Molecular Ecology Notes (2003)

doi: 10.1046/j.1471-8286 .2003.00356.x

PRIMER NOTE
Blackwell Science, Ltd

Polymorphic microsatellite loci for tiger salamanders,
Ambystoma tigrinum
S . G . M E C H ,* A . S T O R F E R ,* J . A . E R N S T ,† M . W . R E U D I N K * and S . C . M A L O N E Y *
*School of Biological Sciences, Washington State University, Pullman, WA 99164– 4236, USA, †Department of Wildlife Ecology and
Conservation, University of Florida, Gainesville, FL32611, USA

Abstract
Microsatellites have been developed for few amphibian species. However, developing
genetic markers for population genetic studies in amphibians is critical because amphibians are declining globally. The tiger salamander, Ambystoma tigrinum, is widespread
throughout the United States and includes the endangered subspecies, A. t. stebbinsi. We
present primers and amplification conditions for 10 polymorphic microsatellite loci that
have produced successful results in three subspecies of A. tigrinum. Number of alleles per
locus ranged from one to 11 and heterozygosity ranged from 0 to 0.815 depending on the
subspecies and locus analysed. These markers should prove useful for future studies of
genetic diversity and population subdivision.
Keywords: Ambystoma tigrinum, amphibian, microsatellite, tiger salamander
Received 23 August 2002; revision received 15 October 2002; accepted 15 October 2002

Microsatellites have been developed for few amphibian
species, including the Pacific chorus frog (Hyla regilla) and
Northern spotted frog (Rana lutiventris; Call & Hallett
1998), the common toad (Bufo bufo; Brede et al. 2001), the
natterjack toad (Bufo calamita; Rowe et al. 1997), newts
(Triturus cristatus, and T. marmoratus; Jehle et al. 2001), and
Pacific giant salamanders (Dicamptodon tenebrosus; Curtis &
Taylor 2000). Developing useful genetic markers for population genetic studies in amphibians is critical because
amphibians are indicator species of habitat degradation
and are now declining globally (Wake 1998).
The tiger salamander, Ambystoma tigrinum, is widespread throughout the United States, southern Canada,
and Mexico (Gehlbach 1967). The species complex contains
eight subspecies, including the endangered A. t. stebbinsi.
Mitochondrial DNA markers, such as the control region
and an insert between proline and threonine tRNAs have
been developed for A. tigrinum, but have not been useful for detecting fine-scale population structure (Shaffer
& McKnight 1996). Thus, genetic markers with high resolution, such as microsatellites, are needed to conduct
population genetic studies within subspecies (Goldstein &
Schlötterer 1999). We developed a microsatellite library for
Correspondence: A. Storfer. Fax: (509) 332 1487; E-mail:
astorfer@wsu.edu
© 2003 Blackwell Publishing Ltd

A. tigrinum that yielded 10 polymorphic loci that successfully amplified in three subspecies.
Genomic DNA was isolated using Puregene® DNA
(Gentra, Inc.) isolation kits following a modified version of
the standard animal tissue isolation protocol. Our modifications followed three basic steps: (i) tissues were homogenized in Puregene® cell lysis buffer and incubated at
55 °C for 1 h with 10 µL of 20 mg/mL Proteinase-K solution;
(ii) tissues were re-homogenized and incubated at 55 °C
overnight; (iii) after incubation, samples were homogenized a third time. Genomic DNA was then digested with
Sau3AI restriction enzyme and 400–1500 bp fragments
were selected via agarose gel electrophoresis and size fractionation using Chromo Spin® columns (Clonetech Laboratories). We then ligated fractionated DNA to Sau3AI
linkers and amplified using standard PCR conditions and
followed a standard enrichment protocol using Vectrex
Avidin D matrix (Vector Laboratories, Inc.) to create a
library enriched for (CA)n and (GA)n repeats. PCR products from the enriched library were then directly ligated to
a TOPO vector (Invitrogen, Inc.). We then transformed
plasmid vectors into One Shot™ E. coli (Invitrogen, Inc.).
Colonies were screened using standard nitrocellulose
membranes and hybridized with a (CA)n chemiluminescent probe (Lifecodes, Inc.) and sprayed with Lumi-Phos
480 (Lifecodes, Inc.). Positive clones were then detected

Heterozygosity†

© 2003 Blackwell Publishing Ltd, Molecular Ecology Notes, 3, 79–81

No. of
cycles

Allele size
range (bp)

mavortium
nebulosum
stebbinsi
321,
mavortium
325–347, 372 nebulosum
stebbinsi
229 – 237
mavortium
nebulosum
stebbinsi
193,
mavortium
199 – 207
nebulosum
stebbinsi
240 –244,
mavortium
252–268, 282 nebulosum
stebbinsi
354 – 366
mavortium
nebulosum
stebbinsi
296 – 302
mavortium
nebulosum
stebbinsi
193 – 200,
mavortium
211– 225
nebulosum
stebbinsi
311– 341, 369 mavortium
nebulosum
stebbinsi
103–109,
mavortium
125–129
nebulosum
stebbinsi

Locus
Repeat
(GenBank ID) Unit

Primer Sequence*

ATS4-11
(AY157747)

(GA)3TT(GA)3AT(GA)5

F: GTGTTACCCACCTTTTTCTATCCC 1.0
R: GGCCGCACAACCTAAGTG

56.5 (30)

50

32

ATS4-20
(AY157748)

(CA)9

F: TGTTTTGCCCTTATGTCG
R: GCCCAAATCCTAAAGAGTAAGT

1.5

59.5 (30)

50

31

ATS4-25
(AY157749)

(CA)7

F: ATAGGGGCCTCAAGTTAAG
R: GGCTACTAGATGGCGTTGT

1.5

56 (30)

50

30

ATS5-6
(AY157750)

(CA)3TA(CA)4TC(CA)7

F: TATGGGTGAGAATCTAGGAG
R: GCACCATCCAGTCAAAC

1.5

50.9 (30)

75

30

ATS5-7
(AY157751)

(CA)10

F: GGGCTTGAATCATGTAGTGG
R: GGGAAGACTAGATGGCAATAAC

1.5

54.7 (50)

70

32

ATS5-8
(AY157752)

(CA)11

F: AGTCCCTCTCTATCTAATCTCG
R: ATTCTCCTGCCTGTATGTTT

1.5

56.5 (50)

50

34

ATS10-7
(AY157753)

(GA)6

F: GAGGCAGGATGATTTAGA
R: CTTGGCATTACTGATTAGG

2.0

58 (50)

70

31

ATS12-3
(AY157754)

(CA)8CG(CA)3CG(CA)14

F: TGTAGCAGAAGACGGGTAT
R: AGTAAAGCGAAGATATGGG

1.5

52.8 (30)

50

34

ATS13-1
(AY157755)

(CA)12TTCGCGCG(CA)17 F: AGGTCTTCTTACAGCACAA
R: TCACCAGGGTAGGGATA

1.5

56.3 (50)

70

33

ATS14-3
(AY157756)

(CA)18

1.5

56 (30)

50

31

F: GGGCACTGAAACGGAACACT
R: CCCCAAATGGCGTCCCT

MgCl2
(mM)

Annealing
Temp (°C)/ Extension
Time (s)
Time (s)

*F and R are forward and reverse primers, respectively.
†HO and HE are observed and expected heterozygosities, respectively, estimated using biosys (Swofford & Selander 1981).
‡A. t. nebulosum sample is fixed for allele 372. Neither other subspecies had this allele.

175, 187–193

Sample
Subspecies Size
18
17
15
17
26
19
20
27
19
20
27
19
19
27
19
20
27
19
20
26
19
20
23
19
17
18
19
19
27
18

No. of
alleles

HE

HO

4
3
2
11
1‡
2
4
3
3
2
6
2
6
8
1
3
4
1
3
1
3
5
4
2
3
9
2
2
4
2

0.640
0.469
0.129
0.889
0.000
0.512
0.706
0.671
0.284
0.481
0.715
0.053
0.647
0.751
0.000
0.445
0.240
0.000
0.678
0.000
0.432
0.501
0.661
0.102
0.314
0.813
0.491
0.149
0.312
0.203

0.722
0.353
0.000
0.765
0.000
0.000
0.500
0.519
0.000
0.350
0.815
0.053
0.105
0.704
0.000
0.400
0.185
0.000
0.500
0.000
0.000
0.400
0.087
0.000
0.235
0.111
0.474
0.053
0.296
0.000

80 P R I M E R N O T E

Table 1 Microsatellite primer information for Ambystoma tigrinum

P R I M E R N O T E 81
using standard autoradiography film and sequenced using
M13 primers on an ABI 377 automated sequencer. Visual
inspection was used to confirm presence of repeats and to
determine whether there was sufficient flanking sequence
to construct PCR primers.
Primer pairs for selected loci were developed using
oligo 6.0 software (Molecular Biology Insights, Inc.). One
primer from each set was fluorescently labelled. Optimization for each primer pair was carried out using individuals
from two common subspecies from Arizona (A. t. nebulosum and A. t. mavortium), and one endangered subspecies
(A. t. stebbensi). DNA was extracted from adult and larval
tail tissue stored in 70% ethanol using Qiagen’s DNAEasy
kit or standard Phenol/Chloroform extractions. Reaction
volumes were optimized using 13 µL TV consisting of
50 mm KCl, 100 mm Tris-HCl, 0.96 mm each dNTP, 25 µm
each oligonucleotide, 1 unit of Taq polymerase (Fisher), 75 –
150 ng template DNA and primer specific MgCl2 concentrations (Table 1).
All PCR profiles began with 2 min at 94 °C (denaturation), followed by a primer-specific number of cycles of 30
s 94 °C denaturation, primer-specific annealing time and
temperature, and primer-specific extension time at 72 °C
(see Table 1 for primer-specific data). Each profile ended
with 5 min at 72 °C to complete the products. Optimization
and amplification reactions were performed on a Bio-Rad
iCycler® gradient thermocycler. All products were run on
an ABI 377 Automated Sequencer using ROX 500 size
standard and analysed using genescan and genotyper
software (ABI).
Of the 15 primer-pairs tested, three were not variable
and two others could not be optimized. The remaining 10
loci showed variation in at least one subspecies (Table 1).
Observed and expected heterozygosities were calculated and tested using the genepop 3.3 software package
(Raymond & Rousset 1995) and biosys (Swofford & Selander
1981). Four loci (ATS4-25, ATS5-7, ATS10-7 and ATS12-3)
had significant heterozygote deficiencies in A. t. mavortium,
and one locus (ATS13-1) had a heterozygote deficiency in
A. t. stebbinsi; note also that this locus works well for A. t.
stebbinsi, but has shown limited success with other tiger

© 2003 Blackwell Publishing Ltd, Molecular Ecology Notes, 3, 79–81

salamander subspecies. Appropriate repeat sizes for alleles
with substantially different lengths were confirmed by
sequencing individuals homozygous for the allele.

Acknowledgements
We thank the BEECS lab at University of Florida for assistance
with initial cloning, and K. Lew for assistance in the lab. This work
was funded by NSF IBN-9977063 to A.S.

References
Brede EG, Rowe G, Trojanowski J, Beebee TJC (2001) Polymerase
chain reaction primers for microsatellite loci in the Common
Toad Bufo bufo. Molecular Ecology Notes, 1, 308–310.
Call DR, Hallett JG (1998) PCR primers for microsatellite loci in the
anurans Rana luteiventris and Hyla regilla. Molecular Ecology, 7,
1085–1087.
Curtis JM, Taylor EB (2000) Isolation and characterization of
microsatellite loci in the Pacific giant salamander, Dicamptodon
tenebrosus. Molecular Ecology, 9, 116–118.
Gehlbach FR (1967) Ambystoma Figrinum (Green). Catalogue of
American Amphibians and Reptiles. pp. 52.1–52.4. American
Society of Ichthyologists and Herpotologists. Kansas City, MO.
Goldstein DB, Schlötterer C (1999) Microsatellites: Evolution and
Applications. Oxford University Press, Oxford.
Jehle R, Arntzen JW, Burke T, Krupa AP, Hoedl W (2001) The
annual number of breeding adults and the effective population
size of syntopic newts (Triturus cristatus, T. marmoratus). Molecular Ecology, 10, 839–850.
Raymond M, Rousset F (1995) genepop Version 1.2.: population
genetics software for exact tests and ecumenicism. Journal of
Heredity, 86, 248–249.
Rowe G, Beebee TJC, Burke T (1997) PCR primers for polymorphic
microsatellite loci in the anuran amphibian Bufo calamita. Molecular Ecology, 6, 401–402.
Shaffer HB, McKnight ML (1996) The polytypic species revisited:
Genetic differentiation and molecular phylogenetics of the tiger
salamander, Ambystoma tigrinum (Amphibia: Caudata) Complex.
Evolution, 50, 417–433.
Swofford DL, Selander RB (1981) biosys-1: a Fortran program
for the comprehensive analysis of electrophoretic data in
population genetics and systematics. Journal of Heredity, 72,
281–283.
Wake DB (1998) Action on amphibians. Trends in Ecology and Evolution, 13, 379–380.