New Species of Nectriaceae (Hypocreales) from China

Species of Nectriaceae commonly occur on living and decaying woody substrates, soil, fruitbodies of other fungi, and insects. Some of them are reported as endophytes, opportunistic pathogens of crops and humans, or producers of mycotoxins. To explore the species diversity of the family, specimens from different regions of China were collected and examined. Four novel taxa of Penicillifer, Pseudocosmospora, and Thelonectria were introduced on the basis of morphological characteristics and DNA sequence analyses of combined datasets of the act, ITS, LSU, rpb1, rpb2, tef1, and tub2 regions. Differences between the new species and their close relatives were compared and discussed.


Introduction
The family Nectriaceae was established in 1865 to accommodate those fungi producing uniloculate perithecia that are yellow, orange-red to purple, or brown; often change color in 3% potassium hydroxide (KOH) and 100% lactic acid (LA); and have a tropical and subtropical distribution [1]. Approximately 69 genera are currently accepted [1][2][3], including genera such as Penicillifer Emden, Pseudocosmospora C.S. Herrera & P. Chaverri, and Thelonectria P. Chaverri & C. Salgado. The generic concepts and phylogenetic relationships of the family were comprehensively stated by Lombard et al. [2].
In our study of the hypocrealean specimens from different regions of China, four unusual fungi were encountered. Judging by their perithecial gross morphology, anatomy, and culture characteristics, they represent four undescribed species of Penicillifer, Pseudocosmospora, and Thelonectria. Their taxonomic placements were further confirmed by multigene phylogenetic analyses of α-actin (act), nuclear ribosomal DNA ITS1-5.8S-ITS2 (ITS), large subunit of nuclear ribosomal DNA (LSU), the largest subunit of RNA polymerase II (rpb1), the second largest subunit of RNA polymerase II (rpb2), translation elongation factor 1-α (tef1), and β-tubulin (tub2). The differences between the novel taxa and their close relatives were compared.
Newly obtained sequences and those retrieved from GenBank are listed in Tables 1-3. The sequences were assembled and aligned, and the primer sequences were trimmed using BioEdit 7.0.5 [29] and converted to nexus files by ClustalX 1.83 [30]. A partition homogeneity test (PHT) was performed with 1000 replicates in PAUP*4.0b10 [31] to evaluate statistical congruence amongst these loci. The aligned sequences were combined in BioEdit and analyzed with Bayesian inference (BI), maximum likelihood (ML), and maximum parsimony (MP) methods to determine the phylogenetic positions of the new species. The BI analysis was conducted by MrBayes 3.1.2 [32] using a Markov chain Monte Carlo algorithm. Nucleotide substitution models were determined by MrModeltest 2.3 [33]. Four Markov chains were run simultaneously for 1,000,000 generations, with the trees sampled every 100 generations. A 50% majority rule consensus tree was computed after excluding the first 2500 trees as 'burn-in'. The Bayesian inference posterior probability (BIPP) was determined from the remaining trees. Branch support measures were calculated with 1000 bootstrap replicates. The ML analysis was performed via IQ-Tree 1.6.12 [34] using the best model for each locus chosen by ModelFinder [35]. The MP analysis was performed with PAUP 4.0b10 [31] using heuristic searches with 1000 replicates of random addition of sequences and subsequent TBR (tree bisection and reconnection) branch swapping. The topological confidence of the resulting trees and the statistical supports of the branches were tested by maximum parsimony bootstrap proportion (MPBP) with 1000 replications, each with 10 replicates of random addition of taxa. Trees were examined by TreeView 1.6.6 [36]. Maximum likelihood bootstrap proportion (MLBP) and MPBP greater than 70% and BIPP greater than 90% were shown at the nodes.

Phylogeny
The sequences of the ITS, LSU, rpb2, and tef1 regions from six Penicillifer species were analyzed. Stachybotrys chartarum (Ehrenb.) S. Hughes was used as the outgroup taxon. The partition homogeneity test (p = 0.01) indicated that the individual partitions were not highly incongruent [37]; thus, these four loci were combined for the phylogenetic analyses. The ML tree is shown in Figure 1. The topologies of the BI and MP trees were similar to that of the ML tree. The isolate CGMCC 3.24130 grouped with other members of Penicillifer and received high statistical support (MLBP/MLBP/BIPP = 96%/100%/100%).  The sequences of ITS, LSU, and tub2 regions from 11 Pseudocosmospora species were analyzed. Corallomycetella repens (Berk. & Broome) Rossman & Samuels and Microcera larvarum (Fuckel) Gräfenhan, Seifert & Schroers were used as outgroup taxa. The partition homogeneity test (p = 0.01) indicated that the individual partitions were not highly incongruent [37]; thus, these three loci were combined for the phylogenetic analyses. The ML tree is shown in Figure 2. The topologies of the BI and MP trees were similar to that of the ML tree. The isolate CGMCC 3.24131 grouped with other species of Pseudocosmospora and received high statistical support (MLBP/MLBP/BIPP = 92%/98%/100%).  The sequences of the act, ITS, LSU, rpb1, and tub2 regions from 13 Thelonectria species were analyzed. Cosmospora coccinea Rabenh. and Nectria cinnabarina (Tode) Fr. were used as outgroup taxa. The partition homogeneity test (p = 0.01) indicated that the individual partitions were not highly incongruent [37]; thus, these five loci were combined for the phylogenetic analyses. The ML tree is shown in Figure 3. The topologies of the BI and MP trees were similar to that of ML tree. The isolates CGMCC 3.24132 and CGMCC 3.24133 were well-located among other Thelonectria species and received high supporting values (MLBP/MLBP/BIPP = 100%/100%/100%). The isolate CGMCC 3.24133 was related to T. rubrococca    GenBank accession numbers: ITS OP223439, LSU OP223435, rpb2 OP272863, tef1 OP272864, rpb1 OP586759, tub2 OP586763.
Notes: Among the known species of Thelonectria, T. globulosa is distinct because of its globose microconidia. Morphologically, T. globulosa resembles T. nodosa C.G. Salgado & P. Chaverri in having solitary to gregarious, globose perithecia that do not collapse upon drying; cylindrical to clavate asci; ellipsoidal to fusiform ascospores; and cylindrical macroconidia. However, T. nodosa differs because of its larger asci (68-115 × 10-17 µm) with an apical ring, macroconidia possessing more septa (up to six septa), and lack of microconidia formation [12]. Moreover, there are 38 bp, 96 bp, 37 bp, and 65 bp divergences in the act, ITS, LSU, and rpb1 regions between the ex-type cultures of the two taxa (CGMCC 3.24132 and GJS 04155). Both the morphology and DNA sequence data distinguish them as different species.

Discussion
The genus Penicillifer is proposed as the preferable name over Viridispora [2], following the International Code of Nomenclature for algae, fungi, and plants [39]. Our analyses, inferred from sequences of ITS, LSU, rpb2, and tef1 and including the new taxon, revealed a tree topology (Figure 1) similar to that given by Lombard et al. [2]. The phylogenetic tree
Notes: The morphological features, such as the superficial, globose to subglobose, broad-pyriform perithecia that do not collapse when dry; ellipsoidal, two-celled, and hyaline ascospores; and curved macroconidia with rounded ends, indicate the placement of T. spinulospora in Thelonectria, which was confirmed by sequence analyses of the act, ITS, LSU, rpb1, and tub2 regions (Figure 3). Amongst the known species of the genus, the new species is morphologically similar and phylogenetically related to T. rubrococca (Brayford & Samuels) C.G. Salgado & P. Chaverri in having solitary to gregarious, globose perithecia that do not collapse upon drying, ellipsoidal ascospores, and cylindrical macroconidia. However, the latter differs in its larger perithecia (200-450 µm in diam.), smaller ascospores (8-14.5 × 3.6-6.6 µm), and macroconidia with more septa (up to five septa) [38]. Sequence comparisons between the ex-type cultures of the two species revealed that 24 bp, 8 bp, 0 bp, 22 bp, and 28 divergences were detected for the act, ITS, LSU, rpb1, and tub2 regions. Both the morphology and DNA sequence data support their distinction at the species level.

Discussion
The genus Penicillifer is proposed as the preferable name over Viridispora [2], following the International Code of Nomenclature for algae, fungi, and plants [39]. Our analyses, inferred from sequences of ITS, LSU, rpb2, and tef1 and including the new taxon, revealed a tree topology (Figure 1) similar to that given by Lombard et al. [2]. The phylogenetic tree shows that Penicillifer species forms a well-supported monophyletic clade (MLBS/MPBP/BIPP/ = 96%/100%/100%) (Figure 1). Penicillifer sinicus is closely related to P. macrosporus (MLBS/MPBP/BIPP/ = 100%/99%/100%). The sequence comparisons revealed that there were 34 bp, 19 bp, 23 bp, 31 bp, and 23 bp differences detected for the ITS, LSU, rpb1, rpb2, and tef1 regions. Therefore, both the molecular and the morphological evidence supports the separation of the two fungi at a specific level. Among the known species of Penicillifer, P. martinii P. Wong, Y.P. Tan & R.G. Shivas is known solely by its sexual stage [5], and only asexual stages of P. japonicus Matsush. and P. pulcher have been discovered [4,40]; the rest species of the genus are holomorphic, including the newly added one.
Historically, nectriaceous species producing small, reddish, smooth, thin-walled perithecia were categorized as Cosmospora Rabenh. sensu lato [1]. The accumulated morpho-logical and phylogenetic information indicated that the genus was not monophyletic [41,42]. Herrera et al. [6] established Pseudocosmospora to accommodate ten cosmospora-like fungi on Eutypa and Eutypella and with acremonium-to verticillium-like asexual stages. Since then, six additional taxa have joined the group [7][8][9]. The genus has become distributed worldwide and displays high species diversity in warm temperate and tropical regions [6]. Species of the genus have the following features in common: they are superficial perithecia, gregarious, KOH+, LA+, laterally collapsed upon drying, and usually less than 250 µm in height; and they have asci containing eight 1-septate ascospores, acremonium-to verticillium-like conidiophores, and non-septate conidia. Pseudocosmospora beijingensis fits well the generic concept. The multigene analyses indicated its distinctions from any other species of the genus (Figure 2).
Members of Thelonectria are often found on twigs and branches, trunks of recently killed or dying trees, and rotting roots; they occasionally cause small cankers and are mainly distributed in tropical, subtropical, and temperate regions [10,11,17]. Among the species of the genus, T. coronata (Penz. & Sacc.) P. Chaverri & C. Salgado, T. discophora, T. lucida (Höhn.) P. Chaverri & C. Salgado, and T. veuillotiana (Roum. & Sacc.) P. Chaverri & Salgado are cosmopolitan and are treated as species complexes [38,43,44]. Salgado-Salazar et al. [11][12][13] carried out a revisionary work on the above species complexes and described 30 cryptic species on the basis of genealogical concordance phylogenetic species recognition. Our phylogenetic results indicated that T. globulosa was associated with but clearly separated from members of the T. veuillotiana complex. Thelonectria aurea, known only by the asexual stage, can be easily distinguished in the absence of microconidia and chlamydospores in culture [17]. Moreover, there were 93 bp and 64 bp divergences in the ITS and tub2 regions between the ex-type culture of the two species.
There are 47 species currently known in this genus, of which 20 species have been reported in China [11,13,15]. Large-scale surveys of fungal resources in various regions with different climates, vegetation, geographic structures, and multiple niches will improve our understanding of the species diversity of nectriaceous fungi in the country.

Conclusions
The species diversity of the family Nectriaceae was investigated, and four novel taxa were discovered. With the joining of the new species, the phylogenetic relationships among species of these three genera were updated.  Data Availability Statement: Names of the new species were formally registered in the database Fungal Names (https://nmdc.cn/fungalnames (accessed on 22 August 2022)). Specimens were deposited in the Herbarium Mycologicum Academiae Sinicae (HMAS). The newly generated sequences were deposited in GenBank (https://www.ncbi.nlm.nih.gov/genbank (accessed on 5 October 2022)).