Vol. 15 - Deutsches Primatenzentrum

Page 60 Lemur News Vol. 15, 2010
of forest cover and fragmentation of suitable habitat (Harper
et al., 2007) which limits the species’ density and range
(Glessner and Britt, 2005). Although protected, the Babakoto
is also threatened by subsistence hunting pressure and
bush meat trade (Golden, 2005). Designated as Endangered
according to the IUCN Red List of Threatened Species
(IUCN, 2008), I. indri is currently split into two subspecies,
Indri indri indri (Gmelin, 1788) and Indri i. variegatus (Gray,
1872). Here, we present population genetic parameter estimates
from populations along the entire range of the species
analyzed from nuclear microsatellite multilocus genotypes.
Methods
Samples were collected from 106 Indri from 10 sites across
the geographical range of the species (Fig. 1). From north to
south,the forests represented in the collection were Anjanaharibe-Sud
Special Reserve, Marotandrano Special Reserve,
Ambatovaky Special Reserve,Zahamena Special Reserve and
National Park, Betampona Nature Reserve, Anjozorobe
Regional Forest Reserve, Mantadia National Park, Analamazoatra
Special Reserve (Andasibe),Maromizaha Classified
Forest, and Anosibe an’ala Classified Forest. The elevations
of the sampling sites ranged from lowland forests (Anosibe
An’ala, 125 m asl) to highland forests (Anjozorobe, 1358 m
asl).
Fig. 1: Map of Madagascar indicating the study areas.
Immobilization and collection
All lemurs investigated in this study were free-ranging and
were immobilized with a CO2 powered DAN-INJECT (Brrkop,
Denmark) Model JM rifle propelling Pneu-Darts (Williamsport,
PA) loaded with 10 mg/kg estimated body weight
of Telazol ® (Fort Dodge).We recorded the location (within 6
m accuracy) of all of the immobilized lemurs using a global
positioning system (GPS) device. Each individual was transported
back to the base camp where complete morphometric
data were taken (Zaonarivelo et al., 2007a). Whole
blood (1.0 cc per kilogram) from the femoral artery and
2.0 mm skin biopsies from the ear pinnae were collected
from each sedated lemur (Junge and Louis, 2002). A Home
Again ® (Home Again Pet Recovery Service, East Syracuse,
NY) microchip was placed subcutaneously between the scapulae
of each lemur to positively identify individuals re-captured
during any future immobilizations. Following data and
sample collection, an injection of lactated Ringer’s solution
was administered subcutaneously to support maintenance
requirements and to dissipate the effect of the Telazol ®.Animals
were monitored for three hours post recovery then
released according to the capture GPS coordinates.
Data generation
Ear punches were dissected into quarters and DNA was
extracted using standard PCI/Chloroform procedures (Sambrook
et al., 1989). Approximately 50 ng of genomic DNA
was used for each PCR reaction. Multilocus genotypes were
generated from a suite of 20 Indri-specific microsatellite loci
as described in Zaonarivelo et al. (2007b). The genotype file
was checked for typographical errors,scoring errors,stutter
bands and allele dropout with Micro-Checker (van Oosterhout
et al., 2004) and Microsatellite Analyser (MSA; Dieringer
and Schlötterer, 2002). We used CERVUS (version 2.0,
Marshall et al.,1998;Slate et al.,2000) to identify loci with excessive
null allele frequency estimates (nf > 0.10) and to estimate
polymorphic information content of the loci.Moderate
(0.05 < nf < 0.20) and high (0.20 < nf) null allele frequencies
can have significant effects on population genetics parameter
estimates (Chapuis and Estoup, 2007). The process of redesigning
primer pairs is both costly and time consuming;
therefore,we opted to delete problematic loci from the data
set. We deleted eight loci with moderate null allele frequencies
(nf > 0.1) to reduce the bias from misclassification of null
heterozygotes as homozygotes (Callen et al., 1993; Hoffman
and Amos, 2005) and to control the variance of parameter
estimates (Chapuis and Estoup, 2007). The accepted loci
were verified for independence of linkage disequilibrium
(with Bonferroni-adjusted P-values) in FSTAT (Goudet,1995,
2001).
Hardy-Weinberg exact tests (Guo and Thompson, 1992)
were performed by locus and population in Genepop (version
4.0, Raymond and Rousset, 1995). Initially, we used the
default settings for the MCMC estimation of HWE then
increased the batch size from 100 to 250 to reduce the standard
error of the P-value to below 0.01. Genetic diversity
was measured as observed heterozygosity (HO) and expected
heterozygosity (HE). In addition, the number of effective
migrants was estimated globally and pair-wise using the private
allele method. We used FSTAT to calculate the total
number of alleles (k), mean number of alleles (MNA), and
rarefacted allelic richness (AR; Leberg, 2002) by locus and
population.Allelic richness estimates the allelic diversity in a
data set based on the population with the fewest number of
individuals contributing genotypes by locus. This is an unbiased
comparison of allelic diversity since populations with
more contributors provide a greater opportunity to capture
more alleles from lower frequency occurrences. Wright’s Fstatistics
were estimated in FSTAT for within population
similarity (FIS) and between population differences (FST)
according to Weir and Cockerham (1984).
The effective population sizes were estimated with the linkage
disequilibrium (LD) option in NeEstimator (Peel et al.,
2004; Hill, 1981; Waples, 1991). We tested all populations
having met the minimum statistical threshold required (n =
20 genes or 10 individuals) for the presence of bottleneck
events using Bottleneck (version 2.0, Cornuet and Luikart,