Cross-species amplification of seventeen polymorphic microsatellite loci in the endangered Crowned...

Cross-species amplification of seventeen polymorphic microsatellite loci in 1

the endangered crowned eagle (Harpyhaliaetus coronatus) 2

3

J. H. Sarasola1-2-3

, D. Canal4, C. Solaro

1-2, M. A. Galmes

1-3, J. I. Zanón-Martínez

1-2 & J. J. 4

Negro4 5

6

1 Centro para el Estudio y Conservación de las Aves Rapaces en Argentina (CECARA), 7

Universidad Nacional de La Pampa – CONICET, Avda. Uruguay 151, 6300 Santa Rosa, La 8

Pampa, Argentina. 9

2 Instituto de las Ciencias de la Tierra y Ambientales de La Pampa (INCITAP), Consejo 10

Nacional de Investigaciones Científicas y Técnicas de Argentina (CONICET), Avda. Uruguay 11

151, 6300 Santa Rosa, La Pampa, Argentina. 12

3 The Peregrine Fund, 5668 West Flying Hawk Lane, Boise, ID 83709 U.S.A. 13

4 Department of Evolutionary Ecology, Estación Biológica de Doñana-CSIC, Avda. Américo 14

Vespucio s/n, 41092 Seville, Spain. 15

16

Keywords: crowned eagle, Harpyhaliaetus coronatus, cross-species amplification, 17

microsatellite, population genetics. 18

19

Corresponding author: José Hernán Sarasola, Phone: +54 2954 430157. Email: 20

[email protected] 21

22

Running title: Microsatellite markers for the crowned eagle23

of 11 Molecular Ecology Resources

Abstract 24

The crowned eagle (Harpyhaliaetus coronatus) is one of the most severely threatened birds of 25

prey in the world for which genetic markers have not been developed. We examined the cross 26

amplification of thirty seven microsatellite loci in this endangered eagle. Seventeen loci were 27

polymorphic and hence valuable as tools for population genetic studies. The number of alleles 28

per locus ranged from 2 to 9, and the average number of alleles across all polymorphic loci 29

was 4.4. The markers tested provide a valuable resource for research in population genetics 30

and the conservation of this species. The success of cross-species amplification suggests that 31

these microsatellites will be useful for studies in a broad range of raptor species. 32

33

of 11Molecular Ecology Resources

The crowned eagle (Harpyhaliaetus coronatus) is one of the rarest and most severely 34

threatened birds of prey in the world. Its range extends from southern Brazil to northern 35

Patagonia, where it inhabits a variety of forested habitats, including woodlands and other 36

savanna-like landscapes (Fergusson-Lees & Christie 2001). The species is listed as 37

endangered under the IUCN Red List with a declining world population estimated at less than 38

1,000 individuals. Crowned eagles are considered extinct in Uruguay where no records have 39

been recorded since 1930 (BirdLife International 2008). Human persecution and other 40

anthropogenic factors seem to be the main threat for the species in semiarid habitats of central 41

and western Argentina (Sarasola & Maceda 2006, Sarasola et al. 2010). Current population 42

status and population trends make the development of molecular tools that can be used to aid 43

the management and conservation of this species crucial. 44

Microsatellite loci are one of the best and more powerful classes of molecular markers 45

for studies of genetic processes in natural populations (Frankham et al. 2004). However, their 46

development maybe costly and a time consuming task. As flanking sequences of 47

microsatellites are conserved region (Dawson et al. 2006, Meglécz et al. 2007) cross-species 48

amplification has been proved, across a range of taxa (insects: Augustinos et al. 2011; 49

amphibians: Hendrix et al. 2010; birds: Dawson et al. 2006), as a cost effective approach to 50

obtain microsatellites markers in additional species. Here we communicate the cross-species 51

amplification of polymorphic microsatellite loci between a phylogenetically diverse group of 52

raptors and the crowned eagle. These microsatellite loci will enable further studies in 53

population genetic structure and genetic diversity to be undertaken on this endangered raptor 54

species. 55

Thirty eight samples of crowned eagles were analyzed in the study: eighteen feathers 56

and twenty blood samples obtained from wild individuals from La Pampa province, 57


Argentina. Genomic DNA was extracted by a standard phenol-chloroform method (Sambrook 58

et al. 1989). Thirty-seven polymorphic microsatellite loci isolated for seven raptor species 59

(order Falconiformes) were chosen for testing amplification in the crowned eagle. All primers 60

were tested for amplification in a preliminary screening with a subset of six blood samples of 61

the species. PCRs were performed in 25-µL reaction volumes containing 1x buffer, 2.0 mM 62

MgCl2, 0.2 mM of each dNTP, 0.5 U Taq Polymerasa, 0.2 µM of each primer and 25 ng of 63

DNA as template. PCR amplifications consisted of initial denaturation (2 min at 94°C) 64

followed by 35 cycles of 30 s at 94°C, 30 s at a annealing temperature between 50ºC and 60 65

ºC and 30 s at 72°C, plus a final extension of 10 min at 72°C. The entire PCR product was run 66

on a 2% agarose gel, and amplicon sizes were determined using a 100-bp-size standard 67

marker (BIOLINE Hyperladder). As some loci either failed to amplify or showed problems in 68

the amplification (i.e. weak or nonspecific bands in gel) we ran new PCRs in order to 69

optimize amplification conditions. One primer of the 35 pairs that apparently amplified a 70

single amplicon was tagged with VIC, FAM, PET or NED fluorescent labels (Applied 71

Biosystems). Fluorescent products were analysed on an ABI377 automated sequencer using 72

Genescan 500-LIZ internal size standard. Polymorphism and alleles sizes were determined 73

with Genemapper 4.0 software (Applied Biosystems). Thirteen loci were monomorphic and 74

four showed unspecific amplifications that, despite new attempts to optimized PCR 75

conditions, could not be eliminated. The remained 18 loci were sequenced to ensure that the 76

homologous microsatellite region was being amplified. This step revealed that locus Hf-C1D2 77

have lost the repetition motif as a consequence of both a deletion in the number of repetitions 78

and substitution of “GA” by “GG” and was therefore discarded for further analyses. Feathers 79

were genotyped three times to evaluate the frequency of genotyping errors (Horváth et al. 80


2004) but no discrepancies among repetitions were found. Genotyped obtained from samples 81

confirmed that each one belonged to different individuals. 82

We developed a multiplexing protocol for the polymorphic markers (Table 1). For 83

multiplex PCR reactions we used Quiagen multiplex PCR Kit following the supplier’s 84

protocol with an annealing temperature of 56ºC. Allele sizes of markers from single locus 85

amplification and multiplex reactions were compared to ensure reliability of multiplexing 86

amplifications. This protocol allowed amplifying 18 loci in five reactions reducing 87

significantly laboratory cost and time. The number of alleles (N), observed and expected 88

heterozygosities (HO and HE) were calculated using CERVUS version 3.0.3 (Marshall 1998). 89

Probability of deviation from Hardy–Weinberg equilibrium and linkage disequilibrium were 90

tested using Genepop 4.0 (Raymond & Rousset 1995). 91

Of the multiple loci tested from seven raptor species, at least some loci amplified from 92

six of the seven species tested. The number of alleles per locus ranged from 2 to 9 with an 93

average of 4.4 alleles per locus (Table 1). All loci but Hal04 and BswD107 conformed to 94

Hardy-Weinberg equilibrium and no pairs of loci showed significant linkage disequilibrium 95

after Bonferroni correction (Table 1). The success of cross amplification in crowned eagles 96

was high in the case of primers described for Aquila heliaca (two of two primers, 100%) 97

followed by Buteo buteo (83% two of three primers, monomorfic locus: Bbu51), Haliaaetus 98

albicilla (60 % three of five; locus Hal03 was monomorfic and Hal01 gave non specific 99

amplification), Falco rusticolus (50% one of two, locus NVHfr195–2 failed to amplify), B. 100

swainsoni (49% six of thirteen; monomorfic loci: BswB220w, BswA204w, BswA303w, 101

BswD327w, BswA312w; loci that failed: BswB221w and BswA110w) and Hieraeetus 102

fasciatus (43% three of seven; monomorfic: Hf-C7G4, Hf-C7E1 and Hf-C1D2; non specific 103

amplification: Hf-C1E6; ). No polymorphic markers were obtained from five primers 104


designed by Martínez-Cruz et al. (2002) for Aquila adalberti (monomorphics: Aa11, Aa26, 105

Aa36 and Aa43; non specific amplification: Aa57). The success of amplification of a locus in 106

cross-species strategy in avian species could be higher when the genetic distance between 107

source and target species is small (Primmer et al. 2005). Primers designed for the two Buteo 108

species, which are included in the same sub-family with Harpyhaliaetus, showed a relative 109

good success of amplification considering the large number of primers tested. Primers 110

designed for Falco genus, however, showed low to moderate success as expected for species 111

included in a different family (Falconidae) than the target species (Accipitridae). 112

The microsatellite markers tested here provide a powerful tool for management and 113

conservation of crowned eagles, allowing further population genetic studies, unambiguous 114

individual identification and paternity assessment in this endangered species. 115

116

Acknowledgements 117

This work was supported by the Agencia Española de Cooperación Internacional para el 118

Desarrollo (AECID, Spain), the Peregrine Fund (USA) and the Universidad Nacional de La 119

Pampa (Argentina). 120

121

References 122

Augustinos AA, Asimakopoulou AK, Papadopoulos NT, Bourtzis K. (2001) Cross-amplified 123

microsatellites in the European cherry fly, Rhagoletis cerasi: medium polymorphic-124

highly informative markers. Bulletin of Entomological Research, 101, 45-52. 125

BirdLife International (2008) Harpyhaliaetus coronatus. In: IUCN 2010. IUCN Red List of 126

Threatened Species. Version 2010.2. www.iucnredlist.org 127


Busch JD, Katzner TE, Bragin E, Keim P (2005) Tetranucleotide microsatellites for aquila 128

and haliaeetus eagles. Molecular Ecology Notes, 5, 39-41. 129

Dawson DA, Burke T, Hansson B, Pandhal J, Hale MC, Hinten GN, Slate J (2006) A 130

predicted microsatellite map of the passerine genome based on chicken- passerine 131

sequence similarity. Molecular Ecology, 15, 1299-1320. 132

Fergusson-Lees J, Christie DA (2001) Raptors of the world. Helm Identification Guides, 133

London, UK. 134

Frankham R, Ballou JD, Briscoe DA (2004) A primer of conservation genetics. Cambridge 135

University Press. 136

Hailer F, Gautschi B, Helander B (2005) Development and multiplex PCR amplification of 137

novel microsatellite markers in the White-tailed Sea Eagle, Haliaeetus albicilla (Aves, 138

Falconiformes, Accipitridae). Molecular Ecology Notes, 5, 938-940. 139

Hendrix R, Hauswaldt JS, Veith M, Steinfartz S (2010) Strong correlation between cross-140

amplification success and genetic distance across all members of ‘True Salamanders’ 141

(Amphibia: Salamandridae) revealed by Salamandra salamandra-specific microsatellite 142

loci. Molecular Ecology Resources, 10, 1038–1047. 143

Hull JM, Tufts D, Topinka JR, May B, Ernest HB (2007) Development of 19 microsatellite 144

loci for Swainson’s hawks (Buteo swainsoni) and other buteos. Molecular Ecology 145

Notes, 7, 346–349. 146

Horváth MB, Martínez-Cruz B, Negro JJ, Kalmàr L, Godoy JA (2004) An overlooked DNA 147

source for non-invasive genetic analysis in birds. Journal of Avian Biology, 36, 84–88. 148

Johnson PCD, Fowlie MK, Amos W (2005) Isolation of microsatellite loci from the common 149

buzzard, Buteo buteo (Aves, Accipitridae). Molecular Ecology Notes, 5, 208-211. 150


Marshall TC, Slate J, Kruuk L, Pemberton JM (1998) Statistical confidence for likelihood 151

based paternity inference in natural populations. Molecular Ecology, 7, 639–65. 152

Martínez-Cruz B, David VA, Godoy JA, Negro JJ, O’Brien SJ, Johnson WE (2002) Eighteen 153

polymorphic microsatellite markers for the highly endangered Spanish imperial eagle 154

(Aquila adalberti) and related species. Molecular Ecology Notes, 2, 323-326. 155

Meglécz, E., Anderson, A., Bourguet, D., Butcher, R., Caldas, A., Cassel-Lundhagen, A., 156

Cœur d’Acier, A., Dawson, A.D., Faure, N., Fauvelot, C., Franck, P., Harper, G., 157

Keyghobadi, N., Kluetsch, C., Muthulakshmi, M., Nagaraju, J., Patt, A., Péténian, F., 158

Silvain JF., Wilcock, HR (2007) Microsatellite flanking region similarities among 159

different loci within insect species. Insect Molecular Biology, 16, 175-185 160

Mira S, Wolff K, Cancela ML (2005) Isolation and characterization of microsatellite markers 161

in Bonelli’s eagle (Hieraaetus fasciatus). Molecular Ecology Notes, 5, 493-495. 162

Nesje M, Røed KH (2000) Microsatellite DNA markers from the gyrfalcon (Falco rusticolus) 163

and their use in other raptor species. Molecular Ecology, 9, 1438-1440. 164

Primmer CR, Painter J N, Koskinen MT, Palo JU, Merilä J (2005) Factors affecting avian 165

cross microsatellite amplification. Journal of Avian Biology, 36, 348-360. 166

Raymond M, Rousset F (1995) Genepop (version 1.2), population genetics software for exact 167

tests and ecumenicism. Journal of Heredity, 86, 248-249. 168

Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual, 2nd ed. 169

Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York. 170

Sarasola JH, Maceda JJ (2006) Past and current evidence of persecution of the endangered 171

Crowned Eagle Harpyhaliaetus coronatus in Argentina. Oryx, 40, 347–350. 172


Sarasola JH, Santillán MA, Galmes MA (2010) Crowned eagles rarely prey on livestock in 173

central Argentina: persecution is not justified. Endangered Species Research, 13, 207-174

213. 175


Table 1. Summary of the polymorphic microsatellite loci developed for six different raptor species and amplified successfully in crowned eagles: 176

original and new Gene Bank Accession Number, annealing temperature in simplex PCR (Ta), alleles per polymorphic locus (N), observed 177

heterozygosity (HO), expected heterozigocity (HE) and source. 178

179

Specie

Locus

Gene Bank

Number

New Gene

bank Number

Ta (ºC)

N

HO

HE

Source

Buteo swainsoni BswB234w S1

DQ988163 JQ309945 56 6 0.393 0.423 Hull et al. (2007)

BswB111aw S4

DQ985713 JQ309946 60 2 0.328 0.341

BswD220w S3

DQ985722 JQ309947 56 5 0.754 0.795

BswD107w S3

DQ985716 JQ309948 56 8 0.793 0.865**

BswA317w S4

DQ985712 JQ309960 56 4 0.333 0.319

BswA302w S1

DQ985709 JQ309961 56 2 0.316 0.292

Falco rusticolus NVHfr206 S5

AF200207 JQ309958 56 3 0.517 0.497 Nesje & Røed (2000)

Aquila heliaca IEAAAG04 S4

AY631063 JQ321581 56 6 0.756 0.716 Busch et al. (2005)

IEAAAG15 S4

AY631070 JQ309959 56 2 0.052 0.051


Haliaeetus albicilla Hal04S1

AY817043 JQ309957 56 7 0.518 0.633 Hailer et al. (2005)

Hal09 S1

AY817048 JQ309956 56 2 0.667 0.538

Hal10 S2

AY817049 JQ309955 56 3 0.375 0.449

Buteo buteo Bbu42 S5

AJ715912 JQ309954 56 9 0.793 0.708 Johnson et al. (2005)

Bbu46 S3

AJ715916 JQ309953 56 7 0.766 0.709

Hieraaetus fasciatus Hf-C1E8S5

AY823587 JQ309952 53 3 0.241 0.592** Mira et al. (2005)

Hf-C3F2 S2

AY823596 JQ309951 56 4 0.500 0.493

Hf-C5D4 S3

AY823597 JQ309950 56 2 0.286 0.319

180

** indicates Hardy-Weinberg disequilibrium at p< 0.01. 181

Multiplex set reactions are denoted by S1, S2, S3, S4 and S5. 182


Cross-species amplification of seventeen polymorphic microsatellite loci in the endangered Crowned...

Documents

Transcript of Cross-species amplification of seventeen polymorphic microsatellite loci in the endangered Crowned...