Tag Archives: Sampling

CA sampling for project phylogeny

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: PotamogetonaceaeAlismataceae, 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 (MyriophyllumCeratophyllum, 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.
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!

Sample preservation experiment

During ZEN DNA extractions we noticed that samples preserved in the Zymo buffer were forming a precipitate with the C1 solution from the MoBio kit. Furthermore, many of the samples also resulted in very low DNA yields, perhaps correlated with the precipitate formation. Any microbiologist will tell you that there are many different ways to preserve samples from the field, but there does not seem to be a universal *best* method.

We decided the best way to approach this problem for the Seagrass Microbiome project was to explore a variety of sample preservation methods and see which approximated the ‘real’ microbiome best (as measured by preservation on dry ice). We chose the following methods to try: Dry ice, Zymo, RNA-later, Drierite, and Ethanol. We also wanted to investigate how different preservation methods performed over time. We chose 4 time points post sampling for our extractions: 24 hours, 1 week, 2 weeks, and 1 month.

We drove to Putah Creek in Winters in search of submerged aquatic plants in the Alismatales (the order that contains the seagrasses). Here is a photo of our study site: 20150611_133052

We found a bed of Elodea canadensis growing near the shore and started sampling.

1460273_10205878523255831_7049527570566647813_n

We pulled out whole plants and divided them into root and leaf for each of our trial preservation methods, with 3 replicates per method per time point. We extracted DNA at each of the different time points, and did PCR of the 16S SSU rRNA gene, with subsequent library preparation and sequencing on the Illumina Mi-Seq. We used QIIME to analyze the sequence data and compare species diversity between our preservation methods.

PCA
Weighted unifrac PCoA

Our PCoA plot shows that preservation in Zymo best approximates the community captured using dry ice (our control), and that Drierite and Ethanol were both very different from anything else. RNA-later appears close to Zymo and dry ice, but we only have 5 data points for it. For the RNA-later and Ethanol extractions, we extracted directly from the solution without pelleting, which may have affected our end result.

Taxonomy summary plot: Order level
Taxonomy summary plot: Order level

The taxonomy table supports the trend seen in our PCoA plot, with Drierite and Ethanol being very different, and Zymo, dry ice, and our 5 RNA-later time points showing similar communities.

Looking at variation in community structure across time points within Zymo we see little to no change.

Weighted unifrac PCoA showing time point variation within Zymo
Weighted unifrac PCoA showing time point variation within Zymo

However, we see some change in community structure by the month mark with dry ice.

Weighted unifrac PCoA showing time point variation within dry ice
Weighted unifrac PCoA showing time point variation within dry ice

Take home messages from our sample preservation experiment:

  • Using Drierite as a preservation technique does not capture the community assembly well.
  • RNA-later and Ethanol could have had better success with pelleting and removing the supernatant prior to extraction. We may investigate this further in the future.
  • In spite of our initial concerns regarding precipitate forming in the Zymo buffer, Zymo is the clear winner in our trial.
  • There are slight changes in the community assembly by the month mark, in all methods (depending on whether you use weighted or unweighted unifrac PCoA).
  • Due to the current ease of sampling using dry ice, we will continue this method except in situations where samples must be kept at room temperature (we will then use Zymo).

20150902_072734

HAPPY SAMPLING 🙂 Please see https://seagrassmicrobiome.org/sample-collection-and-preservation/ for more details regarding sampling and preservation protocols.