Directed seed dispersal: The case of howler monkey latrines

Journal Pre-proof DIRECTED SEED DISPERSAL: THE CASE OF HOWLER MONKEY LATRINES Susana P. Bravo, Victor R. Cueto

PII:

S1433-8319(19)30177-5

DOI:

https://doi.org/10.1016/j.ppees.2019.125509

Reference:

PPEES 125509

To appear in:

Perspectives in Plant Ecology, Evolution and Systematics

Received Date:

5 November 2019

Revised Date:

27 November 2019

Accepted Date:

28 November 2019

Please cite this article as: Bravo SP, Cueto VR, DIRECTED SEED DISPERSAL: THE CASE OF HOWLER MONKEY LATRINES, Perspectives in Plant Ecology, Evolution and Systematics (2019), doi: https://doi.org/10.1016/j.ppees.2019.125509

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DIRECTED SEED DISPERSAL: THE CASE OF HOWLER MONKEY LATRINES

Susana P. Bravoa,1, [email protected] Victor R. Cuetoa,1, [email protected]

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Museo Argentino de Ciencias Naturales “Bernardino Rivadavia” and Instituto de

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Investigación de las Ciencias Naturales, CONICET. Av. Angel Gallardo 470,

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Buenos Aires, Argentina.

Present address: Centro de Investigaciones Esquel de Montaña y Estepa Patagónicas

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(CIEMEP), CONICET-Universidad Nacional de la Patagonia San Juan Bosco. Roca

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780, CP 9200 Esquel, Chubut. Argentina

Highlights

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At consequence of nitrogen availability saplings of the three species studied were bigger and grew up faster in latrines of Alouatta caraya than outside. Each species had a different strategy to improve recruitment in latrines: O. diospyrifolia saplings required good conditions for growth, P. carthagenensis saplings required conditions favoring the saturation of mortality factors, and E. punicifolia saplings required either of both conditions. In conclusion large input of seeds in latrines was not the only reason of sapling recruitment in those sites. The availability of nitrogen in latrines appeared to be the main factor determining sapling recruitment in them, whereas that of phosphorus contributed by river floods would be the limiting factor for sapling growth.

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Abstract

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Latrines of vertebrates are a good example of directed seed dispersal. Although the main problem for seed dispersal at such sites is the low per capita survival expected, because of the high densities of seeds and saplings, saplings of several species recruit in latrines. The aim of the present study was to determine the mechanism involved in the recruitment of saplings in latrines of Alouatta caraya (Primate, Atelidae). To this end, we evaluated the growth and recruitment of saplings of Ocotea diospyrifolia, Eugenia carthagenensis and Psychotria carthagenensis for two years. Our first hypothesis was

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that latrines are suitable for sapling growth and that saplings in latrines are larger, grow faster and have higher nutrient concentration than those outside them, whereas our second (and opposite) hypothesis was that sapling recruitment in latrines is only a

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consequence of a large input of seeds and that the saplings in and outside latrines show similar size, growth rate and nutrient concentration. Considering the variability in the

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number of saplings recruited among latrines, we predicted the following: based on the

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first hypothesis, we predict that the number of saplings recruited in latrines at the end of the study will be positively associated with the per capita survival probability in the latrine and that the survival probability will be related to the particular conditions of

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each latrine, whereas, based on the second hypothesis, we predicted that the number of saplings surviving in a latrine at the end of the study period will be positively associated

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with the number of conspecific saplings present at the beginning of our study. Our

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results showed that the recruitment of saplings in latrines was a consequence not only of the large input of seeds, but also of the fact that latrines are suitable for growth due to the nitrogen availability. However, each species had a different strategy to improve recruitment in latrines: O. diospyrifolia saplings required good conditions for growth, P. carthagenensis saplings required conditions favoring the saturation of mortality factors, and E. punicifolia saplings required either of both conditions. The availability of

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nitrogen in latrines appeared to be the main factor determining sapling recruitment in them, whereas that of phosphorus contributed by river floods would be the limiting factor for sapling growth.

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Key words: Alouatta caraya, Argentinean flooded forest, sapling recruitment, seed dispersal.

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Introduction One of the hypotheses proposed to explain the advantages of seed dispersal is the directed dispersal hypothesis. According to this hypothesis, seed dispersal facilitates recruitment, because seeds are deposited disproportionately at suitable sites (Howe and Smallwood, 1982; Wenny, 2001). Directed seed dispersal is commonly considered rare, but it has been suggested that it may be more common and ecologically relevant than previously believed (Wenny, 2001). The best example

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of directed seed dispersal is that performed by animals, because animals use space in a non-random way. For example, since they move by trails in their home ranges, the seed dispersal pattern generated is non-random (Wenny, 2001; Schupp et al., 2002).

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Also, some behaviors of vertebrates (e.g., use of perches or repeated sleeping sites) contribute to the non-random arrival of seeds and formation of seed patches in

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particular areas (Fragoso, 1997; Julliot, 1997; Krijger et al., 1997; Bravo, 2009;

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Giombini et al., 2009). These seed dispersal patterns in patches impact on the floristic diversity and composition of several communities (Hampe et al., 2008; Stevenson, 2011; González-Zamora et al., 2014, 2015) and the recruitment of

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saplings occurs in these “hotspots” of seed arrival (Julliot, 1997; Russo and Augspurger, 2004; Hampe et al., 2008; Kitamura et al., 2008; Bravo 2012).

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Some of the most common patterns in patches of seed dispersal are latrines

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(Julliot 1997, Bravo 2009, Giombini et al. 2009, Hart and Hart 2018). To reduce the chances of parasitic infections, many mammal species, such as raccoons, tapirs, howler monkeys, and guanacos, show a particular behavior: individuals tend to defecate repeatedly in specific locations (van der Wal et al. 2000, Ezenwa 2004, Hart and Hart 2018). These specific locations are referred to as latrines and, in these sites, it is common to observe accumulated seeds, seedlings and saplings of the

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species consumed by the mammal (Fragoso 1997, Enriquez 2004, Giombini et al. 2009, Bravo 2012, Weinstein et al. 2017). The recruitment of saplings in latrines could be influenced by positive and negative factors (Enriquez 2004, Russo 2005, Dos Santos 2010). Among positive aspects, latrines represent areas with high income of nutrients, particularly nitrogen and phosphorus (Enriquez 2004, Feeley 2005, Dos Santos et al. 2010), whereas, among negative aspects, saplings are in high density and may thus have low chances to survive due to negative density-dependent

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processes (e.g., high herbivory, high competition between seedlings, and high pathogen attacks) (Russo 2005, Russo & Augspurger 2004). So, it is not possible to define “a priori” whether latrines are sites suitable for sapling recruitment or

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whether recruitment is only a consequence of the large input of seeds.

Howler monkeys, which inhabit forests along Central and South America,

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are considered effective seed dispersers, because their latrines are important

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recruitment foci for different tree species (Julliot, 1997; Anzures-Dadda et al., 2011; Bravo 2012). However, the mechanism involved in the recruitment of saplings in latrines remains unknown. Although great mortality of seedlings is observed in

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latrines of howler monkeys after germination (Bravo 2003, Zarate et al. 2018), saplings accumulate in latrines over the years (Julliot 1997, Anzures-Dadda 2011,

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Bravo 2012). In addition, although it is known that saplings of some species rarely

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recruit outside latrines (Bravo 2012, Zarate et al. 2018), it is not known whether the pattern of sapling recruitment is only a consequence of the large input of seeds in latrines or a consequence of the nutrient availability in latrines. The number of saplings varies among latrines, because it is related to the frequency of use by howler monkeys (Bravo 2003, 2012). In addition, the number of saplings of a particular species is also very variable among latrines (Bravo 2012). However, it is

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not known whether this is only a consequence of the variability of seed input among latrines or whether there are particular conditions in each latrine which determine the survival probability of each plant species and the consequent successful recruitment of saplings. In the Argentinean flooded forest, black and gold howler monkeys (Alouatta caraya) are effective seed dispersers and their latrines are recruitment foci for different tree species (Bravo, 2012). Our previous studies in this forest have shown

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that the abundance of saplings in howler monkey latrines is four-fold higher than in other sites of the forest and that saplings of Ocotea diospyrifolia, Eugenia

punicifolia and Psychotria carthagenensis are especially abundant in these latrines

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(Bravo 2012). In the Argentinean flooded forest, these tree species represent 30 % of trees with diameter at breast height (DBH) >5 cm (Bravo and Sallenave, 2003) and

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their fruits are frequently consumed by howlers (Bravo and Sallenave 2003). These

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three species depend heavily on howlers to reproduce: in our study site, more than 90 % of Ocotea diospyrifolia, 70 % of Eugenia punicifolia and 80 % of Psychotria carthagenensis saplings grow in howler monkey latrines (Bravo 2012). The

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recruitment of O. diospyrifolia and E. punicifolia is partly explained by the elimination of larval infestation during the passage through the digestive tract of

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monkeys (Bravo 2008).

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The aim of the present study was to determine the mechanism involved in the

recruitment of saplings of O. diospyrifolia, E. punicifolia and P. carthagenensis in howler monkey latrines. To this end, we evaluated the recruitment and growth of saplings in howler monkey latrines for two years. Our first hypothesis was that latrines are suitable for sapling growth and that saplings in latrines are larger, grow faster and have higher nutrient concentration than those outside them, whereas our

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second (and opposite) hypothesis was that sapling recruitment in latrines is only a consequence of a large input of seeds and that the saplings in and outside latrines show similar size, growth rate and nutrient concentration. Considering the variability in the number of saplings recruited among latrines, we predicted the following: based on the first hypothesis, we predict that the number of saplings recruited in latrines at the end of the study will be positively associated with the per capita survival probability in the latrine and that the survival probability will be related to

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the particular conditions of each latrine, whereas, based on the second hypothesis, we predicted that the number of saplings surviving in a latrine at the end of the study period will be positively associated with the number of conspecific saplings present

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at the beginning of our study.

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Study site

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Methods

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The study was carried out in Brasilera Island (Chaco, Argentina) from June 2000 to August 2002. The island is located at the confluence of the Paraná and

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Paraguay rivers (27° 30’ S, 58° 41’ W, Fig. 1). The climate is subtropical, with an

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annual average temperature of 21°C and an annual thermal amplitude of 12°C. Annual precipitation is approximately 1,500 mm. Although precipitation decreases during the winter, there is no marked dry season because evapotranspiration is lower during this period. Brasilera Island is 280 ha in size, part of which is covered by flooded forests (Fig. 1). This is one of the forest types that form the Atlantic Forest further up the Paraná River, and, at this latitude, represents an intromission of the

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Atlantic Forest into the Humid Chaco (Daly and Mitchell, 2000). Like other flooded forests associated with large rivers (such as the Várzea in the Amazon), the flooded forests of the Paraná River islands are both geomorphologically and biogeographically influenced by fluvial dynamics (Daly and Mitchell, 2000). The forests of Brasilera Island are ideal to study tree regeneration, because periodic floods favor vegetation with high recovery rates and resistance to floods (Eskuche and Fontana, 1996). Although the different forest types and the relative

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abundance of species on islands are defined by topographic, edaphic and age factors, in general, the Paraná River islands tend to have few tree species (Daly and Prance, 1989; Daly and Mitchell, 2000; Casco and Neiff, 2013). In the present study, we

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worked in the highland and lowland forests of the Brasilera Island. The highland

forests occupy 66 ha in the center of the island and are surrounded by lowlands and

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lagoons. Their canopy is dominated by Ocotea diospyrifolia and Albizia inundata

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and the understory by Eugenia punicifolia, Psychotria carthagenensis, and O. diospyrifolia saplings (Bravo and Sallenave, 2003). The lowland forests occupy 62 ha, and their canopy is dominated by Banara arguta (Flacourtaceae) and there is no

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understory stratum.

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Brasilera Island has no permanent human settlement but is impacted by

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neighboring human communities. The main human activities on the island are selective logging, hunting and cattle ranching. The frequency of these activities is variable, and they are greater on the periphery than at the center of the island. These human activities generally increase when the water level is low, although hunting pressure is also high when the river rises and there are only small areas free of water.

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Black and gold howler population and latrine characteristics

The Paraná River islands have the greatest density of howler monkeys within the distribution of the genus Alouatta, from Mexico to Argentina (Crockett, 1998). Density is 4.25 ind/ha in the highland forests and 0.88 ind/ha in the lowland forests and group size ranges between 3 and 25 individuals (Kowalewski, 2007). Black and gold howler monkeys defecate in latrines at the end of a resting period and before or

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after a territorial encounter with another group, so, in the territory of each group there are several latrines (Bravo, 2009). In the Argentinean flooded forest, latrines are very abundant and can be identified just walking through the forest, without

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having to perform any behavioral observations of the monkey groups. Latrines are

approximately 5 m in diameter, feces are accumulated on the ground and saplings of

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different age are growing in them as a result of the large size of monkey groups and

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the small size of their home ranges (Bravo and Sallenave, 2003). Latrines used with less frequency may be smaller than latrines used with more frequency and show

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lower quantity and diversity of growing saplings (Bravo, 2009, Bravo 2012).

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Determination of sapling size and nutrient availability

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To determine sapling size and nutrient availability, we selected 10 latrines

and 10 areas of 5 meters of diameter without the inclusion of any latrine, and at least 10 m away from the border of any latrine (see details in Bravo, 2012). Latrines were in the home range of three groups, and were located during studies of ecology and animal behavior developed from 1997 to 1999 by SPB (Bravo and Sallenave 2003, Bravo 2008, Bravo 2009). Both in each latrine and in the areas outside latrines, we

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randomly selected 20 saplings shorter than 20 cm of each of the three species selected: O. diospyrifolia, E. punicifolia and P. carthagenensis. The size of the saplings growing in and outside the latrines was compared by two variables: the sapling height and the length of the largest leaf. To evaluate the concentration of nitrogen and phosphorus in sapling leaves, we used the micro-Kjeldahl method.

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Recruitment process

To determine survival and growth of saplings of O. diospyrifolia, E.

punicifolia and P. carthagenensis, all saplings of these three species shorter than 25

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cm were evaluated for two years (June 2000 - May 2002), in 12 latrines found along

3 transects of 500 m (Fig. 1). Seedlings (individuals depending on cotyledons and/or

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not lignified) were not included in the study. A plastic stake was placed next to

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every sapling to identify individuals and latrines were monitored at 2, 10, 18 and 23 months. Sapling growth was estimated by means of three variables: sapling height, number of leaves and length of the largest leaf. As the three variables were

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correlated (r >0.65 for each combinations), only height was used for the statistical analysis. The recruitment of saplings of each species in a latrine was estimated by

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the number of saplings of the species that survived the two-year study period, and

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the survival probability was estimated as the number of saplings that survived the two-year study period in relation to the number of individuals of the species at the beginning of the study. All saplings were also examined for damage symptoms. Four types of damage were recorded: herbivory, physical (i.e. cuts in leaves produced by the fall of materials from the canopy, which is very common when monkeys move through

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the branches), dried or burned, and nutritional deficiencies (estimated by discoloration or deformation of young leaves Yeh et al., 2000; Taiz and Zeiger, 2002). Every leaf of a sapling was assigned to one of four levels of damage: < 25 %, 25 – 50 %, 50 – 75 %, and >75 %, determined by visual estimation. If a sapling had more than 25 % of its leaves with more than 25 % of damage, damage was considered “severe”. We calculated the proportion of saplings with severe damage for the four types of damage.

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We also estimated the number of days under water (flooded days) for every latrine, relating the height of flood marks on trees with the Paraná River level. To

this end, first, we measured the height of marks left on the trees nearest every latrine

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by a flood that occurred in October 2000. Secondly, to obtain the height above the

Paraná River of every latrine location, we subtracted the heights of the marks at the

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maximum level reached by the Paraná River during the flood. Paraná River levels

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were obtained from official daily records of the Prefectura Naval Argentina at Isla del Cerrito (5 km up river from our study site). Thirdly, the number of flooded days was estimated for every latrine at the end of the study. To this end, we used the daily

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record of the height of the Paraná River to count the number of days that the river level was higher than the height estimated for every latrine location during our study

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period.

Statistical analysis

The nitrogen and phosphorus contents of P. carthagenensis, O. diospyrifolia and E. punicifolia saplings growing in and outside latrines were compared with a t test. The height and length of the largest leaf of the saplings growing in and outside

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latrines were compared by a two-factor ANOVA, in which the factors were site and species. Height data were log transformed. Data within each latrine were pooled for analysis. The growth of saplings in and outside latrines could not be compared statistically, because of the high mortality outside latrines. To evaluate the recruitment of saplings of each species in the latrines, we used the following variables: number of saplings of all species at the beginning of the study, number of conspecific saplings, proportion of individuals with a severe

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level of damage (including: nutritional deficiency, herbivory, dried or burnt and physical, hereafter “damage”), number of flooded days and survival probability. To

evaluate factors that affected the survival probability of the saplings of each species

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in the latrines, we used the following variables: number of saplings of all species at the beginning of study, number of conspecific saplings, damage, flooded days and

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growth. To test the differences in recruitment and survival probability in different latrines, we applied generalized linear models (GLMs). Outlier data were

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determined by exploring residuals and considering Cook’s distance in a residuals vs leverage plot. We used Akaike’s Information Criterion (AICc) corrected for small

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sample size and deviance squared adjusted to evaluate different models (Guisan and Zimmerman, 2000). To determine the weight of significant variables, we used

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GLMs with standardized variables. To evaluate the effects of the variables on

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recruitment, we used a GLM with Poisson error distribution and log link function, whereas to evaluate the effect of the variables on the survival probability at each latrine, we used a GLM with binomial error distribution and logit link function. The adequacy of error structures used in the GLMs was corroborated with residual analysis (Crawley, 2007). All statistical tests were performed with R 3.3.2 (R Core Team, 2016).

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Results

Nutrient availability and size and growth of saplings in and outside latrines

Saplings of the three species tended to have a higher concentration of

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nitrogen when they grew in latrines than when they grew outside them (Table 1), although only Psychotria carthagenensis saplings showed significant differences.

They had 19.5 % more nitrogen when growing in latrines than outside them (Table

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1). In contrast, the saplings of the three species tended to have lower concentrations

of phosphorus in latrines than outside them (Table 1), although the differences were

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not significant for the three species (Table 1).

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Neither of the factors considered (i.e. site and species) had a significant interaction in relation to sapling height (F = 0.54; p = 0.59; df = 2; Fig. 2a) or length of the largest leaf (F = 0.28; p = 0.75; df = 1; Fig. 2b). Saplings of the three species

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studied were taller (F = 15.96; p = 0.0004; df = 1; Fig. 2a) and their leaves were larger (F = 51.34; p < 0.0001; df = 1; Fig. 2b) in the latrines than outside them. The

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height of saplings was independent of the species (F = 2.11; p = 0.14; df = 2; Fig.

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2a), whereas the length of the largest leaf was dependent on the species (F = 15.96; p = 0.0004; df = 2; Fig. 2b). More than 70 individuals of E. punicifolia and more than 80 of O.

diospyrifolia and P. carthagenensis survived in latrines for the two-year study period. In contrast, 15 saplings of E. punicifolia, but no saplings of O. diospyrifolia or P. carthagenensis survived outside the latrines.

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In the latrines, saplings of E. punicifolia grew at a rate of 0.48 ± 0.2 cm/month, which represented a growth of 114 % in the two-year study period (Fig. 3a). In addition, their leaves enlarged 0.1 ± 0.03 cm/month, which represented 54 % of growth, and reached the size of adult tree leaves (Fig. 3b). The number of leaves increased three fold (Fig. 3c). In contrast, outside the latrines, E. punicifolia saplings showed approximately half the growth rate, i.e. 0.18 ± 0.04 cm/month, which represented a growth of 77 % in the two-year study period. Their leaves grew 0.06 ±

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0.02 cm/month, which represented 76 % of growth, but leaves were still smaller (3.7 ± 2.0 cm) than adult tree leaves (mean value).

Regarding O. diospyrifolia saplings inside the latrines, the growth rate was 0.16

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± 0.26 cm/month, which represented a growth of 32 % (Fig. 3a), and the growth of

leaves was 0.1 ± 0.06 cm/month, which represented a growth of 37 %. The leaves of

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O. diospyrifolia saplings also reached a size similar to that of adult tree leaves (Fig.

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3b) and the number of leaves increased two fold during the study (Fig. 3c). Regarding P. carthagenensis saplings, they grew at a rate of 0.33 ± 0.17 cm/month, which represented a growth of 68 % in the two-year study period (Fig.

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3a). Their leaves enlarged 0.01 ± 0.002 cm/month, which represented 4 % of growth. Sapling leaves also reached a size similar to that of the adult tree leaves (7.21 ± 1.15

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cm) (Fig. 3b), but the number of leaves did not change during the two-year study

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period (Fig. 3c).

During flooding, growth in height and new leaf production stopped in all the

submerged saplings of the three species. However, the three species showed mechanisms that allowed them to be tolerant to submergence. After the flood, E. punicifolia saplings were intact, with sediments deposited on the leaf surfaces. Saplings grew in height and new large leaves sprouted from old and new stem

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portions. Old leaves remained on the saplings together with new leaves throughout the study. Ocotea diospyrifolia saplings showed a phenology similar to that of E. punicifolia, but the old leaves dried up and fell off when the new leaves expanded. Psychotria carthagenensis saplings lost all their leaves and the distal portion of the stem looked rather dried up, but new large leaves sprouted from the remaining stem when the flood ended.

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Recruitment process in latrines

Regarding O. diospyrifolia, the total number of saplings at the beginning of

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the study, the number of conspecific saplings and the survival probability predicted 83 % of variability in the number of saplings recruited (Table 2). The number of

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saplings at the beginning of the study was twice more relevant than the survival

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probability or the number of conspecific saplings (Table 2). The number of conspecific saplings was negatively related to recruitment, while the other variables were positively related (Table 2). The survival probability of O. diospyrifolia

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saplings in latrines was positively related to growth and negatively related to damage, with growth being twice more relevant than damage (Table 2). Among the

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different types of damage, nutritional deficiency was the most important (Fig. 4).

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Regarding E. punicifolia, the number of conspecific saplings and survival

probability predicted 84 % of variability in the number of saplings recruited. Both variables were positively related to recruitment and contributed to the model with a similar weight (Table 2). The survival probability of E. punicifolia saplings in latrines was positively related to the number of days under water and the total number of saplings at the beginning of the study (Table 2).

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Finally, regarding P. carthagenensis, the number of conspecific saplings predicted 93 % of variability in the number of saplings recruited, and this variable was also the only predictor of survival probability (Table 2).

Discussion

The results of the present study indicated that the recruitment of saplings of

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O. diospyrifolia, E. punicifolia and P. carthagenensis in howler monkey latrines was not only a consequence of a large input of seeds in these sites. Saplings of the three

species were larger, grew up faster and tended to show higher nitrogen concentration

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in latrines than outside them, although the difference in nitrogen concentration was significant only for P. carthagenensis. This suggests that latrines were also sites

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suitable for growth, probably as a consequence of the high nitrogen availability in

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these sites. Previous studies in other species and ecosystems in the world have shown an increase in productivity when saplings were supplemented with nitrogen (Pregitzer et al., 2008; Magnani et al., 2007; Wright et al., 2011; Fowler et al.,

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2015). Contrary to that found in Venezuela and French Guiana, where both nitrogen and phosphorus show high concentrations in latrines of Alouatta seniculus (Feeley,

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2005; Dos Santos et al., 2010), in the present study, the phosphorus in Alouatta

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caraya latrines was lower than outside them. This may be explained by the fact that the Paraná River sediments are rich in phosphorus and poor in nitrogen (Carignan et al., 1994; Orfeo and Stevaux, 2002) and that trees species inhabiting flooded forests capitalize the phosphorus and other nutrients provided by the river water (Neiff 2004, Parolin, 2009, Parolin et al., 2016). So, probably, in our study site, the phosphorus supplied by successive floods was accumulated outside latrines.

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However, saplings outside latrines incorporate phosphorus at a low rate due to the few numbers of saplings outside latrines and their limitation in growth due to the low level of nitrogen. In contrast, the high nitrogen availability inside latrines probably favored the growth of the great number of saplings, increasing phosphorus demand until depleting it. In relation to the hypothesis about the variability in the number of saplings recruited among latrines, our data showed that each species had a different strategy,

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probably related to their particular nutritional requirements. In O. diospyrifolia, the mechanism involved in sapling recruitment was

related to the characteristics of each latrine, supporting the hypothesis about latrines

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as sites suitable for sapling growth. Recruitment of O. diospyrifolia was possible under conditions that maximized sapling growth and minimized nutritional

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deficiency. The total number of saplings in a latrine was related to the frequency of

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use of the latrine. This is in agreement with our previous studies showing that the number of saplings is highest when a latrine is used all the year and receives seeds of all the species included in the diet of the monkeys (Bravo 2003). So, these latrines

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with great number of saplings are also latrines with great intake of nitrogen. This may explain the positive relationship between the total number of saplings at the

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beginning of the study and the recruitment of O. diospyrifolia. The negative relation

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between the number of saplings of O. diospyrifolia at the beginning of the study and the recruitment obtained in a latrine after two years is also concordant with the idea of the limitation by nutrients and the probable competition between the conspecific saplings of O. diospyrifolia. Although the growth and size of O. diospyrifolia saplings in latrines were higher than outside them, saplings showed important signs of nutritional deficiencies. The high nitrogen availability was probably the reason

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for the great growth of the saplings in latrines, but, in some species as O. diospyrifolia, it could also highlight deficiencies of other nutrients that were not available in latrines (e.g., phosphorus), because the increase in the growth rate also increases the demand of nutrients (Di Tian et al., 2017; Razzaq et al., 2017). Ocotea diospyrifolia is a shade-tolerant species (Carvalho 2006), and experimental studies with that group of species have shown that they grow faster than less shade-tolerant ones when nitrogen and phosphorus are added (Santiago et al. 2011, Villagra et al.

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2013). Regarding the recruitment of E. punicifolia, our results support both the

hypothesis that latrines with great number of saplings at the beginning of the study

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would show high recruitment after two years and the hypothesis that a high

recruitment would be also possible if latrines have a great survival probability

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related to a high income of nutrients in the latrine. In this species, survival

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probability was related not only to the number of total saplings, as we have previously reported that it is associated with nitrogen supply, but also to the number of flooded days, which could be associated with a high input of phosphorus, being

data.

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an ideal complement to the latrine condition, poor in phosphorus according to our

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Although each tree species has mechanisms that allow them to be tolerant to

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submergence (Neiff 2004, Parolin, 2009, Parolin et al., 2016), our present results showed that floods had a negative effect on sapling survival. Due to the floods, some saplings died or lost height between measurements. However, at the end of the twoyear study period, the net growth was important, particularly for E. punicifolia. In this species, during the post-flood measurement, we detected a decrease in the mean

18

height of saplings of 5 cm (34.5 %) in a latrine flooded for 30 days, but at the end of the study, those saplings had doubled their original mean height, growing by 214 %. Finally, P. carthagenensis recruitment supports the second hypothesis about the role of saturation of mortality factors, because only the number of P. carthagenensis saplings at the beginning of the study determined the number recruited at the end of the two-year study period. So, it can be said that any latrine is suitable for the recruitment of that species.

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The survival probability of seeds or seedlings in monkey latrines is generally estimated as low due to negative density-dependent processes (Russo and

Augspurger, 2004; Russo, 2005). Nonetheless, studies have shown that the

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recruitment of saplings over the years is higher in latrines than outside them (Bravo 2012, Zarate et al., 2018). In the present study we found that negative density-

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dependent processes could be affecting the recruitment of O. diospyrifolia. For this

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species, only some latrines with particular conditions were suitable. In contrast, other species, such as E. punicifolia and P. carthagenensis, could be benefited by the high density conditions, and the great number of conspecific saplings is

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translated in great recruitment. So, our results highlight the importance of focusing on different plant species to increase our understanding of the mechanism involved

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in the seed dispersal process.

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The most important criticism of the directed dispersal hypothesis is that

“most suitable sites” are neither constant nor predictable (Schupp, 2007). Thus, selection should favor the maintenance of diffuse disperser networks that disperse seeds widely rather than specialists that deposit seeds preferentially in specific sites, especially in long-lived plants such as tree species (Schupp, 2007). However, if saturation and the nutritional condition induced by the disperser itself are the main

19

factors operating in the specific site where seeds are deposited (e.g., a latrine), selection would act by favoring the attraction of specialists (“latrine makers”).

Conclusions Our results show that clumped defecation, which leads to the accumulation of a large number of seeds in a specific site, could be an advantage for the recruitment of some species. In addition, our results would be denoting that nutrient

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availability in latrines is also an important factor in the determination of sapling recruitment. The nitrogen supplied in latrines by monkeys’ droppings was

determinant for sapling recruitment, whereas the phosphorus supplied by river

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floods and other nutrients not evaluated by us would be the limiting factor for

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sapling growth. Authors’ contributions

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Susana P. Bravo: contributed to the conception and design of the study, collected, analyzed and interpreted the data, and drafted the article.

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Victor R. Cueto: contributed to the conception and design of the study, collected, analyzed and interpreted the data, and revised the draft manuscript critically.

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Conflict of interest

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None declared

Acknowledgements We thank the several students of the Universidad de Buenos Aires (Buenos Aires, Argentina) and A Pas de Loup volunteers who worked as field assistants. We are especially grateful to Mr. Gallo, his wife Titina, and the Cao family for their logistic support in the field. This study was funded by the National Scientific and Technical 20

Research Council (CONICET), Argentina, and the International Foundation for Science (IFS; D/2686-1 and D/2686-2). We also appreciate the comments of editors

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and reviewers of PPEES.

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Figure captions

Figure 1: Location of Brasilera Island in South America and detail of the island with

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transect locations where the latrines were searched for.

Figure 2: Sapling height (mean ± SE, a) and length of the largest leaf (mean ± SE, b) of

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Ocotea diospyrifolia, Eugenia punicifolia and Psychotria carthagenensis growing in howler monkey latrines (black bars) and outside them (white bars). ** p< 0.001; ***

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p = 0.0001

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Figure 3: Growth of saplings (mean ± SE) in howler latrines (black symbols and solid

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line) and outside them (open symbols and dashed line) along the two-year study

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period. Sapling height (a), length of the largest leaf (b) and number of leaves (c). Triangles represent O. diospyrifolia saplings, circles represent E. punicifolia saplings and squares represent P. carthagenensis saplings.

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Figure 4: Percentage (mean ± SE) of Ocotea diospyrifolia saplings with severe damage of different types recorded in howler monkey latrines. Severe damage: more than 25

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% of leaves with more than 25 % of the surface affected.

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Table 1: Mean concentration of nitrogen and phosphorus (± SE) in saplings of O. diospyrifolia, Eugenia punicifolia and Psychotria carthagenensis that were growing in latrines of Black and Gold Howler monkeys or outside them (10 m away of any latrine). Δ % is the difference of nutrient concentration (In – Out) expressed as percentage of the mean value of saplings growing outside a latrine.

Nitrogen

Phosphorus

In

Out

Δ%

pvalue

Psychotria carthagenensis

3.1 ± 0.4

2.5 ± 0.5

19.5

0.02*

0.29 ± 0.06

0.31 ± 0.05

8.6

0.14

Ocotea diospyrifolia

2.9 ± 0.5

2.7 ± 0.5

4.9

0.3

0.25 ± 0.06

0.28 ± 0.07

9.5

0.2

Eugenia punicifolia

1.6 ± 0.2

1.5 ± 0.2

1.3

0.4

0.15 ± 0.03

0.16 ± 0.03

9.3

0.4

Δ%

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Out

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* t = 2.1; DF = 18

In

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pvalue

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and Gold Howler monkeys in the Argentinean flooded forest. Dependent factor

z

1.07 ± 0.25

4.14

3.4 e-05

Saplings at the beginning

1.95 ± 0.43

4.52

6.2 e-06

Survival probability

0.98 ± 0.29

-3.38

0.0007

-2.30

0.021

Intercept

e-

Ocotea diospyrifolia Recruitment

β ± SE

Independent factor

pr

Species

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Table 2: Results of general linear models on the survival probability and recruitment of saplings of the three species studied in latrines of Black

Pr

O. diospyrifolia saplings Survival probability Intercept

-0.06 ± 0.3

-0.19

0.85

2.92 ± 0.65

4.61

3.98 e-06

Damage

-1.32 ± 0.33

-3.99

6.64 e-05

Intercept

1.41 ± 0.26

5.34

9.22 e-08

E. punicifolia saplings

1.23 ± 0.21

5.34

1.46 e-08

Survival probability

1.03 ± 0.24

4.28

1.85 e-05

-0.72 ± 0.22

-3.23

0.001

Saplings at the beginning

0.90 ±0.21

4.19

2.84 e-05

Flooded days

0.78 ± 0.36

2.14

0.03

Survival probability Intercept

D2 adj 83 %

78 %

Growth

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Eugenia punicifolia Recruitment

-0.71 ± 0.31

P

84%

65 %

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f

Recruitment

Intercept

0.004

1.45 ± 0.26

5.5

3.61 e-08

-2.01 ± 0.33

-5.66

1.48 e-08

0.69 ± 0.31

2.23

0.02

93 %

70 %

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Pr

e-

P. carthagenensis saplings

2.89

pr

P. carthagenensis saplings Survival probability Intercept

0.90 ± 0.31

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Psychotria carthagenensis

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