BIOTROPICA *(*): ***–*** **** 10.1111/j.1744-7429.2006.00217.x

Genetic Variation in Fragmented Forest Stands of the Andean Quercus humboldtii Bonpl. ()1

Juan F. Fernandez-M.´ 2 Laboratoire d’Ecologie, Systematique´ et Evolution, Batˆ 360, Universite´ Paris Sud XI, 91405 Orsay Cedex, France and

Victoria L. Sork Department of Ecology and Evolutionary, The University of California Los Angeles, CA 90095 1606, U.S.A.

ABSTRACT

Quercus humboldtii is a montane forest dominant species in Colombia, which has experienced significant habitat loss. Using three microsatellite loci, we compared the genetic diversity of adults and seedlings in fragments of small and large size. Results show high genetic diversity, comparable to other temperate oak species (Ho = 0.813, He = 0.780, and f =−0.044). However, allelic richness reduction in seedlings of the most fragmented part of the landscape, suggested restricted gene flow and risk of future genetic bottlenecks, if larger tracts of forest disappear.

RESUMEN

Quercus humboldtii es una especie dominante de las montanas˜ colombianas que ha sufrido una importante perdida de habitat.´ Usando marcadores microsatelites,´ comparamos la diversidad genetica´ de adultos y plantulas´ en fragmentos pequenos˜ y grandes. Encontramos una alta diversidad genetica,´ comparable a las especies de robles de zonas templadas (Ho = 0.813, He = 0.780 y f =−0.044). Sin embargo, existe una reduccion´ en la riqueza alelica´ de las plantulas´ de la zona mas´ fragmentada, sugiriendo que deben conservarse grandes areas´ boscosas para evitar riesgos futuros de perdida´ de la diversidad genetica.´

Key words: Andes; Fagaceae; forest fragmentation; genetic diversity; microsatellites; Quercus humboldtii; South America.

HABITAT FRAGMENTATION CAN THREATEN POPULATION VIABILITY adult populations of the Andean oak, which were established prior by altering multiple ecological and genetic processes (Young et al. to landscape fragmentation? (2) What does adult genetic structure 1986, Templeton et al. 1990, Saunders et al. 1991, Ledig 1992, indicate about historical gene flow? and (3) does genetic diversity in Ellstrand & Elam 1993, Frankham et al. 2002). Long-lived woody present day seedling cohorts in fragmented areas show less diversity perennials, in particular, are expected to be resilient to changes than seedlings in large forest stands? in genetic diversity due to ample gene flow and long generation times (Shapcott & Playford 1996, Soejima et al. 1998, White et al. 1999, Merwe et al. 2000, Hamrick 2004). However, if fragmented METHODS populations become isolated and gene flow is reduced, eventually these small populations will be at risk for loss of genetic diversity STUDY SPECIES.—Quercus humboldtii Bonpl. is the southernmost through genetic drift. species of oak in the Western hemisphere and belongs to the group Fragmentation of the forest is widespread in the northern of red oak subgenus Erythrobalanus (Nixon 1993). It is a medium- Andes, where the Colombian oak is a conspicuous element of the to-large size that is endemic to the northern Andes and possibly vegetation forming extensive forest stands of several kilometers long the Darien in Panama. Quercus humboldtii is a characteristic element in the Andean vegetation belt (Hooghiemstra & Sarmiento 1991). of primary Andean vegetation (Cuatrecasas 1958) found in moist Oak are now highly subdivided among pastures, crops, com- forests between 1500- and 3300-m elevation, forming stands of mercial timber, and native forests and virtually all its populations almost a monospecific canopy. Virtually all remnant populations of are in fragments of larger or lesser extent. A critical conservation the Andean oak can be considered fragments varying in size from concern is whether the seedlings that have established in these frag- a fraction of a hectare to probably 5000 ha for the largest tracts of ments are starting to show the isolating effects of fragmentation on forest because of intensive use in the past for construction, fire wood, gene flow, or whether gene flow among extant fragments is suffi- and clearing of the forest for cattle farming. They are considered cient to retain previous levels of genetic diversity. Specific research a vulnerable vegetation type (Fernandez-M´ 1993, Calderon´ 2001) questions we address are: (1) what is the overall genetic diversity of and the species is protected from timber harvesting since 1974 by the Colombian law. Like all , it is a wind-pollinated, monoecious 1 Received 13 May 2005; revision accepted 15 February 2006. tree that produces acorns dispersed mostly by gravity. The oak 2 Corresponding author; e-mail: [email protected] woodpeckers do not store acorns or rely heavily upon them, but feed C 2006 The Author(s) 1 Journal compilation C 2006 by The Association for Tropical Biology and Conservation 2Fernandez-M.´ and Sork

on insects, sap, and fruit year-round (Kattan 1988), which limits and dehydrated in zip-lock bags containing about 90 g of silica long-distance dispersal for the species. Pollen records show that gel. DNA extractions and molecular biology conditions, and data Quercus dispersed into the Colombian Andes at least 340,000 yr BP scoring are described in detail in Fernandez-M. et al. (2000). For from Central America (Hooghiemstra & Sarmiento 1991), which this study, we used three microsatellite loci: QpZAG58, QpZAG15, is relatively recent in comparison to North American modern oaks and QpZAG9. in Western United States, which have been there for approximately 20 million yr (Raven & Axelrod 1978). DATA ANALYSIS.—Number of alleles (A) per locus was determined by direct counting. The effective number of alleles (Ae) was estimated STUDY SITE.—Sampling sites were located in the northeastern Andes ◦   ◦   as the inverse of the expected homozygosity. Observed heterozy- of Colombia, (73 30 13 W, 5 43 14 N) near the towns of Villa gosity (H ), and expected heterozygosity (H ) followed formulas by de Leyva and Arcabuco, Department of Boyaca.´ These locations o e Pons and Chaouche (1995) that account for different sampling size. occurred between 2400 and 2700 m in a fragmented landscape that Inbreeding was estimated as f = 1–H /H for each locus. Stan- was once part of a continuous forest, as judged by several kilome- o e dard deviations of the previous parameters were obtained through ters of continuous oak fragments. The landscape contains multiple resampling individuals with replacement 1000 times. Allele counts stands that range in size from few scattered trees within pastures to are useful direct measures of genetic diversity with highly variable more than 4000 ha. Even this larger area has been subject to logging genetic markers but they are also very sensitive to unequal sample and some parts exhibit small trees that are probably resprouts after sizes (Petit et al. 1998). Therefore, allele count sampling curves logging. Smaller fragments are usually composed of few large trees were simulated for each site by resampling the data with 2, 3,..., surrounded by smaller trees, presumably originated from seeds from N individuals 1000 times for each sampling size. Resampling was these remnant trees. Aerial photographs and topographic maps from performed individually for adults and seedlings classified in two the 1960s show that present day fragments have remained virtually extreme groups of low population density (smaller fragments) and the same for at least 40 yr (Instituto Agust´ın Codazzi map C-241-6, normal population density (plots within the larger fragment). This 1963). Forest fragmentation probably began in the mid 1900s when method provides an estimate of the number of alleles with a 95% trees were cut for charcoal, firewood, and lumber, and rangeland ci for the smallest sample size and allows a comparison of all sam- for cattle, although human disturbance may be at least 500-yr old ples with that minimum sample size. All described analyses were (Molano 1990, Etter & van Wijngaarden 2000).  performed using specific functions written in Matlab R11 by JF. SAMPLE DESIGN.—During 1998 and 1999, we selected forest frag- The genetic structure was described by means of AMOVA ments consisting of small stands of isolated trees and a very large analysis (Excoffier et al. 1992) whose results are summarized in the remnant stand of continuous forest of ca 1200 ha. Separation of the parameter . We used a hierarchical genetic structure model with large fragment to the set of smaller fragments was about 11 km with two types of landscapes relative to the total population (LT )and several intervening oak fragments of variable size. The small stands subpopulations (fragments or plots) within landscape type (SL). were located at varying intersite distances with the closest sites be- Significance of the values was tested by bootstrapping individuals ing 240 m apart and the farthest 1200 m, with average fragment and comparing the upper and lower 95 percentiles. We conducted separation of 598 m (SD = 340; see Fig. 1A). Within the large tract AMOVA analyses using the GeneticStudio software (available by of forests, we selected three plots located in the interior such that request at http://dyerlab.bio.vcu.edu/). the minimum intersite distance was 440 m, the maximum 1400 m, We tested for isolation by distance (IBD) by estimating pairwise and the average = 951 m (SD = 482; see Fig. 1B). Hereafter, we fragment/plot genetic distances using pairwise values of from will consider and refer to the small fragments as representative of the the abovementioned AMOVA analyses. The transformed pairwise “subdivided” or fragmented part of the population, and the plots values of = /(1 – ) were regressed against the logarithm of within the larger remnant stand as representative of the “continu- the pairwise physical distances separating each study site (Rousset ous” part of the landscape. 1997). The significance of the association was tested by computing To compare present day levels of genetic diversity with those of the Z statistic of the Mantel test (Smouse et al. 1986) and by established adults, we sampled both seedlings and adults within all reshuffling 1000 times the genetic distance matrix and recalculating plots. Within the five fragments, we sampled all adult trees, which the parameter. Significance of the Z value was assessed by evaluating yielded a total of 146 adult trees with sample sizes of N = 9, 15, 21, the position of the observed value with respect to the tails of the 95 42, and 59. The area of small stands ranged from 0.05 to 0.2 ha. percentiles. Within the continuous forest plots, we sampled 50 adult trees with DBH greater than 50 cm, each encompassing an area of ca 0.25 ha. Trees were selected in a spiral starting from a focal tree until all RESULTS 50 trees were identified. At each of the eight sites, we sampled all available seedlings resulting in about 10–30 seedlings per sampling GENETIC DIVERSITY.—The three microsatellite loci exhibited 39 al- site, for a total of 162 seedlings, (84 from the fragments and 78 leles in total, distributed as 22 for QpZAG58, 12 for QpZAG15, from the three forest plots). and 5 for QpZAG9 (Table 1). Between 40 and 70 percent of alleles were rare alleles below the 5 percent threshold (73 percent LABORATORY ANALYSES.—A single 6–7 cm mature from each for QpZAG58, 42 percent for QpZAG15, and 40 percent for tree was collected in the field, torn to pieces of approximately 2 cm, QpZAG9, respectively). Average levels of observed heterozygosity Forest Fragmentation and Andean Oak Genetics 3

FIGURE 1. Approximate localization of study site in Northeastern Colombia, South America. (A) Distribution of the oak stands in the most fragmented area of the landscape. Numbers represent the total number of adult trees in each of the fragments. (B) Estimated disposition of plots (stars) within the large track of forest of the Andean oak. Each stand is composed of 50 contiguous trees. Both sites are separated about 11 km from each other, with several intervening oak stands.

(Ho = 0.813) were consistent across loci, although slightly smaller QpZAG15 are not significantly different from zero, but QpZAG9 for QpZAG9 (Ho = 0.720). Expected genetic diversity (mean He = had a significant excess of heterozygosity (f =−0.166, SD = 0.045; 0.780) was similar for QpZAG58 and QpZAG15, and lower for Table 1). Again, seedling values of inbreeding were practically the QpZAG9 (Table 1). Seedling diversity was essentially similar to the same as for the adults. The high number of alleles and high het- values of the adults (results not shown), except for the number of erozygosity indicate no inbreeding for Q. humboldtii. effective alleles that was higher for the seedlings (Ae = 6.40) than = for the adults (Ae 5.75). GENETIC STRUCTURE.—The AMOVA analysis on the adult trees Overall deviation from Hardy–Weinberg equilibrium in Q. suggested that genetic structure is highly significant among the =− humboldtii indicates a small excess of heterozygosity (f 0.044, fragments or plots within the landscapes (SL = 0.128 P < 0.001), = SD 0.107). The inbreeding coefficients for QpZAG58 and but not different among landscapes (LT =−0.021, NS). In other 4Fernandez-M.´ and Sork

TABLE 1. Global genetic diversity results for adult populations of the Andean oak in northeastern Colombia. Numbers in parenthesis are standard deviations from 1000 bootstraps with replacements.

a Locus Size (bp) A Ae Ho He f

QpZAG58 162–208 22 8.5 (0.6) 0.885 (0.018) 0.884 (0.009) −0.002 (0.022) QpZAG15 109–135 12 6.2 (0.4) 0.834 (0.021) 0.839 (0.010) 0.035 (0.030) QpZAG9 241–253 5 2.6 (0.1) 0.720 (0.026) 0.616 (0.012) −0.166 (0.045) Mean - 13.0 (8.5) 5.8 (3.0) 0.813 (0.084) 0.780 (0.144) −0.044 (0.107) a A corresponds to number of alleles, Ae to the effective number of alleles, Ho to observed heterozygosity, He to expected heterozygosity and f to the inbreeding coefficient.

words, at the largest scale, genetic structure is homogeneous for alleles in Q. humboldtii (Ae = 5.8, or 47 percent of all alleles on the region that includes both the fragments and continuous stand, average) is also very similar to temperate oak species. Levels of ob- but, at the local scale, it is highly structured within each landscape served heterozygosity and levels of expected heterozygosity were also type. For the seedling population we found no significant structure within the range of other Quercus species for microsatellite mark- for the between-landscape component (LT =−0.025, NS), and ers (for temperate oak data see: Dow et al. 1995, Dow & Ashley a small but significant structure for the within-landscape variance 1996, Steinkellner et al. 1997, Streiff et al. 1998, Degen et al. 1999, (SL = 0.034, P < 0.1). Lexer et al. 1999, Dutech et al. 2005). These results suggest that, For the adults, IBD was observed at a local scale for the frag- after the genus Quercus migrated from Central America into the ments (Z = 6.64, P = 0.010), as a trend for the control forest plots Andes, the effective population sizes must have either been large (Z = 1.15, P = 0.171), and nonexistent for the ensemble of sampled during migration or increased rapidly while populating the moun- sites (Z = 22.89, P = 0.298). This IBD pattern corroborates the tains. This pattern of migration without any significant bottleneck findings of the AMOVA analysis that suggests that at small scales, is also proposed for Q. petraea in Ireland that has been able to differentiation among adults trees is high (>0), but at larger maintain high levels of diversity after colonization from mainland scales, this differentiation disappears ( ∼ 0) as if there is a patchy Europe only 15,000 yr BP, even though deforestation has occurred distribution of the genetic structure. No significant spatial pattern since the Neolithic (Muir et al. 2004). Given that oaks in Europe was observed for the seedlings at any scale. also show high genetic diversity after the demographic expansion since the Pleistocene, less than 20,000 yr ago (Petit et al. 2002), GENETIC DIVERSITY ACROSS GENERATIONS.—Standardized data of it appears that oaks as taxa are able to maintain the large effective allelic richness A (sample size of N = 78) show that from the three population sizes necessary for the maintenance of genetic diversity utilized loci, only the most variable locus QpZAG58 has differences and Q. humboldtii shows the same tendency toward high genetic among generations. Adults in both landscapes, and seedlings in the diversity that is typical of this genus. most fragmented area, share the same number of alleles (13 ± The finding that raises concern is the lower genetic diversity of 1, Fig. 2). However, seedlings in the plots of the large track of seedlings in the fragments compared to seedlings in the forest plots. forest (ca 1200 ha) have a significantly larger number of alleles than We found that in the forest plots, seedling diversity is greater than any other group (19 ± 1.5) at the standardized size. This result the local adult population, while seedlings from fragments include indicates that the seedlings in fragments are the result of gene flow about the same number of alleles as the set of all adults from the mostly restricted to the fragment, while the seedling in the forest landscape. At a plot or fragment level, seedling diversity is composed plots received gene flow either by pollen or seeds from individuals of local and external alleles indicating that gene flow is feasible in beyond the adult population within the plot. both types of landscapes. So, the greater diversity in the forest plots might be due to greater seed dispersal into those plots than into the small and isolated fragments. In fact, we saw no evidence that acorns DISCUSSION can arrive from outside the fragments (Fernandez-M.´ and V. Sork, pers. obs.). Hence, a continuous population structure of Andean The current adult and seedling populations of Andean oak in this oak is a better conservation strategy than isolated trees in sparse fragmented ecosystem appear to be as genetically diverse and re- landscapes, where gene flow is largely through wind pollination. silient as other temperate oak species. For example, the number of Given the small number of adults in each of these fragments, such alleles for microsatellite loci in different oak species ranges from as restricted gene movement may lead to future genetic bottlenecks few as three in Q. myrsinifolia (Isagi & Suhandono 1997) to 33 among isolated remnant stands if their size continues to diminish in Quercus robur (Steinkellner et al. 1997) and values of at least and if seed dispersal is hampered. 10 alleles per locus are not rare in other American red oak species Gene flow into forest fragments or secondary forest is highly (e.g.,Aldrichet al. 2002). Here we found 5–22 alleles across three variable across studies, and not all studies report reduced genetic loci with a mean of 13 alleles per locus. The effective number of diversity (Sork & Smouse, in press). For instance, for Symphonia Forest Fragmentation and Andean Oak Genetics 5

FIGURE 2. Standardized allele counts for QpZAG58 for adults (upper graphs) and seedlings (lower graphs) for fragmented (right) and continuous (left)partsof the landscape. All samples were standardized to N = 72. globulifera in a 38.5 ha reserve in Costa Rica, Aldrich et al. (1998) than the degree of separation of the sample plots. In fact, other found that 68 percent of the seedlings in remnant forest were de- studies show that Q. humboldtii may have a large local structure as rived from a few pasture trees, creating a genetic bottleneck. Sezen the evaluation of other six populations in Colombia with RAPD et al. (2005) found that founding trees in second growth forest of data and microsatellites also show within population structure val- the Costa Rican palm Iriartea deltoidea were derived from a small ues between 0.1 and 0.2 ( J. Palacio-Mejia et al., pers. comm.). number of trees from the primary forest, but with a similar risk of Interestingly, the values of SL that we found are comparable to a genetic bottleneck. Shorea leprosula in Malaysia (Lee et al. 2000) values for pollen pool structure in savanna oak populations of Q. exhibited lower levels of heterozygosity at the seedling stage in some, lobata where Sork et al. (2002) conclude that the effective number but not every fragmented population, possibly caused by varying of pollen donors is small and effective pollen dispersal is ca 100 m. degrees of isolation. In contrast, genetic diversity of wind-dispersed Thus, the genetic structure of Q. humboldtii indicates that gene Acer saccharum seedlings was higher in fragments than in continu- movement has been local, which explains why the seedling diversity ous forest (e.g.,Fore´ et al. 1992, Young et al. 1993), suggesting that in the fragments is restricted to the levels of the adult population the removal of forest structure might promote gene flow among within the fragments. fragments within gene flow range. Similar results were found across In conclusion, our study provides evidence that the Andean the natural distribution of a rare endemic, bee-pollinated peren- oak still harbors a great wealth of genetic diversity, but the current nial shrub Brongniartia vazquezii in the Central deserts of Mexico, pattern of fragmentation may lead to long-term loss of genetic where the levels of genetic diversity were higher in seedlings than in diversity. Future studies may want to address the interaction between adults (Gonzalez-Astorga´ & Nu´nez-Farf˜ an´ 2001) because of pos- the degree of isolation and fragment size to understand the pattern sible increased post-fragmentation gene flow. In the Andean oak, of landscape mosaic that can sustain genetic diverse populations. the observation that seedling diversity is lower in fragments than in forest plots suggests reduced gene flow, because pollen and/or acorns are not moving among the fragments. ACKNOWLEDGMENTS The genetic structure of adult populations indicates that gene movement in this species has been local historically. Within both The authors wish to thank R. Dyer for his many suggestions through landscape types significant genetic structure among local subpop- the development of the project, the comments of D. Grivet on ulations of the Andean oak adults (SL = 0.128), indicates that this manuscript, and two anonymous reviewers. J. F. M. received historical pollen or seed movement has occurred at a smaller scale support from the International Center for Tropical Ecology and 6Fernandez-M.´ and Sork

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Q1 Author: Please check the page range for Fernandez et al. (2000). Q2 Author: Please check the page range for Nixon (1993). Q3 Author: Please check the page range for Sezen et al. (2005). Q4 Author: Please update reference Sork and Smouse (In press).