Phylogenetic systematics of Yphthimoides Forster, 1964 and related taxa, with notes on the biogeographical history of Yphthimoides species.

Species losses are increasing and may have an impact on our understanding of patterns of evolutionary pathways and phylogenetic relationships among the groups being lost. The knowledge of such patterns can contribute to preventing future losses by identifying which lineages have higher or lower diversification rates, thus informing conservation strategies. Recent years have seen a significant growth in studies of butterfly systematics, allowing a better understanding of evolutionary relationships among most groups and revealing significant taxonomic chaos in several groups. One of the latter groups is the nymphalid subtribe Euptychiina (Satyrinae), which has been shown to include a number of non-monophyletic genera based on recent molecular phylogenetic analyses. Among others, these genera include Yphthimoides, which is widespread throughout the Neotropical region but particularly diverse in the southeastern Neotropics, and a pair of related genera, Pharneuptychia Forster, 1964 and Moneuptychia Forster, 1964. Using molecular data, this study scope and aims were to provide a phylogenetic hypothesis that corroborates Yphthimoides as presently conceived being non-monophyletic, a result reinforced by a comparative study of the male genitalic morphology. Our results also show that Pharneuptychia and Moneuptychia, plus a species misplaced elsewhere in the Euptychiina, Euptychoides castrensis (Schaus, 1902), form a well supported clade, and that the latter 'species' is a complex of cryptic species. We therefore propose a number of taxonomic rearrangements in the present work to resolve these issues: Yphthimoides eriphule (A. Butler, 1867) will be moved to a new genus; Y. affinis (A. Butler, 1867), Y. maepius (Godart, [1824]), Y. mimula (Hayward, 1954), Y. neomaenas (Hayward, 1967) and Y. mythra (Weymer, 1911) are being transferred to Malaveria Viloria & Benmesbah, 2021; Pharneuptychia innocentia (Godart, [1824]) will be moved to another genus to be described; and Euptychoides castrensis, Pharneuptychia romanina (Bryk, 1953) and Yphthimoides viviana (Romieux, 1927) are being moved to Moneuptychia. The dating of divergences points to a split between the ancestral lineage of Yphthimoides and its sister group, Carminda Ebert and Dias, inDias 1998, during the last half of the Miocene, around 11.86 Mya, and to the diversification of the Pharneuptychia during the same time 11.35 (±3.52) Mya. Biogeographic analysis showed that the most recent common ancestor of Yphthimoides started to diversify either in the the Brazilian Cerrado savannas or in a combined area of Cerrado and South Atlantic Forest, with a possible change in the ancestral habitat of Carminda. Furthermore, ancestral character mapping favors a savanna origin hypothesis over a forest origin hypothesis.

High Genetic Diversity and No Population Structure of the New World Screwworm Fly Cochliomyia hominivorax (Diptera: Calliphoridae) on a Microgeographic Scale: Implications for Management Units.

The New World screwworm fly Cochliomyia hominivorax (Coquerel, 1858) (Diptera: Calliphoridae) is an important livestock pest endemic to the Americas that has been eradicated from North and continental Central America with a control program based on the Sterile Insect Technique (SIT). The establishment of target management units is a strategic step in the implementation of new control programs, which can be achieved using genetic studies of natural populations. Previous studies of New World screwworm fly populations were conducted on the continental scale and identified four main groups: two in South America and two in the Caribbean. However, studies within these groups are needed to determine which smaller geographic areas can be treated as management units. Here, we analyze the genetic variability distribution and the population demographic signals of the New World screwworm fly in a 6,000 km2 area located along the border of Brazil and Uruguay. This area was the subject of the first control pilot program conducted in South America. We studied eight microsatellite loci and sequences from two mitochondrial DNA regions in individuals sampled at 20-25 livestock breeding farms. We observed no population structure and found high genetic variability on the geographical scale sampled for both molecular markers. Our microsatellite data suggest that these populations are not in equilibrium, and demographic analyses based on mitochondrial data indicate population expansion. These results suggest that this geographic scale is not adequate for future New World screwworm fly management in South America.

Molecular Characterization of the 2016 New World Screwworm (Diptera: Calliphoridae) Outbreak in the Florida Keys.

New World screwworm (NWS), Cochliomyia hominivorax (Coquerel 1858) (Diptera: Calliphoridae), is a myiasis-causing fly that can be a serious threat to the health of livestock, wildlife, and humans. Its progressive eradication from the southern United States, Mexico, and Central America from the 1950s to 2000s is an excellent example of successful pest management using sterile insect technique (SIT). In late 2016, autochthonous NWS were detected in the Florida Keys, representing this species' first invasion in the United States in >30 yr. Rapid use of quarantine and SIT was successful in eliminating the infestation by early 2017; however, the geographic source of this infestation remains unknown. Here, we use amplicon sequencing to generate mitochondrial and nuclear sequence data representing all confirmed cases of NWS from this infestation, and compare these sequences to preexisting data sets sampling the native distribution of NWS. We ask two questions regarding the FL Keys outbreak. First, is this infestation the result of a single invasion from one source, or multiple invasions from different sources? And second, what is the geographic origin of this invasion? We found virtually no sequence variation between specimens collected from the FL Keys outbreak, which is consistent with a single source of introduction. However, we also found very little geographic resolution in any of the data sets, which precludes identification of the source of this outbreak. Our lack of success in answering our second question speaks to the need for finer-scale genetic or genomic assessments of NWS population structure, which would facilitate source determination of potential future outbreaks.

The microbiomes of blowflies and houseflies as bacterial transmission reservoirs.

Blowflies and houseflies are mechanical vectors inhabiting synanthropic environments around the world. They feed and breed in fecal and decaying organic matter, but the microbiome they harbour and transport is largely uncharacterized. We sampled 116 individual houseflies and blowflies from varying habitats on three continents and subjected them to high-coverage, whole-genome shotgun sequencing. This allowed for genomic and metagenomic analyses of the host-associated microbiome at the species level. Both fly host species segregate based on principal coordinate analysis of their microbial communities, but they also show an overlapping core microbiome. Legs and wings displayed the largest microbial diversity and were shown to be an important route for microbial dispersion. The environmental sequencing approach presented here detected a stochastic distribution of human pathogens, such as Helicobacter pylori, thereby demonstrating the potential of flies as proxies for environmental and public health surveillance.

Meeting the challenge of DNA barcoding Neotropical amphibians: polymerase chain reaction optimization and new COI primers.

Amphibians are one of the most threatened vertebrate classes, yet at the same time new species are being described every year, demonstrating that the number of existing species is grossly underestimated. In groups such as amphibians, with high extinction rates and poorly known species boundaries, DNA barcoding is a tool that can rapidly assess genetic diversity and estimate species richness for prioritizing conservation decisions. However, reliable recovery of the 5' region of the cytochrome c oxidase subunit 1 (COI) gene is critical for the ongoing effort to gather DNA barcodes for all amphibian species. Here, we provide new PCR conditions and tested new primers that increase the efficiency of barcode recovery in amphibians. We found that a low extension temperature for PCR cycles significantly improves the efficiency of amplification for all combinations of primers. Combining low PCR extension temperature and primers AnF1 + AnR1, we were able to recover COI sequences for 100% of the species analysed (N = 161), encompassing ~15% of the species known from Brazil (representing 77 genera and 23 families), which is an important improvement over previous studies. The preliminary assessment of species diversity suggested that number of species might be underestimated by about 25%. We conclude that DNA barcoding is an efficient, simple, and standardized protocol for identifying cryptic diversity in amphibians and advocate for its use in biodiversity inventories and across widespread populations within known species.

Large-scale mitogenomics enables insights into Schizophora (Diptera) radiation and population diversity.

True flies are insects of the order Diptera and encompass one of the most diverse groups of animals on Earth. Within dipterans, Schizophora represents a recent radiation of insects that was used as a model to develop a pipeline for generating complete mitogenomes using various sequencing platforms and strategies. 91 mitogenomes from 32 different species were sequenced and assembled with high fidelity, using amplicon, whole genome shotgun or single molecule sequencing approaches. Based on the novel mitogenomes, we estimate the origin of Schizophora within the Cretaceous-Paleogene (K-Pg) boundary, about 68.3 Ma. Detailed analyses of the blowfly family (Calliphoridae) place its origin at 22 Ma, concomitant with the radiation of grazing mammals. The emergence of ectoparasitism within calliphorids was dated 6.95 Ma for the screwworm fly and 2.3 Ma for the Australian sheep blowfly. Varying population histories were observed for the blowfly Chrysomya megacephala and the housefly Musca domestica samples in our dataset. Whereas blowflies (n = 50) appear to have undergone selective sweeps and/or severe bottlenecks in the New World, houseflies (n = 14) display variation among populations from different zoogeographical zones and low levels of gene flow. The reported high-throughput mitogenomics approach for insects enables new insights into schizophoran diversity and population history of flies.

Four new species of Moneuptychia (Lepidoptera: Satyrinae: Euptychiina) from Brazil.

This paper describes four new species of Moneuptychia as follows: M. montana Freitas, M. vitellina Freitas & Barbosa, M. pervagata Freitas, Siewert & Mielke and M. wahlbergi Freitas, Barbosa, Siewert & Mielke from south and southeastern Brazil. Details are presented on the morphology of adults of all species, and immature stages for two species, and we discuss the taxonomy and identification of the genus Moneuptychia. The mitochondrial CoxI "barcode" region was used for exploring the utility of this DNA marker to identify these species, giving strong support for all new species.

Incongruent nuclear and mitochondrial genetic structure of new world screwworm fly populations due to positive selection of mutations associated with dimethyl- and diethyl-organophosphates resistance.

Livestock production is an important economic activity in Brazil, which has been suffering significant losses due to the impact of parasites. The New World screwworm (NWS) fly, Cochliomyia hominivorax, is an ectoparasite and one of the most important myiasis-causing flies endemic to the Americas. The geographic distribution of NWS has been reduced after the implementation of the Sterile Insect Technique (SIT), being eradicated in North America and part of Central America. In South America, C. hominivorax is controlled by chemical insecticides, although indiscriminate use can cause selection of resistant individuals. Previous studies have associated the Gly137Asp and Trp251Leu mutations in the active site of carboxylesterase E3 to resistance of diethyl and dimethyl-organophosphates insecticides, respectively. Here, we have sequenced a fragment of the carboxylesterase E3 gene (ChαE7), comprising part of intron iII, exon eIII, intron iIII and part of exon eIV, and three mitochondrial gene sequences (CR, COI and COII), of NWS flies from 21 locations in South America. These markers were used for population structure analyses and the ChαE7 gene was also investigated to gain insight into the selective pressures that have shaped its evolution. Analysis of molecular variance (AMOVA) and pairwise FST analysis indicated an increased genetic structure between locations in the ChαE7 compared to the concatenated mitochondrial genes. Discriminant analysis of principal components (DAPC) and spatial analysis of molecular variance (SAMOVA) indicated different degrees of genetic structure for all markers, in agreement with the AMOVA results, but with low correlation to geographic data. The NWS fly is considered a panmitic species based on mitochondrial data, while it is structured into three groups considering the ChαE7 gene. A negative association between the two mutations related to organophosphate resistance and Fay & Wu's H significant negative values for the exons, suggest that these mutations evolved under positive selection.

Applying spatial analysis of genetic and environmental data to predict connection corridors to the New World screwworm populations in South America.

The myiasis causing New World screwworm (NWS) fly is responsible for substantial losses to livestock breeders in the Americas. Due to the negative impact of the NWS fly in animal health, expansion of successful NWS fly eradication programmes is under discussion. However, the effects of geography and environmental diversity on NWS population structure and migration patterns need to be assessed before any political decision is made to implement such a programme. We present a GIS tool to construct potential connection corridors among sampling localities based on genetic and environmental data. We integrate, through a home-made python script, a friction raster based on a Maxent niche model and the pairwise ΦST statistic. Among 38 NWS fly sampling localities from South America, we find a high population connectivity among the sampling localities from the south of the Amazon region. The region along the Atlantic Ocean was identified as the most probable migration corridor between the north (NAG) and the south (SAG) of the Amazon region. The approach highlighted previously undetected population structure within NAG showing low to medium connectivity through the Andes, correlating with current understanding of NWS fly migration in South America. Also, the approach is flexible, allowing future research to incorporate other niche simulations and genetic differentiation metrics. With this flexibility, the tool could become part of any AW-IPM by helping to target regions for control.

Genetic diversity and population structure of the New World screwworm fly from the Amazon region of Brazil.

Cochliomyia hominivorax (Coquerel) is a myiasis fly that causes economic losses to livestock farmers in warmer American regions. Previous studies of this pest had found population structure at north and south of the Amazon Basin, which was considered to be a barrier to dispersal. The present study analyzed three mitochondrial DNA (mtDNA) markers and eight nuclear microsatellite loci to investigate for the first time the genetic diversity and population structure across the Brazilian Amazon region (Amazonia). Both mtDNA and microsatellite data supported the existence of much diversity and significant population structure among nine regional populations of C. hominivorax, which was found to be surprisingly common in Amazonia. Forty-six mtDNA haplotypes were identified, of which 39 were novel and seven had previously been found only at south of Amazonia. Seventy microsatellite alleles were identified by size, moderate to high values of heterozygosity were discovered in all regions, and a Bayesian clustering analysis identified four genetic groups that were not geographically distributed. Reproductive compatibility was also investigated by laboratory crossing, but no evidence of hybrid dysgenesis was found between an Amazonian colony and one each of from Northeast and Southeast Brazil. The results have important implications for area-wide control by the Sterile Insect Technique.

The phylogeographic history of the new world screwworm fly, inferred by approximate bayesian computation analysis.

Insect pest phylogeography might be shaped both by biogeographic events and by human influence. Here, we conducted an approximate Bayesian computation (ABC) analysis to investigate the phylogeography of the New World screwworm fly, Cochliomyia hominivorax, with the aim of understanding its population history and its order and time of divergence. Our ABC analysis supports that populations spread from North to South in the Americas, in at least two different moments. The first split occurred between the North/Central American and South American populations in the end of the Last Glacial Maximum (15,300-19,000 YBP). The second split occurred between the North and South Amazonian populations in the transition between the Pleistocene and the Holocene eras (9,100-11,000 YBP). The species also experienced population expansion. Phylogenetic analysis likewise suggests this north to south colonization and Maxent models suggest an increase in the number of suitable areas in South America from the past to present. We found that the phylogeographic patterns observed in C. hominivorax cannot be explained only by climatic oscillations and can be connected to host population histories. Interestingly we found these patterns are very coincident with general patterns of ancient human movements in the Americas, suggesting that humans might have played a crucial role in shaping the distribution and population structure of this insect pest. This work presents the first hypothesis test regarding the processes that shaped the current phylogeographic structure of C. hominivorax and represents an alternate perspective on investigating the problem of insect pests.

Genetic structure and demographic history of new world screwworm across its current geographic range.

The phylogeographical history of the pest fly screwworm, Cochliomyia hominivorax (Coquerel), was studied using partial mitochondrial DNA sequences of the control region, Cytochrome c oxidase (CO) subunit I and CO subunit II from 361 individuals collected across its current geographic range. Analyses showed marked genetic differentiation on a macrogeographic scale. The genetic diversity in the species is structured into four main "regional groups," corresponding to Cuba, the Dominican Republic, and the North and South Amazon region. Results indicated that the distribution of screwworm genetic diversity was mainly shaped by historical events, i.e., colonization of Caribbean islands, vicariance in the Amazon region and population expansion. Demographic history analyses revealed that the population expansion started approximately 20-25,000 yr ago and recently increased exponentially. We hypothesized that the initial period of expansion was probably associated with environmental amelioration in the late Pleistocene and the exponential increase with resource availability in recent times. The population expansion is probably responsible for the low divergence and the lack of genetic and geographic correlation in the South Amazon region but did not erase the genetic structure pattern on a continental scale. The screwworm is one of the most damaging livestock pests in South and Central America, and the pattern of genetic variability distribution reported here suggests that the Caribbean area and the North and South Amazon regions could be considered as independent units for future pest control programs.

Deep sequencing of New World screw-worm transcripts to discover genes involved in insecticide resistance.

BACKGROUND: The New World screw-worm (NWS), Cochliomyia hominivorax, is one of the most important myiasis-causing flies, causing severe losses to the livestock industry. In its current geographical distribution, this species has been controlled by the application of insecticides, mainly organophosphate (OP) compounds, but a number of lineages have been identified that are resistant to such chemicals. Despite its economic importance, only limited genetic information is available for the NWS. Here, as a part of an effort to characterize the C. hominivorax genome and identify putative genes involved in insecticide resistance, we sampled its transcriptome by deep sequencing of polyadenylated transcripts using the 454 sequencing technology. RESULTS: Deep sequencing on the 454 platform of three normalized libraries (larval, adult male and adult female) generated a total of 548,940 reads. Eighteen candidate genes coding for three metabolic detoxification enzyme families, cytochrome P450 monooxygenases, glutathione S-transferases and carboxyl/cholinesterases were selected and gene expression levels were measured using quantitative real-time polymerase chain reaction (qRT-PCR). Of the investigated candidates, only one gene was expressed differently between control and resistant larvae with, at least, a 10-fold down-regulation in the resistant larvae. The presence of mutations in the acetylcholinesterase (target site) and carboxylesterase E3 genes was investigated and all of the resistant flies presented E3 mutations previously associated with insecticide resistance. CONCLUSIONS: Here, we provided the largest database of NWS expressed sequence tags that is an important resource, not only for further studies on the molecular basis of the OP resistance in NWS fly, but also for functional and comparative studies among Calliphoridae flies. Among our candidates, only one gene was found differentially expressed in resistant individuals, and its role on insecticide resistance should be further investigated. Furthermore, the absence of mutations in the OP target site and the high frequency of mutant carboxylesterase E3 indicate that metabolic resistance mechanisms have evolved predominantly in this species.

The mitochondrial control region of blowflies (Diptera: Calliphoridae): a hot spot for mitochondrial genome rearrangements.

The family Calliphoridae consists of myiasis-causing flies, including species of economic, forensic, and medical importance. In this study, the complete control regions (CRs) of mitochondrial DNA from 15 calliphorid species were sequenced and structurally characterized. The CRs had a high content of adenines (A) and thymines (T) and varied in length from 854 to 2,018 bp, showing intraspecific variations in sequence and length. Two major domains were identified: the conserved domain containing conserved sequence blocks and cis-regulatory structures that may be related to the transcription and the origin of replication of mitochondrial DNA, and the variable domain, containing high sequence and length variation. Within the variable domain, duplication of the tRNA(Ile) gene, previously reported for three Chrysomya species, was identified in two more species of this genus and in two species of two other genera. The structural characterization shows the plasticity of the mitochondrial genome in dipterans. The organizational similarities of the duplicated region found in different species and the possible origin of the duplicated genes are discussed.

Structure and evolution of the mitochondrial genomes of Haematobia irritans and Stomoxys calcitrans: the Muscidae (Diptera: Calyptratae) perspective.

We present the first two mitochondrial genomes of Muscidae dipterans for the species Haematobia irritans (the horn fly) and Stomoxys calcitrans (the stable fly). Typical insect mtDNA features are described, such as a high A+T content (79.1% and 78.9%, respectively), the preference for A+T-rich codons, and the evidence of a non-optimal codon usage. The strong A+T enrichment partially masks another nucleotide content bias maintained by A+C mutation pressure in these Muscidae mtDNAs. The analysis of this data provides a model of metazoans tRNA anticodon evolution, based on the selection hypothesis of anticodon versatility. H. irritans mitochondrial genome (16078 bp) is structurally similar to the hypothetical ancestral mitochondrial genome of arthropods and its control region (A+ T-rich region in insects) organization is consistent with the structure described for Brachycera dipterans. On the other hand, the mitochondrial genome of S. calcitrans is approximately 2kb longer (18 kb), characterized by the presence of approximately 550 bp tandem repeats in the control region, and an extra copy of trnI remarkably similar to a duplicated element of blowflies mtDNA. Putative sequence elements, involved in the regulation of transcription and replication of the mtDNA, were reliably identified in S. calcitrans control region despite the 0.8-1.5 kb gap uncovered from this genome. The use of amino acid and nucleotide sequences of concatenated mitochondrial protein-coding genes (PCGs) in phylogenetic reconstructions of Diptera does not support the monophyly of Muscomorpha, as well as the monophyly of Acalyptratae. Within the Calyptratae group, the inclusion of Muscidae (Muscoidea) as a sister group of Calliphoridae (Oestroidea) implies in a potential conflict concerning the monophyly of the superfamily Oestroidea.

Phylogenetic relationships of butterflies of the tribe Acraeini (Lepidoptera, Nymphalidae, Heliconiinae) and the evolution of host plant use.

The tribe Acraeini (Nymphalidae, Heliconiinae) is believed to comprise between one and seven genera, with the greatest diversity in Africa. The genera Abananote, Altinote, and Actinote (s. str.) are distributed in the Neotropics, while the genera Acraea, Bematistes, Miyana, and Pardopsis have a Palaeotropical distribution. The monotypic Pardopsis use herbaceous plants of the family Violaceae, Acraea and Bematistes feed selectively on plants with cyanoglycosides belonging to many plant families, but preferentially to Passifloraceae, and all Neotropical species with a known life cycle feed on Asteraceae only. Here, a molecular phylogeny is proposed for the butterflies of the tribe Acraeini based on sequences of COI, EF-1alpha and wgl. Both Maximum Parsimony and Bayesian analyses showed that the tribe is monophyletic, once the genus Pardopsis is excluded, since it appears to be related to Argynnini. The existing genus Acraea is a paraphyletic group with regard to the South American genera, and the species of Acraea belonging to the group of "Old World Actinote" is the sister group of the Neotropical genera. The monophyly of South American clade is strongly supported, suggesting a single colonization event of South America. The New World Actinote (s. str.) is monophyletic, and sister to Abananote+Altinote (polyphyletic). Based on the present results it was possible to propose a scenario for the evolution in host plant use within Acraeini, mainly concerning the use of Asteraceae by the South American genera.

Analysis of a conserved duplicated tRNA gene in the mitochondrial genome of blowflies.

The mtDNA control region (CR) and flanking genes of the blowflies Chrysomya albiceps, Chrysomya megacephala and Chrysomya chloropyga (Calliphoridae) were characterized. The most unusual feature found was the presence of duplicated tRNA sequences corresponding to trnI and a portion of trnQ. The partially duplicated trnQ was very likely a pseudogene since most of the sequence of the typical insect trnQ gene was missing. In contrast, the trnI gene had a conserved primary sequence following the duplication event and may represent a functional copy. These results demonstrate the plasticity of the mtDNA molecule in Chrysomya, especially for tRNA genes and the adjacent control region sequences.

The mitochondrial genome of the blowfly Chrysomya chloropyga (Diptera: Calliphoridae).

In view of the medical, sanitary and forensic importance of Chrysomya species, a knowledge of their nucleotide sequences would be useful for the molecular characterization of this genus, and would help in designing primers and in improving the molecular identification of Calliphoridae species. In this work, the mitochondrial genome of the blowfly Chrysomya chloropyga (Diptera: Calliphoridae) was completely sequenced. The entire mitochondrial DNA (mtDNA) molecule was 15,837 bp long and was sequenced using the shotgun approach. The overall nucleotide composition was heavily biased towards As and Ts, which accounted for 76.7% of the whole genome. The cox1 gene had a serine as the start codon, while incomplete termination codons mediated by tRNA signals were found for cox2, nd4 and nd5. The C. chloropyga genes were in the same order and orientation as the mitochondrial genome of other dipteran species, except for the occurrence of a 123 bp region that included a complete duplication of tRNA(Ile) and a partial duplication of tRNA(Gln) genes. C. chloropyga is the first species of Diptera with 23 tRNA genes instead of the usual 22 already described. A phylogenetic analysis showed a split of Brachycera into Calyptratae and Acalyptratae subdivisions. The complete sequence of C. chloropyga mtDNA described here will be a useful source of sequence information for general molecular and evolutionary studies in Diptera.

Levantamento de microhimenópteros parasitóides de dípteros de importância, médico-veterinária no Brasil / Survey of parasitoid microhimenoptera of importance Diptera, veterinarian in Brazil

De larvas e pupas de Musca domestica, Chrysomya albiceps, Cochliomyia homivorax, Stomoxys calcitrans e Syntesiomyia nudiseta coletadas em diversos ambiente, em São Paulo, Paraná, Mato Grosso, Mato Grosso do Sul e Minas Gerais, foram obtidas dez espécies de microhimenópteros parasitóides da supermamília Chalcidoidea, algumas assinaladas pela primeira vez no Brasil.