Molecular and Culture-Based Identification of Aspergillus Species in Water-Impacted Homes Following Hurricane María in Puerto Rico

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Vélez-Torres, Benjamín Bolaños-Rosero, Filipa Godoy-Vitorino, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6786592/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 14 Oct, 2025 Read the published version in Scientific Reports → Version 1 posted 11 You are reading this latest preprint version Abstract Flooding caused by Hurricane María promoted fungal growth in homes across Puerto Rico, raising concerns about indoor air quality and health risks. This study focuses on identifying Aspergillus species from water-impacted homes in San Juan using culture-based and molecular methods. Aspergillus is a common indoor contaminant in moisture-damaged environments, with some species associated with significant health risks. However, species-level identification is often limited. To address this, we collected samples from 14 homes, identifying 28 Aspergillus isolates through morphological examination and gene sequencing of ITS2, beta-tubulin ( benA ), and calmodulin ( CaM ) genes. Species-level identifications of 22 isolates revealed species belonging to the subgenera Aspergillus , Nidulantes , and Circumdanti . We highlighted the CaM gene's importance in molecular identification by phylogenetic analyses, which showed superior resolution in species differentiation. Culture-based methods also played a crucial role in differentiating closely related species, such as A. flavus and A. oryzae , which molecular methods alone could not reliably separate. Our findings underscore the challenges of Aspergillus identification in post-hurricane, water-impacted indoor environments and emphasize the value of integrating phenotypic and genotypic techniques for accurate species identification. These results contribute to a better understanding of fungal diversity and its potential public health implications in disaster-affected settings. Biological sciences/Microbiology Biological sciences/Molecular biology Aspergillus Hurricane María Water-Impacted Homes Species-Level Identification Molecular and Culture Methods Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Hurricane María, which struck Puerto Rico in September 2017, caused widespread devastation, including severe flooding that damaged homes across the island. The floods created the ideal conditions for indoor fungal growth, prompting air quality concerns and potential respiratory health repercussions for the residents. Our previous research confirmed a significant increase in fungal proliferation inside flooded homes in San Juan, with Aspergillus species being the most predominant. 1 This finding aligned with existing literature, which identifies Aspergillus species as common fungi in water-damaged environments. 2–5 Aspergillus is a large genus of filamentous fungi within the Ascomycota division, comprising around 350 species. 6,7 The genus is subdivided into six subgenera, 27 sections, and about 75 series, holding significance as both a pathogen and a source of pharmaceuticals, as well as in agriculture and food production. 8,9 Given its medical and economic importance, accurate identification of Aspergillus species is essential, as different species vary in pathogenicity and ability to produce harmful mycotoxins. Species-level identification is especially crucial in environments affected by flooding, where Aspergillus can pose serious health risks ranging from allergic reactions to severe respiratory infections, depending on the species and the vulnerability of exposed individuals. 9–12 Traditionally, Aspergillus classification has relied on macro- and micromorphological features, such as colony diameter, spore color, and microscopic structures like conidial head arrangement and vesicle size. 13 However, identifying closely related species remains challenging and requires specialized training in fungal taxonomy and microscopy. 14 Since the early 2000s, molecular techniques have increasingly complemented morphological approaches, with DNA sequencing becoming a standard tool for species identification. 6 The internal transcribed spacer (ITS) region is the official DNA barcode for fungi. However, secondary markers like calmodulin ( CaM ) and beta-tubulin ( benA ) are often necessary for distinguishing closely related Aspergillus species. 6,15 These molecular markers offer greater precision in species differentiation, with amplified sequences typically compared against databases like GenBank. 6 Despite the well-documented role of Aspergillus in moisture-damaged indoor environments, research on species-level identification in post-flood conditions remains limited. 5,16,17 Following extreme weather events such as hurricanes, there is a critical need for studies integrating phenotypic and genotypic techniques to accurately identify Aspergillus species in water-impacted homes. This work addresses that gap by using culture-based and molecular methods to characterize Aspergillus diversity in post-Hurricane María homes in San Juan, Puerto Rico. By combining traditional morphological and molecular approaches, it offers a more comprehensive view of fungal presence and diversity in settings susceptible to flooding. This study enhances our understanding of Aspergillus species in water-impacted environments and demonstrates the value of integrating both phenotypic and genotypic methods to improve species identification accuracy. Accurate identification is essential for assessing health risks and informing public health interventions to mitigate fungal contamination in flood-affected areas. Results Morphological characteristics of Aspergillus isolates. We selected 28 Aspergillus isolates from the sampled homes and two positive controls ( A. fumigatus isolate and A. brasiliensis genomic DNA) to achieve species-level identification. The isolates were obtained from 13 different homes (classified as water-impacted, including both flooded and rain-infiltrated homes), with most samples collected approximately one year after the hurricane. Isolates were taken from various locations within the homes (seven from living rooms, seven from kitchens, six from bedrooms, six from bathrooms, and two from outdoor areas) ( Supplementary Table S1 ). To provide a culture-based identification to the species level of the Aspergillus isolates selected, we collected a comprehensive description of macro and micromorphological characteristics applicable to the identification of common Aspergillus species (Tables 1 and 2 ). Colony growth rates on various standardized media and microscopical characteristics are taxonomically informative (Fig. 1 ). Through the culture-based approach and using three different identification keys (Klich, Klich & Pitt, Samson), we were able to identify 22 out of 28 of our Aspergillus isolates and the positive control ( A. fumigatus ) (Table 2 ). The identified isolates belong to different subgenera ( Aspergillus , Circumdanti , and Nidulantes ). Isolates A50, A77, A111, A143, A159, A293 and A299 remained unidentified. Table 2 Micromorphological characteristics of Aspergillus isolates after G25N microcultures observation. Isolate Conidial Heads Seriation Vesicle Shape Vesicle Size Conidial Shape Conidial Size Conidial Texture Identity of Isolates 19 Radiate Biseriate Pyriform 8 µm Globose 3.24 µm Smooth A. versicolor 20 Radiate Biseriate Globose 47 µm Globose 2.65 µm Smooth A. westerdijkiae 27 Radiate Biseriate Globose 81 µm Globose 4.11 µm Finely Roughened A. foetidus 33 Radiate Biseriate Globose 61 µm Globose 4.5 µm Rough A. niger 50 Radiate Biseriate Globose 7.99 µm Globose 2.23 µm Rough Unidentified 73 Radiate Biseriate Globose 34.10 µm Globose 2.04 µm Finely Roughened A. niger var. awamori 75 Radiate Uniseriate Globose 33.49 µm Spherical and Ovoid 5.41 x 4 µm Spinose Eurotium chevalieri = A. chevalieri 77 Radiate Biseriate Sub globose 9.5 µm Globose 2.56 µm Smooth Unidentified 111 Radiate Biseriate Globose 7.28 µm Globose 3.69 µm Smooth Unidentified 137 Radiate Uniseriate Spathulate 10.82 µm Subspherical 4.01 µm Finely Roughened A. penicillioides 139 Radiate Biseriate Sub globose 10.84 µm Spherical 3.56 µm Rough A. sydowii 143 Radiate Uniseriate Pyriform 11.35 µm Globose 3.25 µm Smooth Unidentified 150 Radiate Biseriate Spathulate 12.57 µm Globose 2.9 µm Finely Roughened A. versicolor 155 Radiate to Columnar Uniseriate Elongate 12.69 µm Globose 6.79 µm Finely Roughened A. flavus 159 Radiate Biseriate Globose 36.17 µm Globose 2.12 µm Smooth Unidentified 173 Loosely Columnar Uniseriate Spatulate 7.63 µm Globose 2.72 µm Smooth A. penicillioides 215 Radiate Biseriate Globose 29.81 µm Globose 3.5 µm Finely Roughened A. westerdijkiae 226 Radiate Uniseriate Globose 25 µm Ovoid 4.6 µm Finely Roughened Eurotium chevalieri 229 Radiate Both Globose 6.88 µm Globose 5.0 µm Coarsely Roughened A. tamarii 232 Radiate Uniseriate Pyriform 10.65 µm Globose 2.16 µm Smooth A. penicillioides 273 Radiate Biseriate Globose 55.87 µm Globose 3 µm Smooth A. awamori 276 Radiate Uniseriate Globose 55.56 µm Globose 4.05 µm Coarsely Roughened A. niger var. niger 277 Radiate Uniseriate Globose 76.87 µm Globose 3.83 µm Rough A. aculeatus 293 Radiate Biseriate Globose 13.29 µm Globose 1.24 µm Smooth Unidentified 299 Radiate Uniseriate Globose 54.6 µm 3.82 µm Smooth Unidentified 321 Radiate Uniseriate Sub globose 30.6 µm Sub globose 7.6 µm Finely Roughened Eurotium rubrum 323 Radiate Uniseriate Globose 31.1 µm Globose 4.42 µm Rough A. japonicus 372 Radiate Uniseriate Globose 18.56 µm Globose 4.01 µm Finely Roughened A. oryzae A. fumigatus Columnar Uniseriate Spathulate 17.18 µm Ovoid 2 µm Smooth A. fumigatus DNA extraction and Polymerase Chain Reaction (PCR) of Aspergillus isolates obtained. To begin the molecular identification approach, we successfully extracted the genomic DNA of all 28 Aspergillus isolates and positive control ( A. fumigatus ). The genomic DNA concentration from our samples ranged from 11–120 ng/µL; 50 µL of the product was used for PCR amplification experiments. To achieve fungal identification to the species level of Aspergillus isolates, we amplified the internal transcribed spacer region 2 (ITS2), beta-tubulin ( benA ), and calmodulin ( CaM ) target genes via PCR. Twenty-two out of twenty-eight Aspergillus isolates were positive to PCR amplification of ITS2, benA , and caM genes with amplicon sizes ranging between 300–400 base pairs (bps), 400–5660 bps, and 400–580 bps, respectively. Annealing temperature troubleshooting results for Aspergillus isolates #27, #73, #273, #276, #277, and #299 were unsuccessful. Gene sequence homology of Aspergillus isolates We employed Basic Logical Alignment Search Tool (BLAST) results from the ITS2, benA , and CaM gene sequences of this study on the National Center for Biotechnology Information (NCBI) to assess their sequence similarity with reference sequences in GenBank. The results in Table 3 revealed that most isolates had an identity above 99% similar to reference sequences in GenBank. Table 3 Molecular-based identification of 29 Aspergillus isolates. The percentage of similarity and E-value to reference sequences of GenBank is shown per gene and Aspergillus isolate. “–” denotes that no clear PCR products were obtained using primers from Table 4 . Isolate Code Identity of Isolate Identity (%) to GenBank Sequences and E-Value Molecular Approach ITS2 E-value benA E-value caM E-value 19 A. versicolor 100 7e-163 100 0.0 99.80 0.0 20 A. steynii 99.68 1e-159 100 0.0 100 0.0 27 - - - - 33 A. niger 100 5e-164 100 0.0 100 0.0 50 A. arenarioides 100 1e-159 100 0.0 100 0.0 73 - - - - 75 A. chevalieri 100 3e-161 100 0.0 100 0.0 77 A. petersonii 100 1e-159 100 0.0 99.64 0.0 111 A. subalbidus 100 7e-163 100 0.0 100 0.0 137 A. penicillioides 100 2e-168 99.78 0.0 100 0.0 139 A. sydowii 100 4e-160 100 0.0 99.60 0.0 143 A. hordei 100 2e-168 99.77 0.0 100 0.0 150 A. versicolor 100 3e-161 100 0.0 99.80 0.0 155 A. flavus 100 1e-160 100 0.0 100 0.0 159 A. ochraceopetaliformis 100 1e-159 100 0.0 100 0.0 173 A. gracilis 100 1e-165 100 0.0 100 0.0 215 A. westerdijkiae 100 1e-165 100 0.0 100 0.0 226 A. chevalieri 100 1e-160 100 0.0 100 0.0 229 A. tamarii 100 7e-163 100 0.0 100 0.0 232 A. hordei 100 7e-168 99.36 0.0 100 0.0 273 - - - - 276 - - - - 277 - - - - 293 A. flocculosus 99.38 1e-160 99.82 0.0 100 0.0 299 - - - - 321 A. pseudoglaucus 100 8e-157 99.75 0.0 100 0.0 323 A. brunneoviolaceus 100 2e-153 98.38 0.0 100 0.0 372 A. oryzae 100 6e-148 100 0.0 100 0.0 A. fumigatus (DNA extraction control) A. fumigatus 100 2e-163 100 0.0 99.82 0.0 A. brasiliensis (PCR control) A. brasiliensis 100 3e-161 100 0.0 100 0.0 Comparison between culture and molecular-based methods for Aspergillus isolates identification. Results from culture-based and molecular-based approaches matched for eleven (11) out of the twenty-eight (28) Aspergillus isolates compared (isolates A19, A33, A75, A137, A139, A150, A155, A215, A226, and A229) and the positive control ( A. fumigatus ). The culture-based approach provided preliminary species identification to isolates A27, A73, A273, A276, and A277, which couldn’t be identified through molecular-based approaches (no PCR products were obtained). More importantly, macro and micromorphological characteristics were very useful in distinguishing the closely related species A. flavus and A. oryzae (isolates A155 and A372), where ITS2 , benA , and CaM genes showed > 100% similarity to both A. flavus and A. oryzae . Although culture-based techniques yielded incorrect species identification to five isolates (A20, A173, A232, 321, and 323), the species identified were classified under the same subgenera and/or section ( Supplementary Table S2 ). For example, isolate A20 ( A. steynii ) was incorrectly classified as A. westerdijkiae through culture techniques, but both species belong to the Circumdanti subgenera. The same is observed for isolate A323 ( A. brunneoviolaceus ), identified as A. japonicus through the culture approach, but both species are members of the Nigri section. Isolates A173 ( A. gracilis ) and A232 ( A. hordei ) are both incorrectly classified as A. penicillioides through culture-based techniques, but they all belong to the Restricti section. In terms of isolate A321 ( A. pseudoglaucus ), which was incorrectly identified through macromorphological characterization as A. rubrum (formerly Eurotium rubrum ), a note was highlighted in the identification key stating the close resemblance of this species to Eurotium repens , now known as A. pseudoglaucus . Phylogenetic analysis of Aspergillus isolates. We inferred the evolutionary history of the Aspergillus species isolated from the sampled homes using the Maximum Likelihood method and the Tamura-Nei model in MEGA12. 18 To measure the consistency of the phylogenetic tree, we employed a bootstrap of 1,000 replications. The percentage of trees in which the associated taxa clustered is shown next to the branches. Sequences generated in this study are marked with a red diamond in the phylogenetic tree, while reference sequences obtained from GenBank are unmarked. Hamigera avellanea ( Aspergillaceae ) was used as the outgroup in all trees. The phylogenetic analysis of Aspergillus isolates showed that they grouped into distinct clusters, indicating close genetic relationships. The phylogenetic tree based on the ITS2 gene revealed that the alignment matrix contained 70 sequences, comprising 28 isolates from this study and 42 representative sequences from GenBank ( Supplementary Table S3 ). After applying a 95% site coverage threshold to exclude positions with gaps or missing data in more than 5% of sequences, the final dataset consisted of 242 nucleotide positions. Isolates from Aspergillus species classified under the same taxonomical section had above 83% bootstrap support, with several species-level clusters reaching 99% (e.g., A. versicolor , A. sydowii , A. westerdijkiae ) (Fig. 2 ). The beta-tubulin ( benA ) gene alignment matrix also contained 69 sequences (28 isolates from this study and 41 reference sequences from GenBank). To reduce the influence of poorly aligned regions, the partial deletion option was applied, excluding positions with less than 95% site coverage, resulting in a final dataset of 277 positions. All isolates showed > 83% bootstrap-supported clustering with known reference species from GenBank (Fig. 3 ). High-confidence clusters were observed for species such as A. versicolor , A. sydowii , and the closely related pair A. flocculosus / A. ochraceopetaliformis , each receiving 99–100% support. In contrast, some members of the Nigri section showed weaker support, ranging from 57–70%. The phylogenetic tree based on the CaM gene had an alignment matrix that also included 70 sequences (28 isolates from this study and 42 reference sequences from GenBank). The partial deletion option was applied to eliminate positions with less than 95% site coverage, resulting in a final alignment of 366 positions. All isolates, except for A. petersonii , showed over 80% cluster similarities with representative sequences from GenBank (Fig. 4 ). Different but closely related species like A. flocculosus / A. ochraceopetaliformis (series Steynorium ) and A. flavus / A. oryzae (series Flavi ) were still clustered together in their respective clades. Discussion In this study, we applied both phenotypic and genotypic methods to achieve species-level identification of 28 Aspergillus isolates from homes affected by Hurricane María in San Juan, Puerto Rico. Using molecular techniques, we identified 22 isolates at the species level, categorizing them into the subgenera Circumdanti (n = 12), Aspergillus (n = 8), and Nidulantes (n = 3). The Circumdanti and Nidulantes subgenera, each encompassing over 100 species, represent some of the most diverse Apergillus species . 19,20 Meanwhile, the Aspergillus subgenus, characterized by xerophilic species, thrives in low-moisture environments, making it prevalent indoors. 21,22 Our phylogenetic analysis of ITS2, benA , and CaM sequences showed distinct clustering patterns. ITS2-based trees showed strong bootstrap support (≥ 83%) for most section-level groupings, with species like A. versicolor , A. sydowii , and A. westerdijkiae forming distinct clades supported at 99–100%. The benA gene tree also produced high-confidence clusters, particularly for A. versicolor , A. sydowii , and the pair A. flocculosus / A. ochraceopetaliformis , though bootstrap support was lower (57–70%) for some Nigri section members. The CaM gene tree resolved most isolates at the species level, except for A. petersonii , with species-level clusters for A. flavus , A. oryzae , and A. westerdijkiae supported at 98–100%. These results reinforce the CaM gene’s superior discriminatory power compared to ITS2 and benA, consistent with previous studies. 23,24 However, six isolates, likely belonging to the black Aspergilli in the Nigri section, could not be identified at the species level due to the limitations of both molecular and phenotypic methods. 25,26 While sequence-based identification is the gold standard, morphological traits remain essential for resolving closely related species. For example, in this study, differentiating A. flavus from A. oryzae required morphological analysis, as molecular results alone were inconclusive. 27 Additionally, morphology-based methods allowed for preliminary identification of five black Aspergilli isolates that molecular methods could not fully resolve, underscoring the complementary role of culture techniques in complex identifications. 28 Despite identifying 22 isolates through morphological keys, five showed discrepancies with molecular results but remained in the same subgenus or section, highlighting limitations of morphological identification for certain taxonomic groups. 29 Expanding morphological keys is critical for identifying emerging Aspergillus species in indoor air environments. Most isolates identified in this study, such as A. flavus (A155), A. niger (A33), A. penicillioides (A137), A. sydowii (A139), A. tamarii (A229), A. versicolor (A19, A150), and A. westerdijkiae (A215), have previously been reported as common indoor fungal contaminants. 19,30 Specifically, A. versicolor , A. sydowii , and A. penicillioides , all from xerophilic or primary colonizer sections, are often associated with building dust and are well adapted to indoor environments. 21,22 In Puerto Rico, these species have been detected at high concentrations in indoor fungal population studies. 31,32 Taxonomic classification of our isolates revealed representation across sections, including Circumdanti , Nigri , Petersoniorum , Candidi , and Flavi . For example, isolates from section Circumdanti , such as A. steynii (A20) and A. westerdijkiae (A215), are known producers of the mycotoxin ochratoxin A 33 . Members of the Nigri section, such as A. niger and A. brunneoviolaceus , are also prevalent indoor contaminants. 34,35 Finally, Flavi section members like A. flavus and A. oryzae highlight the health implications of mycotoxigenic Aspergillus species in indoor air. 36 While many of these species are recognized as typical indoor fungi, their frequent recovery in this study suggests they may become more dominant or persistent in post-flood indoor environments, particularly under the warm and humid conditions typical of Puerto Rico. Though Aspergillus species have been broadly reported after hurricanes 2,4,5 , this study provides one of the few species-level analyses of indoor Aspergillus communities in a post-hurricane setting. 37,38 Our findings highlight A. versicolor , A. flavus , A. niger , A. westerdijkiae , and A. sydowii as prevalent species in water-impacted homes, warranting further study into their environmental persistence and potential health risks. This study has several strengths, notably the combined use of phenotypic and genotypic techniques for Aspergillus identification. The integration of morphological methods with molecular analysis using ITS2, ß-tubulin ( benA ), and especially the calmodulin ( CaM ) gene, which provided superior resolution for species differentiation, allowed for more accurate species-level identification and phylogenetic analysis of Aspergillus isolates. Morphological characterization using culture techniques also played a key role in distinguishing closely related species like A. flavus and A. oryzae , which were difficult to tell apart using molecular methods alone. 27 This combined strategy is especially important in post-flood indoor settings, where precise identification can help guide effective public health responses. However, there are limitations to consider. Molecular techniques, while powerful, did not fully resolve all isolates, especially black Aspergilli in the Nigri section, where species-specific differentiation remains challenging. 26,39 Additionally, the final datasets used in our Maximum Likelihood trees were reduced through partial deletion (to 95% site coverage), resulting in 242, 277, and 366 nucleotide positions for ITS2, benA , and CaM , respectively. While these are generally acceptable for fungal phylogenetics, the limited variability in some regions may contribute to lower support values in specific branches. The study's sample size was limited to 28 isolates from a specific geographic area (San Juan, Puerto Rico), potentially limiting the generalizability of findings to broader environments affected by flooding, such as those that are not hot and humid. Additionally, exclusively relying on morphological keys can lead to unsuccessful identifications, especially when dealing with newly described Aspergillus species. The need to revise these keys is indicated as more species are being discovered. In conclusion, this study provides essential insights into the diversity of Aspergillus species in water-impacted homes in a hot and humid environment. It stresses the need for a combination of molecular and morphological techniques for robust fungal identification. Our results on species like A. versicolor , A. flavus , and A. sydowii point to serious implications on indoor air quality and public health in the disaster zone, in favor of extensive identification strategies to inform effective health measures. Methods Characterization of Aspergillus isolates. Collection of Aspergillus isolates. Approximately 400 Aspergillus isolates were cryopreserved in 2.0 mL tubes containing 1 mL of culture media (MEA or G25N) and stored at -80°C until further use. To lower the quantity of Aspergillus isolates to study, we selected Aspergillus isolates from sampled homes that had more than 1000 CFU/m 3 (the recommended limit for fungal contamination in indoor air) of Aspergillus spp. and samples with more than 50% of fungal colonies identified as Aspergillus spp. Approximately 210 Aspergillus isolates recovered from the sampled homes met these criteria. We randomly selected 28 Aspergillus isolates from this list for further testing to balance feasibility and resource constraints, ensuring a manageable sample size for in-depth species identification. These Aspergillus isolates were grown on G25N media for up to 14 days at 25 ± 2°C to generate sufficient growth to identify them at the species level and to perform experiments evaluating their pro-inflammatory potential. Macromorphological characterization of Aspergillus isolates. To identify the selected 28 Aspergillus isolates to species level through culture techniques, we followed Maren A. Klich’s morphologically based system. 14 Colonies were grown on four media to describe the fungal isolates macromorphologically. The media used were the following: Czapek Yeast Agar (CYA, K 2 HPO 4 0.5 g, Czapek concentrate 5.0 mL, yeast extract 2.5 g, sucrose 30.0 g, agar 7.5 g, distilled water 500 mL), Czapek Yeast Agar with 20% sucrose (CY20S, K 2 HPO 4 0.5 g, Czapek concentrate 5.0 ml, yeast extract 2.5 g, sucrose 100.0 g, agar 7.5 g, distilled water 500 mL) and commercial Malt Extract Agar (MEA, Hardy Diagnostics). Twenty-five mL of sterilized media were poured into standard (100 mm) Petri dishes. Four plates were used for each culture: two of CYA and one of CY20S and MEA. To prevent stray colonies on the plates, we prepared spore suspensions using a medium consisting of 0.2% agar and 0.05 Tween 80. Briefly, we pipetted 1 mL aliquots of the sterilized medium into small 2.5 mL cryovials. We mixed conidia from 7 to 14-day-old growth into the medium. Then, we placed 2 µL aliquots at three equidistant points from the center of the plate. We incubated each plate for seven days, with one CYA plate at 37°C and the remaining at 25°C. Incubation at 25°C represents a typical indoor room temperature, whether incubation at 37°C is done to simulate human body temperature. After the seven-day incubation, we collected data on conidial color, colony diameter, mycelial color, exudate presence, reverse color, soluble pigment, sclerotia, and cleistothecia. Micromorphological characterization of Aspergillus isolates. We used the microculture technique to describe the micromorphological features of the selected Aspergillus isolates. Briefly, we inoculated two parallel lines of the Aspergillus conidia from a 7 to 14-day-old colony in a 60 mm Petri Dish with G25N media using an inoculating needle. Then, we inserted a sterile coverslip at a 45-degree angle in each inoculated line. We incubated the microcultures at 25 ± 2°C for seven days. After incubating, we gently collected the coverslips using stained (with lactophenol cotton blue) and unstained slide preparations. We used the NIKON 80i microscope to observe and collect micromorphology features such as seriation type (predominantly uniseriate or biseriate), vesicle shape, conidia characteristics (shape, size, and surface texture), stipe (length, color, and surface texture) and ascospores (color, size, ornamentation, and surface texture) (Fig. 1 ). Molecular identification of Aspergillus isolates. Extraction of fungal genomic DNA. We extracted genomic DNA using the standard protocol of Qiagen DNeasy PowerSoil Pro Kit (QIAGEN LLC, Germantown Road, Maryland, USA). For sample preparation, we grew each fungal isolate on G25N and incubated them at 25 ± 2°C for 7 to 14 days. Then, we transferred fungal growth from the surface of the plate using a sterile swab or a sterile scalpel blade to the bead tube of the DNAeasy PowerSoil Pro Kit and followed the manufacturer’s instructions. We quantified the genomic DNA using the Qubit® dsDNA HS (High Sensitivity) Assay at room temperature (Waltham, Massachusetts, US) and stored at -20°C until genomic DNA amplification and sequencing. PCR Amplification of target genes. For PCR amplification, we used primers specific for Internal Transcribed Spacer Region 2 (ITS2), beta-tubulin gene ( benA ), and calmodulin gene ( CaM ) (Table 4 ). We performed PCR amplification of the extracted DNA in a 100 µL reaction mixture as follows: 5 µL gDNA template, 50 µL Qiagen HotStarTaq Master Mix (QIAGEN LLC, Germantown Road, Maryland, USA), 5 µL of each forward and reverse primers, and 35 µL RNase free water. We included a non-template negative and a positive control (genomic DNA from Aspergillus brasiliensis , ATCC 16404D-2) in each amplification reaction. We programmed the thermocycler to the following PCR conditions: HotStarTaq DNA Polymerase activation incubation step at 95°C for 15 minutes, 35 cycles of denaturation at 94°C for 45 seconds, annealing at 55°C for 45 seconds, and extension at 72°C for 1 minute, with a final extension at 72°C for 10 minutes. After complete amplification, we analyzed the PCR products for gel electrophoresis using 1.5% agarose gel (1.5 grams of agarose in 100 ml of TAE 1x buffer) with ethidium bromide as the staining agent. Table 4 Primers used for the amplification of specific genes in the Aspergillus isolates. Locus Primer Direction Oligonucleotide Sequence (5’-3’) Length (bp) Internal Transcribed Spacer 2 ( ITS2 ) ITS9 Forward GAA CGC AGC RAA IIG YGA 240–460 ITS4 Reverse TCC TCC GCT TAT TGA TAT GC Beta-Tubulin ( benA ) Bt2a Forward GGT AAC CAA ATC GGT GCT GCT TTC 432–560 Bt2b Reverse ACC CTC AGT GTA GTG ACC CTT GGC Calmodulin ( CaM ) CMD5 Forward CCG AGT ACA AGG ARG CCT TC 580 CMD6 Reverse CCG ATR GAC GTC ATR ACG TGG Sequencing and phylogenetic analysis. We purified the PCR products using the Qiagen QIAquick PCR Purification Kit (QIAGEN LLC, Germantown Road, Maryland, USA), according to the manufacturer’s instructions, and sequenced with the primers used for amplification. Sequencing was outsourced using the Big Dye X Terminator Sequencing Kit 3.1 and the ABI 3500 DNA Sequencer (Applied Biosystems) at the Molecular Biology Core Facility from the RCMI Program at the University of Puerto Rico - Medical Sciences Campus. We verified and cleaned the sequences using FinchTV (Geospiza, Inc.) Version 1.5.0 chromatogram viewer software. We assigned species names to the Aspergillus isolates after comparing the contigs (created from forward and reverse complement sequences) with representative sequences available in NCBI (National Center for Biotechnology Information). For phylogenetic analysis, we aligned sequences for each gene region (ITS2, beta-tubulin [benA], and calmodulin [CaM]) using the ClustalW algorithm with default parameters in MEGA version 12. 18 Multiple sequence alignments were visually inspected and trimmed using the partial deletion method with a 95% site coverage cutoff to exclude positions with significant gaps or missing data. Evolutionary relationships were inferred using the Maximum Likelihood method based on the Tamura-Nei model. The best tree topology was selected based on log-likelihood scores from a heuristic search that compared Neighbor-Joining and Maximum Parsimony starting trees. Robustness of the phylogenetic trees was assessed with 1,000 bootstrap replicates. Branches with < 50% support were collapsed.Reference sequences for each gene were downloaded from GenBank and are listed in Supplementary Table S3 . Hamigera avellanea (family Aspergillaceae ) was used as the outgroup in all phylogenetic trees. Declarations Acknowledgments The authors want to thank all the families participating in this research and Dr. Edna E. Aquino for her invaluable support with the molecular-based experiments. This research project partially fulfilled Lorraine N. Vélez-Torres's doctoral dissertation at the University of Puerto Rico—Medical Sciences Campus. Funding This work was supported by a grant from the National Institutes of Health (NIH) number R21 ES029762-0101, and partial funds were received from MBRS-RISE program of UPR-MSC (award number R25GM061838), PR-INBRE BiRC NIH/NIGMS P20 GM103475, and NIMHD CCRHD grant number U54 MD007600. Author contributions Lorraine N. Vélez-Torres contributed substantially to the study's conception and design, performed the primary experiments, acquired, analyzed, and interpreted data, created tables and figures, and drafted the manuscript. Benjamín Bolaños-Rosero supported acquiring and analyzing the culture-based experimental approach and contributed to manuscript revisions. Filipa Godoy-Vitorino assisted in the design of molecular experiments and contributed to manuscript revisions. Félix E. Rivera-Mariani, Juan P. Maestre, Kerry Kinney, and Humberto Cavallin contributed to the review and substantive revisions of the manuscript. Each author has approved the submitted version with their contributions. All authors agree to be personally accountable for their own contributions and to address any questions related to the accuracy or integrity of the study, ensuring any concerns are investigated and appropriately documented in the literature. Data availability statement (mandatory) The data supporting this study's findings, including datasets generated and analyzed during the research, are available upon request. Due to privacy considerations regarding sample locations and specifics, data access is restricted but can be granted to the corresponding author upon reasonable request. Any shared data will include the minimal dataset necessary to interpret, replicate, and build upon the findings reported in this article. Additional Information Competing interests The author(s) declare no competing interests. References Vélez-Torres, L. N. et al. Hurricane María drives increased indoor proliferation of filamentous fungi in San Juan, Puerto Rico: A two-year culture-based approach. PeerJ 10 , 1–24 (2022). (CDC), C. for D. C. and P. Health concerns associated with mold in water-damaged homes after Hurricanes Katrina and Rita--New Orleans area, Louisiana, October 2005. Morb. Mortal. Wkly. Rep. 2 , 41–44 (2006). Barbeau, D. N., Grimsley, L. F., White, L. E., El-Dahr, J. M. & Lichtveld, M. Mold Exposure and Health Effects Following Hurricanes Katrina and Rita. Annu. Rev. Public Health 31 , 165–178 (2010). Chew, G. L. et al. Mold and Endotoxin Levels in the Aftermath of Hurricane Katrina: A Pilot Project of Homes in New Orleans Undergoing Renovation. Environ. Health Perspect. 114 , 1883–1889 (2006). Solomon, G. M., Hjelmroos-Koski, M., Rotkin-Ellman, M. & Hammond, S. K. Airborne Mold and Endotoxin Concentrations in New Orleans, Louisiana, after Flooding, October through November 2005. Environ. Health Perspect. 114 , 1381–1386 (2006). Samson, R. A. et al. Phylogeny, identification and nomenclature of the genus Aspergillus. Stud. Mycol. 78 , 141–173 (2014). Tsang, C.-C., Tang, J. Y. M., Lau, S. K. P. & Woo, P. C. Y. Taxonomy and evolution of Aspergillus, Penicillium and Talaromyces in the omics era – Past, present and future. Comput. Struct. Biotechnol. J. 16 , 197–210 (2018). Houbraken, J. et al. Classification of Aspergillus, Penicillium, Talaromyces and related genera (Eurotiales): An overview of families, genera, subgenera, sections, series and species. Stud. Mycol. 95 , 5–169 (2020). Kendrick, B. Fungi: Ecological Importance and Impact on Humans. eLS (2011) doi:10.1002/9780470015902.a0000369.pub2. Balloy, V. & Chignard, M. The innate immune response to Aspergillus fumigatus. Microbes Infect. 11 , 919–927 (2009). Seyedmousavi, S., Lionakis, M. S., Parta, M., Peterson, S. W. & Kwon-Chung, K. J. Emerging Aspergillus Species Almost Exclusively Associated With Primary Immunodeficiencies. Open Forum Infect. Dis. 5 , ofy213- (2018). Rudramurthy, S. M., Paul, R. A., Chakrabarti, A., Mouton, J. W. & Meis, J. F. Invasive Aspergillosis by Aspergillus flavus: Epidemiology, Diagnosis, Antifungal Resistance, and Management. J. Fungi 5 , 55 (2019). Klich, M. A. Identification of Common Aspergillus Species . (Centraalbureau voor Schimmelcultures, 2002). Klich, M. A. Identification of clinically relevant aspergilli. Med. Mycol. 44 , 127–131 (2006). Schoch, C. L. et al. Nuclear ribosomal internal transcribed spacer (ITS) region as a universal DNA barcode marker for Fungi. Proc. Natl. Acad. Sci. 109 , 6241–6246 (2012). Jakšić, D. et al. Fungi and their secondary metabolites in water‐damaged indoors after a major flood event in eastern Croatia. Indoor Air 31 , 730–744 (2021). Rao, C. Y. et al. Characterization of Airborne Molds, Endotoxins, and Glucans in Homes in New Orleans after Hurricanes Katrina and Rita▿. Appl. Environ. Microbiol. 73 , 1630–1634 (2007). Kumar, S. et al. MEGA12: Molecular Evolutionary Genetic Analysis version 12 for adaptive and green computing. Mol. Biol. Evol. 41 , 1–9 (2024). Chen, A. J. et al. Aspergillus section Nidulantes (formerly Emericella): Polyphasic taxonomy, chemistry and biology. Stud. Mycol. 84 , 1–118 (2016). Sun, B. et al. Four New Species of Aspergillus Subgenus Nidulantes from China. J. Fungi 8 , 1205 (2022). Sklenář, F. et al. Phylogeny of xerophilic aspergilli (subgenus Aspergillus) and taxonomic revision of section Restricti. Stud. Mycol. 88 , 161–236 (2017). Visagie, C. M. et al. A survey of xerophilic Aspergillus from indoor environment, including descriptions of two new section Aspergillus species producing eurotium-like sexual states. MycoKeys 19 , 1–30 (2017). Alshehri, B. & Palanisamy, M. Evaluation of molecular identification of Aspergillus species causing fungal keratitis. Saudi J. Biol. Sci. 27 , 751–756 (2020). Ashtiani, N. M., Kachuei, R., Yalfani, R., Harchegani, A. B. & Nosratabadi, M. Identification of Aspergillus sections Flavi, Nigri, and Fumigati and their differentiation using specific primers. Le Infez. Med. 25 , 127–132 (2017). Abarca, M. L., Accensi, F., Cano, J. & Cabañes, F. J. Taxonomy and significance of black aspergilli. Antonie Van Leeuwenhoek 86 , 33–49 (2004). D’hooge, E. et al. Black aspergilli: A remaining challenge in fungal taxonomy? Med. Mycol. 57 , 773–780 (2018). Nargesi, S. et al. Differentiation of Aspergillus flavus from Aspergillus oryzae Targeting the cyp51A Gene. Pathogens 10 , 1279 (2021). Wickes, B. L. & Wiederhold, N. P. Molecular diagnostics in medical mycology. Nat. Commun. 9 , 5135 (2018). Lass-Flörl, C., Dietl, A.-M., Kontoyiannis, D. P. & Brock, M. Aspergillus terreus Species Complex. Clin. Microbiol. Rev. 34 , e00311-20 (2021). Mousavi, B. et al. Aspergillus species in indoor environments and their possible occupational and public health hazards. Curr. Med. Mycol. 2 , 36–42 (2016). Bolaños-Rosero, B., Betancourt, D., Dean, T. & Vesper, S. Pilot study of mold populations inside and outside of Puerto Rican residences. Aerobiologia (Bologna). 29 , 537–543 (2013). Vesper, S. et al. Mold populations and dust mite allergen concentrations in house dust samples from across Puerto Rico. Int. J. Environ. Health Res. 26 , 198–207 (2016). Visagie, C. M. et al. Ochratoxin production and taxonomy of the yellow aspergilli (Aspergillus section Circumdati). Stud. Mycol. 78 , 1–61 (2014). Jurjević, Ž. et al. Two novel species of Aspergillus section Nigri from indoor air. IMA Fungus 3 , 159–173 (2012). Varga, J. et al. Occurrence of black Aspergilli in indoor environments of six countries. Arch. Ind. Hyg. Toxicol. 65 , 219–223 (2014). Flores, M. E. B. et al. Fungal spore concentrations in indoor and outdoor air in university libraries, and their variations in response to changes in meteorological variables. Int. J. Environ. Health Res. 24 , 320–340 (2013). Andersen, B., Frisvad, J. C., Søndergaard, I., Rasmussen, I. S. & Larsen, L. S. Associations between Fungal Species and Water-Damaged Building Materials. Appl. Environ. Microbiol. 77 , 4180–4188 (2011). Jakšić, D. et al. Post-Flood Impacts on Occurrence and Distribution of Mycotoxin-Producing Aspergilli from the Sections Circumdati, Flavi, and Nigri in Indoor Environment. J. Fungi 6 , 282 (2020). Samson, R. A. et al. Diagnostic tools to identify black aspergilli. Stud. Mycol. 59 , 129–145 (2007). Table 1 Table 1 is available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files SupplementaryTables.docx Table1.docx Cite Share Download PDF Status: Published Journal Publication published 14 Oct, 2025 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 13 Aug, 2025 Reviews received at journal 12 Aug, 2025 Reviews received at journal 06 Aug, 2025 Reviewers agreed at journal 04 Aug, 2025 Reviewers agreed at journal 04 Aug, 2025 Reviewers agreed at journal 04 Aug, 2025 Reviewers invited by journal 04 Aug, 2025 Editor assigned by journal 29 Jul, 2025 Editor invited by journal 05 Jun, 2025 Submission checks completed at journal 04 Jun, 2025 First submitted to journal 04 Jun, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6786592","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":484480387,"identity":"f81728f1-1570-4b27-b34f-55ec14573152","order_by":0,"name":"Lorraine N. 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Microscopic photos from G25N microcultures on 60X.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6786592/v1/b23f82b6b92b1aa0c9f13125.png"},{"id":86923111,"identity":"bc96c314-fdd8-4d40-9022-fd03c433198f","added_by":"auto","created_at":"2025-07-17 08:00:39","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":2052658,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePhylogenetic tree of partial ITS2 gene sequences by maximum likelihood. \u003c/strong\u003eNote: Sequences from this study are highlighted with a red diamond shape. \u003cem\u003eHamigera avellanea\u003c/em\u003e was used as an outgroup. The isolate Section classification is highlighted in color.\u003c/p\u003e","description":"","filename":"Fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-6786592/v1/e8bd82a96796deddf9713e95.png"},{"id":86923116,"identity":"c1c72c1b-572c-4b1a-8d73-b0730f9b4df6","added_by":"auto","created_at":"2025-07-17 08:00:39","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1997589,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePhylogenetic tree of partial \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ebenA\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e gene sequences by maximum likelihood. \u003c/strong\u003eNote: Sequences from this study are highlighted with a red diamond shape. \u003cem\u003eHamigera avellanea\u003c/em\u003e was used as an outgroup.\u003c/p\u003e","description":"","filename":"Fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-6786592/v1/b4712ab83e5a3a1a7f492c15.png"},{"id":86923114,"identity":"c63230bd-5660-4536-b58a-8e6156505cf9","added_by":"auto","created_at":"2025-07-17 08:00:39","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":2058280,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePhylogenetic tree of partial \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ecaM\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e gene sequences by maximum likelihood. \u003c/strong\u003eNote: Sequences from this study are highlighted with a red diamond shape. \u003cem\u003eHamigera avellanea\u003c/em\u003e was used as an outgroup. The isolate Section classification is highlighted in color.\u003c/p\u003e","description":"","filename":"Fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-6786592/v1/3b34a475059530fa9538bb57.png"},{"id":93956681,"identity":"05d34792-42e1-44a1-a213-a6a3efa1521d","added_by":"auto","created_at":"2025-10-20 16:11:54","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":49462327,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6786592/v1/86de9355-61c2-49e0-9148-af5c855f6a37.pdf"},{"id":86923109,"identity":"1fcd30e5-2003-4186-80b0-6592a1ca11bc","added_by":"auto","created_at":"2025-07-17 08:00:39","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":29850,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTables.docx","url":"https://assets-eu.researchsquare.com/files/rs-6786592/v1/6822e13b3529c2eca843a2d7.docx"},{"id":86923112,"identity":"17ddd710-a970-4f86-bd20-29d26b0f3fb8","added_by":"auto","created_at":"2025-07-17 08:00:39","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":26990,"visible":true,"origin":"","legend":"","description":"","filename":"Table1.docx","url":"https://assets-eu.researchsquare.com/files/rs-6786592/v1/d97c6661e0e336816a7fc723.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003eMolecular and Culture-Based Identification of \u003cem\u003eAspergillus\u003c/em\u003e Species in Water-Impacted Homes Following Hurricane María in Puerto Rico \u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eHurricane Mar\u0026iacute;a, which struck Puerto Rico in September 2017, caused widespread devastation, including severe flooding that damaged homes across the island. The floods created the ideal conditions for indoor fungal growth, prompting air quality concerns and potential respiratory health repercussions for the residents. Our previous research confirmed a significant increase in fungal proliferation inside flooded homes in San Juan, with \u003cem\u003eAspergillus\u003c/em\u003e species being the most predominant.\u003csup\u003e1\u003c/sup\u003e This finding aligned with existing literature, which identifies \u003cem\u003eAspergillus\u003c/em\u003e species as common fungi in water-damaged environments.\u003csup\u003e2\u0026ndash;5\u003c/sup\u003e\u003c/p\u003e\u003cp\u003e\u003cem\u003eAspergillus\u003c/em\u003e is a large genus of filamentous fungi within the Ascomycota division, comprising around 350 species.\u003csup\u003e6,7\u003c/sup\u003e The genus is subdivided into six subgenera, 27 sections, and about 75 series, holding significance as both a pathogen and a source of pharmaceuticals, as well as in agriculture and food production.\u003csup\u003e8,9\u003c/sup\u003e Given its medical and economic importance, accurate identification of \u003cem\u003eAspergillus\u003c/em\u003e species is essential, as different species vary in pathogenicity and ability to produce harmful mycotoxins. Species-level identification is especially crucial in environments affected by flooding, where \u003cem\u003eAspergillus\u003c/em\u003e can pose serious health risks ranging from allergic reactions to severe respiratory infections, depending on the species and the vulnerability of exposed individuals.\u003csup\u003e9\u0026ndash;12\u003c/sup\u003e\u003c/p\u003e\u003cp\u003eTraditionally, \u003cem\u003eAspergillus\u003c/em\u003e classification has relied on macro- and micromorphological features, such as colony diameter, spore color, and microscopic structures like conidial head arrangement and vesicle size.\u003csup\u003e13\u003c/sup\u003e However, identifying closely related species remains challenging and requires specialized training in fungal taxonomy and microscopy.\u003csup\u003e14\u003c/sup\u003e Since the early 2000s, molecular techniques have increasingly complemented morphological approaches, with DNA sequencing becoming a standard tool for species identification.\u003csup\u003e6\u003c/sup\u003e The internal transcribed spacer (ITS) region is the official DNA barcode for fungi. However, secondary markers like calmodulin (\u003cem\u003eCaM\u003c/em\u003e) and beta-tubulin (\u003cem\u003ebenA\u003c/em\u003e) are often necessary for distinguishing closely related \u003cem\u003eAspergillus\u003c/em\u003e species.\u003csup\u003e6,15\u003c/sup\u003e These molecular markers offer greater precision in species differentiation, with amplified sequences typically compared against databases like GenBank.\u003csup\u003e6\u003c/sup\u003e\u003c/p\u003e\u003cp\u003eDespite the well-documented role of \u003cem\u003eAspergillus\u003c/em\u003e in moisture-damaged indoor environments, research on species-level identification in post-flood conditions remains limited.\u003csup\u003e5,16,17\u003c/sup\u003e Following extreme weather events such as hurricanes, there is a critical need for studies integrating phenotypic and genotypic techniques to accurately identify \u003cem\u003eAspergillus\u003c/em\u003e species in water-impacted homes. This work addresses that gap by using culture-based and molecular methods to characterize \u003cem\u003eAspergillus\u003c/em\u003e diversity in post-Hurricane Mar\u0026iacute;a homes in San Juan, Puerto Rico. By combining traditional morphological and molecular approaches, it offers a more comprehensive view of fungal presence and diversity in settings susceptible to flooding. This study enhances our understanding of \u003cem\u003eAspergillus\u003c/em\u003e species in water-impacted environments and demonstrates the value of integrating both phenotypic and genotypic methods to improve species identification accuracy. Accurate identification is essential for assessing health risks and informing public health interventions to mitigate fungal contamination in flood-affected areas.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eMorphological characteristics of\u003c/strong\u003e \u003cstrong\u003eAspergillus\u003c/strong\u003e \u003cstrong\u003eisolates.\u003c/strong\u003e We selected 28 \u003cem\u003eAspergillus\u003c/em\u003e isolates from the sampled homes and two positive controls (\u003cem\u003eA. fumigatus\u003c/em\u003e isolate and \u003cem\u003eA. brasiliensis\u003c/em\u003e genomic DNA) to achieve species-level identification. The isolates were obtained from 13 different homes (classified as water-impacted, including both flooded and rain-infiltrated homes), with most samples collected approximately one year after the hurricane. Isolates were taken from various locations within the homes (seven from living rooms, seven from kitchens, six from bedrooms, six from bathrooms, and two from outdoor areas) (\u003cstrong\u003eSupplementary Table \u003cspan class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/strong\u003e). To provide a culture-based identification to the species level of the \u003cem\u003eAspergillus\u003c/em\u003e isolates selected, we collected a comprehensive description of macro and micromorphological characteristics applicable to the identification of common \u003cem\u003eAspergillus\u003c/em\u003e species (Tables \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e and \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e). Colony growth rates on various standardized media and microscopical characteristics are taxonomically informative (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). Through the culture-based approach and using three different identification keys (Klich, Klich \u0026amp; Pitt, Samson), we were able to identify 22 out of 28 of our \u003cem\u003eAspergillus\u003c/em\u003e isolates and the positive control (\u003cem\u003eA. fumigatus\u003c/em\u003e) (Table \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e). The identified isolates belong to different subgenera (\u003cem\u003eAspergillus\u003c/em\u003e, \u003cem\u003eCircumdanti\u003c/em\u003e, and \u003cem\u003eNidulantes\u003c/em\u003e). Isolates A50, A77, A111, A143, A159, A293 and A299 remained unidentified.\u003c/p\u003e\n\u003ctable id=\"Tab2\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eMicromorphological characteristics of \u003cem\u003eAspergillus\u003c/em\u003e isolates after G25N microcultures observation.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eIsolate\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eConidial Heads\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eSeriation\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eVesicle Shape\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eVesicle Size\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eConidial Shape\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eConidial Size\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eConidial Texture\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eIdentity of Isolates\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e19\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eRadiate\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eBiseriate\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePyriform\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8 \u0026micro;m\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eGlobose\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.24 \u0026micro;m\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSmooth\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eA. versicolor\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eRadiate\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eBiseriate\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eGlobose\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e47 \u0026micro;m\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eGlobose\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.65 \u0026micro;m\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSmooth\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eA. westerdijkiae\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e27\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eRadiate\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eBiseriate\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eGlobose\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e81 \u0026micro;m\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eGlobose\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.11 \u0026micro;m\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eFinely Roughened\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eA. foetidus\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e33\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eRadiate\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eBiseriate\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eGlobose\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e61 \u0026micro;m\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eGlobose\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.5 \u0026micro;m\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eRough\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eA. niger\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eRadiate\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eBiseriate\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eGlobose\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7.99 \u0026micro;m\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eGlobose\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.23 \u0026micro;m\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eRough\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eUnidentified\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e73\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eRadiate\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eBiseriate\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eGlobose\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e34.10 \u0026micro;m\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eGlobose\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.04 \u0026micro;m\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eFinely Roughened\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eA. niger var. awamori\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e75\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eRadiate\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eUniseriate\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eGlobose\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e33.49 \u0026micro;m\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSpherical and Ovoid\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5.41 x 4 \u0026micro;m\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSpinose\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eEurotium chevalieri\u0026thinsp;=\u0026thinsp;A. chevalieri\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e77\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eRadiate\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eBiseriate\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSub globose\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e9.5 \u0026micro;m\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eGlobose\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.56 \u0026micro;m\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSmooth\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eUnidentified\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e111\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eRadiate\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eBiseriate\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eGlobose\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7.28 \u0026micro;m\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eGlobose\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.69 \u0026micro;m\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSmooth\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eUnidentified\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e137\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eRadiate\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eUniseriate\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSpathulate\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e10.82 \u0026micro;m\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSubspherical\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.01 \u0026micro;m\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eFinely Roughened\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eA. penicillioides\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e139\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eRadiate\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eBiseriate\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSub globose\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e10.84 \u0026micro;m\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSpherical\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.56 \u0026micro;m\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eRough\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eA. sydowii\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e143\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eRadiate\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eUniseriate\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePyriform\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e11.35 \u0026micro;m\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eGlobose\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.25 \u0026micro;m\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSmooth\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eUnidentified\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e150\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eRadiate\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eBiseriate\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSpathulate\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e12.57 \u0026micro;m\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eGlobose\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.9 \u0026micro;m\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eFinely Roughened\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eA. versicolor\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e155\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eRadiate to Columnar\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eUniseriate\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eElongate\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e12.69 \u0026micro;m\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eGlobose\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6.79 \u0026micro;m\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eFinely Roughened\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eA. flavus\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e159\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eRadiate\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eBiseriate\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eGlobose\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e36.17 \u0026micro;m\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eGlobose\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.12 \u0026micro;m\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSmooth\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eUnidentified\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e173\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eLoosely Columnar\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eUniseriate\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSpatulate\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7.63 \u0026micro;m\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eGlobose\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.72 \u0026micro;m\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSmooth\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eA. penicillioides\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e215\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eRadiate\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eBiseriate\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eGlobose\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e29.81 \u0026micro;m\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eGlobose\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.5 \u0026micro;m\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eFinely Roughened\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eA. westerdijkiae\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e226\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eRadiate\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eUniseriate\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eGlobose\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e25 \u0026micro;m\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eOvoid\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.6 \u0026micro;m\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eFinely Roughened\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eEurotium chevalieri\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e229\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eRadiate\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eBoth\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eGlobose\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6.88 \u0026micro;m\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eGlobose\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5.0 \u0026micro;m\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCoarsely Roughened\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eA. tamarii\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e232\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eRadiate\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eUniseriate\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ePyriform\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e10.65 \u0026micro;m\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eGlobose\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2.16 \u0026micro;m\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSmooth\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eA. penicillioides\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e273\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eRadiate\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eBiseriate\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eGlobose\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e55.87 \u0026micro;m\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eGlobose\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3 \u0026micro;m\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSmooth\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eA. awamori\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e276\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eRadiate\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eUniseriate\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eGlobose\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e55.56 \u0026micro;m\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eGlobose\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.05 \u0026micro;m\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eCoarsely Roughened\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eA. niger var. niger\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e277\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eRadiate\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eUniseriate\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eGlobose\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e76.87 \u0026micro;m\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eGlobose\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.83 \u0026micro;m\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eRough\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eA. aculeatus\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e293\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eRadiate\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eBiseriate\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eGlobose\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e13.29 \u0026micro;m\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eGlobose\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.24 \u0026micro;m\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSmooth\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eUnidentified\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e299\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eRadiate\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eUniseriate\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eGlobose\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e54.6 \u0026micro;m\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3.82 \u0026micro;m\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSmooth\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eUnidentified\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e321\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eRadiate\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eUniseriate\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSub globose\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e30.6 \u0026micro;m\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSub globose\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7.6 \u0026micro;m\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eFinely Roughened\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eEurotium rubrum\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e323\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eRadiate\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eUniseriate\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eGlobose\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e31.1 \u0026micro;m\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eGlobose\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.42 \u0026micro;m\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eRough\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eA. japonicus\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e372\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eRadiate\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eUniseriate\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eGlobose\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e18.56 \u0026micro;m\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eGlobose\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4.01 \u0026micro;m\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eFinely Roughened\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eA. oryzae\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eA. fumigatus\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eColumnar\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eUniseriate\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSpathulate\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e17.18 \u0026micro;m\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eOvoid\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2 \u0026micro;m\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSmooth\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eA. fumigatus\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDNA extraction and Polymerase Chain Reaction (PCR) of\u003c/strong\u003e \u003cstrong\u003eAspergillus\u003c/strong\u003e \u003cstrong\u003eisolates obtained.\u003c/strong\u003e To begin the molecular identification approach, we successfully extracted the genomic DNA of all 28 \u003cem\u003eAspergillus\u003c/em\u003e isolates and positive control (\u003cem\u003eA. fumigatus\u003c/em\u003e). The genomic DNA concentration from our samples ranged from 11\u0026ndash;120 ng/\u0026micro;L; 50 \u0026micro;L of the product was used for PCR amplification experiments. To achieve fungal identification to the species level of \u003cem\u003eAspergillus\u003c/em\u003e isolates, we amplified the internal transcribed spacer region 2 (ITS2), beta-tubulin (\u003cem\u003ebenA\u003c/em\u003e), and calmodulin (\u003cem\u003eCaM\u003c/em\u003e) target genes via PCR. Twenty-two out of twenty-eight \u003cem\u003eAspergillus\u003c/em\u003e isolates were positive to PCR amplification of ITS2, \u003cem\u003ebenA\u003c/em\u003e, and \u003cem\u003ecaM\u003c/em\u003e genes with amplicon sizes ranging between 300\u0026ndash;400 base pairs (bps), 400\u0026ndash;5660 bps, and 400\u0026ndash;580 bps, respectively. Annealing temperature troubleshooting results for \u003cem\u003eAspergillus\u003c/em\u003e isolates #27, #73, #273, #276, #277, and #299 were unsuccessful.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eGene sequence homology of Aspergillus isolates\u003c/em\u003e We employed Basic Logical Alignment Search Tool (BLAST) results from the ITS2, \u003cem\u003ebenA\u003c/em\u003e, and \u003cem\u003eCaM\u003c/em\u003e gene sequences of this study on the National Center for Biotechnology Information (NCBI) to assess their sequence similarity with reference sequences in GenBank. The results in Table \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e revealed that most isolates had an identity above 99% similar to reference sequences in GenBank.\u003c/p\u003e\n\u003ctable id=\"Tab3\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eMolecular-based identification of 29 \u003cem\u003eAspergillus\u003c/em\u003e isolates. The percentage of similarity and E-value to reference sequences of GenBank is shown per gene and \u003cem\u003eAspergillus\u003c/em\u003e isolate. \u0026ldquo;\u0026ndash;\u0026rdquo; denotes that no clear PCR products were obtained using primers from Table \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eIsolate Code\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eIdentity of Isolate\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colspan=\"6\"\u003e\n \u003cp\u003eIdentity (%) to GenBank Sequences and E-Value\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eMolecular Approach\u003c/strong\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eITS2\u003c/strong\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eE-value\u003c/strong\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003ebenA\u003c/strong\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eE-value\u003c/strong\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003ecaM\u003c/strong\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eE-value\u003c/strong\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e19\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eA. versicolor\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7e-163\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e99.80\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eA. steynii\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e99.68\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1e-159\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e27\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e33\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eA. niger\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5e-164\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eA. arenarioides\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1e-159\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e73\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003e-\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e75\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eA. chevalieri\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3e-161\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e77\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eA. petersonii\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1e-159\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e99.64\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e111\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eA. subalbidus\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7e-163\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e137\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eA. penicillioides\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2e-168\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e99.78\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e139\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eA. sydowii\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4e-160\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e99.60\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e143\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eA. hordei\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2e-168\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e99.77\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e150\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eA. versicolor\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3e-161\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e99.80\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e155\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eA. flavus\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1e-160\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e159\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eA. ochraceopetaliformis\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1e-159\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e173\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eA. gracilis\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1e-165\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e215\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eA. westerdijkiae\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1e-165\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e226\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eA. chevalieri\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1e-160\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e229\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eA. tamarii\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7e-163\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e232\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eA. hordei\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e7e-168\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e99.36\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e273\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e276\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e277\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e293\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eA. flocculosus\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e99.38\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1e-160\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e99.82\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e299\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e321\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eA. pseudoglaucus\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8e-157\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e99.75\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e323\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eA. brunneoviolaceus\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2e-153\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e98.38\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e372\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eA. oryzae\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6e-148\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eA. fumigatus\u003c/em\u003e\u003c/p\u003e\n \u003cp\u003e(DNA extraction control)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eA. fumigatus\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2e-163\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e99.82\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eA. brasiliensis\u003c/em\u003e\u003c/p\u003e\n \u003cp\u003e(PCR control)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cem\u003eA. brasiliensis\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e3e-161\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e100\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e0.0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eComparison between culture and molecular-based methods for Aspergillus isolates identification.\u003c/em\u003e Results from culture-based and molecular-based approaches matched for eleven (11) out of the twenty-eight (28) \u003cem\u003eAspergillus\u003c/em\u003e isolates compared (isolates A19, A33, A75, A137, A139, A150, A155, A215, A226, and A229) and the positive control (\u003cem\u003eA. fumigatus\u003c/em\u003e). The culture-based approach provided preliminary species identification to isolates A27, A73, A273, A276, and A277, which couldn\u0026rsquo;t be identified through molecular-based approaches (no PCR products were obtained). More importantly, macro and micromorphological characteristics were very useful in distinguishing the closely related species \u003cem\u003eA. flavus\u003c/em\u003e and \u003cem\u003eA. oryzae\u003c/em\u003e (isolates A155 and A372), where \u003cem\u003eITS2\u003c/em\u003e, \u003cem\u003ebenA\u003c/em\u003e, and \u003cem\u003eCaM\u003c/em\u003e genes showed\u0026thinsp;\u0026gt;\u0026thinsp;100% similarity to both \u003cem\u003eA. flavus\u003c/em\u003e and \u003cem\u003eA. oryzae\u003c/em\u003e. Although culture-based techniques yielded incorrect species identification to five isolates (A20, A173, A232, 321, and 323), the species identified were classified under the same subgenera and/or section (\u003cstrong\u003eSupplementary Table S2\u003c/strong\u003e). For example, isolate A20 (\u003cem\u003eA. steynii\u003c/em\u003e) was incorrectly classified as \u003cem\u003eA. westerdijkiae\u003c/em\u003e through culture techniques, but both species belong to the \u003cem\u003eCircumdanti\u003c/em\u003e subgenera. The same is observed for isolate A323 (\u003cem\u003eA. brunneoviolaceus\u003c/em\u003e), identified as \u003cem\u003eA. japonicus\u003c/em\u003e through the culture approach, but both species are members of the \u003cem\u003eNigri\u003c/em\u003e section. Isolates A173 (\u003cem\u003eA. gracilis\u003c/em\u003e) and A232 (\u003cem\u003eA. hordei\u003c/em\u003e) are both incorrectly classified as \u003cem\u003eA. penicillioides\u003c/em\u003e through culture-based techniques, but they all belong to the \u003cem\u003eRestricti\u003c/em\u003e section. In terms of isolate A321 (\u003cem\u003eA. pseudoglaucus\u003c/em\u003e), which was incorrectly identified through macromorphological characterization as \u003cem\u003eA. rubrum\u003c/em\u003e (formerly \u003cem\u003eEurotium rubrum\u003c/em\u003e), a note was highlighted in the identification key stating the close resemblance of this species to \u003cem\u003eEurotium repens\u003c/em\u003e, now known as \u003cem\u003eA. pseudoglaucus\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePhylogenetic analysis of\u003c/strong\u003e \u003cstrong\u003eAspergillus\u003c/strong\u003e \u003cstrong\u003eisolates.\u003c/strong\u003e We inferred the evolutionary history of the \u003cem\u003eAspergillus\u003c/em\u003e species isolated from the sampled homes using the Maximum Likelihood method and the Tamura-Nei model in MEGA12.\u003csup\u003e18\u003c/sup\u003e To measure the consistency of the phylogenetic tree, we employed a bootstrap of 1,000 replications. The percentage of trees in which the associated taxa clustered is shown next to the branches. Sequences generated in this study are marked with a red diamond in the phylogenetic tree, while reference sequences obtained from GenBank are unmarked. \u003cem\u003eHamigera avellanea\u003c/em\u003e (\u003cem\u003eAspergillaceae\u003c/em\u003e) was used as the outgroup in all trees. The phylogenetic analysis of \u003cem\u003eAspergillus\u003c/em\u003e isolates showed that they grouped into distinct clusters, indicating close genetic relationships.\u003c/p\u003e\n\u003cp\u003eThe phylogenetic tree based on the ITS2 gene revealed that the alignment matrix contained 70 sequences, comprising 28 isolates from this study and 42 representative sequences from GenBank (\u003cstrong\u003eSupplementary Table S3\u003c/strong\u003e). After applying a 95% site coverage threshold to exclude positions with gaps or missing data in more than 5% of sequences, the final dataset consisted of 242 nucleotide positions. Isolates from \u003cem\u003eAspergillus\u003c/em\u003e species classified under the same taxonomical section had above 83% bootstrap support, with several species-level clusters reaching 99% (e.g., \u003cem\u003eA. versicolor\u003c/em\u003e, \u003cem\u003eA. sydowii\u003c/em\u003e, \u003cem\u003eA. westerdijkiae\u003c/em\u003e) (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\n\u003cp\u003eThe beta-tubulin (\u003cem\u003ebenA\u003c/em\u003e) gene alignment matrix also contained 69 sequences (28 isolates from this study and 41 reference sequences from GenBank). To reduce the influence of poorly aligned regions, the partial deletion option was applied, excluding positions with less than 95% site coverage, resulting in a final dataset of 277 positions. All isolates showed\u0026thinsp;\u0026gt;\u0026thinsp;83% bootstrap-supported clustering with known reference species from GenBank (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e). High-confidence clusters were observed for species such as \u003cem\u003eA. versicolor\u003c/em\u003e, \u003cem\u003eA. sydowii\u003c/em\u003e, and the closely related pair \u003cem\u003eA. flocculosus\u003c/em\u003e / \u003cem\u003eA. ochraceopetaliformis\u003c/em\u003e, each receiving 99\u0026ndash;100% support. In contrast, some members of the \u003cem\u003eNigri\u003c/em\u003e section showed weaker support, ranging from 57\u0026ndash;70%.\u003c/p\u003e\n\u003cp\u003eThe phylogenetic tree based on the \u003cem\u003eCaM\u003c/em\u003e gene had an alignment matrix that also included 70 sequences (28 isolates from this study and 42 reference sequences from GenBank). The partial deletion option was applied to eliminate positions with less than 95% site coverage, resulting in a final alignment of 366 positions. All isolates, except for \u003cem\u003eA. petersonii\u003c/em\u003e, showed over 80% cluster similarities with representative sequences from GenBank (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e). Different but closely related species like \u003cem\u003eA. flocculosus\u003c/em\u003e/\u003cem\u003eA. ochraceopetaliformis\u003c/em\u003e (series \u003cem\u003eSteynorium\u003c/em\u003e) and \u003cem\u003eA. flavus\u003c/em\u003e/\u003cem\u003eA. oryzae\u003c/em\u003e (series \u003cem\u003eFlavi\u003c/em\u003e) were still clustered together in their respective clades.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, we applied both phenotypic and genotypic methods to achieve species-level identification of 28 \u003cem\u003eAspergillus\u003c/em\u003e isolates from homes affected by Hurricane Mar\u0026iacute;a in San Juan, Puerto Rico. Using molecular techniques, we identified 22 isolates at the species level, categorizing them into the subgenera \u003cem\u003eCircumdanti\u003c/em\u003e (n\u0026thinsp;=\u0026thinsp;12), \u003cem\u003eAspergillus\u003c/em\u003e (n\u0026thinsp;=\u0026thinsp;8), and \u003cem\u003eNidulantes\u003c/em\u003e (n\u0026thinsp;=\u0026thinsp;3). The \u003cem\u003eCircumdanti\u003c/em\u003e and \u003cem\u003eNidulantes\u003c/em\u003e subgenera, each encompassing over 100 species, represent some of the most diverse \u003cem\u003eApergillus species\u003c/em\u003e.\u003csup\u003e19,20\u003c/sup\u003e Meanwhile, the \u003cem\u003eAspergillus\u003c/em\u003e subgenus, characterized by xerophilic species, thrives in low-moisture environments, making it prevalent indoors.\u003csup\u003e21,22\u003c/sup\u003e\u003c/p\u003e\u003cp\u003eOur phylogenetic analysis of ITS2, \u003cem\u003ebenA\u003c/em\u003e, and \u003cem\u003eCaM\u003c/em\u003e sequences showed distinct clustering patterns. ITS2-based trees showed strong bootstrap support (\u0026ge;\u0026thinsp;83%) for most section-level groupings, with species like \u003cem\u003eA. versicolor\u003c/em\u003e, \u003cem\u003eA. sydowii\u003c/em\u003e, and \u003cem\u003eA. westerdijkiae\u003c/em\u003e forming distinct clades supported at 99\u0026ndash;100%. The \u003cem\u003ebenA\u003c/em\u003e gene tree also produced high-confidence clusters, particularly for \u003cem\u003eA. versicolor\u003c/em\u003e, \u003cem\u003eA. sydowii\u003c/em\u003e, and the pair \u003cem\u003eA. flocculosus\u003c/em\u003e / \u003cem\u003eA. ochraceopetaliformis\u003c/em\u003e, though bootstrap support was lower (57\u0026ndash;70%) for some \u003cem\u003eNigri\u003c/em\u003e section members. The \u003cem\u003eCaM\u003c/em\u003e gene tree resolved most isolates at the species level, except for \u003cem\u003eA. petersonii\u003c/em\u003e, with species-level clusters for \u003cem\u003eA. flavus\u003c/em\u003e, \u003cem\u003eA. oryzae\u003c/em\u003e, and \u003cem\u003eA. westerdijkiae\u003c/em\u003e supported at 98\u0026ndash;100%. These results reinforce the CaM gene\u0026rsquo;s superior discriminatory power compared to ITS2 and benA, consistent with previous studies.\u003csup\u003e23,24\u003c/sup\u003e\u003c/p\u003e\u003cp\u003eHowever, six isolates, likely belonging to the black \u003cem\u003eAspergilli\u003c/em\u003e in the \u003cem\u003eNigri\u003c/em\u003e section, could not be identified at the species level due to the limitations of both molecular and phenotypic methods.\u003csup\u003e25,26\u003c/sup\u003e While sequence-based identification is the gold standard, morphological traits remain essential for resolving closely related species. For example, in this study, differentiating \u003cem\u003eA. flavus\u003c/em\u003e from \u003cem\u003eA. oryzae\u003c/em\u003e required morphological analysis, as molecular results alone were inconclusive.\u003csup\u003e27\u003c/sup\u003e Additionally, morphology-based methods allowed for preliminary identification of five black \u003cem\u003eAspergilli\u003c/em\u003e isolates that molecular methods could not fully resolve, underscoring the complementary role of culture techniques in complex identifications.\u003csup\u003e28\u003c/sup\u003e Despite identifying 22 isolates through morphological keys, five showed discrepancies with molecular results but remained in the same subgenus or section, highlighting limitations of morphological identification for certain taxonomic groups.\u003csup\u003e29\u003c/sup\u003e Expanding morphological keys is critical for identifying emerging \u003cem\u003eAspergillus\u003c/em\u003e species in indoor air environments.\u003c/p\u003e\u003cp\u003eMost isolates identified in this study, such as \u003cem\u003eA. flavus\u003c/em\u003e (A155), \u003cem\u003eA. niger\u003c/em\u003e (A33), \u003cem\u003eA. penicillioides\u003c/em\u003e (A137), \u003cem\u003eA. sydowii\u003c/em\u003e (A139), \u003cem\u003eA. tamarii\u003c/em\u003e (A229), \u003cem\u003eA. versicolor\u003c/em\u003e (A19, A150), and \u003cem\u003eA. westerdijkiae\u003c/em\u003e (A215), have previously been reported as common indoor fungal contaminants.\u003csup\u003e19,30\u003c/sup\u003e Specifically, \u003cem\u003eA. versicolor\u003c/em\u003e, \u003cem\u003eA. sydowii\u003c/em\u003e, and \u003cem\u003eA. penicillioides\u003c/em\u003e, all from xerophilic or primary colonizer sections, are often associated with building dust and are well adapted to indoor environments.\u003csup\u003e21,22\u003c/sup\u003e In Puerto Rico, these species have been detected at high concentrations in indoor fungal population studies.\u003csup\u003e31,32\u003c/sup\u003e Taxonomic classification of our isolates revealed representation across sections, including \u003cem\u003eCircumdanti\u003c/em\u003e, \u003cem\u003eNigri\u003c/em\u003e, \u003cem\u003ePetersoniorum\u003c/em\u003e, \u003cem\u003eCandidi\u003c/em\u003e, and \u003cem\u003eFlavi\u003c/em\u003e. For example, isolates from section \u003cem\u003eCircumdanti\u003c/em\u003e, such as \u003cem\u003eA. steynii\u003c/em\u003e (A20) and \u003cem\u003eA. westerdijkiae\u003c/em\u003e (A215), are known producers of the mycotoxin ochratoxin A\u003csup\u003e33\u003c/sup\u003e. Members of the \u003cem\u003eNigri\u003c/em\u003e section, such as \u003cem\u003eA. niger\u003c/em\u003e and \u003cem\u003eA. brunneoviolaceus\u003c/em\u003e, are also prevalent indoor contaminants.\u003csup\u003e34,35\u003c/sup\u003e Finally, \u003cem\u003eFlavi\u003c/em\u003e section members like \u003cem\u003eA. flavus\u003c/em\u003e and \u003cem\u003eA. oryzae\u003c/em\u003e highlight the health implications of mycotoxigenic \u003cem\u003eAspergillus\u003c/em\u003e species in indoor air.\u003csup\u003e36\u003c/sup\u003e\u003c/p\u003e\u003cp\u003eWhile many of these species are recognized as typical indoor fungi, their frequent recovery in this study suggests they may become more dominant or persistent in post-flood indoor environments, particularly under the warm and humid conditions typical of Puerto Rico. Though \u003cem\u003eAspergillus\u003c/em\u003e species have been broadly reported after hurricanes \u003csup\u003e2,4,5\u003c/sup\u003e, this study provides one of the few species-level analyses of indoor \u003cem\u003eAspergillus\u003c/em\u003e communities in a post-hurricane setting.\u003csup\u003e37,38\u003c/sup\u003e Our findings highlight \u003cem\u003eA. versicolor\u003c/em\u003e, \u003cem\u003eA. flavus\u003c/em\u003e, \u003cem\u003eA. niger\u003c/em\u003e, \u003cem\u003eA. westerdijkiae\u003c/em\u003e, and \u003cem\u003eA. sydowii\u003c/em\u003e as prevalent species in water-impacted homes, warranting further study into their environmental persistence and potential health risks.\u003c/p\u003e\u003cp\u003eThis study has several strengths, notably the combined use of phenotypic and genotypic techniques for \u003cem\u003eAspergillus\u003c/em\u003e identification. The integration of morphological methods with molecular analysis using ITS2, \u0026szlig;-tubulin (\u003cem\u003ebenA\u003c/em\u003e), and especially the calmodulin (\u003cem\u003eCaM\u003c/em\u003e) gene, which provided superior resolution for species differentiation, allowed for more accurate species-level identification and phylogenetic analysis of \u003cem\u003eAspergillus\u003c/em\u003e isolates. Morphological characterization using culture techniques also played a key role in distinguishing closely related species like \u003cem\u003eA. flavus\u003c/em\u003e and \u003cem\u003eA. oryzae\u003c/em\u003e, which were difficult to tell apart using molecular methods alone.\u003csup\u003e27\u003c/sup\u003e This combined strategy is especially important in post-flood indoor settings, where precise identification can help guide effective public health responses.\u003c/p\u003e\u003cp\u003eHowever, there are limitations to consider. Molecular techniques, while powerful, did not fully resolve all isolates, especially black \u003cem\u003eAspergilli\u003c/em\u003e in the \u003cem\u003eNigri\u003c/em\u003e section, where species-specific differentiation remains challenging.\u003csup\u003e26,39\u003c/sup\u003e Additionally, the final datasets used in our Maximum Likelihood trees were reduced through partial deletion (to 95% site coverage), resulting in 242, 277, and 366 nucleotide positions for ITS2, \u003cem\u003ebenA\u003c/em\u003e, and \u003cem\u003eCaM\u003c/em\u003e, respectively. While these are generally acceptable for fungal phylogenetics, the limited variability in some regions may contribute to lower support values in specific branches. The study's sample size was limited to 28 isolates from a specific geographic area (San Juan, Puerto Rico), potentially limiting the generalizability of findings to broader environments affected by flooding, such as those that are not hot and humid. Additionally, exclusively relying on morphological keys can lead to unsuccessful identifications, especially when dealing with newly described \u003cem\u003eAspergillus\u003c/em\u003e species. The need to revise these keys is indicated as more species are being discovered.\u003c/p\u003e\u003cp\u003eIn conclusion, this study provides essential insights into the diversity of \u003cem\u003eAspergillus\u003c/em\u003e species in water-impacted homes in a hot and humid environment. It stresses the need for a combination of molecular and morphological techniques for robust fungal identification. Our results on species like \u003cem\u003eA. versicolor\u003c/em\u003e, \u003cem\u003eA. flavus\u003c/em\u003e, and \u003cem\u003eA. sydowii\u003c/em\u003e point to serious implications on indoor air quality and public health in the disaster zone, in favor of extensive identification strategies to inform effective health measures.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cb\u003eCharacterization of\u003c/b\u003e \u003cb\u003eAspergillus\u003c/b\u003e \u003cb\u003eisolates.\u003c/b\u003e \u003cem\u003eCollection of Aspergillus isolates.\u003c/em\u003e Approximately 400 \u003cem\u003eAspergillus\u003c/em\u003e isolates were cryopreserved in 2.0 mL tubes containing 1 mL of culture media (MEA or G25N) and stored at -80\u0026deg;C until further use. To lower the quantity of \u003cem\u003eAspergillus\u003c/em\u003e isolates to study, we selected \u003cem\u003eAspergillus\u003c/em\u003e isolates from sampled homes that had more than 1000 CFU/m\u003csup\u003e3\u003c/sup\u003e (the recommended limit for fungal contamination in indoor air) of \u003cem\u003eAspergillus\u003c/em\u003e spp. and samples with more than 50% of fungal colonies identified as \u003cem\u003eAspergillus\u003c/em\u003e spp. Approximately 210 \u003cem\u003eAspergillus\u003c/em\u003e isolates recovered from the sampled homes met these criteria. We randomly selected 28 \u003cem\u003eAspergillus\u003c/em\u003e isolates from this list for further testing to balance feasibility and resource constraints, ensuring a manageable sample size for in-depth species identification. These \u003cem\u003eAspergillus\u003c/em\u003e isolates were grown on G25N media for up to 14 days at 25\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C to generate sufficient growth to identify them at the species level and to perform experiments evaluating their pro-inflammatory potential.\u003c/p\u003e\u003cp\u003e\u003cem\u003eMacromorphological characterization of Aspergillus isolates.\u003c/em\u003e To identify the selected 28 \u003cem\u003eAspergillus\u003c/em\u003e isolates to species level through culture techniques, we followed Maren A. Klich\u0026rsquo;s morphologically based system.\u003csup\u003e14\u003c/sup\u003e Colonies were grown on four media to describe the fungal isolates macromorphologically. The media used were the following: Czapek Yeast Agar (CYA, K\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e 0.5 g, Czapek concentrate 5.0 mL, yeast extract 2.5 g, sucrose 30.0 g, agar 7.5 g, distilled water 500 mL), Czapek Yeast Agar with 20% sucrose (CY20S, K\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e 0.5 g, Czapek concentrate 5.0 ml, yeast extract 2.5 g, sucrose 100.0 g, agar 7.5 g, distilled water 500 mL) and commercial Malt Extract Agar (MEA, Hardy Diagnostics). Twenty-five mL of sterilized media were poured into standard (100 mm) Petri dishes. Four plates were used for each culture: two of CYA and one of CY20S and MEA. To prevent stray colonies on the plates, we prepared spore suspensions using a medium consisting of 0.2% agar and 0.05 Tween 80. Briefly, we pipetted 1 mL aliquots of the sterilized medium into small 2.5 mL cryovials. We mixed conidia from 7 to 14-day-old growth into the medium. Then, we placed 2 \u0026micro;L aliquots at three equidistant points from the center of the plate. We incubated each plate for seven days, with one CYA plate at 37\u0026deg;C and the remaining at 25\u0026deg;C. Incubation at 25\u0026deg;C represents a typical indoor room temperature, whether incubation at 37\u0026deg;C is done to simulate human body temperature. After the seven-day incubation, we collected data on conidial color, colony diameter, mycelial color, exudate presence, reverse color, soluble pigment, sclerotia, and cleistothecia.\u003c/p\u003e\u003cp\u003e\u003cem\u003eMicromorphological characterization of Aspergillus isolates.\u003c/em\u003e We used the microculture technique to describe the micromorphological features of the selected \u003cem\u003eAspergillus\u003c/em\u003e isolates. Briefly, we inoculated two parallel lines of the \u003cem\u003eAspergillus\u003c/em\u003e conidia from a 7 to 14-day-old colony in a 60 mm Petri Dish with G25N media using an inoculating needle. Then, we inserted a sterile coverslip at a 45-degree angle in each inoculated line. We incubated the microcultures at 25\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C for seven days. After incubating, we gently collected the coverslips using stained (with lactophenol cotton blue) and unstained slide preparations. We used the NIKON 80i microscope to observe and collect micromorphology features such as seriation type (predominantly uniseriate or biseriate), vesicle shape, conidia characteristics (shape, size, and surface texture), stipe (length, color, and surface texture) and ascospores (color, size, ornamentation, and surface texture) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cem\u003eMolecular identification of Aspergillus isolates.\u003c/em\u003e Extraction of fungal genomic DNA. We extracted genomic DNA using the standard protocol of Qiagen DNeasy PowerSoil Pro Kit (QIAGEN LLC, Germantown Road, Maryland, USA). For sample preparation, we grew each fungal isolate on G25N and incubated them at 25\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C for 7 to 14 days. Then, we transferred fungal growth from the surface of the plate using a sterile swab or a sterile scalpel blade to the bead tube of the DNAeasy PowerSoil Pro Kit and followed the manufacturer\u0026rsquo;s instructions. We quantified the genomic DNA using the Qubit\u0026reg; dsDNA HS (High Sensitivity) Assay at room temperature (Waltham, Massachusetts, US) and stored at -20\u0026deg;C until genomic DNA amplification and sequencing.\u003c/p\u003e\u003cp\u003e\u003cem\u003ePCR Amplification of target genes.\u003c/em\u003e For PCR amplification, we used primers specific for Internal Transcribed Spacer Region 2 (ITS2), beta-tubulin gene (\u003cem\u003ebenA\u003c/em\u003e), and calmodulin gene (\u003cem\u003eCaM\u003c/em\u003e) (Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). We performed PCR amplification of the extracted DNA in a 100 \u0026micro;L reaction mixture as follows: 5 \u0026micro;L gDNA template, 50 \u0026micro;L Qiagen HotStarTaq Master Mix (QIAGEN LLC, Germantown Road, Maryland, USA), 5 \u0026micro;L of each forward and reverse primers, and 35 \u0026micro;L RNase free water. We included a non-template negative and a positive control (genomic DNA from \u003cem\u003eAspergillus brasiliensis\u003c/em\u003e, ATCC 16404D-2) in each amplification reaction. We programmed the thermocycler to the following PCR conditions: HotStarTaq DNA Polymerase activation incubation step at 95\u0026deg;C for 15 minutes, 35 cycles of denaturation at 94\u0026deg;C for 45 seconds, annealing at 55\u0026deg;C for 45 seconds, and extension at 72\u0026deg;C for 1 minute, with a final extension at 72\u0026deg;C for 10 minutes. After complete amplification, we analyzed the PCR products for gel electrophoresis using 1.5% agarose gel (1.5 grams of agarose in 100 ml of TAE 1x buffer) with ethidium bromide as the staining agent.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003ePrimers used for the amplification of specific genes in the \u003cem\u003eAspergillus\u003c/em\u003e isolates.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"5\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eLocus\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003ePrimer\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eDirection\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eOligonucleotide Sequence (5\u0026rsquo;-3\u0026rsquo;)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eLength (bp)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eInternal Transcribed Spacer 2 (\u003cem\u003eITS2\u003c/em\u003e)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eITS9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eForward\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eGAA CGC AGC RAA IIG YGA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e240\u0026ndash;460\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eITS4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eReverse\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eTCC TCC GCT TAT TGA TAT GC\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eBeta-Tubulin (\u003cem\u003ebenA\u003c/em\u003e)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eBt2a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eForward\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eGGT AAC CAA ATC GGT GCT GCT TTC\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e432\u0026ndash;560\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eBt2b\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eReverse\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eACC CTC AGT GTA GTG ACC CTT GGC\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eCalmodulin (\u003cem\u003eCaM\u003c/em\u003e)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCMD5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eForward\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eCCG AGT ACA AGG ARG CCT TC\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e580\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCMD6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eReverse\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eCCG ATR GAC GTC ATR ACG TGG\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eSequencing and phylogenetic analysis.\u003c/b\u003e We purified the PCR products using the Qiagen QIAquick PCR Purification Kit (QIAGEN LLC, Germantown Road, Maryland, USA), according to the manufacturer\u0026rsquo;s instructions, and sequenced with the primers used for amplification. Sequencing was outsourced using the Big Dye X Terminator Sequencing Kit 3.1 and the ABI 3500 DNA Sequencer (Applied Biosystems) at the Molecular Biology Core Facility from the RCMI Program at the University of Puerto Rico - Medical Sciences Campus. We verified and cleaned the sequences using FinchTV (Geospiza, Inc.) Version 1.5.0 chromatogram viewer software. We assigned species names to the \u003cem\u003eAspergillus\u003c/em\u003e isolates after comparing the contigs (created from forward and reverse complement sequences) with representative sequences available in NCBI (National Center for Biotechnology Information). For phylogenetic analysis, we aligned sequences for each gene region (ITS2, beta-tubulin [benA], and calmodulin [CaM]) using the ClustalW algorithm with default parameters in MEGA version 12.\u003csup\u003e18\u003c/sup\u003e Multiple sequence alignments were visually inspected and trimmed using the partial deletion method with a 95% site coverage cutoff to exclude positions with significant gaps or missing data. Evolutionary relationships were inferred using the Maximum Likelihood method based on the Tamura-Nei model. The best tree topology was selected based on log-likelihood scores from a heuristic search that compared Neighbor-Joining and Maximum Parsimony starting trees. Robustness of the phylogenetic trees was assessed with 1,000 bootstrap replicates. Branches with \u0026lt;\u0026thinsp;50% support were collapsed.Reference sequences for each gene were downloaded from GenBank and are listed in \u003cb\u003eSupplementary Table S3\u003c/b\u003e. \u003cem\u003eHamigera avellanea\u003c/em\u003e (family \u003cem\u003eAspergillaceae\u003c/em\u003e) was used as the outgroup in all phylogenetic trees.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors want to thank all the families participating in this research and Dr. Edna E. Aquino for her invaluable support with the molecular-based experiments. This research project partially fulfilled Lorraine N. V\u0026eacute;lez-Torres\u0026apos;s doctoral dissertation at the University of Puerto Rico\u0026mdash;Medical Sciences Campus.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by a grant from the National Institutes of Health (NIH) number R21 ES029762-0101, and partial funds were received from MBRS-RISE program of UPR-MSC (award number R25GM061838), PR-INBRE BiRC NIH/NIGMS P20 GM103475, and NIMHD CCRHD grant number U54 MD007600.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLorraine N. V\u0026eacute;lez-Torres contributed substantially to the study\u0026apos;s conception and design, performed the primary experiments, acquired, analyzed, and interpreted data, created tables and figures, and drafted the manuscript. Benjam\u0026iacute;n Bola\u0026ntilde;os-Rosero supported acquiring and analyzing the culture-based experimental approach and contributed to manuscript revisions. Filipa Godoy-Vitorino assisted in the design of molecular experiments and contributed to manuscript revisions. F\u0026eacute;lix E. Rivera-Mariani, Juan P. Maestre, Kerry Kinney, and Humberto Cavallin contributed to the review and substantive revisions of the manuscript.\u003c/p\u003e\n\u003cp\u003eEach author has approved the submitted version with their contributions. All authors agree to be personally accountable for their own contributions and to address any questions related to the accuracy or integrity of the study, ensuring any concerns are investigated and appropriately documented in the literature.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability statement (mandatory)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data supporting this study\u0026apos;s findings, including datasets generated and analyzed during the research, are available upon request. Due to privacy considerations regarding sample locations and specifics, data access is restricted but can be granted to the corresponding author upon reasonable request. Any shared data will include the minimal dataset necessary to interpret, replicate, and build upon the findings reported in this article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAdditional Information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe author(s) declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eV\u0026eacute;lez-Torres, L. N. \u003cem\u003eet al.\u003c/em\u003e Hurricane Mar\u0026iacute;a drives increased indoor proliferation of filamentous fungi in San Juan, Puerto Rico: A two-year culture-based approach. \u003cem\u003ePeerJ\u003c/em\u003e \u003cstrong\u003e10\u003c/strong\u003e, 1\u0026ndash;24 (2022).\u003c/li\u003e\n\u003cli\u003e(CDC), C. for D. C. and P. Health concerns associated with mold in water-damaged homes after Hurricanes Katrina and Rita--New Orleans area, Louisiana, October 2005. \u003cem\u003eMorb. 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Mycol.\u003c/em\u003e \u003cstrong\u003e59\u003c/strong\u003e, 129\u0026ndash;145 (2007).\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Table 1","content":"\u003cp\u003eTable 1 is available in the Supplementary Files section.\u003c/p\u003e\n"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Aspergillus, Hurricane María, Water-Impacted Homes, Species-Level Identification, Molecular and Culture Methods","lastPublishedDoi":"10.21203/rs.3.rs-6786592/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6786592/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eFlooding caused by Hurricane Mar\u0026iacute;a promoted fungal growth in homes across Puerto Rico, raising concerns about indoor air quality and health risks. This study focuses on identifying \u003cem\u003eAspergillus\u003c/em\u003e species from water-impacted homes in San Juan using culture-based and molecular methods. \u003cem\u003eAspergillus\u003c/em\u003e is a common indoor contaminant in moisture-damaged environments, with some species associated with significant health risks. However, species-level identification is often limited. To address this, we collected samples from 14 homes, identifying 28 \u003cem\u003eAspergillus\u003c/em\u003e isolates through morphological examination and gene sequencing of ITS2, beta-tubulin (\u003cem\u003ebenA\u003c/em\u003e), and calmodulin (\u003cem\u003eCaM\u003c/em\u003e) genes. Species-level identifications of 22 isolates revealed species belonging to the subgenera \u003cem\u003eAspergillus\u003c/em\u003e, \u003cem\u003eNidulantes\u003c/em\u003e, and \u003cem\u003eCircumdanti\u003c/em\u003e. We highlighted the \u003cem\u003eCaM\u003c/em\u003e gene's importance in molecular identification by phylogenetic analyses, which showed superior resolution in species differentiation. Culture-based methods also played a crucial role in differentiating closely related species, such as \u003cem\u003eA. flavus\u003c/em\u003e and \u003cem\u003eA. oryzae\u003c/em\u003e, which molecular methods alone could not reliably separate. Our findings underscore the challenges of \u003cem\u003eAspergillus\u003c/em\u003e identification in post-hurricane, water-impacted indoor environments and emphasize the value of integrating phenotypic and genotypic techniques for accurate species identification. These results contribute to a better understanding of fungal diversity and its potential public health implications in disaster-affected settings.\u003c/p\u003e","manuscriptTitle":"Molecular and Culture-Based Identification of Aspergillus Species in Water-Impacted Homes Following Hurricane María in Puerto Rico","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-17 08:00:34","doi":"10.21203/rs.3.rs-6786592/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-08-13T10:41:16+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-08-12T22:18:57+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-08-06T12:47:44+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"169642665208192882837376640874653450002","date":"2025-08-04T13:13:15+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"246380647588093975834874441280335994707","date":"2025-08-04T09:13:08+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"65523717996168190037812952368254617700","date":"2025-08-04T08:11:06+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-08-04T06:55:19+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-07-29T16:36:40+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-06-05T17:54:41+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-06-04T20:15:18+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-06-04T20:11:28+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"3cc67e12-2bdf-483a-98b3-a9f5fd719748","owner":[],"postedDate":"July 17th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":51671633,"name":"Biological sciences/Microbiology"},{"id":51671634,"name":"Biological sciences/Molecular biology"}],"tags":[],"updatedAt":"2025-10-20T16:07:16+00:00","versionOfRecord":{"articleIdentity":"rs-6786592","link":"https://doi.org/10.1038/s41598-025-19869-9","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2025-10-14 15:58:11","publishedOnDateReadable":"October 14th, 2025"},"versionCreatedAt":"2025-07-17 08:00:34","video":"","vorDoi":"10.1038/s41598-025-19869-9","vorDoiUrl":"https://doi.org/10.1038/s41598-025-19869-9","workflowStages":[]},"version":"v1","identity":"rs-6786592","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6786592","identity":"rs-6786592","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}