The genus Arthrinium (Ascomycota, Sordariomycetes, Apiosporaceae) from marine habitats from Korea, with eight new species

Abstract

Species of Arthrinium are well-known plant pathogens, endophytes, or saprobes found in various terrestrial habitats. Although several species have been isolated from marine environments and their remarkable biological activities have been reported, marine Arthrinium species remain poorly understood. In this study, the diversity of this group was evaluated based on material from Korea, using morphological characterization and molecular analyses with the internal transcribed spacer (ITS) region, β-tubulin (TUB), and translation elongation factor 1-alpha (TEF). A total of 41 Arthrinium strains were isolated from eight coastal sites which represented 14 species. Eight of these are described as new to science with detailed descriptions.

INTRODUCTION

The genus Arthrinium, which belongs to Apiosporaceae in Xylariales (class Sordariomycetes in Ascomycota), was first recognized and established more than 200 years ago, with A. caricicola as type species (Schmidt and Kunze 1817). To date, it comprises approximately 88 species worldwide (Index Fungorum: http://www.indexfungorum.org).

Arthrinium species have traditionally been classified based on morphological characteristics such as conidial shape, conidiophores, and the presence or absence of sterile cells and setae (Schmidt & Kunze 1817; Hughes 1953; Minter 1985). Among these characteristics, conidial shape appears to be diagnostic for distinguishing species (Singh et al. 2013). However, morphological variation is often observed depending on the growth substrate and incubation period (Crous & Groenewald 2013). As such, species identification based on morphological characteristics is problematic and impractical. To address this problem, DNA sequences of the internal transcription spacer (ITS), translation elongation factor 1-alpha (TEF), and β-tubulin gene (TUB) were employed to delimit and recognize closely related Arthrinium species and infer their phylogenetic relationships (Crous & Groenewald 2013).

Arthrinium species have been globally reported as endophytes, plant pathogens, and saprobes and are commonly isolated from various terrestrial environments, including air, plants, and soil (Kim et al. 2011; Crous & Groenewald 2013; Wang et al. 2018). More recently, isolation from various marine environments, including seawater, seaweed, and the inner tissues of marine sponges, has been reported (Miao et al. 2006; Tsukamoto et al. 2006; Suryanarayanan 2012; Flewelling et al. 2015; Hong et al. 2015; Wei et al. 2016; Elissawy et al. 2017; Li et al. 2017). Arthrinium species isolated from sponges, egg masses of sailfin sandfish, and seaweeds showed promising bioactive properties, including high enzymatic activity, antifungal activity, and antioxidant capacity (Elissawy et al. 2017; Li et al. 2017; Park et al. 2018). Some species (A. arundinis, A. phaeospermum, A. rasikravindrae, A. sacchari, and A. saccharicola) have been detected in both marine and terrestrial environments (Wang et al. 2018). Whether these species have specific adaptations to survive in seawater requires further investigation. A recent study showed that marine Arthrinium species developed strategies to adapt to marine environments, such as a symbiotic partnership with seaweed (Heo et al. 2018). In marine systems, dissolved organic matter in seawater can absorb ultraviolet radiation and produce reactive oxygen species (ROS), which cause oxidative stress on marine microorganisms (Mopper & Kieber 2000). Heo et al. (2018) detected relatively high antioxidant activity and radical-scavenging activity in marine-derived Arthrinium species. The antifungal activity of seaweed-pathogenic fungi has also been studied (Hong et al. 2015; Heo et al. 2018). Arthrinium saccharicola (KUC21342) has the potential to inhibit the growth of Asteromyces cruciatus, a pathogenic fungus that attacks brown algae (Heo et al. 2018). The discovery of the promising bioactivities of marine Arthrinium species was one of the reasons motivating our subsequent investigation of the diversity of marine Arthrinium in Korea.

Six species of Arthrinium have previously been reported from marine environments in Korea: A. arundinis, A. marii, A. phaeospermum, A. rasikravindrae, A. sacchari, and A. saccharicola (Hong et al. 2015; Heo et al. 2018; Park et al. 2018). However, many marine species remain unidentified owing to the lack of resolution in ITS-based phylogenies and the paucity of morphological characteristics. The aim of this study was to investigate marine Arthrinium species from coastal environments in Korea and to identify them using morphological characteristics and multigene phylogenies (ITS, TEF, and TUB).

MATERIALS AND METHODS

Sampling and isolation

The seaweed Sargassum fulvellum and unidentified seaweeds were collected from two locations, Taean-gun on the west coast of Korea and Jeju Island south of Korea. To isolate the fungi, the seaweeds were washed with distilled water and cut into small pieces (approximately 5 mm diam) using a sterile surgical blade. The pieces were treated with 70% ethanol for 60 s and washed in sterile distilled water for 10 s. Each piece was placed on 2% malt extract agar (MEA) supplemented with 0.01% streptomycin and 0.01% ampicillin to inhibit bacterial growth. The plates were incubated at 25 °C for 7–15 d. Suspected Arthrinium colonies were transferred onto potato dextrose agar (PDA, Difco, Sparks, MD, USA) plates. The colonies were subsequently identified as belonging to Arthrinium based on ITS sequences (see below). A total of 14 Arthrinium strains were isolated in this study and an additional 27 Arthrinium strains were obtained from the Seoul National University Fungus Collection (SFC), Seoul, Korea. Each strain is stored in 20% glycerol at − 80 °C in the Korea University Fungus Collection (KUC), Seoul, Korea. Type specimens were deposited in the Korean Collection for Type Culture, Daejeon, Korea (KCTC), with ex-type living cultures deposited in KUC.

DNA extraction, PCR amplification, and sequencing

Genomic DNA was extracted using an Accuprep Genomic DNA extraction kit (Bioneer, Korea) according to the manufacturer’s protocol. PCR targeting the ITS, TUB, and TEF regions was carried out according to a previously described method (Hong et al. 2015). For the ITS region, the primers ITS1F and ITS4/LR3 were used (White et al. 1990; Gardes & Bruns 1993); for TUB, we employed Bt2a/T10 and Bt2b/T2 (Glass & Donaldson 1995; O’Donnell & Cigelnik 1997), and for TEF, we used EF1-728F and EF2 (O’Donnell et al. 1998; Carbone & Kohn 1999). All PCR products were checked on a 1% agarose gel and purified with the AccuPrep PCR/Gel DNA Purification Kit (Bioneer, Seoul, Korea). DNA sequencing was performed at Macrogen (Seoul, Korea) on an ABI3730 automated DNA Sequencer (Applied Biosystems, Foster City, CA) using the same set of primers for each locus. Additional DNA sequences of some strains were obtained from previous studies (Hong et al. 2015; Heo et al. 2018). All new sequences generated in this study were deposited in GenBank (Table 1).

Table 1 A list of all the strains included in the phylogenetic analysis

Phylogenetic analysis

ITS sequences were assembled, proofread and edited using MEGA v. 7 (Kumar et al. 2016) and subsequently aligned with Arthrinium reference sequences from GenBank using MAFFT 7.130 (Katoh and Standley 2013). To adjust the ambiguous alignment manually, maximum likelihood analysis was performed using all sequence where ambiguous regions excluded using G-block. Then, the original sequences were aligned based on the supported clades, and ambiguous regions were manually adjusted.

Maximum likelihood (ML) analyses were conducted using RAxML v. 7.03 (Stamatakis 2006) and a GTR + G model with 1000 bootstrap replicates. Bayesian tree inference (BI) was carried out using MrBayes version 3.2 (Ronquist et al. 2012), with the best model (HKY + I + G) selected for each marker based on the Bayesian information criteria using jModeltest v. 2.1.10 (Darriba et al. 2012). To achieve stationary equilibrium, 20 million trees were generated, and trees were sampled every 1000 generations. The first 25% of the trees was discarded as burn-in, and the remaining 75% was used for calculating posterior probabilities (PP) in the majority rule consensus tree. All analyses were performed on the CIPRES web portal (Miller et al. 2010).

The sequences of the other two loci (TEF and TUB) were individually aligned with Arthrinium reference sequences from GenBank using the same approach described for the ITS. ML and BI analyses also followed the above criteria. The models for TEF and TUB were HKY + I + G and K80 + I + G, respectively. The ITS taxa for the multigene tree were different from those of the single ITS tree, so the model test for the ITS region was redone for the multigene analysis. As a result, the SYM + G model was applied to ITS region in the multigene tree. Finally, sequence concatenation was performed using the same methods and models assigned for each locus described above.

Morphological observation

Strains were grown on oatmeal agar (OA, Difco™), PDA, and MEA at 15, 20, and 25 °C in darkness for 14 d. The culture characteristics, such as surface structure, presence of aerial mycelium and the colour of the mycelium, colour of colony or medium, and sporulation (Crous et al. 2009), were recorded. Colors and the corresponding codes were evaluated according to the Munsell color chart (Munsell Color, 2009). To determine fungal growth rates, the diameter of each colony was measured every 24 h, and each measurement was performed in triplicate. Microscopic characters were observed with an Olympus BX51 light microscope (Olympus, Tokyo, Japan). Samples were mounted in water to take pictures of conidiophores and conidia, and pictures were taken using a DP20 microscope camera (Olympus, Tokyo, Japan). At least 30 individuals were measured for each microscopic character. To illustrate the range of variation, 5% of the extreme measurements from each end of the range are given in parentheses.

Scanning electron microscope (SEM) was used to observe detailed morphological characters. Colonies sporulating abundantly on PDA, MEA, and OA were freeze-dried. Ion coating and observation were performed by Wooyoung Solution Inc. (Suwon, Korea), using an S-5200 scanning electron microscope (Hitachi, Tokyo, Japan). The SEM images were taken under 1500x to 8000x magnifications.

RESULTS

A total of 41 Arthrinium strains were identified, representing six known and eight new species. Of these strains, 26 were isolated from various seaweeds, 14 from the eggs of sailfin sandfish, and one from beach sand. The dominant species were three of the new species, A. agari (5 strains), A. arctoscopi (5 strains), and A. marinum (5 strains) (Table 1).

A total of 21 ITS (580–1150 bp), 24 TEF (420–970 bp), and 22 TUB (400–560 bp) sequences were newly generated for the 41 Arthrinium strains. The ITS phylogeny contained 124 terminals, including Nigrospora gorlenkoana as outgroup. The concatenated three-gene phylogeny contained 95 terminals, consisting of 749, 613, and 503 characters respectively, including gaps. Preliminary identification was based on the ITS region, and multigene analysis was used to test the identifications, determine the phylogenetic relationships among the taxa, and to resolve closely related species. Both the ML and Bayesian analyses showed the same tree topologies and the ML tree is represented (Figs. 1, 2).

Fig. 1

ML tree based on the ITS region. The numbers at the nodes indicate ML bootstrap support (BS) > 75% and Bayesian posterior probabilities (PP) > 0.75 as BS/PP. The thickened branches indicate support greater than 85% for BS and 0.95 for PP. A hyphen (‘-‘) indicates values of BS < 70% or PP < 0.75. Ex-holotype strains are indicated with asterisks (‘*’). The fungal cultures examined in this study are shown in bold. Red boxes indicate the novel species. The numbers in the brackets indicate strain number. The scale bar indicates the nucleotide substitutions per position

Fig. 2

ML tree based on the ITS, TUB, and TEF regions combined. The numbers at the nodes indicate ML bootstrap support (BS) > 75% and Bayesian posterior probabilities (PP) > 0.75 as BS/PP. The thickened branches indicate support greater than 85% for BS and 0.95 for PP. A hyphen (‘-‘) indicates values of BS < 70% or PP < 0.75. Ex-holotype strains are indicated with asterisks (‘*’). The fungal cultures examined in this study are shown in bold. Red boxes indicate the novel species. The numbers in the brackets indicate strain number. The scale bar indicates the nucleotide substitutions per position

The 41 Arthrinium strains obtained in this study formed five clades (A, B, C, D, and E), both in the ITS-based and combined phylogeny analyses (Figs. 1, 2). In the ITS tree, many Arthrinium species were distinguished from one another. However, some were not clearly separated (clades B and D) and the relationships of the others (clades C and D) were not resolved. The above problem was solved in the individual trees of TEF and TUB (Figs. 1S, 2S), and the multigene tree based on the ITS, TUB, and TEF regions (Fig. 2). The multigene analysis supported the conclusion that six taxa corresponded to known species. Eight putatively novel species were classified into five clades (Fig. 2). The eight species were clearly separated from the previously sequenced taxa, each forming a clade with high support (over 99% of BS, 0.99 of PP) (Fig. 2). Arthrinium agari and A. koreanum. Were included in clade A, A. piptatheri and A. fermenti were in clade D, and A. pusillispermum and A. taeanense were in clade E. Comparison with morpho-anatomical and other data of species that have so far not been sequenced supported our interpretation of these eight entities representing novel species.

TAXONOMY

Arthrinium agari S.L. Kwon, S. Jang & J.J. Kim, sp. nov.

MycoBank MB834592

(Fig. 3)

Fig. 3

Arthrinium agari (KUC21333). a-c Colonies on PDA (a), MEA (b), and OA (c) (top); d-f, colonies on PDA (d), MEA (e), and OA (f) (bottom); g-h, conidia under SEM; i-k, conidia attached to conidiogenous cells; scale bar = 10 μm

Etymology: ‘agari’ refers to the generic name of Agarum cribrosum, the source of the type strain.

Molecular diagnosis: Arthrinium agari is distinguished from the phylogenetically most closely related species, A. arundinis, by unique single nucleotide polymorphisms in the three loci used in this study (Figs. 3S, 4S, 5S): ITS positions 21 (C), 31 (indel), 36 (C), 38 (T), 93 (C), 111 (C), 113 (T), 122–124 (indel), 190–203 (indel), 205 (indel), 214–223 (indel), 227 (G), 228 (A), 253 (G), 259 (A), 291 (A), 535 (T), and 645 (indel); TEF positions 14 (A), 16 (G), 17 (T), 32 (C), 35 (A), 47 (C), 54 (T), 59–62 (indel), 64 (T), 65 (T), 79 (G), 85 (G), 96 (T), 125 (G), 135 (indel), 151 (C), 173 (G), 174 (A), 176 (G), 192 (T), 213 (C), 249 (G), 265 (C), 271 (C), 288 (G), 302 (T), 306 (G), 312 (indel), 331 (G), and 494 (A); TUB positions 15 (G), 29 (A), 31 (A), 62 (T), 67 (G), 80 (T), 89 (A), 98 (G), 99 (C), 138 (T), 139 (T), 140 (T), 143 (T), 199 (T), 208 (A), 210 (A), 212 (A), 223 (T), 229 (A), 232 (T), 312 (C), 324 (A), 331 (G), 377 (T), 428 (C), 467 (T), and 482 (A).

Type: Korea: Gangwon-do, Yangyang-gun, 38°07′04.8″N, 128°38′00.8″E, isolated from Agarum cribrosum, 11 Sept. 2016, M.S. Park (Herb. KCTC 46909 – holotype preserved in a metabolically inactive state; KUC21333 = NIBRFGC000501588, SFC20161014-M18 – ex-type cultures).

Description: Mycelium of smooth, hyaline, branched, septate, hyphae 2.0–3.5 μm diam. Conidiogenous cells aggregated in clusters on hyphae or solitary, at first hyaline, becoming pale green, cylindrical, sometimes ampulliform. Conidia brown, smooth to granular, globose to subglobose in surface view, (8.5–)9.0–10.5 × (7.0–)7.5–8.5 (− 9.0) μm ($\overline{x}$ = 9.5 × 8.1 μm, n = 30); lenticular in side view, with equatorial slit, 5.5–7.0 μm wide ($\overline{x}$ = 6.4 μm, n = 30), elongated cell observed.

Culture: PDA: colonies thick, concentrically spreading with aerial mycelium, margin irregular; mycelia white to grey and pale brown coloured; sporulation on hyphae; dark olive-brown (2.5Y 3/3) pigment diffused in media; odour indistinct. MEA: colonies low, flat, concentrically spreading with sparse aerial mycelium, margin circular; mycelia white; sporulation not observed; pigment absent in medium; odour indistinct. OA: colonies thick, concentrically spreading with aerial mycelium, margin circular; mycelia white to pink; sporulation was not observed; partially pink (2.5YR 8/3) pigment diffused in media; odour indistinct. Colony diameters (in mm after 120 h): 15 °C PDA 19–20, MEA 15–18, OA 11–13; 20 °C PDA 34–35, MEA 28–34, OA 20–23; 25 °C PDA 24–28, MEA 22–25, OA 19–20.

Additional material examined: Korea: Gangwon-do, Yangyang-gun, 38°07′04.8″N, 128°38′00.8″E, isolated from Agarum cribrosum, 11 Sept. 2016, M.S. Park (KUC21361, KUC21362, KUC21363, and KUC21364).

Notes: Arthrinium agari is phylogenetically related to A. arundinis (over 97.52% similarity in the ITS region, 93.74% in the TEF region, and 93.64% in the TUB region) (Figs. 1, 2). The two species also morphologically resemble each other. The two species have smooth, hyaline, branched, septate mycelium, and ampulliform conidiogenous cells that cluster on hyphae. Arthrinium arundinis and A. agari have similar conidia shape (brown, globose in surface view, lenticular in side view) (Crous & Groenewald 2013). However, A. agari can be distinguished from A. arundinis by its larger conidia (A. agari: 8.5–10.5 × 7.0–9.0 μm, A. arundinis: (5–)6–7 × 3–4 μm diam) (Crous & Groenewald 2013).

Arthrinium agari and A. sinensis (non-sequenced species) also have similar conidia shape (globose in surface view, lenticular in side view). However, they can be distinguished by the shape of conidiogenous cell; cylindrical and sometimes ampulliform in A. agari, whereas lageniform in A. sinensis (Table 2).

Table 2 Summary of conidial morphology of Arthrinium species. Newly established species in this study are shown in bold

Arthrinium arctoscopi S.L. Kwon, S. Jang & J.J. Kim, sp. nov.

MycoBank MB834593

(Fig. 4)

Fig. 4

Arthrinium arctoscopi (KUC21331). a-c Colonies on PDA (a), MEA (b), and OA (c) (top); d-f, colonies on PDA (d), MEA (e), and OA (f) (bottom); g-h, conidia under SEM; I-K, conidia attached to conidiogenous cells; scale bar = 10 μm

Etymology: ‘arctoscopi’ refers to the generic name of Arctoscopus japonicus, the substrate of on which it was found.

Molecular diagnosis: Arthrinium arctoscopi is distinguished from phylogenetically most closely related species, A. obovatum, by unique single nucleotide polymorphisms in the three loci used in this study (Figs. 3S, 4S, 5S): ITS positions 112–124 (indel), 128–137 (indel), 190 (indel), 192 (G), 223 (T), 225 (indel), 226 (indel), 253–254 (indel), 618 (G), 621 (C), 624 (C), and 651 (G); TEF positions 32 (T), 33 (T), 76 (G), 131 (G), 132 (C), 145 (T), 148–150 (indel), 207 (indel), 208 (T), 210 (T), 211 (T), 269 (G), 304 (A), 305 (C), 316 (C), 320 (C), 324 (A), 328 (T), and 333 (A); TUB position 5 (T), 8 (C), 27 (G), 38 (T), 53 (G), 62 (A), 68 (C), 79 (C), 80 (A), 82 (G), 87 (T), 90 (A), 106 (A), 112 (T), 144 (A), 211 (indel), 212 (T), 225 (T), 227 (C), 311 (T), 334 (T), 467 (C), 479 (C), and 506 (C).

Type: Korea: Gangwon-do, Goseong-gun, 38°28′44.0″N, 128°26′18.9″E, isolated from Egg masses of Arctoscopus japonicus, 10 Nov. 2016, M.S. Park (Herb. KCTC 46907 – holotype preserved in a metabolically inactive state; KUC21331 = NIBRFGC000501586, SFC20200506-M05 –ex-type cultures).

Descriptions: Mycelium of smooth, hyaline, branched, septate, hyphae 2.5–4.0 μm diam. Conidiogenous cells aggregated in clusters on hyphae or solitary, at first hyaline, becoming pale green, cylindrical, sometimes ampulliform. Conidia brown, smooth to granular, globose to elongate ellipsoid in surface view, (9.5–)10–12 (− 13) × (7.5–)8.0–11 (− 12) μm ($\overline{x}$ = 11.1 × 10 μm, n = 30); lenticular in side view, with equatorial slit, 5.5–7.5 μm wide ($\overline{x}$ = 6.5 μm, n = 30), elongated cell observed.

Culture: PDA: colonies thick, concentrically spreading with aerial mycelium, margin irregular; mycelia creamy white; sporulation was not observed; pigment absent in medium; odour indistinct. MEA: colonies flat, concentrically spreading with aerial mycelium, margin irregular; mycelia white; sporulation on hyphae after 2 weeks, spores black; pigment absent in medium; odour indistinct. OA: colonies thick, concentrically spreading with aerial mycelium, margin irregular; mycelia creamy pale yellow; sporulation not observed; very dark greyish brown (2.5Y 3/2) pigment diffused from centre into medium; odour indistinct. Colony diameters (in mm after 120 h): 15 °C PDA 9, MEA 13–15, OA 11–13; 20 °C PDA 18–24, MEA 18–22, OA 14–18; 25 °C PDA 5–7, MEA 4–5, OA 7–9.

Additional material examined: Korea: Gangwon-do, Goseong-gun, 38°28′44.0″N, 128°26′18.9″E, isolated from egg masses of Arctoscopus japonicus, 10 Nov. 2016, M.S. Park (KUC21344, KUC21345, KUC21346, and KUC21347).

Notes: Arthrinium arctoscopi is closely related to A. obovatum (98.84% similarity in the ITS region, 96.10% in the TEF region, and 94.31% in the TUB region) and A. aquaticum (99.80% similarity in the ITS region). However, A. arctoscopi can be distinguished from A. obovatum by the conidial shape and growth rate; the conidia of A. arctoscopi are globose to subglobose, whereas those of A. obovatum are obovoid or occasionally elongated to ellipsoid in shape (Wang et al. 2018). In addition, the growth rate of A. arctoscopi (7–9 mm in 7 d at 25 °C, PDA) is slower than that of A. obovatum (covering a 90 mm Petri dish in 7 d at 25 °C, PDA) (Wang et al. 2018). The conidial shape of A. arctoscopi is also slightly different from that of A. aquaticum (globose to subglobose conidia, 9–11 × 8–10 μm, $\overline{x}$ = 10 × 9 μm, n = 20). Two non-sequenced species, A. algicola and A. sinensis, are morphologically similar to A. arctoscopi. The longer length and narrower width of A. algicola conidia (10.5–15 × 6–8 μm) and lageniform conidiogenous cell of A. sinensis distinguish them from A. arctoscopi (Table 2).

Arthrinium fermenti S.L. Kwon, S. Jang & J.J. Kim, sp. nov.

MycoBank MB834594

(Fig. 5)

Fig. 5

Arthrinium fermenti (KUC21288). a-c, Colonies on PDA (a), MEA (b), and OA (c) (top); d-f, colonies on PDA (d), MEA (e), and OA (f) (bottom), g-h, conidia under SEM; i-k, conidia attached to conidiogenous cells; scale bar = 10 μm

Etymology: ‘fermenti’ refers to the yeast-like odour of the cultures.

Molecular diagnosis: Arthrinium fermenti is distinguished from the phylogenetically most closely related species, A. pseudospegazzinii, by unique single nucleotide polymorphisms in the three loci used in this study (Figs. 3S, 4S, 5S): ITS positions 32 (C), 43 (T), 81 (C), 283 (T), 318 (T), 567 (A), and 644 (indel); TEF positions 9 (C), 35 (C), 44 (A), 67 (A), 81–82 (indel), 84 (indel), 87 (C), 92 (G), 93 (A), 114 (G), 126 (C), 133 (T), 134 (G), 140 (T), 154 (G), 170 (C), 171 (T), 172 (T), 178 (indel), 181 (indel), 192 (C), 206 (indel), 208–211 (indel), 213 (T), 239 (G), 243 (T), 252 (A), 264 (C), 288 (G), 305 (C), 311 (C), 322 (indel), 330 (A), 337 (T), 357 (G), 367 (T), 375 (T), 392 (A), and 473 (T); TUB positions 1 (T), 9 (T), 18–22 (indel), 28 (A), 33 (C), 41 (G), 67 (A), 80 (A), 94 (G), 106 (T), 117 (T), 223 (A), 233 (T), 308 (A), 309 (T), 322 (T), 327 (C), 329 (C), 331 (C), 425 (C), and 437 (T).

Type: Korea: Jeollanam-do, Haenam-gun, 34°26′07.2″N, 126°28′16.5″E, isolated from seaweed, 23 Apr. 2014, M.S. Park (Herb. KCTC 46903 – holotype preserved in a metabolically inactive state; KUC21289 = NIBRFGC000501584, SFC20140423-M86 – ex-type cultures).

Description: Mycelium of smooth, hyaline, branched, septate, 2.0–4.0 μm diam. Conidiogenous cells aggregated in clusters on hyphae, at first hyaline, becoming pale brown, polyblastic, discrete, erect, ampulliform. Conidia brown, smooth to granular, globose to elongated ellipsoid, (7.5–)8.0–9.0 × 7.0–8.5 (− 9) μm ($\overline{x}$ = 8.32 × 7.4 μm, n = 30); lenticular in side view, with equatorial slit, 6.0–7.0 μm wide ($\overline{x}$ = 6.6 μm, n = 30).

Culture: PDA: colonies thick, concentrically spreading with aerial mycelium, margin irregular; mycelia white to yellow, becoming pinkish to orange after 2 weeks; sporulation on hyphae, spores black; dark reddish brown (5YR 2.5/2) to yellow (2.5Y 8/8) pigment diffused from centre into media; odour strong baker’s yeast-like. MEA: colonies low, flat, concentrically spreading, thin, margin circular; mycelia white; sporulation was not observed; medium reverse with yellow pigment after 2 weeks; odour strong baker’s yeast–like. OA: colonies thick, concentrically spreading with aerial mycelium, margin irregular; mycelia at first white, reverse randomly pale pink to red-grape and pale yellow to brown after 2 weeks; sporulation on hyphae, spores black; dark yellowish brown (10YR 3/4, 3/6) to dark reddish brown (2.5YR 2.5/4) pigment diffused into the medium; odour strong baker’s yeast–like. Colony diameters (in mm after 120 h): 15 °C PDA 17, MEA 17–18, OA 13–16; 20 °C PDA 27–30, MEA 21–27, OA 15–18; 25 °C PDA 21–23, MEA 18–19, OA 14–16.

Additional material examined: Korea: Jeollanam-do, Haenam-gun, 34°26′07.2″N, 126°28′16.5″E, isolated from seaweed, 23 Apr. 2014, M.S. Park (KUC21288).

Notes: Arthrinium fermenti is closely related to A. pseudospegazzinii (98.96% similarity in the ITS region, 92.47% in the TEF region, and 95.00% in the TUB region) (Figs. 1, 2). It can be distinguished from the latter by conidial shape and colony colour. The conidia of A. fermenti are globose to elongate-ellipsoid, whereas A. pseudospegazzinii has uniformly globose conidia (Crous & Groenewald 2013). Moreover, while the colonies of A. pseudospegazzinii were light orange on PDA and dirty white with an olivaceous grey patch on OA and MEA (Crous & Groenewald 2013), A. fermenti colonies had a yellowish to reddish colour on OA and MEA and a strong yeast odour. Arthrinium globosum (non-sequenced species) has a conidia shape similar to that of A. fermenti – globose to subglobose. However, a lenticular shape in side view was not observed in A. globosum (Table 2).

Arthrinium koreanum S.L. Kwon, S. Jang & J.J. Kim, sp. nov.

MycoBank MB834596

(Fig. 6)

Fig. 6

Arthrinium koreanum (KUC21332). A-C, Colonies on PDA (a), MEA (b), and OA (c) (top); d-f, colonies on PDA (d), MEA (e), and OA (f) (bottom); g-h, conidia under SEM; i-k, conidia attached to conidiogenous cells; scale bar = 10 μm

Etymology: ‘koreanum’ refers to the country in which the type locality is located.

Molecular diagnosis: Arthrinium koreanum is distinguished from the phylogenetically most closely related species, A. qinlingense, by unique single nucleotide polymorphisms in the three loci used in this study (Figs. 3S, 4S, 5S): ITS positions 80 (C), 92 (C), 245 (G), 250 (A), 253 (C), 258 (C), 274 (C), and 293 (G); TEF positions 16 (G), 43 (T), 44 (T), 91 (T), 94 (T), 133 (C), 135 (indel), 149 (indel), 152 (T), 153 (C), 154 (A), 156 (C), 157 (A), 161 (T), 162 (C), 199 (indel), 200 (T), 248 (A), 250 (G), 251 (T), 252 (G), 253 (C), 321 (C), 322 (A), 407 (C); TUB positions 4 (G), 5 (T), 18 (A), 38 (T), 49 (A), 64 (T), 68 (G), 78 (A), 80 (G), 89 (G), 98 (C), 113 (G), 114 (G), 199 (C), 309 (G), 326 (A), 410 (C), 413 (C), and 497 (T).

Type: Korea: Gangwon-do, Goseong-gun, 38°28′44.0″N, 128°26′18.9″E, isolated from egg masses of Arctoscopus japonicus, 10 Nov. 2016, M.S. Park (Herb. KCTC 46908 – holotype preserved in a metabolically inactive state; KUC21332 = NIBRFGC000501587, SFC20200506-M06 – ex-type cultures).

Description: Mycelium consisting of smooth, hyaline, branched, septate, hyphae 1.5–6.0 μm diam. Conidiogenous cells aggregated in clusters on hyphae, hyaline, cylindrical. Conidia brown, smooth to granular, globose to ellipsoid in surface view, (7.5–)8.0–10 (− 11) × (5.5–)6.5–9.5 (− 10) μm ($\overline{x}$ = 9.1 × 8.1 μm, n = 30); lenticular in side view, with equatorial slit, 4.0–6.5 μm wide ($\overline{x}$ = 5.3 μm, n = 30).

Culture: PDA: colonies thick, concentrically spreading with aerial mycelium, margin irregular; mycelia white to pale yellow; sporulation not observed; olive-yellow (2.5Y 6/8) pigment diffused into medium; odour indistinct. MEA: colonies flat, concentrically spreading with sparse aerial mycelium, margin circular; mycelia white; sporulation on hyphae after 2 weeks, spores black; pigment absent in medium; odour indistinct. OA: colonies thick, concentrically spreading with aerial mycelium, margin irregular; mycelia white to orange; sporulation not observed; dark reddish brown (5YR 4/6) pigment diffused in media; odour indistinct. Colony diameters (in mm after 120 h): 15 °C PDA 17–18, MEA 15–19, OA 16–17; 20 °C PDA 27–31, MEA 20–23, OA 27–28; 25 °C PDA 6–7, MEA 3–6, and OA 4–5.

Additional material examined: Korea: Gangwon-do, Goseong-gun, 38°28′44.0″N, 128°26′18.9″E, isolated from egg masses of Arctoscopus japonicus, 10 Nov. 2016, M.S. Park (KUC21348, KUC21349, and KUC21350).

Notes: Arthrinium koreanum is closely related to A. qinlingense (98.48% similarity in the ITS region, 94.92% in the TEF region, and 94.85% in the TUB region) (Figs. 1, 2). They can be distinguished by their conidial sizes; 7.5–11 × 5.5–10 μm in A. koreanum vs. 5–8 μm in diameter in A. qinlingense (Jiang et al. 2018). Arthrinium koreanum has a similar conidia shape to that of the two non-sequenced species, A. globosum and A. sphaerospermum. However, the conidia of the latter two species only have globose to subglobose shape, and lenticular shape is not observed in side view (Table 2).

Arthrinium marinum S.L. Kwon, S. Jang & J.J. Kim, sp. nov.

MycoBank MB834595

(Fig. 7)

Fig. 7

Arthrinium marinum (KUC21328). a-c, Colonies on PDA (a), MEA (b), and OA (c) (top); d-f, colonies on PDA (d), MEA (e), and OA (f) (bottom); g-h, conidia under SEM; i-k conidia attached to conidiogenous cells; scale bar = 10 μm

Etymology: ‘marinum’ refers to the marine origin.

Molecular diagnosis: Arthrinium marinum is distinguished from the phylogenetically most closely related species, A. rasikravindrae, by unique single nucleotide polymorphisms in the three loci used in this study (Figs. 3S, 4S, 5S): ITS positions 100% similarity; TEF positions 191 (T), 253 (C), 256 (A), 319 (A), and 372 (C); TUB positions 2 (T), 15 (A), 20 (G), 30 (C), 69 (G), 111 (indel), 314 (G), 363 (T), 437 (C), and 443 (C).

Type: Korea: Jeollanam-do, Suncheon-si, 34°50′46.9″N, 127°31′31.4″E, isolated from seaweed, 23 Apr. 2014, M.S. Park (Herb. KCTC 46905 – holotype preserved in a metabolically inactive state; KUC21328 = NIBRFGC000501583, SFC20140423-M02 –ex-type cultures).

Description: Mycelium superficial, composed of smooth, hyaline, branched, septate, 3.5–6.0 μm diam. Hyphae. Conidiogenous cells aggregated in clusters on hyphae or solitary, hyaline, erect, ampulliform. Conidia brown, smooth to granular, globose to elongate ellipsoid in surface view, (9.5–)10–12 (− 13) × (7.5–)8.0–10 μm ($\overline{x}$ = 11.1 × 9.4 μm, n = 30); lenticular in side view, with equatorial slit, 6.0–7.5 μm wide ($\overline{x}$ = 7.1 μm, n = 30).

Culture: PDA: colonies thick and dense, concentrically spreading, margin irregular; mycelia white to pale yellow; sporulation was not observed; pale yellow (5Y 8/4) pigment diffused into medium; odour indistinct. MEA: colonies low, flat, concentrically spreading with sparse aerial mycelium, margin circular; mycelia white colored; sporulation on hyphae around centre after 2 weeks, spores black; pigment absent in medium; odour indistinct. OA: colonies thick, concentrically spreading with aerial mycelium, margin circular; mycelia white to pale yellow; sporulation not observed; yellow to pale green (2.5Y 5/6) pigment diffused into medium; odour indistinct. Colony diameters (in mm after 120 h): 15 °C PDA 7–9, MEA 6–12, OA 4–5; 20 °C PDA 16–17, MEA 14–21, OA 7–9; 25 °C PDA 35–47, MEA 32–35, and OA 28–32.

Additional material examined: Korea: Jeollanam-do, Suncheon-si, 34°50′46.9″N, 127°31′31.4″E, isolated from seaweed, 23 Apr. 2014, M.S. Park (KUC21353, KUC21354, KUC21355, and KUC21356).

Notes: Although Arthrinium marinum and A. rasikravindrae were not distinguished on ITS alone (100% similarity in the ITS region), these species formed two distinct clades based on the combined analysis of the ITS, TUB, and TEF regions (99.08% in the TEF region and 97.97% in the TUB region) (Figs. 1, 2). They can also be distinguished by their growth rates: A. marinum (16–17 mm in 5 d on PDA at 20 °C) had a slower growth rate than A. rasikravindrae KUC21327 (34–39 mm in 5 d on PDA at 20 °C).

Non-sequenced species, Arthrinium algicola, has a very similar conidia shape to that of A. marinum, However, they are distinguished by the conidia size; 10.5–15 × 6–8 μm in A. algicola and (9.5–)10–12(− 13) × (7.5–)8–10 μm in A. marinum (Table 2).

Arthrinium pusillispermum S.L. Kwon, S. Jang & J.J. Kim, sp. nov.

MycoBank MB834597

(Fig. 8)

Fig. 8

Arthrinium pusillispermum (KUC21321). a-c, Colonies on PDA (a), MEA (b), and OA (c) (top); d-f, colonies on PDA (d), MEA (e), and OA (f) (bottom); g-h, conidia under SEM; i-k, conidia attached to conidiogenous cells; scale bar = 10 μm

Etymology: ‘pusillus’, tiny and ‘spermum’ spores.

Molecular diagnosis: Arthrinium pusillispermum is distinguished from the phylogenetically most closely related species, A. gutiae, by unique single nucleotide polymorphisms in the three loci used in this study (Figs. 3S, 4S, 5S): ITS positions 43 (C), 260 (T), and 546 (T); TEF positions 1–17 (indel), 26–38 (indel), 43–46 (indel), 64–69 (indel), 76–82 (indel), 84–96 (indel), 112–115 (indel), 125–131 (indel), 137–141 (indel), 151–172 (indel), 173 (C), 174 (A), 175 (G), 178 (G), 180 (T), 192 (T), 193 (indel), 194 (G), 209 (A), 213 (indel), 228 (A), 230 (C), 243 (C), 251 (C), 252 (A), 256 (A), 260 (A), 261 (A), 264 (T), 268 (G), 269 (T), 273–276 (indel), 287–289 (indel), 293 (A), 294 (G), 308 (A), 310 (G), 313 (C), 314 (indel), 315 (C), 321 (T), 325 (indel), 327 (indel), 328 (A), 332 (indel), 337 (T), 356 (C), 358 (A), 360 (T), 364 (C), 374 (A), 395 (C), and 473 (T); TUB position 38 (C), 75 (T), 89 (G), 144 (A), and 498–506 (indel).

Type: Korea: Chungcheongnam-do, Taean-gun, 36°50′14.3″N, 126°11′04.7″E, isolated from Seaweed, 19 Mar. 2016, S. Jang (Herb. KCTC 46906 – holotype preserved in a metabolically inactive state; KUC21321 = NIBRFGC000501585 – ex-type culture).

Description: Mycelium consisting of smooth, hyaline, branched, septate, 1.5–4.5 μm diam. Conidiogenous cells aggregated in clusters on hyphae, hyaline, cylindrical. Conidia brown, smooth to granular, globose to subglobose in surface view, 4.0–6.0 (− 6.5) × (3.0–)3.5–5.0 (− 5.5) μm ($\overline{x}$ = 5.1 × 4.2 μm, n = 30); lenticular in side view, with equatorial slit, 3.5–4.5 μm wide ($\overline{x}$ = 4.1 μm, n = 30), elongated cell present.

Culture: PDA: colonies thick around centre, concentrically spreading with aerial mycelium, margin circular; mycelia white, pale yellow to grey; sporulation was not observed; greenish black (10GY 2.5/1) pigment diffused in medium; odour indistinct. MEA: colonies abundant, flat, concentrically spreading with sparse aerial mycelium, margin irregular; mycelia white to gray colored; sporulation was not observed; pigment absent in medium; odour indistinct. OA: colonies thick, concentrically spreading with aerial mycelium, margin irregular; mycelia white to pale brown and grey to dark grey; sporulation on hyphae around the centre after 2 weeks, spores black; greenish black (10Y 2.5/1) to very dark greenish grey (10Y 3/1) pigment diffused in medium; odour indistinct. Colony diameters (in mm after 120 h): 15 °C PDA 19–25, MEA 10–12, OA 11–12; 20 °C PDA 25–39, MEA 19–25, OA 22–24; 25 °C PDA 9–15, MEA 6–18, and OA 6–20.

Additional material examined: Korea: Chungcheongnam-do, Taean-gun, 36°50′14.3″N, 126°11′04.7″E, isolated from seaweed 19 Mar. 2016, S. Jang (KUC21357).

Notes: Arthrinium pusillispermum is closely related to A. gutiae (99.44% similarity in the ITS region, 88.52% in the TEF region, and 98.98% in the TUB region) (Figs. 1, 2). Arthrinium pusillispermum is distinguished from A. gutiae by the shape of the conidiogenous cells and the substrate: A. pusillispermum has cylindrical conidiogenous cells and was isolated from seaweed, whereas A. gutiae has lageniform conidiogenous cells and was isolated from the gut of grasshoppers (Crous et al. 2015). Arthrinium pusillispermum can be distinguished from the 22 non-sequenced species by its small conidia size (Table 2).

Arthrinium sargassi S.L. Kwon, S. Jang & J.J. Kim, sp. nov.

MycoBank MB834598

(Fig. 9)

Fig. 9

Arthrinium sargassi (KUC21232). a-c, Colonies on PDA (a), MEA (b), and OA (c) (top); d-f, colonies on PDA (d), MEA (e), and OA (f) (bottom); g-h, conidia under SEM; i-k, conidia attached to conidiogenous cells; scale bar = 10 μm

Etymology: ‘sargassi’ refers to the genus name of Sargassum sp., the substrate of the type material.

Molecular diagnosis: Arthrinium sargassi is distinguished from the phylogenetically related species, A. hydei, by unique single nucleotide polymorphisms in the three loci used in this study (Figs. 3S, 4S, 5S): ITS positions 31 (C), 47 (indel), 91 (C), 95 (indel), 309 (T), and 644 (indel); TEF positions 15 (C), 27 (C), 30 (T), 37 (C), 46 (T), 47 (indel), 63 (indel), 64 (C), 66 (T), 67 (A), 92 (C), 93 (A), 95 (G), 140 (G), 152 (C), 153 (A), 155 (G), 160 (T), 193 (T), 222 (C), 224 (A), 225 (C), 253 (C), 254 (C), 262 (C), 265 (T), 293 (A), 328 (A), 336 (A), 358 (T), 367 (A), 371 (T), 374 (C), 376 (A), 386 (C), 392 (A), and 449 (C); TUB positions 10 (C), 18 (C), 22 (T), 23 (G), 30 (T), 45 (T), 47 (A), 50 (G), 52 (A), 69 (A), 70 (C), 80 (G), 106 (T), 133 (A), 145 (A), 225 (A), 230 (G), 380 (T), 416 (T), and 437 (T).

Type: Korea: Jeju-do, 33°23′39.2″N, 126°14′23.0″E, isolated from Sargassum fulvellum, 10 Jan. 2015, S. Jang (Herb. KCTC 46901 – holotype preserved in a metabolically inactive state; KUC21228 = NIBRFGC000501578 – ex-type culture).

Description: Mycelium consisting of smooth, hyaline, branched, septate, 2.0–5.0 μm diam. Conidiogenous cells aggregated in clusters on hyphae or solitary, at first hyaline, becoming pale brown, basauxic, polyblastic, sympodial, erect, cylindrical. Conidia brown, smooth to granular, globose to subglobose in surface view, (8.5–)9.5–11 (− 11.5) × (8.0–)8.5–10 (− 11) μm ($\overline{x}$ = 10.4 × 9.4 μm, n = 30); lenticular in side view, with equatorial slit, 5.5–7.5 μm wide ($\overline{x}$ = 6.5 μm, n = 30), elongated cell present.

Culture: PDA: colonies thick, flat, concentrically spreading with aerial mycelium, margin circular; mycelia white to grey, reverse sparsely pale yellow; sporulation on hyphae and in media after 2 weeks, randomly dense, spores black; yellow (10YR 8/8) pigment diffused in medium from centre, sometimes remaining as dark grey spots; odour indistinct. MEA: colonies slightly thick, flat, concentrically spreading with aerial mycelium, margin circular; mycelia white coloured; sporulation on hyphae and in media after 2 weeks, randomly dense, spores black; pigment absent, sometimes remaining dark grey spots in medium; odour indistinct. OA: colonies thick and dense, flat, concentrically spreading with aerial mycelium, margin circular; mycelia white, reverse usually yellow to green from the centre, sometimes becoming pinkish after 2 weeks; sporulation on hyphae, randomly dense after 2 weeks, spores black; yellow (2.5Y 7/8) pigment diffused in medium; odour indistinct. Colony diameters (in mm after 120 h): 15 °C PDA 10–12, MEA 15–23, OA 14–15; 20 °C PDA 21–26, MEA 20–27, OA 25–27; 25 °C PDA 29–32, MEA 26–28, and OA 30–34.

Additional material examined: Korea: Jeju-do, 33°23′39.2″N, 126°14′23.0″E, isolated from Sargassum fulvellum, 10 Jan. 2015, S. Jang (KUC21232, KUC21284, and KUC21287).

Notes: Arthrinium sargassi has morphological characteristics similar to those of other species in clade B. It can be distinguished from A. aureum (globose to ellipsoid conidia, 10–30 × 10–15 μm) and A. hydei (globose conidia, 17–19 μm diam) in the much smaller conidia, (8.5–)9.5–11 (− 11.5) × (8.0–)8.5–10 (− 11) μm ($\overline{x}$ = 10.4 × 9.4 μm, n = 30) (Calvo 1980; Crous & Groenewald 2013). Arthrinium rasikravindrae KUC21327 (34–39 mm in 5 d on PDA at 20 °C) and A. marinum (16–17 mm in 5 d on PDA at 20 °C) can be distinguished from A. sargassi (21–26 mm in 5 d on PDA at 20 °C) by their growth rate. Unfortunately, there are no data regarding the growth rate of A. chinense, but it can be clearly separated from A. sargassi based on the phylogenetic analysis (Figs. 1, 2). Arthrinium sargassi is morphologically similar to A. sinensis, a non-sequenced species. However, the shape of conidiogenous cell differs between them; lageniform in A. sinensis and cylindrical in A. sargassi (Table 2).

Arthrinium taeanense S.L. Kwon, S. Jang & J.J. Kim, sp. nov.

MycoBank MB834599

(Fig. 10)

Fig. 10

Arthrinium taeanense (KUC21322). A-C, Colonies on PDA (a), MEA (b), and OA (c) (top); d-f, colonies on PDA (d), MEA (e), and OA (f) (bottom); g-h, conidia under SEM; i-k, conidia attached to conidiogenous cells; scale bar = 10 μm

Etymology: ‘taeanense’ refers to the type locality.

Molecular diagnosis: Arthrinium taeanense is distinguished from the phylogenetically most closely related species, A. gutiae, by unique single nucleotide polymorphisms in the three loci used in this study (Figs. 3S, 4S, 5S): ITS positions 22 (A), 32 (indel), 43 (G), 48 (C), 109 (indel), 113 (T), 121 (T), 129–146 (indel), 149–156 (indel), 189–192 (indel), 202–211 (indel), 213 (indel), 221 (T), 227–228 (indel), 248–250 (indel), 253 (C), 257 (T), 263 (A), 283 (G), 300 (T), 308 (C), 535 (C), 536 (G), 546 (T), 591 (A), 592 (T), and 593 (T); TEF positions 173 (T), 174 (C), 175 (A), 176 (C), 179 (C), 180 (T), 189 (G), 194 (G), 200 (indel), 209 (A), 213 (indel), 214 (C), 226 (A), 228 (A), 229 (A), 230 (C), 251 (C), 252 (T), 253 (T), 260 (A), 263 (C), 264 (T), 265 (A), 266 (T), 269 (T), 270 (T), 272 (G), 273–275 (indel), 278 (T), 280 (indel), 281 (A), 287 (G), 289 (C), 293 (A), 302 (indel), 304 (indel), 307 (G), 308 (G), 309 (indel), 310 (A), 313 (A), 314 (indel), 318 (G), 334 (G), 337 (T), 356 (A), 357 (G), 358 (A), 371 (T), 374 (A), 375 (G), 376 (G), 378 (C), 395 (C), 404 (C), 467 (T), and 600 (C); TUB positions 2 (T), 3 (C), 7 (C), 10 (C), 11–12 (indel), 16 (G), 17 (T), 19 (A), 20 (C), 21 (A), 22 (T), 23 (C), 25 (C), 26 (G), 28 (G), 29 (A), 33 (C), 34 (C), 35 (T), 36 (C), 38 (C), 41 (T), 44 (A), 46 (G), 53 (A), 54 (T), 68 (T), 69 (C), 71 (A), 72 (A), 73 (T), 74 (A), 75 (T), 78 (T), 80 (G), 81 (C), 85 (G), 87 (G), 89 (G), 95 (C), 108 (G), 111 (G), 114 (A), 129 (T), 138 (C), 140 (T), 143 (T), 146 (T), 158 (C), 170 (C), 176 (C), 184 (A), 198 (C), 205 (A), 207 (C), 211–212 (indel), 214–216 (indel), 231 (G), 308 (C), 309 (C), 312 (C), 313 (T), 319 (T), 324 (C), 326 (G), 327 (C), 328 (C), 329 (T), 344 (T), 347 (T), 353 (C), 392 (A), 395 (T), 410 (C), 413 (G), 416 (C), 425 (C), 428 (T), 434 (C), 437 (G), 455 (T), 476 (T), 479 (C), and 485 (C).

Type: Korea: Chungcheongnam-do, Taean-gun, 36°50′14.3″N, 126°11′04.7″E, isolated from Seaweed, 19 Mar. 2016, S. Jang (Herb. KCTC 46910 – holotype preserved in a metabolically inactive state; KUC21322 = NIBRFGC000501589 – ex-type culture).

Description: Mycelium consisting of smooth, hyaline, branched, septate, 2.0–4.5 μm diam. Conidiogenous cells aggregated in clusters on hyphae, hyaline, cylindrical. Conidia brown, smooth to granular, globose to elongate ellipsoid in surface view, (5.0–)5.5–6.5 (− 7.0) × 4.0–5.5 (− 6.0) μm ($\overline{x}$ = 6 × 4.7 μm, n = 30); lenticular in side view, with an equatorial slit, 4.0–5.0 μm wide ($\overline{x}$ = 4.7 μm, n = 30), elongated cell observed.

Culture: PDA, colonies thick, concentrically spreading with aerial mycelium, margin circular; mycelia white to yellow, gray and partially pale orange colored; sporulation was not observed; pale yellow (5Y 8/3) pigment to yellow (2.5Y 8/8) pigment diffused in media after 2 weeks; odour indistinct. MEA, colonies thick, flat, concentrically spreading with aerial mycelium, margin circular; mycelia white to yellowish gray colored; sporulation was not observed; pigment absent in medium; odour indistinct. OA, colonies very thick, concentrically spreading with aerial mycelium, margin circular; mycelia white to yellow and orange to brown colored; sporulation was not observed; yellowish brown (10YR 5/8) pigment diffused in media after 2 weeks; odour indistinct. Colony diameters (in mm after 120 h): 15 °C PDA 7–15, MEA 10–20, OA 10–11; 20 °C PDA 28–36, MEA 24–32, OA 21–24; 25 °C PDA 36–39, MEA 34–35, and OA 39–41.

Additional material examined: Korea: Chungcheongnam-do, Taean-gun, 36°50′14.3″N, 126°11′04.7″E, isolated from seaweed, 19 Mar. 2016, S. Jang (KUC21358, KUC21359).

Notes: Arthrinium taeanense is most closely related to A. pusillispermum (95.30% similarity in the ITS region, 80.84% in the TEF region, and 79.30% in the TUB region) and A. gutiae (95.30% similarity in the ITS region, 85.19% in the TEF region, and 78.3% in the TUB region) (Fig. 1). There were no noticeable morphological characters that helped separate these species, but the long stem branches clearly indicate that they represent different, phylogenetically well-separated taxa. Arthrinium taeanense can be distinguished from the 22 non-sequenced species by its small conidia size (Table 2).

DISCUSSION

A total of 14 Arthrinium species associated with marine environments in Korea was identified based on morphological and molecular phylogenetic analyses. Five species, A. arundinis, A. marii, A. rasikravindrae, A. sacchari, and A. saccharicola, had already been reported from marine environments (Hong et al. 2015; Park et al. 2018), whereas A. piptatheri was reported here for the first time from this habitat. The newly recognized taxa represented six species isolated from macroalgae (A. agari, A. fermenti, A. marinum, A. pusillispermum, A. sargassi, and A. taeanense) and two extracted from the egg masses of sailfin sandfish (A. arctoscopi and A. koreanum). To date, the majority of the described Arthrinium species have been isolated from various terrestrial habitats (Tsukamoto et al. 2006; Kim et al. 2011; Crous & Groenewald 2013), whereas only eight Arthrinium species have been reported from marine environments: A. algicola, A. arundinis, A. hispanicum, A. marii, A. phaeospermum, A. rasikravindrae, A. sacchari, and A. saccharicola (Miao et al. 2006; Jones et al. 2009; Crous & Groenewald 2013; Hong et al. 2015; Larrondo 1992; Li et al. 2017; Park et al. 2018; Pintos et al. 2019).

As mentioned, conidial shape, conidiophores, and presence or absence of sterile cells and setae were previously used for the infrageneric classification and delimitation of species (Schmidt & Kunze 1817; Hughes 1953; Minter 1985). However, because these microscopic features often overlap between taxa, it is difficult to solely rely on them to distinguish species. Therefore, the combined use of molecular and morphological characters, in combination with the physiological features of the cultures, is required to identify species in Arthrinium. For example, the newly recognized species, A. marinum, A. pusillispermum, and A. taeanense, cannot be distinguished from their close relatives based on morphology alone; however, the three species could be distinguished by differences in their growth rate and by the molecular data.

Arthrinium species can be divided into two groups based on conidial shape: one group with an irregular conidial shape, similar to a cashew-nut (A. kamischaticum) or a polygon (A. puccinioides), and the other with globose to ellipsoid conidia (Singh et al. 2013). All Arthrinium species in this study produced globose to subglobose or globose to ellipsoid conidia. This corresponds to the conidial shape of other Arthrinium species derived from marine environments (Larrondo 1992; Crous and Groenewald 2013; Singh et al. 2013). Among the species with ellipsoid conidia, those from marine environments generally have more elongated conidia than those from terrestrial environments (Table 2). There are a number of Arthrinium species described only from their sexual morph (e.g., A. balearicum, A. garethjonesii, A. longistromum, A. neosubglobosa, A. subglobosa) (Senanayake et al. 2015; Dai et al. 2016; Dai et al. 2017; Pintos et al. 2019). Unfortunately, no sexual morph is known in any of the marine species. This further increases the difficulty of identifying Arthrinium species through morphological features alone.

DNA sequencing data available for Arthrinium species has been steadily increasing in recent years (Crous and Groenewald 2013; Wang et al. 2018; Pintos et al. 2019). Currently 84 species of Arthrinium are recognized; of these, sequence information on the ITS is available for 62 species, TUB for 51, and TEF for 45 species. This has contributed to an increase in newly recognized species and aids in their accurate and rapid identification (Wang et al. 2018; Pintos et al. 2019). ITS by itself is limited in its ability to identify species within Arthrinium. The use of TUB, TEF, and multigene sequence data (ITS, TUB, and TEF) has increased the accurate identification and phylogenetic relationships in Arthrinium. This study generated 67 sequence datasets for three gene regions (ITS, TUB, and TEF), which will also contribute to furthering the study of the genus Arthrinium.

According to our previous studies on marine Arthrinium species, the 14 identified in this study can be expected to have high biological activity. However, it is not clear whether they are active in the actual marine environment and what the ecological role of Arthrinium species is. We expect to better understand their role in the environment through various studies of Arthrinium species in the future, including the discovery of further novel species and an exploration of their biological properties.

CONCLUSIONS

Our study underlines the notion that the diversity of Arthrinium species is still poorly known. More than half of the Arthrinium species isolated from a limited marine environment resulted to be new to science. According to our results, many more novel taxa are to be expected from marine environments around the world. Further studies in other environments are needed to assess the distribution of these species. Our results also show that a polyphasic approach to the taxonomy of Arthrinium, integrating molecular phylogeny of ITS and protein-coding markers, conidial features and culture characteristics are the most reliable approach to delimit and recognize species in this genus.