Four new additions to Helvella (Helvellaceae, Pezizales) from Northern Thailand

Most species of Helvella have been described from temperate regions in Asia, Europe, and North America, but little is known about the genus from tropical regions. In this report, phylogenetic analyses of 11 newly collected saddle-like fungi from northern Thailand using three genetic markers [the nuclear large subunit ribosomal DNA (LSU), the heat shock protein 90 (HSP90), and the translation elongation factor 1-alpha (TEF)] confirm their assignment in Helvella. Two species were described as new, i.e., Helvella atroides and H. orentitomentosa, and two species, i.e., H. fistulosa and H. rugosa, were reported for the first time in Thailand. Details of macro- and microscopic characters and illustrations were provided for each species. To date, seven species of Helvella have been recorded in Thailand, and a key for identifying the Thai Helvella species was provided here.

In Europe, Skrede et al. (2017Skrede et al. ( , 2020Skrede et al. ( , 2023) ) and Løken et al. (2019) conducted a thorough investigation of saddle fungi and found at least 72 Helvella species are there.In Asia, research on the species diversity of Helvella is mainly concentrated in China, with more than 60 species recorded, mostly from southwest China (Ariyawansa et al., 2015;Zhao et al., 2015Zhao et al., , 2016a,b;,b;Hyde et al., 2016Hyde et al., , 2020;;Wang et al., 2016;Tibpromma et al., 2017;Zhuang et al., 2018;Xu et al., 2022).However, the attention paid to saddle fungi in tropical areas is limited, especially in Thailand.Boonthavikoon (1998) and Tibpromma et al. (2017) reported that three species were recorded in Thailand, i.e., H. crispa, H. crispoides Q. Zhao & K.D. Hyde, and H. elastica Bull.Given the high level of species diversity and provincialism discovered within saddle fungi and the rich fungal diversity in tropical regions, we hypothesize that there will be novel species lineages in these places.
In this study, we reported new knowledge of Helvella species collected from northern Thailand.The 11 Helvella collections are morphologically and phylogenetically analyzed here.The results of four species of Helvella were found, of which two were new species and the other two were recorded for the first time.A taxonomic key to Helvella taxa in Thailand was provided.
. Materials and methods

. . Specimen and morphological studies
Collections were obtained in Chiang Mai and Chiang Rai Provinces, Thailand and photographed in situ.Microscopic observations and photomicrographs were made.The hemiamyloid reaction in Melzer's reagent is as follows: "J + " for a hemiamyloid ("solely red") reaction and "J − " for a negative reaction.For microscopic examination, dried specimens were sliced manually and then rehydrated in water.A Nikon ECLIPSE 80i microscope was used for observation and microphotography.The notations "ascospores (n/m/p)" indicate that the measurements were made on "n" ascospores from "m" ascomata of "p" collections.The measurements of ascospores were indicated in (a-) b-c (-d), where the range b-c represents the 95% confidence interval, and a and b represent the minimum and maximum, respectively.Q refers to the length/breadth ratio of ascospores, and bold Q referred to the average Q of ascospores ± sample standard deviation.Examined specimens were deposited at Mae Fah Luang University, Chiang Rai, Thailand (MFLU).Index Fungorum numbers and Facesoffungi numbers were obtained as detailed in the Index Fungorum (http:// www.indexfungorum.org/names/names.asp)and Jayasiri et al. (2015).

. . DNA sequence data analyses
The phylogenetic trees were constructed using the sequencing data of newly collected Helvella samples and the allied reference sequences of closely related saddle species obtained from the GenBank (Table 2).Dissingia confusa (O-253269, O-253268, KH.12.75) and D. leucomelaena (DMS-9190862, KH.06.01) were used as outgroup taxa.All sequences were assembled and aligned using MAFFT v. 7 (Kuraku et al., 2013;Katoh et al., 2019) and manually edited where necessary using BioEdit version 7.0.9(Hall, 1999).Individual alignments were compiled for LSU, HSP90, and TEF genes.The optimal substitution model for each gene dataset was determined using MrModeltest 2.3 (Nylander, 2004) under the Akaike information criterion (AIC).The results indicated that the GTR+I+G model was optimal for LSU, SYM+G for TEF, and HKY+G for HSP90.Individual datasets were combined to assemble the combined dataset (gene order: LSU, HSP90, and TEF).A maximum likelihood (ML) analysis was performed using IQ-Tree (http://iqtree.cibiv.univie.ac.at/) (Trifinopoulos et al., 2016).The substitution model options for each gene were auto-evaluated according to the provided partition file.Clade support for the ML analysis was assessed using an SH-aLRT test with 1,000 replicates (Guindon et al., 2010) and the ultrafast bootstrap (UFB) (Hoang et al., 2018).In the ML analysis, nodes with support values of both SH-aLRT ≥ 80 and UFB ≥ 95 were considered well-supported, those with either SH-aLRT < 80 or UFB < 95 were weakly supported, and nodes with both SH-aLRT < 80 and UFB < 95 were unsupported.
Bayesian inference (BI) (Rannala and Yang, 1996) was performed with Markov Chain Monte Carlo sampling (MCMC) to evaluate the posterior probability using MrBayes on XSEDE (3.2.7a) with default parameters on CIPRES Science Gateway (https://www.phylo.org/).The number of generations was set at 5,000,000, sampling every 1,000 generations and a burn-in value of 25%.Nodes were considered strongly supported with posterior probability values >0.90.The resulting phylogenetic tree was visualized in Figtree v. 1.4.4 program (Rambaut, 2018).
The phylogeny shows that our newly collected samples formed four evolutionary lineages (Figure 1).Two independent clades were discerned as new species to science, i.e., H. atroides and H. orentitomentosa.They are nested in the fibrosa-macropus lineage, which now consists of seven species.This lineage exhibits a broad spectrum of apothecial shapes, from regularly cupulate to saddleshaped to lobed capitate.Helvella macropus, H. ephippioides, H. convexa, and H. orentitomentosa constitute one lineage and H. fibrosa, H. japonica, and H. atroides constitute a sister lineage.Samples of H. orentitomentosa (Thailand) were inferred as a monophyletic clade, which is a successive sister group to H. convexa (Finland, Sweden), H. ephippioides (Japan, Sweden), and H. macropus [Asia (China), Europe, and North America].Helvella atroides formed an independent lineage separated from H. japonica (Japan, Norway, and Sweden) and H. fibrosa (Asia and Europe).In addition, two geographic distributions of H. fistulosa and H. rugosa are new records in Thailand.Three samples (MFLU23-0093, MFLU23-0094, and MFLU23-0095) were clustered with H. rugosa, indicating that these taxa were homogeneous with H. rugosa.The newly collected samples MFLU23-0096, MFLU23-0097, and MFLU23-0098 were nested in the widely distributed H. fistulosa.
Etymology: atroides named as its gross morphology is similar to H. atra.
The gross morphology of H. atroides is very similar to that of typical H. atra J. König s.s.However, the hymenial surface and stipe of H. atroides are usually dark gray to brownish black when young, while in the latter species, it is generally completely black (Landeros et al., 2012).
Etymology: orentitomentosa refers as its tomentose receptacle surface and stipe surface in oriental region.
Habitat: Solitary, scattered, or gregarious on the ground under Pinus armandii.
Notes: Helvella fistulosa was first reported in Thailand.Skrede et al. (2017) checked some specimen named Helvella fistulosa from Iceland, Japan, Norway, and the United States.They redescribed H. fistulosa and designated a Norwegian specimen labeled O-291887 (H241) as the neotype.In this study, three newly added collections from Thailand were clustered together and nested within H. fistulosa taxa with a strong support value in our phylogeny.Compared with the neotype of H. fistulosa, the hymenium of our samples has a lighter color (Skrede et al., 2017).There is no significant morphological difference between our samples and the neotype in microscopic characteristics.
Habitat: Scattered or gregarious on the ground, and in deciduous forests of Quercus sp.Notes: Helvella rugosa, usually found in deciduous forests, was known only from China (Ariyawansa et al., 2015).Our samples extend their distribution to Thailand.All samples from Thailand are similar to the original description (Ariyawansa et al., 2015), and their ecological preference is associated with the coniferous forest dominated by Quercus spp.

. Discussion
Species delimitation, taxonomy, and typification in saddlelike fungi have always been challenging.Traditionally, the macroscopic characteristics of the hymenium, receptacle surface appendage, excipulum, and stipe have been used as the key diagnostic characters to distinguish Helvella from its allied species (Dissing, 1966;Korf, 1972;Weber, 1972;Harmaja, 1979; Häffner , 1987;Abbott and Currah, 1997;Nguyen et al., 2013).In Dissing's research, the importance of paraphysis pigmentation was emphasized, which was reflected in the color of hymenium.However, the hymenium color varies greatly in some species, pale grayish brown to brown to almost black, e.g., H. elastica, H. lacunosa, and H. rugosa (Dissing, 1966;Ariyawansa et al., 2015).Skrede et al. (2017) indicated that the color of fresh apothecia could change dramatically during drying.In microanatomical features, the characteristics of ectal excipulum, especially the shape, color, and distribution of the outer hyphoid hairs, are of special diagnostic value in morphologically similar species (Landeros et al., 2012).In addition, the ascus base (aporhynchous or pleurorhynchous), as well as the pigmentation and the shape of paraphyses upperpart also helped in species discrimination of some Helvella (Landeros et al., 2012;Skrede et al., 2017).
The development of molecular systematics and the possibility of employing DNA barcode sequences as a more robust tool to identify specimens of closely related species have been applied to taxonomic studies of Helvella (Skrede et al., 2017).As a universal barcode for fungi identification, ITS is not suitable for addressing the phylogeny and species discrimination across the Helvella, because their ITS regions (especially ITS1) are too divergent to be arranged within the whole genus, as well as the limited molecular information of ITS fragments in GenBank (Landvik et al., 1999;Wang et al., 2019).Skrede et al. (2017) showed that genetic markers LSU, RPB2, and HSP90 provided useful barcodes for species delimitation in Helvella, due to their    (2019) focused on the rib-stiped cupulate species of Helvella and revealed that the success rate of using primers ITS3/ITS4 for ITS2 region amplification much higher than that of using primers ITS5/ITS4 for ITS gene amplification.The authors proposed that HSP90 and ITS2 should be used as supplementary DNA barcodes for the cupulate Helvella species with ribbed stipe (Wang et al., 2019).With the help of molecular systematics, in the past decade, some researchers have discriminated some novel species and reevaluated the circumscription of morphologically similar species or pseudo-cryptic species or complex groups (Nguyen et al., 2013;Ariyawansa et al., 2015;Landeros et al., 2015;Zhao et al., 2015Zhao et al., , 2016a,b;,b;Skrede et al., 2017Skrede et al., , 2020;;Hansen et al., 2019;Xu et al., 2022).Ecologically, hosts are also important traits for the taxonomy and phylogeny of ectomycorrhizal and have been used to recognize the species of H. dryophila, H. pseudolacunosa, H. rugosa, and H. vespertina (Nguyen et al., 2013;Ariyawansa et al., 2015).Helvella lacunosa in China is mainly associated with Betula sp., Dryas sp. and Salix sp., and those specimens collected from Sweden are mainly associated with Betula sp., Dryas sp., Fagus sp., Quercus sp., and Malus sp.(Dissing, 1966).Skrede et al. (2017) found that H. arctoalpina and H. dryadophila are always closely associated with Dryas species while H. fusca with Populus species.Our newly described species, H. atroides and H. orentitomentosa, may have mycorrhizal host specificity because they are all found in coniferous forest, such as Pinus spp.However, whether these ectomycorrhizal features can be reflected in the morphology and systematics of saddle fungi remains to be determined.
In this study, based on analyses of combined LSU, HSP90, and TEF sequence data, two new species and two new geolocation records in Thailand were identified and described.In the phylogenetic tree (in Figure 1), the newly collected samples from are scattered among the taxa that are distributed in other continents, mostly from Europe.Previous research also revealed that Helvella samples from different regions encompass many morphologically similar but distinct phylogenetic species (Skrede et al., 2017).In Helvella, some endemic species are only distributed in a narrow area (e.g., H. bachu, H. subspadicea, and H. zhongtiaoensis), while others can be widespread across the world (e.g., H. alpestris, H. capucina, H. solitaria, H. phlebopora, and H. fistulosa) (Zhao et al., 2015(Zhao et al., , 2016a;;Skrede et al., 2017).In tropical Thailand, together with the four newly added species in this study, a total of seven saddle fungi are distributed here.A key to Thai saddle fungi is given.

FIGURE
FIGUREMaximum likelihood (ML) tree of Helvella and its allies within Helvellaceae inferred from combined LSU, TEF, and HSP datasets.Bootstrap support values for ML ≥ of SH-aLRT or of UFB and posterior probability for BI ≥ .are indicated above the nodes and separated by '-/-/-' (SH-aLRT/UFB/BIPP).Specimens of the current study are given in red.Type specimens are in bold.The letter ET stands for epitype, HT for holotype, IET for isoepitype, and NT for neotype.
TABLE Genes and their corresponding primers used in this study.
TABLE Species names, voucher numbers, and corresponding GenBank accession numbers used in this study.