To ensure use of authenticated materials, Lodderomyces elongisporus strain NRRL YB-4239 and Spathaspora passalidarum strain NRRL Y-27907 were obtained from the Agricultural Research Service Culture Collection (Peoria, IL; https://nrrl.ncaur.usda.gov). Meyerozyma guilliermondii strain ATCC 6260 and Scheffersomyces stipitis strain CBS 6054 (ATCC 58785) were purchased from the American Type Culture Collection (https://www.atcc.org). Table 1 lists genome sequences used for this study.

New genome assemblies were constructed for L. elongisporus NRRL YB-4239 (ASM1362098v1), M. guilliermondii ATCC 6260 (ASM6942125v1), S. stipitis CBS 6054 (ASM694211v1), and S. passalidarum NRRL Y-27907 (ASM362096v1) using a combination of Illumina MiSeq and Oxford Nanopore MinION data. Details of the method and genome assembly are in Supplementary Files S1–S4, respectively. Similar methods were described previously (Oh et al., 2019; Oh et al., 2021) and were reproduced here for the reader’s convenience.

Fungal cells were stored at -80°C and streaked to YPD agar plates (per liter: 10 g yeast extract, 20 g Bacto peptone, 20 g dextrose, 20 g Bacto agar). A single colony was inoculated into YPD liquid medium and the culture grown 16 h at 30°C and 200 r/min shaking. Genomic DNA was isolated according to Sherman et al. (1986). Briefly, cells were spheroplasted with zymolyase and lysed with sodium dodecyl sulfate. The lysate was extracted with phenol, DNA precipitated with isopropanol, and the final preparation treated with Proteinase K. DNA shearing was minimized by use of wide-bore pipet tips and gentle mixing. Agarose gel electrophoresis was used to verify the presence of high-molecular-weight DNA prior to subsequent library preparation.

Libraries were constructed and sequenced at the Roy J. Carver Biotechnology Center, University of Illinois at Urbana-Champaign. Individually barcoded shotgun libraries were made using the Hyper Library construction kit (Roche) and pooled in equimolar concentration. The pooled libraries were quantitated by qPCR and sequenced on one MiSeq flowcell for 251 cycles from each end of the fragment using a MiSeq 500-cycle sequencing kit (version 2). PhiX DNA was used as a spike-in control for MiSeq sequencing runs. FASTQ files were generated and demultiplexed with bcl2fastq Conversion Software (Illumina, version 2.17.1.14). MiSeq reads were quality trimmed using Trimmomatic v0.36 (Bolger et al., 2014) to remove adaptors from the 3’ end of the reads prior to assembly, retaining all reads greater than or equal to 50 nt in length.

Libraries for Oxford Nanopore long-read sequencing used 1 μg of genomic DNA that was sheared in a gTube (Covaris, Woburn, MA, United States) for 1 min at 6,000 r/min in a MiniSpin plus microcentrifuge (Eppendorf, Hauppauge, NY, United States). The sheared DNA was converted to a shotgun library with the LSK-108 kit (Oxford Nanopore) with the Expansion barcoding kit (EXP-NBD103). The libraries were pooled in equimolar concentration and the pool was sequenced on two SpotON MK I (R9.5) flowcells for 48 h using a MinION MK 1B sequencer. Basecalling was done with software MinKNOW version 1.7.7 and demultiplexing was performed with the Albacore software version 1.2.4. Sixty bp were trimmed from each end of the reads to remove barcodes using a custom script.

Detailed assembly steps are outlined in Supplemental Files S1–S4 for the four genomes. In brief, trimming was performed using a simple Perl script to remove 60 nt from the ends of the reads, retaining reads longer than or equal to 1000 nt for the final assembly. Canu v1.5 (Koren et al., 2017) was used for assembly with the following parameters: “canu -p asm -d useGrid = false -nanopore-raw genomeSize=<GENOME_SIZE> <TRIMMED_FASTQ>”, using the relevant quality-trimmed reads from Porechop and the following estimated genome sizes as input: S. stipitis = 15.4 Mb, L. elongisporus = 15.5 Mb, M. guilliermondii = 10.6 Mb, S. passalidarum = 13.2 Mb.

Trimmed Oxford Nanopore reads were then aligned against their associated assembly using bwa v0.7.5 (Li, 2018) by first indexing the assembly using “bwa index <ASSEMBLY>”, then aligning reads using the parameters “bwa mem -x ont2d <ASSEMBLY_INDEX> <TRIMMED_READS>”. The alignment was then used to polish the assembly using nanopolish v0.7.1 (Senol Cali et al., 2018). Quality-trimmed MiSeq data were used to further polish the assembly using Pilon v1.22 for error correction (Walker et al., 2014). Assembly names and GenBank accession numbers for the genome sequences for L. elongisporus NRRL YB-4239, M. guilliermondii ATCC 6260, S. passalidarum NRRL Y-27907, and S. stipitis CBS 6054 are located in Table 1.

Methods for identifying ALS genes and deducing predicted protein features were nearly identical to those reported in previous publications (Oh et al., 2019; Oh et al., 2021). Details were reproduced here for the reader’s convenience. BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi) was used to identify potential ALS genes and Als proteins in the genome sequences (Table 1). BLAST searches were also conducted using the Candida Genome Database (www.candidagenome.org; Skrzypek et al., 2017). Query sequences included all C. albicans ALS genes as reported by Oh et al. (2019). As new ALS/Als sequences were identified, they were also used as BLAST queries until search reports failed to reveal new sequences.

ALS gene sequences were verified or corrected by Sanger sequencing of PCR-amplified products. Primers were designed using the Primer Quest Tool (https://www.idtdna.com) and primers purchased from Integrated DNA Technologies (Coralville, IA). PCR used Q5 High Fidelity DNA Polymerase (New England Biolabs) according to the manufacturer’s instructions. Extension time was adjusted to promote amplification of the desired product size. PCR products were purified using the MultiScreen HTS 96-well Filtration System (Millipore), then Sanger sequenced at the Roy J. Carver Biotechnology Center (University of Illinois at Urbana-Champaign). A list of primer sequences is located in Supplementary Table S1.

SignalP-5.0 Server (http://www.cbs.dtu.dk/services/SignalP; Nielsen, 2017) was used to locate putative secretory signal peptides. The Big-PI Fungal Predictor (https://mendel.imp.ac.at/gpi/fungi_server.html; Eisenhaber et al., 2004) identified potential GPI anchor addition sites. Dotmatcher (http://emboss.bioinformatics.nl/cgi-bin/emboss/dotmatcher) was used to detect repeated sequences. The ExPASy Server was used to assess amino acid sequence composition (https://web.expasy.org/protparam; Gasteiger et al., 2005). European Bioinofrmatics Institute (EMBL-EBI) tools were used for translating nucleotide sequences, sequence alignment, and other general processes (https://www.ebi.ac.uk/services; Cook et al., 2017). β-aggregation potential in amino acid sequences was assessed using Tango (http://tango.crg.es; Fernandez-Escamilla et al., 2004; Rousseau et al., 2006). All nucleotide sequences were translated with the alternative yeast nuclear code (translation table 12) except those from C. glabrata and S. cerevisiae which used the standard code (translation table 1). There was disagreement about the translation table for L. elongisporus: the NCBI database used translation table 1 even though the species was recognized as part of the CUG clade that translates CUG as Ser instead of Leu (translation table 12; Santos et al., 2011).

The kingdom Fungi phylogeny of Li et al. (2021), based on 290 genes from 1644 species, was pruned using the R package ape (Paradis and Schliep, 2019). C. metapsilosis was not included in the phylogeny. Because of its close relationship to C. parapsilosis and C. orthopsilosis (Tavanti et al., 2005), C. metapsilosis was attached at the root of these sister species.

Sequences for phylogenetic analysis were trimmed to include only the NT-Als domain, which has adhesive function (Lin et al., 2014). Secretory signal peptide sequences were included in the analysis. Tango (http://tango.crg.es; Rousseau et al., 2006; Fernandez-Escamilla et al., 2004) was used to define the amyloid-forming region (AFR) that marks the C-terminal end of NT-Als (Lin et al., 2014). Sequences that did not encode an AFR were trimmed to a length similar to others in the dataset. Amino acid sequences used for phylogenetic analysis were compiled into Supplementary File S5. Sequences were aligned using MAFFT v7.453 (Katoh and Standley, 2013). A phylogeny was inferred using the best fit model WAG+G+I and subsequent bootstrap analysis was performed using RAxML-NG (Kozlov et al., 2019).

Protein structural predictions were derived using AlphaFold (Jumper et al., 2021), accessed using AlphaFold Colab (https://colab.research.google.com/github/deepmind/alphafold/blob/main/notebooks/AlphaFold.ipynb). ColabFold (Mirdita et al., 2021; https://colab.research.google.com/github/sokrypton/ColabFold/blob/main/AlphaFold2.ipynb) was used for more-rapid generation of structural prototypes. ColabFold utilized MMSeqs2 (UniRef+Environmental), as well as a custom MSA file constructed using Hhblits Toolkit server (Zimmerman et al., 2018; Gabler et al., 2020).

Clustal Omega (Madeira et al., 2019) was used to align amino acid sequences from Supplementary File S5 with those from 4LE8, the Protein Data Bank (https://www.rcsb.org) entry for the crystallographic structure of C. albicans NT-Als3 (chain A, 299 amino acids). This molecule did not include the secretory signal peptide at the N-terminal end nor the amyloid-forming region at the C-terminal end. Sequences in Supplementary File S5 were trimmed according to the Clustal Omega alignment and the trimmed sequences were used for AlphaFold analysis. Sequences for other fungal cell-surface proteins were included as controls in the structural analysis: C. albicans Hyr1 (NCBI Reference Sequence: XP_722183.2, amino acids 21 to 309) and S. cerevisiae Flo1 (NCBI Reference Sequence: NP_009424.1; amino acids 25 to 273). Structural visualization and alignments used the PyMOL Molecular Graphics System, Version 2.5.2 (Schrödinger, LLC).

Examination of new and existing genome sequence data (Table 1) provided the information needed to locate, assemble and/or validate the sequence for 47 ALS loci (5 from Lodderomyces elongisporus NRRL YB-4239; 3 from Candida auris B8441; 3 from Scheffersomyces stipitis CBS 6054; 4 from Meyerozyma guilliermondii ATCC 6260; 29 from Spathaspora passalidarum NRRL Y-27907; and 1 each from Clavispora lusitaniae ATCC 42720, Yamadazyma tenuis ATCC 10573, and Debaryomyces hansenii CBS 767). These sequences are presented here in the context of previously described ALS loci including 8 from Candida albicans SC5314 (Hoyer et al., 2008), 7 from Candida dubliniensis CD36 (Jackson et al., 2009), 13 from Candida tropicalis MYA-3404 (Oh et al., 2021), 5 from Candida parapsilosis CDC 317 (Oh et al., 2019), 3 from Candida orthopsilosis Co 90-125 (Lombardi et al., 2019), and 4 from Candida metapsilosis ATCC 96143 (Oh et al., 2019). Supplementary Table S2 details all gene names, GenBank accession numbers, and predicted protein characteristics. Gene names were taken from locus tags associated with the annotated reference genome for each species. For genes that were not annotated in the reference genome, “0.5” was added to the name, indicating that the ORF was located between two previously annotated genes. Figure 1 displays the phylogenetic relationship between the species described here.

The C. albicans NT-Als3 structure was solved crystallographically (Lin et al., 2014; Protein Data Bank accession number 4LE8), providing experimental information that can be used to produce structural predictions for other NT-Als proteins. A crystallographic structure has not been reported for S. cerevisiae Sag1. The recent development of the highly accurate protein structural prediction program AlphaFold (Jumper et al., 2021) and widespread access to its use (e.g. Mirdita et al., 2021) provided the opportunity to assess predicted structural relatedness between the diverse set of NT-Als sequences and their similarity to Sag1.

Figure 3 shows the crystallographic structure of C. albicans NT-Als3 from 4LE8 (Figure 3A) and the AlphaFold-predicted structure for the same amino acid sequence (Figure 3B). The root-mean-square-deviation (RMSD) value for alignment of the two structures was 0.53, indicating the ability of AlphaFold to reproduce the crystallographic structure of NT-Als3 from its amino acid sequence. AlphaFold was then used to predict the structures of other NT-Als proteins, particularly those with limited sequence identity to C. albicans NT-Als3 (Figures 3C–G). Strikingly, a similar structure was predicted for each protein, including Sag1. Analysis of each NT-Als sequence in Supplementary File S5 produced the same general protein shape (data not shown) suggesting that all, including Sag1, were unified as a family. To demonstrate diversity in structural predictions, the amino acid sequences for the N-terminal domain of the cell-surface proteins C. albicans Hyr1 (amino acids 21 to 309) and S. cerevisiae Flo1 (amino acids 25 to 273) were entered into AlphaFold. The resulting structural predictions were different from the Als family and from each other (Figures 3H, I).

In order to provide biologically meaningful adhesive activity on the fungal cell surface, Als proteins need a secretory signal peptide and GPI anchor addition site to achieve an effective localization (Lu et al., 1994). The crystallographic structure of C. albicans NT-Als3 (Lin et al., 2014) demonstrated the involvement of 8 Cys residues in folding of the mature protein and the function of an invariant Lys at the end of the binding cavity that sinks the negative charge of incoming peptide ligands. Other work demonstrated the importance of the amyloid-forming region (AFR) for increasing the avidity of aggregative interactions (Ho et al., 2019). Supplementary Table S2 evaluated each of these features for the previously characterized and newly introduced Als proteins. The data were summarized in Figure 4 from which various hypotheses emerged.

The cluster of dots in the top center of Figure 4 contained proteins with 8 Cys and an invariant Lys. Each protein represented in the top center cluster of dots also had an AFR with high predicted β-aggregation potential and a location immediately C-terminal to the NT-Als domain as in C. albicans Als proteins (Salgado et al., 2011; Lipke et al., 2018). The top center cluster of dots included 7 of the 8 C. albicans Als proteins, 6 of 7 from C. dubliniensis, and 9 of 13 from C. tropicalis suggesting robust potential for functional adhesive interactions by these species. Also present in the top center cluster of dots were 2 of the S. stipitis Als proteins, 2 from S. passalidarum, and 1 each from C. parapsilosis, C. metapsilosis, L. elongisporus, and Y. tenuis.

Viewing potential Als function according to these criteria provided predictions about the adhesive activity in other species. For example, C. parapsilosis, C. orthopsilosis, and C. metapsilosis Als proteins almost universally had a weaker or absent AFR leading to the hypothesis that these species are functionally less adherent than C. albicans, C. dubliniensis, and C. tropicalis. Data in Figure 4 suggested a similar hypothesis for S. passalidarum: despite its overabundance of ALS-like loci, S. passalidarum encoded only two proteins (SpAls138016 and SpAls156463; Supplementary File S10) that possessed the functional features present in NT-Als3 and therefore may not be a very adherent species. However, nine SpAls proteins had AFR sequences 20-30 amino acids C-terminal to the location where they are found in C. albicans Als3 (Supplementary Table S2). Experimentation is required to evaluate whether sequences with high β-aggregation potential in this alternative location contribute to S. passalidarum aggregative interactions. Using the criteria in Figure 4, C. lusitaniae was an outlier. Its sole Als protein, ClAls3274, had only 4 Cys residues and no AFR. Despite these key differences, the predicted structure for the NT-ClAls3274 domain was similar to those from other species (Figure 3D). The contribution of ClAls3274 remains to be tested, as does the overall adhesion profile for the species.

Proteins with features that more closely resemble Sag1 than C. albicans Als3 segregated to the left column of the diagram. Substitution of Arg (R printed in the dot) in place of the invariant Lys was a common theme. Three of the four M. guilliermondii Als proteins were in the left column. Perhaps these proteins have more-specific, higher-affinity adhesive interactions that more-closely resemble those of Sag1, rather than NT-Als3. The same prediction could be made for two of the C. auris proteins, as well as the sole protein from D. hansenii.

Information presented in Figure 4 and Supplementary Table S2 showed that some species had a complement of Als proteins that possessed features of both S. cerevisiae Sag1 and C. albicans Als3. For example, S. stipitis SsAls4579 had Sag1-like features (lower left, Figure 4) while SsAls2386 and SsAls2786 looked more like C. albicans Als3 (top center, Figure 4). The line diagrams in Supplementary File S9 reinforced this idea, illustrating the extensive regions of tandemly repeated sequences in the latter two proteins (C. albicans Als-like) and lack of a repeated domain in SsAls4579 (Sag1-like). M. guilliermondii was a similar mixing bowl of Als proteins: MgAls673 was the most Sag1-like of the group. Each of the other MgAls proteins had a long stalk of tandemly repeated units with NT domains that perhaps were more like Sag1 than Als3 (Supplementary File S7). L. elongisporus NT-Als proteins also appeared in more than one column in Figure 4 with one (LeAls734) sharing greater similarity with Sag1. The recombinant nature of some of the LeAls proteins presented a more-complex picture than for S. stipitis or M. guilliermondii (Supplementary File S6).

In addition to comparing NT-Als proteins based on the features described above, more-global comparisons were made. Clustal Omega alignment of the NT-Als sequences (Supplementary File S5) showed identity values ranging from approximately 20% to 90% (data not shown). A maximum likelihood tree was drawn to visualize the results (Figure 5).

The topology of the tree reflected the overall diversity in NT-Als sequence identity. For example, NT-Als sequences from the closely related species C. parapsilosis/C. orthopsilosis/C. metapsilosis appeared on short branches in tight groups. In constrast, many branches showing the relationship between the highly diverse S. passalidarum NT-Als sequences were quite long, most notably for SpAls153035.5, which was the least related to other proteins in that species.

Als sequences from the same species tended to cluster together which made the overall tree organization similar to the species tree in Figure 1. The close relationship between C. albicans and C. dubliniensis (Jackson et al., 2009), as well as that between C. parapsilosis/C. orthopsilosis/C. metapsilosis (Tavanti et al., 2005), were reflected by the proteins from these groups being localized to distinct regions of the tree. C. tropicalis sequences were similarly clustered as were most of the S. passalidarum sequences. C. auris and C. lusitaniae sequences were grouped closely, similar to their position on the species tree (Figure 1). Within the groupings of closely related species, orthologs were obvious (e.g. CaAls6/CdAls86290, CaAls7/CdAls86150, CpAls660/CoAls800 and CoAls4210/CmAls4210-1). Examples of paralogs, likely the result of gene duplication, included CpAls4770/CpAls4780/CpAls4800, CtrAls3791/CtrAls2293/CtrAls3786, and SpAls64434/SpAls64435.

Similar to predictions made from the Figure 4 diagram, NT-Als sequences from some species were spread across the tree. The four M. guilliermondii Als sequences were found in three different locations: MgAls673 was grouped more closely to sequences like ScSag1; MgAls2302 and MgAls3259 were clustered together near proteins from C. auris; and MgAls3330 was on its own branch elsewhere on the tree. Low bootstrap support for the latter location suggested that MgAls3330 could easily be located differently on the tree. The three sequences from S. stipitis were also dispersed with one near ScSag1 and the two others closer to the C. albicans/C. dubliniensis proteins. The five L. elongisporus Als proteins were split into two groups: the first including LeAls2536/LeAls5708/LeAls2721 (likely paralogs) and the second including LeAls734/LeAls2716 (recombinants between Als and Iff/Hyr). The Sag1-like features for SsAls4579 and MgAls673 were reflected on the tree (Figure 5), while LeAls734 and LeAls2716 (both Als recombinants with Iff/Hyr) clustered together in a different location.

The tree in Figure 5 was drawn from a portion of each protein sequence. Another way to evaluate phylogenetic relationships is via conservation of physical location (synteny) as depicted using the Candida Gene Order Browser (CGOB; Maguire et al., 2013). CGOB includes all species in Figure 5 except C. glabrata (CAGL0G04125g). A screenshot from one region of the CGOB was included in Figure 2C. It showed genes from several different fungal species that were orthologs of C. albicans ALS6 and ALS7. Orthologs reported by CGOB are shown in Table 3. Yellow hexagons with alphabetical labels were used to display the CGOB relationships on the tree in Figure 5.

CGOB ortholog designations were most reliable in closely related species such as C. albicans and C. dubliniensis: each C. albicans locus had a C. dubliniensis ortholog except CaALS3 and CaALS5 which are missing from the C. dubliniensis genome (Jackson et al., 2009). These ortholog groups were frequently augmented with genes from other species such as Group A which included SsALS2386. Groups E and F were the largest, suggesting ortholog relationships between genes in 7 and 6 species, respectively.

CGOB also proposed ortholog groups that did not include C. albicans or C. dubliniensis sequences. One example was Group J that included the SAG1-like MgALS673 and SsALS4579, as well as DhAls2178, the lone ALS sequence from the primarily haploid D. hansenii. Examination of the predicted protein sequence showed lack of a tandem repeat domain as well as other Sag1-like features (Supplementary File S11 and Figure 4).

Some CGOB ortholog designations were perhaps problematic. For example, PICST_31095 (Group D) belonged to the IFF/HYR family instead of ALS and ScFLO1(Group G) encoded a flocculin with a mannose-binding N-terminal domain that was structurally diverse from the Als family (Figure 3). Perhaps the most intriguing ortholog designation was Group K which included ScSAG1 and CdALS65010. CdALS65010 was nearly 100% identical to CdALS64800, which was included in ortholog Group B. These loci are near each other on C. dubliniensis chromosome 6 and the appear to be paralogs (i.e. recently duplicated; Jackson et al., 2009). As species become more divergent, assessing ortholog designations becomes more challenging. However, the suggestion of orthology between ScSAG1 and a C. dubliniensis ALS gene supported the general idea that the genes were part of the same family and arose from a common ancestor.

The rapidly increasing number of genome sequences present in public databases provided an opportunity to further explore the breadth and potential origin of the ALS family. The NCBI database can be queried using taxonomic identification (taxid) numbers to detect potential ALS loci more broadly across the phylogenetic tree. Table 4 lists the taxids for designations of the major Families within the Order Saccharomycetales. Inputs from NCBI (lifemap-ncbi-univ-lyon1.fr), Shen et al. (2018), and Li et al. (2021) were included although the taxonomic designations were not identical among the sources. The portion of the kingdom Fungi tree (Li et al., 2021) that represented the Subphylum Saccharomycotina was reproduced in Figure 6.

Figure 6 indicated the position of a common ancestor (red dot) for the ALS family that would include both S. cerevisiae SAG1 (in the Saccharomycetaceae) and genes more closely resembling C. albicans ALS loci (in CUG-Ser1). BLAST searching using taxids did not reveal ALS loci in available genome sequences from the Lipomycetaceae, Trignopsidaceae, Dipodascaceae, Trichomonascaceae, Alloascoidaceae, or the Sporopachydermia clade (Table 4). CUG-Ser1 was comprised of the Debaryomycetaceae, Metschnikowiaceae, and Cephaloascaceae (Shen et al., 2018). BLAST searching using taxids suggested that there were numerous additional ALS genes among the 88 Debaryomycetaceae species genomes in NCBI (accessed September 2021). Among the 62 Metschnikowiaceae species, ALS sequences were found primarily in species closely related to C. auris and C. lusitaniae including Candida haemulonii, Candida duobushaemulonis, and Candida pseudohaemulonii, all of which have been observed as pathogens (Jurardo-Martin et al., 2020). C. intermedia, noted for its ability to ferment xylose, as well as a rare cause of catheter-related fungemia (Ruan et al., 2010; Geijer et al., 2020) also had ALS sequences. No ALS sequences were detected in the Cephaloascaceae, although only 2 genomes were available (Table 4).

Pichia kudriavzevii (formerly Candida krusei; Douglass et al., 2018) was included in Table 4 and in Figure 1 since the species is among those associated with clinical disease (Gomez-Gaviria and Mora-Montes, 2020). However, ALS genes were not detected in Pichia kudriavzevii, nor in the genomes of 60 other species from the Family Pichiaceae that were available in the NCBI database (Table 4). No ALS sequences were detected in the species included in the CUG-Ala clade (Figure 6). The lone BLAST hit in the Saccharomycopsidaceae (Table 4; overlap with CUG-Ser2 in Figure 6) was based more on the presence of Ser/Thr-rich repeated sequences than on an NT-Als adhesive domain. Additional work would be required to validate the sequence and determine if an NT-Als domain was present.

Potential ALS genes in the Phaffomycetaceae (Table 4) were located in two species each in the genera Wickerhamomyces and Cyberlindnera; each locus encoded a potential NT-Als domain. Many additional ALS sequences were found among the 104 Saccharomycetaceae genomes available in NCBI. This family included S. cerevisiae, for which SAG1 was the only BLAST hit. For other species such as Torulospora delbrueckii, a species of interest for wine making and baking (Fernandes et al., 2021), more than one ALS sequence was detected in the genome. One (TDEL_0E04030) predicted a protein that more closely resembled S. cerevisiae Sag1, while another (TDEL_0G04810) encoded an NT-Als domain (validated using AlphaFold; data not shown) attached to a complex C-terminal Ser/Thr-rich sequence. This predicted C-terminal domain was notable because it included 5 copies of the 45-amino acid tandem repeat found in S. cerevisiae Flo1 (Soares, 2010), suggesting recombination between the ALS and FLO families. A predicted protein in Lachancea thermotolerans (KLTH0C00440p) suggested an even-more-convincing recombinant that had an NT-Als domain attached to > 30 copies of the Flo1 tandem repeat. These sequences require verification to ensure the recombinants were part of the genome, rather than an artifact of computational genome assembly. ALS sequences were also detected in Kazachstania species extending the representation of ALS sequences throughout the Saccharomycetaceae as defined by Shen et al. (2018). Many of these sequences appeared partial, suggesting the need for PCR amplification and repair to reveal the true nature of their encoded proteins. No ALS sequences were detected among the 21 Saccharomycodaceae species queried.

The taxonomic category Saccharomycetales incerta sedis had four ALS sequences, all in the species Diutina rugosa (formerly Candida rugosa), which is noted as an infrequent human pathogen (Ming et al., 2019). Evaluation of the best hit (DIURU_002000) showed a predicted secretory signal peptide and GPI anchor addition site. Within the NT-Als region, DIURU_002000 was 37% identical to C. albicans NT-Als3, had 8 Cys in the expected locations, Arg instead of the invariant Lys, and a strong predicted AFR. AlphaFold produced the same general structure for the DIURU_002000 NT-Als protein as for those shown in Figure 3 (data not shown).

Collectively, these observations pinpointed the location of the ALS family among currently available genome sequences and suggested potential taxonomic boundaries that will continue to be tested as more genome sequences are added to this region of the phylogenetic tree.