﻿New species and records of Chapsa (Graphidaceae) in China

﻿Abstract We studied the genus Chapsa in China based on morphological characteristics, chemical traits and molecular phylogenetic analysis. One species new to science (C.murioelongata M.Z. Dou & M. Li) and two records new to China were found (C.wolseleyana Weerakoon, Lumbsch & Lücking and C.niveocarpa Mangold). Chapsamurioelongatasp. nov. is characterised by its lobed thalline margin, orange discs with white pruina, clear hymenium, and submuriform and long ascospores. Chapsawolseleyana was recombined into Astrochapsa based on phenotypic traits. Sequences of this species are for the first time reported here and phylogenetic analyses of three loci (mtSSU, ITS and nuLSU) supported the position of this species within Chapsa. A key for the Chapsa species known in China is provided.

Although China is rich in lichenised fungal species (Wei 2020), there are few studies and reports on the genus Chapsa. More than 60 species of Chapsa have been reported in the world, of which only three, C. indica A. Massal, C. mirabilis A. (Zahlbr.) Lücking and C. leprocarpa (Nyl.) Frisch, have so far been found in China (Rivas Plata et al. 2010;Xu et al. 2016;Jia and Lücking 2017;Kalb and Kalb 2017;Wijayawardene et al. 2017;de Lima et al. 2019).
During the study of Chapsa A. Massal. in southern China, one species, C. murioelongata was found new to science, and two species, C. niveocarpa Mangold and C. wolseleyana Weerakoon, Lumbsch & Lücking were found new to China. In our study, 26 sequences were newly generated from freshly collected specimens.

Morphological and chemical analyses
The specimens were collected from southern China and deposited in the Fungarium, College of Life Sciences, Liaocheng University, China (LCUF). Morphological and anatomical characters of thalli and apothecia were examined and photographed under an Olympus SZX16 dissecting microscope and an Olympus BX53 compound microscope. The lichen secondary metabolites were detected and identified by thin-layer chromatography using solvent C (Orange et al. 2010;Jia and Wei 2016).

DNA extraction, PCR sequencing and phylogenetic analysis
Genomic DNA was extracted from ascomata using the Hi-DNA-secure Plant Kit (Tiangen, Beijing, China) according to the manufacturer's protocol. The nuLSU, ITS and mtSSU regions were amplified using the primer pair AL2R/LR6 (Mangold et al. 2008, Vilgalys andHester 1990), ITS1F/ITS4 (Gardes andBruns 1993, White et al. 1990) and mrSSU1/mrSSU3R (Zoller et al. 1999), respectively. The PCR amplification progress followed Dou et al. (2018) and the PCR products were sequenced by Biosune Inc. (Shanghai). The newly generated sequences were submitted to GenBank (Table1). Multi-locus (ITS, mtSSU and nuLSU) phylogenetic analysis was performed. The combined analysis included 70 sequences (Table 1) representing 18 in-group taxa and one out-group taxon. As many species as possible of Chapsa s. lat. were contained in our data matrix including the taxa that were similar in morphology or sequence to the new species and the two records. We blasted sequences of the three species in GenBank and selected sequence-similar taxa on a pre-determined cut-off.
The alignment was undertaken by applying MAFFT 7 with the option of L-INS-I (Katoh and Standley 2013). The three single-locus alignments were concatenated in PhyloSuite v1.2.2 (Zhang et al. 2020). The concatenated data matrix comprised 3188 nucleotide sites (nuLSU 1405 bp, ITS 647 bp and mtSSU 1136 bp). In order to check the consistency between the three loci, incongruence length difference test (ILD Test) was carried out using PAUP. The P value of ILD Test was 0.65 (>0.5), so the three loci were suitable for polygenic phylogeny. Construction of the ML (Maximum Likelihood) tree was undertaken by applying RAxML v.8.2.12 (Stamatakis 2014) and implementing a GTRGAMMA model. For BI (Bayesian Inference) analysis, PartitionFinder 2 (Lanfear et al. 2017) was used to determine the best-fit model for each partition. For the nuLSU region, we used GTR+I+G, for ITS, GTR+G, and for mtSSU, HKY+I+G. BI analysis was performed with MrBayes 3.2.7 (Ronquist et al 2012). Markov Chain Monte Carlo (MCMC) chains were run for 200,000 generations, sampling every 100 th generation, at which point, the average standard deviation of split frequencies was 0.001738. ML bootstrap values (BS) ≥ 75% and Bayesian posterior probabilities (PP) ≥ 0.95 were considered as significantly supported.