Characterization and Phylogeny of Fungi from Industrial Wastewater Using Highly Conserved Multiple Gene Loci

The aim of this study was isolation and molecular characterization of fungi from untreated industrial euent by multigene phylogenetic analyses. The Fungi isolated were characterized based on PCR amplication and genomic sequencing of the internal transcribed spacer region (ITS), partial β-tubulin (Ben A), calmodulin (CaM), and DNA-directed RNA polymerase second large subunit (RPB2) genes, along with morphological characterization and species diversity. Fungal DNA extraction kits and primers sets for the selected genes were purchased and used following the manufacturer’s instructions. The obtained sequences were subjected to BLAST analysis and the corresponding fungal isolates were assigned species names after comparison with representative sequences available in GenBank. All the sequences from this study were deposited in GenBank and the accession number assigned. Phylogenetic trees of the fungal isolates were drawn for each gene by the Maximum Likelihood method using MEGA 7.0 software. Fifteen (15) Fungi species belonging to four genera of Aspergillus, Penicillium, Fusarium and Trichoderma with Aspergillus as the predominant genus were identied.


Introduction
Fungi are diverse group of eukaryotic organisms characterized as heterotrophic, saprophytic, symbiotic and parasitic due to their achlorophyllous nature. Their cell walls are made up of β-glucans and chitin (Manoharachary et al., 2010). They are known as the second largest after kingdom Animalia with estimate of over 5 million species (Raja et al., 2017). The kingdom fungi is composed of nine phyla mamely; Opisthosporidia, Chytridiomycota, Neocallimastigomycota, Blastocladiomycota, Zoopagomycota, Mucoromycota, Glomeromycota, Basidiomycota and Ascomycota (Simeos et al., 2015;Yakop et al., 2019). The Ascomycota or sac fungi, are monophyletic and account for approximately 75% of all described fungi. Generally, fungi are signi cant both economically and medicinally as they are the major decomposers of dead organic matter and contribute signi cantly in recycling of nutrients in natural and modi ed ecosystems. They are also found in edible forms, producers of antibiotics, cheese formers and few of them are pathogenic.
Apart from known enormous importance of Fungi, the taxonomy of these organisms is still challenging due to a lack of reliable and advanced techniques for their identi cation and systematic studies. Morphological or conventional approaches to fungal systematics have some di culties in determining species boundaries as these techniques of identi cation rely primarily on phenotypic analysis. For instance they describe colour, texture, spores, colony size and utilization of carbon and nitrogen compounds (Das et al., 2014). Thus other characteristics are needed for identi cation to species level. Gene sequence based identi cation of fungi has many advantages over the conventional methods as it does not require viable organisms or sporulation.
In order to resolve the di culties on identi cation at species level, many advanced techniques have been employed for the identi cation of fungi with emphasis on house-keeping genes and other target genes through the use of PCR and automated sequencing techniques.
The internal transcribed spacer regions (ITS) are used as o cial universal DNA barcode for fungi (Das et al., 2014;Yin et al., 2017).
The ITS1, ITS2, and ITS4 have been proven to be useful for the identi cation of yeasts and some fungi such Aspergillus, Penicillium, Talaromyces, Cryptococcus, candida and Trichosporon among many others (Adeniyi et al., 2018;Rajeshkumar et al., 2019;Yin et al., 2017). However, ITS sequences cannot be used for phylogenetic analyses of unrelated taxa due to low variability and slow evolution. Also, ITS sequences do not always allow correct species differentiation especially among Aspergillus and Penicillium genera (Houbraken & Samson, 2011;Visagie et al., 2014). Hence, additional gene markers are essential for correct species delineation. Secondary molecular markers such as beta tubulin; calmodulin and RPB2 have been successfully used in fungal genomics (Kolanlarli et al., 2019;Raja et al., 2017;Rajeshkumar et al., 2019;Visagie et al., 2014). Asan et al., 2011 reported that these protein-encoding genes contained highly variable intron regions which contain highly variable introns that evolve at a faster rate compared to ITS.
Beta-tubulin genes are found in all eukaryotes encoding for polypeptide proteins. They have been used for phylogenetic analysis in fungi from the entire kingdom to the species level. Four Beta tubulin genes are found in all fungi; two α-tubulin (tub A) and two β-tubulin (tub B) genes. Tub A is responsible for the production of two alpha tubulin polypeptides (alpha 1 and alpha 2) while Tub B produces one alpha polypeptide (alpha 2) (Das et al., 2014). Reports have it that beta-tubulin gene sequences contain 3.5-fold more phylogenetic information than the small sub-unit (SSU) rRNA gene, thus it has been reported that it is an ideal marker for analysis of deep-level phylogenies and for complex species groups (Raja et al., 2017;Visagie et al., 2014).
Calmodulin (CaM) is a small acidic protein present in all eukaryotic cells and shown to be highly conserved both functionally and structurally (De Carvalho et al., 2003;De Cássia et al., 2001). Its primary role is to serve as an intracellular Ca 2+ receptor which signals proliferation, motility, and cell cyclic progression. Ca 2+ -CaM complexes act by controlling the activity of numerous intracellular proteins such as phosphodiesterase, Ca 2+ -ATPase, serine protein kinases, and protein phosphatases. It also acts on several metabolic pathways and gene expression regulation in many eukaryotic organisms including fungi (De Carvalho et al., 2003).
Transcription of the eukaryotic nuclear DNA into RNA is carried out by RNA polymerases using ribonucleoside triphosphates as substrates (Gerber et al., 2020;Malkus et al., 2006). RNA polymerase II gene (RPB2) encodes for second largest protein subunit in eukaryotes which synthesizes mRNA precursors and functional non-coding RNAs (Malkus et al., 2006). Vetrovsky et al. (2015) reported that RPB2 gene is a viable alternative molecular marker for the analysis of environmental fungal communities due its discriminative power, quantitative representation of community composition and suitability for phylogenetic analyses. Therefore this study was aimed at isolation and molecular identi cation of fungi from waste water using the selected molecular markers discussed.

2.1
Collection of untreated re nery e uents Samples of untreated e uent were collected from waste water channel of Kaduna Re nery and Petrochemical Company (KRPC), Kaduna State, Nigeria. The samples were collected in ten-litre new plastic jerry cans and transported to the laboratory for physicochemical, heavy metal analysis and biosorption studies. The pH and temperature of the e uent were determined at the point of collection.

2.2
Isolation and molecular characterization of test fungi from untreated re nery e uent 2.2.1 Isolation of fungi from untreated waste water The e uent samples collected were kept to stand at room temperature (30 o C) on a sterile laboratory work bench. 10ml of the samples in duplicates were aseptically dispensed in sterile centrifuge tubes and centrifuged at a speed of 250rpm for 10minutes to concentrate the samples. After decanting the supernatant, 0.1ml of the residue of each sample was spread-plated on sterile potato dextrose agar and Malt Extract Agar (MEA) plates in duplicate (containing 50µg/L of chloramphenicol) using sterile bent glass rod.
The plates were incubated at room temperature for 7days.

Colony morphology and microscopic characterization of fungal Isolates
Colonies grown on each medium were distinguished on the basis of their surface characteristics such as texture, colour, zonation, sporulation and diameters. The distinguishable colonies were sub-cultured on PDA slant and incubated at room temperature (25 o C) for 7days to obtain pure isolates. The microscopic characteristics were carried out by mounting small portion of the growing region of the fungi on a clean grease free slide with a drop of lacto phenol cotton blue, covered with a cover slip and examined under electron microscope using ×40 objective lens. The isolates were characterized on the basis of the sexual reproductive structures, presence or absence of septation, presence of foot cells and chlamydospores. The isolates were identi ed using taxonomic guide (Barnett and Hunter;1999;Hakeem and Bhatnagar 2011;Asan et al., 2011 Each of the isolates was grown on potato dextrose agar at room temperature (25 o C) for 5 days. This was followed by sub-culturing each isolates into a 250 mL Erlenmeyer ask containing 100 mL potato dextrose broth and incubating for 5 days. The mycelial mass produced by each isolate were separated from the broth by ltration through sterile No. 5 Whatman lter paper. The mycelial mass were crushed using porcelain mortar and transferred to Eppendorf tubes for extraction.

PCR ampli cation of the target genes
Primers speci c for internal transcribed spacer region (ITS), beta-tubulin gene (benA), calmodulin gene (CaM) and RNA polymerase II second largest subunit (RPB2) loci are presented in Table 1.
PCR ampli cation of the extracted DNA was performed in a 20 µL reaction mixture as follow: 1 µL gDNA template, 0.2 µL DNA polymerase, 0.5 µL each forward and reverse primers, 1µL dNTPs and sterile double distilled water to a nal volume of 20 µL.
The thermocycler was programed for the following PCR conditions: initial denaturation at 94 o C for 5 min, 35 cycles of denaturation at 94 o C for 45 s, annealing at 55 o C for 45s, and extension at 72 o C for 1 min, with a nal extension at 72 o C for 10 min. For the ampli cation of RPB2 gene region, touch-up PCR conditions of 5 cycles with annealing temperature 48 °C followed by 5 cycles at 50 °C and nal 25 cycles at 52 °C were used. After complete ampli cation, the PCR products were analyzed for gel electrophoresis by using 1% agarose gel (1g of agarose in 100ml of Tris buffer) with ethidium bromide as the staining agent (Houbraken & Samson, 2011).

Sequencing and phylogenetic analysis
The fungal isolates were identi ed by DNA sequencing according to standard protocols. Sequencing was carried out in a 28μl reaction mixture as follows: 4μl of each primer, 8μl of puri ed DNA and 16μl of PCR water and the samples was sequenced with the Di-Deoxy Terminator sequencer. The contigs (formed from forward and reverse sequences) obtained were analyzed using BioEdit 7.2.5 software and aligned using Clustal W of MEGA 7.0 software (Goujon et al., 2010;Kumar et al. 2015;Yin et al., 2017). The corresponding fungal isolates were assigned species names after comparison with representative sequences available in NCBI (National Center for Biotechnology Information). The newly obtained sequences were deposited in GenBank and accession number assigned.
The evolutionary history of the fungi was analyzed using the Maximum Likelihood (ML) method based on the Tamura-Nei model of MEGA 7 (Kumar et al. 2015;Sidiq et al., 2016). The bootstrap tree formed from 1000 replicates represents the evolutionary history of the taxa analyzed The percentage associated taxa clustered together in the bootstrap test (1 000 replicates) is shown next to the branches (Houbraken et al., 2010) 3. Results And Discussions 3.1 Cultural and microscopic characteristics of fungal isolates Fifteen (15) fungal isolates consisting of four genera; Aspergillus, Penicillium, Fusarium and Trichoderma were obtained in this study with Aspergillus being the predominant genera (Table 2, Figures 1a and 1b).The results revealed the cultural features of the isolates (F1 to F23) in terms of colour, surface characteristics, reverse, edge and diameter. The microscopic features of the isolates are presented in Figures 2(a) and 2(b) showing the conidia, spores and conidiophores. Aspergillus species had septate hyphae, hyaline conidiophores and radial conidial head bearing the spores (Figure 2a). Penicillium species appeared as septate hyphae with conidiophores and secondary branches (metulae). The metulae bear asked shaped phialides with unbranched chains of round conidia (Figure 2b). Fusarium spp showed septate hyphae, multiseptate canoe shaped macroconidia attached to the conidiophores (Figure 2b). Trichoderma spp appeared as septate hyphae, short conidiophores which are ask shaped clustering together at the end of each phialides (Figure 2b).
The genus Aspergillus is one of the most well researched fungi genera with over 200 o cially recognized species (Oyebanji et al., 2018). The ubiquitous nature of Aspergillus may be due to their saprophytic feeding habit as well as their ability to grow in a wide range of environment (Machido et al., 2014). This observation sturdily indicates that members of these fungal genera isolated, have the capacity to survive and withstand toxic effects of polycyclic aromatic hydrocarbons (Ezeonuegbu et al., 2014).

Polymerase Chain Reaction (PCR) of fungal isolates obtained
The fungal isolates from this study were further subjected to molecular identi cation, by amplifying the internal transcribed spacer (ITS), beta tubulin gene (Ben A), calmodulin gene (CMD) and RNA Polymerase II Second Largest Subunit (RPB2) genes of the fungal isolates (Figures 3-6).

Ampli cation of internal transcribed spacer
Thirteen out of the fteen isolates were positive to PCR ampli cation of ITS regions with amplicon sizes of 600 base pairs ( Figure  3). Aspergillus niger (F3) and P. simplicissimum (F23) represented by lanes 4 and 16 respectively were negative. Although, the ITS region is widely used as universal primers for fungi, it is not su cient for identifying most fungi to specie level due to their low variability and slow evolution (Houbraken & Samson, 2011;Tiwari et al., 2011). Visagie et al. (2014) however suggested the use of other molecular markers for accurate identi cation of fungal species and phylogenetic relationships. Other secondary identi cation markers for Aspergillus and Penicillium species (and other ascomycetes) used in this study were beta tubulin; calmodulin and RPB2. These protein-encoding genes contained highly variable intron regions (Asan et al., 2017;Ozdil et al., 2017;Yin et al., 2017).

Ampli cation of beta-tubulin gene
Ampli cation of the beta tubulin gene revealed isolates represented by lanes 2, 4, 7, 10, 12 respectively were positive with amplicon sizes of 600 base pairs (Figure 4). Isolates represented by lanes 3, 5 and 15 respectively had amplicon sizes of approximately 550bp. Also, isolates represented by lanes 9, 11, 14 and 16 respectively had amplicon sizes of 500bp. Fungi represented by lanes 6, 8 and 13 respectively were negative (Figure 4).
Ampli cation of the partial beta-tubulin gene was successful in thirteen isolates (13)  It is an ideal marker for analysis of deep-level phylogenies and for complex species groups. Reports have shown that beta tubulin genes have more resolution compared to the ITS region. This amount of variation is suitable for phylogenetic relationship among closely related species of Penicillium and Aspergillus species (Asan et al., 2019;Samson et al., 2014).

Ampli cation of RPB2 gene
The ampli ed partial RPB2 genes of the isolates revealed that only two isolates, P. citrinum (F19) and P. citrinum (F19D) represented by lanes 5 and 7 were positive with amplicon sizes of approximately 650 and 600 base pairs respectively ( Figure 5).
This result is in agreement with the studies of Houbraken & Samson, (2011) who identi ed Penicillium citrinum using RPB2 genes.

Ampli cation of Calmodulin gene
The result of the ampli ed calmodulin genes of the isolates showed that A. niger (F5), A. niger (F10) and P. citrinum (F19) represented by lanes 5, 9 and 15 had sizes of 500bp, 550 bp and 500bp respectively ( Figure 6).
The calmodulin gene has been considered important for the identi cation of Aspergillus species, and some reports have even stated it should be used as the primary gene for identi cation of Aspergillus species (Ashtiani et al., 2017;Samson et al., 2014).

Gene Sequences of Fungal Isolates
The identi cations based on cultural features were con rmed by sequence analysis of the isolates. Basic Logical Alignment Search Tool (BLAST) results of ITS region, Beta-tubulin, RPB2 gene and calmodulin gene sequences of this study in National Center for Biotechnology Information (NCBI) provided indications of possible relationships and similarities with reference sequences in GenBank. The ampli ed sequences of each gene were submitted to GenBank, and their accession numbers are shown in Table 3 which revealed that most isolates had above 95% similar identity to reference sequences of GenBank.
There has been little or no extensive research on identi cation of the Fungi using different molecular marker approach in Nigeria. Focus has been more on macroscopic and microscopic features.

Phylogenetic Tree
Phylogenetic trees of the fungal isolates revealed that the isolates were clustered in grouping pattern of close resemblance. The percentage of trees in which the associated taxa are clustered is shown next to the branches. Sequences from this study are shown in red colours while sequences from GenBank are shown in black. Test of phylogeny was bootstrap of 1000 replications. Phylogenetic tree based on ITS gene revealed that the alignment matrix contained 54 nucleotide sequences with 209 positions in the nal dataset. All isolates of Aspergillus and Penicillium species were clustered with ex-type strains from Genbank with cluster identity of above 95%. The tree was out grouped by T. erinaceum (Figure 7).
Beta-tubulin gene alignment matrix contained 52 nucleotide sequences with with 19 positions in the nal dataset. All the fungal species had above 85% cluster similarity with ex-type from GenBank while P. simplicissimum was placed in the out group ( Figure  8).
Phylogenetic tree based on partial RPB2 gene revealed that the alignment matrix involved 19 nucleotide sequences with a total of 404 positions in the nal dataset. The two positive isolates of Penicillium citrinum shared 90% cluster similarities with sequences from GenBank ( Figure 9).

Conclusion
1. There has been little or no extensive research on identi cation of the Fungi using different molecular marker approach in Nigeria. Focus has been more on cultural and microscopic features. The fungal isolates from this study were further subjected to PCR ampli cation coupled with DNA sequencing of four molecular genes markers.
2. The fungal species isolated consist of the following genera; Aspergillus, Penicillium, Fusarium and Trichoderma with Aspergillus being the predominant genera. Sequence results obtained revealed above 95% similarities between the isolates in this study and those found in GenBank.
3. The identi cation and molecular characterization of the fungal isolates to specie level, gave a better results by PCR ampli cation and sequencing of ITS region, partial beta tubulin, calmodulin and RPB2 genes. Therefore should be used as molecular markers for species level identi cation of fungi (especially Aspergillus and Penicillium as proved by this study).

Con ict of interest
The authors declare that they have no competing nancial interests or personal relationships that could have in uence the work reported in this paper.   Phylogenetic tree of partial ITS gene sequences by maximum likelihood Figure 8 Phylogenetic tree of partial β-tubulin gene sequences by maximum likelihood Figure 9 Phylogenetic tree of partial RPB2 gene sequences by maximum likelihood Figure 10 Phylogenetic tree of calmodulin gene sequences by maximum likelihood