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Title: Genetic diversity and population genetic structure in three threatened Ocotea species
(Lauraceae) from Brazil's Atlantic Rainforest and implications for their conservation.
Authors: Martins EM* 12 , Lamont RW 3 , Martinelli G 1 , Lira-Medeiros CF 1 , Quinet A 1 and Shapcott A 3 .
Addresses: 1 Diretoria de Pesquisa, Instituto de Pesquisas Jardim Botânico do Rio de Janeiro, Rio de
Janeiro (RJ), Brasil.
2 Escola Nacional de Botânica Tropical - ENBT, Rio de Janeiro (RJ), Brasil.
3 GeneCology Research Centre, University of the Sunshine Coast, Maroochydore, Queensland, Australia.
Send proofs to:
Author for correspondence: *Eline M. Martins
Mailing address: Diretoria de Pesquisas, Instituto de Pesquisas Jardim Botânico do Rio de Janeiro, Rua
Pacheco Leão 915, 22460-030, Jardim Botânico, Rio de Janeiro, RJ, Brasil. Telephone number: + 55
2132042128; Fax number: +55 2138756206; e-mail: eline@cncflora.net
Keywords: Vulnerable species, conservation genetics, Ocotea catharinensis , Ocotea odorifera , Ocotea porosa , microsatellite markers, in-situ conservation, priority populations.
Running title: Genetic diversity of three Ocotea species
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Abstract
The Atlantic Rainforest in Brazil is currently comprised of small fragments due to the history of conversion and degradation in the last five centuries. The rainforest trees, Ocotea catharinensis , O. odorifera and O. porosa have been heavily harvested because of the high economic value of their timber and essential oils. Their respective habitats have undergone substantial reduction in area due to continuing anthropogenic pressures. As a consequence, these species have suffered large declines in population size and are now considered to be potentially vulnerable to extinction. This study investigated the patterns and levels of genetic diversity and inbreeding of these species using eight microsatellite markers in order to define priority populations for conservation management actions focusing on population enhancement and ex-situ germplasm collections. High genetic diversity was found for each of the species with moderate genetic differentiation among populations. Most populations displayed significant inbreeding and isolation by distance. The results provide important information to choose priority populations for both in situ and ex situ conservation measures.
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Introduction
The Atlantic Rainforest in Brazil, one of the world’s major hotspots of biodiversity (Myers et al.
2000), has suffered a large loss of primary habitat due to continuous anthropogenic pressures, with only
11% of the original area now remaining (Ribeiro et al. 2009). It is mainly comprised of small fragments
(<100 ha), many of which exhibit significant reductions in habitat quality and it is still subjected to processes of degradation (Ribeiro et al. 2009). Consequently, many species have experienced considerable declines in population size due to habitat loss and fragmentation of larger populations. This process has been further exacerbated by the selective logging in the remaining fragments (Lowe et al.
2005). Tree species such as Ocotea catharinensis Mez, O. odorifera (Vellozo) Rohwer and O. porosa
(Nees & Mart.) (Lauraceae) have been heavily harvested because of the high economic value of their timber and/or essential oils. They have undergone particularly large reductions in population size, to the point that they are currently classified as vulnerable to extinction by the IUCN Red List (Varty 1998;
Varty and Guadagnin 1998a; Varty and Guadagnin 1998b), The Official List of Brazilian Threatened
Plant Species (MMA 2008) and O. catharinensis as vulnerable, O. odorifera and O. porosa as endangered by the Red Book of Brazilian Flora (Martinelli and Moraes 2013).
Information regarding the population genetic structure of a species is an essential component for species recovery programs, because genetic-related factors may facilitate the species extinction
(Frankham and Ralls 1998; Oostermeijer et al. 2003; Leimu et al. 2006). For instance, population changes associated with habitat degradation and lack of species connectivity can cause an overall erosion of genetic diversity leading to increased genetic divergence among populations due to random genetic drift, elevated inbreeding and reduced levels of gene flow (Young et al. 1996). Genetic drift has the potential to override natural selection as the main evolutionary process within a species. The inbreeding increases as population sizes decrease, and subsequent deleterious effects on reproductive fitness and the persistence of isolated populations becomes challenging (Frankham 2003). As populations become more isolated, coevolved mutualisms with pollinators and seed-dispersing animals are disrupted altering historical arrangements of genetic subdivision found in continuous populations of tropical tree taxa (Hamilton
1999; Dick 2001).
Appropriate genetic management of fragmented populations therefore requires the identification of populations that need to be prioritized for conservation (Petit et al. 1998; Frankham 2003). The development and implementation of conservation strategies must take into account the species current genetic diversity and population structure to recognize priority areas for both in situ and ex situ conservation, monitoring and protection (Shapcott et al. 2007; Stefenon et al. 2007).
This study investigates the structure and levels of genetic diversity and inbreeding of Ocotea catharinensis , O. odorifera and O. porosa with the aim of indicating priority populations for conservation management actions focusing on population enhancement and ex-situ germplasm collections. The specific objectives were to quantify the genetic diversity within and among populations across the geographic range of each species representing different types of vegetation and land tenure and to test if there is a correlation between genetic and geographic distances among populations.
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Methods
Study Species
Ocotea catharinensis occurs naturally in the South and Southeast of Brazil, being more abundant in montane Atlantic rainforest between 300 - 700 m a.s.l. and less frequent in Araucaria forest (Carvalho
1994; Reitz et al .
1988). The tree reaches more than 25 m in height and up to 1.5 m DBH (diameter at breast height). Its hermaphroditic flowers are pollinated by small insects (Carvalho 1994; Silva et al.
1998; Brotto et al. 2009) and seed dispersal has been reported to be facilitated by monkeys ( Alouatta fusca , Silva et al. 2009; Brachyteles arachnoids, Moraes and Paoli 1999) and the bird, Pipele jacutinga
(Galetti et al. 1997). Both B. arachnoides and P. jacutinga are considered to be endangered by the IUCN
Red List (Mendes et al. 2008; BirdLife International 2012). Its seed germination is known to require high soil moisture with seedlings preferring moderate shade thereafter (Silva and Aguiar 1998; Moraes and
Paoli 1999). The quality timber is highly sought after for building, naval construction, luxury furniture and its essential oil (95% linalol) is used by the perfume industry (Nakaoka Sakita and Yatagai 1992).
Consequently, these species have been heavily harvested in the past with more than 176 000 tons of wood exported from Brazil between 1944 and 1951 (INP 1949-1960).
Ocotea odorifera is found throughout the south of the Bahia State, the Southern region and
Southeastern region of Brazil in Atlantic Rainforest habitat although it also occurs in Araucaria forest, in semi-deciduous and deciduous forests (Carvalho 2005). Its trees grow up to 15 m tall with a DBH up to
1.2 m (Carvalho 1994). Small hermaphroditic flowers are followed by ellipsoid fruit with ~ 2.3 cm in length (Quinet 2008). Seedlings establish best in shade and initial growth rates are extremely slow (Reitz et al. 1978). The species does not reach reproductive maturity until 25 - 40 years of age (Oltramari et al.
2002). Seeds are likely to be dispersed by monkeys and birds (Carvalho 2005). The tree is prized for its essential oil with high teor in Safrol, which was used for cosmetics and folk medicine (Gemballa 1955).
This species trade was centred in the Paraná and Santa Catarina States from where almost the entire production was exported (Raoul and Iachan 1948). By the 1940s the species had undergone a rapid population decline and conservation measures were already suggested (Machado and Souza 1948). The timber also has excellent structural properties and was used for building and naval construction (Pedroso and Mattos 1987).
In contrast, Ocotea porosa occurs naturally mainly in Araucaria forest in the Southern and
Southeastern regions of Brazil, but some populations can also be found in montane rainforest (> 850m a.s.l.). Trees can grow up to 30 m with a DBH of 3.2 m (Carvalho 1994). The small hermaphroditic flowers are self-compatible; however spontaneous self-pollination is very rare (5%) due to protogyny
(Danieli-Silva and Varassin 2012). Pollination is made by thrips (Thysanoptera; Frankliniella gardenia ) which are responsible for either cross-pollination among different plants or geitonogamous pollination between flowers on the same plant (Danieli-Silva and Varassin 2012). Seeds are likely to be dispersed by mammals and birds (Carvalho 2003). The high quality timber was chiefly used for the manufacture of luxury furniture. Approximately ~280 000 m 3 of wood was exported to South Africa and the USA between 1947 – 1967 (INP 1949-1960). Then the intense harvesting of 150 years started to become evident as serious decline in population size (Reitz et al. 1978).
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Field methods and sampling design
Populations of each Ocotea study species were selected from herbarium (JBRJ/JABOT; CRIA database) and floristic inventory data (IFFSC 2012). The sites were selected across the geographic range of each species in order to accurately reflect their distribution, and purposely included different vegetation types in both unprotected and protected areas. Adult trees were sampled at random from a total of 22 populations throughout southern and southeastern regions of Brazil representing a sample of the known populations of each species (Fig. 1). It comprised of six populations of O. catharinensis , nine of O. odorifera and seven of O. porosa.
The collected individuals were identified by a Lauraceae family specialist. Leaf samples of 30 plants were collected from each population. However populations with less than 30 individuals had all adult plants sampled. Individuals with a diameter at breast height (DBH) greater than 5cm were considered to be adults except for two montane O. porosa populations (Op5 and
Op7) that had mature trees significantly smaller as a result of the habitat conditions. Samples were stored in silica gel prior to DNA extraction.
The species’ samples were collected spanning six Brazilian Federation States (Fig. 1; Table 1) in six protected and 11 unprotected areas. Herbarium databases indicated populations of O. catharinensis in
Rio de Janeiro State, O. odorifera populations in Espírito Santo State, and O. porosa in Southern Region, but they could not be located despite an intensive field search effort.
Laboratory methods
Approximately 0.05g of leaf from each sample was grinded using an automated Mixer Mill
(Retsch MM200; Haan, Germany). Total genomic DNA was extracted from samples using CTAB procedure (Doyle and Doyle 1987) modified by Ferreira and Grattapaglia (1998) that uses two extraction stages with CTAB 2% and CTAB 10%. However, this method was not successful for O. catharinensis , which DNA was extracted using DNeasy Plant Mini-kits (Qiagen; Hilden, Germany) following the manufacturer’s instructions. The DNA quantifications were performed by electrophoreses in ethidium bromide-stained 1% agarose gels using genomic lambda DNA as standard.
Microsatellite analysis were conducted using eight markers developed and optimized for O. odorifera (Ood05, Ood07, Ood09, Ood14, Ood15, Ood16, Ood17 and Ood20; Martins et al. “in press”;
Online Resource 1). All markers were subsequently cross-amplified in O. catharinensis and O. porosa samples (Martins et al. “in press”). The locus Ood16 was monomorphic in O. porosa, so it was excluded from the analysis of this species. All PCR reactions were performed in a total volume of 12.5 µL including approximately 20 ng of template DNA, 1 U MyTaq DNA Polymerase (Bioline), 1x MyTaq
Reaction Buffer (Bioline; 1mM dNTPs, 1mM MgCl
2
, stabilizers and enhancers) and 0.2 µM of each primer, with the following cycling conditions: 94°C for 5 min, 35 cycles of 94°C for 1 min, specific annealing temperature for 1 min, 72°C for 1 min and a final extension step at 72°C for 10 min. The amplification products were verified for their expected sizes by electrophoresis using EtBr-stained 2% agarose gels (0.6X TBE) with 100 bp ladder (Axygen Biosciences) as size standard. The forward primer of each marker was directly end-labelled with a fluorescent dye (PET, NED, VIC or FAM) to enable multiplexed reactions for genotyping.
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The genotyping of the microsatellite fragments was conducted on AB 3500 Genetic Analyzer
(Life Technologies Inc., Grand Island, NY, USA). The fragment size was determined based on the size standard GS-600 LIZ (Life Technologies Inc.) using GeneMarker software 1.91 (Softgenetics, State
College PA, USA) and manually checked for consistency and accuracy.
Data analysis
Allelic frequencies were used to calculate species and population measures of genetic diversity including the mean number of alleles per locus ( A ), the mean number of effective alleles per locus ( A e
), observed ( H o
) and expected heterozygosities ( H e
), the allelic fixation index ( F ) and the number of private alleles ( A pr
) using GenAlEx 6.5 (Peakall and Smouse 2006). Tests for departures from Hardy–Weinberg equilibrium were also performed for each locus within each population by estimating F values with 960 randomizations while linkage disequilibrium was tested between pairs of loci using FSTAT 2.9.3.2
(Goudet 2002).
The partitioning of genetic diversity among populations ( F
ST
), within populations ( F
IS
) and across the whole species ( F
IT
) was analyzed with Wright's F statistics (Wright 1965). In addition to test if there were significant differences in genetic partitioning among populations within species, PhiPT was calculated and an AMOVA with 999 permutations was undertaken in GenAlEx 6.5 (Peakall and Smouse
2006). Genetic relationships among individuals of the same species were visualized using Principal
Coordinate Analyses (PCoA; Orloci 1978) with 9999 bootstrap permutations to assess the distinctiveness of genetic groupings between populations which was further analyzed by assignment test with 9999 permutations and 9999 bootstraps in GenAlEx 6.5 (Peakall and Smouse 2006). The correlation between genetic and geographic distance matrices among populations was analyzed using a Mantel test with genetic distance expressed as: F
ST
(1- F
ST
) as described by Rousset (1997) utilizing IBDWS 3.23
(Bohonak 2002) software. The correlations and probabilities were tested using 30000 bootstrap randomizations.
Model-based clustering was performed using the multilocus genotype data of all samples for each species in STRUCTURE 2.3.4 software (Pritchard et al. 2000). The default admixture model settings of the program were used. To determine the most likely number of groups ( K ) in the data, a series of analyses were performed with K=1 to K=7, using a burn-in of 100000 and 800000 Markov Chain Monte
Carlo (MCMC) repetitions with ten iterations per K for O. catharinensis , K=1 to K=10 and 900000
MCMC for O. odorifera and K=1 to K=8 and 1000000 MCMC repetitions for O. porosa . These results were examined using the dK method (Evanno et al. 2005) to identify the true number of clusters in the data as calculated by STRUCTURE Harvester (Earl and VonHoldt 2012).
Priority populations for in situ and ex situ conservation were identified based on genetic diversity within populations, level of inbreeding, number of private alleles and genetic differentiation among populations for each species. Moreover they were considered by the representativeness of the species allelic composition with the selection aiming to include the greatest allelic diversity. This was calculated using the formula (number of alleles in the selected populations - number of loci/number of total alleles of the species - number of loci) proposed by Petit et al. 1998. The highest priority populations thus scored where then ranked in order of conservation value and priority based on their genetic diversity (A, Ae and
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He) and level of genetic uniqueness (presence of private alleles). Populations with lower levels of inbreeding (F) were also given higher ranking. Where a species was found in more than one vegetation type, the highest diversity representatives from each vegetation type were selected for conservation priority. In addition the size of habitat patches was weighted in favor of the largest patches and current level of protection was also taken into account such that unprotected populations were given higher ranking.
Results
Genetic Diversity
All loci displayed independent inheritance and no significant linkage disequilibrium was detected. Microsatellite signatures were highly polymorphic for each species (Table 1). Ocotea catharinensis had the higher number of alleles with 155 alleles found across the eight loci examined
(A
S
=19.4), compared with 143 alleles in O. odorifera (A
S
=17.9), and 137 alleles in O. porosa (A
S
=17.1).
For O. catharinensis , the population with the highest number of effective alleles ( A e
= 7.6) was in Tinguá
Biological Reserve/RJ (Oc1), which was also the largest (34 947ha) and most well-conserved remnant assessed for this species (Table 1). In contrast, the O. odorifera ’s population with the highest number of effective alleles (Colombo/PR Oo4; A e
= 7.52) was located within a small fragment (< 3ha) situated in a degraded unprotected area (Table 1). All Ocotea porosa populations had similar number of effective alleles although they were generally lower than the other two species values (Table 1). Interestingly, O. porosa populations Op5 and Op2 located in the largest protected area and the smallest unprotected remnants, respectively (Op5-Guaratuba/PR, 4670ha and Op2-Bela Vista do Toldo/SC, <3ha) contained equal number of effective alleles ( A e
= 4.75, Table 1), and there was no significant correlation between allelic diversity and patch size (p>0.05) for any of the three species studied.
Private alleles were found in all populations of O. catharinensis and O. porosa and most of O. odorifera with the exception of two populations located on unprotected land (Oo3 and Oo7; Table 1). For
O. catharinensis , the population Oc1 located in a large fragment of protected forest contained the highest number of private alleles (A pr
=20) and the population Oc2 in a small privately-owned fragment had the lowest number of unique alleles (A pr
=2; Table 1). The population Op5 of O. porosa located in a protected montane rainforest fragment had the greatest number of private alleles within this species (A pr
=8). Ocotea odorifera had fewer private alleles (mean A pr
= 2.89) than the other two Ocotea species. The population in
Colombo/PR, Oo4, located within a very small remnant (< 3ha) exhibited the greatest number of private alleles (A pr
=8; Table 1).
All species had high expected heterozygosity ( H e
=0.73; H e
=0.78 and H e
=0.64, for O. catharinensis , O. odorifera and O. porosa , respectively) with similar values among populations (Table 1).
The mean expected heterozygosity ( H e
) was higher than the mean observed heterozygosity ( Ho ) for all species (Table 1). The allelic fixation index was significant greater than zero in most populations of each species indicating a small heterozygote deficit across loci probably due to inbreeding (p<0.5; Table 1).
Ocotea catharinensis had the highest mean value of allelic fixation ( F= 0.21) but this varied considerably among populations ranging from 0.06 to 0.30 (Table 1). Four of its six populations studied were significantly inbred. Mean values for O. odorifera and O. porosa populations were F=0.16 and
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F=0.13 respectively, suggesting overall lower inbreeding levels than O. catharinensis . Only three of seven populations of O. porosa were significantly inbred, the highest allelic fixation index was found in the protected population, Op7 (F=0.36; P<0.05) indicating it was the most inbred population. One population in a protected area (Op6) had negative values of F (-0.01; not significant). Generally negative values of F indicate heterozygote excess, however this was not observed by Ho vs. He on Op6 population. So this value of F , which was very close to zero, could be due to fewer samples and/or missing data for several individuals in this particular population. Eight of the nine populations of O. odorifera were inbred with the highest level occurring in the population Oo7 (F=0.30; P<0.05) and the lowest level in population Oo8 (F=0.02), which is near to random mating (Table 1). The inbreeding was not correlated with patch size (p>0.05) for any of the three species studied.
Population Structure and Isolation by Distance
A moderate genetic differentiation was found among populations of each species; O. catharinensis populations had the greatest divergence ( F
ST
=0.148) and O. odorifera populations were the most similar ( F
ST
=0.086; Table 2). The majority of genetic diversity within each species was found within populations (80%, 88% and 84%, respectively for O. catharinensis , O. odorifera and O. porosa ; Table 2).
These values may indicate effective gene flow among populations before habitat fragmentation.
Ocotea catharinensis
Most of the genetic divergence of O. catharinensis was due to the variation between the samples from Novo Hamburgo (Oc6) and the other populations (Oc6 mean D = 1.943; species mean D = 1.223;
Online Resource 2; Fig. 2). The population Oc5 was also genetically distant from the other populations
(Oc5 mean D = 1.323) but more similar to Oc1 population (Oc5 vs. Oc1 D=0.767; Fig. 2). From all populations analyzed, Oc2 and Oc3 were the most similar genetically ( D = 0.283; Online Resource 2).
The population Oc6 located in Rio Grande do Sul (Fig. 1) is the most southern population and was the most genetically divergent population. It is genetically distinct even from the closely located populations, Oc2, Oc3 and Oc4 (~368 km distant; Online Resource 3). This genetic differentiation was particularly evident at locus Oo17, where the allelic frequencies found in Oc6 were very distinct from the other five populations’ frequencies (Online Resource 4). At first, there was no significant correlation between genetic and geographic distances among O. catharinensis populations ( r = 0.5269, p = 0.08).
Given the genetic distinctiveness of population Oc6 and the possibility of it masking other geneticgeographic relationships, a second Mantel test was run excluding Oc6 population. As predicted, it showed a significant correlation between genetic and geographic distances ( r = 0.7645, p = 0.0351; Online
Resource 5).
The STRUCTURE analysis suggested the existence of five groups ( ∆K =5) with an overlap between populations Oc2, Oc3 and Oc4 (Fig. 3a), consistent with PCoA results. The populations Oc5 and
Oc6 displayed the most distinctive genotypes. The population Oc5 had high genetic diversity (Table 1) and different allelic frequencies as shown at locus Oo17 (Online Resource 4).
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Those results were consistent with the assignment test, which classified most of O. catharinensis individuals to their population of origin (93%), suggesting these populations were distinct genetically but still there was some allelic overlapping specially between populations Oc1, Oc2, Oc3 and Oc4 (Table 3).
Ocotea odorifera
Genetic differentiation among populations of O. odorifera was the lowest of the three Ocotea species analyzed (F
ST
= 0.086) with mean genetic distance (D) among populations of 0.687 (Online
Resource 2). The most genetically distinct was Oo9 which segregated from the other populations (Oo9 mean D =1.536; Online Resource 2; Fig. 4). The population Oo1 was also slightly different from the other populations (Oo1 mean D = 0.701). Both of these populations (Oo1 and Oo9) are also the most distant geographically from the other populations (Fig. 1) and the genetic distance among populations was strongly correlated with the geographic distance for O. odorifera ( r = 0.6672, p = 0.0000; Online
Resource 5).
The STRUCTURE analysis defined four different origins ( ∆K =4; Fig. 3b) with high overlap among populations Oo1 to Oo8 consistent with PCoA analysis (Fig. 4). The genetic distinctiveness of the population Oo9 can be observed in differences in the allelic frequencies at locus Oo14 (Online Resource
6). The assignment test classified most of the individuals to within their population of origin (73%) and again indicated the genetic distinctiveness of the population Oo9 (Table 3). This result also shows a much greater overlap of individuals among most of the populations of O. odorifera .
Ocotea porosa
Ocotea porosa had most of its genetic variation partitioned between the two more southern populations (Op6 and Op7, Fig. 1) and the other populations (between-groups mean D = 0.689; species mean D = 0.521; Online Resource 2). Op6 is a small population (n=8) in a very small fragment (Table 1) and is genetically depleted. The populations Op1 and Op5 were slightly distinct from populations Op2,
Op3 and Op4 and there was a significant correlation between genetic and geographic distances among populations ( r = 0.5902, p = 0.0313; Online Resource 5).
A strong overlap between populations Op2, Op3 and Op4 was detected based on the
STRUCTURE analysis (Fig. 3c) that determined four different origins and on allelic frequencies as observed at locus Oo15 (Online Resource 7). These results are congruent with the PCoA analysis of the distribution of diversity in O. porosa (Fig. 5). The populations Op6 and Op7 though closely located (58 km; Online Resource 3), were slightly distinct (Fig. 3c). The populations Op1 and Op5 showed some overlap with the other populations. Assignment tests classified 91% of the individuals to within their population of origin and indicated the genetic distinctiveness of population Op7 with no individual from other populations (Table 3). The population Op6 was not considered in the assignment test because of its small number of individuals.
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Conservation Issues
A total of 17 populations were indicated as priority for both in situ and ex situ conservation regarding to conserving the genetic diversity of the studied species (Table 4). Populations showing the presence of unique alleles, high genetic diversity, low inbreeding and representative of the different types of vegetation were prioritized. The five O. catharinensis populations selected represent 84% of all allelic diversity sampled in this study. The seven O. odorifera populations have 78,48% of the representativeness of allelic composition and for O. porosa the five priority populations for conservation encompass together 79% of the allelic diversity.
Discussion
Population Genetic Structure of Ocotea species
Reductions in population size and connectivity are known to eventually result in the random loss of rare alleles, while some alleles may become fixed within each fragment by genetic drift. Subsequent inbreeding may accelerate the loss of heterozygosity (Fuchs and Hamrick 2011). Levels of genetic diversity found in the present study for each of the three species were considerably higher than that found in previous studies using allozyme markers at local scale ( Ocotea catharinensis – Tarazi et al. 2010; O. odorifera - Kageyama et al. 2003; Ocotea porosa - Bittencourt 2004; Daros - 2006). This is not surprising since microsatellites are more polymorphic than allozymes (Zhu et al. 2000; Guichoux et al. 2011; Zalapa et al. 2012), and also the present study encompassed a broader geographical scale than the previous ones.
It has been well documented that species with wide geographic distribution frequently possess high genetic variation (Hamrick et al.1979).
Ocotea odorifera has a widespread occurrence from south of Bahia to southern Brazil (~2000 km; Fig. 1) and occurs within different vegetation types and across ill-defined altitudinal gradients. This species showed the highest genetic diversity and lower differentiation among populations in this study, as expected by its broader occurrence. Although O. catharinensis has a similar geographic range, it has quite restricted habitat preferences and it is found mainly in patches of montane rainforest, resulting in the highest genetic differentiation among populations. In contrast, Ocotea porosa is geographically more limited (~900 km) and occurs mostly in Araucaria forest in the southern Brazil. Nevertheless, within its extension of occurrence, suitable habitat is relatively continuous. So this species displayed slightly lower levels of genetic diversity than the levels found for O. catharinensis and O. odorifera in the current study, but still higher than previous studies results for this species.
The structure of genetic diversity in each of the three species was distributed mostly within populations (more than 80%) as expected for outcrossed, long-lived tree species (Hamrick and Loveless
1989; Hamrick and Murawski 1991). There was considerable genetic differentiation among O. porosa
’s studied populations (PhiPT = 0.164). The populations Op6 (São Francisco de Paulo-RS) and Op7
(Cambará do Sul-RS) possessed similar genotypes but were genetically divergent from the other five populations suggesting a geographic isolation. For instance, population Op7 (Cambará do Sul/RS) occurs within small patches of cloud forest set amongst an extensive grassland matrix at high altitude (Pillar and
Quadros 1997).
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The heavily exploited species, O. odorifera , exhibited the highest level of within-population genetic diversity and the lowest but still moderate level of genetic differentiation among populations of the three species (PhiPT = 0.119). This is consistent with the findings using allozyme (Kageyama et al.
2003) which detected a similar pattern of high genetic diversity and genetic structure in two near populations of this species in São Paulo State (θ
P
= 0.028). This is likely due to O. odorifera ’s previous occurrence, prior to logging activities, throughout relatively continuous habitat across a broad geographic range, as well as the fact that long generation times tend to restrict the loss of genetic diversity (Hamrick et al 1979). However, the population Oo9 (Lavras-MG) was genetically eroded compared with all other populations. This population was inbred, genetically dissimilar from other populations and had high allelic fixation index. These results are consistent with loss of genetic diversity due to fragmentation, and it was aggravated due to its patch size of less than three hectares and isolation in a landscape impacted by intense clearing and over-exploitation, with most of the remaining fragments in this region also occupying tracts of less than 10ha (Oliveira-Filho et al. 1994; Teixeira and Barros 1992).
Ocotea catharinensis had the highest genetic differentiation between populations (PhiPT =
0.205) so we suggest that even before logging populations were already differentiated. This can be due to the restricted natural distribution of suitable habitat in patches resulting in limited opportunities for gene flow. A previous study analyzing four populations of O. catharinensis in Santa Catarina State (greater distance between populations: 202 km) had similar genetic differentiation ( F
ST
= 0.1175; Tarazi et al.
2010) corroborating our results. Moraes and Paoli 1999 found that seed dispersal by the monkey
Brachyteles arachnoides may reach over 1Km and each animal disperses ~50 seeds per day. In contrast,
Jacutinga birds ( Pipele jacutinga ) stay up to ten days in the same tree, and then almost all seeds are dispersed under the mother plant (Galetti et al .
1997). Such variation in dispersal mechanisms may explain the patterns of differentiation among populations of O. catharinensis . This species had a high number of private alleles, probably due to patchy population distribution. Interestingly, population Oc1
(Tinguá-RJ) exhibited high numbers of effective and private alleles far above mean values for the species.
This population is highly diverse, but possibly isolated in terms of gene flow. Although the population
Oc5 (Santa Teresa-ES) was more similar to Oc1, it still have a divergent genotype and should be considered a priority population for conservation purposes. The population Oc6 (Novo Hamburgo-RS) had low genetic diversity and highly divergent genotypes, probably due to its geographic isolation. It is located in a depression behind a range of mountains, quite disjunct from other rainforest fragments (E.
Martins, personal communication).
Previous research into O. porosa has found pollen dispersal to be quite restricted (Danieli-Silva and Varassin 2012). All three species in this study displayed moderate to high levels of inbreeding in some of their populations which may be due to restricted pollinator dispersal. However, the levels of inbreeding for O. porosa in this study were lower than found by Bittencourt (2004) which may possibly be due to the higher levels of polymorphism detected by microsatellites. Breeding system and life form are strongly correlated to the genetic structure and diversity in natural plant populations (Hamrick and
Godt 1996). Assuming that O. catharinensis and O. odorifera have similar pollination system of O. porosa (thrips), the highly inbred populations may be a consequence of the limited flight capabilities of
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28 these insect pollinators (Danieli-Silva and Varassin 2012). This may also explain why so many private alleles were found for the three Ocotea species studied here and previously by Bittencourt (2004).
Plant longevity may ensure the representation of many generations in the current population and consequently different genotypes can be maintained more effectively (Aparicio et al. 2012). Therefore, genetic drift would be expected to have the least impact on long-lived species (Hamrick et al.1979; Hartl and Clark 1989; Lowe et al. 2005). The high overall genetic diversity and moderate genetic structure ( F
ST
) found in these three species of Ocotea may be explained by their long generation times counteracting recent habitat loss and population reductions. Due to intense exploration and habitat fragmentation, we would expect higher levels of genetic differentiation among populations for these three species. However our results indicate that there has been considerable gene flow among populations historically, when the biome was more continuous prior to habitat fragmentation and selective logging, except for O. catharinensis studied populations that we believe they were already differentiated before logging.
Aparicio et al. (2012) analyzed the correlation between the effect of habitat fragmentation and plant species’s life span on the genetic diversity and population genetic structure within the same landscape context. They found that diversity measures clearly and consistently increased from the most short-lived species to the longest.
The individuals sampled for this study may belong to the second generation after the period of intensive harvesting. But some individuals could be remnants from before this time and they may be acting as reservoirs of diversity, particularly as overlapping generations are also known to have the potential to mitigate the loss of genetic diversity via within-population gene flow, leading post-logging analyses to underestimate the genetic impacts of such intensive exploitation (Lowe et al. 2005). An analysis of seedlings’ genetic diversity may detect the result of these deleterious effects on the species, as found for O. porosa in Paraná State an excess of homozygotes in the seedlings in relation to adults in the same population (Daros, 2006). However the seedlings’ analysis might be hampered in the studied populations because extremely few seedlings were observed in three populations (Oo1, Oo2 and Oc1), and none in all other populations for the three species (E. Martins, personal communication).
Identification of priority populations for conservation
The findings from this study show that even small forest fragments (eg. Oo4-Colombo/PR; Oo7-
Marcelino Ramos/RS; Table 1) possess high levels of genetic diversity and that both unprotected and protected areas have different allelic frequencies and distinct genotypes (eg. Oc6-Novo Hamburgo/RS;
Oc1-Tinguá/RJ). Thus, regardless of these attributes, each population may have an important function on the maintenance of overall species genetic diversity. The selected populations are described in Table 4 and ranked according to highest value for conservation based on genetic diversity and uniqueness.
Ocotea catharinensis populations were quite differentiated genetically, so all populations, apart from Oc2 (Ituporanga/SC), have high conservation value with regard to preserving the current level of genetic diversity within this species making it hard to identify single populations of higher value (Table
4). The population Oc2 were excluded because it displayed a marked genetic similarity with Oc3
(Taió/SC), however the latter had higher number of private alleles and is located in a larger fragment, therefore it would better represent the genetic diversity of this area. Oc3 has already been indicated by
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40 government agencies as priority area for conservation (MMA 2007), although the site is as yet unprotected area our results will hopefully reinforce the importance of this site as a fragment requiring immediate conservation.
Ocotea odorifera populations were less differentiated; however some populations were quite genetically distinct, such as Oo1-Tinguá/RJ and Oo9-Lavras/MG suggesting their importance for both in situ and ex situ conservation strategies of O. odorifera . Populations Oo7 (Marcelino Ramos/RS) and Oo8
(Três Cachoeiras/RS) were also quite genetically divergent from the other populations and defined as priority populations too. The first one was unique for representing a population in semi-deciduous forest despite being relatively genetically eroded, which urges its necessity of conservation actions. Population
Oo4 is likewise an important population for conservation, although it is located within a small and degraded unprotected fragment (<3ha). The Oo4 plants had many private alleles and displayed high number of effective alleles. The populations Oo2 and Oo3 were genetically similar, but Oo2 have a considerable in situ conservation value, due to its higher number of private alleles and effective alleles than Oo3 and thus has been given higher conservation priority (Table 4).
Ocotea porosa showed distinct genotypes that are represented in the five populations selected for conservation (Table 4) .
Only populations Op2 and Op6 were not assigned as priority for conservation.
Population Op2 genotypes were already represented by populations Op1 and Op4 while population Op6 was genetically similar to Op7. This latter population was deemed more important for conservation as it not only had a higher number of private alleles, but represents a different vegetation ecotype (montane rainforest; Table 4).
Some of the selected priority populations are already protected by Brazilian government laws
(eg. Oc1; Oc4; Oo1; Op7), however they need more effective conservation measures to be implemented.
There are still illegal logging, livestock grazing and illegal hunting happening in many protected areas, due to lack of monitoring and supervision. For instance, in the Tinguá Biological Reserve (created in
1989) has still illegal hunting of some species. The bird Pipele jacutinga , which is seed disperser of many plant species as Ocotea species, is considered extinct there (Fernandez and Travassos 2006). In addition, the conservation of suitable habitat for seedling recruitment and growth needs to be guaranteed. In the populations studied, seedlings were not detected in most of populations and areas nearby, except at
Apiúna/SC (Oo2; one individual of O. odorifera ) and Tinguá/RJ (Oo1 and Oc1). Thus, simply preserving the remaining genetic diversity of the three Ocotea species within protected areas may not be enough to guarantee the persistence of future generations without adequately first investigating factors affecting the recruitment of seedlings.
Conclusions
We have found that three Ocotea species have relatively high levels of genetic diversity with moderate genetic differentiation among populations. As a consequence, we suggest that some populations assessed in this study should be conserved in situ and its plants should be used for ex situ conservation, in order to preserve the species overall genetic diversity and the presence of unique alleles. This study showed that even populations on small parcels of unprotected land and in degraded areas have conservation value and should be preserved. Some populations could act as connectors between
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13 fragments, corridors and/or stepping stones, regarding its location, to promote effective gene flow among populations and minimize inbreeding effects inside populations. Government programs are now essential to ensure the implementation of the recommendations arising from the current and previous studies of these over-exploited Ocotea species, particularly with regard to threat abatement, restoration and conservation management strategies into the future.
Acknowledgments
We would like to thank Centro Nacional de Conservação da Flora (CNCFlora) for financial support.
Thanks also to the GeneCology Research Centre (University of the Sunshine Coast, Australia) and
Instituto de Pesquisas Jardim Botânico do Rio de Janeiro (JBRJ, Rio de Janeiro, Brazil) for technical assistance and the use of their respective laboratory facilities. The first author is grateful for the sandwich scholarship provided by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES).
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Figure titles:
Fig. 1: The location of the (a) Ocotea catharinensis , (b) O. odorifera and (c) O. porosa populations in
South and Southeast regions of Brazil. (a) Oc1, Nova Iguaçu (RJ); Oc2, Ituporanga (SC); Oc3, Taió (SC);
Oc4, Guaratuba (PR); Oc5, Santa Teresa (ES) and Oc6, Novo Hamburgo (RS). (b) Oo1, Nova Iguaçu
(RJ); Oo2, Apiúna (SC); Oo3, Taió (SC), Oo4, Colombo (PR); Oo5, Guaratuba (PR); Oo6, Ponta Grossa
(PR); Oo7, Marcelino Ramos (RS); Oo8, Três Cachoeiras (RS) and Oo9, Lavras (MG). (c) Op1,
Itaiópolis (SC); Op2, Bela Vista do Toldo (SC); Op3, Mafra (SC), Op4, Ponta Grossa (PR); Op5,
Guaratuba (PR) and Op6, São Francisco de Paula (RS); Op7, Cambará do Sul (RS).
Fig.2: Genetic relationship between all Ocotea catharinensis individuals as shown by principal coordinate analysis (PCoA). Symbols indicate the population. Axis 1 accounts for 34.06% and axis 2 for
19.84% of the variation in the data.
Fig. 3: STRUCTURE analysis for (a) Ocotea catharinensis populations based on 8 microsatellite loci.
The K used was from 1 to 7, with the highest ∆K=5. (b)
O. odorifera populations based on eight microsatellite loci. The K used was from 1 to 9, with the highest ∆K=4. (c) O. porosa populations based on seven microsatellite loci. The K used was from 1 to 8, with the highest ∆K =4.
Fig. 4: Genetic relationships between all Ocotea odorifera sampled individuals calculated by principal coordinates analysis (PCoA). Symbols indicate the populations. Axis 1 accounts for 45.32% and axis 2 for 16.10% of the variation in the data.
Fig.5: Genetic relationships between all Ocotea porosa sampled individuals as shown by Principal
Coordinates analysis (PCoA). Symbols indicate the populations. Axis 1 accounts for 26.79% and axis 2 for 19.35% of the variation in the data.
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