Two new species of the genus Drosophila (Diptera: Drosophilidae), in Yanacocha protected forest, Pichincha, Ecuador

Two new species of genus Drosophila were described. These species were collected in Yanacocha Protected Forest, located in the northwest of the Pichincha volcano. Drosophila yanacocha sp. nov., was captured at an elevation of 3 587m and is member of D. annulimana group. Drosophila papaver sp. nov., was collected at an elevation of 3 771m. This species could not be placed with confidence in any species group, therefore it remains ungrouped. Nevertheless the spermatheca of D. papaver sp. nov., has a resemblance to the spermatheca of D. pictifrons Duda (1927) which suggest that both species could be related.


INTRODUCTION
The genus Drosophila contains about 1 146 species (Brake and Bächli 2008). The genus Drosophila includes eight subgenera: Chusqueophila, Dorsilopha, Drosophila, Dudaica, Phloridosa, Psilodorha, Siphlodora and Sophophora (Van der Linde and Houle 2008). The subgenus Drosophila has the largest number of species (Markow and O'Grady 2006). The subgenera are subdivided into groups (i.e related species that do not have a formal taxonomic rank) because the phylogeny of some species are not well characterized (Morales-Hojas and Vieira 2012). In some cases, external morphological characters are similar between or among species and make identification difficult. For this reason additional methods such as polytene chromosome structure and genitalia are necessary for species level determination (Breuer and Pavan 1950).
Ecuador is a tropical country in South America considered one of the most biologically mega-diverse

RESULTS
Two new species of Drosophila were captured in the Protected Forest Yanacocha. One of them, Drosophila papaver sp. nov., was identified from an isofemale line established from a wild caught female in the laboratory. Six adults (four females and two males) of this species were successfully reared to adulthood. The second species Drosophila yanacocha sp. nov., was collected dead from the baited traps. This species was described from one male. Thorax.-Yellow, dusted with setae and hairs emerging from dark spots; length 1.13mm. The anterior dorsocentral seta with a darker area towards the anterior dorsocentral seta, the dark spots join to form a stripe ( Figure 1). Six rows of acrostichal setulae in front of dorsocentral seta; six rows of hairs in disarray between dorsocentral seta. Scutelum yellow, basal scutellar seta divergent. and Rafael 2004band Rafael , 2005, D. morelia (Vela and Rafael 2004b), D. guarani Rafael 2005, 2004c), D. annulimana (Vela and Rafael 2005;Cabezas and Rafael 2013), D. onycophora (Figuero et al. 2012a) and D. asiri (Figuero et al. 2012b).

MATERIALS AND METHODS
Specimens of Drosophila were captured in the Yanacocha Protected Forest (0°6'41.8"S, 78°35'4.9"W) on the northwest flank of the Pichincha volcano (Quito, Pichincha, Ecuador). This forest is managed by the Jocotoco Foundation.
The traps were placed on a transect from 3 560m to 3 798m. This zone is composed of two types of vegetation. In the highest part there is low and scrub montane forest (3 600-4 100m) and in the lower part there is North Andean montane evergreen forest (2 600-4 000m). The traps contained fermented banana with yeast that was prepared the day before use. Specimens of Drosophilaa were collected during the dry season (August 2013 and September 2014) and in wetter season (March and May 2014). Adult Drosophila specimens that were collected alive were placed in tubes with standard Gelatine-Banana culture media (Rafael et al. 2000). The dead Drosophila were placed in microcentrifuge tubes with 75% ethanol. The baits were put inside glass jars sealed with cotton plugs. The material was transported to the laboratory where they were kept until emergence of adults.
In the laboratory, captured females were isolated to eventually produce isofemale lines (placed individually in tubes with Milk-Banana culture medium, Appendix 1), allowing the species identification through the analysis of male offspring. We tried to maintain these females on standard Gelatine-Banana culture but this was unsuccessful; the larvae did not develop in this medium. After several attempts, the Milk-Banana medium was used with excellent results. The larvae fed, pupated and emerged normally as adults.
Eight specimens were measured under a stereomicroscope with built-in camera (Zeiss, Discovery V8). We followed Bächli et al. (2004), for morphological characters indices. The indices and measurements for both species are reported for the holotype and the range of values in parentheses reflect the variation in the measurements of the paratypes. Lengths and dimensions are in mm.
The specimens have been deposited in the Museum of Zoology, Invertebrate Section of the Pontifical Catholic University of Ecuador (QCAZ-I).
Aedeagus.-Slightly curved towards the dorsal part apically and cleft. Ventrally with two fin-like projections with serrate edge. Dorsally with two finlike projections with toothed edges. Paraphysis oval with striations and three setae proximally. Ventral rod well-developed. Apodema sclerotized ( Figures  5-7) Etymology. Name Drosophila yanacocha refers to Protected Forest Yanacocha, where the specimens were collected. Yanacocha in the Quichua language means dark lagoon.
Ecological Data.-Drosophila yanacocha sp. nov., was collected in the protected forest Yanacocha at an elevation of 3 587m in the dry season. The forest is classified as Northern Andean montane evergreen forest. The holotype was collected with the following species: D. asiri, D. ecuatoriana, D. rucux, D. yanayuyu, D. carlosvilelai, D. ninarumi and D. pasochoensis. Transverse distance of dorsocentral setae 0.46 of longitudinal distance, dc index=0.87. Distance between apical scutellar setae 90.62% between apical and basal setae; scut and sterno indices could not be calculated. Medium katepisternal seta 1/3 of the anterior katespisternal seta. Legs yellowish brown.
Abdomen.-Yellowish brown, tergites without dorsal midline and with a dark stripe basally.
Male Terminalia.-Anal plate micropubescent with large setae, partially fused to epandrium. Epandrium micropubescent. Ventral lobe, micropubescent, with two setae and a well sclerotized, beak-shaped structure (p). Surstylus long, micropubescent on the lower part. The right side with 16 inner setae, without outer setae and with 10 prensisetae. The left side with 16 inner setae, without outer setae  Wings. Abdomen.-Brown, first tergite poorly pigmented in the middle part, the second tergite with pigmentation in the middle part surrounded by light areas, laterally pigmented. The rest of the tergites are evenly pigmented. Without lateral pigmentation (Figure 8).
Terminalia.-Anal plate with large setae, micropubescent, partially attached to epandrium. Epandrium micropubescent without setae on both sides. Ventral lobe, micropubescent, with 26 setae on the left side and 20 setae on the right side. Surstylus oval shaped, with microprojections on the most external side; 11 primary prensisetae (teeth) on both sides, without secondary prensisetae, 11 outersetae on left side and 10 outersetae on right side. Descasternum shaped like a hat ( Figure 10).
Aedeagus. Sclerotized, the apex with a slight cleft; in dorsal view with two toothed edges. Ventral rod well developed. Paraphysis square shaped with two setae at the apex. Apodema poorly sclerotized (Figures 11-13). Thorax.-Yellowish brown, length=0.91 (0.91-0.93) mm with six irregular rows of acrostichal setulae between the two anterior dorsocentral setae, h index=1.87. Transverse distance of dorsocentral setae could not be calculated, dc index=0.81 (0.81-1.11). Distance between apical scutellar setae 70% (70%-84.61%) between apical and basal setae. The basal scutellar seta were divergent; scut index=1.72. Sterno index could not be calculated (the posterior katepisternal seta were missing), but in previous photographs the medium katepisternal seta was 1/3 of the length of the anterior katepisternal seta. Legs yellow. Abdomen. Dark brown without dorsal midline, tergites with a posterior stripe in the posterior part darker in the male (Figure 9).
Female terminalia. Oviscapt yellow and sclerotized; with 19 marginal setae in the left valve and 15 marginal setae in the right valve. Two discal outer ovisensilla and a spike with two thin hairs on both valves ( Figure  14). Spermatheca sclerotized, shaped like a capsule with a warty crown on the apex (Figure 15).
Etymology. The name Drosophila papaver, refers to the plant Papaver somniferum because spermatheca of D. papaver sp. nov., is similar to the capsular fruit of this species.
Ecological Data. Drosophila papaver sp. nov., was collected in the Yanacocha protected forest at an elevation of 3 771m in the wet season. The forest is classified as low montane forest and shrub land. The specimens were collected with the following species: D. carlosvilelai, D. ninarumi, D. asiri, D. yanayuyu, D. korefae, D. ecuatoriana, D. mesophragmatica, D. rucux and D. paraguayensis. D. papaver sp. nov., could be related with D. pictifrons Duda, 1927; the spermatheca is morphologically similar to the spematheca of D. papaver sp. nov. 15 2 780m to 3 150m. This species was collected on the Pasochoa volcano (Vela and Rafael 2005). The wind in the mountains is stronger than in the lowlands (Mani 1968), therefore there is a tendency for small insects, including Drosophila, to be blown by the wind to other locations (Hodkinson 2005).
It is possible that the specimen of D. yanacocha sp. nov., reached the collection site by accident and was a temporary visitor to the forest. It is not possible to say with certainly that this species is reproducing in the mountains at this elevation (Mani 1968).

DISCUSSION
In this study we describe two new species of the genus Drosophila. Drosophila papaver sp. nov. has not been grouped as the morphological characters do not have similarities with any species group. Nevertheless D. papaver sp. nov. is probably closely related to Drosophila pictifrons. Both species develop in similar environments, at high altitude, D. pictifrons at 3 500m and D. papaver sp. nov. at 3 771m. Both species differ in certain aspects of external morphology.
Drosophila pictifrons has a yellow body and black ocellar triangle; in the thorax the scutum is slighty pollinose, with six rows of regular acrostical setae. The spermatheca is oval shapped with a cap on the apex (Vilela and Bächli 1990). In 1927, Duda collected this species in the Bolivian Andes at an altitude of 3 500m. The description was based in one female (Duda 1927). In 1988 Bachli redescribed the holotype (Vilela and Bächli 1990).
On the other hand, D. papaver sp. nov., has a brown body and a yellowish brown ocellar triangle; the thorax is yellowish brown and has a light band in the middle. The acrostical setae are arranged in six irregular rows. The oviscapt valve has a spike and two thin hairs. The spermatheca is oval shaped with a warty crown on the apex.
Drosophila yanacocha sp. nov., has been grouped in the D. annulimana species group (Duda 1927). This new species has similarities with several features of this group. Pavan and Cunha (1947) proposed the D. annulimana species group, whose species are characterized by a large, dark, dusted mesonotum, bristles and hairs emerging from dark spots and wing veins R4+5 and M converging at the apex (Freire-Maia and Pavan 1949). The genitalia of D. yanacocha sp. nov. share the features of the anal plate that is partially attached to the epandrium with a micropubescent surstylus. The paraphyses of the aedeagus are oval with three bristles (Cabezas and Rafael 2013). Furthermore, this species also has a sclerotized falciform (beakshaped) projection between the insertion point of the two anal plates as in all species of D. annulimana group (Breuer and Pavan 1950).
Usually species in the D. annulimana group are found in tropical ecosystems, cloud forests at a range of 1 200-2 000m (Santiana et al. 2012;Cabezas and Rafael, 2013). D. yanacocha sp. nov., was collected at 3 587m, in the Yanacocha protected forest. Another species of D. annulimana group that was collected at higher altitudes was D. yanacocha, whose distribution ranges from