﻿Two new species of Scolecobasidium (Venturiales, Sympoventuriaceae) associated with true mangrove plants and S.terrestre comb. nov.

﻿Abstract Scolecobasidium is cosmopolitan and includes species that inhabit a wide range of ecosystems including soil, water, air, plant and cold-blooded vertebrates. During a fungal survey from mangrove, strains of Scolecobasidium occurring on leaf spots of true mangrove plants, Aegicerascorniculatum and Acanthusebracteatus, were isolated from Futian Mangrove in Shenzhen and the Qi’ao-Dangan Island Mangrove in Zhuhai, China. Unlike most species in Scolecobasidium that produce dark conidia, our strains are characterized by hyaline to pale brown conidia and inconspicuous thread-like sterigmata. Further detailed morphological comparison and multi-locus (LSU, ITS, tub2, tef1-α) phylogenetic analyses revealed these collections as two new taxa, namely S.acanthisp. nov. and S.aegiceratissp. nov. We further emend the generic description of Scolecobasidium, propose one new combination, S.terrestre comb. nov., and clarify the taxonomic status of S.constrictum.


Introduction
Scolecobasidium was described based on two species, S. terreum and S. constrictum, with the former as the generic type (Abbott 1927). This genus is slow-growing, and characterized by brownish to black colonies, reduced, hyaline or pigmented conidiophores and septate, smooth-or rough-walled, brown, single, dry and rhexolytic conidia (Abbott 1927; Barron and Busch 1962;Seifert and Gams 2011). A distinguishing feature of Scolecobasidium from other genera is the conidiophores, which are born on aerial hyphae as short non-septate structures producing conidia from the ends of thread-like sterigmata (Abbott 1927). Subsequently, more species with unbranched conidia were described in Scolecobasidium and the conidia of this genus are found to be variable in shape, especially its type species S. terreum producing Y-shaped or T-shaped conidia distinct from other species in the genus. Therefore, Hoog and von Arx (1973) introduced a separate genus Ochroconis, typified by O. constricta (syn. S. constrictum), and transferred many Scolecobasidium species into this genus (De Hoog 1973). At that time, Ochroconis was comprised of species with sympodial conidiogenesis and ellipsoidal, clavate or fusiform conidia, whereas the genus Scolecobasidium was restricted to species with T-or Y-shaped or bilobed, 1-or multiple septate conidia born on ampulliform conidiogenous cells possessing 1-3 conidial-containing denticles at the tip of the conidiophores (De Hoog 1973). Subsequently, Ellis (1976) classified Ochroconis as a synonym of Scolecobasidium, and Gams (2015) and Seifert and Gams (2011) agreed with this interpretation. In addition to the similar morphological characteristics of Scolecobasidium and Ochroconis, recent molecular analyses have clearly shown that the two genera constituted a polyphyletic complex and Ochroconis should not be treated as a separate genus (Hao et al. 2012;Samerpitak et al. 2014). Although the ex-type strains of both S. terreum (CBS 203.27) and O. constricta (CBS 202.27) are sterile after a long period of preservation, they were phylogenetically placed within the Scolecobasidium clade (Shen et al. 2020). Therefore, based on the principle of priority, the older generic name Scolecobasidium was chosen over Ochroconis, and 25 new combinations have been proposed (Shen et al. 2020;Crous et al. 2021;Wei et al. 2022).
Scolecobasidium is the largest genus within Sympoventuriaceae, Venturiales, Dothideomycetes (Shen et al. 2020) and about 98 epithets are currently listed in Index Fungorum (Index Fungorum accession date: 10.01.2023). Scolecobasidium is a cosmopolitan genus of saprotrophic soil hyphomycetes, some of which are also parasitic on plants (De Hoog 1985;Crous et al. 2016), human (Giraldo et al. 2014), fish (Ross and Yasutake 1973) or other animals (VanSteenhouse et al. 1988;Singh et al. 2006). In our study, during the fungal investigations of mangrove plants in China, several strains of Scolecobasidium were isolated from leaf spots on Aegiceras corniculatum and Acanthus ebracteatus, and they were revealed as two novel species through polyphasic analyses. In addition, on the basis of Shen et al. (2020) and Wei et al. (2022), we correct the taxonomic status of ambiguous species in this group.

Sample collection and fungal isolation
During our fungal investigations on mangrove plants in China, 90 strains of 48 species have been isolated from true mangrove plants, Acanthus ebracteatus and Aegiceras corniculatum (Table 1). Among them, Scolecobasidium-like strains piqued our interest

Morphological observations
Colony features including color and growth rate were recorded for the strains grown on oatmeal agar (OA) and malt extract agar (MEA) after 14 days at 25 °C. To enhance sporulation, strains were incubated at 25 °C under near UV light with a 12 h photoperiod for 14 d or longer period. Morphological observations of reproductive structures were made in lactic acid and observed using a Nikon Eclipse 80i microscope using differential interference contrast (DIC) illumination. At least 30 measurements per structure were taken, and the mean value, standard deviation, and minimummaximum values were given.

DNA extraction, PCR amplification and sequencing
Fresh fungal mycelia grown on potato dextrose agar (PDA) for 14 d at 25 °C were scraped from the colony margin and used for genomic DNA extraction using a modified CTAB protocol as described previously (Guo et al. 2000). Genomic DNA was diluted to 1 ng/μL using sterile water as the template for PCR. The amplification of internal transcribed spacer (ITS) region including the flanking 5.8S rRNA gene, was carried using the primer pairs ITS1 and ITS4 (White, Bruns, Lee, Taylor, 1990), the 28S nuclear large subunit (nuLSU) with LR0R/LR5 (White, Bruns, Lee, Taylor, 1990), with EF1-728F/EF-2 (Qiao et al. 2016) for the partial translation elongation factor 1-alpha gene (tef1-α) and Bt2a/Bt2b to amplify the partial beta-tubulin gene (tub2) (Glass and Donaldson 1995), respectively. The reaction volume of 25 μL consisted of 10× PCR buffer 2.5 μL, MgCl 2 2 mM, dNTPs 50 μm/L, forward and reverse primers 0.1 μm/L, DNA polymerase 0.5 U, and DNA template 10 ng. PCR amplification reactions for LSU and ITS were performed as follows: pre-denaturation at 95 °C for 10 min, followed by 35 cycles of denaturation at 95 °C for 45 s, annealing at 52 °C for 45 s, extension at 72 °C for 1 min, and a final extension step at 72 °C for 10 min, but the annealing temperature was adjusted to 56 °C for tef1-α and tub2. PCR products were detected by 1% agarose gel electrophoresis and then sequenced by SinoGenoMax.
MEGA v. 7 was used to obtain consensus sequences from DNA data generated from forward and reverse primers.

Phylogenetic analyses
Phylogenetic analysis was performed using sequences of LSU, ITS, tub2, and tef1-α from 64 type and reference strains of Ochroconis, Scolecobasidium and one outgroup Verruconis calidifluminalis CBS 125818 (Table 2). Single locus alignment was performed using an online version of MAFFT v. 7 (Katoh and Standley 2013) and then concatenated for Maximum Likelihood (ML) and Bayesian analysis (BA). ML and BA were implemented

Phylogeny
The BLAST searches in the NCBI's GenBank nucleotide database using ITS sequences of LC19368, LC19369 and LC19370 showed their closest similarities to Scolecobasidium spp. In the following multi-locus phylogenetic analysis of Scolecobasidium, the dataset comprised 2,932 characters including alignment gaps (LSU: 855 bp, ITS: 818 bp, tub2: 542 bp, tef1-α: 717 bp). The ML search revealed a best tree with an InL of -34731.383727. For the Bayesian inference, a GTR+I+G model was selected for ITS, LSU, tef1-α and tub2. The BA was run for 1,535,000 generations, and a 50% consensus tree and posterior probabilities were calculated from 2,304 trees from two runs. The topologies of phylogenetic trees generated by ML and BA were congruent. Our strains were separated into two distinct clades from all known species of Scolecobasidium (Fig. 1).  Ochroconis de Hoog & Arx, Kavaka 1: 57. 1974. Synonym.
Description. Colonies restricted, slow-growing, brown or olivaceous. Aerial hyphae smooth-or somewhat rough-walled, pigmented. Cleistothecia up to 40 μm in diam, dark brown; peridium wall composed of textura angularis. Ascomata bearing antlershaped appendages, with serrate edges. Asci bitunicate, clavate, 8-spored; ascospores pale brown, verruculose, 1-3-septate. Conidiophores reduced, unbranched or sparingly branched, arising from the aerial hyphae or hyphal ropes, continuous or septate, hyaline or pigmented, ovoid, clavate, wedge-shaped, cylindrical, or irregular. Conidiogenous cells scattered, monoblastic or sympodial, elongate to cylindrical. Conidia produced in clusters or acropetal series from the ends of tubular extensions of the conidiophores; conidia 1-4-celled, pigmented or hyaline, smooth or verrucose, ellipsoidal, ovoid, cylindrical, or T-or Y-shaped. (emended from Abbott 1927; Barron and Busch 1962;Seifert and Gams 2011;Samerpitak et al. 2014). Notes. Abbott (1927) summarized the asexual features of Scolecobasidium as its generic character based on only two species S. terreum and S. constrictum. Over time multiple species of Scolecobasidium have been described, and the boundaries between this genus and closely related genera have also been clarified (Barron and Busch 1962;Seifert and Gams 2011;Samerpitak et al. 2014;Shen et al. 2020;Wei et al. 2022). However, no one has updated the generic character of Scolecobasidium. In this study, we update the generic character of Scolecobasidium based on previous descriptions, especially the morphological features of the sexual stage of the fungus.

Scolecobasidium acanthi
Culture characteristics. Colonies reaching up to 16-20 mm diam after 14 days at 25 °C, producing dense aerial mycelium on MEA and OA. On MEA, surface wheat to greyish brown, reverse saddlebrown, felty, dry, margins smooth. On OA, surface burlywood to peru, reverse brown black, margins smooth.
Culture characteristics. Colonies reaching up to 20-22 mm diam after 14 days at 25 °C, dense aerial mycelium on MEA and OA. On MEA, smooth to felty, dry, surface greyish brown to dark brown, reverse saddle brown. On OA, surface ivory to peru, reverse brown black.
Notes. Scolecobasidium aegiceratis is phylogenetically related to S. dracaenae (Fig. 1) and can be differentiated from the later by DNA sequences of LSU (99.52% similarity), ITS (93.55%) and tef1-α (96.60%) regions. Morphological characters of the two species are overlapping but their conidiophores and conidia show differences. Scolecobasidium aegiceratis can be distinguished from S. dracaenae as it produces hyaline to pale brown (vs. brown in S. dracaenae) conidiogenous cells. In addition, the dimensions of their conidia (8-15(-26.5) × 2.5-3.5(-6.5) μm vs. 6.5-10 × 3-4 μm) and conidiogenous cells (7.5-24 × 1.5-2.5 μm vs. 5-15 × 2.5-3 μm) are different (Crous et al. 2016  Notes. Scolecobasidium constrictum was introduced at the same time as the generic type of Scolecobasidium, S. terreum, by Abbott (1927). Later, Heterosporium terrestre was treated as a synonym of S. constrictum due to their similar morphological characteristics (Barron and Busch 1962). Their original descriptions differ, however, in that H. terrestre produces rough conidia and variable conidiophores both in shape and size, and has occasional phragmospores. Therefore, Barron (1962) thought that Abbott described only a facet of S. constrictum and emended the species description. Subsequently, Ochroconis was introduced to accommodate species with sympodial conidiogenesis and unbranched, subspherical to cylindrical or clavate, melanised conidia, and S. constrictum was transferred to this genus as O. constricta and designated as the generic type (De Hoog 1973).
With the help of molecular analyses, Shen et al. (2020) synonymized Ochroconis under Scolecobasidium, and S. constrictum should be resurrected as a result. We observed that the ex-type cultures of H. terrestre (CBS 211.53) and S. constrictum (CBS 202.27) were separated into two distinct clades with relatively long branches in the current study (Fig. 1) and sequences similarities between the two species were very low (LSU: 98.7%; ITS: 88%, tef1-α: 85%, tub2: 79%). Since S. constrictum (CBS 202.27) is now sterile (Shen et al. 2020), we could only make a detailed morphological comparison among multiple descriptions of ex-type cultures of H. terrestre (CBS 211.53) and S. constrictum (CBS 202.27) (Abbott 1927;Atkinson 1952; Barron and Busch 1962;Samerpitak et al. 2014), and found that the two fungi were morphologically different in the shape (oval to ovate, short-cylindrical and long cylindrical to sole-shaped in H. terrestre vs. echinulate to verrucose in S. constrictum) and number of septa (0-3 vs. 0-1) in the conidia, as well as the size of conidiophores (2-31.5 × 1.5-2.5 μm vs. 5-8 × 2-2.5 μm). Based on morphology combined with the phylogeny, we consider that H. terrestre R.G. Atk. should not be treated as a synonym of S. constrictum, and a new combination Scolecobasidium terrestre comb. nov. is proposed in this study.
In addition, the type strain of S. constrictum should be CBS 202.27, rather than CBS 211.53, which was incorrectly listed in tables 2, 3 and figs 1, 2 in Wei et al. (2022).