Phylogeny of Anophelinae (Diptera: Culicidae) based on nuclear ribosomal and mitochondrial DNA sequences

Abstract Phylogenetic relationships among thirty-two species of mosquitoes in subfamily Anophelinae are inferred from portions of the mitochondrial genes COI and COII, the nuclear 18S small subunit rRNA gene and the expansion D2 region of the nuclear large subunit 28S rRNA gene. Sequences were obtained from the genera Anopheles , Bironella and Chagasia . Representatives of all six subgenera of Anopheles were included: Anopheles , Cellia , Kerteszia , Lophopodomyia , Nyssorhynchus and Stethomyia. Using parsimony and maximum likelihood methods, various combinations of these DNA sequence data were analysed separately: 18S, 28S, combined 18S and 28S, combined COI and COII, and combined 18S, 28S, COI and COII ('total evidence'). The combined rDNA data contain strong phylogenetic signal, moderately to strongly supporting most clades in MP and ML analyses; however, the mtDNA data (analysed as either nucleotide or amino acid sequences) contain little phylogenetic signal, except for relationships of very recently derived groups of species and, at the deepest level, for the monophyly of Anophelinae. The paraphyly of Anopheles relative to Bironella is confirmed by most analyses and statistical tests. Support for the monophyly of subgenera Anopheles , Cellia , Kerteszia and Nyssorhynchus is indicated by most analyses. Subgenus Lophopodomyia is reconstructed as the sister to Bironella , nested within a clade also containing Nyssorhynchus and Kerteszia . The most basal relationships within genus Anopheles are not well resolved by any of the data partitions, although the results of statistical analyses of the rDNA data (S-H-tests, likelihood ratio tests for monophyly and Bayesian MCMC analyses) suggest that the clade consisting of Bironella , Lophopodomyia , Nyssorhynchus and Kerteszia is the sister to the clade containing Cellia and Anopheles .     

Cladistic analysis of the subgenus Anopheles (Anopheles) Meigen (Diptera: Culicidae) based on morphological characters

In the present study, we used morphological characters to estimate phylogenetic relationships among members of the subgenus Anopheles Meigen. Phylogenetic analyses were carried out for 36 species of Anopheles (Anopheles). An. (Stethomyia) kompi Edwards, An. (Lophopodomyia) gilesi (Peryassú), Bironella hollandi Taylor, An. (Nyssorhynchus) oswaldoi (Peryassú) and An. (Cellia) maculatus Theobald were employed as outgroups. One hundred one characters of the external morphology of the adult male, adult female, fourth-instar larva, and pupa were scored and analyzed under the parsimony criterion in PAUP. Phylogenetic relationships among the series and several species informal groups of Anopheles (Anopheles) were hypothesized. The results suggest that Anopheles (Anopheles) is monophyletic. Additionally, most species groups included in the analysis were demonstrated to be monophyletic.     

P elements are found in the genomes of nematoceran insects of the genus Anopheles

We report the identification of genomic sequences in various anopheline mosquitoes (family Culicidae: suborder Nematocera: order Diptera) showing homology to the class II, short inverted-terminal-repeat (ITR) transposable element P from Drosophila melanogaster (family Drosophilidae; suborder Brachycera: order Diptera). Anopheles gambiae appears to have at least six distinct P elements. Other anopheline species, including four additional members of the An. gambiae species complex (An. arabiensis, An. merus, An. melas and An. quadriannulatus), Anopheles stephensi (all subgenus Cellia), An. quadrimaculatus (subgenus Anopheles) and Anopheles albimanus (subgenus Nyssorhynchus) also possess P elements similar to those found in An. gambiae. Ten distinct P element types were identified in the genus Anopheles. At least two of the An. gambiae elements appears to be intact and potentially functional. Phylogenetic analysis of the anopheline P elements reveals them to belong to a distinctly different clade from the brachyceran P elements.     

Reconsideration of anopheline mosquito phylogeny (Diptera: Culicidae: Anophelinae) based on morphological data

The phylogeny of anopheline mosquitoes (Culicidae: Anophelinae) is re‐examined using morphological data derived from adults, fourth‐instar larvae and pupae. Based on the data set of Sallum et al. (2000), we add some previously missing data and simplify and recode characters to eliminate ambiguities and more accurately reflect homologies, with special emphasis on characters of the male genitalia that provide the main criteria for the subgeneric classification of genus Anopheles. The principal aim of the study is to assess objectively the phylogenetic relationships and classification of two taxa not included by Sallum et al. (2000): Anopheles corethroides, a representative of the Australasian Stigmaticus Group, and An. kyondawensis, an unusual Oriental species whose adult and pupal stages were only recently discovered. The revised data set consists of 167 characters for 66 species representing the three traditionally recognised genera of Anophelinae, the six traditionally accepted subgenera of genus Anopheles and all informal series and most species groups of subgenera Anopheles, Cellia and Nyssorhynchus. The data are analysed using equal weighting (EW) and implied weighting (IW). Analysis under EW generates a strict consensus tree with principal lineages consistent with those reported by Sallum et al. (2000). Analysis under IW supports the monophyly of Anophelinae, the basal position of Chagasia, the monophyly of subgenera Cellia, Kerteszia and Nyssorhynchus, and the sister relationship of Kerteszia + Nyssorhynchus, but otherwise yields relationships that differ significantly in one respect or another from those obtained in all previous analyses of both morphological and molecular data. Subgenus Anopheles is arrayed as a polyphyletic lineage basal to a monophyletic clade comprising the Neotropical Kerteszia + Nyssorhynchus and the Old World Cellia in a sister‐group relationship. Bironella, Lophopodomyia and Stethomyia are firmly nested within subgenus Anopheles, which would nevertheless still be paraphyletic if these taxa were subsumed within it. Anopheles kyondawensis is well supported as the sister group of Bironella + all other Anopheles. Bironella, Stethomyia, An. corethroides and several other Anopheles clades are each strongly supported in a pectinate series of relationships, terminating in the clade comprising subgenera Cellia, Kerteszia and Nyssorhynchus. These relationships and other aspects of the phylogeny are discussed in relation to the formal and informal classification of genus Anopheles.     

基于rDNA-ITS2序列的中国按蚊属塞蚊亚属部分种类的系统发育研究(双翅目:蚊科)

应用rDNA-ITS2序列,采用邻接法(NJ)、最大简约法(MP)和最大似然法(ML),以按蚊亚属Anopheles的中华按蚊An.(An.)sinensis和赫坎按蚊An.(An.)hyrcanus为外群,对采自中国的按蚊属Anopheles塞蚊亚属Cellia 21种蚊进行了系统发育分析.结果表明:rDNA-ITS...     

林氏按蚊线粒体全基因组序列的测定及基于线粒体基因组的按蚊属系统发育分析(英文)

【目的】对林氏按蚊Anopheles lindesayi完整的线粒体基因组进行测序及分析,依据已知的线粒体基因组构建并讨论按蚊属蚊虫的分子系统发育关系。【方法】对林氏按蚊线粒体基因组进行测序、注释,并对其基本特征和基本组成进行分析。基于串联的13个蛋白质编码基因的核苷酸序列和氨基酸序列,用ML法和贝叶斯法构建林氏按蚊和按蚊属其他32种蚊虫的系统发育树,据此探讨按蚊属蚊虫的系统发育关系和系统分类。【结果】林氏按蚊线粒体基因组全长为15 366 bp,包含13个蛋白质编码基因,22个tRNA基因,2个rRNA基因和一段控制区。林氏按蚊线粒体基因组呈现明显的AT偏斜和GC偏斜,AT偏斜为正,GC偏斜为负。除了COX1使用TCG和ND5使用GTG作为起始密码子以外,其他蛋白质编码基因的起始密码子均遵循ATN原则;终止密码子为TAA或者T。除了tRNA^Ser(AGN)以外,其他的tRNA基因均呈现典型的三叶草二级结构。控制区AT含量最高,为94.54%。滑窗分析显示蛋白质编码基因是用于构建亚属或属水平系统发育关系的最佳分子标记。系统发育树强烈支持塞蚊亚属Cellia、按蚊亚属Anopheles、徕蚊亚属Nyssorhynchus和柯特蚊亚属Kerteszia均为单系群。小五斑按蚊An. atroparvus和四斑按蚊An. quadrimaculatus A这两个种聚到一起,从传统的形态分类上讲,它们和林氏按蚊均属于按蚊亚属按蚊系蚊虫。但本研究构建的4个系统发育树均显示,(小五斑按蚊An. atroparvus+四斑按蚊An. quadrimaculatus A)和林氏按蚊被属于迈蚊系的中华按蚊分开,这为两个系的分类提供了新的论点。【结论】本研究获得了林氏按蚊的完整的线粒体基因组,探析了按蚊属的线粒体基因组特征和系统发育关系,为进一步研究蚊科线粒体基因组和系统发育关系提供了依据。     

Toward understanding Anophelinae (Diptera,Culicidae) phylogeny: insights from nuclear single-copy genes and the weight of evidence

A phylogeny of the mosquito subfamily Anophelinae was inferred from fragments of two protein-coding nuclear genes, G6pd (462 bp) and white (801 bp), and from a combined data set (2,136 bp) that included a portion of the mitochondrial gene ND5 and the D2 region of the ribosomal 28S gene. Sixteen species from all three anopheline genera and six Anopheles subgenera were sampled, along with six species of other mosquitoes used as an outgroup. Each of four genes analyzed individually recovered the same well-supported clades; topological incongruence was limited to unsupported or poorly supported nodes. As assessed by the incongruence length difference test, most of the conflicting signal was contributed by third codon positions. Strong structural constraints, as observed in white and G6pd, apparently had little impact on phylogenetic inference. Compared with the other genes, white provided a superior source of phylogenetic information. However, white appears to have experienced accelerated rates of evolution in few lineages, the affinities of which are therefore suspect. In combined analyses, most of the inferred relationship were well-supported and in agreement with previous studies: monophyly of Anophelinae, basal position of Chagasia, monophyly of Anopheles subgenera, and subgenera Nyssorhynchus + Kerteszia as sister taxa. The results suggested also monophyletic origin of subgenera Cellia + Anopheles, and the white gene analysis supported genus Bironella as a sister taxon to Anopheles. The present data and other available evidence suggest a South American origin of Anophelinae, probably in the Mesozoic; a rapid diversification of Bironella and basal subgeneric lineages of Anopheles, potentially associated with the breakup of Gondwanaland; and a relatively recent and rapid dispersion of subgenus Anopheles.     

基于mtDNA和rDNA基因序列的中国按蚊属塞蚊亚属种类的系统发育研究（双翅目：蚊科）

【目的】应用mtDNA和rDNA基因特征重建中国按蚊属塞蚊亚属已知种类的系统发育关系, 以阐明亚属内各蚊种的亲缘关系。【方法】对采自中国的按蚊属塞蚊亚属Anopheles (Cellia) 20种蚊的mtDNA-COⅡ和 rDNA-28S-D3序列进行测定和分析, 以按蚊属按蚊亚属Anopheles (Anopheles)的中华按蚊An. (An.) sinensis和赫坎按蚊An. (An.) hyrcanus为外群, 采用COⅡ和D3单基因, 以及“COⅡ+D3”联合数据组以邻接法（NJ）、 最大简约法（MP）、 最大似然法（ML）和贝叶斯法（BI）等重建这些种类的系统发育树。【结果】 mtDNA-COⅡ和rDNA-28S-D3序列的长度范围分别为685 bp和375～410 bp, 在塞蚊亚属蚊种间的遗传距离分别为0.015～0.117和0.003～0.111。各系统树显示外群被合理分开,除在COⅡ树中新塞蚊系为并系外,各系均聚为单系群,新迈蚊系和迈蚊系亲缘关系最近。联合数据组构建的系统合意树显示中国塞蚊亚属各蚊种形成4支,除伪威氏按蚊与多斑按蚊种团未聚为单系群外,其他各种团和复合体成员种均分别聚在一起,各分支的置信值均大于50%。【结论】本研究获得的分子系统发育树清楚地显示了中国按蚊属塞蚊亚属各种类及系之间的系统发育关系, 对其分类和防治研究具有参考价值。     

Molecular identification of mosquito species in the Anopheles annularis group in southern Asia

The Anopheles annularis group of subgenus Cellia Theobald (Diptera: Culicidae) includes five currently recognized species in southern Asia: An. annularis Van der Wulp, Anopheles nivipes (Theobald) and Anopheles philippinensis Ludlow, which are widespread in the region, Anopheles pallidus Theobald, which is known in Sri Lanka, India and Myanmar, and Anopheles schueffneri Stanton, which occurs in Java and Sumatra. Identification of the four mainland species based on morphology is problematic. In view of the fact that the three widespread species are variously involved in malaria transmission in different parts of the region, we developed a species-specific polymerase chain reaction assay based on rDNA internal transcribed spacer 2 (ITS2) sequences to facilitate entomological and epidemiological studies of the four species. The method proved to be reliable when tested over a wide geographical area.     

Mosquito embryos and eggs: polarity and terminology of chorionic layers

The development of genetically modified vectors refractory to parasites is seen as a promising strategy in the future control of endemic diseases such as malaria. Nevertheless, knowledge of mosquito embryogenesis, a pre-requisite to the establishment of transgenic individuals, has been presently neglected. We have here studied the eggs from two neotropical malaria vectors. Eggs from Anopheles (Nyssorhynchus) albitarsis and Anopheles (Nyssorhynchus) aquasalis were analyzed by laser scanning microscopy and scanning electron microscopy and compared to those of Drosophila melanogaster. We verified basic conflicting data such as mosquito egg polarity and ultrastructure of eggshell layers. A 180 degrees rotation movement of the mosquito embryo along its longitudinal axis, a phenomenon not conserved among all Diptera, was confirmed. This early event is not taken into account by several present groups, leading to a non-consensual assignment of eggshell dorsal and ventral poles. Since embryo and egg polarities, defined during oogenesis, are the same, we propose to consider the flattened egg side as the dorsal one. The structure of Anopheles eggshell was also examined. Embryos are covered by a smooth endochorion or inner chorion layer. Outside this coat lies the compound exochorion or outer chorion layer, assembled by a thin basal lamellar layer and external tubercles. The terminology related to eggshell layers is discussed.     

Molecular phylogeny of the Myzorhynchella Section of Anopheles (Nyssorhynchus) (Diptera: Culicidae): genetic support for recently described and resurrected species

Phylogenetic relationships among species of the Myzorhynchella Section of Anopheles (Nyssorhynchus) were investigated using the nuclear ribosomal DNA second internal transcribed spacer (ITS2), the nuclear whitegene and mitochondrial cytochrome oxidase subunit I (COI) regions. The recently described Anopheles pristinus and resurrected Anopheles guarani were also included in the study. Bayesian phylogenetic analyses found Anopheles parvus to be the most distantly related species within the Section, a finding that is consistent with morphology. An. pristinus and An. guarani were clearly resolved from Anopheles antunesi and Anopheles lutzii, respectively. An. lutzii collected in the same mountain range as the type locality were found within a strongly supported clade, whereas individuals from the southern state of Rio Grande do Sul, tentatively identified as An. lutzii based on adult female external morphology, were distinct from An. lutzii, An. antunesi and from each other, and may therefore represent two new sympatric species. A more detailed examination of An. lutzii sensu latoalong its known geographic range is recommended to resolve these anomalous relationships.     

Evolution of mitochondrial and ribosomal gene sequences in anophelinae (Diptera: Culicidae): implications for phylogeny reconstruction

In this study, two mitochondrial genes, cyt b and ND5, and the D2 expansion segment of the 28S nuclear ribosomal gene were used to reconstruct a phylogeny of the mosquito subfamily Anophelinae. The ingroup consisted of all three genera of Anophelinae and five of six subgenera of Anopheles. Six genera of Culicinae were used as the outgroup. Extreme conservation at the protein level coupled with rapid saturation of synonymous positions probably accounted for the lack of meaningful phylogenetic signal in the cyt b gene. In contrast, abundant variation at all codon positions of the ND5 gene allowed recovery of the basal and most of the recent relationships. Phylogenetic analysis of D2 produced results consistent with those of ND5. Combined analysis indicated well-supported monophyletic Anophelinae (with Chagasia basal), Anopheles + Bironella, and subgeneric clades within the genus Anopheles. Moreover, subgenera Nyssorhynchus and Kerteszia were supported as a monophyletic lineage. The Kishino-Hasegawa test could not reject the monophyly of Anopheles, whereas the recently proposed hypothesis of close affinity of Bironella to the subgenus Anopheles was rejected by the analyses of ND5 and combined data sets. The lack of resolution of Bironella and Anopheles clades, or basal relationships among subgeneric clades within Anopheles, suggests their rapid diversification. Recovery of relationships consistent with morphology and previous molecular studies provides evidence of substantial phylogenetic signal in D2 and ND5 genes at levels of divergence from closely related species to subfamily in mosquitoes.     

A photomap of the salivary gland chromosomes of Anopheles stephensi liston (Culicidae: Diptera)

A photomap of the banding pattern of the salivary gland chromosomes of Anopheles stephensi Liston, which is first of its kind, has been prepared. The salivary chromosome complement consists of five arms, the shortest of which represents the telocentric X-chromosome, and the remaining four the autosomal arms. A comparison has been made of the banding pattern of this species with other species of the subgenus Cellia.     

Molecular comparison of topotypic specimens confirms Anopheles (Nyssorhynchus) dunhami Causey (Diptera: Culicidae) in the Colombian Amazon

The presence of Anopheles (Nyssorhynchus) dunhami Causey in Colombia (Department of Amazonas) is confirmed for the first time through direct comparison of mtDNA cytochrome c oxidase I (COI) barcodes and nuclear rDNA second internal transcribed spacer (ITS2) sequences with topotypic specimens of An. dunhami from Tefé, Brazil. An. dunhami was identified through retrospective correlation of DNA sequences following misidentification as Anopheles nuneztovari s.l. using available morphological keys for Colombian mosquitoes. That An. dunhami occurs in Colombia and also possibly throughout the Amazon Basin, is of importance to vector control programs, as this non-vector species is morphologically similar to known malaria vectors including An. nuneztovari, Anopheles oswaldoi and Anopheles trinkae. Species identification of An. dunhami and differentiation from these closely related species are highly robust using either DNA ITS2 sequences or COI DNA barcode. DNA methods are advocated for future differentiation of these often sympatric taxa in South America.     

Egg morphology as an indirect method to identify Anopheles benarrochi, Anopheles oswaldoi and Anopheles rangeli (Diptera: Culicidae)

In the Department of Putumayo in southern Colombia, malaria transmission has continued in the absence of the 4 traditional Latin American vector species--Anopheles darlingi, Anopheles nuneztovari, Anopheles albimanus or Anopheles trinkae. Human bait collections yielded Anopheles mosquitoes and a morphological variant of Anopheles benarrochi, the adult females of which can easily be misidentified as Anopheles oswaldoi. Species identification of females of Anopheles in the subgenus Nyssorhynchus is generally difficult due to overlapping morphological characters; therefore, progeny of field collected females were link-reared to assess species identity. Herein a robust method is presented to identify the species Anopheles benarrochi, Anopheles oswaldoi and Anopheles rangeli from southern Colombia, using the morphology of the eggs induced from wild-caught females. Eggs of A. rangeli and A. benarrochi were differentiated on the basis of the anterior crown. In A. rangeli, this feature is positioned apically with high walls. In A. benarrochi, anterior crown is positioned more ventrally with comparatively shorter walls. No crown is present in A. oswaldoi. These differences are clear with the aid of a dissecting microscope and make accurate species determination possible even in field conditions. Egg morphology is shown to be an accurate, albeit indirect, method for the taxonomic determination for the three southern Colombian species and may also be useful in other regions of Latin America where the morphological variant of A. benarrochi is sympatric with A. oswaldoi.     

Variability and genetic differentiation among Anopheles (Ano.) intermedius Chagas, 1908 and Anopheles (Ano.) mattogrossensis Lutz & Neiva, 1911 (Diptera: Culicidae) from the Brazilian Amazon

Anopheles (Anopheles) intermedius and Anopheles (Ano.) mattogrossensis are Brazilian anopheline species belonging to the scarcely studied Anopheles subgenus. Few studies have been done on the genetic differentiation of these species. Both species have been found infected by Plasmodium and are sympatric with other anopheline species from the Nyssorhynchus subgenus. Eighteen enzymatic loci were analyzed in larval specimens of An. intermedius and An. mattogrossensis aiming to estimate the variability and genetic differentiation between these species. An. mattogrossensis population showed higher genetic variability (P = 44.4 and Ho = 0.081 +/- 0.031) than that of An. intermedius (P = 33.3 and Ho = 0.048 +/- 0.021). Most analyzed loci showed genotypic frequencies according to Hardy-Weinberg equilibrium, except for LAP1 and LAP2 in An. intermedius, and EST1 and PGM loci in An. mattogrossensis. The genetic distance between these species (D = 0.683) was consistent with the inter-specific values reported for Anopheles subgenus. We verified that the polymorphism and heterozygosity percentile values found in both species and compared to those in the literature, showed no relation between the level of isozyme variability and geographical distribution. The low variability found in these two species is probably more related to the niche they occupy than to their geographic distribution.     

Systematics of Anopheles lanei Galvão and Amaral, a species of the subgenus Nyssorhynchus Blanchard (Diptera: Culicidae)

Anopheles (Nyssorhynchus) lanei Galv?o and Amaral is here redescribed using morphological characteristics of adult, male and female, fourth instar larva and pupa. The larva, pupa, and male genitalia are illustrated. Diagnostic morphological characters of adults, male genitalia, fourth instar larva and pupa are provided to distinguish An. lanei from other species of the Argyritarsis section. Species distribution data are based on the published literature records and bionomics data are based on both literature records and field data.     

Intragenomic rDNA ITS2 variation in the neotropical Anopheles (Nyssorhynchus) albitarsis complex (Diptera: Culicidae)

We cloned and sequenced the rDNA internal transcribed spacer 2 (ITS2) of 4 species belonging to the neotropical Anopheles (Nyssorhynchus) albitarsis complex, that is, A. albitarsis; A. albitarsis B; Anopheles marajoara, a proven malaria vector; and Anopheles deaneorum, a suspected vector. Even though the ITS2 sequences of these species were very similar (< or =1.17% divergence), we found differences suitable for species identification and intragenomic variation of possible consequence in phylogenetic reconstruction. Variation came from 2 microsatellite regions and a number of indels and base substitutions. The existence of partially correlated subsets of clones in A. albitarsis is hypothesized either to be separate rDNA loci or to be semi-independently evolving portions of a single rDNA locus. No differences were found between males and females, suggesting that similar rDNA arrays exist on both the X and Y chromosomes. In addition, highly variant clones, possibly pseudogenes, were found in A. marajoara from Venezuela.