﻿New species and new records of Laccaria (Agaricales, Basidiomycota) from Northern Thailand

﻿Abstract Two new species Laccariapseudoalba and L.subroseoalbescens are described and illustrated, based on morphological characteristics and molecular phylogenetic analysis. Two new records, Laccariaumbilicata and L.yunnanensis from Thailand, are also reported. Laccariasubroseoalbescens is characterized by small basidiomata, stipe equal with an enlarged base, and nearly subclavate, pale pink to light orange. Laccariapseudoalba is characterized by pale orange to orange white pileus, has umbo when young on the pileus, and fistulose stipe of the pale to pastel red color. Phylogenetic analysis based on sequence data from rDNA internal transcribed spacer ITS1-5.8S-ITS2 rDNA (ITS), nuc 28S rDNA (28S), RNA polymerase II subunit 2 (rpb2), and translation elongation factor 1-α (tef1-α) are provided as further evidence. Molecular analysis confirms the phylogenetic positions of the two new species and two new records. The differences in characteristics of these two new species and closely related species are discussed herein.

Species of Laccaria are characterized by collybioid to omphaloid basidiomata; echinulate, acyanophilous, and inamyloid basidiospores; and a convex, plane, or umbilicate, hygrophanous pileus.Clamps are present in all parts of the basidiomata (Singer 1986;Mueller 1992;Latha and Raj 2019).Approximately 100 species of Laccaria have been described worldwide (according to Index Fungorum 2024), known to form symbiotic associations with plants of more than 20 genera (including Abies, Castanea, Fagus, Pinus, Picea, Quercus, Larix, Lithocarpus, and others) (Wilson et al. 2017).These associations benefit plant growth and facilitate effective nutrient acquisition (Smith and Read 2008).Therefore, studying Laccaria diversity is crucial for understanding terrestrial ecosystems and forest management.
Laccaria species are globally distributed and have been reported on every continent except Antarctica (Kropp and Mueller 1999).They have been found in association with both angiosperms and gymnosperms worldwide (Wilson et al. 2017) and form ectomycorrhizas (ECM) with many economically important plant species (Kropp and Mueller 1999).However, due to the morphological similarity among Laccaria species, defining species boundaries within the genus is challenging (Sheedy et al. 2013).
The number of Laccaria species described from Asia has been increasing, with more studies focusing on Basidiomycetes.Since 2013, twenty-three new species of Laccaria have been described in Asia (Wilson et al. 2013;Popa et al. 2014;Luo et al. 2016;Popa et al. 2016;Cho et al. 2018;Li 2020;Cui et al. 2021;Zhang et al. 2023).Nevertheless, no Laccaria species were reported or described in Thailand during the same period.
Thailand is renowned as one of the world's most important biodiversity hotspots with high fungal diversity (Hyde et al. 2018;Thongbai et al. 2018).During our recent investigation of Laccaria in Thailand, nine Laccaria specimens were collected.Based on morphological characteristics and phylogenetic analysis, two undescribed species and two new records have been identified.This paper provides detailed descriptions, illustrations, and phylogenetic analyses for these species.

Morphological study
Specimens were collected from Chiang Mai Province, Thailand.They were photographed in the field, then separately wrapped in aluminium foil or kept in a plastic collection box.The fresh basidiomata were macro-morphologically described on the same day of collection.Colour codes were determined following Kornerup and Wanscher (1978).After being thoroughly dried at 50 °C (Hu et al. 2022) in a food drier, the specimens were stored in sealed plastic bags and deposited in Mae Fah Luang University Herbarium (MFLU) and Herbarium of Cryptogams Kunming Institute of Botany, Academia Sinica (KUN-HKAS).Dried materials were sectioned under a stereo microscope, transferred onto slides, and mounted in a 5% KOH solution.For microscopic characteristics, anatomical and cytological characteristics including basidia, basidiospores, and cystidia, were observed and photographed using a Nikon Eclipse 80i microscope at magnifications up to × 1000.For SEM studies, fragments of the lamellae of the dried material were taken, sputter coated with gold, and analysis with a Hitachi S520 (Hitachi Japan).The notation [x/y/z] specifies that measurements were made on x basidiospores measured from y basidiomata of z collections.At least 50 basidiospores and 20 basidia were measured from one basidioma.Basidiospores dimensions are given as (a-) b-c (-d).Where "a" and "d" refer to the minimum and maximum values of all measurements, respectively, b-c presents the range of 95% of the measured values, and Q is the length/width ratio of basidiospores, Q m is the average Q of all basidiospores and is given as Q m ± standard deviation.

Sequence alignment and phylogenetic analysis
The newly generated sequences were checked using BioEdit Sequence Alignment Editor version 7.0.4 and assembled using SeqMan (DNAstar, Madison, WI, USA).The sequences were then blasted using the Basic Local Alignment Search Tool (BLAST) against the GenBank database (Nilsson et al. 2006) to check the most closely related sequences.Reference sequences for a total of 103 specimens representing 55 species were retrieved (Table 2) and minimally adjusted by hand in BioEdit v.7.0.4 (Hall 2007) first, and then aligned using Tri-mAl (Salvador et al. 2009).
Maximum likelihood (ML) analysis was performed separately for each locus and the concatenated dataset using RAxML-HPC2 v. 8.2.12 (Stamatakis 2014) as implemented on the CIPRES portal (Miller et al. 2010), with the GTR+G model for both genes and 1,000 rapid bootstrap (BS) replicates.For Bayesian Inference (BI), the best substitution model for each character set was determined a The three steps given for each primer pair were repeated for 35 cycles, preceded by an initial denaturation step of 5 min at 94 °C, and followed by a final elongation step of 10 min at 72 °C and a final hold at 4 °C.with MrModeltest 2.2 (Nylander 2004) on CIPRES, using the Akaike information criterion.Bayesian analysis was performed using MrBayes ver.3.2.7a(Ronquist et al. 2011) as implemented on CIPRES (Miller et al. 2010).
In MrBayes analysis, two runs of five chains each were run for 2,000,000 generations and sampled every 200 generations.Convergence was further evaluated by checking that the potential scale reduction factor (PSRF) statistic was close to 1 for all parameters.Moreover, the effective sample size (ESS) was much higher than 200 for all parameters.A clade was considered to be supported if showing a bootstrap support value (BS) ≥75% and/or a posterior probability (PP) ≥0.90.Trees were edited in FigTree version 1.4.0 and PowerPoint.
Fig. 2 presents the phylogeny from the combined datasets.Nine specimens collected in northern Thailand formed three monophyletic clades, here described as L. pseudoalba, L. subroseoalbescens, L. umbilicata, and L. yunnanensis, respectively.Each clade was well supported by both ML and BI in the concatenated trees (Fig. 2).In our phylogenetic analysis, the four species clustered as separate clades with high support.Thus, these species are formally described in this paper.Etymology.The epithet "pseudoalba" refers to its similarity to L. alba in their small basidiomata and orange-white to pale orange pileus.
Habitat and phenology.Scattered, gregarious, or caespitose on the ground in the Fagus and Dipterocarpus.
Habitat and phenology.Scattered on the ground in subtropical forests of Dipterocarpus.

Laccaria umbilicata
Habitat and phenology.Gregarious or caespitose on the ground associated with the Fagus and Dipterocarpus.

Discussion
With the development of molecular phylogenetic analysis, many new Laccaria species have been rapidly described (Wilson et al. 2013(Wilson et al. , 2017;;Popa et al. 2014Popa et al. , 2016;;Luo et al. 2016;Cho et al. 2018;Li 2020;Cui et al. 2021;Zhang et al. 2023).Morphological characteristics and systematically informative traits are few in Laccaria; hence, molecular analyses are important for classification and species identification.In this study, we used the molecular phylogenetic analysis (single gene ITS1+ITS2 and 5.8 S Fig. 1), and multi-locus phylogenetic analysis (ITS, LSU, RPB2 and TEF1 sequences Fig. 2) to evaluate the taxonomy of Laccaria in Thailand.We identified 2 new species, namely L. pseudoalba, L. subroseoalbescens, and two new records, L. umbilicata and L. yunnanensis.
Before this study, five Laccaria species, namely L. amethystina Cooke, L. laccata, L. ohiensis (Mont.)Singer, L. proxima (Boud.)Pat., and L. vinaceoavellanea Hongo were reported to occur in Thailand based on morphological characteristics, but the specimens lacked detailed descriptions (Chandrasrikul et al. 2011).In the future, more extensive specimen collection is needed in Thailand to determine whether these species are indeed distributed there.
So far, only Fagus and Dipterocarpus have been found to host L. pseudoalba, L. umbilicata and L. subroseoalbescens.Species in Laccaria are similar in morphology characters, so habitat and host trees can provide important information for species identification.It is clear that several Laccaria species have a wide range of host trees while other species of Laccaria associate with a limited group or single host (Mueller 1992).For example, L. laccata (Scop.)Cooke (hosts: Castanea, Quercus, Pinus) and L. himalayensis A.W. Wilson & G.M. Muell. (hosts: Abies, Pinus, Picea) have been reported with a variety of hosts in forests; whereas L. trichodermophora G.M. Muell.(host: Quercus) and L. masoniae G. Stev.(host: Nothofagus) have only been found with a single host tree species (Mueller 1984 ).
To date, 42 species of Laccaria have been reported in Asia (Wilson et al. 2013(Wilson et al. , 2017;;Popa et al. 2014Popa et al. , 2016;;Luo et al. 2016;Cho et al. 2018;Li 2020;Zhang et al. 2023).These species are described in China (26 species), South Korea (12 species), Japan (seven species), India (seven species), and Thailand (four species, this study).The taxonomy of Laccaria species in Thailand is still poorly understood and unclear.As a result of their very similar morphological characteristics, many Laccaria species are misidentified as the same species.Thus, for a better understanding of the species diversity of Laccaria in Thailand and their relationships within the genus, additional studies and data are required.

Figure 1 .
Figure 1.Maximum likelihood tree based on ITS1-5.8S-ITS2.Bootstrap support values ≥ 70%.The new sequences are highlighted in red, and the holotype of each species is in bold.

Figure 2 .
Figure 2. Maximum likelihood phylogeny using ITS1-5.8S-ITS2,LSU, rpb2, and tef1 sequence data to identify species of Laccaria growing on roots of Mythicomyces corneipes.ML bootstrap (≥70%) and posterior probabilities (≥0.90) are indicated above branches or in front of the branch leading to each node.The new species and a new record are highlighted in red; the holotype of each species is in bold.

Table 1 .
PCR primers and cycling conditions used in the study.

Table 2 .
Laccaria taxa and sample IDs with geographic location and GenBank ID numbers for ITS, LSU, rpb2, and tef1 sequences used in phylogenetic analysis.Sequences produced in this study are marked in bold."*" following a species name indicates that the specimen is the holotype of that species.