This article agrees in part with challenges in plants, though nonetheless discusses values of sequencing there. It is from 1999, there are now newer opportunities with cheaper/easier/deeper/more parallelized long-read and multi-locus sequencing?
https://www.sciencedirect.com/science/article/abs/pii/S1055790399906127
Jessica, I wonder whether you might consider reposting some of these notes as a journal entry on iNat (along with your fantastic idiotās guide to broad fungi classification). There is a lot of interesting and useful information that I think would be valuable for other users and forum posts have a tendency to get buried fairly quickly. (Not that searching for journal posts is much easier, but they do at least provide a more permanent and standalone sort of link and another way to access the material.)
Thatās a good thought; Iāll probably put some thoughts into things and make some journal posts if I can remember to, Iād want to put a little more thought into a journal entry.
I do have one on taking good fungi photos but it is on my work account. I need to copy it over to my personal.
It looks like we just missed the last MycoBlitz of this year. So, if one does not live in one of the open call areas, which fungi are worth having barcoded?
Sure, you can sequence a lot more DNA more easily now, but I would say that if you have to sequence 10s or 100s of genes to get a species ID, thatās not a DNA barcode. The core concept for a DNA barcode is that you only have to sequence one area of DNA to get an ID
Uh, citation needed. While I got burned trying to use a ycf1 barcode on one genus of angiosperms a decade ago, Iād say that species-level barcoding with a few plastid markers is more normal than not for vascular plants.
I think you and I are working with slightly different takes on DNA barcodes. I was thinking in terms of what some people have called āuniversalā DNA barcodes: you have a stretch of DNA that you can sequence for a very broad group of organisms (animals, fungi, bacteria, etc.) and you will usually be able to get a species-level ID just based on that. The cytochrome oxidase 1 gene that we use for DNA barcoding insects works for any order of insect, as well as crustaceans, mollusks, most other invertebrates, and vertebrates.
Based on your description of working with one genus, I think you are talking about specific barcodes or markers that can be used to get species IDs as long as you already know roughly what it is. I agree that those are very commonly used in plants, but they are not the same as universal barcodes, because you wouldnāt be able to use them to easily identify a mystery plant or mystery angiosperm to species.
A 2024 review of plant DNA barcoding* says right in the abstract that āIn plants, the use [of] a universal DNA barcode, such as COI, which is used in animals, has not been achieved so far.ā They do discuss specific barcodes and what they call āsuper barcodesā: apparently if you sequence the entire chloroplast genome, that gives you enough data for a species ID. Personally that seems kind of long (~155,000 DNA base pairs) to be called a barcode, but maybe thatās just me.
*This is supposed to be open-access, but if anyone canāt access it, let me know.
Russulaceae, Inocybaceae, Entolomataceae, Cortinariaceae, Hygrophoraceae and a few others. Itās worth looking through the observations that have the āDNA Barcode ITSā field and seeing if any particular groups have a lot of temp codes or are more solid.
And realstically, some groups are definitely just going to come back as a temp code - Hygrocybe conica is a good example, someone needs to do a serious study on this group and thereās likeā¦ 6 or so different temp codes? Stuff like that it is sometimes just better to stick it under complex and call it a day unless you really want to get into the weeds on things.
Arenāt you in the carribean a bunch? I think Mycota has an open call down there. Honestly, itās probably the most important area to get more sequences from regardless, there is huge biodiversity in the tropics and it is definitely undersampled
At least with fungi, itās fairly easy to pull LSU/SSU if youāre pulling ITS (especially since Nanopore allows for long reads) - functionally, I think thatās still barcoding, since youāre mostly just looking at a slightly longer sequence - still only a couple thousand bp long instead of tens of thousands
IIRC Tef1 and RPB1/2 are the two protein coding regions that folks do sometimes look at as well. (I may be missing some.) Also probably short and simple enough to be considered barcoding
Literally no real clue for other kingdoms of life, honestly,
I do go there frequently. But the rest of the time, I am in North Carolina, where there is no open call. I have copied these families to my iNaturalist-related Notepad file for reference. Thank you.
As to the Caribbean, I will need to be sure of the import/export regulations. International borders do complicate things.
Of course. I donāt have a ton of advice to offer when it comes to customs, unfortunately.
Yeah, NC has had a lot of attention, and I think thatās probably why it isnāt on the open call. Still, if you make any nice collections in the groups Iāve mentioned above, thereās definitely some east coast mycologists that may be interested in looking at them (and a lot are easily contactable via either inat, facebook, or honestly just email.) Shannon Adams for corts, Jean Lodge for Hygrophoraceae, Alfredo Justo for Pluteus, and Brandon Matheny for Inocybaceae - off the top of my head.
Always worth tagging them if you find something really interesting.
Nanopore will give you long reads, and so theoretically if you used single pair of bounding primers you could amplify the entire SSU/ITS1/5.8/ITS2/LSU stretch. The practical problem is the quality of DNA required to do that. You will find the success rate drops significantly and for samples older than a few years drops very low. The alternative is to amplify the markers with a separate sets of tagged primers, with an upfront cost and extra time. Likewise protein encoding genes like RPB1 RPB2 Tef, BTUB, ATP6 etc have low/single copy number (unlike ITS with 10-200 copies). Tef & RPB2 are accepted as good secondary species barcodes for fungi. However, you need good quality/volumes of DNA to successfully amplify single copy genes. Unfortunately, the quick/dirty/cheap methods used for extracting DNA in most ITS-nanopore protocols (like Chelex or high-pH solutions) generally donāt give you the quality you need. Cost and time required for adequate/quality DNA would increase by a factor of 10 probably.
Definitely agree with everything you said.
I know FUNDIS has done a few runs that were LSU in addition to ITS1/2& 5.8s, but it just really wasnāt necessary for the scale we were doing things - if necessary the samples can always be rerun.
With older herbarium specimans, Mycota has been definitely having success with doing shorter reads (just ITS2), and FUNDIS did a bunch of Largentās types with ITS split and then manually connected back together later.
Idk, things definitely seem to be getting more refined with nanopore; I think the run we started at NAMA was averaging around a qscore of twenty, which from what Iām told is decent. Hopefully things just continue to improve.
I think there will be a forced improvement because the cheap flongles that have been used are no longer available, except in limited quantities for existing users. We will all need to move to the standard nanopore MinION flow cells, with higher throughput and potentially much higher RICs coming out. Except they are 10 times more expensive. You may get higher repeat use if flushed until they get clogged.
I know all of the groups I work with are still working ith the flongles - I have no idea how long that is going to last. The full size celsā¦ guh. Itās going to be interesting to see what happens if & when that transition is forced.
I have been keeping tabs on this thread because ā spending a lot of my iNatting time in heavily wooded areas ā I tend to notice wood brackets. Polyporales and Genus Stereum especially. These can be frustrating because there are many that look the same even in different genera ā a lot of my observations have been pushed back to Order Polyporales and marked āas good as it can be.ā Similarly, of my Stereum, only the two identified as S. complicatum have reached Research Grade.
I have been wondering about the potential of DNA barcoding in these two groups.
Iāll be honest, Iām not super confident on most of my stereum IDs so I personally tend to not ID them to species (thereās a few people that are a lot better than me) - but they definitely respond well to barcoding, at least as far as Iāve seen.
That said, Iām surprised stereums are being pushed up -I feel like theyāre at least pretty easy to get to genus? Iām curious, Iāll take a look (no promises though, like I said, itās one of my weaker groups.)
EDIT: but in general, polyporaceae can just be a pain - I sound like a broken record. That said, this time theyāre a pain not because theyāre stupid hard to ID like russula, itās just that they tend to stick around so long on wood that itās hard to get pictures of a fresh speciman with fresh features - sometimes they just get degraded to the point where they are impossible.
Shoot, fuscoporia gilva looks like this fresh
but usually when I see it itās this crusty nonsense
Some of them Iām better at IDing in their partially decayed state than others.
ALSO because I thought of it - Trametes gibbosa/elegans/aesculi can be annoying. Elegans is a name that is almost certainly being entirely misapplied in North America (bar maybe the neotropics); it is a tropical species and I havenāt seen any sequences for north america come back as that - the only two iNat sequences for the species are from South America. Genbank sequences have a lot of South American & Caribbean islands sequences.
Gibbosa and aesculi are ones that I find easy to differentiate in person but can be more annoying to diagnose from images - Gibbosa is usually fatter and has pores that are more slotted, aesculi is usually much thinner and has more maze-like pores (there can be some annoying overlap) - so it can be really important to get both top-down, edge-on, and pore pictures for this group. See mushroom observer comments here for more info about this slightly confusing group https://www.mushroomexpert.com/trametes_species_01.html
https://www.inaturalist.org/observations/182747112 this observation of mine of T. aesculi shows what I mean about the angles you want to try and get. (Iām not always great about this because sometimes I donāt want to pull polypores off logs to get good angles, tbh)
I just saw your comments on those observations. I appreciate the comment regarding the fertile surface. Provided this is a long-lasting fruiting body and not ephemeral like a lot of mushrooms are, I can find it again.
Thatās good to know. Now I need to decide whether to send some in.
Trametes, especially the bigger ones like gibbosa/aesculi, tend to stick around a while for sure.
Well, before this thread fades out completely, Iāll just ask: if I wanted to have Polypores or Trametes barcoded, or Stereums, would it be best to send them to Mycota or a different venue?
