Orthotrichum affine var . bohemicum ( Orthotrichaceae ) , a new variety of epiphytic moss from the Czech Republic

The genus Orthotrichum is a widespread moss group, which includes approximately 155 species [1]. Taxa belonging to this genus are found throughout the world from the Arctic to the Antarctic, avoiding only deserts and wet tropical forests. Species of the genus Orthotrichum grow on trees and rocks to a height of ca. 5000 m a.s.l. [2]. In the Czech Republic there were until recently 20 known taxa [3]. Subsequently in 2009, new species of O. moravicum have been added [4].


Introduction
The genus Orthotrichum is a widespread moss group, which includes approximately 155 species [1].Taxa belonging to this genus are found throughout the world from the Arctic to the Antarctic, avoiding only deserts and wet tropical forests.Species of the genus Orthotrichum grow on trees and rocks to a height of ca.5000 m a.s.l.[2].
In the Czech Republic there were until recently 20 known taxa [3].Subsequently in 2009, new species of O. moravicum have been added [4].

Material and methods
During a bryofloristic survey in the Protected Landscape Area Lužické hory Mts.(NW part of the Czech Republic) in October 2006 a noteworthy epiphytic moss from the genus Orthotrichum was collected (Fig. 1).Upon closer examination the specimen proved to represent a variety new to science.
We are pleased to name the variety after the province Bohemia of the Czech Republic, where the type material has been found.
The nomenclature of the moss taxa in the text below follows Hill et al. [5], hepatics are dealt with Grolle and Long [6] and vascular plants follow Kubát et al. [7].
Orthotrichum affine var.bohemicum is similar to type O. affine in terms of many gametophytic and sporophytic characters, but it can be easily distinguished based on 16 endostome segments (practically as long as exostome teeth) and smaller spore size of 13-15 µm (normal O. affine has spore size of 14-26 µm) in O. affine var.bohemicum.Orthotrichum pallens, which seems superficially related to the above species, can be differentiated from them primarily due to the cryptopore type of stomates and shorter and blunter ended leaves.In addition, O. pallens has endostome segments alternately longer and shorter, whereas in O. affine var.bohemicum all segments are equally long (practically as long as exostome teeth).

Ecology and distribution of O. affine var. bohemicum
The new variety was firstly observed growing on the bark of Fraxinus excelsior, in the lower vertical part of the trunk, on the southwestern side, at a height of 72 cm; the population size was 7.5 cm 2 , and it was accompanied by Brachythecium salebrosum, B. velutinum and Chiloscyphus profundus.The tree was growing within a small group of Fraxinus excelsior in a man-made spruce forest, by the road (Fig. 4).After the specimen had been identified and confirmed as a new species, a detailed survey of the locality was carried out.In July 2009, two new populations of O. affine var.bohemicum were found on another Fraxinus excelsior tree in the proximity of the first one.Both populations were growing at heights of 100 and 120 cm above ground, together with Brachythecium salebrosum.The size of the lower-growing population was 4.5 cm 2 , and that of the upper-growing one was 3 cm 2 .

Molecular analysis
A list of species used in the molecular analysis, details concerning voucher data, and GenBank accession numbers are given in the Appendix S1.Total genomic DNA was extracted using DNeasy Plant Mini Kit (Qiagen).Single stems were ground with silica in a Mini-Beadbeater-1 tissue disruptor for 40 s and subsequently treated with kit reagents following the manufacturer's protocol.Extracted DNA samples were stored at -20°C.For amplification and sequencing of ITS we used the primers of Fiedorow et al. [8], which were successfully used in previous studies on genus Orthotrichum [4,[9][10][11][12].The ITS were amplified in a volume of 25 μl containing 20 mM (NH 4 ) SO 4 , 50 mM Tris-HCl (pH 9.0 at 25°C), 1.5 mM MgCl 2 , 1 μl BSA, 200 μM each dATP, dGTP, dCTP, dTTP, 1.0 μM of each primer, one unit of Taq polymerase (Novazym) and 1 μl of the DNA solution.The reaction was processed at 94°C for 1 min followed by 30 cycles at 94°C for 1 min, 59°C for 1 min, and 72°C for 1.5 min, with a final extension step of 72°C for 5 min.Finally 5 μl of the amplification products were visualized on 1.5% agarose gel with ethidium bromide staining.Purified PCR products were sequenced in both directions using ABI BigDye 3.1 Terminator Cycle Kit (Applied Biosystems) and then visualized using an ABI Prism 3130 Automated DNA Sequencer   (Applied Biosystems).The assembled sequences were aligned using Muscle 3.6 [13] and manually adjusted with BioEdit 7 [14].Phylogenetic analyses were conducted using Minimum Evolution (ME) analysis [15] as implemented in MEGA 4 [16].The pairwise distances were estimated with the Kimura 2-parameter method [17] and initial trees were generated using the neighbor-joining (NJ) method.The ME tree was searched using the Close Neighbor Interchange (CNI) algorithm [18] at a search level of 2, and the maximum number of trees retained at each step was set to 100.Bootstrap analysis [19] was carried out with 1000 replicates.
A combined dataset included 984 characters, of which 849 were constant, 33 variable but parsimony-uninformative and 102 potentially phylogenetically informative.The tree based on ME analysis revealed three well-supported clades (Fig. 5).Clade A (BS 100%) is formed by O. pulchellum and O. stramineum, i.e. species with immersed stomata, which were used as an outgroup based on previous studies [9,11].The specimens of the dioecious O. lyellii formed clade B (BS 100%), which suggests its distinctiveness from monoecious members of the subgenus Gymnoporus, which were included in clade C. Within this group, an analysis of ITS revealed the specimens of O. affine var.bohemicum to represent a sister group to O. affine, but clearly separate (BS 90%) from the species, which  is consistent with morphological data.
The samples of O. affine var.bohemicum differ from the specimens of O. affine from Slovakia and the Czech Republic (specimen 2) by one deletion and two substitutions, and from other specimens of O. affine (Poland and the Czech Republic 1) by additional three substitutions.

Conclusion
According to Lewinsky [2], the morphological features of Orthotrichum affine var.bohemicum, which were supported by the results of our molecular analysis [10], suggest its inclusion within the subgenus Gymnoporus, sect.Affinia.
The new taxon is closely related to type O. affine.These two taxa are morphologically similar with respect to the size of plants, leaf shape, and the type of stomates as well as the hairiness of calyptra.However, they differ significantly in the number of endostome segments (O.affine = 8, O. affine var.bohemicum = 16) and in the shape of leaf cell papillae.Whereas O. affine exceptionally rarely has forked branched papillae, O. affine var.bohemicum almost always develops forked papillae and rarely conical ones.
Subsequently, after we had distinguished the new species from the type-material, we also made a revision of selected Czech herbarium collections.As a result, two more specimens of O. affine var.bohemicum, reported from the territory of the Czech Republic, were found (Fig. 4).The first specimen was collected in the Bílé Karpaty Mts. in a locality near the village Velká nad Veličkou, 513 m a.

Fig. 1
Fig. 1 Photomicrographs of Orthotrichum affine var.bohemicum.a Habit.b Hidden capsule with hairy calyptra.c Superficial stomata.Photos of the holotype specimen were made by V. Plášek.

Fig. 2
Fig. 2 Orthotrichum affine var.bohemicum.a Peristome.b Detail of endostome segments after exostome teeth were cut off.c Calyptra.d Leaves.e Superficial stomata.f Transverse section of leaf.g Habit.Scale bars are given for every picture.Drawings of holotype by Sylwia Wierzcholska.

Fig. 3
Fig. 3 SEM micrographs of Orthotrichum affine var.bohemicum.a Peristome.b Leaves (with recurved margins).c Capsule shape.d,e.External view of peristome showing OPL ornamentation of the exostome and PPL ornamentation of the endostome.f PPL ornamentation of the exostome.g Endostome segment showing IPL ornamentation.h Spore.i Detail of pore.Photos of the holotype specimen were made by V. Plášek and G. Kratošová.
Plášek et al. / New variety of Orthotrichum affine

Fig. 5
Fig. 5 Minimum evolution tree based on ITS sequences.Bootstrap values above 50% are given above branches.