BACKGROUND
the "out of africa" model postulating a single "southern route" dispersal of "modern human" from horn of africa to the persian/arabian gulf and further along the tropical coast of the indian ocean to southeast asia and australasia has largely taken ground in the recent years  <cit> .
this most likely involved the exodus of a founding group of several hundred individuals, who might have made the crossing from northeastern africa, probably over the mouth of the red sea some time after the appearance of lineage l <dig> ~ <dig>  years ago, followed by a period of mutation and drift during which macrohaplogroups m, n, and r evolved and the ancestral l <dig> was lost  <cit> .
subsequently the same three founder macrohaplogroups, with the population expansion most likely occurring on indian coast  <cit>  shows a rapid coastal dispersal from ~ <dig>  years ago around the indian ocean littoral and on to australasia by ~ <dig>  years ago  <cit>  resulting in the non overlapping distribution of the derived haplogroups within m and n and its subclade r in south asia, eastern asia and australasia.
however, the presence of the diversity of basal clades with in mtdna macrohaplogroup m in india exceeds that in eastern eurasia; and numerous so-called m* lineages occur in india but not in east asia.
whereas estimated age of the m macrohaplogroup in india  <dig>  thousand years ;  <cit>  on the other hand is considerably low as compared to its east eurasian counterparts   <cit> .
the reason could be the molecular diversity and so as the coalescence age of the indian m subhaplogroups themselves, which vary substantially as indicated in the studies of sun et al.
<cit>  and thangaraj et al.
<cit> .
nested within this model, there could be two plausible scenarios:
<dig>  the number of drift events in middle/early upper-paleolithic populations  has shaped the present day mtdna phylogenetic structure of indian populations.
<dig>  either the ancestral m existed for a minimum interval of ~ <dig>  to ~ <dig>  years, during which the younger lineages branched off sequentially or second emigrational event most likely occurring ~ <dig>  to ~ <dig>  years ago from the west of the subcontinent has given rise or brought the younger lineages, thereby accounting for the different numbers of mutations accumulated to the present.
the latter has been complicated by the fact that if not all, most of these lineages are autochthonous to india and arose essentially simultaneously from ancestral m as argued by macaulay et al.
<cit> .
furthermore, since the only haplogroup of m lineages found in the substantial number to the west of the subcontinent are members of the m <dig> fragment, it also seems unlikely that the so appeared younger lineage of macrohaplogroup m has originated much farther west.
owing to the aforesaid ambiguity in population structure coupled with the west eurasian contribution into the indian maternal gene pool as a consequence of migrations during the last  <dig>  years before present   <cit>  the origin and settlement of indian people still remains intriguing.
of the known m lineages in india, m <dig> with an estimated age of ~ <dig>  years is the oldest  <cit>  and largest sub-haplogroup, which almost accounting for one tenth of the indian macrohaplogroup m  <cit> .
the distribution of m <dig> is significantly more pronounced in southern part of india as compared to north, a cline similar to that of m in general  <cit> .
moreover metspalu et al.
<cit>  also noted that frequency of m <dig> among the brahmin and kshatriyas of andhra pradesh is not significantly different from that of other caste and tribal populations of the region.
however it is absent among the brahmins and kshatriyas of the northern states of india, while the frequency reaches nearly  <dig> % among other caste and tribal populations of the region.
the high frequency of m <dig> consistent with its greater age and distribution suggests that it may represent the phylogenetic signature of earliest settlers who colonized india through southern route.
to explore the past population dynamics, impact and contribution of middle/earlyupper-paleolithic settlers in shaping the genetic diversity and structure of contemporary indian populations, we have sequenced  <dig> complete mitochondrial genomes of m <dig> lineage from  <dig> relic tribal populations of india.
RESULTS
of the screened  <dig> mtdnas from  <dig> tribes of india the macrohaplogroup m accounted for  <dig>  %, which is consistent with the earlier reports  <cit> .
the frequency distribution of macrohaplogroup m varies significantly  among studied tribes with a cline towards southern and eastern regions of india as shown in table  <dig> and figure  <dig>  in tribes  of western region, macrohaplogroup m frequency is significantly low  as compared to the other studied regions of india.
unexpectedly dungri bhil representing the north-westernmost region shows a high frequency of m  as compared to its other western counterparts.
a as per gordon  <dig> <cit> ; b number of samples screened; c includes all haplogroups within macrohaplogroup m; d includes all other than macrohaplogroup m; * caste populations data from sun et al.
<cit> ; # data not available; "aa" austro-asiatic; "dr" dravidian "ie" indo-european; "mu" mundari; "tb" tibeto-burman.
for the earliest settlers' component among the studied tribes,  <dig> samples of macrohaplogroup m were screened for the motif that confirms haplogroup m <dig> within m as described in methods.
our results indicate that m <dig> is completely absent among the eight tribes of northeast india, expect one m <dig> in sonowal kachari.
avoiding northeast tribes, the m <dig> haplogroup frequency is about  <dig>  % among the studied tribes.
its frequency is ~ <dig> to ~ <dig> % in tribes of western and central india.
the frequency declines gradually to farther north and east.
in southern region tribes, betta kuruba shows highest frequency  whereas the adjacent jenu kuruba tribe shows frequency of only  <dig>  %.
the distribution of subclade m2b varies greatly from complete absence among indo-european speakers of western and central india to as high as  <dig>  % among betta kuruba.
irrespective of region, its frequency is high  in all dravidian speakers, except madia tribe of central region whose linguistic affiliation is not very clear.
similarly it's frequency is high in korku, an austro-asiatic tribe of central india.
in eastern region m2b frequency remains low .
defining the m <dig> substructure
the reconstructed phylogenetic tree based on our newly sequenced  <dig> mtdnas of m <dig> haplogroup and  <dig> additional m <dig> complete sequences from the literature  <cit>  is given in figure  <dig>  out of the four defining mutations of macrohaplogroup m, one transition at nucleotide position   <dig> shows reversion in one of our samples.
besides the commonly occurring  <dig> transition, m <dig> in our samples is defined by the motif 447g-1780-8502-11083-15670- <dig> as also described in kivisild et al., rajkumar et al., sun et al.
and thangaraj et al.
<cit> .
though one major branch in our tree lack mutation at np  <dig> but due to its presence in most m <dig> samples of this study, as well reported elsewhere  <cit>  we considered this mutation as a basal polymorphism of m <dig> as suggested in sun et al.
<cit>  and lack of the  <dig> variant in some samples  indicates a back-mutation event.
similarly lack of mutation at np  <dig> in one of our samples is also treated as reversion event.
the m <dig> tree shows an initial deep split into two sister clades m2a and m2b.
no third clade, as indicated in rajkumar et al.
<cit>  has been found.
the clade m2a is defined by transition at np  <dig>   <dig> and contains three independent basal branches m2a <dig>  m2a <dig> and m2a <dig>  in contrast to the earlier reports  <cit>  where m2a defining motif largely constitute mutations of its sub-braches.
m2a <dig> is defined by the motif 204-5252-8396-9758-16270- <dig>  in which transition at np  <dig> show parallelism in two samples of m2a <dig> branch and transition at np  <dig> shows a reversion event.
m2a <dig> is defined by the motif of four diagnostic 7702-11041a-12657- <dig> and two recurrent 16240c- <dig> mutations.
the branch m2a <dig> is defined by the motif of one recurrent np  <dig> and three specific 5426-5774- <dig> mutations.
the further divergence within these branches of m2a exhibit probable pattern of more shared haplotypes within populations of geographic proximity followed by population specific haplotypes and a few shared haplotypes among geographically apart populations.
unlike m2a, m2b instead of early branching represented by a single deep root defined by the motif 152-182-195- <dig> d-1453-2831t-3630-5744-6647-9899-13254-14766-16183c-16189-16193+c- <dig> which, of late shows branching pattern similar to the sub-branches of m2a.
the m2b <dig> defined by the transition at np 6260- <dig> harbour population of eastern region.
whereas, m2b <dig> defined by transition at np  <dig>  harbours dravidians.
other braches within m2b are more or less population specific.
in this study, spread of m2b by enlarge restricted to dravidians and tribes of eastern region.
the root of m2b in our tree differs in two positions to the earlier definition of sun et al.
<cit>  i.e.
transition at np  <dig> is present in all of our m2b samples so we treated this as basal mutation and lack of this in one sample of sun et al.
<cit>  could be better explained by reversion event, second our all m2b samples has poly 'a' at np 16180– <dig> and twelve 'c's thereafter.
hence in our tree an additional 'c' at np 16184– <dig> has been treated as insertion at np  <dig>  than transversion  reported by sun et al.
<cit> .
age estimates and phylogenetic implications
coalescent age estimates were calculated by rho  statistics  <cit>  using two different mutation rates  <cit>  and  <cit>  shows a marginal time difference when standard deviation is taken into account, the later has been considered because of robustness in view of natural selection  <cit> .
the average sequence divergence of the  <dig> m <dig> coding-region sequences from the root of m <dig> calculated as per  <cit>  corresponds to a coalescence time estimate of  <dig>  ±  <dig>  thousand years .
the founder age estimate for indian mtdna lineages using m <dig> data,  <dig>  ±  <dig>  ky is well within the lower bound range of earlier estimates  of modern human dispersal into arabia and southern asia  <cit> , and perhaps more close to the estimates of  <cit> .
the two clades of m <dig> show differential branching patterns.
m2a with coalescent age  <dig>  ±  <dig>  ky splits into its three deep rooting branches m2a <dig>  m2a <dig> and m2a <dig>  m2a <dig> is specific to kathodi/katkari tribe, whereas m2a <dig> and m2a <dig> encompass almost all the studied tribes.
m2a <dig>  m2a <dig> and m2a <dig> show coalescent estimates of  <dig> to  <dig> ky. the clade m2b doesn't show branching event earlier than estimated coalescence time of  <dig>  ±  <dig>  ky. in our samples we could not find m2b among indo-european speakers of west and central india.
the dravidian speaking tribes of south extending up to central india and tribes of eastern region irrespective of linguistic affiliation, harbour both clades  of m <dig>  presenting a time depth of ~ <dig> ky.
diversity indices
diversity indices and demographic parameters estimated for studied tribes are given in table  <dig>  the m <dig> lineage, haplotype diversity among indian tribes ranged from  <dig>  to  <dig>  and nucleotide diversity from  <dig>  to  <dig> .
though four geographical regions of india did not differ significantly  in haplotype diversity it was comparatively higher in west  followed by central , eastern  and southern tribes .
nucleotide diversity in east  was significantly higher than west  and central tribes , intermediate nucleotide diversity values were observed in south india , they were not significantly different from west and central india  or east india .
these patterns of genetic diversity were further strengthened by the analysis of mean pairwise differences .
mpd of west  and central tribes  were significantly lower  than the mpd from east , whereas mpd from south  were not significantly different from east, west and central tribes .
thus observed mtdna diversity indicate to the fact that haplotype/haplogroup frequency is a poor parameter of deep rooting ancestry rather it is the product of recent population growth.
similarly, the diversity parameters are also influenced by the past demographic events and any phylogenetic inference drawn on such parameter should keep in view the past demographic events, particularly for india where such event has been predicted previously  <cit> .
a number of complete mtdna sequences; b ± standard deviation; c mean pairwise differences; * sequence data from sun et al.
<cit> ; # unable to calculate due to single sequence.
past population dynamics
as indicated in our results and previously  <cit>  the demographic history of populations in different geographic regions might have played pivotal role in shaping the complex net of population phylogenetic relationships in indian subcontinent.
the demographic history of m <dig> lineage as a surrogate of the middle/early upper palaeolithic component of indian populations was reconstructed using bayesian skyline plot   <cit> .
figure  <dig>  shows the bsp of m <dig> lineage produced using  <dig> complete mtdna sequences along with plot  using only coding region.
although the two analyses are very similar, the second is confined to slow evolving region of mtdna  <cit>  which is likely to define lineages that have existed in the population prior to a putative bottleneck, thus increasing the sensitivity of bsp to detect more complex demographic trends.
as the analysis is based on only single lineage it provides insight into the demographic event limiting to the age of the lineage .
most striking is the population decline, observed during last glacial maximum i.e.
<dig> to  <dig> kybp  <cit>  and late glacial aridity i.e.
<dig> to  <dig> kybp  <cit> , followed by many fold population growth in a comparatively short period of time.
if such demographic event had affected the earliest settlers of india it would have resulted in several implications of phylogenetic interest.
firstly, reduction of genetic diversity across all the lineages in which, lineages with a smaller population spread would have been affected the most.
second, it might have mechanized some sort of unifying effect where smaller lineages are eliminated or at least reduced to margins of extinction and lineages of larger spread remained among all the post bottleneck populations.
the post glacial rapid population growth achieved some sort of plateau by  <dig> to  <dig> kybp followed by another decline which was to its maximum around ~ <dig> to  <dig> bp.
now the question is whether the observed demographic trend was uniform throughout india or it was as complex as reported by the earlier studies  <cit> .
a similar analysis for each studied geographical region of india is presented in panel c to f of figure  <dig>  due to the small sample size in each geographic region bsp produces low resolution; however rapid post glacial population growth is evident in east, south and central india, followed by a population decline from  <dig> to  <dig> kybp.
the rapid regain after this period has been observed in central region; however such regains are marginal in other two regions.
the demographic past of ancient lineage among western tribes was quite different- a population growth from ~7– <dig> kybp continued to present.
the negative values of fs that differ significantly from zero indicative of population's demographic expansion  <cit>  also support the recent population expansion in western region .
genetic structure
the above results are indicative of some genetic structure in indian populations, to investigate that, amova was used .
in the total samples   <dig> % of the variance was found within populations and  <dig> % among populations.
studied tribes were then grouped according to geographic proximity , linguistic affinities  and to the results suggested, namely two groups separating indo-european speakers of west and central india from all others .
under the models  <dig> and  <dig>  45– <dig> % of the variance was found within populations, 36–39% among populations within groups and 13– <dig> % among groups.
the model  <dig> more appropriately reflects the genetic structure with variance among groups  <dig> % exceeds the variance among populations within groups  <dig> %.
a variance
discussion
a rapid coastal migration along the "southern route" from africa into southern asia, some time before  <dig> kybp has been strongly suggested by studies on present day world populations   <cit> .
the founder analysis of the mtdnas in this study suggests  <dig>  ±  <dig>  kybp for such arrival which is well within the lower bound range of earlier estimates and perhaps more consistent with the earliest and most pronounced population expansion in southern asia around  <dig> kybp suggested in  <cit> .
magnitude of this southern asian growth phase suggests that over half of the global human population lived in indian subcontinent between ~ <dig> to  <dig> kybp and population size peaked at over 60% around 38kybp  <cit> .
these population expansion estimates are largely in agreement with high mtdna diversity and star like non-overlapping pattern of numerous lineages of macrohaplogroup m reported previously  <cit> .
though the stage upto here is clear, the contribution and role of this sizably large earliest settlers' component in the contemporary indian populations, coupled with later migrations during the last  <dig> thousand years  from west and east of the subcontinent has been an issue of controversy.
cordaux et al.
<cit>  based on the non-overlapping pattern of mtdna phylogeny between india and east eurasia has supported the argument of cavalli-sforza et al.
<cit>  that in india the genetic traces of early migrations along the southern route were erased by the subsequent migrations, which shaped the present-day mtdna gene pool of india.
however presence of numerous autochthonous lineages in india emerging directly from the root of the founder macrohaplogroups m, n and r  <cit>  during the estimated population growth period in southern asia  <cit>  indicates the presence of large component of earliest settlers in the contemporary indian populations.
in the quest of finding the carriers of the genetic legacy of the earliest settlers among the contemporary indian populations, some previous studies on mtdna variation by calculating nucleotide diversity and expansion time  for different linguistic groups of india, distinguished austro-asiatic speaking tribes as the oldest and the carriers of the said legacy  <cit> .
basu et al.
<cit>  also supported the view by reporting that the frequency of the ancient haplogroup m <dig> among the austro-asiatic tribal populations is as high as 19% and they lack the younger haplogroup m <dig>  however metspalu et al.
<cit> , so as this study, rejects such claims as linguistic groups of india do not cluster into distinct branches of the indian mtdna tree,  calculating the beginning of expansion for those groupings is problematic, whereas lack of coding region information in basu et al.
<cit>  have lead to an over estimation of m <dig> frequency.
moreover our results indicate that m <dig> frequency variation among the studied tribes can be better explained by recent population expansion/demographic events than as a function of deep rooting ancestry.
the nucleotide diversity though appears better parameter, is also predisposed to influence of past demographic events.
the phylogenetic inferences based on such parameters should be strictly viewed in reference to the demographic events, particularly for india.
our analysis of mtdna variation in populations of india indicate that the dravidian tribes extending from southern to central india and tribes of eastern india irrespective of the linguistic affiliation shows equally deep rooted m <dig> ancestry ~ <dig> ky , comparable nucleotide diversity  and similar past demographic history .
however indo-european tribes of western and central india except kathodi/katkari and andh tribes harbour only m2a <dig> branch representing a time depth of ~ <dig> ky. kathodi/katkari and andh tribe encompasses other braches of m2a, but lacks m2b.
all these indo-european tribes shows appreciable frequency of m <dig> , but they are low on nucleotide diversity .
thus it would be highly speculative to tag any one or a group of populations based on linguistics or geography as the representatives of earliest settlers, rather it indicate to the fact that earliest settlers' component is more pronounced in the areas extending from southern to eastern india, and shows decline towards north and northwest india, a cline similar to that of m in general .
however a decline of earliest settlers' component across tribe to higher caste gradient may also be accepted in the respective regions as indicated in  <cit> .
the time depth of m <dig> lineage and diversity indices in indo-european speakers of western region extending up to central india posits the expansion of earliest settlers' component into these areas during the post last glacial maximum growth  of population  or perhaps little later .
however this requires further investigation.
it is only during this rapid growth; regional and population specific branching patterns appear on the more or less homogenous m <dig> phylogeny.
the possible explanation would be the earliest settlers of india prior to this rapid population growth had lived in an extended enclave and there had been continuous gene flow across population boundaries.
the second but more plausible reason of such homogeneity could be that earliest settlers by virtue of large population size during ~ <dig> to  <dig> kybp  <cit>  and indian ecological setting which, favoured tendency to isolate and subjugate  <cit>  might have been differentiated into populations distributed far apart as suggested in recent studies  <cit> .
but during the lgm and late glacial aridity, climate across india and south asia generally seems to have been much more arid than present.
geomorphological indicators from the landmass of india suggest dune mobility in the northwest  <cit> , and greatly reduced river flow in north central india during the span of time that covered the full glacial  <cit> .
offshore indicators of salinity  suggest that lgm aridity was substantially greater than at present.
indicators of upwelling intensity in the indian ocean suggest that the summer monsoon was much weaker than present at the lgm, but reaching its weakest at around  <dig>  –  <dig>  c <dig> years ago, that is  <dig> - <dig>  calibrated or 'real' years ago  <cit> .
during this period of cold and more arid conditions rainforest retreated and was replaced by dry grasslands.
however, some monsoon forests and woodlands in southern india and scrub, open woodland in eastern india probably existed in presently moist forest climates.
this appears to be harsh conditions for an hunting gathering based subsistence, thus to fight the adverse, probably shrinking populations might have come close to each other in a more habitable area allowing a free gene flow between populations, whereas ancestral population of the kathodi/katkari m2a <dig> lineage appears to have remained isolated during this period.
in the post lgm growth period, though population spread over wide geographical regions.
maternal gene flow is evident in the geographical neighbors suggesting fluidic population specific boundaries until recently at least among the tribes.
the next important event on the indian scene is the beginnings of agriculture and use of pottery  <cit> .
cultivation of plants/agriculture diffused from the fertile crescent within the past  <dig>  years.
the steady advance beyond this stage seems however to have been primarily driven by the crop-animal complex derived from the mid-east, reaching the tip of southern india around  <dig> to  <dig> kybp  <cit> .
the diffusion of pottery traditions, which arise in response to the need to store and cook grains, shows evidence of the influences from northwest and northeast, with the western influence predominating over much of the country.
thus the black and red ware reflects western, while the corded ware chinese influence  <cit> .
two other technological innovations, known to have originated outside of india, the domestication of horse, around  <dig> kybp on the shores of black sea in present ukraine, and the use of iron around  <dig> kybp in anatolia in present day turkey, appears in the indian archeological records  soon after the agriculture  <cit> .
the recent study investigating the cultural or demic diffusion model of agriculture in india supported the demic diffusion model which predicts a substantial genetic input from migrating agriculturalists  <cit> .
the advent of agriculture and perhaps migrating agriculturists brought about dramatic changes in the economy, technology and demography of human societies.
human habitat in the hunting-gathering stage was essentially on hilly, rocky and forested regions, which had ample wild plant and animal food resources.
agriculture led to the emergence of villages and towns and perhaps brought with it the division of society into occupational groups.
<cit> .
crop cultivation resulted in the loss of the traditional habitat of hunter-gatherers by deforestation, fragmenting and marginalizing numerous such populations, many of whom were assimilated into agriculturally based subsistence economies  <cit> , thereby catalyzing some sort of regional similarities across tribe caste continuum.
our results on reconstructed past population demography indicating decline of earliest settlers' population  during this period in almost all the geographical regions except western  is consistent with the above proposition and suggests that demic diffusion of these technologies were rapid, perhaps involving large migrating populations with these technologies.
the highest frequency of east eurasian- specific mtdna haplogroups  <cit>  and absence of m <dig> an earliest settlers component  among tibeto-burman speaking tribes of northeastern states of india suggests that, despite the more recent migrations to india, these populations remained relatively isolated, explaining the close correlation between genetic and linguistic results  <cit> .
this contrasts with the situation observed in other regions of india, where linguistic structure shows very little concordance with the genetic structures.
CONCLUSIONS
the time depth and diversity of m <dig> lineage among the studied tribes suggests that the tribes of southern and eastern region along with dravidian and austro-asiatic speakers of central india are the modern representatives of earliest settlers of india via proposed southern route.
the lgm and late glacial , climatic conditions across india and south asia seems to be much more arid and harsh for an hunting gathering based subsistence, thus mechanized reduction and bringing earliest settlers' population closer in a more habitable area allowing a free gene flow, followed by a rapid three fold population growth around 12- <dig> kybp when climatic conditions improved, thereby inducing some sort of homogeneity and redistribution of earliest settlers' component in wide geographical regions.
the next important event on the indian scene appears to be demic diffusion of agriculture and associated technologies around  <dig> kybp, resulted in the loss of the traditional habitat of hunter-gatherers by deforestation, fragmenting and marginalizing such populations, many of whom were assimilated into agricultural based subsistence economy, as evident in the decline of earliest settlers' component in all the geographical regions except western.

 the "out of africa" model postulating a single "southern route" dispersal of "modern human" from horn of africa to the persian/arabian gulf and further along the tropical coast of the indian ocean to southeast asia and australasia has largely taken ground in the recent years. this most likely involved the exodus of a founding group of several hundred individuals, who might have made the crossing from northeastern africa, probably over the mouth of the red sea some time after the appearance of lineage l ~ years ago, followed by a period of mutation and drift during which macrohaplogroups m, n, and r evolved and the ancestral l was lost. subsequently the same three founder macrohaplogroups, with the population expansion most likely occurring on indian coast shows a rapid coastal dispersal from ~ years ago around the indian ocean littoral and on to australasia by ~ years ago resulting in the non overlapping distribution of the derived haplogroups within m and n and its subclade r in south asia, eastern asia and australasia. however, the presence of the diversity of basal clades with in mtdna macrohaplogroup m in india exceeds that in eastern eurasia; and numerous so-called m* lineages occur in india but not in east asia. whereas estimated age of the m macrohaplogroup in india thousand years; on the other hand is considerably low as compared to its east eurasian counterparts. the reason could be the molecular diversity and so as the coalescence age of the indian m subhaplogroups themselves, which vary substantially as indicated in the studies of sun et al. and thangaraj et al.. nested within this model, there could be two plausible scenarios: the number of drift events in middle/early upper-paleolithic populations has shaped the present day mtdna phylogenetic structure of indian populations. either the ancestral m existed for a minimum interval of ~ to ~ years, during which the younger lineages branched off sequentially or second emigrational event most likely occurring ~ to ~ years ago from the west of the subcontinent has given rise or brought the younger lineages, thereby accounting for the different numbers of mutations accumulated to the present. the latter has been complicated by the fact that if not all, most of these lineages are autochthonous to india and arose essentially simultaneously from ancestral m as argued by macaulay et al.. furthermore, since the only haplogroup of m lineages found in the substantial number to the west of the subcontinent are members of the m fragment, it also seems unlikely that the so appeared younger lineage of macrohaplogroup m has originated much farther west. owing to the aforesaid ambiguity in population structure coupled with the west eurasian contribution into the indian maternal gene pool as a consequence of migrations during the last years before present the origin and settlement of indian people still remains intriguing. of the known m lineages in india, m with an estimated age of ~ years is the oldest and largest sub-haplogroup, which almost accounting for one tenth of the indian macrohaplogroup m. the distribution of m is significantly more pronounced in southern part of india as compared to north, a cline similar to that of m in general. moreover metspalu et al. also noted that frequency of m among the brahmin and kshatriyas of andhra pradesh is not significantly different from that of other caste and tribal populations of the region. however it is absent among the brahmins and kshatriyas of the northern states of india, while the frequency reaches nearly % among other caste and tribal populations of the region. the high frequency of m consistent with its greater age and distribution suggests that it may represent the phylogenetic signature of earliest settlers who colonized india through southern route. to explore the past population dynamics, impact and contribution of middle/earlyupper-paleolithic settlers in shaping the genetic diversity and structure of contemporary indian populations, we have sequenced complete mitochondrial genomes of m lineage from relic tribal populations of india.  of the screened mtdnas from tribes of india the macrohaplogroup m accounted for %, which is consistent with the earlier reports. the frequency distribution of macrohaplogroup m varies significantly among studied tribes with a cline towards southern and eastern regions of india as shown in table and figure in tribes of western region, macrohaplogroup m frequency is significantly low as compared to the other studied regions of india. unexpectedly dungri bhil representing the north-westernmost region shows a high frequency of m as compared to its other western counterparts. a as per gordon; b number of samples screened; c includes all haplogroups within macrohaplogroup m; d includes all other than macrohaplogroup m; * caste populations data from sun et al.; # data not available; "aa" austro-asiatic; "dr" dravidian "ie" indo-european; "mu" mundari; "tb" tibeto-burman. for the earliest settlers' component among the studied tribes, samples of macrohaplogroup m were screened for the motif that confirms haplogroup m within m as described in methods. our results indicate that m is completely absent among the eight tribes of northeast india, expect one m in sonowal kachari. avoiding northeast tribes, the m haplogroup frequency is about % among the studied tribes. its frequency is ~ to ~ % in tribes of western and central india. the frequency declines gradually to farther north and east. in southern region tribes, betta kuruba shows highest frequency whereas the adjacent jenu kuruba tribe shows frequency of only %. the distribution of subclade m2b varies greatly from complete absence among indo-european speakers of western and central india to as high as % among betta kuruba. irrespective of region, its frequency is high in all dravidian speakers, except madia tribe of central region whose linguistic affiliation is not very clear. similarly it's frequency is high in korku, an austro-asiatic tribe of central india. in eastern region m2b frequency remains low. defining the m substructure the reconstructed phylogenetic tree based on our newly sequenced mtdnas of m haplogroup and additional m complete sequences from the literature is given in figure out of the four defining mutations of macrohaplogroup m, one transition at nucleotide position shows reversion in one of our samples. besides the commonly occurring transition, m in our samples is defined by the motif 447g-1780-8502-11083-15670- as also described in kivisild et al., rajkumar et al., sun et al. and thangaraj et al.. though one major branch in our tree lack mutation at np but due to its presence in most m samples of this study, as well reported elsewhere we considered this mutation as a basal polymorphism of m as suggested in sun et al. and lack of the variant in some samples indicates a back-mutation event. similarly lack of mutation at np in one of our samples is also treated as reversion event. the m tree shows an initial deep split into two sister clades m2a and m2b. no third clade, as indicated in rajkumar et al. has been found. the clade m2a is defined by transition at np and contains three independent basal branches m2a m2a and m2a in contrast to the earlier reports where m2a defining motif largely constitute mutations of its sub-braches. m2a is defined by the motif 204-5252-8396-9758-16270- in which transition at np show parallelism in two samples of m2a branch and transition at np shows a reversion event. m2a is defined by the motif of four diagnostic 7702-11041a-12657- and two recurrent 16240c- mutations. the branch m2a is defined by the motif of one recurrent np and three specific 5426-5774- mutations. the further divergence within these branches of m2a exhibit probable pattern of more shared haplotypes within populations of geographic proximity followed by population specific haplotypes and a few shared haplotypes among geographically apart populations. unlike m2a, m2b instead of early branching represented by a single deep root defined by the motif 152-182-195- d-1453-2831t-3630-5744-6647-9899-13254-14766-16183c-16189-16193+c- which, of late shows branching pattern similar to the sub-branches of m2a. the m2b defined by the transition at np 6260- harbour population of eastern region. whereas, m2b defined by transition at np harbours dravidians. other braches within m2b are more or less population specific. in this study, spread of m2b by enlarge restricted to dravidians and tribes of eastern region. the root of m2b in our tree differs in two positions to the earlier definition of sun et al. i.e. transition at np is present in all of our m2b samples so we treated this as basal mutation and lack of this in one sample of sun et al. could be better explained by reversion event, second our all m2b samples has poly'a' at np 16180– and twelve'c's thereafter. hence in our tree an additional'c' at np 16184– has been treated as insertion at np than transversion reported by sun et al.. age estimates and phylogenetic implications coalescent age estimates were calculated by rho statistics using two different mutation rates and shows a marginal time difference when standard deviation is taken into account, the later has been considered because of robustness in view of natural selection. the average sequence divergence of the m coding-region sequences from the root of m calculated as per corresponds to a coalescence time estimate of ± thousand years. the founder age estimate for indian mtdna lineages using m data, ± ky is well within the lower bound range of earlier estimates of modern human dispersal into arabia and southern asia, and perhaps more close to the estimates of. the two clades of m show differential branching patterns. m2a with coalescent age ± ky splits into its three deep rooting branches m2a m2a and m2a m2a is specific to kathodi/katkari tribe, whereas m2a and m2a encompass almost all the studied tribes. m2a m2a and m2a show coalescent estimates of to ky. the clade m2b doesn't show branching event earlier than estimated coalescence time of ± ky. in our samples we could not find m2b among indo-european speakers of west and central india. the dravidian speaking tribes of south extending up to central india and tribes of eastern region irrespective of linguistic affiliation, harbour both clades of m presenting a time depth of ~ ky. diversity indices diversity indices and demographic parameters estimated for studied tribes are given in table the m lineage, haplotype diversity among indian tribes ranged from to and nucleotide diversity from to. though four geographical regions of india did not differ significantly in haplotype diversity it was comparatively higher in west followed by central, eastern and southern tribes. nucleotide diversity in east was significantly higher than west and central tribes, intermediate nucleotide diversity values were observed in south india, they were not significantly different from west and central india or east india. these patterns of genetic diversity were further strengthened by the analysis of mean pairwise differences. mpd of west and central tribes were significantly lower than the mpd from east, whereas mpd from south were not significantly different from east, west and central tribes. thus observed mtdna diversity indicate to the fact that haplotype/haplogroup frequency is a poor parameter of deep rooting ancestry rather it is the product of recent population growth. similarly, the diversity parameters are also influenced by the past demographic events and any phylogenetic inference drawn on such parameter should keep in view the past demographic events, particularly for india where such event has been predicted previously. a number of complete mtdna sequences; b ± standard deviation; c mean pairwise differences; * sequence data from sun et al.; # unable to calculate due to single sequence. past population dynamics as indicated in our results and previously the demographic history of populations in different geographic regions might have played pivotal role in shaping the complex net of population phylogenetic relationships in indian subcontinent. the demographic history of m lineage as a surrogate of the middle/early upper palaeolithic component of indian populations was reconstructed using bayesian skyline plot. figure shows the bsp of m lineage produced using complete mtdna sequences along with plot using only coding region. although the two analyses are very similar, the second is confined to slow evolving region of mtdna which is likely to define lineages that have existed in the population prior to a putative bottleneck, thus increasing the sensitivity of bsp to detect more complex demographic trends. as the analysis is based on only single lineage it provides insight into the demographic event limiting to the age of the lineage. most striking is the population decline, observed during last glacial maximum i.e. to kybp and late glacial aridity i.e. to kybp, followed by many fold population growth in a comparatively short period of time. if such demographic event had affected the earliest settlers of india it would have resulted in several implications of phylogenetic interest. firstly, reduction of genetic diversity across all the lineages in which, lineages with a smaller population spread would have been affected the most. second, it might have mechanized some sort of unifying effect where smaller lineages are eliminated or at least reduced to margins of extinction and lineages of larger spread remained among all the post bottleneck populations. the post glacial rapid population growth achieved some sort of plateau by to kybp followed by another decline which was to its maximum around ~ to bp. now the question is whether the observed demographic trend was uniform throughout india or it was as complex as reported by the earlier studies. a similar analysis for each studied geographical region of india is presented in panel c to f of figure due to the small sample size in each geographic region bsp produces low resolution; however rapid post glacial population growth is evident in east, south and central india, followed by a population decline from to kybp. the rapid regain after this period has been observed in central region; however such regains are marginal in other two regions. the demographic past of ancient lineage among western tribes was quite different- a population growth from ~7– kybp continued to present. the negative values of fs that differ significantly from zero indicative of population's demographic expansion also support the recent population expansion in western region. genetic structure the above results are indicative of some genetic structure in indian populations, to investigate that, amova was used. in the total samples % of the variance was found within populations and % among populations. studied tribes were then grouped according to geographic proximity, linguistic affinities and to the results suggested, namely two groups separating indo-european speakers of west and central india from all others. under the models and 45– % of the variance was found within populations, 36–39% among populations within groups and 13– % among groups. the model more appropriately reflects the genetic structure with variance among groups % exceeds the variance among populations within groups %. a variance discussion a rapid coastal migration along the "southern route" from africa into southern asia, some time before kybp has been strongly suggested by studies on present day world populations. the founder analysis of the mtdnas in this study suggests ± kybp for such arrival which is well within the lower bound range of earlier estimates and perhaps more consistent with the earliest and most pronounced population expansion in southern asia around kybp suggested in. magnitude of this southern asian growth phase suggests that over half of the global human population lived in indian subcontinent between ~ to kybp and population size peaked at over 60% around 38kybp. these population expansion estimates are largely in agreement with high mtdna diversity and star like non-overlapping pattern of numerous lineages of macrohaplogroup m reported previously. though the stage upto here is clear, the contribution and role of this sizably large earliest settlers' component in the contemporary indian populations, coupled with later migrations during the last thousand years from west and east of the subcontinent has been an issue of controversy. cordaux et al. based on the non-overlapping pattern of mtdna phylogeny between india and east eurasia has supported the argument of cavalli-sforza et al. that in india the genetic traces of early migrations along the southern route were erased by the subsequent migrations, which shaped the present-day mtdna gene pool of india. however presence of numerous autochthonous lineages in india emerging directly from the root of the founder macrohaplogroups m, n and r during the estimated population growth period in southern asia indicates the presence of large component of earliest settlers in the contemporary indian populations. in the quest of finding the carriers of the genetic legacy of the earliest settlers among the contemporary indian populations, some previous studies on mtdna variation by calculating nucleotide diversity and expansion time for different linguistic groups of india, distinguished austro-asiatic speaking tribes as the oldest and the carriers of the said legacy. basu et al. also supported the view by reporting that the frequency of the ancient haplogroup m among the austro-asiatic tribal populations is as high as 19% and they lack the younger haplogroup m however metspalu et al., so as this study, rejects such claims as linguistic groups of india do not cluster into distinct branches of the indian mtdna tree, calculating the beginning of expansion for those groupings is problematic, whereas lack of coding region information in basu et al. have lead to an over estimation of m frequency. moreover our results indicate that m frequency variation among the studied tribes can be better explained by recent population expansion/demographic events than as a function of deep rooting ancestry. the nucleotide diversity though appears better parameter, is also predisposed to influence of past demographic events. the phylogenetic inferences based on such parameters should be strictly viewed in reference to the demographic events, particularly for india. our analysis of mtdna variation in populations of india indicate that the dravidian tribes extending from southern to central india and tribes of eastern india irrespective of the linguistic affiliation shows equally deep rooted m ancestry ~ ky, comparable nucleotide diversity and similar past demographic history. however indo-european tribes of western and central india except kathodi/katkari and andh tribes harbour only m2a branch representing a time depth of ~ ky. kathodi/katkari and andh tribe encompasses other braches of m2a, but lacks m2b. all these indo-european tribes shows appreciable frequency of m, but they are low on nucleotide diversity. thus it would be highly speculative to tag any one or a group of populations based on linguistics or geography as the representatives of earliest settlers, rather it indicate to the fact that earliest settlers' component is more pronounced in the areas extending from southern to eastern india, and shows decline towards north and northwest india, a cline similar to that of m in general. however a decline of earliest settlers' component across tribe to higher caste gradient may also be accepted in the respective regions as indicated in. the time depth of m lineage and diversity indices in indo-european speakers of western region extending up to central india posits the expansion of earliest settlers' component into these areas during the post last glacial maximum growth of population or perhaps little later. however this requires further investigation. it is only during this rapid growth; regional and population specific branching patterns appear on the more or less homogenous m phylogeny. the possible explanation would be the earliest settlers of india prior to this rapid population growth had lived in an extended enclave and there had been continuous gene flow across population boundaries. the second but more plausible reason of such homogeneity could be that earliest settlers by virtue of large population size during ~ to kybp and indian ecological setting which, favoured tendency to isolate and subjugate might have been differentiated into populations distributed far apart as suggested in recent studies. but during the lgm and late glacial aridity, climate across india and south asia generally seems to have been much more arid than present. geomorphological indicators from the landmass of india suggest dune mobility in the northwest, and greatly reduced river flow in north central india during the span of time that covered the full glacial. offshore indicators of salinity suggest that lgm aridity was substantially greater than at present. indicators of upwelling intensity in the indian ocean suggest that the summer monsoon was much weaker than present at the lgm, but reaching its weakest at around – c years ago, that is - calibrated or'real' years ago. during this period of cold and more arid conditions rainforest retreated and was replaced by dry grasslands. however, some monsoon forests and woodlands in southern india and scrub, open woodland in eastern india probably existed in presently moist forest climates. this appears to be harsh conditions for an hunting gathering based subsistence, thus to fight the adverse, probably shrinking populations might have come close to each other in a more habitable area allowing a free gene flow between populations, whereas ancestral population of the kathodi/katkari m2a lineage appears to have remained isolated during this period. in the post lgm growth period, though population spread over wide geographical regions. maternal gene flow is evident in the geographical neighbors suggesting fluidic population specific boundaries until recently at least among the tribes. the next important event on the indian scene is the beginnings of agriculture and use of pottery. cultivation of plants/agriculture diffused from the fertile crescent within the past years. the steady advance beyond this stage seems however to have been primarily driven by the crop-animal complex derived from the mid-east, reaching the tip of southern india around to kybp. the diffusion of pottery traditions, which arise in response to the need to store and cook grains, shows evidence of the influences from northwest and northeast, with the western influence predominating over much of the country. thus the black and red ware reflects western, while the corded ware chinese influence. two other technological innovations, known to have originated outside of india, the domestication of horse, around kybp on the shores of black sea in present ukraine, and the use of iron around kybp in anatolia in present day turkey, appears in the indian archeological records soon after the agriculture. the recent study investigating the cultural or demic diffusion model of agriculture in india supported the demic diffusion model which predicts a substantial genetic input from migrating agriculturalists. the advent of agriculture and perhaps migrating agriculturists brought about dramatic changes in the economy, technology and demography of human societies. human habitat in the hunting-gathering stage was essentially on hilly, rocky and forested regions, which had ample wild plant and animal food resources. agriculture led to the emergence of villages and towns and perhaps brought with it the division of society into occupational groups.. crop cultivation resulted in the loss of the traditional habitat of hunter-gatherers by deforestation, fragmenting and marginalizing numerous such populations, many of whom were assimilated into agriculturally based subsistence economies, thereby catalyzing some sort of regional similarities across tribe caste continuum. our results on reconstructed past population demography indicating decline of earliest settlers' population during this period in almost all the geographical regions except western is consistent with the above proposition and suggests that demic diffusion of these technologies were rapid, perhaps involving large migrating populations with these technologies. the highest frequency of east eurasian- specific mtdna haplogroups and absence of m an earliest settlers component among tibeto-burman speaking tribes of northeastern states of india suggests that, despite the more recent migrations to india, these populations remained relatively isolated, explaining the close correlation between genetic and linguistic results. this contrasts with the situation observed in other regions of india, where linguistic structure shows very little concordance with the genetic structures.  the time depth and diversity of m lineage among the studied tribes suggests that the tribes of southern and eastern region along with dravidian and austro-asiatic speakers of central india are the modern representatives of earliest settlers of india via proposed southern route. the lgm and late glacial, climatic conditions across india and south asia seems to be much more arid and harsh for an hunting gathering based subsistence, thus mechanized reduction and bringing earliest settlers' population closer in a more habitable area allowing a free gene flow, followed by a rapid three fold population growth around 12- kybp when climatic conditions improved, thereby inducing some sort of homogeneity and redistribution of earliest settlers' component in wide geographical regions. the next important event on the indian scene appears to be demic diffusion of agriculture and associated technologies around kybp, resulted in the loss of the traditional habitat of hunter-gatherers by deforestation, fragmenting and marginalizing such populations, many of whom were assimilated into agricultural based subsistence economy, as evident in the decline of earliest settlers' component in all the geographical regions except western. 
accordingly, we attempted to re-evaluate the impact and contribution of earliest settlers in shaping the genetic diversity and structure of contemporary indian populations; using our newly sequenced and published complete mitochondrial genomes of this lineage. our results suggest that tribes of southern and eastern region along with dravidian and austro-asiatic speakers of central india are the modern representatives of earliest settlers of subcontinent. further the reconstructed demographic history of m lineage as a surrogate of earliest settlers' component revealed that the demographic events with pronounced regional variations had played pivotal role in shaping the complex net of populations phylogenetic relationship in indian subcontinent. the demic diffusion of agriculture and associated technologies around kybp, which might have marginalized hunter-gatherer, is coincidental with the decline of earliest settlers' population during this period. the phylogeographic spread and diversity indices of m and its subclades among the tribes of different geographic regions and linguistic phyla were investigated in detail. the high frequency of mitochondrial lineage "m2" consistent with its greater age and distribution suggests that it may represent the phylogenetic signature of earliest settlers. the "out of africa" model postulating single "southern route" dispersal posits arrival of "anatomically modern human" to indian subcontinent around 66– thousand years before present. however the contributions and legacy of these earliest settlers in contemporary indian populations, owing to the complex past population dynamics and later migrations has been an issue of controversy.
 the "out of africa" model postulating single "southern route" dispersal posits arrival of "anatomically modern human" to indian subcontinent around 66– thousand years before present. however the contributions and legacy of these earliest settlers in contemporary indian populations, owing to the complex past population dynamics and later migrations has been an issue of controversy. the high frequency of mitochondrial lineage "m2" consistent with its greater age and distribution suggests that it may represent the phylogenetic signature of earliest settlers. accordingly, we attempted to re-evaluate the impact and contribution of earliest settlers in shaping the genetic diversity and structure of contemporary indian populations; using our newly sequenced and published complete mitochondrial genomes of this lineage.  the m lineage, harbouring two deep rooting subclades m2a and m2b encompasses approximately one tenth of the mtdna pool of studied tribes. the phylogeographic spread and diversity indices of m and its subclades among the tribes of different geographic regions and linguistic phyla were investigated in detail. further the reconstructed demographic history of m lineage as a surrogate of earliest settlers' component revealed that the demographic events with pronounced regional variations had played pivotal role in shaping the complex net of populations phylogenetic relationship in indian subcontinent.  our results suggest that tribes of southern and eastern region along with dravidian and austro-asiatic speakers of central india are the modern representatives of earliest settlers of subcontinent. the last glacial maximum aridity and post lgm population growth mechanised some sort of homogeneity and redistribution of earliest settlers' component in india. the demic diffusion of agriculture and associated technologies around kybp, which might have marginalized hunter-gatherer, is coincidental with the decline of earliest settlers' population during this period. 