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Genetic (AFLP) diversity of nine Cedrela odorata populations in Madre de Dios, southern Peruvian Amazon
Available online at www.sciencedirect.com
Forest Ecology and Management 255 (2008) 334–339 www.elsevier.com/locate/foreco
Genetic (AFLP) diversity of nine Cedrela odorata populations in Madre de Dios, southern Peruvian Amazon Amanda de la Torre a,*, Cesar Lo´pez a, Eliana Yglesias a, Jonathan P. Cornelius b a
Universidad Nacional Agraria La Molina, Av. La Universidad s/n La Molina, Lima 12, Peru b World Agroforestry Centre, CIP-ICRAF, Apartado 1558, Lima 12, Peru
Abstract Cedrela odorata L., one of the most important neotropical timber species, is threatened by deforestation and unsustainable logging in many parts of its natural range. Information on patterns of genetic variation is useful in informing both reforestation and genetic conservation activities. However, to date, no such information is available in Peru or elsewhere in South America. In the present study, genetic diversity between and within nine Peruvian populations of the species, based on amplified fragment length polymorphism (AFLP) markers, is reported. Overall diversity level was high (Ht = 0.22), as expected for a widespread, long-lived tropical species, and consistent with previous studies carried out in Central America. Levels of intrapopulation diversity were higher than those previously reported for the species (Hs = 0.13–0.21). Analysis of molecular variation revealed genetic differences between two population groups located on different rivers and between populations located on the same rivers. Differences between groups were greater than those within groups. Genetic and geographical distances were significantly correlated. The relatively strong genetic differences between populations may be related to the riparian, essentially one-dimensional spatial distribution pattern of the populations studied. No difference was found in percentage of polymorphic loci between relatively undisturbed and logged populations. The existence of appreciable genetic differentiation over a relatively small part of the species range in the Peruvian Amazon suggests the need for caution in use of seed outside its zone of origin. For genetic conservation purposes, it would probably be prudent to sample (ex situ) or conserve (in situ) populations in each of the major watersheds of the Peruvian Amazon. # 2007 Elsevier B.V. All rights reserved. Keywords: Meliaceae; Forest genetic resources; Genetic conservation
1. Introduction Cedrela odorata L. (cedro, Spanish cedar) is a neotropical, broadleaf tree species. Its broad, transequatorial distribution extends from 268N in Mexico to 288S in northern Argentina, and includes moist and seasonally dry forest types, and altitudes ranging from sea level to 1200 m a.s.l. (Pennington and Styles, 1975). The timber of C. odorata, like that of other Swietenioideae (e.g. the American mahoganies, Swietenia spp.), is of high value and is much in demand on domestic and international markets. It is classed as ‘vulnerable’ by IUCN and is threatened both by unsustainable logging (IUCN, 1994; Patin˜o, 1997) and by forest conversion to pasture and other land uses. In Peru, Spanish cedar is considered a threatened
* Corresponding author at: Centre for Forest Conservation Genetics, University of British Columbia, 3041-2424 Main Mall, Vancouver, B.C., Canada V6T 1Z4. Tel.: +51 1 4366804. E-mail address: [email protected] (A. de la Torre). 0378-1127/$ – see front matter # 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.foreco.2007.09.058
species by national authorities (INRENA, 2004) and since 1988 has been listed on CITES Appendix III, under which both export and import require export permits from the producing country (CITES, 2004). However, the species remains relatively common in many parts of its range (including parts of the Peruvian Amazon), including agricultural landscapes, where its heliophytic status and nurturing by farmers appear to have helped to ensure its continued presence in fencerows and forest remnants. In Madre de Dios province, southern Peruvian Amazon, where the present study was carried out, many remaining populations of C. odorata are riparian, i.e. located close to main rivers or to oxbow lakes. Because of their accessibility (i.e. via navigable rivers), many populations in the zone have been heavily depleted. Effective conservation and use of this and other vulnerable species depends partially on knowledge of patterns of genetic variation. For example, the spatial structure of genetic variation should inform sampling strategies for ex situ or in situ conservation. Similarly, marked between-population genetic
A. de la Torre et al. / Forest Ecology and Management 255 (2008) 334–339
differentiation would indicate the need for caution – at least – in use of seed for planting outside its area of origin. This applies even to neutral genetic variation detected by molecular markers, as differentiation may indicate relative absence of migration, which in turn could permit the development of variation in adaptive traits that otherwise could be prevented by gene flow. In the case of Spanish cedar, studies by Gillies et al. (1997), Cavers et al. (2003a) and Navarro (2002) have revealed pronounced genetic differences between populations from the Pacific and Atlantic regions of Costa Rica. However, no information is available for C. odorata in South America, although the region harbours most of the unlogged populations of the species. In the present study, we address two research questions of relevance to the management of Peruvian C. odorata genetic resources. First, we look at the degree of genetic differentiation between populations and between two population groups. Second, we examine whether there is any indication that human disturbance, i.e. logging, has to date affected genetic diversity of intervened populations. In particular, we were interested in examining whether the most highly intervened populations would show lower degrees of polymorphism, due to population bottlenecks (caused by logging) and consequent founder effects. The presence of such effects would imply that heavily logged populations should receive lower prioritization in ex situ and in situ conservation. In order to address these questions, we present information on genetic structure and variation in nine populations of the species. We based our estimates on amplified fragment length polymorphism (AFLP) markers. AFLPs are suitable markers for genetic diversity studies as, although dominant, they permit the assay of a large number of loci.
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2. Materials and methods 2.1. Study sites and sampling Populations were sampled in two protected areas: Manu National Park (six populations) and Los Amigos Conservation Area (three populations) (Fig. 1 and Table 1). The Manu populations are located along an approximately 150 km stretch of the River Madre de Dios. Each population represents an area of 30–450 ha where the species occurs at variable, relatively high densities, i.e. 0.3–7 trees ha 1 (Table 1). With the exception of one additional, unsampled population (Cocha Jua´rez), located between Otorongo and Limonal populations, there are no other significant concentrations of the species either in riparian locations between the sampled populations, or in adjacent non-riparian locations, although it is probable that isolated trees or small groups of trees occur in both situations. We concentrated on riparian populations because, as a common type, their conservation status is important to the continued persistence of the species in Madre de Dios. In addition, they are and have been vulnerable to logging (legal and illegal). The Los Amigos populations are located 0–43 km upstream of the confluence of the Madre de Dios and the Amigos rivers, which occurs approximately 217 km downstream of the closest of the Manu populations. The intervening stretch of the River Madre de Dios flows through heavily deforested areas. In terms of human intervention, the populations fall into two groups: the Los Amigos populations and Limonal in Manu (located close to the park boundary) have been intensively logged. Most wellformed trees have been removed, leaving only low density stands (Table 1) of C. odorata individuals. The remaining populations are relatively undisturbed, including two (Cashu and Maizal) that have never been logged.
Fig. 1. Location of nine populations in two groups sampled in a study of genetic diversity of Cedrela odorata in the Southern Peruvian Amazon. Populations are represented by black dots and located as follows: from the top on the left side: Maizal, Cashu, Gallareta, Salvador, Otorongo and Limonal in Manu; from the bottom on the right side: Cicra TB, Cicra TA and Pv2 in Los Amigos area.
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Table 1 Description of collection areas sampled in a study of genetic variation of Cedrela odorata populations in the southern Peruvian Amazon
2.3. Data analysis
Leaf material was collected from a total of 153 individuals from the nine populations from October to December of 2003; 9–20 individuals were sampled per population (Table 1). Samples were stored in resealable plastic bags with 10–20 g of self-indicating silica gel, following Chase and Hills (1991).
Allele frequencies were estimated using the Bayesian method with non-uniform prior distribution of allele frequencies, as described by Zhivotovsky (1999). We estimated the degree of genetic differentiation between groups (i.e. populations on the River Madre de Dios and those on the River Amigos) and between populations-within-groups using analysis of molecular variance (AMOVA) from ARLEQUIN version 3.0 (Excoffier et al., 2005) and by estimating values of Nei’s F st (Nei, 1973) and related statistics using the AFLP-SURV software (Vekemans et al., 2002), following Lynch and Milligan’s (1994) approach. We tested for a relationship between pairwise physical and genetic distances (Nei, 1972) between the populations by estimating values of Spearman’s rank correlation. Genetic distances were calculated using AFLP-SURV. In order to test for effects on genetic diversity of population bottlenecks caused by logging, we examined the relative degrees of polymorphism of the two groups (logged and unlogged populations), specifically with regard to expectations that the most highly intervened populations would show lower degrees of polymorphism. We did not compare gene diversity between the two groups, because gene diversity is expected to be relatively little affected by bottlenecks (because rare alleles – which are those that are expected to be lost – contribute little to total heterozygosity).
2.2. Laboratory procedures
3. Results
Total genomic DNA was extracted using the CTAB method (Doyle and Doyle, 1987). AFLP protocol followed the standard procedure described by Vos et al. (1995). High quality DNA (i.e. not damaged and free of impurities) was digested using restriction enzymes EcoRI and MseI. DNA fragments were ligated to EcoRI and MseI adapters to generate template DNA for amplification. PCR was performed in two consecutive reactions, according to the Invitrogen protocol (Invitrogen, 2003). In the preamplification, genomic DNAs were amplified with AFLP primers, each having one selective nucleotide. The PCR products of the amplification reaction were diluted and used as a template for the selective amplification using two AFLP primers, each containing three selective nucleotides. Seven primer combinations were probed: EcoRI-ACT/MseI-CAT; EcoRI-AAC/MseI-CAC; EcoRI-ACC/MseI-CAT; EcoRIACC/MseI-CTT; EcoRI-ACC/MseI-CAG; EcoRI-ACA/MseICTC; EcoRI-AGG/MseI-CTC. Those with the best resolution and the greatest number of bands were selected. Thermal cycling conditions for amplification were optimised to 14 cycles of 94 8C for 30 s (denaturation), 65 8C for 30 s (annealing) and 72 8C for 60 s (extension); followed by 23 cycles of 94 8C for 30 s, 56 8C for 30 s, and 72 8C for 60 s. Final temperature was 4 8C. Amplification products were separated on 6% polyacrylamide gels, stained with AgNO3 (according to the standard procedure described by Creste et al., 2001) and visualized through UV light exposure. Subsequently, the gels were recorded using a camera and a scanner before scoring the presence or absence of each scorable band in a binary data matrix.
3.1. General
Collection area
Mean annual precipitation (mm year 1) Mean annual temperature (8C) Number of measured trees Number of sampled trees Mean (range) of densities (trees ha 1) Number of sampled populations Number of individuals sampled per population Degree of disturbance
Manu
Los Amigos
2500
2845
23
22
540
61
103
50
0.85 (0.3–7)
0.09 (0.05–0.1) 3 13–16
6 9–20 Undisturbed (except Limonal population)
Intensively logged
Three of the seven screened AFLP combinations were used in the study (of the others, two were rejected because of low resolution and two because of low numbers of bands). The three AFLP primer combinations produced a total of 258 bands in 137 individuals (from a total of 153 sampled, 137 were successfully amplified (Table 2)), of which 255 (98.8%) were polymorphic (Table 3). The percentage of polymorphism across the nine populations ranged from 41.5% (Salvador) to 66.7% (Otorongo) (Table 2). Levels of genetic diversity within populations (Hs) ranged from 0.13 (Maizal) to 0.21 (Otorongo) (Table 3). Overall gene diversity (Ht) was estimated at 0.22 and overall F st was 0.20. 3.2. Genetic differentiation The AMOVA indicated the presence of significant genetic variation both between the two groups and between populaTable 2 Number of loci evaluated for each of three AFLP primer combinations utilized in assays of 137 individuals of Cedrela odorata from nine southern Peruvian Amazon populations Primer combination
Number of loci
EcoRI-ACT/MseI-CAT EcoRI-AAC/MseI-CAC EcoRI-ACC/MseI-CAT
103 97 58
Total
258
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Table 3 Estimated percentage of polymorphic loci (P) and gene diversity (Hs) within nine populations of Cedrela odorata from the southern Peruvian Amazon Collection area
Population
Manu
Maizal Cashu Gallareta Otorongo Salvador Limonal
Los Amigos
PV2 CicraTA Cicra TB
Sample size
Total
Number of polymorphic loci
P
Hs (S.D.)
18 20 15 13 19 9
112 115 111 172 107 126
43.4 44.6 43 66.7 41.5 48.8
0.13 0.18 0.16 0.21 0.17 0.18
13 14 16
135 120 117
52.3 46.5 45.3
0.17 (0.01) 0.18 (0.01) 0.18 (0.01)
137
255
98.8
0.17 (0.007)
(0.01) (0.01) (0.01) (0.01) (0.01) (0.01)
Table 4 Analysis of molecular variance (AMOVA) between two population groups and nine populations-within-groups of Cedrela odorata from the southern Peruvian Amazon Source of variation
d.f.
Sum of squares
Variance component
Between groups Between populations-within-groups Within populations
1 7 128
367.675 503.09 2830.504
5.01412 3.28699 22.11332
Total
136
3701.272
30.41442
a
Total variance (%) 16.48 10.80 72.70
pa <0.0001 <0.0001 <0.0001
100
Probability of a higher value of F, based on permutation test (1000 permutations).
tions-within-groups. Almost three-quarters of the variation (72.7%) was concentrated within populations, while the between-group component (16.5%) was larger than the between population-within-group component (10.8%) (Table 4). Values of F st for populations-within-groups were similar (0.14 for Manu and 0.15 for Los Amigos) (Table 5). Overall F st was higher (0.20). The correlation between genetic and geographic distance was positive and highly significant (rs = 0.75, p < 0.0005, one-tailed) (Fig. 2). 3.3. Polymorphism in logged and unlogged populations The percentage of polymorphic loci per population varied from 43 to 66.7%. The least disturbed populations were not amongst the most variable and the logged populations had percentages of polymorphic loci similar to or greater than all the other populations except the most variable (Otorongo).
(Ht = 0.27, P = 84.8% (Cavers et al., 2003b); Ht = 0.34, P = 93.8% (Gillies et al., 1997)). However, diversity within populations, varying from 0.13 to 0.21, was higher than reported elsewhere. For example, Cavers et al. (2003b) found that diversity within Costa Rican populations ranged from 0.03 to 0.13. Furthermore, the proportion of polymorphic loci per population was greater than previously evaluated (41.5–66.7% vs. 9–22% found by Cavers et al., 2003b). Either or both of two factors could explain this. First, it might reflect a South American origin of the species (as postulated by Cavers et al., 2003a). This would imply a more recent colonization of Central America, and lower diversity levels could then be explained by founder effects or population bottlenecks during migration events (Rivera-Ocasio et al., 2002; Cavers et al., 2003a,b).
4. Discussion Overall levels of genetic diversity (Ht = 0.22, P = 98.8%) were similar to values reported for C. odorata in Mesoamerica Table 5 Estimates of fixation index (Fst), total gene diversity (Ht) and within population gene diversity (Hs) for two population groups of Cedrela odorata from Madre de Dios, southern Peruvian Amazon Collection area
Hs
Ht
Fst
Manu Los Amigos
0.17 0.17
0.20 0.21
0.14 0.15
0.22
0.20
Total (all populations)
Fig. 2. Scatter plot of genetic distances and geographic distances between nine populations of Cedrela odorata located in Madre de Dios, southern Peruvian Amazon.
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Second, it might reflect different levels of disturbance and human-induced fragmentation, both of which tend to be much greater in Central American populations than in the populations studied here, and may have been sufficiently severe to have led to genetic erosion. Our results, by contrast, indicate that the degree of disturbance and fragmentation found in the studied populations has not resulted in genetic erosion. This probably reflects ongoing gene-flow between the study populations, from upstream intact populations and from other trees in non-riparian situations (because gene flow could restore to individual populations any alleles lost in bottlenecks). Alternatively, it may simply imply that logging intensity was not sufficiently high to cause measurable founder effects. Similar results, attributed to similar causes (i.e. fragmentation, logging and lower long-term effective population sizes), were found by Lemes et al. (2003) and Novick et al. (2003) for Central American and Amazonian populations of big-leaf mahogany (Swietenia macrophylla King), another winddispersed, insect-pollinated, widespread meliaceous species subject to unsustainable intensive logging. According to Yeh’s (2000) criteria our findings on genetic structure indicate large (0.15 < F st 0.25) genetic differentiation at the overall level, and moderate to large (0.05 < F st 0.15) differentiation at the within-group level. It is possible that the presence of appreciable genetic differentiation over this relatively small area may be due to the riparian, essentially one-dimensional spatial distribution pattern of the populations studied here. The positive relationship between genetic and geographical distances suggests that gene flow is likely to be predominantly between adjacent populations, corresponding to Kimura and Weiss’s (1964) stepping stone model. Under the one-dimensional stepping-stone model – appropriate for these linear, riparian populations – more gene flow per generation is required to maintain overall panmixia than in Wright’s (1931) Island Model. For example, in a stepping stone system with migration rates of 0.1 and 2 10 5, respectively for adjacent and long-distance gene flow, ‘considerable’ local differentiation will occur if Ne < 100 (Kimura and Weiss, 1964). In addition, the more pronounced betweengroup variation could not be solely due to greater distance, but also to barriers to hydrochory-mediated gene flow (because the two groups are located upstream of the confluence of their respective rivers). However, based on our data, we cannot separate such effects from those related purely to isolation by distance. Cavers et al. (2003b) reported estimates of fst (an analogue of F st, based on partitioning of molecular variance between and within populations (Excoffier et al., 1992)) for two population groups of C. odorata located in Costa Rica. Our estimates of F st are similar to the fst estimate from Cavers et al.’s first population group of (fst = 0.20). The values we report are much lower than those for Cavers et al.’s (2003b) second group (fst = 0.47). This, however, covered a larger geographic area than that sampled here, including populations physically and reproductively separated by the Costa Rican Central Mountain range.
Taken together, our results and those of Cavers et al. (2003b) appear to indicate that C. odorata may have a tendency towards developing relatively marked differentiation between populations. In part, this could be due to factors specific to the two study sites (riparian distribution in Madre de Dios, human intervention in the case of Costa Rica (see above)). However, it should also be noted that, in general, abiotically dispersed tropical tree species tend to show relatively large population differentiation (Loveless, 1992), i.e. this tendency may be due in part to intrinsic characteristics of the species, and not simply to local ecological or historical factors. The presence of relatively pronounced genetic variation between these relatively closely distributed populations of C. odorata suggests the need for caution in the use of seed outside its zone of origin. This would apply particularly in the case of seed transfers over larger distances than those studied here. The Peruvian Amazon is a vast region, variable in soil types, elevations, and amount and distribution of precipitation. These distances may permit more substantial genetic differentiation than those observed here (as, indeed, seen in Cavers et al.’s (2003b) second group (see above)), particularly if reinforced by a tendency towards linear distributions and, possibly, unidirectional hydrochory. For genetic conservation purposes, it would probably be prudent to sample (ex situ) or conserve (in situ) populations in each the major watersheds of the Peruvian Amazon (e.g. those draining to the Napo, Maran˜on, Huallaga, Ucayali and Madre de Dios rivers). Our results suggest that logged populations should not be excluded from any such measures, as they do not show signs of being genetically depauperate. Future research should seek to clarify genetic structure at these larger geographic scales. Studies of adaptive variation, preferably of traits of commercial significance, should also be undertaken, in order to clarify the implications for planting programmes. Acknowledgments This work forms part of the senior author’s MSc thesis and was done with financial support from ACA (Amazon Conservation Association) and ICRAF (World Agroforestry Centre) through competitive grants for 2003. References Cavers, S., Navarro, C., Lowe, A.J., 2003a. Chloroplast DNA phylogeography reveals colonization history of a Neotropical tree Cedrela odorata L. in Mesoame´rica. Mol. Ecol. 12, 1451–1460. Cavers, S., Navarro, C., Lowe, A.J., 2003b. A combination of molecular markers identifies evolutionarily significant units in Cedrela odorata L. (Meliaceae) in Costa Rica. Conserv. Genet. 4, 571–580. Chase, M., Hills, H., 1991. Silica gel: an ideal material for field preservation of leaf samples for DNA studies. Taxon 40, 215–220. CITES, 2004. Listed species database. http://www.cites.org/eng/resources/species.html. Creste, S., Tulmann, N., Figueira, A., 2001. Detection of single sequence repeat polymorphisms in denaturing polyacrilamide sequencing gels by silver staining. Plant Mol. Biol. Rep. 19, 299–306. Doyle, J.J., Doyle, J.L., 1987. Isolation of plant DNA from fresh tissue. Focus 12, 13–15.
A. de la Torre et al. / Forest Ecology and Management 255 (2008) 334–339 Excoffier, L., Smouse, P.E., Quattro, J.M., 1992. Analysis of molecular variance inferred from metric distances among DNA haplotypes: application to human mitochondrial DNA restriction data. Genetics 131, 479–491. Excoffier, L., Laval, G., Schneider, S., 2005. Arlequin ver 3.0: An integrated software package for population genetics data analysis. Evol. Bioinform. Online 1, 47–50. Gillies, A., Cornelius, J., Newton, A., Navarro, C., Hernandez, M., Wilson, J., 1997. Genetic variation in Costa Rican populations of Spanish Cedar. Mol. Ecol. 6, 1133–1145. INRENA, 2004. Lista de especies de flora bajo categorı´a de amenaza. http:// www.inrena.gob.pe. Invitrogen, 2003. AFLPAnalysis System I, AFLP Starter Primer Kit. Instruction Manual. IUCN, 1994. Americas Regional Workshop (Conservation & Sustainable Management of Trees, Costa Rica) 1998. Cedrela odorata. In: 2004 IUCN Red List of Threatened Species. http://www.redlist.org/. Kimura, M., Weiss, G.H., 1964. The stepping stone model of population structure and the decrease of genetic correlation with distance. Genetics 49, 561–576. Lemes, M., Gribel, R., Proctor, J., Grattapaglia, D., 2003. Population genetic structure of mahogany (Swietenia macrophylla King, Meliaceae) across the Brazilian Amazon, based on variation at microsatellite loci: implications for conservation. Mol. Ecol. 12, 2875–2883. Loveless, M.D., 1992. Isozyme variation in tropical trees: patterns of genetic organization. New Forests 6, 67–94. Lynch, M., Milligan, B.G., 1994. Analysis of population genetic structure with RAPD markers. Mol. Ecol. 3, 1–9. Navarro, C., 2002. Genetic resources of Cedrela odorata and their efficient use in Mesoamerica. Academic Dissertation in Forest Tree Breeding. University of Helsinki, Finland.
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Nei, M., 1972. Genetic distance between populations. Am. Nat. 106, 283–292. Nei, M., 1973. Analysis of gene diversity in subdivided populations. Proc. Natl. Acad. Sci. 70, 3321–3323. Novick, R., Dick, C., Lemes, M., Navarro, C., Caccone, A., Bermingham, E., 2003. Genetic structure of Mesoamerican populations of big-leaf mahogany (Swietenia macrophylla) inferred from microsatellite analysis. Mol. Ecol. 12, 2885–2893. Patin˜o, F., 1997. Los Recursos Gene´ticos de Swietenia macrophylla y Cedrela odorata en los neotro´picos: prioridades para una accio´n coordinada. Recursos Gene´ticos Forestales 25, 21–33. Pennington, T.D., Styles, B.T., 1975. A generic monograph of the Meliaceae. Blumea 22, 419–540. Rivera-Ocasio, E., Aide, T.M., McMillan, O., 2002. Patterns of genetic diversity and biogeographical history of the tropical wetland tree, Pterocarpus officinalis (Jacq.), in the Caribbean basin. Mol. Ecol. 11, 675–683. Vekemans, X., Beauwens, T., Lemaire, M., Roldan-Ruiz, I., 2002. Data from amplified length polymorphism (AFLP) markers show indication of size homoplasy and a relationship between degree of homoplasy and fragment size. Mol. Ecol. 11, 139–151. Vos, P., Hogers, R., Bleeker, M., Reijans, M., Van de Lee, T., Hornes, M., Frijters, A., Pot, J., Peleman, J., Kuiper, M., Zabeau, M., 1995. AFLP: a new technique for DNA fingerprinting. Nucleic Acids Res. 23, 4407–4414. Wright, S., 1931. Evolution in Mendelian populations. Genetics 16, 97– 159. Yeh, F.C., 2000. Population genetics. In: Young, A., Boshier, H., Boyle, T. (Eds.), Forest Conservation Genetics. CABI publishing, Wallingford, England/CSIRO Publishing, Collingwood, Victoria, pp. 21–37. Zhivotovsky, L., 1999. Estimating population structure in diploids with multilocus dominant DNA markers. Mol. Ecol. 8, 907–913.
Forest Ecology and Management 255 (2008) 334–339 www.elsevier.com/locate/foreco
Genetic (AFLP) diversity of nine Cedrela odorata populations in Madre de Dios, southern Peruvian Amazon Amanda de la Torre a,*, Cesar Lo´pez a, Eliana Yglesias a, Jonathan P. Cornelius b a
Universidad Nacional Agraria La Molina, Av. La Universidad s/n La Molina, Lima 12, Peru b World Agroforestry Centre, CIP-ICRAF, Apartado 1558, Lima 12, Peru
Abstract Cedrela odorata L., one of the most important neotropical timber species, is threatened by deforestation and unsustainable logging in many parts of its natural range. Information on patterns of genetic variation is useful in informing both reforestation and genetic conservation activities. However, to date, no such information is available in Peru or elsewhere in South America. In the present study, genetic diversity between and within nine Peruvian populations of the species, based on amplified fragment length polymorphism (AFLP) markers, is reported. Overall diversity level was high (Ht = 0.22), as expected for a widespread, long-lived tropical species, and consistent with previous studies carried out in Central America. Levels of intrapopulation diversity were higher than those previously reported for the species (Hs = 0.13–0.21). Analysis of molecular variation revealed genetic differences between two population groups located on different rivers and between populations located on the same rivers. Differences between groups were greater than those within groups. Genetic and geographical distances were significantly correlated. The relatively strong genetic differences between populations may be related to the riparian, essentially one-dimensional spatial distribution pattern of the populations studied. No difference was found in percentage of polymorphic loci between relatively undisturbed and logged populations. The existence of appreciable genetic differentiation over a relatively small part of the species range in the Peruvian Amazon suggests the need for caution in use of seed outside its zone of origin. For genetic conservation purposes, it would probably be prudent to sample (ex situ) or conserve (in situ) populations in each of the major watersheds of the Peruvian Amazon. # 2007 Elsevier B.V. All rights reserved. Keywords: Meliaceae; Forest genetic resources; Genetic conservation
1. Introduction Cedrela odorata L. (cedro, Spanish cedar) is a neotropical, broadleaf tree species. Its broad, transequatorial distribution extends from 268N in Mexico to 288S in northern Argentina, and includes moist and seasonally dry forest types, and altitudes ranging from sea level to 1200 m a.s.l. (Pennington and Styles, 1975). The timber of C. odorata, like that of other Swietenioideae (e.g. the American mahoganies, Swietenia spp.), is of high value and is much in demand on domestic and international markets. It is classed as ‘vulnerable’ by IUCN and is threatened both by unsustainable logging (IUCN, 1994; Patin˜o, 1997) and by forest conversion to pasture and other land uses. In Peru, Spanish cedar is considered a threatened
* Corresponding author at: Centre for Forest Conservation Genetics, University of British Columbia, 3041-2424 Main Mall, Vancouver, B.C., Canada V6T 1Z4. Tel.: +51 1 4366804. E-mail address: [email protected] (A. de la Torre). 0378-1127/$ – see front matter # 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.foreco.2007.09.058
species by national authorities (INRENA, 2004) and since 1988 has been listed on CITES Appendix III, under which both export and import require export permits from the producing country (CITES, 2004). However, the species remains relatively common in many parts of its range (including parts of the Peruvian Amazon), including agricultural landscapes, where its heliophytic status and nurturing by farmers appear to have helped to ensure its continued presence in fencerows and forest remnants. In Madre de Dios province, southern Peruvian Amazon, where the present study was carried out, many remaining populations of C. odorata are riparian, i.e. located close to main rivers or to oxbow lakes. Because of their accessibility (i.e. via navigable rivers), many populations in the zone have been heavily depleted. Effective conservation and use of this and other vulnerable species depends partially on knowledge of patterns of genetic variation. For example, the spatial structure of genetic variation should inform sampling strategies for ex situ or in situ conservation. Similarly, marked between-population genetic
A. de la Torre et al. / Forest Ecology and Management 255 (2008) 334–339
differentiation would indicate the need for caution – at least – in use of seed for planting outside its area of origin. This applies even to neutral genetic variation detected by molecular markers, as differentiation may indicate relative absence of migration, which in turn could permit the development of variation in adaptive traits that otherwise could be prevented by gene flow. In the case of Spanish cedar, studies by Gillies et al. (1997), Cavers et al. (2003a) and Navarro (2002) have revealed pronounced genetic differences between populations from the Pacific and Atlantic regions of Costa Rica. However, no information is available for C. odorata in South America, although the region harbours most of the unlogged populations of the species. In the present study, we address two research questions of relevance to the management of Peruvian C. odorata genetic resources. First, we look at the degree of genetic differentiation between populations and between two population groups. Second, we examine whether there is any indication that human disturbance, i.e. logging, has to date affected genetic diversity of intervened populations. In particular, we were interested in examining whether the most highly intervened populations would show lower degrees of polymorphism, due to population bottlenecks (caused by logging) and consequent founder effects. The presence of such effects would imply that heavily logged populations should receive lower prioritization in ex situ and in situ conservation. In order to address these questions, we present information on genetic structure and variation in nine populations of the species. We based our estimates on amplified fragment length polymorphism (AFLP) markers. AFLPs are suitable markers for genetic diversity studies as, although dominant, they permit the assay of a large number of loci.
335
2. Materials and methods 2.1. Study sites and sampling Populations were sampled in two protected areas: Manu National Park (six populations) and Los Amigos Conservation Area (three populations) (Fig. 1 and Table 1). The Manu populations are located along an approximately 150 km stretch of the River Madre de Dios. Each population represents an area of 30–450 ha where the species occurs at variable, relatively high densities, i.e. 0.3–7 trees ha 1 (Table 1). With the exception of one additional, unsampled population (Cocha Jua´rez), located between Otorongo and Limonal populations, there are no other significant concentrations of the species either in riparian locations between the sampled populations, or in adjacent non-riparian locations, although it is probable that isolated trees or small groups of trees occur in both situations. We concentrated on riparian populations because, as a common type, their conservation status is important to the continued persistence of the species in Madre de Dios. In addition, they are and have been vulnerable to logging (legal and illegal). The Los Amigos populations are located 0–43 km upstream of the confluence of the Madre de Dios and the Amigos rivers, which occurs approximately 217 km downstream of the closest of the Manu populations. The intervening stretch of the River Madre de Dios flows through heavily deforested areas. In terms of human intervention, the populations fall into two groups: the Los Amigos populations and Limonal in Manu (located close to the park boundary) have been intensively logged. Most wellformed trees have been removed, leaving only low density stands (Table 1) of C. odorata individuals. The remaining populations are relatively undisturbed, including two (Cashu and Maizal) that have never been logged.
Fig. 1. Location of nine populations in two groups sampled in a study of genetic diversity of Cedrela odorata in the Southern Peruvian Amazon. Populations are represented by black dots and located as follows: from the top on the left side: Maizal, Cashu, Gallareta, Salvador, Otorongo and Limonal in Manu; from the bottom on the right side: Cicra TB, Cicra TA and Pv2 in Los Amigos area.
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Table 1 Description of collection areas sampled in a study of genetic variation of Cedrela odorata populations in the southern Peruvian Amazon
2.3. Data analysis
Leaf material was collected from a total of 153 individuals from the nine populations from October to December of 2003; 9–20 individuals were sampled per population (Table 1). Samples were stored in resealable plastic bags with 10–20 g of self-indicating silica gel, following Chase and Hills (1991).
Allele frequencies were estimated using the Bayesian method with non-uniform prior distribution of allele frequencies, as described by Zhivotovsky (1999). We estimated the degree of genetic differentiation between groups (i.e. populations on the River Madre de Dios and those on the River Amigos) and between populations-within-groups using analysis of molecular variance (AMOVA) from ARLEQUIN version 3.0 (Excoffier et al., 2005) and by estimating values of Nei’s F st (Nei, 1973) and related statistics using the AFLP-SURV software (Vekemans et al., 2002), following Lynch and Milligan’s (1994) approach. We tested for a relationship between pairwise physical and genetic distances (Nei, 1972) between the populations by estimating values of Spearman’s rank correlation. Genetic distances were calculated using AFLP-SURV. In order to test for effects on genetic diversity of population bottlenecks caused by logging, we examined the relative degrees of polymorphism of the two groups (logged and unlogged populations), specifically with regard to expectations that the most highly intervened populations would show lower degrees of polymorphism. We did not compare gene diversity between the two groups, because gene diversity is expected to be relatively little affected by bottlenecks (because rare alleles – which are those that are expected to be lost – contribute little to total heterozygosity).
2.2. Laboratory procedures
3. Results
Total genomic DNA was extracted using the CTAB method (Doyle and Doyle, 1987). AFLP protocol followed the standard procedure described by Vos et al. (1995). High quality DNA (i.e. not damaged and free of impurities) was digested using restriction enzymes EcoRI and MseI. DNA fragments were ligated to EcoRI and MseI adapters to generate template DNA for amplification. PCR was performed in two consecutive reactions, according to the Invitrogen protocol (Invitrogen, 2003). In the preamplification, genomic DNAs were amplified with AFLP primers, each having one selective nucleotide. The PCR products of the amplification reaction were diluted and used as a template for the selective amplification using two AFLP primers, each containing three selective nucleotides. Seven primer combinations were probed: EcoRI-ACT/MseI-CAT; EcoRI-AAC/MseI-CAC; EcoRI-ACC/MseI-CAT; EcoRIACC/MseI-CTT; EcoRI-ACC/MseI-CAG; EcoRI-ACA/MseICTC; EcoRI-AGG/MseI-CTC. Those with the best resolution and the greatest number of bands were selected. Thermal cycling conditions for amplification were optimised to 14 cycles of 94 8C for 30 s (denaturation), 65 8C for 30 s (annealing) and 72 8C for 60 s (extension); followed by 23 cycles of 94 8C for 30 s, 56 8C for 30 s, and 72 8C for 60 s. Final temperature was 4 8C. Amplification products were separated on 6% polyacrylamide gels, stained with AgNO3 (according to the standard procedure described by Creste et al., 2001) and visualized through UV light exposure. Subsequently, the gels were recorded using a camera and a scanner before scoring the presence or absence of each scorable band in a binary data matrix.
3.1. General
Collection area
Mean annual precipitation (mm year 1) Mean annual temperature (8C) Number of measured trees Number of sampled trees Mean (range) of densities (trees ha 1) Number of sampled populations Number of individuals sampled per population Degree of disturbance
Manu
Los Amigos
2500
2845
23
22
540
61
103
50
0.85 (0.3–7)
0.09 (0.05–0.1) 3 13–16
6 9–20 Undisturbed (except Limonal population)
Intensively logged
Three of the seven screened AFLP combinations were used in the study (of the others, two were rejected because of low resolution and two because of low numbers of bands). The three AFLP primer combinations produced a total of 258 bands in 137 individuals (from a total of 153 sampled, 137 were successfully amplified (Table 2)), of which 255 (98.8%) were polymorphic (Table 3). The percentage of polymorphism across the nine populations ranged from 41.5% (Salvador) to 66.7% (Otorongo) (Table 2). Levels of genetic diversity within populations (Hs) ranged from 0.13 (Maizal) to 0.21 (Otorongo) (Table 3). Overall gene diversity (Ht) was estimated at 0.22 and overall F st was 0.20. 3.2. Genetic differentiation The AMOVA indicated the presence of significant genetic variation both between the two groups and between populaTable 2 Number of loci evaluated for each of three AFLP primer combinations utilized in assays of 137 individuals of Cedrela odorata from nine southern Peruvian Amazon populations Primer combination
Number of loci
EcoRI-ACT/MseI-CAT EcoRI-AAC/MseI-CAC EcoRI-ACC/MseI-CAT
103 97 58
Total
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Table 3 Estimated percentage of polymorphic loci (P) and gene diversity (Hs) within nine populations of Cedrela odorata from the southern Peruvian Amazon Collection area
Population
Manu
Maizal Cashu Gallareta Otorongo Salvador Limonal
Los Amigos
PV2 CicraTA Cicra TB
Sample size
Total
Number of polymorphic loci
P
Hs (S.D.)
18 20 15 13 19 9
112 115 111 172 107 126
43.4 44.6 43 66.7 41.5 48.8
0.13 0.18 0.16 0.21 0.17 0.18
13 14 16
135 120 117
52.3 46.5 45.3
0.17 (0.01) 0.18 (0.01) 0.18 (0.01)
137
255
98.8
0.17 (0.007)
(0.01) (0.01) (0.01) (0.01) (0.01) (0.01)
Table 4 Analysis of molecular variance (AMOVA) between two population groups and nine populations-within-groups of Cedrela odorata from the southern Peruvian Amazon Source of variation
d.f.
Sum of squares
Variance component
Between groups Between populations-within-groups Within populations
1 7 128
367.675 503.09 2830.504
5.01412 3.28699 22.11332
Total
136
3701.272
30.41442
a
Total variance (%) 16.48 10.80 72.70
pa <0.0001 <0.0001 <0.0001
100
Probability of a higher value of F, based on permutation test (1000 permutations).
tions-within-groups. Almost three-quarters of the variation (72.7%) was concentrated within populations, while the between-group component (16.5%) was larger than the between population-within-group component (10.8%) (Table 4). Values of F st for populations-within-groups were similar (0.14 for Manu and 0.15 for Los Amigos) (Table 5). Overall F st was higher (0.20). The correlation between genetic and geographic distance was positive and highly significant (rs = 0.75, p < 0.0005, one-tailed) (Fig. 2). 3.3. Polymorphism in logged and unlogged populations The percentage of polymorphic loci per population varied from 43 to 66.7%. The least disturbed populations were not amongst the most variable and the logged populations had percentages of polymorphic loci similar to or greater than all the other populations except the most variable (Otorongo).
(Ht = 0.27, P = 84.8% (Cavers et al., 2003b); Ht = 0.34, P = 93.8% (Gillies et al., 1997)). However, diversity within populations, varying from 0.13 to 0.21, was higher than reported elsewhere. For example, Cavers et al. (2003b) found that diversity within Costa Rican populations ranged from 0.03 to 0.13. Furthermore, the proportion of polymorphic loci per population was greater than previously evaluated (41.5–66.7% vs. 9–22% found by Cavers et al., 2003b). Either or both of two factors could explain this. First, it might reflect a South American origin of the species (as postulated by Cavers et al., 2003a). This would imply a more recent colonization of Central America, and lower diversity levels could then be explained by founder effects or population bottlenecks during migration events (Rivera-Ocasio et al., 2002; Cavers et al., 2003a,b).
4. Discussion Overall levels of genetic diversity (Ht = 0.22, P = 98.8%) were similar to values reported for C. odorata in Mesoamerica Table 5 Estimates of fixation index (Fst), total gene diversity (Ht) and within population gene diversity (Hs) for two population groups of Cedrela odorata from Madre de Dios, southern Peruvian Amazon Collection area
Hs
Ht
Fst
Manu Los Amigos
0.17 0.17
0.20 0.21
0.14 0.15
0.22
0.20
Total (all populations)
Fig. 2. Scatter plot of genetic distances and geographic distances between nine populations of Cedrela odorata located in Madre de Dios, southern Peruvian Amazon.
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Second, it might reflect different levels of disturbance and human-induced fragmentation, both of which tend to be much greater in Central American populations than in the populations studied here, and may have been sufficiently severe to have led to genetic erosion. Our results, by contrast, indicate that the degree of disturbance and fragmentation found in the studied populations has not resulted in genetic erosion. This probably reflects ongoing gene-flow between the study populations, from upstream intact populations and from other trees in non-riparian situations (because gene flow could restore to individual populations any alleles lost in bottlenecks). Alternatively, it may simply imply that logging intensity was not sufficiently high to cause measurable founder effects. Similar results, attributed to similar causes (i.e. fragmentation, logging and lower long-term effective population sizes), were found by Lemes et al. (2003) and Novick et al. (2003) for Central American and Amazonian populations of big-leaf mahogany (Swietenia macrophylla King), another winddispersed, insect-pollinated, widespread meliaceous species subject to unsustainable intensive logging. According to Yeh’s (2000) criteria our findings on genetic structure indicate large (0.15 < F st 0.25) genetic differentiation at the overall level, and moderate to large (0.05 < F st 0.15) differentiation at the within-group level. It is possible that the presence of appreciable genetic differentiation over this relatively small area may be due to the riparian, essentially one-dimensional spatial distribution pattern of the populations studied here. The positive relationship between genetic and geographical distances suggests that gene flow is likely to be predominantly between adjacent populations, corresponding to Kimura and Weiss’s (1964) stepping stone model. Under the one-dimensional stepping-stone model – appropriate for these linear, riparian populations – more gene flow per generation is required to maintain overall panmixia than in Wright’s (1931) Island Model. For example, in a stepping stone system with migration rates of 0.1 and 2 10 5, respectively for adjacent and long-distance gene flow, ‘considerable’ local differentiation will occur if Ne < 100 (Kimura and Weiss, 1964). In addition, the more pronounced betweengroup variation could not be solely due to greater distance, but also to barriers to hydrochory-mediated gene flow (because the two groups are located upstream of the confluence of their respective rivers). However, based on our data, we cannot separate such effects from those related purely to isolation by distance. Cavers et al. (2003b) reported estimates of fst (an analogue of F st, based on partitioning of molecular variance between and within populations (Excoffier et al., 1992)) for two population groups of C. odorata located in Costa Rica. Our estimates of F st are similar to the fst estimate from Cavers et al.’s first population group of (fst = 0.20). The values we report are much lower than those for Cavers et al.’s (2003b) second group (fst = 0.47). This, however, covered a larger geographic area than that sampled here, including populations physically and reproductively separated by the Costa Rican Central Mountain range.
Taken together, our results and those of Cavers et al. (2003b) appear to indicate that C. odorata may have a tendency towards developing relatively marked differentiation between populations. In part, this could be due to factors specific to the two study sites (riparian distribution in Madre de Dios, human intervention in the case of Costa Rica (see above)). However, it should also be noted that, in general, abiotically dispersed tropical tree species tend to show relatively large population differentiation (Loveless, 1992), i.e. this tendency may be due in part to intrinsic characteristics of the species, and not simply to local ecological or historical factors. The presence of relatively pronounced genetic variation between these relatively closely distributed populations of C. odorata suggests the need for caution in the use of seed outside its zone of origin. This would apply particularly in the case of seed transfers over larger distances than those studied here. The Peruvian Amazon is a vast region, variable in soil types, elevations, and amount and distribution of precipitation. These distances may permit more substantial genetic differentiation than those observed here (as, indeed, seen in Cavers et al.’s (2003b) second group (see above)), particularly if reinforced by a tendency towards linear distributions and, possibly, unidirectional hydrochory. For genetic conservation purposes, it would probably be prudent to sample (ex situ) or conserve (in situ) populations in each the major watersheds of the Peruvian Amazon (e.g. those draining to the Napo, Maran˜on, Huallaga, Ucayali and Madre de Dios rivers). Our results suggest that logged populations should not be excluded from any such measures, as they do not show signs of being genetically depauperate. Future research should seek to clarify genetic structure at these larger geographic scales. Studies of adaptive variation, preferably of traits of commercial significance, should also be undertaken, in order to clarify the implications for planting programmes. Acknowledgments This work forms part of the senior author’s MSc thesis and was done with financial support from ACA (Amazon Conservation Association) and ICRAF (World Agroforestry Centre) through competitive grants for 2003. References Cavers, S., Navarro, C., Lowe, A.J., 2003a. Chloroplast DNA phylogeography reveals colonization history of a Neotropical tree Cedrela odorata L. in Mesoame´rica. Mol. Ecol. 12, 1451–1460. Cavers, S., Navarro, C., Lowe, A.J., 2003b. A combination of molecular markers identifies evolutionarily significant units in Cedrela odorata L. (Meliaceae) in Costa Rica. Conserv. Genet. 4, 571–580. Chase, M., Hills, H., 1991. Silica gel: an ideal material for field preservation of leaf samples for DNA studies. Taxon 40, 215–220. CITES, 2004. Listed species database. http://www.cites.org/eng/resources/species.html. Creste, S., Tulmann, N., Figueira, A., 2001. Detection of single sequence repeat polymorphisms in denaturing polyacrilamide sequencing gels by silver staining. Plant Mol. Biol. Rep. 19, 299–306. Doyle, J.J., Doyle, J.L., 1987. Isolation of plant DNA from fresh tissue. Focus 12, 13–15.
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