Two new Neotropical species of Drosophilinae ( Diptera : Drosophilidae ) from Uruguay

Two new species of Drosophilidae from Uruguay are described and illustrated: Drosophila montevidensis sp. nov. (Holotype male in MZSP: Facultad de Agronomía, Universidad de la República, Montevideo city, Department of Montevideo), and Scaptomyza pipinna sp. nov. (Holotype male in MZSP: Sarandí del Consejo, near north shore of Laguna de Castillos, Department of Rocha). The former species belongs to the D. tripunctata group and is sibling to Drosophila nappae Vilela, Valente & Basso-da-Silva, 2004, differing mainly in characters of the aedeagus. The latter is closely related to Scaptomyza striaticeps Wheeler & Takada, 1966, from which it can be distinguished by color and terminalia characters. The new Drosophila species was successfully cultured in a modified banana-agar medium which is provided. Photomicrographs of mitotic and meiotic chromosomes of D. montevidensis sp. nov. are also included.

Refer to Bächli et al. (2004) for the terminology used in the descriptions, to Vilela (1983) and Vilela & Bächli (1990) for measurements and indices, to Kaneshiro (1969) and Wheeler & KamBysellis (1966) for details on the methods of preparing the terminalia.Type labels include clarifying notes in square brackets; a slash indicates a label change.In the descriptions, average measurements are followed by range in parentheses.All types were dissected and their terminalia photomicrographed.The disarticulated terminalia are kept in a microvial filled with glycerin and attached by the stopper to the pinned specimen.Photomicrographs of imagoes were taken with an Olympus camera (PM2) loaded with an analog 35 mm Fujichrome Professional 64T film and attached to an Olympus stereomicroscope (SZ11) with a ring illuminator.Photomicrographs of the aedeagi+aedeagal apodeme, inner spermathecal capsules, and oviscapt valves were taken using a black & white APX25 Agfa Pan analog film in Zeiss photomicroscope.The originally analog films (slides and negatives) were later converted to a digital format using an Epson scanner (Perfection 4180 Photo).Line drawings of the terminalia were made using a Zeiss microscope, under an objective 40x, attached to a camera lucida (1.8x).Whenever in the same plate, all line drawings were drawn to the same scale and all photomicrographs were taken and enlarged to the same magnification.
Four Drosophila isofemale lines (coded Q23F2, Q37F53, Q37F56, and Q49F1, see material examined) were used as nontype material to determine the species karyotypes and analyze the male meiotic chromosomes.The latter strain was also used for the analyses of the general body and eye colors, eggs and puparia.Additionally, adult males and females from the line Q37F55 were double mounted and photomicrographed.For comparison purposes, male and female specimens of D. nappae sampled from two isofemale lines (I42F56 and I73F254), derived from wild females collected at the Forest Reserve of IB-USP (São Paulo city, state of São Paulo, Brazil), 26-28.III.1996 and26-28. VIII.1997 respectively, V. Ratcov and C.R. Vilela leg.(MZSP), plus one male of the same species collected at Morro Santana, Porto Alegre (state of Rio Grande do Sul), III.1995, L. Basso da Silva leg.(MZSP) were dissected and their terminalia photomicrographed.The cited isofemale lines were cultured in a cheap, long-lasting, and suitably modified banana-agar culture medium (detailed below) at constant temperature (18 ± 1 °C) and photoperiod (13h light: 10h dark).Because this modified medium is also used to culture many other species of this genus, including Drosophila melanogaster Meigen, 1830, and its mutants, it is worthwhile mentioning here its recipe, as detailed below.
Ingredients.Tap water (1000 ml), agar (10 g), five medium-sized, peeled, and overripe bananas (preferentially Nanica cultivar) blended in advance with 125 ml of tap water (to get a banana purée, which may also be stored at -20 °C for up to one year), aqueous solution of brewer yeast (15 g of powder in 125 ml of tap water), 10% Nipagin ® solution in ethanol (18 ml), both latter solutions prepared in advance.
Cooking instructions.Prepare a wet mixture of tap water and agar, bring it to a boil in a pot, stirring once in a while to prevent clumping, add banana purée and brewer yeast solution and boil it again, stir the mixture, turn off fire and let it cool to about 60 °C before adding Nipagin.Stir well, pour it into cylindrical glass vials (20 x 100 mm) and plug them.After cooling and drying at room temperature the vials medium may be preserved for up to three weeks in the refrigerator (ca.8 °C).After transferring the imagines to a new vial, a tiny ball (about the size of a homeopathic pill) of live baker's yeast should be added.
The following tips may be useful to maintain the strains at a temperature of 18 °C, ideal for keeping specimens of most of the species of the D. tripunctata group in laboratory.The emerged adults are to be transferred to new vials once a week.Turning and keeping the vials upside down until the adult flies reach sexual maturity (ca.two weeks) and also during the subsequent week, reduces the adult mortality caused mainly by the cataleptic behaviour observed in many of these flies whenever they are disturbed (Frota-Pessoa 1954).The vials from the first two weeks are usually devoid of eggs and larvae and must be discarded.The sexually mature flies (ca.14-day old) are transferred into the new vial kept upside down to oviposit for one week.Later on, flies can be either discarded or transferred into new vial, if needed.Alternatively, sexually mature flies could be transferred to vials with powdered milk-agar medium for egg laying and larval development (Bächli et al. 2000).No additional yeast needs to be added at this point.One week later, the medium becomes double-layered due to the presence of large amount of growing larvae.At this stage ca.four V-shaped filter paper strips (ca.1,8 x 10 cm) are inserted into the medium to reduce the excess of humidity.
Mitotic and meiotic chromosomes of the new Drosophila species were obtained by applying the technique of imai et al. (1977, 1988) and matsuda et al. (1983).Cytology procedure follows Vilela & Goñi (2015).Cytological preparations were made from single individuals by using the isofemale lines coded Q23F2 and Q37F53 (see material examined, and observed in an Olympus BX60 ® microscope equipped with an Olympus U-MAD-3 ® camera, under an objective 100x and 1.6x optovar magnification changer.Selected mitotic and meiotic cells were photomicrographed using the Image-Pro Plus ® version 5.1 image analysis software and further edited in GIMP 2.8.14 (GNU Image Manipulation Program).
Abdomen.Shining brownish-yellow, tergites 2-6 with a posterior, medially slightly interrupted dark brown band, not reaching lateral margins, and a variable (diffuse to well delimited) median, light brownish to dark brown longitudinal stripe, which may be completely absent in some specimens.
Remarks.This species belongs to the subgroup I (cf.Vilela The karyotype of these species differ from that of D. platitarsus (n = 6), being 4R, 1V, 1D (V-shaped X and J-shaped Y), with heterochromatic pericentromeric regions in the rod-shaped pairs, the dots, but differ in the shape of the Y, and the location of the heterochromatin in the X chromosome (interstitially located in one arm, and proximally, at the pericentromeric region, in the other arm), in samples of an isofemale line derived from an individual collected at the Forest Reserve of the IB-USP, located in the Cidade Universitária "Armando de Salles  Goñi, unpublished data).However, the apparently allopatric distribution of D. montevidensis sp.nov.and its sibling species, D. nappae, remains to be confirmed.Laboratory Cultures.It is cultivated at 18 °C with a modified banana-agar culture medium (recipe above).
Etymology.The specific name is an adjective in allusion to the type locality (Montevideo city).
Material examined.Type series (8 males, 1 female, as detailed above) plus 318 non-type specimens (142 males, 103 females, and 73 adults not sexed) which were analyzed for distributional and ecological purposes only, as detailed below.Diagnosis.Frons yellowish-brown, dull, lacking defined stripe (present in Scaptomyza striaticeps), orbits and anterior region lighter, ocellar triangle dark brown, antennae brown, medial vertical convergent and conspicuously long (longer than frontal length), vt index 1.63; scutum light brown, with three prominent brown longitudinal stripes, the central one extending to the end of scutellum; one pair of small presutural dorsocentrals; a single dorsal pleural stripe extending to postscutellum; wing clear; epandrium devoid of upper and lower setae (lower setae present in S. striaticeps), cercus slightly fused to epandrium on anteroventral corner (not fused in S. striaticeps), aedeagus straight (bent upwards in S. striaticeps) in lateral view.
Thorax.Length = 0.78 mm.Scutum light brown, subshining, bearing three conspicuous, brown, longitudinal stripes, the central one extending to the tip of scutellum, the two lateral ones lying outside the dorsocentrals, extending onto the sides of scutellum (Fig. 66); one dorsal, brown, conspicuous, pleural stripe, narrow proepisternum, widening at katepisternum, extending to postscutellum and including meso and metathoracic spiracles.Apparently 2 irregular rows of acrostichals (most of them are broken off) at presutural area.Apparently only one postpronotal.Transverse distance of dorsocentral setae 143% of longitudinal distance; dc index undetermined.A pair of additional small dorsocentrals, twice as long as adjacent acrostichal setulae, just anterior to transverse suture.Prescutellar setae absent.Scutellum with a large, diffuse, brown stripe at center, light brown laterally, subshining; distance between apical scutellar setae about 67% of that between apical and basal one, basal setae parallel, apical setae cruciate at median region; scut index undetermined.Pleura yellow at lower half, with a brownish stripe at upper half, subshining, sterno index undetermined; median katepisternal seta 50% of anterior one and noticeably thinner.Proepisternal seta absent.Halter long, pale yellow, contrasting with brownish background of pleural longitudinal stripe.Legs uniformly yellow, except metatarsomere V, brownish.One black seta at base of inner surface of mesotarsomere I, apical setae on protibia and mesotibia, the latter spur-shaped; preapicals on all three.
Abdomen shining dark brown with a dorsal pattern of pale yellow areas except on the last tergite.
Female.Unknown.Distribution.So far only known from its type locality.Remarks.Scaptomyza pipinna sp.nov.belongs to the subgenus Mesoscaptomyza.The male terminalia shows similarities with those of two species depicted by hacKman (1959) and Wheeler & taKada (1966: figs. 15.10 and 18.10).The straight aedeagus, although cylinder-shaped, reminds to those of S. paravittata Wheeler, 1952, from California, and S. setosa Wheeler & Takada, 1966, from Ecuador, which are somewhat cone-shaped; its decasternum and epandrium are similar to those of the Colombian S. striaticeps, from which it differs mainly by the absence of the paralobe sensu hacKman (1959).
Etymology.The specific name pipinna is a noun in apposition, in allusion to the tiny size (~ 81 µm long) of the aedeagus.
Note.While transferring the terminalia sclerites from the microscope slides to the glass microvial, the epandrium and hypandrium have been accidentally lost and only the ae-deagus+aedeagal apodeme and paraphyses, together with the remains of abdominal tergites and sternites, are preserved in the microvial attached to the double-mounted holotype.
(remarkably angled in Drosophila nappae,.Aedeagal apodeme rod-shaped, laterally flattened, slightly shorter than aedeagus; anteriorly expanded dorsoventrally in aged males(Figs.28, 38).Ventral rod completely fused to aedeagal apodeme, relatively long, remarkably right-angled in relation to ventral margin of the aedeagus as seen in lateral view (in D. nappae it is much shorter than aedeagus and obtuse-angled in relation to ventral margin of the aedeagus).
1992: 198) of the Drosophila tripunctata species group of the subgenus Drosophila.It shares with D. angustibucca, D. mediocris, D. medioobscurata, D. nappae, D. neoguaramunu, D. platitarsus, D. rostrata, and D. setula, the following remarkable features: aedeagus dorsally with a small, sclerotized, crescent-shaped plate at subdistal area, and anterodorsally bearing a pair of finger-shaped (sometimes diffuse) and backwards directed processes.The processes are small, diffuse and mostly membranous in all species except in D. nappae, where they are only slightly membranous and proportionally longer; and they are even longer in D. montevidensis sp.nov.Additionally, all of them but the latter two, have in common a sclerotized, inverted T-shaped area partially surrounded by the pair of processes in the anterodorsal, mostly membranous surface of aedeagus.The karyotype of four out of the eight species included in the subgroup I of the Drosophila tripunctata group, namely D. nappae (originally referred to as D. angustibucca) (see FrancK et al. 1984 and Pires 2000 [as Drosophila sp.U3]), D. neoguaramunu (see FrydenBerG 1956), D. platitarsus (see Pires 2000) and D. setula (see clayton & Wasserman 1957), were reported.Apparently, the observed haploid karyotype of D. montevidensis sp.nov.(n = 6) is indistinguishable from D. nappae, with 5R, 1D (Y chromosome is a rod shorter than X); however, it differs remarkably from the haploid karyotype formula of D. neoguaramunu (n = 3), with 3V (X chromosome considerably shorter than rod-shaped Y chromosome), in samples collected from Peru (FrydenBerG 1956).