Morphology and Molecular Phylogeny Reveal Five New Species of Laccaria (Hydnangiaceae, Agaricales) from Southern China

The genus Laccaria is a type of cosmopolitan and ecologically important fungal group. Members can form ectomycorrhizal associations with numerous trees, and some species are common edible fungi in local markets. Although some new species from China are recently published, the species diversity of Laccaria is still unclear in China. In this study, some samples of Laccaria were collected from southern China, and morphological characteristics and phylogenetic analyses based on the multilocus dataset of ITS-LSU-tef1-rpb2 confirmed five new species. Laccaria miniata, L. nanlingensis and L. neovinaceoavellanea were collected from subtropical broad-leaved forests, and L. rufobrunnea and L. umbilicata were collected from subtropical mixed forests of southwest China. Full descriptions, illustrations, comparisons with similar species and phylogenetic analysis are provided.


Introduction
Laccaria Berk.& Broome (Agaricales, Hydnangiaceae) is an important fungal group with high ecological and economic values.Species in Laccaria are widely reported from every continent except Antarctica and can form ectomycorrhizal (ECM) relationships with a wide range of trees, such as Betulaceae, Fagaceae, Myrtaceae, Pinaceae and Salicaceae [1,2].Laccaria is considered a model genus for understanding ectomycorrhizal (ECM) ecology and evolution; some species are reported that can act as pioneer species and can be frequently found in recently disturbed sites and young forest stands, play important roles in primary and secondary succession, and are useful for ecological protection and restoration [1,[3][4][5][6][7][8][9][10][11].In addition, some Laccaria species are edible and common in local markets [12]; for example, L. aurantia, L. bicolor, L. moshuijun and L. vinaceoavellanea are commercial mushrooms in Sichuan Province, China [13].
using IQ-TREE [43] under the Edge-linked partition model (TPM2u+F+R4 for ITS and rpb2, TN+F+R3 for LSU, and TIM2e+G4 for tef 1) for 5000 ultrafast bootstraps [44], as well as the Shimodaira-Hasegawa-like approximate likelihood-ratio test [45].Bayesian Inference (BI) phylogenies were inferred using MrBayes 3.2.6 [46], and the best models of the multilocus datasets were searched via PartitionFinder 2 [47] for each locus, i.e., GTR+F+I+G4 for ITS and tef 1, K80 + I + G for LSU, and SYM + I + G4 for rpb2.BI analysis using four chains was conducted by setting generations to 200,000 and the stoprul command with the value of stopval set to 0.01; trees were sampled every 1000 generations, the initial 25% of sampled data were discarded as burn-in and posterior probabilities (PP) were then calculated from the posterior distribution of the retained Bayesian trees.The phylogenetic trees were visualized in FigTree v1.4.23.
Habitat and distribution-Single, scattered or in groups on soil in subtropical broadleaved forests dominated by Fagaceae (Castanopsis fissa, C. spp.) trees.Currently known to be from southern China.
Notes-Laccaria miniata is mainly characterized by its tiny pileus, relatively slender stipe (stipe length 3-5 times that of pileus diameter), and the absence of pleurocystidia and cheilocystidia.On the base of the morphological features given above, the new species can be placed either in sect.Laccata or in sect.Bisporae of Laccaria [29].
Notes-Laccaria miniata is mainly characterized by its tiny pileus, relatively slender stipe (stipe length 3-5 times that of pileus diameter), and the absence of pleurocystidia and cheilocystidia.On the base of the morphological features given above, the new species can be placed either in sect.Laccata or in sect.Bisporae of Laccaria [29].
Fungal Name: FN571673 Etymology-'nanlingensis' refers to the locality of the type species in Nanling National Nature Reserve.
Habitat and distribution-Single, scattered or in groups on soil in subtropical broadleaved forests mainly dominated by Fagaceae trees.Currently known to be from southern China.
Phylogenetic analysis (Figure 1) showed that two specimens labeled as L. nanlingensis clustered together as an independent clade in the genus Laccaria, although its relationship to other Laccaria species is unclear.
Based on the morphological features, such as the dry or hygrophanous and reddish orange to brownish red pileus, the brownish red to grayish red lamellae, globose basidiospores with Q = 1-1.16,and the four-spored basidia, L. nanlingensis can be placed in the Laccaria sect.Laccata [29].
Habitat and distribution-Single, scattered or in groups on soil in subtropical broadleaved forests mainly dominated by Fagaceae.Currently known to be from southwestern and southern China.
Based on the morphological features, such as the purplish pink to pale violet basidiocarps, globose basidiospores with Q = 1-1.14,four-spored basidia, and the colorless to slightly brownish pileipellis hyphae, L. neovinaceoavellanea can be placed in the Laccaria sect.Violaceae [29].
Fungal Name: FN571674 Diagnosis-Laccaria rufobrunnea is distinctive in the genus Laccaria by its brownish orange to brownish red pileus, pastel red to purplish pink lamellae, white to pinkish white stipe, and the absence of caulocystidia.

Discussion
Laccaria is a monophyletic group in the family Hydnangiaceae [2,23,69].Five specieslevel lineages representing five new species from China were uncovered in the Laccaria clade.
Because the species in Laccaria are very similar in morphology, and the characters of some species change greatly in different growth stages and humidity conditions, there can be difficulties in terms of species identification.The basidiospore dimensions play a crucial role in species identification in most macrofungi groups, but in Laccaria, this characteristic may be confusing because some species share similar size and shape basidiospores.For example, the five new species described above all share with globose to subglobose basidiospores with a Q-value between 1 to 1.1 and the size of basidiospores between 7-10 µm in diameter, except L. nanlingensis.However, the length and density of spines on the spore surface can be a useful distinguishing feature; for example, L. acanthospora, L. alba and L. angustilamella have longer spines that can reach up to 5-6 µm long, while L. miniata and L. prava have shorter spines that are less than 1 µm long (see Table 2); L. miniata, L. neovinaceoavellanea and L. umbilicata have relatively more crowded spines than L. nanlingensis, and L. rufobrunnea (see Figures 3-8).In the description of the above five species, the words 'crowded', 'subdistant', and 'distant' were used to distinguish the different densities of spines on basidiospores surfaces.
In addition, basidia size and whether they are two-or four-spored can be useful distinguishing features.Most species of Laccaria have four-spored basidia, while L. echinospora, L. fraterna and L. nigra have two-spored basidia, L. aurantia, and the new species L. miniata have both two-and four-spored basidia, which are infrequent in Laccaria.
Additionally, ecological information such as host plant species and habitat are also important and can provide useful clues for species identification.Laccaria miniata and L. nanlingensis collected from subtropical broad-leaved forests of southern China and could be associated with trees of Fagaceae; L. rufobrunnea and L. umbilicata distributed in subtropical mixed forests (which mainly dominated by Fagaceae trees and pine trees) of southwest China; and L. neovinaceoavellanea can naturally distributed in subtropical broad-leaved forests of southern and southwestern China, and can form symbiotic relationship with Fagaceae trees.On the basis of our study, the main morphological characters and ecological information of each species in Laccaria described from China are summarized in Table 2.
Although many species of Laccaria have been reported in China, most species are mainly reported from southwestern regions, and the species recognition of Laccaria in China is still in its infancy, especially in tropical and subtropical areas.In the past, many Chinese samples of Laccaria were inaccurately labeled as L. laccata, L. bicolor, L. amethystea and L. vinaceoavellanea [72][73][74][75].Phylogenetic studies have made some species well-understood in China, and the discovery of new species in Laccaria is rapidly increasing [2,17,21,22,[31][32][33][34]50].However, the distribution of L. amethystea, L. bicolor and L. laccata in China still needs to be investigated.A broader taxon sampling coupled with both molecular and morphological data is needed to fully understand the species diversity of Laccaria in China.

L. nanlingensis
In subtropical, broad-leaved forests mainly dominated by Fagaceae trees.Distributed in Guangdong.

L. negrimarginata
In mixed temperate alpine conifer forest with Abies, Acer, Larix, Pinus, Quercus and Salix.Distributed in Tibet.

5-15 mm
Orange-brown fading to buff; squamules dark blackish brown to dark brown.

L. pallidorosea
In subtropical broad-leaved forests with trees of Fagaceae.Distributed in Yunnan.

30-75 mm
Pastel red, pale red to reddish white, even to white with age or on drying.Reddish white to grayish red 6.5-7.5 × 7-8 µm; spines 0.5-1 µm long [33] L. rubroalba In a tropical forest dominated by Fagaceae and other broad-leaf trees.Distributed in Yunnan.

12-35 mm
Brownish orange to brownish red, usually fading to brownish yellow in dry condition Pastel red, pink to purplish pink, usually changing pinkish white to purplish white in dry condition

Figure 1 .
Figure 1.Maximum-likelihood phylogenetic tree of Laccaria generated from the ITS-LSU-tef1-rpb2 dataset.Bootstrap values (ML ≥ 70%) and Bayesian posterior probabilities (BPP ≥ 0.95) are shown around branches.Sequences from type specimens are marked with (T), and the new species is indicated in bold and blue area.

Figure 1 .
Figure 1.Maximum-likelihood phylogenetic tree of Laccaria generated from the ITS-LSU-tef1-rpb2 dataset.Bootstrap values (ML ≥ 70%) and Bayesian posterior probabilities (BPP ≥ 0.95) are shown around branches.Sequences from type specimens are marked with (T), and the new species is indicated in bold and blue area.

Table 1 .
Taxa included in molecular phylogenetic analyses and their GenBank accession numbers.Accession numbers in bold indicate newly generated sequences in this study.Taxa marked with T represent type specimens.

Table 2 .
A morphological comparison of species in Laccaria described from China.