﻿Two new species of Nectriaceae (Hypocreales, Sordariomycetes) from Yunnan, China

﻿Abstract Nectriaceae is a highly diverse family, and members have a worldwide distribution, particularly in warm temperate to tropical regions. During the survey of fungal diversity in different habitats in Yunnan province, China, two new species isolated from soil and air respectively, namely Atractiumyunnanensesp. nov. and Nalanthamalaxishuangbannaensissp. nov., were proposed based on morphological comparisons and the multi-gene phylogenetic analyses of combined ITS, LSU, rpb2, and tub2 sequence data. Phylogenetically, both species clustered in a monophyletic clade within Nectriaceae with strong support. A.yunnanense is characterized by synnematous conidiophores, pale olivaceous-green, clavate to oblong-ellipsoidal, multi-septate conidia, and pale olivaceous-green chlamydospores. N.xishuangbannaensis has acremonium-like or penicillium-like conidiophores and either obovate or ellipsoidal, cylindrical or fusiform conidia. Full descriptions, illustrations, and a phylogenetic tree showing the phylogenetic position of the two new species were provided.


Introduction
Nectriaceae was originally established based on Nectria (Fr.) Fr. and assigned in Hypocreales (Tulasne and Tulasne 1865).Thereafter, the taxonomy of Nectriaceae has undergone several revisions (Dennis 1960;Rossman et al. 1999;Lombard et al. 2015).This family is characterized by uniloculate and verrucose ascomata by reaction in KOH and lactic acid, and unitunicate, 2-8-spored asci with globose, ellipsoid to long-fusiform ascospores.Additionally, it is associated with phialidic asexual morphs producing amerosporous to phragmosporous conidia (Lombard et al. 2015).In a recent study, Perera et al. (2023) proceeded to update DNA sequence-based phylogeny for the order Hypocreales, and accepted 77 genera into Nectriaceae, including Atractium Link and Nalanthamala Subram.Nectriaceae is a highly diverse group, and members have a cosmopolitan distribution, particularly in the warm temperate and tropical regions (Rossman et al. 1999;Chaverri et al. 2011;Lombard et al. 2015).Particularly, this family has ecological and economic significance.The majority of the species are soil-borne saprobes or weak to virulent plant pathogens, and include important plant pathogens of economically important plants worldwide, such as Ilyonectria species and Fusarium species (Jayawardena et al. 2019a(Jayawardena et al. , 2019b)).Moreover, several species have also been reported as important opportunistic pathogens of humans (Chang et al. 2006;Guarro 2013;Köhler et al. 2015Köhler et al. , 2017;;Hoenigl et al. 2021).For instance, a patient with acute myelogenous leukemia experienced a disseminated cerebellar Fusarium infection, which magnetic resonance imaging and a CT scan revealed to be a left cerebellar focus of Fusarium solani (Vincent et al. 2003).
Atractium was introduced by Link (1809) with A. stilbaster Link as the type species.So far, there are 28 epithets for Atractium as listed in the Index Fungorum (https://www.indexfungorum.org;retrieval in June 2024).Of them, five species were accepted in the genus, while the placement of the other 23 species remains uncertain due to disagreement with the emended generic diagnosis or an inability to locate authentic material (Gräfenhan et al. 2011;Bao et al. 2023).Gräfenhan et al. (2011)  Luo was introduced based on morphological and phylogenetic analysis (Bao et al. 2023).The asexual morph of Atractium is characterized by synnematous conidiophores, monophialidic, subulate conidiogenous cells with septate, clavate, obovoid or gently curved conidia, and its sexual morphs fit well with the generic concepts of Nectriaceae (Bao et al. 2023).Currently, single Atractium species (A.fusiformis) was reported as saprobe from China among the five accepted species.
Southwest China is one of the high biodiversity hotspots in the word (Myers et al. 2000;Orme et al. 2005;Mi et al. 2021).Particularly, there is a high fungal diversity in Yunnan province (Feng and Yang 2018;Zheng et al. 2021b).In the last decade or so, our team discovered many new fungal species during the investigation of fungal diversity in different habitats in Yunnan (Zheng et al. 2019(Zheng et al. , 2020(Zheng et al. , 2021a(Zheng et al. , 2022;;Qiao et al. 2020Qiao et al. , 2021;;Yu et al. 2022), which increases knowledge of this important ecological area.In this study, two new taxa in Nectriaceae were discovered from Yunnan, and described as new species of Atractium and Nalanthamala, namely A. yunnanense sp.nov.and N. xishuangbannaensis sp.nov., based on morphological characteristics and multi-gene phylogenetic analysis.

Sample collection, fungal isolation and morphological characterization
Soil samples were collected from Huize county, Yunnan province.Samples were preserved in sterile plastic bags, labeled and transported to the laboratory at 4 °C.The dilution coating method was used to isolate fungal strains from soils as described by Lv et al. (2022).The fungi in air located at Xishuangbanna, Yunnan province, were collected by means of the MAS-100 ISO MH Microbial Air Sampler (Merck Millipore, Germany).Ten liters of air flowed through the surface of each Rose Bengal agar (Guangdong Huankai Microbial Sci and Tech, China) plate which placed in the air sampling equipment.After incubation at 25 °C for 5 days, representative colonies were picked up with a sterilized needle and maintained on potato dextrose agar (PDA; 200 g potato, 20 g dextrose, 18 g agar, 1000 ml distilled water) plates.The pure strains were incubated on PDA and cornmeal agar (CMA; 20 g cornmeal, 18 g agar, 1000 ml distilled water) for observing morphological characteristics of colonies.Microscopic characteristics growing on CMA were examined and captured by an Olympus BX51 microscope connected to a DP controller digital camera, and sterile water was used as a mounting medium for microscopy.
The pure cultures were deposited in the Herbarium of the Laboratory for Conservation and Utilization of Bio-Resources, Yunnan University, Kunming, Yunnan, P.R.China (YMF), China General Microbiological Culture Collection Center (CGMCC), the Guangdong Microbial Culture Collection Center (GDMCC) and Japan Collection of Microorganisms (JCM).MycoBank numbers were obtained in MycoBank database (https://www.mycobank.org/)for the newly-described taxa.

DNA extraction, PCR amplification and sequencing
Total DNA was extracted from fresh mycelia grown on PDA for 7 days, as described by Turner et al. (1997).The primer pairs ITS5/ITS4 (White et al. 1990), LR0R/LR7 (Vilgalys and Hester 1990), rpb2-5F2/rpb2-7CR (Liu et al. 1999), and TUB-2Fd/TUB4RD (Woudenberg et al. 2009) were used for amplification of the internal transcribed spacers (ITS), the large subunit nuclear ribosomal RNA gene (LSU), the RNA polymerase II second largest subunit gene (rpb2), and the beta-tubulin gene (tub2), respectively.The PCR amplifications were performed in 25 µl reaction volumes containing 1.0 µl DNA template, 1.0 µl of each forward and reverse primers, 12.5 µl 2 × Master Mix, and 9.5 µl ddH 2 O.The PCR products were confirmed on 1% agarose electrophoresis gels, and the positive products were sent to Tsingke Biotechnology Company (Kunming, China) for purification and sequencing.Newly obtained sequences were deposited in the GenBank database at the National Center for Biotechnology Information (NCBI) and the accession numbers are listed in Table 1.
Table 1.Species, strains and their corresponding GenBank accession numbers of sequences used for phylogenetic analyses.

Species
Strains GenBank accession no.

Sequence alignment and phylogenetic analysis
Initially, the newly-generated sequences were subjected to the GenBank nucleotide database (https://blast.ncbi.nlm.nih.gov/) using BLAST searches to determine closely related taxa, including Atractium species and Nalanthamala species.To further determine the phylogenetic position of these strains, a multi-locus phylogenetic analysis was performed based on ITS, LSU, rpb2 and tub2.Alignments of different loci, including the sequences obtained from this study and sequences downloaded from GenBank, were initially aligned with ClustalX v1.83 (Thompson et al. 1997).The resulting alignments were subsequently checked and refined using BioEdit v7.0.4.1 (Hall 1999).The concatenation of the four loci was processed with BioEdit, and converted to a NEXUS file using MEGA v10 (Kumar et al. 2018).The concatenated sequence matrix contained 3,064 nucleotide positions from the four loci (633 from ITS, 886 from LSU, 932 from rpb2, and 613 from tub2), and was uploaded to TreeBASE (https://www.treebase.org;submission number: S31491).Maximum Likelihood (ML) and the Bayesian Inference (BI) methods were used for the phylogenetic construction in this study.The ML analysis was performed by RAxML v8.0.9 (Stamatakis 2006) using the GTR-GAMMA model with rapid bootstrap analysis, followed by 1,000 ultrafast bootstrap replicates to estimate bootstrap support values (BS).The BI analysis was conducted with MrBayes v3.2.2 (Ronquist et al. 2012) with NEXUS file.The jModelTest v2.0 (Posada 2008) was used to carry out statistical selection of best-fit models of nucleotide substitution using the corrected Akaike information criterion (AIC), and the estimated best-fit model was GTR+F+I+G4.Markov Chain Monte Carlo (MCMC) simulations were used for 1,000,000 generations with a sampling frequency of every 500 th generations.The initial 25% of sample trees were treated as burn-in and discarded.The remaining trees were used to calculate the Bayesian posterior probabilities (BP).The phylogenetic trees were visualized using FigTree v1.4.3 and subsequently edited in Adobe Photoshop.

Taxonomy
Culture characteristics.Colonies growing on PDA and CMA after 20 days of incubation at 28 °C.Colony on PDA slow-growing, surface thick, rosy buff to white, reverse white, raised, aerial hyphae abundance, reaching 30-33 mm diam., entire margin.Colonies on CMA flat, surface white, reverse translucent to pale white, aerial hyphae sparsely developed, reaching 40-42 mm diam.

Nalanthamala
Culture characteristics.Colonies growing on PDA and CMA after 3 days of incubation at 28 °C.Colony on PDA rapid-growing, surface dusty to fine powdery, white to iron gray due to occurrence of conidial masses, reverse white, aerial hyphae flocculent, reaching 53-57 mm diam., indistinct margin.Colony on CMA thin, surface white, reverse translucent, aerial hyphae sparsely developed, reaching 35-37 mm diam.

Discussion
Yunnan is uniquely situated at the confluence of three climatic zones: the eastern Asian monsoon zone, the Tibetan Plateau zone, and the tropical monsoon zones of southern Asia and Indochina (Yang et al. 2004).This diverse climatic environment and unique geographical position make the province one of the richest sources of fungi, covering over 40% of the known species in China (Feng and Yang 2018).Nectriaceae species are widespread worldwide, but only one Atractium species (A.fusiformis) and one Nalanthamala species (N.psidii) have been recorded from China (Schroers et al. 2005;Gräfenhan et al. 2011;Perera et al. 2023).Therefore, the proposed two new species further showed Yunnan's high fungi diversity.
Most of Nectriaceae species are soil-borne saprobes or plant pathogens (Lombard et al. 2015;Perera et al. 2023;Chen et al. 2024).Nonetheless, Atractium species are commonly associated with water (Gräfenhan et al. 2011).For instance, A. crassum was isolated from drinking water in Germany (Gräfenhan et al. 2011), and the three species A. holubovae, A. aquatica and A. fusiformis have been found on submerged decaying wood in the Philippines, France and China respectively (Seifert et al. 1995;Lechat and Fournier 2015;Bao et al. 2023).Only the type species A. stilbaster was discovered on bark or stump in Canada and Germany (Gräfenhan et al. 2011).Significantly, our proposed species A. yunnanense was discovered from soils in a karst rocky desertification area of Yunnan.This finding extends the habitat of Atractium species.
Currently, five species are accepted in Nalanthamala (Wijayawardene et al. 2020;Crous et al. 2021).The type species N. madreeya was isolated from dead stems in India; unfortunately, the type specimen could not be found in the herbaria and the species has not been recollected.Another four Nalanthamala species were reported as associated with wilt and blight diseases from several economically important crops and were more widespread (Schroers et al. 2005).N. psidii causes a destructive wilt disease to Psidium guajava trees and is distributed in Malaysia, South Africa, and Taiwan, possibly restricted to subtropical or tropical regions.N. vermoesenii could cause necrosis and blight to various Arecaceae species and is distributed in Australia, the Czech Republic, Spain and the Unites States, particularly known from warm temperate, Mediterranean, or (sub)tropical climates.N. diospyri could cause a destructive wilt to Diospyros virginiana and is distributed in the United States.Additionally, N. olivacea was isolated from palm in Mexico, Costa Rica, and the Philippines, possibly restricted to tropical regions.Overall, Nalanthamala species are mainly distributed in the warm temperate to tropical regions (Schroers et al. 2005).Our proposed new species N. xishuangbannaensis was isolated from the air in Xishuangbanna of Yunnan, which is a tropical region, and is also the first Nalanthamala species obtained from air.
Despite having cosmopolitan distribution of Nectriaceae species, Atractium and Nalanthamala species have fewer records in China.Significantly, A. fusiformis and A. yunnanense were first discovered from Yunnan (Bao et al. 2023).Two Nalanthamala species, N. psidii and N. xishuangbannaensis, were found in China.N. psidii, as a widespread species, was found in Taiwan (Rossman et al. 2013), and N. xishuangbannaensis was first reported from Yunnan.The discovery of these Atractium and Nalanthamala species in Yunnan proves once again that it is rich in fungal species.Further studies are needed to understand the pathogenicity of the newly discovered Atractium and Nalanthamala species.

Figure 1 .
Figure1.Phylogenetic tree inferred from a Maximum likelihood analysis based on a concatenated alignment of ITS, LSU, rpb2 and tub2 sequences of some representing species in Nectriaceae.The Bayesian posterior probabilities (BP) ≥ 0.9 and RAxML bootstrap support values (BS) ≥ 75% were shown at the nodes (BP/BS).Strains obtained in this study are shown in blue font.Ex-type strains are marked by a T after the strain number.The tree was rooted to Sarocladium summerbellii (CBS 430.70).0.04 Named after the location Xishuangbanna, where the holotype was collected.
Type.China • Yunnan province, Xishuangbanna Dai Autonomous Prefecture; isolated from air in forest; Apr 2017; Z.F.Yu, preserved by lyophilization (a metabolically inactive state) in State Key Laboratory for Conservation and Utilization of Bio-Resources in Yunnan (YMF 1.05062, holotype); ex-type living culture: CG-MCC 3.27596.