African origin for Madagascan dogs revealed by mtDNA analysis

Madagascar was one of the last major land masses to be inhabited by humans. It was initially colonized by Austronesian speaking Indonesians 1500–2000 years ago, but subsequent migration from Africa has resulted in approximately equal genetic contributions from Indonesia and Africa, and the material culture has mainly African influences. The dog, along with the pig and the chicken, was part of the Austronesian Neolithic culture, and was furthermore the only domestic animal to accompany humans to every continent in ancient times. To illuminate Madagascan cultural origins and track the initial worldwide dispersal of dogs, we here investigated the ancestry of Madagascan dogs. We analysed mtDNA control region sequences in dogs from Madagascar (n=145) and compared it with that from potential ancestral populations in Island Southeast Asia (n=219) and sub-Saharan Africa (n=493). We found that 90% of the Madagascan dogs carried a haplotype that was also present in sub-Saharan Africa and that the remaining lineages could all be attributed to a likely origin in Africa. By contrast, only 26% of Madagascan dogs shared haplotypes with Indonesian dogs, and one haplotype typical for Austronesian dogs, carried by more than 40% of Indonesian and Polynesian dogs, was absent among the Madagascan dogs. Thus, in contrast to the human population, Madagascan dogs seem to trace their origin entirely from Africa. These results suggest that dogs were not brought to Madagascar by the initial Austronesian speaking colonizers on their transoceanic voyage, but were introduced at a later stage, together with human migration and cultural influence from Africa.

2. Introduction

The island of Madagascar is situated less than 500 km off the east coast of Africa (figure 1) and was one of the last large landmasses to be populated, 1500–2000 years before present (YBP) [1]. Despite its proximity to Africa, linguistic and genetic evidence indicate that Madagascar was initially colonized by Austronesian speaking peoples from Indonesia, but subsequent migration from Africa has resulted in approximately equal genetic contributions from both regions [2]. The Madagascan culture is mainly influenced by Africa and, except for the language, few clear contributions remain from the initial Indonesian culture [3]. The domestic dog (together with the pig and the chicken) was an important part of the Austronesian culture [4], suggesting that the dog may be one of few remaining contributions of Austronesian culture in today's Madagascar. However, the origin of Madagascan dogs (as well as other domestic animals) remains largely unstudied, and an ancestry from the Austronesian expansion or from subsequent contacts with Africa, or both, is possible. A study of the ancestry of the dogs in Madagascar is, therefore, of importance for illuminating origins of Madagascan culture, as well as for describing the worldwide dispersal of dogs.

Madagascar is a linguistic outlier with an Austronesian speaking population in the otherwise largely Bantu-speaking sub-Saharan East Africa. Starting from Taiwan approximately 5000 YBP and aided by improvements in boat technology, Austronesian languages spread across more than half the globe, to Madagascar in the west and Easter Island in the east, in one of the most extensive geographical expansions of a human population in history [4]. Linguistic evidence suggests that the migration to Madagascar took place in early seventh century AD [2,5,6]. The modern Malagasy language shares most of its basic vocabulary (approx. 90%) with the languages spoken in the Barito River region in southeastern Borneo, thus distinctly pointing out the probable geographical origin of the first colonizers of Madagascar [5–7]. The remaining vocabulary consists of contributions from, e.g. Bantu, Malay and Sanskrit languages [5]. The words for most domesticates, including the dog, have Bantu origin, indicating that the ancestors of the modern Malagasy adopted new words for these animals, even though they were already familiar to them [2,8,9].

Palaeoecological and archaeological evidence (e.g. human-modified bones from extinct animals) indicate human presence on Madagascar more than 2000 YBP [10,11]. However, the earliest direct evidence for human occupation in the form of charcoal fragments in northern Madagascar was dated to 1300–1680 YBP [1], in good agreement with the linguistic evidence. This was shortly preceded by a decrease in fossil spores of Sporormiella, an obligatory coprophilous fungus occurring exclusively on dung from herbivorous animals, suggesting a decrease in megafauna probably due to hunting [12]. The first permanent settlements in the highlands are indicated by charcoal from increased burning and ceramics, dated to the thirteenth century AD [1,13]. Livestock proliferation on Madagascar is first indicated by a rise in Sporormiella spores ca 1100 YBP [12]. The earliest remains of domestic dog includes bones excavated in Rezoky in western Madagascar, dating to 500–700 YBP [1].

Ancient long-distance contacts across the Indian Ocean are demonstrated by genetic, linguistic and cultural evidence, connecting East Africa with Southeast Asia, the Indian subcontinent and Southwest Asia. However, research is still limited and few details are known about these contacts, particularly before the establishment of the trading societies of the Swahili coast [14]. Early contacts between East Africa and Indonesia are suggested by archaeobotanical and archaeozoological evidence [15]. There is, for example, ancient presence in Africa of Indonesian food plants such as banana (at least 5000 YBP) [16–18], and of water yam and taro [15,19]. At the time when Madagascar was first populated there was extensive trade across the Indian Ocean, which has later left traces in Madagascan culture also from, e.g. Southwest Asia and India [20–22].

Genetic studies based on mitochondrial DNA (mtDNA) and Y-chromosome DNA clearly reveal that Madagascar has an admixed human population of African and Indonesian origins, with approximately equal contribution of both maternal and paternal lineages from each source [23,24]. The Asian ancestry is more conserved among the inland than the coastal populations [23,24], which is consistent with a clear phenotypic difference between highland ‘Indonesian’ and lowland ‘African’ peoples [8]. In agreement with the linguistic evidence for the origin of the Malagasy language, Hurles et al. [23] identified Banjarmasin in southeastern Borneo as the most likely Asian source of the paternal ancestors of the Malagasy people.

At the time when Austronesian people arrived in Madagascar (i.e. 1500–2000 YBP), dogs had been present in East Asia for several millennia [25,26]. Also by this time, the domestic dog had recently arrived in sub-Saharan Africa [27,28]. Madagascan dogs could, therefore, have originated from either of these regions. We have previously shown that dog populations related to Austronesian speaking peoples in Indonesia and ancient Polynesia carry a signature haplotype (Arc2) at high proportions [29]. This haplotype was found in 41% of dogs in Kalimantan and Bali and in 68% of ancient samples from across Polynesia, but was absent outside Southeast Asia and Oceania [25]. Thus, there is a considerable difference between the gene pools of dogs in Indonesia/Polynesia and Africa, which presents us with the opportunity to identify the proportion of genetic ancestry of the Madagascan dogs from either of these two populations.

Here, we perform, to our knowledge, the first study of mtDNA in Madagascan dogs, comparing a large sample from across Madagascar with potential ancestral populations in Island Southeast Asia and sub-Saharan Africa. We investigate the proportion of ancestry from Asian dogs brought with the Austronesian expansion and from African dogs in subsequent contacts, illuminating Madagascan cultural origins as well as the initial dispersal of dogs across the world.

3. Material and methods

3.1 Samples

Three hundred and twenty nine samples of dogs from Africa (145 samples from Madagascar and 184 samples from the African mainland) were sequenced for a 582 bp fragment of the control region of the mitochondrial genome (positions 15 458 to 16 039) and compared with 2470 dogs from across the world published in earlier studies [25,30–34]. Dogs were sampled primarily in rural areas with low influx of foreign dogs, and avoiding known relatedness among individuals. The geographical origin of dogs for regions specifically studied were as follows (see also figure 1, table 1 and the electronic supplementary material, table S1): Africa—Madagascar coastal: northern (n=25), dry southern (n=11), Southeast (n=10), Southwest (n=8); Madagascar highland (n=38), misc. (n=54); East sub-Saharan Africa: Botswana (n=8), Kenya (n=9), Lesotho (n=6), Mozambique (n=8), South Africa (n=119), Tanzania (n=43), Uganda (n=148), Zambia (n=11); West sub-Saharan Africa: Benin (n=3), D. R. Congo (n=4), Gambia (n=4), Namibia (n=119), Sudan (n=3), misc. (n=8); North Africa: Algeria (n=1), Egypt (n=43), Mali (n=1), Morocco (n=19), Tunisia (n=9), misc. (n=12); Indonesia—Bali (n=61), Kalimantan (n=65), Sulawesi (n=3), misc. (n=2); Polynesia (archaeological specimens analysed for a shorter region (263 bp; positions 15 458–15 720))—Cook Islands (n=2), Hawaii (n=4), New Zealand (n=13); India (n=59); Taiwan (n=52); the Philippines (n=36); Australia (Dingo)—Victoria (n=35), New South Wales (n=110), Northern Territory (n=3), Queensland (n=44), South Australia (n=6), Western Australia (n=29), misc. (n=5); Southwest Asia—Afghanistan (n=6), U.A.E. (n=1), Iran (n=150), Israel (n=25), Kazakhstan (n=2), Kyrgyzstan (n=2), Saudi Arabia (n=5), Syria (n=7), Tajikistan (n=1), Turkey (n=111), Uzbekistan (n=1), misc. (n=24); Europe—Britain (n=121), North continent (n=142), South continent (n=117); Scandinavia (n=64), misc. (n=6).

3.2 DNA extraction, amplification and sequencing

Samples were collected as hair samples (n=67) and buccal epithelial cell samples (n=262) on FTA^® cards (Whatman Inc.). Hair samples were extracted as described in Angleby & Savolainen [35] and buccal epithelial cell samples were extracted according to the manufacturer's instructions. PCR amplification, DNA sequencing and sequence analysis were performed as in Angleby & Savolainen [35].

3.3 Analysis of the sequence data

DNA sequences were edited using sequencing analysis (Applied Biosystems, Foster City, CA, USA). Assembly into contigs and further editing was performed using Sequencher 4.1 (Gene Codes Corporation, Ann Arbor, MI, USA). The novel haplotypes found in this study were deposited in GenBank under accession nos. KR069086 (haplotype A250) and KP295480–KP295494 (haplotypes A178, A245, A246, A248, A249, A251, A254, A258, A261, A262, A273, A274, B052, B055, and B056, respectively).

Phylogenetic relations between haplotypes were displayed in minimum-spanning networks drawn by hand based on genetic distances calculated with Arlequin v. 3.11 software [36]. The networks were based on mtDNA sequences from dogs in the specifically studied geographical regions, as well as from dogs sampled across the world [25,30–34], to display the global phylogeny of dogs.

The mutation rate for the 582 bp region was obtained from [25] based on the average genetic distance between dog/wolf and coyote in a phylogenetic tree, calibrated with the time for the separation between the wolf and coyote lineages. The exact calibration point for the wolf/coyote separation is not known, but it may have occurred 1.5–4.5 million YBP. This gives a rate of 1.1×10⁻⁸–4.3×10⁻⁸substitutions per site per year, or 1 substitution per 40 000–155 000 years.

The time of arrival of dogs to Madagascar was estimated using the statistic ρ(the mean number of substitutions for a set of sequences to their common ancestral haplotype) [37], based on the mean distance of Madagascan sequences to the founder haplotypes, and the substitution rate. Because the separation time between wolf and coyote can only be given as a range of possible dates, the time estimate is also obtained as a relatively broad range of possible time.

Genetic distances among geographical regions were estimated as F_ST values based on nucleotide differences including indels using Arlequin v. 3.5.1.2 [36] software. The matrix of pairwise F_ST values was summarized in two dimensions using multidimensional scaling (MDS) analysis implemented in Statistica v. 11 software package (StatSoft, Tulsa, OK, USA).

4. Results

We analysed 582 bp of the mtDNA control region in 145 dogs from across Madagascar in order to investigate their origin (figure 1 and table 1). The Madagascan dog sample was compared with dogs from the two broad source regions for the human Malagasy population, Austronesian speaking populations in Indonesia and African populations in sub-Saharan Africa, and also with ancient samples from across Polynesia linked to the Austronesian expansion. These comparisons were performed in the context of a comprehensive sample of dogs from the Old World, including Europe (to detect the impact of, e.g. the colonial era), and India and Southwest Asia, which have also influenced the Malagasy culture.