Key to the females of Afrotropical Anopheles mosquitoes (Diptera: Culicidae)

Background In 1987, Gillies and Coetzee published a pictorial key for the morphological identification of adult female mosquitoes. Since then, several new species of anopheline mosquitoes have been described. Methods The 1987 key to adult female mosquitoes was used as the template for the current key. Results New species described in the literature over the past 32 years have been included. A list of all currently known Afrotropical species is provided. Anopheles stephensi is included for the first time as occurring on the African continent. Conclusions An updated key for the morphological identification of Afrotropical anopheline species is presented.


Background
Dichotomous keys for the morphological identification of groups of organisms have been used for over 300 years. These keys lead the reader through a series of couplets, each one giving two choices of characters, until a species name is reached. For the anopheline mosquitoes of the Afrotropical Region, which includes some of the most efficient transmitters of malaria parasites in the world, the first key for their identification was published in 1931 [1], and the most recent printed version in 1987 [2], this last being a pictorial key containing line graphics of characters mentioned in each couplet. In the 32 years that have passed since Gillies and Coetzee published their key [2], several new species have been discovered, described and named.

Methods
The pictorial key to adult female anophelines in the Afrotropical Region [2] was used as the template for the current key. New illustrations were produced and new couplets inserted where appropriate to accommodate new species described since 1987.

Results
The present key is a revision of that presented in Gillies and Coetzee [2], with the addition of newly described species and the exclusion of subspecies. Table 1 provides a lists of species, authorship and current classification, while Table 2 gives the number of species described per decade since 1900. The user is encouraged to refer to both the 1968 volume of Gillies and De Meillon [3] and the 1987 supplement of Gillies and Coetzee for full species descriptions, biology and geographic distribution. More recent references include Sinka et al. [4], and Kyalo et al. [5].

Discussion
A major addition to the key is the inclusion of Anopheles stephensi (Section IV), the Asian malaria vector with distribution from the Middle East to China. This species was first detected on the African continent in Djibouti in September 2012 and subsequently in February 2013 [14] through to December 2017 [15]. It has also recently been found in Ethiopia in 2016 [16]. The species is similar to those belonging to the Anopheles gambiae complex-mosquitoes with speckled legs-but differs by having the wing with two pale spots in the 2nd main dark area of the costa and vein 1, thus being  [28] similar to Anopheles maculipalpis and Anopheles pretoriensis, from which it differs by not having hindtarsomeres 4 and 5 all pale.
There are several groups of species where morphological identification is not possible using only the adult females, either because the adults look identical or because of overlap in morphological variation. Some of these species can be identified on immature characters, thus requiring eggs or larvae [3], while others require genetical methods, such as chromosomal inversions [17] or molecular assays [18,19]. Such groups include: (a) the well-known Anopheles gambiae complex (An. gambiae, Anopheles coluzzii [20], Anopheles arabiensis, Anopheles quadriannulatus, Anopheles amharicus [20], Anopheles fontenillei [21], Anopheles bwambae, Anopheles melas, Anopheles merus); The definition of "complex", as applied to the genus Anopheles, is a group of species that are virtually morphologically identical but are otherwise considered valid species. The use of the term "group" denotes species that are morphologically very similar at the adult stage but many can be distinguished at the immature stages.
Except for those of medical importance, the above list is just a small sample of species groups about which we know very little biologically in terms of feeding/resting preferences or their role in malaria parasite transmission. Basic taxonomic research, aligned with molecular analyses, is still very much needed in the Culicidae.

Conclusions
An updated key for the morphological identification of Afrotropical anopheline species is presented. This key should be used in conjunction with earlier works giving full species descriptions, biology, medical importance and distribution.  (6) The key layout Characters are presented in 'couplets' where two options are presented, giving two different outcomes, eventually ending at a species name. The illustration(s) for the first option of each couplet is on the left (or rarely, in the centre) and for the second option on the right. General terminology follows that of Harbach and Knight [22,23]. Terminology of the wing venation has changed over the past 80 years in attempts to align Culicidae with the rest of the Diptera Family ( Table 3). The terminology proposed in the recent Manual of Afrotropical Diptera [24] has been challenged by culicid taxonomists (manuscript reviewers), specifically around the terms used for the posterior veins (veins 5 and 6 in Fig. 1). Since consensus on the terminology has not yet been reached, and given that the malaria vector control field workers in Africa have been using the Anopheles identification keys published 32 years ago [2] that use the numbering shown in Fig. 1, this simplified system is used here. It avoids unnecessary repetition of the various terms in each couplet and reference can be made to Table 3 where recent terminology is required.