DNA isolation, PCR amplification and DNA sequencing followed protocols described in Konstantinova & Vilnet (2011).

Phylogenetic analysis. Three datasets, ITS1-2, trn L- F, trn G-intron, were automatically aligned in BioEdit 7.0.1 (Hall, 1999) with ClustalW option and then manually corrected. The preliminary phylogenetic analyses revealed a lack of incongruence between ITS1-2, trn L-F and trn G-intron and subsequently three datasets were combined. All positions of the final alignment were included in the phylogenetic analysis, lacking parts of sequences were coded as missing.

The combined dataset was analyzed by the maximum likelihood method (ML) with PhyML (Guindon et al., 2010) and the maximum parsimony method (MP) with TNT (Goloboff et al., 2003). The program ModelGenerator (Keane et al., 2004) determined that the GTR+I+G model was the best-fit evolutionary model of nucleotide substitutions. This model was used in ML analysis and the heterogeneity rate among sites was modelled using a gamma distribution with four rate categories. Bootstrap support (BS) for individual nodes was assessed using a resampling procedure with 300 replicates. According to stopping frequency criterion (FC) for bootstrapping procedure (Pattengale et al., 2010) for our dataset even 250 replicates were enough for reaching BS convergence with Pearson average Ñ 100 = 0.997271 realized in RAxML v7.2.6 (Stamatakis, 2006).

The MP analysis involved a New Technology Search with a search for the minimum-length tree by one reiteration and 1000 bootstrap resamplings; the default settings were used for other parameters, indels were taken into account by a modified complex coding algorithm in SeqState (Müller, 2005).

The infraspecific variability of Bucegia romanica was evaluated as the p -distances for ITS1-2, trn L-F and trn Gintron calculated in Mega 5.1 (Tamura et al., 2011) using the pairwise deletion option for counting gaps.

Taxon Specimen voucher GenBank accession number

ITS1-2 trn L-F trn G-intron Bucegia romanica Norway: Svalbard Kinnvika, Konstantinova & Savchenko, K20 / 1-10 (KPABG) KJ195815 KJ195821 KJ195804 Norway: Svalbard Nordvika, Konstantinova & Savchenko, K152 / 1-07 (KPABG) KJ195817 KJ195823 KJ195806 Norway: Svalbard Mimerdalen, Konstantinova & Savchenko, K41 / 2-08 (KPABG) KJ195816 KJ195822 KJ195805 Romania: Bucegi Massif, S. Ștefănuţ, B4322 (BUCA, dupl. KPABG) KJ195812 KJ195825 KJ195807 Romania: Fagaras Mts., S. Ștefănuţ, B4321 (BUCA, dupl. KPABG) KJ195813 KJ195824 KJ195808 Ukraine: Carpathian Mts., D. Zerov, 495 (KW, dupl. KPABG) ITS1 KJ195814

ITS2 KJ195803 KJ195826 KJ195802 Preissia quadrata Russia: Komi Rep., M. Dulin, 113625 (KPABG) KJ195818 KJ195827 KJ195809 Russia: Krasnodar Territory, Fisht Mts.,

Konstantinova & Savchenko, K416 /5-12 (KPABG) KJ195820 KJ195829 KJ195811 Russia: Murmansk Prov., Khibiny Mts., Konstantinova, 18347 (KPABG) KJ195819 KJ195828 KJ195810 Marchantia polymorpha AY 342318 NC001319 NC001319

Total sites Conservative sites Variable sites Parsimony-informative sites

base pairs base pairs % base pairs % base pairs % all ITS+trnL regions 2772 1725 62.23 889 32.07 194 7.00 ITS 1-2 1716 857 49.94 745 43.41 145 8.45 trn L-F 435 404 92.87 27 6.21 12 2.76 trn G-intron 621 464 74.72 117 18.84 37 5.96

Genealogical relationships between Bucegia romanica haplotypes were evaluated in TCS (Clement et al. 2000) based on analysis of the combined data matrix ITS1-2+ trn L-F+ trn G-intron with exclusion of outgroup taxa, gaps were treated as missing data.

RESULTS

The nucleotide sequences of ITS1-2, trn L-F and trn Gintron were obtained for nine specimens. The parameters of produced alignments for each studied loci and combined dataset are shown in Table 2. The trn L-F appears to be the most conservative loci in the studied taxa, whereas both ITS regions are highly variable and difficult to align.

The MP analysis of combined ITS1-2+ trn L-F+ trn Gintron dataset yielded a single tree with a length of 1701 steps, with CI= 0.980249 and RI= 0.952020 calculated in Mega 5.1. The arithmetic means of Log likelihood obtained in ML calculation for combined dataset was –7579.77299. The tree topologies obtained by the two methods are highly congruent, thus on Fig. 1 the MP tree is shown with indication of bootstrap support (BS) values calculated both in MP and ML analyses. The specimens from genera Bucegia and Preissia composed their own robustly separated clades. The specimens of Bucegia romanica were split into two subclades according to their geographical distribution. The subclade I (BS=91% in MP and BS=56% in ML) contains accessions from Europe, but the two Romanian specimens are separated from each other by the Ukrainian one. The subclade II (BS=98% in MP and BS=85% in ML) includes all three specimens from Svalbard.

In Romania, Bucegia romanica is distributed in alpine zone, mostly on calcareous substrates, with some exceptions. In FăgăraȘ Mts, where calcareous substrates occur, Bucegia romanica also colonize crystalline schists, that can be an explanation for the genetic diversification of FăgăraȘ specimens beside Bucegi and Ukraine specimens.

The results of infraspecific and infrageneric p -distance calculations are shown in Table 3. The minute nucleotide variation in ITS1-2 (0.23% and 0.04%) and absence of variation in both trn L-F and trn G-intron were detected in East Europe and Svalbard populations of Bucegia romanica. The p -distance between these populations is 0.28% for ITS1-2, 0.1% for trn L-F and 0.3% for trn G-intron, whereas the level of infraspecific variation in the phylogenetically related Preissia quadrata is more significant (1.8% for ITS1-2, 0.24% for trn L-F and 6.4% for trn G-intron).

TCS analysis of combined dataset produced a network with separation of two haplotype groups (I and II) with specimen distribution corresponding to East Europe and Svalbard populations. The network demonstrated phylogenetic isolation between obtained haplotype groups by four missing haplotypes (Fig. 2). In Europe, the most diverged haplotype belongs to a specimen from FăgăraȘ Mts which is separated from the two others by six missing haplotypes. In the Svalbard population two haplotypes were found: specimens from Nordvika Bay and Kinvika Bay (closely allied territories, see map) posses a common haplotype, whereas specimen from Mimerdalen differs from them by four missing haplotypes.

DISCUSSION

Taxonomic relationship. Schuster (1984) placed Bucegia together with the subantarctic Neohodgsonia Perss. in the separate subfamily Bucegioideae, but that is not supported by molecular data. Study of complex thalloid liverworts based on LSU nrDNA nucleotide sequence of 27 genera provided by Boisselier-Dubayle et al. (2002) resolved Bucegia romanica and Preissia quadrata in one clade with high bootstrap support (98%) in sister relation to the multiply sampled genus Marchantia L., whereas Neohodgsonia was placed in the first diverged phylum of all Marchantiidae, and the genus Dumortiera was found in a more derived clade. Later Long (2006) placed Neohodgsonia in the separate order Neohodgsoniales Value of within group p -distances, %

Value of between groups p -distances, ITS1-2/ trn L-F/ trn G%

Groups

ITS1-2 trn L-F

trn G-intron

BR EE BR S PQ MP

BR EE BR S PQ MP

0.23 0.04 1.8 n /c 0

0 0.24 n /c

0.1 0 6.4 n /c

– 0.28/0.1/0.3 10.8/2.9/6.6 47.4/5.6/11.2 – 11.1/2.8/6.8 44.1/5.7/10.9 – 36.9/4/14.5 – on dorsal side. Thalli flat with elevated sides, V-shaped in cross section. Dorsal epidermis of thin-walled cells, without trigones and oil-cells. Aerenchyma layer is rather thick, usually more than 0.25-0.35(-0.4) of the maximum thallus height and consists of (1-)2-3(-4) layers of large and empty (without any filaments) air-chambers (Figs. 3-5). Pores barrel-shaped, surrounded by 5 superimposed cell rings with (3-)4(-5) cells in each ring. Ventral tissue without oil cells and sclerenchyma cells, often colourless even if the thallus is with red-dark brown dorsal side. Ventral scales in 2 rows, red-violet to blackish violet, but often with colourless margin, large, 1.5-2.4 mm (over 3/4 of a half of ventral thallus) with one linear, oblong, often very short appendage and numerous slime hairs on margin. Cells of ventral scales thin walled, without trigones, large, 36-50×75-100 µm. Dioicous. Female disk conic, to 4-5 mm in diameter, 2-3 mm high, with 3-5 wide lobes. Male receptacles with disk almost circular, not lobed. Spores brown, 45-50 µm, tetrahedral.

At first glance Bucegia romanica (Figs. 6, 7) is similar to the very common and much more widespread arctic-boreomontane Preissia quadrata (Fig. 8) due to similar barrel-shaped pores and stalked male and female receptacles. Both species have similar shape, size and col- or of thalli as well and because of this are often confused. In the field, these species can be distinguished from oth- er thallose liverworts by the verrucose-like side of dorsal thallus, that can be seen without a lens. This is due to the barrel-like pores that are visible like small verruca on the dorsal side of thallus. The color of Preissia and Bucegia thalli is similar, from yellow-green, green to red-violet and red-brown, in arctic often blackish red but never dark-green, like Marchantia polymorpha. Marchantia sometimes has a thallus with dark line in midrib region, that is missing on Preissia and Bucegia. Thallus bearing cup-shaped gemma-receptacles are never present on Preissia and Bucegia thalli, but are quite characteristic for Marchantia.

When sterile, Preissia quadrata and Bucegia romanica could be not be easily distinguished in the field, but if an oblique sectioning of thallus is made, spongiose-like structure can be seen with lens on the dorsal side of Bucegia thallus, because of hollow air-chambers layers. Such spongiose-like structure is not seen on the dorsal side of the Preissia thallus because this species has only one layer of air-chambers and the air-chambers are not hollow: they are filled with uniseriate chlorophyllose filaments growing from the air-chamber floor.

Further distinctions include: 1) absence of oil cells and sclerenchyma cells in cross section of thalli while in P. quadrata they occur, at least scattered; 2) mostly triangular cross section of thallus, usually without long and narrow flanks that are quite characteristic for Preissia; 3) male disk without translucent irregular margin. As it was shown above, these genera are also clearly distinguished molecularly.

From the somewhat similar arcticmontane Asterella lindenbergiana (Corda ex Nees) Arnell Bucegia differs in: 1) barrel shaped pores vs. simple ones in A. lindenbergiana; 2) dioicous sexual condition vs. paroicous or autoicous in A. lindenbergiana; 3) stalked male receptacles vs. sessile ones in A. lindenbergiana; 4) lacking oil cells in ventral scales vs. presence of scattered oil cells in A. lindenbergiana; 5) absence of specific smell vs. a smell of rotten fish of A. lindenbergiana in fresh condition.

Sometimes (for example in specimens from Wrangel Island) Asterella lindenbergiana and Bucegia romanica are mixed in the same samples that sometimes leads to confusion.

Variation. The number of layers of air chambers. It is often stressed (even in keys, see Schljakov, 1982) that Bucegia has 3-4 layers of chambers whereas Preissia has only one. However, in drawing in Müller (1906: fig. 180) only one layer of chambers could be seen in Bucegia. In material from Svalbard the number of air chamber layers vary from one (often) to 2 or 3, and chambers are large and elongated (at least chambers of the upper lay- er) contrary to Preissia with small and mostly isodiametric chambers. In Bucegia, the air chamber layer comprises ca. 1/3 of the height of the thallus (in the middle) whereas in Preissia quadrata it never exceeds 1/4 of the height of the thallus.

Shape of transersal section of thallus is quite peculiar in Bucegia romanica. It is mostly V-shaped (triangular) and similar to that of Asterella lindenbergiana whereas in Preissia quadrata it is U-shaped, often with long narrow flanks. In Svalbard Bucegia sometimes has U-shaped cross-section, but flanks are always very short (Fig. 3).

Size and color of thallus. In collections from Svalbard the width of Bucegia romanica thalli varies from 4- 5 (in Nordaustlandet) to 7-8 mm (in western Spitsbergen). The dorsal side of plants from Nordaustlandet is

mostly red-brown to dark brown. In both localities in

western Spitsbergen dorsal surface of studied plants is

light green with red-colored margins.

KEY FOR THE GENERA OF MARCHANTIACEAE

Key for sterile plants

1. Air-chambers in (1-)2-4(-5) layers, without chlorophyllose filaments ...................................... Bucegia

— Air-chambers in one layer, with chlorophyllose filaments ...................................................................... 2

2. Ventral scales in 4 or 6 rows, oil cells present both in ventral tissues and ventral scales, gemmae cups on dorsal side of thallus often present (sometimes absent) ...................................................... Marchantia

— Ventral scsales in 2 rows, oil cells in ventral scales absent, gemmae cups never present ........... Preissia

Key for fertile plants

1. Plants monoicous, heteroicous or dioicus .. Preissia

— Plants dioicous ...................................................... 2

2. Female receptacle with terete lobes ...... Marchantia

— Female receptacle without terete lobes ................. 3

3. Conic female receptacle with hemispheric apex ..... .................................................................... Bucegia

— Convex female receptacle without hemispheric apex .................................................................... Preissia

Key for Bucegia and Preissia in the field (sterile plants)

1. Thallus with large laminal scales, 3/4 of ventral thallus, scales in 2 rows; no sclerocytes or mucilage cavities in thallus ............................................ Bucegia

— Thallus with vestigial laminal scales, less than 1/2 of ventral thallus, scales in more than 2 rows; frequent sclerocytes and mucilage cavities in thallus. .................................................................... Preissia Key for Bucegia and Preissia in the field (fertile plants)

1. Female receptacle conic with hemispheric apex, plants dioicous ............................................ Bucegia

— Female receptacle convex, with 4 more and less distinct ridges across, plants dioicous, sometime heteroicous (subsp. quadrata) or autoicous (subsp. hyperborea) .................................................... Preissia