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Does Calypogeia azurea (Calypogeiaceae, Marchantiophyta) occur outside Europe? Molecular and morphological evidence

  • Katarzyna Buczkowska ,

    Roles Conceptualization, Formal analysis, Funding acquisition, Investigation, Resources, Writing – original draft

    androsac@amu.edu.pl

    Affiliation Department of Genetics, Faculty of Biology, Adam Mickiewicz University in Poznań, Poznań, Poland

  • Vadim Bakalin,

    Roles Conceptualization, Funding acquisition, Investigation, Methodology, Resources, Writing – original draft

    Affiliation Botanical Garden-Institute, Russian Academy of Science, Vladivostok, Russia

  • Alina Bączkiewicz,

    Roles Funding acquisition, Investigation, Resources

    Affiliation Department of Genetics, Faculty of Biology, Adam Mickiewicz University in Poznań, Poznań, Poland

  • Blanka Aguero,

    Roles Methodology, Resources, Writing – original draft

    Affiliation Duke Herbarium, Department of Biology, Duke University, Durham, NC, United States of America

  • Patrycja Gonera,

    Roles Investigation

    Affiliation Department of Genetics, Faculty of Biology, Adam Mickiewicz University in Poznań, Poznań, Poland

  • Monika Ślipiko,

    Roles Investigation

    Affiliation Department of Botany and Nature Protection, University of Warmia and Mazury in Olsztyn, Olsztyn, Poland

  • Monika Szczecińska,

    Roles Investigation, Methodology

    Affiliation Department of Botany and Nature Protection, University of Warmia and Mazury in Olsztyn, Olsztyn, Poland

  • Jakub Sawicki

    Roles Funding acquisition, Methodology

    Affiliation Department of Botany and Nature Protection, University of Warmia and Mazury in Olsztyn, Olsztyn, Poland

Abstract

Oil bodies are the unique feature of most liverworts. Their shape, color and distribution pattern in leaf and underleaf cells are important taxonomic features of the genus Calypogeia. Most species of the genus Calypogeia have pellucid and colorless oil bodies, whereas colored, including gray to pale brown, purple-brown or blue oil bodies, are rare. To date, C. azurea was the only species with blue oil bodies to have been considered as a species of the Holarctic range. This species has been noted in various parts of the northern hemisphere–from North America, through Europe to the Far East. The aim of this study was to determine the genetic diversity of C. azurea from different parts of its distribution range and to ascertain whether blue oil bodies appeared once or several times in the evolution of the genus Calypogeia. The phylogenetic analyses based on four plastid regions (rbcL, trnG, trnL, trnH-psbA) and one nuclear region (ITS2) revealed that C. azurea is presently a paraphyletic taxon, with other Calypogeia species nested among C. azurea accessions that were clustered into four different clades. Based on the level of genetic divergence (1.03–2.17%) and the observed morphological, ecological and geographical differences, the evaluated clades could be regarded as previously unrecognized species. Four species were identified: C. azurea Stotler & Crotz (a European species corresponding to the holotype), two new species from Pacific Asia—C. orientalis Buczkowska & Bakalin and C. sinensis Bakalin & Buczkowska, and a North American species which, due to the lack of identifiable morphological features, must be regarded as the cryptic species of C. azurea with a provisional name of C. azurea species NA.

Introduction

The genus Calypogeia (Raddi) comprises around 90 described species of leafy liverworts of the suborder Jungermanniineae [1, 2]. Oil bodies, intracellular organelles surrounded by a single unit membrane, are a unique feature of most liverworts [3]. Their shape, color and distribution pattern in leaf and underleaf cells are important taxonomic features of many liverwort species, including the species of the genus Calypogeia. Despite the above, oil bodies are not commonly used in taxonomic identification because they are absent in dried herbarium material [1, 4, 5]. Most species of the genus Calypogeia with identified oil bodies have pellucid and colorless oil bodies, whereas colored, including gray to pale brown, purple-brown or blue oil bodies, are rare [1, 6]. Gray to pale brown oil bodies were detected in C. contracta Inoue, purple-brown oil bodies were noted in C. oblata Herz., whereas deep blue oil bodies were found in five species, i.e. C. aeruginosa Mitten (intergradation between purple and blue), C. goebelii (Schiffn.) Steph., C. granulata Inoue (intergradations of brown within a single leaf), C. lunata Mitt., C. peruviana Ness & Mont., and C. azurea Stotler & Crotz [1, 7, 8, 9]. According to [10, 11], Calypogeia species contain azulene, a natural chromophore whose chemical structure is responsible for the dark blue color of oil bodies. Chemical analyses of C. azurea and C. granulata revealed the presence of 1,4-dimethylazulene and other azulenoids in live plants, but these compounds were not found in species with colorless oil bodies–C. fissa (L.) Raddi and C. suecica (Arnell & J.Perss.) Mull.Frib. [1215].

In the group of species with blue oil bodies, C. azurea is the only widely distributed taxon throughout the northern hemisphere. Calypogeia azurea is regarded as a hemiboreal-montane species. The discussed taxon has been reported from North America, throughout Europe to East Asia, from various habitats ranging from loamy soil, humus, peat, wet stones and rocks to rotten logs [1, 1620]. Calypogeia species are characterized by high environmentally induced variations in morphology [1], and live plants, including C. azurea, are identified based on the presence of blue oil bodies. Calypogeia azurea is the only species with blue oil bodies in Europe, where it occurs mainly in the mountains between low altitude regions to the Alpine belt [17, 19]. Two species with blue oil bodies have been identified in North America, C. azurea and C. peruviana, but they differ in distribution range. Calypogeia azurea is rare, and it occurs mainly in the Pacific Northwest, whereas C. peruviana is found in the southeastern United States from the Carolinas and east Tennessee to Florida and west to Mississippi. In Central and South America, the neotropical species C. peruviana is widespread, while C. azurea is not reported from this region. [1, 6, 21].

In Pacific Asia, blue oil bodies have been identified in C. azurea as well as in C. aeruginosa, C. lunata and C. granulata, but the three latter species clearly differ in the shape of leaves and underleaves and in the structure of the oil bodies [7, 9, 22]. In Asia, C. azurea is restricted to the oroboreal belt of mountain zone, south boreal, hemiboreal and cool temperate zones, and it does not occur in the far north [20, 2324]. Dried specimens, in which oil bodies are absent, may be sometimes mistaken for other Calypogeia species, especially C. muelleriana (Schiffn.) Müll.Frib., C. fissa, C. tosana or C. peruviana [1, 17, 25].

Molecular tools, including fast-growing DNA barcoding proposed by Hebert et al. [26], open new possibilities for identifying plant species based on species-specific differences in short DNA sequences. DNA barcoding is also used to detect genetic diversity and discover new species [27]. Studies where molecular markers were applied to analyze liverwort taxonomy revealed that a morphology-based approach supports only partial detection of species diversity [2833]. Numerous authors have demonstrated that the diversity of liverworts species is underestimated due to hidden genetic diversity [34]. Unrecognized species were discovered especially among liverwort taxa with a world-wide distribution, including Frullania tamarisci [29], Metzgeria furcata and M. conjugata [35], Ptilidium pulcherrimum and P. ciliare [36], Porella platyphylla [30], Cololejeunea lanciloba [37] and Aneura pinguis [3839]. Genetic diversity was also found in Calypogeia species, including C. fissa [40], C. muelleriana [4142] and C. sphagnicola [4344].

The distribution of C. azurea in Holarctic region, its high morphological variability [1, 17], and the results of preliminary population genetic studies [45] support the hypothesis that C. azurea is not genetically homogeneous, but exhibits genetic diversity within its geographical range. In the present study, the DNA sequences of four plastid regions (rbcL, trnG, trnL, trnH-psbA) and one nuclear region (ITS2) were used to assess the genetic diversity of C. azurea from different parts of its distribution range. The study also attempted to determine whether blue oil bodies appeared once or several times during the evolution of the genus Calypogeia.

Materials and methods

Ethics statement

The collection of the material in Vietnam was carried out in accordance with obtained permission for collecting material and transferring of material across national borders provided by the Institute of Ecology and Biological Resources of VAST. The collection of the material in China was accomplished in the course of joint investigation within collaboration project of the Russian Foundation for Basic Researches and Chinese Foundation for Natural Sciences in accordance with permissions for collecting material from the Chinese province of Guizhou and for transfer of the living material across national borders. Suitable permission was also obtained for colleting materials in America. The Polish samples from Tatra and Bieszczady National Parks were collected with the permission given by Ministry of Environment, Department of Nature Conservation and the Directors of these National Parks. For the remaining locations specific permission was not required because species of the genus Calypogeia are neither endangered nor protected species.

Plant material

A total of 40 samples initially identified as C. azurea were examined. Oil bodies were determined in 32 samples of live plants (Fig 1), and they were not identified in 8 herbarium specimens. The analyzed specimens originated from different regions of Europe, North America and Asia. Most samples were collected by authors, and several specimens were obtained from herbarium collections (S1 Table). The specimens of C. azurea were evaluated in molecular studies together with other Calypogeia species harboring blue oil bodies, i.e. C. peruviana, C. granulata, C. aeruginosa and C. lunata, as well as with C. tosana which has colorless oil bodies, but can be easily confused with C. azurea. Most of the Holarctic species with colorless oil bodies, i.e. C. muelleriana, C. integristipula Steph., C. neesiana (Massal. & Carestia) Mull.Frib., C. suecica, C. fissa and C. sphagnicola (Arnell & J.Perss.) Warnst. & Loeske, were used in phylogenetic analyses (Fig 1 and S1 Table).

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Fig 1. Cells of the Calypogeia azurea complex and the related taxa with oil bodies.

(a) C. azurea s. str. (European clade; POZW 41390); (b-c) C. azurea—NA (North American clade; POZW 42444); (d-e) C. orientalis (VBGI P-39-4-15, VBGI P-8-1-16); (f-g) C. sinensis (China-56-78-13, China-56-77-13); (h-i) C. granulata (VBGI V-2-73-16, VBGI China 56-19-13); (j) C. peruviana (POZW 42619); (k) C. aeruginosa (VBGI V-8-75-17); (l) C. tosana (VBGI China-56-86-13). Bar = 50 μm.

https://doi.org/10.1371/journal.pone.0204561.g001

Two species (Calypogeia arguta Ness & Mont and C. sullivantii Austin) from the subgenus Asperifolia were used as an outgroup based on the phylogeny described by Schuster [6]. Although Asperifolia is presently classified as a subgenus of Calypogeia, it is sufficiently genetically distinct to be used as an outgroup for the analyzed species of the subgenus Calypogeia. The sequences of the five studied DNA regions were newly generated for 39–73 specimens, depending on the region. Four DNA sequences of C. azurea were obtained from GenBank, and partial sequences of the remaining species were obtained from our previous studies (GB accession numbers are listed in S1 Table).

DNA analysis

DNA extraction.

Total genomic DNA was extracted from fresh or dried plants. In fresh samples, several stems from one specimen were ground with steel beads in a Bioprep-24 Homogenizer for 35 seconds, and DNA was extracted using the GeneJET Plant Genomic DNA Purification Mini Kit (Thermo Scientific) according to the manufacturer’s protocol. The Novabeads Plant DNA Kit (Novazym, Poland) was used to extract DNA from herbarium samples. The isolated DNA was dissolved in TE buffer and stored at –20°C. The quality of the isolated DNA was evaluated by electrophoresis in 0.8% agarose gel. The concentration and purity of DNA samples were determined using the Epoch Multi-Volume Spectrophotometer System.

PCR amplification and sequencing.

Five DNA regions, including four chloroplast loci (rbcL, trnL, trnG and trnH-psbA) and one nuclear ITS2 region, were sequenced. The standard barcode region [46] was amplified for rbcL [47]. For trnL and trnG, introns were amplified according to Pacak & Szweykowska-Kulińska [48], and intergenic spacer trnH-psbA was amplified according to Sang et al. [49]. The internal transcribed spacer region ITS2 was amplified with the primers described by Sawicki et al. [50]. The same primers were used for the amplification and sequencing (S2 Table). PCR amplification was performed in a total volume of 20 μL, containing 2 μL of 10× PCR buffer with Mg2+ (Novazym; 25 mmol MgCl2), 1 μL of bovine serum albumin (0.25 mg/ml), 200 μmol of each dNTP (Novazym), 0.4 μmol of each primer, 1 U of Taq DNA polymerase (Novazym) and 1 μL of template DNA (about 40 ng). The PCR reaction was performed in the following cycling steps: 4 min of initial denaturation at 94ºC, followed by 30 cycles of 60 s at 94ºC, 30 s at annealing temperature (46–60 ºC, depending on the primer, see S2 Table) and 60 s at 72ºC, with a final extension step of 7 min of 72ºC. Finally, 5 μl of the amplification product was visualized on 1.0% agarose gel by GelView staining, and the remainder was purified with Exo-AP (Thermo Scientific) to remove unincorporated primers and dNTPs. Purified PCR products of the studied DNA regions were sequenced in both directions using the ABI BigDye 3.1 Terminator Cycle Kit (Applied Biosystems) and were visualized with the ABI Prism 3130 Automated DNA Sequencer (Applied Biosystems).

Data analysis.

Chromatograms of DNA sequences were edited and assembled in Geneious R6 (Biomatters, USA). The assembled sequences were aligned with the Muscle algorithm [51] in MEGA 7 [52] using default settings. Regions of ambiguous alignment and incomplete data were excluded from the analysis, and lacking sequences were coded as missing. The differences between the examined specimens were illustrated with the neighbor-joining (NJ) method based on Kimura 2-parametr model [53] for individual and combined DNA regions. Next, phylogenetic trees were generated by maximum likelihood (ML) and maximum parsimony (MP) methods in MEGA 7. First, the analyses were performed for all samples, including samples with incomplete sequences. The analysis was then limited to the accessions with complete sequences of all studied DNA regions. This approach supported evaluations of both old samples that were difficult to sequence as well as GeneBank accessions with missing sequences in some of the analyzed loci. At the beginning, plastid and nuclear loci were analyzed separately. The possible conflicts between the plastid and nuclear data sets were detected in the incongruence length difference (ILD) test [54] with a custom ILD script (http://rossmounce.co.uk/) in TNT 1.1 [5556] with 1000 replicates and default settings for the New Technology Search options. The script is available at http://phylo.wikidot.com/tntwiki#TNT_scripts. We tested for incongruence between data partitions within the chloroplast (4 partitions) and between combined chloroplast and nuclear data sets. The ILD test was run without an outgroup.

In maximum likelihood estimation, the best evolution model for each data set was determined using maximum likelihood model testing and the Bayesian Information Criterion (BIC) in MEGA 7, were discrete gamma distribution was modeled in four categories. The best models for combined plastid and ITS2 data were T92+G and T92, respectively. Maximum Parsimony analyses involved the following tree inference options: Tree-Bisection-Reconnection (TBR) as a search method with 10 number of initial trees (random-addition), search level of 3 and the maximum number of 100 trees to be retained in each step. The confidence of clades within the inferred trees was evaluated by the bootstrap method [57] with 1000 replicates. Genetic distance K2P for the pairs of sequences between and within the studied species was calculated to estimate the degree of evolutionary divergence.

The Automatic Barcode Gap Discovery (ABGD) software was used to group the examined specimens and identify potential hidden species within C. azurea based on pairwise distances by detecting differences between the intraspecific and interspecific variation (barcoding gap) without an a priori species hypothesis. The above program automatically detects the distance where the barcode gap is located, and it can even be used when intraspecific and interspecific distributions overlap to partition the data set into candidate species [58]. The ABGD analyses were performed on a web interface (http://wwwabi.snv.jussieu.fr/public/abgd/abgdweb.html) using all available distance metrics: JC69 [59], K2P and the uncorrected p-distance. Default settings of P value (Pmin = 0.001, Pmax = 0.1) and relative gap width X = 1.5 were used. ABGD relies on a two-steps process. In the first step, the sequences are divided into ABGD units based on a statistically inferred barcode gap as initial partitioning, and in the second step, the same procedure is applied to the groups obtained in the first step to create a recursive partition.

Haplotype networks with the median joining (MJ) option were calculated to examine the variations in the studied C. azurea accessions [60]. The MP option [61] was applied to identify redundant median vectors and links. Haplotype networks were developed in Network 5.0 (Fluxus Technology). Haplotypes were colored according to their geographical origin.

Morphological analysis

The evaluated specimens of the C. azurea complex and the related taxa (S1 Table) were subjected to a morphological analysis. Five stems were collected for analysis from each specimen. Leaves and underleaves were isolated under the Olympus SZX16 dissecting microscope, and digital photographs were taken with the Olympus CX31 translucent microscope with the UC30 camera. Digital photographs were analyzed in Cell D3 Imaging Software (Olympus) to determine morphological variations. In each stem, every trait was measured at least 10 times. Microscopic images of oil bodies were captured in the identified evolutionary lineages of C. azurea (C. azurea s. str., C. azurea–NA, C. sinensis, C. orientalis) and the related taxa. In addition, drawings of gametophytes were made for the newly described species. The new species were described based on fresh specimens with oil bodies. The mean, minimum and maximum values of 47 quantitative traits [62] were computed in STATISTICA 12 (StatSoft. Inc.).

Results

The characteristics of chloroplast and nuclear sequences and incongruence tests

Four plastid regions (rbcL, trnL, trnG, trnH-psbA) and one nuclear region (ITS2) were sequenced. A total of 352 sequences were used in the analysis, including 286 previously unpublished sequences for ingroup and 18 sequences for outgroup. Four sequences were obtained from GenBank and 44 –from our previous studies. DNA sequences revealed that 6 herbarium specimens which were identified as C. azurea without verifying of oil bodies had been misidentified (S1 Table). These specimens were excluded from further analyses. The total alignment of the examined chloroplast regions in the studied Calypogeia species comprised 1814 bp, including 86 variable and 81 parsimony informative sites. The ILD test revealed no conflict between four chloroplast data sets (p = 0.563). The nuclear ITS2 region comprised 513 bp, including 33 variable and 30 parsimony informative sites. The length of the analyzed DNA regions in the studied species, including the numbers of variable and parsimony informative sites, is presented in Table 1. Non-coding loci were more informative. The most parsimony informative locus was trnH-psbA (7.56%) and the least parsimony informative locus was rbcL (3.31%).

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Table 1. The length (in bp) of the analyzed DNA regions in units detected within the C. azurea complex and the related taxa.

https://doi.org/10.1371/journal.pone.0204561.t001

Phylogenetic analysis

A multigene analysis revealed high genetic diversity in plants classified as C. azurea based on the presence of blue oil bodies and morphological features. The phylogenetic analyses (NJ, MP and ML) of the combined plastid loci demonstrated that present C. azurea is a paraphyletic taxon with other species nested in C. azurea accessions. Samples of C. azurea were clustered in four different clades. The remaining species with blue oil bodies were distributed in two clades with C. peruviana and C. granulata in the first clade, and C. lunata and C. aeruginosa in the second clade. However, the second clade was weakly supported due to the presence of accessions with incomplete sequences (Fig 2 and S1 Fig).

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Fig 2. Maximum likelihood consensus tree of the studied Calypogeia species based on a combined chloroplast dataset.

The analysis included all accessions, including those with incomplete sequences. Bootstrap values are given above the branches. Species with blue oil bodies are marked in blue.

https://doi.org/10.1371/journal.pone.0204561.g002

Differences in DNA sequences of Calypogeia azurea

Subsequent analyses were limited to the C. azurea complex and other species with blue oil bodies whose accessions had complete data for all studied DNA regions. Separate analyses of chloroplast and nuclear data revealed several conflicting nodes which were only weakly supported by bootstrap values (Figs 3 and 4). The incongruence between combined plastid and nuclear data sets was confirmed by the ILD test (p = 0.001). The ILD test detected conflict between the nuclear ITS2 region and each of the four plastid regions. The relevant data were not combined, and the analyses were performed separately for chloroplast and nuclear loci.

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Fig 3. Maximum likelihood consensus tree of the Calypogeia azurea complex based on combined chloroplast dataset.

The results of the ABGD analysis are represented by colored stripes on the right side of the diagram. The first bar represents the initial partition, and the second bar represents the recursive partition. Only the accessions containing the sequences of all loci were included in the analysis. The related taxa (C. peruviana, C. granulata, C. tosana) were used for comparison. Calypogeia arguta and C. sullivantii were used as an outgroup. Bootstrap values are given above the branches.

https://doi.org/10.1371/journal.pone.0204561.g003

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Fig 4. Maximum likelihood consensus tree of the Calypogeia azurea complex based on nuclear ITS2 sequences.

The results of the ABGD analysis represented by colored stripes on the right side of the diagram. The first bar of the diagram represents the initial partition, and the second bar represents the recursive partition. Only the accessions containing the sequences of all loci were included in the analysis. The related taxa (C. peruviana, C. granulata, C. tosana) were used for comparison. Calypogeia arguta and C. sullivantii were used as an outgroup. Bootstrap values are given above the branches.

https://doi.org/10.1371/journal.pone.0204561.g004

Two independent main evolutionary lineages were formed based on the combined plastid loci (Fig 3). The first main lineage comprises the European clade of C. azurea, a sister to C. peruviana, Asian-Pacific I clade of C. azurea (described as C. sinensis) and C. granulata. The second main lineage includes the North American clade of C. azurea and Asian-Pacific II clade of C. azurea (described as C. orientalis), which is a sister to C. tosana, with 88–100% bootstrap support. In addition, C. orientalis was divided into two clusters correlated with the geographical region. The first cluster included plants from the Russian Far East and Korea, and the second cluster was composed of plants from Japan (Fig 3). The analysis of the ITS2 region revealed the same four groups of C. azurea, which, however, were bounded by different mutual relationships. The European C. azurea was resolved as a sister of C. granulata; C. sinensis formed a close relationship with C. orientalis, whereas the relationship between the North American clade of C. azurea and C. peruviana remained unresolved. The main differences between the topology of the chloroplast and the nuclear tree was the position of C. azurea KPABG 20–01 and POZW 42447 (Fig 4). These specimens belonged to the European clade and the North American clade of C. azurea, respectively, in the plastid tree, but they formed a separate clade in the nuclear tree.

Each of the four C. azurea evolutionary lineages had specific mutations that distinguished them from each other and from the remaining species with blue oil bodies (S3 Table). In the group of the analyzed C. azurea samples, 11 haplotypes were identified in both the chloroplast and the nuclear genome (Fig 5). The haplotypes were divided into four separate units corresponding to the clades identified within C. azurea in the phylogenetic trees (Figs 3 and 4). All samples from Europe (Poland, Germany, Romania, Austria), and Russia (Caucasus and South Siberia) had the same haplotype of the chloroplast DNA genome. However, only trnL was compared in Russian samples (based on GenBank sequences). Three haplotypes, including two from Poland and one from Russia, were detected in nuclear ITS2. Three haplotypes that differed in single substitutions were identified in the North American clade of C. azurea. Two and five haplotypes were found in C. sinensis and C. orientalis, respectively.

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Fig 5.

A haplotype network of the Calypogeia azurea complex and the related taxa based on combined plastid (A) and ITS2 (B) data. Colored circles represent the geographic origin of the haplotypes. Diameters denote the number of specimens carrying a particular haplotype, where the smallest circle represents a single individual, and the largest circle represents 16 individuals. Black squares represent median vectors.

https://doi.org/10.1371/journal.pone.0204561.g005

The Automatic Barcode Gap Discovery (ABGD) analysis was performed to split the examined specimens of C. azurea into candidate species based on pairwise distances by detecting differences between intraspecific and interspecific variation (i.e. barcoding gap) without an a priori species hypothesis. In the ABGD analysis, data were first divided into groups as the initial partition based on a statistically inferred barcode gap, and the same procedure was then applied to the groups obtained in the first step to form a recursive partition. The ABGD analysis based on the K2P distance model identified four and five units within the examined samples of C. azurea as the initial partition in the combined plastid loci and nuclear ITS2, respectively. This fifth unit in ITS2 comprised one sample from North America (POZW 42447) and one sample from Russia (South Siberia, KPABG 20–01). The units identified in the ABGD corresponded to the groups identified in the phylogenetic analysis (Figs 3 and 4). All examined samples were assigned to the same units as in the phylogenetic analysis. The recursive partitions based on the combined plastid loci produced 5 ABGD units because C. orientalis was split into two groups. In an analysis of individual loci, the examined samples of C. azurea were divided into 3–5 units, depending on the locus (S2 Fig). In the examined DNA regions, C. granulata, C. peruviana, C. lunata, C. aeruginosa and C. tosana formed separate units. Ultimately, four units were identified as the most reliable in the group of the analyzed C. azurea samples, and they corresponded to monophyletic groups in the phylogenetic tree.

The genetic divergence of combined plastid loci between the four lineages of C. azurea identified in the phylogenetic analysis and the ABGD analysis according to the K2P model ranged from 1.03% to 2.17%. Genetic divergence was the lowest (1.03%) between North American C. azurea and C. oreintalis lineages. The highest genetic distances (2.17%) was noted between C. orientalis and C. sinensis. All lineages identified within C. azurea differed from the remaining species, including C. peruviana, C. granulata (blue oil bodies) and C. tosana, and the genetic distances between C. azurea lineages and the above species ranged from 0.73% to 2.23% (Table 2). The lowest genetic distances were noted between the European C. azurea and C. peruviana (0.73%) and between C. orientalis and C. tosana (0.95%). The lineages of C. azurea also differed from C. lunata (3.85–5.07%) and C. aeruginosa (2.42–3.48%); however, only the trnL region was successfully sequenced in the above species. In the nuclear region, genetic differences between C. azurea lineages ranged from 1.00% to 2.48%, and were lowest between the European and North American lineages and highest between North American lineage of C. azurea and C. orientalis (Table 2).

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Table 2. Average genetic differences (%) between the units identified in the C. azurea complex and the related taxa, based on the K2P combined plastid loci (below diagonal) and nuclear ITS2 (above diagonal).

https://doi.org/10.1371/journal.pone.0204561.t002

Morphological differences

The groups identified within C. azurea differed in size and shape of leaves and in the shape of the lateral margin of the underleaf (Table 3, Fig 6, S4 Table, S1 Appendix). All live plants had deep blue oil bodies. The oil bodies of European and North American plants are usually segmented, composed of several 2–4 (up to 9) large and distinct globules, rarely not segmented, 4–7 × 6–12 μm, 2–7 per cell, which are present in all underleaf cells, but are absent in some leaf cells (Fig 1A–1C). The oil bodies in C. azurea were similar to those in C. peruviana (Fig 1J). The oil bodies in C. orientalis were coarsely botryoidal and somewhat similar to those observed in European and North American specimens (Fig 1D and 1E). However, the oil bodies of C. sinensis plants from the Chinese province of Guizhou (China 56-77-13 and China-56-78-13), referred to as C. azurea by Bakalin et al. [63], were quite different from those observed in European, North American and C. orientalis plants. They were composed of numerous small globules and may be called as coarsely granular (Fig 1F and 1G). Interestingly, the dry specimens from North America and China were similar in morphology, but they differed strikingly in the characteristics of oil bodies. A comparison of the species previously referred to as Calypogeia sp. (China-56-19-13; cf. Bakalin et al. [63]) also delivered fascinating results. Based on the results of the DNA sequence analysis, this specimen was included in the C. granulata clade, and it was characterized by gray oil bodies (which is sometimes observed in the species). The oil bodies in the latter species were finely (not coarsely) granular (Fig 1I).

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Fig 6.

Microscopic images of the leaves and underleaves of the Calypogeia azurea complex: C. azurea s. str. (European clade, 1a, 1b –POZW 41390, 2a, 2b –POZW 41746, 3a, 3b –HRE 57); C. azurea—NA (North American clade, 1a, 1b –MO 6005455, 2a, 2b –MO5241997, 3a, 3b POZW 42444); C. sinensis (1a, 1b –VBGI China-56-78-13, 2a, 2b –VBGI China 56-77-13, 3a, 3b –VBGI V-2-73-16) and C. orientalis (1a, 1b –VBGI P-40-1-12, 2a, 2b –VBGI P-39-4-12, 3a, 3b –VBGI J-7-79-14). The samples are numbered in accordance with S1 Table. Bar = 1000 μm for leaves, bar = 500 μm for underleaves.

https://doi.org/10.1371/journal.pone.0204561.g006

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Table 3. Morphological differences between the units detected within the C. azurea complex and the related taxa.

https://doi.org/10.1371/journal.pone.0204561.t003

Taxonomic conclusions and description of new species

The DNA sequence analysis broadened the concept of the Calypogeia azurea complex. Individual clades of C. azurea are more closely related to other recognized species than to each other (Figs 24). The observed level of genetic divergence and the morphological, ecological and geographical differences indicate that these clades can be regarded as previously unrecognized species. The two evolutionary lineages discovered in Pacific Asia differed morphologically and can be described as new Calypogeia species with blue oil bodies. The plants in the Asia-Pacific I clade were described as C. sinensis, whereas the plants in the Asia-Pacific II clade were described as C. orientalis. Due to the lack of diagnostic morphological features, the European and North American lineages, must be temporarily treated as cryptic species of C. azurea. The European plants were named as C. azurea s. str., whereas North American plants as C. azurea species NA.

Calypogeia azurea Stotler & Crotz.

The plants belonging to the European lineage were morphologically similar to the holotype of C. azurea Stotler & Crotz described by Stotler & Crotz [64] based on a specimen of K. Müller from KR herbarium, i.e. Calypogeia trichomanis Corda, Ölkpr. blau! (Auf Erde … am Feldberg, ca 1000 m, 1.5.1946, K. Müller s.n. (KR), Fig 2G–2I) [64]. The examined plants of C. azurea from Europe had cordate to broadly cordate leaves, with usually greater width than length, with a broadly rounded or subacute apex (Fig 6). Underleaves were wider than the stem, suborbicular to ovate, divided by an acute sinus, with lateral margins evenly rounded or rarely with vague angulations (Fig 6). The plants of North American lineage were morphologically similar to the European plants, but the European specimens were larger than North American plants, and the average width of leafy shoots was determined at 2000 and 1678 μm, respectively. The underleaves of European and in North American specimens were wider than the stem at 680 and 580 μm, respectively, on average (Fig 6). Calypogeia azurea was monoicous, mainly autoicous, and often fertile. The studied plants grew mainly on moist humus, peaty, clay or sandy soil, organic debris, rocks with a layer of humus (in Europe) and on decayed logs (in North America). The analyzed specimens were distributed throughout Europe (Poland, Austria, Germany, Romania, European and South Siberian Russia) and North America (Canada—British Columbia, USA—California and Washington). Calypogeia azurea also occurs in the eastern North America, but our samples did not include specimens from this region.

Calypogeia sinensis Bakalin & Buczkowska, sp. nov. (Fig 7).

Description: Plants prostrate, lax and soft, in loose pure mats or with other hepatics or mosses, yellow-brown in a herbarium, blue-green to pale green when alive, 20–50 mm long and 2.0–3.2 mm wide. Rhizoids brownish, erect to obliquely spreading, united in unclear fascicles, rarely separated. Stem rarely to regularly branched, with ventral branches, branch base partly covered by the underleaf, 1(-2) per underleaf. The stem cross-section transversely elliptic, 180–300 × 250–500 μm, with the external wall slightly thickened, cortex not prominent to virtually absent, outer cells on the dorsal side 25–36 μm along the margin (the cells appeared elongated along the margin in cross-section view, and were transversely elliptic in outline), with moderate to small in size trigones and slightly thickened walls (thin walls were rare). Inner cells subisodiametric, 20–50 μm in diameter, thin-walled, with small trigones, ventral epidermal cells similar to dorsal epidermal cells, but cells more or less isodiametric in the cross-section, with thickened and brown-colored walls. Leaves contiguous to loosely imbricate, slightly convex, loosely undulate along the margins, with the apical part turned to the dorsal side, obliquely to subhorizontally inserted, dorsally insertion line arcuate, not decurrent, ovate to widely ovate, apex acute to shortly bidentate, 1.1–1.5 × 1.0–1.5 mm. Underleaves obliquely spreading, transversely elliptic, 300–550 × 400–1000 μm, deeply divided into two triangular lobes, with the lateral side rounded (rarely), crenulate or with distinct but obtuse teeth (then underleaves bisbifid), undivided area between the bottom of the sinus and the initial rhizoid area with 2-3(-4) cell high; lobe apex commonly with obovate slime papilla. Cells in the midleaf shortly oblong to subisodiametric 5-6-gonal, 35–55 × 30–43 μm, trigones absent or vestigial; cells along the leaf margin elongated along margin or isodiametric, 30–50 μm long, cuticle smooth throughout the leaf, oil bodies coarsely granular, deep blue to blue-brown, 3–6 per cell. Asexual reproduction by gemmae at the apices of attenuate tips, spherical unicellular to ellipsoidal bicellular, 20–23 μm in diameter or 20–33 × 20–23 μm, colorless to greenish. Good for holotype: China-56-77-13.

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Fig 7.

Calypogeia sinensis Bakalin & Buczkowska: (a) plant segment, dorsal view; (b-c) plant segment, ventral view; (d) lateral leaves; (e) leaf apex; (f) leaf margin (40×); (g-h) underleaves; (i) stem cross sections (10×); (j) oil bodies (40×). Sample VBGI China-56-77-13.

https://doi.org/10.1371/journal.pone.0204561.g007

Holotype: China. Guizhou Province. Duyun Municipality (26°22’23”N 107°21’21”E), 1300 m alt., VB 22.XI.2013 (China-56-77-13, VBGI, duplicate in POZW).

Paratypes: China. Guizhou Province. Duyun Municipality (26°22’23”N 107°21’21”E), 1300 m alt., VB 22.XI.2013 (China-56-78-13, China-56-96a-13, VBGI, duplicate in POZW).

Vietnam. Lao Cai Province. SaPa, Phan Xi Pan National Park (22°20'18"N 103°46'39"E), 1900–2100 m alt. VB 16.III.2016. (V-2-73-16; VBGI, duplicate in POZW).

DNA sequences: Samples China-56-78-13, China-56-77-13 and V-2-73-16 were used to sequence 4 chloroplast regions (rbcL, trnH-psbA, trnL and trnG) and one nuclear (ITS2) region. The sequences were deposited in GenBank. Accession numbers are given in S1 Table.

Differentiation: In the morphological terms, the most close relative to C. sinensis is C. granulata that moreover has sympatric area with the former species in the Chinese province of Guizhou. The typical C. granulata (including the isotype G00114896!) differed considerably from C. sinensis. The underleaves of C. granulata were narrow (1.5–2.0 times wider than the stem) and obligately bisbifid, whereas in C. sinensis, the underleaves were 2.5–3.5 times wider than the stem, commonly bifid or with smaller additional teeth on each side. Oil bodies were coarsely granular (almost botryoidal) in C. sinensis and finely granular in C. granulata (S1 Appendix).

Ecology: Very little is known about the ecology of C. sinensis. According to the literature, the taxon prefers wet open cliffs and partly shaded cliff caves in the waterfall spray zone, as well as mesic tree trunk bases and decaying wood in broadleaved, evergreen, subtropical to southern subtropical mountane forests.

Distribution: There are only two known localities of the taxon. The first is the subtropical montane forest near Xiniu Waterfall in Chinese province of Guizhou, where the species was collected at the estimated altitude of 1300 m. The second locality is a subtropical montane forest in southern North Vietnam, where the species was collected at the estimated altitude of 2000 m. The taxon probably belongs to the ‘meta-Himalayan’ group of the species [65] that could colonize other parts of the southern-eastern spur of the Tibetan Upland (e.g. Yunnan Province) as well as North Vietnam.

Calypogeia orientalis Buczkowska & Bakalin, sp. nov. (Fig 8).

Description: Plants soft, green to blue-green (blue coloration was particularly visible near the shoot tips) when alive, yellow-green, golden brown to brown-green in a herbarium. Plants grew in dense to loose pure patches or as admixture to mosses and some hepatics. They were 5–30 mm long and 1.5–2.5 mm wide, creeping to loosely ascending near apices. Rhizoids pale brown, obliquely upward spreading (in the direction of the shoot apex, not downward) or erect spreading, mostly united into unclear fascicles. Stems rarely and irregularly ventrally branched; with a transversely elliptic cross-section, 180–220 × 250–300 μm. The cortex not prominent, with epidermal cells on the dorsal side, 15–38 μm in diameter, to the middle, 17–30 μm in diameter; 5-6-gonal, epidermal cells on the ventral side 12–25 μm in diameter; across the entire cross-section, cells thin-walled, and dorsal (rarely ventral) epidermal cells and adjacent cell rows sometimes loosely thickened and brownish in color; trigones small to virtually absent, more pronounced on the dorsal side. Leaves imbricate, obliquely inserted, dorsally insertion line arcuate, not or barely decurrent, leaves nearly plane to slightly concave and loosely canaliculate, with the apex usually slightly turned to the ventral side; widely ovate to slightly obliquely ovate when flattened on the slide -, 0.7–1.2 × 0.7–1.3 mm; the apex acute, sometimes shortly bilobed, rarely rounded. Underleaves loosely appressed to the stem or obliquely spreading, decurrent along 1/3-1/2 of stem width, obliquely elliptic in outline, 200–300 × 400–800 μm, deeply divided into two triangular lobes with obovate slime papillae in the apex, the lateral side entire to loosely crenulate or with an additional obtuse tooth on each side, rarely bisbifid, the undivided portion between the bottom of the sinus and rhizoid initial area with 2-3(-5) cells high. Mid-leaf cells 5-6(-7)-gonal, subisodiametric to shortly oblong, 37–63 × 25–45 μm, thin-walled, trigones vestigial; cells along the leaf margin oblong, 30–48 × 20–30 μm, trigones adjacent to external side, moderate in size and concave; the cuticle smooth across the entire leaf; oil bodies botryoidal, dark blue, 2(-3) per cell, ellipsoidal, irregularly oblong to shortly fusiform, 4–11 × 3–5 μm. Dioicous: only two young archegonia on short ventral branches were observed in the holotype.

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Fig 8.

Calypogeia orientalis Buczkowska & Bakalin: (a) plant segment, dorsal view; (b) plant segment, ventral view; (c) lateral leaves; (d) leaf apex (10×); (e) median leaf cells (10×); (f-i) underleaves; (j-k) stem cross sections (40×). Samples: VBGI P-39-4-12 (parts a, c, d, f, h, i, j, k), VBGI Kor-7-36-11 (parts b, e, g).

https://doi.org/10.1371/journal.pone.0204561.g008

Holotype: Russia. The Russian Far East. Primorsky Territory. Shkotovsky District (43°07’08”N 132°45’45”E), 450 m alt., VB 18.IX.2012 (P-39-4-12, VBGI, duplicate in POZW)

Paratypes: Japan. Fukuoka Prefecture, Tagama-gun (33°28’20”N130°54’07”E), 800–900 m alt., VB 17.III.2014 (J-7-79-14, VBGI, duplicate in POZW).

Republic of Korea. KangWon Province (38°07’40”N 128°26’55”E), 1250 m alt., VB 11.V.2011 (Kor-7-23-11, Kor-7-36-11, VBGI, duplicate in POZW), (38°07’33”N 128°27’22”E), 1550 m alt., VB 12.V.2011 (Kor-10-02-11, VBGI, duplicate in POZW); KyongNam Province (35°19’42”N 127°43’07”E), 1300–1600 m alt., VB 07.V.2015 (Kor-28-1-15, Kor-28-3-15, VBGI).

Russia. The Russian Far East. Primorsky Territory. Khasansky district (43°07’00”N 131°29’15”E), 140 m alt., VB 10.VII.2015 (P-39-4-15, P-39-3-15, VBGI), (43°05’18”N 131°31’19”E), 330 m alt., VB 11.VII.2015 (P-41-8-15, P-41-10-15, VBGI); Shkotovsky District (43°06’57”N 132°47’26”E), 490 m alt., VB & K.G. Klimova 15.X.2016 (Prim-16-10-16, VBGI), (43°07’10”N 132°47’31”E), 550 m alt., VB (P-15-12-12, VBGI, duplicate in POZW), (43°07’08”N 132°45’45”E), 400 m alt., VB (P-40-1-12, VBGI, duplicate in POZW). Sakhalinskaya Province. Iturup island (45°29’46”N 148°49’31”E), 750 m alt., VB 16.IX.2015 (K-77-20-15, VBGI), (45°21’45”N 148°37’13”E), 23 m alt., VB 10.IX.2015 (K-70-3-15, VBGI).

DNA sequences: Samples P-15-12-12, P-39-4-12, Kor-7-23-11, Kor-10-02-11, Kor-7-36-11, J-7-79-14 were used to sequence 4 chloroplast regions (rbcL, trnH-psbA, trnL and trnG) and one nuclear region (ITS2). The sequences were deposited in GenBank. Accession numbers are given in S1 Table.

Differentiation: Calypogeia orientalis is closely morphologically related to C. azurea, but the latter species differed from C. orientalis in the following features: 1) generally wider plants, where well-developed plants had a width of 2.2–2.8 mm wide (smaller if depauperate), 2) more lax-textured plants, with loosely undulate leaves, 3) larger and wider leaves reaching 1.5 × 1.8–2.0 μm in well-developed shoots, 4) larger midleaf cells that commonly reach 52–80 × 45–55 μm, 5) subrounded, not bisbifid underleaf lobe apices, undivided area with 5–6 cells high, size of well-developed underleaves 0.6–0.7 × 0.9–1.0 mm, 6) epidermal cells in the stem cross-section were larger, around 30–45 μm in diameter, inner cells 25–48 μm in diameter, 7) leaves were contiguous, not or very loosely imbricate, 8) leaves subhorizontally (not obliquely) inserted, 9) C. azurea thrives on fine soils along streams or on humificated soil, whereas C. orientalis prefers decaying wood and rarely occurs on soil. The relationships between C. orientalis and C. tosana poses a more intricate question. The two species are the closest genetic relatives despite considerable differences in oil bodies. The oil bodies of C. tosana were colorless, whereas the oil bodies of C. orientalis had a distinct deep blue color. However, oil bodies were absent from dry herbarium specimens, which makes the discussed species difficult to distinguished. The unclear concept of C. tosana further complicates matters. This species is regarded as quite polymorphic, and it has two modifications–with smooth or faintly verruculose cuticle. The analyzed specimens were studied in living conditions and they were characterized by colorless oil bodies (Fig 1l) and smooth cuticle (which was also observed in holotype G00047274/26013!). Therefore, in addition to the color of the oil bodies, as the main distinguishing feature, these two taxa also differed in almost constantly bisbifid underleaves and shortly bilobed leaves in C. tosana (rarely noted in C. orientalis). Thus, the morphological delimitation of these species is difficult, and it could be impossible in the absence of live material.

Ecology: Calypogeia orientalis is an acidophilic mesophyte. It is found mainly on decaying wood in old-growth hemiboreal mixed (coniferous-broadleaved) forests and in corresponding mountain belts. The species spreads widely to oroboreal (corresponding to southern taiga) Abies-Picea forests in the hemiboreal zone. It is less frequently noted in cool-temperate forests, where it is mostly confined to middle mountain belts in the south. The species occurs rarely in upper elevation belts (tundroids, but never true mountain tundra) in the hemiboreal zone, but above the timberline in wet landscapes, commonly surrounded by Pinus pumila (LPall.). In southern Kurils, the species occurs only in Regel thickets. Calypogeia orientalis occasionally colonizes swamps. It is rarely found on fine soils along streams and or on partly shaded moist cliffs in the orotemperate mountain belt of the Korean Peninsula. The species forms pure marts or grows with mosses and other hepatics such as Liochlaena subulata (A.Evans) Schljakov. In swampy habitats, the species sometime overgrows Sphagnum and then associated with Scapania paludicola Loeske et Müll.Frib. In wet fine soils, the species may occurs together with Cephalozia otaruensis Steph.

Distribution: The northernmost localities of the species are known from Southern Kurils, but the species rarely occurred beyond 48°N. The species seems to be more common in the latitude range of 43 to 35°N. The southernmost (confirmed by DNA) locality was determined at 33°N in Hiko-san Mountain, Fukuoka Prefecture, Japan. Site elevation began at near sea level in northern extremes (where the species has been found in localities as high as 750 m), and increased gradually with a decrease in latitude. The highest localities (800–1550 m) were found in the central part of South Korea. According to available data, C. orientalis has not been confirmed in North Korea or north-east China, where it is expected to be found in the future.

Discussion

Molecular evidence for the presence of unrecognized species within C. azurea

Live specimens of C. azurea are easy to identify based on the presence of blue oil bodies. Another characteristic feature are turquoise-blue shoot apices, which make C. azurea easy to recognize at a glance in the field. Due to the ease of identification, C. azurea was not considered as a critical species that required special attention. However, due to high morphological variation within C. azurea, herbarium specimens lacking oil bodies were frequently confused with other species, e.g. C. fissa, C. muelleriana or C. tosana [1, 17, 66].

The results of our study revealed high genetic diversity of C. azurea in the present taxonomic status. The phylogenetic trees constructed based on both combined plastid loci (rbcL, trnL, trnG, trnH-psbA) and the nuclear ITS2 region revealed that other Calypogeia species are nested within the examined C. azurea accessions (Figs 24). The samples that were initially identified as C. azurea were split into four well-supported clades (BS 88–100%) that correlate with geographic distribution. European, North American, and two (I and II) Asian-Pacific evolutionary lineages were distinguished. The same four lineages were detected in the ABGD analysis with the use of the software developed by Puillandre et al. [58], which groups examined samples into several ABGD units (hypothetical species) with the use of pairwise differences. Identical four groups were obtained in network analysis. The genetic divergence between the four lineages of C. azurea ranged from 1.03% to 2.17%. The lowest divergence (1.03%) was noted between the North American lineage (C. azurea—NA) and C. orientalis. Although the divergence between the detected groups did not exceed 2% or 3%, which is often proposed as a threshold in comparisons of congeneric species pairs in barcoding studies [58, 67], we think that the four evolutionary lineages of C. azurea should be considered as separate species. Their species rank is supported by the presence of other species nested between the four C. azurea lineages. Phylogenetic reconstruction suggests that blue oil bodies in Calypogeia appeared at least twice during its evolution (Fig 2).

The relationship between European and North American C. azurea

The morphological features of European plants correspond to the C. azurea holotype described by Stotler & Crotz [64] based on K. Müller specimens. The holotype originated in Germany, therefore, the name C. azurea Stotler & Crotz can only be assigned to the European lineage. The North American lineage is sufficiently genetically different to be distinguished as a separate species and provided with a new name; however, the species cannot be formally described due to the lack of diagnostic morphological features (Fig 6). These plants also had a similar structure of oil bodies (Fig 1A–1C). They differed only in size and ecological preferences. Further research involving more material is needed to describe the morphological features for identifying the discussed species. For this reason, the North American species has to be temporarily named as C. azurea species NA.

The genetic distances between the two lineages of C. azurea in the examined plastid DNA regions was 1.55%. This value approximates the differences reported in other accepted Calypogeia species, including C. azurea and C. sphagnicola [43]. Clear genetic differences between the North American and European lineages of C. azurea were also found in isozyme markers, with limited gene flow between the populations originating from North America and Europe [45]. The existing data also indicate that the species occurred on different substrates. The DNA sequences of the above lineages of C. azurea differed from those of C. peruviana, the second North American species with blue oil bodies which, according to Schuster [1], is sometimes difficult to distinguish based on morphological features. It is worth noting that in terms of chloroplast genome sequences, C. peruviana is a sister group to the European lineage of C. azurea, and the genetic divergence between these species was lowest among all compared species pairs at 0.73% (Table 3). This relationship was not confirmed by ITS2 phylogeny due to the lack of resolution at the backbone. There is a certain degree of discordance in the relationships between the groups distinguished based on plastid and nuclear markers, and the most greatest differences were noted in the location of two C. azurea samples (KPABG 20–01 and POZW 42447) as well as in the phylogenetic relationships between both clades (European and North American) of C. azurea and C. peruviana. Sample KPABG 20–01 had the European haplotype and sample POZW 42447 had the North American haplotype of chloroplast markers, but they were located in a new clade in ITS2 phylogeny. According to Soltis & Soltis [68], topological incongruence between plastid and nuclear phylogeny can be caused by hybridization. Cytological and isozyme studies have demonstrated that European plants of C. azurea have allopolyploid origin [6970], therefore, the obtained results could suggest that C. peruviana and the European C. azurea inherited chloroplasts from a shared parent, whereas the North American and European lineages of C. azurea most likely had different parents as donors of their chloroplast genomes. The European and North American C. azurea most probably evolved during two independent hybridization processes, which would confirm their status of separate species. However, the number of chromosomes (n = 18) was determined only in European material [6971], and the polyploidy status of North American plants remains unknown. The existing data do not support the reconstruction of the hybridization process. Nevertheless, the observed differences in the DNA sequences of European and North American lineages indicate that they are separate species. However, due to the lack of reliable diagnostic morphological features, they are treated as the cryptic species of C. azurea.

Differences between C. azurea and new described species

The two evolutionary lineages discovered in Pacific Asia, C. orientalis and C. sinensis, can be described as new Calypogeia species. Genetic divergence between these species was determined at 2.17%. In terms of DNA sequences, both new species also differed from C. peruviana, C. granulata, C. lunata and C. aeruginosa, other Calypogeia species with blue oil bodies [7, 9, 22], and from the compared species of C. tosana (Figs 24, S1 and S2 Figs). Genetic distances between the newly recognized species ranged from 0.95% (between C. orientalis and C. tosana) to 2.23% (C. sinensis and C. tosana). Further evidence supporting the species rank of C. sinensis and C. orientalis comes from morphological studies, which revealed compatibility between the genotype and the phenotype. A detailed analysis of the samples identified by barcode markers demonstrated clear morphological differences between the distinguished lineages of C. azurea that had been previously overlooked. Both new species (C. sinensis and C. orientalis) can be distinguished from one another and from the European and North American lineages of C. azurea based on the presence of diagnostic traits, both morphological and those related to oil bodies. The major diagnostic features were the shape and the size of leaves and underleaves (Figs 68, S4 Table). Differences in ecological preferences provided additional evidence for the separateness of the taxa. Calypogeia orientalis occurs mainly on decaying wood in old-growth hemiboreal mixed forests (coniferous-broadleaved) at lower altitudes in the north, and in the oro-hemiboreal mountain belt in the south. Calypogeia sinensis generally occupies different habitats than both C. azurea s. str. and C. orientalis. It is an element of evergreen montane subtropical forests, where it colonizes wet open cliffs and partly shaded cliff caves. Very little is known about the ecology of C. sinensis, but one of authors (VB) never observed “C. azurea” s.l. growing on decaying wood in southern China or North Vietnam. Both new species occupy different habitats than the European C. azurea, which grows mainly on moist humus, peaty, clay or sandy soil, organic debris and rocks with a layer of humus, and the North American C. azurea which colonizes decayed logs. In addition, the genetic diversity of C. azurea was also confirmed by differences in the chromosome number which was determined at n = 18 and n = 9 in European and Asian C. azurea, respectively [69, 7276].

Differentiation within species

The four identified lineages of C. azurea differed in the level of genetic variation. European plants did not differ in plastid DNA sequences. The samples of C. azurea originating from different parts of Europe shared one haplotype of all analyzed plastid DNA regions. The sample from the Republic of Buryatiya (KPABG 20–01) had the same haplotype as European plants, but only the sequence of one DNA region (trnL) was available in GenBank. The sample differed in 9 substitutions in the ITS2 sequence. Unlike European specimens, the samples from North America were characterized by 0.12% variation within the lineage, with three different haplotypes in plastid loci and two haplotypes in ITS2. Similar differences between the North American C. azurea were found in isozyme markers [45]. Intraspecific variability was also detected in the two new species, C. sinensis (0.18%), and C. orientalis (0.27%). Within C. orientalis, two sister lineages were observed in the phylogenetic tree. The first consisted of plants from Korea and the Russian Far East, and the second comprised plants from Japan. Our results confirm the observations made by [8] who found that the speciation of the genus Calypogeia occurred on a very regional scale. According to Inoue [8], most of the Japanese species of Calypogeia, including C. nasuensis, C. asakawana, were restricted to very small areas. On the other hand, most European species had much broader ranges [1, 16, 18], which could be attributed to extensive shield glaciations at the end of the Pleistocene which contributed to the homogeneity of the local flora. Our results suggest that only one haplotype of C. azurea could be distributed throughout Europe, whereas several haplotypes are distributed in the Pacific East Asia.

Geographic distribution

To date, C. azurea was generally regarded as a species of the Holarctic range, and it was reported from various parts of the northern hemisphere, from North America throughhout Europe to the Far East [1, 16, 18, 20, 63]. Our results revealed that plants corresponding to C. azurea according to Stotler & Crotz [64] had a more restricted geographical distribution than previously thought. All the species which emerged after the splitting of C. azurea s.l. have allopathic ranges. The actual C. azurea s. str. (corresponding with type) occurs in Europe, whereas the morphologically similar cryptic species provisionally named as C. azurea species NA occurs in the Pacific Northwest. The geographical range of the newly recognized C. orientalis species is probably restricted to hemiboreal and temperate Pacific East Asia. It covers the southernmost flank of the Russian Far East, Korean Peninsula and Japanese archipelago, where insular populations are somewhat different from the continental populations. The distribution of C. sinensis is probably confined to south-eastern Tibetan Plateau in southern China and Vietnam. The existing data indicate that the four species separated from the former C. azurea s.l. had allopatric distribution. The geographic distribution of C. azurea and C. orientalis could potentially coincide in south-eastern Siberia, although the odds are relatively low. There are no reports of C. azurea in the Upper Amur region, also in available herbarium collections from this region. The distribution pattern of many Euro-Siberian taxa, including Pinus silvestris L., is similar, and their geographic range ends in the Upper Amur region and does not continue eastward towards the true East Asian communities. There is growing molecular evidence to suggest that the morphologically defined liverwort species are, in fact, composed of several unrecognized species with a more limited geographic distribution, including Conocephalum conicum [77], Pellia epiphylla [78], Corsinia coriandrina [79], Frullania tamarisci [29], Porella platyphylla [30] and species of the genera Herbertus [32] and Plagiochilla [34]. In some cases, species morphology was re-examined based on the results of recent genetic research, which led to the identification of new species, or the revision of the existing species, such as Conocephalum conicum and C. salebrosum [77], Herbertus borealis and H. norenus [32].

Relationship between newly distinguished and other Calypogeia species

Our studies revealed a close relationship between C. tosana and the newly distinguished species C. orientalis and C. azurea—NA. Genetic divergence between these species in the examined DNA regions is relatively low (0.95% and 1.10%, respectively). However, both new species differ distinctly from C. tosana in the color and structure of oil bodies (colorless and granular in C. tosana, while blue and composed of distinct globules in C. azurea—NA and C. orientalis), which argues for keeping the rank of species for all three taxa. In liverworts, the structure and color of oil bodies are considered a strong and one of the most important taxonomic features used for the delimitation of species. It should be emphasized, however, that the correct delimitation of these species is possible only in the living state, so the species must be identified before drying the plants. However, our results indicate that C. tosana requires further molecular and morphological studies.

The relationship between European C. azurea and C. peruviana has not been resolved either. The species were characterized by low (0.73%) genetic divergence in the analyzed DNA regions, and the question that needs to be answered is whether these differences are sufficient to regard both taxa as separate species. The species status of C. azurea and C. peruviana was supported by differences in isozyme markers, especially the presence of fixed heterozygotes [45, 70] which point to their allopolyploid origin. In this study, C. peruviana material was very limited, which prevented an evaluation of its genetic structure. For this reason, we decided to maintain the species rank for both taxa at the current stage of research. The results of molecular analyses indicate the need for a general revision of the genus Calypogeia.

Supporting information

S1 Fig.

Neighbor joining (A) and maximum parsimony (B) consensus trees of the Calypogeia azurea complex. The analyses were performed separately for a combined plastid loci and nuclear ITS2. Only the accessions with complete sequences of all loci were included in the analysis. The related taxa (C. peruviana, C. granulata, C. tosana) were used for comparison. Calypogeia arguta and C. sullivantii were used as an outgroup. Bootstrap values above 50% are given above the branches.

https://doi.org/10.1371/journal.pone.0204561.s001

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S2 Fig. Neighbor-joining trees of the Calypogeia azurea complex.

The results of the ABGD analysis of individual loci are represented by colored stripes on the right side of the diagram. The first bar of the diagram represents the initial partition, and the second bar represents the recursive partition. The related taxa (C. peruviana, C. granulata, C. aeruginosa, C. lunata and C. tosana) were used for comparison. Calypogeia arguta and C. sullivantii were used as an outgroup. Bootstrap values above 50% are given above the branches.

https://doi.org/10.1371/journal.pone.0204561.s002

(PDF)

S1 Table. Collection details and GenBank accession numbers of the studied Calypogeia samples.

*Samples from the herbarium collection, a-c references to GenBank sequences. Oil bodies 1 –based on the authors’ observations, 2 –based on herbarium data. Shaded lines indicate GenBank samples, and shaded cells indicate sequences from the authors’ previous work.

https://doi.org/10.1371/journal.pone.0204561.s003

(DOCX)

S2 Table. Primer sequences for amplification of Calypogeia specimens in the present study.

https://doi.org/10.1371/journal.pone.0204561.s004

(DOCX)

S3 Table. Species specific sites for C. azurea s. str., C. azurea–NA, C. sinensis and C. orientalis.

The related taxa (C. peruviana, C. granulata, C. tosana) were used for comparison.

https://doi.org/10.1371/journal.pone.0204561.s005

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S4 Table. Descriptive statistics for C. azurea s. str., C. azurea–NA, C. sinensis and C. orientalis.

The means, minimum and maximum values of 47 quantitative traits were calculated according to Buczkowska (2004), N—number of measurements.

https://doi.org/10.1371/journal.pone.0204561.s006

(PDF)

S1 Appendix. The key to Calypogeia with blue oil bodies.

https://doi.org/10.1371/journal.pone.0204561.s007

(PDF)

Acknowledgments

The authors are deeply indebted to Mr. M.A. Bakalin who kindly prepared line drawings for the new species described in the study. The authors would like to thank the Institute of Ecology and Biological Resources of the VAST for assistance in obtaining permission for collecting material in Vietnam and transporting the specimens across national borders. The authors are grateful to Prof. Y. Xiong (Guizhou University) for preparing the permissions for transporting live specimens across national borders and for organizing a field trip in the Guizhou Province. The authors would like to thank supervisor Dr. V.S. Nguyen for his support. The authors are also grateful to A. Schäfer-Verwimp and T. Pócs for providing plant material. We would like to thank the management of Tatra and Bieszczady National Parks for their support during field work and the Curators of KRAM, NYBG, MO, JE, G and GOET herbaria for providing Calypogeia specimens and type materials. Molecular analyses were conducted by the Molecular Taxonomy and Genetics team.

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