Laura Forrest

Laura Forrest

Molecular laboratory technician and bryologist, focusing on liverworts and DNA barcoding, with a PhD in Begoniaceae phylogenetics.

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

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.

 

References:

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 082016
 

One of the main problems with sampling largely from herbarium specimens, rather than from material that has been specifically collected for DNA work (rapidly dried in silica gel then maintained at low humidity), is that the quality of the DNA is unpredictable and usually rather poor. Therefore, despite starting out with 169 accessions and about 20 species of Marchantia, the actual successes, where we were able to get good quality DNA sequence data, were substantially lower. What we currently have is a slightly unbalanced data matrix, with 82 Marchantia accessions for rbcL, and 78 Marchantia accessions for psbA-trnH.

Reboulia hemisphaerica thallus, photographed by David Long (Long 34254)

Reboulia hemisphaerica thallus, photographed by David Long (Long 34254)

We also sequenced both rbcL and psbA-trnH from material of two accessions that we thought were Marchantia but where the sequences turned out to be Reboulia (from Texas) and Wiesnerella (from Bhutan). A quick check of the herbarium voucher specimens for both of these showed that they represented mixed collections of more than one complex thalloid species, for which the “wrong” plant parts had ended up in our silica dried tissue collection. Taking fortune from misfortune, both Reboulia and Wiesnerella form quite adequate outgroups for the phylogeny!

Wiesnerella denuda, photographed by David Long

Wiesnerella denuda thallus, photographed by David Long (Long 36267)

Out of the 20 species that we HAD hoped to sample, we ended up with only 12 named Marchantia species for rbcL (Marchantia polymorpha, M. paleacea, M. linearis, M. papillata, M. inflexa, M. emarginata, M. pinna, M. chenopoda, M. debilis, M. hartlessiana, M. quadrata and M. romanica), and 15 for psbA-trnH (Marchantia polymorpha, M. paleacea, M. linearis, M. papillata, M. inflexa, M. emarginata, M. pinna, M. chenopoda, M. debilis, M. globosa, M. pappeana, M. hartlessiana, M. subintegra, M. quadrata and M. romanica); we also had three Marchantia polymorpha subspecies (polymorpha, ruderalis and montivagans) and two Marchantia paleacea subspecies (paleacea and diptera).

That’s a little disappointing, representing, as it does, fewer than half of the 38 currently recognised species in the genus. However, we did also sequence a number of Marchantia accessions that had not been determined to species, and although many of them were good DNA matches to species that we had sampled, several are clearly different to everything else that we have included: one distinct lineage in Yunnan, China, another that occurs in Yunnan and Nepal, and a third in Indonesia and Malaysia. That’s balanced again by taxa that may not have been identified correctly; the psbA-trnH sequences from African material of M. debilis, M. globosa, M. pappeana and M. polymorpha, for example, are identical.

Intriguingly, in the “Preissia” clade, as well as M. romanica, there appear to be two lineages of Marchantia quadrata, one consisting of accessions from Denmark, Sweden and Sichuan, China, and the other with accessions from Svalbard, Norway and Utah, USA. These may tie in with subspecies quadrata (for the first lineage) and subspecies hyperborea (for the material from Svalbard and Utah), but the degree of genetic divergence is far higher than that found between many of the recognised species in Marchantia. It is a bit disconcerting, however, to notice that we have managed to overlook any Marchantia quadrata material from Scotland in our sampling!

The next step in the project, before it’s time to reveal any of the phylogenetic trees I’ve alluded to, is a phase of reciprocal illumination where we reconcile morphological information from the herbarium specimens with the information derived from the molecular sequence data. In other words, it’s time to double check our plant identifications, a part of the project that’s now in the capable hands of Dr David Long; the pile of Marchantia specimens is already on his desk!

 

 

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

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 302016
 
EDNA label printer

The EDNA label printer in the office

Over the years, many different people have used the molecular laboratories at RBGE, to work on a multitude of projects on a multitude of plants and fungi. Some are staff members who stay for decades, others students who are only in the lab for a matter of months. Every time DNA is extracted and used in a molecular project, the amplified gene regions are processed and then the plastic tubes that they were in are sent for recycling – but the extracted DNA is kept in a DNA bank, in case it is needed for further research. Logistically, managing this DNA can be problematic. Scientists like to use their own numbering systems when they’re working (mine used to be one of the commonest – my initials followed by consecutive numbers, a system which worked perfectly until some of my extractions ended up in the same freezer as extractions by Dr Linda Fuselier), something quick and easy to scrawl onto the plastic tubes. This can link to collection information written in a lab-book, including who collected the plant, what date it was collected, and what country it came from. However, as people move on, and as the years pass, it becomes increasingly difficult to find any particular sample or set of samples, particularly when several sets of people share the same initials – and this is compounded by having to rummage through boxes of frozen DNA samples being kept at either -20° or -80°C. Few places at the Botanics are less pleasant than the dank room that contains our -80°C freezers!

 

Printed labels and EDNA tubes, Lab 32

Printed labels getting stuck onto EDNA tubes, Lab 32

The frustrations associated with rooting through inconsistently labelled DNA collections led Dr Michelle Hart and Alex Clarke, in 2006, to instigate a standardised format for DNA labelling, with samples of DNA identified as part of the RBGE DNA bank and assigned EDNA numbers, the format of which consists of the year the DNA was banked, followed by a multi-digit identification number. For example, the last EDNA number that we have issued is EDNA16-0045851, for DNA extracted from the moss Weissia controversia. Due to uncertainties about institutional databases, in its early years the DNA bank was curated through Excel spreadsheets; this was revamped and upgraded in 2011 to the database that we still use today. Information about the methods and date of DNA extraction, the material’s collector, and the place of collection are all stored and easily retrieved, critical information if the DNA is going to be used to provide data for future publications. The EDNA number stays on all downstream files that are created from the DNA – lab books, raw sequence files, and it is also included as the isolate number in GenBank submissions – meaning that all molecular data generated at RBGE is still valuable after people have moved on and lab books have been mislaid.

 

EDNA tube

A labelled EDNA tube ready for the DNA sample, Lab 32

As to what happens to the actual DNA extraction, long-term storage involves transferring the liquid into a small barcoded and labelled tube in a lockable and numbered 96-tube rack, which will be kept on a labelled shelf in a -80°C freezer. The system is not perfect, however – banking or recovering the DNA samples still involves a trip to our mildewy bank room…

 

Pipelling DNA samples into EDNA tubes, Lab 31

Pipelling DNA samples into labelled EDNA tubes, Lab 31

Aug 232016
 

When people extract DNA in the RBGE molecular lab, we insist that it’s given something we call an EDNA (Edinburgh DNA) number. This links to a database that is internal to RBGE.

evilednaThe EDNA number is used for all internal molecular lab processes – it’s written on the tube of DNA, used to refer to the sample in lab books, and part of the file name for all DNA sequences that are generated from that sample. Using this standard system across all projects means that we can keep track of what DNA we have, we can store it in a way that makes it relatively easy to retrieve, it can be used in other projects, and critical information like which specimen voucher is linked to a DNA extraction is not lost if people move on from RBGE.

Getting an EDNA number involves filling in a simple Excel spreadsheet with some basic collection information, and uploading it to a database. The Excel spreadsheet is accessible to RBGE lab users on an internal server (DNA, Molecular lab registration forms, EDNA (DNA), EDNA_submission_sheet_v01), and has two sets of fields, required and additional. If anything’s missing from the required fields, an EDNA number will not be issued, whereas the additional data is recommended but not essential… However, the more fully complete the data entry is, the faster it is to use it to generate GenBank submissions and publication voucher tables, justifying spending a little extra time on getting the forms completed.

Two points to remember when filling in the spreadsheet are not to use special characters, and not to make any of the entries too long, as there’s a maximum character number.

 

REQUIRED INFORMATION

Taxon name: this should not have authority information (Bellis perennis L.), just the genus and specific epithet (Bellis perennis).

Collector name: this cannot begin with an initial (J. Smith) as it will be rejected by the database; either use a full Christian name (John Smith), or put the surname first (Smith, J.).

Collector number: if there is none, s.n. is accepted.

Country code: two-letter standard codes; when filling in the spreadsheet, there is a tab with all the codes that you can look up (e.g. DE for Germany).

Material type: drop-down menu choices – fresh, frozen, herbarium, seed, silica gel dried.

Extraction type: drop-down menu choices include tissue maceration type, e.g. pestle, or mixer mill, and chemistry used, e.g. CTAB, Plant DNeasy minikits, Qiextractor.

 

ADDITIONAL INFORMATION

User DNA ID: this is the number that was given to the extraction in the lab; it’s extremely useful to have this for various troubleshooting in the lab – it can help match accessions to tubes, sort out issues with sample order, etc.

Extraction Date: entered in standard format year-month-date. Again, this can be useful for later troubleshooting, e.g. for separating batches of extractions by date, in case something went wrong on a particular day.

Herbarium barcode: this is ONLY for RBGE herbarium barcodes, not those from other institutes. If this is available, filling this in will propagate specimen data from the herbarium database. However, the required fields still need to be filled in.

Living Accession Number: this is ONLY for RBGE living accessions, not those from other institutes. If this is available, filling this in will propagate specimen data from the living collection database. However, the required fields still need to be filled in. The qualifier letter should not be filled in here.

Living Qualifier: this field is for any alphabetical character after the Living Accesion Number.

Silica Gel Box Number: this field is best left empty unless silica material came from a box numbered in the same format as “SGN12345”.

Sample note: free field, but there is a limit on how many characters are allowed, so should be kept short, and free from special characters. It may be useful to note e.g. if the extraction was from sporophyte versus gametophyte tissue, or flower versus leaf.

Location: free field, but there is a limit on how many characters are allowed, so should be kept short, and free from special characters.

Coordinates: free field, but there is a limit on how many characters are allowed, so should be kept short.

Decimal longitude:

Decimal latitude:

Collection Date Verbatum: this is for dates that cannot be turned into the correct date format, e.g. “Spring 1920”, “October 1976”.

Collection Date: entered in standard format year-month-date. This can be very useful in relation to DNA quality. If this is filled in, there is no point also filling in the Collection Date Verbatum field.

Note: free field, but there is a limit on how many characters are allowed, so should be kept short, and free from special characters.

 

Once the EDNA form is filled in, it can be uploaded to the EDNA database, which is available to users at RBGE who have a Username and Password.

Once logged on, the tab ‘Importer’ becomes highlighted; at the bottom of the Importer screen is a “Load” button.  The information in the excel sheet should be pasted into the ‘Load data’ window, and mapped to the fields. This will leave four fields that need to be filled in manually, three required fields: User (the lab user’s name, available from a drop-down list); Project (again, from a drop-down, e.g. MSc, barcoding, Leguminosae); Contact (a permanent staff member who will take long-term responsibility for the project, chosen from the drop-down list) – and one optional field, EDBANK Format (how the DNA will be stored long term – Plate, Strip or Tube; for most phylogenetics projects DNA will be stored in individual tubes, while for some population genetic project it will be stored in strips or plates – check with the molecular lab staff if unsure which format to chose).

After this information is filled in, the tab “Validate” becomes available. The entered data is screened for things like collector names that start with initials, accession numbers, dates, latitudes and longitudes that are in the wrong format, or other errors. If any are found, then these need corrected in the excel spreadsheet and the information all needs reloaded and re-entered. If there are no validation errors, the “Import to EDNA” button becomes available. At this point, the data will either successfully import, or other errors will be identified (e.g. non-standard characters, or too many characters). Unfortunately errors identified at this later point only stop EDNA numbers being generated for individual samples rather than for the whole batch, and it is not possible to cancel the issued EDNA numbers. This means that, for example, if entering a plate of 96 DNA extractions to EDNA, it’s quite possible for some samples in the middle of the plate to not be assigned a number. Obviously this becomes a sample labelling headache that is optimally sorted by redoing the entire batch to get consecutive EDNA numbers for all the samples, although this will lead to apparent duplicates of samples in the database. Molecular lab staff should be informed of redundant numbers, so that the duplicates are not also assigned places in the DNA bank.

When the numbers have been generated, they can be downloaded from the database by clicking on the “Tasks” tab, and the “As Spreadsheet” option – this will return all the information that has just been entered, along with the EDNA accession numbers for each sample.

 

See also:

The RBGE DNA bank

Aug 122016
 

University of Edinburgh/RBGE student Kimberley Fackler, studying for the Masters degree in the Biodiversity and Taxonomy of Plants; thesis submitted August 2013.

Supervisors: David Bell, Dr David Long and Dr Laura Forrest.

 

Kim sampled from the six species of Metzgeria generally recognised to occur in the UK. She used standard DNA barcode markers rbcL, matK, psbA-trnH and ITS2.

Metzgeria furcata, vice county 49, Long 8069; photographed by David Long

Metzgeria furcata, vice county 49, Long 8069; photographed by David Long

Metzgeria furcata (L.) Dumort.

M. violaceae (Ach.) Dumort.

M. conjugata Lindb.

M. consanguinea Schiffn.

M. pubescens (Schrank)

M. leptoneura Spruce

Phylogram generated from accessions of Metzgeria species found in the UK

Phylogram generated from accessions of Metzgeria species found in the UK

 

DNA barcoding for all regions but rbcL delimited seven genetic lineages of Metzgeria within the UK. There was a lower amount of sequence variation in rbcL, suggesting that it is suitable for use at a higher taxonomic level than this genus.

Six of the genetic entities correspond to the current species concepts in Metzgeria. Metzgeria furcata was split into two sister groups, in line with the findings of Fuselier et al. 2009; these two groups may correspond to variety ulvula and variety furcata.

Conservation implications: If lineages do not have names, they have no legal recognition, no protection, and we cannot gather information about their rarity or distributions.

 

 

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