Novelties in Fuscosporellaceae (Fuscosporellales): Two New Parafuscosporella from Thailand Revealed by Morphology and Phylogenetic Analyses

: Asexual morphs of freshwater fungi have been mostly reported from tropical and subtropical re-gions. From our ongoing investigation of the diversity and taxonomy of freshwater microfungi in Thailand, a country with rich natural resources and diverse ecosystems, Parafuscosporella ellipsoconidiogena sp. nov. and P. obovata sp. nov., collected from decaying submerged twigs at Phalad Waterfall in a conserved forest in Chiang Mai Zoo, Chiang Mai Province, northern Thailand, are proposed. DNA phylogenies based on a combination of ITS and LSU datasets support the placement of these species in Parafuscosporella (Fuscosporellaceae, Fuscosporellales, Sordariomycetes), and these two novel species differ from known species in terms of morphology. Detailed descriptions, illustrations and a key to Parafuscosporella species are provided, as well as comparisons with other accepted Parafuscosporella species.


Introduction
Parafuscosporella belongs to Fuscosporellaceae (Fuscosporellales, Hypocreomycetidae, Sordariomycetes) [1]. The genus is characterized by sporodochial, black colonies; partly immersed, partly superficial, septate, hyaline to pale brown mycelium; semimacronematous, mononematous, simple or branched, mostly moniliform, smooth-walled, hyaline conidiophores; monoblastic, discrete or integrated, globose, subglobose, ellipsoidal or clavate, smooth-walled, hyaline conidiogenous cells; and conidia that are ellipsoidal to broadly obpyriform, transversely septate, smooth, dark brown to black and pale brown at the basal cell [1]. Based on morphological and molecular data, the type species without sexual morph, P. moniliformis Jing Yang, Bhat & K.D. Hyde, was described on dead and decaying submerged wood in Thailand. To date, five accepted species, namely, P. aquatica H. Yang [1][2][3][4]. In this study, we describe P. ellipsoconidiogena and P. obovata as the sixth and seventh species in the genus, respectively, collected from a waterfall in Chiang Mai Zoo, Chiang Mai Province, Thailand. Morphological descriptions and illustrations of P. ellipsoconidiogena sp. nov. and P. obovata sp. nov., a key to the species and an updated combined gene phylogenetic tree (the internal transcribed spacer (ITS) region of ribosomal DNA and large subunit (LSU) of nuclear ribosomal DNA) are provided to reveal their taxonomic position among taxa in the Fuscosporellaceae (Fuscosporellales).

Sample Collection, Isolation and Morphological Data
Submerged woody material was randomly collected from Phalad Waterfall located in Chiang Mai Zoo (18 •  . Woody samples were placed into plastic bags and transferred to the mycological laboratory at the National Center for Genetic Engineering and Biotechnology (BIOTEC), National Science and Technology Development Agency (NSTDA, Pathum Thani, Thailand), for observation. Decaying wood specimens were incubated in plastic containers with sterile tissue paper soaked with sterile distilled water at room temperature (20-25 • C) for 7-14 days, according to the methods described by Boonyuen et al. [2]. The specimens were observed using a stereomicroscope (Olympus SZ61; Olympus Corporation, Tokyo, Japan) for the presence of freshwater microfungi, and permanent slides were prepared by adding lactoglycerol and sealing with clear nail polish. Morphological characteristics such as conidiophores, conidiogenous cells and conidial dimension were examined. Cultural characteristics such as colony appearance and colour over the plate were also studied. Axenic cultures were obtained by single spore isolation method, following the protocol in Chuaseeharonnachai et al. [5]. Germinated spores were transferred to a potato dextrose agar (PDA, Difco TM , Sparks, MD, USA) plate and incubated at room temperature (20)(21)(22)(23)(24)(25)
PCR amplification was performed in a 50 µL reaction volume containing 25 µL of One PCR TM Ultra (Bio-Helix, New Taipei City, Taiwan; a premix and ready-to-use solution, including Taq DNA polymerase, PCR buffer, dNTPs, gel loading dyes, enhancer, and fluorescence dye), 1 µL of each primer (10 µM), 1 µL of genomic DNA extract and 22 µL of sterile deionized water. The PCR thermal cycle programs of the ITS and LSU were as follows: 94 • C for 2 min, followed by 35 cycles of denaturation at 94 • C for 1 min, annealing at 55 • C for 1 min, elongation at 72 • C for 2 min and a final extension at 72 • C for 10 min. The PCR thermal cycle program of the SSU was as follows: 95 • C for 5 min, followed by 34 cycles of denaturation at 95 • C for 1 min, annealing at 55 • C for 1 min, elongation at 72 • C for 1.5 min and a final extension at 72 • C for 10 min. The PCR thermal cycle program of RPB2 was as follows: 95 • C for 5 min, followed by 34 cycles of denaturation at 95 • C for 1 min, annealing at 58 • C for 1 min, elongation at 72 • C for 1.5 min and a final extension at 72 • C for 10 min. The amplicons of the ITS and LSU were purified and sequenced by Macrogen Inc. (Seoul, South Korea) with the same PCR primer used for DNA amplification. The PCR products of RPB2 were purified using a NucleoSpin ® Gel and PCR Clean-up Kit (Macherey-Nagel, Düren, Germany) and sequenced at Macrogen Inc. (Seoul, South Korea).

Sequence Alignment and Phylogenetic Analyses
The SSU, ITS, LSU and RPB2 sequences of our isolates are provided in this study. Based on previous phylogenetic studies on Fuscosporellaceae (Fuscosporellales) by Yang et al. [3], two combined analyses of the ITS and LSU sequences provided resolution at the species level. In addition, there are only a few SSU and RPB2 sequences of Fuscosporellales available in GenBank. Thus, the ITS and LSU datasets were used only for the combined sequence data analyses in this study.
A maximum likelihood (ML) tree was constructed by RAxML-NG v. 1.0.3 using the GTR+G model and the all-in-one analysis option [11]. The best ML tree was identified using the two-step L-BFGS-B method [12], to optimize the parameters of the LG4X model [13]. ML branch support was obtained using nonparametric bootstrapping with 1000 replications.
A Bayesian inference (BI) phylogenetic tree was constructed with the GTR+G model using the Metropolis-coupled Markov chain Monte Carlo (MCMCMC) method in MrBayes 3.2.7a [14]. The MCMCMC searches were run for 1,000,000 generations with sampling every 100 generations. BI posterior probabilities (BIPPs) were summarized and mapped on the best ML tree using the SumTrees program in DendroPy version 4.5.2 [15]. The first 100 trees were excluded as burn-in. The newly obtained sequences taxa used in phylogenetic analyses were deposited in the GenBank database and are provided in Table 1.
In PDA culture, the sizes of the conidiogenous cells and conidia of both species somewhat overlap, and these two species mainly differ in the shape of the conidiogenous cells as well as the shape and colour of the conidia. Parafuscosporella ellipsoconidiogena has cylindrical or ellipsoidal conidiogenous cells and broadly obpyriform, ellipsoidal, obovoid, medium brown to dark brown conidia, while P. mucosa has doliiform or obovoid conidiogenous cells and globose to subglobose, olivaceous to pale brown conidia [1].

Discussion
In this study, phylogenetic analyses based on the combined ITS and LSU coupled with morphology placed Parafuscosporella species, together with two novel taxa of P. ellipsoconidiogena [1][2][3], and P. obovata within Fuscosporellaceae (Fuscosporellales), in agreement with a previous study [4]. In addition, both novel species described here are clearly separate from the known species in terms of phylogeny and morphology. Thus, two species, P. ellipsoconidiogena and P. obovata, found in Thailand, are newly introduced.
The morphological characters of Parafuscosporella in culture are different from natural material. The culture characteristic of these taxa on PDA is characterized by the absence of conidiomatal colonies; conidiophores reduced to a single conidiogenous cell; integrated or often cylindrical, ellipsoidal, subglobose or dumbbell-shaped conidiogenous cells; and 0-2-septate, pigmented, Humicola-like or Trichocladium-like conidia, as described in Table 3 [1,2,4].
Based on conidial characters, the significant distinctiveness of Parafuscosporella spp. in species identification is mainly on natural material and synthetic media, such as shape, size, septation and conidial formation. To identify Parafuscosporella spp., both morphological description and DNA sequences analyses are needed (i.e., ITS data or the combined analyses of ITS and LSU sequences), so that they can be resolved at the species level [3].
The geographical distribution of Parafuscosporella species show they are only known from Thailand and potentially in China. All Parafuscosporella species are freshwater fungi living on decaying woody material [1][2][3][4]. Parafuscosporella ellipsoconidiogena and P. obovata, introduced here with morphological descriptions and molecular phylogenetic analyses of a multigene DNA sequence dataset, were discovered in Chiang Mai Province in northern Thailand, where previous studies (i.e., from Chiang Dao District, Mae Teang District and Doi Suthep-Pui National Park) have also discovered novel microfungi and new freshwater fungi (i.e., [2,[30][31][32][33]). Compared to other provinces and parts of Thailand, Chiang Mai Province has a tropical savanna climate with low latitudes and moderate elevations and is characterized by days that range between warm and hot year-round and nights that are cool with tolerable temperatures. Furthermore, Chiang Mai has three major seasons, including the cool (November to February), dry-hot (March to May) and rainy (June to October) seasons. In this study, two species, P. ellipsoconidiogena and P. obovata, were collected during the rainy season in August 2018 and August 2019, respectively. This season is characterized by a high level of flowing water and abundant decaying submerged wood at Phalad Waterfall located in Chiang Mai Zoo. Located in a conserved and undisturbed forest in Chiang Mai Zoo, the aquatic environment of Phalad Waterfall is undisturbed by humans; as a consequence, it is probably conducive to the discovery of novel fungal species. In addition, regarding fungal distribution, our results are in accordance with earlier studies [2,4,31], showing that Parafuscosporella species are freshwater hyphomycetes on woody substrates. The main advantage of these fungi on submerged woods is that they have the ability to maintain activity at low temperatures and degrade submerged organic matter under various climatic conditions. These new freshwater asexual fungi add to the increasing number of microfungi known from Thailand, and suggests that numerous new species await discovery in other conserved and undisturbed forests of Thailand. As most Parafuscosporella species are documented from Thailand, wider sampling from other global locations is required.