Dec 022016
Some of the herbarium collections of Marchantia held in the RBGE herbarium

Some of the herbarium collections of Marchantia held in the RBGE herbarium

Many new species are already included in natural history collections around the world, it’s just that nobody has yet got around to examining the material, recognising that it represents something novel, and publishing a name for it. Sometimes these new species are filed under the epithet of a similar named species, sometimes they’re just filed under the genus name with other collections that have not been identified to species, and sometimes they have been annotated to recognise that they’re probably distinct from all the species that have already been described, e.g., as “sp. nov.

David Long has made a huge number of plant collections from around the world in his 40-plus year botanical career, with many of these collections not yet fully examined. Some of this material is being mined for DNA sequencing projects at RBGE, and for some of our key plant groups, as well as sequencing well-identified material, we are also sequencing plants that have not been assigned to species. Molecular lab work is fast compared to close morphological studies of multiple plant specimens; this can therefore speed up the processes of traditional taxonomy, by allowing it to focus on things that are obviously distinct.

One lineage that David Long is particularly involved with, and that remains one of our key plant groups, is the complex thalloid liverworts. Some of our sequencing work has involved Marchantia, which made Xiang et al.‘s recent description of a new species in the genus, Marchantia longii, particularly interesting. In the last few days, the DNA sequences that were included in the paper were made publicly available on the NCBI site, GenBank. One of the regions that was sequenced by Xiang et al., the plastid-encoded RuBisCo Large subunit gene rbcL, was also included in our study, and so I was able to put the two data sets together, and see how the new species fits into our phylogenies.

The results are interesting: When Xiang et al. named M. longii, they did so in part because the area that the plant came from, in northwestern Yunnan, is one in which David has been very active. In fact, at RBGE we had already generated DNA sequence data from nine accessions of Marchantia that David had collected there. I was delighted to find that two of these accessions (collections Long 36155 and Long 34642), which had been filed in our collections without a specific epithet, are an exact genetic match to Marchantia longii. It seems that David really does have an affinity for the plant, having gone out and found some even before it was named for him!


Long’s Marchantia

A rapid phylogeny of Marchantia, from the RBGE collections. II. Illuminating our sampling

A rapid phylogeny of Marchantia, from the RBGE collections. I. Sampling

Sep 122016
Photo 11-09-2016, 12 14 22

The tiny liverwort Colura calyptrifolia (photographed with an iPhone and a x20 handlens!)

Colura calyptrifolia (or to give it its appropriately creepy-sounding common name, the Fingered Cowlwort), is one of our most fascinating UK liverworts. Absolutely tiny (the leaves are about a millimetre long and whole plants often only 2-3 mm), it is heavily modified from the basic leafy-liverwort body plan, the leaves formed into inflated sacs like miniscule balloons with pointed “beaks” at one end. These tiny sacs have even tinier trapdoor-like flaps that only open inwards, allowing them to capture ciliate protozoa and other microscopic creatures (conclusively observed in another species of the same genus). It’s not yet certain if the liverwort gains nutrients from “swallowing” these animals, although this might be a reasonable hypothesis given the similarity of the mechanism to that found in much larger carnivorous plants such as bladderworts.

Photo 11-09-2016, 12 14 20

The leaves of Colura are modified into tiny ballon-like sacs that trap small animals

This colony was spotted yesterday in Anglezarke, Lancashire following the British Bryological Society (BBS) AGM in Manchester. Populations of Colura have undergone a spectacular expansion over the last 10 or 20 years, particularly in conifer plantations where they occur as tiny epiphytes. Previously the plant was rather rare, occurring mostly on rock in humid gullies and restricted to the wetter areas of the far west. This sighting was on the trunk of a willow at the edge of a reservoir.

Something else that has changed rapidly over the last 10 or 20 years is the ease with which small things can be photographed and shared. These pictures were taken simply by pointing the camera of an iPhone through a x20 handlens (the latter much cheaper than an iPhone and even more useful!),  and if we had wished could have been made available online instantaneously. As poorly-known biodiversity is increasingly threatened globally, should we be making better use of cheap imaging and real-time networking of expertise to facilitate species discovery and monitoring?



Barthlott, W., Fischer, E., Frahm, J.-P. & Seine, R. (2000). First Experimental Evidence for Zoophagy in the Hepatic Colura. Plant Biology 2(1):93-97. 

Blockeel, T L, Bosanquet, S D S, Hill, M O and Preston, C D (2014). Atlas of British & Irish Bryophytes. Pisces Publications, Newbury.

Sep 092016
Sphaeropcaros texanus photographed by David Long (Long 33162)

European material of Sphaerocarpos texanus, photographed by David Long (Long 33162)

The Sphaerocarpales (or “Bottle Liverworts”) form a very distinct group in the complex thalloid liverworts, with ca. 30 species in five genera: originally the group just included Geothallus (monospecific), Sphaerocarpos (8-9 species) and Riella (ca. 20 species), with two more monospecific genera, Austroriella and Monocarpus, added within the last few years. All five genera have very unusual, and highly reduced, thallus morphologies. With the exception of Monocarpus, they also all enclose their sex organs (or gametangia – the antheridia and archegonia) in inflated flask-shaped bottles (as can be seen in the accompanying photograph). This feature sets them apart from all other liverworts. All of them are adapted to extreme habitats, including arable fields, hot arid regions, seasonal lakes and pools, and salt pans.

A worldwide revision of the second largest genus of the group, Sphaerocarpos, is over 100 years old (Haynes 1910); other revisional work focuses on individual geographic areas, including South Africa (Proskauer 1955), North America (Haynes & Howe 1923, Frye & Clark 1937, Schuster 1992, Timme 2003), California (Howe 1899), Europe (Reimers 1936, Müller 1954), and France (Douin 1907). No revisions have been made for large areas including Australia, Asia and South America, and most of the work predates any DNA-based concepts of plant identification or species relationships. Bringing the taxonomy of Sphaerocarpos into the 21st century, Dr Daniela Schill spent 18 months (2007-2009) at RBGE on a Sibbald Trust-funded project to compile a world-wide taxonomic revision of the genus. Two field expeditions fed into the project, with Dr David Long collecting European species in Portugal in April 2007, and Daniela collecting North American species in California in March 2008 (funded by the Peter Davis Expedition Fund).

Spore SEMs of Sphaerocarpus drewiae, taken by Daniela Schill

Spore tetrads of Sphaerocarpos drewiae, SEMs taken by, and plate prepared by, Daniela Schill

Daniela’s work is based on morphological and anatomical characters, including spore characters that she observed using Scanning Electron Microscopy (SEM). Her aim has been to produce identification keys to the species, species descriptions, species lists, synonyms, botanical drawings, distribution maps, and ecological, nomenclatural and taxonomical notes. Although the study is not yet published, much of it, including SEM plates for spores from the ca. 9 different species (as seen on the right), is complete.

In parallel, RBGE staff have also been sequencing multiple accessions of all available Sphaerocarpos species, producing data that has helped inform some of Daniela’s taxonomic decisions, and that also allow us to generate a stand-alone phylogeny for the genus.

This research will lead to some taxonomic changes. For example, European Sphaerocarpos texanus plants differ from American S. texanus, both in their DNA sequences and in their spore characters, and so they are likely to be considered a separate species. Furthermore, European Sphaerocarpos michelii material includes three different forms based on spore characters; these are also confirmed by molecular research, and may be recognised at or below the rank of species.



Cargill, D.C. & J. Milne. 2013. A new terrestrial genus and species within the aquatic liverwort family Riellaceae (Sphaerocarpales) from Australia. Polish Botanical Journal 58(1): 71-80.

Douin R. 1907. Les Sphaerocarpus français. Revue Bryologique 34(6): 105-112.

Frye T.C. & L. Clark. 1937. Hepaticae of North America. University of Washington Publications in Biology 6: 105-113.

Haynes C.C. 1910. Sphaerocarpos hians sp. nov., with a revision of the genus and illustrations of the species. Bulletin of the Torrey Botanical Club 37(5): 215-230.

Haynes C.C. & M.A. Howe. 1923. Sphaerocarpales. North American Flora 14: 1-8.

Howe  M.A. 1899. The hepaticae and anthocerotes of California. Memoirs of the Torrey Botanical Club 7: 64-70.

Müller K. 1954. Die Lebermoose Europas. In: Rabenhorst’s Kryptogamenflora von Deutschland, Österreich und der Schweiz. 3. Auflage. Volume VI. Part 1. Leipzig, Akademische Verlagsgesellschaft Geest & Portig K.-G., Johnson Reprint Corporation (1971), New York, London.

Proskauer J. 1955. The Sphaerocarpales of South Africa. The Journal of South African Botany 21: 63-75.

Reimers H. 1936. Revision des europäischen Sphaerocarpus-Materials im Berliner Herbar. Hedwigia 76: 153-164.

Schill D.B., L. Miserere & D.G.Long. 2009. Typification of Sphaerocarpos michelii Bellardi, S. terrestris Sm. and Targionia sphaerocarpos Dicks. (Marchantiophyta, Sphaerocarpaceae). Taxon 58(2): 638-640.

Schuster R.M. 1992. Sphaerocarpales. In: The hepaticae and anthocerotae of North America V. Field Museum of Natural History, Chicago: 799-827.

Timme S.L. 2003. Sphaerocarpaceae. In: Bryophyte Flora of North America, Provisional Publication.


Sep 062016

University of Edinburgh/RBGE student David Bell, studying for the Masters degree in the Biodiversity and Taxonomy of Plants; thesis submitted August 2009.

Supervisors: Dr David Long and Dr Michelle Hart.


David used plastid DNA barcode markers rbcL (from 34 accessions) and psbA-trnH (from 36 accessions) to look at the four species of Herbertus in Europe, H. aduncus subsp hutchinsiae (British Isles, Norway and Faroes), H. stramineus (British Isles, Norway and Faroes), H. borealis (Scotland and Norway) and H. sendtneri (European Alps).

In addition to the four recognised taxa, David’s study identified a fifth species, later named as H. norenus, that occurs in Norway and the Shetland Isles.

A paper based on David’s MSc thesis work was published in Molecular Ecology Resources in 2012.

Herbertus norenus, photographed by David Long

Mixed sward including Herbertus norenus, photographed in Shetland by David Long


Bell et al. 2012, MER



Other student projects at the Gardens:

Student projects at RBGE: DNA barcoding British liverworts: Lophocolea

Student projects at RBGE: Barcoding British Liverworts: Plagiochila (Dumort.) Dumort.

Student projects at RBGE: Barcoding British Liverworts: Metzgeria

Aug 122016

University of Edinburgh Biotechnology student Kenneth McKinlay’s 4th year honours project, 2013. Supervisors: Dr David Long, Dr Laura Forrest

David Long and Kenneth checking out the Lophocolea on a decaying log in the Scottish Borders

David Long and Kenneth check out Lophocolea on a decaying log in the Scottish Borders

Kenneth barcoded all six species of British Lophocolea, L. bidentata, L. bispinosa, L. brookwoodiana, L. fragrans, L. heterophylla and L. semiteres, attempting to get data from three plastid regions (rbcL, matK, psbA-trnH) and one nuclear region (ITS2). The data generated from the rbcL and psbA-trnH regions was effective in discriminating between all the species sampled; however useful data were not obtained from matK or ITS2.

Genetic markers:

1. rbcL: bidirectional sequence data was generated for 38 accessions.

2. matK: amplification was not successful with the primer sets used (LivF1A, LivR1A).

3. psbA-trnH: bidirectional sequence data was generated for 40 accessions.

4. ITS2: although PCR amplification was successful for 35 accessions, the low quality of many of the sequences generated, and the presence of clear heterozygous positions in sequence data from some accessions, made this data set problematic to analyse, so it was excluded from the study.

Lophocolea bispinosa vice county 98 Long 4725

Lophocolea bispinosa vice county 98, Long 4725; photographed by David Long

Lophocolea semiteres vice county 98, Long 0578

Lophocolea semiteres vice county 98, Long 0578; photographed by David Long









Species and trees:

Distance tree generated using rbcL barcode sequence data for UK Lophocolea accessions

Distance tree generated using rbcL barcode sequence data for Lophocolea accessions, rooted on Chiloscyphus

L. fragrans – all accessions were genetically uniform, forming a monophyletic group.

L. heterophylla – although there was a little genetic variation, again, accessions of this species formed a distinct clade for both rbcL and psbA-trnH.

L. semiteres & L. brookwoodiana – these formed a single clade. All the accessions of L. semiteres (including material from the UK and Belgium) were genetically uniform, while two different genotypes were observed for L. brookwoodiana. While L. semiteres is known to be an introduced species in the UK, it’s possible that the three different genotypes in this clade represent separate introductions.

L. bispinosa – species formed a single genetically uniform group; this nests within a L. bidentata grade.

L. bidentata – accessions of this widespread and common species formed a grade, with three genetically distinct groups. One of these groups may represent L. cuspidata, a species that was sunk into L. bidentata by Bates and Walby in 1991, due to a lack of consistently distinguishing morphological characters. The results of this study suggest that a recircumscription of L. bidentata, “probably the commonest leafy liverwort in the British Isles” (Hodgetts, 2010), is required.


Related Posts

Student projects at RBGE: DNA barcoding British liverworts: Lophocolea

Student projects at RBGE: Barcoding British Liverworts: Plagiochila (Dumort.) Dumort.

Student projects at RBGE: Barcoding British Liverworts: Metzgeria

Student projects at RBGE: DNA barcoding of the leafy liverwort genus Herbertus Gray in Europe and a review of the taxonomic status of Herbertus borealis Crundw.



Jul 192016
Decaying wooden fence, between concrete poles, Kufstein, Austria

Decaying wooden fence, between concrete poles, Kufstein, Austria

Recently in Kufstein, the home of Austrian bryologist Wolfgang Hofbauer, the demolition of an attractive old building and clearing of trees and other plants from the land, leaving a bare gravel patch used as a parking space, did have one interesting outcome: The new clearing led Wolfgang’s eye to a decaying wooden fence between concrete posts. Both the posts and the fence are partly covered in bryophytes, but among them, Wolfgang was very surprised to find the moss Schistidium growing on the old wood as well as on the concrete.

Schistidium on fence post, Austria

Schistidium on fence post, AustriaIn the bryological literature, the only reference to the plant growing on wood is a rare occurrence of Schistidium apocarpum, on lime-impregnated tree bark. The situation in this Kufstein parking lot seems unique, with at least two different species of Schistidium on the wood (although species identification is ongoing). Other more typical residents of old wood, which are also present, include Leucodon sciuroides, Orthotrichum affine and Hypnum cupressiforme. However, the unique assemblage is unlikely to last, as the climatic regime at the place will have changed following the removal of the trees, and the newly exposed rotten fence will probably soon be replaced.

Schistidium on fence post, Austria

Schistidium on fence post, Austria

Meanwhile, however, we wonder if similar unlikely assemblages of mosses are being observed elsewhere, and if there is an explanation for any potential changes in habitat?



Botanics Story and images provided by Wolfgang Hofbauer


Related literature

Wolfgang Karl Hofbauer, Laura Lowe Forrest, Peter M. Hollingsworth, Michelle L. Hart. 2016. Preliminary insights from DNA barcoding into the diversity of mosses colonising modern building surfaces. Bryophyte Diversity and Evolution 38(1).

Sam Bosanquet. 2010. Schistidium species reports, in: Atherton, Bosanquet & Lawley, Mosses and Liverworts of Britain and Ireland a field guide, British Bryological Society.

In plain sight – the mosses that grow on British walls.

Hidden diversity in unexpected places – moss growth on modern building surfaces.


May 132016

Plant diversity does not have to be far-flung and exotic to be worth studying; even within Scotland, there are unanswered questions about plant distributions. Growing in our towns and cities, sharing our walls and pavements, there are bryophytes, tiny mosses and liverworts. We pass these every day, step over them, walk past them, hardly noticing that they are there. Miniature ecosystems form in the mosses that grow in the mortar between our bricks, or cling to cement surfaces of our bridges, and yet, partly because they are so commonplace, we don’t usually see them at all. And we have amazingly little understanding of exactly which species are involved, or where they have come from.

Recently, we looked at plants of the common moss Schistidium to find out exactly which species grow on artificial surfaces, like cement, walls and roofs (Hofbauer et al. 2016). Our study included plants from different geographic areas, with many plants collected in Germany and Austria, where Wolfgang Hofbauer, the lead researcher on the study, works and lives. However, a small subset of the plants were collected in the UK, and so also form part of the Royal Botanic Garden Edinburgh’s “Barcoding the British Bryophytes” project. Of 29 Schistidium plants collected in the UK, nine were collected on natural surfaces, like boulders and cliffs, and 17 were collected on artificial surfaces, like walls and roofs (for three accessions we don’t have a record of what kind of surface they were growing on).

Schistidium, photographed by Wolfgang Hofbauer

Schistidium, photographed by Wolfgang Hofbauer

These UK moss samples probably belong to eight species, Schistidium crassipilum, Schistidium pruinosum, Schistidium elegantulum, Schistidium strictum, Schistidium papillosum, Schistidium apocarpum, Schistidium trichodon and Schistidium dupretii, with three of the species, Schistidium crassipilum, Schistidium elegantulum and Schistidium apocarpum, having been collected from both natural and man-made surfaces.

A diagram of genetic relationships between the plants we sampled is shown below.

Schistidium crassipilum – we found three distinct genetic types within this species, which may belong to different species or subspecies. Schistidium crassipilum is known to be common on man-made habitats across Britain and Ireland, and we have collected it on bricks, cement, and even roofs as well as on natural substrates.

Schistidium pruinosum – only one of the moss plants in the study, collected in the Pentlands near Edinburgh, belonged to this species. It’s not known from many collections in the UK, although this may just be because the plants are often overlooked or misidentified, rather than that they are rare.

Schistidium elegantulum – this has been reported from natural and man-made habitats to the south and west of Britain. However, in our study, we have found it growing in the east, on cement in East Lothian and Midlothian, as well as in some more traditionally westerly locations in Scotland.

Schistidium papillosum – only one of the moss plants in this study, collected from limestone in Craig Leek, near Braemar, probably belongs to this species.

Schistidium strictum – again, only one of the plants in our study, collected in Dumfries on rocks, probably belongs to this species.

Schistidium papillosum is sometimes considered to be the same species as S. strictum (e.g. by AJE Smith 1978, The Moss Flora of Britain and Ireland, Cambridge University Press, but not by Bosanquet 2010, p. 515, in Atherton, Bosanquet & Lawley, Mosses and Liverworts of Britain and Ireland a field guide, British Bryological Society), although we did find genetic differences between the two plants that we sampled, consistent with their recognition as two separate species.

Schistidium apocarpum – this is one of the more common Schistidium species, and known to occur on natural and man-made surfaces; we sampled several plants from this species, growing on walls and rocks.

Schistidium trichodon – described as “a rare upland calcicole” by Sam Bosanquet (2010, p. 515, in Atherton, Bosanquet & Lawley, Mosses and Liverworts of Britain and Ireland a field guide, British Bryological Society), both our collections matched the reported habitat, growing on limestone, in Clova and Feith, Scotland.

Schistidium dupretii – we only sampled a single British accession of this species, another rare calcicole, which had been collected at Ben Lawers.

We are still far from having full records of how much genetic diversity there is in Schistidium in the British Isles. Partly because our previous work has focused on mosses on man-made surfaces, we don’t yet have any data for several other species that have been reported from Britain and Ireland (Bosanquet 2010, in Atherton, Bosanquet & Lawley, Mosses and Liverworts of Britain and Ireland a field guide, British Bryological Society). These include Schistidium maritimum (reportedly usually northern and western, in coastal locations), Schistidium rivulare (commonly around water, particularly fast-flowing rivers), Schistidium platyphyllum (another species that grows near rivers), Schistidium agassizii (rare, aquatic and probably often overlooked), Schistidium flexipile (very infrequent, with only one record from recent years), Schistidium robustum (an uncommon upland calcicole), Schistidium confertum (an uncommon upland species), Schistidium frigidum (yet another uncommon reportedly upland species) and Schistidium atrofuscum (a rare moss, only recorded for the UK in the central Highland area).

But at least we are now starting to get a better picture of the mosses that share our towns and cities!


UK Schistidium accessions, parsimony analysis of ITS data with bootstrap support above branches

UK Schistidium accessions: parsimony analysis of nuclear ITS DNA sequence data, with bootstrap support above branches


This work was supported by EU SYNTHESYS project ( gb-taf-3881.
Thanks are also due to David Long for providing many of the specimens.

Wolfgang Karl Hofbauer, Laura Lowe Forrest, Peter M. Hollingsworth, Michelle L. Hart. 2016. Preliminary insights from DNA barcoding into the diversity of mosses colonising modern building surfaces. Bryophyte Diversity and Evolution 38(1)

Sam Bosanquet. 2010. Schistidium species reports, in: Atherton, Bosanquet & Lawley, Mosses and Liverworts of Britain and Ireland a field guide, British Bryological Society.

Mar 172016

One of the most recognisable groups in the bryophytes, the complex thalloid liverwort genus Marchantia, has just become a bit larger. We have sunk Preissia and Bucegia into it, because in molecular phylogenies they are both phylogenetically nested within Marchantia (Villarreal et al. 2015). Although this only adds two species to the genus (Preissia quadrata and Bucegia romanica), taking the total number of species recognised from 36 to 38, it also broadens the range of plant morphologies that occurs in Marchantia.

Marchantia sampling from Villarreal et al. 2015

Maximum likelihood topology for Marchantia from Villarreal et al. 2015; * indicates bootstrap support of 100% and posterior probabilites of 1

Preissia quadrata (Scopoli) Nees returns to an earlier name, Marchantia quadrata Scopoli. This is the name under which it was first described, by Scopoli, in 1772. For Bucegia romanica Rad., first described by Radian (1903), a new combination has been made, Marchantia romanica (Rad.) Long, Crandall-Stotler, Forrest & Villarreal.

A new subgenus, Marchantia subg. Preissia, has been created for these species. A third species that was collected in China by David Long, and was included in the molecular phylogeny, has not yet been named, but will also belong to subgenus Preissia. Morphologically, the new species is most similar to Marchantia quadrata.

bucegia paperMarchantia romanica is known from northern Europe and northern North America (e.g. Evans 1917, Konstantinova et al. 2014). Photographs of fertile plants, and transverse sections showing 2-3 layers of filament-less air chambers, can be seen in Konstantinova et al.’s report. The hollow air chambers they illustrate are one of the clearest features that separated Bucegia from Marchantia and Preissia.

Preissia quadrata carpocephalum, photographed by Des Callaghan

Marchantia quadrata carpocephalum, photographed by Des Callaghan

Marchantia quadrata is also a northern hemisphere species; it is widespread in the British Isles, and a map and photographs are available in the British Bryological Society Field Guide. Like Marchantia romanica, this species lacks gemmae cups. Its two ranks of ventral scales could also be used to separate Preissia from Marchantia, which typically has 4 or more rows of ventral scales. Two subspecies are recognised in Schuster (1992), one more southerly and dioicous (ssp quadrata) and the other northern and monoecious (ssp hyperborea) (Schuster 1985). From our data, using DNA regions from genes that were chosen to show relationships between genera rather than within species, genetic differences can be seen between a collection from the South Ebudes off the west coast of Scotland, and one from Snowbird, Utah in the United States. Molecular sampling from across the range of Marchantia quadrata may reveal a complex of several species. Indeed, this was anticipated by Schuster (1992, p. 366), who described the range of morphological variation within the species as “astonishing”.

Konstantinova et al. 2014: phylogentic variation between accessions of Bucegia and Preissia

Konstantinova et al. 2014: phylogenetic variation between accessions of Bucegia and Preissia

Konstantinova et al. (2014) generated DNA sequences for the nuclear ITS region, and two plastid regions, trnL-F and trnG, for six accessions of Marchantia romanica (“Bucegia”, from Romania, Ukraine and Svalbard), and three accessions of Marchantia quadrata (“Pressia”, from Russia). Unfortunately the regions that they sequenced do not overlap with the sequencing in our study, so we cannot combine the datasets. However, they do rather intriguingly resolve two clades within Marchantia romanica (“Bucegia”), one from mainland Europe, and the other from Svalbard. Again, molecular sampling from across the range of this taxon could reveal several distinct lineages. Adding samples from North America seems a priority.

Thus, although we have only added two species to Marchantia, the number of species is likely to rise with additional molecular sampling, in line with a clearly necessary taxonomic revision of Marchantia subgenus Preissia.


David G. Long, Laura L. Forrest, Juan Carlos Villarreal & Barbara J Crandall-Stotler. 2016. Taxonomic changes in Marchantiaceae, Corsiniaceae and Cleveaceae (Marchantiidae, Marchantiophyta). Phytotaxa 252 (1): 077-080.


Long et al


Jan 302016

This last week I’ve actually managed to spend a bit of time in the lab, trying to get some gaps filled in a DNA barcoding matrix for simple thalloid liverwort Aneura. David Long and I are heading off to Trondheim in just over a week to combine our data set with one generated by Ana Maria Séneca Cardoso, working with Lars Söderström and Kristian Hassel at NTNU.

A fridge-full of DNA at RBGE

A fridge shelf piled with racks and plates of DNA in the RBGE PCR lab

Many of the DNA extractions that I have been trying to amplify are old (with a very few that were extracted 14 years ago at Southern Illinois University). Most of them have already been tried, and have previously failed to amplify, for the four selected barcode markers (three plastid genes, rbcL, rpoC1 and matK, and one plastid intergenic spacer region, psbA-trnH; a nuclear marker, ITS2, was originally included, but proved difficult to get good sequence data from). Most of the DNA extractions I’ve needed were scattered across a large number of Edinburgh DNA bank (EDNA) plates, while some of the rest of it had never been aliquoted out of the original QiaXtractor plates.

Our hard-working PCR machines wait for samples in the molecular lab

Our hard-working PCR machines wait for samples in the RBGE molecular lab

Because the amount of time needed to chase down the DNA samples was more than the amount of time needed to set up the reactions, and because my default protocol has changed, over the course of this project, from using CES as a PCR enhancer to using TBT-PAR as a PCR enhancer, I decided for three of the loci to test the amplification with both CES and TBT-PAR, building on from a previous Botanics Stories posting. (The exception was matK, for which we use a different polymerase, Invitrogen’s Platinum, and 5M betaine as a PCR enhancer; we have found that this gives better sequence reads than amplification with a standard Bioline taq does.)

getting ready for PCR - reagents defrosting on ice

Getting ready for PCR – my reagents defrosting on ice

Reactions were set up in 20 ul, using exactly the same reagents (Sigma water, 5x buffer, magnesium, dNPTs and forward and reverse primers, with 1 ul each of the Aneura DNA extractions), with the exception of the 4 ul of either CES, or TBT-PAR, per sample.

Inside the laminar flow hood and ready to go

Inside the laminar flow hood and ready to go

The PCR products were all run out on standard 1% agarose TBE gels, at 80 volts, for 40 minutes, stained with SybrSafe, and visualised under a blue light, to test for amplification success. As a size standard, 3.5 ul of an Invitrogen 1 kb ladder was loaded at intervals on each gel.

Several of these gels are shown below. In these images, DNA is stained so that it fluoresces in bright blue or UV light. The samples have been loaded into the gel in holes, or wells, that can be seen at the top of, and at regular intervals down, the gel, and migrate through an electric current towards the positive electrode that would be at the bottom of the image. Smaller fragments move faster through the gel, meaning that samples can be separated by the lengths of the DNA fragments.

The comparisons of amplification success with CES and TBT-PAR are unfortunately ambiguous.

For the psbA-trnH region, using TBT-PAR was more successful than using CES – as can be seen particularly clearly in the second row down, where only 3 of the 8 extractions amplified with the CES additive, but all 8 amplified with TBT-PAR.

Aneura DNA amplified for psbA-trnH region: left hand side - with CES additive; right hand side - with TBT-PAR additive

Aneura DNA amplified for the psbA-trnH region: left hand side – with CES additive; right hand side – with TBT-PAR additive

For both rbcL and rpoC1, it’s harder to get a clear picture. Some samples amplified with one additive rather than the other, while most samples amplified with both.

Aneura DNA amplified for rpoC1 region - first two rows with TBT-PAR additive; second two rows with CES additive

Aneura DNA amplified for rpoC1 region – first two rows with TBT-PAR additive; second two rows with CES additive

For a set of the rpoC1 amplifications, although all samples amplified using both additives, the bands from the reactions with CES (the three lowest rows in the gel image) are rather brighter than those from the reactions with TBT-PAR (the upper three rows), meaning that there is more amplified product in them. However, the CES bands do appear slightly more smeary, so it may be worth comparing the quality of sequence data from both sets of reactions as well as just considering amplification success.

The gels for the rbcL samples (shown below) are harder to interpret – the consistent bright bands are not the PCR product that I am looking for, but represent an artefact of the reaction known as “primer dimer”; the PCR product is the second slower (and therefore, longer) DNA fragment that is sometimes present. There seem to be more samples that have amplified with the CES additive, although a few samples that have failed with CES have instead amplified with TBT-PAR, meaning that in this instance, having used both protocols in parallel will allow me to generate DNA sequences from more accessions of Aneura than I would have been able to, had I just used one PCR protocol.

Aneura DNA amplified for rbcL plant barcode region with CES additive

Aneura DNA amplified for rbcL plant barcode region with CES additive

Aneura DNA amplified for rbcL plant barcode region with TBT-PAR additive

Aneura DNA amplified for rbcL plant barcode region with TBT-PAR additive (2 gel images)

160128 Aneura rbcL M745 TBT 7to9 crop

The only recommendation that I can think of from this is that, if time is pressing, the DNA samples are difficult to access, and you need as much amplification as possible as fast as possible, do two sets of PCR reactions, using both additives.

This does, however, have the unfortunate side effect of doubling the cost of your PCR, as well as giving you twice as many samples to load onto your gels…


See also: Botanics Stories: Sparking additions in the Molecular Lab.

Nov 042015

The complex thalloid liverwort Monocarpus sphaerocarpus has been found on two continents, Australia and Africa, separated by around 8,000 km of mostly ocean. The green plants themselves are not particularly easy to compare, as the plants are small and the material that we have to work with has been squashed down and dried to form crumbly herbarium specimens. However, there are differences between the morphological descriptions of plants growing in Australia (Carr 1956; Proskauer 1961) and South Africa (Perold 1999). The characteristics of the plant’s spores are rather easier to observe (using scanning electron microscopy); this shows that spores from collections from Australia and South Africa are quite distinct – although there are also differences between spores from plants from Western Australia and Victoria.

Monocarpus spores: a,d: Victoria, Australia, b,e: Western Australia, c,f: Cape Province, South Africa

From Forrest et al. Fig. 4: Scanning electron micrographs of Monocarpus spores: a,d: Victoria, Australia, b,e: Western Australia, c,f: Cape Province, South Africa.

Although the distances between the three regions in which Monocarpus has been found are huge, it is possible that populations of Monocarpus are more widespread than we might expect. Dust storms could lift spores into the atmosphere, and the dark coloration of the spores suggests some ultraviolet resistance. Proskauer (1961) was able to grow sporelings from four-year-old herbarium collections, showing that they can survive drying out, so may be able to travel a long way in the air currents. However, we would expect genetic isolation over the thousands of kilometres that separate the known populations of these plants.

Given both factors – that there are morphological differences between the plants, and that we expect there to be genetic isolation – we have considered naming the South African material as a separate species, after South African bryologist Sarie Perold, whose publication “Studies in the Sphaerocarpales (Hepaticae) from southern Africa. 1. The genus Monocarpus and its only member, M. sphaerocarpus” is a meticulously detailed piece of work.

However, in order to really understand Monocarpus in South Africa, and decide whether it represents a new species or not, we need to get hold of a bit more material and try some other investigations, including DNA analyses. Perold (1999) got the plant’s South African location from Australian botanist Hellmut Tölken, who had originally found it, and made several trips trying to recollect it; bryologist Terry Hedderson has also been keeping an eye open for samples. But no further material has been forthcoming: the site from which it is known has been developed, and now contains a hotel. And, unfortunately, the Monocarpus specimen from South Africa is scanty. Most of the collection, which was sent out as a loan from the Bolus Herbarium in Cape Town to the National Herbarium, Pretoria (PRE), has, according to T. Trinder-Smith, been lost. All that remains are some fragments of the original collection that were gifted to the National Herbarium of Victoria (MEL), and that we currently have on loan here in Edinburgh:

Monocarpus sphaerocarpus herbarium specimen, photograph by Tiina Sarkinen

Monocarpus sphaerocarpus herbarium specimen, photograph by Tiina Sarkinen

Unless the plant is rediscovered in South Africa, further study of the taxon there is impossible, given the size and fragility of the dust-like remaining specimen (shown in its entirity in the photograph above), as is molecular work to look for genetic differences between Australian and South African material.

It would be unfortunate, though, if the eradication of its only known site caused the extinction of a distinct South African species even prior to its formal description, which in itself is rendered impractical by the loss of nearly all of the existing plant material.


Monocarpus heading




Proskauer J (1961) On Carrpos I. Phytomorphology 11, 359-378.

On Monocarpus –

Finding Monocarpus, in the Herbarium –

Finding Monocarpus, in the field –