How many species of giraffe are there in Africa?David M. Brown1 and Nicholas J. Georgiadis2

1. University of California, Los Angeles; 2. Mpala Research Centre

RATIONALE• The genus Giraffa in Africa is comprised of phenotypically distinct groups, currently classified as nine ‘subspecies’.• Are the different groups reproductively isolated, and perhaps different species?• Or do the different groups interbreed, and will their distinctiveness disappear over time?• Arguments have surrounded these questions for over 100 years.• The different giraffe groups have hybridized in captivity.• What does this variation mean in evolutionary terms?• What are the evolutionary dynamics of giraffes in the wild?

APPROACHWe examined genetic variation in the mitochondrial and nuclear genomes of free-ranging individual giraffes from six taxonomic giraffe subspecies.

We are tested whether the giraffe subspecies:• Are genetically isolated from each other: Is genetic isolation recent or deep in evolutionary time?• Are hybridizing with each other: Is hybridization rare or common?

• Biopsy-dart samples were collected from 397 free-ranging individual giraffes representing six subspecies: * Giraffa camelopardalis peralta (W) from Niger * G.c. rothschildi (N) from Uganda and Western Kenya * G.c. reticulata (R) from Kenya * G.c. tippelskirchi (M) from Kenya and Tanzania * G.c. angolensis (S) from Namibia * G.c. capensis (S) from South Africa

Mitochondrial DNA: MATERNAL HISTORY

Mitochondrial DNA is maternally inherited and does not capture the genetic history of males in a species.• 656 nucleotide fragment starting in cytochrome b extending through tRNAs to control region.• Okapi was used as the ‘outgroup’ for reconstruction of the phylogenetic tree.• There were 37 unique mtDNA sequences (haplotypes) found in 266 individuals.

Peralta Rothschildi Reticulata Tippelskirchi Capensis Angolensis Haplotypes Niger Murchison

Falls Western Kenya

Samburu Meru Ol J ogi Athi Chyulu Naivasha Tarangire

Manyara Serengeti Kruger Etosha Hoanib

1PeraltaA 7 2PeraltaB 15 3RothsA 25 16 4RothsB 8 5RothsC 2 6ReticA 2 7ReticB 1 1 11 8ReticC 7 8 9ReticD 1 10ReticE 1 11ReticF 5 6 12ReticG 2 5 13 13MasaiA 9 14MasaiB 4 13 4 7 5 15MasaiC 1 16MasaiD 2 17MasaiE 2 18MasaiF 1 19MasaiG 2 2 1 2 20MasaiH 1 21MasaiI 2 22MasaiJ 1 23MasaiK 2 14 24MasaiL 1 25MasaiM 3 26MasaiN 1 27MasaiO 3 28CapensisA 11 29CapensisB 1 30AngolensisA 3 31AngolensisB 7 32AngolensisC 2 33AngolensisD 2 34AngolensisE 1 1 35AngolensisF 1 36AngolensisG 17 37AngolensisH 1

Okapi17MasaiE

19MasaiG20MasaiH15MasaiC18MasaiF

14MasaiB21MasaiI

16MasaiD22MasaiJ

24MasaiL23MasaiK25MasaiM

26MasaiN27MasaiO

13MasaiA12ReticG

OlJogi10Hap1510ReticE8ReticC

9ReticD7ReticB

1PeraltaA2PeraltaB

3RothsA5RothsC

4RothsB

32AngolensisC33AngolensisD35AngolensisF31AngolensisB30AngolensisA37AngolensisH

36AngolensisG34AngolensisE

28CapensisA29CapensisB

1 change

NJ

tippelskirchi

6ReticA

reticulata

peralta

rothschildi

angolensis

capensis

Fig. 2. (right) Phylogenetic tree of mitochondrial haplotypes • Each group is monophyletic with exception of one Masai haplotype grouping with reticulated giraffes.• In general, giraffes of different morphotypes are not interbreeding in the wild.

Table 1. Distribution of mtDNA haplotypes by sampling location • peralta (W) N=22; rothschildi (N) N=51; reticulata (R) N=63; tippelskirchi (M) N=83; capensis (S) N=12, angolensis (S) N=35 • Each taxonomic subspecies has a unique set of mtDNA haplotypes• No haplotypes were shared between groups

Nuclear DNA: THE GENETIC HISTORY OF POPULATIONS AND SPECIES

Fig. 3. FES: cladogram of single-copy nuclear intron sequences, representing:

* peralta (W) N=5* rothschildi (N) N=10* reticulata (R) N=14 * tippelskirchi (M) N=27 * capensis (S) N=10 * angolensis (S) N=11

Microsatellites: hypervariable sections of DNA*17 different microsatellite markers *397 individual giraffes from six taxonomic groups: * peralta (W) N=28 * tippelskirchi (M) N=142* rothschildi (N) N=81 * capensis (S) N=27* reticulata (R) N=80 *angolensis (S) N=38

Athi18Athi23ChyuluM2TarangireM2264LoboM2081TarangireM2235TarangireM2249Athi3Athi5Athi6Athi21Athi13Athi2Chyulu17Chyulu25486848565017Chyulu5Chyulu4Chyulu1Athi25227150165026Chyulu20Athi8SamburuRe4812Niger10Niger18RumaRoGR40OlJogiRe2OlJogiRe15OlJogiRe26OlJogiRe3OlJogiRe8Samburu3OlJogi2MFNPF3MFNPM2MFNPM5Kruger12Kruger2Kruger4Niger13GN30Kruger3Niger5Niger14Samburu4GN16GN22MFNPM3MFNPF2MFNPF1481448124810ENPF8Kruger1Kruger11OlJogi16OlJogi18Kruger15Kruger7Kruger13ENPF1ENPF2ENPF3ENPM1ENPM5ENPM6KHBM1HSBM2ENPM3HNBF3Kruger8Okapi1

NJ

tippelskirchi

peralta reticulata rothschildi angolensis capensis

capensis angolensis

Masai (tippelskirchi) giraffes have a unique SNP (single nucleotide polymorphism) in the FES intron sequence, distinguishing them from all others.

Southern Africa capensis+angolensis

Masai(tippelskirchi)

Reticulated (reticulata)

Western Africaperalta+rothschildi

Population clusters of composite microsatellite genotypes

CONCLUSIONSMitochondrial DNA • Each of the sampled groups has a unique set of haplotypes (Reticulated, Rothschilds, Peralta, Masai, Angolensis, Capensis).• Exceptions to monophyly may be due to retention of ancestral polymorphism, or hybridization at range boundaries, for example, between tippelskirchi females and reticulata males in the Athi Plains region.• Since there is no evidence for nuclear DNA hybridization between them, it is likely that recurrent backcrossing of hybrid females to tippelskirchi males replaced the reticulata genome in hybrid individuals over many generations.

Nuclear DNA: Microsatellites• Microsatellite genotypes cluster into 4 major nuclear DNA pools: Reticulated, Rothschilds + Peralta, Masai, Angolensis + Capensis• Each gene pool is equivalent of a monophyletic group (no interbreeding between them)• Some evolutionary force has kept reticulata and tippleskirchi reproductively isolated from each other: *Ecological selection for climate or some other factor? *Sexual selection?• The population genetic structure of giraffes in the wild conflicts with the currently held classification of ‘9 subspecies’.• There are at least four distinct groups of giraffes that, based on genetic evidence, appear to be reproductively isolated from each other in the wild.• These groups may correspond with different species of giraffe.