Undecomposed Twigs in the Leaf Litter as Nest-Building Resources for Ants (Hymenoptera: Formicidae) in Areas of the Atlantic Forest in the Southeastern Region of Brazil

In tropical forests, the leaf-litter stratum exhibits one of the greatest abundances of ant species. This diversity is associated with the variety of available locations for nest building. Ant nests can be found in various microhabitats, including tree trunks and fallen twigs in different stages of decomposition. In this study, we aimed to investigate undecomposed twigs as nest-building resources in the leaf litter of dense ombrophilous forest areas in the southeastern region of Brazil. Demographic data concerning the ant colonies, the physical characteristics of the nests, and the population and structural of the forest were observed. Collections were performed manually over four months in closed canopy locations that did not have trails or flooded areas. A total of 294 nests were collected, and 34 ant species were recorded. Pheidole, Camponotus, and Hypoponera were the richest genera observed; these genera were also among the most populous and exhibited the greatest abundance of nests. We found no association between population size and nest diameter. Only tree cover influenced the nest abundance and species richness. Our data indicate that undecomposed twigs may be part of the life cycle of many species and are important for maintaining ant diversity in the leaf litter.


Introduction
Ants represent only 2% of the described insect fauna; however, they constitute more than 50% of the animal biomass of tropical forests, savannas, fields, and other important habitats [1,2].The majority of ant species inhabit the soil and/or leaf litter [3].In tropical forests, approximately 50% of the ant fauna may be associated with the leaf litter [4], but the factors that structure these communities are poorly understood [5,6], especially for communities that live in the leaf litter of the Brazilian Atlantic Rainforest.One fundamental goal of ecology is to understand the structure and maintenance of diverse tropical assemblages [7].
Studies of the diversity of leaf-litter ants are relatively abundant [8,9], and several studies have been conducted in the Atlantic Rainforest sites selected for the present study (see [10][11][12]).The ant fauna that inhabits the leaf litter is rich in terms of taxonomy, morphology, and function [13][14][15], and its high diversity [5,14,16] has been associated with vegetation structure and the biotic and abiotic characteristics of the habitat [17,18].
In the leaf litter, ants have access to food resources, locations for nest building, and a favorable microclimate [17,[19][20][21][22][23].The variation in nest-building locations among species is a prerequisite for the maintenance of leaf-litter ant species diversity [24].Despite a reasonable amount of ecological knowledge, the biology of the majority of leaflitter species is still largely unknown [25].
Litter-nesting ants are a particularly useful group for community-level comparisons because litter can be exhaustively searched to provide accurate data on nest density and species richness, regardless of weather [6].Ant nests in the leaf litter are located among interstices, in the interior of fruits, between leaves, or in branches and trunks at various Psyche stages of decomposition [26].Among the resources provided by the leaf litter, twigs/trunks are essential for ant nests to occur [27].Small ants and those with little dispersal ability, such as species of Myrmicinae and the tribe Dacetini or species of Ponerinae, can be found by breaking pieces of branches and twigs that have fallen in the leaf litter or by looking in rotten trunks [28].However, fallen trunks are colonized by few species [29]; twigs are important for the nesting of a higher number of species, especially twigs in stages of decomposition [27].This preference may be related to the ants' access to the inside, as digging is easier [27], or to moisture, which is the limiting factor for leaf-litter ants [5].
In the present study, we investigated in detail the ant species that use twigs that are still undecomposed, a subset of twigs that is less exploited by ants [27].Furthermore, to investigate the determinants of community organization, we collected measurements related to twig characteristics, colony structure (workers, alates, and immature ants), canopy cover, and litter depth.

Study Area and Sampling.
The colonies were collected in 10 dense ombrophilous forest fragments [30] located in the Upper Tietê Hydrographic Basin in the state of São Paulo, Brazil (Table 1).The sampling expeditions were only conducted once during the experimental period.The samples were collected between September and December of 2010 (approximately one sampling per week), spanning the region's rainy season [31].Each sampling expedition was conducted one day after rain in locations without trails, without flooded areas and under a closed canopy.The vegetation of the collection areas was similar and composed of trees that were 2 to 20 m in height with trunks that had a diameter at chest height that was equal to or greater than 20 cm.Six 16 m 2 parcels were delimited in each area along a linear transect.The plots were separated by 50 m to ensure the independence of the samples [16,32].
The sampling effort for each plot was constant (collection time = 30 minutes, and number of collectors = 3 per plot).
All of the undecomposed twigs with lengths of 10 to 30 cm and containing ants were manually collected and individually placed in plastic bags for later identification.This twig size was selected because smaller twigs were generally decomposing in the sampling areas.Furthermore, the selected size was the approximate size of most of the ant-colonized twigs [27].Only the twigs that were found on the surface were collected because they are the most recent resources in the leaf litter.
The number of twigs with nests was recorded (=the density of the nests), and the ants were identified to the genus level using identification keys.The species were identified by comparison with the examples deposited in the reference collection of the Zoological Museum of the University of São Paulo (Museu Zoologia da Universidade de São Paulo) (MZSP).The taxonomic scheme proposed by [33] was used.Vouchers are deposited in the collection of the Myrmecology Laboratory of the University of Mogi das Cruzes (Universidade de Mogi das Cruzes) and the MZSP.

Characterization of the Nests, Demographic Data, Canopy
Cover, and Litter Depth.Using a digital pachymeter, the total diameter of the twigs that contained colonies was measured; five measurements were conducted on each twig.The counts of immature ants (eggs, larvae, and pupae) and workers were performed using a manual counter.
The canopy cover was recorded at the center and at the vertices of each of the 16 m 2 plots using a Nikon D80 digital camera with a fish-eye objective.The Gap Light Analyzer (GLA) application version 2.0 was used to analyze the images [34].
The litter depth was measured using calipers at the corners and in the center of each 1 m 2 parcel.These small parcels were randomly marked within each 16 m 2 parcel.The average of five values was used to define the stratum depth.

Data Analysis.
Comparisons among the collection sites were made using the number of occurrences of the species (presence and absence data).The ant richness was compared using accumulation curves based on the number of species Psyche 3  that occurred.The expected number of species was determined using the Chao2 richness estimator, which is based on incidence and uses the number of "uniques" (species found in only one sample) and "duplicates" (species found in only two samples) [35].The curves were obtained using the EstimateS program version 8.2 [35].
We used Spearman correlations to analyze the relationship between the total number of immature ants and workers and the twig diameter, excluding rare species (those with a number of records <10 nests) [27].The relationships between species richness and tree cover and leaf-litter depth were identified using scatter plots and Pearson correlations.The same analyses were performed for nest abundance.All of the analyses were preceded by the Lilliefors test to verify data normality.The BIOESTAT 5.0 software [36] was used for both of the tests with a 5% significance level.

Results
In 960 m 2 of leaf litter, there was a total of 294 nests (0.31 nests/m 2 ), seven subfamilies, 15 genera, and 34 species.The species density was 0.03 species/m 2 .A total of 264 nests (89%) were immature.Eleven species (32%) occupied ≥10 twigs, especially Pheidole sp.13, with 56 recorded nests (Table 2).Each twig was inhabited exclusively by one species (we did not observe the cooccurrence of species in individual twigs).The richness curve did not stabilize, but the estimated curve of the number of species reached a stable level (Figure 1).
Myrmicinae was the richest subfamily both in terms of the number of nests (56%) and the number of species (41%).Ectatomminae was the least rich subfamily, with only one species (Gnamptogenys striatula Mayr) being observed, but it was among the subfamilies with the greatest abundance of nests.Pheidole was the richest genus in terms of species followed by Camponotus.Species of Pheidole, Solenopsis, Myrmelachista, Camponotus and Linepithema represented the greatest numbers of nests and were also the most populous species; the number of workers ranged from 165 to 458 and of immature ants from 108 to 1,317 among these species (Table 2).
The twig diameter ranged from 10.77 (±4.48) to 39.61 (±3.66) mm, and these twigs were occupied by Camponotus sp.9 and Hypoponera sp.10, respectively (Table 2).We did not detect a relationship between the twig diameter and the size of the colonies (workers, immature ants and workers + immature ants) (P > 0.05), except for Pheidole sp.13, for which there was a relationship between the size of the worker population and the twig diameter (r = 0.3283; P < 0.05).
We recorded 17 species with queens, 12 with alates and 8 with queens and alates.We found that more than one queen coexisted in the same twig for five species: L. neotropicum Wild (one nest with five queens), Brachymyrmex incisus Forel (one nest with two queens), Crematogaster sp.1 (one nest with three queens), Pheidole sp.13 (one nest with two queens), and Pheidole sp.43 (one nest with two queens and one with three queens) (Table 3).

Discussion
The results presented in this paper indicate that undecomposed twigs are an important nest-building resource used by many leaf-litter ant species and by several arboreal ant species, as reported in other studies [8,10,37,38].Specifically in the case of arboreal ants, twigs in the leaf litter may represent external nests, as already reported for Azteca [39,40] and Crematogaster torosa Mayr [41].This microhabitat probably originated from trees near the site where colonies resided that fell, bringing the eggs, larvae, alates, and adults to the ground [38].In silk-nesting Camponotus, entire colonies frequently fall, especially during the rainy season [42].
In our study sites, many nests can be characterized as temporary because only workers and immature ants were found.This result suggests the presence of satellite nests, as observed for C. brasiliensis Mayr [27] and Pachycondyla villosa (Fabricius) [43].The existence of satellite nests increases the chances of defending a given territory and increases the survival of the colony itself, which is at the greatest risk of predation when concentrated in one place [42,[44][45][46].When the nest is not centralized, the search for food can be completed by workers found in all of the nest locations, thereby increasing the foraging capacity of the colony [42,47] and reducing the distance to the food supply [48].
Alternatively, we cannot exclude the polyatomic species hypothesis without the presence of queens (see [41]).In the leaf litter of tropical forests, approximately 1/3 of the species are polydomic [5].Although the causes of polydomy are uncertain [41], polydomy may be related to the occupation of new areas [45], increased foraging area [42,47], or increased food supply [41]; there may be yet another cause found for certain arboreal ant species, which have larger colonies, in compensating for restricted space in twigs [27].Polydomy is also observed in polygynous species, such as C. pygmaea Forel [49] and M. schumanni Emery [50,51].Polydomy may be the case for 29% of the nests determined to have more than one queen, especially for Pheidole.
Sampling of ant nests from twigs at different stages of decomposition resulted in abundance measurements of 7.43 nests/m 2 in tropical wet forest (Costa Rica) [16], 0.22 nests/m 2 in the central Amazonian rainforest (Brazil) [27] and 0.88 nests/m 2 in lowland rainforest (Ecuador) [6].Thus, the density of undecomposed twigs with nests recorded in areas of Atlantic Rainforest in southwestern Brazil may be considered high because these twigs represent only a portion of the twigs available in the leaf litter and are probably less attractive for ants due to low moisture [27].Moisture is a limiting factor for ant recruitment in leaf litter, and nest recolonization, and recovery in artificially disturbed parcels is slower, as observed by [5].In contrast, the observed species density was relatively similar to that of the Amazon rainforest, with 0.02 species/m 2 being recorded [27], and the density was similar to that observed in other studies [16,29].However, the observed species curve indicates that rare species were not fully sampled.When using a 1 m 2 sampling unit of dense ombrophilous rainforest, we found that the species richness in fallen twigs on the soil was much lower than that in the leaf litter [8,15].Many species that nest in twigs may be limited by high mortality during alate dispersion or during relocation of colonies from one twig to the next.Changing colonies is a relatively constant factor because twigs are ephemeral [16],  and this relocation may be one of the factors regulating the richness of species that use twigs as resources.
Another factor regulating species richness may be the limited space within twigs.The structure of the ant colonies found in the interiors of twigs and trunks in the leaf litter is relatively simple, due to space limitations [44].However, independent of the twig's dimensions, colonies of L. neotropicum Wild, M. catharinae Mayr, M. ruszkii Forel, Pheidole spp., and Solenopsis spp.had many workers and immature ants.In dense ombrophilous rainforest, records of populous colonies of M. catharinae and M. ruszkii in fallen twigs in the leaf litter seem common and are unrelated to the diameter of the twig where the colony is housed [38].
The presence of a more closed canopy has been associated with habitats richer in resources for ants [23,44].The relationship between the structural complexity of the canopy and the higher number of nests observed in our study also suggests that the canopy can be a determinant of the community structure of the species that use twigs in the leaf litter.Our results also show that leaf-litter depth is not one of these factors.Reference [6] did not find a relationship between tree cover and nest abundance or leaf-litter depth and suggested that predation, prey density, or plant diversity may be responsible for the observed patterns.The ants that build nests in leaf litter do not show preferences for twigs of Psyche 7 specific plant species [5,16], but a high species richness of twigs can favor ant species diversity [22].
Our results expand the current knowledge of ant species that use undecomposed twigs as a nest-building resource in dense ombrophilous forest and show that this habitat has an important role in the biology of certain species, especially those species for which the entire colony development cycle may occur within twigs.Furthermore, our findings suggest that undecomposed twigs, despite containing a low density of species compared to the leaf litter, represent a resource for nest building and consequently are part of the set of structural elements that maintain the diversity of the ants that forage in this layer of tropical forests.Our study also confirms the dominance of the genus Pheidole [27] along with Camponotus and Hypoponera in ant communities that use twigs as a resource in the leaf litter of dense ombrophilous rainforests.

Figure 1 :
Figure 1: Accumulation and estimated species richness curves in undecomposed twigs in the leaf litter of dense ombrophilous forest.

Figure 3 :
Figure 3: Relationship between canopy cover and the nest abundance (a) and species richness (b) in undecomposed twigs in the leaf litter in dense ombrophilous rainforest fragments.

Table 1 :
Locations of sampling sites in southeastern São Paulo state, Brazil.
* Approximate size of the sampling areas.

Table 2 :
The colony and nest structure of species reported in undecomposed twigs in the leaf litter of dense ombrophilous forest.

Table 3 :
Presence of queens and alates among the species reported in undecomposed twigs in the leaf litter of dense ombrophilous forests.