Five new species of the Drosophila tripunctata group ( Diptera : Drosophilidae ) from Podocarpus National Park , Ecuador

Five new species of the genus Drosophila Fallén, 1823 belonging to the tripunctata group are described and illustrated: D. warmi sp. nov., D. kurillakta sp. nov., D. chichu sp. nov., D. saraguru sp. nov. and D. ayauma sp. nov. from the forests of Podocarpus National Park. The fi rst species is ascribed to subgroup II of Frota-Pessoa (1954), the second species to subgroup IV, and the last three species are not assigned to any subgroup. The fl ies were captured using plastic bottles containing pieces of yeast fermented banana.


Introduction
The tripunctata group of Drosophila Fallén, 1823 was proposed by Sturtevant (1942).The group is endemic to the Neotropical Region where it represents the second largest species group, surpassed only by the D. repleta species group (Vilela 1992).The tripunctata group contains 84 species (Bächli 2018).Studies based on morphological characters made by Frota-Pessoa (1954) divided the group into four subgroups.Subgroup I is transitional to the guarani group.Subgroup II is the most typical of the group.Subgroup III has some species that are transitional to the cardini group.Some species belonging to subgroup IV have some affi nities to the cardini group; they have white faces like some members of the cardini group (Frota-Pessoa 1954).Throckmorton (1975) proposed a pattern of radiation of Drosophila that included the tripunctata group, as well as the D. calloptera Freire-Maia & Pavan, 1949, D. cardini Sturtevant, 1942, D. rubifrons Patterson & Wheeler, 1942, D. guarani Dobzhansky & Pavan, 1943, D. macroptera Patterson, 1943, D. pallidipennis Patterson & Stone, 1952and D. sticta Clayton & Wheeler, 1975 species groups.Phylogenetic studies made by Hatadani et al. (2009) included several species of the tripunctata, guarani and other closest species groups.Their results did not support the monophyly of the tripunctata group, agreeing with Throckmorton (1975), Yokoto et al. (2003) and Robe et al. (2005).However, their results support a monophyletic origin for the immigrans-tripunctata radiation.
We describe fi ve new species of Drosophila belonging to the tripunctata group: D. warmi sp.nov., D. kurillakta sp.nov., D. chichu sp.nov., D. saraguru sp.nov.and D. ayauma sp.nov.We include illustrations and photographs of these new species.The similarities between the new species and those within the tripunctata group previously described are discussed.

Material and methods
The fl ies were collected in Loja and Zamora Chinchipe Provinces of Ecuador, in the cloud forests of the Podocarpus National Park and nearby localities.Collections were made at four high altitude localities: Bombuscaro at 1000 m a.s.l.(04°06′59.8″S, 78°58′04.9″W), San Francisco at 2190 m a.s.l.(03°59′16.7″S, 79°05′35″ W) and Cajanuma at 2675 m a.s.l.(04°06′53.7″S, 79°10′54.6″W) and 2800 m a.s.l.(04°06′58.9″S, 79°10′11.9″W).Fifteen fermented banana traps were placed at each location ten meters apart from each other and a maximum of one meter above the base of the trees.Traps were made using recycled 500 ml plastic bottles and baited with banana pieces fermented with yeast for 24 hours.
Living fl ies were captured with an entomological aspirator and transferred to vials with gelatin-banana media (Rafael et al. 2000).Females were individually isolated to produce isofemale lines.Adult males and dead fl ies were preserved in microcentrifuge tubes with ethanol (70-80%) and glycerin (100%) solution (Márquez-Luna 2005).The baits were removed from the traps and put inside glass jars sealed with cotton plugs.This material was transported to the laboratory where the baits were kept until the emergence of adult fl ies.
The external morphology of each fl y was examined under a stereo microscope (Zeiss, Discovery V8).Male and female terminalia were dissected and placed in KOH (10%) and boiled for ten minutes.The genitalia were then placed in 60% glycerin for females and 100% for males.Terminalia were analyzed and compared with the available literature to determine new species.The new species were illustrated using a microscope (Zeiss-46 70 86) with a camera lucida (Zeiss-47 46 20 9900).Structure measurements were made using the Axio Vision V4 software.Descriptive terms and indices follow the system of Bächli et al. (2004).
The holotypes and paratypes of the new species have been deposited in the Museo de Zoología -Invertebrados, Pontifi cia Universidad Católica del Ecuador, Quito (QCAZ-I).

Diagnosis
Aristae with six dorsal and three ventral branches, plus terminal fork.Prominent vibrissa.Thorax yellowish brown.Wings transparent yellow, bM-Cu slightly clouded.Male abdomen yellow with dorsal midline, 2 nd to 4 th tergites with thin pigmented bands, 5 th tergite without pigmentation and 6 th with a median spot.Female abdomen yellow with dorsal midline, 1 st tergite yellow, 2 nd to 4 th tergites with thin pigmented bands, 5 th and 6 th with a median spot between both tergites.Cerci not fused to epandrium.Hypandrium shield-shaped.Gonopod bearing one thick bristle.Paraphyses fused to gonopod bearing one small bristle.Aedeagus tubular and bifi d, with two lateral sclerotized and serrated projections, and two ventral membranous enlargements covered in bright studs.Spermatheca balloon-shaped, with a distal dimple covered in short spines.

Etymology
In the Kichwa language, 'warmi' refers to 'woman'.The species name is in honor of Ecuadorian women.

Description Female
The female was chosen as the holotype for this species because both males are in poor condition.Holotype external morphology: total length (body + wings) 4 mm, body length 3 mm.Body color yellow.HEAD.Aristae with six dorsal and three ventral branches, plus terminal fork and fi ne hairs.Head with orbital plate yellowish brown, frontal length 0.27 mm, frontal index = 0.61, top to bottom width ratio = 0.18.Medial vertical seta closer to lateral vertical seta and slightly towards outer edge of orbital plate, vt index = 0.19; or1-or3 ratio = 1, or2-or1 ratio = 0.91.Ocellar triangle brown, ocellus yellow; frontal triangle yellowish brown.Frontal vitta yellowish brown.Gena and postgena yellow, cheek index = 9.5.Vibrissa prominent, vibrissa index = 0.27.Carina prominent and not sulcate.Proboscis yellow.Eyes scarlet, eye index = 4.02.

Male
Reared from an isofemale line.Only two males emerged and both died crushed into the culture media.Morphologically with same characteristics as female except for abdomen, which is yellow and with a faint tiny spot on 6 th tergite (Fig. 2A).MALE TERMINALIA.Epandrium dorsally microtrichose, ventral lobe with 9 bristles on right and 6 on left side.Cerci not fused to epandrium, dorsally microtrichose.Surstylus with 9 primary teeth and 13 marginal bristles on right side and 14 on left (Fig. 2B).Hypandrium shield-shaped, with sclerotized edge.Gonopod U-shaped, with some bright studs, bearing one thick bristle (Fig. 2C).
AEDEAGUS.Tubular and bifi d, with two lateral sclerotized and serrated projections, below these projections with two ventral, membranous enlargements covered in bright studs.Aedeagal apodeme shorter than aedeagus.Ventral rod slightly developed (Fig. 2D-F).Paraphyses fused to gonopod, bearing one small bristle.

Distribution
Known only from the type locality.

Relationship to other species
This species belongs to subgroup II of the tripunctata group.The most similar species is Drosophila cuaso Bächli, Vilela & Ratcov, 2000.Aedeagus with sclerotized and membranous areas, and two lateral sclerotized and serrated projections, strongly fl attened laterally.

Etymology
In the Kichwa language, 'kurillakta' refers to 'kuri' = 'gold', and 'llakta' = 'land'.Zamora Chinchipe Province is known for its gold mines.It is therefore called the land of gold.

Description Male
Holotype external morphology: total length (body + wings) 3.84 mm, body length 3 mm.Body color yellow.

Distribution
Known only from the type locality.

Relationship to other species
This species belongs to subgroup IV of the tripunctata group.The general shape of the male terminalia suggests a relationship with Drosophila loewi Vilela & Bächli, 2000 from Yucatán, Mexico.

Diagnosis
Aristae with 5 dorsal and 2 ventral branches plus terminal fork.Two prominent oral bristles, 2 nd slightly smaller than 1 st .Thorax yellowish brown.Legs yellowish brown.Wings yellowish brown, dM-Cu slightly clouded.Abdomen yellow with dorsal midline, from 2 nd to 5 th tergites with triangular pigmentation which becomes thinner laterally, 6 th tergite with median spot.Cerci not fused to epandrium.Hypandrium shield-shaped.Gonopod bearing one long bristle.Aedeagus sclerotized and tubular, with a basal ventral bump, ventrally with an apical concavity.Paraphyses fused to gonopod bearing one bristle.

Distribution
Known only from the type locality.

Distribution
Known only from the type locality.

Male
Holotype external morphology: total length (body + wings) 7.43 mm, body length 4.22 mm.Body color yellowish brown.

Distribution
Known from the type locality and from the montane forest of Río Guango at 2548 m a.s.l.

Relationship to other species
The general shape of the male terminalia does not suggest any relationship to other described species of the Drosophila tripunctata group.

Discussion
Drosophila warmi sp.nov. is very similar to D. cuaso Bächli, Vilela & Ratcov, 2000; nevertheless, there are some differences in the aedeagus.Both species share the character of having two lateral sclerotized and serrated projections on the aedeagus.Drosophila warmi sp.nov.has two ventral membranous enlargements covered in bright studs, that noticeably split in two (Fig. 3A-C), while D. cuaso has a ventral membranous rounded surface also covered in bright studs.According to Bächli et al. (2000), D. cuaso always bears, in males and females, one well outlined and relatively large, black or coffee brown spot on the middle area of the 6 th tergite.In D. warmi sp.nov., there is a faint tiny spot on the 6 th tergite of males.In females of D. cuaso, the spot never reaches the anterior or posterior margin, but in D. warmi sp.nov. the median spot is shared between the 5 th and 6 th tergites.The inner spermathecal capsule of D. warmi sp.nov.bears about 15-30 centrodistal short spines (Fig. 3D-E), whereas in D. cuaso it has 5 spines, although the specimen identifi ed as D. cuaso from Bucaramanga, Colombia, illustrated and identifi ed by Bächli et al. (2000), has many more spines.Regarding the similarities in the number of spermathecal distal spines in the females of D. warmi sp.nov.and the specimen from Bucaramanga, Colombia (Bächli et al. 2000), we think that it could have been misidentifi ed, and it could belong to D. warmi sp.nov.Drosophila warmi sp.nov.belongs to subgroup II.This species resembles, in external characters and terminalia, species in the paraguayensis complex (Bächli et al. 2000).The abdominal pigmentation of Drosophila warmi sp.nov. is cryptic with that of species of the paraguayensis complex.
Drosophila kurillakta sp.nov. shares the microtrichose paraphyses and the general shape of the hypandrium with D. loewi Vilela & Bächli, 2000. Although D. kurillakta sp. nov. is similar to D. loewi, the most important difference is in the aedeagus.First, the aedeagus in D. kurillakta sp.nov. is deeply invaginated at the tip, whereas it is just slightly invaginated at the tip in D. loewi (Vilela & Bächli 2000).Second, the aedeagus in D. loewi is wider than in D. kurillakta sp.nov., which is strongly fl attened laterally.Finally, the serrated expansions of the aedeagus in D. loewi are located distally, whereas the serrated expansions in D. kurillakta sp.nov.are subapical.There is a slight difference in the paraphyses; D. loewi bears one short bristle and D. kurillakta sp.nov.does not have this bristle.Drosophila kurillakta sp.nov. is affi liated with subgroup IV as well as the morphologically related species D. loewi.
The similarities between D. chichu sp.nov.and D. cundinamarca Vilela & Bächli, 2000 include external morphological characters and the male terminalia.Both species have a yellowish brown head and thorax; the R-M and dM-Cu cross-veins of the wings are slightly clouded and the abdomen pigmentation pattern in D. cundinamarca and D. chichu sp.nov. is similar.On the 2 nd to 4 th tergites there is a medially interrupted marginal band.The most important similarities of the terminalia, in addition to the general shape of the aedeagus, are in the ventral lobe of the epandrium, which has three grouped setae at the tip, and the aedeagus has a subapical concavity.The main differences between these closely related species are in the terminalia.First, the aedeagus in D. chichu sp.nov.has a particular basal, ventral 'bump'.Second, the aedeagus is membranous dorsally, with bright studs.The paraphyses in D. chichu sp.nov.bear a bristle, but D. cundinamarca does not have this bristle.Drosophila chichu sp.nov. is clearly a member of the tripunctata group because of the external morphology and the general shape of the aedeagus.Despite the general similarities that D. chichu sp.nov.has with D. cundinamarca, the two species bear little resemblance to any other species assigned to a specifi c subgroup.In agreement with the criteria of Vilela & Bächli (2000), we did not assign this species to any subgroup.After a comparison of D. chichu sp.nov.with the paratypes of D. pilaresae Vela & Rafael, 2001 and a review of the published description of D. cundinamarca, we propose placing these species in a cluster formed by D. cundinamarca, D. pilaresae and D. chichu sp.nov., and hereby named the cundinamarca cluster.The diagnostic characteristics of this cluster are the laterally, strongly fl attened aedeagus and the ventrally convex and subapically concave shape of the aedeagus.The ventral lobe of the epandrium of these three species has three grouped bristles, which is unique to these three species.
Drosophila saraguru sp.nov.and Drosophila ayauma sp.nov.are not similar to other species in the tripunctata group.They share the important key characters of the tripunctata group.The genitalia characteristics of D. saraguru sp.nov.and D. ayauma sp.nov., including the cerci not being fused to the epandrium, the shield-shaped hypandrium and the general shape of the aedeagus, are characteristic of species in the tripunctata group (Frota-Pessoa 1954).
Drosophila saraguru sp.nov.and D. ayauma sp.nov.bear little resemblance to any other species assigned to a subgroup; for this reason we are not assigning them to any subgroup.
Finally, the type specimens were found in banana-baited traps placed in the fi eld, or are descendants of isofemale lines, which suggests that these species feed on fermented fruit, as many other species of Drosophila.