Liverwort oil bodies: diversity, biochemistry, and molecular cell biology of the earliest secretory structure of land plants.

Liverworts are known for their large chemical diversity. Much of this diversity is synthesized and enclosed within oil bodies (OBs), a synapomorphy of the lineage. OBs contain the enzymes to biosynthesize and store large quantities of sesquiterpenoids and other compounds while limiting their cytotoxicity. Recent important biochemical and molecular discoveries related to OB formation, diversity, and biochemistry allow comparison with other secretory structures of land plants from an evo-devo perspective. This review addresses and discusses the most recent advances in OB origin, development, and function towards understanding the importance of these organelles in liverwort physiology and adaptation to changing environments. Our mapping of OB types and chemical compounds to the current liverwort phylogeny suggests that OBs were present in the most recent common ancestor of liverworts, supporting that OBs evolved as the first secretory structures in land plants. Yet, we require better sampling to define the macroevolutionary pattern governing the ancestral type of OB. We conclude that current efforts to find molecular mechanisms responsible for the morphological and chemical diversity of secretory structures will help understand the evolution of each major group of land plants, and open new avenues in biochemical research on bioactive compounds in bryophytes and vascular plants.

Defying death: incorporating fossils into the phylogeny of the complex thalloid liverworts (Marchantiidae, Marchantiophyta) confirms high order clades but reveals discrepancies in family-level relationships.

In recent years, the use of extensive molecular and morphological datasets has clarified the phylogenetic relationships among the orders of complex thalloid liverworts (Marchantiidae). However, previous studies excluded extinct taxa; thereby, undersampling the actual taxonomic diversity of the group. Here, we conducted a total-evidence analysis of Marchantiidae incorporating fossils. The combined dataset consisted of 11 genes-sampled from the nuclear, mitochondrial and plastid genomes-and 128 morphological characters. Sixty-two species, representing all classes and orders within Marchantiophyta and genera within Marchantiidae were included in the analyses. Six fossils were scored from literature: two assigned to the outgroup (Metzgeriothallus sharonae and Pallaviciniites sandaolingensis) and four to the ingroup (Marchantites cyathodoides, M. huolinhensis, Ricciopsis ferganica and R. sandaolingensis). Tree searches were conducted using parsimony as the optimality criterion. Clade sensitivity was assessed across a wide range of weighting regimes. Also, we evaluated the influence of fossils on the inferred topologies and branch support. Our results were congruent with previously inferred clades above the order level: Neohodgsoniales was sister to a clade formed by Sphaerocarpales and Marchantiales. However, relationships among families within Marchantiales contradicted recent studies. For instance, a clade consisting of Monosoleniaceae, Wiesnerellaceae and Targioniaceae was sister to the morphologically simple taxa instead of being nested within them as in previous studies. Novel synapomorphies were found for several clades within Marchantiales. Outgroup fossils were more influential than Marchantiidae fossils on overall topologies and branch support values. Except for a single weighting scheme, sampling continuous characters and down-weighting characters improved fossil stability. Ultimately, our results challenge the widespread notion that bryophyte fossils are problematic for phylogenetic inference.

Molecular delimitation of European leafy liverworts of the genus Calypogeia based on plastid super-barcodes.

BACKGROUND: Molecular research revealed that some of the European Calypogeia species described on the basis of morphological criteria are genetically heterogeneous and, in fact, are species complexes. DNA barcoding is already commonly used for correct identification of difficult to determine species, to disclose cryptic species, or detecting new taxa. Among liverworts, some DNA fragments, recommend as universal plant DNA barcodes, cause problems in amplification. Super-barcoding based on genomic data, makes new opportunities in a species identification. RESULTS: On the basis of 22 individuals, representing 10 Calypogeia species, plastid genome was tested as a super-barcode. It is not effective in 100%, nonetheless its success of species discrimination (95.45%) is still conspicuous. It is not excluded that the above outcome may have been upset by cryptic speciation in C. suecica, as our results indicate. Having the sequences of entire plastomes of European Calypogeia species, we also discovered that the ndhB and ndhH genes and the trnT-trnL spacer identify species in 100%. CONCLUSIONS: This study shows that even if a super-barcoding is not effective in 100%, this method does not close the door to a traditional single- or multi-locus barcoding. Moreover, it avoids many complication resulting from the need to amplify selected DNA fragments. It seems that a good solution for species discrimination is a development of so-called "specific barcodes" for a given taxonomic group, based on plastome data.

Mitochondrial genomes of the early land plant lineage liverworts (Marchantiophyta): conserved genome structure, and ongoing low frequency recombination.

BACKGROUND: In contrast to the highly labile mitochondrial (mt) genomes of vascular plants, the architecture and composition of mt genomes within the main lineages of bryophytes appear stable and invariant. The available mt genomes of 18 liverwort accessions representing nine genera and five orders are syntenous except for Gymnomitrion concinnatum whose genome is characterized by two rearrangements. Here, we expanded the number of assembled liverwort mt genomes to 47, broadening the sampling to 31 genera and 10 orders spanning much of the phylogenetic breadth of liverworts to further test whether the evolution of the liverwort mitogenome is overall static. RESULTS: Liverwort mt genomes range in size from 147 Kb in Jungermanniales (clade B) to 185 Kb in Marchantiopsida, mainly due to the size variation of intergenic spacers and number of introns. All newly assembled liverwort mt genomes hold a conserved set of genes, but vary considerably in their intron content. The loss of introns in liverwort mt genomes might be explained by localized retroprocessing events. Liverwort mt genomes are strictly syntenous in genome structure with no structural variant detected in our newly assembled mt genomes. However, by screening the paired-end reads, we do find rare cases of recombination, which means multiple concurrent genome structures may exist in the vegetative tissues of liverworts. Our phylogenetic analyses of the nuclear encoded double stand break repair protein families revealed liverwort-specific subfamilies expansions. CONCLUSIONS: The low repeat recombination level, selection, along with the intensified nuclear surveillance, might together shape the structural evolution of liverwort mt genomes.

Diversity of Secondary Metabolites in the Liverwort Syzygiella rubricaulis (Nees) Stephani (Jamesoniellaceae, Marchantiophyta) from Neotropical High Mountains.

In order to evaluate the chemical diversity of Syzygiella rubricaulis (Nees) Stephani, a species with a disjunct distribution in the neotropical high mountains, a phytochemical study was carried out with samples from 12 different populations of different altitudes from four South American countries. The chemical profiles of lipophilic extracts were analyzed by GC/MS for each population and 50 different compounds were found with the predominance and richness of sesquiterpenes. The majority of the compounds were found only in one population and the total number of substances ranged from 1 to 15 among the populations, but these numbers were not correlated with altitude, and characterize each population as distinct, based on similarity analysis. The qualitative and quantitative variations of metabolites found are a response to different conditions, under which they live, mostly likely altitudinal conditions. Further studies on the quantification of these chemicals may provide information on their ecological roles and importance for the distribution of S. rubricaulis at different altitudes. Despite the known richness of secondary metabolites produced by bryophytes, they are still poorly explored in the context of the ecological expressions.

Chemotypes and Biomarkers of Seven Species of New Caledonian Liverworts from the Bazzanioideae Subfamily.

Volatile components of seven species of the Bazzanioideae sub-family (Lepidoziaceae) native to New Caledonia, including three endemic species (Bazzania marginata, Acromastigum caledonicum and A. tenax), were analyzed by GC-FID-MS in order to index these plants to known or new chemotypes. Detected volatile constituents in studied species were constituted mainly by sesquiterpene, as well as diterpene compounds. All so-established compositions cannot successfully index some of them to known chemotypes but afforded the discovery of new chemotypes such as cuparane/fusicoccane. The major component of B. francana was isolated and characterized as a new zierane-type sesquiterpene called ziera-12(13),10(14)-dien-5-ol (23). In addition, qualitative intraspecies variations of chemical composition were very important particularly for B. francana which possessed three clearly defined different compositions. We report here also the first phytochemical investigation of Acromastigum species. Moreover, crude diethyl ether extract of B. vitatta afforded a new bis(bibenzyl) called vittatin (51), for which a putative biosynthesis was suggested.

Range size heritability and diversification patterns in the liverwort genus Radula.

Why some species exhibit larger geographical ranges than others, and to what extent does variation in range size affect diversification rates, remains a fundamental, but largely unanswered question in ecology and evolution. Here, we implement phylogenetic comparative analyses and ancestral area estimations in Radula, a liverwort genus of Cretaceous origin, to investigate the mechanisms that explain differences in geographical range size and diversification rates among lineages. Range size was phylogenetically constrained in the two sub-genera characterized by their almost complete Australasian and Neotropical endemicity, respectively. The congruence between the divergence time of these lineages and continental split suggests that plate tectonics could have played a major role in their present distribution, suggesting that a strong imprint of vicariance can still be found in extant distribution patterns in these highly mobile organisms. Amentuloradula, Volutoradula and Metaradula species did not appear to exhibit losses of dispersal capacities in terms of dispersal life-history traits, but evidence for significant phylogenetic signal in macroecological niche traits suggests that niche conservatism accounts for their restricted geographic ranges. Despite their greatly restricted distribution to Australasia and Neotropics respectively, Amentuloradula and Volutoradula did not exhibit significantly lower diversification rates than more widespread lineages, in contrast with the hypothesis that the probability of speciation increases with range size by promoting geographic isolation and increasing the rate at which novel habitats are encountered. We suggest that stochastic long-distance dispersal events may balance allele frequencies across large spatial scales, leading to low genetic structure among geographically distant areas or even continents, ultimately decreasing the diversification rates in highly mobile, widespread lineages.

By how much do we underestimate species diversity of liverworts using morphological evidence? An example from Australasian Plagiochila (Plagiochilaceae: Jungermanniopsida).

As a framework for revisionary study of the leafy liverwort Plagiochila in Australia, two methods for species delimitation on molecular sequence data, General Mixed Yule Coalescence model (GMYC) and Automatic Barcode Gap Discovery (ABGD) were applied to a dataset including 265 individuals from Australia, New Zealand, and the Pacific. Groups returned by GMYC and ABGD were incongruent in some lineages, and ABGD tended to lump groups. This may reflect underlying heterogeneity in the history of diversification within different lineages of Plagiochila. GMYC from trees calculated using three different molecular clocks were compared, in some lineages different primary species hypotheses were returned by analyses of trees estimated under different clock models, suggesting clock model selection should be a routine component of phylogeny reconstruction for tree-based species delimitation methods, such as GMYC. Our results suggest that a minimum of 71 Plagiochilaceae species occur in Australasia, 16 more than currently accepted for the region, comprising 8 undetermined species and 8 synonyms requiring reinstatement. Despite modern taxonomic investigation over a four decade period, (1) real diversity is 29% higher than currently recognized; and (2) 12 of 33, or 36%, of currently accepted and previously untested Australasian species have circumscription issues, including polyphyly, paraphyly, internal phylogenetic structure, or combinations of two or more of these issues. These both reflect the many challenges associated with grouping decisions based solely on morphological data in morphologically simple yet polymorphic plant lineages. Our results highlight again the critical need for combined molecular-morphological datasets as a basis for resolving robust species hypotheses in species-rich bryophyte lineages.

[Conserved motifs in the primary and secondary ITS1 structures in bryophytes].

A study of the ITS1 nucleotide sequences of 1000 moss species of 62 families, 11 liverwort species from five orders, and one hornwort Anthoceros agrestis identified five highly conserved motifs (CM1-CM5), which are presumably involved in pre-rRNA processing. Although the ITS1 sequences substantially differ in length and the extent of divergence, the conserved motifs are found in all of them. ITS1 secondary structures were constructed for 76 mosses, and main regularities at conserved motif positioning were observed. The positions of processing sites in the ITS1 secondary structure of the yeast Saccharomyces cerevisiae were found to be similar to the positions of the conserved motifs in the ITS1 secondary structures of mosses and liverworts. In addition, a potential hairpin formation in the putative secondary structure of a pre-rRNA fragment was considered for the region between ITS1 CM4-CM5 and a highly conserved region between hairpins 49 and 50 (H49 and H50) of the 18S rRNA.

A review of molecular-clock calibrations and substitution rates in liverworts, mosses, and hornworts, and a timeframe for a taxonomically cleaned-up genus Nothoceros.

Absolute times from calibrated DNA phylogenies can be used to infer lineage diversification, the origin of new ecological niches, or the role of long distance dispersal in shaping current distribution patterns. Molecular-clock dating of non-vascular plants, however, has lagged behind flowering plant and animal dating. Here, we review dating studies that have focused on bryophytes with several goals in mind, (i) to facilitate cross-validation by comparing rates and times obtained so far; (ii) to summarize rates that have yielded plausible results and that could be used in future studies; and (iii) to calibrate a species-level phylogeny for Nothoceros, a model for plastid genome evolution in hornworts. Including the present work, there have been 18 molecular clock studies of liverworts, mosses, or hornworts, the majority with fossil calibrations, a few with geological calibrations or dated with previously published plastid substitution rates. Over half the studies cross-validated inferred divergence times by using alternative calibration approaches. Plastid substitution rates inferred for "bryophytes" are in line with those found in angiosperm studies, implying that bryophyte clock models can be calibrated either with published substitution rates or with fossils, with the two approaches testing and cross-validating each other. Our phylogeny of Nothoceros is based on 44 accessions representing all suspected species and a matrix of six markers of nuclear, plastid, and mitochondrial DNA. The results show that Nothoceros comprises 10 species, nine in the Americas and one in New Zealand (N. giganteus), with the divergence between the New Zealand species and its Chilean sister species dated to the Miocene and therefore due to long-distance dispersal. Based on the new tree, we formally transfer two species of Megaceros into Nothoceros, resulting in the new combinations N. minarum (Nees) J.C. Villarreal and N. schizophyllus (Gottsche ex Steph.) J.C. Villarreal, and we also newly synonymize eight names described in Megaceros.

Genotypic variation in a foundation tree (Populus tremula L.) explains community structure of associated epiphytes.

Community genetics hypothesizes that within a foundation species, the genotype of an individual significantly influences the assemblage of dependent organisms. To assess whether these intra-specific genetic effects are ecologically important, it is required to compare their impact on dependent organisms with that attributable to environmental variation experienced over relevant spatial scales. We assessed bark epiphytes on 27 aspen (Populus tremula L.) genotypes grown in a randomized experimental array at two contrasting sites spanning the environmental conditions from which the aspen genotypes were collected. We found that variation in aspen genotype significantly influenced bark epiphyte community composition, and to the same degree as environmental variation between the test sites. We conclude that maintaining genotypic diversity of foundation species may be crucial for conservation of associated biodiversity.

Affinities and architecture of Devonian trunks of Prototaxites loganii.

Devonian fossil logs of Prototaxites loganii have been considered kelp-like aquatic algae, rolled up carpets of liverworts, enormous saprophytic fungal fruiting bodies or giant lichens. Algae and rolled liverwort models cannot explain the proportions and branching described here of a complete fossil of Prototaxites loganii from the Middle Devonian (386 Ma) Bellvale Sandstone on Schunnemunk Mountain, eastern New York. The "Schunnemunk tree" was 8.83 m long and had six branches, each about 1 m long and 9 cm diam, on the upper 1.2 m of the main axis. The coalified outermost layer of the Schunnemunk trunk and branches have isotopic compositions (δ(13)CPDB) of -25.03 ± 0.13‰ and -26.17 ± 0.69‰, respectively. The outermost part of the trunk has poorly preserved invaginations above cortical nests of coccoid cells embraced by much-branched tubular cells. This histology is unlike algae, liverworts or vascular plants and most like lichen with coccoid chlorophyte phycobionts. Prototaxites has been placed within Basidiomycota but lacks clear dikaryan features. Prototaxites and its extinct order Nematophytales may belong within Mucoromycotina or Glomeromycota.

A phylogeny of the northern temperate leafy liverwort genus Scapania (Scapaniaceae, Jungermanniales).

Scapania is a northern temperate genus with a few disjunctions in the south. Despite receiving considerable attention, the supraspecific classification of this genus remains unsatisfactorily solved. We use three molecular markers (nrITS, cpDNA trnL-F region, atpB-rbcL spacer) and 175 accessions belonging to 50 species (plus eight outgroup taxa) to estimate the phylogeny and to test current classification systems. Our data support the classification of Scapania into six rather than three subgenera, rearrangements within numerous sections, and inclusion of Macrodiplophyllum microdontum. Scapania species with a plicate perianth form three early diverging lineages; the most speciose subgenus, Scapania s.str., represents a derived clade. Most morphological species concepts are supported by the molecular topologies but classification of sect. Curtae requires further study. Southern lineages are nested in northern hemispheric clades. Palearctic-Nearctic distribution ranges are supported for several species.

A molecular phylogeny of the Lepidozia generic complex supports re-circumscription of the Lepidozioideae.

Five molecular markers (chloroplast rbcL and trnL-trnF, mitochondrial nad5-nad4, and nuclear ITS1 and ITS2) were used to investigate membership of the Lepidoziaceae, subfamily Lepidozioideae and relationships between its constituent species. The Lepidozioideae (comprising Lepidozia, Telaranea, Kurzia, Sprucella, Psiloclada) are polyphyletic as are two of its five constituent genera (Telaranea and Kurzia). We find strong support for a monophyletic lineage comprising Lepidozia, Sprucella (nested within Lepidozia), and part of Telaranea. Within this lineage we find partial support for four main clades. Three clades of Telaranea species form consecutive sister relationships to a monophyletic Lepidozia. Relationships within Lepidozia are incompletely resolved. We provide a re-circumscription of the Lepidozioideae that excludes Kurzia, Psiloclada and elements of Telaranea.

Tramps, narrow endemics and morphologically cryptic species in the epiphyllous liverwort Diplasiolejeunea.

Diplasiolejeunea is a pantropical, epiphytic genus of leafy liverworts that occurs from the lowlands to more than 4000m altitude. Phylogenetic analyses of a molecular dataset consisting of three markers (nuclear ribosomal ITS region, plastidic trnL-F region and rbcL gene) and 122 accessions (plus two outgroups, Colura and Cololejeunea) indicate that the evolutionary diversity of Diplasiolejeunea is underestimated by current morphology-based classification. Four morphologically semi-cryptic species have been recovered. The molecular phylogenies support a deep split into a Neotropical and a Paleotropical clade, the latter structured into Australasian, Asian and Afromadacascan lineages. Presented results confirm the ranges of two pantropical species (D. cavifolia, D. rudolphiana), provide evidence for dispersal from the Neotropics into the Paleotropics, indicate speciation along altitudinal gradients and demonstrate extensive morphological homoplasy. We propose a revised supraspecific classification of Diplasiolejeunea into a predominantly Paleotropical subgenus Physolejeunea and predominantly Neotropical subgenera Austrolejeuneopsis and Diplasiolejeunea, the former containing mainly epiphytic species, the latter mainly epiphylls. Several clades are supported by combinations of morphological character states, and could be assigned to sections at some later point. This is the first comprehensive phylogeny of a largely epiphyllous genus of liverworts.

Evolution of apolar sporocytes in marchantialean liverworts: implications from molecular phylogeny.

In meiosis of basal land plants, meiotic division planes are typically predicted by quadri-lobing of the cytoplasm and/or quadri-partitioning of plastids prior to nuclear divisions. However, sporocytes of several marchantialean liverworts display no indication of premeiotic establishment of quadripolarity, as is observed in flowering plants. In these cases, the shape of sporocytes remains spherical or elliptical and numerous plastids are distributed randomly in the cytoplasm during meiosis. Through a survey of sporocyte morphology in marchantialean liverworts, we newly report the occurrence of apolar sporocytes in Sauteria japonica and Athalamia nana (Cleveaceae; Marchantiales). Molecular phylogenetic analyses revealed that the quadri-lobing of cytoplasm and quadri-partitioning of plastids were lost independently several times during the evolution of marchantialean liverworts. In addition, our phylogenetic analyses indicate that the simplified sporophytes of several marchantialean liverworts are not a primitive condition but rather represent the result of reductive evolution. The loss of the quadripolarity of sporocytes appears to correlate with the evolutionary trend of the sporophyte towards reductions. Through the evolution of the simplified sporophytes, suppression of mitotic divisions of sporogenous cells might had caused not only the modification of sporophyte ontogeny but also the drastic cytological change of sporocyte.

A multi-locus molecular phylogeny of the Lepidoziaceae: laying the foundations for a stable classification.

The Lepidoziaceae, with over 700 species in 30 genera, is one of the largest leafy liverwort families. Despite receiving considerable attention, the composition of subfamilies and genera remains unsatisfactorily resolved. In this study, 10 loci (one nuclear 26S, two mitochondrial nad1 and rps3, and seven chloroplast atpB, psbA, psbT-psbH, rbcL, rps4, trnG and trnL-trnF) are used to estimate the phylogeny of 93 species of Lepidoziaceae. These molecular data provide strong evidence against the monophyly of three subfamilies; Lepidozioideae, Lembidioideae and Zoopsidoideae, and seven of the 20 sampled genera; Lepidozia, Telaranea, Kurzia, Zoopsis, Lembidium, Paracromastigum and Chloranthelia. Several robust clades are recognised that might provide the basis for a revised subfamily circumscription including a narrower circumscription of the Lepidozioideae and a more inclusive Lembidioideae. Neogrollea notabilis is returned to the Lepidoziaceae and Megalembidium insulanum is placed in the Lembidioideae.

A new family of leafy liverworts from the middle Eocene of Vancouver Island, British Columbia, Canada.

PREMISE OF THE STUDY: Morphology is a reflection of evolution, and as the majority of biodiversity that has lived on Earth is now extinct, the study of the fossil record provides a more complete picture of evolution. This study investigates anatomically preserved bryophyte fossils from the Eocene Oyster Bay Formation of Vancouver Island. While the bryophyte fossil record is limited in general, anatomically preserved bryophytes are even more infrequent; thus, the Oyster Bay bryophytes are a particularly significant addition to the bryophyte fossil record. METHODS: Fossils occur in two marine carbonate nodules collected from the Appian Way locality on the eastern shore of Vancouver Island, British Columbia, and were prepared using the cellulose acetate peel technique. KEY RESULTS: The fossils exhibit a novel combination of characters unknown among extinct and extant liverworts: (1) three-ranked helical phyllotaxis with underleaves larger than the lateral leaves; (2) fascicled rhizoids associated with the leaves of all three ranks; (3) Anomoclada-type endogenous branching. CONCLUSIONS: A new liverwort family, Appianacae fam. nov., is established based upon the novel combination of characters. Appiana gen. nov. broadens the known diversity of bryophytes and adds a hepatic component to one of the richest and best characterized Eocene floras.

Formalizing morphologically cryptic biological entities: new insights from DNA taxonomy, hybridization, and biogeography in the leafy liverwort Porella platyphylla (Jungermanniopsida, Porellales).

PREMISE OF THE STUDY: Recognition and formalization of morphologically cryptic species is a major challenge to modern taxonomy. An extreme example in this regard is the Holarctic Porella platyphylla s.l. (P. platyphylla plus P. platyphylloidea). Earlier studies demonstrated the presence of three isozyme groups and two molecular lineages. The present investigation was carried out to elucidate the molecular diversity of P. platyphylla s.l. and the distribution of its main clades, and to evaluate evidence for the presence of one vs. several species. METHODS: We obtained chloroplast (atpB-rbcL, trnL-trnF) and nuclear ribosomal (ITS) DNA sequences from 101 Porella accessions (P. platyphylla s.l., P. × baueri, P. cordaeana, P. bolanderi, plus outgroup species) to estimate the phylogeny using parsimony and likelihood analyses. To facilitate the adoption of Linnean nomenclature for molecular lineages, we chose a DNA voucher as epitype. KEY RESULTS: Phylogenies derived from chloroplast vs. nuclear data were congruent except for P. platyphylla s.l., including a North American lineage that was placed sister to P. cordaeana in the chloroplast DNA phylogeny but sister to the Holarctic P. platyphylla s.str. in the nuclear DNA phylogeny. European and North American accessions of P. cordaeana and P. platyphylla form sister clades. CONCLUSIONS: The genetic structure of P. platyphylla s.l. reflects morphologically cryptic or near cryptic speciation into Holarctic P. platyphylla s.str. and North American P. platyphylloidea. The latter species is possibly an ancient hybrid resulting from crossings of P. cordaeana and P. platyphylla s.str. and comprises several distinct molecular entities.

Frequent pseudogenization and loss of the plastid-encoded sulfate-transport gene cysA throughout the evolution of liverworts.

PREMISE OF THE STUDY: The presence or absence of a functional copy of a plastid gene may reflect relaxed selection, and may be phylogenetically significant, reflecting shared ancestry. In some liverworts, the plastid gene cysA is a pseudogene (inferred to be nonfunctional). We surveyed 63 liverworts from all major clades to determine whether the loss of cysA is phylogenetically significant, whether intact copies of cysA are under selective constraints, and whether rates of nucleotide substitution differ in other plastid genes from taxa with and without a functional copy of cysA. METHODS: Primers annealing to flanking and internal regions were used to amplify and sequence cysA from 61 liverworts. Two additional cysA sequences were downloaded from NCBI. The ancestral states of cysA were reconstructed on a phylogenetic hypothesis inferred from seven markers. Rates of nucleotide substitution were estimated for three plastid loci to reflect the intrinsic mutation rate in the plastid genome. The ratio of nonsynonymous to synonymous substitutions was estimated for intact copies of cysA to infer selective constraints. KEY RESULTS: Throughout liverworts, cysA has been lost up to 29 times, yet intact copies of cysA are evolving under selective constraints. Gene loss is more frequent in groups with an increased substitution rate in the plastid genome. CONCLUSIONS: The number of inferred losses of cysA in liverworts exceeds any other reported plastid gene. Despite frequent losses, cysA is evolving under purifying selection in liverworts that retain the gene. It appears that cysA is lost more frequently in lineages characterized by a higher rate of nucleotide substitutions in the plastid.