Intragenomic Variability of ITS Sequences in Bjerkandera adusta

Bjerkandera adusta is a species of common white rot polyporoid fungi found worldwide. Despite playing an important role in deadwood decay, the species strains are used in bioremediation due to its ability to degrade polycyclic hydrocarbons and some of them are important etiological agents of chronic coughs and are associated with lung inflammations. In our experiments, diversity within the species was investigated using molecular approaches and we found that sequence diversity seen at ITS sequence level is not due to cryptic speciation but to intragenomic variability of ITS sequences in this species.

Moreover, few cases of isolating the Geotrichum-like imperfect stage of B. adusta lacking basidiospores and producing asexual arthroconidia (anamorph) were reported, namely from the fungal culture on agar plate with preferred medium [7,8], the Belize biodeteriorated compact disc [9], cultivated soil using pulp after industrial production of daunomycin as a substrate in Poland [10,11], and from Asian sand dust aerosol [12]. The Geotrichum-like imperfect stage of B. adusta isolated from this latter substrate induces allergic lung diseases.
This fungus has a symptomless endophytic life stage, within the roots of common reed Phragmites australis from Germany [13], the thallus of moss Sphagnum fuscum from Canada [14], xylem of Chilean trees Drimys winteri [15], the lycophyte Huperzia serrata, needles and twigs of Abies beshanzuensis, the roots of Sinosenecio oldhamianus from China [16][17][18], leaves of Sarracenia purpurea from the USA [19], and within sapwood and leaves of Hevea spp. from Brazil, Peru and Mexico [20]. Endophyte sequences identified as B. adusta had a similar pattern, in which these sequences clustered together [20].
B. adusta is an efficient lignin degrader and it is also able to oxidize xenobiotic compounds including some environmental pollutants making it a potential candidate for bio-technological and environmental applications, such as decolorization of commercially used diazo, anthraquinone, triphenylmetane, azo, phthalocyanine and other dyes in textile, leather, cosmetic, pharmaceutical and paper industries [18,[21][22][23][24]. In addition to attractive enzyme activities, B. adusta is capable of producing a wide range of metabolites with interesting biotechnological applications, e.g., disaccharide α-α-trehalose, or antimicrobial activities [21,25].
With the recent development of molecular techniques, cryptic diversity within numerous polypore morphospecies was recognized. Despite the abovementioned biotechnological and environmental importance of the B. adusta, there is only limited data on variability of the species. The morphological variability observed in B. adusta concerns the phenotypic variation of basidiomata, mode of growth, their organization and the degree of tubes development. Several forms (e.g., f. resupinata, f. solubilis, f. tegumentosa) were described during the species history [2,26]. However, molecular analysis did not reveal substantial variability (less than 0.55%) within ITS sequences of Korean B. adusta specimens [27].
The aim of the present study was to analyze the intraspecific genetic variability of Bjerkandera adusta using molecular approaches based on ITS and tef1-α sequences.

Materials and Methods
To analyze the variability in B. adusta specimens from Slovakia (central Europe), multiple basidiomata (18) were collected (see Table S1). For total genomic DNA isolation, fresh basidiocarps were ground by oscillating mill (MM200, Retsch GmbH, Haan, Germany) and DNA was isolated from a small amount of basidiocarp tissue (about 100 mg) using E.Z.N.A. Fungal DNA Mini Kit (Omega Bio-tek, Inc., Norcross, GA, USA). Quality and quantity of isolated DNA was analyzed using electrophoresis in 1.5% agarose gel.
The sequences were checked for similarity against the GenBank database using blastn algorithm and deposited into the database under accession numbers ON391760-ON391777 for ITS sequences and ON411223-ON411229 for tef1-α sequences. For identification of polymorphisms, multiple sequence comparisons using MEGA-X software were used [30].
For RFLP analysis, approximately 0.5 µg of amplified ITS amplicons were cleaved with 5U of BstUI restriction endonuclease (Thermo Fisher Scientific, Inc., Cleveland, OH, USA) at 37 • C in R buffer as specified by the manufacturer. Restriction fragments were analyzed using electrophoresis in 1.5% agarose gel and visualized by Molecular Imager, ChemiDocTM XRS+ system (Bio-Rad Laboratories, Inc., Hercules, CA, USA) using ethidium bromide (0.5 µg/mL).

Results and Discussion
The internal transcribed spacer (ITS) sequence analysis is generally recognized and widely used for identification, systematics and phylogenetics of fungi. Sequence comparisons of B. adusta ITS sequences available in GenBank database (436 sequences by the end of March 2022) indicated the existence of two type of sequences in the analyzed dataset. Two types of sequences (referred to in this paper as type A and type B) differ by five transitions. One of the transitions is located in the ITS1 region, the rest of the transitions are located in the ITS2 region ( Figure 1). In general, the ITS1 region is considered to be more variable than the ITS2 region within the Fungi kingdom; this variation included length and GC content variations as well as polymorphisms [31]. Our comparisons indicate that no clear geographical pattern could be recognized among type A and B sequences, as both types of sequences were reported worldwide. Analysis of available B. adusta ITS sequences (436 GenBank entries) indicated that a weak geographical pattern could be recognized among type A and B sequences. Although both types of sequences were reported worldwide, it seems that type B sequences are more frequently detected in specimens from Europe. For example, no type B sequences were found among 72 entries from the USA and only 2 from 206 entries from China were classified as type B. On the other hand, 13 out of 48 entries from Europe possess GA sequence in 467-468 position in the ITS2 region and can be classified as type B (data not shown). Sequence comparisons of Slovakian B. adusta specimens showed that 7 out of 18 obtained sequences could be classified into type A and 3 into type B sequences. The remaining 8 sequences could not be typed due to the ambiguity of the sequences, observed at all polymorphic positions (marked as AxB type, Table S1). Careful examination of GenBank data showed that similar ambiguity can be seen in several GenBank entries as well. Similar situations as seen in our study have been detected in other research, e.g., in B. adusta sequences from Chile. From the 18 sequences available, 3 can be classified as type B, 13 as type A and the remaining 2 sequences show the same polymorphism as detected in our specimens.
A closer inspection of sequencing chromatograms revealed that the ambiguities occurred in polymorphic but no other sites, and rather than sequencing errors, these basidiomata might possess both types of ITS region sequences (Figure 2). To evaluate the polymorphism at nucleotide position 522 in ITS sequences of B. adusta (G→A transition), a RFLP method was developed. The G→A transition at this position led to the loss or appearance of CGCG tetranucleotide, a recognition site for BstUI restriction endonuclease. RFLP analysis confirmed that both types of ITS sequences are present simultaneously in multiple B. adusta basidiomata ( Figure 3) and no profiles other than the expected restriction profiles were observed. Cleavage by BstUI restriction endonuclease yielded a restriction fragment with size of about 400 bp and two smaller fragments of about 125 and 85 bp in type A isolates. Due to the transition at nucleotide 522 in type B isolates and consequent loss of BstUI site, these specimens produced a banding pattern possessing just two fragments of about 500 bp and 125 bp. In specimens possessing both types of ITS sequences, both 400 and 500 bp fragments were seen simultaneously. Comparison of the intensity of bands indicated that in specimens possessing both types of ITS sequences, type B clearly dominated over type A ITS sequences. Based on the data, it could be concluded that more than half of the basidiomata tested (either by ITS sequencing or RFLP analysis) possessed two different copies of ITS sequences differing by 5 nucleotides (0.9%) in their genome.
The level of sequence polymorphism observed in B. adusta ITS sequences is much higher than the average error rates of Taq polymerase (between 0.1% and less than 0.01%, e.g., Potapov and Ong [32]) and is very close to the threshold values for delimiting fungal species. Some closely related sister species show similarities at ITS level as high as 99.5% [33,34]. Generally, similarity level 98.5% is used for species delineation, e.g., by UNITE and ISHAM databases [35,36]. The presence of intragenomic variability within ITS sequences could hamper correct identification of fungal specimens, especially if ITS amplicons are cloned in Escherichia coli vector prior to sequencing.
Intragenomic variability within ITS sequences of B. adusta was not accompanied by variability at other markers widely used for species delineation in fungi, the partial translation elongation factor (tef1-α) sequence. Partial tef1-α sequences (510 nt) were amplified from both ITS type A and B basidiomata as well as from specimens having hybrid ITS sequences. However, all obtained tef1-α sequences were completely identical, irrespective of ITS type (data not shown). Generally, tef1-α sequences show a higher degree of divergence in closely related species compared to ITS sequences [29,37,38] and the lack of variability seen in B. adusta probably indicates the genetic homogeneity of the species. Based on these data, we conclude that sequence diversity seen at ITS sequence level in B. adusta is not due to cryptic speciation but due to intragenomic variability of ITS sequences in this species.
Intragenomic ITS variability is a well-known phenomenon for bacteria, plants and animals but there is limited evidence on the occurrence of such variability in fungi (e.g., Hughes et al. [39], Stadler et al. [40]), mainly in Ascomycota. In a well-documented example of intragenomic ITS variability in Basidiomycota, Hughes et al. [39] observed variability at 28 ITS positions in Amanita cf. lavendula collections in eastern North America, Mexico and Costa Rica. In the smut fungi Ceraceosorus spp. [41], intragenomic variation of ITS sequences varied in up to four sites only, similar to the extent of ITS variability seen in our experiments. Similarly, in a polypore fungus Trichaptum abietinum, three nonorthologous ITS1 types were detected [42] and Type I and Type II ITS1 sequences were found to coexist in all tested T. abietinum strains.
There is no clear explanation for the observed variability of ITS sequences. Possibly, observed variability could represent an initial state of hybridization event between two divergent taxa as proposed by McTaggart and Aime [43]. However, due to a limited number of specimens possessing pure type B ITS sequences, we were unable to identify some geographical, ecological or substrate specialization differences separating this group from type A group. In our collection, both type A and type B specimens were found within less than 1 km distance in Bratislava municipality and similarly, both type A and type AxB specimens were found in Sala municipality. Likewise, the examination of data from GenBank database indicated that both type A and type B specimens can occur sympatrically.
Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/jof8070654/s1, Table S1: Origin of specimens and variability of Bjerkandera adusta ITS sequences obtained through this study.  Data Availability Statement: All sequence data are available in NCBI GenBank following the accession numbers in the manuscript.