Blepharostoma trichophyllum S.L. (Marchantiophyta): The Complex of Sibling Species and Hybrids

Blepharostoma trichophyllum was found to be a species collectiva formed by several strongly genetically different species. The taxonomic diversity in the group is the possible result of radiation in early stages; then, these taxa likely survived for a long time in similar environmental conditions, which resulted in stasis. Presently, the existing taxa are similar one to another and may be morphologically distinguished with difficulties. The most taxonomically valuable morphological characteristics include oil bodies and cells in the leaf segment features. The most diverse genotypes (the vast majority of which are treated here as distinct species) were found in amphi-Pacific Asia, which may reflect the evolutionary history of the genus or may be the consequence of more profound sampling in the macro-region in comparison with other parts of the Holarctic.


Introduction
Blepharostoma (Dumort.) Dumort. is a non-speciose genus that occurs frequently in various types of humid and mesic vegetation communities. It is largely restricted to the Holarctic [1][2][3][4], where it spreads to high arctic extremes. Southwards, the genus reaches the Paleo-and Neotropics in Africa [5], Mexico and South America [1], and Indochina (the present paper). The genus representatives occur mostly on organic substrates with a slow process of decomposing, such as humus, decaying wood, and dead bryophyte patches. More rarely, Blepharostoma is observed on moist rocky substrates. The genus currently includes four species [6], of which one only-B. trichophyllum (L.) Dumort.-has undoubted status, whereas the others (B. arachnoideum M.Howe, B. indicum G.Asthana, M.Saxena et Maurya, and B. minor Horik.) have a 'knowledge problem' status (two asterisks in l.c.). B. trichophyllym admittedly possesses a wide Holarctic range, whereas other taxa are locally distributed.
Blepharostoma appears to be the one of the oldest genera of leafy liverworts, as shown by Laenen et al. [7]. The estimated age of this genus is approximately 160 Ma. A similar age was suggested for species including the simple thallose Metzgeria Raddi (167 Ma), the oligotypic Phyllothallia E.A.Hodgs. (158 Ma), and the complex thallose monospecific genus Neohodgsonia Perss. (151 Ma). The basal position of the genus Blepharostoma in the suborder Lophocoleineae was repeatedly proven in several molecular phylogenetic studies based on different combinations of DNA loci from a single sampled specimen of B. trichophyllum originating from Finland, Slovenia, and the USA [7][8][9]. The hidden A vast majority of the sequenced specimens were studied when they were alive, and photographs of living cells were obtained, along with photographs of the Blepharostoma patches under living conditions. These photographs were supplemented with photographs of the morphology of the plants based on herbarium specimens and together formed illustrative confirmations of the discussed taxa.

DNA Isolation, Amplification and Sequencing
A NucleoSpin Plant Kit (Macherey-Nagel, Düren, Germany) was used to extract DNA from the dried liverwort tissue. The primers suggested by White et al. [12] for ITS1-2 and Taberlet et al. [13] for trnL-F were implemented for amplification and sequencing.
PCR was carried out in 20 µl volumes with the following procedure: 3 min at 94 • C, 30 cycles (30 s 94 • C, 40 s 56 • C, and 60 s 72 • C), and 2 min for the final extension time at 72 • C. Amplified fragments were visualized on 1% agarose TAE gels by EthBr staining and then purified with GFX PCR DNA and Gel Band Purification Kits (Amersham Biosciences, Chicago, IL, USA). The sequencing reactions were performed with the ABI Prism BigDye Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems, Waltham, MA, USA) following the standard protocol provided for the 3730 DNA Analyzer (Applied Biosystems, Waltham, MA, USA) at the Genome Center of EIMB (Moscow, Russia).

Phylogenetic Analysis
The newly obtained sequences were assembled in BioEdit 7.0.1 [14], and then ITS1-2 and trnL-F nucleotide datasets were automatically aligned using full multiple alignment with default settings for the gaps and extension weights via the ClustalW tool with subsequent manual corrections. Topological incongruence was revealed among the datasets during the preliminary phylogenetic estimations. Thus, the analyses were implemented for each dataset separately. All positions of the final alignments were included in the calculations, and absent data at the ends of the regions were coded as missing.
Phylogenies were tested by the maximum parsimony (MP) method with TNT v. 1.5 [15] (Goloboff and Catalano, 2016), maximum likelihood (ML) with PhyML v. 3.0 [16], and Bayesian reconstruction with MrBayes v. 3.2.1 [17]. The parsimony analysis with TNT involved a New Technology Search for the minimal-length tree using five iterations and 1000 bootstrap replicates. Default settings were used for the other parameters, and gaps were treated as missing.
For the ML analysis, ModelGenerator [18] identified GTR+I+Г as the best-fitting evolutionary model for each dataset. Gamma distribution with four rate categories was used to handle the among-site rate heterogeneity. Bootstrap support (BS) for individual nodes was assessed using a resampling procedure with 500 replicates. According to the stopping frequency criterion (FC) for the bootstrap [19], the ITS1-2 dataset should require 350 replicates to reach convergence with a Pearson average 100 = 0.992385 and trnL-F and 400 replicates with 100 = 0.992489, as estimated by RAxML v. 7.2.6 [20].
The Bayesian estimation for each dataset was done with the GTR+I+Г model, and gamma distributions were approximated using four rate categories. Two independent runs of the Metropolis-coupled MCMC were used to sample the parameter values in proportion to their posterior probability. Each run included three heated chains and one unheated chain, and two starting trees were chosen randomly. Chains were run for ten million generations, and trees were sampled every 1000th generation. The first 2500 trees in each run were discarded as burnin. Thereafter, 15,000 trees were sampled from both runs. For ITS1-2, the software tool Tracer [21] revealed the effective sample size (ESS) as 7050.0732 and the auto-correlation time (ACT) as 2553.4487. The average standard deviation of the split frequencies between two runs was 0.004277. For trnL-F, the ESS was 6427.4931, the auto-correlation time (ACT) was 2800.7809, and the average standard deviation was 0.003623. Bayesian posterior probabilities were calculated from the trees sampled after burn-in.    The average pairwise p-distances were calculated to test the level of the infrageneric and infraspecific variability of ITS1-2 and trnL-F in the genus Blepharostoma using the pairwise deletion option for counting gaps in Mega 5.1 [22].

Results
In total, ITS1-2 nucleotide sequences were newly obtained for 71 specimens, trnL-F sequences were obtained for 77 specimens, and all were deposited into GenBank. The ITS1-2 dataset of Blepharostoma The tree topologies for each dataset revealed under the three criteria appear highly congruent. Figure 1 shows the single tree for the ITS1-2 dataset retained under ML, along with the ML and MP bootstrap values (BS) and the Bayesian posterior probabilities (PP) for each node. Figure 2 shows the tree for the trnL-F dataset.
Eight main clades and phyla could be distinguished on each tree topology, but the affinity of the clades was differing and had poor support. The basal position in the Blepharostoma phylogeny for both topologies belonged to North American B. arachnoideuim (BS = 100% in MP, BS = 100% in ML, PP = 1.00 in BA, and 100/100/1.00 in the ITS1-2 and trnL-F trees) in four/five specimens that composed a strongly supported clade (100/100/1.00 in ITS1-2, 99/97/0.99 in trnL-F). The next divergence (98/95/1.00 in ITS1-2 -/50/0.69 in trnL-F) belonged to the clade with six/seven specimens (100/100/1.00 in ITS1-2, 99/100/1.00 in trnL-F) from Murmansk, Sakhalin Provinces, Primorsky, Khabarovsk Territories, and in a specimen from Finland (DQ293944), tested in the liverwort phylogeny by He-Nygren et al. [9] under B. trichophyllum. In the current study, these specimens relate to a new species, B. prima sp. nov. The following relations in both topologies are different and obtained partial support, so the evolution within each genus could not be clarified directly. The next divergence in the ITS1-2 tree (99/97/1.00 belonged to the clade with three specimens from Primorsky Territory and Kunashir Island (100/100/1.00), classified here as B. pseudominor sp. nov. The divergence of this clade by trnL-F occurred later and was poorly supported (−/72/0.55). This clade united five specimens from the same regions (89/90/0.99), two of them with only trnL-F sequence data. The follow divergence on ITS1-2 (-/56/0.88) showed a clade (100/100/1.00) with six specimens from Svalbard, Murmansk, and Magadan Provinces, Kamchatka, and a specimen from Greenland, provisionally assigned to a new species by Hassel et al. [10] (2013). Under the trnL-F topology, this clade (84/74/0.71) diverged earlier (90/96/0.99). Based on the morphological evidence, all specimens of this clade were assigned to species B. brevirete comb. nov. The next three subsequently divergent clades on the ITS1-2 topology united into one unsupported clade on the trnL-F topology. Two/three specimens from Japan and South Korea composed a clade (100/100/1.00 in ITS1-2 and 86/90/0.99 in trnL-F), and their morphological and ecological features allowed us to describe a new species-B. epilithica sp. nov. The clade (100/100/1.0 in ITS1-2, 96/99/1.00 in trnL-F) composed of six/seven specimens with predominantly Asian distribution (Russian Far East, Japan, South Korea, and Vietnam) that morphologically resembled B. minor. The single specimen from Khabarovsk Territory with a combination of unique features in both the ITS1-2 and trnL-F sequences was distinguished by the cryptic taxon tentatively named B. sp. until further consideration. More than half of the tested B. trichophylum s.l. specimens from the USA, Norway, Greenland, Germany, China, and different regions of Russia take the terminal position on the tree topologies in the two colored clades. The green colored specimens on the ITS1-2 tree intermingled in one clade with the red ones (97/100/1.00), and those on trnL-F intermingled with the blue ones (91/100/1.00). The green colored specimens were morphologically similar to B. trichophyllum and became one of the parent species for "blue" and "red" taxa that hybridized with two unknown species. According to the obtained topology, the green-and red-colored specimens possess a common ITS1-2 (father inheritance), and the green and blue-colored specimens possess a common trnL-F (maternal inheritance). The clade on the trnL-F tree with red-colored specimens called the B. trichophyllum hybrid taxon 1 (87/90/0.99) has a similar morphology to B. brevirete and appears to be a cryptic taxon. The clade on the ITS1-2 tree with blue-colored specimens characterized by unique morphological features is related to the new species B. neglecta sp. nov. (hybrid taxon 2) (100/97/1.00). On the ITS1-2 topology, for three specimens in the "green-red" clade marked by a black color and asterisk, only ITS data were obtained. Among them, the specimens from Hassel et al. [10] originated from Greenland (KC333192) and Norway (KC333193) and were cited previously as B. trichophyllum subsp. brevirete. Two B. trichophyllum specimens from Norway (KC333190, KC333189) tested in this study were placed in the "blue" B. trichophyllum hybrid taxon 2. On the trnL-F tree in the "green-blue" clade, six specimens were marked by a black color and asterisk due to the presence of only cpDNA data among them. As we did not determine the "green parent", we suggest two distinct species of "blue" and "red" that are widely distributed on common areas. The presence of single parental types of ITS1-2 in "red" hybrid accessions indicates complete evolution and could suggest ancient hybridization in the B. trichophyllum complex, as well as the possible extinction of unknown parents compared to the modern hybridization process between two species with sympatric distribution in the phylogenetically young genus of Barbilophozia Loeske [23]. To obtain additional support for the taxon delimitation and trends of evolution in the genus Blepharostoma, the infraspecific and infrageneric p-distances were calculated ( Table 3). The level of infraspecific variability was mainly low in both studied loci (0-1.4% (2% only for B. minor) in ITS1-2, and 0.2-1.1% in trnL-F), whereas the p-distances between the obtained clades were much higher, so such clades could be treated as distinct species. Blepharostoma arachnoideum, the first divergent species, revealed the highest level of nucleotide sequence divergence from other taxa-18.5-21.9% in ITS1-2 and 7.7-13.1% in trnL-F. The second divergent species was B. prima, with 15.3-18.5% in ITS1-2 and 8.8-13.4% in trnL-F. The complex of the following species evidently diverged later and will be discussed separately from the basal species. Two other species with high infrageneric p-distances-B. pseudominor (7.7-11.2% in ITS1-2 and 3.3-9.0% in trnL-F) and B. minor (8.4-11.2% in ITS1-2 and 3.5-7.9% in trnL-F)-passed unclear positions on the phylogenetic trees. Indirectly, both B. minor and B. pseudominor can be characterized by rapidly evolving ITS1-2, whereas the rate of trnL-F's evolution is similar to its related species. B. brevirete has a similar level of differentiation from other species with both loci (5.7-8.4% in ITS1-2 and 3.3-7.4% trnL-F). Two species B. epilitica and "B. sp." evidently originated later then B. minor and B. pseudominor but kept the same characteristics of quickly evolving ITS1-2 (5.8-10.2% and 5.7-9.4%, respectively) and slowly evolving trnL-F (3.2-8.8 and 2.7-6.8%, respectively). The three taxa of the colored B. trichophyllum clades appear to be three distinct species, two of which are characterized by hybrid origins with unknown parents. We considered the absence of secondary parents as additional evidence for the species status of the hybrids, rejecting modern hybridization between existing species. The variability in ITS1-2 between "green" and "red" clades (1.4%) corresponds to the level of infraspecific variability in other Blepharostoma species between the "green+red" and "blue" clades (8.1-8.3%), for the level of infrageneric variability in Blepharostoma; the variability in trnL-F between the "green" and "blue" clades (0.9%) compared to infraspecific variability; and between the "green + blue" and "red" clades (2.6-3.2%) compared to the level of infrageneric variability in Blepharostoma. Below, we provide the revisions of the species descriptions, establish four novel species, new combination, and discuss the evidence of hybridization and cryptic speciation.

Morphology
The morphological similarities of the revealed genotypes compelled us to treat all phylogenetic lines as subspecies or variations of the widely accepted Blepharostoma trichophyllum. However, the high genetic distances (Table 3) are comparable with the inter-generic differences in some groups of hepatics and are certainly higher than the commonly observed inter-specific variations. The latter variations are largely determined by the ancient origin of Blepharostoma. For example, Diplophyllum (Dumort.) Dumort. is ca. 53 Ma old [7] and contains ca. 20 species [6], uniting taxa that are different by 4.3-14.0% in ITS1-2 and by 3.0-6.5% in trnL-F, as estimated by sampling in Bakalin et al. [24]. Scapania (39 Ma, ca. 100 species) possesses inter-specific differences of 5.8-6.9% in ITS1-2 and 4.7-5.6% in trnL-F among the most ancient taxa [25]. Hygrobiella (31 Ma, 4 species) has distances of 4.5-9.0% in ITS1-2 and 2.1-3.6% in trnL-F [26]. Although Blepharostoma features a low evolution rate among its morphological features (or its morphological similarities are the result of convergent evolution), all units found on the phylogenetic trees can be robustly treated as genetically distinct species. The absence of at least two parent species and the restricted areas of existing species could reflect a reduction in previous species diversity over geological time (the same is true for the possible extinctions discussed by Laenen et al. [7]).
Within the genus, B. arachnoideum is only distinctly morphologically different from other congeners. The essential features distinguishing the species were discussed by Wagner [11] and include collapsed leaf segment cells in dry conditions and commonly branched leaf segments that are "seen on every shoot in robust plants, with as many as half the leaves with a forked segment in well-developed material" [11] (p. 697). These two features classify B. arachnoideum distinctly apart from other known congeners. Currently, B. arachnoideum is a locally distributed and rare taxon restricted to the Pacific coast of North America featuring boreal and cool temperate climates. The distance between B. arachnoideum and B. trichophyllum s.l. is the highest in the genus and reaches 18.5-21.9% in ITS1-2 and 7.7-13.1% in trnL-F, which potentially allows segregating the various subgenera for this species. However, we did not perform such a segregation to prevent superfluous complication of the taxonomic structure of the genus.
One more taxon, B. minor, which is recognized at the species level in the last word checklist [6], likely belongs to the B. trichophyllum s.l. complex, as shown in the present analysis. The remaining B. indica [27] is a taxon whose taxonomic position we could not identify with certain confidence. We suggest that this poorly known species may belong to the B. trichophyllum s.l. complex of taxa but does not belong to any of the taxa recognized in the present paper. Blepharostoma indica is characterized by small, smooth-surfaced oil bodies that are few in number (3-4 per cell, ca. 2 µm in the diameter), a combination that is not known in other taxa of the genus. Moreover, the plant's 2-3-lobed leaves (if not the result of plant 'weakness') and verrucose (not striolate or papillose) leaf cuticle differentiate it from other taxa of B. trichophyllum s.l. Considering the revealed genetic diversity within B. trichophyllum s.l. over the course of the present study, B. indica may be treated as a possible Western Himalayan endemic species whose position in the taxonomic structure of the genus needs further consideration.
As seen from the results, the genetic diversity was (and, probably, is) still poorly understood for B. trichophyllum s.l. The phylogenetic analysis showed at least seven well-supported clades, with two of them possessing hybrids based on the analysis of ITS1-2 and trnL-F. The largest variation in the studied material was seen in the numbers, sizes, and surface features of oil bodies in the leaf segment cells; the relative lengths of cells in the cilia; and the ecology and distribution. The vast majority of morphological parameters freely intergrade with one another, which suggests that all revealed taxa should be considered subspecies, not species. However, the latter is not possible if the p-distances are considered. Moreover, to accept the subspecies status of all revealed entities, (1) B. minor should be synonymized with B. trichophyllum s.l. and (2) the information on the distribution of old taxa would be lost, as subspecies are rarely revealed in purely floristic studies. Moreover, taking into account the possible low evolution speed in the genus, each entity may have a unique and long history of dissemination that will help reconstruct the history of its geographic dissemination, not only for the genus by itself but also for some other groups of liverwort in their early stages.

Phytogeographic Considerations
Laenen et al. [7] estimated the approximate origin time of the genus Blepharostoma as far back as 160-198 Ma. The species diversification time within the genus, however, was not suggested. Judging from the obtained tree, the first result of speciation was the splitting of B. arachnoideum, followed by diversification within B. trichophyllum s.l. Since the support of the tree backbone is low while the clades corresponding to the species are highly supported, the taxa within B. trichophyllum s.l. may be regarded as the result of evolutionary radiation at early stages and not associated with morphological disparity. Struck et al. [28] regarded this situation as evidence of stasis, identified as (l.c., p. 155): the "retention of the same ancestral character state over an extended period". Indeed, Figure 2 (D) in [28] shows a scheme similar to our results. This is a widely known problem in taxonomy, where genetically different units are very similar in their morphology (a remarkable example of this problem is Blepharostoma). Minelly [29] mentioned this problem as one of the most vital challenges in the future systematic management of nomenclature for provisionally circumscribed taxa. Moreover, as Minelly [29] (p. 14) wrote (according to [30]), "the status of cryptic species does not describe a natural phenomenon, but only a temporary problematic formalization of species delineation". For the circumscribed situation within Blepharostoma, we described some taxa as new and some as cryptic, understanding that this is not the 'fault' of the species if we could not find robust distinguishing features between them.
Since the current distribution of B. trichophyllum s.l. is not yet well-known, we cannot estimate with certain confidence the reasons, times, and areas that promoted speciation in the group. However, we suggest that some of the extant taxa within B. trichophyllum s.l. are the result of diversification correlated to the collision of the Indian subcontinent with Asia and the splitting of ancient Asian monsoons into East Asian and Southeast Asian monsoons [31][32][33] (likely from the middle Miocene), where the East Asian monsoon collided (and continued to collide) with the humid and mild oceanic climate in the amphi-Pacific Northeast Asia and insular part of East Asia. This sudden change in climate, against a background of uneven cooling from the Miocene (thus nearly synchronous with East Asian monsoon appearance) and the effect of insularity in the current hemiarctic-cool-to warm-temperate edges of East Asia, reinforced by volcanic eruptions in the northwestern flank of the Pacific Ring of Fire, could promote speciation in the treated area as it also occurred in other liverwort groups [34,35]. The coincidence of vegetation zones and even longitudinal segments with the distribution range of some of the revealed taxa may be regarded as a possible confirmation of this hypothesis.
Although the distribution of all recognized taxa is poorly understood, some preliminary observations can be made, taking into account the distribution data obtained in Pacific Asia. In general, the observed regularity is similar to that found in the distribution patterns of three species of Hygrobiella, as we described before [36], including two main features: (1) a distribution of morphologically similar taxa that is partly sympatric and partly allopatric and (2) genetic and taxonomical diversity that is concentrated in the wide contact zone between hemiboreal, boreal, and hemiarctic zones in the amphi-Pacific areas around the Sea of Okhotsk. Conversely, this may be the simple consequence of deeper sampling from that area. Blepharostoma trichophyllum s.l. has a less definite diversity center, and, unlike Hygrobiella, we cannot determine with confidence whether we found the taxonomic diversity center of the genus or simply molecular diversity in the area, which is only one of many areas with high genetic diversity of this genus in the Holarctic.
The most common taxon in the area, recorded from Magadan Province to Southwest China is Blepharostoma neglecta (hybrid 2). Its mother's taxon (B. trichophyllum) is distributed in East Asia more narrowly than its descendant and has been hitherto recorded only around the Sea of Okhotsk (although it is widely distributed in Europe and North America). The B. trichophyllum hybrid 1 is characterized by a similar distribution to its father taxon (B. trichophyllum). Blepharostoma minor is widely distributed on rocky substrates from hemiboreal (in South Kurils) to subtropical zones located southward but restricted to higher elevation belts. This species was not confirmed genetically in southern China, where it should be distributed.
The southern Japanese-southern Korean distribution is characterized by B. epilithica found on cliffs in warm-temperate communities. Blepharostoma brevirete was confirmed only in Northeast Asia (in Pacific Asia). In general, this species is likely distributed and abundant in the areas northward of the Polar Circle in Chukotka, but there are currently no available materials to test this suggestion. The intermediate position (with respect to geography) between the two aforementioned taxa is occupied by B. pseudominor, which may be regarded as a northern derivate of B. minor characterized by dilated cells in the middle of its leaf segments and epixylous occurrence (contrary to the epilithic occurrence in B. minor), albeit with much longer cells in its leaf segments. Blepharostoma prima, as estimated in the present account, was nevertheless confirmed in the continental mainland from hemiarctic (Ayan) to cool-temperate (South Primorye) zones and not observed in East Asia, although it was also confirmed in Murmansk Province of the Russian Northwest and Finland.
The most common taxa recognized in East Asia have relatively large and granulate oil bodies. Frey and Stech [37] estimated Blepharostomataceae as the sister group to Trichocoleaceae. Trichocoleaceae are characterized by having relatively large (2-4 times larger than in Blepharostoma) and finely granulate oil bodies. This may help confirm that the numerous small homogenous-to-obsolete oil bodies among Blepharostoma are an apomorphic feature now present in B. prima.

The Key
The tentative key for the taxa of the "B. trichophyllum s.l." complex is as follows:

Blepharostoma prima Vilnet et Bakalin sp. nov.
Description. Plants (250-)300-500 µm wide. Stem cross section nearly rounded, 80 µm in diameter, merely rigid, 4 cells high, external wall to 5 µm thick, inward cell walls become thinner, although still thick, trigones vestigial to small and concave, cell size nearly equal across the section, 12-20 µm in diameter. Leaves slightly appressed to the stem (feature-a subject of the great variation), in general view straight at almost whole length, leaf segments in the slide straight to slightly falcate, 350-400 µm long. Middle cells in the segment 25 Table 1.
Comments: Blepharostoma prima, in its narrow sense, is characterized by (1) droplet-like oil bodies or oil bodies that are virtually absent, (2) nearly straight outer lines of the segments (not dilated cells), and relatively long (or short in gemmae producing plants) segments. (3) In a stereoscopic (under a dissecting microscope) view, the species is characterized by segments that are nearly entirely straight and sometimes even appressed to the stem, whereas in other locally recognized taxa, the leaf segments are primarily erect and spread for 1/3-2/5 of the length; then, they suddenly curve subparallel to the stem (this feature is subject to great variation). Blepharostoma prima is not common in East Asia and known in relatively few localities. This species is malleable in its ecology and known in both epilithic and epixylous habitats. Based on the data in hand, this species sparsely occurs in the southern half of the Russian Far East and northward to 56 • N (Ayan in the Sea of Okhotsk coast). The parameters of variation in the description are likely underestimated due to a limited number of molecularly confirmed specimens.
Illustrations in the present paper: Figure Table 1.
Comment. This is a narrowly distributed species whose area is chiefly restricted to the contact zone of East Asian and Circumboreal Floristic Regions and morphologically resembles Blepharostoma minor, although with distinctly longer segments (not characteristic for B. minor) and numerous homogenous oil bodies. This species' very soft stems with thin cell walls in the cross-section, height of only four cells, and distinctly dilated cells in the segment middle are different from B. prima. This species is characterized by epixylous ecology, thin segments of moderate length (200-350 µm), slightly dilated leaf segment cells, and numerous and smooth oil bodies. Currently, this species is known in the southern part of Primorsky Territory and Kunashir Island in the southern Kurils.
Comment. This taxon is well-defined both morphologically and geographically. It is restricted to Hemiarctic and Arctic vegetation zones, with the southernmost area reported in Pacific Asia from the Eastern Kamchatka. It is characterized by relatively thick and long (300-500 µm) segments, with non-dilated cells and granulate oil bodies (few per cell). However, this taxon is indistinguishable from the B. trichophyllum hybrid 1 (as shown in the key and the relevant discussion).
The examined specimens are shown in Table 1. Illustrations in the present paper: Figures Table 1.
Comment. This taxon is known from only three specimens from Japan (Shikoku) and South Korea. This taxon is similar to Blepharostoma minor due to its short cilia (120-300 µm) and epilithic habitat. The two species distinctly differ in size (350-600 µm wide in B. epilithica versus 250-350 µm in B. minor) and stem thickness (6 cells versus 4 cells high), with comparatively longer cells in the segment middle (length/width ratio more than 1.5 versus less than 1.4 in B. minor) and longer segment cells. The listed features look as environmentally induced; however, here they were shown to be correlated with genetic differences. The biggest problem is how to distinguish species from B. neglecta due to transitions in many features (differences listed in the key) and the distribution in the warm temperate vegetation zones of Japan and Korean Peninsula. Moreover, this warm temperate taxon, however, may be distributed wider that it is now known.
Illustrations in the present paper: Figure 4B- Figures 1 and 2) Description. Plants 500-800 µm wide. Stem cross section slightly transversely elliptic, 4-5 cells high, ca 110 × 90 µm, to rounded, 90-100 µm in diameter, external wall to 5 µm thick, with surface loosely striolate, cells nearly equal in size across section, (12-)15-30 µm in diameter, outer layer with large concave trigones, inward trigones become small. Leaves 4-lobed, but also commonly 3-lobed (3-ciliate). Leaf segments slightly falcate to nearly straight, 300-420 µm long. Middle cells in the leaf oil bodies in B. trichophyllum, along with a few granulate oil bodies in the same species. Here, the most common taxon, B. trichophyllum s. str., which is known to exist in various parts of the Holarctic, is characterized by a few granulate oil bodies, whereas the 'phases' with numerous and homogenous oil bodies belong to B. prima and B. pseudominor.
Comment. This taxon is very similar to Blepharostoma brevirete due to its short cells in the middle of the leaf segments (however, sometimes these cells are longer than those in typical B. brevirete). The segments are somewhat shorter than those in B. brevirete, but this length parameter in the two taxa is connected by a series of transitional forms (even within the same patch), which represent two extremes. This taxon seems to be indistinctly more southerly distributed and restricted to hemiarctic and boreal zones, although it was also observed in Spitsbergen and, therefore, cannot be distinguished from B. brevirete on a geographical basis with confidence.
Comment. This taxon is characterized by narrow leaf segments composed of relatively long and dilated cells; in this respect, it strongly resembles B. pseudominor, although it distinctly differs in its granulate oil bodies, which are few in number. On the other hand, this taxon is morphologically similar to B. trichophyllum but differs in having narrower cilia with comparatively longer cells. The difference in its spore size is not clear due to the very limited materials available for comparison. Based on the data in hand, this taxon is mostly distributed in hemiboreal landscapes. Within the Russian Far East, it mostly occurs in the insular-peninsular part of the area, from 56 • N in Central Kamchatka to middle Sakhalin and southern Kurils (48 • N). In the mainland, it is known in the southern part of Magadan Province and Primorsky Territory. Southward, it occurs in the Yunnan and Sichuan Provinces of China and is likely distributed more widely than now known. Westward, the taxon is confirmed in eastern Siberia (Zabaikalsky Territory) and Eastern Europe in the Perm Province and Mary-El Republic of Russia.
Comment. This taxon is very similar to Blepharostoma brevirete due to its short cells in the middle of the leaf segments (however, sometimes these cells are longer than those in typical B. brevirete). The segments are somewhat shorter than those in B. brevirete, but this length parameter in the two taxa is connected by a series of transitional forms (even within the same patch), which represent two extremes. This taxon seems to be indistinctly more southerly distributed and restricted to hemiarctic and boreal zones, although it was also observed in Spitsbergen and, therefore, cannot be distinguished from B. brevirete on a geographical basis with confidence.