Descriptions of Four New Species in Cunninghamellaceae (Mucoromycota) from the Brazilian Savanna Through Integrative Taxonomy

Abstract

During a survey on Mucorales fungi from soil in the world’s most biodiverse savanna, the the Brazilian Cerrado, nine specimens belonging to the Cunninghamellace were isolated. Morphological, multiloci analyses (ITS-nLSU-act) and maximum temperature growth data revealed that those specimens represent four new species: two in Absidia and two in Gongronella. Morphological characteristics of the isolates distinguishes them from other species: Absidia rhizoidea sp. nov. forms rhizopodiform rhizoids at the end of stolons, commonly next to the sporangiophores; A. variabilis sp. nov., mostly with slightly dorsiventrally flattened sporangia; Gongronella longapophysata sp. nov., which forms a long apophysis below sporangia; and G. verticilatta sp. nov., with whorled-branched sporangiophores. The maximum temperatures growth (Tmax) of those new species are as follows: A. rhizoidea (33 °C on MEA and 32 °C on PDA), A. variabilis (31 °C on MEA and 32 °C on PDA), G. longapophysata (32 °C on MEA and 33 °C on PDA), and G. verticilatta (31 °C on MEA and PDA). The present study highlights and discusses the micromorphological, physiological (Tmax) and phylogenetic characteristics of the new species.

1. Introduction

The Cerrado biome (Brazilian Savanna), located in the Central Plateau of Brazil, is the second largest biome in extension in the country (over 2,000,000 km², covering about 23% of the Brazilian territory), only surpassed by the Amazon Rainforest [1,2]. Different types of forest formations are observed in the Cerrado, such as riparian forest, gallery forest, dry forest, “cerradão”, savanna and grassland [1,3]. To date (as of 3 March 2026), 613 fungal species have been reported from the Brazilian Cerrado, which corresponds to 0.43% of circa (ca.) 140,000 known fungal species [4] and to 7.74% of the 7918 fungal species registered in Brazil [5]. Regarding the order Mucorales Dumort., only 17 species have been officially registered in this biome [5], from which Backusella gigacellularis J.I. de Souza, Pires-Zottarelli & Harakava [6]; and Isomucor trufemiae J.I. de Souza, Pires-Zottarelli & Harakava [7] were introduced as new species. As for Cunninghamellaceae Naumov ex R.K. Benj., reports of species in this family in the Cerrado are restricted to five species, namely, Absidia caerulea Bainier, A. cylindrospora Hagem, A. spinosa Lendn., Cunninghamella elegans Lendn., and C. phaeospora Boedijn [5]. Despite being one of the largest Brazilian biomes, our knowledge about fungal diversity of the Cerrado is limited, especially regarding Cunninghamellaceae. Furthermore, this biome suffers from constant deforestation, soil degradation, water depletion and fires [8], which makes its unknown mycodiversity even more threatened, making its discovery urgent.

Species in the Cunninghamellaceae form sporangiophores with a columellate sporangium or sporophores with a terminal vesicle with uni- to multispored pedicellate sporangiola. Zygospores are globose with opposed suspensors [9,10]. Naumov [11] accepted Cunninghamella Matr., Sigmoideomyces Thaxt., and Thamnocephalis Blakeslee in this family, and other genera were added to Cunninghamellaceae later, such as Chaetocladium Fresen. [12,13], Mycotypha Fenner [14,15] and Phascolomyces Boedijn [16]. However, Benny et al. [17] and Voigt and Wöstemeyer [18] considered the family as monogeneric, with only Cunninghamella. On the other hand, Voigt [10] and Wijayawardene et al. [4] accepted Absidia Tiegh., Chlamydoabsidia Hesselt. & J. J. Ellis, Cunninghamella, Gongronella Ribaldi, Halteromyces Shipton & Schipper, and Hesseltinella H.P. Upadhyay in the Cunninghamellaceae. Here we are following the classification of Wijayawardene et al. [4].

Absidia species are commonly isolated from herbivore dung, soil, stored food and plant remains [19]. They form stolons and rhizoids not opposed to the sporangium [20,21]. The unbranched or branched sporangiophores arise isolated or in whorls from the stolon. Sporangia are apophysate, globose, subglobose and pyriform (when considering the apophysis), and the varied-shaped columellae commonly present varied-shaped projections on their surface. Zygosporangia are globose to subglobose and born from opposed suspensors with appendages [22]. Some species of this genus are biotechnologically relevant, such as A. caerulea Bainier and A. glauca Hagem, which can produce chitosan and chitin deacetylase [23] and biotransform flavonoids (chrysin, apigenin, luteolin, and diosmetin) and flavanones (pinocembrin, naringenin, eriodictyol, and hesperetin) [24]. Absidia fusca Linnem. and A. cylindrospora Hagem can degrade polycyclic aromatic hydrocarbon compounds [25]. Furthermore, experiments carried out with A. cylindrospora highlighted its capacity to biosorb heavy metals such as cadmium, copper and lead [26]. The genus currently comprises 94 species [27].

Most of the Gongronella species have been reported from soil samples, with only G. namwonensis Hyang B. Lee, A.L. Santiago & H.J. Lim being isolated from fresh water [28]. Species of this genus show morphological characteristics similar to the ones of Absidia species, such as the formation of stolons and rhizoids (although discrete) not opposed to the sporangium and sporangiophores with apophysate sporangia. The columellae are commonly globose, hemispherical and subglobose, and sterile sporangia are formed in some species [9,21]. Sporangiospores are commonly reniform, but may also be lacrimoid, globose and subglobose, and zygosporangia are born from opposed suspensors, without appendages [21,29,30]. Gongronella butleri (Lendn.) Peyronel & Dal Vesco can produce pectinases [31], chitin deacetylase [32], amylases [33], beta-glucosidases [34], and chitosan [35]. This genus currently comprises 28 species [27].

During a survey of Mucorales fungi from the soils of the Chapada das Mesas, an area of Cerrado located in the state of Maranhão, Northeastern Brazil, we discovered four specimens of Gongronella and five of Absidia (Cunninghamellaceae) that differ morphologically and phylogenetically (ITS-LSU-act) from other species. This paper proposes two new species of Absidia and two of Gongronella, as well as discusses their morphology and phylogeny.

2. Materials and Methods

2.1. Sampling Site and Soil Collection

Soil samples were collected in the Chapada das Mesas region (7°17′51.2″ S 47°28′12.2″ W), which has circa (ca.) 160,000 ha and is located within the municipalities of Carolina, Estreito, and Riachão, in the middle of the Tocantins River basin. The vegetation cover of the Chapada das Mesas is typical of the Cerrado biome with formations of gallery forest; riparian forest; dry forest; herbaceous-shrub physiognomy with spaced shrubs and subshrubs, known as “campo sujo”; and herbaceous physiognomy with few shrubs and no trees, known as “campo limpo” [36,37]. The local soil types are cambisols, red-yellow latosols, quartz sands, and the climate is humid and tropical, with temperatures ranging from 26 to 36 °C [37].

Soil collection was carried out randomly in three different areas near the Parque Nacional da Chapada das Mesas: Cachoeira da Mansinha (7°07′54.0″ S 47°26′55.1″ W), Cachoeira do Dodô (7°05′33.2″ S 47°26′35.5″ W), Encanto Azul (7°14′00.6″ S 46°24′24.3″ W) and São Romão (6°02′09.1″ S 46°34′03.5″ W). The soil samples were collected at 5 cm deep, stored in plastic bags and kept in styrofoam boxes with ice during transportation to the Laboratório de Fungos Zigospóricos of the Universidade Federal de Pernambuco, UFPE.

2.2. Isolation, Purification and Deposit

Five milligrams of soil were added to Petri dishes with wheat germ agar medium [38] with chloramphenicol (80 mg L⁻¹), in triplicate. Colony growth was monitored for 96 h at room temperature (28 ± 2 °C). Fragments of the colonies were transferred to Petri dishes with potato dextrose agar (PDA; HIMEDIA, Vadhani, India) [38] with chloramphenicol (80 mg L⁻¹). The lyophilized holotypes and living cultures were deposited at the URM culture collection of the Universidade Federal de Pernambuco (Recife, Brazil).

2.3. Growth Experiments and Micromorphology

Strains were grown in triplicate on potato dextrose agar (PDA) and malt extract agar (MEA) [39], and incubated at 10, 15, 20, 25, 30, and 35 °C. Colony growth was monitored for 8 days, with colony diameters measured every 24 h. Growth results are shown as the arithmetic mean for all isolates of each species on both PDA and MEA. The maximum growth temperature (Tmax) was determined by incubating all the strains on MEA and PDA at temperatures one degree higher than the last temperature with growth. The Tmax of 50 Absidia species retrieved from literature plus four new species (two of Absidia and two of Gongronella) accessed in this work is shown in Table 1.

For observation, mycelial fragments were removed from the cultures, mounted on microscope slides with 3% KOH and/or Amman blue, and observed under a light microscope Leica DM 500 (Leica Microsystems, Wetzlar, Germany). Images were captured using the Leica DM 2500 microscope (Leica Microsystems, Wetzlar, Germany) equipped with a Leica flexacam C3 and processed using the Leica Application Suite X 3.8.1.2 software. One hundred measurements were made for each fungal structure. Measurements of the structures considered the length × width range, including outliers in parentheses. The colony color designations were determined according to Kornerup and Wanscher [40].

Table 1. Maximum growth temperatures of 52 Absidia and two Gongronella species. New species described in this study are in bold.

Species	Country	Tmax (°C)
		MEA	PDA	SMA
Absidia abundans [41]	China	31	-	-
Absidia ampullaceae [42]	China	30	-	-
Absidia arrhiza [43]	China	-	33	-
Absidia biappendiculata [42]	China	35	-	-
Absidia brunnea [42]	China	35	-	-
Absidia cheongyangensis [20]	Korea	33	33	33
Absidia chinensis [42]	China	30	-	-
Absidia cinerea [42]	China	35	-	-
Absidia collariata [44]	China	-	29	-
Absidia crystalloides [45]	China	-	32	-
Absidia digitula [42]	China	32	-	-
Absidia fluvii [20]	Korea	32	32	32
Absidia frigida [46]	China	24	-	-
Absidia gemella [46]	China	29	-	-
Absidia globospora [47]	China	28	28	-
Absidia hainanensis [44]	China	-	34	-
Absidia healeya [48]	Australia	-	30	-
Absidia jiangxiensis [42]	China	31	-	-
Absidia kunryangriensis [20]	Korea	31	32	-
Absidia lobata [41]	China	26	-	-
Absidia longissima [46]	China	36	-	-
Absidia medula [47]	China	32	32	-
Absidia menglianensis [49]	China	-	36	-
Absidia nigra [42]	China	31	-	-
Absidia oblongispora [42]	China	32	-	-
Absidia ovalispora [50]	China	-	-	37
Absidia pacifica [45]	China	-	35	-
Absidia paracylindrospora [20]	Korea	31	32	31
Absidia pararepens [20]	Korea	32	32	32
Absidia pateriformis [45]	China	-	30	-
Absidia purpurea [42]	China	30	-	-
Absidia pyriformis [44]	China	-	33	-
Absidia radiata [41]	China	32	-	-
Absidia rhizoidea	Brazil	33	31	-
Absidia sphaerica [43]	China	-	33	-
Absidia sichuanensis [41]	China	28	-	-
Absidia simplex [43]	China	-	29	-
Absidia sympodialis [42]	China	33	-	-
Absidia tarda [20]	Brazil	36	36	36
Absidia tardiva [44]	China	-	27	-
Absidia terrestris [51]	Mexico	27	-	-
Absidia thailandica [52]	Thailand	-	27	-
Absidia tibetensis [44]	China	-	30	-
Absidia turgida [47]	China	32	32	-
Absidia variabilis	Brazil	31	32	-
Absidia varians [20]	China	29	-	-
Absidia variiprojecta [20]	Brazil	32	32	33
Absidia variispora [20]	Brazil	32	32	32
Absidia virescens [42]	China	33	-	-
Absidia viridis [43]	China	-	29	-
Absidia yunnanensis [41]	China	32	-	-
Absidia zonata [47]	China	37	37	-
Gongronella longapophysata	Brazil	32	33	-
G. verticilatta	Brazil	31	31	-

Legend: PDA—potato dextrose agar; MEA—malt extract agar; SMA—synthetic Mucor agar.

Table 1. Maximum growth temperatures of 52 Absidia and two Gongronella species. New species described in this study are in bold.

Species	Country	Tmax (°C)
		MEA	PDA	SMA
Absidia abundans [41]	China	31	-	-
Absidia ampullaceae [42]	China	30	-	-
Absidia arrhiza [43]	China	-	33	-
Absidia biappendiculata [42]	China	35	-	-
Absidia brunnea [42]	China	35	-	-
Absidia cheongyangensis [20]	Korea	33	33	33
Absidia chinensis [42]	China	30	-	-
Absidia cinerea [42]	China	35	-	-
Absidia collariata [44]	China	-	29	-
Absidia crystalloides [45]	China	-	32	-
Absidia digitula [42]	China	32	-	-
Absidia fluvii [20]	Korea	32	32	32
Absidia frigida [46]	China	24	-	-
Absidia gemella [46]	China	29	-	-
Absidia globospora [47]	China	28	28	-
Absidia hainanensis [44]	China	-	34	-
Absidia healeya [48]	Australia	-	30	-
Absidia jiangxiensis [42]	China	31	-	-
Absidia kunryangriensis [20]	Korea	31	32	-
Absidia lobata [41]	China	26	-	-
Absidia longissima [46]	China	36	-	-
Absidia medula [47]	China	32	32	-
Absidia menglianensis [49]	China	-	36	-
Absidia nigra [42]	China	31	-	-
Absidia oblongispora [42]	China	32	-	-
Absidia ovalispora [50]	China	-	-	37
Absidia pacifica [45]	China	-	35	-
Absidia paracylindrospora [20]	Korea	31	32	31
Absidia pararepens [20]	Korea	32	32	32
Absidia pateriformis [45]	China	-	30	-
Absidia purpurea [42]	China	30	-	-
Absidia pyriformis [44]	China	-	33	-
Absidia radiata [41]	China	32	-	-
Absidia rhizoidea	Brazil	33	31	-
Absidia sphaerica [43]	China	-	33	-
Absidia sichuanensis [41]	China	28	-	-
Absidia simplex [43]	China	-	29	-
Absidia sympodialis [42]	China	33	-	-
Absidia tarda [20]	Brazil	36	36	36
Absidia tardiva [44]	China	-	27	-
Absidia terrestris [51]	Mexico	27	-	-
Absidia thailandica [52]	Thailand	-	27	-
Absidia tibetensis [44]	China	-	30	-
Absidia turgida [47]	China	32	32	-
Absidia variabilis	Brazil	31	32	-
Absidia varians [20]	China	29	-	-
Absidia variiprojecta [20]	Brazil	32	32	33
Absidia variispora [20]	Brazil	32	32	32
Absidia virescens [42]	China	33	-	-
Absidia viridis [43]	China	-	29	-
Absidia yunnanensis [41]	China	32	-	-
Absidia zonata [47]	China	37	37	-
Gongronella longapophysata	Brazil	32	33	-
G. verticilatta	Brazil	31	31	-

Legend: PDA—potato dextrose agar; MEA—malt extract agar; SMA—synthetic Mucor agar.

2.4. DNA Extraction, PCR Amplification and Sequencing

Genomic DNA extraction was carried out with the Wizard Genomic DNA Purification Kit (Promega), following the manufacturer’s guidelines. PCR amplifications for the internal transcribed spacer (ITS), part of the nuclear ribosomal large subunit (nLSU), and actin (act) were performed using primers and PCR conditions, as listed in

Table 2. Molecular markers used in this study with respective primer pairs and PCR protocols.

Markers	PCR Primers	PCR Conditions *	References
ITS	ITS4/ITS5	95 °C, 5 min; (95 °C, 30 s; 55 °C, 45 s; 72 °C, 1:15 min) × 35 cycles; 72 °C, 10 min	White et al. [54]
LSU	LR5/LR0R	95 °C, 5 min; (95 °C, 30 s; 55 °C, 45 s; 72 °C, 1:15 min) × 35 cycles; 72 °C, 10 min	Vilgalys and Hester [55]; Rehner and Samuels [56]
act	Act-1/Act-4R	95 °C, 3 min; (95 °C, 60 s; 55 °C, 60 s; 72 °C, 60 s) × 30 cycles; 72 °C, 10 min	Voigt and Wöstemeyer [18]

* Annealing temperatures are in bold.

. The amplification protocol was performed as described by de Freitas et al. [21,53]. Subsequently, bidirectional sequencing was performed with the same primers using the BigDye^® Terminator v.3.1 Cycle Sequencing Kit (Applied Biosystems Life Technologies, Carlsbad, CA, USA) at the Laboratório de Ecologia e Sistemática de Fungos—LESF, UNESP, Rio Claro, São Paulo and at the Centro Multiusuário de Pesquisa de Bioinsumos e Tecnologias em Saúde (CMBiotecs, IPTSP, UFG), Goiânia, Goiás. The sequences obtained in this study are available in the NCBI GenBank database (

Table 3. GenBank accession numbers of sequences used in this study. Sequences in boldface were obtained during this study. Ex-type strains are marked with (T).

Species	Strains	GenBank Accession Numbers
		ITS	LSU	act
Absidia abundans	XY09265	ON074697	ON074681	–
Absidia abundans	CGMCC 3.16255T	NR_182590	ON074683	–
Absidia abundans	XY09274	ON074696	ON074682	–
Absidia aguabelensis	URM 8213T	NR_189383	NG_241934	–
Absidia alpina	CGMCC 3.16104	OL678133	–	–
Absidia ampullacea	CGMCC 3.16054	MZ354138	MZ350132	–
Absidia anomala	CBS 125.68T	MH859085	MH870799	–
Absidia anomala	FSU5798	EF030523	–	EF030535
Absidia arrhiza	CGMCC 3.28540T = XG09770-7	PQ600860	PQ600254	PQ753533
Absidia arrhiza	XG09770-6	PQ600859	PQ600253	PQ753532
Absidia biappendiculata	CBS 187.64	MZ354153	MZ350147	MZ357438
Absidia bonitoensis	URM 7889T	MN977786	MN977805	–
Absidia brunnea	CGMCC 3.16055T	MZ354139	MZ350133	MZ357421
Absidia brunneola	CGMCC 3.29490T = XG12984-12-1	NMDCN0009VE 6	NMDCN000 9VEM	NMDCN0009 TVO
	XG12984-12-2	NMDCN0009VE 7	NMDCN000 9VEN	NMDCN0009 TVP
Absidia caatinguensis	URM 7156T	NR_154704	NG_058582	–
Absidia caerulea	XY00608	OL620081	–	–
Absidia caerulea	XY00729	OL620082	–	–
Absidia caerulea	CBS101.36	MH855718	MH867230	–
Absidia caerulea	FSU767	AY944870	–	–
Absidia californica	CBS 314.78	JN205816	MH872902	–
Absidia californica	FSU4748	AY944873	EU736301	EU736224
Absidia californica	CBS 126.68 = FSU4747T	AY944872	EU736300	AY944758
Absidia cheongyangensis	CNUFC CY2203T	PP844904	PP852788	PP893196
Absidia cheongyangensis	CNUFC CY2401	–	PP852702	PP893197
Absidia chinensis	CGMCC 3.16057	MZ354141	MZ350135	MZ357422
Absidia chinensis	CGMCC 3.16056T	MZ354140	MZ350134	–
Absidia cinerea	CGMCC 3.16062	MZ354146	MZ350140	MZ357427
Absidia collariata	CGMCC 3.28536T = XG08666-10-1	PQ610533	PQ605104	PQ613279
Absidia collariata	XG08666-10-2	PQ610534	PQ605105	PQ613280
Absidia cornuta	URM 6100T	NR_172976	MN625255	–
Absidia crystalloides	CGMCC3.27496T = SAUCC6948-15	PP377803	PP373736	PP790582
Absidia crystalloides	SAUCC693201	PP377804	PP373736	PP790581
Absidia cuneospora	CBS 102.59	JN205819	–	–
Absidia cylindrospora	CBS 100.08	JN205822	JN206588	–
Absidia digitata	CGMCC 3.29492T = XG18784-2-1	NMDCN0009VE 2	NMDCN000 9VEI	NMDCN0009 TVK
	XG18784-2-2	NMDCN0009VE 3	NMDCN000 9VEJ	NMDCN0009 TVL
Absidia digitula	CGMCC 3.16058T	MZ354142	MZ350136	MZ357423
Absidia edaphica	MFLUCC 20-0088	NR_172305	NG_075367	MT410739
Absidia exilis	CGMCC 3.29493T = XG21013-11-1	NMDCN0009VE 0	NMDCN000 9VEG	NMDCN0009 TVI
	XG21013-11-2	NMDCN0009VE 1	NMDCN000 9VEH	NMDCN0009 TVJ
Absidia fluvi	CNUFC CY2240T	PP844891	PP852703	PP893201
Absidia fluvi	CNUFC CY2241	PP844892	PP852704	PP893202
Absidia fluvi	CNUFC CY2315	PP844893	PP852705	PP893203
Absidia frigida	CGMCC 3.16201T	NR_182565	OM030223	–
Absidia fusca	CBS 102.35T	NR_103625	NG_058552	–
Absidia gemella	CGMCC 3.16202T	OM108488	OM030224	–
Absidia glauca	CBS 129233	MH865253	MH876693	–
Absidia glauca	CBS 101.08T	MH854573	MH866105	–
Absidia glauca	FSU660	AY944879	EU736302	EU736225
Absidia globospora	CGMCC 3.16031T	NR_189829	MW671544	MZ357431
Absidia globospora	CGMCC 3.16035	MW671538	MW671545	MZ357432
Absidia globospora	CGMCC 3.16036	MW671539	MW671546	MZ357433
Absidia healeyae	UoMAU1	MT436028	MT436027	MW861731
Absidia heterospora	SHTH021	JN942683	JN982936	NA
Absidia hainanensis	CGMCC 3.28535T = XG06908-1	PQ610537	PQ605108	PQ613283
Absidia hainanensis	XG06908-4	PQ610538	PQ605109	PQ613284
Absidia jiangxiensis	CGMCC 3.16105T = Ab-216	OL678134	PP780377	PP790577
Absidia jindoensis	CNUFC-PTI1-1	MF926622	MF926616	MF926510
Absidia koreana	EML-IFS45-1T	KR030062	KR030056	KR030058
Absidia koreana	XY00816	OL620083	ON123771	–
Absidia koreana	XY00596	OL620084	–	–
Absidia kunryangriensis	CNUFC CY2230	PP844905	PP956882	PP893198
Absidia lobata	CGMCC 3.16256	ON074690	ON074679	–
Absidia longissima	CGMCC 3.16203T	NR_182566	OM030225	–
Absidia macrospora	FSU4746	AY944882	EU736303	AY944760
Absidia medulla	CGMCC 3.16034	NR_189832	MW671549	MZ357436
Absidia menglianensis	KUNCC 24-18541 = root2-17	PQ594927	PQ594929	–
Absidia menglianensis	KUNCC 24-18542 = MLAS011	PQ594928	PQ594930	–
Absidia montepascoalis	URM 8218T	NR_172995	–	–
Absidia multispora	URM 8210T	MN953780	MN953782	–
Absidia nigra	CBS 127.68T	NR_173068	MZ350146	MZ357437
Absidia nigra	CGMCC 3.16059	MZ354143	MZ350137	MZ357424
Absidia nigra	CGMCC 3.16060	MZ354144	MZ350138	MZ357425
Absidia oblongispora	CGMCC 3.16061T	MZ354145	MZ350139	MZ357426
Absidia ovalispora	CGMCC 3.16019T	NR_176748	MW264131	–
Absidia pacifica	SAUCC6955-16T = CGMCC 3.27497	PP377802	PP373735	PP790579
Absidia pacifica	SAUCC6955-15 = SAUCC 413601	PP377801	PP373734	PP790580
Absidia panacisoli	SYPF 7183T	MF522181	MF522180	–
Absidia paracylindrospora	CNUFC L2207	PP844907	PP956883	PP893204
Absidia pararepens	XY00631	OL620085	ON123774	–
Absidia pararepens	XY00615	OL620086	–	–
Absidia pararepens	XY05899	OL620087	–	–
Absidia pararepens	CBS 146,002 = CCF 6352T	MT193669	MT192308	–
Absidia pateriformis	CGMCC 3.27495 = SAUCC6347D-1T	PP377805	PP373738	PP790583
Absidia pateriformis	SAUCC6347D-2 = SAUCC 634702	PP377806	PP373739	PP790584
Absidia pernambucoensis	URM 7219T	MN635568	MN635569	–
Absidia pseudocylindrospora	EML-FSDY6-2	KU923817	KU923814	KU923815
Absidia psychrophilia	FSU 4745	AY944874	EU736306	AY944762
Absidia purpurea	CGMCC 3.16106	OL678135	–	–
Absidia pyriformis	CGMCC 3.28538T = XG09540-14-1	PQ610531	PQ605102	PQ613277
Absidia pyriformis	XG09540-14-5	PQ610532	PQ605103	PQ613278
Absidia radiata	CGMCC 3.16257	ON074698	ON074684	–
Absidia radiata	XY09330-1	ON074699	ON074685	–
Absidia repens	CBS 115583T	NR_103624	NG_058551	–
Absidia rhizoidea	URM 9234T	PZ234078	PZ227107	PZ227661
Absidia rhizoidea	URM 9233	PZ234079	PZ227108	PZ227662
Absidia rhizoidea	URM 9235	PZ234080	PZ227109	PZ227663
Absidia saloaensis	URM 8209T	MN953781	MN953783	–
Absidia sichuanensis	CGMCC 3.16258T	NR_182589	ON074688	–
Absidia simplex	CGMCC 3.28541T = XG10012-9	PQ600862	PQ600256	PQ686230
Absidia simplex	XG10012-8	PQ600861	PQ600255	PQ686229
Absidia soli	MFLU-20-0414T	MT396373	MT393988	–
Absidia sphaerica	CGMCC 3.28542T = XY00690	PQ600866	PQ600260	PQ777149
Absidia sphaerica	XY00690-1	PQ600865	PQ600259	PQ777148
Absidia spinosa	FSU551	AY944887	EU736307	EU736227
Absidia stercoraria	EML-DG8-1T	KU168828	KT921998	KT922000
Absidia sympodialis	CGMCC 3.16063T	MZ354147	MZ350141	–
Absidia sympodialis	CGMCC 3.16064	MZ354148	MZ350142	–
Absidia tarda	URM 8412	PP844911	PP956884	PP893199
Absidia tardiva	CGMCC 3.28537T = XG08757-4	PQ610529	PQ605100	PQ613275
Absidia tardiva	XG08757-6	PQ610530	PQ605101	PQ613276
Absidia terrestris	ERP-2017T	LT795003	LT795005	–
Absidia tibetensis	CGMCC 3.28534T = XG00415-1	PQ610535	PQ605106	PQ613281
Absidia tibetensis	XG00415-3	PQ610536	PQ605107	PQ613282
Absidia thailandica	MFLUCC:20-0089 = MFLUCC:23-0073T	OR606547	OR606546	NA
Absidia tumida	CGMCC 3.29491T = XG18709-9-1	NMDCN0009VE 4	NMDCN000 9VEK	NMDCN0009 TVM
Absidia tumida	XG18709-9-2	NMDCN0009VE 5	NMDCN000 9VEL	NMDCN0009 TVN
Absidia turgida	CGMCC 3.16032T	NR_189830	NG_241931	MZ357434
Absidia varians	CGMCC 3.16065T	MZ354149	MZ350143	MZ357428
Absidia edaphica	MFLUCC 20-0088T	NR_172305	NG_075367	MT410739
Absidia variabilis	URM 9236T	PZ234081	PZ227110	PZ227664
Absidia variabilis	URM 9237	PZ234082	PZ227111	PZ227665
Absidia variiprojecta	URM 8620T	PP844913	PP956885	PP893200
Absidia variispora	URM 8720	PP844915	PP956886	PP893205
Absidia variicolumellata	URM 8216T = DXL-2021b	MZ331545	MZ331547	–
Absidia virescens	CGMCC 3.16067	MZ354151	MZ350145	MZ357430
Absidia viridis	CGMCC 3.28539T = XG09563-3	PQ600864	PQ600258	PQ753531
Absidia viridis	XG09563-2	PQ600863	PQ600257	PQ753530
Absidia xinjiangensis	CGMCC 3.16107T	OL678136	–	–
Absidia yunnanensis	XY09528	ON074701	ON074686	–
Absidia yunnanensis	CGMCC 3.16259T	NR_182591	NG_149054	–
Absidia zonata	CGMCC 3.16033T	NR_189831	MW671548	MZ357435
Absidia zygospora	RSPG 214	KC478527	–	–
Absidia zygospora	ANG28	DQ914420	–	–
Gongronella abortosporangia	CGMCC 3.27028T	PP195847	PP195948	PP933938
Gongronella abortosporangia	CGMCC 3.27028T	PP195848	PP195949	PP933939
Gongronella apophysata	SAUCC 4846-3	PP195854	PP195955	PP933948
Gongronella apophysata	CGMCC 3.27031T	PP195853	PP195954	PP933947
Gongronella banzhaoae	BRIP 75171aT	OR271908	OR259049	–
Gongronella bartikiae	BRIP 76703a	PQ882522	–	–
Gongronella bawanglingensis	CGMCC 3.27033T	PP195857	PP195958	PP933951
Gongronella bawanglingensis	SAUCC 6946-1	PP195858	PP195959	PP933952
Gongronella brasiliensis	URM 7487T	NR_155148	KY114932	–
Gongronella brasiliensis	URM 7488	KY114931	KY114933	–
Gongronella butleri	CBS 216.58	JN206285	MH869292	–
Gongronella butleri	CBS 179.28	JN206286	–	–
Gongronella butleri	CBS 415.67	MH859014	MH870714	–
Gongronella chlamydospora	CGMCC 3.16118T	OL678157	–	–
Gongronella eborensis	CCMI 1100T	KT809408	MN947301	–
Gongronella eborensis	CCMI 1101	GU244500	MN947302	–
Gongronella fusoacuminata	BCRC 10F0908T	NR_199105	NG_244365	–
Gongronella fusoacuminata	BCRC:10F0918	PQ496504	PQ496507	–
Gongronella fusoacuminata	BCRC:10F0909	PQ496503	PQ496506	–
Gongronella guangdongensis	CGMCC 3.15212T	NR_158464	MN947303	–
Gongronella guangdongensis	LC1994	KC462740	MN947304	–
Gongronella hydei	KUMCC 18.0198T	NR_171964	MT907273	–
Gongronella hydei	BRIP 74937a	OR272184	OR272185	–
Gongronella hydei	KUMCC_18_0204	MT152335	MT907274	–
Gongronella inconstans	CGMCC 3.27029T	PP195849	PP195950	PP933941
Gongronella inconstans	SAUCC 4113-3	PP195850	PP195951	PP933942
Gongronella irregularis	URM9013T	PP923717	PP923718	–
Gongronella koreana	EML-TS2BpT	KP636529	KP636530	KP636527
Gongronella koreana	EML-TS2Bp-2	KP835545	KP835542	KP835543
Gongronella lacrispora	ATCC 24412T	GU244498	JN206609	–
Gongronella longapophysata	URM 9240T	PZ234083	PZ227112	PZ227666
Gongronella longapophysata	URM 9241	PZ234084	PZ227113	PZ227667
Gongronella multiramosa	CGMCC 3.26216T	OR733546	OR733611	PP933937
Gongronella multiramosa	SAUCC 4056-4	OR733545	OR733610	–
Gongronella multispora	CGMCC 3.16119T	OL678158	–	–
Gongronella namwonensis	CNUFC WW2-12T	NR_175640	MN658482	–
Gongronella namwonensis	XY08131	OL620098	–	–
Gongronella oleae	CGMCC 3.26217T	OR742078	OR733608	PP933945
Gongronella oleae	SAUCC 4164-2	OR742079	OR733609	PP933946
Gongronella orasabula	JMRC SF 012180T	NR_148087	KT936263	KT936265
Gongronella orasabula	EML-QF12-2	KT936270	KT936264	–
Gongronella pamphilae	BRIP 74936aT	OR271909	OR259050	–
Gongronella pamphilae	CGMCC 3.27027	PP195845	PP195946	PP933935
Gongronella pamphilae	SAUCC 4031-2	PP195846	PP195947	PP933936
Gongronella pedratalhadensis	URM 8182T	MN912512	MN912508	–
Gongronella pingtangensis	CGMCC 3.27032T	PP195855	PP195956	PP933949
Gongronella pingtangensis	SAUCC 5676-4	PP195856	PP195957	PP933950
Gongronella qichaensis	CGMCC 3.26218T	OR733544	OR733607	–
Gongronella qichaensis	SAUCC 4137-3	OR733543	OR733606	–
Gongronella reniformis	CGMCC 3.27030T	PP195851	PP195952	PP933943
Gongronella reniformis	SAUCC 4142-5	PP195852	PP195953	PP933944
Gongronella sichuanensis	GZUIFR-H25.4.1T	MK813373	MK813855	MK820625
Gongronella sichuanensis	GZUIFR-H25.4.2	MK813374	MK813856	MK820626
Gongronella verticillata	URM 9238T	PZ234085	PZ227114	–
Gongronella verticillata	URM 9239	PZ234086	PZ227115	–
Gongronella zunyiensis	GZUIFR-FX25.1T	MN453856	MN453853	–
Gongronella zunyiensis	GZUIFR-FX25.2	MN453857	MN453854	–
Cunninghamella blakesleeana	CBS 133.27 T	NR_119974	MH866397	KJ156479
Cunninghamella antarctica	CBS 545.75T	JN205893	JN206597	KJ156492

).

2.5. Phylogenetic Analysis

Phylogenetic analyses were performed using sequences generated in this study and reference sequences obtained from the GenBank database, based on markermultiloci concatenated analysis (ITS, LSU, and act). The sequence datasets used in this study were constructed based on studies on Absidia [20,49] and Gongronella [21,30]. The sequences were aligned using the MAFFT v7 online interface [56,57] and manually edited using MEGA v7 software [58]. The loci were concatenated using Mesquite v3.61 software [59].

The data were first analyzed based on Maximum Likelihood (ML) analysis using IQ-TREE v1.6.12 software [60] and RAxML HPC BlackBox v8.2.12 [61]. The RAxML-HPC BlackBox ML analysis was performed using the default system options in the CIPRES Science Gateway [62] with 5000 bootstrap replicates. The IQ-TREE ML analysis involved 5000 replications, and the ultrafast bootstrap (UFboot2) method was used to calculate the branch support [63]. The ModelFinder software (http://iqtree.cibiv.univie.ac.at, accessed on 3 March 2016) included in IQ-TREE v. 1.6.12 [64] was used to determine the partitioning strategy and models based on the Akaike information criterion. The combined datasets were also analyzed based on Bayesian inference (BI) conducted using MrBayes v3.2.7a [65] in the CIPRES Science Gateway, using the same models and partitions as in the ML analysis (IQ-TREE). The BI analysis was conducted with 5 × 10⁷ generations and a burning value of 25%, with chains sampled every 1000 generations. The resulting phylogenetic trees were visualized using FigTree v1.4.4 [66]. Values ≥ 0.95 BI posterior probability (BPP) and bootstrap support (BS) from IQ-TREE UFboot2-BS and RAxML-BS analyses (both BS ≥ 70%) were plotted near the nodes. The final combined alignment was deposited in Figshare (Study ID 10.6084/m9.figshare.31438165).

3. Results

3.1. Phylogenetic Analyses

3.1.1. Absidia

The combined matrix of three markers of Absidia species (ITS, LSU, and act) comprised sequences from 135 strains, including the outgroup Cunninghamella blakesleeana (CBS 133.27) and Cunninghamella antarctica (CBS 545.75), with a total of 2940 characters, including gaps, with 1267 characters for ITS, 823 for LSU, and 850 for act. The models used for IQ-TREE ML and BI were HKY+I+G for ITS, GTR+I+G for LSU and GTR+G for act. For the ML analysis (RAxML-HPC BlackBox) the model GTR+I+G was used and this matrix had 1918 distinct alignment patterns with 49.30% undetermined characters or gaps, and the final ML optimization likelihood value of the best tree was −43,306.257377. The estimated base frequencies were as follows: A = 0.254330, C = 0.209623, G = 0.232160, and T = 0.303887; substitution rates: AC = 1.172145, AG = 2.820942, AT = 1.449869, CG = 0.733730, CT = 4.396602, and GT = 1.000000; gamma distribution shape parameter: α = 0.637676. Based on our multiloci phylogenetic analysis (Figure 1), our isolates were placed as independent lineages with high support values between other species, with one for the isolates URM 9233, URM 9234, and URM 9235 (IQ-TREE-BS = 100%, RAxML-BS = 100%, and BPP = 1) and the other for URM 9236 and URM 9237 (IQ-TREEBS = 100%, RAxML-BS = 100%, and BPP = 1), therefore justifying their statuses as two new species.

3.1.2. Gongronella

The combined matrix of three markers of Gongronella species (ITS, LSU, and act) comprised sequences from 58 strains, including the outgroup Absidia digitula (CGMCC 3.16058) and Absidia turgida (CGMCC 3.16032), with a total of 2426 characters, including gaps, with 760 characters for ITS, 928 for LSU and 738 for act. The model used for IQ-TREE ML and BI was GTR+G for both genes. For the ML analysis (RAxML-HPC BlackBox), the model GTR+I+G was used; this matrix had 904 distinct alignment patterns with 35.71% undetermined characters or gaps, and the final ML optimization likelihood value of the best tree was -11879.360036. The estimated base frequencies were as follows: A = 0.250425, C = 0.216708, G = 0.245264, and T = 0.287603; substitution rates: AC = 0.872173, AG = 2.309143, AT = 1.587038, CG = 0.516161, CT = 4.286676, and GT = 1.000000; gamma distribution shape parameter: α = 0.508375. Based on our multiloci phylogenetic analysis (Figure 2), our isolates were placed as independent lineages with high support values between other species, with one for the isolates URM 9240, and URM 9241 (IQ-TREE-BS = 100%, RAxML-BS = 100%, and BPP = 1) and the other for URM 9238 and URM 9239 (IQ-TREEBS = 99%, RAxML-BS = 100%, and BPP = 1), therefore justifying their status as two new species.

3.2. Taxonomy

3.2.1. Absidia rhizoidea L.W.S. de Freitas & A.L. Santiago, sp. nov. (Figure 3 and Figure 4)

Mycobank number: MB863003.

Etymology: The epitet rhizoidea (Lat.), referring to the rhizopodiform rhizoids formed at the end of solons, commonly next to the sporangiophores.

Diagnosis: The rhizoids are strongly branched, rhizopodiform, commonly formed next to the sporangiophores. Chlamydospores abundant.

Description: Colonies floccose on PDA, initially white (A1), turning brown (6E8), 8.5 cm in diameter after 8 days of incubation at 25 °C; reverse caramel brown (6C6), irregular. Rhizoids caramel brown, commonly formed at the end of stolons and very close to the sporangiophores, rhizopodiform, strongly branched, 2.5–4.5 µm in diameter, smooth-walled. Sporangiophores brown, arising from stolons, isolated or in whorls of up to seven, unbranched, simple or rarely sympodially branched up to three times, (7–)20–350(–575) × (2.5–)5–12(–20) µm, smooth-walled, occasionally with one swelling after 7 days of incubation. One septum is observed near the apophysis. Sporangia light brown, subglobose, 12–40 × 10–40 µm, smooth and deliquescent-walled. Apophysis brownish, short or long, cup or bell-shaped, 5–30 × 6–30 µm. Columellae light brown, hemispherical or subglobose, 2.5–17 × 7–22 µm, occasionally with up to two needle-shaped or filiform with bulbous tip projection, 3–12 × 2–4 µm. Sporangiospores hyaline, cylindrical, some with a slight constriction in the center, ellipsoid and subglobose, 3–8 × 2–6 µm, smooth-walled. Chlamydospores abundant, hyaline, globose and subglobose, many attached to rhizoids or formed in aerial hyphae, 2.5–7 µm in diameter. Zygosporangia not observed.

Growth experiments (8 days): On PDA: At 5 °C—no growth; at 10 °C—1.5 cm diameter; at 15 °C—5.5 cm diameter; at 20 °C—8 cm diameter; at 25 °C—8.5 cm diameter; at 30 °C—8.3 cm diameter; at 35 °C—no growth. On MEA: At 5 °C—no growth; at 10 °C—0.7 cm diameter; at 15 °C—5.3 cm diameter; at 20 °C—8.8 cm diameter; at 25 °C—9 cm diameter; at 30 °C—8.5 cm diameter; at 35 °C—no growth. The Tmax is 33 °C on MEA and 32 °C on PDA.

Habitat and Distribution: Soil from Maranhão state (Brazil).

Specimen examined: BRAZIL, Maranhão, Carolina city, Chapada das Mesas, Cachoeira do Dodô (7°05′33.2″ S 47°26′35.5″ W), soil, 15 May 2023, L.W.S. de Freitas (Holotype URM 9234H metabolically inactive state, Ex-type living culture URM 9234).

GenBank accession numbers: ITS = PZ234078, LSU = PZ227107, act = PZ227661.

Additional specimens examined: BRAZIL, Maranhão, Carolina, Chapada das Mesas, Cachoeira do Dodô (7°05′33.2″ S 47°26′35.5″ W), soil, 15 May 2023, L.W.S. de Freitas (URM 9233); Encanto azul (7°14′00.6″ S 46°24′24.3″ W), soil, 15 May 2023, L.W.S. de Freitas (URM 9235).

GenBank numbers: URM 9233, ITS = PZ234079, LSU = PZ227108, act = PZ227662; URM 9235, ITS = PZ234080, LSU = PZ227109, act = PZ227663.

Notes: In our phylogenetic analysis, Absidia rhizoidea is a sister species of A. variiprojecta (Figure 1). Morphologically, A. rhizoidea forms constant rhizoids close to the sporangiophores, in addition to sporangiophores arising in whorls of up to seven from stolon, while A. variiprojecta has rhizoids not commonly formed close the sporangiophores and forms sporangiophores arising in whorls of up to five from stolon. Furthermore, columellae of the new species are hemispherical or subglobose, occasionally with up to two needle-shaped or filiform with bulbous tip projection, differing from A. rhizoidea that forms columellae subglobose or subglobose to fig-shaped, with one projection generally needle- or triangle-shaped, some bulbous, very rarely feather-shaped. The Tmax of A. variiprojecta is 32 °C, both on PDA and MEA [20], while the Tmax values of the new species are 33 and 32° C on MEA and PDA, respectively.

Figure 3. Absidia rizhoidea (URM 9234, ex-type) on PDA at 25 °C after eight days. (A) Colony verse (left) and reverse (right). (B) Sporangiophore with sporangium. (C) Sporangiophore with a swallow and sporangium. (D) Sporangiophore short branched with sporangium and projection. (E) Two sporangiophores arising from stolon with rhizoid. (F,G) Sporangiophore with apophysis, columella and projection. (H) Sporangiospores. (I) Stolons with sporangiophores and rhizoids (arrows). Scale bars: (B,C) 20 μm; (D,E,I) 15 μm; (F,G) 10 μm; (H) 10 μm.

Figure 3. Absidia rizhoidea (URM 9234, ex-type) on PDA at 25 °C after eight days. (A) Colony verse (left) and reverse (right). (B) Sporangiophore with sporangium. (C) Sporangiophore with a swallow and sporangium. (D) Sporangiophore short branched with sporangium and projection. (E) Two sporangiophores arising from stolon with rhizoid. (F,G) Sporangiophore with apophysis, columella and projection. (H) Sporangiospores. (I) Stolons with sporangiophores and rhizoids (arrows). Scale bars: (B,C) 20 μm; (D,E,I) 15 μm; (F,G) 10 μm; (H) 10 μm.

Figure 4. Absidia variabilis (URM 9236, ex-type) on PDA at 25 °C after eight days. (A) Branched sporangiophore. (B,C) Sporangiophore with a septum below the apophysis, columella and projection. Absidia rizhoidea on PDA at 25 °C after eight days. (D) Sporangiophore with swelling. (E) Sporangiophores in a whorl of four arising from stolon. (F) Sporangiophore with sporangium. (G) Sporangiophore with a long apophysis, columella and projection. (H) Chlamydospores. Scale bars: (A) 5 μm; (B,C) 8 μm; (D,E) 20 μm; (F–H) 10 μm.

Figure 4. Absidia variabilis (URM 9236, ex-type) on PDA at 25 °C after eight days. (A) Branched sporangiophore. (B,C) Sporangiophore with a septum below the apophysis, columella and projection. Absidia rizhoidea on PDA at 25 °C after eight days. (D) Sporangiophore with swelling. (E) Sporangiophores in a whorl of four arising from stolon. (F) Sporangiophore with sporangium. (G) Sporangiophore with a long apophysis, columella and projection. (H) Chlamydospores. Scale bars: (A) 5 μm; (B,C) 8 μm; (D,E) 20 μm; (F–H) 10 μm.

3.2.2. Absidia variabilis L.W.S. de Freitas & A.L. Santiago, sp. nov. (Figure 4 and Figure 5)

Mycobank number: MB863004.

Etymology: The epitet variabilis (lat.) refers to the formation of sporangiophores variable in length.

Diagnosis: Sporangiophores varied in length, arising singular or in whorls of up to five from stolons, commonly unbranched or monopodially branched, never sympodially branched; chlamydospores are abundant.

Description: Colonies floccose on PDA, initially white (A1), becoming light brown (6D8), 8.5 cm in diameter after 8 days of incubation, at 25 °C; reverse light brown (6D8), wave zonate. Rhizoids brown, poorly branched, often with adherent chlamydospores, 2.5–5.5 µm in diameter, with encrusted wall. Sporangiophores erect, light brown, arising from stolons, singular or in whorls of up to five, monopodially, but never sympodially branched, varied in length (15–)60–190(–230) × (2–)3–4(–6) µm, smooth-walled, with no swellings. One septum is observed near the apophysis. Sporangia apophysate, light brown, globose to slightly dorsiventrally flattened, pyriform (when considering the apophysis), (7–)10–20(–30) × (10–)15–30 µm, smooth, with deliquescent wall. Apophysis brownish, short, cup-shaped, (2–)4.5–7(–12) × (3.5–)7.5–15 µm, smooth-walled. Columellae light brown slightly flattened and subglobose (2–)5–17 × (5–)9–22 µm, smooth-walled, collar visible. One filiform, spine-like or occasionally triangular projection, up to 1–4 × 0.5–2.5 µm, can be observed on the columellae surface. Sporangiospores hyaline, mostly cylindrical, constricted in the center, some short cylindrical, 2.5–5.5 × 1.5–2.5 µm, smooth-walled. Chlamydospores abundant, hyaline, globose, subglobose, frequently attached to rhizoids or in aerial hyphae, 2–6 µm in diameter. Zygosporangia not observed.

Growth experiments (8 days): On PDA: At 5 °C—no growth; at 10 °C—1.1 cm diameter; at 15 °C—4.6 cm diameter; at 20 °C—8.6 cm diameter; at 25 °C—9 cm diameter; at 30 °C—8.5 cm diameter; at 35 °C—no growth. On MEA: At 5 °C—no growth; at 10 °C—0.7 cm diameter; at 15 °C—5.1 cm diameter; at 20 °C—8.5 cm diameter; at 25 °C—8.8 cm diameter; at 30 °C—8.6 cm diameter; at 35 °C—no growth. The Tmax is 31 °C on MEA and 32 °C on PDA.

Habitat and Distribution: Soil from Maranhão state (Brazil).

Typification: BRAZIL, Maranhão, Carolina, Chapada das Mesas, São Romão (6°02′09.1′′ S 46°34′03.5′′ W), soil, 15 May 2023, L.W.S de Freitas (Holotype URM 9236H metabolically inactive state, Ex-type living culture URM 9236).

GenBank accession numbers: ITS = PZ234081, LSU = PZ227110, act = PZ227664.

Additional specimen examined: BRAZIL, Maranhão, Carolina, Chapada das Mesas, São Romão (6°02′09.1′′ S 46°34′03.5′′ W), soil, 15 May 2023, L.W.S. de Freitas (URM 9237). GenBank accession numbers: ITS = PZ234082, LSU = PZ227111, act = PZ227665.

Notes: Absidia variabilis is phylogenetically more closely related to A. variispora T.R.L. Cordeiro & A.L. Santiago (Figure 1). The main morphological differences between both species are the formation of sporangiophores arising singular or in whorls of up to six from stolon, as well as the formation of cylindrical, elliptical, globose, and subglobose sporangiospores by A. variispora, while A. variabilis forms sporangiophores single or in whorls of up to five from stolon and produces exclusively cylindrical spores. Furthermore, A. variabilis forms chlamydospores, while A. variispora does not. Absidia variabilis grows up to 31 °C on MEA and 32 °C on PDA, while A. variispora can grow at 32 °C on MEA and PDA [20].

Figure 5. Absidia variabilis (URM 9236, ex-type) on PDA at 25 °C after eight days. (A) Colony verse (left) and reverse (right). (B) Sporangiophore with sporangium. (C–F) Different types of columellae, apophyses, and projections. (G,H) Branched sporangiophore. (I) Two sporangiophores arising from stolon. (J) Rhizoid. (K) Sporangiospores. (L) Chlamydospores. Scale bars: (B) 15 μm; (C–I) 10 μm; (J–L) 5 μm.

Figure 5. Absidia variabilis (URM 9236, ex-type) on PDA at 25 °C after eight days. (A) Colony verse (left) and reverse (right). (B) Sporangiophore with sporangium. (C–F) Different types of columellae, apophyses, and projections. (G,H) Branched sporangiophore. (I) Two sporangiophores arising from stolon. (J) Rhizoid. (K) Sporangiospores. (L) Chlamydospores. Scale bars: (B) 15 μm; (C–I) 10 μm; (J–L) 5 μm.

3.2.3. Gongronella longapophysata L.W.S de Freitas & A.L. Santiago, sp. nov. (Figure 6 and Figure 7)

Mycobank number: MB863005.

Etymology: The epithet longapophysata (Lat.) refers to the sporangiophores with elongated apophysis.

Diagnosis: The species forms sporangiophores with some very elongated apophyses, some with an elongated swelling below apophyses, and chains of 3 to 12 sterile sporangia along sporangiophores.

Description: Colonies white (A1), cottony, with regular margin, 6.5 cm in diameter on PDA after 8 days, at 25 °C. Reverse white (A1). Rhizoids branched, long, often with adherent chlamydospores, 2–10 μm in diameter, and bulbous, 5–38 μm in diameter. Stolons hyaline, branched and coenocytic. Sporangiophores hyaline, erect, curved to sinuous, (10–)35–230(–350) × 2.5–7(–12) μm, with one to two septa below the sporangium, simple or sympodially branched up to four times, wall smooth to slightly incrusted, rarely verticillately branched in whorls of up to five. Fertile sporangia hyaline, subglobose (5–)12–20(–25) × (6.5–)15–20(–25) μm, wall with vitreous aspect, smooth and deliquescent, leaving a collar. Sterile sporangia hyaline, ellipsoid, cylindrical to constricted frequently formed on short sporangiophores branches, 3–20 μm in diameter, occasionally so short that the sterile sporangia appear to be sessile; 3–6, rarely, 7–12 sterile sporangia are also formed along the sporangiophore. Columellae hyaline, hemispherical, subglobose (2–)5–10 × (3.5–)7–11(–15) μm. Apophyses bell-shaped, long or short, vasiform, (2–)3–7(–10) × (5–)7–10(–13) μm. Some sporangiophores may show an elongated swelling below the apophysis, 10–25 × 5–7 μm. Sporangiospores hyaline, reniform, some irregularly-shaped, 2–5.5 × 1.5–2 μm, smooth-walled. Chlamydospores abundant, hyaline, globose, 4–15 μm in diameter. Zygosporangia not observed.

Growth experiments (8 days): On PDA: At 10 °C—no growth; at 15 °C—1.8 cm diameter; at 20 °C—3.1 cm diameter; at 25 °C—6.3 cm diameter; at 30 °C—7.5 cm diameter; at 35 °C—no growth. On MEA: At 10 °C—no growth; at 15 °C—1.9 cm diameter; at 20 °C—4.4 cm diameter; at 25 °C—6.0 cm diameter; at 30 °C—6.8 cm diameter; at 35 °C—no growth. The Tmax is 32 °C on MEA and 33 °C on PDA.

Habitat and Distribution: Soil from Maranhão state (Brazil).

Specimen examined: Brazil, Maranhão, Carolina city, Chapada das Mesas, Cachoeira da Mansinha (7°07′54.0″ S 47°26′55.1″ W), soil, 15 May 2023, L.W.S. de Freitas (Holotype URM 9240H metabolically inactive state, Ex-type living culture URM 9240).

GenBank accession numbers: ITS = PZ234083, LSU = PZ227112, act = PZ227666.

Additional specimen examined: BRAZIL, Maranhão, Carolina city, Chapada das Mesas, Cachoeira da Mansinha (7°07′54.0″ S 47°26′55.1″ W), soil, 15 May 2023, L.W.S de Freitas (URM 9241).

GenBank accession numbers: ITS = PZ234084, LSU = PZ227113, act = PZ227667.

Notes: Gongronella longapophysata is phylogenetically closely related to G. multiramosa and G. abortosporangia (Figure 1). Morphologically, G. multiramosa and G. abortosporangia can be easily differentiated from G. longapophysata due to no formed stolons, no verticillately branched sporangiophores (rare in G. longapophysata), no sporangiophores with a swelling below apophysis, and no chains of 3 to 12 sterile sporangia along sporangiophores [30]. The Tmax of G. longapophysata is 32 °C on MEA and 33 °C on PDA, but Tmax of G. abortosporangia and G. multiramosa is not indicated in the protologues nor elsewhere [29,30].

Figure 6. Gongronella longapophysata (URM 9240, ex-type) on PDA at 25 °C after eight days. (A) Colony verse (left) and reverse (right). (B–D) Columellae and aphophyses. (E) Sporangiophore with elongated swelling below the apophysis and sterile sporangia. (F) Branched sporangiophore. (G) Sporangiospores. Scale bars: (B–E) 10 μm; (F,G) 5 μm.

Figure 6. Gongronella longapophysata (URM 9240, ex-type) on PDA at 25 °C after eight days. (A) Colony verse (left) and reverse (right). (B–D) Columellae and aphophyses. (E) Sporangiophore with elongated swelling below the apophysis and sterile sporangia. (F) Branched sporangiophore. (G) Sporangiospores. Scale bars: (B–E) 10 μm; (F,G) 5 μm.

Figure 7. Gongronella longapophysata (URM 9240, ex-type) on PDA at 25 °C after eight days. (A,B) Branched sporangiophore with fertile sporangia. (C) Sterile sporangium. (D) Simply branched sporangiophore with sporangia. (E) Simply branched sporangiophore with fertile (terminal) and sterile (branch) sporangia. (F) Apophyses and columellae. (G) Sporangiophore with long and short branches. (H) Rhizoid with chlamydospores. (I) Bulbous rhizoid. Scale bars: (A,B) 30 μm; (C–F) 5 μm; (G–I) 10 μm.

Figure 7. Gongronella longapophysata (URM 9240, ex-type) on PDA at 25 °C after eight days. (A,B) Branched sporangiophore with fertile sporangia. (C) Sterile sporangium. (D) Simply branched sporangiophore with sporangia. (E) Simply branched sporangiophore with fertile (terminal) and sterile (branch) sporangia. (F) Apophyses and columellae. (G) Sporangiophore with long and short branches. (H) Rhizoid with chlamydospores. (I) Bulbous rhizoid. Scale bars: (A,B) 30 μm; (C–F) 5 μm; (G–I) 10 μm.

3.2.4. Gongronella verticillata L.W.S. de Freitas & A.L. Santiago, sp. nov. (Figure 8 and Figure 9)

Mycobank number: MB863006.

Etymology: The epithet verticillate (Lat.) refers to the formation of sporangiophores verticillately branched.

Diagnosis: The species commonly forms sporangiophores branched in whorls with up to five branches.

Description: Colonies white (A1), cottony, regular margin, 7 cm in diameter on PDA after 8 days of incubation, at 25 °C; reverse cream (4A3). Rhizoids of two types: root-shaped, hyaline, branched, with spaced septa and chlamydospores attached, and bulbous, without chlamydospores attached, 2.5–8.5 µm in diameter. Stolons hyaline, simple and coenocytic. Sporangiophores simple or sympodially branched up to six times, and commonly verticillately branched, with up to five branches in a whorl, (25–)95–200(–250) × 2.5–5 μm, with one or two septa below the sporangium, rarely forming three septa, smooth-walled. Some sporangiophores are extremely short, 2.5–10 × 2–3 μm, forming sterile apical or lateral sporangia. Fertile sporangia hyaline, globose, (6–)10–17(–22) μm diameter, with vitreous aspect, smooth and deliquescent-walled, leaving a collar. Abortive sporangia ovoid, subglobose and globose 4–18 μm in diameter. Columellae hyaline, mostly globose 1–5 μm in diameter, subglobose, hemispherical, flattened and rounded-topped (1–)4–7(–9) × (2–)6–9(–12) μm, smooth-walled, occasionally so flattened that they seem inconspicuous. Apophyses hyaline, mostly globose, 3–10 μm diameter, subglobose, some bell-shaped and vasiform, (3–)5–10(–13) × (3.5–)6–12(–15) μm, smooth-walled. Sporangiospores hyaline, elliptical to fusiform, irregular or reniform, 2–7.5 × 1.5–4 μm, smooth-walled. Chlamydospores abundant, hyaline, subglobose and ellipsoidal, 4–15 μm diameter. Zygosporangia not observed.

Growth experiments (8 days): On PDA: At 10 °C—no growth; at 15 °C—1.7 cm diameter; at 20 °C—3.2 cm diameter; at 25 °C—6.8 cm diameter; at 30 °C—2.1 cm diameter; at 35 °C—no growth. On MEA: At 10 °C—no growth; at 15 °C—1.9 cm diameter; at 20 °C—4.2 cm diameter; at 25 °C—5.5 cm diameter; at 30 °C—4 cm diameter; at 35 °C—no growth. The Tmax is 31 °C on both MEA and PDA.

Habitat and Distribution: Soil from Maranhão state (Brazil).

Specimen examined: BRAZIL, Maranhão, Carolina city, Chapada das Mesas, Cachoeira da Mansinha (7°07′54.0″ S 47°26′55.1″ W), soil, 15 May 2023, L.W.S. de Freitas (Holotype URM 9238H metabolically inactive state, Ex-type living culture URM 9238).

GenBank accession numbers: ITS = PZ234085, LSU = PZ227114.

Additional specimen examined: BRAZIL, Maranhão, Carolina city, Chapada das Mesas, Cachoeira da Mansinha (7°07′54.0″ S 47°26′55.1″ W), soil, 15 May 2023, L.W.S. de Freitas (URM 9239).

GenBank accession numbers: ITS = PZ234086, LSU = PZ227115.

Notes: Gongronella verticillata is phylogenetically closely related to G. multispora and G. bartikiae (Figure 1). However, G. verticillata forms sporangiophores sympodially and verticillately branched, with up to five branches in a whorl. The apophyses are globose, subglobose, bell-shaped and vasiform; columellae are globose, subglobose, flattened, rounded-topped and inconspicuous, whereas G. multispora forms sporangiophores sympodially and verticillately branched, with 2–3 branches in a whorl. Apophyses are pyriform to subglobose, and the columellae are hemispheric and some inconspicuous. It is curious that both species form sporangiophores branched in whorls, as to the best of our knowledge, this branch pattern has also been reported in G. namwonensis so far [28]. The Tmax of G. verticillata is 31 °C on MEA and PDA, but Tmax of G. multispora has not been given in the protologue [29]. There is no morphological description of G. bartikiae in the literature for comparison [67].

Figure 8. Gongronella verticillata (URM 9238, ex-type) on PDA at 25 °C after eight days. (A) Colony verse (left) and reverse (right). (B,C) Sporangiophore with sporangium. (D–G) Sporangiophore with columella. (H) Simply branched sporangiophore arising from stolon with rhizoid. (I) Sporangiophore with terminal columella and lateral whorled branches with columellae. (J) Rhizoid with chlamydospores attached. (K) Sporangiospores. Scale bars: (B,C) 20 μm; (D,F,J,K) 10 μm; (G,H) 15 μm; (E,I) 5 μm.

Figure 8. Gongronella verticillata (URM 9238, ex-type) on PDA at 25 °C after eight days. (A) Colony verse (left) and reverse (right). (B,C) Sporangiophore with sporangium. (D–G) Sporangiophore with columella. (H) Simply branched sporangiophore arising from stolon with rhizoid. (I) Sporangiophore with terminal columella and lateral whorled branches with columellae. (J) Rhizoid with chlamydospores attached. (K) Sporangiospores. Scale bars: (B,C) 20 μm; (D,F,J,K) 10 μm; (G,H) 15 μm; (E,I) 5 μm.

Figure 9. Gongronella verticillata (URM 9238, ex-type) on PDA at 25 °C after eight days. (A) Sporangiophores with sporangia. (B) Sporangiophores with a septum below the apophysis (left) and columellae. (C) =Bulbous rhizoid. (D) Chlamydospores attached to the mycelium. Scale bars: (A,B) 3 μm; (C) 10 μm; (D) 5 μm.

Figure 9. Gongronella verticillata (URM 9238, ex-type) on PDA at 25 °C after eight days. (A) Sporangiophores with sporangia. (B) Sporangiophores with a septum below the apophysis (left) and columellae. (C) =Bulbous rhizoid. (D) Chlamydospores attached to the mycelium. Scale bars: (A,B) 3 μm; (C) 10 μm; (D) 5 μm.

4. Discussion

Traditionally, new species of Mucorales fungi have been mostly delimited on a morphological basis, leading to further synonymies and generating controversy and doubts among taxonomists. In this context, species of Absidia and Gongronella have been sorted into other genera, as they shared similar morphological characteristics. For instance, G. butleri was first described as A. butleri Lend. in 1926, while Paine [68] proposed A. subpoculata Paine, which was later synonymized with G. butleri. In addition, some species of Absidia have been previously included in Tieghemella, Mycocladus, Proabsidia, Pseudoabsidia, and Protoabsidia [69,70,71]. On the other hand, species of Lichtheimia and Lentamyces have been previously treated as Absidia [72,73].

The advent of the molecular biology allowed the delimitation of new species of Absidia and Gongronella in the last 10–15 years in different countries, such as China, Brazil, South Korea, and Thailand, most of which were proposed based in sequences of ITS and/or LSU rDNA [20,21,29,30,41,42,74,75]. Wang et al. [30] highlighted that the ITS, LSU, Elongation Factor 1-alpha-EF-1α (TEF), act, and RNA Polymerase II Largest Subunit (RPB1) are reliable markers for identification of Gongronella species. Until 1969, only two Gongronella species, namely, G. butleri and G. lacrispora Hesselt. & J. JEllis [76,77], were known, and this number has increased to 28 species (as of 3 March 2026), mostly descried from China. Regarding Absidia, Ji et al. [44] sequenced TEF and act, as well as ITS and LSU, to delimit new species, and 69 new species have been described in the last 15 years [27].

Since Tmax has been successfully used as a discriminative characteristic for delimitation of mucoralean fungi, such as Backusella [39], Cunninghamella [78,79], Lichtheimia [80], Mucor [81], and Rhizopus [82], we believe in the taxonomic relevance of this characteristic for the delimitation of species within Absidia and Gongronella. In this paper, the Tmax of our new Absidia species did not vary much from the phylogenetically close species. However, we retrieved data from the literature on the Tmax of 50 species of Absidia (plus two new species described in this work) and realized that the Tmax varies considerably between species in this genus, from 24 °C (A. frigida) to 37 °C (A. ovalispora and A. zonata) (Table 1); therefore, this feature has taxonomic value. However, as it was also observed in other mucoralean genera [39,78,79,80], some overlapping may occur (e.g., A. menglianensis, A. tarda and A. longissima have a Tmax of 36 °C), which is why this feature should be used along with morphology and phylogeny for delimiting species in Absidia, as well as in other mucoralean genera.

We were unable to compare the Tmax of our new Gongronella species with other species due to the lack of Tmax data in the literature for this genus; therefore, it is still unknown whether this feature is taxonomically informative in Gongronella. Nevertheless, to the best of our knowledge we are providing the first data on Tmax for the genus. Regarding Absidia, Tmax data are available in the literature for only 52% of the known species, mostly for species from China and Korea, with a few species from Brazil and Australia (Table 1). Therefore, studies evaluating the Tmax of all species of both genera should be encouraged.

In this paper, we described, based on an integrated approach, four new species of Cunninghamellaceae from soil samples from the still poorly studied Cerrado biome. As far as we are aware, this is the first report of new Absidia and Gongronella species in this biome. Currently, only 17 species of Absidia have been reported in Brazil, including ten new species [19,20,53,74,75,83,84], whereas only four species of Gongronella were recorded in the country, including three new species [85,86]. All these species were isolated in the Caatinga and Atlantic Forest biomes. This paper increases the number of known species of Absidia and Gongronella to 96 and 30, respectively, as well as the knowledge of these fungi in the Brazilian Savanna. We believe that new inventories of Cunninghamellaceae in other Cerrado areas may unveil additional new species.