Six New Species of Agaricus (Agaricaceae, Agaricales) from Northeast China

Agaricus belongs to Agaricaceae and is one of the most well-known macrofungi, with many edible species. More than 300 Agaricus specimens were collected during a three-year macrofungal resource field investigation in Northeast China. Based on morphological observations and multi-gene (ITS + nrLSU + tef1-a) phylogenetic analyses, six new Agaricus species, i.e., Agaricus aurantipileatus, A. daqinggouensis, A. floccularis, A. griseopileatus, A. sinoagrocyboides, and A. velutinosus, were discovered. These new species belong to four sections within different subgenera: A. (subg. Agaricus) sect. Agaricus, A. (subg. Flavoagaricus) sect. Arvenses, A. (subg. Minores) sect. Minores, and A. (subg. Pseudochitonia) sect. Xanthodermatei. Morphological descriptions, line illustrations, and basidiomata photographs of these new species are provided, and their differences from similar species are discussed.


Introduction
Agaricus L. is a large genus of the family Agaricaceae, with Agaricus campestris L. as the type; it can be distinguished from other genera by its unique characteristics, which include small-sized to large-sized fleshy basidiomata, free lamellae that are white or pink when young but at maturity become brown to dark brown, presence of an annulus on the stipe, brown basidiospores, brown spore prints, pileipellis a cutis of cylindrical hyphae, and absence of capitate cheilocystidia [1][2][3][4][5].The species of Agaricus are saprophytic, distributed on all continents except Antarctica, and commonly found in forests, pastures, or grasslands [6,7].
Agaricus was first described by Linnaeus in 1753; Linnaeus collectively referred to all the large agaric mushrooms found at the time as 'Agaricus' [5].However, the currently defined genus Agaricus was formally established by Karsten (1879) [8].The taxonomic study of Agaricus has a long history, and different taxonomists hold different taxonomy views on the taxonomy system of Agaricus.Before the application of molecular systematics, the taxonomy system proposed by Parra (2008) was widely accepted based on morphology [9].Parra (2008) summarized the previous studies and divided Agaricus into three subgenera: A. subg.Agaricus, A. subg.Conioagaricus and A. subg.Lanagaricus and eight sections, viz., A. sect.Agaricus, A. sect.Arvenses, A. sect.Bivelares, A. sect.Chitonioides, A. sect.Minores, A. sect.Sanguinolenti, A. sect.Spissicaules, and A. sect.Xanthodermatei, based on its macroscopic and microscopic characteristics [1].
In recent years, with the continuous development of biotechnology and scientific means, the taxonomy system of Agaricus has also tended to be stable.Based on the molecular identification of Agaricus species, Zhao et al. (2011) added sequences of specimens from tropical and temperate regions, and molecular phylogenetic results verified the eight known sections proposed by Parra (2008) and found that there were 11 independent clades [7].Additionally, Zhao et al. (2016) proposed a new taxonomic system that classified Agaricus into five subgenera and 20 sections based on divergence times [2].In subsequent studies,

Specimens and Morphological Observations
The examined specimens were mainly collected from Northeast China, including the Inner Mongolia Autonomous Region, Jilin Province, and Heilongjiang Province, and some were studied from the Herbarium of Mycology of Jilin Agricultural University (HMJAU).The voucher specimens are deposited in the Herbarium of Mycology of Jilin Agricultural University.The macromorphological characteristics are based on field records and photographs of fresh basidiomata.The color description of the fresh basidiomata is referenced by Kornerup and Wanscher (1978) [30].Microscopic features were observed based on dry specimens.The corresponding structures were taken and prepared freehand, floated in 5% KOH solution or sterile water, stained with 1% Congo red solution if necessary, and observed through a light microscope (Olympus BX53, Olympus, Tokyo, Japan).The microscopic characteristics of each structure were based on at least 20 measurements.The symbol '(a) b-c (d)' is used to describe the size of basidiospores, where the 'b-c' range represents 90% of the measured values, while the 'a' and 'd' are extreme values.'[Xav = e × f]' indicates the average size of basidiospores.'Q' refers to the ratio of length to width of a single basidiospore from the side view, and 'Qav' refers to the average value of 'Q' of all specimens.Other microstructural measurements include the range between the extreme length and width measurements.

DNA Extraction, PCR Amplification, and Sequencing
Genomic DNA was extracted using the NuClean Plant Genomic DNA kit (CW-BIO, Beijing, China) in strict accordance with the instruction manual.The primer pairs ITS1F/ITS4 [31], LR0R/LR5 [32], and EF1-983F/EF1-1567R [33] were used to amplify the sequences of three DNA regions, ITS, nrLSU, and tef1-a, respectively.The polymerase chain reaction (PCR) procedure was based on the protocol described by Mou and Bau (2021) [34].The PCR products were detected using 1% agarose gel electrophoresis, and the qualified products were sent to Bioengineering (Shanghai) Co., Ltd., Shanghai, China, for sequencing with the same primers.

Sequence Alignment and Phylogenetic Analyses
The chromatograms were checked in BioEdit v.7.1.3.0 [35] to ensure that each sequence had good quality.Then, a BLAST search was carried out in the National Center of Biotechnology Information (NCBI) database (https://www.ncbi.nlm.nih.gov/(accessed on 19-24 October 2023)) to confirm the sequencing results.Finally, the sequences were submitted to GenBank (Table 1 in bold).Based on the BLAST search results, sequences corresponding to the subgenera of the studied species were downloaded for phylogenetic analyses.Subsequently, the multi-gene phylogenetic trees of these subgenera were constructed separately.Particularly, species (Table 1) falling within the clades of the species described in this study were selected and integrated to construct new multi-gene phylogenetic trees.Heinemannomyces sp.ZRL185 was used as an outgroup [2,13].
Maximum likelihood (ML) phylogenies were inferred using IQ-TREE [38] under the TIM2 + I + G4 + F model for 5000 ultrafast bootstraps [39], as well as the Shimodaira-Hasegawa-like approximate likelihood-ratio test.ModelFinder [40] was used to select the best-fit model of ML phylogenies using the BIC criterion.Bayesian Inference (BI) phylogenies were inferred using MrBayes 3.2.6 [41] under the partition model (2 parallel runs, 743,400 generations), in which the initial 25% of sampled data were discarded as burn-in.ModelFinder was again used to select the best-fit partition model (Edge-linked) using the BIC criterion, and the best-fit model according to BIC was HKY + F + G4 for ITS, HKY + F + I for nrLSU and K2P + I for tef1-a.The final trees were visualized using iTOL [42] and edited using Adobe Illustrator 2021 (Adobe, San Jose, CA, USA).
Table 1.Sequences used in the phylogenetic analysis.'T' refers to the type specimen.Bold refers to the sequences produced from this study.Green font refers to the new species.'-' means no relevant genetic information.

Species-Specific ITS Markers
The position of the unique nucleotide (nt) base of a species' ITS sequence in the section to which the species belongs is represented as follows: 'xxxxxXxxxxx @ position', where the capital letter 'X' represents the exclusive or informative character, '@ position' represents the position of 'X', and 'xxxxx' represents the flanking characters.The positions of ITS markers are sequentially numbered starting from the 5 ′ end (ggaaggatcatta).The insertion or deletion (indel) in the ITS alignment was disregarded rather than numbered.It must be noted that the comparisons are made with the currently available sequences of all species in the section and may need to be reassessed when the number of species changes.

Molecular Phylogeny
The dataset used for the phylogenetic analysis consisted of 90 specimens (see Table 1), and 84 sequences were generated for this study, including 37 ITS sequences, 24 nrLSU sequences, and 23 tef1-a sequences.BI and ML analysis resulted in a very similar topology, so the ML tree is provided in this study (Figure 1).Bootstrap support (BS) values ≥ 50% and Bayesian posterior probability (PP) values ≥ 0.70 are indicated on branches (BS/PP).
The phylogenetic tree presents four main clades, corresponding to four sections of different subgenera.Six new species are distributed in these four sections as follows: Agaricus daqinggouensis and A. moelleroides LAPAG319 formed a sister clade with an appreciable support value (BS/PP = 84/0.80) in A. sect.Xanthidermatei.Agaricus sinoagrocyboides and A. albovariabilis TBGT18487/TBGT18462 formed a sister clade with a support value (BS/PP = 97/1) in A. sect.Agaricus.Agaricus velutinosus and A. huijsmanii LAPAG639/HMJAU67712/HMJAU67713 formed a sister clade with a support value       MycoBank: MB 851075 Etymology: 'sinoagrocyboides' refers to the macroscopic characteristics of this Chinese species, which are similar to those of Agrocybe species.
Habit, habitat, and distribution: Solitary or scattered in grassland in summer.Currently, it is only known from the Anhui and Liaoning Provinces, China.

Discussion
The macroscopic and microscopic characteristics of many Agaricus species overlap; therefore, molecular data and phylogenetic analysis are necessary to identify Agaricus species that have similar morphological features [3,7,17].Agaricus griseopileatus, a novel species examined in this study, serves as an exemplary illustration.The morphological distinction between A. griseopileatus and A. arvensis Schaeff.poses challenges, whereas their differentiation in phylogenetic analysis is straightforward.The exclusive base on ITS sequences for phylogenetic analysis of Agaricus is not methodologically rigorous [25]; for example, A. sect.Arvenses and A. sect.Xanthodermatei.The more DNA regions, nrLSU + tef1-a, should be added to ensure the accuracy of species of some sections identification and the stability of phylogenetic relationships.

Figure 1 .
Figure 1.Multi-gene phylogenetic tree of Agaricus obtained from the maximum likelihood analysis (ML) based on ITS, nrLSU, and tef1-a sequence data.Heinemannomyces sp.ZRL185 was used as an outgroup.Bootstrap support (BS) values ≥ 50% and Bayesian posterior probability (PP) values ≥ 0.70 are indicated on branches (BS/PP).'T' refers to the type specimen.Bold refers to the sequences produced from this study.Green font refers to the new species.

Figure 1 .
Figure 1.Multi-gene phylogenetic tree of Agaricus obtained from the maximum likelihood analysis (ML) based on ITS, nrLSU, and tef1-a sequence data.Heinemannomyces sp.ZRL185 was used as an outgroup.Bootstrap support (BS) values ≥ 50% and Bayesian posterior probability (PP) values ≥ 0.70 are indicated on branches (BS/PP).'T' refers to the type specimen.Bold refers to the sequences produced from this study.Green font refers to the new species.