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Revista de Biología Tropical, ISSN: 2215-2075, Vol. 69(3): 865-872, July-September 2021 (Published Aug. 31, 2021)

Ant diversity sampling in the Brazilian Amazon:

a comparison of litter collection and pitfall trapping

Wully B. da Silva

1,2

*; https://orcid.org/0000-0002-6209-4984

Reinaldo L. Cajaiba

; https://orcid.org/0000-0003-0176-9201

Eduardo Périco

; https://orcid.org/0000-0002-2926-6246

1. Laboratory of Ecology and Evolution, University of Taquari Valley, Lajeado, Rio Grande do Sul, Brazil; wully_bio@

hotmail.com (Correspondence*), perico@univates.br

2. Federal Institute of Education, Science and Technology of Pará, Itaituba, Para, Brazil.

3. Laboratory of Ecology and Conservation, Federal Institute of Education, Science and Technology of Maranhão,

Buriticupu, Maranhão, Brazil; reinaldocajaiba@hotmail.com

Received 05-V-2021. Corrected 08-VII-2021. Accepted 19-VIII-2021.

ABSTRACT

Introduction: Ants in tropical forests are a hyper-diverse group that plays important ecological roles. Several

studies on tropical forests have used different sampling protocols to capture soil ants, making it difficult to com-

pare responses and patterns of diversity between studies. Thus, research that compares different well-structured

and standardized sampling methodologies to adequately estimate the richness of ant species in tropical forests

is necessary.

Objective: In this study, we examined the combination of catches with pitfall traps with and without baits and

litter collections for soil ant sampling.

Methods: In the fall traps, we use two baited (sardines and bananas) and one non-baited. For the manual collec-

tions (litter sampling), the litter and only the topsoil of the loose soil were collected.

Results: We found that traps containing sardine baits collected a greater abundance of ants, whereas non-baited

traps collected a greater richness of ant species. On the other hand, litter collections captured the largest number

of exclusive species, presenting a different species composition from the pitfall traps (with and without baits).

In general, baited traps showed greater abundance in more degraded locations, while manual collections and

unbaited pitfalls captured more individuals in preserved environments.

Conclusions: Our results provide evidence that, for accurate sampling of soil ant assemblages in tropical forests,

the use of different methodologies is necessary to capture a greater diversity of species because the methods

differ in effectiveness according to habitat.

Key words: ant community; Formicidae; inventory; sampling methods; tropical forests.

da Silva, W. B., Cajaiba, R. L., & Périco, P. (2021). Ant diversity

sampling in the Brazilian Amazon: a comparison of

litter collection and pitfall trapping. Revista de Biología

Tropical, 69(3), 865-872. https://doi.org/10.15517/rbt.

v69i3.46872

https://doi.org/10.15517/rbt.v69i3.46872

Studies of soil macrofauna require well-

structured and standardized sampling methodo-

logies to adequately estimate species richness

and uniformity (Véle et al., 2009). However,

it is challenging to estimate species richness

in megadiverse regions because the techniques

used by researchers do not always provide a

representative sample of total richness (Agosti

& Alonso, 2001; Orsolon-Souza et al., 2011;

Tista & Fiedler, 2011). Ants are a hyperdiver-

se group in tropical forests and particularly

important in the soil macrofauna, as they are

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Revista de Biología Tropical, ISSN: 2215-2075 Vol. 69(3): 865-872, July-September 2021 (Published Aug. 31, 2021)

highly abundant, have a wide geographical dis-

tribution, and occupy a wide variety of niches

(Andersen & Majer, 2004; Solar et al., 2016;

Tiede et al., 2017). They play critical ecologi-

cal roles, acting as herbivores, seed dispersers,

or predators of other arthropods and scavengers

(Del Toro et al., 2012; Andersen, 2019). In

addition, they contribute in an extraordinary

way to the edaphic processes, such as the

movement of water and soil and the cycling of

nutrients (Sousa-Souto et al., 2007). Because of

these characteristics, ants have frequently been

used as a focal taxon in biodiversity studies or

as bioindicators in soil management studies

(Schimidt & Solar, 2010; Pacheco & Vascon-

celos, 2012).

Several methodologies have been used to

collect ants from the soil, each of which has

limitations. No method can collect all the spe-

cies that inhabit a specific area because these

species usually have a great diversity of fora-

ging and nesting habits (Pacheco & Vasconce-

los, 2012; Salata et al., 2020). Ant community

researchers suggest combining different tech-

niques as the best way to estimate ant richness

and abundance (Delabie et al., 2000a; Longino

et al., 2002; Gotelli et al., 2011). Therefore, it

is important to compare the efficiency of the

various techniques for sampling ant diversity

in one place to advance knowledge about ant

fauna in hyper-diverse regions, especially in

the litter (Castilho et al., 2007; Veiga-Ferreira

et al., 2010; Hanisch et al., 2018). As a result,

many ant inventories employ more than one

sampling technique because their use in com-

bination often increases sampling efficiency

(Wong & Guénard, 2017; Lee & Guénard,

2019; Salata et al., 2020).

Ants have been the subject of several

ecological studies and have been sampled by

various methods, including pitfall traps, bait

traps, litter sampling, fogging, beating the vege-

tation and manual harvesting (Orsolon-Souza

et al., 2011; Yusah et al., 2012; Cajaiba & Silva,

2014; Antoniazzi et al., 2020). In this study,

we compared the performance of two methods

usually used for catching soil ants: manual lit-

ter collection and pitfall traps (Nakamura et al.,

2007). Although the combined use of these two

techniques results in some redundancy (Lopes

& Vasconcelos, 2008; Souza et al., 2012), they

are essential for estimating species richness in

megadiverse regions (Delabie et al., 2000b). In

pitfall traps, we used two types of baits, banana

and sardines, in addition to the non-baited, that

is, pitfall without baits. Our objective was to

test whether there are effects of different sam-

pling techniques on the abundance, richness,

and composition of soil ant species between

each method. In addressing these questions,

we attempt to build a framework of reference,

which should help researchers to evaluate the

trade-offs between sampling completeness and

the costs and time required (Souza et al., 2012).

MATERIALS AND METHODS

Study location: The study was carried out

in the state of Pará, Northern Brazil, in an Ama-

zon rainforest region, during the year 2015, in

the months of February/March (rainy season),

June (final of rainy season and early dry sea-

son) and September/October (dry season). We

conducted collections in areas with five diffe-

rent land uses and land cover, which were clas-

sified into: i) preserved forest – PF (area with

little or no disturbance identified in the last

hundred years, where we used geoprocessing

of satellite images, interviews with residents,

and on-site visits); ii) secondary forest – SF15,

in an intermediate stage of regeneration with 15

years of fallow; iii) secondary forest – SF5, in

an initial stage of regeneration with five years

of fallow; as in preserved forests, we used geo-

processing of satellite images, interviews with

residents, and on-site visits to estimate the age

of secondary forests; iv) agricultural areas – Ag

(cocoa crop, Theobroma cacao), and v) pasture

area – Pa (extensive livestock) - see details of

the study areas in Cajaiba et al. (2020). In each

of these habitats, two areas were sampled, tota-

ling 10 sampled areas.

Ant sampling: Pitfall traps were used

to collect the ants. The traps were 75 mm

in diameter and 110 mm deep, containing

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approximately 500 ml of solution (water, coar-

se salt for preserving dead animals, and neutral

detergent to break the surface tension of the

water), and covered by a suspended roof to pre-

vent rainwater from entering. The traps were

installed at seven random points in each study

site, with a minimum distance of 150 m from

each other, always considering a minimum

distance of 150 m from the edge to prevent

the sample from being influenced by the edge

effect (Lacasella et al., 2015). Three traps were

installed at each point, two baited (sardines

and bananas) and one non-baited, 10 m apart,

totaling 21 pitfall traps at each location. Each

trap remained installed for 48 h, with the pro-

tocol being repeated in all areas and periods

of collection, totaling a sampling effort of 630

traps (see Digital Appendix 1, for details of the

sample design).

For the manual collections (litter sam-

pling), 10 random sampling points of 1 m

(1x1 m - total of 300 sampled points, Digital

Appendix 2) with a distance of 100 m between

them were selected for each ecosystem and

sampling period. At each collection point, the

litter and only the topsoil of the loose soil were

scraped gently with a metal spatula to include

those ants in the samples that fell from the litter

during the collection procedure. The litter here

is defined as the layer of leaves and debris that

can be easily removed from the more compact

soil (Ivanov & Keiper, 2009; Da Silva et al.,

2018). We refrained from simply separating

the litter and the soil, considering that if the lit-

ter is removed, ants can escape and hide in the

topsoil (Nakamura et al., 2007; Cajaiba et al.,

2017). Each soil and litter sample was placed in

a tightly closed tissue bag and kept in an insu-

lated box. All materials were screened in the

laboratory, with detailed searches on the leaves,

branches, and soil. The ants were extracted

manually and preserved in 70 % ethanol solu-

tion. Ants (captured in all collection methods)

were identified according to keys (Bolton,

1994; Palácio & Fernández, 2003; Baccaro et

al., 2015), and reference material in the Labo-

ratory of Ecology and Conservation, Federal

Institute of Education, Science and Technology

of Maranhão.

Data analysis: To analyze the effects

of habitat types on the species richness and

abundance of ants, we performed generalized

linear model (GLM). Models were analyzed

with a Poisson distribution (to analyze species

richness) and a negative binomial distribu-

tion (to analyze abundance), given the high

overdispersion found in ants abundance. We

performed pairwise contrast analyses to detect

differences among treatments (Crawley, 2013).

Statistical analyses were conducted using the

lme4 package in R software version 3.2.0 (R

Core Team, 2016; Bates et al., 2019). The taxo-

nomic composition of ant communities bet-

ween capture methods was compared using a

multivariate analysis of permutational variance

(PERMANOVA). Non-metric multidimensio-

nal scale (NMDS) graphs were used to assist

in the interpretation of the results found with

PERMANOVAs (see Anderson (2001) for a

similar procedure).

RESULTS

Using the various collection techniques

in the different sampled environments, we

collected 9 727 individuals and 131 ant species,

distributed in 31 genera and 8 subfamilies.

The richest subfamilies were Myrmicinae (66

species, 5 029 individuals) and Ponerinae (25

species, 2 786 individuals), while the most

representative genera were Pheidole (15 spe-

cies and 2 872 individuals), Camponotus (12

species and 1 016 individuals) and Solenopsis

(10 species and 1 128 individuals).

Traps containing sardine baits showed

a greater abundance of ants (3 207 indivi-

duals), followed by banana baits (2 529 indi-

viduals). Non-baited traps collected greater

species richness (89 species), followed by

manual collection (83 species). The GLM

tests showed statistically significant differences

of species abundance according to different

collection methods (F = 14.77, P < 0.001). Sig-

nificant differences were found between all the

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collection methods (Fig. 1A). The GLM tests

also showed statistically significant differences

of species richness according to the different

methods (F = 11.98, P < 0.001). According to a

paired test, there were no significant differen-

ces between the non-baited and manual means

(P > 0.05) (Fig. 1B).

In terms of exclusivity, leaf-litter manual

sampling gathered the largest number of exclu-

sive species (21 species). We discovered that

the pitfall, regardless of the baits used, captured

larger ants, while manual collections captured

smaller ants with less foraging capacity. The

species composition differed between the diffe-

rent collection methods (Permanova F = 259.2,

P < 0.001) (Table 1).

These data were corroborated with the

NMDS, where the species collected using

different methods (pitfall and manual) and

different baits differed from each other. Bait

traps have a greater overlap of species (54

shared species), while non-baited pitfalls and

manual collections were separated from the

other methods (Fig. 2).

When ant richness was tested for the diffe-

rent collection methods within each habitat,

significant differences were found (Digital

Appendix 3). In general, baited traps were more

abundant in more degraded places (pasture and

secondary forest with five years of regeneration

and cocoa, respectively), while manual collec-

tions and non-baited pitfall captured more

individuals in more preserved environments.

These differences were confirmed using the

GLM test (Digital Appendix 4). Species rich-

ness followed the same pattern, in which

the different methods were significant within

the sampled environments. Manual collection

and non-baited pitfall showed greater species

richness in primary forests, while sardine and

banana baits collected more species in pasture

and cocoa areas. On the other hand, baited traps

with banana and sardines showed lower species

richness in primary forest areas (Digital Appen-

dix 4). The taxonomic composition of ants cap-

tured by habitat showed significant differences

between the different collection methods, as

demonstrated by PERMANOVA and NMDS

(see Digital Appendix 3 and Digital Appendix

5 for the associated differences between baits

in each studied habitat).

Fig. 1. Box plot expressing the differences in the median values for A. Abundance and B. Richness of the ant community

using the different collection methods. The values followed by the same letters are not significantly different. Sar = sardine,

Ban = banana, Nbt = non-baited, Man = manual.

TABLE 1

PERMANOVA results comparing the composition of ant

assemblages between different sampling methods

Sardine Banana Non-baited Manual

Sardine - 0.001 0.001 0.001

Banana 27.9 - 0.01 0.001

Non-baited 18.8 18.5 - 0.01

Manual 6.80 15.4 28.2 -

PERMANOVA based on the similarity of Bray-Curtis

using 9999 permutations, F = 259.2, P < 0.001.

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DISCUSSION

Our results demonstrate that the inventory

of ant depends on the sampling methods selec-

ted, corroborating other authors’ findings (Lee

& Guénard, 2019; Salata et al., 2020). Thus,

depending on the objectives of each study (eco-

logical studies or species surveys), different

collection methods should be used (Wiezik et

al., 2015). We identified that pitfall traps and

manual collections provided a different species

composition in most of the sampled habitats,

which is in agreement with Salata et al. (2020),

who recommend using an integrated approach

including several complementary methods sui-

table for the studied habitat.

Both collection methods used in this study

have advantages and disadvantages; for exam-

ple, manual collections involve a direct search

for individuals or colonies and are generally

considered the most effective method to cap-

ture the maximum species richness within a

study area (Ellison et al., 2007). However,

manual collections are more time-consuming

and require a greater number of researchers

involved in the fieldwork (Underwood & Fis-

her, 2006), and pitfall traps can be installed in

the field for several days unattended. However,

the use of pitfall traps is limited because they

tend to capture larger individuals with high

foraging capacity (Martelli et al., 2004), a trend

observed in our study. In addition, pitfall traps

do not capture the smaller ants that form small

colonies (Bestelmeyer et al., 2000; Castilho et

al., 2007), so, for these ants, the most suitable

method is manual collection (Lindsey & Skin-

ner, 2001; Véle et al., 2009). We also found that

manual collections are more efficient in envi-

ronments with high litter cover since we obser-

ved that, in pasture areas, manual collection did

not add any species that had not been collected

by pitfall traps (Parr & Chown, 2001). In

fact, we observed that in more anthropogenic

habitats (Agriculture & Pastures), the commu-

nity comprises mainly generalist ants, with the

capacity to use both forest and modified areas,

which can be captured through pitfall traps.

In summary, our findings highlight the

importance of different sampling methods for

collecting ants from the soil, including manual

collection, pitfall traps with and without baits,

as well as other methods not applied here. This

approach can address the different aspects of ant

communities that inhabit different ecosystems

and, therefore, overcome specific disadvanta-

ges of using only a single method to monitor

Fig. 2. Non-metric multidimensional scale (NMDS) showing assemblies of ants grouped according to different

capture methods (using Bray-Curtis similarity; Stress: 0.08). Star = Sardine, Circle = Banana, Triangle = Non-baited,

Square = Manual.

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changes in the ant community across ecosys-

tem boundaries (Bestelmeyer et al., 2000; Tista

& Fiedler, 2011). Although the use of different

sampling methods can be redundant in the ant

fauna obtained from comprehensive surveys, it

is known that each of them registers a certain

fauna, so they are complementary (Wiezik et

al., 2015; Antoniazzi et al., 2020). We also

stress the importance of studying a greater

number of areas with different levels of distur-

bance. It is important to emphasize that new

collections should be standardized for compari-

son with other studies. Finally, it is essential to

consider appropriate methodologies to answer

ecological questions, for example, using the

same sampling methods when the objective is

to compare differences between distinct habi-

tats (Antoniazzi et al., 2020).

Ethical statement: authors declare that

they all agree with this publication and made

significant contributions; that there is no con-

flict of interest of any kind; and that we

followed all pertinent ethical and legal proce-

dures and requirements. All financial sources

are fully and clearly stated in the acknowled-

gements section. A signed document has been

filed in the journal archives.

ACKNOWLEDGMENTS

WBS thank the Coordenação de Aper-

feiçoamento de Pessoal de Nível Superior,

Brazil through a doctorate fellowship (Finance

Code 001). EP thanks Conselho Nacional de

Desenvolvimento Científico e Tecnológico,

Brazil for the research grant (Research Produc-

tivity nº 307303/2019-5).

RESUMEN

Muestreo de la diversidad de hormigas en la Amazonía

brasileña: una comparación de recolección

de hojarasca y trampas de caída

Introducción: Las hormigas en los bosques tropicales

son un grupo hiperdiverso que juega un papel ecológico

importante. Varios estudios en los bosques tropicales han

utilizado diferentes protocolos de muestreo para capturar

las hormigas de suelo, lo que dificulta la comparación

de respuestas y patrones de diversidad entre estudios.

Por lo tanto, es necesaria una investigación que compare

diferentes metodologías de muestreo bien estructuradas

y estandarizadas para estimar adecuadamente la rique-

za y uniformidad de las especies de hormigas en los

bosques tropicales.

Objetivo: En este estudio, examinamos la combinación de

capturas con trampas de caída con y sin cebos y recolec-

ciones manuales para muestreo de hormigas en el suelo.

Métodos: En las trampas de otoño, utilizamos dos cebos

(sardinas y plátanos) y uno sin cebo. Para las recolecciones

manuales (muestreo de hojarasca), se recogió la hojarasca

y solo la capa superior de suelo suelto.

Resultados: Encontramos que las trampas que contenían

cebos de sardina recolectaron una mayor abundancia de

hormigas, mientras que las trampas sin cebo recolectaron

una mayor riqueza de especies de hormigas. Por otro lado,

las recolecciones de hojarasca capturaron el mayor núme-

ro de especies exclusivas, presentando una composición

de especies diferente a las trampas de caída (con y sin

cebo). En general, las trampas con cebo mostraron mayor

abundancia en lugares más degradados, mientras que las

recolecciones manuales y las trampas sin cebo capturaron

más individuos en ambientes preservados.

Conclusiones: Nuestros resultados proporcionan eviden-

cia de que, para un muestreo preciso de ensambles de las

hormigas de suelo en los bosques tropicales, el uso de dife-

rentes metodologías es necesario para capturar una mayor

diversidad de especies, ya que los métodos difieren en la

efectividad de acuerdo con el hábitat.

Palabras clave: comunidad de hormigas; Formicidae;

inventario; métodos de muestreo; bosques tropicales.

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