Identification key to the Anopheles mosquitoes of South America (Diptera: Culicidae). III. Male genitalia

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
General introductory comments, distributions and species authors and publication dates are given in Part I [1] of this series of four articles. Keys to the fourth-instar larvae and adult females are provided in Parts II [2] and IV [3], respectively. A list of species treated here is included in Part I [1].

Methods
The primary types (holotypes and paratypes) and other field-collected specimens deposited in the Coleção Entomológica de Referência, Faculdade de Saúde Pública, Universidade de São Paulo, São Paulo, Brazil (FSP-USP), Museo de Entomología, Universidad del Valle, Santiago de Cali, Colombia (MUSENUV) and the US National Mosquito Collection, Smithsonian Institution, Washington, DC, USA (USNMC) were examined to discover characters to be used in the male genitalia key. For species that we could not access, illustrations were based on published illustrations. Photomicrographs of relevant characters for the male genitalia were taken using a digital Canon Eos T3i (Canon, USA), attached to a Diaplan Leitz microscope, using the program Helicon Focus software (https ://www.helic onsof t.com/helic onsof t-produ cts/helic on-focus /), which was used to build single infocus images by stacking multiple images of the same structure. Photomicrographs were further processed in Adobe Photoshop (https ://www.photo shop.com/en) to embed names and labels. The institutional sources of specimens are recorded on each photograph. The nomenclature adopted is that of Harbach & Knight [4,5]. The water marks embedded in photomicrographs of the male genitalia show the institution where the vouchers are deposited, Universidade de Sao Paulo (USP) and Universidad del Valle.
The key includes most of the species registered in South America, except for 15 species that are poorly known. They are the following: Anopheles (Ano. For these species, it will be necessary to conduct field collections in the type-localities and further taxonomic investigations.

Glossary of morphological terms
The terminology of the male genitalia used in this key follows that of Harbach & Knight [4,5]. Also known as the male terminalia, Harbach & Knight [4] recommended instead to use "the genitalia" to avoid confusion with other terminal structures. The composite male genitalia are structures formed from elements of the posterior segments IX and X of the abdomen. These modified structures are involved in mating, copulation, and insemination. After emergence of the adult male, the posterior part of the abdomen beyond segment VII makes a 180° rotation. Thus, the ventral segmental surfaces become dorsal in relation to the rest of the abdomen, and vice versa. In Culicidae Meigen, 1818, the male genitalia are therefore inverted in relation to the female genitalia. This means that when coupling occurs, both individuals have the same upright orientation, instead of the male ending up vulnerable, and upside down in relation to the female, as is the case in the family Tabanidae Latreille, 1802 (horse flies), which do not have male genital rotation [6]. This phenomenon must be considered when describing the position of the various elements of the genitalia.
Tergum IX, which usually varies little, can exhibit useful morphological variation in certain species. Species of the Arribalzagia Series of the subgenus Anopheles Meigen, 1818 possess ninth tergal lobes (IX-Te lobes) of variable size and development, features which can be useful for species recognition. The internal margin of tergum IX is attached to the proctiger that is formed by tergum X, the cerci, the cercal sclerites and the paraprocts. Dorsally there are two sclerotized plates called the cercal sclerites. The two structures attached laterally on the most posterior part of the abdomen are called the gonocoxopodites. They are adapted to facilitate insemination by grasping the female during copulation. The gonocoxopodites are composed of a proximal gonocoxite and a distal gonostylus.
The gonocoxite (Fig. 1) is a large, relatively long, and somewhat conical structure, much wider than the gonostylus. The external ventral surface is convex, while the internal surface is slightly concave, especially basally at the attachment of the claspettes [7]. The gonocoxites bear a large number of scales and setae, the larger of which are sometimes called spines; for purposes of this key, the term seta will be used. One or two parabasal setae are inserted on the dorsobasal portion of the gonocoxite. In species of the subgenera Nyssorhynchus Blanchard, 1902 and Kerteszia Theobald, 1905, the setae are inserted on prominent parabasal lobes that are situated dorsobasally (in a prerotational sense). The parabasal lobes are absent in Neotropical species belonging to the subgenera Anopheles, Lophopodomyia Antunes, 1937 and Stethomyia Theobald, 1902. The parabasal setae are instead inserted directly on the surface or on relatively small projections on the surface of the gonocoxite. In species of the subgenus Stethomyia, the parabasal setae are absent.
Species of the subgenera Nyssorhynchus (Fig. 2) and Kerteszia have a single dorsally directed seta that is inserted on the parabasal lobe. The Neotropical species of the subgenera Anopheles and Lophopodomyia have two parabasal setae (Fig. 3b, c). The internal seta is inserted on the ventral surface of the gonocoxite, on the distal half or near mid-length. Species of the subgenera Nyssorhynchus and Kerteszia have a pair of accessory setae inserted on the dorsal surface of the gonocoxite (Figs. 2, 3a).
The gonostylus corresponds to the stylus of the gonocoxopodite. It is a well-sclerotized structure, moveable and articulated, on or near the apex of the gonocoxite. It is somewhat thickened and curved. At its apex is a small spiniform structure called the gonostylar claw [8] (Fig. 3).
Attached to the internal surface of the gonocoxite is the claspette. This is a membranous structure, usually divided into ventral and dorsal lobes, both exhibiting great variability according to subgenus and species within the subgenera (Fig. 4). For purposes of this key, the ventral and dorsal lobes of the claspette [4] are referred to   as the ventral claspette and dorsal claspette, respectively. These are terms also used by Faran [9].
In species of the subgenera Anopheles, Lophopodomyia and Kerteszia, the dorsal claspette is divided into ventral and dorsal lobes. These lobes exhibit interspecific variability in the form of the setae, as well as the size and distribution of spicules, which makes them useful in taxonomy. In species of the subgenera Anopheles (Fig. 4), Lophopodomyia and Kerteszia, the ventral lobe is divided apically, with the distance between them being more pronounced in species of the subgenera Kerteszia. In species of the subgenus Nyssorhynchus, the ventral claspette is not subdivided, instead the two ventral claspettes are fused and the composite structure occupies a median position between the gonocoxites. The structure is rich in morphological variation and is therefore useful for species identification. In some taxa, the ventral claspette is smooth (i.e. without spicules) while in others the spicules can be short or long and variously distributed on the claspette.
The apex of the ventral claspette can be rounded, truncated or angular, and, in some species can bear apicolateral expansions that resemble lobes. The presence of these apicolateral lobes in An. Other structures that make up parts of the ventral claspette and are employed in taxonomy treatments include the preapical plate and basoventral lobes (Fig. 5). In species of the subgenera Nyssorhynchus, Kerteszia, Anopheles and Lophopodomyia, there is a short basal portion on the dorsal claspette upon which variable numbers of setae are attached dorsoventrally. These setae are variable in form, point of insertion, development, and quantity ( Fig. 6). In species of the subgenus Stethomyia, the dorsal claspette is absent and the ventral claspette is columnar, with two subdivisions that support apical setae that are variously developed [7].
The aedeagus is part of the phallosome, which includes, in addition to the aedeagus, the parameres and the basal pieces. The aedeagus is articulated basally to the parameres, which are connected to the basal pieces by an acetabulum that is on the median lateral area. The basal pieces, responsible for movement of the aedeagus during copulation, are connected to the gonocoxal apodemes. The aedeagus is the central organ of the phallosome and serves as the intromittent organ [4]. In species of Anopheles, the aedeagus is a tubular structure, dorsally curved, with the walls unequally sclerotized and with a circular opening near or at the apex. The apical part of the aedeagus is variable in form and development. In species of the subgenus Nyssorhynchus, the apical part of the aedeagus is variable, and often used in species identification. The presence of leaflets subapically on the aedeagus, as well as the number of these structures, their form, development, and presence of marginal serrations, permit identification of many species of the genus (Fig. 7).
In species of the subgenus Anopheles, the apical leaflets, when present, can vary in number, position, form, and development. Some species possess a single pair of leaflets that can have smooth or serrate margins, be uniformly or unevenly sclerotized, and be short or long. In species of the subgenera Kerteszia and Nyssorhynchus, the leaflets may be present or absent. When present, they occur as a single subapical pair.