New phytoplankton closed reference database

One of Jonah’s advances in helping clients with metabarcoding was to develop a closed reference database for higher plants to aid in long-term continuity of data. In essence, we permanently assign a consensus sequence to a unique OTU ID. That way, we have continuity over time in what species is/are represented by a particular OTU. When a client comes back a year later and has more diet samples to analyze, we can sequence the samples, match the sequences to our closed reference database, and then seamlessly compare the output over time.

This week we are beta-testing a new closed reference database. This time, for phytoplankton using the 23S region (Sherwood and Presting 2007). Now, clients that wish to examine patterns of phytoplankton in water can be assured that they can compare their results across multiple runs.

We’ve had to tackle a few issues in elevating 23S for long-term usage. 23S is a good marker to use in that it amplifies both cyanobacteria and eukaryotic algae. That way, the relative abundance of the two groups can be assessed. We’ve also assessed the topology of the 23S gene tree to ensure that it broadly represents the phylogeny of plastids**. That way, when we find sequences whose hosts have not been sequenced yet, we have some assurance as to the taxonomic identity. Unknown sequences found in water will be assigned a permanent OTU. When the source for that OTU is sequenced, we update the database to reflect this, improving the results over time.

**It’s interesting to look at the 23S gene tree. You can see where taxa like eustigmatophytes acquired their plastids and the process of endosymbiosis for dinoflagellates (they often nest in with diatoms).

The taxonomic database for 23S is still developing. We have about 1400 taxa in the closed ref database right now, of which about 900 represent phytoplankton. We’re actively working on expanding this database, but in the meantime, analyses of assemblages can occur with taxonomy-free OTUs just as if the taxonomy were assigned.

Final beta-testing with select clients is occurring right now. Once any last issues are worked out, the 23S closed ref approach should be available for long-term and large-scale projects.

Sequencing raptor diet

On the diet side, we have reconstructed diets for a number of birds for clients. We’ve looked at what insects and plants prairie chickens eat. We’ve quantified the plants that geese in Alaska eat. We’ve looked at the insects and other invertebrates that shorebirds and songbirds eat.

We had never tried to sequence the diet of raptors, though. A client recently sent us “hawk chalk” to sequence. The fecal material was collected from a number of different species. We were asked to determine what vertebrates the raptors were eating as well as the identity of the raptors themselves.

The details are not ours to divulge, but it turns out sequencing the fecals for vertebrate DNA worked pretty well. We were able to identify species such as red-tailed hawks, golden eagles, and goshawks. And we identified the rodents, lagomorphs and other birds they were eating.

Another potential opportunity to open the black box on wildlife diet!

Is aquatic eDNA ready?

A short note here to share our thinking around the office and lab…

Here, at Jonah Ventures we’ve spent a fair amount of resources working to understand how to apply next generation sequencing technology to aquatic environmental DNA.

Speaking non-technically for now, if someone wants to assess whether an aquatic ecosystem is impaired, they can either measure a large suite of physical and chemical factors, or quantify the fish or insect species in that water. The latter is what is used most frequently, for good reason. Individual species each have their own tolerances of environmental conditions. As such, their relative abundances tend to reflect environmental conditions.

The hard part is that it isn’t easy to measure the abundances of fish or insects in water. Also mapping individual fish or insect species to environmental conditions is also pretty crude. Right now, these species are used as indicators of “water quality”, but that typically means biological oxygen demand. And the mappings aren’t that tight. Ideally, we’d also be able to use indicator species to assess not only organic pollution, but also availability of different nutrients like nitrogen or phosphorus, pH, salinity, temperature, and heavy metals. All of these deserve to be assessed over time and over broad spatial scales.

In general, it seems clear that measuring fish or insects is not up to (or going to be up to) the task of being able to deliver all of this information. Leaving out details here, it can do some, but not all. And after 50 years of the traditional approaches, no one expects thing to change drastically any time soon.

If traditional approaches are limited, the question then becomes, can eDNA be used to assess environmental conditions better than quantifying the abundances of fish or insects? Is eDNA ready for this?

We think it is…almost.**

**We think bacteria and phytoplankton eDNA is ready for wide-scale use. Insects and fish, not quite.

Regardless of where we are right now, the important part is thinking about what the future might look like.

We think the future of bioassessment is going to be different than what it is today. And we aren’t going to try to do the same things we are doing now with a newer technique. We are going to try to do more.

In the future, we’ll rely on fish and insects/macroinvertebrates as we do today, but also routinely phytoplankton and bacteria.

And we’re not going to get a simple “water quality” index, but an oxygen demand index, a pH index, a phosphorus index, a salinity index, and a heavy metals index.

The databases that we use to assess water quality are going to span across state and country lines, too. Until rivers stop crossing state lines, our assessments will have to cross political lines, too.

And the days of measuring a river every 7 years to assess its quality will be replaced with much more frequent sampling.

We’re writing this today, because at Jonah Ventures we’ll be able to do some things with our modest resources. We’ll figure out how to measure the relative abundance of species in water with eDNA. We’ll put together the infrastructure to collect, analyze and distribute these data.

But, it’s going to take a lot more than that.

We’ll need better reference libraries. That means we’ll need a lot of taxonomists to identify and culture phytoplankton or sort and identify insects on a national scale. We’ll need ecologists to identify reference conditions that can be used to calibrate new indices. We’ll need regulators to determine how to operationalize new indices and determine sampling schemes.

The future of how we use this technique is going to be interesting, but the most important first step is for people to realize that we are largely going to have to start from scratch. Once people commit to that, eDNA’s future will come quicker than people expect.


An aid to help sample water

IMG_8762We were running a pilot project the other day that required us to take 20 aquatic eDNA samples from the same site. The goal of the project is to look at species accumulation curves as we sample more and more water.

The project requires pushing water through our filters again and again.

They say necessity is the mother of invention. After about 8 samples, a new invention  was necessitated. That was starting to hurt.

It turns out that a simple, modified 12″ bar clamp works great. A hole was drilled in one end to hold the end of the syringe filter. That was all the modification necessary.

Our tests have shown that when we push by hand, we can generate ~30 pounds per square inch of pressure. After you hit that point, it’s hard to even push air through the filter.

The bar clamp had no problem with this. Occasionally, there would be a little deformation in the syringe, but that’s not a worry.

Not all inventions are the most technical, but this one will surely help future sampling.



Bird DNA in water

We just finished analyzing ~100 stream eDNA samples for Nebraska DEQ. We ran these samples with 12S primers that amplify vertebrate DNA.

The goal of the project was to look for DNA that might be helpful in determining the sources of fecal coliform bacteria in the water. This part was pretty successful, but what was almost more interesting was the list of bird species whose DNA we found.

wood duck
rock dove
mourning dove
barn swallow
red winged blackbird
melodious blackbird
brown headed cowbird
gray catbird
house sparrow
swainson’s thrush

It’s really an amazing collection of birds that pretty well represents riparian bird assemblages. The chicken DNA is likely coming from agricultural operations, but that remains to be tested..

What’s fascinating is really how much bird DNA is in the water. First obvious question is how it got there. Defecation? And if bird DNA is in there, are their disease-causing organisms in there too? Also, could this tool be used for conservation purposes?

Seems likely.


Sequencing the Potomac River

The Potomac River starts small, at a place called Fairfax Stone. 300 miles later–as it passes by Washington DC and enters the Chesapeake Bay estuary, it’s very large.

Along the way the Potomac does more than pick up water. It picks up nutrients from the surrounding lands and the species that inhabit its waters and banks shift, too.

How much nutrient it picks up and how the species in its waters respond has never been worked out that well though. There have been surveys of rivers like the Potomac. People have looked for insects and fish at different spots. But, as the river gets big, it’s hard to use standard techniques to see what’s in the water.

Jonah Ventures, along with friends at the University of Maryland, tried to see if we could use environmental DNA to put together a biodiversity atlas of the river and whether we could put together a bio assessment index based on the relative abundance of the DNA of bacteria, phytoplankton, macroinvertebrates, and vertebrates along the river.


We just published our results up on the pre-print server Biorxiv (see here). Although the research is still undergoing peer review, we feel pretty confident that the results are pretty amazing (at least for a first shot).

The take home point of the paper is that the assemblages of the headwaters, main river, and estuary were distinct. Not only that, we found a good relationship between increases in phosphorus concentrations in the river and the relative abundance of different species. As the waters picked up phosphorus, a whole suite of bacteria, phytoplankton, and animals became more abundant as another suite dropped out.

By no means is the last word on the question. We still have to develop our techniques a bit more, but it’s hard not to see the potential here. Soon, we should be able to suck up a small amount of water from a stream or river and quantify enough of the organisms that are in the water to infer the status of the waters.

*Bonus fact not discussed to much in the paper. In some parts of the river, almost half of the vertebrate DNA was human DNA. We thought the most extreme parts would be down by DC. Not so. All the sites with a lot of human DNA were actually up in the headwaters, not down by DC.

**Bonus fact not in the paper. We found a lot of different species in the water that we didn’t discuss. The list of species we found was fascinating. Fish like trout, carp, eels and shad. Amphibians like red salamanders and spring peepers. Mammals like pigs, cattle, and fox. The coolest animal: mountain lion (!). At site 5, 2.5% of the mammal DNA was identified as Puma concolor. There are no cats with sequences close to that.







Learning more about diet

Some of our scientific ignorances are so vast we do not even realize it.

What animals eat is one of them.

At Jonah Ventures, we’ve analyzed the diets of over 25 different species and every time we do, we learn something entirely new. But, continuing to learn new things when we analyze the diets of species we’ve measured before, is the best marker of the depth of our ignorance.

Mary Miller at The Nature Conservancy’s Ordway Prairie in South Dakota has been collecting fecal samples for a few years now. The other day, we just ran ~2 more years of samples for her.


Just looking at the three taxa in the diet over the two years reveals insights that we really have not seen before. Here, samples from 2015 are in red and 2016 are in blue.

The most abundant species–a cool-season grass—shows how regularly the bison consume a given species in different years. The proportion of Trisetum (likely spicatum) in the diet follows the same pattern both years: peaking in spring (early May), declining to a minimum in late June, and then peaking again in the fall (mid September). The second most abundant taxa (Poa pratensis and similar species) shows similarities between the two years, but it was consumed a lot more in 2015 than 2016. Some years, it seems that the bison are consuming more of some species than in other years. What is driving this interannual variation just isn’t known. The third most abundant species (a Lotus—bird’s foot trefoil) demonstrates another aspect of bison diet: bison regularly switch the composition of their diet within a year. As spring turns to summer, the bison are switching from cool-season grasses for most of their protein to a legume.

In a previous paper, looking at multiple years of bison diet in Kansas, we saw the same patterns. Consistent shifts among taxa in the diet bison and interannual variation in the reliance on different taxa.

We really don’t have theory to explain the shifts that we’re seeing. Are the herbivores simply following phenological shifts in the plants? We also have yet to incorporate it into our management of herbivores. For example, no bison managers (that we know of) are currently working to promote legumes in their grasslands. In fact, most try to promote grasses.

Hopefully, as more people collect data like these we can produce more theory and then begin to incorporate into our management of wildlife.


Parasites in animals

A lot of things come out with fecals. Not only is their DNA from animals ate and the animals themselves, but also DNA from parasites that infect the animals. Common parasites in the digestive system include nematodes, trematodes, and cestodes. Traditional techniques for parasite assessment have been to look for eggs in the fecals. But this work requires experts (which can mean expensive, slow, inexact) and cannot always differentiate closely related species. Sequencing DNA in fecals also has the potential to identify parasites. For one client, we used 18S sequencing to look for parasites in their birds. To begin, voucher specimens sequenced well. When we looked at DNA in fecals, animals identified as having parasites visually also tested positive using DNA sequencing. One thing we did learn is that for 95% accuracy in testing for the presence of parasites, multiple replicates need to be run (using our standard approach of swabbing the fecal). Still, for those interested in whether their animals might be infected with parasites in the digestive system, this is a simple test to add.

Fish environmental DNA

The technique of assessing the relative abundance of different organisms is one of the strengths of next generation sequencing. For one recent project, we assessed the relative abundance of different fish species across a number of streams in Nebraska by amplifying a region of the mitochondria that is more or less specific to fish. When the percentage data are aggregated, you get a graph something like below. We still have some more work to do to see how well the relative abundance of different fish species is represented by the data below, but the technique identifies a number of the more prevalent taxa found in the streams. We should be introducing a new product for fish eDNA pretty soon as we work out the final details. Stay tuned.


Sampling organisms of Mississippi River

Jonah Ventures is currently working with Cameron Thrash at Louisiana State University to quantify the organisms of the Mississippi River using eDNA. The project is an amazing effort on the part of volunteers that have rowed the entire length of the Mississippi, collecting water samples as they go. Our contribution is to analyze the DNA in the samples for higher organisms such as phytoplankton, insects, and fish.

Something like this has never been done before. Our preliminary results have been encouraging. Analyzing the DNA in the samples, we’ve been able to reconstruct the phytoplankton community along 3000 km of river. Below is the abundance of just one taxa, Skeletonema marinoi (and/or related species), which is a diatom that apparently is more abundant in the larger portions of the river, especially below the confluence with the Missouri.


Skeletonema looks something like this:


Normally, if one wanted to determine its abundance, one would have to take a water sample, filter it, and then examine the contents under a light microscope, counting each shape that resembled something like the SEM above.

Using eDNA, we can do the same counting likely better (because we’re also counting all the green algae and cyanobacteria as we go) and a lot faster.

We’re working on publishing the phytoplankton data and should be acquiring data on insects and fish soon.