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 062017
 

PCR set-up with defrosting reagents in Lab 32

After we extracted a plate’s worth (12 columns by 8 rows, or 96 samples) of Schistidium DNA, the next step in our process is to copy a preselected part of that DNA, using the Polymerase Chain Reaction (PCR). For this study with Wolfgang, we are copying a region of nuclear DNA known as the Internal Transcribed Spacer 2 (ITS2). We set up the reactions in a laminar flow hood, which blows clean air down onto the work surface, keeping everything as clean as possible. Most of the reagents are kept in a -20ºC freezer between uses, so these get set out to defrost before use (time for a coffee break!). The DNA polymerase enzyme, taq, on the other hand, is stored in glycerol so doesn’t freeze at -20ºC; while we’re using it, we keep it on ice so it stays cold.

The reaction components are added into a single “Master-Mix” tube, which includes water, buffer, Magnesium, enhancing additives, short oligonucleotides (primers), and the taq enzyme. Small (19 μl) aliquots of this master mix are then added to a new 96-well plate, and 1 μl of DNA from the extraction plate is transferred across into each of the 96 reactions.

Using the multichannel pipette to transfer DNA

Once the DNA has been added, the plate is sealed (this time with a clear plastic film that sticks firmly in place when heated), briefly spun in a centrifuge to make sure all the reagents are mixed together at the bottom of the 96 plastic wells, and transferred into one of our Thermocyclers, or PCR machines.

PCR plate in Lab 32 thermocycler

The plate sits in a metal block which rapidly “cycles” up and down in temperature, following a predetermined programme. For the gene region that we are copying here, the programme first heats the block to 95ºC for 4 minutes, then starts a cycle of 94ºC for 1 minute, 55ºC for 1 minute, and 72ºC for 45 seconds, repeated 30 times. When the block is at 94ºC, the double-stranded DNA is pulled apart into single strands; when it’s cooled to 55ºC the primers stick on and the taq initiates making copies of the ITS2 region, and when it is heated up again to 72ºC, the copies get completed, before they’re pulled apart again when the block heats up to 94ºC and the cycle starts again…

The thermocycler screen showing the ITS2 PCR programme

A couple of hours later, the reaction is complete, and at this point we HOPE that all the 96 wells in our plate contain millions of copies of the ITS2 DNA molecule from the Schistidium extraction that was added in each case. However, in order to see if any of it has actually worked, we need to stain and visualise the DNA, and for this, we have to run a gel, a process that will be the subject of the next installment.

 

 

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:

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. http://stories.rbge.org.uk/archives/19957

Hidden diversity in unexpected places – moss growth on modern building surfaces. http://stories.rbge.org.uk/archives/17489

 

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

 

Acknowledgments
This work was supported by EU SYNTHESYS project (http://www.synthesys.info) gb-taf-3881.
Thanks are also due to David Long for providing many of the specimens.
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References

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.

Oct 082015
 

Back in 2014, staff in the molecular lab and herbarium at RBGE greatly enjoyed a three-week visit from Austrian Dr Wolfgang Hofbauer. With funding from the EU SYNTHESYS programme, Wolfgang, employed by the Fraunhofer Institute for Building Physics, was here to investigate mosses of modern building surfaces. He brought with him interesting chocolate, a taste for weird sodas, his wife and their son. While his family enjoyed the ambience of Edinburgh, strolling in the city and garden, Wolfgang was hard at work sampling mosses that he had collected in Austria, Germany and the UK, and comparing them to collections in our herbarium.

Schistidium, photographed by Wolfgang Hofbauer

Schistidium, photographed by Wolfgang Hofbauer

Building walls are a bit like cliffs, harsh habitats where only the very hardy can survive, with little shelter, periods of intense light and restricted water availability. Plants that do manage to establish there are frequently stunted individuals, with atypical morphology, which do not always possess the full range of characters (e.g. bryophyte sporophytes and spores) that may be required to identify species reliably. The group of mosses that Wolfgang is particularly interested in, Schistidium, grow slowly on buildings, with frequently incomplete development. In the past, collections of these mosses have mostly been named as Schistidium apocarpum, a species that is thought to be widespread and common. Wolfgang’s work involves comparing well-developed samples of Schistidium species from our herbarium with the stunted individuals he has been collecting from walls, cement and tarmac, both by looking at the plants’ morphological features, and by comparing standard regions of their DNA (plant DNA barcoding). An important part of this work is the development of a “reference” set of sequences from fresh and herbarium samples that possess a full range of morphological characters, allowing them to be identified reliably to species. Many of the Schistidium collections in Edinburgh have been revised by one of the most respected taxonomists working on the group, Dr Hans Blom, allowing us to generate DNA sequences from some of the plants that Blom has studied and named; these should correspond to Blom’s taxonomic ideas about species in Schistidium. This standard reference set will enable future identification of stunted and dwarfed specimens from building substrates, by comparing their DNA sequences to the standards.

Modern building surface with bryophyte growth, photographed by Wolfgang Hofbauer

Modern building surface with bryophyte growth, photographed by Wolfgang Hofbauer

From the work we conducted with Wolfgang during his visit to RBGE, we could see that the first Schistidium colonisers on modern building surfaces actually include several distinct genetic lineages. These lineages have been sampled from several locations around Europe, but in order to define geographic patterns and to evaluate morphological characters, we need more intense sampling, which will allow us to discuss the diversity and ecology of Schistidium colonisers on modern building surfaces. There are two practical outcomes of isolating the lineages that occur on these surfaces. First, if we can identify dominant species, then we may be able to identify selective biocontrol of moss growth on building surfaces, which can be regarded as unsightly. Second, and in contrast, some of these lineages may actually be adapted to grow on modern building surfaces – and therefore could be used for moss gardening. This could be beneficial for insulation purposes, to reduce air pollution and climate gases (CO2 scavenging), to increase biodiversity within an area, and, last but not least, for aesthetic purposes: greening cities and visually breaking up expanses of concrete.

(This Botanics Story has been cowritten by Laura Forrest and Wolfgang Hofbauer.)

 

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)

Apr 152014
 

As someone who has used taxpayers’ money to fund research on bryophytes (the collective term for mosses, liverworts and hornworts), ‘But why do bryophytes actually matter?’ is one of those questions you start to dread. Admittedly it’s not one everyone asks, presumably as many people haven’t realised that there IS a collective term for mosses, liverworts and hornworts. Or possibly because most people have never heard of hornworts at all…. But when we’re competing, for a limited amount of funding, with people who are trying to increase the productivity of wheat, or the nutrient content of rice, or the number of different kinds of chocolate plant, there’s an onus to justify our research. There’s a good stock of standard answers, but although bryophytes are important in nutrient cycling, and although they have all sorts of wonderful secondary chemicals that just might cure cancer, and although they have an understated sort of beauty that comes alive under the lens of a good close-up photographer (a group from which I am entirely and utterly excluded), we don’t eat them. We don’t wear them, we don’t build with them, drink them, feed them to farm animals, power our cars with them. It could be argued that we can use them as fuel, although it’s hard for a bryologist to seriously advocate peat as a major fuel, mainly a) because wars don’t tend to be fought over bogland in the same way as they are over oilfields, and b), fossil fuels are not exactly the greenest deal.

Anthoceros

hornwort Anthoceros, photographed by D.C. Cargill

So why should we care? The converse is simply, ‘Why shouldn’t we?’ – bryophytes represent up to 7/8 of all the evolutionary diversity of land plants. Although they never get very large, they are nearly ubiquitous, present in almost all environments (they don’t grow in the sea). They share most of their genes with other land plants, and there is evidence that genes can have the same functions in mosses (Physcomitrella) and mustards (Arabidopsis). We can use them to advance our knowledge of genetics, a field in which bryophyte studies have led to many important discoveries; this is aided by the fact that the green leafy part of a bryophyte only contains one set of chromosomes (it is haploid), unlike ourselves, or all other land plants, where the green leafy parts have two chromosome copies (they are diploid). A diploid organism contains genes from both of its parents, and how it looks can be an interaction of both genes (like lots of the genes that are responsible for how tall we are), or because one gene is stronger (dominant) than the other (recessive) (a common examples is our brown and blue eye colours). A haploid organism’s appearance is linked far more directly to the genes it contains – if a gene has a mutation, then the result of the mutation can be seen directly – so if a bryophyte gene that let it make little rootlets (rhizoids) got switched off, then the plant wouldn’t be able to make rootlets, and neither would any of its offspring.

In this week’s copy of PNAS is another reason to care about, and conserve, biodiversity regardless of whether we can put an immediate monetary value or use on it – the story, lead-authored by Duke graduate student Fay-Wei Li, of the serendipitous transfer of a gene from a bryophyte to a fern that allowed the subsequent evolutionary radiation of ferns in low-light habitats. Recent genetic work has shown that some plants have a very particular photoreceptor gene that allows them to utilise both red and blue light. Usually, genes are transferred ‘vertically’ – from parent to child (or seed). Very rarely scientists find evidence of ‘horizontal’ transfer, which means that the gene moves sideways and is then passed on to the next generation, as if a virus were to take part of your DNA and put it into the person sitting next to you at lunch, and that person were then to pass it to their child. This is very very very unlikely – but sometimes, it’s the best explanation for a genetic pattern. And in this particular case, if a hornwort had not evolved a red-blue light photoreceptor, and if that changed photoreceptor had not been transfered into ferns, then the diversity of ferns that we see today probably would not have occurred.

Of course, there’s still the question of ‘Why do ferns matter?’ They’re bigger than bryophytes, and people grow them in their gardens, in shady spots that most flowering plants struggle in. People can eat them too. I tried them, once, in New England, and discovered that they taste just like I expected them to taste. I have subsequently stuck to asparagus, superficially ferny looking but a great deal more delicious. Ferns and croziers appear in art, architecture and fashion; Victorians got themselves into frenzies collecting them; in fact they’re all in all a good deal easier to ‘justify’ than bryophytes.

(And for anyone who is not convinced about the value of ferns, my next offering is orchids – a sometimes flamboyant group of over-evolved flowers with dusty seeds so small they need nutrients from a fungal friend to get established. Weirdly, they can use the same fungal partner, Tulasnella, as liverwort genus Aneura.)

 

With thanks to Juan Carlos Villarreal for the PNAS reprint: Horizontal transfer of an adaptive chimeric photoreceptor from bryophytes to ferns, by Fay-Wei Li, Juan Carlos Villarreal, ………… & Kathleen M. Pryer