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Discovery of Backusella paraconstricta sp. nov. (Mucorales, Mucoromycota) in an upland forest in northeastern Brazil with an identification key for Backusella from the Americas

Abstract

During a survey of mucoralean fungi in soil from an upland forest area located in Pernambuco, Brazil, a strain of Backusella (URM 8637) was isolated. Based on morphological, physiological, and molecular data [internal transcribed spacer (ITS) and large subunit (LSU) ribosomal DNA regions], it was recognized that this Backusella differed from all other species in the genus. Morphologically, the new species is characterized as forming varied-shaped columellae, including elongated, basally constricted, unisporate (rare) and multisporate sporangiola, and ellipsoidal sporangiospores. The maximum temperature growth of URM 8637 on malt extract agar and potato dextrose agar was 36 °C. In the phylogram, it was closely related to B. constricta. Based on the evidence from the analyzed datasets, a new species of Backusella is proposed. An updated identification key for Backusella from the Americas is provided.

Keywords:
Backusellaceae; ITS and LSU rDNA; Soil; Taxonomy; New species

Introduction

The genus Backusella was established by Ellis and Hesseltine in 1969Ellis JJ, Hesseltine CW. 1969. Two new members of the Mucorales. Mycologia 61: 863 -872. and typified with B. circina J.J. Ellis and Hesselt. Members of this genus can be found on various substrates, including excrement, invertebrates, leaf litter, soil, toads, and wood (Benny & Benjamin 1975Benny GL, Benjamin RK. 1975. Observations on Thamnidiaceae (Mucorales). New taxa, new combinations, and notes on selected species. Aliso 8: 301-351. doi: 10.5642/aliso.19750803.10
https://doi.org/10.5642/aliso.19750803.1...
; Walther et al. 2013Walther G, Pawłowska J, Alastruey-Izquierdo A et al. 2013. DNA barcoding in Mucorales: an inventory of biodiversity. Persoonia-Molecular Phylogeny and Evolution of Fungi 30: 11-47.; Lima et al. 2016Lima DX, Voigt K, de Souza CAF, Oliveira RJV, Souza-Motta CM, Santiago ALCMA. 2016. Description of Backusella constricta sp. nov. (Mucorales, ex Zygomycota) from the Brazilian Atlantic Rainforest, including a key to species of Backusella. Phytotaxa 289: 59-68. doi: 10.11646/phytotaxa.289.1.4
https://doi.org/10.11646/phytotaxa.289.1...
; Nguyen & Lee 2018Nguyen TTT, Lee HB. 2018. Isolation and characterization of three zygomycetous fungi in Korea: Backusella circina, Circinella muscae, and Mucor ramosissimus. Mycobiology 46: 317-327 doi: 10.1080/12298093.2018.1538071
https://doi.org/10.1080/12298093.2018.15...
; Nguyen et al. 2021Nguyen TTT, Voigt K, Santiago ALCMA, Kirk PM, Lee HB. 2021. Discovery of novel Backusella (Backusellaceae, Mucorales) isolated from invertebrates and toads in Cheongyang, Korea. Journal of Fungi 7: 513. doi: 10.3390/jof7070513
https://doi.org/10.3390/jof7070513...
; Urquhart et al. 2021Urquhart AS, Douch JK, Heafield TA, Buddie A, Idnurm A. 2021. Diversity of Backusella (Mucoromycotina) in south-eastern Australia revealed through polyphasic taxonomy. Persoonia 46: 1-25. doi: 10.3767/persoonia.2021.46.01
https://doi.org/10.3767/persoonia.2021.4...
). Backusella belongs to Backusellaceae K. Voigt & P. M. Kirk, however it was previously associated with Mucoraceae Dumort. Species of this genus were later transferred to Thamnidiaceae Fitzp. due to morphological similarities with Thamnidium Link (Pidoplichko & Milko 1971Pidoplichko NM, Milko AA. 1971. Atlas of mucoralean fungi. Ukranian, Academic Science of the Ukranian SSR.; 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. ). Species of Backusella are known to occur in Australia, Brazil, China, Japan, South Korea, the United States of America, and Thailand (Zheng et al. 2013Zheng RY, Liu XY, Wang YN. 2013. Two taxa of the new record genus Backusella from China. Mycosystema 32: 330-341.; Lima et al. 2016Lima DX, Voigt K, de Souza CAF, Oliveira RJV, Souza-Motta CM, Santiago ALCMA. 2016. Description of Backusella constricta sp. nov. (Mucorales, ex Zygomycota) from the Brazilian Atlantic Rainforest, including a key to species of Backusella. Phytotaxa 289: 59-68. doi: 10.11646/phytotaxa.289.1.4
https://doi.org/10.11646/phytotaxa.289.1...
; Nguyen et al. 2021Nguyen TTT, Voigt K, Santiago ALCMA, Kirk PM, Lee HB. 2021. Discovery of novel Backusella (Backusellaceae, Mucorales) isolated from invertebrates and toads in Cheongyang, Korea. Journal of Fungi 7: 513. doi: 10.3390/jof7070513
https://doi.org/10.3390/jof7070513...
; Urquhart et al. 2021Urquhart AS, Douch JK, Heafield TA, Buddie A, Idnurm A. 2021. Diversity of Backusella (Mucoromycotina) in south-eastern Australia revealed through polyphasic taxonomy. Persoonia 46: 1-25. doi: 10.3767/persoonia.2021.46.01
https://doi.org/10.3767/persoonia.2021.4...
; Hurdeal et al. 2022Hurdeal VG, Jones EG, Santiago ALCMA, Hyde KD, Gentekaki E. 2022. Expanding the diversity of mucoralean fungi from northern Thailand: A novel soil Backusella species. Phytotaxa 559: 275-284.; de Lima et al. 2022De Lima CLF, Lundgren JDAL, Nguyen TTT et al. 2022. Two new species of Backusella (Mucorales, Mucoromycota) from Soil in an Upland Forest in Northeastern Brazil with an Identification Key of Backusella from the Americas. Journal of Fungi 8: 1038. doi: 10.3390/jof8101038
https://doi.org/10.3390/jof8101038...
).

Species of Backusella form sporophores that are transiently curved when young, and erect when mature. They may or not form laterally unispored and multispored pedicellate sporangiola, which have persistent walls. Short, simple, or sympodially branched sporangiophores proliferating only multispored and/or unispored sporangiola may form near the substrate (Benny 2005Benny GL. 2005. Zygomycetes. http://zygomycetes.org. 09 Jan. 2023.
http://zygomycetes.org...
; Walther et al. 2013Walther G, Pawłowska J, Alastruey-Izquierdo A et al. 2013. DNA barcoding in Mucorales: an inventory of biodiversity. Persoonia-Molecular Phylogeny and Evolution of Fungi 30: 11-47.; de Souza et al. 2014de Souza JI, Marano AV, Pires-Zotarelli CLA, Chambergo FS, Harakava R. 2014. A new species of Backusella (Mucorales) from a Cerrado reserve in Southeast Brazil. Mycological Progress 13: 975-980. doi: 10.1007/s11557-014-0981-3
https://doi.org/10.1007/s11557-014-0981-...
). Based on the recognition of transiently curved sporophores as a distinctive feature of Backusella, Walther et al. (2013)Walther G, Pawłowska J, Alastruey-Izquierdo A et al. 2013. DNA barcoding in Mucorales: an inventory of biodiversity. Persoonia-Molecular Phylogeny and Evolution of Fungi 30: 11-47. transferred some species from Mucor to Backusella. As of January 2023, 34 species have been accepted in Backusella (Wijayawardene et al. 2022Wijayawardene NN, Hyde K D, Dai DQ et al. 2022. Outline of fungi and fungus-like taxa -2021. Mycosphere 13: 53-453. doi: 10.5953/mycosphere/13/1/2
https://doi.org/10.5953/mycosphere/13/1/...
; Hurdeal et al. 2022Hurdeal VG, Jones EG, Santiago ALCMA, Hyde KD, Gentekaki E. 2022. Expanding the diversity of mucoralean fungi from northern Thailand: A novel soil Backusella species. Phytotaxa 559: 275-284.; de Lima et al. 2022De Lima CLF, Lundgren JDAL, Nguyen TTT et al. 2022. Two new species of Backusella (Mucorales, Mucoromycota) from Soil in an Upland Forest in Northeastern Brazil with an Identification Key of Backusella from the Americas. Journal of Fungi 8: 1038. doi: 10.3390/jof8101038
https://doi.org/10.3390/jof8101038...
; Cordeiro et al. 2023Cordeiro TRL, Walther G, Souza CAF et al. 2023. A polyphasic approach to the taxonomy of Backusella reveals two new species. Mycological Progress 22: 16 doi: 10.1007/s11557-023-01864-x
https://doi.org/10.1007/s11557-023-01864...
).

During a study on the diversity of mucoralean fungi in an upland forest area in Pernambuco, northeastern Brazil, a strain of Backusella was found. Its identity was confirmed using morphological and molecular data, which included internal transcribed spacer (ITS) and large subunit (LSU) of ribosomal DNA (rDNA). In this work we describe and illustrate this new species. This is not the first new species of Backusella discovered in Brazil; Backusella azygospora, B. brasiliensis, B. constricta, B. obliqua, and B. pernambucensis were previously isolated and described for the first time in this country. This indicates that Brazil is a tropical hotspot for discovering new mucoralean fungi (de Lima et al. 2022De Lima CLF, Lundgren JDAL, Nguyen TTT et al. 2022. Two new species of Backusella (Mucorales, Mucoromycota) from Soil in an Upland Forest in Northeastern Brazil with an Identification Key of Backusella from the Americas. Journal of Fungi 8: 1038. doi: 10.3390/jof8101038
https://doi.org/10.3390/jof8101038...
). In this study, we updated the identification key of Backusella from the Americas provided by de Lima et al. (2022)De Lima CLF, Lundgren JDAL, Nguyen TTT et al. 2022. Two new species of Backusella (Mucorales, Mucoromycota) from Soil in an Upland Forest in Northeastern Brazil with an Identification Key of Backusella from the Americas. Journal of Fungi 8: 1038. doi: 10.3390/jof8101038
https://doi.org/10.3390/jof8101038...
with two additional species.

Materials and methods

Collection site

Soil samples were collected in April 2022 in the district of Jenipapo, municipality of Sanharó (8°17'08.6" S 36°30'53.9" W), located in the state of Pernambuco, Brazil. The local vegetation comprises of subdeciduous and deciduous forests. The climate is tropical and rainy with dry summers. The rainy season starts in January/February and ends in September, but it can continue until October. The average annual temperature is 31 °C, with an average annual rainfall of 496 mm (MME 2005MME - Ministério das Minas e Energia. 2005. Projeto cadastro de fontes de abastecimento por água subterrânea. Diagnóstico do Município de Sanharó, Pernambuco. Companhia de Pesquisa de Recursos Minerais/ Programa de Desenvolvimento Energético dos Estados e Municípios. Recife, CPRM/PRODEEM. https://rigeo.cprm.gov.br/jspui/bitstream/doc/16670/1/Rel_Sanhar%C3%B3.pdf. 24 Dec. 2021.
https://rigeo.cprm.gov.br/jspui/bitstrea...
). Using sterilized spatulas, soil samples were collected at a depth of 5 cm, packed in sterile plastic bags and stored in styrofoam boxes with ice for transport to the Laboratory of Fungos Zigospóricos at the Universidade Federal de Pernambuco (UFPE).

Isolation, purification and morphological description

Five milligrams of soil was added to wheat germ agar culture medium (Benny 2008 Benny GL. 2008. The methods used by Dr. R. K. Benjamin, and other mycologists to isolate zygomycetes. Aliso 26: 37-61. doi: 10.5642/aliso.20082601.08
https://doi.org/10.5642/aliso.20082601.0...
), supplemented with chloramphenicol (80 mg L-1), contained in Petri plates. Colony growth was monitored for 72 h at room temperature (26 ± 2 °C). Mycelial fragments were removed directly from the Petri dishes under a Leica EZ4 stereomicroscope (Leica Microsystems, Wetzlar, Germany) and transferred to malt extract agar (MEA) plates (Benny 2008 Benny GL. 2008. The methods used by Dr. R. K. Benjamin, and other mycologists to isolate zygomycetes. Aliso 26: 37-61. doi: 10.5642/aliso.20082601.08
https://doi.org/10.5642/aliso.20082601.0...
). At least 50 measurements were made for each fungal structure from plates incubated at 25 °C for seven days on MEA in the dark. Mycelial fragments from the specimens were transferred to slides with 2% KOH or lactophenol blue and observed using a light microscope (Leica DM500). A slide corresponding to the holotype of the new species (URM 8367) was deposited in the Herbarium URM, and the ex-type living culture of the new species (URM 8367) was deposited in the URM Culture Collection of the Universidade Federal de Pernambuco.

Growth experiments and macro and microscopy

URM 8637 was grown in triplicate on both MEA and potato dextrose agar (PDA; HiMedia, Vadhani, India) and incubated at 15, 20, 25, 30, 35 and 40 °C in the dark for morphological analysis. Colony growth was measured every 24 h and monitored for 10 d. The maximum growth temperature was determined by growing the strains on MEA at one degree increments. For morphological observation, culture slides (with fragments of the fungal mycelia) were prepared, stained with 2% KOH or lactophenol blue, and observed using a light microscope (Leica DM500). Colony color was determined according to Kornerup and Wanscher (1978)Kornerup A, Wanscher JH. 1978. Methuen handbook of colours. 3rd. edn. London, Eyre Methuen..

DNA extraction, amplification, purification, and sequencing

Fungal biomass was obtained from MEA slant cultures incubated at 28 °C for up to five days and was transferred to 2-mL microtubes with screw caps. To each tube, 0.5 g of acid-washed glass beads (Sigma-Aldrich, Darmstadt, Germany) of two different diameters (150-212 μm and 425-600 μm, 1:1) were added and the fungal biomass was crushed by stirring at high speed in a FastPrep homogenizer (FastPrep-24, MP Biomedicals, California, USA). Genomic DNA was extracted as described by de Oliveira et al. (2016)De Oliveira RJV, Bezerra JL, Lima TEF, Silva GA, Cavalcanti MAQ. 2016. Phaeosphaeria nodulispora, a new endophytic coelomycete isolated from tropical palm (Cocos nucifera) in Brazil. Nova Hedwigia 103: 185-192. doi: 10.1127/nova_hedwigia/2016/0343
https://doi.org/10.1127/nova_hedwigia/20...
, whereby the mycelium was homogenized in CTAB lysis buffer [2% cetyltrimethylammonium bromide, 20 mM EDTA, 0.1 M Tris-HCl (pH 8.0), 1.4 M NaCl (Doyle & Doyle 1987Doyle JJ, Doyle JL. 1987. A rapid DNA isolation procedure for small quantities of fresh leaf tissue. Phytochemical Bulletin 19: 11-15. ; 1990Doyle JJ, Doyle JL. 1990. Isolation of plant DNA from fresh tissue. Focus 12: 13-15. )], and washed with chloroform: isoamyl alcohol (24:1). The DNA-containing supernatant was then separated from the hyphal residues. The supernatant was mixed with an equal volume of isopropanol followed by DNA precipitation after incubation at -20 °C for 30 min. After centrifugation at 13,000 rpm for 15 min, the resulting DNA pellet was washed with 70% ethanol and resuspended in 50 μL ultrapure water.

For the amplification of ITS and LSU rDNA, the primer pairs ITS1/ITS4 and LR1/LSU2 (White et al. 1990White TJ, Bruns T, Lee S, Taylor J. 1990. Amplification and direct sequencing of fungal ribosomal RNA 640 genes for phylogenetics. In: Innis MA, Gelfand DH, Sninsky JJ, White TJ (eds.) PCR protocols: A guide 641 to methods and applications. San Diego, Academic Press. p. 315-322.; van Tuinen et al. 1998van Tuinen D, Zhao B, Gianinazzi-Pearson V. 1998. PCR in studies of AM fungi: From primers to application. In: Varma AK (ed.) Mycorrhizal manual. Berlin, Springer. p. 387-399.; Santiago et al. 2014Santiago ALCMA, Hoffmann K, Lima DX et al. 2014. A new species of Lichtheimia (Mucoromycotina, Mucorales) isolated from Brazilian soil. Mycological Progress 13: 343-352. doi: 10.1007/s11557- 013-0920-8
https://doi.org/10.1007/s11557- 013-0920...
) were used, respectively. The final amplicons were purified with the NucleoSAP enzymatic mix (Molecular Biotecnologia, Belo Horizonte, Brazil) and sequenced at Plataforma de Tecnologia Genômica e Expressão Gênica of the Centro de Ciências Biológicas - UFPE (Pernambuco, Brazil).

Sequence alignment and phylogenetic analysis

The sequences of the URM 8637 strain were used as queries to perform BLASTn in the GenBank database to identify the closest matching sequences. Raw reads were edited to remove ambiguous bases at both ends. Two separate datasets, one for ITS and one for LSU, were assembled using sequences of all available described species in the database. The datasets were aligned using MAFFT v.7 (https://mafft.cbrc.jp/alignment/server) (Katoh & Standley 2013Katoh K, Standley DM. 2013. MAFFT multiple sequence alignment software version 7: Improvements in performance and usability. Molecular Biology and Evolution 30: 772-780. doi: 10.1093/molbev/mst010
https://doi.org/10.1093/molbev/mst010...
) for each molecular marker. The sequences were manually edited using MEGA version 7 (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. doi: 10.1093/molbev/msw054%20
https://doi.org/10.1093/molbev/msw054%20...
). The ITS and LSU rDNA region alignments were concatenated before the phylogenetic analyses (Supplementary Material 01 Supplementary material 01. ). Bayesian inference (BI) and maximum likelihood (ML) analyses were performed with MrBayes v.3.2.2 (Ronquist et al. 2012Ronquist F, Teslenko M, van der Mark P. et al. 2012. MrBayes 3.2: Efficient bayesian phylogenetic inference and model choice across a large model space. Systematic Biology 61: 539-542. doi: 10.1093/sysbio/sys029
https://doi.org/10.1093/sysbio/sys029...
) on XSEDE and RAxML-HPC BlackBox v.8.2.8 (Stamatakis et al. 2008Stamatakis A, Hoover P, Rougemont J. 2008. A rapid bootstrap algorithm for RAxML web-servers. Systematic Biology 57: 758-771. doi: 10.1080/10635150802429642
https://doi.org/10.1080/1063515080242964...
; Stamatakis 2014Stamatakis A. 2014. RAxML version 8: A tool for phylogenetic analysis and post-analysis of large phylogenies. Bioinformatics 30: 1312-1313. doi: 10.1093/bioinformatics/btu033
https://doi.org/10.1093/bioinformatics/b...
), respectively, using the CIPRES Science Gateway (http://www.phylo.org/) (Miller et al. 2010Miller MA, Pfeiffer W, Schwartz T. 2010. Creating the CIPRES Science Gateway for inference of large phylogenetic trees. In: Proceedings of the Gateway Computing Environments Workshop (GCE) , 14 November 2010, New Orleans. p. 1-8. doi: 10.1109/GCE.2010.5676129
https://doi.org/10.1109/GCE.2010.5676129...
). The ML analysis was performed using the GTR+I+G standard nucleotide substitution model, and BI was performed using the best nucleotide model selected by AIC in MrModeltest 2.3 (Nylander 2004Nylander JAA. 2004. MrModeltest v2. Program distributed by the author. Evolutionary Biology Centre, Uppsala University.). Bayesian inference analysis was conducted using 1 × 106 generations with a tree burn-in value of 25%. Phylogenetic trees were viewed and arranged using the Interactive Tree of Life (iTOL) v4 (https://itol.embl.de/) (Letunic & Bork 2019Letunic I, Bork P. 2019. Interactive Tree Of Life (iTOL) v4: Recent updates and new developments. Nucleic Acids Research 47: W256-W259. doi: 10.1093/nar/gkz239
https://doi.org/10.1093/nar/gkz239...
). Values less than 0.95 BI posterior probability and 70% ML bootstrap were not considered. The newly obtained sequences were deposited in the GenBank database. GenBank accession numbers are listed in Tab. 1.

Table 1
Specimens used in the phylogenetic analyses with their GenBank accession numbers.

Results

Phylogenetic analyses

Phylogenetic relationships within Backusella were estimated using BI and ML approaches. The alignment of ITS and LSU consisted of 60 sequences and 1699 characters with 1058 and 641 characters used in the ITS and LSU, respectively. Phylogenetic analysis identified the isolate as a new species that was sister to B. constricta with statistical support (94%ML/1.00PP). The topology of the concatenated tree, as well as the ML bootstrap values and BI posterior probabilities (>70% and >0.95, respectively) are shown in Fig. 1.

Figure 1
Phylogenetic tree of Backusella inferred from the combined internal transcribed spacer (ITS) and large subunit (LSU) ribosomal DNA (rDNA) sequences. Support values on the branches represent maximum likelihood bootstrap support and Bayesian inference posterior probabilities in this order. Bootstrap values lower than 70% or 0.95 are marked with “*”. New taxa are in bold font. Mucor indicus CBS 226.29 was used as outgroup. Ex-type, ex-epitype, and ex-lectotype strains are marked with T, ET, and LT, respectively.

Taxonomy

Backusella paraconstricta F.R.S. Santos, T.R.L. Cordeiro, Hyang B. Lee & A.L. Santiago sp. nov. - Fig. 2

Figure 2
Backusella paraconstrictasp. nov. (URM 8637) A. Colony surface (left) and reverse (right) on malt extract agar (MEA) at 25 °C, B.sporophore with sporangium, C.sporophore branch with sporangium,D -H. sporophore with columella I, J.short sporophore with sporangiola,K. sporangiospores.Scale bars = 20 µm.

MycoBank number: 847766

Colonies rapidly growing, initially white, becoming pale to gray (1-1 B), reaching the entire Petri dish (9 cm diameter and 1.5 cm high) after 4 days at 25 ºC on MEA; reverse yellow (2-8A). Rhizoids well branched. Long sporophores hyaline, curved when young and erect at maturity, frequently simple, infrequently sympodially branched up to three times (rarely four times), with rarely recurved branches, up to 12 μm in diameter. Lateral pedicellate sporangia not formed on sporophores. Sporangia brownish-yellow, globose, smooth-walled with vitreous aspect, 30-70 μm in diameter. Columellae of sporangia light grey, conical, subglobose, infrequently applanate and very rarely elongate to ellipsoid or cylindrical, 20-35 × 15-35 μm, smooth-walled. Some columellae may rarely show a slight constriction at the base. Short sporophores, unbranched, or less commonly sympodially branched (up to three times), bearing only multispored (frequent) and/or unispored sporangiola (rare) formed near the substrate. Multispored sporangiola brownish, globose, 15-40 μm in diameter, containing 3-15 sporangiospores each, persistent and spinulose-walled. Sporangiola unisporate globose, up to 20 μm in diameter, minutely spinulose. Columellae of sporangiola conical to flattened, subglobose, elongated, rarely globose, hyaline or grayish, 15-20 × 12-40 μm, smooth-walled. Collar evident. Sporangiospores hyaline, with greenish contents, ellipsoid (mostly), subglobose, some globose and irregular, 7-16 (-20) × 4.5-9.5 (-12) μm, smooth-walled. No chlamydospores or zygosporangia were observed.

Etymology: referring to the phylogenetic proximity to Backusella constricta.

Material examined: Brazil, Pernambuco; the district of Jenipapo, municipality of Sanharó (8°17'08.6" S 36°30'53.9" W), from soil, 12 Apr. 2022, F.R.S. Santos (Holotype URM 95258; ex-holotype URM 8637). GenBank accessions: OQ625517 (ITS) and OQ625516 (LSU).

Habitat: Soil.

Distribution: Pernambuco state (Brazil).

Media and temperature test: On MEA, at 10 °C - no growth; at 15 °C - slow growth (6 cm in diameter after 168 h); at 20 °C - good growth (8 cm in diameter after 120 h); at 25 °C - excellent growth (9 cm in diameter after 96 h); at 30 °C - good growth (9 cm in diameter after 144 h); at 35 °C - slow growth (9 cm in diameter after 192 h); at 40 °C - no growth. Backusella paraconstricta exhibited similar growth and development of reproductive structures on MEA and PDA culture media. Maximum temperature growth on both MEA and PDA was 36 °C.

Identification key for Backusella species in the Americas

1. Sporangiola formed ………………..……………….. 2

1. Sporangiola not formed ………………..……………….. B. oblongielliptica

2. Unispored sporangiola abundant ………………..……………….. B. circina

2. Unispored sporangiola rare or not formed ………………..……………….. 3

3. Giant cells formed ………………..……………….. 4

3. Giant cells not formed ………………..……………….. 6

4. Columellae of sporangia mostly hemispherical, some applanate, or subglobose; some rhizoids arising from sporophores and surrounding sporangium entangled ………………..……………….. B. pernambucencis

4. Columellae of sporangia not hemispherical; rhizoids never surrounding the sporangium entangled ………………..……………….. 5

5. Columellae of sporangia ellipsoidal, cylindrical, rarely pyriform; chlamydospores

absent ………………..……………….. B. gigacellularis

5. Columellae of sporangia conical (majority), but ellipsoidal with a truncate base, globose to subglobose, subglobose to conical, or rarely conical or cylindrical with slight constriction at the center; chlamydospores abundant ………………..……………….. B. brasiliensis

6. Azygospores formed ………………..……………….. B. azygospora

6. Azygospores not formed ………………..……………….. 7

7. Sporangiospores elipsoidal or mostly ellipsoidal ………………..……………….. 8

7. Sporangiospores not ellipsoidal ………………..……………….. 10

8. Sporangia never extending 70 μm in diameter ………………..……………….. B. paraconstricta

8. Sporangia commonly extending 70 μm in diameter ………………..……………….. 9

9. Sporangia up to 150 (-200) μm diam.; sporangiospores 20-26 × 10-12 μm. ………………..……………….. B. recurva

9. Sporangia up to 100-125 μm in diameter; sporangiospores 11-15 × 7-9 μm ………………..……………….. B. variabilis

10. Sporangiospores with irregular polyhedral shape, with protrusions ………………..……………….. B. tuberculispora

10. Sporangiospores with neither an irregular polyhedral shape nor protrusions ………………..……………….. 11

11. Sporophores forming a terminal sporangium and few lateral pedicellate sporangiola ………………..……………….. B. lamprospora

11. Sporophores forming a terminal sporangium with no lateral pedicellate sporangiola ………………..……………….. 12

12. Columellae of sporangia with varied shapes, some arranged obliquely on sporangiophores, some with one side more swollen than the other; sporangiospores globose to sub-globose ………………..……………….. B. obliqua

12. Columellae of sporangia conical and cylindrical, sometimes constricted at the center, never arranged obliquely on the sporangiophores or with one side more swollen than the other; sporangiospores subglobose to broadly ellipsoidal, some slightly irregular ………………..……………….. B. constricta

Discussion

In this study, we describe the novel species Backusella paraconstricta URM 8637. Our ML and BI phylogenetic analyses demonstrated that this species is genetically distinct from all other species of Backusella and sister to B. constricta in the tree inferred using a concatenated ITS/LSU dataset. The new species belongs to a major clade containing also B. variabilis, B. thermophila and B. indica. Cordeiro et al. (2023)Cordeiro TRL, Walther G, Souza CAF et al. 2023. A polyphasic approach to the taxonomy of Backusella reveals two new species. Mycological Progress 22: 16 doi: 10.1007/s11557-023-01864-x
https://doi.org/10.1007/s11557-023-01864...
proposed that the maximum growth temperature represents a valuable taxonomic feature in Backusella, and that temperature plays an important role in the evolution of this genus. They identified seven Backusella spp. capable of growing at temperatures ≥36 °C, four of which (B. constricta, B. variabilis, B. thermophila and B. indica.) were placed in the same clade in the inferred phylogenies. Herein, we present a new species that grows at 36 °C and also belongs to this clade (Fig. 1). This further confirms that maximum growth temperature is a taxonomically relevant character in Backusella.

Morphologically, B. paraconstricta differs from B. constricta by forming sporangia up to 70 μm in diameter, whereas those of the latter reach 100 μm in diameter. Backusella constricta forms columellae that are conical (majority) or cylindrical, and slightly or strongly constricted in the center (Lima et al. 2016Lima DX, Voigt K, de Souza CAF, Oliveira RJV, Souza-Motta CM, Santiago ALCMA. 2016. Description of Backusella constricta sp. nov. (Mucorales, ex Zygomycota) from the Brazilian Atlantic Rainforest, including a key to species of Backusella. Phytotaxa 289: 59-68. doi: 10.11646/phytotaxa.289.1.4
https://doi.org/10.11646/phytotaxa.289.1...
). Backusella paraconstricta, instead, forms columellae that are mostly conical, subglobose, infrequently applanate, and very rarely elongate to ellipsoid, cylindrical and with a slight constriction at the base. The new species predominantly forms ellipsoidal sporangiospores, although some are subglobose, globose, and irregular, whereas B. constricta only forms sporangiospores that are subglobose to ellipsoidal and slightly irregular. Cordeiro et al. (2023)Cordeiro TRL, Walther G, Souza CAF et al. 2023. A polyphasic approach to the taxonomy of Backusella reveals two new species. Mycological Progress 22: 16 doi: 10.1007/s11557-023-01864-x
https://doi.org/10.1007/s11557-023-01864...
observed that most species of Backusella form subglobose to broadly ellipsoidal sporangiospores, and that ellipsoidal sporangiospores occur in Backusella species that are in the deeper branches of the ITS and RPB1 phylogenetic trees, namely B. indica, B. oblongielliptica, B. oblongispora, B. parvicylindrica, B. recurva, B. thermophila, and B. variabilis. This was also observed in our ITS/LSU phylogeny, which includes B. paraconstricta. Finally, B. paraconstricta can grow at temperatures up to 36 °C, whereas B. constricta can grow up to 39 °C.

In conclusion, our results demonstrate that B. paraconstricta is morphologically and genetically different from the other Backusella species described to date. Therefore, it was described as new. This study contributes to our knowledge of the distribution of mucoralen fungi.

Acknowledgements

We thank the Coordination for the Improvement of Higher Education Personnel (CAPES) for scholarships awarded to Francisca dos Santos and Thalline Cordeiro, and the National Council for Scientific and Technological Development (CNPq) for the research grant provided to André Santiago. This research was supported by the ‘Mucoromycotin Diversity in the different ecosystems of the Atlantic Forest of Pernambuco’ project (FACEPE - APQ - 0842-2.12/14). It was also supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (2022R1I1A3068645).

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Supplementary material

Supplementary material 01.

Publication Dates

  • Publication in this collection
    08 Sept 2023
  • Date of issue
    2023

History

  • Received
    16 Mar 2023
  • Accepted
    30 June 2023
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