Species Diversity of Helvella lacunosa Clade (Pezizales, Ascomycota) in China and Description of Sixteen New Species

Helvella lacunosa and its allies are widely distributed in the Northern Hemisphere and perform important functions in ecosystems. A comprehensive study on 101 collections of Helvella lacunosa, including those deposited in four Chinese fungaria or collected recently from 10 provinces, was conducted based on morphological and molecular characteristics. Phylogenies of “Helvella lacunosa clade” inferred from Hsp90, ITS, LSU, and TEF were reconstructed with 49 lineages recognized, of which 25 lineages occurred in China, and each represented an individual species. Sixteen new species were determined with detailed descriptions and illustrations. Two new Chinese records were reported. Species concepts and their distinctions in macro- and micro-features were discussed.


Introduction
The Helvella lacunosa species complex (Helvellaceae, Pezizales) contains ascomycetous macrofungi having dark, saddle-shaped, lobed, or convex apothecia with sulcate or ribbed stipes. They are widely distributed in the Northern Hemisphere and perform important functions in ecosystems [1,2]. Some Helvella species are taken as precious and delicious food by local people in Shanxi and Xinjiang provinces of China.
The Helvella section Lacunosae was established by Dissing [3] to accommodate a group of operculate cup-fungi, including H. lactea Boud., H. lacunosa Afzel., H. philonotis Dissing, and H. phlebophora Pat. & Doass. The section was accepted by Weber [4] and Häffner [5] with H. palustris Peck and H. sulcata Afzel. added, which were treated as synonyms of H. lacunosa by Dissing [3]. Morphological features of the section include epigeous, stipitate, saddle-shaped or lobed apothecia; ribbed or lacunose stipes; a usually dark-colored hymenium surface; subcylindrical asci; filiform or clavate paraphyses; and ellipsoidal, hyaline, smooth, uniguttulate ascospores. Fragments of small subunit (SSU) and large subunit (LSU) ribosomal RNA genes were first adopted for pezizalean phylogenetic analyses in the 1990s [6] and were followed by subsequent studies [7][8][9][10][11][12][13]. Translation elongation factor 1-α (TEF) and other protein-coding genes were also applied to infer the phylogeny of this group of fungi [10,[14][15][16][17]. As to Helvella, the internal transcribed spacer (ITS) and LSU phylogenies of H. lacunosa species complex were initially reported by Nguyen et al. [18]. And then, phylogenies of the genus based on a single gene, ITS or LSU, were reconstructed [2,19]. Hsp90 was introduced into the analysis of the genus and advocated as a primary DNA barcode [20]. Up to now, LSU is still the most popular locus to be used for species identification of the group, although its capacity is limited. Sequences of Hsp90 and ITS are usually lacking for some Helvella species, which creates barriers for species recognition. Our previous study [21] on the rib-stiped cupulate species of the genus tried to address this aspect.
Through the aid of molecular phylogenetics, species concepts of Helvella became wellestablished. A synopsis of the European species was accomplished by Skrede et al. [20], in which the monophyletic Clade C corresponded to the section containing H. lacunosa and its allies (as Helvella lacunosa clade). A total of 22 lineages were revealed in the clade based on multiple sequence analyses, and epi-/neo-types were designated for H. atra Oeder, H. helvellula (Durieu & Mont.) Dissing, H. lacunosa, H. nigra Bergeret, H. pallescens Schaeff., H. phlebophora, H. queletiana Sacc. & Traverso, and H. sulcata. Thereafter, nine additional species of Helvella were described from Spain [22], one from Mexico [23], and one from China [24], among which nine species belonged to this clade: H. cystidiata R.J. Xu  In China, H. lacunosa was first recorded by Teng [25] from seven provinces, and then H. atra, H. lacunosa, H. pallescens, and H. lacteal were listed by Tai [26]. More taxa of the clade were subsequently reported: H. helvellula, H. lactea, and H. phlebophora by Liu and Cao [27]; H. philonotis by Zhuang and Wang [28]; and H. fusca Gillet by Xu [29]. Recently, H. cystidiata, H. pseudolacunosa Q. Zhao [24,30,31]. A total of 13 species of the group were known in the country.
In connection with our current work on the family Helvellaceae of Flora Fungorum Sinicorum, extensive surveys of Helvella collections from different regions of China have been conducted. The results of a study on the species possessing cupulate apothecia with sulcate or ribbed stipes were previously reviewed [15]. In this work, members of of Helvella lacunosa clade were investigated, and it turned out that species diversity of the clade is extremely high. The current research is a continuation towards the classification of the group based on multigene analysis.

Re-Examination and Collection of Samples
Collections of the Helvella lacunosa clade from China deposited in the following fungaria were re-examined: Herbarium Mycologicum Academiae Sinicae (HMAS), Herbarium of the Microbiology Institute of Guangdong (HMIGD), Herbarium of Mycological Institute of Jilin Agricultural University (HMJAU), and Mycological Herbarium of Chifeng University (CFSZ). Specimens recently collected from 10 cities, provinces, or administrative regions of China (Beijing, Fujian, Guizhou, Jiangsu, Jilin, Shanxi, Sichuan, Tibet, Yunnan, and Zhejiang) were also identified (Table 1). Morphological observations were conducted following the methods indicated in previous studies [21,32].

Phylogenetic Analyses
In this study, newly generated forward and reverse sequences were assembled using Seqman v. 7.1.0 (DNASTAR Inc., Madison, WI, USA). The assembled sequences and those retrieved from GenBank are listed in Table 1. Four single-gene datasets and a multi-locus dataset were compiled. Sequences were aligned using MAFFT v. 7.221 [36] and then manually edited via BioEdit v. 7.1.10 [37] and MEGA v. 6.0.6 [38]. Maximum likelihood (ML) analyses were performed using RAxML-HPC2 [39]

Molecular Phylogenies
Sequences from 49 species of the Helvella lacunosa clade and two outgroup taxa were investigated (Table 1). The characteristics of each dataset, e.g., the number of sequences included, alignment length, and numbers of variable and informative sites, are given in Table 2.
Helvella ravida X.C. Wang  Notes: This species is a sister of H. liui (Figure 1). Their molecular and morphological distinctions were indicated in the notes of the latter. Notes: Helvella terricola was known only from Spain [22]. The Chinese collection extends its distribution to Asia. The Chinese collection is identical with the holotype in the sequence of Hsp90.

Discussion
A total of 101 specimens from four Chinese fungaria (HMAS, HMIGD, HMJAU, and CFSZ) and recent collections from 10 provinces (Beijing, Fujian, Guizhou, Jiangsu, Jilin, Shanxi, Sichuan, Tibet, Yunnan, and Zhejiang) were molecularly and morphologically examined in this study. Four loci were investigated, and 311 sequences were newly generated, including 101 for Hsp90, 82 for ITS, 69 for LSU, and 59 for TEF. A four-locus phylogeny of Helvella lacunosa clade was reconstructed, and 46 lineages were revealed in the tree. All these species occur in the Northern Hemisphere, and 25  Helvella rugosa appears to be the most common species (17 specimens) of this clade in China with the widest distribution in the northeast, south, and southwest of the country ( Table 1). The collections of the fungus formed a monophyletic clade in the phylogenies of ITS, LSU, and TEF. However, they were shown as polyphyletic in the Hsp90 tree and divided into four small groups ( Figure S1). Although different intra-specific clustering of the examined collections existed in the phylogenies ( Figures S1-S4), according to Genealogical Concordance Phylogenetic Species Recognition (GCPSR) [51], all of them should belong to the same species. Specimens from the same locality tended to cluster together (Figure 1), which might give the hint that they may be undergoing speciation. This might involve some underlying mechanism, e.g., incomplete lineage sorting.
Intra-specific variations in macro-and micro-morphology have been observed. For example, one specimen (Wu345) of H. varia has whitish apothecia when fresh ( Figure 2J), while the others (ZRL20150069 and ZRL20191640) are brown to nearly black ( Figure 2K,L), which might influenced by fruitbody age, the ecological niches of a collection, as well as the degree of exposure to light. The collections of H. magna also exhibited apothecial color variations ( Figure 2D-F). Additionally, apothecial size is also variable within individual species: some collections (HMAS 60679, HMAS 61724, and HMAS 66121, all from Gansu Province) of H. magna possess large apothecia (up to 5-6 cm high when dry) and inflated stipes (up to 1.7-2.4 cm diam. when dry, Figure 3F), whereas the others have smaller apothecia (usually less than 2 cm high when dry) and thinner stipes (no more than 0.5 cm diam. when dry); nutrition or texture of the substrates might end up with size changes of apothecia. Microscopically, different lengths of asci were found in H. austrooccidentalis, H. magna, and H. varia (Table 3); the width of the paraphysis apices among collections varied in H. austrooccidentalis, H. cystidiata, H. magna, and H. plateata; the color of paraphyses varied in H. varia and H. vulgata; and variations of the shape of ascospores can be seen in H. magna and H. varia ( Figure 5 and Table 3). These noticeable morphological differences make identifications solely based on morphology unreliable.
Our understanding of the Helvella lacunosa clade in China is significantly renewed through this work. Helvella pseudolacunosa was proved to be a later synonym of H. lacunosa (Figures S2 and S3). Additionally, the previous records of H. fusca, H. helvellula, H. lactea, and H. sulcata in China were based on misidentifications and should be excluded from Chinese fungus flora. The unexpectedly high biodiversity of the lacunosa clade suggests that species diversity of macrofungi in Pezizales may have been underestimated. Further large-scale investigations are desperately needed to examine unexplored pezizalean fungi.