﻿Taxonomic study of Collybiopsis (Omphalotaceae, Agaricales) in the Republic of Korea with seven new species

﻿Abstract Collybiopsis is a genus of the gymnopoid/marasmioid complex of the family Omphalotaceae. The classification system of Collybiopsis has recently undergone large changes through molecular approaches. The new classification system has not been applied for Collybiopsis in the Republic of Korea, and general research on this genus was also lacking. In this study, we analyzed the Collybiopsis species in the Republic of Korea by assessing all gymnopoid/marasmioid specimens collected nationwide for ten years by combining morphological approaches and multilocus (ITS + nrLSU) phylogenetic analysis. We thus confirmed that 16 species of Collybiopsis are present in the Republic of Korea, including two previously unreported species (Co.nonnulla and Co.dichroa) and seven new species (Co.albicantipessp. nov., Co.clavicystidiatasp. nov., Co.fulvasp. nov., Co.orientisubnudasp. nov., Co.subumbilicatasp. nov., Co.undulatasp. nov., and Co.vellereasp. nov.). A thorough examination of the Collybiopsis suggested that it is difficult to distinguish or identify the species based on morphological characteristics only; a combined molecular approach is needed for accurate identification. The Collybiopsis database of the Republic of Korea is updated, and information on the new species is provided. Five new combinations from Marasmiellus to Collybiopsis are also proposed (Co.istanbulensiscomb. nov., Co.koreanacomb. nov., Co.omphalodescomb. nov., Co.pseudomphalodescomb. nov., and Co.ramuliciolacomb. nov.).

Collybiopsis is morphologically similar and phylogenetically close to Gymnopus (Desjardin et al. 1999;Mata 2002;Dutta et al. 2015). Both genera are reported to be distinguishable through like types of the terminal element of pileipellis, attachment of lamellae, the character of stipe, basidiospores, and cheilocystidia. However, as the characteristics of each genus cannot be seen as absolute because exceptions exist, and some characteristics overlap, it is difficult to distinguish Collybiopsis from Gymnopus solely on morphology. Furthermore, the morphological characteristics of their basidiomata vary greatly depending on the environment and developmental stage. Therefore, molecular data play an important role in distinguishing these genera (Antonín and Herink 1999;Hughes et al. 2014;Hughes and Petersen 2015).
Although there have been many taxonomic changes for gymnopoid/marasmioid species, these changes have not been reflected in the gymnopoid/marasmioid species in the Republic of Korea. Since the first report of Collybiopsis confluens (Pers.) R.H. Petersen, as its previous name Collybia confluens Fr. (Kaburagi 1940), nine current Collybiopsis species have been reported until recently (National list of species of Korea 2020). However, they were identified and classified as Collybia, Gymnopus, and Marasmiellus based on their macroscopic morphological features. Owing to the uncertain placement of previous morphologically identified collybioid collections, it was necessary to re-examine Korean collections of collybioids and marasmioids based on molecular data. In this study, we investigated gymnopoid/marasmioid specimens collected over 10 years and deposited in three Korean herbaria based on their molecular analysis. As a result, we provide a list of Collybiopsis species in the Republic of Korea with seven new species.

Collections of specimens
A total of 372 specimens deposited in three Korean fungal herbarium -Seoul National University Fungus Collection (SFC), Korea National Arboretum (KA), and the National Institute of Biological Resources (NIBR) -were used in this study. The specimens were collected from 2012 to 2021 and stored in dried condition. All specimens were identified based on their morphological characteristics by each herbarium. The collection information (e.g. collection date, collection site, collector, etc.) and the notes of fresh basidiomata of each specimen were provided from each herbarium.

Molecular analysis
Genomic DNA was extracted from each specimen using a modified CTAB DNA extraction protocol (Rogers and Bendich 1994). The primer set ITS1F/ITS4B (Gardes and Bruns 1993) was used to amplify the internal transcribed spacer (ITS) region for all specimens, and the primer set LR0R/LR5 (Vilgalys and Hester 1990;Rehner and Samuels 1994) was used to amplify the nuclear large subunit ribosomal RNA (nrLSU) region. PCR was conducted by a C1000 thermal cycler (Bio-Rad, Richmond, CA, USA) using AccuPower PCR master premix (Bioneer Co., Daejeon, the Republic of Korea). PCR conditions for ITS and nrLSU region were: 5 min initial denaturation at 95 ˚C followed by 35 cycles of 40 s at 95 ˚C, 40 s at 55 ˚C and 60 s at 72 ˚C with a final extension step for 7 min at 72 ˚C. The amplifications of the PCR products were verified by visualization using 1% agarose gels with EcoDye DNA staining solution (SolGent Co., Daejeon, the Republic of Korea). The PCR products were purified using the ExpinTM PCR Purification Kit (GeneAll Biotechnology, Seoul, the Republic of Korea) following the manufacturer's instructions. The purified PCR amplicons were sequenced using an ABI Prism 3700 Genetic Analyzer (Life Technologies, Gaithersburg, MD, USA) at Macrogen (Seoul, the Republic of Korea).
All sequences generated in this study were proofread using MEGA version 7 (Kumar et al. 2016). The sequences used for analyses were deposited in GenBank (Table 1). We then selected the closely related sequences from NCBI databases mainly referred to Oliveira et al. (2019) and Petersen and Hughes (2021). After retrieving all published ITS and nrLSU sequences of all Collybiopsis species in GenBank, phylogenetic analyses were performed together with new sequences generated from specimens. The sequences were respectively aligned for each loci using Multiple Alignment Fast Fourier Transform (MAFFT ver. 7) with the L-NSI-I option algorithm (Katoh and Standley 2013). The aligned sequence data were manually checked and edited. The final sequence of each specimen was created as a concatemer by manually attaching the aligned sequences of the two loci. Maximum likelihood (ML) phylogenetic tree was constructed on the CIPRES Science Gateway (Miller et al. 2012) using the GTR+GAMMA model with 1000 bootstrap replicates. Rhodocollybia butyraceae Lennox (TFB14382), Rhodocollybia dotae JL Mata and Halling (REH7007), and Rhodocollybia maculate Singer (TFB13989) were used as outgroups . Bootstraps higher than 70% were considered to support a clade and are shown in the tree (Figure 1).

Morphological observation
All specimens were preliminarily observed and macro/micro-structures of two to four representative specimens, which were in the best condition among the specimens, were presented in figures. Photographs and notes of fresh basidiomata taken at the time of collection were used for macro-morphological description. For micro-morphological observations, tissues of dried specimens were rehydrated in 5% (w/v) KOH and mounted in Congo red solution (Clémençon 1973) and Melzer's reagent. The observation was performed by using a Nikon Eclipse 80i optical microscope (Nikon, Tokyo, Japan) at 20 × to 1000 × magnification. More than thirty basidiospores and more than twenty other microstructures (e.g., basidia, cheilocystidia, etc.) were measured to analyze the microstructures based on the microscopic pictures of specimens stained with Congo red. The Methuen Handbook of Colour (Kornerup and Wanscher 1978) was used for color indications. The following abbreviations and acronyms were used: Co = Collybiopsis; G = Gymnopus; Ma = Marasmiellus; L = the number of complete lamellae; l = the number of lamellulae tiers between neighboring complete lamellae; and Q = the values of the length divided by the width of basidiospores (Petersen and Hughes 2021;Ryoo et al. 2020).

Results
Through ITS sequence analysis of 372 gymnopoid/marasmioid specimens, 201 specimens were confirmed to belong to Collybiopsis. The remaining 160 specimens were identified as members of the following genera: Gymnopus, Marasmius, or Rhodocollybia and were excluded from this study. A total of 201 specimens were segregated into 16 putative taxa based on ITS phylogenetic analyses (Table 2). To confirm the species' identity and to infer the phylogenetic relationships within Collybiopsis, the nrLSU region was amplified and sequenced from 47 representative specimens of 16 taxa ( Table  1). The final phylogenetic analyses were conducted with datasets of two loci from 16 Collybiopsis species (Table 1). In ML analysis, 178 multigene sequences (110 for ITS and 68 for nrLSU) were retrieved from GenBank and used. The adjusted alignments comprised 535 to 794 bases for ITS and 324 to 904 bases for nrLSU. The phylogenetic analysis results of the two combined loci revealed that Collybiopsis specimens from the Republic of Korea were identified as 16 taxa ( Fig. 1   Diagnosis. This species notably has hemispherical to convex, 4-23 mm pileus, distant lamellae, central to eccentric, tomentose, 5-15 × 0.5-1.5 mm stipe with a white base; ellipsoid to ovoid, 5.8-7.4 × 2.8-4 μm basidiospores, clavate (often constricted), 25.5-34.8 × 4.8-6.7 μm basidia, broadly clavate, irregular, sometimes lobed, 26-49 × 5.4-10.6 μm cheilocystidia, and a habit of fruiting on branches.
Habit and habitat. Scattered to gregarious on the branch in mixed forest dominated by Camellia japonica Linne, in summer.
Habit and habitat. Solitary to scattered on dead wood debris of conifers, in summer.
Other Habit and habitat. Scattered or gregarious on the bark of deciduous trees or on the rotting branch of both broadleaf trees and conifers, in summer.
Habit and habitat. Scattered to gregarious on leaf litter in mixed forest dominated with broadleaf trees, in summer.

Proposal for Collybiopsis recombination
In this study, many epithets were found that required an additional transfer of species from Marasmiellus to Collybiopsis apart from the study done by Petersen and Hughes (2021). Oliveira et al. (2019) had previously suggested to replace these species from Gymnopus to Marasmiellus s. str., but this study suggests that these species should be further transferred from Marasmiellus s. str. to Collybiopsis. Taxonomic key to Collybiopsis in Korea

Discussion
Of the 372 gymnopoid/marasmioid specimens, we confirmed 201 specimens (54%) to belong to Collybiopsis. These results indicate that the species of Collybiopsis can be confused with those of similar genera as well as with other Collybiopsis members when identification is based solely on morphological information. This is because some characteristics are overlapped between species (Suppl. material 2: Fig. S2) and the characteristics can be different depending on developmental stage or environmental conditions. Further, the high misidentification ratio may be caused by the slow rate of adoption of the current names. Sequence-based taxonomy has introduced rapid changes in the classification of gymnopoid/marasmioid species (Mata 2002;Mata et al. 2004a;Mata et al. 2004c;Hughes et al. 2010;Oliveira et al. 2019;Hughes 2017, 2021). As such, taxonomic confusion has been resolved in taxa that have been well researched based on molecular data (Desjardin et al. 1999;Mata 2002;Lee et al. 2019).
Nine of the sixteen Collybiopsis species were identified as already known species. Of the nine described species, seven species were identified as the species previously recorded in the Republic of Korea: Collybiopsis biformis, Co. confluens, Co. koreana, Co. luxurians, Co. menehune, Co. polygramma, and Co. ramealis. Two species, Co. dichroa and Co. nonnulla, were reported for the first time in the Republic of Korea. Most of the nine described species formed a monophyletic clade with each corresponding species. However, sequence variations by continent were detected in Co. biformis, Co. confluens, Co. dichroa, and Co. nonnulla. Asian samples, including our specimens, were clearly separated from those of Europe, North America, and Africa. These results have also been reported in previous studies on Collybiopsis biformis (Mata 2002;Petersen and Hughes 2014;Razaq et al. 2020) and Co. confluens (Hughes and Petersen 2015). Especially, Co. confluens is known as a representative example of intra-specific variation between continents. Percent ITS sequence divergence of this species was reported to be 3.25% when comparing the sequences of the North America and Europe (Hughes and Petersen 2015). We confirmed that percent ITS sequence divergence of Asian Co. confluens (our Korean samples and Chinese sequences) were each about 3% when compared to American and European sequences.
Similarly, Co. dichroa showed sequence variations that were previously reported in association with intraspecific hybridization and dramatic sequence variations including frequent nucleotide substitutions of Adenine and Guanine . The Korean Co. dichroa was closely related to Co. dichroa taxa 2 mentioned in . Similarly, the intraspecific genetic variation depending on environmental conditions or geographical distribution has been reported in many other fungal species (Manian et al. 2001;Kauserud et al. 2007;Seierstad et al. 2013). For the last, Korean Co. nonnulla showed high intra-specific divergence when matching with sequences of Co. nonnulla of America and Cameroon. According to the phylogenetic analysis results, there is a slight sequence variation, but it forms a clade supported by a high bootstrap and morphologically almost coincides with the reference. Therefore, we view this sequence variation as due to different environments by continent and identify the specimens as Co. nonnulla. Nevertheless, compared to the fact that it was reported as a new species a long time ago, only seven sequences were deposited in the NCBI, so further study on this species is necessary.
Morphologically, the morphological characteristics of the seven described species were also in agreement with the previous descriptions (Suppl. material 2: Fig. S2). However, Co. luxurians and Co. polygramma found in the Republic of Korea showed few differences compared to the Western descriptions in the previous literature (Mata 2003;Noordeloos et al. 1999). In the case of the Co. luxurians, Korean sequences formed a slightly distinct clade in the phylogenetic tree, along with the Chinese sequence (ZD16102301), from European sequences. In this study, direct morphological comparison studies with European and Chinese samples were difficult and there was no significant morphological difference from the references. For these reasons, we identified Korean specimens as Co. luxurians, but further studies are needed with more samples from other countries for this species.
Seven new species have common characteristics of Collybiopsis such as insititious to subinsititious stipe, ellipsoid to oblong, inamyloid basidiospores, and presence of caulocystidia. However, it is difficult to distinguish them from other Collybiopsis species based on morphological characteristics alone. Upon molecular phylogenetic analyses, each of them clearly formed a distinct clade clearly in the ML phylogenetic tree (Fig. 1). Their morphological features may or may not be distinguished from their phylogenetically close relatives. The morphological differences between new species and morphologically similar or phylogenetically close species are discussed in the remarks for each species.
Two species previously reported in the Republic of Korea, Co. peronata (Cho & Lee, 1979) and Co. subnuda (National list of species of Korea 2020), were not confirmed in this study. Co. peronata and Co. subnuda, which are typical collybioid mushrooms, have been reported in Asia based on their morphological characteristics (Cho and Lee 1979;Kim et al. 1991;Park and Cho 1992;Yoshida and Muramatsu 1998;Tolgor and Yu 2000). Molecular analyses showed that none of the Korean specimens examined in this study could be identified as Co. peronata nor Co. subnuda. Instead, the specimens labelled as Co. peronata or Co. subnuda were identified as different species -Gymnopus similis Antonín, Ryoo & Ka and Co. orientisubnuda. Collybiopsis peronata were originally mostly reported from Europe and America and Co. subnuda were originally reported from America (Desjardin et al. 1999;Mata et al. 2006). Furthermore, there have been no recent sequence uploads to GenBank or reports of Co. peronata and Co. subnuda from Asia, making it difficult to confirm whether they exist in the Republic of Korea. Although Co. orientisubnuda is closely related to Co. peronata and Co. subnuda, there are clear differences in the ITS regions of these three species (Suppl. material 3: Fig. S3). Morphologically, Co. orientisubnuda is highly similar to Co. subnuda and considerably different from Co. peronata. The detailed comparisons of the morphological features are provided in the remarks for each species.
In conclusion, we identified 16 Collybiopsis species in the Republic of Korea through morphological and molecular analyses and we update the Korean inventory of Collybiopsis. Our study showed that the identification of Collybiopsis species requires both morphological and molecular analyses. Further, this study has the following significance as in the previous study conducted by Petersen and Hughes (2021): additional combinations of Marasmiellus species under Collybiopsis, detailed morphological characterization of Collybiopsis species in the Republic of Korea along with photographs and drawings, and specific approaches to species differentiation and identification through morphological and molecular analyses. Furthermore, we believe that this study will be helpful for further studies such as research of Collybiopsis distribution worldwide as it provides additional molecular information about Collybiopsis in the Republic of Korea and proposes seven new species identified from the Republic of Korea. These data will be useful for the identification and taxonomic arrangement of gymnopoid/marasmioid mushrooms.