Systematic reduction of natural enemies and competition across variable precipitation approximates buffelgrass invasiveness (Cenchrus ciliaris) in its native range.

Invasive grasses cause devastating losses to biodiversity and ecosystem function directly and indirectly by altering ecosystem processes. Escape from natural enemies, plant-plant competition, and variable resource availability provide frameworks for understanding invasion. However, we lack a clear understanding of how natural stressors interact in their native range to regulate invasiveness. In this study, we reduced diverse guilds of natural enemies and plant competitors of the highly invasive buffelgrass across a precipitation gradient throughout major climatic shifts in Laikipia, Kenya. To do this, we used a long-term ungulate exclosure experiment design across a precipitation gradient with nested treatments that (1) reduced plant competition through clipping, (2) reduced insects through systemic insecticide, and (3) reduced fungal associates through fungicide application. Additionally, we measured the interaction of ungulates on two stem-boring insect species feeding on buffelgrass. Finally, we measured a multiyear smut fungus outbreak. Our findings suggest that buffelgrass exhibits invasive qualities when released from a diverse group of natural stressors in its native range. We show natural enemies interact with precipitation to alter buffelgrass productivity patterns. In addition, interspecific plant competition decreased the basal area of buffelgrass, suggesting that biotic resistance mediates buffelgrass dominance in the home range. Surprisingly, systemic insecticides and fungicides did not impact buffelgrass production or reproduction, perhaps because other guilds filled the niche space in these highly diverse systems. For example, in the absence of ungulates, we showed an increase in host-specific stem-galling insects, where these insects compensated for reduced ungulate use. Finally, we documented a smut outbreak in 2020 and 2021, corresponding to highly variable precipitation patterns caused by a shifting Indian Ocean Dipole. In conclusion, we observed how reducing natural enemies and competitors and certain interactions increased properties related to buffelgrass invasiveness.

Cis-regulatory modes of Ultrabithorax inactivation in butterfly forewings.

Hox gene clusters encode transcription factors that drive regional specialization during animal development: for example the Hox factor Ubx is expressed in the insect metathoracic (T3) wing appendages and differentiates them from T2 mesothoracic identities. Hox transcriptional regulation requires silencing activities that prevent spurious activation and regulatory crosstalks in the wrong tissues, but this has seldom been studied in insects other than Drosophila, which shows a derived Hox dislocation into two genomic clusters that disjoined Antennapedia (Antp) and Ultrabithorax (Ubx). Here, we investigated how Ubx is restricted to the hindwing in butterflies, amidst a contiguous Hox cluster. By analysing Hi-C and ATAC-seq data in the butterfly Junonia coenia, we show that a Topologically Associated Domain (TAD) maintains a hindwing-enriched profile of chromatin opening around Ubx. This TAD is bordered by a Boundary Element (BE) that separates it from a region of joined wing activity around the Antp locus. CRISPR mutational perturbation of this BE releases ectopic Ubx expression in forewings, inducing homeotic clones with hindwing identities. Further mutational interrogation of two non-coding RNA encoding regions and one putative cis-regulatory module within the Ubx TAD cause rare homeotic transformations in both directions, indicating the presence of both activating and repressing chromatin features. We also describe a series of spontaneous forewing homeotic phenotypes obtained in Heliconius butterflies, and discuss their possible mutational basis. By leveraging the extensive wing specialization found in butterflies, our initial exploration of Ubx regulation demonstrates the existence of silencing and insulating sequences that prevent its spurious expression in forewings.

Sex-linked gene traffic underlies the acquisition of sexually dimorphic UV color vision in Heliconius butterflies.

The acquisition of novel sexually dimorphic traits poses an evolutionary puzzle: How do new traits arise and become sex-limited? Recently acquired color vision, sexually dimorphic in animals like primates and butterflies, presents a compelling model for understanding how traits become sex-biased. For example, some Heliconius butterflies uniquely possess UV (ultraviolet) color vision, which correlates with the expression of two differentially tuned UV-sensitive rhodopsins, UVRh1 and UVRh2. To discover how such traits become sexually dimorphic, we studied Heliconius charithonia, which exhibits female-specific UVRh1 expression. We demonstrate that females, but not males, discriminate different UV wavelengths. Through whole-genome shotgun sequencing and assembly of the H. charithonia genome, we discovered that UVRh1 is present on the W chromosome, making it obligately female-specific. By knocking out UVRh1, we show that UVRh1 protein expression is absent in mutant female eye tissue, as in wild-type male eyes. A PCR survey of UVRh1 sex-linkage across the genus shows that species with female-specific UVRh1 expression lack UVRh1 gDNA in males. Thus, acquisition of sex linkage is sufficient to achieve female-specific expression of UVRh1, though this does not preclude other mechanisms, like cis-regulatory evolution from also contributing. Moreover, both this event, and mutations leading to differential UV opsin sensitivity, occurred early in the history of Heliconius. These results suggest a path for acquiring sexual dimorphism distinct from existing mechanistic models. We propose a model where gene traffic to heterosomes (the W or the Y) genetically partitions a trait by sex before a phenotype shifts (spectral tuning of UV sensitivity).

Pathogen-mediated natural and manipulated population collapse in an invasive social insect.

SignificanceInvasive social insects are among the most damaging of invasive organisms and have proved universally intractable to biological control. Despite this, populations of some invasive social insects collapse from unknown causes. We report long-term studies demonstrating that infection by a microsporidian pathogen causes populations of a globally significant invasive ant to collapse to local extinction, providing a mechanistic understanding of a pervasive phenomenon in biological invasions: the collapse of established populations from endogenous factors. We apply this knowledge and successfully eliminate two large, introduced populations of these ants. More broadly, microsporidian pathogens should be evaluated for control of other supercolonial invasive social insects. Diagnosing the cause of unanticipated population collapse in invasive organisms can lead to applied solutions.

A large deletion at the cortex locus eliminates butterfly wing patterning.

As the genetic basis of natural and domesticated variation has been described in recent years, a number of hotspot genes have been repeatedly identified as the targets of selection, Heliconius butterflies display a spectacular diversity of pattern variants in the wild and the genetic basis of these patterns has been well-described. Here, we sought to identify the mechanism behind an unusual pattern variant that is instead found in captivity, the ivory mutant, in which all scales on both the wings and body become white or yellow. Using a combination of autozygosity mapping and coverage analysis from 37 captive individuals, we identify a 78-kb deletion at the cortex wing patterning locus, a gene which has been associated with wing pattern evolution in H. melpomene and 10 divergent lepidopteran species. This deletion is undetected among 458 wild Heliconius genomes samples, and its dosage explains both homozygous and heterozygous ivory phenotypes found in captivity. The deletion spans a large 5' region of the cortex gene that includes a facultative 5'UTR exon detected in larval wing disk transcriptomes. CRISPR mutagenesis of this exon replicates the wing phenotypes from coding knock-outs of cortex, consistent with a functional role of ivory-deleted elements in establishing scale color fate. Population demographics reveal that the stock giving rise to the ivory mutant has a mixed origin from across the wild range of H. melpomene, and supports a scenario where the ivory mutation occurred after the introduction of cortex haplotypes from Ecuador. Homozygotes for the ivory deletion are inviable while heterozygotes are the targets of artificial selection, joining 40 other examples of allelic variants that provide heterozygous advantage in animal populations under artificial selection by fanciers and breeders. Finally, our results highlight the promise of autozygosity and association mapping for identifying the genetic basis of aberrant mutations in captive insect populations.

Clade-Specific Plastid Inheritance Patterns Including Frequent Biparental Inheritance in Passiflora Interspecific Crosses.

Plastid inheritance in angiosperms is presumed to be largely maternal, with the potential to inherit plastids biparentally estimated for about 20% of species. In Passiflora, maternal, paternal and biparental inheritance has been reported; however, these studies were limited in the number of crosses and progeny examined. To improve the understanding of plastid transmission in Passiflora, the progeny of 45 interspecific crosses were analyzed in the three subgenera: Passiflora, Decaloba and Astrophea. Plastid types were assessed following restriction digestion of PCR amplified plastid DNA in hybrid embryos, cotyledons and leaves at different developmental stages. Clade-specific patterns of inheritance were detected such that hybrid progeny from subgenera Passiflora and Astrophea predominantly inherited paternal plastids with occasional incidences of maternal inheritance, whereas subgenus Decaloba showed predominantly maternal and biparental inheritance. Biparental plastid inheritance was also detected in some hybrids from subgenus Passiflora. Heteroplasmy due to biparental inheritance was restricted to hybrid cotyledons and first leaves with a single parental plastid type detectable in mature plants. This indicates that in Passiflora, plastid retention at later stages of plant development may not reflect the plastid inheritance patterns in embryos. Passiflora exhibits diverse patterns of plastid inheritance, providing an excellent system to investigate underlying mechanisms in angiosperms.

Rampant Nuclear Transfer and Substitutions of Plastid Genes in Passiflora.

Gene losses in plastid genomes (plastomes) are often accompanied by functional transfer to the nucleus or substitution of an alternative nuclear-encoded gene. Despite the highly conserved gene content in plastomes of photosynthetic land plants, recent gene loss events have been documented in several disparate angiosperm clades. Among these lineages, Passiflora lacks several essential ribosomal genes, rps7, rps16, rpl20, rpl22, and rpl32, the two largest plastid genes, ycf1 and ycf2, and has a highly divergent rpoA. Comparative transcriptome analyses were performed to determine the fate of the missing genes in Passiflora. Putative functional transfers of rps7, rpl22, and rpl32 to nucleus were detected, with the nuclear transfer of rps7, representing a novel event in angiosperms. Plastid-encoded rps7 was transferred into the intron of a nuclear-encoded plastid-targeted thioredoxin m-type gene, acquiring its plastid transit peptide (TP). Plastid rpl20 likely experienced a novel substitution by a duplicated, nuclear-encoded mitochondrial-targeted rpl20 that has a similar gene structure. Additionally, among rosids, evidence for a third independent transfer of rpl22 in Passiflora was detected that gained a TP from a nuclear gene containing an organelle RNA recognition motif. Nuclear transcripts representing rpoA, ycf1, and ycf2 were not detected. Further analyses suggest that the divergent rpoA remains functional and that the gene is under positive or purifying selection in different clades. Comparative analyses indicate that alternative translocon and motor protein complexes may have substituted for the loss of ycf1 and ycf2 in Passiflora.

Ritualized aggressive behavior reveals distinct social structures in native and introduced range tawny crazy ants.

How workers within an ant colony perceive and enforce colony boundaries is a defining biological feature of an ant species. Ants fall along a spectrum of social organizations ranging from single-queen, single nest societies to species with multi-queen societies in which workers exhibit colony-specific, altruistic behaviors towards non-nestmate workers from distant locations. Defining where an ant species falls along this spectrum is critical for understanding its basic ecology. Herein we quantify queen numbers, describe intraspecific aggression, and characterize the distribution of colony sizes for tawny crazy ant (Nylanderia fulva) populations in native range areas in South America as well as in their introduced range in the Southeastern United States. In both ranges, multi-queen nests are common. In the introduced range, aggressive behaviors are absent at all spatial scales tested, indicating that within the population in the Southeastern United States N. fulva is unicolonial. However, this contrasts strongly with intraspecific aggression in its South American native range. In the native range, intraspecific aggression between ants from different nests is common and ritualized. Aggression is typically one-sided and follows a stereotyped sequence of escalating behaviors that stops before actual fighting occurs. Spatial patterns of non-aggressive nest aggregation and the transitivity of non-aggressive interactions demonstrate that results of neutral arena assays usefully delineate colony boundaries. In the native range, both the spatial extent of colonies and the average number of queens encountered per nest differ between sites. This intercontinental comparison presents the first description of intraspecific aggressive behavior for this invasive ant and characterizes the variation in colony organization in the native-range, a pre-requisite to a full understanding of the origins of unicoloniality in its introduced range.

Highly accelerated rates of genomic rearrangements and nucleotide substitutions in plastid genomes of Passiflora subgenus Decaloba.

Plastid genomes (plastomes) of photosynthetic angiosperms are for the most part highly conserved in their organization, mode of inheritance and rates of nucleotide substitution. A small number of distantly related lineages share a syndrome of features that deviate from this general pattern, including extensive genomic rearrangements, accelerated rates of nucleotide substitution, biparental inheritance and plastome-genome incompatibility. Previous studies of plastomes in Passiflora with limited taxon sampling suggested that the genus exhibits this syndrome. To examine this phenomenon further, 15 new plastomes from Passiflora were sequenced and combined with previously published data to examine the phylogenetic relationships, genome organization and evolutionary rates across all five subgenera and the sister genus Adenia. Phylogenomic analyses using 68 protein-coding genes shared by Passiflora generated a fully resolved and strongly supported tree that is congruent with previous phylogenies based on a few plastid and nuclear loci. This phylogeny was used to examine the distribution of plastome rearrangements across Passiflora. Multiple gene and intron losses and inversions were identified in Passiflora with some occurring in parallel and others that extended across the Passifloraceae. Furthermore, extensive expansions and contractions of the inverted repeat (IR) were uncovered and in some cases this resulted in exclusion of all ribosomal RNA genes from the IR. The most highly rearranged lineage was subgenus Decaloba, which experienced extensive IR expansion that incorporated up to 25 protein-coding genes usually located in large single copy region. Nucleotide substitution rate analyses of 68 protein-coding genes across the genus showed lineage- and locus-specific acceleration. Significant increase in dS, dN and dN/dS was detected for clpP across the genus and for ycf4 in certain lineages. Significant increases in dN and dN/dS for ribosomal subunits and plastid-encoded RNA polymerase genes were detected in the branch leading to the expanded IR-clade in subgenus Decaloba. This subgenus displays the syndrome of unusual features, making it an ideal system to investigate the dynamic evolution of angiosperm plastomes.

Twittering Pupae of Papilionid and Nymphalid Butterflies (Lepidoptera): Novel Structures and Sounds.

Pupae of numerous Papilionidae and Nymphalidae produce twitter sounds when wriggling in response to mechanical stimulation. The structural basis comprises distinct pairs of sound-producing organs (SPOs) located at intersegmental membranes of the abdomen. They differ-as the twitters do-in sampled taxa of Papilioninae, Epicaliini, and Heliconiini. The opposing sculptured cuticular sound plates (SPs) of each SPO appear structurally the same but are actually mirror-images of each other. Results suggest that sounds are not generated by stridulation (friction of a file and a scraper) but when these inversely sculptured and interlocking surfaces separate during pupal wriggling, representing a stick-slip mechanism. Twitter sounds comprise series of short broadband pulses with the main energy in the frequency range 3-13 kHz; they can be heard by humans but extend into ultrasonic frequencies up to 100 kHz.

Pseudacteon notocaudatus and Pseudacteon obtusitus (Diptera: Phoridae), two new species of fire ant parasitoids from South America.

Ongoing studies in South America of phorid flies of the genus Pseudacteon Coquillett 1907 have revealed two further new species in this genus that are described here: P. obtusitus and P. notocaudatus. Both species are parasitoids of Solenopsis (F.) fire ants.

Widespread Chemical Detoxification of Alkaloid Venom by Formicine Ants.

The ability to detoxify defensive compounds of competitors provides key ecological advantages that can influence community-level processes. Although common in plants and bacteria, this type of detoxification interaction is extremely rare in animals. Here, using laboratory behavioral assays and analyses of videotaped interactions in South America, we report widespread venom detoxification among ants in the subfamily Formicinae. Across both data sets, nine formicine species, representing all major clades, used a stereotyped grooming behavior to self-apply formic acid (acidopore grooming) in response to fire ant (Solenopsis invicta and S. saevissima) venom exposure. In laboratory assays, this behavior increased the survivorship of species following exposure to S. invicta venom. Species expressed the behavior when exposed to additional alkaloid venoms, including both compositionally similar piperidine venom of an additional fire ant species and the pyrrolidine/pyrroline alkaloid venom of a Monomorium species. In addition, species expressed the behavior following exposure to the uncharacterized venom of a Crematogaster species. However, species did not express acidopore grooming when confronted with protein-based ant venoms or when exposed to monoterpenoid-based venom. This pattern, combined with the specific chemistry of the reaction of formic acid with venom alkaloids, indicates that alkaloid venoms are targets of detoxification grooming. Solenopsis thief ants, and Monomorium species stand out as brood-predators of formicine ants that produce piperidine, pyrrolidine, and pyrroline venom, providing an important ecological context for the use of detoxification behavior. Detoxification behavior also represents a mechanism that can influence the order of assemblage dominance hierarchies surrounding food competition. Thus, this behavior likely influences ant-assemblages through a variety of ecological pathways.

Myrmecomorba nylanderiae gen. et sp. nov., a microsporidian parasite of the tawny crazy ant Nylanderia fulva.

A new microsporidian genus and species, Myrmecomorba nylanderiae, is described from North American populations of the tawny crazy ant, Nylanderia fulva. This new species was found to be heterosporous producing several types of binucleate spores in both larval and adult stages and an abortive octosporoblastic sporogony in adult ants. While microsporidia are widespread arthropod parasites, this description represents only the fifth species described from an ant host. Molecular analysis indicated that this new taxon is phylogenetically closely allied to the microsporidian family Caudosporidae, a group known to parasitize aquatic black fly larvae. We report the presence of 3 spore types (Type 1 DK, Type 2 DK, and octospores) with infections found in all stages of host development and reproductive castes. This report documents the first pathogen infecting N. fulva, an invasive ant of considerable economic and ecological consequence.

Chemical warfare among invaders: a detoxification interaction facilitates an ant invasion.

As tawny crazy ants (Nylanderia fulva) invade the southern United States, they often displace imported fire ants (Solenopsis invicta). After exposure to S. invicta venom, N. fulva applies abdominal exocrine gland secretions to its cuticle. Bioassays reveal that these secretions detoxify S. invicta venom. Further, formic acid from N. fulva venom is the detoxifying agent. N. fulva exhibits this detoxification behavior after conflict with a variety of ant species; however, it expresses it most intensely after interactions with S. invicta. This behavior may have evolved in their shared South American native range. The capacity to detoxify a major competitor's venom probably contributes substantially to its ability to displace S. invicta populations, making this behavior a causative agent in the ecological transformation of regional arthropod assemblages.

Multiple recent co-options of Optix associated with novel traits in adaptive butterfly wing radiations.

BACKGROUND: While the ecological factors that drive phenotypic radiations are often well understood, less is known about the generative mechanisms that cause the emergence and subsequent diversification of novel features. Heliconius butterflies display an extraordinary diversity of wing patterns due in part to mimicry and sexual selection. Identifying the genetic drivers of this crucible of evolution is now within reach, as it was recently shown that cis-regulatory variation of the optix transcription factor explains red pattern differences in the adaptive radiations of the Heliconius melpomene and Heliconius erato species groups. RESULTS: Here, we compare the developmental expression of the Optix protein across a large phylogenetic sample of butterflies and infer that its color patterning role originated at the base of the neotropical passion-vine butterfly clade (Lepidoptera, Nymphalidae, Tribe: Heliconiini), shortly predating multiple Optix-driven wing pattern radiations in the speciose Heliconius and Eueides genera. We also characterize novel Optix and Doublesex expression in the male-specific pheromone wing scales of the basal heliconiines Dryas and Agraulis, thus illustrating that within the Heliconinii lineage, Optix has been evolutionarily redeployed in multiple contexts in association with diverse wing features. CONCLUSIONS: Our findings reveal that the repeated co-option of Optix into various aspects of wing scale specification was associated with multiple evolutionary novelties over a relatively short evolutionary time scale. In particular, the recruitment of Optix expression in colored scale cell precursors was a necessary condition to the explosive diversification of passion-vine butterfly wing patterns. The novel deployment of a gene followed by spatial modulation of its expression in a given cell type could be a common mode of developmental innovation for triggering phenotypic radiations.

Pseudacteon tricuspis: its behavior and development according to the social form of its host and the role of interference competition among females.

We studied how the behavior and performance of Pseudacteon tricuspis Borgmeier varies with the social form of its host Solenopsis invicta Buren, in its native range in Argentina where monogyne colonies are more abundant than polygynes (approximately 75 vs. 25%). Female, P. tricuspis took 44% less time (50 vs. 89 s) to attack monogyne than polygyne ants, but oviposition attempts were similar (23 vs. 18 attacks). The presence of the parasitoid affected the average size of foragers on the trail, with the proportion of minor workers increasing on both social forms. In the laboratory, P. tricuspis selected similar host sizes, although pupal survival was 25% higher on monogynes than on polygynes. Developmental times of both genders were similar (33-35 d), although larger females emerged from bigger hosts. The sex ratio of P. tricuspis was more male biased when exploiting polygyne ants. Intraspecific competition significantly affected parasitoid reproductive success, being significantly higher for a solitary female than when three females were present, although the size of workers selected did not vary. The male:female ratio also changed, being 1:1 without competition but 2:1 with competition. We demonstrated for the first time the consequences of interference competition among P. tricuspis females, a common behavior observed in others parasitoids. We discuss why P. tricuspis sex ratios are always biased toward males in both social forms and suggest that similar studies of interference competition within and between already naturalized Pseudacteon species in the United States could help predict establishment patterns.

Imported fire ants near the edge of their range: disturbance and moisture determine prevalence and impact of an invasive social insect.

1. Habitat disturbance and species invasions interact in natural systems, making it difficult to isolate the primary cause of ecosystem degradation. A general understanding requires case studies of how disturbance and invasion interact across a variety of ecosystem - invasive species combinations. 2. Dramatic losses in ant diversity followed the invasion of central Texas by red imported fire ants (Solenopsis invicta). However, recent manipulative studies in Florida revealed no effect on ant diversity following the removal of S. invicta from a disturbed pasture habitat, but moderate loss of diversity associated with their introduction into undisturbed habitat and no invasion occurred without disturbance. Thus, the importance of S. invicta in driving diversity loss and its ability to invade undisturbed systems is unresolved. 3. We examine the distribution and abundance of a large monogyne S. invicta population and its association with the co-occurring ant assemblage at a site in south Texas close to the aridity tolerance limit of S. invicta. 4. We document that moisture modulates S. invicta densities. Further, soil disturbing habitat manipulations greatly increase S. invicta population densities. However, S. invicta penetrates all habitats regardless of soil disturbance history. In contrast, controlled burns depress S. invicta densities. 5. In habitats where S. invicta is prevalent, it completely replaces native fire ants. However, S. invicta impacts native ants as a whole less strongly. Intriguingly, native ants responded distinctly to S. invicta in different environments. In wet, undisturbed environments, high S. invicta abundance disrupts the spatial structure of the ant assemblage by increasing clumping and is associated with reduced species density, while in dry-disturbed habitats, sites with high S. invicta abundance possess high numbers of native species. Analyses of co-occurrence indicate that reduced species density in wet-undisturbed sites arises from negative species interactions between native ants and S. invicta. However, these same data suggest that the high native species density of abundant S. invicta sites in dry-disturbed environments does not result from facilitation. 6. Monogyne S. invicta populations play different roles in different environments, driving ant diversity loss in some, but being largely symptomatic of habitat disturbance in others.

Sexual selection drives the evolution of antiaphrodisiac pheromones in butterflies.

Competition for mates has resulted in sophisticated mechanisms of male control over female reproduction. Antiaphrodisiacs are pheromones transferred from males to females during mating that reduce attractiveness of females to subsequent courting males. Antiaphrodisiacs generally help unreceptive females reduce male harassment. However, lack of control over pheromone release by females and male control over the amount transferred provides males an opportunity to use antiaphrodisiacs to delay remating by females that have returned to a receptive state. We propose a model for the evolution of antiaphrodisiacs under the influence of intrasexual selection, and determine whether changes in this signal in 11 species of Heliconius butterflies are consistent with two predictions of the model. First, we find that as predicted, male-contributed chemical mixtures are complex and highly variable across species, with limited phylogenetic signal. Second, differences in rates of evolution in pheromone composition between two major clades of Heliconius are as expected: the clade with a greater potential for male-male competition (polyandrous) shows a faster rate of divergence than the one with typically monoandrous mating system. Taken together, our results provide evidence that for females, antiaphrodisiacs can be both honest signals of receptivity (helping reduce harassment) and chastity belts (a male-imposed reduction in remating).

Sex chromosome mosaicism and hybrid speciation among tiger swallowtail butterflies.

Hybrid speciation, or the formation of a daughter species due to interbreeding between two parental species, is a potentially important means of diversification, because it generates new forms from existing variation. However, factors responsible for the origin and maintenance of hybrid species are largely unknown. Here we show that the North American butterfly Papilio appalachiensis is a hybrid species, with genomic admixture from Papilio glaucus and Papilio canadensis. Papilio appalachiensis has a mosaic phenotype, which is hypothesized to be the result of combining sex-linked traits from P. glaucus and P. canadensis. We show that P. appalachiensis' Z-linked genes associated with a cooler thermal habitat were inherited from P. canadensis, whereas its W-linked mimicry and mitochondrial DNA were inherited from P. glaucus. Furthermore, genome-wide AFLP markers showed nearly equal contributions from each parental species in the origin of P. appalachiensis, indicating that it formed from a burst of hybridization between the parental species, with little subsequent backcrossing. However, analyses of genetic differentiation, clustering, and polymorphism based on molecular data also showed that P. appalachiensis is genetically distinct from both parental species. Population genetic simulations revealed P. appalachiensis to be much younger than the parental species, with unidirectional gene flow from P. glaucus and P. canadensis into P. appalachiensis. Finally, phylogenetic analyses, combined with ancestral state reconstruction, showed that the two traits that define P. appalachiensis' mosaic phenotype, obligatory pupal diapause and mimicry, evolved uniquely in P. canadensis and P. glaucus, respectively, and were then recombined through hybridization to form P. appalachiensis. These results suggest that natural selection and sex-linked traits may have played an important role in the origin and maintenance of P. appalachiensis as a hybrid species. In particular, ecological barriers associated with a steep thermal cline appear to maintain the distinct, mosaic genome of P. appalachiensis despite contact and occasional hybridization with both parental species.

Fire ant decapitating fly cooperative release programs (1994-2008): two Pseudacteon species, P. tricuspis and P. curvatus, rapidly expand across imported fire ant populations in the southeastern United States.

Natural enemies of the imported fire ants, Solenopsis invicta Buren S. richteri Forel (Hymenoptera: Formicidae), and their hybrid, include a suite of more than 20 fire ant decapitating phorid flies from South America in the genus Pseudacteon. Over the past 12 years, many researchers and associates have cooperated in introducing several species as classical or self-sustaining biological control agents in the United States. As a result, two species of flies, Pseudacteon tricuspis Borgmeier and P. curvatus Borgmeier (Diptera: Phoridae), are well established across large areas of the southeastern United States. Whereas many researchers have published local and state information about the establishment and spread of these flies, here distribution data from both published and unpublished sources has been compiled for the entire United States with the goal of presenting confirmed and probable distributions as of the fall of 2008. Documented rates of expansion were also used to predict the distribution of these flies three years later in the fall of 2011. In the fall of 2008, eleven years after the first successful release, we estimate that P. tricuspis covered about 50% of the fire ant quarantined area and that it will occur in almost 65% of the quarantine area by 2011. Complete coverage of the fire ant quarantined area will be delayed or limited by this species' slow rate of spread and frequent failure to establish in more northerly portions of the fire ant range and also, perhaps, by its preference for red imported fire ants (S. invicta). Eight years after the first successful release of P. curvatus, two biotypes of this species (one biotype occurring predominantly in the black and hybrid imported fire ants and the other occurring in red imported fire ants) covered almost 60% of the fire ant quarantined area. We estimate these two biotypes will cover almost 90% of the quarantine area by 2011 and 100% by 2012 or 2013. Strategic selection of several distributional gaps for future releases will accelerate complete coverage of quarantine areas. However, some gaps may be best used for the release of additional species of decapitating flies because establishment rates may be higher in areas without competing species.