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deviations from HWE revealed no significant departures Excoffier L, Laval G, Schneider S (2005) arlequin ver. 3.0: an inte- (P > 0.05) at eight loci; conversely, a significant excess of grated software package for population genetics data analysis. homozygotes was observed at one locus (ST2-41). The use Evolutionary Bioinformatics Online, 1, 47–50. Goudet J (2001) FSTAT: A program to estimate and test gene diversities of micro-checker software (Van Oosterhout et al. 2004) and fixation indices (v. 2.9.3). Available from http://www2. indicated that the significant deviations from HWE at this unil.ch/popgen/softwares/fstat.htm. locus may be the result of null alleles, although no scoring Grandjean F, Gouin N, Verne S, Delaunay C, Patri S (2005) Char- errors were found associated with stuttering bands or large acterization of polymorphic microsatellite loci in the terrestrial allele dropout. arlequin was also used to test for gametic isopod Porcellionides pruinosus. Molecular Ecology Notes, 5, 503–507. disequilibrium by application of a Bonferroni correction for Haye PA, Marchant S (2007) Newly developed PCR primers for multiple comparisons (Rice 1989). No evidence of gametic polymorphic microsatellite loci from the marine isopod Limnoria sp. Molecular Ecology Notes, 7, 1245–1247. disequilibrium was detected for any pairs of loci. Haye PA, Marchant S (2008) Isolation and characterization of 11 The relatively small number of useful loci for S. terebrans, polymerase chain reaction primers for microsatellite loci for the resulting from an initially promising set of positive clones, Chilean marine isopod Excirolana hirsuticauda. Molecular Ecology suggests that the isolation of suitable microsatellite loci for Resources, 8, 1088–1090. this group of Crustaceans is not easy, as also demonstrated Leese F, Kop A, Agrawal S, Held C (2008) Isolation and character- by the small number of simple sequence repeats isolated ization of microsatellite markers from the marine isopods Serolis in isopods thus far (Grandjean et al. 2005; Verne et al. 2006; Haye paradoxa and Septemserolis septemcarinata (Crustacea: Peracarida). Molecular Ecology Resources, 8, 818–821. & Marchant 2007, 2008; Leese et al. 2008). Nevertheless, we are Rice WR (1989) Analyzing tables of statistical tests. Evolution, 43, confident that the loci characterized in this study are suitable 223–225. to infer parentage, relatedness and phylogeographic patterns Rozen S, Skaletsky H (2000) Primer 3 on the WWW for general users in this taxon, which probably includes some cryptic . and for biologist programmers. Methods in Molecular Biology, 132, 365–386. Van Oosterhout C, Hutchinson WF, Wills DPM, Shipley P (2004) Acknowledgements micro-checker: software for identifying and correcting geno- We thank Sara Fratini and Rocco Rorandelli. We also thank Eleonora typing errors in microsatellite data. Molecular Ecology Notes, 4, Trajano for the help rendered in collecting material. The research 535–538. was funded by the Italian Ministry of University and Research Verne S, Puillandre N, Brunet G et al. (2006) Characterization of (MIUR) (FIRB/2001). polymorphic microsatellite loci in the terrestrial isopod Armadil- lidium vulgare. Molecular Ecology Notes, 6, 328–330. Zane L, Bargelloni L, Patarnello T (2002) Strategies for microsatellites References isolation: a review. Molecular Ecology, 11, 1–16. Baratti M, Goti E, Messana G (2005) High level of genetic differen- tiation in the marine isopod Sphaeroma terebrans (Crustacea Isopoda doi: 10.1111/j.1755-0998.2009.02666.x Sphaeromatidae) as inferred by mitocondrial DNA analysis. Journal of Experimental Marine Biology and Ecology, 315, 225–234. © 2009 Blackwell Publishing Ltd Characterization of parakeet ( eques) microsatellite loci and their cross-utility in other (Psittacidae, Aves)

CLAI RE RAISIN,*†‡§ DEBORAH A. DAWSON,* ANDREW G. GREENWOOD,‡ CARL G . JONES¶§ and JIM J. GROOMBRIDGE† *NERC Molecular Genetics Facility, Department of and Plant Sciences, University of Sheffield, Sheffield S10 2TN, UK, †Durrell Institute of Conservation & Ecology, University of Kent, Canterbury CT2 7NZ, UK, ‡International Zoo Veterinary Group, Keighley Business Centre, South Street, Keighley, W. Yorkshire BD21 1AG, UK, §Mauritian Wildlife Foundation, Grannum Road, Vacoas, Mauritius, ¶Durrell Wildlife Conservation Trust, Les Augres Manor, Trinity, Jersey JE3 5BP, UK

Abstract We characterized 21 polymorphic microsatellite loci in the endangered Mauritius parakeet (Psittacula eques). Loci were isolated from a Mauritius parakeet genomic library that had been enriched separately for eight different repeat motifs. Loci were characterized in up to

Correspondence: Jim Groombridge, Fax: +44 (0)1227 827289; E-mail: [email protected] 1232 PERMANENT GENETIC RESOURCES NOTE

43 putatively unrelated Mauritius parakeets from a single population inhabiting the Black River Gorges National Park, Mauritius. Each locus displayed between three and nine alleles, with the observed heterozygosity ranging between 0.39 and 0.96. All loci were tested in 10 other species. Despite testing few individuals, between seven and 21 loci were poly- morphic in each of seven species tested. Keywords: AVES, cross-species utility, Mauritius parakeet, microsatellite, parrot, Psittacidae, Received 22 December 2008; accepted 3 February 2009

The Mauritius parakeet (Psittacula eques) is the only endemic Each locus was amplified separately. Each 2 μL PCR con- psittacine remaining in the and is classified tained approximately 10 ng of genomic DNA with 0.2 μm as endangered (IUCN 2008). The population declined to less of each primer and 1 μL QIAGEN multiplex PCR mix than 20 individuals (including only three known breeding (QIAGEN Inc.). PCR amplification was performed using a females) in the early 1980s but currently numbers approxi- DNA Engine Tetrad 2 thermal cycler (MJ Research, Bio-Rad). mately 360, all of which are found in the Black River Gorges The PCR profile used was 95 °C for 15 min; then 40 cycles ° ° ° National Park, South West Mauritius (Malham et al. 2006). of 94 C for 30 s, Ta C (Table 1) for 90 s, 72 C for 90 s, We have characterized a set of polymorphic microsatellite followed by one cycle of 10 min at 72 °C. Genotypes were loci for the Mauritius parakeet. scored on an ABI 3730 DNA Analyser with GeneScan Blood was collected from 43 Mauritius parakeet indi- ROX-500 size standard using GeneMapper software (Applied viduals and stored in 70–90% ethanol at room temperature. For Biosystems). Each locus was loaded on the ABI 3730 sepa- library construction, genomic DNA was extracted from blood rately. Multiplexing was not performed pre- or post-PCR using a DNeasy Blood and Tissue Kit (QIAGEN Ltd). Mau- amplification. ritius parakeet microsatellites were isolated from an enriched All 21 loci were polymorphic and the number of alleles genomic library prepared essentially as Jones et al. (2002) by observed when tested in 24–43 individuals from the recov- Genetic Identification Services. The pooled genomic DNA ered population ranged from three to nine (Table 1). The of three Mauritius parakeet individuals (one male and two DNA sequences of the 21 polymorphic loci were confirmed females) was digested with blunt ended-restriction enzymes to be free of vector and unique using blastn version 2.2.4 (AluI, DraI, FspI, HpaI, MscI, NaeI). Fragments (350–700 bp) (Altschul et al. 1997). Loci were checked for sex linkage by were ligated to the double-stranded linker sequence 5′- genotyping individuals of known sex (15 females and 14 AAGCTTTCGTTTTACAACGTCGTGG-3′ (which contains males; sex based on morphology). Two loci (Peq16 and Peq21) a HindIII site at the 5′ end) and hybridized separately against were found to be Z-linked, therefore only males were char- one of eight target repeat sequences: (CA)15, (GA)15, (AAT)15, acterized at these loci as females were always homozygous.

(ATG)12, (CAG)10, (CATC)8, (TAGA)8 and (TGAC)8. The Observed and expected heterozygosities, and null allele enriched fragments were digested at the HindIII site of the frequencies were calculated using Cervus version 2.0 (Mar- linker and then ligated into pUC19-HindIII. Colonies were shall et al. 1998). Tests for departures from Hardy–Weinberg not screened for repeat motifs but directly sequenced. A total and linkage equilibrium were conducted using a Markov of 104 clones were sequenced in the forward orientation chain method implemented in GenePop version 3.4 using an M13-based forward primer (5′-AGGAAACAGC (Raymond & Rousset 1995). One locus (Peq08) deviated TATGACCATG-3′). Primer sets were designed for 48 loci from Hardy–Weinberg equilibrium and also displayed a using DesignerPCR version 1.03 (Research Genetics, Inc.) high predicted null allele frequency (Table 1). and used to amplify eight unrelated Mauritius parakeet Based on sequence homology, 15 of the 21 loci could be individuals to assess polymorphism. When initially ampli- assigned a chromosome location in the zebra finch (Taeniopy- fied in eight unrelated individuals and screened on agarose gia guttata) genome assembly (compiled by the Washington gel, 35 loci appeared polymorphic, eight monomorphic University School of Medicine Genome Sequencing Centre, and five failed to amplify or were unscorable (unpublished http://genome.wustl.edu/genome.cgi?GENOME=Taenio data). Twenty-one polymorphic loci (enriched for (CA)15, pygia%20guttata&SECTION=assemblies) and 11 loci

(GA)15, (CATC)8 and (TAGA)8 motifs) were selected for further were assigned a chicken (Gallus gallus) chromosome location optimization. (using a wu-blast with distant homologies settings imple- Genomic DNA was extracted from blood using ammonium mented on the ENSEMBL webpage www.ensembl.org/ acetate following the method used by Nicholls et al. (2000) Gallus_gallus/index.html; see Dawson et al. 2006; Table 1, and used for PCR. A range of annealing temperatures (50 °C– Fig. 1). Loci Peq16 and Peq21 were assigned to the zebra 68 °C) were tested and the temperature producing the cleanest finch Z chromosome (Table 1, Fig. 1). and strongest PCR product when observed on an 0.8% agarose Fifteen known family groups (both parents and at least gel stained with ethidium bromide was selected for PCR. one offspring) were genotyped and used to test for linkage PERMANENT GENETIC RESOURCES NOTE 1233 Est. null null Est. allele freq. HWE P E H hits also detected; detected; also hits O H , number of unrelated Mauritius n Obs. Obs. allele sizes (bp) §multiple other weaker other weaker §multiple -linked loci and therefore were characterized in characterized were therefore and loci -linked Exp. Exp. allele (bp)‡ size Z 5 180 159–179 0.56 0.55 0.72 — 3 136 131–149 0.50 0.43 1.00 — , annealing temperature; a T nA (males) (males) < 0.001). P

. Peq16 and Peq21 are are Peq21 and Peq16 . C) ° ( a o chromosome assignments conflicted; therefore, when assignmentso chromosome the same chromosome conflicted; therefore, 59 14 Optimised T 636666 4256 43 656 42 202 359 43 145 7 189–20966 42 287 5 0.67 144–15358 0.58 41 287 4 0.87 0.47 274–30056 0.53 42 133 –0.16 7 0.77 0.76 285–30659 0.76 42 216 0.04 3 0.20 0.65 125–13156 0.63 41 128 0.09 9 0.37 0.57 213–24459 0.60 42 255 0.04 6 0.52 0.83 114–12455 0.79 42 218 –0.01 6 0.89 0.40 235–265¶58 0.34 41 133 –0.03 0.48 8 0.64 206–242 0.6859 24 282 –0.06 0.00 6 0.81 109–12963 0.80 23 0.12 279 4 0.78 0.76 253–281€62 0.80 23 125 0.81 0.04 4 0.35 271–291 0.8159 14 219 0.64 0.01 9 0.90 114–12258 0.82 22 206 0.06 0.95 0.67 209–22561 0.65 24 0.00 7 0.97 0.39 205–221€ 0.34 23 228 –0.04 0.96 6 1.00 0.86 23 170 –0.10 0.31 5 196–224 –0.07 230 4 0.85 155–179 0.78 235 0.60 0.78 202–234 0.73 –0.07 0.92 0.87 223–235 0.79 0.00 0.68 0.74 0.71 –0.07 0.51 –0.04 ch genome only (Peq01, Peq03, Peq07, Peq16 and Peq20) and on two occasions, , numberof alleles; ‡expected allelesize basedon length of theMauritius parakeet A ) ′ value as identified by GenePop version 3.4; by 3.4; GenePop version as identified value –3 P ′ ion 3.4 (Raymond & Rousset 1995; & Rousset (Raymond ion 3.4 TGTGCTTTTCCATCACAAG GTGTCGCAACCTCCTTATG ATGGTTCTCCTGTGTCTATCAG TGACAAGTCCACCAAAAATATC AGCTCATAAACAGCCATATCTC GCTAACACCCCCCTATCC GGAGGATAAGCAGAACTTGAG GACATGATGCTGACACAGATAG GGACTGTGGATGGAGAGGT GGATCTTTCCTTTATCTGTGTG TGGGAGGTTAGAGTGAAAAAC CCTTAGTCATCGTGCTTGTAG TTGCTGCACTGTAGTTCTGTGA ACGCTGGATCATAACAGAGTAA GGAGTTTTTTGTCGTTTTCTGT GAAATGATAGGCAACAGACAGT TTCTCCCAGAGCACACAC CTTCAAATTCAGCAAACTTGTC AACCCAATCTGTCTGAGAACAG GCTGTGTTCAAAAGGGTATG CATGTTTGCTTGTCCAGTTTAT AGGCTTAACAGATGTAGGACAC CTTTGAGCACCCACACAAC GGATGTTCAGTTCTCTTGTTTC GGATTTCTCATGCCTAAAATG GGAATTGTAGGTTTTAATGCAC CAAAAGAGCATCAATGGTATG AAACAAACATACCCACAGAAAC AGTCGGGAACAGTTTCATTAG GGTGGGTTGTGTGAAAGAA ACCCTTCCAAGAGAGTTTTAAG CTCAAGGAGAATCTGAAGTCTG CACTCGGGATTCTGTGTTAG GGAACATTCCAAAGCTCAAAGT GGCTACCATCCTATTTGAAAGA CTTCATTTTCCTGTGCAGTCTA GCTCCTAAGCCTTTGTAAAGAC ACCTCACAGACACAAAAGGATA AGCAAGTAGATTTGGTCGTG GAGCCTCCTACCATAGTTGC GATGCAAATGTTACAGCAGTGT CCCAGAGTGGTAGAGAAAGAT R: R: R: R: R: R: R: R: R: R: R: R: R: R: R: R: R: R: R: R: R: R: R: were assigned a location of of the zebra a fin assigned location were , Hardy–Weinberg equilibrium test HEX6-FAM6-FAM F: F: HEX F: 6-FAMHEX F: F: HEX6-FAM F: HEX F: F: 6-FAM6-FAM F: F: HEX F: HEXNED F: NED F: F: 6-FAM F: HEX F: HEX6-FAM F: 6-FAM F: F: 6-FAM F: F: Fluoro-label Fluoro-label (F) (5 sequences Primer HWE 4 10 P

) )

1 2 2 ) )

1 10 ) ) )

5 T ) 1 1 ) ) ATCA CATC 3 7 ( ) TAGA TAGA ) CAGA ( ( CAGA TAGA ( CACC TCTTA & ( ( ( T TC T TAGG m as identified by GenePop vers GenePop by identified m as TCTA TG 10 12 1 3 9 10 6 14 14 2 11 8 13 2 7 6 3 1 10 ( ( CATC TAGA ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ( ( T TAGA 13 11 13 11 8 12 14 la eques microsatellite la eques loci (Psittacidae, Aves) microsatellite ( ) ) ) ) ) ) ) CATC TCTA TAGA TAGA TAGA GATG TAGA TAGG CA TG CA GATG CATC TAGA TCTA TAGA TG CA CA GATG TAGA TCTA TCTA TAGA CA CATC TAA TATC ATCTA TTGA ( ( ( ( ( BLA ST P(N) Repeat motif , expected heterozygosity; E H on in zebra finch is presented. Five loci chickengenome (Peq08 and Peq18). Tgu, zebra finch chromosome; Gga,chicken chromosome Chr. Chr. location (bp) Chr. location Chr. (see footnote and text) , observed heterozygosity; O H Characterization of Mauritius parakeetCharacterization of Psittacu EMBL EMBL accession number parakeet individuals genotyped from the population inhabiting Black River Gorges National Park, South West Mauritius. West South Park, National Gorges River Black inhabiting population the from genotyped individuals parakeet sequenced clone; a location could onlybe assigned inthe Peq02 FM865415Peq03 hits No strong FM865416Peq04 Tgu1 FM865417Peq05 Tgu4 FM865418Peq06 Tgu1 79810332 FM865419Peq07 4E-18 Tgu4 40782899 ( ( FM865420Peq08† 2E-38 Tgu4 39968225 FM865421 ( Gga27Peq09 1E-41 43705460 ( FM865422Peq10 7E-42 Tgu2 59562728 ( FM865423 2668172Peq11 4E-13 Tgu3 ( FM865424 9E-15Peq12 Tgu3 ( 154849943 FM865425 1E-24Peq13 Tgu2§ ( 17147094 FM865426Peq14 2E-48 hits No strong 30982572 ( FM865427Peq15 4E-31 None 154288876 ( FM865428 1E-33Peq16 ( Tgu12 FM865429 TguZPeq18 6379632 ( FM865431Peq19 6E-41 Gga10 43664061 ( FM865432Peq20 1E-16 Tgu1A ( FM865433Peq21 Tgu20 641761 ( FM865434 22948103 TguZ 7E-19 3E-15 ( 2718287 ( Chromosome location: loci could more be assigned a location in the zebra finch genome than the chicken and genome n (15 vs. 11) 1E-08the locati only genomesassigned in both was 612947 ( 9E-13 ( Table 1 Locus Peq01 FM865414 Tgu2 61550289 3E-28 ( ¶someobserved allele sizes by increased only 1 bp andbe mayscore. difficult to male individuals only. †Deviation from Hardy–Weinberg equilibriu †Deviation Hardy–Weinberg from male individuals only. Peq17 FM865430 hits No strong ( 1234 PERMANENT GENETIC RESOURCES NOTE =1 n Crocodylus Crocodylus porosus =1 n Gallus gallus =1 n Anthropoides Anthropoides paradisea Number of Number parrot species polymorphic Blue crane Chicken Crocodile ally cloned and sequenced in the Mauritius parakeet. in the Mauritius and sequenced cloned ally =2 Vasa parrot n Coracopsis vasa =1 Black–lored Black–lored parrot n 208, 219 192 8 176 295 173 Tanygnathus gramineus , number of individualstested; did X, not amplify; #Amp,number of lociamplifying per species; n § ) microsatellite loci* ) microsatellite =2 parrot n Eclectus roratus roratus individuals (see text). Psittacula eques Psittacula ative locus may be amplifying to that which was origin was which to that amplifying be may locus ative =2 n Psittacula eupatria 144, 153144, 144 140, 131, 126 X 4 X X X Psittacula eques Psittacula § =1 Slaty–headed parakeet n Psittacula Psittacula himalayana himalayana llele sizes suggests llele sizes altern an ed with Mauritius ed with Mauritius parakeet =1 Blossom Blossom parakeet n Psittacula Psittacula roseata amplified( using Mauritius parakeet =2 Moustached parakeet n Psittacula Psittacula alexandri reaction constituents as us constituents reaction , the difference, the observed in andexpected a § =1 Derbyan Derbyan parakeet 264 270 267, 243, 239, 268 264 180166, 170 264 260, 256, 249, 287 279, 276 272, 180 252 248, 240, X 7 X 184 X151 146, X 180 180 3 180 180 180 Psittacula Psittacula derbiana n § § =1 Longtailed Longtailed parakeet Psittacula Psittacula longicauda n =10 =10 Ringneck Ringneck parakeet (Rose– ringed) Psittacula Psittacula krameri n (number of alleles) Allele sizes observed in other species when Allele sizes observed in other Peq12Peq13 (4) 275–295 Peq14 (7) 108–123 Peq15 332 243, 222, 226 (2)Peq16 Amp21131415111015151310644 109 (3) 188–217 X 337 283, (3) 135–144 X 134 348 322, 116 114, X# 116 X# Poly 135 X 21 and program PCR same the using amplified were loci *All X per species. of loci polymorphic number #Poly, 135 131, X X X 8 161 127 104, X X 299 291, 210 8 108 X X 270 234, 11 308 284, 108 104, X X X 108 5 X 100 7 4 X 4 X X X 12 122 116, X X X 12 123 116, X 123 1 1 7 X X 4 X X X X 2 1 1 Table 2 Locus Peq05Peq06 (3) 121–125 Peq07 (5) 217–238 Peq08 123 122–126 (3)Peq09 203 (3) 251–259 Peq10 112 195–235 (6)Peq11 277 272, (4) 117–129 120 242, 246 241–273 (7) 203 X 280 276, X 308, 343 X 116 288 263, 200 125 109, 128 116, 257, 261, 265 136 132, 259 251, 126 124, 234, 258 X X 110 122 X X X 236 255 251, 125 123, 129 113, 191, 222 238 234, 125 121, X 291 279, 259, 138 136, 235 175 120 201 100 263 255, 168, 172 X 131 120, 203 129 113, 8 X 5 X 113 X X 173, 175 6 131 5 4 2 X X 129 113, X X X X X X X X 129 113, X X X X Peq01Peq02 (7) 189–203 Peq03 144–190 (4)Peq04 X (7) 282–306 149, 157 297, 317 (2) X 194, 219 X 203 199, 190 210 195, X 184, X 219 211, 199 195, X 197 213,223 X 192, 202, 282 199 195, X 189,213 X 209 205, 193, X 104,108 203 195, X 194 X 7 X X X X X 195 X X X 2 0 X X X X X X Peq17Peq18 (6) 189–223 Peq19 (4) 162–178 Peq20 241 233, (4) 218–234 Peq21 X 214–242 (6) 231 207, 205 201, (3) 168–188 X 248 286208 266, 205, 201, X 277 265, 261, 234 X 230 X X X 257 X 221 217, 242 X X 209 205, 229, 245 281 225, 217 213, X 259, 267 X 270 258, 246 X 7 X 6 X 128 X X X X X X X 3 1 X X X X X X X PERMANENT GENETIC RESOURCES NOTE 1235

Fig. 1 Chromosome locations in the zebra finch (Taeniopygia guttata) and chicken (Gallus gallus) genome of 15 microsatellite loci characterized in the Mauritius parakeet (Psittacula eques). using CriMap software (Lander & Green 1987). Loci were was supported by the Natural Environment Research Council, UK also checked for linkage disequilibrium in unrelated indi- and Wildlife Vets International, Keighley, UK. viduals using GenePop version 3.4 and applying a sequential Bonferroni correction (Rice 1989). One pair of loci was assigned References to closely neighbouring locations on the same zebra finch Altschul SF, Madden TL, Schäffer AA et al. (1997) Gapped blast chromosome (Peq09 and Peq12, located 0.5 Mb apart on and psi-blast: a new generation of protein database search Tgu2; Table 1, Fig. 1) and displayed both linkage (LOD = programs. Nucleic Acids Research, 25, 3389–3402. 10.24) and linkage disequilibrium. The second nearest known Dawson DA, Burke T, Hansson B et al. (2006) A predicted micro- neighbouring pair of loci, Peq04 and Peq06 were located satellite map of the passerine genome based on chicken–passerine 3 Mb apart on Tgu4 and did not display either significant sequence similarity. Molecular Ecology, 15, 1299–1321. linkage or linkage disequilibrium. Significant linkage was IUCN (2008) Red List of Threatened Species. Available from URL: http://www.iucnredlist.org. detected between Peq03 and Peq05 (LOD = 4.16) although Jones KC, Levine KF, Banks JD (2002) Characterization of 11 poly- these loci mapped to different zebra finch chromosomes; morphic tetranucleotide microsatellites for forensic applications these loci were not in linkage disequilibrium. Linkage in California elk (Cervus elaphus canadensis). Molecular Ecology disequilibrium was additionally indicated between Peq08 and Notes, 2, 435–427. Peq09, which were assigned to different avian chromosomes. Lander ES, Green P (1987) Construction of multilocus genetic All 21 loci were tested in 13 other species including 10 linkage maps in human. Proceedings of the National Academy of parrot species (Table 2). Between seven and 21 loci were Sciences, USA, 84, 2363–2367. Malham J, Reuleaux A, Freeman P et al. (2006) Echo Parakeet Man- polymorphic in each of seven parrot species despite testing agement Report, 2006. Mauritian Wildlife Foundation. Vacoas, only one to two individuals. Six loci show particular Mauritius. potential for genetic studies of other parrot species. These Marshall TC, Slate J, Kruuk LEB, Pemberton JM (1998) Statis- six loci were each polymorphic in at least seven other parrot tical confidence for likelihood-based paternity inference in species (Peq01, Peq04, Peq08, Peq11, Peq12 and Peq17). natural populations. Molecular Ecology, 7, 639–655. (http:// We have successfully characterized 21 polymorphic helios.bto.edsac.uk/evolgen/cervus/cervus.html). microsatellite markers for the Mauritius parakeet. These Nicholls JA, Double MC, Rowell DM, Magrath RD (2000) The evolu- tion of cooperative and pair breeding in thornbills Acanthiza loci will be used to study the genetic variability of the exist- (Pardalotidae). Journal of Avian Biology, 31, 165–176. ent population and the species’ mating system. Raymond M, Rousset F (1995) GenePop (version 1.2): population genetics software for exact tests and ecumenism. Journal of Acknowledgements Heredity, 86, 248–249. (http://genepop.curtin.edu.au/). Rice WR (1989) Analysing tables of statistical tests. Evolution, 43, Hans Cheng, Jon Ekstrom, Winston Kaye, Kate Meares and Tiawanna 223–225. Taylor kindly provided blood and/or DNA samples. Andy Krupa and Gavin Horsburgh provided technical assistance. We thank Jason doi: 10.1111/j.1755-0998.2009.02621.x Malham and all the Mauritian Wildlife Foundation staff who assisted with the collection of blood samples over several years. This work © 2009 Blackwell Publishing Ltd