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 requirements of conducting such a study.
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). 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.
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.
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.
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.  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. The optimal eDNA collection conditions are during periods of high macro organism activity in an environment which is low temperature, alkaline, low UV radiation. 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, while cooling the samples aids in DNA preservation. 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. 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. 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. 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.
 Philip Francis Thomsen, Eske Willerslev, 2015 “Environmental DNA – An emerging tool in conservation for monitoring past and present biodiversity”, Biological conservation, 183, 4-18,
 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
 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
 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.
 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.
 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.
 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
 Melanie et al. 2002 “an evaluation of long term preservation methods for brown bear (Ursus arctos) faecal samples” Conservation Genetics 3: 435–440, 2002.
 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