Leafy Liverworts Research Articles

Super-Mitobarcoding in Plant Species Identification? It Can Work! The Case of Leafy Liverworts Belonging to the Genus Calypogeia

Molecular identification of species is especially important where traditional taxonomic methods fail. The genus Calypogeia belongs to one of the tricky taxons. The simple morphology of these species and a tendency towards environmental plasticity make them complicated in identification. The finding of the universal single-locus DNA barcode in plants seems to be 'the Holy Grail'; therefore, researchers are increasingly looking for multiloci DNA barcodes or super-barcoding. Since the mitochondrial genome has low sequence variation in plants, species delimitation is usually based on the chloroplast genome. Unexpectedly, our research shows that super-mitobarcoding can also work! However, our outcomes showed that a single method of molecular species delimitation should be avoided. Moreover, it is recommended to interpret the results of molecular species delimitation alongside other types of evidence, such as ecology, population genetics or comparative morphology. Here, we also presented genetic data supporting the view that C. suecica is not a homogeneous species.

Seasonal changes in the storage compounds and enzymatic activities in three Indian leafy liverworts

Seasonal changes in the storage compounds and enzymatic activities in three Indian leafy liverworts

Epiphytic Bryodiversity of Cedrus deodara (Roxb. ex D.Don) G.Don Forest at Lohaghat (Champawat), Uttarakhand

The present bryoexploratory study deals with the documentation of epiphytic bryophytes species and communities flourishing on the Cedrus deodara (Roxb. ex D.Don) G.Don phorophytes at Lohaghat. The study site is located near the bank of Lohawati River, with an altitudinal range of 1600-1754 m at the North-East aspect of district Champawat. A total of 42 bryotaxa were recognized from the acidic (pH 4.20 - 4.74) bark of C. deodara phorophytes of varied girth classes ranging from saplings to giant ones. In all, 09 leafy liverworts and 33 mosses were recorded, belonging to 27 families. Amongst leafy liverworts, Frullaniaceae turned out to be the dominant family followed by Lejeuneaceae. Two species of Frullania viz., F. benjaminiana Inoue and F. udarii V. Nath & Ajit P. Singh have been identified as new records for Western Himalayas. Pleurocarpous epiphytic mosses were found to dominate over the acrocarpous ones. Pylaisiadelpha capillacea (Griff.) B.C. Tan & Y. Jia, (Pylaisiadelphaceae) is identified as a new record for Western Himalayan moss flora. The members of the family Meteoriaceae and Anomodontaceae were recognized as the dominant pleurocarps. Amongst acrocarpous mosses, the family Bryaceae was noticed as the most diversified epiphytes. Morphologically, the majority of epiphytic bryophytes were found to be mat-forming followed by the tuft. Leafy liverworts namely Lophocolea minor Nees and Radula complanata (L.) Dum., as well as the mosses viz., Brothera leana (Sull.) Mull. Hal. and Pylaisiadelpha capillacea emerged as gemmiferous. The north and south aspects of the forest site were discovered to have the highest species richness. The water trickling site of the tree trunks, junction of the broad branches, humus accumulated areas, as well as giant tree bases, were observed to be the suitable habitat and microhabitats for the colonization of epiphytic bryophytes on Cedar phorophytes. The present study will provide firm bases for comparative and quantitative bryosociological investigations and would be useful to future workers.

Taxonomic notes and range extension of the monospecific genus Ramudaria (Lophocoleaceae, Marchantiophyta) endemic to India

Ramudaria is a monospecific genus well-characterized by the longitudinal, serially arranged lamelliform strips on the surface of the stem. This character seems to be unique amongst the leafy liverworts. Ramudaria lamellicaulis is so far known only from Arunachal Pradesh and Sikkim in the Eastern Himalaya, India. The species is reported here for the first time for China. The locality recorded here for China is the northernmost and highest in altitude. A description and illustrations of R. lamellicaulis based on the Chinese specimen are provided, along with a distribution map. A discussion about the status of such a morphologically remarkable genus is presented and an author citation of Ramudaria is corrected.

<I>Plagiochila pseudopoeltii</I> Inoue (Plagiochilaceae), a new distribution record to the bryoflora of Kashmir Himalaya, India

Plagiochila pseudopoeltii, a leafy liverwort is reported here for the first time from Kashmir Himalaya. The species was collected from Dachigam National Park, Srinagar district, and grows on moist soil as well as on the tree trunk. A detailed illustrated account of the species has been provided to validate the new distribution record and facilitate its easy identification.

Nanopore sequencing of chloroplast genome of Scapania undulata (L.) Dumort., 1835 (Scapaniaceae, Jungermanniales)

We performed long-read sequencing of the Scapania undulata (L.) Dumort. chloroplast genome with Oxford Nanopore Technology using improved flow cells (10.3). S. undulata is a leafy liverwort living on rocks in or near water in mountains. The size of the complete plastome sequence is 117,651 bp long, the large single copy (LSC) region is 80,606 bp long, the small single copy (SSC) region is 19,401 bp long. The nanopore sequencing allowed for the distinction of inverted repeats A and B (IRA, IRB) which were not identical. Guanine cytosine (GC) ratio is 33.7% for the complete chloroplast genome, for LSC and SSC are 31.7% and 30.6%. The phylogeny reconstruction shows that Scapania undulata is the sister to S. ciliata and S. griffithii.

Impact of in vitro phytohormone treatments on the metabolome of the leafy liverwort Radula complanata (L.) Dumort.

Impact of in vitro phytohormone treatments on the metabolome of the leafy liverwort Radula complanata (L.) Dumort.

Multiple paternity in the unisexual, haploid, leafy liverwort Porella platyphylloidea

We used isozyme markers to obtain an estimate of multiple paternity (mating of females with multiple males) in the unisexual, haploid leafy liverwort Porella platyphylloidea. From a sample of 119 plants, 15 (12.6%) were non-sex-expressing, 16 (13.4%) were females without sporophytes, 38 (31.9%) were males, and 50 (42.0%) were females bearing sporophytes. Thus, 87.4% of all plants expressed sex, and 73.5% of females bore sporophytes. The overall observed sex ratio was female-biased at 1.74:1. We extracted a total of 121 sporophytes from the 50 fertile females. Using the strict criterion that only female plants bearing two or more sporophytes with different multilocus genotypes represent cases of multiple paternity, our overall estimate is 56.8% (46 out of 81). Spearman rank-order correlation coefficients revealed that plants growing lower on trees were significantly larger than those higher on the tree and that female plants were significantly larger than male plants. Multivariate analysis showed, however, that the primary determinant underlying these correlations was the larger mean size of females.

Radula heinrichsii (Radulaceae, Porellales), a leafy liverwort from the mid-Cretaceous of Myanmar

With ca. 250 extant species, Radula is one of the largest genera of the Porellales and the sole extant genus of the leafy liverwort family Radulaceae, widely distributed around the world. The earliest fossils of Radula were described from the mid-Cretaceous Kachin amber of Myanmar, namely R. cretacea and R. heinrichsii. Here, we report two new exquisitely preserved specimens of R. heinrichsii from Kachin amber, yielding new morphological characters for an extended description of this taxon. Cladistic analysis based on a matrix of fourteen morphological characters and a molecular backbone constraint suggests its close relationship with extant species of Radula subgenus Amentuloradula. But due to distinctive morphological characters in R. heinrichsii and the younger estimated age for the crown group, it is likely that R. heinrichsii represents a stem group of the subgenus Amentuloradula. The genus Radula is characterized by a high degree of morphological homoplasy, which makes the assignment of fossils to this extant taxon difficult and requires profound knowledge of the morphology of fossils.

Re-appraisal of lacewing mimicry of liverworts from the mid-Cretaceous Kachin amber, Myanmar with a description of Selaginella cretacea sp. nov. (Selaginellales, Selaginellaceae)

A unique case of liverwort mimesis in lacewing larvae of Phyllochrysa huangi has been reported from the mid-Cretaceous Kachin amber, Myanmar. However, we caution to take this interpretation for granted because of the still incomplete assessment of the plant diversity preserved in the Kachin amber inclusions. Our re-examination of the plant fossils regarded as models for Phyllochrysa huangi received evidence for a new species of the lycophyte genus Selaginella, namely S. cretacea sp. nov., and two indeterminate species of Selaginella, besides four species of the leafy liverwort family Frullaniaceae, including Frullania baerlocheri, F. kachinensis, F. partita, and Protofrullania cornigera. Careful morphological comparison recovered that the flat larvae of Phyllochrysa huangi closely match the dorsiventrally complanate strobili of Selaginella subgenus Stachygynandrum (lycophytes) represented by S. cretacea sp. nov. In turn, the larval morphology did not show obvious similarities with the shoots of the leafy liverworts. In the context of the improved taxonomic survey of the plant inclusions, it is evident that the so-called liverwort mimesis in Cretaceous lacewing larvae was actually lycophyte mimesis. The larvae of Phyllochrysa huangi provided the earliest evidence of a putative extinct Phyllochrysa-Selaginella mimetic system.

Obtusifoliaceae, a new family of leafy liverworts to accommodate Konstantinovia, newly described from the Hengduan Mts. (South China) and Obtusifolium (Cephaloziineae, Marchantiophyta)

Obtusifoliaceae, a new family of leafy liverworts to accommodate Konstantinovia, newly described from the Hengduan Mts. (South China) and Obtusifolium (Cephaloziineae, Marchantiophyta)

Leaf Morphogenesis: Insights From the Moss Physcomitrium patens.

Specialized photosynthetic organs have appeared several times independently during the evolution of land plants. Phyllids, the leaf-like organs of bryophytes such as mosses or leafy liverworts, display a simple morphology, with a small number of cells and cell types and lack typical vascular tissue which contrasts greatly with flowering plants. Despite this, the leaf structures of these two plant types share many morphological characteristics. In this review, we summarize the current understanding of leaf morphogenesis in the model moss Physcomitrium patens, focusing on the underlying cellular patterns and molecular regulatory mechanisms. We discuss this knowledge in an evolutionary context and identify parallels between moss and flowering plant leaf development. Finally, we propose potential research directions that may help to answer fundamental questions in plant development using moss leaves as a model system.

A review of frameworks for using bryophytes as indicators of climate change with special emphasis on Sri Lankan bryoflora.

The tropical island of Sri Lanka, with a land area of 65,610 km2 and 1340 km of coastline, is highly vulnerable to impacts of climate change, with detrimental effects on agriculture, water resources, human health, coastal zones, infrastructure, industry, and biodiversity. A general increase in temperature and precipitation patterns, rising sea levels, and increase in weather-related natural disasters, such as floods and droughts, have been traced over the years. Bryophytes (liverworts, mosses, hornworts) occupy a pivotal position in the land plant evolution and form a unique part of the vegetation. Many taxa of bryophytes exhibit observable, distinct adaptations in response to changes in environmental conditions quickly. Bryophytes can be used to monitor climate change in two ways; (i) presence or absence in the ecosystem and (ii) changes in morphology and physiology that can be used for monitoring. Sri Lanka has a rich bryophyte flora consisting of 575 species of mosses, 338 species of liverworts, and 07 species of hornworts. It is estimated that 11% of mosses are endemic; there are no endemic thalloid liverworts or hornworts found in Sri Lanka, and the endemicity of leafy liverworts is yet to be investigated. The taxonomic status of endemic taxa and the biogeographic affinities of many taxa remain unexplored. Further, the potential use of bryophytes as indicators of climate change in Sri Lanka has not yet been investigated. This paper compiles the information on morphological and physiological responses of bryophytes to elevated temperature, increase in greenhouse gases, increased ultraviolet-B (UV-B) radiation, and fluctuations in humidity. In the light of this gathered global knowledge, possible species of bryophytes to be used in assessing and predicting climate change and developing a climate change model in Sri Lanka are proposed. Asian bryophytes, in general, have poorly been represented in climate change literature. We believe that this knowledge will form the foundation for future research focused on climate change mitigation in other tropical and Asian countries.

Liverworts frequently form mycothalli on Spitsbergen in the High Arctic

Mycothalli, symbioses between liverworts and soil fungi, have not previously been recorded in the Arctic. Here, 13 species of leafy liverwort from west Spitsbergen in the High Arctic are examined for the symbiosis using epifluorescence microscopy and sequencing of fungal ribosomal (r)RNA genes amplified from plant tissues. Microscopy showed that intracellular hyphal coils, key indicators of the symbiosis, were frequent (>40% stem length colonized) in nine species of liverwort in the families Anastrophyllaceae, Lophoziaceae, Cephaloziellaceae, Cephaloziaceae and Scapaniaceae, with hyphae occurring frequently (>40% cells occupied) in the rhizoids of 10 species in the same families. Dark septate hyphae, apparently formed by ascomycetes, were frequent on the stems of members of the Anastrophyllaceae, Cephaloziellaceae and Cephaloziaceae, and typically those growing on acidic mine tailings. Sequencing of fungal rRNA genes showed the presence of nine distinct groups (based on a 3% cut-off for ITS sequence divergence) of the basidiomycete Serendipita in the Anastrophyllaceae and Lophoziaceae, with ordinations and correlative analyses showing the presence of the genus to be positively associated with the frequency of hyphal coils, the occurrence of which was positively associated with edaphic factors (soil δ15N value and concentrations of moisture, nitrogen, carbon and organic matter). We propose that the frequency of mycothalli in leafy liverworts on west Spitsbergen, which is an order of magnitude higher than at lower latitudes, may arise from benefits conferred by mycobionts on their hosts in the harsh environment of the High Arctic.

Miocene Ethiopian amber: A new source of fossil cryptogams

AbstractAmber is renowned for the exceptional preservation state of its inclusions, allowing detailed morphological analysis and providing relevant environmental, palaeoecological, geographical, and geological information. Amber deposits are predominantly known from North America, Europe, and Asia, and are considered to be rare on the continents that formed Gondwana. The recent discovery of fossiliferous amber deposits in Ethiopia, therefore, provides an inimitable opportunity to close gaps in the fossil record of African terrestrial biota and to study organisms which are otherwise rare in the fossil record. Here we show that diverse cryptogams are preserved in highest fidelity in Miocene Ethiopian amber. We describe gametophyte fragments of four liverworts: Thysananthus aethiopicus sp. nov. (Porellales, Lejeuneaceae), Lejeunea abyssinicoides sp. nov. (Porellales, Lejeuneaceae), Frullania shewanensis sp. nov. (Porellales, Frullaniaceae), and Frullania palaeoafricana sp. nov. (Porellales, Frullaniaceae). Furthermore, we describe a pleurocarpous moss of the extant genus Isopterygium (Hypnales, Pylaisiadelphaceae) and a lichen representing the order Lecanorales. These new specimens represent the first amber fossils of liverworts, mosses, and lichens from the African continent and render Ethiopian amber as one of the few worldwide amber deposits preserving bryophytes (mosses and liverworts) or lichens. Fossil species of Thysananthus were recorded in Eocene Baltic and Oligocene Bitterfeld as well as Miocene Dominican and probably also Miocene Mexican ambers. Fossils that can unequivocally be assigned to Lejeunea have only been found in Dominican amber so far. Neotropical ambers contain only one taxon of Frullania to date, while the genus is most diverse in Baltic, Bitterfeld, and Rovno ambers, formed in temperate regions. The new fossils support a tropical to subtropical origin of Ethiopian amber. The new African liverwort fossils are included in an updated list of leafy liverworts described from worldwide Cenozoic ambers to date.

The evolution and biogeographic history of epiphytic thalloid liverworts

Among liverworts, the epiphytic lifestyle is not only present in leafy forms but also in thalloid liverworts, which so far has received little attention in evolutionary and biogeographical studies. Metzgeria, with about 107 species worldwide, is the only genus of thalloid liverworts that comprises true epiphytes. In the present study, we provide the first comprehensive molecular phylogeny, including estimated divergence times and ancestral ranges of this genus. Analyses are based on a plastid marker dataset representing about half of the Metzgeria species diversity. We show for the first time with molecular data that Austrometzgeria is indeed a member of Metzgeria and that two morpho-species M. furcata and M. leptoneura are not monophyletic, but rather represent geographically well-defined clades. Our analyses indicate that Metzgeria started to diversify in the Cretaceous in an area encompassing today’s South America and Australasia. Thus, Metzgeria is one of the few known epiphytic liverwort genera whose biogeographic history was directly shaped by Gondwana vicariance. Subsequent dispersal events in the Cenozoic resulted in the colonization of Asia, Africa, North America, and Europe and led to today’s worldwide distribution of its species. We also provide the first reliable stem age estimate for Metzgeria due to the inclusion of its sister taxon Vandiemenia in our dating analyses. Additionally, this stem age estimate of about 240 million years most likely marks the starting point of a transition from a terrestrial to an epiphytic lifestyle in thalloid liverworts of the Metzgeriales. We assume that the Cretaceous Terrestrial Revolution played a key role in the evolution of epiphytic thalloid liverworts similar to that known for leafy liverworts.

Liverworts from Cretaceous amber

DNA-based analyses as well as the fossil record indicate that liverworts date back to the early Paleozoic, but diverse specimens of lifelike three-dimensional fossils only occur as amber inclusions from the mid-Cretaceous onwards. Some Albian–Cenomanian ambers preserve elements of a diverse biota that existed during the rise of angiosperms, a time of fundamental change in terrestrial ecosystems that likely affected epiphytic taxa. Recent analyses have provided important insight into the phylogenetic diversification and morphological evolution of liverworts. They suggest that the mid- to Late Cretaceous was a transitional time with a significant species turnover. Here, we review all currently recognized leafy liverwort species from Cretaceous ambers based on the examination of previously described fossil specimens as well as newly discovered amber inclusions. Our survey includes a determination key. The study of 26 new fossils from Kachin amber, including fertile material, enabled us to emend descriptions for Frullania cretacea, F. baerlocheri, and Protofrullania cornigera. Mid-Cretaceous Frullaniaceae often possess some characters and character combinations that are absent in extant representatives, substantiating the assumption of a lineage turnover in the Cretaceous. Among the new fossils is Radula heinrichsii sp. nov., which expands the number of Cretaceous, amber-preserved liverwort species to eight.

A new leafy liverwort of Frullania (Frullaniaceae, Porellales) from the mid‐Cretaceous Kachin amber, Myanmar

With over 300 extant species, the nearly worldwide‐distributed genus Frullania is one of the most species‐rich genera of leafy liverworts and the sole extant genus of Frullaniaceae. Amber fossil record of Frullania is also rather diverse, with inclusions known from several amber deposits. The earliest fossils are F. baerlocheri, F. cretacea, and F. partita from the mid‐Cretaceous Kachin amber, Myanmar. Here, we describe a new fossil species of Frullania as F. kachinensis sp. nov., based on seven sterile gametophyte fragments from three pieces of Kachin amber. This new species is characterized by leaf dorsal lobes mostly with rounded apexes, leaf ventral lobules inserted in approximately 30–90 μm distance to stem, lanceolate styli, and deeply bilobed underleaves with entire‐margined to toothed lobes and V‐shaped sinuses. Morphological comparisons indicate that F. kachinensis shows mosaic character combinations of two coeval species F. cretacea and F. partita. The leaf lobes of F. kachinensis resemble those of F. cretacea, while the underleaves are more similar to those of F. partita. But in taphonomy, F. kachinensis is preserved together with Protofrullania cornigera or F. baerlocheri in the same amber, which could indicate that these three species might live in the same habitat and even on the same tree. The fossils presented in this study provide additional evidence for the presence of a diverse liverwort flora, mostly composed of Frullaniaceae lineages in the mid‐Cretaceous Kachin amber forest.

Sesquiterpene biosynthesis in a leafy liverwort Radula lindenbergiana Gottsche ex C. Hartm

Liverworts (Marchantiophyta) are among the earliest diverging lineages of extant land plants. Among their unique features, most liverworts contain membrane-bound oil bodies, organelles that accumulate diverse secondary metabolites, especially terpenoids. In contrast to the rich information on liverwort terpenoid chemistry, little is known about their biosynthesis. Recently, terpenoid biosynthesis was studied in a model thalloid species Marchantiapolymorpha, in which sesquiterpenes and monoterpenes are biosynthesized by a new type of terpene synthases termed microbial terpene synthase-like (MTPSL) proteins. Here we study terpenoid biosynthesis in a leafy liverwort Radula lindenbergiana. Vegetative plants of R.lindenbergiana were found to contain a mixture of sesquiterpenes, with (E,E)-α-farnesene/β-curcumene and (Z)-β-bisabolene being the most abundant constituents. From the analysis of the R. lindenbergiana transcriptome, five full-length MTPSL genes were identified. They were designated RlMTPSL1-5, respectively. Recombinant RlMTPSL proteins were produced in Escherichia coli and tested for sesquiterpene synthase activities using farnesyl diphosphate (FPP) as substrate. All except RlMTPSL5 were demonstrated to catalyze the formation of different sesquiterpenes. RlMTPSL1 produced multiple sesquiterpenes with eremophilene and an unidentified sesquiterpene as major products. The major products of RlMTPSL2 and RlMTPSL3 were β-elemene and an unidentified sesquiterpene, respectively. RlMTPSL4 was also a multi-product sesquiterpene synthase with an unidentified sesquiterpene being the major product. Homology-based structural modeling was performed to understand the structural basis underlying different product profiles of the RlMTPSLs proteins. Most of the sesquiterpene products of the four active RlMTPSLs were also detected in R. lindenbergiana plants. Expression levels of the four RlMTPSL genes encoding active enzymes in vegetative plants were compared. In phylogenetic analysis, RlMTPSL genes were found to cluster together, indicating lineage-specific expansion of MTPSL genes in lineages leading to R.lindenbergiana and M. polymorpha. This study strengthens evidence for the contribution of MTPSL genes to terpenoid biosynthesis in liverworts.

Advances in understanding of mycorrhizal-like associations in bryophytes

Mutually beneficial associations between plants and soil fungi, mycorrhizas, are one of the most important terrestrial symbioses. These partnerships are thought to have propelled plant terrestrialisation some 500 million years ago and today they play major roles in ecosystem functioning. It has long been known that bryophytes harbour, in their living tissues, fungal symbionts, recently identified as belonging to the three mycorrhizal fungal lineages Glomeromycotina, Ascomycota and Basidiomycota. Latest advances in understanding of fungal associations in bryophytes have been largely driven by the discovery, nearly a decade ago, that early divergent liverwort clades, including the most basal Haplomitriopsida, and some hornworts, engage with a wider repertoire of fungal symbionts than previously thought, including endogonaceous members of the ancient sub-phylum Mucoromycotina. Subsequent global molecular and cytological studies have revealed that Mucoromycotina symbionts, alongside Glomeromycotina, are widespread in both complex and simple thalloid liverworts and throughout hornworts, with physiological studies confirming that, in liverworts at least, these associations are mycorrhizal-like, and highlighting important functional differences between Mucoromycotina and Glomeromycotina symbioses. Whether a more prominent role of Mucoromycotina symbionts in plant nitrogen nutrition, as identified in liverworts, extends to other plant lineages, including the flowering plants, is a major topic for future research.
 The latest finding that ascomycete symbionts of leafy liverworts are not restricted to one fungus, Rhizoscyphus ericae, but include species in the genus Meliniomyces, as shown here in Mylia anomala, together with the recent demonstration that R. ericae forms nutritional mutualisms with the rhizoids of Cephalozia bicuspidata, fill other major gaps in our growing knowledge of fungal associations across land plants.