Category Archives: Plant phylogeny

Adventures in SEM

April 14, 2016Plant phylogeny, Project Updateslauraevann

SEM demo room @ UCDavis. Hitachi TableTop is the large rectangular machine on the left.

Some of us at team Seagrass participated in a workshop on SEM (scanning electron microscopy) sponsored by the Electron Microscopy Lab at UC Davis. For this week only Hitachi Tabletop SEM was made available to researchers to test out. SEM is a great tool to produce high resolution images, especially for samples for which preparation techniques would otherwise alter the sample. We decided this would be a great opportunity to visually explore microbial diversity on seagrass roots/leaves/rhizomes. The amount of sample and preparation is minimal relative to other techniques. We’ve been particularly concerned with our previous FISH images, as FISH requires many washes, and may be removing microbes from the surface of our samples.

We put a minimal amount of sample of root (top), fresh leaf (right), decaying leaf (bottom), and rhizome cross section (left) on a piece of carbon conductive paper (sticky on both sides), which was stuck on top of the SEM sample mount. We then inserted the mount into the SEM and turned on the vacuum. The vacuum for this particular SEM is actually a partial vacuum, which maintains a small amount of air molecules within the chamber, which produces a better image in the absence of coating your sample with conductors.

The results were great! We were able to see a lot of diatoms spread out across the leaf surface, as well as some interesting plaque (?) formations on the root tips. We also noticed differences in diatom abundance between the live and decaying leaves, with the live leaf being completely covered with diatoms. Diatoms are marine microbial eukaryotes (Heterokonts) that form silica based outer layers, often resembling complex geometric patterns. Diatom assemblages have previously been characterized in Thalassia testudinum and in Zostera marina. We’re excited to use the SEM to explore microbial diversity on additional seagrass species and freshwater relatives within the Alismatales. Hopefully these results can help inform our future culturing experiments.

CA sampling for project phylogeny

October 2, 2015Field Work, Plant phylogeny, Project Updates, Uncategorizedfieldwork, project phylogeny, Samplinglauraevann

A few of us at Team Seagrass have been doing fieldwork in Northern CA in order to collect seagrass relatives. We have specifically been targeting freshwater species (highlighted in blue in the below phylogeny). We will continue with marine and brackish species when the tides in the San Francisco Bay are lower.

From https://phylogenomics.wordpress.com/people/post-docs/jenna-lang/seagrass/photo-4/: This is a phylogeny of the seagrasses and their aquatic relatives. This tree was built using parsimony and ~1200bp alignment of the rbcL gene. From Les and Cleland, 1997. — From https://phylogenomics.wordpress.com/people/post-docs/jenna-lang/seagrass/photo-4/: This is a phylogeny of the Alismatales, including the seagrasses and their aquatic relatives. This tree was built using parsimony and ~1200bp alignment of the rbcL gene. From Les and Cleland, 1997.

We have been following the methods detailed in https://seagrassmicrobiome.org/sample-collection-and-preservation/ and have managed to collect a few representative species from the following clades: Potamogetonaceae, Alismataceae, Hydrocharitaceae, Najadaceae, and the Lilaceae.

crap.001 — Example leaf and root from Alisma species, collected at Cosumnes River

For further plant identification and the construction of a ‘host phylogeny’ we will be using chloroplast DNA markers.

In addition to what we have sampled thus far, we have also managed to sample some outgroups (Myriophyllum, Ceratophyllum, and Camboba). We will hopefully be able to sample additional outgroup species shown on the above tree.

We have been successful in 7 out of the 10 locations that we have tried. The lack of success so far can be attributed to drought conditions in CA and the increase in invasive submerged aquatic vegetation (e.g. Myriophyllum, Water Milfoil; and Eichhornia crassipes, Water Hyacinth). Water Milfoil and Water Hyacinth form dense mats below and above water, respectively, outcompeting native vegetation. Even in sites where we been successful, much of the area we surveyed has been overrun with these two invasive species.

Obviously drought has also been an issue for us, as many of the lakes and tributaries have little to no water in them this year, resulting in a lack of any aquatic vegetation. Water levels in Folsom Lake, for example, are so far reduced that the Park Service has had to build additional parking lots on the water side of the boat ramp in order for people to be able to get close to the new shoreline.

Map of Folsom Lake, with parking instructions drawn on by park official. X marks location of new parking lot, which is still at 15 minute walk to the shoreline.

In the following weeks we will head up to the foothills in search of remaining freshwater species, as well as explore the Bay Area coastline and salt marsh/Delta area for marine and brackish species. Stay tuned!

Species to target for evolutionary analysis

February 21, 2015Field Work, Microbiome Evolution, Plant phylogenyjennomics

Right now, we have a lot of Zostera marina microbiome samples from around the world. So, pairing that with the ZEN data, we should have a pretty nice ecological/biogeographical story, and hopefully we will soon have a postdoc to help us address questions about community assembly with those data.

Now, my attention is turning more earnestly towards the big evolutionary questions, and how to obtain the data we need to answer those. For the most part, because we are plugged in to a nice network of seagrass researchers, I don’t feel like getting all of the seagrass species that we’ll need is going to be too difficult. However, we are kinda lost when it comes to the fresh and brackish water and terrestrial relatives. I wouldn’t say that I’m panicking about it yet, but I am starting to feel like the right way to tackle the problem of collecting those samples is going to be to do it myself. In order to ask for help in collecting those samples, I’d have to:

1. Make a list of target species.

2. Find out their ranges and who is likely to have access to samples.

3. Contact the person/s who might be willing to go grab a sample for me.

4. Wait (hope) for that person to get back to me.

5. Send sampling supplies, and hope that they will be able to freeze samples for me (because the Zymo buffer is pretty sucky.)

6. Wait for the samples to be collected and returned to me.

The problem is that doing this for all of the target species could take forever. I have control over 1-3, but absolutely none over 4-6.

In the past, I’ve had success taking epic road trips and meeting researchers along the way who were willing to help me collect local species. So, I’m thinking that an approach like that might work here.

Here’s a list of the taxa of which I’d like to have representatives, and a tree of them below. I’ll start compiling range and contact information.

Potamogetonaceae
-Groenlandia
-Potamogeton

Zannichelliaceae
-Zannichellia
-Lepilaena

Zosteraceae
-Zostera
-Phyllospadix
-Heterozostera

Cymodoceaceae
-Thalassodendron
-Amphibolis
-Cymodocea
-Sryingodium
-Halodule

Ruppiaceae
-Ruppia

Posidonaeceae
-Posidonia

Lilaeaceae
-Lilaea

Juncaginaceae
-Triglochin
-Cycnogeton

Aponogetonaceae
-Aponogeton

Scheuchzeriaceae
-Scheuchzeria

Najadaceae
-Najas

Hydrocharitaceae
-Thalassia
-Enhalus
-Halophila
-Vallisneria
-Nechamandra
-Hydrilla
-Blyxa
-Ottelia
-Elodea
-Apalanthe
-Lagarosiphon
-Statiotes
-Hydrocharis
-Limnobium

Butomaceae
-Butomas

Limnocharitaceae
-Limnocharis
-Hydrocloys

Alismataceae
-Alisma
-Baidellia (or Baldellia)
-Damasonium
-Luronium
-Ranalisma
-Sagittaria
-Wiesneria
-Echinodorus

OUTGROUP:

Araceae/Lemnaceae
-Lasia
-Xanthosma
-Montrichardia
-Ariopsis
-Pistia
-Lemna
-Gymnostachys
-Symplocarpus

Acoraceae
-Acorus

Seagrass Phylogeny

April 24, 2014Plant phylogenybioinformatics, Order Alismatales, rbcL phylogenyjennomics

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.