A Noninvasive Sampling Method Utilizing Small Avian Museum Specimens for Historic Genetic Analysis
Gregory O. Cain, Rebecca A. Eckroad & Igor V. Ovchinnikov
Department of Biology University of North Dakota
Introduction
Results
Preservation methods for avian museum specimens have changed greatly over the years. In the past, arsenic, a known PCR inhibitor (Töpfer et al., 2011), was used as a
preservation agent. The use of arsenic is now prohibited in the museum community (Marte et al., 2006). However, arsenic could still be present on many specimens. For this
reason, it is critical that we understand the arsenic concentration in the specific area of the museum specimen to be sampled. To our knowledge no one has ever tested arsenic
concentrations of avian museum specimens that have been sampled for genetic analysis.
Sampling of avian museum specimens for DNA studies are often destructive in nature. Sampling should be done to avoid damage to the museum specimen, especially
destruction of material that may have possible taxonomic value such as toe pads (Clark, 1973; Payne and Sorenson, 2002). Samples taken should ensure that museum specimens
can be sampled repeatedly to confirm results. Currently used methods of sampling avian museum specimens for genetic material do not meet these standards.
The goal of this study is to develop a method of sampling that:
A.) Ensures as little possible material that is not taxonomically important is destroyed.
B.) Ensures that a museum specimen can be re-sampled for conformation of results, as well as remain available for future genetic and morphological studies.
Successful amplification of PCR products from museum specimens ranged from 53.33% with Direct PCR to 60%
with Chelex -100 and the Zymo Kit.
Sampling Method
- 5mm X 1mm incision was made into the lower proximal rachis of a primary
flight feather.
- All utensils were bleached and allowed to air dry in-between specimens. The
DNA extraction hood was irradiated with UV-light after every use to insure no
amplifiable DNA was present.
Franklin’s Gull
Leucophaeus pipixcan
Horned Lark
Eremophila alpestris
Red-winged Blackbird
Agelaius phoeniceus
Arsenic Testing
- It was thought that our museum specimens may contain arsenic as a possible
explanation for failure of some PCR reactions, especially in older specimens.
- Arsenic Paper Test Kit (Macherey-Nagel Corporation)
Figure 1. Noninvasive sampling method used to obtain DNA from the museum specimens in this study.
This specimen is a Franklin’s Gull collected in 1905 from Stump Lake, ND. A). Full body of the Franklin’s
Gull specimen after sampling. B). Approximate location of sampling of a feather removed from this
specimen for better visual illustration. C). Shows three feather cuttings removed from this specimen
without removal of feathers from the museum specimen.
Laboratory Methods
Extraction Methods
- EDTA with proteinase K followed by
column-based purification (Genomic
DNA Tissue Micro Prep,Zymo Research)
- Chelating resin with dithiothreitol
(Chelex-100, Bio-Rad)
- Direct PCR (Terra Direct PCR,
ClonTech)
-The best quality PCR products for each
method was selected for DNA
sequencing.
- Test strips were visually compared
vs. known arsenic concentration
controls and photographed
Lucus
CR
CR
COI
COI
ND2
ND2
Primer Name
Betty
Fred
RWCOIF1
RWCOIR1
HLND2F7
HLND2R7
Sequence 5' to 3’
TCCACCTGGTACGAAGC
GTCCAACAGTCATTAATAT
CGGTGCATGAGCCGGAATAG
TTCGTCCCAATGCTATGTCA
GTGCAATGCTCATACTAACC
TGTTGCACAGTATGCGAGGC
1892
1892
1892
1892
1905
181
181
181
181
330
Y
N
N
N
Y
N
N
N
N
Y
Y
N
N
N
N
Year
1966
1973
1975
1975
1975
Size (bp)
181
224
181
181
181
Chelex
Y
N
Y
Y
Y
Zymo
Y
N
N
Y
Y
Direct
Y
Y
Y
Y
Y
Year
Size (bp)
Chelex
Zymo
Direct
Red-winged Blackbird
Franklin's Gull
Red-winged Blackbird
Horned Lark
Red-winged Blackbird
1977
1978
1978
1979
1991
224
330
224
181
224
N
N
Y
Y
Y
Y
Y
Y
Y
Y
Y
N
N
Y
N
Further Conformation of Viability of Sampling Method
To confirm that this sampling method not only works with specimens from the University of North
Dakota, specimens Red-winged Blackbirds were sampled from University of Minnesota Duluth, PCR success
was achieved with 7 of 8 museum specimens.
Sequence Results
Sequencing reactions were made with BigDye Terminator v3.1 Cycle Kit (Applied Biosystems).
Sequences were run on an Applied Biosystems 3100 Genetic Sequencer.
Forward COI Mitochondrial Sequence from a Horned Lark
Collected from Christine, North Dakota in 1966 – Vertebrate Museum University of North Dakota
- Detectable in only 2 Horned Lark Specimens
Collected in 1892 and 1979
Discussion
Lab assistant Rebecca Eckroad swabs the head of a
Red-Winged Blackbird for arsenic testing.
This method uses only a small part of the bird specimen to cut down on the destructive nature of sampling.
Using only a small feather cutting, the bird can then be re-sampled by later for verification of results. We
demonstrate that this method can be applied to a variety of small bird species and will produce quality PCR bands,
that can be sequenced. To our knowledge this is the first time Direct PCR has been used on historic bird
specimens. Direct PCR allows researchers to avoid DNA extraction, which is a possible source of cross-transfer of
DNA between samples and modern DNA contamination.
Genetic change over time will continue to be a important factor in worldwide conservation and ecosystem
restoration efforts. Historic genetic information will be important to consider when determining contemporary
changes in genetic diversity of wildlife populations .
References
Swabs soaking in distilled water.
Coplin jars with As test strips.
Table 1. This table shows all primes used for PCR amplification in this study. Each species
primer is from a separate mtDNA gene: Franklin’s Gull (Krmpotich, unpublished) – The
Control Region, Red-winged Blackbird - Cytochrome Oxidase 1(COI) gene, Horned Lark –
Nitrogen Dehydrogenase (ND2) gene .
Species
Franklin's Gull
Franklin's Gull
Red-winged Blackbird
Red-winged Blackbird
Horned Lark
Horned Lark
Horned Lark
Horned Lark
Horned Lark
Horned Lark
Franklin's Gull
Age Group ~50 Years
Species
Horned Lark
Red-winged Blackbird
Horned Lark
Horned Lark
Horned Lark
Age Group ~25 Years
Species
- Three areas (head, sampling area, and toe pad) on each of our 15 specimens were
tested
- PCR products were run out on an
agarose gel for identification of products
of the correct size.
The Ovchinnikov Laboratory’s special set up for
work with low copy and degraded DNA.
Table 2. Successful PCR reactions with each extraction method listed by museum specimen. Museum specimens are listed by species and year collected. Size
indicated the ideal PCR product size from primer design. Successful PCR reactions (Y) and unsuccessful PCR reactions(N) are listed under the designated
extraction method.
Age Group ~100 Years
PCR Success
Species
Year
Size (bp)
Chelex
Zymo
Direct
Clark, G. A. 1973. Notched toe pads in climbing oscines. Condor 75: 119-120.
Marte, F., Pequignot, A., Von Endt, D.W. 2006. Arsenic in Taxidermy collections: History, Detection , and Management. Collection Forum 21:143-150.
Payne, R.B. and Sorenson, M.D. 2002. Museum collections as sources of genetic data. Bonn. Zool. Beitr. 51:97–104.
Töpfer, T., Gamaug, A., Haring, E. 2011. Utility of Arsenic – Treated Bird Skins for DNA Extraction. BMC Research Notes 4:197.
Acknowledgements
Funding was provided by: ND EPSCoR Faculty Seed Money to IO, Ovchinnikov Lab Start-Up Fund, Ester Wadsworth Hall Wheeler Award
to GC. We would like to thank University of Minnesota Duluth (Gerald Niemi), for allowing us to sample museum specimens. Special
thanks to Rebbeca Simmons and AnnMarie Krmpotich for Franklin’s Gull primers, and Ken Drees for technical advice in the lab.
Test Strips for FG1 and HL1.
Known concentration test strips.