Abstract
The purpose of this post propose an pilot study to collect environmental DNA (eDNA) on the tepui summits of the Guyana Shield, and to quickly address the
Introduction to Environmental DNA
Environmental DNA (eDNA) collection and surveying is a
fairly new technique in wildlife biology. The foundation of eDNA is based on
the principle that organisms produce and loose enough cells containing DNA that
these cells can be retrieved from the surrounding environment without the need
to procure specimens for DNA extraction. The applications and possibilities of
eDNA collection and analysis are just now coming to the forefront of wildlife
and conservation biology. It is in fact such a new and important method that an entire open access special issue of the journal Biological Conservation has been dedicated to the topic. Studies conducted using eDNA extraction range from
extraction and analysis of current DNA samples (i.e. produced by currently
living organisms) to DNA extraction of paleo samples (i.e. DNA retrieved from
long dead and extinct organisms).[1]
While there is a considerable application in using the technique to investigate
and catalog microbial communities in a variety of environments, the
significant breakthrough has been DNA retrieval from macro fauna, which
continually loose cells and DNA into their surrounding ecosystem. The ability
to rapidly obtain and process this latent DNA has allowed for the study of not
just individual animals and target species, but biodiversity monitoring on an ecosystem
level. The applications for this new technique are emerging currently, and the
technique is growing in popularity and importance.
Methodological and technical benefits of eDNA
1 Broad spectrum sampling: eDNA collection and analysis allows for the
sampling and collection of a wide range of macro-organism who continually shed
genetic material into the environment, as well as potentially surveying the sum totality of micro-organism present in
a specific location
2 Identification of cryptic species:
species which are difficult to obtain, observe, or catalog in the environment
can be identified by latent eDNA, with multiple location sampling efforts range
and population size may be extrapolated.
3 Cost/field time reduction: DNA can be
obtained with minimal field time, as it does not rely on lengthy field
observations. Reduction in field time and and active or passive searching means
that relevant studies can be done with fewer man hours, reducing the cost of
personnel and field equipment.
4 It’s non-invasive: As the DNA collected
comes from environmental sources, macro-organism do not need to be removed from
the field or tranquilized to have tissue samples removed. Reduction in field
time contributes to a less invasive field surveys, decreasing the human
footprints in wild environments.
Applications for Biokryptos
The tepuis are notoriously difficult to access and study.
They are topographically discontinuous, difficult and /or expensive to access,
suffer from virtually continual precipitation, and incredibly remote. Their summit
biotas and the ecological dynamics present on the summits are little studied across the totality of the
Guiana Shield. They are also in extreme
danger of major extinction driven by human caused climate change over the next
100 years: we have little way to stop this process, and can only hope that
increased conservation efforts will help mitigate this impending catastrophe. Yet, in order to preserve their biodiversity,
first we have to obtain it in totality, and understand the ecological dynamics of the
tepuis are, preferably down to the molecular level. Until recently, this
research has been accomplished by GIS and meta-analysis when not in the field,
and with expensive short term collection expeditions when field access is
possible. Horizontal and long term studies do not exist, yet. We need a rapid sampling methodology which can be repeated over long periods of time, and this can be accomplished with eDNA analysis.
Tepui ecology
sampling and monitoring: a new methodology
Biokryptos team members are, so far (and still) the first
explorers to place camera traps on the talus and summit slopes of any Tepui. So
far, we have had camera traps active on the talus of Auyan from April 2014 to
July 2014, and at campo Naranja on the summit from August 2014 to April 2015.
This has been a minimally invasive study- we collect photos, and periodically
go up to service and check the cameras, collect data, and explore and
photograph the environment and ecosystems of the summit, making transect
surveys as we go. However, at this point, with eDNA collection and analysis
possible, it is time to integrate this new method with our current field
surveys.
Collecting eDNA on the Tepui summits
The study area for the pilot program for eDNA collection
will be the summit of Auyan Tepui. Auyan can be divided into five vegetaional zones which correspond heavily to surface
topography and substrate. In the case of this first study, we will be studying
samples from drainage areas representing all five major zones, as dictated by
topography and ease of access. In this manner, we should be able to obtain an
overview of the entire tepui as heavy year round precipitation flushes animal
cells and DNA into the major river systems on Auyan. If successful, we will expand our efforts
beyond this sampling method and focus on areas of Auyan which are subsets of
these broadly defined zones.
For the pilot study, we will divide Auyan Tepui into three
collection areas along the sampling route which corresponds to our camera tarp
sites:
1)Lecho/Dragon
camp, and surrounding water shed: The area is a drainage basin for three major types of vegetation: rocky
mesothermic shrubs lands, forests, and pioneer assemblages.
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2) Churum river oxbow system: This is the outlet
point for the vast majority of drainage which occurs across central Auyan tepui.
The drainage area and watershed is the largest area on Auyan covered almost
exclusively by mesothermic swamp and shrubby vegetation, with forest pockets.
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3) Lagoon system and Angel Falls River: Sampling from this large river system, which
feeds a series of lagoons and Angel Falls itself, should provide a fairly
accurate survey of the dense swamp and forests which comprise a densely
forested vegetation profile in the north of Auyan.
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Technical issues
involved
Methods and protocols for eDNA acquisition and analysis are
still being refined and perfected. eDNA analysis, in the specific application
for Biokryptos research, was first undertaken in 2008 with the successful
identification of vertebrate DNA in a freshwater environment. [2]
Biokryptos will be focused on freshwater
eDNA collection and analysis, there are a several variables which must be
analyzed and included in order to make the pilot study successful.
1 DNA production and environmental considerations
When we are looking at eDNA acquisition, local conditions
and methodological principles in wildlife biology are more relevant than ever.
Forensic levels of DNA may be present in the environment in certain
concentrations at all times, but collecting larger amounts of DNA is
preferable. Thus, collection efforts should focus on time periods during which
eDNA is either being released in high volumes (breeding seasons, particularly
for amphibians and reptiles), or when surface water flow is continuous but not
extreme ( beginning and end of dry and wet seasons). eDNA sampling locations will comprise a
combination of lentic and lotic ecosystems, so that comparisons can be made
between eDNA retention in these environments. So far, a variety of eDNA retention
rates have been examined in various types of streams and lakes, indicating that
retention and accumulation of eDNA is not a variable, and that DNA itself is
only removed from gradual break down, physical damage, or inaccessibility due
to settlement in sediments. eDNA
production rates may vary with the ecological cycle of the target organism, such
as reproduction, death, health, and abiotic factors such as seasonality and
water conditions.[3] The
optimal eDNA collection conditions are during periods of high macro organism activity
in an environment which is low temperature, alkaline, low UV radiation.[4] In the case of the tepuis, the abiotic factors
are seasonality, water acidity, and UV radiation. The tepuis are approximately
2000 meters above sea level, and UV radiation which damages DNA is higher at
this elevation. Equally, the water ph. is higher due to large quantities of tannic
acids from decaying plant material in the water. Finally, dry season conditions
will affect the wash out rates from riparian watershed, potentially limiting
terrestrial eDNA present in streams and lakes. Seasonality will also affect the
reproductive cycles of aquatic and semi aquatic organisms, and eDNA production
is expected to be lower during the dry season. Alternatively, dry season concentrations of
terrestrial fauna near perennial water sources may increase the amount DNA from
terrestrial macro-fauna in aquatic environments. While the abiotic factors seem daunting, eDNA
has been recovered from fossiliferous sources, permafrost, and a variety of
hostile environments. The variables such as seasonality, biotic/abiotic factors
must be noted in the study and explored in the future. Correlating these and
other variables with sampling methodology will improve eDNA analysis and sampling techniques in the
rest of the Pan-Tepui.
2 DNA collection in field conditions and post processing
Optimal collection and preservation of eDNA collected is a
significant variable in conducting an eDNA analysis. While most protocols and
studies involve keeping samples at below freezing temperatures, there is recent
evidence demonstrating the freezing and thawing of eDNA samples damages target
DNA[5],
while cooling the samples aids in DNA preservation.[6] In the tepui environment, as in most field
condition in remote areas, preserving samples at very low temperatures is next
to impossible. Recent studies have addressed this problem, and through comparative
methodological analysis determined that eDNA collection and preservation can be
optimized at room temperatures using cellulose nitrate filters, a DNA
preservation buffer to preserve the filter in a 5 ml tube, followed by
extraction with the use of commercially available DNA analysis kits.[7]
Controls need to be produced as well, in the case of aquatic DNA sampling a
negative control needs to be produced using distilled water in the field, with
the same equipment used to collect DNA. As transporting distilled water in
quantity is not possible, distilled water must be produced on site using a
distillation method.
Biokryptos eDNA methodology- outline
The difficulties in launching an eDNA field study are based
in two primary factors- optimizing sampling times and environments, and
implementing a field study in the tepui environment which maintains scientific standards
for collection.
1 Sampling time and locations
The proposed sampling locations – Lecho, Rio Churum, and
Salto Angel Lagoons- should provide a broad spectrum sample of the aquatic
environments on the summit of Auyan at this time. They are also bodies of water
which either border a series of vegetaional zones (Campo Lecho) or represent
wide areas of little explored regions, with significant drainage (Rio Churum).
As differing levels of precipitation effect both DNA wash in to these areas,
dry season and wet season sampling will be conducted. Dry season faunal
composition should be around perennial water, theoretically concentrating eDNA.
Wet season sampling will coincide with the breeding season of amphibians,
allowing for a broad spectrum sampling of species producing large amounts of
cells, and by extension DNA. Sampling at
both times will allow for spatial correlation of organisms present in a specific
ecosystem at a specific time. Tepui
Watch camera traps will be present in these locations concurrently with
sampling efforts, allowing Biokryptos to collect data that can give us an
example of both aquatic environments, terrestrial environments, and the flow
rate of eDNA into aquatic environments.
2 Collection methods
As we are producing a pilot field study in an environment
with limited access to resources, we are going to have to alter standard field
methodologies to conform to the realities of what we can do on a tepui summit.
Luckily, the equipment required can be obtained with little expense, and
shipped into the field with few problems.
As per the results obtained in Renshaw et al 2015, we will
use 0.45 nano-meter cellulose nitrate filters to filter two liters of water, and
extract the filter for preservation in ethanol, a proven method for preserving
DNA for extended periods of time in room temperature and cool environments[8].
Water will be obtained using a hand powered siphon pump, which can be cleaned
after use using a 10% bleach solution. The control will be made using distilled
water produced at the sampling locations, distilled in a custom built field
ready water distillation apparatus. Five samples will be collected from Lecho
and five from the Rio Churum, with five samples collected in the Salto Angel
Lagoon, and five downstream and five upstream
from the Lagoons, producing 25 total eDNA samples, with five controls. The samples will then be
stored for shipping at approximate room temperature, kept cool using exothermic
chemical packs. Once the samples are shipped, the DNA will be extracted using
Quiagen’s DNeasy Blood and Tissue Kit, and replicated using PRC techniques.
Conclusion: eDNA Pilot study
In terms of utility as a sampling methodology, eDNA
collection and analysis in the tepui environments fits perfectly into the goals
and agenda of Biokryptos: it is low impact, inexpensive, easily deployed, and
can be replicated across the entirety of the Pant-Tepui. eDNA analysis has the capability of advancing
the mission of Biokryptos- “to collect and understand the sum totality of tepui
biodiversity to aid in conservation activity”. When combined with camera
trapping, eDNA collection can help us understand the spatial arrangement of
tepui biota, allow us to examine how the ecosystem is changing in response to
global warming, and let Biokryptos field experts refine their methods. There
may be advantages to eDNA analysis
compared to current Biokryptos methods: eDNA studies conducted in the UK using
a volunteer collectors reported success rates above traditional sampling
methods[9].
Biokryptos field operatives, highly trained Pemon guides intimately familiar
with the tepui environment, are more than capable of producing the data for
this pilot study, combining eDNA collection with their extensive field
knowledge. eDNA may allow us to solve some of the mysteries of the tepui fauna,
from cryptic amphibians, to reports of larger animals, and track the presence
of invasives as tepui ecology changes with global climate change.
[1] Philip
Francis Thomsen, Eske Willerslev, 2015 “Environmental
DNA – An emerging tool in conservation for monitoring past and present
biodiversity”, Biological conservation, 183, 4-18,
[2] G.F.
Ficetola, C. Miaud, F. Pompanon, P. Taberlet, 2008. “Species detection using
environmental DNA from water samples” Biol. Lett., 4 (2008), pp. 423–425
[3] G.F.
Ficetola, C. Miaud, F. Pompanon, P. Taberlet, 2008.” Species detection using
environmental DNA from water samples”, Biol. Lett., 4 (2008), pp. 423–425
[4] Strickler,
K.M., Fremier, A.K., Goldberg, C.S., 2015.” Quantifying the effects of UV,
temperature, and pH on degradation rates of eDNA in aquatic microcosms”. Biol.
Conserv. 183, 85–92.
[5] Takahara,
T., Minamoto, T., Doi, H., 2015. “Effects of sample processing on the detection
rate of environmental DNA from the Common Carp (Cyprinus carpio)”. Biol.
Conserv. 183, 64–69.
[6] Strickler,
K.M., Fremier, A.K., Goldberg, C.S., 2015. “Quantifying the effects of UV,
temperature, and pH on degradation rates of eDNA in aquatic microcosms”. Biol.
Conserv. 183, 85–92.
[7]
Mark A. Renshaw, Brett P. Olds, Christopher L. Jerde, Margaret M. Mcveigh and David
M. Lodge. 2015 “The room temperature preservation of filtered environmental DNA
samples and assimilation into a phenol–chloroform–isoamyl alcohol DNA
extraction Molecular Ecology Resources” 15, 168–176
[8]
Melanie et al. 2002 “an evaluation of long term preservation methods for brown
bear (Ursus arctos) faecal samples” Conservation Genetics 3: 435–440, 2002.
[9]
Biiggs, J. et al. 2015. Using eDNA to develop a national citizen science-based
monitoring programme for the great crested newt (Triturus cristatus). Biological
Conservation 183: 19-28
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