All posts by jennomics

About jennomics

I am a Postdoc in Jonathan Eisen's Lab at UC Davis. jennomics@gmail.com

What the fungi do I do with my ITS library? (Part 2)

What the fungi do I do with my ITS library (Part 2)
Originally posted on jennomics.com on May 22, 2014

Previously, I expressed some concern about size variation in my environmental fungal ITS PCR libraries. I’m still concerned about that, but I have an additional concern. The ITS region can’t be aligned, and I’m partial to phylogenetic approaches to pretty much everything. So maybe ITS is not for me?

So, I asked Twitter again…

In summary, I don’t think that I can use ITS given the size variation that I see, and I’m not sure that I want to, given the fact that you cannot align it to do phylogeny-based analyses.

28S (or LSU) is a reasonable alternative to ITS that has two big downsides: 1) the reference database is much smaller than the ITS reference database and 2) it does not provide the fine-scale taxonomic resolution that ITS does.

Rachel Adams referred me to Amend et al, in which they use both. I’ll have to look into this approach…

Seagrass Microbe Sampling Kit

Here’s the version of the sampling kit that’s being sent to 25 ZEN partner sites throughout the world:

Photo: Madison Dunitz
Photo: Madison Dunitz

Each ZEN partner (site) is working in two Zostera marina beds (subsites.) At each subsite, the partners will take three water, sediment, and plant (root+leaf tissue) samples.

On the left, is the AeroPress coffee maker that will function as a water filtration device. The plunger is hollow, so inside of that, I put plastic forceps and 6 gloves. A piece of tape over the top will make sure that it doesn’t all slide out.

On the right is a 500mL plastic bottle for water collection.

In the middle is a plastic, hinged tube storage box. There are 30 2mL tubes inside. The tops of the tubes are pre-labeled:

Lavender: R1.1, R1.2, R1.3 for the three root collections at subsite 1.

Lavender: R2.1, R2.2, R2.3 for the three root collections at subsite 2.

Green: L1.1, L1.2, L1.3 for the three leaf collections at subsite 1.

Green: L2.1, L2.2, L2.3 for the three root collections at subsite 2.

Orange: S1.1, S1.2, S1.3 for the three sediment collections at subsite 1.

Orange: S2.1, S2.2, S2.3 for the three sediment collections at subsite 2.

Blue (2 of each): W1.1, W1.2, W1.3 for the three water collections at subsite 1.

Blue (2 of each): W2.1, W2.2, W2.3 for the three water collections at subsite 2.

There are two blue tubes for each water collection because the filter is to be cut in half and put into two tubes. There are also 30 Tough Tag labels for the sides of the tubes that are to be filled out by the ZEN partners with their site ID, subsite ID, plot ID, and date.

Also, six 0.22micron filters that have been LASER cut to fit the AeroPress, a small pair of scissors for cutting leaves and roots and water filters, a 6mL syringe (top cut off) for taking sediment cores, plastic spatulas for scooping a little bit of sediment out of the corer, a Sharpie, 24 individually-wrapped alcohol swabs, and the 3D-printed stand for the Aeropress.

Pretty cool, huh? Hope I didn’t forget anything!!

I will update with the sampling protocol when it has been finalized.

3D printed AeroPress base

While in Cocoa Beach, waiting for the rocket to launch, we decided to test out Russell Neches’s idea for filtering seawater with an AeroPress coffee maker. We were also shooting the instructional video for the Seagrass Microbiome sampling kits that include the AeroPress for this purpose.

Forcing water through a 0.2 micron filter with the AeroPress was easier than I thought it would be, but pretty awkward. It would have been much easier to put that thing over something like – oh I don’t know – a coffee cup to hold it in place while pressing down. I’d decided that if I put a 500mL wide-mouth plastic collection bottle (for the seawater collections) in the kit, that they might also be able to use the bottle for that purpose. But, I was having a hard time figuring out if the mouth of the bottle would be wide enough. So, I went next door to complain to Russell Neches and David Coil. And David said, “you should just have Russell 3D print something,” and Russell said, “Sure, I can do that!” and then 30 minutes later an email had been sent to Madison Dunitz, an undergrad in the lab, and within a couple of hours, it’s done! It’s really simple, just a ring that serves the same purpose as the rim of the coffee cup, with channels in the sides for the water to go through, that is just tall enough to keep the bottom of the AeroPress off the table (or whatever surface.)

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Visit to the Smithsonian Marine Station

Armed with some instructions for where to find a few different seagrass species and a couple of names to ask for, I headed to the Smithsonian Marine Station (SMS) in Fort Pierce, FL on Thursday, April 17th.

The SMS was kind of deserted, but there was a behind the scenes tour scheduled for 2pm, so I waited around for that. In the meantime, I went to meet with Bill Hoffman, the Exhibit Manager at the St. Lucie County Aquarium, across the street from the SMS. He shared with me his experience with setting seagrasses up in his aquarium there. He said that at first, he would try to lay a patch of seagrass, with surrounding sediment into the tanks (like sod,) but that they kept dying. Eventually, he had success when he planted the plants into the tanks that contained the dead “sod.” But still, not every species he tried will grow in the tanks.

Bill loaned me some snorkeling gear so that I could go hunt down the seagrass beds that Robert Virnstein described to me. My only experience of seagrass is the Zostera marina at Bodega Bay, so I was interested in laying eyes on some other species. I found the dense Syringodium bed next to the boat ramp at the end of the Aquarium’s parking lot. It was remarkable to me how variable the density of both the seagrass as well as the algae were over a really small area. Sometime, the algae was so abundant and dense that I couldn’t even see the seagrasses. Other times, it was easy to see individual seagrass shoots poking out in the sandy sediment. Syringodium has cylindrical leaves, so that was different. The leaves were only about 8 inches long, and the rhizomes were very delicate compared to Z. marina.

Then, I went over to Chuck’s Steak House. There, I found a mixed bed with two kinds; I think Halophila and Halodule. They were about the same size, but their leaves were shaped differently. These were remarkably tiny! I will definitely have to re-write the sampling protocol for these.

The three species I found in Indian River Lagoon. My guess is Top: Halodule, Right: Syringodium, Bottom: Halophila
The three species I found in Indian River Lagoon. My guess is Top: Halodule, Right: Syringodium, Bottom: Halophila

After my little snorkeling adventure, I went to the SMS “Behind the Scenes” tour. I learned a lot about the Indian River Lagoon, which has significant temperature and salinity gradients, making it an incredibly biodiverse ecosystem. The tour was useful and interesting. Especially, the Mangrove experiments – couldn’t help but wonder about the phylosphere microbial communities on these salty leaves.

Baby Mangroves! Some deal with salt by excreting it from the leaf surface, others have specialized salt excretion nodules at the base of the leaves.
Baby Mangroves! Some deal with salt by excreting it from the leaf surface, others have specialized salt excretion nodules at the base of the leaves.

Eventually, I connected with Niclas Engene, who is interested in collaborating with us on a GoLife proposal to do Cyanobacterial genome sequencing/phylogenetics. I also chatted with a few people, including Jenny Sneed about which would be the best primers to use for algal diversity surveys. No one seemed to know, but at least I’m not alone in my uncertainty. I gather that the person I really needed to talk to is Justin Campbell, but he was probably on a plane coming back from Belize while I was there. Oh well.

Seagrass Phylogeny

We need a comprehensive phylogeny that includes all of the seagrasses. All of the seagrass lineages are within the Order Alismatales. The best available Alismatales phylogeny only resolves lineages to genus, and uses a combination of morphological characters and rbcL. So, here’s what I did to produce a phylogeny of all available Alismatales with rbcL:

1. Search the NCBI Nucleotide database for “rbcl” and “partial cds”

2. Use the Taxonomic Groups filter (box on the right side of the results page) to get only the Alismatales

3. Export (Send to > file > FASTA) the 1649 Alismatales sequences in fasta format

4. Fix the sequence identifiers.

5. Align those sequences with Muscle.

6. Build tree with FastTree.

Then, I had to do a lot of manual editing to 1) highlight the seagrass species, 2) remove some matK sequences that made their way in there somehow, 3) de-replicate redundant species (when they formed monophyletic groups), and 4) reduce the tree to the smallest monophyletic clade that contained all of the seagrasses.

I would not use this tree in an analysis or publication, but for our current needs, I think this will be sufficient. Producing a high-quality, comprehensive phylogeny of the monophyletic clade that contains all seagrasses is going to be a big job.

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When life gives you a failed rocket lauch…

Make fieldwork!

I was in the Cape Canaveral area to view our Space Microbes getting launched to the ISS this past Monday, April 14th. Unfortunately, the launch was canceled due to a helium leak in the SpaceX Falcon 9 rocket. The next launch was scheduled for Friday the 18th, so we decided to stick around and wait for it. Jonathan suggested that I rent a car and head down to the Smithsonian Marine Station in Fort Pierce.

That’s where Valerie Paul works, and we’ve been talking to her about incorporating a microbial sampling component in the Thallassia Experimental Network that she’s currently setting up. This is modeled after ZEN, and they will be doing experiments in which they will be modifying grazing and nutrient levels at four sites (Indian River Lagoon, FL Keys, Carrie Bow Bay in Belize, and Bocas del Toro in Panama.) Unfortunately, Valerie is in Belize right now, so I scrambled to find other contacts in the area, or at least get some tips about where I could find some seagrasses. I decided to post some of the information I got here.

First, from Jay Stachowicz:

“I checked out google earth and it looks like there might be some patchy seagrass beds just north of the Cocoa Beach causeway that you might be able to see from the beach or walk out to if the water is super shallow if you are feeling adventurous. Looking on google earth, the large dark areas look to me like deep water and that there are some small greenish dots that look like clumps of seagrass potentially.”

Cool! I would NOT have thought to use Google Earth for this purpose.

Then, Pamela Reynolds suggested that I contact Erik Sotka (who I’d met at the ZEN pertners meeting in March) and he wrote:

I know a few seagrass beds in the Indian River lagoon (south of Cape Canaveral). I’ve collected amphipods from seagrass beds at the park across the road from the Smithsonian Marine Station. That’s a good place to start. Another place with accessible seagrass is at the Jaycee Park boat launch on the outer barrier island near Fort Pierce. Val (and some of her postdocs and staff) will know other accessible locations. Other seagrass specialists in Indian River Lagoon to ask:
– at harbor branch marine institute (~20 miles from SMS), you can try to contact Dennis Hanisak
– Robert Virnstein, Seagrass Ecosystems Analysts, seagrass3@gmail.com (386-546-0204)

Also, see some other contacts within this PDF of abstracts from a recent conference (http://www.unf.edu/uploadedFiles/aa/coas/biology/BENTHIC/BEM2014OralAbstracts.pdf). Download and search for “Indian River”

as a side note, if you want to goto Tampa Bay, I can point you to good, walkable sites for seagrass.
good luck seagrass-hunter.

Awesome! So, I contacted Dennis and Robert and (per Jonathan’s suggestion) a postdoc of Valerie’s (Justin Campbell).

I got this from Robert Virnstein:

Attached is a map with some labels.

At the “SI” site, where the Smithsonian Marine Ecosystems Exhibit is, there are grassbeds that extend about 40 m offshore. Most/All will be Halodule wrightii. You should be able to wade most of it. The Smithsonian lab/office is 250 yards east of the exhibit. And 560 yards east of the exhibit is Chuck’s Steak House (“SI2”), the first building on the left. From the restaurant parking lot should be better grassbeds. On the flats look for Halophila johnsonii. It might be only 1-2 cm tall, so look closely.

On the North Causeway (A1A North) stop at the first park/boat ramp you come to. There may be some grass there. For really good grassbeds, scramble south through the mangroves. There is a huge flat that has Halodule, Syringodium filiforme, and some Thalassia testudinum if you look hard, but you might have to wade/swim south about 100 m. Look at the Google Earth imagery and go back one time step to 2010.
Or you can swim straight out from the boat ramp, across the narrow boat channel, and 50 m from the boat ramp you should find dense grass beds, dominated by Syringodium.

At the “3a” and “3b” sites, if you can get south of the marina onto flats, I feel confident you will find Halophila johnsonii, a federally threatened species listed under the Endangered Species Act.

Or, if you go to the SI lab/office and can find Sherry Reed or Woody Lee, they have worked there for decades and really know the Lagoon. They know where everything is. Tell them I said hi.

SMS_seagrasses

Are you kidding me right now? How amazingly helpful everyone has been! So, tomorrow, I’ll head over to the SMS and try to find Sherry and Woody. Hopefully, they can lend me some snorkeling gear so I can go check out all of these sites. Hooray for fieldwork!