Environmental degradation impact on native communities of arbuscular mycorrhizal fungi in an urban fragment of semideciduous plateau forest

(Environmental degradation impact on native communities of arbuscular mycorrhizal fungi in an urban fragment of semideciduous plateau forest). Three forest reserves, with highly degraded areas, are open to visitors in Maringá, Paraná, Brazil. Impact caused by tree cutting, heavy traffic and visitors on the establishment of arbuscular mycorrhizal fungi (AMF) was evaluated in two areas with different degradation stages of the Dr. Luis Teixeira Mendes Forest Garden, a remnant of semideciduous forest. Soil samples were removed from three locations within each area; spores were isolated from the soil by wet sieving and sucrose centrifugation and mounted on permanent slides. Spores were counted and identified taxonomically under a microscope. Diversity, dominance, equitability and similarity indexes were calculated from abundance data. The degraded area had the highest number of spores and featured communities with the lowest rates in richness, diversity and equitability. However, high spore density was caused by the frequent presence of G. sinuosum sporocarps. Ten to 12 species were verified in each site from the preserved area while this number varied from 6 to 12 in the degraded area. In the degraded area, Site II, lying in the most protected area of the forest fragment, diversified and equilibrated communities existed, similar to sites in the preserved area. Results suggest that environmental degradation had negative effects on the establishment and diversity of AMF.


Introduction
Arbuscular mycorrhizal fungi (AMF), linked to improvements in vegetation growth for more than one hundred years (Trappe 1987), are of paramount importance to recover disturbed natural areas.Th e number of plants dependent on arbuscular mycorrhizal (AM) association is generally high and nutrient defi ciencies are an important restriction in their growth (Li et al. 1991).
Considered to be the most relevant components of soil microbiota (Oehl et al. 2003) and present in various regions of the world, AMF are involved in ecological succession and fl ora rebuilding, contributing to the diversifi cation, productivity and stability of natural ecosystems (Allen et al. 1995;Klironomos et al. 2000).
Th ere are three forest reserves in Maringá with remnant native vegetation representing sites of biological reserve.Conservation areas have been defi cient and several sites are highly degraded.
Impact caused by pathways and tree removal within forest fragments, car traffi c and visitors have caused a series of environmental changes (Kattan & Alvaréz-López 1996), which interfere in AMF communities and in the functioning of symbiosis.Microclimate variations (mainly temperature rise and decrease in humidity), loss of macro-and micronutrients, changes in fl oristic composition and genetic erosion are the consequences of forest clearings (Kattan & Alvaréz-López 1996;Schellas & Greenberg 1996).Such modifi cations may negatively aff ect establishment and diversity of AMF communities (Cuenca et al. 1998;Stahl et al. 1988;Zhang et al. 2004).Consequently, knowledge of several factors that aff ect AMF diversity is of great importance for the management, recovery and conservation of the environment.
Several studies have used population data, such as distribution and abundance of spores of species, and sociological data of AMF communities in cultivated and natural ecosystems, in Brazil and in other countries.Still, there are still few studies focusing on AMF diversity in urban forest fragments predominantly constituted by native vegetation, and in Brazil assessments have started in Paraná state, specifi cally in Maringá.In this municipality, two of seventeen parks have been evaluated (Dr.Luiz Teixeira Mendes Forest Garden and Cinquentenário Park), and in these, diff erent communities of AMF were verifi ed in terms of composition as well as diversity.In the fi rst fragment, considered to be a forest reserve, two studies were made over a two-year period in which 114 soil samples were evaluated.During the fi rst year, the diversity of AMF was examined under two environmental conditions, preserved and degraded (this paper), using samples with diff erent volumes of soil.Since the recovery of species was not better in the biggest samples and data dispersal was higher, we considered only data from the smallest ones.In the second year, Carrenho & Santos (2006) investigated the edge eff ect on the composition of species in those areas and verifi ed lower number of species (1 to 6 in the preserved area and 2 to 4 in the degraded area) and spores (averages of 36.46 and 29.2 in the preserved and degraded areas, respectively).It is interesting to stress that the fi rst study was done in the summer and the second in the winter, period which no additional species was identifi ed.In the second fragment (Cinquentenário Park), smaller and in a less advanced stage of succession and anthropization, 85 samples were collected from diff erent sites in an unique period from summer, resulting in two studies whose main objectives were to verify the effi ciency of diff erent trap plants (Lippert 2009) or homeopathic solutions on peanut (Santos 2009) under pot culture in detecting the diversity of species coming from fi eld.
Little is known about the eff ects of urbanization on AMF established in the referred fragments, but results from other studies have evidenced negative responses when plants imported from other areas are introduced (Helgason et al. 2002) or when the area is exposed to common pollutants, such as nitrogenous compounds, toxic metals and ozone (Cairney & Meharg 1999;Egerton-Warburton & Allen 2000).Th us, current research evaluates if the loss of environmental quality, caused by deforestation, paths and visitors infl uences AMF communities.

Site characterization
Th e Forest Garden is delimited by the coordinates Latitude 22 o 30'-24 o 30'S and Longitude 51 o 30'-54 o W, at a mean altitude of 556 meters, featuring tropical deciduous plateau vegetation (Embrapa/Iapar 1984).Th e soil in Maringá is a dystrophic Dusky-Red Latosol, originated from basalt decomposition (www.codem.org.br).According to Köppen classifi cation, a humid tropical mesothermal climate, or Cw'h, predominates.Data from the Main Climatology Station of Maringá at the State University of Maringá show that in 1999 mean, maximum and minimum temperatures were 28.1 o C and 17.4 o C, respectively; relative humidity was 68%; total rainfall was 1,412.8mm; total evaporation was 1,920.4mm; and total sunshine reached 2,701.6 h (www.codem.org.br).
Two distinguishable areas may be detected in the Forest Garden: a protected area, prohibited to visitors, and a more exposed area with several sites lacking vegetation, in which visitors and cars are allowed.Soil chemical properties of the two areas show statistically signifi cant diff erences, seemingly related to vegetation (Tab.1).

Sampling
Soil was collected in PVC tubes, 15 cm long and 5 cm diameter, with a volume of 294.37 mL.Samples were removed at three randomized sites (I, II, III) from the two areas (preserved and degraded) of the Dr. Luis Teixeira Mendes Forest Garden on December 13, 1999.Th ree replications from each site, totaling 18 samples, were taken in order to extract the spores, and another six samples were collected to be used as source of propagules for culture in pots.

Trap culture
For culturing the native AM fungal communities, traps were established by placing 200 g of soil samples with root pieces as layers sandwiched between two layers of sterile soil (methyl bromide).Plastic pots used were 10 cm wide and 15 cm tall having the capacity to accommodate 1 kg of soil.Pots were fi rst fi lled with sterile soil up to 8 cm, the soil inoculum was added and topped with another layer of sterile soil.Four replications from each site, totaling 24 samples, were used to multiply the AMF.Sorghum bicolor (L.) Mönch was used as bait plant, and at the end of the four-month growing cycle, 100 g soil was sampled from each pot for AMF spore extraction, counting and identifi cation.Nevertheless, this trial failed to establish a successful production of spores, although the roots exhibited some sites colonized by AMF (unpublished data) 3 .It is interesting to emphasize that those roots were also colonized by dark septate fungi, which were more frequently observed than AMF (unshown data).

Soil processing and preservation of spores
Soil from tubes was dried at room temperature and AMF spores were isolated by wet sieving technique (Gerdemann & Nicolson 1963) and sucrose centrifugation (Jenkins 1964).
Spores were mounted on semi-permanent slides in two separate groups: one group with PVLG resin and the other with PVLG resin + Melzer, and counted under a microscope (Morton et al. 1993).Th e sporocarps were carefully broken and the spores were counted.All of the spores are kept in the personal collection from Rosilaine Carrenho, and those in good state of conservation are mounted on slides which are being incorporated into Herbário da Universidade Estadual de Maringá (HUEM).Photographs can be obtained from the responsible author.

Sociological parameters
Behavior of AMF communities was evaluated by spore density and ecological indexes (richness, diversity, dominance, equitability, similarity), following Magurran (1988).Total density of spores consists of spores found in the tubes (samples); species density is the number of spores of each species.Frequency of occurrence refers to the number of samples (replications) in which a certain species occurred.Richness was represented by number of species.Dominance was shown by Simpson's index; diversity was determined by Shannon's index; equitability was calculated by Pielou's index.Similarity of communities in the diff erent environments was determined by Sorensen's qualitative coeffi cient.

Statistic analysis
Data on spore density were analyzed by non-parametric ANOVA and Mann-Whitney test.

Results and discussion
When soil volume analyzed in each collecting site (883.11mL) is taken into account, total number of spores was low (Table 2).In studies related to fragmented forest environment, means ranged between 288 and 2,797 in 100 g of dry soil (Santos 2001;Alves 2004) and in urban forest fragments, means ranged from 83.67 to 165 (Lippert 2009; (1) S I, S II and S III correspond to the sampling sites.
3 Th e data were not shown or evaluated by statistical tests because many plants died, mainly those cropped on soil from degraded areas, thereby weakening the statistical analysis.Santos 2009).In those studies, the highest numbers of spores were verifi ed in fragments of larger dimensions and under lesser anthropic intervention, diff erent from this study, whose higher number of spores occurred in the degraded area (Tab.2).Th is fact was, however, due to the constant presence of sporocarps of Glomus sinuosum, whose spore numbers were considered to calculate population abundance and related ecological indexes (Tab.3).When the above species is not taken into account, AMF communities in sites I and III have very low number of spores (18 and 16, respectively).Th ese sites, close to paths with traffi c of cars and people, show poor vegetation as well as low soil fertility (Tab. 1) diff erent from site II, that lies in the middle of the fragment and exhibits better environmental conditions (higher humidity; richer and denser vegetation; higher rates of phosphorus and carbon).Stahl et al. (1988) noted fewer propagules and lower species richness of AMF in environmentally disturbed areas that is partially concordant with the present data.Considering only spore numbers, which are one of the types of mycorrhizal propagules, and excluding Glomus sinuosum from samples, it is possibl e to observe the referred relationship.Since soil organic matter is derived main ly from plant residues, it cont ains all of the essential plant nutrient s.Accumulated organic matter, therefore, is a storehouse of plant nutrients.Loss of plant cover aff ects both quality and quantity of soil organic matter, which modify many chemical and physical properties, including soil pH.Organic matter decomposition generally increases soil acidity by addition of hydrogen and this will depend on input rates.Th e less input on soil, the less acidifi cation, as verifi ed in the present study and pH has been considered a modulating factor on AMF growth.Santos (2001) and Barbosa (2004) found that spore numbers and soil pH were negatively related and corroborate data in current study (excluding G. sinuosum from the samples).It is possible that species forming free spores or in small groups and species forming dense sporocarps are diff erently aff ect by soil properties, that can explain the opposite responses verifi ed for G. sinuosum.
All sites in the preserved area are well structured with many herbaceous plants in the lower layer and well-closed top covering.In this area, soil properties were generally evaluated as uniform with sharp diff erences only in phosphorus and carbon rates.Site I, more exposed to light and wind, had a lower P rate while the innermost site (Site III), more shaded and with extensive vegetation, had the highest C and P rates (Table 1).However these contrasts in environ-mental variables and edaphic properties were not enough to change the structure of AMF communities, which showed higher richness, diversity and equability (Tab.4).In this area, interspecifi c competition seemed to be symmetric and diff erences in spore abundance may be due to reproductive eff ort of each species.
Intensity, frequency and duration of natural or anthropic disturbances are directly related to restoration and regeneration capacity of the ecosystem.Low environmental perturbations may stimulate proliferation and diversifi cation of biotic communities and cause subtle changes in their composition.Serious disturbances produce heterogeneous environments with communities exhibiting mosaicism, which may induce unrepairable metabolic errors and development instability in these organisms, causing loss of genetic variability (White & Jentsche 2001).However, it is important to keep in mind that in clonal organisms, such AMF, the genetic heterogeneity originating from somatic mutations, mitotic recombinations, mitotic gene conversions, genome duplications, or transfer of nuclei between hyphae can represent an important source of variation within a population.Experimental studies on this topic are lacking and genetic processes in AMF are critically discussed by Pawlowska (2005).Burrows & Pfl eger (2002) reported that an increase in plant diversity in a given environment may cause a increase in the number of spores (30% to 150%) and in AMF spore volume (40% to 70%), or rather, a greater quantity of large spores.Table 2 shows that species with the above qualities (A. laevis, A. tuberculata, G. macrocarpum, G. versiforme and Scutellospora rubra) sporulated preferentially in the preserved area with richer and more diversifi ed vegetation, supporting the above authors' report.Zhang et al. (2004) recorded that unfavorable soil conditions for plant growth also decrease the density of propagules of the organisms.Soil samples of the degraded area had quite lower rates of Ca, Mg, K, P and C than those of the preserved area and this condition seemed to limit the sporulation of almost all species (Tab.1).On the other hand, G. sinuosum occurred with high population abundances and high frequency on those conditions, which increased the dominance on AMF communities from those sites (Tab.4) Glomus macrocarpum and G. versiforme were also abundant at Site II of the degraded area, which is the most protected site, with higher P and C rates in the soil.Glomus macrocarpum is widely distributed in Brazil and its sporulation has been positively related to soils with high (1).All of the AMF communities showed log distribution.  (1n=3. (2)AD means the accumulated number of spores in each one of the sampling sites. (3)number inside parenthesis correspond to the frequency of occurrence.
phosphorus levels (Carrenho 1998).According to Bononi & Trufem (1983) the generalized occurrence of a specifi c AMF taxon may indicate greater ecological disturbance in the environment.Likewise, restricted occurrence may indicate lesser disturbances and a trend towards AMF association with specifi c phytobionts.
Although G. versiforme is a restricted species in Brazil, a high representation of the species in protected natural forest areas and in deforested areas may indicate, according to Zhang et al. (2004), a wide adaptive and tolerance capacity to environmental disturbances.
Acaulosporaceae and Glomeraceae predominate in AMF communities in terms of spore abundance and in species richness.Six species of Acaulospora, one species of Entrophospora, ten species of Glomus and one species of Scutellospora (Tab.2) were reported.Sporulation of Acaulospora was more extensive in the preserved area and seemed to be related to high acid pH in the soil.Above data have been confi rmed by Johnson et al. (1991) and Carrenho et al. (2002).Since a similar behavior has been verifi ed for Glomus, C rate of soil is probably the modulating factor of sporulation of these organisms, as has been previously reported by Johnson & Wedin (1997) and Carpenter et al. (2001).Within the most frequent and abundant species, Acaulospora laevis, Glomus etunicatum, G. macrocarpum and G. versiforme showed great diff erences in total number of spores and were dominant in the preserved area.Forest fragment size (more extensive in this area) may have contributed to such behavior, as Mangan et al. (2004) have reported in their studies in Panama.
Fift een AMF species have been reported in the preserved area with slight variations (9, 11 and 10 species for sites I, II and III respectively) among the sampling sites.Further, the number of species restricted to a single site was also low (G.clavisporum at site I and G. microaggregatum and Scutellospora rubra at site II).Th is fact induced the formation of similar communities, as Sorensen's similarity coeffi cients have shown (Tab.4).A similar behavior has been reported for total density of spores (Tab.3).Since communities are assessed according to the proportionality of species occurrence and abundance, site II had the highest richness, diversity and equitability and site III had the highest dominance.However, diff erences were slight and revealed good AMF performance in this area (Tab.4).It was also observed that the similarity between the sites was lower when Site I was confronted (Tab.4) and this can be due to the edge eff ect on plant communities and their associated AMF.
Th irteen species, distributed in a variety of manners among the sampling sites (6, 13 and 18 respectively at sites I, II and III), were found in the degraded area.Site II (less disturbed and with higher P and C rates in soil) revealed communities with the highest richness, diversity and equitability.Th is fact contrasted greatly with communities at sites I and III (Tab.4).In spite of such unequal distributions, similarity indexes between sites were high, or rather, between 61.5% and 73.7%, being the lowest ones related with Site I, located on the edge of the fragment that indicates the edge eff ect altered the distribution and densities of AMF, a fact also verifi ed in the preserved area.It must also be emphasized that dominance in sites I and III is accountable to G. sinuosum (Tab.3), which occurs in compact sporocarps, and assumes a typical "phalanx" growth strategy, whose tightly packed arrangement of spores and profuse population aggregation restrict local occupation by other species, that evidences a clear example of asymmetric competition.Th is can be due to direct competition for space and resources or by indirect infl uence, through the production of high concentrations of extracellular enzymes (related to mineralization of organic P compounds, that may enhance mycorrhizal utilization of an important nutrient pool in soil) or pigments such as melanin (given the supposed involvement of melanin in protecting fungal structures, improving hyphal longevity), which may interfere negatively with the spread of other species.
Current data show that the production of spores of most species and the diversity of AMF communities were jeopardized by stresses produced by disturbances in the original environment.Acaulospora laevis, Glomus etunicatum, G. macrocarpum and G. versiforme were highly aff ected by deforestation, forest paths and visitors, while G. sinuosum was resistant to the conditions of the degraded area.

Table 1 .
Chemical and physical-chemical soil characteristics in diff erent sites of Dr. Luis Teixeira Mendes Forest Garden in Maringá, PR.

Table 2 .
Infl uence of environmental degradation on spore density of AMF established in two areas of Dr. Luis Teixeira Mendes Forest Garden.

Table 4 .
Ecological indexes for communities of AMF established in two areas of Dr. Luis Teixeira Mendes Forest Garden, Maringá -PR, under diff erent environmental conditions.(1)

Table 3 .
Total (TD) and specifi c density (SD) of spores and frequency of occurrence of arbuscular mycorrhizal fungi in diff erent areas of Dr. Luis Teixeira Mendes Forest Garden according to level of anthropic perturbation.