Drosophilidae ( Diptera ) associated to fungi : differential use of resources in anthropic and Atlantic Rain Forest areas

This study investigates the Drosophilidae species associated to fruiting bodies of fungi in forested and anthropized environments of the Atlantic Rain Forest Biome, in south and southeastern Brazil. We collected samples of imagoes flying over and emerging from fruiting bodies of species of five fungi families, in six collection sites. We obtained 18 samples, from which emerged 910 drosophilids of 31 species from the genera Drosophila Fallen, 1823, Hirtodrosophila Duda, 1923, Leucophenga Mik, 1886, Mycodrosophila Oldenberg, 1914, Scaptomyza Hardy, 1849, Zaprionus Coquillett, 1901 and Zygothrica Wiedemann, 1830. The Drosophila species collected on fungi, as well as Zaprionus indianus Gupta, 1970, had previously been recorded colonizing fruits, demonstrating their versatility in resource use. Most of these species belong to the immigrans-tripunctata radiation of Drosophila. Our records expands the mycophagous habit (feeding or breeding on fungi) to almost all species groups of this radiation in the Neotropical region, even those supposed to be exclusively frugivorous. Assemblages associated to fungi of forested areas were more heterogeneous in terms of species composition, while those associated to fungi of anthropized areas were more homogeneous. The drosophilids from anthropized areas were also more versatile in resource use.

Almost all studies on mycophagous drosophilids have been conducted in North America, Europe and Japan (COURTNEY et al., 1990;BURLA et al., 1991;TODA et al., 1999;YAMASHIDA & HIJII, 2007), and indicate that Drosophila is a genus that abounds in fungi fruiting bodies, and that it may even represent the majority of Diptera that emerge from this substrate (TODA et al., 1999).These investigations also offer evidences that most mycophagous Drosophila species known belong to the pinicola, quinaria, testacea and tripunctata species groups, all of which are included in the immigranstripunctata radiation (REMSEN & O'GRADY, 2002;MARKOW & O'GRADY, 2005).
Some of these temperate zone species were used as model organisms in several studies on field and experimental ecology and offered significant contributions.However, concerning the Neotropical region, the information available about this guild is scarce.Samples were obtained from the 1940's up to the 1960's (FROTA-PESSOA, 1945, 1951;CORDEIRO, 1952;BURLA, 1956;MOURÃO et al., 1965), in studies whose aims were not to investigate the ecology of these species.Later, in a revision of the Zygothrica genus, GRIMALDI (1987) collected individuals in Peru and observed that few species of the genus oviposit in fungi, while some use them as courtship site.VAL & KANESHIRO (1988) carried out a survey in the Atlantic Rain Forest with commercial fungi and banana baits, and Zygothrica was the most abundant genus after Drosophila, with almost 50 species identified.Recently, ROQUE et al. (2006) collected drosophilids flying over and emerging from fruiting bodies of Pleurotus sp.(Tricholomataceae, Agaricales) and another undetermined fungus at the Cerrado Biome, Central Brazil, where 20 species from five genera of Drosophilidae were collected, and Drosophila was the most abundant.
Although there have been important studies on the ecology of mycophagous drosophilids, they were conducted in temperate zones, where the diversity of Drosophilidae are lower than those found inhabiting other regions.Therefore, it becomes necessary to discover whether the processes and patterns found in temperate zones are applicable in tropical zones.In this context, the aim of the present study is to contribute to the understanding of the processes driving the colonizing patterns of mycophagous drosophilids, by analyzing and comparing the drosophilid assemblage associated to fruiting bodies of fungi in the Atlantic Rain Forest stricto sensu and anthropic areas, in south and southeastern Brazil.

MATERIAL AND METHODS
Drosophilids and fungi were collected in six sites in south (States of Rio Grande do Sul and Santa Catarina) and southeast regions of Brazil (State of São Paulo) (Fig. 1).Samplings were conducted at three environments of the Atlantic Rain Forest stricto sensu and three anthropic areas, near buildings or gardens (Tab.I).
To evaluate the probable feeding sites of mycophagous Drosophilidae species, adult flies flying over fructification bodies of fungi were captured in the field by sweeping with an entomological net or with an entomological aspirator.To assess the breeding sites of Drosophilidae species, fructification bodies of fungi were collected, brought to the laboratory and kept in glass vials with sterilized sand.Vials were maintained in controlled temperature at 24 ± 1°C.Water was added whenever necessary to prevent dehydration.Emerged adults were aspired daily.
To test the association between drosophilid species and feeding sites, we conducted a Detrended Correspondence Analysis (DCA) (HILL & GAUCH, 1980), where the drosophilid species that flew over the fruiting bodies were the descriptors and fungi taxa were the objects.Another DCA was used to test an association between drosophilid species and breeding sites (fungi taxa), where the former were the descriptors and the latter the objects.DCA is an ordination analysis and, similarly to the Correspondence Analysis, it has been proposed as a way to analyze two-way contingence tables.However, DCA removes the arch effect found in most ordination analysis.DCA was conducted on the software R 2.5.0 (R DEVELOPMENT CORE TEAM, 2007), with the VEGAN package 1.8-8 (OKSANEN et al., 2007), and it was based on 26 segments, as recommended by HILL & GAUCH (1980).

RESULTS
A total of 910 individuals belonging to 31 Drosophilidae species were collected (Tabs.I, II).Among the drosophilid genera collected, Drosophila was the richest (17 species) and the most abundant in all environments sampled, even when compared to other exclusively mycophagous species, like Hirtodrosophila (five species), Leucophenga (two species) and Mycodrosophila (two species).Considering all sites sampled, 90 individuals were collected flying over fructification bodies, totaling 21 fly species, of which 14 belonged to the genus Drosophila (Table III).At least four fungi species (probably used as feeding sites) were identified: Coprinus comatus (Agaricaceae, Agaricales), Phallus sp.(Phallaceae, Agaricales) and one species of Agaricales sampled in anthropic areas; one member of the Polyporaceae family and a group of several undetermined fungi species sampled in forest areas.In the DCA conducted for the specimens collected flying over fruiting bodies, the components 1 and 2 accounted for 40.9 % of data variation.Our analysis distinguished between generalist and specialist fly species (Fig. 2).
Drosophilidae (Diptera) associated to fungi: differential use of... Generalist Drosophilidae species generated dots closer to the point of origin of axes, and occurred mainly in association with C. comatus samples, that was found in the anthropized environments and which had also more homogeneous assemblages.The dots of the more specialist drosophilids are farther away from the point of origin, just as the dots representing more heterogeneous assemblages associated to feeding sites.
Specimens of four fungi families (Agaricaceae, Auriculariaceae, Marasmiaceae and Polyporaceae) were collected and brought to the laboratory, of which 820 drosophilids emerged (Tab.IV).Drosophila was once again the richest and most abundant genus, with ten species and 273 individuals.In the DCA for species that emerged from the fructification bodies (breeding sites) (Fig. 3), the components 1 and 2 accounted for 14.8% of data variation.Exactly as observed in feeding sites, the composition of breeding Drosophilidae from fungi of anthropized areas (C.comatus, Macrolepiota sp., Marasmius sp. and Agaricales latu sensu) was more homogeneous.Similar situation was observed regarding the presence of more generalist species.This is highlighted by the clustering of dots next to the point of origin of the chart's axes.The fauna emerged from Polyporacea, that was found mainlly in the forest areas, was typical and distinct from that of other fungi, including specialized Drosophilidae genera, as Hirtodrosophila, Mycodrosophila and Zygothrica.The Atlantic Rain Forest samples were likewise more heterogeneous regarding composition of colonizing species (evidenced by respective dots distant from the point of origin), and the species that colonized fungi from the woods were observed exclusively in these samples.

DISCUSSION
As a whole, our results indicate that mycophagy is far more common than previously thought, since the present records expands the mycophagous habit (feeding or breeding on fungi) to almost all species groups of immigrans-tripunctata in the Neotropical region, even those supposed to be exclusively frugivorous.The species of calloptera, cardini, guarani and tripunctata species groups of Drosophila, all belonging to the immigrans-tripunctata radiation (REMSEN & O'GRADY, 2002;MARKOW & O'GRADY, 2005), were frequent in our samples (except for Polyporaceae).The same had been previously observed in a study conducted in central Brazil by ROQUE et al. (2006).Most of the species belonging to the testacea and quinaria species groups of Drosophila, which also belong to the immigrans-tripunctata radiation, are obligatory mycophagous flies.The characteristics related to the use of fungi by species of the immigranstripunctata radiation may thus be synapomorphic, since all its species groups, with the exception of pallidipennis, were recorded colonizing fungi.However, the homoplasy hypothesis cannot be rejected.
The occurrence of Hirtodrosophila, Leucophenga, Mycodrosophila, Scaptomyza and Zygothrica in fungi lends more consistency to the existing literature records (GRIMALDI, 1987;VAL & KANESHIRO, 1988;COURTNEY et al., 1990).Nevertheless, the presence of Zaprionus Coquillett, 1901 in our samples attracted our interest, as the only species observed in the Neotropics, Z. indianus, is considered to be frugivorous.Adult specimens of Z. indianus were collected over fruiting bodies of Phallus sp.(Phallaceae, Phallales) in anthropized areas of Ribeirão Preto (São Paulo, Brazil), and two individuals emerged from fungi collected by ROQUE et al. (2006).Taken together, these data demonstrate the versatility of this species, which recently invaded the American continent and is considered a pest for fruit production (VILELA et al., 2000).
Drosophilid assemblages associated to Polyporaceae were distinct from that associated to fungi of other families, considering both breeding and feeding sites.The dominance of Hirtodrosophila, Mycodrosophila and Zygothrica species may be linked to higher frequencies of this fungus family in Atlantic Rain Forest areas, since it is possible that the richness of these drosophilid genera is higher in forests, as suggested by SPIETH (1987) andCOURTNEY et al. (1990).In these forested areas, the assemblages were more heterogeneous regarding their composition.In contrast, the fauna emerging from the Agaricaceae, Auriculariaceae, Marasmiaceae and Phallaceae families, more abundant at anthropized areas, was far more homogeneous, with dominance of Drosophila species.
In the present study, the cardini species group of Drosophila (D. cardini Sturtevant, 1916, D. cardinoides Dobzhansky & Pavan, 1943and D. neocardini Streisinger, 1946) was restricted to the anthropized environments, since these species were observed only in fungi collected in these areas.The low abundance of such species in forested areas, in contrast with the high abundance observed in anthropic ones, was also recorded by GOTTSCHALK et al. (2007), though using banana baits.
Similarlly, the calloptera group, in spite of the low abundance observed, was restricted to forest areas, what have also been observed in banana-baited collections (DE TONI et al., 2007).On the other hand, species of the tripunctata and guarani species groups were observed in fungi collected in both forested and anthropized environments.
In the present study, 31 Drosophilidae species were collected associated to fungi fruiting bodies in the Atlantic Rain Forest and urbanized areas.Of these species, those belonging to the Drosophila and Zaprionus genera were also collected associated to fruits in other studies (CARSON, 1971;SILVA et al., 2005;GARCIA et al., 2008), demonstrating their versatility in resource use.The use of fungi as a trophic resource has probably played a more significant role, in comparison to previous notions, in the evolutive success of Neotropical species belonging to the immigrans-tripunctata radiation of Drosophila.Assemblages associated to fungi of forested areas were more heterogeneous in terms of species composition, while those associated to fungi of anthropized areas were more homogeneous at species level.The species of the cardini groups of Drosophila was restricted to anthropized areas, while the calloptera species group was restricted to forested areas.The drosophilids from anthropized areas were also more versatile in resource use, since they were collected in several samples of feeding and breeding sites.

Figure 1 .
Figure 1.South America map indicating the sampled sites in Brazil of the Drosophilidae species associated to fruiting bodies of fungi in forested and anthropized environments (A, Campus of Universidade Federal do Rio Grande do Sul, Porto Alegre, RS; B, Campus of Universidade de São Paulo, Ribeirão Preto, SP; C, Campus of Universidade Federal de Santa Catarina, Florianópolis, SC; D, Morro da Lagoa da Conceição, Florianópolis, SC; E, Biguaçu, SC; F, Piraí, Joinville, SC).

Figure 2 .
Figure 2. Detrended Correspondence Analysis results for feeding sites data.DCA1, component 1; DCA2, component 2; circles, Drosophilidae species; cross and underlined text: fungi species collected in anthropized environment; cross and underlined text in bold: fungi species collected in Atlantic Rain Forest.

Figure 3 .
Figure 3. Detrended Correspondence Analysis results for breeding sites data.DCA1, component 1; DCA2, component 2; circles, Drosophilidae species; cross and underlined text: fungi species collected in anthropized environment; cross and underlined text in bold: fungi species collected in Atlantic Rain Forest.

Table I .
Sampling sites of the Drosophilidae species associated to fruiting bodies of fungi, co-ordinates, and their respective environment types.

Table II .
Fungi taxa, dates of collections, sampled sites (A, Campus of Universidade Federal do Rio Grande do Sul, Porto Alegre, RS; B, Campus of Universidade de São Paulo, Ribeirão Preto, SP; C, Campus of Universidade Federal de Santa Catarina, Florianópolis, SC; D, Morro da Lagoa da Conceição, Florianópolis, SC; E, Biguaçu, SC; F, Piraí, Joinville, SC) and size (number of fruiting bodies) of each sample.