Character displacement among bat-pollinated flowers of the genus Burmeistera: analysis of mechanism, process and pattern

Coexisting plants that share pollinators can compete through interspecific pollen transfer. A long-standing idea holds that divergence in floral morphology may reduce this competition by placing pollen on different regions of the pollinator's bodies. However, surprisingly little empirical support for this idea exists. Burmeistera is a diverse neotropical genus that exhibits wide interspecific variation in the degree to which the reproductive parts are exserted outside the corolla. Coexisting Burmeistera share bats as their primary pollinators, and the degree of exsertion determines the site of pollen deposition on the bats' heads. Here we study the mechanism, process and pattern of floral character displacement for assemblages of coexisting Burmeistera. Flight cage experiments with bats and pairs of Burmeistera species demonstrate that the greater the divergence in exsertion length, the less pollen transferred interspecifically. Null model analyses of exsertion lengths for 19 species of Burmeistera across 18 sites (each containing two to four species) demonstrate that observed assemblage structure is significantly overdispersed relative to what would be expected by chance. Local evolution, rather than ecological sorting, appears to be the primary process driving this pattern of overdispersion because local adaptation of the nine widespread species accounts for a large portion of the observed pattern. Taken together, results of this study provide strong support for the idea that competition through interspecific pollen transfer can drive character displacement in plants.     

Minor quantitative trait loci underlie floral traits associated with mating system divergence in Mimulus

The genetic basis of species differences provides insight into the mode and tempo of phenotypic divergence. We investigate the genetic basis of floral differences between two closely related plant taxa with highly divergent mating systems, Mimulus guttatus (large-flowered outcrosser) and M. nasutus (small-flowered selfer). We had previously constructed a framework genetic linkage map of the hybrid genome containing 174 markers spanning approximately 1800 cM on 14 linkage groups. In this study, we analyze the genetics of 16 floral, reproductive, and vegetative characters measured in a large segregating M. nasutus x M. guttatus F2 population (N = 526) and in replicates of the parental lines and F1 hybrids. Phenotypic analyses reveal strong genetic correlations among floral traits and epistatic breakdown of male and female fertility traits in the F2 hybrids. We use multitrait composite interval mapping to jointly locate and characterize quantitative trait loci (QTLs) underlying interspecific differences in seven floral traits. We identified 24 floral QTLs, most of which affected multiple traits. The large number of QTLs affecting each trait (mean = 13, range = 11-15) indicates a strikingly polygenic basis for floral divergence in this system. In general, QTL effects are small relative to both interspecific differences and environmental variation within genotypes, ruling out QTLs of major effect as contributors to floral divergence between M. guttatus and M. nasutus. QTLs show no pattern of directional dominance. Floral characters associated with pollinator attraction (corolla width) and self-pollen deposition (stigma-anther distance) share several pleiotropic or linked QTLs, but unshared QTLs may have allowed selfing to evolve independently from flower size. We discuss the polygenic nature of divergence between M. nasutus and M. guttatus in light of theoretical work on the evolution of selfing, genetics of adaptation, and maintenance of variation within populations.     

Pleiotropic quantitative trait loci contribute to population divergence in traits associated with life-history variation in Mimulus guttatus

Evolutionary biologists seek to understand the genetic basis for multivariate phenotypic divergence. We constructed an F2 mapping population (N = 539) between two distinct populations of Mimulus guttatus. We measured 20 floral, vegetative, and life-history characters on parents and F1 and F2 hybrids in a common garden experiment. We employed multitrait composite interval mapping to determine the number, effect, and degree of pleiotropy in quantitative trait loci (QTL) affecting divergence in floral, vegetative, and life-history characters. We detected 16 QTL affecting floral traits; 7 affecting vegetative traits; and 5 affecting selected floral, vegetative, and life-history traits. Floral and vegetative traits are clearly polygenic. We detected a few major QTL, with all remaining QTL of small effect. Most detected QTL are pleiotropic, implying that the evolutionary shift between these annual and perennial populations is constrained. We also compared the genetic architecture controlling floral trait divergence both within (our intraspecific study) and between species, on the basis of a previously published analysis of M. guttatus and M. nasutus. Eleven of our 16 floral QTL map to approximately the same location in the interspecific map based on shared, collinear markers, implying that there may be a shared genetic basis for floral divergence within and among species of Mimulus.     

Nectar reward and advertisement in hummingbird-pollinated Silene virginica (Caryophyllaceae)

We tested for an association between nectar and various floral traits and investigated their roles as primary and secondary pollinator attractants in hummingbird-pollinated Silene virginica. Our goal was to gain insight into the mechanisms of pollinator-mediated selection that underlies floral trait divergence within the genus. In a field population of S. virginica, we measured five floral and eight vegetative traits and quantified nectar volume, nectar sugar concentration, and total sugar reward (nectar volume × nectar sugar concentration). All three components of nectar reward were positively correlated to flower size, and nectar volume varied significantly among individuals within the population. To ascertain whether the correlation of specific floral traits with nectar reward influences the behavior of the primary pollinator of S. virginica, the ruby-throated hummingbird, Archilochus colubris, we investigated whether A. colubris preferred the expression of floral traits associated with high nectar volume and total sugar reward. We accomplished this by constructing floral arrays consisting of artificial flowers that had equal nectar quantity and total sugar reward but that differed in petal area and corolla tube diameter, which were positively correlated with nectar quantity and total sugar reward in our field study. In observations of visitation frequencies to the various floral-trait combinations, hummingbirds preferentially visited artificial floral phenotypes with larger petal displays, with the greatest preference for floral phenotypes with both larger petals and wider corolla-tube diameters. This association between primary and secondary floral attractants and hummingbird discrimination of floral features supports the concept that the floral traits of S. virginica reflect pollinator-mediated selection by the principal pollinator.     

Reproductive character displacement and environmental filtering shape floral variation between sympatric sister taxa

Divergence in reproductive traits between closely related species that co‐occur contributes to speciation by reducing interspecific gene flow. In flowering plants, greater floral divergence in sympatry than allopatry may reflect reproductive character displacement (RCD) by means of divergent pollinator‐mediated selection or mating system evolution. However, environmental filtering (EF) would prevail for floral traits under stronger selection by abiotic factors than pollination, and lead to sympatric taxa being more phenotypically similar. We determine whether floral UV pigmentation and size show signatures of RCD or EF using a biogeographically informed sister taxa comparison. We determine whether 35 sister pairs in the Potentilleae tribe (Rosaceae) are allopatric or sympatric and confirm that sympatric sisters experience more similar bioclimatic conditions, an assumption of the EF hypothesis. We test whether interspecific differences are greater in allopatry or sympatry while accounting for divergence time. For UV pigmentation, sympatric sisters are more phenotypically similar than allopatric ones. For flower size, sympatric sisters show increased divergence with time since speciation but allopatric ones do not. We conclude that floral UV pigmentation shows a signature of EF, whereas flower size shows a signature of RCD. Discordant results between the traits suggest that the dominant selective agent differs between them.     

THE INFLUENCE OF POLLINATOR PHYLOGEOGRAPHY AND MATE PREFERENCE ON FLORAL DIVERGENCE IN A SEXUALLY DECEPTIVE DAISY

Divergent mate preferences and subsequent genetic differentiation between populations has been demonstrated, but its effects on interspecific interactions are unknown. Associated species exploiting these mate preferences, for example, may diverge to match local preferences. We explore this idea in the sexually deceptive, fly‐mimicking daisy, Gorteria diffusa, by testing for association between genetic structure in the fly pollinator (a proxy for mate preference divergence) and geographic divergence in floral form. If genetic structure in flies influences interactions with G. diffusa, we expect phylogeographically distinct flies to be associated with different floral forms. Flies associated with forms exploiting only feeding behavior often belonged to several phylogeographic clades, whereas flies associated with forms exploiting male‐mating behavior always belonged to distinct clades, indicating the possibility of pollinator‐mediated floral divergence through phylogeographic variation in mating preferences of male flies. We tested this hypothesis with reciprocal presentations using male flies from distinct clades associated with separate floral forms. Results show that males from all clades exhibit similar preferences, making pollinator driven divergence through geographic variation in mate preference unlikely. Males, however, showed evidence of learned resistance to deceptive traits, suggesting antagonistic interactions between plants and pollinators may drive deceptive floral trait evolution in G. diffusa.     

Plant–pollinator interactions and floral convergence in two species of <Emphasis Type="Italic">Heliconia</Emphasis> from the Caribbean Islands

Variation in interspecific interactions across geographic space is a potential driver of diversification and local adaptation. This study quantitatively examined variation in floral phenotypes and pollinator service of Heliconia bihai and H. caribaea across three Antillean islands. The prediction was that floral characters would correspond to the major pollinators of these species on each island. Analysis of floral phenotypes revealed convergence among species and populations of Heliconia from the Greater Antilles. All populations of H. caribaea were similar, characterized by long nectar chambers and short corolla tubes. In contrast, H. bihai populations were strongly divergent: on Dominica, H. bihai had flowers with short nectar chambers and long corollas, whereas on Hispaniola, H. bihai flowers resembled those of H. caribaea with longer nectar chambers and shorter corolla tubes. Morphological variation in floral traits corresponded with geographic differences or similarities in the major pollinators on each island. The Hispaniolan mango, Anthracothorax dominicus, is the principal pollinator of both H. bihai and H. caribaea on Hispaniola; thus, the similarity of floral phenotypes between Heliconia species suggests parallel selective regimes imposed by the principal pollinator. Likewise, divergence between H. bihai populations from Dominica and Hispaniola corresponded with differences in the pollinators visiting this species on the two islands. The study highlights the putative importance of pollinator-mediated selection as driving floral convergence and the evolution of locally-adapted plant variants across a geographic mosaic of pollinator species.     

Character displacement drives floral variation in Pelargonium (Geraniaceae) communities

Interactions between plant community members are an underexplored driver of angiosperm floral variation. We investigate character displacement as a potential contributor to floral variation in Pelargonium communities. Pelargoniums all place pollen on the ventral sides of their pollinators, potentially leading to interspecific pollen transfer (IPT) in sympatry. We show that the positions of pollen placement and receipt are determined by anther and style exsertion lengths. Using field experiments, we demonstrate that heterospecific species experience higher IPT if they have similar style lengths than when they have greater style length differences. Using crosses, we show that IPT has negative consequences on seed set. In combination, these results suggest that character displacement in style length is likely to reduce IPT and increase female fitness in sympatry. Patterns of style length variation across 29 different Pelargonium communities suggest that character displacement has occurred in multiple communities. Furthermore, analyses using a wide-ranging species pair show that style lengths are more different between sympatric populations than they are between allopatric populations. In addition to pollinators as agents of floral divergence, this study suggests that variation in Pelargonium community structure has driven style length variation through character displacement.     

Divergence on floral traits and vertebrate pollinators of two endemic Encholirium bromeliads

Shifts in pollen vectors favour diversification of floral traits, and differences in pollination strategies between congeneric sympatric species can contribute to reproductive isolation. Divergence in flowering phenology and selfing could also reduce interspecific crossing between self‐compatible species. We investigated floral traits and visitation rates of pollinators of two sympatric Encholirium species on rocky outcrops to evaluate whether prior knowledge of floral characters could indicate actual pollinators. Data on flowering phenology, visitation rates and breeding system were used to evaluate reproductive isolation. Flowering phenology overlapped between species, but there were differences in floral characters, nectar volume and concentration. Several hummingbird species visited flowers of both Encholirium spp., but the endemic bat Lonchophylla bokermanni and an unidentified sphingid only visited E. vogelii. Pollination treatments demonstrated that E. heloisae and E. vogelii were partially self‐compatible, with weak pollen limitation to seed set. Herbivores feeding on inflorescences decreased reproductive output of both species, but for E. vogelii the damage was higher. Our results indicate that actual pollinators can be known beforehand through floral traits, in agreement with pollination syndromes stating that a set of floral traits can be associated with the attraction of specific groups of pollinators. Divergence on floral traits and pollinator assemblage indicate that shifts in pollination strategies contribute to reproductive isolation between these Encholirium species, not divergence on flowering phenology or selfing. We suggest that hummingbird pollination might be the ancestral condition in Encholirium and that evolution of bat pollination made a substantial contribution to the diversification of this clade.     

Standard percent DNA sequence difference for insects does not predict species boundaries

Diagnosis and assessment of species boundaries of economically important insects are often problematic because of limited morphological and/or biological characters. DNA data can help to identify and revise species. Nonoverlapping intra- and interspecific sequence divergences are often used as evidence for species. Thus, the establishment of a standardized percent nucleotide divergence to predict species boundaries would aid in cases where species status is suspect. However, given variation in nucleotide mutation rates and species concepts, association between a standard percent sequence divergence and species is questionable. This review surveys the percent DNA sequence difference found between sister-species of economically important insects, to assess whether a standard divergence associates with all taxa. Sixty-two comparisons of intra- and interspecific pairwise DNA differences were made for mitochondrial and nuclear loci spanning families of Isoptera, Phthiraptera, Hemiptera, Coleoptera, Lepidoptera, Diptera, and Hymenoptera. Intra- and interspecific sequence divergences varied widely among insects, 0.04-26.0 and 1.0-30.7%, respectively. The ranges of intra- and interspecific sequence divergences overlapped in 28 of 62 comparisons. This implies that a standardized percent sequence divergence would fail to correctly diagnose species for 45% of the cases. Common occurrence of nonmonophyly among closely related species probably explains this observation. Nonmonophyly and overlap of intra- and interspecific divergences were significantly associated. The reviewed studies suggest that a standard percent sequence divergence does not predict species boundaries among economically important insects. DNA data can help best to predict species boundaries via its inclusion in nonphenetic phylogenetic analysis and subsequent systematic expert scrutiny.     

Multiple strong postmating and intrinsic postzygotic reproductive barriers isolate florally diverse species of Jaltomata (Solanaceae)

Divergence in phenotypic traits often contributes to premating isolation between lineages, but could also promote isolation at postmating stages. Phenotypic differences could directly result in mechanical isolation or hybrids with maladapted traits; alternatively, when alleles controlling these trait differences pleiotropically affect other components of development, differentiation could indirectly produce genetic incompatibilities in hybrids. Here, we determined the strength of nine postmating and intrinsic postzygotic reproductive barriers among 10 species of Jaltomata (Solanaceae), including species with highly divergent floral traits. To evaluate the relative importance of floral trait diversification for the strength of these postmating barriers, we assessed their relationship to floral divergence, genetic distance, geographical context, and ecological differences, using conventional tests and a new linear‐mixed modeling approach. Despite close evolutionary relationships, all species pairs showed moderate to strong isolation. Nonetheless, floral trait divergence was not a consistent predictor of the strength of isolation; instead this was best explained by genetic distance, although we found evidence for mechanical isolation in one species, and a positive relationship between floral trait divergence and fruit set isolation across species pairs. Overall, our data indicate that intrinsic postzygotic isolation is more strongly associated with genome‐wide genetic differentiation, rather than floral divergence.     

A preliminary study on pollination biology of Omphalogramma souliei Franch. (Primulaceae), a species endemic to China

The pollination biology of Omphalogramma souliei Franch., a species endemic to southwestern China, was investigated. Floral phenology, flower visitors, pollen/ovule ratio, attractants and rewards to the visitors were observed, measured, and recorded. Bagging experiments to exclude pollinators were carried out in the wild for two years. Our results revealed five important aspects of the reproductive biology of Omphalogramma souliei. 1) The pollen-ovule ratio was 1748±233. The breeding system was self-compatible, with facultative xenogamy. 2) The pollination syndrome is entomophily, and this species could not be pollinated by wind if the pollinators were unavailable. 3) Six insect species were observed visiting the flowers of Omphalogramma souliei in the wild, of them, three species of hymenoptera, Lasioglossum sp., Heriades parvula Cockerell and Micrapis florae Fabricius, are the principal floral visitors and effective pollinators. 4) The visual attractants to the visitors are floral color and shape, the large yellow anthers, and the rewards for visitors are pollen and nectar. 5) Cleistogamy may also occur, since the anthers of some flowers dehisced before opening of the corolla. The results of floral biology and pollination characters suggest that xenogamy predominated and autogamy played an assistant role in the evolution of reproduction and breeding system of O. souliei.     

Post-pollination reproductive isolation between diurnally and nocturnally flowering daylilies, Hemerocallis fulva and Hemerocallis citrina

To examine whether floral and post-pollination isolation develops independently or not, we conducted a crossing experiment between Hemerocallis fulva and Hemerocallis citrina that shows large floral divergence adapted for diurnal and nocturnal pollinators that have been believed to be fully cross-fertile. Flowers of the two species from sympatric populations were hand-pollinated with conspecific pollen from the same population (control), interspecific pollen from the same area (sympatric cross), and interspecific pollen from the different area (allopatric cross). After capsule dehiscence, the fruit set, seed set per fruit and seed set per flower were determined among three cross categories. The seed sets per flower were 32 and 77% lower in sympatric and allopatric crosses than in the control when H. fulva was the pollen recipient. There was no difference in three reproductive measures among the cross categories when H. citrina was the pollen recipient. This finding indicates that post-pollination isolation does exist between H. fulva and H. citrina, although it is partial, asymmetric, and weakened in sympatry. Our result suggests that floral and post-pollination isolation may develop independently, and reinforcement may not be a general phenomenon in plants.     

Sexual Attraction and Mating Patterns in Cylindrocorpus longistoma and C. curzii (Nematoda: Cylindrocorporidae)

Females of Cylindrocorpus longistoma and C. curzii excrete attractants which probably function to bring the sexes together before mating. Intraspecific and interspecific heterosexual and homosexual pairing experiments showed the attractants to be species specific as well as sex specific. Observations on mating behavior support the hypothesis that sexual attraction and copulation require independent stimuli.     

The role of mating preferences in shaping interspecific divergence in mating signals in vertebrates

Vertebrates represent one of the best-studied groups in terms of the role that mating preferences have played in the evolution of exaggerated secondary sexual characters and mating behaviours within species. Vertebrate species however, also exhibit enormous interspecific diversity in features of mating signals that has potentially led to reproductive isolation and speciation in many groups. The role that sexual selection has played in interspecific divergence in mating signals has been less fully explored. This review summarizes our current knowledge of how mating preferences within species have shaped interspecific divergence in mate recognition signals among the major vertebrate groups. Certain signal modalities appear to characterize mating signal diversification among different vertebrate taxa. Acoustic signals play an important role in mating decisions in anuran amphibians and birds. Here, different properties of the signal may convey information regarding individual, neighbor and species recognition. Mating preferences for particular features of the acoustic signal have led to interspecific divergence in calls and songs. Divergence in morphological traits such as colouration or ornamentation appears to be important in interspecific diversity in certain groups of fishes and birds. Pheromonal signals serve as the primary basis for species-specific mating cues in many salamander species, most mammals and even some fishes. The evolution of interspecific divergence in elaborate courtship displays may have played an important role in speciation of lizards, and particular groups of fishes, salamanders, birds and mammals. While much research has focused on the importance of mating preferences in shaping the evolution of these types of mating signals within species, the link between intraspecific preferences and interspecific divergence and speciation remains to be more fully tested. Future studies should focus on identifying how variation in mating preferences within a species shapes interspecific diversity in features of mating signals in order to better understand how sexual selection may have led to speciation in vertebrates.     

Widespread interspecific divergence in cis-regulation of transposable elements in the Arabidopsis genus

Transposable elements (TEs) are so abundant and variable that they count among the most important mutational sources in genomes. Nonetheless, little is known about the genetics of their variation in activity or silencing across closely related species. Here, we demonstrate that regulation of TE genes can differ dramatically between the two closely related Arabidopsis species A. thaliana and A. lyrata. In leaf and floral tissues of F1 interspecific hybrids, about 47% of TEs show allele-specific expression, with the A. lyrata copy being generally expressed at higher level. We confirm that TEs are generally expressed in A. lyrata but not in A. thaliana. Allele-specific differences in TE expression are associated with divergence in epigenetic modifications like DNA and histone methylation between species as well as with sequence divergence. Our data demonstrate that A. thaliana silences TEs much better than A. lyrata. For long terminal repeat retrotransposons, these differences are more pronounced for younger insertions. Interspecific differences in TE silencing may have a great impact on genome size changes.     

The genomic bases of morphological divergence and reproductive isolation driven by ecological speciation in Senecio (Asteraceae)

Ecological speciation, driven by adaptation to contrasting environments, provides an attractive opportunity to study the formation of distinct species, and the role of selection and genomic divergence in this process. Here, we focus on a particularly clear‐cut case of ecological speciation to reveal the genomic bases of reproductive isolation and morphological differences between closely related Senecio species, whose recent divergence within the last ~200 000 years was likely driven by the uplift of Mt. Etna (Sicily). These species form a hybrid zone, yet remain morphologically and ecologically distinct, despite active gene exchange. Here, we report a high‐density genetic map of the Senecio genome and map hybrid breakdown to one large and several small quantitative trait loci (QTL). Loci under diversifying selection cluster in three 5 cM regions which are characterized by a significant increase in relative (F_ST), but not absolute (d_XY), interspecific differentiation. They also correspond to some of the regions of greatest marker density, possibly corresponding to ‘cold‐spots’ of recombination, such as centromeres or chromosomal inversions. Morphological QTL for leaf and floral traits overlap these clusters. We also detected three genomic regions with significant transmission ratio distortion (TRD), possibly indicating accumulation of intrinsic genetic incompatibilities between these recently diverged species. One of the TRD regions overlapped with a cluster of high species differentiation, and another overlaps the large QTL for hybrid breakdown, indicating that divergence of these species may have occurred due to a complex interplay of ecological divergence and accumulation of intrinsic genetic incompatibilities.     

Comparative intra‐ and interspecific sexual organ reciprocity in four distylous Primula species in the Himalaya‐Hengduan Mountains

• Distyly is a mechanism promoting cross‐pollination within a balanced polymorphism. Numerous studies show that the degree of inter‐morph sexual organ reciprocity (SOR) within species relates to its pollen‐mediated gene flow. Similarly, a lower interspecific SOR should promote interspecific isolation when congeners are sympatric, co‐blooming and share pollinators. In this comparative study, we address the significance of SOR at both intra‐ and interspecific levels.
• Seventeen allopatric and eight sympatric populations representing four Primula species (P. anisodora, P. beesiana, P. bulleyana and P. poissonii) native to the Himalaya‐Hengduan Mountains were measured for eight floral traits in both long‐ and short‐styled morphs. GLMM and spatial overlap methods were used to compare intra‐ and interspecific SOR.
• While floral morphology differed among four Primula species, SOR within species was generally higher than between species, but in species pairs P. poissonii/P. anisodora and P. beesiana/P. bulleyana, the SOR was high at both intra‐ and interspecific levels. We did not detect a significant variation in intraspecific SOR or interspecific SOR when comparing allopatric versus sympatric populations for all species studied.
• As intraspecific SOR increased, disassortative mating may be promoted. As interspecific SOR decreased, interspecific isolation between co‐flowering species pairs also may increase. Hybridisation between congeners occurred when interspecific SOR increased in sympatric populations, as confirmed in two species pairs, P. poissonii/P. anisodora and P. beesiana/P. bulleyana.

     

Spiral phyllotaxis underlies constrained variation in <Emphasis Type="Italic">Anemone</Emphasis> (Ranunculaceae) tepal arrangement

Stabilization and variation of floral structures are indispensable for plant reproduction and evolution; however, the developmental mechanism regulating their structural robustness is largely unknown. To investigate this mechanism, we examined positional arrangement (aestivation) of excessively produced perianth organs (tepals) of six- and seven-tepaled (lobed) flowers in six Anemone species (Ranunculaceae). We found that the tepal arrangement that occurred in nature varied intraspecifically between spiral and whorled arrangements. Moreover, among the studied species, variation was commonly limited to three types, including whorls, despite five geometrically possible arrangements in six-tepaled flowers and two types among six possibilities in seven-tepaled flowers. A spiral arrangement, on the other hand, was unique to five-tepaled flowers. A spiral phyllotaxis model with stochasticity on initiating excessive primordia accounted for these limited variations in arrangement in cases when the divergence angle between preexisting primordia was less than 144°. Moreover, interspecific differences in the frequency of the observed arrangements were explained by the change of model parameters that represent meristematic growth and differential organ growth. These findings suggest that the phyllotaxis parameters are responsible for not only intraspecific stability but interspecific difference of floral structure. Decreasing arrangements from six-tepaled to seven-tepaled Anemone flowers demonstrate that the stabilization occurs as development proceeds to increase the component (organ) number, in contrast from the intuition that the variation will be larger due to increasing number of possible states (arrangements).     

Speciation history of the North American funnel web spiders, Agelenopsis (Araneae: Agelenidae): phylogenetic inferences at the population-species interface

Intra- and interspecific relationships of 12 out of 13 described species as well as a potential new species in the spider genus Agelenopsis (Araneae: Agelenidae) were analyzed using sequence data from two mitochondrial genes, cytochrome oxidase I (COI) and 16S ribosomal RNA. Approximately half of the species examined formed well-supported monophyletic groups, whereas the rest of the species were part of well-supported monophyletic species groups. Rather than viewing cases where species were not identified as being monophyletic as poor taxonomy, these cases more likely represent recent speciation and offer insights into the process of speciation. The clade with the lowest levels of interspecific sequence divergence was found in eastern North America, whereas western species displayed much higher levels of interspecific divergence. These patterns appear to extend below the species level as well, with southwestern species exhibiting the highest levels of intraspecific sequence divergence and geographic structuring. The relationship between Agelenopsis and Barronopsis, a genus once considered a sub-genus of Agelenopsis, was also examined. The two genera are reciprocally monophyletic but more generic level sampling is needed to confirm an apparent sister relationship between the two.