Identification keys to the Anopheles mosquitoes of South America (Diptera: Culicidae). I. Introduction.

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

Malaria continues to be a serious public health problem. In 2016, there were an estimated 216 million cases of malaria in 91 countries, an increase of 5 million cases over 2015. Malaria deaths reached 445,000 in 2016, a similar number (446,000) to 2015 (http://www.who.int/news-room/fact-sheets/detail/malaria) [1]. In 2017, there were an estimated 219 million cases of malaria worldwide, mostly in developing countries, resulting in an estimated 435,000 deaths of which 266,000 deaths were children under 5 years of age [2].

In Central and South America, malaria transmission occurs primarily at altitudes of less than 1000 meters above sea level. It is not surprising therefore that the incidence of malaria is highest in lowland countries, such as those situated in the Amazon basin: Brazil, Colombia, Venezuela, Ecuador, Peru, and the Guianas. Brazil, Colombia, Peru, and Venezuela contributed approximately 82% of the 875,000 malaria cases reported in the region in 2016: Brazil (18.0%); Colombia (15.3%); Peru (14.3%) and Venezuela (34.4%) [1]. In 2017, an increase in malaria cases was reported in Brazil, Ecuador, Mexico, Nicaragua, and Venezuela [2].

Variability in the epidemiological components of malaria transmission (host(s), parasites, environment) varies widely by locality, with differences in physiography, regional and local ecological characteristics, vector competence and vectorial capacity of individual species of Anopheles Meigen, 1818. Mosquito vectors can differ in physiology, behavior, and ecology, as well as in morphological characters used for their identification. These differences taken together can facilitate the definition of species and are the source of characteristics for species identification. The most accessible method of identification is by morphological characteristics. However, if basic (alpha) morphological taxonomy is incomplete, it is possible to make incorrect identifications of morphologically similar species of very different public health importance. This is especially the case for species complexes that include both vector and non-vector species. The definition and delineation of morphologically similar species often requires auxiliary tools, for example molecular markers, which include DNA sequences. DNA not only has given us evolutionary insights but also many new characters for the recognition and identification of species. However, molecular methods are not available in many malaria-endemic areas in South America. Recently, multiple studies (e.g. [3-9]) have reported satisfactory resolution of taxonomic problems using a combination of molecular markers, and/or morphological characters from all developmental stages. These studies have led to description or re-description of multiple species, and the recognition of complexes of morphologically similar species, which are summarized here.

In spite of technological and analytical advances, the identification of mosquito species using external morphological characters is still a preferred method since microscopes are easy to use, relatively inexpensive and can also be employed to study live specimens for ecological studies, such as capture-mark-release-recapture. Even though there are many keys addressing morphological identification of Neotropical Anopheles, they are limited in geographical or taxonomic scope and/or are outdated. For this reason, we present comprehensive keys (based on the morphology of male genitalia, females, and fourth-instar larvae) to all Anopheles species recorded in South America.

Mosquitoes (family Culicidae Meigen, 1818), belong to order Diptera (true flies, i.e. insects with two wings), and like many other insect orders (Coleoptera, Hymenoptera, etc.) they are holometabolous, meaning they have distinctly different egg, larval, pupal and adult stages. The culicids are recognized as a monophyletic group (with a single common ancestor) [10-12], that diverged from its nearest relative, family Chaoboridae Edwards, 1912, about 255 million years ago (mya) [13]. The two culicid subfamilies, Anophelinae Grassi, 1900 and Culicinae Meigen, 1818, diverged 229–192 mya [14].

Adult mosquitoes (Fig. 1) can be distinguished [15] by: scales on wing veins and usually also on the head, legs, thorax and abdomen; proboscis long, extending well beyond the clypeus; characteristic wing venation (also found, however, in families Dixidae Schiffner, 1868 and Chaoboridae) [16] (i.e. subcostal (Sc) vein ending near or beyond midpoint of the costal vein (C), Sc and vein R1 both reach C in the apical half of the wing in front of R2 and R3, and vein M three-branched); antennal pedicel prominent and nearly always larger than the scape. Mosquito (and chaoborid) larvae [16, 17] have the three thoracic segments fused into a rounded composite structure that is wider than the head or abdomen; abdominal segment X with a fan-like ventral brush; lateral tufts of long setae on most thoracic and abdominal segments; labrum with a distinctive brush of long setae on either side (Culicidae only); and antenna moderately long, usually with a number of apical setae (Culicidae only).

Fig. 1

Female anopheline mosquito, lateral view. Abbreviations: Ap, antepronotum; C-I, forecoxa; C-II, midcoxa; C-III, hindcoxa; Fe-I, forefemur; Fe-II, midfemur; Fe-III, hindfemur; Hl, halter; La, labellum; Mks, mesokatepisternum; Mm, mesepimeron; MPlp1–5, maxillary palpus, segments 1–5; Mpn, mesopostnotum; MS, mesothoracic spiracle; Mts, metepisternum; P, proboscis; Pa, paratergite; PA, postspiracular area; Ppn, postpronotum; Ps, proepisternum; S-I-VIII, sterna I-VIII; Scu, scutum; Stm, scutellum; Ta-III1–5, hindtarsomeres 1–5; Te-I-VIII, terga I-VIII; Ti-III, hindtibia; Tr-I, foretrochanter; Tr-II, midtrochanter; Tr-III, hindtrochanter

While the above characters serve for identification, another overlapping set of characters further characterize the family and can be used for phylogenetic analyses [18]. According to [10], the synapomorphies of Culicidae are internal premandibles in the larvae, apparently without residual external sclerites, and a long proboscis, with stilettes corresponding to the maxillae, mandibles and the labrum-hypopharynx, all encased in the labium [16]. In a study using characters of the adults and fourth-instar larvae, Harbach et al. [12] defined three synapomorphies for adult Culicidae: presence of erect scales on the head, dorsally; mouthparts forming a long proboscis; and the presence of prealar setae. In the larval stage, mosquitoes can be identified by: a well-sclerotized head that is clearly separated from the thorax; legless thoracic segments that are little differentiated from each other; and an abdomen made up of 10 segments, but with only nine apparent. The larvae are metapneustic (single spiracle in the post-abdominal region) with a spiracular plate that opens at the end of a siphon or on a small dorsal lobe that has a narrow, sclerotized posterior band in the dorsal region of abdominal segment VIII [12], and frequently with two pairs of anal papillae inserted at the end of segment X.

The Culicidae, and related families (Dixidae, Chaoboridae and Corethrellidae Edwards, 1932), pass through four larval instars (first-, second-, third- and fourth-instar) before developing into the pupal stage. The pupal and larval stages have serially (segmentally) homologous setae that have also been shown to be homologous between the larval and pupal stages (e.g. [19-21]). A common numbering system for these setae makes comparison of taxa straightforward.

Currently, the family Culicidae includes nearly 3600 valid species and a number of subspecies, which would add up well over 3600 [22] within two subfamilies (Anophelinae and Culicinae). Subfamily Anophelinae (genera Anopheles, Chagasia Cruz, 1906 and Bironella Theobald, 1905) contains about 500 described species. Most species of the Anophelinae can be distinguished in the adult stage from those of the subfamily Culicinae by the female maxillary palpus about as long as the proboscis, the male palpus also long and usually clubbed apically, a rounded (not tri-lobed) scutellum (except genus Chagasia), and the characteristic elevated angle of the abdomen at rest or when feeding. The larvae have no respiratory siphon and characteristically lie parallel to the water surface, although some other mosquitoes, such as genus Uranotaenia Lynch Arribálzaga, 1891, have a very short siphon and can be mistaken for Anopheles at first glance. The larvae of most species of Anopheles have at least some abdominal seta 1 broadened and leaf-like, which allows the larva to take advantage of surface tension to remain parallel to the surface.

The internal classification and phylogeny of the genus Anopheles has been reported by Harbach & Kitching [23], Harbach [24], Sallum et al. [25, 26], Foster et al. (2017) [27]. Worldwide there are three genera in the subfamily Anophelinae; Anopheles, Bironella and Chagasia (but see [27]). Most species belong to genus Anopheles while Bironella (Australasian) and Chagasia (Neotropical) have eight and five species respectively, which are not of medical importance. Genus Anopheles has seven subgenera (but see [27] and [28]): Anopheles (183 species); Baimaia Harbach, Rattanarithikul & Harrison, 2005 (1 species); Cellia Theobald, 1902 (224 species); Christya Theobald, 1903 (2 species); Kerteszia Theobald, 1905 (12 species); Lophopodomyia Antunes, 1937 (6 species); Nyssorhynchus Blanchard, 1902 (40 species); Stethomyia Theobald, 1902 (5 species) (Table 1), and approximately 13 recently discovered new species of the subgenera Anopheles and Nyssorhynchus [29]. Species of all subgenera except for Baimaia and Cellia occur in South America, and species of the subgenera Kerteszia, Lophopodomyia, Nyssorhynchus and Stethomyia are only found in the Neotropics. The largest cosmopolitan genera are Anopheles and Cellia. From a malaria transmission standpoint, a relatively small number of species of subgenus Cellia (i.e. the Gambiae Complex) are responsible for much of world’s malaria transmission [8]. The subgenera are further subdivided into informal morphologically similar groupings, usually for convenience, but which often lack phylogenetic significance [24].

Table 1

Valid species of the genus Anopheles of the subgenera Anopheles, Kerteszia, Lophopodomyia, and Stethomyia found in South America, grouped by subgenus and series

Subgenus/Series	Species, authorship, date
Anopheles Meigen, 1818 Series Arribalzagia	anchietai Corrêa & Ramalho, 1968a
	annulipalpis Lynch Arribálzaga, 1878
	apicimacula Dyar & Knab, 1906
	bustamantei Galvão, 1955
	calderoni Wilkerson, 1991b
	costai da Fonseca & da Silva Ramos, 1940a
	evandroi da Costa Lima, 1937
	fluminensis Root, 1927a
	forattinii Wilkerson & Sallum, 1999a
	guarao Anduze & Capdevielle, 1949
	maculipes (Theobald, 1903)a
	malefactor Dyar & Knab, 1907
	mattogrossensis Lutz & Neiva, 1911a
	medialis Harbach, 2018 (new name for An. intermedius Chagas, 1908)a
	mediopunctatus (Lutz, 1903)a
	minor da Costa Lima, 1929a
	neomaculipalpus Curry, 1931b
	peryassui Dyar & Knab, 1908a
	pseudomaculipes (Chagas in Peryassú, 1908)
	punctimacula Dyar & Knab, 1906
	rachoui Galvão, 1952
	shannoni Davis, 1931
	vestitipennis Dyar & Knab, 1906
Series Anopheles	eiseni eiseni Coquillett, 1902
	eiseni geometricus Corrêa, 1944a
	pseudopunctipennis levicastilloi Levi Castillo, 1944
	pseudopunctipennis neghmei Mann, 1950
	pseudopunctipennis noei Mann, 1950
	pseudopunctipennis patersoni Alvarado & Heredia, 1947
	pseudopunctipennis pseudopunctipennis Theobald, 1901b
	pseudopunctipennis rivadeneirai Levi Castillo, 1945
	tibiamaculatus (Neiva, 1906)a
Kerteszia Theobald, 1905	auyantepuiensis Harbach & Navarro, 1996
	bambusicolus Komp, 1937a
	bellator Dyar & Knab, 1906a
	boliviensis (Theobald, 1905)
	cruzii Dyar & Knab, 1908a
	gonzalezrinconesi Cova-García, Pulido F. & Escalante de Ugueto, 1977
	homunculus Komp, 1937a
	laneanus Corrêa & Cerqueira, 1944a
	lepidotus Zavortink, 1973
	neivai Howard, Dyar & Knab, 1913b
	pholidotus Zavortink, 1973b
	rollai Cova-García, Pulido F. & Escalante de Ugueto, 1977a
Lophopodomyia Antunes, 1937	gilesi (Neiva in Peryassú, 1908)a
	gomezdelatorrei Leví-Castillo, 1955a
	oiketorakras Osorno-Mesa, 1947a
	pseudotibiamaculatus Galvão & Barretto, 1941a
	squamifemur Antunes, 1937a
	vargasi Gabaldón, Cova García & López, 1941a
Stethomyia Theobald, 1902	acanthotorynus Komp, 1937a
	canorii Floch & Abonnenc, 1945
	kompi Edwards, 1930a
	nimbus (Theobald, 1902)a
	thomasi Shannon, 1933a

Note: Sources of specimens used to photograph fourth-instar larvae and male genitalia are denoted as: a FSP-USP, Brazil; b MUSNUVE, Colombia

Recently, Foster et al. [27] proposed that the Neotropical subgenera of Anopheles should be elevated to genus rank, see [28] for discussion of these changes, which were proposed subsequent to the writing of our keys. If these subgenera are considered by future authors to be genera, agreement of genus-species gender and inclusion or deletion of parentheses around author names as appropriate will be needed. For example, An. (Ker.) boliviensis (Theobald, 1905) would be Kerteszia boliviensis Theobald since it was originally described in genus Kerteszia.

In South America, the genus Anopheles has approximately 86 formally named species, with many yet to be named. Some Anopheles are associated with undisturbed forested areas while others are more abundant in areas that have been severely altered ecologically as a result of human activities, such as farming and logging. In the Neotropics, the genus Anopheles consists mostly of subgenera not found elsewhere in the world (see Table 1 for species, authors, and publication dates). Even the widespread subgenus Anopheles is comprised mostly of a group of species unique to the region (Series Arribalzagia). Phylogenetic studies have shown that the genus Chagasia, the earliest extant branch in the subfamily Anophelinae, is found only in Latin America. This has led to the quite plausible assertion that genus Anopheles originally evolved in this region of the world [24].

Subgenus Anopheles

Subgenus Anopheles (with 183 species) is represented by 29 species in the Neotropics and an additional number of putative species, which were newly discovered by Bourke et al. [29]. Most belong to Arribalzagia Series (23 species), a group only found in Central and South America. Most of the species of the series are forest mosquitoes, found in swamps or slow-moving streams. Anopheles (Anopheles) pseudopunctipennis Theobald, 1901 (Anopheles Series, Pseudopunctipennis Group) is widespread, found in freshwater sometimes at higher elevations, usually in association with the green alga Spyrogyra Nees, 1820. Some species in this subgenus are important in malaria transmission, e.g. An. (Anopheles) calderoni Wilkerson, 1991, An. (Anopheles) fluminensis Root, 1927, An. (Anopheles) pseudopunctipennis, An. (Anopheles) punctimacula Dyar & Knab, 1906, and An. (Anopheles) vestitipennis Dyar & Knab, 1906.

Subgenus Kerteszia

Larvae of the subgenus Kerteszia (12 species) almost exclusively utilize water collections in species of the mostly Neotropical plant family Bromeliaceae (bromeliads). They are most similar to larvae of the subgenus Nyssorhynchus but can be distinguished by a number of morphological characters [30], including differing setae on the gonocoxites [24]. Some species of Kerteszia have historically been quite important malaria vectors (e.g. An. bellator Dyar & Knab, 1906, An. cruzii Dyar & Knab, 1908, An. homunculus Komp, 1937, An. neivai Howard, Dyar & Knab, 1913, and An. pholidotus Zavortink, 1973). Logging activities diminish larval habitats, as many bromeliads are only found in trees. Anopheles bambusicolus Komp, 1937, as its name might imply, are the only Kerteszia spp. not to occupy bromeliads, but instead are found in unbroken bamboo internodes. One of us (RW) also found larvae in Amazonian nut pods in Peru (unpublished observation), and in a discarded tire in Brazil [31].

Subgenus Lophopodomyia

Larvae of the subgenus Lophopodomyia (six species) are found in forested, shaded habitats in small slow-moving streams. They are similar to larvae of the subgenus Anopheles, but can be distinguished by characteristics of the male genitalia [24], long setae on the prothoracic and mesothoracic pleural groups (P,M-9-12), well-developed palmate setae, and fringed pupal paddles. Females are not known to be of medical importance but can blood-feed on humans when they enter the forest environment [32].

Subgenus Nyssorhynchus

Larvae of the subgenus Nyssorhynchus (40 species with numerous cryptic unnamed species) are found in a large variety of open or partly shady areas [32]. Species included in the Nyssorhynchus are most closely related to species of the subgenus Kerteszia but can be distinguished by several morphological characters [30]. The subgenus contains many important malaria vectors including An. albimanus Wiedemann, 1820 (malaria vector in Central America and northern South America), species of the An. albitarsis Lynch Arribálzaga, 1878 complex (9 species), An. aquasalis Curry, 1932 (found in brackish waters), An. darlingi Root, 1926 (commonly associated with rivers and streams), An. nuneztovari Gabaldon, 1940, An. triannulatus (Neiva & Pinto, 1922), and many comprise species complexes.

Subgenus Stethomyia

Larvae of the subgenus Stethomyia (5 species) are, as in Lophopodomyia, forest species found in well-shaded small streams and swampy areas. Adults are mostly dark-colored but have a characteristic silvery stripe on the scutum. The setae of the gonocoxite are distinctive [24]. In the larva, head seta 2-C are widely separated, reminiscent of subgenus Cellia, thoracic setae P,M,T-9–12 have thorn-like branches, and the abdomen lacks distinct palmate setae. The species are not known to be of medical importance, but females will bite humans [32].

Distribution of Neotropical Anopheles spp.

The distributions of the species treated here are summarized in Table 2. Location should be considered as one of the most important “characters” used for identification. For example, if a species is only known from east of the Andes mountains but is reported from the Pacific side of the cordillera, it should be flagged as a possible misidentification or perhaps evidence of cryptic diversity. Likewise, species reported well outside known recorded ranges should be carefully evaluated. The combination of species found in a given country is usually unique since ecologies are heterogeneous among countries (or states, provinces, etc.). In fact, most identifiers will first ask the question “Where was it collected?” before selecting a key.

Table 2

Distributions of the Anopheles species, sorted by subgenus, based on data contained in the on-line catalog of the Walter Reed Biosystematics Unit (http://www.mosquitocatalog.org/) and more recent publications [65–81]

Species	AR	BO	BR	CL	CO	EC	GF	GY	PY	PE	SR	UY	VE
Stethomyia
acanthotorynus							×			×
canorii							×
kompi			×		×		×			×	×		×
nimbus		×	×		×		×	×		×			×
thomasi			×		×					×			×
Anopheles
anchietai	×		×
annulipalpis	×											×
apicimacula (s.l.)	×	×			×	×		×			×		×
bustamantei			×
calderoni					×	×				×			×
costai (s.l.)		×	×		×		×			×	×		×
eiseni eiseni		×	×		×	×	×	×		×	×		×
eiseni geometricus			×
evandroi	×		×
fluminensis (s.l.)	×	×	×		×	×			×	×			×
forattinii			×		×	×	×			×			×
guarao													×
maculipes	×	×	×				×		×			×
malefactor					×	×							×
mattogrossensis		×	×		×	×				×			×
medialis	×	×	×		×	×	×	×	×	×	×		×
mediopunctatus	×		×		×		×	×		×	×
minor	×		×				×		×		×
neomaculipalpus	×	×	×		×	×			×	×			×
peryassui		×	×		×		×	×		×	×		×
pseudomaculipes			×		×					×		×
pseudopunctipennis	×	×	×	×	×	×	×	×	×	×	×		×
levicastilloi
p. neghmei				×
p. noei				×
p. patersoni	×
p. pseudopunctipennis	×	×	×	×	×	×	×	×	×	×	×		×
p. rivadeneirai						×
punctimacula	×	×	×		×	×		×				×	×
rachoui			×
shannoni		×	×		×	×		×		×	×		×
tibiamaculatus	×	×	×							×
vestitipennis					×			×	×
Lophopodomyia
gilesi		×	×		×	×		×					×
gomezdelatorrei						×
oiketorakras					×
pseudotibiamaculatus			×
squamifemur		×	×		×	×	×			×			×
vargasi													×
Nyssorhynchus
albertoi			×
albimanus			×		×	×				×	×		×
albitarsis (s.s.)	×	×	×		×				×			×	×
albitarsis F					×								×
albitarsis G			×
albitarsis H			×
albitarsis I			×		×								×
antunesi	×		×		×		×					×
aquasalis	×		×		×	×	×	×	×		×	×	×
argyritarsis	×	×	×		×	×	×	×	×	×		×	×
arthuri (s.s.)			×
arthuri B			×
arthuri C			×
arthuri D			×
atacamensis				×
benarrochi		×	×		×		×	×	×			×	×
benarrochi B					×					×
braziliensis	×	×	×		×		×	×		×	×		×
darlingi	×	×	×		×	×	×	×	×	×	×	×	×
deaneorum	×	×	×						×
dunhami			×		×					×
evansae (s.l.)	×	×	×		×	×			×	×		×	×
galvaoi	×		×							×
goeldii			×								×
guarani	×		×
halophylus			×
ininii	×	×	×				×	×	×
janconnae			×										×
konderi (s.l.)		×	×							×
lanei			×
lutzii (s.l.)	×	×	×						×	×
marajoara		×	×
nigritarsis	×		×
nuneztovari (s.l.)		×	×		×	×	×	×		×	×		×
oryzalimnetes			×						×
oswaldoi (s.s.)			×
oswaldoi A			×		×
oswaldoi B					×	×				×
oswaldoi (s.l.)	×	×	×		×	×	×	×	×	×	×	×	×
parvus	×	×	×	×	×				×	×			×
pictipennis	×		×	×
pristinus			×
rangeli		×	×		×	×				×			×
rondoni	×	×	×						×				×
sanctielii							×
sawyeri			×
striatus			×
strodei (s.l.)	×	×	×		×	×	×	×	×	×	×		×
triannulatus (s.s.)	×	×	×		×	×	×	×	×	×	×		×
triannulatus C			×
trinkae		×			×	×				×
Kerteszia
auyantepuiensis													×
bambusicolus	×	×	×		×	×	×	×		×	×		×
bellator (s.l.)			×					×			×		×
boliviensis		×	×		×	×	×	×	×	×	×		×
cruzii (s.l.)	×	×	×		×	×	×	×		×	×		×
gonzalerinconesi													×
homunculus (s.l.)		×	×		×		×	×		×			×
laneanus	×	×	×							×
lepidotus		×			×	×				×
neivai (s.l.)		×	×		×	×	×	×		×	×		×
pholidotus		×			×	×				×			×
rollai													×

Abbreviations: AR, Argentina; BO, Bolivia; BR, Brazil; CL, Chile; CO, Colombia; EC, Ecuador; GF, French Guiana; GY, Guyana; PY, Paraguay; PE, Peru; SR, Suriname; UY, Uruguay; VE, Venezuela

Methods

Illustrations for keys are photomicrographs or drawings. Photomicrographs were taken using a digital Canon Eos T3i, attached to a light Diaplan Leitz microscope, using the program Helicon Focus, which was used to build single in-focus images by stacking multiple images of the same structure. Photomicrographs of the male genitalia were taken from specimens from FSP-USP and MUSENUV. For few species, illustrations were reproduced from published literature. Keys are presented in Part II for the fourth-instar larvae [33], Part III for the male genitalia [34], and Part IV for the adult females [35].

Results and discussion

As a basis for these keys, the primary types (holotypes and paratypes) and other specimens deposited in the Coleção Entomológica de Referência, Faculdade de Saúde Pública, Universidade de São Paulo, São Paulo, Brazil (FSP-USP) [36-38], Museo de Entomología, Universidad del Valle, Santiago de Cali, Colombia (MUSENUV) and the US National Mosquito Collection, Smithsonian Institution, Washington, DC, USA (USNMC), and original descriptions, keys, summaries and revisions from the published literature were examined. Primary publications for identification of Neotropical Anopheles include: key to genus Anopheles based on the morphology of male genitalia (Amazonian Brazil) [39]; keys to genus Anopheles based on the morphology of male genitalia, females and larvae (Venezuela) [40-44]; keys to Anopheles females and larvae (Brazil and other countries of South America) [45, 46]; key to Anopheles subgenus Nyssorhynchus Blanchard, 1902 females (western Venezuela) [47]; keys to Anopheles subgenus Nyssorhynchus based on the morphology of male genitalia, females and larvae (Amazonian Region) [48]; revision of all stages of Anopheles subgenus Nyssorhynchus, Albimanus Section [49]; genus Anopheles, comprehensive (North and South America) [32, 50]; keys to genus Anopheles females and larvae (Colombia) [51]; keys to genus Anopheles females and larvae (Venezuela, eastern and western South America, Central America and Panama) [52-55]; key to Anopheles subgenus Kerteszia Theobald, 1905 females [56]; keys to genus Anopheles male genitalia, larvae and eggs (Neotropics) [57]; revision of all stages of Anopheles subgenus Nyssorhynchus, Argyritarsis Section [58]; key to larvae (Venezuela) [59]; summary of Anopheles subgenus Nyssorhynchus, with definition of the Myzorhynchella Series [30]; key to females of Anopheles subgenus Nyssorhynchus (Venezuela) [60]; key to Anopheles females (Central America) [61]; key to Anopheles females (in Spanish) (Central America) [62]; and, revision of Anopheles subgenus Kerteszia [63]. From these available published literature records, we initiated the keys using [32, 49, 50, 56, 63] and [61].

Based on the published literature and specimens of additional species deposited in the previously mentioned collections [58-60], we present a list of species included in the identification keys. Table 1 shows the traditional classification of the genus Anopheles, including formal and informal groups, species authorship, and the date of publication.

The subgenus Nyssorhynchus includes the Albimanus [49], Argyritarsis [58], and Myzorhynchella Sections [30, 64]. The Albimanus and Argyritarsis Sections further include series, groups, subgroups, and species complexes [65-81]. The classification into section categories does not indicate monophyletic groups as shown in phylogenetic studies using morphology [25] and sequence data [26, 27]. Currently, the internal classification of Nyssorhynchus is primarily based on morphological similarities of the adults, males, and females, and it is herein adopted for convenience (Table 3). Species complex, such as An. arthuri Unti, 1941, includes additional three phylogenetic taxa, which were found in studies using mitochondrial and nuclear genes [69, 81]. Despite these taxa have not been formally described, they are included in Table 2, and details for species identification are provided in Sallum et al. [33, 34].

Table 3

Valid species of the subgenus Nyssorhynchus of Anopheles found in South America, grouped by series and informal groupings

Series	Group	Subgroup	Complex	Species, authorship, date
Albimanus [20]				albimanus Wiedemann, 1820b
Oswaldoi [20]	Oswaldoi [20]	Oswaldoi [20]		aquasalis Curry, 1932a
				evansae (Brèthes, 1926)a
				galvaoi Causey, Deane & Deane, 1943a
				ininii Senevet & Abonnenc, 1938
				konderi Galvão & Damasceno, 1942 (s.s.)a
				oswaldoi (Peryassú, 1922) (s.s.)a
				rangeli Gabaldon, Cova Garcia & López, 1940a
				sanctielii Senevet & Abonnenc, 1938
				trinkae Faran, 1979
			Nuneztovari [65, 66]	dunhami Causey, 1945a
				goeldii Rozeboom & Gabaldon, 1941a
				nuneztovari Gabaldon, 1940 (s.s.)a
		Strodei [20]		albertoi Unti, 1941a
				arthuri Unti, 1941 (s.s.)a
				rondoni (Neiva & Pinto, 1922)a
				striatus Sant’Ana & Sallum, 2016a
				strodei Root, 1926a
			Benarrochi [67]	benarrochi Gabaldon, Cova Garcia & López, 1941 (s.s.)a
		Triannulatus [20]		halophylus Silva-do-Nascimento & Lourenço-de-Oliveira, 2002a
				triannulatus (Neiva & Pinto, 1922) (s.s.)a
Albitarsis [30]	Albitarsis [30]		Albitarsis [68]	albitarsis Lynch Arribálzaga, 1878 (s.s.)a
				deaneorum Rosa-Freitas, 1989a
				janconnae Wilkerson & Sallum, 2009a
				marajoara Galvão & Damasceno, 1942a
				oryzalimnetes Wilkerson & Motoki, 2009a
	Braziliensis [30]			braziliensis (Chagas, 1907)a
Argyritarsis	Argyritarsis [30]			argyritarsis Robineau-Desvoidy, 1827a
				sawyeri Causey, Deane, Deane & Sampaio, 1943a
	Darlingi [30]			darlingi Root, 1926a
	Lanei [30]			lanei Galvão & Franco do Amaral, 1938a
	Pictipennis [30]			atacamensis González & Sallum, 2010a
				pictipennis (Philippi, 1865)a
Myzorhynchella				antunesi Galvão & Franco do Amaral, 1940a
				guarani Shannon, 1928a
				lutzii Cruz, 1901 (s.s.)
				nigritarsis (Chagas, 1907)a
				parvus (Chagas, 1907)a
				pristinus Nagaki & Sallum, 2010a

Note: Sources of fourth-instar larvae and male genitalia photographed for the illustrations are denoted as: a FSP-USP, Brazil; b MUSNUVE

Conclusion

Taxonomic information and identification keys for species of South American Anopheles were updated and revealed the need for further morphology-based studies and descriptions of species of several complexes, species which have been defined on the basis of DNA sequence data but have not been formally named.