Amphibia-Reptilia 33 (2012): 533-536

New highly polymorphic microsatellite loci for the Galápagosmarine iguana, Amblyrhynchus cristatus

Amy MacLeod1,∗, Volker Koch2,3, Carolina García-Parra2, Fritz Trillmich1, Sebastian Steinfartz1

Abstract. We describe the development and characterisation of six new dinucleotide motif microsatellite loci for populationsof marine iguanas (Amblyrhynchus cristatus), endemic to the Galápagos archipelago. Primers were based on microsatellite-bearing sequences and initially developed using universally labelled primers. When analysed across 5 populations (repre-senting 150 individuals), new loci displayed, on average, high levels of genetic diversity (range: 2-13 alleles, mean: 5.73)and values of heterozygosity (range: 0.0-0.906, mean: 0.605). No consistent deviations from Hardy-Weinberg equilibriumor significant linkage disequilibrium were observed, and all loci were shown to be free of common microsatellite errors.Utilising the 13 previously available microsatellite loci for this species, we describe here four multiplex combinations for thesuccessful amplification of 19 microsatellite loci for marine iguanas. This powerful set of highly polymorphic markers willallow researchers to explore future questions regarding the ecology, evolution, and conservation of this unique species.

Keywords: Population genetics, genetic effective population size, island populations, primer development.

Among the many biological fascinations of theGalápagos Islands, the endemic marine iguana,Amblyrhynchus cristatus, counts among themost ancient (Rassmann, 1997) and famous ver-tebrates. As the world’s only lizard with an am-phibious lifestyle, A. cristatus occurs on 13 ma-jor islands of the archipelago, where it can befound basking along the shoreline and feedingalmost exclusively on algae in the intertidal andshallow subtidal zone (Trillmich and Trillmich,1986). The unique life history and island de-pendent distribution of this iguana provides anexceptional system to explore ecological andevolutionary questions. To date, analysis of 13microsatellite loci have revealed historical pat-terns of progressive colonisation from older toyounger islands across the archipelago, and al-most no contemporary gene flow between is-land populations (Steinfartz et al., 2009). Al-

1 - Department of Behavioural Biology, Unit of MolecularEcology and Behaviour, University of Bielefeld, Mor-genbreede 45, D-33619 Bielefeld, Germany

2 - Charles Darwin Research Station, Puerto Ayora, SantaCruz Island, Galápagos, Ecuador

3 - Investigación para la Conservación y el Desarrollo, LaPaz, BCS, México∗Corresponding author; e-mail:ms.amymacleod@gmail.com

though the total number of animals is thoughtto be hundreds of thousands, there is mountingconcern for some genetically distinct popula-tions (Steinfartz et al., 2009), where census sizenumber estimates may be as low as 50 (Wikel-ski and Nelson, 2004). At present, discrete dataon census population sizes are severely lacking,and existing genetic estimates of effective popu-lation size (e.g. Steinfartz et al., 2007) couldstill be improved. Increasing the number of mi-crosatellite loci reduces bias and improves pre-cision in genetic effective population size es-timation, without the need to increase samplesize (Palstra and Ruzzante, 2008). Here we de-scribe the development and characterisation ofprimers to amplify six new polymorphic mi-crosatellite loci, which can be subsequently em-ployed in combination with the 13 existing locito explore the population genetics, evolution,and conservation status of marine iguanas withmuch greater resolution.

Based on genomic sequences bearing microsatellite din-ucleotide motifs deposited in GenBank (see table 1 for Gen-Bank accession numbers), primers were designed for 12 po-tential loci using PRIMER 3 software (Rozen and Skaletsky,2000). These 12 loci were subsequently tested for polymor-phisms using the ‘M13 (-21)-tail’ method (Schuelke, 2000).In order to ensure that the new loci will be polymorphicfor all sampled marine iguana populations, we performedthe initial test of polymorphism for twelve individuals of

© Koninklijke Brill NV, Leiden, 2012. DOI:10.1163/15685381-00002854

534 Short Notes

Table 1. Characterisation of the full set of 19 available microsatellite loci for the Galápagos marine iguana (Amblyrhynchuscristatus). Loci are grouped in amplified multiplex combinations, using the 6 newly developed primer pairs from this study(highlighted in bold) along with the 13 previously published loci (Steinfartz and Caccone, 2006).

Locus GenBank Fluorescence labelling & Repeat No. Size Taaccession primer sequences (5′-3′) motif alleles range (°C)number (bp)

Mul

tiple

x1

MIG-E3 DQ376112.1 F: HEX-GTGTGAGTGACATTTCTGCA (GAAA)4 10 137-159 60R: TGAAAGTATGCTTTGCTCCCTTTGC (GA)2

MIG-E4 DQ376113.1 F: HEX-TTGAGCTAAGTGGGAAAAGAAGAC (GT)22 20 226-276 57R: AAAGTCTTCCCAGGAGATCACAC

MIG-E6 DQ376115.1 F: NED-ACGTCACTGGAGCTGACACA (TG)20 12 150-172 57R: GAACAGTATCTAGGCACTCTCCAAA

MIG-E11 DQ376118.1 F: FAM-CAGTCCATTCTGCTTCCTCA (GT)19 11 163-183 57R: CCTCAAACTCTGCCCTCTTG

MIG-E14 DQ376121.1 F: FAM-AAATTTTCTGCAGTTCTGTTGTCAT (AAGG)20 19 234-326 57R: AGAATCATAGAAGTGGAAGGGACTC

MIG-E1612 DQ376123.1 F: NED-ACTAGCATAATCAGAGGTCATCCTG (GTT)13 9 222-240 57R: ACCAGAGTTCGATTCTCCATTTAG

Mul

tiple

x2

MIG-E8 DQ376116.1 F: HEX-ACCAAGCAAATGGTTTCCAG (AAGG)14 11 152-184 57R: TTGTTCCAAATAGCATAAAATATCA

MIG-E12 DQ376119.1 F: FAM-GGAAGACACTTCAGGCAGCACTTTG (GT)20 13 169-195 57R: TTAGTCAAACCTTTACTCCGACCTG

MIG-E13 DQ376119.1 F: FAM-GAGGATGAACAGATGGTAAGTCAAT (CA)18 9 265-277 57R: AGAACTCTGAGGTATGGAGGAAGAT

MIG-E19 AF452621.1 HEX-TGTGCCAAATGAAGATGAGC (GT)n 10 234-254 60MIG-E19R GCTACTCAATAATCTATGGCATGAAMIG-E22 AY035303.1 NED-AAATTGGCATAGCTGAGAAACA (CT)n(GT)n 8 154-168 59MIG-E22R AATCACTTTCCCAAGCCAAGMIG-E23 AY035299.1 NED-GCATGTCTAATTTCCACTGTGC (GT)n 7 213-225 60MIG-E23R TTCCTAATACCTACAAGTGCCTTAG

Mul

tiple

x3

MIG-E2 DQ376111.1 F: FAM-GTGTGAGTGACATTTCTGCA (GAAA)4 10 229-265 57R: TGAAAGTATGCTTTGCTCCCTTTGC (GA)2

(GAAA)10(GA)2

MIG-E10 DQ376117.1 F:HEX-CCTTATAAATGCTGATCTGGAGCTGT (AAGG)14 13 194-250 57R: CTTTTGCAGTGTTTACTTTTTCCAT

MIG-E15 DQ376122.1 F: FAM-AGACAGGACTGATGTCCTCTAAGAA (TG)19 11 140-160 57R: GGTTGACAACTTATAAGCCTGAAGA

MIG-E21 AY035302.1 F: NED-TTGGCTTTGTAAACTAACACAGTTTC (CA)n 2 160-162 60MIG-E21R R: TGAGCCTACACCATTGGAGA

Mul

tiple

x4

Am(GT)4 F: FAM-TTATGGATGAGCAATAC (GT)n 8 207-223 54Am(GT)4b R: GTATATATGCCTTGTAGMIG-E18 AF297084.1 F: HEX-CATCTGTCCCTACCCTTGCT (GT)n 15 184-216 59MIG-E18R R: TGCTGAACATATGCTTCTCATGTMIG-E20 AY035304.1 NED-TGCAATCATTTTTAAACATTCACA (CT)n 14 195-243 59MIG-E20R AGTGTTCCCAGTTGGACAGC

Locus designation, accession number of associated GenBank sequence, labelling dye, direction (F is foward, R is reverse),primer sequence, microsatellite motif, number of alleles (maximum number across all tested populations), amplified fragmentsize range and annealing temperature (Ta) of the primer for PCR reactions are provided.

Short Notes 535

Table 2. Locus specific characteristics of 6 new microsatellite loci found across 5 populations of Amblyrhynchus cristatus.Sample size (n), observed (Ho) and expected (He) values of heterozygosity are reported. Significant deviations from linkagedisequilibrium (sequential Bonferroni correction: α = 0.05, k = 6) are reported by use of shared superscript letter, anddeviations from Hardy-Weinberg equilibrium (sequential Bonferroni correction: α = 0.05, k = 6) are shown with an asterisk(∗). Locus MIG-E21 was monomorphic in two populations, and thus heterozygosities were not calculated in this case, asindicated by a hash (#) symbol.

Locus Population n Ho He Locus Population n Ho He

MIG-E18 San CristóbalA 30 0.633 0.710 MIG-E21 San Cristóbal 31 # #Santa CruzC 31 0.871 0.808 Santa Cruz 32 0.188 0.173Marchena 26 0.692 0.665 MarchenaD 24 0.125 0.191Fernandina 30 0.833 0.909 Fernandina 30 # #Floreana 30 0.733 0.658 FloreanaE 29 0.310 0.354

MIG-E19 San CristóbalB 30 0.800 0.729 MIG-E22 San Cristóbal 31 0.613 0.717Santa Cruz 32 0.656 0.767 Santa CruzC 32 0.844 0.773MarchenaD 24 0.583 0.558 Marchena 26 0.462 0.452Fernandina 30 0.800 0.742 Fernandina 30 0.633 0.804FloreanaE 28 0.500 0.554 Floreana 30 0.800 0.794

MIG-E20 San Cristóbal*A 30 0.500 0.658 MIG-E23 San CristóbalB 30 0.800 0.735Santa Cruz 32 0.906 0.873 Santa Cruz 31 0.613 0.700Marchena 26 0.692 0.643 Marchena 26 0.654 0.630Fernandina 30 0.800 0.828 Fernandina 30 0.533 0.491Floreana 30 0.867 0.854 Floreana 25 0.720 0.745

the Punta Pitt population on San Cristóbal island (89°36′W0°56′S), which harbours the lowest genetic diversity of anypopulation analysed so far (Steinfartz et al., 2009). Polymor-phic loci were then further tested with genomic DNA from150 individuals, collected between 1991-93 from 5 distinctisland populations (Steinfartz et al., 2009) of marine igua-nas: Santa Cruz (Caamaño; 90°17′W 0°46′S), Fernandina(Punta Espinosa; 91°27′W 0°16′S), Floreana (Punta Mon-tura; 90°30′W 1°19′S), Marchena (Bahia Negra; 90°31′W0°18′N) and San Cristóbal (Loberia; 89°36′W 0°56′S). La-belled primers were combined with Type-it multiplex PCRkit (Qiagen) in a 10 μl multiplex PCR reaction containing1 μl of genomic DNA, which was extracted from blood us-ing the SDS-Proteinase K/phenol-chloroform method andstored in 200 μl of Tris-HCl (10 mM Tris-HCl, pH 8). Thenew primers were combined in 4 multiplexes (MP1-MP4)along with 13 published loci (Steinfartz and Caccone, 2006;see table 1 for further details). Applied PCR parameterswere as follows: (I) an initial Taq polymerase activation stepof 5 min at 95°C, (II) 30 s at 95°C, (III) 90 s at an annealingtemperature (Ta) of 60°C, (IV) 30 s extension at 72°C; stepsII-IV were repeated for 30 cycles then step (V), a final ex-tension phase of 30 min at 60°C, completed the PCR. MP4was the only exception to these parameters, where a Ta of55.5°C was used to accommodate the low melting point ofprimer Am(GT)4. Obtained PCR products were diluted with200 μl of water, and to 1 μl of each multiplex reaction, 20 μlof Genescan 500-Rox size standard (Applied Biosystems)was added before analysis on an ABI 3730 96-capillary au-tomated DNA sequencer. Scoring of alleles was performedwith GENEMAPPER (version 1.95; Applied Biosystems).

For the six new loci, the number of alle-les varied from 1 (MIG E-21 on Fernandina

and San Cristóbal) to 13 (MIG-E20 on SantaCruz) with a mean of 5.73 ± SD = 3.0. Ob-served heterozygosity values (Ho) ranged from0.0 (MIG-E21 on San Cristóbal and Fernand-ina) to 0.906 (MIG-E20 on Santa Cruz) with anaverage of 0.605 ± 0.253 (mean ± SD). Devia-tions from Hardy-Weinberg equilibrium (HWE)and significant levels of linkage disequilib-rium (LD) were tested in ARLEQUIN (version3.11; Excoffier, Laval and Schneider, 2005) foreach locus (parameters used: 100 000 Markov-chain steps; 10 000 dememorization steps). Af-ter sequential Bonferroni-correction, only lo-cus MIG-E20 on San Cristóbal deviated sig-nificantly from HWE (table 2). Additionally,screening with MICRO-CHECKER (Van Oost-erhout et al., 2004) indicated no evidence forstuttering, null alleles or large allele drop atany of the six loci across the 5 populations. Al-though efforts were taken to reduce genotypicerror, including repeated PCR and allele scor-ing, this remains a potential source of error.However, since the possibility of large alleledrop out (a potentially large source of error ingenotypic scoring; Bonin et al., 2004) was ex-cluded, the error rate is likely to be low.

536 Short Notes

Recent approaches in Galapagos marineiguana research indicate that population differ-entiation has been not only vastly underesti-mated in this species, but remains far from be-ing understood (see Steinfartz et al., 2009). Thisunique species is doubtless of great interest tobiologists, and with a total number of 19 poly-morphic microsatellite loci now available, in-creasingly detailed population biology and evo-lutionary studies will be possible in the nearfuture.

Acknowledgements. This publication is contribution num-ber 2053 of the Charles Darwin Foundation for the Gala-pagos Islands. The authors thank the Swiss Association ofFriends of the Galápagos Islands for providing funding tocarry out this study. We also thank J. Sonnentag, R. Carter,J. Sands, M. Wikelski and W. Hayes for depositing the se-quences on which the primers were based with GenBank.Finally, we thank K. Rassmann, M. Wikelski and T. Roedlfor collecting blood samples of A. cristatus, Elke Hippauffor help in the laboratory, and the Servicio Parque NacionalGalápagos and the Charles Darwin Research Station forgranting the sampling permits and for providing necessaryinfrastructure.

References

Bonin, A., Bellemain, E., Bronken Eidesen, P., Pompanon,F., Brochmann, C., Taberlet, P. (2004): How to track andassess genotyping errors in population genetics studies.Mol. Ecol. 13: 3261-3273.

Excoffier, L., Laval, G., Schneider, S. (2005): Arlequin (ver-sion 3.0): an integrated software package for populationgenetics data analysis. Evol. Bioinf. Online 1: 47-50.

Palstra, F.P., Ruzzante, D.E. (2008): Genetic estimates ofcontemporary effective population size: what can they

tell us about the importance of genetic stochasticity forwild population persistence? Mol. Ecol. 17: 3428-3447.

Rassmann, K. (1997): Evolutionary age of the Galápagosiguanas predates the age of the present Galápagos Is-lands. Mol. Phylogenet. Evol. 7: 158-172.

Rozen, S., Skaletsky, H. (2000): PRIMER3 on the WWWfor general users and for biologist programmers. In:Bioinformatics Methods and Protocols, p. 365-386.Krawetz, S., Misener, S., Eds, Humana Press, Totowa,NJ.

Schuelke, M. (2000): An economic method for the fluores-cent labeling of PCR fragments. Nat. Biotechnol. 18:233-234.

Steinfartz, S., Caccone, A. (2006): A set of highly dis-criminating microsatellite loci for the Galápagos ma-rine iguana Amblyrhynchus cristatus. Mol. Ecol. Notes6: 927-929.

Steinfartz, S., Glaberman, S., Lanterbecq, D., Marquez, C.,Rassmann, K., Caccone, A. (2007): Genetic impact ofa severe El Niño event on Galápagos marine iguanas(Amblyrhynchus cristatus). PLoS One 12: e1285.

Steinfartz, S., Glaberman, S., Lanterbecq, D., Russello,M.A., Rosa, S., Hanley, T.C., Marquez, C., Snell, H.L.,Snell, H.M., Gentile, G., Dell’Olmo, G., Powell, A.M.,Caccone, A. (2009): Progressive colonization and re-stricted gene flow shape island-dependent populationstructure in Galápagos marine iguanas (Amblyrhynchuscristatus). BMC Evol. Biol. 9: 297.

Trillmich, K.G.K., Trillmich, F. (1986): Foraging strategiesof the marine iguana, Amblyrhynchus cristatus. Behav.Ecol. Sociobiol. 18: 259-266.

Van Oosterhout, C., Hutchinson, W.F., Wills, D.P.M., Ship-ley, P. (2004): Micro-Checker: software for identifyingand correcting genotyping errors in microsatellite data.Mol. Ecol. Notes 4: 535-538.

Wikelski, M., Nelson, K. (2004): Conservation of Galapa-gos marine iguanas (Amblyrhynchus cristatus). Iguana11: 191-197.

Submitted: August 27, 2012. Final revision received: Sep-tember 17, 2012. Accepted: September 26, 2012.Associated Editor: Sylvain Ursenbacher.