Two new species of the industrially relevant genus Absidia (Mucorales) from soil of the Brazilian Atlantic Forest

Thalline Rafhaella Leite Cordeiro Thuong Thuong Thi Nguyen Diogo Xavier Lima Suzana Brito Gomes da Silva Catarina Ferreira de Lima Joana D’Arc Alves Leitão Luciana Melo Sartori Gurgel Hyang Burm Lee André Luiz Cabral Monteiro de Azevedo Santiago About the authors

ABSTRACT

During a survey of Mucorales in soil from an upland forest area in Pernambuco, Brazil, two specimens were isolated and characterized based on their morphological, physiological, and molecular data (ITS and LSU rDNA). Phylogenetic analyses of the isolates revealed that the strains URM 8209 and URM 8210 are closely related to species of Absidia. URM 8209 forms conical, subglobose, and strawberry-shaped columellae and the sporangiospores are cylindrical and ellipsoid. URM 8210 produces hemispheric, subglobose, and strawberry-shaped columellae and the sporangiospores are globose, subglobose, ellipsoid, and short cylindrical. Based on evidence obtained through analysis of datasets (LSU and ITS rDNA regions), A. saloaensis sp. nov. (URM 8209) and A. multispora sp. nov. (URM 8210) are proposed here as novel species of Absidia. A table with morphological characteristics of Neotropical Absidia spp. is provided.

Keywords:
Cunninghamellaceae; Mucoromyceta; rDNA; soil; taxonomy

Introduction

The genus Absidia is composed of cosmopolitan fungal species commonly isolated from soil, herbivorous dung and decaying substrates (van Tieghem 1878van Tieghem P. 1878. Troisième mémoire sur les Mucorinées. Annales des Sciences Naturelles Botanique 4: 312-399.). Species of this genus commonly produce sporangiophores in whorls, arising from stolons that bear apophysate and pyriform sporangia with a deliquescent wall. Rhizoids are never opposed to sporangiophores (Benny 2001Benny GL, Humber R, Morton J. 2001. The Zygomycota: Zygomycetes. In: McLaughlin DJ, McLaughlin EG, Lemke PA (eds.) The Mycota. Systematics and Evolution. Verla, Springer. p. 84-195.), and columellae may be conical, subglobose, or aplanate, frequently showing an apical projection (van Tieghem 1878van Tieghem P. 1878. Troisième mémoire sur les Mucorinées. Annales des Sciences Naturelles Botanique 4: 312-399.; Hoffmann et al. 2007Hoffmann K, Discher S, Voigt K. 2007. Revision of the genus Absidia (Mucorales, Zygomycetes): based on physiological, phylogenetic and morphological characters: thermotolerant Absidia spp. form a coherent group, the Mycocladiaceae fam nov. Mycologycal Research 111: 1169-1183. ). Absidia species reproduce asexually by the formation of sporangiospores and sexually through the formation of zygospores within zygosporangia supported by opposite suspensory cells that have appendages (Hoffmann et al. 2007Hoffmann K, Discher S, Voigt K. 2007. Revision of the genus Absidia (Mucorales, Zygomycetes): based on physiological, phylogenetic and morphological characters: thermotolerant Absidia spp. form a coherent group, the Mycocladiaceae fam nov. Mycologycal Research 111: 1169-1183. ; Hoffmann 2010Hoffmann K. 2010. Identification of the genus Absidia (Mucorales, Zygomycetes): a comprehensive taxonomic revision. In: Gherbawy Y, Voigt K. (eds.) Molecular identification of fungi. Berlin, Springer. p. 439-460. ).

Among mucoralean species of Absidia that have been studied for their industrial importance, A. coerulea specimens are capable of transforming saponins that show high yield and regioselectivity to 20 (S)-protopanaxatriol (Chen et al. 2007Chen G, Yang M, Lu Z, et al. 2007. Microbiol transformation of 20(S)-protopanaxatriol-type saponins by Absidia coerulea. Journal of Natural Products 70: 1203-1206.). Absidia glauca performs biotransformation of 3-Oxo-Oleanolic acid resulting in hydroxylated metabolites and both species mentioned above are excellent chitosan producers used in food processing, antimicrobial production and biotransformation of steroid products (Abdel-Fattah et al.1984Abdel-Fattah AF, Ismail AMS, El-Aasar SA. 1984. Production of rennin-like enzyme by Absidia cylindrospora. Agricultural Wastes 11: 125-131.; Smith et al. 1989Smith KE, Latif S, Kirk DN, White KA. 1989. Microbial transformation of steroids IV. 6,7-dehydrogenation; a new class of fungal steroid transformation product. Journal of Steroid Biochemistry 33: 271-276. ; Muzzarelli et al.1994Muzzarelli RAA, Ilari P, Tarsi R, Dubini B, Xia W. 1994. Chitosan from Absidia coerulea. Carbohydrate Polymers 25: 45-50. ; Brzezowska et al. 1996Brzezowska E, Dmochowska-Gladysz J, Kołek T. 1996. Biotransformation XXXIX. Metabolism of testosterone, androstenedione, progesterone and testosterone derivatives in Absidia coerulea culture. The Journal of Steroid Biochemistry Molecular Biology 57: 357-362. ; Huszcza & Gladysz 2003Huszcza E, Gladysz JD. 2003. Transformations of testosterone and related steroids in Absidia glauca culture. Journal of Basic Microbiology 43: 113-120. Rungsardthong et al.2006Rungsardthong V, Wongvuttanakul N, Kongpien N, Chotiwaranon P. 2006. Application of fungal chitosan for clarification of apple juice. Process Biochemistry 41: 589-593. ;Dai et al.2009Dai T, Tegos GP, Burkatovskaya M, Castano AP, Hamblin MR. 2009. Chitosan acetate bandage as a topical antimicrobial dressing of infected burns. Antimicrobial Agents and Chemotherapy 53: 393-400.). Absidia griseola has biotransformation capacity, carrying out microbial hydroxylation of progesterone resulting in 14α-hydroxyprogesterone and 6β, 11α-dihydroxyprogesterone (Habibi et al. 2012Habibi Z, Yousefi M, Ghanian S, Mohammadi M, Ghasemi S. 2012. Biotransformation of progesterone by Absidia griseolla var. igachii and Rhizomucor pusillus. Steroids 77: 1446-1449. ). Absidia fusca and A. cylindrospora are used in bioremediation processes due to their ability to degrade polycyclic aromatic compounds, such as hydrocarbons (Guiraud et al.2008Guiraud P, Bonnet JL, Boumendjel A, et al. 2008. Involvement of Tetrahymena pyriformis and selected fungi in the elimination of anthracene, and toxicity assessment of the biotransformation products. Ecotoxicology Environmental Safety 69: 296-305. ). Specimens of A. cylindrospora have also been used for biosorption of Cadmiun, Copper and Lead metals under experimental conditions (Albert et al. 2018Albert Q, Leleyte L, Lemoine M, et al. 2018. Comparison of tolerance and biosorption of three trace metals (Cd, Cu, Pb) by the soil fungus Absidia cylindrospora. Chemosphere 196: 386-392. ).

Hesseltine & Ellis (1964Hesseltine CW, Ellis JJ. 1964. The genus Absidia: Gongronella and cylindrical-spored species of Absidia. Mycologia 56: 568-601.; 1966Hesseltine CW, Ellis JJ. 1966. Species of Absidia with ovoid sporangiospores I. Mycologia 58: 761-785.) and Ellis & Hesseltine (1965)Ellis JJ, Hesseltine CW. 1965. The genus Absidia: globose-spored species. Mycologia 57: 222-235. monographed the genus Absidia and grouped its species based on the shape of sporangiospores. Later, in a molecular-physiology and micromorphology study of Absidia, Hoffmann et al. (2007Hoffmann K, Discher S, Voigt K. 2007. Revision of the genus Absidia (Mucorales, Zygomycetes): based on physiological, phylogenetic and morphological characters: thermotolerant Absidia spp. form a coherent group, the Mycocladiaceae fam nov. Mycologycal Research 111: 1169-1183. ) showed that the species of this genus basically consisted of three groups separated according to the growth temperatures: (1) mesophilic species, with ideal growth between 25 and 34 °C (includes all currently accepted Absidia s.s species), (2) mycoparasite species of other mucoralean fungi, with optimal growth between 14 and 25 ºC (species after being transferred to Lentamyces), and (3) thermotolerant species, with optimal growth between 37 and 45 °C [species after being transferred to Lichtheimia by Hoffmann et al. (2009)Hoffmann K, Walther G, Voigt K. 2009. Mycocladus vs. Lichtheimia: a correction (Lichtheimiaceae fam. nov., Mucorales, Mucoromycotina). Mycologycal Research 113: 275 -278.]. In the last 10 years, six new Absidia species have been reported worldwide: A. caatinguensis, A. jindoensis, A. koreana, A. panacisoli, A. stercoraria, and A. terrestris (Ariyawansa et al. 2015Ariyawansa HA, Hyde KD, Jayasiri SC, et al. 2015. Fungal diversity notes 111-252-taxonomic and phylogenetic contributions to fungal taxa. Fungal Diversity 75: 27-274. ; Li et al. 2016Li GJ, Hyde KD, Zhao RL, et al. 2016. Fungal diversity notes 253-366: taxonomic and phylogenetic contributions to fungal taxa. Fungal Diversity 78: 1-237. ; Crows et al. 2018Crows PW, Luangsa-Ard JJ, Wingfield MJ, et al. 2018. Fungal Planet description sheets: 785-867. Persoonia 41:1-180. ; Wanasinghe et al. 2018Wanasinghe DN, Phukhamsakda C, Hyde KD, et al. 2018. Fungal diversity notes 709-839: taxonomic and phylogenetic contributions to fungal taxa with an emphasis on fungi on Rosaceae. Fungal Diversity 89: 1-236. ; Zhang et al. 2018Zhang TY, Yu Y, Zhu H, et al. 2018. Absidia panacisoli sp. nov., isolated from rhizosphere of Panax notoginseng. International Journal of Systematic and Evolutionary Microbiology 68: 2468-2472. ).

During a survey on the diversity of mucoralean fungi in soils of upland forest fragments in the semiarid area of Brazil, two specimens of Absidia that varied morphologically and genetically in comparison with the other species of the same genus were isolated. Based on morphological, physiological, and molecular analyses (LSU and ITS rDNA regions), two new species of Absidia are being proposed here.

Materials and methods

Sampling sites

Soil samples were collected from the Brejo Nature Reserve (09º00.418´ S 036º46.898´ W) located in Saloá municipality, Pernambuco State, Brazil. The average annual temperature in this region is 20 ºC, with the rainy season beginning in January/February and ending in September/October and precipitation ranging between 0 to 50 mm in the driest months and 50 to 100 mm in the wettest months. The vegetation is a predominant trait of the species of the Atlantic ombrophilous and semi-deciduous forests, being found in the herbaceous areas composed of litter, shrubby vegetation, grasses, and shrubs. The soil has a podzolic characteristic, with areas ranging from being clayey to containing granite blocks (Silva-Júnior et al. 2012Silva-Júnior AP, Silva CIA. 2012. Plano de Manejo da RPPN. Reserva Natural Brejo. http://www.icmbio.gov.br/portal/images/stories/docs_planos_de_manejo/rppn_reserva_natural_brejo_pm.pdf.
http://www.icmbio.gov.br/portal/images/s...
).

Isolation and purification of Absidia spp.

Five milligrams of soil were inoculated directly into Petri dishes containing wheat germ agar culture medium (Benny 2008Benny GL. 2008. The methods used by Dr. R.K. Benjamin, and other Mycologists to isolate Zygomycetes. Aliso: A Journal of Systematic and Evolutionary Botany 26: 37-61.) plus chloramphenicol (80 mg.L-1), in triplicate. Growth was observed for seven days at room temperature (28 °C) under alternating light and dark conditions. Fragments of mycelium were removed directly from the Petri dishes under a Leica EZ4 stereomicroscope and transferred to malt extract agar plates (MEA) (Benny 2008Benny GL. 2008. The methods used by Dr. R.K. Benjamin, and other Mycologists to isolate Zygomycetes. Aliso: A Journal of Systematic and Evolutionary Botany 26: 37-61.). Slides corresponding to the holotypes of A. saloaensis sp. nov. (URM 94180) and A. multispora sp. nov. (URM 94181) where deposited in the Herbarium URM of the Universidade Federal de Pernambuco. Ex-type living cultures of A. saloaensis sp. nov. (URM 8209) and A. multispora sp. nov. (URM 8210) where deposited in the culture collection Micoteca URM of the Universidade Federal de Pernambuco. Pure cultures were also deposited in the culture collection (CNUFC) of the Environmental Microbiology Laboratory Fungarium, Chonnam National University, Gwangju, Korea (A. saloaensis sp. nov. CNUFC B190012 and A. multispora sp. nov. CNUFC B190013).

Growth experiments

Pure cultures were grown in triplicates in MEA and potato dextrose agar (PDA) and incubated at 15, 20, 25, 28, 30, and 35 °C for 15 days. For morphological identification, fragments were removed from the cultures and observed under a stereomicroscope (Carl Zeiss Axioscope 40) and light microscope (Leica DM500). The color designation of the colonies was performed according to previous literature (Maerz & Paul 1950Maerz AJ, Paul MR. 1950. A Dictionary of Color. 2nd. edn. New York, McGraw-Hill Book Company. ).

Molecular analysis (DNA extraction, amplification, cloning and sequencing)

Genomic DNA was extracted from fresh fungal mycelia that were grown on cellophane at 25 °C for four days using the SolgTM Genomic DNA Preparation Kit (Solgent Co. Ltd., Daejeon, Korea) according to the manufacturer's instructions with a few modifications. The modifications included the DNA precipitated overnight at -20 oC using an equal volume of ice-cold 100 % isopropanol and the DNA pellet was washed twice using 500 μL of ice-cold 70 % ethanol. The rDNA ITS region was amplified using the ITS1 and ITS4 primers (White et al. 1990White TJ, Bruns T, Lee S, Taylor J. 1990. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: Innis MA, Gelfand DH, Sninsky JJ, White TJ. (eds.) PCR protocols: a guide to methods and applications. San Diego, Academic Press. p. 315-322. ), and LROR and LR3 were used to amplify the large subunit (LSU) rDNA (Vilgalys & Hester 1990Vilgalys R, Hester M. 1990. Rapid genetic identification and mapping of enzymatically amplified ribosomal DNA from several Cryptococcus species. Journal of Bacteriology 172: 4238-4246. ; Rehner & Samuels 1995Rehner SA, Samuels GJ. 1995. Molecular systematics of the Hypocreales: a teleomorph gene phylogeny and the status of their anamorphs. Canadian Journal of Botany 73: 816-823.). The PCR products were purified using an Accuprep PCR Purification Kit (Bioneer Corp.). PCR products of the LSU region were used for direct sequencing on the ABI PRISM 3730XL Genetic Analyzer (Applied Biosystems, California, USA) (Macrogen, Daejeon, Korea).

Since direct sequencing of the ITS region from PCR products was unsuccessful, PCR products were cloned using the pGEM-T Easy Vector System (Promega) cloning kit, following the manufacturer’s instructions. These clones were sequenced using primers M13F forward (5’- GTAAAACGACGGCCAGT-3’) and M13R-pUC reverse (5’-CAGGAAACAGCTATGAC-3’) by ABI PRISM 3730XL Genetic Analyzer.

Sequence alignment and phylogenetic analyses

Raw sequences were assembled and edited (edges trimmed) using BioEdit (Hall 1999Hall TA. 1999. BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symposium Series 41: 95-98.). All sequence data used in this study were obtained from GenBank (https://www. ncbi.nlm.nih.gov). Sequences were aligned using MAFFT 7 (https://mafft.cbrc.jp/alignment/server) (Katoh et al. 2019Katoh K, Rozewicki J, Yamada KD. 2019. MAFFT online service: multiple sequence alignment, interactive sequence choice and visualization. Briefings in Bioinformatics 20: 1160-1166. ) and then manually refined in MEGA7 (Kumar et al. 2016Kumar S, Stecher G, Tamura K. 2016. MEGA7: Molecular Evolutionary Genetics Analysis version 7.0 for bigger datasets. Molecular Biology and Evolution 33: 1870-1874.). Bayesian inference (two runs over 3 × 106 generations with a burn-in of 2500) and maximum likelihood (with support estimated by bootstrap analysis with 1000 replicates) analyses were performed with Mrbayes 3.2.2 (Ronquist et al. 2012Ronquist F, Teslenko M, Mark PVD, et al. 2012. MrBayes 3.2: efficient Bayesian phylogenetic inference and model choice across a large model space. Systematic Biology 61: 539 -542. ) and PhyML 3.0 (Guindon et al. 2010Guindon S, Dufayard JF, Lefort V, Anisimova M, Hordijk W, Gascuel O. 2010. New algorithms and methods to estimate maximum-likelihood phylogenies: assessing the performance of PhyML 3.0. Systematic Biology 59: 307-21. ), respectively. The best-fit model of nucleotide substitution for each data set was obtained using jModelTest v.2.1.10 software (Guindon & Gascuel 2003Guindon S, Gascuel O. 2003. A simple, fast, and accurate algorithm to estimate large phylogenies by maximum likelihood. Systematic Biology 52: 696-704. ; Darriba et al. 2012Darriba D, Taboada GL, Doallo R, Posada D. 2012. jModelTest 2: more models, new heuristics and parallel computing. Nature Methods 9: 772. http://dx.doi.org/10.1038/nmeth.2109
http://dx.doi.org/10.1038/nmeth.2109...
)

Sequence data were compared with those of similar sequences available in the National Center for Biotechnology Information GenBank database using BLASTn. The newly obtained sequences were deposited in the GenBank database: A. saloaensis sp. nov.: ITS (MN953781), LSU (MN953783), and A. multispora sp. nov: ITS (MN953780), LSU (MN953782) (Tab. 1).

Table 1
Accession numbers in collection cultures and voucher numbers of sequences used for the phylogenetic analysis

Results

Phylogenetic analyses

The phylogenetic relationship of two novel species and related species was determined by analysis of concatenated sequences datasets of two loci (ITS and LSU) (Fig.1). The concatenated alignment consisted of 1516 characters (including alignment gaps) with 816 and 700 characters used in the ITS and LSU, respectively. TIM2+I+G was found to be the most suitable model for the analysis of the concatenated ITS-LSU sequences. Absidia multispora URM 8210 was closely related to A. anomala CBS 125.68. The BLASTn search revealed that the ITS and LSU sequences of URM 8210 strain were 92.7 % and 97.7 % identical with A. anomala (GenBank accession numbers: EF030523 and JN982937), respectively. Absidia saloaensis URM 8209 was clustered together with A. koreana EML-IFS45-1 in the concatenated ITS-LSU tree. In addition, the BLASTn search showed that ITS and LSU sequences of URM 8209 were 83.14 % and 93.9 % homologous with A. koreana (GenBank accession numbers: KR030062 and KR030056), respectively.

Figure 1
Phylogenetic tree of Absidia multispora URM 8210 and Absidia saloaensis URM 8209 and related species based on maximum likelihood (ML) analysis of a combined DNA data set of ITS and LSU sequences. Bootstrap values for Bayesian posterior probabilities (BYPP) over 0.95 and maximum likelihood greater than 70 % are placed above the branches. Bootstrap values lower than 0.95 and 70 % are marked with “*”, and absent are marked with “-”. The bar indicates the number of substitutions per position. Cunninghamella phaeospora CBS 692.68 and Cunninghamella vesiculosa CBS 989.96 were used as outgroups. The new species are in blue and the type species are indicated with T (ex-type) or NT (neotype).

Taxonomy

Absidia multispora T.R.L. Cordeiro, D.X. Lima, Hyang B. Lee & A.L. Santiago sp. nov. (Fig. 2A-I).

Figure 2
Absidia multispora (URM 8210). A. Surface of colony on PDA at 28°C; B, C. Branched sporangiophore with fertile sporangia and an abortive sporangium; D. Branched sporangiophore with fertile sporangia; E. Unbranched sporangiophore with sporangium; F, G. Unbranched sporangiophore with a columella with one projection on its surface; H. Rhizoids; I. Sporangiospores. Bars: B, C, D, E, F, G, I = 10 µm; H = 20 µm.

Etymology: multispora. Reference to variable-shaped sporangiospores that are produced.

Diagnosis: Differs from other species of Absidia by the combination of the following characters: globose, subglobose, ellipsoid, cylindrical, short-cylindrical, and irregular sporangiospores. Sporangia subglobose and pyriform and columellae which are hemispheric, subglobose and strawberry-shaped, some of them with a projection on their surface.

Type: Brazil, Pernambuco: Saloá, Fazenda Brejo Nature Reserve (09º00.418´ S 036º46.898´ W) isolated from soil samples, 10 Nov 2018, T.R.L. Cordeiro (Holotype: URM 94181, Herbarium URM; Ex-type: URM 8210, Micoteca URM). Index Fungorum number: IF557224. GenBank accession numbers: MN953780 and MN953782 (ITS and LSU, respectively).

Description: Colony brownish gray turning dark gray (MP21 A1, gray-drab), zoned, colonizing the entire Petri dish (9 cm in diam) within five days at 28 °C; light gray reverse (MP20 A1, minera-grey). Odor absent. Rhizoids present, short or long, branched or unbranched. Stolons light gray, with slightly encrusted wall. Sporangiophores slightly brownish-gray, arising from stolons, usually unbranched, single or in whorls of 2 (4), occasionally with aging, up to 270 µm in length and 5 µm in width, some with one swelling, thick-walled; successive branches may originate from abortive sporangia; one septum was observed near the apophysis, two septa were rarely present. Sporangia brownish-gray, apophysate, subglobose, and pyriform, up to 30 µm in diam, multisporated, and smooth-walled. Columellae hyaline, hemispheric, subglobose, and strawberry-shaped, (7-) 10-16 × (-7) 10-15 (-20) µm, smooth-walled. Projection on columellae present or absent; when present, mostly conical, short or thin, elongated, needle-like, up to 5 × 2.5 µm. Collar usually evident. Sporangiospores brownish-gray, globose, subglobose (2.5-) 5-7.5 (-9), ellipsoid, short cylindrical, broadly-ellipsoidal, irregular, 5-9.5 (-12) × (3.5) 5-7.5 (-9) µm, smooth, and thick-walled. Chlamydospores absent. Zygospores not observed.

Absidia saloaensis T.R.L. Cordeiro, D.X. Lima, Hyang B. Lee & A.L. Santiago sp. nov. (Fig. 3A-I).

Figure 3
Absidia saloaensis (URM 8209). A. Surface of colony on PDA at 28°C; B. Two sporangiophores in a whorl with sporangia and rhizoids; C, D, E, F, G. Unbranched sporangiophore with a columella with one projection on its surface; H. Rhizoids; I. Sporangiospores. Bars: B = 50 µm; C, D, E, F, G, I = 10 µm; H = 100 µm.

Etymology: saloaensis. Reference to the city (Saloá) from where the species was first isolated.

Diagnosis: Differs from other species of Absidia by the combination of the following characters: strawberry-shaped columellae, and cylindrical and elliptical sporangiospores.

Type: Brazil, Pernambuco: Saloá, Fazenda Brejo Nature Reserve, 09º00.418´ S 036º46.898´ W, isolated from soil samples, 10 Nov 2018, T.R.L. Cordeiro (Holotype: URM 94180, Herbarium URM; Ex-type: URM 8209, Micoteca URM). Index Fungorum number: IF557227. GenBank accession: MN953781 and MN953783 (ITS and LSU, respectively).

Description: Colony grayish-brown (MP22 C1, dusty-gr.) colonizing the entire Petri dish (9 cm in diam) within five days at 28 °C; reverse grayish-white zoned (MP21 B2, olive-gray). Odor absent. Rhizoids present, weakly branched. Stolons hyaline, smooth-walled. Sporangiophores hyaline, long and short, growing along the stolons and terminally, up to 280 µm in length and 6 µm in width, erect, slightly encrusted walled, with one septum near the apophysis, and occasionally with one swelling; solitary or more often in whorls of 5 (6); sporangia hyaline, pyriform (15-) 20-35 µm in diam, multisporate, deliquescent, smooth-walled, apophysate. When the sporangium wall liquefies, some sporangiospores may remain attached to the columellae. Columellae hyaline, conical to subglobose and strawberry-shaped, (4.5-) 7-22 × (5-) 8.5-20 (-25) µm, smooth-walled; collar visible. Projection on the columella generally elliptical, conical, or needle-shaped, up to 5 × 3.5 µm, occasionally short, almost inconspicuous. Sporangiospores hyaline, mostly cylindrical and elliptical, (3.5-) 5-7 (9.5) × 2.5-3.5 (-5) µm, some slightly constricted in the center, smooth-walled. Chlamydospores absent. Zygospores not observed.

Discussion

Morphologically, A. saloaensis sp. nov. and A. multispora sp. nov. present characteristics of the Absidia sensu stricto group, such as apophysate sporangiophores arising from stolons, rhizoids never opposed to sporangiophores, and pyriform sporangia (Benny 2001Benny GL, Humber R, Morton J. 2001. The Zygomycota: Zygomycetes. In: McLaughlin DJ, McLaughlin EG, Lemke PA (eds.) The Mycota. Systematics and Evolution. Verla, Springer. p. 84-195.). For the Neotropical region, only nine species had been reported (Tab. 2).

Table 2
Morphological characteristics of Absidia species from Neotropics plus A. koreana.

Absidia multispora sp. nov. is phylogenetically closely related to A. anomala (Fig. 1). However, morphologically, the former differs from A. anomala in the size and shape of sporangiospores and columellae. Absidia anomala produces cylindrical sporangiospores with 3-4 × 2.2 µm, differing from A. multispora sp. nov. that produces sporangiospores globose, subglobose (2.5-) 5-7.5 (-9) µm, ellipsoidal, short cylindrical, broadly-ellipsoidal, and irregular with 5-9.5 (-12) × (3.5) 5-7.5 (-9) µm. Additionally, the columellae of A. multispora are hemispherical, subglobose, and strawberry-shaped, unlike the ones of A. anomala that are hemispherical only (Hesseltine & Ellis 1964Hesseltine CW, Ellis JJ. 1964. The genus Absidia: Gongronella and cylindrical-spored species of Absidia. Mycologia 56: 568-601.).

Absidia saloaensis sp. nov. is phylogenetically related to A. koreana (Fig. 1), but morphologically both species are different. A. saloaensis produces bigger columellae and sporangiospores than those observed in A. koreana. Furthermore, A. saloaensis produces strawberry-shaped columellae, while A. koreana has globose columellae. The sporangiospores of A. koreana are cylindrical with 2.07-4.28 × 1.73-1.98 µm (Ariyawansa et al. 2015Ariyawansa HA, Hyde KD, Jayasiri SC, et al. 2015. Fungal diversity notes 111-252-taxonomic and phylogenetic contributions to fungal taxa. Fungal Diversity 75: 27-274. ), unlike A. saloaensis sp. nov. sporangiospores that are cylindrical and elliptical with (3.5-) 5-7 (9.5) × 2.5-3.5 (-5) µm.

In conclusion, our molecular analyses (ITS and LSU rDNA) show that A. multispora sp. nov. and A. saloaensis sp. nov. are genetically different from other Absidia species. Additionally, both novel isolates exhibit a combination of morphological traits that are not yet described for other Absidia species. In addition to their bioremediation capacity, Absidia species present great potential as chitosan producers and biotransformers of saponins, organic acids, and steroids. Therefore, future experiments to elucidate whether A. saloaensis and A. multispora exhibit industrial potential should be highly encouraged.

Acknowledgements

The authors express their gratitude to Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) for the scholarships provided to authors and for the research grant awarded to A.L. Santiago. This manuscript was financed by the project ‘Diversity of Mucoromycotina in the different ecosystems of the Atlantic Rainforest of Pernambuco’ (FACEPE- APQ - 0842-2.12/14) and was supported in part by the Graduate Program for the Undiscovered Taxa of Korea funded by NIBR of the Ministry of Environment (MOE) of Korea.

References

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  • Albert Q, Leleyte L, Lemoine M, et al 2018. Comparison of tolerance and biosorption of three trace metals (Cd, Cu, Pb) by the soil fungus Absidia cylindrospora Chemosphere 196: 386-392.
  • Ariyawansa HA, Hyde KD, Jayasiri SC, et al 2015. Fungal diversity notes 111-252-taxonomic and phylogenetic contributions to fungal taxa. Fungal Diversity 75: 27-274.
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  • Benny GL. 2008. The methods used by Dr. R.K. Benjamin, and other Mycologists to isolate Zygomycetes. Aliso: A Journal of Systematic and Evolutionary Botany 26: 37-61.
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Publication Dates

  • Publication in this collection
    02 Oct 2020
  • Date of issue
    Jul-Sep 2020

History

  • Received
    11 Feb 2020
  • Accepted
    12 June 2020
Sociedade Botânica do Brasil SCLN 307 - Bloco B - Sala 218 - Ed. Constrol Center Asa Norte CEP: 70746-520 Brasília/DF - Alta Floresta - MT - Brazil
E-mail: acta@botanica.org.br