CEPHALOZIELLA KONSTANTINOVAE (CEPHALOZIELLACEAE, A NEW LEAFY LIVERWORT SPECIES FROM RUSSIA AND MONGOLIA IDENTIFIED BY INTEGRATIVE TAXONOMY

. In the course of a taxonomic study of the genus Cephaloziella (Spruce) Schiffn. (Cephaloziellaceae, Marchantiophyta) in Asia, the new species Cephaloziella konstantinovae Mamontov & Vilnet, sp. nov ., from the eastern regions of Russia and from the Republic of Mongolia was discovered. The new species is formally described and illustrated here. Morphologically it is similar to C. divaricata var . asperifolia (Taylor) Damsh. , but differs in its leaf shape and thin-walled, inflated stem and leaf cells. The new species can be distinguished from other Cephaloziella taxa by the following characters: ( i ) female bracts entirely free from each other and from bracteole, ( ii ) perianth campanulate, ( iii ) cells of perianth mouth subquadrate, ( iv ) capsule spherical, ( v ) seta with 8–10 + 4–6-seriate morphology, and ( vi ) elaters with 1–2 spiral bands. Molecular phylogenetic analyses of nrITS1-5.8S-ITS2 and chloroplast trn L-F sequences from 63 samples (34 species, 23 genera) confirm the taxonomical status of the new species. Five specimens of C. konstantinovae form a clade placed sister to a clade of C. elachista (J. B. Jack) Schiffn. and C. rubella (Nees) Warnst.


Introduction
1 Corresponding author Schiffner (1893) established the leafy liverwort genus Cephaloziella (Spruce) Schiffn. to accommodate species of minute size having ventral and lateral branches, small leaf and stem cells, and occasionally underleaves. Schiffner included 20 species in the genus. Cephaloziella is virtually cosmopolitan; its species occur on rotten wood, peat bogs, soil and rocks, but a few taxa in tropical forests are corticolous (Schuster 2002).
Recently intensified floristic studies in remote regions of Russia -the Arctic, Caucasus, Siberia and the Far East -produced new records for a number of Cephaloziella species ), allowed revision of numerous specimens, and confirmed some opinions of hepaticologists on the circumscription of several taxa. Here we apply an integrative approach, considering evidence from DNA sequence variation as well as morphology, ecology and phytogeography, with a special focus on some morphologically unique specimens collected by the senior author in the Trans-Baikal Region of Siberia. These specimens resembled Cephaloziella but showed some morphological overlap with Scapaniaceae + Anastrophyllaceae in branching mode and generative features. Phylogenetic analyses of nuclear ribosomal ITS and chloroplast DNA trnL-F sequences support the suggestion that these Trans-Baikalian plants belong to Cephaloziella and are distinct from all putative relatives; hence they are described as a new species, Cephaloziella konstantinovae.

Materials and methods taxon sampling
Species representing the families and genera of Jungermanniales suborder Cephaloziineae (Сrandall-Stotler et al. 2009) were chosen to test the morphology-based attribution of the new species to Cephaloziella. A total of 63 accessions representing 34 species were used for this study (Table 1). We analyzed 12 species (41 samples) from the family Cephaloziellaceae, 10 species (10 samples) from Scapaniaceae, 8 species (8 samples) from Anastrophyllaceae, 2 species (2 samples) from Cephaloziaceae, and 2 species (2 samples) from Odontoschismataceae. We chose Cephalozia affinis Lindb. ex Steph. as outgroup taxon based on the phylogenetic hypotheses presented by Feldberg et al. (2013).
The ingroup includes 11 taxa from the genus Cephaloziella and one species of the genus Cylindrocolea R. M. Schust (Cephaloziellaceae). Ingroup taxa are represented by multiple accessions from different regions. The nucleotide data for 9 specimens were taken from earlier studies and downloaded from GenBank (Vilnet et al. 2012;Bakalin & Vilnet 2014). DNA vouchers are listed in Table 1, including GenBank accession numbers and voucher details. dna isolation, pcr amplification and dna sequencing DNA was extracted from dried liverwort tissue using the NucleoSpin Plant Kit (Macherey-Nagel, Germany). Amplification and sequencing were performed using primers suggested by White et al. (1990) for nrITS1 -2 and Taberlet et al. (1991) for trnL-F. PCRs were carried out in 20 µl volumes according to the following procedure: 3 min at 94°C, 30 cycles (30 s 94°C, 40 s 56°C, 60 s 72°C) and 2 min extension time at 72°C. Amplified fragments were visualized on 1% agarose TAE gels by EthBr staining, purified using the GFX ™ PCR DNA and Gel Band Purification Kit (Amersham Biosciences, USA), and then used as a template in sequencing reactions with the ABI Prism BigDye Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems, USA) following the standard protocol for the 3100 Avant Genetic Analyzer (Applied Biosystems, USA).

phylogenetic analysis
Two datasets (ITS1-2, trnL-F) were automatically aligned in BioEdit 7.0.1 (Hall 1999) using the ClustalW option and then manually corrected. The preliminary phylogenetic analyses revealed no incongruence between the nuclear and the chloroplast DNA datasets; hence they were combined. Lacking sequences were coded as missing.
The ModelGenerator program (Keane et al. 2006) determined the GTR+I+Γ model as the best-fit evolutionary model of nucleotide substitutions for the combined alignment. In the BA, four partitions of the combined alignment (ITS1-2, trnL-F) were separately assigned the GTR+I+Γ model, and gamma distributions were approximated using four categories. Two independent runs of the Metropolis-coupled ΜCMC were used to sample parameter values in proportion to their posterior probability. Each run included three heated chains and one unheated chain, and the two starting trees were chosen randomly. The number of generations was 10,000,000, and trees were saved once every 10,000 th generation. Average standard deviation of split frequencies between two runs was 0.009191. Bayesian posterior probabilities were calculated from trees sampled after burn-in (Fig. 1).
Jackknife support was calculated in NONA for 1000 replications (number of search reps 10, hold 10, max tree 100, do max) and shown in Figure 2.
Due to the unusual morphology of the studied Trans-Baikalian plants here assigned to Cephaloziella konstantinovae, Bayes factor comparison was additionally used to test whether the new species could be related to Anastrophyllaceae or Scapaniaceae rather Table 1. List of taxa, voucher specimens and GenBank accession numbers. The sequences from our previous study are in italics. Initial species identifications from herbarium vouchers are indicated for specimens reclassified in this study based on results of DNA analyses.

Taxon name
Voucher specimens GenBank accession number ITS1-2 trnL-F Alobiellopsis parvifolia (Steph.) R. M. Schust. Japan,Bryophytes of Asia Fasc. 8,No. 191 (KPABG) JX629894 JX630020 than to Cephaloziella. Specifically, we contrasted the hypothesis that the new species and Anastrophyllaceae + Scapaniaceae form a monophyletic group (M1) with the hypothesis that they do not form a monophyletic group (M2). In order to do this, the ratio of the marginal likelihoods of the models M1 and M2, the Bayes factor, was computed. The logarithm of the Bayes factor is the difference in the logarithms of the marginal model likelihoods (Kass & Raftery 1995). A rough estimate of the marginal model likelihoods can be obtained as a harmonic mean of the likelihood values of the MCMC samples (Newton & Raftery 1994). MrBayes 3.2.1 provides that method for estimating the logarithm of the marginal model likelihoods (Ronquist et al. 2011). The harmonic mean of the likelihood values of the MCMC samples is the logarithm of the marginal model likelihood (Ronquist et al. 2011).
To test the hypotheses, a hard constraint (M1) and a negative constraint (M2) were specified. In the step of the hard constraint, the constrained tree was defined wherein Cephaloziella konstantinovae was forced to belong to clade Anastrophyllaceae + Scapaniaceae.
It allowed us to force a partition to always be present in the sampled trees. The harmonic mean from the MCMC-based search was recorded under M1. In the step of the negative constraint, the constrained tree was defined wherein Cephaloziella konstantinovae was forced not to belong to Anastrophyllaceae + Scapaniaceae. It allow sampling across all trees that do not contain the specified partition. The harmonic mean from the MCMC-based search was recorded under M2. The difference between harmonic means from both runs, the logarithm of the Bayes factor, was calculated. The corresponding interpretation was found in the table of Kass and Raftery (1995). To use this table, twice the difference in the harmonic means was calculated (Ronquist et al. 2011).

Results
Nuclear ribosomal ITS sequences and chloroplast DNA trnL-F sequences were newly generated for 31 specimens. The ITS1-2+trnL-F alignment  (63 specimens) included 1822 character sites, among them 852 constant positions; 296 were variable and 674 parsimony-informative. Arithmetic means of log likelihoods in the BA analysis in both runs sampled were -15, 143.52 and -15,145.63. The Bayes factor calculation revealed the harmonic mean estimate for model 383.98 in log units, which is ca 179 log units worse than the previous model. Twice the difference between the harmonic means is 358 log units. A log difference above 5 is considered to be very strong evidence in favor of the better model (Kass & Raftery 1995). Thus, the harmonic mean estimator gives very strong evidence in favor of Cephaloziella konstantinovae being distant from both Anastrophyllaceae and Scapaniaceae.
The Bayesian and the parsimony topologies were largely similar (Figs 1 & 2). The majority of Cephaloziella accessions are found in three clades -A, B and C -which are stable in both BA and MP trees but differently supported in the cal- Although the present analyses did not resolve deeper nodes of Cephaloziella, it is clear that our new species is nested within this genus.
Being first recognized from the Trans-Baikal Territory in 2009, the new species was found subsequently in the Republic of Buryatia, southern Siberia, in Primorsky Territory, the Russian Far East, and the Republic of Mongolia. Due to the presence of well-developed underleaves and multicellular outgrowths on the abaxial leaf face, the sterile specimens of this species were initially identified as Cephaloziella divaricata var. asperifolia (Taylor) Damsh. or C. byssacea var. asperifolia Macvicar (in herb. VBGI) and published as C. aspericaulis Jørg. or C. divaricata var. asperifolia (Taylor) Damsh. for the Trans-Baikal Territory, Russia (Konstantinova & Afonina 2009;Afonina et al. 2012;Czernyadjeva et al. 2013) or C. microphylla (Steph.) Douin for Tuv Aimag of Mongolia (Tsegmed & Bai 2013). The senior author's collections contain fertile plants with mature androecia, perianthia and sphorophytes. These plants' characteristics are as follows: (i) female bracts entirely free from each other and from bracteole, (ii) perianth campanulate, (iii) cells of perianth mouth rounded-rectangular to subquadrate, (iv) capsule spherical, (v) seta with 8-10 + 4-6-seriate morphology, and (vi) elaters with 1-2 spiral bands. According to Schuster (2002), none of the genera of Cephaloziellaceae have this combination of character states of the reproductive structures. In Chonecolea, which is considered an element of Cephaloziellaceae by Patzak et al. (2016), all of those characteristics are present, but the spore surface in Chonecolea is reticulate, whereas in Cephaloziella konstantinovae as well as other Cephaloziellaceae (except Cylindrocolea reticulata) the spore surface is finely granulate, papillose or verruculose.
It should be noted that the 8-10 outer seta cells of Cephaloziella konstantinovae do not occur in other members of Cephaloziellaceae s.str. In this character the new species is similar to some elements of Lophoziaceae sensu Schuster (1969), where the seta consists of numerous cell rows or is reduced to (2-3)4 inner and (6-7)8 outer rows in some taxa (Schuster 1969: 219). According to Schuster (2002)  Crossocalyx hellerianus (Nees ex Lindenb.) Meyl.], but it is never fixed at the 8+4 state in these taxa (Schuster 2002).
On the other hand, in Lophozioideae sensu Schuster (1969) the area of one inner cell of the seta (in cross section) approximates that of one outer cell of the seta (Schuster 1969: Fig. 163: 2, Fig. 235: 7-10, Fig. 242: 2, Fig. 244: 4). In all species of Cephaloziellaceae s.str., especially in Cephalojonesia, Cephaloziella, Cephaloziopsis, Cylindrocolea, Kymatocalyx and Phycolepidozia, the inner cells of the seta are reduced in size, so that the area of inner cells (in cross section) does at best reach the size of one outer cell (Douin 1908(Douin , 1920Grolle & Vanden Bergen 1970;Schuster 2002). This character is also observed in Cephaloziella konstantinovae where the collective area of inner seta cells (in cross section) is almost equal to that of one outer seta cell (Figs 25 & 27).
Within Lophozioideae, Gymnocoleopsis capensis (S. W. Arnell) R. M. Schust. has a seta morphology of the 8 + 4-type, but the inner cells (in cross section) are reduced in size (Arnell 1963;Schuster 2002). On the other hand, a close affinity between Gymnocoleopsis and Cephaloziella was shown in a molecular phylogenetic study of Lophoziaceae (De Roo et al. 2007), and the genus Gymnocoleopsis is now placed within Cephaloziellaceae s.str. (Crandall-Stotler et al. 2009;Váňa et al. 2013).
The genera Protolophozia (R. M. Schust.) Schljakov and Pseudocephaloziella R. M. Schust. of the former Lophoziaceae resemble Cephaloziella konstantinovae in the absence of nodular thickenings on the cell walls and the presence of well-developed underleaves. However, the phylogenetic relationships of Pseudocephaloziella remain unclear because the sporophyte characters are unknown and nucleotide sequence data are not available. The genus Protolophozia differs from Cephaloziella konstantinovae in seta morphology. According to Schuster (1988: 63, Fig. 5: 10), the seta in Protolophozia elongata (Steph.) Schljakov consists of 17 outer and 20 inner cells that do not differ in size. Moreover, the phylogenetic position of Protolophozia is not yet established. Schuster (2002) considers Protolophozia (incl. P. elongata) to be a subgenus within the genus Lophozia. Heinrichs et al. (2005) proposed to merge Lophoziaceae into Scapaniaceae; in , Protolophozia was placed within Scapaniaceae s.str. (excl. genera later assigned to Anastrophyllaceae). Vilnet et al. (2012) found inconsistent positions of Protolophozia elongata in a series of phylogenetic analyses; however, Váňa et al. (2013) proposed to include Protolophozia in Cephaloziellaceae. In the obtained phylogenetic trees (Figs 1 & 2), Protolophozia elongata is related to the clade of Anastrophyllaceae + Scapaniaceae rather than to Cephaloziellaceae.
For the abovementioned reasons, the seta structure as well as the shape of bracts and bracteole and the degree of their connection probably are not sufficient to delimitate genera of Cephaloziellaceae s.str. It may be that vegetative characters, particularly the branching mode and the presence/ absence of underleaves, are of greater taxonomic importance.
The campanulate perianth and the subiso- The combination of characters of Cephaloziella konstantinovae is unusual for the family Cephaloziellaceae s.str. Some of these characters are presented in isolated species of oligospecific genera and subgenera -Allisoniella, Amphicephalozia, Cephalojonesia, Cephaloziopsis, Cylindrocolea subg. Cylindroscyphus -but have been found collectively (excl. spore surface) only in Chonecolea. Furthermore, the type of reproductive structures found in Cephaloziella konstantinovae is unknown in other species of the genus Cephaloziella.

phylogenetic relationships and morphological features within holarctic cephaloziella
Our molecular phylogenies compelled us to reexamine the morphology of the included Cephaloziella accessions and to revise species identifications. Plants initially identified as Cephaloziella arctogena (R. M. Schust.) Konstant. ('Trans-Baikal Territory') with large underleaves on sterile shoots and without gemmae were found in one clade in the Bayesian tree together with plants determined as C. elegans (Heeg) Schiffn. ('Republic of Altai') characterized by purplish and violet pigmentation, broad leaf lobes (up to 9 cells wide), distinct underleaves, the presence of gemmae, and C. rubella ('Altai Territory') with narrow leaf lobes (5-7 cells wide), claret red to violet pigmentation, and small indistinct underleaves on both fertile and gemmiferous shoots. We consider our molecular data to be supportive of a treatment of C. arctogena and C. elegans as varieties of C. rubella: C. rubella var. arctogena R. M. Schust. and C. rubella var. elegans (Heeg) R. M. Schust. respectively. It should be noted that the specimen 'Trans-Baikal Territory' of C. rubella forms a long branch in the Bayesian tree and a separate subclade in the MP tree.
The C. rubella clade is resolved as sister to C. elachista in both BA and MP topologies. Cephaloziella elachista is the type of section Schizophyllum (Müll. Frib.) Jørg., and C. rubella is the type of section Rubellae R. M. Schust. Hence, section Rubellae probably should be reduced to a synonym of section Schizophyllum.
The close affinity between Cylindrocolea recurvifolia and other species of Cephaloziella is evident from the presented phylogenetic trees. This species is nested within clade A together with C. kiaeri and C. microphylla, being related to them in several identical nucleotide substitutions as well as unique insertions and deletions in both ITS1-2 and trnL-F. Accordingly, Cylindrocolea recurvifolia should be treated as Cephaloziella recurvifolia (Steph.) S. Hatt. Members of clade A share autoicy and an East Asian origin.
Cephaloziella spinicaulis is resolved within the group of clades A, B, and C. konstantinovae in the Bayesian tree, or a well-supported subclade distant to clades A, B, and C in the MP topology. It resembles species of clade A in its tendency to form flagelliform shoot apices and frequent axillary postical intercalary branches. However, it is a dioecious species with (i) a disjunctive, probably relictual distribution in East Asia and eastern North America, and (ii) a conspicuously roughened stem with numerous spine-like, simple or bifurcate projections of 1-4 cells arranged in longitudinal lines.
Cephaloziella turneri is separated from all other Cephaloziellaceae elements in the BA and MP topologies. The remote affinity of C. turneri to other congeneric species was also shown in Feldberg et al. (2013). This species produces angulate gemmae, whereas other analyzed species of Cephaloziella have smooth elliptical gemmae.
Cephaloziella varians appears to be a highly variable species that is widely distributed in Russia, especially in the mountains of southern Siberia and the Russian Far East. All studied specimens are identical in trnL-F and have minor differences in ITS sequences. All specimens have irregular and entire (not bifid) underleaves of different sizes. The majority of these specimens consist of sterile plants only, and were thus initially assigned to C. divaricata or C. polystratosa (R. M. Schust. & Damsh.) Konstant. Some autoecious specimens were initially determined as C. grimsulana due to having obtuse or rounded leaves and large leaf cells, or as C. uncinata and C. spinigera due to the presence of elongated, somewhat hooked leaves and small leaf cells.
The 'true' Cephaloziella divaricata seems to be rare in Russia. Only a single specimen from Sakhalin ) is found in one clade with the specimen identified earlier as C. stellulifera (Taylor) Schiffn. from the Netherlands (Konstantinova 1е-2-99). The plants of C. divaricata from Sakhalin are sterile and have regular well-developed (ca 0.5 of leaf length) bifid underleaves, whereas the specimen of C. stellulifera contains plants bearing only paroecious inflorescences, well-developed bifid underleaves up to 0.7 of leaf length, and large cells at the base of leaf lobes, 17-19 µm.
Members of clade C are morphologically heterogeneous yet show only low levels of ITS and trnL-F sequence variation. Follow-up studies need to include more variable marker systems and more extensive sampling.
Etymology. The species is named in honor of Prof. Dr. Nadezhda A. Konstantinova, eminent Russian hepaticologist. Distribution. In Siberia, Cephaloziella konstantinovae is common in low mountain areas in the south of the Republic of Buryatia and Trans-Baikal Territory, and possibly common in the north of Mongolia (Bulgan and Tuv Provinces), but becomes more rare towards the north. It was collected sporadically on the Barguzin and South Muya ranges in the north of Buryatia, and on Kodar Range in the north of Trans-Baikal Territory, but was not found north of 57°N. In Primorye Territory, the Russian Far East, C. konstantinovae was collected only once.
Ecology. In Siberia, C. konstantinovae occurs in low mountain steppe communities and forests dominated by Larix dahurica Turcz. and L. sibirica Ledeb., but was not found in mountain forests where Pinus pumila (Pall.) Regel occurs. It grows in pure dense patches on soil in rock niches or on thin soil overlying vertical rock surfaces in partial shade, and also as an admixture to other saxicolous hepatics, especially of the genus Frullania Raddi. particularly Figure 19: 2, 7-11, Figure 25: 3,7 and Figure 26: 1,2,4,16, where the leaves are toothed at the base or along the whole leaf margin.