PCR: Please Chug Rapidly

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Gel of the Day: FAIL

My new primers didn't work. At all.

Bleah.

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Gel of the Day: A Horny Little Bug That's Pissing Me Off

Just for the record, I would like it known that the 3-4mm long diaperine beetles lumped together into the species Neomida bicornis are stinky stupid-heads and I don't want to play with them anymore.

What can I say after that deeply professional and scientific appraisal? Well, I can offer up this Gel of the Day as evidence of my contentions:

The first two lanes are 28S and 5.8S nuclear ribosomal subunits and the third is cox1 from Neomida bicornis. The last three are the same genes from a positive control, Bolitotherus cornutus. Everything else is, of course, the same. The same aliquots of primers, the same Taq polymerase, the same dNTPs, the same thermocycler protocol.

Now, if this were the first time I'd gotten a result like this, it would be no problem. And, in fact, it wasn't a problem four attempts ago. This gel is my fifth attempt at getting these sequences! I've tried tweaking everything I can think of with my existing protocol, but nothing changes the result. My next step will be to use a forensic DNA extraction and amplification kit, which I've been trying not to do because of the expense involved.

If not for the fact that I have some evidence that N. bicornis is more than one species, I would have given up on this by now. The thing is, it's worth it to me because part of my research has to do with the evolution of host specificity and I've collected this beetle from four different fungi now. Not only is the coloration different in these populations, but there are more subtle morphological differences as well (for example, the proportions of the tarsomeres of the metatarsi). I've also used the large subunit sequences from two populations — one from Ganoderma lucidum in Florida and one from Pycnoporus cinnibarinus in Massachusetts — in a molecular phylogenetic analysis and guess what... they turn out to fall into two separate clades with 99% bootstrap support in a neighbor-joining tree after 500 replications. In other words, they're taxonomically classified as a single species, but considering how slowly this sequence evolves it's highly unlikely that they really are. I need to get the more rapidly-evolving cox1 sequence to clinch this, along with analyzing a few more populations and I can't get the damned thing.

What a tease.

I mean, just look...

If I had hair, I'd be pulling it out right now. I can take or leave the small subunit, but I must have the cox1 sequence.

OK, that's it, "Neomida bicornis," if that is your real name. You've pushed me too far. Next time you see me on the street, you'd better run. I'm gonna be packing Raid.

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Well, Hello There

And this is what I look like while I'm dissecting beetles. My adviser thinks it's a "good look for me." Am I a geek yet?

Yeah, yeah.

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Wachusett Wednesday Cancelled: Fall Semester, Florida Expedition and Shifting Priorities

Due to a backlog of lab work in the face of the upcoming semester and the fact that I'll be doing field work in Florida all next week, there will be no Wachusett Wednesday this week. As perfect as the weather is and as much as I will miss this part of my weekly routine, I must put my priorities elsewhere over the next few days.

It's hard to believe that summer has come and gone so quickly, but the fact is that this is the last week before the fall semester begins. I will once again be teaching two lab sections for Introductory Biology this semester. As much as I enjoy teaching (although honesty compels me to admit that I'm not too fond of grading), this essentially means that there will be some combination of a reduction of my research time and late nights at the lab. The classroom portion of my teaching assistantship alone entails six hours each week. Add in grading, office hours, lab setup and miscellaneous issues that arise and a day disappears from my schedule. Additionally, I will be auditing an entomology course at another local university this semester; that adds up to about another six hours each week. It's going to be another very busy semester. With this in mind, I'm trying to spend the rest of this week tying up a bunch of loose ends.

A week from today, of course, I leave for the North Florida field expedition. The loss of my Wednesday on the mountain this week helps to relieve the pressure that a full week out of town will create. On the other hand, all of the planning for that trip has continued to come together very well. The biology faculty at Florida State has been exceedingly accommodating to this alumnus. My colleague and I have been furnished with laboratory space in the new biology building and the head of the university's herbarium has allowed us use of the dehydration equipment for the preservation of specimens. I still need to set up temporary accounts at FSU so that we'll have computer access from the lab.

Florida's DEP and parks management have also been incredibly helpful. In fact, I feel compelled to write a thank you to state biologist Harold Mitchell here. He has been a tremendous help in making the upcoming trip possible and interacting with him has been a pleasure. Moreover, the park managers at both Torreya and Lake Talquin State Parks have likewise been kind enough to provide my colleague and I with free and open access to their land anytime we wish, even going so far as to give us the combinations to open the gates to our sites before regular hours so that we can start as early as we like in order to beat the heat as much as possible in the Big Bend/Panhandle in early September.

Wachusett Wednesday has become one of this blog's most popular recurring features, so to readers who will miss it this week, all I can say is that it's a trade off. Wait until you see what's popping up in Florida this time of year. With the recent and unfortunate stormy weather, I expect that you'll be seeing a bumper crop of amazing and beautiful fungi in this space in the coming week.

On the other hand, Gel of the Day fans (because some people just can't get enough electrophoresis) will get a second, and quite possibly third, installment this week as I attempt to sequence a Ganoderma in the G. lucidum complex whose morphology alone hasn't been enough to provide a positive identification. I also intend to do the same with a sap beetle (Nitidulidae) collected earlier from Laetiporus sulphureus; while not in my core family, I've noticed something interesting about its foot that makes me think that having molecular data from these insects will be informative about one of the evolutionary hypotheses in my study. Markedly reduced fourth tarsomere, anyone? I have so far been unable to adequately resolve the genus of this beetle, though, and genetic sequencing should clarify the picture. Agarose lovers, rejoice!

In the words of Willie Wonka, "So much time, so little to do! Strike that. Reverse it."

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Gel of the Day: What the Heck Happened Here?

Sometimes, a perfectly good gel gets funkified in the process of handling. I think that's what happened with this gel, which was otherwise indicative of a near-perfect PCR:
The specimens in this case were three adult and one larval Bolitotherus cornutus which I collected at the MSA foray in Pennsylvania on August 10. The last eight lanes, 25-32, are from the larva. I haven't successfully sequenced a larva before; maybe my dissection technique is improving. The cox1 bands (31) look a bit weak, but the product should do for sequencing.

If you're familiar with electrophoresis gels or have at least been peeking at previous gels of the day, you're probably asking about now, "Mike, what the heck happened to lanes 21, 22 and 27?"

Well, I'll tell you. In order to take this photo using a UV light, I have to remove the gel from the casting tray, put it in a weigh boat and take it to a dark room upstairs from my lab. In the process of transferring the gel from tray to boat to camera, it gets a little scrape over an edge or two. This time, for whatever reason, that caused the bands in three lanes to get smeared. Nonetheless, there are bands there and since they are duplicates from two different dilutions (as far as specimen and gene go, 21=17, 22=18 and 27=31), it's not a problem.

Also, every fourth well is a control; there shouldn't be anything there. There seems to be a very faint band in lane 4, but it's nothing to worry about. I may just try and sequence it to see what the contamination is, since the position of that band is unlike that of any of the legitimate bands on this gel. I suspect that the culprit is a dirty microtube; if my primers, buffers, etc. had been contaminated, I would expect to see that phantom band in all of the control lanes.

The reason I'm running another batch of B. cornutus is that these are the first specimens I've collected from a site outside of Massachusetts. It's worth looking at them just to see if there are any polymorphisms present in introns in the sequences I'm analyzing.

I also read a very interesting paper today:

Leschen RAB and TR Buckley. 2007. Multistate characters and diet shifts: evolution of Erotylidae (Coleoptera). Systematic Biology 56(1):97-112. DOI: 10.1080/10635150701211844.

It's very much relevant to my own research. In this paper, Leschen and Buckley analyze the evolution of Erotylidae, a family of beetles only distantly related to the family I'm studying, and link their hyperdiversity to a shift to a diet of macrofungi. It sounds rather familiar, no? It's a very interesting read if you're into this sort of thing. It's based on morphology, not molecular biology, and finds a good link between diet and the external reproductive structure (gonocoxa and ovipositor) of Coleopteran females. Mycologists (at least those with an interest in basidiomycetes) and coleopterists alike will undoubtedly find something of interest in the paper. Enjoy.

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Neomida bicornis: in ur pycnoporus tunnelin ur context

While dissecting some galleried Pycnoporus cinnibarinus this morning, I found another teneb that I hadn't encountered before. It's a horned member of the tribe Diaperini called Neomida bicornis (more info here). It looks exactly like the photo shown here except, of course, much much smaller. The male I found was 3.25mm long, 1.75mm wide and 1.15mm deep. It's the first beetle in Tenebrionidae I've collected from a Pycnoporus. Previously, I've collected a few Anthribinae from that fungus. The sporocarp from which I collected this beetle came from a spot in Wachusett that I've been checking since April.

While examining the insect, taking measurements and such, I dropped it on the black-speckled linoleum floor. I then spent about 20 minutes crawling around looking for the thing. You have no idea how quickly a tiny, dark-colored beetle can disappear, and it's the only one I've recovered so far. Since it's in Diaperinae, is apparently a context tunneler and doesn't damage the hymenium, it may prove to be an important data point for my overall work. Next week, I'll sequence it and see if it has a mutation in common with it's relative, Diaperis maculata.

Now that I know where to find them, I'll keep going back to that spot for more specimens. As small as they are, it will probably take a few tries to get clean DNA sequences and build solid consensus contigs. We shall see.

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Gel of the Day: Half a Forked Fungus Beetle is Better Than None

Not a particularly successful PCR run today. For some reason, the ribosomal small subunit DNA didn't get amplified. I suspect that either I may have used an incorrect primer by mistake or else one of the pair may have gone bad. It happens. Luckily, I still have plenty of stock DNA and can have another crack at it. At least I got the large subunit and one of the cox1 genes from all of the specimens (the last eight wells are just a negative control; it's a good thing there's nothing there). I got nucLSU DNA, so it must be an error on my part that explains why the nucSSU didn't show up.

Oddly, the last two wells were taken from a 1:100 dilution and smeared; the previous two bands from a 1:50 dilution prepared from the same specimen didn't. Smearing usually indicates an excess of product. I'm not sure why that would have happened in this case. It's a duplicate, anyhow, so that's not important, just a bit strange.

The first sixteen wells in this gel are local specimens, collected at Wachusett Mountain. The third set of eight are from one of the specimens I picked up during the MSA foray in Pennsylvania last week. I have three more of those to sequence, too, once I get everything straight with these three.

As for tomorrow's field work, any B. cornutus I find will be shipped off live to Louisiana State for investigation of fungal symbionts in their gut. I don't really need to sequence anymore of them for the time being. Hopefully, I'll get lucky and find species other than this one and Diaperis maculata tomorrow. Those two seem to be dominant around here, but I'm going to try looking in a couple of different niches and see what turns up.

The North Florida field trip is two weeks from tomorrow. I know there will be more diversity down there, but I'd still like to come up with a few more local tenebs before winter sets in.

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No Wachusett Wednesday This Week

Due to inclement weather, this week's Wachusett Wednesday has been canceled. I am rather bummed about it. Instead of field work, I'll instead be purifying the PCR products from yesterday's gel and cleaning up/aligning the Sarcodon sequences, all of which is OK but hardly a substitute for being in the field.

In all likelihood, my next venture into the field will be on Sunday. That's the day of the Mycological Society of America foray at Lock Haven University. A lab and equipment is being graciously made available after the foray and throughout the duration of the conference, and I expect that I'll have wireless access at my hotel, so I'll be able to blog about the foray and provide photos and info about my collections as usual at that time.

sigh.

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Gel of the Day: Meat the Beetles!

This Gel of the Day comes with a dedication to the unnamed UC Santa Cruz researcher whose car was blown up on Saturday by fruitcakes who want to liberate Drosophila.

The Fab Four in this case are four specimens of Diaperis maculata; John, Paul and George were collected together from a decomposing Piptoporus betulinus fruiting body. Ringo joined them later, having been recovered from a Ganoderma applanatum sporocarp. In each of the bosex, the first four lanes represent a 1:50 dilution and the second four a 1:100 dilution of the initial DNA solution. Lanes 1, 5, 9, 13, 17, 21, 25 and 29 are the nucLSU; 2, 6, 10, 14, 18, 22, 26 and 30 are nucSSU; 3, 7, 11, 15, 19, 23, 27 and 31 are cox1 amplified using the general Coleopteran PAT/JER primer set; 4, 8, 12, 16, 20, 24, 28 and 32 were an attempt to amplify cox1 using a custom-designed primer set that works on the other Tenebrionidae on which I've used it but apparently not on D. maculata. This is the same result I got last when I tried a competitive test of the cox1 primer sets on several species which also included another member of the same subfamily.

This might be a useful phylogenetic signal; it could very well indicate a mutation in the binding site for the new primer set which is conserved in all the other Tenebs for which I could find sequences (I could find none for D. maculata when I designed the primers). Moreover, an alignment from my previous attempt came up with an insertion and two transitions for D. maculata at a binding site that wasn't present in the other tenebs. I can't tell yet whether this is a species-specific situation or if the mutations are present in all members of the genus. I'm hoping to collect specimens of D. maculata' cousin, D. nigronotata, in Florida next month. If I can get sequences out of it, I will be able to say that much more about the evolution of genus Diaperis and perhaps about tribe Diaperini as well. That is, if Big Coleopterists don't Expel me over raising the slight possibility that the tribe might not be monophyletic and if no wackadoos with IEDs decide to set me on fire in my sleep.

Have I mentioned lately how cool it is to find something that nobody (as far as I can find in the literature) has seen before? This is the good stuff right here. This is why I get up in the morning and slave over a hot thermocycler.

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Good Day with a Rough Start

This morning started off with a good deal of confusion over where to deposit my samples on a sequencer plate, but it all worked out in the end. I should be getting sequence reads back tomorrow that will tell me something about the identity of the two Sarcodon specimens I collected. I won't get to work on them until Thursday, though, as tomorrow is my day for field work.

I received the approved permits for the research sites in North Florida at which I and my colleague will be working on our upcoming Florida expedition. We've got everything set now — field sites, flights, a place to stay and a lab to work in. The only thing left to do is put together a schedule for when we'll be at specific sites, as the terms of the permit requires that we notify the state biologist two weeks in advance so that any necessary arrangements can be made.

I also received 25g of 2-sulfobenzaldehyde sodium salt when I came back from lunch. I've been waiting for that to show up. One of the compounds used in identifying fungi particularly russula, is sulfovanillin. It's a pain to work with as it can't be stored for long periods of time and mixing up a batch of the stuff requires messing about with sulfuric acid. SBA is a good substitute that stains gloeocystidia and can be stored and doesn't involve acid and fume hoods. I can't wait to try it out (yes, I know how geeky that sounds). Perhaps I'll have the chance when I get back to the lab tomorrow.

Finally, today is my kid sister's birthday. She turns 37, so I guess "kid" isn't really applicable. I did get her a present that replaces something of hers that I melted into little blue puddles when she was about 7, though. I hope she gets a chuckle out of it.

Not a bad day, all in all. I've got potential PCR product from four Diaperis maculatus on the thermocycler as I write this. It should be ready for electrophoresis at about 2:45 PM, so all (five of) you "gel of the day" fans might be in for a good one today. 32 samples comprised of large and small rDNA and two different cox1 segments may be ready before the day's out.

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Gel of the Day: Sarcodon what?

You might recall that I've twice now collected what I suspect to be a rarely-collected fungus, Sarcodon underwoodii, in the past few weeks. The first collection was made inside Worcester city limits on July 11 and the second time from the Wachusett State Reservation on July 17. I should mention that on my last trip to Wachusett all of the prospective S. underwoodii was gone and the more common S. imbricatum has replaced it entirely. The latter fungus has quite different coloration, the background being a red-brown instead of S. underwoodii's buff.

I've been trying to get enough DNA out of my collections to sequence and so make a positive identification so that I can let the people who manage the land on which I've made these collections know that they have an unusual species present. I tried earlier this week and got nothing. The context of the fungus is loaded with pigments that interfere with DNA amplification. I took a second pass at the problem, though, and let my samples digest in proteinase K for more than two hours during extraction. That seems to have done the trick. I've got PCR product galore and expect to sequence the nuclear ribosomal large subunit and ITS early next week. Here's the gel:


Lanes 3, 5 and 6 are from the fruiting body collected on July 11 and 7 through 12 are from the Wachusett specimen.

By middle of next week, I should know whether both of these really are S. underwoodii and increase what's known about the ecology of the two areas from which I collected them just a tiny bit.

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The Very Long Sunday

It's 9:35 PM as I write this. It's been a long day. My sequences, however, are ready to go on the plate in the morning. I hope it's worth it. If I'm really lucky, I may find a mutation at the primer binding site of one or two species under tribe Diaperini when I get the reads back on Tuesday. If there is one there, it could turn out to be a very useful character in reconstructing Tenebrionidae phylogeny.

Or it could all be pure chance.

Like a slightly schizophrenic ex-girlfriend of mine once told me on an evening when her pupils were the size of basketballs, "You never know until you find out."

Me, I've got a scotch and soda in one hand and a remote control in the other. Time to turn the brain off for the night.

My day ended with an act of great generosity being made toward me. I ran out of cigarettes hours earlier but I was too busy with DNA-related stuff to leave and get some. On my way out the door, I bumped into CaKh, the Argentinian entomologist/biogeographer. He insisted on giving me 3/4 of a pack of Marlboros for the ride home. That might not be a big deal in some places, but in Massachusetts I believe that qualifies as a dowry.

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Gel of the Day: Moderate Success

Sound the trumpets! I actually got some PCR products out of today's experiment with COI primers.

It looks from the results like both primer sets work, although my new Tenebrionidae-specific primers (PATten and JERten) amplifies a smaller fragment than the traditional Coleoptera COI primers. Also, it seems that the traditional primer set worked for one species but not for another in the same tribe (Diaperini), and the new primers worked for the other member of that tribe (both are Diaperini; the new primers worked on Pentaphyllus but not Diaperis and the old ones on Diaperis but not Pentaphyllus). Why? I dunno. I still have my suspicions about how monophyletic tribes, and even subfamilies, really are in this family, and maybe that's part of what I'm seeing in these results. I may know more once I try aligning these sequences and especially when I add this data together with the large and small subunit stuff.

Otherwise, both primer sets worked on everything except one miss by the new set on a single Bolitotherus cornutus, but it worked on the other specimens of the same species, so I really have no idea with that one.

Still, here's the gel. There are a couple of faint bands in the lower half that may be hard to see in the reduced photo, but they're in there. Hopefully I'll still be able to get sequences out of those:

I used several different dilutions (1:100, 1:10, 1:5) and got some products from all of them. It all comes down to using a lower annealing temperature for the COI stuff. From here on in, I should be able to get those sequences reliably.

Now I'm just hanging around and waiting for my sequencing reactions to finish up, which they should do in another 2.5 hours. Thankfully, that Del Tenney DVD I have is a double feature; I can watch Curse of the Living Corpse while I'm waiting. I'll be here late on a Sunday, but I should be ready to go when tomorrow's sequencer run starts up at 11:00 AM. Go, me.

If anybody wants me, I'll be in the conference room.

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Amplify Naked: In the Lab on a Sunday

I'm here at the lab this afternoon. See?

You can tell it's a lab because there are microscopes and other sciencey-looking things in the picture.

It being Sunday, I'm the only one in the lab, which is a bit lonely but also very nice. During the week, we have an undergrad here on a summer project in which he's working under the supervision of a post-doc to sequence a whole bunch of Boletales specimens from another university to make sure that they're actually what they're supposed to be. Most type specimens were collected before the days of molecular analysis and so in many cases nobody is as sure of their identities as they could be. It's a worthwhile project, but with everybody else doing their molecular thing, too, the clean lab is often at capacity. I have a sizable experiment I'm working on, testing a couple of primer sets for my so far-elusive cytochrome oxidase subunit 1 genes under a new protocol on the thermocycler. It's not that complicated a project, really, but getting it all set up requires a bit more physical room than is usually available. I'm just now waiting for the cycler to finish before I run a gel and hope that I actually get some bands. Then I'll be able to see if one of the primer set works better than the other, if both sets work well and the difficulty has been all about the annealing temperature, or if the whole thing just isn't going to work for some reason. I suspect the combination of a new, family-specific primer set and a different thermocycler regimen will solve the problem. We'll see.

Because there's nobody else here today, though, I also got to watch an old Del Tenney drive-in flick after getting the thermocycler set up. I watched the amazingly goofy Horror of Party Beach in a conference room. There's something particularly gratifying about kicking one's feet up and watching a cheezy flick about radioactive jellyfish-zombie-monsters that eat only nubile young beach party girls and the scientists trying to devise ways to destroy them when one is sitting in a biological science center and waiting for PCR products to be amplified. You can always tell the scientist in a 1960's rubber suit horror flick; he's the guy in the white coat, smoking a pipe and saying things like, "The organs of these beings are too decomposed to retain oxygen so they've been replaced by sea anemones. They're giant protozoa!" Errrr... yeah. I hope I don't give an answer like that when I take my quals.

I did want to get this experiment run today for another reason, too. With LL coming home, I won't be here most of next week. If I get everything ready for the sequencer by the end of the day today, I can have it on a plate tomorrow and get my sequences back on Tuesday. Or I can have it ready for the plate on Tuesday and then download them and toss them into an alignment next Monday. Either way works, really.

34 more minutes left on the thermocycler now. You know, if I'd have known I'd be the only person here today, I'd have amplified naked.

Nobody touch that line. It's going on a t-shirt.

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Mystery Mushroom: Evolutionary Theory in Action

You might remember that a few days ago I collected some mystery mushrooms that I couldn’t identify based solely on macroscopic and microscopic characteristics. This morning, I got my rDNA sequences back and I’m happy to report that I have now been able to identify the fungus based on molecular characters. This is evolutionary theory put to practical application. Waddya know, this evolution stuff really works!

Here’s my final phylogenetic tree (although I could have collapsed the weakly-supported nodes if I'd wanted):


I produced this by matching my sequence against highly similar sequences in BLAST. I aligned these using MAFFT and then cleaned up the alignment in MacClade. The cladogram shown is a neighbor-joining tree created in MAFFT with branch lengths proportionate to phylogenetic distance. Bootstrap support is shown at all nodes and is the result of 500 replications. The taxon highlighted in red is my mystery mushroom and the clade containing its closest relatives is outlined in green.

As you can see, the unknown mushroom is clearly of the genus Gymnopus and is closest to, but not identical with, Gymnopus brunneigracilis. It may be that the mystery mushroom has not been sequenced before or I might need more characters to correctly identify it to species. Nonetheless, I do now know its genus and a photograph of one of its close relatives, G. luxurians, bears a striking resemblance. Moreover, the morphology and growth habits of G. luxurians are extremely similar to those of the mushroom I collected.

Imagine for a moment that this wasn’t a mushroom I found growing in wood chips. If this had been a mysterious fungus growing out of Aunt Tilly’s ear, I would have used the exact same techniques to diagnose our (thankfully) pretend parasite. None of this would have been possible without the principles of evolutionary theory, which informed every step of the process.

Of course, if you would like to try to identify the source of my sequence without resorting to evolutionary theory, by all means feel free. Here’s a link to the actual sequence from the fungus. I would be very interested in hearing all about how you made sense of it without involving mutations or knowing the genus and its relationship to other genera sharing a common ancestor.

Or, if you feel like trying your hand at this sort of thing, you can see pretty much the same things that I saw on the way to this result by using the links provided. If you don't feel like buying a copy of MacClade, you can substitute the freeware program BioEdit. It's all evolutionary theory in action embodied in software that makes life a lot easier than it used to be in the days of dot-plots and slide rules!

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Gel of the Day: Mystery Mushroom

One of my lablings pointed out a cluster of mushrooms growing near the loading dock attached to our building the other day. Despite macroscopic and microscopic examination, we've been unable to identify it satisfactorily even to genus. The fact that it wasn't producing enough spores to obtain a print didn't help, and the few spores (fewer than two dozen) that I could find on the lamellae all had the same shape but their size and color varied. Some are colorless (hyaline), some are brown.

The gel above shows PCR products amplified from a small bit of the mushroom after drying. The first two bands are ITS and the second two are large subunit rDNA. Now that I have DNA, I can get the sequence and match it to known sequences. If anyone has sequenced this fungus before, we can identify it via molecular biology. If there's no exact match, the game still isn't over. Thanks to evolution, the sequences will still be closer to some fungi than others. If need be, I can do a quick alignment of similar sequences and then use phylogenetic software to determine to what the specimen is closest. With that much information, there's a very good chance it will be identified to species.

Next time someone tells you that they don't know anything practical that's based on evolutionary principles, ask them how they would have solved a problem like this if the fungus were eating the beams that support their house.

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Reasons to Be Cheerful: DNA, Expeditions and Mycological Society Meeting

It's the little things in life sometimes that bring the greatest pleasures... at least when one revels in being a geek.

The first thing I need to do today is to clean up some DNA I have waiting in the freezer. Among them are the rDNA sequences from Diaperis maculata. Once I've cleaned the DNA I'll be able to load it onto a plate and will get the sequences back and be able to add them to my alignments. I'll know a tiny bit more about the evolution of fungus-associated Coleoptera. This is enough to make me look forward to a Monday. It's the little things... and D. maculata is only 5-6 mm long. That qualifies as "little."

I also have to start work on securing permits for the collecting expedition in September. Two months should be enough lead time, hypothetically, but one never knows with bureaucracies. We just need a couple of good sites to work at, though. I'll probably put feelers out on more than we'll actually use. That way if the process gets too long and we get turned down for a couple of sites at the last minute, we'll still have a couple of back-ups. Better to have it and not need it than to need it and not have it.

Speaking of science-related travel, it's only a month until I hit the road for Penn State and the annual Mycological Society of America meeting. It's the largest gathering of professional and amateur mycologists in the nation and promises to be a great time. I'm looking forward to the whole program, but I have to admit that I'm particularly tickled by the opportunity to collect in Pennsylvania, a state in which I've never forayed before. I'm even hauling my industrial-strength dehydrator on the trip so that folks can preserve specimens. The abstracts look like there will be some very interesting talks on fungal evolution and informatics tools. Folks from my lab are doing four of the presentations, I think. I'm not one of them yet (I've just started gathering data!) There are also a couple of talks by David Maddison that look like they'll be particularly interesting stuff. Days of full immersion... I can hardly wait.

Are any readers planning to attend MSA this year? If so, I'd love to meet up for a drink or two.

If you're new to this stuff and this is the first you're hearing about it and you'd like to attend, you can still register for the meeting. Even if you're a novice, you couldn't ask for a better opportunity to learn about fungi. Please consider joining us! If you're a newbie and can make it in time for the foray on August 10, I'd be more than happy to hang out with you in the field and help you get better acquainted with the various chitinous life-forms you'll encounter. I'd consider it a privilege to do so, in fact.

Today, and the next couple of months, are going to be great. Wonder-Geek Powers Activate!

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Gel of the Day: Ambitious but Only Slightly Successful

I was feeling a bit ambitious today; my new COI primers arrived. They didn't work, but I did try them. Amplifying COI is turning out to be a bit tricky. I'm going to have to do some digging on this problem.

The mystery COIs from last time are still more or less a mystery... but they are at least Coleoptera and they are cytochrome oxidases. That much is good. There's nothing with better than 86% identity to them on GenBank, though, including what is supposed to be a Penthe species; I have my doubts as to whether that sequence is actually from that genus.

Anyhow, today's gel:


007 is my putative Pentaphyllus. I tried to amplify lsu and ssu from it. Didn't work. Neither did the same genes from Penthe pimelia (004). 009 and 010 are a pair of Bolitotherus cornutus; I got lsu and ssu from them but no COI. Why? I don't know. For some reason, I can't get any ribosomal sequences out of 007 and 004, only COI... and I can't get COI out of B. cornutus, even though they should all have the same conserved sequences. 015 is a new addition, Diaperis maculatus, a teneb from the subfamily Diaperinae. I only got the ribosomal sequences out of that one, too. Why? I don't know.

I'm neither entirely happy nor entirely unhappy with these products. I at least get to add some more data to my set, and that's something, anyhow. I'll clean and sequence these on Monday and chuck 'em into my alignment. I do wish I had gotten the Penthe and Pentaphyllus rDNA, though. It would have cleared up a couple of questions I still have about these two insects. I may be able to do another extraction from Penthe; it's a big bug and there's still enough left of my one specimen to try one more time. My Pentaphyllus stock is used up, though. They're tiny insects, 3-4 mm long, and I can only use heads and pronota for extraction to avoid contamination by gut symbionts. It's a bit like trying to suck the juice out of a pinhead.

Think I'll go have a nice Scotch now.

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Gel of the Day: ...and Then a Miracle Happens

If you're the kind of person who reads this blog then you have probably seen the cartoon with two physicists standing in front of a chalkboard. In the middle of a bunch of equations are the words "and then a miracle occurs" and one of the physicist is saying "I think you should be more explicit in step 2."

Today I had a similar thing happen with one of my gels:


The first two are DNA from taxa that didn't come out last time I tried. The last group of four is a positive control. I used stock DNA and the same primers for all. Now, here's the really weird part: lanes 4 and 8 shouldn't have a product in them at all. No bands should be there. They're the products of a cytochrome oxidase I primer that I discovered today should not work. The primer I was given is for the wrong beetle family; the one I'm studying is too divergent. I was just investigating the problem this morning, in fact, right after I put these samples in the thermocycler. I wound up designing two new Tenebrionidae-specific COI primers and sending an email off to two of my committee members telling them that I'd done so.

So, of course, the primers that have never worked before, and shouldn't have worked this time, are the only ones that produced bands in the first two taxa... but NOT in the positive control. The only band I got in that one is for the small subunit. I should have one for the large subunit as well. It worked last time using the same dilution and primers.

I would just like to say "Argh." Really, what else can I say?

Now, it may be that I used the wrong primers. I've been very out of sorts after not getting enough sleep last night and I could have screwed up. Still, the way I load my tubes, I should have gotten the same result in the positive control (#001) if that were the case. But I didn't.

Tomorrow, I'll clean the PCR products and get them ready for sequencing using the old COI primers. My bet is that I'll get back nothing. And then I'll just start all over again. Maybe someone tampered with the annealing temperature on the thermocycler or something.

I dunno.

I need a drink.

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