A new species of Dictyochaeta (Sordariomycetes, Chaetosphaeriales, Chaetosphaeriaceae) from freshwater habitats in China

Abstract Background Freshwater fungi refer to the fungi that depend on the freshwater habitats for the whole life cycle or part of their life cycle. In this context, a new aquatic hyphomycete was isolated from decaying wood in a freshwater habitat in Jiangxi Province, China. New information Dictyochaetajiangxiensis sp. nov., a new aquatic hyphomycete, is characterised by its unbranched, septate, base-fertile conidiophores with multisepta and single phialide at the apex, brown, sterile seta, monophialidic, subcylindrical conidiogenous cells narrowing below the funnel-shaped collarette, hyaline, unicellular, thin-walled, smooth, guttulate, falcate to subclavate conidia narrowly rounded at both ends with hair-like appendages. Phylogenetically, the new species Dictyochaetajiangxiensis clustered together with Dictyochaetabrevis MFLU 19-0216 in a well-supported clade, but formed a separate branch. In order to better define the taxonomic status of the new species, a phylogenetic tree of most closely-related taxa in Chaetosphaeriaceae was established, based on multi-locus sequences (ITS and LSU). The novel species is described and illustrated. Newly-generated molecular data of Dictyochaetajiangxiensis is also provided.


Introduction
established the genus Dictyochaeta with D. fuegiana as type species, which was isolated from fallen leaves of Nothofagus betuloides (Mirb.)Oerst.Gamundí et al. (1977) and Godeas et al. (1977) verified the holotype and redescribed it.Later, Réblová (2004) re-examined the type species and gave more detailed description about the genus Dictyochaeta, as two-layer conidiophores, the upper layer setiform, when sterile, monophialidic or rarely polyphialidic, the lower layer always fertile, monophialidic, rarely polyphialidic, collarette on conidiogenous cells and aseptate, hyaline, falcate conidia without setulae.Since then, more and more species have been discovered and classified as or transferred to Dictyochaeta and its molecular and morphological data have been expanded.According to literature and herbarium records, the Dictyochaeta-like fungi are globally distributed in the Holarctic Region and the Tropics and grow on decaying plant, such as bark, wood, bamboo culms, palm fronds, fallen leaves and petioles in freshwater and terrestrial environments.They also occur as plant pathogens or endophytes in living plants.(Agnihothrudu 1968, Lunghini et al. 1971, Shearer and Crane 1971, Sutton and Hodges 1975, Hewings and Crane 1981, Holubová-Jechová 1984, Kuthubutheen 1987, Kuthubutheen and Nawawi 1990, Kuthubutheen and Nawawi 1991a, Kuthubutheen and Nawawi 1991b, Réblová et al. 1999, Kirschner and Chen 2002, Crous et al. 2014, Crous et al. 2015, Maharachchikumbura et al. 2016).
There are always different opinions on the classification of Dictyochaeta, which is considered as a synonym of several genera, such as Codinaea Maire., Menispora Pers.and Menisporopsis S. Hughes.Previously, Dictyochaeta was usuallly regarded as a synonym of Codinaea ( Maire 1937), because they share greatly similar features on phialidic conidiogenous cells and setae, but differ mainly in the conidia without setulae (Cai et al. 2006).Crous et al. (2018) thought that priority should be given to the older name, Dictyochaeta.Réblová (2000) recommended that species with setulae should be classified into Codinaea and those without setulae into Dictyochaeta.Réblová et al. (2021a) reevaluated the concept of Dictyochaeta and revised species delimitation, based on six loci (ITS, LSU, SSU, RPB2, TEF1-α, TUB2) along with comparative morphological and cultivation studies.In their study, some species of Dictyochaeta, such as D. siamensis, D. simplex etc. clustered within the clade Codinaea with a high support.As for the demarcation between Dictyochaeta and Codinaea, Réblová et al. (2021a) supported using conidial appendages as a classification criterion to distinguish Dictyochaeta from Codinaea (Réblová and Winka 2000).Based on revised species, morphological characteristics of conidia (shape, septation, absence or presence of setulae), collarettes (shape) and setae (presence or absence) and extension of the conidiogenous cell proved to be important at the generic level.To date, Dictyochaeta-like fungi, together with Codinaea-like fungi, were divided into five lineages in the phylogenetic analyses (Réblová et al. 2021b).Dual DNA barcoding and ancestral reconstruction of ecological and geographic distribution facilitated re-assessment of Dictyochaeta-like fungi.Réblová et al. (2021b) introduced five genera (Codinaeella, Nimesporella, Stilbochaeta, Tainosphaeriella and Xyladelphia) to accommodate Codinaea-like fungi and retained the taxonomic status of Dictyochaeta sensu stricto.

Sample collection and specimen examination
Submerged wood samples were collected in a stream from Jishui County, Ji'an City, Jiangxi Province, China on 9 April 2018.The samples were taken to the laboratory in ziplock bags and placed in plastic boxes.The microscopic analysis was performed by a stereomicroscope to observe the fungal fruiting body on a natural substrate.Microexamination and photomicrographs were taken under a compound microscope (Nikon Ni).The specimens were deposited in the Herbarium of Fungi, Jiangxi Agricultural University (HFJAU), Nanchang, China.

DNA extraction, PCR amplification and sequencing
Cultures were grown at room temperature on potato-dextrose agar (PDA).Mycelia were directly scraped off from plates and transferred into centrifugal tube after fragmentation.DNA was extracted with the CTAB method following Doyle and Doyle (1987).Approximately 500 mg of mycelium was mixed with ca.0.2 g of white quartz sand and ground with preheated (ca.65°C) 2 × CTAB buffer [2% (w/v) CTAB; 100 mM Tris-HCl; 1.4 M NaCl; 20 mM EDTA, pH 8.0].DNA was extracted by chloroform:isoamyl alcohol (24:1) and precipitated by isopropanol at -20°C.The DNA precipitation was purified by 70% ethanol to remove remaining impurities.Approximately 50-100 μl TE buffer or deionised water were added and stored at -20°C.Dried DNA was dissolved in deionised water at 37°C and stored at -20°C.

Phylogenetic analyses
The novel sequences and reference sequences collected from GenBank were aligned with MAFFT v.7.036 (http://mafft.cbrc.jp/alignment/server,Katoh et al. (2019)).The multilocus sequences were concatenated by PhyloSuite v. 1.2.2 (Zhang et al. 2020).The concatenated aligned datasets were analysed separately using Maximum Likelihood (ML) and Bayesian Inference (BI).The best-fit models of evolution for the two loci tested were estimated by PhyloSuite v. 1.2.2 (Zhang et al. 2020).The ML analyses were conducted with RAxML v.7.2.6 (Stamatakis and Alachiotis 2010) using a GTRGAMMA substitution model with 1000 bootstrap replicates.The robustness of the analyses was evaluated by bootstrap support (MLBS).Markov Chain Monte Carlo (MCMC) methods in MrBayes was used to estimate the posterior probabilities (PP) (Zhaxybayeva and Gogarten 2002).Trees were sampled every 100 generations.The MCMC sampling was set as four chains (three hot chains and one cold chain) running 2,000,000 generations simultaneously, resulting in 20001 total trees.The first 25% of trees were discarded as burn-in trees and the remaining trees were used to calculate posterior probabilities.Posterior probabilities values of the BI analyses (BPP) over 0.95 were regarded to be important.Sequences generated in this study were displayed in GenBank (Table 1).4.9 μm at the opening, 0.8-1.1 μm at deep.Conidia accumulating at the heads white, 23-32 × 2.5-3.2μm (av.= 26.1 × 2.9 µm, n = 30), hyaline, unicellular, thin-walled, smooth, abundant guttulate, falcate to subclavate, rarely straight, narrowly rounded at the both ends, with 6-11 μm long hair-like appendages at both ends, smooth (Fig. 2).

Species
Culture characteristics: Conidia germinating on PDA within 12 h.Colonies growing on PDA, reaching 20-30 mm diam.after 3 weeks at 28°C, circular, white to pale grey mycelium with hyaline margin, centre lightly raised, pale brown to dark brown in reverse, with smooth margin.

Etymology
'jiangxiensis' refering to the host location, Jiangxi Province, where the holotype was collected.

Notes
Dictyochaeta jiangxiensis is a distinct species in the genus as supported by molecular phylogenetic analysis and it clusters with D. brevis, but the latter has smaller conidia (7.5-11.4µm long, 2.0-2.9 μm wide; Lin et al. ( 2019)).We found that there was 8% nucleotide difference of ITS sequences and about 2% nucleotide difference between

Analysis Phylogenetic analyses
Based on ITS and LSU, a multi-locus phylogenetic tree was established to demonstrate the relationships between the new species and related taxa in Chaetosphaeriaceae (Fig. 1).
The alignment has 1767 characters (including alignment gaps), with 715 characters for ITS and 1052 characters for LSU.The ML analysis result showed coincident topology with BI.Fig. 1 shows the ML tree based on the combined dataset, along with the fully supported bootstrap values and Bayesian posterior probabilities.All phylogenetic trees were similar in topologies.
The new species Dictyochaeta jiangxiensis, together with Dictyochaeta brevis MFLU 19-0216, formed a well-supported clade (BPP = 1.00;MLBS = 100%), but formed a separate branch and there were obvious differences between them.By comparing the ITS and LSU sequences of Dictyochaeta jiangxiensis sp.nov.JAUCC2824 and Dictyochaeta brevis MFLU 19-0216 respectively in NCBI, we found that there was 8% nucleotide difference of ITS sequences and about 2% nucleotide difference between the LSU sequences of Dictyochaeta jiangxiensis sp.nov JAUCC2824 and Dictyochaeta brevis MFLU 19-0216.

Discussion
Freshwater fungi refer to the fungi that rely on the freshwater habitats for the whole life cycle or part of the life cycle.Phylogenetic studies on freshwater ascomycetes have shown that some species cluster with terrestrial ascomycete lineages, while others cluster with exclusive aquatic lineages (Raja et al. 2018).However, Shearer (1993) defined freshwater fungi as "fungi that must rely on the freshwater environment to complete their life cycle".The concept of aquatic fungi in a broad sense was adopted in this study.
Chaetosphaeriaceae is a huge and diverse group with overwhelmingly phialidic fungi and some members of Chaetosphaeriaceae possess known teleomorphs.The family has a world-wide distribution.They are predominantly isolated from soil and plant debris, some are endophytic and have been isolated from herbaceous plants (Hughes and Kendrick 1968, Réblová 2004, Fernández and Huhndorf 2005, Huhndorf and Fernández 2005, Crous et al. 2012, Yang et al. 2018, Lin et al. 2019, Luo et al. 2019).In this study, the new Dictyochaeta species in the family Chaetosphaeriaceae was isolated from a freshwater environment.Most known species in Dictyochaeta were reported from rotting parts of plants, such as decaying leaf, bark or stem and submerged wood, partly from soil.Previously, Dictyochaeta and Codinaea were hard to demarcate, not only because of their morphological and ecological similarities, but their closely-related phylogenetic relationship.
The difference between Dictyochaeta and Codinaea lies in the presence or absence of setae.The taxonomy of these fungi has relied mainly on morphological criteria.However, it is hard to treat setae as a criterion for identification as setae are always irregular amongst these similar taxa as mentioned above.Codinaea was introduced to accommodate a single species, C. aristata.Since then, the type species of Codinaea has become a taxonomic bottleneck.This species has not been recorded in any literature since its initial description.
The holotype material and molecular data could not be traced.That is why the phylogenetic statuses of the Dictyochaeta-like fungi are still ambiguous.Hughes and Kendrick (1968) made an attempt at using the name Dictyochaeta instead of Codinaea on account of the principle of priority and suggested to adopt the name Codinaea as the type material for D. fuegiana of Dictyochaeta.Since Gamundí et al. (1977) redescribed D. fuegiana from fresh material, Dictyochaeta became a precedently used name.Crous et al. (2018) accepted this treatment.Simultaneously, the name Codinaea was suggested to be treated as the type material for D. fuegiana of Dictyochaeta by Hughes and Kendrick (1968), but this view has great limitations.Shortage of abundant original descriptions and loss of the type material of Codinaea were the factors (Liu et al. 2016) which indicated that the molecular phylogeny of Dictyochaeta has not been solved due to the small number of sequences in GenBank.The species, thus, need recollecting, epitypifying and sequencing to establish which morphological characters are of taxonomic significance and generic boundaries.
Recently, Dictyochaeta has still not been classified as monophyletic even though most Dictyochaeta-like and Codinaea-like species were re-assessed and recognised as five genera: Codinaeella, Nimesporella, Stilbochaeta, Tainosphaeriella and Xyladelphia (Réblová et al. 2021b).Réblová et al. (2021b) indicated Codinaea is a highly polyphyletic taxon unrelated to Dictyochaeta and that its original delimitation, based on a single morphotype of C. aristata, is too narrow and unsustainable and they emphasise the importance of combination of microscopic morphological characters developed in culture and under a natural substrate for identification.In our analysis, the phylogenetic position of some taxa, such as Zignoëlla and Menispora, are unclear in the tree with low BS and PP values.Kionochaeta formed a sister clade with Dictyochaeta and they were clustered together, but with low BS and PP values.Quite a number of species have not been adopted to establish the phylogenetic tree on account of the absence of molecular data and type materials.The molecular database and materials of Chaetosphaeriaceae species needs to be supplemented and improved.

Figure 1 .
Figure 1.Phylogenetic tree based on combined ITS, LSU sequences of most taxa of the Chaetosphaeriaceae.Species name given in bold red is a new taxon in this study; species name given in bold indicates a type strain, respectively.Asterisk (*) indicates branches with MLBS = 100% and PP value = 1.0.The ML bootstrap support values and Bayesian posterior probabilities are given above the branches (MLBS/BPP).The tree is rooted to Tracylla aristata CPC 25500 and Tracylla eucalypti CPC:31806.