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 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

Jul 202016
 

Not long ago, the only non-crop plant that the mainstream scientific community seemed to be aware of was the brassica Arabidopsis thaliana – easily cultivated, with a short generation time and small nuclear genome, it seemed the perfect plant model. The model moss Physcomitrella patens came along some years later, again with a short generation time and small genome and with easy protocols for cultivation, but with the addition of a haploid dominant lifecycle and the ability to undergo efficient homologous recombination. A far more recent addition to the pool of available model organisms is the complex thalloid liverwort Marchantia polymorpha. Like Physcomitrella, the haploid part of the plant’s lifecycle is dominant, the nuclear genome is small, the plants are easy to cultivate and fast growing, and there are established protocols to get them into sexual phases. Genetic transformation is also straightforward, and because the thalloid plant is haploid, the effects of mutations are easily visible and can be propagated via vegetative clones.

model system posterIndicative of its growing popularity as a model organism, the February 2016 edition of Plant and Cell Physiology, edited by Professors John Bowman, Takashi Araki and Takayuki Kohchi, was a special focus edition on Marchantia polymorpha, while in June this year, EMBO hosted an international workshop on new model systems for early land plant evolution, wherein 21 of 36 speakers (58%) and 43 of 67 poster presenters (64%) mentioned Marchantia in their abstracts.

Marchantia sampling from Villarreal et al. 2015

Marchantia species sampling from Villarreal et al. 2015, New Phytologist

With publications starting to emerge from the longstanding RBGE project on the phylogeny of complex thalloid liverworts, we felt it was time to also explore some of the species level variation within genera. Previous published work from RBGE examines species relationships in other complex thalloid groups, Dumortiera, Cleveaceae and Mannia, while we are currently working on manuscripts for Sphaerocarpos and for the Aytonicaeae.

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

Some of the collections of Marchantia held in the RBGE herbarium

Boxes of silica-dried liverworts in RBGE's tissue storage room

Boxes of silica-dried liverworts in RBGE’s tissue storage room

Considering the current levels of interest in Marchantia, and in light of our recent paper sinking both Preissia and Bugecia into the genus, we did a rapid survey of our silica-dried tissue and herbarium collections, to see how feasible it was to flesh out the sampling in our 2015 paper (which included about 8 species), to produce the first phylogeny for species of Marchantia. In 2013-2014, we extracted DNA from all our silica-dried tissue collections, and from many of the most recent herbarium collections, which in combination with sampling for our complex thalloid phylogeny, various EU-funded SYNTHESYS projects, and ongoing DNA barcoding work, meant that we now have DNA extracted from 169 Marchantia accessions, which have been determined to represent ca. 20 species, out of a total of 38 accepted species in the genus.

Marchantia polymorpha ssp montivagans, photographed in Merida, Venezuela by David Long

Marchantia polymorpha ssp montivagans, photographed in Merida, Venezuela by David Long (Long 33064)

However, nearly 45% of the accessions (76) are detted as Marchantia polymorpha, as some of our previous work has focused on investigating the genetic differences between its three recognised subspecies, ruderalis (the common, weedy form), polymorpha (from damp natural habitats) and montivagans (frequently an upland plant). Most of the Marchantia polymorpha accessions are from the UK, but there are also samples from Ireland, Portugal, Sweden, Latvia, Switzerland, France, China, Malaysia, Pakistan, Tanzania, the US, Canada, Argentina, Ecuador and Peru.

The other species that we have extracted DNA for are M. alpestris, from Greenland; M. berteroana, from Chile and New Zealand; M. chenopoda, from Ecuador and Mexico; M. debilis from Cameroon, Ghana and South Africa; M. emarginata, from China and Japan; M. foliacea, from New Zealand; M. globosa, from Reunion; M. hartlessiana, from Bhutan, India and Nepal; M. inflexa, from Mexico; M. linearis, from Bhutan and Nepal; M. paleacea, from Italy, Portugal, the US, Mexico, Yemen, India, Bhutan, China and Japan; M. papillata from Bhutan, Bangladesh, Nepal, Rhodesia, Tanzania and Malawi; M. pappeana from Lesotho, Tanzania and Malawi; M. pinnata from Japan; M. plicata from Ecuador, and M. subintegra from Bhutan, India and Nepal.

Marchantia quadrata, photographed in the US by David Long (Long 35670)

Marchantia quadrata, photographed in the US by David Long (Long 35670)

In addition, we have accessions of Marchantia romanica (formerly Bucegia romanica) from Romania and Slovakia, and accessions of Marchantia quadrata (formerly Preissia quadrata), from the UK, Ireland, France, Germany, Sweden, Norway, the Czech Republic, China and the US.

The choice of which DNA regions would be best to work with was straightforward – given that many of our DNA extractions were from herbarium specimens, we were limited to short regions of sequence, and to regions that are present in multiple copies within the plant genome – effectively, plastid and nuclear ribosomal regions. When specimens are preserved for the herbarium, the quality of their DNA drops considerably compared to fresh material; eventually it can break into many short fragments. In order to make copies of regions of DNA using standard laboratory PCR methods, there has to be an unbroken piece of DNA to act as the template, and the shorter the region that’s being copied, and the more copies of it that are present in the genome, the more likely it is to work. Because we routinely sequence the two standard plant DNA barcode loci, from the plastid rbcL and matK genes, and two supplementary DNA barcode regions, the plastid psbA-trnH spacer region and the nuclear ribosomal spacer ITS2, these relatively short regions were obvious choices. However, we found two of these regions, matK and ITS2, consistently problematic to amplify and to sequence for Marchantia polymorpha, and so decided to focus on just the rbcL and psbA-trnH regions for this initial study.

 

References:

David G. Long. 2010. Marchantia polymorpha subsp. montivagans/polymorpha/ruderalis, in: Atherton, Bosanquet & Lawley, Mosses and Liverworts of Britain and Ireland a field guide, British Bryological Society.

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.

Juan Carlos Villarreal, Barbara J. Crandall-Stotler, Michelle L. Hart, David G. Long & Laura L. Forrest. 2015. Divergence times and the evolution of morphological complexity in an early land plant lineage (Marchantiopsida) with a slow molecular rate. New Phytologist. 209: 1734–46, doi: 10.1111/nph.13716.

Relevant posts:

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

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

Apr 262016
 
Setting up PCRs in our laminar flow hood

Setting up Aneura PCRs in our laminar flow hood

Sitting in Edinburgh airport on a Monday morning, waiting for David Long to join me, checked in through to Trondheim via Copenhagen, I felt completely unprepared. The previous week had been a fluster of lab work and reading DNA sequences, trying to get everything ready in time – a stressful Friday evening, trying to copy all the Aneura files into my Dropbox and onto flash drives before the building shut down at 6pm, willing all the file transfers to go faster and faster… but in the end having to leave many of the images that I had planned to take with me behind in the office. Despite a relatively early start on the Monday, we had a 7 hour layover in Copenhagen,

The Lego shop, Copenhagen

The Lego shop, Copenhagen

time for a train ride into the city, lunch, and a meander through the downtown streets, so didn’t get to Trondheim until late. From the airport bus we could make out snow and birch trees, before getting off on a near-deserted icy street. A short walk to the Comfort Hotel Park, an easy check-in, and sleep.

Swedish bryologist Lars Söderström picked us up in the foyer at about 9am. The university is only 20 minutes or so walk away, but the icy pavements made that impractical, so we were taking the bus. Lars had our bus tickets on his phone, cheaper and easier than using cash on the bus. Once purchased, they’re good for an hour and a half, with a spinning bar that gets shorter over the time period until it eventually disappears and the ticket has gone. It was a short ride, across the river and uphill, through mostly painted wooden buildings. Ana Séneca, our Portugese team-Aneura colleague, met us on the bus.

The university building is modern and airy, with open atriums the height of the building, planted with dead bamboo. Ana and I made our way to the Herbarium, a windowless room filled with cupboards of bryophytes that had mostly been collected by herself and by Lars. This was the day that the two of us had put aside for compiling and analysing our Aneura data. I’d begun sending sequences over to Ana on the Friday, so the datasets were already joined together. We had sequences from just over 300 accessions of Aneura, mostly from the British Isles and Norway, but with representatives from Albania, Sweden, Iceland, Portugal, Belgium, Austria, Latvia, the Faroe Islands, China, Fiji, India, the Falklands, Reunion, South Africa, the US, Canada, Panama, Peru…

Building phylogenetic trees in the University Herbarium, Trondheim

Building phylogenetic trees for Aneura in the University Herbarium, Trondheim

We used PAUP to run some quick parsimony analyses, printing out multi-page phylogenetic trees for each of four gene regions that we had been sequencing.

Papering over the table with our Aneura data

Papering over the table with our Aneura data

Clades that were in common between all four trees were marked on using some provisional, and informal, clade names, and after a search for coloured crayons, Ana undertook the serious business of marking geography onto the trees. Although she tracked down a pack of 12 coloured crayons, that wasn’t quite enough to separate the regions we were interested in, so we ended up with a key that combined colour and symbols.

Back to basics - colouring in the trees

Back to basics – colouring in the trees

A little after 5pm, it was time to call it a day, roll up the trees we’d made, and head out into the cold and dark to catch the bus back into town; the four of us headed to the Microbrewery in town for beer and burgers, then a nightcap of whisky at the hotel before Lars and Ana caught the bus out to their home.

Lars, David and Ana pick their way across the least icy route to the Museum

Lars, David and Ana pick their way across the least icy route to the Museum

The next morning, Lars and Ana met us at the hotel again, but this time instead of a bus, we were walking to the Museum, only five minutes or so from where we were staying. The paths were icy, but the views across the river were beautiful in the sunlight. We signed in at the Museum, where our Norwegian friend and colleague, Kristian Hassel, was waiting. First we headed up to the Herbarium, with views out across the city, before going downstairs for coffee, and settling on a sofa in the library to roll out our trees and start the conversation – what are we going to do with this data?

View from the Herbarium, NTNU Museum

View from the Herbarium, NTNU Museum

Luckily, we all agreed on the next actions – we are going to give names to a set of new species, based on molecular characters. We won’t name things that have only been collected and sequenced once, but if there are 4 or more accessions that form a lineage, then they will get named. Because of the focus of our sampling, we will restrict the taxonomy to taxa that occur in Europe. We also have to deal with the species of Aneura that have already been described. Because we are planning to use DNA for taxonomy, then we need to also have sequence data for all the existing names in the genus, even those that were described before anyone know what DNA was. This can be done retrospectively, using epitypification.

Ana and Lars compare names in the Museum library

Ana and Lars compare names in the Museum library

When a species name is published, it is linked to something known as a ‘type’. Usually this is a physical specimen, botanically, a dried out plant sample, although historically, illustrations were also used. The specimens are particularly important, often placed in special red folders, and treated with great respect. Methods like DNA extraction, which involve physically destroying parts of the material, are frowned upon. Given that some of the material can be over a hundred years old, DNA methods can also have very low success.

Instead of trying to get hold of old plant types and grind them up, we intend to use an alternative, which is the designation of new good-quality plant material as ‘epitypes’ – explanatory types that have more characteristics than the original material had, and so allow a better understanding of the correct application of the plant name. The material that we will designate as epitypes will be from large collections, with associated DNA material, and will have been sequenced for the set of four DNA markers in our project.

Trondheim, by the river

Trondheim

Trondheim

Trondheim

Thursday saw us back in the University, continuing discussions about data handling, dealing with mundane tasks like tracking down specimen information and compiling tables of data. More excitingly, bringing together collection details for plants in different evolutionary groups in our trees started to reveal some biology behind our proposed new species, with different ones occurring in different habitats. Although our departure on the Friday morning can only be described as totally uncivilised, with a 6.30 am flight from Trondheim to Oslo, a short stopover then an arrival in Edinburgh at approximately breakfast time, at least we had the satisfaction that the story of Aneura is finally beginning to come together – and an agreement that the next time we meet, it will be somewhere a bit less frozen, like Portugal…

A land of snow and ice - Norway from the plane

A land of snow and ice – Norway from the plane