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 (B) Y chromosome AMELY 1551 bp 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 DS1537 DS1556 LO3517 distinct groups (left panel; Groves and Grubb, 2000). Nuclear gene analyses using both slower-evolving Maternally and paternally GR (1) 63/80/64 DS1504 DS1521 DS1523 DS1524 DS1555 OD0001 nuclear gene sequences (center; Roca et al., 2001) and more rapidly evolving microsatellites (right; Comstock inherited markers demonstrate DS (8) 99/99/98 DS1503 DS1505 DS1511 DS1527 GR0016 GR0022 cytonuclear dissociation. DS1528 DS1530 II et al., 2002) demonstrated a deep genetic split between forest and savanna elephants, estimated at 3.5 million DS1532 DS1543 LO3505 62/74/78 DS (1), OD (2) BE (2), WA (1) AM0003 AM0005 Phylogenetic relationships for Asian, AM0007 AM0015 years. Only a few morphological intermediates and genetic hybrids were detected in a zone of mixed habitat AM0018 IIB AM0019 AM0020 AM0021 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) AM0030 AM0035 AM4551 AM4576 AM4583 AM4584 AM4585 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) AM4587 BE4035 BE4053 CH0882 CH0885 CH0895 CH0931 genetic studies, analyses using mitochondrial DNA (mtDNA) have detected genetic diversity in savanna the maternally inherited II HW0062 HW0067 HW0076 HW0082 HW0086 HW0092 HW0112 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) HW0115 HW0117 HW0120 HW0122 HW0124 HW0151 KE4501 between forest and savanna elephants. To investigate this apparent disparity, we sampled wild elephant tissue individuals with identical haplotypes 94/96/98 IIA LO (2) KE4509 KE4511 KE4516 KE4517 KE4549 KE4550 KE4601 from 21 African locations to determine the DNA sequences for 1642 biparentally inherited chromosomal indicated by location), and from (B) BE (1), WA (2) KE4607 KE4609 KE4614 KE4617 KE4620 KE4621 KE4623 segments in 3 X-linked genes, and 302 mtDNA and 128 Y chromosome sequences. 1551 bp of the paternally inherited Y KR0114 MA0807 NA4653 NA4660 NA4665 NA4668 NA4669 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) NA4670 NA4671 NA4675 NA4678 NA4688 NA4697 NA4699 individual shown separately). Forest 70/66/63 AB (1), AM (2), KE (4) 87/93/90 I NA4702 NA4704 NA4710 NG2178 NG2180 NG2181 NG2182 populations or individuals are CH (3), HW (4), KR (25), MA (3), SW (5) NG2191 NG2192 NG2193 NG2194 NG2214 NG2215 NG2229 indicated in green; savanna in blue; 100/100/100 SA0972 SA0993 SA0994 SA1002 SA1004 I SA1005 SA1009 Asian elephants in red. Garamba AB (1), KE (17), WA (4) SE2051 SE2098 SE2101 SE2103 SE2104 SE2106 64/64/63 SE2165 (GR) is a mixed habitat zone and is GR (1) TA1144 TA1145 TA1431 TA1443 TA1450 TA1458 BE (1), WA (1) not colored. WA4020 WA4022 WA4027 ZZ0145 ZZ0148 Ema006 Elephas maximus (1) 0.001 substitutions/site 0.005 substitutions/site mtDNA biparental Y chro. AB mtDNA biparental Y chro. FIGURE 4 LO WA KE MK n=11 n=65 n=2 BE n=58 n=280 n=15 Distribution of cytonuclear disequilibrium. OD 3 7 1 AM Pie charts indicate by locale the distribution of R 35 149 17 DS KE MK CH 8 genetic markers that are inherited maternally OD AB SA 1 26 3 171 15 SE AM 4 11 154 11 (left pie chart in each set of three), paternally GR NG TA 11 48 1 3 HW 8 71 13 (right), or biparentally (center). Totals indicate 1 1 BE SW ZZ 5 the number of individuals (mtDNA, Y Savannah elephants Savannah elephants Savannah elephants Forest elephants SOUTHERN EASTERN NORTH-CENTRAL DS-Dzanga Sangha 5 4 26 1 2 10 46 2 chromosomes) or combined number of CH-Chobe AB-Aberdares BE-Benoue LO-Lope WA MA HW-Hwange KR-Kruger AM-Amboseli MK-Mount Kenya WA-Waza GA-Garamba 1 5 94 3 ZZ SW 4 56 1 chromosome segments (biparental genes) NA-Namibia KE-Central Kenya NA CH HW 16 microsatellite loci MA-Mashatu SA-Savuti NG-Ngorongoro SE-Serengeti Asian elephants SE SA KR examined. SW-Sengwa TA-Tarangire Comstock et al. 2002 MA ZZ-Zambezi 3 9 44 7 KR 37 170 2 Map indicates locations of sampled elephant NG NA 4 1 32 11 21 157 15 populations in Africa. Green circles are forest TA ND5 BGN PLP AMELY locations. Blue circles are savanna locations. 6 67 7 ND5 BGN AMELY PHKA2 Garamba (GR) includes both habitats. Orange PLP 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, African 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 Forest 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 D retreats (or when forest herds move into savanna habitats), larger male African 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 Savanna habitat retained in the now-savanna herds. Haplotypes for three biparentally-inherited nuclear genes display almost complete separation among E Forest mtDNA, savanna nuclear DNA Savanna mtDNA 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. Cameroon.
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