ZOOLOGIA 33(6): e20160142 ISSN 1984-4689 (online) urn:lsid:zoobank.org:pub:52E45989-B00A-403F-9DA9-6D9553C313BB www.scielo.br/zool TAXONOMY AND NOMENCLATURE Two new Neotropical species of Drosophilinae (Diptera: Drosophilidae) from Uruguay Beatriz Goñi1* & Carlos R. Vilela2 1Sección Genética Evolutiva, Instituto de Biología, Facultad de Ciencias, Universidad de la República. Iguá 4225, Montevideo 11400, Uruguay. 2Departamento de Genética e Biologia Evolutiva, Instituto de Biociências, Universidade de São Paulo. Caixa Postal 11461, 05422-970 São Paulo, SP, Brazil. E-mail: [email protected] *Corresponding author. E-mail: [email protected] ABSTRACT. 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, De- partment 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. KEY WORDS. Argentina, Drosophila tripunctata group, karyotypes, Mesoscaptomyza, taxonomy. For decades, Drosophila nappae Vilela, Valente & Bas- in southern Uruguay by net sweeping over fallen, decaying fleshy so-da-Silva, 2004, a South American species belonging to the D. seeds of maidenhair tree (Ginkgo biloba L.) and decaying fruits of tripunctata group, was misidentified under the nominal species both native and exotic plants or emerging from such fruits (GOÑI Drosophila angustibucca Duda, 1925 described from Central et al. 1997, 1998), or attracted to dung and carrion baited pitfall America. Drosophila angustibucca is apparently endemic to Costa traps (GOÑI et al. 2012). Only by checking the male terminalia, Rica and adjacent countries, whereas D. nappae occurs in the mainly the shape of their aedeagi, of this pair of sibling species Atlantic forest of southeastern and southern Brazil and most it is possible to tell them apart. Additionally, a single male of an probably also in Paraguay (VILELA et al. 2004). undescribed species of Scaptomyza Hardy, 1849, closely related to During a preliminary survey of the drosophilid fauna of the Colombian Scaptomyza striaticeps Wheeler & Takada, 1966, Uruguay, conducted during the last two decades, the first author was collected by net sweeping over grass by our colleague Maria collected some specimens of an unknown Drosophila Fallén, 1823 E. Martínez at the eastern wetlands (bañados in Spanish) of the species which had been cited by GOÑI et al. (1997, 1998) as D. Uruguayan Department of Rocha. gr. tripunctata and as an unidentified species of theD. tripunctata The purpose of the present paper is to erect names to group, respectively. At that time, Goñi and colleagues believed these two species new to science, and to present their formal that their unknown specimens of Drosophila belonged to an descriptions, including the karyotype description of the new undescribed species, which would be later described as D. nappae Drosophila species. (VILELA et al. 2004) from flies collected at Porto Alegre, state of Rio Grande do Sul, southern Brazil. More recently, we became MATERIAL AND METHODS convinced that the unidentified specimens from Uruguay cited by GOÑI et al. (1997, 1998) do not belong either to D. angustibucca Specimens examined are deposited in the Entomological or to Drosophila nappae, but are members of a sibling and still Collection (Diptera), Facultad de Ciencias, Universidad de la undescribed species referred to as D. aff. nappae in GOÑI et al. República, Montevideo, Uruguay (FCE-D) and the Museu de (2012). The specimens unidentified to species rank were collected Zoologia, Universidade de São Paulo, São Paulo, Brazil (MZSP). ZOOLOGIA 33(6): e20160142 | DOI: 10.1590/S1984-4689zool-20160142 | December 15, 2016 1 / 13 B. Goñi & C.R. Vilela Refer to BÄCHLI et al. (2004) for the terminology used in Cooking instructions. Prepare a wet mixture of tap water the descriptions, to VILELA (1983) and VILELA & BÄCHLI (1990) for and agar, bring it to a boil in a pot, stirring once in a while to measurements and indices, to KANESHIRO (1969) and WHEELER prevent clumping, add banana purée and brewer yeast solution & KAMBYSELLIS (1966) for details on the methods of preparing and boil it again, stir the mixture, turn off fire and let it cool to the terminalia. Type labels include clarifying notes in square about 60 °C before adding Nipagin. Stir well, pour it into cylin- brackets; a slash indicates a label change. In the descriptions, drical glass vials (20 x 100 mm) and plug them. After cooling average measurements are followed by range in parentheses. All and drying at room temperature the vials medium may be pre- types were dissected and their terminalia photomicrographed. served for up to three weeks in the refrigerator (ca. 8 °C). After The disarticulated terminalia are kept in a microvial filled with transferring the imagines to a new vial, a tiny ball (about the glycerin and attached by the stopper to the pinned specimen. size of a homeopathic pill) of live baker’s yeast should be added. Photomicrographs of imagoes were taken with an Olympus cam- The following tips may be useful to maintain the strains era (PM2) loaded with an analog 35 mm Fujichrome Professional at a temperature of 18 °C, ideal for keeping specimens of most of 64T film and attached to an Olympus stereomicroscope (SZ11) the species of the D. tripunctata group in laboratory. The emerged with a ring illuminator. Photomicrographs of the aedeagi+aedea- adults are to be transferred to new vials once a week. Turning gal apodeme, inner spermathecal capsules, and oviscapt valves and keeping the vials upside down until the adult flies reach were taken using a black & white APX25 Agfa Pan analog film sexual maturity (ca. two weeks) and also during the subsequent in Zeiss photomicroscope. The originally analog films (slides week, reduces the adult mortality caused mainly by the cataleptic and negatives) were later converted to a digital format using behaviour observed in many of these flies whenever they are an Epson scanner (Perfection 4180 Photo). Line drawings of disturbed (FROTA-PESSOA 1954). The vials from the first two weeks the terminalia were made using a Zeiss microscope, under an are usually devoid of eggs and larvae and must be discarded. The objective 40x, attached to a camera lucida (1.8x). Whenever in sexually mature flies (ca. 14-day old) are transferred into the new the same plate, all line drawings were drawn to the same scale vial kept upside down to oviposit for one week. Later on, flies can and all photomicrographs were taken and enlarged to the same be either discarded or transferred into new vial, if needed. Alterna- magnification. tively, sexually mature flies could be transferred to vials with pow- Four Drosophila isofemale lines (coded Q23F2, Q37F53, dered milk-agar medium for egg laying and larval development Q37F56, and Q49F1, see material examined) were used as non- (BÄCHLI et al. 2000). No additional yeast needs to be added at this type material to determine the species karyotypes and analyze point. One week later, the medium becomes double-layered due the male meiotic chromosomes. The latter strain was also used to the presence of large amount of growing larvae. At this stage for the analyses of the general body and eye colors, eggs and ca. four V-shaped filter paper strips (ca. 1,8 x 10 cm) are inserted puparia. Additionally, adult males and females from the line into the medium to reduce the excess of humidity. Q37F55 were double mounted and photomicrographed. For Mitotic and meiotic chromosomes of the new Drosophila comparison purposes, male and female specimens of D. nappae species were obtained by applying the technique of IMAI et al. sampled from two isofemale lines (I42F56 and I73F254), derived (1977, 1988) and MATSUDA et al. (1983). Cytology procedure from wild females collected at the Forest Reserve of IB-USP (São follows VILELA & GOÑI (2015). Cytological preparations were Paulo city, state of São Paulo, Brazil), 26-28.III.1996 and 26-28. made from single individuals by using the isofemale lines cod- VIII.1997 respectively, V. Ratcov and C.R. Vilela leg. (MZSP), plus ed Q23F2 and Q37F53 (see material examined, and observed ® one male of the same species collected at Morro Santana, Porto in an Olympus BX60 microscope equipped with an Olympus ® Alegre (state of Rio Grande do Sul), III.1995, L. Basso da Silva leg. U-MAD-3 camera, under an objective 100x and 1.6x optovar (MZSP) were dissected and their terminalia photomicrographed. magnification changer. Selected mitotic and meiotic cells were ® The cited isofemale lines were cultured in a cheap, long-lasting, photomicrographed using the Image-Pro Plus version 5.1 image analysis software and further edited in GIMP 2.8.14 (GNU Image and suitably modified banana-agar culture medium (detailed Manipulation Program). 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 TAXONOMY melanogaster Meigen, 1830, and its mutants, it is worthwhile Drosophilidae Rondani, 1856 mentioning here its recipe, as detailed below. Ingredients. Tap water (1000 ml), agar (10 g), five medi- Drosophila (Drosophila) montevidensis sp. nov. um-sized, peeled, and overripe bananas (preferentially Nanica Figs. 1-8, 17-49, 54-55, 58-63 cultivar) blended in advance with 125 ml of tap water (to get a urn:lsid:zoobank.org:act:DDAFE6AD-5838-4141-84F9-2DB89A1DE856 banana purée, which may also be stored at -20 °C for up to one D.