Finding complexity in complexes: Assessing the causes of mitonuclear discordance in a problematic species complex of Mesoamerican toads

Mitonuclear discordance is a frequently encountered pattern in phylogeographic studies and occurs when mitochondrial and nuclear DNA display conflicting signals. Discordance among these genetic markers can be caused by several factors including confounded taxonomies, gene flow, and incomplete lineage sorting. In this study, we present a strong case of mitonuclear discordance in a species complex of toads (Bufonidae: Incilius coccifer complex) found in the Chortís Block of Central America. To determine the cause of mitonuclear discordance in this complex, we used spatially explicit genetic data to test species limits and relationships, characterize demographic history, and quantify gene flow. We found extensive mitonuclear discordance among the three recognized species within this group, especially in populations within the Chortís Highlands of Honduras. Our data reveal nuclear introgression within the Chortís Highlands populations that was most probably driven by cyclical range expansions due to climatic fluctuations. Though we determined introgression occurred within the nuclear genome, our data suggest that it is not the key factor in driving mitonuclear discordance in the entire species complex. Rather, due to a lack of discernible geographic pattern between mitochondrial and nuclear DNA, as well as a relatively recent divergence time of this complex, we concluded that mitonuclear discordance has been caused by incomplete lineage sorting. Our study provides a framework to test sources of mitonuclear discordance and highlights the importance of using multiple marker types to test species boundaries in cryptic species.     

Mitonuclear coevolution as the genesis of speciation and the mitochondrial DNA barcode gap

Mitochondrial genes are widely used in taxonomy and systematics because high mutation rates lead to rapid sequence divergence and because such changes have long been assumed to be neutral with respect to function. In particular, the nucleotide sequence of the mitochondrial gene cytochrome c oxidase subunit 1 has been established as a highly effective DNA barcode for diagnosing the species boundaries of animals. Rarely considered in discussions of mitochondrial evolution in the context of systematics, speciation, or DNA barcodes, however, is the genomic architecture of the eukaryotes: Mitochondrial and nuclear genes must function in tight coordination to produce the complexes of the electron transport chain and enable cellular respiration. Coadaptation of these interacting gene products is essential for organism function. I extend the hypothesis that mitonuclear interactions are integral to the process of speciation. To maintain mitonuclear coadaptation, nuclear genes, which code for proteins in mitochondria that cofunction with the products of mitochondrial genes, must coevolve with rapidly changing mitochondrial genes. Mitonuclear coevolution in isolated populations leads to speciation because population‐specific mitonuclear coadaptations create between‐population mitonuclear incompatibilities and hence barriers to gene flow between populations. In addition, selection for adaptive divergence of products of mitochondrial genes, particularly in response to climate or altitude, can lead to rapid fixation of novel mitochondrial genotypes between populations and consequently to disruption in gene flow between populations as the initiating step in animal speciation. By this model, the defining characteristic of a metazoan species is a coadapted mitonuclear genotype that is incompatible with the coadapted mitochondrial and nuclear genotype of any other population.     

Delimitation despite discordance: Evaluating the species limits of a confounding species complex in the face of mitonuclear discordance

The delimitation of species is an essential pursuit of biology, and proper taxonomies are crucial for the assessment and conservation management of organismal diversity. However, delimiting species can be hindered by a number of factors including highly conserved morphologies (e.g., cryptic species), differences in criteria of species concepts, lineages being in the early stages of the speciation or divergence process, and discordance between gene topologies (e.g., mitonuclear discordance). Here we use a taxonomically confounded species complex of toads in Central America that exhibits extensive mitonuclear discordance to test delimitation hypotheses. Our investigation integrates mitochondrial sequences, nuclear SNPs, morphology, and macroecological data to determine which taxonomy best explains the divergence and evolutionary relationships among these toads. We found that a three species taxonomy following the distributions of the nuclear SNP haplotypes offers the best explanation of the species in this complex based off of the integrated data types. Due to the taxonomic instability of this group, we also discuss conservation concerns in the face of improper taxonomic delimitation. Our study provides an empirical and integrative hypothesis testing framework to assess species delimitation hypotheses in the face of cryptic morphology and mitonuclear discordance and highlights the importance that a stable taxonomy has over conservation‐related actions.     

Speciation along a latitudinal gradient: The origin of the Neotropical cycad sister pair Dioon sonorense–D. vovidesii (Zamiaceae)

Latitude is correlated with environmental components that determine the distribution of biodiversity. In combination with geographic factors, latitude‐associated environmental variables are expected to influence speciation, but empirical evidence on how those factors interplay is scarce. We evaluated the genetic and environmental variation among populations in the pair of sister species Dioon sonorense–D. vovidesii, two cycads distributed along a latitudinal environmental gradient in northwestern Mexico, to reveal their demographic histories and the environmental factors involved in their divergence. Using genome‐wide loci data, we determined the species delimitation, estimated the gene flow, and compared multiple demographic scenarios of divergence. Also, we estimated the variation of climatic variables among populations and used ecological niche models to test niche overlap between species. The effect of geographic and environmental variables on the genetic variation among populations was evaluated using linear models. Our results suggest the existence of a widespread ancestral population that split into the two species ~829 ky ago. The geographic delimitation along the environmental gradient occurs in the absence of major geographic barriers, near the 28th parallel north, where a zonation of environmental seasonality exists. The northern species, D. vovidesii, occurs in more seasonal environments but retains the same niche of the southern species, D. sonorense. The genetic variation throughout populations cannot be solely explained by stochastic processes; the latitudinal‐associated seasonality has been an additive factor that strengthened the species divergence. This study represents an example of how speciation can be achieved by the effect of the latitude‐associated factors on the genetic divergence among populations.     

Geographic variation and divergence of songs in the Olive Sparrow species complex

Phenotypic traits such as songs are important in species recognition. Variation in acoustic traits can form barriers to gene flow and promote speciation. Therefore, understanding song divergence is crucial in groups with controversial taxonomy such as Olive Sparrows (Arremonops rufivirgatus), a widespread Neotropical species of songbird with multiple allopatric populations. Taxonomic authorities disagree on the number of Olive Sparrow subspecies, placing them into either two or three main groups. These groups may represent separate species based on morphological traits, but trait divergence within the complex has not been examined. We studied geographic variation in the characteristics of the songs of Olive Sparrows at two geographical levels: among three proposed groups and among five allopatric populations. In a second analysis, we evaluated the strength of acoustic divergence within the complex by comparing acoustic distances among groups and allopatric populations of Olive Sparrows with the acoustic distance among three recognized species in the genus Arremonops. We analyzed 802 songs from 174 individuals across 81 locations and measured 12 variables to describe the fine structural characteristics of the songs of Olive Sparrows, Green-backed Sparrows (A. chloronotus), Black-striped Sparrows (A. conirostris), and Tocuyo Sparrows (A. tocuyensis). We found significant acoustic variation in the Olive Sparrow complex at both geographical levels. Our divergence analysis also revealed that vocal divergence within the complex is similar to or greater than that found between recognized species in the genus. Together, these results suggest that acoustic diversity within the Olive Sparrow complex probably originated by isolation in tandem with selective and/or non-selective factors.     

Current geography masks dynamic history of gene flow during speciation in northern Australian birds

Genome divergence is greatly influenced by gene flow during early stages of speciation. As populations differentiate, geographic barriers can constrain gene flow and so affect the dynamics of divergence and speciation. Current geography, specifically disjunction and continuity of ranges, is often used to predict the historical gene flow during the divergence process. We test this prediction in eight meliphagoid bird species complexes codistributed in four regions. These regions are separated by known biogeographical barriers across northern Australia and Papua New Guinea. We find that bird populations currently separated by terrestrial habitat barriers within Australia and marine barriers between Australia and Papua New Guinea have a range of divergence levels and probability of gene flow not associated with current range connectivity. Instead, geographic distance and historical range connectivity better predict divergence and probability of gene flow. In this dynamic environmental context, we also find support for a nonlinear decrease of the probability of gene flow during the divergence process. The probability of gene flow initially decreases gradually after a certain level of divergence is reached. Its decrease then accelerates until the probability is close to zero. This implies that although geographic connectivity may have more of an effect early in speciation, other factors associated with higher divergence may play a more important role in influencing gene flow midway through and later in speciation. Current geographic connectivity may then mislead inferences regarding potential for gene flow during speciation under a complex and dynamic history of geographic and reproductive isolation.     

Hard and soft allopatry: physically and ecologically mediated modes of geographic speciation

Aim Three common patterns have emerged in comparative phylogeographic analyses at many barriers: (1) a potential geographic pseudocongruence of lineage divergences; (2) a disconnect between the inference of temporally clustered, relatively recent timing for observed speciation events, and dates spanning a broader, apparently random time‐scale; and (3) an apparent prevalence of speciation with recent or continuing gene flow. It is unclear if there is a unifying explanation for these phenomena. We argue that the interaction between geographic barriers to dispersal and ecological limits on the distribution of species can explain these patterns. We suggest that these patterns can be explained by the presence of a continuum between two underlying processes, here termed ‘hard’ and ‘soft’ allopatric divergence, which result from the interplay between organismal ecology and the physioecological nature of geographic barriers. Location Examples from North America. Methods We examine comparative phylogeographic divergences in 18 groups of terrestrial vertebrates at two major biogeographic features in North America – the Mississippi River Embayment and the Cochise Filter Barrier – to test predictions made by this hypothesis. Results We find support for the two distinct processes of hard and soft allopatry, and note several examples exhibiting characteristics of both. Hard allopatry is caused by physical barriers promoting divergence as a function of consistent geographic isolation. Soft allopatry is caused by ecological processes that isolate populations geographically in allopatric refugia through niche conservatism, or across ecological transition zones through niche divergence, but which may be periodic or inconsistent through time. Main conclusions Viewing geographic speciation as a continuum between hard and soft allopatry can explain all three patterns as a consequence of the physical and ecological mechanisms that isolate populations, and provides an alternative perspective on the impact of ecological factors and physical barriers on lineage formation.     

Maintenance of species boundaries in a Neotropical radiation of Begonia

A major goal of evolutionary biology is to determine the mechanisms generating biodiversity. In Begonia, one of the largest plant genera (1900+ species), it has been postulated that the high number of endemic species is a by‐product of low gene flow among populations, which predisposes the group to speciation. However, this model of divergence requires that reproductive barriers accumulate rapidly among diverging species that overlap in their geographic ranges, otherwise speciation will be opposed by homogenizing gene flow in zones of secondary contact. Here, we test the outcomes of secondary contact in Begonia by genotyping multiple sympatric sites with 12 nuclear and seven plastid loci. We show that three sites of secondary contact between B. heracleifolia and B. nelumbiifolia are highly structured, mostly containing parental genotypes, with few F1 hybrids. A sympatric site between B. heracleifolia and B. sericoneura contains a higher proportion of F1s, but little evidence of introgression. The lack of later‐generation hybrids contrasts with that documented in many other plant taxa, where introgression is extensive. Our results, in conjunction with previous genetic work, show that Begonia demonstrate properties making them exceptionally prone to speciation, at multiple stages along the divergence continuum. Not only are populations weakly connected by gene flow, promoting allopatric speciation, but species often show strong reproductive barriers in secondary contact. Whether similar mechanisms contribute to diversification in other large genera remains to be tested.     

Patterns of morphological and ecological similarities of small-eared shrews (Soricidae,Cryptotis) in tropical montane cloud forests from Mesoamerica

It has been proposed that high morphological similarity between closely related species of small-eared shrews resulted from a recent divergence and intermittent population connectivity, presumably due to Pleistocene climatic fluctuations and associated changes in forest habitat distribution. Here we examined the morphological variation of two sister species of small-eared shrews inhabiting cloud forests from Mexico, Cryptotis obscurus and C. mexicanus. We then used ecological niche modelling to provide compelling evidence for current environmental barriers for population connectivity, and for detecting divergent ecological niches between candidate species. Our results indicated that the species boundaries in this clade should be subject to change. High morphological similarity suggested that populations of C. obscurus and C. mexicanus located west of the Isthmus of Tehuantepec, a major geographic barrier for montane species, are conspecific. Niche divergence between these two putative species was not supported indicating niche conservatism across the evolutionary history of these small-eared shrews. In addition, several barriers seem to play a main role for current lineage divergence between populations within this clade. The population located east of the Isthmus, previously referred to C. mexicanus, might prove to represent a new species based on morphological distinction and current geographic isolation. We have highlighted that estimating species’ potential distributions provides insights to evaluating the effect of geographic barriers on lineage divergence and making stronger inferences when delimiting species.     

Song variation in an avian ring species

Abstract.— Divergence of mating signals can occur rapidly and be of prime importance in causing reproductive isolation and speciation. A ring species, in which two reproductively isolated taxa are connected by a chain of intergrading populations, provides a rare opportunity to use spatial variation to reconstruct the history of divergence. I use geographic variation in the song of a likely ring species, the greenish warbler ( Phylloscopus trochiloides ) to reconstruct the microevolutionary steps that occurred during divergence of a trait that is often important in speciation in birds. Populations of a western Siberian ( P. t. viridanus ) and an eastern Siberian ( P. t. plumbeitarsus ) form of the greenish warbler meet, but do not interbreed in central Siberia; these forms are connected by a chain of interbreeding populations extending in a ring to the south around the treeless Tibetan Plateau. I show that: (1) song structure differs greatly between the two Siberian forms, which share the same habitat; (2) song structure changes gradually around the ring; (3) singing behavior is relatively simple in the Himalayas, but becomes increasingly complex to the north, both to the west and east of the Tibetan Plateau; and (4) song varies along independent axes of complexity in the western and eastern south-north clines. By comparing geographic variation in singing behavior and ecological variables, I distinguish among possible causes of song divergence, including selection based on the acoustic environment, stochastic effects of sexual selection, and selection for species recognition. I suggest that parallel south-to-north ecological gradients have caused a greater intensity of sexual selection on song in northern populations and that the stochastic effects of sexual selection have led to divergence in song structure.     

Adaptation and the origin of species

As reflected in the title of his masterwork On the Origin of Species, Darwin proposed that adaptation is the primary mechanism of speciation. On this, Darwin was criticized for his neglect of reproductive isolation, his lack of appreciation for the role of geographic barriers, his failure to distinguish varieties from species, and his typological species concept. Two developments since Darwin, the biological species concept of Ernst Mayr and the methods of Coyne and Orr for estimating the contribution of different barriers to the total reproductive isolation, provide a framework for reconciling Darwin's view on the primacy of adaptation in speciation with later proposals that emphasize reproductive isolation. A review of the few studies that have estimated the contributions of multiple isolating barriers suggests that habitat isolation and other barriers that operate before hybrid formation are much stronger than intrinsic postzygotic isolation. In light of these data, I suggest that Darwin's focus on adaptation in the origin of species was essentially correct, a conclusion that calls for future studies that explore the links between adaptation and speciation, in particular, ecogeographic isolating barriers that result from adaptive divergence in habitat use. The recent revival in thinking about ecological factors and adaptive divergence in the origin of species echoes Darwin's much-criticized "principle of divergence" and suggests that the emerging views from today's naturalists are not so different from those espoused by Darwin some 150 years ago.     

Microgeographic evolution of snail shell shape and predator behavior

Genetic divergence in geographically isolated populations is a prerequisite for allopatric speciation, one of the most common modes of speciation. In ecologically equivalent populations existing within a small, environmentally homogeneous area, an important role for environmentally neutral divergence is often found or inferred. We studied a species complex of conspicuously shaped Opisthostoma land snails on scattered limestone outcrops within a small area of lowland rainforest in Borneo. We used shell morphometrics, mitochondrial and nuclear DNA sequences, and marks of predation to study the factors involved in allopatric divergence. We found that a striking geographic divergence exists in shell morphology, which is partly associated with neutral genetic divergence. We also found geographic differentiation in the behavior of the snails' invertebrate predator and evidence of an evolutionary interaction between aspects of shell shape and predator behavior. Our study shows that adaptation to biotic aspects of the environment may play a more important role in allopatric speciation than previously suspected, even on a geographically very small scale.     

Ecological divergence and speciation between lemur (Eulemur) sister species in Madagascar

Understanding ecological niche evolution over evolutionary timescales is crucial to elucidating the biogeographic history of organisms. Here, we used, for the first time, climate‐based ecological niche models (ENMs) to test hypotheses about ecological divergence and speciation processes between sister species pairs of lemurs (genus Eulemur) in Madagascar. We produced ENMs for eight species, all of which had significant validation support. Among the four sister species pairs, we found nonequivalent niches between sisters, varying degrees of niche overlap in ecological and geographic space, and support for multiple divergence processes. Specifically, three sister‐pair comparisons supported the null model that niches are no more divergent than the available background region. These findings are consistent with an allopatric speciation model, and for two sister pairs (E. collaris–E. cinereiceps and E. rufus–E. rufifrons), a riverine barrier has been previously proposed for driving allopatric speciation. However, for the fourth sister pair E. flavifrons–E. macaco, we found support for significant niche divergence, and consistent with their parapatric distribution on an ecotone and the lack of obvious geographic barriers, these findings most strongly support a parapatric model of speciation. These analyses thus suggest that various speciation processes have led to diversification among closely related Eulemur species.     

Comparison of gene flow among species that occur within the same geographic locations versus gene flow among populations within species reveals introgression among several Elymus species

One of the challenges in evolutionary biology is to understand the evolution of speciation with incomplete reproductive isolation as many taxa have continued gene flow both during and after speciation. Comparison of population structure between sympatric and allopatric populations can reveal specific introgression and determine if introgression occurs in a unidirectional or bidirectional manner. Simple sequence repeat markers were used to characterize sympatric and allopatric population structure of plant species, Elymus alaskanus (Scribn. and Merr.) Löve, E. caninus L., E. fibrosus (Schrenk) Tzvel., and E. mutabilis (Drobov) Tzvelev. Our results showed that genetic diversity (H_E) at species level is E. caninus (0.5355) > E. alaskanus (0.4511) > E. fibrosus (0.3924) > E. mutabilis (0.3764), suggesting that E. caninus and E. alaskanus are more variable than E. fibrosus and E. mutabilis. Gene flow between species that occurs within the same geographic locations versus gene flow between populations within species was compared to provide evidence of introgression. Our results indicated that gene flow between species that occur within the same geographic location is higher than that between populations within species, suggesting that gene flow resulting from introgressive hybridization might have occurred among the sympatric populations of these species, and may play an important role in partitioning of genetic diversity among and within populations. The migration rate from E. fibrosus to E. mutabilis is highest (0.2631) among the four species studied. Asymmetrical rates of gene flow among four species were also observed. The findings highlight the complex evolution of these four Elymus species.     

Phylogeography of Sardinian cave salamanders (genus Hydromantes) is mainly determined by geomorphology

Detecting the factors that determine the interruption of gene flow between populations is key to understanding how speciation occurs. In this context, caves are an excellent system for studying processes of colonization, differentiation and speciation, since they represent discrete geographical units often with known geological histories. Here, we asked whether discontinuous calcareous areas and cave systems represent major barriers to gene flow within and among the five species of Sardinian cave salamanders (genus Hydromantes) and whether intraspecific genetic structure parallels geographic distance within and among caves. We generated mitochondrial cytochrome b gene sequences from 184 individuals representing 48 populations, and used a Bayesian phylogeographic approach to infer possible areas of cladogenesis for these species and reconstruct historical and current dispersal routes among distinct populations. Our results show deep genetic divergence within and among all Sardinian cave salamander species, which can mostly be attributed to the effects of mountains and discontinuities in major calcareous areas and cave systems acting as barriers to gene flow. While these salamander species can also occur outside caves, our results indicate that there is a very poor dispersal of these species between separate cave systems.     

The on‐again,off‐again relationship between mitochondrial genomes and species boundaries

The study of reproductive isolation and species barriers frequently focuses on mitochondrial genomes and has produced two alternative and almost diametrically opposed narratives. On one hand, mtDNA may be at the forefront of speciation events, with co‐evolved mitonuclear interactions responsible for some of the earliest genetic incompatibilities arising among isolated populations. On the other hand, there are numerous cases of introgression of mtDNA across species boundaries even when nuclear gene flow is restricted. We argue that these seemingly contradictory patterns can result from a single underlying cause. Specifically, the accumulation of deleterious mutations in mtDNA creates a problem with two alternative evolutionary solutions. In some cases, compensatory or epistatic changes in the nuclear genome may ameliorate the effects of mitochondrial mutations, thereby establishing coadapted mitonuclear genotypes within populations and forming the basis of reproductive incompatibilities between populations. Alternatively, populations with high mitochondrial mutation loads may be rescued by replacement with a more fit, foreign mitochondrial haplotype. Coupled with many nonadaptive mechanisms of introgression that can preferentially affect cytoplasmic genomes, this form of adaptive introgression may contribute to the widespread discordance between mitochondrial and nuclear genealogies. Here, we review recent advances related to mitochondrial introgression and mitonuclear incompatibilities, including the potential for cointrogression of mtDNA and interacting nuclear genes. We also address an emerging controversy over the classic assumption that selection on mitochondrial genomes is inefficient and discuss the mechanisms that lead lineages down alternative evolutionary paths in response to mitochondrial mutation accumulation.     

Genetic divergence and speciation in lowland and montane peruvian poison frogs

Amazonia is famous for high biodiversity, and the highlands of the transition zone between the Andes and the lowlands of the Amazon basin show particularly high species diversity. Hypotheses proposed to explain the high levels of diversity in the highlands include repeated parapatric speciation across ecological gradients spanning the transition zone, repeated allopatric speciation across geographic barriers between the highlands and lowlands, divergence across geographic barriers within the transition zone, and simple lineage accumulation over long periods of time. In this study, we investigated patterns of divergence in frogs of the genus Epipedobates (family Dendrobatidae) using phylogenetic and biogeographic analyses of divergence in mitochondrial DNA (1778 aligned positions from genes encoding cyt b, 12S and 16S rRNA for 60 Epipedobates and 11 outgroup specimens) and coloration (measured for 18 specimens representing nine species in Epipedobates). The majority of phenotypic and species diversity in the poison frog genus Epipedobates occurs in the transition zone, although two morphologically conserved members of the genus are distributed across the lowlands of the Amazon basin. Phylogenetic analysis reveals that there is a single highland clade derived from an ancestral colonization event in northern Peru by a population of lowland ancestry. Epipedobates trivittatus, a widespread Amazonian species, is a member of the highland clade that reinvaded the lowlands. Comparative analyses of divergence in coloration and mtDNA reveals that divergence in coloration among populations and species in the highlands has been accelerated relative to the lowlands. This suggests a role for selection in the divergence of coloration among populations and species.     

Combining Biogeographic and Phylogenetic Data to Examine Primate Speciation: An Example Using Cercopithecin Monkeys

We combined phylogenetic and biogeographic data to examine the mode of speciation in a group of African monkeys, the Cercopithecini. If allopatric speciation is the major force producing species, then there should be a positive relationship between the relative divergence time of taxa and their degree of geographic range overlap. Alternatively, an opposite relationship between divergence time and geographic range overlap is consistent with sympatric speciation as the main mechanism underlying the cercopithecin radiation. We collected biogeographic and phylogenetic data for 19 guenon species from the literature. We digitized geographic range maps and utilized three different phylogenetic hypotheses based on Y chromosome, X chromosome, and mitochondrial (mtDNA) data. We used regressions with Monte Carlo simulation to examine the relationship between the relative time since divergence of taxa and their degree of geographic range overlap. We found that there was a positive relationship between relative divergence time and the proportion of geographic range overlap between taxa using all three molecular data sets. Our findings provide evidence for allopatric speciation being the common mode of diversification in the cercopithecin clade. Because most of these primates are forest adapted mammals, the cyclical contraction and expansion of African forests from the late Miocene to the present has likely been an important factor driving allopatric speciation. In addition, geographic barriers such as the Congo and Sanaga rivers have probably played a complementary role in producing new species within the clade.     

Hawthorn-infesting populations of Rhagoletis pomonella in Mexico and speciation mode plurality

Categorizing speciation into dichotomous allopatric versus nonallopatric modes may not always adequately describe the geographic context of divergence for taxa. If some of the genetic changes generating inherent barriers to gene flow between populations evolved in geographic isolation, whereas others arose in sympatry, then the mode of divergence would be mixed. The apple maggot fly, Rhagoletis pomonella, has contributed to this emerging concept of a mixed speciation mode "plurality." Genetic studies have implied that a source of diapause life-history variation associated with inversions and contributing to sympatric host race formation and speciation for R. pomonella in the United States may have introgressed from the Eje Volcanico Trans Mexicano (EVTM; a.k.a. the Altiplano) in the past. A critical unresolved issue concerning the introgression hypothesis is how past gene flow occurred given the current 1200-km disjunction in the ranges of hawthorn-infesting flies in the EVTM region of Mexico and the southern extreme of the U.S. population in Texas. Here, we report the discovery of a hawthorn-infesting population of R. pomonella in the Sierra Madre Oriental Mountains (SMO) of Mexico. Sequence data from 15 nuclear loci and mitochondrial DNA imply that the SMO flies are related to, but still different from, U.S. and EVTM flies. The host affiliations, diapause characteristics, and phylogeography of the SMO population are consistent with it having served as a conduit for gene flow between Mexico and the United States. We also present evidence suggesting greater permeability of collinear versus rearranged regions of the genome to introgression, in accord with recent models of chromosomal speciation. We discuss the implications of the results in the context of speciation mode plurality. We do not argue for abandoning the terms sympatry or allopatry, but caution that categorizing divergence into either/or geographic modes may not describe the genetic origins of all species. For R. pomonella in the United States, the proximate selection pressures triggering race formation and speciation stem from sympatric host shifts. However, some of the phenological variation contributing to host-related ecological adaptation and reproductive isolation in sympatry at the present time appears to have an older history, having originated and become packaged into inversion polymorphism in allopatry.