Apr 122017

Once we realised that most of our plate of Schistidium ITS2 amplifications had been successful, it was an easy decision to process them all for DNA sequencing. If a higher proportion had failed, we would have had to “cherry pick”, selecting and transferring the successful reactions into new tubes. Every sequencing reaction has a cost, and so deliberately sending a lot of failed reactions through the process, knowing that they won’t generate DNA sequences, is worth avoiding. However, transferring the reactions into new tubes is a meticulous job. In a plate of reactions, there are 96 wells arranged in 12 columns (1-12) and eight rows (A-H).  The first sample is at 1A, then 1B, 1C, 1D, 1E, 1F, 1G, 1H, 2A… all the way to 12H. Imagine if the first failed sample is at 1B and the second at 1G – then we’d transfer 1A to 1A, 1C to 1B, 1D to 1C, 1E to 1D, 1F to 1E, 1H to 1F, 2A to 1G, etc… it’s easy to see that any interruptions can be fatal (this sort of task is why people in the lab sometimes have a “Do Not Disturb” sign stuck on the back of their lab coats). Thus, with only a few failures, we’re better keeping any liquid transfers simple (and manageable using a multichannel pipette).

A 96-well plate on ice

After electrophoresis and visualisation of the Schistidium ITS PCR products under blue, or U.V., light, the next step is to clean up these PCR products. This is where we remove unincorporated primers and dNTPs from the reactions. In our PCRs, we added all the ingredients in excess, so that there was more of everything than the reaction needed. This might seem wasteful, but compared to the costs of the time and plastics that would be required to carefully optimise each reaction, throwing lots in and getting quicker results really isn’t too profligate.

When a PCR is successful, the primer (an oligonucleotide, or short single stranded DNA molecule) binds to the end of the region that is getting copied, and a new strand of DNA is synthesised from the end of the primer, incorporating the primer into the new strand. Thus, primers get used up. The building blocks for DNA synthesis, the nucleotides (As, Ts, Gs and Cs), are the dNPTs (deoxynucleoside triphosphates) that we added to the PCR reaction, again in excess. So even when we’ve built a lot of copies, we still have some primers and dNTPS that weren’t used up.

This didn’t matter when it came to looking at the results on the gel, but does become important in our next reaction, the sequencing PCR. In the sequencing PCR, we add just a single primer, and we add a very precise blend of nucleotides. So we don’t want to carry over primers and dNPTs from the previous reaction. There are several ways of removing these.

The cheapest is through ethanol precipitation of the synthesised DNA, where the unincorporated primers and dNTPs stay in solution and are thrown away. This is less easy to scale up to plates, and the moment where you turn it all upside down and toss the liquid out is rather worrying – the cost of plates of subsequent sequencing failure if the DNA pellets were lost is huge, and the pellets tend not to be visible.

For several years we used a column-based approach, a bit like our DNA extraction method, where the PCR product is bound to a membrane, and the unincorporated primers and dNTPs are flushed off it, before the PCR product is eluted back into solution.

However, the method we now use most regularly involves a combination of two enzymes, Eco1, and Shrimp Alkaline Phosphatase (SAP), which work elegantly in combination, and which involves methodology that is easily scalable for working with plates and multichannel pipettes. The Eco1 enzyme digests single-stranded DNA, so it cuts the primers up into individual nucleotides. The SAP enzyme dephosphorylates the nucleotides (the dNTPs) so that they are no longer functional. Thus after using these enzymes, although nothing has been physically removed from the tubes, the unwanted reagents have been rendered unusable. For convenience, we buy in a commercial combination of the two enzymes called ExoSAP-IT™, and add a small amount of that to our reactions (in this case, around 1 μl ExoSAP-IT to about 15 μl of PCR product).

The enzymes work best at 37ºC, and so the reactions were put on a heating block for 15 minutes. The enzymes are killed at a higher temperature, and so the next step was to heat the reactions to 80ºC for 15 minutes, to make sure that no viable enzymes are left to interfere with the sequencing reaction. After that, the plate of cleaned Schistidium ITS2 amplicons was left in the fridge until it was needed for the sequencing reactions.


Links to reports on Moss diversity in an artificial landscape, an EU Synthesys Access project with Dr Wolfgang Hofbauer at RBGE:

Apr 112017


Gel loading in Lab 33

Once the polymerase chain reaction is over, it’s time to Run The Gel; this is make-or-break time, when we find out if our PCR amplification has actually worked.

The first step is to prepare and pour the gel. The gel is a 1-2% mix of agarose in a salt-containing buffer solution (for us, this is usually 1x TBE); the agarose is dissolved into the buffer using heat, in a microwave, and a bit of stain is added. We use about 5 μl of SYBRsafe for a 100 ml gel. The SYBRsafe binds to DNA and fluoresces under blue light, so we can see if we have produced amplified DNA.

After the agarose gel has cooled down to a comfortable temperature (so we don’t risk warping our ridiculously expensive plastic gel set-up with it!) we pour it onto a plate, and add a “comb” which sits in the gel and around which it will set, forming lines of small oblong holes in the gel’s upper surface into which we will later pipette our PCR product.

Setting up the loading buffer and PCR product in Lab 31

While waiting for the gel to set, we mix a small amount of the Schistidium ITS PCR product (3-5 μl) with a little loading dye (1-2 μl). The loading dye serves two purposes: it contains glycerol, which is rather dense and helps the liquid drop into the holes (wells) in our gel, and it also contains a colour (in this case, bromophenol blue), which means that we can see what we’re doing – dropping a colourless liquid through another colourless liquid into a colourless hole would otherwise be rather more an act of faith!

Using a manual multichannel to load the PCR product/loading solution

When the gel has set hard, we lift off the comb, taking care not to rip the gel (which is rather more brittle than gelatine-based jellies), and put the gel into a gel tank, covered with a thin layer of the TBE buffer that was used to make the gel. Then it’s time to load. Down one side, we put 3.5 μl of a “ladder”, a bought-in product containing DNA fragments of known lengths and quantities, which will act as a standard for the gel and let us estimate the sizes of any PCR products that show up. Adding the ladder, in which we know there is DNA, is also like a “positive control” that we’ve made our gel properly – if nothing at all shows up in the image of the gel, then it’s not proof that our PCR reaction failed, but that there was something wrong with our gel.

With the gel set-up that we are using for our plate of 96 Schistidium amplifications, we can load 8 samples at a time using one of the multichannel pipettes. For this, instead of our usual electric multichannels, we use a manual one so that we have more control over the rate of dispensation, and so we can stop if some of the liquid is not going in the right place: it’s quite a precarious operation!

Running our gel

Because the sugar-phosphate exerior of a DNA molecule has a negative electric charge, DNA molecules migrate through an electric field towards the positive electrode, and so if we apply a current to the gel, we can get the DNA to move through it. The filtering effect of the set agarose in the gel means that the DNA molecules migrate at a rate proportional to their size, with short molecules moving quickly, and longer ones more slowly. Generally, 30-45 minutes at 80 volts is enough to see if our PCR is successful.

After electophoresis, the gel is placed on the transilluminator

After the gel electrophoresis, the gel is carefully lifted out of the buffer, off the gel tank, and placed on a plate in a light box. (At this point with larger thinner gels like the one we ran for the Schistidium PCR products, it’s quite easy to break the gel.) Because the shape of the gel and of the light box are different, these gels need to be cut in two. Luckily, the halves fit nicely side by side in a single image.

Our gel beneath the filter

An orange filter is added, and when it’s turned on, the transilluminator shines with blue light that will make any DNA stained with SYBRsafe fluorescent, at which point a digital image is taken of the gel.


The first image of our Schistidium PCR products

To see what’s on the gel, we use an automated setting, where the computer choses the best exposure for the image. However, a bit of subsequent cropping and fiddling with the image itself can make for a far better gel picture. And the really good news for us is that, looking at our gel, it is clear that most of our Schistidium DNA extractions were successfully amplified for the ITS2 region in our PCR reaction!

The wells can be seen at the top of each row, as darker shadows; for the purposes of this image, the DNA has migrated towards the bottom of the screen. For only about seven of the 96 reactions is there no visible band of PCR product on the gel, and in the vast majority of cases a clear bright single band is present. (The fuzzier band that is sometimes visible a little further down than the ITS product, especially for the second last sample on this gel, is primer-dimer, an artifact caused during PCR, and not the amplified product we want; it’s far far shorter than the region we’re working with, as can be seen by comparing it with the size of our 1 kilobase DNA ladder).

Cropped and adjusted gel image picture, with a ladder on the right hand side of every third row

Links to reports on Moss diversity in an artificial landscape, an EU Synthesys Access project with Dr Wolfgang Hofbauer at RBGE:

Apr 052017

Wolfgang hard at work in the Cryptogam Workroom at RBGE

Just over a week into our current Synthesys-funded Schistidium project, and Wolfgang has picked through piles of packets of mosses, selecting the 96 that we would most like to get DNA sequences for, and putting tiny pieces of them into plastic tubes for DNA extraction.

Ready to start our DNA extractions in Lab 30

Down in the basement, we have a storeroom with racks of lab coats, so we ventured down and found one that was a good fit for Wolfgang, to keep him from spilling chemicals down his regular clothes. We also got him some gloves; at RBGE, instead of latex, to which some people are allergic, we use nitrile. These serve two purposes – one, clearly, to protect our hands from any hazardous materials we might use, but in many cases in the molecular lab, the gloves are actually to protect our samples from us, so that we do not get our DNA or enzymes into the tubes we’re working with.

Our 96 moss samples ready for grinding

The first step of the extraction is the mechanical disruption of the plant tissue. Back to Biology 101, and a major difference between animal and plant cells is that plant cells have a tough cellulose-based cell wall around the outside of the cell membrane. To break through this cell wall, we add small tungsten beads to our tubes, and pop them into a TissueLysis machine which vibrates the tubes rapidly back and forth until the plants are rendered to a fine powder.

About half a millilitre (420 μl) of a salty, soapy buffer solution is added to this powder. Because we’re using tubes that are in strips of eight, we can use a multichannel pipette to dispense eight aliquots of the buffer at a time, reducing the amount of handling time. This first buffer helps break open the plant cell membranes, releasing the Schistidium DNA into solution. We put the tubes on a heated shaking block, leaving the samples for 1.5 hours at 65°C.

Schistidium DNA extractions with added binding buffer

After the lysis step, the strips are centrifuged to remove bits of plant debris, 220 μl of the liquid Schistidium extraction is pipetted into a new deep 96-well plate, and 440 μl of a binding buffer is added, before 600 μl of the solution is transferred to yet another plate… This next plate contains a membrane to which the Schistidium DNA binds. While the DNA is stuck to the membrane, we pass four wash buffers over it, to remove compounds that might have co-extracted with the DNA, but that might inhibit some of our downstream reactions. These wash buffers contain quite high proportions of alcohol, so they don’t remove the DNA from the membrane (because DNA is not soluble in alcohol it doesn’t come out of precipitation).  The final step in the process is to remove the DNA from the membrane in an elution step, using 100 μl of a water-based elution buffer per sample, in which the DNA can dissolve.

Our plate of Schistidium DNA, sitting in the Lab 32 fridge

Once the 96 Schistidium DNA extractions are finished, the plate is sealed (with a thin sticky metal film), and left in the fridge (in this case, as we are going to use the DNA the next day), or in the freezer, until we are ready to move on to the next step.


Links to reports on Moss diversity in an artificial landscape, an EU Synthesys Access project with Dr Wolfgang Hofbauer at RBGE:


Mar 302017

The moss Campylopus introflexus, native to the southern hemisphere, is now considered an invasive plant in parts of Europe and North America. While it occurs on some natural sites within Edinburgh, notably on Arthur’s Seat, it is also no stranger to man-made habitats. At the Botanics, the species forms large tactile ball-like clumps between the glass panes of the Research House roof. However, as it can damage the roof, it is one of our less welcome bryological volunteers.

Gunnar Ovstebo holding a tactile moss ball from the Research House roof


Wolfgang Hofbauer and Gunnar Ovstebo checking out the bigger and better Campylopus moss-balls that are out of reach further up the roof


Furry Campylopus plants growing on the Research house roof

Campylopus introflexus moss ball

Links to reports on Moss diversity in an artificial landscape, an EU Synthesys Access project with Dr Wolfgang Hofbauer at RBGE:

Mar 292017

There are very few bryophytes growing in the living collections of the Royal Botanic Garden, Edinburgh. What I mean by this is that there are very few bryophytes that we have carefully selected as wild plants, and have planted and nurtured as part of our curated collection, databased and in possession of accession numbers. On the other hand, there are lots and lots of different bryophytes growing here, having volunteered to become part of our living landscape. Given that part of the work that we are doing with visiting Synthesys researcher Dr Wolfgang Hofbauer is looking at ways to increase and promote the growing of mosses, and to better understand their place in the built environment, horticulturalist Gunnar Ovstebo and I started off Wolfgang’s visit with an excursion into the garden, to see what we already have.

Wolfgang and Gunnar in the Arabian Research House

We began behind the scenes in the Arabian Research House, where a light moss cover is used to help the establishment of some arid-land ferns.

A mossy bed for young ferns growing in the Arabian Research House

It’s bryophytes galore in the Gesneriaceae Research House, even over the plastic under-bench tanking

We also visited the Gesneriaceae and fern research collections, warm moist glasshouses that are incidentally filled with bryophytes. Some of these might be native to Scotland and have come in from the surrounding area; others could have come from around the world as we have made additions to our living collection.

Gunnar and Wolfgang check out one of the crevices in the Arid House

Moving into one of our public display houses, some cool shaded crevices between the rocks of the Arid House provide habitats for algae, mosses and liverworts, which in turn provide germination sites for some of the larger plants like ferns and begonias. The Arid House is also home to three accessioned species of the complex thalloid genus Plagiochasma, which have established really well between some of the rocks.

The tufa wall in the new Alpine House

The third place we visited is the new Alpine house, which has a tufa rock wall dotted with little rosette-forming plants. Mosses are also growing on the wall, but are not particularly welcome!

Mosses spread along damp mortar-work at the back of the new Alpine House

Venturing round the back of the new building, patches of moss are happily spreading in patches along damp cement-work between the bricks.

Behind-the-scenes again, in the Alpine Nursery, although the tunnels are remarkably bryophyte-free, hundreds of pots fill a series of cold frames, and in and amongst these, many moss volunteers thrive.

The Alpine Nursery – the only glasshouse where we didn’t see any bryophytes

Mosses volunteering in the cold-frames of the Alpine Nursery

Having obtained an idea of what we currently have here, over the next few weeks we will be starting up some moss transplant and cultivation experiments, because although our volunteers are very much part of our garden landscape, as scientists and horticulturalists we also want to be able to grow and display particular selected species and accessions; these may then form part of our research collection or our education programme.


Links to reports on Moss diversity in an artificial landscape, an EU Synthesys Access project with Dr Wolfgang Hofbauer at RBGE:

Mar 292017

Some Schistidium collections from the RBGE Herbarium

Monday 27th March was the start of a month-long visit to RBGE by the Fraunhofer Institute for Building Physics‘s Dr Wolfgang Hofbauer, funded by the EU Synthesys Access programme. This funding enables researchers from other institutes to get their hands on the natural history collections that they need to see and understand, but it is equally vital for collections-based institutes like ourselves, as it promotes the use and curation of some of the material that we conserve.

DNA barcoding publication resulting from Wolfgang’s previous visit to RBGE

Wolfgang first visited us at RBGE as part of an earlier Synthesys programme, in 2014, which initiated a very useful collaborative project looking at the growth of species from the moss genus Schistidium on the built environment. We used DNA sequence data to try to identify some of these mosses, because the harsh environment in which they grow means that the plants are often malformed or underdeveloped, and difficult to identify using morphology.

Wolfgang has come back to RBGE in order to continue this work, in part by adding to our “Reference Library” of DNA sequences from different Schistidium species, but also to look at ways of developing our ability to grow some of these mosses where and how we want them.

We hope that by the end of this visit, we will be closer to answering the five following questions:

a) What is the taxonomy of Schistidium diversity on modern building surfaces?
b) Can we show geographic patterns of morphological and genetic variation in Schistidium on modern buildings in different European regions?
c) Are certain Schistidium taxa confined to special ecological situations (material, exposure, etc.) and can we use this for management of moss growth on buildings?
d) Which taxa of Schistidium are the best candidates for moss gardening, and is there potential for specially developed masonry/techniques to facilitate their growth?
e) Does improving the taxonomy of Schistidium on building surfaces allow us to find specific natural antagonists that could be used for its biocontrol?

Wolfgang hard at work in the Cryptogam Workroom at RBGE

The outcomes that we would like to see from this work are:

1) A complete baseline DNA-barcode library of Schistidium species as a tool for identification of sterile or morphologically atypical material.
2) Increased insight into the ecology and taxonomy of Schistidium species that grow on modern building structures.
3) A common project with the theme of deliberate growth of suitable cryptogams on building surfaces, as a collaboration between Science and Horticulture Divisions here at RGBE, and the Fraunhofer Institute for Building Physics, where Wolfgang is based.
4) Preliminary information on potentially specific biocontrol of unwanted growth on building surfaces, by identification of the moss lineages involved.
5) Development of an accessioned living collection of Schistidium species that have been identified using DNA barcoding and cultivated at RBGE, to be used for moss cultivation experiments and for public display.
6) Working with RBGE’s bryologists and horticulturalists, the development of a living display of moss colonisation (a kind of “living poster”) that can be used for outreach activities.
7) Published research, both on the DNA barcoding of Schistidium and the diversity of the genus on building surfaces, and also on bryophyte cultivation methods.


Links to reports on Moss diversity in an artificial landscape, an EU Synthesys Access project with Dr Wolfgang Hofbauer at RBGE:


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

Nov 222016
David Long in Gaoligong Shan; photo by Dong Lin

Dr David Long in Gaoligong Shan; photo by Dong Lin

Formerly the head of our Cryptogam section, and currently an extremely active RBGE Research Associate, David Long is well known and respected for his botanical work in the Himalayas, and for his bryological research. He has collected a huge number of taxonomically and phylogenetically interesting bryophytes on numerous plant collecting expeditions, collaborating with researchers around the world. His 2006 monograph on Eurasia Asterella reflects a special interest in the complex thalloid liverworts (Marchantiopsida), which has formed a focal point for subsequent research at RBGE on the systematics of the group (e.g., Villarreal et al. 2015).

Marchantia longii, from Fig. 1, Xiang et al. 2016, The Bryologist

Marchantia longii, from Fig. 1, Xiang et al. 2016, The Bryologist

In October this year, Chinese colleagues You-Liang Xiang, Lei Shu and Rui-Liang Zhu, using morphological and molecular evidence, described a new species of Marchantia from the northwestern region of Yunnan. Their paper, in the American Bryological and Lichenological Society journal The Bryologist, suggests that this is a distinct species, phylogenetically related to Marchantia inflexa, M. papillata and M. emarginata.

Xiang et al. 2016, The Bryologist

Fig. 4, Xiang et al. 2016, The Bryologist

The new species differs morphologically from other Marchantia species in the area by a suite of pore, thallus and receptacle characters, one of the most obvious of which is its very large epidermal pores, which can clearly be seen in the photographs presented by Xiang et al. The authors have named their new plant Marchantia longii R.L.Zhu, Y.L.Xiang et L.Shu, in honour of David, because he is “the specialist of complex thalloid liverworts and made several bryological expeditions in northwestern Yunnan, China”.

On these expeditions to the area, David collected extensively. It remains to be seen, however, whether his own collections include any plants of the newly named Long’s Marchantia!

Nov 162016
Telaranea tetradactyla, photographed by David Long (Long 37778)

Telaranea tetradactyla at Benmore, photographed by David Long (Long 37778)

Murphy’s threadwort (Telaranea murphyae) has had a singular position in the British flora. The species was described by renowned bryologist Jean Paton in 1965, from plants collected in the south of England. It’s a tiny leafy liverwort that is found in only four locations, at Tresco and St Mary’s on the Isles of Scilly, Branksome Chine, Poole in Dorset and Alum Chine, Bournemouth. Murphy’s threadwort has always been known to be an alien species in our flora, and yet because it’s never been found elsewhere, the sole responsibility for conserving the species lay with the UK. Being non-native, however, it was not considered a priority for UK Biodiversity Action Plans.

Telaranea tetradactyla from the RBGE fern house, photographed by Lynsey Wilson

Telaranea tetradactyla from the RBGE fern house, growing with Conocephalum conicum; photographed by Lynsey Wilson

Using DNA sequence data from the plant, and comparing it to sequences from other related species, we showed that genetically, the English plants are the same species as a New Zealand plant, Long’s threadwort (Telaranea tetradactyla, synonmy Telaranea longii). Long’s threadwort was already known from several locations in the UK, including inside the fernhouse at RBG Edinburgh, and near the fernery in Benmore. These habitats are not entirely coincidental – the Victorian craze for ferns saw many gardens import living tree ferns from countries such as Australia and New Zealand, with many smaller plants hitching a ride along on their trunks. Today, conscious of plant health issues and the potential transport of pathogens, new plant living collections have to spend time in quarantine before being planted out; past gardeners were less careful, and some of these hitchhikers have subsequently escaped into the local landscape.

Telaranea tetradactyla from the RBGE fern house, photographed by Lynsey Wilson

Telaranea tetradactyla from the RBGE fern house, photographed by Lynsey Wilson

Sinking our UK Murphy’s threadwort plants into the New Zealand species means that any conservation requirements now rest instead with New Zealand, although we can continue to enjoy seeing this diminutive mat-forming liverwort in its select few UK locations.



Key reference: Porley, R.D., 2013, England’s Rare Mosses and Liverworts. Princeton University Press.



Villarreal et al. 2014, Journal of Bryology 36(3): 191-199

Villarreal et al. 2014, Journal of Bryology 36(3): 191-199


Sep 142016
Sphaerocarpos texanus and S. michelii, from the British Bryological Society Field Guide (see references for link)

Sphaerocarpos texanus and S. michelii, from the British Bryological Society Field Guide (see references for link)

In conjunction with Dr Daniela Schill’s monographic work on Sphaerocarpos, we’ve been building a molecular phylogeny for the genus. We have attempted to extract DNA from 66 accessions, including three S. cristatus, all from California, seven S. donnellii from the US, five S. drewei from California, two S. hians, 13 S. michelii from France, Great Britain, Italy, Malta and Portugal, two S. muccilloi, five S. stipitatus from Nepal, Portugal and South Africa, and 25 S. texanus from Belguim, France, Great Britain, Italy, Portugal, Turkey, California and Illinois. We have also included some as yet unidentified material, including an accession from Chile.

Because much of our work at RBGE focuses on plant DNA barcoding and the protocols are established and frequently successful, we have chosen to use sequence data from some of these barcoding regions for this project. However, the liverwort matK primer sets were not very successful in Sphaerocarpos, with a very limited number of good quality sequences generated. The nuclear ITS2 region had its own issues, with many of the sequence reads being difficult to interpret due to overlapping peaks. In the end we focused on the three most successful plastid loci, the rbcL and rpoC1 barcoding amplicons, and a region that encompasses part of the psbA gene and the psbA-trnH intergenic spacer.

Unfortunately, we have not been able to amplify DNA from all the samples we extracted, with failures particularly for some of the older specimens. One of the species we attempted to sequence, Sphaerocarpos muccilloi, has not worked for any of the gene regions that we have been using, while another species, Sphaerocarpos hians, has so far only amplified for a single region, rbcL.

Sample phylogenetic tree for Sphaerocarpos, based on rbcL sequence data

Sample phylogenetic tree for Sphaerocarpos, based on rbcL sequence data

Although each species seems to be genetically distinguishable from the other species sampled, two of the most widespread species, S. michelii and S. texanus, are resolving as para- or polyphyletic. The phylogenetic tree contains three distinct groups of S. michelii accessions, and two distinct groups of S. texanus, one from Europe and the other from California. An Illinois accession that has been published as S. texanus resolves here with S. donnellii. The Illinois material lacks spores and is thus difficult to identify morphologically, but is outwith the Southeastern Coastal Plain area where S. donnellii is thought to occur.

The next steps in this study involve a second pass through the DNA extractions, to see if using other PCR additives will help increase the sequence success rate, then combining the sequence data from the three sequenced loci into a single matrix, to produce a more robust and supported phylogeny. Description of new species, where required, will fall under Daniela’s remit, in line with the comprehensive taxonomic revision that she has carried out.


Links & References:

Sphaerocarpos, preview to a monograph

BFNA | Family List | BFNA Vol. 3 | Sphaerocarpaceae

BBS Field Guide Sphaerocarpos michelii / texanus