Geographic distribution of M1

Figure 2 shows the reduced median network obtained from the 261 M1 haplotypes found in a global search comprising more than 38,713 HVSI sequences. In Africa, haplogroup M1 has supra-equatorial distribution (see additional files 1 and 2). As previously reported its highest frequencies and diversities (Table 2) are found in Ethiopia in particular and in East Africa in general. Two appreciable gradients exist. Frequencies significantly diminished from East to West and also going South to sub-Saharan areas. M1 is not uncommon in the Mediterranean basin showing a peak in the Iberian Peninsula. However, it is rare in continental Europe. Although in low frequencies, its presence in the Middle East has been well established from the South of the Arabian Peninsula to Anatolia and from the Levant to Iran. The central HVSI haplotype (16129–16189–16223–16249–16311) has been found only once in northwestern India [27]. Another possible Indian M1 candidate is the derived sequence: 16086–16129–16223–16249–16259–16311 [28]. However, in two recent studies in which 24 [24] and 56 [25] Indian M complete sequences were analyzed no ancestral M1 lineages have been found. M1 haplotypes have also been occasionally spotted in the Caucasus and the Trans Caucasus [23, 29] and in Central Asia [30]. It seems that, going east, M1 even reached the Tibet as the HVSI diagnostic motif was sampled there [31]. However, although haplotypes sharing four of the five HVSI transitions defining M1 (16129–16223–16249–16278–16311–16362; 16129–16223–16234–16249–16311–16362) have been sampled in Thailand and Han Chinese [32, 33], complete sequencing have unequivocally allocated them in the D4a branch of D, the most abundant haplogroup representing M in East Asia. As commented previously, this is a clear example of the danger of establishing affinities between geographically distant areas only on the basis of HVSI homologies as, often, they are the product of geographic isolation and molecular convergence [18]. Within this sparse but geographically wide range of M1 distribution its three identified branches also had uneven radiations. Although M1a (HVSI identified by the 16359 transition) is present in all the M1 range, its greatest frequencies and diversities are found in Ethiopia and eastern Africa (Table 2), pointing to this area as the most probable origin of the M1a expansion in all directions, with particular incidence in western Asia and sub-Saharan Africa. Not all the M1b lineages can be HVSI identified; however, several specific subclades have different locations. Those characterized by transitions 16260–16320 [21], and by presence of 16182 transition and 16265C transversion [22] are restricted to Ethiopia with occasional spreads to eastern Africa. In addition, there is an M1b branch, identified by 16185 transition and 16190 deletion that has a northwestern distribution excepting a Jordan haplotype (Fig. 2). Despite that M1c cannot be unequivocally defined by transition 16185, it can be stated that M1c is an overwhelmingly Northwest African clade which spreads to the Mediterranean and West sub-Saharan Africa areas. Finally, other unclassified M1 branches have also different geographic ranges. Those identified by the presence of 16357 transition and by the reversion of the diagnostic position 16129 are of Ethiopian eastern Africa adscription, while clusters characterized by loss of the diagnostic position 16223 and by the 16399 transition have a northwestern distribution (Fig. 2). However, M1 assignation of haplotypes, which lack any of the basic positions, based only on HVSI information is risky when they share other diagnostic positions with different haplogroups. For instance, the Russian haplotype 16183C–16189–16249–16311, classified as M1 on the basis of its HVSI sequence [34] also matches with haplotypes assigned to the U1a clade [35].

a)	Locals
namea	sample	nM1b	%M1	pi*1000	h(%)	nM1a	%M1a	pi*1000	h(%)
Europe
IPE	5007	34	0.7	5.6 ± 3.8	84 ± 3	10	29	1.4 ± 1.7	38 ± 18
MEU	3278	15	0.5	5.5 ± 3.9	76 ± 10	9	60	3.0 ± 2.7	42 ± 19
REU	10735	1	0.0	-	-	1	100	-	-
TEU	19020	50	0.3	5.8 ± 3.9	84 ± 3	20	40	2.5 ± 2.2	45 ± 14
Africa
NWA	1175	38	3.2	3.9 ± 2.9	70 ± 7	2	5	0.0 ± 0.0	0 ± 0
CWA	2351	7	0.3	7.2 ± 5.3	86 ± 14	1	14	-	-
NEA	288	23	8.0	5.7 ± 3.9	85 ± 5	13	57	4.9 ± 3.7	78 ± 10
ETH	344	53(78)	15.4	9.4 ± 5.6	92 ± 2	45	58	5.5 ± 3.7	82 ± 5
CEA	533	61(86)	11.4	9.1 ± 5.5	92 ± 2	48	56	5.4 ± 3.7	81 ± 5
WA	3526	45	1.3	4.5 ± 3.2	75 ± 6	3	7	0.0 ± 0.0	0 ± 0
EA	821	84(109)	10.2	8.5 ± 5.2	92 ± 2	61	56	5.5 ± 3.7	81 ± 5
SEA	4347	129(154)	3.0	7.9 ± 4.9	91 ± 1	64	42	5.2 ± 3.6	79 ± 5
Asia
WAS	7589	36(28)	0.5	5.5 ± 3.8	78 ± 8	18	64	2.8 ± 2.4	49 ± 14
Total
TOT	30956	215(232)	0.7	7.2 ± 4.5	89 ± 1	102	44	4.4 ± 3.1	69 ± 5
EAs	5912	0	0.0	-	-	-	-	-	-
SAf	596	0	0.0	-	-	-	-	-	-
b)	Jews
namea	sample	nM1b	%M1	pi*1000	h(%)	nM1a	%M1a	pi*1000	h(%)
Europe
JIP	48	2	4.2	3.6 ± 5.1	100 ± 50	2	100	3.6 ± 5.1	100 ± 50
JRE	663	6	0.9	0.0 ± 0.0	0 ± 0	6	100	0.0 ± 0.0	0 ± 0
JTE	711	8	1.1	2.5 ± 2.4	46 ± 20	8	100	2.5 ± 2.4	46 ± 20
Africa
JNWA	135	0	0.0	-	-	-	-	-	-
JNEA	20	0	0.0	-	-	-	-	-	-
JET	69	14	20.3	5.7 ± 4.0	81 ± 7	12	86	3.8 ± 3.1	76 ± 8
JCEA	69	14	20.3			12	86
JEA	89	14	15.7			12	86
JSEA	224	14	6.3			12	86
Asia
JAS	473	7	1.5	0.0 ± 0.0	0 ± 0	7	100	0.0 ± 0.0	0 ± 0
Total
JEW	1408	29	2.1	6.3 ± 4.2	86 ± 3	27	93	5.6 ± 3.8	84 ± 3
c)	Totals
namea	sample	nM1b	%M1	pi*1000	h(%)	nM1a	%M1a	pi*1000	h(%)
Europe
IPEj	5055	36	0.7	5.7 ± 3.8	84 ± 3	12	33	1.7 ± 1.8	44 ± 16
REUj	11398	7	0.1	2.1 ± 2.2	29 ± 20	7	100	2.1 ± 2.2	29 ± 20
TEUj	19731	58	0.3	6.2 ± 4.1	86 ± 3	28	48	3.3 ± 2.6	64 ± 9
Africa
NWA	1310	38	2.9			2	5
NEA	308	23	7.5			13	57
ETHj	413	67(92)	16.2	9.1 ± 5.5	92 ± 2	57	62	5.4 ± 3.6	82 ± 4
CEAj	602	75 (100)	12.5	8.9 ± 5.4	92 ± 2	60	60	5.3 ± 3.6	81 ± 4
EAj	910	98(123)	10.8	8.4 ± 5.1	92 ± 2	73	59	5.4 ± 3.7	82 ± 4
SEAj	4571	143(168)	3.1	8.0 ± 4.9	92 ± 1	78	45	5.5 ± 3.7	81 ± 4
Asia
WASj	8062	43(35)	0.5	5.7 ± 3.9	82 ± 5	25	71	3.6 ± 2.3	67 ± 9
Total
TOTj	32364	244(261)	0.8	7.4 ± 4.6	90 ± 1	129	49	4.8 ± 3.3	77 ± 4

^a Name for locals, Jews and total for different areas: Europe: Iberian Peninsula and related Islands (IPE, JIP, IPEj), Europe Mediterranean area (MEU), Rest of Europe (REU, JRE, REUj), and total Europe (TEU, JTE, TEUj); Africa: Northwest Africa (NWA, JNWA), Central West Africa (CWA), Northeast Africa (NEA, JNEA), Ethiopia (ETH, JET, ETHj), Central East Africa (CEA, JCEA, CEAj), East Africa (EA, JEA, EAj), West Africa (WA) and Supra-Equatorial Africa (SEA, JSEA, SEAj); Asia: West Asia (WAS, JAS, WASj); Total of these samples (without South Africa and East Asia) (TOT, JEW, TOTj); East Asia (EAs); South Africa (SAf).

^b Number outside and inside the parenthesis were used to estimate the M1 and M1a frequency, respectively.

M1 haplotypes in Jews

Several M1 haplotypes have been detected in Jewish communities albeit in low frequencies [36, 37]. However, when compared with non-Jew populations they show significantly higher frequencies for the whole M1 haplogroup (p = 33.54***) and for M1a in particular (p = 24.90***). The only striking exception is that of the Moroccan Jews for which no M1 lineages have been detected at all [36]. Interestingly, all M1 lineages found in Jews, except two, belong to the eastern clade M1a (Fig. 2). Therefore, as for the bulk of the M1 Near East haplotypes, the most probable origin of these Jewish M1 lineages is the result of an eastern African expansion around 5000 years ago. Another peculiarity of M1 in Jewish communities is its reduced haplotypic diversity (Table 2) which has been already detected for other mtDNA lineages [36, 38]. In addition, there is a strong M1 geographic differentiation among Jewish communities. For example, all European Ashkenazi Jews have only one M1a lineage characterized by a transition in the 16289 position that has not been detected in other Jew or non-Jew populations. Similarly, all West Asian Jews shared an identical M1a motif characterized by a transition in the 16209 position that has been detected only once in Ethiopia. These results are congruent with the proposition that, in the majority of the cases, Jewish migrations implied strong maternal founder effects [36–38]. Nevertheless, as M1a Jewish lineages are unique and different in different groups, we think that its source Near East population should not suffer strong genetic bottlenecks. Finally, it is worth mentioning that M1 frequencies of Jewish groups and their host populations are significantly correlated (r = 0.942**) which suggests that some genetic interchange must have happened between them as already proposed by others authors [36, 37].

Radiation ages and evolution of lineages

Phylogeographic parallelism between M1 and U6 haplogroups

There are striking similarities between the geographical dispersals and radiation ages observed here for M1 lineages and those previously published for the North African U6 haplogroup [40]. It was proposed that U6a first spread was in Northwest Africa around 30,000 ya. Coalescence ages for M1 also fit into this period and the oldest clade M1c has an evident northwestern Africa distribution; however it had to have a wide geographic range as some M1c lineages are today still present in Jordanians (Figs. 1 and 2). It is curious that this prehistoric Near Eastern colonization was also pointed out by the uniqueness of the U6a haplotypes detected in that area. A posterior East to West African expansion around 17,000 ya was indicated by the U6a1 relative diversity and distribution. Again, age, relative East to West diversities and geographic range accurately correspond with the M1a1 expansion detected here. More recent local spread of lineages U6b and U6c also parallel the M1b and M1c1 distributions. Furthermore, these similarities also hold outside Africa. U6 lineages in the Iberian Peninsula have been considered traces of northward expansions from Africa. Based on the uneven distribution of U6a and U6b lineages in Iberia, with the former predominating in southern and the latter in northern areas, it was proposed that U6b in Iberia represents a signal of a prehistoric North African immigration whereas the presence of U6a could be better attributed to the long lasting historic Arab/Berber occupation [40]. Again, this pattern is accurately repeated by the M1c and M1a distribution in the Iberian Peninsula, the northwest African M1 being more abundant in northern areas (56%) and the East African M1a in southern areas (85%) although, due to the small sample size, difference does not reach a significant level (p = 0.07). Additional support to the hypothesis of a prehistoric introduction are the recently detected presence of a Northwest African M1c lineage in a Basque cemetery dated to the 6^th–7^th centuries AD, prior to the Moorish occupation [42], and the ancestral phylogenetic position of another Basque M1d sequence (Fig. 1) that does not match any African sequence. Finally, two autochthonous U6 lineages (U6b1 and U6c1) traced the origin of the Canary Islands prehispanic aborigines to Northwest Africa [43]. Although exclusive M1 lineages have not been detected in the Canary Islands, it is worth mentioning that those sampled belong to the Northwest African area [44]. Outside Africa and the Iberian Peninsula, as with U6, M1 has been mainly detected in other Mediterranean areas with main incidences in islands such as Sicily. It is customary to attribute these incidences to the above mentioned Arab/Berber historic occupations. However, taking into account the major Jewish assignation for all the M1a haplotypes detected in Europe, the possibility of a Jewish maternal ascendance for at least some of these lineages should not be rejected.

Note that the two M1 lineages sampled in the Balearic isles were of Jewish adscription [45]. Also, there were well documented Jewish settlements in Sicily since early Roman times [46] and, coincidentally, half of the M1 lineages sampled in that island [47, 48] belong to the M1a cluster. Finally, the Atlantic archipelagos of Canaries and Madeira, where the rigor of the Spanish Inquisition was stronger, only have M1c representatives. In contrast, in the Azores Islands, that were used as a refuge by Sephardim Jews expelled from the Iberian Peninsula, half of the M1 sequences detected are of M1a assignation [49, 50]. These possible Jewish contributions might be also extended to the U6 lineages of eastern origin because all U6 haplotypes detected in Ashkenazim and other Jewish groups, excepting one that is a basal U6a (16172–16219–16278), belong to the eastern Africa clade U6a1 [36, 26]. An additional proof of the striking parallelism between M1 and U6 lineages is the fact that, as for M1, no U6 representatives were sampled in Moroccan Jews in spite of the high frequency of this clade in the Moroccan and Berber host populations [36].

Most probable origin of M1 ancestors

Mitochondrial M lineages in Ethiopia were first detected by RFLP analyses [51]. To explain its presence in that area the authors suggested two possibilities: 1) the marker was acquired by Ethiopians through interchanges with Asians or 2) it was present in the ancient Ethiopian population and was carried to Asia by groups who migrated out of Africa. Later, the second hypothesis was favored and a single origin of haplogroup M in Africa was suggested, dating the split between Asian and African M branches older than 50,000 ya [22]. Although not completely discarding this last scenario other authors considered that the disjunctive was unsettled. The vast diversity of haplogroup M in Asia compared to Africa pointed to the possibility that M1 is a branch that traces a backflow from Asia to Africa [7, 23]. Due to the scarcity of M lineages in the Near East and its richness in India, this region was proposed as the most probable origin of the M1 ancestor [7, 52]. However, recent studies based on Indian mtDNA sequences [24, 25] have not found any positive evidence that M1 originated in India. Nevertheless, the inclusion of M1 complete mtDNA lineages in the construction of the macrohaplogroup M phylogeny clearly established that the antiquity of Indian lineages, as M2, as compared to Ethiopian M1 lineages support an Asian origin of macrohaplogroup M [24]. Furthermore, the comparison within Africa of eastern and western M1 sequences left the origin of M1 in Africa uncertain [21]. On the light of our and other authors results, it seems clear that by their respective coalescence ages and diversities, M1 is younger than other Asiatic M lineages. Although it is out of doubt that the L3 ancestor of M had an African origin, macrohaplogroup M radiated outside Africa and M1 should be considered an evolved branch that signals its return to this continent. Even more, as the coalescence ages of the northwestern M1c clade is older than the eastern M1a clade, we think that the most ancient dispersals of M1 occurred in northwestern Africa, reaching also the Iberian Peninsula, instead of Ethiopia. The detection of an ancestral M1c sequence in Jordanians could be explained by two alternative hypotheses: 1) that the Near East was the most probable origin of the primitive M1 dispersals, West into Africa and East to Central Asia. This supposition would explain the presence of basic M1 lineages, instead of the most common M1a derivates, as far as the Tibet. The actual scarcity of these types in eastern areas could be explained by posterior migrations that erased these primitive lineages. The absence of these ancestral M1c lineages in Ethiopia would point to the Sinai Peninsula as the most probable gate of entrance of this backflow to Africa. 2) That M1 is an autochthonous North African clade that had its earliest spread in northwestern areas marginally reaching the Near East and beyond. This would explain the shortage of basic M1 lineages in the Near East but would leave the Asiatic origin of the M1 ancestor undetermined. In any case, both alternatives envisaged M in Africa as an offshoot of the Asiatic M trunk. The striking phylogeographic parallelism between U6 and M1 haplogroups adds additional support to these hypotheses. It is possible to correlate the dispersion ages of the different M1 clades with their contemporary climatic, archaeological, paleoanthropological and linguistic information. For instance, the first M1 backflow to Africa, dated around 30,000 ya, is coincidental with a harsh glacial period which suggests that this human retreat to Africa could be forced by climatic conditions. The low sea level in the Gibraltar Strait at that time could also facilitate the Iberian Peninsula colonization. The northwestern African M1c and the probable north central M1b expansions are coincidental with the Iberomaurusian and Capsian industries. The anomalous evolution of M1a2 lineages left the coalescence ages of the eastern Africa M1a expansion uncertain, but as suggested for the sister U6a1 radiation; these movements could be correlated in time with an African origin and expansion of Afroasiatic languages [40]. Finally, from a maternal genetic perspective it seems that Neolithic occupation of the Sahara had both eastern and western influences. Most probably other mtDNA lineages participated in this human back flow to Africa. It has been suggested that the North African X1 branch of the Euroasiatic haplogroup X could be one of them [63].

Whilst this paper was under review, a new paper also dealing with U6 and M1 haplogroups was published [53]. Haplogroup topologies and phylogeographic conclusions proposed by Olivieri et al. [53] are highly coincidental with those proposed by us in our previous paper on U6 [40] and in the present paper, dealing with M1. Regrettably, there are differences in nomenclature for M1. Whereas our M1 phylogeny adhered to that proposed previously by other authors [21], Olivieri et al. [53] chose to apply their own. Nevertheless, the diagnostic positions for the different M1 subhaplogroups allowed us to establish subhaplogroup homologies between the two works. Clearly their M1b subgroup (defined by transition 13111) corresponds to our M1c subgroup; their M1a2 subgroup (defined by transition 15884) corresponds to our M1b subgroup. Finally, their M1a1 subgroup (defined by transitions at 3705, 12346 and 16359) corresponds to our M1a subgroup. In addition to the reinforcing overlap of ideas, it is worthwhile mentioning the high coincidence for the coalescence ages of M1 and the majority of its subhaplogroups, when the same substitution rate [8] is used. Olivieri et al. [53] calculated a coalescence time estimate of 36.8 ± 7.1 ky for the entire haplogroup M1 that matches our estimate of 35.2 ± 7.1 ky. Our coalescence time for M1c (25.7 ± 6.6 ky) also overlaps with Olivieri et al. [53] haplogroup M1b (23.4 ± 5.6 ky). Likewise, the coalescence age calculated for our M1a subhaplogroup (22.6 ± 8.1 ky) is in the range of the Olivieri et al. [53] estimation for their M1a1 subhaplogroup (20.6 ± 3.4 ky). The only discrepancy is about the coalescence time estimate between our M1b subhaplogroup (13.7 ± 4.8 ky) that is younger than that calculated by Olivieri et al. [53] for their homologous M1a2 (24.0 ± 5.7 ky). As our calculations are based only on three lineages and that of Oliveri et al [53] on six, we think that their coalescence time estimation should be more accurate that ours. In fact, when time estimation is based on the eight different lineages (AFR-KI43 is common to both sets) a coalescence age of 20.6 ± 5.0 ky is obtained. Although with overlapping errors, these results, together with the relative ancestral positions of each subgroup in the phylogenetic tree (Fig. 1), would suggest that the northwestern M1c clade radiation was older than those for the ubiquitous M1b and the eastern M1a clades, as also proposed by Olivieri et al. [53].

Lineages

We have analyzed thirteen complete or nearly complete mtDNA sequences belonging to the M1 subhaplogroup. Eight of them are new sequences. Two were published previously [7] but have been partially re-sequenced to confirm some dubious diagnostic positions. One additional Asiatic [54] and two Ethiopian M1 sequences [55] are from other authors. In addition we analyzed 261 partial M1 sequences gathered, in a global search, from more than 38,713 published or unpublished subjects sequenced mainly for the mtDNA HVSI segment.

Complete mtDNA sequencing

Complete mtDNA were amplified in 32 overlapping fragments with primers and PCR conditions previously described [7]. The same primers were utilized to directly sequence both strands of the fragments on an ABI 3100 analyzer using Big-Dye Terminator chemistry (Applied Biosystems). Sequence data were assembled and compared with the SeqScape software (Applied Biosystems), and all chromatograms were visually inspected.

M1 phylogenetic analyses

Phylogenetic relationships among complete mtDNA sequences were established using the reduced median network algorithm [56]. In addition to the thirteen complete or nearly complete M1 sequences, a complete M Indian sequence [25], assigned to the Indian subhaplogroup M30, was used as an outgroup.

M1 phylogeographic analyses

Relationships among the different M1 haplotypes were inferred using the reduced median network algorithm [56]. To resolve reticulations, the highly recurrent mutations, 16182C, 16183C, 16093 and the in M1 recurrent 16249, were half weighted.

Differences in accumulated mutations among M1 branches

The non-parametric test, resampling probability estimates for the difference between the means of two independent samples (http://feculty.vassar.edu/lowry/VassarStats.html) was used to calculate the significance level of accumulated mutations between the different M1 subclades. Synonymous vs. nonsynonymous substitutions between lineages were compared by the exact-Fisher test. Differences between mean number of substitutions for coding and non-coding regions were t-Student tested.

M1 diversity and differentiation within and between areas

Arlequin package [57] was used to evaluate the M1 diversity within areas using molecular nucleotide diversity (π) and gene diversity (h). AMOVA was used to test population structure. Pairwise differences between areas were obtained by means of linearized F_ST. Contingence tests were used to compare differences in M1 and M1a frequencies between groups.

Time estimates

For the complete sequences only substitutions in the coding region, excluding indels, were taken into account. The mean number of substitutions per site to the most common ancestor (ρ) of each clade [58] was estimated, and converted into time using two substitution rates: 1.7 × 10^-8 [6] and 1.26 × 10^-8 [8]. For HVSI, the age of clusters or expansions was calculated as the mean divergence (ρ) from inferred ancestral sequence types [58] and converted into time by assuming that one transition within np 16090–16365 corresponds to 20,180 years [59]. The standard deviation of the ρ estimator was calculated as previously described [60]. Ages for HVSI of complete sequences were also independently calculated.

Accesion numbers

The eight new complete mitochondrial DNA sequences are registered under GenBank accession numbers: DQ779925–32.