Additions to Occultibambusaceae ( Pleosporales , Dothideomycetes ): Unrevealing Palmicolous Fungi in China

: During a survey of microfungi associated with palms from karst formations, three novel ascomycetes were found from decaying petioles of Trachycarpus fortunei ( Arecaceae ) in Guizhou, China. Multi-gene phylogenetic analyses based on a combined SSU, ITS, LSU, RPB2 and TEF1 α sequence data showed that these collections were afﬁliated to Brunneofusispora and Neooccultibambusa in the family Occultibambusaceae . A new species Brunneofusispora inclinatiostiola is introduced. It is phylogenetically close to B. clematidis but represents a distinct lineage. Morphologically, it differs from the latter in having immersed ascomata with eccentric, periphysate ostiole and smaller ascospores. Morpho-phylogenetic evidence also revealed two new Neooccultibambusa species, N. kaiyangensis and N. trachycarpi . Together with the generic type N. chiangraiensis , they formed a distinct lineage within the genus Neooccultibambusa . Three novel palmicolous fungi of Occultibambusaceae are described, illustrated and notes on their identiﬁcation are provided. The ecological signiﬁcance of the new taxa and the phylogenetic relationship of genera in Occultibambusaceae is discussed.


Introduction
Occultibambusaceae was established by Dai et al. [1] and typified by Occultibambusa D.Q. Dai [1][2][3][4]. Versicolorisporium is the genus that only has an asexual morph in the family but is clustered with Occultibambusa in recent phylogenetic studies [2,5,6]. The difference between Seriascoma and the other four genera is that its sexual morphs have stromatic ascomata under a clypeus [1,7]. Brunneofusispora was considered to be different because of phylogenetic distinction and the ascomata of the type species have long necks [4]. Neooccultibambusa resembles Occultibambusa, however they are phylogenetically distinct, and the former has hyphomycetous asexual morphs or produces chlamydospores in culture [3,[8][9][10].

Phylogenetic Analyses
The reference dataset included representatives of the five accepted genera in Occultibambusaceae, as well as three Massarina rubi, which were detected by blastn search of our strains. Six simultaneous Markov chains were run for 595,000 generations and trees were sampled every 1000 generation and 595 trees were obtained. The first 119 trees representing the burn-in phase of the analyses were discarded, while the remaining 476 trees were used for calculating posterior probabilities in the majority rule consensus tree (critical value for the topological convergence diagnostic is 0.01).
The six isolates obtained in this study grouped with Brunneofusispora and Neooccultibambusa based on the multi-gene phylogeny analyses. Two of them were sister to Brunneofusispora clematidis with strong support and can be recognized as a new species B. inclinatiostiola. The other four strains were closely related to the generic type N. chiangraiensis and formed a well-supported clade (Figure 1), of which two new species N. kaiyangensis and N. trachycarpi were recognized. Neooccultibambusa kaiyangensis clustered together with N. chiangraiensis but can be morphologically and phylogenetically distinguished, while N. trachycarpi showed to be sister to N. chiangraiensis and N. kaiyangensis. mony analysis of the remaining 868 parsimony-informative characters resulted in 1000 trees with TL = 2859, CI = 0.567, RI = 0.674, RC = 0.382, and HI = 0.433. Six simultaneous Markov chains were run for 595,000 generations and trees were sampled every 1000 generation and 595 trees were obtained. The first 119 trees representing the burn-in phase of the analyses were discarded, while the remaining 476 trees were used for calculating posterior probabilities in the majority rule consensus tree (critical value for the topological convergence diagnostic is 0.01).   an immersed ostiolar neck. Ostiole are usually ecentric, brown to black and periphysate. Peridium 15-55 µm wide, sometimes up to 70 µm at the side, composed of several layers of hyaline to brown, thick-walled cells of textura angularis, darker and thicker outwardly. Hamathecium are 2.0-3.5 µm wide, cellular pseudoparaphyses, anastomosing above the asci, embedded in a gelatinous matrix. Asci 60-145 × 7.5-13.5 µm (x = 100.6 × 10.3 µm, n = 20), 8-spored, bitunicate, fissitunicate, cylindrical or cylindric-clavate, straight or slightly curved, short pedicellate, rounded at the apex, with an ocular chamber. Ascospores are (13.5-)14.0-19.0(-27.0) × 4.0-6.5 µm (x = 17.4 × 4.7 µm, n = 50), uniseriate to partially biseriate, fusiform to ellipsoidal, hyaline, becoming pale brown at maturity, 1-septate, the upper cell slightly wider than the lower cell, guttulate when young, smooth-walled, surrounded by a mucilaginous sheath. Asexual morph: Undetermined.

Discussions
A recent contribution to Occultibambusaceae is the introduction of new species for Occultibambusa and Serisacoma [30]. It also discussed that Occultibambusa and Notes: Neooccultibambusa trachycarpi resembles N. chiangraiensis and N. kaiyangensis in having immersed ascomata, fusiform, hyaline to pale brown, septate ascospores with mucilaginous sheath. However, N. trachycarpi differs from the latter ones in having hyaline, 1-septate ascospores, which become pale brown when senescent and present two pseudosepta. The ascospores dimensions are also different ( Table 2). The phylogenetic results (Figure 1) showed that Neooccultibambusa trachycarpi clustered with N. chiangraiensis and N. kaiyangensis and formed a distinct lineage within Neooccultibambusa. The establishment of the new species Neooccultibambusa trachycarpi is justified by morphological and phylogenetic evidence.

Discussions
A recent contribution to Occultibambusaceae is the introduction of new species for Occultibambusa and Serisacoma [30]. It also discussed that Occultibambusa and Versicolorisporium are phylogenetically related, but they are different genera due to their significant difference in asexual morphs [30]. In this study, multi-gene phylogenetic analyses showed our samples were affiliated to Brunneofusispora and Neooccultibambusa, respectively.
Based ). Therefore, phylogenetic analysis was performed based on different matrices with the inclusion of those Massarina sp. and/or Massarina rubi (data not shown) and the multi-gene phylogeny result (Figure 1) supports the recognition of three new species. On the other hand, the genus Neooccultibambusa is not monophyletic (Figure 1), of which N. thailandensis formed an independent lineage in Occultibambusaceae. However, this is consistent with recent relevant studies [6,30] and further studies are needed to provide a better understanding towards the classification of Neooccultibambusa with more sampling and taxa population included in the analysis.
Karst landscape is attractive to tourists but also inhabits rich biodiversity due to its typical low temperature and high humidity environment. The fan palm Trachycarpus fortunei (Arecaceae) is native to subtropical and temperate mountain forests of China and 23 fungal species have been described from this plant [31,32]. The three new species introduced herein were found on Trachycarpus fortunei in a karst region. Additionally, Occultibambusa jonesii and O. maolanensis were also reported from karst landforms in China [12]. It is noteworthy that they are distributed in three different genera in Occultibambusaceae. Although this is only a small sample of data, it reflects somehow the hidden fungal diversity of palms and the karst formations.

Data Availability Statement:
The data is available upon request form the corresponding authors. The data is not publicly available due to its usage in the ongoing study.