Establishment of Tree Seedlings in the Understory of Restoration Plantations: Natural Regeneration and Enrichment Plantings Maria Isabel F

RESEARCH ARTICLE Establishment of tree seedlings in the understory of restoration plantations: natural regeneration and enrichment plantings Maria Isabel F. Bertacchi1, Nino T. Amazonas2, Pedro H. S. Brancalion2,3, Gilvano E. Brondani4, Anderson C. S. de Oliveira5, Marcelino A. R. de Pascoa5, Ricardo R. Rodrigues1

Little is known about the potential of restoration plantations to provide appropriate understory conditions to support the establishment of seeds arriving from neighboring native forests. In this article, we investigated how seedling establishment is affected in the understory of restoration sites of different ages and assessed some of the potential environmental factors controlling this ecological process. We first compared the density and richness of native tree seedlings among 10-, 22-,and 55-year-old restoration plantations within the Atlantic Forest region of southeastern Brazil. Then, we undertook a seed addition experiment in each study site, during the wet season, and compared seedling emergence, survival, and biomass on local versus old-growth forest soil (transferred from a reference ecosystem), in order to test whether local substrate could hamper seedling establishment. As expected, the oldest restoration site had higher density and richness of spontaneously regenerating seedlings. However, seedling establishment was less successful both in the oldest restoration planting and using substrate transferred from a reference ecosystem, where emergence and survival were lower, but surviving seedlings grew better. We attribute these results to lower light availability for seedlings in the understory of the oldest site and speculate that higher incidence of pathogens on old-growth forest soil may have increased seedling mortality. We conclude that the understory of young restoration plantations provides suitable microsite conditions at the early establishment phases for the spontaneous regeneration or enrichment planting of native trees. Key words: direct seeding, forest regeneration, light limitation, microsite limitation, seed addition, tropical forest restoration

agriculture—a classic “biodiversity unfriendly landscape” Implications for Practice (Melo et al. 2013). These ecological restrictions hamper the • Enrichment plantings in seasonal semideciduous tropical potential of the landscape to foster the recolonization of a large forests are more likely to succeed if implemented during array of native species within any areas targeted for ecological early stages of forest restoration or in small gaps, when restoration, either through barriers to seed dispersal or by higher canopy openness can enhance seedling establish- limiting seedling establishment (Brancalion et al. 2013; Reid & ment and growth, and seedling mortality is lower. Holl 2013). • When needed, enrichment plantings in young restoration The lack of nearby seed sources has been considered as a plantations can help reduce restoration costs due to low limiting factor to seedling recruitment in various restoration seedling mortality under shade conditions. projects across tropical regions (Donath et al. 2003; Elmars- • Substrate and light conditions of young restoration plan- dottir et al. 2003; White et al. 2004; Reid & Holl 2013). tations are suitable for direct seeding of canopy species,

so translocation of non-local topsoil is not necessary. Author contributions: RR, PB designed the research; MB performed the experiments; GB, AO, MP, PB analyzed the data; NA, PB wrote, edited, and reviewed most of the manuscript.

1Departamento de Ciências Biológicas, Escola Superior de Agricultura “Luiz de Introduction Queiroz”, Universidade de São Paulo, Piracicaba, SP 13418-900, Brazil 2Departamento de Ciências Florestais, Escola Superior de Agricultura “Luiz de In several regions of the tropics, landscapes are highly frag- Queiroz”, Universidade de São Paulo, Piracicaba, SP 13418-900, Brazil mented and have low forest cover, thus hampering biodiversity 3Address correspondence to P. H. S. Brancalion, email [email protected] 4Departamento de Engenharia Florestal, Universidade Federal do Mato Grosso, conservation and the provision of ecosystem services (Wright Cuiabá, MT 78060-900, Brazil 2005; FAO 2010; Hansen et al. 2013). Such landscapes typically 5Departamento de Estatística, Universidade Federal do Mato Grosso, Cuiabá, MT contain natural forest remnants that have become biologically 78060-900, Brazil impoverished after a historical process of fragmentation, © 2015 Society for Ecological Restoration degradation, and massive defaunation. These fragments are doi: 10.1111/rec.12290 Supporting information at: commonly embedded in a harsh matrix dominated by intensive http://onlinelibrary.wiley.com/doi/10.1111/rec.12290/suppinfo

100 Restoration Ecology Vol. 24, No. 1, pp. 100–108 January 2016 Seedling establishment in restoration plantations

Some restoration techniques such as the establishment of bat Such lack of knowledge on the factors controlling the estab- roosts (Reid et al. 2013), the use of fruit essential oils to lishment of native seedlings in the understory of restoration attract dispersers (Bianconi et al. 2012), the reintroduction of plantations may constrain sowing seeds and transplanting animal-dispersed trees (Lindell et al. 2013), applied nucleation seedlings and other strategies to foster the increase of taxo- methods (Zahawi et al. 2013), and the high diversity plantations nomic and functional diversity of restoration projects embedded of native species (Rodrigues et al. 2009) have been evaluated in landscapes with dispersal limitation. Enrichment plantings regarding the increase in the amount and diversity of seed rain of areas undergoing restoration are usually done by direct over restoration sites in highly fragmented tropical landscapes. seeding or seedling plantation. On one hand, planting seedlings Overall, it is expected that, if restoration interventions succeed, is more costly, but leads to higher survival and growth rates seed arrival and the diversity of species dispersed to restora- (Fields-Johnson et al. 2010). On the other hand, direct sowing tion sites will increase with time (Reid et al. 2015). With the is cheaper (Farlee 2013), results in seedlings with better root temporal development of vegetation in restoration sites, seed system (Zadworny et al. 2014), and allows estimating the suit- dispersers may be more attracted as a consequence of the higher ability of microsite conditions for seeds arrived through natural availability of nesting places, refugees from predators, and food, dispersal. Doubts regarding the appropriate moment to imple- especially fleshy fruits (Wunderle 1997; Munro et al. 2011; Cat- ment enrichment plantings through direct seeding, the substrate terall et al. 2012). Many of the planted trees may also become conditions to sown native seeds, and the potential strategies reproductive some time after plantation, representing an addi- to favor reintroduced individuals may hamper the adoption tional seed source to the regeneration of seedlings in the under- and effectiveness of enrichment plantings as an adaptive story (Brancalion et al. 2010). management intervention to improve restoration trajectory. However, one cannot assume seed arrival to be a predictor of Here, we sought to investigate how the structural develop- regeneration success, because dispersed seeds must overcome ment and soil conditions of restoration plantations with dif- many ecological filters in order to establish (Reid & Holl 2013). ferent ages influence the establishment of seedlings in their Microsite conditions for seedling establishment may even be understory through changes in microsite conditions. Our main harsher in sites where the plantation of native tree species is assumption is that older restoration plantations have undergone improvements in microsite conditions for seedling establish- the main restoration method, since plantation is typically used ment. Considering this and the cumulative effect of seed arrival, within sites of intense degradation and reduced resilience, as as more individuals/species reach reproductive age with time, a consequence of historical land use for agriculture (Holl & we hypothesize that: Aide 2011; Rodrigues et al. 2011). Commonly, restoration plantations have been implemented in soils highly modified by agri- 1. The regeneration community, formed by newly established culture, which are treated only at the tree-planting holes/lines seedlings resulted from seeds produced by planted trees via fertilization and localized decompaction to provide suitable or arriving from neighboring remnants, is more dense and microsite conditions for the planted seedlings. This localized species-rich in older restoration plantations. soil amelioration by forestry techniques may well be enough 2. Seedling establishment is more successful in older restoration for fostering the initial growth of planted trees (Campoe et al. plantations. 2010), but the regeneration of seedlings within the understory 3. Seedling establishment is favored by environmental condi- of these plantations may be hampered by unsuitable microsite tions typical of older restoration plantations (closed canopy, conditions. Consequently, restoration plantations may not pro- higher soil organic matter content, and higher soil nutrient vide suitable microsite conditions to seedling establishment in availability). the first year following implementation. But this limitation may 4. Substrate from forests undergoing restoration prevent optimal change over time. seedling establishment, which will be more successful in the Following the gradual increase in biomass and canopy cover substrate translocated from an old-growth forest remnant. in tropical forest restoration plantations (Suganuma & Durigan 2015), nutrient cycling is enhanced (Amazonas et al. 2011), and overall environmental conditions for seedling establishment can Methods be ameliorated. However, while environmental filtering may be attenuated during succession, biotic interactions mediated by Study Sites plant pathogens and herbivores may increase their influence We performed this study in three differently aged sites under- in forest regeneration dynamics over time (Chazdon 2014). going restoration and one reference ecosystem, located in São Soil pathogens in particular have played an important role in Paulo state, southeastern Brazil (Table 1; Fig. 1). We acknowl- the regulation of tree species abundance in tropical forests edge the limitation of not having replicates for each age class, (Mangan et al. 2010). In the case of restoration plantations, which is a consequence of reduced availability of old restora- little is currently known regarding the potential of this process tion plantations in our study region. However, our study sites to modulate environmental filtering and ecological interactions have a very similar land use history and were set in simi- associated with the improvement of microsite conditions for lar conditions, which increases the reliability of their use for naturally colonizing or planted mid- to late-successional native assessing age effects in seedling establishment. The sites are species in the understory. geographically close to each other, with a 27-km maximum

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Table 1. Characteristics of study sites where the regenerating community, seedling establishment, and microsite conditions were assessed in the understory of restoration plantations in the Atlantic Forest of SE Brazil. aSistema de Informações Florestais do Estado de São Paulo (2010). bBertacchi et al. (2012).

Site 10 Years Old 22 Years Old 55 Years Old Reference Ecosystem

Location Santa Bárbara d’Oeste, SP Iracemápolis, SP Cosmópolis, SP Mombuca, SP (22∘43′S–47∘20′W) (22∘35′S–47∘31′W) (22∘39′S–47∘12′W) (22∘55′S–47∘39′W) Native forest cover of 5.0% 5.6% 10.5% 13.2% the municipalitya Area 10 ha 50 ha 9 ha 213 ha Altitude 540 m 608 m 546 m 546 m Vegetation 50-m wide forest belt 50-m wide forest belt 70-m wide riverine forest Old-growth, planted around a water planted around a water along the Jaguari River. well-preserved remnant reservoir for public reservoir for public Moderately high with dense vegetation water supply. High water supply. High species diversity (71 and a 15–25 m high species diversity species diversity native and 21 exotic). canopy, with emergent (approximately 80 (140—mostly native Random spacing and trees that reach over native species). 3 × 2m trees). 3 × 3 m spacing. grouping. Canopy 30 m in height. spacing. Canopy height: Canopy height: 20 m.b height: 20 m.b Canopy 20 m.b Canopy Canopy coverage: 84%. coverage: 92%. Dense, coverage: 52%. Almost Low-density understory diverse, and stratified no natural regeneration with many Piper spp., understory. No soil in the understory with Cestrum spp., and translocation reported. some invasive grasses. planted species No soil translocation individuals. No soil reported. translocation reported.

distance between the two most distant areas, and have a Cwa cli- The characterization revealed patterns of change for canopy mate type (Köppen 1948) and a moderately hilly relief. They are openness, soil moisture, soil chemistry, and soil density. Canopy all embedded in an agricultural matrix with very low native for- openness was nearly 50, 20, and 10% at our 10-, 22-, and est cover remaining (<10%) dominated by sugarcane fields and 55-year-old forests, respectively. Soil moisture was lowest at the pasturelands (Rodrigues et al. 2011). The sites are also isolated youngest site (12.3 ± 4%), intermediate at the 22-year-old site from natural forest remnants (apart for at least 200 m), but have (20.9 ± 7%), and highest at the oldest site (26.8 ± 5%). Soil den- some connections with young secondary natural riparian forests sity was higher in the youngest site and lower in the older sites. that are growing along streams and rivers. The present research Soil chemistry revealed that the 55-year-old forest has higher complements previous studies that used the same sites in a pH, P,Ca, Mg, and organic matter content; the 22-year-old forest chronosequence scheme (Amazonas et al. 2011; Garcia et al. has higher Al and H + Al; and the youngest forest had the high- 2014, 2015) and, associated with these other reports, allows an est K content. We paid special attention to canopy openness and integrative vision on the effects of restoration plantations in eco- soil moisture data, chemistry, and density in the discussion of logical processes. our results. More details are provided in Bertacchi et al. (2012). All of the sites are included within the original bound- aries of seasonal semideciduous forest (Veloso 1992), within Seedling Bank Assessment the Atlantic Forest biome. In this forest type, the canopy of old-growth forests is dominated by large wind-dispersed, In each site, we established 40 plots (1 × 1 m) located 4 m apart semideciduous tree species (Souza et al. 2014), and some of the from one another, aligned about 15 m parallel to water edge, most representative species were selected for the direct seeding to assess the individuals density and the number of species experiment, as further described. In the state of São Paulo, only per plot of spontaneously regenerating seedlings. We sampled 8.2% of this forest type remains, predominantly in heavily frag- all of the regenerating individuals less than 0.5 m high within mented landscapes (Rodrigues & Bononi 2008). In Brazil as a each plot, and then separated them into morphospecies. At the whole, the surviving remnants add up to just 12% of the original Iracemápolis site, we did not establish plots within portions biome domain (Ribeiro et al. 2009). of the understory dominated by the invasive species Clausena excavata Burm. f. (Rutaceae) and excluded the individuals of Microsite Characteristics this species from the analysis when they were sampled, in order to avoid any biased results. We used data from Bertacchi et al. (2012) obtained from the same plots in this study to characterize microsites. We evaluated several microsite indicators (i.e. canopy openness, litter Direct Seeding Experiment biomass, soil organic matter, density, porosity, humidity, sand, Near to the sampling plots, at each study site, we established silt, clay, pH, K, P, Ca, Mg, and Al) in order to characterize the 10 experimental blocks consisting of three 1 × 1 m subplots conditions where seedlings establish and grow in such areas. each (split-plot design), to test the following treatments: (1)

102 Restoration Ecology January 2016 Seedling establishment in restoration plantations

Figure 1. Location of study sites in São Paulo state, southeastern Brazil. direct seeding in natural substrate conditions (local substrate Myroxylon peruiferum L. f (Fabaceae, late-successional), with direct seeding); (2) direct seeding on litter and soil from Cedrela fissilis Vell. (Meliaceae, mid-successional), a nearby old-growth forest remnant of semideciduous seasonal Gallesia integrifolia (Spreng.) Harms (Phytolaccaceae, forest (old-growth forest substrate with direct seeding); and (3) early/mid-successional), and Piptadenia gonoacantha (Mart.) control (local substrate without direct seeding, to assess if any J.F. Macbr. (Fabaceae, early/mid-successional). Such species of the species sown were also spontaneously regenerating from are greatly represented in the canopy of semideciduous sea- seed rain). We collected litter and surface soil (7 cm depth) sonal forest in the study region, where most canopy trees from 30 randomly distributed 1 × 1 m plots in the reference site, have wind-dispersed, orthodox seeds, differently than in ever- and these samples were subsequently mixed in order to obtain green tropical forests, where the canopy is dominated by a composite sample. We transferred the substrate to the restora- animal-dispersed, recalcitrant seeds. We obtained seeds from tion sites and used to fill the 1.0 × 1.0 × 0.1 m plots, where soil a nursery in Piracicaba, SP, which collects seeds in the same had previously been removed to the depth of 10 cm. We also region as our sites. As the selected species do not have seed manipulated local substrate as old-growth forest substrate in dormancy, we applied no pre-treatment before sowing. We order to minimize possible interference from soil manipulation sowed 25 seeds of each species within treatments A and B on results. at the beginning of the rainy season, in December 2009, and We selected nine wind-dispersed, native tree species data collection was done until the beginning of the dry sea- that regenerate in understory conditions, but grow faster son, in June 2010, when seedling mortality is expected to be in open areas: Ceiba speciosa (A.St.-Hil.) Ravenna (Mal- higher. Each species was sown in 0.3 × 0.3–m subplots, and vaceae, early/mid-successional), Anadenanthera macro- the distribution of species in each plot was randomized. We carpa (Benth.) Brenan (Fabaceae, early/mid-successional), also evaluated the seed bank in subsamples of the soil removed Aspidosperma polyneuron Mull. Arg. (Apocynaceae, from the old-growth forest remnant, which was translocated late-successional), Handroanthus impetiginosus (Mart. Ex to the restoration sites. Seed bank was assessed in greenhouse DC.) Mattos (Bignoniaceae, late-successional), Machaerium conditions with no seeding (control) to test whether they stipitatum (DC.) Vogel (Fabaceae, early/mid-successional), had viable seeds of the nine species sown in the experiment.

January 2016 Restoration Ecology 103 Seedling establishment in restoration plantations

This experiment complements the control treatment in field seedlings, although no difference in species number per plot conditions that also assessed the presence of the targeted was found between the 10-and the 22-year-old sites. The old- species in the seed rain and soil. As no seedlings of these est site had also the highest seedling density, the 22-year-old species emerged in the tests, we assumed that all seedlings of forest had an intermediate value, and the youngest site had the the nine species found in the experimental plots resulted from lowest seedling density (Fig. 2). direct seeding. Contrary to Hypothesis 2, seedling emergence was not We collected data of seedling emergence and mortality eight different among restoration sites (p = 0.11), regardless of their times over 6 months. We made the first four evaluations at 15, age, but survival differed (p < 0.0001) and was lower at the 30, 45, and 60 days after sowing and the other four on a monthly oldest site. In general, shoot (p < 0.0001) and total biomass basis. We tagged every seedling that emerged individually and (p < 0.0001) differed among sites and were higher in seedlings located its position with a wooden stake in order to monitor that grew in the youngest restoration plantation (Table 2). mortality. At the end of the evaluation period, we assessed the Survival and growth were highly variable among species survival rate of each species. We carefully removed seedlings (least significant difference, LSD, p = 0.05, Tables S1 & S2, from the ground using a garden shovel and dried them at 80∘C Supporting Information). Contrary to Hypothesis 3, when all for 48 hours, using the oven method, to obtain root, shoot, and sites were analyzed together, we found a positive correlation total dry biomass per seedling. between canopy coverage and seedling mortality (r = 0.49; p = 0.006) and a negative correlation between canopy cover- Data Analysis age and root biomass (r =−0.71; p = 0.0001), shoot biomass (r =−0.70; p = 0.0001), and total dry biomass per seedling We used the randomized block design to evaluate richness and (r =−0.72; p = 0.0001). There was no correlation of any other density of spontaneously regenerating seedlings. Treatments parameter with litter biomass. When analyzing data for each were composed of 10-, 22-, and 50-year-old planted native site, we found no strong patterns of microsite parameters forests, 40 blocks total. To test for among sites differences in influencing seedling establishment (Table S3), which maybe seedling density and species number per plot in the seedling caused by experimental design limitations (poor replication, bank (Hypothesis 1), we used analysis of deviance (ANDEV) inadequate sampling scale, and reduced local environmental for generalized linear models (GLM) of Poisson or negative heterogeneity). binomial distributions, and applied the contrasts afterwards. Seedling emergence (p < 0.0001) and survival (p < 0.0001) For the evaluation of emergence (%), survival (%), and biomass differed between local and old-growth substrate conditions but, (shoot, root, and total biomass) of seedlings resulting from contrary to our expectations, were greater in the local substrate direct seeding in the local substrate (Hypothesis 2), we used a (Table 2). Seedling root (p = 0.69), shoot (p = 0.17), and total randomized 3 × 2 factorial experiment without interactions in (p = 0.28) were similar in both substrates within the younger which the factors were the three sites (10-, 22-, and 50-year-old restoration plantations, whilst, in the 55-year-old forest, planted native forests) and two substrates (local or old-growth seedling biomass differed between substrates (p < 0.0001) forest). For each combination of site and soil, we had 10 and was higher within the old-growth forest substrate replicates (225 seeds each). For all variables measured, we (Table 2). used analysis of variance (ANOVA) and Tukey’s post hoc test. The Kolmogorov–Smirnov test was used to test for the assumptions of normality of errors; the Lavene test was used to test for homoscedasticity; and the Durbin–Watson Discussion test was used for independence of errors. We tested the cor- The hypothesis that the regenerating community would be relation between microsite characteristics and data of the denser and species richer in the older restoration plantations direct seeding experiment via a Pearson correlation analysis was supported by our results. Although age effects may be (Hypothesis 3). biased by site-to-site variations, other work carried out in the We used the same approach to compare the parameters same region and using a higher number of plantations also mentioned above for local versus old-growth forest substrates found the same trend (Suganuma et al. 2014). The greater (Hypothesis 4). As none of the sown species emerged in any density and species number per plot of seedlings regenerating of the control plots, as a result of natural seed dispersal, we in the understory of the oldest reforestation project, in spite of did not include the control plots in the statistical analysis. the absence of differences for species density between the two Statistical analyses were done with the software R (R Core youngest sites, could be initially explained by better microsite Team 2015). conditions for seedling establishment combined with a higher seed deposition over time. In older sites, the present seedling bank may be a result of the accumulated seed deposition of the Results last years or even decades, because shade-tolerant species may As expected for spontaneously regenerating seedlings, species survive as seedlings in the forest understory for long periods, number per plot (p < 0.0001) and seedling density (p < 0.0001) until small gaps are opened to favor their growth (Kitajima differed among restoration sites. The 55-year-old forest con- & Fenner 2000). In addition, older sites may have more trees tained the highest number of species per plot of regenerating producing seeds, which may have an important contribution

104 Restoration Ecology January 2016 Seedling establishment in restoration plantations

A 16 A B 16 14 14 12 12

-2 B -2 m C 10 10 m

gs s A

lin 8 8 d ecie p

6 S 6 See B B 4 4 2 2 0 0 10-year old 22-year old 55-year old 10-year old 22-year old 55-year old

Figure 2. Mean values (±SE) of seedlings density (A) and species number per plot (B) in 10-, 22-, and 55-year-old restoration plantations in the Atlantic Forest. Mean values followed by the same letters are not different by contrast test at 5% probability.

Table 2. Comparison of seedling emergence (E, %) between local (LS) and further discussed. It is important to mention that our plots were old-growth forest substrate (OGFS) in each restoration plantations (10-, 20-, not colonized by invasive fodder grasses, a common problem and 55-year-old forests) located in the Atlantic Forest of São Paulo state, Brazil. aIn the row, mean values followed by the same capital letter, and facing young restoration plantations in Brazil (Campoe et al. in the column, mean values followed by the same lower case letter, are not 2010). Invasive species may impose additional constrains to the different by Tukey’s test at 5% probability. Data are showed as mean value establishment of native species in areas undergoing restoration (±SE). with more opened canopy, and future studies on this issue are Restoration Plantations LSa OGFS needed. Indeed, for many species, the conditions for seedling sur- Emergence (%) vival must be less harsh than those required for germination Aa Ba 10 years old 35.3 (±3.4) 20.5 (±3.6) (Turnbull et al. 2000), and microsite limitations have been found 20 years old 39.3Aa (±1.9) 27.4Ba (±1.8) to strongly determine seedling survival and growth following 55 years old 37.1Aa (±4.5) 17.4Ba (±2.9) Survival (%) emergence (Munzbergová & Herben 2005). In particular, safe 10 years old 24.0Aa (±2.6) 11.2Ba (±2.6) sites for seedling establishment in tropical forests are highly 20 years old 20.3Aa (±2.3) 11.5Ba (±1.7) influenced by light regimes in the understory (Nicotra etal. 55 years old 12.2Ab (±1.8) 3.6Bb (±0.7) 1999; Chazdon 2014). In this study, we found that seedling sur- Shoot biomass (g) vival and growth were variable among species but overall higher Aa Aa 10 years old 0.143 (±0.012) 0.164 (±0.015) in younger restoration plantations and we attribute this to higher Ab Ab 20 years old 0.094 (±0.011) 0.103 (±0.010) canopy openness in younger forests, in spite of the better nutri- 55 years old 0.065Ac (±0.008) 0.075Ac (±0.011) Root biomass (g) ent and water availability in the older restoration plantations 10 years old 0.083Aa (±0.012) 0.081Aa (±0.015) (Bertacchi et al. 2012). 20 years old 0.036Ab (±0.004) 0.041Ab (±0.004) Our results are similar to those found by other investiga- 55 years old 0.019Ab (±0.002) 0.025Ab (±0.003) tions carried out in natural remnants, where seedling survival Total biomass (g) was favored by higher canopy openness and, consequently, Aa Aa 10 years old 0.226 (±0.022) 0.246 (±0.026) light availability (LePage et al. 2000; Bellingham & Richard- Ab Ab 20 years old 0.130 (±0.015) 0.144 (±0.013) son 2006). However, survival rates may change with time as a 55 years old 0.085Ac (±0.010) 0.110Ac (±0.013) result of seasonal variations in water availability in soil, which can increase seedling mortality in sites with higher canopy openness in the dry season. In addition, the effect of canopy open- to seed rain abundance in landscapes with dispersal limitation ness in seedling survival and growth could have been differ- (Rodrigues et al. 2011). ent in species from other ecological groups, as we predomi- Although we did not assess seed rain in the studied sites, we nantly sowed light-demanding species that regenerate in less controlled for this factor via a seed addition experiment in order shaded understory conditions typical from seasonal semidecid- to assess whether the successional development of restora- uous forests. Such results have then to be considered with cau- tion plantings would improve the microsite conditions for tion, since our experiment focused on the early establishment regeneration, as was predicted by one of our hypotheses. This of light-demanding seedlings, and thus did not consider how hypothesis was not confirmed, however, as seedling emergence shade-tolerant species would perform at the same conditions, was found to be similar at all sites, survival was lower in the old- neither monitored seedlings for a longer time and throughout est reforestation project and seedling biomass was higher at the rainy and dry seasons. youngest forest. Such results were mainly driven by differences Although the species used in our experiment are typical in canopy cover and, probably, due to the possibly highest inci- canopy trees in old-growth remnants, which have a more dence of pathogens in the oldest site (Packer & Clay 2000), as shaded understory, their recruitment may be favored by higher

January 2016 Restoration Ecology 105 Seedling establishment in restoration plantations light incidence in small gaps and “gaps of deciduousness,” planting of native trees. Further studies with greater site replica- that is canopy openings created by deciduous species in the tion are necessary to draw stronger conclusions about the effects dry season, as already demonstrated (Souza et al. 2014). Such of forest age and structure on the limitations for natural recruit- ecological considerations may provide valuable guidelines ment of native trees and their use in enrichment plantings. It for enrichment plantings in semideciduous forests embedded remains to be understood if enrichment would be more success- in highly fragmented tropical landscapes, like those found in ful if seedlings were used instead of direct seeding. We also southeastern Brazil. In this region, some previous research recommend future research to investigate the causes of seedling have already reported the reversion of forests back to a grass mortality, including soil pathogens, to guide microsite manipu- state due to the senescence of the initial pioneer planted lation to favor seedling establishment in restoration sites. species, and lack of mature forest seedling recruitment (Souza & Batista 2004; Rodrigues et al. 2009). Those guidelines may include the implementation of enrichment interventions Acknowledgments in young restoration plantations—before pioneer species We thank S. Dunster for language checking, R. B. de Carvalho senescence and when the canopy is not so closed—in natural for providing the map of studied sites, three anonymous review- gaps or in gaps artificially created by thinning tree branches ers, L. Reid, and J. Aronson for their contribution in reviewing or entire individuals to favor canopy species recruitment this manuscript. M. I. F. B. thanks the São Paulo State Research in the understory, and beneath deciduous or semideciduous Foundation (FAPESP) for MSc scholarship (2009/12663-9) and canopy species. However, our results and recommendations National Council of Technological and Scientific Development may not apply to typical understory species and to evergreen (CNPq) for research grant (2013/50718-5), and R. R. R. thanks tropical forests, which have more specialist species regard- the CNPq for his research grant. ing light requirements than semideciduous forests (Chazdon et al. 2011). LITERATURE CITED Our final hypothesis, which stated that substrate from forests Amazonas NT, Martinelli LA, Piccolo MC, Rodrigues RR (2011) Nitrogen undergoing restoration prevent optimal seedling establishment dynamics during ecosystem development in tropical forest restoration. when compared to reference ecosystem substrates, was not Forest Ecology and Management 262:1551–1557 supported either. In all cases, emergence and survival were Augspurger CK (1983) Seed dispersal of the tropical tree, Platypodium elegans, observed to be greater in local versus old-growth forest sub- and the escape of its seedlings from fungal pathogens. Journal of Ecology strate. Such results may be a consequence of higher seedling 71:759–771 mortality in the topsoil from the old-growth natural forest. Basu S, Behera N (1993) The effects of tropical soil conversion on soil microbial biomass. Biology and Fertility of Soils 16:302–304 It is well-known that higher soil organic matter content, as Bell T, Freckleton RP, Lewis OT (2006) Plant pathogens drive density-dependent observed in old-growth forests, increases microbial biomass seedling mortality in a tropical tree. Ecology Letters 9:569–574 and activity (Basu & Behera 1993; Phasad et al. 1994; Har- Bellingham PJ, Richardson SJ (2006) Tree seedling growth and survival over ris 2009). Soil microorganisms play an important role in trop- 6 years across different microsites in a temperate rain forest. Canadian ical forest communities, promoting organic matter decompo- Journal of Forest Research 36:910–918 sition and nutrient cycling, although they may be responsible Bertacchi MIF, Brancalion PHS, Brondani GE, Medeiros JC, Rodrigues RR (2012) Caracterização das condições de microssítio de áreas em restau- for much of the mortality rate of seedlings in tropical forests ração com diferentes idades. Revista Árvore 36:895–905 (Bell et al. 2006; Mangan et al. 2010; Chanthorn et al. 2013). Bianconi GV, Suckow U, Cruz-Neto AP, Mikich SB (2012) Use of fruit essential Pathogen development is favored by low levels of solar radi- oils to assist forest regeneration by bats. Restoration Ecology 20:211–217 ation and high soil moisture, as found at our oldest restoration Brancalion PHS, Melo FPL, Tabarelli M, Rodrigues RR (2013) Restora- site. As a result, seedling mortality from pathogens is likely to be tion reserves as biodiversity safeguards in human-modified landscapes. greater in areas beneath more closed canopies than in forest gaps Natureza e Conservação 11:1–5 Brancalion PHS, Rodrigues RR, Gandolfi S, Kageyama PY, Nave AG, Gandara (Augspurger 1983). Despite higher mortality, seedlings that sur- FB, Barbosa LM, Tabarelli M (2010) Instrumentos legais contribuem para a vived on old-growth forest substrate in the oldest restoration restauração de florestas tropicais biodiversas. Revista Árvore 34:455–470 plantation produced more biomass, probably because of better Campoe OC, Stape JL, Mendes JCT (2010) Can intensive management accelerate soil nutrient and water availability. Little is known, however, the restoration of Brazil’s Atlantic forests? Forest Ecology and Manage- regarding the intricate interaction of soil pathogens and plants ment 259:1808–1814 in restoration sites, which requires further investigations in order Catterall CP, Freeman AND, Kanowski J, Freebody K (2012) Can active restoration of tropical rainforest rescue biodiversity? A case with bird community to shed light onto the influence of microorganisms on restora- indicators. Biological Conservation 146:53–61 tion trajectory (Harris 2009). However, our results indicate that Chanthorn W, Caughlin T, Dechkla S, Brockelman WY (2013) The relative the local substrate of restoration plantations may not hamper importance of fungal infection, conspecific density and environmental the establishment of native tree species in the understory, thus heterogeneity for seedling survival in a dominant tropical tree. Biotropica enrichment plantings may be implemented without concerns 45:587–593 about it. Chazdon RL (2014) Second growth: the promise of tropical forest regeneration in an age of deforestation. University of Chicago Press, Chicago, Illinois We conclude that the understory of young restoration planta- Chazdon RL, Chao A, Colwell RK, Lin SY, Norden N, Letcher SG, Clark DB, tions provides suitable microsite conditions at the early estab- Finegan B, Arroyo JP (2011) A novel statistical method for classifying lishment phases for spontaneous regeneration or enrichment habitat generalists and specialists. Ecology 92:1332–1343

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Donath T, Holzel N, Otte A (2003) The impact of site conditions and seed R Core Team (2015) R: a language and environment for statistical com- dispersal on restoration success in alluvial meadows. Applied Vegetation puting. R Foundation for Statistical Computing, Vienna, Austria. Science 6:13–22 http://www.R-project.org/ (accessed 15 Feb 2015) Elmarsdottir A, Aradottir AL, Trlica MJ (2003) Microsite availability and estab- Reid JL, Holl KD (2013) Arrival≠survival. Restoration Ecology 21:153–155 lishment of native species on degraded and reclaimed sites. Journal of Reid JL, Holl KD, Zahawi RA (2015) Seed dispersal limitations shift over time Applied Ecology 40:815–823 in tropical forest restoration. Ecological Applications 25:1072–1082 FAO (2010) Global forest resources assessment 2010. Food and Agricultural Reid JL, Holste EK, Zahawi RA (2013) Artificial bat roosts did not accelerate for- Organization of the United Nations, Rome, Italy est regeneration in abandoned pastures in southern Costa Rica. Biological Farlee LD (2013) Direct seeding of fine hardwood tree species. Pages 31–47. In: Conservation 167:9–16 Van Sambeek JW, Jackson EA, Coggeshall MV,Thomas AL, Mischler CH. Ribeiro MC, Metzger JP, Martsen AC, Ponzoni FJ, Hirota MM (2009) The (eds) Managing fine hardwoods after a half century of research. Proceed- Brazilian Atlantic Forest: how much is left, and how is the remaining ings of the Seventh Walnut Council Research Symposium. Department of forest distributed? Implications for conservation. Biological Conservation Agriculture, Forest Service, Northern Research Station, Pennsylvania 142:1141–1153 Fields-Johnson C, Burger JA, Evans DM, Zipper CE (2010) Response of Rodrigues RR, Bononi VLR (2008) Diretrizes para a conservação e restauração improved American chestnuts to planting practices on reclaimed surface da biodiversidade no estado de São Paulo. Instituto de Botânica, São Paulo, mined land. Pages 319–336. In: Barnhisel RI (ed) Proceedings of 27th Brazil Meeting of the American Society of Mining Reclamation. Pittsburgh, Rodrigues RR, Gandolfi S, Nave AG, Aronson J, Barreto TE, Vidal CY, Bran- Pennsylvania calion PHS (2011) Large-scale ecological restoration of high-diversity Garcia LC, Cianciaruso MV, Ribeiro DB, Santos FAM, Rodrigues RR (2015) tropical forests in SE Brazil. Forest Ecology and Management Flower functional trait responses to restoration time. Applied Vegetation 261:1605–1613 Science 18:402–412 Rodrigues RR, Lima RAF, Gandolfi S, Nave A (2009) On the restoration of high Garcia LC, Hobbs RJ, Santos FAM, Rodrigues RR (2014) Flower and fruit diversity forests: 30 years of experience in the Brazilian Atlantic Forest. availability along a forest restoration gradient. Biotropica 46:114–123 Biological Conservation 142:1242–1251 Hansen M, Potapov P, Moore R, Hancher M, Turubanova S, Tyukavina A, Thau Sistema de Informações Florestais do Estado de São Paulo (2010) Inventário D, Stehman S, Goetz S, Loveland T (2013) High-resolution global maps Florestal. http://www.iflorestal.sp.gov.br/sifesp/ (accessed 21 Jan 2014) of 21st-century forest cover change. Science 342:850–853 Souza FM, Batista JLF (2004) Restoration of seasonal semideciduous forests in Harris J (2009) Soil microbial communities and restoration ecology: facilitators Brazil: influence of age and restoration design on forest structure. Forest or followers? Science 325:573–574 Ecology and Management 191:185–200 Holl KD, Aide TM (2011) When and where to actively restore ecosystems? Souza FM, Gandolfi S, Rodrigues RR (2014) Deciduousness influences the Forest Ecology and Management 261:1558–1563 understory community in a semideciduous tropical forest. Biotropica Kitajima K, Fenner M (2000) Ecology of seedlings regeneration. Pages 331–360. 46:512–515 In: Fenner M (ed) Seeds: the ecology of regeneration in plant communities. Suganuma MS, Assis GB, Durigan G (2014) Changes in plant species compo- Commonwealth Agricultural Bureau International, Wallingford, United sition and functional traits along the successional trajectory of a restored Kingdom patch of Atlantic Forest. Community Ecology 15:27–36 Köppen W (1948) Climatologia: con un estudio de los climas de la tierra. Fondo Suganuma MS, Durigan G (2015) Indicators of restoration success in riparian de Cultura Econômica. México. tropical forests using multiple reference ecosystems. Restoration Ecology LePage PT, Canham CD, Coates KD, Bartemucci P (2000) Seed abundance 23:238–251 versus substrate limitation of seedling recruitment in northern temperate Turnbull LA, Crawley MJ, Rees M (2000) Are plant populations seed-limited? forest of British Columbia. Canadian Journal of Forest Research 30: A review of seed sowing experiments. Oikos 88:225–238 415–427 Veloso HP (1992) Manual técnico da vegetação brasileira. IBGE, Rio de Janeiro, Lindell CA, Reid JL, Cole RJ (2013) Planting design effects on avian seed Brazil dispersers in a tropical forest restoration experiment. Restoration Ecology White E, Tucker N, Meyers N, Wilson J (2004) Seed dispersal to revegetated 21:515–522 isolated rainforest patches in North Queensland. Forest Ecology and Man- Mangan SA, Schnitzer SA, Herre SA, Mack KML, Valencia MC, agement 192:409–426 Sanchez EI, Beyer JD (2010) Negative plant–soil feedback pre- Wright SJ (2005) Tropical forests in a changing environment. Trends in Ecology dicts tree-species relative abundance in a tropical forest. Nature 466: and Evolution 20:553–560 752–755 Wunderle JM Jr (1997) The role of animal seed dispersal in accelerating native Melo FP, Arroyo-Rodríguez V, Fahrig V, Martínez-Ramos M, Tabarelli M forest regeneration on degraded tropical lands. Forest Ecology and Man- (2013) On the hope for biodiversity-friendly tropical landscapes. Trends agement 99:223–235 in Ecology & Evolution 28:462–468 Zadworny M, Jagodzinski´ AM, Łakomy P, Ufnalski K, Oleksyn J (2014) The Munro NT, Fischer J, Barrett G, Wood J, Leavesley A, Lindenmayer DB (2011) silent shareholder in deterioration of oak growth: common planting prac- Bird’s response to revegetation of different structure and floristics—are tices affect the long-term response of oaks to periodic drought. Forest “restoration plantings” restoring bird communities? Restoration Ecology Ecology and Management 318:133–141 19:223–235 Zahawi RA, Holl KD, Cole RJ, Reid JL (2013) Testing applied nucleation as a Munzbergová Z, Herben T (2005) Seed, dispersal, micro-site, habitat and recruit- strategy to facilitate tropical forest recovery. Journal of Applied Ecology ment limitation: identification of terms and concepts in studies of limita- 50:88–96 tions. Oecologia 145:1–8 Nicotra AB, Chazdon RL, Iriarte SV (1999) Spatial heterogeneity of light and woody seedling regeneration in tropical wet forests. Ecology 80:1908–1926 Supporting Information Packer A, Clay K (2000) Soil pathogens and spatial patterns of seedling mortality The following information may be found in the online version of this article: in a temperate tree. Nature 404:278–281 Phasad P, Basu S, Behera N (1994) A comparative account of the microbiological Table S1. Seedling survival (%) of species grown on local (LS) versus old-growth characteristics of soils under natural forest, grassland and crop field from forest substrate (OGFS) in each restoration plantation (10-, 20-, and 55-year-old Eastern India. Plant and Soil 175:85–91 forests) located in the Atlantic Forest of São Paulo state, Brazil.

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Table S2. Total dry biomass per seedling (g) for species grown on local (LS) Mg, and Al) and data of emergence, mortality, root biomass (RB), aerial biomass (AB), versus old-growth forest substrate (OGFS) in each restoration plantation (10-, 20-, and total biomass (TB), and final number of seedlings (FNS) from data collected in the 10-, 55-year-old forests) located in the Atlantic Forest of São Paulo state, Brazil. 22-, and 55-year-old study sites. Table S3. Pearson correlation between microsite attributes (canopy coverage, litter biomass, soil organic matter, density, porosity, humidity, sand, silt, clay, pH, K, P, Ca,

Coordinating Editor: James Aronson Received: 21 July, 2014; First decision: 20 August, 2014; Revised: 3 September, 2015; Accepted: 4 September, 2015; First published online: 21 October, 2015

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