Immature Stages, Natural History, Systematics and Conservation of an Endangered Neotropical Butterfly: the Case of Scada karschina delicata (Nymphalidae: Ithomiini).

The endangered butterfly Scada karschina delicata Talbot, 1932 (Nymphalidae: Danainae: Ithomiini) is endemic to northeastern Brazil, occurring in very few forest remnants of the 'Pernambuco Center of Endemism'. Larvae feed on Solanaceae and are very similar to those of other species in the subtribe Mechanitina, with lateral projections on body, one of the main synapomorphies of this subtribe. Based on molecular data, S. k. delicata clustered together with S. karschina karschina, as a monophyletic group sister to the amazon clade of S. reckia. Based on all available data, S. k. delicata is known from only five localities of mid- to high-altitude forests (from 500 to 1000 m of altitude) in northeastern Brazil. Grounded on available data, a new assessment of extinction risk is proposed, and S. k. delicata is now considered Vulnerable (VU) taxon.

A New and Rare Actinote Hübner (Lepidoptera: Nymphalidae: Heliconiinae: Acraeini) from Southeastern Brazil.

The present paper describes a new species of Actinote (Nymphalidae, Heliconiinae, Acraeini), Actinote keithbrowni Freitas, Francini & Mielke sp. nov., from southeastern Brazil, based on morphological and molecular data. The new species is very similar in wing pattern to Actinote eberti, within what we term the "light-gray mimicry complex." The host plants and immature stages of the new species are unknown. Actinote keithbrowni sp. nov. is one of the rarest species of Actinote from Brazil: in total, only eight individuals of this species are known from a narrow region of the Itatiaia massif despite years of collecting effort in that region since the 1940s.

A New Subspecies of Heliconius hermathena (Nymphalidae: Heliconiinae) from Southern Amazonia.

The present paper describes Heliconius hermathena curua Freitas & Ramos ssp. nov. This subspecies exhibits a non-mimetic phenotype typical of H. hermathena, but is characterized by the merging of the yellow streak over the forewing cubitus with the red postmedian band in the dorsal forewing. The subspecies is known from two localities in the south of Altamira, Pará State, Brazil, where it inhabits an isolated patch of "campina" vegetation more than 600 km from the nearest known H. hermathena populations. Geographic isolation of the population is supported by molecular data; based on the mitochondrial gene COI, all individuals of H. hermathena curuassp. nov. form a monophyletic group and all haplotypes found in it are unique, suggesting that gene flow is not currently on-going. Given the fragile situation of Amazonian white sand forests and the proximity of the population to areas of intensive agriculture, this new subspecies and its habitat deserve attention.

Comparison of bacterial 16S rRNA variable regions for microbiome surveys of ticks.

Ticks vector diverse pathogenic bacteria that are important to identify in public health and veterinary contexts. Technological advances in high throughput sequencing have given an unprecedented opportunity to comprehensively characterize bacterial associates of ticks, but recent studies have used different 16S rRNA variable regions and sequence read lengths with little consideration of whether they reveal the same bacterial diversity. We compare the effectiveness of bacterial surveys using three library preparations across nine 16S variable regions and a set of 12 tick specimens (Acari: Ixodidae). We identify the bacterial assemblages present in extractions from wild-collected Ixodes scapularis from two regions of Canada, and provide the first microbiome survey for Ixodes angustus. Four bacterial families accounted for most diversity, with Rickettsiaceae being replaced as most common by Enterobacteriaceae or Pseudomonadaceae in some I. scapularis, and Francisellaceae being most abundant in I. angustus. The commercially available Ion 16S kit, based on 6 amplicons representing 16S regions V2, V3, V4, V67, V8 and V9, gave the most comprehensive estimates of bacterial families, with the Ion V4 amplicon generally giving the highest estimated diversity. Sequencing of the V4 amplicon by the MR DNA commercial service also provided cost effective assays of tick microbiomes that were within the range of results from the Ion 16S kit. Subtraction of the number of reads found in an extraction control sample lowered estimates of the number of bacterial families by approximately half. Our study shows that diversity patterns obtained from 16S microbiome surveys depend on the amplicon and protocol used, demonstrating that more than one marker region is needed to provide reliable inferences.

Paulogramma hydarnis (n. comb.) (Nymphalidae: Biblidinae): Distribution, Systematic Position, and Conservation Status of a Rare and Endangered Butterfly.

The nymphalid Paulogramma hydarnis (Godart) (n. comb., previously in the genus Callicore) is an endangered butterfly present in a few montane sites in the Atlantic Forest in the Southeastern Brazil. The precise systematic position of P. hydarnis was previously unknown. Based on molecular data, we find that it is sister to Paulogramma pygas (Godart) (n. comb., also previously in Callicore), a common and widespread species in the Neotropics. In addition, we find that Callicore is not monophyletic and that "Callicore" hydarnis (along with other species) is more related to the genus Paulogramma, and should thus be placed in that genus. The genus Paulogramma is now composed by the following species: Paulogramma pyracmon (Godart), Paulogramma eunomia (Hewitson) n. comb., Paulogramma hydarnis (Godart) n. comb., Paulogramma hystaspes (Fabricius) n. comb., Paulogramma pygas (Godart) n. comb., and Paulogramma tolima (Hewitson, 1852) n. comb. Museum specimens and field data report P. hydarnis in four sites in Southeastern Brazil. Recently, P. hydarnis was recorded for the first time at Parque Nacional do Caparaó, states of Espírito Santo and Minas Gerais, expanding its distribution about 200 km northward of the previously known limit. Although regularly recorded in some sites, most records are historic, before the 1960s, and the current conservation situation of this species is delicate, deserving attention.

Morphological and molecular marker contributions to disentangling the cryptic Hermeuptychia hermes species complex (Nymphalidae: Satyrinae: Euptychiina).

The genus Hermeuptychia is common and widespread through the Americas, from Argentina to the southern United States of America. All eight recognized species within Hermeuptychia are small and brown, with very similar interspecific external morphologies and intraspecifically variable ocelli patterns that render taxonomic identification based on morphology difficult. In our study, we surveyed variability within Hermeuptychia, and evaluated species boundaries based on molecular data (sequences of the 'barcode' mitochondrial DNA COI gene) and morphology (mainly male genitalia), using a phylogenetic approach. We found eight DNA-based and 12 morphological groups in our sampling. Species names were assigned based mainly on comparisons with male genitalia morphology descriptions corresponding to name-bearing type specimens. Morphological and DNA variability were highly congruent, with the exception of group H, the Hermeuptychia cucullina complex. Also, the barcode region showed a clear threshold for intra- and interspecific mean distances around 2%. Based on these results, we circumscribe the species boundaries in the genus Hermeuptychia and discuss conflicts between mitochondrial genes and classic morphological approaches for identifying and delimiting species. Our study revealed cryptic diversity within an ubiquitous genus of Neotropical butterflies.

Development and characterization of microsatellite loci for genetic studies of the sugarcane borer, Diatraea saccharalis (Lepidoptera: Crambidae).

We present polymorphic microsatellite markers isolated for genetic studies of the sugarcane borer, Diatraea saccharalis (Fabricius). We isolated 16 microsatellite loci through an enriched genomic library protocol. After characterization, 12 markers showed polymorphic information expressed in the observed number of alleles (ranging from 2 to 7; 5 on average) and in the polymorphism information content (ranging from 0.292 to 0.771; 0.535 on average). These markers can be used in further studies to understand the basic ecological characteristics of the sugarcane borer, e.g., dispersion patterns and population genetic differentiation, associated with distinct geographic scales and host plants.

Using population genetic methods to identify the origin of an invasive population and to diagnose cryptic subspecies of Telchin licus (Lepidoptera: Castniidae).

Telchin licus, the giant sugarcane borer, is an important pest species of sugarcane in northeast Brazil. Four subspecies of Telchin licus are recognized in Brazil based on their geographic distribution and subtle differences in wing colour pattern. Some taxa are morphologically indistinguishable, and their accurate identification is key to their efficient control. Mitochondrial genes sequences (cytochrome oxidase I and subunit 6 of the nicotinamide adenine dinucleotide dehydrogenase) were applied to delimit taxonomic entities of T. licus, and to infer the origin of a newly established population in the state of São Paulo. The molecular data indicated that specimens sampled at different regions in Brazil are morphologically cryptic but genetically isolated entities, and at least three subspecies were assigned to the sampled localities. These data also suggested that the population collected from the state of São Paulo must have a common origin with populations from northeast Brazil, which corroborate the hypothesis that ornamental plants infested with larvae of T. licus might have been transported from the northeast to the southeast regions.

Genetic variability and demographic history of Heliothis virescens (Lepidoptera: Noctuidae) populations from Brazil inferred by mtDNA sequences.

Intra- and inter-population genetic variability and the demographic history of Heliothis virescens (F.) populations were evaluated by using mtDNA markers (coxI, coxII and nad6) with samples from the major cotton- and soybean-producing regions in Brazil in the growing seasons 2007/08, 2008/09 and 2009/10. AMOVA indicated low and non-significant genetic structure, regardless of geographical scale, growing season or crop, with most of genetic variation occurring within populations. Clustering analyzes also indicated low genetic differentiation. The haplotype network obtained with combined datasets resulted in 35 haplotypes, with 28 exclusive occurrences, four of them sampled only from soybean fields. The minimum spanning network showed star-shaped structures typical of populations that underwent a recent demographic expansion. The recent expansion was supported by other demographic analyzes, such as the Bayesian skyline plot, the unimodal distribution of paired differences among mitochondrial sequences, and negative and significant values of neutrality tests for the Tajima's D and Fu's F(S) parameters. In addition, high values of haplotype diversity (H) and low values of nucleotide diversity (π), combined with a high number of low frequency haplotypes and values of θ(π)<θ(W), suggested a recent demographic expansion of H. virescens populations in Brazil. This demographic event could be responsible for the low genetic structure currently found; however, haplotypes present uniquely at the same geographic regions and from one specific host plant suggest an initial differentiation among H. virescens populations within Brazil.

A new genus and species of Euptychiina (Lepidoptera: Nymphalidae: Satyrinae) from southern Brazil.

This paper describes a new genus and a new species of Euptychiina from open grassland habitats (campos de cima da serra) in southern Brazil. The systematic position of this new taxon is discussed based on morphological and molecular data, and it is considered sister to Taydebis Freitas. Since the campos vegetation is considered endangered due to anthropogenic activities, this butterfly species deserves attention and should be included in future conservation plans for this biome.

Out of the Andes: patterns of diversification in clearwing butterflies.

Global biodiversity peaks in the tropical forests of the Andes, a striking geological feature that has likely been instrumental in generating biodiversity by providing opportunities for both vicariant and ecological speciation. However, the role of these mountains in the diversification of insects, which dominate biodiversity, has been poorly explored using phylogenetic methods. Here we study the role of the Andes in the evolution of a diverse Neotropical insect group, the clearwing butterflies. We used dated species-level phylogenies to investigate the time course of speciation and to infer ancestral elevation ranges for two diverse genera. We show that both genera likely originated at middle elevations in the Andes in the Middle Miocene, contrasting with most published results in vertebrates that point to a lowland origin. Although we detected a signature of vicariance caused by the uplift of the Andes at the Miocene-Pliocene boundary, most sister species were parapatric without any obvious vicariant barrier. Combined with an overall decelerating speciation rate, these results suggest an important role for ecological speciation and adaptive radiation, rather than simple vicariance.

New evidence on the systematic and phylogenetic position of Parides burchellanus (Lepidoptera: Papilionidae).

Parides burchellanus is considered a rare and threatened swallowtail species restricted to central Brazil. It shows considerable morphological similarity to Parides panthonus jaguarae, with which it shares both geographical range and larval host plant. At present, P. burchellanus and P. panthonus are believed to be distinct species, based on minor differences in male genitalia. In this study, the phylogenetic and systematic position of P. burchellanus in relation to three subspecies of P. panthonus (P. p. jaguarae, P. p. lysimachus and P. p. aglaope) was evaluated using molecular evidence: the complete sequence of the mtDNA genes COI and COII and of the nuclear gene EF-1α (c. 3300 bp). In addition, the informativeness of the 'barcode' region next to the 5' end of COI (c. 650 bp) was evaluated for delimiting these taxa. Individual analysis by neighbour-joining, using Kimura 2-parameter distance model, and by maximum parsimony showed that P. p. jaguarae + P. p. lysimachus + P. p. aglaope + P. burchellanus form a strongly supported monophyletic clade, and all molecular regions consistently recovered P. p. jaguarae and P. burchellanus as sister species. The genetic divergence among the subspecies of P. panthonus and P. burchellanus is equivalent to the divergence among conspecifics of other species of Parides, and smaller than the interspecific divergence among different sister species of this genus. The results support the proposal that P. p. jaguarae and P. burchellanus are likely to be synonymous, and suggest that P. burchellanus can be considered conspecific with P. panthonus. The taxonomic classification of P. burchellanus should be revised on the basis of the molecular data.