Observation 32228: Rhodocybe Maire
When: 2010-01-18
No herbarium specimen

Proposed Names

7% (2)
Recognized by sight

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Eye3 = Observer’s choice
Eyes3 = Current consensus


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By: Johannes Harnisch (Johann Harnisch)
2010-05-09 14:11:57 PDT (-0700)

Yes I think you should make a article on it (sequencing Fungi) I have Googled it and didn’t find any thing much on how to do it.
So do you have this one sequenced then?
BTW How does sequencing help you ID Fungi? do species in the same Genera have similar Sequence ?
BTW thanks for you time, this is intriguing!

Dan -
By: Christian (Christian Schwarz)
2010-05-08 22:03:31 PDT (-0700)

That’s correct. The tags are simply added markers – they don’t change the order of the code.

However, for some (usually older) techniques, ddNTs are used – these are nucleotide bases that have had both their 2’ and 3’ OH groups chemically removed (dd stands for di deoxy). Since the ribose-sugar backbone of DNA is formed by repeated links between the 5’ phosphate groups and 2’ or 3’ OH groups, the building of a new (complementary) strand requires the presence of free 2’ or 3’ OH groups.

If a DNA polymerase is given a mix of ddNTs and normal nucleotide bases, it will sometimes incorporate ddNTs which terminate the construction of the growing strand (because they don’t provide a free hydroxyl group to attach the next phosphate group to). In this method, all you have to do is tag the ddNTs with the fluorescent tag, separate the different strands by length (by gel electrophoresis), and read the tag for each fragment size.

If all the fragments were synthesized from the same starting point, the shortest possible fragment is one nucleotide long (ie. the first base added by DNA polymerase was of the ddNT form). If this was an adenine ddNT tagged red, you would see a one-NT long fragment fluorescing red. And so forth, up to the longest strand – the one in which normal nucleotides were added up until the very last position.

In reality, the statistical gremlins make the first and last handful(s) of bases more uncertain than the intermediate-length fragments.

The cutting-edge technologies are much more accurate, but currently give much shorter read lengths (no more than 300 or so bases, I think). This simply requires some computationally intensive (and boring) stitching-together of the fragments in post-processing.

454 sequencing, the sequel
By: Chris Parrish (kitparrish)
2010-05-08 21:27:32 PDT (-0700)

Recent developments at Stanford are aimed at developing a super-sequencer that could “sequence one human genome in a single run with 20x coverage, The researchers … hope to achieve this goal within three years” Awesome technology.

454 sequencing
By: Chris Parrish (kitparrish)
2010-05-08 21:06:29 PDT (-0700)

Fascinating, Christian. Roche offers an explanation of 454 sequencing and the Multimedia Presentations" available from the side panel (esp. the second presentation on the workflow) give an idea of how it is done by fragmenting the DNA and sticking the fragments onto 400,000 beads and then processing the lot in parallel … resulting in the sequencing of 100 million base pairs in 8 hours! Amazing!

Tagged nucleotides
By: Dan Molter (shroomydan)
2010-05-08 19:47:07 PDT (-0700)

Thanks Christian. That was very informative. Can you tell us how the nucleotides are tagged.

The nucleotides are altered to make them visible to a laser scanner, but the change in molecular structure of the nucleotide does not affect a change in the code. Is that right?

Okay, here goes…
By: Christian (Christian Schwarz)
2010-05-07 21:11:50 PDT (-0700)

Nucleotide sequencing happens like this:

DNA extracted from the mushroom is heated by a machine called a thermocycler, causing it to separate into two strands from its double-stranded, stable, ‘resting’ state.
This ‘melted’ sample is exposed to DNA polymerase – an enzyme that makes each of the melted strands complete by building a new complementary strand onto each of them.
BUT! (this is the important part)
That DNA polymerase is provided with fluorescently-tagged bases (ie. A is tagged with red, G is blue, C is green, T is black) to add. So, when the enzyme adds an A (adenine), a laser can read that position on by sensing a red wavelength, and the computer outputs an ‘A’ for that position on the strand.
I am taking a genomics/bioinformatics class right now, and the current edge of the field (454, Illumina, pyrosequencing) is actually much more advanced than the type of sequencing described above (Sanger sequencing).
Even Sanger sequencing is (a bit) more complicated than what I described. If folks here want me to write a short article on it, I will – maybe Dimitar can host it on his site, or possibly Mykoweb.
Hope this helps and please feel free to ask me specific questions.

By: Roy Halling (royh)
2010-05-07 08:44:44 PDT (-0700)

that’s the article from the Mycologia site. Please send me an email if you can’t get the PDF from my website! Thanks.

I do not know how DNA sequencers determine nucleotide order, and that kind of info won’t be in the Bothia paper.

Wait a minute
By: Johannes Harnisch (Johann Harnisch)
2010-05-07 08:42:18 PDT (-0700)
By: Johannes Harnisch (Johann Harnisch)
2010-05-07 08:35:13 PDT (-0700)

Roy, but can’t seem to find the PDF file…does it tell how sequencing is done?

Sequencing protocols
By: Roy Halling (royh)
2010-05-07 06:38:01 PDT (-0700)

are a bit lengthy to post as a Comment. In short, DNA is extracted, purified and then amplified. Then specific genes are sequenced using primers specific for those genes (e.g., nuclear ribosomal, mitochondrial). A specific example from a bolete can be found in: Mycologia, 99(2), 2007, pp. 310–316. A free PDF of this paper by Halling, Baroni, & Binder is the one you want (http://www.nybg.org/bsci/res/hall/boletes/publications.html). Even though that paper is about a bolete, the protocols are pretty much the same, although certain methods are tweaked depending on the beast being examined.

By: Dan Molter (shroomydan)
2010-05-06 19:28:11 PDT (-0700)

The nucleotide sequence could in theory be read with an atomic force microscope, but I’m ignorant of the methods actually used. If Christian or anyone else has the time to explain the method, I’d be very interested to learn about it.

if you don’t mind
By: Johannes Harnisch (Johann Harnisch)
2010-05-06 17:05:53 PDT (-0700)

actually I am quite curiously interested.

A sequence vs. TO sequence
By: Christian (Christian Schwarz)
2010-05-06 15:33:52 PDT (-0700)

In this case ‘a sequence’ means a string of nucleotides (the building blocks of DNA) in a particular (and hopefully unique and informative) order.

‘To sequence’ means to determine this order. This is accomplished by somewhat complicated biotechnological methods that I won’t go into unless you want me to explain…

Very interesting
By: Johannes Harnisch (Johann Harnisch)
2010-05-06 14:14:11 PDT (-0700)

I have a question,
What does sequence mean in this case?

Specimens received.
By: Dan Molter (shroomydan)
2010-05-06 08:56:43 PDT (-0700)

Thanks to Christian for forwarding these to Tim Baroni. Dr. Baroni has examined them and plans to sequence them. I’ll update this observation with a name as soon as he gives me one.

faceted spores
By: Dan Molter (shroomydan)
2010-01-30 09:07:43 PST (-0800)

I found my 15x eye piece and took another look at the spores. They appear to be faceted.

specimens sent
By: Dan Molter (shroomydan)
2010-01-24 09:03:28 PST (-0800)

I spent about four hours combing the woods for these a few days ago and returned with a single new specimen. It is very small, about 1.5 cm in diameter. I saw the little mushroom when I collected the larger specimen from the initial observation. Heading out, I knew the little one was there, but I was worried that something might have eaten it. I was happy to see it still there.

Both mushrooms were dried and are on their way to Christian Schwarz.

Yes Richard, it was good to be back in the woods for a few days. The top photo turned out really neat. Fill flash makes the mushroom appear to glow against the dull winter background. Thanks for the compliment, I’m glad you like the photo.

I have also found Pluteus romelii. The mushroom in this observation is a bit meatier and has attached gills, though one could easily be mistaken for the other.

Pluteus romellii looks very like this?
By: Richard Sullivan (enchplant)
2010-01-22 05:21:03 PST (-0800)

So is it the difference of the attached gills, and the faceting on the spores?

BTW Dan, It didn’t take you long to get out there and start taking great photos again after the snow melted.
I was in Ohio last week for business and it was covered in nasty white stuff.

Entoloma molteri
By: Irene Andersson (irenea)
2010-01-20 23:43:32 PST (-0800)

I wouldn’t be surprised if this is a new species.
Christian, make sure to describe and publish it validly ;-)

I looked closer at the spores again, and I beleive there are true facets on them, though not seen clearly in outline.

By: Dan Molter (shroomydan)
2010-01-20 18:10:47 PST (-0800)

Cool Christian, I’ll check the patch again tomorrow and I’ll send you what I find the following day.

Do you still have my address?
By: Christian (Christian Schwarz)
2010-01-20 17:49:11 PST (-0800)

You can send me some of these – I will certainly do a full micro description, and I may also be able to get sequence for it.

Do you still have my address?
By: Christian (Christian Schwarz)
2010-01-20 17:48:09 PST (-0800)

You can send me some of these – I will certainly do a full micro description, and I may also be able to get sequence for it.

specimen available
By: Dan Molter (shroomydan)
2010-01-20 17:32:45 PST (-0800)

This mushroom still exists, and I might be able to collect a few more. I’ll happily send a collection to anybody who wants to sequence them.

Spore shape
By: Christian (Christian Schwarz)
2010-01-20 16:13:03 PST (-0800)

Molecular phylogeny and spore evolution of Entolomataceae
D. Co-David, D. Langeveld, M.E. Noordeloos, Persoonia 23, 2009.

The authors used electron microscopy to show that there are a number of Entoloma with spores of “irregular entolomatoid” structure (ie. with unconnected bumps), however all of the Entoloma clade species (as opposed to the Rhodocybe clade species) had at least some true “facets” on the spores – faces bounded by angular, connected ridges.

Your 1000x shot doesn’t appear to show any true facets. My vote is for a species in the Rhodocybe clade

I hope
By: Irene Andersson (irenea)
2010-01-20 13:44:28 PST (-0800)

you have taken good care of this collection, and eventually will be able to send it to someone who knows what to do with it, maybe Machiel Noordeloos?

Typical Rhodocybe spores should be more verrucose, I think. These are also too smooth to fit an ordinary Entoloma, but the pointed tip where they were attached to the basidia looks typical for the Entolomatacea. There are a few Entoloma species with almost smooth and subglobose spores, but I haven’t found anyone looking like this species. I have no idea if this should be placed in Entoloma or Rhodocybe.

Hey Eddee
By: Dan Molter (shroomydan)
2010-01-19 09:54:43 PST (-0800)

I think yours are Flammulina. I found a few of those yesterday as well. The mushroom in this OB does not have a slimy cap, and the spore are pinkish brown.

Yes, very grateful for the mid winter thaw! I might even head back out today. :)

Cool Dan
By: Eddee (eddeeee)
2010-01-19 09:48:03 PST (-0800)

Good to see winter is not stopping us. Like you i have been under snow for the last month and finally there is some land to walk on. Ok just found these guys this morning (http://mushroomobserver.org/32235 ) looks kind of similar to yours but These are very slimy. What do you think? Think they might be Rhodocybe? I thought them to be Flammulina but if you notice no velvet stem.

Yep, same as last year
By: Dan Molter (shroomydan)
2010-01-19 09:12:00 PST (-0800)

This mushroom was found in the same general area as last year’s collection:


The spore print is the same color. Very strange to find an agaric that fruits only in winter. We’ve a had a few days above freezing, but before that Ohio was covered in snow for a few weeks. The snow melts and these guys pop up.

Spore shots on the way.

By: Irene Andersson (irenea)
2010-01-19 07:29:15 PST (-0800)

Is this the same as the pink-spored guys you found last winter?
Such an annoying species.. is it possible to get a microshot of the spores this time?

Created: 2010-01-19 05:58:04 PST (-0800)
Last modified: 2010-05-07 06:17:06 PDT (-0700)
Viewed: 501 times, last viewed: 2016-10-22 06:32:41 PDT (-0700)
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