DOI: 10.5061/dryad.cfxpnvxjp
title: Data from: Lower-intensity restoration interventions drive greater seedling establishment for later-successional tree species

description: Recovery of tree community composition in restored tropical forests relies
 on successful recruitment of later-successional species. However, the
 long-term effects of different restoration interventions on establishment
 success of arriving seeds are poorly understood. We evaluated the
 effects of three restoration treatments on the seed-to-seedling transition
 for later-successional tree species in a fragmented agricultural landscape
 in southern Costa Rica. Restoration plots (0.25 ha) were established in a
 block design nearly two decades prior and spanned a gradient of
 intervention intensity: natural regeneration (not planted), applied
 nucleation (planted tree clusters), and plantation (fully planted). We
 conducted seed addition experiments from 2021-2023 using eight species at
 seven replicate restoration sites and in four nearby remnant forests.
description: <strong><em>Study
 region</em></strong> We conducted this
 study from June 2021 to September 2023 in southern Costa Rica near Las
 Cruces Biological Station. The forests in this region are at the boundary
 between Tropical Premontane Wet and Rain Forest zones (Holdridge et al.,
 1971), and the study sites range from ~1100 to 1200 m in elevation. Soils
 are volcanic in origin, mildly acidic (pH ~5.5), low in P, and high in
 organic matter. Mean annual temperature is ~21°C, and annual precipitation
 is between 2700 and 5000 mm, with a dry season from December to March. The
 landscape was largely deforested between 1960 and 1980 and is now a
 fragmented mosaic of remnant forest, secondary forest, cattle pastures and
 agricultural fields. <strong><em>Design of
 long-term restoration experiment</em></strong>
 We used seven restoration sites set up in 2004-2006 (Figure 1), a
 subset of the 15 sites described in Holl et al. (2020). All sites were
 previously used for ≥ 18 years for grazing or coffee production, and most
 are steeply sloped (15-35º). Minimum distance between sites is 700 m. Each
 restoration site had three 50 × 50 m (0.25 ha) plots: natural
 regeneration, applied nucleation, and plantation. Natural regeneration
 plots were not planted, whereas applied nucleation and plantation plots
 were planted with seedlings of four tree species: <em>Erythrina
 poeppigiana </em>(Walp.) Skeels<em>, Inga</em>
 <em>edulis </em>Mart.<em>, Terminalia amazonia
 </em>(J.F. Gmel.) Exell<em>, </em>and <em>Vochysia
 guatemalensis</em> Donn. Sm.<em> E. poeppigiana
 </em>and<em> I. edulis </em>are naturalized,
 fast-growing N-fixers commonly used in agricultural intercropping, whereas
 <em>T. amazonia</em> and <em>V. guatemalensis</em>
 are native timber species. In applied nucleation plots trees were planted
 in six nuclei or patches, two each of 4 × 4, 8 × 8, and 12 × 12 m, whereas
 plantation plots<em> </em>were<em> </em>planted
 uniformly throughout; the number of trees planted in applied nucleation
 plots was 27% of the total in plantation plots. Four sites also have
 paired remnant forest (2 to &gt;300 ha) located 5-50 m from
 restoration plots, which serve as a reference system for comparing the
 conditions and ecological processes of restoration plots. On average,
 percent canopy cover was lower and cover of exotic pasture grasses was
 much greater in natural regeneration than planted treatments (Kulikowski
 et al., 2023). <strong><em>Seed addition
 experiment</em></strong> We selected eight
 tree species (Table 1; Figure S1) for seed addition experiments, based on:
 (a) their later-successional status, categorized by expert opinion as
 occurring exclusively in late-successional forest or both early- and
 late-successional forest (Schubert et al., 2025); (b) a continuum of seed
 sizes; and (c) regional availability of seeds (&gt;700) for the
 experiment. Study species were all animal-dispersed and ranged from 2.4 to
 24.8 mm seed width. We collected seeds from at least
 three mother trees per species, encountered in conditions that differed by
 species: (1) mature fruits/nuts from the ground (<em>Pseudolmedia
 mollis</em>, <em>Quercus benthamii</em>,
 <em>Trophis mexicana</em>, and some <em>Erythroxylum
 macrophyllum </em>and<em> Otoba
 novogranatensis</em>),<em> </em>(2) seeds with pulp
 already removed by birds from the ground (<em>Ocotea</em>
 <em>puberula</em> and some <em>O.
 novogranatensis</em>), and (3) ripe fruit directly from trees
 (<em>Lacistema aggregatum</em>, <em>Palicourea
 padifolia</em>, some <em>E.
 macrophyllum</em>).<em> </em>We manually removed any
 fruit pulp from seeds and briefly submerged all seeds in water to check
 for insect damage. Seeds that floated were assumed to be non-viable and
 not used. We measured fresh mass and width for ≥50 seeds per species
 (Table 1; Figure S1). We thoroughly mixed conspecific seeds to avoid bias
 in seed source or quality among sites or habitat types (hereafter
 ‘treatments’). We conducted concurrent germination trials to confirm that
 no species completely failed to germinate in a nursery setting.
 Within each restoration plot and remnant forest, we established
 four sets (hereafter ‘stations’) of three 1-m<sup>2</sup>
 quadrats separated by ≥15 m. In natural regeneration, plantation, and
 reference forest plots, one station was located in each quarter of the
 plot (Figure 2a). In applied nucleation plots, stations were
 systematically distributed with one station at each of four positions
 relative to the initial planting design (Figure 2b): (1) within a large
 nucleus; (2) at the edge of a medium nucleus; (3) between two nuclei, and
 (4) far from any nucleus. Nuclei have spread considerably beyond the
 planted area due to crown expansion and natural recruitment; however,
 these positions spanned a representative range of microsite conditions.
 Two quadrats per station were used for seed additions to accommodate all
 eight species, and one non-sown control quadrat was surveyed to assess
 background abundance of naturally recruited seedlings of the study species
 (Figure 2c). Each control quadrat was located 2 m from a seed addition
 quadrat and on the uphill side if on a slope. We placed quadrats to avoid
 large tree trunks but did not remove existing vegetation or leaf
 litter. At each station, we sequentially added seeds of
 three species (<em>P. mollis</em>, <em>O.
 novogranatensis</em>, and <em>O. puberula</em>) to one
 quadrat (26 total seeds m⁻²) and five species (<em>Q. benthamii, E.
 macrophyllum, L. aggregatum, T. mexicana, </em>and<em> P.
 padifolia)</em> to the second quadrat (47 total seeds m⁻²) as seeds
 of each species became available between July 2021 and August 2022. The
 number of seeds added per quadrat ranged from 7 to 11 seeds per species
 (Table 1) because of varying seed availability. Realized densities of
 total added individuals were lower than the cumulative densities because
 of (a) temporal staggering of additions by species and (b) seed mortality.
 We placed seeds on top of the litter or soil to simulate how seeds would
 naturally be deposited from primary dispersal, slightly pressing down to
 minimize rolling on steeper slopes. We secured a roll of fine mesh ~5 cm
 high on the downhill side of each seed addition quadrat to catch seeds
 washed downslope by runoff. Seeds were placed in rows with 10-20 cm
 minimum spacing and assigned an identifier based on grid position. For the
 five smallest-seeded species, locations were marked with popsicle sticks
 to facilitate monitoring. We monitored seedling
 emergence, survival, and height (to nearest 0.5 cm) for each species at
 least four times. Census intervals were timed to: (a) to capture seedling
 emergence approximately 1-2 months post seed addition and every 2-4 months
 thereafter, and (b) measure the first-year survival percentage of emerged
 seedlings 12-15 months after seed addition. Because some seedlings may
 have emerged and died between censuses or prior to the first census, we
 may have underestimated emergence and overestimated seedling survival.
 This uncertainty was mitigated by our observations of obviously dead seeds
 (indicating failure to emerge) or dead seedlings (indicating emergence but
 subsequent mortality). For the four larger-seeded species for which
 ungerminated seeds could be reliably re-found, missing seeds were
 interpreted as pre-emergence mortality. Assuming that missing seeds were
 predated was reasonable because a companion study found that almost all
 seeds removed by vertebrates were eventually predated (Joyce et al.,
 2024). Variable durations of the “first year” period are typical in
 tropical tree seed addition experiments involving multiple species with
 differing phenologies (e.g. Svenning & Wright, 2005). One of the
 seven sites where we added seeds in 2021 was then cleared by the
 landowner, so in 2022 we added seeds of the remaining six species to an
 alternate site. The other six sites were used for the duration of the
 study. Accordingly, <em>P. mollis</em> and <em>Q.
 benthamii, </em>were tested at six sites rather than
 seven. We did not tag seedlings but were able to
 distinguish between natural recruits and seedlings that recruited from
 added seeds based on the location of seedlings relative to the grid and
 the size of seedlings compared to the experimental cohort. We sampled the
 non-sown control quadrat at each station at a single timepoint (July 2023)
 for seedlings similarly sized to our added individuals.
 <strong><em>References</em></strong> Holdridge, L. R., Grenke, W. C., Hatheway, W. H., Liany, T., & Tosi Jr, J. A. (1971). <em>Forest environments in tropical life zones: A pilot study</em>. Pergamon Press. Holl, K. D., Reid, J. L., Cole, R. J., Oviedo‐Brenes, F., Rosales, J. A., & Zahawi, R. A. (2020). Applied nucleation facilitates tropical forest recovery: Lessons learned from a 15-year study. <em>Journal of Applied Ecology</em>, <em>57</em>(12). https://doi.org/10.1111/1365-2664.13684 Joyce, F. H., Ramos, B. M., Zahawi, R. A., & Holl, K. D. (2024). Vertebrate seed predation can limit recruitment of later-successional species in tropical forest restoration. <em>Biotropica</em>, <em>56</em>(6), e13381. https://doi.org/10.1111/btp.13381 Kulikowski, A. J., Zahawi, R. A., Werden, L. K., Zhu, K., & Holl, K. D. (2023). Restoration interventions mediate tropical tree recruitment dynamics over time. <em>Philosophical Transactions of the Royal Society B: Biological Sciences</em>, <em>378</em>(1867), 20210077. https://doi.org/10.1098/rstb.2021.0077 Schubert, S. C., Zahawi, R. A., Oviedo-Brenes, F., Rosales, J. A., & Holl, K. D. (2025). Active restoration increases tree species richness and recruitment of large-seeded taxa after 16–18 years. <em>Ecological Applications</em>, <em>35</em>(1), e3053. https://doi.org/10.1002/eap.3053 Svenning, J.-C., & Wright, S. J. (2005). Seed limitation in a Panamanian forest. <em>Journal of Ecology</em>, <em>93</em>(5), 853–862. https://doi.org/10.1111/j.1365-2745.2005.01016.x
description: # Data from: Lower-intensity restoration interventions drive greater
 seedling establishment for later-successional tree species Dataset DOI:
 [10.5061/dryad.cfxpnvxjp](10.5061/dryad.cfxpnvxjp) ## Description of the
 data and file structure Data from a field experiment in which seeds of
 eight later-successional tree species were added to restoration plots and
 remnant forests in southern Costa Rica to assess barriers to seedling
 establishment.   ### Files and variables #### File: canopy\_cover.csv
 **Description:** canopy cover above each seed addition quadrat, calculated
 from four densiometer measurements ##### Variables * plot_id: (character)
 Unique identifier for each plot, combining site and treatment (see below).
 * site: (character) two-letter code identifying each study site *
 treatment: (character) two-letter code for treatment. NR = natural
 regeneration, AN = applied nucleation, PL = plantation, RF = reference
 forest * station: factor (integer 1-4) identifying the set of plots
 (“station”) within each plot. * station_id: (character) uniquely
 identifying each station; combination of site, treatment, and station. *
 quadrat: character variable identifying the quadrat within each station *
 quadrat_id: (character) unique identifier for each seed addition quadrat *
 densi_1 to densi_4 : Four numeric variables with raw densiometer
 measurements (number of densiometer points not open sky, range 0-96). *
 mean_densi: (numeric) mean of densi_1, densi_2, densi_3, and densi_4 *
 canopy_cover: mean_densi measurement converted to percent canopy cover
 (mean_densi/96*100), rounded to 1 decimal point #### File:
 litter\_depth.csv **Description:** leaf litter (or thatch) depth in each
 seed addition quadrat ##### Variables * plot_id: (character) Unique
 identifier for each plot, combining site and treatment (see below). *
 station_id: (character) Unique identifier for each station * quadrat_id:
 (character) unique identifier for each seed addition quadrat *
 site: (character) two-letter code identifying each study site *
 treatment: (character) two-letter code for treatment. NR = natural
 regeneration, AN = applied nucleation, PL = plantation, RF = reference
 forest * station: factor (integer 1-4) identifying the set of plots
 (“station”) within each plot. * quadrat: character variable (A or B)
 identifying the quadrat within each station. * date: date of measurement;
 format is YYYY-MM-DD * pt1-pt5: litter depth in cm at each of 5 points
 laid out in an X-shaped pattern within each 1-m quadrat, measured using a
 marked pin-flag (nearest 0.5 cm). * quadrat_mean_depth: mean of 5 depth
 measurements in each quadrat (pt1-pt5) #### File:
 natural\_recruit\_frequency.csv **Description:** Summary of naturally
 recruiting seedlings of study species recorded in non-seeded quadrats at
 each station. Each row is a combination of study species and treatment.
 ##### Variables * treatment: (character) two-letter code for treatment. NR
 = natural regeneration, AN = applied nucleation, PL = plantation, RF =
 reference forest * species: (character) 6-letter code for species *
 quads_with_recruits: (integer) number of sampled non-seeded quadrats with
 natural recruits of the specified species * n_quads: (integer) total
 number of non-seeded quadrats that were sampled for each species and
 treatment combination * freq: relative frequency of natural recruits
 within sampled non-seeded quadrats. Equals quads_with_recruits divided by
 n_quads. #### File: seedling\_emergence.csv **Description:** summary of
 seedling emergence, where each row is a combination of study species and
 station ##### Variables * species: (character) 6-letter code for species.
 Corresponding study species are as follows: * PAL_PAD = *Palicourea
 padifolia* * TRO_MEX = *Trophis mexicana* * LAC_AGG = *Lacistema
 aggregatum* * ERY_MAC = *Erythroxylum macrophyllum* * OCO_PUB = *Ocotea
 puberula* * OTO_NOV = *Otoba novogranatensis* * QUE_BEN = *Quercus
 benthamii* * PSE_MOL = *Pseudolmedia mollis* * plot_id: (character) Unique
 identifier for each plot, combining site and treatment (see below). *
 site: character) two-letter code identifying each study site * treatment:
 (character) two-letter code for treatment. NR = natural regeneration, AN =
 applied nucleation, PL = plantation, RF = reference forest * station:
 integer (1-4) identifying the set of plots (“station”) within each plot. *
 station_id: (character) uniquely identifying each station; combination of
 site, treatment, and station. * added_seeds: (integer) number of
 conspecific seeds added to each quadrat * live_stems: (integer) number of
 recorded seedlings that emerged within the quadrat * failed: (integer)
 number of seeds that failed to emerge. Equal to difference between
 added_seeds and live_stems. * prop_emerged: proportion of added seeds that
 emerged. Equal to live_stems/added_seeds. #### File: seedling\_heights.csv
 **Description:** median seedling height for seedlings of each species
 surviving to one year in each station ##### Variables *
 species: (character) 6-letter code for species * site: (character)
 two-letter code identifying each study site * treatment: (character)
 two-letter code for treatment. NR = natural regeneration, AN = applied
 nucleation, PL = plantation, RF = reference forest * station: integer
 (1-4) identifying the set of plots (“station”) within each plot. *
 station_id: (character) uniquely identifying each station; combination of
 site, treatment, and station. * median_height_cm: (numeric) median height
 of conspecific seedlings that survived to the end of the 1-year
 experimental period. NA = not available (used when sample_size = 0; i.e.,
 when no seedlings survived). * sample_size: (integer) number of seedlings
 that survived to the end of the 1-year period; n for calculation of
 median_height_cm. #### File: seedling\_survival.csv
 **Description:** proportion of seedlings surviving to one year for each
 combination of species and station. ##### Variables * species: (character)
 6-letter code for species. * plot_id: (character) Unique identifier for
 each plot, combining site and treatment (see below). * site: (character)
 two-letter code identifying each study site * treatment: (character)
 two-letter code for treatment. NR = natural regeneration, AN = applied
 nucleation, PL = plantation, RF = reference forest * station: integer
 (1-4) identifying the set of plots (“station”) within each plot. *
 station_id: (character) uniquely identifying each station; combination of
 site, treatment, and station. * germ_seeds: (integer) number of added
 seeds that emerged * survived_seedlings: (integer) number of seedlings
 that survived to the end of the ~1-year experimental period. *
 dead_seedlings: (integer) number of emerged seedlings that died *
 prop_surv: proportion of emerged seedlings that survived to the end of the
 ~1-year experimental period, rounded to two decimal places. Equal to
 survived_seedlings/germ_seeds. ## Code/software Any software capable of
 opening csv files. ## Access information Other publicly accessible
 locations of the data: * NA Data was derived from the following sources: *
 NA