Research Article
Print
Research Article
Neodactylariales, Neodactylariaceae (Dothideomycetes, Ascomycota): new order and family, with a new species from China
expand article infoMin Qiao, Hua Zheng, Ruili Lv, Zefen Yu
‡ Yunnan University, Kunming, China
Open Access

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. simaoensis sp. nov., and placed in a new family Neodactylariaceae fam. nov. and a new order Neodactylariales ord. nov.

Keywords

Dothideomycetes, new family, new order, new species, phylogenetic analysis, taxonomy

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. 2011, 2013a, b; Manamgoda et al. 2012), human and animal pathogens (Siu and Lzumi 2004; da Cunha et al. 2012, 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 (Crous et al. 2017). The genus is characterized by having integrated, polyblastic and sympodial extended conidiogenous cells producing solitary, septate, obpyriform or rostrate conidia (Crous et al. 2017). 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 dothideomycetous 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.

Materials and methods

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 using 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. (2019, 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.

Table 1.

Species, strains, and their corresponding GenBank accession numbers of sequences used for phylogenetic analyses.

Species Straina,b GenBank accession numbersc
LSU SSU tef1
Acanthostigma chiangmaiense Boonmee & K.D. Hyde MFLUCC 10-0125T JN865197 JN865185 KF301560
Allophaeosphaeria muriformis Ariyaw., Camporesi & K.D. Hyde MFLUCC 13-0349T KP765681 KP765682
Bambusaria bambusae (J.N. Kapoor & H.S. Gill) Jaklitsch, D.Q. Dai, K.D. Hyde and Voglmayr CBS 139763 KP687813 KP687962 KP687983
Botryobambusa fusicoccum Phook., Jian K. Liu & K.D. Hyde MFLUCC 11-0143T JX646809 JX646826
Botryosphaeria agaves (Henn.) E.J. Butler MFLUCC 11-0125T JX646808 JX646825
Botryosphaeria dothidea (Moug.) Ces. & De Not. CBS 115476 DQ678051 DQ677998 DQ767637
Cophinforma atrovirens (Mehl & Slippers) A. Alves & A.J.L. Phillips MFLUCC 11-0425T JX646817 JX646833
Dematiopleospora mariae Wanas., Camporesi, E.B.G. Jones & K.D. Hyde MFLUCC 13-0612T KJ749653 KJ749652 KJ749655
Dothidea hippophaes Fuckel CBS 188.58 DQ678048 U42475 DQ677887
Dothidea insculpta Wallr. CBS 189.58 DQ247802 DQ247810 DQ471081
Gloniopsis praelonga (Schwein.) Underw. & Earle CBS 112415 FJ161173 FJ161134 FJ161090
Helicangiospora lignicola Boonmee, Bhat & K.D. Hyde MFLUCC 11-0378T KF301531 KF301539 KF301552
Helicoma chiangraiense Boonmee & K.D. Hyde MFLUCC 10-0115 JN865188 JN865176 KF301551
Helicoma fagacearum Boonmee & K.D. Hyde MFLUCC 11-0379 KF301532 KF301540 KF301553
Hysterium angustatum Alb. & Schwein. CBS 236.34 FJ161180 GU397359 FJ161096
Hysterobrevium smilacis (Schwein.) E. Boehm & C.L. Schoch CBS 114601 FJ161174 FJ161135 FJ161091
Hysteropatella clavispora (Peck) Höhn. CBS 247.34 AY541493 DQ678006 DQ677901
Kellermania macrospora (Durieu & Mont.) Minnis & A.H. Kenn. CBS 131716T JX444874 JX444902
Kellermania yuccigena Ellis & Everh. CBS 131727 JX444883 JX444908
Minutisphaera aspera Raja, Oberlies, Shearer & A.N. Mill. DSM 29478T KP309993 KP309999
Minutisphaera fimbriatispora Shearer, A.N. Mill. & A. Ferrer A242-8a HM196367 HM196374
Minutisphaera japonica Kaz. Tanaka, Raja & Shearer JCM 18560T AB733440 AB733434
Murispora rubicunda (Niessl) Y. Zhang ter, J. Fourn. & K.D. Hyde IFRD 2017 FJ795507 GU456308 GU456289
Myriangium duriaei Mont. & Berk. CBS 260.36 DQ678059 AY016347 DQ677900
Myrmaecium rubrum (Aptroot, Aa & Petrini) Jaklitsch & Voglmayr CBS 109505 GU456324 GU456303 GU456260
Myrmaecium fulvopruinatum (Berk.) Jaklitsch & Voglmayr CBS 139058 KP687861 KP687968 KP688030
Myrmaecium rubricosum (Fr.) Fuckel CBS 139068 KP687885 KP687979 KP688053
Neodactylaria obpyriformis Guevara-Suarez, Deanna A. Sutton, Wiederh. & Gené CBS 142668 MK562751 MK562750
Neodactylaria simaoensis H. Zheng & Z.F. Yu YMF 1.3984 MH379210 MK562747 MK562748
Oedohysterium insidens (Schwein.) E. Boehm & C.L. Schoch CBS 238.34 FJ161182 FJ161142 FJ161097
Parawiesneriomyces syzygii Crous & M.J. Wingf. CBS 141333T KX228339
Patellaria atrata (Hedw.) Fr. CBS 958.97 GU301855 GU296181 GU349038
Phaeotrichum benjaminii Malloch & Cain CBS 541.72 AY004340 AY016348 DQ677892
Phyllosticta ampelicida (Engelm.) Aa CBS 237.48 DQ678085 DQ678034
Phyllosticta citricarpa (McAlpine) Aa CBS 102374 GU301815 GU296151 GU349053
Populocrescentia forlicesenensis Wanas., Camporesi, E.B.G. Jones & K.D. Hyde MFLUCC 14-0651T KT306952 KT306955
Pseudogliophragma indica Phadke & V.G. Rao MTCC 11985T KM052851 KM052852
Psiloglonium araucanum (Speg.) E. Boehm, Marinc. & C.L. Schoch CBS 112412 FJ161172 FJ161133 FJ161089
Saccharata proteae (Wakef.) Denman & Crous CBS 115206 GU301869 GU296194 GU349030
Schismatomma decolorans (Erichsen) Clauzade & Vězda DUKE 47570 AY548815 AY548809 DQ883725
Speiropsis pedatospora Tubaki CBS 397.59 KR869797
Trematosphaeria pertusa Fuckel CBS 122368 FJ201990 FJ201991 GU456276
Trematosphaeria pertusa Fuckel CBS 122371 FJ201992 FJ201993 GU349085
Trichodelitschia bisporula (P. Crouan & H. Crouan) Munk CBS 262.69 GU348996 GU349000 GU349020
Trichodelitschia munkii N. Lundq. Kruys 201 DQ384096 DQ384070
Tubeufia chiangmaiensis Boonmee & K.D. Hyde MFLUCC 11-0514T KF301538 KF301543 KF301557
Tubeufia javanica Penz. & Sacc. MFLUCC 12-0545T KJ880036 KJ880035 KJ880037
Valsaria insitiva (Tode) Ces. & De Not. CBS 127882T KP687886 KP687980 KP688054
Valsaria lopadostomoides Jaklitsch & Voglmayr CBS 139062T KP687868 KP687972 KP688037
Valsaria neotropica Jaklitsch, J. Fourn. & Voglmayr CBS 139064T KP687874 KP687974 KP688042
Valsaria robiniae (Schwein.) Cooke CBS 139063 KP687870 KP687973 KP688039
Valsaria rudis (P. Karst. & Har.) Theiss. & Syd. ex Petr. & Syd. CBS 139066T KP687879 KP687976 KP688047
Valsaria spartii Maubl. CBS 139070T KP687843 KP687964 KP688013

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).

Results

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 parsimony-informative, 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.

Figure 1. 

Maximum likelihood (RAxML) tree obtained by phylogenetic analyses of the combined LSU, SSU, and tef1 sequence alignment of 53 taxa belonging to the 11 orders shown to the right of the tree. The numbers of nodes in clades represent Maximum likelihood bootstrap support values (ML-BS, 0–100) and Bayesian posterior probabilities (BPP, 0–1.0). ML-BS greater than 50% and BPP above 0.5 are indicated at the nodes (ML-BS/BPP). The scalebar represents the number of changes. Schismatomma decolorans DUKE 47570 was used as outgroup. The strain numbers are noted after the species names with ex-type strains indicated with T. The proposed new order is in boldface.

Taxonomy

Neodactylariales H. Zheng & Z.F. Yu, ord. nov.

MycoBank No: MycoBank No: 830161

Type family

Neodactylariaceae H. Zheng & Z.F. Yu.

Description

Asexual morph from human-associated organs or saprobic on plant debris. Conidiophores acroauxic, macronematous, mononematous, branched or unbranched. Conidiogenous cells mono- and polyblastic, sympodially extended. Conidia solitary, hyaline or pale pigmented, smooth, verrucous or echinulate. Sexual morph not observed.

Neodactylariaceae H. Zheng & Z.F. Yu, fam. nov.

MycoBank No: MycoBank No: 830162

Type genus

Neodactylaria Guevara-Suarez, Deanna A. Sutton, Wiederh. & Gené.

Description

Mycelium superficial or immersed, composed of branched, septate, hyaline to subhyaline hyphae. Conidiophores macronematous, mononematous, straight or flexuous, septate, unbranched. Conidiogenous cells terminal or intercalary, polyblastic, sympodial, with short-cylindrical denticles. Conidial secession schizolytic. Conidia solitary, smooth or finely echinulate. Sexual morph not observed.

Neodactylaria Guevara-Suarez, Deanna A. Sutton, Wiederh. & Gené, in Crous et al. Persoonia 38: 345 (2017)

Type species

Neodactylaria obpyriformis Guevara-Suarez, Deanna A. Sutton, Wiederh. & Gené.

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 mid-brown. 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.

Neodactylaria simaoensis , H. Zheng & Z.F. Yu, sp. nov.

MycoBank No: MycoBank No: 830160
Fig. 2

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.

Type

China, Yunnan Province, Simao country, 100°59'19"N, 22°46'38"E, ca 1330 m alt., from submerged unidentified dicotyledonous leaves, 28 Oct 2013, Z.F. Yu, live culture YMF 1.03984 – holotype, dried slide YMFT 1.03984.

Description

Mycelium partly superficial or partly immersed, composed of branched, septate, hyaline to subhyaline, creeping, 1.0–2.0 μm wide hyphae. Conidiophores macronematous, mononematous, straight or flexuous, slightly geniculate towards the apex, 2–4-septate, unbranched, hyaline or pale brown, 38–86 (–129) × 3–4 μm, arising from the creeping hyphae pale brown. Conidiogenous cells polyblastic, indeterminate, sympodial extended, integrated, terminal or intercalary, denticulate with protuberant cylindrical denticles. Conidia solitary, obclavate to long obpyriform, subulate or slightly rostrate towards the obtuse or rounded apex, lumina micro-guttulate, 1–2 (–3)-septate, constricted at the septa, pale to mid brown, 15–40 × 3.6–6.5 μm, with a subhyaline, protuberant basal hilum up to 1 μm long.

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.

Habitat and distribution

In submerged dicotyledonous leaves from south-western China.

Teleomorph

Not known.

Etymology

The species epithet indicates its occurrence in the county of Simao, China.

Notes

Based on a Blast search of NCBIs GenBank nucleotide database, the closest hits using the ITS sequences of N. simaoensis (GenBank MH379209) is N. obpyriformis (GenBank NR_154267, Identities = 545 / 569(96%), Gaps = 4 / 569(0%)). Morphologically, the new species, N. simaoensis, shares several characters with N. obpyriformis (type species): both have white to cream-coloured colonies, with short-cylindrical denticles as conidiogenous cells and obpyriform to slightly rostrate conidia (Crous et al. 2017). However, N. simaoensis differs from N. obpyriformis by having obviously longer and slightly wider conidia (15–40 × 3.6–6.5 μm vs 10–14 × 3–5 μm) and more septa.

Figure 2. 

Culture and anamorph of Neodactylaria simaoensis (YMF 1.03984) A culture on CMA B–D conidia E conidia and conidiophores F immature conidium and conidiogenous cells G conidiophores and conidia under low power microscope. Scale bars: 1 cm (A); 10 μm (B–F); 50 μm (G).

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 (1942, 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 (Crous et al. 2017). 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, 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 (Crous et al. 2017). 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.

Acknowledgements

This work was financed by the National Natural Science Foundation Program of PR China (31760012, 31770026). We are grateful to two reviewers for critically reviewing the manuscript and for providing helpful suggestions to improve this paper.

References

  • Cortinas MN, Burgess T, Dell B, Xu D, Crous PW, Wingfield BD, Wingfield MJ (2006) First record of Colletogloeopsis zuluense comb. nov., causing stem canker of Eucalyptus in China. Mycological Research 110: 229–236. https://doi.org/10.1016/j.mycres.2005.08.012
  • Crous PW, Wingfield MJ, Burgess TI, Hardy GESTJ, Barber PA, Alvarado P, Barnes CW, Buchanan PK, Heykoop M, Moreno G, Thangavel R, van der Spuy S, Barili A, Barrett S, Cacciola SO, Cano-Lira JF, Crane C, Decock C, Gibertoni TB, Guarro J, Guevara-Suarez M, Hubka V, Kolařík M, Lira CRS, Ordoñez ME, Padamsee M, Ryvarden L, Soares AM, Stchigel AM, Sutton DA, Vizzini A, Weir BS, Acharya K, Aloi F, Baseia IG, Blanchette RA, Bordallo JJ, Bratek Z, Butler T, Cano-Canals J, Carlavilla JR, Chander J, Cheewangkoon R, Cruz RHSF, da M, Dutta AK, Ercole E, Escobio V, Esteve-Raventós F, Flores JA, Gené J, Góis JS, Haines L, Held BW, Jung MH, Hosaka K, Jung T, Jurjević Ž, Kautman V, Kautmanova I, Kiyashko AA, Kozanek M, Kubátová A, Lafourcade M, La Spada F, Latha KPD, Madrid H, Malysheva EF, Manimohan P, Manjón JL, Martín MP, Mata M, Merényi Z, Morte A, Nagy I, Normand AC, Paloi S, Pattison N, Pawłowska J, Pereira OL, Petterson ME, Picillo B, Raj KNA, Roberts A, Rodríguez A, Rodríguez-Campo FJ, Romański M, Ruszkiewicz-Michalska M, Scanu B, Schena L, Semelbauer M, Sharma R, Shouche YS, Silva V, Staniaszek-Kik M, Stielow JB, Tapia C, Taylor PWJ, Toome-Heller M, Vabeikhokhei JMC, van Diepeningen AD, Van Hoa N, Van Tri M, Wiederhold NP, Wrzosek M, Zothanzama J, Groenewald JZ (2017) Fungal Planet description sheets: 558–624. Persoonia 38: 240–384. https://doi.org/10.3767/003158517X698941
  • Da Cunha KC, Sutton DA, Fothergill AW, Cano J, Gené J, Madrid H, De Hoog S, Crous PW, Guarro J (2012) Diversity of Bipolaris species in clinical samples in the United States and their antifungal susceptibility profiles. Journal of Clinical Microbiology 50: 4061–4066. https://doi.org/10.1128/JCM.01965-12
  • Da Cunha KC, Sutton DA, Fothergill AW, Gené J, Cano J, Madrid H, De Hoog S, Crous PW, Guarro J (2013) In vitro antifungal susceptibility and molecular identity of 99 clinical isolates of the opportunistic fungal genus Curvularia. Diagnostic Microbiology and Infectious Disease 76: 168–174. https://doi.org/10.1016/j.diagmicrobio.2013.02.034
  • Damm U, Verkley GJM, Crous PW, Fourie PH, Haegi A, Riccioni L (2008) Novel Paraconiothyrium species on stone fruit trees and other woody hosts. Persoonia 20: 9–17. https://doi.org/10.3767/003158508X286842
  • De Wit PJGM, Van der Burgt A, Ökmen B, Stergiopoulos I, Abd-Elsalam KA, Aerts AL, Bahkali AH, Beenen HG, Chettri P, Cox MP, Datema E, De Vries RP, Dhillon B, Ganley AR, Griffiths SA, Guo Y, Hamelin RC, Henrissat B, Kabir MS, Jashni MK, Kema G, Klaubauf S, Lapidus A, Levasseur A, Lindquist E, Mehrabi R, Ohm RA, Owen TJ, Salamov A, Schwelm A, Schijlen E, Sun H, Van den Burg HA, Van Ham RCHJ, Zhang S, Goodwin SB, Grigoriev IV, Collemare J, Bradshaw RE (2012) The genomes of the fungal plant pathogens Cladosporium fulvum and Dothistroma septosporum reveal adaptation to different hosts and lifestyles but also signatures of common ancestry. PLOS Genetics 8(11): 1–22. https://doi.org/10.1371/journal.pgen.1003088
  • Don R, Cox P, Wainwright B, Baker K, Mattick J (1991) ‘Touchdown’ PCR to circumvent spurious priming during gene amplification. Nucleic Acids Research 19: 4008. https://doi.org/10.1093/nar/19.14.4008
  • Gams W, Hoekstra ES, Aptroot A (1998) CBS Course of Mycology, Fourth Edition. Centraalbureau voor Schimmelcultures, Baarn.
  • Guo MT, Qian WY, Li JY, Yu ZF (2015) One genus and three species of aquatic hyphomycetes new to china. Mycosystema 2015(6): 1205–1208.
  • Guo JS, Zhang Z, Qiao M, Yu ZF (2019) Phalangispora sinensis sp. nov. from Yunnan, China and two new members of Wiesneriomycetaceae. International Journal of Systematic and Evolutionary Microbiology 69(10): 3207–3213. https://doi.org/10.1099/ijsem.0.003612
  • Hall TA (1999) BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symposium Series 41: 95–98.
  • Hernandez-Restrepo M, Gené J, Castaneda-Ruiz RF, Mena-Portales J, Crous PW, Guarro J (2017) Phylogeny of saprobic microfungi from Southern Europe. Studies in Mycology 86: 53–97. https://doi.org/10.1016/j.simyco.2017.05.002
  • Hirayama K, Tanaka K, Raja HA, Miller AN, Shearer CA (2010) A molecular phylogenetic assessment of Massarina ingoldiana sensu lato. Mycologia 102: 729–746. https://doi.org/10.3852/09-230
  • Hyde KD, Nilsson RH, Alias SA, Ariyawansa HA, Blair JE, Cai L, De Cock AWAM, Dissanayake AJ, Glockling SL, Goonasekara ID, Gorczak M, Hahn M, Jayawardena RS, Van Kan JAL, Laurence MH, Lévesque CA, Li XH, Liu JK, Maharachchikumbura SSN, Manamgoda DS, Martin FN, McKenzie EHC, McTaggart AR, Mortimer PE, Nair PVR, Pawłowska J, Rintoul TL, Shivas RG, Spies CFJ, Summerell BA, Taylor PWJ, Terhem RB, Udayanga D, Vaghefi N, Walther G, Wilk M, Wrzosek M, Xu JX, Yan JY, Zhou N (2014) One stop shop: backbones trees for important phytopathogenic genera: I. Fungal Diversity 67: 21–125. https://doi.org/10.1007/s13225-014-0298-1
  • Inderbitzin P, Landvik S, Abdel-Wahab MA, Berbee ML (2001) Aliquandostipitaceae, a new family for two new tropical ascomycetes with unusually wide hyphae and dimorphic ascomata. American Journal of Botany 88: 52–61. https://doi.org/10.2307/2657126
  • Jiang SH, Hawksworth DL, Lücking R, Wei JC (2020) A new genus and species of foliicolous lichen in a new family of Strigulales (Ascomycota: Dothideomycetes) reveals remarkable class-level homoplasy. IMA Fungus 11: 1–1. https://doi.org/10.1186/s43008-019-0026-2
  • Katoh K, Standley DM (2013) MAFFT multiple sequence alignment software version 7: improvements in performance and usability. Molecular Biology and Evolution 30: 772–780. https://doi.org/10.1093/molbev/mst010
  • Kindermann J, El-Ayouti Y, Samuels GJ, Kubicek CP (1998) Phylogeny of the genus Trichoderma based on sequence analysis of the internal transcribed spacer region 1 of the rDNA clade. Fungal Genetics and Biology 24: 298–309. https://doi.org/10.1006/fgbi.1998.1049
  • Liu JK, Hyde KD, Jeewon R, Phillips AJL, Maharachchikumbura SSN, Ryberg M, Liu ZY, Zhao Q (2017) Ranking higher taxa using divergence times: a case study in Dothideomycetes. Fungal Diversity 84: 75–99. https://doi.org/10.1007/s13225-017-0385-1
  • Manamgoda DS, Cai L, McKenzie EHC, Crous PW, Madrid H, Chukeatirote E, Shivas RG, Tan YP, Hyde KD (2012) A phylogenetic and taxonomic re-evaluation of the Bipolaris-Cochliobolus-Curvularia complex. Fungal Diversity 56: 131–144. https://doi.org/10.1007/s13225-012-0189-2
  • Mapook A, Hyde KD, Dai D, Li J, Jones EBG, Bahkali AH, Boonmee S (2016) Muyocopronales, ord. nov., (Dothideomycetes, Ascomycota) and a reappraisal of Muyocopron species from northern Thailand. Phytotaxa 265(3): 225–237. https://doi.org/10.11646/phytotaxa.265.3.3
  • Matsushima T (1981) Matsushima Mycological Memoirs 2. Matsushima Fungus Collection, Kobe, 68 pp.
  • Myers N, Mittermeier RA, Mittermeier CG, da Fonseca GA, Kent J (2000) Biodiversity hotspots for conservation priorities. Nature 403: 853–858. https://doi.org/10.1038/35002501
  • Nieuwenhuijzen VE, Miadlikowska J, Houbraken J, Adan OO, Lutzoni F, Samson RA (2016) Wood staining fungi revealed taxonomic novelties in Pezizomycotina: New order Superstratomycetales and new species Cyanodermella oleoligni. Studies in Mycology 85: 107–124. https://doi.org/10.1016/j.simyco.2016.11.008
  • Nilsson RH, Larsson K-H, Taylor AFS, Bengtsson-Palme J, Jeppesen TS, Schigel D, Kennedy P, Picard K, Glöckner FO, Tedersoo L, Saar I, Kõljalg U, Abarenkov K (2019) The UNITE database for molecular identification of fungi: handling dark taxa and parallel taxonomic classifications. Nucleic Acids Research 47: 259–264. https://doi.org/10.1093/nar/gky1022
  • Ohm RA, Feau N, Henrissat B, Schoch CL, Horwitz BA, Barry KW, Condon BJ, Copeland AC, Dhillon B, Glaser F, Hesse CN, Kosti I, LaButti K, Lindquist EA, Lucas S, Salamov AA, Bradshaw RE, Ciuffetti L, Hamelin RC, Kema GHJ, Lawrence C, Scott JA, Spatafora JW, Turgeon BG, De Wit PJGM, Zhong S, Goodwin SB, Grigoriev IV (2012) Diverse life styles and strategies of plant pathogenesis encoded in the genomes of eighteen Dothideomycetes Fungi. PLoS Pathogens 8(12): e1003037. https://doi.org/10.1371/journal.ppat.1003037
  • Rao V, Reddy KA, Hoog GSD (1984) Heliocephala, a new genus of dematiaceous Hyphomycetes. Persoonia 12: 239–242.
  • Rayner RW (1970) A Mycological Colour Chart. Commonwealth Mycological Institute and British Mycological Society, Kew.
  • Santos MGS, Bezerra JDP, Svedese VM, Sousa MA, Silva DCV, Maciel MHC, Paiva LM, Porto ALF, Souza-Motta CM (2015) Screening of endophytic fungi from cactus of the Brazilian tropical dry forest according to their L-asparaginase activity. Sydowia 67: 147–156.
  • Schoch CL, Crous PW, Groenewald JZ, Boehm EW, Burgess TI, De Gruyter J, De Hoog GS, Dixon LJ, Grube M, Gueidan C, Harada Y, Hatakeyama Hirayama SK, Hosoya T, Huhndorf SM, Hyde KD, Jones EBG, Kohlmeyer J, Kruys A, Li YM, Lücking R, Lumbsch HT, Marvanová L, Mbatchou JS, McVay AH, Miller AN, Mugambi GK, Muggia L, Nelsen MP, Nelson P, Owensby CA, Phillips AJL, Phongpaichit S, Pointing SB, Pujade-Renaud V, Raja HA, Plata ER, Robbertse B, Ruibal C, Sakayaroj J, Sano T, Selbmann L, Shearer CA, Shirouzu T, Slippers B, Suetrong S, Tanaka K, Volkmann-Kohlmeyer B, Wingfield MJ, Wood AR, Woudenberg JHC, Yonezawa H, Zhang Y, Spatafora JW (2009b) A class-wide phylogenetic assessment of Dothideomycetes. Studies in Mycology 64: 1–15. https://doi.org/10.3114/sim.2009.64.01
  • Schoch CL, Sung GH, Lopez-Giraldez F, Townsend JP, Miadlikowska J, Hofstetter V, Robbertse B, Matheny PB, Kauff F, Wang Z, Gueidan C, Andrie RM, Trippe K, Ciufetti LM, Wynns A, Fraker E, Hodkinson BP, Bonito G, Groenewald JZ, Arzanlou M, De Hoog GS, Crous PW, Hewitt D, Pfister DH, Peterson K, Gryzenhout M, Wingfield MJ, Aptroot A, Suh SO, Blackwell M, Hillis DM, Griffith GW, Castlebury LA, Rossman AY, Lumbsch HT, Lücking R, Büdel B; Rauhut A, Diederich P, Ertz D, Geiser DM, Hosaka K, Inderbitzin P, Kohlmeyer J, Volkmann-Kohlmeyer B, Mostert L, O’Donnell K, Sipman H, Rogers JD, Shoemaker RA, Sugiyama J, Summerbell RC, Untereiner W, Johnston PR, Stenroos S, Zuccaro A, Dyer PS, Crittenden PD, Cole MS, Hansen K, Trappe JM, Yahr R, Lutzoni F, Spatafora JW (2009a) The Ascomycota tree of life: a phylum-wide phylogeny clarifies the origin and evolution of fundamental reproductive and ecological traits. Systematic Biology 58: 224–239. https://doi.org/10.1093/sysbio/syp020
  • Siu K, Lzumi AK (2004) Phaeohyphomycosis caused by Coniothyrium. Cutis 73: 127–130.
  • Stergiopoulos I, Kourmpetis YAI, Slot JC, Bakker FT, De Wit JPGM, Rokas A (2012) In silico characterization and molecular evolutionary analysis of a novel superfamily of fungal effector proteins. Molecular Biology and Evolution 29: 3371–3384. https://doi.org/10.1093/molbev/mss143
  • Suetrong S, Boonyuen N, Pang K, Ueapattanakit J, Klaysuban A, Sri-indrasutdhi V, Sivichai S, Jones EBG (2011) A taxonomic revision and phylogenetic reconstruction of the Jahnulales (Dothideomycetes), and the new family Manglicolaceae. Fungal Diversity 51: 163–188. https://doi.org/10.1007/s13225-011-0138-5
  • Turner D, Kovacs W, Kuhls K, Lieckfeldt E, Peter B, Arisan-Atac I, Strauss J, Samuels GJ, Börner T, Kubicek CP (1997) Biogeography and phenotypic variation in Trichoderma sect. Longibrachiatum and associated Hypocrea species. Mycological Research 101: 449–459. https://doi.org/10.1017/S0953756296002845
  • Verkley GJM, da Silva M, Wicklow DT, Crous PW (2004) Paraconiothyrium, a new genus to accommodate the mycoparasite Coniothyrium minitans, anamorphs of Paraphaeosphaeria, and four new species. Studies in Mycology 50: 323–335.
  • Vilgalys R, Hester M (1990) Rapid genetic identification and mapping of enzymatically amplified ribosomal DNA from several Cryptococcus species. Journal of Bacteriology 172: 4238–4246. https://doi.org/10.1128/JB.172.8.4238-4246.1990
  • Voglmayr H, Gardiennet A, Jaklitsch WM (2016) Asterodiscus and Stigmatodiscus, two new apothecial Dothideomycete genera and the new order Stigmatodiscales. Fungal Diversity 80(1): 271–284. https://doi.org/10.1007/s13225-016-0356-y
  • Wijayawardene NN, Crous PW, Kirk PM, Hawksworth DL, Boonmee S, Braun U, Dai DQ, D’souza MJ, Diederich P, Dissanayake A, Doilom M, Hongsanan S, Jones EBG, Groenewald JZ, Ruvishika Jayawardena R, Lawrey JD, Liu JK, Lücking R, Madrid H, Manamgoda DS, Muggia L, Nelsen MP, Phookamsak R, Suetrong S, Tanaka K, Thambugala KM, Wanasinghe DN, Wikee S, Zhang Y, Aptroot A, Ariyawansa HA, Bahkali AH, Bhat DJ, Gueidan C, Chomnunti P, De Hoog GS, Knudsen K, Li WJ, McKenzie EHC, Miller AN, Phillips AJL, Piątek M, Raja HA, Shivas RS, Slippers B, Taylor JE, Tian Q, Wang Y, Woudenberg JHC, Cai L, Jaklitsch WM, Hyde KD (2014) Naming and outline of Dothideomycetes – 2014 including proposals for the protection or suppression of generic names. Fungal Diversity 69: 1–55. https://doi.org/10.1007/s13225-014-0309-2
  • Wijayawardene NN, Hyde KD, Lumbsch HT, Liu JK, Maharachchikumbura SSN, Ekanayaka AH, Tian Q, Phookamsak R (2018) Outline of Ascomycota: 2017. Fungal Diversity 88: 167–263. https://doi.org/10.1007/s13225-018-0394-8
  • Wikee S, Lombard L, Crous PW, Nakashima C, Motohshi K, Chukeatirote E, Alias SA, McKenzie EHC, Hyde KD (2013a) Phyllosticta capitalensis, a widespread endophyte of plants. Fungal Diversity 60: 91–105. https://doi.org/10.1007/s13225-013-0235-8
  • Wikee S, Lombard L, Nakashima C, Motohashi K, Chukeatirote E, Alias SA, McKenzie EHC, Hyde KD (2013b) A phylogenetic re-evaluation of Phyllosticta (Botryosphaeriales). Studies in Mycology 76: 1–29. https://doi.org/10.3114/sim0019
  • Wikee S, Udayanga D, Crous PW, Chukeatirote E, Mckenzie EHC, Bahkali AH, Dai DQ, Hyde KD (2011) Phyllosticta – an overview of current status of species recognition. Fungal Diversity 51: 43–61. https://doi.org/10.1007/s13225-011-0146-5
  • Woudenberg JHC, Seidl MF, Groenewald JZ, de Vries M, Stielow JB, Thomma BPHJ, Crous PW (2015) Alternaria section Alternaria: Species, formae speciales or pathotypes? Studies in Mycology 82: 1–21. https://doi.org/10.1016/j.simyco.2015.07.001
  • Zhang Y, Qiao M, Xu JP, Baral HO, Zhang KQ, Yu ZF (2020) Morphological and molecular characterization of two new species of Orbilia (Orbiliomycetes) from China. International Journal of Systematic and Evolutionary Microbiology 70: 2664–2676. https://doi.org/10.1099/ijsem.0.004088
  • Zheng H, Yang XQ, Xu JP, Yu ZF (2020a) Beltrania sinensis sp. nov., a new endophytic fungus from China and a key to species of the genus. International Journal of Systematic and Evolutionary Microbiology 70(2): 1178–1185. https://doi.org/10.1099/ijsem.0.003897
  • Zheng H, Yu ZF, Xu JP, Castañeda-Ruiz RF, Qiao M (2020b) Ramichloridium endophyticum sp. nov., a new species of endophytic fungi from Potamogeton pectinatus in Tibet, China. International Journal of Systematic and Evolutionary Microbiology 70: 3433–439. https://doi.org/10.1099/ijsem.0.004190
  • Zheng H, Zhang Z, Liu DZ, Yu ZF (2019) Memnoniella sinenesis sp. nov., a new species from China and a key to species of the genus. International Journal of Systematic and Evolutionary Microbiology 69(10): 3161–3169. https://doi.org/10.1099/ijsem.0.003605
login to comment