Genetic Evidence for Two Species of Elephant in Africa Alfred L. Roca, Nicholas Georgiadis, Jill Pecon-Slattery, and Stephen J. O'Brien

Variable nucleotide sites among African savannah, Garamba, African forest, and Asian elephants for four intronic gene segments. Fixed differences between Asian and African elephants are indicated by boxes in the Asian elephant sequences. Shaded nucleotides in the African forest elephant sequences indicate fixed differences between them and African savannah elephants. Sites are numbered from the first aligned nucleotide used in the analysis for each gene. Lines separate the four categories of elephants. Asian elephants are labeled Ema (Elephas maximus); African elephant labels are in Fig. 1. Key: ".", identical with reference sequence; *, indel AAACC present; #, indel TTTTTTTTTATTTGCT present; "@", deletion. The combined sequence of the four gene segments was unique for each forest and Garamba individual. For savannah and Asian elephants, the number of individuals with identical sequences is in parentheses. One individual is listed for each unique combined sequence as follows: AM0003 was identical with AB4521, AB4522, AB4524, AB4525, AB4526, AB4530, AB4531, AB4532, AB4535, AB4541, AB4542, AM0023, AM0033, BE4035, BE4036, BE4053, BE4059, CH0881, CH0892, HW0059, KE4505, KE4518, KE4519, KE4601, KE4602, KE4603, KR0014, KR0090, MK4544, MK4545, NA4651, NA4657, NA4658, NA4659, NA4666, NA4688, NG2179, NG2215, NG2229, SA1029, SE2100, SW0910, TA1450, WA4004, WA4022, ZZ0155, and ZZ0163; CH0885 with KE4506, MA0804, NA4673, NG2208, SA0990, SA0994, TA1431, WA4027, and ZZ0162; HW0067 with NA4680; KE4516 with AB4523, AB4533, AB4534, KE4540, NG2194, and WA4029; KR0099 with AB4527, MA0816, and NG2191. BE4058, KR0138, NA4669, and WA4024 were unique. Ema001 was identical with Ema 004, Ema006, and Ema007; Ema008 with Ema010.

Supplemental Table 1. Distinctiveness among Asian and African elephants based on GBA haplotype frequencies and selected VIM/CHRNA1 variant nucleotide site occurrence.
		Population
GBA	[20.79] Haplotype/ Frequency:	Asian	Forest	Garamba	Savannah
I	[C.C]	100%	63%	41%	4%
II	[C.T]	0.0%	37%	50%	0%
III	[T.C]	0.0%	0.0%	9%	96%
No. of chromosomes:		n = 14	n = 70	n = 34	n = 262
VIM	Position/ Nucleotide*	Asian	Forest	Garamba	Savannah
	71	G	G/(A)	G/A	G
	115	G	G/A	G/A	G
	126	C	C/T	C/T	C
	149	C	C/T	C/T	C
	162	T	T/C	T/C	T
	185	G	G/C	G/C	G
	217(Indel)H	-	±	±	-
	265	A	A/G	A/G/(C)	G/(A)
CHRNA1	Position/ Nucleotide*
	250	C	C/T	C/T	C
	251	G	G/(A)	G/A	A
	312	G	G/C	G/C	G
	418	G	G	G	G/A
	460(Indel)H	+	±	±	+
	514	A	A	A	A/G
*Listed in order of frequency; very low frequency in parentheses. HIndels defined as in Fig. 2: -, deletion; +, insertion.