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Using next generation sequencing of alpine plants to improve fecal metabarcoding diet analysis for Dall’s sheep

December 2021
BMC Research Notes 14(1)

DOI: 10.1186/s13104-021-05590-z

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CC BY 4.0

Authors:

Kelly Williams

University of Chicago

Damian Menning

U.S. Geological Survey

Sandra L. Talbot

United States Geological Survey

Show all 6 authorsHide

Objectives Dall’s sheep ( Ovis dalli dalli ) are important herbivores in the mountainous ecosystems of northwestern North America, and recent declines in some populations have sparked concern. Our aim was to improve capabilities for fecal metabarcoding diet analysis of Dall’s sheep and other herbivores by contributing new sequence data for arctic and alpine plants. This expanded reference library will provide critical reference sequence data that will facilitate metabarcoding diet analysis of Dall’s sheep and thus improve understanding of plant-animal interactions in a region undergoing rapid climate change. Data description We provide sequences for the chloroplast rbcL gene of 16 arctic-alpine vascular plant species that are known to comprise the diet of Dall’s sheep. These sequences contribute to a growing reference library that can be used in diet studies of arctic herbivores.

Overview of data files for arctic plant rbcL sequencing

…

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Williamsetal. BMC Res Notes (2021) 14:173

https://doi.org/10.1186/s13104-021-05590-z

DATA NOTE

Using nextgeneration sequencing ofalpine

plants toimprove fecal metabarcoding diet

analysis forDall’s sheep

Kelly E. Williams1,4* , Damian M. Menning2, Eric J. Wald3, Sandra L. Talbot2, Kumi L. Rattenbury3 and

Laura R. Prugh1

Abstract

Objectives: Dall’s sheep (Ovis dalli dalli) are important herbivores in the mountainous ecosystems of northwestern

North America, and recent declines in some populations have sparked concern. Our aim was to improve capabilities

for fecal metabarcoding diet analysis of Dall’s sheep and other herbivores by contributing new sequence data for

arctic and alpine plants. This expanded reference library will provide critical reference sequence data that will facilitate

metabarcoding diet analysis of Dall’s sheep and thus improve understanding of plant-animal interactions in a region

undergoing rapid climate change.

Data description: We provide sequences for the chloroplast rbcL gene of 16 arctic-alpine vascular plant species that

are known to comprise the diet of Dall’s sheep. These sequences contribute to a growing reference library that can be

used in diet studies of arctic herbivores.

Keywords: Alpine, Arctic, Boreal, Dall’s sheep, Diet, Fecal, Metabarcoding, Chloroplast, Plant

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Objective

Dall’s sheep (Ovis dalli dalli) are endemic to alpine eco-

systems of northwestern North America, and their pop-

ulations have been declining in recent decades [1–4].

Climate change may be altering alpine plant communi-

ties and contributing to these declines. Dall’s sheep have

a generalist plant diet; they were observed eating 110

diﬀerent plant species in the Yukon Territory, Canada

through traditional observational methods [5]. However,

the diet of Dall’s sheep remains relatively poorly char-

acterized and represents a gap in understanding how

climate change is aﬀecting plant-animal interactions in

alpine ecosystems.

e level of taxonomic resolution of items consumed

in a diet study greatly aﬀects ecological analysis [6].

DNA based tools can infer diet composition with higher

resolution and reduces cost, time, and eﬀort compared

to observational, morphological, and microhistological

methods [7, 8]. Speciﬁcally, DNA metabarcoding uses

universal primers for multispecies identiﬁcation to mass-

amplify DNA barcodes using PCR that are then read

using next generation sequencing and assigned to the

appropriate taxon [9]. DNA barcoding includes a refer-

ence database of potential diet components, providing

the capability to identify diet items to a desirable taxo-

nomic resolution, ensuring that all components will be

detected and assigned [10]. Next generation sequencing

of DNA from fecal samples has been successfully used to

characterize diets of a variety of species, including ungu-

lates [11, 12]. However, metabarcoding has not yet been

used to assess the diet of Dall’s sheep. Lack of sequence

data for some arctic/alpine plants known to be grazed

Open Access

BMC Research Notes

*Correspondence: kel.elizabeth.williams@gmail.com

1 School of Environmental and Forest Sciences, University of Washington,

Seattle, USA

Full list of author information is available at the end of the article

Page 2 of 4

Williamsetal. BMC Res Notes (2021) 14:173

upon by Dall’s sheep currently limits the development

and application of metabarcoding for alpine herbivore

diet studies.

To improve capabilities for diet analysis of Dall’s sheep

and other arctic herbivores, we used a python script [13]

to identify gaps in archived nucleotide sequence data for

species known to comprise the diet of Dall’s Sheep, then

obtained specimens of 16 species of arctic/alpine vascu-

lar plants for which sequence information was missing

or underrepresented in publicly archived databases. We

then sequenced the rbcL gene of the plant chloroplast

genome, which is one of the most commonly used bar-

coding regions for plants [9, 14].

Data description

Plant specimens were obtained from herbarium speci-

mens collected from the various arctic or alpine sites

across mainland Alaska (Additional ﬁle 1). Plant tissue

was extracted at the U. S. Geological Survey Alaska Sci-

ence Center, employing a CTAB-PVP protocol modiﬁed

from Stewart and Via [15] as reported by Muñiz-Sala-

zar et al. [16]. Extracts were quantiﬁed and shipped

to the School of Environmental and Forest Sciences

Genetics Lab at the University of Washington for PCR

ampliﬁcation and NexteraXT library preparation for

sequencing. e rbcL gene region of each specimen was

ampliﬁed via a two-step PCR protocol [17] with a pri-

mary ampliﬁcation with tailed primers (rbcLaf + adap-

tor, rbcLr506 + adaptor) followed by a second round of

ampliﬁcation to anneal NexteraXT indices. Amplicons

were quantiﬁed using a Qubit 4 Fluorometer (er-

moFisher) and diluted with dH2O to the recommended

starting concentration for library preparation, 0.2 ng/

μL (Illumina). Tagmentation, library ampliﬁcation, and

clean-up steps were completed according to the Nexter-

aXT library preparation protocol (Illumina) with a vari-

ation of using New England Biolabs AMPure XP beads

for cleanup instead of Agentcourt AMPure beads. e

libraries were normalized and pooled prior to sequencing

on an Illumina Miseq platform. Samples were paired-end

sequenced in a 2 × 300bp format .

Illumina sequence reads were processed in Geneious

Prime 2020.2.4. Forward and reverse read ﬁles (fastq)

were paired upon import, then quality trimmed with

BBDuk trimmer (minimum quality 20, minimum over-

lap 20, minimum length 20). Sequences were normalized,

then aligned and assembled using the de novo assembly

tool (Geneious Prime). Assembled contigs were uploaded

and annotated using BankIt, then submitted to GenBank

[18] (Table1).

Limitations

e following are limitations for these data ﬁles:

1. We sequenced one DNA extraction from each plant

species.

2. e sequencing project was funded through a grant

to train new users on Illumina Nextera sequencing.

Abbreviations

rbcL: Large subunit of ribulose 1, 5 bisphosphate carboxylase/oxyge-

nase (RUBISCO or RuBPCase); CTAB-PVP: DNA extraction method using

Table 1 Overview of data ﬁles for arctic plant rbcL sequencing

Label Name of data le/data set File types Data repository and identier

Data ﬁle 1 Elymus borealis rbcl contig *.gb https:// ident iﬁers. org/ ncbi/ insdc: MW538 513 [19]

Data ﬁle 2 Gentiana propinqua rbcl contig *.gb https:// ident iﬁers. org/ ncbi/ insdc: MW538 515 [20]

Data ﬁle 3 Juncus mertensianus rbcl contig *.gb https:// ident iﬁers. org/ ncbi/ insdc: MW548 523 [21]

Data ﬁle 4 Luzula arctica rbcl contig *.gb https:// ident iﬁers. org/ ncbi/ insdc: MW548 524 [22]

Data ﬁle 5 Ranunculus kamchaticus rbcl contig *.gb https:// ident iﬁers. org/ ncbi/ insdc: MW548 525 [23]

Data ﬁle 6 Oxytropsis scammaniana rbcl contig *.gb https:// ident iﬁers. org/ ncbi/ insdc: MW548 526 [24]

Data ﬁle 7 Packera ogotorukensis rbcl contig *.gb https:// ident iﬁers. org/ ncbi/ insdc: MW548 527 [25]

Data ﬁle 8 Penstemon gormanii rbcl contig *.gb https:// ident iﬁers. org/ ncbi/ insdc: MW548 528 [26]

Data ﬁle 9 Saxifraga caespitosa rbcl contig *.gb https:// ident iﬁers. org/ ncbi/ insdc: MW548 529 [27]

Data ﬁle 10 Silene tayloriae rbcl contig *.gb https:// ident iﬁers. org/ ncbi/ insdc: MW548 530 [28]

Data ﬁle 11 Smelowskia integrifolia rbcl contig *.gb https:// ident iﬁers. org/ ncbi/ insdc: MW548 531 [29]

Data ﬁle 12 Stellaria alaskana rbcl contig *.gb https:// ident iﬁers. org/ ncbi/ insdc: MW548 532 [30]

Data ﬁle 13 Taraxacum lyratum rbcl contig *.gb https:// ident iﬁers. org/ ncbi/ insdc: MW548 533 [31]

Data ﬁle 14 Anemone lithophilia rbcl contig *.gb https:// ident iﬁers. org/ ncbi/ insdc: MW526 257 [32]

Data ﬁle 15 Carex pyrenaica rbcl contig *.gb https:// ident iﬁers. org/ ncbi/ insdc: MW538 514 [33]

Data ﬁle 16 Elymus latiglumis rbcl contig *.gb https:// ident iﬁers. org/ ncbi/ insdc: MW537 582 [34]

Page 3 of 4

Williamsetal. BMC Res Notes (2021) 14:173

cetyltrimethylammonium bromide as a detergent-based extraction buﬀer

and polyvinylpyrrolidone, which is added to remove phenolic compounds

from plant DNA extracts [15, 16]; PCR: Polymerase chain reaction; NexteraXT:

NexteraXT DNA library preparation kit enables sequencing of small genomes,

PCR amplicons, and plasmids (Illumina); Miseq: Illumina Miseq Next Genera-

tion Sequencer is an integrated instrument that performs clonal ampliﬁcation,

genomic DNA sequencing, and data analysis with base calling, alignment,

variant calling, and reporting in a single run (Illumina).

Supplementary Information

The online version contains supplementary material available at https:// doi.

org/ 10. 1186/ s13104- 021- 05590-z.

Additional le1. Table of information about the plant specimens used

for rbcl sequencing.

Acknowledgements

The Illumina team at the University of Washington provided valuable training

on Illumina sequencing technology. Plant specimens were obtained from the

U.S. Geological Survey and the University of Alaska, Anchorage herbarium. Any

use of trade, ﬁrm, or product names is for descriptive purposes only and does

not imply endorsement by the U.S. Government.

Authors’ contributions

EW, KR, DM, and ST identiﬁed need and with KW and LP contributed to study

design. ST obtained plant specimens and performed DNA extraction at the

U.S. Geological Survey Alaska Science Center, Alaska. DM scanned sequence

data archived in GenBank to identify data gaps for candidate plant species.

KR and DM chose the ﬁnal list for analysis based on these data gaps, available

information on Dall’s sheep diets, and expected plants in habitat where

populations have declined (e.g., Brooks Range). KW performed laboratory

work for library preparation and sequencing and assembled sequences in

Geneious Prime. KW and LP wrote the manuscript, and DM, EW, and ST edited

the manuscript. All authors read and approved the ﬁnal manuscript.

Funding

Illumina and NASA’s Arctic and Boreal Vulnerability Experiment program (Grant

NNX15AU21A to LRP). The United States Geological Survey and National Park

Service provided funding in terms of salary and ﬁeld and laboratory processes.

Availability of data and materials

Please see Table 1 and references [19–34] for details and links to the data.

Please see Additional ﬁle 1 for a table of information about the plant speci-

mens used for sequencing.

Declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

There are no competing interests.

Author details

1 School of Environmental and Forest Sciences, University of Washington,

Seattle, USA. 2 U.S. Geological Survey, Alaska Science Center, Anchorage,

USA. 3 Arctic Network Inventory & Monitoring Division, National Park Service,

Fairbanks, USA. 4 Present Address: Department of Evolutionary Anthropology,

Duke University, Durham, USA.

Received: 11 February 2021 Accepted: 28 April 2021

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23. Williams K, et al. Ranunculus kamchaticus rbcl partial GenBank https://

ident iﬁers. org/ ncbi/ insdc: MW548 525. 2021.

24. Williams K, et al. Oxytropsis scammaniana rbcl partial GenBank https://

ident iﬁers. org/ ncbi/ insdc: MW548 526. 2021.

25. Williams K, et al. Packera ogotorukensis rbcl partial GenBank https:// ident

iﬁers. org/ ncbi/ insdc: MW548 527. 2021.

26. Williams K, et al. Penstemon gormanii rbcl partial GenBank https:// ident

iﬁers. org/ ncbi/ insdc: MW548 528. 2021.

27. Williams K, et al. Saxifraga caespitosa rbcl partial GenBank https:// ident

iﬁers. org/ ncbi/ insdc: MW548 529. 2021.

28. Williams K, et al. Silene tayloriae rbcl partial GenBank https:// ident iﬁers.

org/ ncbi/ insdc: MW548 530. 2021.

29. Williams K, et al. Smelowskia integrifolia rbcl partial GenBank https:// ident

iﬁers. org/ ncbi/ insdc: MW548 531. 2021.

30. Williams K, et al. Stellaria alaskana rbcl partial GenBank https:// ident iﬁers.

org/ ncbi/ insdc: MW548 532. 2021.

31. Williams K, et al. Taraxacum lyratum rbcl partial GenBank https:// ident

iﬁers. org/ ncbi/ insdc: MW548 533 (2021).

32. Williams K, et al. Anemone lithophilia rbcl partial GenBank https:// ident

iﬁers. org/ ncbi/ insdc: MW526 257. 2021.

33. Williams K, et al. Carex pyrenaica rbcl partial GenBank https:// ident iﬁers.

org/ ncbi/ insdc: MW538 514. 2021.

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Jun 2015
P NATL ACAD SCI USA

Significance Theory holds that sympatric large mammalian herbivores (LMH) must partition food resources to coexist, and traditional frameworks categorize LMH along a spectrum from grass-eating grazers to non–grass-eating browsers. Yet it has never been clear how finely LMH partition the enormous species diversity subsumed within these two broad plant types. By sequencing plant DNA from LMH fecal samples, we analyzed the diets of an LMH assemblage in Kenya. Diet composition was similar within species and strongly divergent across species, irrespective of feeding guild: Grazers ate similar total amounts of grass but different suites of grass species. These results suggest that species-specific plant traits may be key to understanding the dietary differences thought to underpin LMH diversity.

Who is eating what: diet assessment using Next Generation Sequencing

Article

Full-text available

Dec 2011
MOL ECOL

The analysis of food webs and their dynamics facilitates understanding of the mechanistic processes behind community ecology and ecosystem functions. Having accurate techniques for determining dietary ranges and components is critical for this endeavour. While visual analyses and early molecular approaches are highly labour intensive and often lack resolution, recent DNA-based approaches potentially provide more accurate methods for dietary studies. A suite of approaches have been used based on the identification of consumed species by characterization of DNA present in gut or faecal samples. In one approach, a standardized DNA region (DNA barcode) is PCR amplified, amplicons are sequenced and then compared to a reference database for identification. Initially, this involved sequencing clones from PCR products, and studies were limited in scale because of the costs and effort required. The recent development of next generation sequencing (NGS) has made this approach much more powerful, by allowing the direct characterization of dozens of samples with several thousand sequences per PCR product, and has the potential to reveal many consumed species simultaneously (DNA metabarcoding). Continual improvement of NGS technologies, on-going decreases in costs and current massive expansion of reference databases make this approach promising. Here we review the power and pitfalls of NGS diet methods. We present the critical factors to take into account when choosing or designing a suitable barcode. Then, we consider both technical and analytical aspects of NGS diet studies. Finally, we discuss the validation of data accuracy including the viability of producing quantitative data.

Promises and pitfalls of using high-throughput sequencing for diet analysis

Article

Mar 2019
MOL ECOL RESOUR

The application of DNA sequencing‐based approaches to DNA extracted from environmental samples such as gut contents and faeces has become a popular tool for studying dietary habits of animals. Due to the high resolution and prey detection capacity they provide, both metabarcoding and shotgun sequencing are increasingly used to address ecological questions grounded in dietary relationships. Despite their great promise in this context, recent research has unveiled how a wealth of biological (related to the study system) and technical (related to the methodology) factors can distort the signal of taxonomic diversity. Here, we review these studies in light of high throughput sequencing‐based assessment of trophic interactions. We address how the study design can account for distortion factors, and how acknowledging limitations and biases inherent to sequencing‐based diet analyses are essential for obtaining reliable results, thus drawing appropriate conclusions. Furthermore, we suggest strategies to minimise the effect of distortion factors, measures to increase reproducibility, replicability and comparability of studies, and options to scale up DNA sequencing‐based diet analyses. In doing so, we aim to aid end‐users in designing reliable diet studies by informing them about the complexity and limitations of DNA sequencing‐based diet analyses, and encourage researchers to create and improve tools that will eventually drive this field to its maturity. This article is protected by copyright. All rights reserved.

Ecological investigation of a population of Dall sheep (Ovis dalli dalli Nelson)

Article