Introduction

Molecular phylogenetic studies have substantiated the position of liverworts (Marchantiophyta) as the earliest diverging extant main lineage of land plants (Qiu et al. 2006) and provided numerous new insights into their classification (Crandall-Stotler et al. 2009). The leafy liverworts represent a derived lineage that basically splits into two main clades assigned to as Jungermanniales and Porellales (Heinrichs et al. 2005, 2007; He-Nygrén et al. 2006). Molecular data allowed for a subdivision of Jungermanniales into four suborders (He-Nygrén et al. 2006) and pointed at numerous incongruences of morphology-based classification schemes and topologies derived from DNA sequence evidence (Heinrichs et al. 2004; Hentschel et al. 2006, 2007a; De Roo et al. 2007; Hendry et al. 2007; Vilnet et al. 2008). Schill et al. (2004) demonstrated that the family Lophoziaceae is nested in Scapaniaceae. Heinrichs et al. (2005) formalized the synonymy of Lophoziaceae and Scapaniaceae and showed that Lophoziaceae subfam. Jamesonielloideae (Inoue 1966) forms a separate lineage. Subsequently, this subfamily was included in Adelanthaceae (Heinrichs et al. 2007) or treated as a family of its own (He-Nygrén et al. 2006).

In the framework of a study on the molecular phylogeny of Adelanthaceae s. l. (Feldberg et al., unpublished), we produced chloroplast DNA rbcL sequences of several genera that earlier were assigned to Lophoziaceae subfam. Jamesonielloideae or related clades (Horikawa 1934; Schuster 2002; Long and Váňa 2007). Here we present our results with regard to the genera Gottschelia Grolle and Scaphophyllum Inoue. Gottschelia was set up to include two species with a jungermannialean habit that have exceedingly elongated, cylindrical perianths with the female bracts not accompanied by a bracteole, and slenderly ellipsoidal to cylindrical capsules with linear valves, and angular-stellate gemmae (Grolle 1968; Schuster 2002). Grolle et al. (2003) and Long and Váňa (2007) added two further species that have broadly ellipsoid to ellipsoid-cylindric perianths and do not produce gemmae. Scaphophyllum includes a single species with canoe-shaped, strongly concave leaves (Schuster 1998).

Materials and methods

DNA extraction, PCR amplification and sequencing

DNA extraction, PCR amplification and sequencing were carried out as described in Hentschel et al. (2006). If no product was detected, a second (nested) PCR was performed using the forward primer rbcL-210-F and the reverse primer rbcL-1200-R (Gradstein et al. 2006).

Taxon sampling and phylogenetic analyses

Nineteen new sequences of the genera Adelanthus Mitt., Gottschelia, Scaphophyllum, Syzygiella Spruce and Wettsteinia Schiffn. were generated. The new rbcL sequences were compared with GenBank sequences using the BLASTN program (Altschul et al. 1990) and incorporated into a large alignment of rbcL sequences of various taxa of Jungermanniopsida. The BLAST searches and preliminary maximum parsimony (MP) analyses of the Jungermanniopsida dataset (results not shown) indicated an affiliation of the new sequences to either Jungermanniales suborder Cephaloziineae or Jungermanniineae sensu He-Nygrén et al. (2006). Further sequences of representatives of these suborders were sampled based on the results of Hentschel et al. (2007a). Representatives of Jungermanniales suborder Lophocoleineae [Bazzania Gray, Chiloscyphus Corda, Mastigophora Nees, Plagiochila (Dumort.) Dumort., He-Nygrén et al. 2006] were chosen as outgroups.

The sequences (Table 1) were manually aligned in BioEdit version 5.09 (Hall 1999) resulting in a dataset including 1.053 homologous sites. Missing parts of sequences were coded as “N” (A, C, G or T). Phylogenetic trees were inferred using maximum parsimony as implemented in PAUP* version 4.0b10 (Swofford 2000). MP analyses were performed with the following options implemented: heuristic search mode with 1,000 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 (BS) values were estimated by calculating 1,000 bootstrap replicates (Felsenstein 1985), each with 10 random-addition-sequence replicates, TBR branch swapping, and MULTrees on. Rearrangements were limited to 10 million per replicate. Bootstrap percentage values above 70 were regarded as good support (Hillis and Bull 1993). Bayesian inference of phylogeny was conducted using a general time reversible model (GTR) as implemented in MrBayes version 3.1.2 (Huelsenbeck and Ronquist 2001). A Bayesian search was carried out using four simultaneous Markov chains, 5 million generations and sampling every 100th generation. The software tool Tracer version 1.3 (Rambaut and Drummond 2003) was used to examine the parameters and determine the number of trees needed to reach stationarity (burn-in). Bayesian posterior probability (BPP) confidence values were generated from trees found after this initial burn-in period. Bayesian clade credibility values were regarded as significant when exceeding BPP ≥ 0.95 (Larget and Simon 1999).

Table 1 Taxa used in the present study, including information about the origin of the studied material, voucher information and the herbaria where the vouchers are deposited, as well as GenBank accession numbers
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Results

Three hundred and eight of the 1,053 aligned characters were parsimony informative, 90 autapomorphic and 655 constant. The strict consensus of 432 maximally parsimonious trees (length 1,659 steps, consistency index excluding uninformative characters 0, 29) is depicted in Fig. 1. Bayesian searches sampled 45,001 trees (mean ln = −9.465955). The Bayesian topology (Fig. 2) is largely congruent with the MP topology. The ingroup is divided into Cephaloziineae (BS 76, BPP 1.00) and Jungermanniineae (BS 52, BPP 1.00). Seven accessions of the generitype Gottschelia schizopleura (Spruce) Grolle form a monophyletic lineage (BS 100, BPP 1.00) that is placed sister to Chaetophyllopsis whiteleggei (Carrington and Pearson) R. M. Schust. (BS 96, BPP 1.00). Asian and African accessions of G. schizopleura are separated by a deep split (Fig. 2). The G. schizopleura/Chaetophyllopsis clade is placed sister to a clade with representatives of Scapaniaceae (Cephaloziineae) (BS 98, BPP 1.00). Two accessions of Gottschelia patoniae Grolle, Schill and D. G. Long and three accessions of Scaphophyllum speciosum (Horik.) Inoue form monophyletic lineages within the Solenostoma Mitt. clade (Jungermanniineae) (BS 77, BPP 1.00). An accession (type material) of Gottschelia grollei D. G. Long and Váňa is placed sister to Solenostoma sphaerocarpum (Hook.) Steph.

Fig. 1
figure 1

Strict consensus of the 432 most parsimonious trees recovered during 1,000 random-taxon-addition heuristic searches of the rbcL data set. Bootstrap support is indicated at branches. The family classification is based on Heinrichs et al. (2007, Adelanthaceae), Hentschel et al. (2007a, Jungermanniaceae) and Crandall-Stotler et al. (2009)

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Fig. 2
figure 2

Majority-rule consensus tree of trees recovered in stationary phase of Bayesian search. Bayesian posterior probabilities (≥0.95) are indicated at branches

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Based on the molecular topologies, we propose a transfer of G. grollei, G. patoniae, and S. speciosum to Solenostoma:

Solenostoma grollei (D. G. Long and Váňa) K. Feldberg, J. Hentschel, A. Bombosch, D. G. Long, Váňa and J. Heinrichs, comb. nov. Bas.: Gottschelia grollei D. G. Long and Váňa, J. Bryol. 29: 167. 2007.

Solenostoma patoniae (Grolle, Schill and D. G. Long) K. Feldberg, J. Hentschel, A. Bombosch, D. G. Long, Váňa and J. Heinrichs, comb. nov. Bas.: Gottschelia patoniae Grolle, Schill and D. G. Long, J. Bryol. 25: 3. 2003.

Solenostoma speciosum (Horik.) J. Hentschel, K. Feldberg, A. Bombosch, D. G. Long, Váňa and J. Heinrichs, comb. nov. Bas.: Anastrophyllum speciosum Horik., J. Sci. Hiroshima Univ., Ser. B, Div. 2, Bot. 2: 147. 1934.

Solenostoma speciosum subsp. villosum (R. M. Schust.) J. Hentschel, K. Feldberg, A. Bombosch, D. G. Long, Váňa and J. Heinrichs, comb. nov. Bas.: Scaphophyllum speciosum subsp. villosum R. M. Schust., Bryologist 101: 434. 1998.

Discussion

The recent progress in liverwort phylogeny is reflected in the new liverwort classification of Crandall-Stotler et al. (2009). However, several genera have not yet been sequenced or been tested for their monophyly. Therefore we anticipate that modifications of the current classification will prove to be necessary to arrive at a natural subdivision.

Several recent molecular phylogenetic studies demonstrated that genus concepts need to be modified both in Scapaniaceae (De Roo et al. 2007; Vilnet et al. 2008) and Adelanthaceae s. l. (De Roo et al. 2007; He-Nygrén et al. 2006). Recent molecular work also demonstrated the need for modified family concepts in Jungermanniineae (Hentschel et al. 2007a). The present study adds to growing evidence that many current genus concepts do not reflect ancestral relationships within these clades. Accessions of three species currently assigned to Gottschelia (Long and Váňa 2007) are placed in two different suborders of Jungermanniales, Cephaloziineae (G. schizopleura) and Jungermanniineae (G. grollei, G. patoniae). The generitype G. schizopleura is resolved in Scapaniaceae (including Lophoziaceae, Heinrichs et al. 2005) as suggested by earlier authors (Grolle et al. 2003; Long and Váňa 2007). This position is supported by the stellate gemmae, and the long-cylindrical, slender perianth with a plicate, crenulate-dentate mouth (Grolle 1968). G. grollei and G. patoniae lack gemmae and are provided with broadly ellipsoid to ellipsoid-cylindric perianths (Long and Váňa 2007). These characters link the two species with Solenostoma. The concave, unlobed leaves with rough cuticle are also known from some representatives of Solenostoma (Váňa 1973, 1974). Based on the outcome of the molecular phylogenetic analyses and the morphological similarities, we transfer G. grollei and G. patoniae to Solenostoma. Attempts to sequence the fourth Gottschelia species, G. maxima (Steph.) Grolle (Long and Váňa 2007), remained unsuccessful. This species [epitype, designated by Grolle et al. (2003: 6), JE!] closely resembles G. schizopleura but differs in the crenulate-dentate leaf margins. Judging from morphology, it can remain in Gottschelia.

The monospecific genus Scaphophyllum (Schuster 1998) differs from Gottschelia and Solenostoma by canoe-shaped, strongly concave leaves, and a loosely to densely ciliate stem surface. Based on these differences, Schuster (1998, 2002) placed Scaphophyllum in its own subfamily, Scaphophylloideae. However, already Váňa (1973) pointed at striking similarities of Scaphophyllum and several species of Jungermannia L. s. l. that are now placed in Solenostoma. The present study supports Váňa’s (1973) view: Scaphophyllum is nested in a robust subclade of Solenostoma. Based on the molecular topologies (Figs. 1, 2) and the morphological similarities of Scaphophyllum and Solenostoma, we transfer Scaphophyllum to the latter genus.

The African and Asian accessions of G. schizopleura are separated by long branches (Fig. 2), indicating a considerable period of isolation of the respective populations. Genetic variation within morphologically circumscribed liverwort species seems to be a common phenomenon and has been observed for several clades, e.g., Bryopteris (Nees) Lindb. (Hartmann et al. 2006), Frullania Raddi (Hentschel et al. 2009), Herbertus Gray (Feldberg et al. 2007), Porella L. (Hentschel et al. 2007b) and Ptychanthus Nees (Ahonen et al. 2005). In many cases, the phylogenetic structure follows a geographical pattern and could be indicative of cryptic speciation (Heinrichs et al. 2009). The Madagascan and Réunion accessions of G. schizopleura are more robust than the accessions from Indonesia, Malaysia and Sri Lanka. A detailed morphological investigation could possibly reveal morphological characters that would allow for a separation of the main clades of G. schizopleura. The type of G. schizopleura originates from Madagascar (Spruce 1885). Grolle (1968, 1971) lowered several (Austral-)asian taxa to synonyms of G. schizopleura. If two species are actually at hand, the basionym Jungermannia colorata δ microphylla Nees should be picked up to name the Asian populations.