Pl. Syst. Evol. 262: 125–137 (2006) DOI 10.1007/s00606-006-0463-4 Taxonomic studies in Chiloscyphus Corda (Jungermanniales: Lophocoleaceae) based on nrITS sequences and morphology J. Hentschel1,3, H.-J. Zündorf1, F. H. Hellwig1, A. Schäfer-Verwimp2, and J. Heinrichs3 1 Institut für Spezielle Botanik mit Herbarium Haussknecht und Botanischem Garten, Universität Jena, Germany 2 Herdwangen-Schönach, Germany 3 Abteilung Systematische Botanik, Universität Göttingen, Germany Received March 8, 2006; accepted June 26, 2006 Published online: October 12, 2006 Ó Springer-Verlag 2006 Abstract. Maximum parsimony and likelihood analyses of 40 Lophocoleaceae nrITS sequences and 6 Plagiochilaceae sequences (outgroup) lead to a robust phylogeny of Chiloscyphus. Four main lineages are assigned to as Chiloscyphus subgenera Chiloscyphus, Lophocolea, Connati and Notholophocolea. Chiloscyphus subgen. Connati is resolved sister to the remainder of this genus. Chiloscyphus subgenus Lophocolea is subdivided into sections Heterophylli (incl. sect. Semiteretes, syn. nov.), Lophocolea, Microlophocolea, and Novae-Zeelandiae. Five accessions of Chiloscyphus pallescens with a chromosome number of n = 18 form a robust monophyletic lineage that is placed sister to a well supported clade with 4 accessions of C. polyanthos [n = 9]. Chiloscyphus mandonii is placed in the synonymy of C. latifolius. Key words: Jungermanniales, Lophocoleaceae, Chiloscyphus, nrITS, phylogeny, chromosome counts. Introduction Jungermanniales with a diversity of about 3000 species are the largest extant lineage of liverworts (Heinrichs et al. 2005a, He-Nygrén et al. 2006). Representatives of Jungermanniales inhabit a wide range of humid habitats ranging from the Antarctics to the northernmost landmasses (Bednarek-Ochyra et al. 2000). This speciose clade is characterized by succubous or incubous leaves, lack of watersacs, occurrence of ventral branches and non-fasciculate rhizoids, exosporous protonemata, and association with Asco- and Basidiomycota (Nebel et al. 2004, Heinrichs et al. 2005a). Classification of Jungermanniales has been hampered by a low number of taxonomically informative characters and extensive morphological homoplasy (Crandall-Stotler et al. 2005, Hentschel et al. 2006) that led to varied interpretations. Many recent authors included the strictly perianth bearing Lophocoleaceae in the marsupia/perigynia carrying Geocalycaceae (e.g. Grolle 1965, 1983; Schuster 1980; CrandallStotler and Stotler 2000; Srivastava and Srivastava 2002) whereas Müller (1951–1958) and Frahm and Frey (2004) kept the taxa as separate entities. Recent molecular data have helped resolve this long lasting controversy. RbcL sequence data support a separation of the strictly perianth bearing Lophocoleaceae from 126 the Geocalycaceae with female involucres originating at least partly from stem tissue (Hentschel et al. 2006). Lophocoleaceae are characterized by succubous, undivided to bilobed, entire or toothed, almost horizontally inserted leaves, conspicuous underleaves often fused with the adjacent leaves, terminal, Frullania-type branching as well as lateral and ventral intercalary branching (Plagiochilaand Bazzania-type), stems without hyalodermis and well differentiated cortex, rhizoids in bundles from underleaf bases or rarely scattered, gametoecia on leading shoots or on short branches, and a 0-3-keeled perianth. Another controversial subject of discussion is the separation of the vast Lophocoleaceae genera Chiloscyphus Corda and Lophocolea (L.) Dumort. Engel and Schuster (1984) merged both genera under Chiloscyphus, based on a broad overlap in gametophytic and sporophytic features but not all later authors adopted this concept (Grolle 1995, Paton 1999, Gradstein et al. 2001). However, the generitype Chiloscyphus polyanthos (L.) Corda was resolved within Lophocolea in analyses of chloroplast DNA markers (He-Nygrén and Piippo 2003, Heinrichs et al. 2005a, Hentschel et al. 2006), supporting Engel and Schusters’ broad genus concept. While genus and family circumscription thus become clearer, many questions related to the infrageneric subdivision of Chiloscyphus and to species boundaries remain unanswered. Circumscription of the generitype Chiloscyphus polyanthos is a controversial issue since the description of Chiloscyphus pallescens (Hoffm.) Dumort., a dioecious C. polyanthos segregate that otherwise differs from C. polyanthos s.str. by slightly larger leaf cells and a normally toothed perianth (Paton 1999). Schuster (1980), Paton (1999), Damsholt (2002) and Erzberger and Papp (2004) distinguish the taxa at species level whereas Smith (1990) accepts only varieties. Järvinen (1983) and Gradstein and van Melick (1996) lower C. pallescens to a synonym of C. polyanthos. In this paper we utilize nrITS sequences, chromosome counts and cell measurements to evaluate the different concepts. J. Hentschel et al.: Taxonomic studies in Chiloscyphus Materials and methods Plant materials for sequencing. Sampling of DNA specimens was based on material available to represent the morphological diversity and the range of Chiloscyphus (Engel and Schuster 1984). The ingroup was completed with several accessions of the closely related Lophocoleaceae genus Heteroscyphus Schiffn. (Hentschel et al. 2006). The outgroups Chiastocaulon dendroides (Lindenb.) Carl, Plagiochila alternans Lindenb. & Gottsche, P. bifaria (Sw.) Lindenb., P. porelloides (Nees) Lindenb., Pedinophyllum interruptum (Nees) Kaal. and Plagiochilion mayebarae S.Hatt were selected based on previous phylogenetic studies (Groth and Heinrichs 2003; Heinrichs et al. 2005a, b). Details of the plant material, including voucher information, accession provenance, and nucleotide sequence identification, are listed in Table 1. DNA extraction, PCR amplification and sequencing. Upper parts of a few shoots of up to 25 years old herbarium specimens were extracted with Invisorb Spin Plant Mini Kit (Invitek, Berlin, Germany). The 50 primer Hep2F and the 30 primer HepCR (Groth and Heinrichs 2003) were used to amplify the internal transcribed spacer (ITS) region of the nuclear ribosomal DNA, containing ITS1, the 5.8S gene, and ITS2. Polymerase chain reaction (PCR, Saiki et al. 1988) was performed in a total volume of 50 ll, containing one unit TaqDNA polymerase (BioLine, Berlin, Germany), 5 ll PCR buffer (BioLine, Berlin, Germany), 2 ll MgCl2 (50 mM, BioLine, Berlin, Germany), 1 ll dNTP-mixture (10 mM, Fermentas, St. Leon-Rot, Germany), 2 ll dimethylsulfoxide, 1 ll of both forward and reverse primer (10 lM), and 1 ll template. PCR amplification was carried out using the following program: 120 s initial denaturation at 92°C, followed by 25 cycles of 60 s denaturation at 92°C, 50 s annealing at 51°C, and 90 s elongation at 72°C. Final elongation was carried out in one step (10 min 72°C). Afterwards two (nested) PCRs were performed using the above protocol with 30 cycles and the 50 primer Hep3F (Groth et al. 2003) and the 30 primer 5.8SR (Feldberg et al. 2004) (ITS1) as well as the 50 primer 5.8SAF (CAA CGA TGA AGA ACG CAG) and the 30 primer HepAR (Groth et al. 2003) (ITS2). Sequencing was carried out on a LI-COR DNA sequencer 4000L or a MegaBACE 1000 capillary sequencer. J. Hentschel et al.: Taxonomic studies in Chiloscyphus 127 Table 1. Geographic origins, voucher numbers, and GenBank/EMBL accession numbers of the investigated taxa Taxon Origin Voucher Accession number Chiastocaulon dendroides (Nees) Carl Chiloscyphus austrigenus (Hook.f. & Taylor) J.J.Engel & R.M.Schust. Chiloscyphus connatus (Sw.) J.J.Engel & R.M.Schust. Chiloscyphus costatus (Nees) J.J.Engel & R.M.Schust. Chiloscyphus fragmentissimus (R.M.Schust.) J.J.Engel & R.M.Schust. Chiloscyphus fragrans (Moris & De Not.) J.J.Engel & R.M.Schust. Chiloscyphus gottscheoides (Besch. & C.Massal.) J.J.Engel & R.M.Schust. Chiloscyphus gottscheoides Chiloscyphus guadeloupensis (Steph.) J.J.Engel & R.M.Schust. Chiloscyphus latifolius (Nees) J.J.Engel & R.M.Schust. Chiloscyphus latifolius [C. mandonii (Steph.) J.J.Engel & R.M.Schust.] Chiloscyphus latifolius Chiloscyphus liebmannianus (Gottsche) J.J.Engel & R.M.Schust. Chiloscyphus martianus (Nees) J.J.Engel & R.M.Schust. (I) Chiloscyphus martianus (II) Chiloscyphus minor (Nees) J.J.Engel & R.M.Schust. Chiloscyphus muricatus (Lehm.) J.J.Engel & R.M.Schust. Chiloscyphus novae-zeelandiae (Lehm. & Lindenb.) J.J.Engel & R.M.Schust. Chiloscyphus pallescens (Hoffm.) Dumort. (I) Chiloscyphus pallescens (II) Japan Ohnishi 5770 (HIRO) AY438233 Chile Hyvönen et al. 5793 (JE) AM282805 Costa Rica Gradstein 9404 (GOET) AM282806 Malaysia AM282807 Venezuela Schäfer-Verwimp & Verwimp 18724/A (JE) Frahm 97/5/N (GOET) AM282809 Azores Schwab 113 (JE) AM282810 Chile Drehwald & Mues 3239 (GOET) AM282811 Argentina Costa Rica Roivainen 835 (JE) Gradstein & Mues 9630 (GOET) AM282812 AM282813 Germany Hentschel Bryo 01416 (JE) AM180587 Bolivia Churchill et al. 21808 (GOET) AM282814 South Africa Mexico Arts RSA 22/28 (JE) Burghardt Bryo 01655 (GOET) AM282815 AM282816 Ecuador Gradstein 10119 (GOET) AM282817 Mexico Germany Gradstein & Equihua 7920 (GOET) AM282808 Hentschel Bryo 01006 (JE) AM282818 Australia Streimann 51629 (JE) AM282819 Australia Eggers AUS 3/81 (JE) AM282820 Chiloscyphus pallescens (III) Germany, Hentschel Bryo 01418 (JE) Thuringia Germany, Hentschel Bryo 01415 (JE) Thuringia Germany, Hentschel Bryo 01811 (GOET) Saxony-Anhalt AM282821 AM282822 AM282823 128 J. Hentschel et al.: Taxonomic studies in Chiloscyphus Table 1. (Continued) Taxon Origin Voucher Accession number Chiloscyphus pallescens (IV) Germany, Lower Saxony Bulgaria Guyana Hentschel Bryo 01590 (GOET) AM282824 Hentschel Bryo 0772 (JE) Gradstein 4890 (GOET) AM282825 AM282826 Gradstein 5042 (GOET) Hentschel Bryo 01308 (JE) AM282827 AM282828 Hentschel Bryo 01758 (GOET) AM282829 Hentschel Bryo 01792 (GOET) AM282830 Hentschel Bryo 0318 (JE) Hentschel Bryo 01414 (JE) AM282831 AM282832 Chiloscyphus Chiloscyphus J.J.Engel & Chiloscyphus Chiloscyphus Corda (I) Chiloscyphus pallescens (V) perissodontus (Spruce) R.M.Schust. (I) perissodontus (II) polyanthos (L.) polyanthos (II) Chiloscyphus polyanthos (III) Chiloscyphus polyanthos (IV) Chiloscyphus profundus (Nees) J.J.Engel & R.M.Schust. Chiloscyphus randii (S.W.Arnell) J.J.Engel & R.M.Schust. Chiloscyphus sabuletorum (Hook.f. & Taylor) J.J.Engel & R.M.Schust. (I) Chiloscyphus sabuletorum (II) Chiloscyphus semiteres (Lehm.) Lehm. & Lindenb. (I) Chiloscyphus semiteres (II) Chiloscyphus spiniferus (Hook.f. & Taylor) J.J.Engel & R.M.Schust. Heteroscyphus aselliformis (Reinw., Blume & Nees) Schiffn. Heteroscyphus coalitus (Hook.) Schiffn. Heteroscyphus cuneistipulus (Steph.) Schiffn. Heteroscyphus fissistipus (Hook.f. & Taylor) Schiffn. Heteroscyphus splendens (Lehm. & Lindenb.) Grolle Pedinophyllum interruptum (Nees) Kaal. Plagiochila alternans Lindenb. & Gottsche Plagiochila bifaria (Sw.) Lindenb. Plagiochila porelloides (Nees) Lindenb. Plagiochilion mayebarae S.Hatt. Guyana Germany, Thuringia Germany, Lower Saxony Germany, Hesse Slovakia Germany Prince Edward Isles Argentina Gremmen 98-63 (JE) AM282833 Hyvönen 3233 (JE) AM282834 Chile Australia Busch et al. Bryo 01396 (JE) Streimann 58464 (GOET) AM282835 AM282836 The Netherlands Stieperaere 8611 (JE) New Zealand Schäfer-Verwimp & Verwimp 13808 (JE) AM282837 AM282838 Indonesia Gradstein 10240 (GOET) AM180588 Nepal New Zealand Long 17402 (JE) Frahm 9-15 (GOET) AM282839 AM282840 Ireland Long H4064 (JE) AM282841 Malaysia Schäfer-Verwimp & Verwimp 18905 (GOET) United Kingdom Rycroft 020907 (GOET) AM180589 Bolivia Heinrichs et al. GP 16 (GOET) AY550130 Bolivia Germany Japan Heinrichs et al. 4076 (GOET) AJ620674 Heinrichs & Groth 4340 (GOET) AJ414633 Ohnishi 5588 (HIRO) AY438238 Phylogenetic analyses. Thirty seven new ITS15.8S- and ITS2 sequences and 9 sequences from Renker et al. (2002), Groth and Heinrichs (2003), AY438234 Groth et al. (2004) and Heinrichs et al. (2004, 2006) were aligned manually in BioEdit version 7.0.5.2 (Hall 1999), resulting in an alignment including 806 J. Hentschel et al.: Taxonomic studies in Chiloscyphus putatively homologous sites (alignment available upon request). Phylogenetic trees were inferred using maximum parsimony (MP) and maximum likelihood (ML) criteria as implemented in PAUP version 4.0b10 (Swofford 2003). MP analyses were performed with the following options implemented: heuristic search mode with 1000 random-addition sequence replicates, tree bisection-reconnection branch swapping (TBR), MULTrees option on, and collapse zero-length branches off. All characters were treated as equally weighted and unordered. Bootstrap support values were estimated by calculating 1000 bootstrap replicates (Felsenstein 1985), each with 10 random-addition-sequence replicates, TBR branch swapping, and MULTrees on. To decide on the nucleotide substitution model with the smallest number of parameters that best fits the data, the program Modeltest 3.06 (Posada and Crandall 1998) was employed. The hierarchical likelihood ratio tests selected the TrN model (Tamura and Nei 1993) with gamma shape parameter for among site variation and proportion of invariable sites. This model was implemented in ML analyses carried out as heuristic search with 20 random-addition-sequence replicates. Branching confidence was assessed using 200 bootstrap resamplings in ML analysis using a neighbor joining tree as starting tree and allowing for 3000 rearrangements under the TrN+I+G model. Chromosome counts. Gametophytes of Chiloscyphus polyanthos s.l. were cultivated in petri dishes at room temperature. Fixation and staining were performed following Auer et al. (1998), Darlington and La Cour (1963), Newton (1989), and Fritsch (1981). A few cleaned shoot tips were fixed in equal amounts of acetic acid, chloroform, and absolute ethanol and kept for one or two days in a refrigerator. The shoots were stained with lacto-orcein (50 ml lactate, 50 ml acetic acid, 2.5 g orcein) for one or two days. Chromosome numbers were identified using a light microscope Leica DMLS (magnification 1000x – 2500x). Leaf cell measurements. Ten vigorous shoots of each voucher of C. polyanthos and C. pallescens were moistened. Two leaves from the middle sector of every shoot were separated. Medium leaf cell size was determined from 10 cells from the central portion of every leaf, leading to 200 measurements/ voucher (light microscope magnification 400x). Mean leaf cell diameter and standard deviation were determined. 129 Results Of 806 investigated characters of the nrITS region, 351 were parsimony informative, 107 autapomorphic and 348 constant. The heuristic search recovered 2 equally most parsimonious trees with a length of 1343 steps, a consistency index (CI) of 0.50, a CI excluding uninformative characters of 0.45, a retention index of 0.73, and a rescaled consistency index of 0.36. The strict consensus of these trees is depicted in Fig. 1. The well bootstrap supported genus Chiloscyphus is placed sister to Heteroscyphus. Chiloscyphus is subdivided into four robust main lineages assigned to as C. subgen. Connati (Lindenb.) J.J.Engel., C. subgen. Notholophocolea (R.M.Schust.) J.J.Engel & R.M.Schust., C. subgen. Chiloscyphus, and C. subgen. Lophocolea (Dumort.) J.J.Engel & R.M.Schust. Chiloscyphus subgen. Connati is resolved sister to a well supported polytomous clade with the remaining subgenera of Chiloscyphus. The ML phylogram (ln=)7309.744, Fig. 2) is largely congruent to the MP tree. Contrary to the MP tree, C. subgen. Notholophocolea is resolved in a moderately supported sister relationship with C. subgen. Chiloscyphus. Five sectional clades are identified in C. subgen. Lophocolea, namely C. sects. Novae-Zeelandiae (‘‘Novae-zeelandii’’) J.J. Engel, Microlophocolea Spruce, Heterophylli (R.M.Schust.) J.J.Engel & R.M.Schust., and Chiloscyphus sect. Lophocolea (Dumort.) J.Hentschel, comb. nov. Bas.: Jungermannia sect. Lophocolea Dumort., Syll. Jungerm. Europ.: 59. 1831. Type: Jungermannia bidentata L. Chiloscyphus (sect. Semiteretes J.J.Engel) semiteres (Lehm.) Lehm. & Lindenb. is placed sister to C. minor (Nees) J.J.Engel & R.M.Schust. and C. profundus (Nees) J.J.Engel & R.M.Schust. of C. sect. Heterophylli. Based on this robust sister relationship and morphological similarities (see discussion chapter) we propose to include C. sect. Semiteretes in C. sect. Heterophylli: =Chiloscyphus sect. Heterophylli (R.M.Schust.) J.J.Engel & R.M.Schust., Nova 130 J. Hentschel et al.: Taxonomic studies in Chiloscyphus Fig. 1. Strict consensus of the two most parsimonious trees recovered during 1000 random-taxon-addition heuristic searches of the nrITS sequence alignment. Bootstrap percentage values (>50) are indicated at branches Hedwigia 39: 409. 1984; Lophocolea sect. Heterophylli R.M.Schust., Hepat. Anthocerotae N. Amer. 4: 214. Type: Jungermannia heterophylla Schrad. = Chiloscyphus sect. Semiteretes (‘‘Semiteres’’) J.J.Engel, Novon 9: 22. 1999, syn. nov. Type: Jungermannia semiteres Lehm. J. Hentschel et al.: Taxonomic studies in Chiloscyphus 131 Fig. 2. Phylogram resulting from a maximum likelihood analysis of 46 nrITS1-, 5.8S- and ITS2-sequences using the TrN+I+G model. Bootstrap percentage values (>50) were determined for ML (using TrN+I+G). Successful chromosome counts are indicated within Chiloscyphus subgen. Chiloscyphus. Bottom left: Chiloscyphus polyanthos A sequence of C. mandonii (Steph.) J.J.Engel & R.M.Schust. from Bolivia is nested in a robust clade made up of C. latifolius (Nees) J.J.Engel & R.M.Schust. sequences from Germany and South Africa. Morphologically, C. mandonii differs from C. latifolius only weakly by the somewhat shorter leaf teeth and more asymmetrical leaf shape. Based on the molecular topologies (Figs. 1 and 2) and the morphological similarities we lower C. mandonii to a synonym of C. latifolius: Chiloscyphus latifolius (Nees) J.J.Engel & R.M.Schust., Nova Hedwigia 39: 418. 1984; Lophocolea latifolia Nees, Naturg. Eur. Leberm. 2: 234. 1836. 132 = Chiloscyphus mandonii (Steph.) J.J.Engel & R.M.Schust., Nova Hedwigia 39. 419. 1984; Lophocolea mandonii Steph., Spec. Hep. 3: 149. 1907. syn. nov. Five accessions of Chiloscyphus pallescens from four different localities form a monophyletic lineage with a bootstrap support of 100. The sister clade is made up of four accessions of C. polyanthos s.str.; this clade also achieves a bootstrap support of 100. Chromosome counts succeeded for three accessions identified as C. polyanthos in the molecular analyses and four accessions of C. pallescens (Fig. 2); the C. polyanthos accessions had a chromosome number of n = 9, C. pallescens of n = 18. The mean leaf cell size of the investigated C. polyanthos accessions varied between 34 lm and 37 lm, of the investigated C. pallescens accessions between 42 lm and 50 lm (Fig. 3). J. Hentschel et al.: Taxonomic studies in Chiloscyphus Discussion Müller (1941) provided evidence for C. pallescens with n = 18 chromosomes usually having larger leaf cells than C. polyanthos s.str. with n = 9 chromosomes. This finding is confirmed in the present study, however, based on cytometry and cytology it is unclear whether C. pallescens consistently evolves from C. polyanthos or whether it forms a separate lineage. According to the molecular topologies (Figs. 1 and 2) Chiloscyphus polyanthos s.str. and C. pallescens form robust monophyletic lineages, indicating a longer separation and warranty of the morphologically circumscribed taxa. Congruence of morphological, cytological and molecular phylogenetic data supports species rank. The finding that Chiloscyphus plants with double chromosome sets usually have larger leaf cells than plants with single Fig. 3. Leaf cell size and chromosome number of the investigated Chiloscyphus polyanthos and C. pallescens accessions (in each case 10 leaf cell measurements from each 2 leaves from medium sector of 10 vigorous shoots; altogether 200 measurements per accession). Square: mean value. Bar: standard deviation J. Hentschel et al.: Taxonomic studies in Chiloscyphus chromosome sets is comparable to von Wettstein’s (1938) observation that polyploid moss gametophytes are usually more vigorous than gametophytes with simple chromosome sets. Different ploidy levels have been observed in several morphologically similar liverworts, e.g. Metzgeria simplex Müll.Frib. (n = 9) and M. conjugata Lindb. (n = 18), Pellia epiphylla (L.) Corda (n = 9) and P. borealis Lorb. (n = 18), Riccia fluitans L. (n = 8) and R. rhenana Lorb. (n = 16), Plagiochila porelloides (Nees) Lindenb. (n = 9) and P. britannica Paton (n = 18) as well as Cephalozia bicuspidata (L.) Dumort. (n = 18) and C. lammersiana (Huebener) F.Lees (n = 27) (Paton 1999, Frahm and Frey 2004). Järvinen (1983) and Paton (1999) point out that cell size of Chiloscyphus polyanthos and C. pallescens is subject to variation and may occasionally lead to erroneous identifications, especially of C. pallescens morphotypes from dry locations that often have rather small leaf cells. Cell size should be regarded as the first indication for species affiliation, in addition to leaf shape, number of oil bodies per cell, shape of the perianth mouth, as well as ecological parameters. Chiloscyphus polyanthos usually has broadly rounded or subtruncate leaves, 2–4 oil bodies per leaf cell, and undivided, often entire perianth lobes. Leaves of C. pallescens are subtruncate rather than rounded, have 2–5 oil bodies per cell and a plurilobed perianth with a toothed mouth (Paton 1999). Chiloscyphus polyanthos is a mainly hygrophytic species growing normally submerged on rocks, tree roots and rotting wood at the edges of streams or ponds. This species is usually lacking at strongly calcareous sites where it is replaced by C. pallescens, a mesophytic species occurring on base-rich to circumneutral substrates including damp clay and chalk, decaying logs, and other organic materials (Schuster 1980). In contrast to C. polyanthos, C. pallescens usually does not grow submerged on rocks and stones in running water (Schuster 1980, Paton 1999). The molecular data support a synonymy of Chiloscyphus latifolius and C. mandonii, a 133 taxon differing from C. latifolius s.str. mainly by the somewhat shorter leaf teeth and the more asymmetrical leaf shape. The limited value of leaf dentition for species taxonomy has already been demonstrated in the sister family Plagiochilaceae (Heinrichs 2002). Schuster (1980) regarded species of Chiloscyphus as polymorphous and the number of described binomials not reflecting the actual situation. The identical nrITS sequences of C. austrigenus (Hook.f. & Taylor) J.J.Engel & R.M.Schust. and C. gottscheoides (Besch. & C.Massal.) J.J.Engel & R.M.Schust. point to a possible conspecificity of these taxa. The molecular data also point at close relationships of C. muricatus (Lehm.) J.J.Engel & R.M.Schust., C. liebmannianus (Gottsche) J.J.Engel & R.M.Schust., and C. fragrans (Moris & De Not.) J.J.Engel & R.M.Schust. Schuster (1980) regarded C. liebmannianus with non-spinescent vegetative leaves as an extreme morphotype of the completely spinescent C. liebmannianus s.str. According to Paton (1999), Chiloscyphus fragrans has a completely smooth surface but is otherwise similar to the former taxa. If the topology holds true in an extended sampling including accessions from South Africa (type localities of C. muricatus and C. fragrans), C. fragrans and C. liebmannianus could be treated as varieties of a polymorphic C. muricatus. The southern hemispherical Chiloscyphus semiteres was introduced in Europe in the 1950s (Paton 1965, Stieperaere 1994). This range extension is supported by the molecular investigation with accessions from Australia and the Netherlands in a robust monophyletic lineage. Taxon sampling is still inadequate to provide a revised supraspecific classification of Chiloscyphus based on molecular and morphological evidence but the present data already allow some statements on previous morphological concepts. Following Hentschel et al. (2006) main clades of Chiloscyphus are here assigned to subgenera. Engel and Schuster (1984) assumed the ancestor of Chiloscyphus and Heteroscyphus 134 to have subisophyllous foliation, an erect growth, and gametangia on undifferentiated axes. They considered the species of Chiloscyphus subgen. Notholophocolea as the closest extant relatives of this hypothetical ancestor (Engel and Schuster 1984, Engel 1992a). However, C. subgen. Notholophocolea represents a derived clade in the molecular investigation and is identified sister to C. subgen. Chiloscyphus (Fig. 2) or in a polytomous lineage with C. subgen. Chiloscyphus and subgen. Lophocolea (Fig. 1). The latter three clades share mostly alternate leaves and underleaves that are not, or only inconspicuously, fused with the adjacent leaves. Chiloscyphus subgen. Notholophocolea includes species with uni- or oligoseriate antheridial stalks whereas species of subgenera Chiloscyphus and Lophocolea have uniseriate antheridial stalks. Chiloscyphus subgenus Chiloscyphus includes species with usually entire, unlobed, alternate leaves, usually free underleaves, terminal gynoecia on abbreviated lateral branches, and inconspicuous trigonous perianths (Schuster 1980, Engel and Schuster 1984). Chiloscyphus subgen. Lophocolea [including subgen. Microlophocolea (Spruce) J.J.Engel, Hentschel et al. 2006] is characterized by broad-based, alternate leaves, gynoecial branches of variable length, and trigonous perianths with often winged keels (Schuster 1980, Engel and Schuster 1984). The nrITS topologies support the position of Chiloscyphus fragmentissimus (R.M.Schust.) J.J.Engel & R.M.Schust. [= Campanocolea fragmentissima (R.M.Schust.) R.M.Schust.] within C. subgen. Lophocolea (Hentschel et al. 2006). Extension of the taxon sampling of Hentschel et al. (2006) allows for some comments on the sectional subdivision of this subgenus. Species belonging to C. sect. Microlophocolea are characterized by their small size, indistinctly bilobed, irregularly toothed leaves, lack of gemmae, and a laterally compressed, unwinged perianth (Vogelpoel 1977, Schuster 1980, Paton 1999). Chiloscyphus sect. Novae- J. Hentschel et al.: Taxonomic studies in Chiloscyphus Zeelandiae is resolved sister to C. sect. Microlophocolea. This section differs from sect. Microlophocolea by winged perianths and the frequent occurrence of gemmae (Engel 1999). Chiloscyphus (sect. Semiteretes) semiteres is placed sister to C. minor and C. profundus of sect. Heterophylli. These three taxa share the occurrence of propagules on the leaf margins, alternate, +/) rectangular leaves, bilobed underleaves that are inconspicuously fused with one of the adjacent leaves, and a camphor-like aroma (Arnell 1963, Schuster 1980, Scott 1985, Engel 1992b, Stieperaere 1994, Paton 1999, Damsholt 2002). With regard to these similarities we lower C. sect. Semiteretes to a synonym of C. sect. Heterophylli. Chiloscyphus sect. Lophocolea is the sister of C. sect. Heterophylli. The related species have bilobed to rounded leaves, perianths terminal on main shoots, and lack vegetative distribution by propagules. Future prospects The molecular data so far available lead to a partial revision of previous morphological subdivisions of Chiloscyphus. Extension of the taxon and marker set is necessary to arrive at a natural subdivision of this speciose liverwort genus and to identify the morphological characters that support the lineages. Some of the molecular results point at intercontinental gene flow or recent long distance dispersal of disjunct taxa. Sequencing of multiple accessions of the Chiloscyphus latifolius and C. muricatus complex may lead to a better understanding of both species boundaries and biogeographical patterns. We thank the late Riclef Grolle, Hermann Manitz, Ludwig Martins, Jochen Müller (all Jena), Reinhard Fritsch (Gatersleben), Rüdiger Mues (Saarbrücken), as well as Harald Schneider (Göttingen) for helpful discussions and advice. Financial support of the German Research Foundation (DFG grant HE 3584/1) is gratefully acknowledged. J. Hentschel et al.: Taxonomic studies in Chiloscyphus References Arnell S. (1963) Hepaticae of South Africa. Norstedt & Söners Förlag, Stockholm. Auer C., Hanck-Huth E., Anton H., Lion U., Mues R. (1998) Chromosomenzahlen heimischer Moose. Aus: Natur Landschaft Saarland 24: 11–24. Bednarek-Ochyra H., Váňa J., Ochyra R., Lewis Smith R. I. (2000) The liverwort flora of Antarctica. Polish Academy of Sciences, Cracow. Crandall-Stotler B. J., Forrest L. L., Stotler R. E. (2005) Evolutionary trends in the simple thalloid liverworts (Marchantiophyta, Jungermanniopsida subclass Metzgeriidae). Taxon 54: 299–316. Crandall-Stotler B. J., Stotler R. E. (2000) Morphology and classification of the Marchantiophyta. In: Shaw A. J., Goffinet B. (eds.) Bryophyte Biology. Cambridge University Press, Cambridge, pp. 21–70. Damsholt K. (2002) Illustrated flora of nordic liverworts and hornworts. Nordic Bryological Society, Lund. Darlington C. D., La Cour L. F. (1963) Methoden der Chromosomenuntersuchung. Franckh’sche Verlagshandlung. Stuttgart. Engel J. J. (1992a) Studies on Geocalycaceae (Hepaticae). VIII. A revision of Chiloscyphus subg. Notholophocolea (Schust.) Engel & Schust. J. Hattori Bot. Lab. 75: 105–115. Engel J. J. (1992b) Studies on Geocalycaceae VII. Subspecific differentiation of Chiloscpyhus semiteres together with further refinements in Chiloscyphus (s.lat.). Contr. Univ. Michigan Herb. 18: 107–111. Engel J. J. (1999) Studies on Geocalycaceae (Hepaticae). XI. Supraspecific new taxa and new combinations in Chiloscyphus Corda for Australasia. Novon 9: 22–24. Engel J. J., Schuster R. M. (1984) An overview and evaluation of the genera of Geocalycaceae subfamily Lophocoleoideae (Hepaticae). Nova Hedwigia 39: 385–463. Erzberger P., Papp B. (2004) Annotated checklist of Hungarian bryophytes. Stud. Bot. Hung. 35: 91–149. Feldberg K., Groth H., Wilson R., Schäfer-Verwimp A., Heinrichs J. (2004) Cryptic speciation in Herbertus (Herbertaceae, Jungermanniopsida): range and morphology of Herbertus 135 sendtneri inferred from nrITS sequences. Pl. Syst. Evol. 249: 247–261. Felsenstein J. (1985) Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39: 783–791. Frahm J.-P., Frey W. (2004) Moosflora, 4th ed. Ulmer, Stuttgart. Fritsch R. (1981) Methoden der Chromosomenzählung bei Bryophyten. In: Szweykowski J. (ed.) New perspectives in bryotaxonomy and bryogeography. Ser. Biol. Poznan. 20: 61–72. Gradstein S. R., Churchill S. P., Salazar-Allen N. (2001) Guide to the bryophytes of tropical America. Mem. New York Bot. Gard. 86: 1–577. Gradstein S. R., van Melick H. (1996) De Nederlandse Levermossen & Hauwmossen. Flora en verspreidingsatlas van de Nederlandse Hepaticae en Anthocerotae. Lecturis, Eindhoven. Grolle R. (1965) Harpanthus drummondii - ein Lebermoosendemit des östlichen Nordamerika. Oesterr. Bot. Z. 112: 268–284. Grolle R. (1983) Nomina Generica Hepaticarum; references, types and synonymies. Acta Bot. Fenn. 121: 1–62. Grolle R. (1995) The Hepaticae and Anthocerotae of the East African Islands. An annotated catalogue. Bryophyt. Biblioth. 48: 1–178. Groth H., Heinrichs J. (2003) Reinstatement of Chiastocaulon, based on evidence from nuclear ribosomal ITS and chloroplast gene rps4 sequences. Pl. Biol. 5: 615–622. Groth H., Lindner M., Heinrichs J. (2004) Phylogeny and biogeography of Plagiochila (Plagiochilaceae) based on nuclear and chloroplast DNA sequences. Monogr. Syst. Bot. Missouri Bot. Gard. 98: 365–387. Groth H., Lindner M., Wilson R., Hartmann F. A., Schmull M., Gradstein S. R., Heinrichs J. (2003) Biogeography of Plagiochila (Hepaticae): natural species groups span several floristic kingdoms. J. Biogeogr. 30: 965–978. Hall T. A. (1999) BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symp. Ser. 41: 95–98. Heinrichs J. (2002) A taxonomic revision of Plagiochila sect. Hylacoetes, sect. Adiantoideae and sect. Fuscoluteae in the Neotropics with a preliminary subdivision of Neotropical Plagiochilaceae into nine lineages. Bryophyt. Biblioth. 58: 1–184, Append. 1–5. 136 Heinrichs J., Gradstein S. R., Wilson R., Schneider H. (2005a) Towards a natural classification of liverworts (Marchantiophyta) based on the chloroplast gene rbcL. Cryptogam. Bryol. 26: 131–150. Heinrichs J., Lindner M., Gradstein S. R., Groth H., Buchbender V., Solga A., Fischer E. (2005b) Origin and subdivision of Plagiochila in tropical Africa based on evidence from nuclear and chloroplast DNA sequences and morphology. Taxon 54: 317–333. Heinrichs J., Lindner M., Groth H., Hentschel J., Feldberg K., Renker C., Engel J. J., von Konrat M., Long D. G., Schneider H. (2006) Goodbye or welcome Gondwana? - insights into the phylogenetic biogeography of the leafy liverwort Plagiochila with a description of Proskauera, gen. nov. (Plagiochilaceae, Jungermanniales). Pl. Syst. Evol. 258: 227–250. Heinrichs J., Lindner M., Pócs T. (2004) nrDNA internal transcribed spacer data reveal that Rhodoplagiochila R.M.Schust. (Marchantiophyta: Jungermanniales) is a member of Plagiochila sect. Arrectae Carl. Organisms Diversity Evol. 4: 109–118. He-Nygrén X., Juslén A., Ahonen A., Glenny D, Piippo S. (2006) Illuminating the evolutionary history of liverworts (Marchantiophyta) - towards a natural classification. Cladistics 22: 1–31. He-Nygrén X., Piippo S. (2003) Phylogenetic relationships of the generic complex Chiloscyphus-Lophocolea-Heteroscyphus (Geocalycaceae, Hepaticae): insights from three chloroplast genes and morphology. Ann. Bot. Fenn. 40: 317–329. Hentschel J., Wilson R., Burghardt M., Zündorf H.-J., Schneider H., Heinrichs J. (2006) Reinstatement of Lophocoleaceae (Jungermanniopsida) based on chloroplast gene rbcL data: exploring the importance of female involucres for the systematics of Jungermanniales. Pl. Syst. Evol. 258: 211–226. Järvinen I. (1983) Taxonomy and distribution of the European taxa of the genus Chiloscyphus (Hepaticae, Geocalycaceae). Ann. Bot. Fennici 20: 87–99. Müller K. (1941) Revision der europäischen Arten der Lebermoosgattung Chiloscyphus auf Grund des Chromosomensatzes und von Kulturen. Ber. Deutsch. Bot. Ges. 59: 428–436. Müller K. (1951–1958) Die Lebermoose Europas. In: Rabenhorst’s Kryptogamen-Flora von Deu- J. Hentschel et al.: Taxonomic studies in Chiloscyphus tschland, Österreich und der Schweiz. Vol. 6, 3rd ed. Geest & Portig, Leipzig. Nebel M., Kreier H.-P., Preussing M., Weiss M., Kottke I. (2004) Symbiotic fungal associations of liverworts are the possible ancestors of mycorrhizae. In: Agerer R., Piepenbring M., Blanz P. (eds.) Frontiers in Basidiomycote Mycology. IWH-Verlag, Eching, pp. 339–360. Newton M. E. (1989) A practical guide to bryophyte chromosomes. Brit. Bryol. Soc. Special 2: 1–19. Paton J. A. (1965) Lophocolea semiteres (Lehm.) Mitt. and Telaranea murphyae sp. nov. established on Tresco. Trans. British Bryol. Soc. 5: 232–236. Paton J. A. (1999) The liverwort flora of the British Isles. Harley Books, Colchester. Posada D., Crandall K. A. (1998) Modeltest: testing the model of DNA substitution. Bioinformatics 14: 817–818. Renker C., Heinrichs J., Pröschold T., Groth H., Holz I. (2002) ITS sequences of nuclear ribosomal DNA support the generic placement and the disjunct range of Plagiochila (Adelanthus) carringtonii. Cryptog. Bryol. 23: 23–29. Saiki R. K, Gelfand D. H., Stoffel S., Scharf S. J., Higuchi R., Horn G. T., Mullis K. B., Erlich H. A. (1988) Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239: 487–491. Schuster R. M. (1980) The Hepaticae and Anthocerotae of North America. East of the hundredth meridian, vol. 4. Columbia University Press, New York. Scott G. A. M. (1985) Southern Australian liverworts. Austral. Fl. Fauna Ser. 2: 1–216. Smith A. J. E. (1990) The liverworts of Britain and Ireland. Cambridge University Press, Cambridge. Srivastava A., Srivastava S. C. (2002) Indian Geocalycaceae (Hepaticae). A taxonomic study. Bishen Singh Mahendra Pal Singh, Dehra Dun. Stieperaere H. (1994) Lophocolea semiteres (Lehm.) Mitt. in Belgium and The Netherlands, another antipodal bryophyte spreading on the European continent. Lindbergia 19: 29–36. Swofford D. L. (2003) PAUP*, phylogenetic analysis using parsimony (*and other methods). Sunderland, MA: Sinauer Associates. Tamura K., Nei M. (1993) Estimation of the number of nucleotide substitutions on the J. Hentschel et al.: Taxonomic studies in Chiloscyphus control region of mitochondrial DNA in humans and chimpanzees. Molec. Biol. Evol. 10: 512–526. Vogelpoel D. A. J. (1977) Some typifications and a new subgenus of Lophocolea (Dum.) Dum. (Hepaticae). Acta Bot. Neerl. 26: 493–495. Wettstein F. von (1938) Experimentelle Untersuchungen zum Artbildungsproblem I. Zellgrössenregulation und Fertilwerden einer polyploiden Bryum-Sippe. Z. Indukt. Abstammungs- Vererbungsl. 74: 34–53. 137 Addresses of the authors: J. Hentschel, H.-J. Zündorf, F. H. Hellwig, Institut für Spezielle Botanik mit Herbarium Haussknecht und Botanischem Garten, Friedrich-Schiller-Universität, Fürstengraben 1, 07740 Jena, Germany. Alfons Schäfer-Verwimp, Mittlere Letten 11, 88634 Herdwangen-Schönach, Germany. J. Heinrichs (e-mail: jheinri@uni-goettingen.de), Abteilung Systematische Botanik, Albrecht-von-Haller-Institut für Pflanzenwissenschaften, Untere Karspüle 2, 37073 Göttingen, Germany. Verleger: Springer-Verlag GmbH, Sachsenplatz 4–6, 1201 Wien, Austria. – Herausgeber: Prof. Dr. F. H. Hellwig, Institut für Spezielle Botanik, Botanischer Garten und Herbar Haussknecht, Universität Jena, Philosophenweg 16, 07743 Jena, Germany. Redaktion: Philosophenweg 16. 07743 Jena, Germany. – Satz und Umbruch: Scientific Publishing Services (P) Ltd., Madras. – Druck: Holzhausen Druck & Medien GmbH, Holzhausenplatz 1, 1140 Wien, Austria. – verlagsort: Wien. – Herstellungsort: Wien. – Printed in Austria.