Divergent host-plant use promotes reproductive isolation among cynipid gall wasp populations

Ecological speciation occurs when reproductive isolation evolves as a consequence of divergent natural selection among environments. A direct prediction of this process is that ecologically divergent pairs of populations will exhibit greater reproductive isolation than ecologically similar pairs of populations. By comparing allopatric populations of the cynipid gall wasp Belonocnema treatae infesting Quercus virginiana and Quercus geminata, we tested the role that divergent host use plays in generating ecological divergence and sexual isolation. We found differences in body size and gall structure associated with divergent host use, but no difference in neutral genetic divergence between populations on the same or different host plant. We observed significant assortative mating between populations from alternative host plants but not between allopatric populations on the same host plant. Thus, we provide evidence that divergent host use promotes speciation among gall wasp populations.     

Perspective: Reproductive isolation caused by natural selection against immigrants from divergent habitats

The classification of reproductive isolating barriers laid out by Dobzhansky and Mayr has motivated and structured decades of research on speciation. We argue, however, that this classification is incomplete and that the unique contributions of a major source of reproductive isolation have often been overlooked. Here, we describe reproductive barriers that derive from the reduced survival of immigrants upon reaching foreign habitats that are ecologically divergent from their native habitat. This selection against immigrants reduces encounters and thus mating opportunities between individuals from divergently adapted populations. It also reduces the likelihood that successfully mated immigrant females will survive long enough to produce their hybrid offspring. Thus, natural selection against immigrants results in distinctive elements of premating and postmating reproductive isolation that we hereby dub "immigrant inviability." We quantify the contributions of immigrant inviability to total reproductive isolation by examining study systems where multiple components of reproductive isolation have been measured and demonstrate that these contributions are frequently greater than those of traditionally recognized reproductive barriers. The relevance of immigrant inviability is further illustrated by a consideration of population-genetic theory, a review of selection against immigrant alleles in hybrid zone studies, and an examination of its participation in feedback loops that influence the evolution of additional reproductive barriers. Because some degree of immigrant inviability will commonly exist between populations that exhibit adaptive ecological divergence, we emphasize that these barriers play critical roles in ecological modes of speciation. We hope that the formal recognition of immigrant inviability and our demonstration of its evolutionary importance will stimulate more explicit empirical studies of its contributions to speciation.     

Parallel Patterns of Morphological and Behavioral Variation among Host-Associated Populations of Two Gall Wasp Species

A powerful approach to address the general factors contributing to ecological speciation is to compare distantly related taxa that inhabit the same selective environments. In this design, similarities among taxa can elucidate general mechanisms of the process whereas differences may uncover specific factors important to the process for individual taxa. Herein, we present evidence of parallel patterns of morphological and behavioral variation among host-associated populations of two species of cynipid gall wasps, Belonocnema treatae and Disholcaspis quercusvirens, that each exhibit a life cycle intimately tied to the same two host plant environments, Quercus geminata and Q. virginiana. Across both gall-former species we find consistent differences in body size and gall morphology associated with host plant use, as well as strong differences in host plant preference, a measure of habitat isolation among populations. These consistent differences among taxa highlight the important role of host plant use in promoting reproductive isolation and morphological variation among herbivorous insect populations–a prerequisite for ecological speciation.     

Immigrant reproductive dysfunction facilitates ecological speciation

The distributions of species are not only determined by where they can survive – they must also be able to reproduce. Although immigrant inviability is a well‐established concept, the fact that immigrants also need to be able to effectively reproduce in foreign environments has not been fully appreciated in the study of adaptive divergence and speciation. Fertilization and reproduction are sensitive life‐history stages that could be detrimentally affected for immigrants in non‐native habitats. We propose that “immigrant reproductive dysfunction” is a hitherto overlooked aspect of reproductive isolation caused by natural selection on immigrants. This idea is supported by results from experiments on an externally fertilizing fish (sand goby, Pomatoschistus minutus). Growth and condition of adults were not affected by non‐native salinity whereas males spawning as immigrants had lower sperm motility and hatching success than residents. We interpret these results as evidence for local adaptation or acclimation of sperm, and possibly also components of paternal care. The resulting loss in fitness, which we call “immigrant reproductive dysfunction,” has the potential to reduce gene flow between populations with locally adapted reproduction, and it may play a role in species distributions and speciation.     

Partitioning of herbivore hosts across time and food plants promotes diversification in the Megastigmus dorsalis oak gall parasitoid complex

Communities of insect herbivores and their natural enemies are rich and ecologically crucial components of terrestrial biodiversity. Understanding the processes that promote their origin and maintenance is thus of considerable interest. One major proposed mechanism is ecological speciation through host‐associated differentiation (HAD), the divergence of a polyphagous species first into ecological host races and eventually into more specialized daughter species. The rich chalcid parasitoid communities attacking cynipid oak gall wasp hosts are structured by multiple host traits, including food plant taxon, host gall phenology, and gall structure. Here, we ask whether the same traits structure genetic diversity within supposedly generalist parasitoid morphospecies. We use mitochondrial DNA sequences and microsatellite genotypes to quantify HAD for Megastigmus (Bootanomyia) dorsalis, a complex of two apparently generalist cryptic parasitoid species attacking oak galls. Ancient Balkan refugial populations showed phenological separation between the cryptic species, one primarily attacking spring galls, and the other mainly attacking autumn galls. The spring species also contained host races specializing on galls developing on different host‐plant lineages (sections Cerris vs. Quercus) within the oak genus Quercus. These results indicate more significant host‐associated structuring within oak gall parasitoid communities than previously thought and support ecological theory predicting the evolution of specialist lineages within generalist parasitoids. In contrast, UK populations of the autumn cryptic species associated with both native and recently invading oak gall wasps showed no evidence of population differentiation, implying rapid recruitment of native parasitoid populations onto invading hosts, and hence potential for natural biological control. This is of significance given recent rapid range expansion of the economically damaging chestnut gall wasp, Dryocosmus kuriphilus, in Europe.     

Immigrant inviability in yellow pecan aphid

1. Host‐associated differentiation (HAD) may be an important driver of parasite biodiversity. The sympatric occurrence of host‐associated populations features prominently in studies documenting HAD but this neither confirms nor denies that the lineages arose in sympatry. It does raise the question of what maintains such structure despite the proximity of populations in space and time. 2. We tested for immigrant inviability in yellow pecan aphid Monelliopsis pecanis Bissell, a species previously shown to be host associated, using a laboratory reciprocal transplant experiment on pecan and water hickory. 3. Immigrant inviability on the alternative host is present in yellow pecan aphid. Immigrants to both hosts were significantly less fecund than residents and their nymphs were significantly less viable. Future studies should establish the genetic and non‐genetic (maternal effects) basis of immigrant inviability in yellow pecan aphid in order to be certain about its contribution to pre‐mating reproductive isolation. 4. Since aphids are cyclic parthenogens, inviability of both immigrants and their offspring has the potential to contribute to total pre‐mating reproductive isolation. This is different than the case of obligate sexually reproducing organisms where offspring viability is a post‐mating reproductive isolating mechanism. We discuss the implications of this for estimating the contributions of immigrant and offspring inviability to total reproductive isolation in aphids.     

Immigrant and extrinsic hybrid seed inviability contribute to reproductive isolation between forest and dune ecotypes of Epipactis helleborine (Orchidaceae)

Reproductive isolation caused by divergent natural selection arising from differences between ecological environments or ecological interactions represents a major mechanism contributing to speciation, but its relative importance is poorly known. In this study, controlled reciprocal crossings and seed germination experiments were combined with genetic and morphometric analyses to test the hypothesis that previously described differences in mycorrhizal communities between forest and dune ecotypes of Epipactis helleborine were sufficiently strong to create complete reproductive isolation between the two ecotypes. Molecular analyses using 770 SNP markers showed that the two ecotypes were genetically distinct and that populations of the dune ecotype were genetically impoverished compared to populations of the forest ecotype. Morphologically, the two ecotypes were also significantly different, with plants of the dune ecotype generally being smaller than plants of the forest ecotype. The results further showed that immigrant seeds had a significantly lower probability of protocorm formation than native seeds, indicating strong immigrant inviability. Although both ecotypes were able to cross easily and to produce a large number of viable seeds, hybrid seeds showed significantly lower protocorm formation than pure seeds, further contributing to reproductive isolation. Overall, these results indicate that interfertile populations of a widespread orchid adapting to contrasting environments diverge as a consequence of concurrent selection acting against immigrants and hybrids and suggest that mycorrhizal fungi can play a role in the early stages of plant speciation.     

Cascading reproductive isolation: Plant phenology drives temporal isolation among populations of a host‐specific herbivore

All organisms exist within a complex network of interacting species, thus evolutionary change may have reciprocal effects on multiple taxa. Here, we demonstrate “cascading reproductive isolation,” whereby ecological differences that reduce gene flow between populations at one trophic level affect reproductive isolation (RI) among interacting species at the next trophic level. Using a combination of field, laboratory and common‐garden studies and long‐term herbaria records, we estimate and evaluate the relative contribution of temporal RI to overall prezygotic RI between populations of Belonocnema treatae, a specialist gall‐forming wasp adapted to sister species of live oak (Quercus virginiana and Q. geminata). We link strong temporal RI between host‐associated insect populations to differences between host plant budbreak phenology. Budbreak initiates flowering and the production of new leaves, which are an ephemeral resource critical to insect reproduction. As flowering time is implicated in RI between plant species, budbreak acts as a “multitrophic multi‐effect trait,” whereby differences in budbreak phenology contribute to RI in plants and insects. These sister oak species share a diverse community of host‐specific gall‐formers and insect natural enemies similarly dependent on ephemeral plant tissues. Thus, our results set the stage for testing for parallelism in a role of plant phenology in driving temporal cascading RI across multiple species and trophic levels.     

Divergent natural selection drives the evolution of reproductive isolation in an Australian wildflower

Ecological speciation occurs when reproductive isolation evolves between populations adapting to contrasting environments. A key prediction of this process is that the fitness of hybrids between divergent populations should be reduced in each parental environment as a function of the proportion of local genes they carry, a process resulting in ecologically dependent reproductive isolation (RI). To test this prediction, we use reciprocal transplant experiments between adjacent populations of an Australian wildflower, Senecio lautus, at two locations to distinguish between ecologically dependent and intrinsic genetic reproductive barriers. These barriers can be distinguished by observing the relative fitness of reciprocal backcross hybrids, as they differ in the contribution of genes from either parent while controlling for any intrinsic fitness effects of hybridization. We show ecologically dependent fitness effects in establishment and survival of backcrosses in one transplant experiment, and growth performance in the second transplant experiment. These results suggest natural selection can create strong reproductive barriers that maintain differentiation between populations with the potential to interbreed, and implies a significant role for ecology in the evolutionary divergence of S. lautus.     

Detection of ecological hybrid inviability in a pair of sympatric phytophagous ladybird beetles (Henosepilachna spp.)

Ecological speciation is a process by which reproductive isolation evolves as the result of divergent natural selection between populations inhabiting distinct environments or exploiting alternative resources. Ecological hybrid inviability provides direct evidence for ecological speciation. To detect ecological hybrid inviability, we examined survival rates to the second instar of F1 hybrids and backcross hybrids in a pair of sympatric phytophagous ladybird beetles, Henosepilachna niponica Lewis and Henosepilachna yasutomii Katakura (Coleoptera: Coccinellidae: Epilachninae), reared on their respective host plants, thistle [Cirsium alpicola Nakai (Asteraceae)] and blue cohosh [Caulophyllum robustum Maxim. (Berberidaceae)], and on a common food plant, Japanese nightshade [Solanum japonense Nakai (Solanaceae)]. Hybrid larvae reared on leaves of the Japanese nightshade always had high rates of survival, irrespective of the crossing type of their parents, suggesting a lack of intrinsic hybrid inviability between the two species. In contrast, survival rates on thistle and blue cohosh varied greatly. On blue cohosh, the survival rate of F1 hybrids was nearly as high as that of H. yasutomii, but on thistle, survival was significantly lower than of H. niponica. Survival rates of backcross hybrids on the two host plants were intermediate between those of the parents, showing a reversed rank order of different types of backcross hybrids on the two food plant species. These results suggest that ecological hybrid inviability exists between H. niponica and H. yasutomii, although the two species do not show intrinsic hybrid inviability. Thus, our study supports the hypothesis that H. niponica and H. yasutomii underwent ecological speciation by divergent selection.     

Divergent natural selection promotes immigrant inviability at early and late stages of evolutionary divergence

Natural selection's role in speciation has been of fundamental importance since Darwin first outlined his theory. Recently, work has focused on understanding how selection drives trait divergence, and subsequently reproductive isolation. “Immigrant inviability,” a barrier that arises from selection against immigrants in their nonnative environment, appears to be of particular importance. Although immigrant inviability is likely ubiquitous, we know relatively little about how selection acts on traits to drive immigrant inviability, and how important immigrant inviability is at early‐versus‐late stages of divergence. We present a study evaluating the role of predation in the evolution of immigrant inviability in recently diverged population pairs and a well‐established species pair of Brachyrhaphis fishes. We evaluate performance in a high‐predation environment by assessing survival in the presence of a predator, and swimming endurance in a low‐predation environment. We find strong signatures of local adaptation and immigrant inviability of roughly the same magnitude both early and late in divergence. We find remarkably conserved selection for burst‐speed swimming (important in predator evasion), and selection for increased size in low‐predation environments. Our results highlight the consistency with which selection acts during speciation, and suggest that similar factors might promote initial population differentiation and maintain differentiation at late stages of divergence.     

Do assortative mating and immigrant inviability help maintain population genetic structuring of an herbivore on a crop and a wild relative?

Population genetic structuring is common among herbivorous insects and frequently is associated with divergent host plants, such as crops and their wild relatives. Previous studies showed population genetic structuring in corn leafhopper Dulbulus maidis in Mexico, such that the species consists of two sympatric, host plant-associated populations: an abundant and widespread "pestiferous” population on maize (Zea mays mays), and a small and localized "wild" population on perennial teosinte (Zea diploperennis). a maize wild relative with a limited distribution. This study addressed whether assortative mating and immigrant inviability mediate genetic structuring of corn leafliopper by comparing the mating and reproductive successes of pestiferous and wild females that colonize their nonassociated host plants against the successes of females colonizing their associated host plants. Assortative mating was assessed by comparing mating frequencies and premating and mating times among females of each population on each host plant: immigrant inviability was assessed by comparing, across two generations, the fecundity, survival, development time, sex ratio, and population growth rate among leafhopper populations and host plants. Our results showed that on maize, and compared to resident, pestiferous females, wild females were more likely to mate, and greater proportions of their offspring survived to adult stage and were daughters;consequently, the per-generation population growth rate on maize was greater for immigrant, wild leafhoppers compared to resident, pestiferous leafhoppers. Our results suggested that wild leafhoppers emigrating to maize have a fitness advantage over resident, pestiferous leafhoppers, while immigrant pestiferous and resident wild leafhoppers on teosinte have similar fitnesses.     

Batesian mimicry promotes pre‐ and postmating isolation in a snake mimicry complex

We evaluated whether Batesian mimicry promotes early‐stage reproductive isolation. Many Batesian mimics occur not only in sympatry with their model (as expected), but also in allopatry. As a consequence of local adaptation within both sympatry (where mimetic traits are favored) and allopatry (where nonmimetic traits are favored), divergent, predator‐mediated natural selection should disfavor immigrants between these selective environments as well as any between‐environment hybrids. This selection might form the basis for both pre‐ and postmating isolation, respectively. We tested for such selection in a snake mimicry complex by placing clay replicas of sympatric, allopatric, or hybrid phenotypes in both sympatry and allopatry and measuring predation attempts. As predicted, replicas with immigrant phenotypes were disfavored in both selective environments. Replicas with hybrid phenotypes were also disfavored, but only in a region of sympatry where previous studies have detected strong selection favoring precise mimicry. By fostering immigrant inviability and ecologically dependent selection against hybrids (at least in some habitats), Batesian mimicry might therefore promote reproductive isolation. Thus, although Batesian mimicry has long been viewed as a mechanism for convergent evolution, it might play an underappreciated role in fueling divergent evolution and possibly even the evolution of reproductive isolation and speciation.     

Reproductive isolation due to prezygotic isolation and postzygotic cytoplasmic incompatibility in parasitoid wasps

The reproductive barriers that prevent gene flow between closely related species are a major topic in evolutionary research. Insect clades with parasitoid lifestyle are among the most species‐rich insects and new species are constantly described, indicating that speciation occurs frequently in this group. However, there are only very few studies on speciation in parasitoids. We studied reproductive barriers in two lineages of Lariophagus distinguendus (Chalcidoidea: Hymenoptera), a parasitoid wasp of pest beetle larvae that occur in human environments. One of the two lineages occurs in households preferably attacking larvae of the drugstore beetle Stegobium paniceum (“DB‐lineage”), the other in grain stores with larvae of the granary weevil Sitophilus granarius as main host (“GW‐lineage”). Between two populations of the DB‐lineage, we identified slight sexual isolation as intraspecific barrier. Between populations from both lineages, we found almost complete sexual isolation caused by female mate choice, and postzygotic isolation, which is partially caused by cytoplasmic incompatibility induced by so far undescribed endosymbionts which are not Wolbachia or Cardinium. Because separation between the two lineages is almost complete, they should be considered as separate species according to the biological species concept. This demonstrates that cryptic species within parasitoid Hymenoptera also occur in Central Europe in close contact to humans.     

Reproductive system,social organization,human disturbance and ecological dominance in native populations of the little fire ant,Wasmannia auropunctata

The invasive ant species Wasmannia auropunctata displays both ecologically dominant and non‐dominant populations within its native range. Three factors could theoretically explain the ecological dominance of some native populations of W. auropunctata: (i) its clonal reproductive system, through demographic and/or adaptive advantages; (ii) its unicolonial social organization, through lower intraspecific and efficient interspecific competition; (iii) the human disturbance of its native range, through the modification of biotic and abiotic environmental conditions. We used microsatellite markers and behavioural tests to uncover the reproductive modes and social organization of dominant and non‐dominant native populations in natural and human‐modified habitats. Microsatellite and mtDNA data indicated that dominant and non‐dominant native populations (supercolonies as determined by aggression tests) of W. auropunctata did not belong to different evolutionary units. We found that the reproductive system and the social organization are neither necessary nor sufficient to explain W. auropunctata ecological dominance. Dominance rather seems to be set off by unknown ecological factors altered by human activities, as all dominant populations were recorded in human‐modified habitats. The clonal reproductive system found in some populations of W. auropunctata may however indirectly contribute to its ecological dominance by allowing the species to expand its environmental niche, through the fixation over time of specific combinations of divergent male and female genotypes. Unicoloniality may rather promote the range expansion of already dominant populations than actually trigger ecological dominance. The W. auropunctata model illustrates the strong impact of human disturbance on species’ ecological features and the adaptive potential of clonal reproductive systems.     

Ecological speciation in an island snail: evidence for the parallel evolution of a novel ecotype and maintenance by ecologically dependent postzygotic isolation

Speciation is the process by which reproductive isolation evolves between populations. Two general models of speciation have been proposed: ecological speciation, where reproductive barriers evolve due to ecologically based divergent selection, and mutation‐order speciation, where populations fix different mutations as they adapt to similar selection pressures. I evaluate these alternative models and determine the progress of speciation in a diverse group of land snails, genus Rhagada, inhabiting Rosemary Island. A recently derived keeled‐flat morphotype occupies two isolated rocky hills, while globose‐shelled snails inhabit the surrounding plains. The study of one hill reveals that they are separated by a narrow hybrid zone. As predicted by ecological speciation theory, there are local and landscape level associations between shell shape and habitat, and the morphological transition coincides with a narrow ecotone between the two distinct environments. Microsatellite DNA revealed a cline of hybrid index scores much wider than the morphological cline, further supporting the ecological maintenance of the morphotypes. The hybrid zone does not run through an area of low population density, as is expected for mutation‐order hybrid zones, and there is a unimodal distribution of phenotypes at the centre, suggesting that there is little or no prezygotic isolation. Instead, these data suggest that the ecotypes are maintained by ecologically dependent postzygotic isolation (i.e. ecological selection against hybrids). Mitochondrial and Microsatellite DNA indicate that the keeled‐flat form evolved recently, and without major historical disruptions to gene flow. The data also suggest that the two keeled‐flat populations, inhabiting similar rocky hills, have evolved in parallel. These snails provide a complex example of ecological speciation in its early stages.     

Divergent host plant specialization as the critical driving force in speciation between populations of a phytophagous ladybird beetle

Detecting the isolating barrier that arises earliest in speciation is critically important to understanding the mechanism of species formation. We tested isolating barriers between host races of a phytophagous ladybird beetle, Henosepilachna diekei (Coleoptera: Coccinellidae: Epilachnine), that occur sympatrically on distinct host plants. We conducted field surveys for the distribution of the beetles and host plants, rearing experiments to measure six potential isolating factors (adult host preference, adult and larval host performance, sexual isolation, egg hatchability, F(1) hybrid inviability, and sexual selection against F(1) hybrids), and molecular analyses of mitochondrial ND2 and the nuclear ITS2 sequences. We found significant genetic divergence between the host races, and extremely divergent host preference (i.e. habitat isolation) and host performance (i.e. immigrant inviability), but no other isolating barriers. The fidelity to particular host plants arises first and alone can prevent gene flow between differentiating populations of phytophagous specialists.     

Developmental plasticity and reduced susceptibility to natural enemies following host plant defoliation in a specialized herbivore

Host-specific phytophagous insects that are short lived and reliant on ephemeral plant tissues provide an excellent system in which to investigate the consequences of disruption in the timing of resource availability on consumer populations and their subsequent interactions with higher tropic levels. The specialist herbivore, Belonocnema treatae (Hymenoptera: Cynipidae) induces galls on only newly flushed leaves of live oak, Quercus fusiformis. In central Texas (USA) episodic defoliation of the host creates variation in the timing of resource availability and results in heterogeneous populations of B. treatae that initiate development at different times. We manipulated the timing of leaf flush in live oak via artificial defoliation to test the hypothesis that a 6- to 8-week delay in the availability of resources alters the timing of this gall former’s life cycle events, performance and survivorship on its host, and susceptibility to natural enemies. B. treatae exhibits plasticity in development time, as the interval from egg to emergence was significantly reduced when gallers oviposited into the delayed leaf flush. As a consequence, the phenologies of gall maturation and adult emergence remain synchronized in spite of variation in the timing of resource availability. Per capita gall production and gall-former performance are not significantly affected by the timing of resource availability. The timing of resource availability and natural enemies interact, however, to produce strong effects on survivorship: when exposed to natural enemies, B. treatae developing in galls initiated by delayed oviposition exhibited an order-of-magnitude increase in survivorship. Developmental plasticity allows this gall former to circumvent disruptions in resource availability, maintain synchrony of life cycle events, and results in reduced vulnerability to natural enemies following defoliation of the host plant.     

Chromosomal variation segregates within incipient species and correlates with reproductive isolation

Reproductive isolation is a critical step in the process of speciation. Among the most important factors driving reproductive isolation are genetic incompatibilities. Whether these incompatibilities are already present before extrinsic factors prevent gene flow between incipient species remains largely unresolved in natural systems. This question is particularly challenging because it requires that we catch speciating populations in the act before they reach the full‐fledged species status. We measured the extent of intrinsic postzygotic isolation within and between phenotypically and genetically divergent lineages of the wild yeast Saccharomyces paradoxus that have partially overlapping geographical distributions. We find that hybrid viability between lineages progressively decreases with genetic divergence. A large proportion of postzygotic inviability within lineages is associated with chromosomal rearrangements, suggesting that chromosomal differences substantially contribute to the early steps of reproductive isolation within lineages before reaching fixation. Our observations show that polymorphic intrinsic factors may segregate within incipient species before they contribute to their full reproductive isolation and highlight the role of chromosomal rearrangements in speciation. We propose different hypotheses based on adaptation, biogeographical events and life history evolution that could explain these observations.     

A test of adaptive divergence in a newly discovered host association of the soapberry bug Jadera haematoloma on Mexican buckeye,Ungnadia speciosa

Host races represent an important step in the speciation process of phytophagous insects as they reflect the maintenance of genetically divergent host-associated populations in the face of appreciable gene flow. The red-shouldered soapberry bug, Jadera haematoloma (Herrich-Schäffer) (Hemiptera: Rhopalidae), is an oligophagous seed predator with a history of host race evolution on plant associations in the (soapberry) family Sapindaceae. Soapberry bugs are a model group for understanding rapid ecological adaptation to their hosts, and hence good candidates for investigating evolutionary divergence in host associations over short timescales. Here, we describe the recent discovery of Mexican buckeye, Ungnadia speciosa Endl., as a host of J. haematoloma in a region of the Chihuahuan desert including west Texas and southeastern New Mexico, USA. This host differs from J. haematoloma’s previously recorded hosts in the Sapindaceae in seed chemistry, ecology, and phylogeny. The tendency toward rapid, host-associated adaptations by populations of J. haematoloma and the unique biology of the newly discovered Ungnadia host create the opportunity for potential host race formation, as it overlaps geographically with two previously recorded host plants in this region – the native western soapberry tree, Sapindus saponaria var. drummondii (Hook & Arn.), and the non-native goldenrain tree, Koelreuteria paniculata Laxm. We explore the possibility of host race formation on Ungnadia-associated insects by testing for host-associated differentiation in morphology and feeding behaviors. We find evidence of differentiation in the length of the mouth parts, which is an ecologically relevant feeding trait between host plant species with larger or smaller seed capsules. This divergence is maintained in the face of potential gene flow by reproductive isolation in the form of habitat isolation, which we detect in host preference trials. Together, our results demonstrate that soapberry bugs associated with this newly discovered host exhibit morphological and behavioral traits consistent with host race formation, but additional work is required to confirm its state along the speciation continuum.