Umbilicaria aprina and U. rhizinata (Umbilicariaceae, lichenized Ascomycota) in Russia

The distribution of Umbilicaria aprina and U. rhizinata in Russia was revised basing on morphological and molecular-phylogenetic data. Umbilicaria aprina is new to Alexandra Land Island (Franz Josef Land Archipelago), Sverdrup Island (Taimyr Dolgano-Nenets District), the republics of Altai, North Ossetia-Alania and Sakha-Yakutia. Umbilicaria rhizinata is new to the republics of Tyva and Buryatia, Putorana Plateau, Kamchatka Peninsula, and the Magadan Region and is therefore reported for the first time to the Asian Subarctic. New localities of the species in other regions are cited. The ITS\5.8S phylogenetic tree supports the distinctness of U. aprina and U. rhizinata. The distribution map is drawn and patterns of distribution of two species are discussed. Umbilicaria aprina occurs in the high Arctic between 74°N and 81°N, whereas the northernmost locality of U. rhizinata is in the vicinity of Norilsk on the Putorana Plateau (ca. 70°N). In mountains U. aprina grows at higher maximal elevations compared to U. rhizinata.


Introduction
Species of Umbilicariaceae Chevall.are predominantly saxicolous lichens mostly found in higher latitudes or at higher elevations worldwide.Umbilicaria aprina Nyl. and U. rhizinata (Frey et Poelt) Krzewicka are related species from the 'Umbilicaria aprina group' -a monophyletic batch of morphologically similar taxa within Umbilicaria Hoffm.subg.Umbilicaria.Species of the Umbilicaria aprina group have a slightly areolate or reticulate center of the grey-coloured upper thallus surface, scarce to dense, simple or branched rhizinomorphs, non-septate to 10-septate thalloconidia, and omphalodisc apothecia with asci containing hyaline, unicellular ascospores.Species of the Umbilicaria aprina group are characteristic to harsh environments in polar and high mountain regions (Davydov et al., 2017).
Umbilicaria rhizinata is closely related to U. aprina and differs mainly by a smaller size of 3-4 cellular (vs.1-2 cellular) thalloconidia.The species boundaries are not clear for many taxa, and U. rhizinata is represented in the GenBank by a single ITS sequence.The aim of the study was to provide additional data on the distribution of two species in Russia mostly basing on recently collected specimens and to test the monophyly of U. rhizinata by molecular phylogenetic method using ITS sequences.

Sampling and phenotypic studies
The core material for the study was collected by authors between 2009 and 2022 in different regions of Russia and deposited in herbaria ALTB, LE, LECB, MAG, UHU, and private collections of the authors.
Additionally, specimens were studied from the herbaria HMAS, KPABG, KW, LE, and NS.
The specimens were examined using a stereomicroscope (Zeiss Stemi 2000-C) and a compound microscope (Zeiss Axio Lab.A1).Anatomical examination was undertaken using hand-cut sections mounted in water.Lichen substances were studied using high performance thin-layer chromatography (HPTLC) with solvent systems B' (hexane: methyl tert-butyl ether: formic acid, 140 : 72 : 18) and C (toluene: acetic acid = 170 : 30) (Culberson, Kristinsson, 1970;Culberson, Johnson, 1982;Orange et al., 2001).We used a scanning electron microscope (SEM) to visualize thalloconidia.Microphotographs were obtained using a Carl Zeiss EVO MA 10 SEM.The samples were dried in air and fixed on aluminum stubs with double-sided sticky film, and then, gold was sprayed on them.

DNA extraction, amplification and sequencing
Single thallus parts (100-200 mg) were carefully checked for fungal infections and thoroughly cleaned of extraneous matter, excised under a magnifying lens, and transferred to sterile 1.5 ml reaction tubes.The samples were frozen in liquid nitrogen and powderized in the tubes using sterile pestles.DiamondDNA Plant Kit (ABT) was used for DNA extraction as recommended by the manufacturer.
To test the phylogenetic relationships within the species, the internal transcribed spacer region of nuclear ribosomal DNA (ITS) was amplified in a single reaction using ITS 1F-5' as a forward primer and ITS 4-3' or LR3-3' as a reverse primes (Vilgalys, Hester, 1990;White et al., 1990;Gardes, Bruns, 1993).Same primers were used for sequencing.Cycling conditions included initial denaturation at 94 °C for 35 cycles of 95 °C for 20 s, 52 °C for 40 s, 72 °C for 60 s, and a final extension step at 72 °C for 7 min.The program Geneious 6.0 (Biomatters Ltd., New Zealand) was used for assembling sequence reads and datasets.Consensus sequences were compiled from double-stranded sequenced parts of the sequences.

Sequences and phylogenetic reconstructions
To test the phylogenetic relationships of Umbilicaria aprina and U. rhizinata collected in Russia, the ITS region of the nrDNA (ITS1, 5.8S, and ITS2 nrDNA) was sequenced (Table ).These markers were chosen because they were used in phylogenetic analyses recently (Davydov et al., 2017).
All newly obtained sequences were supplemented with sequences obtained during our previous study of Umbilicariaceae phylogeny (Davydov et al., 2017), representing species of the U. aprina group, as well as selection of sequences from different subgenera of Umbilicaria (Fig. 1); Xylopsora friesii (Ach.)Bendiksby et Timdal was used as the outgroup.Selection of the outgroup is based on our previous study (ibid.).GenBank Accession numbers are provided on Fig. 1.Newly generated sequences are listed in Table .ITS\5.8S 484 bp matrix were aligned using the MAFFT algorithm (Katoh et al., 2005).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 ITS1, 5.8S, and ITS2 subsets.
The tree topologies are taken from RAxML (Fig. 1).Bootstrap support values were noted onto the best scoring tree.

Results
Specimens of U. aprina and U. rhizinata were identified basing on their morphology and anatomy.Phylogenetic analysis supported the identifications.Thus, septation of thalloconidia is a constant diagnostic trait for U. aprina and U. rhizinata.

The phylogenetic study.
According to the ITS/5.8Sphylogram (Fig. 1), the sequences of species of Umbilicaria aprina group cluster together with high statistical support (RAx-ML 100 % BS).Within U. aprina group, sequences of U. aprina and U. rhizinata clustered separately, which indicates distinctness of two species.Nine sequences of Umbilicaria rhizinata from different regions of Russia clustered together with high support (RAxML 87% BS), which indicates their identity.Six sequences of Umbilicaria aprina clustered in three subclades at the basal part of U. aprina group, but this segregation lacks bootstrap support.

The secondary chemistry.
All studied specimens were investigated by TLC.Both species showed the same spots pattern in TLC, and contain gyrophoric acid as a major and lecanoric as a minor compound.

Discussion
We proved the distinction of two species of Umbilicaria aprina group: U. aprina and U. rhizinata.Umbilicaria aprina is a well-known species from harsh Polar and glacial high mountain habitats.
Locally, especially in regions with a maritime climate, e.g.Kamchatka or Kola Peninsula, U. aprina can grow on rocks near the timberline or even in upper part of the forest belt.It is a common trait for other arctic alpine species and depends on a local climate.The species is widespread in continental Antarctica Umbilicaria aprina and U. rhizinata (Umbilicariaceae, lichenized Ascomycota) in Russia and is a model for studies in physiology of lichens in harsh condition, e. g. photosynthetic activity in different water content (Kappen, Breuer, 1991), CO 2 exchange and water relations in lichens at subzero temperatures (Schroeter et al., 1994;Schroeter, Scheidegger, 1995), photosynthesis and respiration at low temperatures and strong sun irradiance conditions (Green et al., 1998;Kappen et al., 1998;Sancho et al., 2003;Singh et al., 2012), seasonal activity (Schroeter et al., 2011), and dehydration resistance (Harańczyk et al., 2008).The distribution of U. aprina has been extensively studied (see the introduction).Nevertheless, here we report the species for the first time to Alexandra Land Island (Franz Josef Land Archipelago), Sverdrup Island (Taimyr Dolgano-Nenets District), and Yakutia.
In contrast to the previous species, the ecology and the distribution of U. rhizinata have so far been insufficiently studied, and the available data are scattered to discuss the range and ecological preferences of the species.The knowledge on the distribution of U. rhizinata has been increasing significantly; here we report the species as new to such macro regions as the republics of Tyva and Buryatia, Putorana Plateau, Kamchatka Peninsula, and the Magadan Region.Thus, for the first time, we have the opportunity to assess differences in ecology and distribution of two species.In Russia both species occur in Arctic-Subarctic or high mountain ('alpine') saxicolous communities above the timberline, but we never collected both species in the same locality.This fact may point at different distributional patterns of the species.Umbilicaria aprina occurs in high Arctic between 74°N and 81°N, whereas the northernmost locality of U. rhizinata is in the vicinity of Norilsk in Putorana Plateau (ca.70°N).Both species, U. aprina and U. rhizinata, occurred in different localities in the same mountain region, in the republics of Altai, Buryatia, the Magadan Region, and Kamchatka Peninsula.Similarly, in the mountainous regions, U. aprina grows in harsher environment conditions -higher elevations or north-exposed slopes.

Fig. 3 .
Fig. 3.The known distribution of Umbilicaria aprina (red) and U. rhizinata (black) in Russia based on the investigated specimens.