Keywords: Tuareg, genetic diversity, phylogeography

Subjects

The biological samples (buccal swabs) were obtained from three different groups of self-identified Tuareg (90 unrelated individuals in total). One population sample (n=38) was secured in Burkina Faso around the village Gorom-Gorom (further referred to as TGor). The second sample (n=31) was taken in the Republic of Niger in the vicinity of Tanut (TTan). The third sample (n=21) was collected in Mali near Gossi (TGos). The samples from Mali and Burkina Faso are geographically relatively close to each other as they are located within the bend of the Niger River, whereas the sample collected in the central part of the Republic of Niger is located some 1500 km eastward (Figure 1). The field sampling was undertaken with the collaboration of local Tuareg assistants. Of these 90 healthy and unrelated individuals, 47 were male and 43 were female. Oral informed consent was obtained from all participants in the study and research permits were obtained from the Ministries of Education and/or Health in all the three countries.

Laboratory analyses

DNA extractions and PCR amplifications of mtDNA hypervariable segments I and II (HVS-I and HVS-II) were carried out as in earlier studies.^6,7 Amplicons were purified and sequenced using forward PCR primers. In some cases the reverse primer was also used for sequencing (due to for example, the presence of poly-C stretches between nt 16184 and 16193). In some samples, SNP testing was analysed through matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and minisequencing.^7,8 Whole genome sequencing of mtDNAs affiliated by D-loop to the haplogroup M1 was undertaken following the protocols reported elsewhere.^7,9,10,11

For the detection of Y chromosome SNP polymorphisms, the Signet Y-SNP Identification System v 2.0 (Marligen, Rockville, MD, USA) was used. All the samples were first analysed by A-R multiplex (polymorphisms M122, M168, M175, M207, M304, M343, M45, M89, M96 and M9), differentiating main evolutionary branches of Y chromosome phylogeny.¹² Subsequently, samples belonging to haplogroup E were further analysed for polymorphisms DYS391, M2, M33, M35, M58, M75, M78, M81 and M123.

Haplogroup nomenclature

mtDNA classification into haplogroups L, H, U and M was carried out in accordance with the most recent phylogenetic studies of Salaset al,¹³ Kivisildet al¹⁴ and Beharet al¹⁵ for L; Olivieriet al¹⁶ for U and M; and Achilliet al¹⁷ for H (see also van Oven and Kayser, 2009).¹⁸ The numbering is consistent with the revised Cambridge Reference Sequence.¹⁹ The three complete mtDNA sequences have been deposited in GenBank (accession numbersGQ377749;GQ377750; andGQ377751). For the Y chromosome SNP affiliation, the nomenclature of Karafetet al¹² was followed.

Statistical and phylogenetic analyses

Analysis of population structure and molecular diversity measures was calculated by using Arlequin software version 3.0.²⁰ Two-tailed Fisher's exact testP-values of 2 × 2 contingency tables were calculated in DnaSP.²¹F_ST genetic distances calculated by Arlequin were subsequently visualized in multidimensional scaling (MDS) by means of SPSS 10.0 software (SPSS Inc, Chicago, IL, USA). Extensive data sets of both mtDNA sequences and Y-SNPs were used to characterize the Tuareg diversity within Mediterranean and sub-Saharan population contexts (see Supplementary Material SM1 and SM2).

Phylogenetic reconstruction of mtDNA diversity was based on both HVS-I and complete sequences. The dates of the most recent common ancestor of specific subclusters in the phylogeny were estimated usingρ.²² The average number of transitions from the ancestral haplotype to all haplotypes in the cluster, for both coding (between positions 577 and 16 023) and HVS-I (between positions 16 090 and 16 365) regions, was considered with respect to mutation rate estimates of 5138 years²³ and 20 180 years per transition²⁴ within the region, respectively. Standard errors were calculated as in Saillardet al.²⁵ Recently, updated mutation rates published by Soareset al²⁶ were also used for the entire molecule (1 mutation every 3624 years) and synonymous substitutions (1 mutation every 7884 years). As the mutation rate determined by these authors for the HVS-I (between positions 16 090 and 16 365) region, however, is very similar to the one used above (1 transition every 20 129 yearsversus 20 180 years) the calculations overlapped and we present only those estimated with the originalρ mutation rate of 1 substitution every 20 180 years.

Interpolation maps

To determine and visualize the geographical distribution of haplogroups H1, H3 and V, we drew interpolation maps using the ‘Spatial Analyst Extension' of ArcView version 3.2 (http://www.esri.com/software/arcview/). Inverse distance weighted option that we used assumes that each input point has a local influence that diminishes with distance. The geographic location used is the centre of the distribution area, from where the individual samples of each population were collected. Comparative data for H1 and H3 were taken from Finnilaet al,²⁷ Herrnstadtet al,²⁸ Pereiraet al,²⁹ Cherniet al³⁰ and Ennafaaet al;³¹ and those for haplogroup V from Torroniet al,¹⁰ Pereiraet al,³² Beharet al³³ and Cherniet al.³⁰

The mtDNA pool of the Tuareg

The polymorphisms present in the 90 Tuareg individuals led to the identification of 53 different D-loop haplotypes (Table 1). As can be seen in the network based only on HVS-I diversity (Supplementary Material SM3), for which only 33 different haplotypes are observed, there are varying degrees of sharing of haplotypes among the analysed groups: only one belonging to haplogroup H was shared by all three groups; two haplotypes were shared by TGos–TGor and TGos–TTan; and three haplotypes were shared by TGor–TTan. Only 18 of the 33 haplotypes are unique, what is a rather low proportion when compared to most African samples.³² This is further corroborated by haplotype diversities in the three Tuareg samples, which are lower as compared with other populations – especially in the two groups of the Niger bend (0.861±0.027 in TGor; 0.910±0.037 in TGos; and 0.963±0.020 in TTan; see Supplementary Material SM4).

A total of 48% of the mtDNA haplotypes observed in the Tuareg populations could be ascribed to sub-Saharan haplogroups. Another 39%, however, were of West Eurasian ancestry (non-L types inTable 1), which is a substantial proportion considering the sub-Saharan geographical location. In fact, it has been observed that in typical North African populations there is a gradient of increasing frequency of West Eurasian lineages ranging from around 50–75% in the northernmost locations.³⁴ The Tuareg's neighbours, however, have a markedly smaller proportion of West Eurasian haplotypes (22% in Western Chad Arabs, 8% in Shuwa Arabs from North-eastern Nigeria, 7% in the Buduma from South-eastern Niger and 6% in the Kanuri from North-eastern Nigeria).³⁵ The remaining 13% of Tuareg haplotypes belong to the typical East African haplogroup M1.

Furthermore, we noticed some differences in the distribution of West Eurasian mtDNA haplogroups between Tuareg groups. Most of the West Eurasian haplogroups (30 out of 35 sequences, amounting to 6 out of 9 HVS-I haplotypes) and the East African M1 (11 out of 12 sequences but amounting to only 2 out of 3 HVS-I haplotypes) are observed in the two Tuareg populations – TGos and TGor – located within the bend of the Niger. Tuareg from the Republic of Niger, TTan, have much higher proportion of sub-Saharan (81%) haplogroups than of West Eurasian (16%) and East African (3%) ones. These differences in haplogroup distribution led to statistically significant genetic distances when comparing HVS-I haplotypes between Tuareg from Mali (TGos) with those from the Republic of Niger (TTan) (F_ST=0.048; unadjustedP-value=0.009), as well as Tuareg from Burkina Faso (TGor) with those from the Republic of Niger (TTan) (F_ST=0.064; unadjustedP-value=0.000), whereas Tuareg from Mali (TGos) and from Burkina Faso (TGor) are not statistically different (F_ST=0.012; unadjustedP-value=0.234). Similarly, analysis of MDS based onF_ST distances and using a large database of West Eurasian and African mtDNA sequences has shown a very good separation of the sub-Saharan and West Eurasian-North African gene pools (Figure 2). Only some East African populations are closer to the West Eurasian samples, respectively, to the North African populations analysed here. This picture is a good representation ofF_ST values as the normalized raw stress is very low (0.01165). However, the analysed Tuareg populations are divided between two gene pools: like the sample from Libya,⁵ the groups located within the bend of Niger (TGor and TGos) fall into the West Eurasian gene pool, whereas the Tuareg from the Republic of Niger (TTan) and the Tuareg sample from the Watson's data set^3,4 are permeated by the sub-Saharan mtDNA gene pool.

The West Eurasian component observed in the Tuareg is highly interesting. A major proportion (94%) could be allocated to haplogroups H1, H3 and V, West Eurasian lineages of Iberian origin that spread to Europe^{7,10,17,26,29,36} and most probably North Africa^30,31 with the improvement of the climatic conditions after the retreat of the ice sheets 15 000–13 000 years ago. The interpolation maps of these lineages across North Africa and Europe (Supplementary Material SM5) clearly place the Tuareg population in the path of the southern African edge of post-Last Glacial Maximum expansions. The H1 haplogroup (Supplementary Material SM5A and SM5B, with and without the outlier Norway, respectively) is as frequent in our southern Tuareg groups as in Libya and the centre of the dispersion within the Iberian Peninsula. The H3 haplogroup is almost vestigial in Tuareg (Supplementary Material SM5C), having the highest observed frequencies outside of Iberia in Algeria and Tunisia. Again for haplogroup V, Tuareg present frequencies as high as in the Basque country (Supplementary Material SM5D).

Both H1 and H3 commonly display rather low diversity in the D-loop region, but the Tuareg haplotypes belonging to haplogroup V have a specific diagnostic mutation – the transition at position 16 234. All the Tuareg V haplotype samples collected in Burkina Faso and the Republic of Niger (three haplotypes observed in 11 individuals) bear this mutation together with the defining substitution at position 16 298, This polymorphism is present in two of the five V haplotypes observed in the recently published Libyan Tuaregs.⁵ This fact seems to point to a founder effect in haplogroup V occurring in our southern Tuareg population; the further presence of two other polymorphisms in two V samples (substitutions at positions 16 189 and 16 293) allows a very preliminary estimation for the Time to the Most Recent Common Ancestor (TMRCA) of this Tuareg V sub-lineage at around 3600±2600 years ago or at a maximum of 8800 years ago if using a 95% confidence interval (see Supplementary Material SM6 for the network).

Another very interesting characteristic of the West Eurasian mtDNA pool in the Tuareg population as a whole (including Watson's and Ottoni's data sets) is the total absence of so-called Neolithic haplogroups derived from the branch JT, which are otherwise common in Near Eastern, North African, Mediterranean and even some East African populations. The virtual absence of these lineages in the Tuareg is statistically significant when comparing the frequency of these lineages in Morocco³⁴ (18% unadjustedP-value=0.000), Tunisia³⁰ (12% unadjustedP-value=0.000) and Egypt³⁷ (29% unadjustedP-value=0.000). Notice also the absence of the haplogroup U6, which is present mainly in Berbers but also in several others North African groups.^13,16,38

The sub-Saharan mtDNA pool of the Tuareg is composed of various lineages from the major L-type haplogroups including: 2.3% of L0; 14.0% of L1; 58.1% of L2; 23.3% L3; and 2.3% of L4. We assayed to search for haplotype matches in an extensive database of 7211 individuals from all over Africa (Table 2). The most ancient lineages L0a1a and L1c, characteristic of east/southeast Africa¹³ and the Pygmies,³⁹ respectively, were each observed in only one individual. The highly frequent African haplogroup L2, and specifically its dominant clade L2a, is also dominant in Tuareg – it is probable that some branches of L2a were involved in the Bantu expansion towards the African south^13,40 and many matches are observed for these haplotypes all over the continent. Curiously, the two L2a lineages having substitutions at positions 16 192 and 16 193, respectively, have no match in Africa. As far as the L3 macrohaplogroup is concerned, the two L3b haplotypes observed in the Tuareg are widespread throughout the continent, but one of the L3f1 haplotypes (T47 inTable 1) has no matches. Both are included in the L3f1 sub-haplogroup, which is quite frequent and widespread, and which very probably originated in East Africa. No L3f3, a typical marker of the Chadic migration,⁴¹ has been observed in the Tuareg.

In summary, the matches between Tuareg sub-Saharan haplotypes and the diverse African regions were, after correcting for the size for each region, 5.6% with Africa-Central; 4.3% with Africa-East; 3.4% with Africa-North; 1.1% with Africa-South; 4.3% with Africa-southeast; 4.5% with Africa-southwest; 12.7% with Africa-West; and 13.4% with Africa-westcentral. The West Africa or West-Central African lineages thus are clearly dominant in the extant Tuareg.

The influence of East Africa in the Tuareg can be investigated more directly through haplogroup M1.¹⁶ As concerns the finer classification of Tuareg M1 haplotypes, two of them (5 sequences out of 12) belong to M1b, which has a clear Mediterranean distribution, pointing to North Africa as its most probable gateway to the Tuareg. This finding is inconsistent with the absence of U6, which is believed to have entered Africa together with M1 in a back migration from western Eurasia around 45 000 years ago. The time estimate for M1b, based on the coding region, is 23 400±5600 years,¹⁶ placing its origin in the Early Upper Palaeolithic. More promising in ascertaining Eastern African origin is another haplotype observed in seven Tuareg individuals from Burkina Faso belonging to haplogroup M1a, which, though being considered dominant in East Africa⁴² also spread to the Mediterranean, and which has a total age of 28 800±4900 years.¹⁶ We performed the complete sequencing of three individuals, which despite not displaying any difference at HVS-I and HVS-II, might present some substitutions in the coding region, allowing for a better estimate of a TMRCA. These three samples, however, did not bear any difference even when sequencing the complete genome. Nonetheless, when taken together with the other M1a2a individuals (Figure 3) reported in Olivieriet al¹⁶ (sample 1 inFigure 3, accession numberEF060335; sample 2, accession numberEF060336), Gonzálezet al⁴³ (sample 3; accession numberDQ779927) and Maca-Meyeret al⁴⁴ (sample 4; accession numberAF381984) allowed an age estimation for this sub-haplogroup at 8000±2400-years old based on diversity in the coding region. We checked the TMRCA using Soareset al²⁶ mutations rates for the entire molecule and for the synonymous substitutions, obtaining, respectively, the following concordant dates: 10 400±2300 and 10 200±3400. Notice, however, that all the other four M1a2a complete sequences were observed in the Mediterranean region and inTable 2 the HVS-I motif observed in Tuareg has 10 perfect matches in the Africa-North data set and one in Africa-westcentral.

Y chromosome pool in Tuareg

From the 20 branches of the Y chromosome tree, which could be discriminated by the analyses performed, only 7 were observed in our Tuareg population sample (Supplementary Material SM7). Again, from this perspective of Y chromosome diversity, TTan is closer to sub-Saharan populations than the other two Tuareg populations, presenting 5.6% of the old AB lineages and 44.4% of E1b1a, whereas TGor and TGos have, respectively, 16.7 and 9.1% of E1b1a. Curiously, TTan also presents the highest frequency (33.3%) of West Eurasian R1b lineages whereas TGor presents only 5.6% of lineage K^* (xO,P), and TGos presents none. There were no instances of the Eurasian J haplogroup in the Tuareg, which is otherwise frequent in North Africa (an average of 20% see Arrediet al⁴⁵), and attains the highest frequency in the Middle East (around 50% see Seminoet al)⁴⁶.

The dominant haplogroup in TGor (77.8%) and TGos (81.8%) is E1b1b1b, which has a much lower frequency in TTan (11.1%). This haplogroup reaches a mean frequency of 42% in North Africa, decreasing in frequency from 76% in Morocco to ∼10% in Egypt.⁴⁵ Arrediet al⁴⁵ dated this haplogroup in North Africa from 2800 to 9800 YBP, associating its expansion with the Neolithic demic diffusion of Afro-Asiatic-speaking pastoralists from the Middle East.

The low level of diversity attained in the Tuareg populations (see Supplementary Material SM8) is consistent with a model of population constancy, although it can also be due in part to the ascertainment bias in the selection of a few Y-SNPs. Haplotype diversities and mean number of pairwise differences were very low in TGor and TGos, being among the lowest values observed in many populations, but TTan showed much higher levels of diversity.

MDS ofF_ST distances based on available Y-SNP West Eurasian and African population data sets shows, as in the case of mtDNA, separation of the West Eurasian-North African and sub-Saharan populations (Figure 4). A certain separation between the Iberian and Near Eastern groups can be explained by the absence of samples from the Central Mediterranean for the Y-NRY data set. However, though the Tuareg groups from the Niger bend (TGor and TGos) belong clearly on the West Eurasian side, the Tuareg from central Niger lean towards sub-Saharan variability.

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