1 23

Conservation Genetics Resources ISSN 1877-7252Volume 4Number 3 Conservation Genet Resour (2012)4:815-819DOI 10.1007/s12686-012-9642-5

The use of cross-species testing ofmicrosatellite markers and sibship analysisin ex situ population management

Eva Ringler

1 23

Your article is protected by copyright and

all rights are held exclusively by Springer

Science+Business Media B.V.. This e-offprint

is for personal use only and shall not be self-

archived in electronic repositories. If you

wish to self-archive your work, please use the

accepted author’s version for posting to your

own website or your institution’s repository.

You may further deposit the accepted author’s

version on a funder’s repository at a funder’s

request, provided it is not made publicly

available until 12 months after publication.

APPLICATION ESSAYS

The use of cross-species testing of microsatellite markersand sibship analysis in ex situ population management

Eva Ringler

Received: 7 January 2012 / Accepted: 30 March 2012 / Published online: 18 April 2012

� Springer Science+Business Media B.V. 2012

Abstract Breeding programs that are aimed for ex situ

conservation of endangered species should assure minimal

inbreeding among individuals in order to maintain the

genetic variation within captive populations. In the absence

of pedigree information, e.g. in cases of confiscated indi-

viduals, rapid, easily applicable and cost-efficient methods

are needed for the establishment of optimal breeding pro-

tocols. The present paper reports the cross-species testing

of already published microsatellite markers from other

closely related species and their use in two critically

endangered Neotropical parrots, Amazona collaria and

Amazona agilis. The aim was to identify full sibling groups

among individuals that had been confiscated due to illegal

trade and were kept at the Vienna Zoo. Of the eight tested

loci only one failed to amplify in both of the two species,

while all other loci produced consistent products. All of the

seven successfully amplifying loci proved to be polymor-

phic in A. collaria, while one locus was monomorphic in A.

agilis. The loci yielded a mean numbers of alleles per locus

of 7.43 (range 4–14) and 8.71 (range 1–14), and a mean

expected heterozygosity of 0.744 and 0.683, respectively.

The identification of several full sibling groups among

birds in both species indicates that the poachers had taken

whole nests. This finding emphasizes that ex situ man-

agement programs have to implement accurate relatedness

or pedigree information in their breeding designs, partic-

ularly if confiscated, illegally traded eggs are involved.

Keywords Amazon parrot � Amazona �Microsatellite markers � Relatedness � Illegal trade �Captive breeding

Introduction

Neotropical parrots (family Psittacidae) are among the

most threatened bird species in the world (Collar et al.

1994). Beside environmental factors such as deforestation,

resource extraction, and climate change (Laurance et al.

2002), also illegal trapping and nest poaching for the pet

trade have become major reasons for the decline of many

natural populations (Munn 1992; Wright et al. 2001). The

popularity of parrots in the pet trade however is in contrast

with the lack of knowledge about their biology, population

genetic structure, and phylogenetic relationships (Masello

and Quillfeldt 2002; Munn 1992). In cases where birds or

eggs of endangered species are confiscated and individuals

cannot immediately be released in their natural habitat, ex

situ breeding programs can play an important role in the

conservation of these species. Captive programs that are

designated for ex situ conservation should aim to establish

optimal breeding protocols in order to preserve the genetic

variation of the founder population (Witzenberger and

Hochkirch 2011). To this end, precise assessment of

relatedness among the focal individuals is required. In

cases where confiscated eggs are involved, it is highly

likely that due to the poaching of whole nests, the sample

contains various groups of full siblings. Thus, accurate

assessment of relatedness may be of particular importance

in order to avoid inbreeding in such cases. However, ex situ

breeding programs are often confronted with financial but

also temporal constraints, as confiscation events cannot be

predicted and management decisions need to be taken

E. Ringler (&)

Department of Evolutionary Biology, University

of Vienna, Althanstraße 14, 1090 Vienna, Austria

e-mail: eva.ringler@univie.ac.at

123

Conservation Genet Resour (2012) 4:815–819

DOI 10.1007/s12686-012-9642-5

Author's personal copy

immediately. Rapid, easily applicable and cost-efficient

methods are thus needed to accurately assess genetic

relatedness among individuals of unknown ancestry (cf.

Russello and Amato 2004).

Microsatellite markers are a powerful tool to assess

genetic differences on the individual and population level

(Jehle and Arntzen 2002; Selkoe and Toonen 2006). Thus

they can be used to investigate genetic diversity within

species, genetic distance and gene flow between popula-

tions, but also to reveal pedigree information about the

individuals within a population, such as as parent-offspring

and sibling relationships (Jones and Ardren 2003). As the

development of suitable microsatellite markers is costly and/

or time-consuming, cross-species testing of already avail-

able markers for closely related species is often used to

obtain suitable markers for species where no species-spe-

cific markers have been developed. Cross-testing thus

minimizes financial expenses and time spent for de novo

microsatellite development. Additionally, cross-species

amplification success of microsatellite markers gives indi-

cation for close phylogenetic relationship between different

species (Freeland 2005; Hendrix et al. 2010). In Neotropical

parrots, several microsatellite loci are already available with

some also having been tested for cross-species amplification

(Gebhardt and Waits 2008). In the genus Amazona, micro-

satellite markers have only been developed for the two

species Amazona guildingii (Russello et al. 2001, 2005) and

A. leucocephala (Taylor and Parkin 2007a), but so far, these

markers were rarely applied to individuals in natural or

captive populations (but see Russello and Amato 2004).

The Yellow-billed Parrot Amazona collaria and the

Black-billed Parrot A. agilis are endemic to Jamaica (For-

shaw 1989). Both species show a severe decline in their

population size due to loss and fragmentation of natural

forests (Cruz and Gruber 1981; Varty 1991). Therefore both

species are given the IUCN status ‘Vulnerable’ and listed on

CITES Appendix II (Snyder et al. 2000).

This paper reports the testing of eight microsatellite

markers that were originally designed for the endangered St.

Vincent Parrot, A. guildingii and for the Moluccan Cockatoo,

Cacatua moluccensis. This choice was based on information

about cross-species amplification and polymorphy in con-

generic species (Gebhardt and Waits 2008; Russello et al.

2001, 2005). Seven of the eight tested loci that succeeded to

amplify in A. agilis and A. collaria were then used to identify

groups of full siblings among the birds in both species.

Methods

Blood samples of 23 A. collaria and 22 A. agilis were

obtained from birds kept at the zoological garden of Vienna

(Tiergarten Schonbrunn) and were preserved in 96 %

alcohol. These birds are the hatchlings of 74 illegally traded

eggs that had been confiscated at the Vienna Airport. Given

the circumstance that the eggs had been subject to illegal nest

poaching, a high likelihood of close relatedness among birds,

i.e. the presence of several full sibling groups (nests), was

expected. Information about the sex of all birds was already

available (genetic analysis of sex chromosomes using DNA

from feather tips). The sex ratio of individuals was almost

balanced in A. agilis (0.83 m/f), and slightly female biased in

A. collaria (0.64 m/f). Genomic DNA was isolated using a

Proteinase K digestion followed by a standard phenol–chlo-

roform protocol (Sambrook et al. 1989). All individuals were

genotyped at 8 microsatellite loci that were previously

described for the species Amazona guildingii (AgGT04,

AgGT07, AgGT12, AgGT19, AgGT21, AgGT42, AgGT81;

Russello et al. 2001, 2005; primer pairs taken from Gebhardt

and Waits 2008) and C. moluccensis (Cmol02; Taylor and

Parkin 2007a). PCR reactions were performed using

20–50 ng DNA, 0.5 U Taq polymerase (Axon), 19 PCR

Reactionbuffer B (Axon), 1.5 mM MgCl2, 2 mM each dNTP

and 5 pmol of each primer in a final volume of 10 ll. PCR

cycles consisted of an initial denaturation at 94 �C for 5 min,

39 cycles at 94 �C for 45 s, 52 �C (57 �C for Cmol02) for

45 s, and 72 �C for 45 s, followed by a final extension of

72 �C for 5 min. Differences in the final allele sizes and in the

fluorescent dye labels of the primers allowed for pooling of

multiple loci. The pooled products were then diluted with

water 1:25, mixed with HiDiformamid (Applied Biosystems)

and the internal size standard ROX500, and run on an ABI

3130xl sequencer. All loci were visually identified using the

program PeakScanner 1.0 (Applied Biosystems). Final allele

sizes were determined using the binning software Tandem

1.01 (Matschiner and Salzburger 2009). Ambiguous samples

were re-genotyped up to three times. CERVUS 3.0 (Kali-

nowski et al. 2007) was used to determine expected (HE) and

observed (HO) heterozygosities, and the overall probability of

identity (PID) in both species. FSTAT v2.9.3.2 (Goudet 2001)

was used to and to determine departures from Hardy–Wein-

berg equilibrium at each locus, to calculate probability tests

for genotypic linkage disequilibrium between loci, and to

determine the inbreeding coefficient FIS. MICROCHECKER

v.2.2.3 (van Oosterhout et al. 2004) was used to test for the

possibility of scoring errors, allelic dropout, and null alleles.

Sibship analysis was carried out with the software COLONY

v.2 (Wang 2009), a likelihood-based method implementing a

group-wise approach for sibship reconstruction. For these

sibship inferences the full likelihood model was used.

Results and discussion

Of the eight loci tested, only one locus (AgGT04) failed to

amplify in both species. In A. collaria, polymorphism was

816 Conservation Genet Resour (2012) 4:815–819

123

Author's personal copy

Ta

ble

1M

icro

sate

llit

ech

arac

teri

sati

on

inA

.co

lla

ria

and

A.

ag

ilis

on

the

bas

iso

fse

ven

mic

rosa

tell

ite

loci

Lo

cus

Pri

mer

seq

uen

ce(50 –

30 )

A.

coll

ari

aA

.a

gil

isG

enb

ank

acce

ssio

nn

o.

Ref

eren

ce

NA

HO

HE

Pnull

NA

HO

HE

Pnull

Cm

ol0

2F

:G

GG

TG

AG

GG

AG

AA

AG

GG

AA

G5

0.6

52

0.5

1-

0.3

55

30

.72

70

.50

3-

0.4

44

DQ

35

41

26

Tay

lor

and

Par

kin

(20

07

a)

R:

AG

CA

GG

TA

TG

GG

GA

CA

GC

AG

Ag

GT

07

F:

CC

TC

CC

CT

AA

AT

AC

GC

AC

AC

60

.65

20

.71

50

.03

59

10

00

AF

33

97

56

Ru

ssel

loet

al.

(20

01

),G

ebh

ard

tan

dW

aits

(20

08

)

R:

AG

CC

AA

AT

CT

GA

CC

AC

CA

GA

Ag

GT

12

F:

TT

CA

GG

AC

AG

CA

CA

GG

TG

AG

40

.52

20

.68

90

.10

61

80

.86

40

.78

2-

0.0

67

AF

33

97

58

Ru

ssel

loet

al.

(20

01

),G

ebh

ard

tan

dW

aits

(20

08

)

R:

GC

TT

GG

GG

TT

TT

GT

TT

TT

GT

Ag

GT

19

F:

GT

CC

TG

CC

TC

CC

AA

AA

AG

A5

0.6

09

0.7

47

0.0

86

69

0.9

55

0.8

72

-0

.06

4A

Y8

78

24

8R

uss

ello

etal

.(2

00

5),

Geb

har

dt

and

Wai

ts(2

00

8)

R:

CA

AC

AT

TG

AC

TC

CT

GG

CA

AA

Ag

GT

21

F:

TT

CA

AA

GG

TG

TC

TG

TA

TG

CA

AT

C1

00

.69

60

.81

40

.05

86

12

0.7

73

0.8

26

0.0

18

7A

F3

39

76

0R

uss

ello

etal

.(2

00

1),

Geb

har

dt

and

Wai

ts(2

00

8)

R:

TC

AG

CC

AG

TT

TC

AG

GC

AC

TA

Ag

GT

42

F:

CA

AA

AT

AC

AC

CT

TA

AA

CC

TG

CA

CA

80

.87

00

.81

1-

0.0

48

14

0.9

09

0.9

04

-0

.01

7A

Y8

78

25

2R

uss

ello

etal

.(2

00

5),

Geb

har

dt

and

Wai

ts(2

00

8)

R:

TG

GG

GT

AA

TG

GA

AG

GA

GT

GA

Ag

GT

81

F:

AG

CT

CT

GG

AG

GG

AG

GA

AA

GA

14

0.5

65

0.9

22

0.1

87

61

40

.59

10

.89

60

.16

58

AF

33

97

61

Ru

ssel

loet

al.

(20

01

),G

ebh

ard

tan

dW

aits

(20

08

)

R:

CA

AG

TA

TC

CA

TG

GG

AT

TT

GG

A

Mea

n7

.43

0.6

70

.74

0.0

18

.71

0.7

00

.68

-0

.06

Ffo

rwar

dp

rim

er,

Rre

ver

sep

rim

er,

NA

nu

mb

ero

fal

lele

s,H

Oo

bse

rved

het

ero

zyg

osi

ty,

HE

exp

ecte

dh

eter

ozy

go

sity

,P

null

pro

bab

ilit

yo

fn

ull

alle

les

bo

ldv

alu

esin

dic

ate

sig

nifi

can

th

om

ozy

go

te

exce

ss(B

on

ferr

on

ico

rrec

ted

)

Conservation Genet Resour (2012) 4:815–819 817

123

Author's personal copy

observed at all seven tested loci with the number of alleles

per locus ranging from four to 14 (mean = 7.43) and

observed heterozygosities ranging from 0.522 to 0.870

(Table 1). In A. agilis one locus (AgGT07) was found to be

monomorphic, while all other loci had between three and

14 alleles (mean = 8.71) with observed heterozygosities

ranging from 0.591 to 0.955 (Table 1). On the basis of

female heterozygotes, all loci are assumed to be autosomal.

In both species, one locus displayed significant deviations

from HWE (after Bonferroni corrected P level of 0.00714;

AgGT81: P = 0.0001; Table 1), due to an excess of

homozygotes. This deviation might either be attributable to

the presence of null alleles, as also the MICROCHECKER

analysis revealed a high probability of null alleles at this

locus in both species (see Table 1), or to the high relatedness

among the birds sampled. Linkage disequilibrium was sig-

nificant for one out of 21 pairwise comparisons in A. collaria

(AgGT07 9 AgGT19: P \ 0.0001; Bonferroni corrected

P level = 0.000238), while there was no evidence for

linkage between any locus pair in A. agilis. The inbreeding

coefficient was much higher in A. collaria (FIS = 0.126)

than in A. agilis (FIS = –0.007). The overall probability of

identity was 6 9 10-8 for A. collaria and 4 9 10-8 for A.

agilis.

Although cross-species polymorphism of microsatellite

loci is assumed generally low in parrots (Hughes et al. 1998;

Taylor and Parkin 2007b), I found quite high numbers of

alleles per locus (Table 1). These loci were used to detect

sibship groups among the birds in each of the samples.

According to the results in the Best(ML) Configuration under

the full likelihood model in COLONY, 12 clusters of full

siblings were identified within the 23 individuals of A. col-

laria: two groups that consisted of three individuals, seven

groups of two full siblings, and for three individuals no full

siblings were identified. The 22 individuals of A. agilis were

identified to constitute 11 clusters of full siblings: four groups

that consisted of three individuals, three groups of two full

siblings, and four single individuals without full siblings.

These numbers conform to the natural clutch sizes in both

species (A. collaria: mean ± SD = 3.0 ± 0; A. agilis:

mean ± SD = 3.1 ± 0.5; Koenig 2001), which supports my

hypothesis that whole nests were taken by the poachers. This

finding critically emphasizes the importance of accurate

relatedness or pedigree information to be implemented in

captive breeding protocols, particularly if individuals had

been confiscated due to illegal trade.

In the course of searching for suitable primers I detected

that the A. guildingii clone AgGT07 (GenBank accession

no. AF339756) is identical with the clone AgGT90 (Gen-

Bank accession no. AY878255), as they constitute inverse

locus sequences of one another.

The set of seven microsatellite markers used in this

study were sufficiently powerful for sibship analysis in the

two species A. agilis and A. collaria. This information will

serve to prioritize future breeding decisions in both species.

The successful cross-species amplification of the micro-

satellite markers in this and previous studies indicates a

close phylogenetic relationship among Amazon parrots (cf.

Sibly and Alquist 1990). Concluding, I highly recommend

cross-testing across different species as a time and cost-

efficient approach to de novo development of species-

specific markers. Such markers can then be used to identify

groups of full siblings among individuals of unknown

relatedness in order to design effective breeding protocols

in a wide variety of species.

Acknowledgments This study was funded by the Vienna Zoo

‘Tiergarten Schonbrunn’. Thomas Voracek provided the blood sam-

ples of all individuals. Sampling and all molecular analyses were

conducted under the current Austrian laws and following the ASAB

guidelines. Thanks to Christian Baranyi and Max Ringler for com-

ments on the manuscript.

References

Collar NJ, Crosby MJ, Stattersfield AJ (1994) Birds to watch. 2 the

world list of threatened birds. Bird Life Internatn, Washington,

DC

Cruz A, Gruber S (1981) The distribution, ecology, and breeding

biology of Jamaican amazon parrots. In: Pasquier RF (ed)

Conservation of new world parrots, techn. publ. 1. Smithsonian

Institution Press for the International Council of Bird Preserva-

tion, Washington, DC, pp 103–131

Forshaw JM (1989) Parrots of the world, 3rd edn. Lansdowne

Editions, Melbourne

Freeland JR (2005) Molecular ecology. Wiley, West Sussex

Gebhardt KJ, Waits LP (2008) Cross-species amplification and

optimization of microsatellite markers for use in six neotropical

parrots. Mol Ecol Res 8:835–839

Goudet J (2001) FSTAT, a program to estimate and test gene

diversities and fixation indices (version 2.9.3). Available from

http://www2.unil.ch/popgen/softwares/fstat.htm

Hendrix R, Hauswaldt JS, Veith M, Steinfartz S (2010) Strong

correlation between cross-amplification success and genetic

distance across all members of ‘‘True Salamanders’’

(Amphibia:Salamandridae) revealed by Salamandra salaman-dra-specific microsatellite loci. Mol Ecol Res 10:1038–1047

Hughes CR, Melland RR, Beissinger SR (1998) Polymorphic trinu-

cleotide microsatellite loci for a neotropical parrot, the green-

rumped parrotlet, Forpus passerinus. Mol Ecol 7:1247–1263

Jehle R, Arntzen JW (2002) Review: microsatellite markers in

amphibian conservation genetics. Herp J 12:1–9

Jones AG, Ardren WR (2003) Methods of parentage analysis in

natural populations. Mol Ecol 12:2511–2523

Kalinowski ST, Taper ML, Marshall TC (2007) Revising how the

computer program CERVUS accommodates genotyping error

increases success in paternity assignment. Mol Ecol 16:1099–

1106

Koenig SE (2001) The breeding biology of black-billed parrot

Amazona agilis and yellow-billed parrot Amazona collaria in

cockpit country, Jamaica. Bird Conserv Internatn 11:205–225

Laurance WF, Albernaz AKM, Schroth G, Fearnside PM, Bergen S,

Venticinque EM, Da Costa C (2002) Predictors of deforestation

in the Brazilian Amazon. J Biogeogr 29:737–748

818 Conservation Genet Resour (2012) 4:815–819

123

Author's personal copy

Masello JF, Quillfeldt P (2002) Chick growth and breeding success of

the burrowing parrot. Condor 104:574–586

Matschiner M, Salzburger W (2009) TANDEM: integrating auto-

mated allele binning into genetics and genomics workflows.

Bioinformatics 25:1982–1983

Munn CA (1992) Macaw biology and ecotourism, or ‘when a bird in

the bush is worth two in the hand’. In: Beissinger SR, Snyder

NFR (eds) New world parrots in crisis: solutions from conser-

vation biology. Smithsonian Institution Press, Washington, DC,

pp 47–72

Russello M, Amato G (2004) Ex situ population management in the

absence of pedigree information. Mol Ecol 13:2829–2840

Russello M, Calcagnotto D, DeSalle R, Amato G (2001) Character-

ization of microsatellite loci in the endangered St. Vincent parrot

Amazona guildingii. Mol Ecol Notes 1:162–164

Russello MA, Lin K, Amato G, Caccone A (2005) Additional

microsatellite loci for the endangered St. Vincent Parrot

Amazona guildingii. Conserv Genet 6:643–645

Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a

laboratory manual, 2nd edn. Cold Spring Harbor Laboratory

Press, New York

Selkoe KA, Toonen RJ (2006) Microsatellites for ecologists: a

practical guide to using and evaluating microsatellite markers.

Ecol Lett 9:615–629

Sibly CG, Alquist JE (1990) Phylogeny and classification of birds—a

study in molecular evolution. Yale University Press, New Haven

Snyder NFR, McGowan P, Gilardi J, Grajal A (2000) Parrots: status,

survey, and conservation action plan 2000–2004. IUCN, Gland

Taylor TD, Parkin DT (2007a) Characterisation of 13 microsatellite

loci for the Moluccan Cockatoo, Cacatua moluccensis, and

Cuban Amazon, Amazona leucocephala, and their conservation

and utility in other parrot species (Psittaciformes). Conserv

Genet 8:991–994

Taylor TD, Parkin DT (2007b) Characterization of 12 microsatellite

primer pairs for the African grey parrot, Psittacus erithacus and

their conservation across the Psittaciformes. Mol Ecol Notes

7:163–167

van Oosterhout C, Hutchinson WF, Wills DPM, Shipley P (2004)

Micro-checker: software for identifying and correcting genotyp-

ing errors in microsatellite data. Mol Ecol Notes 4:535–538

Varty N (1991) The status and conservation of Jamaica’s threatened

and endemic forest avifauna and their habitats following

Hurricane Gilbert. Bird Conserv Int 1:135–151

Wang J (2009) A new method for estimating effective population

sizes from a single sample of multilocus genotypes. Mol Ecol

18:2148–2164

Witzenberger KA, Hochkirch A (2011) Ex situ conservation genetics:

a review of molecular studies on the genetic consequences of

captive breeding programmes for endangered animal species.

Biodivers Conserv 20:1843–1861

Wright TM, Toft CA, Enkerlin-Hoeflich E et al (2001) Nest poaching

in Neotropical parrots. Conserv Biol 15:710–720

Conservation Genet Resour (2012) 4:815–819 819

123

Author's personal copy