Spatiotemporally consistent genomic signatures of reproductive isolation in a moving hybrid zone

Studies of hybrid zone dynamics often investigate a single sampling period and draw conclusions from that temporal snapshot. Stochasticity can, however, result in loci with spurious outlier patterns, which is exacerbated by limited temporal or geographic sampling. Comparing admixed populations from different geographic regions is one way to detect repeatedly divergent genomic regions potentially involved in reproductive isolation. Temporal comparisons also allow us to control partially for the role of stochasticity, but the power of temporal sampling has not yet been adequately explored. In North America, black‐capped (Poecile atricapillus) and Carolina (P. carolinensis) chickadees hybridize in a contact zone extending from New Jersey to Kansas. The hybrid zone is likely maintained by strong intrinsic selection against hybrids, and it is moving north. We used a reduced representation genomic approach and temporally spaced sampling—two samples of ～80 individuals separated by a decade—to determine the pattern and consistency of selection and genomic introgression in the chickadee hybrid zone. We report consistently low introgression for highly divergent loci between P. atricapillus and P. carolinensis in this moving hybrid zone. This is strong evidence that these loci may be linked to genomic regions involved in reproductive isolation between chickadees.     

SPECIATION IN NORTH AMERICAN CHICKADEES: I. PATTERNS OF mtDNA GENETIC DIVERGENCE

We surveyed mitochondrial DNA haplotype divergence within and between populations of six species of North American chickadees (Parus, Subgenus Poecile) with the following results. (1) Genotype diversities (range 0.3 to 0.7) and low nucleotide diversities (range 3 to 27 × 10^?4) within populations were typical of known vertebrates. (2) The two widespread, northern species (atricapillus and hudsonicus) exhibit little mtDNA genetic differentiation throughout their previously glaciated continental distributions, most likely because of recent, postglacial range expansions. (3) Newfoundland populations of atricapillus and maritime province (Newfoundland plus Nova Scotia) populations of hudsonicus have distinct mtDNA haplotypes which differ from continental haplotypes by single restriction site changes. (4) Haplotypes of the southeastern U.S. species P. carolinensis divide into eastern and western sets which have diverged by three percent. This heretofore unrecognized, divided population structure may correspond to the Tombigbee River/ Mobile Bay disjunction known in some other vertebrate taxa. (5) Allopatric populations of the southwestern species sclateri and gambeli exhibit divergences of one and three percent respectively. (6) Prevailing interspecific divergence distances of three to seven percent suggest speciation early in the Pleistocene rather than during late (e.g., Wisconsin) glaciations. (7) Phylogenetic analyses suggest that North American taxa include two clades, hudsonicus-rufescens-sclateri versus carolinensis-atricapillus-gambeli and that carolinensis and atricapillus are not sister species.     

Hybrid chickadees are deficient in learning and memory

Identifying the phenotypes underlying postzygotic reproductive isolation is crucial for fully understanding the evolution and maintenance of species. One potential postzygotic isolating barrier that has rarely been examined is learning and memory ability in hybrids. Learning and memory are important fitness‐related traits, especially in scatter‐hoarding species, where accurate retrieval of hoarded food is vital for winter survival. Here, we test the hypothesis that learning and memory ability can act as a postzygotic isolating barrier by comparing these traits among two scatter‐hoarding songbird species, black‐capped (Poecile atricapillus) and Carolina chickadees (Poecile carolinensis), and their naturally occurring hybrids. In an outdoor aviary setting, we find that hybrid chickadees perform significantly worse on an associative learning spatial task and are worse at solving a novel problem compared to both parental species. Deficiencies in learning and memory abilities could therefore contribute to postzygotic reproductive isolation between chickadee species. Given the importance of learning and memory for fitness, our results suggest that these traits may play an important, but as yet overlooked, role in postzygotic reproductive isolation.     

Conspecific olfactory preferences and interspecific divergence in odor cues in a chickadee hybrid zone

Understanding how mating cues promote reproductive isolation upon secondary contact is important in describing the speciation process in animals. Divergent chemical cues have been shown to act in reproductive isolation across many animal taxa. However, such cues have been overlooked in avian speciation, particularly in passerines, in favor of more traditional signals such as song and plumage. Here, we aim to test the potential for odor to act as a mate choice cue, and therefore contribute to premating reproductive isolation between the black‐capped (Poecile atricapillus) and Carolina chickadee (P. carolinensis) in eastern Pennsylvania hybrid zone populations. Using gas chromatography–mass spectrometry, we document significant species differences in uropygial gland oil chemistry, especially in the ratio of ester to nonester compounds. We also show significant preferences for conspecific over heterospecific odor cues in wild chickadees using a Y‐maze design. Our results suggest that odor may be an overlooked but important mating cue in these chickadees, potentially promoting premating reproductive isolation. We further discuss several promising avenues for future research in songbird olfactory communication and speciation.     

Variation in reproductive isolation across a species range

Reproductive isolation is often variable within species, a phenomenon that while largely ignored by speciation studies, can be leveraged to gain insight into the potential mechanisms driving the evolution of genetic incompatibilities. We used experimental greenhouse crosses to characterize patterns of reproductive isolation among three divergent genetic lineages of Campanulastrum americanum that occur in close geographic proximity in the Appalachian Mountains. Substantial, asymmetrical reproductive isolation for survival due to cytonuclear incompatibility was found among the lineages (up to 94% reduction). Moderate reductions in pollen viability, as well as cytoplasmic male sterility, were also found between some Mountain populations. We then compared these results to previously established patterns of reproductive isolation between these Mountain lineages and a fourth, widespread Western lineage to fully characterize reproductive isolation across the complete geographic and genetic range of C. americanum. Reproductive isolation for survival and pollen viability was consistent across studies, indicating the evolution of the underlying genetic incompatibilities is primarily determined by intrinsic factors. In contrast, reproductive isolation for germination was only found when crossing Mountain populations with the Western lineage, suggesting the underlying genetic incompatibility is likely influenced by environmental or demographic differences between the two lineages. Cytoplasmic male sterility was also limited in occurrence, being restricted to a handful of Mountain populations in a narrow geographic range. These findings illustrate the complexity of speciation by demonstrating multiple, independent genetic incompatibilities that lead to a mosaic of genetic divergence and reproductive isolation across a species range.     

Genomic regions underlying metabolic and neuronal signaling pathways are temporally consistent in a moving avian hybrid zone

The study of hybrid zones can provide insight into the genetic basis of species differences that are relevant for the maintenance of reproductive isolation. Hybrid zones can also provide insight into climate change, species distributions, and evolution. The hybrid zone between black-capped chickadees (Poecile atricapillus) and Carolina chickadees (Poecile carolinensis) is shifting northward in response to increasing winter temperatures but is not increasing in width. This pattern indicates strong selection against chickadees with admixed genomes. Using high-resolution genomic data, we identified regions of the genomes that are outliers in both time points and do not introgress between the species; these regions may be involved in the maintenance of reproductive isolation. Genes involved in metabolic regulation processes were overrepresented in this dataset. Several gene ontology categories were also temporally consistent—including glutamate signaling, synaptic transmission, and catabolic processes—but the nucleotide variants leading to this pattern were not. Our results support recent findings that hybrids between black-capped and Carolina chickadees have higher basal metabolic rates than either parental species and suffer spatial memory and problem-solving deficits. Metabolic breakdown, as well as spatial memory and problem-solving, in hybrid chickadees may act as strong postzygotic isolation mechanisms in this moving hybrid zone.     

Failed species,innominate forms,and the vain search for species limits: cryptic diversity in dusky salamanders (Desmognathus) of eastern Tennessee

Cytochrome B sequences and allozymes reveal complex patterns of molecular variation in dusky salamander (Desmognathus) populations in eastern Tennessee. One group of allozymically distinctive populations, which we refer to as the Sinking Creek form (SCF), combines morphological attributes of Desmognathus fuscus with cytB sequences characteristic of Desmognathus carolinensis. This form is abruptly replaced by D. fuscus just north of Johnson City, TN with no evidence of either sympatry or gene exchange. To the south, allozymic markers indicate a broad zone of admixture with populations characterized by distinct cytB sequences and that may or may not be ultimately referable to Desmognathus conanti. A third distinctive group of populations, which we refer to as the Lemon Gap form (LGF), occurs in the foothills of the Great Smoky and southern Bald Mountains and exchanges genes with Desmognathus santeetlah along the escarpment of the Great Smokies, D. carolinensis in the southern Bald Mountains, and populations of a different haplotype clade in the Ridge and Valley. We treat all these as innominate forms that may represent “failed species,” recognizing that it may never be possible to reconcile species limits with patterns of phylogeny, morphology, and gene exchange in these salamanders.     

Hybridization between two parapatric ranid frog species in the northern Sierra Nevada,California, USA

Contact zones between species provide a unique opportunity to test whether taxa can hybridize or not. Cross‐breeding or hybridization between closely related taxa can promote gene flow (introgression) between species, adaptation, or even speciation. Though hybridization events may be short‐lived and difficult to detect in the field, genetic data can provide information about the level of introgression between closely related taxa. Hybridization can promote introgression between species, which may be an important evolutionary mechanism for either homogenization (reversing initial divergence between species) or reproductive isolation (potentially leading to speciation). Here, we used thousands of genetic markers from nuclear DNA to detect hybridization between two parapatric frog species (Rana boylii and Rana sierrae) in the Sierra Nevada of California. Based on principal components analysis, admixture, and analysis of heterozygosity at species diagnostic SNPs, we detected two F1 hybrid individuals in the Feather River basin, as well as a weak signal of introgression and gene flow between the frog species compared with frog populations from two other adjacent watersheds. This study provides the first documentation of hybridization and introgression between these two species, which are of conservation concern.     

Can alternative mating tactics facilitate introgression across a hybrid zone by circumventing female choice?

Reproductive barriers and divergence in species’ mate recognition systems underlie major models of speciation. However, hybridization between divergent species is common, and classic mechanisms to explain permeable reproductive barriers rarely consider how an individual may attain reproductive success. Alternative mating tactics (AMTs) exist in various forms across animal taxa. Such tactics may allow poorer quality individuals to gain mating opportunities and facilitate introgression either through asymmetrical positive selection or by circumventing female choice altogether in areas of secondary contact. One such tactic is satellite behaviour in frogs, where silent males perch near advertisers in an attempt to intercept females. To test whether such satellite male tactics are context‐dependent and favoured by hybrids, we genotyped and quantified the morphology of 80 male spring peeper (Pseudacris crucifer) individuals involved in caller–satellite associations from a secondary contact zone between two intraspecific mitochondrial lineages. Irrespective of population, satellite behaviour was best predicted by size but not body condition. Within the contact zone, pure individuals showed a significantly greater probability of being active callers, whereas hybrids of one lineage were more likely to adopt the satellite tactic. We suggest that satellite behaviour in P. crucifer promotes introgression, breaks down reproductive isolating barriers and contributes to asymmetrical introgression in this secondary contact zone. AMTs may thus be an underexplored but important alternative to oft‐discussed causes of genetic discordance found in hybrid zones.     

Flexible responses to stage‐specific offspring threats

When caring for their young, parents must compensate for threats to offspring survival in a manner that maximizes their lifetime reproductive success. In birds, parents respond to offspring threats by altering reproductive strategies throughout the breeding attempt. Because altered reproductive strategies are costly, when threats to offspring are limited, parents should exhibit a limited response. However, it is unclear if response to offspring threat is the result of an integrated set of correlated changes throughout the breeding attempt or if responses are a flexible set of dissociable changes that are stage‐specific. We test these hypotheses in a system where house wrens (Troglodytes aedon) compete for nesting cavities with Carolina chickadees (Poecile carolinensis) by usurping and destroying their nests during the early stage of the breeding attempt (the egg stage). Due to the specificity of the house wren threat, we can test whether parental responses to an offspring threat show flexibility and stage specificity or if parental strategies are an integrated and persistent response. We monitored nests in a natural population to compare life history traits of chickadees nesting in boxes that were in the presence of house wrens to chickadees nesting in boxes that did not overlap with house wrens. Carolina chickadees that nested near house wrens laid significantly smaller clutch sizes (early change in reproductive strategy) but did not alter nestling provisioning or nestling stage length (late change in reproductive strategy), suggesting that chickadees respond in a flexible and stage‐specific manner to the threat of house wrens. By responding only when a threat is highest, parents minimize the cost of antithreat responses. Our study suggests that parents can respond in subtle and nuanced ways to offspring threats in the environment and specifically alter reproductive behaviors at the appropriate stage.     

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

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

Mate preference: a possible causal mechanism for a moving hybrid zone

The study of patterns and underlying mechanisms within hybrid zones may provide insight into speciation. Black-capped (Poecile atricapillus; BC) and Carolina (P. carolinensis; CA) chickadees hybridize in an east-west band in the U.S.A. from New Jersey to Kansas. Within the past century, the Ohio portion of this hybrid zone and the CA range to the south have been moving northward while the BC range has retracted. We examined mate preference in females of both species as one possible causal mechanism for this shift. To be conservative about the nomenclature and results, the samples are referred to as either ‘BC-like’ or ‘CA-like’ due to the observed genetic introgression in the study individuals. Given a choice within an aviary setting, in the aggregate, BC-like and CA-like females that had not observed the direct social interactions between a dyad of a BC-like male and a CA-like male preferred to associate with the BC-like male. In nature, both species form within-sex dominance hierarchies. In the aviary, CA-like males dominated BC-like males unless a CA-like male was substantially smaller. Once females of both types had observed the physical interaction of a particular heterospecific dyad, they associated preferentially with the dominant male, regardless of species type. Thus, the effect of CA male intrasexual dominance on female mate preference may be contributing to the northward movement of the hybrid zone. Copyright 2003 Published by Elsevier Science Ltd on behalf of The Association for the Study of Animal Behaviour.     

Carex fumosimontana (Cyperaceae), a new endemic from the Great Smoky Mountains National Park, North Carolina and Tennessee

Carex fumosimontana (Great Smoky Mountain Sedge), a new and narrowly endemic species of section Phacocystis, is described from the southern Appalachian Mountains of North Carolina and Tennessee, in the southeastern United States. It differs from the closely related C. gynandra in its dark reddish-brown pistillate scales that are strongly retuse apically, shorter spikes, narrower leaves, and strongly red-scabrous proximal sheath faces. It is locally abundant and sometimes the dominant sedge where it occurs in spite of being completely restricted to high-elevation spruce-fir forests and associated environs of Great Smoky Mountains National Park.     

Sharp acoustic boundaries across an altitudinal avian hybrid zone despite asymmetric introgression

Birdsong is a sexually selected trait that could play an important evolutionary role when related taxa come into secondary contact. Many songbird species, however, learn their songs through copying one or more tutors, which complicates the evolutionary outcome of such contact. Two subspecies of a presumed vocal learner, the grey‐breasted wood‐wren (Henicorhina leucophrys), replace each other altitudinally across the western slope of the Ecuadorian Andes. These subspecies are morphologically very similar, but show striking differences in their song. We examined variation in acoustic traits and genetic composition across the altitudinal range covered by both subspecies and between two allopatric populations. The acoustic boundary between the subspecies was found to be highly abrupt across a narrow elevational range with virtually no evidence of song convergence. Mixed singing and use of hetero‐subspecific song occurred in the contact zone and was biased towards the use of leucophrys song types. Hetero‐subspecific song copying by hilaris and not by leucophrys reflected a previously found asymmetric pattern of response to song playback. Using amplified fragment length polymorphisms (AFLP) markers, we detected hybridization in the contact zone and asymmetric introgression in parapatric populations, with more leucophrys alleles present in hilaris populations than vice versa. This pattern may be a trail of introgression due to upslope displacement of leucophrys by hilaris. Our data suggest that song learning may impact speciation and hybridization in contrasting ways at different spatial scales: although learning may speed up population divergence in songs, thereby enhancing assortative mating and reducing gene flow, it may at a local level also lead to the copying of heterospecific songs, therefore allowing some level of hybridization and introgression.     

Differential introgression of a female competitive trait in a hybrid zone between sex‐role reversed species

Mating behavior between recently diverged species in secondary contact can impede or promote reproductive isolation. Traditionally, researchers focus on the importance of female mate choice and male–male competition in maintaining or eroding species barriers. Although female–female competition is widespread, little is known about its role in the speciation process. Here, we investigate a case of interspecific female competition and its influence on patterns of phenotypic and genetic introgression between species. We examine a hybrid zone between sex‐role reversed, Neotropical shorebird species, the northern jacana (Jacana spinosa) and wattled jacana (J. jacana), in which female–female competition is a major determinant of reproductive success. Previous work found that females of the more aggressive and larger species, J. spinosa, disproportionately mother hybrid offspring, potentially by monopolizing breeding territories in sympatry with J. jacana. We find a cline shift of female body mass relative to the genetic center of the hybrid zone, consistent with asymmetric introgression of this competitive trait. We suggest that divergence in sexual characteristics between sex‐role reversed females can influence patterns of gene flow upon secondary contact, similar to males in systems with more typical sex roles.     

Hybridization and rapid differentiation after secondary contact between the native green anole (Anolis carolinensis) and the introduced green anole (Anolis porcatus)

In allopatric species, reproductive isolation evolves through the accumulation of genetic incompatibilities. The degree of divergence required for complete reproductive isolation is highly variable across taxa, which makes the outcome of secondary contact between allopatric species unpredictable. Since before the Pliocene, two species of Anolis lizards, Anolis carolinensis and Anolis porcatus, have been allopatric, yet this period of independent evolution has not led to substantial species‐specific morphological differentiation, and therefore, they might not be reproductively isolated. In this study, we determined the genetic consequences of localized, secondary contact between the native green anole, A. carolinensis, and the introduced Cuban green anole, A. porcatus, in South Miami. Using 18 microsatellite markers, we found that the South Miami population formed a genetic cluster distinct from both parental species. Mitochondrial DNA revealed maternal A. porcatus ancestry for 35% of the individuals sampled from this population, indicating a high degree of cytonuclear discordance. Thus, hybridization with A. porcatus, not just population structure within A. carolinensis, may be responsible for the genetic distinctiveness of this population. Using tree‐based maximum‐likelihood analysis, we found support for a more recent, secondary introduction of A. porcatus to Florida. Evidence that ~33% of the nuclear DNA resulted from a secondary introduction supports the hybrid origin of the green anole population in South Miami. We used multiple lines of evidence and multiple genetic markers to reconstruct otherwise cryptic patterns of species introduction and hybridization. Genetic evidence for a lack of reproductive isolation, as well as morphological similarities between the two species, supports revising the taxonomy of A. carolinensis to include A. porcatus from western Cuba. Future studies should target the current geographic extent of introgression originating from the past injection of genetic material from Cuban green anoles and determine the consequences for the evolutionary trajectory of green anole populations in southern Florida.     

Distance,elevation and environment as drivers of diversity and divergence in bumble bees across latitude and altitude

Identifying drivers of dispersal limitation and genetic differentiation is a key goal in biogeography. We examine patterns of population connectivity and genetic diversity using restriction site‐associated DNA sequencing (RADseq) in two bumble bee species, Bombus vosnesenskii and Bombus bifarius, across latitude and altitude in mountain ranges from California, Oregon and Washington, U.S.A. Bombus vosnesenskii, which occurs across a broader elevational range at most latitudes, exhibits little population structure while B. bifarius, which occupies a relatively narrow higher elevation niche across most latitudes, exhibits much stronger population differentiation, although gene flow in both species is best explained by isolation with environmental niche resistance. A relationship between elevational habitat breadth and genetic diversity is also apparent, with B. vosnesenskii exhibiting relatively consistent levels of genetic diversity across its range, while B. bifarius has reduced genetic diversity at low latitudes, where it is restricted to high‐elevation habitat. The results of this study highlight the importance of the intersect between elevational range and habitat suitability in influencing population connectivity and suggest that future climate warming will have a fragmenting effect even on populations that are presently well connected, as they track their thermal niches upward in montane systems.     

Regional Differentiation in Genetic Components for the American Beech, Fagus grandifolia Ehrh., in Relation to Geological History and Mode of Reproduction

Fagus grandifolia , were investigated throughout its geographical range, using allozyme polymorphisms. A total of 1,131 trees from 21 populations were examined for 32 alleles of 10 polymorphic and two monomorphic loci in eight enzyme systems. The mean expected heterozygosity was 0.186, which indicates a relatively high genetic diversity within the populations. The levels of population differentiation were high, as revealed by genetic parameters, i.e., G _ST =0.168 and F _ST =0.167. The results of principal component analysis on allele frequencies clearly revealed unique regional patterns of differentiation in genetic components among populations “with” and “without” vegetative regeneration by root suckers. The American beech populations consist of two genetically distinct clusters, one from the Gulf-coastal plain, eastern coastal plain, Piedmont Plateau and Ozark Plateau; and the other from the remaining northern glaciated territories. Populations from the Blue Ridge and Great Smoky Mountains turned out to belong to the latter cluster, which is also characterized by extensive regeneration via root suckers. The consequences of regional differentiation in genetic components are discussed in relation to the postglacial spread from refugia to the current geographic distributions and the mode of reproduction. Received 8 August 2000/ Accepted in revised form 29 May 2001     

Contrasting patterns of genetic structure and phylogeography in the marine agarophytes Gelidiophycus divaricatus and G. freshwateri (Gelidiales,Rhodophyta) from East Asia

The evolutionary and population demographic history of marine red algae in East Asia is poorly understood. Here, we reconstructed the phylogeographies of two upper intertidal species endemic to East Asia, Gelidiophycus divaricatus and G. freshwateri. Phylogenetic and phylogeographic inferences of 393 mitochondrial cox1, 128 plastid rbcL, and 342 nuclear ITS2 sequences were complemented with ecological niche models. Gelidiophycus divaricatus, a southern species adapted to warm water, is characterized by a high genetic diversity and a strong geographical population structure, characteristic of stable population sizes and sudden reduction to recent expansion. In contrast, G. freshwateri, a northern species adapted to cold temperate conditions, is genetically relatively homogeneous with a shallow population structure resulting from steady population growth and recent equilibrium. The overlap zone of the two species roughly matches summer and winter isotherms, indicating that surface seawater temperature is a key feature influencing species range. Unidirectional genetic introgression was detected at two sites on Jeju Island where G. divaricatus was rare while G. freshwateri was common, suggesting the occurrence of asymmetric natural hybrids, a rarely reported event for rhodophytes. Our results illustrate that Quaternary climate oscillations have left strong imprints on the current day genetic structure and highlight the importance of seawater temperature and sea level change in driving speciation in upper intertidal seaweed species.     

Neophylax kolodskii (Trichoptera: Uenoidae), A New Species from the Great Smoky Mountains National Park,U.S.A.

Neophylax kolodskii sp. n. from Great Smoky Mountains National Park is described and illustrated from adult specimens.