2013 Stech PLOS ONE Barcoding Racomitrium canescens

Molecular Species Delimitation in the Racomitrium canescens Complex (Grimmiaceae) and Implications for DNA Barcoding of Species Complexes in Mosses Michael Stech1*, Sarina Veldman1, Juan Larraı́n2, Jesús Muñoz3,4, Dietmar Quandt5, Kristian Hassel6, Hans Kruijer1 1 Naturalis Biodiversity Center, Section National Herbarium of the Netherlands, Leiden University, Leiden, The Netherlands, 2 Departamento de Botánica, Universidad de Concepción, Concepción, Chile, 3 Real Jardı́n Botánico, Consejo Superior de Investigaciones Cientı́ficas (RJB-CSIC), Madrid, Spain, 4 Centro de Investigación de la Biodiversidad y Cambio Climático, Universidad Tecnológica Indoamérica, Quito, Ecuador, 5 Nees-Institute for Plant Biodiversity, Rheinische Friedrich-Wilhelms-Universität, Bonn, Germany, 6 Museum of Natural History and Archaeology, Norwegian University of Science and Technology, Trondheim, Norway Abstract In bryophytes a morphological species concept is still most commonly employed, but delimitation of closely related species based on morphological characters is often difficult. Here we test morphological species circumscriptions in a species complex of the moss genus Racomitrium, the R. canescens complex, based on variable DNA sequence markers from the plastid (rps4-trnT-trnL region) and nuclear (nrITS) genomes. The extensive morphological variability within the complex has led to different opinions about the number of species and intraspecific taxa to be distinguished. Molecular phylogenetic reconstructions allowed to clearly distinguish all eight currently recognised species of the complex plus a ninth species that was inferred to belong to the complex in earlier molecular analyses. The taxonomic significance of intraspecific sequence variation is discussed. The present molecular data do not support the division of the R. canescens complex into two groups of species (subsections or sections). Most morphological characters, albeit being in part difficult to apply, are reliable for species identification in the R. canescens complex. However, misidentification of collections that were morphologically intermediate between species questioned the suitability of leaf shape as diagnostic character. Four partitions of the molecular markers (rps4-trnT, trnT-trnL, ITS1, ITS2) that could potentially be used for molecular species identification (DNA barcoding) performed almost equally well concerning amplification and sequencing success. Of these, ITS1 provided the highest species discrimination capacity and should be considered as a DNA barcoding marker for mosses, especially in complexes of closely related species. Molecular species identification should be complemented by redefining morphological characters, to develop a set of easy-to-use molecular and non-molecular identification tools for improving biodiversity assessments and ecological research including mosses. Citation: Stech M, Veldman S, Larraı́n J, Muñoz J, Quandt D, et al. (2013) Molecular Species Delimitation in the Racomitrium canescens Complex (Grimmiaceae) and Implications for DNA Barcoding of Species Complexes in Mosses. PLoS ONE 8(1): e53134. doi:10.1371/journal.pone.0053134 Editor: Dmitry A. Filatov, University of Oxford, United Kingdom Received February 10, 2012; Accepted November 26, 2012; Published January 14, 2013 Copyright: ß 2013 Stech et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: MS and HK received financial support from the European Centre for Arctic Environmental Research (projects ARCFAC-026129-2008-31, ARCFAC026129-2009-123) and the Dutch Organization for Scientific Research (NWO, project ALW-NAP/08-01). JM received financial support from the Ministry of Science and Technology of Spain (grant CGL2009-09530-BOS). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: [email protected] populations that are morphologically distinguishable from other groups [1]. DNA sequence analyses allow testing the morphological species circumscriptions and providing new insights into species relationships. An increasing number of studies of bryophyte species, especially moss species, has already revealed incongruence between morphological species circumscriptions and molecular data (see [1,2] for review). In particular, heterogeneity between rates of molecular versus morphological evolution seems to be evident in bryophytes, partly leading to a hidden molecular diversity and cryptic speciation (e.g. [3,4]). DNA sequence analyses can provide new tools for species identification as well by comparing sequences from unidentified specimens with a reference database of sequences of identified specimens (DNA barcoding), which can also be used by researchers not specialized in bryology. Although rbcL and matK were advocated as core DNA barcoding markers for land plants [5], the identification success Introduction Mosses (Bryophyta) represent the most species rich of the three lineages of bryophytes, and the second most species rich lineage of land plants after angiosperms [1]. Mosses contribute significantly to the biodiversity of terrestrial ecosystems. For example, species of the moss genus Racomitrium s.l. (Grimmiaceae, Bryophyta) are important components of many terrestrial and saxicolous habitats, from coastal sand dunes to mountain ecosystems to the arctic tundra. However, ecological and biodiversity research aiming at including bryophytes is often hampered by unclear species circumscriptions and identification difficulties of bryophyte taxa based on morphological characters. This is especially true for complexes of closely related, morphologically similar species. In bryophytes, a morphological species concept is still most commonly employed, i.e., species are groups of individuals or PLOS ONE | www.plosone.org 1 January 2013 | Volume 8 | Issue 1 | e53134 Species Delimitation in Racomitrium canescens studies was too limited to infer species delimitations within the R. canescens complex with confidence. In the present study, molecular species delimitations and relationships in the R. canescens complex are assessed based on accessions from all species of the complex, including R. varium. According to previous analyses [9,14,15], the plastid (cpDNA) rps4-trnT-trnL and nuclear ribosomal ITS regions were chosen as most promising markers in terms of potential sequence variability and sequencing success. We aim to infer whether (i) the morphological taxa of the R. canescens complex can be distinguished at the molecular level and whether the degree of genetic differentiation supports their recognition at the species level, (ii) morphological characters used for species identification are suitable in the light of the molecular data, and (iii) intraspecific molecular diversity corresponds to hitherto recognized intraspecific taxa. We will discuss which part of the sequenced DNA regions is most suitable for molecular species identification and the implications thereof for DNA barcoding of mosses, in particular in complexes of closely related species. using these two markers has been demonstrated to be below 70% in angiosperms (e.g. [6]). Therefore, other potential barcoding markers need to be tested and discussed, especially for bryophytes (e.g. [7–11]), which have lower molecular rates compared to angiosperms [12]. The taxonomy of the genus Racomitrium exemplifies the need of testing morphological taxon circumscriptions by molecular data. Morphologically distinguishable groups have been treated either at different taxonomic levels within a broadly defined genus Racomitrium, or as four separate genera [13], which led to considerable changes in species names. For example, the R. canescens species complex was treated as genus Niphotrichum [13]. First molecular phylogenetic reconstructions, however, supported a monophyletic Racomitrium clade [14,15]. The Racomitrium canescens complex is of Holarctic distribution and widespread in most parts of the northern temperate to arctic zones. It is easily distinguished from the other Racomitrium species by a combination of morphological characters, which include strongly papillose laminal cells with the tall papillae situated over the cell lumina, very long peristome teeth that are regularly cleft to base in 2–3 filiform prongs, and hyaline alar cells forming often decurrent auricles. The extensive morphological variability within the complex has led to different opinions in the older literature about the number of species and intraspecific taxa that should be distinguished (cf. [16,17]). Consequently, Frisvoll [17] aimed at finding stable morphological characters that should represent different species, and distinguishing these characters from environmental modifications. Based mainly on leaf characters (leaf shape, hairpoint morphology, nerve length, alar cells and basal marginal leaf cells), Frisvoll [17] carried out a comprehensive taxonomic revision, in which eight species in two subsections were recognised, a classification still accepted to date (e.g. [18]). Frisvoll [17] argued that his morphologically defined species represent different genotypes, due to the frequent occurrence of mixed populations of two or more types of morphologically distinguishable plants (for diagnostic morphological characters of the species see [17,18]). Four species are widespread across the Holarctic, namely R. canescens s.str., R. elongatum, R. ericoides, and R. panschii, the latter being mainly confined to the Arctic, whereas the other four species occur mainly in western North America (R. pygmaeum, R. muticum) or East Asia (R. barbuloides, R. japonicum), respectively. In addition, Frisvoll [17] distinguished two subspecies within R. canescens s.str., subsp. canescens and subsp. latifolium. They are found in mixed populations in an overlapping part of their otherwise separated distribution areas, but are morphologically intergrading and were therefore not considered separate species by Frisvoll [17]. The available taxonomic revisions and floristic treatments (e.g. [16–18]) provided a sound basis for testing morphological species circumscriptions in the R. canescens complex. Molecular phylogenetic reconstructions supported the monophyly of the complex [15,19] and indicated that another species, the western North American R. varium, belongs to the complex as well [15]. The latter was considered to belong to the Racomitrium segregate Codriophorus [13], which differs from Niphotrichum by having leaf papillae situated over the cell walls, undifferentiated or coloured alar cells, and shorter peristome teeth. In R. varium, however, the peristome teeth are long, which supports its position in the R. canescens complex. A clade composed of two species, R. fasciculare and R. laevigatum, was resolved as sister group of the R. canescens complex [15,19]. A first case study to evaluate molecular markers for species identification in Grimmiaceae indicated that DNA barcoding can facilitate species identification in the R. canescens complex [9]. However, the taxon sampling of these molecular PLOS ONE | www.plosone.org Results The rps4-trnT-trnL region could be amplified and sequenced from 68 out of the 70 newly analyzed Racomitrium specimens. From two specimens only the rps4-trnT part could be amplified. The complete ITS region (ITS1-5.8S-ITS2) was obtained from all 70 specimens, except that in one specimen the 59 end of ITS1 remained incomplete. Considering also the six additional specimens which could not or only partially be amplified and were excluded from further analyses, amplification and sequencing success was 90% for the complete rps4-trnT-trnL region and 92% for ITS. The alignment of the rps4-trnT-trnL region comprised 992 positions and included the 39 end of the rps4 gene (positions 1– 124), rps4-trnTUGU intergenic spacer (125–462), trnTUGU gene (463–536), trnTUGU-trnLUAA spacer (537–870), trnLUAA 59 exon (871–905), and the 59 part of the trnLUAA intron (906–992). The nrITS alignment comprised 1418 positions and included the internal transcribed spacer 1 (positions 1–817), 5.8S rRNA gene (818–975), internal transcribed spacer 2 (976–1409), and the 59end of the 26S rRNA gene (1410–1418). Parsimony-informative positions were 48 in the plastid region (26 substitutions/22 indels coded by simple indel coding [SIC]) and 308 (134/174) in ITS, resulting in a total of 356 parsimony-informative positions including indels. Ranges of intraspecific versus interspecific pairwise nucleotide distances according to the Kimura 2-parameter (K2P) model overlapped in the combined dataset and the ITS partitions (Table 1). This was due to small interspecific distances between R. muticum and R. pygmaeum on the one hand and rather large intraspecific distances within R. japonicum on the other. To further compare the different partitions, maximum intraspecific divergences were plotted against minimum interspecific divergences for all possible species pairs of the five species with more than one specimen sequenced (R. canescens, R. elongatum, R. ericoides, R. japonicum, R. muticum). The resulting graphs (Fig. 1A–F) showed that interspecific divergences were clearly greater than intraspecific variation except for few pairwise comparisons in trnT-trnL and ITS2, i.e., that a barcoding gap was present (data points above the 1:1 line). Tables of all nucleotide distances measured are available on request. Significant p-values were obtained for all pairwise comparisons of Fst estimates based on plastid and ITS haplotypes for the the five species with more than one specimen sequenced (Table 2), except for ITS of R. japonicum–R. panschii. 2 January 2013 | Volume 8 | Issue 1 | e53134 Species Delimitation in Racomitrium canescens Table 1. Intra- versus interspecific pairwise distances of rps4-trnT-trnL and ITS sequences in the Racomitrium canescens complex. combined rps4-trnT-trnL rps4-trnT trnT-trnL ITS ITS1 ITS2 intra 0–0.0068 0–0.0011 0 0–0.0025 0–0.0135 0–0.0173 0–0.0223 inter 0.0056–0.0511 0.0022–0.0132 0.0023–0.0161 0.0025–0.0151 0.0094–0.0913 0.0149–0.1686 0.0069–0.1006 overlap 0.0012 0 0 0 0.0041 0.0024 0.0154 Kimura 2-parameter (K2P) distances are shown for the combined molecular markers and different partitions thereof. The upper two rows indicate the ranges of intraspecific and interspecific distances for all eight species of the R. canescens complex plus R. varium. The last row indicates the overlap between the maximum intraspecific and minimum interspecific distances. doi:10.1371/journal.pone.0053134.t001 canescens, namely R. barbuloides (R. canescens var. epilosum H. Müll. ex Milde, fide [20]), R. ericoides, and R. muticum [17,18], and the species newly described by Frisvoll [17], viz. R. elongatum and R. pygmaeum, are molecularly clearly separated from R. canescens s.str. Despite low phylogenetic distances in the R. canescens complex, genetic differentiation between species is generally larger than within species, as inferred from the generally smaller intraspecific than interspecific distances (Fig. 1, branch lengths in Fig. 2) and the significant pairwise Fst comparisons for five species of the complex (Table 2). It has to be noted, however, that the Fst estimates are based on a small sampling and only a small part of the genomes with few haplotypes. Furthermore, no incongruence between the plastid and ITS sequences was found, rejecting the occurrence of hybridization in the present dataset. The overlap in the total sequence distance ranges (Table 1) is due to an exception of the general observation of smaller intraspecific than interspecific distances, as the molecular divergence in ITS between R. muticum and R. pygmaeum is smaller than the intraspecific divergence within R. japonicum. It could therefore be argued that the former two should be treated as one species, whereas R. japonicum should be split into two. However, R. muticum and R. pygmaeum are easily distinguishable by morphological characters [15–18]. Racomitrium japonicum, in contrast, shows only little morphological variation, and if split, distinguishing the two segregate taxa might be only possible by molecular characters and possibly a geographic separation (China versus Japan, cf. Fig. 2). This needs to be tested by analyzing a larger number of specimens from the entire East Asian distribution area of R. japonicum. Based on the present inferences we argue that the molecular data support Frisvoll’s [17] thorough revision and that his morphological species circumscriptions should be maintained, as they correspond to well-supported clades in the molecular phylogenetic reconstructions. Molecular species delimitation in the R. canescens complex is thus straightforward, in contrast to several other genera of liverworts and mosses analyzed recently, where the presence of para- or polyphyletic species, cryptic speciation, incongruence between molecular markers, or incongruence between molecular and morphological characters complicated species delimitation (e.g. [2,4,21–24]). In line with distinguishing the taxa of the R. canescens complex at the species level, intraspecific molecular divergence could be treated taxonomically at the subspecies level, especially when groups of specimens form at least moderately supported subclades, such as within R. canescens, R. ericoides, and R. japonicum (Fig. 2). In fact, one of the subclades within R. canescens corresponds to R. canescens subsp. latifolium (Fig. 2), which is morphologically and geographically separated from R. canescens subsp. canescens, although both morphological characters and the distribution range overlap between the two subspecies [17]. The taxonomic status of other intraspecific clades that are molecularly distinguishable within the widespread Racomitrium species remains to be Separate phylogenetic reconstructions under maximum parsimony (MP) of the rps4-trnT-trnL versus ITS sequences and of the four smaller partitions (rps4-trnT, trnT-trnL, ITS1, and ITS2) resulted in differently resolved trees, but did not show incongruence with respect to significantly supported clades (Supporting Figs. S1, S2, S3, S4, S5, S6). The partition homogeneity test (ILD test) between the ITS and plastid alignments did not indicate the presence of incongruence (P = 0.8). Bootstrap support values of the clades of the Racomitrium canescens complex, including R. varium, which were obtained in the separate analyses, are compared in Table 3. In the MP-PRAP analyses of the combined dataset, 525 trees (lengths 351 steps, consistency index CI = 0.815, retention index RI = 0.964) were retained without indels and 1113 trees (lengths 750, CI = 0.700, RI = 0.953) with indels coded by SIC included. A maximum likelihood calculation recovered a single optimal tree (lnL = 25429.03137), which is depicted in Fig. 2, with statistical support from MP analyses (bootstrap support values, BS) and Bayesian inference (BI; posterior probability values, PP), both without and with indels, indicated. As shown in Fig. 2, the Racomitrium canescens complex, including R. varium, is well supported in all MP and BI analyses. Within the complex, all species with more than one specimen included (R. canescens, R. elongatum, R. ericoides, R. japonicum, and R. muticum) form clades with significant statistical support. The relationships of R. barbuloides as sister to R. japonicum, R. varium as sister to these two species, and R. pygmaeum as sister to R. muticum receive significant support as well. Relationships between these clades and the other species, i.e., the backbone of the phylogenetic reconstruction, however, remain unsupported or receive significant support in the Bayesian analyses only. Intraspecific variation is observed in R. canescens s.str., R. ericoides, and R. japonicum, whereas sequences are almost or even completely identical in R. elongatum and R. panschii. One clade within R. canescens s.str. corresponds to R. canescens subsp. latifolium. Discussion Species circumscriptions and relationships in the Racomitrium canescens complex All eight morphologically defined species of the R. canescens complex plus R. varium are easily distinguishable from each other based on the combined rps4-trnT-trnL and ITS sequence data (Fig. 2). Much confusion about species delimitations in the R. canescens complex arose from early taxonomic attempts to subdivide R. canescens s.l. into various varieties or forms (overview in [17]), which were often based on environmental modifications of morphological characters only. Frisvoll [17] emphasized the frequent presence of mixed populations (‘mixed stands’) of morphologically distinguishable plants, which indicated that a number of genetically distinct taxa do exist in the R. canescens complex. In fact, the species formerly treated as varieties of R. PLOS ONE | www.plosone.org 3 January 2013 | Volume 8 | Issue 1 | e53134 Species Delimitation in Racomitrium canescens Figure 1. Sequence divergence percentages between species pairs in the Racomitrium canescens complex. Comparison of maximum intraspecific versus minimum interspecific divergence percentages for species pairs of five species (R. canescens, R. elongatum, R. ericoides, R. japonicum, R. muticum) with more than one specimen sequenced for the plastid rps4-trnT-trnL region (A) and its partitions rps4-trnT (B) and trnT-trnL (C) as well as the nrITS region (D) and its partitions ITS1 (E) and ITS2 (F). doi:10.1371/journal.pone.0053134.g001 tested. As in R. japonicum, subclades in R. canescens seem to be geographically separated (circum-North Pacific, western North America, Europe), which would provide support for treating them as separate subspecies in addition to subsp. latifolium, but more specimens, especially from outside Europe, need to be analyzed. At the supraspecific level, the present molecular data do not support the division of the R. canescens complex into two groups of PLOS ONE | www.plosone.org species, which were formerly treated as Racomitrium subsections Canescens and Ericoides [17] or Niphotrichum sections Niphotrichum and Elongata [18], respectively. According to these classifications, R. canescens s.str. and R. panschii should be closely related (both placed in subsect. Canescens) and separated from the remaining species of the complex (classified in subsect. Ericoides). However, the present molecular data indicate a closer relationship of R. panschii with R. 4 January 2013 | Volume 8 | Issue 1 | e53134 Species Delimitation in Racomitrium canescens Table 2. Calculations of pairwise Fst estimates and p-values for five species of the Racomitrium canescens complex. R. japonicum R. elongatum 0.907/0.001* R. japonicum R. panschii R. ericoides R. canescens 0.253/0.096* 0.286/0.039* 0.253/0.078* 0.796/0.000* 0.619/0.000* 0.549/0.000* 0.243/0.002* 0.214/0.006* R. elongatum 0.465/0.002* R. panschii 1.000/0.166* 0.515/0.000* R. ericoides 1.000/0.001* 0.661/0.000* 1.000/0.000* R. canescens 0.502/0.000* 0.335/0.000* 0.537/0.000* 0.244/0.000* 0.641/0.000* Group comparisons based on the ITS sequence and indel data are indicated above the diagonal, whereas group comparisons based on the plastid sequence and indel data are shown below the diagonal. Significant p-values (significance level 0.05) are indicated by an asterisk. doi:10.1371/journal.pone.0053134.t002 barbuloides, R. japonicum, and R. varium (Fig. 2). Racomitrium japonicum, which was considered by Frisvoll [17] as distantly related to the remaining taxa of the R. canescens complex, is found here closely related to R. barbuloides (Fig. 2), with which it frequently grows together in mixed stands [17]. Both R. barbuloides and R. japonicum are predominantly East Asian species, the latter reaching southwards to Vietnam and Australia [25,26]. Similarly, a close relationship is indicated between the circum-North Pacific R. muticum, which is most frequent in western North America [18], and the western North American endemic R. pygmaeum. Further inferences on species relationships as well as analyses of phylogeographic patterns in the R. canescens complex need further study based on a denser sampling especially in the North American-East Asian region and sequencing of additional markers to increase support for the backbone of the phylogenetic reconstruction (Fig. 2). The clear molecular distinction between the species of the R. canescens complex indicates that the morphological characters used to identify them (leaf shape, hairpoint structure and stance, nerve length, alar cells and basal marginal leaf cells), albeit being in part rather small and difficult to apply, are reliable for species identification. On the other hand, misidentification of collections seems to be a severe problem in the R. canescens complex. If the molecular phylogenetic reconstructions accurately reflect species delimitations, 14 out of the 70 newly sequenced specimens (20%) were misidentified based on morphological characters (Figs. 2, 3). This could partly be due to mixed collections or field determinations without later checking the material microscopically (e.g. collections provisionally named R. canescens [sensu lato] to indicate that they belong to the complex), but might also be an inherent problem of the diagnostic characters. The majority of the 14 misidentified specimens belonged to species of subsection Ericoides sensu Frisvoll [17], not to R. canescens s.str. or subsp. latifolium (Fig. 3). Morphological re-investigation revealed that some of the collections named R. canescens, but actually belonging to R. ericoides or R. elongatum, were intermediate in terms of the diagnostic characters separating these three species. They showed rather broad and obtusely keeled leaves, typical for R. canescens, but a single, non-forked nerve reaching at least L up the leaf, typical for the two latter species, members of subsect. Ericoides. The suitability of leaf shape as a diagnostic character hence is questionable, but this needs to be confirmed by analysis of a larger number of collections labelled R. canescens. Implications for DNA barcoding of species complexes in mosses Species identification by DNA barcoding seems to be more difficult in bryophytes than in other land plant lineages or in animals. Instead of using a single, short piece of DNA to discriminate species across a wide range of lineages, barcoding in land plants is supposed to be based on one or two core markers, plus additional information from other DNA regions where Table 3. Comparison of maximum parsimony bootstrap support (in %) for clades of species of the Racomitrium canescens complex. rps4-trnT-trnL rps4-trnT trnT-trnL ITS ITS1 ITS2 R. canescens 94/88 64/– 88/88 100/100 100/100 63/85 R. elongatum 85/90 62/73 62/66 100/100 100/100 81/100 R. ericoides 93/99 83/91 63/89 96/100 92/100 59/79 R. panschii 87/95 88/86 –/84 99/100 99/99 84/99 R. varium+(R. barbuloides+R. japonicum) –/– –/– –/– 95/85 –/– 99/92 R. barbuloides+R. japonicum 86/100 84/100 –/– 100/100 100/100 94/100 R. japonicum 77/81 63/68 74/92 100/100 100/100 100/100 R. pygmaeum+R. muticum 63/54 –/– 64/– 100/100 97/100 56/64 R. muticum –/– –/– –/– 95/100 94/100 –/56 The bootstrap analyses were performed using parts of the analyzed DNA regions rps4-trnT-trnL and nrITS, which can be amplified separately with established primers for species identification purposes (DNA barcoding). Two specimens with missing sequences were excluded from the analysis of trnT-trnL. Values before and after the dash are from analyses without and with indels included by simple indel coding (SIC), respectively; values .70% are in bold. Dashes denote clades that were not resolved in the respective phylogenetic reconstructions. doi:10.1371/journal.pone.0053134.t003 PLOS ONE | www.plosone.org 5 January 2013 | Volume 8 | Issue 1 | e53134 Species Delimitation in Racomitrium canescens Figure 2. Molecular species circumscriptions and relationships in the Racomitrium canescens species complex. Single optimal maximum likelihood tree of 73 specimens based on combined plastid rps4-trnT-trnL and nrITS sequences. Racomitrium fasciculare and R. laevigatum were used as outgroup representatives. Thick lines indicate bootstrap support (BS) values from respective maximum parsimony and significant posterior probabilities (PP) from respective Bayesian analysis: BS.95% and PP.95 (black), BS.70% and PP.95 (dark grey), BS.70% or PP.95 (light grey). Asterisks indicate specimens whose identification was revised according to their position in the molecular phylogenetic reconstructions. doi:10.1371/journal.pone.0053134.g002 PLOS ONE | www.plosone.org 6 January 2013 | Volume 8 | Issue 1 | e53134 Species Delimitation in Racomitrium canescens clade support (Table 3). Here a different indel coding strategy might be desired. Advantages and disadvantages of using the nrITS region as DNA barcode have been discussed, e.g., by [29,32]. Apart from inherent problems such as the possible presence of paralogous ITS copies or incongruence between ITS1 and ITS2 (e.g. [33]), amplification difficulties of the whole ITS1-5.8S-ITS2 region need to be taken into account. The latter, however, can often be solved by amplifying ITS1 and ITS2 separately, which has also been done in phylogenetic analyses of bryophytes (e.g. [34]). Until now, mostly ITS2 has been considered as plant DNA barcode marker [32,35]. Bell et al. [7] considered the whole ITS region for the liverwort genus Herbertus. However, amplification and sequencing success was lower than in the employed plastid markers. With respect to the results of the present study, we argue that ITS1, which is generally more variable than ITS2 [36], should also be considered as potential barcode marker in complexes of closely related species of mosses. The preliminary observation in the Racomitrium canescens complex that ITS1 seems to outperform ITS2 should be tested in further species complexes to decide which part of the nrITS region works best as potential core barcoding marker for species identification in mosses. Furthermore, DNA barcoding in difficult species complexes should be complemented by redefining morphological characters, to develop a set of easy-touse molecular and non-molecular identification tools for improving biodiversity assessments and ecological research, including mosses. Figure 3. Revised species identifications in the Racomitrium canescens species complex based on molecular data. Changes in species identification of 14 specimens analyzed in the present study are indicated by arrows. Arrow thickness is equivalent to the number of specimens transferred from one species to another (one, two, or four specimens, respectively). Grey or black arrows indicate changes within or between subsections Canescens and Ericoides [15], respectively. doi:10.1371/journal.pone.0053134.g003 necessary (e.g. [11,27,28]). Different marker combinations, mostly from the plastid genome, have been proposed (see [11] for review), such as rbcL and matK as core markers for land plants [5], or the psbA-trnH spacer together with the nrITS region for angiosperms [29]. In bryophytes, especially mosses, however, the respective plastid markers either tend to be short (psbA-trnH spacer, trnL-F spacer, [30,31]), their discrimination capacity at the species level is still debated (rbcL, [8,10]), or efforts are still needed concerning primer design and amplification strategy (trnK/matK, e.g. [7]). A stand-alone barcoding marker is unlikely to be found among the standard plastid markers used for phylogeny reconstruction [11]. Liu et al. [8] identified five plastid markers with amplification success .90% and taxon assignment success .80% (in different combinations up to 92%), viz. rbcL, rpoC1, rps4, psbA- trnH, and trnL-F. The suitability of these markers for discriminating closely related moss species in species complexes, however, remained difficult to assess, as only few species from selected genera were compared. The subsequent study focusing on Grimmiaceae [9] already showed that (partial) rbcL sequences did not perform well, and the discrimination capacity of the best marker (psbA- trnH) did not exceed 65% in the whole family. In Racomitrium, all 11 species included in Liu et al. [9] could be distinguished by rps4, and with .80% success by rbcL and psbA-trnH, but again the question arises whether these markers would provide sufficient information when further closely related species are compared. In the present study of the Racomitrium canescens complex, almost no differences were observed between the plastid rps4-trnT-trnL and nuclear ribosomal ITS regions concerning amplification and sequencing success. Both regions could be sequenced completely for at least 90% of the analyzed specimens. The ITS region showed a clear gap between intra- and interspecific sequence divergence for the species represented by more than one accession (Fig. 1) and allowed to resolve clades of all species of the complex with significant statistical support (Table 3). The same was true for ITS1 alone (the clade of R. varium, R. barbuloides and R. japonicum was not resolved, but R. varium still formed a clade separate from the other species). Although the different parts of the rps4-trnT-trnL region and the ITS2 performed well, too (Fig. 1), they failed to discriminate R. pygmaeum and R. muticum (Table 3). Including indels by simple indel coding did not significantly improve discrimination capacity of any marker and in some cases even resulted in lower PLOS ONE | www.plosone.org Materials and Methods Ethics statement All necessary permits were obtained for field studies to collect material used for molecular analysis in Svalbard (Governor of Svalbard, references 2008/00688-2, 2009/00412), Greenland (BioBasis project, Dr. N.M. Schmidt, Aarhus University), Madeira (Dr. S. Fontinha, National Park of Madeira Services/Madeira University), Azores (Secretaria Regional da Agricultura e Florestas Dos Açores), UK (H. Cole, Manager/Senior Ranger Naturalist, Ben Lawers National Nature Reserve), and The Netherlands (Dr. H. van der Hagen, Dunea N.V.). Further material was collected in areas without specific permissions required or concerned herbarium collections from G, KRAM, L, and S, which were used according to regulations of the respective herbaria. Areas without permission needed were neither privately owned nor protected. The species sampled are neither protected nor listed by CITES (Convention on the International Trade in Endangered Species). Taxon sampling, DNA extraction, PCR and sequencing The present taxon sampling and outgroup selection was based on recent molecular phylogenetic reconstructions of Racomitrium s.l., which showed that R. fasciculare and R. laevigatum (both treated as Codriophorus by [13]), formed a sister clade to the R. canescens complex [15,19] (treated as Niphotrichum by [13]), the latter including R. varium [15] (also treated as a Codriophorus in [13]). Sequences from the plastid rps4-trnT-trnL and nuclear ribosomal ITS regions were newly generated for 70 specimens of the Racomitrium canescens complex. Voucher information and Genbank accession numbers are listed in Appendix S1. Six further collections could not or only partially be sequenced or the sequences were of insufficient quality. In addition, sequences of five Racomitrium specimens were taken from earlier studies, namely R. elongatum (Spain) from [14] and R. ericoides (both samples from Poland) as well as R. fasciculare and R. laevigatum as outgroup representatives from [19]. 7 January 2013 | Volume 8 | Issue 1 | e53134 Species Delimitation in Racomitrium canescens Distal parts of single shoots were thoroughly cleaned with distilled water. Total genomic DNA was extracted using the DNeasyH Plant Kit (Qiagen) or the NucleoSpinH Plant II Kit (Macherey-Nagel). The employed molecular markers were amplified by PCR using protocols and primers as described in [14] for rps4-trnT-trnL (primers rps4-166F and P6/7) and [37] for nrITS (primers 18F and 25R). In a few cases of difficulties with obtaining PCR products, the rps4-trnL part was split into two halves, which were amplified and sequenced separately with primers rps4-166F and A-Rbryo or A-Fbryo and P6/7, respectively [14]. PCR products were purified using the WizardH DNA Clean-up kit (Promega) or by Macrogen Inc. (www.macrogen.com), where the automated sequencing was performed as well. Sequencing primers were those used for PCR. (MCMCMC) method, using MrBayes v3.1 [49]. In a second set of Bayesian analyses the indels coded by SIC were included, with sequence and indel data treated as separate and unlinked partitions, employing the restriction site model (‘F81’) for the indel matrix. The a priori probabilities supplied were those specified in the default settings of the program. Four runs with four chains (106 generations each) were run simultaneously, with the temperature of the single heated chain set to 0.2. Chains were sampled every 1000 generations and the respective trees written to a tree file. Fifty percent majority rule consensus trees and posterior probabilities of clades were calculated by combining the four runs and using the trees sampled after the chains converged. Trace plots generated in Tracer v1.5 [50] were used to check for convergence of the runs (plateaus of all runs at comparable likelihoods) and to infer the ‘burnin’, which ranged approximately between the first 100000 and 120000 generations (first 100–120 sampled trees). Consequently, the first 150 trees (15%) were deleted to be sure that only trees of the stationary phase were included. Alignment, sequence analysis and phylogenetic reconstructions DNA sequences were manually aligned in PhyDEH v0.995 [38]. Phylogenetic reconstructions of taxon circumscriptions and relationships were performed based on the maximum parsimony (MP) principle and using two model-based approaches, maximum likelihood (ML) and Bayesian inference (BI). Separate MP analyses of the rps4-trnT-trnL versus ITS sequences were performed and the resulting tree topologies were checked for possible incongruence between the plastid and nuclear markers by visual inspection and by applying a partition homogeneity test (ILD test) [39,40] as implemented in PAUP* 4.0b10 [41] (100 replicates). Calculations of pairwise Fst estimates and p-values based on plastid and ITS haplotypes were performed using Arlequin v3.5.1.3 [42], with haplotypes (including indel characters) delineated in TCS v1.2.1 [43]. Phylogenetic analyses of the combined plastid and nuclear markers were run using MP, ML, and BI. To evaluate the employed markers for DNA barcoding, further MP analyses were performed of four partitions for which primers pairs are available, viz. rps4-trnT, trnT-trnL, ITS1, and ITS2. In addition, pairwise nucleotide distances between all sequences were calculated according to the K2P model (cf. [8,9]) for the combined dataset and all partitions, and compared between and within the species of the R. canescens complex. Calculation of pairwise distances as well as MP and ML analyses were performed in PAUP. Heuristic searches under parsimony were implemented using random sequence addition with 1000 replicates and tree bisection-reconnection (TBR) branch swapping. All MP analyses were run with gaps (indels) either treated as missing data or coded as informative by a simple indel coding (SIC) strategy [44] as implemented in SeqState [45]. Heuristic bootstrap searches under parsimony were performed with 1000 replicates and 10 random addition cycles per bootstrap replicate with the same options in effect. To search the tree space for islands of more parsimonious trees, parsimony ratchet analyses were performed with PRAP2 [46] in combination with PAUP, employing the default options (200 iterations, 25% of randomly chosen positions up-weighted to 2) and superimposed 10 random addition cycles. For the model-based approaches, model testing was performed in Modeltest 3.7 [47] employing MrMTgui [48]. GTR+C+I was indicated as best-fit model of the combined dataset according to the Akaike information criterion (AIC). Consequently, the settings basefreq = (0.3011 0.2002 0.2254), nst = 6, Rmat = (1.5235 5.1208 0.6544 2.3313 7.1000), rates = gamma, shape = 0.7849, and pinvar = 0.6263 were used for ML, and nst = 6 and rates = invgamma for BI. Bayesian posterior probabilities were calculated based on the Metropolis-coupled Markov chain Monte Carlo PLOS ONE | www.plosone.org Supporting Information Maximum parsimony phylogenetic reconstruction of the Racomitrium canescens species complex based on plastid rps4-trnT-trnL sequences. Indels coded by simple indel coding were included. Bootstrap support values of the respective analyses without indels (before the slash) and with indels (after the slash) are indicated. (TIF) Figure S1 Maximum parsimony phylogenetic reconstruction of the Racomitrium canescens species complex based on plastid rps4-trnT sequences. Indels coded by simple indel coding were included. Bootstrap support values of the respective analyses without indels (before the slash) and with indels (after the slash) are indicated. (TIF) Figure S2 Maximum parsimony phylogenetic reconstruction of the Racomitrium canescens species complex based on plastid trnT-trnL sequences. Indels coded by simple indel coding were included. Bootstrap support values of the respective analyses without indels (before the slash) and with indels (after the slash) are indicated. (TIF) Figure S3 Maximum parsimony phylogenetic reconstruction of the Racomitrium canescens species complex based on nuclear ribosomal ITS sequences. Indels coded by simple indel coding were included. Bootstrap support values of the respective analyses without indels (before the slash) and with indels (after the slash) are indicated. (TIF) Figure S4 Maximum parsimony phylogenetic reconstruction of the Racomitrium canescens species complex based on nuclear ribosomal ITS1 sequences. Indels coded by simple indel coding were included. Bootstrap support values of the respective analyses without indels (before the slash) and with indels (after the slash) are indicated. (TIF) Figure S5 Maximum parsimony phylogenetic reconstruction of the Racomitrium canescens species complex based on nuclear ribosomal ITS2 sequences. Indels coded by simple indel coding were included. Bootstrap support values of Figure S6 8 January 2013 | Volume 8 | Issue 1 | e53134 Species Delimitation in Racomitrium canescens the respective analyses without indels (before the slash) and with indels (after the slash) are indicated. (TIF) Acknowledgments Sincere thanks are due to N. Cox (NERC Station, Ny-Ålesund), M. Loonen (Arctic Centre Groningen/Netherlands Arctic Station, NyÅlesund), and M. Sim-Sim (Lisbon) for collaboration and support during fieldwork, to L. Hedenäs (S), R. Ochyra and H. Bednarek-Ochyra (KRAM), and M. Price (G) for loan of herbarium collections, and to M.C.M. Eurlings for technical assistance. Appendix S1 Geographic origin (with numbering corresponding to Fig. 1 of the manuscript), voucher information and herbarium locations (in brackets), and GenBank accession numbers (rps4-trnT-trnL, nrITS) of 70 Racomitrium specimens newly sequenced for the present study. (DOC) Author Contributions Conceived and designed the experiments: MS HK. Performed the experiments: MS SV JL HK. Analyzed the data: MS SV. Contributed reagents/materials/analysis tools: JM DQ KH. Wrote the paper: MS. References 26. Gao C, Crosby MR, He S (2003) Moss Flora of China, Vol. 3. Grimmiaceae– Tetraphidaceae. Beijing, New York & St. Louis: Science Press & Missouri Botanical Garden. 27. Pennisi E (2007) Taxonomy – Wanted: A barcode for plants. Science 318: 190– 191. 28. Fazekas AJ, Kesanakurti PR, Burgess KS, Percy DM, Graham SW, et al. (2009) Are plant species inherently harder to discriminate than animal species using DNA barcoding markers? Mol Ecol Resources 9 (Suppl. 1): 130–139. 29. 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