Molecular Phylogeny Of Cheilolejeunea (Lejeuneaceae)

Cheilolejeunea is one of the three largest genera in Lejeuneaceae. With ca.170 accepted names, Cheilolejeunea makes a major component of the liverwort flora in tropical and subtropical regions around the world. Traditional taxonomic studies found Cheilolejeunea and its relative genera possess several intermediate characters of Lejeuneoideae and Ptychanthoideae. But the lack of molecular studies and worldwide taxonomic treatments blurred the definition of Cheilolejeunea complex, and could not clarify the phylogeny of these genera. The present study sampled 122 accessions representing 53 taxa from Cheilolejeunea, Aureolejeunea, Evansiolejeunea, Leucolejeunea, and Omphalanthus as ingroups, as well as 12 taxa from other genera in Lejeuenaceae as outgroups. Two chloroplast genes(trnL, trnG) were amplified from the genomic DNA extraction. The phylogeny of Cheilolejeunea was reconstructed by maximum parsimony (MP) analysis, maximum likelihood (ML) analysis and Bayesian interference (BI). The position of Cheilolejeunea was explored by analyzing accessible data from Genbank. We chose two amber fossils to estimate the diverging time of Cheilolejeunea and the clades within it based on our dataset from phylogenic study. Seven characters were also chosen and analyzed based on the same dataset to reconstruct the ancient characters. Our major conclusions are as follows:1. Most species from Cheilolejeunea were grouped with Aureolejeunea, Evansiolejeunea, Leucolejeunea and Omphalanthus, forming a monophyletic clade resulted from trnL, trnG, two plastid genes matrix, as well as trnL matrix for Lejeunaceae. This clade is strongly supported by ML (bootstrap=75), MP (bootstrap= 75) and BI (=0.95) results. Based on our knowledge about the morphology of the above five genera and the present molecular data, the latter four genera are reduced to new synonyms of Cheilolejeunea. The traditional Cheilolejeunea is a paraphyletic group, Ch. decurviloba and Ch. gaoi should be excluded from the new Cheilolejeunea.2. According to our results and traditional treatments, at least five subgenera exist in Cheilolejeunea:subg. Cheilolejeunea, subg. Leucolejeunea, subg. Euosmolejeunea, subg. Omphalanthus and subg. Xenolejeunea. They differ from each other by the form of segmentation on the surface of their oil bodies, the margin of their underleaves, the length of the lobules, the length and number of cells of the second tooth of the lobule, and the pigmentation in the cell walls.3. Common ancestor of living Cheilolejeunea species occurred in late Cretacenous (66.74 mya). The first segregated clade within the genus was subg. Cheilolejeunea, which separated with other subgenera in early Eocene (54.65 mya). The major divergence of subgenera and species in Cheilolejeunea happened densely within late Oligocene and early Miocene (26.18-18.85 mya). The last 37 mya contributed a significant part of the diversity among Cheilolejeunea species.4. Ancestral state reconstructions revealed that entire underleaves are homoplastic, and have been involved several times in different clades of Cheilolejeunea. It also revealed that single cell or even reduced second tooth of the lobule is an independent event during the Cheilolejeunea evolution process. The reconstruction favored the coarsely segmented compound-type as the ancestral state of oil bodies.5. Regarding the numerous taxonomic treatment and potential nomenclature problems, we therefore proposed to reject the name "Omphalanthus" with priority, and conserve Cheilolejeunea as the name of the broad concept "Cheilolejeunea". The following new combinations are proposed:Cheilolejeunea aurifera (R.M.Schust.) W.Ye & R.L.Zhu, Ch. caducifolia (Gradst.& Schaf.-Verw.) W.Ye & R.L.Zhu, Ch. clypeata (Schwein.) W.Ye & R.L.Zhu, Ch. conchifolia (A.Evans) W.Ye & R.L.Zhu, Ch. filiformis (Sw.) W.Ye & R.L.Zhu, Ch. fulva (R.M.Schust.) W.Ye & R.L.Zhu, Ch. huanucensis (Gradst.) W.Ye & R.L.Zhu, Ch. ovalis (Lindenb. & Gottsche) W.Ye & R.L.Zhu, Ch. paramoensis (R.M.Schust.) W.Ye & R.L.Zhu, Ch. platycoleus (Herzog) W.Ye & R.L.Zhu.6. Cheilolejeunea decurviloba is morphologically different from other species of Cheilolejeunea. With the support from our phylogenetic study and the similarity found between Ch. decurviloba and Pycnolejeunea species, the former is moved back into Pycnolejeunea. 7. The unique Allorgella-type of denticulation of the leaf margin, the blunt apex of the leaf lobule, the shape and situation of the hyaline papilla distinguished Cheilolejeunea gaoi from any species in Cheilolejeunea. Taken under consideration of its distribution as endemic to China, as well as the support from our molecular data, we suggest that Ch. gaoi should be raised to the genus level, and a new genus, Sinolejeunea R.L.Zhu et al., is proposed.