Two decades of colonization of the urban environment of Porto Alegre, southern Brazil, by Drosophila paulistorum (Diptera, Drosophilidae)

Drosophila paulistorum Dobzhansky & Pavan, 1949 had initially been considered absent in anthropogenically disturbed environments, but in 1985 the detection of the species in Porto Alegre city, southern Brazil, suggested its potential to colonize new habitats and laid the foundations for ecologic studies on this species’ populations. This study followed the variations in D. paulistorum populations in this town almost 20 years after its first local record. Drosophilid specimens were collected in sites with different urbanization grades and the results point to the expressive decline in D. paulistorum populations in Porto Alegre. This decline may be linked to urban growth and to naturally driven population decline, as imputed to climatic changes like variations in maximum and minimum temperatures as a consequence of a global climate warming. Also, the recent introduction of exotic species Zaprionus indianus Gupta, 1970 seems to play a role in this scenario, changing the interactions between native species.


Drosophila paulistorum
is a superspecies of the D. willistoni subgroup, comprising six races or semispecies (DOBZHANSKY & SPASSKY, 1959;PÉREZ-SALAS et al., 1970). The geographic distribution of the taxon is vast, stretching from Guatemala to southern Brazil, where only the Andean-Brazilian semispecies occurs. In southern Brazil, this semi-species coexists sympatrically with its sibling species, D. willistoni Sturtevant, 1916. In several tropical zones of the American continent, D. paulistorum comes second in abundance within the D. willistoni subgroup (SPASSKY et al., 1971).
Until the publication of the study by SPASSKY et al. (1971), the southernmost limit of D. paulistorum geographic distribution was the city of Osório (29°54'S; 51°16'W), state of Rio Grande do Sul, southern Brazil. But in May 1985, D. paulistorum was captured in the urban environments of the city of Porto Alegre (30°02'S; 51°14'W), 90 km south of Osório (SANTOS & VALENTE, 1990). Though D. paulistorum had been considered incapable of surviving in anthropogenically disturbed environments (EHRMAN & POWELL, 1982) upon its capture in Porto Alegre city, the sampling of the species in urban environments suggests its high colonization potential, and certainly laid the foundations for ecologic studies about these populations.
The importance of studies on urban faunas lies in the fact that urbanization exposes organisms that formerly inhabited natural environments to a novel ecological dynamic. Among these disturbance factors is the depletion of forest areas observed as a consequence of the growth of cities to meet the needs of one single species, Homo sapiens (MCKINNEY, 2006). Another ecological consequence operated by the expansion of urbanization is pollution, which significantly alters the environment by impairing the quality of air, of water and of natural resources (MARCUS & DETWYLER, 1972). The massive disturbance set off by urban growth not only spoils the natural habitat of native species, but also generates new environments that favour the establishment of exotic species capable of adapting to this new urban setting. The major consequence of urbanization progression for species conservation is that exotic species may actually enrich local biodiversity, as often observed (SAX & GAINES, 2003). Global diversity, nevertheless, is narrowed due to the subsequent extinction of endemic native species, lost to the overall species set (MCKINNEY, 2006).
In the recent year of 2000, the systematic study of drosophilids within the urban environment of the city of Porto Alegre afforded to detect the introduction of Zaprionus indianus Gupta, 1970(CASTRO & VALENTE, 2001. The arrival of this invader species to that environment seems to be promoting adjustments in the survival strategies of native species. Such adaptations are held to bring about certain changes in richness and abundance of the species that make up these drosophilid assemblies, as recently suggested by SILVA et al. (2005a,b).
Except for the studies focused on pest or epidemic control, research papers on arthropod populations in urban environments are scarce (MCINTYRE, 2000). Although drosophilids constitute a suitable animal model to this line of research -an advantage imputed to the organism's abundance in cities -much is yet to be established regarding the ways urbanization affects the group (KREMEN et al., 1993;VALIATI & VALENTE, 1996;LUCCHESE et al., 2003). Studies on urban populations of D. willistoni and D. paulistorum using different genetic markers have revealed far-reaching genetic changes, seemingly forced upon species by urbanization (REGNER & VALENTE, 1993;VALENTE et al., 1993;VALIATI & VALENTE, 1997;SAAVEDRA et al., 2001). Between 1991and 1992, VALIATI & VALENTE (1996 carried out a second comprehensive study on the ecology of D. paulistorum in Porto Alegre city revealing that the species' frequency had increased since its first record in the city. Both the studies by SANTOS & VALENTE (1990) and by VALIATI & VALENTE (1996) indicated the preference expressed by D. paulistorum for exotic fruits as breeding and feeding sites, whereas D. willistoni was observed to settle for native fruits. These observations led to the inference that such tendencies may be part of a likely strategy adopted by species to avoid competition.
Such body of research has broadened the understanding of Drosophila adaptation to different environmental gradients. Also, these studies have demonstrated the particular importance of the genera as animal model in investigations of urbanization impact over the biodiversity of native species (PARSONS, 1991;POWELL, 1997;AVONDET et al., 2003;FERREIRA & TIDON, 2005).
With a view to shedding new light on the path followed by D. paulistorum as a colonizing species, the present study monitored the occurrence and frequency of the species 20 years after the its first record in the urban environment of Porto Alegre, southern Brazil.
Two sampling methods were employed. In the first method, an entomological net was used to capture adult flies over different kinds of decomposing fruits found in the collection sites, as well as over banana and orange baits placed on the ground and topped with yeast. Bait amount was standardized as 5 Kg bananas and 5 Kg oranges at each collection site. Baits were distributed at different points 100 m away from one another along each collection site, in all sample collections. The second method used rotten fruits that were brought to the laboratory and kept in tubes containing vermiculite under controlled temperature and humidity (25°C ± 1°C, 60% R.H.) upon the emergence of the imagoes. Adult specimens were aspirated and transferred to bottles containing standard growth medium (MARQUES et al., 1966). Adult flies were identified and counted using keys available from the Drosophila Laboratory, Federal University of the State of Rio Grande do Sul. In some cases, male genitalia were dissected. The sibling species D. paulistorum and D. willistoni were identified by electrophoresis of the Acid Phosphatase-1 enzyme (Acph-1), according to GARCIA et al. (2006). Drosophilid populations were compared using the numeric and frequency records for the species studied. The records were made for sites BOG and MTS in years 1986and 1987by SANTOS & VALENTE (1990 and, for sites FAP, BOG and MTS in years 1991and 1992, by VALIATI & VALENTE (1996. The data published by SANTOS & VALENTE (1990) list only D. paulistorum and D. willistoni frequencies; the other species were not discriminated and therefore could not be employed in some of the analyses carried out. Drosophilid data collected in years 2001 and 2002 and described by SILVA et al. (2005b), for the same places evaluated in this study, except MTS (MarioTotta street). Nevertheless, as specimens of the D. willistoni subgroup were not discriminated for species in this last paper mentioned, we resorted to these data only in the cases when D. willistoni and D. paulistorum clustered under the D. willistoni subgroup.
In order to evaluate the variations in species frequencies for the different collection sites and seasons, results were statistically analyzed using the Kruskall-Wallis Multiple Comparison Test. In turn, the Spearman's Correlation Test was used to assess the role played by temperature in species frequency. Temperature records were obtained from the National Meteorology Institute (INMET), 8 th District, a division of the Ministry of Agriculture, Cattle-Breeding and Supplies (MAPA), based in Porto Alegre, RS, Brazil.

RESULTS AND DISCUSSION
Throughout 2004, 27,985 drosophilids were collected in the four sampling sites of Porto Alegre (Tab. I). Of these, 13,461 were classified as Drosophila simulans Sturtevant, 1919, 5,956 as Z. indianus and 4,365 as D. willistoni. These occurrence figures make them the three most abundant species, and taken together account for 85% of the total number of individuals captured. Drosophila paulistorum, with 246 specimens caught, represented a mere 0.9% of the total number of drosophilids collected.  VALENTE (1990) and VALIATI & VALENTE (1996) for the same collection sites. Samples were sorted for collection site, year and season. Trophic resources employed to capture specimens were in turn organized under the categories native fruits (from Neotropical plants), and exotic fruits.
For years prior to 2004, D. paulistorum frequency had always been higher than D. willistoni frequency in exotic fruits such as Averrhoa carambola (L.) (Oxalidaceae) and Maclura pomifera (Raf.) C. K. Schneider (Moraceae). Oppositely, D. willistoni had constantly been more abundant in native fruits, as Syagrus romanzoffiana (Cham.) Glassmann (Arecaceae), for that same period. In 2004, D. paulistorum was basically not observed in native fruits, and sustained its preference for some exotic fruits, as Eriobotrya japonica (Lindl.)  (Rosaceae), verified in the GKP spring record. In this particular trophic resource, the species frequency was substantially high as compared to D. willistoni (28.4% and 5.4%, respectively). Nevertheless, a significant alteration was observed in A. carambola as compared to records of previous years, made clear by the most surprising observation that D. paulistorum frequencies were actually lower than D. willistoni frequencies (exception is made for the MTS spring collection). It may be hypothesized that this change in trophic resource preference by D. willistoni has interfered in the composition of drosophilid assemblies that feed on these fruits, thus affecting D. paulistorum abundance. Apart from this, the decrease in D. paulistorum frequency values may also be the result of the pressure exerted by Z.
indianus. This last species was present in 13 of the 14 trophic resources available (the species was not observed in E. japonica, exclusively). Further investigations should be conducted to ascertain whether these notions are correct or not. Figure 1 illustrates the variation of mean frequencies of D. paulistorum, D. willistoni, Z. indianus and of the other drosophilid species collected between the years 1986 and 2004 in sites BOG and MTS. The analytic panorama obtained suggests that D. paulistorum successfully started the colonization of Porto Alegre at some moment in years 1986 and 1987, with a mean baseline frequency of 11% within drosophilid assemblies (SANTOS & VALENTE, 1990). The species managed to maintain frequencies round that value (13%) in samples collected five years later (VALIATI & VALENTE, 1996); yet, a considerable decrease in abundance was observed in 2004, as revealed by the maximum frequency of 1.4%. Figure 1 also shows that Z. indianus was not observed in collections carried out between 1986 and 1992. In fact, the species was recorded in Porto Alegre for the first time only in the year 2000 (CASTRO & VALENTE, 2001). Zaprionus indianus is an invader species native to Africa, which very speedily has been colonizing South America (DE TONI et al., 2001;GOÑI et al., 2001;TIDON et al., 2003;SILVA et al., 2005a,b) since its first record in the continent, made in São Paulo city (Brazil) in March 1999 (VILELA, 1999). Figure 2 illustrates the mean frequencies of drosophilid assemblies sampled between 1986 and 2004, irrespective of geographic site, season, and trophic resource used. The most representative groups were D. melanogaster Meigen, 1830 subgroup (represented by D. melanogaster and by D. simulans, this last with abundance over 90%), D. willistoni subgroup (represented by D. willistoni and by D. paulistorum), followed by the species Z. indianus. Noticeable is the fact that the frequencies of cosmopolitan D. melanogaster subgroup species and those of Z. indianus varied in reverse order. It was thus that the D. melanogaster subgroup came to represent 75% of the total number of individuals collected in 1991, when Z. indianus had not yet been recorded in Porto Alegre. However, this frequency was lowered to 16.7% in 2001, contrasting with the 44.2% frequency then recorded for Z. indianus. Still another reversal in abundance values was observed in 2004, when D. melanogaster subgroup frequency rose again (49%), accompanied by a decrease in Z. indianus frequency (21%). An explanation for these oscillating frequencies may lie in a likely competition between these species, as Z. indianus records in Brazil were carried out in disturbed environments (TIDON et al., 2003;SILVA et al., 2005a,b;TIDON, 2006), which is similar to what has been observed for the D. melanogaster subgroup (PARSONS & STANLEY, 1981).

D. willistoni
The present study afforded, for the first time, to detect the oscillations in D. paulistorum frequencies since the organism's arrival in Porto Alegre, before and after the arrival of Z. indianus. Figure 4 illustrates the occurrences of D. paulistorum, D. willistoni, Z. indianus, and of the remaining species for the 2004 seasons, irrespective of collection site. The highest D. paulistorum frequency (2.2% in spring) matches the lowest Z. indianus frequency (3.5%), and specially the lowest D. willistoni frequency (3.4%). It may be advocated that the low 2004 D. paulistorum frequency relates indirectly with the presence of Z. indianus. This assumption is based on the probable interaction between Z. indianus and D. willistoni, as the latter species and D. paulistorum are not found in different trophic resources -an observation made by SANTOS & VALENTE (1990) and by VALIATI & VALENTE (1996). Figure 5 illustrates D. paulistorum and D. willistoni frequencies for years 1986 to 1992 in three Porto Alegre sites with different urbanization grades. We observe that D. paulistorum was differentially distributed in these environments. The species preferred low urbanization grade sites, specially MTS, in which the number of individuals collected was significantly higher in contrast with BOG values (Z=2.98; p<0.01) and FAP (Z=2.08; p<0.05). Figure 6 is a representation of the 2004 frequencies of D. paulistorum, D. willistoni, and Z. indianus for the collections made in sites FAP, BOG, and MTS, which were also evaluated in previous years, and in GKP, likewise investigated by SILVA et al. (2005b). The distribution pattern of D. paulistorum frequencies in low urbanization grade sites was observed to have been constant throughout 2004; however, a marked decrease in the species' abundance is now observed in all environments studied (Figs. 5 and 6). The Kruskall-Wallis analysis revealed that this decrease in D. paulistorum abundance was significant in sites FAP (Z=3.16; p<0.01), BOG (Z=3.24; p<0.01), and MTS (Z=4.44; p<0.01) throughout 2004. It is remarkable that D. paulistorum had previously been sampled in MTS at a frequency that reached 43.3% (Tab. II).
Several aspects may lie behind this decline in D. paulistorum populations. For instance, the urban growth experienced in Porto Alegre city since the 1980's might have altered urbanization grades after its first classification, made by RUSZCZYK (1986). Perhaps the area where BOG is located -in which tall buildings have been erected and a wide avenue has recently been builtshould be re-classified as a medium urbanization grade area, while MTS and GKP are still low urbanization grade sites. Contrary to what was observed for D. paulistorum, the likely alterations in urbanization grades in Porto Alegre have not affected D. willistoni frequencies to a significant extent (p<0.05), since the species has been shown to inhabit all these different collection sites. DOBZHANSKY (1965) compared D. willistoni and D. paulistorum capacities to explore novel environments, and suggested that D. willistoni alone, due to the species' genetic characteristics, revealed a trend to become a colonizing species. Though this notion has been challenged by the data in SANTOS & VALENTE (1990) and in VALIATI & VALENTE (1996) -obtained when D. paulistorum expansion was clearly taking place in Porto Alegre -our present findings have led us to consider the DOBZHANSKY (1965) hypothesis, as D. paulistorum is once again found in less urbanized environments only.
Just like D. willistoni, Z. indianus has been captured in almost all Porto Alegre collection sites, with almost all trophic resources. This evinces its success as invader species in the town ( Fig. 6 and Tab. II). FERREIRA & TIDon (2005), while studying drosophilid potentials to colonize sites of different urbanization grades, came to similar results for Z. indianus.
Among the sites evaluated in the present study, special attention is due to GKP, where the lowest Z.
indianus frequencies were observed. Even so, this difference was statistically significant only when the species frequency were compared to its occurrence in the highest urbanization grade site, FAP (Z=3.06; p<0.01). Among the places investigated, GKP has some thickly forested spots that may explain the decreased Z. indianus frequencies there observed. In Central Brazil, TIDON et al. (2003) have reported higher abundance values for the species in disturbed environments limited to the outer stretches of gallery forests. FERREIRA & TIDON (2005) verified that the majority of endemic Drosophilidae species are not capable of colonizing urban environments. The conclusion was similar to that in the study by AVONDET et al. (2003): some Drosophilidae species may indicate not only the occurrence of environmental changes, but also the degree of environmental disturbance revealed by the alterations in species frequencies. Thus, these species are efficient indicators of ecological changes brought about by urbanization.
According to PARSONS (1987), cities are relatively unfavourable habitats to native species. This is because these species cannot rely on the adaptive conditions necessary to thrive in a disturbed setting which, as a consequence, is suitable to the colonization by exotic species. The decline in arthropod richness observed with    increased urbanization grades has been imputed to a series of factors, such as the isolation of native populations into smaller zones within their natural environment, as well as to pollution and to the destruction of feeding and breeding sites (MCINTYRE, 2000). This drop in species richness within an urbanization gradient has also been observed for birds (MARZLUFF, 2001), mammals (MACKIN-ROGALSKA et al., 1988), and butterflies (BLAIR & LAUNER, 1997). As a rule, more intensely urbanized areas are the home of a small group of exotic species and of an even smaller number of native species, as compared to the suburbs. In our study, the only high urbanization grade site (FAP) presented the lowest species diversity (20), with 16 native and 4 exotic species captured. In low urbanization grade sites (BOG, MTS, GKP), the number of exotic species oscillated between 4 and 5. Drosophila simulans, D. immigrans Sturtevant, 1921, and Z. indianus were common to all these sites, and were also captured in FAP. Nevertheless, native species diversity (21 to 23) in less urbanized locations was over 30% higher (31.2% to 43.7%) than in FAP. These suburban areas have green spots, remnants of the natural habitats of these native species and that may work as ecological refuges, which diminish local extinctions and contribute to greater richness of native species. Figure 7 shows the frequency oscillations for Z. indianus and for the D. willistoni subgroup, as well as the mean maximum and minimum temperatures and respective standard deviations for the seasons in years 2001, 2002, and 2004 in Porto Alegre city. In 2001 and 2002, D. willistoni subgroup frequency was observed to decrease significantly (r=0.91; p<0.01) in the seasons when Z. indianus frequency increased, which however was not observed in 2004 (r=0.60; p<0.05). The growth of Z. indianus populations was observed in summer and spring, when mean temperatures were higher. Yet, Z. indianus frequencies are more intimately correlated to mean minimum temperatures (r=0.61; p<0.01) than to mean maximum temperatures (r=0.32; p<0.05); still, when global mean temperatures are considered, such correlation is not always observed. For instance, it was only in the 2002 collections that a correlation between mean temperatures and higher Z. indianus frequencies was established (r=0.99; P<0.01). TIDON et al. (2003) did not find any correlation between Z. indianus frequencies and mean temperatures, while TIDON (2006) reported a significant though low correlation with the temperature in some study sites in the Brazilian Cerrado biome. So, mean temperatures do not always reflect what actually happens with temperature extremes (maximum and minimum values), which may be more informative to evaluate oscillations in drosophilid assemblies. Therefore, very low temperatures constitute limiting factors to the population size of Z. indianus, as observed in winters, specially in 2004, when minimum temperatures were the lowest recorded throughout the study period. In like manner, D. willistoni subgroup frequencies are temperature-dependent also in Porto Alegre city. However, differently from what was observed for Z. indianus, the subgroup presented a negative correlation with maximum temperatures (r=0.72; p<0.01), and had the lowest frequencies in hotter seasons. In a recent paper that studied drosophilid composition across an urban gradient in Florianópolis city, state of Santa Catarina, southern Brazil, M. S. Gottschalk (pers. comm.), observed exotic species to predominate in periods of moderate and high temperatures, while native species prevailed in colder weather.
As a final point, the expressive decrease in population size suffered by D. paulistorum in the past few years is also reflected in chromosomal inversion polymorphisms. In the study by SANTOS & VALENTE (1990), the number of different heterozygous chromosomal inversions detected in all populations of Porto Alegre was 18; but VALIATI & VALENTE (1997) detected 23, of which only six had been found in the first study mentioned. In the present study, the analysis of chromosomal polymorphism in D. paulistorum populations in Porto Alegre revealed the occurrence of only three heterozygous chromosomal inversions (data not shown).
The temperature of the planet has risen roughly 0.8°C in the past 100 years, and around 0.2°C every decade in the past 30 years (HANSEN et al., 2006). Recent studies have shown that several genetic changes in different organisms seem to be associated to these climate changes, called global warming (PARMESAN & YOHE, 2003;COTTON, 2003;LEVITAN & ETGES, 2005;BRADSHAW & HOLZAPFEL, 2006). BALANYÀ et al. (2006BALANYÀ et al. ( , 2007 studied 26 populations of D. subobscura Collin & Gordon, 1936(COLLIN, 1936 distributed across Europe and South and North Americas. The data obtained for the past 24 years demonstrate that the temperature has risen in 22 of the places investigated. The authors verified that the frequency of chromosomal inversions typically observed in species inhabiting low latitudes, i.e., warmer regions, has increased swiftly in 25 populations studied. These fast alterations in chromosomal arrangement frequency reflect the adaptation response by some genes to climate change across the geographic distribution of D. subobscura (HEERWAARDEN & HOFFMANN, 2007) and D. melanogaster (UMINA et al., 2005).
All in all, there appears to be a decrease in the colonizing potential of D. paulistorum in Porto Alegre, made evident by the drop in the species' frequency in  recent years. This fact may be linked to the city's growth, as well as to the natural population decline that may possibly take place in the long run. This decline may also bear a relationship with climatic changes, such as variations in minimum and maximum temperature amplitudes. Moreover, the presence of Z. indianus seems to contribute to this scenario, as the species might be interfering in the interactions between native species.