CORRELATION BETWEEN ANOLIS LIZARD DEWLAP PHENOTYPE AND ENVIRONMENTAL VARIATION INDICATES ADAPTIVE DIVERGENCE OF A SIGNAL IMPORTANT TO SEXUAL SELECTION AND SPECIES RECOGNITION

Although the importance of signals involved in species recognition and sexual selection to speciation is widely recognized, the processes that underlie signal divergence are still a matter of debate. Several possible processes have been hypothesized, including genetic drift, arbitrary sexual selection, and adaptation to local signaling environments. We use comparative analyses to investigate whether the remarkable geographic variation of dewlap phenotype in a Hispaniolan trunk Anolis lizard (A. distichus) is a result of adaptive signal divergence to heterogeneous environments. We recover a repeated pattern of divergence in A. distichus dewlap color, pattern, and size with environmental variation across Hispaniola. These results are aligned with ecological models of signal divergence and provide strong evidence for dewlap adaptation to local signaling environments. We also find that A. distichus dewlaps vary with the environment in a different manner to other previously studied anoles, thus expanding upon previous predictions on the direction dewlaps will diverge in perceptual color space in response to the environment.     

Genetic differentiation among populations of a Hispaniolan trunk anole that exhibit geographical variation in dewlap colour

Long neglected by classic island biogeographical theory, speciation within and among islands is increasingly recognized as a major contributor to insular diversity. Although the factors responsible for island speciation remain poorly understood, this process appears critically dependent on geographical variation and speciation in allopatry or parapatry. Here, we investigate geographical variation and speciation in a complex of Hispaniolan trunk anoles (Anolis distichus), where populations with strikingly distinct dewlap colours and patterns correspond with deeply divergent mtDNA structure. Using a multilocus, population‐level analysis, we investigate whether these phenotypically and mitochondrially distinct populations exhibit the type of nuclear differentiation expected among species or incipient species. Along a transect that extends across a recently recessed marine barrier, our results are consistent with the persistence of an abrupt phenotypic and mitochondrial transition following secondary contact, in spite of little or no evidence for a reduction in nuclear gene flow. Along a second transect extending across a steep environmental gradient, our phenotypic and microsatellite data suggest a sharp genetic break with little or no admixture, whereas mtDNA recovers a signature of extensive unidirectional introgression. Together, these results are consistent with previous studies of Lesser Antillean anoles, suggesting that allopatric divergence alone is insufficient for speciation, whereas reduced gene flow and partial reproductive isolation may accumulate in the presence of ecological gradients.     

Clustering of loci controlling species differences in male chemical bouquets of sympatric Heliconius butterflies

The degree to which loci promoting reproductive isolation cluster in the genome—that is, the genetic architecture of reproductive isolation—can influence the tempo and mode of speciation. Tight linkage between these loci can facilitate speciation in the face of gene flow. Pheromones play a role in reproductive isolation in many Lepidoptera species, and the role of endogenously produced compounds as secondary metabolites decreases the likelihood of pleiotropy associated with many barrier loci. Heliconius butterflies use male sex pheromones to both court females (aphrodisiac wing pheromones) and ward off male courtship (male‐transferred antiaphrodisiac genital pheromones), and it is likely that these compounds play a role in reproductive isolation between Heliconius species. Using a set of backcross hybrids between H. melpomene and H. cydno, we investigated the genetic architecture of putative male pheromone compound production. We found a set of 40 significant quantitative trait loci (QTL) representing 33 potential pheromone compounds. QTL clustered significantly on two chromosomes, chromosome 8 for genital compounds and chromosome 20 for wing compounds, and chromosome 20 was enriched for potential pheromone biosynthesis genes. There was minimal overlap between pheromone QTL and known QTL for mate choice and color pattern. Nonetheless, we did detect linkage between a QTL for wing androconial area and optix, a color pattern locus known to play a role in reproductive isolation in these species. This tight clustering of putative pheromone loci might contribute to coincident reproductive isolating barriers, facilitating speciation despite ongoing gene flow.     

Reproductive isolation via divergent genital morphology due to cascade reinforcement in Ohomopterus ground beetles

Secondary contact between incipient species and selection against maladaptive hybridization can drive reinforcement between populations in contact and result in reproductive character displacement (RCD). Resultant divergence in mating traits within a species may generate downstream reproductive isolation between populations with displaced and non-displaced traits, referred to as the cascade reinforcement hypothesis. We examined this hypothesis using three allopatric populations of the ground beetle Carabus maiyasanus with a genital lock-and-key system. This species shows RCD in male and female genital morphologies in populations in contact with the sister species C. iwawakianus. In a reciprocal mating experiment using three allopatric populations with differences in male and female genital sizes, insemination failure increased as the difference in genital size increased. Based on the reproductive isolation index, insemination failure was the major postmating-prezygotic isolation barrier, at least in one population pair with comparable total isolation to those of other species pairs. By contrast, there was only incomplete premating isolation among populations. These results suggest that RCD in genital morphologies drives incipient allopatric speciation, supporting the cascade reinforcement hypothesis. These findings provide insight into the roles of interspecific interactions and subsequent trait diversification in speciation processes.     

Geographic variation in hybridization across a reinforcement contact zone of chorus frogs (Pseudacris)

Reinforcement contact zones, which are secondary contact zones where species are diverging in reproductive behaviors due to selection against hybridization, represent natural laboratories for studying speciation‐in‐action. Here, we examined replicate localities across the entire reinforcement contact zone between North American chorus frogs Pseudacris feriarum and P. nigrita to investigate geographic variation in hybridization frequencies and to assess whether reinforcement may have contributed to increased genetic divergence within species. Previous work indicated these species have undergone reproductive character displacement (RCD) in male acoustic signals and female preferences due to reinforcement. We also examined acoustic signal variation across the contact zone to assess whether signal characteristics reliably predict hybrid index and to elucidate whether the degree of RCD predicts hybridization rate. Using microsatellites, mitochondrial sequences, and acoustic signal information from >1,000 individuals across >50 localities and ten sympatric focal regions, we demonstrate: (1) hybridization occurs and (2) varies substantially across the geographic range of the contact zone, (3) hybridization is asymmetric and in the direction predicted from observed patterns of asymmetric RCD, (4) in one species, genetic distance is higher between conspecific localities where one or both have been reinforced than between nonreinforced localities, after controlling for geographic distance, (5) acoustic signal characters strongly predict hybrid index, and (6) the degree of RCD does not strongly predict admixture levels. By showing that hybridization occurs in all sympatric localities, this study provides the fifth and final line of evidence that reproductive character displacement is due to reinforcement in the chorus frog contact zone. Furthermore, this work suggests that the dual action of cascade reinforcement and partial geographic isolation is promoting genetic diversification within one of the reinforced species.     

A genomic assessment of species boundaries and hybridization in a group of highly polymorphic anoles (distichus species complex)

Delimiting young species is one of the great challenges of systematic biology, particularly when the species in question exhibit little morphological divergence. Anolis distichus, a trunk anole with more than a dozen subspecies that are defined primarily by dewlap color, may actually represent several independent evolutionary lineages. To test this, we utilized amplified fragment length polymorphisms (AFLP) genome scans and genetic clustering analyses in conjunction with a coalescent‐based species delimitation method. We examined a geographically widespread set of samples and two heavily sampled hybrid zones. We find that genetic divergence is associated with a major biogeographic barrier, the Hispaniolan paleo‐island boundary, but not with dewlap color. Additionally, we find support for hypotheses regarding colonization of two Hispaniolan satellite islands and the Bahamas from mainland Hispaniola. Our results show that A. distichus is composed of seven distinct evolutionary lineages still experiencing a limited degree of gene flow. We suggest that A. distichus merits taxonomic revision, but that dewlap color cannot be relied upon as the primary diagnostic character.     

Are subspecies of Anolis lizards that differ in dewlap color and pattern also genetically distinct? A mitochondrial analysis

Subspecies of Anolis lizards are often defined on the basis of geographic variation in the color and pattern of the dewlap, an extensible throat fan considered central to species recognition and sexual selection. Among the most impressive examples of this phenomenon are two species of trunk anoles found across Hispaniola and the Bahamas: Anolis distichus is divided into 16 subspecies with dewlap colors ranging from deep wine red to pale yellow while Anolis brevirostris is divided into three subspecies with dewlaps ranging from pale yellow to orange. Limited sampling of allozyme data indicates some genetic divergence among subspecies and suggests that they may deserve recognition at the species-level. Our goal here is to use more comprehensive geographic sampling of mtDNA haplotypes to test whether the five subspecies of A. distichus and three subspecies of A. brevirostris that occur in the Dominican Republic correspond with genetically distinct populations that may warrant recognition under the general lineage concept. We obtain an aligned dataset of 1462bp comprised of the genes encoding ND2 and adjacent tRNAs from 76 individuals of A. distichus from 28 localities and 12 individuals of A. brevirostris from five localities. We find that haplotypes sampled from each Dominican subspecies of A. distichus form well-supported and deeply divergent clades (>10% uncorrected sequence divergence). Strong concordance between mtDNA haplotype structure and previously diagnosed phenotypic variation in traits central to interspecific communication (i.e., the dewlap) leads us to hypothesize that each of the presently recognized Dominican subspecies of A. distichus and A. brevirostris deserves elevation to full species status under the general lineage concept.     

Climate‐related environmental variation in a visual signalling device: the male and female dewlap in Anolis sagrei lizards

Animals communicate using a variety of signals that differ dramatically among and within species. The astonishing dewlap diversity in anoles has attracted considerable attention in this respect. Yet, the evolutionary processes behind it remain elusive and have mostly been explored for males only. Here, we considered Anolis sagrei males and females to study signal divergence among populations. First, we assessed the degree of variation in dewlap design (size, pattern and colour) and displays by comparing 17 populations distributed across the Caribbean. Second, we assessed whether the observed dewlap diversity is associated with variation in climate‐related environmental conditions. Results showed that populations differed in all dewlap characteristics, with the exception of display rate in females. We further found that males and females occurring in ‘xeric’ environments had a higher proportion of solid dewlaps with higher UV reflectance. In addition, lizards inhabiting ‘mesic’ environments had primarily marginal dewlaps showing high reflectance in red. For dewlap display, a correlation with environment was only observed in males. Our study provides evidence for a strong relationship between signal design and prevailing environmental conditions, which may result from differential selection on signal efficacy. Moreover, our study highlights the importance of including females when studying dewlaps in an evolutionary context.     

Rapid sexual and genomic isolation in sympatric Drosophila without reproductive character displacement

The rapid evolution of sexual isolation in sympatry has long been associated with reinforcement (i.e., selection to avoid maladaptive hybridization). However, there are many species pairs in sympatry that have evolved rapid sexual isolation without known costs to hybridization. A major unresolved question is what evolutionary processes are involved in driving rapid speciation in such cases. Here, we focus on one such system; the Drosophila athabasca species complex, which is composed of three partially sympatric and interfertile semispecies: WN, EA, and EB. To study speciation in this species complex, we assayed sexual and genomic isolation within and between these semispecies in both sympatric and allopatric populations. First, we found no evidence of reproductive character displacement (RCD) in sympatric zones compared to distant allopatry. Instead, semispecies were virtually completely sexually isolated from each other across their entire ranges. Moreover, using spatial approaches and coalescent demographic simulations, we detected either zero or only weak heterospecific gene flow in sympatry. In contrast, within each semispecies we found only random mating and little population genetic structure, except between highly geographically distant populations. Finally, we determined that speciation in this system is at least an order of magnitude older than previously assumed, with WN diverging first, around 200K years ago, and EA and EB diverging 100K years ago. In total, these results suggest that these semispecies should be given full species status and we adopt new nomenclature: WN—D. athabasca, EA—D. mahican, and EB—D. lenape. While the lack of RCD in sympatry and interfertility do not support reinforcement, we discuss what additional evidence is needed to further decipher the mechanisms that caused rapid speciation in this species complex.     

Divergence history of the Carpathian and smooth newts modelled in space and time

Information about demographic history is essential for the understanding of the processes of divergence and speciation. Patterns of genetic variation within and between closely related species provide insights into the history of their interactions. Here, we investigated historical demography and genetic exchange between the Carpathian (Lissotriton montandoni, Lm) and smooth (L. vulgaris, Lv) newts. We combine an extensive geographical sampling and multilocus nuclear sequence data with the approximate Bayesian computation framework to test alternative scenarios of divergence and reconstruct the temporal and spatial pattern of gene flow between species. A model of recent (last glacial period) interspecific gene flow was favoured over alternative models. Thus, despite the relatively old divergence (4–6 mya) and presumably long periods of isolation, the species have retained the ability to exchange genes. Nevertheless, the low migration rates (ca. 10^?6 per gene copy per generation) are consistent with strong reproductive isolation between the species. Models allowing demographic changes were favoured, suggesting that the effective population sizes of both species at least doubled as divergence reaching the current ca. 0.2 million in Lm and 1 million in Lv. We found asymmetry in rates of interspecific gene flow between Lm and one evolutionary lineage of Lv. We suggest that intraspecific polymorphism for hybrid incompatibilities segregating within Lv could explain this pattern and propose further tests to distinguish between alternative explanations. Our study highlights the importance of incorporating intraspecific genetic structure into the models investigating the history of divergence.     

Species Recognition, Dewlap Function and Faunal Size

Although having a number of functions in reproductive, territorialand aggressive behavior, Anolis dewlaps are only one means ofintraspecific signaling and they are always present in smallanole faunas (e.g., those of one or two species islands) buthere, in contrast to the situation in large faunas, their colorand pattern appear usually very similar and appear to be unimportantfor species (or population) recognition. The latter functionis then performed by such characteristics as adult size andbody color and pattern. Where, however, numerous species abutor overlap, dewlap color and pattern tend to be diverse anddiagnostic (particularly between overlapping forms). Even here,however, adult size, body shape and body color often redundantlyreinforce the species and population recognition function ofeven marked dewlap difference. In certain cases in complex faunas,size and/or body pattern substitute for the species recognitionfunction of reduced or absent dewlaps.     

Highly variable reproductive isolation among pairs of Catostomus species

Hybridization between diverged taxa tests the strength of reproductive isolation and can therefore reveal mechanisms of reproductive isolation. However, it remains unclear how consistent reproductive isolation is across species' ranges and to what extent reproductive isolation might remain polymorphic as species diverge. To address these questions, we compared outcomes of hybridization across species pairs of Catostomus fishes in three rivers in the Upper Colorado River basin, where an introduced species, C. commersoni, hybridizes with at least two native species, C. discobolus and C. latipinnis. We observed substantial heterogeneity in outcomes of hybridization, both between species pairs and across geographically separate rivers within each species pair. We also observed hybridization of additional related species with our focal species, suggesting that reproductive isolation in this group involves interactions of multiple evolutionary and ecological factors. These findings suggest that a better understanding of the determinants of variation in reproductive isolation is needed and that studies of reproductive isolation in hybrids should consider how the dynamics and mechanisms of reproductive isolation vary over ecological space and over evolutionary time. Our results also have implications for the conservation and management of native catostomids in the Colorado River basin. Heterogeneity in outcomes of hybridization suggests that the threat posed by hybridization and genetic introgression to the persistence of native species probably varies with extent of reproductive isolation, both across rivers and across species pairs.     

Male and female contributions to behavioral isolation in darters as a function of genetic distance and color distance

Determining which reproductive isolating barriers arise first between geographically isolated lineages is critical to understanding allopatric speciation. We examined behavioral isolation among four recently diverged allopatric species in the orangethroat darter clade (Etheostoma: Ceasia). We also examined behavioral isolation between each Ceasia species and the sympatric rainbow darter Etheostoma caeruleum. We asked (1) is behavioral isolation present between allopatric Ceasia species, and how does this compare to behavioral isolation with E. caeruleum, (2) does male color distance and/or genetic distance predict behavioral isolation between species, and (3) what are the relative contributions of female choice, male choice, and male competition to behavioral isolation? We found that behavioral isolation, genetic differentiation, and male color pattern differentiation were present between allopatric Ceasia species. Males, but not females, discerned between conspecific and heterospecific mates. Males also directed more aggression toward conspecific rival males. The high levels of behavioral isolation among Ceasia species showed no obvious pattern with genetic distance or male color distance. However, when the E. caeruleum was included in the analysis, an association between male aggression and male color distance was apparent. We discuss the possibility that reinforcement between Ceasia and E. caeruleum is driving behavioral isolation among allopatric Ceasia species.     

Phenotypic and genetic diversity in aposematic Malagasy poison frogs (genus Mantella)

Intraspecific color variation has long fascinated evolutionary biologists. In species with bright warning coloration, phenotypic diversity is particularly compelling because many factors, including natural and sexual selection, contribute to intraspecific variation. To better understand the causes of dramatic phenotypic variation in Malagasy poison frogs, we quantified genetic structure and color and pattern variation across three closely related species, Mantella aurantiaca, Mantella crocea, and Mantella milotympanum. Although our restriction site‐associated DNA (RAD) sequencing approach identified clear genetic clusters, they do not align with current species designations, which has important conservation implications for these imperiled frogs. Moreover, our results suggest that levels of intraspecific color variation within this group have been overestimated, while species diversity has been underestimated. Within major genetic clusters, we observed distinct patterns of variation including: populations that are phenotypically similar yet genetically distinct, populations where phenotypic and genetic breaks coincide, and populations that are genetically similar but have high levels of within‐population phenotypic variation. We also detected admixture between two of the major genetic clusters. Our study suggests that several mechanisms—including hybridization, selection, and drift—are contributing to phenotypic diversity. Ultimately, our work underscores the need for a reevaluation of how polymorphic and polytypic populations and species are classified, especially in aposematic organisms.     

Analysis of color spectra in comparative evolutionary studies: molecular phylogeny and habitat adaptation in the St. Vincent Anole (Anolis trinitatis)

The use of color (as distinct from color pattern) in comparative evolutionary studies is important, and objective, independent characters are needed. A new method was employed to investigate geographic color variation in the small arboreal lizard Anolis trinitatis on the island of St. Vincent. The simple delta analysis (based on the difference between eigenvector coefficients for adjacent regions of the spectrum) is aimed at increasing the objectivity with which a spectrum is cut into independent segments and does not predetermine segment width or number. There are distinct habitat types within this small island and distinct phylogenetic lineages (based on a kilobase of cytochrome b sequence) within this species. A series of matrix correspondence (Mantel) tests indicate that aspects of color are associated with habitat type (e.g., green dorsum in rain forest lizards), molecular phylogeny, or both. Hence, both adaptation by selection and historical processes are implicated as causes of geographic variation in color. The dewlap variation (e.g., strong ultraviolet reflectance in some Atlantic coastal sites) is very pronounced and, contrary to some expectations, may result in reproductive isolation even within small Lesser Antillean islands.     

Does breeding season variation affect evolution of a sexual signaling trait in a tropical lizard clade?

Sexually selected traits can be expected to increase in importance when the period of sexual behavior is constrained, such as in seasonally restricted breeders. Anolis lizard male dewlaps are classic examples of multifaceted signaling traits, with demonstrated intraspecific reproductive function reflected in courtship behavior. Fitch and Hillis found a correlation between dewlap size and seasonality in mainland Anolis using traditional statistical methods and suggested that seasonally restricted breeding seasons enhanced the differentiation of this signaling trait. Here, we present two tests of the Fitch–Hillis Hypothesis using new phylogenetic and morphological data sets for 44 species of Mexican Anolis. A significant relationship between dewlap size and seasonality is evident in phylogenetically uncorrected analyses but erodes once phylogeny is accounted for. This loss of strong statistical support for a relationship between a key aspect of dewlap morphology and seasonality also occurs within a species complex (A. sericeus group) that inhabits seasonal and aseasonal environments. Our results fail to support seasonality as a strong driver of evolution of Anolis dewlap size. We discuss the implications of our results and the difficulty of disentangling the strength of single mechanisms on trait evolution when multiple selection pressures are likely at play.     

Persistent postmating,prezygotic reproductive isolation between populations

Studying reproductive barriers between populations of the same species is critical to understand how speciation may proceed. Growing evidence suggests postmating, prezygotic (PMPZ) reproductive barriers play an important role in the evolution of early taxonomic divergence. However, the contribution of PMPZ isolation to speciation is typically studied between species in which barriers that maintain isolation may not be those that contributed to reduced gene flow between populations. Moreover, in internally fertilizing animals, PMPZ isolation is related to male ejaculate—female reproductive tract incompatibilities but few studies have examined how mating history of the sexes can affect the strength of PMPZ isolation and the extent to which PMPZ isolation is repeatable or restricted to particular interacting genotypes. We addressed these outstanding questions using multiple populations of Drosophila montana. We show a recurrent pattern of PMPZ isolation, with flies from one population exhibiting reproductive incompatibility in crosses with all three other populations, while those three populations were fully fertile with each other. Reproductive incompatibility is due to lack of fertilization and is asymmetrical, affecting female fitness more than males. There was no effect of male or female mating history on reproductive incompatibility, indicating that PMPZ isolation persists between populations. We found no evidence of variability in fertilization outcomes attributable to different female × male genotype interactions, and in combination with our other results, suggests that PMPZ isolation is not driven by idiosyncratic genotype × genotype interactions. Our results show PMPZ isolation as a strong, consistent barrier to gene flow early during speciation and suggest several targets of selection known to affect ejaculate‐female reproductive tract interactions within species that may cause this PMPZ isolation.     

A test of alternative hypotheses for the evolution of reproductive isolation between spadefoot toads: support for the reinforcement hypothesis

Abstract How do species that interbreed become reproductively isolated? If hybrids are less fit than parental types, natural selection should promote reproductive isolation by favoring the evolution of premating mechanisms that prevent hybridization (a process termed reinforcement). Although reinforcement should generate a decline in hybridization over time, countervailing forces of gene flow and recombination are thought to preclude natural selection from enhancing and finalizing reproductive isolation. Here, I present recent estimates of hybridization frequency between two species of spadefoot toad, Spea multiplicata and S. bombifrons. I compare these recent measures of hybrid frequency with previously published estimates and show that hybridization between these species has declined precipitously over the past 27 years. Although previous studies suggest that reinforcement possibly accounts for this decline in hybrids over time, three alternative hypotheses also can explain the observed decrease in hybridization. First, if one of the two interacting species becomes rare, opportunities for and incidence of hybridization may decrease. Second, if one of the two interacting species is initially rare, hybridization may be initially common if the rare species has difficulty locating conspecific mates. Third, if hybrids are produced only in particular environments, hybrid frequency may decline if habitat changes result in loss of those environments that promote hybrid formation. I found no support for these three alternative explanations of the decline in hybrids. Instead, reinforcement appears to best account for the evolution of enhanced reproductive isolation between these species. Moreover, the finding that hybridization declined precipitously in only 27 years suggests that many systems that have undergone reinforcement may be overlooked because reproductive isolation between the interacting populations or species may already be complete.     

A tree of tree frogs around the Black Sea

Speciation, the process by which one species evolves into two or more, is a major focus of ongoing debate, particularly regarding the geographic context in which it occurs. Geographic models of speciation tend to fall into discrete categories, typically referred to as allopatric, parapatric and sympatric speciation, according to whether two groups evolve reproductive isolation while geographically isolated, differentiated but connected by gene flow, or completely co‐occurring. Yet molecular studies indicate that full development of reproductive isolation can take very long compared with the timescale at which climatic oscillations occur, such that the geographic context of differentiating forms might change often during the long process to full species. Studies of genetic relationships across the ranges of organisms with low‐dispersal distances have the potential to reveal these complex histories. In a particularly elegant example in this issue, Dufresnes et al. ( 2016 ) use genetic variation and ecological niche modelling to show that a ring of populations of the eastern tree frog (Hyla orientalis) surrounding the Black Sea had a complex history of geographic differentiation. Alternating phases of geographic fragmentation and phases of gene flow between neighbouring populations have produced a pattern of gradual genetic change connecting the western, southern and eastern sides of the ring, with the northwestern and northeastern forms being most differentiated. In the north, a population in Crimea appears to have been produced through mixture of the two extreme forms. The overall genetic relationships are reminiscent of those found in ring species, which have been used as prime demonstrations of the process of speciation. The difference, however, is that the terminal forms appear to have mixed rather than be reproductively isolated, although more research is needed to infer whether there might be some reproductive isolation on the northern side of the ring.