Directory UMM :Data Elmu:jurnal:B:Biochemical Systematics and Ecology:Vol29.Issue2.Feb2001:
Biochemical Systematics and Ecology 29 (2001) 125}136
Species discrimination and population
di!erentiation in ants using microsatellites
Julie M. Macaranas1, Donald J. Colgan*, Richard E. Major,
Gerasimos Cassis, Michael R. Gray
Centre for Biodiversity and Conservation Research, Australian Museum, 6 College Street, Sydney 2010 NSW,
Australia
Received 24 November 1999; accepted 22 March 2000
Abstract
This study was conducted to establish the regional scale of population di!erentiation of ants
in the wheat belt of central western New South Wales. Microsatellite variation was surveyed at
"ve loci in two morphologically similar ant species (designated `Aa and `Ba) from the
Camponotus ephippium complex. Three of the "ve scored microsatellite loci were highly variable
with totals, in the two species, of 11, 13 and 42 alleles. The other loci had two and three alleles.
The mean number of alleles per locus per sample ranged from 2.0 to 4.6 for species A and from
1.4 to 3.8 in species B. Mean observed heterozygosity was 0.385 for species A and 0.363 for
species B. The geographic distribution of genotypes was signi"cantly non-random for all tested
loci in both species. Eight of 47 alleles in species A and 15 of 28 in species B were restricted to
a single site. Allelic accumulation percentages were calculated for several orderings of samples
* level of heterozygosity, sample size and geographic position. In all orderings three or more
samples must be included for more than three-quarters of alleles to be represented. ( 2001
Elsevier Science Ltd. All rights reserved.
Keywords: Camponotus; Ant; Microsatellite; Species discrimination; Genetic variation
1. Introduction
There has been a long and continuing interest in the genetics of ants, with
eager application of new techniques to questions about colony structure and nest
1 Present address: DFID Fish Genetics Program at Asian Institute of Technology, Aquaculture & Aquatic Resources Management Program (AARM). PO Box 4, Klong Luang, Pathumthani 12120, Thailand.
* Corresponding author. Tel.: #61-2-9320-6000.
E-mail address: [email protected] (D.J. Colgan).
0305-1978/01/$ - see front matter ( 2001 Elsevier Science Ltd. All rights reserved.
PII: S 0 3 0 5 - 1 9 7 8 ( 0 0 ) 0 0 0 3 8 - 7
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J.M. Macaranas et al. / Biochemical Systematics and Ecology 29 (2001) 125}136
inter-relationships (Pamilo et al., 1997). In general, signi"cant genetic structuring
in ant species has been observed at scales of tens of metres and of a few kilometres.
At these scales, the amount of di!erentiation is strongly associated with the
sociogenetic organisation of a species' colonies and its means of dispersal
(Sundstrom, 1993; Seppa and Pamilo, 1995; Chapuisat et al., 1997; Tay et al.,
1997; Ross et al., 1997). Monogynous species, which generally take part in mating
#ights and found new nests independently, are less di!erentiated than polygynous
species. These are characterised by reduced queen dispersal during mating with
budding as a major mode of nest founding (Pamilo et al., 1997; Pedersen and
Boomsma, 1999).
In contrast to the level of interest in local genetic di!erentiation, there has
been relatively little emphasis on regional level di!erentiation in ants. This level
would be important for the extension of ants' use as ecological species indicators
(Andersen, 1990; Bestelmeyer and Wiens, 1996) to conservation genetics. This
paper examines genetic variation at the regional level in ants of the Camponotus
ephippium species group in the wheat belt of central western New South Wales.
Microsatellites were chosen for study, as they have proven generally applicable
to analyses of "ne-scale di!erentiation and gene #ow studies in ants (Pamilo
et al., 1997). Moreover, primers have been developed for Camponotus by Gertsch et al.
(1995) and Crozier et al. (1999), the latter already having proven successful in
ampli"cation of DNA from species other than C. consobrinus in which they were
developed.
Habitat fragmentation potentially causes di$culties for the maintenance of genetic
and species diversity by restricting gene #ow and lowering the likelihood of reestablishment of locally extinct populations through immigration (reviewed in Forman, 1995; Setterle et al., 1996). Questions of concern include the degree to which
fragmentation has reduced intra-population variation, whether this has impinged on
variation in the regional population considered as a whole, and what strategy should
be adopted for reservation practices to maximise the probability of maintaining
genetic variability.
2. Materials and methods
2.1. Study site
The study area (Fig. 1) lies in the wheatbelt of New South Wales between
West Wyalong and Condobolin. Sivertsen and Metcalfe (1995) describe its vegetation.
Only 16% of the native vegetation in the region has escaped the clearing that
began on an extensive scale in the late 1800s. Much of the remnant native vegetation
is on public land such as state forests and roadside reserves. The sample sites
comprise six state forests between 640 and 1860 ha in area and six long roadside
reserves of approximately 20 m in width. The length of the study area is about 40 km
from north to south and its breadth about 14 km, with 1.2 km between the nearest pair
of sites.
J.M. Macaranas et al. / Biochemical Systematics and Ecology 29 (2001) 125}136
127
Fig. 1. Map of the study area. The inset shows the location of the area in the central wheat belt (shaded).
The state forests are: Weelah, Manna, Euglo South, Nerang Cowal, Lake View and Corringle. The road
reserves are numbered from 1 to 6 and are indicated by "lled circles. Other areas of native vegetation are
shown by light shading.
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J.M. Macaranas et al. / Biochemical Systematics and Ecology 29 (2001) 125}136
2.2. Study species
C. ephippium group species are widely distributed in the area (Major et al., 1999).
Worker ants, of what was believed to be one species based on morphological
taxonomy (voucher specimen numbers K150547-9, K150550-1 and K150552-3), were
sampled by hand. One ant was taken from each 20}30 m length (approximated) along
the entire distance of a 500 m transect bisected by a road or, for the state forests,
a management trail. Ants were missing from some parts of the transects. Samples were
stored in ethanol. This procedure was adopted to improve estimation of population
genetic parameters by reducing multiple sampling from single nests. Multiple sampling from a single colony might still occur if it were polydomous and its nests were
highly dispersed.
2.3. Molecular methods
Individual ants were fully rehydrated in distilled water and their gasters removed
before DNA extraction using the Rapidgene Genomic DNA puri"cation kit (Amresco
USA). Five microsatellite primer pairs developed for other Camponotus species
(Gertsch et al., 1995: Camp 4 and Camp 8; Crozier et al., 1999: Ccon 12, Ccon 70 and
Ccon 79) readily generated PCR products (see Table 1).
PCR was performed under the following conditions: 5 min at 963C, 1 min at 563C,
1 min at 723C (1 cycle), followed by 1 min at 943C, 1 min at 563C, 1 min at 723C (34
cycles) and "nally 1 min at 943C, 1 min at 563C, 5 min at 723C. PCR reaction mixtures
(25 ll) contained 25}200 ng of genomic DNA, dCTP, dGTP and dTTP at 200 lM,
dATP at 10 lM, 25 pmoles of primer, 1.5 mM MgCl , 1 lCi of a33P-dATP (1500 Ci
2
mmol~1, Bresatec Australia), 1]reaction bu!er and 0.5 U of Thermostable DNA
polymerase from Thermus &icelandicus' (Advanced Biotechnologies, UK). Each reaction was covered with 50 ll mineral oil. PCR products (excluding oil overlay) were
mixed with 7 ll of Stop solution (United States Biochemical), heated to 943C for 2 min
and immersed in ice water before application to a sequencing gel (6% 19 : 1 acrylamide:bis-acrylamide, 7 M urea). Electrophoretic runs using 1]TBE bu!er (89 mM
Tris-HCl pH 8.3, 89 mM boric acid, 5 mM EDTA) lasted from 1.5 to 3.5 h according
to the size of the microsatellites. At least three reference microsatellites were included
in every run. After vacuum-drying, the gel was used to expose X-ray "lm for at least
16 h. Microsatellite size and sequence were determined from non-radioactive PCR
products of known homozygotes. For sequencing using dye terminator technology on
a PE 377, products were cleaned using the QIAQUICK PCR puri"cation kit (Qiagen,
Germany).
Allelic size estimates were based on the relative mobility of sequenced alleles and/or
microsatellite ladders. For Ccon 79, sizes could only be roughly estimated from the
sequenced alleles as bands at this locus did not produce a ladder-like pattern.
Generally, (but see the `Resultsa section below), alleles were designated alphabetically
in order of decreasing mobility, with the most anodal being designated A.
Estimates of genetic variability were obtained using the BIOSYS 1.7 program
(Swo!ord and Selander, 1989). Clustering based on Nei's unbiased genetic distance
J.M. Macaranas et al. / Biochemical Systematics and Ecology 29 (2001) 125}136
129
and population structure statistics using Weir's (1990) approach were estimated with
GDAWIN version d10 (Lewis, 1996) which incorporates the analytical approach of
Weir (1990). Mantel tests of the correlation between genetic distance and the geographic
separation of sample sites were based on the Spearman rank correlation coe$cient and
used GENEPOP (Raymond and Rousset, 1995) with 1000 permutations.
3. Results
The absence of many heterozygote classes for each of the loci suggests that two
genetic species were present in our Camponotus ephippium samples (Table 1). These
taxa were not distinguished morphologically in the "eld or in previous collections.
However, the two taxa could usually be distinguished by size with the bene"t of the
hindsight provided by genetic criteria. The species were designated as `Aa and `Ba.
Ants could de"nitely be assigned to species A or species B according to their
phenotypes at Ccon 12, Ccon 79 and Ccon 70, as none were heterozygous at any of
these loci for alleles from each of the two taxa. Except for Ccon 70, all alleles from both
species were included in the nomenclatural ordering for each locus. The alleles present
in species A are detailed in Table 2 and those present in species B in Table 3. For Ccon
70, alleles with apparently similar fragment sizes had di!erent numbers of dinucleotide repeats. For this locus, alleles were designated according to ordering within
species, with upper case letters in species A and lower case in species B.
Microsatellite sequences (GENBANK Accession numbers (AF062568-AF0625582)
were highly conserved within and between species for Ccon 12, with only one base
substitution apart from the number of dinucleotide repeats. Camp 4, Camp 8, Ccon 70
and Ccon 79 sequences were conserved within species, but di!ered between them. At
Camp 4, base changes between species occurred in eight positions. At Camp 8, indels
were observed at two positions. At Ccon 79, species B had a large deletion in the
microsatellite repeat region. Complete sequences for Ccon 70 from species B have not
been obtained but a part sequence of allele D of species A (215 bp) has 40 GA repeats
while allele i of species B (216 bp) had only 32.
Table 1
Characterization of microsatellite alleles for Camponotus ephippium group species A and B
Species A
Species B
Locus
Repeat type
No. of alleles Allele size range (bp) No. of alleles Allele size range (bp)
Camp 4!
Camp 8!
Ccon12!
Ccon70"
Ccon79"
TA
AC
GA
GA
GA
1
12
2
23
9
210
114}148
172}174
175}227
310}350
2
4
1
19
2
210}212
122}128
164
166}246
304}306
!Primers were developed by Gertsch et al. (1995) for C. ligniperda and C. herculeanus.
"Primers were developed by Crozier et al. (1999) for C. consobrinus.
130
Population
Mean sample size
per locus
Mean no. of
alleles per locus
Sites
Manna
Euglo South
Nerang Cowal
Lake View
Corringle
Road 1
Road 2
Road 3
Road 4
Road 5
8.6 (0.4)
11.8 (0.2)
12.0 (0)
10.0 (0)
11.0 (0)
8.6 (0.2)
4.0 (0)
9.2 (0.4)
10.8 (0.2)
6.0 (0.4)
3.6
3.6
4.4
2.0
3.6
2.2
3.0
4.2
4.6
2.6
Total
92.0 (0.9)
9.4 (4.0)
(1.2)
(1.6)
(1.5)
(0.4)
(1.2)
(0.6)
(0.9)
(1.2)
(1.3)
(0.8)
Ccon12
AB
AB
B
B
AB
B
B
AB
AB
AB
Camp8
HJL
MHK
ABDEGH
BLH
HML
HM
CDH
EHJLM
CFHK
H
Ccon79
DFGH
CG
ACDFG
BH
ACFG
DFG
CEDG
CDEGHI
ACDEGH
CEGH
Ccon70
DGHJKMQT
ABEFGMNQRS
EILMOPRSW
GJL
JKMORSTU
IMPT
FHQSUW
BCDHQR
KMNRSP
LOSTV
Mean heterozygosity
Observed
Expected
0.543
0.356
0.233
0.540
0.364
0.172
0.300
0.488
0.425
0.425
(0.17)
(0.13)
(0.12)
(0.23)
(0.16)
(0.09)
(0.15)
(0.20)
(0.17)
(0.23)
0.514(0.14)
0.385 (0.14)
0.423 (0.18)
0.367 (0.15)
0.448 (0.16)
0.246 (0.14)
0.400 (0.18)
0.577 (0.16)
0.464 (0.18)
0.353 (0.19)
0.385 (0.14)
0.499 (0.17)
J.M. Macaranas et al. / Biochemical Systematics and Ecology 29 (2001) 125}136
Table 2
Measures of variability for each site sample of Camponotus ephippium group species A. Alleles found in a sample are indicated for each locus except Camp4 which
was "xed for allele B in species A. Alleles that were found only in a single sample are indicated in bold letters. Standard errors are given in parentheses
Population
Mean sample size
per locus
Sites
Weelah
Manna
Lake View
Corringle
Road 2
Road 3
Road 6
11.8
3.0
1.0
1.0
7.2
1.8
12.0
Total
37.8 (0.6)
(0.2)
(0)
(0)
(0)
(0.4)
(0.2)
(0)
Mean no. of
alleles per locus
3.8
2.4
1.4
1.4
1.8
1.6
1.8
(1.6)
(0.7)
(0.2)
(0.2)
(0.4)
(0.4)
(0.4)
5.6 (3.4)
Camp4
AB
AB
B
B
AB
B
AB
Camp8
IJKL
IJKL
JK
JK
JK
JK
JL
Ccon79
JK
K
K
K
K
K
K
Ccon70
bcdehijkqr
acin
io
jl
ims
fgk
eip
Mean heterozygosity
Observed
Expected
0.415
0.333
0.400
0.400
0.395
0.400
0.300
0.439
0.413
0.400
0.400
0.291
0.300
0.215
(0.18)
(0.18)
(0.24)
(0.24)
(0.19)
(0.24)
(0.19)
0.363 (0.13)
(0.16)
(0.20)
(0.24)
(0.24)
(0.13)
(0.19)
(0.12)
0.390 (0.16)
J.M. Macaranas et al. / Biochemical Systematics and Ecology 29 (2001) 125}136
Table 3
Measures of variability for each site sample of Camponotus ephippium group species B. Alleles found in a sample are indicated for each locus except Ccon12 that
was "xed for allele C in species B. Alleles that were found only in a single sample are indicated in bold letters. Standard errors are given in parentheses
131
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J.M. Macaranas et al. / Biochemical Systematics and Ecology 29 (2001) 125}136
Microsatellite genotypes for all scored individuals are available from the "rst or
second authors. Statistical analyses for species B were limited due to its low sample
size at a number of sites. The mean number of alleles per locus (APL) within samples
ranged from 2.0 to 4.6 for species A (Table 2) and from 1.4 (in the two singleton
samples) to 3.8 in species B (Table 3).
At each highly polymorphic locus in species A, some alleles, such as allele H at
Camp 8, allele G at Ccon 79 and allele M at Ccon 70, were found in almost all
sites. Other alleles were restricted to particular sites (Table 2). Variation among
sites for species B was mostly observed at Ccon 70. Except for allele i, found in "ve of
seven sites, there was very little similarity of allelic pro"les between sites. In total,
there are eight alleles from a total of 47 in species A which are found in only one
sample and 15 of 28 in species B including 14 of 19 at Ccon 70. In species B, this
may be partly an artefact of low sample numbers. Notably, however, both singleton
samples in this species (Corringle and Lake View) have Ccon 70 alleles that are seen
nowhere else.
The percentage of variation, considered as the number of alleles, included in
a selection of populations was calculated for orderings of population addition based
on observed heterozygosity, sample size and position on a north-to-south
transect. For instance, the order of addition of populations by heterozygosity in
species B was Weelah SF, Manna SF, Lake View SF, Corringle SF, Roads 3, 2 and 6.
With only one population, 18 of 27 ("67%) of alleles in species B were included.
The addition of subsequent populations gives 74, 78, 81, 88, 96 and 100% of alleles.
For heterozygosity in species A, the accumulation percentages are: 49, 57, 72, 77, 91,
94, 96, 98, 100 and 100. For the north}south transect in species A the percentages are:
30, 53, 68, 77, 81, 85, 94, 94, 96 and 100. For the transect in species B, the percentages
are: 30, 74, 81, 89, 93, 96 and 100. For sample size in species A, the percentages are 46,
65, 76, 85, 87, 98, 98, 98, 100 and 100 and in species B, these are: 30, 70, 78, 85, 93, 96
and 100.
s2 tests of heterogeneity suggest that the geographic distribution of genotypes is
signi"cantly non-random for all loci in species A (P"0.046 for Camp 4, P(0.005 for
the others). This was also the case for all loci except Ccon 12, where the test was not
possible, in species B (P(0.005 for all).
Table 4
Levels of di!erentiation in Camponotus ephippium group species A and B. The parameters f, F and /P (Weir,
1990) are analogous to Wright's F , F and F , respectively. Ranges in brackets are the con"dence limits
is it
st
from 999 bootstrap pseudosample replicates using GDAWIN (Lewis, 1996). Estimates of gene #ow are
calculated from the relation Nm+(1//P}1)/4
Species
f
F
/P
Nm
A
0.094
[0.049}0.180]
!0.130
[!0.455}0.057]
0.243
[0.217}0.330]
0.140
[!0.074}0.236]
0.164
[0.131}0.200]
0.239
[0.176}0.317]
1.27
B
0.80
J.M. Macaranas et al. / Biochemical Systematics and Ecology 29 (2001) 125}136
133
Fig. 2. Neighbor-joining tree of populations of C. ephippium species A based on Nei's unbiased genetic
distance.
Measures of di!erentiation using population structure statistics are shown for
species A and B in Table 4 (for species B, the singleton samples Lake View and
Corringle SFs were omitted from calculations). The values of /P (analogous to
Wright's F ) for species A (0.164) and B (0.239) indicate signi"cant genetic di!erentiST
ation within the total population of each species. Estimating migration rates by the
formula Nm+(1//P}1)/4, gives values of 1.27 for species A and 0.80 for species B.
GENEPOP was used for the private alleles method of migration rate estimation
corrected for sample size Barton and Slatkin, 1986). Nm estimated from this approach
is 0.99 in species A and 0.33 in species B.
Neighbor-joining phenograms based on Nei's unbiased genetic distance
(Fig. 2 * for species A, species B not illustrated) show some groupings of proximal
populations (e.g. Manna and Weelah SFs in species B), but most clusters have little
apparent relationship to geography. We conducted Mantel tests of the correlation of
genetic di!erentiation, measured as F /(1!F ) and the natural logarithmic transST
ST
formation of geographic distance. Under the null hypothesis of no association between the matrices, the probability of observing a correlation greater than that
actually seen is 0.28 for species A and 0.56 for species B.
4. Discussion
The microsatellite data are unequivocal that there are two species of ants in our
study sample of Camponotus ephippium. This is not surprising as the taxon is known to
be a species complex (Shattuck, pers. comm.). The potential of such variable loci for
species discrimination is, however, notable and encourages their use in systematic
134
J.M. Macaranas et al. / Biochemical Systematics and Ecology 29 (2001) 125}136
studies. The species have no alleles in common at three loci. One of these is highly
variable in both species and one is highly variable in one of the species. Although
the use of microsatellites for systematic purposes has been suggested (Chapuisat,
1996; Crozier et al., 1999), the "eld remains relatively neglected as it is usually
supposed (e.g. Bruford et al., 1996) that the high mutability of microsatellite loci would
obscure interspeci"c di!erences.
Neither species A nor B is genetically homogeneous over the sampled region. Gene
#ow within the species is low, with the higher estimates of Nm of 1.27 (for species A)
and 0.8 (for species B) being at the lower bound of values required for population
homogenisation (Slatkin, 1987, 1994). Non-signi"cant Mantel tests of the correlation
of genetic di!erentiation and geographic distance suggest, however, that isolation by
distance is not playing a predominant role in causing this heterogeneity in either
species.
The sampling design of the present survey was intended to study a di!erent
spatial scale to the microgeographic, colony-level di!erentiation that is often
observed in polygynous ant species (Chapuisat et al., 1997). With this design,
a proportion of colonies could, theoretically, be represented by more than one
individual. Even highly polydomous species have nest aggregations generally separated by less than 10 m (Seppa and Pamilo, 1995; Pedersen and Boomsma, 1999).
Examples of larger aggregations are known, however, and in one extreme instance,
a `supercolonya of the ant Formica paralugubris has a linear extent of 0.7 km
(Chapuisat et al., 1997). The e!ects of scoring multiple individuals from a colony
might reduce estimates of gene #ow or intrapopulation variability but would not
signi"cantly alter our general conclusions that there is regional heterogeneity in allelic
frequencies.
Accumulation percentages suggest that almost all alleles that would be discovered
by extensive sampling are already found in our samples. A corollary is that the
samples contain most alleles that would have been present had there been no habitat
loss or fragmentation. Apparently, population sizes within fragments have been
su$cient to prevent major losses of variation due to inbreeding. We do not, however,
have baseline data that would permit estimation of the loss of variation within
individual populations. The overall heterogeneity of allelic frequencies in both species
and the geographically restricted range of many alleles has implications for the design
of reserves for invertebrate conservation. In no tested ordering of the samples for allele
accumulation for either species would it be possible to represent more than three
quarters of the genetic variation without reservation of three or more sites. Consequently, multiple reserves would be required for any comprehensive attempt to
conserve genetic diversity at the regional level.
Acknowledgements
We gratefully acknowledge the assistance of Derek Smith and Rebecca Harris
in collecting the ants and Greg Gowing for preparing Fig. 1. Steve Shattuck of
CSIRO Division of Entomology, Canberra, did species group identi"cation of the
J.M. Macaranas et al. / Biochemical Systematics and Ecology 29 (2001) 125}136
135
samples. Ross Crozier kindly provided microsatellite primer sequences prior to
publication and commented on an earlier version of the manuscript. We also thank
the State Forests of New South Wales for allowing us to conduct research on their
estate. This project was funded by the Centre for Biodiversity and Conservation
Research.
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J.M. Macaranas et al. / Biochemical Systematics and Ecology 29 (2001) 125}136
SundstroK m, L., 1993. Genetic population structure and sociogenetic organisation in Formica truncorum
(Hymenoptera; Formicidae). Behav. Ecol. Sociobiol. 33, 345}354.
Swo!ord, D. L., Selander, R. B., 1989. BIOSYS-1: a computer program for the analysis of allelic variation in
population genetics and biochemical systematics. Release 1.7. Illinois Natural History Survey, Champaign, USA.
Tay, W.T., Cook, J.M., Rowe, D.J., Crozier, R.H., 1997. Migration between nests in the Australian arid-zone
ant Rhytidoponera sp. 12 revealed by DGGE analyses of mitochondrial DNA. Molec. Ecol. 6, 403}411.
Weir, B.S., 1990. Genetic Data Analysis. Sinauer Associates, Sunderland.
Species discrimination and population
di!erentiation in ants using microsatellites
Julie M. Macaranas1, Donald J. Colgan*, Richard E. Major,
Gerasimos Cassis, Michael R. Gray
Centre for Biodiversity and Conservation Research, Australian Museum, 6 College Street, Sydney 2010 NSW,
Australia
Received 24 November 1999; accepted 22 March 2000
Abstract
This study was conducted to establish the regional scale of population di!erentiation of ants
in the wheat belt of central western New South Wales. Microsatellite variation was surveyed at
"ve loci in two morphologically similar ant species (designated `Aa and `Ba) from the
Camponotus ephippium complex. Three of the "ve scored microsatellite loci were highly variable
with totals, in the two species, of 11, 13 and 42 alleles. The other loci had two and three alleles.
The mean number of alleles per locus per sample ranged from 2.0 to 4.6 for species A and from
1.4 to 3.8 in species B. Mean observed heterozygosity was 0.385 for species A and 0.363 for
species B. The geographic distribution of genotypes was signi"cantly non-random for all tested
loci in both species. Eight of 47 alleles in species A and 15 of 28 in species B were restricted to
a single site. Allelic accumulation percentages were calculated for several orderings of samples
* level of heterozygosity, sample size and geographic position. In all orderings three or more
samples must be included for more than three-quarters of alleles to be represented. ( 2001
Elsevier Science Ltd. All rights reserved.
Keywords: Camponotus; Ant; Microsatellite; Species discrimination; Genetic variation
1. Introduction
There has been a long and continuing interest in the genetics of ants, with
eager application of new techniques to questions about colony structure and nest
1 Present address: DFID Fish Genetics Program at Asian Institute of Technology, Aquaculture & Aquatic Resources Management Program (AARM). PO Box 4, Klong Luang, Pathumthani 12120, Thailand.
* Corresponding author. Tel.: #61-2-9320-6000.
E-mail address: [email protected] (D.J. Colgan).
0305-1978/01/$ - see front matter ( 2001 Elsevier Science Ltd. All rights reserved.
PII: S 0 3 0 5 - 1 9 7 8 ( 0 0 ) 0 0 0 3 8 - 7
126
J.M. Macaranas et al. / Biochemical Systematics and Ecology 29 (2001) 125}136
inter-relationships (Pamilo et al., 1997). In general, signi"cant genetic structuring
in ant species has been observed at scales of tens of metres and of a few kilometres.
At these scales, the amount of di!erentiation is strongly associated with the
sociogenetic organisation of a species' colonies and its means of dispersal
(Sundstrom, 1993; Seppa and Pamilo, 1995; Chapuisat et al., 1997; Tay et al.,
1997; Ross et al., 1997). Monogynous species, which generally take part in mating
#ights and found new nests independently, are less di!erentiated than polygynous
species. These are characterised by reduced queen dispersal during mating with
budding as a major mode of nest founding (Pamilo et al., 1997; Pedersen and
Boomsma, 1999).
In contrast to the level of interest in local genetic di!erentiation, there has
been relatively little emphasis on regional level di!erentiation in ants. This level
would be important for the extension of ants' use as ecological species indicators
(Andersen, 1990; Bestelmeyer and Wiens, 1996) to conservation genetics. This
paper examines genetic variation at the regional level in ants of the Camponotus
ephippium species group in the wheat belt of central western New South Wales.
Microsatellites were chosen for study, as they have proven generally applicable
to analyses of "ne-scale di!erentiation and gene #ow studies in ants (Pamilo
et al., 1997). Moreover, primers have been developed for Camponotus by Gertsch et al.
(1995) and Crozier et al. (1999), the latter already having proven successful in
ampli"cation of DNA from species other than C. consobrinus in which they were
developed.
Habitat fragmentation potentially causes di$culties for the maintenance of genetic
and species diversity by restricting gene #ow and lowering the likelihood of reestablishment of locally extinct populations through immigration (reviewed in Forman, 1995; Setterle et al., 1996). Questions of concern include the degree to which
fragmentation has reduced intra-population variation, whether this has impinged on
variation in the regional population considered as a whole, and what strategy should
be adopted for reservation practices to maximise the probability of maintaining
genetic variability.
2. Materials and methods
2.1. Study site
The study area (Fig. 1) lies in the wheatbelt of New South Wales between
West Wyalong and Condobolin. Sivertsen and Metcalfe (1995) describe its vegetation.
Only 16% of the native vegetation in the region has escaped the clearing that
began on an extensive scale in the late 1800s. Much of the remnant native vegetation
is on public land such as state forests and roadside reserves. The sample sites
comprise six state forests between 640 and 1860 ha in area and six long roadside
reserves of approximately 20 m in width. The length of the study area is about 40 km
from north to south and its breadth about 14 km, with 1.2 km between the nearest pair
of sites.
J.M. Macaranas et al. / Biochemical Systematics and Ecology 29 (2001) 125}136
127
Fig. 1. Map of the study area. The inset shows the location of the area in the central wheat belt (shaded).
The state forests are: Weelah, Manna, Euglo South, Nerang Cowal, Lake View and Corringle. The road
reserves are numbered from 1 to 6 and are indicated by "lled circles. Other areas of native vegetation are
shown by light shading.
128
J.M. Macaranas et al. / Biochemical Systematics and Ecology 29 (2001) 125}136
2.2. Study species
C. ephippium group species are widely distributed in the area (Major et al., 1999).
Worker ants, of what was believed to be one species based on morphological
taxonomy (voucher specimen numbers K150547-9, K150550-1 and K150552-3), were
sampled by hand. One ant was taken from each 20}30 m length (approximated) along
the entire distance of a 500 m transect bisected by a road or, for the state forests,
a management trail. Ants were missing from some parts of the transects. Samples were
stored in ethanol. This procedure was adopted to improve estimation of population
genetic parameters by reducing multiple sampling from single nests. Multiple sampling from a single colony might still occur if it were polydomous and its nests were
highly dispersed.
2.3. Molecular methods
Individual ants were fully rehydrated in distilled water and their gasters removed
before DNA extraction using the Rapidgene Genomic DNA puri"cation kit (Amresco
USA). Five microsatellite primer pairs developed for other Camponotus species
(Gertsch et al., 1995: Camp 4 and Camp 8; Crozier et al., 1999: Ccon 12, Ccon 70 and
Ccon 79) readily generated PCR products (see Table 1).
PCR was performed under the following conditions: 5 min at 963C, 1 min at 563C,
1 min at 723C (1 cycle), followed by 1 min at 943C, 1 min at 563C, 1 min at 723C (34
cycles) and "nally 1 min at 943C, 1 min at 563C, 5 min at 723C. PCR reaction mixtures
(25 ll) contained 25}200 ng of genomic DNA, dCTP, dGTP and dTTP at 200 lM,
dATP at 10 lM, 25 pmoles of primer, 1.5 mM MgCl , 1 lCi of a33P-dATP (1500 Ci
2
mmol~1, Bresatec Australia), 1]reaction bu!er and 0.5 U of Thermostable DNA
polymerase from Thermus &icelandicus' (Advanced Biotechnologies, UK). Each reaction was covered with 50 ll mineral oil. PCR products (excluding oil overlay) were
mixed with 7 ll of Stop solution (United States Biochemical), heated to 943C for 2 min
and immersed in ice water before application to a sequencing gel (6% 19 : 1 acrylamide:bis-acrylamide, 7 M urea). Electrophoretic runs using 1]TBE bu!er (89 mM
Tris-HCl pH 8.3, 89 mM boric acid, 5 mM EDTA) lasted from 1.5 to 3.5 h according
to the size of the microsatellites. At least three reference microsatellites were included
in every run. After vacuum-drying, the gel was used to expose X-ray "lm for at least
16 h. Microsatellite size and sequence were determined from non-radioactive PCR
products of known homozygotes. For sequencing using dye terminator technology on
a PE 377, products were cleaned using the QIAQUICK PCR puri"cation kit (Qiagen,
Germany).
Allelic size estimates were based on the relative mobility of sequenced alleles and/or
microsatellite ladders. For Ccon 79, sizes could only be roughly estimated from the
sequenced alleles as bands at this locus did not produce a ladder-like pattern.
Generally, (but see the `Resultsa section below), alleles were designated alphabetically
in order of decreasing mobility, with the most anodal being designated A.
Estimates of genetic variability were obtained using the BIOSYS 1.7 program
(Swo!ord and Selander, 1989). Clustering based on Nei's unbiased genetic distance
J.M. Macaranas et al. / Biochemical Systematics and Ecology 29 (2001) 125}136
129
and population structure statistics using Weir's (1990) approach were estimated with
GDAWIN version d10 (Lewis, 1996) which incorporates the analytical approach of
Weir (1990). Mantel tests of the correlation between genetic distance and the geographic
separation of sample sites were based on the Spearman rank correlation coe$cient and
used GENEPOP (Raymond and Rousset, 1995) with 1000 permutations.
3. Results
The absence of many heterozygote classes for each of the loci suggests that two
genetic species were present in our Camponotus ephippium samples (Table 1). These
taxa were not distinguished morphologically in the "eld or in previous collections.
However, the two taxa could usually be distinguished by size with the bene"t of the
hindsight provided by genetic criteria. The species were designated as `Aa and `Ba.
Ants could de"nitely be assigned to species A or species B according to their
phenotypes at Ccon 12, Ccon 79 and Ccon 70, as none were heterozygous at any of
these loci for alleles from each of the two taxa. Except for Ccon 70, all alleles from both
species were included in the nomenclatural ordering for each locus. The alleles present
in species A are detailed in Table 2 and those present in species B in Table 3. For Ccon
70, alleles with apparently similar fragment sizes had di!erent numbers of dinucleotide repeats. For this locus, alleles were designated according to ordering within
species, with upper case letters in species A and lower case in species B.
Microsatellite sequences (GENBANK Accession numbers (AF062568-AF0625582)
were highly conserved within and between species for Ccon 12, with only one base
substitution apart from the number of dinucleotide repeats. Camp 4, Camp 8, Ccon 70
and Ccon 79 sequences were conserved within species, but di!ered between them. At
Camp 4, base changes between species occurred in eight positions. At Camp 8, indels
were observed at two positions. At Ccon 79, species B had a large deletion in the
microsatellite repeat region. Complete sequences for Ccon 70 from species B have not
been obtained but a part sequence of allele D of species A (215 bp) has 40 GA repeats
while allele i of species B (216 bp) had only 32.
Table 1
Characterization of microsatellite alleles for Camponotus ephippium group species A and B
Species A
Species B
Locus
Repeat type
No. of alleles Allele size range (bp) No. of alleles Allele size range (bp)
Camp 4!
Camp 8!
Ccon12!
Ccon70"
Ccon79"
TA
AC
GA
GA
GA
1
12
2
23
9
210
114}148
172}174
175}227
310}350
2
4
1
19
2
210}212
122}128
164
166}246
304}306
!Primers were developed by Gertsch et al. (1995) for C. ligniperda and C. herculeanus.
"Primers were developed by Crozier et al. (1999) for C. consobrinus.
130
Population
Mean sample size
per locus
Mean no. of
alleles per locus
Sites
Manna
Euglo South
Nerang Cowal
Lake View
Corringle
Road 1
Road 2
Road 3
Road 4
Road 5
8.6 (0.4)
11.8 (0.2)
12.0 (0)
10.0 (0)
11.0 (0)
8.6 (0.2)
4.0 (0)
9.2 (0.4)
10.8 (0.2)
6.0 (0.4)
3.6
3.6
4.4
2.0
3.6
2.2
3.0
4.2
4.6
2.6
Total
92.0 (0.9)
9.4 (4.0)
(1.2)
(1.6)
(1.5)
(0.4)
(1.2)
(0.6)
(0.9)
(1.2)
(1.3)
(0.8)
Ccon12
AB
AB
B
B
AB
B
B
AB
AB
AB
Camp8
HJL
MHK
ABDEGH
BLH
HML
HM
CDH
EHJLM
CFHK
H
Ccon79
DFGH
CG
ACDFG
BH
ACFG
DFG
CEDG
CDEGHI
ACDEGH
CEGH
Ccon70
DGHJKMQT
ABEFGMNQRS
EILMOPRSW
GJL
JKMORSTU
IMPT
FHQSUW
BCDHQR
KMNRSP
LOSTV
Mean heterozygosity
Observed
Expected
0.543
0.356
0.233
0.540
0.364
0.172
0.300
0.488
0.425
0.425
(0.17)
(0.13)
(0.12)
(0.23)
(0.16)
(0.09)
(0.15)
(0.20)
(0.17)
(0.23)
0.514(0.14)
0.385 (0.14)
0.423 (0.18)
0.367 (0.15)
0.448 (0.16)
0.246 (0.14)
0.400 (0.18)
0.577 (0.16)
0.464 (0.18)
0.353 (0.19)
0.385 (0.14)
0.499 (0.17)
J.M. Macaranas et al. / Biochemical Systematics and Ecology 29 (2001) 125}136
Table 2
Measures of variability for each site sample of Camponotus ephippium group species A. Alleles found in a sample are indicated for each locus except Camp4 which
was "xed for allele B in species A. Alleles that were found only in a single sample are indicated in bold letters. Standard errors are given in parentheses
Population
Mean sample size
per locus
Sites
Weelah
Manna
Lake View
Corringle
Road 2
Road 3
Road 6
11.8
3.0
1.0
1.0
7.2
1.8
12.0
Total
37.8 (0.6)
(0.2)
(0)
(0)
(0)
(0.4)
(0.2)
(0)
Mean no. of
alleles per locus
3.8
2.4
1.4
1.4
1.8
1.6
1.8
(1.6)
(0.7)
(0.2)
(0.2)
(0.4)
(0.4)
(0.4)
5.6 (3.4)
Camp4
AB
AB
B
B
AB
B
AB
Camp8
IJKL
IJKL
JK
JK
JK
JK
JL
Ccon79
JK
K
K
K
K
K
K
Ccon70
bcdehijkqr
acin
io
jl
ims
fgk
eip
Mean heterozygosity
Observed
Expected
0.415
0.333
0.400
0.400
0.395
0.400
0.300
0.439
0.413
0.400
0.400
0.291
0.300
0.215
(0.18)
(0.18)
(0.24)
(0.24)
(0.19)
(0.24)
(0.19)
0.363 (0.13)
(0.16)
(0.20)
(0.24)
(0.24)
(0.13)
(0.19)
(0.12)
0.390 (0.16)
J.M. Macaranas et al. / Biochemical Systematics and Ecology 29 (2001) 125}136
Table 3
Measures of variability for each site sample of Camponotus ephippium group species B. Alleles found in a sample are indicated for each locus except Ccon12 that
was "xed for allele C in species B. Alleles that were found only in a single sample are indicated in bold letters. Standard errors are given in parentheses
131
132
J.M. Macaranas et al. / Biochemical Systematics and Ecology 29 (2001) 125}136
Microsatellite genotypes for all scored individuals are available from the "rst or
second authors. Statistical analyses for species B were limited due to its low sample
size at a number of sites. The mean number of alleles per locus (APL) within samples
ranged from 2.0 to 4.6 for species A (Table 2) and from 1.4 (in the two singleton
samples) to 3.8 in species B (Table 3).
At each highly polymorphic locus in species A, some alleles, such as allele H at
Camp 8, allele G at Ccon 79 and allele M at Ccon 70, were found in almost all
sites. Other alleles were restricted to particular sites (Table 2). Variation among
sites for species B was mostly observed at Ccon 70. Except for allele i, found in "ve of
seven sites, there was very little similarity of allelic pro"les between sites. In total,
there are eight alleles from a total of 47 in species A which are found in only one
sample and 15 of 28 in species B including 14 of 19 at Ccon 70. In species B, this
may be partly an artefact of low sample numbers. Notably, however, both singleton
samples in this species (Corringle and Lake View) have Ccon 70 alleles that are seen
nowhere else.
The percentage of variation, considered as the number of alleles, included in
a selection of populations was calculated for orderings of population addition based
on observed heterozygosity, sample size and position on a north-to-south
transect. For instance, the order of addition of populations by heterozygosity in
species B was Weelah SF, Manna SF, Lake View SF, Corringle SF, Roads 3, 2 and 6.
With only one population, 18 of 27 ("67%) of alleles in species B were included.
The addition of subsequent populations gives 74, 78, 81, 88, 96 and 100% of alleles.
For heterozygosity in species A, the accumulation percentages are: 49, 57, 72, 77, 91,
94, 96, 98, 100 and 100. For the north}south transect in species A the percentages are:
30, 53, 68, 77, 81, 85, 94, 94, 96 and 100. For the transect in species B, the percentages
are: 30, 74, 81, 89, 93, 96 and 100. For sample size in species A, the percentages are 46,
65, 76, 85, 87, 98, 98, 98, 100 and 100 and in species B, these are: 30, 70, 78, 85, 93, 96
and 100.
s2 tests of heterogeneity suggest that the geographic distribution of genotypes is
signi"cantly non-random for all loci in species A (P"0.046 for Camp 4, P(0.005 for
the others). This was also the case for all loci except Ccon 12, where the test was not
possible, in species B (P(0.005 for all).
Table 4
Levels of di!erentiation in Camponotus ephippium group species A and B. The parameters f, F and /P (Weir,
1990) are analogous to Wright's F , F and F , respectively. Ranges in brackets are the con"dence limits
is it
st
from 999 bootstrap pseudosample replicates using GDAWIN (Lewis, 1996). Estimates of gene #ow are
calculated from the relation Nm+(1//P}1)/4
Species
f
F
/P
Nm
A
0.094
[0.049}0.180]
!0.130
[!0.455}0.057]
0.243
[0.217}0.330]
0.140
[!0.074}0.236]
0.164
[0.131}0.200]
0.239
[0.176}0.317]
1.27
B
0.80
J.M. Macaranas et al. / Biochemical Systematics and Ecology 29 (2001) 125}136
133
Fig. 2. Neighbor-joining tree of populations of C. ephippium species A based on Nei's unbiased genetic
distance.
Measures of di!erentiation using population structure statistics are shown for
species A and B in Table 4 (for species B, the singleton samples Lake View and
Corringle SFs were omitted from calculations). The values of /P (analogous to
Wright's F ) for species A (0.164) and B (0.239) indicate signi"cant genetic di!erentiST
ation within the total population of each species. Estimating migration rates by the
formula Nm+(1//P}1)/4, gives values of 1.27 for species A and 0.80 for species B.
GENEPOP was used for the private alleles method of migration rate estimation
corrected for sample size Barton and Slatkin, 1986). Nm estimated from this approach
is 0.99 in species A and 0.33 in species B.
Neighbor-joining phenograms based on Nei's unbiased genetic distance
(Fig. 2 * for species A, species B not illustrated) show some groupings of proximal
populations (e.g. Manna and Weelah SFs in species B), but most clusters have little
apparent relationship to geography. We conducted Mantel tests of the correlation of
genetic di!erentiation, measured as F /(1!F ) and the natural logarithmic transST
ST
formation of geographic distance. Under the null hypothesis of no association between the matrices, the probability of observing a correlation greater than that
actually seen is 0.28 for species A and 0.56 for species B.
4. Discussion
The microsatellite data are unequivocal that there are two species of ants in our
study sample of Camponotus ephippium. This is not surprising as the taxon is known to
be a species complex (Shattuck, pers. comm.). The potential of such variable loci for
species discrimination is, however, notable and encourages their use in systematic
134
J.M. Macaranas et al. / Biochemical Systematics and Ecology 29 (2001) 125}136
studies. The species have no alleles in common at three loci. One of these is highly
variable in both species and one is highly variable in one of the species. Although
the use of microsatellites for systematic purposes has been suggested (Chapuisat,
1996; Crozier et al., 1999), the "eld remains relatively neglected as it is usually
supposed (e.g. Bruford et al., 1996) that the high mutability of microsatellite loci would
obscure interspeci"c di!erences.
Neither species A nor B is genetically homogeneous over the sampled region. Gene
#ow within the species is low, with the higher estimates of Nm of 1.27 (for species A)
and 0.8 (for species B) being at the lower bound of values required for population
homogenisation (Slatkin, 1987, 1994). Non-signi"cant Mantel tests of the correlation
of genetic di!erentiation and geographic distance suggest, however, that isolation by
distance is not playing a predominant role in causing this heterogeneity in either
species.
The sampling design of the present survey was intended to study a di!erent
spatial scale to the microgeographic, colony-level di!erentiation that is often
observed in polygynous ant species (Chapuisat et al., 1997). With this design,
a proportion of colonies could, theoretically, be represented by more than one
individual. Even highly polydomous species have nest aggregations generally separated by less than 10 m (Seppa and Pamilo, 1995; Pedersen and Boomsma, 1999).
Examples of larger aggregations are known, however, and in one extreme instance,
a `supercolonya of the ant Formica paralugubris has a linear extent of 0.7 km
(Chapuisat et al., 1997). The e!ects of scoring multiple individuals from a colony
might reduce estimates of gene #ow or intrapopulation variability but would not
signi"cantly alter our general conclusions that there is regional heterogeneity in allelic
frequencies.
Accumulation percentages suggest that almost all alleles that would be discovered
by extensive sampling are already found in our samples. A corollary is that the
samples contain most alleles that would have been present had there been no habitat
loss or fragmentation. Apparently, population sizes within fragments have been
su$cient to prevent major losses of variation due to inbreeding. We do not, however,
have baseline data that would permit estimation of the loss of variation within
individual populations. The overall heterogeneity of allelic frequencies in both species
and the geographically restricted range of many alleles has implications for the design
of reserves for invertebrate conservation. In no tested ordering of the samples for allele
accumulation for either species would it be possible to represent more than three
quarters of the genetic variation without reservation of three or more sites. Consequently, multiple reserves would be required for any comprehensive attempt to
conserve genetic diversity at the regional level.
Acknowledgements
We gratefully acknowledge the assistance of Derek Smith and Rebecca Harris
in collecting the ants and Greg Gowing for preparing Fig. 1. Steve Shattuck of
CSIRO Division of Entomology, Canberra, did species group identi"cation of the
J.M. Macaranas et al. / Biochemical Systematics and Ecology 29 (2001) 125}136
135
samples. Ross Crozier kindly provided microsatellite primer sequences prior to
publication and commented on an earlier version of the manuscript. We also thank
the State Forests of New South Wales for allowing us to conduct research on their
estate. This project was funded by the Centre for Biodiversity and Conservation
Research.
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