Amphibia-Reptilia 33 (2012): 533-536

New highly polymorphic microsatellite loci for the Galpagos marine iguana, Amblyrhynchus cristatus
Amy MacLeod1, , Volker Koch2,3 , Carolina Garca-Parra2 , Fritz Trillmich1 , Sebastian Steinfartz1
Abstract. We describe the development and characterisation of six new dinucleotide motif microsatellite loci for populations of marine iguanas (Amblyrhynchus cristatus), endemic to the Galpagos archipelago. Primers were based on microsatellitebearing sequences and initially developed using universally labelled primers. When analysed across 5 populations (representing 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 equilibrium or signicant 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 the successful amplication of 19 microsatellite loci for marine iguanas. This powerful set of highly polymorphic markers will allow 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 the Galpagos Islands, the endemic marine iguana, Amblyrhynchus cristatus, counts among the most ancient (Rassmann, 1997) and famous vertebrates. As the worlds only lizard with an amphibious lifestyle, A. cristatus occurs on 13 major islands of the archipelago, where it can be found basking along the shoreline and feeding almost exclusively on algae in the intertidal and shallow subtidal zone (Trillmich and Trillmich, 1986). The unique life history and island dependent distribution of this iguana provides an exceptional system to explore ecological and evolutionary questions. To date, analysis of 13 microsatellite loci have revealed historical patterns of progressive colonisation from older to younger islands across the archipelago, and almost no contemporary gene ow between island populations (Steinfartz et al., 2009). Al-

though the total number of animals is thought to be hundreds of thousands, there is mounting concern for some genetically distinct populations (Steinfartz et al., 2009), where census size number estimates may be as low as 50 (Wikelski and Nelson, 2004). At present, discrete data on census population sizes are severely lacking, and existing genetic estimates of effective population size (e.g. Steinfartz et al., 2007) could still be improved. Increasing the number of microsatellite loci reduces bias and improves precision in genetic effective population size estimation, without the need to increase sample size (Palstra and Ruzzante, 2008). Here we describe the development and characterisation of primers to amplify six new polymorphic microsatellite loci, which can be subsequently employed in combination with the 13 existing loci to explore the population genetics, evolution, and conservation status of marine iguanas with much greater resolution.
Based on genomic sequences bearing microsatellite dinucleotide motifs deposited in GenBank (see table 1 for GenBank accession numbers), primers were designed for 12 potential loci using PRIMER 3 software (Rozen and Skaletsky, 2000). These 12 loci were subsequently tested for polymorphisms using the M13 (-21)-tail method (Schuelke, 2000). In order to ensure that the new loci will be polymorphic for all sampled marine iguana populations, we performed the initial test of polymorphism for twelve individuals of
DOI:10.1163/15685381-00002854

1 - Department of Behavioural Biology, Unit of Molecular Ecology and Behaviour, University of Bielefeld, Morgenbreede 45, D-33619 Bielefeld, Germany 2 - Charles Darwin Research Station, Puerto Ayora, Santa Cruz Island, Galpagos, Ecuador 3 - Investigacin para la Conservacin y el Desarrollo, La Paz, BCS, Mxico Corresponding author; e-mail: ms.amymacleod@gmail.com
Koninklijke Brill NV, Leiden, 2012.

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Short Notes

Table 1. Characterisation of the full set of 19 available microsatellite loci for the Galpagos marine iguana (Amblyrhynchus cristatus). Loci are grouped in amplied 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 accession number Fluorescence labelling & primer sequences (5 -3 ) Repeat motif (GAAA)4 (GA)2 (GT)22 (TG)20 (GT)19 (AAGG)20 (GTT)13 No. alleles 10 20 12 11 19 9 Size range (bp) 137-159 226-276 150-172 163-183 234-326 222-240 Ta (C) 60 57 57 57 57 57

MIG-E3 MIG-E4 Multiplex 1 MIG-E6 MIG-E11 MIG-E14

DQ376112.1 F: HEX-GTGTGAGTGACATTTCTGCA R: TGAAAGTATGCTTTGCTCCCTTTGC DQ376113.1 F: HEX-TTGAGCTAAGTGGGAAAAGAAGAC R: AAAGTCTTCCCAGGAGATCACAC DQ376115.1 F: NED-ACGTCACTGGAGCTGACACA R: GAACAGTATCTAGGCACTCTCCAAA DQ376118.1 F: FAM-CAGTCCATTCTGCTTCCTCA R: CCTCAAACTCTGCCCTCTTG DQ376121.1 F: FAM-AAATTTTCTGCAGTTCTGTTGTCAT R: AGAATCATAGAAGTGGAAGGGACTC

MIG-E1612 DQ376123.1 F: NED-ACTAGCATAATCAGAGGTCATCCTG R: ACCAGAGTTCGATTCTCCATTTAG MIG-E8 DQ376116.1 F: HEX-ACCAAGCAAATGGTTTCCAG R: TTGTTCCAAATAGCATAAAATATCA MIG-E12 DQ376119.1 F: FAM-GGAAGACACTTCAGGCAGCACTTTG R: TTAGTCAAACCTTTACTCCGACCTG MIG-E13 DQ376119.1 F: FAM-GAGGATGAACAGATGGTAAGTCAAT R: AGAACTCTGAGGTATGGAGGAAGAT MIG-E19 AF452621.1 HEX-TGTGCCAAATGAAGATGAGC MIG-E19R GCTACTCAATAATCTATGGCATGAA MIG-E22 AY035303.1 NED-AAATTGGCATAGCTGAGAAACA MIG-E22R AATCACTTTCCCAAGCCAAG MIG-E23 AY035299.1 NED-GCATGTCTAATTTCCACTGTGC MIG-E23R TTCCTAATACCTACAAGTGCCTTAG DQ376111.1 F: FAM-GTGTGAGTGACATTTCTGCA R: TGAAAGTATGCTTTGCTCCCTTTGC

(AAGG)14 (GT)20 (CA)18 (GT)n (CT)n (GT)n (GT)n

11 13 9 10 8 7

152-184 169-195 265-277 234-254 154-168 213-225

57 57 57 60 59 60

Multiplex 2

MIG-E2 Multiplex 3

(GAAA)4 (GA)2 (GAAA)10 (GA)2 MIG-E10 DQ376117.1 F:HEX-CCTTATAAATGCTGATCTGGAGCTGT (AAGG)14 R: CTTTTGCAGTGTTTACTTTTTCCAT MIG-E15 DQ376122.1 F: FAM-AGACAGGACTGATGTCCTCTAAGAA (TG)19 R: GGTTGACAACTTATAAGCCTGAAGA MIG-E21 AY035302.1 F: NED-TTGGCTTTGTAAACTAACACAGTTTC (CA)n MIG-E21R R: TGAGCCTACACCATTGGAGA Am(GT)4 F: FAM-TTATGGATGAGCAATAC Am(GT)4b R: GTATATATGCCTTGTAG MIG-E18 AF297084.1 F: HEX-CATCTGTCCCTACCCTTGCT MIG-E18R R: TGCTGAACATATGCTTCTCATGT MIG-E20 AY035304.1 NED-TGCAATCATTTTTAAACATTCACA MIG-E20R AGTGTTCCCAGTTGGACAGC (GT)n (GT)n (CT)n

10

229-265

57

13 11 2

194-250 140-160 160-162

57 57 60

8 15 14

207-223 184-216 195-243

54 59 59

Multiplex 4

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), amplied fragment size range and annealing temperature (Ta ) of the primer for PCR reactions are provided.

Short Notes

535

Table 2. Locus specic 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. Signicant deviations from linkage disequilibrium (sequential Bonferroni correction: = 0.05, k = 6) are reported by use of shared superscript letter, and deviations from Hardy-Weinberg equilibrium (sequential Bonferroni correction: = 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, as indicated by a hash (#) symbol. Locus MIG-E18 Population San Santa CruzC Marchena Fernandina Floreana San CristbalB Santa Cruz MarchenaD Fernandina FloreanaE San Cristbal*A Santa Cruz Marchena Fernandina Floreana CristbalA n 30 31 26 30 30 30 32 24 30 28 30 32 26 30 30 Ho 0.633 0.871 0.692 0.833 0.733 0.800 0.656 0.583 0.800 0.500 0.500 0.906 0.692 0.800 0.867 He 0.710 0.808 0.665 0.909 0.658 0.729 0.767 0.558 0.742 0.554 0.658 0.873 0.643 0.828 0.854 Locus MIG-E21 Population San Cristbal Santa Cruz MarchenaD Fernandina FloreanaE San Cristbal Santa CruzC Marchena Fernandina Floreana San CristbalB Santa Cruz Marchena Fernandina Floreana n 31 32 24 30 29 31 32 26 30 30 30 31 26 30 25 Ho # 0.188 0.125 # 0.310 0.613 0.844 0.462 0.633 0.800 0.800 0.613 0.654 0.533 0.720 He # 0.173 0.191 # 0.354 0.717 0.773 0.452 0.804 0.794 0.735 0.700 0.630 0.491 0.745

MIG-E19

MIG-E22

MIG-E20

MIG-E23

the Punta Pitt population on San Cristbal island (8936 W 056 S), which harbours the lowest genetic diversity of any population analysed so far (Steinfartz et al., 2009). Polymorphic loci were then further tested with genomic DNA from 150 individuals, collected between 1991-93 from 5 distinct island populations (Steinfartz et al., 2009) of marine iguanas: Santa Cruz (Caamao; 9017 W 046 S), Fernandina (Punta Espinosa; 9127 W 016 S), Floreana (Punta Montura; 9030 W 119 S), Marchena (Bahia Negra; 9031 W 018 N) and San Cristbal (Loberia; 8936 W 056 S). Labelled primers were combined with Type-it multiplex PCR kit (Qiagen) in a 10 l multiplex PCR reaction containing 1 l of genomic DNA, which was extracted from blood using the SDS-Proteinase K/phenol-chloroform method and stored in 200 l of Tris-HCl (10 mM Tris-HCl, pH 8). The new 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 parameters were as follows: (I) an initial Taq polymerase activation step of 5 min at 95C, (II) 30 s at 95C, (III) 90 s at an annealing temperature (Ta ) of 60C, (IV) 30 s extension at 72C; steps II-IV were repeated for 30 cycles then step (V), a nal extension phase of 30 min at 60C, completed the PCR. MP4 was the only exception to these parameters, where a Ta of 55.5C was used to accommodate the low melting point of primer Am(GT)4. Obtained PCR products were diluted with 200 l of water, and to 1 l of each multiplex reaction, 20 l of Genescan 500-Rox size standard (Applied Biosystems) was added before analysis on an ABI 3730 96-capillary automated DNA sequencer. Scoring of alleles was performed with GENEMAPPER (version 1.95; Applied Biosystems).

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

and San Cristbal) to 13 (MIG-E20 on Santa Cruz) with a mean of 5.73 SD = 3.0. Observed heterozygosity values (Ho ) ranged from 0.0 (MIG-E21 on San Cristbal and Fernandina) to 0.906 (MIG-E20 on Santa Cruz) with an average of 0.605 0.253 (mean SD). Deviations from Hardy-Weinberg equilibrium (HWE) and signicant levels of linkage disequilibrium (LD) were tested in ARLEQUIN (version 3.11; Excofer, Laval and Schneider, 2005) for each locus (parameters used: 100 000 Markovchain steps; 10 000 dememorization steps). After sequential Bonferroni-correction, only locus MIG-E20 on San Cristbal deviated signicantly from HWE (table 2). Additionally, screening with MICRO-CHECKER (Van Oosterhout et al., 2004) indicated no evidence for stuttering, null alleles or large allele drop at any of the six loci across the 5 populations. Although efforts were taken to reduce genotypic error, including repeated PCR and allele scoring, this remains a potential source of error. However, since the possibility of large allele drop out (a potentially large source of error in genotypic scoring; Bonin et al., 2004) was excluded, the error rate is likely to be low.

536 Recent approaches in Galapagos marine iguana research indicate that population differentiation has been not only vastly underestimated in this species, but remains far from being understood (see Steinfartz et al., 2009). This unique species is doubtless of great interest to biologists, and with a total number of 19 polymorphic microsatellite loci now available, increasingly detailed population biology and evolutionary studies will be possible in the near future.

Short Notes tell us about the importance of genetic stochasticity for wild population persistence? Mol. Ecol. 17: 3428-3447. Rassmann, K. (1997): Evolutionary age of the Galpagos iguanas predates the age of the present Galpagos Islands. Mol. Phylogenet. Evol. 7: 158-172. Rozen, S., Skaletsky, H. (2000): PRIMER3 on the WWW for 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 uorescent labeling of PCR fragments. Nat. Biotechnol. 18: 233-234. Steinfartz, S., Caccone, A. (2006): A set of highly discriminating microsatellite loci for the Galpagos marine iguana Amblyrhynchus cristatus. Mol. Ecol. Notes 6: 927-929. Steinfartz, S., Glaberman, S., Lanterbecq, D., Marquez, C., Rassmann, K., Caccone, A. (2007): Genetic impact of a severe El Nio event on Galpagos 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., DellOlmo, G., Powell, A.M., Caccone, A. (2009): Progressive colonization and restricted gene ow shape island-dependent population structure in Galpagos marine iguanas (Amblyrhynchus cristatus). BMC Evol. Biol. 9: 297. Trillmich, K.G.K., Trillmich, F. (1986): Foraging strategies of the marine iguana, Amblyrhynchus cristatus. Behav. Ecol. Sociobiol. 18: 259-266. Van Oosterhout, C., Hutchinson, W.F., Wills, D.P.M., Shipley, P. (2004): Micro-Checker: software for identifying and correcting genotyping errors in microsatellite data. Mol. Ecol. Notes 4: 535-538. Wikelski, M., Nelson, K. (2004): Conservation of Galapagos marine iguanas (Amblyrhynchus cristatus). Iguana 11: 191-197.

Acknowledgements. This publication is contribution number 2053 of the Charles Darwin Foundation for the Galapagos Islands. The authors thank the Swiss Association of Friends of the Galpagos Islands for providing funding to carry out this study. We also thank J. Sonnentag, R. Carter, J. Sands, M. Wikelski and W. Hayes for depositing the sequences on which the primers were based with GenBank. Finally, we thank K. Rassmann, M. Wikelski and T. Roedl for collecting blood samples of A. cristatus, Elke Hippauf for help in the laboratory, and the Servicio Parque Nacional Galpagos and the Charles Darwin Research Station for granting the sampling permits and for providing necessary infrastructure.

References
Bonin, A., Bellemain, E., Bronken Eidesen, P., Pompanon, F., Brochmann, C., Taberlet, P. (2004): How to track and assess genotyping errors in population genetics studies. Mol. Ecol. 13: 3261-3273. Excofer, L., Laval, G., Schneider, S. (2005): Arlequin (version 3.0): an integrated software package for population genetics data analysis. Evol. Bioinf. Online 1: 47-50. Palstra, F.P., Ruzzante, D.E. (2008): Genetic estimates of contemporary effective population size: what can they

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