Revision of the afrotropical species of Zaprionus (Diptera, Drosophilidae), with descriptions of two new species and notes on internal reproductive structures and immature stages

Abstract A new classification of the subgenus Zaprionus is proposed in light of recent phylogenetic findings. The boundaries of the armatus and inermis species groups are redefined. The vittiger subgroup is upgraded to the level of a species group. The tuberculatus subgroup is transferred from the armatus to the inermis group. A new monotypic group, neglectus, is erected. Full morphological descriptions of four species belonging to the vittiger group are given: Zaprionus lachaisei sp. n. from Tanzania and Zaprionus santomensis sp. n. from São Tomé and Principé, and two cryptic species of the indianus complex, Zaprionus africanus Yassin & David and Zaprionus gabonicus Yassin & David. Three nominal species are synonymised: Zaprionus beninensis Chassagnard & Tsacas, syn. n. with Zaprionus koroleu Burla, Zaprionus simplex Chassagnard & McEvey, syn. n. with Zaprionus neglectus Collart, and Zaprionus megalorchis Chassagnard & Tsacas, syn. n. with Zaprionus ornatus Séguy. Half of the 46 species of the subgenus are available as laboratory strains and this has allowed full descriptions of the internal structure of their reproductive systems and their immature stages.


Introduction
Th e 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 classifi ed 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). Th e 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) classifi ed 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(Yassin et al. , 2010. 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.

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, Z. (Anaprionus) bogoriensis Mainx, was added to the analysis.  Chassagnard, 1996 h, and Z. (Z.) davidi Chassagnard & Tsacas, 1993 i.

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.

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. Th is 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. Th ey 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. Th e 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 diff erences were only found within those for which multiple measurements were taken.

. Z. gabonicus
[Gabon] -♂ aedeagal fl ap serrated basally (Fig. 12g) (43)  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. Th e 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 fi ndings, a new classifi cation scheme is proposed (Table 2). Table 2 also shows the breeding niche and the possibility to rear in the laboratory for some species. Th ese 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, fl ower and decaying tree trunk breeding. Most species are fruit breeders. Some species (e.g., Z. badyi, Z. momorticus, and Z. neglectus) are generalist fl ower-breeders, whereas two species of the armatus group (Z. fumipennis and Z. vrydaghi) breed exclusively in fl owers of Costus afer (Tsacas and Chassagnard 1990). Records of Z. montanus suggest this species to mine bamboo leaves or stems (Graber 1957;Chassagnard 1989). Th e breeding niche of its sibling species, Z. 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 diffi culty of rearing). Other Zaprionus  Chassagnard, 1989 c, Z. serratus Chassagnard, 1989d Z. proximus Collart, 1937e, and Z. indianus Gupta, 1970 species that were also bred from cut tree trunks included Z. armatus, Z. inermis and Z. ghesquierei.
It is still diffi cult to estimate with certainty the niches for some of the problematic species in Lachaise and Tsacas's (1983) review. For example, Z. 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) re view 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 Z. 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 Z. sepsoides and Z. tuberculatus. Records on the Gabonese strain of Z. ornatus in Lachaise and Tsacas (1983) were assigned to Z. taronus since Chassagnard and Tsacas (1993) showed this strain to be misidentifi ed with Z. ornatus by Tsacas (1980).

Th e armatus group
Th e armatus group was initially erected to include three subgroups: armatus, tuberculatus and vittiger (Chassagnard and Tsacas 1993   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 diff erentiation 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, Z. campestris, and its phylogenetic position did not confi rm Yassin et al.'s (2008a) placement (Yassin et al., in press). Th erefore, Tsacas and Chassagnard's (1990) subclassifi cation will be retained with slight modifi cations until new molecular sequences become available. Th e 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 diff erentiated F1 spines (Ensemble II); and the armatus subgroup with nine species bearing a row of undiff erentiated F1 spines (Ensemble III). Th e armatus subgroup is further subdivided into three complexes: the hoplophorus complex with two species bearing diff erentially oriented strong F1 spines; the armatus complex with fi ve species bearing undiff erentially oriented strong F1 spines; and the vrydaghi complex with two species bearing undiff erentially oriented fi ne F1 spines and wings blackened anteriorly.

Th e inermis group
Th e inermis group comprises species with spineless F1 (Figs 2a, b). Th e 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 Z. litos and Z. neglectus being closely related to the arma- tus and the vittiger groups. Th ese suggestions were confi rmed by later molecular analyses (Yassin et al., in press) which also suggested that two other species (Z. sexstriatus and Z. sexvittatus) formed the sister clade with the vittiger group. Four species of the inermis group (Z. arduus, Z. badyi, Z. momorticus and Z. 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 classifi ed 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. Th e F1 of several species of tuberculatus subgroup carries a tubercule (Fig. 2b). Th ese two subgroups are closely related to each other as they share the bare and bristleness epandrium (Fig. 7)  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.

Discussion.
Zaprionus neglectus is a continental species lacking F1 ornamentation and the hairy brush on F1 basitarsus in males (Collart 1937b ; Fig. 5a). It is the only species previously belonging to the inermis group to lack such a secondary sexual character. Two species of the spinosus subgroup of the armatus group also lack the male hairy brush. Burla (1954) and Lachaise and Tsacas (1983) described that Z. neglectus bred on decaying fruits and in fl owers of Ipomoea and Crinum. Chassagnard and McEvey (1992) described a species, Z. simplex, lacking F1 ornamentation and the male hairy brush from Madagascar. Th ey also noted that some specimens were "collected from Crinum sp. fl owers but no evidence was found that it bred therein" (p. 322).
We have recently collected a strain of Z. simplex from Crinum sp. in Madagascar and reared it in the laboratory. Burla (1954) noted the presence of two long caecae around  Tsacas & Chassagnard, 1990 f, and Z. serratus Chassagnard, 1989g [From Chassagnard 1989, 1996Tsacas and Chassagnard 1990; courtesy of M. T. Chassagnard]. the ejaculatory bulb in males of Z. neglectus. Dissection of cultured males of Z. simplex also revealed the presence of long caecae in the Malagasy strain. Wing shape indices were also strongly similar in the original descriptions of the two species. Hence, Z. simplex Chassagnard & McEvey is considered a junior synonym to Z. neglectus Collart. Yassin et al. (2008a) suggested in light of morphological characters Z. simplex, syn. n. to belong to the armatus group, but in the lack of molecular data of any species of this group such relation remains questionable. Indeed, the species has more than 2 epandrial bristles and lacks any F1 ornamentation. Molecular analysis of the Malagasy strain showed the species to be the earliest branch of the subgenus not belonging to any of the three other species groups (Yassin et al., in press). Th us, a group is erected for this single species.

Th e vittiger group
Th e 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 fl ap. Th e 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 ). Th ree species (Z. sexstriatus, Z. sexvittatus and Z. litos) have the unarmed F1 and have been classifi ed in the inermis group (Chassagnard and Tsacas 1993;Chassagnard 1996). Species with F1 bearing composite spines are classifi ed into six complexes: the sexvittatus complex with three species having two additional submedian silvery longitudinal stripes on the thorax (Fig. 1); the ornatus complex   Chassagnard, 1990 c, andZ. tuberarmatus Tsacas &Chassagnard, 1990 d. with two species having the aedeagal fl ap 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 fi ve species having the partially hairy epandrium and the smooth spermatheca. Chassagnard and Tsacas 1993, syn. n.

Z. megalorchis
Discussion. Séguy (1933) described a species of the vittiger group from Côte d'Ivoire, which has diff erentiated F1 composite spines; i.e. the spines are borne on protruding tubercules that decrease in size distally. He called the species Z. ornatus. Collart (1937a) considered this character an intraspecifi c variation and synonymised Z. ornatus with Z. vittiger. Chassagnard and Tsacas (1993) redescribed Séguy's female holotype and illustrated the distinctive elongated spermatheca that had also been previously illustrated by Burla (1954) for Z. aff . vittiger. In the same paper, they also described a new species from Congo with the distinctive elongated spermatheca and F1 ornamentation. Th ey called the new species Z. megalorchis and noted that the only diff erence between it and Z. ornatus was the presence of silver pilosity on the inner side of fl agellomere I in Z. ornatus. Yassin et al. (2008a) erected the megalorchis species complex for the two species. However, Figure 10. Lateral and dorsal views of Zaprionus koroleu Burla, 1954a, b, Z. vittiger Coquillett, 1902 n. e, Z. santomensis Yassin & David, sp. n. f, and Z. camerounensis Chassagnard & Tsacas, 1993 g, h. we have examined a number of strains collected from the type locality of Z. megalorchis and found the fl agellomere I pilosity to be polymorphic. We consider thus Z. megalorchis Chassagnard & Tsacas, syn. n. and Z. aff . vittiger Burla, syn. n. to be junior synonyms to Z. ornatus Séguy. Yassin et al. (2008b) have also considered Z. megalorchis (and thus Z. ornatus) a member of the indianus species complex, but it is considered here as belonging to an independent, monophyletic complex along with Z. litos (Yassin et al., in press).

Zaprionus (Zaprionus) africanus Yassin & David in Yassin et al. 2008b
Diagnosis. Th is species resembles Z. indianus and Z. gabonicus, but can be distinguished from them by the deep serration of the apical margin of the aedeagal fl ap, the shape of the spermatheca being wider than long and the presence of 8 (rarely 7) peglike ovisensilla on the oviscape, which is constricted ventrally (Fig. 12). Th orax. Scutum brown, darker than frons, with 2 silvery white stripes. acs in 6 rows in front of adc; adc:pdc = 0.8. Scutellum darker than scutum, with black borders of the stripes expanded posteriorly; bsc:asc = 0.7. Pleura yellow; sterno-index = 0.38. Forefemur with 4-5 spines borne on warts on the anteroventral margin. Male basitarsus with a hairy brush.
Abdomen. Entirely yellow with deep dark spots at the bases of tergal setae. Terminalia. Epandrium densely pubescent throughout its entire length; posterior margin pubescent at dorsal portion with 4 long setae; epandrial ventral lobe with 3 long setae. Surstylus quadrate with two rows of prensisetae. Cercus triangular laterally. Hypandrium densely pubescent at the lateral portion of the paraphyses. Aedeagus expanded apically with a hook-like appendix; aedeagal fl ap expanded and deeply serrated. Apodeme subequal in length to aedeagus.
Egg. Elliptical with 4, equally long and fi ne fi laments. Larva. Escaping the culture medium when crowded.
Abdomen. Entirely yellow with deep dark spots at the bases of tergal setae. Terminalia. Epandrium densely pubescent throughout its entire length; posterior margin pubescent at dorsal portion with 4 long setae; epandrial ventral lobe with 3 long setae. Surstylus quadrate with two rows of prensisetae. Cercus triangular laterally. Hypandrium densely pubescent at the lateral portion of the paraphyses. Aedeagus slender expanded apically without a hook-like appendix; aedeagal fl ap expanded and not serrated. Apodeme subequal in length to aedeagus.
Egg. Elliptical with 4 equally long and fi ne fi laments. Larva. Escaping the culture medium when crowded.

Discussion
. Th e identity of the dark species Z. koroleu has long been problematic since its description by Burla (1954) from lowland rainforests in Côte d'Ivoire. It had often been confused with another montane dark species in Uganda (Buruga 1976) and Cameroon (Tsacas 1980;Bennet-Clark et al. 1980), which was later described as Z. camerounensis by Chassagnard and Tsacas (1993). Chassagnard and Tsacas (1993) re-examined Burla's type and considered the enlargement and fusion of BV on the scutellum a characteristic trait of Z. koroleu in the lack of distinctive features of the male genitalia. However, the examination of diff erent strains of Z. vittiger has shown this character to be polymorphic and not exclusive to Z. koroleu. Chassagnard and Tsacas (1993) also noted that Z. koroleu is distinguishable from Z. beninensis in having the thorax and abdomen darker than the frons, whereas in Z. beninensis the abdo- Figure 15. Larval cephalopharyngeal skeleton of Zaprionus sepsoides Duda, 1939. men is darker than the frons and the thorax as confi rmed by re-examining the type series of Z. beninensis. All species of the vittiger complex are found in high latitudes or altitudes with the exception of Z. koroleu and Z. beninensis. Burla (1954) noted that Z. koroleu was bred in Côte d'Ivoire from decaying Raphia trunk along with other palm breeding drosophilids of the genera Chymomyza and Scaptodrosophila, and this was similar to the breeding niche of Z. beninensis in Benin (fallen trunks of coconut palm; J. R. David, unpublished observations). Both species are, however, generalists as Burla (1954) bred Z. koroleu also from fermenting fruits and as Z. beninensis was maintained in laboratory for almost ten years (Chassagnard and Tsacas 1993 Diagnosis. Th is species resembles Z. vittiger, but has the bigger body size (TL > 1.60 mm), spiniform spines enlarged and blackened on the fi rst two tarsomeres of the foreleg (Fig. 5), and shorter puparial anterior spiracles (H = 5) (Fig. 11). It is also distinguishable by a peculiar behavior of the larvae which do not leave the culture bottle when disturbed or crowded. Description. ♂. TL = 1.68 mm. Head. Arista with 3 dorsal and 2 ventral rays plus a terminal fork, pedicel tan. Frons orange-tan with lateral white stripes; median white stripe absent; ocellar triangle raised and darker; hw:fw = 2.04, fw:fl = 1.05. Face pale; carina large; palpus yellow. Gena broad, o:j = 10.2, o:ch = 5.2. Orbital bristles in straight line; or2 very minute, or1:or2:or3 = 7:2:5, orbito-index = 1.4. Ocellar setae long, divergent; oc:or1 = 1.3, poc:oc = 0.5, iv:ov = 0.6. Eye red and densely pilose.
Th orax. Scutum tan, darker than frons, with four white longitudinal stripes continuing on scutellum; white stripes narrow, bordered with large black stripes, especially on the inner side; acs in 6 regular rows anterior to adc and 4 irregular rows between them; psc enlarged, adc:psc = 1.5; adc:pdc = 0.6. Scutellum slightly pointed at the apex, where white spot absent; bsc:asc = 1.3. Sterno-index = 0.6. F1 with 4 setiferous spines not borne on tubercules on the anteroventral margin. Basitarsus of the foreleg with a hairy brush on the ventral margin. Spiniform spines of the fi rst and second tarsomeres of the foreleg enlaged and blackened.
Abdomen. Uniformly tan, with dark spots at the bases of tergal bristles. Terminalia (Fig. 13a). Epandrium densely pubescent at ventral portion; posterior margin pubescent at dorsal portion with 5 long bristles; anterior phragma narrow; epandrial ventral lobe with 3 long bristles. Surstylus quadrate with two rows of prensisetae. Cercus triangular laterally. Hypandrium with a small pubescent patch at the lateral portion of the paraphyses. Aedeagus expanded apically; aedeagal fl ap expanded and deeply serrate. Apodeme subequal in length to aedeagus.
Egg. Elliptical with 4 equally long and fi ne fi laments. Larva. Not escaping the culture medium when disturbed or crowded.
Discussion . Attempts to hybridize this strain with others belonging to the vittiger complex have all failed. Th e species is very prolifi c and easy to breed in the laboratory.
Etymology . Patronym, in honor of the French Drosophila systematist Dr. Daniel Lachaise (1948Lachaise ( -2006, collector of the types of two new species described here. Zaprionus sp. B in Araripe et al. 2004 Diagnosis. Th is species resembles those of the indianus complex in having abdominal tergal spots and F1 spines not borne on protruding tubercule. It can be distinguished from them by the bigger body size, the darker body color mainly in contrast with the frons which is bright orange (Fig. 10f ), the wings being dusky rather than hyaline, the smaller hairy brush of the male basitarsus (1/3 of basitarsus) (Fig. 5e), and the lack of an apical introvert in the spermatheca (Fig. 13d).
Abdomen. Entirely yellowish, lighter than thorax, with faint dark spots at the bases of tergal setae.
Egg. Elliptical with 4 equally long and fi ne fi laments.
Larva. Escaping the culture medium when crowded.
Discussion. Th is species resembles Z. proximus, from which it can be distinguished on the basis of F1 ornamentation. An important physiological diff erence also exists between these species, as Z. santomensis is a very heat-sensitive species since a growth temperature of 25°C is lethal for both sexes and males are sterile at 23 and 24°C (cf. Araripe et al. 2004).
Etymology. Th e species epithet is in reference to the type locality.

Comparative anatomy of reproductive system
Many authors described the internal anatomy of some Zaprionus species that can be grown in laboratory (Burla 1954;Th rockmorton 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 diff erences 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 diff erences.  rionus. 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 Z. ornatus). Th e vas deferens (VD) ranges from 0.04 mm in Z. ghesquierei to 2.20 mm in Z. ornatus. Th e quasi-absence of VD in Z. ghesquierei is exceptional as the next value to it is 0.20 mm in a number of species of the inermis group (Z. inermis, Z. kolodkinae and Z. sepsoides). Indeed, Th rockmorton (1962) described VD morphology in a laboratory strain of Z. ghesquierei. Th e 12 males he dissected "were variable, showing two major types with only slight integradation between them" (pp. 232). Th e 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 fi nd this polymorphism in the few individuals dissected. Th e longest VD in the inermis group is found in Z. burlai (VD = 1.1 mm), and it is greater than VDs of its two relatives (0.7 mm in Z. tuberculatus and 0.8 mm in Z. verruca).

Male reproductive system
Th e ejaculatory bulb of Zaprionus species is moderately large, rounded and bearing long posterior caecae (Th rockmorton 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. Th e length of the caecae (CAE) ranges from 0.1 mm in Z. sepsoides to 2.0 mm in Z. neglectus. Th e long CAE of Z. neglectus is exceptional (Burla 1954) and it was used as one of the arguments to synonymize Z. neglectus Burla with Z. simplex Chassagnard & McEvey. CAE can also be used to distinguish Z. cercus (CAE = 1.6 mm) from its sibling species Z. inermis (CAE = 0.4 mm), which has particularly small CAE. Lachaise (1972) also noted that CAE of Z. 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 Z. tuberculatus and Z. burlai (CAE = 0.3 mm).

Female reproductive system
Th e seminal receptacle (SR) ranges from 0.8 mm in Z. kolodkinae to 12.0 mm in Z. ornatus. As with TST, species of the vittiger group tend to have larger SR than those of the inermis group. Th e 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). Th is correlation is obvious in Zaprionus (r = 0.93; P < 0.001). SR can distinguish Z. burlai females (SR = 6.3 mm) from Z. tuberculatus (SR = 3.6 mm), and Z. indianus (SR = 4.8 mm) from Z. africanus (SR = 3.8 mm) and Z. gabonicus (SR = 3.5 mm). Burla (1954) provided the fi rst 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). Th e elongate form of the spermatheca of Z. ornatus is characteristic and it was one of the arguments for considering Z. megalorchis Chassagnard and Tsacas syn. n. and Z.
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 Z. africanus the width of the spermatheca was always relatively greater than its length, whereas in its two cryptic species Z. indianus and Z. 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 Th e eggs of species of the Oriental subgenus Anaprionus have two fi laments (Bock 1966;Bock and Baimai 1967), whereas in African Zaprionus s.s. they have four fi laments. A single exception in Zaprionus s.s. is Z. davidi whose eggs have also two fi laments (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.
Th e length of the fi laments varies between species (Table 3). In Z. momorticus, the four fi laments are very short (Graber 1957). In most species, however, the posterior (dorsal) fi laments are usually longer than the anterior (ventral) ones. In some species (Z. mascariensis, Z. kolodkinae, Z. sepsoides and Z. tsacasi) of the Z. tuberculatus species subgroup (Fig. 3), the posterior fi laments are usually elongated and spatulate near the apex.

Larva
Larvae of the genus Zaprionus are all of the amphipneustic type as in other drosophilid fl ies (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). Th e puparial length (PL) ranges from 2.82 mm in Z. gabonicus to 4.58 mm in Z. inermis, in complete concordance with the diff erences of body size in the adults (Yassin and David, in prep.). Th e only other species with PL exceeding 4.00 mm are Z. lachaisei sp. n. (PL = 4.30 mm) and Z. bogoriensis (PL = 4.20 mm). Th e puparial shape (PL:Pl) ranges from 2.24 in Z. santomensis sp. n. to 2.65 in Z. vittiger. Interestingly this ratio can serve in discriminating puparia of some close species such as between: Z. inermis (2.62) and Z. cercus (2.40), and Z. tuberculatus (2.59) and Z. burlai (2.29).
Th e horn-index (H) is a classical taxonomic measurement in drosophilid systematics. H ranges from 5.0 in Z. lachaisei sp. n. (Fig. 11D) to 15.3 in Z. neglectus (Fig. 11A) with the mean of 9.7 ± 0.4 in African Zaprionus (9.3 in the Oriental species Z. bogoriensis). With the exception of the two extremes, H ranges from 6.8 to 13.1. In the tuberculatus species complex, H discriminates Z. verruca (H = 10.6) from its two sibling species, Z. tuberculatus (H = 7.0) and Z. 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). Th e arrangement of the branches on the stalk is of the type Y in which pseudocentral branches (sensu Okada 1968) are absent. Th e 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 Z. inermis where the number of branches ranges from 18 to 21 (Fig. 11b). Th is facilitates the discrimination of its puparia from those of its sibling species, Z. cercus, which has 11 to 13 branches (Fig. 11c).