Transcript Roca - Smithsonian Tropical Research Institute

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
319 bp
Multiple lines of evidence suggest that forest and savanna elephants are distinct species.
Measurements from 295 skulls demonstrated that forest and savanna elephants fall into two morphologically
distinct groups (left panel; Groves and Grubb, 2000). Nuclear gene analyses using both slower-evolving
nuclear gene sequences (center; Roca et al., 2001) and more rapidly evolving microsatellites (right; Comstock
et al., 2002) demonstrated a deep genetic split between forest and savanna elephants, estimated at 3.5 million
years. Only a few morphological intermediates and genetic hybrids were detected in a zone of mixed habitat
that surrounds the tropical forests of Africa.
In contrast to the distinctions between forest and savanna elephants detected by morphological and nuclear
genetic studies, analyses using mitochondrial DNA (mtDNA) have detected genetic diversity in savanna
elephants high enough to appear incongruent with nuclear DNA studies, and suggested greater mixing
between forest and savanna elephants. To investigate this apparent disparity, we sampled wild elephant tissue
from 21 African locations to determine the DNA sequences for 1642 biparentally inherited chromosomal
segments in 3 X-linked genes, and 302 mtDNA and 128 Y chromosome sequences.
FIGURE 3
(B) Y chromosome AMELY
1551 bp
DS (5)
DS (11)
Maternally
and
paternally
inherited markers demonstrate
cytonuclear
dissociation.
Phylogenetic relationships for Asian,
African forest, and African savanna
elephants inferred from (A) 319 bp of
the
maternally
inherited
mitochondrial ND5 gene (number of
individuals with identical haplotypes
indicated by location), and from (B)
1551 bp of the paternally inherited Y
chromosome gene AMELY (each
individual shown separately). Forest
populations or individuals are
indicated in green; savanna in blue;
Asian elephants in red. Garamba
(GR) is a mixed habitat zone and is
not colored.
BE (1)
BE4059
DS1555
OD0001
GR0016
GR0022
DS (8)
99/99/98
II
DS (1), OD (2)
62/74/78
BE (2), WA (1)
IIB
CH (1), HW (6), SA (8), ZZ (4)
HW (2), NG (4), SA (2), SE (9), TA (1), ZZ (1)
89/92/97
GR (10)
II
DS (1), LO (9), OD (1)
BE (1), WA (1)
94/96/98
DS1504
DS1521
DS1523
DS1524
63/80/64
GR (1)
DS1537
DS1556
LO3517
LO (2)
IIA
BE (1), WA (2)
AB (6), AM (34), CH (5), KE (16), KR (12) MA (1),
MK (1), NA (21), NG (3), SE (1), SW (5), TA (6)
90/91/87
87/93/90
70/66/63
AB (1), AM (2), KE (4)
I
CH (3), HW (4), KR (25), MA (3), SW (5)
100/100/100
I
AB (1), KE (17), WA (4)
64/64/63
GR (1)
BE (1), WA (1)
DS1503
DS1505
DS1511
DS1527
DS1528
DS1530
DS1532
DS1543
LO3505
AM0003
AM0005
AM0007
AM0015
AM0018
AM0019
AM0020
AM0021
AM0023
AM0030
AM0035
AM4551
AM4576
AM4583
AM4584
AM4585
AM4587
BE4035
BE4053
CH0882
CH0885
CH0895
CH0931
HW0062
HW0067
HW0076
HW0082
HW0086
HW0092
HW0112
HW0115
HW0117
HW0120
HW0122
HW0124
HW0151
KE4501
KE4509
KE4511
KE4516
KE4517
KE4549
KE4550
KE4601
KE4607
KE4609
KE4614
KE4617
KE4620
KE4621
KE4623
KR0114
MA0807
NA4653
NA4660
NA4665
NA4668
NA4669
NA4670
NA4671
NA4675
NA4678
NA4688
NA4697
NA4699
NA4702
NA4704
NA4710
NG2178
NG2180
NG2181
NG2182
NG2191
NG2192
NG2193
NG2194
NG2214
NG2215
NG2229
SA0972
SA0993
SA0994
SA1002
SA1004
SA1005
SA1009
SE2051
SE2098
SE2101
SE2103
SE2104
SE2106
SE2165
TA1144
TA1145
TA1431
TA1443
TA1450
TA1458
WA4020
WA4022
WA4027
ZZ0145
ZZ0148
Ema006
0.001 substitutions/site
Elephas maximus (1)
0.005 substitutions/site
mtDNA biparental Y chro.
mtDNA biparental Y chro.
AB
KE
MK
WA
LO
n=11
n=65
n=2
BE
OD
1
7
R
DS
26
171
KE
MK
AB
SE AM
NG TA
OD
15
3
1
GR
1
48
11
1
Savannah elephants
NORTH-CENTRAL
BE-Benoue
WA-Waza
Asian elephants
Forest elephants
DS-DzangaSangha
LO-Lope
GA-Garamba
16 microsatellite loci
CH
SA
35
149
17
11
154
11
8
71
13
10
46
2
4
56
1
37
170
2
21
157
15
4
HW
1
5
SW
ZZ
5
26
4
1
2
WA
MA
1
5
94
3
NA
SE
Comstock et al. 2002
9
44
3
7
NG
ZZ SW
CH HW
SA
MA
KR
KR
NA
32
4
11
1
TA
ND5
6
ND5
67
7
BGN
AMELY
PLP
PHKA2
BGN
AMELY
PLP
PHKA2
DS-Dzanga Sangha, LO-Lope, OD-Odzala, GR-Garamba, AB-Aberdares, AMAmboseli, BE-Benoue, CH-Chobe, HW-Hwange, KE-Central Kenya, KR-Kruger,
MA-Mashatu, MK-Mount Kenya, NA-Namibia, NG-Ngorongoro, SA-Savuti, SESerengeti, SW-Sengwa, TA-Tarangire, WA-Waza, ZZ-Zambezi.
METHODS
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
maximum parsimony (MP), neighbor joining (NJ), and maximum likelihood (ML) methods implemented in PAUP*4.0b10.
African
Forest
8
3
BE
Savannah elephants
EASTERN
AB-Aberdares
AM-Amboseli
MK-Mount Kenya
KE-Central Kenya
NG-Ngorongoro
SE-Serengeti
TA-Tarangire
n=280
AM
3
Savannah elephants
SOUTHERN
CH-Chobe
HW-Hwange
KR-Kruger
NA-Namibia
MA-Mashatu
SA-Savuti
SW-Sengwa
ZZ-Zambezi
n=15
n=58
(B)
(A)
(C)
FIGURE 4
Distribution of cytonuclear disequilibrium.
Pie charts indicate by locale the distribution of
genetic markers that are inherited maternally
(left pie chart in each set of three), paternally
(right), or biparentally (center). Totals indicate
the number of individuals (mtDNA, Y
chromosomes) or combined number of
chromosome segments (biparental genes)
examined.
Map indicates locations of sampled elephant
populations in Africa. Green circles are forest
locations. Blue circles are savanna locations.
Garamba (GR) includes both habitats. Orange
indicates current African elephant range; historic
range includes entire land area shown.
FIGURE 5: CONCLUSIONS
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
inferred from differing patterns detected by maternally-inherited versus paternally- or biparentally-inherited
genes are as follows:
Herd#2
A
Asian
African
Savanna
BGN, 646 bp, n=556
PHKA2, 1012 bp, n=440
PLP, 479 bp, n=657
FIGURE 2
Haplotypes for three biparentally-inherited nuclear genes display almost complete separation among
three elephant taxa. MP trees are shown. The length of each gene segment and number of chromosomes
examined are indicated for each gene. Number of chromosomes per haplotype is proportional to the size of
circles; differences between alleles are proportional to the distance between circles. Haplotypes/alleles found
in Asian elephants are red; African forest haplotypes are green; and African savanna haplotypes are blue. (A)
BGN haplotypes are completely distinct between forest and savanna populations. (B) PHKA2 proved to be the
most diverse nuclear gene segment; the chromosomes examined were completely distinct between forest and
savanna populations.(C) PLP haplotypes were distinct between forest and savanna elephants except for one
haplotype, indicated by the arrow. This common forest elephant haplotype is present in two individuals from
Cameroon.
(A) Male-mediated gene flow occurs between adjacent forest elephant
herds, and between adjacent savanna elephant herds; however (B)
interbreeding between savanna and forest elephants at the contact zone
between forest and savanna habitats is rare. (C) As forest habitat
retreats (or when forest herds move into savanna habitats), larger male
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
dominance by larger unhybridized savanna males. (E) After multiple
generations of unidirectional hybridization, nuclear genes alleles are
those of savanna elephants, although a forest mitochondrial haplotype is
retained in the now-savanna herds.
Herd#1
A
B
Forest
habitat
C
D
Savanna
habitat
E
Forest mtDNA,
savanna nuclear DNA
Savanna mtDNA
and nuclear DNA
References and Acknowledgments
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.
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.
Roca, A. L., Georgiadis, N. and O'Brien, S. J. (2003) Male-driven genomic chimerization of elephant herds in Africa. In preparation.
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.
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
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,
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.