Cyto-nuclear genomic dissociation in African elephant species
Alfred L. Roca*, Nicholas Georgiadis‡ and Stephen J. O’Brien†
*Basic Research Program, SAIC-Frederick, and †Laboratory of Genomic Diversity, National Cancer Institute, Frederick, MD 21702, USA
‡Mpala Research Center, PO Box 555, Nanyuki, Kenya
FIGURE 1: INTRODUCTION (A) mtDNA ND5
(B) Y chromosome AMELY
Multiple lines of evidence suggest that forest and savanna elephants are distinct species. FIGURE 3 DS (5)
Measurements from 295 skulls demonstrated that forest and savanna elephants fall into two morphologically DS (11)
BE (1) BE4059
distinct groups (left panel; Groves and Grubb, 2000). Nuclear gene analyses using both slower-evolving Maternally and paternally GR (1) 63/80/64 DS1504
nuclear gene sequences (center; Roca et al., 2001) and more rapidly evolving microsatellites (right; Comstock inherited markers demonstrate DS (8)
et al., 2002) demonstrated a deep genetic split between forest and savanna elephants, estimated at 3.5 million
62/74/78 DS (1), OD (2) BE (2), WA (1) AM0003
Phylogenetic relationships for Asian,
years. Only a few morphological intermediates and genetic hybrids were detected in a zone of mixed habitat
CH (1), HW (6), SA (8), ZZ (4) AM0023
that surrounds the tropical forests of Africa. African forest, and African savanna HW (2), NG (4), SA (2), SE (9), TA (1), ZZ (1)
In contrast to the distinctions between forest and savanna elephants detected by morphological and nuclear elephants inferred from (A) 319 bp of 89/92/97 GR (10)
genetic studies, analyses using mitochondrial DNA (mtDNA) have detected genetic diversity in savanna the maternally inherited II HW0062
elephants high enough to appear incongruent with nuclear DNA studies, and suggested greater mixing mitochondrial ND5 gene (number of DS (1), LO (9), OD (1)
BE (1), WA (1)
between forest and savanna elephants. To investigate this apparent disparity, we sampled wild elephant tissue individuals with identical haplotypes 94/96/98
from 21 African locations to determine the DNA sequences for 1642 biparentally inherited chromosomal indicated by location), and from (B) BE (1), WA (2)
segments in 3 X-linked genes, and 302 mtDNA and 128 Y chromosome sequences. 1551 bp of the paternally inherited Y KR0114
chromosome gene AMELY (each 90/91/87
AB (6), AM (34), CH (5), KE (16), KR (12) MA (1),
MK (1), NA (21), NG (3), SE (1), SW (5), TA (6)
individual shown separately). Forest 70/66/63 AB (1), AM (2), KE (4)
populations or individuals are CH (3), HW (4), KR (25), MA (3), SW (5)
indicated in green; savanna in blue; 100/100/100
Asian elephants in red. Garamba AB (1), KE (17), WA (4)
(GR) is a mixed habitat zone and is GR (1) TA1144
BE (1), WA (1)
Elephas maximus (1) 0.001 substitutions/site
mtDNA biparental Y chro.
mtDNA biparental Y chro. FIGURE 4
LO WA KE
n=11 n=65 n=2
BE n=58 n=280 n=15 Distribution of cytonuclear disequilibrium.
3 7 1
AM Pie charts indicate by locale the distribution of
R 35 149 17
genetic markers that are inherited maternally
OD AB SA
SE AM 4
11 154 11
(left pie chart in each set of three), paternally
GR NG TA
8 71 13
(right), or biparentally (center). Totals indicate
the number of individuals (mtDNA, Y
Savannah elephants Savannah elephants Savannah elephants Forest elephants
SOUTHERN EASTERN NORTH-CENTRAL DS-Dzanga Sangha
10 46 2
chromosomes) or combined number of
CH-Chobe AB-Aberdares BE-Benoue LO-Lope WA MA
5 94 3 ZZ SW 4 56 1
chromosome segments (biparental genes)
NA-Namibia KE-Central Kenya NA CH HW
16 microsatellite loci
SW-Sengwa TA-Tarangire Comstock et al. 2002 MA
9 44 7
KR 37 170 2
Map indicates locations of sampled elephant
21 157 15
populations in Africa. Green circles are forest
TA ND5 BGN
AMELY locations. Blue circles are savanna locations.
6 67 7
ND5 BGN AMELY
Garamba (GR) includes both habitats. Orange
PHKA2 indicates current African elephant range; historic
DS-Dzanga Sangha, LO-Lope, OD-Odzala, GR-Garamba, AB-Aberdares, AM- range includes entire land area shown.
Amboseli, BE-Benoue, CH-Chobe, HW-Hwange, KE-Central Kenya, KR-Kruger,
MA-Mashatu, MK-Mount Kenya, NA-Namibia, NG-Ngorongoro, SA-Savuti, SE-
METHODS Serengeti, SW-Sengwa, TA-Tarangire, WA-Waza, ZZ-Zambezi.
DNA was extracted from samples from wild African elephants and captive Asian elephants (Elephas maximus). Three nuclear
gene segments (BGN, PHKA2 and PLP); a portion of the mitochondrial gene ND5, and a Y-chromosome gene fragment (AMELY)
were amplified and sequenced. Sequences were aligned using CLUSTALX. Phylogenetic analyses were performed using
FIGURE 5: CONCLUSIONS
maximum parsimony (MP), neighbor joining (NJ), and maximum likelihood (ML) methods implemented in PAUP*4.0b10.
Cytonuclear disequilibrium suggests historic unidirectional hybridization (i.e., savanna males and forest
females) with subsequent unidirectional backcrossing to larger reproductively successful savanna males,
swamping the forest nuclear genomic contribution. The interactions between forest and savanna elephants
(A) (B) (C)
Forest inferred from differing patterns detected by maternally-inherited versus paternally- or biparentally-inherited
genes are as follows: Herd#2
A Herd#1 A
(A) Male-mediated gene flow occurs between adjacent forest elephant B
Asian herds, and between adjacent savanna elephant herds; however (B) habitat
interbreeding between savanna and forest elephants at the contact zone C
between forest and savanna habitats is rare. (C) As forest habitat
retreats (or when forest herds move into savanna habitats), larger male
Savanna savanna elephants have increased opportunity to hybridize with forest
female elephants. However, (D) the smaller forest and hybrid males do
not reproduce due either to outbreeding depression or to reproductive
BGN, 646 bp, n=556 PHKA2, 1012 bp, n=440 PLP, 479 bp, n=657
dominance by larger unhybridized savanna males. (E) After multiple
generations of unidirectional hybridization, nuclear genes alleles are
FIGURE 2 those of savanna elephants, although a forest mitochondrial haplotype is
retained in the now-savanna herds.
Haplotypes for three biparentally-inherited nuclear genes display almost complete separation among E Forest mtDNA,
savanna nuclear DNA
and nuclear DNA
three elephant taxa. MP trees are shown. The length of each gene segment and number of chromosomes
References and Acknowledgments
examined are indicated for each gene. Number of chromosomes per haplotype is proportional to the size of
Comstock, K. E., Georgiadis, N., Pecon-Slattery, J., Roca, A. L., Ostrander, E. A., O'Brien, S. J. and Wasser, S. K. (2002) Patterns of molecular genetic variation among African elephant populations. Mol Ecol.11:2489-98.
circles; differences between alleles are proportional to the distance between circles. Haplotypes/alleles found Groves, C. P. and Grubb, P. (2000) Do Loxodonta cyclotis and L. africana interbreed? Elephant 2(4):4-7.
Roca, A. L., Georgiadis, N., Pecon-Slattery, J. and O'Brien, S. J. (2001) Genetic evidence for two species of elephant in Africa. Science 293(5534):1473-7.
in Asian elephants are red; African forest haplotypes are green; and African savanna haplotypes are blue. (A) Roca, A. L., Georgiadis, N. and O'Brien, S. J. (2003) Male-driven genomic chimerization of elephant herds in Africa. In preparation.
BGN haplotypes are completely distinct between forest and savanna populations. (B) PHKA2 proved to be the We thank R. Ruggiero, W. J. Murphy, E. Eizirik, A. Brandt, M. P. Gough, B. Gough, M. J. Malasky, J. Arthur, R. L. Hill, D. Munroe, S. Cevario, N. J. Crumpler, G. K. Pei, K. M. Helgen. For elephant samples, we thank A.
Turkalo, J. M. Fay, 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.
most diverse nuclear gene segment; the chromosomes examined were completely distinct between forest and Pierre, C. Tutin, M. Fernandez, R. Barnes, B. Powell, G. Doungoubé, M. Storey, M. Phillips, B. Mwasaga, A. Mackanga-Missandzou, B. York and A. Baker at the Burnet Park Zoo, and M. Bush at the National Zoological
Park. We thank the governments of Botswana, Cameroon, the Central African Republic, Congo (Brazzaville), Congo (Kinshasa), Gabon, Kenya, Namibia, South Africa, Tanzania, and Zimbabwe for permission to collect
savanna populations.(C) PLP haplotypes were distinct between forest and savanna elephants except for one samples. Tissues were obtained in full compliance with specific Federal Fish and Wildlife Permits (endangered/threatened species and CITES Permits US 750138 and US 756611 to N.G.). For funding we thank the U. S.
Fish and Wildlife Service, National Geographic Society, and European Union (through the Wildlife Conservation Society). This publication has been funded in part with Federal funds from the National Cancer Institute,
haplotype, indicated by the arrow. This common forest elephant haplotype is present in two individuals from National Institutes of Health, under Contract No. N01-CO-12400. The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade
names, commercial products, or organizations imply endorsement by the U.S. Government.