ABSTRACT

Zygothrica is a genus of Drosophilidae (Diptera) whose species utilize flowers and fungi for breeding sites, with records of fungi being used as courtship arena. Due to this habit, its representation in Drosophilidae surveys using banana-baited traps is generally low. However, Zygothrica orbitalis was well represented in a few samples with these traps. In this study, we report for the first time the breeding site of Z. orbitalis in living fruits of Psychotria brachyceras (Rubiaceae), noting that the use of living fruits is rare among Drosophilidae. The fructification of the plant occurs in the area of study from May to August, with previous collection records of the species in the Restinga (sandbank or strand) forest. Additionally, the emergence of some individuals of the invasive species Drosophila suzukii was observed, which highlights the necessity for continuous study of this plant to understand the dynamics between a native and an exotic species. Besides the ecological importance, our results are relevant for understanding the evolution of trophic resource use by the Zygothrica genus.

Keywords:
Drosophila suzukii; host plant association; larval breeding; oviposition site; trophic resource

Introduction

Drosophilidae is a family of Diptera that is distributed worldwide and is primarily known for the genetic and evolutionary studies conducted with Drosophila melanogasterMeigen, 1830Meigen, J.W., 1830. Systematische Beschreibung der Bekannten Europäischen Zweiflügeligen Insekten. Schulzische Buchhandlung, Hamm.. Beyond genetics, however, Drosophilidae species have emerged as valuable scientific models in various biological fields. To date, there have been 4,692 species of Drosophilidae described, which are organized into 75 genera (Bächli, 2023Bächli, G., 2023. TaxoDros: The Database on Taxonomy of Drosophilidae, Database 2022/6, v1.04. Available in https://www.taxodros.uzh.ch/ (accessed 06 January 2023).
https://www.taxodros.uzh.ch/... ). The genus Drosophila itself comprises 1,676 nominal species. There are 10 genera with species numbers ranging from 128 to 300, while the remaining genera contain fewer than 77 species each (Bächli, 2023Bächli, G., 2023. TaxoDros: The Database on Taxonomy of Drosophilidae, Database 2022/6, v1.04. Available in https://www.taxodros.uzh.ch/ (accessed 06 January 2023).
https://www.taxodros.uzh.ch/... ).

ZygothricaWiedemann, 1830aWiedemann, C.R.W., 1830a. Achias Dipterorum Genus a Fabricio conditum; illustratum novisque auctum et conventui physicorum germanorum oblatum, Kiliae Holsatorum, Mohr, Kiliae., with its 128 species predominantly found in the Neotropical Region (Bächli, 2023Bächli, G., 2023. TaxoDros: The Database on Taxonomy of Drosophilidae, Database 2022/6, v1.04. Available in https://www.taxodros.uzh.ch/ (accessed 06 January 2023).
https://www.taxodros.uzh.ch/... ), is essentially mycophagous or anthophilic (Grimaldi, 1987Grimaldi, D.A., 1987. Phylogenetics and taxonomy of Zygothrica (Diptera, Drosophilidae). Bull. Am. Mus. Nat. Hist. 186, 103–268. Available in: http://hdl.handle.net/2246/913 (accessed 23 October 2023).
http://hdl.handle.net/2246/913... ; Gautério et al., 2020Gautério, T.B., Machado, S., Loreto, E.L.S., Gottschalk, M.S., Robe, L.J., 2020. Phylogenetic relationships between fungus-associated Neotropical species of the genera Hirtodrosophila, Mycodrosophila and Zygothrica (Diptera, Drosophilidae), with insights into the evolution of breeding sites usage. Mol. Phylogenet. Evol. 145, 106733. https://doi.org/10.1016/j.ympev.2020.106733.
https://doi.org/10.1016/j.ympev.2020.106... ). Grimaldi (1987)Grimaldi, D.A., 1987. Phylogenetics and taxonomy of Zygothrica (Diptera, Drosophilidae). Bull. Am. Mus. Nat. Hist. 186, 103–268. Available in: http://hdl.handle.net/2246/913 (accessed 23 October 2023).
http://hdl.handle.net/2246/913... notes that flowers, particularly large and fleshy ones, serve as the primary larval food resource for Zygothrica and some species utilize fungi as a courtship arena. More recently, Valer et al. (2016)Valer, F.B., Bernardi, E., Mendes, M.F., Blauth, M.L., Gottschalk, M.S., 2016. Diversity and associations between Drosophilidae (Diptera) species and Basidiomycetes in a Neotropical forest. An. Acad. Bras. Cienc. 88, 705–718. https://doi.org/10.1590/0001-3765201620150366.
https://doi.org/10.1590/0001-37652016201... recorded the emergence of different species of Zygothrica from fructification bodies of fungi. Given their feeding habits, Zygothrica species are only occasionally collected using standard banana-baited traps with fermented fruits, a methodology that captures preferentially species attracted by decaying fruits (Gottschalk et al., 2008Gottschalk, M.S., Hofmann, P.R.P., Valente, V.L.S., 2008. Diptera, Drosophilidae: historical occurrence in Brazil. Check List 4, 485–518. https://doi.org/10.15560/4.4.485.
https://doi.org/10.15560/4.4.485... ).

As expected, Zygothrica orbitalis (Sturtevant, 1916Sturtevant, A.H., 1916. Notes on north american Drosophilidae with descriptions of twenty-three new species. Ann. Entomol. Soc. Am. 9, 323–343. https://doi.org/10.1093/aesa/9.4.323.
https://doi.org/10.1093/aesa/9.4.323... ) has been sporadically observed in surveys in various regions of Brazil. Most studies with banana-baited traps that have reported the presence of Z. orbitalis managed to collect only a few dozen individuals, even though tens or hundreds of thousands of Drosophilidae were sampled (De Toni et al., 2007De Toni, D.C., Gottschalk, M.S., Cordeiro, J., Hofmann, P.P.R., Valente, V.L.S., 2007. Study of the Drosophilidae (Diptera) Communities on Atlantic Forest Islands of Santa Catarina State, Brazil. Neotrop. Entomol. 36, 356-375. https://doi.org/10.1590/S1519-566X2007000300004.
https://doi.org/10.1590/S1519-566X200700... ; Gottschalk et al., 2007Gottschalk, M.S., De Toni, D.C., Valente, V.L.S., Hofmann, P.R.P., 2007. Changes in Brazilian Drosophilidae (Diptera) assemblages across an urbanization gradient. Neotrop. Entomol. 36, 848–862. https://doi.org/10.1590/S1519-566X2007000600005.
https://doi.org/10.1590/S1519-566X200700... ; Schmitz et al., 2007Schmitz, H.J., Valente, V.L.S., Hofmann, P.R.P., 2007. Taxonomic Survey of Drosophilidae (Diptera) from Mangrove Forests of Santa Catarina Island, Southern Brazil. Neotrop. Entomol. 36, 53–64. https://doi.org/10.1590/S1519-566X2007000100007.
https://doi.org/10.1590/S1519-566X200700... ; Döge et al., 2007Döge, J.S., Gottschalk, M.S., Bizzo, L.E.M., Oliveira, S.C.F., Schmitz, H.J., Valente, V.L.S., Hofmann, P.R.P., 2007. The genus Zygothrica Wiedemann 1830 (Diptera, Drosophilidae) in Santa Catarina state, southern Brazil: distribution and ecological notes. Biota Neotrop. 7, 33–36. https://doi.org/10.1590/S1676-06032007000300003.
https://doi.org/10.1590/S1676-0603200700... , 2008Döge, J.S., Valente, V.L.S., Hofmann, P.R.P., 2008. Drosophilids (Diptera) from an Atlantic Forest Area in Santa Catarina, southern Brazil. Rev. Bras. Entomol. 52, 615–624. https://doi.org/10.1590/S0085-56262008000400013.
https://doi.org/10.1590/S0085-5626200800... ; Bizzo et al., 2010Bizzo, L., Gottschalk, M.S., De Toni, D.C., Hofmann, P.R.P., 2010. Seasonal dynamics of a drosophilid (Diptera) assemblage and its potential as bioindicator in open environments. Iheringia Ser. Zool. 100, 185–191. https://doi.org/10.1590/S0073-47212010000300001.
https://doi.org/10.1590/S0073-4721201000... ; Poppe et al., 2014Poppe, J.L., Schmitz, H.J., Grimaldi, D., Valente, V.L.S., 2014. High diversity of Drosophilidae (Insecta, Diptera) in the Pampas Biome of South America, with descriptions of new Rhinoleucophenga species. Zootaxa 3779, 215–245. https://doi.org/10.11646/zootaxa.3779.2.6.
https://doi.org/10.11646/zootaxa.3779.2.... ; Coutinho-Silva et al., 2017Coutinho-Silva, R.D., Montes, M.A., Oliveira, G.F., de Carvalho-Neto, F.G., Rohde, C., Garcia, A.C.L., 2017. Effects of seasonality on drosophilids (Insecta, Diptera) in the northern part of the Atlantic Forest, Brazil. Bull. Entomol. Res. 107, 634–644. https://doi.org/10.1017/S0007485317000190.
https://doi.org/10.1017/S000748531700019... ; Duarte et al., 2018Duarte, L.B., Gottschalk, M.S., Robe, L.J., 2018. Assemblage of drosophilids (Diptera, Drosophilidae) inhabiting flooded and nonflooded areas in the extreme South of Brazil. Rev. Bras. Entomol. 62, 29–35. https://doi.org/10.1016/j.rbe.2017.11.005.
https://doi.org/10.1016/j.rbe.2017.11.00... ). Furthermore, with studies that specifically targeted Drosophilidae associated with fungi, Z. orbitalis was seldom captured: one specimen was collected visiting Auricularia auricula-judae (Bulliard) Quélet, but there are no records of Z. orbitalis emerging from fungi, in a study carried out by Valer et al. (2016)Valer, F.B., Bernardi, E., Mendes, M.F., Blauth, M.L., Gottschalk, M.S., 2016. Diversity and associations between Drosophilidae (Diptera) species and Basidiomycetes in a Neotropical forest. An. Acad. Bras. Cienc. 88, 705–718. https://doi.org/10.1590/0001-3765201620150366.
https://doi.org/10.1590/0001-37652016201... with 26 fungi species (Table S1).

We previously hypothesized that Z. orbitalis might be anthophilic like other species within its genus, but articles that report emergence of Drosophilidae from flowers in Brazil did not record the species, even though other Zygothrica species were (Santos and Vilela, 2005Santos, R.C.O., Vilela, C.R., 2005. Breeding sites of Neotropical Drosophilidae (Diptera). IV. Living and fallen flowers of Sessea brasiliensis and Cestrum spp. (Solanaceae). Rev. Bras. Entomol. 49, 544–551. https://doi.org/10.1590/S0085-56262005000400015.
https://doi.org/10.1590/S0085-5626200500... ; Schmitz and Valente, 2019Schmitz, H.J., Valente, V.L.S., 2019. The flower flies and the unknown diversity of Drosophilidae (Diptera): a biodiversity inventory in the Brazilian fauna. Pap. Avulsos Zool. 59, e20195945. https://doi.org/10.11606/1807-0205/2019.59.45.
https://doi.org/10.11606/1807-0205/2019.... ; Cordeiro et al., 2020Cordeiro, J., de Oliveira, J.H.F., Schmitz, H.J., Vizentin‐Bugoni, J., 2020. High niche partitioning promotes highly specialized, modular and non‐nested florivore-plant networks across spatial scales and reveals drivers of specialization. Oikos 129, 619–629. https://doi.org/10.1111/oik.06866.
https://doi.org/10.1111/oik.06866... ). Santos and Vilela (2005)Santos, R.C.O., Vilela, C.R., 2005. Breeding sites of Neotropical Drosophilidae (Diptera). IV. Living and fallen flowers of Sessea brasiliensis and Cestrum spp. (Solanaceae). Rev. Bras. Entomol. 49, 544–551. https://doi.org/10.1590/S0085-56262005000400015.
https://doi.org/10.1590/S0085-5626200500... described Drosophilidae species associated with fallen and living flowers of Solanaceae from nine species; Schmitz and Valente (2019)Schmitz, H.J., Valente, V.L.S., 2019. The flower flies and the unknown diversity of Drosophilidae (Diptera): a biodiversity inventory in the Brazilian fauna. Pap. Avulsos Zool. 59, e20195945. https://doi.org/10.11606/1807-0205/2019.59.45.
https://doi.org/10.11606/1807-0205/2019.... investigated flowers from 125 plant species across 18 botanical families widely distributed in Brazil; and Cordeiro et al. (2020)Cordeiro, J., de Oliveira, J.H.F., Schmitz, H.J., Vizentin‐Bugoni, J., 2020. High niche partitioning promotes highly specialized, modular and non‐nested florivore-plant networks across spatial scales and reveals drivers of specialization. Oikos 129, 619–629. https://doi.org/10.1111/oik.06866.
https://doi.org/10.1111/oik.06866... investigated 82 plant species from 33 botanical families in Southern Brazil (Table S2).

In contrast, three studies conducted in Brazil reported a significantly higher number of Z. orbitalis individuals. Santa-Brígida et al. (2019)Santa-Brígida, R., Wartchow, F., Medeiros, P.S., Gottschalk, M.S., Martins, M.B., Carvalho, C.J.B., 2019. Mycophagous Drosophilidae (Diptera) guild and their hosts in the Brazilian Amazon. Pap. Avulsos Zool. 59, e20195920. https://doi.org/10.11606/1807-0205/2019.59.20.
https://doi.org/10.11606/1807-0205/2019.... collected 140 individuals flying over the fructification bodies of fungi in the Caxiuanã National Forest in the Amazon Biome. Monteiro et al. (2014)Monteiro, L.S., Cabral, W.B.M., Montes, M.A., Garcia, A.C.L., Rohde, C., 2014. Occurrence of the genus Zygothrica (Diptera, Drosophilidae) in a high-altitude forest in northeastern Brazil. Drosoph. Inf. Serv. 97, 15–17. collected 1,749 individuals using banana-baited traps in a high-altitude forest in the Atlantic Forest Biome. Finally, Mendes et al. (2017)Mendes, M.F., Valer, F.B., Aleixo, J.G., Blauth, M.L., Gottschalk, M.S., 2017. Diversity of Drosophilidae (Insecta, Diptera) in the Restinga forest of southern Brazil. Rev. Bras. Entomol. 61, 248–256. https://doi.org/10.1016/j.rbe.2017.05.002.
https://doi.org/10.1016/j.rbe.2017.05.00... sampled 307 specimens with banana-baited traps in a Restinga (sandbank or strand) forest in the Pampa Biome.

Considering the records of Z. orbitalis obtained by Mendes et al. (2017)Mendes, M.F., Valer, F.B., Aleixo, J.G., Blauth, M.L., Gottschalk, M.S., 2017. Diversity of Drosophilidae (Insecta, Diptera) in the Restinga forest of southern Brazil. Rev. Bras. Entomol. 61, 248–256. https://doi.org/10.1016/j.rbe.2017.05.002.
https://doi.org/10.1016/j.rbe.2017.05.00... , as well as the database of Drosophilidae assemblages in the Restinga forest in Southern Brazil using traps (Valer et al., 2013Valer, F.B., Neutzing, A.S., Garcia, F.R.M., Gottschalk, M.S., Blauth, M.L., 2013. The first record of Zygothrica orbitalis (Sturtevant, 1916) for the state of Rio Grande do Sul and the southernmost limits for seven species of Drosophilidae (Insecta: diptera). Drosoph. Inf. Serv. 96, 120–123.; Mendes et al., 2017Mendes, M.F., Valer, F.B., Aleixo, J.G., Blauth, M.L., Gottschalk, M.S., 2017. Diversity of Drosophilidae (Insecta, Diptera) in the Restinga forest of southern Brazil. Rev. Bras. Entomol. 61, 248–256. https://doi.org/10.1016/j.rbe.2017.05.002.
https://doi.org/10.1016/j.rbe.2017.05.00... ) or associated resources (Valer et al., 2016Valer, F.B., Bernardi, E., Mendes, M.F., Blauth, M.L., Gottschalk, M.S., 2016. Diversity and associations between Drosophilidae (Diptera) species and Basidiomycetes in a Neotropical forest. An. Acad. Bras. Cienc. 88, 705–718. https://doi.org/10.1590/0001-3765201620150366.
https://doi.org/10.1590/0001-37652016201... ; Mendes et al., 2019Mendes, M.F., Machado, D.R., Garcia, F.R.M., Blauth, M.L., Gottschalk, M.S., 2019. The use of the trophic resource by exotic and native species Drosophilidae: fruit colonization on the plant. Drosoph. Inf. Serv. 102, 18–20.; Cordeiro et al., 2020Cordeiro, J., de Oliveira, J.H.F., Schmitz, H.J., Vizentin‐Bugoni, J., 2020. High niche partitioning promotes highly specialized, modular and non‐nested florivore-plant networks across spatial scales and reveals drivers of specialization. Oikos 129, 619–629. https://doi.org/10.1111/oik.06866.
https://doi.org/10.1111/oik.06866... ), we focused on native fruits in this region as a potential trophic resource. The present study aims to describe the utilization of living fruits of Psychotria brachyceras Müll. Arg. (Rubiaceae) by Z. orbitalis and discuss the ecological implications of this atypical association.

Methodology

Host Plant and Study Area

The Horto Botânico Irmão Teodoro Luis (HBITL) is an approximately 23-hectares Federal Conservation Unit of Restinga forest within the Pampa Biome, located in Southern Brazil (31°48’54’’S, 52°25’48’’W). Psychotria brachyceras is an abundant and widely dispersed understory shrub in this area, characterized by its small purple fruits and white flowers (Figure 1). The plant is endemic to Brazil, encompassing both tropical and subtropical areas. It is distributed across Atlantic Forest and rainforests (Taylor and Gomes, 2015Taylor, C.M., Gomes, Z.D., 2015. Psychotria in lista de espécies da Flora do Brasil. Available in: http://floradobrasil2015.jbrj.gov.br/jabot/floradobrasil/FB14160 (accessed 23 October 2023).
http://floradobrasil2015.jbrj.gov.br/jab... ). Fewer records have been documented in the Amazon, Caatinga and Cerrado Biomes (EOL, 2023Encyclopedia of Life - EOL, 2023. Available in: https://eol.org/ (accessed 23 October 2023).
https://eol.org/... ; GBIF, 2023Global Biodiversity Information Facility - GBIF, 2023. Available in: https://www.gbif.org/ (accessed 23 October 2023).
https://www.gbif.org/... ; SIBBR, 2023Sistema de Informação sobre a Biodiversidade Brasileira - SIBBR, 2023. Available in: https://www.sibbr.gov.br/ (accessed 23 October 2023).
https://www.sibbr.gov.br/... ). At HBITL, we observed fructification in February, with ripening starting from May to August.

Figure 1
Psychotria brachyceras at the Horto Botânico Irmão Teodoro Luis (HBITL), located in the municipality of Capão do Leão, state of Rio Grande do Sul, Brazil. (A) Shrub of P. brachyceras bearing fruits; (B) Close-up of the ripe fruits; (C) Overall view of the flowers.

Sampling

Ripe fruits of P. brachyceras were collected from the plant on April 14^th, July 2^nd, and July 6^th, 2021. The ripe fruits were recognized by their intense violet color (Figure 1). After collection, the fruits were kept in three different ways for Drosophilidae emergence:

1. April 14^th: Ten fruits were collected from each of 20 different sites within HBITL, with each site at least 10 meters apart from one another. Each fruit was placed individually in an autoclaved test tube containing one previously frozen fruit of P. brachyceras and non-hydrated Formula 4-24® Instant Drosophila Medium (Carolina Biological). The fruits were frozen to eliminate previous colonization. This procedure involved a total of 200 fruits.

2. July 02^nd: Seven vials contained a variable number of ripe fruits (149, 207, 207, 212, 239, 249 and 322 fruits), taken from the plant, totaling 1,585 fruits. These fruits were kept in autoclaved sand. The number of fruits was obtained counting the seeds after the emergence of insects.

3. July 06^th: Similar to the procedure described previously in method 1, but the vials contained only the autoclaved and non-hydrated Formula 4-24® Instant Drosophila Medium (Carolina Biological), without frozen fruits of P. brachyceras. This change in methodology was made following the observation that Z. orbitalis completes its development inside a single fruit. This procedure involved a total of 200 fruits.

The inclusion of nutritional supplementation (additional fruits and/or Formula 4-24® Instant Drosophila Medium) was conducted to optimize adult insect emergence by reducing larval competition (Grimaldi and Jaenike, 1984Grimaldi, D.A., Jaenike, J., 1984. Competition in Natural Populations of Mycophagous Drosophila. Ecology 65, 1113–1120. https://doi.org/10.2307/1938319.
https://doi.org/10.2307/1938319... ; Marco Silva Gottschalk, unpublished data). As the biology of Z. orbitalis was unknown and seemed to be very specific, we augment the trophic resource with more P. brachyceras fruits and/or a culture medium commonly used for breeding a large number of Drosophila species.

Additional pilot tests utilized autoclaved fruits or hydrated 4-24® Instant Drosophila Medium. However, these methods were discontinued as the autoclaved fruits fell apart, and hydrating the 4-24® Instant Drosophila Medium resulted in increased mold growth. Regarding the second methodology, it is one of the most commonly used procedures to study the fauna of fruit-emerging Drosophilidae, typically yielding higher richness and abundance of Drosophilidae. This method was employed to confirm the prevalence of Z. orbitalis in the studied plant species.

Following the emergence of the flies, the fruits were dissected, and the medium was stirred to locate insects, puparia, and larvae, which could indicate fruit colonization.

Taxonomic Identification

The Drosophilidae were identified based on their external morphology (Burla, 1956Burla, H., 1956. Die Drosophilidengattung Zygothrica und ihre Beziehung zur Drosophila-Untergattung Hirtodrosophila. Mitt. Zool. Mus. Berl. 32, 189–321.; Bächli et al., 2004Bächli, G., Viljoen, F., Escher, S.A., Saura, A., 2004. The Drosophilidae of Fennoscandia and Denmark. Leiden, Brill.; Vilela and Mori, 2014Vilela, C.R., Mori, L., 2014. The invasive spotted-wing Drosophila (Diptera, Drosophilidae) has been found in the city of São Paulo (Brazil). Rev. Bras. Entomol. 58, 371–375. https://doi.org/10.1590/S0085-56262014000400004.
https://doi.org/10.1590/S0085-5626201400... ). The terminalia of some Z. orbitalis male were dissected following the procedures of Bächli et al. (2004)Bächli, G., Viljoen, F., Escher, S.A., Saura, A., 2004. The Drosophilidae of Fennoscandia and Denmark. Leiden, Brill. to confirm species identification according to Burla (1956)Burla, H., 1956. Die Drosophilidengattung Zygothrica und ihre Beziehung zur Drosophila-Untergattung Hirtodrosophila. Mitt. Zool. Mus. Berl. 32, 189–321.. When the spiracles were intact, the puparium was also utilized for Drosophilidae identification. Voucher specimens were deposited in Museu de Ciências Naturais Carlos Ritter, UFPel, with the codes DRO-00537 to DRO-00551. Other insects were identified at the family taxa (Rafael et al., 2012Rafael, J.A., Melo, G.A.R., Carvalho, C.J.B., Casari, S.A., Constantino, R., 2012. Insetos do Brasil: diversidade e taxonomia, Holos, Ribeirão Preto.).

The plant identification was conducted based on general morphology and floral characteristics. A voucher specimen of the plant is deposited in the HerbárioPel, UFPel, with the code MBM00254514.

Analysis of fruit colonization

An Excel table was organized to catalog each fruit, whether isolated or grouped in vials, along with the taxa and number of emerging insects.

The rate of fruit colonization for isolated fruits was calculated by dividing the number of fruits with Drosophilidae emergence by the total number of fruits. In some cases, the puparium was counted as “Drosophilidae colonization” without the species identification. The rate of fruit colonization was also calculated separately for methods 1 and 3, as well as for each vial used in method 2. The larvae were not counted for colonization because they could not be identified.

The incidence of each species was determined by dividing the number of colonized fruits by the total number of fruits with Drosophilidae emergence.

Image capture and processing methods

The plants were photographed in the field using a Samsung Galaxy S10e SM-G970F cell phone camera.

The adults and fruit with puparia were photographed at several depths of focus using a Zeiss Discovery V.20 photomicroscope. Also, the male terminalia structures of Z. orbitalis, prepared according Frech-Telles et al. (2023)Frech-Telles, M.H.F., Valente, V.L.S., Gottschalk, M.S., 2023. Three new Neotropical species of Hirtodrosophila Duda, 1923 (Diptera: drosophilidae). Rev. Bras. Entomol. 67, e20230083. https://doi.org/10.1590/1806-9665-RBENT-2023-0083.
https://doi.org/10.1590/1806-9665-RBENT-... , were photographed at several depths of focus using a Canon EOS REBEL T3 camera coupled to an Olympus BX51 microscope with a 20x objective lens. To indicate the scale bar on the pictures, an objective micrometer was also photographed at the same magnification. All the pictures were saved at the maximum resolution in JPEG format. The pictures taken at different depths of focus were stacked into an all-in-focus composite image by the rendering method “C” (pyramid) with the minimal smoothing setting of the Helicon Focus 7.7.5 Pro lifetime software (www.heliconsoft.com).

All figures boards were created in Adobe Photoshop CS6 v. 13.0 64x and the photos of terminalia were edited to remove the background.

Results

From the 400 isolated fruits (methods 1 and 3), pupae or adult Drosophilidae were observed from 75 fruits (19%). Among these, 87% were colonized by Z. orbitalis (Figure 2), while about 9% were colonized by D. suzukii (Matsumura, 1931Matsumura, S., 1931. 6000 Illustrated Insects of Japan-empire. The Toko-Shoin, Tokio.) (Figure 3). For the remaining colonized fruits, the determination of the species identity was not possible. From two fruits, we observed the emergence of two Z. orbitalis individuals, and from one fruit we observed the emergence of four D. suzukii individuals. In all other cases, the emergence of only one fly per fruit was observed.

Figure 2
Male of Zygothrica orbitalis collected emerging from a fruit of Psychotria brachyceras. (A) Lateral view; (B) Frontal view of head; (C) Dorsal view of head and thorax; (D) Dorsal view of abdomen; (E) Posteroventral view of articulated periphallic and phallic organs; (F) Lateral view of hypandrium, phallus and associated sclerites; (G) Ventral view of hypandrium, phallus and associated sclerites. Abbreviations: cerc,cercus; epand, epandrium; hpp, hypoproctal plate; hypd, hypandrium; pgt, postgonite; ph, phallus; phapod, phallapodeme; pregt, pregonite; sur, surstylus.

Figure 3
Drosophila suzukii collected emerging from a fruit of Psychotria brachyceras. (A) Male in lateral view (arrow indicating the dark spot at wing apex, at the intersection of veins R₂₊₃ and C, typical of males of the species); (B) Detail of sex combs in foretarsus (arrows); (C) Posteroventral view of male periphallic organs; (D) Lateral view of the apex of abdomen of a female, showing the serrated oviscapt. Abbreviations: cerc, cercus; epand, epandrium; ovscp, oviscapt; ph, phallus; sur, surstylus.

The emergence of flies from grouped fruits (method 2) was either equivalent to or greater in number than the emergence from isolated fruits (Table 1). We also observed two males and one female belonging to an undetermined species of the genus CladochaetaCoquillett, 1900Coquillett, D.W., 1900. Report on a collection of dipterous insects from Puerto Rico. Proc. U. S. Natl. Mus. 22, 249–270. https://doi.org/10.5479/si.00963801.22-1198.249.
https://doi.org/10.5479/si.00963801.22-1... , seven microhymenopterans parasitoids, and one neuropteran larva. If we assume that typically only one fly emerges from each fruit, the colonization rate for the samples collected on June 2^nd can vary between 0.18 and 0.45. However, this estimation might be on the higher side, as instances were noted where a single fruit housed multiple larvae. Additionally, a higher mortality rate should be taken into account for samples in which the fruits were isolated in tubes.

The fruits colonized by Drosophilidae became dry, and the seeds could be easily detached, whereas the fruits without fly colonization retained their pulp even months after collection. Some pupae were observed inside the fruits (Figure 4A), and by isolating the fruits, we were able to identify the puparium of Z. orbitalis (Figure 4B) due to its distinctive anterior spiracles with radially arranged branches resembling thin spines, while the anterior spiracles of D. suzukii have fewer radially arranged branches.

Figure 4
Unopened puparia of Zygothrica orbitalis. (A) Red arrow pointing to puparium in Psychotria brachyceras fruit; (B) Dorsal view.

Discussion

Although Zygothrica has been characterized as a mycophagous and anthophilic genus (Grimaldi, 1987Grimaldi, D.A., 1987. Phylogenetics and taxonomy of Zygothrica (Diptera, Drosophilidae). Bull. Am. Mus. Nat. Hist. 186, 103–268. Available in: http://hdl.handle.net/2246/913 (accessed 23 October 2023).
http://hdl.handle.net/2246/913... ), there have been no observations of Z. orbitalis utilizing fungi or flowers as breeding sites for their larvae (dos Santos and Vilela, 2005Santos, R.C.O., Vilela, C.R., 2005. Breeding sites of Neotropical Drosophilidae (Diptera). IV. Living and fallen flowers of Sessea brasiliensis and Cestrum spp. (Solanaceae). Rev. Bras. Entomol. 49, 544–551. https://doi.org/10.1590/S0085-56262005000400015.
https://doi.org/10.1590/S0085-5626200500... ; Valer et al., 2016Valer, F.B., Bernardi, E., Mendes, M.F., Blauth, M.L., Gottschalk, M.S., 2016. Diversity and associations between Drosophilidae (Diptera) species and Basidiomycetes in a Neotropical forest. An. Acad. Bras. Cienc. 88, 705–718. https://doi.org/10.1590/0001-3765201620150366.
https://doi.org/10.1590/0001-37652016201... ; Schmitz and Valente, 2019Schmitz, H.J., Valente, V.L.S., 2019. The flower flies and the unknown diversity of Drosophilidae (Diptera): a biodiversity inventory in the Brazilian fauna. Pap. Avulsos Zool. 59, e20195945. https://doi.org/10.11606/1807-0205/2019.59.45.
https://doi.org/10.11606/1807-0205/2019.... ; Cordeiro et al., 2020Cordeiro, J., de Oliveira, J.H.F., Schmitz, H.J., Vizentin‐Bugoni, J., 2020. High niche partitioning promotes highly specialized, modular and non‐nested florivore-plant networks across spatial scales and reveals drivers of specialization. Oikos 129, 619–629. https://doi.org/10.1111/oik.06866.
https://doi.org/10.1111/oik.06866... ). Capturing Z. orbitalis in traditional banana-baited traps is also rare, despite numerous studies using this methodology. However, in some instances, adults of this species have been captured in high numbers flying over the fungi (Grimaldi, 1987Grimaldi, D.A., 1987. Phylogenetics and taxonomy of Zygothrica (Diptera, Drosophilidae). Bull. Am. Mus. Nat. Hist. 186, 103–268. Available in: http://hdl.handle.net/2246/913 (accessed 23 October 2023).
http://hdl.handle.net/2246/913... ; Santa-Brígida et al., 2019Santa-Brígida, R., Wartchow, F., Medeiros, P.S., Gottschalk, M.S., Martins, M.B., Carvalho, C.J.B., 2019. Mycophagous Drosophilidae (Diptera) guild and their hosts in the Brazilian Amazon. Pap. Avulsos Zool. 59, e20195920. https://doi.org/10.11606/1807-0205/2019.59.20.
https://doi.org/10.11606/1807-0205/2019.... ), or in banana-baited traps (Monteiro et al., 2014Monteiro, L.S., Cabral, W.B.M., Montes, M.A., Garcia, A.C.L., Rohde, C., 2014. Occurrence of the genus Zygothrica (Diptera, Drosophilidae) in a high-altitude forest in northeastern Brazil. Drosoph. Inf. Serv. 97, 15–17.; Mendes et al., 2017Mendes, M.F., Valer, F.B., Aleixo, J.G., Blauth, M.L., Gottschalk, M.S., 2017. Diversity of Drosophilidae (Insecta, Diptera) in the Restinga forest of southern Brazil. Rev. Bras. Entomol. 61, 248–256. https://doi.org/10.1016/j.rbe.2017.05.002.
https://doi.org/10.1016/j.rbe.2017.05.00... ). The highest abundance of Z. orbitalis was recorded in the months of July and August in Amazon (Santa-Brígida et al., 2019Santa-Brígida, R., Wartchow, F., Medeiros, P.S., Gottschalk, M.S., Martins, M.B., Carvalho, C.J.B., 2019. Mycophagous Drosophilidae (Diptera) guild and their hosts in the Brazilian Amazon. Pap. Avulsos Zool. 59, e20195920. https://doi.org/10.11606/1807-0205/2019.59.20.
https://doi.org/10.11606/1807-0205/2019.... ), Atlantic Forest (Monteiro et al., 2014Monteiro, L.S., Cabral, W.B.M., Montes, M.A., Garcia, A.C.L., Rohde, C., 2014. Occurrence of the genus Zygothrica (Diptera, Drosophilidae) in a high-altitude forest in northeastern Brazil. Drosoph. Inf. Serv. 97, 15–17.) and Pampa Biomes (Mendes et al., 2017Mendes, M.F., Valer, F.B., Aleixo, J.G., Blauth, M.L., Gottschalk, M.S., 2017. Diversity of Drosophilidae (Insecta, Diptera) in the Restinga forest of southern Brazil. Rev. Bras. Entomol. 61, 248–256. https://doi.org/10.1016/j.rbe.2017.05.002.
https://doi.org/10.1016/j.rbe.2017.05.00... ). Also, Döge et al. (2007)Döge, J.S., Gottschalk, M.S., Bizzo, L.E.M., Oliveira, S.C.F., Schmitz, H.J., Valente, V.L.S., Hofmann, P.R.P., 2007. The genus Zygothrica Wiedemann 1830 (Diptera, Drosophilidae) in Santa Catarina state, southern Brazil: distribution and ecological notes. Biota Neotrop. 7, 33–36. https://doi.org/10.1590/S1676-06032007000300003.
https://doi.org/10.1590/S1676-0603200700... , who analyzed samples from Atlantic Forest areas, recorded Z. orbitalis primarily from April to September. The sampling of Z. orbitalis coincided with the ripening of P. brachyceras fruits in HBITL from May to August aligning with the described phenology of the plant. Its fructification period ranges from May to July (Taylor and Gomes, 2015Taylor, C.M., Gomes, Z.D., 2015. Psychotria in lista de espécies da Flora do Brasil. Available in: http://floradobrasil2015.jbrj.gov.br/jabot/floradobrasil/FB14160 (accessed 23 October 2023).
http://floradobrasil2015.jbrj.gov.br/jab... ). The sampling in the Amazon, Atlantic Forest, and Pampa Biomes is in concordance with the distribution of the plant in Brazil (EOL, 2023Encyclopedia of Life - EOL, 2023. Available in: https://eol.org/ (accessed 23 October 2023).
https://eol.org/... ; GBIF, 2023Global Biodiversity Information Facility - GBIF, 2023. Available in: https://www.gbif.org/ (accessed 23 October 2023).
https://www.gbif.org/... ; SIBBR, 2023Sistema de Informação sobre a Biodiversidade Brasileira - SIBBR, 2023. Available in: https://www.sibbr.gov.br/ (accessed 23 October 2023).
https://www.sibbr.gov.br/... ). However, two issues remain unresolved: P. brachyceras has only been recorded in Brazil, unlike Z. orbitalis, which has also been recorded in Panama (Bächli, 2023Bächli, G., 2023. TaxoDros: The Database on Taxonomy of Drosophilidae, Database 2022/6, v1.04. Available in https://www.taxodros.uzh.ch/ (accessed 06 January 2023).
https://www.taxodros.uzh.ch/... ). Furthemore, the type species was collected on Taboga Island, Panama, in 1907, and initially named Drosophila orbitalis (Sturtevant, 1916Sturtevant, A.H., 1916. Notes on north american Drosophilidae with descriptions of twenty-three new species. Ann. Entomol. Soc. Am. 9, 323–343. https://doi.org/10.1093/aesa/9.4.323.
https://doi.org/10.1093/aesa/9.4.323... ). This suggests that the species might be capable of utilizing other plant for larval breeding. Secondly, since the fruit is not available year-round, the persistence of Z. orbitalis could depend on the use of other plants, or the species might undergo diapause. In D. melanogaster, diapause is triggered by shorter day lengths, which block vitellogenesis (Anduaga et al., 2018Anduaga, A.M., Nagy, D., Costa, R., Kyriacou, C.P., 2018. Diapause in Drosophila melanogaster. Photoperiodicity, cold tolerance and metabolites. J. Insect Physiol. 105, 46–53. https://doi.org/10.1016/j.jinsphys.2018.01.003.
https://doi.org/10.1016/j.jinsphys.2018.... ). Unlike D. melanogaster, Z. orbitalis may respond to longer days or higher temperatures, but this requires further investigation.

Beyond the ecological importance of the association between P. brachyceras and Z. orbitalis, this relationship is essential for understanding the evolution of the genus Zygothrica. Grimaldi (1987)Grimaldi, D.A., 1987. Phylogenetics and taxonomy of Zygothrica (Diptera, Drosophilidae). Bull. Am. Mus. Nat. Hist. 186, 103–268. Available in: http://hdl.handle.net/2246/913 (accessed 23 October 2023).
http://hdl.handle.net/2246/913... proposed a phylogenetic hypothesis for Zygothrica species using morphological traits, characterizing the genus as predominantly anthophilic, and associating traits such as heavily sclerotized, narrow, and elongated oviscapes with oviposition in flowers. He suggested that mycophagy in Zygothrica is a retention of a primitive trait. In Grimaldi's phylogenetic hypothesis, Z. orbitalis was placed in a separate group (Grimaldi, 1987Grimaldi, D.A., 1987. Phylogenetics and taxonomy of Zygothrica (Diptera, Drosophilidae). Bull. Am. Mus. Nat. Hist. 186, 103–268. Available in: http://hdl.handle.net/2246/913 (accessed 23 October 2023).
http://hdl.handle.net/2246/913... ), suggesting that it represents a distinct lineage. Gautério et al. (2020)Gautério, T.B., Machado, S., Loreto, E.L.S., Gottschalk, M.S., Robe, L.J., 2020. Phylogenetic relationships between fungus-associated Neotropical species of the genera Hirtodrosophila, Mycodrosophila and Zygothrica (Diptera, Drosophilidae), with insights into the evolution of breeding sites usage. Mol. Phylogenet. Evol. 145, 106733. https://doi.org/10.1016/j.ympev.2020.106733.
https://doi.org/10.1016/j.ympev.2020.106... presented a molecular phylogenetic hypothesis in which Z. orbitalis was found clade together with Z. dispar (Wiedemann, 1830bWiedemann, C.R.W., 1830b. Aussereuropaische Zweiflugelige Insekten, Schulzischen Buchhandlung, Hamm.), Z. mesopoeyi Burla, 1956, Z. vittimaculosa Burla, 1956, Z. vittinubila Burla, 1956, Z. vittipunctata Burla, 1956, and Z. zygopoeyi Burla, 1956, most of which are anthophilic species. This suggests that Z. orbitalis may have either acquired a new habit within the group or developed it in parallel with the origin of anthophilic habits. Zhang et al. (2021)Zhang, Y., Katoh, T.K., Finet, C., Izumitani, H.F., Toda, M.J., Watabe, H., Katoh, T., 2021. Phylogeny and evolution of mycophagy in the Zygothrica genus group (Diptera: drosophilidae). Mol. Phylogenet. Evol. 163, 107257. https://doi.org/10.1016/j.ympev.2021.107257.
https://doi.org/10.1016/j.ympev.2021.107... presented similar results to those of Gautério et al. (2020)Gautério, T.B., Machado, S., Loreto, E.L.S., Gottschalk, M.S., Robe, L.J., 2020. Phylogenetic relationships between fungus-associated Neotropical species of the genera Hirtodrosophila, Mycodrosophila and Zygothrica (Diptera, Drosophilidae), with insights into the evolution of breeding sites usage. Mol. Phylogenet. Evol. 145, 106733. https://doi.org/10.1016/j.ympev.2020.106733.
https://doi.org/10.1016/j.ympev.2020.106... , but with different taxonomic sampling.

Notably, Z. orbitalis uses living fruits, which is an uncommon feature for frugivorous Drosophilidae, as they typically utilize ripe and fallen fruits. The exceptions are D. suzukii and potentially D. subpulchrella Takamori and Watabe in Takamori et al., 2006Takamori, H., Watabe, H.-A., Fuyama, Y., Zhang, Y.-P., Aotsuka, T., 2006. Drosophila subpulchrella, a new species of the Drosophila suzukii species subgroup from Japan and China (Diptera: drosophilidae). Ent. Sci. 9, 121–128. https://doi.org/10.1111/j.1479-8298.2006.00159.x.
https://doi.org/10.1111/j.1479-8298.2006... , which can pierce and oviposit in fruits with soft skin (Atallah et al., 2014Atallah, J., Teixeira, L., Salazar, R., Zaragoza, G., Kopp, A., 2014. The making of a pest: the evolution of a fruit-penetrating ovipositor in Drosophila suzukii and related species. Proc. Biol. Sci. 281, 20132840. https://doi.org/10.1098/rspb.2013.2840.
https://doi.org/10.1098/rspb.2013.2840... ). The ability of Z. orbitalis to pierce P. brachyceras fruits warrants further investigation. Lastly, the presence of D. suzukii in P. brachyceras fruits raises questions regarding competition between species, as this fruit is the only known breeding site for Z. orbitalis. Drosophila suzukii, an exotic species first recorded in South Brazil in 2013 (Deprá et al., 2014Deprá, M., Poppe, J.L., Schmitz, H.J., De Toni, D.C., Valente, V.L.S., 2014. The first records of the invasive pest Drosophila suzukii in South American Continent. J. Pest Sci. 87, 379–383. https://doi.org/10.1007/s10340-014-0591-5.
https://doi.org/10.1007/s10340-014-0591-... ; Mendes et al., 2017Mendes, M.F., Valer, F.B., Aleixo, J.G., Blauth, M.L., Gottschalk, M.S., 2017. Diversity of Drosophilidae (Insecta, Diptera) in the Restinga forest of southern Brazil. Rev. Bras. Entomol. 61, 248–256. https://doi.org/10.1016/j.rbe.2017.05.002.
https://doi.org/10.1016/j.rbe.2017.05.00... ), is considered a pest due to its ability to use non-rotten fruits for rearing larvae by piercing soft-skinned fruits. Drosophila suzukii is polyphagous (Poyet et al., 2015Poyet, M., Le Roux, V., Gibert, P., Meirland, A., Prévost, G., Eslin, P., Chabrerie, O., 2015. The wide potential trophic niche of the Asiatic fruit fly Drosophila suzukii: the key of its invasion success in temperate Europe? PLoS One 10, e0142785. https://doi.org/10.1371/journal.pone.0142785.
https://doi.org/10.1371/journal.pone.014... ), a trait that contributes to the species' successful dispersion into different areas and complicates management strategies. Poyet et al. (2015)Poyet, M., Le Roux, V., Gibert, P., Meirland, A., Prévost, G., Eslin, P., Chabrerie, O., 2015. The wide potential trophic niche of the Asiatic fruit fly Drosophila suzukii: the key of its invasion success in temperate Europe? PLoS One 10, e0142785. https://doi.org/10.1371/journal.pone.0142785.
https://doi.org/10.1371/journal.pone.014... also described the ability of D. suzukii to utilize fruits from various plant families and to establish itself in natural ecosystems and gardens. Given that Z. orbitalis and D. suzukii did not emerge from the same fruit and that the number of D. suzukii emergence was lower, direct competition between the species seems unlikely. However, long-term monitoring is essential to understand the potential competitive interaction between these species.

This study revealed that the fruits of P. brachyceras are predominantly colonized by the Drosophilidae species Z. orbitalis, and occasionally by D. suzukii. The ability of Z. orbitalis to utilize living fruits for breeding suggests a specific ecological niche and an uncommon behavior among frugivorous Drosophilidae, while the presence of D. suzukii, an exotic species, raises questions about potential competitive and ecological impacts in the long term, emphasizing the need for ongoing monitoring and further research.

Supplementary material

Supplementary material accompanies this paper.

Table S1 - Associations between fungi and Drosophildae specimens documented in studies conducted in Brazil. Fungi, refers to the taxonomical identification of fungi; Reference, indicates the bibliographical reference where the record is documented; Collection Methodology, specifies whether the adults were collected with a sweep net or entomological aspirator (Flying Over), or were collected upon emergence from mushrooms used as breeding sites (Emergence).

Table S2 - List of species of plants sampled in Brazil which had flowers examined by the Drosophilidae emergence. Zygothrica orbitalis were not recorded emerging from any sample.

This material is available as part of the online article from https://doi.org/10.1590/1806-9665-RBENT-2023-0059

Acknowledgments

The authors are grateful to the anonymous reviewers for their valuable comments and to the area editor for their editorial guidance, both of which have greatly contributed to the improvement of this manuscript.

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