A04_STATEMENT_OF_WORK.pdf

STATEMENT OF WORK

Background

Description of the requirement:

o Great Smoky Mountains National Park is colloquially referred to as the ‘Salamander Capital of the World’ due to its renowned salamander diversity, one of many reasons it was designated as a World Heritage Site and International Biosphere Reserve. The largest of GRSM’s salamanders is the eastern hellbender, an imperiled species of aquatic amphibian found within the park’s streams and rivers. Hellbenders were recently proposed for listing as federally endangered by the USFWS and are listed as state-endangered in TN and a Species of Special Concern in NC. Last year, GRSM was awarded national funds to conduct a Natural Resource Condition Assessment (NRCA) on its hellbender populations.

This project will fund the field work and analyses to better understand the status and trends of hellbender populations and genetics across the park in order to conserve their habitat and inform their management. As an NPS unit, GRSM is tasked with preserving unimpaired the natural and cultural resources of the National Park System for the enjoyment, education, and inspiration of this and future generations. This contract will provide GRSM with the tools and data to make the best decisions regarding the conservation of eastern hellbenders for the enjoyment of local communities and visiting public into the future. GRSM is in need of a vendor with the capacity to process high volumes of environmental genetic samples, tissue samples for next generation genomics and transcriptomics, and the academic interest and capability of developing novel genetic tools. Preferably, this vendor will be familiar with amphibian genomes and have the proper biological knowledge and physical primers to ensure quality data. We expect this vendor to sequence 95 environmental DNA (eDNA) water samples, sequence 96 genomic tissue samples, and develop environmental RNA (eRNA) sampling as a method of detecting larval eastern hellbenders in lotic systems. It is expected that this methodology will be developed by using captive eastern hellbenders in a controlled environment and can be field tested in GRSM. All samples will be collected by park partners and NPS field staff and then shipped to said vendor following their protocols.

o

Background materials:

o From the NPS description of the grant: “The Natural Resource Condition

Assessment (NRCA) Program evaluates the condition of natural resources in parks. Condition assessments document current conditions, evaluate trends, identify critical data gaps. Project findings provide credible science to assist park managers with planning, management, and stewardship activities.”

Objectives

Goals and objectives of the contract effort:

o The contractor will work with the National Park Service to sequence direct and environmental genetic material from eastern hellbenders as well as adapt eRNA methodology to this species to inform their management within GRSM.

o The contractor will work with the National Park Service to fulfill the requirements of a Natural Resource Condition Assessment on eastern hellbenders.

Intended use of the results or end products:

o Results from eDNA sequencing will be used to assess presence/absence of eastern hellbenders in 31 waterways across GRSM. Genomic data from the 96 tissue samples will assess the relatedness of hellbenders found in populations geographically isolated by the damming of larger rivers. This data can be used to assess the risk of genetic bottlenecking due to damming and inform possible translocations. The eRNA methodology will be implemented on samples collected for eDNA sequencing, when feasible, and will provide GRSM with the means to quickly assess the presence/absence of larval hellbenders. Through this detection of larvae, GRSM can determine which streams and rivers have actively reproducing hellbenders. Results will be incorporated into the final NRCA report and at least one scientific journal article prepared by the principal investigator and GRSM. Additionally, the data will be used to create scientific communication materials for the NPS about eastern hellbender conservation and supplied to the USFWS to inform their listing status. All materials will be used to inform management of eastern hellbenders, and their habitat, in GRSM including the use of passive and active management tools such as monitoring, translocations, and re-introductions.

Scope Scope of the contract: The National Park Service at GRSM hopes to receive the resultant presence/absence data from 95 filtered eDNA samples, analyzed genetic data from next generation 3RAD sequencing on 96 tissue samples, and a peer reviewed report, scientific article, or manuscript describing the adaptation of eRNA methodology to eastern hellbenders for use in the field and lab. All aforementioned materials should be used to inform a NRCA Report following standard Natural Resources Science and Stewardship guidelines.

Tasks

3.1.1 Task 1 – Analyze environmental samples (eDNA/eRNA) for presence of eastern hellbender (Cryptobranchus alleganiensis alleganiensis) genetic material

The contractor shall extract, replicate, and quantify eastern hellbender DNA from 95 aquatic samples following peer-reviewed methodologies to determine the presence of hellbender DNA within a given aquatic system. The contractor shall provide their preferred SOP on aquatic sample collection in the field which best supports their lab protocols. The contractor shall provide GRSM with the resultant presence/absence data per sample. Should the experimental eRNA methodology be successfully adapted to eastern hellbenders, then these same aquatic samples will be used again to detect the presence/absence of larval eastern hellbender RNA.

3.1.2 Task 2 – Utilize next generation sequencing techniques to analyze the genomics of 96 eastern hellbenders

The contractor shall use 3RAD sequencing, or another peer-reviewed methodology, to analyze the genomics of 96 eastern hellbender tissue samples. The contractor shall provide their preferred SOP on tissue collection in the field which best supports their lab protocols. The contractor shall analyze and format the resultant data into phylogenetic trees or principal components, or another method which best represents the final dataset, following peer-reviewed methodologies to indicate the relatedness of the individuals within each tributary to their conspecifics within the Little TN River/Hiwassee clade. The contractor shall provide their expertise on population genetics as to any indications of genetic bottlenecking within each population. The contractor shall provide all data and figures to GRSM pursuant to their National Park Service Research Permit.

3.1.3 Task 3 – Adapt eRNA methodology to eastern hellbenders for use in detecting larval eastern hellbender RNA

The contractor shall conduct experiments to determine the efficacy of using the published eRNA methodology, see Parsley and Goldberg 2023 (“Environmental RNA can distinguish life stages in amphibian populations”), in detecting larval eastern hellbenders within their natural habitat. The contractor is expected to follow the Parsley and Goldberg methodology, where applicable, but has the latitude to adapt and adjust as needed. Methodological steps should include developing RNA assays of larval eastern hellbenders, understanding if this RNA is expressed within cells which would be shed into the environment, testing the extraction, replication, and detection of RNA in a controlled environment with both larval and adult hellbenders, and testing this method in a stream with and without known larval hellbenders.

The contractor shall submit a peer-reviewed scientific manuscript, or NPS Natural Resources, Science, and Stewardship report, detailing their experimental process and the efficacy of eRNA in larval eastern hellbender detection.

Task Number

Work Milestones

Projected Completion Date

3.1.1 Received all physical samples for analysis. 12/31/2026

Complete all lab work, data analysis, and return a dataset indicating eDNA, and eRNA if applicable, presence/absence per sample.

12/31/2027

3.1.2 Received all tissue samples for analysis. 12/31/2026

Complete all lab work and return a raw dataset, figures, and analyzed data.

12/31/2027

3.1.2 Produce a peer-reviewed scientific manuscript or report on the efficacy of eRNA in detecting larval eastern hellbenders in their natural habitat.

12/31/2027

Delivery

Deliverables:

o Dataset of eDNA presence/absence indicated by sample.

o Dataset of eRNA presence/absence, if applicable, indicated by sample.

o Raw dataset, analyzed dataset, figures, and short report with an interpretation of the eastern hellbender genomics in relation to genetic bottlenecking and relatedness.

o Scientific manuscript or report outlining the experimental design and efficacy of eRNA in detection of larval eastern hellbenders.

Delivery time:

o All materials must be submitted by 23:59 EST 12/31/2027

Delivery location:

o 1316 Cherokee Orchard Rd., Gatlinburg, TN 37738

Government-Furnished Property, Material, Equipment, or Information (GFP, GFM, GFE, GFI)

95 aquatic environmental samples (filters) and 96 tissue samples from eastern hellbenders will be provided to the contractor for analysis. Sample outcomes will be outlined in the corresponding NPS Research Permit.

Special Material Requirements and Considerations

All data, both analyzed, raw, and their interpretations, should be reviewed by NPS before public release to protect the location information for this rare and threatened species pursuant to the NPS Research Permit and agency guidelines.

Place of Performance Contractor facility.

Period of Performance Three calendar years from the date of contract award.