SYSTEMATICS Molecular and Morphometrical Revision of the tuberculatus Species Subgroup (Diptera: ), with Descriptions of Two Cryptic Species

AMIR YASSIN1

Laboratoire Evolution, Ge´nomes et Spe´ciation, Centre National de la Recherche ScientiÞque, av. de la Terrasse, 91198 Gif-sur-Yvette, France Downloaded from https://academic.oup.com/aesa/article/101/6/978/2758498 by guest on 27 September 2021

Ann. Entomol. Soc. Am. 101(6): 978Ð988 (2008) ABSTRACT Zaprionus is an important drosophilid genus in the Afrotropical region. Here, two new species, Z. burlai n. sp. and Z. tsacasi n. sp., are described from Tanzania and Sa˜o Tome´, respectively. The two species show incomplete reproductive isolation with Z. tuberculatus Malloch and Z. sepsoides Duda, respectively, with intercrosses producing fertile females but sterile males. The latter two have long been considered sibling species and together with three other species (Z. mascariensis Tsacas & David, Z. kolodkinae Chassagnard & Tsacas, and Z. verruca Chassagnard & McEvey) form the tuberculatus subgroup. The phylogenetic relationships of these seven species of the subgroup were revised in light of mitochondrial (COII) gene sequences and wing morphometrics. Mitochondrial DNA Þrmly distinguished most of the species, except for a triad of Z. tuberculatus, Z. verruca, and Z. burlai. Wing morphometrics was able to distinguish between closely related species and also indicated the altitudinal origin of each species. Most species can be identiÞed through internal anatomy of the reproductive system (testis and seminal recep- tacle lengths), and the discovery of the new species with incomplete reproductive isolation may help in understanding the genetic basis of this variation through interspeciÞc hybridization. The molecular phylogeny reconÞrmed the Malagasy origin of the subgroup during the Late Pliocene. Colonization of probably involved two independent events during the Pleistocene.

KEY WORDS COII barcoding, Africa paleobiogeography, wing shape, cryptic speciation, interspe- ciÞc hybridization

Zaprionus (Coquillett 1901) is a pan-tropical dros- species (Tsacas et al. 1981, Chassagnard and Tsacas ophilid genus divided into two geographically disjunc- 1993). Even within the most widespread and most tive subgenera: the subgenus Zaprionus s.s. (46 spe- studied species, Z. indianus, two cryptic species en- cies) located in the Afrotropical region, and the demic in tropical Africa were discovered using mo- subgenus Anaprionus (10 species) found in the Ori- lecular and morphometrical analyses (Yassin et al. ental and Australasian region. Yassin et al. (2008a) 2008b). revised the phylogeny of the genus based on molec- The Z. tuberculatus species subgroup has been ular markers, and they suggested a recent origin of the erected as a member of the armatus group of the genus in the Oriental region of Ϸ10Ð13 million years subgenus Zaprionus s.s. (Chassagnard and Tsacas ago (MYA), followed by the colonization of Africa via 1993). It was originally created as a complex of three the maritime route of the islands of the Indian Ocean species characterized by the presence of a spur borne Ϸ7 MYA. The subgenus Zaprionus is very common in on a salient tubercle on the medioventral margin of the Africa (Tsacas et al. 1981). Recently, three African forefemur (Tsacas et al. 1977), namely, Z. tuberculatus, species (Z. indianus Gupta, Z. tuberculatus Malloch, Z. sepsoides Duda, and Z. mascariensis Tsacas & David. and Z. ghesquierei Collart) have been introduced into The two former species are widespread throughout the Palearctic region (Chassagnard and Kraaijeveld the Afrotropical region and the islands of the Indian 1991), with Z. indianus further expanding its range in Ocean, whereas Z. mascariensis is restricted to the India and the Americas (David et al. 2006, Yassin et al. insular Indian Ocean. A molecular phylogenetic revi- 2008b). Due to its lack of sexual dimorphism, high sion of the subgenus (Yassin et al. 2008a) revealed that ecological diversity, and its great morphological uni- 1) the subgroup is a member of the inermis group, and formity, Zaprionus is thought to harbor many cryptic its forefemoral ornamentation is not homologous to that of other species of the armatus group; 2) the subgroup is polyphyletic with two species from the 1 Current address: De´partement Syste´matique et Evolution, Mu- se´um National dÕHistoire Naturelle, UMR 5202 GPS3, 16 rue Buffon, inermis group, Z. kolodkinae Chassagnard & Tsacas 75005 Paris, France (e-mail: [email protected]). and Z. verruca Chassagnard & McEvey, and endemic

0013-8746/08/0978Ð0988$04.00/0 2008 Entomological Society of America November 2008 YASSIN:SYSTEMATICS OF Z. tuberculatus SUBGROUP 979 to Madagascar. In addition, two new species, Z. burlai Molecular Analysis. Mitochondrial DNA was ex- n. sp. from Tanzania and Z. tsacasi n. sp. from Sa˜o tracted from single ßies by using a DNA extraction kit Tome´, with incomplete reproductive isolation with Z. (QAIGEN, Hilden, Germany). Polymerase chain re- tuberculatus and Z. sepsoides, respectively, are de- action ampliÞcation, COII primers, and sequencing scribed here. This increases the number of members protocols were as described in Yassin et al. (2008b). of the tuberculatus subgroup from three to seven spe- Nucleotide sequences were viewed and manually ed- cies. ited using Molecular Evolutionary Genetics Analysis Z. tuberculatus and Z. sepsoides are similar at the (MEGA) version four (Tamura et al. 2007). Table 1 morphological level, and they can only be distin- shows GenBank accession numbers for the different guished on the basis of internal anatomical structures species and populations used in this study. MEGA also (testis length and shape of egg velae). This requires was used to reconstruct phylogenies using neighbor- freshly killed specimens or laboratory cultures, which

joining (NJ) (Saitou and Nei 1987) and maximum Downloaded from https://academic.oup.com/aesa/article/101/6/978/2758498 by guest on 27 September 2021 may eliminate the use of museum-pinned or alcohol- parsimony (MP). Maximum likelihood (ML) and Bayes- preserved material. Recent advances in molecular and ian inference (BI) were performed using PHYML morphometrical tools should help to delimit cryptic (Guindon and Pascual 2003) and MrBayes version 3.1 species easier (see Bickford et al. 2007). Here, the (Ronquist and Huelsenbeck 2003), respectively, by us- seven species of the tuberculatus subgroup were re- ing the GTR ϩ⌫substitution model (Tavare´ 1986) as vised in light of molecular analysis of the mitochon- proposed by the FindModel program (Tao 2005). drial cytochrome oxidase subunit two (COII) gene Monophyly-conÞdence limits were determined at 50% and morphometrical analysis of wing venation. It has cut-off after 500 bootstrap iterations (NJ, MP, and recently argued that the species identiÞcation utility ML) or 50% posterior probability after a run of of COII is equivalent to that of COI in the DNA 2,000,000 generations (BI). Divergence times were barcoding project in Diptera (Roe and Sperling 2007). estimated using the mutational rate of Because neither DNA sequences nor morphometrical 1.1 ϫ 10Ϫ8 substitution per site per year (Tamura et al. analyses are substitutes for morphological alpha-tax- 2004) under a relaxed UCLN clock model (Drum- onomy (Schlick-Steiner et al. 2007), detailed descrip- mond et al. 2006) as implemented in the BEAST ver- tions of the two new cryptic species are included here. sion 1.4 package (Drummond and Rambaut 2007). The morphological evolution and historical biogeog- Morphometrical Analysis. Right wings of 10 males raphy of the subgroup also are discussed. per species were mounted in 20% glycerol and pho- tographed using a digital camera attached to a stereo- Materials and Methods scope (Lecia, Wetzlar, Germany). Only Þve males were available for Z. verruca. For Z. tuberculatus and Specimen Preparation and Morphological Descrip- Z. sepsoides, individuals from the same geographical tion. were collected using fermenting banana traps or by net sweeping over rotten fruit. Laboratory location (Eshowe, South Africa) were used. Ten vein cultures were maintained at 21ЊC on standard Dro- intersections and terminations were landmarked by sophila medium, following the precautions described using the TpsDig 2 software package (Rohlf 2006) as for Zaprionus in David et al. (2006). Z. kolodkinae and shown in Fig. 2. Interlandmark distances were esti- Z. verruca ßies were only available as frozen material mated using PAST version 1.68 (Hammer et al. 2001) of the type strains. package. Formulae for Drosophila wings (McEvey Formal morphological description of the new spe- 1990) were estimated from interlandmark distances as ϭ ϭ ϭ cies followed standard Drosophila terminology and follows: C-index a/b, 4v-index c/d, 4c-index ϭ ϭ ϭ index formulae (McEvey 1990). Specimens were de- b/d, 5x-index e/f, M-index e/d, and ac-index posited in Laboratoire Evolution, Ge´nomes et Spe´cia- b/i (Fig. 2). The C3-fringe index, the ratio of the tion, Gif-sur-Yvette, France (LEGS), as living cul- length of the heavy to the light setation in third costal tures, frozen and alcohol-preserved material, and in section, was included in the morphological descrip- the Muse´um National dÕHistoire Naturelle, Paris, tion but not in the morphometrical analysis. Overall France (MNHN) as pinned material. For comparative shape variation among species was analyzed using purposes, male genitalia of the seven species were multivariate analysis of variance (MANOVA) with dissected, mounted on microscopic slides (Fig. 1), and species as the main effect as implemented in PAST. A preparations are stored in LEGS. Anatomical struc- multivariate version of the discriminant function anal- tures were abbreviated as follows: fw, front width; ß, ysis is the canonical variate analysis (CVA) which front length; hw, head width; o, maximum diameter of produces a scatter plot of individuals along the two the eye; j, width of gena in line with o; ch, maximum Þrst canonical axes, producing maximal and second to width of gena; or1, proclinate orbital seta; or2, anterior maximal separation between all species. The canonical reclinate orbital seta; or3, posterior reclinate orbital axes are linear combinations of the original variables, seta; oc, ocellar seta; poc, postocellar seta; iv, inner and eigenvalues indicate amount of variation ex- vertical seta; ov, outer vertical seta; acs, acrostichal plained by these axes. The biological meaning of each setulae; adsc, anterior dorsocentral; pdsc, posterior canonical axis can be interpreted by estimating its dorsocentral; psc, prescutullar seta; bsc, basal scutellar morphometrical character loadings, i.e., the correla- seta; asc, apical scutellar seta; L, wing length; and w, tion between each index and the scores along each wing width. canonical axis. 980 ANNALS OF THE ENTOMOLOGICAL SOCIETY OF AMERICA Vol. 101, no. 6 Downloaded from https://academic.oup.com/aesa/article/101/6/978/2758498 by guest on 27 September 2021

Fig. 1. Photomicrographs of male genitalia of the seven species of the Z. tuberculatus subgroup. Z. mascariensis. (a) Phallic organs (lateral view): af, aedeagal ßap; aa, aedeagal apodeme; hyp, hypandrium. (b) Phallic organs (ventral view): pph, paraphysis; gp, gonopod. Z. kolodkinae. (c) Phallic organs (lateral view). (d) Phallic organs (ventral view). Z. sepsoides. (e) Paraphallic organs: ep, epandrium; eph, epandrial phragma; evl, epandrial ventral lobe; su, surstylus; ce, cercus. (f) Phallic organs (lateral view). (g) Phallic organs (ventral view). Z. tsacasi n. sp. (h) Epandrium. (i) Phallic organs (lateral view). (j) Surstylus. Z. tuberculatus. (k) Epandrium. (l) Phallic organs (lateral view). (m) Phallic organs (ventral view). Z. verruca. (n) Phallic organs (ventral view). Z. burlai n. sp. (o) Surstylus. (p) Phallic organs (lateral view). (q) Phallic organs (ventral view).

Results egg longer with preapical velae as long as or longer than egg. Holotype Male. Body length 4.6 mm (thorax length Zaprionus (Zaprionus) burlai,n.sp. 1.7 mm). Diagnosis. Resembles Z. tuberculatus, from which it Head. Arista black with three dorsal and two ventral can be distinguished by body darker and bigger, the rays plus one terminal fork. Frons orange tan with a three faint longitudinal vittae between the mesoscutal median white vitta in addition to the two large orbital white vittae only at vertex; aedeagal dorsal margin ones, anterior region in front of or1 lighter, bearing 18 sharply curved; testis and ventral receptacle longer; symmetrical frontal setulae, followed by 9Ð10 larger November 2008 YASSIN:SYSTEMATICS OF Z. tuberculatus SUBGROUP 981

Table 1. List of species and populations of the Z. tuberculatus subgroup used in this study with GenBank accession number for the COII gene

Species Pop Coordinates Altitude COII burlai n. sp. Amani, Tanzania 39.05, Ϫ5.07 870 EU95363 kolodkinae Tsacas and Chassagnard Tsimbazaza, Madagascar 45.10, Ϫ22.40 1300 EF453715 mascariensis Tsacas & David La Runion Island 56.40, Ϫ20.40 300 EF453714 sepsoides Duda Brazzaville, Congo 15.14, Ϫ4.14 300 EU95375 Eshowe, South Africa 31.25, Ϫ28.52 500 EU95370 Kibale, Uganda 32.35, 0.46 1100 EU95376 Uganda 32.00, 1.00 1000 EU95372 tsacasi n. sp. So Tom, So Tom and Principe 6.43, 0.19 1500 EU95374 tuberculatus Malloch Alexandria, Egypt 29.55, 31.15 10 EU95368 Brazzaville, Congo 15.14, Ϫ4.14 300 EU95369 Crete, Greece 25.00, 35.10 400 EU95365 Downloaded from https://academic.oup.com/aesa/article/101/6/978/2758498 by guest on 27 September 2021 Eshowe, South Africa 31.25, Ϫ28.52 500 EU95370 Kibale, Uganda 32.35, 0.46 1100 EU95372 Makoukou, Gabon 12.47, 0.38 500 EU95371 Niamey, Niger 2.05, 13.32 200 EU95367 So Tom, So Tom and Principe 6.43, 0.19 600 EU95366 Zimbabwe 32.40, Ϫ19.00 1200 EU95373 verruca Chassagnard & McEvey Mandraka, Madagascar 45.00, Ϫ20.00 1200 EU95364

Underlined accession numbers are from Yassin et al. (2008a). setulae between or1; ocellar triangle reddish brown deÞned (adc:psc ϭ 2.2); adc:pdc ϭ 0.7, bsc parallel to and raised, fw:ß ϭ 0.9, hw:fw ϭ 2.2. Pedicel silvery slightly divergent (bsc:asc ϭ 0.7), pdc:asc ϭ 0.7. Pleura white in line with orbital vittae, ßagellomere I dusky concolorous dark, with large white vitta at the ventral tan, with short dark pile. Facial carina broad, high and margin of anepisternum. Sterno-index ϭ 0.26, two bulbous. Face subshining tan. Palpus tan with one short median katepisternal setae, sternopleural setulae prominent apical seta. Gena broad, uniformly tan; in two rows between katepisternal setae. Fore femur o:ch ϭ 4.6; o:j ϭ 6.4. Eye sepia, oblong and covered with a salient tubercle ventrally, pointing distally and with dense bright pile. Orbital setae in straight line, bearing one stout seta, one minute setula, and a spur, or1:or2:or3 ϭ 1.2:0.5:1.0, orbito-index ϭ 1.3; oc:or1 ϭ the three ventro-distal setae of equal size. Preapical 1.2, poc convergent and well developed ϭ 0.85 (poc: spurs on three tibiae, apical spur on midleg tibia; tar- oc ϭ 0.6); or3:iv ϭ 1.0. somere I of the foreleg with a tuft of pale tan sensilla, Thorax. Scutum dark brown, darker than frons, with tarsomere V blackish. two longitudinal silvery white vittae on each side: one Wing. Yellowish iridescent, veins tan. L:w ϭ 2.6, lying lateral to dsc setae and in line with orbital vitta, C-index ϭ 1.9, 4v-index ϭ 1.6, 4c-index ϭ 1.1, 5x- and continuing on scutellum, the other beginning at index ϭ 1.2, M-index ϭ 0.4, ac-index ϭ 3.6, C3-fringe ϭ the anterior part of the postpronotum and ending in 0.59, and length ϭ 2.7 mm. Halter tan. the wing axillary area; each vitta bordered with black Abdomen. Uniformly yellow, with a faint brownish especially internally; area between the dorsocentral line at the posterior margin of each tergite. vittae uniformly dark brown, except at vertex with two Male Terminalia (Fig. 1o–q; holotype not dis- whitish spots; scutellum concolorous, with small white sected). Epandrium longitudinally narrow, posterior patch apically. Postpronotal setae 3, acs setulae black, margin hirsute with Þne setulae, anterior phragma in six rows anterior to dsc and between them, psc well humped at dorsal part, epandrial ventral lobe with

Fig. 2. Wing of Z. sepsoides showing shape coordinate landmarks (numbered ●) and lettered interlandmark dis- tances. 982 ANNALS OF THE ENTOMOLOGICAL SOCIETY OF AMERICA Vol. 101, no. 6 three long setae. Surstylus rectangular with two rows Msingi (1400 m), on coffee cultivation in front of a of prensisetae, the dorsal row with 4Ð5 long setae, the European house, 22Ð28-I-1952 (E. Lindner). ventral row with 7Ð8 smaller setae. Cercus pointed Etymology. Patronym, in honor of the Swiss Dro- laterally, with a tuft of short setulae at the hypoproctal sophila systematist Dr. Hans Burla (Zoological Mu- plate. Hypandrium semiquadrate, paraphyses en- seum, Zurich), the Þrst to suggest the presence of larged bearing two short setae. Aedeagus short, robust, cryptic species in Z. tuberculatus. deeply curved at apex, aedeagal ßap Þnely serrated. Apodeme subequal in length to aedeagus. Testis long (4.4 mm), caecum 0.3 mm. Zaprionus (Zaprionus) tsacasi, n. sp. Allotype Female. Resembles male. Female Terminalia. (Fig. 2; allotype not dissected) Holotype Male. Body length 4.4 mm (thorax ϭ

Oviscapt tan, with 12 peg-like and three Þne, marginal length 1.8 mm). Downloaded from https://academic.oup.com/aesa/article/101/6/978/2758498 by guest on 27 September 2021 setae plus three to four supernumerary nonmarginal Diagnosis. Resembles Z. sepsoides Duda, from which setae. Seminal receptacle very long (6.3 mm). Sper- it can be distinguished by distal forefemoral setulae un- matheca brown, quadrate and papillate at the upper differentiated; aedeagal curvature deeper, testis very half. Egg long, preapical velae as long as or longer short (1.3 mm), caecum very long. than egg. Head. Arista black with three dorsal and two ventral Distribution. TANZANIA, endemic. rays plus one terminal fork. Frons orange tan with a Type Material. Holotype (male) and allotype (fe- narrow, median white vitta not bordered with black, male), TANZANIA, East-Usambara Mountains, Amani in addition to the two large orbital ones slightly bor- (870 m), ex type strain ZMT.1Ð6, 8-III-2008, founder dered in black, anterior region in front of or1 lighter, female coll. 25-IX-2002 (D. Lachaise). Paratypes: 10 bearing 12 symmetrical frontal setulae, followed by males and 10 females with same label. Types deposited 9Ð10 larger setulae between or1; ocellar triangle in MNHN. brown and slightly raised, fw:ß ϭ 0.8, hw:fw ϭ 2.1. Remarks. In general, this species resembles Z. tu- Pedicel silvery white in line with orbital vittae, ßag- berculatus, with which it can produce some hybrid ellomere I dusky tan, with short dark pile. Facial carina progeny, although the cross is difÞcult. F1 hybrid fe- broad, high and bulbous. Face subshining tan, clypeus males are fertile, but F1 males are completely sterile. brownish. Palpus tan with one prominent apical seta. To avoid contamination in the lab, the strain was Gena broad, uniformly whitish yellow; o:ch ϭ 5.2; marked by an eye-color mutation analogous to sepia of o:j ϭ 6.9. Eye red, oblong and covered with dense (Meigen). Some males have bright pile. Orbital setae in straight line, or1:or2:or3 ϭ an additional or3, located in front of or2. Burla (1957) 1.3:0.6:1.0, orbito-index ϭ 1.4; oc:or1 ϭ 1.1, poc con- described three specimens from Msingi, southwest of vergent and well developed (poc:oc ϭ 0.6); iv shorter Mt. Kilimanjaro (1,400 m) to be very similar to Z. than or3 (or3:iv ϭ 1.1). tuberculatus without deciding if they were of a distinct Thorax. Scutum tan, concolorous with frons, with species. At the time of the publication by Burla (1957), two longitudinal silvery white vittae on each side: one the two sibling species Z. tuberculatus and Z. sepsoides lying lateral to dsc setae and in line with orbital vitta, were considered synonyms (Collart 1948). Burla and continuing on scutellum, and the other beginning (1954) described a male tuberculatus and a female at the anterior part of the postpronotum and ending in sepsoides from Abidjan, Coˆte dÕIvoire, under the name the wing axillary area; each vitta bordered with black of Z. tuberculatus inferred from his description (p. 88) especially interiorly; area between the dorsocentral of the testis “Hoden gelb, mit 2one-half inneren und vittae uniformly tan, darker between and posterior to 4a¨usseren Windungen”, and from the description of adc, where anterior edge forms a smooth W; scutellum the egg velae “Die hinteren Velae sind in ihren dis- concolorous, with no apical white patch. Postpronotal talen drei Vierteln stark verbreitert, die vorderen setae 2, subequal; acs setulae black, in six rows anterior Velae sind fadenfo¨rmig.” It is difÞcult to judge from his to dsc and between them, psc well deÞned (adc:psc ϭ illustration of the male genitalia of the questionable 2.6), adc:pdc ϭ 0.8, pdc:asc ϭ 0.9. Pleura tan, with specimens (Þgs. 11Ð14, Burla, 1957: 40Ð41) if they large white vitta at the ventral margin of anepister- were Z. tuberculatus, Z. sepsoides, or Z. burlai n. sp. num. Sterno index ϭ 0.31, two short median katepis- However, Z. sepsoides has not been collected from ternal setae. Forefemoral medioventral tubercle not Tanzania, whereas a strain of Z. tuberculatus was col- very salient, bearing one stout seta, one minute setula, lected from the same type locality of Z. burlai n. sp. and a spur, the three ventro-distal setae of equal size. (Mt. Amani) but was lost. Judging from the descrip- Preapical spurs on three tibiae, apical spur on fore and tion of Burla (1957): 44 of the egg velae and body size mid tibiae; tarsomere I of the foreleg with a tuft of pale of the questionable species “Vom gro¨sseren der beiden tan sensilla. ǨǨ wurden Eier gewonnen; sie zeigen fu¨ r Z. tuber- Wing. Yellowish iridescent, veins tan. L:w ϭ 2.8, culatus typische Velaform; das Vela ist aber etwas C-index ϭ 2.9, 4v-index ϭ 1.6, 4c-index ϭ 0.9, 5x- la¨nger, indem es die Eila¨nge erreicht” they might be- index ϭ 0.9, M-index ϭ 0.3, ac-index ϭ 2.2, C3-fringe ϭ long to Z. burlai n. sp. The three specimens (1 Z and 0.4; length 3.0 mm. Halter tan. 2 Ǩ) are in the Zoological Museum of Zurich, labeled Abdomen. Uniformly tan, almost with no brownish TANZANIA, Southwest-Kilimanjaro Mountains, line at the posterior margin of each tergite. November 2008 YASSIN:SYSTEMATICS OF Z. tuberculatus SUBGROUP 983 Downloaded from https://academic.oup.com/aesa/article/101/6/978/2758498 by guest on 27 September 2021

Fig. 3. Aged Bayesian phylogeny of the species of the Z. tuberculatus subgroup inferred from COII sequences. Internal nodes are numbered.

Male Terminalia (Fig. 1h–j; holotype not dis- to be better adapted to cold temperatures in the lab- sected). Epandrium longitudinally narrow, posterior oratory than is Z. sepsoides. margin hirsute but with no long setae, anterior Etymology. Patronym, in honor of the Greek-born, phragma slightly humped at dorsal part, epandrial ven- French Drosophila systematist, Dr. Leonidas Tsacas tral lobe with 2Ð3 long setae. Surstylus quadrate with (Muse´um National dÕHistoire Naturelle, Paris), dis- two rows of prensisetae, the dorsal row with four long tinguisher of the two sibling species Z. tuberculatus setae, the ventral row with 7Ð8 smaller setae. Cercus and Z. sepsoides, and the Þrst to mention the utility of pointed laterally. Hypandrium semiquadrate, paraph- the subgroup in evolutionary studies. yses enlarged bearing two short setae. Aedeagus short, Molecular Phylogeny. Figure 3 shows the BI phy- robust, deeply curved at apex, aedeagal ßap with Þne logeny estimated for the species (and populations interspaced serration. Apodeme subequal in length to aedeagus. Testis very short (1.3 mm), caecum long -Table 2. Support values and divergence time (؎95% confi (0.4 mm). dence interval) estimates for nodes presented in Fig. 3: bootstrap Allotype Female. Resembles male. values after 500 iterations for neighbor-joining (NJ), maximum Female Terminalia (Fig. 3; allotype not dissected). parsimony (MP), and maximum-likelihood (ML) phylogenies, and Oviscapt yellow, with 10 peg-like and Þve Þne, mar- posterior probability estimates for and Bayesian (BI) phylogenies ginal setae plus two supernumerary, robust nonmar- Divergence time ginal setae. Seminal receptacle short (1.2 mm). Sper- Support value Node no. (in MYA) matheca brown, campanulate and papilate. Egg NJ MP ML BI Mean Min. Max preapical velae spatulate. Distribution. SAO TOME AND PRINCIPE, en- 1 100 100 100 100 2.300 1.106 12.289 2 100 100 100 100 1.288 0.738 2.568 demic. 3 84 83 71 93 0.733 0.498 1.700 Type Material. Holotype (male) and allotype (fe- 4 98 98 96 100 0.371 0.053 1.136 male), SAO TOME AND PRINCIPE, Pico de Sa˜o 5 95 87 99 99 0.065 0.002 0.444 Tome Park (1,500 m), ex type strain ZSS, 8-III-2008, 6 Ñ Ñ 88 20 0.009 Ñ Ñ ´ 7 Ñ Ñ Ñ 20 0.017 Ñ Ñ founder female coll. III-2001 (D. Lachaise). Paratypes: 8 73 75 67 99 0.218 0.132 0.878 10 males and 10 females with same label. Types de- 9 51 50 50 97 0.161 0.025 0.616 posited in MNHN. 10 49 42 46 54 0.114 0.005 0.429 Remarks. In general, this species resembles Z. sep- 11 33 16 27 11 0.190 Ñ Ñ 12 54 10 8 Ñ 0.148 Ñ Ñ soides, with which it mates easily and produces a large 13 Ñ 10 26 5 0.079 Ñ Ñ number of female and male progeny. Hybrid females 14 Ñ Ñ 14 11 0.048 Ñ Ñ are fertile, whereas males are sterile. It differs from Z. 15 66 67 70 99 0.029 0.000 0.230 sepsoides by the characters given in the Diagnosis. 16 56 37 37 98 0.134 0.004 0.265 17 49 21 46 33 0.058 0.000 0.260 Zaprionus sepsoides lives in humid forest at low alti- tudes and it is rarely encountered above 800 m. Zaprio- ConÞdence intervals were not estimated for nodes with BI poste- nus tsacasi n. sp. was collected at 1,500 m, and it seems rior probabilities Յ30. 984 ANNALS OF THE ENTOMOLOGICAL SOCIETY OF AMERICA Vol. 101, no. 6

Table 3. Mean and range ͓in brackets͔ of wing indices and centroid size of the Z. tuberculatus subgroup species

Species N C 4v 4c 5x M ac Centroid size mascariensis 10 2.7 ͓2.3Ð3.0͔ 1.5 ͓1.4Ð1.6͔ 0.9 ͓0.8Ð1.0͔ 1.0 ͓0.9Ð1.2͔ 0.4 ͓0.4Ð0.4͔ 2.6 ͓2.3Ð2.6͔ 458 ͓441Ð474͔ kolodkinae 10 3.0 ͓2.7Ð3.1͔ 1.4 ͓1.3Ð1.5͔ 0.8 ͓0.7Ð0.9͔ 1.1 ͓0.9Ð1.2͔ 0.4 ͓0.3Ð0.4͔ 2.4 ͓2.2Ð2.5͔ 468 ͓457Ð482͔ sepsoides 10 2.5 ͓2.4Ð2.7͔ 1.5 ͓1.4Ð1.5͔ 0.9 ͓0.8Ð1.0͔ 1.0 ͓0.9Ð1.1͔ 0.4 ͓0.4Ð0.5͔ 2.7 ͓2.5Ð2.8͔ 451 ͓440Ð469͔ tsacasi n. sp. 10 2.8 ͓2.5Ð3.0͔ 1.5 ͓1.5Ð1.6͔ 0.9 ͓0.8Ð1.0͔ 0.9 ͓0.8Ð1.0͔ 0.4 ͓0.3Ð0.4͔ 2.2 ͓2.1Ð2.4͔ 428 ͓415Ð442͔ tuberculatus 10 2.5 ͓2.3Ð2.8͔ 1.5 ͓1.4Ð1.7͔ 0.9 ͓0.9Ð1.0͔ 1.0 ͓0.9Ð1.2͔ 0.4 ͓0.4Ð0.5͔ 2.7 ͓2.4Ð3.0͔ 434 ͓411Ð447͔ burlai n. sp. 10 2.4 ͓2.3Ð2.6͔ 1.5 ͓1.4Ð1.6͔ 0.9 ͓0.8Ð1.0͔ 1.0 ͓1.1Ð0.9͔ 0.4 ͓0.4Ð0.4͔ 2.7 ͓2.3Ð3.1͔ 444 ͓427Ð464͔ verruca 5 2.6 ͓2.5Ð2.8͔ 1.7 ͓1.7Ð1.8͔ 1.0 ͓1.0Ð1.0͔ 0.9 ͓0.9Ð1.1͔ 0.5 ͓0.4Ð0.5͔ 2.4 ͓2.2Ð2.6͔ 425 ͓419Ð445͔ when possible) of the Z. tuberculatus subgroup in- MYA). Zaprionus kolodkinae formed the second early Downloaded from https://academic.oup.com/aesa/article/101/6/978/2758498 by guest on 27 September 2021 ferred from COII sequences. Internal nodes were branch (node 2, 1.29 MYA). After these early numbered and their support values (bootstrap and branches, the two sibling species Z. tuberculatus and Z. posterior probabilities) and ages (mean divergence Ϯ sepsoides form two distinct phylads (node 3, 0.73 minimum and maximum boundaries in million years MYA). The sepsoides phylad is monophyletic and con- ago) are given in Table 2. Although the tree was not sists of two species Z. sepsoides and Z. tsacasi n. sp. rooted, Z. mascariensis seems distant enough to be (node 4, 0.37 MYA), with the four populations of Z. considered as the earliest offshoot (node 1, 2.30 sepsoides sharing the same haplotype and thus were

Fig. 4. Analysis of wing shape indices for the species of the Z. tuberculatus subgroup. (a) CVA. (b) Unweighted pair-group method with arithmetic average phenogram. November 2008 YASSIN:SYSTEMATICS OF Z. tuberculatus SUBGROUP 985 Downloaded from https://academic.oup.com/aesa/article/101/6/978/2758498 by guest on 27 September 2021

Fig. 5. Phylogeographical correlated evolution of the wing C-index in the Z. tuberculatus species subgroup. For each species, the a and b interlandmark distances are plotted. Bars indicate standard errors. grouped together in a single, monophyletic polytome of the total variance in wing indices, which is usually (node 5, 0.07 MYA). The tuberculatus phylad is a size vector. This index was estimated from two in- polyphyletic (node 8, 0.22 MYA). Although most Z. terlandmark distances: a (the second costal section tuberculatus populations form a monophyletic clade between the subcostal break and R2 ϩ 3) and b (the (node 11, 0.19 MYA), a single population (Zimbabwe) third costal section between R2 ϩ 3 and R4 ϩ 5). When belongs to another clade, along with the two allied plotting the two distances, as shown in Fig. 5, the species, Z. verruca and Z. burlai n. sp. (node 9, 0.16 variance of wing size can be explained in light of two MYA). The low bootstrap values of the tuberculatus forces: a phylogenetic force working on the x-axis phylad (node 8, NJ ϭ 73, MP ϭ 75, and ML ϭ 67) are (distance a), and an environmental (altitude of ori- in concordance with the paraphyly of the Z. tubercu- gin) force working on the y-axis (distance b). This latus species (Funk and Omland 2003). means that the simultaneous analysis of the size com- Morphometrical Analysis. Standard morphometrics ponents of the C-index in these drosophilids can suc- of wing indices and centroid size estimated from in- cessfully distinguish between phylogenetic groups terlandmark distances was analyzed and summary sta- and between geographical populations within groups tistics (means and ranges) are given in Table 3. Al- as well. though MANOVA, with species as the main effect, was highly signiÞcant (WilkÕs ␭ ϭ 0.022; F ϭ 9.11; P Ͻ 36,235.5 Discussion 0.001), it was difÞcult to discriminate each species by wing indices alone as almost all index ranges over- and the Genetics of Speciation in the Z. lapped. However, CVA shown in Fig. 4a discriminated tuberculatus Subgroup. Modern systematics has the two new cryptic species, Z. burlai n. sp. and Z. been challenged with the increasing number of newly tsacasi n. sp., from their nominal relatives, Z. tubercu- discovered cryptic species due to advances in molec- latus and Z. sepsoides, respectively. The unweighted ular and morphometrical techniques (DeSalle et al. pair-group method with arithmetic average tree of 2005; Bickford et al. 2007). This will impose the ap- wing indices (Fig. 4b) did not follow the molecular plication of species concepts and delimitation criteria phylogeny, showing a single well supported cluster of in the diagnosis of new species beyond simple mor- three species (Z. burlai n. sp., Z. tuberculatus, and Z. phological characters. Species of the Z. tuberculatus sepsoides), with a bootstrap value of 90% after 1,000 subgroup are a good example of the limitation of mor- iterations. phological diagnoses, and the present revision high- Wing (centroid) size seemed to decrease following lights the great utility of molecular and morphometri- the molecular phylogeny because early branching cal tools. COII sequences can now be used to assign species, Z. mascariensis and Z. kolodkinae, have bigger any new specimen to most of the species with great wings, whereas the sepsoides and tuberculatus phylads success, with the exception of the Z. tuberculatus phy- have smaller wings, respectively. The costal index (C- lad. Moreover, wing morphometrical analysis was not index) was the main factor of CVA1 explaining 63.7% only able to discriminate cryptic species, but could 986 ANNALS OF THE ENTOMOLOGICAL SOCIETY OF AMERICA Vol. 101, no. 6 Downloaded from https://academic.oup.com/aesa/article/101/6/978/2758498 by guest on 27 September 2021

Fig. 6. Geographical distribution of species of the Z. tuberculatus subgroup. even assign specimen to a geographical locality (alti- termined by simple genetic systems, perhaps by few tude). genes. Unfortunately, the lack of hybrid progeny from Nonetheless, crossability is still the most direct test their interspeciÞc crosses prevented further genetic of the boundaries of sexually reproducing species. analysis. Interestingly, the two cryptic species described Indeed, results of hybrid incompatibility analyses sug- here show the opposite extremes of the evolution of gested that Z. sepsoides should no longer be consid- testis and seminal receptacle lengths. In Z. burlai n. sp. ered a junior synonym of Z. tuberculatus (Barker they are even longer than in Z. tuberculatus, whereas in 1975). The ad hoc revision of the type specimens of Z. tcacasi n. sp. they are even shorter than in Z. sepsoides. both species by Tsacas et al. (1977) conÞrmed the Moreover, both species can produce fertile hybrid fe- bona Þde species status of Z. sepsoides. When Z. mas- males that can be used to further analyze the genetic cariensis was discovered on Mauritius, its inability to basis of these characters in Zaprionus. produce hybrids with both Z. tuberculatus and Z. sep- Geography of Speciation in the Z. tuberculatus Sub- soides preceded the examination of its genitalia group. The COII phylogeny (Fig. 3) is in concordance (Tsacas and David 1975). The two new species de- with that proposed by Yassin et al. (2008a) on the basis scribed here were also discovered by crossing analy- of COII and a nuclear gene Amyrel. Nonetheless, the ses. In contrast to the previous cases, crosses are pos- divergence times estimated here are slightly lower sible, yet resulting in sterile hybrid males. than in Yassin et al. (2008a), probably due to the faster Tsacas et al. (1977) conducted the Þrst integrative evolution of mitochondrial genes. The early branching comparison of the then three species of the Z. tuber- pattern in both studies suggests a Malagasy origin of culatus complex in light of adult and immature exter- the subgroup as Z. mascariensis is only found in the nal morphology, development, biometry and karyol- islands of the Indian Ocean (Mauritius, La Reunion, ogy. Their main conclusion was that Z. mascariensis and Madagascar), whereas Z. kolodkinae is endemic to occupies an intermediate position between Z. tuber- Madagascar. Figure 6 shows the geographical distri- culatus and Z. sepsoides. It shares with Z. sepsoides two bution of the species of the tuberculatus subgroup. The immature characters: spatulate preapical egg vela and two sibling species, Z. tuberculatus and Z. sepsoides are shorn pupal anterior spiracle; and with Z. tuberculatus widespread throughout the African continent south of two adult characters: long testis in males and long the Sahara. Although they were collected also from seminal receptacle in females. Tsacas et al. (1977) Madagascar, their introduction there seemed to be suggested that such qualitative differences (long ver- recent (Chassagnard and McEvey 1992). Moreover, sus short and spatulate versus tapered) might be de- the two new cryptic species were both discovered November 2008 YASSIN:SYSTEMATICS OF Z. tuberculatus SUBGROUP 987 from the mainland which may be taken as further References Cited evidence of their continental origin. However, Z. ver- Barker, J. F. 1975. Preliminary note on the identity of ruca, a member of the tuberculatus phylad, is endemic Malloch (Dipt., Drosophilidae). to Madagascar. Entomol. Mon. Mag. 110: 95. Two hypotheses of speciation and colonization of Bickford, D., D. J. Lohman, N. S. Sodhi, P.K.L. Ng, R. Meier, mainland Africa can thus be proposed. First, a single K. Winker, K. K. Ingram, and I. Das. 2007. 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