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Background: Some of the most important malaria vectors in South America belong to the Albitarsis Complex (Culicidae; Anophelinae; Anopheles). Understanding the origin, nature, and geographical distribution of species diversity in this important complex has important implications for vector incrimination, control, and management, and for modelling future responses to climate change, deforestation, and human population expansion. This study attempts to further explore species diversity and evolutionary history in the Albitarsis Complex by undertaking a characterization and phylogenetic analysis of the mitogenome of all 10 putative taxa in the Albitarsis Complex., Methods: Mitogenome assembly and annotation allowed for feature comparison among Albitarsis Complex and Anopheles species. Selection analysis was conducted across all 13 protein-coding genes. Maximum likelihood and Bayesian inference methods were used to construct gene and species trees, respectively. Bayesian methods were also used to jointly estimate species delimitation and species trees., Results: Gene composition and order were conserved across species within the complex. Unique signatures of positive selection were detected in two species-Anopheles janconnae and An. albitarsis G-which may have played a role in the recent and rapid diversification of the complex. The COI gene phylogeny does not fully recover the mitogenome phylogeny, and a multispecies coalescent-based phylogeny shows that considerable uncertainty exists through much of the mitogenome species tree. The origin of divergence in the complex dates to the Pliocene/Pleistocene boundary, and divergence within the distinct northern South American clade is estimated at approximately 1 million years ago. Neither the phylogenetic trees nor the delimitation approach rejected the 10-species hypothesis, although the analyses could not exclude the possibility that four putative species with scant a priori support (An. albitarsis G, An. albitarsis H, An. albitarsis I, and An. albitarsis J), represent population-level, rather than species-level, splits., Conclusion: The lack of resolution in much of the species tree and the limitations of the delimitation analysis warrant future studies on the complex using genome-wide data and the inclusion of additional specimens, particularly from two putative species, An. albitarsis I and An. albitarsis J., (© 2021. The Author(s).)

The Neotropical Albitarsis Group is a complex assemblage of essentially isomorphic species which currently comprises eight recognized species-five formally described (Anopheles albitarsis Lynch-Arribalzaga, An. deaneorum Rosa-Freitas, An. janconnae Wilkerson and Sallum, An. marajoara Galvao and Damasceno, An. oryzalimnetes Wilkerson and Motoki) and three molecularly assigned (An. albitarsis F, G & I)-and one mitochondrial lineage (An. albitarsis H). To further explore species recognition within this important group, 658 base pairs of the mitochondrial DNA cytochrome oxidase subunit I (COI) were analyzed from 988 specimens from South America. We conducted statistical parsimony network analysis, generated estimates of haplotype, nucleotide, genetic differentiation, divergence time, and tested the effect of isolation by distance (IBD). Ten clusters were identified, which confirmed the validity of the eight previously determined species, and confirmed the specific status of the previous mitochondrial lineage An. albitarsis H. High levels of diversity were highlighted in two samples from Pará (= An. albitarsis J), which needs further exploration through additional sampling, but which may indicate another cryptic species. The highest intra-specific nucleotide diversity was observed in An. deaneorum, and the lowest in An. marajoara. Significant correlation between genetic and geographical distance was observed only in An. oryzalimnetes and An. albitarsis F. Divergence time within the Albitarsis Group was estimated at 0.58-2.25 Mya, during the Pleistocene. The COI barcode region was considered an effective marker for species recognition within the Albitarsis Group and a network approach was an analytical method to discriminate among species of this group., (Published by Oxford University Press on behalf of Entomological Society of America 2020.)

Background: The worldwide genus Anopheles Meigen, 1918 is the only genus containing species evolved as vectors of human and simian malaria. Morbidity and mortality caused by Plasmodium Marchiafava & Celli, 1885 is tremendous, which has made these parasites and their vectors the objects of intense research aimed at mosquito identification, malaria control and elimination. DNA tools make the identification of Anopheles species both easier and more difficult. Easier in that putative species can nearly always be separated based on DNA data; more difficult in that attaching a scientific name to a species is often problematic because morphological characters are often difficult to interpret or even see; and DNA technology might not be available and affordable. Added to this are the many species that are either not yet recognized or are similar to, or identical with, named species. The first step in solving Anopheles identification problem is to attach a morphology-based formal or informal name to a specimen. These names are hypotheses to be tested with further morphological observations and/or DNA evidence. The overarching objective is to be able to communicate about a given species under study. In South America, morphological identification which is the first step in the above process is often difficult because of lack of taxonomic expertise and/or inadequate identification keys, written for local fauna, containing the most consequential species, or obviously, do not include species described subsequent to key publication., Methods: Holotypes and paratypes and other specimens deposited in the Coleção Entomológica de Referência, Faculdade de Saúde Pública (FSP-USP), Museo de Entomología, Universidad del Valle (MUSENUV) and the US National Mosquito Collection, Smithsonian Institution (USNMC) were examined and employed to illustrate the identification keys for female, male and fourth-instar larvae of Anopheles., Results: We presented, in four concurrent parts, introduction and three keys to aid the identification of South American Anopheles based on the morphology of the larvae, male genitalia and adult females, with the former two keys fully illustrated., Conclusions: Taxonomic information and identification keys for species of the genus Anopheles are updated. The need for further morphology-based studies and description of new species are reinforced.

Background: Morphological identification of adult females of described species of the genus Anopheles Meigen, 1818 in South America is problematic, but necessary due to their differing roles in the transmission of human malaria. The increase in the number of species complexes uncovered by molecular taxonomy challenges accurate identification using morphology. In addition, the majority of newly discovered species have not been formally described and in some cases the identities of the nominotypical species of species complexes have not been resolved. Here, we provide an up-to-date key to identify Neotropical Anopheles species using female external morphology and employing traditionally used and new characters., Methods: Morphological characters of the females of South American species of the genus Anopheles were examined and employed to construct a species/group identification key. Photographs of key characters were obtained using a digital Canon Eos T3i, attached to a microscope. The program Helicon Focus was used to build single in-focus images by stacking multiple images of the same structure., Results: A morphological identification key to the adult females of species of the genus Anopheles described in South America is presented. Definitions and illustrations of the key characters are provided to facilitate use of key., Conclusions: Identification of species of the genus Anopheles based on female morphology is challenging because some key characters can be variable and overlapping among species. In addition, the majority of key characters are linked to color and shape of scales, their distribution on the head, scutum, abdomen, maxillary palpi, labium and legs, and pattern of pale and dark scales on dorsal and ventral surfaces of the wing veins. Thus, it is understandable that a specimen needs to be in good condition to be accurately identified. Morphologically similar species, such as those of the Konderi, Oswaldoi, Nuneztovari, Benarrochi and Albitarsis Complexes, and the Triannulatus and Strodei Groups, among others, cannot be accurately identified using characters included in the key. Further investigation will be required to exploit morphological characteristics for identification of members of those complexes, with formal description of new species.

Background: Accurate species identification of South American anophelines using morphological characters of the fourth-instar larva is problematic, because of the lack of up-to-date identification keys. In addition, taxonomic studies, employing scanning electron microscopy of the eggs and DNA sequence data, have uncovered multiple complexes of morphologically similar species, and resulted in the resurrection of other species from synonymy, mainly in the subgenus Nyssorhynchus. Consequently, the identification keys urgently need to be updated to provide accurate morphological tools to identify fourth-instar larvae of all valid species and species complexes., Methods: Morphological characters of the fourth-instar larvae of South American species of the genus Anopheles were examined and employed to elaborate a fully illustrated identification key. For species for which no specimens were available, illustrations were based on published literature records., Results: A fully illustrated key to the fourth-instar larvae of South American species of the genus Anopheles (Diptera: Culicidae) is presented. Definitions of the morphological terms used in the key are provided and illustrated., Conclusions: Morphological identification of South American Anopheles species based on the fourth-instar larvae has been updated. Characters of the spiracular apparatus were determined useful for the identification of morphologically similar species, in the Strodei Group and some taxa in the Myzorhynchella Section. The single versus branched abdominal seta 6-IV used to differentiate Myzorhynchella species from other Nyssorhynchus species was shown to be variable in Myzorhynchella species. Also, the abdominal setae 1-IV,V of Anopheles atacamensis and Anopheles pictipennis were shown to be slightly serrate at the edges. Recognition of this character is important to avoid inaccurate identification of these species as members of the subgenus Anopheles.

Background: Accurate identification of the species of Anopheles Meigen, 1818 requires careful examination of all life stages. However, morphological characters, especially those of the females and fourth-instar larvae, show some degree of polymorphism and overlap among members of species complexes, and sometimes even within progenies. Characters of the male genitalia are structural and allow accurate identification of the majority of species, excluding only those in the Albitarsis Complex. In this key, based on the morphology of the male genitalia, traditionally used important characters are exploited together with additional characters that allow robust identification of male Anopheles mosquitoes in South America., Methods: Morphological characters of the male genitalia of South American species of the genus Anopheles were examined and employed to construct a comprehensive, illustrated identification key. For those species for which specimens were not available, illustrations were based on published illustrations. Photographs of key characters of the genitalia were obtained using a digital Canon Eos T3i attached to a light Diaplan Leitz microscope. The program Helicon Focus was used to build single in-focus images by stacking multiple images of the same structure., Results: An illustrated key to South American species of Anopheles based on the morphology of the male genitalia is presented, together with a glossary of morphological terms. The male genitalia of type-specimens of previously poorly documented species were also examined and included in the key, e.g. Anopheles (Anopheles) tibiamaculatus (Neiva, 1906) which has a unique quadrangular-shaped aedeagus with an apical opening., Conclusions: Male genitalia of South American species of Anopheles possess robust characters that can be exploited for accurate species identification. Distortion that can occur during the dissection and mounting process can obstruct accurate identification; this is most evident with inadvertent damage or destruction of unique features and interferes with correctly assigning shapes of the features of the ventral claspette. In some species, the shape, and anatomical details of the aedeagus also need to be examined for species identification. For members of the Myzorhynchella Series, both ventral and dorsal claspettes possess multiple characteristics that are herein used as reliable characters for species identification.

Background: Malaria remains a significant public health issue in South America. Future climate change may influence the distribution of the disease, which is dependent on the distribution of those Anopheles mosquitoes competent to transmit Plasmodium falciparum. Herein, predictive niche models of the habitat suitability for P. falciparum, the current primary vector Anopheles darlingi and nine other known and/or potential vector species of the Neotropical Albitarsis Complex, were used to document the current situation and project future scenarios under climate changes in South America in 2070., Methods: To build each ecological niche model, we employed topography, climate and biome, and the currently defined distribution of P. falciparum, An. darlingi and nine species comprising the Albitarsis Complex in South America. Current and future (i.e., 2070) distributions were forecast by projecting the fitted ecological niche model onto the current environmental situation and two scenarios of simulated climate change. Statistical analyses were performed between the parasite and each vector in both the present and future scenarios to address potential vector roles in the dynamics of malaria transmission., Results: Current distributions of malaria vector species were associated with that of P. falciparum, confirming their role in transmission, especially An. darlingi, An. marajoara and An. deaneorum. Projected climate changes included higher temperatures, lower water availability and biome modifications. Regardless of future scenarios considered, the geographic distribution of P. falciparum was exacerbated in 2070 South America, with the distribution of the pathogen covering 35-46% of the continent. As the current primary vector An. darlingi showed low tolerance for drier environments, the projected climate change would significantly reduce suitable habitat, impacting both its distribution and abundance. Conversely, climate generalist members of the Albitarsis Complex showed significant spatial and temporal expansion potential in 2070, and we conclude these species will become more important in the dynamics of malaria transmission in South America., Conclusions: Our data suggest that climate and landscape effects will elevate the importance of members of the Albitarsis Complex in malaria transmission in South America in 2070, highlighting the need for further studies addressing the bionomics, ecology and behaviours of the species comprising the Albitarsis Complex.

Background: Effective malaria control relies on accurate identification of those Anopheles mosquitoes responsible for the transmission of Plasmodium parasites. Anopheles oswaldoi s.l. has been incriminated as a malaria vector in Colombia and some localities in Brazil, but not ubiquitously throughout its Neotropical range. This evidence together with variable morphological characters and genetic differences supports that An. oswaldoi s.l. compromises a species complex. The recent fully integrated redescription of An. oswaldoi s.s. provides a solid taxonomic foundation from which to molecularly determine other members of the complex., Methods: DNA sequences of the Second Internal Transcribed Spacer (ITS2 - rDNA) (n = 192) and the barcoding region of the Cytochrome Oxidase I gene (COI - mtDNA) (n = 110) were generated from 255 specimens of An. oswaldoi s.l. from 33 localities: Brazil (8 localities, including the lectotype series of An. oswaldoi), Ecuador (4), Colombia (17), Trinidad and Tobago (1), and Peru (3). COI sequences were analyzed employing the Kimura-two-parameter model (K2P), Bayesian analysis (MrBayes), Mixed Yule-Coalescent model (MYC, for delimitation of clusters) and TCS genealogies., Results: Separate and combined analysis of the COI and ITS2 data sets unequivocally supported four separate species: two previously determined (An. oswaldoi s.s. and An. oswaldoi B) and two newly designated species in the Oswaldoi Complex (An. oswaldoi A and An. sp. nr. konderi). The COI intra- and inter-specific genetic distances for the four taxa were non-overlapping, averaging 0.012 (0.007 to 0.020) and 0.052 (0.038 to 0.064), respectively. The concurring four clusters delineated by MrBayes and MYC, and four independent TCS networks, strongly confirmed their separate species status. In addition, An. konderi of Sallum should be regarded as unique with respect to the above. Despite initially being included as an outgroup taxon, this species falls well within the examined taxa, suggesting a combined analysis of these taxa would be most appropriate., Conclusions: Through novel data and retrospective comparison of available COI and ITS2 DNA sequences, evidence is shown to support the separate species status of An. oswaldoi s.s., An. oswaldoi A and An. oswaldoi B, and at least two species in the closely related An. konderi complex (An. sp. nr. konderi, An. konderi of Sallum). Although An. oswaldoi s.s. has never been implicated in malaria transmission, An. oswaldoi B is a confirmed vector and the new species An. oswaldoi A and An. sp. nr. konderi are circumstantially implicated, most likely acting as secondary vectors.

Background: The molecular phylogenetic relationships and population structure of the species of the Anopheles triannulatus complex: Anopheles triannulatus s.s., Anopheles halophylus and the putative species Anopheles triannulatus C were investigated., Methods: The mitochondrial COI gene, the nuclear white gene and rDNA ITS2 of samples that include the known geographic distribution of these taxa were analyzed. Phylogenetic analyses were performed using Bayesian inference, Maximum parsimony and Maximum likelihood approaches., Results: Each data set analyzed septely yielded a different topology but none provided evidence for the seption of An. halophylus and An. triannulatus C, consistent with the hypothesis that the two are undergoing incipient speciation. The phylogenetic analyses of the white gene found three main clades, whereas the statistical parsimony network detected only a single metapopulation of Anopheles triannulatus s.l. Seven COI lineages were detected by phylogenetic and network analysis. In contrast, the network, but not the phylogenetic analyses, strongly supported three ITS2 groups. Combined data analyses provided the best resolution of the trees, with two major clades, Amazonian (clade I) and trans-Andean + Amazon Delta (clade II). Clade I consists of multiple subclades: An. halophylus + An. triannulatus C; trans-Andean Venezuela; central Amazonia + central Bolivia; Atlantic coastal lowland; and Amazon delta. Clade II includes three subclades: Panama; cis-Andean Colombia; and cis-Venezuela. The Amazon delta specimens are in both clades, likely indicating local sympatry. Spatial and molecular variance analyses detected nine groups, corroborating some of subclades obtained in the combined data analysis., Conclusion: Combination of the three molecular markers provided the best resolution for differentiation within An. triannulatus s.s. and An. halophylus and C. The latest two species seem to be very closely related and the analyses performed were not conclusive regarding species differentiation. Further studies including new molecular markers would be desirable to solve this species status question. Besides, results of the study indicate a trans-Andean origin for An. triannulatus s.l. The potential implications for malaria epidemiology remain to be investigated.

Background: Mosquitoes belonging to the Albitarsis Group (Anopheles: Nyssorhynchus) are of importance as malaria vectors across the Neotropics. The Group currently comprises six known species, and recent studies have indicated further hidden biodiversity within the Group. DNA barcoding has been proposed as a highly useful tool for species recognition, although its discriminatory utility has not been verified in closely related taxa across a wide geographic distribution., Methods: DNA barcodes (658 bp of the mtDNA Cytochrome c Oxidase--COI) were generated for 565 An. albitarsis s.l. collected in Argentina, Brazil, Colombia, Paraguay, Trinidad and Venezuela over the past twenty years, including specimens from type series and type localities. Here we test the utility of currently advocated barcoding methodologies, including the Kimura-two-parameter distance model (K2P) and Neighbor-joining analysis (NJ), for determining species delineation within mosquitoes of the Neotropical Albitarsis Group of malaria vectors (Anopheles: Nyssorhynchus), and compare results with Bayesian analysis., Results: Species delineation through barcoding analysis and Bayesian phylogenetic analysis, fully concur. Analysis of 565 sequences (302 unique haplotypes) resolved nine NJ tree clusters, with less than 2% intra-node variation. Mean intra-specific variation (K2P) was 0.009 (range 0.002-0.014), whereas mean inter-specific divergence were several-fold higher at 0.041 (0.020-0.056), supporting the reported "barcoding gap". These results show full support for separate species status of the six known species in the Albitarsis Group (An. albitarsis s.s., An. albitarsis F, An. deaneorum, An. janconnae, An. marajoara and An. oryzalimnetes), and also support species level status for two previously detected lineages--An. albitarsis G &An. albitarsis I (designated herein). In addition, we highlight the presence of a unique mitochondrial lineage close to An. deaneorum and An. marajoara (An. albitarsis H) from Rondônia and Mato Grosso in southwestern Brazil. Further integrated studies are required to confirm the status of this lineage., Conclusions: DNA barcoding provides a reliable means of identifying both known and undiscovered biodiversity within the closely related taxa of the Albitarsis Group. We advocate its usage in future studies to elucidate the vector competence and respective distributions of all eight species in the Albitarsis Group and the novel mitochondrial lineage (An. albitarsis H) recovered in this study.

One of the major obstacles for studies of the biology, ecology, and behavior of Neotropical vectors of human Plasmodium has been the lack of reliable and efficient means of identifying many species. Although the subgenus Nyssorhynchus includes most species responsible for human transmission in South America, there are no polymerase chain reaction (PCR)-based techniques for identifying members of this taxon. We describe the first multiplex PCR for identifying four species in the subgenus Nyssorhynchus that are vectors of human Plasmodium spp. Four species specific primers, together with a universal primer that anneals to the 5.8S rDNA region, produce amplicons of the internal transcribed spacer two with base pair sizes of 131,308,371, and 441 for An. triannulatus, An. trinkae, An. strodei, and An. rangeli, respectively.

A genetic and morphologic survey of Anopheles darlingi populations collected from seven countries in Central and South America was performed to clarify the taxonomic status of this major malaria vector species in the Americas. Population genetics was based on three techniques including isozyme, random amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR), and internal transcribed spacer 2 (ITS2) markers. The results of the isozyme analysis indicated moderate differences in the allele frequencies of three putative loci (glutamate oxalaoacetate transaminase-1, isocitrate dehydrogenase-1, and phosphoglucomutase) of the 31 analyzed. No fixed electromorphic differences separated the populations of An. darlingi, which showed little genetic divergence (Nei distances = 0.976-0.995). Fragments produced by RAPD-PCR demonstrated evidence of geographic partitioning and showed that all populations were separated by small genetic distances as measured with the 1 - S distance matrix. The ITS2 sequences for all samples were identical except for four individuals from Belize that differed by a three-base deletion (CCC). The morphologic study demonstrated that the Euclidean distances ranged from 0.02 to 0.14, with the highest value observed between populations from Belize and Bolivia. Based on these analyses, all the An. darlingi populations examined demonstrated a genetic similarity that is consistent with the existence of a single species and suggest that gene flow is occurring throughout the species' geographic range.

Based on similarity of male genitalia, the malaria vector Anopheles trinkae Faran from the eastern Andean piedmont of Colombia, Ecuador, Peru, and Bolivia was determined by Peyton (1993) to be a junior synonym of An. dunhami Causey, then known from a single locality in Amazonian Brazil. Following an appraisal of molecular, chromosomal, and morphological characters, we conclude herein that the 2 taxa are specifically distinct and remove An. trinkae from synonymy with An. dunhami. Eggs of the 2 species are distinguished easily by the anterior crown, long floats, and closed deck that occur only in An. trinkae. The X chromosome of larval polytenes is divisible into R and L arms in An. dunhami, but not in An. trinkae. A phenogram based on banding pattern scores from 18 random amplified polymorphic DNA primers separated with 100% resolution An. dunhami, An. trinkae, Anopheles nuneztovari Gabaldón and Anopheles darlingi Root. In the ITS2 region of rDNA, 25% of base sites distinguished An. trinkae from An. dunhami and 21% from the related An. nuneztovari; males of these 3 species had accessory glands of significantly different sizes. Preliminary isoenzyme screening indicated that 3 of 11 loci were diagnostic for separating An. trinkae from An. dunhami. The results indicate that An. dunhami is related more closely to An. nuneztovari than to An. trinkae and illustrate the merits of a multidisciplinary approach to mosquito systematics.