Generation, Transfer, and Loss of Alternative Oxidase Paralogues in the Aspergillaceae Family

Alternative oxidase (Aox) is a terminal oxidase operating in branched electron transport. The activity correlates positively with overflow metabolisms in certain Aspergilli, converting intracellular glucose by the shortest possible path into organic acids, like citrate or itaconate. Aox is nearly ubiquitous in fungi, but aox gene multiplicity is rare. Nevertheless, within the family of the Aspergillaceae and among its various species of industrial relevance—Aspergillus niger, A. oryzae, A. terreus, Penicillium rubens—paralogous aox genes coexist. Paralogous genes generally arise from duplication and are inherited vertically. Here, we provide evidence of four independent duplication events along the lineage that resulted in aox paralogues (aoxB) in contemporary Aspergillus and Penicillium taxa. In some species, three aox genes are co-expressed. The origin of the A. niger paralogue is different than that of the A. terreus paralogue, but all paralogous clades ultimately arise from ubiquitous aoxA parent genes. We found different patterns of uncorrelated gene losses reflected in the Aspergillus pedigree, albeit the original aoxA orthologues persist everywhere and are never replaced. The loss of acquired paralogues co-determines the contemporary aox gene content of individual species. In Aspergillus calidoustus, the two more ancient paralogues have, in effect, been replaced by two aoxB genes of distinct origins.


Introduction
Cyanide-resistant terminal oxidase (alternative oxidase, Aox; ubiquinol:oxygen oxidoreductase, non-electrogenic; EC 1.10.3.11) has been reported in many organisms, especially in higher plants and fungi [1][2][3][4][5].Aox is a mitochondrial inner membrane enzyme encoded in the nuclear genome and an 'alternative' to electron flow via the cytochromedependent respiratory pathway [3].The site of the branching point from the main chain is at the level of Coenzyme-Q.Therefore, the alternative path is resistant to the inhibitors of Complex III and IV, such as cyanide, nitric oxide or azide, but can be blocked selectively by aromatic hydroxamic acids like salicylic-hydroxamate (SHAM [6]).Compared to the cytochrome pathway, the Aox pathway moves fewer protons across the inner mitochondrial membrane to generate a proton motive force to be used to synthesize ATP, as Complexes III and IV of the mitochondrial electron transport system are bypassed, and Aox lacks proton pumping activity.As a consequence, this pathway provides only 40% of the normal levels of ATP for energy conservation via oxidative phosphorylation.
Aox is induced by stresses such as wounding, chilling, drought, osmotic stress and pathogen attack, in addition to treatment with salicylic acid, hydrogen peroxide or with inhibitors of the cytochrome-dependent respiratory chain.During fungal overflow metabolism in fungi, such as citric acid fermentation by Aspergillus niger, Aox uncouples the re-oxidization of NADH from ATP synthesis, thereby allowing carbon catabolism to continue even when mycelia do not require high energy levels [7,8].The recycling of reducing equivalents (NAD + ) for primary catabolism is the predicted role of Aox in Microsporidia, a phylum of obligate endoparasitic fungi that have no mitochondrial DNA and, hence, no cytochrome electron transport chain [9].In Cryptomycota, Complex I of the respiratory chain is absent, and the alternative respiratory pathway is composed of alternative (type-2) NADH dehydrogenases, and Aox operates to recycle NAD + without any protons translocated [10].
Alternative oxidase is nearly ubiquitous in fungal phyla with representative genomesequenced.There are some ascomycete taxa lacking the alternative mitochondrial terminal oxidase, including the family of Saccharomycetideae and the taxa of unicellular Taphrinomycotina.Fungal Aox has sparsely been studied as; it is absent from the fungal "model organisms" of Saccharomyces cerevisiae and Schizosaccharomyces pombe.
On the other hand, aox gene multiplicity appears to be rare in fungi.Three occasions of concurring paralogues have been documented.In the yeast Candida albicans (Debaryomycetideae family, Saccharomycotina subphylum), two differentially expressed, neighbouring genes are orientated in tandem [11].Furthermore, sequence-related but genetically unlinked genes called aod-1 and aod-3 have been described in Neurospora crassa [12].We recently described a third instance, where a rare second aox paralogue named aoxB (the primary enzyme AoxA encoded by the ubiquitous aoxA gene) was found scattered in four species of Aspergillaceae amongst hundreds of genome-sequenced member species [13].Importantly, in the Aspergillus niger sensu stricto complex-where more than 80 genome sequences are available for comparison-we identified five mutations in this acquired aoxB gene that could be used to distinguish six taxa in this species complex known to be notoriously difficult to subdivide [13].
An indication that this rare aoxB paralogue may have been transferred horizontally is the presence of a divergently transcribed, equally rare paralogue gene for an alternative (type-2) NADH dehydrogenase (non-electrogenic), called andB, which is exclusive to the same four species.To broaden these efforts, a more detailed investigation into the origins of aox paralogues in species of Aspergillus and Penicillium was undertaken in this paper.We will show that paralogous aox genes have been generated at least four times during the evolution of Aspergillaceae.

Mining of Alternative Oxidase Genes, Intron-Exon Structure Conservation, and Gene Synteny
The coding sequences of alternative oxidase genes (ATG-stop codon) were mined upon TBLASTN screening of the DNA databases on the National Center for Biotechnology Information (NCBI) servers, primarily the Whole-Genome Shotgun contigs (WGS) database, using the available online tools [14].Using the AoxA protein from Aspergillus tubingensis strain WU-2223L (Previously known as Aspergillus niger WU-2223L) [15,16] as the query, sequence-similar alternative oxidase genes were searched for, apparently encoding peptides with 55-65% amino acid identity to the query protein, in particular, with the enzyme domains encoded in exons 2 and 3.The N-terminal mitochondrial signal and membrane anchor (helix domain) 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; Extension 1, while composition adjustment was omitted and low complexity regions were not filtered.For a few fungi, the genome sequences are located in the Refseq genome database.We did not use the results of automated annotation at NCBI ("Models" or "mRNA") nor protein databases.To generate estimations of sequence similarity (% of amino acid or DNA identity), we ran Clustal Omega multiple sequence alignments [17] that produce percent identity matrices as part of the output.
We included Aspergillus species exclusively available from the Mycocosm depository of fungal genome sequences of the US Dept. of Energy (DOE) Joint Genome Institute (JGI; https://mycocosm.jgi.doe.gov/mycocosm/home,accessed on 23 April 2023) [18], initially for the lack of genome sequences of section Usti species in the NCBI databases.We obtained permission to use JGI-lodged genome assemblies generated by the Aspergillus whole-genus sequencing project (Principal Investigator: Dr. Scott Baker at the US Dept. of Energy Joint Bioenergy Institute) to identify any paralogous aox genes in Aspergilli.
To corroborate orthology and paralogy among sequence-similar genes, TBLASTN hits and their local environment were inspected for intron positional conservation and for the local synteny around the aox genes.With few exceptions, all aox genes in the taxonomic order of the Eurotiales-aoxA orthologues and paralogues alike-have two phase-two introns bounding a central exon with a length of about 300 nt, usually 291 nt.The genome browsers of the respective JGI genomes of species with publicly available genomes (i.e., those highlighted in light green) were used to inspect the direct environments of the paralogous aox loci.Here, we use the JGI-based annotation to determine the orientation of the neighbouring genes and their predicted function to achieve indications in terms of possible gene synteny.

Maximum-Likelihood Phylogenetic Analysis
Eurotiales, Onygenales and Lecanoromycetes Aox proteins were first aligned using Multiple Sequence Alignment with Fast Fourier Transform (MAFFT, version 7) [19,20] using E-INS-i iterative refinement and the BLOSUM45 scoring matrix (fixed variables).The resulting multiple sequence alignments (MSAs) were subsequently trimmed using BMGE (Block Mapping and Gathering using Entropy: [21]) to condense and optimize the ensemble of the highly informative regions, utilizing the substitution matrix BLOSUM55 and a block size of 4 (fixed settings).BMGE-trimmed alignments were then used to infer Maximum Likelihood (ML) trees with PhyML (version 3; online module) [22] employing the general replacement matrix LG [23] with the following settings: invariable sites, estimated; substitution rate, gamma; number of substitutions, 4; gamma-shape, estimated.ML trees were drawn with FigTree version 1.4.3 and rooted in the designated outgroup of Lecanoromycetes AoxA: the outgroup was subsequently eliminated via subtree selection.Branch stability was assessed with approximate Likelihood Ratio Tests (aLRTs) [24] integral to PhyML operation using default settings.
The presence of paralogues in phylogenetic analyses often leads to distortion of the underlying topology of orthologue sequences.We studied each of the aox multiplication events individually to obtain an indication of the origin of that particular aox gene duplication, notwithstanding the existence of other paralogues.Considerable topological instability becomes apparent after the addition of Aox paralogues into mixed phylogenies with the AoxA proteins, mostly affecting the interrelations amongst the Aspergillus sections.By contrast, paralogous Aox clades themselves appear monophyletic.

Confirmation of Expression with Extant RNA Sequence Reads
We identified perfectly matching RNA sequence reads confirming intron excision via BLASTN screening of the species-designated Sequence Read Archives (SRAs) deposited at NCBI.We did not need to create SRA resources ourselves to confirm the gene models and transcript splicing of the aox genes identified in this work.We employed 60 nt-long sequences covering the exon fusion site produced by the predicted intron excision as query material.Imperfect SRA reads (<98% identity) were generally ignored as evidence of splicing.

Expression Verification of aox Paralogous Genes
The fungi used to typify the various independent aox duplication events reported in this work, giving rise to extant aox paralogues (i.e., aoxB1; aoxB2-1; aoxB2-2; aoxB3; aoxB4) in species of the sister genera Aspergillus and Penicillium are listed in Table 1, along with their original sources and the accession numbers of their determined cDNA sequences.The list includes a specimen of the Trichoderma asperellum/asperelloides taxon participating in the lateral transfer of an Aspergillus-born aox gene (see Results and Discussion section).The rare aoxB gene found intact in one clade of the A. niger sensu stricto complex (typified by strains ATCC 1015 and CBS 147482), as well as in A. calidoustus, A. implicatus and Penicillium swiecickii, was previously identified as part of a gene couple with an equally rare type-2 NADH dehydrogenase paralogue gene (andB), unique to these four divergent taxa [13].In the gene nomenclature adopted to describe the multiple aox gene duplication events in the lineage of the Aspergillaceae, this previously described paralogue is dubbed "aoxB1".cDNA analysis proves the excision of introns from pre-mRNA, hence, the expression of the studied gene.* cDNA was generated and sequenced as described in the Material and Methods section.cDNA sequences from ATG to stop codon were deposited at GenBank.** Penicillium rubens strains were previously incorrectly called P. chrysogenum, a closely related but different species (recently reviewed by [31]).*** CBS: Centraal Bureau voor Schimmelcultures, currently known as the Westerdijk Fungal Biodiversity Institute (Utrecht, The Netherlands).

Isolation of Total RNA for cDNA Sequence Analyses
Total RNA for first strand cDNA synthesis was isolated from the biomass of submerged cultures.Fungal biomass was generated in 500 mL Erlenmeyer flasks (VWR International Kft., Debrecen, Hungary) containing 100 mL of a synthetic growth medium in a rotary shaker (Infors AG, Basel, Switzerland) at 200-250 revolutions per minute (rpm) and 28-37 • C for 24-48 h, dependent on the fungus.Trichoderma asperellum, A. wentii and A. oryzae were grown on PDB (potato dextrose broth); the medium for A. oryzae additionally contained 1% Triton X-100.For Penicillium rubens and A. calidoustus, a minimal medium was used consisting of 2 g/L KH 2 PO 4 ; 8 g/L Na 2 HPO 4 ; 0.25 g/L MgSO 4 ; 10 g/L (NH 4 ) 2 SO 4 ; trace element solution (0.1 g/L CaCl 2 ; 1 mg/L CoCl 2 ; 8.8 mg/L ZnSO Liquid cultures were inoculated with freshly prepared, high-density conidiospore suspensions in a 0.01% Tween-20 solution.Mycelia for RNA isolation were harvested from three independent liquid cultures (i.e., three independent biological replicates) via filtration over Miracloth (Millipore, Merck KGaA, Darmstadt, Germany), washed with distilled water and deep frozen in liquid nitrogen for further processing.Total RNA was isolated from powdered deep frozen biomass using the RNA Plant kit (Macherey-Nagel GmbH & Co., KG, Düren, Germany).Genomic DNA (gDNA) was isolated from the same biomass using the Macherey-Nagel NucleoSpin Plant II kit (Macherey-Nagel GmbH & Co., KG, Düren, Germany).

Polymerase Chain Reaction (PCR) and cDNA Sequence Determination
First-strand cDNA was synthesized from total RNA with an Oligo(dT) primer using the RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific, Thermo Fisher Scientific, Waltham, MA, USA).The produce from first-strand cDNA synthesis was then used as the template for PCR reaction(s).PCRs were performed with gene-specific oligonucleotide primer pairs (Supplementary Table S1; 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).The designed primer pairs were verified for their performance on the gDNA template.The cycling conditions after initial denaturation at 95 • C (3 min) were: 35 cycles of 95 • C for 30 s, 54 • C for 1 min, and 72 • C for 30 s/min, followed by one post-cyclic elongation at 72 • C (5 min).Purified PCR fragments (NucleoSpin Gel & PCR Clean-up, Macherey-Nagel GmbH & Co., KG, Düren, Germany) were cloned into the bacterial vector 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 GmbH & Co., KG, Düren, Germany).Plasmid 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 open reading frame of the determined cDNA sequences (i.e., ATG to stop codon) were deposited at GenBank (see Table 1 for the respective accession numbers).

aox Paralogous Genes Have Been Generated Independently at Four Different Occasions in the Aspergillaceae
A survey of the whole fungal kingdom suggested that aox gene multiplicity-generally a rather rare condition-occurs multiple times in the Eurotiales order (Eurotiomycetes class, Pezizomycotina subphylum), particularly in the Aspergillaceae family.Preliminary phylogenetic analysis implied that most aox paralogous genes in Aspergillaceae have their origin within the larger clade of the Eurotiomycetidae subclass constituting the sister orders Eurotiales and Onygenales.We omitted one mixed group of Eurotiales secondary Aox proteins from Rasamsonia emersonii, Evansstolkia leycettana, Monascus, Aspergillus clavatus, A. cejpii and A. thermomutatus, that consistently clustered deep in the Dothideomycetes AoxA clade (i.e., another taxonomic class) from further analysis.
To investigate the origins of aox paralogues in species of Aspergillus and Penicillium, we collected the DNA sequences coding for more than 500 alternative oxidases from about 350 species of Eurotiomycetidae and Lecanoromycetes, mostly from the freely accessible DNA databases at the NCBI in April 2023.Almost all Eurotiomycetidae aox genes we collected typically have two phase-two introns bounding a central exon of about 300 nt, usually 291 nt (exceptions to the conserved gene model are mentioned below in Sections 3.2 and 3.5).After manual deduction of the intron-exon structures and subsequent translation of the coding sequences after removal of the introns, a maximum likelihood (ML) tree for the updated alternative oxidase complement of 531 proteins in 351 mainly Eurotiales species was generated and then rooted with a homogeneous clade of uniquely Lecanoromycetes AoxA proteins.From this tree, we deduced that paralogous aox genes have been generated (at least) four times during the evolution of the Aspergillaceae.Figure 1a graphically shows the four independent paralogous clades (alternating in red) and the approximate sites of their respective connection with the AoxA backbone (351 proteins AoxA).Supplementary Figure S1 shows the full, circular version of the ML tree (531 Aox) without clade collapses, used as the basis of the cladograms in Figure 1. Figure 1b schematically summarizes the relations between the AoxA pedigree and the four independent duplication events of which descendants persist to this present day.Figure 1c highlights the considerable sequence similarity between each of the Aox paralogues for the conserved enzyme domains encoded by the second and third exons.One duplication event appears to have occurred before the separation of the Aspergillus and Penicillium genera and comprises about a hundred paralogous Aox proteins (see below, Section 3.2).Subsequent gene loss of the acquired paralogues must have taken place frequently in evolution of the extended Aspergillus genus as there are whole sections (and series) of Aspergillus where only the ubiquitous AoxA (aoxA gene) is present.
We have described one of the four events in our previous paper on the rare aoxB paralogue found in the Aspergillus niger sensu stricto complex and in three other highly divergent species of Aspergillaceae [13].In the context of this current work, we renamed this rare aox paralogue aoxB1 (Figure 1a, scheme at the left) because its origin is the furthest away from the tips of the terminal branches (i.e., the present), the small, seemingly homogeneous paralogous AoxB1 clade consistently appearing as the sister clade of the Onygenales AoxA in alternative ML phylogenies (For clarity, all Onygenales species only specify one Aox, AoxA).

Ancient Gene Duplication in an Aspergillaceae Ancestor of Aspergillus and Penicillium
Most of the paralogous Aox in the family, including virtually all paralogous aox genes present in the Penicillium genus, appear to derive from a gene duplication event that must have occurred in early Aspergillaceae before the divergence of the sister genera (Figure 2).Most Penicillium genomes investigated feature two aox genes, although some Penicillium taxa (e.g., all species of the series Roquefortorum) only have aoxA.There were almost 100 paralogues of AoxB2-1 in our collection of 531 proteins.The basal branch of the AoxB2-1 clade is by far the longest in the ML tree, indicative of increased genetic variation.AoxB2-1 has also survived in the majority of species in the Aspergillus sections of Flavi, Terrei and Candidi included in the phylogeny (i.e., Supplementary Figure S1) as well as in the series Versicolores, one of the taxonomic series defined in the section Nidulantes (cf.[32]).Two early divergent section Flavi species, A. avenaceus and A. coremiiformis, do not have AoxB2-1 (at present).Nevertheless, typical for section Flavi, the gene encoding the ubiquitous aoxA has lost the phase-two intron at the second conserved position.Two out of seven species from our initial collection of 351 species in the early divergent section Aspergillus-A.chevalieri and A. montevidendis-harbour a genuine AoxB2-1 paralogue.In Aspergillus cristatus (two genomes public), the paralogue is a recognizable pseudogene, but A. glaucus and A. ruber only feature aoxA.In other sections of Aspergillus-Fumigati, Nigri, Circumdati, Cremei, and Usti, and in the series of section Nidulantes other than series Versicolores-the duplication AoxB2-1 is absent (at the present).The available species in the series Unguium, Multicolores and Nidulantes (including A. nidulans) only harbour the ubiquitous aoxA gene and thus must have lost B2-1.  Figure 1.Overview of the independent origins of alternative oxidase paralogous genes in the Eurotiales order and the sequence similarity between the individual paralogous Aox's.(a) A section of a ML tree of 531 Aox proteins from 351 species of Eurotiales, Onygenales and Lecanoromycetes was used.All species in the survey have the ubiquitous aoxA gene, but some species of Aspergillus and Penicillium have one or two additional paralogous (aoxB) genes named after the duplication event they emerged from (i.e., B1, B2, B3 or B4).The distinction between AoxB2-1 and AoxB2-2 is explained in the Results and Discussion section.The rooted section with only the Eurotiales-Onygenales proteins was drawn as a cladogram of collapsed taxa.Clades of related proteins collapsed at the level of Sections for Aspergillus, at the genus level for other Aspergillaceae or at the family level for the other taxa of Eurotiales.In the four schemes, the cladogram is the same, but in each, one of the four events duplications is named and highlighted by the red triangle.(b) Graphic summary of the ML analysis (Supplementary Figure S1).The source of each contemporary paralogous aoxB pedigree is the ubiquitous aoxA gene.Four events of aox gene duplication were identified and numbered from 1 to 4. The contemporary paralogue genes emerging therefrom are named accordingly.Some representative species are given for each of the paralogous clades.(c) Alignment of the peptides encoded by the three paralogous aox genes in A. calidoustus (i.e., aoxA; aoxB1; aoxB4) and the three paralogous aox genes in A. sydowii (i.e., aoxA; aoxB2-1; aoxB3).Aligned peptides start with a conserved tyrosine [Y] coded by the first intact codon in exon 2 of each of the encoding genes, all with fully conserved three-exon gene model.Identical amino acids are shaded on the black background.The amino acid similarity figures (% ID) between each couple of Aox proteins can be extracted from the matrix.The orientation and deduced function description of the product of the neighbouring genes were collected from the JGI genome browsers for freely accessible Aspergillus and Penicillium annotations.The aox paralogue gene in the centre is represented by the light blue arrows that always point to the right (5 to 3 ).The different colors represent different predicted functions for the neigbouring gene products.For the sake of clarity, the differently colored arrows are all equal in size and thus do not represent the real size of the coding regions of the neigbouring genes.

Similarity (ID %)
This big paralogue AoxB2-1 clade is linked with a satellite clade of another Aox paralogue, AoxB2-2, which currently persists in only three species of Section Flavi-A.caelatus, A. bombycis (aka A. luteovirescens), and A. arachidicola (Figure 2b; Supplementary Figure S2).Due to a lack of sequences, it is impossible to determine whether there is direct descendancy or parallel evolution of the two aoxB2 paralogues, but the three Flavi species mentioned above have both AoxB2-1 and AoxB2-2.Omission of the small satellite clade (AoxB2-2) from phylogenies of AoxA-plus-AoxB2-1 resulted in a dramatic shift in the topology of Aspergillus AoxA considering the perceived evolutionary relations amongst the Aspergillus sections.Closer inspection of section Flavi genome data suggested that AoxB2-2 paralogues in three more species, A. pseudocaelatus, A. transmontanensis, and A. novoparasiticus, are heavily degenerated.The six species are not clustered in one and the same clade (cf.[33]) but are distributed over three discernible terminal clades, series Flavi, Kitamyces and Nomiarum (cf.[32]).This unequal distribution implies that other species in these three series have been rid of AoxB2-2, all the while maintaining the AoxB2-1 paralogue.These observations may suggest that the AoxB2-2 paralogue is doomed to disappear.

Lateral Transfer of a Fungal aox Gene between Species of Different Taxonomic Classes
Contemporary paralogous genes are generally the product of gene duplication.The newly acquired copy is passed on vertically to yield defined lineages with the dual presence of paralogues.The absence of the acquired copy from taxa within such taxonomic clades can be explained by gene loss.A rare, alternative path to acquire paralogous genes is horizontal gene transfer (HGT) or lateral transfer.In the current work, we fortuitously identified an unambiguous lateral transfer between long divergent filamentous fungal taxa involving an aox gene from an Aspergillus section Flavi donor and a narrow taxon in Trichoderma (Hypocreaceae family, Hypocreales order, Sordariomycetes class; Supplementary Figure S2).Unique to two closely related cryptic species-Trichoderma asperellum and T. asperelloides [34]-we noticed a second aox gene in all 12 genome sequences available at NCBI, of which the protein product consistently and tightly clusters with the evanishing small clade of paralogues AoxB2-2 (described above).The ubiquitous aoxA gene in Trichoderma species has two phase-zero introns bounding a central exon of 399 nt, an intron-exon structure conserved in all Hypocreaceae and in almost all genome-sequenced Hypocreales.However, the intron-exon structure of the extra (second) aox gene in T. asperellum/asperelloides includes two phase-two introns binding to a central exon of 297 nt: this is exactly the gene model of the large majority of aox genes in the Aspergillus and Penicillium genera (as well as in other Eurotiales; Supplementary Figure S2a).Intron position conservation is diagnostic of orthology (cf.[35]).Clearly, the second aox gene is not the product of a duplication in the Trichoderma aoxA lineage.The protein most similar to the second Aox in T. asperellum/ asperelloides (345 amino acids) is the AoxB2-2 paralogue of equal length in Aspergillus arachidicola (Section Flavi, series Flavi; Supplementary Figure S2b).These two proteins should be considered orthologues; >86% identical over the complete gene product and >93% identical when the exon-1-encoded amino acids-including the mitochondrial signal peptide-are omitted.Limited gene synteny is conserved upstream of the aoxB2-2 gene in the opposite participants of the plausible transfer.

A Gene Duplication Seemingly Arising from within the Clade of Penicillium aoxA
Another independent event has given rise to a monophyletic clade of aox paralogues in ten taxa of Aspergillus and one species of Penicillium, P. brevicompactum (Figure 3).The ten taxa in Aspergillus (from the 351 used in Supplementary Figure S1) represent five different taxonomic sections.In section Circumdati, three species available at NCBI have the AoxB3 paralogue (Figure 3b), but A. steynii, A. sclerotiorum and A. persii lack it.On the contrary, Aspergillus uvarum (subgenus Circumdati, section Nigri, series Japonici) seems to be the only species of section Nigri to feature duplication aoxB3.A close inspection of the gene sequences identified A. sydowii (CBS 593.65) as the principal Versicolores that (still) features the AoxB3 paralogue.Gene sequence alignments imply that strains ATCC 9577, AS33 and Z5 co-identify as variants of A. sydowii (CBS 593.65).The unassigned strain Aspergillus sp.MA 6041 is arguably the only other Versicolores available at NCBI with an intact aoxB3 gene.Interestingly, A. jensenii, A. tennesseensis and A. creber feature a small 3 terminal remnant of the coding region, implying that the aoxB3 gene was present but then lost from (these) other Versicolores.Due to the lack of available sequences, we observe that AoxB3 occurrence in section Aspergillus appears to be restricted to A. chevalieri while Aspergillus wentii is the only representant of section Cremei at NCBI.Phylogenetic analyses (Figure 3b) suggest that the aoxB3 paralogue arose from a Penicillium parent rather than from an Aspergillus taxon after the separation of the two genera and independent of the earlier aoxB2-1 duplication.Changing the protein input or the substitution matrix used to build alternative AoxA-plus-AoxB3 phylogenetic trees (with the NCBI-based sequences) does have an influence on the exact "connection" of the monophyletic AoxB3 clade within the Penicillium AoxA clade.Figure 3b shows four possible "points of origin" amongst taxa of subgenus Aspergilloides or as a sister clade to subgenus Penicillium AoxA.The presence of AoxB3 in species of five diverse Aspergillus sections-Cremei, Aspergillus, Circumdati, Nigri-and in the series Versicolores of Section Nidulantes remains consistent with vertical inheritance of AoxB3 after acquisition of the paralogue by a common ancestor upon lateral transfer from a Penicillium host, followed by numerous independent gene loss events in divergent taxa.Nevertheless, the existence of a third Penicillium brevicompactum protein locked within the seemingly monophyletic AoxB3 clade may best be explained by a secondary lateral transfer of the aoxB3 paralogue from an unknown Aspergillus host.Recently, the transfer of a complete gene cluster from a Penicillium host to a narrow taxon of Aspergillus section Flavi species was reported [36].

Recent Gene Duplication at the Basis of Section Usti (Subgenus Nidulantes)
Paralogue AoxB4 is confined to species of the section Usti with the illogical exception of the third aox gene found in multiple Penicillium brasilianum genomes (Figure 4).As there are only two named section Usti genomes available at NCBI, we enlarged the sample with the aox complement in five closely related species from the Mycocosm whole-genome depository at the US Department of Energy Joint Genome Institute [18] after obtaining permission to use their unpublished genome data to identify their alternative oxidase gene content (see Material and Methods section).The evolutionary relations revealed by alternative AoxA-plus-AoxB4 phylogenies appear to be consistent with a recent gene duplication event near the basis of the section Usti (Figure 4b) after the divergence of the section Ochraceorosei.This is consistent with the conservation of gene synteny around the locus of aoxB4 integration (Figure 4c), something that was not observed amongst the present-day produce of the much older duplication events 2 and 3. Remarkably, the original ubiquitous aoxA gene has acquired a third intron (phase zero) 55 nt downstream of the 3 position-conserved phase-two intron: this phase-zero intron is unique to section Usti aoxA and does not occur section Nidulantes.The most parsimonious explanation for the presence of aoxB4 in P. brasilianum would be a recent lateral transfer from a section Usti host after the earlier aoxB4 gene duplication.From Figure 4c, one can appreciate that the piece of DNA transferred to P. brasilianum is considerably bigger than the aoxB4 gene, comprising at least four neighbouring genes.The amino acid similarity between Aspergillus ustus aoxB4 and P. brasilianum aoxB4 is 84.5% identity over the complete width of the protein and almost 90% identity for the peptide product, disregarding exon 1.This last figure is considerably higher than the similarity between the ubiquitous AoxA proteins from A. ustus and P. brasilianum, ~74% identity (disregarding exon 1).(b) relevant clade of a mixed-input maximum likelihood tree with Subgenus Nidulantes AoxA proteins and the paralogous AoxB4 proteins to localize the origin of event 4 in the Subgenus.Clades consisting of sections other than section Usti were all collapsed; (c) the contemporary environment of the aoxB4 locus.The orientation and deduced function description of the product of the neighbouring genes were collected from the JGI genome browsers (see legend to Figure 2).The aox paralogue gene in the centre is represented by the turquoise arrows.The different colors represent different predicted functions for the neigbouring gene products.

Verification of the Expression of Alternative Oxidase Paralogous (aoxB) Genes
In our ML phylogenies, no instances of loss of the original aoxA gene were observed in the set of 351 species investigated.This suggests that the function of the omnipresent aoxA gene cannot be fully replaced by paralogous aox genes wherever paralogous aox genes coexist.Regardless, a few evolutionary scattered Aspergillus and Penicillium species have "accumulated" three aox genes.We sought to establish whether these paralogue aoxB genes are factually expressed or even co-expressed with aoxA, or not.Seven species with either two or three aox genes in their genome were selected (Table 1): five Aspergilli, one Penicillium and one Trichoderma-the latter species involved in a lateral transfer of a rare Aspergillus aox paralogue (see Section 3.3).Paralogue genes originating from each of the duplication events identified (cf. Figure 1) were covered at least once in this set of species.The expression of aoxA was also assessed.First, we looked for direct evidence of RNA splicing in extant RNA sequence read archives (SRAs; Supplementary Table S2).Eurotiales and Onygenales aox genes generally have two position-conserved phase-two introns, bounding a central exon of ~300 nt (usually 291 nt).For Aspergillus wentii, we screened computer-assembled RNA contigs covering the predicted exon-exon fusions at the appropriate JGI genome browser for the absence of RNA SRAs in that species.With one exception-the first intron in the aoxA transcript in A. wentii-we found sequence reads (or EST contigs) for each of the selected aox genes, covering the predicted exon-exon fusions within the mRNAs.Thus, all nine aoxB paralogues tested were expressed by this criterion.Interestingly, exonexon fusions covering the predicted introns in both or all three aox genes in each species were encountered in the same species-specific SRA database.This implied that the tested paralogue aoxB genes were all co-expressed with their original aoxA genes (at least in the seven fungi assessed).These include both introns of the laterally transferred, Aspergillusborn aox gene (i.e., aoxB2-2) in T. asperellum (see Supplementary Figure S2).We have verified and confirmed the conclusions from the SRA screen by performing targeted RT-PCRs with gene-specific oligonucleotide primers on total RNA samples isolated from fresh liquid cultures of the seven fungi and subsequent sequence analysis of cloned cDNAs (see Materials and Methods section).The complete coding regions in the obtained cDNA sequences were deposited at GenBank (see Table 1 for the accession numbers).

Different Patterns of aoxB Gene Loss in the Aspergillus Genus
The limited representation of Aspergillus sections other than Flavi and Nigri in the public databases (NCBI) resulted in an incomplete view of the inheritance of the aoxB2-1 and aoxB3 paralogues.It is plausible to assume that duplication event 2 took place before the divergence of Aspergillus and Penicillium (Figure 2b) and thus that all emerging lineages must have had the aoxB2-1 paralogue at their onset (including Penicillium).Event 3 must have an independent origin from a Penicillium aoxA donor (Figure 3b), explicitly after the separation of the genera.Currently, aoxB3 is present in taxa belonging to the long divergent subgenera Cremei (A. wentii), Aspergillus (A. chevalieri), Nidulantes (A. sydowii) and Circumdati (e.g., A. westerdijkiae) strongly suggesting it has its origin in a common ancestor of these subgenera, putatively an ancestor to all current species in the genus.Crucially, no paralogue Aox can apparently fully replace AoxA, which appears omnipresent throughout Pezizomycotina.On the other hand, it would appear that duplication aoxB2-2 is on the edge of extinction in a few now separated narrow taxa in section Flavi that explicitly maintain the more sequence-variant aoxB2-1 paralogue (see Figure 2a).Regardless of its broad occurrence in both subgenera of Penicillium, in Aspergillus sections Fumigati, Nigri, Circumdati, Cremei, and Usti, the duplication AoxB2-1 is absent (at the present).The section Fumigati stands out for having only the ubiquitous aoxA gene, and all transient aoxB paralogues from past duplications are lost at present.
It is likely that independent episodes of paralogue aoxB gene loss have taken place in defined pedigrees of Aspergillus, eventually resulting in the present-day distribution of these two aox paralogues, aoxB2-1 and aoxB3.To track patterns of gene loss of aox paralogues in the Aspergillus genus more confidently, we expanded our data set with information about aoxB genes in the whole-genome sequences deposited at the Mycocosm webpage of the Joint Genome Institute (US Department of Energy) [18].We gained the necessary permission (see Material and Methods) to use whole-genome data (i.e., DNA contigs) from more than 170 species of Aspergillus (situation on 6 September 2023), all part of the Aspergillus whole-genus sequencing project (JGI Proposal ID: 1307).Figure 5a summarizes the status of aox paralogue genes in eight Aspergillus sections with multiple aox genes based on this wider set of Aspergillus whole-genome sequences.More complete information is found in Supplementary Table S3, deduced from reanalyses, including the extra JGI-lodged genomes.

Subgenus Section Series Paralogs
Schematic overview of detectable aox gene loss patterns in lineages of Aspergilli.(a) Occurance and distribution of multiple paralogous aox genes in eight Aspergillus sections points towards independent gene loss events; (b) patterns of aox gene loss (B2, B3) and gain (B4, B1: in Usti) in sections and series of the subgenus Nidulantes.The ubiquitous aoxA gene is always present.In both panels, the rectangles at the left of each of the schemes represent the taxonomic lineage, sections and series.The series within each of the sections are given in alternating shades of grey, and their names are abbreviated to a two-or three-letter code; No: Nomiarum; Ki: Kitamyces; Fl: Flavi; Le: Leporum; Ca: Calidousti; Us: Usti; Ve: Versicolores; St: Stellati; Ni: Nidulantes; Mu: Multicolores; Un: Unguium; Ch: Chevalierorum; As: Aspergillus (series); Ru: Rubri; We: Wentiorum; Ar: Arxiorum; In: Inflati; Te: Terrei; Am: Ambigui; Can: Candidi; Ci: Circumdati.To their right, the colored dots represent the presence of maximal three aox genes in one species, each of the six aox paralogues with its unique color: aoxA: black; aoxB1: turquoise; aoxB2-1: green; aoxB2-2: violet; aoxB3: yellow; aoxB4: magenta.In (b), three sections of the Nidulantes subgenus are abbreviated with a one-letter code; O: Ochraceorosei; U: Usti; N: Nidulantes (section).In addition, Oc: Ochraceorosei (series without aox paralogous genes not mentioned in (a)).
Inspection of the extended set showed the presence of aoxB3 beyond A. wentii in the section Cremei, with presence in three series of that early divergent section.Likewise, additional species in series Circumdati have the aoxB3 gene, albeit not all species, while other series in this section did not feature aoxB3 at all (at present).In these diverse sections, the aoxB3 gene proves to be more persistent than the aoxB2-1 paralogue.On the contrary, in sections Flavi, Terrei and Candidi-which like Circumdati belong to extended subgenus Circumdati (cf.[32])-the aoxB2-1 paralogue is preserved while aoxB3 is absent (at present).
There are also multiple Aspergillus taxa that feature both aoxB2-1 and aoxB3 at present, although not in all related species grouped in those taxa.This situation concurs with the early divergent section Aspergillus and in the series Versicolores of the section Nidulantes, taxa unambiguously belonging to different subgeneras (Figure 5a).In our initial analysis, we found one named species of each taxon with three aox genes, namely A. chevalieri and A.sydowii (respectively).One can deduce that in the section Aspergillus, the series Aspergillus and Rubri species have lost aoxB2-1 after the separation from the series Chevalierorum.Interestingly, the aoxB3 paralogue seems more vulnerable to elimination than aoxB2-1 in Chevalierorum; in all available species but A. chevalieri, aoxB3 is lost while aoxB2-1 persists in all.However, independent gene loss must have occurred recently as one species of series Rubri in our extended set-Aspergillus cumulatus-features both aoxB paralogues, i.e., the original context.
A complex pattern of aoxB gene loss and gain can be observed in the evolution and divergence of the sections and series within the subgenus Nidulantes (Figure 5b).Both paralogues arising from events 2 and 3, aoxB2-1 and aoxB3, must have been present in the last common ancestor to the whole subgenus.After the divergences of the Ochraceorosei and Usti, both paralogues aoxB2-1 and aoxB3 have been lost during independent events in both those two sections but were maintained in section Nidulantes.In section Usti, a fourth duplication event took place, giving rise to the new aoxB4 paralogous gene.(see also Figure 5a).In addition, Aspergillus calidoustus-all six genomes in the databases-trapped a rare copy of the aoxB1 duplication event (cf.[13]).In A. calidoustus, both paralogous aoxB genes are thus effectively "replaced" by two other aoxB paralogues originating from different duplication events.In the section Nidulantes, the species in most constituent series for which genome sequences are available have lost aoxB2-1 and aoxB3 on a third occasion (i.e., the fifth and sixth independent gene loss events in the subgenus), including Aspergillus nidulans, A. mulundensis and A. unguis, which all are left with just the ubiquitous aoxA gene.Indeed, the series Stellati clusters with the series Nidulantes, Multicolores and Unguium (cf.[32]), but Aspergillus angustatus has conserved its three aox genes (aoxA; aoxB2-1; aoxB3).Finally, the species in the series Versicolores all persist with the aoxB2-1 paralogue, but most of them have lost aoxB3 (i.e., the seventh independent gene loss event in the subgenus).The exceptions are A. sydowii and Aspergillus sp.MA 6041 (see Sections 3.2 and 3.4), who have both retained aoxB2-1 and aoxB3.Hence, in the two distally related series Versicolores and Chevalierorum, aoxB3 seems more transient and aoxB2-1 more persistent.
Thus, different patterns of consecutive, independent gene loss events are equally crucial to contemporary aox gene content as the original duplication or transfer events, giving rise to aox paralogue genes in the extended Aspergillus genus.

Conclusions
Alternative oxidase (Aox) is a non-electrogenic terminal oxidase operating in branched electron transport, oxidizing ubiquinol and reducing molecular oxygen without generating proton motive force over the mitochondrial inner membrane.Aox lowers the energy yield of respiration compared to the canonical electron transport chain and oxidative phosphorylation while dissipating the excess reducing power generated by fast carbon catabolism and moderating oxidative stress via reactive oxygen species, an inevitable byproduct of cytochromic electron transport.In fungi, these activities are positively correlated with sustained overflow metabolism, a feature of immense biotechnological importance.Aox protein is nearly ubiquitous in the fungal kingdom (aoxA gene), but aox gene multiplicity is rare.Nevertheless, within the Aspergillaceae family, and amongst its various industrial cell factories, like Aspergillus oryzae, A. terreus, A. niger, A. wentii and Penicillium rubens, paralogous aox genes coexist.Paralogous genes generally originate from duplication and are inherited vertically.Our study provides evidence for four independent duplication events at different points in evolution that resulted in aox (aoxB) paralogues in contemporary Aspergilli and Penicillia.The paralogous clades all arise from ubiquitous aoxA parent genes but never replace the latter: aoxA is actually persistent across filamentous fungi.The

Figure 2 .
Figure 2. Duplication event 2 and the aoxB2 paralogous genes emerging from it.(a) Amino acid alignment of the conserved C-terminal enzymatic domains of AoxB2-1 and the ubiquitous AoxA proteins from Aspergillus terreus (section Terrei).Two phase-two introns are position conserved in Eurotiales and Onygenales, in aoxA as well as in the various paralogous aoxB genes.Aligned peptides start with a conserved tyrosine [Y] coded by the first intact codon in exon 2. Identical amino acids are shaded in black background; (b) relevant mixed maximum likelihood tree for the ubiquitous AoxA proteins and the paralogous AoxB2 proteins.On the left, a summary tree with Aspergillus sections,

Figure 3 .Figure 4 .
Figure 3. Duplication event 3 and the aoxB3 paralogous genes emerging from it.(a) Amino acid alignment of the conserved C-terminal enzymatic domains of AoxB3 and the ubiquitous AoxA proteins from Aspergillus westerdijkiae (section Circumdati).Aligned peptides start with a conserved tyrosine [Y] coded by the first intact codon in exon 2. Identical amino acids are shaded in black background; (b) Mixed maximum likelihood trees with Penicillium AoxA proteins and the paralogous AoxB3 proteins; (c) The contemporary environment of the aoxB3 paralogous locus.The orientation and deduced function of the product of the neighbouring genes were collected from the JGI genome browsers (see legend to Figure 2).The aox paralogue gene in the centre is represented by the light green-blue arrows.The different colors represent different predicted functions for the neigbouring gene products.

Table 1 .
Aspergillus and Penicillium species used to confirm the expression of paralogous aoxB genes originating from the four independent duplication events and the lateral transfer to Trichoderma asperellum.