original article nematode trophic structure in ... file 2 robaina et al. nematodes in atlantic...

1http://dx.doi.org/10.4322/nematoda.01615 Nematoda, 2015;2: e162015

Nematoda. ISSN 2358-436X. This work is licensed under a Creative Commons Attribution International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted non-commercial use, distribution, and reproduction in any medium provided the original work is properly cited.

Nematode trophic structure in phytotelmata of Canistropsis billbergioides and Nidularium procerum (Bromeliaceae) in the Atlantic Forest - variability in relation to climate variables and plant architectureRenata Rodrigues Robainaa, Ricardo Moreira Souzaa*, Vicente Martins Gomesa, Denise Oliveira Cardosoa and Alexandre Macedo Almeidaa

a Grupo de Pesquisa em Nematologia, Laboratório de Entomologia e Fitopatologia (LEF), Centro de Ciências e Tecnologias Agropecuárias (CCTA), Universidade Estadual do Norte Fluminense Darcy Ribeiro (UENF), Campos dos Goytacazes (RJ) Brazil

*[email protected]

HIGHLIGHTS• Bromeliad phytotelmata host a wide range of nematodes belonging to different genera and trophic groups.• The trophic structure presented some distinctions between the two bromeliad species studied.• The trophic structure presented distinctions between leaf axils located in different positions on the plants.• The nematofauna presented a seasonality related to rainfall and temperature fluctuations.

ABSTRACT: In the first ecological study ever conducted on the nematofauna associated with natural phytotelmata, nematodes were systematically sampled during a 24 month-period in the phytotelmata of two bromeliad species in the Atlantic Forest, Rio de Janeiro state, Brazil. Samplings revealed a wide range of nematodes from the bacteriophagous, mycophagous, predator and ingestor of unicellular eukaryotes trophic groups. Plant-parasitic forms, probably contaminants, were only occasionally found. The trophic structure – assessed by the total abundance of nematodes and the abundance of nematodes per trophic group - was significantly distinct among the leaf axils located at the three architectonic levels of the plants - base, middle and upper - particularly in Canistropsis billbergioides. The leaf axils located at the base and middle levels seemed more conducive to nematofauna. The nematofauna presented a seasonality significantly related to rainfall and temperature fluctuations, but this seasonality was not related to the amount of organic matter – mostly canopy litter – deposited in artificial collectors positioned next to the bromeliads.

Keywords: phytotelma, bromeliads, meiofauna, nematofauna, rainforest.

Cite as Robaina RR, Souza RM, Gomes VM, Cardoso DO, Almeida AM, Gonçalves LSA. Nematode trophic structure in phytotelmata of Canistropsis billbergioides and Nidularium procerum (Bromeliaceae) in the Atlantic Forest - variability in relation to climate variables and plant architecture. Nematoda. 2015;2: e162015. http://dx.doi.org/10.4322/nematoda.01615

Received: Nov. 3, 2015 Accepted: Dec. 4, 2015

ORIGINAL ARTICLE

INTRODUCTIONThe term phytotelma (phyton= plant, telma= puddle) designates a small body of water retained in

a living or dead plant structure. Phytotelmata are colonized by micro-, meso- and macro-organisms, specialists or generalists in this habitat, which present different levels of interaction with the phytotelma for their survival, reproduction and dispersal. Phytotelmata may also play an important role in storing water, benefiting the non-resident fauna[1]. Due to these singularities and the relative ease in experimental manipulation, phytotelmata have been studied for their biodiversity and as microcosms

2http://dx.doi.org/10.4322/nematoda.01615

Nematodes in Atlantic Forest bromeliadRobaina et al.

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for testing ecological hypotheses[2]. The phytotelmata that are most studied are those associated with carnivorous plants, tree-holes, bamboos and bromeliads (Bromeliaceae).

Bromeliaceae include about 3,000 species that are almost exclusively endemic to the tropical and subtropical regions of the Americas, between parallels 37o N and 44o S. Bromeliads occur in the most varied conditions of altitude, temperature and humidity, and they present terrestrial, epiphytic, rupicolous and saxicolous types[3]. Depending on their architecture, bromeliads may form a phytotelma that stores up to 30 liters of water, with organic matter and associated biota[4]. The phytotelma may be restricted to a single tubular structure, formed by an almost vertical arrangement of leaves, or may be dispersed in the axils of all the leaves. Most of the fauna that live in the phytotelmata of bromeliads specialize in this kind of environment and belong to the class Insecta, with more than 70 families in 11 orders reported, of which Diptera is the most abundant[5]. Platyhelminthes, oligochaetes, crustaceans, myriapods, arachnids, pseudoscorpions, scorpions, mites, mollusks and nematodes are also found[2].

Despite the many inventories that have been carried out in different types of phytotelmata, nematodes have generally received little attention from ecologists. When studying the food web of phytotelmata in Nepenthes alata Blanco, Sota et al.[6] did not identify the nematodes they observed, and Mestre et al.[7] and Armbruster et al.[8] did not identify the nematodes found in epiphytic bromeliads. Devetter[9] reported that nematodes predominate in the invertebrate fauna in tree-holes, but did not identify them.

Few studies have focused on the nematofauna of phytotelmata, and most were inventories. The genera Diplogaster, Dorylaimus, Plectus, Rhabditis, Molgolaimus, Actinonema, Dominicactinolaimus, Paractinolaimus, Tylocephalus, Plectus, Anaplectus, Tripylella, Pelodera and a taxon of the family Dorylaimidae have been reported from phytotelmata of Nepenthes spp. (Menzel, 1922, cited by Quisado[10]; Quisado[10]). From the phytotelmata of bromeliads various genera were reported by Zullini (1977) and Jacobs (1984) (cited by Hodda et al.[11]) and Zullini et al.[12]. Five nematode species have been described from phytotelmata in bromeliads and Nepenthes spp. (Meyl[13]; Bert et al.[14]; Menzel, 1922 and Holovachov et al., 2004, cited by Quisado[10]).

Apparently, the only studies that deal with ecological aspects of the nematofauna of phytotelmata are restricted to artificial tree-holes. Ristau et al.[15, 16] observed alterations in the diversity, trophic structure and biomass of the nematofauna in artificial phytotelmata that had been nutritionally enriched. The application of different quantities of leaf litter in artificial phytotelmata correlated positively with nematofauna abundance and diversity, and with the abundance of the bacteriophagous forms[17].

Studies in natural ecosystems have revealed the influence of nematodes on the mineralization rate of organic matter, by means of the interference of bacteriophagous and mycophagous forms on primary decomposers. Nematodes also contribute to the nitrogen cycle[18]. In ecosystems altered by humans – fresh water, marine and soil – nematodes are used as indicators of pollution and other disturbances, with advantages in relation to physical-chemical monitoring or other invertebrates[19, 20, 21].

The Atlantic Forest (AF) in Brazil is one of the world´s most disturbed biomes by human interference[22]. Its dimension is currently reduced to about 12% of its original area[23]. Although there are various governmental and private initiatives to preserve and restore the AF, few inventories of its microbiota have been conducted and even fewer studies have focused on the response of the microbiota to anthropic disturbance (Brasil[24]; Lewinsohn & Prado, 2004, cited by Maia[25]).

Due to their abundance, diversity and sensitivity to environmental variations, it is hypothesized that nematodes are valuable bioindicators in the AF. Nematodes associated with the phytotelmata of bromeliads seem an interesting subgroup to test this hypothesis because: i) bromeliads are the second most diverse botanical group in the AF and the most abundant[26], ii) there are indications that bromeliads are sensitive to anthropic disturbances[27] and iii) each bromeliad constitutes a microcosm - which defines the sampling space – and their abundance offers easy experimental replicates. Aiming to test this hypothesis, the present work involved characterizing the nematofauna in the bromeliads of a preserved area of AF, and noting its responses to climatic variations and plant architecture.

MATERIAL AND METHODS

Sampling areaThe sampling area was about 500 x 100 m along a stream on São Julião farm (21°48’336’’ S and

41°38’317’’ W) in the Desengano State Park, near the city of Campos dos Goytacazes, Brazil. The area presents a submontane dense tropical rainforest, and the hot, wet climate is classified as Af (Köppen). During summer there are constant short bursts of rain, and even in the driest month the rainfall is



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over 60 mm, while the mean temperature is equal to or above 18 ºC in the coldest month[28]. The area is located at about 80 m above sea level. In 2012 the data on monthly accumulated rainfall and monthly mean temperature were collected at the Instituto Nacional de Meteorologia meteorological station, located at about 15 Km in a straight line from the sampling area[29]. In 2013 and 2014, data collection was local, using a Watchdog® pluviograph and temperature sensor coupled to a datalogger (Figure 1).

Optimization of samplingFor the characterization of the biota associated with the phytotelmata of bromeliads, it is common

to take the plant out of its site. The leaves are removed and their proximal (axil) region is rinsed. The rinsing water is pooled with the water present in the phytotelma to count all the biota. However, in protected areas the environmental legislation may not allow the removal of bromeliads, and this is the case in the Desengano State Park, the place chosen for this study. In these cases, it is usual to adopt the suction of water contained in the phytotelma (e.g. Jocque et al.[30]). Therefore, for the present study, the preliminary stage was to optimize the methods for sampling the nematofauna, because i) it is difficult to remove all the water from the phytotelma, as its volume may be small during the dry season, ii) there is little space available in the leaf axils in which to insert the suction equipment, and iii) there is often a blockage in this equipment, due to the large quantity of organic matter in suspension.

The species Canistropsis billbergioides (Schult.f.) Leme and Nidularium procerum Lindm were selected, both being widely present in the Park and presenting formation of phytotelmata and a rupiculous habit. Typically, these bromeliads form groups of individuals separated from other species. Samplings were carried out in 2012, in a single day in the months of February and September for C. billbergioides, and April and October for N. procerum.

At each sampling time, eight bromeliads were sampled. The basic sampling method (M1) was the suction of the water retained in the phytotelma (in all leaf axils) with the aid of an automatic pipette linked to a rubber tube. The sucked water was stored in a flask. M2 consisted of flushing the same phytotelma with about 100 mL of water, applied under pressure with a wash bottle, followed by suction of the entire volume with a pipette and storage in another flask. M3 to M7 consisted of successive reflushings and suctions, to a total of seven samples for each of the eight sampled bromeliads. The water for flushing the bromeliads was free of nematodes, being carried to the sampling area in back sprayers.

The 56 samples were individually processed by centrifuging and sieving, using 60 and 500-mesh sieves. To count the nematodes, three aliquots of 1 mL from each sample were observed in a Peter’s slide under a stereoscopic microscope. The total cumulative abundance of the nematodes, the cumulative number of genera and the cumulative abundance per trophic group, from M1 to M7, were evaluated. The original data, without transformation, were submitted to regression analysis using the program R[31].

Figure 1. Monthly accumulated rainfall and monthly mean temperatures from 2012 through 2014, in Campos dos Goytacazes (RJ), Brazil. Circles indicate the sampling times for the phytotelmata of Canistropsis billbergoides and asterisks indicate the sampling times for Nidularium procerum.



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In this and the other experiments, the trophic group nomenclature was that of Moens et al.[32], based on the morphology of the esophageal and mouth regions: mycophagous, bacteriophagous, predator, phytoparasite and ingestor of unicellular eukaryotes. For generic identification, several taxonomy keys were used[33, 34, 35, 36, 37, 38].

Optimization of sample processingFor the extraction of nematodes present in samples from soil and plant tissue the methods of

Jenkins[39] and Coolen & D`Herde[40] are widely used. However, the losses in the stages of floating and centrifuging may be high if a large amount of organic matter is present in the sample. Coolen & D`Herde recommend adding 1 cm3 (approximately 0.833 g) of kaolin to the sample in the centrifuge tube, which adsorbs and precipitates the organic matter. However, this procedure has not been validated for samples with a high content of particulate organic matter in suspension, as occurs in phytotelmata in bromeliads. Therefore, the efficiency of extraction of nematodes from phytotelma water was also evaluated, adding different quantities of kaolin to the centrifuge tubes.

About 2 L of water collected from the phytotelmata of imperial bromeliads [Alcantarea imperialis (Carrie) Harms] was passed through 60 and 500 mesh sieves and collected in a bucket. Several aliquots of 5 mL were poured into a Petri dish and observed under an inverted optical microscope, to confirm the absence of nematodes. Next, 48 samples of 30 mL were prepared in centrifuge tubes, to which were added 200 infective juveniles (IJs) of Heterorhabditis baujardi Phan, Subbotin, Nguyen & Moens LPP7 (Heterorhabditidae) per sample. To the samples were added different quantities (treatments) of kaolin, with six repeats (tubes) per treatment: 0.03, 0.06, 0.12, 0.25, 0.5, 1.0 or 2.0 g. Samples with no kaolin added served as control. The samples were centrifuged at 760.24 g for 3 min and 190.06 g for 1 minute and passed through 60 and 500 mesh sieves. To count the recovered IJs, three aliquots of 1 mL from each sample were observed on a Peter’s slide under the stereoscopic microscope. The original data, without transformation, were submitted to regression analysis, using the program R.

Nematode trophic structure in relation to climate variables and plant architectureEight sampling times were defined (T1 through T8) throughout the seasons of the years for each

bromeliad species: C. billbergioides was sampled in December 2012, April, July and October of 2013, and January, March, July and November of 2014. N. procerum was sampled in January, May, August and November of 2013, and February, April, August and December of 2014 (Figure 1).

To avoid resampling the plants and to minimize local interferences, eight groups of plants were chosen in the 50,000 m2 sampling area, for each bromeliad species. At each sampling time, one bromeliad was sampled from each group, for a total of eight bromeliads per species.

Using the sampling methods defined in the item above (see Results and Discussion), samples were collected at three architectonic levels: in the leaf axils at the base, middle and upper levels of the plant (Figure 2), being careful not to allow water to overflow from the upper levels of the plant to the lower. Also, based on the methods for sample processing defined in the item above (see Results and Discussion), the 24 samples were processed separately. For the counting and identification of the

Figure 2. Bromeliad Nidularium procerum, with arrows indicating the leaf axils at the upper, middle and base architectonic levels that were sampled to obtain nematofauna.



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nematodes, the entire volume of the resulting samples was observed in Petri dishes under an inverted microscope. The total abundance of nematodes and the abundance per trophic group were computed.

As it was impossible to remove bromeliads from the Park, the amount of organic matter deposited in the phytotelmata – mostly canopy litter - was estimated by installing collectors made out of a metal ring (0.2 m2 of area) coupled to a collection cone made of cloth, 1 m in depth. The collectors were positioned in the sampling area at 1.6-1.8 m in height, near the bromeliads. At each sampling time, all the organic material collected from eight collectors was removed, dried in an oven for 72 hours at 45°C, weighed and expressed as organic matter dry mass (OMDM), in grams.

The data analyses were carried out using the program R; the data were submitted to non-parametric analysis of variance (Anova-type statistic) and the differences between the architectonic levels of the bromeliads were analyzed by the standard error calculated by bootstrap at 95% probability (10,000 re-samplings) and by Bonferroni’s test. The data were also submitted to principal components analysis (PCA).

RESULTS AND DISCUSSION

Optimization of samplingFigure 3 shows that for both bromeliad species and both sampling times the simple suction of water

contained in the phytotelma (M1) recovered only a small part of the nematofauna. It is probable that most nematodes live in among the small particles of organic matter deposited in the small space that exists in the leaf axils. The total cumulative abundance of nematodes recovered increased with successive flushings of the phytotelma with water applied under pressure (M2-M7), which resuspended the organic matter and the nematofauna, allowing it to be suctioned out. It has already been suggested that greater recovery of the microbiota of phytotelmata in bromeliads could be achieved by flushing out and suction, as a non-destructive method[30].

Figure 3. Total cumulative abundance of nematodes recovered from phytotelmata of Canistropsis billbergioides, in February and September of 2012 (a and c, respectively) and of Nidularium procerum, in April and October of 2012 (b and d, respectively), in function of the number of times the phytotelma flushing and suction were carried out (M1 to M7). Values are the mean of eight bromeliads for each species, at each sampling time. Asterisks indicate significance at P<0.01.



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The curves for recovery of the nematofauna present a tendency to level out between M5 and M7, in particular for N. procerum, independently of the differences in nematode abundance between species of bromeliad and between sampling times. This suggests that saturation of the sampling method may occur after nine or 10 successive flushes and suctions.

With regard to the cumulative number of genera recovered (Figure 4), a saturation of the sampling method was seen in M5, except for the sampling done in N. procerum in October 2012. The accumulation curves were similar, independently of the differences in diversity between the species of bromeliad and the sampling times. For cumulative abundance per trophic group (Table 1), there was saturation of the sampling method in M5 for almost all the curves. Collectively, these results led the authors to adopt M5 as the standard method for the present study.

Optimization of sample processingProcessing without adding kaolin to the centrifuge tubes presented about 60% efficiency in the

recovery of H. baujardi LPP7 IJs (Figure 5). As a result, we recognize that our samplings underestimate nematode community abundance and diversity. Although a direct interaction was observed between the doses of kaolin applied to centrifuge tubes and the clearness of samples post-processing, there was a steady fall in recovery of IJs. Therefore, for the present study, no kaolin was added to the samples being processed.

Nematode trophic structure in relation to climate variables and plant architectureIn general, the nematode community in the phytotelmata of both bromeliad species is composed of

bacteriophagous, mycophagous, predators and ingestor of unicellular eukaryotes genera (Table 2). Some phytoparasitic specimens were found occasionally, being considered contamination originating from the shallow layer or soil / litter existent over the boulder, by means of rain splashes, or from arboreal

Figure 4. Cumulative number of genera of nematodes recovered from phytotelmata of Canistropsis billbergioides, in February and September of 2012 (a and c, respectively) and from Nidularium procerum, in April and October of 2012 (b and d, respectively), in function of the number of times the phytotelma flushing and suction were carried out (M1 to M7). Values are the mean of eight bromeliads for each species, at each sampling time. Asterisks indicate significance at P<0.01.



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Table 1. Cumulative abundance per trophic group of nematodes recovered from phytotelmata of Canistropsis billbergioides and Nidularium procerum, in function of the number of times the phytotelma flushing and suction were carried out (M1 to M7), at two sampling times.

Trophic groups M1 M2 M3 M4 M5 M6 M7 Equations R2

C. billbergioides – February/2012

Bacteriophagous 0.88* 1.75 2.00 2.13 2.13 2.13 2.13 ( )1.18 /0.54)

2.121 xy

exp −=

+0.99

Predator 0.38 3.25 6.63 8.13 10.13 10.38 10.38 ( )2.68 /0.76)

10.371 xy

exp −=

+0.99

C. billbergioides – September/2012

Bacteriophagous 2.38 3.63 6.38 7.88 8.50 9.00 9.75 ( )2.38 /1.13)

9.611 xy

exp −=

+0.98

Predator 0.75 1.38 3.50 4.88 5.25 5.63 5.63 ( )2.10 /0.76)

5.621 xy

exp −=

+0.99

Ingestor** 0.13 0.50 0.63 0.88 0.88 0.88 1.00 ( )2.16 /0.83)

0.931 xy

exp −=

+0.96

Mycophagous 0.25 1.25 1.63 1.88 2.25 2.25 2.25 ( )2.10 /0.79)

2.231 xy

exp −=

+0.96

N. procerum – April/2012


48.011 xy

exp −=

+0.99

Predator 0.38 1.63 1.88 2.50 2.50 2.63 2.63 ( )1.92 /0.71)

2.581 xy

exp −=

+0.96

Ingestor 0.63 0.75 1.25 1.25 1.25 1.50 1.50 ( )1.60 /1.43)

1.511 xy

exp −=

+0.92

Mycophagous 3.25 10.25 13.88 14.38 15.38 15.63 16.25 ( )1.68 /0.55)

15.461 xy

exp −=

+0.99

N. procerum – October/2012


22.861 xy

exp −=

+0.99

Predator 3.13 5.50 11.25 15.00 19.50 20.00 21.25 ( )3.01 /1.05)

21.611 xy

exp −=

+0.99

Ingestor 1.00 2.50 4.38 5.50 5.88 6.88 7.00 ( )2.60 /1.20)

6.931 xy

exp −=

+0.98

Mycophagous 1.50 7.00 9.38 11.25 11.75 11.88 12.25 ( )1.93 /0.62)

11.841 xy

exp −=

+0.98

*Values are means of eight bromeliads for each species, at each sampling time. **Ingestor= ingestors of unicellular eukaryotes.



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soil, by means of rain dripping. The total abundance of nematodes varied from five to 35 specimens in C. billbergioides and from two to 50 in N. procerum. In one bromeliad many specimens of H. baujardi were found, probably due to the death of an infected insect in the phytotelma.

Non-parametric analysis revealed that in the phytotelmata of C. billbergioides there were significant differences for the sources of variation architectonic level of the leaf axils (upper, middle and base) and sampling times (T1-T8) for total abundance of nematodes and abundance per trophic group, except for the ingestors of unicellular eukaryotes in relation to the levels (Table 3).

The significance for the architectonic levels suggests that they present micro-climatic differences that determine differences in the resident nematofauna. Sensors that were inserted in the leaf axils at the three architectonic levels for constant monitoring of the water (temperature, pH and O2 concentration)

Figure 5. Number of infective juveniles (IJs) of Heterohabiditis baujardi recovered from samples with 200 specimens processed by centrifugation and sieving in function of different quantities of kaolin added to centrifuge tubes. Values are means of six repeats (centrifuge tubes) per treatment.

Table 2. Orders, genera and trophic groups recovered from phytotelmata of Canistropsis billbergioides and Nidularum procerum (Bromeliaceae) in the Atlantic Forest, Campos dos Goytacazes, Brazil.

Order Genera Trophic group

Rhabditida Criconema, Criconemoides, Helicotylenchus, Hemicycliophora, Nothocriconemoides, Rotylenchus, Tylenchus, Xiphinema

Phytoparasite

Aphelenchus, Aphelenchoides, Paraphelenchus Mycophagous

Acrobeles, Bunonema, Cephalobus, Macrolaimus, Mesorhabditis, Odontopharynx, Rhabditis, Tylocephalus, Wilsonema, Zullinius

Bacteriophagous

Dorylaimida Actinca, Dorylaimus, Dorylaimoides, Eudorylaimus, Laimydorus Predator

Mononchida Mononchus Predator

Enoplida Anoplostoma Ingestor of unicellular eucaryotes

Araeolaimida Not identified Bacteriophagous

Monhysterida Not identified Ingestor of unicellular eucaryotes



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failed to collect data on various occasions due to the small amount of water present in the phytotelmata during the long drought in 2014. It is known, however, that the architecture of bromeliads determines physical aspects of phytotelmata, such as tubular form vs. dispersed arrangement in the leaf axils. In the bromeliads with dispersed phytotelmata, the dimension of leaves and their angle of insertion in the stalk, at the different architectonic levels of the plant, determine the volume of water retained in the leaf axil and its surface to volume ratio, as well as the level of solar incidence (in bromeliads in the open). These variations may result in differences in water pH, temperature and concentration of O2 and nutrients[1, 41, 42]. Such variations may affect the structure of communities in the phytotelmata (e.g. Montero et al.[43]; Torreias et al.[44]). The present study suggests that, in the phytotelma of C. billbergioides, the middle and base architectonic levels are more favorable to the nematofauna, because when significant differences occurred between the levels these presented the greatest mean values for total abundance and abundance per trophic group (Figures 6, 7).

The significance for sampling times suggests that seasonal factors influenced the nematofauna. The monthly mean temperature fluctuated in a way typical of the region, with higher values from September to March (Spring and Summer) and lower from April to August (Autumn and Winter) (Figure 1). On the other hand, the rainfall was atypical and irregular from 2012 to 2014, with a prolonged drought in the latter year.

It is worth noting that the highest means for total abundance of the nematodes were observed from October/2013 through March/2014, associated with monthly accumulated rainfall of over 450 mm

Table 3. Non-parametric statistical test for the total abundance of nematodes and the abundance per trophic group associated with the different architectonic levels of the bromeliads (L) and different sampling times (T), in phytotelmata of Canistropsis billbergioides.

EffectANOVA-type statistic (ATS)

Statistic GL p-value

Total abundance

Levels (L) 15.21 1.93 <0.001

Times (T) 7.76 4.06 <0.001

L × T 2.68 6.74 0.009

Bacteriophagous

Levels (L) 13.29 1.93 <0.001

Times (T) 8.83 4.62 <0.001

L × T 1.86 7.26 0.068

Mycophagous

Levels (L) 9.83 1.85 <0.001

Times (T) 12.36 4.73 <0.001

L × T 2.20 7.46 0.027

Ingestor of unicellular eukaryotes

Levels (L) 3.40 1.99 0.053

Times (T) 4.12 3.33 0.004

L × T 1.77 5.58 0.107

Predator

Levels (L) 3.94 1.94 0.020

Times (T) 5.12 3.65 <0.001

L × T 1.83 6.44 0.083



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(Figures 1, 6). Higher rainfall was also associated with more significant distinctions between the architectonic levels (October/2013 through July/2014) and with higher abundance of bacteriophagous nematodes (January through July/2014) (Figure 7). A similar pattern was reported by, for example, Sodré et al.[45], who observed greater abundance of Chironomidae (Insecta: Diptera) in periods with higher rainfall and volume of water in the phytotelmata of the bromeliads Neoregelia concentrica (Vellozo) L.B. Smith and Aechmae nudicaulis (L.) Grisebach.

On the other hand, it is plausible to suppose that periods of less rainfall result in a lower quantity of water deposited in the phytotelma of rupiculous, saxicolous and terrestrial bromeliads, with repercussions for its biota, because the forest canopy retains a large part of the rainfall before it drips on the ground[46]. Indeed, in the present work the volume of water retained in the phytotelmata was minimal at some of the sampling times during the drought. Lower mean temperatures were recorded from April through July/2013, which may have contributed to the lower total abundance of nematodes.

Other macro- and micro-climatic variables with seasonal variation may have contributed to the trophic structure of nematofauna in the phytotelmata, such as water temperature in the phytotelma and the incidence and intensity of winds, which can affect the amount of organic matter (mostly canopy litter) deposited in the phytotelma. Direct and indirect interference of various seasonal factors has been reported for phytonematodes and for free-living nematodes in soil, freshwater and marine environments. In relation to the interaction architectonic levels × sampling times, there was significance only for total abundance of nematodes and abundance of the mycophagous forms.

Principal components analysis indicated that the first two components explained 80.75% of the variability of the total abundance of nematodes and the abundance per trophic group, in relation to the architectonic levels of C. billbergioides and the sampling times (Figure 8). There was a positive interaction between the abundance of mycophagous and bacteriophagous nematodes at the middle architectonic level and the sampling times T5 to T7. The higher rainfall and mean temperatures for T4 to T6 may have favored the activity and density of decomposer fungi and bacteria, favoring those trophic groups. Predator nematodes and those that ingest unicellular eukaryotes showed a positive interaction with the base level in T4 and T5.

For the phytotelmata of N. procerum, there were significant differences for the source of variation sampling time for all variables (Table 4). For the architectonic levels, there were significant differences

Figure 6. Comparison of means of total abundance of nematodes between the architectonic levels of Canistropsis billbergioides (base, middle and upper) at different sampling times. By Bonferroni’s test * < 0.05, ** < 0.01. The vertical bar indicates the confidence interval of 95% via bootstrap (10,000 resamplings). Values are means of eight bromeliads per sampling time.



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for the total abundance of nematodes and the abundance of bacteriophagous and predator trophic groups. There were no significant differences for the interaction of architectonic levels × sampling times.

The sampling times with greatest total abundance of nematodes in the phytotelmata of N. procerum (November/2013 through April/2014) (Figure 9) were the same as observed in C. billbergioides. However, there were fewer significant distinctions between the architectonic levels in N. procerum. The same

Figure 7. Comparison of means of abundance of nematodes per trophic group between the architectonic levels of Canistropsis billbergioides (base, middle and upper) at different sampling times. By Bonferroni’s test * < 0.05, ** < 0.01. The vertical bar indicates the confidence interval of 95% via bootstrap (10,000 re-samplings). Values are means of eight bromeliads per sampling time.



Nematoda, 2015;2: e162015

Figure 8. Principal components analysis for the total abundance (Ab. total) and abundance per trophic group of nematodes associated with the phytotelmata of Canistropsis billbergioides in different architectonic levels (N1= base; N2= middle; N3= upper) and sampling times (T1-T8). Bact= Bacteriophagous, Myc= Mycophagous, Uni= ingestors of unicellular eukaryotes, Pred= Predator.

Figure 9. Comparison of means of total abundance of nematodes between the architectonic levels of Nidularium procerum (base, middle and upper) at different sampling times. By Bonferroni’s test ** < 0.01. The vertical bar indicates the confidence interval of 95% via bootstrap (10,000 re-samplings). Values are means of eight bromeliads per sampling time.



Nematoda, 2015;2: e162015

tendency occurred for the abundance of bacteriophagous and predator nematodes (Figure 10). It is possible that the architecture of N. procerum, with the leaves being inserted at a greater angle, providing more “open” phytotelma, resulted in smaller distinctions between the architectonic levels in terms of physical-chemical characteristics of the water, producing a more uniform nematofauna.

In N. procerum, PCA explained 80.1% of the total variability (Figure 11). In relation to abundance per trophic group, a positive interaction was observed between bacteriophagous nematodes and the sampling time T5, at the three architectonic levels. There were also positive interactions between predator and mycophagous nematodes at the sampling time T2 (at the base and middle levels), T4 (at the base and upper levels) and T6 (at the base level).

The non-parametric analysis revealed that there were significant differences in the OMDM obtained from the collectors positioned close to the plants, for the sources of variation bromeliad species and sampling times, and their interaction (Table 5; Figure 12). The significance for bromeliad species is counter-intuitive, because the forest canopy above the bromeliads was uniform and the collectors were all identical. The significance for sampling time is probably due to seasonal variations in the incidence and intensity of winds, as well as in the senescence of leaves in the forest canopy.

However, a Spearman correlation analysis did not detect a correlation between the OMDM and the total abundance of nematodes or the abundance per trophic group, in both species of bromeliad (results not shown). This result is counter-intuitive, since Brouard et al.[47] reported a correlation between the amount of organic matter deposited in the phytotelmata and the functional diversity of the biota

Table 4. Non-parametric statistical test for the total abundance of nematodes and the abundance per trophic group associated with the different architectonic levels of the bromeliads (L) and different sampling times (T), in phytotelmata of Nidularium procerum.


Statistic GL p-value

Total abundance

Levels (L) 5.75 1.92 0.004

Times (T) 11.25 3.73 <0.001

L × T 1.95 5.91 0.071

Bacteriophagous

Levels (L) 6.20 1.92 0.002

Times (T) 18.21 4.36 <0.001

L × T 0.96 6.41 0.451

Mycophagous

Levels (L) 1.12 1.75 0.320

Times (T) 8.13 4.09 <0.001

L × T 0.97 5.72 0.442

Ingestor of unicellular eukaryotes

Levels (L) 1.79 1.89 0.169

Times (T) 7.47 3.37 <0.001

L × T 1.52 5.59 0.173

Predator

Levels (L) 6.85 1.98 0.001

Times (T) 9.90 4.00 <0.001

L × T 1.82 6.45 0.084



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in different species of bromeliads. Also, Ristau et al.[15, 16] observed changes in nematode diversity, trophic structure and biomass as artificial phytotelmata were nutritionally enriched. The application of different quantities of leaf litter in artificial phytotelmata also showed a direct correlation with the total abundance of nematodes and their diversity, and with the abundance of bacteriophagous forms[17]. It is possible that in bromeliads growing in the AF, the interaction between the amount of organic matter deposited in the phytotelma and the nematode trophic structure is mediated by factors that were not monitored in this study.

This is apparently the first study to deal with ecological aspects of the nematofauna associated with natural phytotelmata. As well as establishing methodological parameters for the quantitative sampling of the nematofauna associated with the phytotelmata of bromeliads, data were collected in a native AF area over a period of 24 months, a longer and more representative period than that normally used in studies of the biota of phytotelmata. The decision to perform a thorough examination of the samples eliminated the errors inherent to the examination of aliquots, such as the sub-representation of less abundant genera and trophic groups. On the other hand, this prevented a quantitative analysis of the

Figure 10. Comparison of means of abundance of nematodes per trophic group between the architectonic levels of Nidularium procerum (base, middle and upper) at different sampling times. By Bonferroni’s test ** < 0.01. The vertical bar indicates the confidence interval of 95% via bootstrap (10,000 re-samplings). Values are means of eight bromeliads per sampling time.



Nematoda, 2015;2: e162015

nematofauna at the genus and species levels, due to the abundance of specimens, especially in the juvenile stages.

The monitoring of total abundance of nematodes and abundance per trophic group revealed significant distinctions in the nematofauna of different architectonic levels of the phytotelma in both bromeliad species, with a tendency to greater abundance at the base and middle levels. It is known that the litter deposited in bromeliads is progressively fragmented and it becomes an energy source for fungi and bacteria in the lower axils of the plants. Therefore, mycophagous, bacteriophagous, ingestor of unicellular eukaryotes - such as algae, which are abundant in bromeliads in full sunlight – and possibly omnivorous nematodes have more food available in the lower axils of the plants. The results of this study also suggest that the water present in the lower axils of bromeliads does not present pH and O2 levels which are restrictive to the nematofauna.

This study also revealed a significant seasonality in the nematofauna of both bromeliad species, related to rainfall and mean temperatures. The climatic seasonality possibly acted primarily on the base trophic level – algae, fungi and bacteria – affecting the nematofauna in an indirect manner.

Figure 11. Principal components analysis for the total abundance (Ab. total) and abundance per trophic group of nematodes associated with the phytotelmata of Nidularium procerum at different architectonic levels (N1= base; N2= middle; N3= upper) and sampling times (T1 through T8). Bact= Bacteriophagous, Myc= Mycophagous, Uni= ingestors of unicellular eukaryotes, Pred= Predator.

Table 5. Non-parametric test for the organic matter dry mass obtained from collectors (C) installed next to the bromeliads Canistropsis bilbergioides and Nidularium procerum, at eight sampling times (T).


Statistic Df p-value

C 4.81 1.00 <0.028

T 12.05 2.83 <0.001

C × T 4.99 2.83 <0.002



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As mentioned before, bromeliads are the most abundant botanical group in the AF biome, maintaining important interactions with various functional groups of the biota. The AF provides various ecosystem services to Brazilian society and is home to an immense endemic biodiversity[23]. In recent years the functional role of nematodes in the mineralization of organic matter and nutrient cycling – especially nitrogen – has been revealed, as well as their potential as bioindicators for human disturbances. Therefore, the study of the nematofauna associated with the phytotelmata of bromeliads in the AF and in other ecosystems seems to be an area with much potential for basic and applied ecological studies, despite the complexity of the environmental factors involved.

CONCLUSIONSThis work revealed that bromeliad phytotelmata in the AF host a wide range of nematodes belonging

to different trophic groups, with some distinctions between the two bromeliad species studied. The nematode trophic structure was significantly distinct among the leaf axils located at the different architectonic levels of the plants, particularly in C. billbergioides. The leaf axils located at the base and middle levels seemed more conducive to nematofauna. The nematofauna presented a seasonality significantly related to rainfall and temperature fluctuations.

ACKNOWLEGEMENTSThe authors are indebted to Ralph Belletti Guzzo and Eleonora Sardinha Aguiar, the owners of São

Julião farm, for providing access to their land and general support for this work.

REFERENCES1. Kitching RL. 2000. Food webs and container habitats: the natural history and ecology of phytotelmata. Cambridge

University Press, New York, 431 pp.

2. Kitching RL. 2001. Food webs in phytotelmata: “bottom-up” and “top-down” explanations for community structure. Annual Review of Entomology, 46: 729-760. http://dx.doi.org/10.1146/annurev.ento.46.1.729. PMid:11112185.

3. Benzing DH. 2000. Bromeliaceae: profile of an adaptative radiation. Cambridge University Press, Cambridge, 710 pp.

4. Martinelli G, Vieira CM, Gonzalez M, Leitman P, Piratininga A, Costa AF, Forzza RC. 2008. Bromeliaceae da mata atlântica brasileira: lista de espécies, distribuição e conservação. Rodriguésia, 59: 209-258.

Figure 12. Comparison of means of organic matter dry mass (g) obtained from collectors installed near the bromeliads Canistropsis bilbergioides (in blue) and Nidularium procerum (in red), at different sampling times. By Bonferroni’s test ** < 0.01. The vertical bar indicates the confidence interval of 95% via bootstrap (10,000 re-samplings). Values are means of eight collectors per sampling time, for each bromeliad species.

http://dx.doi.org/10.1146/annurev.ento.46.1.729

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=11112185&dopt=Abstract



Nematoda, 2015;2: e162015

5. Greeney HF. 2001. The insects of plant-held waters: a review and bibliography. Journal of Tropical Ecology, 17: 241-260. http://dx.doi.org/10.1017/S026646740100116X.

6. Sota T, Mogi M, Kato K. 1998. Local and regional-scale food web structure in Nepenthes alata pitchers. Biotropica, 30: 82-91. http://dx.doi.org/10.1111/j.1744-7429.1998.tb00371.x.

7. Mestre LAM, Aranha JMR, Esper MLP. 2001. Macroinvertebrate fauna associated to the bromeliad Vriesea inflata of the Atlantic Forest (Paraná state, southern Brazil). Brazilian Archives of Biology and Technology, 44: 89-94. http://dx.doi.org/10.1590/S1516-89132001000100012.

8. Armbruster P, Hutchinson RA, Cotgreave P. 2002. Factors influencing community structure in a South American tank blomeliad fauna. Oikos, 96: 225-234. http://dx.doi.org/10.1034/j.1600-0706.2002.960204.x.

9. Devetter M. 2004. Invertebrate fauna of treeholes in relation to some habitat conditions in southern Bohemia (Czech Republic). Acta Societatis Zoologicae Bohemicae, 68: 161-168.

10. Quisado SJ. 2013. Nematode diversity of phytotelmata of Nepenthes spp. in Mount Hamiguitan Range Wildlife Sanctuary, Philippines [dissertation]. Ghent University, Ghent, Belgium, 74 pp.

11. Hodda M, Ocaña A, Traunspurger W. 2006. Nematodes from extreme freshwater habitats. In: Eyualem-Abebe, Traunspurger W, Andrássy I (eds.) Freshwater nematodes: ecology and taxonomy. CABI, Wallingford, p. 179-210.

12. Zullini A, Loof PAA, Bongers T. 2002. Free-living nematodes from nature reserves in Costa Rica. 2. Mononchina. Nematology, 4: 1-23. http://dx.doi.org/10.1163/156854102760082168.

13. Meyl AH. 1957. Two new freeliving nematodes, found in the rain-water reserve of Quesnelia arvensis (Vell.) Mez. (Bromeliaceae) from Brazil. Proceedings of the Helminthological Society of Washington, 24: 62-66.

14. Bert W, De Ley I, Van Driessche R, Segers H, De Ley P. 2003. Baujardia mirabilis gen. n., sp. n. from pitcher plants and its phylogenetic position within Panagrolaimidae (Nematoda:Rhabditida). Nematology, 5: 405-420. http://dx.doi.org/10.1163/156854103769224395.

15. Ristau K, Faupel M, Traunspurger W. 2012. The effects of nutrient enrichment on a freshwater meiofaunal assemblage. Freshwater Biology, 57: 824-834. http://dx.doi.org/10.1111/j.1365-2427.2012.02750.x.

16. Ristau K, Faupel M, Traunspurger W. 2013. Effects of nutrient enrichment on the trophic structure and species composition of freshwater nematodes – a microcosm study. Freshwater Science, 32: 155-168. http://dx.doi.org/10.1899/11-104.1.

17. Ptatscheck C, Traunspurger W. 2014. The meiofauna of artificial water-filled tree holes: colonization and bottom-up effects. Aquatic Ecology, 48: 285-295. http://dx.doi.org/10.1007/s10452-014-9483-2.

18. Neher DA. 2010. Ecology of plant and free-living nematodes in natural and agricultural soil. Annual Review of Phytopathology, 48: 371-394. http://dx.doi.org/10.1146/annurev-phyto-073009-114439. PMid:20455699.

19. Höss S, Traunspurger W, Zullini A. 2006. Freshwater nematodes in environmental science. In: Abebe E, Traunspurger W, Andrássy I (eds.). Freshwater nematodes: ecology and taxonomy. CABI, Wallingford, p. 144-162.

20. Ekschmitt K, Korthals GW. 2006. Nematodes as sentinels of heavy metals and organic toxicants in the soil. Journal of Nematology, 38: 13-19. PMid:19259425.

21. Wilson MJ, Kakouli-Duarte T. 2009. Nematodes as environmental indicators. CABI, Wallingford, 326 pp.

22. Myers N, Mittenmeier RA, Mittenmeier CG, Fonseca GAB, Kent J. 2000. Biodiversity hotspots for conservation priorities. Nature, 403: 853-858. http://dx.doi.org/10.1038/35002501. PMid:10706275.

23. Scarano FR. 2014. Mata atlântica – uma história do futuro. Edições de Janeiro and Conservation International - Brasil, Rio de Janeiro, 272 pp.

24. Brasil. Ministério do Meio-Ambiente. 2000. Avaliação de ações prioritárias para a conservação da biodiversidade da mata atlântica e campos sulinos. Ministério do Meio Ambiente, Brasília, 40 pp.

25. Maia MP. 2005. Políticas ambientais e a conservação da biodiversidade no Brasil. In: Franke CR, Rocha PLB, Klein W, Gomes SL (eds.). Mata atlântica e biodiversidade. Editora da Universidade Federal da Bahia, Salvador, p. 379-407.

26. Guedes MLS, Batista MA, Ramalho M, Freitas HMB, Silva EM. 2005. Breve incursão sobre a biodiversidade da mata atlântica. In: Franke CR, Rocha PLB, Klein W, Gomes SL (eds.). Mata Atlântica e Biodiversidade. Editora da Universidade Federal da Bahia, Salvador, p. 39-92.

27. Zotz A, Bader MY. 2009. Epiphytic plants in a changing world-global: change effects on vascular and non-vascular epiphytes. In: Luttge U, Beyschlag W, Budel B, Francis D (eds.). Progress in botany. Springer, Dordrecht, p. 147-170.

28. Kottek M, Grieser J, Beck C, Rudolf B, Rubel F. 2006. World map of the Köppen-Geiger climate classification updated. Meteorologische Zeitschrift, 15: 259-263. http://dx.doi.org/10.1127/0941-2948/2006/0130.

29. Instituto Nacional de Metereologia. 2012 [on line]. Acessed on May 24, 2014. Available from: http://www.inmet.gov.br/portal.

30. Jocque M, Kernahan A, Nobes A, Willians C, Field R. 2010. How effective are non-destructive sampling methods to assess aquatic invertebrate diversity in bromeliads? Hydrobiologia, 649: 293-300. http://dx.doi.org/10.1007/s10750-010-0272-1.

31. The R Foundation. 2015. The R Project for Statistical Computing [on line]. Accessed on July 1, 2013. Available from: http://www.r-project.org.

32. Moens T, Traunspurger W, Bergtold M. 2006. Feeding ecology of free-living benthic nematodes. In: Abebe E, Traunspurger W, Andrássy I (eds.). Freshwater Nematodes: ecology and taxonomy. CABI, Wallingford, p. 105-129.

33. Hunt DJ. 1993. Aphelenchida, Longidoridae and Trichodoridae: their systematics and bionomics. CABI, Wallingford, 352 pp.

34. Siddiqi MR. 2000. Tylenchida: parasites of plants and insects. CABI, Wallingford, 864 pp.

35. De Ley P, Mundo-Ocampo M, De Ley IT. 2003. Identification of free living nematodes (Secernentea). University of California Riverside University Extension, Riverside, 129 pp.

http://dx.doi.org/10.1017/S026646740100116X

http://dx.doi.org/10.1111/j.1744-7429.1998.tb00371.x

http://dx.doi.org/10.1590/S1516-89132001000100012

http://dx.doi.org/10.1590/S1516-89132001000100012

http://dx.doi.org/10.1034/j.1600-0706.2002.960204.x

http://dx.doi.org/10.1163/156854102760082168

http://dx.doi.org/10.1163/156854103769224395

http://dx.doi.org/10.1163/156854103769224395

http://dx.doi.org/10.1111/j.1365-2427.2012.02750.x

http://dx.doi.org/10.1899/11-104.1

http://dx.doi.org/10.1899/11-104.1

http://dx.doi.org/10.1007/s10452-014-9483-2

http://dx.doi.org/10.1146/annurev-phyto-073009-114439



http://dx.doi.org/10.1038/35002501


http://dx.doi.org/10.1127/0941-2948/2006/0130

http://dx.doi.org/10.1007/s10750-010-0272-1

http://dx.doi.org/10.1007/s10750-010-0272-1



Nematoda, 2015;2: e162015

36. Abebe E, Traunspurger W, Andrássy I. 2006. Freshwater nematodes: ecology and taxonomy. CABI, Wallingford, 752 pp.

37. Geraert E. 2008. The Tylenchidae of the world – identification of the family Tylenchidae (Nematoda). Academia Press, Gent, 540 pp.

38. Jairajpuri SM, Ahmad W. 1992. Dorylaimida - free-living, predaceous and plant-parasitic nematodes. E.J. Brill, Leiden, 458 pp.

39. Jenkins WR. 1964. A rapid centrifugal-flotation technique for extracting nematodes from soil. Plant Disease Report, 48: 692.

40. Coolen WA, D’Herde CJ. 1972. A method for the quantitative extraction of nematodes from plant tissue. State Agricultural Research Centre, Ghent, 77 pp.

41. Laessle AM. 1961. A micro-limnological study of Jamaican bromeliads. Ecology, 42: 499-517. http://dx.doi.org/10.2307/1932236.

42. Fincke AM. 1999. Organization of predator assemblages in Neotropical tree holes: effects of abiotic factors and priority. Ecological Entomology, 24: 13-23. http://dx.doi.org/10.1046/j.1365-2311.1999.00166.x.

43. Montero G, Feruglio C, Barberis IM. 2010. The phytotelmata and foliage macrofauna assemblages of a bromeliad species in different habitats and seasons. Insect Conservation and Diversity, 3: 92-102. http://dx.doi.org/10.1111/j.1752-4598.2009.00077.x.

44. Torreias SRS, Ferreira KRR, Spacek GR, Hamada N. 2010. Mosquitoes (Diptera, Culicidae) inhabiting foliar tanks of Guzmania brasiliensis Ule (Bromeliaceae) in central Amazonia, Brazil. Revista Brasileira de Entomologia, 45: 618-628. http://dx.doi.org/10.1590/S0085-56262010000400013.

45. Sodré VM, Rocha O, Messias MC. 2010. Chironomid larvae inhabiting bromeliad phytotelmata in a fragment of the Atlantic rainforest in Rio de Janeiro state. Brazilian Journal of Biology Online, 70: 587-592. http://dx.doi.org/10.1590/S1519-69842010000300015. PMid:20730345.

46. Madigosky SR. 2004. Tropical microclimatic considerations. In: Lowman MD, Rinker HB (eds.). Forest canopies. 2nd ed. Elsevier Academic Press, Burlington, p. 24-48.

47. Brouard O, Céréghino R, Corbara B, Leroy C, Pelozuelo L, Dejean A, Carrias J.-F. 2012. Understorey environments influence functional diversity in tank-bromeliad ecosystems. Freshwater Biology, 57: 815-823. http://dx.doi.org/10.1111/j.1365-2427.2012.02749.x.

http://dx.doi.org/10.2307/1932236

http://dx.doi.org/10.2307/1932236

http://dx.doi.org/10.1046/j.1365-2311.1999.00166.x

http://dx.doi.org/10.1111/j.1752-4598.2009.00077.x

http://dx.doi.org/10.1111/j.1752-4598.2009.00077.x

http://dx.doi.org/10.1590/S0085-56262010000400013

http://dx.doi.org/10.1590/S1519-69842010000300015

http://dx.doi.org/10.1590/S1519-69842010000300015


http://dx.doi.org/10.1111/j.1365-2427.2012.02749.x

http://dx.doi.org/10.1111/j.1365-2427.2012.02749.x

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