Introduction

The drosophilid genus Zaprionus Coquillett, 1902 is characterized by the presence of longitudinal white stripes on the frons and the mesonotum (Fig. 1). It is a Paleotropical genus whose species are classified under two subgenera: Zaprionus sensu stricto in the Afrotropical region (48 species), and Anaprionus in the Oriental and Australasian regions (11 species) (Okada and Carson 1983; Markow and O’Grady 2006; Brake and Bächli 2008). The two subgenera are distinguished on the basis of the number of their mesonotal stripes, being even in Zaprionus s.s. and odd in Anaprionus. Flies of the subgenus Zaprionus form an important component of the Afrotropical drosophilid fauna, in terms of number of species, relative abundance and large body size (Tsacas et al. 1981; Yassin and David in press). Chassagnard and Tsacas (1993) classified those species under two groups: the armatus group with ornamented forefemora, and the inermis group with unornamented forefemora. Recent phylogenetic revisions using molecular and morphological characters have shown Zaprionus s.s. species to be monophyletic, but both species groups to be polyphyletic (Yassin et al. 2008a, 2010, in press).

In this paper, we propose a new classification based on recent phylogenetic findings, describe two new species, and provide a taxonomic key to all African Zaprionus species. In the early 1990s, several taxonomic keys were published for African Zaprionus (Tsacas and Chassagnard 1990; Chassagnard and McEvey 1992; Chassagnard and Tsacas 1993), but these usually treated some species subgroups or geographical localities and covered only 76% of the then known species. Since 1993, eight species were described including the two new ones described here. Twenty three species were available as laboratory strains, and this allowed us to also provide descriptions of internal reproductive system and premature morphology.

Materials and methods Specimens examined

Examined specimens were museum-preserved material or laboratory strains. Laboratory strains in the Laboratoire Evolution, Génomes et Spéciation (LEGS) belonged to 23 species (Table 1), and they were used in describing internal structures of the male and female reproductive systems and immature stages. As shown in Table 1, a congeneric Oriental species, Zaprionus (Anaprionus) bogoriensis Mainx, was added to the analysis.

Morphological description

Formal morphological description of the new species followed standard Drosophila terminology and index formulae as in McEvey (1990). Specimens were deposited in Laboratoire Evolution, Génomes et Spéciation, Gif-sur-Yvette, France (LEGS) as living cultures, frozen and alcohol-preserved material and microscopic preparations, and in Muséum National d’Histoire Naturelle, Paris, France (MNHN) as pinned material.

Morphological structures are abbreviated as: fw = front width; fl = front length; hw = head width; o = maximum diameter of the eye; j = width of gena in line with o; ch = maximum width of gena; or1 = proclinate orbital seta; or2 = anterior reclinate orbital seta; or3 = posterior reclinate orbital seta; oc = ocellar seta; poc = post-ocellar seta; iv = inner vertical seta; ov = outer vertical seta; acs = acrostichal setulae; adc = anterior dorsocentral; pdc = posterior dorsocentral; psc = prescutullar seta; bsc = basal scutellar seta; asc = apical scutellar seta; F1 = forefemur; WL = wing length; Wl = wing width; TL = thorax length; WV = width of white vittae at adc; BV = width of black vittae surrounding WV at adc; A = number of abdominal bristles summed over successive sternites. Measurements on immature stages were taken from uncrowded cultures grown under the same conditions (at 21°C). Measurements are abbreviated as: EL = egg length; El = egg width; PF = length of egg posterior filament; PL = puparium length; Pl = puparium width; H (horn index) = the ratio of the length of the anterior spiracles to the total length of the puparium × 100.

Anatomy of the internal reproductive system

Mature, about 10 days old adults were dissected in a Drosophila Ringer solution. For the male reproductive system (see drawings in Lachaise 1972; Araripe et al. 2004), testes were uncoiled before a linear measurement could be done. This operation was facilitated by allowing the Ringer solution to evaporate a little so that the testis loses its rigidity. Linear measurements were done with a stereomicroscope equipped with a micrometer. Six lengths were measured: TST = testis; SV = seminal vesicle; VD = vas deferens; PAR = paragonia (accessory gland); EC = ejaculatory canal; EB = ejaculatory bulb; and CAE = caecum. PAR and EB are glandular structures and their measurements are variable according to the reproductive status of the dissected male. They do not provide thus reliable taxonomic information. For the female (cf. Lachaise 1972), the lengths of two organs were measured after dissection: SR = seminal receptacle and SP = spermatheca length. The SR also makes irregular coils at the junction between the oviduct and uterus, and was uncoiled with tiny needles before measurement. As with immature stages, two or three individuals from almost each species were measured and the results were very similar. Multiple measurements were not taken for all species, but slight differences were only found within those for which multiple measurements were taken.

A key to African Zaprionus Revised classification of Zaprionus s.s.

Chassagnard and Tsacas (1993) divided Zaprionus s.s. into two groups: inermis and armatus, the latter comprising three subgroups: armatus, tuberculatus and vittiger. The phylogenetic revision of Yassin et al. (2008a) revealed both groups and subgroups to be polyphyletic. However, almost half of the species used in their study lacked DNA sequences, and the discovery and the subsequent molecular analysis of some of these species revealed some new insights (Yassin et al., in press). In light of these findings, a new classification scheme is proposed (Table 2).

Table 2 also shows the breeding niche and the possibility to rear in the laboratory for some species. These two attributes are interrelated, as generalist fruit-breeding species are usually those that can be reared with ease on standard Drosophila medium. Lachaise and Tsacas (1983) reviewed the breeding niche for 12 Zaprionus s.s. species. With the exception of the curious entomophagous ecology of some Afrotropical drosophilids, Zaprionus species share almost all of the known breeding niches of the Afrotorpical fauna, i.e. fruit, flower and decaying tree trunk breeding. Most species are fruit breeders. Some species (e.g., Zaprionus badyi, Zaprionus momorticus, and Zaprionus neglectus) are generalist flower-breeders, whereas two species of the armatus group (Zaprionus fumipennis and Zaprionus vrydaghi) breed exclusively in flowers of Costus afer (Tsacas and Chassagnard 1990). Records of Zaprionus montanus suggest this species to mine bamboo leaves or stems (Graber 1957; Chassagnard 1989). The breeding niche of its sibling species, Zaprionus campestris, is unknown as it was collected by non-selective light or Malaise traps. Zaprionus koroleu was bred from cut palm trunks along with other palm breeding drosophilids of the genera Chymomyza and Scaptodrosophila. However, it appears that no strict association with palm trees has yet evolved in this species as it was able to be reared in the laboratory (although the strain has been lost due to the difficulty of rearing). Other Zaprionus species that were also bred from cut tree trunks included Zaprionus armatus, Zaprionus inermis and Zaprionus ghesquierei.

It is still difficult to estimate with certainty the niches for some of the problematic species in Lachaise and Tsacas’s (1983) review. For example, Zaprionus indianus had almost 80 host plants being the most ecologically diverse drosophilid in the Afrotropical fauna. However, most of the ecological records prior to Tsacas’ (1980) review confused this species with other species of the vittiger group, and even after its identity has been established (Tsacas 1985) the recent discovery of two cryptic species, one of which is also widespread in tropical Africa (Yassin et al. 2008b), sheds doubt on its hosts there. Indeed, Lachaise and Tsacas (1983) described three native host plants from Makokou (Gabon), a locality where the two cryptic species coexist (Yassin et al. 2008b). Although the breeding niches of Zaprionus indianus have been properly determined in its introduced regions in Brazil (Silva et al. 2005; Tidon 2006; Garcia et al. 2008) and the Palearctic region (Yassin et al. 2009), attention has to be paid in the future to determine its breeding niche in its zone of origin. We excluded also the records on the tuberculatus subgroup predating Tsacas et al.’s (1977) discrimination of two sibling species Zaprionus sepsoides and Zaprionus tuberculatus. Records on the Gabonese strain of Zaprionus ornatus in Lachaise and Tsacas (1983) were assigned to Zaprionus taronus since Chassagnard and Tsacas (1993) showed this strain to be misidentified with Zaprionus ornatus by Tsacas (1980).

The armatus group

The armatus group was initially erected to include three subgroups: armatus, tuberculatus and vittiger (Chassagnard and Tsacas 1993). We transferred the tuberculatus subgroup to the inermis group and upgraded the vittiger subgroup to a species group hence restricting the armatus group to the 14 species of the previous armatus subgroup bearing a simple row of spines on F1 (Tsacas and Chassagnard 1990; Fig. 2c, d; Fig. 8). Tsacas and Chassagnard (1990) further subdivided the 14 species of the armatus subgroup to three ‘Ensembles’ I, II and III on the basis of the differentiation of the F1 spines. Yassin et al. (2008a) suggested, using morphological characters of the male genitalia, this subgroup to be polyphyletic. Nonetheless, molecular sequences became later available from a single species, Zaprionus campestris, and its phylogenetic position did not confirm Yassin et al.’s (2008a) placement (Yassin et al., in press). Therefore, Tsacas and Chassagnard’s (1990) subclassification will be retained with slight modifications until new molecular sequences become available. The armatus group is now subdivided into three subgroups: the montanus subgroup with two species bearing two oppositely oriented F1 spines (Ensemble I); the spinosus subgroup with three species bearing a row of differentiated F1 spines (Ensemble II); and the armatus subgroup with nine species bearing a row of undifferentiated F1 spines (Ensemble III). The armatus subgroup is further subdivided into three complexes: the hoplophorus complex with two species bearing differentially oriented strong F1 spines; the armatus complex with five species bearing undifferentially oriented strong F1 spines; and the vrydaghi complex with two species bearing undifferentially oriented fine F1 spines and wings blackened anteriorly.

The inermis group

The inermis group comprises species with spineless F1 (Figs 2a, b). The F1 spinelessness is also found in the Oriental subgenus Anaprionus, suggesting a plesiomorphy, and the monophyly of this group was questionable (Chassagnard and Tsacas 1993). Yassin et al. (2008a) suggested on the basis of morphological characters that this group was polyphyletic with two species Zaprionus litos and Zaprionus neglectus being closely related to the armatus and the vittiger groups. These suggestions were confirmed by later molecular analyses (Yassin et al., in press) which also suggested that two other species (Zaprionus sexstriatus and Zaprionus sexvittatus) formed the sister clade with the vittiger group. Four species of the inermis group (Zaprionus arduus, Zaprionus badyi, Zaprionus momorticus and Zaprionus niabu) have not been included in any of these previous studies and their phylogenetic placement remains thus uncertain. Zaprionus ghesquierei forms the earliest branch for the remaining species that are classified here under two subgroups: the inermis subgroup with two species having the short straight aedeagus; and the tuberculatus subgroup with seven species having the curved robust aedeagus. The F1 of several species of tuberculatus subgroup carries a tubercule (Fig. 2b). These two subgroups are closely related to each other as they share the bare and bristleness epandrium (Fig. 7) and the fine serration on the dorsal margin of the aedeagus. These synapomorphies are absent in Zaprionus ghesquierei, Zaprionus arduus, Zaprionus badyi and Zaprionus momorticus. No male specimen has ever been collected for Zaprionus niabu. The tuberculatus subgroup contains two species complexes as suggested by Yassin (2008): the sepsoides complex with two species having short testicules; and the tuberculatus complex with three species having long testicules.

The neglectus group The vittiger group

The vittiger group comprises 17 species with usually hairy epandrium carrying more than 2 posterior bristles (Fig. 14d, f). It is mainly characterized by the relatively deep serration of the aedeagal flap. The F1 of most of its species carry composite spines that have bristles fused at their bases and usually are borne on protruding tubercules (Fig. 2e, f). Three species (Zaprionus sexstriatus, Zaprionus sexvittatus and Zaprionus litos) have the unarmed F1 and have been classified in the inermis group (Chassagnard and Tsacas 1993; Chassagnard 1996). Species with F1 bearing composite spines are classified into six complexes: the sexvittatus complex with three species having two additional submedian silvery longitudinal stripes on the thorax (Fig. 1); the ornatus complex with two species having the aedeagal flap weakly serrate apically and smooth basally and greatly extended basally and tapering to a point; the indianus complex with three species having the entirely hairy epandrium and hypandrium and the smooth spermatheca (Fig. 12); the davidi complex with two species having the partially hairy epandrium and rough spermatheca (Fig. 14); the proximus complex with two species having the epandrium enlarged dorsally and tapered ventrally (Fig. 14), the broadened hypandrium and the voluminous cercus lobate at the dorsal margin; and the vittiger complex with five species having the partially hairy epandrium and the smooth spermatheca.

Comparative anatomy of reproductive system

Many authors described the internal anatomy of some Zaprionus species that can be grown in laboratory (Burla 1954; Throckmorton 1962; Lachaise 1972; Araripe et al. 2004); but with the exception of Tsacas et al.’s (1977) study on the tuberculatus subgroup, little attention has been paid to quantify the differences between the species. Table 3 shows the measurements of some structures in the laboratory strains used in this study. As shown, many measurements give insightful taxonomic differences.

Male reproductive system

Testis length (TST) ranges from 1.0 mm in Zaprionus kolodkinae to 12.4 mm in Zaprionus ornatus. The Oriental species, Zaprionus (Anaprionus) bogoriensis, has TST of 4.4 mm which approaches that of the mean of the African species (3.7 ± 0.5 mm). Species of the inermis group can be classidfied under two categories: those with small testis ranging from 1.0 to 2.0 mm (Zaprionus inermis, Zaprionus cercus, Zaprionus kolodkinae, Zaprionus sepsoides and Zaprionus tsacasi), and those with large testis ranging from 3.2 to 4.4 mm (Zaprionus mascariensis, Zaprionus tuberculatus, Zaprionus burlai and Zaprionus verruca). Species of the last category are all members of the tuberculatus subgroup which also include some species of the first category, and TST presents a very informative taxonomic clue (Fig. 3; Tsacas et al. 1977; Yassin 2008). In the vittiger group, Zaprionus ornatus with its very long testis (TST = 12.4 mm) is particular. The remaining species can be classified under four discontinuous categories: Zaprionus gabonicus and Zaprionus davidi with TST from 2.5 to 2.6 mm; Zaprionus proximus and Zaprionus santomensis sp. n. with TST of 3.6 mm; Zaprionus capensis, Zaprionus camerounensis, Zaprionus vittiger and Zaprionus lachaisei sp. n. with TST from 4.0 to 4.4 mm; and Zaprionus indianus, Zaprionus africanus and Zaprionus taronus with TST from 5.2 to 5.4 mm. Unlike in the inermis group, the categories of the vittiger group do not reflect any phylogenetic trend.

The seminal vesicle (SV) is the part of the vas deferens that has undergone a differentiation for sperm storage. It ranges from 0.6 mm in Zaprionus ghesquierei and Zaprionus sepsoides to 7.2 mm in Zaprionus ornatus, with the mean of 1.6 ± 0.3 mm in African Zaprionus. Species of the inermis group tend to have small SV, ranging from 0.6 to 1.6 mm, whereas species of the vittiger group have larger SV, ranging from 0.7 to 2.4 mm (excluding Zaprionus ornatus).

The vas deferens (VD) ranges from 0.04 mm in Zaprionus ghesquierei to 2.20 mm in Zaprionus ornatus. The quasi-absence of VD in Zaprionus ghesquierei is exceptional as the next value to it is 0.20 mm in a number of species of the inermis group (Zaprionus inermis, Zaprionus kolodkinae and Zaprionus sepsoides). Indeed, Throckmorton (1962) described VD morphology in a laboratory strain of Zaprionus ghesquierei. The 12 males he dissected “were variable, showing two major types with only slight integradation between them” (pp. 232). The VDs of three males were quasi-absent like the one described here, whereas those of the remaining nine males were “somewhat longer and associates closely with the ventral surface of the paragonia.” We did not find this polymorphism in the few individuals dissected. The longest VD in the inermis group is found in Zaprionus burlai (VD = 1.1 mm), and it is greater than VDs of its two relatives (0.7 mm in Zaprionus tuberculatus and 0.8 mm in Zaprionus verruca).

The ejaculatory bulb of Zaprionus species is moderately large, rounded and bearing long posterior caecae (Throckmorton 1962). In the vittiger species group, the posterior caecae are branched several times, whereas in the remaining African and Oriental species the caecae are unbranched. The length of the caecae (CAE) ranges from 0.1 mm in Zaprionus sepsoides to 2.0 mm in Zaprionus neglectus. The long CAE of Zaprionus neglectus is exceptional (Burla 1954) and it was used as one of the arguments to synonymize Zaprionus neglectus Burla with Zaprionus simplex Chassagnard & McEvey. CAE can also be used to distinguish Zaprionus cercus (CAE = 1.6 mm) from its sibling species Zaprionus inermis (CAE = 0.4 mm), which has particularly small CAE. Lachaise (1972) also noted that CAE of Zaprionus inermis was about 0.6 mm. Zaprionus verruca has exceptional long CAE of 1.2 mm in the tuberculatus subgroup, that can easily distinguish it from its two sibling species Zaprionus tuberculatus and Zaprionus burlai (CAE = 0.3 mm).

Female reproductive system

The seminal receptacle (SR) ranges from 0.8 mm in Zaprionus kolodkinae to 12.0 mm in Zaprionus ornatus. As with TST, species of the vittiger group tend to have larger SR than those of the inermis group. The correlation between TST and SR is a well-established fact in the Drosophilidae, although the correlation is thought to be functional rather than genetic (Joly and Bressac 1994). This correlation is obvious in Zaprionus (r = 0.93; P < 0.001). SR can distinguish Zaprionus burlai females (SR = 6.3 mm) from Zaprionus tuberculatus (SR = 3.6 mm), and Zaprionus indianus (SR = 4.8 mm) from Zaprionus africanus (SR = 3.8 mm) and Zaprionus gabonicus (SR = 3.5 mm).

Burla (1954) provided the first account of the morphology of the spermatheca (SPR) in Zaprionus species from Côte d’Ivoire, and illustrations of spermathecae became a taxonomic routine in all descriptions following his study (Figs 3, 6, 12, 13). The elongate form of the spermatheca of Zaprionus ornatus is characteristic and it was one of the arguments for considering Zaprionus megalorchis Chassagnard and Tsacas syn. n. and Zaprionus aff. vittiger Burla as junior synonyms for this species (Fig. 13). We dissected 10 females per species in the indianus complex and found that in Zaprionus africanus the width of the spermatheca was always relatively greater than its length, whereas in its two cryptic species Zaprionus indianus and Zaprionus gabonicus, the spermatheca length and width were subequal (Fig. 12). In the tuberculatus species subgroup, it is the shape rather than the length of the spermatheca which provides the best taxonomic clues (Fig. 3).

Immature stages Egg

The eggs of species of the Oriental subgenus Anaprionus have two filaments (Bock 1966; Bock and Baimai 1967), whereas in African Zaprionus s.s. they have four filaments. A single exception in Zaprionus s.s. is Zaprionus davidi whose eggs have also two filaments (Chassagnard and Tsacas 1993). However, they still can be distinguished from those of the Oriental species by the presence in the latter of a thin, chitinized crest at the apex of the operculum.

The length of the filaments varies between species (Table 3). In Zaprionus momorticus, the four filaments are very short (Graber 1957). In most species, however, the posterior (dorsal) filaments are usually longer than the anterior (ventral) ones. In some species (Zaprionus mascariensis, Zaprionus kolodkinae, Zaprionus sepsoides and Zaprionus tsacasi) of the Zaprionus tuberculatus species subgroup (Fig. 3), the posterior filaments are usually elongated and spatulate near the apex.

Larva

Larvae of the genus Zaprionus are all of the amphipneustic type as in other drosophilid flies (Okada 1968). In all instars of both subgenera, the larval cephalopharyngeal skeleton is smooth lacking any dentition (Fig. 15). In all species, when cultures are crowded, the mature larvae climb up the bottle and often escape through the plug, and die from desiccation (Bock 1966; David et al. 2006). Zaprionus lachaisei sp. n. is the only species of which larvae do not show this peculiar behavior, and this makes its laboratory culture an easier.

Puparium

Puparia of the two subgenera are reddish brown in color (Fig. 11). The puparial length (PL) ranges from 2.82 mm in Zaprionus gabonicus to 4.58 mm in Zaprionus inermis, in complete concordance with the differences of body size in the adults (Yassin and David, in press). The only other species with PL exceeding 4.00 mm are Zaprionus lachaisei sp. n. (PL = 4.30 mm) and Zaprionus bogoriensis (PL = 4.20 mm). The puparial shape (PL:Pl) ranges from 2.24 in Zaprionus santomensis sp. n. to 2.65 in Zaprionus vittiger. Interestingly this ratio can serve in discriminating puparia of some close species such as between: Zaprionus inermis (2.62) and Zaprionus cercus (2.40), and Zaprionus tuberculatus (2.59) and Zaprionus burlai (2.29).

The horn-index (H) is a classical taxonomic measurement in drosophilid systematics. H ranges from 5.0 in Zaprionus lachaisei sp. n. (Fig. 11d) to 15.3 in Zaprionus neglectus (Fig. 11A) with the mean of 9.7 ± 0.4 in African Zaprionus (9.3 in the Oriental species Zaprionus bogoriensis). With the exception of the two extremes, H ranges from 6.8 to 13.1. In the tuberculatus species complex, H discriminates Zaprionus verruca (H = 10.6) from its two sibling species, Zaprionus tuberculatus (H = 7.0) and Zaprionus burlai (H = 7.2).

Another important taxonomic character of the puparium is the branches of the anterior spiracle. In all Zaprionus species, these branches are of the clubbed type (Okada 1968). The arrangement of the branches on the stalk is of the type Y in which pseudocentral branches (sensu Okada 1968) are absent. The number of branches tends to vary from 11 to 14 in the inermis species group, and from 15 to 17 in the vittiger group. A particular exception is found in Zaprionus inermis where the number of branches ranges from 18 to 21 (Fig. 11b). This facilitates the discrimination of its puparia from those of its sibling species, Zaprionus cercus, which has 11 to 13 branches (Fig. 11c).