TY - CONF AU - Karaffa, Levente AU - Flipphi, Michel Johannes Anthonie AU - Márton, Alexandra AU - Bíró, Vivien AU - Bakondi-Kovács, István AU - Ág-Rácz, Viktória AU - Ág, Norbert AU - Fekete, Erzsébet TI - Second Alternative Oxidase Genes in Aspergillaceae: Genesis, Loss and Mutations T2 - The 20th International Aspergillus Meeting,Asperfest20 Program and Abstract Book PY - 2024 SP - 26 EP - 27 PG - 2 UR - https://m2.mtmt.hu/api/publication/34751176 ID - 34751176 AB - Alternative oxidase (Aox) is a terminal oxidase in branched mitochondrial electron transport that provides a non-electrogenic alternative to canonical cytochrome-mediated electron flow, bypassing the proton- pumping complexes III and IV. The consequence of the direct transfer of electrons from ubiquinol to oxygen without concomitant build up of proton motive force is the uncoupling of ATP synthesis via oxidative phosporylation from NADH reoxidation, to allow carbon catabolism to continue unabated even when ATP demand is low or when non-carbon nutrients become limiting. Thus, Aox plays an important role in the energetics of overflow metabolism-based bioprocesses such as Aspergillus niger citric acid fermentation and Aspergillus terreus itaconate production. Aox (aoxA gene) is near ubiquitous in the fungal kingdom, but coexistence of multiple aox genes is rare. However, a second aox gene (aoxB) is present in some taxa of Aspergillaceae. Paralogous genes generally originate from duplication and inherit vertically; we provide evidence for four independent duplication events at different points in evolution that resulted in aoxB paralogs in contemporary Aspergilli and Penicillia. The paralog in A. niger has a different origin than the paralog in A. terreus, while a third independently formed paralog is found in A. wentii. All paralogous clades arise from original aoxA parent genes but never replace them. Few species have accumulated three co-expressed aox genes. Therefore, loss of once acquired paralogs co-determines contemporary aox gene content in individual species. For instance, section Fumigati has lost all its transient paralogs. In the subgenus Nidulantes, we identified seven independent occasions of aoxB gene loss and two gains. In A. calidoustus, both more ancient aoxB paralogs present in the last common ancestor of the subgenus have been substituted by two other aoxB genes of completely distinct origins. We found that the paralogous aoxB gene in some 75 genome-sequenced A. niger strains features variation at a level not detected for the ubiquitous aoxA gene. Five mutations were identified that plausibly affect transcription, function, or terminally modify the gene product. A full-length AoxB is encoded in the acid producer ATCC 1015. Hence, the A. niger sensu stricto complex can be subdivided into six taxa according to the resident aoxB allele. To date, confident separation could only be accomplished after comparative analyses of whole genome sequences. LA - English DB - MTMT ER - TY - GEN AU - Karaffa, Levente AU - Flipphi, Michel Johannes Anthonie AU - Márton, Alexandra AU - Bíró, Vivien AU - Bakondi-Kovács, István AU - Ág-Rácz, Viktória AU - Ág, Norbert AU - Fekete, Erzsébet TI - Second Alternative Oxidase Genes in Aspergillaceae: Genesis, Loss and Mutations CY - Asperfest20 PY - 2024 UR - https://m2.mtmt.hu/api/publication/34751163 ID - 34751163 AB - Alternative oxidase (Aox) is a terminal oxidase in branched mitochondrial electron transport that provides a non-electrogenic alternative to canonical cytochrome-mediated electron flow, bypassing the proton- pumping complexes III and IV. The consequence of the direct transfer of electrons from ubiquinol to oxygen without concomitant build up of proton motive force is the uncoupling of ATP synthesis via oxidative phosporylation from NADH reoxidation, to allow carbon catabolism to continue unabated even when ATP demand is low or when non-carbon nutrients become limiting. Thus, Aox plays an important role in the energetics of overflow metabolism-based bioprocesses such as Aspergillus niger citric acid fermentation and Aspergillus terreus itaconate production. Aox (aoxA gene) is near ubiquitous in the fungal kingdom, but coexistence of multiple aox genes is rare. However, a second aox gene (aoxB) is present in some taxa of Aspergillaceae. Paralogous genes generally originate from duplication and inherit vertically; we provide evidence for four independent duplication events at different points in evolution that resulted in aoxB paralogs in contemporary Aspergilli and Penicillia. The paralog in A. niger has a different origin than the paralog in A. terreus, while a third independently formed paralog is found in A. wentii. All paralogous clades arise from original aoxA parent genes but never replace them. Few species have accumulated three co-expressed aox genes. Therefore, loss of once acquired paralogs co-determines contemporary aox gene content in individual species. For instance, section Fumigati has lost all its transient paralogs. In the subgenus Nidulantes, we identified seven independent occasions of aoxB gene loss and two gains. In A. calidoustus, both more ancient aoxB paralogs present in the last common ancestor of the subgenus have been substituted by two other aoxB genes of completely distinct origins. We found that the paralogous aoxB gene in some 75 genome-sequenced A. niger strains features variation at a level not detected for the ubiquitous aoxA gene. Five mutations were identified that plausibly affect transcription, function, or terminally modify the gene product. A full-length AoxB is encoded in the acid producer ATCC 1015. Hence, the A. niger sensu stricto complex can be subdivided into six taxa according to the resident aoxB allele. To date, confident separation could only be accomplished after comparative analyses of whole genome sequences. LA - English DB - MTMT ER - TY - CONF AU - Karaffa, Levente AU - Bíró, Vivien AU - Márton, Alexandra AU - Bakondi-Kovács, István AU - Nicholas, Geoffrion AU - Adrian, Tsang AU - Christian, P. Kubicek AU - Fekete, Erzsébet TI - Transcriptome analysis of manganese(II) ion depletion during high-yield citric acid fermentation in Aspergillus niger T2 - The 20th International Aspergillus Meeting,Asperfest20 Program and Abstract Book PY - 2024 SP - 27 EP - 27 PG - 1 UR - https://m2.mtmt.hu/api/publication/34751135 ID - 34751135 AB - Structural and Functional Genomics, Concordia University, Montreal, QC, Canada, 3Institute of Chemical, Environmental and Bioscience Engineering, TU Wien, Vienna, Austria Citric acid (citrate) is amongst the most important bulk products of industrial biotechnology. The discovery of its accumulation by the filamentous fungus Aspergillus niger led to the development of a large-scale submerged fermentation process employing stirred tank reactors, which today accounts for 95% of the world’s production. High-yield citric acid production requires a combination of unusual nutritional conditions, of which establishing a low (<5 ppb) manganese(II) ion concentration at the onset of the fermentation is a key feature. Changes in the metabolism of A. niger prompted by Mn(II)-deficiency has not been investigated on a functional genomics level so far. In order to get an insight into how Mn(II) deficiency triggers citric acid accumulation, we compared the transcriptome of the citric acid hyperproducer A. niger NRRL 2270 strain at three time points (24-hour, 48-hour, and 72-hour) at Mn(II)-deficient (=5 ppb) and Mn(II)-sufficient (=100 ppb) fermentation conditions. This experimental design emanates from our previous observation that the effect of Mn(II) ions on citric acid accumulation is dependent on the cultivation time: it has the strongest effect at the onset of the cultivation and then gradually decreases (Fekete et al., 2022). Comparison of the transcriptomes of Mn(II)-deficient and Mn(II)-sufficient fermentation conditions revealed that 1436 transcripts are differentially regulated (log2> 2 at p <0.05). Of these, 629 transcripts are upregulated and 807 transcripts are downregulated under Mn(II)-deficient condition. Of the transcripts that displayed differential regulation only at the 24-hour timepoint, 101 transcripts were upregulated and 101 were downregulated. Among the 133 transcripts that were upregulated at all three timepoints under Mn(II)-deficient condition, the majority (= 97) transcripts are predicted to encode secreted or membrane-bound proteins. Notably, cexA, encoding the major citrate exporter in A. niger (Reinfurt et al., 2023), was upregulated 75-, 15- and 2-fold at the three respective timepoints of cultivation under Mn(II)-deficient condition. LA - English DB - MTMT ER - TY - CONF AU - Karaffa, Levente AU - Flipphi, Michel Johannes Anthonie AU - Márton, Alexandra AU - Bíró, Vivien AU - Bakondi-Kovács, István AU - Ág-Rácz, Viktória AU - Ág, Norbert AU - Fekete, Erzsébet TI - Second Alternative Oxidase Genes in Aspergillaceae: Genesis, Loss and Mutations T2 - 32nd Fugal Genetics Conference Abstract Book PY - 2024 SP - 300 EP - 301 PG - 2 UR - https://m2.mtmt.hu/api/publication/34750649 ID - 34750649 AB - Alternative oxidase (Aox) is a terminal oxidase in branched mitochondrial electron transport that provides a non-electrogenic alternative to canonical cytochrome-mediated electron flow, bypassing the proton-pumping complexes III and IV. The consequence of the direct transfer of electrons from ubiquinol to oxygen without concomitant build up of proton motive force is the uncoupling of ATP synthesis via oxidative phosporylation from NADH reoxidation, to allow carbon catabolism to continue unabated even when ATP demand is low or when non-carbon nutrients become limiting. Thus, Aox plays an important role in the energetics of overflow metabolism-based bioprocesses such as Aspergillus niger citric acid fermentation and Aspergillus terreus itaconate production. Aox (aoxA gene) is near ubiquitous in the fungal kingdom, but coexistence of multiple aox genes is rare. However, a second aox gene (aoxB) is present in some taxa of Aspergillaceae. Paralogous genes generally originate from duplication and inherit vertically; we provide evidence for four independent duplication events at different points in evolution that resulted in aoxB paralogs in contemporary Aspergilli and Penicillia. The paralog in A. niger has a different origin than the paralog in A. terreus, while a third independently formed paralog is found in A. wentii. All paralogous clades arise from original aoxA parent genes but never replace them. Few species have accumulated three co-expressed aox genes. Therefore, loss of once acquired paralogs co-determines contemporary aox gene content in individual species. For instance, section Fumigati has lost all its transient paralogs. In the subgenus Nidulantes, we identified seven independent occasions of aoxB gene loss and two gains. In A. calidoustus, both more ancient aoxB paralogs present in the last common ancestor of the subgenus have been substituted by two other aoxB genes of completely distinct origins. We found that the paralogous aoxB gene in some 75 genome-sequenced A. niger strains features variation at a level not detected for the ubiquitous aoxA gene. Five mutations were identified that plausibly affect transcription, function, or terminally modify the gene product. A full-length AoxB is encoded in the acid producer ATCC 1015. Hence, the A. niger sensu stricto complex can be subdivided into six taxa according to the resident aoxB allele. To date, confident separation could only be accomplished after comparative analyses of whole genome sequences. LA - English DB - MTMT ER - TY - CONF AU - Fekete, Erzsébet AU - Ág, Norbert AU - Ág-Rácz, Viktória AU - Márton, Alexandra AU - Bíró, Vivien AU - Flipphi, Michel Johannes Anthonie AU - Karaffa, Levente TI - Continual propagation of [D1,2] stwintrons in divergent Xylariales T2 - 32nd Fugal Genetics Conference Abstract Book PY - 2024 SP - 158 EP - 158 PG - 1 UR - https://m2.mtmt.hu/api/publication/34750608 ID - 34750608 AB - Spliceosomal twin introns consist of two nested U2 introns excised consecutively. In a [D1,2] stwintron, an internal intron interrupts the 5’-donor of an external intron between the 1st and 2nd nucleotide (nt) (5’-G 1|U 2). For Hypoxylon sp. CO27-5, one can classify [D1,2]’s in two groups. Of these, sequence-similar “sister” stwintrons cross-identify by blastn, and occur at new intron positions in narrow taxa (species, variants). When reciprocal blastn screens were performed in genomes of other Xylariales species–using proven CO27-5 sister stwintrons as primary queries–258 new sequence-similar stwintrons were revealed in 12 species. Some species contain > 50 sister stwintrons, others < 10. All of them are integrated seamlessly in seemingly random exonic sequences, excluding transposon-driven mechanisms or splice-site co-option for their propagation. One observes essentially species-specific clades of sister stwintrons in maximum likelihood trees, implying vertical transmission of sequence-diverging duplication- competent [D1,2]’s. Xylaria sp. MSU SB201401 and X. striata RK1-1 are intimately related–like strains of the same species–albeit isolated from very different plant species, growing on different continents. 4 stwintrons unique to MSU SB201401 are phylogenetically clustered and the 3 most similar ones are >99 % identical, while the genes harboring them are completely unrelated in sequence, intron-exon structure and function. This lineage involves consecutive strain-specific stwintron duplication events, arguably the most recent duplications in our set. Although the continuous 11-nt near-terminal inverted repeat in these 4 MSU SB201401 stwintrons is not as long as those in prototypical CO27-5 sisters, the near-terminal stem structure is the stand-out common feature. We propose that this stem structure can bring the donor G1 of the internal- and the acceptor G3 of the external intron in close proximity, necessary for the rare duplication of [D1,2] stwintrons into double-stranded DNA breaks, including those with smaller or less perfect terminal inverted repeats. We also identified one potential lateral transfer: one MSU SB201401 stwintron is >93 % sequence-identical with two different [D1,2]’s in Xylariaceae sp. FL1651. The 3 genes harboring them are completely unrelated. This may imply that duplication-competent stwintrons can be (re)acquired by lateral transfer. Such rare events could contibute to the periodicity of overall (stw)intron gain and loss. LA - English DB - MTMT ER - TY - CONF AU - Fekete, Erzsébet AU - Ág, Norbert AU - Ág-Rácz, Viktória AU - Márton, Alexandra AU - Bíró, Vivien AU - Flipphi, Michel Johannes Anthonie AU - Karaffa, Levente TI - Continual propagation of [D1,2] stwintrons in divergent Xylariales T2 - 32nd Fugal Genetics Conference Abstract Book PY - 2024 SP - 24 EP - 24 PG - 1 UR - https://m2.mtmt.hu/api/publication/34750415 ID - 34750415 AB - Spliceosomal twin introns consist of two nested U2 introns excised consecutively. In a [D1,2] stwintron, an internal intron interrupts the 5’-donor of an external intron between the 1st and 2nd nucleotide (nt) (5’-G 1|U 2). For Hypoxylon sp. CO27-5, one can classify [D1,2]’s in two groups. Of these, sequence-similar “sister” stwintrons cross-identify by blastn, and occur at new intron positions in narrow taxa (species, variants). When reciprocal blastn screens were performed in genomes of other Xylariales species–using proven CO27-5 sister stwintrons as primary queries–258 new sequence-similar stwintrons were revealed in 12 species. Some species contain > 50 sister stwintrons, others < 10. All of them are integrated seamlessly in seemingly random exonic sequences, excluding transposon-driven mechanisms or splice-site co-option for their propagation. One observes essentially species-specific clades of sister stwintrons in maximum likelihood trees, implying vertical transmission of sequence-diverging duplication- competent [D1,2]’s. Xylaria sp. MSU SB201401 and X. striata RK1-1 are intimately related–like strains of the same species–albeit isolated from very different plant species, growing on different continents. 4 stwintrons unique to MSU SB201401 are phylogenetically clustered and the 3 most similar ones are >99 % identical, while the genes harboring them are completely unrelated in sequence, intron-exon structure and function. This lineage involves consecutive strain-specific stwintron duplication events, arguably the most recent duplications in our set. Although the continuous 11-nt near-terminal inverted repeat in these 4 MSU SB201401 stwintrons is not as long as those in prototypical CO27-5 sisters, the near-terminal stem structure is the stand-out common feature. We propose that this stem structure can bring the donor G1 of the internal- and the acceptor G3 of the external intron in close proximity, necessary for the rare duplication of [D1,2] stwintrons into double-stranded DNA breaks, including those with smaller or less perfect terminal inverted repeats. We also identified one potential lateral transfer: one MSU SB201401 stwintron is >93 % sequence-identical with two different [D1,2]’s in Xylariaceae sp. FL1651. The 3 genes harboring them are completely unrelated. This may imply that duplication-competent stwintrons can be (re)acquired by lateral transfer. Such rare events could contibute to the periodicity of overall (stw)intron gain and loss. LA - English DB - MTMT ER - TY - GEN AU - Karaffa, Levente AU - Bíró, Vivien AU - Márton, Alexandra AU - Bakondi-Kovács, István AU - Nicholas, Geoffrion AU - Adrian, Tsang AU - Christian, P. Kubicek AU - Fekete, Erzsébet TI - Transcriptome analysis of manganese(II) ion depletion during high-yield citric acid fermentation in Aspergillus niger CY - March 11-12, 2024 PY - 2024 UR - https://m2.mtmt.hu/api/publication/34748228 ID - 34748228 AB - Citric acid (citrate) is amongst the most important bulk products of industrial biotechnology. The discovery of its accumulation by the filamentous fungus Aspergillus niger led to the development of a large-scale submerged fermentation process employing stirred tank reactors, which today accounts for 95% of the world’s production. High-yield citric acid production requires a combination of unusual nutritional conditions, of which establishing a low (<5 ppb) manganese(II) ion concentration at the onset of the fermentation is a key feature. Changes in the metabolism of A. niger prompted by Mn(II)-deficiency has not been investigated on a functional genomics level so far. In order to get an insight into how Mn(II) deficiency triggers citric acid accumulation, we compared the transcriptome of the citric acid hyperproducer A. niger NRRL 2270 strain at three time points (24-hour, 48-hour, and 72-hour) at Mn(II)-deficient (=5 ppb) and Mn(II)-sufficient (=100 ppb) fermentation conditions. This experimental design emanates from our previous observation that the effect of Mn(II) ions on citric acid accumulation is dependent on the cultivation time: it has the strongest effect at the onset of the cultivation and then gradually decreases (Fekete et al., 2022). Comparison of the transcriptomes of Mn(II)-deficient and Mn(II)-sufficient fermentation conditions revealed that 1436 transcripts are differentially regulated (log2> 2 at p <0.05). Of these, 629 transcripts are upregulated and 807 transcripts are downregulated under Mn(II)-deficient condition. Of the transcripts that displayed differential regulation only at the 24-hour timepoint, 101 transcripts were upregulated and 101 were downregulated. Among the 133 transcripts that were upregulated at all three timepoints under Mn(II)-deficient condition, the majority (= 97) transcripts are predicted to encode secreted or membrane-bound proteins. Notably, cexA, encoding the major citrate exporter in A. niger (Reinfurt et al., 2023), was upregulated 75-, 15- and 2-fold at the three respective timepoints of cultivation under Mn(II)-deficient condition. LA - English DB - MTMT ER - TY - GEN AU - Karaffa, Levente AU - Flipphi, Michel Johannes Anthonie AU - Márton, Alexandra AU - Bíró, Vivien AU - Bakondi-Kovács, István AU - Ág-Rácz, Viktória AU - Ág, Norbert AU - Fekete, Erzsébet TI - Second Alternative Oxidase Genes in Aspergillaceae: Genesis, Loss and Mutations CY - Wednesday, March 13, 2024 | 7:00 PM - 10:30 PM PY - 2024 UR - https://m2.mtmt.hu/api/publication/34747301 ID - 34747301 AB - Alternative oxidase (Aox) is a terminal oxidase in branched mitochondrial electron transport that provides a non-electrogenic alternative to canonical cytochrome-mediated electron flow, bypassing the proton-pumping complexes III and IV. The consequence of the direct transfer of electrons from ubiquinol to oxygen without concomitant build up of proton motive force is the uncoupling of ATP synthesis via oxidative phosporylation from NADH reoxidation, to allow carbon catabolism to continue unabated even when ATP demand is low or when non-carbon nutrients become limiting. Thus, Aox plays an important role in the energetics of overflow metabolism-based bioprocesses such as Aspergillus niger citric acid fermentation and Aspergillus terreus itaconate production. Aox (aoxA gene) is near ubiquitous in the fungal kingdom, but coexistence of multiple aox genes is rare. However, a second aox gene (aoxB) is present in some taxa of Aspergillaceae. Paralogous genes generally originate from duplication and inherit vertically; we provide evidence for four independent duplication events at different points in evolution that resulted in aoxB paralogs in contemporary Aspergilli and Penicillia. The paralog in A. niger has a different origin than the paralog in A. terreus, while a third independently formed paralog is found in A. wentii. All paralogous clades arise from original aoxA parent genes but never replace them. Few species have accumulated three co-expressed aox genes. Therefore, loss of once acquired paralogs co-determines contemporary aox gene content in individual species. For instance, section Fumigati has lost all its transient paralogs. In the subgenus Nidulantes, we identified seven independent occasions of aoxB gene loss and two gains. In A. calidoustus, both more ancient aoxB paralogs present in the last common ancestor of the subgenus have been substituted by two other aoxB genes of completely distinct origins. We found that the paralogous aoxB gene in some 75 genome-sequenced A. niger strains features variation at a level not detected for the ubiquitous aoxA gene. Five mutations were identified that plausibly affect transcription, function, or terminally modify the gene product. A full-length AoxB is encoded in the acid producer ATCC 1015. Hence, the A. niger sensu stricto complex can be subdivided into six taxa according to the resident aoxB allele. To date, confident separation could only be accomplished after comparative analyses of whole genome sequences. LA - English DB - MTMT ER - TY - GEN AU - Fekete, Erzsébet AU - Ág, Norbert AU - Ág-Rácz, Viktória AU - Márton, Alexandra AU - Bíró, Vivien AU - Flipphi, Michel Johannes Anthonie AU - Karaffa, Levente TI - Continual propagation of [D1,2] stwintrons in divergent Xylariales CY - Wednesday, March 13, 2024 | 7:00 PM - 10:30 PM PY - 2024 UR - https://m2.mtmt.hu/api/publication/34746992 ID - 34746992 AB - Spliceosomal twin introns consist of two nested U2 introns excised consecutively. In a [D1,2] stwintron, an internal intron interrupts the 5’-donor of an external intron between the 1st and 2nd nucleotide (nt) (5’-G1|U2). For Hypoxylon sp. CO27-5, one can classify [D1,2]’s in two groups. Of these, sequence-similar “sister” stwintrons cross-identify by blastn, and occur at new intron positions in narrow taxa (species, variants). When reciprocal blastn screens were performed in genomes of other Xylariales species–using proven CO27-5 sister stwintrons as primary queries–258 new sequence-similar stwintrons were revealed in 12 species. Some species contain > 50 sister stwintrons, others < 10. All of them are integrated seamlessly in seemingly random exonic sequences, excluding transposon-driven mechanisms or splice-site co-option for their propagation. One observes essentially species-specific clades of sister stwintrons in maximum likelihood trees, implying vertical transmission of sequence-diverging duplication-competent [D1,2]’s. Xylaria sp. MSU SB201401 and X. striata RK1-1 are intimately related–like strains of the same species–albeit isolated from very different plant species, growing on different continents. 4 stwintrons unique to MSU SB201401 are phylogenetically clustered and the 3 most similar ones are >99 % identical, while the genes harboring them are completely unrelated in sequence, intron-exon structure and function. This lineage involves consecutive strain-specific stwintron duplication events, arguably the most recent duplications in our set. Although the continuous 11-nt near-terminal inverted repeat in these 4 MSU SB201401 stwintrons is not as long as those in prototypical CO27-5 sisters, the near-terminal stem structure is the stand-out common feature. We propose that this stem structure can bring the donor G1 of the internal- and the acceptor G3 of the external intron in close proximity, necessary for the rare duplication of [D1,2] stwintrons into double-stranded DNA breaks, including those with smaller or less perfect terminal inverted repeats. We also identified one potential lateral transfer: one MSU SB201401 stwintron is >93 % sequence-identical with two different [D1,2]’s in Xylariaceae sp. FL1651. The 3 genes harboring them are completely unrelated. This may imply that duplication-competent stwintrons can be (re)acquired by lateral transfer. Such rare events could contibute to the periodicity of overall (stw)intron gain and loss. LA - English DB - MTMT ER - TY - GEN AU - Fekete, Erzsébet AU - Ág, Norbert AU - Ág-Rácz, Viktória AU - Márton, Alexandra AU - Bíró, Vivien AU - Flipphi, Michel Johannes Anthonie AU - Karaffa, Levente TI - Continual propagation of [D1,2] stwintrons in divergent Xylariales CY - Wednesday, March 13, 2024 5:20 PM - 5:40 PM PY - 2024 UR - https://m2.mtmt.hu/api/publication/34746327 ID - 34746327 AB - Spliceosomal twin introns consist of two nested U2 introns excised consecutively. In a [D1,2] stwintron, an internal intron interrupts the 5’-donor of an external intron between the 1st and 2nd nucleotide (nt) (5’-G1|U2). For Hypoxylon sp. CO27-5, one can classify [D1,2]’s in two groups. Of these, sequence-similar “sister” stwintrons cross-identify by blastn, and occur at new intron positions in narrow taxa (species, variants). When reciprocal blastn screens were performed in genomes of other Xylariales species–using proven CO27-5 sister stwintrons as primary queries–258 new sequence-similar stwintrons were revealed in 12 species. Some species contain > 50 sister stwintrons, others < 10. All of them are integrated seamlessly in seemingly random exonic sequences, excluding transposon-driven mechanisms or splice-site co-option for their propagation. One observes essentially species-specific clades of sister stwintrons in maximum likelihood trees, implying vertical transmission of sequence-diverging duplication-competent [D1,2]’s. Xylaria sp. MSU SB201401 and X. striata RK1-1 are intimately related–like strains of the same species–albeit isolated from very different plant species, growing on different continents. 4 stwintrons unique to MSU SB201401 are phylogenetically clustered and the 3 most similar ones are >99 % identical, while the genes harboring them are completely unrelated in sequence, intron-exon structure and function. This lineage involves consecutive strain-specific stwintron duplication events, arguably the most recent duplications in our set. Although the continuous 11-nt near-terminal inverted repeat in these 4 MSU SB201401 stwintrons is not as long as those in prototypical CO27-5 sisters, the near-terminal stem structure is the stand-out common feature. We propose that this stem structure can bring the donor G1 of the internal- and the acceptor G3 of the external intron in close proximity, necessary for the rare duplication of [D1,2] stwintrons into double-stranded DNA breaks, including those with smaller or less perfect terminal inverted repeats. We also identified one potential lateral transfer: one MSU SB201401 stwintron is >93 % sequence-identical with two different [D1,2]’s in Xylariaceae sp. FL1651. The 3 genes harboring them are completely unrelated. This may imply that duplication-competent stwintrons can be (re)acquired by lateral transfer. Such rare events could contibute to the periodicity of overall (stw)intron gain and loss. LA - English DB - MTMT ER -