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Evolutionary and demographic processes shaping geographic patterns of genetic diversity in a keystone species, the African forest elephant (Loxodonta cyclotis)

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Abstract

The past processes that have shaped geographic patterns of genetic diversity may be difficult to infer from current patterns. However, in species with sex differences in dispersal, differing phylogeographic patterns between mitochondrial (mt) and nuclear (nu) DNA may provide contrasting insights into past events. Forest elephants (Loxodonta cyclotis) were impacted by climate and habitat change during the Pleistocene, which likely shaped phylogeographic patterns in mitochondrial (mt) DNA that have persisted due to limited female dispersal. By contrast, the nuclear (nu) DNA phylogeography of forest elephants in Central Africa has not been determined. We therefore examined the population structure of Central African forest elephants by genotyping 94 individuals from six localities at 21 microsatellite loci. Between forest elephants in western and eastern Congolian forests, there was only modest genetic differentiation, a pattern highly discordant with that of mtDNA. Nuclear genetic patterns are consistent with isolation by distance. Alternatively, male‐mediated gene flow may have reduced the previous regional differentiation in Central Africa suggested by mtDNA patterns, which likely reflect forest fragmentation during the Pleistocene. In species like elephants, male‐mediated gene flow erases the nuclear genetic signatures of past climate and habitat changes, but these continue to persist as patterns in mtDNA because females do not disperse. Conservation implications of these results are discussed.
Ecology a nd Evolution . 201 8 ;1–1 3 .    
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www.ecolevol.org
1 | INTRODUCTION
Morphological and genetic studies have strongly suppor ted rec-
ognition of t wo African elephant species: the African savanna ele-
phant (Loxodonta africana) and African forest elephant (L. cyclotis)
(Comstock et al., 2002; Groves & Grubb, 2000; Ishida et al., 2011;
Roca, Georgiadis, Pecon- Slattery, & O’Brien, 2001; Rohland et al.,
2010). While many studies have indicated that the forest elephant is
a species distinct from the savanna elephant, the analysis of genetic
diversit y below the species level has been limited. Mitochondrial
DNA (mtDNA) patterns have been examined in African forest el-
ephants across their range (Debruyne, 2005; Debruyne, Van Holt,
Barriel, & Tassy, 2003; Eggert, Rasner, & Woodruff, 2002; Ishida,
Georgiadis, Hondo, & Roca, 2013; Johnson et al., 2007; Nyakaana,
Arctander, & Siegismund, 2002). Five distinct mitochondrial sub-
clades have been detected among forest elephants, each of which
Received:15September2017 
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  Revised:15March2018 
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  Accepted:19March2018
DOI: 10.100 2/ece3.4062
ORIGINAL RESEARCH
Evolutionary and demographic processes shaping geographic
patterns of genetic diversity in a keystone species, the African
forest elephant (Loxodonta cyclotis)
Yasuko Ishida1| Natalie A. Gugala1| Nicholas J. Georgiadis2| Alfred L. Roca1,3
This is an op en access article under t he terms of t he Creat ive Commons Attr ibutio n License , which pe rmits u se, dist ributi on and rep roduc tion in any m edium,
provide d the orig inal work is proper ly cited.
© 2018 The Aut hors. Ecology an d Evolution pu blished by John Wiley & Sons Ltd .
1Department of Animal Sciences, University
of Illinois at Urbana-Champaign, Urbana,
Illinois
2Puget Sound Institute, Universit y of
Washington, Tacoma, Washington
3Carl R . Woese Ins titute for Genomic
Biolog y, Universi ty of Illinois at Urb ana-
Champaign, Urbana, Illinois
Correspondence
Alfred L . Roca and Yasuko Ishida ,
Department of Animal Sciences, University
of Illinois at Urbana-Champaign, Urbana, IL.
Emails: roca@illinois.edu; yishida@illinois.
edu
Funding information
USFWS African Elephant Conservation
Fund, Grant/Award Number: AFE-0778-
F12AP01143andAFE1606-F16AP009 09
Abstract
The past processes that have shaped geographic patterns of genetic diversity may be
difficult to infer from current patterns. However, in species with sex differences in
dispersal, differing phylogeographic patterns between mitochondrial (mt) and nu-
clear (nu) DNA may provide contrasting insights into past events. Forest elephants
(Loxodonta cyclotis) were impacted by climate and habitat change during the
Pleistocene, which likely shaped phylogeographic patterns in mitochondrial (mt)
DNA that have persisted due to limited female dispersal. By contrast, the nuclear (nu)
DNA phylogeography of forest elephants in Central Africa has not been determined.
We therefore examined the population structure of Central African forest elephants
bygenot yping94individualsfromsixlocalitiesat21microsatelliteloci.Betweenfor-
est elephants in western and eastern Congolian forests, there was only modest ge-
netic differentiation, a pattern highly discordant with that of mtDNA. Nuclear genetic
patterns are consistent with isolation by distance. Alternatively, male- mediated gene
flow may have reduced the previous regional differentiation in Central Africa sug-
gested by mtDNA patterns, which likely reflect forest fragmentation during the
Pleistocene. In species like elephants, male- mediated gene flow erases the nuclear
genetic signatures of past climate and habitat changes, but these continue to persist
as patterns in mtDNA because females do not disperse. Conservation implications of
these results are discussed.
KEYWORDS
Congolian forest block, conservation, isolation by distance, landscape genetics, microsatellites
2 
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   ISHIDA et Al.
has a different geographically restricted distribution (Ishida et al.,
2013). However, several factors can lead to discordant patterns in
the phylogeography of nuclear and mitochondrial DNA markers,
both with in and across spe cies (Petit & Exco ffier, 2009; Toews &
Brelsford, 2012), and in elephant s, there is evidence that female
philopatry and male- biased dispersal combine to produce incon-
gruent mitonuclear patterns (Debruyne, 2005; Roca, Georgiadis, &
O’Brien, 2005).
Field studies have shown strong evidence that, despite living in
a fission–fusion society, female elephants remain with their clos-
est kin after they mature (Archie, Moss, & Alberts, 2011). Genetic
analyses have suppor ted female philopatry by demonstrating al-
most complete uniformity of mtDNA haplotypes within families
(Archie, Fitzpatrick, Moss, & Alberts, 2011). By contrast, male el-
ephants upon reaching maturity disperse from their natal herds
(Lee, Poole, Njiraini, Sayialel, & Moss, 2011) and enter periods of
musth characterized by competitive interactions with other males
for reproductive access to females (Poole, Lee, Njiraini, & Moss,
2011). The dispersal of male elephants from their natal social groups
thus mediates nuclear gene flow (Ishida et al., 2011; Nyakaana &
Arctander,1999;Rocaetal.,2005,2015).Thephylogeographicpat-
terns revealed by analyses of Y- chromosome sequences is similar to
the pattern for other nuclear markers, but different from patterns
shown by mtDNA, supporting the role of males in establishing nu-
clear phylogeographic patterns (Roca, Georgiadis, & O’Brien, 2007;
Roca et al., 2005).
Because mitochondrial phylogeographic patterns are often
discordant from nuclear patterns in species in which only males
disperse(Petit&Excoffier,2009;Toews&Brelsford,2012),includ-
ing elephants (Roca et al., 2005, 2007), there is a strong need to
analyze nuclear markers among forest elephants to examine more
completely their evolutionary history and population structure.
Furthermore, Central Africa, the region in which most forest ele-
phants live, has suffered from the highest levels of elephant poach-
ing of any subregion within the continent (CITES, 2012), and has
been the main source for the illegal trade in elephant bushmeat
and ivory (Wasser et al., 2015). Forest elephant numbers declined
by ca. 62% bet ween 2002 and 2011, to <10% of their estimated
historical population size, mainly due to illegal poaching for their
tusks (Maisels et al., 2013). There is thus a strong need to exam-
ine fine- scale population substructure within forest elephants
using nuclear markers, for proper conservation management of the
species.
Here, we use microsatellite markers to examine nuclear genetic
structure in the forest elephant. Ninety- three individuals from five
localities in Central Africa and one individual from Sierra Leone were
genotyped using 21 microsatellite markers. We examined nuclear
genetic markers for geographic differences among forest elephant
localities. We discuss the extent to which regional populations may
or may not be genetically distinctive, and the implications of these
findings for forest elephant conservation. We also specifically ex-
amine the degree of discordance between the phylogeographic pat-
terns inferred using microsatellite markers and patterns previously
reported for forest elephant mtDNA across the same tropical forest
localities within Central Africa.
2 | MATERIALS AND METHODS
2.1 | Samples
This stu dy was conducte d under the Univer sity of Illinois In stitutional
Animal Care and Use Committee (IACUC)- approved protocol num-
ber 15053. Samples were collected in full compliance with required
Convention on International Trade in Endangered Species of Wild
Fauna and Flora and other institutional permits. Wild African for-
est elephants (L. cyclotis) were sampled from six localities (Figure 1).
Tissue samples were collected primarily by biopsy darting from ele-
phants in Lope (LO) in Gabon, Odzala (OD) in the Republic of Congo,
Dzanga Sangha (DS) in the Central African Republic, and Garamba
(GR) in the Democratic Republic of Congo. Dung samples of el-
ephants were collected from the Bili Forest (BF) in the Democratic
Republic of Congo. A blood sample was obtained from a forest
FIGURE1 The map shows the sampling locations of forest elephants. Abbreviations are as follows: DS—Dzanga Sangha, Central African
Republic; OD—Odzala, Republic of Congo; BF—Bili Forest, Democratic Republic of Congo; LO- Lope, Gabon; and SL—Sierra Leone (one zoo
individual). GR—Garamba in Democratic Republic of Congo is located in the Guinea–Congolian/Sudanian transition zone of vegetation
(Olson et al., 2001) that historically included a mix ture of forest and secondary grasslands suitable for both African elephant species (Groves
& Grubb, 2000)
DS
OD
LO
GR
BF
SL
1,000 km
    
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ISHIDA et A l.
elephant kept at the Paris Zoo that originated in Sierra Leone (SL).
These localities represent different geographic regions: Sierra Leone
(SL) is located in West Africa; the others are in the Congolian forest
block, with LO, OD, and DS to the west, and BF and GR to the east
(Figure 1).
In total, 94 fo rest elepha nts from six lo calities (Fig ure1) were
successfully genot yped at the 21 microsatellite loci (SL: n = 1, LO:
n = 15, OD: n = 3, DS: n = 53, BF: n = 3, GR: n = 19)(Tables1andS1).
As only one sample was available from SL, it was not included in
some statistical analyses. Garamba has historically included mixed
forest and savanna habitats suitable for both species of African ele-
phant (Groves & Grubb, 2000). Most of our samples from Garamba
are forest elephants, although a few are hybrids of savanna and for-
est elephants based on nuclear genotypes (Comstock et al., 2002;
Groves & Grubb, 200 0; Ishida et al., 2011; Roca et al., 2001; Rohland
et al., 2010).
In addition to the forest elephants, 15 African savanna ele-
phants (L. africana) were genotyped, one each from 15 localities
(CH—Chobe and MA—Mashatu in Botswana; BE—Benoue and
WA—Waza in Cameroon; AB—Aberdares, AM—Amboseli, and KE—
Central Kenya/Laikipia in Kenya; NA—Northern Namibia/Etosha;
KR—Kruger in South Africa; NG–Ngorongoro, SE–Serengeti, and
TA—Tarangire in Tanzania; HW—Hwange, SW—Sengwa, and ZZ—
Zambezi in Zimbabwe). Savanna elephant samples were included
in light of previous findings that forest and savanna elephants are
genetically distinct species with a narrow region of hybridization
(Comstock et al., 20 02; Groves & Grubb, 20 00; Ishida et al., 2011;
Roca et al., 2001; Rohland et al., 2010). Because our samples con-
tain forest elephants from Garamba where a few hybrids of sa-
vanna and forest elephants have been identified based on nuclear
genotypes (Comstock et al., 2002; Ishida et al., 2011; Mondol
et al., 2015; Roca et al., 20 01), it was determined that the micro-
satellites would be amplified in savanna elephants, as they would
be needed to identif y hybrids of savanna and forest elephants.
Details on the sampling and DNA extraction have been previously
published (Ishida et al., 2011).
2.2 | Microsatellite genotyping
Allelic variation was examined at 21 microsatellite loci. These
markers have been previously developed by Gugala et al. (Gugala,
Ishida, Georgiadis, & Roca, 2016). Primer sequences are listed in
Table S1. All forward primers included the M13 forward sequence
(TGTAA AACGACGGCCAGT ) at the 5′ end. The PCR pr imer mix
consiste d of a 5′ FAM- or VIC-fluorescent-labeled M13 for ward
primer (to label the PCR amplicon), along with the forward primer
(with M13 forward sequence at the 5′ end) and reverse prim-
ers. The PCR mix included 1× PCR buffer II (Life Technologies,
Carlsbad, CA , USA), 2 mmol/l MgCl2, 200 μmol/l of each dNTP (Life
Technologies) with 0.04 units/μl final concentration of AmpliTaq
Gold DNA Polymerase (Life Technologies) along with 1.2 μl of the
primer mix. For the DNA samples from BF that had been extrac ted
from dung, 1 μg/μl final concentration of bovine serum albumin
(New England BioLabs Inc.) was also included. The PCR cycling pro-
gram consi sted of an initial 95°C fo r 10min; with cycles of 15s
denaturingat95°C,followedby30sannealingat60,58,56,54,or
52°C(two cycleseachtemperature); or 50°C(last 30 cycles),fol-
lowedby45s extensionat72°C;withafinalextension of30min
at72°C.For locus Lcy-M45,the PCR cycling program was modi-
fied as described previously (Gugala et al., 2016). PCR amplicons
were visualized on a 1.5%–2% agarose gel with ethidium bromide
under ultraviolet light. Amplicons of two different loci labeled with
different fluorescent dyes (FAM and VIC) were diluted and mixed
depending on the intensity of the signal on the agarose gel photo-
graph. Fragment analysis was conducted on the ABI 3730XL capil-
lary sequencer at the University of Illinois at Urbana- Champaign
High- Throughput Sequencing and Genotyping Unit. The soft ware
Genemapper version 3.7 (Life Technologies) was used to call alleles.
Relying on a standard of known size, the binning function of the
software Genemapper was used to determine fragment lengths,
following the procedures indicated in the manual. For the DNA
samples extracted from dung (BF), at least four independent ampli-
fications were repeated to confirm homozygotes and three amplifi-
cations for heterozygotes (Allentoft et al., 2011).
2.3 | Characterization of microsatellites
Arlequin version 3.5.1.3 (Excoffier & Lischer, 2010) and GenAlEx 6.5
(Peakall & Smouse, 2012) were used to calculate expected heterozy-
gosity (He) and observed heterozygosit y (Ho). The sof tware GenAlEx
6.5 was also used to calculate Shannon’s diversity index (I) and to
make allele f requency distribution histograms for each locus for each
locality. Tests for Hardy–Weinberg equilibrium (HWE) and linkage
disequilibrium (LD) were conducted on the forest elephant micro-
satellite data. A Markov chain algorithm was used to test for HWE
using 10,000 dememorization steps, 1,00 0 batches, and 10,000 it-
erations per batch using the software Genepop 4. 2 (Rousset, 2008),
and 1,000,0 00 steps and 100,000 dememorization steps were used
with Arlequin version 3.5.1.3. LD was examined using 10,000 de-
memorization steps, 1,000 batches and 10,000 iterations per batch
TABLE1 Comparison of pairwise FST values calculated using
nuclear and mitochondrial DNA
LO (17) OD (3) DS (54) BF (0) GR (20)
LO (15) 0.54 0.87 NA 0 .76
OD (3) 0.02 0.49 NA 0.38
DS (53) 0.02 0.02 NA 0.61
BF (3) 0.06 0.06 0.04 NA
GR(19) 0.05 0.07 0.03 0.00
Pairwise FST is shown for comparisons between localities using nuclear
microsatellites (below diagonal) and mtDNA (above diagonal). FST values
of mtDNA are from Ishida et al. (2013). The values that are significant are
indicated in boldface. Sample sizes are in parenthesis for mtDNA (top
row) and microsatellites (first column). Localities corresponding to the
abbreviations are shown in Figure 1. FST values calculated using mtDNA
are not shown for BF as comparable mtDNA sequences are not available
fo r B F.
4 
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   ISHIDA et Al.
for each pairwise comparison between loci for Genepop 4.2 and
10,000 permutations for Arlequin version 3.5.1.3.
2.4 | Population genetic analyses
Analysis of molecular variation (AMOVA) was conducted in Arlequin
version 3.5.1.3 (Excof fier & Lischer, 2010) using 10,0 00 permuta-
tions. For each pair of localities, Arlequin was also used to calculate
pairwise FST values and statistical support using 10,00 0 permuta-
tions, and to calculate the inbreeding coefficient for each locality,
except SL, as only one sample was available from SL.
STRUCTURE 2.3.4 (Pritchard, Stephens, & Donnelly, 2000),
which applies a model- based clustering algorithm to multilocus gen-
otype data, was used to infer population structure using datasets
that included or excluded the savanna elephants. STRUCTURE was
run eight times for each value of K from 1–10, without the use of
prior information on localit y, under the admixture- correlated model,
with each iteration using at least 1 million Markov chain Monte
Carlo generations following a burn- in of at least 100,000 steps.
The uppermost hierarchical level of population structure was ex-
amined using an ad hoc statistic ∆K based on the rate of change in
the log probability of the data for a given K between successive K
values, implemented in Structure Harvester (Earl & vonHoldt, 2012).
Average coef ficients were estimated for each K value that was es-
timated to be uppermost, and for lower values of K, employing the
Greedy algorithm with 1,000 random input orders as implemented
in the program CLUMPP version 1.1.2 (Jakobsson & Rosenberg,
2007). These outputs were visualized using DISTRUCT version 1.1
(Rosenberg, 2004). After identif ying the hybrid elephants in GR, we
also conducted STRUCTURE analyses excluding them to remove
the influence of savanna elephant genotypes. We also conducted
STRUCTURE analyses to compare each pair of localities.
Factorial correspondence analyses (FCA) were implemented
in GENETIX version 4.05 (Belkhir, Borsa, Chikhi, Raufaste, &
Bonhomme, 200 4) to graphically plot the distribution of genetic
variation for each locality with forest elephants (excluding the hy-
brids). Principal coordinate analyses (PCoAs) were implemented in
GenAlEx 6.5 (Peakall & Smouse, 2012) to visualize the genetic rela-
tionships among individual elephants, both including and excluding
the savanna elephants and hybrid GR elephants.
2.5 | Isolation by distance
The coordinate information of each localit y was estimated using the
LatLong.net website (http://www.latlong.net/), and the pairwise
distances between each locality were calculated using the coordi-
nate calculators and distance tools in GPS Visualizer (http://www.
gpsvisualizer.com/calculators). To examine the relationship between
genetic distances and geographic distances among forest elephants
at the five localities in Central Africa, a spatial autocorrelation anal-
ysis was implemented in GenAlEx 6.5 (Peakall & Smouse, 2012).
The spatial autocorrelation analysis divided the pairwise distances
intofour ordinalclasses andused 9,999random permutationsand
9,999 bootstrap iterations. Isolation-by-distance (IBD) analyses
were conducted to test the relationships between genetic differ-
ences bet ween each pair of localities in Central Africa and the geo-
graphic distance between them. These analyses used the Isolation
By Distance Web Service Version 3.23 (Jensen, Bohonak, & Kelley,
2005). Two different measures of genetic distance were calculated:
FST and Rousset’s distance FST/(1- FST). Mantel tests were run with
30,000 randomizations (one- tailed test). For Slatkin’s similarity
index, we used the recommended log- transformation of both M and
geographic distance. As we had only one sample from West Africa
(from Sierra Leone, SL), we excluded this sample from analyses.
2.6 | Phylogenetic analyses
We inferred the phylogenetic relationships among localities using
the neighbor–joining (NJ) method implemented in POPTREEW
(Takezaki, Nei, & Tamura, 2014). As the number of samples was
different among localities, we used DST (Nei, 1972)andFST (Latter,
1972)withsamplesizebiascorrec tioninadditiontoDA (Nei, Tajima,
&Tateno,1983)(forwhichsamplesizebiascorrectionwasnotavail-
able) to calculate genetic distances among localities. Support for the
nodes in each analysis was assessed using 10,0 00 bootstrap pseu-
doreplicates. To exclude the influence of savanna elephant geno-
types on GR, forest–savanna hybrid elephants were not included
in these analyses. The program FigTree v1.4.2 (Rambaut, 2014) was
used to draw trees. To assess the influence of the small sample size
of SL (n = 1), we also conducted additional analyses using only one
sample from an alternative location (LO). The single LO sample was
chosen randomly by RESEARCH RANDOMIZER (https://www.rand-
omizer.org/). Three iterations were run in which a single sample from
LO was chosen at random, and the NJ tree was reconstruc ted with
the single LO sample.
3 | RESULTS
3.1 | Characterization of forest elephants using
microsatellites
Although 21 microsatellite markers were genotyped, three were re-
moved before analyses. Marker Lc y- M4 had a low genotyping suc-
cess rate due potentially to null alleles. In marker Lcy- M15, a 1- bp
indel was detected in some savanna elephants; this marker was
removed from the forest elephant analyses as some elephants in
Garamba are savanna–forest elephant hybrids. The marker Lcy- M52
showed a significant deviation from HWE even after Bonferroni cor-
rection (p < .0026), and was monomorphic in three localities. The re-
maining 18 microsatellite loci (Table S2) did not show deviation from
HWE in forest elephants and were used in the population analyses.
No significant linkage disequilibrium was detected between pairs of
loci after Bonferroni correction.
The mean number of alleles, the mean Ho, and the mean He of the
18 markers am ong forest e lephants we re 7.44±0.79, 0.58±0.05 ,
and 0.61 ± 0.05 respectively. The allele frequencies for each locus
    
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 5
ISHIDA et A l.
are shown in Figure S1 and allele number, heterozygosit y, and other
information for each marker are listed in Table S2. The markers did
not show high diversity in savanna elephant s. This would be ex-
pected for two reasons. First, the markers had been designed based
only on the presence of polymorphisms among forest elephants
(Gugala et al., 2016) that are 4–7 million years divergent from sa-
vanna elephants (Brandt, Ishida, Georgiadis, & Roca, 2012; Rohland
et al., 2010). Additionally, savanna elephant s are known to have re-
duced nuclear genetic diversity relative to forest elephants (Roca
et al., 2001; Rohland et al., 2010). In savanna elephants, allele num-
bers for the 18 microsatellite loci ranged from 1 to 4, with the mean
of2.39±0.24.ThemeanHo was 0. 21 ± 0.04, and the mean He was
0.25 ± 0.05. Allele numbers, heterozygosity, and other information
for each marker are listed for the savanna elephants in Table S3.
3.1.1| Analyses of population structure across
forest elephant localities
Population genetic analyses involved only forest elephants, except
as otherwise indicated. Many analyses were run separately for data-
sets that included or excluded elephants in GR that were identified
as hybrids between forest and savanna elephants, although the out-
comes of these analyses were not greatly affected by including or
excluding hybrids. Analysis of molecular variance (AMOVA) found
that only 3.04% of the variance was accounted for by differences
among localities (Table S4). FIT was 0.054 (p < .005) with significant
deviation from HWE while FIS was 0.024 but did not deviate signifi-
cantly from HWE. The value calculated for FST was low at 0.030, and
this value was statistically significant (p < .001).
FIGURE2 Bayesian clustering
approach implemented in STRUCTURE
(Pritchard et al., 2000) using 18
microsatellite genotypes, including both
forest and savanna elephants or only
forest elephants. (a) When both savanna
and forest elephants were included, the
uppermost K value was estimated as four
(Earl & vonHoldt, 2012). At K = 2, the
forest (Loxodonta cyclotis) and savanna
(L. africana) elephants were almost
completely separated into different
partitions, with a few hybrids in GR,
consistent with previous reports (Ishida
et al., 2011). At higher levels of K, the
additional partitions do not completely
separate elephants from different
localities, although forest elephants from
the eastern Congolian forest (BF, GR)
show different patterns in their partitions
than localities further west. (b) When
only forest elephants were analyzed,
the uppermost K value was estimated
as three. Partitioning within the forest
elephants resembles that seen in panel A,
with a distinctive but incomplete pattern
of partitioning between eastern and
western localities. Note that additional
analyses (Figure S2) excluding the forest–
savanna hybrid elephants from GR did not
greatly af fect the patterns of partitioning
(a)
(b)
Loxodontacyclotis L. africana
SL
LO
OD
DS
BF
GR
Laf
K = 4
SL
LO
OD
DS
BF
GR
Laf
K = 3
SL
L
O
O
D
D
S
BF
G
R
L
af
K = 2
L
O
D
G
L
K = 3
GR0021GR0020
K = 2
SL
LO
OD
DS
BF
GR
SL
LO
O
D
DS
BF
G
R
6 
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   ISHIDA et Al.
For each pair of forest localities, FST was also calculated ( Tables 1
and S5) for pairs of localities (excluding SL for which the sample size
was one). We identified five elephants in Garamba as hybrids using
STRUCTURE (see below) and removed these five elephants from
the analyses. Pairwise FST values were high when each forest local-
ity was compared to savanna elephants, which for these analyses
were grouped together (Laf in Table S5). In the comparisons involv-
ing a forest locality and the grouped savanna elephants, pairwise
FST ranged from 0.40 to 0.65, with a statistically significant result
for each comparison. FST was also calculated between each pair of
localities containing forest elephants (Tables 1 and S5), with values
ranging from zero (BF and GR) to 0.07 (GR and OD). FST values were
low between pairs of forest elephant localities although there were
modest but statistically significant dif ferences between some lo-
calities in the eastern and western regions of the Congolian forest
block (Tables 1 and S5). Localities with larger sample sizes (DS, GR,
and LO) tended to yield statistically significant values. Even when
the pair wise differences were found to be statistically significant,
the low values for FST suggested that genetic differentiation among
forest elephants in the Congolian forest block is modest. When sam-
ples from localities in the western forest block were combined and
compared to samples combined across the eastern Congolian forest
block, the calculated FST was low (0.035), although this value was
statistically highly supported (p < .001). By contrast, FST values that
had been previously estimated using only mitochondrial DNA (Ishida
et al., 2013) were much higher (Table 1). The FST values calculated
FIGURE3 Analyses of forest elephants grouped by locality. Five forest–savanna elephant hybrids from GR were excluded from the
analyses. (a) Factorial correspondence analyses implemented in GENETIX version 4.05 (Belkhir et al., 20 04) were used to graphically
represent the distribution of genetic variation among forest elephant localities. Coordinate 1 explained 30.81% of the genetic variation
and separated the Congolian block forest elephants into western (DS, OD, and LO; indicated using darker green) and eastern (BF and GR;
indicatedusinglightgreen)groups.Coordinate3explained19.51%ofthegeneticvarianceandseparatedasingleWestAfricanGuinean
forest block elephant originating in Sierra Leone (SL) from the Central African Congolian forest block elephants. (b) Neighbor–joining trees
based on DA (top), sample size bias- corrected DST (middle), and sample size bias- corrected FST (bottom) showed consistent topologies.
Western Congolian forest block localities (DS, OD, and LO, highlighted in darker green) and eastern Congolian forest block localities (BF and
GR,highlightedwithlightgreen)groupedseparately.Bootstrapvalues≥70%areshown.Onallthreetrees,acladeconsistingofthetwo
localities in the eastern Congolian forest block (BF and GR) showed relatively high bootstrap support. Interestingly, a West Afric an elephant
from Sierra Leone was separated from the other localities by a long branch, which proved robust regardless of method used to calculate
distance or attempts to account for the limited sample size (see Figure S6). Although the distant placement of the Sierra Leone elephant is
intriguing, we caution that no strong conclusion can be drawn from a single individual
(a) (b)
SL
DS
LO
OD
LO
DS OD
SL
BF
GR
GR
BF
85
86
72
0.03
0.02
0.01
    
|
 7
ISHIDA et A l.
using mtDNA ranged from 0.38 to 0.87 for each pair of localities,
while pairwise FST determined using microsatellite markers was 0.07
or less.
Analyses using Structure Harvester suggested that the upper-
most clustering level was K = 4 when we included savanna ele-
phants and K = 3 when we analyzed using only forest elephants data
(Figure 2). The savanna and forest elephants fell into two distinct
partitions, with hybrids detected in GR (Figure 2a), consistent with
previous report s (Ishida et al., 2011). At higher levels of K, the addi-
tional partitioning tended to occur between the three localities on
the western side of the Congolian forest block (LO, OD, and DS) and
the two localities in the eastern side of the Congolian forest block
(BF and GR), although partitioning between west and east was in-
complete (Figure 2a: K = 3, Figure 2b: K = 2). The STRUCTURE analy-
ses excluding 5 hybrid GR elephants produced similar results (Figure
S2). We also conduc ted STRUCTURE analyses for each pair of lo-
calities. We detected differences but incomplete partitioning be-
tween GR and the two western localities, DS and LO in the pairwise
comparisons (Figure S3). Different patterns of par titioning were not
observed between BF and other localities in these pair wise compar-
isons, presumably due to the small sample size for BF (Figure S3).
A principal coordinates analysis (PCoA) conducted using
GenAlEx showed that 25.03% of the genetic variance was explained
by coordinate 1, which revealed clear separation between savanna
and forest elephant (Figure S5a), except for a GR elephant (GR0021)
that was also identified as a forest–savanna elephant hybrid using
STRUCTURE (Figure 2a). Factorial correspondence analyses (FCA)
impleme nted using GENETI X (Belkhir et al., 2004) also d emonstrated
distinc tiveness between forest and savanna elephants (Figure S4a).
Coordinate 1 explained 64.32% of the genetic variance and clearly
separated savanna and forest elephant s. In this FCA, western
Congolian forest elephants separated from eastern Congolian forest
elephants along Coordinate 2 (Figure S4a).
In PCoAs using only forest elephant data, distinctiveness among
localities was not evident when every individual elephant was
plotted (Figure S5b). However, when forest genotypes were com-
bined within each locality, FCA separated localities into two groups
(Figures 3a, S 4a). Coordinate 1 of the FCA explained 30.81% of the
genetic variance, separating forest elephant localities into a western
Congolian forest group (DS, OD, and LO) and an eastern Congolian
forest group (BF and GR). The single sample from Sierra Leone clus-
tered with BF and GR along coordinate 1, but coordinate 3 separated
SL from all other forest elephant localities (Figure 3a). Although co-
ordin at e3explain ed on ly19.51%ofthegenet ic va ri an ceandSL con-
sisted of only one elephant sample, this is an intriguing result given
that SL consisted of a single sample from the only one of our local-
ities within the Guinean forest block of West Africa, which is not
contiguous with the Congolian forest block of Central Africa.
Neighbor–joining (NJ) analyses of forest elephant genotypes
grouped by localit y produced consistent topologies using three
genetic distance calculations: DA, bias- corrected DST, and bias-
corrected FST. The lat ter two methods correct for biases caused by
sample size differences among localities. The analyses involving DA
tended to show a longer branch for the localities with small sample
sizes (Figures 3b, S 4b). This tendency was not evident using bias-
corrected DST and FST (Figures 3b, S4b). In all three trees, savanna
elephants (Laf) formed a lineage distinct from forest elephants
(Figure S4b). Among forest elephants, the eastern Congolian forest
localities (DS, OD, and LO) formed a clade that was distinct from a
clade formed by the western Congolian forest localities (BF and GR),
while SL (in the Guinean forest block) had a long branch separat-
ing it from all other forest elephant localities (Figures 3b, S4b). To
examine whether the separation of SL on the tree was merely due
to it having the smallest sample size of n = 1, we reran the analyses
while also limiting the sample size of LO to a single individual (Figure
S6). Reducing the sample size of LO to one individual affected the
trees based on DA, with the terminal branch length of LO becoming
relatively longer. By contrast, trees based on DST and FST that were
corrected for sample size bias consistently showed a relatively long
branch for SL but not for LO when a single individual was used for
each locality (Figure S6). This was true for three dif ferent individuals
from LO, randomly chosen and used in separate analyses in which
the sample size of LO was limited to one. For the two bias- corrected
methods, the long branch length was consistently present for SL
with n = 1, but not for LO with n = 1, which may suggest that the
long separation between SL and the other populations may be due
to actual genetic differences, and not be a mere artifact of the small
sample size.
3.1.2 | Evidence for isolation by distance among
forest elephants in the Congolian forest block
We examined the degree to which genetic differences among for-
est elephants at different localities varied with the geographic
distances separating them. Geographic distances were computed
between each pair of elephant s, with the distances placed into
quartiles (x- axis in Figure 4a) to implement a spatial autocorrela-
tion analysis (Peakall & Smouse, 2012). Genetic distances between
pairs of elephants were also determined (y- axis in Figure 4a). A
spatial autocorrelation analysis showed a correlation between
genetic distance and geographic distance. Forest elephants that
were close to each other geographically were also more similar
genetically than were the geographically distant forest elephants
(Figure 4a).
Suppor t for isolation by distance (Jensen et al., 2005) was exam-
ined. When the single sample from Sierra Leone (SL) was included in
the analyses, only a marginally significant correlation between ge-
netic distance and geographic distance was detected for FST (r = .45,
p = .049, Mantel test ) and for Rousset ’sd istance (r = .45, p = .052,
Mantel test) (Figure S7). However, as SL included only one sample,
and SL is from a different and geographically discontinuous forest
block, a more conser vative approach excluded the single sample
from SL for these analyses. Within the Congolian forest block, isola-
tion by dist ance received strong suppor t, as determined using both
FST (r = .86, p < .01, Mantel test) (Figure 4b) and Rousset’s distance
(r = .85, p < .01, Mantel test) (Figure 4c).
8 
|
   ISHIDA et Al.
4 | DISCUSSION
4.1 | Current population structure among forest
elephants
Nuclear genetic dif ferences were detected among Central African
forest locations. Specifically, genetic partitioning by STRUCTURE
identified that elephants in western (LO, OD, and DS) and in eastern
(BF and GR) Congolian forests were detectably different, although
partitioning was far from complete (Figure 2). The same groupings
were also evident in clustering analyses (Figure 3). However, as indi-
cated in the PCoA, microsatellite profiles from individuals in eastern
and western Central Africa showed a great deal of overlap, and FST
values were modest. All of these result s point to only limited genetic
differentiation between eastern and western localities within the
Central African forests.
Hybrids between forest and savanna elephants have been docu-
mented, but only within relatively narrow transition zones bet ween
forest and savanna habitats (Comstock et al., 2002; Ishida et al.,
2011; Mondol et al., 2015; Roca et al., 2001). One impor tant point
regards the number of markers needed to estimate the relative
contributions of the t wo species to hybrid individuals. In a previous
analysis (Ishida et al., 2011), the hybrid individual shown to have
the greatest proportion of savanna elephant alleles was GR0020,
whereas the current study identified GR0021 as having the highest
savanna elephant contribution (GR0020 had the second highest pro-
portion; Figures 2a and S5a). This likely reflect s stochasticity in the
genomic distribution of genetic markers from forest or savanna lin-
eages in hybrids, and in the proportion of each genotype attributed
to either lineage by the assignment software. The use of a larger
number of microsatellite markers, combining the novel markers used
here with those previously developed, is likely to provide greater
FIGURE4 The relationship between genetic distance and geographic distance among forest elephants from different localities. (a)
Spatial autocorrelation analysis found that genetic distance was correlated with geographic distance (r is the spatial autocorrelation
coefficient; Uistheupper95%randomizationlimitsofr; Listhelower95%randomizationlimitsofr). (b) Results consistent with potential
isolation by distance were obtained by comparing genetic distance (FST) to geographic distance (km) in pairwise comparisons of forest
elephant localities (r = .86, p < .01). (c) Result s consistent with potential isolation by distance were obtained when genetic distances were
calculated using Rousset’s distance FST/(1- FST) and compared to geographic distances (km) in pairwise comparisons of the genotypes of
forest elephants grouped by localit y (r = .85, p < .01). Five forest–savanna elephant hybrids from GR were excluded from the analysis shown
in each panel; Sierra Leone (SL) was not used in the pairwise comparisons because the sample size was one. For results including the SL
sample, see Figure S7
0.04
–0.02
–0.04
0.00
0.02
r
r
U
–0.06 161284
Distance class (end point)
L
0.070
0.062
0.080000
0.071000
(c)(b)
(a)
0.046
0.054
0.053000
0.062000
0.014
0.022
0.030
0.038
Genetic distance (
FST)
0.017000
0.026000
0.035000
0.044000
Genetic distance ((FST/(1-FST)))
0599 1199 1799 2399 2999
–0.010
–0.002
0.006
–0.010000
–0.001000
0.008000
Geographic distance
0599 1199 1799 2399 2999
Geographic distance
    
|
 9
ISHIDA et A l.
precision in estimating the degree to which hybrids received alleles
fromonelineageor theother(Boecklen&Howard,1997).Itwould
also be likely to further increase the accuracy and precision of esti-
mating the provenance of confiscated ivory using nuclear markers
(Wasser et al., 2015).
The distinctiveness of elephants from West Africa has been pro-
posed (Eggert et al., 2002), but based largely on mitochondrial DNA
data, which can be misleading in elephants (due to maternal inher-
itance and female philopatr y) (Ishida et al., 2011, 2013). The single
individual from West Africa (Sierra Leone) in this study appeared
to anchor one of the axes in the FCA analysis, and also generated a
long branch in the phylogeny, when compared to other isolated in-
dividuals (Figures 3 and S4). While suggestive, this is not conclusive
evidence for the distinctiveness of West African elephants (a larger
sample set is required). However, the Benin/Dahomey Gap sepa-
rating the Congolian from Guinean forest blocks may hinder gene
flow in forest elephants, as it has affected the distribution of other
forest- dwelling taxa (Linder, 2014). For this reason, we previously
recommended that a conservative approach would treat elephants
on either side of the Gap as deserving of separate conservation sta-
tus (Roca et al., 2015). Whether Guinean and Congolian forest ele-
phants form genetically distinctive groups (based on nuclear DNA
analysis) remains one of the most important unanswered questions
in elephant conservation genetics (Roca et al., 2015).
4.2 | Discordant mitonuclear patterns and the
role of range expansion
Mitochondrial DNA patterns have previously been examined in
African forest elephants (Debruyne, 2005; Debruyne et al., 2003;
Egger t et al., 20 02; Ishida et al., 2013; Johnson et al., 20 07; Ny akaana
et al., 2002), revealing five mitochondrial subclades with distinctive
geographically restricted distributions (Ishida et al., 2013). In a pre-
vious study (Ishida et al., 2013), pairwise FST values calculated using
mtDNA were quite high, ranging from 0.38 to 0.87 when estimated
pairwise between localities (Table 1). However, in taxa with male-
biased dispersal, phylogeographic patterns are often discordant
betweennuclearand mitochondrial DNA (Petit & Excof fier, 2009;
Toews & Brelsford, 2012), and discordant mitonuclear patterns have
been reported among living and extinct elephantid species (Enk
et al., 2011; Lei, Brenneman, Schmitt, & Louis, 2012; Meyer et al.,
2017; Palkopoulou et al., 2015; Roca, 2015; Roca et al., 2005). This
is consistent with the current analysis, which found nuclear genetic
differentiation among forest elephants to be much lower than what
might be inferred using mtDNA alone, with all values of FST≤0.07
among forest elephant populations across Central Africa (Table 1).
We would note that the high mutation rate of mtDNA would not ac-
count for the discrepant patterns, because a faster- evolving marker
would only reveal greater resolution than a slower one; by contrast
mtDNA demonstrates a strikingly different phylogeographic pat-
tern than nuclear DNA in forest elephant s (Figure 2 vs. Figure S8)
as in other elephantids (Enk et al., 2011; Lei et al., 2012; Meyer et al.,
2017; Palkopoulou et al., 2015; Roca et al., 2005, 2007, 2015).
Mitonuclear discordant patterns in most cases have been at-
tributed to adaptive introgression of mtDNA , demographic dispar-
ities and sex- biased asymmetries, with some studies also implicating
habitat changes and hybrid zone movements ( Toews & Brelsford,
2012). In the case of forest elephants, adaptive introgression of
mtDNA is unlikely, not only because selective sweeps are unlikely
to occur in markers carried only by the nondispersing sex (Petit &
Excoffier,2009),butbecause mtDNAshowsgreaterdifferentiation
across the forest elephant range than does nuDNA ( Table 1). Instead,
sex- based differences in gene flow appear to be responsible for the
discordant mitonuclear patterns, with some impact likely due to
changes in habitat across geological time. Because female elephants
are matrilocal and remain with their natal social group (Archie et al.,
2007; Hollister- Smith et al., 2007), this behavior can account for the
persistence of geographic structuring in forest elephant mtDNA
(Ishida et al., 2013). By contrast, male elephants disperse from their
natal social groups and mediate nuclear gene flow across the land-
scape(Nyakaana&Arctander,1999;Rocaetal.,20 05,2015).
In forest elephant s, the mitonuclear patterns were likely im-
pacted by habitat changes across geological time, and discordant
mitonuclear patterns may provide a means for studying their range
expansion after the end of the last glacial period. Genetic patterns
largely depend on the demographic and ecological characteristics of
a species (C astric & Bernatchez, 2003). Spatial patterns of genetic
diversit y may also reflect past changes in climate and habitats that
expanded and contracted the ranges of species, sometimes at a
fast pace (Hewitt, 2000). Current spatial genetic diversity may re-
flect such past events rather than species demography, with geo-
graphic differences in genetic diversity reflecting the effects of past
climate- driven range dynamics (Hewitt, 2000). Range expansion can
lead to patchiness after migration, due to long- distance movements
followed by population expansions (i.e., a leptokurtic distribution of
dispersal distances during colonization) (Ibrahim, Nichols, & Hewitt,
1996;Klopfstein,Currat,&Excoffier,2006;McInerny,Turner,Wong,
Travis,&Benton, 2009).These compounded foundation processes
can lead to increased genetic differentiation, although such effects
are negatively correlated with migration rate, because migration
decreases lags in colonization and reduces the strength of founder
effects(Klopfsteinetal.,2006;McInernyetal.,2009).
In many species, it is often difficult or impossible to infer pro-
cesses that are not directly observable from the current spatial ge-
netic structure, especially as various processes may create similar
patterns(McIntire&Fajardo,2009).However,inelephantsextreme
sex differences in dispersal may allow for the study of both current
demographic effects through the examination of male- mediated nu-
clear patterns (this study), and for the study of ancient landscape
effects through analysis of mitochondrial pat terns mediated by fe-
males (Figure S8) (Ishida et al., 2013), which may retain signatures
of leptokurtic dispersal and compounded foundation processes.
Pleistocene glacial cycles caused habitat changes that temporarily
isolated populations of some species, with repeated cycles of isola-
tion followed by expansion and contraction (Hewitt, 200 0). During
periods of spatial expansion, alleles present at the expanding edge
10 
|
   ISHIDA et Al.
of the species range can reach high frequencies (Klopfstein et al.,
2006), with expanding populations potentially subject to iterated
founder effects (Klopfstein et al., 2006). During expansions, rare
long- distance dispersal events followed by exponential population
growth can generate long- term patchiness in population structure
(Hewitt , 2000; Ibr ahim etal., 1996). Such ancient e vents may be
preserved in elephant mitochondrial geographic patterns, which are
likely to be stable due to female philopatr y.
Further, studies of forest elephants would also avoid a common
pitfall of using too small a geographic scale (Jenkins et al., 2010)
while benefiting from a very large number of museum samples that
are available for mtDNA analyses and also have precise provenance
information. Species refugia during glacial cycles are better charac-
terized in Europe and North America than elsewhere, and the forest
elephant may provide novel insights into the impact of global glacial
cycles in the African tropics (Hewitt, 200 0).
4.3 | The potential role of isolation by distance
Distinguishing between discrete population genetic structure
(Figures 2 and 3) and isolation by distance (Figure 4) can be difficult
(Meirmans, 2012). Models of isolation by distance are often used
to approach the balance between drift and dispersal (Jenkins et al.,
2010;Wright,1943).Limitedmigrationpermitsgeneticdrift,increas-
ing population differentiation and leading to a correlation between
neutral genetic differentiation and geographic distance (Jenkins
etal.,2010;Wright,1943).IBDdevelopsincontinuouslydistributed
species when divergence accumulates due to genetic drift between
locations separated by geographic distances large enough to over-
comethehomogenizingeffectsofgeneflow(Chesser,1983;Crispo
&Hendry,2005;Hewitt,20 00;Jenkinsetal.,2010;Wright,1943),
and is common in natural populations (Jenkins et al., 2010). IBD can
remain at equilibrium, after sufficient time has elapsed for genetic
patterns to be established and stabilized (Castric & Bernatchez,
2003).
Forest elephants have been contiguously distributed across
Central Africa from the start of the Holocene (Plana, 20 04), until
atleast themid-to late 1900s (Douglas-Hamilton, 1987). The cor-
relation between genetic and geographic distances among localities
in Central Africa (Figure 4) may be attributable to isolation by dis-
tance (IBD)(Jenkinsetal., 2010; Wright, 1943).However,because
forest elephant populations expanded from discrete glacial refugia,
it would be difficult to distinguish a role of IBD from the persistence
of discrete population structure (Meirmans, 2012) that would have
been diminished but perhaps not erased by postglacial gene flow.
4.4 | Conservation implications
Given the endangered status of forest elephants, and their role as
a keystone species, discussion of the conser vation implications of
our results is warranted. An import ant step for the conservation of
forest elephants would be universal recognition of its status as a
separate species in need of species- specific conservation measures
(Roca et al., 2015). Central Africa, the region in which most forest el-
ephants live, has suf fered from the highest levels of elephant poach-
ing of any subregion (CITES, 2012) and has been the main source for
the illegal trade in elephant bushmeat and ivory ( Wasser et al., 2015)
leading to massive declines in their numbers (Maisels et al., 2013).
The increase in human numbers and activities has caused fragmen-
tation of elephant habitats and range (Blake et al., 2007, 2008). Such
fragmentation reduces genetic connectivit y, which can lead to loss
of genetic variation, and ultimately to inbreeding and increased drift.
Recognition both of species divisions and of population genetic
patterns below the species level is essential for the maintenance of
biodiversity, and an important conser vation principle is to retain
populat ions representi ng existing genet ic variation (Mori tz, 1994;
Ryder, 1986). Our findings suggest that West African, western
Congolian, and eastern Congolian forest elephants should be man-
aged separately. For species such as the forest elephant that exhibit
patterns indicative of limited population structure (Figures 2 and
3) and possibly isolation by distance (Figure 4), Chesser (Chesser,
1983)hassuggesteddividingtherangeintomanagementunits,with
greater genetic exchanges within units than across units in order to
balance the need for connectivity with the need to prevent loss of al-
leles. Within regions, it is important to prevent ex treme habitat frag-
mentation and retain connec tivity so that gene flow can continue
amongpopulations(Chesser,1983).Shouldthedestructionofele-
phants cease while habitats remain, populations could expand from
multiple locations. Should active management become necessary for
recolonization, the best source populations for translocations would
be those that are geographically close, as these would be most simi-
lar to any extirpated population (Monsen & Blouin, 20 04).
Forest elephants play a critical role in shaping their ecosystem,
maintaining tree diversity, dispersing seeds in greater quantities
and distances than most other fauna, and improving rates of seed
germination following passage through the gut (Campos- Arceiz &
Blake, 2011). In the central Congo Basin, seventy- eight percent of
the larger tree species in the rain forest are dependent on forest ele-
phants for seed dispersal (Blake et al., 2007). Thus, it is important to
consider their ecological role in seed germination and dispersal when
determining conservation priorities. Terrestrial ecoregions of the
world have been mapped to identif y areas of high biodiversity and
representative communities (Olson et al., 2001). The range encom-
passed by Central African forest elephants includes five tropical for-
est ecoregions, three ecoregions of forest–savanna mosaic, as well
as the Albertine Rift montane forests and Cameroonian highlands
forests(FigureS9)(Olson etal., 2001). Given limitedgeneticdiffer-
entiation among elephants across the Congo Basin, these ecoregions
could ser ve as management units for the forest elephants, reflecting
the dependence of many plant species on elephants for seed ger-
mination and dispersal. Within regions, the extirpation of local ele-
phant populations should be minimized, to minimize effects on the
regional flora.
An impor tant further consideration is the impact of forest ele-
phant conservation on those plant species that are rare or regionally
restricted. A survey of 5,881 species of plants across sub- Saharan
    
|
 11
ISHIDA et A l.
Africa has been used to map endemism richness, which is defined as
the sum of species present at a geographic loc ation, but with the occur-
rence of each plant species inversely weighted by the size of its range
(Figure S10) (Linder, 2014; Linder et al., 2012). The endemic richness of
plants has been found to be highly congruent with the endemic rich-
ness of frogs, snakes, birds, and mammals, which when combined indi-
cated that the Benin Gap has influenced these patterns (Linder, 2014).
Congruence across these groups has been attributed to (1) a similar
influence across ver tebrates of the vegetation and flora, (2) common
responses to the same climatic parameters, and (3) a common underly-
ing histor y (Linder, 2014). For plants, regions with highest levels of en-
demism have been identified (Linder, 2014; Linder et al., 2012) and are
indicated in Figure S10. These regions of endemism may be considered
when setting conservation priorities for forest elephants, although a
more precise mapping of endemism among those plants that are de-
pendent on elephants would be helpful. Conserving elephants in the
regions rich in plant endemism would directly benefit the conservation
of many plant species dependent on elephants, and indirectly benefit
other vertebrate and nondependent plant species for which levels of
endemism are geographically congruent.
ACKNOWLEDGMENTS
The work was funded by USFWS African Elephant Conservation
Fund Grants AFE-0778-F12AP01143 and AFE1606-F16AP00909.
The study was condu cted under the Universit y of Illinois Institu tional
Animal Care and Use Committee (IACUC) approved protocol number
12040. Samples were impor ted using appropriate CITES permits.
For technic al or other assistance, we thank M. Malask y, R. Hanson,
and the UIUC High- Throughput Sequencing and Genotyping Unit.
We are grateful to the governments of Bot swana, Cameroon,
Central African Republic, Democratic Republic of Congo, Gabon,
Kenya, Namibia, Republic of Congo, South Africa, Tanzania, and
Zimbabwe for permission to collect samples. For help with sam-
ple collection, we thank A. Turkalo, M. Fay, L. White, R. Weladji,
W. Karesh, M. Lindeque, W. Versvelt, K. Hillman Smith, F. Smith,
M. Tchamba, S. Gartlan, P. Aarhaug, A. M. Austmyr, Bakari, Jibrila,
J. Pelleteret, L. White, M. Habibou, M. W. Beskreo, D. Pierre, C.
Tutin, M. Fernandez, R. Barnes, B. Powell, G. Doungoubé, M. Storey,
M. Phillips, B. Mwasaga, A. Mackanga- Missandzou, K. Amman, K.
Comstock, M. Keele, D. Olson, B. York, and A. Baker at the Burnet
Park Zoo, M. Bush at the National Zoological Park, and A. Lécu at
Zoo de Vincennes (Paris Zoo).
CONFLICT OF INTEREST
None declared.
AUTHOR CONTRIBUTIONS
YI and ALR designed the study. YI and NAG performed experiment s
and analyses. NJG provided samples. YI, NJG, and ALR contributed
to writing the manuscript.
DATA ACCESSIBILITY
The sequences of microsatellites used in this manuscript were estab-
lishedanddepositedatNCBIGenBank(KU947083–KU947105),and
primer sequences are available in Gugala et al. (Gugala et al., 2016).
ORCID
Alfred L. Roca http://orcid.org/0000-0001-9217-5593
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SUPPORTING INFORMATION
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How to cite this article: Ishida Y, Gugala NA, Georgiadis NJ,
Roca AL. Evolutionary and demographic processes shaping
geographic patterns of genetic diversit y in a keystone
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Evol. 2018;00:1–13. https://doi.org/10.1002/ece3.4062

Supplementary resource (1)

... But dispersing males cannot propagate mtDNA because offspring of both sexes inherit mtDNA only from their mothers. Therefore, mutations in mtDNA are likely to persistently remain geographically localized (Roca et al. 2015;Ishida et al. 2018). The genetic diversity of mtDNA may be slower to erode among populations than nuclear DNA diversity, because the latter would be affected by a high degree of male-male competition among elephants (Petit and Excoffier 2009;Roca et al. 2015). ...
... Discrepant mtDNA and nuclear DNA phylogeographic patterns suggest that analyses including both nuclear and mitochondrial genetic markers would provide improvements in the accuracy and precision of geographic assignment of provenance, and that mtDNA data can reduce uncertainty of assignment for some samples examined using nuclear DNA (Ishida et al. 2013). The LL could also be used to examine in greater detail the evolutionary history of African elephants, which were heavily impacted by climate and habitat changes during the Pleistocene (Roca et al. 2015;Ishida et al. 2018). These events likely shaped phylogeographic patterns in mtDNA, which continue to persist because females do not disperse (Roca et al. 2015;Ishida et al. 2018). ...
... The LL could also be used to examine in greater detail the evolutionary history of African elephants, which were heavily impacted by climate and habitat changes during the Pleistocene (Roca et al. 2015;Ishida et al. 2018). These events likely shaped phylogeographic patterns in mtDNA, which continue to persist because females do not disperse (Roca et al. 2015;Ishida et al. 2018). ...
Article
Illegal hunting is a major threat to the elephants of Africa, with more elephants killed by poachers than die from natural causes. DNA from tusks has been used to infer the source populations for confiscated ivory, relying on nuclear genetic markers. However, mitochondrial DNA (mtDNA) sequences can also provide information on the geographic origins of elephants due to female elephant philopatry. Here, we introduce the Loxodonta Localizer (LL; www.loxodontalocalizer.org), an interactive software tool that uses a database of mtDNA sequences compiled from previously published studies to provide information on the potential provenance of confiscated ivory. A 316 bp control region sequence, which can be readily generated from DNA extracted from ivory, is used as a query. The software generates a listing of haplotypes reported among 1917 African elephants in 24 range countries, sorted in order of similarity to the query sequence. The African locations from which haplotype sequences have been previously reported are shown on a map. We demonstrate examples of haplotypes reported from only a single locality or country, examine the utility of the program in identifying elephants from countries with varying degrees of sampling, and analyze batches of confiscated ivory. The LL allows for the source of confiscated ivory to be assessed within days, using widely available molecular methods that do not depend on a particular platform or laboratory. The program enables identification of potential regions or localities from which elephants are being poached, with capacity for rapid identification of populations newly or consistently targeted by poachers.
... There are a number of reputed geographic differences in the morphology of forest and of savanna elephants within their respective species ranges. For example, the desert dwelling elephants of Namibia are said to be taller and leaner, with longer legs and larger feet than other populations of savanna elephants, and they have been proposed as a distinct subspecies (Ishida et al. 2018). Several taxa of pygmy elephants have been said to inhabit the tropical forests of Africa (Frade 1955;Groves and Grubb 2000a, b;Debruyne et al. 2003). ...
... Likewise, there is evidence from the distribution of indels (insertion-deletion variants) that gene flow has been extensive among the forest elephants across central Africa (Roca et al. 2001. The estimated F ST between forest elephant populations in the eastern Congolian forest block and those in the western Congolian forest block is just 0.035 (Ishida et al. 2018), and populations across the central African rainforest do not display great genetic distinctiveness (figure 2) (Ishida et al. 2018). These low estimates for limited geographic differentiation within each species are supported by various studies using different types of nuclear DNA data (Roca et al. 2001;Ishida et al. 2011;Ahlering et al. 2012). ...
... Likewise, there is evidence from the distribution of indels (insertion-deletion variants) that gene flow has been extensive among the forest elephants across central Africa (Roca et al. 2001. The estimated F ST between forest elephant populations in the eastern Congolian forest block and those in the western Congolian forest block is just 0.035 (Ishida et al. 2018), and populations across the central African rainforest do not display great genetic distinctiveness (figure 2) (Ishida et al. 2018). These low estimates for limited geographic differentiation within each species are supported by various studies using different types of nuclear DNA data (Roca et al. 2001;Ishida et al. 2011;Ahlering et al. 2012). ...
Article
During the last two decades, our understanding of the genetics of African elephant populations has greatly increased. Strong evidence, both morphological and genetic, supports recognition of two African elephant species: the savanna elephant (Loxodonta africana) and the forest elephant (L. cyclotis). Among elephantids, phylogeographic patterns for mitochondrial DNA are highly incongruent with those detected using nuclear DNA markers, and this incongruence is almost certainly due to strongly male-biased geneflow in elephants. As our understanding of elephant population genetics has grown, a number of observations may be considered enigmatic or anomalous. Here, several of these are discussed. (i) There are a number of within-species morphological differences purported to exist among elephants in different geographic regions, which would be difficult to reconcile with the low genetic differentiation among populations. (ii) Forest elephants have a higher effective population size than savanna elephants, with nuclear genetic markers much more diverse in the forest elephants than savanna elephants, yet this finding would need to be reconciled with the life history of the two species. (iii) The savanna and forest elephants hybridize and produce fertile offspring, yet full genome analysis of individuals distant from the hybrid zone suggests that gene flow has been effectively sterilized for atleast ∼500,000 years. (iv) There are unexplored potential ramifications of the unusual mito-nuclear patterns among elephants. These questions are considered in light of highmale and low female dispersal in elephants, higher variance of reproductive success among males than females, and of habitat changes driven by glacial cycles and human activity.
... Ref. 40 . As a result, the mtDNA phylogeography differs from the nuclear one, as the mitochondrial genome is tied to the geographic range of the herd, whereas nDNA phylogeography is subject to the male-mediated gene flow among herds 13,41 . The fact that our specimen was identified as female, strengthens its provenance estimation. ...
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Abstract Molecular species identification plays a crucial role in archaeology and palaeontology, especially when diagnostic morphological characters are unavailable. Molecular markers have been used in forensic science to trace the geographic origin of wildlife products, such as ivory. So far, only a few studies have applied genetic methods to both identify the species and circumscribe the provenance of historic wildlife trade material. Here, by combining ancient DNA methods and genome skimming on a historical elephantid tooth found in southwestern Portugal, we aimed to identify its species, infer its placement in the elephantid phylogenetic tree, and triangulate its geographic origin. According to our results the specimen dates back to the eighteenth century CE and belongs to a female African forest elephant (non-hybrid Loxodonta cyclotis individual) geographically originated from west—west-central Africa, from areas where one of the four major mitochondrial clades of L. cyclotis is distributed. Historical evidence supports our inference, pointing out that the tooth should be considered as post-Medieval raw ivory trade material between West Africa and Portugal. Our study provides a comprehensive approach to study historical products and artefacts using archaeogenetics and contributes towards enlightening cultural and biological historical aspects of ivory trade in western Europe.
... But in recent years, fragmentation has escalated across their range largely restricting many mega-herbivores to protected areas (Graham et al., 2009;Jenkins & Joppa, 2009), which represent fragments of the once continuous historic ranges (Ripple et al., 2015). Habitat fragmentation and illegal hunting for ivory may lead to inbreeding depression (Allendorf, Luikart, & Aitken, 2013;Ishida, Gugala, Georgiadis, & Roca, 2018) and loss of genetic variation (Gobush, Kerr, & Wasser, 2009;Wasser et al., 2015), especially when the oldest individuals (who are often the target) are involved (Archie et al., 2008). This poses a question of whether populations that once ranged across the continent are becoming genetically isolated because of ongoing habitat destruction, fragmentation and illegal killings. ...
Preprint
Habitat fragmentation plays a major role in the reduction of genetic diversity among wildlife populations. The African savannah elephant population of the Ruaha-Rungwa and Katavi-Rukwa ecosystems in south-western Tanzania, comprises one of the world’s largest remaining elephant populations, but is increasingly threatened by loss of connectivity and poaching for ivory. We investigate whether there are incipient signs of genetic isolation (loss of heterozygosity) within the younger cohort as a result of habitat loss between the two ecosystems. To investigate the genetic structure of populations, we compared the genotypes for 11 microsatellite loci in the western (n = 81 individuals from Katavi-Rukwa), central (n = 36 individuals from Lukwati and Piti), and eastern populations (n = 193, individuals from Ruaha-Rungwa). We found evidence of significant genetic differentiation among the three populations, but the levels were low, suggesting recent divergence. Furthermore, we identified weak isolation by distance, suggesting higher gene flow among nearer individuals with samples within 50km of each other being more genetically similar to one another than beyond. Although sample sizes were small, a further analysis of genetic differences across populations and in separate age classes revealed evidence of increasing genetic structure among younger age classes across the landscape. In a long-lived species with overlapping generations, it takes a long time to develop genetic substructure even when there are substantial obstacles to migration. Thus, in these recently fragmented populations, inbreeding (and the loss of heterozygosity) may be less of an immediate concern than demography (the loss of adults due to illegal hunting).
... However, population genetic analysis carried out in a forensic context is increasingly being applied in large African mammals, for example in both the forest and savannah elephant to identify the origins of seized animal products [44] and to identify demographic units for conservation management (e.g. [45]). Forensic studies require large genetic reference databases, thus a large and growing number of white rhinoceros have been routinely genotyped for forensic purposes [46], and we advocate making use of this unique genetic resource to aid the management of genetic diversity. ...
Article
The white rhinoceros (Ceratotherium simum) has a discontinuous African distribution, which is limited by the extent of sub-Saharan grasslands. The southern population (SWR) declined to its lowest number around the turn of the nineteenth century, but recovered to become the world's most numerous rhinoceros. In contrast, the northern population (NWR) was common during much of the twentieth century, declining rapidly since the 1970s, and now only two post-reproductive individuals remain. Despite this species's conservation status, it lacks a genetic assessment of its demographic history. We therefore sampled 232 individuals from extant and museum sources and analysed ten microsatellite loci and the mtDNA control region. Both marker types reliably partitioned the species into SWR and NWR, with moderate nuclear genetic diversity and only three mtDNA haplotypes for the species, including historical samples. We detected ancient interglacial demographic declines in both populations. Both populations may also have been affected by recent declines associated with the colonial expansion for the SWR, and with the much earlier Bantu migrations for the NWR. Finally, we detected post-divergence secondary contact between NWR and SWR, possibly occurring as recently as the last glacial maximum. These results suggest the species was subjected to regular periods of fragmentation and low genetic diversity, which may have been replenished upon secondary contact during glacial periods. The species's current situation thus reflects prehistoric declines that were exacerbated by anthropogenic pressure associated with the rise of late Holocene technological advancement in Africa. Importantly, secondary contact suggests a potentially positive outcome for a hybrid rescue conservation strategy, although further genome-wide data are desirable to corroborate these results. © 2018 The Author(s) Published by the Royal Society. All rights reserved.
... However, population genetic analysis carried out in a forensic context is increasingly being applied in large African mammals, for example in both the forest and savannah elephant to identify the origins of seized animal products [44] and to identify demographic units for conservation management (e.g. [45]). Forensic studies require large genetic reference databases, thus a large and growing number of white rhinoceros have been routinely genotyped for forensic purposes [46], and we advocate making use of this unique genetic resource to aid the management of genetic diversity. ...
Article
The white rhinoceros (Ceratotherium simum) has a discontinuous African distribution, which is limited by the extent of sub-Saharan grasslands. The southern population (SWR) declined to its lowest number around the turn of the nineteenth century, but recovered to become the world's most numerous rhinoceros. In contrast, the northern population (NWR) was common during much of the twentieth century, declining rapidly since the 1970s, and now only two post-reproductive individuals remain. Despite this species's conservation status, it lacks a genetic assessment of its demographic history. We therefore sampled 232 individuals from extant and museum sources and analysed ten microsatellite loci and the mtDNA control region. Both marker types reliably partitioned the species into SWR and NWR, with moderate nuclear genetic diversity and only three mtDNA haplotypes for the species, including historical samples. We detected ancient interglacial demographic declines in both populations. Both populations may also have been affected by recent declines associated with the colonial expansion for the SWR, and with the much earlier Bantu migrations for the NWR. Finally, we detected post-divergence secondary contact between NWR and SWR, possibly occurring as recently as the last glacial maximum. These results suggest the species was subjected to regular periods of fragmentation and low genetic diversity, which may have been replenished upon secondary contact during glacial periods. The species's current situation thus reflects prehistoric declines that were exacerbated by anthropogenic pressure associated with the rise of late Holocene technological advancement in Africa. Importantly, secondary contact suggests a potentially positive outcome for a hybrid rescue conservation strategy, although further genome-wide data are desirable to corroborate these results.
... But in recent years, fragmentation has escalated across their range largely restricting many mega-herbivores to protected areas (Graham et al., 2009;Jenkins & Joppa, 2009), which represent fragments of the once continuous historic ranges (Ripple et al., 2015). Habitat fragmentation and illegal hunting for ivory may lead to inbreeding depression (Allendorf, Luikart, & Aitken, 2013;Ishida, Gugala, Georgiadis, & Roca, 2018) and loss of genetic variation (Gobush, Kerr, & Wasser, 2009;Wasser et al., 2015), especially when the oldest individuals (who are often the target) are involved (Archie et al., 2008). This poses a question of whether populations that once ranged across the continent are becoming genetically isolated because of ongoing habitat destruction, fragmentation and illegal killings. ...
Article
Habitat fragmentation can play a major role in the reduction of genetic diversity among wildlife populations. The Ruaha-Rungwa and Katavi-Rukwa ecosystems in south-western Tanzania comprise one of the world's largest remaining African savannah elephant metapopulations but are increasingly threatened by loss of connectivity and poaching for ivory. To investigate the genetic structure of populations, we compared the genotypes for nine microsatellite loci in the western, central and eastern populations. We found evidence of genetic differentiation among the three populations, but the levels were low and mostly concerned the younger cohort, suggesting recent divergence probably resulting from habitat loss between the two ecosystems. We identified weak isolation by distance, suggesting higher gene flow among individuals located less than 50 km apart. In a long-lived species with overlapping generations, it takes a long time to develop genetic substructure even when there are substantial obstacles to migration. Thus, in these recently fragmented populations, inbreeding (and the loss of heterozygosity) may be less of an immediate concern than the loss of adults due to illegal hunting. 2018 John Wiley & Sons Ltd.
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Asian elephant (Elephas maximus) plays a significant role in natural ecosystems and it is considered as an endangered animal. Molecular genetics studies on elephants’ dates back to 1990s. Microsatellite markers have been the preferred choice and have played a major role in ecological, evolutionary and conservation research on elephants over the past 20 years. However, technical constraints especially related to the specificity of traditionally developed microsatellite markers have brought to question their application, specifically when degraded samples are utilized for analysis. Therefore, we analyzed the specificity of 24 sets of microsatellite markers frequently used for elephant molecular work. Comparative wet lab analysis was done with blood and dung DNA in parallel with in silico work. Our data suggest cross-amplification of unspecific products when field-collected dung samples are utilized in assays. The necessity of Asian elephant specific set of microsatellites and or better molecular techniques are highlighted.
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The Asian elephant is at risk of extinction due to anthropogenic pressures, and remaining populations are often small and fragmented remnants, occupying a fraction of the species’ former range. Once widely distributed across China, only a maximum of 245 elephants are estimated to survive across seven small populations. We assessed the Asian elephant population in Nangunhe National Nature Reserve in Lincang Prefecture, China using camera traps between May to July 2017, to estimate the population size and structure of thisgenetically important population. Our results indicate that whilst detection probability was 29 low (0.31), we estimated a total population size of approximately 20 individuals, and an effective density of 0.39 elephants per km2. Social structure indicated a strong sex ratio bias towards females, with only one adult male detected within the population. Most of the elephants associated as one herd but three adult females remained separate from the herd throughout the trapping period. These results highlight the fragility of remnant elephant populations such as Nangunhe and we suggest options such as a managed metapopulation approach for their continued survival in China and more widely.
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The straight-tusked elephants Palaeoloxodon spp. were widespread across Eurasia during the Pleistocene. Phylogenetic reconstructions using morphological traits have grouped them with Asian elephants ( Elephas maximus ), and many paleontologists place Palaeoloxodon within Elephas . Here, we report the recovery of full mitochondrial genomes from four and partial nuclear genomes from two P. antiquus fossils. These fossils were collected at two sites in Germany, Neumark-Nord and Weimar-Ehringsdorf, and likely date to interglacial periods ~120 and ~244 thousand years ago, respectively. Unexpectedly, nuclear and mitochondrial DNA analyses suggest that P. antiquus was a close relative of extant African forest elephants ( Loxodonta cyclotis ). Species previously referred to Palaeoloxodon are thus most parsimoniously explained as having diverged from the lineage of Loxodonta , indicating that Loxodonta has not been constrained to Africa. Our results demonstrate that the current picture of elephant evolution is in need of substantial revision.
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Background African elephants comprise two species, the savanna elephant (Loxodonta africana) and the forest elephant (L. cyclotis), which are distinct morphologically and genetically. Forest elephants are seriously threatened by poaching for meat and ivory, and by habitat destruction. However, microsatellite markers have thus far been developed only in African savanna elephants and Asian elephants, Elephas maximus. The application of microsatellite markers across deeply divergent lineages may produce irregular patterns such as large indels or null alleles. Thus we developed novel microsatellite markers using DNA from two African forest elephants. Findings One hundred microsatellite loci were identified in next generation shotgun sequences from two African forest elephants, of which 53 were considered suitable for testing. Twenty-three microsatellite markers successfully amplified elephant DNA without amplifying human DNA; these were further characterized in 15 individuals from Lope National Park, Gabon. Three of the markers were monomorphic and four of them carried only two alleles. The remaining sixteen polymorphic loci carried from 3 to 8 alleles, with observed heterozygosity ranging from 0.27 to 0.87, expected heterozygosity from 0.40 to 0.86, and the Shannon diversity index from 0.73 to 1.86. Linkage disequilibrium was not detected between loci, and no locus deviated from Hardy–Weinberg equilibrium. Conclusions The markers developed in this study will be useful for genetic analyses of the African forest elephant and contribute to their conservation and management. Electronic supplementary material The online version of this article (doi:10.1186/s13104-016-2167-3) contains supplementary material, which is available to authorized users.
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This chapter reviews the role that kinship plays in the spatial and social relationships that occur among adult female African elephants in Amboseli. First, it describes the range of genetic relationships that occur among adult females that live in the same family group. Long-term observations indicate that nearly all female elephants are matrilocal. The chapter then describes the correlation between kinship and the patterns of fission and fusion within families. The degree to which kin are predictably together in the same group determines the opportunities that individuals have to influence their indirect fitness. Next, the chapter moves beyond family-level association patterns and describes the degree to which kinship predicts fission and fusion between family groups across the population. Finally, it investigates whether kinship influences affiliative, cooperative, and competitive social relationships by first discussing whether female elephants seem to discriminate among affiliative and cooperative social partners on the basis of kinship and then investigating whether kinship predicts dominance-rank relationships.
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We describe a model-based clustering method for using multilocus genotype data to infer population structure and assign individuals to populations. We assume a model in which there are K populations (where K may be unknown), each of which is characterized by a set of allele frequencies at each locus. Individuals in the sample are assigned (probabilistically) to populations, or jointly to two or more populations if their genotypes indicate that they are admixed. Our model does not assume a particular mutation process, and it can be applied to most of the commonly used genetic markers, provided that they are not closely linked. Applications of our method include demonstrating the presence of population structure, assigning individuals to populations, studying hybrid zones, and identifying migrants and admixed individuals. We show that the method can produce highly accurate assignments using modest numbers of loci—e.g., seven microsatellite loci in an example using genotype data from an endangered bird species. The software used for this article is available from http://www.stats.ox.ac.uk/~pritch/home.html.
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Male elephants interact frequently with other elephants. Young males face feeding competition from females and other males, and all males compete over access to females. Rates of aggression among mature adult males tend to be high. Competition obviously affects male grouping and associations, which are explored further in this chapter. This chapter first addresses how young, sexually active, non-musth males associate with other elephants and then discusses how being an older musth male affects sociality. Early in life, males have high energy costs due to growth and a high rate of mortality; with age, the growth costs diminish, but competitive costs increase. The chapter explores social dynamics from a developmental perspective—as a process of long duration—and with respect to the attainment of full socio-sexual functioning.
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The African elephant consists of forest and savanna subspecies. Both subspecies are highly endangered due to severe poaching and habitat loss, and knowledge of their population structure is vital to their conservation. Previous studies have demonstrated marked genetic and morphological differences between forest and savanna elephants and despite extensive sampling, genetic evidence of hybridization between them has been restricted largely to a few hybrids in the Garamba region of northeastern Democratic Republic of Congo (DRC). Here we present new genetic data on hybridization from previously unsampled areas of Africa. Novel statistical methods applied to these data identify 46 hybrid samples - many more than have been previously identified - only two of which are from the Garamba region. The remaining 44 are from three other geographically-distinct locations: a major hybrid zone along the border of the DRC and Uganda, a second potential hybrid zone in Central African Republic, and a smaller fraction of hybrids in the Pendjari-Arli complex of West Africa. Most of the hybrids show evidence of interbreeding over more than one generation, demonstrating that hybrids are fertile. Mitochondrial and Y chromosome data demonstrate that the hybridization is bidirectional, involving males and females from both subspecies. We hypothesize that the hybrid zones may have been facilitated by poaching and habitat modification. The localized geography and rarity of hybrid zones, their possible facilitation from human pressures, and the high divergence and genetic distinctness of forest and savanna elephants throughout their ranges, are consistent with calls for separate species classification. This article is protected by copyright. All rights reserved.