FIGURE 1: INTRODUCTION

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.

Cyto-nuclear genomic dissociation in African elephant speciesAlfred 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

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.

AfricanForest

Asian

AfricanSavanna

BGN, 646 bp, n=556 PHKA2, 1012 bp, n=440 PLP, 479 bp, n=657

(A) (B) (C)

Herd#2

Forest mtDNA,savanna nuclear DNA

Herd#1

Foresthabitat

Savannahabitat

Savanna mtDNAand nuclear DNA

A A

D

C

E

B

CHNAZZ

SA

NG

SEAB

KEMK

AMTA

BE

WA

OD

R

SW

MAKR

HW

157 1521

AMELYBGNPLP

PHKA2

ND5

mtDNA biparental Y chro.

AMELYBGNPLP

PHKA2

ND5

GR

3

48

BE

1

26

WA

1

94

LO

n=65

OD

7

DS

171

n=2

1

3

15

2

3

1

SE

44 7

NG

32

TA

67

11

7

n=11

3

261

11

5

1

5

4

9

1

4

3

1

6

mtDNA biparental Y chro.

CHSA

154

HW

71

11

13

SWZZ

MA

KR

NA

46

56

170

2

2

1

AM

149 17358

11

8

4

10

5

4

37

ABKEMK

n=280 n=15n=58

(A) mtDNA ND5319 bp

AB (6), AM (34), CH (5), KE (16), KR (12) MA (1), MK (1), NA (21), NG (3), SE (1), SW (5), TA (6)

0.005 substitutions/site

AB (1), AM (2), KE (4)

CH (3), HW (4), KR (25), MA (3), SW (5)

AB (1), KE (17), WA (4)

GR (1)

BE (1), WA (1)

Elephas maximus (1)

DS (1), LO (9), OD (1)

BE (1), WA (1)

LO (2)

BE (1), WA (2)

HW (2), NG (4), SA (2), SE (9), TA (1), ZZ (1)

GR (10)

CH (1), HW (6), SA (8), ZZ (4)

DS (5)

DS (8)

DS (1), OD (2) BE (2), WA (1)

GR (1)

BE (1)

DS (11)

100/100/100

64/64/63

70/66/63

90/91/87

89/92/97

94/96/98

62/74/78

I

II

IIA

IIB

(B) Y chromosome AMELY1551 bp

Ema006

0.001 substitutions/site

AM0003AM0005AM0007AM0015AM0018AM0019AM0020AM0021AM0023AM0030AM0035AM4551AM4576AM4583AM4584AM4585AM4587BE4035BE4053CH0882CH0885CH0895CH0931HW0062HW0067HW0076HW0082HW0086HW0092HW0112HW0115HW0117HW0120HW0122HW0124HW0151KE4501KE4509KE4511KE4516KE4517KE4549KE4550KE4601KE4607KE4609KE4614KE4617KE4620KE4621KE4623KR0114MA0807NA4653NA4660NA4665NA4668NA4669NA4670NA4671NA4675NA4678NA4688NA4697NA4699NA4702NA4704NA4710NG2178NG2180NG2181NG2182NG2191NG2192NG2193NG2194NG2214NG2215NG2229SA0972SA0993SA0994SA1002SA1004SA1005SA1009SE2051SE2098SE2101SE2103SE2104SE2106SE2165TA1144TA1145TA1431TA1443TA1450TA1458WA4020WA4022WA4027ZZ0145ZZ0148

DS1503DS1505DS1511DS1527DS1528DS1530DS1532DS1543LO3505

DS1537DS1556LO3517

BE4059

DS1504DS1521DS1523DS1524

DS1555

GR0016GR0022

OD0001

63/80/64

99/99/98

87/93/90

I

II

FIGURE 2Haplotypes 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.

FIGURE 3Maternally 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.

FIGURE 4Distribution 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.

(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.

16 microsatellite loci

Comstock et al. 2002

Forest elephantsDS-Dzanga SanghaLO-LopeGA-Garamba

Savannah elephantsNORTH-CENTRALBE-BenoueWA-Waza

Savannah elephantsEASTERNEASTERNAB-AberdaresAM-AmboseliMK-Mount KenyaKE-Central KenyaNG-NgorongoroSE-SerengetiTA-Tarangire

Savannah elephantsSOUTHERNCH-ChobeHW-HwangeKR-KrugerNA-NamibiaMA-MashatuSA-SavutiSW-SengwaZZ-Zambezi

Asian elephants

METHODSDNA 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.

FIGURE 5: CONCLUSIONSCytonuclear 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:

DS-Dzanga Sangha, LO-Lope, OD-Odzala, GR-Garamba, AB-Aberdares, AM-Amboseli, BE-Benoue, CH-Chobe, HW-Hwange, KE-Central Kenya, KR-Kruger, MA-Mashatu, MK-Mount Kenya, NA-Namibia, NG-Ngorongoro, SA-Savuti, SE-Serengeti, SW-Sengwa, TA-Tarangire, WA-Waza, ZZ-Zambezi.