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 1969 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 1975; Walther et al. 2013; Lima et al. 2016; Nguyen & Lee 2018; Nguyen et al. 2021; Urquhart et al. 2021). 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 1971; Wanasinghe et al. 2018). Species of Backusella are known to occur in Australia, Brazil, China, Japan, South Korea, the United States of America, and Thailand (Zheng et al. 2013; Lima et al. 2016; Nguyen et al. 2021; Urquhart et al. 2021; Hurdeal et al. 2022; de Lima et al. 2022).

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 2005; Walther et al. 2013; de Souza et al. 2014). Based on the recognition of transiently curved sporophores as a distinctive feature of Backusella, Walther et al. (2013) transferred some species from Mucor to Backusella. As of January 2023, 34 species have been accepted in Backusella (Wijayawardene et al. 2022; Hurdeal et al. 2022; de Lima et al. 2022; Cordeiro et al. 2023).

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. 2022). In this study, we updated the identification key of Backusella from the Americas provided by de Lima et al. (2022) 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 2005). 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), 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). 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).

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), 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 1987; 1990)], 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. 1990; van Tuinen et al. 1998; Santiago et al. 2014) 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 2013) for each molecular marker. The sequences were manually edited using MEGA version 7 (Kumar et al. 2016). The ITS and LSU rDNA region alignments were concatenated before the phylogenetic analyses (^{Supplementary Material 01}). Bayesian inference (BI) and maximum likelihood (ML) analyses were performed with MrBayes v.3.2.2 (Ronquist et al. 2012) on XSEDE and RAxML-HPC BlackBox v.8.2.8 (Stamatakis et al. 2008; Stamatakis 2014), respectively, using the CIPRES Science Gateway (http://www.phylo.org/) (Miller et al. 2010). 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 2004). 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 2019). 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.

Species	Strain number	GenBank accession No.		References
		ITS	LSU
Backusella australiensis	UoMAU34 T	MK959062	MK958800	Urquhart et al. (2021)
Backusella australiensis	UoMAU90	MK959064	MK958797	Urquhart et al. (2021)
Backusella azygospora	URM 8065 T	MK625216	MK625222	Crous et al. (2019)
Backusella brasiliensis	URM 8395 T	OM458082	OM458083	de Lima et al. (2022)
Backusella circina	CBS 128.70 T	JN206258	JN206529	Ellis and Hesseltine (1969)
Backusella circina	CBS 129.70	JN206257	MH871299	Walther et al. (2013)
Backusella chlamydospora	CNUFC PS1 T	MZ171385	MZ148709	Nguyen et al. (2021)
Backusella chlamydospora	CNUFC HL7	MZ171386	MZ148710	Nguyen et al. (2021)
Backusella constricta	URM 7322 RV05	KT937157	-	Lima et al. (2016)
Backusella constricta	URM 7322 RV06	KT937158	-	Lima et al. (2016)
Backusella constricta	URM 7322 RV07	KT937159	-	Lima et al. (2016)
Backusella constricta	URM 8701	OQ354764	-	This study
Backusella paraconstricta	URM 8637 T	OQ625517	OQ625516	This study
Backusella dispersa	CBS 107.09 T	JN206269	MH866118	Urquhart et al. (2021)
Backusella dispersa	CBS 195.28	JN206271	JN206530	Urquhart et al. (2021)
Backusella gigacellularis	CCIBt 3866 T	KF742415	-	de Souza et al. (2014)
Backusella gigaspora	CBS 538.80 T	HM999964	HM849692	Cordeiro et al. (2023)
Backusella ‘group X’	UoMAU121	MK959103	MK958792	Urquhart et al. (2021)
Backusella ‘group X’	UoMAU152	MK959102	MK958791	Urquhart et al. (2021)
Backusella indica	CBS 786.70	JN206255	MH871743	Walther et al. (2013)
Backusella koreana	CNUFC CM05 T	MZ171387	MZ148711	Nguyen et al. (2021)
Backusella koreana	CNUFC CM06	MZ171388	MZ148712	Nguyen et al. (2021)
Backusella lamprospora	CBS 118.08 T	NR_145291	NG_058650	Benny and Benjamin (1975)
Backusella liffmaniae	UoMAU58 T	MK959065	MK958734	Urquhart et al. (2021)
Backusella liffmaniae	UoMAU128	-	MK958735	Urquhart et al. (2021)
Backusella locustae	EML-SFB2 T	KY449291	KY449292	Wanasinghe et al. (2018)
Backusella locustae	EML-SFB4	KY449293	KY449290	Wanasinghe et al. (2018)
Backusella luteola	UoMAU6 T	MK959058	MK958795	Urquhart et al. (2021)
Backusella luteola	UoMAU36	-	MK958794	Urquhart et al. (2021)
Backusella macrospora	UoMAU7 T	MK959107	MK958628	Urquhart et al. (2021)
Backusella macrospora	UoMAU54	-	MK958629	Urquhart et al. (2021)
Backusella mclennaniae	UoMAU11	MK959077	MK958776	Urquhart et al. (2021)
Backusella mclennaniae	UoMAU12 T	MK959087	MK958777	Urquhart et al. (2021)
Backusella morwellensis	UoMAU14	-	MK958806	Urquhart et al. (2021)
Backusella morwellensis	UoMAU16 T	MK959059	MK958808	Urquhart et al. (2021)
Backusella obliqua	URM 8427T	ON858475	ON858467	de Lima et al. (2022)
Backusella oblongielliptica	CBS 568.70 LT	JN206278	JN206533	Walther et al. (2013)
Backusella oblongielliptica	CNUFC IL02	MZ171391	MZ148715	Nguyen et al. (2021)
Backusella oblongispora	CBS 569.70 T	JN206251	JN206407	Walther et al. (2013)
Backusella oblongispora	CNUFC TKB11	MZ420786	MZ148717	Nguyen et al. (2021)
Backusella parvicylindrica	UoMAU35 T	MK959109	MK958727	Urquhart et al. (2021)
Backusella parvicylindrica	UoMAU39	-	MK958728	Urquhart et al. (2021)
Backusella pernambucensis	URM 7647 T	OP339860	OP339863	Cordeiro et al. (2023)
Backusella pernambucensis	URM 7648	OP339861	OP339864	Cordeiro et al. (2023)
Backusella psycrophilia	UoMAU26	-	MK958748	Urquhart et al. (2021)
Backusella psycrophilia	UoMAU55 T	MK959093	MK958749	Urquhart et al. (2021)
Backusella recurva	CBS 196.71	JN206265	JN206523	Walther et al. (2013)
Backusella recurva	CBS 317.52	JN206262	MH868593	Walther et al. (2013)
Backusella recurva	CBS 318.52 ET	JN206261	JN206522	Walther et al. (2013)
Backusella solicola	MFLUCC 22-0067 T	ON899832	ON892503	Hurdeal et al. (2022)
Backusella tarrabulga	UoMAU5 T	MK959060	MK958804	Urquhart et al. (2021)
Backusella tarrabulga	UoMAU187	-	MK958805	Urquhart et al. (2021)
Backusella thermophila	CNUFC CS02 T	MZ171389	MZ148713	Nguyen et al. (2021)
Backusella thermophila	CNUFC CS03	MZ171390	MZ148714	Nguyen et al. (2021)
Backusella tuberculispora	CBS 562.66 LT	JN206267	JN206525	Walther et al. (2013)
Backusella tuberculispora	CBS 570.70	JN206266	MH871631	Walther et al. (2013)
Backusella variabilis	CBS 186.87T of B. grandis	JN206252	JN206527	Walther et al. (2013)
Backusella variabilis	CBS 564.66 LT	JN206254	JN206528	Walther et al. (2013)
Backusella westeae	UoMAU4 T	MK959061	MK958796	Urquhart et al. (2021)
Mucor indicus	CBS 226.29 ET	NR_077173	NG_057878	Walther et al. (2013)

• Bold letters indicate the strains obtained in this study. CBS culture collection of the Westerdijk Fungal Biodiversity Institute, The Netherlands; CNUFC Chonnam National University Fungal Collection, Gwangju, South Korea; UoMAU National Herbarium of Victoria, Australia; MFLUCC Mae Fah Luang University Culture Collection. Ex-type, ex-epitype, and ex-lectotype strains are marked with T, ET, and LT, respectively.

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) 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. 2016). 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) 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