Orthotrichum (Bryophyta) Inferred from Nuclear ITS Sequences

Ann. Bot. Fennici 46: 507–515 ISSN 0003-3847 (print) ISSN 1797-2442 (online) Helsinki 18 December 2009 © Finnish Zoological and Botanical Publishing Board 2009

Preliminary studies on the phylogeny of Orthotrichum (Bryophyta) inferred from nuclear ITS sequences

Jakub Sawicki1,*, Vítězslav Plášek2 & Monika Szczecińska1

1) Deparment of Botany and Nature Protection, University of Warmia and Mazury in Olsztyn, Plac Łódzki 1, PL-10-727 Olsztyn, Poland (*corresponding author’s e-mail: [email protected]) 2) Department of Biology and Ecology, University of Ostrava, Slívova 32, CZ-710 00 Slezská Ostrava, Czech Republic

Received 8 July 2008, revised version received 1 Dec. 2008, accepted 3 Dec. 2008

Sawicki, J., Plášek, V. & Szczecińska, M. 2009: Preliminary studies on the phylogeny of Orthotri- chum (Bryophyta) inferred from nuclear ITS sequences. — Ann. Bot. Fennici 46: 507–515.

The study presents a phylogenetic analysis of species of the moss genus Orthotrichum. ITS1 and ITS2 for 30 species were sequenced. The results do not fully reflect the current division of Orthotrichum into subgenera and sections. Molecular data divide the genus into two groups of species with superficial and immersed stomata, and indicate a clear distinctness of dioecious species. This suggests that a previous concept postulat- ing that the subgenus Orthophyllum should be excluded from the genus Orthotrichum might be justified.

Key words: Orthotrichum, ITS, molecular systematics, mosses, phylogeny

Introduction included in and excluded from the genus in the attempts to divide it into lower taxonomic units, The genus Orthotrichum is a widespread moss subgenera and sections. The basis for the classi- group, which includes approximately 155 spe- fication of Orthotrichum in a historical perspec- cies (Goffinet et al. 2007) distributed throughout tive was described in detail by Lewinsky (1993) the world from the Arctic to the Antarctic, except and Lewinsky-Haapasaari and Hedenäs (1998). in deserts and wet tropical forests. Species of According to the latest revision, the genus Orthotrichum grow on trees and rocks to a height Orthotrichum is divided into seven subgenera of ca. 5000 m a.s.l. (Lewinsky 1993). In the most (Lewinsky 1993): Callistoma, Exiguifolium, recent revision, Orthotrichaceae was divided into Gymnoporus, Orthotrichum, Orthophyllum, Pha- two subfamilies, each comprising two tribes: the neroporum and Pulchella. Based on morphologi- Schlotheimieae and Macromitrieae (Macromitri- cal and molecular data, Goffinet et al. (2004) oideae), and the Zygodonteae and Orthotricheae excluded the subgenus Exiguifolium from Orthot- (Orthotrichoidae), and Orthotrichum was placed richum and transferred it to the genus Letaria. The in the latter group (Goffinet & Vitt 1998, Goffi- subgenera are distinguished based on the follow- net et al. 2004). The subdivision of Orthotri- ing criteria: stoma type (superficial vs. immersed), chum has been a moot point since the end of the details of peristome teeth, presence or absence of 19th century. Certain taxa have been alternately connecting membrane, cell division of the inner 508 Sawicki et al. • Ann. BOT. Fennici Vol. 46 peristome layer, and ecology. Two of the sub- influenced by the changing environmental con- genera, Gymnoporus and Pulchella, are further ditions in which the plants have grown. Hence, divided into sections, Affinia and Leiocarpa, and molecular data can be used as a powerful tool in Pulchella, Diaphana and Rivularia, respectively. resolving taxonomic and systematic problems. The features determining species affiliation to Using nuclear ITS sequences from 28 Orthotri- particular sections are usually the details of the chum species, we investigated the phylogeny of structure of the endostome and leaves. Orthotrichum. The aim of the study was to find Despite numerous controversies and ambigu- out if the genus Orthotrichum and its subgenera ities regarding its division (Vitt 1971, Lewinsky and sections are monophyletic based on this data. 1993, Lewinsky-Haapasaari & Hedenäs 1998), Orthotrichum has never been subject to a phylogenetic analysis. The only available informa- Material and methods tion was provided by an analysis of the family Orthotrichaceae, which comprised only several Material species of the genus Orthotrichum (Goffinet et al. 1998, 2004). Our analyses included 30 species representing The internal transcribed spacer (ITS) region three genera of the family Orthotrichaceae. The is commonly used in phylogenetic and popula- genus Orthotrichum was represented by 28 spe- tion genetic studies on bryophytes (Fiedorow cies representing five subgenera. Zygodon rup- et al. 1998, Shaw 2000, Shaw & Allen 2000, estris was used as outgroup, based on a previous Shaw et al. 2005, Juslén 2006, Goryunov et al. higher-level analysis (Goffinetet al. 1998, Goffi- 2007, Sawicki & Zieliński 2008, Plášek et al. net et al. 2004). 2009). In plants, the ITS region is grouped into The list of species used in a molecular analy- arrays consisting of hundreds to thousands of sis, the details concerning voucher data and tandem repeats. This region includes two spac- the GenBank accession numbers are given in ers, ITS1 and ITS2, that separate the 18S, 5.8S Table 1. Since O. diaphanum has two sub- and 26S genes of nuclear ribosomes (Baldwin stantially different ITS sequences (J. Sawicki et al. 1995). A review of the applications of the unpubl. data), only the sequence showing greater ITS region in bryophyte systematics is given similarity to other species of the genus Orthot- in Vanderpoorten et al. (2006). Although ITS richum was used for this analysis. The recently sequences have proved effective in phyloge- described species O. moravicum (Plášek et al. netic studies on bryophytes, it should be noted 2009) and the doubtful species O. fastigiatum, that single gene phylogeny often disagrees with often treated as O. affine, were also analyzed. species phylogeny (Miyamoto & Fitch 1995, Maddison 1997). Those differences may stem not only from differing rates of evolution of DNA extraction particular genome regions (Graur & Li 2000), but also from such phenomena as hybridization, Total genomic DNA was extracted from her- common in bryophytes (Natcheva & Cronberg barium material. Single stem was ground with 2004), or horizontal gene transfer (Gustavsson silica beads in a FastPrep tissue disruptor for et al. 2005). This concerns primarily chloroplast 20 seconds and subsequently treated processed genes (Shaw et al. 2005, Sawicki et al. 2008), using the DNAEasy® Plant Mini Kit (Qiagen) but cases of such incompatibility among nuclear following the manufacturer’s protocol. Extraced genes have been also reported (Gustavsson et al. DNA samples were stored at –20 °C. 2005, Ghatnekar et al. 2006). Current morphological revisions of various moss taxa are often supported by molecular data ITS amplification and sequencing (Virtanen 2003, Hyvönen et al. 2004, Pedersen & Hedenäs 2005, Cano et al. 2005). As compared For amplification and sequencing of ITS we with morphological data, DNA sequences are not used the primers of Fiedorow et al. (1998). The Ann. BOT. Fennici Vol. 46 • Phylogeny of Orthotrichum inferred from nuclear ITS sequences 509 447/480 428/452 429/453 429/452 420/452 427/452 416/452 462/484 643/484 428/452 398/435 405/441 404/434 396/435 396/435 408/431 403/432 396/435 402/435 396/437 397/436 398/436 429/452 431/454 396/435 396/435 396/435 469/441 304/466 397/ 436 ITS1/ITS2 Sequence length ITS1/ITS2 Accession number E U871638 E U863211/ E U863214/ U871640 E U863206/ U072689 E U443993/ U072697 E U860400/ U072690 E U863212/ U871639 E U863204/ U072687 E U863213/ U072695 E U863208/ U072693 E U863210/ U871637 E U443997/ U484066 E U443996/ U484065 E U484063/ U484070 E U443998/ U484067 E U484077/ U484073 E U443994/ U035537 E U484081/ U484068 E U443999/ U072696 E U443995/ U484064 E U863207/ U072688 E U490618/ U072694 E U863209/ U871636 E U863205/ U871635 E U443992/ U072691 E U484072/ U484071 E U484062/ U484069 E U863216/ U871642 e U863215/ E U871641 e U860401/ E U072692 e U443991/ E U072686

specimens used in analysis. Herbaria: NYBG = New Botanical York Garden, Orthotrichum

Provenance, herbarium Slovakia, Poloniny Mts., Ruský potok village, herb .OP C zech Rep.,. Moravia, Jihlavské vrchy hills, herb. OP Ramit, herb. NYBG Dushanbe, in Valle Tadzhikistan, zech Rep. , Prov. Bohemia, Pivoň village, herb. OP C zech Rep. , Prov. zech Rep. , Prov. Moravia, Lázně Jeseník town, herb. OP C zech Rep. , Prov. Armenia, Yerevan, Pambakskij Khrebet, herb. NYBG Yerevan, Armenia, Bohemia, Ostrůvek, herb. OP C zech Rep. , Prov. herb NYBG C anada, Newfoundland, Notre Dame Bay, herb. OP Mts., Liptovský dvôr, Tatry Slovakia, Nízké Grenlandia, Godthab distr. Kilearsarfik, herb. NYBG Grenlandia, Godthab distr. USA, WA, Grays Harbor C o., Lake Quinault, herb NYBG USA, WA, USA, C lallam o., Little River Road, herb. NYBG Bohemia, Český les Mts., herb. OP C zech Rep. , Prov. Great Britain, S. Devon, R. Bovey, herb. NYBG Great Britain, S. Devon, R. Bovey, USA, C A, Mono o. Inyo National Forest, herb. NYBG iechanów, on Salix sp., herb OLS Poland, Młock near C iechanów, Moravia, 3 km N E of Bílá, herb. OP C zech Rep. , Prov. Poland, Góry Bialskie, Bielice village, herb. OP Moravia, Zábřeh na Moravě, herb. OP C zech Rep. , Prov. herb. OLS Mazowieckie, C iechanów, Poland, Prov. Putnam C o, Fields Farmstead herb. NYBG USA, NY, Bohemia, Rybník, herb. OP C zech Rep. , Prov. C anary Islands, umpre Neuva, umpre, herb. NYBG anada, Alberta, 3 km E of Rock Lake, herb. NYBG C anada, zech Rep. , Prov. Bohemia, Branov, Malá Pleš,, herb. OP Bohemia, Branov, C zech Rep. , Prov. herb. OP Poland, Góry Bialskie Mts., Stary Gieraltów, Poland, Pieniny Mts., Kornajowska skała, herb NYBG USA, Gunnison C o., ement reek, herb NYBG Moravia, Moravský kras karst, herb. OP C zech Rep. , Prov. Silesia, Salajka reserve, herb. OP C zech Rep. , Prov.

section Leiocarpa section Affinia section Pulchella section Rivularia section Diaphana

Gymnoporus Gymnoporus Orthophyllum Pulchella Pulchella Pulchella Phaneroporum Orthotrichum O. striatum O. vladikavkanum O. lyellii O. affine O. fastigiatum O. sordidum O. speciosum O. gymnostomum O. obtusifolium O. pylaisii O. consimile O. pulchellum O. scanicum O. rivulare O. alpestre O. diaphanum O. moravicum O. pallens O. patens O. pumilum O. stellatum O. stramineum O. tenellum O. laevigatum O. rupestre O. anomalum O. cupulatum O. pellucidum Ulota crispa Zygodon rupestris USA; OP = Opava, C zech Republic; OLS Olsztyn, Poland. USA; OP

Table 1. Table GenBank Locality, accession numbers and ITS sequence length of Species Subgenus Subgenus

Subgenus Subgenus

Subgenus

Subgenus Genus Ulota Genus Zygodon 510 Sawicki et al. • Ann. BOT. Fennici Vol. 46 sequences of the applied primers were as follow: the bootstrap method (Felsenstein 1985) with ITS1-forward 5´ CAAGGTTTCCGTAGGTG 1000 replicates. AAC 3´; ITS1-reverse 5´ CAAGAGCCAAGA- Bayesian inference was performed using TATCCG 3´; ITS2-forward 5´ CGGATATCTT- MrBayes 3.12 (Huelsenbeck & Ronquist 2001). GGCTCTTG 3´; ITS2-reverse 5´ CCGCTTAG The parameters of the likelihood model were TGATATGCTTA 3´. The ITS region was ampli- those of the general time reversible model (nst fied in a volume of 25 µl containing 20 mM = 6) with the proportion of invariable sites in

(NH4)SO4, 50 mM Tris-HCl (pH 9.0 at 25 °C), accordance with the best fitted nucleotide evolu-

1.5 mM MgCl2, 1 µl BSA , 200 µM each dATP, tion model selected on the basis of the Akaike dGTP, dCTP, dTTP, 1.0 µM of each primer, one Information Criterion (Akaike 1974) scores in unit of Taq polymerase (Qiagen) and 1 µl of the the Modeltest 3.7 (Posada and Crandall 1998). DNA solution. The reaction was processed at The MCMC algorithm was run for 1 000 000 94 °C for 1 min. followed by 30 cycles at 94 °C generations with four incrementally heated for 1 min., 59 °C for 1 min., and 72 °C for 1.5 chains, starting from random trees and sampling min., with a final extension step of 72 °C for 5 one out of every 100 generations. Trees were min. Finally 5 µl of the amplification products checked for stability, which appeared at around were visualized on 1.5% agarose gel with ethid- 40 000–50 000 chains, therefore, the first 1000 ium bromide staining. Purified PCR products trees were discarded as burn-ins. Remaining trees were sequenced in both directions using ABI were used to construct the Bayesian consensus BigDye 3.1 Terminator Cycle Kit (Applied Bio- tree. We consider good bootstrap support > 70% systems) and then visualized using an ABI Prism and weak support < 70%. In case of the Bayesian 3130 Automated DNA Sequencer (Applied Bio- clade, credibility values, significant support was systems). estimated as ≥ 95%.

Data analyses Results

Electropherograms were edited and assembled The length of the ITS1 spacer ranged from 304 using Sequencher 4.5 (Genecodes Inc.). The bp in Zygodon rupestris to 643 bp in O. obtusifo- assembled sequences were aligned using Muscle lium. The shortest ITS1 sequence among Orthot- 3.6 (Edgar 2004) and manually adjusted with richum species, 396 bp in length, was found in BioEdit 7 (Hall 1999). Phylogenetic analyses several taxa with immersed stomata: O. alpestre, were conducted using maximum parsimony O. anomalum, O. cupulatum, O. pallens, O. pel- (MP), minimum evolution (ME) and Bayesian lucidum and O. rivulare. The length of the ITS2 inference. Gaps were excluded from all phy- spacer ranged from 432 bp in O. moravicum to logenetic analyses. MEGA 4 (Tamura et al. 484 bp in O. gymnostomum and O. obtusifolium. 2007) was used for the Minimum Evolution ITS2 was ca. 20 bp longer in species with superfi- (ME) analysis and Maximum Parsimony (MP) cial stomata than in taxa with immersed stomata. analysis. The pairwise distances were estimated The alignment had a total length of 1266 with the Maximum Composite Likehood method bases. The ITS dataset contained 414 variable (Tamura et al. 2004) and initial trees gener- sites, of which 169 were parsimony informative. ated using a neighbour-joining (NJ) method. A maximum parsimony (MP) analysis resulted in The ME tree was searched using the Close 19 most parsimonious trees of 396 steps, with a Neighbor Interchange (CNI) algorithm (Nei & consistency index (CI) of 0.7761 and a retention Kumar 2000) at the search level of 2, and the index (RI) of 0.8368. The Minimum Evolution maximum number of trees retained at each step (ME) method (figure not shown) and Bayesian was set to 100. For parsimony analyses, we interference (Fig. 1) resulted in very similar trees, an applied branch and bound search as imple- differing mostly in the position of O. lyellii and mented in MEGA 4. Statistical significance of Ulota crispa. Three main clades were formed. clades within inferred trees was evaluated using Species of the subgenus Orthophyllum formed Ann. BOT. Fennici Vol. 46 • Phylogeny of Orthotrichum inferred from nuclear ITS sequences 511

Zygodon rupestris O. lyellii O. fastigiatum 99 O. striatum 100 O. sordidum O. affine Gymnoporus O. pylaisii 97 O. speciosum 97 100 O. vladikavkanum O. rupestre Phaneroporum O. laevigatum Ulota crispa 100 O. obtusifolium Orthophyllum 99 O. gymnostomum 100 O.pellucidum 96 99 O. cupulatum Orthotrichum 87 O. anomalum O. patens 98 O. stramineum 95 O. stellatum O. tenellum O. consimile O. scanicum O. pulchellum Pulchella 100 O. diaphanum 100 100 O. pumilum 100 O. moravicum 99 O. rivulare O. alpestre O. pallens 0.05 substitution/site Fig. 1. Phylogram based on the Bayesian approach for 28 Orthotrichum species with ITS sequence data. Clade credibility values above 95% are given above the branches. Bootstrap values of clades supported under parsimony and Minimum Evolution (set in italics) are given under the branches. Species with immersed stomata are under- lined and names of dioecious species are set in bold face. a distinct, well-supported clade (MP 99% and does not reflect the current division of this sub- ME 100% bootstrap support and Bayesian infer- genus into sections. The third main clade was ence 100% clade credibility). The second clade formed by species with immersed stomata. Three was formed by species with superficial stomata, species of the subgenus Orthotrichum, O. anom- including Ulota crispa (in the case of MP and alum, O. cupulatum and O. pellucidum, formed ME analysis) which seems to be more closely a distinct, well-supported (ME 84% bootstrap related to the monoecious species than O. lyellii. support, Bayesian inference 99% clade credibil- Within this clade, monoecious species formed ity) to poorly-supported (MP 62% bootstrap sup- a distinct, well-supported group (MP 97% and port) clade. Among sections of the subgenus ME 99% bootstrap support and Bayesian infer- Pulchella, only the section Diaphana seems to be ence 97% clade credibility), however showing no polyphyletic. Three representatives of the section distinct division into subgenera. Only in the case Pulchella, O. consimile, O. pulchellum and O. of the minimum evolution analysis, members scanicum, formed their own but poorly supported of the subgenus Phaneroporum, O. laevigatum clade (ME 68% and MP 30% bootstrap support). and O. rupestre, formed their own, but poorly supported clade (ME 44% bootstrap support). Among species of the subgenus Gymnoporus, Discussion a well-supported clade (MP 92% and ME 93% bootstrap support and Bayesian inference 100% Molecular data do not support the current divi- clade credibility) was formed by O. affine, O. sion of the genus Orthotrichum into subgenera. fastigiatum, O. sordidum and O. striatum, which It seems that the only distinguishable subgenus is 512 Sawicki et al. • Ann. BOT. Fennici Vol. 46

Orthophyllum, whose taxonomic distinctness has ing cells (immersed, cryptopore). The occur- been frequently postulated before (Hagen 1908, rence of both stomata types on all continents Damsholt et al. 1969, Goffinet et al. 2004). Its suggests that both types have a long history. two species O. gymnostomum and O. obtusi- However, their historical relationships are still folium were indeed placed in a separate genus, unclear (Lewinsky 1977). Vitt (1971) tried to Stroemia, by Hagen (1908). They were separated explain the fact as a genetic change, because the by obtuse leaves with incurved or plane leaf species with superficial stomata have a haploid margins and incrassate leaf cells with a stout, chromosome number, n = 6, whereas cryptopore central papilla on each side. Since Stroemia species have n = 11. However, autopolyploidy was an illegitimate name, it was later replaced plus the loss of one chromosome seem to be by Nyholmiella (Damsholt et al. 1969). A later unlikely to create the immersed type of stomata, revision of the genus Orthotrichum resulted in because it would mean that this feature is con- the inclusion of O. gymnostomum and O. obtusi- trolled by only one chromosome. The separation folium into Orthotrichum (Vitt 1973), as the of the two types of stomata is probably due to a above features were also observed in other rep- much more complex mechanism, dependent on resentatives of this genus. The affiliation of these the interaction between genes in different chro- species to the genus Orthotrichum was tested by mosomes (Lewinsky 1977). According to Paton Lewinsky-Haapasaari and Hedenäs (1998) with and Pearce (1957), the development of cryp- the use of cladistic methods. However, an analy- topore stomata as a result of adaptation to xeric sis of the selected morphological characters of conditions seems unlikely because many species the above taxa did not confirm their distinctness with immersed stomata are found in moist habi- sufficiently to place them into a separate genus. tats (O. pulchellum, O. consimile). The position On the other hand, an analysis of a nuclear of stomata provided a basis for the division of ITS sequence conducted in this study suggested this genus by Lindberg (1879), who divided it that the members of the subgenus Orthophyllum into two subgroups, Gymnoporus and Calyp- are genetically distinct from the other species toporus, comprising species with superficial and of Orthotrichum. In addition, the investigated immersed stomata respectively. Within the first sequence revealed a closer relationship between of those subgroups, taxa belonging to two sub- Ulota crispa and other Orthotrichum species genera, Gymnoporus and Phaneroporum, did than between these species and O. gymnosto- not form clades in our study. A clear division mum and O. obtusifolium, which strongly sup- of the genus Orthotrichum into groups of spe- ports separation of the latter two species from cies with immersed and superficial stomata was Orthotrichum. The distinctness O. obtusifolium also confirmed by chloroplast and mitochondrial from the other species of Orthotrichum was also sequences (Goffinet et al. 2004). An analysis of revealed by an analysis of sequences from four the nuclear locus 26S, mitochondrial locus nad5 loci (26S, nad5, rps4 and trnL–trnF region), and chloroplast loci rps4 and trnL–trnF revealed which provided a basis for excluding species of the distinctness of O. affine, O. laevigatum and the subgenus Orthophyllum from Orthotrichum O. lyellii from O. alpestre, O. anomalum, O. (Goffinet et al. 2004). Certainly, such a deci- assimile and O. macrocephalum, placing these sion should be based on an analysis of a greater species in two separate clades. number of genes and populations of these spe- Molecular data do not support the division cies. of the subgenus Gymnoporus into the sections Our results also support considerable dis- Leiocarpa and Affinia, as proposed by Lewinsky tinctness of other species with superficial sto- (1993) and Lewisky-Haapasaari and Hedenäs mata from those with immersed stomata. Stoma (1998). The type taxon of the section Leiocarpa, type is important in the taxonomy of Orthotri- O. striatum, was included in a moderately sup- chum. They are either level, with surrounding ported clade together with O. affine, O. fastig- exothecial cells (superficial, phaneropore), or iatum and O. sordidum of the section Affinia. immersed and almost covered by the surround- Another member of the section Leiocarpa, the Ann. BOT. Fennici Vol. 46 • Phylogeny of Orthotrichum inferred from nuclear ITS sequences 513 dioecious O. lyellii, was clearly distinct from to these groups are genetically different, despite the other species of the subgenus Gymnoporus. certain similarities such as the preferred habitats A large number of fixed differences in relation and the characteristics of peristome and calyptra. to the other species in the subgenus suggests For the subgenus Pulchella, in contrast to that this taxon should be placed into a separate the subgenus Gymnoporus, our study resulted in infrageneric taxon. Its exclusion from the sec- a moderately supported division into sections. tion Leiocarpa and the formation of the section The only clearly distinct section was Pulchella, Lyelliana by Schimper (1876, followed Lewin- whose species were grouped within a single sky 1993), seems justified. The distinctness of clade in all analyses. The section Pulchella could O. lyellii from members of the section Leiocarpa be considered monophyletic, while the larg- was also noted by Vitt (1971), who however did est section, Diaphana, is rather paraphyletic. not classify this taxon into a separate section or The position of O. rivulare may also indicate a subgenus. Based on the ITS sequences Ulota genetic distinctness of the section Rivularia. crispa is closely related to Orthotrichum species The number and ornamentation of exostome of the subgenera Gymnoporus and Phaneropo- teeth and endostome segments have a central role rum. Gymnoporus species were found to be more in the taxonomy of Orthotrichum. The ancestors genetically similar to U. crispa than to the dioe- of the genus probably had a very well devel- cious O. lyellii belonging to the same subgenus. oped double peristome (with 16 exostome teeth Similar results were obtained while analyzing and endostome segments) covered with papillae the chloroplast rbcL sequence which showed a (Lewinsky 1977). Vitt (1973) suggests that a close relationship between Ulota and Orthot- reduction in peristome characters (mainly in the richum species of the subgenus Gymnoporus number of endostome segments and exostome (Goffinet et al. 1998). An analysis of four loci teeth, from 16 to 8 or 0) may result from adapta- from the nuclear (26S), mitochondrial (nad5) tion to different ecological conditions. According and chloroplast (rps4, trnL–trnF) genome also to that author, specialization to xeric habitats, showed that Orthotrichum species with superfi- such as the trunks of trees and dry rock surfaces, cial stomata are closer to species of Ulota than is the driving force. This view, however, cannot to Orthotrichum species with immersed stomata be regarded as generally valid, as a reduction of of the subgenera Orthotrichum and Pulchella endostome segments has not been observed in (Goffinet et al. 2004). many xeric species growing in dry places (O. A further group was formed by species of the diaphanum, O. striatum), whereas a species (O. subgenera Orthotrichum and Pulchella. Unlike affine) of shaded and moist places has a reduced species with superficial stomata, representa- peristome of eight teeth and segments. The pres- tives of the subgenus Orthotrichum formed a ence of a preperistome, considered by Vitt (1973) clade. As regards morphological characteristics, as an advanced character, was subsequently subgenus Orthotrichum differs from subgenus regarded as a primitive state, because it is present Pulchella mostly in having straighter peristome in many species with a well-developed, not teeth and no connecting membrane (Lewinsky reduced peristome (Lewinsky 1989). We found 1993). The placing of species of this subgenus no correlation between particular peristome char- within the ‘Pulchella’ group is consistent with acters and adaptive evolution in Orthotrichum. the results of Vitt (1971) and with one of the Although our results do not fully reflect the three relationship ideas proposed by Lewinsky- current taxonomic division, it points to the fact Haapasaari and Hedenäs (1998). In both cases, that the former taxonomic concepts were often the authors placed the subgenus Orthotrichum correct. This refers in particular to the divi- within the Pulchella clade, based on morpho- sion proposed by Lindberg (1879) and to the logical characters. However, our results did not separation of the genus Stroemia (Hagen 1908), confirm a close relationship between members of which seems justified. However, this remains a the subgenera Orthotrichum and Phaneroporum, hypothesis with some support and it should be suggested by Lewinsky (1993). Taxa belonging confirmed by further studies. 514 Sawicki et al. • Ann. BOT. Fennici Vol. 46

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