Appendix S1. Conceptual flowchart for developing new microsatellite markers based on the enrichment
technique (one of many methods that are in use – see Zane et al. 2004 and Glenn 2005), and primer
optimization steps.
A. Extract DNA from a single tissue sample.
B. Create a DNA library:
1. Cut the genome into 500 bp fragments pieces with a restriction enzyme digest.
2. Attach ‘linker’ DNA to the ends of each fragment – linker DNA has a known sequence so that
primers can be designed to bind to them.
3. Amplify the DNA fragments using primers for the linker ends with PCR.
C. Separate out fragments with repeat sequences:
1. Mix the DNA fragments with a microsatellite probe (an oligonucleotide made of a repeat
sequence of your choice) that can be recovered magnetically.
2. Promote the hybridization of probes to any complementary repeat sequences in the DNA
fragments by heating to denature the DNA and cooling slowly.
3. Hold a magnet to the tube to attract the probes (now bound to the DNA), and wash away the
rest of the unbound DNA with a series of rinses.
D. Sequence the fragments to find microsatellite loci:
1. Using primers for the linker DNA, amplify DNA with PCR to concentrate it.
2. Clone the DNA to prepare it for sequencing - insert it into a plasmid, inoculate bacteria with the
plasmid, grow the bacteria to replicate the DNA.
3. Isolate the DNA from the bacteria.
4. Sequence microsatellite DNA in the plasmid with primers targeted to the insertion points on the
plasmid.
E. Examine the sequences to find microsatellite repeats.
F. Design primers for the flanking region of the microsatellites (with help from a primer selection software
program such as Primer3 which selects optimal primer sites) and have them made.
G. Attempt amplification of loci with the new primers. Use a gradient of PCR conditions in which the
temperatures, times, magnesium and primer concentrations vary to find optimal conditions.
H. Use gel electrophoresis to confirm the presence of PCR products. Discard primer pairs that fail to
amplify after several attempts.
I. Check for polymorphism by running the successful primer pairs on 10-20 individuals. Estimate allelic
diversity and heterozygosity levels. Discard invariant loci.
J. Check for reliability. Rerun the successful primer pairs on the same individuals twice more to ensure
that genotype scoring is consistently reproducible. Discard loci with unreliable amplification.
H. Order fluorescently labeled primers for the remaining loci. Complete the full screening process detailed
in the text. Discard problematic loci.
K. Streamline the genotyping of the full dataset with the remaining loci by establishing a “multiplex” PCR
protocol – primers for multiple loci (labeled with different dyes) are amplified in a single PCR reaction.
1
Appendix S2. Scoring microsatellite genotypes from sequencer data output.
Background on microsatellite genotyping with a DNA sequencer
A DNA sequencer is a highly precise gel electrophoresis apparatus. PCR products are loaded
onto the gel and separated by size by applying a charge. A laser scans the gel to detect bands
containing fluorescent dye. The primers used in the PCR reaction are tagged with different fluorescent
dyes to enable this detection. The sequencer software converts the banding pattern into a plot with
peaks corresponding to the width and intensity (height) of each band. The position of the peak along the
x-axis corresponds to the size of the DNA product in the band measured in base pairs (BP). The
height/intensity corresponds to the concentration of the DNA product, which is a consequence of the
efficiency of the amplification process in PCR. One color is used for a size standard to calibrate the band
positions with the size of the DNA product (here it is red). An asterisk identifies all true microsatellite
alleles in the figures below.
A
B
C
D
100
BP
125
150
175
200
A An ideal output: The two alleles of this heterozygote are even in height and easy to distinguish from
the “stutter peaks” adjacent to them – during PCR some products are 1, 2 or 3 repeats short due to errors
in replication (similar to step-wise mutation) and show up as evenly spaced peaks with decreasing height
to the left of the true peak. Some loci show extensive stuttering and others show virtually none. The
stutter bands are useful for distinguishing microsatellite products from non-specific or non-target products.
Notice that there are “pull-up peaks” in the green section of the spectrum. Pull-up is due primarily to
spectral overlap in the emission spectra of the dyes which the sequencer records as a false peak in a
different color. This is a common artifact of the DNA sequencer’s analysis process that can create
confusion in some situations.
B Two examples of trickier outputs: The blue genotype is a heterozygote but the 2 alleles are only 1
repeat different in size. This creates a characteristic pattern for loci with stutter in which the second peak
is higher than the first because of the additive intensity of the larger allele’s first stutter peak and the
smaller allele’s true peak. If the first two peaks were equal in height it would be difficult to determine if the
2
genotype has one or two alleles. One clue is that there are 4 stutter peaks to the right of the largest peak
instead of 3 (assuming the pattern from plot A is characteristic of the blue locus). The second locus in
this plot is shown with black dye and has larger alleles. This locus has no stutter perhaps because there
are few repeats so that the Taq polymerase doesn’t make stepwise errors. This genotype is also a
heterozygote, but the larger allele is faint. Larger alleles usually show at least slightly shorter peaks
because PCR is less efficient for longer products. Here, the larger allele is so small that it could be easily
overlooked or mistaken for noise. If any less PCR product were loaded onto the gel it might not show up
at all – in which case it would be an example of “large allele drop-out,” another common source of inflated
homozygosity counts.
C More examples: When multiple loci are loaded in the same gel lane for efficiency, or amplified together
in one PCR “multiplex” reaction, allele peaks can overlap and be sometimes easy to miss. Here the
green and blue loci share an allele size. Distinguishing the true alleles is made even more difficult due to
the occurrence of green pull-up peaks. If the green allele product were less intense than the blue allele
product (instead of equal as shown here), it might be mistaken for a pull up peak. Re-running the green
locus separately in such cases will minimize scoring error. Here again, the blue locus shows two alleles
that differ by one base pair. The heights are the same because this locus does not show strong stutter.
But the blue locus does show small flanking peaks – the left side is a very faint stutter so only the largest
stutter peak is visible, and the right side might be an “A-Tail,” when the Taq adds an extra adenine
nucleotide onto some copies of the product, increasing it by 1 bp. It will not be mistaken for a
microsatellite allele because an extreme height difference would not occur for 2 alleles so close in size,
as their amplification efficiencies should be similar. The black locus is a homozygote. Even though there
is a small black peak on the left side of the plot, a smaller allele is almost never shorter than a larger
allele. An additional clue is that the larger black peak is quite fat and tall -- PCR produces approximately
double the amount of product when an allele is homozygous because it does not compete for the Taq
with a second allele. The purple locus represents a “split peak” problem that occurs from high rates of ATailing by the Taq. The “+A” peaks occur for all of the stutter peaks, making the scoring difficult,
especially for heterozygotes with closely sized alleles. Although the true allele is denoted here, a locus
with alleles that look like the purple one would be too difficult to score reliably. Usually the problem can be
corrected by adding an extra extension step to the PCR program that gives the Taq time to add an A-Tail
to all copies of the product consistently (bumping all alleles and stutter peaks up in size by 1 bp from their
true length).
D Unscorable loci: The blue locus is a “stegosaur” with unacceptably high stutter. The black locus has
too many non-specific artifact peaks to reliably choose the microsatellite alleles. The green locus has
been overloaded on the gel or has unusually high PCR product concentration and is smearing in the lane.
3
Appendix S3. Citations for the papers used to generate Table 3. We examined the most recent
papers in the journals Molecular Ecology and Evolution and chose the first 25 from each journal,
in reverse chronological order from July 2005 issues, that used microsatellites to assess
ecological and genetic traits of single or multiple species. We excluded studies that used
microsatellite markers taken from previously published research studies to minimize the chance
that some tests were done previously and therefore not reported in the current study. We then
surveyed these papers to estimate the frequency and results of reported marker screening as
explained in Part III of the text and Table 2.
Molecular Ecology
Astanei, I., Gosling, E., Wilson, J. & Powell, E. (2005). Genetic variability and phylogeography
of the invasive zebra mussel, Dreissena polymorpha (Pallas). Molecular Ecology, 14,
1655-1666.
Baums, I.B., Miller, M.W. & Hellberg, M.E. (2005). Regionally isolated populations of an
imperiled Caribbean coral, Acropora palmata. Molecular Ecology, 14, 1377-1390.
Bottin, L., Verhaegen, D., Tassin, J., Olivieri, I., Vaillant, A. & Bouvet, J.M. (2005). Genetic
diversity and population structure of an insular tree, Santalum austrocaledonicum in New
Caledonian archipelago. Molecular Ecology, 14, 1979-1989.
Bowen, B.W., Bass, A.L., Soares, L. & Toonen, R.J. (2005). Conservation implications of
complex population structure: lessons from the loggerhead turtle (Caretta caretta).
Molecular Ecology, 14, 2389-2402.
Charmantier, A. & Reale, D. (2005). How do misassigned paternities affect the estimation of
heritability in the wild? Molecular Ecology, 14, 2839-2850.
Colautti, R.I., Manca, M., Viljanen, M., Ketelaars, H.A.M., Burgi, H., Macisaac, H.J. & Heath,
D.D. (2005). Invasion genetics of the Eurasian spiny waterflea: evidence for bottlenecks
and gene flow using microsatellites. Molecular Ecology, 14, 1869-1879.
Fredsted, T., Pertoldi, C., Schierup, M.H. & Kappeler, P.M. (2005). Microsatellite analyses
reveal fine-scale genetic structure in grey mouse lemurs (Microcebus murinus).
Molecular Ecology, 14, 2363-2372.
Funk, W.C., Blouin, M.S., Corn, P.S., Maxell, B.A., Pilliod, D.S., Amish, S. & Allendorf, F.W.
(2005). Population structure of Columbia spotted frogs (Rana luteiventris) is strongly
affected by the landscape. Molecular Ecology, 14, 483-496.
Goossens, B., Chikhi, L., Jalil, M.F., Ancrenaz, M., Lackman-Ancrenaz, I., Mohamed, M.,
Andau, P. & Bruford, M.W. (2005). Patterns of genetic diversity and migration in
increasingly fragmented and declining orang-utan (Pongo pygmaeus) populations from
Sabah, Malaysia. Molecular Ecology, 14, 441-456.
Hauswaldt, J.S. & Glenn, T.C. (2005). Population genetics of the diamondback terrapin
(Malaclemys terrapin). Molecular Ecology, 14, 723-732.
Jones, K.L., Krapu, G.L., Brandt, D.A. & Ashley, M.V. (2005). Population genetic structure in
migratory sandhill cranes and the role of Pleistocene glaciations. Molecular Ecology, 14,
2645-2657.
Keeney, D.B., Heupel, M.R., Hueter, R.E. & Heist, E.J. (2005). Microsatellite and mitochondrial
DNA analyses of the genetic structure of blacktip shark (Carcharhinus limbatus)
nurseries in the northwestern Atlantic, Gulf of Mexico, and Caribbean Sea. Molecular
Ecology, 14, 1911-1923.
4
Magalon, H., Adjeroud, M. & Veuille, M. (2005). Patterns of genetic variation do not correlate
with geographical distance in the reef-building coral Pocillopora meandrina in the South
Pacific. Molecular Ecology, 14, 1861-1868.
Maki-Petays, H., Zakharov, A., Viljakainen, L., Corander, J. & Pamilo, P. (2005). Genetic
changes associated to declining populations of Formica ants in fragmented forest
landscape. Molecular Ecology, 14, 733-742.
McRae, B.H., Beier, P., Dewald, L.E., Huynh, L.Y. & Keim, P. (2005). Habitat barriers limit
gene flow and illuminate historical events in a wide-ranging carnivore, the American
puma. Molecular Ecology, 14, 1965-1977.
Mesquita, N., Hanfling, B., Carvalho, G.R. & Coelho, M.M. (2005). Phylogeography of the
cyprinid Squalius aradensis and implications for conservation of the endemic freshwater
fauna of southern Portugal. Molecular Ecology, 14, 1939-1954.
Michaux, J.R., Hardy, O.J., Justy, F., Fournier, P., Kranz, A., Cabria, M., Davison, A., Rosoux,
R. & Libois, R. (2005). Conservation genetics and population history of the threatened
European mink Mustela lutreola, with an emphasis on the west European population.
Molecular Ecology, 14, 2373-2388.
Otero-Arnaiz, A., Casas, A., Hamrick, J.L. & Cruse-Sanders, J. (2005). Genetic variation and
evolution of Polaskia chichipe (Cactaceae) under domestication in the Tehuacan Valley,
central Mexico. Molecular Ecology, 14, 1603-1611.
Oyler-McCance, S.J., Taylor, S.E. & Quinn, T.W. (2005). A multilocus population genetic
survey of the greater sage-grouse across their range. Molecular Ecology, 14, 1293-1310.
Shrivastava, J., Qian, B.Z., McVean, G. & Webster, J.P. (2005). An insight into the genetic
variation of Schistosoma japonicum in mainland China using DNA microsatellite
markers. Molecular Ecology, 14, 839-849.
Spear, S.F., Peterson, C.R., Matocq, M.D. & Storfer, A. (2005). Landscape genetics of the
blotched tiger salamander (Ambystoma tigrinum melanostictum). Molecular Ecology, 14,
2553-2564.
Sutherland, D.R., Spencer, P.B.S., Singleton, G.R. & Taylor, A.C. (2005). Kin interactions and
changing social structure during a population outbreak of feral house mice. Molecular
Ecology, 14, 2803-2814.
Westneat, D.F. & Mays, H.L. (2005). Tests of spatial and temporal factors influencing extra-pair
paternity in red-winged blackbirds. Molecular Ecology, 14, 2155-2167.
Whitehead, A., Anderson, S.L., Kuivila, K.M., Roach, J.L. & May, B. (2003). Genetic variation
among interconnected populations of Catostomus occidentalis: implications for
distinguishing impacts of contaminants from biogeographical structuring. Molecular
Ecology, 12, 2817-2833.
Wright, T.F., Rodriguez, A.M. & Fleischer, R.C. (2005). Vocal dialects, sex-biased dispersal,
and microsatellite population structure in the parrot Amazona auropalliata. Molecular
Ecology, 14, 1197-1205.
Evolution
Allendorf, F.W. & Seeb, L.W. (2000). Concordance of genetic divergence among sockeye
salmon populations at allozyme, nuclear DNA, and mitochondrial DNA markers.
Evolution, 54, 640-651.
5
Arnegard, M.E., Bogdanowicz, S.M. & Hopkins, C.D. (2005). Multiple cases of striking genetic
similarity between alternate electric fish signal morphs in sympatry. Evolution, 59, 324343.
Bacles, C.F.E., Burczyk, J., Lowe, A.J. & Ennos, R.A. (2005). Historical and contemporary
mating patterns in remnant populations of the forest tree Fraxinus excelsior L. Evolution,
59, 979-990.
Castric, V., Bonney, F. & Bernatchez, L. (2001). Landscape structure and hierarchical genetic
diversity in the brook charr, Salvelinus fontinalis. Evolution, 55, 1016-1028.
Chapuisat, M., Bocherens, S. & Rosset, H. (2004). Variable queen number in ant colonies: No
impact on queen turnover, inbreeding, and population genetic differentiation in the ant
Formica selysi. Evolution, 58, 1064-1072.
Clarke, K.E., Rinderer, T.E., Franck, P., Quezada-Euan, J.G. & Oldroyd, B.P. (2002). The
Africanization of honeybees (Apis mellifera L.) of the Yucatan: A study of a massive
hybridization event across time. Evolution, 56, 1462-1474.
Dutech, C., Maggia, L., Tardy, C., Joly, H.I. & Jarne, P. (2003). Tracking a genetic signal of
extinction-recolonization events in a neotropical tree species: Vouacapoua americana
aublet in french guiana. Evolution, 57, 2753-2764.
Evans, B.J., Supriatna, J. & Melnick, D.J. (2001). Hybridization and population genetics of two
macaque species in Sulawesi, Indonesia. Evolution, 55, 1686-1702.
Goodisman, M.A.D. & Crozier, R.H. (2002). Population and colony genetic structure of the
primitive termite Mastotermes darwiniensis. Evolution, 56, 70-83.
Hansson, B., Westerdahl, H., Hasselquist, D., Akesson, M. & Bensch, S. (2004). Does linkage
disequilibrium generate heterozygosity-fitness correlations in great reed warblers?
Evolution, 58, 870-879.
Hendry, A.P., Taylor, E.B. & McPhail, J.D. (2002). Adaptive divergence and the balance
between selection and gene flow: Lake and stream stickleback in the misty system.
Evolution, 56, 1199-1216.
Heuertz, M., Hausman, J.F., Hardy, O.J., Vendramin, G.G., Frascaria-Lacoste, N. & Vekemans,
X. (2004). Nuclear microsatellites reveal contrasting patterns of genetic structure between
western and southeastern European populations of the common ash (Fraxinus excelsior
L.). Evolution, 58, 976-988.
Hoffman, J.I., Boyd, I.L. & Amos, W. (2003). Male reproductive strategy and the importance of
maternal status in the antarctic fur seal Arctocephalus gazella. Evolution, 57, 1917-1930.
Hufford, K.M. & Hamrick, J.L. (2003). Viability selection at three early life stages of the tropical
tree, Platypodium elegans (Fabaceae, Papilionoideae). Evolution, 57, 518-526.
Lampert, K.P., Lamatsch, D.K., Epplen, J.T. & Schartl, M. (2005). Evidence for a monophyletic
origin of triploid clones of the Amazon molly, Poecilia formosa. Evolution, 59, 881-889.
Noor, M.A.F., Pascual, M. & Smith, K.R. (2000). Genetic variation ln the spread of Drosophila
subobscura from a nonequilibrium population. Evolution, 54, 696-703.
Peakall, R., Ruibal, M. & Lindenmayer, D.B. (2003). Spatial autocorrelation analysis offers new
insights into gene flow in the Australian bush rat, Rattus fuscipes. Evolution, 57, 11821195.
Pujolar, J.M., Maes, G.E., Vancoillie, C. & Volckaert, F.A.M. (2005). Growth rate correlates to
individual heterozygosity in the european eel, Anguilla anguilla L. Evolution, 59, 189199.
6
Scribner, K.T., Arntzen, J.W., Cruddace, N., Oldham, R.S. & Burke, T. (2001). Environmental
correlates of toad abundance and population genetic diversity. Biol. Conserv., 98, 201210.
Storz, J.F. (2002). Contrasting patterns of divergence in quantitative traits and neutral DNA
markers: analysis of clinal variation. Molecular Ecology, 11, 2537-2551.
Storz, J.F., Bhat, H.R. & Kunz, T.H. (2001). Genetic consequences of polygyny and social
structure in an Indian fruit bat, Cynopterus sphinx. I. Inbreeding, outbreeding, and
population subdivision. Evolution, 55, 1215-1223.
Thelen, G.C. & Allendorf, F.W. (2001). Heterozygosity-fitness correlations in rainbow trout:
Effects of allozyme loci or associative overdominance? Evolution, 55, 1180-1187.
Turgeon, J. & Bernatchez, L. (2001). Clinal variation at microsatellite loci reveals historical
secondary intergradation between glacial races of Coregonus artedi (Teleostei:
Coregoninae). Evolution, 55, 2274-2286.
Vargo, E.L. (2003). Hierarchical analysis of colony and population genetic structure of the
eastern subterranean termite, Reticulitermes flavipes, using two classes of molecular
markers. Evolution, 57, 2805-2818.
Vassiliadis, C., Saumitou-Laprade, P., Lepart, J. & Viard, F. (2002). High male reproductive
success of hermaphrodites in the androdioecious Phillyrea angustifolia. Evolution, 56,
1362-1373.
7