In bromeliad phytotelma, anthropic disturbance does not affect the nematode trophic structure

The study was conducted in Restinga de Jurubatiba National Park (RJNP) (http://www.icmbio.gov.br/parnajurubatiba/), in the state of Rio de Janeiro, Brazil. The park protects a large area of restinga, an Atlantic Forest ecosystem that includes beaches, dunes, lagoons and areas subject to periodic flooding, in which herbaceous and shrubby species predominate. The region’s climate is AW (tropical savanna) according to the Köppen classification, with an average annual temperature of 23°C and a yearly rainfall ranging from 1,000 to 1,350 mm.

The RJNP encompasses areas which have been impacted by human activities before the park’s creation. Areas 1, 2, and 3 were selected according to their level of disturbance (Table 1). Area 1 (22°11′09.3″ S; 41°25′50.8″ W) suffered removal of vegetation and impact from a nearby coconut cultivation. Now and then cattle trespass the park’s limits and graze in the area (Fig. 1A, B). Area 2 (22°10′34.7″ S; 41°24′42.0″ W) is located 4 Km away from area 1 and it has suffered relatively less impact. Occasional cattle grazing occurs during the dry season only (Fig. 1C, D). Area 3 (22°09′54.1″ S 41°24′03.1″ W) is located 5 Km away from area 2 and suffered no disturbance, except for the occasional transit of the park’s surveillance vehicles along narrow trails (Fig. 1E, F). These areas are roughly rectangular, ranging from 500 to 600 m × 200 to 300 m (10-18 ha).

Figure 1:

Samples were collected in the terrestrial bromeliad Neoregelia cruenta (R. Graham) L. B. Smith, which thrives abundantly in the RJNP, shaded by short trees and shrubs. Only mature plants with inflorescence were sampled because this phenological stage is known to support a more abundant nematode community (Almeida and Souza, 2020).

In areas 1, 2, and 3, samplings were carried out from June 2014 through March 2016, once every season, for a total of eight sampling dates. On each sampling date, in each area, eight bromeliads were chosen along a random path. This process was carefully done not to repeat previous paths. The water retained in the bromeliads was suctioned and submitted to the following measurements: volume (mL); temperature (^oC); pH; dissolved oxygen (DO₂, in mg/L); dissolved solids (DS, in mg/L); and electrical conductivity (EC, in mS/cm). These were measured in the field with an Icel Manaus^® meter (model PH–1500) with appropriate sensors. A 10 mL aliquot was collected in a new, sterile penicillin flask and used for measuring dissolved organic carbon (DOC, in mg/L) and dissolved nitrogen (N, mg/L), using a Shimadzu meter (model TOC–Vcph). Macroscopic organic debris (OD) fallen into the phytotelma was collected in a paper bag, dried for 24 hr in an oven at 80°C, and expressed in grams.

For nematode sampling, the 24 bromeliads were removed from the soil and placed in plastic bags, with care to return the water initially sucked from the phytotelma for the PCP measurements. For nematode extraction, each plant was individually defoliated and washed in tap water, collecting all the water in a 5 L bucket. The water was passed through 60 and 500 mesh sieves (250 and 25 µm opening, respectively). The resulting suspension was submitted to the method of Coolen and D’Herde (1972), with modification (no previous grinding in a blender), with centrifugation at 760.24 G for 3 min and 190.06 G for 2 min.

The 24 suspensions were reduced to a volume of 5 ml and observed entirely in Peter’s slides for nematode counting. The total abundance of nematodes and the abundance per trophic group were computed. Nematodes were assigned as plant, hyphal, or bacterial feeders, unicellular eukaryote feeders, or carnivores (Moens et al., 2006) by examination in a Nikon Eclipse^® microscope with Nomarski interference contrast in the 40 and 100X lenses.

The assays’ raw data are publicly available at https://doi.org/10.6084/m9.figshare.12925571. Unicellular eukaryote feeders were found only occasionally and plant feeders were not found. Hence, no statistics were applied to these groups. Nematode counts – total, hyphal, and bacterial feeders, and carnivores – were tested for homogeneity of variances (Cochran and Bartlett tests) and for normality of errors (Lilliefors test), at 5% probability. Since the assumptions were satisfied, nematode counts were evaluated through a two factor-ANOVA using the R language v.3.6.1 (R Core Team, 2019). The linear model accounted nematode counts as response variables, and the areas, seasons, and water PCPs as explanatory variables. The residual distribution was examined and data with residues higher than three standard deviations (|x| > 3 SD) were considered outliers and removed from the dataset. The pairwise comparison among least square means was done by Tukey test at 5% probability using the R language v.3.6.1. These comparisons were made by pooling data from all eight sampling dates (seasons), and by decomposing the data per season.