Architrypethelium murisporum (Ascomycota, Trypetheliaceae), a remarkable new lichen species from Thailand challenging ascospore septation as an indicator of phylogenetic relationships

Abstract Architrypethelium murisporum Luangsuphabool, Lumbsch & Sangvichien is described for a crustose lichen occurring in dry evergreen forest in Thailand. It is characterised by a green to yellow-green corticated thallus, perithecia fused in black pseudostromata with white rim surrounding the ostiole and small, hyaline and muriform ascospores. Currently, all species in the genus Architrypethelium have transversely septate ascospores, hence the discovery of this new species indicates that ascospore septation is variable within the genus, similar to numerous other groups of lichen-forming ascomycetes. Phylogenetic analyses of two loci (mtSSU and nuLSU) supported the position of the new species within Architrypethelium. This is the first report of the genus in Southeast Asia.


Introduction
The genus Architrypethelium Aptroot (Ascomycota, Dothideomycetes, Trypetheliales) includes crustose lichens with perithecioid ascomata growing on tree bark in the tropics (Aptroot 1991, Aptroot et al. 2008, Aptroot and Lücking 2016. The genus accommodates species with a corticate thallus, solitary or aggregate ascomata with apical or eccentric ostioles, a clear or inspersed hymenium and hyaline or brown, 3-5 septate, transversely septate ascospores (Aptroot et al. 2008, Aptroot andLücking 2016). Although, Architrypethelium is morphologically similar to Astrothelium species, the two genera have been shown to be distantly related. The latter genus fell into two clades (Lücking et al. 2016) with one being a sister group to Architrypethelium. Phenotypically Architrypethelium differs from Astrothelium in having predominantly large ascospore without diamond-shaped lumina when mature (Aptroot 1991, Aptroot et al. 2008, Nelsen et al. 2014, Aptroot and Lücking 2016, Lücking et al. 2016b). Another genus with muriform ascospores is Aptrootia, which also shares an astrothelioid stage in the young ascospores (Lücking et al. 2016) and the genus formed a sistergroup to a clade including Architrypethelium and Astrothelium p.pt. further calling the generic delimitation in the family in question. Morphologically, Aptrootia differs from Astrothelium in having dark brown ascospores with a hard outer shell (Lücking et al. 2016). While most genera in Trypetheliaceae, such as Astrothelium s.str., Bathelium, Polymeridium and Viridothelium include species with various ascospore types (Hyde et al. 2013, Nelsen et al. 2014, Aptroot and Lücking 2016, Lücking et al. 2016b, the species of Architrypethelium shared a similar ascospore morphology (Nelsen et al. 2014, Lücking et al. 2016b. Previously, three species were accepted in Architrypethelium (Aptroot 1991, Aptroot et al. 2008. Recently, the numbers of species increased with the description of two new species and two combinations into the genus (Aptroot and Lücking 2016, Flakus et al. 2016, Lücking et al. 2016a (Aptroot 1991, Aptroot et al. 2008, Aptroot and Lücking 2016, Flakus et al. 2016, Lücking et al. 2016a). All species are known from the Neotropics, except A. uberinum, which is also known from Oceania (Aptroot and Lücking 2016, Flakus et al. 2016, Lücking et al. 2016a, suggesting a pantropical distribution (Aptroot and Lücking 2016). Until now, the genus Architrypethelium has not been known from southeast Asia. Here we describe a new species from Thailand, which has a rich pyrenocarpous lichen flora (Buaruang et al. 2017), with muriform ascospores, confirming its presence in southeast Asia. Further, we provide phylogenetic evidence to support its placement in the genus Architrypethelium and hence demonstrating that the ascospore septation is also variable in this genus.

Specimen collection and phenotypical studies
The material of the new species was found in a dry evergreen forest of the north-eastern region in Thailand. Morphology was studied using an Olympus SZ11 dissecting microscope and free hand sections were mounted in distilled water and studied using an Olympus BX53 compound microscope with differential interference contrast (DIC) (Olympus U-DICT), connected to a Canon EOS650 digital camera. Secondary metabolites were studied using thin-layer chromatography (TLC) with standard solvent A (Orange et al. 2001, Lumbsch 2002.

Molecular data
Genomic DNA of the holotype was extracted from the dried lichen thallus using the CTAB method with chloroform precipitation (Cubero and Crespo 2002). DNA amplification was performed for mitochondrial small subunit ribosomal DNA (mtSSU) and nuclear large subunit ribosomal DNA (nuLSU) using primer pairs mrSSU1 (Zoller et al. 1999) with MSU7 (Zhou and Stanosz 2001) and LR0R with LR3 (Vilgalys and Hester 1990), respectively. PCR reaction mixture was prepared in a total volume of 50 μl, consisting of 5 μl of 10× Pfu Buffer with MgSO 4 , 2mM of dNTP mix, 20 μM of each primer, 1·25 U of Pfu DNA Polymerase (Thermo Fisher Scientific Inc.) and 5 μl of 1/10 dilution of DNA solution. PCR was performed using a thermal cycler Life ECO (Hangzhou Bioer Technology Co., China) as follows: initial denaturation for 1 min at 94 °C and 38 cycles of 94 °C for 1 min, 52 °C for 45 s (LR0R/LR3) and 53 °C for 45 s (mrSSU1/ MSU7), followed by an extension at 72 °C for 1 min and a final extension at 72 °C for 5 min. DNA purification and sequencing methods followed Luangsuphabool et al. (2016).

Phylogenetic analysis
The new sequences were aligned with other species of Architrypethelium and other Trypetheliaceae from GenBank (Table 1). Aptrootia and Astrothelium s. lat. have been shown to be the sister groups to Architrypethelium (Lücking et al. 2016b) and two taxa of Bathelium madreporiforme were used as the outgroup. The DNA datasets (mtSSU and nuLSU) were aligned separately using MUSCLE (Edgar 2004) and improved manually using MEGA v.7 (Kumar et al. 2016). The nucleotide substitution model for maximum likelihood (ML) and Bayesian inference (BI) analyses was chosen using jModelTest v.2.1.4 (Darriba et al. 2012) with the Akaike Information Criterion (AIC). The ML tree was performed on the CIPRES supercomputer using the programme RAxML-HPC2 v.8.2.10 on XSEDE (Miller et al. 2010) and bootstrap values were estimated with 1000 pseudo- replicates. Bayesian inference analysis and posterior probabilities were calculated using MrBayes v.3.2.1 (Ronquist and Huelsenbeck 2003) with the Markov chain Monte Carlo (MCMC) algorithm. Four chains and two independent runs were carried out with 10 million generations. Every 100th tree was saved into a file and aborting the analysis was set at the mean standard deviation < 0·01. Tree topology of both ML and BI analyses was illustrated using FigTree v.1.4.2 (http:// tree.bio.ed.ac.uk/software/figtree/).

Results and discussion
Two new DNA sequences of mtSSU and nuLSU were generated for this study (Table 1). The alignment matrix contained 609 unambiguously aligned nucleotide position characters, including 200 mtSSU and 409 nuLSU positions. The GTR+I+G model was chosen as the best-fit model for phylogenetic analyses. The topology of single locus analyses did not show any conflicts and hence the combined data set was used for the analysis. The posterior probabilities of the BI analysis together with the ML bootstrap values are both shown in the ML tree (Fig. 1). The tree topology supported the fact that the new species is part of the genus Architrypethelium with strong support values (Fig. 1). Although the morphological characters of the new species would place it in the genus Astrothelium (Fig. 2), the shape of ascospore lumina is somewhat different from Astrothelium in having rounded-shaped  (Aptroot and Lücking 2016). This new species seems to be closer related to species with hyaline ascospore (Architrypethelium lauropaluanum) than brown ascospores (A. nitens and A. uberinum) (Aptroot 1991, Aptroot et al. 2008, Lücking et al. 2016a. So far, all species in Architrypethelium had large, transversely septate ascospores (Aptroot and Lücking 2016). However, our new species has small, muriform ascospores ( Fig. 2B-C). The ascospore ontogeny in the new species resembles that of Architrypethelium spp. (Sweetwood et al. 2012), but continues septation to form muriform spores and the endospore is reduced when mature.
The variation of ascospore size and septation in Architrypethelium is not surprising given the variation of ascospores in other genera of Trypetheliaceae. This phenomenon is also commonly found in many genera in families of non-lichenised ascomycetes, viz. Lophiostomataceae and Melanommataceae (Mugambi and Huhndorf 2009) and lichenised families, such as Graphidaceae and Pyrenulaceae (Lücking 2009, Aptroot 2012, Weerakoon et al. 2012, Aptroot and Lücking 2016, Gueidan et al. 2016, which supports the fact that ascospore characters are often poor predictors of phylogenetic relationships (Nelsen et al. 2014, Lücking et al. 2016b).
Diagnosis. Characterised within the genus by having small, hyaline and muriform ascospores.
Etymology. The specific epithet refers to the muriform ascospore character of the new species.
Distribution and ecology. The new species was found in north-eastern Thailand, growing in a dry evergreen forest on tree bark. It is only known from the type locality.