Diversity of mycorrhizal Tulasnella associated with epiphytic and rupicolous orchids from the Brazilian Atlantic Forest, including four new species.

The genus Tulasnella often forms mycorrhizas with orchids and has worldwide distribution. Species of this genus are associated with a wide range of orchids, including endangered hosts. Initially, species identification relied mostly on morphological features and few cultures were preserved for later phylogenetic comparisons. In this study, a total of 50 Tulasnella isolates were collected from their natural sites in Minas Gerais, Brazil, cultured, and subjected to a phylogenetic analysis based on alignments of sequences of the internal transcribed spacer (ITS) of the nuclear ribosomal DNA. Our results, based on phylogeny, integrated with nucleotide divergence and morphology, revealed the diversity of isolated Tulasnella species, which included four new species, namely, Tulasnella brigadeiroensis, Tulasnella hadrolaeliae, Tulasnella orchidis and Tulasnella zygopetali. The conservation of these species is important due to their association with endangered orchid hosts and endemic features in the Brazilian Atlantic Forest.

Introduction

Orchidaceae (or orchids) is the largest family of flowering plants, with approximately 27,000 species described[1]. The Neotropics is the region of greatest orchid diversity[2] and approximately 205 genera and 2,650 species occur in Brazil, of which about 1,800 are endemic[3]. Many orchid species are endangered, mainly due to anthropogenic pressure and dependency between orchids and other organisms, i.e. pollinators or mycorrhizal fungi[4,5].

Several endangered orchid species are listed in the Livro Vermelho da Flora do Brasil[6]. Among them, Hadrolaelia jongheana is an epiphytic orchid found in the Zona da Mata and Quadrilátero Ferrífero, two areas severely affected by anthropogenic activity. Zygopetalum maxillare is an epiphytic species which, although not officially endangered, grows almost exclusively in tree ferns[7], which limits its distribution. Cattleya cinnabarina and Cattleya caulescens are rupicolous (i.e. grow on bare rocks) and endemic to the Southeastern Brazil[8]. These species belong to Brazilian Atlantic Forest, a highly diverse but endangered hotspot of biodiversity[9]. Like all orchids, they need mycorrhizal fungi for germination due to the limited reserves in seeds[10]. The symbiotic fungus supplies the embryo with carbon and other nutrients, which enable the germination and establishment of the orchid[11]. Orchids associate mainly with Basidiomycota often called rhizoctonia, a polyphyletic that includes taxa belonging to the families Sebacinaceae, Serendipitaceae, Ceratobasidiaceae and Tulasnellaceae[12,13].

The specificity of orchid–mycorrhizal fungi varies among species[12,14] and the distribution of mycorrhizal fungi can affect the patterns of distribution of orchids[15]. Species with low specificity for their fungal partner may be more successful in conservation strategies, such as assisted migration[8]. Despite this, specialist orchids might be widely distributed if their fungal partners are broadly distributed[14,16]. Indeed, the ecology of Tulasnella species orchid roots apart remains poorly known and even though they are often considered saprotrophic[11] they may also colonize the roots of non-orchid plants[17]. The availability of compatible symbionts may directly impact the conservation of species[4].

The genus Tulasnella is often observed as orchid mycorrhizal fungi in temperate and tropical regions[12,18,19], and several isolates have been reported to increase seed germination and seedling growth[20-25]. Identification of mycorrhizal fungi in South American orchids, mostly conducted in Brazil, has often revealed Tulasnella symbionts: Tulasnella species were isolated from Epidendrum secundum[26,27], Epidendrum dendrobioides and Sophronits milleri[28], Oeceoclades maculata, Epidendrum rigidum and Polystachya concreta[29], E. rigidum and P. concreta[30]. Yet little is known about Tulasnella in the hotspot of biodiversity of the Brazilian Atlantic Forest.

Tulasnella species have complex morphological characteristics, but rarely form fruitbodies in situ or sexual structures in vitro[29-33]. As morphological characteristics are not sufficient to describe Tulasnella species[34], molecular approaches have been used too[32,33,35-38]. Species identification is mostly based on phylogenetic concordance of multiple unrelated genes/regions, but for this complex genus, the internal transcribed spacer (ITS) of the nuclear ribosomal DNA was shown to be highly suitable for species delimitation in Tulasnella[31,38].

In a survey of cultivable mycorrhizal fungi associated with the roots of the rare-to-endangered Brazilian orchids H. jongheana, C. cinnabarina, C. caulescens and Z. maxillare, we obtained 50 isolates of Tulasnella. Herein, based on morphological and molecular analyses, we have evaluated the diversity of Tulasnella associated with these four orchids and describe potentially new Tulasnella species.

Results

Tulasnella isolates from Brazilian Atlantic Forest

Fifty isolates of the genus Tulasnella were obtained in this study (Table 1), namely, twenty isolates from C. cinnabarina roots, fourteen from C. caulescens roots, nine from H. jongheana (eight from Parque Estadual da Serra do Brigadeiro (PESB) and one from Parque Estadual da Serra Negra (PESN)) and seven isolates from Z. maxillare. As they were isolated from pelotons dissected from roots, they all are likely orchid mycorrhizal fungi. All isolates from C. cinnabarina and C. caulescens were identified as Tulasnella calospora, whereas isolates obtained from H. jongheana and Z. maxillare are described below as four new Tulasnella species.

Table 1

Tulasnella isolates obtained in this study. Ex-type strains are indicated in bold face.

Identity	Culture accession no.	Orchid Host	Origin	Habitat	GenBank accession no.
Tulasnella calospora	COAD 2850	Cattleya caulescens	Mariana - MG	Rupicolous	MK192009
	COAD 2851	Cattleya caulescens	Mariana - MG	Rupicolous	MK192010
	COAD 2852	Cattleya caulescens	Mariana - MG	Rupicolous	MK191991
	COAD 2853	Cattleya caulescens	Mariana - MG	Rupicolous	MK191993
	COAD 2854	Cattleya caulescens	Mariana - MG	Rupicolous	MK191994
	COAD 2855	Cattleya caulescens	Mariana - MG	Rupicolous	MK192007
	COAD 2856	Cattleya caulescens	Mariana - MG	Rupicolous	MK191995
	COAD 2857	Cattleya caulescens	Mariana - MG	Rupicolous	MK191996
	COAD 2858	Cattleya caulescens	Mariana - MG	Rupicolous	MK191997
	COAD 2859	Cattleya caulescens	Mariana - MG	Rupicolous	MK191998
	COAD 2860	Cattleya caulescens	Mariana - MG	Rupicolous	MK191999
	COAD 2861	Cattleya caulescens	Mariana - MG	Rupicolous	MK192000
	COAD 2862	Cattleya caulescens	Mariana - MG	Rupicolous	MK192005
	COAD 2863	Cattleya caulescens	Mariana - MG	Rupicolous	MK192003
	COAD 2864	Cattleya cinnabarina	Mariana - MG	Rupicolous	MK191974
	COAD 2865	Cattleya cinnabarina	Mariana - MG	Rupicolous	MK191975
	COAD 2866	Cattleya cinnabarina	Mariana - MG	Rupicolous	MK192006
	COAD 2867	Cattleya cinnabarina	Mariana - MG	Rupicolous	MK191976
	COAD 2868	Cattleya cinnabarina	Mariana - MG	Rupicolous	MK191977
	COAD 2869	Cattleya cinnabarina	Mariana - MG	Rupicolous	MK191978
	COAD 2870	Cattleya cinnabarina	Mariana - MG	Rupicolous	MK191979
	COAD 2871	Cattleya cinnabarina	Mariana - MG	Rupicolous	MK191980
	COAD 2873	Cattleya cinnabarina	Mariana - MG	Rupicolous	MK191981
	COAD 2874	Cattleya cinnabarina	Mariana - MG	Rupicolous	MK191982
	COAD 2875	Cattleya cinnabarina	Mariana - MG	Rupicolous	MK191983
	COAD 2876	Cattleya cinnabarina	Mariana - MG	Rupicolous	MK191984
	COAD 2877	Cattleya cinnabarina	Mariana - MG	Rupicolous	MK191985
	COAD 2878	Cattleya cinnabarina	Mariana - MG	Rupicolous	MK191986
	COAD 2879	Cattleya cinnabarina	Mariana - MG	Rupicolous	MK192004
	COAD 2880	Cattleya cinnabarina	Mariana - MG	Rupicolous	MK191987
	COAD 2881	Cattleya cinnabarina	Mariana - MG	Rupicolous	MK191988
	COAD 2882	Cattleya cinnabarina	Mariana - MG	Rupicolous	MK192008
	COAD 2883	Cattleya cinnabarina	Mariana - MG	Rupicolous	MK191989
Tulasnella brigadeiroensis sp. nov.	COAD 2884	Hadrolaelia jongheana	Araponga - MG	Epiphytic	MK192001
	COAD 3007	Hadrolaelia jongheana	Araponga - MG	Epiphytic	MT090025
	COAD 3008	Hadrolaelia jongheana	Araponga - MG	Epiphytic	MT090026
Tulasnella hadrolaeliae sp. nov.	COAD 2887	Hadrolaelia jongheana	Araponga - MG	Epiphytic	MN385724
	COAD 2888	Hadrolaelia jongheana	Araponga - MG	Epiphytic	MN385725
	COAD 2889	Hadrolaelia jongheana	Araponga - MG	Epiphytic	MN385726
	COAD 2890	Hadrolaelia jongheana	Araponga - MG	Epiphytic	MN385727
	COAD 2891	Hadrolaelia jongheana	Araponga - MG	Epiphytic	MN385728
Tulasnella orchidis sp. nov.	COAD 2893	Zygopetalum maxillare	Araponga - MG	Epiphytic	MN385729
	COAD 2894	Zygopetalum maxillare	Araponga - MG	Epiphytic	MN385731
	COAD 2895	Zygopetalum maxillare	Araponga - MG	Epiphytic	MN385730
Tulasnella zygopetali sp. nov.	COAD 2896	Zygopetalum maxillare	Araponga - MG	Epiphytic	MN385732
	COAD 2897	Zygopetalum maxillare	Araponga - MG	Epiphytic	MN385733
	COAD 2898	Zygopetalum maxillare	Araponga - MG	Epiphytic	MN385734
	COAD 2899	Zygopetalum maxillare	Araponga - MG	Epiphytic	MN385735
Tulasnella sp.	COAD 2885	Hadrolaelia jongheana	Itamarandiba - MG	Epiphytic	MK192002

Phylogeny

The ITS alignment consisted of 93 strains (including the outgroup sequence), of which 43 are from NCBI or UNITE and 50 from this study (Tables 1 and 2) and had a total length of 583 characters (including alignment gaps). Among these, 371 characters were parsimony-informative, 419 were variable and 147 were conserved.

Table 2

GenBank and UNITE accession numbers of additional Tulasnella isolates included in the phylogenetic analysis. Ex-type strains are indicated in bold face.

Species	Strain No.	Origin	GenBank accession No.	UNITE accession No.
Epulorhiza amonilioides	3S	Brazil	JF907600
Epulorhiza amonilioides	aero8	Brazil	KC928335
Epulorhiza anaticula	UAMH 5428	Canada	EU218891
Epulorhiza anaticula	13O004	South Korea	KT164598	SH1174351.08FU
Tulasnella albida	KC110	Unknown	AY373294
Tulasnella asymmetrica	MAFF 305808 clone C001	Australia	KC152356
Tulasnella asymmetrica	AL.LM4.4.1	Australia	MH134544	SH1541682.08FU
Tulasnella bifrons	BPI 724849	Canada	AY373290
Tulasnella calospora	MAFF P305801	Ecuador	DQ388041
Tulasnella calospora	MAFF P305802	Ecuador	DQ388042
Tulasnella calospora	MAFF P305803	Ecuador	DQ388043
Tulasnella calospora	MAFF P305804	Ecuador	DQ388044
Tulasnella calospora	MAFF P305805	Ecuador	DQ388045
Tulasnella calospora	FCb4	China	KC796458	SH1554832.08FU
Tulasnella danica	KC388	USA	AY373297
Tulasnella eichleriana	KC852	Unknown	AY373292
Tulasnella eichleriana	K(M)143600	United Kingdom	KC152381
Tulasnella irregularis	JHW 0632	Australia	EU218889
Tulasnella irregularis	D1-KT-TC-1	Thailand	GU166413
Tulasnella irregularis	C3-DT-TC-2	Thailand	GU166423	SH1561236.08FU
Tulasnella prima	CLM159	Australia	KF476556
Tulasnella prima	07033-45	Australia	HM196800
Tulasnella pruinosa	DAOM 17641	Unknown	AY373295
Tulasnella pruinosa	AFTOL ID610	Unknown	DQ457642	SH1549691.08FU
Tulasnella secunda	CLM009	Australia	KF476575
Tulasnella secunda	CLM222	Australia	KF476568
Tulasnella sp.	141	USA	AY373264
Tulasnella sp.	10 MM-2016	USA	KU664580
Tulasnella sphagneti	CLM541	Australia	KY095117
Tulasnella sphagneti	CLM583	Australia	KY445922
Tulasnella tomaculum	KC429	Unknown	AY373296
Tulasnella tubericola	EP-15	Spain	KX929166
Tulasnella tubericola	EP-1	Spain	KX774345
Tulasnella violea	FO24380a	Germany	KC152439	SH1555437.08FU
Tulasnella violea	DC292	Germany	KC152432
Tulasnella warcupii	CLM027	Australia	KF476596
Tulasnella warcupii	CLM007	Australia	KF476600
Uncultured Tulasnella	Clone 33tu-12	China	HM230652
Botryobasidium botryosum	AFTOL ID604	Germany	DQ267124

Our phylogenetic analyses confirmed that mycorrhizal fungi isolated from the studied orchid species were Tulasnella (Fig. 1). Among these, four species are new in this genus and are described below, namely, Tulasnella hadrolaeliae, Tulasnella brigadeiroensis, Tulasnella orchidis and Tulasnella zygopetali. The newly proposed species are based on phylogenetic analyses, pairwise sequence divergence and morphological features (see below). The clades containing the Brazilian Tulasnella isolates are highlighted in the phylogenetic tree (Fig. 1).

Figure 1

Bayesian phylogenetic tree for Tulasnella based on ITS alignment. Maximum likelihood bootstrap support (ML > 60) and Bayesian posterior probabilities (PP) values are indicated next to the nodes (ML/PP). Species from Brazil are in the colored block and the new species described in this paper are indicated in bold face. Botryobasidium botryosum (AFTOL604) was used as the outgroup.

Phylogenetically, all isolates of Tulasnella from C. caulescens and C. cinnabarina are grouped in a clade including T. calospora isolates, close to another group composed of T. tubericola and T. bifrons (Fig. 1). The new species Tulasnella hadrolaeliae formed a well-supported clade (Maximum likelihood (ML)/Posterior probabilities (PP) = 100/1), which is a sister group of T. albida and T. pruinosa. Tulasnella brigadeiroensis isolates were grouped in a monophyletic clade. Tulasnella orchidis, isolated from Z. maxillare, clustered in a sister clade to T. brigadeiroensis and Tulasnella sp. COAD 2885. Finally, isolates of Tulasnella zygopetali obtained from Z. maxillare formed a strongly supported clade (ML/PP = 100/1), distinct from other Tulasnella species. Although the phylogenetic analyzes indicate that Tulasnella sp. COAD 2885 may represent a new species, it will not be formally described here since only one isolate was obtained during our study.

Divergence within and between clades

The Kimura-2-parameter distances between Tulasnella species ranged from 1.9 to 65.2% (Table 3). The divergence within Tulasnella species described here was lower than 0.6%. The nucleotide divergence between Tulasnella sp. COAD 2885 and T. brigadeiroensis was 7.5%, far above the 3% threshold suggested by Linde et al.[31] in Tulasnella, and supposedly belong to two different species. For some species it was not possible to calculate the divergence within the clade, because only one isolate was used in analysis.

Table 3

Estimates of percentage nucleotide divergence by the Kimura-2P distances for Tulasnella within and between species. There was a total of 272 positions in the final dataset. All positions containing gaps and missing data were eliminated.

	Within taxa	1	2	3	4	5	6	7	8	9	10	11	12	13	14	15	16	17	18	19	20	21
1	2.2
2	—	8.2
3	1.2	16.8	14.4
4	0.4	16.4	14.9	2.5
5	—	18.1	15.1	4.3	4.4
6	0.4	32.9	32.7	33.7	33.6	33.5
7	0.0	33.4	33.2	38.8	38.4	37.4	10.9
8	—	32.9	33.9	37.3	38.2	38.7	12.6	9.0
9	0.2	33.0	30.8	33.2	34.4	34.4	5.9	11.0	12.1
10	—	38.2	36.6	38.8	40.4	40.1	11.7	15.9	15.9	7.5
11	0.0	31.5	30.2	32.8	32.6	32.2	8.8	11.2	12.0	8.8	14.6
12	0.5	34.4	32.6	34.4	35.1	33.0	8.2	10.4	13.6	5.4	10.5	8.1
13	0.0	33.9	33.3	32.9	32.4	35.0	27.9	28.7	31.3	26.2	28.9	22.8	26.9
14	3.4	34.4	35.0	35.9	35.1	38.1	33.6	33.1	36.1	30.1	34.8	29.1	31.0	8.6
15	0.0	47.1	44.0	43.5	42.6	42.7	37.0	36.6	44.6	37.3	41.6	40.0	36.2	41.0	48.0
16	0.7	58.8	57.7	60.4	61.6	63.6	49.4	48.0	52.4	46.3	51.4	54.0	49.1	51.3	52.5	50.2
17	0.0	57.1	55.2	59.4	60.7	60.9	48.7	46.6	50.8	45.7	48.6	54.2	47.7	51.4	56.0	51.7	7.0
18	—	57.3	56.2	59.5	60.9	61.1	48.0	46.7	49.4	45.1	50.1	51.1	47.2	50.7	53.7	48.8	3.8	5.0
19	0.4	60.9	59.8	63.0	64.0	65.2	49.9	51.4	49.6	47.4	50.4	52.4	49.0	53.7	61.8	54.7	8.6	9.7	8.0
20	0.0	61.3	60.2	62.6	64.1	64.3	54.6	52.4	55.3	50.6	56.1	55.7	54.3	54.4	56.7	51.0	8.8	10.7	8.2	11.8
21	0.0	59.2	57.7	61.6	63.1	63.3	52.3	50.1	53.0	48.4	53.7	53.3	52.0	55.1	55.8	48.7	9.2	11.2	8.6	12.2	1.9
22	0.4	62.9	62.6	59.4	60.4	59.8	53.9	49.3	51.3	52.1	54.5	51.4	52.2	52.5	56.5	54.8	18.0	17.6	16.1	19.0	21.1	20.5

1 = Tulasnella anaticula, 2 = T. danica, 3 = T. calospora, 4 = T. tubericola, 5 = T. bifrons, 6 = T. asymmetrica, 7 = T. pruinosa, 8 = T. albida, 9 = T. brigadeiroensis, 10 = Tulasnella sp. COAD 2885, 11 = T. hadrolaeliae, 12 = T. orchidis, 13 = T. irregulares, 14 = T. amonilioides, 15 = T. zygopetali, 16 = T. eichleriana, 17 = T. secunda, 18 = T. tomaculum, 19 = T. wacupii, 20 = T. prima, 21 = T. sphagneti, 22 = T. violea.

Taxonomy

E.F.S. Freitas, Meir. Silva & M.C.M. Kasuya, sp. nov. (Fig. 2)

Figure 2

Tulasnella brigadeiroensis (COAD2884). (a) Eight-day-old PDA culture. (b) Hyphae with branching at right angles. (c) Hyphae stained with SYBR Green I showing binucleate cells (N = nuclei; S = septa). Bars = 50 µm.

Mycobank: MB832785

Etymology:— Referring to Parque Estadual Serra do Brigadeiro, where the type species was isolated.

Diagnosis: Tulasnella brigadeiroensis is phylogenetically closely related to T. orchidis. In a comparison of the 583 ITS nucleotides, T. brigadeiroensis differs from T. orchidis by 47 bp (8.1%).

Type:—BRAZIL: Minas Gerais: Parque Estadual Serra do Brigadeiro, isolated from roots of the orchid Hadrolaelia jongheana, February 2018, E.F.S. Freitas (holotype VIC47299, ex-type culture COAD2884).

Description: Colonies on PDA attaining 31 mm diam after 8 d at 25 °C, white to cream, with undulate and submersed edge, aerial mycelium present. Reverse of the colony white to cream. Hyphae are regularly septate with branching at right angles, 1.5–2.5 µm diam ( ± SD = 2 ± 0.3 μm), hyaline, with binucleate cells. Molinioid cells not observed. Sexual morph not observed.

Substrate or host: Roots of Hadrolaelia jongheana.

Additional material examined.—BRAZIL: Minas Gerais: Parque Estadual Serra do Brigadeiro, from roots of Hadrolaelia jongheana, October 2019, E.F.S. Freitas (COAD3007, COAD3008). This species was isolated three times from two roots. There was no difference between the morphology of the isolates.

Juel, Bih. K. svenska Vet-Akad. Handl. 23: 23 (1897). (Fig. 3)

Figure 3

Tulasnella calospora (COAD2869). (a) Eight-day-old PDA culture. (b) Hyphae stained with SYBR Green I showing binucleate cells (M= monilioid cell; N = nuclei; S = septa). (c) Hyphae with branching at right angles. (d) Monilioid cell chains in CMA. Bars = 50 µm.

Description: Colonies on PDA attaining 45–67 mm diam after 8 d, at 25 °C, white to cream, with undulate and submersed edge, some cultures showing aerial mycelium. Hyphae from cultures are regularly septate, with branching at right angles, 3–4 µm diam ( ± SD = 3.5 ± 0.3 μm), hyaline, with binucleate cells. Molinioid hyaline, barrel to elongated barrel-shaped, in branched chains with more than five cells. Sexual morph not observed.

Substrate or host: Roots of Cattleya caulescens and Cattleya cinnabarina.

Additional material examined—BRAZIL. Minas Gerais, Mariana, Mina da Alegria, Vale S.A., isolated from roots of Cattleya caulescens, COAD 2850–COAD2863; and from roots of Cattleya cinnabarina, COAD2864–2883, 2010, Bocayuva, M.F. There was no difference between the morphology of the isolates.

E.F.S. Freitas, Meir. Silva & M.C.M. Kasuya, sp. nov. (Fig. 4)

Figure 4

Tulasnella hadrolaeliae (COAD2889). (a) Thirty-day-old PDA culture. (b) Hyphae with branching at right angles. (c) Hyphae stained with SYBR Green I showing binucleate cells (N = nuclei; S = septa). Bars: B = 50 µm; C = 10 µm.

Mycobank: MB832786

Etymology: — Name derived from the plant host genus Hadrolaelia.

Diagnosis: Tulasnella hadrolaeliae is phylogenetically closely related to T. albida and T. pruinosa. In a comparison of the ITS nucleotides, T. hadrolaeliae differed from T. albida by 64 bp (11%) and from T. pruinosa by 73 bp (12.5%).

Type:—BRAZIL: Minas Gerais: Parque Estadual Serra do Brigadeiro, isolated from roots of orchid Hadrolaelia jongheana, February 2018, E.F.S. Freitas (holotype VIC47304, ex-type culture COAD2889).

Description: Colonies on PDA showed very slow-growing (56–59 mm diam after 30 d at 25 °C), white to cream, showing concentric rings, with undulate and submersed edge, aerial mycelium present. Reverse of the colony white to cream. Hyphae are regularly septate with branching at right angles, 2–3.5 µm diam ( ± SD = 2.5 ± 0.3 μm), hyaline, with binucleate cells and thin-walled. Molinioid cells not observed. Sexual morph not observed.

Substrate or host: Roots of Hadrolaelia jongheana.

Additional material examined.—BRAZIL: Minas Gerais: Parque Estadual Serra do Brigadeiro, from roots of Hadrolaelia jongheana, February 2018, E.F.S. Freitas (COAD2887, COAD2888, COAD2890, COAD2891). This species was isolated five times from three roots. There was no difference between the morphology of the isolates.

E.S. Cruz, E.F.S. Freitas, Meir. Silva & M.C.M. Kasuya, sp. nov. (Fig. 5)

Figure 5

Tulasnella orchidis (COAD2893). (a) Fourteen-day-old PDA culture. (b) Hyphae stained with SYBR Green I showing binucleate cells (N = nuclei; S = septa). (c) Hyphae with branching at right angles. (d) Monilioid cell chains in CMA. Bars = 50 µm.

Mycobank: MB832787

Etymology:— Name derived from the nature of host, an orchid, from which it was isolated.

Diagnosis: Tulasnella orchidis differs from T. brigadeiroensis by the culture characteristics on PDA, colonies forming concentric rings with undulate edge, whereas T. brigadeiroensis show uniform colonies with regular edge. In a comparison of the 583 ITS nucleotides, T. orchidis differed from T. brigadeiroensis by 47 bp (8%).

Type:—BRAZIL: Minas Gerais: Parque Estadual Serra do Brigadeiro, isolated from roots of Zygopetalum maxillare, February 2019, E.S. Cruz (holotype VIC47308, ex-type culture COAD2893).

Description: Colonies on PDA attaining 62–71 mm diam after 14 d, at 25 °C, white to cream, with undulate and submersed edge, showing concentric rings, no formation of aerial mycelium. Reverse of the colony white to cream. Hyphae are regularly septate with branching at right angles, 2.5–4.5 µm diam ( ± SD = 3.5 ± 0.5 μm), hyaline, with binucleate cells and thin-walled. Molinioid cells hyaline, barrel to elliptical-shaped, 5–11.5 µm diam ( ± SD = 8 ± 2 μm) and in branched chains. Sexual morph not observed.

Substrate or host: Roots of Zygopetalum maxillare.

Additional material examined.—BRAZIL: Minas Gerais: Parque Estadual Serra do Brigadeiro, from roots of Zygopetalum maxillare, February 2019, E.S. Cruz (COAD2894, COAD289). This species was isolated three times from the same root. There was no difference between the morphology of the isolates.

E.S. Cruz, E.F.S. Freitas, Meir. Silva & M.C.M. Kasuya, sp. nov. (Fig. 6)

Figure 6

Tulasnella zygopetali (COAD2896). (a) Eight-day-old PDA culture. (b) Hyphae stained with SYBR Green I showing binucleate cells (N = nuclei; S = septa). (c) Hyphae with branching at right angles. (d) Monilioid cell chains in CMA. Bars = 50 µm.

Mycobank: MB832789

Etymology: — Name derived from the plant host genus Zygopetalum, from which it was first collected.

Diagnosis: Tulasnella zygopetali is phylogenetically different from other Tulasnella species. Morphologically, T. zygopetali differs from other Tulasnella species described here as it has wider hyphae (3–6 µm diam) and monilioid cells (6.5–12.5 µm diam). In a comparison of the 583 ITS nucleotides, T. zygopetali differed from T. brigadeiroensis by 134 bp (23%), from T. hadrolaeliae by 148 bp (25.4%) and from T. orchidis by 135 bp (23%).

Type:—BRAZIL: Minas Gerais: Parque Estadual Serra do Brigadeiro, isolated from roots of Zygopetalum maxillare, February 2019, E.S. Cruz (holotype VIC47311, ex-type culture COAD2896).

Description: Colonies on PDA attaining 86 mm diam after 8 d, at 25 °C, white to cream, with regular and submersed edge, dense aerial mycelium. Reverse of the colony white to cream. Hyphae are regularly septate with branching at right angles, 3–6 µm diam ( ± SD = 4 ± 0.9 μm), hyaline, with binucleate cells and thin-walled. Molinioid cells hyaline, elongated barrel-shaped, 6.5–12.5 µm diam ( ± SD = 10 ± 1.5 μm), in branched chains with more than five cells. Sexual morph not observed.

Substrate or host: Roots of Zygopetalum maxillare.

Additional material examined—BRAZIL: Minas Gerais: Parque Estadual Serra do Brigadeiro, from roots of Zygopetalum maxillare, February 2019, E.S. Cruz (COAD2897, COAD2898, COAD2899). This species was isolated four times from the same root. There was no difference between the morphology of the isolates.

Discussion

We investigated Tulasnella species associated with the roots of four Brazilian orchids from different vegetations of the Atlantic Forest, where this fungal genus is little known. A previous study of the same area, based only on the molecular approach, observed high fungal community diversity in roots of H. jongheana, C. caulescens and C. cinnabarina orchids, but no Tulasnella was identified[8]. The authors suggested that Tulasnella sequences were not detected due to the primers used. Indeed, universal fungal primers such as ITS1F/ITS4 often do not detect Tulasnella species due to a high rate of molecular evolution of nuclear rDNA genes in this genus[35,39].

The genus Tulasnella (Tulasnellaceae) was described in 1888 by Schröter, with Tulasnella lilacina J. Schröt. as the type species, and nowadays there are 73 accepted species in Index Fungorum[40]. Due to the lack of molecular data from the type specimen, many Tulasnella species are described only by morphological-based approaches[38]. Morphological characters, such as size and shape of hyphae, basidia, sterigmata and basidiospore, when used alone, may lead to incorrect species identification[34], e.g. because they are affected by cultural conditions. For species delimitation, we have combined both molecular and morphological data as recommended by Cruz et al.[34,36], using ITS as suggested by Linde et al.[38].

Among the species of the genus Tulasnella, T. calospora is considered as a nearly universal orchid symbiont[41]. It has been isolated from orchids in Asia[42,43], Australia[44,45], Europe[46] and South America[47,48]. However, the definition of T. calospora species is still unclear, since phylogenies have shown taxonomic problems concerning this species[35]. In Brazil, T. calospora was obtained from the roots of the orchids Oeceoclades maculata[29], Epidendrum secundum, Acianthera limae and Polystachya concreta[48] in the Zona da Mata and Quadrilátero Ferrífero regions of the state of Minas Gerais. Herein, T. calospora was isolated from C. caulescens and C. cinnabarina roots also sampled in the Quadrilátero Ferrífero region. These results suggest that T. calospora is a nonspecific orchid symbiont broadly distributed in the studied region.

The present study also yielded information for four species, which likely are only a small fraction of the unknown Tulasnella species diversity. Tulasnella hadrolaeliae and T. brigadeiroensis are mycorrhizal fungi isolated from pelotons in the roots of H. jongheana, an endangered epiphytic orchid. Tulasnella brigadeiroensis was collected at two different times: first (February 2018) just one isolate was obtained, and second (October 2019) two additional isolates of the new species T. brigadeiroensis were collected. Tulasnella zygopetali and T. orchidis were isolated from pelotons from the same individual of Zygopetallum maxillare. Zygopetalum maxillare is an epiphytic orchid with high specificity in a host tree relationship[7]. In PESB, Z. maxillare grows exclusively on the stems of tree ferns.

The new Tulasnella species studied here were described using a polyphasic approach. Phylogenetically, T. hadrolaeliae formed a sister clade with T. albida and T. pruinosa. However, the definition of the phylogenetic species of T. albida cannot be confirmed due to the absence of molecular data from the type specimen[49]. Additionally, morphological characters cannot distinguish T. albida and T. pruinosa[34]. Therefore, as for T. calospora, molecular data from the type specimen are required to confirm the delimitation of the species T. albida and T. pruinosa[49].

Tulasnella brigadeiroensis and T. orchidis formed well-supported sisters clades. Tulasnella brigadeiroensis and Tulasnella sp. COAD 2885 showed high percentage sequence divergence between clades (7.5%). This value is higher than the 3% sequence divergence cut-off value proposed for species delimitation[50] or 3–5% divergence used for Tulasnella species[38]. Regarding the other new species described here, the interspecific nucleotide divergence ranged from 5.4 to 41.6%. These values are comparable to or even higher than those found in previous studies on Tulasnella[33,34,38].

Knowledge of the diversity of orchid mycorrhizal fungi is important for successful conservation strategies[4], together with their maintenance in culture collection. Our study contributes to the description of diversity of Tulasnella associated with orchids of the Brazilian Atlantic Forest, which is relevant for conservation of these orchids and for knowledge of fungal richness in this hotspot of biodiversity. Further studies are required to verify the potential of new species to support seed germination, seedling development and, consequently, orchid conservation programs.

Conclusions

Phylogenetic analyses, integrated with nucleotide divergence and morphological characteristics, showed the diversity of Tulasnella species associated with orchids of the Brazilian Atlantic Forest, including the description of four novel Tulasnella species. This is the first study using a polyphasic approach to the description of Tulasnella in Brazil, and it suggests that further studies will uncover more diversity. The cultivation of these species may help the strategies of conservation of endangered Brazilian orchids.

Methods

Sample collection and isolates

Root samples of the epiphytic orchid H. jongheana were collected from the PESB (Araponga – MG, Brazil) and PESN (Itamarandiba – MG, Brazil) (Fig. 7). Zygopetalum maxillare samples were also obtained from PESB, while C. cinnabarina and C. caulescens were sampled from iron mining areas in the Quadrilátero Ferrífero region (Mariana – MG, Brazil) (Fig. 7). Apparently healthy roots were analyzed at the Laboratório de Associações Micorrízicas (DMB/UFV). The root samples were gently washed under running tap water, cut into pieces of transversal root fragments, 2–3 mm thick, surface-sterilized in 70% ethanol for 1 min, 2% sodium hypochlorite for 3 min, followed by two successive rinses of sterile distilled water. These fragments were then examined under a stereomicroscope, after slicing into several thin transversal slices. Cells containing pelotons were placed on potato dextrose agar (PDA) medium without antibiotics and then incubated at 25 °C in the dark. Axenic cultures were preserved on rice grains in an ultrafreezer at −72 °C or silica gel and were deposited in the Coleção Oswaldo Almeida Drummond collection (COAD) at the Universidade Federal de Viçosa. Representative specimens were deposited at the Fungarium of the Universidade Federal de Viçosa (VIC).

Figure 7

Investigated orchids: (a), flower of Hadrolaelia jongheana; (b), Zygopetalum maxillare; (c), flower of Cattleya cinnabarina; (d), flower of Cattleya caulescens.

Morphology

The fungus and colony characteristics were described from cultures grown on PDA at 25 °C in the dark for 1–4 weeks depending on their growth rate. Measurements of colony diameters were taken using digital calipers. Color terminology followed Rayner[51]. The nuclear condition was observed from young hyphae after staining with SYBR Green I according to Meinhardt et al.[52]. The isolates were transferred to Corn Meal Agar (CMA) medium and incubated at 25 °C in the dark, for 4–6 weeks, to induce monilioid cell formation[29]. Observations, measurements and photographic images of microscopic fungal structures were recorded using an Olympus BX53 light microscope, with an Olympus Q-Color5TM digital high-definition color camera and differential interference contrast (DIC) illumination. Adobe Photoshop CS5 was used for the final editing of the acquired images and photographic preparations.

DNA extraction, PCR amplification and sequencing

The genomic DNA was extracted from fungal mycelia grown on PDA at 25 °C for 4 weeks, using the Nucleospin® Soil (MACHEREY-NAGEL GmbH & Co. KG), in accordance with the manufacturer’s instructions. The nuclear ribosomal internal transcribed spacer (ITS) region was amplified using primer pairs ITS1 and ITS4[53]. Each polymerase chain reaction (PCR) was performed in 50 µL containing 10–20 ng of DNA template, 1× Taq buffer, 2 mM MgCl2, 0.2 μM of each primer, 0.4 mM of each dNTP, and 1.0 U Taq DNA polymerase (Cellco Biotec do Brasil Ltda, São Paulo, Brazil). PCR was carried out using a MyCyclerTM Thermal Cycler (Bio-Rad Laboratories B.V., Veenendal, The Netherlands) with an initial denaturation at 95 °C, for 2 min, followed by 39 PCR cycles (denaturation at 95 °C for 1 min; annealing at 50 °C for 1 min; extension at 72 °C for 1 min) before a final extension at 72 °C for 10 min.

The PCR products were visualized on 1% agarose gels stained with ethidium bromide to assess product size and quality, purified and then sequenced from the two strands using the primers ITS1 and ITS4[53]. Consensus sequences were generated using the MEGA v.7.0.26 software tool[54]. All sequences were checked manually, and nucleotides with ambiguous positions were clarified using both primer direction sequences. The sequences were deposited in GenBank (see accession numbers in Table 1).

Phylogenetic analyses

Consensus sequences were compared against NCBI’s GenBank nucleotide databases by using the BLASTn algorithm. The most similar sequences were downloaded in FASTA format and aligned with our sequences by using the MAFFT v. 7 online portals[55]. The resulting sequence alignments were manually checked and adjusted in MEGA v.7.0.26 software tool[54].

Bayesian inference (BI) analyses employing a Markov Chain Monte Carlo method were performed on all sequences. Nucleotide substitution models were determined using the MrModeltest 2.3 program[56] and, once the likelihood scores had been calculated, the models were selected according to the Akaike information criterion (AIC). The results of MrModeltest recommended a GTR + G model for ITS, and a dirichlet (1,1,1,1) state frequency distribution and a gamma distributed rate variation were set. The phylogenetic analysis was performed using the CIPRES web portal[57] and the MrBayes program v.3.1.1[58]. Two sets of four MCMC chains were run simultaneously, starting from random trees for 1,000,000 generations and sampling every 1,000th generation. The first 25% of the trees were discarded as the burn-in phase for each analysis. Posterior probabilities[59] were determined from the remaining trees and are presented on the left of each node. Maximum likelihood (ML) analysis was implemented using the RAxML-HPC v.8 on XSEDE (8.2.12) available on the CIPRES web portal. Parameters for maximum likelihood were set to rapid bootstrapping and the analysis was carried out using 1000 replicates. Alignments and trees were deposited in TreeBASE (http://treebase.org/treebase-web/) (25158). The trees were visualized in FigTree V1.4.4[60] and the layout of the tree for publication was done using Adobe Illustrator v. CC.

Divergence between clades and haplotype network

In order to assess the sequence divergence between and within the clades obtained in the phylogeny tree, the Kimura-2-parameter distances were calculated as implemented in MEGA v.7.0.26[61]. The analysis involved 85 nucleotide sequences. All positions containing gaps and missing data were eliminated. There was a total of 272 positions in the final dataset.