Supergene evolution via gain of auto-regulation.

The development of complex phenotypes requires the coordinated action of many genes across space and time, yet many species have evolved the ability to develop multiple discrete, alternate phenotypes1-3. Such polymorphisms are often controlled by supergenes, sets of tightly-linked mutations in one or more loci that function together to produce a complex phenotype4. Although theories of supergene evolution are well-established, the mutations that cause functional differences between supergene alleles remain essentially unknown. doublesex is the master regulator of insect sexual differentiation but functions as a supergene in multiple Papilio swallowtail butterflies, where divergent dsx alleles control development of discrete non-mimetic or mimetic female wing color patterns5-7. Here we demonstrate that the functional elements of the mimetic allele in Papilio alphenor are six new cis-regulatory elements (CREs) spread across 150 kb that are bound by DSX itself. Our findings provide experimental support to classic supergene theory and suggest that the evolution of auto-regulation may provide a simple route to supergene origination and to the co-option of pleiotropic genes into new developmental roles.

Speciation in kleptoparasites of oak gall wasps often correlates with shifts into new tree habitats, tree organs, or gall morphospace.

Host shifts to new plant species can drive speciation for plant-feeding insects, but how commonly do host shifts also drive diversification for the parasites of those same insects? Oak gall wasps induce galls on oak trees and shifts to novel tree hosts and new tree organs have been implicated as drivers of oak gall wasp speciation. Gall wasps are themselves attacked by many insect parasites, which must find their hosts on the correct tree species and organ, but also must navigate the morphologically variable galls with which they interact. Thus, we ask whether host shifts to new trees, organs, or gall morphologies correlate with gall parasite diversification. We delimit species and infer phylogenies for two genera of gall kleptoparasites, Synergus and Ceroptres, reared from a variety of North American oak galls. We find that most species were reared from galls induced by just one gall wasp species, and no parasite species was reared from galls of more than four species. Most kleptoparasite divergence events correlate with shifts to non-ancestral galls. These shifts often involved changes in tree habitat, gall location, and gall morphology. Host shifts are thus implicated in driving diversification for both oak gall wasps and their kleptoparasitic associates.

Acute and Long-Term Consequences of Co-opted doublesex on the Development of Mimetic Butterfly Color Patterns.

Novel phenotypes are increasingly recognized to have evolved by co-option of conserved genes into new developmental contexts, yet the process by which co-opted genes modify existing developmental programs remains obscure. Here, we provide insight into this process by characterizing the role of co-opted doublesex in butterfly wing color pattern development. dsx is the master regulator of insect sex differentiation but has been co-opted to control the switch between discrete nonmimetic and mimetic patterns in Papilio alphenor and its relatives through the evolution of novel mimetic alleles. We found dynamic spatial and temporal expression pattern differences between mimetic and nonmimetic butterflies throughout wing development. A mimetic color pattern program is switched on by a pulse of dsx expression in early pupal development that causes acute and long-term differential gene expression, particularly in Wnt and Hedgehog signaling pathways. RNAi suggested opposing, novel roles for these pathways in mimetic pattern development. Importantly, Dsx co-option caused Engrailed, a primary target of Hedgehog signaling, to gain a novel expression domain early in pupal wing development that is propagated through mid-pupal development to specify novel mimetic patterns despite becoming decoupled from Dsx expression itself. Altogether, our findings provide multiple views into how co-opted genes can both cause and elicit changes to conserved networks and pathways to result in development of novel, adaptive phenotypes.

New molecular insights into butterfly pigmentation.

Hanly et al.1 and Nishida et al.2 use distinct approaches to provide exceptional lessons regarding the genetic, molecular, morphological, and biochemical bases of butterfly wing pigmentation. These mechanistic insights collectively have important implications for our understanding of phenotype evolution.

Speciation in Nearctic oak gall wasps is frequently correlated with changes in host plant, host organ, or both.

Quantifying the frequency of shifts to new host plants within diverse clades of specialist herbivorous insects is critically important to understand whether and how host shifts contribute to the origin of species. Oak gall wasps (Hymenoptera: Cynipidae: Cynipini) comprise a tribe of ∼1000 species of phytophagous insects that induce gall formation on various organs of trees in the family Fagacae-primarily the oaks (genus Quercus; ∼435 sp.). The association of oak gall wasps with oaks is ancient (∼50 my), and most oak species are galled by one or more gall wasp species. Despite the diversity of both gall wasp species and their plant associations, previous phylogenetic work has not identified the strong signal of host plant shifting among oak gall wasps that has been found in other phytophagous insect systems. However, most emphasis has been on the Western Palearctic and not the Nearctic where both oaks and oak gall wasps are considerably more species rich. We collected 86 species of Nearctic oak gall wasps from most of the major clades of Nearctic oaks and sequenced >1000 Ultraconserved Elements (UCEs) and flanking sequences to infer wasp phylogenies. We assessed the relationships of Nearctic gall wasps to one another and, by leveraging previously published UCE data, to the Palearctic fauna. We then used phylogenies to infer historical patterns of shifts among host tree species and tree organs. Our results indicate that oak gall wasps have moved between the Palearctic and Nearctic at least four times, that some Palearctic wasp clades have their proximate origin in the Nearctic, and that gall wasps have shifted within and between oak tree sections, subsections, and organs considerably more often than previous data have suggested. Given that host shifts have been demonstrated to drive reproductive isolation between host-associated populations in other phytophagous insects, our analyses of Nearctic gall wasps suggest that host shifts are key drivers of speciation in this clade, especially in hotspots of oak diversity. Although formal assessment of this hypothesis requires further study, two putatively oligophagous gall wasp species in our dataset show signals of host-associated genetic differentiation unconfounded by geographic distance, suggestive of barriers to gene flow associated with the use of alternative host plants.

Delimiting the cryptic diversity and host preferences of Sycophila parasitoid wasps associated with oak galls using phylogenomic data.

Cryptic species diversity is a major challenge regarding the species-rich community of parasitoids attacking oak gall wasps due to a high degree of sexual dimorphism, morphological plasticity, small size and poorly known biology. As such, we know very little about the number of species present, nor the evolutionary forces responsible for generating this diversity. One hypothesis is that trait diversity in the gall wasps, including the morphology of the galls they induce, has evolved in response to selection imposed by the parasitoid community, with reciprocal selection driving diversification of the parasitoids. Using a rare, continental-scale data set of Sycophila parasitoid wasps reared from 44 species of cynipid galls from 18 species of oak across the USA, we combined mitochondrial DNA barcodes, ultraconserved elements (UCEs), morphological and natural history data to delimit putative species. Using these results, we generate the first large-scale assessment of ecological specialization and host association in this species-rich group, with implications for evolutionary ecology and biocontrol. We find most Sycophila target specific subsets of available cynipid host galls with similar morphologies, and generally attack larger galls. Our results suggest that parasitoid wasps such as Sycophila have adaptations allowing them to exploit particular host trait combinations, while hosts with contrasting traits are resistant to attack. These findings support the tritrophic niche concept for the structuring of plant-herbivore-parasitoid communities.

The Arthropod Associates of 155 North American Cynipid Oak Galls.

The identities of most arthropod associates of cynipid-induced oak galls in the western Palearctic are generally known. However, a comprehensive accounting of associates has been performed for only a small number of the galls induced by the estimated 700 species of cynipid gall wasps in the Nearctic. This gap in knowledge stymies many potential studies of diversity, coevolution, and community ecology, for which oak gall systems are otherwise ideal models. We report rearing records of insects and other arthropods from more than 527,306 individual galls representing 201 different oak gall types collected from 32 oak tree species in North America. Of the 201 gall types collected, 155 produced one or more arthropods. A total of 151,075 arthropods were found in association with these 155 gall types, and of these 61,044 (40.4%) were gall wasps while 90,031 (59.6%) were other arthropods. We identified all arthropods to superfamily, family, or, where possible, to genus. We provide raw numbers and summaries of collections, alongside notes on natural history, ecology, and previously published associations for each taxon. For eight common gall-associated genera (Synergus, Ceroptres, Euceroptres, Ormyrus, Torymus, Eurytoma, Sycophila, and Euderus), we also connect rearing records to gall wasp phylogeny, geography, and ecology -including host tree and gall location (host organ), and their co-occurrence with other insect genera. Though the diversity of gall wasps and the large size of these communities is such that many Nearctic oak gall-associated insects still remain undescribed, this large collection and identification effort should facilitate the testing of new and varied ecological and evolutionary hypotheses in Nearctic oak galls.