The genetic history of Peninsular Malaysia

Gene 586 (2016) 129–135

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Gene journal homepage: www.elsevier.com/locate/gene

Review

The genetic history of Peninsular Malaysia Hanim Kamis Norhalifah a, Fatnin Hisham Syaza a, Geoffrey Keith Chambers b, &, Hisham Atan Edinur a,⁎ a b

Forensic Science Programme, School of Health Sciences, Universiti Sains Malaysia, Health Campus, Kelantan, Malaysia School of Biological Sciences, Victoria University of Wellington, Wellington, New Zealand

a r t i c l e

i n f o

Article history: Received 25 August 2015 Received in revised form 17 March 2016 Accepted 5 April 2016 Available online 7 April 2016 Keywords: Genetics Orang Asli Austronesians Malays Peninsular Malaysia

a b s t r a c t This article explores the genetic history of the various sub-populations currently living in Peninsular Malaysia. This region has received multiple waves of migrants like the Orang Asli in prehistoric times and the Chinese, Indians, Europeans and Arabs during historic times. There are three highly distinct lineages that make up the Orang Asli; Semang, Senoi and Proto-Malays. The Semang, who have ‘Negrito’ characteristics, represent the first human settlers in Peninsular Malaysia arriving from about 50,000 ya. The Senoi later migrated from Indochina and are a mix between an Asian Neolithic population and the Semang. These Asian genomes probably came in before Austroasiatic languages arrived between 5000 and 4000 years ago. Semang and Senoi both now speak Austro-Asiatic languages indicative of cultural diffusion from Senoi to Semang. In contrast, the Proto-Malays who came last to the southern part of this region speak Austronesian language and are Austronesians with some Negrito admixture. It is from this group that the contemporary Malays emerged. Here we provide an overview of the best available genetic evidences (single nucleotide polymorphisms, mitochondrial DNA, Y-chromosome, blood groups, human platelet antigen, human leukocyte antigen, human neutrophil antigen and killer-cell immunoglobulin-like receptor) supporting the complex genetic history of Peninsular Malaysia. Large scale sampling and high throughput genetic screening programmes such as those using genome-wide single nucleotide polymorphism analyses have provided insights into various ancestral and admixture genetic fractions in this region. Given the now extensive admixture present in the contemporary descendants of ancient sub-populations in Peninsular Malaysia, improved reconstruction of human migration history in this region will require new evidence from ancient DNA in well-preserved skeletons. All other aspects of the highly diverse and complex genetic makeup in Peninsular Malaysia should be considered carefully for genetic mapping of disease loci and policy formation by health authorities. © 2016 Elsevier B.V. All rights reserved.

Contents 1. Introduction . . . . . . . . . . . . . . . . . . 2. Waves of prehistoric migration build the layer cake 3. More recent historic events add further complexity 4. The new genetic evidence . . . . . . . . . . . . 5. Findings from immunological markers . . . . . . 6. Concluding observations and future directions . . Conflict of interest . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . .

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1. Introduction Abbreviations: ISEA, Island Southeast Asia; HLA, human leucocyte antigen; mtDNA, mitochondrial DNA; SNP, single nucleotide polymorphism; NGS, next generation sequencer; ya, years ago. ⁎ Corresponding author at: Forensic Science Programme, School of Health Sciences, Health Campus, Universiti Sains Malaysia, 16150 Kubang Kerian, Kelantan, Malaysia. E-mail address: [email protected] (H.A. Edinur).

http://dx.doi.org/10.1016/j.gene.2016.04.008 0378-1119/© 2016 Elsevier B.V. All rights reserved.

Peninsular Malaysia lies along a major trading route between East and West. It has long been a crossroad between widely differing peoples, languages and cultures (Diamond, 2014). It is not surprising, therefore, that the history of its population has been one of complex blending of

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H.K. Norhalifah et al. / Gene 586 (2016) 129–135

disparate elements. What is perhaps surprising is that to date there have been few complete accounts of this story. Some are now beginning to emerge e.g. from Deng et al. (2014, 2015), Hatin et al. (2014) and Aghakhanian et al. (2015) and these include testing of anthropological hypotheses using high resolution genetic data. Today the Malays form the majority of the population (54.6%) together with Chinese (24.6%) and Indians (7.3%) – see Population and Housing Census of Malaysia (2010). The Malays have a formal political definition (Article 160(2), Constitution of Malaysia) as a: ‘Malaysian citizen born to a Malaysian citizen who professes the religion of Islam, habitually speaks the Malay language, conforms to Malay custom and is domiciled in Malaysia’. However practical this definition might be, it does not take full account of their anthropological status as an ethnic group of the Austronesian peoples who speak a Malayo-Polynesian language (Bellwood, 1993, 2007). Neither does it take account of the various constituent Malay subethnic groups or their genetic ancestry and their place alongside the indigenous Orang Asli (Nagata, 1974; Fernando, 2002). In turn, the Orang Asli themselves consist of at least three genetically distinct lineages (Semang, Senoi and ProtoMalays) of people (Fix, 2008; JAKOA — see Table 1 for their subgroups, geographical locations and languages). Presently they make up only approximately 0.5% of national population of Peninsular Malaysia. Some might say it would be better to classify these three populations linguistically, Aslian versus Malayo-Chamic; the Senoi plus the Semang and the Proto-Malays plus the Malays respectively. However, this would be to neglect the deep time origin of the Semang and their presumed lost Proto-Papuan language. Nonetheless, the significance of the Orang Asli should not be understated. The Malays who make up the majority of population living Peninsular Malaysia are thought to be admixed descendants of Proto-Malays by intermarriage with other late arrival ethnicities, including Chinese and Indians (Simon, 2012). 2. Waves of prehistoric migration build the layer cake It is now appropriate to construct a historic account of the settlement of the region as these successive waves of people arrived. Not only did they follow one another in time, but also they came from quite different directions. The Semang are the smallest Orang Asli group and are believed to be the very first settlers (~74–40 kya) in Peninsular Malaysia (Zuraina,

Table 1 Orang Asli groups and sub-groups in Peninsular Malaysia. Groupsa

Semang

Sub-groupsa Locationsa

Languageb

Kensiu Kintak Lanoh Jahai Mendriq Bateq Che Wong

Austro-Asiatic Austro-Asiatic Austro-Asiatic Austro-Asiatic Austro-Asiatic Austro-Asiatic Austro-Asiatic

Mah Meri Senoi

Jahut Semoq Beri Semai Temiar Kuala Kanaq Seletar Proto-Malay Jakun Semelai Temuan a

Baling, Kedah Gerik, Hulu Perak Perak Remote areas of Perak and Kelantan Gua Musang, Kelantan Pahang, Kelantan and Terengganu Raub and Temerloh, Pahang Coastal areas of Selangor, Putrajaya, and Negeri Sembilan Temerloh and Jerantut, Pahang Pahang and Terengganu Pahang, Perak and Selangor Perak, Kelantan, and Pahang Batu Pahat and Pontian, Johor Kota Tinggi, Johor Coastal regions of Johor Southern parts of Peninsular Malaysia Pahang, Negeri Sembilan and Johor Negeri Sembilan, Selangor and Johor

Austro-Asiatic Austro-Asiatic Austro-Asiatic Austro-Asiatic Austro-Asiatic Austronesian Austronesian Austronesian Austronesian Austro-Asiatic Austronesian

Jabatan Kemajuan Orang Asli (JKOA; http://www.jakoa.gov.my, accessed 5th May 2015). Ethnologue languages of the World (http://www.ethnologue.com, accessed 5th May 2015). b

1990; Barker et al., 2002; Barker, 2005; Hill et al., 2006; Bellwood, 2007; Oppenheimer, 2012; Baer, 2014). Today these people remain as small bodied survivors in the forests of central Malaya. They are associated with the first of ‘Out of Africa’ dispersal wave by Anatomically Modern Humans. Other branches of this lineage stretch even further to Australia and Papua New Guinea (i.e. Papuans) (Bellwood, 2007). In Malaysia, these phenotypically dark-skinned, frizzy haired Semang speak Aslian (a branch of Mon–Khmer), which is believed to have first been introduced to them by the Senoi (Austroasiatic speaking peoples who entered Malaya from the north, perhaps around 4000 years ago if we link their arrival with the arrival of Neolithic cultures from southern Thailand) leading eventually to linguistic replacement; see Blust, 2013 and Peter Bellwood, personal communication). The Senoi are physically taller and lighter than Semang and migrated south from mainland of Southeast Asia to Peninsular Malaysia (Hill et al., 2006). They were thought to have arrived as late as 4000 ya, but see Jinam et al. (2012) for genetic evidence suggesting the possibility of an even earlier arrival from around 10,000 ya. A date this early is totally impossible for the Aslian languages themselves. The Austroasiatic language family has an agricultural proto-language with terms for rice, first attested in the general region around 4000 years ago. People living 10,000 years were Hoabinhians, hunter-gatherers, with Australo-Papuan craniofacial features (Peter Bellwood, personal communication). Neolithic material culture including cord-marked pottery and rice cultivation has been described for several archaeological sites linked the Senoi way of life in Southern China, Vietnam and Thailand (Bellwood, 2005). These linguistic and archaeological reconstructions match well with those early studies on mtDNA and Y-chromosome markers (Ballinger et al., 1992; Melton et al., 1995, 1998; Kayser et al., 2000; Su et al., 2000; Macaulay et al., 2005; Trejaut et al., 2005; Hill et al., 2006; Ricaut et al., 2006). The Austronesians make up the final wave of the major human migration into the Island South East Asia (ISEA) region extended to include Peninsular Malaysia. The original story that developed from genetic studies generally pointed to either Taiwan or ISEA as the homeland of the Austronesians. These apparently competing accounts depended to some extent on which populations were being studied and what genes were tested (Chambers, 2006; Chambers and Edinur, 2015). Sometimes completely different pictures emerged because different genetic loci were used. For instance, data generated from a maternal marker (i.e. mitochondrial DNA) seemed to contradict those from paternal Y-chromosome DNA polymorphisms. The matrilineal mtDNA data suggested a Taiwanese ancestry for Austronesians in accord with ‘Out of Taiwan’ hypothesis (Hill et al., 2006; Melton et al., 1995). On the other hand, Y-chromosome patrilines indicated ISEA as the distal source of the great Austronesian Diaspora (Underhill et al., 2001). The controversy has been clarified by Tabbada et al. (2010) and Ko et al. (2014). These authors showed clear evidence for a southward dispersal of Austronesian mtDNA haplotype lineages, consistent with the ‘Out of Taiwan’ migration pattern leading into ISEA and across the Melanesia (Near Oceania) and Polynesia (Remote Oceania). Movement of the Austronesian-speaking populations is coupled with genderbiased introgression of paternal markers from indigenous Semang and Papuan populations, respectively. This is a particularly important process and arises because the Austronesian populations practise matrilocal marriages which greatly contribute towards the gender-biased gene flow (Jordan et al., 2009; Hage and Marck, 2003). It must be admitted that genetic evidence has its own limitations particularly when it arises from sex-limited genepools. Pre-historic reconstruction using these markers is much more affected by founder events and genetic drift than autosomal genes. It is fortunate, then, that several large scale, high resolution studies of autosomal loci have recently been reported; see the HUGO Pan-Asian SNP Consortium (2009), Deng et al. (2014, 2015), Hatin et al. (2011, 2014), Jinam et al. (2012), Lipson et al. (2014). These new works have provided a reliable genetic framework upon which to test historic reconstructions like the one advanced here. We will return to these later.


3. More recent historic events add further complexity We have shown that three highly differentiated genetic lineages make up the basic Malaysian Genetic Layer Cake, as we have chosen to call their national genepool. Now we come to several historic events that have contributed even further genetic complexity and admixture in Peninsular Malaysia. Starting from the first century till before the Empire of Malacca, Penisular Malaysia contained several independent empires, many of which had a strong influence from Indian and Buddhist culture (as evidenced by the Lembah Bujang remains; see Chun et al., 2005). The Champa Empire was established between 3rd and 15th century effectively and area including all of Southern of Vietnam and much of Cambodia. The Chams spoke Malayo-Polynesians and socio-economic interations did take place between the Chams and those people living on the Eastern Coast of Peninsular Malaysia and Northern coast of Borneo. It is apparent that only limited genetic admixture took place and similarities Malays and Cambodians due to cultural diffusion and linguistic replacement in the latter (Baker, 1999; Peng et al., 2010). Between the 6th and 7th century, the Melaka and Sunda Straits had became an important maritime trade routes following the fall of the Indianized Funan, Kedah and Langkasuka Empires, which previously controlled portage trade across the Isthmus of Kra (Baker, 1999). The Straits of Malacca, lie to the west of Peninsular Malaysia and sea traffic through there contributed greatly to the early establishment of the region as the major entreport for international traders from India, China and South East Asia. Due to the strategic location between the South and East Asia continents, the trade network reached as far as costal Africa, Arabia and Europe (Belle, 2014). The later Malay Empires, including Srivijaya (6th–13th century) in Sumatra, Majapahit (13th – 15th century) in Java and Melaka (15th–17th century) in Peninsular Malaysia then regulated trading activities in this part of Southeast Asia (Baker, 1999; Mohd Jali et al., 2003). These historical events not only involve interactions between Malays and India, Arabs and China traders, but also Orang Asli. The rapid rise of international trade activities in the Peninsular Malaysia led to increase demand for forest products which largely depended on supplies from Orang Asli (Andaya, 2002). This period was followed by European colonisations (Portuguese, Dutch and British) starting from the early of sixteenth century (see Swee-Hock, 2007). The European colonial powers brought in large groups of immigrant labours. For example, Chinese came to work in tin mines and Indians were recruited for work on tea and rubber plantations. In addition, the high reputations of successful trading ports in Peninsular Malaysia during the Malacca and Johor Empires attracted other Malay subethnic groups from nearby islands (Sumatra; Aceh, and Minangkabau Malays, Java; Jawa Malays, Sulawesi; Bugis Malays and Kalimantan; Banjar Malays) to move there during the last 300 years (Hussein et al., 2007). Despite the seemingly overwhelming frequency and magnitudes of these migrations over the past several hundred years, these events are not expected to cause genetic replacement in the contemporary Malays and Orang Asli tribes themselves. However, inflow of genes between these sub-populations of the Peninsular Malaysia and from the Indians, Arabs and Chinese traders plus the European colonists over most over the last 500–600 years is likely to have significant effects on their genepools (Hatin et al., 2014; Deng et al., 2015 and see later). Therefore, we can expect that they will have added minor/ major genetic noise to the mainstream prehistoric signals in particular sub-populations (see Fig. 1a). Therefore whilst it is likely true that the modern Malays are the descendant of the indigenous Proto-Malays, admixture components from Indian, East Asian and Indo-China populations should not be overlooked. The same genetic admixture effects were reported earlier for the Semang and Senoi (Deng et al., 2015). Minor exceptions can be seen in the Portuguese–Eurasian and Baba-Nyonya communities in Malacca. Both these groups emerged as new ethnicities with their own social/cultural identity but also absorbed some practises from their neighbouring sub-populations such as Malays, rather than integrating into the more uniform set

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of existing settlers in Peninsular Malaysia (Tan, 1979; Lee, 2008; Pillai et al., 2015). 4. The new genetic evidence Currently, it is possible to carry out large scale genetic screening; i.e. including single nucleotide polymorphism (SNP) genome wide association studies and whole genome sequencing. These two types of approach provide the most reliable data presently imaginable because they capture such extensive amounts of genetic information. However, even then improper interpretation of these data can also mislead one's perception (Lu and Xu, 2013). In this respect, SNP data reported by the HUGO Pan-Asian SNP Consortium (2009) was first taken to indicate a single wave of migration to ISEA. However, this study did not have an opportunity to include the most distant putative ancestors (i.e. Taiwanese aborigines and Papuans) of Australo-Papuan and Austronesian descendants currently living in ISEA. In addition, inferences were made largely based on the assumption of a strictly tree-like evolutionary process, which is simply not true for the reticulate mode of human evolution in ISEA. This region contains many different lineages of people with gene flow between populations. Thus, the most appropriate statistical method for analysis of such information should be able to partition populations into ancestral fractions and allow for gene flow between populations. Therefore, re-analysis of whole genome SNP data using such methods can facilitate and enhanced interpretations. Recently, several whole genome SNP analyses have been conducted on various populations in Peninsular Malaysia (Aghakhanian et al., 2015; Hatin et al., 2011, 2014; Jinam et al., 2013; Deng et al., 2014, 2015; Hoh et al., 2015; Liu et al., 2014, 2015). In general, these studies demonstrated several genetically differentiated layers in these populations (e.g. Malays, Proto-Malays, Senoi and Semang) in Peninsular Malaysia, but with significant admixture which reflect gene flow, at least since the first millennium (see Deng et al., 2015 and Fig. 1a). The study conducted by Deng et al. (2015) showed little admixture in Semang, but a larger scale of gene flow in Senoi and Proto-Malays/Malays. The genepool of the contemporary Senoi showed a greater gene flow from the Semang, as might be expected due to long term of contact between them, which lead to linguistic replacement in Semang. However, multiple ancestral fractions were reported for Proto-Malays/Malays, with the largest being from Taiwanese aborigines and Senoi and a lesser amount from Indians and Indo China populations. This is not surprising due to international trade activities in the ISEA, starting from Isthmus of Kra from 500BC and later shifted to Melaka and Sunda Straits around mid-ninth to fifteenth century (Andaya, 2002). Several entrepots in these regions emerged as re-distribution centres for forest products (e.g. resins and gaharu wood) collected by Orang Asli and exchanged for spices and silk which arrived from Maluku and China (Andaya, 2002). Further, the long-standing argument about Taiwan as a proximal point of Austronesians origin was recently examined in great detail by Lipson et al. (2014). These authors found strongly in favour of the ‘Out of Taiwan’ model, but with unexpectedly high levels of admixture in descendant migrants during the subsequent stages of the Austronesian Diaspora. The migration patterns inferred by these authors are presented in Fig. 1b, but have been re-drawn by the present authors to provide a better match between the voyaging pathways and local geography. The re-drawn map has Semang/Australo-Papuan going to the Philippines and towards Taiwan. The Austro-Asiatic ancestry component is geographically limited to the mainland and the Austro-Asiatic languages never spread into Indonesia as far as one can tell and there is no good trace of them there now. Finally, Austronesians first migrated from west to east along the northern coast of New Guinea, but not through the New Guinea Highlands at all. Linguistic and archaeological evidence takes them via islands to the north – Marianas, Palau, Admiralties and on to the Bismarcks, from where they colonized northern coastal mainland PNG itself (Peter Bellwoood, personal communication). Lipson et al. (2014) have identified a Formosan ancestry component which they

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Fig. 1. a. Gene pool of various populations inferred using whole genome single nucleotide polymorphism (SNP) analyses. This figure shows a clear description of unexpectedly high levels of admixture between genetically distinct lineages of people (Semang, Senoi and Proto-Malays) currently living in Island of South East Asia (ISEA). Adapted by permission from Macmillan Publishers Ltd.: [Scientific Reports] (Deng et al., 2015), copyright (2015). b. Multiple waves of human migrations into Island of South East Asia (ISEA) inferred from linguistic, archaeological and genetic data. Settlement of Peninsular Malaysia by Semang, Senoi and Proto-Malays are shown using red, green and blue arrows, respectively. Adapted by permission from Macmillan Publishers Ltd.: [Nature Communications] (Lipson et al., 2014), copyright (2014).


class as Austronesian, another for Philippine Negritos, and another for Papuans. During their expansion, Austronesians admixed with these two other indigenous Australo-Papuan groups (Peter Bellwood, personal communication). The third (shown in green) was identified by Lipson et al. (2014) as Htin, or Austroasiatic (AA), presumably because that language group is spoken by some mainland peoples today. In our opinion, the so-called Austro-Asiatic ancestry component in Indonesians might be of older mainland Asian origin since Austro-Asiatic languages and culture never spread down to Indonesia. At 10 kya, the whole today's ISEA was Australo-Papuan and many groups must have moved around. Only the Semang are left — now just a tiny and rather peripheral group. Ancient DNA analysis from well-preserved skeletons would help to clarify this story (Peter Bellwood, personal communication). Today, as indicated earlier, the Malays form the major population element of Peninsular Malaysia, followed by Chinese, Indians and other native groups in Borneo. Recently, Wong et al. (2013) reported whole genome sequencing data for Malay, Chinese and Indian individuals using next generation sequencing (NGS). Their data showed that Malays are significantly different from both Chinese and Indians. So the NGS technology now gives investigators capability to screen entire genomes at population genetic scale. It is confidently expected that this type of approach will now be applied to large numbers of populations in the near future, including for highly populated Southeast Asia region (Lu and Xu, 2013). In the interim, we hold that Malays are one set of descendants from the remarkably bold and successful prehistoric Austronesian Diaspora. Contemporary Deutero-Malays have an equal share of Semang heritage with Proto-Malays which distinguishes them from the uniquely Austronesian genepools found in the natives of Taiwan who have never been in significant contact with the Australo-Papuan populations as compared with their descendants in the ISEA (Chambers and Edinur, 2013, 2015). Thus, the DeuteroMalays remain quite similar to the ancestral Proto-Malays, but with varying levels of admixture from neighbouring sub-populations (Deng et al., 2015). 5. Findings from immunological markers Previous genetic surveys on human leukocyte antigen (HLA) genes have made significant contributions to our present understanding of the great Austronesian Diaspora (Edinur et al., 2012; Hagelberg et al., 1999; Riccio et al., 2013; Serjeantson et al., 1982; Zimdahl et al., 1999). The HLA loci are among the most polymorphic genetic markers available for use in ancestry studies and investigators can often draw on abundant data produced by tissue typing laboratories during routine typing work. For instance, Edinur et al. (2009) used low resolution molecular typing HLA data to show genetic relationships between Malay sub-ethnic groups. They were able to trace them back to their points of origin in Sumatra (Rawa and Minangkabau Malays), Sulawesi (Banjar Malays), Java (Jawa Malays). Except for Kelantan Malays, the Malay sub-ethnic groups all arrived fairly recently in Peninsular Malaysia (2000–600 ya) and are only slightly differentiated from the otherwise ancestrally related Proto-Malays who had settled earlier (3000 ya) and their descendants the modern Malays (Andaya, 2002). Edinur et al. (2009) were also able to show the influence of neighbouring Thai populations on Kelantan Malays. This earlier HLA study did not have the opportunity to include data from other relevant populations in their ethnogenesis analysis. These include HLA data for Orang Asli groups Semang (Jehai and Kensiu) and Proto-Malays (Temuan) as reported later by Jinam et al. (2010). The study conducted by Jinam et al. (2010) does not show marked separation between some of populations e.g. those residents in Java, Borneo (Bidayuh) and The Philippines, Malays, Temuan, Jehai and Kensiu. The Semang and Proto-Malays appear to be quite closely related to Austronesian populations, including Malays, Javanese and Filipinos. This observation may indicate historic gene flow between various sub-populations in Peninsular Malaysia. The same effect

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can be seen in the Kelantan Malays who might well have received genetic influence from populations to their north (i.e. as suggested by Edinur et al., 2009). We should note one caveat here; specifically that HLA loci can be affected by local selective forces, which reflects their key role in the immune system (see Edinur et al., 2013; Sanchez-Mazas et al., 2011; Solberg et al., 2008 for more details). The same local selective forces may have shaped the genepool of Semang, Senoi, Proto-Malays since they first settled in Peninsular Malaysia (Liu et al., 2013, 2015). We have also tested this Malaysian Genetic Layer Cake using several markers that are important in transfusion (blood groups, human platelet antigen and human neutrophil antigen) and transplantation (killer-cell immunoglobulin-like receptor and cytokine genes) medicine using DNA samples collected from several Malay subethnic volunteers (e.g. Banjar, Bugis, Champa, Jawa and Kelantan, Minangkabau, Mandailing and Patani Malays) currently living in various places in Peninsular Malaysia (see Abd Gani et al., 2015; Manaf et al., 2015; Norhalifah et al., 2015; NurWaliyuddin et al., 2014; Wan Syafawati et al., 2015). Generally, our statistical comparisons showed a common Austronesian origin for the studied Malay subethnic groups and minor exceptions may be associated with population histories such as founder effects, isolation and admixture. We are now screening the same sets of genetic markers across several other Orang Asli groups and sub-groups (Semang; Lanoh, Kensiu and Batek, Senoi; Che Wong and Semai and Proto-Malays; Orang Kanaq). It is expected that the new population data that will emerge from our work on these medically important markers will help to increase further our understanding of the complex genetic histories and health of several distinct sub-populations/groups in Malaysia. 6. Concluding observations and future directions The Malaysian Genetic Layer Cake has three basic tiers (i.e. the Orang Asli groups; Semang, Senoi and Proto-Malays) and two toppings (Malay subethnic groups plus worldwide immigrants). The foundation is formed by ancient Semang people followed by the Senoi who form the second tier (Bellwood, 2005). The third layer is made up of admixed Austronesians (Tumonggor et al., 2013). The Malay subethnic groups arrived in fairly recent historical times from neighbouring regions, mainly Sulawesi, Java and Sumatra and may perhaps arguably be considered to form a fourth layer to the national cake rather than a topping. In genetic terms, these later immigrant populations are almost all mainstream Austronesians stock (Edinur et al., 2009) and so do not add greatly to the genetic diversity already present in the three foundation layers. Later episodes of admixture of the now-settled peninsular peoples took place with Arab, Chinese and Indian traders and provide the final topping of new genetic variation. Thus, we conclude that one must agree with the proposition that human history in Malaysia is complex. It involves at least three independent founding waves with each one bringing their own novel genetic material into the region (Semang, Senoi and Proto-Malays/Austronesians). They have been followed by later settlers from many other places who have enjoyed lesser or greater admixture with existing residents. This picture is now well supported by the extensive high resolution genetic analyses reviewed here. Overall, we feel that it is important to recognize that Malaysia (Borneo and Peninsular Malaysia) is a multi-ethnic country which comprises of people from very different genetic lineages; Semang, Senoi, Proto-Malays, Malays, Chinese and Indians. Given the extensive admixture in the descendants of ancient sub-populations in Peninsular Malaysia/ISEA, reconstruction of human migration history in this region require a new evidence from study of ancient DNA and well preserved skeletons (Peter Bellwood, personal communication). Until then, any findings from genetic study of modern DNA should be interpreted with caution particularly those that may contradict archaeological findings and chronology. This is because culture and genetics can change in remarkably different ways and a quite disconnected rates leading to potential confusion. This lesson can learned well from genetic

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reconstruction of population history in Peninsular Malaysia as reviewed here. In other aspects, the highly diverse and complex genetic makeup in Peninsular Malaysia should be considered carefully for any genetic mapping of disease and in new policies set up by health authorities (e.g. see Abd Gani et al., 2015; Hoh et al., 2015). Future genetic surveys should include loci of medical significance e.g. candidate genes for gout diabetes etc. and investigators should be aware that differences in frequencies of candidate loci may help to explain differential disease schedules between ethnic groups. Conflict of interest The authors declare that they have no conflict of interest. Acknowledgements The authors acknowledge support given by Universiti Sains Malaysia (Short Term Grant Scheme; 304/PPSK/61313062) and Geoffrey Keith Chambers thanks Victoria University of Wellington for alumnus scholar support. 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