Multilocus Genotyping and Intergenic Spacer Single Nucleotide Polymorphisms of Amylostereum areolatum (Russulales: Amylostereacea) Symbionts of Native and Non-Native Sirex Species

Sirex noctilio along with its mutualistic fungal symbiont, Amylostereum areolatum (a white rot fungus), is an invasive pest that causes excessive damage to Pinus plantations in Northeast China. In 2015, S. noctilio were found to attack Pinus sylvestris var. mongolica, and often share larval habitat with the native woodwasp, S. nitobei. The objective of this study was to determine the possible origin(s) of the introduced pest complex in China and analyse the genetic diversity between A. areolatum isolated from invasive S. noctilio, native S. nitobei and other woodwasps collected from Europe (native range) and other countries. Phylogenetic analyses were performed using the intergenic spacer (IGS) dataset and the combined 4-locus dataset (the internal transcribed spacer region (ITS), translation elongation factor alpha 1 (tef1), DNA-directed ribosomal polymerase II (RPB2), and mitochondrial small subunit (mtSSU)) of three Amylostereum taxa. The multilocus genotyping of nuclear ribosomal regions and protein coding genes revealed at least three distinct multilocus genotypes (MLGs) of the fungus associated with invasive S. noctilio populations in Northeast China, which may have come from North America or Europe. The IGS region of A. areolatum carried by S. noctilio from China was designated type B1D2. Our results showed a lack of fidelity (the paradigm of obligate fidelity to a single fungus per wasp species) between woodwasp hosts and A. areolatum. We found that the native S. nitobei predominantly carried A. areolatum IGS-D2, but a low percentage of females instead carried A. areolatum IGS-B1D2 (MLG A13), which was presumably due to horizontal transmission from S. noctilio, during the sequential use of the same wood for larval development. The precise identification of the A. areolatum genotypes provides valuable insight into co-evolution between Siricidae and their symbionts, as well as understanding of the geographical origin and history of both Sirex species and their associated fungi.


Sample Collection and Symbiont Isolation
Amylostereum samples were collected from Sirex females between 2017 and 2020 from five sites, namely, Jinbaotun (JBT), Yushu (YS), Linyi (LY), Dumeng (DM), and Hegang (HG) (Figure 1a). The sampling sites also included the sites of co-occurrence (JBT and YS) of S. noctilio and S. nitobei in Northeast China. Data for 71 Sirex females (female wasps carry the fungus in internal mycangia) were sources for the Amylostereum species and genotypes were used in the present study.
Living Sirex samples were collected using two methods: (1) mature P. sylvestris var. mongolica, P. tabuliformis, and P. thunbergii with signs of infestation (e.g., resin beads in Figure 1b) were cut down in spring or early summer; portions of the trees were placed in individual mesh-bags under ambient conditions. Logs were cut into 70-cm long bolts, and the ends were waxed to prevent contamination by other wood-rot fungi, and to conserve moisture. The S. noctilio and S. nitobei that emerged from the bolts were collected daily. For method (2), samples were collected weekly from lure-based black panel traps. collected from pine trees in other continents attacked by S. noctilio. Here, we investigated the geographical origin of A. areolatum clonal lineages in the areas of Northeast China recently colonized by the S. noctilio-A. areolatum complex. We believe that such studies would help determine the extent of the level of specificity between the symbiont and the host.

Sample Collection and Symbiont Isolation
Amylostereum samples were collected from Sirex females between 2017 and 2020 from five sites, namely, Jinbaotun (JBT), Yushu (YS), Linyi (LY), Dumeng (DM), and Hegang (HG) (Figure 1a). The sampling sites also included the sites of co-occurrence (JBT and YS) of S. noctilio and S. nitobei in Northeast China. Data for 71 Sirex females (female wasps carry the fungus in internal mycangia) were sources for the Amylostereum species and genotypes were used in the present study.
Living Sirex samples were collected using two methods: (1) mature P. sylvestris var. mongolica, P. tabuliformis, and P. thunbergii with signs of infestation (e.g., resin beads in Figure 1b) were cut down in spring or early summer; portions of the trees were placed in individual mesh-bags under ambient conditions. Logs were cut into 70-cm long bolts, and the ends were waxed to prevent contamination by other wood-rot fungi, and to conserve moisture. The S. noctilio and S. nitobei that emerged from the bolts were collected daily. For method (2), samples were collected weekly from lure-based black panel traps. S. noctilio causes damage to P. sylvestris var. mongolica and sometimes co-infests trees with S. nitobei, although emergence of the relatively short-lived adult females of these two species is often separated by at least a month in Northeast China [14]. Sirex species were identified using the morphological method (the color of male abdomen and female legs) described by Schiff et al. [31]. Fungal symbiont samples could be collected only from adult female Sirex, and mycangia were removed using tweezers; the spore masses were transferred aseptically to Petri dishes with potato dextrose agar (PDA), following the method reported by Thomsen [23]. To establish fungal cultures, the contents of mycangium were transferred to a Petri dish containing PDA supplemented with antibiotics (300 mg/L streptomycin sulfate), whereas subsequent cultures were performed S. noctilio causes damage to P. sylvestris var. mongolica and sometimes co-infests trees with S. nitobei, although emergence of the relatively short-lived adult females of these two species is often separated by at least a month in Northeast China [14]. Sirex species were identified using the morphological method (the color of male abdomen and female legs) described by Schiff et al. [31]. Fungal symbiont samples could be collected only from adult female Sirex, and mycangia were removed using tweezers; the spore masses were transferred aseptically to Petri dishes with potato dextrose agar (PDA), following the method reported by Thomsen [23]. To establish fungal cultures, the contents of mycangium were transferred to a Petri dish containing PDA supplemented with antibiotics (300 mg/L streptomycin sulfate), whereas subsequent cultures were performed on PDA without antibiotics. Cultures were incubated in the dark at 25 • C ± 1 • C for two weeks.

Colony Polymerase Chain Reaction (PCR) and Sequencing
Fifty microliter of lysis buffer (the Lysis Buffer for Microorganism to Direct PCR, Takara) was added to each 1.5-mL microtube. Single colonies were picked using sterile pipette tips, stirred in a microtube for about five seconds and then removed. Pipette tips were not kept in the microtube for too long to avoid effects on the lysate volume and PCR amplification. After thermal denaturation at 80 • C for 15 min (ThermoUnit, CHB-100, Shanghai, China), the supernatant was centrifuged (Thermo Fresco21, 75002425, Shanghai, China) at a low speed (3000 r/3 min), and 1-5 µL of the supernatant was taken as the template for PCR. The PCR reaction mix (T100 Thermal Cycler, Beijing, China) (25 µL) is presented in Table 1, and primer sequences are presented in Table 2. Extracted DNA was stored at 4 • C until use.

Multilocus Genotyping
Electrophoresis was performed to examine the amplified products ( Figure 2). The PCR amplicons were sent to Beijing Ruibo Biotech Co., Ltd. (Beijing, China) and sequenced using the ABI Prism™ 3730 × l automated DNA sequencer (Applied Biosystems USA, Foster City, California). Although the internal transcribed spacer region (ITS), translation elongation factor alpha 1 (tef1), DNA-directed ribosomal polymerase II (RPB2), and mitochondrial small subunit (mtSSU) data were obtained for all species, the four loci could not always be amplified for the same isolates. DNA sequences of the amplicons generated by forward and reverse primers were used to obtain consensus sequences by using SeqMan version 7.1.0 in DNAStar Lasergene Core Suite software (DNAStar Inc., Madison, WI, USA). Sequences were aligned using ClustalW [36] with default parameters and manually adjusted using a BioEdit Sequence Alignment Editor [37]. BLAST searches were performed in GenBank to identify related sequences. The degree of mutational saturation was evaluated using the substitution saturation index I ss in DAMBE version 6 [38][39][40]. The estimates of I ss were lower than the critical value I ss.c for all datasets (p < 0.05). All unique sequence data were submitted to GenBank (ITS: OL307781, OL307782; tef1: OL410536, OL410537; RPB2: OL410538-OL410540; mtSSU: OL323050, OL323051).

Fragment Analysis of Intergenic Spacer (IGS) Types
The nuc-IGS-rDNA region between the nuclear large subunit (LSU) and the 5S gene of ribosomal RNA (rRNA) operon was amplified using PCR primers, specific for the basidiomycetes P-1 [41] and 5S-2B [42]. All the isolates identified (by looking for oidia in culture [43,44]) as A. areolatum were verified through direct sequencing with Applied Biosystems 3730×l DNA Analyzer (Foster City, CA, USA) at Beijing Ruibo Biotech Co., Ltd., and most isolates identified as A. chailletii were also verified through sequencing. The nuc-IGS-rDNA region may be present in A. areolatum as multiple copies. The primer IGS-intF (5′-GTTTCTTAGGGCTGTTCCAGACTTGTG-3′) included a 7-bp "pigtail" (GTTTCTT) in the 5′ end. The primer 5S-2B was labeled with a FAM fluorescent marker [45]. PCR for fragment analysis was run under the following conditions: one cycle at 94 °C for 4 min; 35 cycles of 94 °C for 50 s, 55 °C for 45 s, and 72 °C for 45 s; a final extension at 72 °C for 10 min. The PCR temperature was maintained at 4 °C until gel visualization. Samples were mixed with formamide and LIZ500 size standard and then electrophoresed with the Applied Biosystems 3730×l DNA Analyzer (Foster City, CA, USA) at Beijing Ruibo Biotech Co., Ltd.; fragment sizes were determined using GeneMarke version 2.2.0 (SoftGenetics LLC) ( Figure S1).
In case of heterozygosity (at least one heterozygous site) or length heterogeneity, cloning was performed. To interpret the double product amplified for some isolates of A. areolatum and to obtain sequence for the whole amplified fragment, secondary PCR products were cloned for sequencing. PCR fragments were purified using the pCloneEZ-NRS-Omni AMP/HC Cloning Kit. Then, it was ligated to the TOPO-cloning vector and inserted into chemically competent cells of Escherichia coli. Positive colonies containing the insert were screened through PCR by using the TOPO-F and TOPO-R primers. Cloned products were precipitated and purified using the aforementioned method and sequenced using the primers TOPO-F (5′-GAGCCAGTGAGTTGATTGTG-3′) and TOPO-R (5′-CAGGAAACAGCTATGACC-3′).

Data Analysis
Heterozygous positions were coded using degenerate bases, according to the IUPAC-IUB nomenclature. For the phylogenetic analysis, the five newly generated sequences were combined with 17 publicly available sequences (Table 3). A. chailletii and A. laevigatum were selected as outgroups. Bayesian inference (BI) in combination with MrBayes helped in partitioning the combined datasets and concomitantly applying an independent model of evolution to each partition, because unlinked genes often have different evolutionary constraints. The combined dataset was divided into four unlinked partitions (ITS, tef1, RBB2, and mtSSU). BI analysis was performed under the Kimura 2parameter model (Kimura, 1981) plus invariant sites (K2P + I) [46]: ITS + RPB2; Kimura-2- Figure 2. Visualization of the amplification of the four genes (the internal transcribed spacer region (ITS), translation elongation factor alpha 1 (tef1), DNA-directed ribosomal polymerase II (RPB2), and mitochondrial small subunit (mtSSU)) of Amylostereum isolates from Sirex, on a 1.5% agarose gel stained with ethidium bromide.

Fragment Analysis of Intergenic Spacer (IGS) Types
The nuc-IGS-rDNA region between the nuclear large subunit (LSU) and the 5S gene of ribosomal RNA (rRNA) operon was amplified using PCR primers, specific for the basidiomycetes P-1 [41] and 5S-2B [42]. All the isolates identified (by looking for oidia in culture [43,44]) as A. areolatum were verified through direct sequencing with Applied Biosystems 3730×l DNA Analyzer (Foster City, CA, USA) at Beijing Ruibo Biotech Co., Ltd., and most isolates identified as A. chailletii were also verified through sequencing. The nuc-IGS-rDNA region may be present in A. areolatum as multiple copies. The primer IGS-intF (5 -GTTTCTTAGGGCTGTTCCAGACTTGTG-3 ) included a 7-bp "pigtail" (GTTTCTT) in the 5 end. The primer 5S-2B was labeled with a FAM fluorescent marker [45]. PCR for fragment analysis was run under the following conditions: one cycle at 94 • C for 4 min; 35 cycles of 94 • C for 50 s, 55 • C for 45 s, and 72 • C for 45 s; a final extension at 72 • C for 10 min. The PCR temperature was maintained at 4 • C until gel visualization. Samples were mixed with formamide and LIZ500 size standard and then electrophoresed with the Applied Biosystems 3730×l DNA Analyzer (Foster City, CA, USA) at Beijing Ruibo Biotech Co., Ltd.; fragment sizes were determined using GeneMarke version 2.2.0 (SoftGenetics LLC) ( Figure S1).
In case of heterozygosity (at least one heterozygous site) or length heterogeneity, cloning was performed. To interpret the double product amplified for some isolates of A. areolatum and to obtain sequence for the whole amplified fragment, secondary PCR products were cloned for sequencing. PCR fragments were purified using the pCloneEZ-NRS-Omni AMP/HC Cloning Kit. Then, it was ligated to the TOPO-cloning vector and inserted into chemically competent cells of Escherichia coli. Positive colonies containing the insert were screened through PCR by using the TOPO-F and TOPO-R primers. Cloned products were precipitated and purified using the aforementioned method and sequenced using the primers TOPO-F (5 -GAGCCAGTGAGTTGATTGTG-3 ) and TOPO-R (5 -CAGGAAACAGCTATGACC-3 ).

Data Analysis
Heterozygous positions were coded using degenerate bases, according to the IUPAC-IUB nomenclature. For the phylogenetic analysis, the five newly generated sequences were combined with 17 publicly available sequences (Table 3). A. chailletii and A. laevigatum were selected as outgroups. Bayesian inference (BI) in combination with MrBayes helped in partitioning the combined datasets and concomitantly applying an independent model of evolution to each partition, because unlinked genes often have different evolutionary constraints. The combined dataset was divided into four unlinked partitions (ITS, tef1, RBB2, and mtSSU). BI analysis was performed under the Kimura 2-parameter model (Kimura, 1981) plus invariant sites (K2P + I) [46]: ITS + RPB2; Kimura-2-parameter (K2P): tef1; and the likelihood model Felsenstein 1981 plus fixed (empirical) and invariant sites (Felsenstein 1981) (F81 + F + I) [47]: mtSSU nucleotide substitution models, which were determined through ModelFinder by using the Bayesian information criterion (BIC) implemented in PhyloSuite 1.21 [48]. Stationarity (chain convergence) was accessed by examining the average standard deviations of split frequencies (0.002246). Maximum likelihood (ML) phylogenetic trees were constructed using IQ-TREE [49], implemented in PhyloSuite 1.21, with 10,000 replicates of ultrafast [50] bootstrap (UFBoot) and 1000 replicates of the Shimodaira-Hasegawa-like approximate-likelihood ratio test (SH-aLRT) [51]. Nodes receiving ML bootstrap values of ≥70% and Bayesian posterior probabilities more than 0.95 were considered significantly supported. As asexual reproduction is the predominant form of reproduction in A. areolatum, genetic analyses were conducted with a clone-corrected dataset. The relatedness of haplotype sequences to A. areolatum strains and their specificity to native or non-native Sirex species (Table S1) were analyzed through hierarchical clustering analysis (Ward method) in SAS-JMP pro version 16.0.0 software (SAS Institute Inc., Cary, NC, USA); each haplotype was scored as present (1) or absent (0). The haplotype of global A. areolatum (IGS region) distributions was mapped using POPART [52].  1 Each letter represents a unique sequence for each locus, and sequence data for representative strains with different types were deposited to GenBank.

Phylogenetic Relationship in Amylostereum
The final multilocus dataset utilized for ML and BI analyses consisted of 2116 characters (ITS: 574 characters; tef1: 345 characters; RPB2: 684 characters; mtSSU: 513 characters). Of all, 1935 characters were constant, 51 characters were variable and parsimony-uninformative, and 130 characters were parsimony-informative. The topology of the ML tree was mostly congruent with that of the BI tree, and the tree derived by ML was shown in Figure 3. In the A. areolatum clade, a subclade of multilocus genotypes (MLGs), A13, A14, and A15, associated with the non-native S. noctilio-A. areolatum from China was well supported. For IGS-D (a subclade consisting of MLGs), A4, A16, and A17 from Japan and China were the native isolates. Three strains of A. areolatum collected from S. noctilio in China had identical mtSSU and ITS loci ( Table 3).
The parts of A. areolatum samples from the mycangia of S. noctilio that provided insufficient amounts of DNA for sequencing all four loci were excluded from the analysis. A total of 5 MLGs (MLGs A13-17, unique sequence) were observed in all 26 A. areolatum isolates from China (Table 3), which suggested the occurrence of at least five clonal lineages of A. areolatum in Northeast China. These MLGs did not match perfectly with those observed in other continents.

Phylogenetic Relationship in Amylostereum
The final multilocus dataset utilized for ML and BI analyses consisted of 2116 characters (ITS: 574 characters; tef1: 345 characters; RPB2: 684 characters; mtSSU: 513 characters). Of all, 1935 characters were constant, 51 characters were variable and parsimony-uninformative, and 130 characters were parsimony-informative. The topology of the ML tree was mostly congruent with that of the BI tree, and the tree derived by ML was shown in Figure 3. In the A. areolatum clade, a subclade of multilocus genotypes (MLGs), A13, A14, and A15, associated with the non-native S. noctilio-A. areolatum from China was well supported. For IGS-D (a subclade consisting of MLGs), A4, A16, and A17 from Japan and China were the native isolates. Three strains of A. areolatum collected from S. noctilio in China had identical mtSSU and ITS loci ( Table 3).
The parts of A. areolatum samples from the mycangia of S. noctilio that provided insufficient amounts of DNA for sequencing all four loci were excluded from the analysis. A total of 5 MLGs (MLGs A13-17, unique sequence) were observed in all 26 A. areolatum isolates from China (Table 3), which suggested the occurrence of at least five clonal lineages of A. areolatum in Northeast China. These MLGs did not match perfectly with those observed in other continents. Based on the sequences of ITS, mtSSU, RPB2, and tef1, the A. areolatum samples were grouped into 17 MLGs (Table 3, Figure 3). Ten of these MLGs had only one sample, whereas the remaining MLGs consisted of 2-16 samples. A1, the most widespread MLG, was observed in the S. juvencus, S. noctilio, Urocerus albicornis, and U. gigas samples collected from Denmark, Hungary, or Spain. The other two widespread MLGs, A5 and A15, were also detected; A5 was observed in the S. juvencus and S. noctilio samples collected from five countries, including Europe, the United States, and Australia, whereas A15 was detected in the S. noctilio sample only collected from China, which is a unique and dominant haplotype not found elsewhere in the rest of the world. Comparison between DNA sequences of these MLGs, and those of the samples from pine trees invaded by S. noctilio in other countries, revealed that the MLGs A13, A14 and A15 (China) were highly similar to the MLG A8 reported by Castrillo et al. (2015) in the US and the MLG A7 reported in European samples. The two strains of A. areolatum (MLGs A16 and A17) collected from S. nitobei in China revealed identical RPB2 and mtSSU loci, and alignments showed the presence of these gene haplotypes in the Japanese samples (MLG A4) (Table 3, Figure 3).

Haplotype Relationships Based on IGS Sequences and Fragment Analysis Data
Two different sequences (hereafter referred to as sequence B and D) for the nuc-IGS-rDNA region were observed in the A. areolatum isolates from China. The heterogeneity in the IGS region sequences of the A. areolatum isolates was not observed in those of the A. chailletii isolates. The IGS region of A. areolatum sequences was obtained from 18 strains, with each strain carrying a single haplotype. According to a panel of rDNA intergenic spacer-single nucleotide polymorphisms (haplotype-specific markers) focused on the nucleotide position in the range from 206 to 276, those were designated as type D2 (Supplementary Figure S2) [53]. Fragment analysis of these strains also revealed only one peak of approximately 470 bp. The remaining 28 of the A. areolatum strains had heterogenic sequences, and their combination haplotypes were determined to be type B1D2 Cluster analysis of the relatedness of haplotype sequences for A. areolatum strains and their specificity to native or non-native Sirex species (Figure 4, Supplementary Table S1) showed that the haplotype D2 was associated with the native S. nitobei isolates (M1 and L29) in China. IGS-BE and E were found only in A. areolatum from S. nigricornis (native to North America). Other haplotypes were found in A. areolatum, associated with S. noctilio. The haplotype D2 was consistently associated with the haplotype B1 found in A. areolatum from non-native S. noctilio (except for the isolate GR94-11_IGS-B1D1 from New York, United States). The loci of ITS and mtSSU consistent with IGS haplotype were observed in all A. areolatum isolates (D3, D18, D10, M1 and L29) from China (Table 3, Figure 4).  Table S1, unique sequence), and relatedness of haplotypes in the Amylostereum areolatum isolates from native and non-native Sirex specimens, from multiple locations in China. The plot arranges the A. areolatum isolates as endpoints, and each cluster join as a new point, whereas the lines represent membership in a cluster. The length of a line between cluster joins approximates the distance between the clusters that were joined, where X-and Y-axes enable comparison of the relative distance between clusters; the longer the lines, the greater the distance between the clusters. The axis scaling, orientation of points, and angles of the lines on the constellation plot are arbitrary, with no assigned unit in SAS-JMP pro version 16.0.0.

Co-Infestation of S. noctilio and S. nitobei in Pinus
Among the Sirex species that emerged, S. noctilio and S. nitobei co-occurred in 41.   Table S1, unique sequence), and relatedness of haplotypes in the Amylostereum areolatum isolates from native and non-native Sirex specimens, from multiple locations in China. The plot arranges the A. areolatum isolates as endpoints, and each cluster join as a new point, whereas the lines represent membership in a cluster. The length of a line between cluster joins approximates the distance between the clusters that were joined, where X-and Y-axes enable comparison of the relative distance between clusters; the longer the lines, the greater the distance between the clusters. The axis scaling, orientation of points, and angles of the lines on the constellation plot are arbitrary, with no assigned unit in SAS-JMP pro version 16.0.0.

Co-Infestation of S. noctilio and S. nitobei in Pinus
Among the Sirex species that emerged, S. noctilio and S. nitobei co-occurred in 41.4% of the 29 pine trees. For the 12 trees where co-occurrence within sections of trees could be determined, 74.7% (n = 269) of S. nitobei emerged from sections of the trees where S. noctilio emerged, and 41.2% (n = 466) of S. noctilio emerged from sections of the trees where S. nitobei emerged. S. nitobei mainly carried either IGS-D2 A. areolatum or A. chailletii. All S. noctilio females emerging from P. sylvestris var. mongolica tree carried A. areolatum IGS-B1D2 in different sites (DM, HG, JBT, YS) ( Figure 5). Two female S. nitobei from the same sections of co-infested trees carried A. areolatum IGS-B1D2 in JBT (Table 4).

Discussion
S. noctilio was first found in the samples collected from Northeast China in 2013 [57]. Based on COI sequence, Sun et al. (2020) have reported the genetic diversity and structure of S. noctilio populations [58]. In some regions, S. noctilio and S. nitobei can infest the same trees [14]. To better understand the phylogenetic relationships within A. areolatum in China, we analyzed sequences from 27 strains (59%) of A. areolatum isolates from sympatric distributions, plus 19 strains (41%) of isolates from independent distributions. Isolates from the native and exotic countries were included to determine the patterns of geographical distribution and the origin of the introduced pest species in China.

Multilocus Genotyping and IGS Heterogeneity of A. areolatum
Multilocus sequencing of the A. areolatum isolates revealed three distinct S. noctilioassociated A. areolatum MLGs in Northeast China (Table 3), which have not been detected in S. noctilio-native countries. The MLGs A13, A14, and A15 exhibited sequence similarities with A8 (AH1-17) and A7 (B1385) reported by Castrillo et al. (2015), indicating that multiple S. noctilio species might have been introduced to China from Europe or North America (Figure 3, Table 3) [59]. These species originated probably from unrepresented source populations. Our results are consistent with the patterns of multiple invasions and the spread of S. noctilio across China [58]. Additional samples and heterogeneous genetic markers for assessing the population genetics of A. areolatum are required to determine whether these introductions occurred either through a direct route from Europe, or indirectly from North America, or a combination of both routes.
In addition to the observation of fragment size differences among the A. areolatum IGS rRNA sequences, we also paid attention to single nucleotide polymorphisms (SNPs). Two different-sized PCR products indicated the presence of a heterogenic sequence in the

Discussion
S. noctilio was first found in the samples collected from Northeast China in 2013 [57]. Based on COI sequence, Sun et al. (2020) have reported the genetic diversity and structure of S. noctilio populations [58]. In some regions, S. noctilio and S. nitobei can infest the same trees [14]. To better understand the phylogenetic relationships within A. areolatum in China, we analyzed sequences from 27 strains (59%) of A. areolatum isolates from sympatric distributions, plus 19 strains (41%) of isolates from independent distributions. Isolates from the native and exotic countries were included to determine the patterns of geographical distribution and the origin of the introduced pest species in China.

Multilocus Genotyping and IGS Heterogeneity of A. areolatum
Multilocus sequencing of the A. areolatum isolates revealed three distinct S. noctilioassociated A. areolatum MLGs in Northeast China (Table 3), which have not been detected in S. noctilio-native countries. The MLGs A13, A14, and A15 exhibited sequence similarities with A8 (AH1-17) and A7 (B1385) reported by Castrillo et al. (2015), indicating that multiple S. noctilio species might have been introduced to China from Europe or North America ( Figure 3, Table 3) [59]. These species originated probably from unrepresented source populations. Our results are consistent with the patterns of multiple invasions and the spread of S. noctilio across China [58]. Additional samples and heterogeneous genetic markers for assessing the population genetics of A. areolatum are required to determine whether these introductions occurred either through a direct route from Europe, or indirectly from North America, or a combination of both routes.
In addition to the observation of fragment size differences among the A. areolatum IGS rRNA sequences, we also paid attention to single nucleotide polymorphisms (SNPs). Two different-sized PCR products indicated the presence of a heterogenic sequence in the IGS of the nuclear rDNA locus in the A. areolatum isolates. This finding was confirmed by cloning and sequencing these fragments. We found only one genotype of A. areolatum in Northeast China was associated with S. noctilio, that is, the heterogeneous strain (BD), in contrast with the invasion of the southern hemisphere (AB). The strain carried the B sequence, previously reported in Europe and the southern hemisphere [42], along with the D sequence. IGS-BD is widely distributed across five continents ( Figure 6). The homogeneous strain (D) of A. areolatum associated with S. nitobei, which is found only in the northern hemisphere, was equivalent to an isolate obtained from S. nitobei in Japan (B1395) [59].
Northeast China was associated with S. noctilio, that is, the heterogeneous strain (BD), in contrast with the invasion of the southern hemisphere (AB). The strain carried the B sequence, previously reported in Europe and the southern hemisphere [42], along with the D sequence. IGS-BD is widely distributed across five continents ( Figure 6). The homogeneous strain (D) of A. areolatum associated with S. nitobei, which is found only in the northern hemisphere, was equivalent to an isolate obtained from S. nitobei in Japan (B1395) [59].

Sirex noctilio and S. nitobei-A. areolatum Association
In this study, S. nitobei was found to carry A. areolatum-D, which were all collected from outside the known geographic repartition of S. noctilio (in Linyi), while a very small percentage of individuals carried A. areolatum-BD in sympatric distribution (S. noctilio and S. nitobei) and shared the same MLG A13 with S. noctilio. Moreover, S. noctilio and S. nitobei were sometimes observed as co-occurring on P. sylvestris var. mongolica, thereby providing the potential for horizontal transmission (S. noctilio share larval habitat with the native woodwasp when infesting the same trees) of fungal symbionts from sympatric S. noctilio. In addition, studies have shown the evolution of associations between these woodwasps and fungal symbionts allows plasticity [60][61][62]. Ann et al. (2018) found the spillover of A. areolatum from invasive S. noctilio to native Urocerus spp. [63], and the horizontal transmission of fungal strains from S. noctilio to S. nigricornis [45] in North America. In Spain, the newly introduced U. albicornis (well known to be associated with A. chailletii) was found to carry a European strain, A. areolatum [63]. Introduced fungi would definitely have vectored to new trees during oviposition by new woodwasp hosts; thus, horizontal transmission in associations would assist in the establishment and spread of the newly introduced wood-decay fungus.

Sirex noctilio and S. nitobei-A. areolatum Association
In this study, S. nitobei was found to carry A. areolatum-D, which were all collected from outside the known geographic repartition of S. noctilio (in Linyi), while a very small percentage of individuals carried A. areolatum-BD in sympatric distribution (S. noctilio and S. nitobei) and shared the same MLG A13 with S. noctilio. Moreover, S. noctilio and S. nitobei were sometimes observed as co-occurring on P. sylvestris var. mongolica, thereby providing the potential for horizontal transmission (S. noctilio share larval habitat with the native woodwasp when infesting the same trees) of fungal symbionts from sympatric S. noctilio. In addition, studies have shown the evolution of associations between these woodwasps and fungal symbionts allows plasticity [60][61][62]. Ann et al. (2018) found the spillover of A. areolatum from invasive S. noctilio to native Urocerus spp. [63], and the horizontal transmission of fungal strains from S. noctilio to S. nigricornis [45] in North America. In Spain, the newly introduced U. albicornis (well known to be associated with A. chailletii) was found to carry a European strain, A. areolatum [63]. Introduced fungi would definitely have vectored to new trees during oviposition by new woodwasp hosts; thus, horizontal transmission in associations would assist in the establishment and spread of the newly introduced wood-decay fungus.
According to Slippers et al. (2002) [42], the divergence of A. areolatum can be attributed to the obligate relationship of A. areolatum with its insect vectors, and to the predominance of asexual reproduction compared with other Amylostereum species. This study shows that the A. areolatum (MLG A15) isolates from China, collected over four years (from 2017 to 2020), represent a lineage of MLG A15 linked with the IGS-BD. There is no sequence variation in the nuclear IGS rRNA region. The results indicated that vegetative reproduction in its symbiosis with S. noctilio was the predominant or only form of reproduction in A. areolatum in northern China. Basidiocarps of A. areolatum have not been reported in these areas. In the ecology of Amylostereum spp., sexual reproduction could allow genetic recombination, leading to polymorphisms. However, asexual reproduction of A. areolatum and vertical transmission across generations by the woodwasp vector would result in the extensive spread of cloned fungus [64]. The sample size of the present study was insufficient for fully assessing the population genetics of the A. areolatum associated with S. noctilio in China. The close similarity of A14 and A15 from S. noctilio with A13 from S. noctilio and S. nitobei, and its associated IGS type in Northeast China, warrants further examination of A. areolatum genotypes associated with more native siricid species and additional European and North American samples.

Focus for Further Research
Symbiont fidelity is the main mechanism in the evolution and stability of mutualisms. Heterogenic sequences in the nuc-IGS-rDNA region of A. areolatum isolates make it possible to compare and characterize populations of these fungi that are associated with different wasp species. The occurrence and combination of these sequences provides insight into both the geographical distribution and evolutionary relationships of populations of fungus. Effective IPM strategies rely on determining the species and genetic variants present in a given population or region, especially where both native and invasive species are coinfesting. This study can fill in this knowledge gap by characterizing the population structure of A. areolatum in Northeast China. Our understanding of the A. areolatum population structure is limited, which further impedes the development and application of effective IPM approaches. Identification studies utilizing DNA sequence data on a large number of isolates from native areas, where diverse wasp species/populations co-exist, would be valuable. Such studies would make it possible to determine the extent of the levels of specificity between the symbiont and host. Funding: This research was funded by Chinese National Natural Science Foundation (31870642) and Beijing's Science and Technology Planning Project "Z201100008020001".