﻿Paramphibambusabambusicola gen. et. sp. nov., Arecophilaxishuangbannaensis and A.zhaotongensis spp. nov. in Cainiaceae from Yunnan, China

﻿Abstract Morphological comparisons and multi locus phylogenetic analyses (base on the combined genes of ITS, LSU, rpb2 and tub) demonstrated that three new saprobic taxa isolated from bamboo belong to Cainiaceae. These taxa comprise a novel genus Paramphibambusa (P.bambusicolasp. nov.) and two new species, Arecophilaxishuangbannaensis and A.zhaotongensis. The three new taxa belong to Cainiaceae (Xylariales, Sordariomycetes) a poorly studied family, which now comprises eight genera. Paramphibambusa can be distinguished from other Cainiaceae genera in having ascomata with a neck and ascospores lacking longitudinal striation, germ slits or germ pores. The two new Arecophila species clustered in a clade with Arecophila sp. and A.bambusae. Detailed morphological descriptions, illustrations, and an updated phylogenetic tree are provided for the new taxa.

Arecophila was introduced by Hyde (1996) with A. gulubiicola K.D. Hyde as the type species.The genus Arecophila was initially regarded as a member of Amphisphaeriaceae G. Winter based on the morphology.Subsequently, Kang et al. (1999) accepted Arecophila as a member of Cainiaceae.Afterwards, the placement of Arecophila within the Cainiaceae has been confirmed based on analyses of partial LSU gene sequences (Jeewon et al. 2003;Senanayake et al. 2015;Li et al. 2022).Currently, 18 epithets are listed under Arecophila based on morpho-molecular study (Li et al. 2022;Index Fungorum 2023), and 15 epithets are listed under Arecophila in Species Fungorum (2023).

Sample collection, single spore isolation and morphological study
Bamboo culms were collected in northeastern (Zhaotong), northwestern (Shangri-La), and southwestern (Xishuangbanna) Yunnan Province, China, stored in disposable plastic Ziplock bags and brought back to the laboratory for examination and study.Morphological observation and single spore isolation were followed as described in Dai et al. (2017).The ascomata on the host surface were observed by Leica using a S8AP0 microscope and photographed by HDMI 200C.Micro-morphological features were observed using an Olympus BX53 compound microscope and captured with an Olympus DP74 camera (Olympus SZ61; Olympus Corporation, Tokyo, Japan).The asci were stained by Meltzer's reagent to examine the J-/J+ ring at the tip of the asci.India ink was used to stain the ascospores for checking the mucilaginous sheath.The micro-morphological features and fruiting bodies were measured by Tarosoft (R) Image FrameWork (IFW).The photo plates were created by Adobe Photoshop CS6 software (Adobe Systems Inc., San Jose, CA, USA).Herbarium material and living cultures were deposited at the Herbarium of Guizhou Medical University (GMB), Guizhou Medical University Culture Collection (GMBCC) Guiyang, Zhongkai University of Agriculture and Engineering (ZHKU), Zhongkai University of Agriculture and Engineering Culture Collection (ZHKUCC) Guangdong, China, and the Guizhou Culture Collection (GZCC), Guiyang, China.MycoBank numbers were obtained from MycoBank database (https://www.mycobank.org/;accessed on 23 January 2024) to register the newly described taxa (MycoBank 2024).

Paramphibambusa bambusicola
Culture characters.Ascospores germinating within 24 h.Colonies reaching 45 mm diam. in 20 days under dark and at 28 °C conditions, circular, flocculent, yellowish from above and below.
Notes.In the phylogenetic tree, Paramphibambusa bambusicola formed a stable clade basal to the other species of Cainiaceae with 90% ML, and 1.00 PP statistical supports (Fig. 1).In morphology, Paramphibambusa bambusicola has Cainiaceae species typical characteristics that are cylindrical asci, with a J+, apical ring, and ellipsoidal ascospores surrounded by a mucilaginous sheath.However, the spores of Cainiaceae species have the ornamented walls with longitudinal striations or germ slits or germ pores.Paramphibambusa bambusicola differs from the current Cainiaceae species by having smooth-walled ascospores.Notes.The genus Arecophila is characterized by immersed ascomata, usually with a clypeus, unitunicate, cylindrical asci, commonly producing an apical ring, and ascospores with longitudinal striation or a verrucose wall, and surrounded by a mucilaginous sheath (Hyde 1996;Li et al. 2022).Li et al. (2022) provided a morphological comparison of the main characters of Arecophila species.The asexual morph of Arecophila has not been reported.According to Li et al. (2022), this genus is distributed across 12 countries and is reported from 16 host species.Etymology.Named after the location "Xishuangbanna" where the new taxon was discovered.
Culture characters.Ascospores germinating within 24 h.Colonies reach 20 mm diam. in 15 days under dark and at 28 °C conditions, circular, hairy, white from above, and yellow to yellowish from below.

Discussion
Paramphibambusa forms deeply immersed, dark ascomata, with a long neck, J+ asci and smooth-walled ascospores.Interestingly, genera in Cainiaceae usually form ascospores with longitudinal striations or germ slits or germ pores, however, these characters were not observed in our new collection (GMB-W1350).Hence, we introduced the new genus Paramphibambusa in Cainiaceae based on morphological characteristics and phylogenetic analyses (Fig. 1).Moreover, we introduced two new Arecophila species in Cainiaceae.The establishment of Paramphibambusa and the introduction of two new Arecophila species enriches the species diversity of the family Cainiaceae and the diversity of bambusicolous fungi.
Currently, some species in the Cainiaceae are monospecific, such as Longiappendispora (Mapook et al. 2020), and Paramphibambusa (this study), while Amphibambusa, and Atrotorquata each contain only two species (Kohlmeyer and Volkmann-Kohlmeyer 1993;Liu et al. 2015;Jiang et al. 2021).Hence, more samples are needed to better understand each genus.Wijayawardene et al. (2022b) mentioned that it is essential to carry out more studies on host plants (that have been extensively studied for fungi, such as bamboo) in biodiversity-rich regions to reveal more novel species.Yunnan is exceedingly rich in fungal diversity, especially in higher level taxa, such as ascomycetes and basidiomycetes (Wijayawardene et al. 2021b;Dai et al. 2022).Hence, we believe that future studies on bamboo-associated fungi in Yunnan Province would disclose more novel taxa.
Atrotorquata was introduced as a monotypic genus by Kohlmeyer and Volkmann-Kohlmeyer (1993) to accommodate A. lineata Kohlm.& Volkm.-Kohlm.Subsequently, Liu et al. (2015) introduced A. spartii Thambug et al. as the second species.These two species share similar morphology, but their phylogenetic relationship was not well-resolved by Liu et al. (2015).Due to a lack of sequence data in GenBank, Atrotorquata clusters outside of Cainiaceae.More sequences especially protein genes loci are needed, to clarify its family placement.
Eighteen epithets were listed in Arecophila (Li et al. 2022), but only four taxa and a unnamed species have available molecular data, viz., A. australis Q.R. Li et al. (GZUCC0112,GZUCC0124), A. bambusae (HKUCC 4794), A. clypeata Q.R. Li et al. (GZUCC0110,GZUCC0127), A. miscanthi Q.R Li & J.C. Kang (GZUCC0122, MFLU 19-2333), and Arecophila sp.(HKUCC 6487).Thus, it is necessary to recollect fresh specimens and designate epitypes or reference specimens.Li et al. (2022) divided Arecophila into two clades based on phylogenetic analyses.We obtained the same results in our study, probably because most species of Arecophila lack protein genes regions in GenBank.We may need to design more suitable primers for sequencing protein genes fragments of Arecophila to support phylogenetic study.
, only four Cainiaceae species are associated with bamboo (Amphibambusa hongheensis H.B. Jiang & Phookamsak, Arecophila bambusae Umali & K.D. Hyde, A. coronata (Rehm) Umali & K.D. Hyde and A. nypae K.D. Hyde) in China.In this study, we aim to collect bamboo samples in Yunnan, China, describe and introduce a new genus Paramphibambusa to accommodate P. bambusicola, and two new species Arecophila xishuangbannaensis and A. zhaotongensis in the family of Cainiaceae.This study enriches the species diversity of bambusicolous Cainiaceae species in China.

Table 1 .
Sequences used for phylogenetic analyses in this study.The newly generated sequences are in bold.Type strains or type specimens are labelled with HT (holotype), ET (epitype), IT (isotype), and PT (paratype), T (Type), "N/A" indicates no available sequences.
ATCC: American Type Culture Collection; BISH: Bishop Museum, Department of Natural Sciences; CAI: Cairo University, Botany Department; CBS: