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One Year of DNA Barcoding

Update May 28, 2012: My project and, I assume those for other amateurs (Canadians might still be on board), has been suspended due to reduction of funds to operate iBOL. They posted costs for researchers who can pay for the analyses. I will always be grateful for the opportunity to participate over the last four years. If the funding situation improves, the project might be reactivated. However, it had become increasingly difficult for me to avoid duplication of species and needless expense on their part. So, my project is complete as far as I am concerned.

This is a review of my experiences over the year that I have been submitting moth specimens to Paul Hebert at the University of Guelph for the International Barcode of Life Project. See request for participation at All Leps
The research team at Guelph is not simply interested in building barcode coverage for Lepidoptera, but also for other groups of insects.

Bob Patterson suggested that I open a topic here on DNA barcoding of moths.

I responded to their request for specimens because of my frustration with the identifications of micro moths and from a comment once received that there were likely many undiscovered micro species in my area (east central Great Plains)

Paul Hebert responded with interest in collecting specimens from this area and offered to send all necessary materials and to pay shipping. I took the materials but pay the shipping. The ~$8 every couple of months is a small price to pay for such an improvement in the hobby. They assigned a contact and created an account where I can follow the progress and see the results - here - BOLD

I pin the moths without spreading. I have no doubt that they would be thrilled with properly pinned specimens. The tiniest micros are put into 2" x 2" Ziplock bags. There is a backlog there on pinning micros so they sent me a shipment of minuten pins, curved tweezers and foam strips. If I do that pinning the specimens can be advanced in the queue by avoiding their need to do that step.

Initially, I tried to rely on a match of my images with their photographs after sequencing. This was not a good way to go. I had lost some of my images and their images were many times taken at an angle and quality that made the correlation very difficult. This year, I have been transmitting my images by ftp to their computer soon after the photography. I identify each photo by a sample number that includes the date and logbook number. This has eliminated confusion at my end. They are now using those images in the BOLD - such as BOLD - Acontia

Initially, I submitted almost everything found leading to excess duplication for some common species. The bad part was that it might have been some wasted effort on their part. The good part was that some of these look like local variations - such as BOLD - Galphyria

I see my role as just one who submits specimens and then am on my own. It seems to me that the priority there should be to improve the database with properly identified specimens. This may delay my results but improves the chances for my identifications.

Next year of I will be dropping way back on submissions. I will try to avoid species already sent unless they are under-represented in BOLD or have already shown up as possible local variations.

I keep summaries of a few of the many interesting cases here: pbase . Feel free to make comments there.

Here are some rough categories of results - pretty much what one would expect.
Common moths with dead-on identification.
Common moths that seem to show a local variation by my interpretation of the taxonomy relationship tree.
Common moths that seem to show a local variation by their expert and have been given a code (see the Galphyria above)
Moths that are difficult to pin down by wing pattern and for which the taxonomy identification tree shows the same confusion.
Moths that I have identified but not in their database. (Their taxonomy experts take a look and sometimes agree as in the Acontia above))
Species that are not yet in their database or are just new - such as pbase

I try to report to BugGuide based using reasonable interpretations of the relationship tree results and using a good image.
After some discussion with Bob it seems that an appropriate way of reporting these could follow these guidelines.
"Supported by DNA at the species level (or as species name) by BOLD (specimen ID)"
"Suggested by DNA as very near (species name) by BOLD (specimen ID)"
"Identified only at the genus/family level by BOLD (specimen ID)" – only when other moths of that genus are nearby in the relationship tree.
"At present, identified only at the genus/family level by BOLD but appears to be (species name) based on photos in Guide or at MPG"
My specimen IDs look like "LPOKA111-08" or "LPOKB222-09"

Variations of these descriptions have been used depending on the situation. When the specimen has been included in BOLD as a reference, I include a link and a pointer to the image - such as BG

I have restricted my submissions to BugGuide to those where species identification are difficult by wing pattern such as - BG , where the species is new to BugGuide or MPG, or where only the genus is identified and identification here could be passed on as a suggestion to BOLD.

I would encourage anyone interested to apply for a project but would suggest reading up some on the technique. I have done this but by no means have become an expert. My career was in experimental solid-state physics and I still feel like a novice about the DNA interpretations.

Project suspended
See noted edited in at the top of the forum topic.

Too bad. In Germany this is just getting started and they are _paying_ selected collectors as much as $6/specimen for submissions!


New platform at BOLD
There is a new platform at BOLD , . In the Public Portal, enter the species name under Taxonomy, Public Data, or BINs options. A specimen process identifcation number (such as LPOKD343-09 for my project) can be entered in Public Data and BINs. Much more data is publicly available in the new system as well as new information about collectors, identifiers and geographical locations in the BINs.

A virtual PCR process that you can try yourself
OpenPCR has a virtual demonstration of the PCR process (one of the steps* that happens to our samples after they arrive in the barcoding lab) that you can try yourself. As you'll see, it's a relatively simple process that other than the appropriate primers, etc. only really requires a thermocycler (think testtube incubator with programmable timer). These apparently have become much less expensive in recent years.

The virtualization does a great job (at least I think so) of demonstrating how the PCR process (whose purpose is to largely isolate and magnify the desired DNA segment) works:

* I clearly do not yet understand all the steps involved -- I believe that first DNA extraction is necessary, which apparently also requires centrifuging, and there may be more steps as well.

Any tips on participation? I believe that John S. is also involved (I'm already picking his brain about this in the Spider Forum), but it finally is clear to me this methodology will only become more important (waiting for the first field analyzer).

How does one become involved? Is it an obligation that the specimens remain in the barcoding archive? (This does indeed make sense, I must admit). Do you get the raw data back or do they also do the divergence analysis, etc.?

We see relatively few _collected_ spider specimens here, most keys require mature specimens, and often one simply has a single specimen that is 'uncertain' (particularly when one has not seen too many specimens of a suspected species). Thus, such an analytical approach could be a big help.

Are there various organizations doing this now? Or is it best to contact the University of Guelph directly?

DNA barcoding participation
Tips: Read up on DNA barcoding. Submit your own images ( I upload them to my folder on their computer by ftp). Keep good records. Accumulate at least 95 specimens for each shipment - to fill a standard tray for the DNA analysis. Otherwise it will take longer to get the results. Avoid duplications to save on their costs - except, I think, for possible new species and those that are not in currently in BOLD. You can survey what is there by searching spider species here: BOLD
Involvement: Look at the last part of this summary by Bob Patterson "Getting Started with BOLD" MPG
- that suggests to contact Paul Hebert at Guelph directly to get started.
Once you have been accepted and have an account, you will have tools available to look at divergences within your specimens and with respect to public data in BOLD.
You can provide identifications if you are sure of them. Uncertain ones might match with some that have been submitted and will provide an identification. Or, you will have to wait until matching reference specimens are submitted. I do not submit identifications for my specimens.
Other suggestions will be available through your contact at BOLD.

Thanks, Mark!
Thanks, Mark!

Looking at regional variation
Here are a few case studies aimed at showing regional DNA divergence for some of my moths. These use my data and publicly available data from BOLD which is currently mainly from Canada, Tennessee and Costa Rica. The images and captions tell the story for each one. When more data is publicly available, my interpretations might change.
There are others of my species that might be added.
Local DNA variations

Fissicrambus mutabilis
Callopistria floridensis
Chionodes mediofuscella
Lochmaeus bilineata
Acronicta ovata
Argyrotaenia velutinana
Decantha boreasella

It is now nearly three years of this activity. It has become difficult to find new candidates here on my patio at the black light. I recently sent what might be the last shipment to Guelph. However, a move to the eastern San Diego area near my son and family looks likely and maybe a survey out there would be interesting. They do not have as many bugs as here.

fabulous work, Mark. Thanks a lot for your contributions!

Plug at the Rockefeller Univ. DNA barcoding blog
Here is a comment supporting the efforts: Rockefeller DNA barcoding blog

NYT article mentioning DNA barcoding and Rockefeller
A bit of DNA bacoding history here: NYT

Thank you!
Hi Mark, I worked at BIO for 8 months in the collections with Jayme and I would just like to thank you for all your hard work!

Some details
Here are some methods developed during the year in cooperation with my contact at the U. of Guelph.

An example of the images that are sent by ftp to their computer soon after they are made. For lateral views the head is pointed to the left. The specimen ID (same as the file name) is added to the image. They will designate it as something like 'LPOKB234-09' when logged in. That will be used in taxonomy identification trees. I do not send some specimens when it is later determined that they are unnecessary duplications.

The start of a box of specimens is shown here. The specimens are arranged in chronological order for the benefit of the technicians at Guelph. The box is shipped inside a larger one with cushioning.

Two pinned specimens are shown here. The micro moth on the left has been attached with a minuten pin to a foam strip which is then pinned through the .5" x .5" label cut from card stock paper. The moth on the right is typical of larger moths. The very large moths have a couple of large pins crossed over to keep them from getting loose and damaging the others during shipment (as shown above).

An example of a resultant taxonomy identification tree is shown here. The red label refers to the specimen of interest.

About 700 sequenced specimens have completed the process including review. About 290 different species have been identified. Many more are expected after full reviews are completed at Guelph. I'll gradually submit some of these as suggested by Bob Patterson in his comment here.

Contributing to BOLD
This is a very interesting page, Mark. Thank you for putting it together. You have obviously put a lot of work into submitting specimens, but the results will be very valuable for everyone. It seems to be one area in which amateur enthusiasts can make a real contribution. I hope I can submit some specimens this year too.


Recent discussion about microcaddisflies
There is some recent discussion regarding microcaddisflies here: BG

Great Start on Topic!
I was bogged down in posting to the MPG database the many photos received during the last month and didn't have time to immediately comment on your draft. I have only a couple of comments at this time.

Consider posting here, or linking directly to, your photos of a box of specimens to be sent to BOLD, and also the photo of the tree showing results for your specimens. Please also create a photo of one pinned specimen sitting beside the label that you prepare, clearly showing what the label data look like.

I recommend that you post at BugGuide (hence making the photo available to MPG) at least one good photo of each species for which you have gotten supporting ID data from DNA. You might have noticed that, at MPG, I have begun crediting photos as "© Mark Dreiling - DNA," and have done/will do this for photos from Dick Wilson, Gary McDonald, and anyone else collaborating with BOLD. To me, a DNA-supported photo is a gold-standard photo, even if it is of a common species for which we have lots of unambiguous photos. We ought to have at least one such photo for every species, and several for species with extreme variation in maculation.

I'm looking forward to learning a lot more from you "DNA Pioneers" at BugGuide and to your asistance in helping to create a page on this topic to reside at MPG.

Mark, I think what you are doing is very important in helping ID moths and and clarify some of the issues we have with identification of moths from photos. Several other contributors are also participating in this effort. As results come back this will help provide a more definitive source of IDs for comparison with new ID requests. Probably nothing is 100% with species because of variation in time and place, but it's a step forward. Most people just want to be able to ID a moth from a photo, but without verified specimens it is often just an educated guess. I know I have been frustrated with trying to ID many of the moths I and others find. I sent in some this year and Dick Wilson sent in many individuals, so more data should be coming in slowly. Thanks, John.

Dick Wilson article in Barcode Bulletin
Here is a recent article about Dick Wilson and barcoding. iBOL

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