one gene pool. The prevalent view is that reproductive barriers evolve gradually as a by‐product of genetic changes accumulated by natural selection and genetic drift in groups that are segregated spatially and/or temporally. Reproductive barriers, however, can also be reinforced by natural selection against maladaptive hybridization. These mutually compatible theories are both empirically supported by studies, analysing relationships between intensity of reproductive isolation and genetic distance in sympatric taxa and allopatric taxa. Here, we present the – to our knowledge – first comparative study in a haplodiploid organism, the social spider mite Stigmaeopsis miscanthi, by measuring premating and post‐mating, pre‐ and post‐zygotic components of reproductive isolation, using three recently diverged forms of the mite that partly overlap in home range. We carried out cross‐experiments and measured genetic distances (mitochondrial DNA and nuclear DNA) among parapatric and allopatric populations of the three forms. Our results show that the three forms are reproductively isolated, despite the absence of premating barriers, and that the post‐mating, prezygotic component contributes most to reproductive isolation. As expected, the strength of post‐mating reproductive barriers positively correlated with genetic distance. We did not find a clear pattern of prezygotic barriers evolving faster in parapatry than in allopatry, although one form did show a trend in line with the ecological and behavioural relationships between the forms. Our study advocates the versatility of haplodiploid animals for investigating the evolution of reproductive barriers.     

Karyotype differentiation and reproductive isolation among natural populations of Drosophila lacertosa

Drosophila lacertosa is widely distributed from northern India to Far East of Russia throughout China. We have studied geographical distributions of three kinds of chromosomal karyotypes, type D (2n=10, 4 pairs of V-shaped metacentric chromosomes and a pair of micro-chromosomes), type L (2n=10, 5V with a pair of large-sized hetero-chromatic autosomes) and type M (2n=10, 5V with middle-sized ones). Type D was found exclusively in local populations of D. lacertosa distributed in Yun-Gui Plateau, southwestern China. Both type L and M have a wide range of distribution, and the former occurred in subtropical regions of China including Taiwan Island, whereas the latter in cool temperate regions of East Asia covering Far East of Russia, Korea and the Japan Islands. A strong premating isolation was detected between flies with type D and those with type L or M. These data demonstrate that genetic differentiation leading to cryptic speciation might have occurred in natural populations of Drosophila lacertosa.     

Morphological and environmental differentiation as prezygotic reproductive barriers between parapatric and allopatric Campanula rotundifolia agg. cytotypes

Background and AimsReproductive isolation and local establishment are necessary for plant speciation. Polyploidy, the possession of more than two complete chromosome sets, creates a strong postzygotic reproductive barrier between diploid and tetraploid cytotypes. However, this barrier weakens between polyploids (e.g. tetraploids and hexaploids). Reproductive isolation may be enhanced by cytotype morphological and environmental differentiation. Moreover, morphological adaptations to local conditions contribute to plant establishment. However, the relative contributions of ploidy level and the environment to morphology have generally been neglected. Thus, the extent of morphological variation driven by ploidy level and the environment was modelled for diploid, tetraploid and hexaploid cytotypes of Campanula rotundifolia agg. Cytotype distribution was updated, and morphological and environmental differentiation was tested in the presence and absence of natural contact zones.MethodsCytotype distribution was assessed from 231 localities in Central Europe, including 48 localities with known chromosome counts, using flow cytometry. Differentiation in environmental niche and morphology was tested for cytotype pairs using discriminant analyses. A structural equation model was used to explore the synergies between cytotype, environment and morphology.Key ResultsTremendous discrepancies were revealed between the reported and detected cytotype distribution. Neither mixed-ploidy populations nor interploidy hybrids were detected in the contact zones. Diploids had the broadest environmental niche, while hexaploids had the smallest and specialized niche. Hexaploids and spatially isolated cytotype pairs differed morphologically, including allopatric tetraploids. While leaf and shoot morphology were influenced by environmental conditions and polyploidy, flower morphology depended exclusively on the cytotype.ConclusionsReproductive isolation mechanisms vary between cytotypes. While diploids and polyploids are isolated postzygotically, the environmental niche shift is essential between higher polyploids. The impact of polyploidy and the environment on plant morphology implies the adaptive potential of polyploids, while the exclusive relationship between flower morphology and cytotype highlights the role of polyploidy in reproductive isolation.     

Divergence in the calling songs between sympatric and allopatric populations of the southern wood cricket Gryllus fultoni (Orthoptera: Gryllidae)

In the eastern United States the wood cricket Gryllus fultoni (Orthoptera: Gryllidae) occurs in sympatry with G. vernalis in an area between eastern Kansas and west of the Appalachian Mountains. Calling songs were recorded from 13 sympatric and allopatric localities. Both field and laboratory recordings showed that chirp rate (CR) and pulse rate (PR) overlapped extensively between allopatric populations of G. fultoni and sympatric populations of G. vernalis; by contrast, there was little or no overlap in these variables between sympatric populations of these two species. Divergence in PR and CR between the two species was thus greater in areas of sympatry than in areas of allopatry. Our field and laboratory studies of G. fultoni calling songs thus demonstrate the pattern expected of character displacement and support the genetic assumptions of this hypothesis. Other possible explanations for the sympatric divergence such as ecological character displacement and clinal variation are discussed.     

Rapid evolution of prezygotic barriers in non‐territorial damselflies

A central question in evolutionary biology concerns the accumulation of reproductive barriers during speciation. However, separating the reproductive barriers that have led to speciation from those that have secondarily accumulated (i.e. after initial divergence) is a widely recognized problem. Ideal candidate species for overcoming this problem are young species, where time for additional barriers to accrue has been limited. In the present study, we add to previous studies investigating the strength of reproductive barriers between the parapatric damselflies Ischnura elegans and Ischnura graellsii by quantifying seven prezygotic barriers between the allopatric pairs of I. elegans and Ischnura genei, as well as I. graellsii and I. genei. Specifically, we measured four premating (temporal, sexual, mechanical _I, and mechanical _II) and three postmating (oviposition success, fecundity, and fertility) barriers using experimental approaches and, for first time, we investigated the mechanisms causing mechanical isolation, which is the strongest reproductive barrier in ischnurans. The findings of the present study support the notion that premating barriers are generally strong and contribute significantly to total reproductive isolation in young lineages (65–98%), although they never solely lead to complete isolation. Asymmetry was generally stronger in premating than in postmating barriers, and was driven mostly through asymmetry in mechanical isolation, which is caused by morphological divergence of secondary sexual appendages. We found that barriers act multiplicatively in all species combinations tested, with the exception of sexual isolation, which was not detected. Our results are consistent with a recent allopatric speciation scenario driven by differences in male anal appendages, either impeding copulation or affecting female preferences. Taken together, the results from this and previous studies in diverse odonate genera suggest that premating barriers have evolved rapidly in ischnuran damselflies and, although reproductive isolation in ischnurans is more commonly the result of several barriers acting together, morphological divergence of secondary sexual appendages appears to be a common factor facilitating premating isolation in this group. © 2014 The Linnean Society of London, Biological Journal of the Linnean Society, 2014, 113 , 485–496.     

Asymmetric reproductive barriers and mosaic reproductive isolation: insights from Misty lake–stream stickleback

Ecological speciation seems to occur readily but is clearly not ubiquitous – and the relative contributions of different reproductive barriers remain unclear in most systems. We here investigate the potential importance of selection against migrants in lake/stream stickleback (Gasterosteus aculeatus) from the Misty Lake system, Canada. This system is of particular interest because one population contrast (Lake vs. Outlet stream) shows very low genetic and morphological divergence, whereas another population contrast (Lake vs. Inlet stream) shows dramatic genetic and morphological divergence apparently without strong and symmetric reproductive barriers. To test whether selection against migrants might solve this “conundrum of missing reproductive isolation”, we performed a fully factorial reciprocal transplant experiment using 225 individually marked stickleback collected from the wild. Relative fitness of the different ecotypes (Lake, Inlet, and Outlet) was assessed based on survival and mass change in experimental enclosures. We found that Inlet fish performed poorly in the lake (selection against migrants in that direction), whereas Lake fish outperformed Inlet fish in all environments (no selection against migrants in the opposite direction). As predicted from their phenotypic and genetic similarity, Outlet and Lake fish performed similarly in all environments. These results suggest that selection against migrants is asymmetric and, together with previous work, indicates that multiple reproductive barriers contribute to reproductive isolation. Similar mosaic patterns of reproductive isolation are likely in other natural systems.     

The diapause response of Rhagoletis pomonella to varying environmental conditions and its significance for geographic and host plant‐related adaptation

The recent shift of Rhagoletis pomonella Walsh (Diptera: Tephritidae) from its ancestral host hawthorn to apple is a model for incipient sympatric speciation in action. Previous studies have shown that changes in the over‐wintering pupal diapause are critical for differentially adapting R. pomonella flies to a difference in the fruiting times of apples vs. hawthorns, generating ecologically based reproductive isolation. Here, we exposed pupae of the hawthorn race to various combinations of pre‐ and over‐wintering rearing conditions and analyzed their effects on eclosion time and genetics. We report certain unexpected results in regards to a combination of brief pre‐winter and over‐wintering periods indicative of gene*environment interactions requiring a reassessment of our current understanding of R. pomonella diapause. We present a hypothesis that involves physiological factors related to stored energy reserves in pupae that influences the depth and duration of Rhagoletis diapause. This ‘pupal energy reserve’ hypothesis can account for our findings and help clarify the role host plant‐related life history adaptation plays in phytophage biodiversity.     

Migratory divides coincide with reproductive barriers across replicated avian hybrid zones above the Tibetan Plateau

Migratory divides are proposed to be catalysts for speciation across a diversity of taxa. However, it is difficult to test the relative contributions of migratory behaviour vs. other divergent traits to reproductive isolation. Comparing hybrid zones with and without migratory divides offers a rare opportunity to directly examine the contribution of divergent migratory behaviour to reproductive barriers. We show that across replicate sampling transects of two pairs of barn swallow (Hirundo rustica) subspecies, strong reproductive isolation coincided with a migratory divide spanning 20 degrees of latitude. A third subspecies pair exhibited no evidence for a migratory divide and hybridised extensively. Within migratory divides, overwintering habitats were associated with assortative mating, implicating a central contribution of divergent migratory behaviour to reproductive barriers. The remarkable geographic coincidence between migratory divides and genetic breaks supports a long‐standing hypothesis that the Tibetan Plateau is a substantial barrier contributing to the diversity of Siberian avifauna.     

Quantifying patterns in the evolution of reproductive isolation

We present a likelihood-based statistical method for examining the pattern or rate of evolution of reproductive isolation. The method uses large empirical datasets to estimate, for a given clade, the average duration of two phases in the divergence of populations. The first phase is a lag phase and refers to the period during which lineages diverge but no detectable reproductive isolation evolves. The second is an accumulation phase, referring to the period during which the magnitude of reproductive isolation between diverging lineages increases. The pattern of evolution is inferred from the relative durations of these two phases. Results of analyses of postzygotic isolation data indicate significant differences among taxa in the pattern of evolution of postzygotic isolation that are consistent with predictions based on genetic differences among these groups. We also examine whether the evolution of postzygotic isolation is best explained by either of two models for the rate of accumulation: a linear model or a quadratic function as may be suggested by recent studies. Our analysis indicates that the appropriateness of either model varies among taxa.     

Life history affects the evolution of reproductive isolation among species of Coreopsis (Asteraceae)

The evolution of reproductive isolation within Coreopsis is investigated by integrating phylogenetic data with estimates of pollen viability of plants from inter- and intraspecific crosses. Three different models that predict F1 fitness are compared. The first uses ITS pairwise distances, the second is based on phylogenetic branch lengths derived from DNA sequences, and the third elaborates on the second model by dividing branch length according to reconstructions of the evolution of life history. This is the first study to use phylogenetic branch-length estimates for predicting levels of reproductive isolation. Estimated branch lengths (model 2) predict hybrid fitness more accurately than simply genetic distance (model 1) but only very slightly. This is probably because the two variables are strongly correlated in Coreopsis. Prediction is substantially improved by allowing evolutionary rates to differ between annual and perennial branches (model 3). A bootstrapping procedure indicates that the life-history effect is statistically significant. The more rapid evolution of reproductive isolation within annual species of Coreopsis may be due to differing mechanisms of reproductive isolation, that is, chromosomal rearrangements rather than genetic incompatibilities.     

A second case of genetic host races in Rhagoletis? A population genetic comparison of sympatric host populations in the European cherry fruit fly,Rhagoletis cerasi

Despite an increasing acceptance in the biological community for sympatric speciation as a mode of species formation, well documented examples of sympatrically evolved ‘incipient species’ remain rare. The sympatric host races of apple maggot, Rhagoletis pomonella (Walsh), represent one of the most prominent case studies for sympatric speciation via a host shift. The European cherry fruit fly, R. cerasi (L.), shows strong ecological similarities to R. pomonella: (1) infestation of two different host plants, Lonicera xylosteum L. and Prunus avium L., and (2) divergent phenological and behavioral adaptations of flies on different hosts. The population genetic study presented here addresses whether the host associated populations of R. cerasi also represent genetically differentiated true host races. Out of a total of 29 allozyme loci examined, six were polymorphic and used to analyze six sympatric pairs of R. cerasi populations on Lonicera and Prunus from Switzerland and Germany. A direct comparison of allele frequencies between sympatric sites showed no pattern indicative of host races in R. cerasi. However, the hierarchical F‐statistic for one locus, mannose 6‐phosphate isomerase (Mpi), showed significant population differentiation that was in accordance with host race differentiation. Mpi is one of several loci that are also diagnostic for host race differentiation in R. pomonella. Results from Mpi suggest the formation of sympatric host races in R. cerasi, but additional polymorphic markers are necessary.     

Flies across the water: genetic differentiation and reproductive isolation in allopatric desert Drosophila

Between sister species of Drosophila, both pre- and postzygotic reproductive isolation commonly appear by the time a Nei's genetic distance of 0.5 is observed. The degree of genetic differentiation present when allopatric populations of the same Drosophila species exhibit incipient reproductive isolation has not been systematically investigated. Here we compare the relationship between genetic differentiation and pre- and postzygotic isolation among allopatric populations of three cactophilic desert Drosophila: D. mettleri, D. nigrospiracula, and D. mojavensis. The range of all three is interrupted by the Gulf of California, while two species, D. mettleri and D. mojavensis, have additional allopatric populations residing on distant Santa Catalina Island, off the coast of southern California. Significant population structure exists within all three species, but only for allopatric populations of D. mojavensis is significant isolation at the prezygotic level observed. The genetic distances for the relevant populations of D. mojavensis were in the range of 0.12, similar to that for D. mettleri whose greatest D = 0.11 was unassociated with any form of isolation. These observations suggest further investigations of Drosophila populations with genetic distances in this range be undertaken to identify any potential patterns in the relationship between degree of genetic differentiation and the appearance of pre- and/or postzygotic isolation.     

On the evolution of heavy-metal tolerant populations in Armeria maritima: evidence from allozyme variation and reproductive barriers

The process of ecological differentiation leading to the evolution of heavy-metal tolerant populations in Armeria maritima was studied by comparing population genetic structure and pattern of gene flow between populations growing on heavy-metal contaminated against non-contaminated sites using allozyme markers. In addition the evolution of reproductive isolation among populations was studied by measuring pollen fertility in interpopulational hybrids. The allozyme data suggested that in A. maritima multiple independent evolutionary origins of heavy-metal tolerant populations have occurred in the absence of strong genetic bottlenecks. The pattern of gene flow among populations was consistent with the model of isolation by distance with considerable gene flow between neighbor populations, and no reduction of gene flow between tolerant and non-tolerant populations. Hence it appears that substantial gene flow has not hampered genetic differentiation, probably because of the high selection pressure for heavy-metal tolerance. The pattern of reproductive isolation among populations suggests that evolution of heavy-metal tolerant populations has not triggered the development of reproductive barriers against non-tolerant populations. However, partial reproductive isolation has occurred under geographic separation.     

Evolution of intrinsic reproductive isolation among four North American populations of Rhagoletis pomonella (Diptera: Tephritidae)

Across its range in North America, four geographically separated, ecologically and genetically diverged populations of hawthorn (Crataegus)‐infesting Rhagoletis pomonella (Diptera: Tephritidae) flies inhabit the Eje Volcánico Trans Mexicano (EVTM), the Sierra Madre Oriental (SMO), the Chiapas Highlands (CHIS) and the USA. Here, we tested whether these four populations are reproductively isolated by any intrinsic, nonhost‐related, pre‐ or postmating barriers to gene flow. Crossing experiments suggested that a low level of host‐independent prezygotic isolation may exist between hawthorn flies from EVTM and the three other populations, but only with respect to a slight reduction in copulation duration in EVTM matings. Some evidence for postmating isolation was found, again primarily involving EVTM crossed to SMO, CHIS and US flies. Certain crosses produced no (SMO male × EVTM female) or few (EVTM male × CHIS female; CHIS male × SMO female) F1 hybrid offspring. F2 crosses were generally fertile, except for US male × CHIS female matings. Inherent reproductive isolation therefore appears to be quantitative rather than absolute between populations, as the possibility for gene flow exists through at least some combinations of mating among EVTM, SMO, CHIS and US flies. Our results are consistent with a recently advanced hypothesis that episodic introgression from Mexico into the USA has played a role in providing genetic variation, facilitating sympatric host race formation and the adaptive radiation of the R. pomonella sibling species' complex in the USA. © 2010 The Linnean Society of London, Biological Journal of the Linnean Society, 2010, 100 , 213–223.