Aspicilia stalagmitica (Megasporaceae) – a new lichen species with isidia-like thalline outgrowths

Summary . Aspicilia stalagmitica Paukov et Davydov from the Altai Mts , a species with isidia-like outgrowths on areoles, is described as new to science. From other species of the genus Aspicilia stalagmitica differs by the fol lowing set of characters: short narrow marginal lobes, conidiomata in the isidia-like outgrowths, appressed to almost substipitate apothecia, long picnoconidia, and stictic acid as a main secondary metabolite. A phylogenetic analysis of Aspicilia stalagmitica (ITS) showing its relationships within Aspicilia is presented.


Introduction
Family Megasporaceae (or Aspicilia s. l.) is remarkable in its morphological diversity and comprises taxa of different life forms from those having immersed thalli to dwarf-fruticose and vagrant species (Sohrabi et al., 2013). Saxicolous and some terricolous representatives of the fruticose life form start their ontogenesis from formation of areolate thalli followed by the development of straight or contorted, simple or branched outgrowths up to several millimeters high (Oxner, 1971). A small number of Aspicilia species develop only tiny isidia-like structures which subsequently never form 6 Paukov A. G., Davydov E. A. Aspicilia stalagmitica (Megasporaceae) -a new lichen species from Altai fruticose thalli (Poelt, 1961). During field work in the northern China in 2005 Evgeny Davydov collected several specimens with such morphology, which were recognized as representatives of a previously undescribed species. Here we describe this species as new to science.

Specimens and phenotype studies
The core material for this study was collected by Evgeny Davydov during the expedition to Xinjiang (China) in 2005 and deposited in herbaria LE, ALTB, UFU, and PE. Morphological observations were made using a dissecting microscope. Crosssections of apothecia and thalli were cut by hand with a razor blade and observed after mounting in water, K, N and iodine solutions. Measurements of spores and conidia are presented as follows: (smallest value recorded) (X-SE) -X -(X+SE) (largest value recorded), where X is the (arithmetic) sample mean, and SE the sample error of mean. The measurements were made with the precision of 0.5 μm.

Sequences and phylogenetic reconstructions
To test phylogenetic relations to other species, nuclear internal transcribed spacers and 5.8S rDNA (ITS) sequences of the putative new species and other sequences retrieved from the NCBI database (GenBank) were used for a molecular phylogenetic analysis. Our sampling comprised 14 species of Aspicilia including a putative new species, species of Oxneriaria, Lobothallia and Circinaria, as well as Ochrolechia parella (L.) A. Massal as an outgroup. This selection is based on the studies of Nordin et al. (2007,2008,2010), Kondratyuk et al. (2016), Haji Moniri (2017) and a five-gene analysis by Miądlikowska et al. (2014). The information on the samples with the GenBank accession numbers are given in Table. Table Species of lichens used in the phylogenetic analysis in this study together with specimen information and GenBank Accession numbers. New specimen and its sequence are in bold.   Ronquist et al., 2012). Three parallel Bayesian analyses were run using six chains and every 200th generation was sampled. Convergence of the chains was inferred by calculating the average standard deviation of split frequencies every 100,000 generations using a burn-in fraction of 0.5, and the runs terminated when the standard deviation of split frequencies dropped below 0.001. This was the case after 7.1M generations. The first 50 % of the trees were discarded as burn-in and a 50 % majority rule consensus tree calculated from the remaining trees of three runs with the sumt command implemented in MrBayes 3.2.3. The most likely tree and 1000 rapid bootstrap replicates were calculated using RAxML 8.0.26 (Stamatakis, 2014) by raxmlGUI software version 1.3.1 (Silvestro, Michalak, 2012) applying the GTRGAMMA model of substitution to the subsets. The tree topologies were taken from RAxML. Bootstrap support values and BMCMC posterior probability were noted onto the best scoring tree (Fig. 1).

Results
An ITS sequence was successfully obtained from one specimen of the putative new species, described below as Aspicilia stalagmitica. The Bayesian 50 % majority-rule consensus tree had the same topology as the maximum likelihood tree generated by RAx-ML. The phylograms are combined in Fig. 1. According to the ITS sequence the new taxon belongs to Aspicilia and is the closest relative to the North-American Aspicilia cuprea Owe-Larss. et A. Nordin. These two taxa form a clade well-supported by MrBayes (0.97PP), but only weakly by RAxML (62 % BS) and rather long branches lengths. The sister clade contains the type species of the genus, Aspicilia cinerea (L.) Körb.   Table). Originally produced sequence is marked in bold. Ochrolechia parella was used as an outgroup. Branch lengths represent the estimated number of substitutions per site assuming the respective models of substitution. Exception is the branch with a black dot, which was shortened to reduce the overall figure size.
Chemistry. Thallus K+ yellow, C−; medulla K+ yellow, C−; stictic acid complex by TLC, norstictic acid as a minor substance in all specimens.
Etymology. The name refers to the vertical outgrowths on areoles which resemble stalagmites.
Ecology. Aspicilia stalagmitica was found in arid conditions on exposed siliceous rocks (granite and schistose) in steppe communities at elevations 880-1600 m a. s. l. The following species cooccurred with

Discussion
Aspicilia stalagmitica is a peculiar species which can be easily distinguished from other Aspicilia s. l. by its isidia-like outgrowths, which contain conidiomata. Along with this character the species has long pycnoconidia, up to 33 μm, and contains the stictic acid complex. All the found specimens contain vertical outgrowths but the combination of long conidia and stictic acid may be a separate character which segregates this species from all known taxa within the genus.