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Author(s): Ke Zhang, Berit Samils ,Juha Kaitera & Åke Olson Title: Low disease incidence and cone bagging in Picea abies are associated with low genotypic diversity in Thekopsora areolata Year: 2022 Version: Published version Copyright: The Authors 2022 Rights: CC BY-NC-ND 4.0 Rights url: http://creativecommons.org/licenses/by-nc-nd/4.0/ Please cite the original version: Zhang, K., Samils, B., Kaitera, J. & Olson, Å. (2022). Low disease incidence and cone bagging in Picea abies are associated with low genotypic diversity in Thekopsora areolata. Plant Pathology, 71, 1743– 1756. https://doi.org/10.1111/ppa.13602 Plant Pathology. 2022;71:1743–1756.  | 1743wileyonlinelibrary.com/journal/ppa 1  |  INTRODUC TION Norway spruce (Picea abies) produces abundant female flowers and seeds in mast years. The interval between mast years is usually about 7 years, and the frequency is influenced by weather (Nussbaumer et al., 2016). The supply of genetically improved high- quality seeds produced in seed orchards is often in shortage due to this irregu- lar flowering (Lundströmer et al., 2020). Furthermore, insects and fungal diseases can decrease the seed yield significantly in a mast year, increasing the imbalance between the supply and demand of seeds. Cherry spruce rust is a rust disease that associates with the primary host, Norway spruce, and the alternate host, mainly bird Received: 19 February 2022  | Accepted: 23 May 2022 DOI: 10.1111/ppa.13602 O R I G I N A L A R T I C L E Low disease incidence and cone bagging in Picea abies are associated with low genotypic diversity in Thekopsora areolata Ke Zhang1  | Berit Samils1 | Juha Kaitera2 | Åke Olson1 This is an open access article under the terms of the Creative Commons Attribution- NonCommercial- NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non- commercial and no modifications or adaptations are made. © 2022 The Authors. Plant Pathology published by John Wiley & Sons Ltd on behalf of British Society for Plant Pathology. 1Department of Forest Mycology and Plant Pathology, Swedish University of Agricultural Sciences, Uppsala, Sweden 2Natural Resources Institute Finland, Oulu, Finland Correspondence Ke Zhang, Department of Forest Mycology and Plant Pathology, Swedish University of Agricultural Sciences, Almas Allé 5, 75651, Uppsala, Sweden. Email: ke.zhang@slu.se Funding information Kungl. Skogs- och Lantbruksakademien, Grant/Award Number: Tandem Forest Values TFV 2018- 0001; Svenska Forskningsrådet Formas, Grant/Award Number: 2017- 01489 Abstract Thekopsora areolata infects pistillate cones of Picea spp. with monokaryotic basidi- ospores in the spring. Receptive monokaryotic hyphae in the cones are fertilized by monokaryotic spermatia in the summer, and dikaryotic aecia are produced in cones in late summer. Infected cones produce no fertile seeds, meaning the disease causes large reductions in seed production. To understand the seasonal variation of T. areo- lata genotypic diversity, 548 aecia from 55 infected cones were sampled from multiple seed orchards in 2015, 2019 and 2020. Cone bagging experiments were performed during two seasons to investigate the sexual reproduction of T. areolata. In addition to the published simple- sequence repeat (SSR) markers, we developed 10 new polymor- phic SSR markers to improve the resolution of population genetic analysis. Aecia were genotyped with 18 SSR markers in total. In 2015, when disease incidence was high in the seed orchards, the T. areolata populations had high genotypic diversity (H = 4.69). In 2019 and 2020, when disease incidence was low, the T. areolata populations had lower genotypic diversity (H = 3.88 and 3.85) and several cones were dominated by a single multilocus genotype. The genotypic diversity of T. areolata in a recently estab- lished seed orchard was exceptionally low (H = 2.01). Seven bagged cones that were infected produced either aecial primordia or aecia with lower diversity than exposed cones. The results indicate that cross- fertilization is important for sexual reproduction and aecia formation of T. areolata, and genotypic diversity of T. areolata increased with higher disease prevalence. K E Y W O R D S heterothallism, Norway spruce, population genetics, rust fungi, spermatia 1744  |    ZHANG et Al. cherry (Prunus padus) in Scandinavia (Kaitera et al., 2014, 2017, 2019; Kaitera, Aarnio, Ylioja,et al., 2021; Kaitera, Kauppila et al., 2021; Zhang et al., 2021). The typical symptoms of infected spruce cones are reddish- brown to dark brown aecia that fully cover both sides of the scales (Kaitera et al., 2009a). Therefore, seeds in infected cones are either missing or poorly developed (Kaitera & Tillman- Sutela, 2014). This disease can cause a 10- fold reduction in seed germination (Kaitera & Tillman- Sutela, 2014) and as high as 89% of seed yield loss (Kaitera, 2013). The causal agent of cherry spruce rust is the rust fungus Thekopsora areolata. This pathogen is widely distributed in Europe and Asia, but absent in North and South America, Africa and Oceania (CABI, 2022). T. areolata is heteroecious; both Norway spruce and bird cherry are required to complete its macrocyclic life cycle (Kaitera et al., 2017, 2019). During its 2- year life cycle, T. areolata pro- duces five types of spores (Littlefield & Heath, 2012; Olive, 1943). Teliospores are the overwintering structures in bird cherry leaf lit- ter. In the early stage, they are dikaryotic, having two haploid nu- clei in each cell. During the germination of teliospores in the spring, karyogamy and meiosis occur, after which haploid monokaryotic basidiospores are produced and then spread under optimal weather conditions to infect pistillate cones (Kuprevich & Transchel, 1957; Zhang et al., 2022). From late spring to early summer, T. areolata hy- phae colonize the developing cones and produce spermogonia with haploid monokaryotic receptive hyphae and spermatia. The mating of receptive hyphae and spermatia occurs in mid- summer, after which aecia with dikaryotic aeciospores are produced in the cones in mid- to late summer (Kaitera et al., 2009a; Kaitera, Aarnio, Karhu, et al., 2021). After overwintering, aeciospores are released to infect the bird cherry, where uredinia with dikaryotic urediniospores are produced from late spring to late summer (Kaitera, Aarnio, Karhu, et al., 2021), and teliospores are produced in autumn. Maintaining the mechanism of sexual reproduction has a cost to the pathogen and two individuals with different mating- type genes are required (Lee et al., 2010); however, it is considered advanta- geous to plant pathogens, because the mating of two individuals cre- ates new combinations of genetic alleles that help adaption to new environments (Drenth et al., 2019). In rust fungi, including T. areo- lata, sexual reproduction is achieved by the transfer of spermatia to receptive hyphae, which requires insect vectors or rain. In cones infected by T. areolata, a sugary liquid is produced by spermogonia in mid- summer (Kaitera, Aarnio, Karhu, et al., 2021; Murray, 1955). No study has addressed the relationship between this sugary liquid and the visitation by insects leading to fertilization in T. areolata, but this type of insect attraction has been demonstrated in other rusts (Alexopoulos et al., 1996). For example, Puccinia arrhenatheri infects Berberis vulgaris leaves, where insects can be attracted by the bright yellow colour of the diseased leaves and the sugary nectar and vola- tiles produced by the fungal spermogonia. Subsequently, the insect transfers spermatia in the sugary nectar between leaves and enables successful outcrossing (Naef et al., 2002). Most studied species in Basidiomycota are heterothallic, that is, two individuals with different mating types are required for sexual reproduction (Whitehouse, 1949). In Pucciniomycotina, most known mating systems are bipolar (such as Puccinia graminis) or tetrapolar (such as Melampsora lini), although some strains are homothallic or anamorphic (Kües et al., 2011). The mating system of T. areolata is not well understood. Capador et al. (2018) developed eight polymor- phic microsatellite/simple- sequence repeats (SSR) markers and an- alysed the diversity of T. areolata individuals from Finland, Norway and Sweden. The results suggested high gene flow, sexual reproduc- tion and multiple infections of Norway spruce cones by T. areolata. However, a few homozygous individuals were found from four cones (Capador et al., 2020). This homozygosity could be explained either by self- fertilization between genetically identical spermatia and re- ceptive hyphae or by the limited resolution of the molecular markers. The temporal dynamic of pathogen populations often undergoes "boom and bust" cycles, especially for crop pathogens. These fluctu- ations are the result of variations in environmental factors, pathogen inoculum dispersal, and host susceptibility and senescence (Burdon et al., 1989; Mundt et al., 2011). The genetic and genotypic diversity of pathogen populations can be greatly affected by population fluc- tuation through random genetic drift (McDonald & Linde, 2002). In the case of T. areolata, the population fluctuation in seed orchards can be influenced by additional factors, such as the change of availabil- ity of alternate hosts and long mast year intervals in Norway spruce. Information about the variation in pathogen genotypic diversity be- tween years with different disease prevalence would improve our understanding of the infection and reproduction processes of this pathogen and support the selection of strategies for disease control. Norway spruce seed orchards in Sweden had high cherry spruce rust disease incidence in 2015 (Olle Rosenberg, Skogforsk, olle.rosenberg@ skogforsk.se, personal communication), providing material for previous studies (Capador et al., 2018, 2020). Population genetic analysis with SSR markers revealed a high genotypic diversity of T. areolata aecia at the levels of seed orchards, trees, cones and even scales (Capador et al., 2020). The disease incidences of cherry spruce rust in 2019 and 2020 in the studied seed orchards were lower than in the previous years (Rosenberg, personal communication). We hypothesized that during these years with low cherry spruce rust incidences, T. areolata populations would have lower genotypic diversity. The previous study by Capador et al.(2020) provided evidence for multiple infection and cross- fertilization events in single cones. Accordingly, we hypothesized that bagging a cone before the pro- duction of the sugary fluid containing spermatia would lead to lower genetic diversity of T. areolata aecia by reducing the frequency of the mating between receptive hyphae and alien spermatia, therefore reducing the sexual reproduction frequency between cones. The re- sults from such cone bagging would increase understanding of the biology of the rust and provide new evidence for the involvement of insects in the sexual reproduction of T. areolata. In this study, we developed new SSR markers to improve the resolution in the population genetics analysis of T. areolata. The ob- jectives of this research were (a) to study the variation in genotypic diversity of T. areolata populations in Swedish and Finnish seed or- chards during years of high and low disease incidences; and (b) to     |  1745ZHANG et Al. investigate the effect of bagging of Norway spruce cones on the in- fection, fructification, and genetic diversity of T. areolata. 2  |  MATERIAL S AND METHODS 2.1  |  Fieldwork and sample collection Because of the irregular flowering of Norway spruce, it was impos- sible to collect samples every year from all seed orchards included in this study. The cone bagging experiments were conducted in seed orchards in Rörby, Sweden, and Suhola (sv 403), Finland, in 2019, and Söregärde, Sweden, and Vuolenkoski (sv 445), Finland, in 2020. The seed orchards were established in 1967 (Rörby), 2009 (Söregärde), 1997 (Suhola) and 2014 (Vuolenkoski). In May, 2019 and 2020, 300 to 500 pistillate Norway spruce cones were bagged with paper steriliza- tion pouches and sealed with zip- ties in each seed orchard. Pistillate cones were bagged individually unless it was impractical to do so (if two or more cones were tightly aggregated). All the cones were bagged before the formation of T. areolata spermatia, and therefore, the mating of spermatia and receptive hyphae between cones dur- ing the summer was inhibited. Pistillate cones were covered in the bags until mature cones with aecia had developed. All bagged cones were harvested in early September and examined in the laboratory: four cones were infected in Suhola, Finland, in 2019; two cones were infected in Vuolenkoski, Finland, in 2020; and one cone was infected in Söregärde, Sweden, in 2020. Additional infected exposed (un- bagged) cones were collected from Vuolenkoski (6) and Suhola (8 and 6), Finland, and Söregärde (7), Sweden, in 2019 and 2020 (Table 1). These bagged and exposed cones from 2019 and 2020 represented samples from seed orchards with low disease incidence. One special case was Suhola 2020, where very few cones were produced in the seed orchard (in total about 20), but among these, approximately 90% were infected, meaning that the disease incidence was high but the occurrence of disease was very low. Therefore, in this study, we re- garded Suhola as having low disease incidence. In 2015, high disease incidences of cherry spruce rust were observed in many Swedish seed orchards including Ålbrunna (es- tablished in 1982) and Söregärde (O. Rosenberg, personal commu- nication). Scale samples and aecial DNA samples from eight cones from Söregärde and five cones from Ålbrunna collected in 2015 (Capador et al., 2020) were retrieved from −20°C storage. These samples represent aecia from seed orchards with high disease in- cidence. In total, samples from 55 cones were investigated in this study (Table 1). 2.2  |  Single aecium sampling and DNA extraction From each infected cone, aecia were collected as described by Capador et al. (2020): each cone was split vertically into two halves and then horizontally into five sections in each half, and one scale with aecia was selected from each section. From each scale, one aecium T A B L E 1   Li st o f N or w ay s pr uc e sa m pl es in fe ct ed w it h Th ek op so ra a re ol at a th at w er e pr oc es se d fo r g en ot yp in g Ye ar Se ed o rc ha rd C oo rd in at es C on e pr od uc ti on D is ea se in ci de nc ea Ex po se d or ba gg ed c on e N o. o f co ne s Sa m pl in g N o. o f ae ci a 20 15 Sö re ga rd e, S w ed en 56 °4 7′ N , 1 6° 21 ′ E M as t ye ar H ig h, 1 0% E xp os ed 8 O ne a ec iu m p er s ca le , 1 0 sc al es 80 Å lb ru nn a, S w ed en 59 °3 0′ N , 1 7° 32 ′ E M as t ye ar H ig h, n a E xp os ed 5 O ne a ec iu m p er s ca le , 9 o r 10 s ca le s 48 20 19 Su ho la , F in la nd 62 °1 5′ N , 2 7° 42 ′ E M as t ye ar Lo w , ≤ 1% E xp os ed 6 O ne a ec iu m p er s ca le , 1 0 sc al es 60 Ba gg ed 4 O ne a ec iu m p er s ca le , 1 0 sc al es 40 Å lb ru nn a, S w ed en 59 °3 0′ N , 1 7° 32 ′ E M as t ye ar Lo w , 3 % E xp os ed 8 O ne a ec iu m p er s ca le , 1 0 sc al es 80 20 20 V uo le nk os ki , F in la nd 61 °0 5′ N , 2 6° 0 8′ E Lo w p ro du ct io n Lo w , ≤ 1% E xp os ed 6 O ne a ec iu m p er s ca le , 1 0 sc al es 60 Ba gg ed 2 O ne a ec iu m p er s ca le , 1 0 sc al es 20 Su ho la , F in la nd 62 °1 5′ N , 2 7° 42 ′ E Lo w p ro du ct io n Lo w , 9 0% b E xp os ed 8 O ne a ec iu m p er s ca le , 1 0 sc al es 80 Sö re gä rd e, S w ed en 56 °4 7′ N , 1 6° 21 ′ E M as t ye ar Lo w , 0 % E xp os ed 7 O ne a ec iu m p er s ca le , 1 0 sc al es 70 Ba gg ed 1 O ne a ec iu m p er s ca le , 1 0 sc al es 10 a T he d is ea se in ci de nc es w er e ca lc ul at ed e ith er b y th e ow ne rs o f t he s ee d or ch ar ds a ft er h ar ve st o r b y th e au th or s du rin g th e fin al s am pl e co lle ct io n. b D ue t o th e lo w f lo w er in g ra te , v er y fe w n ew c on es (a bo ut 2 0) w er e pr od uc ed in S uh ol a 20 20 , a nd a lt ho ug h ab ou t 90 % o f t he se c on es w er e in fe ct ed , t he o cc ur re nc e of d is ea se w as s ti ll ve ry lo w a nd s o w e re ga rd d is ea se in ci de nc e as lo w fo r t he p ur po se o f t hi s st ud y. 1746  |    ZHANG et Al. was picked up carefully with a scalpel and transferred into a 1.5- ml tube. In this way, 10 evenly distributed aecia were sampled from each cone. In the 1.5- ml tube, the aecium was precleaned with 0.2% Tween 20 (Merck), then surface sterilized with 1.5% sodium hypochlorite for 1 min, 70% ethanol for 1 min and rinsed with sterilized water three times. DNA from each aecium was extracted with a NucleoSpin Plant DNA extraction kit (Macherey- Nagel) according to the manufactur- er's manual. In total, 548 aecia were included in the study. 2.3  |  Development of SSR markers and screening for polymorphism A draft genome assembly of T. areolata isolate SB- 2076- 252 was ob- tained by next- generation sequencing (unpublished data). The original assembly consisted of 165,963 contigs with lengths from 200 to 31,680 base pairs (bp). The assembly was filtered with Geneious Prime 2019.1 (https://www.genei ous.com); subsequently, 413 contigs longer than 10,000 bp were screened with BatchPrimer3 v. 1.0 (You et al., 2008) for SSR loci that were longer than 12 bp and with corresponding primer pairs that yielded a product of optimal size (100– 300 bp). The preliminary screening yielded 359 SSR loci that contained repeated motifs: 51 dinucleotide, 97 trinucleotide, 141 tetranucle- otide, 41 pentanucleotide and 29 hexanucleotide loci. Thirty- three loci and corresponding primers (Table S1) were selected to test the polymorphism in standard PCR with 12 randomly selected aecial DNA samples from Sweden and Finland collected in 2019. The se- lection was made according to the following criteria: (a) only one SSR locus was selected from each contig, (b) primer GC content was be- tween 40% and 60%, and (c) longer SSR loci with more repeats were favoured. Standard PCR was performed in 20 μl reaction volumes (2 μl DNA template, 0.5 μM forward primer, 0.5 μM reverse primer, 0.2 mM each dNTP, 1× PCR buffer, 0.5 mM MgCl2 and 1 U DreamTaq DNA polymerase [Thermo Fisher Scientific]), under the following cycling conditions: 95°C for 5 min; 40 cycles of 95°C for 30 s, 56°C for 30 s and 72°C for 30 s; followed by a final extension of 10 min at 72°C. Amplification products were loaded on a 3% agarose gel for electrophoresis at 100 V for 2 h, then examined in a gel imaging system. SSR loci that were polymorphic among tested samples were identified based on the variation of amplicon sizes. Of the 33 loci tested for polymorphism (Table S1), 13 loci pro- duced detectable polymorphic amplicons on 3% agarose gel, but three of them had either weak amplicon bands or the amplicon size significantly differed from the estimated size based on NGS contigs. Therefore, 10 new SSR markers were included in the genotyping along with eight published markers from Capador et al. (2018) (Table 2). 2.4  |  Genotyping A total of 18 SSR markers were divided into six groups accord- ing to the amplicon size, and the forward primers were labelled with fluorescein amidite (FAM) or hexachloro- fluorescein (HEX) (Table 2). Standard PCR of each locus was performed individually in 15 μl reaction volumes (2 μl of DNA template, 0.5 μM forward primer, 0.5 μM reverse primer, 0.2 mM each dNTP, 1× PCR buffer and 0.75 U DreamTaq DNA polymerase [Thermo Fisher Scientific]), with cycling conditions as described above for SSR marker develop- ment. The PCR products of three markers in the same group were mixed together and then diluted 50 times for fragment length analy- sis (Macrogen Europe) with an ABI 3730xl DNA analyzer (Applied Biosystems) and GeneScan 400HD ROX as size standard. Raw data were scanned with GeneMarker v. 3.0.1 (SoftGenetics) to determine the SSR allele sizes. 2.5  |  Population genetics analysis Genotypic diversity analysis and haplotype reconstruction were performed with non- clone- corrected data, while clone- corrected data was used to analyse the SSR loci statistics. The genotyping data of 18 SSR loci of all aecium individuals were summarized in GenAlEx (Peakall & Smouse, 2006). The data were stratified and analysed according to year, seed orchard, bagged or exposed, and individual cone. The SSR loci statistics and genotypic diversity were analysed with R in Rstudio and poppr v. 2.8.0 (Kamvar et al., 2014). Data of the 478 samples from 48 exposed cones were used to compare the variation of T. areolata genotypic diversity be- tween years. Data of the 70 samples from seven bagged cones and 190 samples from 19 unbagged cones from the same year and seed orchard were compared to study the effect of cone bagging. Indices of genotypic diversity included the number of observed multilocus genotypes (MLG), the number of expected multilocus genotypes based on rarefaction (eMLG), Shannon– Wiener diversity index (H) (Shannon, 2001), Stoddard and Taylor's index (G) (Stoddart & Taylor, 1988) and evenness (Grünwald et al., 2003). The haplotypes of individuals from the same cones were inferred in Arlequin v. 3.5 (Excoffier & Lischer, 2010) with Excoffier– Laval– Balding (ELB) algorithm, where parameters used in the analysis were Dirichlet prior α = 0.01, γ = 0, ε = 0.1, sampling interval = 500, number of samples = 20,000, burn- in steps = 10,000, recombina- tion steps = 0. The numbers of T. areolata haplotypes in aecia from the same cone were summarized and visualized in R. The haploid diversity (HD) was represented by the average haplotype number per aecium. 2.6  |  Microscopy of aecial primordia from bagged cones In 2020, poorly developed fruiting bodies identified as aecial primordia were found in three infected cones from the cone bagging experiment in both Sweden and Finland. Scales of these cones were examined under a dissecting microscope. Individual aecial primordia were picked up from the scale with a scalpel and fixed in formaldehyde– acetic acid– ethanol (FAA) solution for 24 h. Cross- sections of the fruiting     |  1747ZHANG et Al. bodies were prepared by cryostat (CM1850; Leica) and microtome, and stained with lactophenol cotton blue. To quantify the number of nu- clei in the fungal tissue, cross- sections of the aecial primordia and the hyphae around the fruiting bodies were stained with the fluorescent dye 4′,6- diamidino- 2- phenylindole (DAPI) for staining DNA (Samils et al., 2011), and then examined under a fluorescence microscope. TA B L E 2  Characteristics of polymorphic simple sequence repeat (SSR) markers and primers, and their fluorescent dye labelling for determining multilocus genotypes of Thekopsora areolata Marker Primer sequence (5′– 3′) Motif No. of repeats in T. areolata reference genomea Amplicon size in T. areolata reference genome (bp)a Fluorescent dye Assigned groupb SSR1 F AGATCACCAGTTTAGTGACCA TCATCT 4 146 FAM 1 R AACCTAATTCTGCTGTCCTTC SSR24 F CGTTGAGATGAGACTGGATTA GA 9 154 HEX R GCGCTGTGTGTATGTAAGTATT SSR4 F AAGGAGGGGGATTTTAGTAGT TCAA 6 235 FAM R TACCACCATCTCAACCTTTTA SSR5 F TTCTCATTCTCATTCTCATCC CTTTTT 6 150 FAM 2 R CGTCATTAGCAAATCAAATTC SSR13 F TCTCGTGTGTTCATAACTGTG TG 9 172 HEX R AGTAAGGATGGAAATGGAGTC Tha9 F AAGGCAGATGACAGTCGTGA AC 17– 21 296– 300 FAM R TCCTCTGTCCAAAGCGTCTT SSR10 F CAAAACAGGTGTCAAAGTGAT CT 12 149 FAM 3 R CGGAGAGAGAGAAAAGAAAAA Tha91 F GTCTGTGTCTCTGGTGTCGA AG 3– 8 181– 191 HEX R ACCAAAGTTCCCTGATATCCC Tha61 F TGGGTAATTTGGGGTGTTTGT AC 15– 17 337– 341 FAM R ACAGAAGTTACTCCGCCCTT SSR16 F AGGAACAAGTTGACTTGCAG GAGAAT 5 140 FAM 4 R TAATCGATCAGACAACCCTTA SSR18 F GAGATATGTATGCAGGCAAAG GA 15 155 HEX R GTCTATCTGGTCTTCCCAAAC Tha92 F TTCTCGGGAATGGTGTGGAA AACAAAAT 9– 10 355– 363 FAM R CCCCACAAATCTTACGAGCTG SSR19 F GATCACGGATCAAGTTATCAA TCA 7 147 FAM 5 R GTGTTTAGGAGGTTGGTTTTT Tha96 F ATCACAACGCCTGATGG AAG 12– 21 176– 203 HEX R GCTCACAACATTCGCAATCC Tha136 F CAAGCACAACCTTCACCACA AG 10– 40 231– 261 FAM R TGGGTCATCAGCTTTACGGA SSR22 F ATCCAAGTCTTTTCTTCACCT CT 8 150 FAM 6 R TCGAAAAGTGTGAGAGAGTTT Tha105 F GCCGATTCTCAAACCTACACC AG 7– 17 184– 204 HEX R TGCTGCCAACTTTTCACGTT Tha137 F AAAGGGTTTTCAGGAGGGGC AG 7– 11 225– 233 FAM R CCAGTGCAAATCAAACGTCC Note: The markers developed in this study are in bold. aNo. of repeats of the motif and amplicon size in the T. areolata reference genome were calculated for the SSR markers developed in this study. Information about the published markers was taken from Capador et al. (2020). bSSR amplicons in the same group of the same aecia sample were pooled in one tube for one fragment length analysis reaction. The signal of each SSR can be identified according to the fragment length and fluorescent label. 1748  |    ZHANG et Al. 3  |  RESULTS 3.1  |  Diversity of SSR markers in individual aecial samples collected in 2015, 2019 and 2020 All SSR markers included in this study were polymorphic in the entire set of 548 aecial samples, as well as in the subsets of samples col- lected in 2015, 2019 and 2020; the only exception was locus Tha92 in samples collected in 2019, which was monomorphic (Table 3). In total, these markers detected 123 alleles with a variation from 2 (Tha92) to 14 (Tha96) alleles at each locus. The samples from 2015 had the highest average number of alleles (5.167 alleles per locus), even though the sample size (n = 128) was smaller than the subsets from 2019 and 2020 (n = 180 and 240, respectively). However, sam- ples from 2019 and 2020 had marginally higher expected heterozy- gosity (Hexp = 0.479 and 0.477, respectively) and alleles had more even distributions (evenness = 0.677 and 0.636). Almost all aecial samples (518 out of 548) had at least one het- erozygous locus among the 18 SSR loci. Exceptions were the three bagged cones collected in Söregärde and Vuolenkoski in 2020, from which aecial primordia instead of mature aecia were identified. These samples showed only one allele at all SSR loci, and therefore, they were excluded from the population genetics analysis. 3.2  |  Variation of T. areolata genotypic diversity between years SSR markers used in the study achieved high resolution that iden- tified 276 multilocus genotypes (MLGs) from the 548 samples (Figure S1); the genotype accumulation increased linearly and later a plateau was reached. Among the 478 T. areolata aecia samples from 48 exposed cones, a total of 259 MLGs were identified (Table 4). Aecia that shared the same MLG were always from the same cone, whereas aecia from different cones never shared the same MLG. A significantly higher number of MLGs was found in aecia sam- ples collected in 2015 compared to 2019 and 2020 (MLG = 115 vs 71 and 73, respectively) despite a smaller sample size (N = 128 vs 140 and 210; Table 4). Most aecial samples (n = 106) from 2015 had a unique MLG, while two to four aecia from the remaining samples (n = 22) shared the same MLGs (Figure S2). All exposed cones from 2015 had at least seven MLGs when 9 to 10 aecia were sampled (Figure 1). Eight out of 14 exposed cones from 2019 and 2020 had only one to six MLGs when 10 aecia were sampled (Figure 1), and it was common that aecia from the same cone shared identical MLGs (Figure S2). The genotypic diversity indices calculated in this study, Shannon– Wiener index (H) and Stoddart and Taylor's index (G), were higher in 2015 than in the other years. In addition, the MLGs had a more even distribution in 2015 compared to 2019 and 2020 (Table 4). Samples from the same seed orchard followed the general trend mentioned above: the genotypic diversity was higher in 2015 when disease incidence was generally higher than in 2019 and 2020. For example, with lower numbers of aecial samples, rust populations in 2015 in Ålbrunna and Söregärde had a much higher number of MLGs and eMLGs, and higher genotypic diversity indices than in 2019 in Ålbrunna and 2020 in Söregärde (Table 5). In Suhola, Finland, sam- ples were collected in 2019 and 2020 when the disease incidences were low. The 2020 Suhola population had an even lower number of eMLGs and genotypic diversity than the 2019 Suhola population (Table 5). Within the same sampling years, the diversity of populations from different seed orchards had similar levels of genotypic diver- sity: the diversity was the highest in 2015, lower in 2019, and the lowest in 2020. For example, Shannon– Wiener indices (H) were 3.84 and 4.14 in Ålbrunna and Söregärde in 2015, 3.13 and 3.24 in Suhola and Ålbrunna in 2019, and 2.95 and 3.17 in Suhola and Söregärde in 2020 (Table 5). The population in the youngest seed orchard, Vuolenkoski in 2020, was the least diverse (H = 2.01). Only nine MLGs were identified from 60 aecia samples from exposed cones, and the 10 aecia sampled from the same cones were always domi- nated by one MLG (Figure S3). The differences in genotypic diversity between years and seed orchards were shown in the rarefaction analysis (Figure 2): the ex- pected MLGs increased almost linearly as the sample size grew in the two 2015 populations. The rarefaction curves of the populations from 2019 and 2020 were grouped together below the 2015 curves, which indicated lower genotypic diversity. The low plateau of the rarefaction curves of 2020 Vuolenkoski suggested a very low geno- typic diversity, and the levelling out in the curve indicated that most genotypes existing in the seed orchard were sampled. 3.3  |  The effect of cone bagging on T. areolata genotypic diversity Seven infected cones were found among about 1200 bagged cones in 2019 in Suhola, 2020 in Söregärde and 2020 in Vuolenkoski. In general, aecia from bagged cones had lower numbers of MLGs (Figure 1): one to three MLGs were usually identified from the bagged cones, except for one bagged cone in Suhola 2019, which had six MLGs. In total, 17 MLGs were identified from 70 samples from seven bagged cones (Figure 3). Among exposed cones from the same years and seed orchards, four to nine MLGs could be usu- ally identified from the 10 aecia, except for cones from 2020 in Vuolenkoski, where one to two MLGs were identified in each cone (Figure 1). In total, 74 MLGs were identified from 190 samples from 19 exposed cones from the same seed orchards and years. The four bagged cones in Suhola 2019 had on average 3.5 MLGs, while the exposed cones had on average 5.3 MLGs. In the four bagged cones from Suhola 2019, fully developed aecia with multiple heterozygous MLGs coexisted in the same cone. However, all fruiting bodies (N = 30) from the three bagged cones collected from Söregärde and Vuolenkoski in 2020 were poorly de- veloped and identified as aecial primordia. All the aecial primordia samples from these bagged cones had only one allele at all SSR loci,     |  1749ZHANG et Al. T A B L E 3   Su m m ar y of a lle lic r es ul ts f or e ac h si m pl e- se qu en ce r ep ea t (S SR ) l oc us in s am pl es o f T he ko ps or a ar eo la ta a ec ia c ol le ct ed in 2 01 5, 2 01 9 an d 20 20 , a nd in t he t ot al p op ul at io n SS R 20 15 20 19 20 20 To ta l A lle le n o. H ex pa Ev en ne ss A lle le n o. H ex p Ev en ne ss A lle le n o. H ex p Ev en ne ss A lle le n o. H ex p Ev en ne ss SS R1 6 0. 57 1 0. 59 1 4 0. 61 2 0. 74 6 5 0. 58 3 0. 64 9 7 0. 60 1 0. 56 6 SS R2 4 3 0. 49 7 0. 86 9 3 0. 49 6 0. 82 1 4 0. 55 1 0. 79 3 4 0. 54 0 0. 84 8 SS R4 9 0. 67 1 0. 71 4 6 0. 74 6 0. 79 3 6 0. 69 6 0. 76 6 11 0. 70 5 0. 70 2 SS R5 4 0. 58 6 0. 81 9 6 0. 60 0 0. 75 9 4 0. 56 7 0. 79 1 7 0. 60 7 0. 80 8 SS R1 3 6 0. 65 2 0. 66 0 8 0. 75 1 0. 73 0 6 0. 69 9 0. 73 6 9 0. 75 5 0. 71 0 Th a9 3 0. 25 0 0. 58 8 3 0. 40 6 0. 72 2 3 0. 30 5 0. 66 0 3 0. 31 8 0. 65 0 SS R1 0 7 0. 73 8 0. 76 0 5 0. 57 6 0. 67 3 7 0. 75 8 0. 79 3 8 0. 77 9 0. 80 2 Th a9 1 6 0. 61 8 0. 72 1 5 0. 64 5 0. 72 2 4 0. 57 5 0. 69 9 7 0. 62 0 0. 65 2 Th a6 1 3 0. 18 3 0. 47 8 3 0. 09 1 0. 40 1 6 0. 29 6 0. 43 1 8 0. 19 0 0. 36 5 SS R1 6 3 0. 27 4 0. 63 7 3 0. 36 7 0. 62 4 3 0. 36 2 0. 64 8 3 0. 32 8 0. 62 2 SS R1 8 6 0. 62 2 0. 74 6 5 0. 72 3 0. 83 6 6 0. 41 5 0. 47 7 6 0. 63 6 0. 68 5 Th a9 2 2 0. 16 2 0. 55 2 1 na na 2 0. 07 6 0. 45 4 2 0. 09 1 0. 47 2 SS R1 9 3 0. 30 9 0. 61 5 3 0. 52 5 0. 86 1 3 0. 46 3 0. 68 2 3 0. 44 9 0. 71 2 Th a9 6 11 0. 58 2 0. 51 3 10 0. 64 8 0. 57 4 6 0. 66 8 0. 73 8 14 0. 63 2 0. 54 9 Th a1 36 7 0. 27 6 0. 40 5 5 0. 24 2 0. 41 5 5 0. 24 4 0. 43 8 10 0. 25 7 0. 36 8 SS R2 2 3 0. 18 7 0. 52 4 3 0. 36 2 0. 69 6 5 0. 47 9 0. 54 8 5 0. 33 0 0. 51 0 Th a1 05 9 0. 83 9 0. 81 3 9 0. 78 5 0. 74 9 9 0. 82 7 0. 82 8 12 0. 85 6 0. 84 3 Th a1 37 2 0. 00 9 0. 30 8 2 0. 03 5 0. 38 7 2 0. 01 3 0. 32 8 2 0. 01 8 0. 34 5 M ea n 5. 16 7 0. 44 6 0. 62 9 4. 66 7 0. 47 9 0. 67 7 4. 77 8 0. 47 7 0. 63 6 6. 72 2 0. 48 4 0. 62 3 N ot e: T he a na ly si s w as p er fo rm ed w ith c lo ne - c or re ct ed d at a fr om 5 48 s am pl es . a E xp ec te d he te ro zy go si ty . 1750  |    ZHANG et Al. and all samples from the same cone belonged to the same haploid MLG (Figures 1 and 3). 3.4  |  Haplotype inference The inferred number of haplotypes included both the haplotypes of basidiospores that infected the cones and the haplotypes of the spermatia that mated with the receptive hyphae (Figure 1). 3.4.1  |  Variation between years High numbers of haplotypes (HD of 1.86 and 1.48) were inferred from each exposed cone in Swedish seed orchards in 2015 (Figure 1), when there was high disease incidence. This indicated that all cones were infected by multiple basidiospores and/or received multiple spermatia. In 2019 and 2020, with low disease incidence, the number of haplotypes in exposed cones varied. For example, four exposed cones in Suhola 2019 and 2020 had as many as 16– 18 haplotypes TA B L E 4  Population diversity of Thekopsora areolata at the level of collection year Year (disease severity) Na MLGb eMLGc SEc Hd Ge Evenness 2015 (high) 128 115 115.0 0.00 4.69 99.9 0.915 2019 (low) 140 71 66.6 1.59 3.88 32.6 0.668 2020 (low) 210 73 54.7 2.92 3.85 34.7 0.733 Total 478 259 93.7 4.32 5.16 112.2 0.644 Note: The analysis was performed on samples from exposed cones. aNumber of samples. bNumber of multilocus genotypes. cExpected number of MLGs and standard error at the smallest sample size (128) based on rarefaction. dShannon– Wiener index. eStoddart and Taylor's index. F I G U R E 1  Number of multilocus genotypes (MLGs) of Thekopsora areolata aecia and inferred number of haplotypes of T. areolata in each infected cone of Norway spruce collected from four seed orchards in 2015, 2019 and 2020. The haploid diversity (HD) is represented by average haplotype number per aecium. Bagged cones are labelled with asterisks. The three bagged cones in 2020 only produced haploid aecial primordia     |  1751ZHANG et Al. (Figure 1, Table S2). However, eight exposed cones from 2019 and 2020 were dominated by only one MLG and two haplotypes (Figure 1, Table S2). On average, samples from 2019 and 2020 had lower haplotype diversity (HD <1) than those of 2015 (HD = 1.86 and 1.48). 3.4.2  |  Variation between exposed and bagged cones The number of haplotypes per cone, seed orchard and year varied from 2 to 20 in exposed cones and from 1 to 8 in bagged cones (Figure 1, Table S2). The haplotype diversity of samples from four infected bagged cones from Suhola in 2019 (HD = 0.53) was lower than that of samples from the exposed cones (HD = 0.93). Three to eight haplotypes were inferred from these bagged cones (Figure 1, Table S2), which indicated that multiple basidiospores with differ- ent haplotypes had already infected the pistillate cones before the bagging. Only one haplotype was inferred from each of the bagged cones from 2020, which was supported by the observation that only aecial primordia were found in these cones. 3.5  |  Microscopy of aecial primordia in infected bagged cones In infected cones of T. areolata with mature aecia, the aecia that de- veloped on the inner/adaxial side of the scale were hemispherical or polygonal, reddish- brown to dark brown, and with a smooth surface (Figure 4a). The three cones in bags collected in 2020 only devel- oped aecial primordia, and aeciospores were not observed. These early- stage structures were hemispherical or polygonal, 0.8– 1.2 mm in diameter, 80– 100 μm high, disk- shaped, white (Figure 4b), and sometimes light brown because of the coverage of Norway spruce seed wings (Figure 4c,d). Aecial primordia were surrounded by white mycelia (Figure 4b,d). Cross- sections of the aecial primordia showed a dense layer of fungal basal cells growing closely to the cone scale epidermis. Chains of swollen fertile cells were produced above the basal cells and covered by a gelatinous matrix (Figure 4e– h). After DAPI staining of aecial primordia, only one nucleus could be found in each fungal hyphal cell (Figure 4i– l). 4  |  DISCUSSION In this study, 10 new polymorphic SSR markers were developed for T. areolata based on its genome sequence. Most of the SSR markers TA B L E 5  Population diversity of Thekopsora areolata at the level of collection year and Norway spruce seed orchard Year Seed orchard Na MLGb eMLGc SEc Hd Ge Evenness 2015 Ålbrunna 48 47 47.00 0.000 3.84 46.08 0.988 Söregärde 80 68 43.07 1.581 4.14 56.14 0.889 2019 Suhola 60 32 27.41 1.485 3.13 15.79 0.673 Ålbrunna 80 39 26.80 2.093 3.24 17.11 0.658 2020 Suhola 80 31 22.21 1.943 2.95 13.39 0.682 Söregärde 70 33 25.93 1.782 3.17 17.88 0.737 Vuolenkoski 60 9 8.73 0.476 2.01 6.95 0.918 Total 478 259 41.64 2.252 5.16 112.22 0.644 Note: The analysis is based on samples from exposed cones. aNumber of samples. bNumber of multilocus genotypes. cExpected number of MLGs and standard error at the smallest sample size (48) based on rarefaction. dShannon– Wiener index. eStoddart and Taylor's index. F I G U R E 2  Rarefaction curve of Thekopsora areolata aecia collected from four seed orchards in 2015, 2019 and 2020. Vertical line indicates minimum sample size, horizontal lines indicate expected number of multilocus genotypes (MLGs) with the minimum sample size 1752  |    ZHANG et Al. were highly polymorphic, with 2– 14 alleles per marker identified in the studied populations. The genotype accumulation curve sug- gested a high resolution of MLGs: 276 MLGs were identified from the 548 samples; the genotype accumulation showed a linear in- crease for the first few loci, and later a plateau was reached. In a previous study (Capador et al., 2018) using eight polymorphic SSR markers, one MLG was found from different cones from three seed orchards, and another four samples were homozygous at all tested loci (Capador et al., 2020). However, in the present study, individual aecia that shared the same MLG were always from the same cone, that is, no MLGs were found twice from different cones. The four samples that were homozygous for the eight SSR markers in the pre- vious study, were shown to be heterozygous at two to five loci when tested with the new expanded set of SSR markers (data not shown). Results from aecia samples investigated in this study agree with the previous study by Capador et al. (2020) that the Ålbrunna 2015 population had high genotypic diversity and multiple basidiospore in- fection in the same cone. The Söregärde 2015 population, which was not included in the previous study, showed a similar level of diversity as well as multiple basidiospore infections. The T. areolata popula- tions had low genotypic diversity in 2019 and even lower diversity in 2020 in all seed orchards in this study. In both years, cherry spruce rust incidence in seed orchards was usually low and infected cones were scarce to find as samples. In Finland, the low disease incidence in 2019 was due to a low Prunus infection rate on overwintered leaves and poor flowering (Kaitera, Aarnio, Karhu, et al., 2021). In 2020, the weather conditions were unfavourable for the spread of rust, and the flowering of P. abies was also poor (authors' personal observations). The lower disease incidence and low genotypic diver- sity of aecia is most probably a result of infection by a limited num- ber of basidiospores with very few haplotypes. However, there is still a possibility that cones could be infected by many basidiospores but the basidiospores shared fewer haplotypes, such as occurred in Suhola 2020: the amount of cones harvested from the seed orchard was extremely low despite a high infection rate, and T. areolata di- versity was low. This could be explained by population bottlenecks: according to the life cycle of T. areolata, if any biotic or abiotic factor caused a lower infection rate by basidiospores on Norway spruce cones in 2018 or by aeciospores on Prunus in 2019, fewer alleles and haplotypes would be inherited by basidiospores in 2020. Thorough monitoring of T. areolata haplotype diversity and infection rate on Norway spruce and Prunus will help to investigate their association. F I G U R E 3  Multilocus genotype (MLG) frequency of Thekopsora areolata aecia in exposed and bagged cones from Norway spruce seed orchards in Suhola in 2019, Söregärde in 2020 and Vuolenkoski in 2020. Each number on the x axis indicates one MLG     |  1753ZHANG et Al. F I G U R E 4  Microscopy of cone scales of Norway spruce infected with Thekopsora areolata showing normal and abnormal fruiting bodies. (a) Typical infected scale with mature hemispherical aecia. (b– d) Infected scales with abnormal flat fruiting bodies surrounded by white hyphae, from cones collected in Sweden (b) and Finland (c,d), bar = 2 mm. (e– h) Cross- section of abnormal fruiting bodies, stained with lactophenol cotton blue. (i– l), 4′,6- diamidino- 2- phenylindole (DAPI) staining of hyphae from aecial primordial structures in bright field and fluorescence, showing only one nucleus in each fungal cell (arrows indicate septa) [Colour figure can be viewed at wileyonlinelibrary.com] 1754  |    ZHANG et Al. The extremely low genotypic diversity of the Vuolenkoski 2020 population is unique compared to the other studied seed orchards. Only two to four haplotypes were identified for each exposed cone, indicating that each cone was only infected by basidiospores of one or two haplotypes and fertilized with spermatia of one or two haplo- types. One explanation could be the young age of the seed orchard that created a shortage of genetic material and a lack of genetic variation. This was supported by the low observed number of old infected cones in the seed orchard indicating that previous rust ep- idemics were rare in Vuolenkoski before 2020 (J. Kaitera, unpub- lished observations). In macrocyclic rust fungi, sexual reproduction is initiated by the plasmogamy of spermatia and receptive hyphae in the aecial host, and sexual recombination of genes occurs during karyogamy and meiosis in teliospores in the telial host (Figueroa et al., 2020; Rodriguez- Algaba et al., 2014). Accordingly, the mating of T. areolata occurs in Norway spruce, and the recombination of gene alleles occurs in bird cherry. The Vuolenkoski seed orchard was established in 2014, and due to its young age and long interval of flowering of spruce, only one cycle of mating and recombination of the fungus might have been completed between spruces and bird cherry surrounding the seed orchard. Many rust fungi produce spermatia in sugary nectar to attract in- sects, which act as vectors to transfer spermatia, such as in Puccinia spp. (Leonard & Szabo, 2005; Naef et al., 2002). T. areolata sperma- tia in fluid have frequently been reported (Kaitera, Aarnio, Karhu, et al., 2021; Murray, 1955) or observed by researchers over time, but the contribution of insects in the mating process has not been con- firmed experimentally. Cone bagging has previously been tested to inhibit the T. areolata basidiospore infection of spruce cones (Kaitera et al., 2009b). In the present study, cones were bagged before the production of spermogonia, and therefore, these cones were isolated while the other exposed cones were visited by insects. Only a limited number of bagged infected cones were collected in this study, and the genotypic diversity of aecia and aecial primordia from bagged cones was lower than that of aecia from exposed cones in the same seed orchard. In 2019, bagged cones were infected by basidiospores of multiple haplotypes: gravity or shaking of the branch may have caused dripping of spermatia nectar within the bag, enabling com- patible haplotypes to mate (Figure S4, case3). In exposed cones from the same seed orchard, cones were usually infected by more basid- iospore haplotypes, and subsequently mated with more spermatia haplotypes. This study was not able to prove conclusively that insect vectors contributed to the higher diversity of aecia in exposed cones because the ability of insects to carry T. areolata spermatia was not directly tested. However, alien spermatia increase aecia diversity in cones and basidiospore infection in isolated cones results in either no aecia or aecia with reduced diversity, confirming the significance of cross- fertilization in sexual reproduction. In 2020, only aecial pri- mordia of one haplotype were identified from all bagged cones. This indicated that each of these cones was only infected by one basid- iospore, or several basidiospores of the same haplotype. Because no spermatia of other haplotypes were available, sexual reproduc- tion never occurred. Therefore, alien spermatia can be critical for production of aecia; whether the spread of spermatia usually occurs via insects (Naef et al., 2002) or other factors such as water splashes (Tadych et al., 2014) requires further study to determine. T. areolata aecial primordia have never been described in detail although they have been reported earlier (Kaitera et al., 2009a). Infected cones with mature aecia are usually easy to identify in trees because the cone scales open prematurely (Kaitera et al., 2009a; Murray, 1955) and their appearance is already clearly distinguish- able from healthy cones by late summer (Kaitera, Aarnio, Karhu, et al., 2021). In this study, the scales of cones with aecial primordia remained closed, similar to healthy cones, but these structures could be found by pulling up the scales of the cone. Thus, aecial primordia are cryptic and hard to locate in exposed cones in seed orchards. For this reason, population genetics statistics were not performed for bagged cones and exposed cones collected in 2020 due to the risk of biased sampling. However, aecial primordia are more likely to be discovered when the cones are isolated in bags, because, unlike exposed cones infected with a single haplotype, they are less likely to receive alien spermatia, possibly by attracting insects with sper- matial fluid, which produce mature aecia after sexual reproduction. The absence of homozygous aecia and the presence of aecial primordia in bagged cones collected in Vuolenkoski and Söregärde in 2020 suggested that T. areolata is a heterothallic species and self- fertilization is unlikely to happen. In Pucciniales, the mating system of Puccinia spp., Melampsora spp. and Cronartium spp. are better un- derstood. All these species are heterothallic according to the sper- matia transfer experiment (Craigie, 1927) and/or genetic structure of mating- type genes (Duplessis et al., 2011). Their mating processes are controlled by one locus or two unlinked loci (Kües et al., 2011). Future characterization of the gene structure of mating- type loci will provide more information about the mating system of T. areolata. In the previous study by Capador et al. (2020), it was proposed that basidiospore infections were local and vegetative spreading oc- curred only after dikaryotization/plasmogamy because only a few dominant MLGs were found throughout the cones. In this study, some cones were found to be colonized by several MLGs in dif- ferent parts of the cones, and these MLGs shared one haplotype (Figure S4, case3). Similar cases were also observed in Cronatium pini and Puccinia spp.: different MLGs that share the same haplotype were often found within one aecial cluster (Berlin et al., 2017; Samils et al., 2011). It is possible that the cones in our study were infected by several basidiospores' haplotypes in the spring, and these hap- lotypes were fertilized by spermatia of the same haplotype in early summer. Another possible scenario is that these cones were infected by one basidiospore, after which the monokaryotic hyphae grew vegetatively to colonize the whole cone, and hyphae with the same haplotype were fertilized by different spermatia. Strong evidence for the vegetative growth of monokaryotic hyphae before dikaryoti- zation was that one homogeneous haplotype was found throughout the same bagged cone in 2020. In conclusion, this research increased our understanding of the infection, colonization, and sexual reproduction of T. areolata by population genetics analyses based on 18 polymorphic SSR markers.     |  1755ZHANG et Al. In the year with lower disease incidence, cones were infected by fewer fungal haplotypes, that is, fewer basidiospores and/or sper- matia, which resulted in lower genotypic diversity of aecia. In the youngest seed orchard, cones were infected by basidiospores of very few haplotypes, probably because of low disease prevalence in the local rust population. Mature aecia in cones isolated in bags had lower diversity than aecia in exposed cones, which suggested that transfer of spermatia by vectors promotes genotypic diversity of the rust fungus. Homogenous aecial primordia of T. areolata that occupy the entire cones were reported, and homothallic mating of the same gametes is less likely to occur in T. areolata. ACKNOWLEDG EMENTS We thank Siemen Forelia Oy, Sveaskog, Södra skogsägarna and Stora Enso for the use of their seed orchards for the fieldwork and sample collections of this study. We also thank Hernán Capador for shar- ing samples and data, and Olle Rosenberg for sharing the historical disease incidence data from Swedish seed orchards. This project was funded by The Royal Swedish Academy of Agriculture and Forestry (KSLA) under Tandem Forest Values research program, grant num- ber TFV 2018- 0001, and the Swedish Research Council FORMAS (grant 2017- 01489). The authors have no conflicts of interest to de- clare. Open access funding enabled and organized by ProjektDEAL. DATA AVAIL ABILIT Y S TATEMENT The data that support the findings of this study are available from the corresponding author upon reasonable request. ORCID Ke Zhang https://orcid.org/0000-0003-4204-1516 R E FE R E N C E S Alexopoulos, C.J., Mims, C.W. & Blackwell, M. (1996) Introductory mycol- ogy, 4th edition. 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