Population and genetic structure of two dioecious timber species Virola surinamensis and Virola koschnyi (Myristicaceae) in southwestern Costa Rica

Forest Ecology and Management 323 (2014) 168–176

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Population and genetic structure of two dioecious timber species Virola surinamensis and Virola koschnyi (Myristicaceae) in southwestern Costa Rica Pablo Riba-Hernández a,⇑, Jorge Lobo Segura b, Eric J. Fuchs b, Juan Moreira a a b

Proyecto Carey, Península de Osa, Puntarenas, Costa Rica. Apdo, 10672-1000 San José, Costa Rica Escuela de Biología, Universidad de Costa Rica, 2600 San Pedro, Costa Rica

a r t i c l e

i n f o

Article history: Received 22 December 2013 Received in revised form 6 March 2014 Accepted 10 March 2014 Available online 31 March 2014 Keywords: Dieocy Flowering sex ratios Myristicaceae Osa Peninsula Selective logging Spatial distribution

a b s t r a c t Selective logging regulations generally fail to account for sex ratios, sex size distribution, spatial patterns and genetic structure in dioecious timber species. Furthermore, sympatric congeneric dioecious tropical species are harvested under the same vernacular name, failing to account for potential variation in species population traits. This practice is expected to have deleterious consequences in the population density and reproduction of the least abundant species. Here we document density, sex ratios, sex size distribution, spatial patterns and genetic structure in two dioecious timber tree species, Virola surinamensis and V. koschnyi in southwestern Costa Rica. In addition, we assessed the probability that harvesting these two species under the same vernacular name will cause a significant decline in either sex density of the least abundant species, which is expected to unbalance sex ratios, therefore, reducing the reproductive potential of the species. In a 62 ha plot we tagged, geo-referenced and sampled for cambium tissue all adults of the two species (dbh > 30 cm) for genetic analyses. Microsatellites loci were used to describe genetic diversity parameters and spatial genetic structure. In a nuclear subplot (42 ha) we measured dbh and monitored sex expression during two reproductive events to describe population density, sex ratios, sex size distribution and spatial patterns. Adult density was twofold higher for V. surinamensis than V. koschnyi. The proportion of flowering males and females and diametric size distribution did not differ within species. Adults of both Virola species were spatially aggregated, but sexes were distributed randomly. We found a significant but weak spatial genetic structure for V. surinamensis, but not for V. koschnyi. Finally, there is a high probability (Multivariate hypergeometric distribution, p = 0.47) that harvesting these two species under the same vernacular name will cause a drastic decline in the density of male or female trees of V. koschnyi. Overall our results suggest that dioecy does not influence tree size or spatial distribution of these two timber species. The weak spatial genetic structure in V. surinamensis is likely due to clumped seed dispersal and absence of thinning during the recruitment of genetically related seeds to the adult stage. Harvesting these two species under the same vernacular name will have important consequences in the reproduction of V. koschnyi. We suggest that selective logging regulations for dioecious species should encourage appropriated species identification, ascertain the sex of reproductive individuals, harvest these species in proportion to their sex ratios and reduce the proportion of harvested individuals in the population. Ó 2014 Elsevier B.V. All rights reserved.

1. Introduction Dioecy is the separation of male and female functions in different individuals. This reproductive strategy has higher incidence in woody plants from tropical area and island (Bawa, 1980; Sobrevila and Kalin Arroyo, 1982; Kress and Beach, 1994). Conservation and ⇑ Corresponding author. Tel.: +506 88506222; fax: +506 2330151. E-mail address: [email protected] (P. Riba-Hernández). http://dx.doi.org/10.1016/j.foreco.2014.03.018 0378-1127/Ó 2014 Elsevier B.V. All rights reserved.

management strategies of tropical forest areas must take into account this reproductive strategy as a factor that influences population structure parameters and reproduction of many tropical plants, in particular, tree species. In undisturbed conditions the population structure of dioecious plants is expected to be influenced by intersexual differences on life history traits (Lloyd and Webb, 1977). For instances, it has been shown that females allocate more resources (e.g. biomass) to reproduction than males (see Obeso, 2002, for a review). This sex-biased resources

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allocation is predicted to affect not only flowering frequency (e.g. Bullock and Bawa, 1981; Armstrong and Irvine, 1989; Thomas and LaFrankie, 1993), but also female resources investment for growth and protection against herbivores. Generally, female plants are expected to have lower growth rates (e.g. Cipollini and Whigham, 1994) or higher mortality (e.g. Allen and Antos, 1988) than males. In addition, it is expected that females predominate were resources are limited to compensate for the investment on reproduction, which may lead to generate spatial segregation of sexes (SSS) along environmental gradients (Bierzychudek and Eckhart, 1988). For tropical dioecious trees, biased and balanced sex ratios have been found to be equally likely (Opler and Bawa, 1978; Ackerly et al., 1990; Wheelwright and Bruneau, 1992; Thomas and LaFrankie, 1993; Queenborough et al., 2007a; Amorim et al., 2011; Fernandez-Otarola et al., 2013). Whenever sex ratio is unbalanced, male bias is expected (Opler and Bawa, 1978; Armstrong and Irvine, 1989; Ackerly et al., 1990; Thomas and LaFrankie, 1993; Nicotra, 1998; Somanathan and Borges, 2000; Lenza and Oliveira, 2006; Queenborough et al., 2007a). However, female-biased sex ratios have also been reported (Melampy and Howe, 1977; Thomas, 1997; Forero-Montaña et al., 2010). Males have not shown either ‘‘precocious’’ reproduction (Thomas and LaFrankie, 1993; ForeroMontaña et al., 2010), or size differences relative to female plants (Queenborough et al., 2007a; Pavón and Ramírez, 2008; ForeroMontaña et al., 2010). Finally, spatial segregation of sexes (SSS) seems to be rare in tropical tree species (Bullock, 1982; Queenborough et al., 2007a; Forero-Montaña et al., 2010). In general, the above exceptions to theoretical expectations regarding sex ratios, plant size and spatial segregation of sexes suggest that not all tropical dioecious trees species respond similarly to reproduction costs, rather it appears that other sex-independent factors may shape the population structure of dioecious trees (Thomas and LaFrankie, 1993; Forero-Montaña et al., 2010; Field et al., 2012). Spatial aggregation is a common feature of tropical trees, including dioecious species (Condit et al., 2000; Queenborough et al., 2007b). Spatial aggregation is also common between sexes in tropical dioecious species (Bullock, 1982; Mack, 1997; Queenborough et al., 2007a; Forero-Montaña et al., 2010). In tropical tree species it has been attributed to seed dispersal limitation, as most seeds fall in close proximity of maternal trees (Janzen, 1970; Schupp et al., 2002). One potential consequence of an aggregated spatial arrangement is the non-random distribution of related individuals over short distances, resulting in spatial genetic structure (SGS). In spite of the potential of SGS on inbreeding and genetic drift in plant populations (Schnabel et al., 1998), few studies have evaluated the presence of SGS in tropical dioecious trees. Current evidences suggest weak SGS in adults of tropical vertebrate-dispersed dioecious trees (Hardesty et al., 2005; Hardy et al., 2006). Dioecy is expected to increase the vulnerability of trees threatened by anthropogenic disturbances (Vamosi and Vamosi, 2005). In species with this obligated outcrossing breeding system, reproduction is susceptible to changes in population structure such as sex ratio (House, 1992; Osunka, 1999), male–female distance (de Jong et al., 2005), individual size (Bullock and Bawa, 1981; Somanathan and Borges, 2000; Fernandez-Otarola et al., 2013) and pollinator abundance or behavior (House, 1993; Somanathan and Borges, 2000). Therefore, any anthropogenic activity that modifies any of these factors could affect the long-term viability of dioecious plants. Selective logging is by far the most common management strategies to exploit commercial timber trees in tropical regions (Putz et al., 2012). Harvesting strategies are based on two main parameters, the proportion of adult trees extracted per species per area over a minimum dbh size threshold and the frequency of cutting cycles. Frequently, these technical parameters are applied regardless of the species reproductive biology; as a consequence it is difficult to

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predict the viability of residual populations after logging. This problem is emphasized in dioecious trees, because selective logging is conducted without any information on the sex of harvested individuals. Information on the effect of selective logging on tropical dioecious timber trees is still limited, but a few studies suggest that sex ratios, size and spatial distributions are modified in residual populations after extraction (Macedo and Anderson, 1993; Somanathan and Borges, 2000; Sebbenn et al., 2008). The use of vernacular names is a common non-technical tree identification system used by logging companies to conduct forestry inventories. This system is well established in a few tropical regions (Swaine and Agyeman, 2008), however it has been shown to be highly inaccurate (Lacerda and Nimmo, 2010). One of the fundamental flaws of this system is the variation of local knowledge on tree species identification; common names may represent several species within a genus or even represent species from different genera (Barrantes et al., 1999; Lacerda and Nimmo, 2010). Consequently, the use of vernacular names generates misleading harvesting parameters, compromising the long-term viability of harvested species. In particular, its impact is expected to be higher on timber species that occurs at lower densities (Lacerda and Nimmo, 2010). In this study, we describe the population structure and adult spatial genetic structure of two timber dioecious tree species in the Virola genus in a continuous forest. In particular we evaluate the following questions; 1) Do population sex ratios deviate from unity? 2) Do males have a different size distribution than females? 3) Are sexes spatially segregated? 4) Are these two species spatially aggregated at the population level? and 5) Is there any evidence of spatial genetic structure in the adult population of these two dioecious trees species? Finally, we estimated the likelihood that harvesting these two species under the same vernacular name will cause a significant decline in the density of male or female trees of the less abundant species. 2. Methods 2.1. Study site This study was conducted in a tropical humid forest (Holdridge et al., 1971), located at the Punta Rio Claro Wildlife Refuge, Osa Peninsula, southwestern Costa Rica (8°390 N, 83°440 E), adjacent to the Golfo Dulce Forest Reserve (61,702 ha). This Refuge encompasses a total area of 247 ha, with about 90% of it covered by mature forest, while the rest is represented by secondary forest in advance stages of regeneration and open areas. The climate of the Osa Peninsula is characterized by high precipitation levels (3500–5000 mm), with a dry season between January and March, and the highest precipitation occurs during August to October. The temperature ranges from 21 to 33.5 °C (Lobo et al., 2008). Steep slopes dominate the topography and soils are mostly ultisols (Weissenhofer and Huber, 2001). 2.2. Study species Virola surinamensis (Rol.) Warb., is a dioecious canopy tree distributed from Costa Rica to the Amazon basin. In Costa Rica it is found in both the Pacific and Atlantic slopes (Quesada et al., 1997). In Panama this species is associated with slopes and streams (Harms et al., 2001). Flowering occurs between November and January (P. Riba-Hernández, unpubl. data). In Brazil, flowers are reported to be pollinated by two species of flies, Copestylum sp. and Erystalys sp. in the Syrphidae family (Jardim and Mota, 2007). In the Osa Peninsula fruits mature between May and July (P. RibaHernandez, unpubl. data). Fruits are woody capsules containing a

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single arillated large seed (mean size  23.3 mm). In our study site, vertebrates such as the Chestnut-mandibled toucans (Ramphastos ambiguus), spider monkeys (Ateles geoffroyi), and crested guans (Penelope purpurascens) are the predominant seed dispersers (P. Riba-Hernandez, unpubl. data), however hydrochory has also been suggested as a likely mean of seed dispersal (Moegenburg, 2002). This species is listed as endangered by IUCN (IUCN 2010). Virola koschnyi Warb., is distributed throughout Central America, from Guatemala to Panamá and Ecuador. In Costa Rica it is found in both the Pacific and Atlantic slopes (Quesada et al., 1997). In the Osa Peninsula flowering occurs between January and February (Lobo et al., 2008). Pollination is probably conducted by small insects (Bawa et al., 1985). Fruiting occurs between June and August (Lobo et al., 2008). Fruits are woody capsules, containing a single arillated large seed (mean size  20.4 mm). In our study site, seeds are dispersed by the same assemblage of vertebrates that disperse V. surinamensis (P. Riba-Hernandez, unpubl. data) and those reported for other Virola species (Howe and Van De Kerckhove, 1981; Russo, 2003; Holbrook and Loiselle, 2009). The Virola genus has been extensively exploited for timber over its geographic distribution (Macedo and Anderson, 1993; Barrantes et al., 1999; Piña-Rodrigues, 1999; Nebel and Meilby, 2005). In southwestern Costa Rica, specifically in the Osa Peninsula, 475 Virola trees (dbh > 50 cm), representing about 2065 m3 of wood, were legally logged under selective logging procedures between 1996 and 1999 (Barrantes et al., 1999). 2.3. Logging practices and regulations in Costa Rica In Costa Rica, selective logging is based on harvesting marketable tree species (dbh > 50 cm) represented locally at densities greater than 0.3 individuals ha1. Harvestable stock densities per species are estimated based on a forestry inventory on the property to be managed. Up to 50% of the local population can be extracted in a single cutting episode, with following cutting cycles every 15 years (Lobo et al., 2007). Vernacular names are commonly used to identify commercial timber species, both at forestry inventories and in the government resolution that authorizes wood harvesting. V. surinamensis and V. koschnyi are grouped under the same vernacular name (‘‘fruta dorada’’) in forest inventories and legal permits for timber extraction (Barrantes et al., 1999). 2.4. Sampling All individuals (dbh > 30 cm) within a 62 ha plot (1000 m  620 m) were tagged and geo-referenced. We used this minimum dbh threshold, because smaller individuals have not been observed to flower (P. Riba-Hernandez, personal observation). In a nuclear subplot of 42 ha (700 m  600 m), all individuals were sexed, measured and monitored for sex expression for two consecutive flowering events (2011 and 2012). Sexual expression was assessed by examining flower structures under a stereoscope. At least ten flowers were collected under different sections of the canopy for each individual. Trees were monitored twice during flowering seasons to ascertain their sex. 2.5. DNA extraction and microsatellite amplification All individuals located in the 62 ha plot were sampled for genetic analyses. A small piece of cambium (1 cm2) was collected from each tree bole and stored in a sealed tube filled with silica gel. DNA was extracted using the plant DNEasy™ kits (QIAGEN). Microsatellites developed by Draheim et al. (2009) were amplified by PCR multiplex reactions using QIAGENÒ Multiplex PCR (QIAGEN) with fluorochromated oligonucleotides. Six microsatellites were amplified for V. koschnyi and five for V. surinamensis. 13 ll reaction

volumes were used in two amplification sets. For V. surinamensis the first amplification set contained a mix of primers of the loci Vsur34 and Vsur58, and the other set contained the primers Vsur2-35, Vsur56 and Vsur2-41, each at concentration 0.2 lM. For V. koschnyi, the first amplification set contained a mix of primers Vsur34, Vmul2-65, and Vmul2-66, and the other set contained the primers Vsur58, Vseb3 and Vsur2-55, each at a concentration of 0.2 lM. PCR reactions included an initial activation step of 95 °C at 15 min, followed by 30 cycles of 30 s of 94 °C, 90 s of 57 °C, 90 s at 72 °C, with a final extension step at 72 °C 10 min. PCR products were visualized in an ABI 310 Sequencer (Applied Biosystems). Fragment sizes were determined using GeneScan Liz 500 (Applied Biosystems). Alleles were scored manually using GeneMaker Software version 1.80 (SoftGenetics). 2.6. Statistical analysis Following Wilson and Hardy (2002), sex ratios were expressed as the proportion of males in the population: [males/(females + males)]). Deviation from equal sex proportions was tested with a G-test goodness of fit test. Cumulative sex ratios represented the total number of flowering individuals over the two reproductive events. For each sex, we determined the frequency of individuals in diameter categories in 10 cm increments. Deviation from unity in sex ratios by diametric classes was tested with a Chi-square test of heterogeneity. Differences in diameter size distribution between sexes were tested with a Kolgomorov-Smirnov two-sample test. Statistical analyses were performed in Statistica 7 (Statsoft, 2004). 2.7. Spatial analysis Spatial patterns at the population level and spatial interaction between males and female individuals were analyzed using Ripley’s K(t) and K12(t) functions. The K(t) function estimates the average number of trees in a given distance t of each point. The average number estimated by the K(t) function is compared to expected values under the null hypothesis of Complete Spatial Randomness (CSR). Monte Carlo procedures are used to create confidence envelopes to test if K(t) values are congruent with CSR. To reduce scale dependencies and stabilize variances, we used the L(t) square root transformation of K(t) suggested by Besag (1977). We evaluated the L(t) function in 10 m intervals for t distances ranging from 1 to 350 m, which is half the distance of the shortest side of our rectangular plot, isotropic edge correction was used in all cases (Ripley, 1991). Confidence envelopes were constructed from 999 realizations of the Poisson process (i.e. CSR). In L(t) plots spatial aggregation is assumed if L(t) > 0, while negative values indicate a tendency towards regularity. L(t) values within the confidence envelopes are consistent the null hypothesis of CSR (Bivariate Poisson process). The spatial interaction (association or repulsion) between males and female trees was analyzed with Ripley’s second order K12(t) function. K12(t) estimates the expected number of type 1 points (males in this study) within a t distance of a randomly chosen type 2 point (females). As in previous analyses, K12(t) functions were square root transformed to L12(t). Spatial interaction was evaluated for t distances between 0 and 350 m and an isotropic edge correction was also applied. For bivariate Ripley’s K12(t) we followed the procedures implemented in Nanami et al. (2005), Schmidt (2008), Zhang et al. (2010) and chose ‘random labeling’ as the null hypothesis to construct confidence envelopes (Goreaud and Pélissier, 2003). Confidence envelopes were constructed from 999 permutations. In each permutation females and males were assigned randomly to the spatial position of reproductive individuals. Spatial association between males and females is suggested if values of L(t) > 0. Conversely, L (t) < 0 indicates repulsion between male

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and female trees. If L(t) values fall within the confidence envelope, the spatial relationship between males and females is deemed random. All spatial analyses were conducted using the spatstat library (Baddeley and Turner, 2005) implemented in the R programming language (R Development Core Team, 2012). In all cases we tested curve-wise significance of deviations from null hypotheses with the goodness-of-fit test proposed by Loosmore and Ford (2006) and also described in Diggle (2003) and implemented in the dclf.test() function in spatstat. In all accounts 999 realizations of a Poisson random process were used to assess the significance of the dclf test. 2.8. Genetic analyses Genetic diversity in V. surinamensis and V. koschnyi populations was quantified using the following parameters: allele number averaged across loci (Na), the number of effective alleles averaged across loci (Ne), observed (Ho) and expected heterozygosities (He). These parameters were estimated using SPAGeDI (Hardy and Vekemans, 2002). To study the relationship between spatial distance and genetic relatedness in these two species spatial autocorrelation analysis were used as implemented in the software SPAGeDi (Hardy and Vekemans, 2002). Autocorrelation analyses measure genetic similarity between all pairs of individuals separated by a specific geographic distance. For each distance class the mean and the confidence intervals of genetic relatedness were calculated and its statistical significance was assessed. Distance classes ranged from 0 to 250 m, in 25 m intervals, including a final interval of all pairwise comparisons between trees separated by more than 250 m. As a measure of genetic similarity, the relationship coefficient (rij) proposed by Queller and Goodnight (1989) was used, that measures the proportion of gene copies in one individual that are identical by descent to those of a reference individual. To test the hypothesis of random distribution of genotypes in space, observed rij values were compared to the 95% confidence interval of rij values under the assumption of no spatial genetic structure, estimated through random shuffling of all individuals among all geographic locations. We performed 10,000 random permutations for null distributions of rij values for each distance class. 2.9. Evaluating selective logging practices on sex frequency We used the multivariate hypergeometric distribution to evaluate the probability that selective logging will decrease the frequency of either sex to values lower than 0.1 individuals ha1 in the least abundant Virola species. This value represents a tenfold reduction of the estimated population size of V. koschnyi in this study (see results). In dioecious tree species a significant reduction in sex density, together with an increase in distance between

sexes, may result in a significant decline of fruit production (Mack, 1997; Somanathan and Borges, 2000; de Jong et al., 2005). This effect is expected in the study species, because apomictic fruit production has been reported as limited in this genus (Lenza and Oliviera, 2006). The multivariate hypergeometric probability distribution calculates the probability of the different outcomes of random sampling without replacement from a finite population (i.e. selective logging). Given the total population of Virola individuals (N, (i.e. pooled harvestable population of the two studied species (dbh > 50 cm) in the 42 ha plot), the number of males and females of the rarer species, K1 and K2 respectively, the number of individuals (males and females) of the most common species N  K1  K2, and the size of the population to be harvested (n = 0.5 * N); we can calculate the probability of obtaining k1 males and k2 females of the rarer species in a sample of size n of this population by:

 Prðk1 ; k2 Þ ¼

K1



k1

K2 k2



N  K1  K2



n  k1  k2 

N

 ð1Þ

n Then, the remaining number of males (K1  k1) and females (K2  k2) in the population of the rare species can be calculated. We added the probability of all logging events that results in (K1  k1)/42 or (K2  k2)/42 values of less than 0.1, where 42 represents the area extension of the nuclear plot. 3. Results 3.1. Density and flowering constancy We mapped 235 V. surinamensis individuals and 90 V. koschnyi individuals (dbh > 30 cm) in the 62 ha plot. A subset of 154 V. surinamensis trees and 66 V. koschnyi trees were monitored for sex expressions in two consecutive reproductive events (2011 and 2012) within the 42 ha plot. V. surinamensis had twice as much stem density than V. koschnyi, both stem density and harvestable stock (dbh > 50 cm) (Table 1). In two reproductive episodes we observed 33 (21.4%) V. surinamensis trees and 14 (22.4%) of V. koschnyi trees that did not flower; these individuals were considered as non-reproductive members of the population (Table 1). Out of 121 V. surinamensis reproductive individuals (dbh > 30 cm), 91 (76%) flowered over the two reproductive episodes, 41 were females and 50 were male trees, which represented 75% and 78% of all reproductive females and males, respectively. Likewise, 37 V. koschnyi trees flowered in both sampled years; 16 females and 21 males flowered consecutively in both reproductive events, representing 64% and 84% of all reproductive females and males, respectively. In both species, none of the reproductive individuals switched sex over the two reproductive events.

Table 1 Population density, annual flowering frequency and cumulative sex ratios of two dioecious timber tree species (Virola, Myristicaceae) during two reproductive events (2011– 2012) in a 42 ha plot located at the Osa Peninsula in southwestern Costa Rica. Density values represents a) the number of adults trees with dbh > 30 cm. This size threshold represents the minimum flowering dbh size observed in our study b) the number of harvestable individuals (dbh > 50 cm). Numbers in parenthesis represent the total number of individuals in each size category. Non-fl is the number of individual (dbh > 30 cm) that did not flower in a given year. Nrep indicates the number of reproductive individuals in the harvestable size category. Sex ratio is estimated as = males/(males + females). NS denotes p > 0.05 (G-test goodness of fit). Year

Cumulative

2011 Species

Virola surinamensis Virola koschnyi a

Density (ind/ha) dbh > 30 cm

dbh > 50 cm

3.7 (154) 1.6 (66)

1.5 (64) 0.6 (24)

Individuals dbh > 30 cm.

2012

Sex Ratioa

Non-fla

Nrep dbh > 50 cm

Sex ratioa

Non-fla

Nrep dbh > 50 cm

Sex ratioa

Non-fla

0.57NS 0.50

57 15

56 24

0.61NS 0.60NS

52 26

55 21

0.54 0.50

33 15

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3.2. Sex ratios We failed to observe inter-annual variation in sex expression in both species (Table 1). V. surinamensis had a slightly greater proportion of flowering males than females in both reproductive events, particularly in 2012, however this pattern did not significantly deviate from unity (Table 1). V. koschnyi showed a similar proportion of flowering males and females in 2011, with a slight but not significant male-biased in 2012 (Table 1). The cumulative sex ratio for both species did not deviate significantly from a 1:1 ratio (Table 1). In addition, sex ratios were approximately 1:1 across diameter classes in both species (V. surinamensis, X 2heterogeneity ¼ 6:96, d.f. = 5, p > 0.05; V. koschnyi, X 2heterogeneity ¼ 8:99, d.f. = 5, p > 0.05) (Fig. 1).

3.3. Diametric size distribution Most non-reproductive individuals in both species were trees smaller than 40 cm dbh (28 and 8, for V. surinamensis and V. koschnyi, respectively) (Fig. 1). The mean diameter size (mean ± SD) of reproductive individuals was 52.6 ± 17.3 for V. surinamensis and 53.8 ± 13.5 for V. koschnyi. The total number of reproductive individual in the population with a dbh > 50 cm (i.e., minimum cutting diameter of selective logging in Costa Rica) was 62 for V. surinamensis and 24 trees for V. koschnyi, which respectively

represents 47% and 49%, of reproductive individuals. Reproductive individuals represent the majority of the harvestable stock (V. surinamensis (85–87%) and V. koschnyi (100–87%)) (Table 1). We did not find significant differences in diameter size distribution between sexes in any of the two study species (V. surinamensis (D = 0.13, p > 0.1), V. koschnyi (D = 0.15, p > 0.1) (Fig. 1). 3.4. Spatial distribution At the species level, both V. surinamensis and V. koschnyi were spatially aggregated (Fig. 2). Curve-wise goodness-of-fit test (dclf. test) rejected the CSR null hypothesis in both accounts (p < 0.001) (Supp. Material, Fig. 1). We did not find any evidence of spatial interaction between males and females neither for V. surinamensis nor for V. koschnyi (Fig. 2). In both species, spatial interactions between males and females did not deviate from random expectations (Goodness-of-fit test for V. surinamensis (p = 0.4) and for V. koschnyi (p = 0.2)) (Supp. Material, Fig. 2). 3.5. Genetic diversity and structure We observed a total of 123 alleles (range: 20–34 alleles per locus) in V. surinamensis and 98 microsatellite alleles (range: 9–39 alleles per locus) in V. koschnyi (Table 2). Both species showed high expected heterozygosities (Table 2), with lower genetic

(a) Non-reproductive Females

Individuals

Males

30-40

40-50

50-60

60-70

70-80

80-90

90-100

Diameter class (cm)

(b) 20

individuals

18

Non-reproductive

16

Females

14

Males

12 10 8 6 4 2 0

30-40

40-50

50-60

60-70

70-80

80-90

Diameter class (cm) Fig. 1. Frequency distribution of stem diameters (dbh > 30 cm) for each sex and for non-reproductive individuals during two reproductive events of two dioecious timber species (Virola, Myristicaceae) in a 42 ha old growth forest plot in the Osa Peninsula, Southwestern Costa Rica (2011–2012). The graph is based on cumulative sex expression for two consecutive reproductive events (2011–2012), a) V. surinamensis, and b) V. koschnyi.

Fig. 2. Spatial distribution of males (s), females (d) and non-reproductive individuals (D) with dbh > 30 cm, for two timber tree species of the genus Virola in a 42 ha plot located at the Osa Peninsula, Costa Rica. (a) Virola surinamensis and (b) V. koschnyi. Point size is proportional to diameter size.

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diversity levels in V. koschnyi (He = 0.793, s.e. = 0.033) relative to V. surinamensis (He = 0.896, s.e. = 0.018). In V. surinamensis, fixation indexes (F) were low for all loci with a mean value (F = 0.044) close to their standard error (s.e. = 0.020), suggesting Hardy Weinberg equilibrium. However, we found a significant deficit of heterozygotes for most loci in V. koschnyi, resulting in a high mean F value (F = 0.132, s.e. = 0.036), more than two times higher than its standard error (Table 2). Because Hardy–Weinberg is not a prerequisite to use the Queller and Goodnight relationship coefficient (Hardy and Vekemans, 2002), we decided to conduct the analysis for V. koschnyi. No spatial genetic structure was detected in V. koschnyi adults (Fig. 3). In contrast, relatedness in V. surinamensis between individuals located between 25 and 50 m was low (0.04) but statistically significant. However, relatedness values decreased, as expected, as distance between trees increased (Fig. 3). This pattern suggests a weak genetic structure in the adult population of V. surinamensis.

(a)

(b)

3.6. Selective logging effect on sex ratios In the 42 ha study plot, Costa Rican selective logging practices would allow the cutting of 43 out of 86 Virola trees identified under the same vernacular name (50% of all trees dbh > 50 cm named ‘‘fruta dorada’’) (Table 1). In this plot only 13 males and 11 females V. koschnyi trees have dbh > 50 cm, in contrast to higher sexes densities found in V. surinamensis (28 males and 34 females). There is a high probability (multivariable hypergeometric distribution, p = 0.47) that removing 43 harvestable trees without distinguishing between both Virola species will drastically reduce the residual male or female density in V. koschnyi to values lower than 0.1 ha1. 4. Discussion Given the extension of the surveyed area (50 ha), this study represents an accurate report of the population density and sex ratios of these two Virola species in the Osa Peninsula, Costa Rica. Furthermore, differences in population densities for these two species are congruent with abundance estimates at other sites within the Osa peninsula and Golfo Dulce region (Thomsen, 1997; E. Chacón, (1–6 ind ha1 V. surinamensis and 0–4 ind ha1 for V. koschnyi), personal communication), confirming that V. surinamensis is more common that V. koschnyi in this area. Additionally, this study represents the first report of sexual expression and hence sex ratios of these two species in the region.

Fig. 3. Spatial genetic structure autocorrelograms for adult trees (dbh > 30 cm) of (a) Virola surinamensis (N = 235) and (b) Virola koschnyi (N = 90), in a 62 ha plot located in the Osa Peninsula, Costa Rica. Distance values represent the end point of intervals at 25 m increments. Black squares represent relatedness coefficients (r) values for each distance class. Dotted lines represent 95% confidence intervals for a spatially non-structured population.

We found that dioecy does not have an effect on the population structure of these two timber species. Our findings suggest that males and females have similar sizes (i.e. dbh), flowering frequencies and are random distributed. It has been suggested that tropical dioecious trees commonly show male biased sex ratios. Contrariwise, there is also evidence of sex ratios that not deviate from unity (e.g. Opler and Bawa, 1978; Bullock, 1982; Bullock et al., 1983; Morellato, 2004; Pavón and Ramírez, 2008). Several proximate causes explain maleness in dioecious species: (a) males can flower

Table 2 Genetic diversity estimates for adults (dbh > 30 cm) of two dioecious timber tree species (Virola, Myristicaceae) in the Peninsula de Osa, Costa Rica. Individuals were surveyed in a 62 ha mature forest plot. Diversity parameters are shown by locus, using microsatellites primers developed by Draheim et al. (2009). A total of 235 individuals of V. surinamensis and 90 of V. koschnyi were surveyed. Sample size (N), number of alleles averaged across loci (Na), number of effective alleles averaged across loci (Ne), observed heterozygosity (Ho), genetic diversity (He), and fixation index (F). Mean values are reported with standard errors in parentheses. **p < 0001. Locus

N

Na

Ne

Ho

He

F

Virola surinamensis Vsur34 Vsur58 Vsur56 Vsur2–35 Vsur2–41 Mean (SE)

211 214 203 203 190

20 24 20 25 34 24.6(2.5)

9.07 6.05 9.78 11.93 17.44 10.8 (1.9)

0.891 0.762 0.833 0.857 0.953 0.858 (0.031)

0.890 0.835 0.898 0.916 0.943 0.896 (0.018)

-0.001 0.088⁄⁄ 0.073⁄⁄ 0.064⁄⁄ -0.011 0.044 (0.020)

Virola koschnyi Vsur34 Vmul2.65 Vmul2.66 Vsur58 Vseb3 Vsur2.55 Mean (SE)

84 89 87 89 90 89

39 12 9 11 9 18 16.3 (4.7)

12.97 4.91 3.44 4.06 3.64 6.72 5.9 (1.4)

0.905 0.775 0.552 0.596 0.611 0.719 0.693 (0.054)

0.923 0.797 0.710 0.754 0.726 0.851 0.793 (0.033)

0.020⁄⁄ 0.027⁄⁄ 0.223⁄⁄ 0.210⁄⁄ 0.158 0.155⁄⁄ 0.132 (0.036)

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more frequently than females (e.g. Bullock and Bawa, 1981; Bullock, 1982; Thomas and LaFrankie, 1993; Nicotra, 1998), (b) males are able to flower at a smaller diametric size than females (‘‘precocious males’’) (e.g. Opler and Bawa, 1978; Bullock and Bawa, 1981; Armstrong and Irvine, 1989; Ackerly et al., 1990; Queenborough et al., 2007a), (c) females have greater mortality rates than males (e.g. Allen and Antos, 1988) and (d) sexes are spatially segregated (e.g. Bierzychudek and Eckhart, 1988). We found that sex ratios in our two study species did not deviate significantly from unity; therefore none of the above expectations were supported by our findings. Flowering frequency was similar for both sexes and for both species. Both study species showed similar size distribution of flowering individuals between sexes, indicating that males are not precocious. Also, similar size distributions in both male and female plants suggest that mortality may not be greater for females. In fact, there is little evidence in tropical regions that mortality differs between sexes in dioecious trees (Bullock, 1992; Nicotra, 1998). Finally, in agreement with other tropical dioecious species, we found no evidence of spatial segregation of the sexes (SSS) in both Virola species. In contrast, we found that males are randomly distributed relative to females (Nicotra 1998; Queenborough et al., 2007a; Forero-Montaña et al., 2010). Spatial segregation is suggested to result from resources that show an extreme gradient in their availability, thus it is expected that sex frequencies segregate in this gradient according to their resources needs (Freeman et al., 1976; Bierzychudek and Eckhart, 1988). The distribution of nutrients in the study plot is unknown, however another study site estimated nutrients distribution and in this case males and females trees did not show any spatial association with nutrient gradient (Forero-Montaña et al., 2010). Additionally, in a different site, the relationship between habitat preference by one sex (i.e. topography) and fecundity, as a proxy of SSS (Bullock, 1982), has also failed to find a clear effect on spatial segregation of the sexes (Queenborough et al., 2007a). Thus, the general evidences suggest that SSS seems to be a rare phenomenon for tropical dioecious trees. Random spatial distribution of sexes has also been found in four other Virola species (Queenborough et al., 2007a). The random spatial distribution between sexes even if species are spatially aggregated at the population level, may be explained because sex determination is likely to be genetic in origin (Ainsworth, 2000). The sex of the seed is determined pre-dispersal and sexual expression does not influence seed dispersal nor germination and recruitment of adults from seeds. The similarities in life history traits between sexes suggest that females can compensate for the cost of reproduction and/or male reproduction is as costly as female reproduction. Several compensation mechanisms have been proposed for females in tropical dioecious species, such as resource reabsorption (Pavón and Ramírez, 2008), longer leaf live span or higher rates of resources assimilation (Nicotra, 1999). However, these strategies need to be validated in our study species. In agreement with other studies on tropical tree species, the two Virola tree species in our study are spatially aggregated (Condit et al., 2000). This pattern has been described for other species in the genus (Russo and Augspurger, 2004; Queenborough et al., 2007b). Contagious seed dispersal by vertebrate frugivores may maintain this clumped distribution (Schupp et al., 2002). Spider monkeys (Ateles geoffroyi) and toucans (Ramphastos ambiguus) are the most important seed dispersers in our study site for these two species (P. Riba-Hernandez, unpubl. data). These seed dispersers may cause an aggregated spatial distribution by depositing seeds close to females and sleeping/roosting sites, as reported for other Virola species (Russo and Augspurger, 2004). Although density and distance dependent mechanism may reduce the survival of seeds and seedling in close proximity of adults and sleeping sites, the clumped distribution persists to older life stages even after thinning of the population through mortality (Russo and

Augspurger, 2004; Fuchs et al., 2013). The persistence of this clumped distribution may open the possibility that related individuals in close proximity survived to adulthood. However, only V. surinamensis showed significant spatial genetic structure. A greater density of reproductive females of V. surinamensis compared with V. koschnyi and larger seeds in V. surinamensis probably promote distance-restricted seed dispersal from the same maternal tree (Manasse and Howe, 1983). The low mean coefficient of relatedness observed for V. surinamensis individuals located between 25 and 50 m, suggests that only a few individuals within this distance interval are related. Variation on selective thinning (e.g. mortality of juveniles), long-distance seed dispersal events and extended pollen flow may contribute to shape the heterogeneity in family structure. Finally this study follows the general idea that tropical trees are characterized by having high levels of genetic diversity (Dick et al., 2008). Although genetic diversity data on dioecious tropical trees is still limited, the adult population of the study species showed higher values of genetic diversity compared to the population of adults of other dioecious tropical species with similar life history traits (Hardesty et al., 2005). 4.1. Implications for selective logging in dioecious species Our results indicated that population densities and genetic structure of these two congeneric sympatric species are very different and the consequences of logging these species under the same vernacular name will affect mostly the rarer species, in this case V. koschnyi. Given the unbiased sex ratio parameters observed for V. koschnyi and the annual variation in flowering frequency, we predict that selective logging procedures can have important implications on the reproduction of this species by reducing the frequency of either of the two sexes in the residual population and the random removal of a large fraction of reproductive individuals. Significant male or female-biased sex ratios can result after logging dioecious species, as is evidence by our findings and others (e.g. Somanathan and Borges, 2000; Sebbenn et al., 2008). A biased sex ratio may hamper reproduction by reducing the effective reproductive population, influencing female fecundity (i.e. production of flowers and fruits) and/or increase pollen limitation through an increase in the distance among reproductive individuals, as shown for other dioecious trees in altered habitats (Somanathan and Borges, 2000). The possible deleterious effects of selective logging are intensified by the relative short time between cutting cycles (15 year) (Sebbenn et al., 2008), which will prevent the recruitment of juveniles into the reproductive cohort, given the slow growth rate reported for V. koschnyi, 0.3–9 mm year1 (Lieberman et al., 1985). Therefore, altering sex ratios through harvesting without prior knowledge of sex expression is more likely to affect the less dense species. This is more likely to occur if selective logging practices fail to differentiate the rare species from the abundant one. Thus, misidentification of species is particularly detrimental in dioecious species where relative densities may seriously impact sex ratios in the population. In addition selective logging can have an important effect on the potential regeneration of the population of both species, because it targets the reproductive cohorts in the population (Franco and Silvertown, 2004). A potential consequence will be a reduction in the number of ‘‘seed trees’’ in the residual population. If fruit production is related to tree size (e.g. dbh), as it has been describe for other Virola species (Fernandez-Otarola et al., 2013), there is unlikely to be enough time between cutting cycles for the residual population to grow and become reproductive, resulting in reduced fruit production (Plumptre, 1995). On the other hand, if reproduction (i.e. pollen and fruit production) is concentrated on a few dominant individuals (Bullock, 1982) and if these individuals are

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included in the harvestable stock and are thus extracted, this could have a predominant and negative effect on the reproduction of the population. These effects will be stronger in V. koschnyi as the density of larger individuals (>80 cm) declines as size increases in the population. 5. Conclusions Our results suggest that incorrect identification or the use of vernacular names to identify timber species can lead to logging sympatric congenerics with very different population and genetic structure, under the same harvesting parameters. This practice can affect the future reproduction of the least abundant species, especially in the case of dioecious trees. Therefore, we suggest that selective logging regulations for dioecious species should include: (i) proper identification of timber species during forest inventories, (ii) ascertain sexual expression of reproductive individuals, (iii) harvest comparable proportions of reproductive individuals from both sexes as to maintain sex ratios, and (iv) a reduction in the proportion of harvestable individuals; extraction parameters should consider the number of reproductive individuals in the population and sex ratios of flowering individuals in order to preserve the potential of regeneration and population grow after extraction. Finally more information is needed regarding the relationship between fecundity, growth rates and size (e.g. dbh) to determine the effect of minimum felling diameter on the long-term regeneration capacity of timber species. Acknowledgments This study was supported by grants from International Student Volunteers, Inc. (ISV), Consejo Nacional para investigaciones Científicas y Tecnología (CONICIT), Ministerio de Ciencia y Tecnología (MICIT), Vicerrectoría de Investigación de la Universidad de Costa Rica (PIOSA Project Nos. 605-A4-954 and 111-B1-149), Universidad Nacional Autónoma de México (UNAM: PAPIIT project IN201011), and the Consejo Nacional de Ciencia y Tecnología (CONACyT 2009–131008 and 2010–155016). We thank Evelio Alvarez ‘‘Waneger’’ for his helpful field assistance during the establishment, measuring and tree censuses and Jenny Muñoz for her assistance on laboratory analysis. We appreciate the comments of two anonymous reviewers that improved early versions of the manuscript. Also we thank all volunteers and interns of Proyecto Carey that collaborate with the annual tree censuses. In addition, we highly appreciate the permission given from the owner of Punta Rio Claro Wildlife refuge to conduct this research in his property. Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.foreco.2014.03. 018. References Ackerly, D., Rankin De Merona, J.M., Rodrigues, W.A., 1990. Tree densities and sex ratios in breeding populations of dioecious Central Amazonian Myristicaceae. J. Trop. Ecol. 6, 239–248. Ainsworth, C., 2000. Boys and Girls come out to play: the molecular biology of dioecious plants. Ann. Bot. 86, 211–221. Allen, G.A., Antos, J.A., 1988. Relative reproductive effort in males and females of the dioecious shrub Oemleria cerasiformis. Oecologia 76, 111–118. Amorim, F.W., Mendes-Rodrigues, C., Maruyama, P.K., Oliveira, P.E., 2011. Sexual ratio and floral biology of the dioecious Neea theifera Oerst. (Nyctaginaceae) in a cerrado rupestre of central Brazil. Acta. Botanica. Brasilera. 25, 785–792. Armstrong, J.E., Irvine, K.A., 1989. Flowering, sex ratios, pollen-ovule ratios, fruit set: and reproductive effort of a dioecious tree, Myristica insipida (Myristicaceae), in two different rain forest communities. Am. J. Bot. 76, 74–85.

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