BACKGROUND
the democratic republic of timor-leste  is located in the lesser sunda islands  of island  southeast asia , between mainland southeast asia  and australia, the indian and the pacific ocean.
it extends over the eastern part of timor, the adjacent islands ataúro and jaco, and oecusse, an exclave within western timor  .
under portuguese rule since the 16th century, east timor declared its independence in  <dig> and obtained it in  <dig> after indonesian occupation.
the country has a population of ~ <dig>  million, a total area of  <dig>  km <dig> and its highest peak reaches  <dig>  meters.
thirty-two languages are spoken in the  <dig> districts; four working languages are used  <cit> .
archaeological, ethnographic, linguistic and genetic investigations of isea have outlined colonization on the crossroads of multiple migrations between today’s ea mainland, australia and the pacific islands.
the area of eastern indonesia  has been described as a “melting pot” and a migratory “highway” based on genetic data  <cit> .
initial dna studies have provided insights into east timor’s complex composition  <cit> .figure  <dig>
the geographic location of east timor and the populations included in this study.
<dig> - east timor,  <dig> - nusa tenggara,  <dig> - bali,  <dig> - java,  <dig> - sumatra,  <dig> - mentawai,  <dig> - nias,  <dig> - peninsular malaysia,  <dig> - borneo,  <dig> - sulawesi,  <dig> - moluccas,  <dig> - philippines,  <dig> - vietnam,  <dig> - laos,  <dig> - thailand,  <dig> - south korea,  <dig> - taiwan,  <dig> - hainan,  <dig> - mixed han ,  <dig> - wng,  <dig> - png,  <dig> - admiralty islands,  <dig> - solomon islands,  <dig> – polynesia/fiji,  <dig> - australia,  <dig> - cambodia,  <dig> - myanmar,  <dig> - new britain and new ireland ,  <dig> - bougainville,  <dig> - vanuatu,  <dig> - new caledonia,  <dig> - micronesia,  <dig> - new zealand maori .
populations 2– <dig>  9– <dig>  and  <dig> are in indonesia;  <dig>   <dig> and  <dig> in png.
the population codes were retained throughout this study.
for references see additional file  <dig> where subpopulations are indicated with suffixes.
the routes and timing of human dispersal in the area remain to be fully clarified .
in any assumption, the influence of a changing climate needs to be considered.
the sea level in isea rose after ~ <dig> thousand years ago , then sank from ~ <dig> kya during the last glacial maximum  until ~ <dig> kya, whereupon the landmasses of sunda  and sahul  became partly submersed again by the rising sea.
dramatic coastline shifts occurred ~ <dig> ,  <dig> , and  <dig>  kya, creating the extant archipelago topography  <cit> .
timor was an island even during the lgm.
the pioneer migration out of africa brought the first anatomically modern humans, likely hunter-gatherers and beachcombers able to perform  voyages, to sahul ~60- <dig> kya  <cit> .
the expansion from sunda to sahul might have occurred along northern  or southern wallacea   <cit> .
the so-called austro-melanesian, “negrito”, or australoid first settlers are associated with non-austronesian  languages, spoken as far east as the solomon islands  <cit> .
their initial “fast train” migration likely left individuals carrying deep-rooting mtdna founder haplotypes along its course.
autochthonous mtdna lineages of isea, melanesia and australia support a long-term in situ development and date the first modern human arrival to > <dig> kya  <cit> .
the presence of upper paleolithic settlers in the region is confirmed by findings in east timor   <cit> , borneo   <cit> , eastern papua new guinea    <cit> , melanesia and australia   <cit> .
submergence could have caused a scarcity of sites .
subsequent migrations led to admixture with or replacement of the present foragers and shaped the genetic diversity of isea.
a major southward expansion out of taiwan is postulated from mid-holocene  linguistic and cultural changes associated with the spread of livestock domestication, agriculture and austronesian  speakers.
their arrival in eastern indonesia  is estimated to ~ <dig> kya from neolithic findings.
this migration has found support by dispersal patterns of mtdna lineages with ample basal diversity in taiwan, and is connected to the east asian  proportion of the genetic pool in isea and an speaking populations  <cit> .
however, the so-called “out of taiwan”  event has been questioned both in linguistics as single source of an languages  <cit>  and in genetics, since the model does not predict a direction and an speakers share few mtdna lineages despite matrilocality  <cit> .
moreover, archaeobotanic research indicates plant domestication in isea before this presumed advent  <cit> .
a two-step arrival, ~60- <dig> kya and ~8- <dig> kya, does not appear to fully reflect the demographic history of the region  <cit> .
mtdna coalescence ages, phylogenetic and dispersal patterns that neither fit with long term in situ development > <dig> kya nor southward population expansions < <dig> kya indicate a broader timeframe and geographic origin of migrations  <cit> .
additional mid-holocene expansions, labelled “express train” and “slow boat”  <cit> , might have originated elsewhere, possibly within isea, msea or near oceania  <cit> .
to account for these inconsistencies and to integrate the expected effects of a changing environment, such as facilitated migration by exposed and forced displacement by submersing land  <cit> , a more comprehensive model needs to be outlined to explain the extant maternal genetic landscape of isea.
the classical “waves” might rather represent longer periods of migration than short, distinct events.
the second “wave” may be extended to a period of ongoing gene flow in the late pleistocene and early holocene between ~40/30-10/ <dig> kya, with substantial, recurrent population expansions and shifts between asia and sahul and within isea, that may include the dispersals postulated from lineage-specific investigations  <cit> .
these intermediate migrations are supported by archaeological, linguistic, y-chromosomal and autosomal dna evidence .
further complexity might be added by gene flow from india into australia ~ <dig>  kya  <cit>  and “historic” movements from china, arabia and india associated with trade and the spread of religions  <cit> .
even in the classical scenario, east timor is of high genetic interest: it could have accommodated the “last step” before the colonization of sahul, westward  migrations have been suggested, and the southward oot migration could have extended there  <cit> .
nevertheless, mtdna composition of its population is scarcely described: the available data comprise  <dig> hypervariable segment i  sequences  <cit>  and  <dig> not individually reported haplotypes  <cit> .
we here present the first representative complete mtdna control region  reference dataset for east timor, comprising  <dig> country-wide samples  sequenced according to highest forensic quality standards.
our aim was to explore the potential of this geographically restricted sample in providing insights into the history of  human dispersal into and over the entire region.
we thus also generated complete mitogenome sequences of  <dig> samples and fundamentally refined the phylogeny of mtdna haplogroup p <dig> that appeared most promising for phylogeographic reconstructions.
we used the ion torrent personal genome machine  on an mtdna population sample in a forensic environment as stand-alone approach for the first time; this study therefore significantly contributes towards the implementation of massively parallel sequencing  into high quality mtdna typing routine.
RESULTS
ample mtdna variation of the east timor population
the diverse haplogroup spectrum of east timor revealed in this first representative mtdna study illustrates its position at the crossroads of several migrations between isea, melanesia and australia – and likely also the area’s rapid change from a large continental landmass to an archipelago  <cit> .
we detected  <dig> different cr haplotypes in the  <dig> mtdnas ;  <dig> of these  were unique in the dataset.
macro-haplogroup m comprised  <dig> % of the samples in  <dig> haplogroups; macro-haplogroup n  <dig> % in  <dig> haplogroups .
most frequent were q <dig>  and m7c <dig> .
using all information,  <dig> % of the samples could be assigned to a terminal “twig” of the mtdna phylotree  .
the most frequent cr haplotypes, relative to the revised cambridge reference sequence   <cit> , were 73g 146c 199c 263g  <dig> c  <dig> c 489c 523del 524del 16223t 16295t 16362c 16519c  and 73g 152c 249del 263g  <dig> c  <dig> c 521del 522del 523del 524del 16129a 16172c 16294t 16304c 16362c 16519c , with  <dig> % each .
figure  <dig> depicts the proportions of haplogroups, their phylogenetic relations and postulated geographic origin.
the latter reflects influence from two geographic macro-regions: mainly ea lineages were detected .
a considerable proportion indicated expansions of ea precursors in isea and polynesia , followed by mtdnas with indigenous melanesian/eastern indonesian/near oceanian origin .
admixture from outside eastern eurasia could only be concealed among the unresolved m* and r* mitogenomes .
the  <dig> novel east timor mtdna haplotypes  are illustrated in additional files  <dig> and  <dig>  available in genbank  and included in the european dna profiling group mtdna population database    <cit> .table  <dig>
mtdna haplogroup frequencies in our east timor sample
haplogroup
n
frequency
“p1e”§
total
#classified after complete mitogenome sequencing.
§postulated novel clade.
n - number of mtdnas.
haplogroups according to  <cit> , build  <dig>
schematic phylogenetic tree of all haplogroups observed in the  <dig> east timor samples.
their phylogenetic relation and postulated geographic origin are indicated.
the sizes of the circles correspond to haplogroup frequencies.
stem lengths are of no information content.
the tree is rooted in the mrca.
all information available was used.
haplogroups are according to  <cit> , build  <dig>
new insights from complete mitogenomes
our analyses at the highest resolution substantially contributed to elucidate and refine the phylogeny of haplogroup p <dig>  this is not “monotypic” as previously described , which would be uncommon for a successful founder.
the most parsimonious tree reconstructed from the available  <dig>  mitogenomes  confirmed subclade p1d and added considerable internal variation to p1d <dig> with five additional basal branches .
five east timor samples and a png singleton  <cit>  defined a novel branch characterized by the motif p1-152-13722-@ <dig>  that we tentatively called “p1e”, expanding the current mtdna nomenclature  <cit> .
the east timor p1e quintet additionally shared the transition np  <dig>  resulting in a previously studied polycytosine stretch .
two mitogenomes  <cit>  indicated additional basal p <dig> branches.
the earlier proposed subclades p1a-c  were defined partly by homoplasic markers and group with p1d and p1e lineages of our novel phylogeny.
multiple back-mutations of transitions currently defining haplogroup p <dig> , viz.
nps  <dig>   <dig>  and  <dig>  indicate the need for an updated nomenclature relying solely on the transition at np  <dig> as cr marker.figure  <dig>
the refined phylogeny of haplogroup p <dig>  the  <dig> novel completely sequenced haplogroup p <dig> mitogenomes from east timor are shown together with the eight previously published.
all differences are indicated  relative to the rcrs  <cit> .
haplogroups are according to  <cit> , build  <dig>  “p1e” is a novel suggestion.
bases are indicated according to the iubmb nucleotide code.
the prefix @ indicates the reversion of a mutation occurring earlier in the phylogeny.
underlined mutations are recurrent in the tree.
for details and references, see additional file  <dig>
the five completely sequenced non-p <dig> mitogenomes revealed novel lineages within haplogroups d6a, m21b, m73a, q <dig>  and r9c1b <dig> .
the genetic position of east timor
based on hvs-i and hvs-ii data, east timor  was amid the other island population in the unique haplotype proportions  and the intra-population random match probabilities  .
mainland populations yielded higher proportions of unique haplotypes  and lower rmps, likely due to smaller sample sets from islands, colonization bottlenecks and founder effects/isolation .
in total,  <dig> % of the east timor haplotypes  were also found in other populations.
the philippine population shared  <dig> % of its haplotypes  with east timor, followed by taiwan , all other populations were ≤ <dig> | <dig> %.
east timor ranked second  in the intra-population numbers of mean pairwise differences   -  <dig>  ) .
the inter-population mpd maxima of  <dig>   and  <dig>   derived from comparisons with the png sample.
for east timor, the inter-population mpd were found highest also with png and lowest with the philippines.
the pairwise fst values for east timor indicate a pronounced genetic differentiation if compared with png  and minor ones with all other populations .
the values including png were  <dig> - <dig> , while all other comparisons yielded ≤ <dig> .
amova revealed that the observed genetic variation was mainly attributable to variance within populations  .table  <dig>
genetic diversity of the east timor population and  <dig> surrounding populations using hvs-i and hvs-ii
population
reference
n
a
hts
b
unique hts
mpd
c
rmp
d
1a - east timor
<dig> - peninsul.
malaysia*
<dig> - philippines
<dig> - vietnam
<dig> - laos
<dig> - thailand
<dig> - south korea
<dig> - taiwan
<dig> - hainan
<dig> - mixed han
<dig> - papua new guinea
*.
anumber of mtdnas.
bnumber of haplotypes.
cmean number of pairwise differences.
drandom match probability.
reading frame: nps 16080– <dig>  16194– <dig>  73–300; total sample size: n =  <dig> .
for geographic locations, see figure  <dig>
restricting the comparisons to hvs-i to expand the population number, east timor  again positioned among the island populations  in the unique haplotype proportions .
the values for mainland populations, except australia and peninsular malaysia, were generally higher also here .
east timor yielded an rmp of 1: <dig>  similar to other isea populations  , and  <dig> % of its haplotypes  were also found in the  <dig> surrounding populations.
in general, iea and oceanian populations shared greater proportions with east timor than mainland asians.
the moluccas ranked highest  in the shared haplotype proportions and second in the proportions of individuals, where polynesia was highest .
this and similarly high values in other oceanian populations were caused by the outstanding prevalence of shared b4a <dig> haplotypes.
the influence of greatly differing haplotype numbers and haplogroup spectra  is also visible for nusa tenggara and new guinea  that shared rather low  haplotype proportions with east timor compared to the other  isea populations, but large sample proportions .
australia ranked lowest in both aspects  .
the highest numbers of intra-population mpd were obtained from ng , polynesia was lowest .
east timor again ranked high , closest to nusa tenggara .
the inter-population mpd maxima for hvs-i were  <dig>   and  <dig>  .
for east timor, the lowest  number of inter-population mpd was calculated with sulawesi  and the highest with wng .
based on the corrected numbers, mpd of east timor were lowest with the moluccas  and nusa tenggara ; the remaining values were more than double, up to  <dig>  .
amova revealed that  <dig> % of the variation was attributable to differences within populations .
we used the pairwise hvs-i fst values  for an mds analysis to depict genetic distances between populations .
a limited genetic differentiation indicating high gene flow between most ea populations was reflected by the resulting large main cluster.
east timor located in a separate cluster with the nusa tenggara and the moluccan samples, slightly shifted towards the outlying “eastern” populations from ng and the admiralties.
little gene flow was also indicated with the outlying populations from polynesian and the solomon islands; this mirrored the distant position shown by the genetic indices.
closer to the main cluster, about equidistant to east timor, were the isea outliers nias, java, mentawai and, interestingly, also the australian sample in the small range analyzed.
all outlier islands were characterized by low haplotype diversity/high rmp .table  <dig>
genetic diversity of the east timor population and  <dig> surrounding populations using hvs-i
population
reference
n
a
hts
b
unique hts
mpd
c
rmp
d
<dig> - east timor
1a - east timor
<dig> - nusa tenggara
<dig> - bali
<dig> - java
<dig> - sumatra
<dig> - mentawai
<dig> - nias
<dig> - peninsular malaysia*
<dig> - borneo
<dig> - sulawesi
<dig> - moluccas
<dig> - philippines
<dig> - vietnam
<dig> - laos
<dig> - thailand
<dig> - south korea
<dig> - taiwan
<dig> - hainan
<dig> - mixed han
<dig> - west new guinea
<dig> - papua new guinea
<dig> - admiralty islands
<dig> - solomon islands
<dig> - polynesia
<dig> - australia
*including singapore.
anumber of mtdnas.
bnumber of haplotypes.
cmean number of pairwise differences.
drandom match probability.
reading frame: nps 16080– <dig>  16195–16354; total sample size: n =  <dig> .
for geographic locations, see figure  <dig>
mds plot displaying the genetic distances between east timor and  <dig> surrounding populations.
based on pairwise f
st values in hvs-i .
for population codes, see figure  <dig>  for details and references, see table  <dig>  additional file  <dig>
elucidating the initial settlement from its maternal footprints
haplogroup p exhibited widespread distribution in both northern and southern sahul  as the only autochthonous lineage.
in our efforts to reconstruct the initial migrations between sunda and sahul we therefore particularly focussed on this haplogroup.
p generally occurred at frequencies <10%, but central and southern populations revealed proportions up to  <dig> %.
in most cases , these mtdnas completely or mainly consisted of p <dig> representatives; the highest p <dig> proportion was reached in png .
p <dig> lineages not further resolved by hvs-i  and the derived p1d <dig> mostly occurred together.
the remaining p lineages were less widespread even if combined and always occurred in parallel with and in smaller proportions than p <dig> , except in australia  and the philippines, as well as new caledonia, the admiralty islands and the kula ring .
pxp <dig> proportions > <dig> % were only revealed in the philippines, ng, australia, and new caledonia  .figure  <dig>
distribution of haplogroup p mtdnas in east timor and surrounding populations.
p <dig> and p1d <dig>  these categories discernible with hvs-i data enabled equal treatment of all populations included.
all other, including unassigned, p clades are combined into “pxp1”;  p1-p <dig>  all available information was considered .
the total of haplogroup p has been set to 100% for every population to better depict  proportions.
inconclusive p samples were denoted as “p*”.
frequencies are indicated by colored circle segments.
see legends for color codes.
population reference numbers located within a circle indicate that the analyzed haplogroup were not found in this population.
populations:  <dig> - east timor,  <dig> - nusa tenggara ,  <dig> - bali,  <dig> - java,  <dig> - sumatra,  <dig> - mentawai,  <dig> - nias,  <dig> - peninsular malaysia,  <dig> - borneo,  <dig> - sulawesi,  <dig> - moluccas ,  <dig> - philippines,  <dig> - vietnam,  <dig> - laos,  <dig> - thailand,  <dig> - south korea,  <dig> - taiwan,  <dig> - hainan,  <dig> - mixed han ,  <dig> - wng ,  <dig> – png ,  <dig> - admiralty islands,  <dig> - solomon islands,  <dig> - polynesia/fiji,  <dig> - australia ,  <dig> - cambodia,  <dig> - myanmar,  <dig> - new britain and new ireland,  <dig> - bougainville,  <dig> - vanuatu,  <dig> - new caledonia,  <dig> - micronesia,  <dig> - new zealand maori.
for more information, see additional file  <dig>
both an eastern indonesian and a melanesian  origin for haplogroup p have been proposed  <cit> .
the high-resolution phylogeographic reconstruction using the  <dig> available complete mitogenomes already indicates that not even the basal p <dig> diversity is concentrated in eastern indonesia : all sublineages were also found in ng, and samples from that island and the philippines pointed out additional branches at all hierarchical levels  <cit> .
a western melanesian cradle is also supported by published cr data.
various p <dig> sublineages are dispersed in eastern indonesia and beyond, but both the frequency and diversity peak lay in ng, whose central role was confirmed by the remaining p clades.
besides the more widespread p4a, ng shared p <dig> with australian populations, and p <dig> only found in taiwan and the philippines .
the only p lineages not found in ng were p <dig> from the philippines, and those restricted to australia, viz.
p4b and p5- <dig>  vice versa, p <dig> is only reported in ng, but has not been assessed in most other populations .
the coalescent ages of the autochthonous southwest pacific haplogroup p fit intriguingly well with the archaeological dating of the first settlements in this area : it was estimated to  <dig> - <dig>  ky based on cr  <cit>  and  <dig>  ky using complete mitogenomes.
the latter calculations yielded  <dig> - <dig>  ky for p2’ <dig>  p <dig>  p <dig> and p6; estimates for the remaining clades were not made due to paucity of data  <cit> .
our maximum likelihood  point estimate for haplogroup p <dig> considering the entire mtdna molecule and all  <dig> sequences was  <dig>  ky, p1d was dated at  <dig>  ky, p1d <dig> at  <dig>  ky and p1e at  <dig>  ky. rho  statistics yielded remarkably similar estimates completely overlapping with the ml ranges .
thus, there is no indication for an age underestimation by ρ statistics, an effect noticed using low-resolution data  <cit> .
the novel dating of p <dig> was consistent with results using fewer samples  <cit> .
partial mitogenome-based age reports for p <dig> were  <dig> - <dig>  ky  <cit> .
it appears that haplogroup p and its sublineages arose in a successful period at the cusp of or shortly after the first arrival of anatomically modern humans, merely the most successful clade p <dig> occurred separately and later.table  <dig>
molecular divergences and age estimates obtained by maximum likelihood and rho statistics for haplogroup p1
haplogroup
n
a
all nucleotide substitutions
ml
b
s.e.
c
t
d
Δt
d
ρ
σ
t
d
Δt
d
p1
> p1d
>> p1d1
> p1e
anumber of complete mtdna sequences.
bmaximum likelihood molecular divergence.
cstandard error.
dage estimates using the corrected molecular clock  <cit> .
the p lineage spectrum in australia is distinct from those from all other areas .
it is made up almost completely of the most ancient clades ; p <dig>  rare and shared with ng at a very low level , is also rather old .
despite a singular report of the only other shared p lineage, the youngest and elsewhere predominant p <dig>  this contradicts an origin of haplogroup p in central/southern sahul  followed by migrations to northern sahul .
rather it favours a southward colonization prior to ~ <dig> kya, when p <dig> developed, and little later north–south exchange even though a landbridge existed until ~ <dig> kya  <cit> .
developing this scenario further, the more recent p clades, such as p <dig>  would have arisen later during an “incubation” stage in northern sahul from root p* mtdna carriers left behind.
therefore, they were not or very limitedly represented on the forefront of settlers that proceeded southward and gave rise to australian mtdna diversity .
our age estimates also contraindicate that haplogroup p <dig> is old but simply did not move on  <cit> .
intriguingly, the singular reported australian p <dig> haplotype was revealed in a northeast aboriginal population considered closer to sea natives than other australian groups in oral history and due to anthropological traits  <cit>  .
apart from that, it could also derive from more recent, possibly individual, migration or displacement – a general caveat when equating lineage ages with timing of migration.
the synopsis of haplogroup p data indicates that eastern indonesia, including east timor, was not the cradle of this founder lineage, thus probably did not lie on the initial main route that likely led into northern sahul, but was rather populated from there after haplogroup p <dig> , p1d <dig> , and likely even p1e  , had arisen.
the landmass increase from ~ <dig> kya  could have triggered the demographic expansion at ~28- <dig> kya indicated in the bayesian skyline plot  from western melanesian p <dig> sequences , the time when all known p <dig> subclades emerged   - and could also have led to a geographic expansion that brought, among others, carriers of haplogroup p <dig> westward.
the lack of pxp <dig> lineages in east timor may be caused by the fact that a finite sample does not cover each and every rare lineage, or by drift, as they are found in and around eastern indonesia  <cit> .
for the same reasons - and the lack of mitogenomic data - we would not consider a more detailed dating of the westward movement reliable as it is based on the presence of only a p1e sublineage and the lack of p1d <dig> in east timor as termini ante resp.
post quem.
the second most widespread p lineage, p4a, is much younger   <cit> , absent from australia, could also have arisen in northern sahul, thus supporting an ongoing dynamic phase .figure  <dig>
bayesian skyline plot of mtdna haplogroup p <dig>  the hypothetical effective female population size is based on the complete p <dig> mitogenomes  assuming a generation time of 25 years.
the distribution of haplogroups q and q <dig> resembled that of p and p <dig> to a very high degree.
q carriers were only absent in the southern- and northernmost populations included; the possible cradle of this indigenous haplogroup, again lay in ng: proportions reached >70%, and only there and in the bismarck archipelago, all three q clades were found.
proportions >10% were revealed in the moluccas , east timor  and its northwest nusa tenggara neighbor islands , as well as in western melanesia .
haplogroup q proportions consisted predominantly of q <dig>  the most frequent “pioneer” lineage of eastern indonesia in general  <cit>  and east timor, highly diverse even in the cr .
q <dig> is widespread, and generally rare .
lineage q <dig> was found on the latter, in this study , and in ng , however, only one subclade carries hvs-i polymorphisms.
a single q sample was reported from australia, a q <dig> – again from the north  <cit>  .
haplogroup q has been dated to  <dig> - <dig>   ky using partial  <cit>  and  <dig>  ky using complete mitogenomes  <cit> .
the age estimates for q <dig>  q <dig>  and q <dig> from the latter study were  <dig> ,  <dig>  and  <dig>  ky, respectively, and widely ranged  when partial mitogenomic data were employed  <cit> .figure  <dig>
distribution of mtdna haplogroups q and n <dig> in east timor and surrounding populations.
haplogroups q1-q <dig>  unassigned/inconclusive q samples were denoted as “q*”;  haplogroup n <dig>  all available information was considered .
frequencies in populations are indicated by colored circle segments.
see legends for color codes.
population reference numbers located within a circle indicate that the analyzed haplogroup were not found in this population.
for the populations included, see figure  <dig>  for details and references, see additional file  <dig>
the complete absence of haplogroup q carriers from australia  indicates that southern sahul was populated before this lineage arose , as we postulate for haplogroup p <dig>  the outlined westward movements might also have caused the wide spread of the predominant q <dig>  the youngest q subclade, after  <dig>  kya - the timing we hypothesised from haplogroup p4a  .
these coincidences support a common history of haplogroups p <dig>  p4a and q and their spread probably even within the same group of settlers - separated from the events that led to the colonization of today’s australia.
westward movements after a stall in migration in northern sahul are the most likely scenario that explains our findings .
they have also been postulated in archaeology from old australian vs. younger timorese sites.
our genetic dating is however much younger than the first known settlements in timor , but older than those of modern humans in flores   <cit> .
a northward migration after an initial colonization of the south  appears less likely because of the complete lack of old australian p lineages everywhere else.
several mtdna genetic indices confirm that the relation between east timor and ng is indeed not a recent one .
ng populations, due to their extremely high frequencies of haplogroups p and q, appear generally far from all isea populations in these analyses.
still, east timor yielded the least genetic differentiation from ng of all samples included, possibly because of its most proximal location in westward expansions .
the “almost complete  isolation between the two regions”  <cit>  is also seen in nuclear dna investigations.
these support a continuous population history for sahul dating to probably ≥ <dig> kya demonstrating a deep common origin of australians and papua new guineans with little later migration  <cit> .
the often depicted split of initial sahul settlers into a northern and southern group after arrival on the continent, followed by isolation , would not explain the age gap in mtdna lineages between south and north alone, unless an “incubatory” phase is taken into consideration.
separate settlement waves from sunda to ng and to australia  <cit>  would also need to integrate the later development of p <dig> and q from root haplotypes.figure  <dig>
migration routes and their contribution to the east timor mtdna pool.
the major postulated migration events into isea and our novel findings are depicted.
the asterisk highlights east timor.
the darker grey areas indicate the predicted late pleistocene coastline.
the initial human settlement carrying haplogroups p, q, n <dig> and others that arrived  between 60– <dig> kya.
our results indicate  a colonization of australia  before  <dig> kya and  an incubation period in northern sahul  followed by westward expansions after  <dig> kya;  the holocene  southward out of taiwan movement marked by haplogroups m7c <dig>  d <dig>  f1a <dig>  f1a <dig> between 8– <dig> kya followed a  western or  eastern route , and  a local arisal, possibly connected to , of the “polynesian motif” ~ <dig> kya followed by west- and eastward migrations;  the postglacial expansion of haplogroup e   originating in eastern sunda and a dispersal 30– <dig> kya that  reached eastern indonesia ~ <dig> kya.
the inlay pie chart in  depicts the proportions of haplogroups associated with ,  and  within the extant east timor population.
the dashed line separates the proportion of the “polynesian motif”, as it has also been described to derive from a separate event.
see text for details.
from the similarly small proportions of autochthonous haplogroups along both, we cannot favour a northern or southern route through wallacea for the initial settlers  <cit> .
the clustering of the sulawesi and msea populations in the mds plot rather derives from the footprints of later inputs in the mtdna pool .
haplogroup n <dig>  another postulated “first settlement” marker, found in proportions ≤ <dig> % in msea and isea, has been dated to  <dig>  ky  <cit> .
east timor was its easternmost location.
this evidence is not easily compatible with migration scenarios .
in total, the autochthonous lineages considered to have arisen in the course of the initial dispersal, i.e.
p, q, m <dig>  m <dig>  n <dig>  r <dig>  and possibly further unresolved m* and r* clades  <cit> , account for  <dig> % of our east timor sample.
this confirms previous estimates for isea  <cit> .
traces of later settlements
we do not find that “the mitochondrial genomes of timorese women predominantly derive from papuan progenitors”  <cit> , but rather that additional migration after initial colonization is necessary to explain the predominant proportion of ea lineages, mirrored by high haplotype sharing rates and relatively small genetic, despite large geographic, distance .
we investigated east timor’s mtdna composition  in the light of four particular “later” migratory events postulated to have occurred between ea and melanesia during late pleistocene and mid-holocene in previous studies, often overlapping in terms of time and geography.
when their contributions are merged,  <dig> % of the east timor population  can be explained.
figure  <dig> depicts the migration routes we found to be relevant to the extant east timorese mtdna pool.
a neolithic oot expansion into isea in mid-holocene ~8- <dig> kya  <cit> : its postulated mtdna marker, the former haplogroup entity m7c3c , reached a frequency of  <dig> % in east timor .
also haplogroups d <dig> , f1a <dig> , f1a <dig> , and f3b  have been associated with this migration  <cit> .
thus,  <dig> %  of the east timor mtdnas could be related to this dispersal.
a ~20% contribution for isea was previously estimated  <cit> .
the oot-related mtdna haplogroups  peak in the philippines and eastern isea, but are almost absent from msea , melanesia, polynesia, and australia : this migration might thus have ended in eastern indonesia and not proceeded eastward as hypothesized  <cit>  .
a western  and an eastern  route into isea have been suggested  <cit> , as well as several subevents via more than a single route  <cit> .
our results support an eastern route into east timor: the combined marker haplogroups’ proportions were larger along this route , the western route marker y <dig>  <cit>  was absent in east timor, and the haplotype-based genetic parameters put east timor closest to nusa tenggara , and sulawesi and the moluccas, proximal only on the eastern route.
in the mds plot, sulawesi located at some distance from the cluster of east timor-nusa tenggara-moluccas, but closest to the philippines and taiwan, the postulated origin of the migration.
larger distances from the latter two, but similar ones to east timor, were yielded by the “western route” populations .
a pre-mid-holocene expansion in isea and polynesia  <cit> : marker haplogroup b4a1a <dig> probably originated in the vicinity of the bismarck archipelago and might have reached timor westward along a “voyaging corridor”  <cit> : the wide spread of this lineage   <cit> , highlights early navigating routes and capabilities.
the so-called “polynesian motif”  comprises  <dig> % of the east timor sample  .
notably, b4a1a has also been considered an oot marker  <cit> ; hence, the “maternal” contribution of that event to east timor would rise to  <dig> % .
the two dispersals would be strongly interwoven, and eastern isea  would not only represent the last section of a southward corridor , but also the assumed bottleneck through which only few haplogroups made it to melanesia and polynesia  <cit> .
a major postglacial eastward expansion into isea in late pleisto- and early holocene ~30- <dig> kya : accordingly, lineage e originated on the eastern sunda coastline and during this expansion, caused by the rising sea, reached eastern indonesia ~ <dig> kya  <cit> .
carriers were widespread in isea and near oceania, with a focus off the former eastern rim of sunda.
the second highest frequency was observed in east timor .
the second marker lineage, b4c <dig>  was generally rare.
altogether,  <dig> % of the east timor dataset  could be traced back to this neolithic dispersal .
remarkably, also haplogroup e has been linked to the oot expansion  <cit> .
a holocene arrival ~ <dig> kya from msea to isea  <cit>  may have played a minor role in populating its far east.
marker haplogroup f1a1*, frequent throughout eastern indonesia, msea, and on taiwan, is a singleton in the philippines  and east timor , where the second marker haplogroup, n9a <dig>  was absent .
CONCLUSIONS
our results from the oldest indigenous mtdna lineages of the southwest pacific region indicate migratory events that commenced < <dig>  kya, more than 15– <dig> ky after the initial arrival to sahul.
we thus confer the extension of the rather strictly timed classical two stages  also upon the “initial wave” of colonization and suggest a much broader timescale of events connected to indigenous haplogroups.
secondary expansions of initial settlers and westward migrations have been postulated before  and dated at ~ <dig> kya  <cit> .
this is the first clear genetic indication of a temporal proximity and possible coincidence of dispersals which encompassed autochthonous haplogroups with postulated “later” events of ea origin reconstructed from younger lineages.
in our scenario, it seems now probable that the “initial” and “later” migration routes, or even the population groups themselves, were or became intertwined.
ongoing genetic  “diffusion” of humans  was likely facilitated once trade corridors were established  <cit> .
the peak of sahul’s surface area ~30- <dig> kya coincides with the highly dynamic migratory period we report and might have triggered it.
population dispersals  do not necessarily also mean population expansion  <cit> , but bsps from thousands of samples in the hvs-i range  and tens of complete mitogenomes  from mixed haplogroups have  indicated a slowly growing population in the pleistocene that peaked at 20– <dig> kya and declined thereafter  <cit> .
this seems to pinpoint “substantial impact of common environmental forces” in isea  <cit> .
in  <dig>  ar wallace noted that the timorese appeared closer to inhabitants of the moluccas and ng than sea and speculated about admixture  <cit> .
almost one and a half century later, we tried to shed light on these relations by characterizing the mtdna composition of east timorese.
the blurring of older “footprints” by subsequent migrations, and the paucity of available  mitogenomic data may have confounded our assumptions on dispersal and time estimates.
in any event, the migration history  reconstructed from mtdna is only the “female side” of the medal, but “necessarily coupled” to the history of the entire population  <cit> .
other genetic markers  will support or modify our proposals, but contribute to the complete picture only together with non-genetic disciplines, such as archaeology  and linguistics - particularly, since the geographically overlapping an and papuan language families are thought to be separable in time; with the reservation of difficulties in speaker affiliation and language grouping .
all-embracing studies on human colonization will need to consider also other hominin species or populations  <cit> .

 the democratic republic of timor-leste is located in the lesser sunda islands of island southeast asia, between mainland southeast asia and australia, the indian and the pacific ocean. it extends over the eastern part of timor, the adjacent islands ataúro and jaco, and oecusse, an exclave within western timor. under portuguese rule since the 16th century, east timor declared its independence in and obtained it in after indonesian occupation. the country has a population of ~ million, a total area of  km and its highest peak reaches meters. thirty-two languages are spoken in the districts; four working languages are used. archaeological, ethnographic, linguistic and genetic investigations of isea have outlined colonization on the crossroads of multiple migrations between today’s ea mainland, australia and the pacific islands. the area of eastern indonesia has been described as a “melting pot” and a migratory “highway” based on genetic data. initial dna studies have provided insights into east timor’s complex composition.figure the geographic location of east timor and the populations included in this study. - east timor, - nusa tenggara, - bali, - java, - sumatra, - mentawai, - nias, - peninsular malaysia, - borneo, - sulawesi, - moluccas, - philippines, - vietnam, - laos, - thailand, - south korea, - taiwan, - hainan, - mixed han, - wng, - png, - admiralty islands, - solomon islands, – polynesia/fiji, - australia, - cambodia, - myanmar, - new britain and new ireland, - bougainville, - vanuatu, - new caledonia, - micronesia, - new zealand maori. populations 2– 9– and are in indonesia; and in png. the population codes were retained throughout this study. for references see additional file where subpopulations are indicated with suffixes. the routes and timing of human dispersal in the area remain to be fully clarified. in any assumption, the influence of a changing climate needs to be considered. the sea level in isea rose after ~ thousand years ago, then sank from ~ kya during the last glacial maximum until ~ kya, whereupon the landmasses of sunda and sahul became partly submersed again by the rising sea. dramatic coastline shifts occurred ~,, and kya, creating the extant archipelago topography. timor was an island even during the lgm. the pioneer migration out of africa brought the first anatomically modern humans, likely hunter-gatherers and beachcombers able to perform voyages, to sahul ~60- kya. the expansion from sunda to sahul might have occurred along northern or southern wallacea. the so-called austro-melanesian, “negrito”, or australoid first settlers are associated with non-austronesian languages, spoken as far east as the solomon islands. their initial “fast train” migration likely left individuals carrying deep-rooting mtdna founder haplotypes along its course. autochthonous mtdna lineages of isea, melanesia and australia support a long-term in situ development and date the first modern human arrival to > kya. the presence of upper paleolithic settlers in the region is confirmed by findings in east timor, borneo, eastern papua new guinea, melanesia and australia. submergence could have caused a scarcity of sites. subsequent migrations led to admixture with or replacement of the present foragers and shaped the genetic diversity of isea. a major southward expansion out of taiwan is postulated from mid-holocene linguistic and cultural changes associated with the spread of livestock domestication, agriculture and austronesian speakers. their arrival in eastern indonesia is estimated to ~ kya from neolithic findings. this migration has found support by dispersal patterns of mtdna lineages with ample basal diversity in taiwan, and is connected to the east asian proportion of the genetic pool in isea and an speaking populations. however, the so-called “out of taiwan” event has been questioned both in linguistics as single source of an languages and in genetics, since the model does not predict a direction and an speakers share few mtdna lineages despite matrilocality. moreover, archaeobotanic research indicates plant domestication in isea before this presumed advent. a two-step arrival, ~60- kya and ~8- kya, does not appear to fully reflect the demographic history of the region. mtdna coalescence ages, phylogenetic and dispersal patterns that neither fit with long term in situ development > kya nor southward population expansions < kya indicate a broader timeframe and geographic origin of migrations. additional mid-holocene expansions, labelled “express train” and “slow boat”, might have originated elsewhere, possibly within isea, msea or near oceania. to account for these inconsistencies and to integrate the expected effects of a changing environment, such as facilitated migration by exposed and forced displacement by submersing land, a more comprehensive model needs to be outlined to explain the extant maternal genetic landscape of isea. the classical “waves” might rather represent longer periods of migration than short, distinct events. the second “wave” may be extended to a period of ongoing gene flow in the late pleistocene and early holocene between ~40/30-10/ kya, with substantial, recurrent population expansions and shifts between asia and sahul and within isea, that may include the dispersals postulated from lineage-specific investigations. these intermediate migrations are supported by archaeological, linguistic, y-chromosomal and autosomal dna evidence. further complexity might be added by gene flow from india into australia ~ kya and “historic” movements from china, arabia and india associated with trade and the spread of religions. even in the classical scenario, east timor is of high genetic interest: it could have accommodated the “last step” before the colonization of sahul, westward migrations have been suggested, and the southward oot migration could have extended there. nevertheless, mtdna composition of its population is scarcely described: the available data comprise hypervariable segment i sequences and not individually reported haplotypes. we here present the first representative complete mtdna control region reference dataset for east timor, comprising country-wide samples sequenced according to highest forensic quality standards. our aim was to explore the potential of this geographically restricted sample in providing insights into the history of human dispersal into and over the entire region. we thus also generated complete mitogenome sequences of samples and fundamentally refined the phylogeny of mtdna haplogroup p that appeared most promising for phylogeographic reconstructions. we used the ion torrent personal genome machine on an mtdna population sample in a forensic environment as stand-alone approach for the first time; this study therefore significantly contributes towards the implementation of massively parallel sequencing into high quality mtdna typing routine.  ample mtdna variation of the east timor population the diverse haplogroup spectrum of east timor revealed in this first representative mtdna study illustrates its position at the crossroads of several migrations between isea, melanesia and australia – and likely also the area’s rapid change from a large continental landmass to an archipelago. we detected different cr haplotypes in the mtdnas; of these were unique in the dataset. macro-haplogroup m comprised % of the samples in haplogroups; macro-haplogroup n % in haplogroups. most frequent were q and m7c. using all information, % of the samples could be assigned to a terminal “twig” of the mtdna phylotree. the most frequent cr haplotypes, relative to the revised cambridge reference sequence, were 73g 146c 199c 263g c c 489c 523del 524del 16223t 16295t 16362c 16519c and 73g 152c 249del 263g c c 521del 522del 523del 524del 16129a 16172c 16294t 16304c 16362c 16519c, with % each. figure  depicts the proportions of haplogroups, their phylogenetic relations and postulated geographic origin. the latter reflects influence from two geographic macro-regions: mainly ea lineages were detected. a considerable proportion indicated expansions of ea precursors in isea and polynesia, followed by mtdnas with indigenous melanesian/eastern indonesian/near oceanian origin. admixture from outside eastern eurasia could only be concealed among the unresolved m* and r* mitogenomes. the novel east timor mtdna haplotypes are illustrated in additional files and available in genbank and included in the european dna profiling group mtdna population database.table mtdna haplogroup frequencies in our east timor sample haplogroup n frequency “p1e”§ total #classified after complete mitogenome sequencing. §postulated novel clade. n - number of mtdnas. haplogroups according to, build schematic phylogenetic tree of all haplogroups observed in the east timor samples. their phylogenetic relation and postulated geographic origin are indicated. the sizes of the circles correspond to haplogroup frequencies. stem lengths are of no information content. the tree is rooted in the mrca. all information available was used. haplogroups are according to, build new insights from complete mitogenomes our analyses at the highest resolution substantially contributed to elucidate and refine the phylogeny of haplogroup p this is not “monotypic” as previously described, which would be uncommon for a successful founder. the most parsimonious tree reconstructed from the available mitogenomes confirmed subclade p1d and added considerable internal variation to p1d with five additional basal branches. five east timor samples and a png singleton defined a novel branch characterized by the motif p1-152-13722-@ that we tentatively called “p1e”, expanding the current mtdna nomenclature. the east timor p1e quintet additionally shared the transition np resulting in a previously studied polycytosine stretch. two mitogenomes indicated additional basal p branches. the earlier proposed subclades p1a-c were defined partly by homoplasic markers and group with p1d and p1e lineages of our novel phylogeny. multiple back-mutations of transitions currently defining haplogroup p, viz. nps and indicate the need for an updated nomenclature relying solely on the transition at np as cr marker.figure the refined phylogeny of haplogroup p the novel completely sequenced haplogroup p mitogenomes from east timor are shown together with the eight previously published. all differences are indicated relative to the rcrs. haplogroups are according to, build “p1e” is a novel suggestion. bases are indicated according to the iubmb nucleotide code. the prefix @ indicates the reversion of a mutation occurring earlier in the phylogeny. underlined mutations are recurrent in the tree. for details and references, see additional file the five completely sequenced non-p mitogenomes revealed novel lineages within haplogroups d6a, m21b, m73a, q and r9c1b. the genetic position of east timor based on hvs-i and hvs-ii data, east timor was amid the other island population in the unique haplotype proportions and the intra-population random match probabilities. mainland populations yielded higher proportions of unique haplotypes and lower rmps, likely due to smaller sample sets from islands, colonization bottlenecks and founder effects/isolation. in total, % of the east timor haplotypes were also found in other populations. the philippine population shared % of its haplotypes with east timor, followed by taiwan, all other populations were ≤ | %. east timor ranked second in the intra-population numbers of mean pairwise differences - ). the inter-population mpd maxima of and derived from comparisons with the png sample. for east timor, the inter-population mpd were found highest also with png and lowest with the philippines. the pairwise fst values for east timor indicate a pronounced genetic differentiation if compared with png and minor ones with all other populations. the values including png were -, while all other comparisons yielded ≤. amova revealed that the observed genetic variation was mainly attributable to variance within populations.table genetic diversity of the east timor population and surrounding populations using hvs-i and hvs-ii population reference n a hts b unique hts mpd c rmp d 1a - east timor - peninsul. malaysia* - philippines - vietnam - laos - thailand - south korea - taiwan - hainan - mixed han - papua new guinea *. anumber of mtdnas. bnumber of haplotypes. cmean number of pairwise differences. drandom match probability. reading frame: nps 16080– 16194– 73–300; total sample size: n = . for geographic locations, see figure  restricting the comparisons to hvs-i to expand the population number, east timor again positioned among the island populations in the unique haplotype proportions. the values for mainland populations, except australia and peninsular malaysia, were generally higher also here. east timor yielded an rmp of 1: similar to other isea populations, and % of its haplotypes were also found in the surrounding populations. in general, iea and oceanian populations shared greater proportions with east timor than mainland asians. the moluccas ranked highest in the shared haplotype proportions and second in the proportions of individuals, where polynesia was highest. this and similarly high values in other oceanian populations were caused by the outstanding prevalence of shared b4a haplotypes. the influence of greatly differing haplotype numbers and haplogroup spectra is also visible for nusa tenggara and new guinea that shared rather low haplotype proportions with east timor compared to the other isea populations, but large sample proportions. australia ranked lowest in both aspects. the highest numbers of intra-population mpd were obtained from ng, polynesia was lowest. east timor again ranked high, closest to nusa tenggara. the inter-population mpd maxima for hvs-i were and. for east timor, the lowest number of inter-population mpd was calculated with sulawesi and the highest with wng. based on the corrected numbers, mpd of east timor were lowest with the moluccas and nusa tenggara; the remaining values were more than double, up to. amova revealed that % of the variation was attributable to differences within populations. we used the pairwise hvs-i fst values for an mds analysis to depict genetic distances between populations. a limited genetic differentiation indicating high gene flow between most ea populations was reflected by the resulting large main cluster. east timor located in a separate cluster with the nusa tenggara and the moluccan samples, slightly shifted towards the outlying “eastern” populations from ng and the admiralties. little gene flow was also indicated with the outlying populations from polynesian and the solomon islands; this mirrored the distant position shown by the genetic indices. closer to the main cluster, about equidistant to east timor, were the isea outliers nias, java, mentawai and, interestingly, also the australian sample in the small range analyzed. all outlier islands were characterized by low haplotype diversity/high rmp.table genetic diversity of the east timor population and surrounding populations using hvs-i population reference n a hts b unique hts mpd c rmp d - east timor 1a - east timor - nusa tenggara - bali - java - sumatra - mentawai - nias - peninsular malaysia* - borneo - sulawesi - moluccas - philippines - vietnam - laos - thailand - south korea - taiwan - hainan - mixed han - west new guinea - papua new guinea - admiralty islands - solomon islands - polynesia - australia *including singapore. anumber of mtdnas. bnumber of haplotypes. cmean number of pairwise differences. drandom match probability. reading frame: nps 16080– 16195–16354; total sample size: n = . for geographic locations, see figure  mds plot displaying the genetic distances between east timor and surrounding populations. based on pairwise f st values in hvs-i. for population codes, see figure  for details and references, see table  additional file elucidating the initial settlement from its maternal footprints haplogroup p exhibited widespread distribution in both northern and southern sahul as the only autochthonous lineage. in our efforts to reconstruct the initial migrations between sunda and sahul we therefore particularly focussed on this haplogroup. p generally occurred at frequencies <10%, but central and southern populations revealed proportions up to %. in most cases, these mtdnas completely or mainly consisted of p representatives; the highest p proportion was reached in png. p lineages not further resolved by hvs-i and the derived p1d mostly occurred together. the remaining p lineages were less widespread even if combined and always occurred in parallel with and in smaller proportions than p, except in australia and the philippines, as well as new caledonia, the admiralty islands and the kula ring. pxp proportions > % were only revealed in the philippines, ng, australia, and new caledonia.figure distribution of haplogroup p mtdnas in east timor and surrounding populations. p and p1d these categories discernible with hvs-i data enabled equal treatment of all populations included. all other, including unassigned, p clades are combined into “pxp1”; p1-p all available information was considered. the total of haplogroup p has been set to 100% for every population to better depict proportions. inconclusive p samples were denoted as “p*”. frequencies are indicated by colored circle segments. see legends for color codes. population reference numbers located within a circle indicate that the analyzed haplogroup were not found in this population. populations: - east timor, - nusa tenggara, - bali, - java, - sumatra, - mentawai, - nias, - peninsular malaysia, - borneo, - sulawesi, - moluccas, - philippines, - vietnam, - laos, - thailand, - south korea, - taiwan, - hainan, - mixed han, - wng, – png, - admiralty islands, - solomon islands, - polynesia/fiji, - australia, - cambodia, - myanmar, - new britain and new ireland, - bougainville, - vanuatu, - new caledonia, - micronesia, - new zealand maori. for more information, see additional file both an eastern indonesian and a melanesian origin for haplogroup p have been proposed. the high-resolution phylogeographic reconstruction using the available complete mitogenomes already indicates that not even the basal p diversity is concentrated in eastern indonesia: all sublineages were also found in ng, and samples from that island and the philippines pointed out additional branches at all hierarchical levels. a western melanesian cradle is also supported by published cr data. various p sublineages are dispersed in eastern indonesia and beyond, but both the frequency and diversity peak lay in ng, whose central role was confirmed by the remaining p clades. besides the more widespread p4a, ng shared p with australian populations, and p only found in taiwan and the philippines. the only p lineages not found in ng were p from the philippines, and those restricted to australia, viz. p4b and p5- vice versa, p is only reported in ng, but has not been assessed in most other populations. the coalescent ages of the autochthonous southwest pacific haplogroup p fit intriguingly well with the archaeological dating of the first settlements in this area: it was estimated to - ky based on cr and ky using complete mitogenomes. the latter calculations yielded - ky for p2’ p p and p6; estimates for the remaining clades were not made due to paucity of data. our maximum likelihood point estimate for haplogroup p considering the entire mtdna molecule and all sequences was ky, p1d was dated at ky, p1d at ky and p1e at ky. rho statistics yielded remarkably similar estimates completely overlapping with the ml ranges. thus, there is no indication for an age underestimation by ρ statistics, an effect noticed using low-resolution data. the novel dating of p was consistent with results using fewer samples. partial mitogenome-based age reports for p were - ky. it appears that haplogroup p and its sublineages arose in a successful period at the cusp of or shortly after the first arrival of anatomically modern humans, merely the most successful clade p occurred separately and later.table molecular divergences and age estimates obtained by maximum likelihood and rho statistics for haplogroup p1 haplogroup n a all nucleotide substitutions ml b s.e. c t d Δt d ρ σ t d Δt d p1 > p1d >> p1d1 > p1e anumber of complete mtdna sequences. bmaximum likelihood molecular divergence. cstandard error. dage estimates using the corrected molecular clock. the p lineage spectrum in australia is distinct from those from all other areas. it is made up almost completely of the most ancient clades; p rare and shared with ng at a very low level, is also rather old. despite a singular report of the only other shared p lineage, the youngest and elsewhere predominant p this contradicts an origin of haplogroup p in central/southern sahul followed by migrations to northern sahul. rather it favours a southward colonization prior to ~ kya, when p developed, and little later north–south exchange even though a landbridge existed until ~ kya. developing this scenario further, the more recent p clades, such as p would have arisen later during an “incubation” stage in northern sahul from root p* mtdna carriers left behind. therefore, they were not or very limitedly represented on the forefront of settlers that proceeded southward and gave rise to australian mtdna diversity. our age estimates also contraindicate that haplogroup p is old but simply did not move on. intriguingly, the singular reported australian p haplotype was revealed in a northeast aboriginal population considered closer to sea natives than other australian groups in oral history and due to anthropological traits. apart from that, it could also derive from more recent, possibly individual, migration or displacement – a general caveat when equating lineage ages with timing of migration. the synopsis of haplogroup p data indicates that eastern indonesia, including east timor, was not the cradle of this founder lineage, thus probably did not lie on the initial main route that likely led into northern sahul, but was rather populated from there after haplogroup p, p1d, and likely even p1e, had arisen. the landmass increase from ~ kya could have triggered the demographic expansion at ~28- kya indicated in the bayesian skyline plot from western melanesian p sequences, the time when all known p subclades emerged - and could also have led to a geographic expansion that brought, among others, carriers of haplogroup p westward. the lack of pxp lineages in east timor may be caused by the fact that a finite sample does not cover each and every rare lineage, or by drift, as they are found in and around eastern indonesia. for the same reasons - and the lack of mitogenomic data - we would not consider a more detailed dating of the westward movement reliable as it is based on the presence of only a p1e sublineage and the lack of p1d in east timor as termini ante resp. post quem. the second most widespread p lineage, p4a, is much younger, absent from australia, could also have arisen in northern sahul, thus supporting an ongoing dynamic phase.figure bayesian skyline plot of mtdna haplogroup p the hypothetical effective female population size is based on the complete p mitogenomes assuming a generation time of 25 years. the distribution of haplogroups q and q resembled that of p and p to a very high degree. q carriers were only absent in the southern- and northernmost populations included; the possible cradle of this indigenous haplogroup, again lay in ng: proportions reached >70%, and only there and in the bismarck archipelago, all three q clades were found. proportions >10% were revealed in the moluccas, east timor and its northwest nusa tenggara neighbor islands, as well as in western melanesia. haplogroup q proportions consisted predominantly of q the most frequent “pioneer” lineage of eastern indonesia in general and east timor, highly diverse even in the cr. q is widespread, and generally rare. lineage q was found on the latter, in this study, and in ng, however, only one subclade carries hvs-i polymorphisms. a single q sample was reported from australia, a q – again from the north. haplogroup q has been dated to - ky using partial and ky using complete mitogenomes. the age estimates for q q and q from the latter study were, and ky, respectively, and widely ranged when partial mitogenomic data were employed.figure distribution of mtdna haplogroups q and n in east timor and surrounding populations. haplogroups q1-q unassigned/inconclusive q samples were denoted as “q*”; haplogroup n all available information was considered. frequencies in populations are indicated by colored circle segments. see legends for color codes. population reference numbers located within a circle indicate that the analyzed haplogroup were not found in this population. for the populations included, see figure  for details and references, see additional file the complete absence of haplogroup q carriers from australia indicates that southern sahul was populated before this lineage arose, as we postulate for haplogroup p the outlined westward movements might also have caused the wide spread of the predominant q the youngest q subclade, after kya - the timing we hypothesised from haplogroup p4a. these coincidences support a common history of haplogroups p p4a and q and their spread probably even within the same group of settlers - separated from the events that led to the colonization of today’s australia. westward movements after a stall in migration in northern sahul are the most likely scenario that explains our findings. they have also been postulated in archaeology from old australian vs. younger timorese sites. our genetic dating is however much younger than the first known settlements in timor, but older than those of modern humans in flores. a northward migration after an initial colonization of the south appears less likely because of the complete lack of old australian p lineages everywhere else. several mtdna genetic indices confirm that the relation between east timor and ng is indeed not a recent one. ng populations, due to their extremely high frequencies of haplogroups p and q, appear generally far from all isea populations in these analyses. still, east timor yielded the least genetic differentiation from ng of all samples included, possibly because of its most proximal location in westward expansions. the “almost complete isolation between the two regions” is also seen in nuclear dna investigations. these support a continuous population history for sahul dating to probably ≥ kya demonstrating a deep common origin of australians and papua new guineans with little later migration. the often depicted split of initial sahul settlers into a northern and southern group after arrival on the continent, followed by isolation, would not explain the age gap in mtdna lineages between south and north alone, unless an “incubatory” phase is taken into consideration. separate settlement waves from sunda to ng and to australia would also need to integrate the later development of p and q from root haplotypes.figure migration routes and their contribution to the east timor mtdna pool. the major postulated migration events into isea and our novel findings are depicted. the asterisk highlights east timor. the darker grey areas indicate the predicted late pleistocene coastline. the initial human settlement carrying haplogroups p, q, n and others that arrived between 60– kya. our results indicate a colonization of australia before kya and an incubation period in northern sahul followed by westward expansions after kya; the holocene southward out of taiwan movement marked by haplogroups m7c d f1a f1a between 8– kya followed a western or eastern route, and a local arisal, possibly connected to, of the “polynesian motif” ~ kya followed by west- and eastward migrations; the postglacial expansion of haplogroup e originating in eastern sunda and a dispersal 30– kya that reached eastern indonesia ~ kya. the inlay pie chart in depicts the proportions of haplogroups associated with, and within the extant east timor population. the dashed line separates the proportion of the “polynesian motif”, as it has also been described to derive from a separate event. see text for details. from the similarly small proportions of autochthonous haplogroups along both, we cannot favour a northern or southern route through wallacea for the initial settlers. the clustering of the sulawesi and msea populations in the mds plot rather derives from the footprints of later inputs in the mtdna pool. haplogroup n another postulated “first settlement” marker, found in proportions ≤ % in msea and isea, has been dated to ky. east timor was its easternmost location. this evidence is not easily compatible with migration scenarios. in total, the autochthonous lineages considered to have arisen in the course of the initial dispersal, i.e. p, q, m m n r and possibly further unresolved m* and r* clades, account for % of our east timor sample. this confirms previous estimates for isea. traces of later settlements we do not find that “the mitochondrial genomes of timorese women predominantly derive from papuan progenitors”, but rather that additional migration after initial colonization is necessary to explain the predominant proportion of ea lineages, mirrored by high haplotype sharing rates and relatively small genetic, despite large geographic, distance. we investigated east timor’s mtdna composition in the light of four particular “later” migratory events postulated to have occurred between ea and melanesia during late pleistocene and mid-holocene in previous studies, often overlapping in terms of time and geography. when their contributions are merged, % of the east timor population can be explained. figure  depicts the migration routes we found to be relevant to the extant east timorese mtdna pool. a neolithic oot expansion into isea in mid-holocene ~8- kya: its postulated mtdna marker, the former haplogroup entity m7c3c, reached a frequency of % in east timor. also haplogroups d, f1a, f1a, and f3b have been associated with this migration. thus, % of the east timor mtdnas could be related to this dispersal. a ~20% contribution for isea was previously estimated. the oot-related mtdna haplogroups peak in the philippines and eastern isea, but are almost absent from msea, melanesia, polynesia, and australia: this migration might thus have ended in eastern indonesia and not proceeded eastward as hypothesized. a western and an eastern route into isea have been suggested, as well as several subevents via more than a single route. our results support an eastern route into east timor: the combined marker haplogroups’ proportions were larger along this route, the western route marker y was absent in east timor, and the haplotype-based genetic parameters put east timor closest to nusa tenggara, and sulawesi and the moluccas, proximal only on the eastern route. in the mds plot, sulawesi located at some distance from the cluster of east timor-nusa tenggara-moluccas, but closest to the philippines and taiwan, the postulated origin of the migration. larger distances from the latter two, but similar ones to east timor, were yielded by the “western route” populations. a pre-mid-holocene expansion in isea and polynesia: marker haplogroup b4a1a probably originated in the vicinity of the bismarck archipelago and might have reached timor westward along a “voyaging corridor”: the wide spread of this lineage, highlights early navigating routes and capabilities. the so-called “polynesian motif” comprises % of the east timor sample. notably, b4a1a has also been considered an oot marker; hence, the “maternal” contribution of that event to east timor would rise to %. the two dispersals would be strongly interwoven, and eastern isea would not only represent the last section of a southward corridor, but also the assumed bottleneck through which only few haplogroups made it to melanesia and polynesia. a major postglacial eastward expansion into isea in late pleisto- and early holocene ~30- kya: accordingly, lineage e originated on the eastern sunda coastline and during this expansion, caused by the rising sea, reached eastern indonesia ~ kya. carriers were widespread in isea and near oceania, with a focus off the former eastern rim of sunda. the second highest frequency was observed in east timor. the second marker lineage, b4c was generally rare. altogether, % of the east timor dataset could be traced back to this neolithic dispersal. remarkably, also haplogroup e has been linked to the oot expansion. a holocene arrival ~ kya from msea to isea may have played a minor role in populating its far east. marker haplogroup f1a1*, frequent throughout eastern indonesia, msea, and on taiwan, is a singleton in the philippines and east timor, where the second marker haplogroup, n9a was absent.  our results from the oldest indigenous mtdna lineages of the southwest pacific region indicate migratory events that commenced < kya, more than 15– ky after the initial arrival to sahul. we thus confer the extension of the rather strictly timed classical two stages also upon the “initial wave” of colonization and suggest a much broader timescale of events connected to indigenous haplogroups. secondary expansions of initial settlers and westward migrations have been postulated before and dated at ~ kya. this is the first clear genetic indication of a temporal proximity and possible coincidence of dispersals which encompassed autochthonous haplogroups with postulated “later” events of ea origin reconstructed from younger lineages. in our scenario, it seems now probable that the “initial” and “later” migration routes, or even the population groups themselves, were or became intertwined. ongoing genetic “diffusion” of humans was likely facilitated once trade corridors were established. the peak of sahul’s surface area ~30- kya coincides with the highly dynamic migratory period we report and might have triggered it. population dispersals do not necessarily also mean population expansion, but bsps from thousands of samples in the hvs-i range and tens of complete mitogenomes from mixed haplogroups have indicated a slowly growing population in the pleistocene that peaked at 20– kya and declined thereafter. this seems to pinpoint “substantial impact of common environmental forces” in isea. in ar wallace noted that the timorese appeared closer to inhabitants of the moluccas and ng than sea and speculated about admixture. almost one and a half century later, we tried to shed light on these relations by characterizing the mtdna composition of east timorese. the blurring of older “footprints” by subsequent migrations, and the paucity of available mitogenomic data may have confounded our assumptions on dispersal and time estimates. in any event, the migration history reconstructed from mtdna is only the “female side” of the medal, but “necessarily coupled” to the history of the entire population. other genetic markers will support or modify our proposals, but contribute to the complete picture only together with non-genetic disciplines, such as archaeology and linguistics - particularly, since the geographically overlapping an and papuan language families are thought to be separable in time; with the reservation of difficulties in speaker affiliation and language grouping. all-embracing studies on human colonization will need to consider also other hominin species or populations. 
in this study, one of the first in a forensic environment, we used the ion torrent personal genome machine for generating complete mitogenome sequences via stand-alone massively parallel sequencing and describe a standard data validation practice. in this first representative investigation on the mitochondrial dna variation of east timor population including > individuals, we put special emphasis on the reconstruction of the initial settlement, in particular on the previously poorly resolved haplogroup p an indigenous lineage of the southwest pacific region. east timor island southeast asiamitochondrial dnamtdna haplogroup phuman migrationfirst settlerspopulation geneticsforensic mtdna analysisnext generation sequencingion torrent pgmissue-copyright-statement© the author 2015 the temporal proximity and possible coincidence of these latter dispersals, which encompassed autochthonous haplogroups, with the postulated “later” events of east asian origin pinpoints a highly dynamic migratory phase. our results suggest a colonization of southern sahul > kya, limited subsequent exchange, and a parallel incubation of initial settlers in northern sahul followed by westward migrations < kya.
 distinct, partly competing, “waves” have been proposed to explain human migration in today’s island southeast asia and australia based on genetic evidence. the paucity of high quality and high resolution data has impeded insights so far. in this study, one of the first in a forensic environment, we used the ion torrent personal genome machine for generating complete mitogenome sequences via stand-alone massively parallel sequencing and describe a standard data validation practice.  in this first representative investigation on the mitochondrial dna variation of east timor population including > individuals, we put special emphasis on the reconstruction of the initial settlement, in particular on the previously poorly resolved haplogroup p an indigenous lineage of the southwest pacific region. our results suggest a colonization of southern sahul > kya, limited subsequent exchange, and a parallel incubation of initial settlers in northern sahul followed by westward migrations < kya.  the temporal proximity and possible coincidence of these latter dispersals, which encompassed autochthonous haplogroups, with the postulated “later” events of east asian origin pinpoints a highly dynamic migratory phase. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords east timor island southeast asiamitochondrial dnamtdna haplogroup phuman migrationfirst settlerspopulation geneticsforensic mtdna analysisnext generation sequencingion torrent pgmissue-copyright-statement© the author 2015 