Journal of Heredity 2011:102(5):610–616
doi:10.1093/jhered/esr073
Advance Access publication July 20, 2011
Ó The American Genetic Association. 2011. All rights reserved.
For permissions, please email: [email protected].
Distinguishing Forest and Savanna
African Elephants Using Short Nuclear
DNA Sequences
YASUKO ISHIDA, YIRMED DEMEKE, PETER J. VAN COEVERDEN
E.A. LEGGETT, VIRGINIA E. FOX, AND ALFRED L. ROCA
DE
GROOT, NICHOLAS J. GEORGIADIS, KEITH
From the Department of Animal Sciences, University of Illinois at Urbana-Champaign, Urbana, IL 61801 (Ishida and Roca);
the Wildlife for Sustainable Development, Addis Ababa, Ethiopia (Demeke); the Department of Biology, Queen’s
University, Kingston, Ontario, Canada (Van Coeverden de Groot); the Director, Bole and Klingenstein Foundation, Cody,
WY (Georgiadis); the Namibian Elephant and Giraffe Trust, Outjo, Namibia (Leggett); the Elephant Human Relations Aid
(EHRA), Swakopmund, Namibia (Fox); and the Institute for Genomic Biology, University of Illinois at Urbana-Champaign
Urbana, IL 61801 (Roca). Keith E. A. Leggett is now at UNSW Arid Zone Research Station, Fowlers Gap, via Broken Hill,
2880 NSW, Australia.
Address correspondence to Alfred L. Roca at the address above, or e-mail: [email protected].
A more complete description of African elephant phylogeography would require a method that distinguishes forest
and savanna elephants using DNA from low-quality samples.
Although mitochondrial DNA is often the marker of choice
for species identification, the unusual cytonuclear patterns in
African elephants make nuclear markers more reliable. We
therefore designed and utilized genetic markers for short
nuclear DNA regions that contain fixed nucleotide differences between forest and savanna elephants. We used M13
forward and reverse sequences to increase the total length
of PCR amplicons and to improve the quality of sequences
for the target DNA. We successfully sequenced fragments
of nuclear genes from dung samples of known savanna and
forest elephants in the Democratic Republic of Congo,
Ethiopia, and Namibia. Elephants at previously unexamined
locations were found to have nucleotide character states
consistent with their status as savanna or forest elephants.
Using these and results from previous studies, we estimated
that the short-amplicon nuclear markers could distinguish
forest from savanna African elephants with more than 99%
accuracy. Nuclear genotyping of museum, dung, or ivory
samples will provide better-informed conservation management of Africa’s elephants.
Key words: conservation, fecal DNA, forensics, Loxodonta,
systematics, taxonomy
African elephants were long considered to comprise a single
species. Recent morphological studies (Groves and Grubb
2000; Grubb et al. 2000) and nuclear DNA analyses (Roca
et al. 2001, 2005; Comstock et al. 2002; Rohland et al. 2010)
have supported their classification into 2 highly divergent
610
species: savanna elephant (Loxodonta africana) and forest
elephant (L. cyclotis). Several studies that examined African
elephant phylogeography have based their conclusions
largely or completely on mitochondrial DNA (mtDNA)
analyses (Eggert et al. 2002; Debruyne 2005; Johnson et al.
2007). Other studies have reported that the phylogeography
of African elephant mtDNA differs from the partitions
found using nuclear DNA (Roca et al. 2005, 2007; Roca and
O’Brien 2005). The mtDNA is subject to the demographic
constraint of being retained within matrilocal social groups
because females do not typically migrate between elephant
herds (Archie et al. 2008). By contrast, nuclear DNA is
subject to widespread and rapid male-mediated transmission
across herds and populations (Roca et al. 2005, 2007; Roca
and O’Brien 2005). Conclusions about the systematics or
population genetics of African elephants based solely on
mtDNA present an incomplete picture if nuclear DNA
patterns are not also examined (Roca et al. 2005; Lei et al.
2008, 2009).
mtDNA exists in greater copy numbers and is often easier
to amplify from fecal or museum samples for which
amplification of nuclear DNA is in many cases more
problematic. Although dung samples collected under ideal
conditions may produce good quality DNA (Fernando et al.
2003; Wasser et al. 2004, 2007; Okello et al. 2005, 2008),
samples of dung cannot always be collected in a manner that
would provide reliable nuclear DNA amplification. In the case
of museum specimens (or of confiscated ivory), the researcher
often has no control over the condition of the samples. Thus,
one impediment to generating comparable data across
elephant studies would be the difficulty of generating nuclear
DNA sequences from samples of degraded or low quality.
Brief Communications
We therefore sought to develop and utilize nuclear DNA
markers effective for elephant samples of low-quality DNA,
without the use of expensive or labor-intensive procedures,
such as subcloning or gel purification. The 2 methods tested
included, first, using a forward primer with a 40-bp
‘‘neutral’’ sequence, defined as not complementary to
known published sequences (Binladen et al. 2007). The
use of this sequence had been reported as improving the
sequencing of extremely short amplicons (Binladen et al.
2007). Second, we tried adding M13 forward and reverse
sequences to the 5# ends of, respectively, forward and
reverse primers designed to amplify short amplicons, in
order to overcome the poor quality at the 5# end that is
typical for Sanger sequences of short amplicons. The
primers were designed to include species-diagnostic nucleotide site differences for 3 X-linked genes. Using the second
method, M13-tailed primers, we successfully produced good
quality sequences that demonstrated species-specific nucleotide differences between forest and savanna elephants in 3
elephant populations, 2 of which had not been previously
examined for nuclear DNA sequences.
Materials and Methods
2001), AY045053–AY045173 (CHRNA1) (Roca et al. 2001),
AY045174–AY045363 (GBA) (Roca et al. 2001), AY823326–
AY823336 (BGN) (Roca et al. 2005), and AY823352–
AY823387 (PHKA2, PLP) (Roca et al. 2001, 2005).
Unpublished flanking sequences were also included in some
alignments. Nucleotide sites with fixed or nearly fixed
differences between African forest elephants, African savanna
elephants, and Asian elephants were identified within the
genes (Roca et al. 2001, 2005). Primers to amplify short
regions surrounding fixed nucleotide sites were designed using
the software Primer3 (http://fokker.wi.mit.edu/primer3/
input.htm) (Koressaar and Remm 2007). The primer
sequences were based on conserved sequences (Murphy and
O’Brien 2007) across elephant species (Roca et al. 2001,
2005). The 2 primers in each pair were closely spaced so that
PCR would amplify extremely short regions of 21–47 bp
(excluding the lengths of the primers) for the 40-bp tailed
sequencing primers method (below). For the M13 forward–
and reverse–tailed primer method (below), primers were
designed to amplify 47–114 bp regions of DNA (excluding
primer lengths) for 2 regions of BGN (primer sets BGN-s1
and s2) and PHKA2 (primer sets PHKA2-s1 and s2) and one
region of PLP.
Sample Collection and DNA Extraction
Use of 40-bp Tailed Sequencing Primers
The study was conducted in compliance with the University
of Illinois Institutional Animal Care and Use Committed
approved protocol number 09036. Samples were collected
with appropriate permits and the permission of local
authorities. Dung samples from wild African elephants
were collected from 3 locations in Africa. Dung from
savanna elephants was collected in northern Namibia
(designated ‘‘LAF-NA’’ or ‘‘NA’’) and in the Babille
Elephant Sanctuary (LAF-BA or BA), eastern Ethiopia.
Dung from forest elephants was collected in the Bili Forest
(LCY-BF or BF) of the Bas-Uele Province of the northern
Democratic Republic of Congo. NA, BA, and BF dung
samples were collected in ethanol (NA) or in a salt-saturated
dimethyl sulfoxide solution (BA, BF). After shipment,
samples were stored at 80 °C. DNA was extracted using
the QIAamp DNA stool mini kit (Qiagen) from 8 savanna
elephants (4 BA and 4 NA) and 4 forest elephants (BF). For
positive controls, DNA was also extracted from blood
samples drawn from 2 elephants: a male wild savanna
elephant from the Babille Elephant Sanctuary (BA0001) and
a male Asian elephant (Elephas maximus) born in the wild and
currently at the Tulsa Zoo and Living Museum, Tulsa,
Oklahoma, USA (Asian Elephant North American Regional
Studbook No. 160; our designation ‘‘EMA-SN160’’). The
blood samples were extracted using the QIAamp DNA
Blood Mini Kit (Qiagen).
The quality of sequences produced for short PCR amplicons
has been reported to improve by addition of a 40- or 60-bp
sequence ‘‘tail,’’ not complementary to published sequences,
to the 5# end of the sequencing primers (Binladen et al. 2007).
Following this approach, a 40-bp tail (Binladen et al. 2007)
was added to forward primers in sequencing reactions. PCR
was attempted using the conventional forward and reverse
primer pair (without tails) and conducted as described below,
with products visualized and examined under ultraviolet light
on a 1.5–2% ethidium bromide stained agarose gel. The PCR
products were purified using the QIAquick gel extraction kit
(Qiagen). Sequencing reactions were then performed using
the 40-bp tailed forward primer or a reverse primer that did
not have a tail. Sequencing was conducted in duplicate, using
2.5 and 5 ll of gel-purified PCR products, with sequencing
conducted as described below. Additionally, we also
separately tried using the 40-bp tailed forward primer in
PCR reactions (along with a reverse primer that did not
include a tail). Sequencing reactions were conducted with the
same primers used for PCR for this case.
Oligonucleotide Primer Design
Previously published DNA sequences from Asian and African
elephants were aligned for the genes BGN, CHRNA1, GBA,
PHKA2, and PLP and included the following GenBank
accession numbers: AY044919–AY044925 (BGN) (Roca et al.
Use of M13 Forward– and Reverse–Tailed Primers
M13-tailed primers have previously been used in microsatellite
studies (Boutin-Ganache et al. 2001) and have also been applied
to sequencing (Ishida et al. 2006). For this approach, M13 tails
(18 bp for M13F and 17 bp for M13R) were added at the 5# end
of both primers in each pair, to increase the lengths of PCR
amplicons: forward primers included the M13 forward
sequence (M13F: TGTAAAACGACGGCCAGT), whereas
reverse primers included the M13 reverse sequence M13R:
(CAGGAAACAGCTATGAC). Use of the M13-tailed primers
also simplified the sequencing reactions because sequencing
611
Journal of Heredity 2011:102(5)
primers consisting of only the M13 forward or only the M13
reverse sequence could be used to sequence all PCR products
across the different genes in which the PCR primers each
included the shared common M13 motifs. Sequencing was
conducted as described below, with 3 different amounts of
purified PCR products tested in the sequencing reactions.
Three sequencing reactions were run, using 0.5, 1, or 2.5 ll of
purified PCR product (of an original PCR total volume of 25
ll), below, to determine the amount that generated the best
quality sequences, as apparent in electropherograms (in 10 ll
total volume sequencing reactions).
PCR Amplification
Unless otherwise specified, PCR amplifications (see Detailed
Protocols in Supplementary files) were performed using 0.4 lM
final concentration of each primer, 1.5 mM MgCl2, 200 lM
of each dNTP (Applied Biosystems Inc. [ABI]), and 1 lg/ll
final concentration of bovine serum albumin (New England
BioLabs Inc.) with 0.04 units/ll final concentration of
AmpliTaq Gold DNA Polymerase (ABI). However, in order
to reduce primer–dimer formation, which occurred only
when the BGN-s2 primer pair was used, primer concentrations for BGN-s2 were reduced to 0.2 lM. PCR consisted
of a touchdown protocol with an initial denaturation at 95 °C
for 9:45 min; cycles of denaturation for 20 s at 94 °C,
annealing for 3 cycles of 30 s at 60 °C, then decreasing the
annealing temperature by 2 °C for 5 cycles each at 58, 56,
54, and 52 °C with a final annealing temperature of 50 °C
for 22 cycles, a 30 s extension at 72 °C, with a final
extension of 7 min at 72 °C. In all cases, DNA extracted
from blood from savanna and Asian elephants provided
a positive control, while 2 negative control samples were
run: one with human genomic DNA (Promega) to assure
that primers would not amplify human DNA (because
contamination by human DNA is often a concern in PCR
using degraded target DNA); and another with water to
make sure that all reagents were free from contamination.
PCR amplicons were examined on a 1.5–2% agarose gel that
contained ethidium bromide under ultraviolet light. PCR
products amplified using the M13 forward–and reverse–tailed
primer approach were treated as follows to remove excess
primers and unincorporated dNTPs prior to sequencing:
Each 20 ll of PCR product received a mix of 0.38 ll of 10
U/ll exonuclease I (USB Corporation), 0.74 ll of 1 U/ll
shrimp alkaline phosphate (USB Corporation), and 3.8 ll of
water (Hanke and Wink 1994). Incubation followed at 37 °C
for 75 min (to enable enzymatic activity) followed by 95 °C
for 5 min (to inactivate the enzymes; see Detailed Protocols
in Supplementary file) (Hanke and Wink 1994).
Sequencing
Cycle sequencing reactions were performed with the BigDye
Terminator v3.1 Cycle Sequencing Kit (ABI), using 0.12 lM
of primer with standard protocols modified to reduce the
amount of BigDye Terminator (see Detailed Protocols in
Supplementary file). The sequencing algorithm consisted of
an initial step of 95 °C for 1 min, followed by 34 cycles of
612
15 s at 95 °C, 5 s at 45 °C, and 4 min at 60 °C. Sequence
products were purified and analyzed on an ABI 3730XL
capillary sequencer at the University of Illinois Core DNA
Sequencing Facility. The software Sequencher 4.5 (Gene
Codes Corp.) was used to examine the electropherograms
and edit sequences. Sequences were verified using NCBI
BLAST (http://www.ncbi.nlm.nih.gov/blast/Blast.cgi) and
also with reference sequences previously published (Roca
et al. 2001, 2005). Species-diagnostic sites were identified
based on previous results on hundreds of elephants.
Because homozygous individuals are counted as 1 X
chromosome when the sex is unknown, the numbers listed
under species-specific diagnostic site likely underestimate
the totals, suggesting that support for species-specific
differences is likely to be even more robust than indicated.
Detailed Laboratory Protocols
Detailed step-by-step protocols for use of the M13-tailed
primers to amplify degraded DNA are included as
supplementary information. These cover in specific detail
the ordering of primers, setup of PCR reactions, purification
of PCR products, and setup of sequencing reactions.
Results
We initially adopted an approach previously reported to
improve sequencing quality for short amplicons, in which
a 40 or 60 bp length sequence tail is added to the 5# end of
an oligonucleotide primer used for sequencing (Supplementary Table 1) (Binladen et al. 2007). Primers without tails
were used in PCR on 4 samples of DNA extracted from
dung from elephants in the Babille Elephant Sanctuary
(BA). We obtained faint bands with strong primer–dimer
formation and some background bands (‘‘ghost’’ bands).
Sequencing results were of poor quality, both when the
conventional forward primer without a tail was used or
when a forward primer with a 40-bp tail was used.
Sequences were poor even after bands were eluted from
gels to avoid background bands. Using the 40-bp tailed
forward primer as part of the initial PCR reaction (as well as
for sequencing) also produced poor results. Although
further troubleshooting could have improved results for
this method, we did not pursue this because a subsequent
alternative approach proved successful.
The alternative approach sought to overcome the
difficulty of Sanger sequencing of short PCR amplicons by
adding M13 sequences to the 5# end of each primer of a pair.
Primers were designed to produce short amplicons to be able
to amplify degraded DNA. The M13 forward or reverse
sequence was added to the forward or reverse primer,
respectively, in order to increase the lengths of PCR
amplicons. An M13 forward or reverse oligonucleotide was
then used as the primer in Sanger sequencing of the PCR
amplicon. This allowed the target-specific 3# region of the
PCR primer sequence to comprise part of the poor quality 5#
end of the sequence, thereby reducing the portion of the poor
quality sequence consisting of the target gene sequence.
Brief Communications
We designed the primers to amplify short nuclear DNA
fragments that included diagnostic sites for African elephant
species (Roca et al. 2001, 2005). The target sequences
included fixed or nearly fixed nucleotide site differences
between African forest, African savanna, or Asian elephants
that have been reported within the nuclear genes BGN,
PHKA2, and PLP (Roca et al. 2001, 2005). The oligonucleotide primers targeting these regions were based on flanking
sequences conserved across the elephant species (Roca et al.
2001, 2005). A total of 17 primers were designed (Table 1),
targeting 2 regions in the gene BGN (designated primer sets
BGN-s1 and BGN-s2 with 1 and 4 informative nucleotide
sites, respectively), 2 in PHKA2 (primer sets PHKA2-s1 and
PHKA2-s2 with 2 and 1 informative sites, respectively), and
1 in PLP (primer set PLP-s1 with 2 informative sites). PCR
was run using all combinations of forward and reverse primer
pairs that targeted a given gene region.
Each of the primer pairs tested was successful at
amplifying the targeted region. Sequencing using the M13
forward or reverse primers was successful and identified
species-diagnostic nucleotide differences in each of the gene
segments (Table 2). The newly designed primers were much
more effective at amplifying DNA extracted from dung
samples than those previously developed for use with highquality DNA, which had been designed to produce much
longer amplicons (Roca et al. 2001, 2005). As an example,
Supplementary Figure 1 shows PCR results for the gene
PHKA2. Primers previously designed for use with high-
quality nuclear DNA (Roca et al. 2005), which amplified
a 1002 bp region of PHKA2 (excluding primer lengths), were
successful in amplifying only 3 of 12 DNA samples extracted
from dung. The newly designed primers, which targeted DNA
regions of 48 and 72 bp (excluding primer lengths), amplified
10 of the same 12 samples (Supplementary Figure 1).
In an attempt to improve the quality of sequences for the
short amplicons, different amounts of PCR product per
sequencing reaction were tested for Sanger sequencing.
From PCR reactions of 25 ll, sequencing reactions (of 10 ll
volume) were conducted using PCR amplicon product
volumes of 0.5, 1.0, or 2.5 ll. Sequence quality was better
when 0.5 or 1 ll of PCR amplicon product was used rather
than 2.5 ll. Furthermore, disassociated primer dyes (‘‘dye
blobs’’) were common in reactions involving 2.5 ll of PCR
product and sometimes appeared when using 1 ll of
template PCR product (Supplementary Figure 2). Thus,
using 0.5 ll of PCR product appears to be ideal for use in
Sanger sequencing of the short amplicons, and this is the
amount recommended in the detailed protocols included as
supplementary information. Sequence quality differences
appeared to be greater for different volumes of PCR
product among those primers that displayed greater primer–
dimer formation. By contrast, sequence quality was similar
for different pairs of primers used to amplify the same genic
region. The more successful pair of primers for each locus is
indicated (asterisks) in Table 1. The electropherograms
covering diagnostic sites—fixed differences between forest
Table 1 PCR primers used to generate short amplicons for BGN, PHKA2, and PLP
Gene/primers
BGN
BGN-s1 (268–331)
BGN-s1F-M13F
BGN-s1F2-M13F*
BGN-s1R-M13R
BGN-s1R2-M13R*
BGN-s2 (472–586)
BGN-s2F-M13F*
BGN-s2R-M13R
BGN-s2R2-M13R*
PHKA2
PHKA2-s1 (26–73)
PHK-s1F-M13F*
PHK-s1F2-M13F
PHK-s1R-M13R*
PHKA2-s2 (853–923)
PHK-s2F-M13F*
PHK-s2F2-M13F
PHK-s2R-M13R
PHK-s2R2-M13R*
PLP
PLP-s1 (312 369)
PLP-s1F-M13F*
PLP-s1R-M13R
PLP-s1R2-M13R*
Pimer sequence
TGTAAAACGACGGCCAGTATAGGGGGAGGTCCTTCCTT
TGTAAAACGACGGCCAGTCGTGACTATAGGGGGAGGTC
CAGGAAACAGCTATGACAGTGTGGAATAGACACAGACACG
CAGGAAACAGCTATGACACGCACGGGAAGACACAG
TGTAAAACGACGGCCAGTTTCCAAACAGGGTCACATCC
CAGGAAACAGCTATGACGCCAATTTCCTTTTGTCTGTTT
CAGGAAACAGCTATGACAGCCAATTTCCTTTTGTCTGTT
TGTAAAACGACGGCCAGTTGTGCTCTCCTGTTGCTTTG
TGTAAAACGACGGCCAGTGTGCTCTCCTGTTGCTTTGA
CAGGAAACAGCTATGACACCTACTTGTTGCTGACTTTTGAA
TGTAAAACGACGGCCAGTTCCTGCAATAAGCAGCAACA
TGTAAAACGACGGCCAGTCCTGCAATAAGCAGCAACAT
CAGGAAACAGCTATGACTTTCCCAAAGATGATGAAAACAT
CAGGAAACAGCTATGACTTTCCCAAAGATGATGAAAACA
TGTAAAACGACGGCCAGTGATTGAGTTGGAGCCTCTGC
CAGGAAACAGCTATGACCTGAGAAGACTCAAGCAGTGTCA
CAGGAAACAGCTATGACCTCAAGCAGTGTCAAATCAAAA
Forward primers have M13 forward sequence as a 5# tail; reverse primers have M13 reverse sequence as a 5# tail. Tail sequences are italicized. Asterisks
denote the pair of primers best for amplifying each region, with the region of the gene amplified by the best primer pair indicated within parentheses.
Numbering for each gene follows Roca et al. (2005).
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Journal of Heredity 2011:102(5)
Table 2
Variable nucleotide sites in amplified regions of 3 nuclear genes in elephants
BGN-s1
Sample ID
Loxodonta africana
BA0101
BA0102
BA0201
BA0202
NA5121
NA5125
NA5154
NA5155
BA0001
L. cyclotis
BF0008
BF0009
BF0012
BF0014
Elephas maximus
SN-160
BGN-s2
PHKA2-s1
Sample type 287 304 308 472 485 499 508 513 515 516 570 39
Dung
Dung
Dung
Dung
Dung
Dung
Dung
Dung
Blood
Dung
Dung
Dung
Dung
Blood
G
—
G
G
G
—
G
G
G
G
GA
A
G
G
G
G
G
T
—
T
T
T
—
T
T
T
T
C
C
C
C
C
C
C
G
—
G
G
G
—
G
G
G
G
GC
G
G
G
G
G
G
A
—
A
A
A
A
A
A
A
A
AG
A
A
A
A
A
A
T
—
T
T
T
T
T
T
T
T
C
C
C
C
C
C
C
DT
—
D
D
D
D
D
D
D
D
D
D
D
D
D
T
T
G
—
G
G
G
G
G
G
G
G
T
T
T
T
T
G
G
G
—
G
G
G
G
G
G
G
G
G
G
G
G
G
A
A
GD
—
G
G
G
G
G
G
G
G
D
D
D
D
D
G
G
DG
—
D
D
D
D
D
D
D
D
D
D
D
D
D
D
D
C
—
C
C
C
C
C
C
C
C
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
C
C
C
C
C
C
C
PHKA2-s2
PLP-s1
71
871
872
319 345 361
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
G
G
T
T
T
T
T
T
T
T
T
T
C
C
C
C
C
C
C
G
G
G
G
G
G
—
G
G
G
G
GA
G
G
G
G
G
G
G
G
G
G
G
G
G
G
GA
A
G
A
A
G
G
T
T
T
T
T
—
T
T
T
T
T
T
T
T
T
C
C
A
A
A
A
A
—
A
A
A
A
G
G
G
G
G
G
G
Reference sequences and position numbers are from Roca et al. (2005); both bases are shown for variable sites and heterozygote individuals. Speciesspecific nucleotide sites (Roca et al. 2005) are indicated by boldface. D indicates a deletion; dash indicates sequences not generated for a sample. BA: Babille
Elephant Sanctuary, Ethiopia; NA: Namibia; BF: Bili Forest, Democratic Republic of Congo; SN: Studbook number (North American Zoos).
and savanna elephants—that were amplified by these
primer pairs are shown in Figure 1. For all 3 nuclear genes,
species-specific diagnostic sites were identified in elephants
from 3 populations: forest elephants in Bili Forest (BF) in
the Democratic Republic of Congo, savanna elephants in
the Babille Elephant Sanctuary (BA) in Ethiopia, and
savanna elephants in Namibia (NA). No elephants in BF or
BA had been previously examined for nuclear gene
sequences, and the elephants from NA also represented
previously unexamined individuals. The expected species-
specific nucleotide character states were detected in these
individuals and populations.
Discussion
Because it is often difficult to collect tissue or blood samples
from large numbers of animals in the wild, many elephant
studies have relied on noninvasively collected samples, such as
dung or museum specimens (Eggert et al. 2002; Nyakaana
Figure 1. Short sequences of X-linked genes included species-specific diagnostic sites. The electropherograms (screenshots of
Sequencher software) show nucleotide differences present in African savanna (Loxodonta africana), African forest (L. cyclotis), and
Asian (Elephas maximus) elephants. For 3 X-linked genes, arrows indicate nucleotide sites in which the character state distinguishes
between savanna and forest elephants. Position numbers for the gene sequences follow Roca et al. (2005). For each gene, the
species-diagnostic sites are listed with character states for savanna (left) and forest (right) African elephants. All African elephant
electropherograms shown were produced using DNA from dung samples (the Asian elephant sequences are from DNA extracted
from blood). The primers used for each gene segment were those indicated by asterisks in Table 1.
614
Brief Communications
Table 3 Species-diagnostic nucleotide sites amplified by the novel short-amplicon nuclear gene primers
BGN-s1
BGN-s2
304
485
T
C
T
PHKA2-s1
508
C
T
513
G
A
Savanna elephants
This study
7
0
9
0
0
9 0
Roca et al. (2005)
598
0 598
0
0 598 0
Lei et al. (2009)
201
0 201
0
0 201 0
Total
806
0 808
0
0 808 0
Forest elephants
This study
0
4
0
4
4
0 0
Roca et al. (2005)
0 112
0 112 112
0 0
Total
0 116
0 116 116
0 0
Asian elephants
This study
0
1
0
1
0
1 1
Roca et al. (2001, 2005)
0 11
0 11
0 11 11
Total
0 12
0 12
0 12 12
570
G
C
T
9
9
598 598
201 201
808 808
4
112
116
0
0
0
39
T
A
0 10
0 511
0 201
0 722
0
0
0
0
PHKA2-s2
PLP-s1
71
871
345
G A
T
C
0 10
9
0 511 511
0 201 201
0 722 721
0
0
0
0
0
4
0 112
0 116
0 4
0 67
0 71
0
0
0
4
67
71
0
0
0
4
67
71
0
0
0
0 1 1
0 11 11
0 12 12
0
0
0
0
0
0
1
11
12
1
11
12
C
361
T
A
G
0
8
8
0
0 653 651
2
— — — —
0 661 659
2
0
4
0 114
0 118
1
11
12
0
0
0
0
4
0 114
0 118
0
0
0
1
11
12
Chromosome frequencies of Lei et al. (2009) were estimated from their sample number: 93 females and 15 males. PLP savanna elephant results include 2
putative hybrid elephants from Cameroon (Roca et al. 2005). PLP was not sequenced by Lei et al. (2009).
et al. 2002; Debruyne et al. 2003; Debruyne 2005; Johnson
et al. 2007). This facilitates the sampling of large numbers of
individuals for molecular genetic studies. However, reliance on
low-quality DNA from dung, or museum specimens, has also
led to a reliance on mtDNA haplotypes as the basis for
inferring the population structure or taxonomy of Africa’s
elephants, as mtDNA can be amplified more readily than
nuclear DNA in samples of low quality. In many cases, nuclear
DNA markers from the samples have not been examined,
even though a more complete picture of elephant phylogeography would be provided by examining nuclear DNA (Roca
et al. 2005, 2007; Roca and O’Brien 2005; Lei et al. 2008,
2009). This strategy can provide an incomplete picture of
elephant population structure, because mtDNA follows
a different evolutionary trajectory than all other genetic
markers in elephants (Roca 2008), because female elephants
remain with their matrilocal core social groups, whereas males
leave natal herds and mediate gene flow of nuclear DNA
(Sukumar 2003; Archie et al. 2007; Hollister-Smith et al. 2007;
Roca et al. 2007). We developed a method for amplifying and
sequencing short nuclear DNA regions using highly degraded
DNA samples, without the use of expensive or labor-intensive
procedures, such as subcloning or gel purification. The
approach of adding an M13 sequence to both the forward
and the reverse primers to increase PCR amplicon sizes was
successful for each of the gene regions attempted, suggesting
that the method could be generally applied across nuclear loci
for use with samples of degraded DNA.
Species-diagnostic sites between forest and savanna
elephants had been previously established using much longer
genic sequences from hundreds of individual elephants (Roca
et al. 2005). Table 3 summarizes species-diagnostic nucleotide
sites detected by the current study using dung samples,
combined with the results of previous studies that relied on
much longer amplicons from high-quality DNA (Roca et al.
2005; Lei et al. 2009). The elephant dung DNA samples in
the current study show results consistent with those among
elephants in the 2 previous studies and carry the expected
fixed species-specific nucleotides for BGN and PHKA2 as
well as the nearly fixed nucleotide difference in PLP (Table
3). Given the hundreds of African elephants genotyped in
previous studies, we calculate that each of the currently
developed primers will be effective with greater than 99%
accuracy at distinguishing forest from savanna African
elephants (Table 3). For PLP, 2 elephants in savanna
locations in Cameroon had been found in a previous study
to carry a forest-typical PLP allele. These were believed to
be of hybrid origin, and the composite data from novel
nuclear markers should allow identification of populations
containing hybrids between the 2 species in regions where
forest and savanna habitats meet (Groves and Grubb 2000;
Roca et al. 2001, 2005; Comstock et al. 2002).
The nuclear markers that we developed in this study
would enable African elephant samples in which the DNA is
degraded to be much more readily screened for nuclear loci.
Even though the PCR amplicons generated by our novel
primers are very short, the targeted DNA segments were
designed to contain highly informative fixed nucleotide
character state differences that have distinguished forest
from savanna elephants across hundreds of individuals from
dozens of locations. The current study establishes the
presence of these diagnostic sites at 3 locales within Africa.
Identification of these sequences across a larger number of
samples that include existing collections of dung samples,
museum specimens, and confiscated ivories could establish
the genetic affinities and degree of hybridization for
elephants across the continent, assisting in their effective
conservation management (Blanc et al. 2007).
Supplementary Material
Supplementary material can be found at http://www.jhered.
oxfordjournals.org/.
615
Journal of Heredity 2011:102(5)
Funding
U.S. Fish and Wildlife Service African Elephant Conservation Fund, grant no. AFE-0458-98210-8-G743.
Acknowledgments
We thank K. Amman for African elephant samples and E. Kock at the
Tulsa Zoo and Living Museum, Tulsa, Oklahoma, USA for Asian elephant
samples. We thank T. Honda for technical assistance. We thank the
governments of the Democratic Republic of Congo, Ethiopia, and Namibia
for permission to collect samples.
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Received April 11, 2011; Revised June 21, 2011;
Accepted June 23, 2011
Corresponding Editor: William Murphy