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The vast amount of biodiversity on Earth cannot be detected with the naked eye, yet even macro-organisms can harbor diversity that is not readily observable. This so-called cryptic diversity can be detected using DNA-based methods. By targeting specific portions of the genomes that make up different species of plants, animals, and microbes, a more complete picture of biodiversity can be obtained. These DNA 'fingerprints' also can be used to cross-check visual species identifications and help ensure consistency in taxonomic data sets over time. They may also detect evolutionary processes - such as speciation - while they are happening, because changes in the DNA of a population may be evident much sooner than any visual changes can be measured.

Looking beyond DNA fingerprinting, DNA provides insights into ecosystem functioning. For example, whole-genome shotgun DNA sequencing can determine the DNA sequences of entire genomes by randomly selecting fragments of DNA from a complex environmental sample. This method can detect the presence of entire communities, as well as the genes coded for in the genomes of those communities. Such information can be used to understand how biogeochemical processes such as nitrogen cycling are related to gene functions and community structure. And because shotgun DNA sequencing does not target any organism or gene in particular, it can detect novel diversity, gene functions, and even viruses that are present in the environment.

During observational sampling bouts of certain organisms, NEON field ecologists select a subset of aquatic and terrestrial organisms that receive genetic analysis. The resulting genomic extracts of many of the samples are also archived into the NEON Biorepository. DNA analysis serves several purposes, including verification of taxonomy of specimens that do not receive expert identification, clarification of the taxonomy of rare or cryptic species, characterization of diversity using molecular markers, and functional gene characterization. DNA sequences of organisms vary by site type and include:

• Metacommunity sequences of environmental samples, particularly from surface water, benthic samples, and terrestrial soils
• Metacommunity sequences of macroinvertebrate samples and zooplankton samples
• DNA barcoding of tissue from individual organisms; target taxa are fish, ground beetles, mosquitoes, and small mammals

Shotgun Metagenome Sequences

The following data products are created from collected samples that are frozen on dry ice and shipped to an analytical facility for DNA extraction, sample preparation, shotgun metagenomic sequencing, and high-throughput sequence analysis using primer sets targeting the small subunit of the ribosomal RNA gene.

Benthic Microbe Marker Genes and Metagenomes

Site Type: Streams

Benthic microbe samples are collected at the same time and location as periphyton samples three times per year in wadeable streams during aquatic biology bout windows, roughly in spring, summer, and fall. Samples are scrubbed from rock surfaces (epilithon) and filtered, or collected from the sediments (epipsammon, epipelon) or plant surface (epiphyton). A duplicate set of filtered samples collected from rock scrubs are sent to the NEON Biorepository.

Surface Water Microbe Marker Genes and Metagenomes

Site Type: Streams, Rivers, Lakes

Surface water microbe samples are collected at the same time and location as surface water cell count samples and surface water chemistry samples once per month in wadeable streams (12 times per year) and every other month in lakes and rivers (six times per year). In wadeable streams, surface water microbe samples are collected near the downstream surface water sensor location. In lakes and rivers, microbial samples are collected near the buoy-mounted sensor station, where sampling depth is dependent on stratification. At flow-through lakes, surface water microbes are also collected near the inlet and outlet sensors. A duplicate set of samples are sent directly to the archive facility.

Soil Microbe Marker Genes and Metagenomes

Site Type: Terrestrial

Soil sampling occurs annually during the historic peak in vegetation greenness at three randomly assigned base plots are terrestrial sites. Soil samples are collected to a maximum depth of 30 cm, with organic and mineral soils sampled separately. Subsamples of homogenized soil (rocks, roots, and organic debris removed) from each of the three sampling locations are combined to form a plot-level composite sample that is used for metagenomics analysis.

Metabarcodes

The following data products are created from samples that are shipped to an external facility for homogenization and high-throughput sequencing (metabarcoding) targeting two regions of the COI gene.

Macroinvertebrate Metabarcoding

Site Type: Streams, Rivers, Lakes

Benthic macroinvertebrate DNA samples are collected three times per year at wadeable stream, river, and lake sites during aquatic biology bout windows, roughly in spring, summer, and fall, at the same time and location as morphological taxonomy samples. Samples from the spring and fall bouts are shipped directly to the Biorepository. Macroinvertebrate samples are collected using the most appropriate sampler for the habitat type. In wadeable streams, samples are collected in the most dominant habitat type (e.g. riffles, runs, pools, step pools). In lakes and rivers, samples are collected in littoral areas. Samples are preserved in ethanol in the field and returned to the Domain support facility for a preservative change before shipping to an external facility. Samples from the mid-summer bout are used for DNA extraction and metabarcoding.

Zooplankton Metabarcoding

Site Type: Lakes

Zooplankton DNA samples are collected three times per year at lake sites during aquatic biology bout windows, roughly in spring, summer, and fall. Samples from the mid-summer bout are used for metabarcoding. Samples from the spring and fall bouts are shipped directly to the Biorepository. Samples are collected using a tow net (water deeper than 4 m) or a Schindler-Patalas trap (water shallower than 4 m) depending on the depth at the sampling location. Zooplankton samples are collected near the buoy-mounted sensor station, as well as the inlet and outlet sensor sets. Samples are preserved in ethanol in the field and returned to the Domain support facility for a preservative change before shipping to an external facility.

DNA Barcode Sequences

Live fish sample from a NEON site

The following data products are created from tissue samples that are shipped to an external lab for DNA extraction. Target sequences are amplified via PCR. Barcodes of cytochrome oxidase I are generated per specimen.

Fish DNA Barcodes

Site Type : Streams, Lakes

Fish sampling is conducted twice a year at most wadeable streams and lakes, roughly in the fall and spring. During each sampling bout, fin clips are taken from up to five specimens per species to be used for DNA barcoding. The primary objective for DNA barcoding fish is to elucidate cryptic and hybrid species as well as for taxonomic verification.

Ground Beetle DNA Barcodes

Site Type: Terrestrial

At each terrestrial site, ground beetle samples are collected biweekly throughout the growing season using pitfall traps. During these sampling bouts, a subset of beetle legs is collected from up to ten individuals per species per site. Up to 95 of these samples per site per year are used for DNA barcoding. Beetles that are rare, particularly difficult to identify, or poorly represented in previous collection events are prioritized for DNA sequencing. Only beetle specimens that have been identified by an expert taxonomist are eligible for DNA barcoding.

Pinned mosquito sample of Mansonia titillans

Mosquito DNA Barcodes

Site Type: Terrestrial

At each terrestrial site, mosquito samples are collected periodically throughout the active mosquito season using CO₂ traps. During these sampling bouts, a subset of mosquito legs is collected from up to ten individuals per species per NEON Domain. Up to 95 of these samples per Domain per year are used for DNA barcoding.

Small Mammal DNA Barcodes

Site Type: Terrestrial

At terrestrial field sites, live trapping is conducted 4-6 times per year to collect small mammal observations. During these sampling bouts, up to 240 ear punches are collected from live specimens at each site per year. Up to 95 of these samples per Domain per year are used for DNA barcoding.

Data Products

Shotgun Metagenome Sequences

• (DP1.20279.001)
• (DP1.10107.001)
• (DP1.20281.001)

Metabarcodes

• (DP1.20126.001)
• (DP1.20221.001)

DNA Barcode Sequences

• (DP1.20105.001)
• (DP1.10020.001)
• (DP1.10038.001)
• (DP1.10076.001)

Archival Samples

Shotgun Metagenome Sequences

Benthic Microbe Marker Genes and Metagenomes

NEON archives unprocessed Sterivex filters (suitable for researchers to process for genetic analyses) in addition to genomic extracts that remain after DNA sequencing is complete. See the aquatic microbes data collection page for a high-level summary. Archival samples of water collected at NEON aquatic field sites are available from the .

Surface Water Microbe Marker Genes and Metagenomes

NEON archives unprocessed Sterivex filters (suitable for researchers to process for genetic analyses) in addition to genomic extracts that remain after DNA sequencing. See the aquatic microbes data collection page for a high-level summary. Archival samples of water collected at NEON aquatic field sites are available from the NEON Biorepository.

Soil Microbe Marker Genes and Metagenomes

Archival soil samples collected from NEON terrestrial field sites are available from the NEON Biorepository. Both organic and mineral horizons are archived if present at the site location. NEON archives unprocessed frozen soils (suitable for researchers to process for genetic analyses) in addition to genomic extracts that remain after DNA sequencing. See the soil microbe data collection page for a high-level summary.

Metabarcodes

Macroinvertebrate and Zooplankton Metabarcoding Archival Samples

All genomic extracts remaining after the metabarcoding analysis is complete are archived at the NEON Biorepository. In addition to samples that are directly related to the DNA sequence data on the portal, NEON also archives sorted and identified macroinvertebrates and bulk unsorted samples of macroinvertebrates that researchers can request if they want to do additional genetic analyses.

DNA Barcode Sequences

Fish DNA Barcodes

The NEON program archives both whole fish vouchers (some of which are DNA barcoded, but not always) and genomic extracts remaining from DNA barcode analysis. Both are available from the NEON Biorepository.

Ground Beetle DNA Barcodes

All DNA extracts are archived at the NEON Biorepository. Legacy beetle vouchers are maintained at a few collections, but all beetle vouchers collected after 2018 are located at the NEON Biorepository.

Legacy collection**:

Mosquito DNA Barcodes

A subset of identified mosquitoes (up to 20 individuals of each species) is mounted on points as vouchers. DNA barcoding is performed on some of these vouchers; any surplus genomic extracts from this analysis are also archived.

Small Mammal DNA Barcodes

Both whole specimen and tissue samples are available for use from the NEON Biorepository. Samples may include preserved whole specimens (for trait analysis), ear punches, hair and whisker samples, biological tissues (skin, organs), and feces. Some samples are available for destructive purposes and others for non-destructive research purposes only. NEON also archives small mammal DNA extractions.

Legacy collections**:

* Select specimens collected from 2013-2017 and most specimens collected after 2018 are archived at the NEON Biorepository at Arizona State University. See table below for information about the archive locations of early (2013-2017) material.

� Quantities represented in this table are annual quantities expected from sampling conducted throughout the Observatory during full Operations. Actual quantities (particularly from years in early construction) will differ from these projections

� Quantities are number of samples (not number of well plates); up to 96 samples are contained on the same well plate

** Some legacy material is curated at other institutions, where material was archived before the creation of the NEON Biorepository. NEON is required to continue archiving some materials (e.g., mammal specimens collected in Florida) at these institutions due to permitting requirements that specify certain material must reside in the state where it was originally collected.