Mutations in the Second Alternative Oxidase Gene: A New Approach to Group Aspergillus niger Strains

Alternative oxidase is a terminal oxidase in the branched mitochondrial electron transport chain of most fungi including Aspergillus niger (subgenus Circumdati, section Nigri). A second, paralogous aox gene (aoxB) is extant in some A. niger isolates but also present in two divergent species of the subgenus Nidulantes—A. calidoustus and A. implicatus—as well as in Penicillium swiecickii. Black aspergilli are cosmopolitan opportunistic fungi that can cause diverse mycoses and acute aspergillosis in immunocompromised individuals. Amongst the approximately 75 genome-sequenced A. niger strains, aoxB features considerable sequence variation. Five mutations were identified that rationally affect transcription or function or terminally modify the gene product. One mutant allele that occurs in CBS 513.88 and A. niger neotype strain CBS 554.65 involves a chromosomal deletion that removes exon 1 and intron 1 from aoxB. Another aoxB allele results from retrotransposon integration. Three other alleles result from point mutations: a missense mutation of the start codon, a frameshift, and a nonsense mutation. A. niger strain ATCC 1015 has a full-length aoxB gene. The A. niger sensu stricto complex can thus be subdivided into six taxa according to extant aoxB allele, which may facilitate rapid and accurate identification of individual species


Introduction
For over a century, the filamentous Ascomycete fungus Aspergillus niger (subgenus Circumdati, section Nigri, series Nigri), a ubiquitous soil-borne saprophyte, has played a pivotal role in industrial biotechnology. Besides citric acid, A. niger also produces compounds such as gluconate [1], oxalate [2] and malate [3] and various native or heterologous enzymes [4] for diverse industrial applications. In addition to the native or heterologous production of single proteins, A. niger has become a platform for the synthesis of enzyme mixtures such as cocktails used for the degradation of plant polysaccharides [5]. The prominence of A. niger as a platform organism is not independent of the fact that it is classified as "generally regarded as safe" (GRAS) in the USA. However, black aspergilli, like most species of the extensive genus Aspergillus, are opportunistic fungi capable of colonizing the breathing tracts, trachea, nose and lungs of higher animals and humans, and can cause severe non-invasive mycoses and invasive aspergillosis in immunocompromised individuals and people suffering from respiratory maladies. Black aspergilli rank third among the causants of invasive aspergillosis in immunocompromised individuals; the (secondary) fungal infection complicates severe cases of COVID-19 in hospital treatment regimes. These fungi are notoriously difficult to separate by classical means, while the usual molecular taxonomy markers do not aid in reliable identification within the series Nigri of the section Nigri. Black aspergilli are cosmopolitan fungi, and clinical incidence of the respiratory disease symptoms is rising (e.g., [6][7][8]).
Joint molecular phylogeny of the calmodulin and beta-tubulin loci improved the resolution to a level which allowed for distinction with increased confidence to be made between Aspergillus welwitschiae and A. niger (sensu stricto), although some isolates could not be unambiguously assigned to the one or the other of these sibling taxa [8][9][10]. However, this resolution was clearly insufficient to identify diverse clades within the A. niger sensu stricto complex, although rapid and accurate identification of individual species could be crucial in clinical settings.
The alternative terminal oxidase (systematic name: ubiquinol:oxygen oxidoreductase, non-electrogenic; EC 1.10.3.11) is encoded in the nuclear genome but located in the mitochondrion, anchored in the inner mitochondrial membrane (e.g., [11,12]). It provides an alternative for the cytochromic electron flow and bypasses the proton-pumping complexes III and IV of the canonical electron transfer chain [13] (Supplementary Figure S1). While alternative oxidase (Aox) has been associated with various apparently nonessential physiological functions, the consequence of the direct transfer of electrons from ubiquinol to oxygen without concomitant proton translocation is the uncoupling of ATP synthesis via oxidative phosporylation from NADH reoxidation, to allow carbon catabolism to continue unabated when ATP demand is low (e.g., during slow growth: [14]). For this reason it plays an important role in the energetics of overflow metabolisms: alternative oxidase activity is positively correlated with the final yield of industrial bioprocesses as A. niger citric acid [15,16], Aspergillus terreus itaconic acid [17] or Acremonium chrysogenum cephalosporin-C fermentation [18], but also with the detrimental production of polyketide mycotoxins (e.g., sterigmatocystin, aflatoxin) by, e.g., Aspergillus nidulans [19] and Aspergillus flavus [12]. Understanding the origin, structure and evolution of alternative respiratory pathways and their key non-electrogenic enzymes could therefore contribute to strain improvement efforts in industrial biotechnology.
Alternative oxidase (Aox) is near ubiquitous in the fungal kingdom, although it is not an essential enzyme activity under all growth conditions and lifestyles. For instance, the so-called genetic model yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe do not have Aox. Conversely, the co-existence of paralogue alternative oxidase genes is rarely described in fungi. Two neighbouring genes, AOX1a andAOX1b, are orientated in tandem in the ascomycete yeast Candida albicans [20,21]. On the other hand, the filamentous model fungus Neurospora crassa has two sequence-related but genetically unlinked alternative oxidase genes, aod-1 and aod-3 [22,23].
In Aspergillus niger strain WU-2223L (recently reappreciated as an isolate of Aspergillus tubingensis), a full-length alternative oxidase cDNA was reported [24,25] (accession number of the cDNA: AB016540). The identified aoxA gene encodes a protein of 351 amino acids (AA) (BAA32033 and BAB03469) with a predicted N-terminal mitochondrial target sequence of 53 amino acids. aoxA has two phase-two introns bounding an exon of 291 base pairs (bp). This gene model is conserved in all Eurotiales and Onygenales for which genome sequences are available (in June 2022; results not shown). With the publication of the first two Aspergillus niger whole genome sequences in 2007 and 2011 [26,27], it became evident that there are also paralogous aox sequences in some strains of Aspergillus niger that were apparently absent from other Aspergillus niger isolates. The current availability of dozens of whole genome sequences of Aspergillus niger strains and other species in the Nigri series of the section Nigri (for molecular taxonomic relations within the Aspergillaceae, see [28]) allows for investigation of the presence, the status and the origin of the second alternative oxidase gene in the A. niger complex (A. niger sensu stricto: cf. [10,29]). In the current work, we show that the paralogous aoxB gene in some 75 genome-sequenced A. niger strains features variations which rationally affect the transcription or the function of aoxB, or at the least, alter the encoded protein at one of its termini. Based on five aoxB mutant alleles, one could subdivide the A. niger sensu stricto complex into six taxa. Moreover, the presence of an aoxB pseudogene distinguishes A. tubingensis strains (A. niger sensu lato) from other named species in the A. luchuensis clade. aoxB could, thus, be used as a new diagnostic marker unique to a narrow taxon of black aspergilli, which allows for distinction between multiple clades within the A. niger sensu stricto complex in clinical and industrial settings. To date, confident separation could only be accomplished after comparative analyses of whole genome sequences.

Mining of Alternative Oxidase and Type-II NADH:Ubiquinone Oxidoreductase Genes, Gene Synteny around Gene Loci and Confirmation of Expression
The coding sequences of alternative oxidase and type-II NADH:ubiquinone oxidoreductase genes (ATG-STOP) were mined following TBLASTN screening of DNA databases on the National Center for Biotechnology Information (NCBI) servers (primarily the Whole Genome Shotgun contigs (WGS) database, which includes the genomes of approximately 75 different A. niger strains and isolates) using the available online tools [30]. The TBLASTN query sequences are specified in the Results and Discussion section of this paper. Using the AoxA protein from WU-2223L [24,25] as the query, a second alternative oxidase gene was searched for, apparently encoding peptides with 55-65% amino acid identity to the query protein (in particular, in exons 2 and 3). The N-terminal mitochondrial signal and membrane anchor in exon 1 are far less sequence-conserved between Aox paralogues coexisting in the same fungus. TBLASTN screens were run with near-default settings, although the Expect threshold stringency was lowered to 1000 and the Gap Cost reduced to Existence 10 and Extension 1, while composition adjustment was omitted, and low complexity regions were not filtered. For a few fungi (including A. niger CBS 513.88), the genome sequences are located in the Refseq genome database, and these were screened using the BLAST Genomes module. We did not use the results of automated annotation at NCBI ("Models" or "mRNA" at nr/nt), nor did we mine deduced protein databases for computer-annotated sequences.
We included species exclusively available from the MycoCosm depository of fungal genome sequences of the U.S. Dept. of Energy Joint Genome Institute (JGI) (https: //mycocosm.jgi.doe.gov/mycocosm/home) (accessed on 5 November 2022) [31]. For the JGI screens, search criteria were narrowed and second alternative oxidase genes-having 50-75% identity at the amino acid level with A. niger ATCC 1015 AoxB-were searched for that neighbour divergently orientatedparalogues (AndB) of type-II alternative NADH dehydrogenase genes, which (themselves) are 45-55% (amino acid level) identical to the ubiquitous alternative NADH dehydrogenase (AndA), present in all genome-sequenced species of the Eurotiomycetidae subclass. For two species of Aspergillaceae, we obtained permission to use their JGI-lodged genome assemblies to identify the paralogous aox and and genes and to deduce their gene models (Supplementary Table S1) guided by the conservation of intron positions observed in these paralogues in Eurotiomycetidae taxa. These two species were Aspergillus implicatus strain CBS 484.95 (project ID: 1052426) and Penicillium swiecickii strain 182_6C1 (project ID: 1144761). We confirmed the absence of the aoxB-andB gene couple from a group of species closely related to Aspergillus calidoustus (same series) and exclusively available at the JGI genome sequence depository: A. To corroborate orthology amongst homologous genes, TBLASTN hits and their local environment were inspected for intron positional conservation (cf. [32]) and for colinearity with neighbouring genes that could imply gene clustering. The genome browsers of the respective JGI genomes were used to inspect the direct environments of the aoxB locus for gene synteny in Supplementary Table S2) by BLASTN screening the species-designated Sequence Read Archives at NCBI, using 60 nt long query sequences covering the exon fusion site produced by the predicted intron excision. To generate estimations of sequence similarity (% of amino acid or DNA identity) we ran Clustal Omega multiple sequence alignments [33] that produce percent identity matrices as part of the output.

Maximum-Likelihood Phylogenetic Analysis
Different selections of Eurotiomycetidae and Lecanoromycetes Aox proteins were first aligned using multiple sequence alignment with fast Fourier transform (MAFFT, version 7) [34,35] using E-INS-i iterative refinement, trained to recognize multiple conserved domains with large spacing, and BLOSUM45 as the scoring matrix (fixed variables). The resulting alternative alignments were subsequently trimmed using BMGE (block mapping and gathering using entropy: [36]) to optimize the ensemble of the highly informative regions while deleting less similar areas harbouring many of the gaps introduced during alignment, utilizing the amino acid substitution matrix BLOSUM55 and a block size of 4 (fixed settings). BMGE-trimmed alignments were subsequently used to construct maximumlikelihood (ML) trees with PhyML (version 3) (online module) [37], initially employing the general amino acid replacement matrix LG [38] with the following settings: invariable sites, estimated; substitution rate, gamma; number of substitutions, 4; gamma-shape, estimated. However, using the Aikake information criterion (AIC), the updated automated Smart Model Selection module of PhyML [39] currently selects the kingdom-specific amino acid replacement matrix, Q.plant [40], as the recommended model. We addressed the apparent inconsistency of using a "plant-specific" amino acid substitution model by estimating the evolutionary relationships between filamentous fungal AoxA and AoxB paralogues from the same trimmed alignment in parallel with either the LG or the Q.plant matrices. Maximumlikelihood estimations were used consistently in an effort to make our analyses compatible with published molecular taxonomy studies of black aspergilli (for example, [8-10]).
ML trees were drawn with FigTree version 1.4.3 and rooted in the designated outgroup of Lecanoromycetes AoxA; the outgroup was subsequently eliminated from view by subtree selection in the tree-drawing program. For the convenience of the reader, the selected maximum-likelihood tree shows the stable placement of the paralogous AoxB clade across the alternative phylogenies along the collapsed monophyletic AoxA clades for species belonging to each of the Eurotiales families Aspergillaceae, Thermoascaceae, Elaphomycetaceae, or Trichocomaceae, or species belonging to the Onygenales order. Branch stability was assessed with approximate Likelihood Ratio Tests (aLRTs) [41] integral to PhyML operation using the default settings.

Aspergillus niger Strains Used for Sequence Verification of aoxB Alleles Reported in this Study
The strains used to identify the six aoxB alleles are listed in Table 1, along with their original source and the accession numbers of their sequences. The sequences we determined and submitted to GenBank are from ATG to STOP for the aoxB alleles, except for allele II from which the 5 -quarter (23%) of the aoxB coding region has been removed by the chromosomal deletion of 2294 nt typical for allele II (see Results and Discussion section of this paper). cDNA proves excision of two introns from the aoxB pre-mRNA (ATCC 1015) which are position-conserved with those in the aoxA pre-mRNA in WU-2223L.  Targeted PCR reactions using a gDNA template were performed with gene-specific oligonucleotide primer pairs (Supplementary Table S3) (Integrated DNA Technologies, Leuven, Belgium) and DreamTaq DNA Polymerase (Thermo Scientific, Thermo Fisher Scientific, Waltham, MA, USA) in a T100TM Thermal Cycler (Bio-Rad, Bio-Rad Hungary Ltd., Budapest, Hungary). Cycling conditions after initial denaturation at 95 • C for 3 min: 35 cycles of 95 • C for 30 s, 54 • C for 1 min and 72 • C for 0.5-1 min, followed by post-cyclic elongation at 72 • C for 5 min. Purified PCR fragments (NucleoSpin Gel & PCR Clean-up, Macherey-Nagel) were cloned into pGEM-T Easy (pGEM-T Easy Vector System I, Promega Corporation, Madison, WI, USA). Plasmid DNA was isolated using the NucleoSpin Plasmid EasyPure kit (Macherey-Nagel). DNA from three independent clones (three technical replicates) was sequenced over both strands using universal primers hybridizing to the vector (Eurofins Genomics, Ebersberg, Germany). The determined aoxB and andB sequences were deposited at GenBank (see Table 1 for the respective accession numbers).

Isolation of A. niger Total RNA for cDNA Sequence Analysis
Total RNA for first-strand cDNA synthesis was isolated from cultures of ATCC 1015. The biomass was grown under manganese paucity in 500 mL Erlenmeyer flasks (VWR International Kft., Debrecen, Hungary) with 100 mL of growth medium (2.50 g (NH 4 ) 2 SO 4 ; 0.15 g KH 2 PO 4 ; 0.15 g NaCl; 2.25 g MgSO 4 ·7H 2 O; 1.50 mg Zn 2+ ; 140 g Dglucose; 0.10 mg Fe 2+ and 0.06 mg Cu 2+ , per litre) (i.e., no MnCl 2 added) in a rotary shaker (Infors AG, Basel, Switzerland) at 250 rpm at 30 • C. Total RNA was isolated using the RNA Plant kit (Macherey-Nagel GmbH & Co., KG, Düren, Germany). First-strand cDNA was synthesized from a total RNA template with Oligo(dT) as the primer using the RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific, Thermo Fisher Scientific, Waltham, MA, USA). First strand cDNA was subsequently used as the template for the PCR reaction, and the cycling conditions after initial denaturation at 95 • C (3 min) were as follows: 35 cycles of 95 • C for 30 s, 54 • C for 1 min, and 72 • C for 0.5-1 min, followed by one post-cyclic elongation at 72 • C (5 min). The downstream sequencing procedure after cloning of the RT-PCR amplification was the same as described for gDNA (above). All RT-PCR experiments were performed in triplicate, starting with biomass from three independent liquid cultures.

A Rare Alternative Oxidase Paralogous Gene (aoxB) Occurs in the Aspergillus niger Species Complex (Sensu Stricto) as well as in Distally Related Aspergillus calidoustus
Comparative analysis allowed for resolution of the gene model of a full-length gene (aoxB) for a paralogue alternative oxidase of 347 amino acids in A. niger strain ATCC 1015 as well as in the sibling species Aspergillus welwitschiae (strain CCMB 674) and Aspergillus awamori (strain IFM 58123). The predicted gene models of the three aoxB genes are detailed in Supplementary Table S1. A schematic overview of the aoxB locus in ATCC 1015 is shown in Supplementary Figure S2, including its intron-exon structure and that of the divergently orientated neighbouring gene. The aoxB gene in these three genomes has two phase-two introns, strictly position-conserved with those in the ubiquitous aoxA gene in the same fungi, bounding the central exon of 291 bp in both aoxA and aoxB (Supplementary Figure S2a). Moreover, the coding part of the 3 exon is 511 bp in both paralogous genes. The two-intron aoxB gene model in ATCC 1015 was confirmed with overlapping cDNA clones obtained after RT-PCR-amplification (GenBank accession OQ686795). The aoxB DNA sequences (ATG-STOP) are 100% identical in ATCC 1015, NRRL3 (also known as N400) and N402, 98% identical to those in IFM 58123 and 96 % identical to those in CCMB 674. The paralogous AoxB protein in A. niger ATCC 1015 (347 residues) is >57% identical to the ubiquitous AoxA (351 residues) in the same strain. The similarity increases to >63% when the predicted N-terminal mitochondrial transfer peptide (~50 AA) is removed from both proteins.
A very similar aoxB paralogue (1159 bp start-to-stop, including introns) is found in all six accessible genomes of Aspergillus calidoustus (strain SF006504 [42]: WGS Master Accession CDMC), a species of series Calidousti, section Usti, in the subgenus Nidulantes (rather than Circumdati, the subgenus to which A. niger belongs). The two phase-two introns are positionconserved; exon 2 and the coding part of exon 3 are exactly the same size as in A. niger ATCC 1015, at 291 and 511 bp, respectively. The overall similarity between aoxB in ATCC 1015 and in A. calidoustus at the DNA level (including introns) is~70% identical. The A. calidoustus AoxB protein is predicted to be 345 AA long and is almost 74% identical to the orthologous A. niger ATCC 1015 AoxB over the complete sequence. We did not encounter full-length aoxB orthologues in other Black Aspergilli (for which the genome sequence has been published), all of which do harbour the ubiquitous aoxA gene. Moreover, we did not find an aoxB orthologue in the other species of section Usti available at NCBI, neither in Aspergillus ustus nor in Aspergillus sp. ADI-1, nor in five species of the series Calidousti for which genome sequences have been lodged at the JGI, namely A. carlsbadensis, A. germanicus, A. insuetus, A. keveii and A. pseudodeflectus.

aoxB Is Divergently Orientated from a Rare Paralogous Gene Encoding a Type-II Alternative NADH:Ubiquinone Reductase
When we inspected the direct environment of the locus of the rare aoxB orthologue in A. niger ATCC 1015 and A. calidoustus, it was found that aoxB is divergently orientated from a gene encoding an equally rare paralogue of a type-II NADH dehydrogenase or alternative NADH:ubiquinone reductase (non-electrogenic). Figure 1 schematically shows the divergently orientated genes in A. niger ATCC 1015. In Supplementary Figure S2, the proposed intron-exon structure (4 introns) of the divergently orientated gene has been depicted for A. niger ATCC 1015 opposite that of aoxB, and in Supplementary Table S1 the coordinates of the exons are provided. The andB intron-exon structure was confirmed by comparative analysis and by cDNA sequencing (GenBank OQ689783). This divergent gene couple appears to be unique in the NCBI databases for the two different narrow Aspergillus taxa mentioned above.
A. niger ATCC 1015 and A. calidoustus, it was found that aoxB is divergently orientated from a gene encoding an equally rare paralogue of a type-II NADH dehydrogenase or alternative NADH:ubiquinone reductase (non-electrogenic). Figure 1 schematically shows the divergently orientated genes in A. niger ATCC 1015. In Supplementary Figure  S2, the proposed intron-exon structure (4 introns) of the divergently orientated gene has been depicted for A. niger ATCC 1015 opposite that of aoxB, and in Supplementary Table S1 the coordinates of the exons are provided. The andB intron-exon structure was confirmed by comparative analysis and by cDNA sequencing (GenBank OQ689783). This divergent gene couple appears to be unique in the NCBI databases for the two different narrow Aspergillus taxa mentioned above. Type-II NADH dehydrogenase (EC 1.6.5.9) re-oxidizes NADH to NAD + but transfers the electrons to ubiquinone without pumping protons out of the mitochondrion (see [43] for a recent review). The rare alternative NADH dehydrogenase paralogue in the divergent gene couple extant in {A. niger ATCC 1015-A. welwitschiae-A. awamori} and in A. calidoustus we have named andB, and the unlinked ubiquitous gene for the enzyme activity present in all species of Eurotiomycetidae and Lecanoromycetes we have named andA. The A. niger ubiquitous andA gene is the structural orthologue of the A. nidulans gene at locus AN1094 (~85% AA identity) and the N. crassa gene at locus NCU08980 (~65% AA identity). The A. nidulans and N. crassa orthologous enzymes oxidize cytosolic NADH, i.e., they are non-proton-pumping type-II NADH dehydrogenases located on the cytosolic side of the mitochondrial inner membrane. [44,45].
The two paralogue alternative NADH dehydrogenases (AndA and AndB) are ~51% identical in both A. niger ATCC 1015 and A. calidoustus (for the full-length gene products). The two orthologous AndB peptides are ~67% identical, much lower than > 83% in the Type-II NADH dehydrogenase (EC 1.6.5.9) re-oxidizes NADH to NAD + but transfers the electrons to ubiquinone without pumping protons out of the mitochondrion (see [43] for a recent review). The rare alternative NADH dehydrogenase paralogue in the divergent gene couple extant in {A. niger ATCC 1015-A. welwitschiae-A. awamori} and in A. calidoustus we have named andB, and the unlinked ubiquitous gene for the enzyme activity present in all species of Eurotiomycetidae and Lecanoromycetes we have named andA. The A. niger ubiquitous andA gene is the structural orthologue of the A. nidulans gene at locus AN1094 (~85% AA identity) and the N. crassa gene at locus NCU08980 (~65% AA identity). The A. nidulans and N. crassa orthologous enzymes oxidize cytosolic NADH, i.e., they are non-proton-pumping type-II NADH dehydrogenases located on the cytosolic side of the mitochondrial inner membrane [44,45].
The two paralogue alternative NADH dehydrogenases (AndA and AndB) are~51% identical in both A. niger ATCC 1015 and A. calidoustus (for the full-length gene products). The two orthologous AndB peptides are~67% identical, much lower than >83% in the case of the AndA gene products. A stand-out difference between the paralogous and genes is the absence of the third (phase zero) intron from the ubiquitous "A" genes (cf. Supplementary Figure S2b).

The aoxB and andB Gene Couple Occurs Sporadically in Four Dispersed Taxa of Aspergillaceae
The rare occurrence of the coupled paralogous aoxB and andB genes was further investigated by searching the MycoCosm genome sequence depository at the Joint Genome Institute (U.S. Department of Energy) [31]. Amongst the~300 species of Aspergillus and Penicillium deposited, we found two additional species in which the rare gene couple aoxB-andB was present. Aspergillus implicatus belongs to the section Sparsi, series Implicati, an early divergent taxon of the subgenus Nidulantes. On the other hand, Penicillium swiecickii is a species of the Penicillium subgenus section Ramosum, series Lanosa, long separated from the three Aspergillus species that also harbour the rare gene couple. The gene models of the coupled genes we predict in these two Aspergillaceae are detailed in Supplementary  Table S1, giving the JGI scaffold numbers on which they occur and the coordinates of the exons in between the startcodon and the stopcodon. See [28] for the assignment of these four species to Aspergillus/Penicillium subgenera, sections and series.
Further inspection of the direct environment of the coupled aoxB-andB loci (Figure 2a) suggests that the two genes directly neighbouring the divergent couple could be part of a four-gene cluster found in {A. niger ATCC 1015-A. welwitschiae-A. awamori} as well as A. calidoustus. The seven-exon gene of a putative zinc-cluster transcriptional factor is located downstream of aoxB and transcribed from the opposite strand (pink gene labelled "gene-1" in Figure 2a): the zinc-cluster regulator proteins are 60% identical over their complete width. Downstream of andB and on the same strand is a two-exon gene for a protein which is~600 amino acids long with an N-terminal 3-hydroxyacyl-CoA dehydrogenase domain (yellow gene labelled "gene-1" in Figure 2a). The orthologues in ATCC 1015 and A. calidoustus are >80% identical. implicatus and P. swiecickii. The three auto-annotated loci (filtered models) on each side of aoxB were taken from the respective genome browsers on the JGI website (see Materials and Methods section for details of the appropriate websites). The orientation of the neighbouring genes was indicated by the arrow, but for the sake of simplicity, the absolute size of the predicted coding regions, the intergenic distances and the deduced intron-exon structures were ignored in the schematic summary shown here. The predicted functions of the neighbouring genes were colour-coded such that genes with identical annotated function could easily be discerned. The gene at the left of aoxB is the divergently orientated alternative NADH dehydrogenase andB. In A. niger ATCC 1015 and A. calidoustus, the two genes directly neighbouring the aoxB-andB gene couple (divergent blue arrows) are also . The orientation of the neighbouring genes was indicated by the arrow, but for the sake of simplicity, the absolute size of the predicted coding regions, the intergenic distances and the deduced intron-exon structures were ignored in the schematic summary shown here. The predicted functions of the neighbouring genes were colour-coded such that genes with identical annotated function could easily be discerned. The gene at the left of aoxB is the divergently orientated alternative NADH dehydrogenase andB. In A. niger ATCC 1015 and A. calidoustus, the two genes directly neighbouring the aoxB-andB gene couple (divergent blue arrows) are also orthologous, encoding a zinc-cluster transcriptional regulator (~700 AA) next to aoxB, and a~600 AA-sized protein with an N-terminal 3-hydroxyacyl-CoA dehydrogenase domain downstream of andB. To test whether the coupled aoxB-andB genes are co-expressed and thus potentially part of a functional gene cluster, we screened extant sequence read archives available at NCBI (generated by third parties but freely accessible). We screened SRA libraries (RNA reads) in A. calidoustus and A. implicatus for sequence reads that cover the predicted exonexon fusions after intron excision of pre-mRNAs for aoxB and andB. We also identified SRA reads that confirm intron excision for both genes in a set of A. niger gene deletion strains, generated in the NRRL3 background which is in the same pedigree as ATCC 1015 (i.e., wild type for aoxB). Supplementary Table S2 lists individual reads which cover exon-exon fusions. We found pre-mRNA splicing for all six predicted introns in A. calidoustus as well as in A. niger (strains delta-araR, delta-gaaR, delta-galX, delta-rhaR, delta-xlnR in the NRRL3 background) grown on complex carbon sources. In A. implicatus, we found SRA reads that cover the excision of either of the aoxB introns and reads that cover the exon-exon fusions of introns 2 and 4 of the andB gene. Note that the expression rate is highly dependent of the growth conditions under which these sequence read resources were created, and therefore quantitative analyses of covering SRAs are not informative.

The Rare aoxB Paralogue Appears to Originate from an Unidentified Host Taxon within the Eurotiomycetidae but Not an Aspergillaceae Taxon
To investigate the origins of the aoxB paralogue in the rare aoxB-andB gene couple, we collected the DNA coding for some 350 Aoxs from Eurotiomycetidae and Lecanoromycetes genomes, following TBLASTN screening of the NCBI's Whole Genome Shotgun database in May 2022, using the ubiquitous A. niger ATCC 1015 AoxA protein as the query sequence. After manual deduction of the intron-exon structures and subsequent translation of the open reading frame after in silico intron removal, we generated alternative maximumlikelihood trees with variably sized AoxA content and 12 full-length AoxB paralogues (the latter specified in Figure 2b). Two different substitution models, Q.plant and LG, were applied in parallel in estimating multiple maximum-likelihood phylogenies from multiple sequence alignments of differently defined batches of Aox proteins using the PhyML (v3.0) online facilities (see Materials and Methods section for more details). The alternative trees were all rooted on the clade of Lecanoromycetes AoxA proteins as the default outgroup. The relevant section of one representative maximum-likelihood tree is shown in Figure 2b. Despite the scattered and sporadic occurrence, all alternative AoxA-AoxB phylogenies consistently predict essentially the same origin for aoxB, with the grouped AoxB proteins acting as a sister clade to the Onygenales AoxA proteins in a distinctive subtree further consistent with the AoxAs from species in the (other) Eurotiales families-Trichocomaceae, Elaphomycetaceae and Thermoascaceae. The genome-sequenced species in the aforementioned four taxa (i.e., those outside Aspergillaceae) do not feature aox paralogues, with the exception of Rasamsonia emersonii, whose paralogue is not intimately related with AoxB. In A. implicatus, a secondary duplication is likely at the origin of the two paralogue Aoxs that evolved within the diminutive but welldefined AoxB clade. In Figure 2b, the third paralogue not linked to any alternative NADH dehydrogenase gene is called AoxB2. This third aox paralogous gene in A. implicatus is also expressed (Supplementary Table S2).
The underlying topology of the ubiquitous AoxA was revealed by inferring maximumlikelihood trees from orthologous "A" proteins only (i.e., without AoxB) using exactly the same settings for alignment, trimming and tree inference as for the mixed paralogue phylogenies. Supplementary Figure S3 shows that the taxonomically aberrant position of the Onygenales AoxA clade, clustering amongst the Trichocomaceae, Elaphomycetaceae and Thermoascaceae family clades of the Eurotiales instead of forming a discernible outgroup to all Eurotiales, is not due to the presence of the AoxBs in the mixed phylogenies.
One characteristic differentiating the ubiquitous andA gene from its andB paralogue is the intron-exon structure (cf. Supplementary Figure S2b). In A. niger ATCC 1015, A. calidoustus and A. implicatus, andB has four introns at strictly conserved gene positions; P. swiecickii lacks an intron at the first of those positions. By contrast, the ubiquitous andA gene lacks the phase-zero intron at the third conserved position in all four species harbouring the aoxB-andB gene couple. In fact, the third intron position (phase zero) is not occupied in the andA orthologous gene in all genome-sequenced species of Penicillium as well as in the overwhelming majority of genome-sequenced Aspergillus species: the exceptions are A. wentii (section Cremei), Aspergillus sp. HF37 and A. sclerotiales (section Polypaecilum). In all other taxa of Eurotiomycetidae, including the remaining Aspergillaceae taxa (i.e., Penicilliopsis, Evansstolkia, Monascus, Xeromyces and "Byssochlamys" sp. BYSS01) as well as all the sequenced Thermoascaceae, Trichocomaceae, Elaphomycetaceae and Onygenales, the ubiquitous andA gene also has all four intron positions occupied. The change in the gene model of the andA gene-the loss of the phase-zero intron at conserved position 3 in all Penicillia and in almost all Aspergilli-provides circumstantial evidence that the origin of the present day aoxB-andB couple is amongst the divergent families of Eurotiales beyond the divergence of the Aspergillaceae.

Multiple Alleles for the Acquired aoxB Paralogue in the Aspergillus niger Sensu Stricto Complex
Interestingly, the acquired aoxB gene in genome-sequenced strains of A. niger sensu stricto features considerable genetic variation. Amongst the genome-sequenced strains, we found five different mutations that severely affect the encoded enzyme or, at the least, terminally modify the aoxB gene product. Figure 3 provides details about the six alleles and the mutations at their basis. Table 2 lists the A. niger strains that carry the six aoxB alleles identified. Group VI consists of two strains that have accumulated a nonsense mutation in the background of the pre-extant frameshift mutant allele V. All five other groups contain strains of opposite mating type (cf. [29,46]). We have confirmed by PCR amplification and sequencing that five of these aoxB alleles are indeed identical to those originally identified in silico in the corresponding genome sequences. In Table 1, the corresponding GenBank accession numbers certifying various aoxB alleles (including the wild type allele) are listed together with the strains taken as typical for each of these aoxB alleles. Nine A. niger strains listed in Table 2 in Group IV-sequenced by three different groups of scientists-carry the defining A-C transversion that would eliminate the original start codon. Their aoxB sequences (1185 bp) are identical except for one nucleotide deep within the first intron (i.e., position 280).
Genomes of grey-marked clinical isolates were made public recently at NCBI. Their sequences are not used in aoxB phylogenies. * Genome sequences contaminated with Aspergillus DNA; two different aoxA genes present. # 5′-and 3′ parts separated by the transposon are split over two sequence contigs.  Table 2 and include strain ATCC 1015 (GenBank OQ606865). The positions of the mutation or interruption in the aoxB coding region are indicated by the boxed % of CDS. Alleles IV, V and VI are the consequences of point mutations shown in the three sequence inlets. For Allele IV, the use of a downstream start codon (relevant ATGs in green) is the predictable consequence of the observed missense mutation (A-C transversion) that eliminates the wild-type start codon, N-terminally truncating the mitochondrial target sequence (53 AA) by one third. For Allele V, the deletion of one T frameshifts the CDS near its 3 end, indicated by alternate underlining of the used codons in the wild-type and the mutant alleles, the red sequence occurring after the frameshift in aoxB alleles V and VI. Allele VI is a double mutant allele derived from allele V, as it features an additional nonsense mutation (in codon number 38) near the 5 of the aoxB coding region. strain S1 strain Y2001A1 Genomes of grey-marked clinical isolates were made public recently at NCBI. Their sequences are not used in aoxB phylogenies. * Genome sequences contaminated with Aspergillus DNA; two different aoxA genes present. # 5 -and 3 parts separated by the transposon are split over two sequence contigs.
The wild-type aoxB allele potentially produces the full-length AoxB paralogue of 347 AA in length in, amongst others, A. niger strain ATCC 1015 [27] and in the laboratory strains NRRL 3 and N402. Full-length AoxB is marginally shorter at the N-terminus than the ubiquitous AoxA protein (351 AA), there where the mitochondrial target sequence is located. This full-length allele is also present in the sibling species A. welwitschiae [47] (5 strains) and A. awamori IFM 58123 [48], in addition to 14 genome-sequenced Aspergillus niger sensu stricto strains. The genome sequences of "Penicillium fimorum" strain S/N-308-OC-P1 (Master accession number JACVQR) are likely contaminated with the DNA of an A. niger-like fungus, specifying two non-identical aoxA genes and one full-length aoxB orthologue.
The most dramatic of the mutant aoxB alleles concerns a chromosomal deletion of 2294 bp that removed exon 1 and intron 1 (i.e., everything before codon Tyr82) (Figure 3, group II), but also the complete intergenic region (626 bp) as well as approximately 76% of the coding regions of the divergently orientated andB gene. This deletion thus eliminates expression of both genes. Figure 1 outlines the situation in CBS 513.88, the first A. niger strain that was genome-sequenced [26]. This severe dual-loss-of-function mutation is also present in the neotype strain CBS 554.65 [46] (verified: GenBank OQ590013), as well as in A. lacticoffeatus (CBS 101883) and A. niger Van Tieghem (ATCC 13496) [49]. The aoxB deletion (allele II) is present in 13 genome-sequenced strains.
The second aoxB mutation involves the integration of a non-autonomous copy of a retrotransposon in the third exon at~80% of the coding sequences ( Figure 3: Group III). The codons of amino acids R276 and M277 are included in the 7 nt long target-site duplication (TSD) 5 -GAGAATG generated by the integration of an~13 kb long type-1 element. The aoxB transposon interruption occurs in 16 genome-sequenced strains. The retrotransposon features long terminal repeats (LTRs: direct repeats) of~158-162 bp which are 72.84% identical. Supplementary Figure S4 shows the LTRs of the eight copies of this transposon (eight different TSDs) found in one or more of the 16 strains in which the aoxB gene is retrotransposon interrupted. In CBS 147324, an autonomous copy is present, 5127 nt long with 100% identical, 159 nt long LTRs between hexamer TSDs 5 -AGAAAC. This RNA transposon identifies as a member of the Ty1/copia family [50]. The autonomous copy contains the 4512 nt long open-reading frame encoding a multifunctional protein (1503 AA) with integrase, reverse transcriptase and RNase H domains in the order typical of the Ty1/copia family (Supplementary Figure S5). The Ty1/copia transposon in CBS 147324 has not been reported before: its LTRs are quite distinct from those of the A. niger CBS 513.88 ANiTA1 retrotransposon (cf. [51]). We have given the new A. niger Ty1/copia transposon in CBS 147324 the name ANiTA2. In strain CBS 133816, a solo LTR-100% identical to those of the autonomous ANiTA2 copy in CBS 147324-is observed at the same integration site between the (same) hexamer TSDs 5 -AGAAAC, suggesting that the retrotransposon is eliminated from the CBS 133816 genome by recombination.
The third mutant aoxB allele is the result of a point mutation that changes the start codon into CUG (Met1Leu) (Figure 3: Group IV). We found this missense mutation in nine strains (September 2022). If a protein can be produced using the next-in-frame AUG in the aoxB transcript (either Met18 or Met23) as its start codon, the mitochondrial target peptide is truncated by at least 17 AAs, i.e., one-third of the predicted mitochondrial target peptide would be lost, including a trimer of positively charged amino acids {RKR}, and, consequently, it may no longer function properly as a mitochondrial target sequence.
The fourth mutant aoxB allele we identified is due to a single-base deletion of a T in the DNA at the wobble position of the Val328 codon near the 3 end of exon 3, at~95% of the coding sequences (Figure 3: Groups V and VI). Consequently, a frameshift occurs after Pro329, yielding a 345 AA long protein with a completely different C-terminus (16 AA). We found four strains with this frameshift mutation, all of them sequenced recently [29]. Two of these strains, CBS 131.52 and CBS 769.97, also carry a nonsense mutation at the other end of the coding region in exon 1, resulting in a premature ochre stop codon at the position of Ser38 at~11% of the gene product, within the mitochondrial target signal. These two latter strains thus harbour the sixth and only double-mutant allele of aoxB.

aoxB Pseudogenes Occur in (Most) Aspergillus tubingensis but Not in Other Named Members of the Aspergillus luchuensis Clade
The acquisition of the four-membered gene cluster with the divergent aoxB-andB genes in its centre is likely to have taken place earlier then the speciation events separating the Aspergillus niger clade in the series Nigri from the Aspergillus luchuensis clade [28,52]. A TBLASTN screen for the presence of the ensemble of the four genes (NB. Queries were the four gene products from strain ATCC 1015) resulted in the identification of a fraction of the A. luchuensis clade in which a remnant of the gene cluster has been retained in most isolates named Aspergillus tubingensis, including the strain in WU-2223L ( [53]: WGS Master Accession BLWE). In addition, this clade also contains multiple isolates of "Aspergillus niger sensu lato", such as strain An76 ( [54], WGS: BCMY) and strain 3.316 (WGS: JAALJE). Within the identified group of isolates of black aspergilli, the two outward genes of the four-membered cluster (cf. Figure 2a), encoding for a conserved zinc-cluster transcriptional regulator and a protein with an N-terminal 3-hydroxyacyl-CoA dehydrogenase domain, respectively, are intact in the genomes listed in Table 2 (sensu lato isolates, separate column at the right of the table). However, the andB gene is severely degenerated in all listed genomes with numerous frameshifts, and a sizeable deletion is apparent between introns 2 and 4. The aoxB pseudogene features two or three frameshifts in exons 2 and 3, respectively. Nevertheless, the similarity between the full-length aoxB gene (A. awamori) and the pseudogene apparent in a fraction of isolates in the A. luchuensis clade (Supplementary Figure S6) remains high at~85% (introns included).
Other named genome-sequenced species in the A. luchuensis clade (A. luchuensis, A. piperis, A. vadensis, A. eucalypticola and A. neoniger) and at the basis of the series Nigri (A. brasiliensis) do not have orthologues of any of the four genes of the plausible cluster (not shown), suggesting a prominent separation within the A. luchuensis clade based on the presence or absence of these four neighbouring genes, including aoxB.

Conclusions
The Aspergillus niger aoxB gene encoding a second alternative oxidase is divergently orientated from the andB gene encoding a paralogue of a type-II NADH dehydrogenase, constituting the aoxB-andB gene couple. These two genes are likely to be involved in parallel pathways to uncouple ATP synthesis by means of oxidative phosphorylation from NADH reoxidation, coordinated expression of which could in extremis allow control of metabolic reducing power without any concomitant build-up of proton motive force. A plausible distal origin of the aoxB paralogue outside the Aspergillaceae family coincides with a sporadic, scattered occurrence (i.e., in four species, three Aspergilli of different sections and one Penicillium) of the gene couple within the Aspergillaceae. This situation is congruent with the implication of one or more horizontal transfer events in the cluster's dispersal as a credible alternative to vertical inheritance with myriad occasions of independent cluster loss in many diverged clades, even though the actual sources of the transferred DNA remain obscure. Different mutations in one gene, the aoxB gene, enable the categorization of the formal species A. niger (sensu stricto) into six groups of variants or cryptic species. Section Nigri rank third as identifiable causants of pulmonary aspergillosis, closely behind species of the section Flavi [6,[8][9][10]55]. However, in some clinical studies causal Aspergillus spp. were not identified further. The existence of different aoxB alleles may contribute to a deeper resolution of the A. niger sensu stricto complex by molecular taxonomy in clinical settings, for example in cases of COVID-19-associated pulmonary aspergillosis [55].
Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/jof9050570/s1, Figure S1: Schematic overview of branched respiratory electron-transport chain in the mitochondrial inner membrane in filamentous fungi to illustrate the metabolic function of alternative oxidase (Aox) and type-II NADH dehydrogenase (And); Figure S2: The intron-exon structures of the two paralogous genes for alternative oxidase and for alternative NADH dehydrogenase (oxidizing cytosolic NADH) extant in Aspergillus niger strain ATCC 1015; Figure S3: The taxonomically aberrant position of the Onygenales AoxA clade is not a con-sequence of joint analysis of (orthologous) AoxA proteins with AoxB paralogues; Figure S4: Alignment of opposite LTRs of the non-autonomous and autonomous copies of ANiTA2 found amongst sixteen A. niger sensu stricto isolates with aoxB disrupted by retrotransposon insertion; Figure S5: Ty1/copia retrotransposon ANiTA2, autonomous copy 5129 nt; Figure S6: DNA similarity between the coding region of A. awamori aoxB and the aoxB pseudogene in Aspergillus niger (sensu lato) strain An76; Table S1: The intron-exon structure of aoxB and divergently orientated andB genes; Table S2: NCBI-lodged RNA sequence reads that cover exon-exon fusions in aoxB and andB in A. calidoustus, A. implicatus and A. niger strains (with full-length aoxB and andB genes); Table S3: Oligonucleotide primers used for PCR amplification of the aoxB and andB genes and cDNA prior to sequence verification of mutant alleles.