Neodactylariales, Neodactylariaceae (Dothideomycetes, Ascomycota): new order and family, with a new species from China

Abstract During a mycological survey of aquatic hyphomycetes on submerged decaying leaves in southwest China, a distinct new fungus was collected. The collection was cultured and sequenced and a BLAST search of its ITS and LSU sequence against data in GenBank revealed a dothideomycetous affiliation, with the closest related taxa in the genus Neodactylaria. Phylogenetic analyses of a multigene matrix containing sequences from four genes (LSU, SSU, rpb2, and tef1), representing broad groups of Dothideomycetes, revealed its placement within Dothideomycetes, but without a supported familial or ordinal affiliation. Based on further phylogenetic analyses and morphological investigations, the new fungus is described here as a new species of Neodactylaria, N. simaoensissp. nov., and placed in a new family Neodactylariaceaefam. nov. and a new order Neodactylarialesord. nov.


Introduction
The kingdom Fungi contains an estimated 700,000 to over 5 million species, amongst which only about 120,000 have been described (Lynne 2016). Dothideomycetes is one of the largest and most significant classes of fungi within Ascomycota (Kirk et al. 2008;Schoch et al. 2009a;Hyde et al. 2013). Thousands of species have been included in the class Dothideomycetes, and many of them are important plant pathogens (Cortinas et al. 2006;Crous et al. 2007;Wikee et al. 2011Wikee et al. , 2013aManamgoda et al. 2012), human and animal pathogens (Siu and Lzumi 2004;da Cunha et al. 2012da Cunha et al. , 2013, or used in biotechnological applications (Verkley et al. 2004;Damm et al. 2008;de Wit et al. 2012;Ohm et al. 2012;Stergiopoulos et al. 2012;Hyde et al. 2014). The members of Dothideomycetes are still increasing with the discovery of many novel species and inclusion of DNA sequence data. In the past few years, molecular phylogenetic studies have advanced our understanding of the systematics of Dothideomycetes (Inderbitzin et al. 2001;Schoch et al. 2009b;Hirayama et al. 2010;Suetrong et al. 2011;Hyde et al. 2013;Wijayawardene et al. 2014;Liu et al. 2017;Jiang et al. 2020). Wijayawardene et al. (2014) recommended 23 orders and 110 families in Dothideomycetes based on culture characteristics and molecular phylogenetic analyses. More recently, Liu et al. (2017) provided an updated phylogenetic assessment of Dothideomycetes at the order level by using molecular clock methods and accepted 29 orders. However, the latest research by Wijayawardene et al. (2018) expanded this to 33. Despite the progress in our understanding of the systematics of Dothideomycetes, a number of newly described and/or previously reported taxa are currently incertae sedis and their family and order level positions within the Dothideomycetes remain obscure; many taxa lack sequencing data or appropriate classification rank to accommodate them (Hyde et al. 2013;Wijayawardene et al. 2018).
The genus Neodactylaria Guevara-Suarez et al., typified by N. obpyriformis Guevara-Suarez et al., was originally described from human bronchoalveolar lavage in the USA ). The genus is characterized by having integrated, polyblastic and sympodial extended conidiogenous cells producing solitary, septate, obpyriform or rostrate conidia . Morphologically, Neodactylaria is similar to two Dactylaria species, D. kumamotoensis Matsush. and D. madrasensis Matsush., and several Pyricularia species, such as P. grisea Cooke ex Sacc. and P. pennisetigena Klaubauf, M.-H. Lebrun & Crous. However, in the phylogeny inferred from sequences of the large subunits of nuclear ribosomal DNA (LSU), Neodactylaria was placed within Dothideomycetes, but the ordinal and familial position was unresolved.
Southwestern China is one of the world's 34 biodiversity hotspots (Myers et al. 2000;Zhang et al. 2020). During a survey of aquatic hyphomycetes on submerged decaying leaves from this area, several new species have been reported (Guo et al. 2019;Qiao et al. 2019a, b;Yu et al. 2019). In a further study, an unidentified fungus was collected, which had a similar morphology to Heliocephala proliferans V. Rao et al. (Pezizomycotina incertae sedis;Rao et al. 1984;Mel'nik et al. 2013), but detailed morphological examination showed that the conidiogenous cells were terminal or intercalary, with short-cylindrical denticles, and the conidia were 1-or 2-septate and constricted at the septum. Sequence data obtained from cultures of conidia confirmed that this species does not belong in Heliocephala. A BLAST search of its LSU gene sequences against the public sequence records in GenBank (Sayers et al. 2019) confirmed its dothideo-mycetous affinity and that it was closely related to members of the genus Neodactylaria. Subsequently, we obtained the type species of Neodactylaria, N. obpyriformis Guevara-Suarez et al., from the CBS-KNAW Fungal Biodiversity Centre (Netherlands) and processed it with full morphological and phylogenetic analyses. Our new collection prompted the study of the molecular phylogenetic relationships of taxa within Neodactylaria, as well as the higher order phylogenetic relationship of Neodactylaria within the Dothideomycetes.
Our comparative analyses identified that the newly collected fungus is a species of Neodactylaria, N. simaoensis. However, due to their significant divergence, there was no apparent family or order for placement of Neodactylaria. We propose that the genus be placed in a new family and new order within Dothideomycetes.

Isolation and morphological study
Submerged dicotyledonous leaves were collected from a stream in Simao, Yunnan Province, southern China. Samples were preserved in zip-lock plastic bags, labelled and transported to the laboratory. Each rotted leaf was cut into several 3-4 × 4-5 cm sized fragments, and these were incubated on CMA (20 g cornmeal, 18 g agar, 1000 ml distilled water), supplemented by two antibiotics (penicillin G, 0.04 g/l; and streptomycin, 0.03 g/l; Gams et al. 1998), for 5 days at room temperature. Individual conidia were isolated using a sterilised toothpick under a BX51 microscope and cultivated on CMA plates. Morphological observations were conducted on cultures growing on CMA after incubation at 25 °C for 1 week. Colony colour was based on the colour charts of Rayner (1970).
Pure cultures have been deposited in the Herbarium of the Laboratory for Conservation and Utilization of Bio-resources, Yunnan University, Kunming, Yunnan, P.R. China (YMF, formerly Key Laboratory of Industrial Microbiology and Fermentation Technology of Yunnan).

DNA extraction, polymerase chain reaction (PCR) amplification and sequencing
Pure cultures were grown on PDA for 5 days at 25 °C. Actively-growing mycelia were scraped off the surface of a culture and transferred to 2 ml Eppendorf micro-centrifuge tubes. Total genomic DNA was extracted according to the procedures in Turner et al. (1997). To determine the phylogenetic position of Neodactylaria, we amplified five nuclear genomic loci, including the internal transcribed spacer (ITS), the 28S large subunit ribosomal RNA (LSU), the 18S small subunit ribosomal RNA (SSU), the translation elongation factor1-alpha partial gene (tef1) and the RNA polymerase II subunit 2 (rpb2). The following primers were used: the ITS region was amplified us-ing the primers ITS1 and ITS4 (White et al. 1990); the LSU nuc rDNA region was amplified with primers LROR and LR7 (Vilgalys and Hester 1990); the SSU nuc rDNA region was amplified with primers NS1 and NS4 (White et al. 1990); an approx. 1.1 kb fragment of the rpb2 gene was amplified using the primer pair fRPB2-5f and fRPB2-7cr (Liu et al. 1999); an approximately 1.0 kb fragment of the tef1 gene was amplified with the primers TEF983F and TEF2218R (initially obtained from S. Rehner: http://ocid.nacse.org/research/deephyphae/EF1primer.pdf ).
PCR reactions were prepared in a 25 μl final volume as described by Zheng et al. (2019Zheng et al. ( , 2020a. PCR amplifications were performed in an Eppendorf Mastercycler thermal cycler. PCR conditions were as follows: an initial 4 min denaturing step at 94 °C, followed by 35 cycles of 75 s at 94 °C, 90 s at 52 °C (for rpb2, LSU, and SSU) and 100 s at 72 °C. After a final extension step of 7 min at 72 °C, the samples were stored at 4 °C. Conditions for amplification of the ITS and tef1 regions were an initial step of three cycles at an annealing temperature of 54 °C, followed by 30 cycles with the annealing temperature set at 48 °C. When needed, a 'touchdown' (Don et al. 1991) protocol preceded the PCR cycle. PCR products were then purified using a commercial kit (Bioteke Biotechnology Co. Ltd, China). Each fragment was sequenced from both directions using the forward and reverse primers in separate reactions using a LI-COR 4000L automatic sequencer as described by Kindermann et al. (1998). The sequences obtained have been submitted to GenBank at the National Center for Biotechnology Information (NCBI) and the accession numbers are listed in Table 1.

Sequence alignment and phylogenetic analysis
Preliminary BLAST searches with ITS, SSU, LSU, rpb2, and tef1 gene sequences of the new isolate against GenBank and UNITE databases (Nilsson et al. 2019) identified sequences closely related to our isolates. However, we were only able to robustly determine their placements within the class Dothideomycetes. To infer a phylogenetic relationship for our strain, an initial alignment of the newly generated sequences (SSU, LSU, rpb2, and tef1) and 74 representatives belonging to 33 orders of the Dothideomycetes, extracted from recent studies (Mapook et al. 2016;Nieuwenhuijzen et al. 2016;Voglmayr et al. 2016;Hernandez-Restrepo et al. 2017;Liu et al. 2017;Wijayawardene et al. 2018) with a species from the sibling class, Arthoniomycetes, as the outgroup, was performed using the online MAFFT interface (Katoh and Standley 2013; http://mafft.cbrc.jp/alignment/ server). This alignment was used to infer a preliminary phylogenetic relationship for the new sequences based on Bayesian inference (BI) analyses (data not shown).
Based on the initial analysis, a second alignment combined SSU, LSU, and tef1 sequence data were constructed from the closest relatives to our strain in Botryosphaeriales, Dothideales, Hysteriales, Minutisphaerales, Myriangiales, Patellariales, Phaeotrichales, Pleosporales, Tubeufiales, and Venturiales. In the second alignment, Schismatomma decolorans (DUKE 47570) was used as an outgroup taxon. All sequence data were aligned using MAFFT (v. 7.110) online program (http://mafft.cbrc.jp/alignment/server/) (Katoh and Standley 2013). The alignments were checked and uninformative gaps minimized manually where necessary in BioEdit 7.0.1 (Hall 1999). Maximum likelihood (ML) and BI were used in the analyses following the methodology as described in Mapook et al. (2016). The nucleotide substitution models use for analyses was determined using jModelTest 2.0 (Posada 2008). The GTR+I+G model with inverse gamma rate were selected for individual data from each partition with the combined aligned dataset. The phylogenetic tree was visualized in FigTree v. 1.4 (Rambaut 2012) and the layout of the tree was done in Adobe Illustrator v. CS5.1. The alignment of phylogenetic analyses was deposited in TreeBASE (https://www.treebase.org, submission number 24051).

Molecular phylogeny
Following the results of preliminary phylogenetic analysis of the initial alignment (data not shown), the phylogenetic reconstruction of the second alignment was performed including SSU, LSU, and tef1sequences from 53 strains representing 10 different orders in the Dothideomycetes and one order in the Arthoniomycetes (Table 1). The three-gene dataset comprised of LSU sequences for all 52 ingroup sequences, 50 SSU sequences, and 36 tef1 sequences. After exclusion of ambiguous regions and introns, the combined dataset included 2555 characters (826 for LSU, 1012 for SSU, and 717 for tef1). In the BI analysis, the alignment has 952 distinct patterns, 600 parsimonyinformative, 205 singleton sites, and 1750 constant sites. The best tree (RAxML) obtained using the ML analysis is shown as Fig. 1, with the support values from the ML and BI analyses plotted at the nodes. In this tree, our newly proposed species and N. obpyriformis formed a distinct clade within Dothideomycetes with significant ML bootstrap support (100%) and Bayesian sposterior probability (1.0). Moverover, the Neodactylaria clade is sister to the Pleosporales clade, but only with low bootstrap support values (51%) and Bayesian posterior probabilities (0.72). The results suggested that our strain belongs to the genus Neodactylaria. The order Pleosporales has characters that are very different from those of species of Neodactylaria and, therefore, we introduce a new order and new family, Neodactylariales and Neodactylariaceae, respectively, for this group of fungi. In addition, combined with morphological differences, our strain was described and illustrated herein as a new species of Neodactylaria.  Description. Mycelium superficial or immersed, composed of branched, septate, smooth-walled, hyaline to subhyaline hyphae. Conidiophores macronematous, mononematous, straight or flexuous, septate, unbranched, smooth-walled, pale to midbrown. Conidiogenous cells polyblastic, sympodial extended, integrated, terminal or intercalary, denticulate, with short cylindrical denticles, pale to medium-brown. Conidial secession schizolytic. Conidia obpyriform to obclavate, unicellular or septate, attenuate, subulate or rostrate toward the obtuse apex, with a tiny, protuberant basal hilum, smooth or finely echinulate, subhyaline or pale brown. Sexual morph not observed. Diagnosis. It is characterised by straight or flexuous, 2-4-septate, unbranched conidiophores, with denticulate conidiogenous cells and obclavate to long obpyriform, subulate or slightly rostrate towards the obtuse or rounded apex and 1-2 (-3)-septate conidia. Differs from N. obpyriformis by longer and slightly wider conidia and more septa.
Culture characteristics. Colonies attaining 1 cm in diameter on CMA after 7 days at 25 °C. On CMA, colonies flat, floccose at the centre, lacking aerial mycelium towards periphery, white to cream-coloured, reverse same colour, sporulation abundant. On PDA, colonies flat, white to cream-coloured, margin entire; sporulation sparse.

Discussion
Aquatic hyphomycetes, which have always been important members of the Dothideomycetes, play critical roles in the decomposition of organic compounds and nutrient cycling in aquatic habitats. Since Ingold (1942Ingold ( , 1943 first reported aquatic hyphomycetes in the 1940s, research on this group have been steadily increasing throughout the world. It was estimated that over 300 species of over 80 genera of aquatic hyphomycetes are reported worldwide (Kirk et al. 2008;Guo et al. 2015). Studies of aquatic hyphomycetes have revealed a huge fungal diversity. Our study again underlined the importance of these microorganisms for fungal taxonomic discovery.
In this study, a preliminary phylogenetic analysis combined SSU, LSU, rpb2, and tef1sequences from 74 representative taxa of Dothideomycetes and Arthoniomycetes revealed the Neodactylaria as a unique clade within Dothideomycetes (data not shown). The second phylogenetic analyses using three loci (SSU, LSU, tef1) also showed our new collected strain and N. obpyriformis form a strongly supported monophyletic and distinct clade (ML-BS = 100%, BPP = 1.0) within the Dothideomycetes (Fig. 1). In this tree, the Neodactylaria clade is close to the Pleosporales but with low support (ML-BS = 51%, BPP = 0.72). The original study on N. obpyriformis, which conducted a phylogenetic analysis of the LSU sequence, also showed that Neodactylaria is related to Dothideomycetes, but with an uncertain taxonomic position at the ordinal level and family level ). Thus, we establish a new order (Neodactylariales) and family (Neodactylariaceae) within the Dothideomycetes for this unique clade.
The genus Neodactylaria is morphologically similar to two species of the genus Dactylaria, D. kumamotoensis and D. madresensis, which were described by Matsushima from soil and plant debris in Japan and India, respectively (Matsushima 1981(Matsushima , 1984. Although these two fungi in Dactylaria could be congeneric with N. simaoensis, they are only known from the type collection and no living cultures are available for molecular comparison. Morphologically, the conidia of N. simaoensis are smaller than D. kumamotoensis and are distinguished from D. madresensis by their size and the number of septa. In addition, the genus Dactylaria is heterogeneous. Related information showed that the classification position of D. kumamotoensis was in the order Helotiales, the class Leotiomycetes (http://www.indexfungorum.org/Names/NamesRecord. asp?RecordID=111390), but most Dactylaria species were placed in the Sordariomycetes . Thus, although the genus Neodactylaria shares some morphological characters with the genus Dactylaria, Neodactylaria was placed in the Dothideomycetes by phylogenetical analysis and was phylogenetically distant from Dactylaria.
In the Dothideomycetes, many orders show various morphological characteristics and lifestyles, such as the order Pleosporales. In our new order, the two species within genus Neodactylaria also have different habitats: N. obpyriformis was found from human bronchoalveolar lavage in the USA, but N. simaoensis was found from submerged decaying leaves in China. Therefore, it seems fungi in this genus may be broadly distributed in different habitats.
The class Dothideomycetes is one of the most important and diverse classes in the phylum Ascomycota. It comprises pathogenic fungi, aquatic hyphomycetes, fungi with different life cycles and habitats, and also fungi with biotechnological potential (Wijayawardene et al. 2014;Santos et al. 2015;Woudenberg et al. 2015;Zheng et al. 2020b). In recent years, this class has received significant attention, and several papers have highlighted its importance to fungal taxonomy, based on its fungal diversity and on new studies performed to improve the classification of dothideomycetous fungi (Schoch et al. 2009a;Hyde et al. 2013;Wijayawardene et al. 2014). In Dothideomycetes, most families comprise both sexual genera and asexual genera and only a few families are totally comprised of asexual genera, such as Cladosporiaceae Nann., which contains seven asexual hyphomycetous genera and Neodevriesiaceae Quaedvlieg & Crous, which contains one asexual hyphomycetous genus (Wijayawardene et al. 2014). However, the order Lichenoconiales, only comprising one family, was also established with an asexual genus (Hyde et al. 2013). Here, we added a new order containing only an asexual genus to Dothideomycetes. These results show asexual genera have equal status to sexual genera at various taxon ranks. In addition, the description of Neodactylariales, as a new order in this study, highlights the need to collect fungal biodiversity from a range of diverse environments and substrates, as these diverse niches frequently harbour fungal lineages that are still missing in current phylogenetic studies.