Guidelines for Seed Collection of Araucaria Angustifolia (Bertol

Forest Ecology and Management 438 (2019) 10–17

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Forest Ecology and Management

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Guidelines for seed collection of Araucaria angustifolia (Bertol.) Kuntze: A genetic, demographic and geographic approach T ⁎ Tiago Montagnaa, , Miguel Busarello Lauterjunga, Newton Clóvis Freitas da Costaa, Alison Paulo Bernardia, Rafael Candido-Ribeirob, Maurício Sedrez dos Reisa a Núcleo de Pesquisas em Florestas Tropicais, Universidade Federal de Santa Catarina, Rodovia Admar Gonzaga, 1346, Florianópolis, Santa Catarina, Brazil b Department of Forest and Conservation Sciences, University of British Columbia, Vancouver, BC V6T 1Z4, Canada

ARTICLE INFO ABSTRACT

Keywords: Restoration of degraded ecosystems is essential for the conservation of global biodiversity. In southern Brazil, the Araucaria forest Araucaria forest is a highly reduced and fragmented ecosystem of the Atlantic Forest Biome, and restoration is Effective population size recommended for the conservation of both Araucaria forest and Araucaria angustifolia, an endangered conifer of Fragmentation this ecosystem. Thus, knowledge about seed collection sites, criteria for the maintenance of genetic diversity, Genetic diversity and adaptive potential of seeds is crucial to restoration success. Therefore, this study aimed to identify priority Restoration areas for seed collection of A. angustifolia based on its previously known genetic, demographic and geographic characteristics. To accomplish this, the density of reproductive individuals of A. angustifolia associated with a probability of reproduction (50% or 90%) was extrapolated to all the Araucaria forest fragments from Santa Catarina state (SC), Brazil. Then, we estimated which forest fragments would be suitable for seed collection based on average values of fine scale genetic structure (FSGS) and the number of sampled seed trees for which different effective population sizes (100 or 1000) could be retained. Despite high fragmentation, some forest fragments remain suitable as seed source for restoration. However, using strict criteria, only 4.1% of the fragments could be deemed suitable. Collectively, our results represent a starting point for policymakers concerned with identifying restoration areas for endangered species, in particular, the criteria for seed collection of A. angustifolia.

1. Introduction and the evolutionary potential of restored populations (Breed et al., 2018; Broadhurst et al., 2008). In this sense, appropriate selection of Restoration of at least 15% of degraded ecosystems by 2020 is one seed collection sites should minimize the possible risks of introducing of the 20 Aichi Biodiversity Targets (CDB, 2010). Accordingly, the non-adapted genotypes into the new populations, which could lead to Brazilian target for restoration by 2020 was also set to 15% of degraded maladaptation and outbreeding/inbreeding depression, both resulting ecosystems, with the most degraded biomes, hydrographic regions, and in lower adaptive potential (McKay et al., 2005; Stingemore and Krauss, ecoregions prioritized for repair (Brazil, 2017). To accomplish these 2013; Vander Mijnsbrugge et al., 2010). Furthermore, in order to avoid targets, seeds from several species are required. However, improving a loss of genetic diversity (bottlenecks), seed collections should be the chance of successful restoration depends on a comprehensive un- performed under criteria that determine the minimum number of seed derstanding of seed availability, seedling propagation, and source of trees to be sampled and the proper distances between seed trees to propagules (Hoﬀmann et al., 2015; Luna-Nieves et al., 2017; McKay avoid the collection of seeds from closely related individuals (Degen et al., 2005). Given the scale of such ambitious restoration target, the and Sebbenn, 2016; Sebbenn, 2006, 2002). complexity involved in the process of selecting areas for collection The Araucaria forest, a highly fragmented ecosystem in the Atlantic (Broadhurst et al., 2008), collecting seeds in large quantities, and de- Forest Biome (Ribeiro et al., 2009; Vibrans et al., 2012b) is so noted livering seeds in the right time for restoration (Merritt and Dixon, because of the predominance of Araucaria angustifolia (Bert.) O. Kuntze, 2011), may be signiﬁcantly high. a dioecious wind pollinated conifer (Reitz and Klein, 1966), in the Another layer of complexity added to the seed collection in the upper canopy of the forest. This ecosystem was reduced to 12.6% of its context of restoration is related to the maintenance of genetic diversity original area (Ribeiro et al., 2009) through anthropogenic activities

⁎ Corresponding author at: Rodovia Admar Gonzaga, 1346, Florianópolis, Zip Code: 88034-000, Santa Catarina, Brazil. E-mail address: [email protected] (T. Montagna). https://doi.org/10.1016/j.foreco.2019.02.006 Received 11 December 2018; Received in revised form 25 January 2019; Accepted 4 February 2019 0378-1127/ © 2019 Elsevier B.V. All rights reserved. T. Montagna et al. Forest Ecology and Management 438 (2019) 10–17 such as logging and agriculture (Guerra et al., 2002; Reitz and Klein, 2. Material and methods 1966). One of the most exploited tree species in the Araucaria forest was A. angustifolia, mainly harvested for timber production (Guerra 2.1. Demographic data acquisition et al., 2002; Reitz and Klein, 1966). Because of a large reduction in population sizes and habitat, A. angustifolia is classified as critically Demographic data were obtained from a systematic survey per- endangered in the IUCN Red List of Threatened Species (Thomas, formed by the IFFSC group, following the methodology described by 2013). Given this picture of fragmentation and threat, efforts towards Vibrans et al. (2010). Specifically, clusters of four crosswise 1000 m2 conservation of the Araucaria forest, including restoration are necessary plots (20 × 50 m) were systematically distributed at the intersections of and recommended (Hoffmann et al., 2015; Montagna et al., 2017). a 10 × 10 km grid all over the Araucaria forest domain in SC (Fig. 1) Araucaria angustifolia is considered to be a long-lived pioneer spe- (Vibrans et al., 2010). In each cluster, all individuals of A. angustifolia cies with potential for establishment in open areas (Reitz and Klein, with diameter at breast height (DBH) greater than 10 cm were counted 1966; Souza et al., 2008). This species is also considered to be a nurse and had their height and DBH measured. Intersections without forest plant (Duarte et al., 2006) since it facilitates the growth of forest flora cover were not evaluated. This systematic survey produced information under its canopy. Furthermore, A. angustifolia is animal-dispersed and on demographic structure and density of A. angustifolia in 143 Arau- plays a crucial role as a food source for several species of mammals caria forest remnants. (e.g., Dasyprocta azarae, Delomys dorsalis, Oligoryzomys nigripes, Pecari Using DBH data, we estimated the density of reproductive in- tajacu, Procyon cancrivorus, Sciurus ingrami, and Tayassu pecari) and dividuals in each forest fragment based on the probability of an in- birds (e.g., Amazona vinaceae, A. pretrei, Cyanocorax caeruleus, and C. dividual being reproductive according to its size (DBH). For A. angu- chrysops), with some of them being dispersers (Iob and Vieira, 2008; stifolia, the probability of reproduction equals 50% at 26.3 cm of DBH Solórzano-Filho, 2001; Vieira et al., 2011). Thus, the use of A. angu- and 90% at 35.2 cm of DBH (Paludo et al., 2016). Therefore, we esti- stifolia is highly recommended in restoration of degraded areas (Brack mated the density of reproductive A. angustifolia individuals in each and Grings, 2011; Carvalho, 2003). However, to restore degraded areas forest fragment based on these two probabilities, i.e., excluding in- of Araucaria forest will demand large amounts of A. angustifolia seeds dividuals with DBH < 26.3 cm or excluding individuals with DBH < given its broad natural range and the large proportion of the ecosystem 35.2 cm. loss. Aspects related to the mating system of A. angustifolia and the dis- 2.2. Mapping tribution of genetic diversity among and within populations are well documented (Auler et al., 2002; Bittencourt and Sebbenn, 2007; Area and location of each forest fragment of Araucaria forest in SC Medina-Macedo et al., 2015; Sant’Anna et al., 2013; Stefenon et al., were obtained from a shapefile created by the SOS Mata Atlântica 2007, among many others). Mating system parameters can be used to Foundation and the National Institute for Space Research (INPE) and estimate the number of seed trees from which it would be necessary to based on Landsat 8 OLI images from 2015 and 2016 (SOS Mata Atântica collect seeds, aiming to retain a given reference effective population and INPE, 2017). The geographical limits of the Araucaria forest were size (see Medina-Macedo et al., 2016; Montagna et al., 2018; Silva extracted from the phytogeographic map of SC (Klein, 1978), which is et al., 2008). In turn, the knowledge of the distribution of genetic di- available in shapefile format on the IFFSC site (www.iff.sc.gov.br). In versity within populations (fine-scale genetic structure) inform contrast to point-based demographic information, forest fragments are minimum distances between seed trees that could prevent the collection based on area information. Therefore, we extrapolated the observations of seeds from closely related individuals (Sebbenn, 2006). Moreover, in of A. angustifolia (143 plots) to the area of Santa Catarina, using Inverse Santa Catarina state (SC), southern Brazil (Fig. 1), the Floristic and Distance Weighted (IDW) interpolation to a 1 km resolution raster from Forest Inventory of SC (IFFSC) was created to survey the remaining which we then extracted the average value of the species population forests of the state. This inventory was carried out through all SC state density for each fragment shapefile. All these operations were per- from 2007 to 2011, investigating forest aspects such as floristic com- formed in R (R Development Core Team, 2015), using raster (Hijmans, position, structure and mapping of forest fragments (Vibrans et al., 2015), sp (Bivand et al., 2013; Pebesma and Bivand, 2005), and gstat 2012a). The major outcome of this effort, a database to support forest (Pebesma, 2004) packages. and conservation policies, is now available (Vibrans et al., 2012a). This program has generated valuable information concerning the location 2.3. Genetic data acquisition and analysis and size of Araucaria forest fragments as well as demographic structure (i.e., number of seed trees) of the remaining A. angustifolia populations. We next searched the literature for studies reporting on the mating Therefore, integrating such information with the available genetic data system and fine-scale genetic structure (FSGS) of A. angustifolia,a can help one to generate guidelines for seed collection of A. angustifolia dioecious species. More specifically, we sought average values of to restore degraded areas. coancestry within progeny arrays (Θ), obtained from codominant gene Owing to fragmentation and exploitation of the Araucaria forest, a markers and analyzed under the mixed-mating model (Ritland and Jain, lack of appropriate sites with enough A. angustifolia individuals may 1981) and the correlated mating model (Ritland, 1989). Following hinder the collection of seeds with the appropriate genetic diversity and Sousa et al. (2005), Θ for a dioecious species can be obtained as adaptive potential for the species. Therefore, using previously docu- Θ=++ 0.125(1Frppm )(1 ()), where Fp is the inbreeding coefficient in mented knowledge about genetic, demographic, and geographic char- the reproductive population, and rpm()is the multilocus paternity cor- acteristics of A. angustifolia populations, our major aim in this study was relation, which represent the proportion of full-sibs within progeny. to answer the following questions: (a) to what extent is the SC state From the Θ estimate, it is possible to obtain the effective variance size suitable for seed collection of A. angustifolia observing genetic and de- of an open-pollinated progeny array (Nev()= 0.5/Θ)(Cockerham, 1969). mographic criteria? (b) which are the priority areas for seed collection? This value represents how many different individuals a given progeny and (c) which are the limiting factors for the seed collection? The ap- array can represent. The maximum value within a family is 4, in case of proached questions will guide A. angustifolia restoration and con- half-sib progenies. Therefore, in accordance with Sebbenn (2002), the servation programs as well as help policymakers in establishing para- number of seed trees (m) necessary for seed collection in order to retain meters for other endangered species. a given effective population size (Ner()) can be obtained as mN= er()/ N ev (). For instance, Bittencourt and Sebbenn (2008) reported Θ = 0.174 for an A. angustifolia population. Thus, the effective variance

size of the progeny is equal to 2.87 individuals (Nev()= 0.5/0.174), and

11 T. Montagna et al. Forest Ecology and Management 438 (2019) 10–17

Fig. 1. Map showing the Araucaria forest (dark-grey) and the Grasslands (light-grey) domains. The black dots represent the 143 forest remnants where demographic data of Araucaria angustifolia were gathered in Santa Catarina.

the number of seed trees to be sampled in order to retain an Ne = 500 is 2.4. Forest fragments identification around to 174 (m = 500/2.87). Naturally, the reported Nev()can be used in combination with other Ner()values. Here, we used Ner()values of 100 To declare a fragment as adequate for seed collection, it must meet and 1,000 individuals. The first is expected to avoid inbreeding de- two criteria: (i) have a minimum number of A. angustifolia trees from pression in the short term (five generations), while the second is re- which the seed collection would retain a reference effective size of 100 quired to maintain initial evolutionary potential in perpetuity or 1000 individuals (Ner()) and (ii) have individuals spaced by a (Frankham et al., 2014). minimum distance to avoid the sampling of closely related individuals Nevertheless, several tree species tend to present significant levels owning to FSGS. of FSGS (Hardy et al., 2006), i.e., the nonrandom distribution of the Criterion i will be met if the number of individuals in a fragment genotypes across the landscape (Vekemans and Hardy, 2004). The main (Nobs) is higher than the required number of individuals (m). The implication of significant FSGS is that spatially closer individuals are number of individuals in a fragment was calculated as N = DA· , where obs 2 more likely to be closely related. Therefore, collecting seeds from trees D is density of reproductive individuals (male and female) of the − close to each other may represent the sampling of genetically related fragment (ind ha 1), and A is area of the fragment (ha). We divided individuals, which phenomenon can reduce the effective size of the density by two because A. angustifolia is a dioecious species (Reitz and collected seeds (Degen and Sebbenn, 2016; Sebbenn, 2006). FSGS levels Klein, 1966), under the assumption that the sex ratio did not differ from are often expressed in meters, i.e., the distance up to which the statistic 1:1 (Paludo et al., 2016, 2009), and we need only female trees for seed describing pairwise genetic relatedness or differentiation is significant. collection. Therefore, FSGS values should be used as a minimum distance between Criterion ii will be met if the actual area of the fragment (Aobs)is seed trees in order to avoid collecting seeds from closely related in- greater than the area that would be occupied by female trees spaced by dividuals (Sebbenn, 2006). Hence, we searched for literature explicitly FSGS (Areq). We assumed that individuals would be uniformly spaced as 2 reporting distances up to which FSGS was significant in A. angustifolia. 2·FSGS · Ner() in a squared grid. Therefore, Areq = , where Areq is the re- 10000·Nev() quired area for collection (ha); FSGS is the distance with significant

12 T. Montagna et al. Forest Ecology and Management 438 (2019) 10–17

FSGS (m); Ner()is the desired effective population size (100 or 1000); Table 1 ff and Nev() is the effective variance within progeny. For every female Coancestry within progeny arrays (Θ) and e ective variance size (Nev( )) in po- (seed) tree, we have a male tree, occupying the same area, explaining pulations of Araucaria angustifolia distributed over three Brazilian states. the multiplication by two. We constructed CI (95%) for m and Areq with Author State Marker Θ Nev()= 0.5/Θ 10,000 bootstraps, resampling values of Nev() and FSGS used in their calculations. If m > Nobs (criterion i) and Aobs > Areq (criterion ii), then Bittencourt and Sebbenn (2007) PR SSR 0.155 3.23 the fragment can be appropriated for seed collection. Bittencourt and Sebbenn (2008) PR SSR 0.174 2.87 All genetic information gathered for this study came from presumed Cristofolini (2017) SC SSR 0.132 3.79 Ferreira et al. (2012) SC allozymes 0.150 3.33 neutral markers. Consequently, this tells us nothing about the extent Ferreira et al. (2012) SC allozymes 0.130 3.85 and scale of local adaptation of A. angustifolia populations. Ferreira et al. (2012) SC allozymes 0.180 2.78 Furthermore, the available information about adaptive variation in A. Mantovani et al. (2006) SP allozymes 0.150 3.33 angustifolia is scarce and often reported for narrow proportions of its Medina-Macedo et al. (2016) SC SSR 0.135 3.70 Medina-Macedo et al. (2016) SC SSR 0.134 3.73 geographic range when compared to the amount of data regarding Medina-Macedo et al. (2016) SC SSR 0.142 3.52 neutral variation (see Duarte et al., 2012; Sebbenn et al., 2003; Medina-Macedo et al. (2016) SC SSR 0.169 2.96 Shimizu, 1999). To indirectly overcome this lack of information, all the Sousa et al. (2005) SP allozymes 0.151 3.31 identified forest fragments were geographically located allowing pos- Sousa et al. (2005) SP allozymes 0.174 2.87 sible further decisions on seed collection based on the distance between Sousa et al. (2005) PR allozymes 0.179 2.79 Sousa et al. (2005) PR allozymes 0.181 2.76 the source of seeds and the place where seeds would be sown (e.g., for Sousa et al. (2005) PR allozymes 0.151 3.31 restoration). Sousa et al. (2005) PR allozymes 0.189 2.65 Sousa et al. (2005) PR allozymes 0.200 2.50 3. Results Sousa et al. (2005) SC allozymes 0.155 3.23 Sousa et al. (2005) SC allozymes 0.147 3.40 Sousa et al. (2005) SC allozymes 0.139 3.60 3.1. Demographic and mapping data Sousa et al. (2005) SC allozymes 0.153 3.27 Sousa et al. (2005) SC allozymes 0.138 3.62 According to the shapefile from the SOS Mata Atlântica Foundation Sousa et al. (2005) SC allozymes 0.145 3.45 and INPE (SOS Mata Atântica and INPE, 2017), the remaining area of Zechini et al. (2018) SC allozymes 0.137 3.65 Zechini et al. (2018) SC allozymes 0.143 3.49 Araucaria forest in SC is 809,932 ha or 18.9% of the maximum area. Zechini et al. (2018) SC allozymes 0.166 3.01 The average area of each fragment is 57.4 ha (median = 15.3 ha, Mean 0.156 3.26 minimum = 0.0004 ha, maximum = 64288.821 ha); however, 86% of these fragments are less than 50 ha. The average A. angustifolia density PR: Paraná; SC: Santa Catarina; SP: São Paulo. − (dbh > 10 cm) from the surveyed points was 24.2 ind⋅ha 1. After ex- tracting information from the interpolated raster corresponding to a Table 2 fi reproductive probability of 50% (dbh ≥ 26.3 cm) and 90% Extension of ne-scale genetic structure (FSGS) in populations of Araucaria angustifolia distributed over four Brazilian states. (dbh ≥ 35.2 cm) (Supplementary material 1), these fragments had an − average density of 9.3 and 5.5 ind⋅ha 1, respectively. Author State Marker FSGS (m)

Bittencourt and Sebbenn (2007) PR SSR 50 3.2. Mating system and FSGS Bittencourt and Sebbenn (2008) PR SSR 75 Cristofolini (2013) SC SSR 37 We were able to gather information of progeny arrays from 27 A. Mantovani et al. (2006) SP allozymes 70 angustifolia populations, as reported in eight publications (Table 1). On Medina-Macedo et al. (2015) SC SSR 90 average, the coancestry within progeny arrays (Θ) was equal to 0.156, Patreze and Tsai (2010) SP SSR 25 Sant'Anna et al. (2013) SC SSR 20 slightly higher than the expected from half-sibs (Θ = 0.125). Therefore, Stefenon et al. (2008) SP SSR 0 the average effective variance size (Nev()) was equal to 3.26, ranging Stefenon et al. (2008) PR SSR 33 from 2.5 up to 3.85. Stefenon et al. (2008) PR SSR 27 The average distance with significant FSGS was equal to 37 m, Stefenon et al. (2008) SC SSR 36 Stefenon et al. (2008) SC SSR 20 ranging from 0 up to 90 m (Table 2). This average is based on in- Stefenon et al. (2008) RS SSR 0 formation from 13 populations of A. angustifolia, as published in eight Mean 37 studies. In one of these studies (Stefenon et al., 2008), the FSGS analysis was performed for six populations using two molecular markers, SSR PR: Paraná; RS: Rio Grande do Sul; SC: Santa Catarina; SP: São Paulo. and AFLP, thus producing 12 results. We retained the six SSR studies only to avoid any duplication of data. reproduction of 50%, if the seed collection aims to retain a Ner()of 1000 individuals, only 5.7% of the forest fragments could possibly support 3.3. Forest fragment identification seed collection. A shapefile containing the information summarizing fragments indicated for seed collection (Fig. 2) can be found in The percentage of forest fragments indicated for seed collection Supplementary material 2. varied widely according to different assumptions (combination of probability of reproduction and the desired (Ner()). Under more relaxed 4. Discussion assumptions, i.e., considering a probability of reproduction equal to

50% and aiming to retain a Ner()of 100 individuals, seed collection can A steep reduction in the number of fragments and, to a lesser extent, be performed in 64.1% (95% CI = 43.1–71.2%) of the Araucaria forest in the area suitable for seed collection was observed when Ner() was fragments, or 94.3% of the remaining Araucaria forest area (Table 3). increased from 100 to 1000 individuals, under any scenario of prob-

However, this percentage drops to 4.1% (95% CI = 2.5–5.7%) of ability of reproduction. To increase Ner()from 100 to 1000 individuals fragments, or 59.8% of remaining area (95% CI = 56.5%–61.7%), implies multiplying by a factor of 10 the number of required individuals when the more restricted assumptions are applied, such as probability for seed collection, reducing to less than 10% the number of fragments of reproduction equal to 90% and aiming to retain a Ner() of 1000 in- deemed as suitable for seed collection. This is a clear indication of dividuals (Table 3, Fig. 2). Even considering a probability of threat for the long-term conservation of A. angustifolia genetic diversity,

13 T. Montagna et al. Forest Ecology and Management 438 (2019) 10–17

Table 3 South Brazilian grasslands (Fig. 1). The grasslands in southern Brazil Percentage of number and area (ha) of forest fragments fulfilling the established are composed of a vegetation mosaic mixing dominant herbaceous * criteria for seed collection under different assumptions of probability of re- species with scarce forest patches where A. angustifolia commonly oc- ff production (P(repr)) and reference e ective size (Ner( )) in Santa Catarina. curs. Consequently, it should be noted that the estimated remaining

Ner()= 100 Ner()= 1,000 area of Araucaria forest does not take into account these forest patches with A. angustifolia from the grasslands. Even though these grassland P (repr) Mean (95% CI) Mean (95% CI) patches were not computed in our study, they may also be important sources of seeds since gene flow via pollen tends to be higher for the 50% Number 64.1 (43.1–71.2) 5.7 (3.1–9.6) Area 94.3 (89.6–95.2) 64.4 (59.1–68.3) studied species in discontinuous landscapes, such as grasslands, than in continuous forests fragments (see Bittencourt and Sebbenn, 2008, 2007; – – 90% Number 48.8 (37.1 51.8) 4.1 (2.5 5.7) ’ Area 89.4 (86.7–89.9) 59.8 (56.5–61.7) Medina-Macedo et al., 2015; Sant Anna et al., 2013). Thus, because of the possible long distances of pollen dispersal, even isolated A. angu- CI low/upp: lower and upper 95% confidence interval limits. stifolia trees can produce seeds with high genetic diversity, as long as * criterion (i): have a minimum number of A. angustifolia trees from which these are near from other patches or forest fragments (Medina-Macedo the seed collection would retain a reference effective size of 100 or 1000 in- et al., 2016). For instance, gene flow distances through pollen can dividuals (Ner( )) and criterion (ii): be individuals spaced by a minimum distance surpass 2 km (Bittencourt and Sebbenn, 2007). The high levels of gene to avoid the sampling of closely related individuals by FSGS. flow via pollen can also allow seed collection from several nearby forest fragments, as they can be connected by gene flow. This implies that our but it should also alert policymakers about the conservation of larger methods may have underestimated the real number of fragments fragments. For instance, discounting the probability of reproduction, deemed as adequate for collection, which is important to notice since assuming Ner()of 1000, and using the average gathered values of FSGS most fragments (86%) are smaller than 50 ha. and Θ, the required area (Areq) obtained is equal to 84 ha. However, We assumed that all seed trees would produce an adequate number 86% of the Araucaria forest fragments in SC are currently smaller than of seeds each year. However, some fragments deemed as appropriate for 50 ha. Therefore, it is crucial to conserve large forests fragments in seed collection may not contain all seed-producing individuals in a order to maintain populations of A. angustifolia with the potential to given collection period, because A. angustifolia presents considerable produce seeds useful in restoration. variation with respect to seed maturation and seed fall (Adan et al., The proposed limits for the Araucaria forest in SC (Klein, 1978)do 2016; Mattos, 1994; Reitz and Klein, 1966). Furthermore, the annual not consider another ecosystem with occurrence of A. angustifolia: the

Fig. 2. Forest fragments where it is possible for seed collection (red-colored) to meet the established criteria* for seed collection under different assumptions of probability of reproduction (P(repr)) and reference effective size (Ner( )) in Santa Catarina. Light-yellow area represents Araucaria forest limits. * criterion (i): have a minimum number of A. angustifolia trees from which the seed collection would retain a reference effective size of 100 or 1000 individuals (Ner( )) and criterion (ii): be individuals spaced by a minimum distance to avoid the sampling of closely related individuals by FSGS.

14 T. Montagna et al. Forest Ecology and Management 438 (2019) 10–17 amount of seed produced can vary between individuals, years, and seed collection of A. angustifolia is clearly one of the main effects of regions (Mantovani et al., 2004; Zechini, 2012). Variation in the fragmentation. The genetic diversity of the species may also have been number of seeds produced can also result from the amount of rain that negatively affected, even with the species presenting high levels of gene occurs during the pollination period, particularly since pollen is carried flow. The maintenance of high levels of fixation index (FIS) when by the wind (Mattos, 1994). Therefore, the number of fragments with comparing seedlings with reproductive individuals (Lauterjung et al., enough seeds for collection could be lower than what we estimated or, 2019) is a strong indication of that. In addition, other negative effects at least, this number will vary between years. can be expected with the reduction of forest fragments, such as higher Despite variation in the seed production, some additional re- frequency of mating among closely related individuals, ultimately recommendations for seed collection should be noted. Ideally, seeds in sulting in higher inbreeding levels in the progeny and loss of genetic bulk (> 1,000, for instance) should be collected from each seed tree in diversity (Aguilar et al., 2008; Young et al., 1996). Thus, in order to order to maximize the chances that different genotypes will be re- avoid the collection of seeds in genetically depleted populations, any presented. Nevertheless, this deserves further studies on the possible existing knowledge on genetic diversity levels is important. impacts on the fauna that feed on A. angustifolia seeds, and on the To be able to use published data, we had to make several assump- turnover of natural populations. Furthermore, the seed production per tions, such as uniform spatial distribution of the individuals in each individual in A. angustifolia varies widely, from 50,7 (Vieira-da-Silva remnant, constant reproductive activity over the years, constant 1:1 sex and Reis, 2009) up to 1573,6 seeds per tree (Mantovani et al., 2004). ratio over all remnants, constant probability of reproduction according Thus, we recommend collecting an equal, or nearly equal, amount of to DBH, and the reliability of interpolated densities. Consequently, our seeds from each seed tree to avoid a reduction in the effective size of the results should be interpreted by taking into consideration these as- sample produced by unbalanced female gamete contribution sumptions. In this sense, our study can be treated as indicative of which

(Vencovsky, 1987). As the average Nev()was equal to 3.26, ranging from fragments may potentially serve for the collection of A. angustifolia 2.5 up to 3.85, we do not recommend collecting fewer than four seeds seeds, observing the discussed genetic criteria. from each seed tree. However, any effort to increase the number of In spite of the sharp reduction and fragmentation inflicted on the collected seeds is important, because four seeds per seed tree is the Araucaria forest, our study shows that some forest fragments are still minimum number to allow the sampling of the entire Nev()of a progeny suitable as a seed source for restoration projects in SC. However, re- array. Furthermore, A. angustifolia seeds are grouped in cones dis- storation activities must be initiated by local authorities. Perhaps, tributed around the tree crown. As the pollination is mediated by wind, considering the history of the Araucaria forest during its last expansion, different cones facing different directions are likely pollinated by dif- we will find inspiration for the conservation and restoration of this ferent males. Therefore, whenever possible, we recommend the col- important ecosystem, even in its current fragmented state. Growing lection of seeds from different cones at different positions of the crown. evidence from several disciplines, such as archeology, genetics, The Araucaria forest has been extremely reduced and fragmented, ecology, and palynology, supports the hypothesis that the last expan- and A. angustifolia does not occur in any other Brazilian ecosystem, sion of the Araucaria forest was highly mediated by human groups and except in forest patches from the grasslands. Therefore, activities un- that it occurred mainly in the recent past (see Behling, 1997, 1995, dertaken towards restoration of degraded ecosystems depend on the Behling et al., 2004, 2001; Bitencourt and Krauspenhar, 2006; Iriarte collection of seeds in the remaining fragments. Consequently, our re- and Behling, 2007; Lauterjung et al., 2018; Reis et al., 2014; Robinson sults can be a good example of the potential integration of genetic and et al., 2018). Therefore, at the same time that humans are capable of demographic data for conservation purposes. Combining published and threatening ecosystems, they can also take responsibility for restoring, available data has advantages. Firstly, we were able to identify areas for expanding and conserving them. seed collection for the entire Araucaria forest domain in SC, which is around 4.3 million of ha (Klein, 1978; Vibrans et al., 2012b). However, this result was only possible because of the data generated by the IFFSC, 5. Conclusions highlighting the importance of forest inventories and other systematic monitoring programs to underpin conservation strategies. Collectively, our results represent a starting point for policymakers The criteria proposed in our study allowed us to identify sites were concerned with identifying restoration areas for endangered species, in it would be possible to collect seeds. However, as the assembled genetic particular, the criteria for seed collection of A. angustifolia. Even taking information came from presumed neutral markers, we were not able to into account the severe reduction and fragmentation perpetrated on the define the meaning of “local” for the genotypes of A. angustifolia, nei- Araucaria forest, and the strictest criteria proposed in this study, there ther to recommend maximum or minimum distances between the are still forest fragments suitable as a seed source (4.1% of the frag- source of seeds and the place where seeds would be sown. Nevertheless, ments). Perhaps, the main challenge for policymakers and forest man- there is some important information to bring to this discussion. agers is how to prompt the use of A. angustifolia in restoration projects Populations of reproductive individuals of A. angustifolia in southern or plantations for timber and seed production. Therefore, the use will be Brazil consistently present low genetic divergence (e.g., FST and GST ), a tool for the expansion of the Araucaria forest. i.e., lower than 5% (Auler et al., 2002; Lauterjung et al., 2018; Montagna et al., 2017). Moreover, seedling individuals, in a set of 21 populations in SC presented FST = 0.017 (Lauterjung et al., 2019), Acknowledgements suggesting high levels of gene flow via pollen, which can connect populations. Apart from this, considerable palynological evidence sup- We gratefully acknowledge the financial support of Fundação de ports that the last expansion of the Araucaria forest occurred mainly Amparo à Pesquisa e Inovação do Estado de Santa Catarina (FAPESC) between 1500 and 900 years before present (Behling, 1997; Robinson for the entire IFFSC Project in its several steps. We are also very grateful et al., 2018) from the valleys in the east replacing the grasslands to the to all the IFFSC crew members, especially the researchers from west. Thus, we are probably dealing with a species with recent and Universidade Regional de Blumenau who were responsible for col- rapid expansion from a single refugium, at least in southern Brazil lecting and sharing the demographic data used in this article. We also (Lauterjung et al., 2018). Therefore, the extension of a “local” genotype thank the Coordenação de Aperfeiçoamento de Pessoal de Nível for A. angustifolia may be broader than a few kilometers, and, as such, Superior (CAPES) for the fellowships to TM, MBL, NCFC, and APB and this remains an open question. the Conselho Nacional de Desenvolvimento Científico e Tecnológico As mentioned, 86% of the Araucaria forest fragments in SC present (CNPq) for granting a research fellowship to MSR (304724/2010-6). less than 50 ha. The restriction on the amount of appropriate areas for Finally, we thank David Martin for editing this manuscript.

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