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GARDNER, R. A. W., D. G. OSCROFT and A. K. NTOMBELA development of a non-destructive near infrared (NIR) (2001): Final report on the 1992 Zululand site-species calibration model for screening of pulping properties. interaction trial series SGE32 to SGE34. ICFR Bulletin MSc Thesis, University of KwaZulu-Natal, Pietermar- Series 17/2001. Pietermaritzburg, Institute for Com- itzburg. mercial Forestry Research. SAS INSTITUTE (2002-2003): SAS/STAT Computer Soft- HARRAND, L., J. J. VARGAS HERNANDEZ, J. LOPEZ UPTON ware. Release 9.1.3. SAS Institute Inc. Cary, North Car- and G. RAMIREZ VALVERDE (2009). Genetic parameters of olina, U.S.A. growth traits and wood density in Eucalyptus grandis SQUILLACE, A. E. (1974): Average genetic correlations progenies planted in Argentina. Silvae Genetica 58: among offspring from open pollinated forest trees. Sil- 11–19. vae Genetica 23: 149–156. LEE, D. (2007): Relative performance of Corymbia hybrids SWAIN, T-L. and D. G. OSCROFT (2003): First year height and parental open-pollinated families in subtropical measurements of Eucalyptus henryi and E. longirostra- Queensland. In: Proceedings of “Eucalypts and Diversi- ta provenance trials in Zululand. ICFR Bulletin ty: Balancing Productivity and Sustainability”, IUFRO 06/2003. Institute for Commercial Forestry Research, Working Party 2.08.03, Durban, South Africa, 22–26 Pietermaritzburg. October 2007. SWAIN, T-L. and D. G. OSCROFT (2008): Performance of LITTELL, R. C., G. A. MILLIKEN, W. W. STROUP and R. D. Eucalyptus longirostrata and Corymbia henryi on the WOLFINGER (1996): SAS System for Mixed Models. SAS Zululand Coastal Plain: Four year provenance/ Institute Inc., Cary, NC, U.S.A. progeny trial results. ICFR Technical Note 04/2008. LOUW, A. K. (2006): Investigation of the application of best Pietermaritzburg, Institute for Commercial Forestry linear prediction for breeding and clonal production Research. purposes in a Eucalyptus grandis population. MSc The- VERRYN, S. D., C. L. SNEDDEN and R. C. PARFITT (2000): sis, University of KwaZulu-Natal, Pietermaritzburg. Program for the deterministic modelling of genetic MAHMOOD, K., N. E. MARCAR, M. H. NAQVI, R. J. ARNOLD, gains of tree breeding and seed and clone produc- D. F. CRAWFORD, S. IQBAL and K. M. AKEN (2003): Genet- tion strategies. Southern African Forestry Journal 189: ic variation in Eucalyptus camaldulensis Dehnh. For 3–9. growth and stem straightness in provenance-family VERRYN, S. D., C. L. SNEDDEN and K. A. EATWELL (2007): A trial on saltland in Pakistan. Forest Ecology and Man- comparison of deterministically predicted genetic gains agement 176: 405–416. with those realised in a South African E. grandis breed- NAMKOONG, G. (1979): Introduction to Quantitative Genet- ing program. In: Proceedings of “Eucalypts and Diversi- ics in Forestry. U.S. Department of Agriculture. Techni- ty: Balancing Productivity and Sustainability”, IUFRO cal Bulletin No.1588. Working Party 2.08.03, Durban, South Africa, 22–26 NDLOVU, Z. T. L. (2009): Breeding of advanced generation October 2007. of Eucalyptus macarthurii – growth parameters and

Changes in Genetic Diversity of Whitebark Pine (Pinus albicaulis Engelm.) Associated with Inbreeding and White Pine Blister Rust Infection

By A. D. BOWER1),2) and S. N. AITKEN

Centre for Forest Conservation Genetics, Department of Forest Sciences, University of British Columbia, 3041-2424 Main Mall, Vancouver, B.C. V6T 1Z4, Canada

(Received 9th April 2010)

Abstract sampled from 14 sites in British Columbia, Oregon, We investigated the association of inbreeding and Idaho, and Montana. Comparison of genetic diversity infection by the introduced disease white pine blister parameters among cohorts within a site was used to assess the extent and persistence of inbreeding with rust (caused by the fungus Cronartium ribicola J. C. Fisch) with genetic diversity of whitebark pine ( age, while comparisons of parameters among sites with- Pinus in a cohort were used to assess the impact of the disease albicaulis Engelm.) by genetically comparing cohorts of different ages in natural stands. Isozyme analysis of bud on genetic diversity. Significant evidence of inbreeding (F >0) was found in all age cohorts. When sites were tissue was used to estimate expected and observed het- is erozygosity (H and H ), and Wright’s fixation index (F ) stratified by level of blister rust infection, differences in e o is F and H among cohorts were only significant when for three age cohorts (seedling, young, and mature), is o level of infection was low. A significant negative associa- 1) Current Address: USDA Forest Service, Olympic National For- tion was found between level of blister rust infection est, 1835 Black Lake Blvd. SW, Olympia, WA 98512. Phone and Ho in the mature cohort. This suggests that when 360-956-2405, Fax 360-956-2330. E-mail: [email protected] differential selection due to blister rust is weak, more 2) Communicating author: ANDREW D. BOWER. heterozygous individuals may be favored; however, more

Silvae Genetica 60, 3–4 (2011) 113

DOI:10.1515/sg-2011-0016 edited by Thünen Institute of Forest Genetics Bower et. al.·Silvae Genetica (2011) 60-3/4, 113-123 homozygous individuals may have higher fitness under infection by the introduced disease white pine blister higher blister rust levels rust (caused by the fungus Cronartium ribicola J. C. Fisch.) in British Columbia also increases from west to Key words: whitebark pine, Pinus albicaulis, white pine blister east (ZEGLEN, 2002), although infection varies widely rust, genetic diversity, isozymes, inbreeding, cohort analysis, heterozygosity. among stands even within a mountain range (CAMPBELL and ANTOS, 2000). Trends in genetic diversity observed in British Columbia populations could be the result of Introduction either postglacial colonization patterns or selection by Exotic diseases can have a devastating impact on pop- the disease against more homozygous, inbred individu- ulations that have not coevolved with pathogens and als (KRAKOWSKI et al., 2003). However, in that study, any have little or no natural resistance, potentially leading potential effects of inbreeding are confounded with the to rapid declines in effective population size and loss of level of disease, due to the field sampling design. genetic variation. They pose unusual challenges to con- The goals of this study were to examine the individual servation because of their potential to drive rapid effects of inbreeding and blister rust infection on genetic changes in host abundance and genetic composition diversity of whitebark pine. The effects of inbreeding (ALTIZER et al., 2003). Species in many genera of forest were assessed by comparing Fis and Ho among three age trees have been severely impacted by introduced dis- cohorts within populations, and the effects of blister rust eases (e.g. Pinus, Chamaecyparis, Quercus, Castanea, were assessed by comparing these genetic diversity val- Ulmus, Fagus, Juglans, Cornus) (BINGHAM et al., 1971; ues for a given cohort among populations distributed LEDIG, 1991; DAUGHTREY ans HIBBEN, 1994; MALONEY et across a wide geographic area. al., 2005; LOO, 2009), but data on the effects of these dis- eases on genetic structure and diversity is sparse. It is well known that disease can have a significant impact Materials and Methods on both individual species and on the structural compo- Sample Materials sition of forests (BURDON et al., 2006); however, empiri- Thirty individuals from each of three age cohorts were cal demonstrations of the effect of diseases on host sampled from 14 sites in British Columbia, Idaho, Mon- demographics are difficult to conduct (ALEXANDER et al., tana, and Oregon in 2002 and 2003 (Table 1 and Figure 1996). In addition, studies of the effects of diseases on 1). The seedling cohort was represented by three-year- genetic diversity are difficult because baseline, pre-epi- old trees grown from seed collected from natural popula- demic genetic data are impossible to collect after a dis- tions at these sites and grown in a common garden ease has swept through (MCDONALD et al., 1998). experiment in Vancouver, British Columbia. The young Conifer populations often show increasing levels of and mature cohorts were sampled in the field from the heterozygosity with age, as embryos typically have a same natural stands where seeds were previously col- deficiency of heterozygotes, yet populations of mature lected for the common garden experiment. The young trees usually have an excess (PLESSAS and STRAUSS, cohort comprised trees estimated to be less than 30 1986; POLITOV et al., 1992; LEDIG et al., 2000). The years old, while the mature cohort included larger, older excess homozygosity in seeds is most likely due to trees of reproductive age. To assess any potential genetic inbreeding (LEDIG et al., 2000), including self-pollination differences between putatively resistant (clean) and sus- and consanguineous matings, and selection against ceptible (infected) trees, the health status of each indi-

inbreds is the most likely reason for the reduction in Fis vidual was recorded. Whitebark pine often grows in with age (KRUTOVSKII et al., 1995). For example, a study multistemmed clusters, and without genetic analysis, it of Bosnian pine (Pinus leucodermis Ant.), which has is impossible to determine if these comprise one or more reported selfing rates in the same range as whitebark genotypes (TOMBACK and SCHUSTER, 1994). Therefore, pine (18–28%), found that inbred individuals were not the stems in each cluster were conservatively regarded efficiently selected against during seed maturation and as comprising a single genotype and only a single stem germination, but were eliminated by age five (MORGANTE was sampled from each cluster. If any stems in a sam- et al., 1993). A heterozygote deficiency may be the result pled cluster were infected, that sample was considered of genetic substructuring within stands producing a to be from an infected individual. Buds were sampled Wahlund effect, but in most cases, inbreeding appears to from all individuals in both the field and the common be the primary cause and this declines with age due to garden experiment, and placed separately into separate selection over time against more homozygous inbred plastic vials for transportation and subsequent storage individuals (PLESSAS and STRAUSS, 1986). at –80°C. Whitebark pine (Pinus albicaulis Engelm.) experi- A disease infection survey was performed on each site ences inbreeding from selfing, and to a lesser extent, to estimate the level of white pine blister rust infection. consanguineous mating (KRAKOWSKI et al., 2003; BOWER The survey protocol generally followed SMITH and HOFF- and AITKEN, 2007). In addition, the inbreeding coeffi- MAN (2000). Strip transects were used to estimate stand

cient (Fis) of seedlings is higher than that of mature infection levels by categorizing 50 trees on each site as trees, indicating selection against inbred individuals 1) alive and clean: no evidence of blister rust; 2) infected over time (BOWER and AITKEN, 2007). A previous study and alive: alive with cankers, flagged or dead branches,

reported that Fis decreased and observed heterozygosity or dead stems in multiple stemmed clumps with some (Ho) increased from west to east in whitebark pine in living stems; 3) infected and dead: dead with clear evi- British Columbia (KRAKOWSKI et al., 2003). Stand-level dence of blister rust cankers; or 4) dead unknown: dead

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DOI:10.1515/sg-2011-0016 edited by Thünen Institute of Forest Genetics Bower et. al.·Silvae Genetica (2011) 60-3/4, 113-123 but unable to determine cause of death. Four to six tran- Isozyme Analysis sects were assessed on most sites, except one site with Whole buds were ground in a buffer slightly modified very low whitebark pine density, where only one tran- from MITTON et al. (1977) and the supernatant absorbed sect was completed which encompassed most of the trees onto 3 x 15 mm Whatman filter paper wicks. Samples on this site (Lunch Peak). Infection percentage was cal- were analyzed using starch gel electrophoresis on gels of culated as: # of infected trees (both alive and dead)/ 10.5% starch and 7.5% sucrose (w/v). Two buffer and (total # of trees assessed – # dead from unknown caus- electrode systems were used to analyze 10 loci as fol- es). The mean infection percentage over all transects on lows: 1) lithium borate/tris-citrate at a pH of 8.3 a site was used to estimate the level of blister rust infec- (SELANDER et al., 1971) was used for PGI (E.C. 5.3.1.9), tion. For comparative purposes, sites were classified by SKDH 1, 2 (E.C. 1.1.1.25), PGM (E.C. 2.7.5.1), and TPI infection level as low (<50% infection, = 5), moderate n (E.C. 5.3.1.1); 2) histadine-EDTA (CHELIAK and PITEL, (50–75% infection, = 5), or high (>75% infection, = 4). n n 1984) was used for IDH (E.C. 1.1.1.42), MDH 1, 2 (E.C. 1.1.1.37), GDH (E.C. 1.4.1.3) and ALD (E.C. 4.1.2.13). Gels were electrophoresed with wicks in place at half voltage for 30 minutes, then wicks were removed and the lithium borate gels run at 300 V and the histadine- EDTA gels run at 225 V. Electrophoresis continued until a marker dye reached to within 1 cm of the edge of the gel. Gels were sliced and stained using recipes as detailed in CHELIAK and PITEL (1984), then fixed in a mix ture of 1 : 5 : 5 glacial acetic acid : distilled water : methanol.

Data Analysis The Fstat program (GOUDET, 1995) was used to calcu-

late expected (He) and observed (Ho) heterozygosities and Wright’s fixation index (Fis) (WRIGHT, 1951) for each cohort on each site. WEIR and COCKERHAM’s (1984) ⌰

(Fst) and f (Fis) were estimated with Fstat for each cohort, and for sites grouped by infection level within each cohort. Fstat calculates F-statistics per locus and globally over all loci for a site or age cohort (GOUDET, 1995).

SAS Version 8 (SAS INSTITUTE, 1999) was used for all statistical analysis. Means and standard errors were calculated for genetic parameters, and the differences among cohorts tested using PROC GLM for one-way analysis of variance (ANOVA) with a Duncan’s multiple range test. The ANOVA was also conducted separately on groups of sites stratified by level of infection. Simple Figure 1. – Map of the range of whitebark pine and locations and level of blister rust infect of 14 sample locations. Infection regression with PROC REG was used to determine if level: low = <50%, moderate = 50–75%, high = >75%. there was a significant association between level of blis-

Table 1. – Study site names, geographic locations, and white pine blister rust infection.

a Refers to number on figure 1.

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DOI:10.1515/sg-2011-0016 edited by Thünen Institute of Forest Genetics Bower et. al.·Silvae Genetica (2011) 60-3/4, 113-123 ter rust infection and site latitude and longitude or genetic parameters. Paired t-tests were performed on data from the mature cohort to determine if clean and infected trees

paired by site differed in Ho and Fis. Sites varied in the number of clean and infected trees in this cohort (Table 1). To eliminate potential confounding due to variation in sample size between clean and infected trees on each

site, Fis was calculated as 1- (Ho for the group of clean or infected trees/He calculated for all trees on the site). The t-test was limited to sites that had a minimum of four individuals in each group.

Results Significant inbreeding (F > 0 at = 0.05) was detect- is ␣ Figure 3. – Mean observed heterozygosity (Ho) by cohort for ed in all cohorts when analyzed across all sites, even low, moderate, and high rust infection level sites (error bars when sites were stratified by level of rust infection (Fig- are 95% confidence intervals determined by bootstrapping over loci 1000 times in Fstat). ure 2). Across all sites, differences in mean Fis values were marginally significant among cohorts (p = 0.056) but in the multiple range test, only means of the seedling and young cohorts were significantly different higher in the Rocky Mountains than in the Coast Range at ␣ = 0.05. By locus, the difference among cohorts was (0.156 vs. 0.144); however, no differences were signifi- only significant for SKD 1 (p = 0.014), with only the cant in T-tests of group means. No significant associa- tions were found between stand level blister rust infec- seedling and young cohort differing. Differences in Ho among sites were not significant when analyzed over all tion and latitude (R2 = 0.081, p = 0.324) or longitude loci, but differed significantly for PGM and SKD 1 when (R2 = 0.003, p = 0.843). Regressions of the genetic para-

loci were analyzed individually. In both cases, the meters on latitude were only significant for He in the seedling cohort differed from both the young and mature young cohort (R2 = 0.334, p = 0.031), and all regressions cohorts, When sites were stratified by the level of rust on longitude were not significant (p > 0.05). Regressions infection, the mean of the seedling cohort was signifi- of the genetic parameters for each of the cohorts with cant different from the young and mature cohorts for blister rust infection levels showed a significant associa- tion only for H in the mature cohort (p = 0.018). Plots of both Fis (p = 0.025) and Ho (p = 0.039) when the rust level o was low. However, no differences were detected among Fis and Ho versus infection percentage indicated that the cohorts when the rust level was moderate or high (Fig- association between Fis and infection increased with ures 2 and 3). cohort age while the association of Ho with infection When populations were analyzed by mountain range, decreases (Figures 4a–f). Figure 4d shows one outlier site with high infection but low Ho. This point repre- average He and Ho were slightly higher in the Coast Range (populations 1–7 in Figure 1) (H = 0.222 and sents Lunch Peak (site #8 Figure 1, Table 1). This site e had a very low density of whitebark pine, with large dis- Ho = 0.188) than in the Rocky Mountains (populations 8–14 in Figure 1) (H = 0.210 and H = 0.177). F was tances between trees. Only a single infection survey e o is transect could be performed on this site because of the small population size. It is likely that the low density and wide spacing among whitebark pine trees at this site resulted in a higher rate of selfing. This is reflected

in the high Fis (Figure 4a – filled circle and table S1-sup- plimentary data) and low Ho values (Figure 4d – filled circle and table S2 – supplementary data) in the seedling cohort. Cook’s distance can be used to measure the influence of a data point on a regression (WEISBERG,

1985), and the Cook’s distance value for Ho from this site was an order of magnitude higher than most of the other sites, indicating that this outlier had a large influ- ence on the regression. When this data point was

removed from the analysis, the regression of Ho on infec- tion percentage for the seedling cohort was also signifi- cant (p = 0.033), but with a positive slope (Figure 4d). Comparison of clean and infected trees within a site

showed that for nine of the fourteen sites, Ho was higher Figure 2. – Mean fixation index (Fis) by cohort for low, moder- ate, and high rust infection level sites (error bars are 95% con- on average in the infected trees than in clean trees. fidence intervals determined by bootstrapping over loci 1000 However, paired T-tests for both Fis and Ho showed that times in Fstat). the differences between the group means were not sta-

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Figure 4. – Scatterplot of (a-c) fixation index (Fis) and (d-f) observed heterozygosity (Ho) of whitebark pine versus infection percent for 14 sites.

Table 2. – Fixation Indices (Fis) at three life stages for several conifers.

a Determined from inferred maternal genotype.

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Table S1. – Fis values for 10 individual isozyme loci and over all loci from 14 sites.

tistically significant (p = 0.297 and 0.247 for Fis and Ho, Discussion respectively). We observed a significant decrease in Fis between the Genetic differentiation among populations varied seedling and young cohort when the level of rust infec-

among cohorts (Fst = 0.075, 0.025, and 0.040 for the tion was low, but no differences among cohorts when the seedling, young, and mature cohorts, respectively), but level of rust was moderate or high. In our study, all values are within the range previously reported for inbreeding and blister rust affect the genetic diversity of whitebark pine (YANDELL, 1992; JORGENSEN and HAM- whitebark pine in different ways. Inbreeding usually RICK, 1997; STUART-SMITH, 1998; RICHARDSON et al., 2002; leads to lower genetic diversity within individuals and a KRAKOWSKI et al., 2003). heterozygote deficiency in seeds and seedlings, but this

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DOI:10.1515/sg-2011-0016 edited by Thünen Institute of Forest Genetics Bower et. al.·Silvae Genetica (2011) 60-3/4, 113-123 effect decreases over time as trees age and density- against heterozygotes by the rust may be a reason why

dependent selection occurs (Table 2). This pattern can be Fis values stay high in the young and mature cohorts on seen when the level of rust infection is low (Figure 2). sites with higher infection levels, and why Ho decreases However, when selection pressure due to blister rust is with level of rust infection in the mature cohort. There higher, heterozygosity levels are nearly the same across are other reports of differentiation of enzyme loci associ- cohorts, and the heterozygote deficiency typical of ated with environmental variation which suggest that seedlings was maintained in both young and mature genotypes for specific enzymes may result in physiologi- trees in the more infected populations studied. Selection cal differences that are acted on by natural selection

Table S2. – Ho values for 10 individual isozyme loci and over all loci from 14 sites.

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DOI:10.1515/sg-2011-0016 edited by Thünen Institute of Forest Genetics Bower et. al.·Silvae Genetica (2011) 60-3/4, 113-123 (MOPPER et al., 1991; MITTON, 1995). An alternate expla- can chestnut (Castanea dentata) that have persisted nation may be that where selection or local mating sys- through the chestnut blight epidemic through sprouting tem has produced highly heterozygous stands, rust suggests that selection has favored more heterozygous infection levels do not reach moderate or high levels. individuals (STILWELL et al., 2003). This could be due to local environmental conditions in Introduced pathogens can result in high selection which more heterozygous individuals have higher fit- pressure when levels of natural resistance are low, and ness. However, while they were not statistically signifi- evolution towards increased resistance could result in cant, our results showed that on individual sites, infect- decreased levels of diversity due to a genetic bottleneck ed trees often have higher heterozygosity than clean or selective sweep. In a comparison of populations of trees, which argues against this hypothesis. western white pine (Pinus monticola), a closely related As the level of rust infection increases, overall H of o species in the Pinus subsection Strobus, KIM et al. the mature trees within the stands we assessed (2003) found that a natural population under low rust decreased, yet the seed cohort produced from these pressure had higher polymorphism (P) and heterozygos- stands had higher H than stands with lower rust infec- o ity (He), and more private alleles than a population with tion. One potential explanation for this is that if rust is high rust pressure. Assuming that levels of genetic killing stems or even just the cone-bearing branches of diversity in the two populations were similar prior to individuals within clusters, this could result in a reduc- invasion by blister rust, this suggests that selection by tion in matings among related individuals on sites with rust resulted in reduced diversity. Inferences regarding higher rust infection. This would lead to higher het- the level of resistance in the natural whitebark pine erozygosity in the seed than in the mature individuals populations sampled in this study are not possible; how- that are reproducing. A decrease in density leading to a ever, if there is stronger differential selection on high loss of spatial genetic structure (e.g. due to harvesting) rust sites, this may lead to higher levels of overall resis- often has been reported to result in increases in inbreed- tance in the remaining population, albeit at the risk of ing (MARQUARDT et al., 2007), and wind-pollinated dramatic reductions in population size and genetic species would be expected to show a positive relation- diversity, and even local extirpations if resistance genes ship between population density and outcrossing rate are recessive. (FARRIS and MITTON, 1984). However, empirical tests of Both dominant and recessive resistance genes have this relationship are inconsistent (DYER and SORK, 2001), and studies of the effect of different silvicultural been identified in a variety of crop plants (TOYODA et al., treatments on genetic diversity showed an increase in 2002; CHU et al., 2004). To date, only one dominant ver- heterozygosity in naturally regenerated seedlings of tical resistance mechanism to blister rust (hypersensi- tivity) has been identified in the white pines (KINLOCH Pinus strobus after harvesting compared to old-growth populations (MARQUARDT et al., 2007). The authors sug- and DUPPER, 2002), while several horizontal mecha- gested that logging decreased the spatial genetic struc- nisms have been identified with dominant, recessive, ture, reducing spatially caused biparental inbreeding for and additive modes of inheritance hypothesized for a the seedlings produced post-disturbance. The relation- number of pine-rust pathosystems (see references in KINLOCH, 1982 and KINLOCH et al., 2008). Evidence of ship we observed in Ho in the seedling cohort may also reflect a decrease in selfing or biparental inbreeding in recessive gene segregation was found in early inocula- stands with higher rust infection, however, this cannot tion trials of western white pine (BINGHAM et al., 1960; be tested with our data. This hypothesis is speculative, BINGHAM, 1966) and recessive inheritance of genes con- but warrants further investigation. A study to investi- ferring high resistance or immunity in Idaho western gate genetic diversity in mature parent trees with their white pine were hypothesized by MCDONALD and HOFF offspring that includes both a measure of local tree den- (1970) and HOFF and MCDONALD (1971). Disease resis- sity and level of blister rust infection may be able to test tance genes are often dominant (BURDON, 2001) this hypothesis. although about 10% of resistance genes in crops are recessive (BURDON and THOMPSON, 1995). In a large, Previous studies exploring the effects of disease epi- truly panmictic population, recessive genes would rarely demics on genetic diversity have yielded varying results be expressed and thus would be of little selective value in both forest trees and agricultural crops (MCDONALD (BURDON, 2001), but forest trees typically have mixed et al., 1998; OUBORG et al., 2000; KIM et al., 2003). In mating systems with some selfing and bi-parental wild plant-pathogen populations, hosts should evolve inbreeding (LEDIG, 1998). In wild populations, this towards increased resistance (GILBERT, 2002). However, mixed mating will facilitate the expression of recessive it is not always possible to determine if changes in phenotypes produced by such alleles in some individu- genetic diversity are correlated with increases in resis- als. Although this will likely be associated with some tance (MCDONALD et al., 1998) and depending on the inbreeding depression (WILLIAMS and SAVOLAINEN, 1996), pathosystem, genetic control of resistance varies. For the cost could be small compared with the selective example, in a study of American beech (Fagus grandifo- advantage of disease resistance (BURDON, 2001). lia Ehrh.), trees susceptible to beech bark disease had The relationship between H and level of blister rust higher Ho values than resistant trees and showed an o excess of heterozygotes, while resistant trees showed a infection found in our study suggests, but does not con- deficiency of heterozygotes, suggesting recessive control firm, that more homozygous individuals have a higher of resistance (HOUSTON and HOUSTON, 2000). Conversely, fitness when challenged by blister rust possibly due to a heterozygote excess detected in populations of Ameri- recessive genes for resistance being expressed under

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inbreeding. Our results suggest that, contrary to our CAMPBELL, E. M. and J. A. ANTOS (2000): Distribution and expectations, more heterozygous individuals may be severity of white pine blister rust and mountain pine more likely than more homozygous whitebark pine geno- beetle on whitebark pine in British Columbia. Can. J. types to suffer mortality due to the introduced pathogen For. Res. 30: 1051–1059. white pine blister rust. Whitebark pine may have some CHELIAK, W. M. and J. A. PITEL (1984): Techniques for type of resistance to white pine blister rust that is reces- starch gel electrophoresis of enzymes from forest tree sively controlled. However, this must be confirmed by species. Can. For. Serv., Petawawa Natl. For. Inst. Rep. No. Pi-X-42. identification of specific resistance mechanisms and CHELIAK, W. M., B. P. DANCIK, K. MORGAN, F. C. YEH and determination of their genetic control in common garden C. STROBECK (1985): Temporal variation of the mating experiments with controlled exposure to spores of this system in a natural population of jack pine. Genetics introduced disease. 109: 569–584. CHU, Z., Y. OUYANG, J. ZHANG, H. YANG and S. WANG (2004): Genome-wide analysis of defense-responsive Acknowledgements genes in bacterial blight resistance of rice mediated by The authors thank the USDA Forest Service regions 1, the recessive R gene xa13. Mol. Genet. Genomics 271: 111–120. and 6, the British Columbia Ministry of Forests, B.C. DAUGHTREY, M. L. and C. R. HIBBEN (1994): Dogwood Parks E. C. Manning and Tweedsmuir Provincial Parks, anthracnose: a new disease threatens 2 native Cornus and BOB BRETT of Snowline Ecological Consulting, species. Annu. Rev. Phytopathol. 32: 61–73. Whistler, B.C. for providing seed for this study. DOROTHY DYER, R. J. and V. L. SORK (2001): Pollen pool heterogene- WATSON, DANE SZOHNER, JODIE KRAKOWSKI, and MILENA ity in shortleaf pine, Pinus echinata Mill. Mol. Ecol. 10: SEMPRONI helped in the field and lab and CHRISTINE 859–866. CHOURMOUZIS created the map. Funding for this study EL-KASSABY, Y. A., M. D. Meagher, J. PARKINSON and F. T. came from the British Columbia Forestry Investment PORTLOCK (1987): Allozyme inheritance, heterozygosity Account through the Forest Genetics Council of B.C. to and outcrossing rate among Pinus monticola near Lady- the Centre for Forest Conservation Genetics. Thank you smith, British Columbia. Heredity 58: 173–181. to Drs. MICHAEL WHITLOCK, ALVIN YANCHUK, JEANETTE FARRIS, M. A. and J. B. MITTON (1984): Population density, WHITTON, ANDREW ECKERT, JEFF MITTON, and SIERRA outcrossing rate, and heterozygote superiority in pon- derosa pine. Evolution : 1151–1154. MCLANE for their helpful comments on an earlier draft 38 of this manuscript. FINS, L. and W. J. LIBBY (1982): Population variation in Sequoiadendron: seed and seedling studies, vegetative propagation, and isozyme variation. Silvae Genet. 31: 102–110. 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Genetic diversity of Anadenanthera colubrina Vell. (Brenan) var cebil, a tree species from the South American subtropical forest as revealed by cpSSR markers

By M. E. BARRANDEGUY1),3),*), M. V. GARCÍA1),3),4),*), C. F. ARGÜELLES1) and G. D. L. CERVIGNI2),3)

(Received 14th April 2010)

Abstract sis indicated that substructure population is present. Jost s differentiation global index (D ) was 0.049 while var is a tree species ’ est Anadenanthera colubrina cebil D pairwise comparison reflected a certain level of native to the Upper Parana Atlantic Forest where est human activities have severely impacted causing deep genetic structure. fragmentation. Microsatellites are not available in this The high diversity level detected in the adult trees of species. Therefore, the first objective of this study was to A. colubrina var cebil from the populations under study generate chloroplast simple sequence repeats (cpSSR) could be due to recent human influence. In this way, the by cross-species transfer. Understanding the evolution- reduction in population size caused a reduction in ary dynamics of subdivided populations is an important the number of trees leading to surviving trees showing matter. In this way, a first approach to the characteriza- the historical diversity of the populations analyzed. tion of the haplotypic diversity within and between pop- ulations as well as the genetic structure of native Argen- Key words: Upper Parana Atlantic Forest, Curupay, intraspe- tinean populations were the main goals of this study. cific genetic diversity, cpSSR cross-species transfer. Twenty four individuals from two populations of the Misiones province were studied and four cpSSR loci Introduction were tested. Two of them exhibited polymorphic pat- The study of the genetic diversity distribution, popula- terns leading to the identification of 11 cpDNA haplo- tion structure and evolutionary features of a species types with high mean genetic diversity (GD = 0.73). The minimum spanning network defined three clear groups native range is of particular relevance when evaluating which can be assigned to at least three subpopulations. their long-term variability and fitness (GÓMEZ et al., AMOVA indicated that the total variance showed the 2002). highest percentage of variation (48%) within subpopula- Anadenanthera colubrina Vell. Brenan var cebil, tions with a fixation index (FST) statistically significant (Fabaceae, Mimosoideae) known locally as curupay, is a (F = 0.520; p < 0.05). Brown’s two component analy- ST loci tree species native to the South American subtropical forest. Curupay trees can be found in the Upper Parana 1) Departamento de Genética. Facultad de Ciencias Exactas, Atlantic Forest, which extends from Brazil into the Químicas y Naturales. Universidad Nacional de Misiones. northern part of Argentina, where human activities 2) Centro de Estudios Fotosintéticos y Bioquímicos. have had a severe impact leading to deep fragmentation. 3) Consejo Nacional de Investigaciones Científicas y Técnicas These trees can reach up to 35 m in height, with flowers (CONICET). arranged in hermaphrodite inflorescences and long 4 ) Corresponding author: MARIA VICTORIA GARCÍA. Genética de legume fruits with narrow and flattened seeds (JUSTI- Poblaciones y Cuantitativa, Departamento de Genética, Facul- tad de Ciencias Exactas, Químicas y Naturales, Universidad NIANO and FREDERICKSEN, 1998; CIALDELLA, 2000). Their Nacional de Misiones, Félix de Azara 1552, (3300) Posadas. floral traits show that pollination is performed by bees Misiones, Argentina. E-Mail: [email protected]. and seed dissemination occurs over short distances by *) These authors contributed equally to this work. anemochory – autochory after pod dehiscence (JUSTI-

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DOI:10.1515/sg-2011-0016 edited by Thünen Institute of Forest Genetics