ON YEASTS ASSOCIATED WITH NECROTIC CACTUS STEMS FROM DIFFERENT LOCALITIES IN BRAZIL

The yeast species found in necrotic stems of three columnar cacti (Pilosocereus machrisii, Pilosocereus vilaboensis, and Praecereus euchlorus) at eight localities in Brazil were described and a similarity analysis using Sorensen distances was used to compare the composition of yeast species at these localities. Of 56 necrotic cactus stems sampled, 32 produced yeast colonies. Ten species of yeast or yeast-like microorganisms were identified from 53 isolates, with Pichia cactophila, Candida sonorensis, Geotrichum sp., and Sporopachydermia cereana being the most common. The remaining species occurred in low proportions in the cacti surveyed. The similarity analysis provided a dendogram (UPGMA) that clustered the yeast communities from different cactus species and indicated that host cactus species was unimportant in this clustering.


INTRODUCTION
The cactus-yeast-Drosophila system represents a three-way ecological association in which necrotic cactus tissue serves as a substrate for a diverse and often highly specific yeast flora.These yeasts, in turn, constitute an important food source for larval and adult stages of Drosophila, which often have specific relationships with the substrate used for breeding and feeding (Sang, 1978;Fogleman, 1982;Phaff et al., 1985;Ganter et al., 1986;Starmer et al., 1990).The interdependence of yeast and Drosophila on decaying tissues of cactus also occurs because the flies serve to disperse yeasts to new cactus habitats (Gilbert, 1980).This interactive system provides an interesting scenario for ecological and evolutionary studies.
Previous studies on yeasts associated with cacti have revealed a spectrum of host plant specificities.For instance, Pichia heedii is specific to only one cactus species in the North American Sonoran desert (Phaff et al., 1978), Candida deserticola is restricted to several species of the genus Stenocereus (Phaff et al., 1985), and Pichia cactophila and Candida sonorensis are found in almost all cactus species from which yeasts have been collected (Lachance et al., 1988;Starmer et al., 1990).Studies on the spatial heterogeneity in species composition and relative abundance in yeast communities associated with necrotic cacti have shown differences within and among geographic areas (Starmer et al., 1990;Barker et al., 1983).
Studies of the cactus-yeast-Drosophila system in Brazil have used mainly the Drosophila buzzatii species cluster and the cactus genera Opuntia, Cereus, and Pilosocereus (Sene et al., 1988;Rosa et al., 1994Rosa et al., , 1995)).In this work we describe the yeast species found in the necrotic stems of three columnar cactus species at eight localities in Brazil and report a similarity analysis for these localities based on the species composition.This survey is part of an extensive study on the evolutionary history of D. gouveai, a member of the D. buzzatii cluster that breeds exclusively in decaying cactus stems and inhabits most of the geographical areas discussed here.

METHODS
Fifty-eight necrotic cactus stems were sampled from eight localities in Brazil (Table 1), but only 32 provided yeast colonies.Three columnar cacti distributed in South America, Pilosocereus machrisii, Pilosocereus vilaboensis, and Praecereus euchlorus, occurred at these localities, with no overlap in distribution.P. machrisii and P. vilaboensis are typical of quite barren rocky outcrops associated with savannah and campos rupestres vegetation, whereas Praecereus euchlorus occurs in slopes with dry, shallow soil amidst gallery forest.
Necrotic stems were sampled aseptically by suspending about one gram of material (from various positions within the necrosis, and only one necrotic stem per plant) in 5 ml of sterile distilled water.The samples were vortexed in aseptic conditions and one loop full was streaked onto YM agar (0.3% yeast extract, 0.3% malt extract, 0.5% peptone, 1% glucose, and 2% agar) supplemented with 100 mg of cloramphenicol/L.The plates were incubated at room temperature (27 ± 3ºC) for three days.Individual yeast colonies were purified and characterized using standard methods (van der Walt & Yarrow, 1984;Lachance et al., 1988).The yeast species were identified according to the keys of Kurtzman & Fell (1998).When two or more cultures isolated from one sample were identified as the same species, they were considered as a single isolate.
The similarity between pairs of yeast communities from different localities was calculated by using the Sorensen distance (Ludwig & Reynolds, 1988) based on the absence or presence of each species at each locality.Clustering was done using the unweighted pair-group arithmetic average (UPGMA) procedure (Sneath & Sokal, 1973).

RESULTS AND DISCUSSION
A total of 53 isolates were obtained and ten species of yeast or yeast-like microorganisms were identified (Table 2).Pichia cactophila was the most commonly isolated yeast species, followed by Candida sonorensis, Geotrichum sp., and Sporopachydermia cereana.The PIR locality was the only one where P. cactophila was not isolated.The yeast-like microorganism Geotrichum sp. was the prevalent species at Altinópolis, SP (FOR) and Brotas, SP (BRO), where the host cactus was P. machrisii.
P. cactophila is the most widely distributed cactophilic yeast (Lachance et al., 1988) and the most frequent yeast in necrotic cactus samples (Starmer et al., 1990).This species is rarely found in habitats other than necrotic cactus stems (Starmer et al., 2003).C. sonorensis has only been found in cacti and occurs in rotting somatic tissue more or less independently of the host species or geographic location (Starmer et al., 1990).The species of Geotrichum are prevalent in the yeast community associated with the cactus Pilosocereus arrabidae in southeastern Brazil (Rosa et al., 1994;Morais et al., 1994;Rosa et al., 1995).S. cereana is a complex of 10 related species (Lachance et al., 2001) and, in Brazil, occurs as two species: S. cereana "australis" in P. arrabidae (Rosa et al., 1994) and S. cereana "brasiliensis" in Cereus pernambucensis (Rosa et al., 1995).P. cactophila, S. cereana, and C. sonorensis are the principal species of most cactus yeast communities (Starmer et al., 1990;Starmer et al., 2003).P. cactophila, Geotrichum spp., and C. sonorensis are among the most prevalent yeast species present in the intestinal tract of Drosophila serido larvae collected in necrotic stems of P. arrabidae (Morais et al., 1994).D. serido is a cactophilic species of the D. buzzatii cluster in South America.
The yeast species found in lowest proportions in the cacti surveyed included Clavispora lusitaneae, which was found only at Furnas, MG (FUR) and has frequently been isolated from Pachycereinae cacti species in southern Mexico (Starmer et al., 1990).This species may also occur in habitats other than cactus (Lachance et al., 1986).This is the first published record of C. lusitaneae isolated from Cactaceae in Brazil.Cryptococcus humicola also was found only at FUR, and species of this genus generally occur at a low frequency in cacti (Lachance et al., 1988;Rosa et al., 1994).Pichia membranifaciens, a yeast species that is not strictly cactophilic (Lachance et al., 1988), was found only at FOR.This species has been found in necrotic stems of P. arrabidae from southeastern Brazil (Rosa et al., 1994).Pichia novergensis is an exogenous component of the yeast flora of necrotic cactus tissues (Lachance et al., 1988).In Brazil, P. novergensis has been found in stems of Opuntia spp.(Rosa et al., 1995) and of P. arrabidae (Rosa et al., 1994), as well as in flowers of C. pernambucensis (Rosa et al., 1995).Rhodotorula spp.have been isolated at very low frequencies from yeast communities associated with cactus (Lachance et al., 1988), and have been found on the stems and flowers of P. arrabidae and Opuntia spp.from Brazil (Morais et al., 1994;Rosa et al., 1995).Pichia guilliermondii occurred only at Pirenópolis, GO (PIR), and was the sole species isolated at this locality.This species has been observed only sporadically in the somatic tissue and fruit of cactus (Lachance et al., 1988).In previous surveys, only the anamorph of this species, Candida guilliermondi, has been isolated from necrotic cactus tissues (Rosa et al., 1994;Morais et al., 1994;Starmer et al., 1990).
Figure 1 shows the dendogram of similarity among the yeast communities based on the composition of yeast species at each locality.The yeast communities from FOR, BRO (host cactus P. machrisii), and São Carlos, SP (SAS) (host cactus P. euchlorus) formed a cluster with high similarity.The yeast community from FUR clustered with that from Cristalina, GO (CRI), mainly because of the presence of the yeast species P. novergensis and Rhodotorula sp., which occurred only at these localities.The PIR locality was isolated on a branch by itself because of the presence of the yeast P. guillermondii, which was the only species isolated from this locality.Because yeast communities from different cactus species were clustered together in the dendogram, the host cactus species appeared to be unimportant in this clustering.This result agrees with the wide cactus niche occupied by the most of the species isolated (Starmer et al., 2003).We have made extensive collections of cactophilic Drosophila from the localities studied in this survey and, with the exception of SAS and Dois Córregos, SP (DOC), all of the other sites had D. gouveai and D. buzzatii, two members of the D. buzzatii cluster.P. euchlorus is the only host cactus found in São Simon, SP (SAS) and DOC.The absence of cactophilic Drosophila at these two sites suggests that P. euchlorus is an unsuitable breeding site for these Drosophila species.The lack of D. gouveai and D. buzzatii in necrotic stems of P. euchlorus could reflect the absence of suitable nutrients provided by the yeast species associated with this host.However, based on the similarity in the composition of the yeast species at the different localities (Fig. 1), this hypothesis appears to be unlikely.The yeast communities at SAS and DOC were similar to those of other localities where the cacti present belonged to the genus Pilosocereus, a well-defined breeding site for the D. buzzatii species cluster.  1 for locality identification).Similarity was calculated using the Sorensen distance (Ludwig & Reynolds, 1988) based on the presence or absence of yeast species at each locality.The host cactus present at each locality is shown in parentheses.Abbreviations: PM, Pilosocereus machrisii; PE, Praecereus euchlorus; PV, Pilosocereus vilaboensis.

Fig. 1 -
Fig. 1 -UPGMA dendogram showing the similarity in the composition of cactus yeast species at different localities (see Table1for locality identification).Similarity was calculated using the Sorensen distance(Ludwig & Reynolds, 1988) based on the presence or absence of yeast species at each locality.The host cactus present at each locality is shown in parentheses.Abbreviations: PM, Pilosocereus machrisii; PE, Praecereus euchlorus; PV, Pilosocereus vilaboensis.