Biological Conservation 191 (2015) 159–167

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Biological Conservation

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Overfishing disrupts an ancient mutualism between frugivorous fishesand plants in Neotropical wetlands

Sandra Bibiana Correa a, Joisiane K. Araujo b, Jerry M.F. Penha b, Catia Nunes da Cunha b,Pablo R. Stevenson c, Jill T. Anderson a,⁎a Department of Genetics, Odum School of Ecology, University of Georgia, 120 Green St., Athens, GA 30602, USAb Instituto de Biociências, Universidade Federal de Mato Grosso, Ave. Fernando Correia 2367, Cuiabá, MT, Brazilc Departamento de Ciencias Biológicas, Universidad de los Andes, Carrera 1 No. 18A-12, Bogotá, Colombia

⁎ Corresponding author: Tel.: +1 706 542 0853.E-mail address: jta24@uga.edu (J.T. Anderson).

http://dx.doi.org/10.1016/j.biocon.2015.06.0190006-3207/© 2015 Elsevier Ltd. All rights reserved.

a b s t r a c t

a r t i c l e i n f o

Article history:Received 7 May 2015Received in revised form 13 June 2015Accepted 14 June 2015Available online xxxx

Keywords:Seed dispersalDefaunationFlooded forestFlooded savannahAmazonPantanal

Defaunation is disrupting plant–animal interactions worldwide. The overhunting of frugivores disrupts seeddispersal and diminishes plant regeneration, yet investigations of frugivore overexploitation neglect an ancientguild: fruit-eating fish. For nearly five decades, Neotropical frugivorous fishes have been intensively harvested.These fishing activities have reduced population sizes of some species by up to 90% and have likely alteredpopulations to younger, smaller individuals. Here we evaluate potential ecological consequences of overfishingfrugivores for seed dispersal and recruitment dynamics. We analyzed dietary data from seven fruit-eating fishspecies in Amazonian and Pantanal wetlands to test the hypothesis that seed dispersal effectiveness increaseswith fish size within and across species. Relative to small individuals, larger fish dispersed large numbers ofseeds of a higher diversity of plants and a greater range of seed sizes. For some seed species, dispersal by largerfish augmented germination success, relative to seeds dispersed by smaller fishes. Large Piaractus mesopotamicusin the Pantanal disperse seeds of 27%more species than fishes under theminimum size limit for this fishery. Ourresults indicate that the ongoing overexploitation of multiple frugivorous fish species could depress the quantityand diversity of seeds dispersed, as well as the quality of seed dispersal in wetland habitats that extend over 15%of the area of South America.

© 2015 Elsevier Ltd. All rights reserved.

1. Introduction

The disruption of pollination and seed dispersal mutualisms directlyaffects plant reproductive success and threatens biodiversity (Aslanet al., 2013; Dirzo et al., 2014; Valiente-Banuet et al., 2015). The vastmajority of plants rely on animals to disperse their genes and progeny(Jordano, 2000;Ollerton et al., 2011). For example, vertebrate frugivoresdisperse the seeds of 70–95% of woody plant species in tropical forests(e.g., Jordano, 2000). Seed dispersal sets the initial template of plant dis-tribution in the landscape, shapes the pool of interacting plant species,and potentially facilitates species coexistence through competition/colonization tradeoffs (Howe and Smallwood, 1982; Seidler andPlotkin, 2006).

Frugivorous vertebrates are overhunted in tropical forests across theglobe, which reduces plant recruitment, alters plant species composi-tion, diminishes biodiversity, and causes numerous indirect changes tocommunities (Caughlin et al., 2015; Markl et al., 2012; Poulsen et al.,2013). As hunters prefer big prey, overharvesting is particularly

problematic for large-seeded plant species, which require seed dispersalby sizable frugivores (Caughlin et al., 2015; Effiom et al., 2014). Localextinction of large frugivores can even induce rapid evolutionary changesin seed size (Galetti et al., 2013). Aburgeoning bodyofwork evaluates theecological repercussions of hunting frugivores (Aslan et al., 2013), butneglects the largest clade of vertebrates: fish.

Worldwide, over 275 species of fish consume fruits and disperseseeds; of these, at least 150 inhabit South American wetlands (Hornet al., 2011), where they disperse seeds of at least 566 plant speciesfrom 82 families (Correa et al., 2015). Neotropical wetlands extendacross eight countries and the three largest South American river basins(Amazon, Orinoco, and Paraná–Paraguay), occupying at least 15% ofthe continent (Junk and Piedade, 2010). Fruiting is synchronized withthe annual flood, lasting up to seven months, and many fruits andseeds exhibit adaptations for dispersal by water (hydrochory) or fish(ichthyochory; Ferreira et al., 2010; Kubitzki and Ziburski, 1994).During lengthy flooded seasons, fishes spend ~87% of their time infloodplain habitats (Anderson et al., 2011) where seeds can germinateafter floodwaters recede (Ferreira et al., 2010).

Globally, selective harvesting of fish concentrates on large individ-uals, inducing changes in population structure by favoring the survivalof smaller fishes that reproduce earlier (Allan et al., 2005; Palkovacs,

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2011). A fishery-induced reduction in body size of frugivorous specieswould have profound effects on plant regeneration if larger fishes arebetter seed dispersers than smaller fishes (Anderson et al., 2011;Correa et al., 2015; Galetti et al., 2008; Kubitzki and Ziburski, 1994).Large Neotropical frugivorous fishes are prized in commercial, subsis-tence, and recreational fisheries, and are threatened by ongoing overex-ploitation (Isaac and Ruffino, 1996; Peixer et al., 2007). Frugivorousfishes are heavily consumed in the Amazon, Orinoco and Pantanal, con-tributing to food security and economic growth (Barthem andGoulding,2007; Mateus et al., 2004; Rodriguez et al., 2007). Intensive commercialfisheries developed in the 1970s across the Amazon and Pantanal withthe introduction of nylon gill nets and large capacity freezer rooms(Mateus et al., 2004; Santos and Santos, 2005). By the early 1990s,stocks of Colossoma macropomum in the Central Amazon were alreadyoverexploited (Isaac and Ruffino, 1996) and commercial and recrea-tional fishing activities have reduced population sizes of Piaractusmesopotamicus in the Pantanal by 90% (Albuquerque et al., 2012;Peixer et al., 2007). In addition to population declines, overfishing likelyhas shifted body size of these species to smaller individuals that repro-duce earlier (Palkovacs, 2011), however the lack of basin-wide continu-ous historicfishery statistics (Mateus et al., 2004; Ruffino, 2008) hindersour ability to assess changes in population structure of overexploitedpopulations. Striking population declines in other large species such ascatfishes have shifted fishing pressure to under-exploited stocks oflarge frugivorous fishes in remote locations (Agudelo et al., 2012;Albuquerque et al., 2012; Mateus et al., 2004; Reinert and Winter,2002). Small- andmedium-sized frugivorous fish species are also inten-sively exploited in the Amazon and Pantanal (Mateus et al., 2004;Santos and Santos, 2005). These species are heavily consumed by river-ine people; therefore, a large portion of the capture is not accounted forin commercial fishery statistics, leading to an underestimation of thereal fishing pressure (Castello et al., 2013).

Here, we examine the ecological consequences of overfishing in twoexpansive and diverse Neotropical wetlands: the Pantanal and theAmazon. In both regions, we predict that the largest fishes dispersemore seeds of a greater diversity of plant species, and that large-bodied fish species are among the primary vectors of dispersal forplant species with big seeds (e.g., Stevenson, 2011). Additionally, weexamine size-dependent shifts in seed predation, testing the predictionthat larger fishes depredate fewer seeds than smaller individuals. In apreliminary analysis, Correa et al. (2015) found that smaller fishes hada greater probability of destroying seeds than larger fishes acrossthree species of Brycon, presumably because big fishes are more adeptat swallowing fleshy fruits entire. Finally, we tested whether seedgermination success increaseswithfish size in a greenhouse experiment.Overexploitation could fundamentally alter fish–fruit interactionsfrom high quality seed dispersal by large fishes to greater rates of seedpredation by smaller fishes.

2. Materials AND Methods

2.1. Study areas

2.1.1. Brazilian PantanalSpanning over 160000 km2, the Pantanal is one of the largest

tropical wetlands in the world (Junk and Nunes da Cunha, 2005). Weconducted our study in the SESC Pantanal Private Natural HeritageReserve (1063 km2; 16°30′51″S, 56°22′38″W; Fig. 1b) in the northernPantanal. The Pantanal Reserve constitutes a mosaic of seasonallyflooded savannas with small patches of semi-deciduous mono-dominant forests of cambará (Vochysia divergens, Vochysiaceae) andgallery forests. Tree diversity in forests is low (up to 10 species withdbh N 10 cm per 0.1 ha; Arieira et al., 2011) and the flooding regime ismoderate (2 m depth for 5 months annually, Junk and Nunes daCunha, 2005).

2.1.2. Colombian AmazonThe lowlands of the Amazon River and its tributaries contain vast

expanses of seasonally flooded forests. Amazonian floodplains coverapproximately 250000 km2, constituting the largest wetland on earth(Junk, 1993). We sampled the lower Caquetá River (1°16′32″S, 69°43′50″W; Fig. 1a), where the floodplain supports a continuous, relativelyundisturbed evergreen forest with high tree diversity (up to 54 specieswith dbh N 10 cm per 0.1 ha; Duivenvoorden, 1996) that floods up to9 m depth for 6 months annually (Rodriguez, 1991).

2.2. Fish sampling

To sample frugivorous fishes of varying sizes, we baited hooks ofdifferent sizes with ripe local fruits, as well as with dough made ofcooked manioc flour and artificial fruit flavors, and fished with poleand line. In the Amazon, hooks were also suspended from vegetationin the flooded forest and monitored hourly (Correa and Winemiller,2014), which is not feasible in the Pantanal due to abundant Caimanpopulations. Our sampling selectively captures fruit-eating fish species(Correa and Winemiller, 2014). Immediately after removal from thehook, we euthanized fishes with Tricaine methanesulfonate (MS-222).We recorded species identity, standard length (SL—length from the tipof the snout to the posterior end of the hypural bones, excluding thecaudal fin), weight, and mouth gape of the fish, and collected stomachand intestinal samples by dissection. This research complies withanimal use guidelines (AUP# 2194-100789-011314).

In the Pantanal, we sampled fishes in four habitats (river channels,savannas, mono-dominant and gallery forests) during the floodingseason of 2014 (February–May) for a total of n = 374 individuals offour species (Serrasalmidae: P. mesopotamicus, n = 217, 14–59 cm SL;Mylossoma duriventre, n = 50, 11–21 cm SL; Myloplus tiete, n = 40,11–16 cm SL. Characidae: Brycon hilarii, n = 67, 11–36 cm SL; Fig. 2).In the Amazon, we sampled fishes in three floodplain forest habitats(river channels, islands within the river channel, and floodplainchannels) during the peak flooding (June–August 2014) for a total ofn= 285 individuals of four species (Serrasalmidae:Myloplus torquatus,n = 69, 12–28 cm SL; M. duriventre, n = 34, 15–27 cm SL. Characidae:Brycon amazonicus, n = 52, 23–35 cm SL; Brycon melanopterus, n =130, 12–27 cm SL; Fig. 2). These sample sizes exclude fishes withempty digestive tracts (n = 20 in the Pantanal and n = 6 in the Ama-zon). Fish nomenclature follows Reis et al. (2003).

2.3. Diet estimation

We separated stomach (excluding bait) and intestinal contents intofood categories (intact seeds,masticated seeds, and fruit pulp), calculat-ed the volume of each category by water displacement, and identifiedand quantified intact seeds. Intact seeds pass through the digestivesystem undamaged, and are likely viable, whereas masticated seedsare destroyed during consumption and digestion. We measured lengthand width of at least 3 intact seeds per taxa when possible.

To assess the number of species fruiting during the flooded season,we established 100m long × 10mwide transects in all sampled habitattypes: savanna (n = 3), mono-dominant (n = 3) and gallery forest(n= 4) in the Pantanal; and floodplain forest parallel (n= 5) and per-pendicular to the rivers' edge (n = 3) and along floodplain channels(n = 2) in the Amazon. Biweekly, we recorded the number of plantsper species with ripe and immature fruits. Botanical vouchers were de-posited at the Herbarium of the Universidade Federal de Mato Grosso,Cuiabá, Brazil and the Colombian Amazon Herbarium (COAH),Colombia. Plant nomenclature follows Angiosperm Phylogeny Groupand Tropicos (http://www.tropicos.org).

We tested the hypothesis that seed dispersal effectiveness increaseswith fish size within and across fish species using four metrics ofdispersal quality and quantity: (1) proportion of intact to total seedmatter (the probability of dispersing rather than destroying seeds),

Fig. 1.Map of the study sites. (a) Río Caquetá, Amazonas, Colombia, Northwestern Amazonia. Sampling was conducted between the confluence with the RíoMirití (A) and the floodplainforest below the Córdoba rapids (B). (b) Reserva Particular do Patrimônio Natural (RPPN) SESC Pantanal, Mato Grosso, Brazil, Northern Pantanal. Sampling was conducted in Riozinho(C) and the Rio Cuiabá (D). Dark green areas in (b) are monodominant forests of Vochysia divergens. Satellite images were retrieved from ESRI. (For interpretation of the references tocolor in this figure legend, the reader is referred to the web version of this article.)

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(2) number of seeds, (3) seed species richness, and (4) seed size. Wemodeled these response variables as a function of individual fish bodysize (SL), fish species, and the size by species interaction. When theinteraction was non-significant, we removed it. We analyzed thesefour response variables separately because of different statistical distri-butions, and sites separately because of differences in fish and plantspecies compositions between the Pantanal and Amazon. We adjustedprobability values for multiple tests of the same dataset (Pantanal vs.Amazon) using the Benjamin and Hochberg (1995) correction. Modelswere implemented in R (version 3.1.2).

To test if the probability of dispersing seeds increases with fish size,we calculated the proportion of seeds that were intact in the digestivetract of each fish relative to total seed matter (intact seed volume:intact + masticated seed volume), which is directly related to theprobability of seed predation (seed predation = 1 − seed dispersal).Fishes with entirely masticated seeds had values of 0, whereas fisheswith entirely intact seeds had values of 1. We limited this analysis tofisheswith seedmatter (intact ormasticated) in their digestive contents(n= 325 fishes of 4 species in the Pantanal, and 261 fishes of 4 speciesin the Amazon). We implemented zero-one inflated beta models inGAMLSS (version 4.3-1 Rigby and Stasinopoulos, 2005) with the BEINF

family to test the proportion of intact seeds as a function of fish size(SL), fish species, and the interaction. Zero-one inflated beta regressionanalyzes proportions as a mixture of Bernoulli and beta distributions,and is the only approach currently capable of accommodating propor-tional data that include values of 0 and 1, i.e., on the interval [0,1].These models simultaneously estimate 3 parameters: (1) the probabili-ty that this proportion has a value of 0 (nu); (2) the expected value forthe beta component (values between 0 and 1, mu); and (3) the proba-bility that this proportion has a value of 1 (tau). If larger fishes are betterseed dispersers, we expect negative relationships between fish size andnu (the probability that digestive contents contained 0 intact seeds),and positive relationships between fish size and mu (proportionsbetween 0 and 1) and tau (probability that digestive contents containedonly intact seeds).

We then restricted the datasets to individuals with intact seeds intheir digestive contents to assess the quantity and quality of dispersalamong fishes serving as seed dispersers (n = 229 fishes of 4 species inthe Pantanal, and 157 fishes of 4 species in the Amazon). The numberof intact seeds in fish digestive tracts was modeled with a quasipoissonregression, which fitted the data better than a Poisson model givenoverdispersion in the dataset. We analyzed seed species richness with

Fig. 2. Variation in body size across species in local assemblages of frugivorous fish species in the Pantanal (Mt—Myloplus tiete, Md—Mylossoma duriventre, Bh—Brycon hilarii, andPm—Piaractus mesopotamicus) and Amazon (Md—Mylossoma duriventre, Mtq—Myloplus torquatus, Bm—Brycon melanopterus, and Ba—Brycon amazonicus). Photographs represent thelargest adult individuals per species captured during the study, except for Piaractus mesopotamicus (largest individual: 59 cm SL) and Mylossoma duriventre in the Amazon (largestindividual: 27 cm SL). SL—standard length. Photos by S.B. Correa and J.K. Araujo.

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a Poisson regression of the number of seed species present in individualgut contents. Finally, since gape size constrains the size of the seeds thata frugivore can consume (Wheelwright, 1985), we hypothesized thatlarge fishes are unique vectors of dispersal for the biggest seeds. Wecalculated the size (length × width) of the largest intact seed in thedigestive tract of each fish to estimate the upper bound of seed sizesthat a fish can consume. Mouth gape was tightly correlated with bodylength (R2 = 0.89, F1,526 = 4092, p b 0.0001) across our 7 fish species;therefore, we used fish size as a proxy for gape size to test the hypothesisthat the size of dispersed seeds increaseswith fish size. Since large fishescan swallow both small and large seeds, heterogeneity in seed sizeincreased with fish size in our datasets; therefore, we conducted ageneralized least squares regression in which variance in seed size isproportional to fish size (Zuur et al., 2009) in the R package nlme(version 3.1-118, Pinheiro et al., 2014).

2.4. Germination experiment

To test whether fishes enhance germination, we conducted agreenhouse experiment comparing seeds removed from fish digestivetracts vs. local control seeds without fruit pulp. Removing the pulpfrom control seeds tested if (1) fish consumption increases germination

success via scarification, in which case gut-processed seeds should havea greater probability of germinating than control seeds or (2) fishconsumption influences germination simply by eliminating fruit pulp,in which case gut-processed seeds should have equivalent germinationsuccess as control seeds (Traveset et al., 2008). We randomly plantedindividual seeds of the most abundant species in fish diets at eachsite (Pantanal: 11 species, total n = 1660; Amazon: 5 species, totaln = 746; see Appendix A for additional details) into pots with 40, 60or 80mL of soil, depending on seed size. Pots were filled with commer-cial potting soil for forest plants (Pantanal) or local forest soil that wassieved to remove seeds and large debris (Amazon). Trays were housedin a screened greenhouse (50% shade) at each field site. We monitoredseeds for germination daily (Pantanal: 8 months, Amazon: 6 months)and watered as needed.

We conducted a logistic regression to test the effects of seed species,treatment (de-pulped control seeds vs. gut treatment by fish) andtheir interaction on germination success in separate analyses forthe Pantanal and Amazon datasets (Proc Logistic, SAS ver. 9.3). Owingto quasi-separation of data points, we used a penalized likelihoodmethod (Firth, 1993). A second logistic regression examinedwhether germination success of seeds processed by fish increasedwith fish size.

Fig. 3. Proportion of dispersed seeds [volume of intact to total seeds on the interval (0–1),mu] modeled as a function of fish size (SL—standard length). Pantanal: (a) Piaractusmesopotamicus. Amazon: (b) Brycon amazonicus, and (c) Myloplus torquatus.

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3. Results

We found 42938 intact seeds of 53 species in the digestive tractsof 229 individual fishes in the Pantanal and 150624 intact seeds of70 species in the digestive tracts of 163 fishes in the Amazon (seeAppendix B for additional details). Fishes dispersed 52% and 14% ofthe fleshy-fruited plant species recorded during vegetation transectsin the Pantanal and Amazon, respectively (Appendix C). However,several seed species found intact in fish guts were not observed inthe vegetation transects (Pantanal: 11 species, Amazon: 54 species;Appendix B–C), suggesting that these highly mobile fishes may haveconsumed fruits in distant wetlands (Anderson et al., 2011). A singlefish carried up to 3554 (mean seed length and width =5.5 mm × Proc Logistic, SAS 4.8 mm; fish size: 40.5 cm SL) and 8386(mean seed length and width = 4.4 mm × 2.0 mm; fish size: 23 cmSL) seeds in its digestive tract, in the Pantanal and Amazon, respectively.Fish-dispersed seeds ranged in size from 0.8 mm × 0.6 mm (Banaraarguta, Flacourtiaceae) to 40.7 mm × 19.6 mm (Couepia uiti,Chrysobalanaceae) in the Pantanal, and 0.6 mm × 0.5 mm (Ficus sp.Moraceae) to 27.1 mm × 11.9 mm (Pouteria sp., Sapotaceaceae) in theAmazon. In the Pantanal and Amazon, fishes dispersed 10 and 12 large-seeded species (N10 mmwide, Stevenson, 2011), respectively.

3.1. Seed dispersal effectiveness

Concordant with predictions, the probability of dispersing intactseeds increased with body size (see Appendix D for full model results).We observed the expected negative relationship between fish size andthe probability afish destroyed all consumed seeds (nu) for two species:a 1 cm increase in fish standard length decreased the odds of completeseedmastication (nu) by 6.6% for P. mesopotamicus in the Pantanal (95%CI: 2.6, 10.3; p adjusted for multiple tests = 0.008) and by 28.0% forB. melanopterus in the Amazon (95% CI: 9.1, 43.0; adjusted p = 0.028).Furthermore, we observed the expected positive relationship betweenfish size and mu [volume of intact to total seeds on the interval (0,1)]for three species: a 1 cm increase in fish standard length increased theodds of seed dispersal (mu) by 2.7% for P.mesopotamicus in the Pantanal(95% CI: 0.74%, 4.77%; adjusted p = 0.031; Fig. 3a) and by 58.0%for B. amazonicus in the Amazon (95% CI: 11.3%, 125.4%; adjustedp = 0.035, Fig. 3b). M. torquatus followed a similar overall pattern(Fig. 3c), but the p-value was non-significant after correction for multi-ple tests (raw p = 0.034, adjusted p = 0.081).

The abundance, species richness and size of dispersed seeds in-creasedwithfish size in bothwetlands. In the Pantanal, largerfishes dis-persed more seeds (Fig. 4a, Appendix E) irrespective of fish species(SL × species, F3,221. = 0.65, p = 0.59). In the Amazon, a 1 cm increasein standard length increased the number of intact seeds by 1.18 (95%CI: 1.02, 1.35; p=0.026) forM. torquatus (Fig. 4b), but there was no re-lationship between fish size and intact seed abundance forB. amazonicus, B. melanopterus and M. duriventre (SL × species,F3,149 = 4.76, adjusted p = 0.021). In both wetlands, the species rich-ness of dispersed seeds increased with fish size (Fig. 4c, d; AppendixE), independent of fish species (SL × species, Pantanal: F3,221 = 0.11,p=0.95; Amazon: F1,149 = 1.79, p= 0.15), as did the size of dispersedseeds (Fig. 4e, f; Appendix E; SL × species, Pantanal: F3,221 = 0.71, p =0.55; Amazon: F3,149 = 0.85, p = 0.47).

In the Pantanal, the effect of treatment (de-pulped control seeds vs.gut treatment by fish) on germination success differed across seed spe-cies (seed species × treatment:χ2= 32.96, p=0.048; Appendix F). For8 of 11 seed species, there was no difference in germination success be-tween seed treatments. For B. arguta (Flacourtiaceae), control seedswere more likely to germinate than those dispersed by B. hilarii. ForDuroia duckei (Rubiaceae) and Mouriri guianensis (Melastomataceae),control seeds had higher germination success than those dispersedby P. mesopotamicus, but not those dispersed by other fish species(Appendix F). When we analyzed the effect of fish size on germination

success of fish-ingested seeds, we found that larger fishes increasedthe odds of germination for four out of 11 seed species (Fig. 5, fishsize × seed species: χ2 = 29.95, p = 0.001; Appendix G–H), and therewas no relationship between germination success and fish size for theremaining 7 species.

In the Amazon, germination success differed between seed species(χ2 = 23.04, p = 0.0001), but did not differ between seed treatment(gut passage by various fish species vs. control seeds: χ2 = 5.07,p = 0.17). Nor did we find evidence for a seed species by treatmentinteraction (χ2 = 4.79, p = 0.44; Appendix F); therefore, in theAmazon, fishes neither enhance nor depress seed germination successrelative to seeds removed from fruit pulp manually. However, whenwe assessed the effect of fish size on germination of fish-ingested

Fig. 4. Abundance, diversity and maximum size of intact seeds dispersed by fishes in two Neotropical wetlands (Pantanal: a, c, e; Amazon: b, d, f) modeled as a function of fish size(SL—standard length). p values were corrected for multiple comparisons per data set. Black bars represent the range of body sizes per species of individuals included in the analyses.Species codes follow those in Fig. 2.

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seeds, we found that a 1 cm increase in standard length enhanced theodds of germination by 16.7% (95% CI: 4–31%, χ2 = 6.9, p = 0.009;Fig. 6). This effect held across seed species, as we found no significantinteraction between fish size and seed species (fish size × seed species:χ2 = 5.92, p = 0.21).

4. Discussion

Seed dispersal effectiveness increased with fish body size in twodistinct regions with different flora, fauna and landscape features. Atany given time, fishes had access to multiple species of fruits of variablemorphologies (Appendix B). In addition, the species composition of

fruits changed across the season in both regions; fruits available to fish-es captured early in the season were different from those available tofishes captured late in the season (Correa et al., in prep.). Despite thevariability in the diets of individual fish sampled in different habitatsat different times, we found remarkably consistent results in thePantanal and Amazon: relative to small individuals, bigger fishes masti-cated a lower proportion of the seeds they consume, instead dispersinglarger numbers of intact seeds of a greater plant diversity and of a widerrange of seed-sizes.

Furthermore, big fishes enhanced germination success acrossmultiple seed species. Frugivores influence the probability of seedgermination by removing the pulp and/or scarifying the seed coat

Fig. 5.Germination probabilitymodeled as a function of fish size (SL—standard length) for four plant species dispersed byfishes in the Pantanal: (a) Banara arguta, (b) Cayaponia podantha,(c) Eugenia inundata, and (d) Passiflora cf. edulis.

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(Traveset et al., 2008). In our study, ingestion by fishes did not augmentor inhibit germination for most seed species relative to manually de-pulped seeds, suggesting that fish consumption influences germinationsimply by eliminating fruit pulp. However, germination success of

Fig. 6.Germination probabilitymodeled as a function of fish size (SL—standard length) forfive plant species dispersed by fishes in the Amazon. The five species are shown in onepanel because the fish size by seed species interaction was non-significant.

multiple seed species increased with fish size. This positive relationshipbetween germination success and fish size could be related to longerretention time in larger intestinal tracts, as intestinal length increaseswith fish body size in all of our focal fish species except M. tiete(R2=0.91, F13,444=341.4, p b 0.0001). Laboratory experiments feedingseeds of various species to fishes of different body sizes could help elu-cidate possible physical or chemical changes to seed coats in response tolonger retention times (Pollux, 2011; Traveset et al., 2008).

Effective seed dispersal enhances seedling recruitment and directlycontributes to plant community structure (Jordano, 2000; Wang andSmith, 2002). Most studies assessing seed dispersal effectiveness focuson birds and mammals (Schupp et al., 2010) as these are the classicmodel systems for endozoochorous seed dispersal (Fleming and Kress,2013). Only relatively recently have empiricists turned their attentionto quantifying seed dispersal effectiveness of frugivorous fishes.Previous studies of three of the largest frugivorous fish species in theNeotropics found that big P. mesopotamicus disperse a greater numberof intact seeds of a common palm in the Pantanal (Galetti et al., 2008),that the volume of intact seeds increased with body size forC. macropomum and P. brachypomus in the Peruvian Amazon, andpassage through guts of adult C. macropomum accelerated seed germi-nation for a common pioneer tree in Amazonian flooded forests(Anderson et al., 2009). Here, we demonstrate that these patterns,observed in a handful of population-level studies, can scale to localcommunities of frugivorous fishes containing small- and large-sized

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species. Within and across small- to large-sized fish species, largeindividuals are better seed dispersers.

4.1. Consequences of overharvesting frugivorous fishes

Big frugivores are key components of seed dispersal networks intropical wetlands because of their consumption of large numbers offruits, their long seed-retention times, extensive movement patterns,and unique ability to disperse large-seeded species (Anderson et al.,2011; Galetti et al., 2008; Kitamura, 2011; Stevenson et al., 2014;Woodward et al., 2005; Wotton and Kelly, 2012). Given that large fishspecies are the main target of commercial fishing operations (Allanet al., 2005), overexploitation of large frugivorous fishes likely has pro-found implications for the recruitment of large-seeded canopy speciesand the maintenance of diversity in wetland forests. For example, inour study, large P. mesopotamicus in the Pantanal disperse 27% moreseed species than individuals under theminimum size limit for this fish-ery (total length = 45 cm). Simulation models suggest that the loss oflarge-mammal seed dispersers from Thai forests depressed survivaland growth at multiple life stages of a canopy tree species, and reducedpopulation viability (Caughlin et al., 2015). Population declines of forestelephants in the Congo eliminated seedling recruitment for 12 speciesof elephant-dependent large seeded-trees due to increased predationof undispersed seeds (Beaune et al., 2013). Finally, inwestern Amazonia,intact forests with large-bodied primates had higher seedling diversityand better recruitment of medium- and large-seeded canopy speciesrelative to overhunted sites (Stevenson, 2011).

In our study, the probability of seed predation decreased with fishsize for P. mesopotamicus, B. melanopterus and B. amazonicus, and weobserved a non-significant trend in that direction for M. torquatus.Such size-dependent shifts in seed predation could be difficult to reverseif commercial fisheries induce evolutionary changes in frugivorous fishpopulations to smaller individuals that mature early (Palkovacs, 2011).Overfishing could change the nature of fruit–fish relationships fromprimarily mutualistic to increasingly antagonistic with negativeconsequences for plant community structure and diversity. Trees inAmazonian flooded forest typically do not form seed banks, as theirseeds germinate shortly after the water recedes (Ferreira et al., 2010).Plant communitieswithnegligible seed banks are particularly susceptibleto increased seed predation, which diminishes seedling recruitment(Vaz Ferreira et al., 2011).

Our study also demonstrates that largerfishes are capable of dispers-ing seeds of a broader range of sizes than smaller fishes (Fig. 4e, f). Gapesize limits the size of seeds a frugivore can consume and disperse(Wheelwright, 1985), which means that large primates and birdsare typically the primary vectors of dispersal for large-seeded plantspecies in terrestrial systems (Kitamura, 2011; Stevenson, 2011).P. mesopotamicus is the biggest frugivorous fishes in the Pantanal. Itcan achieve more than five times the length of reproductive adults ofM. tiete (15 cm SL), the smallest frugivorous species therein (Fig. 2).The size of the largest seed (M. guianensis: 7.6mm× 5.6mm) dispersedby M. tiete, is merely 8% of the size of the largest seed (C. uiti:40.7 mm × 19.6 mm) dispersed by P. mesopotamicus. Within assem-blages of frugivorous fishes, adults of the largest fish species dispersesuites of seed species that are unavailable to smaller individuals ofthose fish species and to adults of smaller fish species.

Mutualistic interactions between frugivorous fishes and tropicalplants date back to the Late Cretaceous (~70 Ma) in South America(Correa et al., 2015; Thompson et al., 2014), which coincides with theradiation of angiosperms (Berendse and Scheffer, 2009), and pre-datesmost bird- and mammal–fruit interactions (Correa et al., 2015).Knowledge of how seed dispersal services of fishes compare withthose of non-aquatic frugivores in wetlands is rather limited, althoughobservations from one fish species in the Pantanal suggest that fishesdisperse a different suite of species (Donatti et al., 2011). If indeed,fishes, birds and mammals disperse different seed species, the loss of

one or several key frugivorous fishes may not be mitigated by theremaining frugivore groups (e.g., Effiom et al., 2014).

4.2. Conclusions

Through a comprehensive multi-system, multi-species study of therole of fish size on seed dispersal, we documented that the quality andquantity of seed dispersal increases with body size within and acrossfish species. Larger individuals disperse a greater number of viableseeds of a higher diversity of plants, and are unique vectors of dispersalfor big seeds. As fishes of varying size classes differ in their seed dispers-al services, we predict that small fisheswill not be able tomaintain seeddispersal in wetlands if populations of large fishes continue to decline.Future empirical studies on recruitment dynamics and spatial aggrega-tion of fish-dispersed seed species in areas with intact fish populationsvs. overfished areas should be conducted to test the consequences ofseed dispersal limitation for plant population viability (e.g., Caughlinet al., 2015). Overexploitation of fruit-eating fishes could disrupt an an-cient mutualism, potentially leading to declines in plant recruitmentsuccess and diversity in wetlands that rely on fruit-eating fishes. Toconserve wetland plant communities, we need to protect not justthe habitat, but the interactions that occur within that habitat. Plantspecies composition could change dramatically in floodplain forests ifoverfishing removes the fishes that provide the greatest seed dispersalquantity and quality. Although minimum legal capture sizes havebeen established to preserve reproductive potential, this managementstrategy does not consider the role of these fishes in wetland plantregeneration. A scientifically robust management plan that preserveslarger individuals, such as maximum size thresholds (e.g., Pierce,2010), can help safeguard a key ecosystem process.

Acknowledgments

We thank Jessika Sanabria, Tafnys Hadassa, Érika de-Faria, Pedrode-Anunciaçao, Ademar, Rodrigo Brandāo, Lázaro Ramos, AndersonAlvarenga, Ivo Brandāo, Oilton de-Moraes, Márcio Correa, JhonPatarroyo, Margarita Roa, Jarvis Rodriguez and Benedicto Neira for fieldassistance. We thank Fernando Barbosa, Helio Ferreira, Norida Canchalaand Dairon Cárdenas for plant identifications and Ivonne Vargas forseed identifications.We thank Tainá F.D. Rodrigues (Universidade Feder-al de Mato Grosso) for preparing the map. We thank Seth Wenger,Thomas Pendergast, Mauro Galetti, and two anonymous reviewers forvaluable comments on a previous draft. Permits for this research weregranted by the Chico Mendes Institute of Biodiversity Conservation,Brazil (License# 42085-1) and the National Authority of EnvironmentalLicenses, Colombia (Act# 1177 to Universidad de los Andes). We thankthe Eppley Family Foundation, SESC Pantanal, the University of SouthCarolina, and the University of Georgia for support of this research.Equipment donated by IdeaWild (to S.B.C.) was used in this research.

Appendix A. Supplementary data

Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.biocon.2015.06.019.

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