The Male Hindwing Costal Roll in Cochylina (Lepidoptera: Tortricidae): Morphological Variation, Phylogenetic Distribution, and Relationship to Host Utilization

This is the first morphological study of the male hindwing costal roll (CR), a scent organ of tortricid moths of the subtribe Cochylina (Lepidoptera, Tortricidae). This composite organ varies from a simple membranous roll of the hindwing costa to a complex roll that incorporates a hairpencil and two types of microscales. All the components show taxon-dependent traits. Both optical and electron microscopy are used to elucidate the structure. The costal roll is associated primarily with the Aethes Billberg, Saphenista Walsingham, Phalonidia Le Marchand, and Cochylis Treitschke groups of genera. The general notions that wing male scent organs are phylogenetically labile and that they may have arisen among closely related species as a consequence of habitat sharing are explored using network analysis and phylogenetic signal. Taxa with a costal roll certainly support a more complex area of the food web, however, the character shows a strong phylogenetic signal and is not the result of a sporadic evolution.

Systematic Revision of a New Butterfly Genus, Cisandina Nakahara & Espeland, n. gen., with Descriptions of Three New Taxa (Lepidoptera: Nymphalidae: Satyrinae)

We here establish a new genus in the nymphalid butterfly subtribe Euptychiina, Cisandina Nakahara & Espeland, n. gen. to harbor five species hitherto placed within two polyphyletic genera, namely Magneuptychia Forster, 1964 and Euptychoides Forster, 1964. We compiled data from over 350 specimens in 17 public and private collections, as well as DNA sequence data for all relevant species, to revise the species-level classification of this new genus. According to our multi-locus molecular phylogeny estimated with the maximum likelihood approach, Cisandina lean. comb., Cisandina philippan. comb. & reinst. stat., Cisandina fidan. comb., Cisandina sanmarcosn. comb., and Cisandina trinitensisn. comb. are proposed as new taxonomic combinations, since these species are distantly related to the type species of Magneuptychia and Euptychoides and cannot reasonably be accommodated in any other genus. Lectotypes are designated for Papilio lea Cramer, 1777, Papilio junia Cramer, 1780, Euptychia philippa Butler, 1867, and Eupytchia fida Weymer, 1911. Two new species of Cisandinan. gen. are named and described herein, C. esmeralda Nakahara & Barbosa, n. sp. and C. castanya Lamas & Nakahara, n. sp., increasing the described species diversity of the genus to seven. The immature stages of C. castanyan. sp. and C. philippan. comb. & reinst. stat. are documented along with their natural hostplants, representing the first two species of the genus with known life history information. We describe a new subspecies, Cisandina fida directa Nakahara & Willmott, n. ssp., based on a limited number of specimens from southern Ecuador and central Peru. We were unable to obtain genetic data for the nominate race of C. fidan. comb., and thus, this taxonomic hypothesis is currently based solely on phenotypic characters.

High-Throughput Sequencing for Life-History Sorting and for Bridging Reference Sequences in Marine Gerromorpha (Insecta: Heteroptera)

Accurate identification and association of larval specimens with adults is a major challenge in insect taxonomy. Fortunately, it is now possible for nonexperts to sort collections of bulk samples with DNA barcodes rapidly and cost-effectively. We demonstrate this process using nanopore barcoding of 757 marine insects (Insecta: Gerromorpha), of which 81% were nymphs and many samples did not have co-occurring adult males for specific identification. We successfully associated 738 specimens (97%) to nine gerromorphan species, which would have been impossible to identify using morphological characters alone. This improved ability to incorporate information from all life-history stages has led to greater precision of species distributional ranges—knowledge that will be crucial for a more complete understanding of marine insects. We also highlighted two distinct, nonoverlapping Gerromorpha COI sequence databases on GenBank—a consequence of using two different primer sets to amplify different regions of COI. This issue inevitably hinders species identification with DNA-based methods, particularly for poorly represented groups such as marine insects. We bridged these databases by analyzing full-length COI sequences. We believe this will inspire future studies to incorporate DNA-based methods for more adult–larval association studies and for enhancing existing genetic resources, especially in understudied groups.

The Genealogical Divergence Index Across a Speciation Continuum in Hercules Beetles

The genealogical divergence index (gdi) was developed to aid in molecular species delimitation under the multispecies coalescent model, which has been shown to delimit genetic structures but not necessarily species. Although previous studies have used meta-analyses to show that gdi could be informative for distinguishing taxonomically good species, the biological and evolutionary implications of divergences showing different gdi values have yet to be studied. I showed that an increase in gdi value was correlated with later stages of divergence further along a speciation continuum in an Amazonian Hercules beetle system. Specifically, a gdi value of 0.7 or higher was associated with diverge between biological species that can coexist in geographic proximity while maintaining their evolutionary independence. Divergences between allopatric species that were conventionally given subspecific status, such as geographic taxa that may or may not be morphologically divergent, had gdi values that fell within the species delimitation ambiguous zone (0.2 < gdi < 0.7). However, the results could be drastically affected by the sampling design, i.e., the choice of different geographic populations and the lumping of distinct genetic groups when running the analyses. Different gdi values may prove to be biologically and evolutionarily informative should additional speciation continua from different empirical systems be investigated, and the results obtained may help with objectively delimiting species in the era of integrative taxonomy.

Divergence in Body Mass, Wing Loading, and Population Structure Reveals Species-Specific and Potentially Adaptive Trait Variation Across Elevations in Montane Bumble Bees

Biogeographic clines in morphology along environmental gradients can illuminate forces influencing trait evolution within and between species. Latitude has long been studied as a driver of morphological clines, with a focus on body size and temperature. However, counteracting environmental pressures may impose constraints on body size. In montane landscapes, declines in air density with elevation can negatively impact flight performance in volant species, which may contribute to selection for reduced body mass despite declining temperatures. We examine morphology in two bumble bee (Hymenoptera: Apidae: Bombus Latreille) species, Bombus vancouverensis Cresson and Bombus vosnesenskii Radoszkowski, across mountainous regions of California, Oregon, and Washington, United States. We incorporate population genomic data to investigate the relationship between genomic ancestry and morphological divergence. We find that B. vancouverensis, which tends to be more specialized for high elevations, exhibits stronger spatial-environmental variation, being smaller in the southern and higher elevation parts of its range and having reduced wing loading (mass relative to wing area) at high elevations. Bombus vosnesenskii, which is more of an elevational generalist, has substantial trait variation, but spatial-environmental correlations are weak. Population structure is stronger in the smaller B. vancouverensis, and we find a significant association between elevation and wing loading after accounting for genetic structure, suggesting the possibility of local adaptation for this flight performance trait. Our findings suggest that some conflicting results for body size trends may stem from distinct environmental pressures that impact different aspects of bumble bee ecology, and that different species show different morphological clines in the same region.

Phylogenomic Variation at the Population-Species Interface and Assessment of Gigantism in a Model Wolf Spider Genus (Lycosidae, Schizocosa)

Animal body size has important evolutionary implications. The wolf spider genus Schizocosa Chamberlin, 1904 has developed as a model for studies on courtship, with visual and vibratory signals receiving attention; however, body size has never been carefully evaluated. Although species of Schizocosa can be distinguished from their close relatives by differences in genitalic structures, male ornamentation, and behavior, some species are morphologically similar, making diagnosis, and identification difficult. Evaluation of species boundaries using genetic data across Schizocosa is limited. The similar species S. maxima Dondale & Redner, 1978 and S. mccooki (Montgomery, 1904) are separated predominantly on the basis of size differences, with S. maxima being larger. We evaluate the evolution of size in these two Schizocosa species distributed in western North America, where gigantism of S. maxima is hypothesized to occur, particularly in California. We sampled subgenomic data (RADseq) and inferred the phylogeny of S. mccooki, S. maxima, and relatives. We apply a variational autoencoder machine learning approach to visualize population structuring within widespread S. mccooki and evaluate size within the context of a comparative phylogenetic framework to test the hypotheses related to genetic clustering of populations and gigantism. Our data show S. mccooki populations are not genealogically exclusive with respect to S. maxima. Likewise, S. maxima individuals are not recovered as a lineage and do not form an isolated genetic cluster, suggesting that the observed differences in size cannot be used to accurately delimit species. The cause of gigantism in S. maxima remains unexplained, but provides a framework for future studies of size variation and speciation.

ddRAD Sequencing Sheds Light on Low Interspecific and High Intraspecific mtDNA Divergences in Two Groups of Caddisflies

Large-scale global efforts on DNA barcoding have repeatedly revealed unexpected patterns of variability in mtDNA, including deep intraspecific divergences and haplotype sharing between species. Understanding the evolutionary causes behind these patterns calls for insights from the nuclear genome. While building a near-complete DNA barcode library of Finnish caddisflies, a case of barcode-sharing and some cases of deep intraspecific divergences were observed. In this study, the Apatania zonella (Zetterstedt, 1840) group and three Limnephilus Leach, 1815 species were studied using double digest RAD sequencing (ddRAD-seq), morphology, and DNA barcoding. The results support the present species boundaries in the A. zonella group species. A morphologically distinct but mitogenetically nondistinct taxon related to parthenogenetic Apatania hispida (Forsslund, 1930) got only weak support for its validity as a distinct species. The morphology and genomic-scale data do not indicate cryptic diversity in any of the three Limnephilus species despite the observed deep intraspecific divergences in DNA barcodes. This demonstrates that polymorphism in mtDNA may not reflect cryptic diversity, but mitonuclear discordance due to other evolutionary causes.

Extremely Endangered Butterflies of Scattered Central European Dry Grasslands Under Current Habitat Alteration

Central European dry grasslands represent extrazonal patches of the Eurasian steppe biome. They suffer from severe habitat alterations due to land-use changes, abandonment, or inappropriate management. The butterflies Chazara briseis (Linnaeus, 1764) (Lepidoptera: Nymphalidae), Polyommatus damon (Denis & Schiffermüller, 1775) (Lepidoptera: Lycaenidae), and Polyommatus dorylas (Denis & Schiffermüller, 1775) (Lepidoptera: Lycaenidae), specialized inhabitants of these steppe patches, are all swiftly disappearing from Central Europe. We reviewed data on the recent history of their population retractions in the region, including conservation efforts. Using samples from their whole distribution ranges, we sequenced and analyzed COI and wingless genes and together with Species Distribution Modelling reconstructed their biogeographic histories. Populations of C. briseis expanded over the Eurasian steppe biome, where large ungulates maintained extensive grasslands with short open sward. Polyommatus damon became widespread in the steppes during glacial times, and retracted during interglacials, resembling cold-adapted species. It is limited by too dry weather, and it requires disturbed grassland followed by temporal abandonment. Its present genetic structure was induced by the major Pleistocene Mountain glaciations. Polyommatus dorylas prefers an oceanic climate and populated Central Europe from the Balkans during the Holocene. The species depends on disturbed ground. Currently, all three species inhabit only a few remnant sites in Central Europe, and their populations have been further declining in recent years. Targeted conservation actions, including habitat management at remaining sites, ex situ breeding, and (re)introductions, are being taken in Austria, the Czech Republic, and Germany.

Is Phylogeographic Congruence Predicted by Historical Habitat Stability, or Ecological Co-associations?

Comparative phylogeographic studies can distinguish between idiosyncratic and community-wide responses to past environmental change. However, to date, the impacts of species interactions have been largely overlooked. Here we used non-genetic data to characterize two competing scenarios about expected levels of congruence among five deadwood-associated (saproxylic) invertebrate species (i.e., a wood-feeding cockroach, termite, and beetle; a predatory centipede, and a detritivorous millipede) from the southern Appalachian Mountains—a globally recognized center of endemism. Under one scenario, abiotic factors primarily drove species’ responses, with predicted congruence based on the spatial overlap of climatically stable habitat areas estimated for each species via ecological niche modeling. The second scenario considered biotic factors to be most influential, with proxies for species interactions used to predict congruence. Analyses of mitochondrial and nuclear DNA sequences focused on four axes of comparison: the number and geographic distribution of distinct spatial-genetic clusters, phylogeographic structure, changes in effective population size, and historical gene flow dynamics. Overall, we found stronger support for the ecological co-associations scenario, suggesting an important influence of biotic factors in constraining or facilitating species’ responses to Pleistocene climatic cycles. However, there was an imperfect fit between predictions and outcomes of genetic data analyses. Thus, while thought-provoking, conclusions remain tentative until additional data on species interactions becomes available. Ultimately, the approaches presented here advance comparative phylogeography by expanding the scope of inferences beyond solely considering abiotic drivers, which we believe is too simplistic. This work also provides conservation-relevant insights into the evolutionary history of a functionally important ecological community.

Estimating Migration of Gonioctena quinquepunctata (Coleoptera: Chrysomelidae) Inside a Mountain Range in a Spatially Explicit Context

The cold-tolerant leaf beetle Gonioctena quinquepunctata displays a large but fragmented European distribution and is restricted to mountain regions in the southern part of its range. Using a RAD-seq-generated large single nucleotide polymorphism (SNP) data set (> 10,000 loci), we investigated the geographic distribution of genetic variation within the Vosges mountains (eastern France), where the species is common. To translate this pattern of variation into an estimate of its capacity to disperse, we simulated SNP data under a spatially explicit model of population evolution (essentially a grid overlapping a map, in which each cell is considered a different population) and compared the simulated and real data with an approximate Bayesian computation (ABC) approach. For this purpose, we assessed a new SNP statistic, the DSVSF (distribution of spatial variation in SNP frequencies) that summarizes genetic variation in a spatially explicit context, and compared its usefulness to standard statistics often used in population genetic analyses. A test of our overall strategy was conducted with simulated data and showed that it can provide a good estimate of the level of dispersal of an organism over its geographic range. The results of our analyses suggested that this insect disperses well within the Vosges mountains, much more than was initially expected given the current and probably past fragmentation of its habitat and given the results of previous studies on genetic variation in other mountain leaf beetles.