Botanical Journal of the Linnean Society, 2016, 180, 427–451. With 5 figures Integrative taxonomy sheds light on an old problem: the Ulota crispa complex (Orthotrichaceae, Musci) 1
Departamento de Biolog
ıa (Botanica), Facultad de Ciencias, Universidad Aut
onoma de Madrid, calle Darwin 2, E-28049 Madrid, Spain 2
Departamento de Botanica, Facultad de Farmacia, Universidad de Valencia, Avda. Vicente Andr
es Estell
es s/n, E-46100 Burjassot, Spain Received 4 November 2015; revised 23 January 2016; accepted for publication 31 January 2016 The combined use of morphological and molecular analyses has been proven to be useful in resolving taxonomic complexes with hidden diversity. In bryology, however, integrative taxonomy has rarely been employed to revisit relevant old, unresolved problems. One of these classical controversies is whether the Ulota crispa complex comprises one or three species. To elucidate this, an exhaustive morphological revision, based on numerous herbarium and fresh specimens from most of the Holarctic areas in which U. crispa has been reported, and molecular analyses, using one nuclear (ITS2) and three plastid (trnG, trnL-trnF, atpB-rbcL) loci on a selection of representative specimens, have been performed. The results unambiguously point to the existence of three morphotypes, ascribable to the previously described Ulota crispa s.s., U. crispula and U. intermedia, which can be differentiated by an ample set of qualitative and quantitative morphological characters. A phylogenetic reconstruction based on molecular data is proposed, including samples of these morphotypes in a framework of another ten Ulota spp. and two Orthotrichum and two Zygodon spp. According to the results, the samples belonging to these morphotypes are placed in three independent and coherent monophyletic clades, indicating that they represent three closely related, but independent, species. The origin and development of the taxonomic debate around U. crispa and related taxa are discussed to illuminate the reasons for this historical confusion. The analysis of the geographical origin of the studied samples shows that all three species are widely distributed throughout the Northern Hemisphere, although with different ranges. Ulota crispula has an amphi-Atlantic range, whereas U. crispa and U. intermedia display broader disjunct ranges; only in Europe do the three species co-occur. A key to the three species and updated detailed descriptions are provided. © 2016 The Linnean Society of London, Botanical Journal of the Linnean Society, 2016, 180, 427–451 ADDITIONAL KEYWORDS: Bryophyta – cryptic species – disjunct distributions – molecular phylogeny – morphology – sibling species – Ulota crispula – Ulota intermedia. INTRODUCTION Integrative taxonomy (Dayrat, 2005; Padial et al., 2010; Schlick-Steiner et al., 2010) and, in particular, the combined approach of thorough morphological studies and DNA sequence-based phylogenetic analysis for the determination of species boundaries are allowing taxonomic problems in various biological groups to be solved satisfactorily (e.g. Grismer et al., 2013; Malekzadeh-Viayeh et al., 2014; Vigalondo et al., 2015; Zamora, Calonge & Mart
ın, 2015). *Corresponding author. E-mail: ricardo.garilleti@uv.es Among bryophytes, many cryptic species (sensu Bickford et al., 2007) have been uncovered thanks to this methodology (e.g. Vanderpoorten et al., 2010; Damayanti et al., 2012; Medina et al., 2012, 2013; Buchbender et al., 2014; Heden€ as et al., 2014; Draper et al., 2015; Heinrichs et al., 2015). In these organisms, integrative taxonomy has been used mainly for the study of complexes in which a hidden diversity has been suspected, but for which no critical morphological analyses have been undertaken in recent times. In that sense, the combined use of morphological and molecular analyses has rarely been applied to old taxonomic conflicts. The target of this © 2016 The Linnean Society of London, Botanical Journal of the Linnean Society, 2016, 180, 427–451 427 Downloaded from https://academic.oup.com/botlinnean/article-abstract/180/4/427/2416569 by guest on 23 May 2020
1,2, FRANCISCO LARA1, ISABEL DRAPER1, VICENTE MAZIMPAKA1 RUT CAPARROS and RICARDO GARILLETI2* 428
ET AL. R. CAPARROS commonest traits that define a large number of frequently sympatric species in the genus, the differences among them sometimes being subtle. Because of such morphological variability and wide distribution, many species have been described or discussed by comparison (e.g. U. fulva Brid., U. bruchii Hornsch.) and some have been synonymized (e.g. U. intermedia Schimp.). An inevitable consequence of this has been that the concept of the species has become confused and many synonymizations have been made. In its wider concept, U. crispa is the species in the genus with by far the longest list of synonyms. In recent years, some confusion has been resolved on the basis of classic morphological studies alone, by the analysis of characters not or only rarely used previously, e.g. in cases of U. bruchii (Garilleti et al., 2000) and U. crispula Bruch (Caparr
os, Garilleti & Lara, 2014). Despite these advances, a degree of uncertainty remains over the general validity of the morphological characters used to distinguish these species and on the relationships among them. Some doubtful synonyms that have elusive morphological differences remain under U. crispa and a new integrative approach is needed to resolve the true nature of these names. Two of them represent the most important cases: U. crispula and U. intermedia. The protologue of U. crispa by Hedwig (1801), as Orthotrichum crispum Hedw., is somewhat ambiguous, as this moss was described as having crowded, crisped when dry linear leaves with a wide base, and a long exserted ovate capsule, a vague set of traits that fit most of the currently recognized species in the genus. Some years later, Bruch (in Bridel, 1827) was able to recognize a form sufficiently different to define a new species, U. crispula Bruch in Brid., considering his new species as being close to U. crispa. Later, Schimper (1876) proposed a new species, U. intermedia Schimp., explicitly stating its intermediate morphology between U. crispa and U. crispula. Although the traits used by these authors when describing their species are still used to discriminate Ulota spp., the extension and limits of their variability are imprecise in the protologues, which makes the species discrimination difficult. Moreover, the three supposedly different mosses share ecological preferences and it is common to find two growing together, which has made the interpretation of the true nature of these taxa even more difficult. This situation, with two species apparently discriminated only by subtle morphological differences, and a third with intermediate character states, has created a long-standing controversy around the nature of the two latter taxa. In fact, this old problem has been revisited several times over the last century (Smith & Hill, 1975; © 2016 The Linnean Society of London, Botanical Journal of the Linnean Society, 2016, 180, 427–451 Downloaded from https://academic.oup.com/botlinnean/article-abstract/180/4/427/2416569 by guest on 23 May 2020 study is precisely one of these old problems: the delimitation of taxa in the Ulota crispa (Hedw.) Brid. complex. Ulota D.Mohr is a large genus with 69 recognized taxa (Garilleti, Mazimpaka & Lara, 2015), mainly distributed in temperate oceanic areas all around the globe, but with a few species living in montane habitats in tropical or subtropical regions. These taxa mostly grow as epiphytes on a wide diversity of trees and shrubs, although some species are primarily saxicolous or can live on rocks. They usually form part of diverse epiphytic communities and can be the dominant species on trees or shrubs under oceanic hyper-humid conditions (Garilleti, Mazimpaka & Lara, 2012; Garilleti et al., 2015). The nomenclatural history of the genus is complicated, as the strong morphological similarity with Orthotrichum Hedw. has led to numerous changes in taxa between both genera, including the repeated consideration of Ulota as a section or subgenus of Orthotrichum (M€ uller, 1849; Mitten, 1869; Boulay, 1884). Even the most outstanding monographer of Ulota, N. Malta, considered that the generic separation was doubtful, although he maintained it (Malta, 1927, 1932). Since then, few taxonomic treatments of the genus have been published, and all have been regional revisions without a wide territorial or systematic scope that could contribute to a better understanding of the inner structure or relationships of the genus, and thus we have no constancy of any posterior questioning of the status of this genus as an independent entity. Moreover, molecular data also point to the close relationship of Ulota with Orthotrichum spp. with superficial stomata. According to Goffinet et al. (2004) and Sawicki, Pl
asek & Szczeci
nska (2010), the monophyly of Ulota and the phaneroporous Orthotrichum spp. is ambiguous. The fact that the molecular evidence gathered so far is not taxonomically conclusive with regard to Ulota emphasizes the need for the combination of molecular and morphological approaches for any taxonomic or systematic study. Ulota crispa is not only the type of the genus, but also one of the species with the widest range, as it has been reported from both the Palaearctic and the Nearctic. It seems to be a common species in humid oceanic forests in parts of Asia and North America, and it has been extensively reported from Europe, where it is considered to be the most common Ulota sp., and its predominance over other species of this genus has been regionally demonstrated (Albertos et al., 2000). This dual condition of type species with an extensive range has contributed to make U. crispa a model species in the genus. This moss, so variable in its current concept, presents most of the INTEGRATIVE STUDY OF ULOTA CRISPA MATERIAL AND METHODS MORPHOLOGICAL STUDIES The morphological approach was an unbiased study including a large number of samples matching the concept of the problem group of species. Specimens from herbaria all around the world (BCB, BM, BP, DUKE, FCO, G, H, HIRO, LISU, MA, MAUAM, MHA, MO, MUB, NICH, NY, S, TU and VIT, and personal herbaria of P. Boudier and D. Callaghan) were included in the analyses. In addition, specimens doubtfully identified as belonging to different species of this complex were also included. As the synonymization of U. crispula and U. intermedia with U. crispa is an old topic, many of the herbarium samples were kept under the last name and only in rare cases were they found under the other names. In addition to herbarium specimens, the research team collected a large number of fresh material from the west coast of North America, Japan and Europe. The scope of this broad specimen sampling was intended to represent the entire distribution and ecological range of the species. In total, c. 800 samples were studied and a selection is listed in Appendix 1. Nearly 100 morphological traits were scored, selected on the basis of the experience of the research team on Ulota and related groups of Orthotrichaceae (Medina et al., 2012, 2013; Caparr
os et al., 2014). The variation in these characters across the studied samples led to the identification of different morphotypes. These were considered as potential species candidates and their characteristics were cross-checked with the type material available for U. crispa and related taxa (for the detailed study, see Caparr
os et al., in press) to give the nomenclaturally correct name to each morphotype. DNA EXTRACTION, POLYMERASE CHAIN REACTION (PCR) AMPLIFICATION AND SEQUENCING For the molecular study, we sampled several specimens covering the geographical range of each morphotype: 11 specimens morphologically assigned to U. crispa s.s.; seven specimens assigned to U. crispula; and seven specimens assigned to U. intermedia. Other Northern Hemisphere Ulota spp. were also included to provide a phylogenetic framework: U. bruchii, U. coarctata (P.Beauv.) Hammar, U. curvifolia (Wahlenb.) Lilj., U. drummondii (Hook. & Grev.) Brid., U. macrospora Baur & Warnst., U. megalospora Venturi, U. obtusiuscula M€ ull. Hal. & Kindb., U. phyllantha Brid., U. rehmannii Jur. and U. reptans Mitt. Specimens of other members of Orthotrichaceae were also included to complete the outgroup sampling: Orthotrichum affine Brid., O. rupestre Schleich. ex Schw€ agr., Zygodon pentastichus (Mont.) M€ ull. Hal. and Z. viridissimus (Dicks.) Brid. A complete list and details of the specimens can be found in Appendix 2. Total DNA was extracted from a single individual in each sample using a Plant DNeasy Mini Kit from Qiagen. PCR was performed in an Eppendorf Mastercycler using PuReTaqTM Ready-To-GoTM PCR Beads (GE Healthcare, Madrid, Spain) in a reaction volume of 25 lL, according to the manufacturer’s instructions. Four molecular regions were amplified, one from the nuclear genome (ITS2) and three from the plastid genome (trnG, trnL-trnF and atpB-rbcL). For ITS2, the primers used were ITS2 forward and ITS2 reverse (Ziolkowski & Sadowski, 2002), and the amplification protocol included one melting step of 1 min at 94 °C, followed by 30 cycles (up to 35 in samples that were difficult to amplify) of 1 min at 94 °C, 1 min at 59 °C and 1 min 30 s at 72 °C, and a final extension period of 5 min at 72 °C. For trnG, the primers used were trnGf_leu (Stech et al., 2011) and trnGr (Pacak & Szweykowska-Kulinska, 2000), and the PCR program included one melting step of 5 min at 94 °C, followed by 40 cycles of 30 s at 94 °C, 40 s at 52 °C and 1 min 30 s at 72 °C, and a final extension period of 8 min at 72 °C. For trnL-trnF, the primers used were trnLc-104 and trnFF-425 (Vigalondo et al., 2016). The PCR program included one melting step of 5 min at 94 °C, followed by 38 cycles of 30 s at 94 °C, 1 min at 47 °C, 30 s at 72 °C and 30 s at 94 °C, and a final extension period of 1 min at 72 °C. For atpB-rbcL, the primers used were atpB and rbcL (Chiang, Schaal & Peng, 1998), and the PCR program included one melting step of 5 min at 94 °C, followed by 30 cycles of 30 s at 94 °C, 1 min at 52 °C and 30 s at 68 °C, and a final extension period of 7 min at 68 °C. © 2016 The Linnean Society of London, Botanical Journal of the Linnean Society, 2016, 180, 427–451 Downloaded from https://academic.oup.com/botlinnean/article-abstract/180/4/427/2416569 by guest on 23 May 2020 Rosman-Hartog & Touw, 1987) without achieving an entirely satisfactory solution. The goal of the present work is to determine whether the forms grouped into the so-called U. crispa complex correspond to a single species or whether it is possible to differentiate definitively several taxa within it. To this end, a detailed morphological study has been made to evaluate the traits traditionally used in the taxonomy of the genus, and to find new characters to define the variability and distinction of the different species more accurately. We then contrasted the morphological entities against clades inferred from phylogenetic analyses of molecular data to infer the existence of monophyletic, morphologically diagnosable taxa (see Medina et al., 2012, 2013). 429 430
ET AL. R. CAPARROS After visualization on 1% agarose gels, successful amplifications were purified using the Exo/SAP protocol (Thermo Fisher Scientific, Madrid, Spain). Samples were incubated with 1 lL Exo1 enzyme and 4 lL FastAP following the manufacturer’s instructions. Cleaned PCR products were sequenced by Macrogen (www.macrogen.com). EDITING, ALIGNMENT AND PHYLOGENETIC ANALYSIS Nucleotide sequences were edited and assembled for each DNA region in PhyDE v0.9971 (M€ uller et al., 2006). The assembled sequences were manually aligned and the primer annealing sites and regions with incomplete data at the 50 and 30 ends of the sequences were excluded from subsequent analyses: for ITS2, 36 positions at the 30 end; for trnG, 26 positions at the 50 end/72 positions at the 30 end; for trnL-trnF, 12/20 positions; and for atpB-rbcL, 73/60 positions. Phylogenetic reconstructions were made on the basis of three different optimality criteria [maximum parsimony (MP), maximum likelihood (ML) and Bayesian inference (BI)] to assess whether there were any differences in the trees recovered with regard to the method used. The analyses were performed with the programs TNT 1.1 (Goloboff, Farris & Nixon, 2008) for MP, MEGA 6 (Tamura et al., 2013) for ML and MrBayes 3.2.2 (Huelsenbeck & Ronquist, 2001; Ronquist & Huelsenbeck, 2003; Ronquist et al., 2012) for BI. Among the outgroup taxa (Appendix 2), Z. pentastichus was considered to be the furthest outgroup and was used to root the consensus tree recovered by BI. The swapping algorithm selected in TNT was tree bisection–reconnection (TBR), with ten trees held in memory. All characters were equally weighted. Clade support in MP analyses was assessed via non-parametric bootstrapping (bootstrap support, BS) using the default settings in TNT, except for the number of replicates, which was set to 1000. The output results requested were the absolute frequencies. For all generated MP trees, the consistency index (CI), retention index (RI) and tree length were calculated. The nucleotide substitution models used for the ML and BI analyses were selected for each locus separately using jModeltest 2.1.4 (Posada, 2008; Darriba et al., 2012) and based on the Akaike and Bayesian information criteria. These were HKY + I + G for ITS2 and HKY + G for the three plastid markers (trnG, trnL-trnF, atpB-rbcL). On the basis of these results, the matrix for the final analyses was partitioned in two, one partition corresponding to the nuclear and the other to the plastid regions. Clade support in ML was assessed using 1000 bootstrap RESULTS MORPHOLOGICAL APPROACH The analysis of morphological variability led to the identification of three different morphotypes in the U. crispa complex. The study of the appropriate type material enabled each of these three morphotypes to be related to one of the three principal names of the complex: Orthotrichum crispum ( U. crispa), U. crispula and U. intermedia. Results from the study of the type material, including the detailed lectotypification, are presented in Caparr
os et al. (in press). The differentiation of these species © 2016 The Linnean Society of London, Botanical Journal of the Linnean Society, 2016, 180, 427–451 Downloaded from https://academic.oup.com/botlinnean/article-abstract/180/4/427/2416569 by guest on 23 May 2020 SEQUENCE replications, and the ML heuristic method selected was nearest-neighbour interchange. The BI analyses used one cold and three incrementally heated Monte Carlo Markov chains (MCMCs) on two simultaneous runs. MCMC runs continued until the standard deviation of the split frequencies was below 0.01 (3 000 000 generations), with one tree sampled every 1000th generation, each using a random tree as a starting point and a temperature parameter value of 0.2 (the default in MrBayes). The first 25% of the total sampled trees of each run were discarded as ‘burnin’ to achieve the MCMC log-likelihoods that had become stationary and converged. Clade support in these analyses was assessed as the posterior probability (PP). Insertions and deletions (indels) in non-coding regions are sometimes difficult to assess (e.g. Kelchner, 2000) and can lead to ambiguous alignments. This is the case in part of the ITS regions, and to determine the effect of their inclusion, all the analyses were run with the indels considered as missing information and with indels coded as informative characters. The indel coding strategy was the simple method of Simmons & Ochoterena (2000), as implemented in SeqState (M€ uller, 2004), and the model selected for the indel partition in MrBayes was F81, as recommended by Ronquist, Huelsenbeck & Teslenko (2011). The analyses were run with the same parameters indicated for the datasets without coded indels. Initially, the four regions (ITS2, trnG, trnL-trnF and atpB-rbcL) were analysed separately, using the three different optimality criteria (MP, ML and BI). The matrices used have been deposited in TreeBASE (http://purl.org/phylo/treebase/phylows/study/ TB2:S18716). The results were visually compared to try to find possible incongruence among regions, between the datasets (coding or treating the indels as missing data) or among the different reconstruction methods. After this inspection, the four regions were combined in a single matrix. INTEGRATIVE STUDY OF ULOTA CRISPA is based on a broad set of characters (see Taxonomic section and Table 1), including several qualitative macro- and microscopic characters. MOLECULAR 431 U. rehmannii, U. megalospora, U. bruchii, U. coarctata and U. macrospora are gathered in a wellsupported sister clade (1/1/94/–). Finally, U. phyllantha is located in an early-branching grade according to BI (0.94/0.99). SEQUENCE VARIATION PHYLOGENETIC RECONSTRUCTION The MP analyses yielded 96 (without considering the indels) or 16 (with the indels coded) most parsimonious trees (length: 725/440; CI = 0.78/0.81; RI = 0.83/0.84). The consensus tree had a congruent topology with that obtained in the ML and BI analyses, although both MP and ML resolved fewer clades than BI, as shown in Figure 1. We hereafter refer to the support values in the text following the scheme (PPindels/PP/BSindels in MP/BS in MP). ML analyses recovered trees with low support, which are not shown. The molecular data resolve all of the included samples of Ulota in a monophyletic well-supported group according to BI (0.94/0.99/–/–), which is placed as the sister clade of the outgroup species belonging to Orthotrichum. In Ulota, the ingroup samples are gathered in three separate well-supported clades that correspond to the three morphotypes: U. crispa (1/1/79/78), U. crispula (1/1/79/79) and U. intermedia (0.99/0.95/64/65). The molecular data gathered for the present study are not sufficient to fully resolve the relationships within Ulota. According to the tree recovered when the indels are considered as informative, U. crispula and U. crispa could be closely related. This sister relationship is poorly supported (0.7) and, when the indels are treated as missing information, the tree recovered suggests a closer relationship between U. crispula and U. intermedia (0.86). All of the analyses indicate that sister species to the clade including the ingroup samples, are U. curvifolia and U. obtusiuscula (1/0.77/82/79). Other species closely related to this group are U. reptans and U. drummondii (1/1/86/–). However, DISCUSSION The species belonging to the U. crispa complex are conspicuous mosses common on trees in temperate regions of Europe and North America, and hence they have been extensively collected, including in the early days of bryology. Dillenius (1742) and Linnaeus (1763) had already referred to these plants, and the first species validly published of what became the genus Ulota, Orthotrichum crispum, was in the opus that has been chosen as the nomenclatural starting point for most mosses (Hedwig, 1801). Before long, Mohr (1806) created the genus Ulota and Bridel (1818) made the combination U. crispa. Subsequently, U. crispula was described (Bruch in Bridel, 1827), accurately discussing its most outstanding differences from U. crispa. Bruch, the author of that species, had a clear concept of these two species and their differences, including the characters that later led him to recognize in schedula (BM 000852210!) a new moss, first considered as an Orthotrichum sp. (O. intermedium, cf. Caparr
os et al., in press). Schimper published the valid description of U. intermedia after the death of Bruch (Schimper, 1876), with whom he had collaborated intensively. Schimper partially based the description of the species on the above-mentioned specimens of Bruch, but he added several materials identified by himself that were not always correctly interpreted. Although U. intermedia became well defined, Schimper’s incorrect mixture of materials complicated the establishment of its clear morphological delimitation. Moreover, this could maintain or increase the existing uncertainties about the distinction of U. crispa and allied forms, and revitalize the increasing controversy on the taxonomic value of these forms. Indeed, U. crispula had already been proposed at variety rank (Hammar, 1857). After the work of Schimper (1876), some classical authors recognized all three species (Limpricht, 1890–1895; Husnot, 1892–1894), but others regarded them as forming a single species with varieties (Braithwaite, 1888– 1895; Dixon, 1924) or two different species with a transitional form corresponding to U. intermedia (Boulay, 1884). Paradoxically, most of these authors properly differentiated the three taxa but, independent of the taxonomic rank considered, they regarded ‘crispula’ and ‘intermedia’ as forms of doubtful value because of the difficulties in a clear allocation of © 2016 The Linnean Society of London, Botanical Journal of the Linnean Society, 2016, 180, 427–451 Downloaded from https://academic.oup.com/botlinnean/article-abstract/180/4/427/2416569 by guest on 23 May 2020 The length variation in the four DNA regions is shown in Table 2, with the information content of each data partition. No incongruence in terms of well-supported clades was observed in separate analyses of the four molecular loci, and therefore all the markers were combined in a single matrix that had a total length of 1944 bp, with 206 variable sites (95 potentially parsimony informative, 19 in U. crispa, U. crispula and U. intermedia). Simple indel coding increased the number of potentially parsimonyinformative characters by three within the ingroup and recovered trees with identical topology, but slightly higher support, than those obtained when the indels were treated as missing data. Urn shape when dry and empty Constriction below mouth when dry and empty Capsule furrows (spaces between ribs) Width of furrows when dry and empty Exothecial bands Number of cell rows Cell colour Differentiation at capsule mouth Peristome: exostome teeth Tendency of teeth pairs to split External visibility of principal peristomial layer (PPL) at marginal parts of teeth Peristome: endostome segments Segment appearance and durability Segment position when dry Ulota crispa Ulota crispula Ulota intermedia (3.0–)3.2–6.8(–7.4) (1.8–)2.0–3.8(–4.0) (3.0–)3.2–6.5 (12–)14–23(–25) (18–)20–29(–30) (15–)18–35(–38) Cylindrical to ellipsoid Short cylindrical to ovoid or somewhat urceolate Short cylindrical to somewhat urceolate or obconic Not or slightly constricted Ellipsoid to cylindrical-ellipsoidal, rarely long ovoid Cylindrical, oblong-cylindrical, obconic or elongate-ovoid Not constricted Commonly uniformly narrow, collapsed at the constricted area of the urn Usually broad, somewhat sinuous or irregular, not collapsing anywhere Broad, not collapsing anywhere 4–5(–6) Evenly yellow to pale orange 2–4 Evenly pale yellow Neatly reaching the mouth or obscurely separated by a ring of small, thin-walled cells in 1–3(4) layers Not reaching the mouth, visibly separated by a ring of small, thinwalled cells in (1)2–4(5) layers 2–4(–5) Hyaline with pale yellow incrassated lateral walls Not reaching the mouth, visibly separated by a ring of small, thin-walled cells in (1)2–6(7) layers No, most teeth remain tightly fused in pairs even in old capsules Yes, as a smooth hyaline halo (sometimes not visible in deteriorate teeth) Yes, teeth split easily after being recurved No Yes, teeth tend to split after being recurved No Robust and persistent Incurved Slender and fragile Irregular (some incurved, other erect or patent) Almost linear with a more or less widened base Uniseriate or irregularly biseriate, with thin transversal walls Robust and usually persistent Incurved Urceolate Strongly constricted Segment shape Long triangular to subulate Segment cell pattern at the inner peristome layer (IPL) Uniseriate, with transversal walls variably incrassate Broadly linear with a more or less widened base or subulate Uniseriate, with incrassate and prominent transversal walls
ET AL. R. CAPARROS Downloaded from https://academic.oup.com/botlinnean/article-abstract/180/4/427/2416569 by guest on 23 May 2020 © 2016 The Linnean Society of London, Botanical Journal of the Linnean Society, 2016, 180, 427–451 Sporophyte Sporophyte total length (mm) Spores Spore size (lm) Capsule shape Capsule when dry and full of spores 432 Table 1. Main morphological characters for the differentiation of Ulota crispa, U. crispula and U. intermedia Internal ornamentation (IPL) Operculum Colour Gametophyte Habit and aspect when dry Leaves Leaf shape when dry Leaf base Leaf basal marginal cells Width of band with differentiated cells Ulota crispa Ulota crispula Ulota intermedia Smooth to finely papillose below, variably papillose above, but never opaque because of the papillae density Densely covered by a reticulum of striae, perceived above as an opaque, almost smooth ornamentation Smooth to very finely papillose below, variably reticulate above, but never opaque because of the ornamentation density Yellowish, without a differentiate basal rim Yellowish, usually with an orange to reddish basal rim Yellowish, without a differentiate basal rim Medium to large cushions, generally strongly crisped Small to large cushions, slightly to markedly crisped Medium to large cushions, markedly crisped Tortuose or circinate, occasionally falcate Mostly obovate, distinctly concave, abruptly narrowing into the lamina Curved to tortuose, more rarely circinate Mostly elliptical, not obviously concave, gradually narrowing into the lamina Tortuose or circinate, occasionally falcate-tortuous Mostly obovate, distinctly concave, abruptly narrowing into the lamina Broad, (5–)7–16(–20) cell rows Usually narrow, (1–)3–8(–10) cell rows Narrow to broad, (2–)3–14(–16) cell rows INTEGRATIVE STUDY OF ULOTA CRISPA 433 Downloaded from https://academic.oup.com/botlinnean/article-abstract/180/4/427/2416569 by guest on 23 May 2020 © 2016 The Linnean Society of London, Botanical Journal of the Linnean Society, 2016, 180, 427–451 Table 1. Continued 434
ET AL. R. CAPARROS Table 2. Length variation, number of variable and potentially informative sites and most suitable substitution model for the nuclear (ITS2) and plastid (trnG, trnL-trnF and atpB-rbcL) sequences used in this study trnG trnL-trnF atpB-rbcL 460 460 452 474 449–450 449 449 474 449–450 488 471 Incomplete Incomplete 515 485 Incomplete 446 80 6 33 5 68 3 31 0 1–585 HKY + I + G 512 514 513 513 511–517 511 512 512 511–512 513 513 512 509 513 512 518 524 52 5 22 4 25 1 11 1 586–1115 HKY + G 305 305 306 Incomplete 304–305 303–305 306 306 305–306 Incomplete 306 306 305 306 307 319 318 36 3 20 3 11 2 6 2 1116–1443 HKY + G Incomplete 493 494 494 484–485 484–495 484 483–484 484 494 494 485 493 494 484 491 494 38 9 20 7 9 0 5 0 1444–1944 HKY + G some exemplars to any of the three taxa. The situation worsened during the 20th century, when U. crispula and U. intermedia were generally considered as unimportant forms or merely synonyms of U. crispa (Grout, 1935; Nyholm, 1956; Smith & Hill, 1975; Rosman-Hartog & Touw, 1987). Finally, Smith & Proctor (1993) did not consider U. crispula or U. intermedia in their revision of the U. crispa complex, which, in fact, consisted of the distinction of U. crispa and U. bruchii, a question later resolved by Garilleti et al. (2000). Only recently, Caparr
os et al. (2014) reinstated U. crispula based on significant morphological differences, leading to the current study. Qualitative macroscopic characters that differentiate the three species can be observed with a hand lens, even in the field, whenever samples are in dry conditions and have ripe capsules (Table 1). These traits include: the capsule shape both when full of spores and when dry and empty; the eventual constriction of the urn below the mouth when dry and empty and the subsequent collapse of the capsule ribs at the constricted area or the more or less uniform separation of ribs by broad furrows; in the peristome, the possible tendency of teeth pairs to split, the segment shape and its durability in empty capsules; and the potential differentiation of a coloured basal rim in the operculum. Additional quantitative macroscopic characters, such as the sporophyte total length, the size of the cushions and the degree of crispature of leaves when dry, eventually can help in the identification of these species, even in the field. A complementary set of diagnostic microscopic characters allows a safe discrimination in the laboratory of specimens in a good state of preservation and with the necessary morphological structures. These include some qualitative traits related to the peristome, such as the existence or not of a thin hyaline membrane-like margin bordering the exostome teeth, or the precise structure, ornamentation and position of the endostome segments when dry. To this type of discriminant characters also belong the form of the leaf bases (shape, type of narrowing towards the lamina and concavity), the structure of the exothecial © 2016 The Linnean Society of London, Botanical Journal of the Linnean Society, 2016, 180, 427–451 Downloaded from https://academic.oup.com/botlinnean/article-abstract/180/4/427/2416569 by guest on 23 May 2020 Length variation Orthotrichum affine Orthotrichum rupestre Ulota bruchii Ulota coarctata Ulota crispa Ulota crispula Ulota curvifolia Ulota drummondii Ulota intermedia Ulota macrospora Ulota megalospora Ulota obtusiuscula Ulota phyllantha Ulota rehmannii Ulota reptans Zygodon pentastichus Zygodon viridissimus Variable sites Variable sites (ingroup) Informative sites Informative sites (ingroup) Indel sites Indel sites (ingroup) Indel informative sites Indel informative sites (ingroup) Positions in data matrix Substitution model ITS2 INTEGRATIVE STUDY OF ULOTA CRISPA 435 bands (number of cell rows and cell colour) and their potential reach to the mouth. Finally, some microscopic quantitative traits have representative ranges of values for the different species, namely the spore size and the width of the band of leaf basal marginal cells (Table 1). Our phylogenetic reconstruction based on molecular data places samples identified as U. crispa, U. crispula or U. intermedia in independent clades that are coherent with the morphological placement. The congruent results obtained with all the different phylogenetic methods employed (BI, MP and ML) and from the morphological study increase the support for the recognition of the three morphotypes at the species level. Despite the lack of a complete phylogenetic analysis of the genus, the results suggest that these species are closely related. According to Bickford et al. (2007), this could be considered as a true complex of cryptic species, in the sense that three distinct species have been erroneously classified (and hidden) under one species name (U. crispa) by most bryologists during the last century. The results of specific studies carried out by Smith & Hill (1975) and Rosman-Hartog & Touw (1987), searching for differences between these species, also point in the same direction. However, according to our results, each of these three entities can be diagnosed on the basis of morphological traits (Table 1), several of them being decisive. In consequence, specimens in good condition can be unequivocally ascribed to the corresponding species in the complex (see Taxonomic section). Not all the differential traits highlighted here for the differentiation of the species in the U. crispa complex are new. Indeed, many have been used previously for the distinction of these taxa, although not always exactly as we do. It is unclear why the delimitation of the species in the complex has been unsatisfactory in the past, but it could rely on difficulties related to the interpretation of characters. On the one hand, it seems that some of the most useful traits considered here were poorly or ambiguously defined. This was the case for the features related to the capsule shape and appearance. Capsule outline and colour, its constriction below the mouth and the aspect of the capsule ribs, although of great interest for the distinction of these species (Table 1), show a wide variation that depends on the capsule condition (full or empty of spores, dry or wet state and degree of ripeness). Our conclusion is that, to be informative, these features must be referred to a specific condition (wet or dry, full of spores or empty). A similar situa- © 2016 The Linnean Society of London, Botanical Journal of the Linnean Society, 2016, 180, 427–451 Downloaded from https://academic.oup.com/botlinnean/article-abstract/180/4/427/2416569 by guest on 23 May 2020 Figure 1. Consensus phylogram based on Bayesian inference (BI) resulting from the analysis of the matrix combining ITS, trnG, trnL-trnF and atpB-rbcL datasets, treating indels as missing information. The numbers above the branches indicate node supports when indels were coded and considered in the analyses; the numbers below the branches indicate node supports when the indels were treated as missing data. The first value indicates BI support (posterior probability, values shown are those > 0.9); the second value indicates maximum parsimony (MP) (bootstrap support, values shown are those > 80). The tree was rooted using Zygodon pentastichus as outgroup. Sample numbers are indicated in Appendix 2. 436
ET AL. R. CAPARROS also highlighted by other bryologists (Wilson, 1855; Schimper, 1876) and specifically treated by Jones (1951), who confirmed previous data and further suggested that the maturation of capsules in U. intermedia and U. crispa occurred at the same time. Although Smith & Hill (1975) later provided data that seemed to blur these phenological trends, our observations agree with those of Jones (1951). TAXONOMIC SECTION As discussed above, the three species considered here have an important set of differential characters. However, the identification of Ulota samples can be a complex task depending on the degree of development of the plants, the availability of mature sporophytes and the status of preservation of the material under study. The following artificial key can be used for the identification of samples in good condition, i.e. well-developed cushions with mature capsules bearing complete peristomes, belonging to the involved species. For samples in suboptimal conditions, a wider set of characters might be needed. Table 1 summarizes the most useful traits to be taken into account when discerning between U. crispa, U. crispula and U. intermedia, but careful evaluation of the full set of traits provided in the descriptions of these species could also be necessary before naming some particularly difficult samples. KEY TO SPECIES 1 Capsules when dry and empty strongly constricted below mouth, with ribs separated by narrow furrows, collapsed at the constricted area of the urn; exothecial bands formed by four to six rows of cells, reaching the mouth or nearly so; exostome teeth remaining tightly fused in pairs in empty capsules, bordered by a hyaline halo. . . . . . . . . . . . . . . . . . . . . . . U. crispa s.s. 10 Capsules when dry and empty not or slightly constricted below mouth, with ribs separated by more or less broad furrows in the upper half of urn; exothecial bands formed by two to four (five) rows of cells, visibly separated from the mouth by a ring of small thin-walled cells; pairs of exostome teeth partially splitting in empty capsules, teeth not bordered by a hyaline halo . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 2 Endostome segments incurved when dry, uniseriate with incrassate and prominent transverse walls; all the cells of the exothecial bands hyaline with pale yellow incrassate lateral walls; leaves markedly crisped when dry, abruptly narrowing from a concave base . . . . . . . . . . . . . U. intermedia © 2016 The Linnean Society of London, Botanical Journal of the Linnean Society, 2016, 180, 427–451 Downloaded from https://academic.oup.com/botlinnean/article-abstract/180/4/427/2416569 by guest on 23 May 2020 tion has occurred with the leaf base, as its shape must be specified in terms of outline and, in particular, by the degree of narrowing towards the lamina and concavity. However, most quantitative traits used previously, such as the capsule and seta lengths or leaf crispature, show a broad variability and should not be used as main differential characters, although they can help in the identification as complementary features in many cases. The degree of leaf crispature when dry could be useful to separate U. crispula, which normally is evidently less crisped than U. crispa and U. intermedia. However, it is not uncommon to find more strongly crisped exemplars of U. crispula and exceptionally slightly crisped forms of U. crispa, which seem to be especially frequent in Japan. Most of the authors who supported the distinction of the taxa in the U. crispa complex based it on a misleading interpretation of the morphological variability, without finding significant qualitative characters for a sharp distinction. As a consequence, they could only establish typical trends of morphological variation for each taxon and, at the same time, indicated the existence of intermediate forms. This fact was decisive for more synthetic taxonomists or those who knew the complex less well and gave little relevance to the differences shown by the extreme forms. In both cases, the result was the classification of all forms under a single species name. However, as pointed out above, qualitative differential characters for the distinction of the three Ulota spp. exist and, in most cases, they are related to structures that were studied only superficially, e.g. the peristome and the exothecial bands. Other differences have been completely overlooked, e.g. spore size and the differentiation of a basal rim in the operculum. In our opinion, the sum of differential morphological traits found in this study should be sufficient for the definitive recognition of U. crispula and U. intermedia as distinct species. The molecular evidence provided in this study adds support to the recognition of the three taxa. Even in the cases in which thorough morphological investigations provide convincing taxonomic evidence, integrative taxonomy offers the opportunity to obtain strong support for the conclusions (e.g. Medina et al., 2013; Heden€ as et al., 2014). For such a purpose, molecular evidence is the most used and convincing today (Heinrichs et al., 2015). Nevertheless, other sources of evidence are also possible (Bickford et al., 2007). In the case of the U. crispa complex, an additional taxonomic signal comes from phenology and has been known for a long time. Bruch & Schimper (1837) stated that sporophytes of U. crispa and U. crispula mature at different times: in late summer for the former vs. usually in late spring for the latter. This was INTEGRATIVE STUDY OF ULOTA CRISPA 20 Endostome segments variably bent when dry, uniseriate or irregularly biseriate with thin transverse walls; cells of the exothecial bands evenly pale yellow, at least in the two central rows; leaves slightly or moderately crisped when dry, gradually narrowing from a plane to slightly concave base . . . . . . . . . . . . . . . . . . . . U. crispula DESCRIPTIONS A  Orthotrichum crispum Hedw., Sp. musc. frond. 162. 1801 (basionym).  Bryum crispum (Hedw.) With., Syst. arr. Brit. pl. (ed. 4) 3: 810. 1801.  Weissia crispa (Hedw.) P.Gaertn., B.Mey. & Scherb., Oekon. Fl. Wetterau 3(2): 94. 1802.  Systegium crispum (Hedw.) Schur, Enum. pl. Transsilv. 866. 1866. hom. illeg. [non S. crispum (Hedw.) Schimp. - 1860]. Type: In saxis, arborum truncis per omnem Europam. Lectotype: s. loc., s. coll. (G 00040001!, herb. B C D E F Figure 2. Comparison of general habit and capsule shape when dry and empty of Ulota crispa (A, B; MAUAM-Brio 4441), U. crispula (C, D; MAUAM-Brio 4439) and U. intermedia (E, F; MAUAM-Brio 4749). © 2016 The Linnean Society of London, Botanical Journal of the Linnean Society, 2016, 180, 427–451 Downloaded from https://academic.oup.com/botlinnean/article-abstract/180/4/427/2416569 by guest on 23 May 2020 SPECIES Ulota crispa (Hedw.) Brid., Muscol. recent. suppl. 4: 112. [1818] 1819. 437 438
ET AL. R. CAPARROS B C D E F G H I Figure 3. Comparison of Ulota crispa (A–C; MAUAM-Brio 4441), U. crispula (D–F; MAUAM-Brio 4439) and U. intermedia (G–I; MAUAM-Brio 4749). A, D, G, Details of the capsule mouth with well-preserved peristome teeth and segments; in D, the characteristic teeth splitting of U. crispula can be observed in the frontal tooth pair. B, E, H, Images of the exothecial bands; note the thinnest band in U. crispula, only two cells wide, and the suboral ring of short cells in U. crispula and U. intermedia. C, F, I, Different transitions from leaf base to lamina: abruptly narrowing in U. crispa and U. intermedia, and gradual in U. crispula. © 2016 The Linnean Society of London, Botanical Journal of the Linnean Society, 2016, 180, 427–451 Downloaded from https://academic.oup.com/botlinnean/article-abstract/180/4/427/2416569 by guest on 23 May 2020 A INTEGRATIVE STUDY OF ULOTA CRISPA C E B D F Figure 4. Comparison of peristome of Ulota crispa (A, B; MAUAM-Brio 4441), U. crispula (C, D; MAUAM-Brio 4439) and U. intermedia (E, F; MAUAM-Brio 4749). A, C, E, Views of exostome tooth pairs and a principal endostome segment. B, D, F, Details of an endostome segment; in B and F, transversal walls are clearly incrassate. The hyaline halo of the peristome of U. crispa can be observed in the central part of the tooth (A); in this case, the halo is not particularly well developed. The trabeculae of the primary peristomial layer (PPL) of U. crispa (A) and U. intermedia (E) cause the retention of the air droplets that appear in these photographs; the trabeculae are also apparent in Figure 3A, G. Hedwig-Schw€ agrichen). Lectotypified by Caparr
os et al. (in press). Figures 2A, B; 3A–C; 4A, B. Plants to 3.3 cm high, usually strongly crisped, growing in dense, medium to large cushions, olivaceous to dark green above, brownish to reddish below. Stems abundantly branched. Rhizoids common at stem base, frequently also scattered along stems, brown-reddish, smooth. Leaves mostly tortuose or circinate, occasionally falcate when dry, erect-patent to squarrose when wet, (1.3–)1.4–3.7 (–3.8) 9 0.3–0.9(–1.0) mm; leaf lamina lanceolate to narrowly lanceolate, unistratose, variably keeled; leaf base wide, obovate, rounded or oblate, distinctly concave, abruptly narrowing into lamina. Apex acuminate, sometimes channelled or with one margin irregularly incurved. Margins weak and variably recurved on one or both sides of lamina. Costa ending at some distance from apex. Median and upper leaf cells rounded to elliptic, (6–)9–16(– © 2016 The Linnean Society of London, Botanical Journal of the Linnean Society, 2016, 180, 427–451 Downloaded from https://academic.oup.com/botlinnean/article-abstract/180/4/427/2416569 by guest on 23 May 2020 A 439 440
ET AL. R. CAPARROS Distribution: West coast of North America, common and widespread throughout Europe, south-west Asia (Pontic Mountains, Caucasus), China, Russian Far East, Japan and Taiwan. Ulota crispula Bruch, Bryol. Univ. 1: 793. 1827.  Orthotrichum crispulum (Bruch) Hornsch., Bryol. Eur. fasc. 2-3: 23. 1837.  Ulota crispa var. crispula (Bruch) Hammar, Monogr. Orthotrich. Ulot. Suec. 24. 1852.  Orthotrichum crispum ssp. crispulum (Bruch) Nyl. & Saelan, Herb. Mus. Fenn. 66. 1859.  Systegium crispula (Bruch) Schur, Enum. pl. Transsilv. 866. 1866.  Weissia crispula (Bruch) Lindb., Acta Soc. Sci. Fenn. 10: 12. 1871. Illegitimate, later homonym [non W. crispula Hedw. - 1801].  Orthotrichum ulophyllum ssp. crispulum (Bruch) Kindb., Bih. Kongl. Svenska Vetensk.-Akad. Handl. 7(9): 121. 1883.  Orthotrichum cupulatum ssp. crispulum (Bruch) Boulay, Musc. France, mousses 344. 1884.  Weissia ulophylla var. crispula (Bruch) Braithw., Brit. moss Fl. 2: 94. 1889.  Orthotrichum cupulatum var. crispulum (Bruch) Dalla Torre, Z. Ferdinandeums Tirol 35: 241. 1891. Hom. illeg.  Ulota crispa ssp. crispula (Bruch) H
erib., M
em. Acad. Sci. Clermont-Ferrand 14: 333. 1899.  Ulota ulophylla var. crispula (Bruch) Weim., F€orteckn. Skand. V€ axt., moss. (ed. 2) 40. 1937.  Ulota ulophylla ssp. crispula (Bruch) Giacom., Ist. Bot. Reale Univ. Reale Lab. Crittog. Pavia, Atti 4: 249. 1947. Type: In arboribus sylvaticis c. Bipontium clar. Bruch detexit; a quo ut et a Funckio amice communicatum habemus. Lectotype: [Germany, RheinlandPfalz: Zweibr€ ucken]. ‘An Waldb€ aumen bey Zweybr€ ucken/(Bruch) [s.n.]’ (B 31 0251 01!). Lectotypified by Caparr
os et al. (in press). Figures 2C, D; 3D–F; 4C, D. Plants to 4.2 cm high, variably crisped, growing in dense, small to large cushions, olivaceous above, brown-reddish to dark brown below. Stems abundantly branched. Rhizoids abundant at stem base and frequently ascending along stems, brownreddish, smooth. Leaves curved to tortuose, more rarely circinate when dry, erect-patent to squarroserecurved when wet, (0.7–)1.2–2.8(–3.0) 9 (0.1–) 0.3–0.6(–0.7) mm long; leaf lamina lanceolate to narrowly lanceolate, unistratose, variably keeled; leaf base elliptical, sometimes oblong to obovate, slightly concave, usually gradually narrowing into lamina. Apex acuminate to long acuminate, frequently channelled or with one margin irregularly © 2016 The Linnean Society of London, Botanical Journal of the Linnean Society, 2016, 180, 427–451 Downloaded from https://academic.oup.com/botlinnean/article-abstract/180/4/427/2416569 by guest on 23 May 2020 22) 9 (6–)9–12(–17) lm, with moderately incrassate cell walls, with one or two low and simple, frequently inconspicuous papillae; basal leaf cells long rectangular to linear, (15–)25–45(–74) 9 (5–)7– 11(–14) lm, with incrassate, sinuous, sometimes porose cell walls; differentiated basal marginal cells in (5–)7–16(–20) rows, forming a broad marginal band along leaf base. Goniautoicous. Perichaetial leaves differentiated, clearly longer and with wider and more elongated base than vegetative leaves, apex acute to acuminate, and costa frequently percurrent. Vaginula naked or variably hairy, with hairs partially two- to four-seriate, papillose, hyaline. Sporophyte (3.0–)3.2–6.8(–7.4) mm long. Seta (0.3–)0.5–4.4(–5.0) mm long. Capsule long exserted. Urn cylindrical to ellipsoid when dry and full of spores; clearly urceolate, strongly constricted below mouth when dry and empty, then with eight long and prominent ribs separated by narrow furrows, collapsed at the constricted area of urn; (0.5–) 0.8–1.7(–2.0) mm long, yellowish brown to reddish brown with concolorous ribs. Neck usually long, 0.5–3.0 times as long as urn, gradually tapering into seta. Exothecial bands four or five (six) cells wide, almost as long as urn, reaching the mouth or obscurely separated from it by a thin ring, 8–33(–38) lm tall, formed by one to three (four) layers of small, thin-walled cells; differentiated cells of the band evenly yellow to pale orange, not concolorous with other exothecial cells. Stomata located in urn base and upper part of neck. Peristome double. Prostome frequent, usually incomplete and low, more rarely well developed. Exostome of eight pairs of teeth remaining tightly fused even in old capsules, yellowish to orange, recurved and lying on the exothecium when dry; outer surface outer peristome layer (OPL) densely ornamented by thin papillae, weakly striate in the distal portion; inner surface (exostomial Principal Peristome Layer [PPL]) smooth or finely papillose, frequently with distinct trabeculae; PPL visible externally as a hyaline halo as OPL edges partially retract. Endostome of eight robust and persistent segments, occasionally with up to eight additional intermediate, sometimes shorter processes; principal segments c. two-thirds the length of teeth, long triangular to subulate, uniseriate with transversal walls variably incrassate, incurved when dry, translucent and shiny; external surface (endostomial PPL) smooth; internal surface inner peristome layer (IPL) smooth to finely papillose below, variably papillose above, but never opaque because of the papillae density. Operculum plane-convex to conic, rostrate, yellowish without differentiated basal ring. Calyptra hairy, hairs one- to four- (to five-) seriate, exceeding the beak. Spores (12–)14–23(–25) lm in diameter. INTEGRATIVE STUDY OF ULOTA CRISPA with a dense reticulum of striae which, in extreme cases, may look like an opaque and almost smooth ornamentation. Operculum plane to convex, rarely conic, rostrate, yellowish, usually with an orange to reddish basal ring. Calyptra hairy, hairs two- or three- (four-) seriate, variably exceeding the beak. Spores (18–)20–29(–30) lm in diameter. Distribution: Amphi-Atlantic, widespread throughout eastern North America, Europe and south-western Asia (Pontic Mountains, Caucasus). Ulota intermedia Schimp. Syn. musc. Eur. (ed. 2): 305. 1876.  Orthotrichum ulophyllum var. intermedium (Schimp.) Kindb., Bih. Kongl. Svenska Vetensk.Akad. Handl. 7(9): 121. 1883.  Ulota crispa var. intermedia (Schimp.) Cardot, Bull. Soc. Roy. Bot. Belgique 24(2): 26. 1885.  Weissia ulophylla var. intermedia (Schimp.) Braithw., Brit. moss Fl. 2: 94. 1889. Illegitimate, earlier name included.  Weissia intermedia (Schimp.) Tolf, Bih. Kongl. Svenska Vetensk.-Akad. Handl. 16 Afd. 3(9): 70. 1891.  Orthotrichum ulophyllum ssp. intermedium (Schimp.) Kindb., Eur. N. Amer. Bryin. 2: 307. 1897.  Ulota crispa ssp. intermedia (Schimp.) H
erib., M
em. Acad. Sci. Clermont-Ferrand 14: 333. 1899.  Orthotrichum intermedium (Schimp.) Kindb. ex Paris, Index bryol. (ed. 2) 3: 331. 1905. nom. inval.  Ulota ulophylla var. intermedia (Schimp.) Jansen & Wacht., Ned. Kruidk. Arch. 53: 215. 1943. Type: Hic illic per Europam D. crispae et crispulae associata: in Hassia superior ad Fagos male evolutas (Bruch), prope Jever Oldenburgiae, ipse copiose legi ad arborum, praeprimis Fraxini et Aceris truncos in valle Scotiae Loch Lomond. Lectotype: [Germany, Hesse: Marburg-Biedenkopf]. ‘Ludwigsh€ utte. Jul Aug. 41. Bruch m. Nov. 41’ (BM 000852210!). Lectotypified by Caparr
os et al. (in press). Figures 2E, F; 3G–I; 4E, F. Plants to 3.5 cm high, crisped, growing in dense, medium to large cushions, olivaceous to dark green above, dark brown below. Stems abundantly branched. Rhizoids at stem and branch bases, sometimes ascending along stems, brown-reddish, smooth. Leaves tortuose or circinate, occasionally falcate-tortuose when dry, erect-patent to squarroserecurved when wet, (1.0–)1.5–3.4(–3.7) 9 0.3–0.7(– 0.8) mm; leaf lamina lanceolate to narrowly lanceolate, unistratose, variably keeled; leaf base obovate to orbicular, rarely oblate or elliptic, distinctly concave, abruptly narrowing into lamina. Apex acuminate to long acuminate, sometimes acute, frequently channelled. Margins weak and irregularly recurved on one or both sides, crenulate to crenulate-papillose. © 2016 The Linnean Society of London, Botanical Journal of the Linnean Society, 2016, 180, 427–451 Downloaded from https://academic.oup.com/botlinnean/article-abstract/180/4/427/2416569 by guest on 23 May 2020 incurved. Margins weak and variably recurved on one or both sides. Costa ending at some distance from apex, sometimes close to it. Median and upper leaf cells rounded to elliptic, (5–)8–18 (–31) 9 (5–)8–12(–18) lm, with moderately incrassate walls, smooth or with one or two low and simple, frequently inconspicuous papillae; basal leaf cells long rectangular to linear, (11–)25–40(–60) 9 (5–)6– 10(–13) lm with incrassate, sinuous, sometimes porose cell walls; differentiated basal marginal cells in (1–)3–8(–10) rows, forming a relatively narrow marginal band along leaf base. Goniautoicous. Perichaetial leaves somewhat differentiated, lanceolate to widely lanceolate, longer and wider than regular leaves, leaf base oblong to obovate, variably sheathing. Vaginula hairy, sometimes densely, with hairs partially two- or three-seriate, papillose, hyaline. Sporophyte (1.8–)2.0–3.8(–4.0) mm long. Seta 0.9–2.1(–3.0) mm long. Capsule exserted. Urn short cylindrical to ovoid or slightly urceolate when dry and full of spores; short cylindrical, somewhat urceolate, or obconic (gradually tapering from mouth to seta), not or slightly constricted below mouth when dry and empty, then with eight prominent ribs usually separated by broad, somewhat sinuous or irregular furrows; (0.6–)0.7–1.2 mm long, pale yellow to brownish yellow, with darker ribs. Neck long, 0.4–2.0 times as long as urn, gradually tapering into seta. Exothecial bands two to four cells wide, almost as long as urn, separated from the mouth by a conspicuous ring, (12–)18–65(–75) lm tall, formed by (one–)two to four(–five) layers of small, thinwalled cells; differentiated cells of the band evenly pale yellow and not concolorous with other exothecial cells, rarely hyaline and concolorous (eastern North America). Stomata located in urn base and upper part of neck. Peristome double. Prostome frequent, usually incomplete and low, sometimes developed up to one-third to one-half of teeth length. Exostome of eight pairs of teeth with marked tendency to split into 16, yellowish to pale orange, recurved and lying on the exothecium when dry; outer surface (OPL) ornamented by thick papillae, sometimes becoming longitudinally striate or with crests in upper one-half to one-third; inner surface (exostomial PPL) smooth, occasionally with sparse, sometimes aligned, papillae in upper half; PPL not externally visible at tooth margins. Endostome of eight slender and fragile segments, occasionally with up to eight additional intermediate, sometimes shorter processes; principal segments almost as long as teeth, almost linear with variably widened base, irregularly biseriate, with thin transversal walls, incurved, erect or patent when dry, variably translucent at base or lower half, more or less opaque in upper part; external surface (endostomial PPL) smooth; internal surface (IPL) 441 442
ET AL. R. CAPARROS or finely papillose in lower half, variably reticulate above but never opaque because of the ornamentation density. Operculum plane to convex, sometimes conic, rostrate, yellowish without differentiated basal ring. Calyptra variably hairy, hairs (one-) two- to four- (five-) seriate, not or scarcely exceeding the beak. Spores (15–)18–35(–38) lm in diameter. Distribution: Western and eastern North America, northern Europe and southern European cordilleras, Altai, Russian Far East and Japan. SPECIES DISTRIBUTION Ulota crispa s.l. has been reported from several, mainly oceanic, areas in both the Nearctic and Palaearctic. In North America, it is currently recognized for the eastern third of the continent (Vitt, 2014), although Grout (1935, 1946) also signalled records from the west coast in southern Alaska. In Europe, it is considered to be widespread, being scarce or absent in different inland and Mediterranean territories (Lewinsky-Haapasaari, 1998; Ignatov et al., 2006; Ros et al., 2013). In Asia, U. crispa has been reported from four disjunct territories (Noguchi, 1989; Ignatov & Ochyra, 1994; Ignatov et al., 2006; Jia, He & Guo, 2011; Wang & Jia, 2012): south-western Asia, in mountainous areas bordering the Black and Caspian seas; Altai and the neighbouring Central Asian mountains; Russian Far East and Japan; and subtropical China and surroundings. Thus, U. crispa is present in all or most of the centres of diversity of Ulota in the Holarctic kingdom, as defined in Garilleti et al. (2015). In addition, for North America and Europe, a number of mainly ancient records have reported U. intermedia or U. crispula at specific or variety levels (e.g. Lesquereux & James, 1884; Husnot, 1892–1894; Grout, 1935; Nyholm, 1956). The results of our taxonomic revision draw a substantially different biogeographical panorama, as each of the three recognized species shows a different Holarctic disjunct distribution (Fig. 5). Ulota crispula displays an amphi-Atlantic area, being widespread throughout eastern North America (southern Canada and USA, except southernmost areas), most of Europe (rare in northernmost and Mediterranean areas) and south-western Asia (Pontic Mountains, Caucasus). Ulota intermedia shows a much more wider and fragmented area, being present in western North America (mainly Alaska and Canada), eastern North America (mainly Canada), northern Europe, European cordilleras (North Iberian ranges, Alps, Carpathians, etc.), Altai, Russian Far East and Japan (most main islands, not recorded from Kyushu). This species seems to prefer high elevational © 2016 The Linnean Society of London, Botanical Journal of the Linnean Society, 2016, 180, 427–451 Downloaded from https://academic.oup.com/botlinnean/article-abstract/180/4/427/2416569 by guest on 23 May 2020 Costa ending at some distance from apex. Median and upper leaf cells rounded to elliptic, 7–16(– 17) 9 (6–)7–13 lm, with moderately incrassate walls, with one or two simple or bifurcate papillae; basal leaf cells long rectangular to linear, (18–)20–63(– 68) 9 5–10(–12) lm, with incrassate, sinuous, sometimes porose cell walls; differentiated basal marginal cells in (2–)3–14(–16) rows, forming a broad marginal band along leaf base. Goniautoicous. Perichaetial leaves differentiated, clearly longer than regular leaves, leaf base long ellipsoidal to oblong-ellipsoidal, sometimes obovate or almost orbicular, frequently sheathing, apex acute to shortly acuminate. Vaginula naked or hairy, with hairs partially one- or two- (three-) seriate, papillose, hyaline. Sporophyte (3.0–)3.2–6.5 mm long. Seta 1.0–4.0(–5.5) mm long. Capsule long exserted. Urn ellipsoidal to cylindrical-ellipsoidal, rarely long ovoid when dry and full of spores; cylindrical, oblongcylindrical, obconic or elongate-ovoid, not constricted below the mouth when dry and empty, then with prominent ribs separated by broad furrows; (0.6–) 0.8–1.5(–1.7) mm long, pale yellow to brownish yellow, with darker ribs. Neck long, 0.5–2.0 times as long as urn, gradually tapering into seta. Exothecial bands two to four (five) cells wide, almost as long as urn, separated from the mouth by a conspicuous ring, (15–)18–113(–238) lm tall, formed by (one-) two to six (-seven) layers of small, thin-walled cells; differentiated cells of the band hyaline with pale yellow incrassate lateral walls, concolorous with other exothecial cells. Stomata located in urn base and upper part of neck. Peristome double. Prostome frequent, incomplete and low or with fragments irregularly sparse along teeth, sometimes well developed. Exostome of eight teeth pairs with tendency to split into 16, pale yellow with bases sometimes becoming darker, brown–orange, recurved and lying on the exothecium, except sometimes at their bases, when dry; outer surface (OPL) with a dense papillose reticulum, sometimes the papillae fused into transverse striae in lower part or into longitudinal weak striae in upper third; inner surface (exostomial PPL) smooth or finely papillose, sometimes the papillae forming thin lines in lower half, frequently with marked trabeculae and fenestrate in upper third; PPL not visible externally at tooth margins. Endostome of eight robust and usually persistent segments, occasionally with up to eight additional intermediate, sometimes shorter processes; principal segments three-quarters as long as teeth, broadly linear with variably widened base to subulate, uniseriate, usually with incrassate and prominent transverse walls, incurved when dry, hyaline and usually shiny; external surface (endostomial PPL) smooth; internal surface (IPL) smooth INTEGRATIVE STUDY OF ULOTA CRISPA 443 or altitudinal areas in the temperate zone. Finally, U. crispa s.s., although displaying a wide and disjunct distribution throughout the Holarctic, is not present in all the territories in which it has formerly been recognized. Although uncommon, it is present along the west coast of North America (from British Columbia to Oregon), where no previous records seem to exist. In contrast, we have not found any sample ascribable to this species from eastern North America, which, in turn, always corresponded either to U. crispula or, usually in northern areas, to U. intermedia. In Europe, U. crispa s.s. is a common and widespread species and frequently coexists with U. crispula, as has been verified recently for the Iberian Peninsula (Caparr
os et al., 2014). Finally, in Asia, U. crispa s.s. is confirmed for the south-western part of the continent (Pontic Mountains, Caucasus), China, Russian Far East, Japan (all main islands) and Taiwan; we have no evidence of its presence in Central Asian mountains where, however, U. intermedia occurs. For a taxonomic work like this, the revision of all existing records related to the U. crispa complex is an unattainable goal. However, the importance of these species for most North American and Eurasian floras implies that, before long, a much more accurate drawing of the distribution of these three Ulota spp. will be available, based on the re-evaluation of samples under the new taxonomic light by trained bryologists around the world. ACKNOWLEDGEMENTS The authors gratefully thank the curators and staff of the following herbaria: BCB, BM, BP, DUKE, FCO, G, H, HIRO, LISU, MA, MHA, MO, MUB, NICH, NY, S, TU and VIT. Thanks are also due to Pierre Boudier and Des Callaghan for loaning material from their personal herbaria. In addition, the curators and staff of BM, NY and G are especially acknowledged for hosting RC and providing her with all facilities during her stay in these institutions. Two anonymous referees and the associate editor are also acknowledged for their useful comments on an earlier version of the manuscript. This work was funded by the Spanish Ministries of Economy and Competitiveness (CGL2013-43246-P) and Science and Innovation (CGL2011-28857/BOS). RC benefitted from a doctoral grant from the Ministry of Education and Culture (Grant AP2007-04693). REFERENCES Albertos B, Lara F, Garilleti R, Mazimpaka V. 2000. Distribution and abundance of corticolous species of the genus Ulota Mohr (Orthotrichaceae, Musci) in the NW Iberian Peninsula. Nova Hedwigia 70: 461–470. Bickford D, Lohman DJ, Sodhi NS, Ng PKL, Meier R, Winker K, Ingram K, Das I. 2007. Cryptic species as a window on diversity and conservation. Trends in Ecology and Evolution 22: 148–155. © 2016 The Linnean Society of London, Botanical Journal of the Linnean Society, 2016, 180, 427–451 Downloaded from https://academic.oup.com/botlinnean/article-abstract/180/4/427/2416569 by guest on 23 May 2020 Figure 5. World approximate distribution of the different species in the Ulota crispa complex, based on the studied material (see Appendix 1). 444
ET AL. R. CAPARROS chaceae: Bryophyta) based on variation in four loci from all genomes. Monographs in Systematic Botany from the Missouri Botanical Garden 98: 270–289. Goloboff P, Farris J, Nixon K. 2008. TNT, a free program for phylogenetic analyses. Cladistics 24: 774–786. Grismer LL, Wood PL Jr, Anuar S, Muin MA, Quah ESH, Mcguire JA, Brown RM, van Tri N, Thai PH. 2013. Integrative taxonomy uncovers high levels of cryptic species diversity in Hemiphyllodactylus Bleeker, 1860 (Squamata: Gekkonidae) and the description of a new species from Peninsular Malaysia. Zoological Journal of the Linnean Society 169: 849–880. Grout AJ. 1935. Moss flora of North America north of Mexico, vol. 2(2). Newfane, VT: The author. Grout AJ. 1946. North American Flora, vol. 15A(1), Orthotrichaceae. New York: New York Botanical Garden. Hammar O. 1857. Monographia Orthotricharum et Ulotarum Sueciae, Pars III. Lund. Heden€ as L, D
esamor
e A, Laenen B, Papp B, Quandt D, Gonz
alez-Mancebo JM, Pati~ no J, Vanderpoorten A, Stech M. 2014. Three species for the price of one within the moss Homalothecium sericeum s.l. Taxon 63: 249–257. Hedwig J. 1801. Species muscorum frondosorum. Leipzig. Heinrichs J, Feldberg K, Bechteler J, Scheben A, Czumaj A, P
ocs T, Schneider TH, Sch€ afer-Verwimp A. 2015. Integrative taxonomy of Lepidolejeunea (Jungermanniopsida: Porellales): Ocelli allow the recognition of two neglected species. Taxon 64: 216–228. Huelsenbeck JP, Ronquist F. 2001. MrBayes: Bayesian inference of phylogeny. Bioinformatics 17: 754–755. Husnot T. 1892–1894. Muscologia Gallica. Descriptions et figures des mousses de France et des contr
ees voisines. Paris. Ignatov MS, Afonina OM, Ignatova EA, Abolina A, Akatova TV, Baisheva EZ, Bardunov LV, Baryakina EA, Belkina OA, Bezgodov AG, Boychuk MA, Cherdantseva VYA, Czernyadjeva IV, Doroshina GYA, Dyachenko AP, Fedosov VE, Goldberg IL, Ivanova EI, Jukoniene I, Kannukene L, Kazanovsky SG, Kharzinov ZKH, Kurbatova LE, Maksimov AI, Mamatkulov UK, Manakyan VA, Maslovsky OM, Napreenko MG, Otnyukova TN, Partyka LYA, Pisarenko OYU, Popova NN, Rykovsky GF, Tubanova DYA, Zheleznova GV, Zolotov VI. 2006. Check-list of mosses of East Europe and North Asia. Arctoa 15: 1–130. Ignatov MS, Ochyra R. 1994. Bryophytes of Altai Mountains. III. The genus Ulota (Orthotrichaceae, Musci). Arctoa 3: 59–66. Jia Y, He S, Guo S-L. 2011. Orthotrichaceae. In: PengCheng W, Crosby MR, He S, eds. Moss Flora of China, English version. Volume 5: Erpodiaceae-Climaciaceae. Beijing and St. Louis, OH: Science Press and Missouri Botanical Garden Press. Jones EW. 1951. The fruiting time of Ulota bruchii Hornsch. and U. crispa Brid. Transactions of the British Bryological Society 1: 20–21. Kelchner SA. 2000. The evolution of non-coding chloroplast DNA and its application in plant systematics. Annals of the Missouri Botanical Garden 87: 482–498. © 2016 The Linnean Society of London, Botanical Journal of the Linnean Society, 2016, 180, 427–451 Downloaded from https://academic.oup.com/botlinnean/article-abstract/180/4/427/2416569 by guest on 23 May 2020 Boulay JN. 1884. Muscin
ees de la France. Premi ere partie. Paris: Mousses. Braithwaite R. 1888–1895. The British moss-flora, vol. 2. London. Bridel SE. 1818. Muscologia recentiorum, Suppl. 4. Gotha. Bridel SE. 1827. Bryologia Universa, Vol. 1, Suppl. J. Leipzig. Bruch P, Schimper WP. 1837. Bryologia Europaea seu genera muscorum Europaeorum monographice illustrata, Fasc. 2 et 3. Stuttgart. Buchbender V, Hespanhol H, Krug M, S
ergio C, S
eneca A, Maul K, Heden€ as L, Quandt D. 2014. Phylogenetic reconstructions of the Hedwigiaceae reveal cryptic speciation and hybridisation in Hedwigia. Bryophyte Diversity and Evolution 36: 1–21. Caparr
os R, Garilleti R, Lara F. 2014. Ulota D. Mohr. In: Guerra J, Cano MJ, Brugu
es M, eds. Flora briof
ıtica Ib
erica, vol. V. Murcia: Universidad de Murcia, Sociedad Espa~ nola de Briolog
ıa. Caparr
os R, Garilleti R, Price MJ, Mazimpaka V, Lara F. Typification of Orthotrichum crispum Hedw., Ulota crispula Bruch and Ulota intermedia Schimp. (Bryopsida: Orthotrichaceae). Taxon in press. Chiang T-Y, Schaal BA, Peng C-I. 1998. Universal primers for amplification and sequencing a noncoding spacer between the atpB and rbcL genes of chloroplast DNA. Botanical Bulletin of Academia Sinica 39: 245–250. Damayanti L, Mu~ noz J, Wicke S, Symmank L, Shaw B, Frahm J-P, Quandt D. 2012. Common but new: Bartramia rosamrosiae, a “new” widespread species of apple mosses (Bartramiales, Bryophytina) from the Mediterranean and western North America. Phytotaxa 73: 37–59. Darriba D, Taboada GL, Doallo R, Posada D. 2012. jModelTest 2: more models, new heuristics and parallel computing. Nature Methods 9: 772. Dayrat B. 2005. Towards integrative taxonomy. Biological Journal of the Linnean Society 85: 407–415. Dillenius U. 1742. Historia muscorum. Oxford. Dixon HN. 1924. The student’s handbook of British mosses, 3rd edn. Sumfield. Draper I, Heden€ as L, Stech M, Pati~ no J, Werner O, Gonz
alez-Mancebo JM, Sim-Sim M, Lopes T, Ros RM. 2015. How many species of Isothecium (Lembophyllaceae, Bryophyta) are there in Macaronesia? A survey using integrative taxonomy. Botanical Journal of the Linnean Society 177: 418–438. Garilleti R, Lara F, Albertos B, Mazimpaka V. 2000. Peristomial ornamentation, a precise character for discrimination of Ulota bruchii and U. crispa (Orthotrichaceae, Bryopsida). Journal of Bryology 22: 273–278. Garilleti R, Mazimpaka V, Lara F. 2012. New Ulota species with multicellular spores from southern South America. Bryologist 115: 585–600. Garilleti R, Mazimpaka V, Lara F. 2015. Ulota larrainii (Bryophyta, Orthotrichaceae) a new species from Chile, with comments on the worldwide diversification of the genus. Phytotaxa 217: 133–144. Goffinet B, Shaw AJ, Cox CJ, Wickett NJ, Boles S. 2004. Phylogenetic inferences in the Orthotrichoideae (Orthotri- INTEGRATIVE STUDY OF ULOTA CRISPA maries. Available at: http://mrbayes.sourceforge.net/mb3.2_manual.pdf (accessed 3 June 2015). Ronquist F, Huelsenbeck P. 2003. MrBayes 3: Bayesian phylogenetic inference under mixed models. Bioinformatics 19: 1572–1574. Ronquist F, Teslenko M, van der Mark P, Ayres DL, Darling A, H€ ohna S, Larget B, Liu L, Suchard MA, Huelsenbeck JP. 2012. MrBayes 3.2: efficient Bayesian phylogenetic inference and model choice across a large model space. Systematic Biology 61: 539–542. Ros RM, Mazimpaka V, Abou-Salama U, Aleffi M, Blockeel TL, Brugu
es M, Cros RM, Dia NG, Dirkse GM,  A, Ganeva A, Gabriel Draper I, El-Saadawi W, Erdag R, Gonz
alez-Mancebo JM, Granger C, Herrnstadt I, Hugonnot V, Khalil K, K€ urschner H, Losada-Lima A, Lu
ıs L, Mifsud S, Privitera M, Puglisi M, Sabovljevi
c M, S
ergio C, Shabbara HM, Sim-Sim M, Sotiaux A, Tacchi R, Vanderpoorten A, Werner O. 2013. Mosses of the Mediterranean, an annotated checklist. Cryptogamie, Bryologie 34: 99–283. Rosman-Hartog N, Touw A. 1987. On the taxonomic status of Ulota bruchii Hornsch. ex Brid., U. crispa (Hedw.) Brid. and U. crispula Bruch ex Brid. Lindbergia 13: 159–164. Sawicki J, Pl
asek V, Szczeci
nska M. 2010. Molecular studies resolve Nyholmiella (Orthotrichaceae) as a separate genus. Journal of Systematics and Evolution 48: 183–194. Schimper WP. 1876. Synopsis muscorum europaeorum, 2nd edn. Stuttgart. Schlick-Steiner BC, Steiner FM, Seifert B, Stauffer C, Christian E, Crozier RH. 2010. Integrative taxonomy: a multisource approach to exploring biodiversity. Annual Review of Entomology 55: 421–438. Simmons MP, Ochoterena H. 2000. Gaps as characters in sequence-based phylogenetic analyses. Systematic Biology 49: 369–381. Smith AJE, Hill MO. 1975. A taxonomic investigation of Ulota bruchii Hornsch. ex Brid., U. crispa (Hedw.) Brid. and U. crispula Bruch ex Brid. European material. Journal of Bryology 8: 423–433. Smith AJE, Proctor MCF. 1993. Further observations on the Ulota crispa complex. Journal of the Hattori Botanical Laboratory 74: 171–182. Stech M, Werner O, Gonz
alez-Mancebo JM, Pati~ no J, Sim-Sim M, Fontinha S, Hildebrandt L, Ros RM. 2011. Phylogenetic inference in Leucodon Schw€ agr. subg. Leucodon (Leucodontaceae, Bryophyta) in the North Atlantic region. Taxon 60: 79–88. Tamura K, Stecher G, Peterson D, Filipski A, Kumar S. 2013. MEGA6: Molecular Evolutionary Genetics Analysis version 6.0. Molecular Biology and Evolution 30: 2725– 2729. Vanderpoorten A, Sch€ afer-Verwimp A, Heinrichs J, Devos N, Long DG. 2010. The taxonomy of the leafy liverwort genus Leptoscyphus (Lophocoleaceae) revisited. Taxon 59: 176–186. Vigalondo B, Fern
andez-Mazuecos M, Vargas P, S
aez L. 2015. Unmasking cryptic species: morphometric and phylogenetic analyses of the Ibero-North African Linaria © 2016 The Linnean Society of London, Botanical Journal of the Linnean Society, 2016, 180, 427–451 Downloaded from https://academic.oup.com/botlinnean/article-abstract/180/4/427/2416569 by guest on 23 May 2020 Lesquereux L, James TP. 1884. Manual of the mosses of North America. Boston. Lewinsky-Haapasaari J. 1998. Orthotrichales. In: E Nyholm, ed. Illustrated flora of Nordic mosses. Fascicle 4. Copenhagen and Lund: Nordic Bryological Society. Limpricht KG. 1890–1895. Die Laubmoose Deutschlands, € Osterreichs und der Schweiz. In: A Fischer, F Hauck, G Limpricht, Ch Luerssen, W Migula, H Rehm, P Richter, G Winter eds. Dr. L. Rabenhorst’s Kryptogamen-Flora von € Deutschland, Osterreich und der Schweiz ed. 2, 4(2). Leipzig. Linnaeus C. 1763. Species plantarum. Vol. 2. Ed. 2. Stockholm. Malekzadeh-Viayeh R, Pak-Tarmani R, Rostamkhani N, Fontaneto D. 2014. Diversity of the rotifer Brachionus plicatilis species complex (Rotifera: Monogononta) in Iran through integrative taxonomy. Zoological Journal of the Linnean Society 170: 233–244. Malta N. 1927. Die Ulota-Arten Sud-Amerikas. Acta Horti Botanici Universitatis Latviensis 2: 165–208. Malta N. 1932. A survey of the Australasian species of Ulota. Acta Horti Botanici Universitatis Latviensis 7: 1–24. Medina R, Lara F, Goffinet B, Garilleti R, Mazimpaka V. 2012. Integrative taxonomy successfully resolves the pseudo-cryptic complex of the disjunct epiphytic moss Orthotrichum consimile (Orthotrichaceae). Taxon 61: 1180–1198. Medina R, Lara F, Goffinet B, Garilleti R, Mazimpaka V. 2013. Unnoticed diversity within the disjunct moss Orthotrichum tenellum s.l. validated by morphological and molecular approaches. Taxon 62: 1133–1152. Mitten W. 1869. Musci austro-americani Enumeratio omnium Austro-americanorum auctori hucusque congitorum. Journal of the Linnean Society, Botany 12: 1–659. Mohr DMH. 1806. Observations on Orthotrichum and Neckera, together with some other genera of mosses. Annals of Botany (K€ onig & Sims) 2: 532–548. M€ uller K. 1849. Synopsis muscorum frondosorum omnium hucusque cognitorum. Berlin. M€ uller K. 2004. SeqState – primer design and sequence statistics for phylogenetic DNA data sets. Applied Bioinformatics 4: 65–69. M€ uller K, M€ uller J, Neinhuis C, Quandt D. 2006. PhyDE – phylogenetic data editor, v0.995. Available at: http:// www.phyde.de Noguchi A. 1989. Illustrated moss flora of Japan. Part 3. Nichinan: Hattori Botanical Laboratory. Nyholm E. 1956. Illustrated moss flora of Fennoscandia. II. Musci. Fasc. 2. Lund: Gleerups. Pacak A, Szweykowska-Kulinska Z. 2000. Molecular data concerning alloploid character and the origin of chloroplast and mitochondrial genomes in the liverwort Pellia borealis. Plant Biotechnology Journal 2: 101–108. Padial JM, Miralles A, de la Riva I, Vences M. 2010. The integrative future of taxonomy. Frontiers in Zoology 7: 16. Posada D. 2008. jModelTest: phylogenetic model averaging. Molecular Biology and Evolution 25: 1253–1256. Ronquist F, Huelsenbeck JP, Teslenko M. 2011. MrBayes version 3.2 manual: tutorials and model sum- 445 446
ET AL. R. CAPARROS Appendix 1 All specimens listed below are a selection of the samples used for morphological analyses. Ulota crispa (Hedw.) Brid. - AUSTRIA, Ober€osterreich: beim Ht. Langbathsee, alt. 750 m, S-B177772; Ober€osterreich: im unteren Stammbachtal bei Goisern, c. 510 m, S-B225053; BELGIUM, Li ege, Reinardstein, (pres Malmeady), 2.xi.1913, Toussaint s.n., BM; CANADA, British Columbia, Regional District of Mount Waddington, Vancouver Island, Winter Harbour, Kwaksistah Regional Park, around the campsite, 50°310 0100 N, 128°020 2300 W, alt. 2 m, MAUAM-Brio 4806; Clayoquot District, Ucluelet, Vancouver Island, 48°550 0000 N, 125°340 0000 W, NY 156768; CHINA, Hunan, Sangzhi Co., Badagongshan, Liaoyewan, c. 1 km NE of the administration office, collecting locality 43, 29°460 N, 110°030 E, alt. 1400 m, S-B176297; CROATIA, Lika-Senj, Velebit in sylva Jasikovac, prope Gospi
c, BP 43305; FRANCE, Aquitaine, La Rhume, alt. 900 m, BCB 19166; Centre, Saint-Denis-des-Puits, Etangs de la Gatine, en rive droite du dernier
etang, alt. 200 m, Herb. Boudier 2389; Haute-Normandie, For^et de Brotonne (Seine inf
erieure), 1891, Th
eriot s.n., BM; Lorraine, Hooker s.n., BM; Midi-Pyr
en
ees, Luchon, Vall
ee de Lys, BCB 19165; Poitou-Charentes, F^orets des Lussac et de Ch^ ateauneuf, Schimper 457, BM; R
egion Basse Normandie, 1.iv.1912, Douin s.n., BM; Vogesengebiet, An Naldlaeument, P. Beaub s.n., BM; GEORGIA, distr. Chokhatauri, montes Meskhedski khrebet, in viciniis pagi Nabeglavi, alt. 1000 m,
s.n., G 00124254; Abkhazia, distr. 21.vii.1979, Vas̆ ak Gulripsh, in vicinitate pagi Lata, in valle fluminis Kodori, alt. 500 m, G 00124247; GERMANY, Baden- W€ urttemberg, Baden-Baden, Cascade de Geroldsau pr es de Baden-Baden, 17.ix.1858, Jack s.n., BM; Oberbayern, Baiern: Reichenhall, alt. 480 m, 10.ix.1909, Schiffner s.n., BM; Turingia, Saaldfeld, Thuiringes Wald, Reinsch s.n., BM; HUNGARY, Fejer, In vallis Hat
arv€ olgy prope pag P
alh
ara, BP 26366; Somogy, In silvis vs. stationem pagi Szenta, alt. 157 m, BP 121748; Szabolcs-Szatm
ar Bereg, In sylva Megykerek-erd} o prope K€ om€ or} o, alt. 110 m, BP 121586; Vas, In silva Nagy-erd
o ad M
etnekpuszta orh
aza, alt. 250 m, BP 121736; IRELAND, od€ prope G€ Leinster, aparcamiento del centro de visitantes Glendalough, MAUAM-Brio 4802; Munster, Tork Cascade, m. Killarney (Hibernia), vi.1865, BM; ITALY, Trentino-S€ udtirol, Kohlern Wald, BM; JAPAN, Kyoto, Honshu, Ine-cho, Yoza-gun, Kamanyu, alt. 100 m, Deguchi 34159, HIRO; Fukushima, Honshu, Bandaisan National Park, Urubandai, alrededores del Urubandai Royal Hotel, 37°390 1700 N, 140°050 3700 E, alt. 775 m, MAUAM-Brio 4744; Hiroshima, Honshu, Geihoku-cho, Yamagata-gun, Higashi-yawatabara, Mt. Garyu, alt. 1200–1223 m, 19.x.1999, Kitamura 410; HIRO; Tottori, Honshu, Chizuch
o, Sugijinja, MAUAM-Brio 4805; Yamanashi, Honshu, Fuji Hakone Izu National Park, vertiente norte del Monte Fuji, 35°230 2600 N, 138°420 5900 E, alt. 2270 m, MAUAM-Brio 4749; Iburi, Hokkaido, Shikotsu-Toya National Park, lago Toya, Toyako Onsen, parque junto a la orilla del lago, 42°340 0000 N, 140°480 2900 E, alt. 87 m, MAUAM-Brio 4742; Kushiro, Hokkaido, Akan National Park, orillas del lago Akan, alrededores de Akanko Onsen, 43°260 1500 N, 144°050 5100 E, alt. 436 m, MAUAM-Brio 4804; Kumamoto, Kyushu, Aso Kuju National Park, carretera 265, c. Soyo-machi, 32°410 1400 N, 131°080 3100 E, alt. 582 m, MAUAM-Brio 4759; Miyazaki, Kyushu, Kunishi Dake Area, Motoyashiki, estaci
on de esqu
ı Gokase Highland, 32°340 4400 N, 131°070 0500 E, alt. 1290 m, MAUAM-Brio 4760; Tokushima, Shikoku, Mt. Tsurugisan, 33°510 5900 N, 134°050 3300 E, alt. 1420 m, MAUAM-Brio 4756; Ehime, Shikoku, Ishizuchisan Quasi National Park, Mt. Kamegamori, inicio del camino de subida junto a la carretera, 33°470 1100 N, 133°110 2500 E, alt. 1690 m, MAUAM-Brio 4765; Yagoshima, Yakushima, Kirishima-Yaku National Park, Hadogawa Trail, Hananoego, inicio del camino a Ishizuka hat, 30°190 2000 N, 130°300 4000 E, € alt. 1650 m, MAUAM-Brio 4762; NORWAY, Hubener s.n., BM; POLAND, Podkarpackie, East Carpathians, Polish Eastern Beskids, Slonne G
ory Hills, in hornbeam forest near Mt. Sobi
en, alt. 400 m, 13.v.1973, Ochyra s.n., BM; PORTUGAL, Beira Litoral, Lous~ a, Espig~ ao, Volta da Lomba, UTM 29TNE6435, alt. 890 m, LISU 204647; Douro Litoral, Serra da Freita, Senhora da Lapa, UTM 29TNF5825, alt. 820 m, LISU 204593; Estremadura, Serra de Sintra, Pen- © 2016 The Linnean Society of London, Botanical Journal of the Linnean Society, 2016, 180, 427–451 Downloaded from https://academic.oup.com/botlinnean/article-abstract/180/4/427/2416569 by guest on 23 May 2020 incarnata complex. Botanical Journal of the Linnean Society 177: 395–417. Vigalondo B, Lara F, Draper I, Valc
arcel V, Garilleti R, Mazimpaka V. 2016. Is it really you, Orthotrichum acuminatum? Ascertaining a new case of intercontinental disjunction in mosses. Botanical Journal of the Linnean Society 180: 30–49. Vitt DH. 2014. Orthotrichaceae Schimper. In: Flora of North America Editorial Committee, ed. Flora of North America north of Mexico. Vol 28: Bryophyta, part 2. New York and Oxford: Oxford University Press. Wang Q-H, Jia Y. 2012. A taxonomic revision of the Asian species of Ulota Mohr (Orthotrichaceae). Bryologist 115: 412–443. Wilson W. 1855. Bryologia Britannica. London. Zamora JC, Calonge FD, Mart
ın MP. 2015. Integrative taxonomy reveals an unexpected diversity in Geastrum section Geastrum (Geastrales, Basidiomycota). Persoonia 34: 130–165. Ziolkowski PA, Sadowski J. 2002. FISH-mapping of rDNAs and Arabidopsis BACs on pachytene complements of selected Brassicas. Genome 45: 189–197. INTEGRATIVE STUDY OF ULOTA CRISPA 123°470 1600 W, alt. 190 m, MAUAM-Brio 4808; Gifford Pinchot National Forest, Sunset Campground, along E Fork of Lewis River, upriver from campground, alt. 325 m, NY 432308; Ulota crispula Bruch AUSTRIA, Burgenland, s€ o von G€ ussing bei Gr. M€ urbisch, alt. 275–300 m, S-B177769; Ober€ osterreich, Ob.Ost.: im unteren Stammbachtal bei Goisern, alt. 510 m, S-B177773; BELGIUM, Namur, LouetteSaint-Pierre, vi.1872, Gravet s.n., BM; BULGARIA, Burgas, Southern Black Sea coast, Strandzha National Park, Marina reka reserve at Balgari village, 42°070 100 N, 27°450 54.400 E, alt. 255 m, BP 180189; Montana, Stara Planina Mts, Varsec, Zanozhene village, valley of Stara reka river, above Byalata voda shelter house, under Todorini Kukli peak, 43°080 1.500 N, 23°120 4500 E, alt. 1190 m, BP 173885; CANADA, British Columbia, Westminster Junction, NY 224932; New Brunswick, Albert Co., Fundy National Park, trail to Third Vault Falls, 45°380 N, 65°000 W, NY 224956; Newfoundland & Labrador, Avalon Peninsula, Whitbourne, NY 156715; Nova Scotia, Annapolis Co., Kejimkujik National Park, Mill Falls on Mersey River, 44°290 N, 65°080 W, NY 224977; Ontario, Bruce Co., Fathom National Marine Park, Flowerpot Island, along trail from Beachy Cove to Marl Bed, 45°170 5000 N, 81°370 3800 W, NY 1206493; Prince Edward Island, Queens Co., 1 mile north of Argyle Shore, 46°100 N, 63°210 W, NY 224983; Quebec, Laurentian Mts., Vicinity of St. Jerome, NY 152119; CAUCASUS, 1836, Dollinger s.n., BM; DENMARK, Zealand, G 00124259; ESTONIA, P€ arnu, Kreis Pernau, Fich Tenhochwalde 1 km SO von der Station Surrie, 21.viii.1909, Mikutowicz s.n., BM; FRANCE, Aquitaine, Gan, v.1847, Spruce s.n., BM; Bretagne, C^ otes-du-Nord, Gorges du Corong, W ene, alt. 200 m, G 00047155; Centre, Saint-Denis-des-Puits, Etangs de la Gatine, en rive droite du dernier
etang, alt. 200 m, BOUDIER 2389; Jura, Les Fourgs, entre la Petit Vitiau et les Prises, formation du pre-bois, alt. 1140 m, BOUDIER 3905B; Limousin, For^ et de Crozat, 7.vii.1871, BM; Pays de la Loire, For^ et de Perseignes, BM; Poitou-Charentes, Rougnac, Comm. De Charras, SE Angoul^ eme, Charente, alt. 130 m, G 00124241; Pyr
en
ees-Atlantiques, La Rhume, alt. 900 m, BCB 19166; Rh^ one-Alpes, carretera de N.A Bellecombe (entre Chamonix y Albertuille), 45°470 56.7″N, 6°290 15.0″E, alt. 832 m, MAUAM-Brio 4710; GEORGIA, distr. Chokhatauri, in viciniis pagi Nabeglavi, montes Meskhedski khrebet, alt. 1000 m, G 00124254; Abkhazia, distr. Gulripsh, in vicinitate pagi Lata, in valle fluminis Kodori, alt. 500 m, G 00124247; GERMANY, Baden-W€ urttemberg, Emmendingen, alt. 260 m, G 2747; Bayern, Waldm€ unchen, G 00124261; Hesse, Ludwigshutte, Bruch, vii.1841, BM; Rheinland-Pfalz, in valle Rhenana prope St Goar, 1874, Herpell s.n., BM; Sylva © 2016 The Linnean Society of London, Botanical Journal of the Linnean Society, 2016, 180, 427–451 Downloaded from https://academic.oup.com/botlinnean/article-abstract/180/4/427/2416569 by guest on 23 May 2020 inha, UTM 29TMC5991, alt. 417 m, LISU 222040; Minho, Corno do Bico, Travassos, pr
oximos de Coutos, UTM 29TNG4137, alt. 640–650 m, LISU 198665; Tras-os-Montes, Vila Real, Parque Natural do Alv~ ao, ^ pr
oximo de Lamas de Olo, UTM 29TNF9878, alt. 1092 m, MAUAM-Brio 4783; ROMANIA, Alba, in valle K}ok€oz prope Nyirmez}o = Poiana Aiudului, montes Bihar, BP 77356; Beszterce-Nasz
od, Mt. Radnay havasok, in ramis Piceae excelsae declivium valis V. Rosu, pr, pag. Borberek, BP 8437; Buz au, Mts. Apuseni, Pietroase, desv
ıo a Poiana Florilor, alt. 400 m, MAUAM-Brio 4810; Sibiu, Carpat. Merid. In valle Podragu, montes Fogarasi havasok, alt. 1400 m, BP 72068; RUSSIA, Caucasus, Kapa-Cy-Bezengi River, 43°180 N, 43°260 E, alt. 850 m, 3.viii.2004, Ignatov, Ignatova & Kharzinov s.n., MHA; Caucasus occidentalis, Sochi, distr. Sochi, in colle Bytkha, in valle fluminis Macesta ad cataractas rivi Zmeika prope pagun, Kraievo Armianskoye, alt. 300–400 m, G 00124249; SLOVAKIA, Kosice, Mts Slovensk
y Raj.Vel‘k
y Sokol dolina Valley, 20°200 500 N,  in ramu48°550 53.400 E, alt. 700 m, BP 176055; Zilina, lis Piceae in Piceetis vallis, alt. 1000–1100 m, BP 121697; SPAIN, Asturias, R
ıo Pur
on, MAUAM-Brio 4800; Burgos, Angostina, ribera del Arceniega, UTM 30TVN8573, alt. 290 m, VIT5789; Cantabria, Picos de Europa, Sª de la Corta, Monte de la Llama, MAUAM-Brio 4809; Le
on, Sierra de Ancares, MAMusci 4223; Girona, Vilallonga de Ter, Torrent de Vallvigil, UTM DG48, alt. 1200 m, BCB 24461; Castell
on, El Esprio, Penyagolosa, BCB 10211; Orense, Devesa do Caurel, Sierra do Caurel, 42°360 N, 07°60 W, alt. 900–1000 m, MUB 43446; Navarra, Orbaiceta, Bosque de Irati, Bco. Erlan, UTM 30TXN4863, alt. 980 m, VIT 34922; Vizcaya, Abadiano, Gorosarri, UTM 30TWN3075, alt. 230 m, VIT 14697; SWITZERLAND, Jura, La D^ ole, 18.vii.1869, Bernet s.n., BM; Vaud, Hinter St. Cergue, G 00124244; TAIWAN, Nantou, Ren-ai Village, Mei-feng Shan (Mt.), 24°070 N, 121°100 E, alt. 2000– 2200 m, MO 5355215; TURKEY, Rize, near mainroad about 4 km W of Ardesen, alt.  20 m, S-B99508; Trabzon, carretera de Macka al Monasterio de S€ umela, 40°410 0600 N, 39°390 5600 E, alt. 1500 m, MAUAM-Brio 4801; carretera de Macßka a Torul, 40°420 3200 N, 39°270 5600 E, alt. 1450 m, MAUAM-Brio 4770; UK, Scotland, New Galloway (73) Scotland, SB17776; Wales, c. 3.5 km SSW Machynlleth, Llyfnant Valley, UTM SN753975, alt. 91 m, BCB 12364; UKRAINE, Transcarpathia, In silvis vallis Hoverla prope Tiszabogd
any, alt. 1–1400 m, BP 121548; USA, Oregon, Tillamook State Forest, Elk Creek, 46°360 4300 N, 123°280 0000 W, alt. 300 m, MAUAM-Brio 4807; Corbett, Columbia Gorge, Along Big Cr., NY 524159; Washington, Olympic National Park, Lake Crescent, Storm King Ranger Station, 48°030 3000 N, 447 448
ET AL. R. CAPARROS de la Corta, Monte de la Llama, MAUAM-Brio 4813; Le
on, Los Ancares, valle del Cui~ na, UTM PH84, BCB 19875; Girona, Sant Llorencß de la Muga, BCB 6332; A Coru~ na, Parque Natural de las Fragas del Eume, MAUAM-Brio 4794; Huesca, Argu
ıs, Sierra de Gratal, hayedo calc
ıcola de Peir
o, UTM 30TYM0788, alt. 1400 m, VIT 27399; Navarra, Lapiaz de Larra a 800 m. al norte del refugio, alt. 1335 m, MAUAM
Brio 4787; Alava, Sierra de Urbasa, bajando del puerto de Opakua, alt. 980 m, MAUAM-Brio 4780; SWITZERLAND, Soleure, Hagendorf, 23.v.1896, Dresler s.n., BM; Ticino, Roncaccio, preso Locarno, BM; Vaud, Hinter St. Cergue, G 00124244; TURları Milli Parkı, Ayder, KEY, Rize, Kacßkar Dag 40°560 5200 N, 41°080 0800 E, alt. 1550 m, MAUAM-Brio 4811; Trabzon, carretera de Of a Uzung€ ol, en la salida de Tasßhanpazari, 40°500 1500 N, 40°160 3300 E, alt. 140 m, MAUAM-Brio 4774; UK, North West England, Windermere, Westmoreland, Amie’s wood, BM; UKRAINE, Transcarpathia, in monte Magurica prope Turjaremete, alt. 350 m, BP 64679; USA, Alleghanay Mts., NY 507792; Connecticut, Mansfield, Joshua’s Trust, next to Knowlton Pond, MAUAMBrio 4766; Delaware, Farmhurst Station near Wilmington, NY 991745; Georgia, Chattahoochee National Fores, Cohutta Wilderness, Tearbritches Trail from Forest Serv. Rd. 68 to Bald Mountain, NY 507804; Indiana, Clifty Falls State Park, NY 507812; Kentucky, Ezel Quad, Rough Br. (tributary of Spaws Cr.), 1.1 miles north of Dan, 37°580 1200 N, 83°270 5100 W, alt. 274.32 m, DUKE 0127685; Maine, Spruce Point c. 1.5 miles SE of Boothbay Harbor, NY 507783; Grafton Notch State Park, 44°250 24″N, 70°560 44″W, MAUAM-Brio 4768; Massachusetts, Cape Cod. Brewster, MAUAM-Brio 4767; Michigan, along Carp Creek at the Iron Bridge Douglas Lake Region, NY 795404; Big Stone, NY 507721; New Hampshire, Mt. Willard, NY 507799; New York, Adirondack Mts., along trail east of Henderson Lake, 30 miles N. Tahawus, NY 507845; Catskill Mountains, NY 507719; North Carolina, Tanawha Trail, near Wilson Creek overlook, Blue Ridge Parkway, NY 507776; Great Smoky Mountains National Park, Andrews Bald Trail, NY 507814; Ohio, Bog north of Springfield, NY 507817; Pennsylvania, between Mt. Hope and Penryn, on the Lancaster and Lebanon County Line, NY 507801; Rhode Island, border of towns of Exeter and South Kingstown, Marion Eppley Wildlife Sanctuary, c. 1.5 miles NE of Usquepaug, along Queens River, 41°310 0600 N, 71°350 3300 W, NY 840619; Vermont, Green Mountain National Forest, between Woodford and Bennington, 42°530 1200 N, 73°060 1500 W, alt. 419 m, MAUAM-Brio 4812; Virginia, Jefferson National Forest, Whitetop Mountain, 36°380 1700 N, 81°360 2100 W, alt. 1675 m, NY 986405; West Virginia, Monongahela National Forest, upper © 2016 The Linnean Society of London, Botanical Journal of the Linnean Society, 2016, 180, 427–451 Downloaded from https://academic.oup.com/botlinnean/article-abstract/180/4/427/2416569 by guest on 23 May 2020 negra, prope Griesbach, Jun, Sch. L s.n., BM; HUNGARY, Baranya, alt. 125 m, BP 168437; Heves, in silvis montis Hosszub
erc supra vallem Ilona-v€ olgy prope Par
adf€ urd€o, alt. 330 m, BP 121758; Somogy, in silvis ‘Uras
agi-erd}o’ prope Z
ak
any, alt. 200 m, BP
121722; Szabolcs-Szatm
ar-Bereg, ‘Eretthegyi-erd} o’ prope M
and, alt. 110 m, BP 121588; Zala, in silva ‘Nyirlakosai-erd}o’ prope V
arf€olde, alt. 200 m, BP 121723; IRELAND, Leinster, aparcamiento del centro de visitantes Glendalough, MAUAM-Brio 4814;  MONTENEGRO, Zabljak, Durmitors Mts, Durmitor  NP, Zabljak, between Crno jezero and Poljana, 43°080 52,000 N, 19°050 4800 E, alt. 1421 m, BP 178715; NORWAY, Telemark, Sannidal, Lislau (farmer), 23.vii.1946, Lid s.n., BM; POLAND, Malopolskie, Tatry zachodnie: Dolina Ko
scieliska, na zeschlych galeziach
swierku, BP 83187; Podkarpackie, East Carpathians, Polish Eastern Beskids, Slonne G
ory Hills, alt. 400 m, BP 160736; PORTUGAL, Beira Litoral, Lous~ a, Antenas, UTM 29TNE6939, alt. 1115 m, LISU 205975; Douro Litoral, Serra da Freita, Senhora da Lapa, UTM 29TNF5825, alt. 820 m, LISU 204593; Minho, Corno do Bico, Travassos, pr
oximos de Coutos, UTM 29TNG4137, alt. 640650 m, LISU 198665; Tras-os-Montes e Alto Douro, Carvalhal de Sezelhe, UTM 29TNG9228, alt. 1000 m, LISU 236060; ROMANIA, Alba, in valle K}ok€oz prope Nyirmez}o-Poiana Aiudului, montes Bihar, BP 77356; Buz au, Mts. Apuseni, Pietroase, desv
ıo a Poiana Florilor, alt. 400 m, MAUAM-Brio 4815; Harghita, ad ‘Medvebarlang’ prope Borsz
ek, alt. 900 m, BP 121669; Sibiu, in valle Podragu, montes Fogarasi havasok, alt. 1400 m, BP 72067; Szeben, in rupibus schist. Silvat. Supra Zibinklamm/Cheia, Riulmare/infra Hohe Rinne = Paltinis, alt. 900– 1000 m, BP 121813; RUSSIA, Caucasus, Cherek Bezengijskij River, 1 km upstream Dumala Creek mouth, 43°100 N, 43°140 E, alt. 1650 m, 1.viii.2004, Ignatov, Ignatova & Kharzinov s.n., MHA; Caucasus occidentalis, (Sochi) in faucibus fluminis. Psakho infra vicum Kamenka, alt. 200–300 m, G 00124251; ad cataractas in faucibus rivi Agura, alt. 350–400 m, G 00124252; in vicinitate pagi Loo, 6 km ab ostio fluminis Loo, alt. 30–150 m, G 00124257; SERBIA, Golija Biosphera Reserve, Golija Biosphera Reserve, at Orlov kamen, between Bele Vode and Kumanica villages, 43°270 3800 N, 20°150 42.800 E, alt. 950 m, BP 171951; Tara National Park, Joki
ca potok, Ljuti breg reserve area, alt. 960 m, BP 168279; SLOVAKIA, Bansk
a Bystrica, Tatra Humiliore, in ripa rivi Bisztra prope T
ale, alt. 800 m, BP 121717; Presov, prope pag T
atraf
ured (Smokovec) monte Visoky Tatry, alt.  1000 m, BP 66758; Zilina, in ramulis Piceae in Piceetis vallis, alt. 1000–1100 m, BP 121697; SPAIN, Asturias, pr
oximo al nacimiento del r
ıo Pur
on, MAUAM-Brio 4788; Cantabria, Picos de Europa, Sª INTEGRATIVE STUDY OF ULOTA CRISPA Zacopane (al pie de los Tatra), Dolina Strazinska, MAUAM-Brio 4711; PORTUGAL, Minho, Viana do Castelo, Melgacßo, Lamas de Mouro, UTM 29TNG6654, alt. 863 m, LISU 215261; Tras-os-Montes, Vila Real, Parque Natural do Alv~ ao, pr
oximo de ^ Lamas de Olo, UTM 29TNF9878, alt. 1092 m, MAUAM-Brio 4785; Tras-os-Montes e Alto Douro, Serra do Alv~ ao, Lamas de Olo, pr. Boucßa do Ribeiro, UTM PF0081, alt. 1000–1050 m, LISU 213870; ROMANIA, Alba, in valle K} ok€ oz prope Nyirmez} o = Poiana Aiudului, montes Bihar, BP 77356; Brașov, in faucem Taminaszakad
ek in pede montis Nagyk} ohavas, alt. 1100 m, BP 69049; Buz au, Apuseni Mountains, c. Plateau de adis, alt. 1200 m, MAUAMBrio 4824; Cluj, ad caverna Skerisora prope pag. Aranyosf€ o = Scarisoara, BP 77351; Szeben, montes Szeben, valle del r
ıo Degeneasa cerca de Paltinis, alt. 1300 m, BP 74062; RUSSIA, Altai, northern shore of Teletzkoye, Yailyu, 51°450 N, 87°350 E, alt. 450 m, 24.vi.1991, Ignatov s.n., MHA; Kamchatskaya oblast,
Syd-Kamtchatka, Nikolajevsk, S-B193307; Oblast de Sajal
ın, viii.1933, Sakurai s.n., BM; SERBIA, Tara National Park, at Tepih Livada, Crveni potok reserve area, alt. 1050 m, BP 168240; SLOVAKIA, Kosice, Mts Slovensk
y Raj.Vel‘k
y Sokol dolina Valley, 20°200 500 N, 48°550 53.400 E, alt. 700 m, BP 176055; Presov, ‘H
arom t
ocsa’ supra T
atralomnic = Tatranska  Lomnica, alt. 990 m, BP 121707; Zilina, Porubka Liptovska, alt. 681 m, BP 76404; SLOVENIA, Notranjska, In monte Sn ezn
ık supra Lasac, Baum
gartner, BP 43224; SPAIN, Alava, Salvatierra, Ubirin, UTM 30TWN5538, alt. 980 m, VIT 13953; Asturias, Sierra del Sueve, MAUAM-Brio 4798; Cantabria, Cahecho, La ermita del Santo, alt. 825 m, MAUAM-Brio 4778; A Coru~ na, Parque Natural de las Fragas del Eume, MAUAM-Brio 4795; Huesca, Linza, UTM 30TXN8051, alt. 1500 m, VIT 29563; Navarra, Lapiaz de Larra a 800 m. al norte del refu
gio, alt. 1335 m, MAUAM-Brio 4786; Alava, San Mill
an, Oc
ariz, Puerto Opacua, UTM 30TWN5441, alt. 920 m, VIT 3547; SWEDEN, S€ odermanland, Ut€ o,  Al€ o nature reserve, S€ oderskog, 200 m S of Vreta, 58°55,2250 N, 18°12,6600 E, S-B176729; Bahusiae, Thedenius s.n., BM; Blekinge, R€ odeby, Spjutsbygd, 56°150 N, 15°360 E, 25.vii.1888, Arnell 120, BM; € Osterg€ otland, Borgs Udde, S-B81846; Sk ane, Hj€ ars as, Sibbhult, 0.5 km NE of Tykatorp, S€ B179352; S€ odermanland, Osmo, Hammersta. Just N of Frugrottan, 59°1.3230 N, 18°0.3160 E, S-B184625; SWITZERLAND, Fribourg, Foret des Reposoirs, near la Valsainte, alt. 1030 m, 29.vi.1923, Rhodes 1961, BM; Jura, La D^ ole, 18.vii.1869, Bernet s.n., BM; UK, Escocia, New Galloway (73) Scotland, S-B177766; USA, Alaska, Kenai Lake cerca de Primrose (1.7 km al NW), en la 9 Hwy, 60°230 0700 N, 149°210 1100 W, alt. 157 m, MAUAM-Brio 4715; picnic site, Lowe River, © 2016 The Linnean Society of London, Botanical Journal of the Linnean Society, 2016, 180, 427–451 Downloaded from https://academic.oup.com/botlinnean/article-abstract/180/4/427/2416569 by guest on 23 May 2020 slopes of Spruce Mountain, vicinity of Blue Knob, Lobelia Quad., 38°100 3200 N, 80°160 4700 W, alt. 1158–1311 m, NY 954721; Wisconsin, Highlands, NY 507808; Ulota intermedia Schimp. - AUSTRIA, K€ arnten, K€ a.: n€ achst Ossia. . . w des Meierhof. . . Tauern, alt. 890 m, S-B177775; Salzburg, am Ufer des Attersees zw. Burgau u. Unterach, alt. 480 m, SB177776; CANADA, British Columbia, Central Coast Regional District, Bella Coola, pista que sube por la Clynton Fall Creek, 52°190 5800 N, 126°490 1900 W, alt. 335 m, MAUAM-Brio 4720; New Brunswick, Lepreau Parish, New River Beach Provincial Park, Barnaby Head Trail between Raspberry Cove and Chitticks Beach, 46°070 5000 N, 66°310 3100 W, NY 01220922; Newfoundland & Labrador, Bay Bulls, 47°180 57″N, 52°480 37″W, 1.x.1892, Aron 22, BM; Nova Scotia, Lake Weir, 7.5 miles NW of Halifax, NY 152129; Ontario, Ottawa, 45°250 00″N, 75°420 00″W, Macoun s.n., BM; Quebec, Jupiter River, 49°280 01.200 N, 63°370 01.200 W, NY 152104; Gasp-Sud Co., Ile Bonaventure, NY 224972; Saskatchewan, Glen Harbour, 15.v.1883, Waghorne s.n., BM; CROATIA, Lika-Senj, Velebit, Rusajca Crakoviae prope Mesak, BP 43226; DENMARK, Copenhague, Dyrehaven, 4.viii.1902, Hesselbo s.n., BM; FRANCE, Aquitania, subida a Col d’Aubisque, carretera de 918, alt. 1500 m, MAUAMBrio 4818; Miquelon Island, 47°050 40″S, 56°220 00″W, vii.1883, Delamare s.n., BM; Rh^one-Alpes, Villard de Lans, For^ets des Jarrands, G 00124268; GERMANY, Bayern, Ludwigsh€ utte, Bruch, viii.1837, BM; Hesse, Braunfels, Faurie s.n., BM; HUNGARY, BorsodAba
uj-Zempl
en, in sylva . . .. . .prope pag Telkib
anya, montes S
atorhegys
eg, BP 62776; Nogr
ad, in valle rivi . . . prope Kir
alyh
ara, montes Borzs€ony, BP 59613; Zala, in silva ‘Nyirlakosai-erd}o’ prope V
arf€olde, alt. 200 m, BP 121723; IRELAND, Munster, Muckross, Braithwaite s.n., BM; JAPAN, Hokkaido, prov. S^ oya, Isl. Rishiri, Mt. Rishiri, alt. 100–500 m, 8.viii.1954, Hasegawa s.n., H; Kamikawa, Hokkaido, Daisetsuzan National Park, Sounkio valley, al SO de Sounkio, Obako, 43°410 5800 N, 143°000 5700 E, alt. 715 m, MAUAM-Brio 4817; Kushiro, Hokkaido, Akan National Park, laderas del monte del lago Mashu, carretera 52 hacia Teshikaga, 43°320 2300 N, 0 00 144°30 23 E, alt. 370 m, MAUAM-Brio 4736; Tokachi, Hokkaido, Daisetsuzan National Park, Nukabira National Hwy, Horoka, unos 15 km al NNW de Nukabira, 43°290 4100 N, 143°080 4400 E, alt. 650 m, MAUAM-Brio 4730; Yamanashi, Honshu, Fuji Hakone Izu National Park, vertiente norte del Monte Fuji, 35°230 2600 N, 138°420 5900 E, alt. 2270 m, MAUAM-Brio 4750; MAUAM-Brio 4746; Tokushima, Shikoku, Ichiu-mura, Mima-gun, Mt. Tsurugi, alt. 1800–1900 m, Taoda 5621, HIRO; NORWAY, Telemark, Sannidal, Lislau (farmer), 23.xii.1946, Lid s.n., BM; POLAND, voivodato de la Peque~ na Polonia, 449 450
ET AL. R. CAPARROS 12 miles E of Valdez, alt. 609.6 m, NY 156791; Oregon, Tillamook State Forest, Elk Creek, 46°360 4300 N, 123°280 0000 W, alt. 300 m, MAUAM-Brio 4713; Wash- ington, Junction of Little River Road and Olympic Hot Springs Road, along Little River, 48°070 N, 123°350 W, alt. 70 m, NY 524160. Appendix 2 Specimens included in the molecular analyses, with corresponding GenBank accession numbers. Taxon Geographical origin Herbarium voucher MAUAM-Brio 4805 (ID304) MAUAM-Brio 4808 (ID305) MAUAM-Brio MAUAM-Brio MAUAM-Brio MAUAM-Brio 4809 4810 4802 4806 (ID315) (ID320) (ID321) (ID323) MAUAM-Brio 4804 (ID330) MAUAM-Brio 4807 (ID336) Ulota crispula S-B225053 (ID337) MAUAM-Brio 4801 (ID352) MAUAM-Brio 4800 (ID353) MAUAM-Brio 4812 (ID302) MAUAM-Brio 4813 (ID303) MAUAM-Brio 4811 (ID319) MAUAM-Brio 4815 (ID333) S-B177773 (ID338) MAUAM-Brio 4814 (ID355) NY 1206493 (ID363) Ulota intermedia MAUAM-Brio 4715 (ID300) MAUAM-Brio 4824 (ID301) VIT 3547 (ID342) MAUAM-Brio 4817 (ID343) MAUAM-Brio 4818 (ID346) S-B176729 (ID357) NY 01220922 (ID362) Outgroup taxa Orthotrichum affine Orthotrichum rupestre Ulota bruchii Ulota coarctata Ulota curvifolia Ulota drummondii Ulota Ulota Ulota Ulota Ulota Ulota macrospora megalospora obtusiuscula phyllantha rehmannii reptans MAUAM-Brio 4329 (O-108) VAL-Briof 11631 (ID368) MAUAM-Brio 4444 (ID383) MAUAM-Brio 4438 (ID360) MAUAM-Brio 4826 (ID359) MAUAM-Brio 4816 (ID356) MAUAM-Brio 4825 (ID361) P. Boudier 7595-B (ID365) MAUAM-Brio 4819 (ID358) MAUAM-Brio 4820 (ID329) MAUAM-Brio 4821 (ID331) MAUAM-Brio 4822 (ID328) MAUAM-Brio 4823 (ID349) Japan USA, Western Coast Spain Romania Ireland Canada, West. Coast Japan USA, Western Coast Austria Turkey Spain USA, Eastern Coast Spain Turkey Romania Austria Ireland Canada, East Coast USA, Western Coast Romania Spain Japan France Sweden Canada, East Coast Spain Cyprus Spain Spain Russia Japan Canada France Canada Canada USA Turkey Japan trnG trnL-trnF atpB-rbcL KT804262 KT804266 KT804301 KT804305 KT804340 KT804344 KT804380 KT804383 KT804267 KT804270 KT804269 KT804264 KT804306 KT804309 KT804308 KT804303 KT804345 KT804348 KT804347 KT804342 KT804384 KT804386 – – KT804261 KT804265 KT804300 KT804304 KT804339 KT804343 KT804379 KT804382 KT804271 KT804263 KT804268 KT804272 KT804310 KT804302 KT804307 KT804311 KT804349 KT804341 KT804346 KT804350 – KT804381 KT804385 KT804387 KT804273 KT804274 – KT804276 KT804275 KT804277 KT804312 KT804313 KT804315 – KT804314 KT804316 KT804351 KT804352 KT804354 KT804355 KT804353 KT804356 KT804388 – KT804390 KT804391 KT804389 – KT804283 KT804322 KT804362 KT804397 KT804286 KT804284 KT804281 KT804285 KT804287 KT804282 KT804325 KT804323 KT804320 KT804324 KT804326 KT804321 KT804365 KT804363 KT804360 KT804364 KT804366 KT804361 KT804400 KT804398 KT804395 KT804399 KT804401 KT804396 KT804255 KT804256 KT804259 KT804260 KT804278 KT804280 KT804279 KT804288 KT804289 KT804290 KT804291 KT804292 KT804293 JQ836900 KT804295 KT804298 KT804299 KT804317 KT804319 KT804318 KT804327 KT804328 KT804329 KT804330 KT804331 KT804332 JQ836985 KT804334 KT804337 KT804338 KT804357 KT804359 KT804358 KT804367 KT804368 KT804369 KT804370 KT804371 KT804372 JQ836694 KT804374 KT804377 KT804378 KT804392 KT804394 KT804393 KT804402 KT804403 KT804404 KT804405 KT804406 KT804407 © 2016 The Linnean Society of London, Botanical Journal of the Linnean Society, 2016, 180, 427–451 Downloaded from https://academic.oup.com/botlinnean/article-abstract/180/4/427/2416569 by guest on 23 May 2020 Ingroup taxa Ulota crispa ITS2 INTEGRATIVE STUDY OF ULOTA CRISPA 451 Table . Continued Taxon Zygodon pentastichus Zygodon viridissimus Herbarium voucher Geographical origin ITS2 trnG trnL-trnF atpB-rbcL MAUAM-Brio 2981 (ID207) MAUAM-Brio 2910 (ID208) Argentina UK, England KT804257 KT804258 KT804296 KT804297 KT804335 KT804336 KT804375 KT804376 Downloaded from https://academic.oup.com/botlinnean/article-abstract/180/4/427/2416569 by guest on 23 May 2020 © 2016 The Linnean Society of London, Botanical Journal of the Linnean Society, 2016, 180, 427–451