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Genetic analysis of inherited reduced susceptibility of Fraxinus excelsior L. seedlings in Austria to ash dieback

Genetic analysis of inherited reduced susceptibility of Fraxinus excelsior L. seedlings in... Downloaded from https://academic.oup.com/forestry/article/91/4/514/4962186 by DeepDyve user on 20 July 2022 Forestry An International Journal of Forest Research Forestry 2018; 91, 514–525, doi:10.1093/forestry/cpy012 Advance Access publication 5 April 2018 Genetic analysis of inherited reduced susceptibility of Fraxinus excelsior L. seedlings in Austria to ash dieback 1 2 1 Alexandra Wohlmuth , Franz Essl and Berthold Heinze Department of Forest Genetics, Austrian Federal Research Centre for Forests (BFW), 1130 Vienna, Austria Division of Conservation, Vegetation and Landscape Ecology, University Vienna, Rennweg 14, 1030 Vienna, Austria *Corresponding author. Tel: +431878382219; Fax: +431878382250; Email: [email protected] Received 28 March 2017 Hymenoscyphus fraxineus causes massive dieback of common ash (Fraxinus excelsior L.) across populations. Previous common garden trials have revealed differences in susceptibility among individuals, suggesting a genetic basis for reduced susceptibility to the pathogen. The aim of the study was to identify any correlation between damage intensity of mature trees and their offspring in natural ash stands. Crown and shoot dam- age of naturally infected trees and saplings were assessed in two geographically isolated stands in Austria, and parentage analysis was carried out with molecular markers. No significant correlation could be detected using Spearman’s rank correlation analysis, suggesting that this approach would need higher numbers of adult–offspring pairs present to compensate for environmental and genetic variability at the sites. Likewise, an in situ estimate of heritability was nearly zero. The results thus support the results of other studies, i.e. that highly resistant individuals occur only at low frequency within European ash populations. While most of the previous studies were conducted in progeny trails or seed orchards and suggested a fairly strong genetic com- ponent, results from our investigation support a more complex mechanism of susceptibility differences under natural, heterogeneous conditions. Further analyses are needed to obtain a better understanding of gene– environment interactions and individual infection pressure of ash dieback in natural environments; such studies would need to be based on much higher sample numbers. Identification and propagating of non-susceptible ash trees is an important challenge to halt large-scale dieback of common ash. (Kowalski, 2006; Queloz et al.,2011). The pathogen was most likely Introduction introduced to Europe from East Asia (Zhao et al.,2012; Gross et al., Common ash (Fraxinus excelsior L.) is an ecologically and eco- 2014)and was firstobservedin Polandinthe early1990s (Przybyl, nomically important hardwood tree species widely distributed 2002; Pautasso et al., 2013). Since then, the pathogen has spread throughout temperate Europe. The species tolerates a wide quickly towards Western and Southern Europe. The first ash range of environmental conditions from riparian to mountain dieback symptoms were observed in Austria in 2005 at a few sites in Lower Austria, Upper Austria and Styria, and subse- habitats (Dobrowolska et al., 2011). Compared with beech (Fagus sylvatica L.), common ash is adapted to sites that are quently the disease spread to all federal provinces of Austria either moister or drier and it prefers more nutrient-rich soils by 2009 (Cech, 2006a, b; Cech et al., 2012). (Marigo et al., 2000; Thomas, 2016). Although the seedlings are The symptoms of ash dieback range from necrotic lesions and wilting on ash leaves and petioles to necrotic lesions on relatively shade tolerant, good light conditions are needed to compete with other broadleaved tree species (Marigo et al., branches, shoots and stem, leading to wood discoloration and 2000). Common ash has a complex reproductive system with crown dieback, and in most severe cases to death of the tree male, female and hermaphrodite individuals and its pollen and (Cech, 2006b; Bakys et al., 2009). Through wind dispersal, the seeds (samaras) are wind-dispersed (Morand-Prieur et al., 2003; fungus infects ash leaves during summer, growing into petioles Wallander, 2008). and shoots and overwintering on infected petioles in the ground litter (Kräutler and Kirisits, 2012; Landolt et al., 2016). Ash die- Recently, common ash has become highly threatened by a fungal disease (ash dieback) caused by the ascomycete back attacks trees of all age classes, although symptoms pro- Hymenoscyphus fraxineus (Gross et al., 2014), previously known gress more severely and more rapidly in younger individuals, as H. pseudoalbidus, with its anamorph stage Chalara fraxinea causing problems for natural regeneration (Keßler et al., 2012; © Institute of Chartered Foresters, 2018. All rights reserved. For Permissions, please e-mail: [email protected]. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. 514 Downloaded from https://academic.oup.com/forestry/article/91/4/514/4962186 by DeepDyve user on 20 July 2022 Genetic analysis of inherited reduced susceptibility of Fraxinus excelsior L. seedlings Heinze et al., 2017). As the pathogen causes high mortality of the effects. Several possible mechanisms of the genetic basis of ash trees, ash dieback has become a serious problem raising tolerance have very recently also been described with the help concerns about the future of the species. of whole-genome sequencing (Sollars et al., 2017). We thus In several European countries, recent studies in seed orch- looked at our data again, with a view on how to improve our ards, progeny trials and in natural stands have aimed to approach, if possible at all. increase understanding of the pathogen and its impact on com- In this study, we investigated if transmission of resistance to mon ash. Although there is an overall progression of the disease ash dieback from mature trees to saplings is detectable in two over the years in individual trees, several studies have revealed naturally regenerating common ash populations in Austria. We differences in symptom intensity among individuals in clonal or used parentage analysis and analysed if and how closely symp- progeny trials, providing evidence for genetically based variation tom intensity of saplings and their parents are correlated by dir- in susceptibility, and have suggested the existence of resistance ect parent–offspring comparisons, and by analysing correlation or tolerance (McKinney et al., 2011; Pliūra et al., 2011; Kjær et al. between kinship and damage intensity of trees and saplings. 2012; Stener, 2013). Results from inoculation studies showed Positive correlations should imply a likely genetic resistance to that the pathogen was less penetrative in tolerant individuals ash dieback and reveal its relative contribution to observed sus- because of a better response mechanism of the host (McKinney ceptibility under field conditions. As this is likely a strategy with et al., 2012; Cleary et al., 2014). The extent of possible resistance several crucial issues in comparison to offspring testing under against ash dieback was previously estimated based on covari- common garden conditions (e.g. environmental and age hetero- ance and estimated heritability among half-sibs and full-sibs in geneity, and numbers of related seedlings present), our study is progeny trails, suggesting that parents that are less susceptible a first attempt to identify if the necessary conditions for this transmit the resistance to their offspring (Kjær et al. 2012; Lobo approach are given in forest situations in Austria. et al., 2014). We planned and executed most of the study in the year 2015, when this was the state of knowledge. We thus expected Methods that some insight may be gained from directly observing and comparing trees and their natural offspring in woodlands. It is Study sites and sampling as yet not entirely clear whether expression of tolerance in nur- Requirements were defined a priori for selecting suitable sampling sites. sery seedlings is sufficiently related to that under natural site We searched for optimal sites representing fairly isolated ash stands (so conditions. This would possibly allow us to see whether genetic that seedlings would likely be descendents of the local adults) with sub- selection is already at work there, i.e. if the seedlings that are stantial natural regeneration and a range of healthy to severely present and healthy are so because they derive from tolerant damaged mature trees. Among several candidates (suggested by collea- adults. A positive result would significantly speed up the identifi- gues involved in monitoring ash dieback and in research in natural forest cation of tolerant genotypes, because the field testing of off- reserves in Austria), two study sites were finally chosen (Figure 1). spring, possibly over several years, requires significant resources The first sampling site is a small ash stand within Johannser Kogel, and time. Clearly, being able to make inferences without having which is a strict natural forest reserve of ~45 ha located in the north- to grow seedlings and assessing them for several consecutive western part of Lainzer Tiergarten (Türk and Pfleger, 2008, Table 1). The Lainzer Tiergarten is a protected Natura 2000 site of 2460 ha with near- years would therefore be an advantage. Phenotypic selection of natural old-growth forests and interspersed, extensively used grass- adults is likely not sufficient for identification of tolerant geno- lands; it is located in the western outskirts of Vienna and belongs to the types; but combined assessment of adults and their offspring at biosphere reserve Wienerwald (Forst-und Landwirtschaftsbetrieb der the same sites may increase precision (and so decrease the Stadt Wien, 2017). The natural forest reserve Johannser Kogel is domi- amount of field testing of seedlings), we thought. nated by an oak (Quercus sp.)-hornbeam (Carpinus betulus L.) forest We also wanted to see whether the presence of healthy with iconic old oak trees which are up to 400 years old (Türk and Pfleger, seedlings at a site would correlate with the presence of related, 2008). The ash stand covers a core area of ~2 ha and is found on the tolerant adults, i.e. whether genetic relationships can be estab- top of the hill in the centre of the reserve, it is further described as a so lished among young and adult plants in similar disease classes called ‘hilltop ash forest’ (‘Gipfeleschenwald’). Hilltop ash forests are (like in a sort of inverse Janzen–Connell effect, see e.g. Comita often pure stands on hilltops and on northern slopes (Willner, 1996). The et al., 2014), and thus check for any juvenile–adult correlations. hilltops may represent atypical sites where ash finds better (locally and Alternatively, site conditions may contribute strongly to disease temporarily moister) conditions and out-competes other forest trees (Willner, 1996). The core ash forest at Johannser Kogel is mixed with prevalence; genotype by environment interaction (G×E) could field maple (Acer campestre L.) and some hornbeam, and surrounded by interfere with this analysis; or size effects could also play a role old-age oak-hornbeam forest. Ash is rare in the immediately surround- (bigger trees might be more tolerant than smaller or non- ing oak-hornbeam woods (at the scale of hundreds of metres), and dominant ones). We explored whether sites in Austria would be parts of Johannser Kogel are bordered by meadows. However, ash is a small and isolated enough so that sufficient numbers of adult- common component of the further surrounding broadleaf forest land- sapling pairs can be detected. scapes on a kilometre scale. Because of the strong interest in this pathogen system, new The second sampling site Siegenfeld is a small, nearly pure ash stand insights have been gained on heritability and on the genetic of ~1 ha area located in a forest between Siegenfeld and Heiligenkreuz structure of tolerance since we conducted our study. Lobo et al. at the eastern rim of the Alps, c. 30 km south of Vienna (Figure 1 and (2015) and Muñoz et al. (2016) have furthered our understand- Table 1). It is surrounded by a spruce (Picea abies Karst.) forest in the ing and found out that while there is a family component of tol- north, west and south and adjacent to a forest road and meadow in the erance in open-pollinated offspring, there is also great variability east. Other tree species in the ash stand are rare and include single within families, thus requiring large sample numbers for sizing beech, maple (Acer sp.) and larch (Larix decidua Mill.) trees. The altitude 515 Downloaded from https://academic.oup.com/forestry/article/91/4/514/4962186 by DeepDyve user on 20 July 2022 Forestry Figure 1 Location of the study sites in eastern Austria; 1: Johannser Kogel (Lainzer Tiergarten, southwest Vienna); 2: Siegenfeld (Heiligenkreuz, Wienerwald) Table 1 Study sites, stand information and sampling Site Johannser Kogel Siegenfeld Geographical coordinates Lat. N 48°11′; Long. E 16°12′ Lat. N 48°11′; Long. E 16°12′ Elevation (above sea level) 290–377 m ~400 m Precipitation ~650 mm 688 mm Exposition South-west and hilltop East, nearly flat Approx. size of plot 2 ha 1 ha Density of target species (adult common 41 trees/ha 53 trees/ha ash, Fraxinus excelsior L.) Diameter (dbh) range of adult ash trees 25–65 cm 20–55 cm Stand top height 25–30 m 20–25 m Other species present Field maple (Acer campestre L.) and hornbeam Single European beech (Fagus sylvatica L.), maple (Carpinus betulus L.), approximate collective share (Acer sp.), European larch (Larix decidua Mill.) in – 0.3; single, big decaying oak (Quercus sp.) trees the western border of stand Adult trees sampled and assessed for 82 53 damage DNA successfully genotyped 81 52 Saplings sampled and assessed for 80 80 damage DNA successfully genotyped 80 72 is ~400 m and mean annual precipitation is ~688 mm (data from on 20 trees per plot. Monitoring was continued on a sub-set of 16 plots Climate-data.org, 2017). In 2007, the Federal Research Centre for in later years. In 2009, the Austrian Forest Inventory included 1200 plots Forests (BFW) conducted a monitoring program on 50 plots in Lower in an effort to assess the status of ash dieback all over Austria. Austria where crown dieback intensity of common ash was estimated Siegenfeld in Lower Austria was included in all these assessments (Cech 516 Downloaded from https://academic.oup.com/forestry/article/91/4/514/4962186 by DeepDyve user on 20 July 2022 Genetic analysis of inherited reduced susceptibility of Fraxinus excelsior L. seedlings et al., 2012). Most of the 20 trees assessed in detail at Siegenfeld DNA amplification and fragment analysis showed decreasing symptom intensity from 2007 to 2009 (Cech et al., −1 DNA concentrations for all samples were below 20 ng μL . Therefore, 2012), which singled out Siegenfeld as one of the relatively healthier DNA extracts were employed undiluted for polymerase chain reaction monitoring plots by now (trees at many other sites were not in a good (PCR). Nine nuclear microsatellite loci were used for amplification (with health state at all; Katharina Schwanda pers. comm.). annealing temperatures: Femsatl-4 – 60°C, Femsatl-10 – 50°C, Femsatl- Saplings occurred in small patches at both sites that seemed to 11 – 55°C, Femsatl-12 – 55°C, Femsatl-16 – 62°C, Femsatl-19 – 58°C, depend on penetrance of the canopy by sunlight. Biological material for M230 – 57°C, FR639485 – 55°C and FR646655 – 60°C; Brachet et al., DNA analysis was collected in June, July and August 2015 at both sites 1999; Lefort et al., 1999; Beatty et al., 2015). Each forward primer was as follows. One leaf was sampled from randomly chosen saplings across labelled with a fluorescent dye. PCRs were carried out using the QIAGEN the site (resulting in 80 saplings per site). Either a leaf, brought down by Type It Microsatellite Kit. The amplification reactions were performed on a slingshot if this was possible, or otherwise a bark plug per mature tree a PTC-100 Thermal Cycler (BIO-RAD, Vienna, Austria) under the following (from all mature ash trees at the site – resulted in a total of 134 trees) conditions: An initial denaturation step of 5 min at 95°C, 28 cycles of was collected (Table 1). The bark plug, from which cambium tissue can denaturation at 95°C for 30 s, annealing with corresponding tempera- be sliced, was cut out of the trunk with a 1 cm diameter leather punch. tures (see above) for 90 s and extension at 72°C for 30 s, and a final Cambium tissue as well as leaf tissue contains the same genomic DNA. extension step at 60°C for 30 min. The material was immediately dried in silica gel and kept at room tem- A CEQ 8000 Beckman-Coulter (Vienna, Austria) Sequencer was used perature prior to DNA extraction. to visualize the PCR products based on fragment length polymorphism, Ash dieback was recorded as the degree of crown dieback (loss of crown as compared with CEQ DNA Size Standard Kit-400 (Beckman-Coulter). foliage; see Table 2). Mature trees were classified into one of six damage Allele assessment, calling and binning were carried out using the frag- classes by visual inspection (Figure 2A). There were also six damage classes ment analysis tool of GenomeLab GeXP Beckman-Coulter software (ver- for sapling assessment, but these were based on shoot damage instead sion 10.2.3), with additional visual inspection and binning of peaks. (percentage of the shoots of a sapling affected by the disease; Figure 2B and Table 2). Additionally the diameter at breast height (DBH) of each tree and the height of each sapling were measured. Saplings were only sampled when they exceeded the height of ~60 cm, so that symptom identification Parentage analysis and damage class categorization were possible with higher confidence. CERVUS 3.0.7 software (Marshall et al., 1998; Kalinowski et al., 2007; Field Genetics Ltd, London, UK) was used for parentage analysis and for calculating other parameters, such as expected (He) and observed (Ho) heterozygosity, polymorphic information content (PIC), average non- DNA extraction exclusion probability for one candidate parent (NE-P1), average non- On average, 25–45 mg dried leaf or cambium tissue per individual was exclusion probability for a candidate parent pair (NE-PP), Hardy put into 2 mL tubes together with two glass balls of 3 mm diameter and Weinberg equilibrium (HW) and estimated null allele frequency (NULL). one of 4 mm (for cambium tissue, steel balls were used), one spatula tip All of these parameters give information about the loci and their suit- each of glass powder, activated charcoal, polyvinyl pyrrolidon (PVP ability for parentage analysis. 40 000) and sodium metabisulfite (pro analysi grades were used for all For parentage assignment, the CERVUS program uses likelihood ratio, chemicals, most of which were purchased from Sigma-Aldrich, St. Louis, a well-established statistical method (Marshall et al., 1998). Parentage is MO, USA). For homogenization, the material was frozen in liquid nitrogen assigned to a candidate parent if the likelihood is large relative to the for two minutes and ground with a TissueLyser shaking mill (QIAGEN, likelihood of alternative candidate parents. The likelihood ratio is Hilden, Germany) at 25 Hz for 2 min. The process was repeated a second expressed as LOD scores (logarithm of the likelihood ratio; Marshall time. The DNA was extracted using the Invisorb Spin Plant Mini Kit et al., 1998). Candidate parents with positive LOD scores are more likely (STRATEC Molecular, Birkenfeld, Germany), applying the protocol recom- to be the true parents, and candidate parents with negative LOD scores mended for the kit, but replacing Lysis Buffer P (provided by the kit) with are less likely to be the true parents. If two or more parents have posi- a mixture of 800 μL2× CTAB Buffer (20 g/L cetyl trimethyl ammonium tive LOD scores, Marshall et al. (1998) defined delta (difference in LOD bromide [CTAB], 100 mM Tris–HCL pH 8.0, 1.4 M NaCl, 25 mM EDTA pH scores) as an assessment criterion. 8.0) plus 1.6 μL β-mercaptoethanol and 1 μL proteinase K (QIAGEN) for Delta is the difference in LOD scores between the most likely candi- lysis. Additionally 40 μL RNAse A (10 mg/mL; QIAGEN) were added to date parent and the second most likely candidate parent. This param- each sample before the binding process. To determine DNA concentra- eter is useful when two candidate parents have a positive LOD score. If tions, a NanoDrop 1000 Spectrophotometer (Thermo Fischer Scientific, delta is high enough, parentage can be assigned to the candidate par- Ulm, Germany) was used. ent with the higher LOD score. The advantage in the use of CERVUS lies in the allowance for mistyping and missing data for individuals at a spe- cified number of loci. Parentage analysis for one parent (implemented by the ‘maternity Table 2 Definition of damage classes analysis’ function of CERVUS) and parent-pair analysis were carried out for both sites separately. Prior to parentage analysis, the simulation of Damage Range of crown foliage loss (trees) or percentage parentage was performed. This is important, because it is used to check class of damaged shoots (saplings) the feasibility of the parentage analysis and it calculates values of likeli- hood ratios, so that the confidence of parentage assignment can be 1 No or few symptoms; < 10% loss/damage determined. In short, in a simulation appropriate LOD and delta scores 2 Between 10 and 25% loss/damage for valid parentage assignment are generated for the parentage analysis 3 Between 25 and 50% loss/damage with the real data from genotyping. The simulation in this study was 4 Between 50 and 75% loss/damage performed with 10 000 offspring, an error rate of 0.01 at strict (95 per 5 Between 75 and < 100% loss/damage cent) and relaxed (80 per cent) confidence levels. As additional para- 6 Trees/saplings died from infection meters of the simulation, the number of candidate parents was set to 100 for Johannser Kogel with a 0.75 proportion of candidate parents 517 Downloaded from https://academic.oup.com/forestry/article/91/4/514/4962186 by DeepDyve user on 20 July 2022 Forestry Figure 2 Illustration of the visually assessed damage classes (d.c.) (A) for mature trees, no tree was assessed for damage class 6 (photos from Siegenfeld by Alexandra Wohlmuth); (B) for saplings (d.c. 5 is not shown) (Photos by Thomas Kirisits, University of Natural Resources and Life Sciences, Vienna, Austria). sampled, and 60 candidate parents with a 0.90 proportion of candidate confounds health state correlations. The damage classes of trees in dif- parents sampled for Siegenfeld. The proportion of sampled candidate ferent DBH classes at both sites were plotted as a ‘box and whisker plot’, parents were estimated by field observation, as the occurrence of with a similar intention (to check for possible covariance). We tabulated unsampled trees in the proximity of the stands cannot be determined the number of offspring per damage class for parents in each damage with certainty. Unsampled candidate parents were assumed (for the class. We also calculated the frequency distribution of the damage purpose of the CERVUS simulation) to be present in moderate frequency classes of local saplings (one or both parents assigned locally) and at Johannser Kogel and at low frequency at Siegenfeld. immigrant saplings (no local parents assigned, thus descents exclusively of trees outside of the stand) to see whether there was a difference in the health performance between these cohorts (local and immigrant), using a Kolmogorov–Smirnov test to check for significance. The boxplot Data analysis (DBH for damage classes) was done in SPSS, whereas the tables and the We performed a correlation analysis on the relationship between the bar diagram were done in Microsoft Excel 2013 (Redmond, WA, USA). DBH of mature ash trees and the number of their offspring in order to To estimate the significance of correlation between damage class of see whether tree size explains seedling numbers and thus possibly parent and offspring (categorial data), a one-tailed Spearman’s rank 518 Downloaded from https://academic.oup.com/forestry/article/91/4/514/4962186 by DeepDyve user on 20 July 2022 Genetic analysis of inherited reduced susceptibility of Fraxinus excelsior L. seedlings correlation analysis was calculated using SPSS Statistics 23 software (IBM, Vienna, Austria). The calculation was applied separately for those offspring where both parents were assigned (Spearman’s rank correl- ation coefficient between the damage class of the offspring and the averaged mean damage class of both parents) and for those where only one parent was assigned (Spearman’s rank correlation coefficient between the damage classes of the offspring and the one parent). Alternatively, an attempt was made to estimate heritability in situ in the sense of Ritland (2000): a pairwise matrix of differences in damage class for each possible pair of individuals in our study was regressed onto a pairwise kinship coefficient matrix, using the program SpaGeDi 1.05 (Hardy and Vekemans, 2002). The slope (b) and intercept of the regression, as well as the coefficient of determination (r ) were calcu- lated. This coefficient estimates the degree to which similarity in dam- age classes is determined by kinship, thus resembles a heritability value. The data set was permutated 1000 times in order to estimate P-values. Figure 3 The percentage of ash individuals that belong to damage classes 1–6 shown separately for mature trees and saplings from each Results study site; SJ: saplings from Siegenfeld; SA: mature trees from Siegenfeld; JJ: saplings from Johannser Kogel; JA: mature trees from Tree and sapling dimensions, and damage assessment Johannser Kogel. The mean diameter of the mature trees at breast height (DBH) was 39.7 cm at Johannser Kogel and 35 cm at Siegenfeld. Johannser Kogel hosts some particularly old trees with DBH dia- meters of around 60 cm (Table 1). At Johannser Kogel mean height of saplings was 96.5 cm with a range from 65 to 190 cm, and at Siegenfeld, 99.3 cm ranging from 70 to 170 cm. Disease symptoms were present at both sites in adults and saplings. Shoot dieback and crown defoliation were detectable, but there were few, if any, stem collar necroses. Both sites show a similar range of slightly to severely damaged individuals, with most mature trees having a crown damage intensity between 10 and 50 per cent and assigned to damage classes 2 and 3 (67 per cent of the trees at Johannser Kogel and 54 per cent at Siegenfeld). Percentages of assessment to damage class 1 were higher in Siegenfeld than in Johannser Kogel (Figure 3). However, the distribution of damage classes of saplings and mature trees showed no significant differences across both sampling sites according to Kolmogorov–Smirnov tests (saplings: P = 0.692; trees: P = 0.897). No tree was observed to have lost all its crown foliage (no damage class 6). Trees in the healthiest damage class at Johannser Kogel had higher DBH, but the ranges of DBH in the other damage classes at both sites were overlapping (Figure 4). Allele frequencies A total of 285 individuals of 294 sampled were successfully geno- Figure 4 Boxplot of the assignment of mature ash trees of different size typed for the nine microsatellite loci. DNA from one ash tree and (measured by their diameter at breast height, DBH) to different damage eight saplings (including all of the six dead saplings from Siegenfeld) classes at the two study sites. could not be amplified, likely due to insufficient DNA quality. These individuals had to be excluded from further analysis. The loci showed high allele numbers (N)and most of them were highly polymorphic (Table 3). The highest allele variation Observed heterozygosity (Ho) ranged from 0.38 to 0.85 for was found at locus Femsatl-10 with 41 alleles, and the lowest Johannser Kogel, and from 0.29 to 0.87 for Siegenfeld. Expected variation with six alleles at locus Femsatl-16. In total, 183 heterozygosity was higher than observed in most of the loci and alleles were detected in 161 individuals from Johannser Kogel in both sites. Mean polymorphic information content was 0.71 for with an average number of 20.3 alleles per locus, and 166 Johannser Kogel and 0.77 for Siegenfeld. Deviations from Hardy– alleles were detected for 124 individuals from Siegenfeld (aver- Weinberg equilibrium were detected at five loci in Johannser age 18.4 alleles per locus; Table 3). The mean proportion of loci Kogel and at one locus in Siegenfeld. The estimated frequency of typed exceeded 0.99 for both sites. Mean expected heterozygos- null alleles ranged from −0.003 (FR639485, Johannser Kogel) to ity (He) was 0.75 for Johannser Kogel and 0.80 for Siegenfeld. 0.5 (Femsatl-12, Siegenfeld) with a mean value of 0.072. 519 Downloaded from https://academic.oup.com/forestry/article/91/4/514/4962186 by DeepDyve user on 20 July 2022 Forestry Table 3 Information about the microsatellite loci used and the analysed parameters Locus Analysed parameters Johannser Kogel Analysed parameters Siegenfeld NHo He PIC NE-1 P NE-PP HW Null NHo He PIC NE-1 P NE-PP HW Null Femsatl-4 26 0.665 0.734 0.695 0.65 0.275 NS 0.052 21 0.734 0.808 0.787 0.53 0.162 NS 0.0437 Femsatl-10 41 0.745 0.895 0.884 0.347 0.067 * 0.0924 37 0.772 0.935 0.927 0.24 0.031 ND 0.0913 Femsatl-11 22 0.776 0.873 0.858 0.406 0.096 *** 0.0589 19 0.789 0.883 0.869 0.384 0.085 NS 0.0561 Femsatl-12 17 0.376 0.737 0.701 0.646 0.268 *** 0.3333 15 0.286 0.855 0.837 0.446 0.114 *** 0.5006 Femsatl-16 7 0.547 0.526 0.454 0.858 0.588 NS −0.0227 6 0.459 0.524 0.489 0.85 0.507 NS 0.0853 Femsatl-19 16 0.85 0.802 0.776 0.549 0.186 ** −0.0328 17 0.79 0.877 0.861 0.403 0.096 NS 0.0515 M230 37 0.844 0.891 0.881 0.349 0.066 * 0.0234 36 0.871 0.94 0.933 0.225 0.027 ND 0.0366 FR639485 9 0.638 0.583 0.524 0.816 0.496 NS −0.051 8 0.677 0.654 0.601 0.757 0.405 NS −0.0258 FR646655 8 0.696 0.695 0.647 0.719 0.36 NS −0.0029 7 0.697 0.71 0.66 0.701 0.342 NS 0.008 total 183 166 mean 20.3 0.682 0.748 0.713 0.593 0.267 0.0501 18.4 0.675 0.798 0.774 0.504 0.197 0.0941 Number of alleles (N); Ho, observed heterozygosity; He, expected heterozygosity; PIC, polymorphic information content; NE-1 P, average non- exclusion for one candidate parent; NE-PP, average non-exclusion probability for a candidate parent-pair; HW, significance of deviation from Hardy– Weinberg equilibrium; NS = not significant, ND = not determined. *Significant at 5 per cent level, **significant at 1 per cent level, ***significant at 0.1 per cent level. Null: Estimated null allele frequency. Parentage assignment there were 11 more saplings assigned at 80 per cent, one more at 95 per cent, four improved, one worsened in significance, and The assignments suggested by CERVUS (including LOD scores three seedlings were assigned to different parent pairs (numbers and delta values) are given in Supplementary Data File S1. and details in Table 4 and Supplementary Data File S1). Combined non-exclusion probabilities for the first parent and for the parent pair were 5.70E-03 and 6.00E-07 for Johannser Kogel and 8.92E-04 and 8.73E-09 for Siegenfeld if Femsatl-12 Data analysis was included. Without Femsatl-12, the values were less favour- able (by approximately one order of magnitude in most cases): The data show a very weak positive correlation of parents with higher DBH with the number of their offspring (R = 0.0101; 8.82E-03 and 2.24E-06 (Johannser Kogel first parent/parent pair) Supplementary Figure S1; single-parent and parent-pair results and 2.00E-03 and 8.00E-08 (Siegenfeld first parent/parent pair). combined; FEMSATL 12 included). The numbers of offspring per Maternity analysis, i.e. the assignment of only one parent, for Johannser Kogel was successful in 42 cases or 52.5 per cent disease class of adult trees are rather equally distributed across (20 of the cases also at high stringency); for Siegenfeld, it resulted damage classes (Supplementary Table S1). The frequency distri- bution of the damage classes of local and immigrant saplings in a 58.3 per cent assignment rate (42 saplings; all at strict confi- showed no apparent differences in the health status between dence level). The assignment rate for the parent-pair analysis in these cohorts at both sites (Supplementary Table S2; not signifi- Johannser Kogel was 7.5 per cent, implying that five saplings from Johannser Kogel were assigned to both parents (among the local cantly different in Kolmogorov–Smirnov tests). Spearman’s rank adult trees) at relaxed confidence level (80 per cent) and one add- correlation for parentage vs damage class was generally low (−0.293 to 0.039, Table 4) and thus showed no apparent associ- itional sapling at strict confidence level (95 per cent). In Siegenfeld, ation between damage classes of offspring and parents, neither the assignment rate for the parent-pair analysis was higher with for parent-pair, nor for maternity analysis, and only one of the 27.7 per cent – both parents were determined for 20 saplings from Siegenfeld at a relaxed confidence level (10 of which also at coefficients – with a negative sign! – was significantly different strict confidence level). from zero (−0.293 without Femsatl-12 for single parent/mater- nity, P = 0.039; Table 4). Parentage analysis (‘maternity’ or one parent only) without The value for in situ heritability (r ) obtained by the regres- marker Femsatl-12 (at less stringent non-exclusion probabilities) sion of damage class differences on kinship (calculated includ- resulted in 39 saplings assigned to candidate parents at Johannser Kogel at 80 per cent confidence level (19 of which at ing Femsatl-12; distribution shown in Supplementary Figure S2) strict level). For Siegenfeld, the numbers increased as well, two was very close to zero (4.69E-006) and not statistically signifi- cant after permutations (P = 0.34). additional saplings were assigned to a single parent (at 80 per cent), and eight more at 95 per cent, but one of the original ones lost significance of assignment (total, 50 saplings assigned), while for two saplings, alternative parents were assigned. Parent- Discussion pair assignment without Femsatl-12 resulted in the following Genetic parameters and parentage assignment changes: Johannser Kogel, six more saplings assigned at 80 per cent, one more at 95 per cent, one improved from 80 to 95 per The majority of the loci used in this study were highly poly- cent, but three lost at 80 per cent significance. At Siegenfeld, morphic, and expected heterozygosity was high. Similar 520 Downloaded from https://academic.oup.com/forestry/article/91/4/514/4962186 by DeepDyve user on 20 July 2022 Genetic analysis of inherited reduced susceptibility of Fraxinus excelsior L. seedlings Table 4 Spearman’s rank-correlation analyses for offspring for which one parent was assigned, and for offspring for which both parents were assigned, calculated including and excluding marker Femsatl-12 Comparison Number of Spearman’s rank P-value and cases correlation coefficient significance Offspring assigned to parent pairs at 80% confidence including Femsatl- 26 0.098 0.318 NS 12 – damage class offspring to average of parent pairs Additional offspring assigned to one parent at 80% confidence including 58 −0.082 0.272 NS Femsatl-12 – damage classes compared Offspring assigned to parent pairs at 80% confidence excluding Femsatl- 42 0.059 0.710 NS 12 – damage class offspring to average of parent pairs Additional offspring assigned to one parent at 80% confidence excluding 50 −0.293 0.039* Femsatl-12 – damage classes compared NS: not significant, *significant at the 0.05 level. observations were made for the same microsatellite loci in pre- than half of the saplings under various conditions of the ana- vious population genetic studies in common ash, suggesting lysis, and only 17 per cent of the saplings were assigned to both high genetic diversity within populations across Europe (Heuertz parents within their stands, despite their relative isolation. A et al., 2001, Hebel et al., 2006; Beatty et al., 2015). The values possible technical explanation for the low assignment rate could for genetic variation are typical for studies with these microsat- be genotyping or binning errors which would probably lead to ellite markers in European ash (see Heinze and Fussi, 2017 for a undetected parent–offspring trios or duos, although CERVUS comparison). While the deviations from Hardy–Weinberg equilib- uses likelihood equations that take account of such errors. High rium, and the high frequency of null alleles in Femsatl-12 are combined exclusion probability (99 per cent) across all loci for not ideal, the combined non-exclusion probabilities are very unrelated parents suggests low error rate in parentage assign- much appropriate (given the numbers of candidate parents pre- ment and shows that the microsatellite set used was adequate. sent). Femsatl-12 seems to show an increase of null alleles Nevertheless, we tested our data analysis procedure omitting from Western to Eastern Europe (Heinze and Fussi, 2017, their the critical Femsatl-12 marker. The parent non-exclusion prob- Table 4). Null alleles do not amplify consistently during PCR and abilities then changed to less stringent values (Supplementary thus cannot be reliably detected, leading to false assessments Data File S1) and slightly higher rates for parentage assignment of heterozygotes as homozygotes (Kalinowski and Taper, 2006). resulted, so that more saplings could be assigned to parents. They are problematic in parentage analysis and appear some- However, the correlation (Spearman’s rank) did not change times simultaneously with deviations from the Hardy–Weinberg in a meaningful way. Thus our conditions are conservative equilibrium (Pemberton et al., 1995), which was also the case in the sense that more relaxed assignment conditions, if they for five loci in Johannser Kogel and at one locus in Siegenfeld. lead to higher errors in parent–offspring assignments, possibly However, estimation of null allele frequencies does not necessar- underestimate correlations (but provide a wider data basis for ily imply that a null allele is present. If no parent–offspring rela- them). tionship is definitely known and there is no Hardy–Weinberg Although we made an effort to sample all putative parents equilibrium, it is difficult (or impossible) to decide about the pres- in both fairly isolated stands, it is possible that these missing ence of null alleles with certainty (Dakin and Avise, 2004). We do parents are actually scattered trees outside the stands. not expect strict Hardy–Weinberg equilibria at our sites, as we Therefore, a more likely explanation for the low assignment observed incoming seed and pollen in the young generation. rates is that the rest of the saplings had been sired by these But even Femsatl-12 adds power to this analysis (better non- unsampled trees. High seed and pollen inflow from the sur- exclusion probabilities), because undetected null alleles do not roundings is a phenomenon often detected in forest tree stands, lead to (wrong) exclusions of candidate parents; so the number even if we did not expect it here due to the scarcity of ash trees of parent–offspring pairs is not underestimated because of the in the surroundings, and because of the generally low physical null alleles. It could be overestimated, but the simulations in disposition of ash seed and pollen for passive transport beyond CERVUS take the null frequencies into account for suggesting a few hundred metres (Richards, 1997). Nevertheless, in this thresholds of LOD and delta values. The deviations from Hardy– respect our findings resemble those of Lobo et al. (2015), where Weinberg equilibria are already a hint for absence of panmixia paternity analysis in two Danish seed orchards resulted in an (which would require equal parental contributions to the off- assignment rate of less than 40 per cent. They suggested the spring generation) and/or the presence of immigrant saplings small size of the seed orchard as one possible explanation (12 (see below). putative parents in the orchard). Analysis of seed dispersal in Both our sites had nearly the same set of alleles with few pri- two natural ash woodlands in Ireland carried out by Beatty vate alleles (alleles that only occur in one population) at each et al. (2015) indicates the potential for seed dispersal over sev- site, and little differentiation (like in Heuertz et al., 2001; Hebel eral hundreds of metres. Bacles et al. (2005) and Bacles and et al., 2006; Ballian et al., 2008). Surprisingly, positive assign- Ennos (2008) estimated pollen dispersal in fragmented ash ments of at least one parent were made only for slightly more populations in Scotland, suggesting effective pollination within 521 Downloaded from https://academic.oup.com/forestry/article/91/4/514/4962186 by DeepDyve user on 20 July 2022 Forestry 300 m, but detected rare pollination events over distances of up cultivated in a nursery, and rather intensively managed after to 3 km. While these studies on pollination were carried out in planting (with wider spacing; McKinney et al., 2011; Stener, isolated tree stands in open landscapes, where pollen can be 2013). Family size in the study of Muñoz et al. (2016) was transported easier with less barriers being present, a study by between 8 and 68 half-sibs per mother tree. Lobo et al. (2015) Heuertz et al. (2003) in a Romanian continuous ash woodland used 2–48 offspring per half-sib family in inoculation tests (8–48 estimated average distances of 14 m between seeds and their in maternal families). We expected to arrive at numbers similar female parents, and average pollen flow below 140 m distance to the lower part of this range in our settings, but that was not (between seed mothers and pollinating trees). However, their the case. Progeny trails and seed orchards (and especially sap- common ash stand was much denser (~200 mature stems per ling inoculations) represent more controlled environments and ha), and their gene flow estimates were indirectly derived from they often do not fully resemble natural woodland conditions. genetic data of adults only (decrease of kinship with increasing In contrast, this study was conducted in naturally regenerating distance), while we analysed saplings (and thus the ‘realized ash stands, and the more heterogeneous conditions there likely gene flow’ of the transition of one generation to the next). We led to higher genotype × (micro-)environment interactions., which infer from our low assignment rate for parent-pair analysis and in turn lowered the genetic correlations observed (see also below) the moderate assignment rate for maternity analysis that seed Lobo et al. (2014) estimated the heritability of damage inten- and pollen dispersal within mixed landscapes is probably higher sity in two progeny trails in Denmark. Both trials included pro- than the estimates of Heuertz et al. (2003) for continuous genies from the same mother trees, but one trial was left woodlands and more in the range of the estimates of Beatty unfenced and progenies were exposed to strong competition et al. (2015) in Irish woodlands (but lower than those for very from vegetation and to browsing animals, whereas the other open landscapes). It is also the experience of foresters in was protected by fencing. Lower estimated heritability values of Austria in similar forest types that (i) either a single or a few ash the unfenced (0.2) compared with the fenced site (0.42–0.53) trees are able to produce dense seedling patches, if light and indicated that estimates of heritability under heterogeneous other conditions are good (‘seed shadows’), and (ii) these conditions may be lower (Lobo et al., 2014). Johannser Kogel is patches can be found up to a few hundred metres away from also fenced, but the fence is obviously penetrable at some the source (especially following the main wind direction; points, as wild boars were observed inside the fence during Herfried Steiner, Werner Ruhm, pers. comm.). Further analysis is sampling. Pliūra et al. (2014) tested clones from seven different needed in order to infer the precise influence of landscape fea- sites from Lithuania exposed to different ash-dieback infection tures on these parameters. pressure. They calculated the genotype–environment interaction on phenotypic variation and found significant contributions of genetic variation in plasticity and reaction norms of clones across a range of infection pressure environments (Pliūra et al., Variation in susceptibility 2014). This indicates the presence of variation of individual The presence of seeds that originate entirely from outside the response to the disease across sites depending on site condi- stands investigated allowed us to calculate if there are ‘popula- tions (Pliūra et al., 2011, 2014), or different levels of disease tion differences’ in susceptibility between offspring of the local pressure. Results from previous studies, together with our cur- trees vs immigrant saplings. The frequency distribution of the rent ones, suggest that precise estimates of heritability are only damage classes between these cohorts showed no apparent possible in more controlled environments, or with much higher differences (Supplementary Table S2). This is a further indication sample numbers (sapling half-sib families detected and that there is no selection (yet) for highly tolerant saplings at our assigned to parents). Therefore, environmental conditions and sites (or for more tolerant adults with a consequent higher suc- sample sizes are likely more relevant and need to be strongly cess in contributing to seed production in the wider surround- considered for inferences on susceptibility in field observation ings). It also follows that there is no reason to believe that studies. different susceptibilities of trees outside our stands (compared It seems from all cited studies that up to now, only a small with the adults within our stands) have led to any systematic fraction of ash individuals maintain potential resistance to the error in the correlations. disease, as practically all trees are infected at some point. Kjær No significant relationship (with the exception of one nega- et al. (2012) found high susceptibility and mortality among trees tive) was found based on Spearman’s rank correlation between in two progeny trails in Denmark. They further estimated that damage intensity of parents and their offspring (Table 4), nei- only 1 per cent of the trees have the potential to pass lower ther for parent–offspring duos (offspring with one parent), nor susceptibility to their offspring. In three Lithuanian progeny trails for parent–offspring trios (offspring with both parents). Previous almost 90 per cent of the trees died during the observation peri- studies suggested genetically based variation as an explanation od of eight years (Pliūra et al., 2011). In Austria, such high mor- for observed differences in susceptibility to dieback symptoms tality rates have not yet been reported from observation sites based on the observation of clones and single-tree offspring in (Heinze et al., 2017). In 50 ash dieback monitoring plots in progeny tests, and they concluded that these differences are Lower Austria (mainly adult stands), mean crown dieback inten- transferred across ash generations (Bakys et al., 2009; McKinney sity reached 18.1 per cent in 2009 and 17.6 per cent in 2010 et al., 2011; Lobo et al., 2014; Muñoz et al., 2016). Most of these (Kirisits and Freinschlag, 2012). Three seed orchards in Austria studies were performed in clonal seed orchards or established showed moderate mean crown dieback intensity in 2011 (14.2 per progeny trials. The seedlings were raised in a nursery prior to cent, 13.5 and 31 per cent), but no clone was totally unaffected planting (Kjær et al., 2012; Muñoz et al., 2016) and the clones (Heinze et al., 2017). Crown and shoot dieback intensity was also for seed orchards were grafted onto rootstocks and also further relatively moderate at Johannser Kogel and Siegenfeld (66 and 72 522 Downloaded from https://academic.oup.com/forestry/article/91/4/514/4962186 by DeepDyve user on 20 July 2022 Genetic analysis of inherited reduced susceptibility of Fraxinus excelsior L. seedlings per cent of the individuals, respectively, showed lower than 50 per substances that have a higher prevalence in ‘resistant’ vs sus- cent damage intensity). It is likely that the mature trees that were ceptible tree. identified as parents did not express assessable resistance due to Despite these advances, the exact genetic background(s) of lower infection pressure (especially for the higher crowns of thicker low susceptibility still remains unclear and it may well be that trees, Figure 4), and consequently produced more offspring more than one gene network or metabolic pathway is involved, becauseof their greatersizeand thebetter healthcondition asso- or that different such mechanisms are at work in different ciated with it (Supplementary Figure S1), while infection pressure regions of Europe. The robustness of the observed potential was higher for saplings close to the forest floor, where the pres- resistance in previous field studies needs to be re-evaluated. ence of petioles and more humid conditions would favour high Precise genetic tests of their offspring are necessary to deter- spore concentrations. This could be one reason for the low correl- mine which of the field-resistant trees really transmit this trait ation between damage classes of parents and offspring in our to seeds. Large offspring numbers are necessary for this pur- study. However, this effect may impact the severity, but not so pose, which make such tests costly. The alternative approach much the direction of susceptibility; healthier mature trees should we tested would, however, require sites with high numbers of then still produce healthier offspring, though the average damage seedlings with established parentage, much higher than the class rating would be shifted between old trees and young sap- ones we identified. Thus, such sites should have many seedlings, lings. Probably the small sample size of parent–offspring trios but few immigrants among them. This may be difficult to find. (N = 26–50) and of half-sib families (only nine mature trees Although the distribution data of ash seedlings in the wider area produced more than two offspring) may have led to underesti- of Johannser Kogel suggest the presence of ‘seed shadows’ of mation of actual correlations of damage intensity between single adults (Herfried Steiner, pers. comm.), it remains to be parent and offspring for possible reduced susceptibility. Muñoz seen whether numbers are high enough at a particular site. The et al. (2016) found that there are family effects in open-pollinated higher workload of laboratory testing would require more time offspring, but they also calculated individual breeding values for (and it would increase costs), but the approach may still be fas- the offspring, and most of the genetic variation was found within ter in total than field planting of seedlings to be tested for dis- families. Thus, our small family sizes may not allow estimating her- ease tolerance. itabilities with great precision. The in situ heritability estimation It is uncertain whether low susceptibility in single, but infre- basedonall kinship relationships among trees and saplings also quent individuals could hinder massive decline of ash in Europe, resulted in a coefficient of determination of very close to zero. It and if that resistance would sustain massive infection pressure may not necessarily be the case that related trees have very simi- at natural forest sites. However, selecting and breeding non- lar disease tolerance levels. susceptible trees in a timely manner throughout Europe seems Breeding values, which represent the average effect of the the only way to overcome the disease; the approach we discuss parent genotype, estimated from the performance of its off- here may help to accelerate selection. Alternatively, selecting spring (McKinney et al., 2014), were calculated by Kjær et al. ash stands that combine many environmental and demographic (2012) in a Danish progeny trail, and by Muñoz et al. (2016) in factors that disadvantage high infection pressure and the pro- France. Only one of 101 tested mother trees in Denmark had gress of the disease (e.g. hot and dry summers, dry soils during breeding values for ‘susceptibility’ below 10 per cent and was the sporulation period of the fungus, big trees with large, dom- estimated to produce healthy offspring (and four trees with inating crowns or stands were leaf litter does not persist until breeding values below 20 per cent were estimated to produce the next summer) may emerge as a conservation strategy in fairly healthy offspring). Another study carried out by McKinney situations where genetic tolerance is generally low (Heinze et al. (2011) supported these findings, with only one of 39 et al., 2017). tested clones exhibiting breeding values below 10 per cent. Breeding values for susceptibility in Kjær et al. (2012) were nor- mally distributed, suggesting that possible resistance is based Conclusion on expression of several genes rather than on one alone (McKinney et al., 2014). The data from France (Muñoz et al., In this study, no significant correlation could be found between 2016) also suggest that many genes contribute to resistance, as ash dieback damage intensity of parent and offspring in two the largest part of genetic variation was found within families. natural stands in Austria. This can reflect low power of estimat- This also means that much larger numbers of saplings are ing heritability under natural in situ conditions, where the micro- necessary to estimate heritability in our approach. Harper et al. environment may have a stronger influence on the trait than in (2016), in contrast, discovered a number of single nucleotide planted tests. Given this, and the recent insights into the genet- polymorphism (SNP) and gene expression markers that are ics of disease tolerance, much higher numbers of offspring with associated with crown damage in infected trees using identified parenthood (than the ones we could find) are neces- Associated Transcriptomics. With three markers (concerning sary for our approach. Other possible causes could be that there transcription factor genes) they were able to predict individuals were no very tolerant parent trees at our sites, or that juvenile– with a low level of susceptibility to the disease (combined R adult correlations are low for this trait. We further suggest that value of 0.28). Sollars et al. (2017) very recently published the the genetic basis for variation in susceptibility might be more sequence of a low-heterozygosity ash tree, and further improved complex under natural heterogeneous conditions than in more these markers (which, in their data, explained Danish disease controlled environments like planted progeny trials or seed orch- scores with r = 0.25) and suggested that according to these ards, where most of the previous studies were implemented. markers, native trees in Great Britain would have a lower chance The robustness of the observed potential low susceptibility in of becoming susceptible. They also reported on biochemical previous studies should therefore be critically re-evaluated after 523 Downloaded from https://academic.oup.com/forestry/article/91/4/514/4962186 by DeepDyve user on 20 July 2022 Forestry Cech, T.L. 2006b Eschenschaden in Österreich [Ash dieback and prema- planting offspring from tolerant trees in production forests. The ture leaf shedding in Austria]. Forstschutz Aktuell (BFW) 37,18–20. influence of environmental conditions should be considered when inferring on susceptibility (i.e. when assessing field resist- Cech, T.L., Kessler, M. and Brandstetter, M. 2012 Monitoring des ance). A scenario of large-scale decline of common ash trees in Zurücksterbens der Esche in Österreich [Monitoring ash dieback in Austria]. Forstschutz Aktuell (BFW) 55,56–58. Europe is becoming more and more likely. It will be important to identify and propagate healthy ash individuals throughout Cleary, M.R., Andersson, P.F., Broberg, A., Elfstrand, M., Daniel, G. and Stenlid, J. 2014 Genotypes of Fraxinus excelsior with different susceptibil- Europe, to find out about the genetic mechanism of any resist- ity to the ash dieback pathogen Hymenoscyphus pseudoalbidus and their ance in more detail and about the environmental conditions response to the phytotoxin viridiol—a metabolomic and microscopic that favour low susceptibility, in order to implement a manage- study. Phytochemistry 102, 115–125. ment program that accounts for these aspects. Climate-data.org. 2017 Climate Data for Siegenfeld. https://de.climate- data.org/search/?q=Siegenfeld (accessed on 22 August, 2017). Comita,L.S., Queenborough, S.A.,Murphy, S.J.,Eck,J.L., Xu,K., Supplementary data Krishnadas, M., et al. 2014 Testing predictions of the Janzen-Connell Supplementary data are available at Forestry online. hypothesis: a meta-analysis of experimental evidence for distance- and density-dependent seed and seedling survival. J. Ecol. 102, 845–856. Acknowledgements Dakin, E.E. and Avise, J.C. 2004 Microsatellite null alleles in parentage analysis. Heredity 93, 504–509. Special thanks to Renate Slunsky, Daniela Jahn, MSc, and the Genome Research unit at the Federal Research Centre for Forests (BFW) for their Dobrowolska, D., Hein, S., Oosterbaan, A., Wagner, S., Clark, J. and kind support and also to the Natural Forest Reserve and the Skovsgaard, J.P. 2011 A review of European ash (Fraxinus excelsior L.): Phytopathology units at BFW, especially to Dr Katharina Schwanda, implications for silviculture. Forestry 84, 133–148. Christian Neureiter and Mag. Herfried Steiner. ‘European Cooperation in Forst-und Landwirtschaftsbetrieb der Stadt Wien. 2017 Lage, Größe, Science and Technology (COST)’ Action FP1103 ‘FRAXBACK’ is acknowl- Geologie und Klima—Lebensraum Lainzer Tiergarten [Situation, size, geol- edged for providing a stimulating environment of meetings and discus- ogy and climate—habitat Lainzer Tiergarten; online; in German]. https:// sions. Furthermore we would like to thank Dr Thomas Kirisits, University www.wien.gv.at/umwelt/wald/erholung/lainzertiergarten/lebensraum/lage. of Natural Resources and Life Sciences in Vienna, who provided his pho- html (accessed on 22 August, 2017). tos of the damage classes of saplings, and the Editor in Chief and three Gross, A., Holdenrieder, O., Pautasso, M., Queloz, V. and Sieber, T.N. 2014 reviewers for their helpful comments and suggestions. Hymenoscyphus pseudoalbidus, the causal agent of European ash die- back. Mol. Plant Pathol. 15 (1), 5–21. 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Przybyl, K. 2002 Fungi associated with necrotic apical parts of Fraxinus excelsior shoots. For. Pathol. 32 (6), 387–394. Lefort, F., Brachet, S., Frascaria-Lacoste, N., Edwards, K.J. and Douglas, G.C. 1999 Identification and characterization of microsatellite loci in ash Queloz, V., Grünig, C.R., Berndt, R., Kowalski, T., Sieber, T.N. and (Fraxinus excelsior L.) and their conservation in the olive family (Oleaceae). Holdenrieder, O. 2011 Cryptic speciation in Hymenoscyphus albidus. For. Mol. Ecol. 8 (6), 1088–1089. Pathol. 41 (2), 133–142. Lobo, A., Hansen, J.K., McKinney, L.V., Nielsen, L.R. and Kjær, E.D. 2014 Richards, A.J. 1997 Plant breeding systems. 2nd edn. Chapman & Hall, Genetic variation in dieback resistance: growth and survival of Fraxinus p. 529. excelsior under the influence of Hymenoscyphus pseudoalbidus. Scand. J. Sollars, E.S.A., Harper, A.L., Kelly, L.J., Sambles, C.M., Ramirez-Gonzalez, R. For. Res. 29 (6), 519–526. H., Swarbreck, D., et al 2017 Genome sequence and genetic diversity of Lobo, A., McKinney, L.V., Hansen, J.K., Kjær, E.D. and Nielsen, L.R. 2015 European ash trees. Nature 541 (7636), 212–216. Genetic variation in dieback resistance in Fraxinus excelsior confirmed by Stener, L.-G. 2013 Clonal differences in susceptibility to the dieback of progeny inoculation assay. For. Pathol. 45 (5), 379–387. Fraxinus excelsior in southern Sweden. Scand. J. For. Res. 28 (3), Marigo, G., Peltier, J.-P., Girel, J. and Pautou, G. 2000 Success in the 205–216. demographic expansion of Fraxinus excelsior L. Trees 15 (1), 1–13. Thomas, P.A. 2016 Biological flora of the British isles: Fraxinus excelsior. Marshall, T.C., Slate, J., Kruuk, L.E.B. and Pemberton, J.M. 1998 Statistical J. Ecol. 104 (4), 1158–1209. confidence for likelihood-based paternity inference in natural popula- Türk, R. and Pfleger, H.S. 2008 Die Flechtenflora am Johannser Kogel im tions. Mol. Ecol. 7 (5), 639–655. Lainzer Tiergarten und in den Steinhofgründen (Wien, Österreich) [The McKinney, L.V., Nielsen, L.R., Collinge, D.B., Thomsen, I.M., Hansen, J.K. lichen flora at Johannser Kogel in Lainzer Tiergarten and at and Kjær, E.D. 2014 The ash dieback crisis: genetic variation in resistance Steinhofgründe (Vienna, Austria)]. Verh. Zool. Bot. Ges. Österr. Österr. can prove a long-term solution. Plant Pathol. 63 (3), 485–499. 145,83–95. McKinney, L.V., Nielsen, L.R., Hansen, J.K. and Kjær, E.D. 2011 Presence of Wallander, E. 2008 Systematics of Fraxinus (Oleaceae) and evolution of natural genetic resistance in Fraxinus excelsior (Oleraceae) to Chalara dioecy. Plant Syst. Evol. 273 (1–2), 25–49. fraxinea (Ascomycota): an emerging infectious disease. Heredity 106 (5), Willner, W. 1996 Die Gipfeleschenwälder des Wienerwaldes [The hilltop 788–797. ash forests of Wienerwald]. Verh. Zool. Bot. Ges. Österr. 133, 133–184. McKinney, L.V., Thomsen, I.M., Kjær, E.D. and Nielsen, L.R. 2012 Genetic Zhao, Y.-J., Hosoya, T., Baral, H.-O., Hosaka, K. and Kakishima, M. 2012 resistance to Hymenoscyphus pseudoalbidus limits fungal growth and Hymenoscyphus pseudoalbidus, the correct name for Lambertella albida symptom occurrence in Fraxinus excelsior. For. Pathol. 42 (1), 69–74. reported from Japan. Mycotaxon 122,25–41. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Forestry Oxford University Press

Genetic analysis of inherited reduced susceptibility of Fraxinus excelsior L. seedlings in Austria to ash dieback

Forestry , Volume 91 (4) – Oct 1, 2018

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Oxford University Press
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Copyright © 2022 Institute of Chartered Foresters
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0015-752X
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1464-3626
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10.1093/forestry/cpy012
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Abstract

Downloaded from https://academic.oup.com/forestry/article/91/4/514/4962186 by DeepDyve user on 20 July 2022 Forestry An International Journal of Forest Research Forestry 2018; 91, 514–525, doi:10.1093/forestry/cpy012 Advance Access publication 5 April 2018 Genetic analysis of inherited reduced susceptibility of Fraxinus excelsior L. seedlings in Austria to ash dieback 1 2 1 Alexandra Wohlmuth , Franz Essl and Berthold Heinze Department of Forest Genetics, Austrian Federal Research Centre for Forests (BFW), 1130 Vienna, Austria Division of Conservation, Vegetation and Landscape Ecology, University Vienna, Rennweg 14, 1030 Vienna, Austria *Corresponding author. Tel: +431878382219; Fax: +431878382250; Email: [email protected] Received 28 March 2017 Hymenoscyphus fraxineus causes massive dieback of common ash (Fraxinus excelsior L.) across populations. Previous common garden trials have revealed differences in susceptibility among individuals, suggesting a genetic basis for reduced susceptibility to the pathogen. The aim of the study was to identify any correlation between damage intensity of mature trees and their offspring in natural ash stands. Crown and shoot dam- age of naturally infected trees and saplings were assessed in two geographically isolated stands in Austria, and parentage analysis was carried out with molecular markers. No significant correlation could be detected using Spearman’s rank correlation analysis, suggesting that this approach would need higher numbers of adult–offspring pairs present to compensate for environmental and genetic variability at the sites. Likewise, an in situ estimate of heritability was nearly zero. The results thus support the results of other studies, i.e. that highly resistant individuals occur only at low frequency within European ash populations. While most of the previous studies were conducted in progeny trails or seed orchards and suggested a fairly strong genetic com- ponent, results from our investigation support a more complex mechanism of susceptibility differences under natural, heterogeneous conditions. Further analyses are needed to obtain a better understanding of gene– environment interactions and individual infection pressure of ash dieback in natural environments; such studies would need to be based on much higher sample numbers. Identification and propagating of non-susceptible ash trees is an important challenge to halt large-scale dieback of common ash. (Kowalski, 2006; Queloz et al.,2011). The pathogen was most likely Introduction introduced to Europe from East Asia (Zhao et al.,2012; Gross et al., Common ash (Fraxinus excelsior L.) is an ecologically and eco- 2014)and was firstobservedin Polandinthe early1990s (Przybyl, nomically important hardwood tree species widely distributed 2002; Pautasso et al., 2013). Since then, the pathogen has spread throughout temperate Europe. The species tolerates a wide quickly towards Western and Southern Europe. The first ash range of environmental conditions from riparian to mountain dieback symptoms were observed in Austria in 2005 at a few sites in Lower Austria, Upper Austria and Styria, and subse- habitats (Dobrowolska et al., 2011). Compared with beech (Fagus sylvatica L.), common ash is adapted to sites that are quently the disease spread to all federal provinces of Austria either moister or drier and it prefers more nutrient-rich soils by 2009 (Cech, 2006a, b; Cech et al., 2012). (Marigo et al., 2000; Thomas, 2016). Although the seedlings are The symptoms of ash dieback range from necrotic lesions and wilting on ash leaves and petioles to necrotic lesions on relatively shade tolerant, good light conditions are needed to compete with other broadleaved tree species (Marigo et al., branches, shoots and stem, leading to wood discoloration and 2000). Common ash has a complex reproductive system with crown dieback, and in most severe cases to death of the tree male, female and hermaphrodite individuals and its pollen and (Cech, 2006b; Bakys et al., 2009). Through wind dispersal, the seeds (samaras) are wind-dispersed (Morand-Prieur et al., 2003; fungus infects ash leaves during summer, growing into petioles Wallander, 2008). and shoots and overwintering on infected petioles in the ground litter (Kräutler and Kirisits, 2012; Landolt et al., 2016). Ash die- Recently, common ash has become highly threatened by a fungal disease (ash dieback) caused by the ascomycete back attacks trees of all age classes, although symptoms pro- Hymenoscyphus fraxineus (Gross et al., 2014), previously known gress more severely and more rapidly in younger individuals, as H. pseudoalbidus, with its anamorph stage Chalara fraxinea causing problems for natural regeneration (Keßler et al., 2012; © Institute of Chartered Foresters, 2018. All rights reserved. For Permissions, please e-mail: [email protected]. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. 514 Downloaded from https://academic.oup.com/forestry/article/91/4/514/4962186 by DeepDyve user on 20 July 2022 Genetic analysis of inherited reduced susceptibility of Fraxinus excelsior L. seedlings Heinze et al., 2017). As the pathogen causes high mortality of the effects. Several possible mechanisms of the genetic basis of ash trees, ash dieback has become a serious problem raising tolerance have very recently also been described with the help concerns about the future of the species. of whole-genome sequencing (Sollars et al., 2017). We thus In several European countries, recent studies in seed orch- looked at our data again, with a view on how to improve our ards, progeny trials and in natural stands have aimed to approach, if possible at all. increase understanding of the pathogen and its impact on com- In this study, we investigated if transmission of resistance to mon ash. Although there is an overall progression of the disease ash dieback from mature trees to saplings is detectable in two over the years in individual trees, several studies have revealed naturally regenerating common ash populations in Austria. We differences in symptom intensity among individuals in clonal or used parentage analysis and analysed if and how closely symp- progeny trials, providing evidence for genetically based variation tom intensity of saplings and their parents are correlated by dir- in susceptibility, and have suggested the existence of resistance ect parent–offspring comparisons, and by analysing correlation or tolerance (McKinney et al., 2011; Pliūra et al., 2011; Kjær et al. between kinship and damage intensity of trees and saplings. 2012; Stener, 2013). Results from inoculation studies showed Positive correlations should imply a likely genetic resistance to that the pathogen was less penetrative in tolerant individuals ash dieback and reveal its relative contribution to observed sus- because of a better response mechanism of the host (McKinney ceptibility under field conditions. As this is likely a strategy with et al., 2012; Cleary et al., 2014). The extent of possible resistance several crucial issues in comparison to offspring testing under against ash dieback was previously estimated based on covari- common garden conditions (e.g. environmental and age hetero- ance and estimated heritability among half-sibs and full-sibs in geneity, and numbers of related seedlings present), our study is progeny trails, suggesting that parents that are less susceptible a first attempt to identify if the necessary conditions for this transmit the resistance to their offspring (Kjær et al. 2012; Lobo approach are given in forest situations in Austria. et al., 2014). We planned and executed most of the study in the year 2015, when this was the state of knowledge. We thus expected Methods that some insight may be gained from directly observing and comparing trees and their natural offspring in woodlands. It is Study sites and sampling as yet not entirely clear whether expression of tolerance in nur- Requirements were defined a priori for selecting suitable sampling sites. sery seedlings is sufficiently related to that under natural site We searched for optimal sites representing fairly isolated ash stands (so conditions. This would possibly allow us to see whether genetic that seedlings would likely be descendents of the local adults) with sub- selection is already at work there, i.e. if the seedlings that are stantial natural regeneration and a range of healthy to severely present and healthy are so because they derive from tolerant damaged mature trees. Among several candidates (suggested by collea- adults. A positive result would significantly speed up the identifi- gues involved in monitoring ash dieback and in research in natural forest cation of tolerant genotypes, because the field testing of off- reserves in Austria), two study sites were finally chosen (Figure 1). spring, possibly over several years, requires significant resources The first sampling site is a small ash stand within Johannser Kogel, and time. Clearly, being able to make inferences without having which is a strict natural forest reserve of ~45 ha located in the north- to grow seedlings and assessing them for several consecutive western part of Lainzer Tiergarten (Türk and Pfleger, 2008, Table 1). The Lainzer Tiergarten is a protected Natura 2000 site of 2460 ha with near- years would therefore be an advantage. Phenotypic selection of natural old-growth forests and interspersed, extensively used grass- adults is likely not sufficient for identification of tolerant geno- lands; it is located in the western outskirts of Vienna and belongs to the types; but combined assessment of adults and their offspring at biosphere reserve Wienerwald (Forst-und Landwirtschaftsbetrieb der the same sites may increase precision (and so decrease the Stadt Wien, 2017). The natural forest reserve Johannser Kogel is domi- amount of field testing of seedlings), we thought. nated by an oak (Quercus sp.)-hornbeam (Carpinus betulus L.) forest We also wanted to see whether the presence of healthy with iconic old oak trees which are up to 400 years old (Türk and Pfleger, seedlings at a site would correlate with the presence of related, 2008). The ash stand covers a core area of ~2 ha and is found on the tolerant adults, i.e. whether genetic relationships can be estab- top of the hill in the centre of the reserve, it is further described as a so lished among young and adult plants in similar disease classes called ‘hilltop ash forest’ (‘Gipfeleschenwald’). Hilltop ash forests are (like in a sort of inverse Janzen–Connell effect, see e.g. Comita often pure stands on hilltops and on northern slopes (Willner, 1996). The et al., 2014), and thus check for any juvenile–adult correlations. hilltops may represent atypical sites where ash finds better (locally and Alternatively, site conditions may contribute strongly to disease temporarily moister) conditions and out-competes other forest trees (Willner, 1996). The core ash forest at Johannser Kogel is mixed with prevalence; genotype by environment interaction (G×E) could field maple (Acer campestre L.) and some hornbeam, and surrounded by interfere with this analysis; or size effects could also play a role old-age oak-hornbeam forest. Ash is rare in the immediately surround- (bigger trees might be more tolerant than smaller or non- ing oak-hornbeam woods (at the scale of hundreds of metres), and dominant ones). We explored whether sites in Austria would be parts of Johannser Kogel are bordered by meadows. However, ash is a small and isolated enough so that sufficient numbers of adult- common component of the further surrounding broadleaf forest land- sapling pairs can be detected. scapes on a kilometre scale. Because of the strong interest in this pathogen system, new The second sampling site Siegenfeld is a small, nearly pure ash stand insights have been gained on heritability and on the genetic of ~1 ha area located in a forest between Siegenfeld and Heiligenkreuz structure of tolerance since we conducted our study. Lobo et al. at the eastern rim of the Alps, c. 30 km south of Vienna (Figure 1 and (2015) and Muñoz et al. (2016) have furthered our understand- Table 1). It is surrounded by a spruce (Picea abies Karst.) forest in the ing and found out that while there is a family component of tol- north, west and south and adjacent to a forest road and meadow in the erance in open-pollinated offspring, there is also great variability east. Other tree species in the ash stand are rare and include single within families, thus requiring large sample numbers for sizing beech, maple (Acer sp.) and larch (Larix decidua Mill.) trees. The altitude 515 Downloaded from https://academic.oup.com/forestry/article/91/4/514/4962186 by DeepDyve user on 20 July 2022 Forestry Figure 1 Location of the study sites in eastern Austria; 1: Johannser Kogel (Lainzer Tiergarten, southwest Vienna); 2: Siegenfeld (Heiligenkreuz, Wienerwald) Table 1 Study sites, stand information and sampling Site Johannser Kogel Siegenfeld Geographical coordinates Lat. N 48°11′; Long. E 16°12′ Lat. N 48°11′; Long. E 16°12′ Elevation (above sea level) 290–377 m ~400 m Precipitation ~650 mm 688 mm Exposition South-west and hilltop East, nearly flat Approx. size of plot 2 ha 1 ha Density of target species (adult common 41 trees/ha 53 trees/ha ash, Fraxinus excelsior L.) Diameter (dbh) range of adult ash trees 25–65 cm 20–55 cm Stand top height 25–30 m 20–25 m Other species present Field maple (Acer campestre L.) and hornbeam Single European beech (Fagus sylvatica L.), maple (Carpinus betulus L.), approximate collective share (Acer sp.), European larch (Larix decidua Mill.) in – 0.3; single, big decaying oak (Quercus sp.) trees the western border of stand Adult trees sampled and assessed for 82 53 damage DNA successfully genotyped 81 52 Saplings sampled and assessed for 80 80 damage DNA successfully genotyped 80 72 is ~400 m and mean annual precipitation is ~688 mm (data from on 20 trees per plot. Monitoring was continued on a sub-set of 16 plots Climate-data.org, 2017). In 2007, the Federal Research Centre for in later years. In 2009, the Austrian Forest Inventory included 1200 plots Forests (BFW) conducted a monitoring program on 50 plots in Lower in an effort to assess the status of ash dieback all over Austria. Austria where crown dieback intensity of common ash was estimated Siegenfeld in Lower Austria was included in all these assessments (Cech 516 Downloaded from https://academic.oup.com/forestry/article/91/4/514/4962186 by DeepDyve user on 20 July 2022 Genetic analysis of inherited reduced susceptibility of Fraxinus excelsior L. seedlings et al., 2012). Most of the 20 trees assessed in detail at Siegenfeld DNA amplification and fragment analysis showed decreasing symptom intensity from 2007 to 2009 (Cech et al., −1 DNA concentrations for all samples were below 20 ng μL . Therefore, 2012), which singled out Siegenfeld as one of the relatively healthier DNA extracts were employed undiluted for polymerase chain reaction monitoring plots by now (trees at many other sites were not in a good (PCR). Nine nuclear microsatellite loci were used for amplification (with health state at all; Katharina Schwanda pers. comm.). annealing temperatures: Femsatl-4 – 60°C, Femsatl-10 – 50°C, Femsatl- Saplings occurred in small patches at both sites that seemed to 11 – 55°C, Femsatl-12 – 55°C, Femsatl-16 – 62°C, Femsatl-19 – 58°C, depend on penetrance of the canopy by sunlight. Biological material for M230 – 57°C, FR639485 – 55°C and FR646655 – 60°C; Brachet et al., DNA analysis was collected in June, July and August 2015 at both sites 1999; Lefort et al., 1999; Beatty et al., 2015). Each forward primer was as follows. One leaf was sampled from randomly chosen saplings across labelled with a fluorescent dye. PCRs were carried out using the QIAGEN the site (resulting in 80 saplings per site). Either a leaf, brought down by Type It Microsatellite Kit. The amplification reactions were performed on a slingshot if this was possible, or otherwise a bark plug per mature tree a PTC-100 Thermal Cycler (BIO-RAD, Vienna, Austria) under the following (from all mature ash trees at the site – resulted in a total of 134 trees) conditions: An initial denaturation step of 5 min at 95°C, 28 cycles of was collected (Table 1). The bark plug, from which cambium tissue can denaturation at 95°C for 30 s, annealing with corresponding tempera- be sliced, was cut out of the trunk with a 1 cm diameter leather punch. tures (see above) for 90 s and extension at 72°C for 30 s, and a final Cambium tissue as well as leaf tissue contains the same genomic DNA. extension step at 60°C for 30 min. The material was immediately dried in silica gel and kept at room tem- A CEQ 8000 Beckman-Coulter (Vienna, Austria) Sequencer was used perature prior to DNA extraction. to visualize the PCR products based on fragment length polymorphism, Ash dieback was recorded as the degree of crown dieback (loss of crown as compared with CEQ DNA Size Standard Kit-400 (Beckman-Coulter). foliage; see Table 2). Mature trees were classified into one of six damage Allele assessment, calling and binning were carried out using the frag- classes by visual inspection (Figure 2A). There were also six damage classes ment analysis tool of GenomeLab GeXP Beckman-Coulter software (ver- for sapling assessment, but these were based on shoot damage instead sion 10.2.3), with additional visual inspection and binning of peaks. (percentage of the shoots of a sapling affected by the disease; Figure 2B and Table 2). Additionally the diameter at breast height (DBH) of each tree and the height of each sapling were measured. Saplings were only sampled when they exceeded the height of ~60 cm, so that symptom identification Parentage analysis and damage class categorization were possible with higher confidence. CERVUS 3.0.7 software (Marshall et al., 1998; Kalinowski et al., 2007; Field Genetics Ltd, London, UK) was used for parentage analysis and for calculating other parameters, such as expected (He) and observed (Ho) heterozygosity, polymorphic information content (PIC), average non- DNA extraction exclusion probability for one candidate parent (NE-P1), average non- On average, 25–45 mg dried leaf or cambium tissue per individual was exclusion probability for a candidate parent pair (NE-PP), Hardy put into 2 mL tubes together with two glass balls of 3 mm diameter and Weinberg equilibrium (HW) and estimated null allele frequency (NULL). one of 4 mm (for cambium tissue, steel balls were used), one spatula tip All of these parameters give information about the loci and their suit- each of glass powder, activated charcoal, polyvinyl pyrrolidon (PVP ability for parentage analysis. 40 000) and sodium metabisulfite (pro analysi grades were used for all For parentage assignment, the CERVUS program uses likelihood ratio, chemicals, most of which were purchased from Sigma-Aldrich, St. Louis, a well-established statistical method (Marshall et al., 1998). Parentage is MO, USA). For homogenization, the material was frozen in liquid nitrogen assigned to a candidate parent if the likelihood is large relative to the for two minutes and ground with a TissueLyser shaking mill (QIAGEN, likelihood of alternative candidate parents. The likelihood ratio is Hilden, Germany) at 25 Hz for 2 min. The process was repeated a second expressed as LOD scores (logarithm of the likelihood ratio; Marshall time. The DNA was extracted using the Invisorb Spin Plant Mini Kit et al., 1998). Candidate parents with positive LOD scores are more likely (STRATEC Molecular, Birkenfeld, Germany), applying the protocol recom- to be the true parents, and candidate parents with negative LOD scores mended for the kit, but replacing Lysis Buffer P (provided by the kit) with are less likely to be the true parents. If two or more parents have posi- a mixture of 800 μL2× CTAB Buffer (20 g/L cetyl trimethyl ammonium tive LOD scores, Marshall et al. (1998) defined delta (difference in LOD bromide [CTAB], 100 mM Tris–HCL pH 8.0, 1.4 M NaCl, 25 mM EDTA pH scores) as an assessment criterion. 8.0) plus 1.6 μL β-mercaptoethanol and 1 μL proteinase K (QIAGEN) for Delta is the difference in LOD scores between the most likely candi- lysis. Additionally 40 μL RNAse A (10 mg/mL; QIAGEN) were added to date parent and the second most likely candidate parent. This param- each sample before the binding process. To determine DNA concentra- eter is useful when two candidate parents have a positive LOD score. If tions, a NanoDrop 1000 Spectrophotometer (Thermo Fischer Scientific, delta is high enough, parentage can be assigned to the candidate par- Ulm, Germany) was used. ent with the higher LOD score. The advantage in the use of CERVUS lies in the allowance for mistyping and missing data for individuals at a spe- cified number of loci. Parentage analysis for one parent (implemented by the ‘maternity Table 2 Definition of damage classes analysis’ function of CERVUS) and parent-pair analysis were carried out for both sites separately. Prior to parentage analysis, the simulation of Damage Range of crown foliage loss (trees) or percentage parentage was performed. This is important, because it is used to check class of damaged shoots (saplings) the feasibility of the parentage analysis and it calculates values of likeli- hood ratios, so that the confidence of parentage assignment can be 1 No or few symptoms; < 10% loss/damage determined. In short, in a simulation appropriate LOD and delta scores 2 Between 10 and 25% loss/damage for valid parentage assignment are generated for the parentage analysis 3 Between 25 and 50% loss/damage with the real data from genotyping. The simulation in this study was 4 Between 50 and 75% loss/damage performed with 10 000 offspring, an error rate of 0.01 at strict (95 per 5 Between 75 and < 100% loss/damage cent) and relaxed (80 per cent) confidence levels. As additional para- 6 Trees/saplings died from infection meters of the simulation, the number of candidate parents was set to 100 for Johannser Kogel with a 0.75 proportion of candidate parents 517 Downloaded from https://academic.oup.com/forestry/article/91/4/514/4962186 by DeepDyve user on 20 July 2022 Forestry Figure 2 Illustration of the visually assessed damage classes (d.c.) (A) for mature trees, no tree was assessed for damage class 6 (photos from Siegenfeld by Alexandra Wohlmuth); (B) for saplings (d.c. 5 is not shown) (Photos by Thomas Kirisits, University of Natural Resources and Life Sciences, Vienna, Austria). sampled, and 60 candidate parents with a 0.90 proportion of candidate confounds health state correlations. The damage classes of trees in dif- parents sampled for Siegenfeld. The proportion of sampled candidate ferent DBH classes at both sites were plotted as a ‘box and whisker plot’, parents were estimated by field observation, as the occurrence of with a similar intention (to check for possible covariance). We tabulated unsampled trees in the proximity of the stands cannot be determined the number of offspring per damage class for parents in each damage with certainty. Unsampled candidate parents were assumed (for the class. We also calculated the frequency distribution of the damage purpose of the CERVUS simulation) to be present in moderate frequency classes of local saplings (one or both parents assigned locally) and at Johannser Kogel and at low frequency at Siegenfeld. immigrant saplings (no local parents assigned, thus descents exclusively of trees outside of the stand) to see whether there was a difference in the health performance between these cohorts (local and immigrant), using a Kolmogorov–Smirnov test to check for significance. The boxplot Data analysis (DBH for damage classes) was done in SPSS, whereas the tables and the We performed a correlation analysis on the relationship between the bar diagram were done in Microsoft Excel 2013 (Redmond, WA, USA). DBH of mature ash trees and the number of their offspring in order to To estimate the significance of correlation between damage class of see whether tree size explains seedling numbers and thus possibly parent and offspring (categorial data), a one-tailed Spearman’s rank 518 Downloaded from https://academic.oup.com/forestry/article/91/4/514/4962186 by DeepDyve user on 20 July 2022 Genetic analysis of inherited reduced susceptibility of Fraxinus excelsior L. seedlings correlation analysis was calculated using SPSS Statistics 23 software (IBM, Vienna, Austria). The calculation was applied separately for those offspring where both parents were assigned (Spearman’s rank correl- ation coefficient between the damage class of the offspring and the averaged mean damage class of both parents) and for those where only one parent was assigned (Spearman’s rank correlation coefficient between the damage classes of the offspring and the one parent). Alternatively, an attempt was made to estimate heritability in situ in the sense of Ritland (2000): a pairwise matrix of differences in damage class for each possible pair of individuals in our study was regressed onto a pairwise kinship coefficient matrix, using the program SpaGeDi 1.05 (Hardy and Vekemans, 2002). The slope (b) and intercept of the regression, as well as the coefficient of determination (r ) were calcu- lated. This coefficient estimates the degree to which similarity in dam- age classes is determined by kinship, thus resembles a heritability value. The data set was permutated 1000 times in order to estimate P-values. Figure 3 The percentage of ash individuals that belong to damage classes 1–6 shown separately for mature trees and saplings from each Results study site; SJ: saplings from Siegenfeld; SA: mature trees from Siegenfeld; JJ: saplings from Johannser Kogel; JA: mature trees from Tree and sapling dimensions, and damage assessment Johannser Kogel. The mean diameter of the mature trees at breast height (DBH) was 39.7 cm at Johannser Kogel and 35 cm at Siegenfeld. Johannser Kogel hosts some particularly old trees with DBH dia- meters of around 60 cm (Table 1). At Johannser Kogel mean height of saplings was 96.5 cm with a range from 65 to 190 cm, and at Siegenfeld, 99.3 cm ranging from 70 to 170 cm. Disease symptoms were present at both sites in adults and saplings. Shoot dieback and crown defoliation were detectable, but there were few, if any, stem collar necroses. Both sites show a similar range of slightly to severely damaged individuals, with most mature trees having a crown damage intensity between 10 and 50 per cent and assigned to damage classes 2 and 3 (67 per cent of the trees at Johannser Kogel and 54 per cent at Siegenfeld). Percentages of assessment to damage class 1 were higher in Siegenfeld than in Johannser Kogel (Figure 3). However, the distribution of damage classes of saplings and mature trees showed no significant differences across both sampling sites according to Kolmogorov–Smirnov tests (saplings: P = 0.692; trees: P = 0.897). No tree was observed to have lost all its crown foliage (no damage class 6). Trees in the healthiest damage class at Johannser Kogel had higher DBH, but the ranges of DBH in the other damage classes at both sites were overlapping (Figure 4). Allele frequencies A total of 285 individuals of 294 sampled were successfully geno- Figure 4 Boxplot of the assignment of mature ash trees of different size typed for the nine microsatellite loci. DNA from one ash tree and (measured by their diameter at breast height, DBH) to different damage eight saplings (including all of the six dead saplings from Siegenfeld) classes at the two study sites. could not be amplified, likely due to insufficient DNA quality. These individuals had to be excluded from further analysis. The loci showed high allele numbers (N)and most of them were highly polymorphic (Table 3). The highest allele variation Observed heterozygosity (Ho) ranged from 0.38 to 0.85 for was found at locus Femsatl-10 with 41 alleles, and the lowest Johannser Kogel, and from 0.29 to 0.87 for Siegenfeld. Expected variation with six alleles at locus Femsatl-16. In total, 183 heterozygosity was higher than observed in most of the loci and alleles were detected in 161 individuals from Johannser Kogel in both sites. Mean polymorphic information content was 0.71 for with an average number of 20.3 alleles per locus, and 166 Johannser Kogel and 0.77 for Siegenfeld. Deviations from Hardy– alleles were detected for 124 individuals from Siegenfeld (aver- Weinberg equilibrium were detected at five loci in Johannser age 18.4 alleles per locus; Table 3). The mean proportion of loci Kogel and at one locus in Siegenfeld. The estimated frequency of typed exceeded 0.99 for both sites. Mean expected heterozygos- null alleles ranged from −0.003 (FR639485, Johannser Kogel) to ity (He) was 0.75 for Johannser Kogel and 0.80 for Siegenfeld. 0.5 (Femsatl-12, Siegenfeld) with a mean value of 0.072. 519 Downloaded from https://academic.oup.com/forestry/article/91/4/514/4962186 by DeepDyve user on 20 July 2022 Forestry Table 3 Information about the microsatellite loci used and the analysed parameters Locus Analysed parameters Johannser Kogel Analysed parameters Siegenfeld NHo He PIC NE-1 P NE-PP HW Null NHo He PIC NE-1 P NE-PP HW Null Femsatl-4 26 0.665 0.734 0.695 0.65 0.275 NS 0.052 21 0.734 0.808 0.787 0.53 0.162 NS 0.0437 Femsatl-10 41 0.745 0.895 0.884 0.347 0.067 * 0.0924 37 0.772 0.935 0.927 0.24 0.031 ND 0.0913 Femsatl-11 22 0.776 0.873 0.858 0.406 0.096 *** 0.0589 19 0.789 0.883 0.869 0.384 0.085 NS 0.0561 Femsatl-12 17 0.376 0.737 0.701 0.646 0.268 *** 0.3333 15 0.286 0.855 0.837 0.446 0.114 *** 0.5006 Femsatl-16 7 0.547 0.526 0.454 0.858 0.588 NS −0.0227 6 0.459 0.524 0.489 0.85 0.507 NS 0.0853 Femsatl-19 16 0.85 0.802 0.776 0.549 0.186 ** −0.0328 17 0.79 0.877 0.861 0.403 0.096 NS 0.0515 M230 37 0.844 0.891 0.881 0.349 0.066 * 0.0234 36 0.871 0.94 0.933 0.225 0.027 ND 0.0366 FR639485 9 0.638 0.583 0.524 0.816 0.496 NS −0.051 8 0.677 0.654 0.601 0.757 0.405 NS −0.0258 FR646655 8 0.696 0.695 0.647 0.719 0.36 NS −0.0029 7 0.697 0.71 0.66 0.701 0.342 NS 0.008 total 183 166 mean 20.3 0.682 0.748 0.713 0.593 0.267 0.0501 18.4 0.675 0.798 0.774 0.504 0.197 0.0941 Number of alleles (N); Ho, observed heterozygosity; He, expected heterozygosity; PIC, polymorphic information content; NE-1 P, average non- exclusion for one candidate parent; NE-PP, average non-exclusion probability for a candidate parent-pair; HW, significance of deviation from Hardy– Weinberg equilibrium; NS = not significant, ND = not determined. *Significant at 5 per cent level, **significant at 1 per cent level, ***significant at 0.1 per cent level. Null: Estimated null allele frequency. Parentage assignment there were 11 more saplings assigned at 80 per cent, one more at 95 per cent, four improved, one worsened in significance, and The assignments suggested by CERVUS (including LOD scores three seedlings were assigned to different parent pairs (numbers and delta values) are given in Supplementary Data File S1. and details in Table 4 and Supplementary Data File S1). Combined non-exclusion probabilities for the first parent and for the parent pair were 5.70E-03 and 6.00E-07 for Johannser Kogel and 8.92E-04 and 8.73E-09 for Siegenfeld if Femsatl-12 Data analysis was included. Without Femsatl-12, the values were less favour- able (by approximately one order of magnitude in most cases): The data show a very weak positive correlation of parents with higher DBH with the number of their offspring (R = 0.0101; 8.82E-03 and 2.24E-06 (Johannser Kogel first parent/parent pair) Supplementary Figure S1; single-parent and parent-pair results and 2.00E-03 and 8.00E-08 (Siegenfeld first parent/parent pair). combined; FEMSATL 12 included). The numbers of offspring per Maternity analysis, i.e. the assignment of only one parent, for Johannser Kogel was successful in 42 cases or 52.5 per cent disease class of adult trees are rather equally distributed across (20 of the cases also at high stringency); for Siegenfeld, it resulted damage classes (Supplementary Table S1). The frequency distri- bution of the damage classes of local and immigrant saplings in a 58.3 per cent assignment rate (42 saplings; all at strict confi- showed no apparent differences in the health status between dence level). The assignment rate for the parent-pair analysis in these cohorts at both sites (Supplementary Table S2; not signifi- Johannser Kogel was 7.5 per cent, implying that five saplings from Johannser Kogel were assigned to both parents (among the local cantly different in Kolmogorov–Smirnov tests). Spearman’s rank adult trees) at relaxed confidence level (80 per cent) and one add- correlation for parentage vs damage class was generally low (−0.293 to 0.039, Table 4) and thus showed no apparent associ- itional sapling at strict confidence level (95 per cent). In Siegenfeld, ation between damage classes of offspring and parents, neither the assignment rate for the parent-pair analysis was higher with for parent-pair, nor for maternity analysis, and only one of the 27.7 per cent – both parents were determined for 20 saplings from Siegenfeld at a relaxed confidence level (10 of which also at coefficients – with a negative sign! – was significantly different strict confidence level). from zero (−0.293 without Femsatl-12 for single parent/mater- nity, P = 0.039; Table 4). Parentage analysis (‘maternity’ or one parent only) without The value for in situ heritability (r ) obtained by the regres- marker Femsatl-12 (at less stringent non-exclusion probabilities) sion of damage class differences on kinship (calculated includ- resulted in 39 saplings assigned to candidate parents at Johannser Kogel at 80 per cent confidence level (19 of which at ing Femsatl-12; distribution shown in Supplementary Figure S2) strict level). For Siegenfeld, the numbers increased as well, two was very close to zero (4.69E-006) and not statistically signifi- cant after permutations (P = 0.34). additional saplings were assigned to a single parent (at 80 per cent), and eight more at 95 per cent, but one of the original ones lost significance of assignment (total, 50 saplings assigned), while for two saplings, alternative parents were assigned. Parent- Discussion pair assignment without Femsatl-12 resulted in the following Genetic parameters and parentage assignment changes: Johannser Kogel, six more saplings assigned at 80 per cent, one more at 95 per cent, one improved from 80 to 95 per The majority of the loci used in this study were highly poly- cent, but three lost at 80 per cent significance. At Siegenfeld, morphic, and expected heterozygosity was high. Similar 520 Downloaded from https://academic.oup.com/forestry/article/91/4/514/4962186 by DeepDyve user on 20 July 2022 Genetic analysis of inherited reduced susceptibility of Fraxinus excelsior L. seedlings Table 4 Spearman’s rank-correlation analyses for offspring for which one parent was assigned, and for offspring for which both parents were assigned, calculated including and excluding marker Femsatl-12 Comparison Number of Spearman’s rank P-value and cases correlation coefficient significance Offspring assigned to parent pairs at 80% confidence including Femsatl- 26 0.098 0.318 NS 12 – damage class offspring to average of parent pairs Additional offspring assigned to one parent at 80% confidence including 58 −0.082 0.272 NS Femsatl-12 – damage classes compared Offspring assigned to parent pairs at 80% confidence excluding Femsatl- 42 0.059 0.710 NS 12 – damage class offspring to average of parent pairs Additional offspring assigned to one parent at 80% confidence excluding 50 −0.293 0.039* Femsatl-12 – damage classes compared NS: not significant, *significant at the 0.05 level. observations were made for the same microsatellite loci in pre- than half of the saplings under various conditions of the ana- vious population genetic studies in common ash, suggesting lysis, and only 17 per cent of the saplings were assigned to both high genetic diversity within populations across Europe (Heuertz parents within their stands, despite their relative isolation. A et al., 2001, Hebel et al., 2006; Beatty et al., 2015). The values possible technical explanation for the low assignment rate could for genetic variation are typical for studies with these microsat- be genotyping or binning errors which would probably lead to ellite markers in European ash (see Heinze and Fussi, 2017 for a undetected parent–offspring trios or duos, although CERVUS comparison). While the deviations from Hardy–Weinberg equilib- uses likelihood equations that take account of such errors. High rium, and the high frequency of null alleles in Femsatl-12 are combined exclusion probability (99 per cent) across all loci for not ideal, the combined non-exclusion probabilities are very unrelated parents suggests low error rate in parentage assign- much appropriate (given the numbers of candidate parents pre- ment and shows that the microsatellite set used was adequate. sent). Femsatl-12 seems to show an increase of null alleles Nevertheless, we tested our data analysis procedure omitting from Western to Eastern Europe (Heinze and Fussi, 2017, their the critical Femsatl-12 marker. The parent non-exclusion prob- Table 4). Null alleles do not amplify consistently during PCR and abilities then changed to less stringent values (Supplementary thus cannot be reliably detected, leading to false assessments Data File S1) and slightly higher rates for parentage assignment of heterozygotes as homozygotes (Kalinowski and Taper, 2006). resulted, so that more saplings could be assigned to parents. They are problematic in parentage analysis and appear some- However, the correlation (Spearman’s rank) did not change times simultaneously with deviations from the Hardy–Weinberg in a meaningful way. Thus our conditions are conservative equilibrium (Pemberton et al., 1995), which was also the case in the sense that more relaxed assignment conditions, if they for five loci in Johannser Kogel and at one locus in Siegenfeld. lead to higher errors in parent–offspring assignments, possibly However, estimation of null allele frequencies does not necessar- underestimate correlations (but provide a wider data basis for ily imply that a null allele is present. If no parent–offspring rela- them). tionship is definitely known and there is no Hardy–Weinberg Although we made an effort to sample all putative parents equilibrium, it is difficult (or impossible) to decide about the pres- in both fairly isolated stands, it is possible that these missing ence of null alleles with certainty (Dakin and Avise, 2004). We do parents are actually scattered trees outside the stands. not expect strict Hardy–Weinberg equilibria at our sites, as we Therefore, a more likely explanation for the low assignment observed incoming seed and pollen in the young generation. rates is that the rest of the saplings had been sired by these But even Femsatl-12 adds power to this analysis (better non- unsampled trees. High seed and pollen inflow from the sur- exclusion probabilities), because undetected null alleles do not roundings is a phenomenon often detected in forest tree stands, lead to (wrong) exclusions of candidate parents; so the number even if we did not expect it here due to the scarcity of ash trees of parent–offspring pairs is not underestimated because of the in the surroundings, and because of the generally low physical null alleles. It could be overestimated, but the simulations in disposition of ash seed and pollen for passive transport beyond CERVUS take the null frequencies into account for suggesting a few hundred metres (Richards, 1997). Nevertheless, in this thresholds of LOD and delta values. The deviations from Hardy– respect our findings resemble those of Lobo et al. (2015), where Weinberg equilibria are already a hint for absence of panmixia paternity analysis in two Danish seed orchards resulted in an (which would require equal parental contributions to the off- assignment rate of less than 40 per cent. They suggested the spring generation) and/or the presence of immigrant saplings small size of the seed orchard as one possible explanation (12 (see below). putative parents in the orchard). Analysis of seed dispersal in Both our sites had nearly the same set of alleles with few pri- two natural ash woodlands in Ireland carried out by Beatty vate alleles (alleles that only occur in one population) at each et al. (2015) indicates the potential for seed dispersal over sev- site, and little differentiation (like in Heuertz et al., 2001; Hebel eral hundreds of metres. Bacles et al. (2005) and Bacles and et al., 2006; Ballian et al., 2008). Surprisingly, positive assign- Ennos (2008) estimated pollen dispersal in fragmented ash ments of at least one parent were made only for slightly more populations in Scotland, suggesting effective pollination within 521 Downloaded from https://academic.oup.com/forestry/article/91/4/514/4962186 by DeepDyve user on 20 July 2022 Forestry 300 m, but detected rare pollination events over distances of up cultivated in a nursery, and rather intensively managed after to 3 km. While these studies on pollination were carried out in planting (with wider spacing; McKinney et al., 2011; Stener, isolated tree stands in open landscapes, where pollen can be 2013). Family size in the study of Muñoz et al. (2016) was transported easier with less barriers being present, a study by between 8 and 68 half-sibs per mother tree. Lobo et al. (2015) Heuertz et al. (2003) in a Romanian continuous ash woodland used 2–48 offspring per half-sib family in inoculation tests (8–48 estimated average distances of 14 m between seeds and their in maternal families). We expected to arrive at numbers similar female parents, and average pollen flow below 140 m distance to the lower part of this range in our settings, but that was not (between seed mothers and pollinating trees). However, their the case. Progeny trails and seed orchards (and especially sap- common ash stand was much denser (~200 mature stems per ling inoculations) represent more controlled environments and ha), and their gene flow estimates were indirectly derived from they often do not fully resemble natural woodland conditions. genetic data of adults only (decrease of kinship with increasing In contrast, this study was conducted in naturally regenerating distance), while we analysed saplings (and thus the ‘realized ash stands, and the more heterogeneous conditions there likely gene flow’ of the transition of one generation to the next). We led to higher genotype × (micro-)environment interactions., which infer from our low assignment rate for parent-pair analysis and in turn lowered the genetic correlations observed (see also below) the moderate assignment rate for maternity analysis that seed Lobo et al. (2014) estimated the heritability of damage inten- and pollen dispersal within mixed landscapes is probably higher sity in two progeny trails in Denmark. Both trials included pro- than the estimates of Heuertz et al. (2003) for continuous genies from the same mother trees, but one trial was left woodlands and more in the range of the estimates of Beatty unfenced and progenies were exposed to strong competition et al. (2015) in Irish woodlands (but lower than those for very from vegetation and to browsing animals, whereas the other open landscapes). It is also the experience of foresters in was protected by fencing. Lower estimated heritability values of Austria in similar forest types that (i) either a single or a few ash the unfenced (0.2) compared with the fenced site (0.42–0.53) trees are able to produce dense seedling patches, if light and indicated that estimates of heritability under heterogeneous other conditions are good (‘seed shadows’), and (ii) these conditions may be lower (Lobo et al., 2014). Johannser Kogel is patches can be found up to a few hundred metres away from also fenced, but the fence is obviously penetrable at some the source (especially following the main wind direction; points, as wild boars were observed inside the fence during Herfried Steiner, Werner Ruhm, pers. comm.). Further analysis is sampling. Pliūra et al. (2014) tested clones from seven different needed in order to infer the precise influence of landscape fea- sites from Lithuania exposed to different ash-dieback infection tures on these parameters. pressure. They calculated the genotype–environment interaction on phenotypic variation and found significant contributions of genetic variation in plasticity and reaction norms of clones across a range of infection pressure environments (Pliūra et al., Variation in susceptibility 2014). This indicates the presence of variation of individual The presence of seeds that originate entirely from outside the response to the disease across sites depending on site condi- stands investigated allowed us to calculate if there are ‘popula- tions (Pliūra et al., 2011, 2014), or different levels of disease tion differences’ in susceptibility between offspring of the local pressure. Results from previous studies, together with our cur- trees vs immigrant saplings. The frequency distribution of the rent ones, suggest that precise estimates of heritability are only damage classes between these cohorts showed no apparent possible in more controlled environments, or with much higher differences (Supplementary Table S2). This is a further indication sample numbers (sapling half-sib families detected and that there is no selection (yet) for highly tolerant saplings at our assigned to parents). Therefore, environmental conditions and sites (or for more tolerant adults with a consequent higher suc- sample sizes are likely more relevant and need to be strongly cess in contributing to seed production in the wider surround- considered for inferences on susceptibility in field observation ings). It also follows that there is no reason to believe that studies. different susceptibilities of trees outside our stands (compared It seems from all cited studies that up to now, only a small with the adults within our stands) have led to any systematic fraction of ash individuals maintain potential resistance to the error in the correlations. disease, as practically all trees are infected at some point. Kjær No significant relationship (with the exception of one nega- et al. (2012) found high susceptibility and mortality among trees tive) was found based on Spearman’s rank correlation between in two progeny trails in Denmark. They further estimated that damage intensity of parents and their offspring (Table 4), nei- only 1 per cent of the trees have the potential to pass lower ther for parent–offspring duos (offspring with one parent), nor susceptibility to their offspring. In three Lithuanian progeny trails for parent–offspring trios (offspring with both parents). Previous almost 90 per cent of the trees died during the observation peri- studies suggested genetically based variation as an explanation od of eight years (Pliūra et al., 2011). In Austria, such high mor- for observed differences in susceptibility to dieback symptoms tality rates have not yet been reported from observation sites based on the observation of clones and single-tree offspring in (Heinze et al., 2017). In 50 ash dieback monitoring plots in progeny tests, and they concluded that these differences are Lower Austria (mainly adult stands), mean crown dieback inten- transferred across ash generations (Bakys et al., 2009; McKinney sity reached 18.1 per cent in 2009 and 17.6 per cent in 2010 et al., 2011; Lobo et al., 2014; Muñoz et al., 2016). Most of these (Kirisits and Freinschlag, 2012). Three seed orchards in Austria studies were performed in clonal seed orchards or established showed moderate mean crown dieback intensity in 2011 (14.2 per progeny trials. The seedlings were raised in a nursery prior to cent, 13.5 and 31 per cent), but no clone was totally unaffected planting (Kjær et al., 2012; Muñoz et al., 2016) and the clones (Heinze et al., 2017). Crown and shoot dieback intensity was also for seed orchards were grafted onto rootstocks and also further relatively moderate at Johannser Kogel and Siegenfeld (66 and 72 522 Downloaded from https://academic.oup.com/forestry/article/91/4/514/4962186 by DeepDyve user on 20 July 2022 Genetic analysis of inherited reduced susceptibility of Fraxinus excelsior L. seedlings per cent of the individuals, respectively, showed lower than 50 per substances that have a higher prevalence in ‘resistant’ vs sus- cent damage intensity). It is likely that the mature trees that were ceptible tree. identified as parents did not express assessable resistance due to Despite these advances, the exact genetic background(s) of lower infection pressure (especially for the higher crowns of thicker low susceptibility still remains unclear and it may well be that trees, Figure 4), and consequently produced more offspring more than one gene network or metabolic pathway is involved, becauseof their greatersizeand thebetter healthcondition asso- or that different such mechanisms are at work in different ciated with it (Supplementary Figure S1), while infection pressure regions of Europe. The robustness of the observed potential was higher for saplings close to the forest floor, where the pres- resistance in previous field studies needs to be re-evaluated. ence of petioles and more humid conditions would favour high Precise genetic tests of their offspring are necessary to deter- spore concentrations. This could be one reason for the low correl- mine which of the field-resistant trees really transmit this trait ation between damage classes of parents and offspring in our to seeds. Large offspring numbers are necessary for this pur- study. However, this effect may impact the severity, but not so pose, which make such tests costly. The alternative approach much the direction of susceptibility; healthier mature trees should we tested would, however, require sites with high numbers of then still produce healthier offspring, though the average damage seedlings with established parentage, much higher than the class rating would be shifted between old trees and young sap- ones we identified. Thus, such sites should have many seedlings, lings. Probably the small sample size of parent–offspring trios but few immigrants among them. This may be difficult to find. (N = 26–50) and of half-sib families (only nine mature trees Although the distribution data of ash seedlings in the wider area produced more than two offspring) may have led to underesti- of Johannser Kogel suggest the presence of ‘seed shadows’ of mation of actual correlations of damage intensity between single adults (Herfried Steiner, pers. comm.), it remains to be parent and offspring for possible reduced susceptibility. Muñoz seen whether numbers are high enough at a particular site. The et al. (2016) found that there are family effects in open-pollinated higher workload of laboratory testing would require more time offspring, but they also calculated individual breeding values for (and it would increase costs), but the approach may still be fas- the offspring, and most of the genetic variation was found within ter in total than field planting of seedlings to be tested for dis- families. Thus, our small family sizes may not allow estimating her- ease tolerance. itabilities with great precision. The in situ heritability estimation It is uncertain whether low susceptibility in single, but infre- basedonall kinship relationships among trees and saplings also quent individuals could hinder massive decline of ash in Europe, resulted in a coefficient of determination of very close to zero. It and if that resistance would sustain massive infection pressure may not necessarily be the case that related trees have very simi- at natural forest sites. However, selecting and breeding non- lar disease tolerance levels. susceptible trees in a timely manner throughout Europe seems Breeding values, which represent the average effect of the the only way to overcome the disease; the approach we discuss parent genotype, estimated from the performance of its off- here may help to accelerate selection. Alternatively, selecting spring (McKinney et al., 2014), were calculated by Kjær et al. ash stands that combine many environmental and demographic (2012) in a Danish progeny trail, and by Muñoz et al. (2016) in factors that disadvantage high infection pressure and the pro- France. Only one of 101 tested mother trees in Denmark had gress of the disease (e.g. hot and dry summers, dry soils during breeding values for ‘susceptibility’ below 10 per cent and was the sporulation period of the fungus, big trees with large, dom- estimated to produce healthy offspring (and four trees with inating crowns or stands were leaf litter does not persist until breeding values below 20 per cent were estimated to produce the next summer) may emerge as a conservation strategy in fairly healthy offspring). Another study carried out by McKinney situations where genetic tolerance is generally low (Heinze et al. (2011) supported these findings, with only one of 39 et al., 2017). tested clones exhibiting breeding values below 10 per cent. Breeding values for susceptibility in Kjær et al. (2012) were nor- mally distributed, suggesting that possible resistance is based Conclusion on expression of several genes rather than on one alone (McKinney et al., 2014). The data from France (Muñoz et al., In this study, no significant correlation could be found between 2016) also suggest that many genes contribute to resistance, as ash dieback damage intensity of parent and offspring in two the largest part of genetic variation was found within families. natural stands in Austria. This can reflect low power of estimat- This also means that much larger numbers of saplings are ing heritability under natural in situ conditions, where the micro- necessary to estimate heritability in our approach. Harper et al. environment may have a stronger influence on the trait than in (2016), in contrast, discovered a number of single nucleotide planted tests. Given this, and the recent insights into the genet- polymorphism (SNP) and gene expression markers that are ics of disease tolerance, much higher numbers of offspring with associated with crown damage in infected trees using identified parenthood (than the ones we could find) are neces- Associated Transcriptomics. With three markers (concerning sary for our approach. Other possible causes could be that there transcription factor genes) they were able to predict individuals were no very tolerant parent trees at our sites, or that juvenile– with a low level of susceptibility to the disease (combined R adult correlations are low for this trait. We further suggest that value of 0.28). Sollars et al. (2017) very recently published the the genetic basis for variation in susceptibility might be more sequence of a low-heterozygosity ash tree, and further improved complex under natural heterogeneous conditions than in more these markers (which, in their data, explained Danish disease controlled environments like planted progeny trials or seed orch- scores with r = 0.25) and suggested that according to these ards, where most of the previous studies were implemented. markers, native trees in Great Britain would have a lower chance The robustness of the observed potential low susceptibility in of becoming susceptible. They also reported on biochemical previous studies should therefore be critically re-evaluated after 523 Downloaded from https://academic.oup.com/forestry/article/91/4/514/4962186 by DeepDyve user on 20 July 2022 Forestry Cech, T.L. 2006b Eschenschaden in Österreich [Ash dieback and prema- planting offspring from tolerant trees in production forests. The ture leaf shedding in Austria]. Forstschutz Aktuell (BFW) 37,18–20. influence of environmental conditions should be considered when inferring on susceptibility (i.e. when assessing field resist- Cech, T.L., Kessler, M. and Brandstetter, M. 2012 Monitoring des ance). A scenario of large-scale decline of common ash trees in Zurücksterbens der Esche in Österreich [Monitoring ash dieback in Austria]. Forstschutz Aktuell (BFW) 55,56–58. Europe is becoming more and more likely. It will be important to identify and propagate healthy ash individuals throughout Cleary, M.R., Andersson, P.F., Broberg, A., Elfstrand, M., Daniel, G. and Stenlid, J. 2014 Genotypes of Fraxinus excelsior with different susceptibil- Europe, to find out about the genetic mechanism of any resist- ity to the ash dieback pathogen Hymenoscyphus pseudoalbidus and their ance in more detail and about the environmental conditions response to the phytotoxin viridiol—a metabolomic and microscopic that favour low susceptibility, in order to implement a manage- study. Phytochemistry 102, 115–125. ment program that accounts for these aspects. Climate-data.org. 2017 Climate Data for Siegenfeld. https://de.climate- data.org/search/?q=Siegenfeld (accessed on 22 August, 2017). Comita,L.S., Queenborough, S.A.,Murphy, S.J.,Eck,J.L., Xu,K., Supplementary data Krishnadas, M., et al. 2014 Testing predictions of the Janzen-Connell Supplementary data are available at Forestry online. hypothesis: a meta-analysis of experimental evidence for distance- and density-dependent seed and seedling survival. J. Ecol. 102, 845–856. Acknowledgements Dakin, E.E. and Avise, J.C. 2004 Microsatellite null alleles in parentage analysis. Heredity 93, 504–509. Special thanks to Renate Slunsky, Daniela Jahn, MSc, and the Genome Research unit at the Federal Research Centre for Forests (BFW) for their Dobrowolska, D., Hein, S., Oosterbaan, A., Wagner, S., Clark, J. and kind support and also to the Natural Forest Reserve and the Skovsgaard, J.P. 2011 A review of European ash (Fraxinus excelsior L.): Phytopathology units at BFW, especially to Dr Katharina Schwanda, implications for silviculture. Forestry 84, 133–148. Christian Neureiter and Mag. Herfried Steiner. ‘European Cooperation in Forst-und Landwirtschaftsbetrieb der Stadt Wien. 2017 Lage, Größe, Science and Technology (COST)’ Action FP1103 ‘FRAXBACK’ is acknowl- Geologie und Klima—Lebensraum Lainzer Tiergarten [Situation, size, geol- edged for providing a stimulating environment of meetings and discus- ogy and climate—habitat Lainzer Tiergarten; online; in German]. https:// sions. Furthermore we would like to thank Dr Thomas Kirisits, University www.wien.gv.at/umwelt/wald/erholung/lainzertiergarten/lebensraum/lage. of Natural Resources and Life Sciences in Vienna, who provided his pho- html (accessed on 22 August, 2017). tos of the damage classes of saplings, and the Editor in Chief and three Gross, A., Holdenrieder, O., Pautasso, M., Queloz, V. and Sieber, T.N. 2014 reviewers for their helpful comments and suggestions. Hymenoscyphus pseudoalbidus, the causal agent of European ash die- back. Mol. Plant Pathol. 15 (1), 5–21. 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Published: Oct 1, 2018

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