Coetzee Et Al 2013.Pdf

Zootaxa 3619 (3): 246–274 ISSN 1175-5326 (print edition) www.mapress.com/zootaxa/ Article ZOOTAXA Copyright © 2013 Magnolia Press ISSN 1175-5334 (online edition) http://dx.doi.org/10.11646/zootaxa.3619.3.2 http://zoobank.org/urn:lsid:zoobank.org:pub:88063F32-3BAE-4B2C-B694-45D649F1FA65 Anopheles coluzzii and Anopheles amharicus, new members of the Anopheles gambiae complex

MAUREEN COETZEE1,2,7, RICHARD H. HUNT1,2, RICHARD WILKERSON3, ALESSANDRA DELLA TORRE4, MAMADOU B. COULIBALY5 & NORA J. BESANSKY6 1Malaria Entomology Research Unit, School of Pathology, Faculty of Health Sciences, University of the Witwatersrand, 7 York Ave- nue, Parktown, Johannesburg 2001, South Africa. E-mail: [email protected]; [email protected] 2Vector Control Reference Laboratory, Centre for Occupational Tropical and Hospital Infections, National Institute for Communicable Diseases of the National Health Laboratory Service, 1 Modderfontein Road, Sandringham, Johannesburg 2131, South Africa 3Department of Entomology, National Museum of Natural History, Museum Support Center, 4210 Silver Hill Rd, Suitland, Maryland, 20746, USA. E-mail: [email protected] 4Istituto Pasteur-Fondazione Cenci Bolognetti, Dipartimento di Scienze di Sanità Pubblica, Universita di Roma “La Sapienza”, Rome, Italy. E-mail: [email protected] 5Malaria Research and Training Centre, University of Bamako, Bamako, Mali. Email: [email protected] 6Eck Institute for Global Health, Department of Biological Sciences, 317 Galvin Life Sciences Bldg, University of Notre Dame, Notre Dame IN 46556, USA. E-mail: [email protected] 7Corresponding author

Abstract

Two new species within the Anopheles gambiae complex are here described and named. Based on molecular and bionomical evidence, the An. gambiae molecular "M form" is named Anopheles coluzzii Coetzee & Wilkerson sp. n., while the "S form" retains the nominotypical name Anopheles gambiae Giles. Anopheles quadriannulatus is retained for the southern African populations of this species, while the Ethiopian species is named Anopheles amharicus Hunt, Wilkerson & Coetzee sp. n., based on chromosomal, cross-mating and molecular evidence.

Key words: Subgenus Cellia, morphology, taxonomy, systematics, description

Introduction

Historically, the taxon Anopheles (Cellia) gambiae Giles was known as a major vector of malaria in Africa, exhibiting a wide range of biological attributes (de Meillon 1947; Gillies & de Meillon 1968). Ecologically, it was highly diverse, reportedly feeding indoors or outdoors on humans or cattle, resting indoors or outdoors, and its larvae were found in a wide variety of habitats including rain pools, hoof prints, rice paddies, mineral springs and saline water. In some places it did not even transmit malaria (Mastbaum 1957). Taxonomy. Work in the 1940s and 50s provided clear evidence for the species distinctness of the salt-water breeding forms in East and West Africa (Ribbands 1944; Muirhead-Thompson 1947, 1951), but this was ignored for over 15 years (see Hunt & Coetzee 1995). From 1962 to 1964, a series of papers appeared that provided genetic evidence for "freshwater" An. gambiae being a species complex (Paterson 1962, 1963a, 1964; Davidson & Jackson 1962) explaining many of the earlier conundrums that faced malariologists in those days (see Gillies & Coetzee 1987). By 1967, the complex was recognized as comprising three fresh-water and one mineral-water breeding species (designated as species A, B, C and D) and the two salt-water breeders (retaining the early names of An. merus for the East African species and An. melas for the West African species) (Davidson et al. 1967). All could be distinguished on the characteristics of inter-species hybridization resulting in sterile male progeny (Davidson et al. 1967) and, later, the banding patterns of the giant polytene chromosomes found in the larval salivary glands and the female ovaries (Coluzzi & Sabatini 1967, 1968a, 1968b, 1969). Several publications appeared proposing names for various members of the An. gambiae complex (Paterson

246 Accepted by G. Courtney: 23 Jan. 2013; published: 28 Feb. 2013 1963b, 1968; White 1975), culminating in the adoption of those proposed by Mattingly (1977) and White (1985) as follows: Anopheles gambiae s.s. Giles—(originally species A). The nominotypical member of the complex, described by Giles (1902) from specimens collected in 1900 on McCarthy Island, Georgetown, the Gambia, 150 miles inland from the coast (and therefore a fresh-water breeder, ruling out the West African saltwater breeder, An. melas). Species B was not considered due to its preference for more arid environments and the fact that Dr MT Gillies had found only species A in a survey of Georgetown at the appropriate time of the year and in a variety of habitats (Mattingly 1977). Species C and D were also not considered for the name “gambiae” due to their restricted distribution, neither of which occurs in West Africa. The following two synonyms are now listed under An. gambiae: (1) costalis Loew—(nomen dubium Gillies & de Meillon 1968, p.263), incorrectly assigned and probably a member of the Series Paramyzomyia (Mattingly 1977); (2) gracilis Dönitz—description probably published after that of gambiae Giles, no specimens extant, and at least one specimen might have been melas based on the palpal index given in the description, but no indication of how many specimens were examined. Recent collections carried out in the Gambia (Caputo et al. 2008) found one specimen of An. melas at Teneng Fara, about 20 km west of Georgetown and An. arabiensis was common at several of the sites sampled in close proximity to Georgetown. Mattingly's conclusions, however, are still reasonable given the origin of the name arabiensis. Anopheles arabiensis Patton—(originally species B). The only member of the complex found in the Aden hinterland and preferring a more arid climate compared with An. gambiae. One synonym appears under An. arabiensis: quadriannulatus davidsoni Ribeiro et al. (Ribeiro et al. 1979)—shown by cytogenetics (Cambournac et al. 1982) to be a synonym of An. arabiensis (Gillies & Coetzee 1987). Anopheles quadriannulatus Theobald—(originally species C). Described from a single specimen from Onderstepoort, South Africa, it was considered to be a highland species because of its distribution in Ethiopia (White 1985). Subsequently, however, the Ethiopian population was shown to be a separate species based on cross-mating and chromosomal studies (Hunt et al. 1998) and is formally assigned a new name below. Anopheles melas Theobald—long known as the West African saltwater breeder. Despite intermediate morphological variation of key characters, Mattingly (1977) favored the retention of the name. The name gracilis would have had precedence over melas if original specimens had been available for examination, but as they were not, the name melas has been retained for the West African saltwater breeder. Anopheles merus Dönitz—the East African saltwater breeder. This is the only member of the An. gambiae complex that could be reasonably associated with the actual type specimen (Paterson 1963b; Coluzzi 1964; Mattingly 1977). Two synonyms are associated with An. merus: (1) gambiae litoralis Halcrow, 1957; (2) tangensis Kuhlow, 1962. Anopheles bwambae White—(originally species D). Mattingly (1977) mentions this species only once where he considered it to be “entirely new and thus presents no nomenclatorial problem”. The formal description and naming of species D was left to White (1985). It is known only from the geothermal hot springs in the Semliki forest of Uganda's Bwamba county (White 1985). In addition to the above, Anopheles comorensis Brunhes, Le Goff & Geoffroy was described and named by Brunhes et al. (1997) based on the morphology of a single specimen from the Indian Ocean islands of the Comoros in the Mozambican channel. Until such time as adequate genetic confirmation of species distinctness is forthcoming, the specific status of An. comorensis and its association with the An. gambiae complex remain questionable. Cytogenetics. A series of extensive studies on the chromosomal polymorphism of members of the An. gambiae complex initiated more than 30 years ago highlighted a non-random distribution of chromosome inversions within An. gambiae, An. arabiensis and An. melas, and suggested genetic discontinuities within these species (Coluzzi et al. 1979, 2002). While genetic discontinuities within the last two species involve geographically allopatric populations, five sympatric and syntopic “chromosomal forms” (informally named Mopti, Savanna, Bamako, Forest and Bissau) were described within An. gambiae (Coluzzi et al. 1977, 1979, 1985; Toure et al. 1998). These were characterized by different inversion arrangements on the right arm of chromosome- 2 that are believed to have facilitated high ecological flexibility and more efficient exploitation of different ecological niches, through the capture and stabilization within inversions of locally adapted genes (Coluzzi et al. 1985, 2002; Ayala & Coluzzi 2005).

NAMING MEMBERS OF THE GAMBIAE COMPLEX Zootaxa 3619 (3) © 2013 Magnolia Press · 247 Molecular systematics. In the past 15 years or so, the availability of molecular tools provided further insight into the complexity of An. gambiae. Genetic differences outside of the chromosome-2 rearrangements were found in non-coding sequences of the ribosomal DNA, a multigene family located near the centromere of the X chromosome, distinguishing between the Mopti chromosomal form and Bamako/Savanna populations (Favia et al. 1994, 1997, 2001; Gentile et al. 2001, 2002; della Torre et al. 2001). Della Torre et al. (2001) first introduced the concept of molecular forms, using the terms M and S to describe assortative mating populations defined by form- specific SNPs in the rDNA IGS region, in an extensive study correlating rDNA genotypes with chromosomal karyotypes in 10 African countries, from Senegal in the West to Madagascar in the East. This study emphasized the lack of association between chromosomal constitution, which seems to be involved in ecotypic adaptation, and speciation. Although chromosomal inversions and even chromosome-2 karyotypes are shared between molecular forms, significant lack of gene flow between M and S based on the absence or rarity of hybrid rDNA genotypes (using PCR diagnostic methods of Favia et al. 1997, 2001; Fanello et al. 2002) has been demonstrated through geographically comprehensive surveys across West Africa (della Torre et al. 2005; Esnault et al. 2008) and in individual countries, e.g. Cameroon (Wondji et al. 2002; Simard et al. 2009; Lee et al. 2009; Aguilar et al. 2010), Burkina Faso (Costantini et al. 2009), Mali (Slotman et al. 2007; Manoukis et al. 2008), Ghana (Yawson et al. 2007) and The Gambia (Caputo et al. 2008). For the most part, hybridization between the M and S forms is extremely low, with frequencies of M-S hybrids below 1% (della Torre et al. 2005), clearly indicating positive assortative mating over their extensive distribution. However, molecular data suggest that incompletely reproductively isolated forms might have come into secondary contact recently in an area at the westernmost extreme of An. gambiae range (Caputo et al. 2008, 2011; Oliveira et al. 2008; Marsden et al. 2011; Weetman et al. 2012). Premating barriers exist between M and S (Diabate et al. 2009; Pennetier et al. 2010), and although these are not absolute, ecologically based divergent selection may contribute substantially to the lack of gene flow, as suggested by genome-wide M-S divergence detected by whole genome sequencing (Lawniczak et al. 2010) and SNP genotyping (Neafsey et al. 2010; Weetman et al. 2010, 2012). Most recently, Reidenbach et al. (2012) analysed 400,000 SNPs across the genomes of paired population samples of M and S from Mali, Burkina Faso and Cameroon, concluding that the two taxa are evolving collectively on independent evolutionary trajectories. Based on the above population genomic evidence that M and S are cohesive and exclusive taxonomic groups (de Queiroz 2007) across their shared range, we assign the name An. gambiae Giles to the S molecular form and An. coluzzii Coetzee & Wilkerson sp.n. to the M form. At the same time, conclusive evidence was provided by Hunt et al. (1998) for the specific distinctness of An. quadriannulatus species B from Ethiopia compared with populations from southern Africa. Cross-mating studies showed hybrid male sterility and the chromosomes of hybrid females were extensively asynapsed. The reason that the Ethiopian species was not recognized as a new species earlier was because no laboratory colony of An. quadriannulatus was available in the 1960s and 70s to carry out the crossing experiments between the two geographical forms (Davidson et al. 1967) and the chromosomal banding patterns of Species B are homosequential with An. quadriannulatus from southern Africa (Hunt et al. 1998). The Ethiopian member of the An. gambiae complex is named here as An. amharicus Hunt, Wilkerson & Coetzee sp.n.

Materials and methods

Molecular diagnostic procedures and sequences for An. coluzzii and An. gambiae (GenBank AF470093-AF470116, from Gentile et al. 2002) can be found in Favia et al. (1997, 2001), Fanello et al. (2002), Wilkins et al. (2006) and Santolamazza et al. (2008, 2011). The recommended methods for discriminating between An. gambiae and An. coluzzii are those of Fanello et al. (2002), Wilkins et al. (2006) and Santolamazza et al. (2008). See Santolamazza et al. (2011) for discussion on the sensitivity and appropriateness of the different methods. Primer sequences for differentiating An. amharicus from An. quadriannulatus can be found in Fettene et al. (2002) and Fettene & Temu (2003). Morphological descriptions are taken from Coluzzi (1964), Reid (1975), Ribeiro et al. (1979), White (1985), Coetzee (1987, 1989) and Petrarca et al. (1998). The morphological terminology follows that of Harbach & Knight (1980, 1982). Abbreviations used here for specimens are: F, female; M, male; G, genitalia; Pe, pupal exuviae; Le, larval exuviae; and Pa, pupal paddle.

248 · Zootaxa 3619 (3) © 2013 Magnolia Press COETZEE ET AL. The holotypes of both species are deposited in the National Museum of Natural History, Smithsonian Institution, Washington DC. Paratypes have been deposited in the NMNH and the Natural History Museum, London. Additional specimens of An. coluzzii and An. amharicus have been deposited in the collections of the National Institute for Communicable Diseases (previously the South African Institute for Medical Research), Johannesburg, South Africa.

Taxonomic treatment

Anopheles (Cellia) gambiae Giles

Anopheles costalis Loew, 1866 Anopheles gambiae Giles, 1902 Anopheles gracilis Dönitz, 1902 Anopheles gambiae molecular S form, della Torre et al. 2001

Anopheles (Cellia) coluzzii Coetzee & Wilkerson, sp.n.

Anopheles gambiae molecular M form, della Torre et al. 2001

Holotype female and associated pupal and larval exuviae. Essentially as in the descriptions of Anopheles gambiae given by Gillies & de Meillon (1968, pp. 208–209) and as figured by White (1985) for An. bwambae, with the following additional observations. Description. Female (Figs. 1A–F), holotype. Head: Frontal tuft of long white scales extending well beyond base of antenna; interocular space above frontal tuft with small golden appressed fusiform scales; ocular setae pale brown near vertex, dark laterally; scales of vertex erect, long slender, pale yellow anteriorly, becoming abruptly shorter, broader and dark brown laterally. Antenna about 1.2 mm; pedicel with short white fusiform scales mesally, dorsally and laterally; flagellomere 1 with distinct basomesal patch of white fusiform scales. Proboscis 1.95 mm; prementum dark with dark brown scales, scales on basal 0.25 semi-erect; labella yellowish brown. Maxillary palpus 2.4 mm, dark brown-scaled with three white-scaled bands: narrow band at apex of palpomere 2, wider band on apex of palpomere 3 and base of 4, and third band on apical half of palpomere 4 and all of 5. Palpomere lengths:

1, 0.125 mm; 2, 0.5 mm; 3, 0.8 mm; 4, 0.375 mm; 5, 0.235 mm. Palpal index (MPlp4 + MPlp5 / MPlp3) 0.75.

Subapical dark band (dark portion of MPlp4) 0.2 mm; apical pale band (apical pale of MPlp4 + MPlp5) 0.325 mm

(see White 1985). Ventral surface of palpus bare; MPlp2 with erect scales, those on basal 0.5 about twice length of apical scales. Clypeus bare. Thorax: Integument pale yellowish brown, scutum and scutellum silvery pruinose, pleura silvery pruinose with 3 irregular transverse dark areas. Anterior promontory with few pale yellow erect falcate scales, mostly appressed falcate scales continue onto anterior dorsocentral area and on to prescutellar area; scutal, fossal and supraalar areas with sparse golden yellow spatulate and fusiform scales and sparse brown setae; dorsocentral and acrostichal setae brown; scutellum with row of long brown and golden-brown setae and a few pale falcate scales. Mesopostnotum and postpronotum bare. Antepronotum with few long yellowish-brown and brown setae. Pleura with pale yellow setae on: proepisternum (1), prespiracular area (4), prealar knob (7), upper (3) and lower (5) mesokatepisternum and upper mesepimeron (5). Wing: Length 3.4 mm. Pale scales yellowish. Following costal pale spots absent: basal pale, presector pale and accessory sector pale. Preapical dark spot on vein

R1 with pale interruption on one wing but not other (i.e. proximal dark scales absent). Scales of prehumeral pale spot yellowish-brown, slightly darker than other pale spots. Halter: Scabellum and pedicel pale yellow, capitellum dark with dark brown scales. Legs: Forecoxa with about 15 dark dorsal and anterior setae and few small dark spatulate scales; mid- and hindcoxae as in forecoxa but with fewer setae and scales. Trochanters with dark brown scales and pale yellow setae. Femora and tibiae mostly dark with sparse, mostly irregularly shaped speckling. Incomplete narrow pale bands on bases of midfemur and mid- and hindtibiae. Complete narrow pale bands on: apices of all tibiae; basal 0.3 of foretarsomere 1, apices of foretarsomeres 1–3 and base of foretarsomere 4; apices of midtarsomeres 1–3 and apices of hindtarsomeres 1–4; pale band at apex of hindtarsomere 3 0.05 mm. Pale markings of foreleg more extensive than on other legs. Midtarsomere 4 and tarsomere 5 of all legs dark.

NAMING MEMBERS OF THE GAMBIAE COMPLEX Zootaxa 3619 (3) © 2013 Magnolia Press · 249 Abdomen: Very pale with golden-yellow setae dorsally, venter with golden-yellow setae changing to brown mesally, small tuft of posteromesal dark setae on sternum VII. Cerci dark brown with dark scales and setae.

FIGURE 1. Anopheles (Cellia) coluzzii sp. n., adult female (holotype, WRBU digital image prep #2127): A, habitus; B, head, lateral view showing detail of vertex and antennae; C, maxillary palpi; D, thorax, dorsal view; E, abdomen, dorsal view; F, wing.

250 · Zootaxa 3619 (3) © 2013 Magnolia Press COETZEE ET AL. FIGURE 2. Anopheles (Cellia) coluzzii sp. n., pupa (holotype exuviae, WRBU digital image prep #2124): A, trumpet showing lengths of meatus and pinna; B, segment VIII, distal, showing form of seta 9; C, paddles.

Male, allotype. Similar to holotype except for the usual sexual differences. Head: Flagellomere 1 with few pale scales, pedicel without scales. Proboscis slightly longer than in female (2.25 mm). Maxillary palpus: palpomeres 3 and 4 with intermixed dark and pale slender appressed fusiform scales, darker at base and distal 0.67 of palpomere 3 and sub-apically on 4; distal most part of 4 with discrete pale yellow band contrasting with dark band at base of 5; palpomere 5 with dark-scaled basal band and median dark band connected by dark ventral line, remainder pale yellow. Combined segments 4 and 5 (club) with basal narrow pale band, followed toward apex by dark band, pale band, median broad pale area, narrow dark band and pale apex. Thorax: Wing scales more sparse, roughly 0.67 of female. Genitalia (specimen MaliM 2.29): As figured in White (1985) for An. bwambae. Most distal parabasal seta (Harbach & Kitching 2005) gradually tapered and then more abruptly tapered to fine point; parabasal closest to distal parabasal broadened toward apex and then abruptly attenuated to fine point. Apex of gonostylus beyond subapical seta slightly flattened. Pupa (Figs. 2A–C). Positions of setae essentially as illustrated for An. bwambae (White 1985). Branch numbers for the holotype of An. coluzzii in Table 1. Counts that differ of possible diagnostic use in Table 2. Cephalothorax: Evenly lightly pigmented except for darker areas beneath trumpet and mesally on metathoracic wing. Trumpet: Slightly pigmented. Length 0.46 mm; meatus 0.23 mm; pinna 0.23 mm. Abdomen: Length 1.37 mm; evenly lightly pigmented. Seta 9-II–IV peg-like and progressively longer; seta 9-V–VII slender, short to very long, curved inward; seta 9-VIII with many branches or long aciculae. Seta 9 lengths: II, 0.012 mm; III, 0.02 mm; IV, 0.032 mm; V, 0.12 mm; VI, 0.15 mm; VII, 0.16 mm; VIII, 0.12 mm. Paddle: Length from base to seta 1-Pa 0.38 mm, width 0.28 mm. Paddle lightly pigmented; midrib well developed basally becoming weak about 0.7 from base. Marginal serrations begin about 0.23 mm from base (straight line) abruptly changing to short filaments about 0.75 from base; filaments inconspicuous beyond seta1-Pa. Larva (Figs. 3A–F). Positions of setae essentially as illustrated for An. bwambae (White 1985). Branch numbers for holotype of An. coluzzii in Table 3. Counts that differ of possible diagnostic use in Table 4. Head: Width 0.77 mm, length 0.59 mm. Evenly pigmented but ventrally with darkened area with irregular border roughly at level of anterior tentorial arms. Antenna: Length 0.27 mm, width at widest point 0.04 mm; seta 1-A inserted 0.33 from base, minute, about as long as longest antennal spicule; spicules ventral, sparse, small. Distance between setae 2-C about twice distance between setae 2-C and 3-C on one side. Seta 2-C nearly as long as antenna, about 3 times longer than seta 3-C. Seta 2-C branches could be interpreted as aciculae. Thorax and abdomen: Positions and form of setae essentially as in White (1985) for An. bwambae. Seta 1-I reduced palmate with about half number of leaflets as on following segments. Pecten plate: Pecten spines as follow; anterior spine longest, 3 short, 1 long, 2 short, 1 long, 2 short, 1 long, 1 short, 1 long, 1 short; members of each size class not necessarily of equal length (subequal). Denticles of small spines sparse and basal, or absent.

NAMING MEMBERS OF THE GAMBIAE COMPLEX Zootaxa 3619 (3) © 2013 Magnolia Press · 251 TABLE 1. Comparison of full pupal setal counts of Anopheles coluzzii sp. n. and An. amharicus sp. n. with three other species of the An. gambiae complex reported in the literature. Body region/segment; Seta An. coluzzii An. amharicus An. quadriannulatus An. bwambae An. arabiensis composite counts number (Coetzee 1987, n = (White 1985, (Ribeiro et al. 30) illustration) 1979, illustration) Cephalothorax 1 3/5 3/2 2–3 2 2 2 2/2 2/2 2–3 2 2 3 3/2 2/2 3–5 3 3 4 3/2 2/3 3–5 4 3 5 2/4 6/6 3–7 4 4 6 2/2 2/2 2–3 1 3 7 1/1 2/2 2–3 2 3 8 2/1 1/1 1–3 1? 1 9 1/2 1/1 2–3 1 2 10 2/1 2/2 1–4 1? 2 11 1/1 2/2 2–4 2 3 12 1/1 1/1 1–3 1 1

Abdomen I 1 9/9 10/12 nc 9 8 2 4/4 4/4 3–9 1 4 3 1/1 1/1 1 3 1 4 3/3 2/1 4–10 3 5 5 3/3 2/1 1–4 2 4 6 1/1 1/? 1–2 1 6 7 3/1 2/3 2–7 1? 4 9 1/1 1/1 1 1 1

Abdomen II 0 1/1 1/1 1 1? nn 1 3/4 4/4 5–7 5 6 2 5/5 6/5 5–7 8 4 3 1/1 1/1 1–3 1 1 4 3/2 2/2 4–9 4? 5 5 3/2 2/2 3 4? 2 6 2/3 1/1 1 1 1 7 1/1 4/1 4–12 2 2 8 ?/? ?/? 0–3 ? 2 9 1/1 1/1 1 1 1 10 ?/? ?/? 1–3 1? 3 11 ?/? ?/? - - 1

Abdomen III 0 1/1 1/1 1 1 nn 1 4/5 4/4 3–6 5 5 2 5/4 4/4 3–5 9? 4 3 4/2 1/1 1–4 1 1 4 1/1 2/1 4–5 3 3 5 7/7 5/4 4–7 6 5 ...... continued on the next page

252 · Zootaxa 3619 (3) © 2013 Magnolia Press COETZEE ET AL. TABLE 1. (Continued) Body region/segment; Seta An. coluzzii An. amharicus An. quadriannulatus An. bwambae An. arabiensis composite counts number (Coetzee 1987, n = (White 1985, (Ribeiro et al. 30) illustration) 1979, illustration) 6 m/1 2/2 1–5 2 1 7 1/1 1/1 4–5 2? 4 8 1/1 1/1 2–4 ? 3 9 1/1 1/1 1 1 1 10 2/1 1/2 3–4 2 3 11 1/1 1/1 1 1? 1 14 1/1 1/1 1 1 nn

Abdomen IV 0 1/1 3/3 1–2 1 nn 1 4/3 3/4 3–4 3 5 2 3/4 3/3 3–5 5 3 3 4/2 2/3 5–7 4 5 4 4/1 4/4 3–5 1? 3 5 4/3 1/1 3–6 4 5 6 m/1 2/2 1–3 1 1 7 2/1 1/3 3–5 2 2 8 1/1 1/1 2–3 2? 3 9 1/1 1/1 1 1 1 10 2/1 1/1 1–2 1 1 11 1/1 1/1 1 1 1 14 1/1 1/1 1 1 nn

Abdomen V 0 1/1 1/1 1 1 nn 1 1/1 1/m 1–3 1 1 2 4/4 3/3 3–4 4 3 3 1/1 1/1 1–3 1 1 4 3/2 2/1 4–5 3 4 5 5/5 3/4 3–4 3 4 6 1/1 1/1 1–2 1 1 7 2/2 1/1 2–4 2 3 8 1/1 1/1 1–2 ? 2 9 1/1 1/1 1 1 1 10 1/1 1/1 1 1 1 11 1/1 1/1 1 1? 1 14 1/1 1/1 1 1 nn

Abdomen VI 0 1/1 1/1 1 1 nn 1 1/1 3/3 1 1 1 2 3/3 1/1 3–4 3? 3 3 1/1 1/1 1–3 1 1 4 1/1 3/3 1–2 1 1 ...... continued on the next page

NAMING MEMBERS OF THE GAMBIAE COMPLEX Zootaxa 3619 (3) © 2013 Magnolia Press · 253 TABLE 1. (Continued) Body region/segment; Seta An. coluzzii An. amharicus An. quadriannulatus An. bwambae An. arabiensis composite counts number (Coetzee 1987, n = (White 1985, (Ribeiro et al. 30) illustration) 1979, illustration) 5 3/4 1/1 3–5 3 3 6 1/1 1/1 1–3 1 1 7 1/1 1/1 1–2 1 1 8 1/1 1/1 1–3 1? 1 9 1/1 1/1 1 1 1 10 1/1 1/1 2 2 1 11 1/1 1/1 1–2 1? 1 14 1/1 1/1 1 1 nn

Abdomen VII 0 1/1 1/1 1 1 nn 1 1/1 1/1 1 1 1 2 3/3 3/3 2–4 5? 2 3 2/2 2/2 2–4 3 2 4 1/1 1/1 1–2 1 1 5 3/4 4/4 2–4 3 4 6 1/1 1/1 1–2 1 1 7 1/1 1/1 1–2 1 1 8 3/2 1/1 4–5 3 3 9 1/1 1/1 1–2 1 1 10 4/1 1/1 2–4 1 2 11 2/3 1/1 2 2? 2 14 1/1 1/1 1 1 nn

Abdomen VIII 0 1/1 1/1 1 1? nn 4 1/1 1/1 1–3 1 2 9 12/m 16/14 9–15 13? 17 14 alv – 1 1 nn

Abdomen IX 1 1/1 1/1 - 1 nn

Paddle 1 1/1 1/1 1 1 1 2 3/2 1/1 2–4 2? 3

4-II + 5-II 10 8 - - - 1-III + 1-IV 16 14 - - - 2-I + 2-II + 2-III 27 26 - - - 2-IV + 2-V + 2-VI + 27 25 23–40 - - 2-VII 4-II + 2-V + 10-VII 18 12 - - - 4-II - 2-VII 11 11 - - - 9-VIII - 4-II 29 34 - - - Not noted = nn; Not counted = nc; Missing = m; Alveolus = alv.

254 · Zootaxa 3619 (3) © 2013 Magnolia Press COETZEE ET AL. NAMING MEMBERS OF THE GAMBIAE COMPLEX Zootaxa 3619 (3) © 2013 Magnolia Press · 255 256 · Zootaxa 3619 (3) © 2013 Magnolia Press COETZEE ET AL. FIGURE 3. Anopheles (Cellia) coluzzii sp. n. larva (holotype exuviae, WRBU digital image prep #2124): A, head, dorsal view; B, head, relative sizes and positions of setae 2-C and 3-C; C, antenna, position of seta 1-A; D, abdomen, development of palmate setae (1-I–III); E, thorax, branching of setae 1–3-P; F, pecten plate.

NAMING MEMBERS OF THE GAMBIAE COMPLEX Zootaxa 3619 (3) © 2013 Magnolia Press · 257 TABLE 3. Comparison of full larval setal counts of Anopheles coluzzii sp. n. and An. amharicus sp. n. with two other species of the An. gambiae complex reported in the literature. Seta number An. coluzzii An. amharicus An. quadriannulatus An. bwambae (White (Coetzee 1987, n = 30) 1985, illustration) Range Head 0 - - 1 nn 1 1/1 1/1 1 1 2 5/3 1/1 4–13 5 3 1/1 1/1 1–5 3 4 1/1 1/1 1–2 1 5 16/16 16/19 12–24 19 6 16/17 20/18 16–24 19 7 15/16 13/15 19–29 21 8 m/1 1/1 1–2 1 9 3/3 3/3 2–7 5 10 2/2 2/2 2–3 2 11 >20/>20 >20/>20 58–81 >20 12 1/3 3/3 2–4 3 13 4/5 4/3 3–7 4 14 5/4 3/3 10+/- 8 15 6/m 4/5 11-15 10

Prothorax 0 ?/? ?/? 1 nn 1 5/5 12/9 5–18 10 2 7/7 13/9 10–21 10 3 1/1 1/1 1 1 4 23/18 19/21 20–26 19 5 21/18 22/22 36+/- >20 6 1/1 1/1 1 1 7 29/20 m/18 24+/- 20 8 >20/>20 m/>20 43+/- >20 9 17/15 m/>20 14-19 18 10 1/1 1/m 1 1 11 3/3 3/2 2–4 2 12 1/1 m/m 1 1 13 ?/? 3/? 3–5 3 14 3/3 3/3 3–4 3

Mesothorax 1 >20/>20 >20/>20 26–40 >20 2 3/1 3/3 1–3 1 3 1/1 1/1 1 1 4 3/3 2/2 2–3 2 5 1/1 1/1 1 1 6 3/3 3/3 3–4 3 7 2/2 6/3 3–4 3 ...... continued on the next page

258 · Zootaxa 3619 (3) © 2013 Magnolia Press COETZEE ET AL. TABLE 3. (Continued) Seta number An. coluzzii An. amharicus An. quadriannulatus An. bwambae (White (Coetzee 1987, n = 30) 1985, illustration) 8 m/28 >20/>20 28+/- >20 9 1/1 1/1 1 1 10 1/1 1/1 1 1 11 1/1 1/1 1 1 12 1/1 1/1 1 1 13 5/? 6/3 6–11 3 14 11/10 12/10 11–16 11

Metathorax 1 2/3 2/2 2–4 2 2 1/1 1/1 1 1 3 2/2 2/3 1–4 4 4 3/3 4/3 3–4 2 5 >20/>20 >20/>20 49+/- >20 6 2/4 2/2 3–5 4 7 m/>20 >20/>20 44+/- >20 8 >20/>20 >20/m 42+/- >20 9 >20/>20 >20/>20 17-25 >20 10 1/1 1/? 1 1 11 1/1 1/1 1 1 12 2/2 2/m 2–3 2 13 3/3 3/3 3 3

Abdomen I 1 5/5 8/7 4–10 8 2 2/3 2/2 2–3 2 3 1/1 1/1 1 1 4 2/5 5/3 4–10 8 5 3/3 4/4 3–4 4 6 >20/>20 >20/>20 40+/- >20 7 >20/>20 >20/>20 41+/- >20 9 4/5 5/5 4-6 4 10 1/1 1/1 1 1 11 3/3 3/3 3 3 12 3/2 2/3 2–3 3 13 5/6 4/4 3–10 5

Abdomen II 0 1/1 1/1 1 1 1 10/11 14/14 8–13 13 2 4/4 4/4 4–6 2 3 1/1 1/1 1 1 4 7/6 5/9 7–12 8 5 5/5 6/6 4–7 5 ...... continued on the next page

NAMING MEMBERS OF THE GAMBIAE COMPLEX Zootaxa 3619 (3) © 2013 Magnolia Press · 259 TABLE 3. (Continued) Seta number An. coluzzii An. amharicus An. quadriannulatus An. bwambae (White (Coetzee 1987, n = 30) 1985, illustration) 6 >20/>20 >20/>20 42+/- >20 7 >20/>20 >20/>20 39+/- >20 8 2/2 2/2 2 2 9 9/8 11/8 7–15 10 10 4/4 3/2 3–6 3 11 1/1 2/1 1 1 12 1/1 1/1 1 1 13 7/7 6/6 4–12 7

Abdomen III 0 1/1 1/1 1 1 1 15/13 15/13 14–17 17 2 3/3 3/3 3–5 3 3 1/1 1/1 1 1 4 2/3 4/3 3–6 3 5 4/4 5/3 3–4 3 6 >20/>20 >20/>20 23–36 >20 7 5/3 5/3 3–7 4 8 3/2 2/2 2 2 9 7/8 8/6 7–12 10 10 2/2 1/1 1–2 1 11 1/2 2/2 3–4 2 12 2/2 1/2 2–3 2 13 3/3 3/3 3–4 3 14 1/1 1/1 1 1

Abdomen IV 0 1/1 1/1 1 1 1 14/18 16/16 15–19 18 2 1/1 1/1 1 1 3 4/? 3/3 3–5 3 4 3/3 3/3 3–5 3 5 3/3 3/3 3 3 6 m/2 2/2 2–3 2 7 6/6 5/5 6–8 4 8 3/3 2/2 2–3 2 9 5/7 6/7 5–10 7 10 1/1 1/1 1 1 11 2/3 2/3 3–4 2 12 3/3 2/2 1–3 2 13 3/3 3/3 3–4 3 14 1/1 1/1 1 1

...... continued on the next page

260 · Zootaxa 3619 (3) © 2013 Magnolia Press COETZEE ET AL. TABLE 3. (Continued) Seta number An. coluzzii An. amharicus An. quadriannulatus An. bwambae (White (Coetzee 1987, n = 30) 1985, illustration) Abdomen V 0 1/1 ?/? 1 1 1 11/15 16/16 15–19 17 2 1/1 1/3 1–2 1 3 1/1 1/1 1 1 4 3/3 3/3 3–5 3 5 3/3 2/4 3–4 3 6 2/2 3/2 2–3 2 7 2/? 3/4 5–6 4 8 3/3 2/2 2–3 3 9 5/5 8/5 5–9 7 10 1/1 1/1 1 1 11 2/3 3/2 2–4 2 12 3/3 2/3 2–4 2 13 3/3 3/3 3 3 14 1/1 1/1 1 1

Abdomen VI 0 1/1 1/1 1 1 1 11/14 16/18 14–19 17 2 3/3 3/3 2–3 3 3 1/1 1/1 1–2 1 4 1/1 1/1 1 1 5 3/4 5/5 4–6 4 6 3/4 3/3 3–4 2 7 4/? 4/3 3–4 3 8 3/? 3/3 2–3 3 9 3/? 5/7 6–9 6 10 2/? 2/2 2–3 2 11 2/? 2/2 3–4 2 12 1/1 1/1 1 1 13 11/? 11/12 9–14 10 14 1/1 1/1 1–2 1

Abdomen VII 0 1/1 1/1 1 1 1 12/13 18/16 12–15 16 2 4/3 4/6 4–6 4 3 4/3 3/4 3 3 4 1/1 2/1 1 1 5 6/4 4/5 5–7 5 6 4/? 4/3 5–6 3 7 3/? 4/3 3–4 3 8 5/? 6/5 4–7 6 ...... continued on the next page

NAMING MEMBERS OF THE GAMBIAE COMPLEX Zootaxa 3619 (3) © 2013 Magnolia Press · 261 TABLE 3. (Continued) Seta number An. coluzzii An. amharicus An. quadriannulatus An. bwambae (White (Coetzee 1987, n = 30) 1985, illustration) 9 5/? 6/4 5–9 4 10 5/? 4/5 6–9 5 11 2/? 2/2 3–4 3 12 1/? 1/1 1 1 13 3/? 3/3 9–14 3 14 - - 1–2 1

Abdomen VIII 0 1/1 1/1 1 1 1 - 1/1 2–3 2 2 - 8/5 9–14 8 3 5/? 11/13 7–12 7 4 1/1 1/1 1 1 5 6/5 3/3 3–6 5 14 - - 1 1

S1 5/4 6/5 4–8 7 S2 5/4 5/6 6–8 5 S6 2/2 2/3 2–3 2 S7 1/1 ?/? - 2 S8 4/2 2/2 3–6 4 S9 2/2 2/4 3–7 3

Abdomen X 1 1/1 1/1 1 1 2 22/22 - - 23 3 12/11 - - 12 4 6-14 - - 9pr Not noted = nn; Missing = m.

Egg. Unknown. The descriptions given by Lounibos et al. (1990) for An. gambiae are derived from a very old colony from the type locality in The Gambia (very possibly a mixture of M and S forms) and a colony from Tanzania that is almost certainly An. gambiae (S form). Comparative analysis of these two samples showed that the Gambian colony eggs were significantly longer and wider than the Tanzanian eggs. Significant differences were also seen in the mean float length, float length per rib, anterior tubercle density and mean number of lobed tubercles (Lounibos et al. 1990). Bionomics. Anopheles coluzzii is associated with longer lasting breeding sites resulting from human activity. In the savannah, these tend to be irrigated habitats such as rice fields, reservoirs and drainage ditches. In forest areas, they tend to be urban pools, sometimes polluted. Anopheles gambiae, on the other hand, prefers habitats that are more ephemeral and rain-dependent (della Torre et al. 2005; Lehmann & Diabate 2008; Kamdem et al. 2012). Distribution. The distribution of An. coluzzii extends from northern Senegal in the west to east-central Africa and south to coastal Angola, with one specimen identified from the Zambezi Valley in Zimbabwe. Anopheles gambiae shares the same distribution but extends across the continent and into Madagascar. (See Fig. 1, della Torre et al. 2005.) Etymology. This species is named in honor of the late Professor Mario Coluzzi, former Director of the Institute of Parasitology, University “La Sapienza” in Rome, Italy, who dedicated his life to the study of the Anopheles gambiae complex.

262 · Zootaxa 3619 (3) © 2013 Magnolia Press COETZEE ET AL. NAMING MEMBERS OF THE GAMBIAE COMPLEX Zootaxa 3619 (3) © 2013 Magnolia Press · 263 Type series. Anopheles coluzzii was collected by indoor resting catches from Soukourani (14°13’13.99”N, 6°03’02.83”W), Niono District, 270 km NE of the capital Bamako in Mali in July 2010 and identified as the molecular M form of An. gambiae by PCR (Fanello et al. 2002). Pinned adults and associated mounted exuviae from the progeny of a single wild female “MaliM 1” is designated as the type series. HOLOTYPE female (MaliM 1.27) with associated LePe, WRBU digital prep # 2128. Paratypes: Males (with associated LePe unless otherwise noted): MaliM 1.1–1.5 (allotype), 1.7, 1.10, 1.14 (Pe), 1.21, 1.22, 1.24, 1.26, 1.30, 1.31, 1.33, 1.34, 1.37, 1.41, 1.43(Pe), 1.47, 1.52, 1.54, 1.56, 1.57; females (with associated PeLe unless otherwise noted): MaliM 1.6, 1.8, 1.9, 1.11, 1.12 (PeLe missing), 1.13, 1.16, 1.17, 1.18 (Pe), 1.19, 1.20, 1.23, 1.25, 1.28, 1.29, 1.32, 1.35, 1.36, 1.38–1.40 (Pe), 1.42, 1.44–1.46, 1.48–1.51(1.50 no adult), 1.53, 1.55 (PeLe missing). Paratypes not mounted, stored dry: MaliM 1.60–1.69, 7 females, 2 males, mixed exuviae in ethyl alcohol. Other material examined. Progeny brood from female “MaliM 2.” Same data as for type series. Males (with associated PeLe unless otherwise noted): MaliM 2.1–2.3, 2.8 (Pe), 2.9 (Pe), 2.11–2.14, 2.17, 2.18, 2.23, 2.29 (genitalia dissected). Females (with associated PeLe unless otherwise noted): MaliM 2.4–2.7, 2.10, 2.15, 2.16, 2.19–2.22, 2.24–2.28.

Anopheles (Cellia) amharicus Hunt, Wilkerson & Coetzee, sp.n.

Anopheles quadriannulatus species B, Hunt et al. 1998.

Holotype female and associated pupal and larval exuviae. Essentially as in the description of Anopheles coluzzii above with the following additional observations. Female (Figs. 4A–E). Head: Antenna about 1.2 mm. Proboscis 2.05 mm. Maxillary palpus 1.83 mm.

Palpomere lengths: 1, 0.1 mm; 2, 0.5 mm; 3, 0.675 mm; 4, 0.35 mm; 5, 0.2 mm. Palpal index (MPlp4 + MPlp5 /

MPlp3) 0.81. Subapical dark band (dark portion of MPlp4) 0.25 mm; apical pale band (apical pale of MPlp4 +

MPlp5) 0.275 mm (see White 1985). Thorax: Pleura with pale yellow setae on proepisternum (3) prespiracular area (2), prealar knob (4), upper (2) and lower (5) mesokatepisternum and upper mesepimeron (2). Wing: Length 3.75 mm. Pale scales pale yellowish-white. Following costal pale spots absent: basal pale, accessory sector pale.

Preapical dark spot on vein R1 with pale interruption. Scales of prehumeral pale spot yellowish brown, slightly darker than other pale spots. Halter: Scabellum and pedicel pale yellow, capitellum dark with dark brown scales. Legs: Forefemur with ventral silvery-white scales and golden-yellow setae. Other than sparse speckling, femora dark. Midtibia with basal pale band; mid- and hindtibiae with apical pale bands; foretibia with apical anterodorsal pale spot. Apices of all tarsomeres with narrow pale bands; bases of foretarsomeres 2–4 with pale bands; fore- and midtarsomere 5 dark, hindtarsomere 5 yellowish. Abdomen: Very pale with golden-yellow setae dorsally, venter with golden-yellow setae changing to brown mesally, lacking small tuft of posteromesal dark setae on sternum VII. Cerci dark brown with dark scales and setae. Male, allotype. As in An. coluzzii. Proboscis slightly longer than in female (2.5 mm). Pale scales of maxillary palpus yellowish white, less extensive than in An. coluzzii (possibly rubbed). Male genitalia: As in White (1985) except subapical seta of gonostylus less distal than figured. Pupa (Figs. 5A–C). Positions of setae essentially as illustrated for An. bwambae (White 1985). Branch numbers for holotype in Table 1. Counts of possible diagnostic use in Table 2. Cephalothorax: Evenly lightly pigmented except for darker areas beneath trumpet and mesally on metathoracic wing. Trumpet: Lightly pigmented. Length 0.5 mm; meatus 0.26 mm; pinna 0.24 mm. Abdomen: Length 1.46 mm; evenly lightly pigmented. Seta 9-II–IV peg-like and progressively longer; seta 9-V–VII slender, short to very long, curved inward; seta 9-VIII with many branches or long aciculae. Seta 9 lengths: II, 0.01 mm; III, 0.02 mm; IV, 0.024 mm; V, 0.12 mm; VI, 0.17 mm; VII, 0.19 mm; VIII, 0.17 mm. Paddle: Length from base to seta 1-Pa 0.39 mm, width 0.31 mm. Paddle lightly pigmented; midrib well developed basally, becoming weak about 0.7 from base. Marginal serrations begin about 0.18 mm from base (straight line), abruptly changing to short filaments about 0.75 from base; filaments inconspicuous beyond seta 1-Pa. Larva (Figs. 6A–F). Positions of setae essentially as illustrated for An. bwambae (White 1985). Branch numbers for holotype in Table 3. Counts of possible diagnostic use in Table 4. Head: Width 0.82 mm, length 0.68 mm. As in An. coluzzii except dark ventral area not evenly pigmented, with intermixed pale areas. Antenna: Length 0.31 mm, width at widest point 0.04 mm; seta 1-A inserted 0.39 and 0.47 from base, longer than for

264 · Zootaxa 3619 (3) © 2013 Magnolia Press COETZEE ET AL. FIGURE 4. Anopheles (Cellia) amharicus sp. n., adult female (holotype, WRBU digital image prep #2128): A, habitus; B, head, dorsolateral view showing detail of vertex and antennae; C, maxillary palpi; D, thorax, dorsal view; E, wing.

NAMING MEMBERS OF THE GAMBIAE COMPLEX Zootaxa 3619 (3) © 2013 Magnolia Press · 265 An. coluzzii, about half antennal width; spicules ventral, sparse, small. Thorax and abdomen: Positions and form of setae essentially as in White (1985) for An. bwambae. Branch numbers for holotype in Table 3. Seta 1-I reduced palmate with about half number of leaflets as on following segments. Pecten plate: Pecten spines as follow: anterior spine longest, 6 short, 1 long, 3 short, 1 long, 2 short, 1 long; members of each size class not necessarily of equal length (subequal). Denticles of short spines more abundant than in An. coluzzii, some extending nearly to apices of spines. Egg. Unknown. Bionomics. Ethiopian populations are strongly associated with cattle, being found abundantly in animal shelters, or mixed human/animal dwellings (White 1974; Hunt et al. 1998; Fettene et al. 2004). Females are rarely found in human only dwellings or artificial pit shelters and are not known to transmit malaria parasites (White 1974; Fettene et al. 2004). Nothing is known about the larval habitats. Distribution. Known from the following localities in Ethiopia: Bahir Dar 11°36'N, 37°22E, Bako 5°47'N, 36°33E, Bedele 8°27'N, 36°21E, Dejen 10°10'N, 38°08E, Jimma 7°40'N, 36°50E, Omo River 8°13'N, 37°35E. Etymology. This species is named after Amharic, the official Semitic language of Ethiopia and is derived as a Latinized adjective. Type series. Type material of An. amharicus was collected resting inside a cattle structure in Babo village (7°42’45.00”N, 3658’00.00”E) outside Jimma, Ethiopia in 01–05.xii.1997. HOLOTYPE female with the following labels: An. quadriannulatu (sic) sp B; Ethiopia, Babo: 01–05.xii.1997/ Cattleshed; Coll. R. Hunt; Det. M. Coetzee; ETH 445.1/ WRBU Digital Image Prep # 2127. Paratypes with the same data as the holotype: collection code 388.1 (4 males, 5 females); collection code 445.1 (3 males, 13 females), including a male labeled as the allotype and a male with dissected slide-mounted genitalia with a WRBU prep label of 12/2. Slide-mounted LePe not directly associated with the adult holotype: collection code 388.1 (3 males, 4 females), collection code 445.1 (2 males, 4 females), a female with WRBU Digital Image Prep #2125 and a male with WRBU Digital Image Prep #2126.

FIGURE 5. Anopheles (Cellia) amharicus sp. n., pupa (paratype, WRBU digital image prep #2125): A, trumpet showing lengths of meatus and pinna; B, segment VIII, distal, showing form of seta 9; C, paddles.

266 · Zootaxa 3619 (3) © 2013 Magnolia Press COETZEE ET AL. FIGURE 6. Anopheles (Cellia) amharicus sp. n., larva (paratypes, WRBU digital image preps #2125 (A–D), #2126 (F): A, head, dorsal view; B, pecten plate; C, antenna, position of seta 1-A; D, abdomen, development of palmate setae (1-I–III); E, head, relative sizes and positions of setae 2-C and 3-C; F, thorax, branching of setae 1–3-P.

Systematics. Assigning formal names to biological species is an integral part of describing the earth's biodiversity. It allows sensible discourse amongst scientists, avoiding ambiguity and confusion between biological taxa. Definition of the species in the An. gambiae complex has been based on genetical criteria starting with cross- mating experiments supported by molecular, chromosomal, distributional and biological differences. The An. gambiae complex has had its fair share of confusion and controversy, with species initially being labeled A, B, C and D in the 1960s and more recently A (=An. gambiae) being split into M and S ‘forms’ in the past decade. The A, B, C and D issue was resolved in 1977 with Mattingly's (1977) publication of formal names for these species widely accepted as An. gambiae, An. arabiensis, An. quadriannulatus and An. bwambae respectively. Anopheles gambiae M and S ‘forms’ are now recognized as good biological species and here we assign formal names so that further research into each taxon can be carried out without unnecessary reference to the other. In accordance with the International Code for Zoological Nomenclature, the molecular M form of An. gambiae is named in honor of the late Professor Mario Coluzzi who devoted much of his life to the study of this species complex in West Africa. The S form retains the name An. gambiae. Further subdivision of An. coluzzii is possible given recent evidence from Cameroon and Mali indicating significant population structuring between the M forms at these localities (Wondji et al. 2002; Simard et al. 2009; Lee et al. 2009; Costantini et al. 2009; Slotman et al. 2007). Comparisons between Gambian and Cameroon “M forms” will need to be undertaken to assess their specific status. Further subdivision of An. gambiae, not correlated with either M or S form, has been reported from Burkina Faso and this also requires taxonomic investigation (Riehle et al. 2011). Anopheles quadriannulatus was said to have a relict distribution (White 1974) occurring in Ethiopia, Zanzibar and South Africa. Subsequent studies in Ethiopia and South Africa clarified the status of these populations (Hunt et al. 1998), but no records of the species have been published from Zanzibar since its original record in 1968 (Odetoyinbo & Davidson 1968). Given that these authors managed to cross one specimen from Zanzibar with An. quadriannulatus from southern Mozambique (Odetoyinbo & Davidson 1968) and found no male sterility in the resultant offspring, we conclude that the Zanzibar population was An. quadriannulatus. Recent unpublished reports of the Zanzibar Malaria Control Program recorded a small number of An. quadriannulatus on the island but more recent mosquito surveys in 2011 failed to collect this species (K. Haji, unpublished data). Morphology. It is essential to remember that the historical descriptions given for An. gambiae are quite possibly a mixture of not only An. gambiae and An. coluzzii, but also An. gambiae and An. arabiensis, especially where ‘freshwater gambiae’ was lumped or colony material was used. Given this caveat, and based on data from studies where wild-caught material was used, it is clear that the morphological overlap remains a hurdle to the effective use of classical taxonomy for the identification of members of the complex. While some success was attained using statistical analysis of combinations of morphological characters (reviewed by Coetzee 1989), and some authors were able to separate populations at a given locality, no characters have been shown to be consistently diagnostic. As a result, what could be considered “complete” descriptions are limited. Partial exceptions are: Gillies & de Meillon (1968) (brief descriptions of the adult male and female, the egg and larva of An. gambiae); Coetzee (1987) (redescription of An. quadriannulatus with analysis of wing spots, coeloconic sensillae and setal counts for both larvae and pupae); White (1985) (complete illustrations for An. bwambae of the adult female, male genitalia, larva, pupa and egg); Lounibos et al. (1999) (description of the eggs of six members of the complex from laboratory colony material showing significant differences between two samples of An. gambiae); and Ribeiro et al. (1979) (description of An. arabiensis as An. quadriannulatus davidsoni n. sp., illustration of the pupa). To completely describe and compare all the known species is beyond the scope of this paper but more thorough descriptions are needed before a conclusion can be reached indicating that morphological characters are not useful for species identifications (see White (1977) for in depth consideration of this subject). Here we describe and compare the adult female holotypes and associated immatures of two new species in the Gambiae Complex and summarize and compare these with observations others have published for members of the complex. We only present data that apparently refer to a single species and are not from colony specimens. There are no comprehensive morphological studies available but there is some suggestion that additional characters (Tables 2 and 4) could aid in future studies, especially of larvae and pupae. White (1985), in addition to referring to the palpal index proposed by Coluzzi (1964), measured the

268 · Zootaxa 3619 (3) © 2013 Magnolia Press COETZEE ET AL. relationship of the basal dark band on palpomere 4 to the apical pale band on palpomeres 4 + 5. Anopheles bwambae usually has more pale and fewer dark scales but overlaps with An. gambiae s.l., An. arabiensis, An. quadriannulatus and An. melas. Average mean ratios of pale to dark of White (1985, fig. 4) are: An. gambiae, 1.04; An. arabiensis, 0.89; An. quadriannulatus, 1.07; and An. bwambae 2.53. Holotypes of An. coluzzii and An. amharicus were 1.62 and 0.85 respectively. Anopheles coluzzii fell a bit outside of the cluster made up of all other species reported by White (1985) but An. amharicus fell near the center of the cluster. Note that the An. quadriannulatus sample used by White (1985) included specimens from Ethiopia and Zimbabwe and was therefore a combination of An. quadriannulatus and An. amharicus. The palpal index proposed by Coluzzi (1964) for separation of species in the complex, defined as MPlp4 + MPlp5 / MPlp3, has been investigated for its diagnostic value by many researchers (e.g. Gillies & Wilkes 1969; Bryan 1980; Bushrod 1981; Ribeiro et al. 1979; Coetzee 1987). The general result has been that the saltwater species (An. melas, mean 0.87, An. merus, mean 0.85) can usually be separated from the freshwater species (An. gambiae, An. arabiensis, An. quadriannulatus, overall mean 0.75), with An. bwambae falling somewhere in between (mean 0.81) (White 1985). Anopheles coluzzii (0.75) and An. amharicus (0.81) holotypes are consistent with indices for other freshwater species. A commonly measured character is the size of the pale band at the apex of hindtarsomere 3, which is proportional to the size of bands on other segments. The band for both An. coluzzii and An. amharicus is about 0.05 mm. This character was used in a key to the southern African species to separate An. arabiensis / An. gambiae, with a band of 0.09 mm or less, from An. quadriannulatus / An. merus where the band width was 0.1 mm or more (Coetzee et al. 1982; Coetzee 1989) (also see White 1985, p. 517). This character proved not to be useful for identification of An. arabiensis in DDT sprayed areas of South Africa (Sharp et al. 1989). Given that An. amharicus has a band size of 0.05 mm and occurs in sympatry with An. arabiensis in Ethiopia, this character has no diagnostic value. Larval seta 1-P is one of few setae used alone and in combination to separate species in the complex. Coetzee (1989) reviewed what was studied up to that time, most of which was based on laboratory colony specimens. No diagnostic features have so far been found. Seta 1-P counts for An. coluzzii (5/5) are on the low end of reported data (Ribeiro 1980). A count of 20 setae from 20 larval paratypes (same progeny brood) showed a range of 4–7. Counts for An. amharicus were 6–12 (7 individuals, 11 setae) (holotype 12/9). Most values reported by Coetzee (1989) are similar to An. amharicus but lower counts, more like An. coluzzii, were found in An. gambiae s.l. The pecten plates of both species described here have the alternating subequal long and short spines found in other freshwater species. Individual denticles found on the short teeth of An. coluzzii are sparse and basal or absent, whereas in An. amharicus there are about twice as many and extend in some spines to near the apex. This character has not been explored for use in identification. The position and length of larval antennal seta 1-A has not been documented, except in the illustration of An. bwambae (White 1985). We measured a small sample of An. coluzzii and An. amharicus and found possible differences as follows: Anopheles coluzzii (24 antennae, 12 individuals): antenna mean width at widest point 0.039 mm, range 0.036–0.04 mm; seta 1-A mean length 0.012 mm, range 0.005–0.016 mm; ratio of length to antennal width, 0.295 ± 0.082, range 0.2–0.44; mean distance of seta 1-A from antennal base as a fraction of antennal length, 0.34 ± 0.064, range 0.261–0.476. Anopheles amharicus (24 antennae, 12 individuals): antenna mean width at widest point 0.041 mm, range 0.036–0.044 mm; seta 1-A mean length 0.021 mm, range 0.012–0.032 mm; ratio of length to antennal width, 0.515 ± 0.114, range 0.33–0.8; mean distance of seta 1-A from antennal base as a fraction of antennal length, 0.437 ± 0.044, range 0.333–0.513. While there is overlap in the length of seta 1-A and its distance from the antennal base between these two species, it is possible that the two measures used in combination with other known variable characters could be used to separate other species. Further study is needed to confirm the measures in these two species, and then compare them with all the other members of the complex. Key characters for the An. gambiae complex applied to An. coluzzii and An. amharicus are listed here. Character numbers are taken in sequence (using current morphological terms) from Gillies & Coetzee (1987). Adult female: 1) abdominal segments without laterally projecting tufts of scales; 2) hindtarsus without entirely pale tarsomeres; 3) legs sparsely speckled; 4) (Section IV) maxillary palpus with 3 pale bands; 5) scaling on abdominal terga sparse or absent; 6) preapical dark spot on vein R1 with or without a pale interruption. Or, if

NAMING MEMBERS OF THE GAMBIAE COMPLEX Zootaxa 3619 (3) © 2013 Magnolia Press · 269 palpus with 4 pale bands, an uncommon variant in the complex, then 1) tarsomeres 1–4 with conspicuous pale bands on at least the apices; 2) wing with pale fringe spots up to veins CuA or 1A; and 3) preapical dark spot on vein R1 with or without a pale interruption. In An. coluzzii the apical pale band is quite narrow or absent on several tarsomeres, at least in the holotype. In addition, the pale interruption in the preapical dark spot on vein R1 is present on only one wing of the holotype of An. coluzzii. Pupa: 1) median keel absent; 2) cephalothorax without rows of conspicuous hooks; 3) trumpet without ribs; 4) seta 9-VIII not short and blunt; 5) paddle fringe with spines more or less abruptly changing to slender filaments; 6) paddle fringe continued beyond seta 1-Pa; 7) seta 1-VI and 1-VII not in the form of a tuft; 8) setae 1-VI, 1-VII, 5-VI and 5-VII single or branched; 9) seta 1-Pa more than quarter length of paddle, hooked (since this seta is in the form of a hook determination of its length is problematic); 10) meatus about half length of trumpet. The paddle fringe continued beyond seta 1-Pa is difficult to see and can be interpreted as not continuing beyond seta 1-Pa. When interpreted in this way, a second path in Gillies & Coetzee (1987) could lead to the An. gambiae complex, except that one encounters an ambiguous couplet possibly leading to An. cinereus: 1) setae 1-V and/or 1-VI about as long or longer than (following) segment; 2) (couplet 57) “Setae 1-V to 1-VII longer than following segment” (cinereus) or “Setae 1-V to 1-VII not normally longer than following segment.” Larva: 1) distance between setae 2-C about the same or greater than the distance between setae 2-C and 3-C on one side; 2) seta 3-C simple or with fewer than 8 branches; 3) seta 2-C simple, “frayed” or lightly feathered (= aciculate? These aciculae usually have been interpreted as branches); 4) seta 1-X single; 5) sides of thorax and abdomen without spicules; 6) tergal plate on abdominal segment V less than 0.66 distance between palmate setae; 7) setae 9-M and 10-M usually single but with at most 3 branches (Section VII); 8) seta 1-P poorly developed, basal tubercles very small or absent; 9) basal spine of mesothoracic pleural setae (9–12-M) large, curved and sharply pointed. The character “1-P poorly developed” is open to misinterpretation. In An. amharicus and other species (Table 3), seta 1-P is multi-branched and on an obvious tubercle. If this character were misinterpreted, one would be led to couplet 17 (An. cydippis, An. argenteolobatus). In addition, the illustration of seta1-P in Gillies & Coetzee (1987) has a main stalk with branches along its length. We observed, however, that this seta had multiple branches mostly originating from at or near the base.

Conclusions

Based on strong genetic evidence, the An. gambiae M and S molecular ‘forms’ and An. quadriannulatus sp. B are assigned formal names: M form = An. coluzzii, S form = An. gambiae and An. quadriannulatus sp. B = An. amharicus. As demonstrated in other studies of the taxonomy of this complex of species, no distinct characters are available for rapid, accurate, morphological identification of the species and identifications must be based on molecular methods.

Acknowledgements

Jim Pecor is thanked for mounting type material and Judy Stoffer for the photographs. The An. amharicus specimens were collected by Dr M Fettene Gebremariam in Ethiopia. Dr Y Linton and two anonymous reviewers are thanked for constructive criticisms of the manuscript. MC is funded by the DST/NRF South African Research Chairs Initiative.

References cited

Aguilar, R., Simard, F., Kamdem, C., Shields, T., Glass, G.E., Garver, L.S. & Dimopoulos, G. (2010) Genome-wide analysis of transcriptomic divergence between laboratory colony and field Anopheles gambiae mosquitoes of the M and S molecular forms. Insect Molecular Biology, 19, 695–705. http://dx.doi.org/10.1111/j.1365-2583.2010.01031.x Ayala, F. & Coluzzi, M. (2005) Chromosome speciation: humans, Drosophila, and mosquitoes. Proceedings of the National Academy of Sciences, USA, 102, Suppl 1: 6535–6542. http://dx.doi.org/10.1073/pnas.0501847102 Brunhes, J., Le Goff, G. & Geoffroy, B. (1997) Anopheles Afro-Tropicaux. I—Descriptions d’especes nouvelles et

270 · Zootaxa 3619 (3) © 2013 Magnolia Press COETZEE ET AL. changements to statuts taxonomiques (Diptera: Culicidae) Annales de la Societe Entomologique de France, 33, 173–183. Bryan, J.H. (1980) Use of palpal ratio and the number of pale bands on the palps in separating Anopheles gambiae Giles s.s. and Anopheles melas Theobald (Diptera: Culicidae). Mosquito Systematics, 12, 155–163. Bushrod, F.M. (1981) The Anopheles gambiae Giles complex and Bancroftian filariasis transmission in a Tanzanian coastal village. Annals of Tropical Medicine and Hygiene, 75, 93–100. Cambournac, F.J.C., Petrarca, V. & Coluzzi, M. (1982) Anopheles arabiensis in the Cape Verde archipelago. Parassitologia, 24, 265–267. Caputo, B., Nwakanma, D., Jawara, M., Adiamoh, M., Dia, I., Konate, L., Petrarca. V., Conway, D.J. & della Torre, A. (2008) Anopheles gambiae complex along The Gambia river, with particular reference to the molecular forms of An. gambiae s.s. Malaria Journal, 7, 182. http://dx.doi.org/10.1186/1475-2875-7-182 Caputo, B., Santolamazza, F., Vicente, JL., Nwakanma, DC., Jawara, M., Palsson, K., Jaenson, T., White, BJ., Mancini, E., Petrarca, V., Conway, DJ., Besansky, NJ., Pinto, J. & della Torre, A. (2011) The "far-west" of Anopheles gambiae molecular forms. PLoS One, 6, e16415. http://dx.doi.org/10.1371/journal.pone.0016415 Coetzee, M. (1987) A taxonomic description of Anopheles quadriannulatus Theobald (Diptera: Culicidae) from southern Africa. Systematic Entomology, 12, 305–312. http://dx.doi.org/10.1111/j.1365–3113.1987.tb00203.x Coetzee, M. (1989) Comparative morphology and multivariate analysis for the discrimination of four members of the Anopheles gambiae group in southern Africa. Mosquito Systematics, 21, 100–116. Coetzee, M., Newberry, K. & Durand, D. (1982) A preliminary report on a morphologicalcharacter distinguishing important malaria vectors in the Anopheles gambiae Giles complex in southern Africa. Mosquito Systematics, 14, 88-93. Coluzzi, M. (1964) Morphological divergences in the Anopheles gambiae complex. Rivista di Malariologia, 43, 197–232. Coluzzi, M. (1992) Malaria vector analysis and control. Parasitology Today, 8, 113–118. http://dx.doi.org/10.1016/0169- 4758(92)90277-9 Coluzzi, M., Petrarca, V. & Di Deco, M.A. (1985) Chromosomal inversion intergradation and incipient speciation in Anopheles gambiae. Bollettino di Zoologia, 52, 45–63. http://dx.doi.org/10.1080/11250008509440343 Coluzzi, M. & Sabatini, A. (1967) Cytogenetic observations on sp. A and B of the Anopheles gambiae complex. Parassitologia, 9, 73–88. Coluzzi, M. & Sabatini A, (1968a) Cytogenetic observations on species C of the Anopheles gambiae complex. Parassitologia, 10, 155–165. Coluzzi, M. & Sabatini, A. (1968b) Cytogenetic observations on species C, merus and melas of the Anopheles gambiae complex. Cahiers ORSTOM Entomologie Medicale, 6, 211–213. Coluzzi, M. & Sabatini, A. (1969) Cytogenetic observations on the salt-water species Anopheles merus and Anopheles melas, of the gambiae complex. Parassitologia, 11, 177–187. Coluzzi, M., Sabatini, A., Petrarca, V. & Di Deco, M.A. (1977) Behavioural divergences between mosquitoes with different inversion karyotypes in polymorphic populations of the Anopheles gambiae complex. Nature, 266, 832–833. http:// dx.doi.org/10.1038/266832a0 Coluzzi, M., Sabatini, A., Petrarca, V. & Di Deco, M.A. (1979) Chromosomal differentiation and adaptations to human environments in the Anopheles gambiae complex. Transactions of the Royal Society of Tropical Medicine and Hygiene, 73, 483–497. http://dx.doi.org/10.1016/0035-9203(79)90036-1 Coluzzi, M., Sabatini, A., della Torre, A., Di Deco, M.A. & Petrarca, V. (2002) A polytene chromosome analysis of the Anopheles gambiae species complex. Science, 298, 1415–418. http://dx.doi.org/10.1126/science.1077769 Costantini, C., Ayala, D., Guelbeogo, W.M., Pombi, M., Some, C.Y., Bassole, I.H.N., Ose, K., Fotsing, J.M., Sagnon, N.F., Fontenille, D., Besansky, N.J. & Simard, F. (2009) Living at the edge: biogeographic patterns of habitat segregation conform to speciation by niche expansion in Anopheles gambiae. BMC Ecology, 9, 16. http://dx.doi.org/10.1186/1472- 6785-9-16 Davidson, G. & Jackson, C.E. (1962) Incipient speciation in Anopheles gambiae Giles. Bulletin of the World Health Organization, 27, 303–305. Davidson, G., Paterson, H.E., Coluzzi, M., Mason, G.F. & Micks, D.W. (1967) The Anopheles gambiae complex. In: Wright, J.W. & Pal, R. (Eds), Genetics of Insect Vectors of Disease. Elsevier Publishers, Amsterdam, pp. 211–250. della Torre, A., Fanello, C., Akogbeto, M., Dossou-yovo, J., Favia, G., Petrarca, V. & Coluzzi, M. (2001) Molecular evidence of incipient speciation within Anopheles gambiae s.s. in West Africa. Insect Molecular Biology, 10, 9–18. http://dx.doi.org/ 10.1046/j.1365-2583.2001.00235.x della Torre, A., Tu, Z. & Petrarca, V. (2005) On the distribution and genetic differentiation of Anopheles gambiae s.s. molecular forms. Insect Biochemistry and Molecular Biology, 35, 755–769. http://dx.doi.org/10.1016/j.ibmb.2005.02.006 de Meillon, B. (1947) The Anophelini of the Ethiopian Geographical Region. Publications of the South African Institute for Medical Research, Johannesburg, no. 49. de Queiroz, K. (2007) Species concepts and species delimitation. Systematic Biology, 56, 879–886. http://dx.doi.org/10.1080/ 10635150701701083 Diabate, A., Dao, A., Yaro, A.S., Adamou, A., Gonzalez, R., Manoukis, N.C., Traore, S.F., Gwadz, R.W. & Lehmann, T. (2009) Spatial swarm segregation and reproductive isolation between the molecular forms of Anopheles gambiae. Proceedings of the Royal Society B: Biological Sciences, 276, 4215–4222. http://dx.doi.org/10.1098/rspb.2009.1167 Esnault, C., Boulesteix, M., Duchemin, J.B., Koffi, A.A., Chandre, F., Dabire, R., Robert, V., Simard, F., Tripet, F., Donnelly,

NAMING MEMBERS OF THE GAMBIAE COMPLEX Zootaxa 3619 (3) © 2013 Magnolia Press · 271 M.J., Fontenille, D. & Biemont, C. (2008) High genetic differentiation between the M and S molecular forms of Anopheles gambiae in Africa. PLoS ONE, 3(4), e1968. http://dx.doi.org/10.1371/journal.pone.0001968 Fanello, C., Santolamazza, F. & della Torre, A. (2002) Simultaneous identification of species and molecular forms of the Anopheles gambiae complex by PCR-RFLP. Medical and Veterinary Entomology, 16, 461–464. http://dx.doi.org/10.1046/ j.1365-2915.2002.00393.x Favia, G., della Torre, A., Bagayoko, M., Lanfrancotti, A., Sagnon, N.F., Toure, Y.T. & Coluzzi, M. (1997) Molecular identification of sympatric chromosomal forms of Anopheles gambiae and further evidence of their reproductive isolation. Insect Molecular Biology, 6, 377–383. http://dx.doi.org/10.1046/j.1365-2583.1997.00189.x Favia, G., Dimopoulos, G. & Louis, C. (1994) Analysis of the Anopheles gambiae genome using RAPD markers. Insect Molecular Biology, 3, 149–159. http://dx.doi.org/10.1111/j.1365-2583.1994.tb00162.x Favia, G., Lanfrancotti, A., Spanos, L., Siden-Kiamos, I. & Louis, C. (2001) Molecular characterization of ribosomal DNA polymorphisms discriminating among chromosomal forms of Anopheles gambiae s.s. Insect Molecular Biology, 10, 19–23. http://dx.doi.org/10.1046/j.1365-2583.2001.00236.x Fettene, M., Hunt, R.H., Coetzee, M. & Tessema, F. (2004) Behaviour of Anopheles arabiensis and An. quadriannulatus sp. B mosquitoes and malaria transmission in south-western Ethiopia. African Entomology, 12, 83–87. Fettene, M., Koekemoer, L.L., Hunt, R.H. & Coetzee, M. (2002) PCR assay for identification of Anopheles quadriannulatus species B from Ethiopia and other sibling species of the Anopheles gambiae complex. Medical and Veterinary Entomology, 16, 214–217. http://dx.doi.org/10.1046/j.1365-2915.2002.00354.x Fettene, M. & Temu, E.A. (2003) Species–specific primer for identification of Anopheles quadriannulatus sp. B (Diptera: Culicidae) from Ethiopia using a multiplex polymerase chain reaction assay. Journal of Medical Entomology, 40, 112–115. http://dx.doi.org/10.1603/0022-2585-40.1.112 Gentile, G., della Torre, A., Maegga, B., Powell, J.R. & Caccone, A. (2002) Genetic differentiation in the African malaria vector, Anopheles gambiae s.s., and the problem of taxonomic status. Genetics, 161, 1561–1578. Gentile, G., Slotman, M., Ketmaier, V., Powell, J.R. & Caccone, A. (2001) Attempts to molecularly distinguish cryptic taxa in Anopheles gambiae s.s. Insect Molecular Biology, 10, 25–32. http://dx.doi.org/10.1046/j.1365-2583.2001.00237.x Giles, G.M. (1902) A Handbook of the Gnats or Mosquitoes. 2nd Edition. John Bale, Sons & Danielsson, London. Gillies, M.T. & Coetzee, M. (1987) Supplement to the Anophelinae of Africa South of the Sahara. Publications of the South African Institute of Medical Research, Johannesburg, no. 55. Gillies, M.T. & de Meillon, B. (1968) The Anophelinae of Africa South of the Sahara. Publications of the South African Institute of Medical Research, Johannesburg, no. 54. Gillies, M.T. & Wilkes, T.J. (1969) A comparison of the range of attraction of animal baits and of carbon dioxide for some West African mosquitoes. Bulletin of Entomological Research, 59, 441–456. http://dx.doi.org/10.1017/S0007485300003412 Harbach, R.E. & Kitching, I.J. (2005) Reconsideration of anopheline phylogeny (Diptera: Culicidae: Anophelinae) based on morphological data. Systematics and Biodiversity, 3, 345–374. http://dx.doi.org/10.1017/S147720000500174X Harbach, R.E. & Knight, K.L. (1980) Taxonomists' Glossary of Mosquito Anatomy. Plexus Publishing, Marlton, New Jersey. Harbach, R.E. & Knight, K.L. (1982) Corrections and additions to Taxonomists' glossary of mosquito anatomy. Mosquito Systematics, 13, 201–217. Hunt, R.H. & Coetzee, M. (1995) Species concepts: their effect on the understanding of malaria transmission in Africa. In: Lambert, D.M. & Spencer, H.G. (Eds), Speciation and the Recognition Concept: Theory and Application. Johns Hopkins University Press, Baltimore, pp. 90–102. Hunt, R.H., Coetzee, M. & Fettene, M. (1998) The Anopheles gambiae complex: a new species from Ethiopia. Transactions of the Royal Society of Tropical Medicine and Hygiene, 92, 231–235. http://dx.doi.org/10.1016/s0035-9203(98)90761-1 Kamdem, C., Tene Fossog, B., Simard, F., Etouna, J., Ndo, C., Kengne, P., Bousses, P., Etoa, F.X., Awono-Ambene, P., Fontenille, D., Antonio-Nkondjio, C., Besansky, N.J. & Costantini, C. (2012) Anthropogenic habitat disturbance and ecological divergence between incipient species of the malaria mosquito Anopheles gambiae. PLoS One, 7(6), e39453. http://dx.doi.org/10.1371/journal.pone.0039453 Lawniczak, M.K.N., Emrich, S.J., Holloway, A.K., Regier, A.P., Olson, M., White, B., Redmond, S., Fulton, L., Appelbaum, E., Godfrey, J., Farmer, C., Chinwalla, A., Yang, S.P., Minx, P., Nelson, J., Kyung, K., Walenz, B.P., Garcia-Hernandez, E., Aguiar, M., Viswanathan, L.D., Rogers, Y.H., Strausberg, R.L., Saski, C.A., Lawson, D., Collins, F.H., Kafatos, F.C., Christophides, G.K., Clifton, S.W., Kirkness, E.F. & Besansky, N.J. (2010) Widespread divergence between incipient Anopheles gambiae species revealed by whole genome sequences. Science, 330, 512–514. http://dx.doi.org/10.1126/ science.1195755 Lee, Y., Cornel, A.J., Meneses, C.R., Fofana, A., Andrianarivo, A.G., McAbee, R.D., Fondjo, E., Traore, S.F. & Lanzaro, G.C. (2009) Ecological and genetic relationships of the Forest-M form among chromosomal and molecular forms of the malaria vector Anopheles gambiae sensu stricto. Malaria Journal, 8, 75. http://dx.doi.org/10.1186/1475-2875-8-75 Lehmann, T. & Diabate, A. (2008) The molecular forms of Anopheles gambiae: a phenotypic perspective. Infection, Genetics and Evolution, 8, 737–746. http://dx.doi.org/10.1016/j.meegid.2008.06.003 Lounibos, L.P., Coetzee, M., Duzak, D., Nishimura, N., Linley, J.R., Service, M.W., Cornel, A.J., Fontenille, D. & Mukwaya, L.G. (1999) A description and morphometric comparison of eggs of species of the Anopheles gambiae complex. Journal of the American Mosquito Control Association, 15, 157–185. Manoukis, N.C., Powell, J.R., Toure, M.B., Sacko, A., Edillo, F.E., Coulibaly, M.B., Traore, S.F., Taylor, C.E. & Besansky, N.J.

272 · Zootaxa 3619 (3) © 2013 Magnolia Press COETZEE ET AL. (2008) A test of the chromosomal theory of ecotypic speciation in Anopheles gambiae. Proceedings of the National Academy of Sciences, USA, 105, 2940–2945. http://dx.doi.org/10.1073/pnas.0709806105 Marsden, C.D., Lee, Y., Nieman, C.C., Sanford, M.R., Dinis, J., Martins, C., Rodrigues, A., Cornel, A.J. & Lanzaro, G.C. (2011) Asymmetric introgression between the M and S forms of the malaria vector, Anopheles gambiae, maintains divergence despite extensive hybridization. Molecular Ecology, 20, 4983–4994. http://dx.doi.org/10.1111/ j.1365–294X.2011.05339.x Mastbaum, O. (1957) Past and present position of malaria in Swaziland. Journal of Tropical Medicine and Hygiene, 60, 3–11. Mattingly, P.F. (1977) Names for the Anopheles gambiae complex. Mosquito Systematics, 9, 323–328. Muirhead–Thomson, R.C. (1947) Studies on Anopheles gambiae and A. melas in and around Lagos. Bulletin of Entomological Research, 38, 527–558. http://dx.doi.org/10.1017/S0007485300023221 Muirhead–Thomson, R.C. (1951) Studies on salt–water and fresh–water Anopheles gambiae on the East African coast. Bulletin of Entomological Research, 41, 487–502. http://dx.doi.org/10.1017/S0007485300027772 Neafsey, D.E., Lawniczak, M.K., Park, D.J., Redmond, S.N., Coulibaly, M.B., Traore, S.F., Sagnon, N., Costantini, C., Johnson, C., Wiegand, R.C., Collins, F.H., Lander, E.S., Wirth, D.F., Kafatos, F.C., Besansky, N.J., Christophides, G.K. & Muskavitch, M.A. (2010) SNP genotypying defines complex gene-flow boundaries among African malaria vector mosquitoes. Science, 330, 514–517. http://dx.doi.org/10.1126/science.1193036 Odetoyinbo, J.A. & Davidson, G. (1968) The Anopheles gambiae complex and its role in the malaria transmission in the islands of Zanzibar and Pemba, United Republic of Tanzania. Unpublished World Health Organization Document, WHO/Mal/ 68.660. Oliveira, E., Salgueiro, P., Palsson, K., Vicente, J.L., Arez, A.P., Jaenson, T.G., Caccone, A. & Pinto, J. (2008) High levels of hybridization between molecular forms of Anopheles gambiae from Guinea Bissau. Journal of Medical Entomology, 45, 1057–1063. http://dx.doi.org/10.1603/0022-2585(2008)45[1057:hlohbm]2.0.co;2 Paterson, H.E. (1962) Status of the East African salt–water–breeding variant of Anopheles gambiae Giles. Nature, 195, 469–470. http://dx.doi.org/10.1038/195469a0 Paterson, H.E. (1963a) The species, species control and antimalarial spraying campaigns. Implications of recent work on the Anopheles gambiae complex. South African Journal of Medical Science, 28, 33–34. Paterson, H.E. (1963b) On the naming of the East African salt–water species of the Anopheles gambiae complex. Unpublished World Health Organization Document, WHO/Mal/421, pp. 7–10. Paterson, H.E. (1964) Direct evidence for the specific distinctness of Forms A, B and C of the Anopheles gambiae complex. Rivista di Malariologia, 43, 191–196. Paterson, H.E. (1968) Evolutionary and population genetical studies of certain Diptera. University of the Witwatersrand, PhD thesis, pp. 211–213. Pennetier, C., Warren, B., Dabire, K.R., Russell, I.J. & Gibson, G. (2010) “Singing on the wing” as a mechanism for species recognition in the malarial mosquito Anopheles gambiae. Current Biology, 20, 131–136. http://dx.doi.org/10.1016/ j.cub.2009.11.040 Petrarca, V., Sabatinelli, G., Toure, Y.T. & Di Deco, M.A. (1998) Morphometric multivariate analysis of field samples of adult Anopheles arabiensis and An. gambiae s.s. (Diptera: Culicidae). Journal of Medical Entomology, 35, 16–25. Reid, J.A. (1975a) Pupal differences between species A and B of the Anopheles gambiae group from Kisumu, East Africa. Mosquito Systematics, 7, 1–8. Reid, J.A. (1975b) Pupal differences between species A and B of the Anopheles gambiae group from Kaduna, West Africa. Mosquito Systematics, 7, 299–302. Reidenbach, K.R., Neafsey, D.E., Costantini, C., Sagnon, N’F., Simard, F., Ragland, G.J., Egan, S.P., Feder, J.L., Muskavitch, M.A.T. & Besansky, N.J. (2012) Patterns of genomic differentiation between ecologically differentiated M and S forms of Anopheles gambiae in West and Central Africa. Genome, Biology and Evolution, 4, 1202–1212. http://dx.doi.org/10.1093/ gbe/evs095 Ribbands, C.R. (1944) Differences between Anopheles melas (A. gambiae var. melas) and Anopheles gambiae. Annals of Tropical Medicine and Parasitology, 38, 85–99. Ribeiro, H. (1980) A biometric study of the taxonomy of the Anopheles gambiae Giles complex (Diptera: Culicidae). Garcia de Orta, Séries Zoológico, 9, 139–154. Ribeiro, H., Ramos, H. da C., Pires, C.A. & Capela, R.A. (1979) Description and biometric study of Anopheles (Cellia) quadriannulatus davidsoni sp.n., a seventh member of the Anopheles gambiae Giles complex (Diptera: Culicidae) endemic to the Cape Verde archipelago. Garcia de Orta, Séries Zoológico, 8, 75–88. Riehle, M.M., Guelbeogo, W.M., Gneme, A., Eiglmeier, K., Holm, I., Bischoff, E., Garnier, T., Snyder, G.M., Li, X., Markianos, K., Sagnon, N. & Vernick, K.D. (2011) A cryptic subgroup of Anopheles gambiae is highly susceptible to human malaria parasites. Science, 331, 596–598. http://dx.doi.org/10.1126/science.1196759 Santolamazza, F., Mancini, E., Simard, F., Qi, Y., Tu, Z. & della Torre, A. (2008) Insertion polymorphisms of SINE200 retrotransposons within speciation islands of Anopheles gambiae molecular forms. Malaria Journal, 7, 163. http:// dx.doi.org/10.1186/1475-2875-7-163 Santolamazza, F., Caputo, B., Calzetta, M., Vicente, J.L., Mancini, E., Petrarca, V., Pinto, J. & della Torre, A. (2011) Comparative analyses reveal discrepancies among results of commonly used methods for Anopheles gambiae molecular form identification. Malaria Journal, 10, 215. http://dx.doi.org/10.1186/1475-2875-10-215

NAMING MEMBERS OF THE GAMBIAE COMPLEX Zootaxa 3619 (3) © 2013 Magnolia Press · 273 Sharp, B.L., Le Sueur, D. & Ridl, F. (1989) The value of hindleg banding patterns in the identification of species of the Anopheles gambiae complex in Natal, South Africa. Mosquito Systematics, 21, 77–82. Simard, F., Ayala, D., Kamdem, G.C., Pombi, M., Etouna, J., Ose, K., Fotsing, J.M., Fontenille, D., Besansky, N.J. & Costantini, C. (2009) Ecological niche partitioning between Anopheles gambiae molecular forms in Cameroon: the ecological side of speciation. BMC Ecology, 9, 17. http://dx.doi.org/10.1186/1472-6785-9-17 Slotman, M.A., Tripet, F., Cornel, A.J., Meneses, C.R., Lee, Y., Riemer, L.J., Thiemann, T.C., Fondjo, E., Fofana, A., Traore, S.F. & Lanzaro, G.C. (2007) Evidence for subdivision within the M molecular form of Anopheles gambiae. Molecular Ecology, 16, 639–649. http://dx.doi.org/10.1111/j.1365-294X.2006.03172.x Toure, Y.T., Petrarca, V., Traore, S.F., Coulibaly, A., Maiga, H.M., Sankare, O., Sow, M., Di Deco, M.A. & Coluzzi, M. (1998) The distribution and inversion polymorphism of chromosomally recognized taxa of the Anopheles gambiae complex in Mali, West Africa. Parassitologia, 40, 477–511. Weetman, D., Wilding, C.S., Steen, K., Morgan, J.C., Simard, F. & Donnelly, M.J. (2010) Association mapping of insecticide resistance in wild Anopheles gambiae populations: major variants identified in a low-linkage disequilibrium genome. PLoS One, 5(10), e13140. http://dx.doi.org/10.1371/journal.pone.0013140 Weetman, D., Wilding, C.S., Steen, K., Pinto, J. & Donnelly, M.J. (2012) Gene flow-dependent genomic divergence between Anopheles gambiae M and S forms. Molecular Biology & Evolution, 29, 279–291. http://dx.doi.org/10.1093/molbev/ msr199 White, G.B. (1974) Anopheles gambiae complex and disease transmission in Africa. Transactions of the Royal Society of Tropical Medicine and Hygiene, 68, 278–301. http://dx.doi.org/10.1016/0035-9203(74)90035-2 White, G.B. (1975) Notes on a catalogue of Culicidae of the Ethiopian Region. Mosquito Systematics, 7, 303–344. White , G.B. (1977) The place of morphological studies in the investigation of Anopheles species complexes. Mosquito Systematics, 9, 1–24. White, G.B. (1985) Anopheles bwambae n.sp., a malaria vector in the Semliki Valley, Uganda, and its relationships with other sibling species of the An. gambiae complex (Diptera: Culicidae). Systematic Entomology, 10, 501–522. http://dx.doi.org/ 10.1111/j.1365-3113.1985.tb00155.x Wilkins, E.E., Howell, P.I. & Benedict, M.Q. (2006) IMP PCR primers detect single nucleotide polymorphisms for Anopheles gambiae species identification, Mopti and Savanna rDNA types, and resistance to dieldrin in Anopheles arabiensis. Malaria Journal, 5, 125. Wondji, C., Simard, F. & Fontenille, D. (2002) Evidence for genetic differentiation between the molecular forms M and S within the Forest chromosomal form of Anopheles gambiae in an area of sympatry. Insect Molecular Biology, 11, 11–19. http://dx.doi.org/10.1046/j.0962-1075.2001.00306.x Yawson, A.E., Weetman, D., Wilson, M.D. & Donnelly, M.J. (2007) Ecological zones rather than molecular forms predict genetic differentiation in the malaria vector Anopheles gambiae s.s. in Ghana. Genetics, 175, 751–761. http://dx.doi.org/ 10.1534/genetics.106.065888