Forest regeneration and seed rain in the conversion of a stand of Pinus sp. into native forest

In Southern Brazil, the National Forests (NF) are protected areas of sustainable use; however, most of them present a significant cover with old plantations of Pinus spp. established to foster commercial forestry. Nowadays, the NF management plans propose the conversion of Pinus stands into native forests. Pinus spp. are worrisome


Introduction
Invasive plants promote the reduction in the abundance and diversity of native plant and animal communities and can critically modify the hydrology and the physical-chemical environment (SIMBERLOFF et al., 2010 and references therein). The magnitude and the direction of these impacts are widely variable, depending on the biological species attributes (VILÀ et al., 2011;RICCIARDI et al., 2013;MOYANO et al., 2020) and community traits .
In Brazil, Pinus species are considered worrisome invasive plants of wide ecological adaptability (BECHARA et al., 2013), occurring spontaneously even in protected areas, generating many impacts on flora, fauna, and biogeochemical processes (BURGUEÑO et al., 2013;DECHOUM, 2013). Pinaceae is one of the ten most invasive plant families globally, and Pinus is the genera of wood species with the greatest invasiveness (PYŠEK, 1998;REJMÁNEK, 2004 (Federal law 9,985, 2000). Most of them started as experimental areas with non-native (introduced) and native species to stimulate commercial forestry. However, the contribution of the NF to commercial forestry became negligible, and the scope of the NF changed in the last decades contemplating conservation goals and promoting ecosystem services. The four NFs in the state of Santa Catarina encompass 7,216 ha, including the Chapecó National Forest (CNF), which covers 1,591 ha. According to the change in objectives, the Management Plan of the CNF contemplates managing actions such as "… to restore areas back to their natural condition with native forests, in a spontaneous or induced way" (ICMBIO, 2013). Based on that assumption, the conversion of Pinus stands to native forests was started with clear-felling and extraction of Pinus wood in the last decade.
The CNF is located in the Atlantic Forest in the transition of the Araucaria Forest and Seasonal Forest domains. The Atlantic Forest is a highly reduced and fragmented biodiversity hotspot (RIBEIRO et al., 2009). Due to the considerable reduction and conversion of the forests, ecological restoration has been considered among the most critical initiatives for the conservation and recovery of the native biodiversity in the Atlantic Forest (SCARANO; CEOTTO, 2015).
Ecological restoration methods applied in the Atlantic Forest include seedling planting, nucleation, direct seeding, assisted natural regeneration, as well as mixed methods. Active methods, including direct seeding and seedling planting, are the most popular (BRANCALION et al., 2016;GUERRA et al., 2020). However, the methods that take advantage of spontaneous forest regeneration, where there is a potential for it, are considered more effective for tropical forests (CROUZEILLES et al., 2017;SHIMAMOTO et al., 2018) and can be highly cost-effective for forest restoration in the Atlantic Forest (DE REZENDE et al., 2015).
On the other hand, the presence of non-native invasive species may require eradication or suppression measures (MCGEOCH et al., 2010;PRIOR et al., 2018). Considering the invasiveness of the Pinus spp., simply abandoning a fallow after Pinus spp production would not achieve satisfactory restoration standards. In sandy coastal ecosystems, clear-felled Pinus plantations presented a strong tendency towards the regeneration of Pinus species, both in the Southern and Northern hemispheres (STURGESS; ATKINSON, 1993;BECHARA et al., 2013). Based on studies focused on the coastal Atlantic Forest over sandy soils where Pinus invasiveness is massive, Bechara et al. (2013; recommended monitoring Pinus recruitment during forest restoration. However, in other ecoregions within the Atlantic Forest, such as the Araucaria Forest and Seasonal Forest, information about Pinus recurrence is scarce. The study of Guidini et al. (2014) in the Santa Catarina Araucaria Forest indicates that the invasiveness of Pinus taeda L. in the mature forest is low due to its pioneer features, which was confirmed by a long-term survey at the same site; however, the established trees in pioneer communities tend to remain alive (SPIAZZI et al., 2017). Scipioni et al. (2018) also confirmed that the presence of P. taeda in an Araucaria Forest remnant is associated with a pioneer community and the neighborhood of a Pinus stand. Besides, specimens planted inside the Araucaria forest tend to die under the closed canopy of a mature forest (EMER; FONSECA,

2011).
Our goal was to quantify and compare the seed rain in a stand where Pinus was eliminated to drive passive regeneration (REG) and in a remaining Pinus plantation stand (PIN). The seed rain in REG can indicate if dispersion is a limitation to spontaneous regeneration and if the Pinus seeds originated in the neighboring Pinus stands still reached the area. Besides, we aimed to describe the spontaneous regeneration of native-woody species, and to compare the woody community in the early restoration phase (one year after Pinus harvesting) in the REG with PIN woody composition, and also with a native mature Araucaria forest (NAT) in the vicinities. Another goal was to quantify Pinus species recruitment in the REG site. This information is relevant since there is a possibility that Pinus regeneration can threaten native forest regeneration.
This information could support forest management decisions in other Pinus plantations to be converted into forests with native species in the Araucaria Forest and Subtropical Seasonal Forest geographic coverage.

Study area
The study site is located in the Chapecó National Forest (CNF), whose headquarters are under the coordinates 27°5'18.54"S and 52°46'49.62"W. The CNF has 1,591 ha. The climate is subtropical, with well-distributed rains during the year and an average temperature of 22°C in the warmest month. The Our study counted on three sample locations as treatments. First, the area undergoing restoration (REG) with 10.82 ha in the southwest end of the CNF 27°6'4.00"S and 52°47'31.10"W ( Figure 1), where the Pinus trees were removed. Pinus harvesting was carried out starting in 2013 and ending in 2015.
Second, an adjacent Pinus spp. (mixed P. taeda and P. elliottii) stand (PIN) without recent (i. e., in the last two Forest passive restoration in Pinus stand

Seed rain
In August 2016 (one year after the end of the Pinus harvesting), 12 circular seed traps with 0.48 m of diameter and height of 10 cm above the ground were installed at the REG, and the other 10 seed traps inside the adjacent Pinus stand (PIN), with a spacing of 20 m between them. The seed traps corresponded to a total horizontal area of 2.17 and 1.81 m², respectively.
The seeds were gathered at intervals of 29 to 35 days, between September 2016 and August 2017, performing a total of eleven collections in one year. The dates of seed collection during 2016 were September 21, October 26, November 24, December 23; during 2017, they were January 24, February 24, March 31, May 05, June 09, July 14, and August 17. Results are presented as seeds m -2 day -1 for comparisons.
The seeds were counted and the species were identified according to the literature (LORENZI, 2008;-2016 and by the authors, compared with individuals in the surroundings. The dispersal syndromes were classified according to Van der Pijl (1982). The similarity regarding the composition of the seed rain was calculated with the Morisita index.

Woody community structure
Despite the importance of all plant growth forms for the restoration, the analyses of vegetation were restricted to the woody species, considering the more precise taxonomic knowledge available for this synusia and the possibility of comparison with other studies. The woody synusia was described through 12 contiguous 20 x 25 m plots arranged in two parallel lines of six plots in each vegetation type. Only trees and palms with a diameter at breast height (dbh) ≥ 5 cm were included. The sampled area was 0.6 ha in each stand (REG, PIN, and NAT). The species richness of the woody communities of REG, PIN, and NAT were compared using species accumulation curves performed through the software EstimateS 9.1.0 (COLWELL, 2013). All the data were obtained between September 2016 and May 2017. The scientific names were checked for synonyms and accepted names in the electronic databases "Lista de Espécies da Flora do Brasil" (FLORA DO BRASIL, S. d.) and The Plant List v. 1.1 (THE PLANT LIST, 2020). Family classification followed APG IV (ANGIOSPERM PHYLOGENY GROUP, 2016).
The relative density (RD) of each species was determined according to RD = n i /AD where n i is the number of individuals of the species i per unit area and AD is the absolute density (number of individuals of all species per unit area). The AD corresponds to the total number of sampled individuals in each vegetation category. The basal area was obtained from each individual's circumference, measured at 20 cm above ground level with a measuring tape.
The diversity in the woody communities was evaluated using the indexes of Shannon (H'), evenness (J), Simpson (D), and the Rényi index profile (RÉNYI, 1961) with the software PAST 3.25 (HAMMER et al. 2001). The Rényi index curve is a method used to compare the different indexes of diversity in a synthetic approach (MELO, 2008). Woody communities were compared using the Morisita index. Unless otherwise stated, Pinus spp. was included in the synecological analysis and data presentation.
The communities were compared statistically regarding Basal Area (m 2 .ha -1 ) and Density (ind. ha -1 ), considering each plot as a replication (n=12). Data of basal area were not normally distributed for the three vegetation categories and for the density of individuals from the Pinus area (Shapiro-Wilk test [SHAPIRO;WILK, 1965], P > 0.05), therefore the vegetation categories were compared with a Kruskal-Wallis test followed by a Mann-Whitney pairwise test.
The successional classification of each species was determined based on the criteria of Coelho et al. (2016), and also consulting Ruschel et al. (2007) and Carvalho (2003Carvalho ( -2014. The dispersal syndromes were classified according to Van der Pijl (1982). The proportions of individuals presenting different dispersal syndromes and different successional categories were compared considering the treatments altogether, using the Chisquare model, followed by a Monte Carlo permutation with 9,999 replications. The statistical procedures were executed in the software PAST 3.25 (HAMMER et al.
In addition, to the data of the woody community structure in the three vegetation categories (REG, PIN, and NAT), the recruits of Pinus spp. (seedlings at least 5 cm high) were quantified in REG to verify the regeneration of these non-native species in the restoring area.

Seed rain
In the regeneration area (REG), 3,130 seeds.m -2 of 19 species and ten botanic families were found during the entire collection period (one year). No seeds of Pinus or other introduced species were observed. In the Pinus stand (PIN) 1,608 m -2 .year -1 seeds from six species (including Pinus spp.) and five families were observed. A complete list of species in the seed rain is presented in the supplementary material (Appendix -Table S1). Hereafter, only the data of the woody species will be presented in detail and discussed. In REG and PIN, 57.5% and 93.4 % of the seeds were from woody species, respectively (Table 1). REG presented eight woody species in the seed rain while the PIN presented only two. Pinus seeds were 95.6% of the PIN seed rain.
Seeds of the anemochoric Baccharis dracunculifolia DC. were 87.0%, and the zoochoric species accounted for 12.3% of the total REG seed rain. Anemochory also dominated seed rain in the PIN area, however it corresponded only to the Pinus seeds (Table 1). The amount of the seed rain corresponding to zoochoric species was 221.3 and 66.4 seeds m -².yr -1 in REG and PIN, belonging to six and one species, respectively. The group of zoochoric species was widely dominated by the seeds of Solanum mauritianum Scop., which reached 93.7% of the zoochoric seeds in the REG seed rain ( Table 1). The Morisita index between the seed rain composition of the two areas was 0.0031.
Seed rain in the REG was heavier between March and August 2017 reaching its maximum in March of that year. In PIN, the peak was in July 2017, widely dominated by the Pinus seed rain (Figure 2).

Tree community
Taken as a whole, the 3 areas presented 74 different tree species, including one not identified. The composition of the communities (REG, PIN, and NAT)    The restoring area (REG) and the old-growth forest (NAT) presented very similar diversity profiles in terms of richness, Shannon and Simpson indexes, and evenness (Table 5). On the other hand, PIN present lower values of diversity considering the same indexes. REG and NAT are also similar regarding the species accumulation curve (Figure 3) and the Rényi diversity profile (Figure 4). In turn, the species accumulation curve and the Rényi profile of PIN indicate a lower diversity. Despite the similar diversity between REG and NAT, REG exhibit the lowest density of individuals (363 ind ha -1 ), which corresponds to 41% in relation to the density of the individuals in the NAT (Table 5).
Although REG and NAT were similar regarding diversity, the communities were very distinct in composition. Whereas pioneer and mid-successional species dominated REG, late-successional species dominated NAT. Pioneer individuals predominated in PIN (excluding Pinus spp.) with 54%, and also in the REG, with 45%. The individuals of mid-successional species reached almost the same value of the pioneer individuals in the REG site (45% and 44% respectively, Table 5). The pioneer individuals were only 13% in NAT, where the late-successional individuals predominated with 59% (

Forest passive restoration in Pinus stand
The zoochory predominates in all vegetations, with 93% of the individuals in PIN (excluding Pinus spp.), 89% for the REG, and 82% for the NAT. Anemochoric individuals were 5%, 11% and 16%, respectively ( Table 5). The proportion of the different dispersal syndromes in the areas differed significantly in the Chisquare test (Monte Carlo P = 0.0051).
The REG community assemblage presented a higher similarity with the PIN assemblage (Morisita index of 0.140) than with NAT (Morisita index of 0.046).
The former areas also have the presence of M. umbellata among the four dominant species (Table 5) in common. The Morisita index between PIN and NAT was 0.110.
The value could also be considered high compared with the value of 1.72 seeds m -2 .day -1 observed by Barbosa and Pizo (2006) in a galley forest within the Seasonal Semideciduous Forest domain, eight years after restoration began. Notwithstanding, REG is a regenerating area at a very early stage, making it difficult to compare with other studies. Besides, all the zoochoric species in the seed rain of REG are small-seeded, according to the criteria of Costa et al. (2012), that is, measuring less than 15 mm. In addition, all of them are already present in the woody sinusya. Therefore, evidence that the seed rain can aggregate diversity in this regeneration stage is lacking, suggesting seed rain limitation. Such seed limitation could be a widespread phenomenon in tropical forests, especially regarding the large-seeds species, even when a higher forest cover in the vicinities as source propagules is available (DALLING et al., 2002;COSTA et al., 2012).
The anemochoric dispersion predominated in the seed rain of REG. However, it was represented by only one species (B. dracunculifolia). As a general rule, zoochoric dispersion dominates the seed rain of woody species in the Atlantic Forest (TOMAZI et al., 2010;DOS SANTOS et al., 2011;COSTA et al., 2012). Nevertheless, in a restoration context, the anemochory can predominate (BARBOSA; PIZO, 2006).
The absence of seeds of other anemochoric species also suggests seed limitation; however, the influence of this limitation in the future of the restored forest remains an open question. It should be stressed that the individuals of anemochoric species in the woody strata of REG were only 11%, which could be considered low values for the non-riparian forests in the Southern Atlantic Forest. NAT presented almost 16% of anemochoric individuals. In mature forests of the region, Ruschel et al. (2007) and Benvenuti-Ferreira and Coelho (2009) recorded respectively 19 and 41% of anemochoric individuals. In turn, Maçaneiro et al. (2018) recorded 27% of anemochoric trees in the upper layer and 24% in the lower layer in an Araucaria Forest in the state of Santa Catarina. The anemochoric species tend to be the tallest trees in dense forests, facilitating seed dispersion (HORN et al., 2001;GIEHL et al., 2007). Besides, they represent the predominant fraction of basal area in the hinterland forests of Southern Brazil (e. g., RUSCHEL et al., 2007;COELHO, 2009), and therefore have a crucial contribution to the structure of the forest.
The absence of Pinus in the seed rain of REG suggests that the Pinus recruits (and the one individual with dbh ≥ 5.0 cm) were more probably originated from the seed bank or remnant juvenile individuals that survived Pinus cutting and harvesting. Although the seeds of P. taeda can surpass 11 km when dispersed, most released seeds are susceptible to local neighborhood dispersion in distances ranging from 5 to 140 m (WILLIAMS et al., 2006). However, Jankovski (1985), in a test to check dispersion capacity, had obtained only 3.7% of the total amount of Pinus seeds from a P. taeda stand in seed traps 55-60 m apart. Cooper (1957) reported that more than 90% of the P. elliottii seeds fall within 45 m from the seed source. Therefore, the distance between REG and the source of Pinus seeds (at least 250 m) could explain why Pinus seeds were not detected in the seed rain of the REG.
The seed rain of Pinus spp. exhibited a peak between May and August. The viability of the seeds of P. elliottii in the seed bank fall under 2% after 180 days (BECHARA et al., 2013). Seeds of Pinus taeda L. retain viability for a few months, and few seeds remain viable (not more than 0.1 percent) in the second year after seed fall (LITTLE; SOMES, 1959;BAKER;LANGDON, 1990). The retention of the viability of Pinus seeds in Brazilian sites requires additional studies. Nevertheless, Pinus clearing and extraction could be executed outside the Pinus seed crop peak, opening an alternative for the management to forest conversion. Further studies considering the seed bank of Pinus stands can contribute to the improvement of these methods.
The zoochoric dispersion group was predominant in the woody communities studied here, with proportion above 82%. Zoochory is typically dominant in the woody communities of forests in the austral Atlantic Forest, with proportions ranging between 55 and 90% of the tree individuals (RUSCHEL et al., 2007;COELHO, 2009;DUARTE et al., 2009).
The result corroborates the importance assigned to the zoochory in the forest succession and ecological restoration in the Southern Atlantic Forest (TRES; REIS, 2009b;DOS SANTOS et al., 2011).
As expected, the predominance of pioneer species in REG is coherent with an early phase of secondary succession. However, the presence of the latesuccessional species in the tree synusia in REG (10.6% of relative density) is noteworthy, which probably includes native individuals that survived Pinus cutting and harvesting, since that proportion is very similar to that of the community in the Pinus stand (11.8%). The presence of late-successional species only one year after Pinus harvesting can contribute to the effective recovery of the tree community in the long term. The tree community in the mature forest or advanced successional stages in the South Atlantic Forest usually present dominance of late-successional species, at least concerning relative density (RUSCHEL et al., 2007;COELHO, 2009), which was confirmed by our data from the old-growth forest (NAT). Moreover, the individuals of mid-successional species also exhibit an expressive value (44%) in REG, which can contribute to secondary succession. As pioneer individuals die, mid-successional species can replace them in the canopy, while the forest grows and slowly acquires late-successional species.
One year after the conclusion of Pinus harvesting, the spontaneous regeneration could be considered satisfactory, presenting a higher diversity compared to the PIN stand. Species diversity of natural regeneration in REG reached values close to the undisturbed native forest, besides the community composition differences, which presented more similarity with the Pinus stand community. However, this may be a consequence of the initial stage of succession in REG and the predominance of typical species from the early stages of ecological succession. Continued monitoring can be a relevant contribution to know the successional potential in this context.
The high density of recruiting Pinus sp. plants in the restoration site is equivalent to 75% of the Pinus adult trees in the stand plantation. Although the survival potential of these individuals is unknown, the finding indicates the necessity of complementary actions of control to avoid its reestablishment. Bechara et al. (2013) observed massive recruitment in the P. elliottii after harvesting an adult pine stand in a coastal sandy plain in Southern Brazil, attaining 27 plants m -2 after 2.5 years. However, the mortality rate was also high, reaching 70% after 2.5 years. On the other hand, Spiazza et al. (2017) observed 100% survival for P. taeda (dbh ≥ 5 cm) after four years in a Mixed Ombrophilous Forest in an advanced successional stage. This suggests a higher survival potential of the P. taeda individuals, once established. Besides, future natural gap openings in the secondary forest (as the pioneer species die) can offer opportunities for Pinus recruitment (SPIAZZA et al., 2017). Therefore, the recruiting Pinus sp. plants in REG deserves attention.
The analyses carried out suggest that spontaneous regeneration can potentially replace the Araucaria Forest from Pinus plantations without neglecting actions to control Pinus recruiting individuals. G. C. Coelho et al.