196.

201 Biodiversity of New Zealand

eDNA
what’s out there?

This week we are looking at the eDNA approach to studying biodiversity. Using DNA
sequences to study biodiversity relies on databases to match sequences from samples with
names of taxa and taxonomy. To investigate the quality of the databases we will begin by
looking at how many common moth species have DNA sequence data associated with their
name on GenBank databases.

When DNA is extracted from a mixed sample (e.g. water, soil, leaf-litter) many individuals
are combined in the environmental DNA (eDNA) approach. Millions of DNA sequences per
sample are then generated using high-through-put methods. This method usually incorporates
metabarcoding which is the identification of large numbers of specimens from multiple
taxonomic groups simultaneously.

PART A: Specimen identification

Up-load your moth photograph to iNaturalist
Add the link for your iNaturalist photo to the class table- Wednesday’s
Table[https://docs.google.com/spreadsheets/d/1hVC7v52USW5SuNGH6HxWZFxreKBS1E
wi4S7qvggD-SI/edit#gid=0].

Your moth observation will have an identification. Hopefully to the level of species but
possible only the genus or only the family or order (Lepidoptera).
Even if you start without much information, often observations will be identified by experts.
For example, this observation: https://www.inaturalist.org/observations/131689054
Now has been identified as Pseudocoremia fenerata

Moths come from caterpillars – and caterpillars need plants to eat. So what did your moth eat
as a youngster?

1. Go to Plant-SyNZ™: an invertebrate herbivore biodiversity assessment tool

https://plant-synz.landcareresearch.co.nz/index.asp

Search for associations. Add details to the class table [link here]

Have you found a rare endemic species?

Go to the New Zealand Threat Classification System (NZTCS) database
2. https://nztcs.org.nz/

Search using the genus name. If your species is on that list click on its name. Add details to
table. Is it endemic? What is the threat status of your species?

3. Go to GenBank: https://www.ncbi.nlm.nih.gov/genbank/

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Do you know what NIH and NCBI stand for?

Add them here: NIH: National Institute of health

NCBI: National centre for biotechnology information

Use the search tool to see if the moth you photographed has data on this database

For example, I’d pasted the moth genus and species name above Pseudocoremia fenerata

The answer is no, this species is not in the database

If I search with just the genus

I find

Click on this name to see more details. This is a New Zealand moth species but not the one
photographed. The DNA sequenced is 28S ribosomal RNA gene. This gene is not usually
used for eDNA studies.

Search for your moth and add data to the google table.

A1. How many moth observations were identified to species level? (24/24 or 100%)

A2. How many moth species observed have DNA sequences in GenBank? (6/24 or 25%)

A3. What genes have been sequenced

……………… …………………… …………………. ………………… ……………

……………… …………………… …………………… ……………… …………….

A4. If we extracted DNA, amplified a gene and sequenced the product we could add our data
directly to GenBank. How many new moth species could our class observations provide?

…………………………………

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PART B: eDNA of soil invertebrates

Below are a set of questions relating to a study using large DNA sequence datasets, published
in 2020. The paper is available on the course stream site

Dopheide A, Makiola A, Orwin KH, Holdaway RJ, Wood JR, Dickie IA (2020) Rarity is a
more reliable indicator of land-use impacts on soil invertebrate communities than other
diversity metrics. eLife 9:e52787 DOI: https://doi.org/10.7554/eLife.52787

B1. In this study, what was sampled for DNA extraction? Soil invertebrates communities, soil
cores
Invertebrates were extracted from a 1 litre subsample of homogenised soil material from each
site using berlese
B2. What do the authors suggest are advantages to using a DNA metabarcoding method?
Made it easier to distinguish between species and calculate the biodiversity of entire
populations. Rapid and detailed identification of large numbers of inverts from multi tax
groups simuktaneously
More efficient calculation of biodiversity metrics

B3. How many sampling sites were in “Natural Forest”? 14/15 sampling sites

B4. How many natural forest sites sampled were very close to (paired with) grassland sites
that were also sampled? (within ~30km)?
26 samples

B5. The authors wrote (page 3)

“We used these metrics [species richness, effective species numbers, rarity, phylogenetic diversity] to
assess the impacts of land use on a comprehensive range of soil invertebrate taxa”

When comparing land-use impacts on biodiversity why would the spatial distribution of
sampling matter?

Other factors that might impact land use are Geological history, Climate and elevation
Sampling matters with eDNA

B6. Paired sampling statistical design

Paired samples (also called dependent samples) are samples in which natural or matched couplings
occur. This generates a data set in which each data point in one sample is uniquely paired to a data
point in the second sample. Paired t-tests are considered more powerful than unpaired t-tests
because using the same participants or item eliminates variation between the samples that could be
caused by anything other than what's being tested.

a. Would a paired sampling design to assess the impacts of land use be appropriate here? Yes
…

b. Did they use a paired sampling design to assess the impacts of land? No Comparison and

B7. What gene was amplified for DNA sequencing? COI - Cytopromoxadase

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B8. What does OTU stand for? Operational Taxonomic Unit

B9. In the methods section the authors wrote:

“We excluded any OTUs that were not identified as belonging to an expected
terrestrial invertebrate phylum”
a. Do you think it is normal to remove data from a dataset before analysis? No

b. Why might it be considered inappropriate to remove data before analysis? Because we

might be doing in non random matter sampling is better to be random so its not skewed.

c. What type of sequences would come under this excluded category? Contaminated
samples

d. Could these DNA sequences be due to contamination? Yes

e. Could these DNA sequences be due to mistakes of identification in GenBank accessions?

Yes

f. What do the authors conclude about how land-use has impacted soil invertebrates in New
Zealand?

Pervasive impacts on ag use impacts soil invertebrates negatively affecting them. Horticulture
eg orchards and vineyards have severe impact with low species compared to grasslands and
forests.
Commonly used Diversity metrics may underestimate