Microglossum capitatum: taxonomy and conservation

Abstract

Microglossum capitatum was originally described based on specimens collected from tropical China. The name was previously considered a synonym for M. longisporum due to similar ascospore characteristics. A recently described species, M. macrosporum, from northern Thailand is very similar to M. capitatum in morphology. Using four recently collected samples from the type locality of M. capitatum, the systematic relationship and taxonomic position of this species were reanalyzed based on morphological and three-locus evidences. Our data suggested that M. longisporum is clearly different from M. capitatum by its cinnamon-brown clavate apothecia and subtropical to temperate ecological habitats. Further studies indicated that M. capitatum and M. macrosporum belong to the same species, and, thus, the latter should be treated as a synonym of the former. Our field observations suggested that M. capitatum has been facing severe threats of habitat loss and degradation, and it is urgent to protect this species.

Earth tongue fungi generally refer to those inoperculate discomycetes that have terrestrial, club-shaped and stipitate ascomata, and include species of genera such as Geoglossum Pers., Trichoglossum Boud., Microglossum Gillet, Leotia Pers., and Cudonia Fr. (Persoon, 1794; Schumacher & Sivertsen, 1987; Spooner, 1987; Wang et al., 2006a). These fungi are widely distributed from peat bogs to grasslands and sandy habitats (Fedosova et al., 2018; Kučera & Lizoň, 2012; Kučera et al., 2014a, 2014b, 2021).

Earth tongues exhibit a variety of trophic modes, encompassing the widely recognized saprophytic lifestyle as well as the mycorrhizal symbiosis recently identified in various studies (Baba et al., 2021; Baba & Hirose, 2023; Kučera et al., 2017; Nitare, 1984; Ohenoja, 1995; Wang et al., 2006a). They have high environmental requirements and are often regarded as indicator species of environmental quality; thus, they are of great conservation significance. Hustad et al. (2013) have noted that Geoglossomycetes Zheng Wang, C.L. Schoch & Spatafora species are representative of undisturbed grasslands. The presence of earth tongues, along with associated fungi, is deemed a clear sign of grassland health (Genney et al., 2009; Hustad et al., 2013; Newton et al., 2003). With changes in habitat conditions, especially in land-use patterns, earth tongue fungi are experiencing serious threats in many parts of Europe, and an increasing number of them are included on the IUCN Red List (Gärdenfors, 2010; Jordal, 2019; Kålås et al., 2010; Senn-Irlet et al., 2007). For example, the habitat of M. atropurpureum (Batsch) Pers. has declined by 30%-50% over the last 50 y, and seven species of Geoglossaceae Corda adapted to grasslands have experienced a 90% decline in western Europe over the last 75 y, primarily as a result of land-use changes (Griffith et al., 2013; Jordal, 2019).

Historically, earth tongue fungi have been assigned to the genus Geoglossum, which belongs to the class Leotiomycetes O.E. Erikss. & Winka and the family Geoglossaceae, based on morphological features (Korf, 1973; Persoon, 1794; Spooner, 1987). However, the genus has undergone several revisions (Geesteranus, 1964; Imai, 1941; Korf, 1973; Pfister & Kimbrough, 2001; Schoch et al., 2009; Spooner, 1987; Wang et al., 2006a). With the wide application of molecular approaches, it has been revealed that the Geoglossaceae is not monophyletic. Geoglossum, Trichoglossum, and Sarcoleotia S. Ito & S. Imai form a separate monophyletic clade (Schoch et al., 2009; Wang et al., 2006a, 2006b). Schoch et al. (2009) erected the class Geoglossomycetes and the order Geoglossales Zheng Wang, C.L. Schoch & Spatafora, and the family Geoglossaceae was included in the new class (Hustad et al., 2013; Schoch et al., 2009). However, Microglossum, Spathularia Pers. and Cudonia, previously treated in the Geoglossaceae, the former was placed in the family Leotiaceae Corda (Leotiomycetes) and the latter two were placed in the family Cudoniaceae (Rhytismatales) (Ge et al., 2014; Gernandt et al., 2001; Hustad et al., 2013; Johnston et al., 2019; Schoch et al., 2009; Wang et al., 2006a).

The genus Microglossum was established by Gillet (1879) to accommodate G. viride (Schrad. ex J. F. Gmel.) Pers. and G. olivaceum Pers., which have green ascomata. Subsequently, Saccardo (1884) attributed the species of earth tongues with white spores to Microglossum. Durand (1908) selected M. viride as the lectotype of the genus. Microglossum is characterized by club-like fruiting bodies, blue ascus pore iodine reactions, and often multiguttulate, hyaline, and small ascospores (Zhuang, 2004).

Within the genus Microglossum, a few species may develop two types of ascospores, such as M. longisporum E.J. Durand, M. tetrasporum F.L. Tai, M. capitatum and M. macrosporum Ekanayaka & K.D. Hyde. The former was described from and is distributed in North America (Durand, 1908; Mains, 1955), while M. tetrasporum and M. capitatum were described from tropical Yunnan, southwestern China (Tai, 1944) and M. macrosporum was originally described from northern Thailand (Ekanayaka et al., 2019). Zhuang and Wang (1997) and Zhuang (2004) suggested M. capitatum, M. tetrasporum, and M. longisporum should be considered as the same species. However, further differentiation among the four species requires the utilization of morphological characteristics and molecular data.

During the field survey of macrofungi in tropical Yunnan, we have made several collections of Microglossum, which is very similar to M. macrosporum. This study explores: 1) the taxonomic relationships of M. longisporum, M. capitatum, and M. macrosporum based on four new samples collected from the type locality of M. capitatum and morphological characteristics of M. longisporum and M. macrosporum have been reported previously (Durand, 1908; Ekanayaka et al., 2019; Mains, 1955); 2) the threats currently facing M. capitatum and the necessary conservation actions.

Type specimen of M. capitatum were collected by H. S. Yao from Jinghong in 1939, and four more samples were collected near the type localities in 2019 (101.568583 °E, 21.93866111 °N; alt. 843 m). Additionally, one specimen was collected from Mengla in 1988 and was subsequently identified as M. longisporum by Zhuang (2004). Macromorphological characteristics and habitat information were recorded based on the fresh apothecia in the field. DNA extraction was based on silica-dried material and specimens. The specimens were deposited in the Herbarium of Cryptogams at the Kunming Institute of Botany, Chinese Academy of Sciences (KUN-HKAS). The microscopic characteristics of the specimens were observed under a light microscope. The dried materials were sectioned and rehydrated in 5% NH_3·H₂O. The microscopic characteristics were observed using a Nikon ECLIPSE 80i compound microscope and photomicrographs were captured using a Canon 450D digital camera. The paraphyses, asci, and ascospores were measured from materials mounted in water, and the mean values were used in the descriptions. Apical amyloid of the asci was observed in Melzer's reagent.

Genomic DNA was extracted from herbarium specimen and four new samples of M. longisporum and M. capitatum collected in 1988 and 2019, respectively, using an Ezup Column Fungi Genomic DNA Purification Kit (Sangon Biotech, Shanghai, China). The polymerase chain reactions (PCR) system of 25 μL was selected for amplification: BioTaq master mix (BioTeke, Beijing, China) 12.5 μL, forward primer and reverse primer 1 μL each, DNA template 1 μL, and ddH₂O up to 25 μL. Three fragments, the large subunit nuclear ribosomal RNA (nrLSU), using LROR/LR5 primers (Vilgalys & Hester, 1990), the internal transcribed spacer (ITS), using ITS1/ITS4 primers (White et al., 1990), and the second largest subunit of RNA polymerase II (rpb2), using fRPB2-5F/fRPB2-7cR primers (Liu et al., 1999), were amplified. The amplified products were viewed on 1% agarose gel and electrophoresis was conducted in 1.0 × TAE buffer and were sent to Kunming Qingke Bio-Engineering Co,. Ltd for sequencing.

The 12 newly obtained sequences in this study have been deposited in GenBank. Eighty-nine sequences belonging to Microglossum species and the outgroup taxon Mitrula paludosa Fr. were retrieved from GenBank and included in the analyses (Table 1, newly generated sequences are in boldface). Consensus DNA sequences for each gene were edited, aligned using Multiple Alignment using Fast Fourier Transform v.7.490 with Q-INS-i strategy (http://mafft.cbrc.jp/alignment/server/index.html), and manually modified using BioEdit where necessary (Hall, 1999). Maximum likelihood (ML) phylogenetic analyses were performed using the randomized accelerated maximum likelihood method based on the combined nuclear dataset (Stamatakis et al., 2008). Bootstrap analysis for the ML tree was calculated with 1000 replicates using the GTR+I+G substitution model. ML bootstrap values equal to or exceeding 50% are labeled above the nodes in Fig. 1. The tree obtained from the phylogenetic analyses was viewed with FigTree v.1.4.2. and modified manually with Adobe Illustrator 2021. The combined aligned data matrix was deposited to the TreeBASE (https://treebase.org/) under the accession number TB2:S31563 for the tree in Fig. 1.

Table 1. GenBank accession numbers for sequences used in phylogenetic analyses of Microglossum that retrieved from GenBank.

Species names	Strain number	GenBank accession
		ITS	nrLSU	rpb2
Microglossum aff. nudipes	SAV F-11053	KX382838	KX382867	KX382886
M. aff. nudipes	SAV F-11274	KX382836	KX382836	KX382888
M. aff. nudipes	SAV F-11285	KX382859	KX382869	KX382887
M. capitatum	HKAS135652	PP982470	PP968430	PP971758
M. capitatum	HKAS135653	PP982471	PP968431	PP971759
M. capitatum	HKAS135654	PP982472	PP968432	PP971760
M. capitatum	HKAS135655	PP982473	PP968433	PP971761
M. clavatum	SAV F-11276type	KX382864	KX382864	KX382884
M. clavatum	SAV F-11074	KX382865	KX382865	KX382885
M. fuscorubens	SAV F-11275	KX382834	KX382834	KX382883
M. griseoviride	SAV 9920 type	NR_132025	NG_060290	-
M. griseoviride	SAV 10699	KC595261	KC595262	-
M. macrosporum	MFLU 18-1830 type	-	MK591992	MK614724
M. olivaceum	SAV 9967	KC595255	KC595256	-
M. olivaceum	SAV 9902	KC595251	KC595252	-
M. parvisporum	SAV 10998 type	KM114901	KM114901	-
M. parvisporum	SAV F-11283	KX382860	KX382870	KX382878
M. pratense	SAV 11021	KJ513008	KJ513008	-
M. pratense	SAV 10567	KJ513007	KJ513007	-
M. pratense	SAV 11020	KJ513006	KJ513006	-
M. pratense	O 294564	KJ513005	KJ513005	-
M. pratense	O 64797	KJ513004	KJ513004	-
M. rufescens	SAV F-11204	KX382835	KX382866	KX382893
M. rufescens	SAV F-11282	KX382858	KX382868	KX382892
M. rufescens	Lueck19	KP965782	KP965798	-
M. rufescens	SAV 9921	KC595257	KC595258.2	-
M. rufum	AFTOL-ID 1292	-	DQ470981	DQ470933
M. tenebrosum	SAV F-11278 type	NR_148113	KX382845	KX382891
M. tenebrosum	SAV F-11070	KX382846	KX382846	KX382890
M. tenebrosum	SAV F-11072	KX382844	KX382844	KX382889
M. tenebrosum	SAV F-11279	KX382843	KX382843	-
M. tenebrosum	SAV F-11273	KX382842	KX382842	-
M. truncatum	SAV F-11280 type	KX382861	KX382861	KX382875
M. truncatum	SAV 11022	KJ513011	KJ513011	-
M. truncatum	O 224247	KJ513010	KJ513010	-
M. truncatum	SAV 11023	KJ513009	KJ513009	-
M. truncatum	SAV F-11262	KX382862	KX382862	KX382877
M. truncatum	LE 291847	KX382863	KX382871	KX382876
M. viride	SAV 10249 type	NR_132026	NG_060291	-
M. viride	SAV 10697	KC595265	KC595266	-
M. viride	23-VIII-97	AY144534	-	AY144499
Mitrula paludosa	MBH50636	AY789424	AY789423	-
Mitrula paludosa	113754	AY789320	AY789319	-

Newly generated sequences are in bold, authentic specimens are highlighted with type. The absence of corresponding DNA sequence information is indicated by -.

Fig. 1 - Maximum likelihood phylogenetic tree generated from a three-locus (ITS, nrLSU, and rpb2) dataset of the Microglossum species. ML bootstrap (BS > 50%) are shown above or below the branches. Voucher specimens are provided. Microglossum capitatum is in bold.

DNA was successfully extracted from all specimens, and three loci (ITS, nrLSU, and rpb2) were successfully amplified and sequenced. One specimen of M. capitatum collected in 1988 and treated as M. longisporum by Zhuang (2004) was too degraded to be successfully extracted; thus, only microscopic observations were made. In addition, 40 sequences representing 14 taxa were downloaded from GenBank for phylogenetic analysis (Table 1).

The aligned dataset comprised 2149 characters: a phylogenetic tree for Microglossum was generated based on this dataset. The resulting tree solved the relationship between M. capitatum and M. macrosporum, which clustered together.

Microglossum capitatum F. L. Tai, Llodia 7 (2): 147, figs. 2, 17, 1944.

Synonyms: Ochroglossum capitatum (F.L. Tai) S. Imai, Sci. Rep. Yokohama Natl. Univ., Sect. 2 4: 7, 1955; Microglossum tetrasporum F.L. Tai, Llodia 7(2): 147, figs. 1, 16, 1944; Ochroglossum tetrasporum (F.L. Tai) S. Imai, Sci. Rep. Yokohama Natl. Univ., Sect. 2 4: 8, 1955; M. macrosporum Ekanayaka & K.D. Hyde, Mycosphere 10 (1): 408, fig. 46, 2019

Index Fungorum number: IF 288355.

Etymology - refers to capitate fertile ascogenous part.

Holotype - China, Yunnan Province, Jinghong City, 10 Aug 1939, H.S. Yao 7422 (HMAS 3422).

Epitype - The type specimen, collected >85 y ago, complicates DNA sequencing for phylogenetic analysis. Therefore, we designate here L.K. Jia 412 (HKAS 135652), found near the type locality, as epitype.

Description: Ascomata 10-30 mm high, clavate-capitate, stipitate. Ascogenous portion 7-15 mm long and 5-10 mm wide, capitate to flattened capitate, or with depressed areas, smooth and glabrous; surface dull yellow to brownish yellow when fresh, becoming brown when dry. Stipe 10-20 mm long and 3-5 mm wide, cream-colored to yellowish, nearly glabrous, clearly distinguishable from ascogenous portion (Fig. 2A, B).

Fig. 2 - Morphology of Microglossum capitatum (KUN-HKAS135653, photos by Gen-Shen Wang). A, B: Apothecia. C: Cross section of an apothecium. D: Filiform paraphyses. E: Amyloid ascus apices. F-H: Cylindric-clavate asci. I-K: Cylindrical big-ascospores. L-P: Cylindrical to shortly-clavate small-ascospores. Bars: C 100 μm, D, E, I-K 25 μm, F-H 20 μm, L-P 5 μm.

Asci 90-125 × 10-12 μm ($\overline{x}$ = 110.3 × 14.8 μm, n = 30), cylindric-clavate, 8-spored, base with crozier, apex amyloid (Fig. 2E-H). Ascospores dimorphic, large-ascospores 4, lying side-by-side, multiguttulate and continuous firstly and 14-16 septate finally, 43-85 × 4-6(-7.4) μm ($\overline{x}$ = 62.5 × 5.6 μm, n = 60), ends rounded, cylindrical to curved, rarely straight, hyaline, multiguttulate (Fig. 2I-K); small-ascospores 4, located near the ascus apex, at first continuous and 2-4 septate finally, (8.3-)11-15(-19.8) × (2.5-)3-4 μm ($\overline{x}$ = 12.8 × 3.3 μm, n = 30), cylindrical to shortly-clavate, hyaline, multiguttulate, commonly located near the ascus apex (Fig. 2L-P). Paraphyses numerous, filiform, aseptate, apically enlarged and obtuse (Fig. 2D). Medullary tissue composed of thin-walled hyaline hyphae (textura intricata) (Fig. 2C).

Habitat and distribution: scattered on the ground in tropical seasonal rain forests dominated by Castanopsis or Lithocarpus species, mostly associated with mosses, such as Dicranum scoparium, Fissidens exilis, and Calypogeia fissa. Currently known from China's Yunnan, and Thailand's Chiang Rai.

Specimens examined:-CHINA. Yunnan Province: Xishuangbanna Dai Autonomous Prefecture, Mengla County, in tropical seasonal rainforest with Lithocarpus species, alt. 700 m, 20 Aug 1988, Z. L. Yang 636 (KUN-HKAS 21471); Jinghong City, on the ground in tropical seasonal rainforest with Castanopsis species near a rubber plantation, alt. 843 m, 25 Aug 2019, L. K. Jia 412 (KUN-HKAS 135652, epitype, designated here), G. S. Wang 732 (KUN-HKAS 135653), R. J. Xu 212 (KUN-HKAS135654), and M. Zeng 313 (KUN-HKAS 135655).

Species of Microglossum often have bright colors, including green, yellow or yellowish, but also dull colors such as brown to dark brown (Kučera et al., 2017; Kuntze, 1891; Ohenoja et al., 2010; Zhuang, 2004). Among them, M. capitatum and M. longisporum differ from other species within the genus in that they possess spores of two different sizes (Durand, 1908; Tai, 1944; Zhuang, 2004; Zhuang & Wang, 1997). The morphological characteristics of the ascomata, asci, and spores of the materials used in this study were identical to those described by Tai (1944) for M. capitatum (Table 2). The original description of M. capitatum included four spores, which align with the number of large spores observed in this study. Additionally, Tai (1944) noted the presence of smaller spores but did not specify their count.

Table 2. Morphologies of Microglossum longisporum, M. capitatum, M. tetrasporum, and M. macrosporum (Small-ascospores data for M. macrosporum not available in original literature, indicated by -).

Species names	Ascomata		Asci size (μm)	Ascospores (μm)		Paraphyses size (μm)	Reference
	Size (mm)	Color		Large-ascospores	Small- ascospores
M. longisporum	30-60	cinnamon to umber-brown	100-130 × 10-12	60-100 × 4-5	12-18 × 3	2.0	Durand (1908)
M. capitatum	30-35	smoky yellow	140-178 × 11-14	64-81 × 3-5	10-25 × 2-3	1.5-2.0	This study; Tai (1944)
M. tetrasporum	25-60	dark beaver	100-117 × 12-14	38-69 × 3.5-5.0	10-25 × 2-3	1.5-2.0	Tai (1944); Zhuang (2004)
M. macrosporum	10-20	greenish brown	85-105 × 10-12.5	50-75 × 4.5-5.5	-	2.5-3.2	Ekanayaka et al. (2019)

Previous studies treated M. capitatum, M. tetrasporum, and M. longisporum as the same species because they possess two types of ascospores, and their asci and ascospores are similar in size and shape (Zhuang, 2004; Zhuang & Wang, 1997). This study discussed the relationships between them using newly collected materials and it was observed that the size differences in the asci and spores between M. capitatum and M. tetrasporum fell within the range of normal intraspecific variation (Table 2). The asci and spores of both species were within the size range measured in this study; therefore, they should be treated as the same species.

However, significant morphological differences were observed between M. capitatum and M. longisporum. Microglossum capitatum has relatively smaller ascomata with dull yellow to brownish yellow capitate to capitate-clavate ascogenous portion and a cream-colored to yellowish stipe, whereas M. longisporum has larger ascomata with rich cinnamon-brown oblong to elliptical ascogenous portion and a cinnamon stipe with small scales (Durand, 1908; Mains, 1955; observations of the authors). Although both species have two types of ascospores, M. capitatum has four large ascospores lying side-by-side, and four small-ascospores located near the ascus apex, whereas M. longisporum typically had two large ascospores (rarely 3 or 4) and six small-ascospores near the ascus apex. In addition, M. capitatum has a typical tropical distribution range, while M. longisporum is distributed in subtropical to temperate ecological habitats in North America.

Phylogenetic analysis reveals that M. macrosporum, published by Ekanayaka et al. (2019), clusters together with M. capitatum with almost the same DNA sequences (Fig. 1). In the protologue of M. macrosporum, Ekanayaka et al. (2019) did not mention smaller ascospores, which might have been due to limitations in the available material. During the current observations, small-ascospores were not observed in some young specimens.

Field observations have shown that M. capitatum usually occurs at the edges of tropical seasonal rainforests at altitudes of 600-800 m and is associated with mosses. Combined with the work of Ekanayaka et al. (2019), this species has only been found in China's Yunnan and Thailand's Chiang Rai Province. To date, only ten specimens have been collected in the over 80 y since 1939 and nine distributed in China.

In the last 17 y, the natural forest is 71.07% of the total land (19124.5 km²) in 2002, while this was decreased to 51.98% in 2018 in Xishuangbanna (Zhang et al., 2019). Consequently, the geographic range of the species must be roughly decreased about 20% in the 17 y and severely fragmented. Considering the similar environmental conditions, occurrence of this species in Burma can also be anticipated. But the vegetation situations in Burma and Thailand are similar to those in Xishuangbanna. Half of the tropical Asian nations, including the aforementioned two ones, have already experienced severe (>70%) forest loss (Laurance et al., 2007). With the expansion of rubber plantations over the past few decades, the habitat of M. capitatum has become significantly compressed and this species have been facing severe threats of habitat loss and degradation (Fig. 2A). Based on the categories and criteria of IUCN (2012), we have assessed M. capitatum as “endangered” species [C2a(ii)]. Given the species' importance in indicating ecological conditions, it is urgent to raise awareness of the need to conserve this species.

Disclosure

The authors declare no conflicts of interest. All the experiments undertaken in this study comply with the current laws of the country where they were performed.

Acknowledgments

The authors are grateful to the curator of TENN for loan of specimens and color images of Microglossum longisporum for comparison. We thank Dr. Gregory M. Mueller, Chair of the IUCN SSC Fungal Conservation Committee, for his comments on the red list assessment of M. capitatum. We sincerely thank Dr. G.-S. Wang (Kunming Institute of Botany, Chinese Academy of Science, KIB, CAS), R.-J. Xu (King Saud University) and M. Zeng (King Saud University) for providing related materials and photos, thank Dr. Y.-B. Wang (KIB, CAS) and H.-F. Liao (Yunnan University) for the assistance in microscopic observations. We would like to thank Editage company for English language editing. This study was supported by the Yunnan Revitalization Talent Support Program: Science & Technology Champion Project (202305AB350004).

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