Five new species of Graphidaceae ( Ascomycota , Ostropales ) from Thailand

Five new species of Graphidaceae are described from Thailand. Molecular evidence and phenotypical characters support their independent status from related and similar species. Glaucotrema thailandicum Naksuwankul, Lücking & Lumbsch is unique within the genus in having submuriform ascospores. Ocellularia klinhomii Naksuwankul, Lücking & Lumbsch is characterized by having a whitish gray, rimose thallus with ascomata in verrucae and surrounded by a black ring and lack of secondary metabolites. Ocellularia phatamensis Naksuwankul, Parnmen & Lumbsch has a grayish, thick and rimose thallus, differing from O. klinhomii in lacking a dark apothecial rim and having ascomata that are not immersed in verrucae. Ocellularia thailandica Naksuwankul, Kraichak & Lumbsch differs from O. albocincta in lacking a columella. Ocellularia rotundifumosa Naksuwankul, Lücking & Lumbsch differs from O. fumosa in having ascospores with rounded ends. An epitype for O. krathingensis is selected.


Introduction
Phenotypical characters, such as morphology of the thallus and ascomata and anatomy of the ascomata as well as secondary chemistry have traditionally guided species delimitation in lichenized ascomycetes.However, especially crustose lichens often exhibit only few traits and without independent markers, such as DNA sequence data, it is often difficult to assess whether variation is due to genetic differences or plasticity.Indeed, recent phylogenetic studies suggest high amounts of homoplasy in phenotypical characters used to delimit taxa in lichenized fungi (Grube et al. 2004;Tehler and Irestedt 2007;Schmitt et al. 2009;Rivas Plata and Lumbsch 2011;Lumbsch et al. 2014a).Hence, molecular data have greatly increased our ability to identify distinct lineages, including the detection of numerous cryptic lineages (Crespo and Lumbsch 2010;Lumbsch and Leavitt 2011;Leavitt et al. 2015).While numerous foliose and fruticose lichen groups have been studied in some detail, especially in the diverse Parmeliaceae, our knowledge on species delimitation in crustose lichens is still in its infancy.However, among predominantly crustose families, Graphidaceae is now relatively well known.
Graphidaceae constitutes the largest family of crustose tropical lichens with about 2100 accepted species (Rivas Plata et al. 2012;Lücking et al. 2013;Cáceres et al. 2014; Van den Broeck et al. 2014;Lumbsch et al. 2014b;Kraichak et al. 2014).The family has its center of distribution in the tropics, but also occurs in temperate regions with a smaller number of species, in some cases even extending towards the Sub-Antarctic region.The family is most common, however, in the tropics where its species occur often on bark, but can also be found on rocks, wood or soil and sometimes on leaves.Recently, the first author started a project on the diversity of thelotremoid Graphidaceae in East Asia (Papong et al. 2014).Thelotremoid Graphidaceae have rounded ascomata (formerly placed in Thelotremataceae), in contrast to species with lirellate ascomata.The group is still relatively poorly known in Thailand and generally in south-east Asia, but preliminary studies have provided important baseline data for the distribution of species and have indicated that numerous additional species can be expected in Thailand (Homchantara and Coppins 2002;Papong et al. 2010;Sutjaritturakan and Kalb 2015).Molecular data have been used to identify distinct lineages in this group of lichenized fungi and subsequent re-analysis of phenotypical characters often allowed identification of morphological or chemical traits to separate those species (Lumbsch et al. 2008;Mangold et al. 2014;Poengsungnoen et al. 2014;Medeiros et al. 2016).This paper employs molecular, morphological and chemical data to identify six distinct lineages of thelotremoid lichens from Thailand and to describe them as species new to science.Based on our limited sampling of thelotremoid Graphidaceae from other regions of southeast Asia, we expect the new species described here from Thailand to occur in other countries of the region.

Material and methods
This study is mainly based on new collections made by the first two authors deposited in F and MSUT.Sections of thalli and apothecia were cut using a razor blade and examined in water, a solution of KOH, and Lugol's solution using a ZEISS Axioscope 2 plus compound microscope.Chromatography (HPTLC) was performed with standard solvent systems A and C (Culberson 1972;Arup et al. 1993).
We performed two different phylogenetic analyses: 1) sequences of six samples of the genus Glaucotrema were aligned with two outgroup taxa (Leptotrema wightii, Reimnitzia santensis) and 2) sequences of 35 samples of Ocellularia s. str.were aligned with O. cavata as outgroup.Selection of samples was done using Blast searches and included best hits to ensure that all similar sequences were included.In addition sequences of morphologically similar species were added to the data set.Sequences of mtSSU rDNA, nuLSU rDNA, and the protein-coding RPB2 gene were used for this study.Voucher information and Genbank numbers are listed in Table 1.DNA isolation, PCR, and direct cycle sequencing conditions were described previously (Kraichak et al. 2014).
For the phylogenetic analyses, the alignment of the nucleotide sequences for each dataset was performed separately using Geneious version 8.0.3 (Drummond et al. 2014) and manually inspected for removal of any ambiguous characters.We then performed a maximum likelihood analysis, using RAxML-HPC Blackbox version 8.2.8 (Stamatakis 2006) with the default rapid hill-climbing algorithm and the GTRGAM-MA model of nucleotide substitution.The analysis was carried out on the online server CIPRES science Gateway version 3.3 (Miller et al. 2010) with a total of 1,000 pseudoreplicates to assess the rapid bootstrap value support.A bootstrap support value of 70 and above was considered a strong support for a clade.The resulting bipartitioned trees were visualized with the program FigTree version 1.4.2(Rambaut 2012).

Phylogenetic analysis
The final alignment of the combined data set for the Glaucotrema analysis consisted of 802 unambiguously aligned nucleotide positions for mtSSU, 865 for nuLSU, and for 985 RPB2.The final alignment of the dataset for the Ocellularia taxa consisted of 787 unambiguously aligned nucleotide positions for mtSSU, 879 for nuLSU, and for 913 RPB2.As the topologies of the single locus phylogenies for these two datasets did not show any conflicts, they were analyzed in a concatenated matrix.
In the Glaucotrema tree (Fig. 1), the Thai material formed an unsupported sistergroup relationship with G. glaucophaenum, and G. stegoboloides.The latter two species were not separated in our analysis but were supported as different species in a broader   relationship of these three taxa, which are not only phenotypically disparate but also have distinct geographic distributions, suggests that the loci here used may be of limited use for species delimitation in recently evolved complexes, which has already been discussed for mtSSU by Kraichak et al. (2014).Two samples, included as spec.nov.8 in Kraichak et al. (2014), formed an unsupported sister-group relationship with O. albocincta, a species that differs morphologically (see below) and so the Thai material is described as a new species (O.siamensis) below.Nine samples included as spec.nov.6 in Kraichak et al. (2014) from Thailand clustered together, related to O. diacida, which is readily distinguished by the presence of the hirtifructic acid chemosyndrome.
The species is described new to science below as O. phatamensis.A single specimen, included as spec.nov.7 in Kraichak et al. (2014), is also related to O. diacida but differs -among other characters -by the absence of secondary metabolites.Etymology.The specific epithet refers to the country where the type specimen was collected.
Distribution and ecology.The new species was found in northeastern Thailand, growing on bark in a dry evergreen forest.It is known only from the type locality.
Remarks.This new species is unique within the genus in having submuriform ascospores, whereas all other described species have transversely septate ascospores.In addition, the ascospores in G. bahianum, G. costaricense and G. stegoboloides are smaller than in the new species.Molecular data support the distinction of the new taxon.In morphology it resembles G. bahianum and G. stegoboloides.Diagnosis.Differing from the similar O. krathingensis in having a whitish grey, rimose thallus.
Etymology.The specific epithet refers to the collector Mr. Winia Klinhom, mycologist from Thailand.
Secondary chemistry.No substances detected by TLC.Distribution and ecology.The new species was collected in northeastern Thailand, growing on bark in a dry evergreen forest.It is known only from the type locality.
Remarks.Similar in ascospore size, lack of secondary metabolites and only apically carbonized exciple to O. krathingensis but differing in having a whitish gray, rimose thallus with ascomata in verrucae and surrounded by a black ring, reminiscent of O. wirthii (Mangold et al. 2008).The latter species is readily distinguished by having a broader, carbonized columella and the presence of the psoromic acid chemosyndrome.The species would key out at alternative 60 in the Ocellularia key for Thailand (Sutjaritturakan & Kalb 2015).

Ocellularia phatamensis
Etymology.The specific epithet refers to the name of the Pha Tam National Park in Ubon Ratchathani Province, Thailand.
Secondary chemistry.No substances detected by TLC.Distribution and ecology.The new species was collected in northeastern Thailand, growing on bark in a dry evergreen forest.It is known only from the type locality.
Remarks.The new species is similar to O. krathingensis in having an apically carbonized exciple and columella, transversely septate, amyloid ascospores, and lacking secondary metabolites, but differs in having a grayish and thicker thallus (Homchantara and Coppins 2002).Another similar species is O. klinhomii, but differs in lacking a dark apothecial rim and the ascomata are not immersed in verrucae.Molecular data support the distinction of these two species (Fig. 2).Another similar and related species is O. diacida, which is readily distinguished by the presence of the hirtifructic acid chemosyndrome.The species would key out at alternative 60 in the Ocellularia key for Thailand (Sutjaritturakan and Kalb 2015).Diagnosis.Differing from O. fumosa in having ascospores with rounded ends.Etymology.The specific epithet refers to the ascospore shape with rounded ends and to the similarity with O. fumosa.
Secondary chemistry.No compounds detectable by TLC.Distribution and ecology.The new species was collected in northeastern Thailand, growing on bark in a dry evergreen forest.It is known only from the type locality.
Remarks.Similar to O. fumosa, but differing in having rounded ends of the ascospores instead of acute ones in O. fumosa.Molecular data support the distinction of the species (Fig. 2).Characters to separate the related O. natashae and O. thryptica are discussed above.The species would key out at alternative 23 in the Ocellularia key for Thailand (Sutjaritturakan and Kalb 2015).Diagnosis.Differing from the similar O. viridipallens in having broader ascospores with up to 7 septa.

Ocellularia thailandica
Etymology.The specific epithet refers to the country where the type specimen was collected.
Secondary chemistry.No substances detected by TLC.Distribution and ecology.The new species was collected in northeastern Thailand, growing on bark in a dry evergreen forest.It is known only from the type locality.
Remarks.This new species is closely related to O. albocincta (Fig. 2).However, this species differs in lacking a columella (Papong et al. 2010).Morphologically it resembles O. viridipallens, which differs in having narrower ascospores.The species would key out at alternative 60 in the Ocellularia key for Thailand (Sutjaritturakan & Kalb 2015).

Figure 1 .
Figure 1.Phenogram depicting phylogenetic relationships of Glaucotrema species.Only bootstrap support values above 70 are displayed on the nodes.

Figure 2 .
Figure 2. Phenogram depicting phylogenetic relationships of Ocellularia species.Only bootstrap support values above 70 are displayed on the nodes.

Table 1 .
Genbank numbers and voucher information of specimens used in this study.For author names see Index Fungorum (http://www.indexfungorum.org).Missing data are indicated by[-].