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Resolving the ancestry of Austronesian-speaking populations
There are two very different interpretations of the prehistory of Island Southeast Asia (ISEA), with genetic evidence invoked in support of both. The "out-of-Taiwan" model proposes a major Late Holocene expansion of Neolithic Austronesian speakers from Taiwan. An alternative, proposing that Late Glacial/postglacial sea-level rises triggered largely autochthonous dispersals, accounts for some otherwise enigmatic genetic patterns, but fails to explain the Austronesian language dispersal. Combining mitochondrial DNA (mtDNA), Y-chromosome and genome-wide data, we performed the most comprehensive analysis of the region to date, obtaining highly consistent results across all three systems and allowing us to reconcile the models. We infer a primarily common ancestry for Taiwan/ISEA populations established before the Neolithic, but also detected clear signals of two minor Late Holocene migrations, probably representing Neolithic input from both Mainland Southeast Asia and South China, via Taiwan. This latter may therefore have mediated the Austronesian language dispersal, implying small-scale migration and language shift rather than large-scale expansion.
Austronesian languages are spoken throughout Taiwan, Island Southeast Asia (ISEA), parts of New Guinea and most of the Pacific Islands. The high linguistic diversity observed in the aboriginal groups of Taiwan, compared to the single language branch (Malayo-Polynesian) spoken throughout the remainder of this vast distribution (Ross 2005), has suggested to historical linguists a homeland on the island of Taiwan (Blust 1976, 1995). The Taiwanese linguistic homeland model has received further support in recent years from the work of Ross (2009), which effectively nests Malayo-Polynesian within the Formosan language tree, and has led in turn to the prevailing "out-of-Taiwan" model (Bellwood 1997; Hung et al. 2011) for the spread by demic diffusion of farming—referring specifically to rice agriculture—and red-slipped pottery from 4000 years ago (4 ka) in ISEA, culminating in the spread of Austronesian Oceanic speakers into the Pacific within the last 3 ka (Bellwood 1997; Spriggs 2003, 2007). In practice, the Neolithic in ISEA is defined by the appearance of ceramics, and less ubiquitously new shell artefacts and cloth and barkcloth technologies, and any role of rice or other introduced agriculture has proved much more contentious (Spriggs 2011).
The "out-of-Taiwan" model has been dominant for 30 years, despite challenges on many fronts. Languages can be transmitted horizontally, so a root in Taiwan need not automatically imply a demic diffusion model. More complex pictures are emerging for ISEA, in which coastal language shift and language transmissions play the major role (Donohue and Denham 2010, 2015). This paves the way for a modified small-scale "out-of-Taiwan" model that retains the linguistic argument for the origin of the Austronesian family in Taiwan without assuming any large-scale population movement or replacement, or that rice agriculture is the driving force (Diamond and Bellwood 2003; Oppenheimer 2004; Spriggs 2011).
In fact, a key driver of human mobility may have been the dramatic transformation in the landscape of ISEA in the late Pleistocene/early Holocene. Sea-level rises due to global warming at the end of the last glaciation separated the ancient Sunda continent—for millennia an extension of mainland Asia—into present-day ISEA and Mainland Southeast Asia (MSEA). These are thought to have been concentrated in three major episodes, from 15 to 13.5 ka, 11.5 to 10 ka and 7 to 8 ka (Pelejero et al. 1999). Alternative models to "out-of-Taiwan" have argued that it may have been the rapid coastal transformation and resulting land-loss (Solheim 2006) that had the most profound effect on genetic patterns in the region, rather than a more recent expansion from Taiwan (Oppenheimer 1998; Oppenheimer and Richards 2001; Soares et al. 2008, 2011) However, although such models attempt to explain the current population structure in ISEA, they have been less successful in incorporating the linguistic evidence suggesting an Austronesian origin in Taiwan (Barker and Richards 2013).
On the genetic side, many seemingly contradictory results have been published in recent years, shifting the perspective back and forth between a strong Neolithic expansion and minor or non-existent dispersals from Taiwan. The two-layer colonization model (Pleistocene colonization and mid-Holocene "out-of-Taiwan" expansion) (Bellwood 1997) often remains the lens through which data are interpreted. Thus, genetic variation is often categorised either as autochthonous (first colonization or "Melanesian") or as a later Asian input interpreted as "Austronesian" (Friedlaender et al. 2008; Kayser et al. 2008b). The "Asian" signal on the mtDNA is generated by the so-called "Polynesian motif" (Delfin et al. 2012; Melton et al. 1995; Redd et al. 1995; Soares et al. 2011; Sykes et al. 1995; Trejaut et al. 2005), which approaches 100 % in many Remote Pacific islands.
However, Soares et al. (2011) estimated an arrival of this clade (or its ancestor) in Near Oceania ~6 to 10 ka. Although the motif has an ultimately mainland Asian ancestry (in haplogroup B4a1a) sometime in the last 10–20 ka (Soares et al. 2011), it was already well established in Near Oceania by the mid-Holocene. This implies that the contribution of a Neolithic "out-of-Taiwan" migration to Remote Pacific Islanders is negligible in the mtDNA [as well as Y-chromosome (Capelli et al. 2001; Kayser et al. 2000)] variation in the last 3 ka. But, for ISEA, too, the picture is far from consistent with an "out-of-Taiwan" demic expansion. The largest surveys consistently suggest a far more complex picture than the two-layer model (Capelli et al. 2001; Hill et al. 2007; Karafet et al. 2010; Trejaut et al. 2014; Tumonggor et al. 2013). Sea-level rises probably shaped much of the genetic structure of ISEA (Hill et al. 2007; Karafet et al. 2010), with major dispersals originating in what is now the mainland [including mtDNA haplogroup B4a1a (Soares et al. 2011)] as well as across what is now ISEA [including haplogroup E (Soares et al. 2008)].
Genome-wide data have also led to challenges to the "out-of-Taiwan" model, albeit with caveats (discussed below). The Pan-Asian SNP Consortium (Abdulla et al. 2009) suggested that the diversity of Taiwanese aboriginals is likely a sub-set of the ISEA diversity, implying that dispersals between Taiwan and ISEA took place in the reverse direction. This would match the situation seen in mtDNA haplogroup E, inferred to have expanded in ISEA in the postglacial period and reached Taiwan within the last 8 ka (Soares et al. 2008).
Here we perform founder analyses with large new mtDNA datasets, both control-region and whole-genome sequences, and—for the first time—Y-chromosome data. Founder analysis estimates dispersal times and quantifies the contribution of each migration to the present-day population. We develop an explicit set of criteria by which to evaluate candidate "out-of-Taiwan" markers, and show that haplogroup M7c3, analysed here at the maximal resolution level of whole-mtDNAs, and found in aboriginal Taiwanese and the Philippines at moderate frequencies, but only low frequencies in ISEA and the western Pacific, fulfils these criteria almost perfectly. However, the other major candidates proposed for the "out-of-Taiwan" dispersal, haplogroups E and B4a1a, fail to meet any of them.
Single-locus studies of the uni-parental marker systems can today provide exquisite resolution, but they are, of course, subject to greater stochastic effects than the autosomal genome. We therefore here back up mtDNA and Y-chromosome variation with fresh analyses of the autosomal genome-wide structure of Southeast Asians. These multi-locus analyses support the view that the spread of the red-slipped pottery Neolithic and Austronesian languages in ISEA were indeed accompanied by dispersals of sea-farers from Taiwan, but beyond the Philippines the primary mechanism for the spread of both was acculturation. In fact, a slightly earlier Neolithic dispersal from MSEA, involving paddle-impressed ceramics and possibly accompanied by Austroasiatic languages, had a substantially greater genetic impact on much of ISEA, especially in the south.
mtDNA founder analysis
Founder analysis (Richards et al. 2000) works by identifying founder types (the result of individual migration events from source to sink in the past) and then partitioning clusters derived from them in the sink population on the basis of their coalescence times, to estimate arrival times. This mtDNA founder analysis departed from reduced-median networks (Bandelt et al. 1995) of data from HVS-I (the first hypervariable segment) of the mtDNA control region (between nucleotide positions 16,051 and 16,400), usually augmented by haplogroup-diagnostic coding-region variants.
For our sink region, we analysed 2216 mtDNA sequences from Island Southeast Asia (556 from the Philippines, 340 from Borneo, and 1320 from the rest of Indonesia), including 320 new sequences from ISEA (183 from Sabah, Brunei and Kalimantan in Borneo) and published data (Table S1). For the source we included 6070 Chinese sequences, 1429 from MSEA, 827 aboriginal Taiwanese and 4573 sequences throughout North and Central Asia. We included additional data from Malaysia (519 Malays and 308 Orang Asli), 55 unpublished sequences from Singapore and published data from other regions to further resolve the phylogenetic networks (Table S2). A second database was created with the aim of performing a founder analysis for Remote Oceania. Adding to the sequences above, datasets for New Guinea (846), the Karkar Islands (47), the Solomon Islands (258), the Bismarck Archipelago (1005) and Bougainville (255) were included in the new source population. Sequences from Vanuatu (130) and throughout Polynesia (148) were included in the sink population (Table S3).
We analysed the data phylogenetically haplogroup by haplogroup, and carried out the founder analysis as before (Rito et al. 2013), including a 200-year scan as a preliminary step. We estimated errors using the approach of Saillard et al. (2000), to allow for non-star-like founder clusters. In this approach, we replaced the number of samples in the ρ estimation by an effective number of samples based on the number of samples that would be present in a completely star-like network associated with the same level of uncertainty as we have implemented before (Soares et al. 2012) including at the whole-population level for South African populations (Rito et al. 2013). We employed a mutation rate of one mutation every 16,677 years for the range 16,051–16,400 (Soares et al. 2009) in the founder analysis.
We regard the scan as a heuristic approach to detecting and dating peaks of immigration, and the partition analysis as an attempt to quantify and place confidence limits upon them. Following examination of the heuristic scan, we then used archaeological evidence to finalize the dates chosen for the partition analysis, which should compensate for any systematic bias in the HVS-I mutation rate we adopted. 4.5 ka approximates the earliest likely arrival of both the putative "out-of-Taiwan" Neolithic and the paddle-impressed-ware Neolithic from MSEA in ISEA and is likely to be conservative in the case of the former—an age of 4.5 ka is more persuasive for the influence of MSEA than Taiwan, which more likely postdates 4 ka (Anderson 2005; Spriggs 2007). The putative episode of sea-level rise at ~8 ka (Pelejero et al. 1999) was initially the least well-established of the three episodes (Blanchon and Shaw 1995), but a rapid rise between 7.4 and 6.5 ka has recently received clear support (Bird et al. 2010). The existence of human dispersals at around this time is, however, supported by whole-mtDNA genome evidence from haplogroup E in ISEA and Taiwan (Soares et al. 2008), and by the founder analysis scan. For the primary settlement, we used 50 ka as a rough approximation; evidence from Niah Cave and New Guinea suggest an age upwards of 49 ka (Hunt et al. 2007; Summerhayes et al. 2010).
Y-chromosome founder analysis
We enhanced the resolution of previously available data (Capelli et al. 2001) by augmenting with 10 more Y-STR (short tandem repeat) markers and five more SNPs. Adding to the ten previous Y-chromosome STRs (DYS388, DYS393, DYS392, DYS19, DYS390, DYS391, DYS425, DYS426, DYS389I and DYS389II), we included an additional ten: DYS460, DYS461, DYS438, DYS448, DYS458, DYS437, DYS439, H4, A10 and DYS635. We typed the ten Y-STRs in a multiplex that also included the previously typed Y-STRs, DYS388 and DYS425 (Capelli et al. 2001), to control against mixing of samples across the studies, and as a result we excluded several samples. The DYS426 marker gave inconsistent typing results across the dataset and we therefore excluded this locus from further analyses. Table S4 shows a list of the primers used in the multiplex. We ran the samples on an ABI 16-capillary 3130XL DNA Analyser at the University of Leeds.
Five SNPs were added that included: M230, which allowed the identification of the Pacific haplogroup S; M324, which allowed the identification of O3a against O3* in the previous dataset (Capelli et al. 2001); M50, which allowed the identification of subclade O1a2 within O1a, which were to prove highly relevant to identifying "out-of-Taiwan" founders; and M38 and M208, which permit the identification of subclades C-M38 and the Pacific C-M208 in samples previously labelled C* haplogroup only (Capelli et al. 2001). We typed M230, M324 and M208 using restriction analysis (Table S5 indicate primers and enzymes used), and M50 and M38 using denaturing high-performance liquid chromatography (dHPLC), as described previously (Underhill et al. 2000). We ran the samples on an automated 3500HT Wave (Transgenomic) dHPLC instrument and analyzed the results with the Navigator software. Fig. S1 summarises the SNPs analysed and the distribution of the clades identified across the sampling locations.
We used the SNP data to test the reliability of the STR analyses by constructing a median-joining network (Bandelt et al. 1999) of the overall Y-STR data. Initially we calculated a highly reticulated network using the same standard weight (10) for all STRs. We registered the number of occurrences of each STR change, and used these values to generate a graduated weighting scheme for a new network reconstruction (weight of 1 for the STR showing the highest number of occurrences and a weight of 10 for the lowest). We repeated this process until the phylogeny became stable with further iterations. The final weighting scheme gave DYS458 a weight of 1, while DYS388, DYS425, DYS437 and DYS438 were each given a weight of 10. The new STR network showed a remarkable congruence with the clustering of SNP-defined lineages (Fig. S2), with just a few minor exceptions (most particularly, ten C-M38 lineages that separated from the main cluster), showing that, although the SNP tree is limited, the Y-STR data, suitably analysed, can provide an unbiased and reliable reconstruction of ancestry. STR markers are, of course, far more unstable than bi-allelic variants but, if we consider the difficulties associated with generating a reliable tree of mtDNA haplogroups based on HVS-I data alone, we can assume we have a comparable resolution to the maternal founder analysis counterpart—possibly higher.
Zhivotovsky et al. (2004) calculated an effective mutation rate of 6.9 × 10−4 mutations in 25 years per Y-STR, or an average Y-STR mutation rate of 2.76 × 10−5 mutations per year, which is slower than estimated Y-STR pedigree rates by an order of magnitude (Gusmão et al. 2005). Since our dataset included a star-like clade that is confined to Remote Oceania (Fig. S3), and considering that the colonization time of this region is well established by radiocarbon evidence, we opted for re-calibrating the specific 19 Y-STR average mutation rate using the settlement of the Remote Pacific as the calibration point. To this end, we constructed a network of haplogroup C-M208 and applied weights obtained previously in the general Y-STR network (Fig. S4) to the network. C-M208 shows three main branches, two present in Madang and Vanuatu and a third present only in Remote Oceania (excluding Vanuatu) from Fiji to French Polynesia. The expansion into Remote Oceania began as early as ~3.3 ka, indicated by radiocarbon estimates at a Lapita site in Vanuatu (Bedford et al. 2006), while Western Polynesia (Tonga and Samoa) was colonised ~2.9 ka (Rieth and Hunt 2008). The star-like subclade, C-M208, was not present in Vanuatu and we therefore assumed an estimate of 3000 years for the age of this subclade, whose westernmost location was Fiji [see discussion in Clark and Anderson (2009)]. We estimated an average Y-STR mutation rate of 4.08 × 10−5 mutations per year, meaning nearly 50 % faster than the one calculated by Zhivotovsky et al. (2004). We must emphasize that this average mutation rate corresponds specifically to this group of STRs, and also that we are dealing with a very recent human event in the calibration. Our estimated mutation rate is still substantially slower when compared with father–son transmission studies (Ballantyne et al. 2010; Gusmão et al. 2005).
As for the HVS-I mtDNA data, we calculated networks for each clade. We rooted the networks by estimating the root through midpoint rooting and using an outgroup of the consensus STR length of the closest available clade to further pinpoint a hypothetical root. Again we selected founders using both f1 and f2 criteria (Richards et al. 2000). We used the Bayesian migration partition tool (Richards et al. 2000) in the same two ways: a scan of equally distant intervals of 200 years (Rito et al. 2013; Soares et al. 2012) and a model of migration with migration time windows based on the scan and archaeological and climatological data. This is the first direct application of founder analysis to Y-chromosome data.
The partition model included, as for mtDNA, migrations at 0.5 ka (for recent gene flow), 4.5 ka (Neolithic) and 8 ka (postglacial migrations) differing only in the time of the older migration (20 ka instead of 50 ka). We do not expect the ρ statistic (Forster et al. 1996) to be a good estimator of age for more ancient lineages in a highly mutating system such as STRs. Because of this, using a realistic time of first settlement based on archaeology of ~50 ka (Hunt et al. 2007) in the partition model would cause the older lineages dating to about 20 ka to be statistically allocated in the postglacial migration at 8 ka, to which they are closer, which we believe would be quite misleading. We therefore opted for including a migration at the time of the peak, 20 ka, even though the age of the peak probably does not correspond to the time of migration.
Validation: founder analyses for Remote Oceania
To evaluate the performance of the methodology, we also executed a founder analysis from ISEA/Near Oceania into Remote Oceania. The well-characterized time of expansion into the Remote Pacific islands provides a valuable framework for testing the clock and the founder analysis methodology that we employed. The expansion into Remote Oceania may have begun before 3 ka into Vanuatu (Bedford et al. 2006), but the major migration into Western Polynesia began only within the last 3 ka (Rieth and Hunt 2008). In our founder analysis using a 200-year scan we obtained the same pattern for the two criteria: a single peak at 3000 years (Fig. S5), fitting very well the archaeological data.
We obtained a similar pattern for the Y-chromosome analysis (Fig. S5). However, contrary to the mtDNA analysis, we should re-emphasize that this is very far from a completely independent check for the methodology, since the rate of the Y-STRs we employed was calibrated assuming 3000 years for the major founder clade entering Remote Oceania. It is nevertheless reassuring that the time of the peak was not affected by the inclusion of the other clades in the analysis. We should note also that the estimated mutation rate is to be employed in determining migratory fractions in ISEA and not the Pacific, so its use does not provide circular evidence, only the cross-checking.
Genome-wide analysis
We used 1251 samples taken from the Pan-Asian SNP Genotyping Database (Abdulla et al. 2009; Ngamphiw et al. 2011) (Table S6). We used the West African Yoruba data as an outgroup, as well as a South Asian group. The objective of the analysis is to compare the genome-wide patterns with the haploid marker results.
The initial dataset contained 54,794 SNPs. We pruned this dataset for linkage disequilibrium (LD) using PLINK (Purcell et al. 2007). One SNP in pairs with LD higher than r2 = 0.1 was removed in windows of 50 SNPs shifted five SNPs each time, as used before (Pierron et al. 2014; Verdu et al. 2014). We used a total of 23,332 SNPs in further analyses. We employed ADMIXTURE (Alexander et al. 2009) to estimate population structure using a maximum likelihood approach, assuming different numbers (2–15) of ancestral populations or genetic components (K). A few populations, namely the "Negrito" groups in Malaysia, displayed a single private component for very low values of K that is not present elsewhere, most probably due to the effect of strong genetic drift. As the objective of the analysis was to display overall geographic patterns to provide a frame of comparison with the uniparental markers' phylogeography, and since these populations did not provide any relevant information in this regard, we opted for excluding them from the final analysis. We performed a cross-validation by inspecting the cross-validation error (CVE) in the analyses with different values of K (2–15). Theoretically, the one with the lowest CVE should be the most accurate. A graphic of the variation of the CVE obtained is shown in Fig. S6. Although the CVE does not vary much above K = 5, K = 10 displays the lowest value.
To visualize the distribution of specific mtDNA clades or autosomal components, we displayed the frequency distributions using the Kriging algorithm of Surfer 8. Data points used for the mtDNA and the genome-wide components are shown in Fig. S7.
Whole-mitochondrial genomes
We analysed lineages from across the range of variation in mtDNA haplogroup M7, but with a particular focus on the candidate "out-of-Taiwan" marker, M7c3c. We generated a total of 114 new M7 sequences, including 38 Taiwanese, 20 Vietnamese, 16 Indonesians, 12 Peninsular Malaysians, 7 East Malaysians, 6 Laotians, 4 Chinese, 4 Micronesians (from Nauru and Kiribati), 3 Bruneians, 2 Filipinos, 1 Burmese and 1 Thai. We also extracted 51 new M7 sequences from the raw data of the 1000 Genomes project (unavailable at the start of this study). We performed whole-mtDNA sequencing as previously described (Torroni et al. 2001) using an ABI 48-capillary 3730 DNA Analyser (Taipei) an ABI 16-capillary 3130XL DNA Analyser (Leeds) and an ABI 16-capillary 3100 DNA Analyser (Porto). Details on the new and published sequences used in the phylogenetic reconstruction of haplogroup M7 are indicated in Table S7. We deposited the new whole-mtDNA sequences in GenBank (accession numbers JX987440-JX987470 and KU131308-KU131390). For comparison with mtDNA haplogroup M7, we also re-analysed haplogroups B4a1 and E using all the available published whole-mtDNA genome sequences. We list the samples we used in the analyses in Tables S8 and S9.
We reconstructed phylogenies of haplogroups M7, B4a1a and E using Network 4.6 software with the reduced-median algorithm (Bandelt et al. 1995), resolving reticulations on the basis of the relative rates of the mutations involved (Soares et al. 2009). We estimated ages for the different phylogenies using both the ρ statistic (Forster et al. 1996) and maximum likelihood (ML), using the mtDNA clock of Soares and collaborators that corrects for purifying selection from the long-term phylogenetic rate of one mutation every 3624 years (Soares et al. 2009). We estimated branch lengths in ML using PAML 3.13 (Yang 1997) assuming the HKY85 substitution model with gamma-distributed rates (32 categories). We also employed a synonymous mutation rate of one substitution every 7884 years (Soares et al. 2009) using the ρ statistic (Forster et al. 1996). We have discussed in some detail previously the potential impact of mutation rate uncertainty on phylogeographic conclusions (Mellars et al. 2013) and we re-calculated the confidence intervals in similar fashion here.
We obtained Bayesian skyline plots (BSPs) (Drummond et al. 2005) using BEAST 1.4.6. (Drummond and Rambaut 2007) to detect signatures of population increment associated with the haplogroups under analysis. We employed a relaxed molecular clock (lognormal in distribution across branches and uncorrelated between them), a mutation rate of 2.514 × 10−8 mutations per site per year for the whole-mtDNA genome (Pereira et al. 2010) and the HKY model of nucleotide substitutions with gamma-distributed rates, assuming a generation time of 25 years. We compared different signatures of population growth in ISEA and aboriginal Taiwanese data for the three haplogroups analysed, M7c3c, B4a1a and E.
Given a recent age estimate for haplogroup E based on a single ancient DNA sequence (Ko et al. 2014) that diverges significantly from previous estimates for this haplogroup, we performed a new estimate that was also based on ancient DNA sequences. Ancient DNA calibration is becoming a widely used approach (Ho et al. 2007), but estimates based on a single sequence are highly divergent and present large confidence intervals (Fu et al. 2013b). Calibrating a clock with only a single ancient DNA sequence can be misleading, particularly for recent samples for which even the stochastic presence of one more or one less mutation than the expected clade average can lead to strong departures from a realistic mutation rate. For calibration purposes, we added two more ancient East Asian sequences to the haplogroup E sequence described by Ko et al. (2014). One dates to the same time-frame as the E sequence: Boshan 11, from north-east China, at 8.18 ka (Fu et al. 2013b). The second one is older: Tianyuan 1301, also from north-east China, at 39.5 ka (Fu et al. 2013a); it is important to include this in the analysis, since haplogroup E as a whole will necessarily be older than the ancient haplogroup E sequence. In the analysis, we used a tree that represents a snapshot of human diversity (Table S10), which allows a better estimate of the evolutionary parameters in mtDNA, with all the main haplogroup E branches represented. We employed a relaxed molecular clock (lognormal in distribution across branches and uncorrelated between them) using BEAST 1.4.6. (Drummond and Rambaut 2007), with a constant population size and the HKY model of nucleotide substitutions, with gamma-distributed rates.
mtDNA control-region and Y-chromosome founder analyses
To investigate the genetic input into ISEA through time, we carried out founder analyses with both mitochondrial DNA (mtDNA) control-region sequences—for the maternal line of descent—and Y-chromosome variation using a 19 Y-STR dataset within SNP-defined lineages—for the paternal line of descent. Founder analysis is a quantitative phylogeographic approach developed to evaluate the diversity of lineage clusters that has arisen within a particular geographic sink region (in this case, ISEA), following migration from a specified (assumed) source region (in this case, MSEA/China/Taiwan). Using the molecular clock to convert to time depth, these values are a proxy for the minimum arrival age of each founder cluster in the sink (Richards et al. 2000).
For Y-chromosome variation (Fig. 1b), we obtained very similar peaks with both criteria (one at 4–5 ka and a second at ~8 ka). Rather remarkably, the two main peaks in two different genetic systems with distinct mutation rates and estimated using two distinct founder criteria are consistent across each of these different analyses. In addition, we observed an increment representing very recent migrations and, with the f2 criterion, a further extra peak at 10–11 ka. This peak might signal the second well-defined episodic flood immediately after the Younger Dryas (Pelejero et al. 1999). We did not include it in the migration model, however, for two reasons: it was detected only under a single criterion and with one genetic system; and, in any case, founders at this peak will be included statistically in the ~8 ka migration that overall can be defined as postglacial migrations. The oldest arrivals here date to ~20 ka, largely haplogroup K and C lineages. This may well correspond with the ancient minor peak for mtDNA; we expect ρ dating with STRs to provide severe underestimates for ancient clades because of mutational saturation. However, for the present analysis this is a minor issue, since we are concerned primarily with events in the Holocene. Particularly in the case of K, an older age than this could be expected, considering that K probably evolved in the region since the first settlement as displayed by the high prevalence of K* and K subclades in the ancient Sahul populations, including Aboriginal Australians (Hudjashov et al. 2007).
We then partitioned the founders in ISEA using a migration model informed not only by the scan results in the two genetic systems, but also archaeological and palaeoclimatological evidence, to quantify the contribution of each immigration event to the extant mtDNA and Y-chromosome gene pools in ISEA. The model from mtDNA data here assumes migrations at 4.5, 8 and 50 ka, corresponding to Neolithic immigration, postglacial expansions and first Pleistocene settlement. We assumed a further dispersal at 0.5 ka to allow for any recent/historical gene flow.
For Y-chromosome variation, we used a more recent age of 20 ka to cover the more ancient migrations, as mentioned above. However, the matching of peaks at 4–5 ka and 8–10 ka for both the paternal and maternal line of descent is striking. The overall contribution at each proposed migration time for each of the two founder criteria in the mtDNA and Y-chromosome variation is shown graphically in Fig. 1c, d. The mtDNAs coalescing at the time of the first settlement (~50 ka) accounted for ~10 to 20 % of modern mtDNA lineages in ISEA. Note that many lineages from the ancient Sunda continent would very likely be present across both ISEA and MSEA, which were only finally separated by sea-level rise ~8 ka. However, MSEA is a source region in this analysis, so this value in the founder analysis corresponds to ancient lineages private to ISEA only. In the mtDNA analysis, lineages descending directly from the haplogroups carried by the first settlers correspond to M*, N*, R* and possibly haplogroup F3 (Fig. 1d). Although a recently published ancient mtDNA haplogroup E sequence (Ko et al. 2014) was used to suggest a Taiwanese source for this clade, an early origin in ISEA (Soares et al. 2008) remains more likely, as discussed below. At this ancient time-frame, Y-chromosome lineages (with STR ρ dating) are uninformative due to saturation, but haplogroups K* and even C may date to the first colonization at that time. These are above 30 % in the Y-chromosome analysis.
Overall, the migration at ~8 ka contributes the most lineages to the current gene pool of ISEA with a fraction of ~40–50 % in both mtDNA and Y-chromosome variation (Fig. 1c). We stress again that, statistically, this migration time could include lineages entering ISEA throughout the period of sea-level rises, from 14 to 8 ka, covering all three flooding episodes (Pelejero et al. 1999). This partition probabilistically includes major and well-studied haplogroups such as B4a1a (Soares et al. 2011), subclades of haplogroup E (Soares et al. 2008), F1a*, and subclades of haplogroup M shared between ISEA and MSEA, with B4a1a and E the major contributors. In Y-chromosome variation, this migration includes most clusters within haplogroups O2a1 and O3 and a subclade of O1a (Fig. S4), matching to some extent the results of Karafet et al. (2010) indicating that O1a* entered ISEA before the Neolithic. We should note that in our recent Y-chromosome survey (Trejaut et al. 2014), O2 and O3 clades declined in frequency moving north from ISEA towards Taiwan, the opposite of what one might expect from an "out-of-Taiwan" movement. A previous survey (Karafet et al. 2010) also suggested that O3, O2a1 and O1a* entered ISEA from the mainland before the Neolithic period.
The contribution at the time of the Neolithic, at 4–5 ka, varied with the criterion and the genetic system, but 25–35 % is probably the best estimate (Fig. 1c). (The f1 criterion in mtDNA probably overestimates recent migration due to the large size of the source sample used.) Only one major founder presented significant differences between the analyses: B4b appears Neolithic in f1 criterion and part of the postglacial migration in the f2 criterion (Fig. 1d). This haplogroup deserves further attention in the future. The widely held model for the spread of the Neolithic in ISEA implicates expanding pre-Austronesian/Austronesian speakers from South China/Taiwan (Bellwood 1997); but in fact not all of the Neolithic founders we identify support this hypothetical "out-of-Taiwan" dispersal. A large fraction of Neolithic mtDNA founder clusters from haplogroups B5a1 and F1a1a (~10 % out of the 25–35 % Neolithic lineages in the analysis) appear to have originated in MSEA, and are rare or absent in either Taiwan or the Philippines.
On the male line of descent, the Neolithic contribution is lower (15–20 %) but, since MSEA is not represented in the Y-chromosome dataset, all these Neolithic founders are likely to represent the putative "out-of-Taiwan" dispersal, mirroring closely the ~20 % "out-of-Taiwan" founders for mtDNA. Most of O1a and all of O1a2 likely represent signals of Neolithic migrants from Taiwan, confirming earlier suggestions (Karafet et al. 2010; Trejaut et al. 2014). A portion of O3a (~10 % in the f1 criterion) was also partitioned into the Neolithic in our analysis.
Corroboration of founder analyses with genome-wide evidence
We next compared these results with patterns observed in autosomes, using genome-wide data from the Pan-Asian SNP Genotyping Database (Abdulla et al. 2009; Ngamphiw et al. 2011) and the ADMIXTURE software.
Analyses from K = 6 to K = 15 generate additional components by further sub-dividing these Northeast and Southeast Asian components, whilst maintaining the African, South Asian and Melanesian/Near Oceanian components intact across the analyses. The autosomal estimate with ten ancestral populations, theoretically the best estimate of ancestry for the data as it has the lowest cross-validation error, includes seven components with discernible frequencies in at least one location in Austronesian-speaking populations (Fig. S9). The South Asian component (white) is present at low frequencies only in the Malay Peninsula and Sumatra, matching the historical record (Manguin et al. 2011). The Northeast Asian component (red) is seen at appreciable frequency only in the Philippines (at only very low rates). A Near Oceanian component (pale blue) dominates many of the populations of Eastern Indonesia, as expected. Two minor components (dark green and yellow) are virtually specific to ISEA, mainly in what was western Sundaland (Java/Sumatra/Malay Peninsula), with one (yellow) markedly elevated in Aboriginal Malays.
One important component (grey) is both specific for Austronesian-speaking populations and highly frequent across ISEA (Fig. S9). It reaches 60–70 % in the two aboriginal Taiwanese groups in the sample—the equivalent cluster in Pan-Asian SNP data approaches 100 % (Abdulla et al. 2009)—peaking in our dataset in the Philippines, Sumatra and Sulawesi (70–90 %), and is virtually absent from Continental Asia, suggesting an insular origin. Comparison between the analyses with five and ten ancestral populations also suggests that this was part of the larger Southeast Asian component in the former. Considering the major postglacial signal observed in mtDNA and Y-chromosome variation in both our founder analysis and in earlier analyses (Hill et al. 2007; Karafet et al. 2010; Soares et al. 2008, 2011; Trejaut et al. 2014), and the sharing of many lineages between ISEA and Taiwan (Soares et al. 2008, 2011), this autosomal component may correspond to an ancestral cluster common to both Taiwan and ISEA that was established before the hypothetical dispersal of Austronesian. Even if we consider that there is likely a signal of Austronesian expansion "out-of-Taiwan" in the genome-wide data (see below), this component, which is most frequent in Taiwan, the Philippines, the Mentawai Islands and Sulawesi, disparate islands at opposite extremes of the Sunda shelf, could explain why a maximum likelihood population tree of the Pan-Asian SNP data indicated Taiwan as an offshoot of ISEA diversity (Abdulla et al. 2009). Such population trees only depict broad patterns and, although a minor component could show an ancestry in Taiwan when compared with ISEA, the most frequent component could show the overall opposite ancestry.
Two autosomal components that might signal Neolithic dispersals can be compared with the patterns obtained from Neolithic founder candidates in the mtDNA analysis. One of these components (paler green in Fig. 3b) is frequent in MSEA/Southwest China (up to ~70 %) and varies from 5 to 40 % in Indonesia (Fig. 2d), but is absent from Taiwan and rare in the Philippines. It is probably a relatively recent arrival as it is not evenly distributed across ISEA. The MSEA Neolithic candidates in the mtDNA also show a strong peak of frequency in MSEA and frequencies of 5–30 % in Indonesia, but are rare in the Philippines and Taiwan (Fig. 2c). We can also match these distributions with the presence of basket-marked and carved paddle-impressed pottery: in Sarawak, assemblages at ~4.5 ka with carved cord-wrapped or basketry-wrapped paddle-impressed pottery (Bellwood 1997; Bulbeck 2008) show the influence of an early Neolithic from MSEA in Western Indonesia.
The final component (dark blue in Fig. 3b) has a high frequency in South China (Fig. 2b) and is also seen in Taiwan at ~25–30 %, in the Philippines at ~20–30 % (except in one location which is almost zero) and across Indonesia/Malaysia at 1–10 %, declining overall from Taiwan within Austronesian-speaking populations. The mtDNA candidates for "out-of-Taiwan" markers (Fig. 2a) also show an overall frequency of up to ~35 % in Taiwan and the Philippines, but are almost absent in parts of Borneo, Java and Eastern Indonesia. Sumatra superficially presents a more discordant picture between genome-wide and mtDNA results, but the sampling of the Pan-Asian SNP dataset involves only Batak people whilst our mtDNA sampling involved the wider Sumatran population. We should also bear in mind that the genome-wide sampling lacks major areas of ISEA, including the whole of Borneo.
Therefore, the overall picture from the ADMIXTURE analysis with 10 ancestral populations where the cross-validation error was the lowest, is concordant with the mtDNA and Y-chromosome pattern, with a minor Neolithic input from MSEA, probably immediately preceding a Neolithic input from Taiwan (Anderson 2005) that had a strong demographic impact in the Philippines, but a much more minor genetic input elsewhere in the Indo-Malaysian Archipelago.
Confirmation with whole-mtDNA genome data
Although providing much larger sample sizes, the low phylogenetic resolution of mtDNA HVS-I data can create problems for phylogeographic analyses such as founder analysis, for example by conflating distinct founders. In parallel, we therefore checked the phylogeographic signal with the much better resolved whole-mtDNA genomes for the major "out-of-Taiwan" haplogroup in the founder analysis, M7c3c. In particular, we wished to compare the results for M7c3c with the two putative postglacial signals for haplogroups E and B4a1a (Soares et al. 2008, 2011).
The overall phylogenetic and phylogeographic pattern is strikingly clear: both aboriginal Taiwanese and Island Southeast Asian-specific lineages are close to the tips of an overall mainland Eastern Asian distribution. The major subclade of M7b3, M7b3a, is only present in Taiwan and ISEA. It is frequent in Taiwan (at ~10 %) and considering its age (~6 ka) seems likely to have arrived in Taiwan with the rice Neolithic from South China; but it is vanishingly infrequent across ISEA. In M7b1, M7b1d3 is also restricted to Taiwan, and with a similar age may also have arrived from China with the Neolithic, but again it is virtually absent from ISEA.
In M7c′e′f, the three subclades branch from a single node and all show evidence of East Asian ancestry. Within M7c, M7c3 is by far the most frequent and the only one to disperse significantly into Taiwan and ISEA. This clade probably had an origin in South China, with several subclades also present in Taiwan. Its major subclade, M7c3c [M7c1c in Hill et al. (2007)], here re-dated with whole-mtDNA genomes to ~5 ka, is restricted to Austronesian-speaking populations (both Taiwan and ISEA). Given the presence of other subclades of M7c3 in Taiwan and South China, the most probable source for M7c3c is in Taiwan (amongst M7c3 arrivals from China, again perhaps with the rice Neolithic), with subsequent dispersal into ISEA. Several subclades of M7c3c exist throughout Taiwan and ISEA, and there is also one in the Pacific (M7c3c2, found in both Micronesia and the Solomon Islands), dating to less than 3 ka. This pattern confirms M7c3c as a strong candidate for an "out-of-Taiwan" marker, as indicated by the HVS-I founder analysis.
- (a)
If the haplogroup was carried into Taiwan from South China by rice-agriculturists ~6 to 8 ka, the dispersal's timing should be bracketed by the age of the ancestral clade seen in South China (upper bound) and the insular Austronesian-specific subclade (lower bound);
- (b)
the insular and Austronesian-specific subclade should date to after the arrival of rice-agriculturists from China ~5.5 ka, but before the "out-of-Taiwan" migration ~4.5 ka;
- (c)
the founder age in ISEA for the subclade should date to ~4.5 ka, the time of the "out-of-Taiwan" dispersal;
- (d)
the founder age from Taiwan/Philippines to the rest of ISEA should be lower than the date of the "out-of-Taiwan" migration, ~4 ka; and
- (e)
the expansion of the clade in Taiwan should predate the expansion in ISEA.
Point (b) stipulates that the insular subclade should originate after the hypothetical arrival of rice-agriculturists in Taiwan and before the dispersal "out-of-Taiwan". M7c3c, at 5.2 [4.0; 6.5] ka, follows this pattern; B4a1a, at 9.9 [5.5; 14.5] ka, and haplogroup E, at 24.0 [14.5; 33.9] ka, both suggest an earlier origin within currently Austronesian-speaking populations.
Taking point (c), an average founder age for M7c3c from Taiwan into ISEA is 4.4 [3.2; 5.7] ka, matching the 4.5 ka prediction of the "out-of-Taiwan" model. Haplogroups E and B4a1a yield 8.8 [6.0; 11.6] ka and 7.3 [5.2; 9.4] ka, respectively, suggesting earlier postglacial expansions. When including the Philippines along with Taiwan as part of the source for the dispersal—point (d)—the founder for haplogroup M7c3c dated a little lower at 4.2 [2.5; 5.9] ka—a striking match to the hypothetical Austronesian arrival in the Indo-Malaysian archipelago. Haplogroup E, by contrast, yielded 6.4 [4.8; 8.0], and the B4a1a point estimate actually increased to 8.5 [4.8; 12.3] ka, when compared with the previous founder age estimate into ISEA as a whole, clearly indicating that the "out-of-Taiwan" assumption of the founder model in this case is likely to be false.
Finally (e), we used BSPs to estimate the expansion time of each haplogroup. Figure 5c–e show the increment or rate of expansion (corresponding skyline plots in Fig. S10; data in Table S13). The B4a1a data for Taiwan and ISEA (Fig. 5c) suggest a very similar time of expansion, starting ~10 ka (with a second expansion restricted to Taiwan ~2000 years ago). However, haplogroup E expanded in ISEA before Taiwan (Fig. 5d), starting ~8 ka for ISEA and ~7 ka for Taiwan. Finally, for M7 we see a first expansion in Taiwan starting ~7.5 ka, peaking at 5.2 ka, while for ISEA the expansion starts later at 5.2 ka with peak at ~4 ka, corresponding closely to the "out-of-Taiwan" model.
Therefore, haplogroup M7c3c meets all the criteria expected for an "out-of-Taiwan" marker, whereas haplogroups E and B4a1a meet none of them. Yet a haplogroup E lineage recently recovered from human remains in the Strait of Taiwan, dating to ~8 ka, evidently represents a sequence ancestral to the E1 subclade, leading Ko et al. (2014) to suggest an origin of haplogroup E ~10 ka ago in China or Taiwan and a Neolithic migration into ISEA (based on a Bayesian analysis). This compares with our estimate for the age of haplogroup E with the time-dependent clock (Soares et al. 2009) of ~24 ka (Fig. 5). Previous age estimates based on the time-dependent clock and Bayesian ancient DNA calibrations do not differ to this extent (Fu et al. 2013b), despite some claims to the contrary. The authors of one recent estimate based on several ancient sequences claim that their estimated rate is 45 % faster than the one we estimated (Brotherton et al. 2013), but this arises from their comparing their estimated rate with our inter-specific phylogenetic rate rather than the time-dependent rate. For the time-frame of the European Neolithic and Bronze Age with which they were concerned, our curve indicates a mutation rate of 2.307 × 10−8 substitutions per site per year for the time of 6.15 ka (their oldest sample), only 4 % slower than the one they estimated. The difference would be even less for the age of their other, younger samples.
Here, indeed, we estimate an age for haplogroup E of 29.7 [18.5; 43.9] ka and an average mutation rate of 2.041 × 10−8 [1.54 × 10−8; 2.48 × 10−8] substitutions per site per year using a Bayesian estimate with two additional East Asian ancient DNA sequences. Given that the root of haplogroup E is seven mutations from the root of the "out-of-Africa" haplogroup M (Macaulay et al. 2005; Mellars et al. 2013) which has an average branch length to the present-day (~50,000 years) of ~20 mutations, age estimates for E more recent than ~20 ka seem implausible.
Involving haplogroup E in a wide-scale Neolithic dispersal across and out of mainland China also ignores the evidence that haplogroup E is restricted to the off-shore islands and has never been seen in any extant Chinese populations. Its age of >20 ka and insular distribution rather suggest an origin on the eastern side of the Sunda shelf. Although the early Holocene haplogroup E sequence creates a deeper link within E1, extant diversity haplogroup E diversity nevertheless remains deeper in ISEA, for both E1 and E2 (Soares et al. 2008). Moreover, a large mtDNA survey of aboriginal Taiwanese groups, which probably diverged early in Austronesian history, but were subsequently isolated and experienced drift very differently from other Austronesian populations, failed to detect any novel haplogroup E diversity, finding the same sub-set of ISEA diversity (Ko et al. 2014). The 8-ka age of the sample would place it in a period of intense postglacial expansions, due to huge sea-level changes resulting from global warming, and might be better explained as an offshoot from the south, where many lineages were lost in the postglacial period. We would caution against drawing strong conclusions from a single sample. Nevertheless, regardless of its point of origin, our analyses show that haplogroup E most probably expanded in ISEA well before the Neolithic period.
Settlement models of ISEA that emphasize climate change and drastic shifts in the population in the early postglacial period have tended to side-step the linguistic evidence for a Taiwanese origin of the Austronesian languages (Donohue and Denham 2010). Although languages may sometimes be transmitted solely horizontally, for example by trade, this seems unlikely to explain the pattern of the Austronesian languages in ISEA as a whole, in the context of such a wide and ecologically complex region (Blench 2012). We address this issue here from the standpoint of genetic variation across the genome.
Previous results have shown a strong common ancestry between ISEA and Taiwan populations predating the pottery Neolithic period for mtDNA and Y-chromosome variation (Capelli et al. 2001; Hill et al. 2007; Karafet et al. 2010; Trejaut et al. 2014; Tumonggor et al. 2013), as well as indications that some minor lineages entered ISEA during the Neolithic. Here we show that two Neolithic waves entered ISEA, as previously suggested on the basis of pottery comparisons (Anderson 2005) and recently from autosomal analyses (Lipson et al. 2014), but that both were small-scale affairs.
The first Neolithic migration, from MSEA ["Neolithic I" in the scheme of Anderson (2005)], reflected in the distribution of haplogroups B5a1 and F1a1a and the "pale green" genome-wide component, took place ~4.5 ka and affected mainly Western Indonesia/Borneo—although it extended as far as Eastern Indonesia, particularly in the south, even reaching regions of contact with Papuan populations. A signal for this dispersal was also recently proposed by Lipson et al. (2014), although they favoured admixture with Austronesian agriculturists dispersing around the coasts of MSEA as an explanation, which our results render unlikely.
The second Neolithic wave ["out-of-Taiwan" or Anderson's "Neolithic II" (Anderson 2005)] is marked by the appearance of red-slipped pottery ~4 ka (Spriggs 2007, 2011) and impacted strongly on the Philippines (accounting for 30–40 % of current genetic diversity), where domesticated rice does indeed appear relatively early in the archaeological record (Paz 2002). However, for the rest of ISEA (the Indo-Malaysian archipelago), the demographic impact was much lower—often negligible. The overall fractions of "out-of-Taiwan" immigrants in the founder analysis for both mtDNA and Y-chromosome variation are very similar at ~15 to 20 %, suggesting that previous models inferring highly divergent male and female contributions are incorrect (similarly to the Pacific). The mtDNA haplogroup M7c3c, in particular, closely matches the expected pattern for an "out-of-Taiwan" marker.
Thus, although the Neolithic dispersal from Taiwan suggested by red-slipped pottery proves not to have been a large-scale demographic event (at least, beyond the Philippines), it did indeed occur, and followed an expansion into Taiwan from South China, as one archaeological model predicts (Bellwood 1997). However, we must be careful what we mean by the term "Neolithic", since the archaeological record for most of ISEA primarily indicates the appearance of various novel ceramics, and provides little or no evidence for large changes in the subsistence base. The low level of settlement across ISEA at this time accords not with large-scale demic diffusion fuelled by rice agriculture, but with more with archaeological views that stress the transition from grain cultivation to the root and arboreal crops that dominate agricultural systems in the western Pacific (Donohue and Denham 2010; Paz 2002). It is clearly parsimonious to conclude that these sea-faring settlers spoke Austronesian and spread their languages across ISEA, but they may have had rather little to do with either rice farming or arboriculture/vegeculture (aspects of which originated much earlier, in part diffusing from Near Oceania (Barker and Richards 2013; Blench 2012).
The low scale of the migrations overall concurs with recent archaeological evaluations (e.g. Spriggs 2011), but contrasts sharply with the recent interpretation of Lipson et al. (2014). However, their assumption that aboriginal Taiwanese represent the source for ISEA, their use of only three autosomal source clusters and their extremely recent age estimates for admixture times (within the last 2200 years) compromise their conclusions. Our analysis supports a scenario in which language shift played the major role, rather than large-scale population replacement (Donohue and Denham 2010, 2015).
The genetic situation further east seems to require a model where language was transmitted mostly horizontally across the north coast of New Guinea. Curiously, M7c3c (most or all probably belonging to the subclade M7c3c2 dating to ~2.6 ka) and some other putative "out-of-Taiwan" subclades (like B4b1) are detected at relatively high frequencies in Eastern Micronesia/Northwest Polynesia. These lineages may have been carried directly through Western Micronesia from the vicinity of the Philippines (Fitzpatrick and Callaghan 2013; Hung et al. 2011). This migration was, however, distinct from the primary spread of the Austronesian languages into the Pacific, and would be expected to have affected mainly the Marianas.
Otherwise, whilst languages may have moved alongside other lineages integrated within ISEA, "out-of-Taiwan" haplogroups are virtually undetected across the north coast of New Guinea, the Bismarck Archipelago or the Solomon Islands. Minor exceptions include 1.4 % M7c3c in the Admiralty Islands (Kayser et al. 2008a), <0.2 % in New Britain (Friedlaender et al. 2007) and two closely related whole-mtDNA M7c3c sequences (~2 %) in the Solomon Islands (Duggan et al. 2014). M7c3c sequences, all within M7c3c2, are also seen in Ontong Java, a Polynesian outlier in the north Solomons. M7c3c and the other probable "out-of-Taiwan" clades have not been detected in Vanuatu, Fiji or Samoa, despite very extensive sampling.
Most of the present-day diversity in Near and Remote Oceania was established in New Guinea by ~10 ka (Soares et al. 2011), a fraction of which was carried by Austronesian speakers into the Remote Pacific. Powerful, long-established spheres of interaction may have facilitated the spread of the Austronesian languages in the south (Bulbeck 2008; Terrell and Welsch 1997; Torrence and Swadling 2008). They may thus have spread stepwise from the north and west via small-scale interactions and waves of acculturation. There appears to have been no "Austronesian farming-dispersal" in any meaningful sense across ISEA—early Austronesian speakers were more likely fisher–foragers—opening up the discussion to a range of innovative archaeological and linguistic models (Barker and Richards 2013; Donohue and Denham 2010, 2015). As both archaeologists and linguists have suggested, alluding to the spread of the early Metal Age in Europe, it may be that what began to spread across ISEA around 4000 years ago was primarily a new way of thinking—the adoption of a new ideology and perhaps even a new religion (Blench 2012; Spriggs 2011).
This work was supported by FCT, the Portuguese Foundation for Science and Technology, through the project PTDC/IVC-ANT/4917/2012. PS is supported by FCT, European Social Fund, Programa Operacional Potencial Humano and the FCT Investigator Programme and acknowledges FCT/MEC for support to CBMA through Portuguese funds (PIDDAC)—PEst-OE/BIA/UI4050/2014. PS was also supported through the course of this work by a Marie Curie Early Stage Training Grant and FCT grant SFRH/BPD/64233/2009. MBR thanks the British Academy for financial support through project LRG-42440 and PS and MBR also thank the de Laszlo Foundation and the British Academy (project BARDA-48208). IPATIMUP is an Associate Laboratory of the Portuguese Ministry of Science, Technology and Higher Education and is partially supported by FCT, the Portuguese Foundation for Science and Technology.
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All procedures performed in studies involving human participants were approved by the University of Leeds, Faculty of Biological Sciences Ethics Committee, and that of the University of Huddersfield, School of Applied Sciences.
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I want to make a complete but simple meal from my good friends and neighbors who are moving (just a street away), next week. My plan was to take them a complete meal with paper plates, napkins, main course, over to their new home on the day they move in so they don't have to worry about what to eat after all that moving work. There will be the couple, 2 children, and probably a set of grandparents at the house to help with the move. None of them are real foodies, and my friend is a self admitted terrible cook. So, nothing extravagant. I was thinking about a chicken piccata, a nice salad, bread, and maybe some type of pasta dish. I would like to bring everything over in containers that they can heat the food up in, and hopefully throw away.
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2013 - What do u class it as so far
Started by Tough titties , Jan 04 2013 12:17 PM
58 replies to this topic
Posted 04 January 2013 - 12:17 PM
So far I class 2013 as horrible. Last year was crap and so far 2013 has not had a good start.
* Found out that my mums cousin committed suicide
* Just went to my freezer and it seems someone left the freezer door ajar overnight (defrosted meat)
* DS just broke a plate (not really that big a deal but just thought I would add it.)
So has the new year been a good start or bad start for you?
Posted 04 January 2013 - 12:24 PM
Bad. Sitting on the couch on New Years eve reflecting on 2012, and that even though we'd had a rough time on the TTC front, the other areas of our life were great.
Then one of my pets ran away. We spent New Years eve searching instead of partying and I've spent every day since searching. This year has been sucky so far
Posted 04 January 2013 - 01:45 PM
Bad here also.
New years day, a good friends was killed in a car accident,
Washing machine died
Car battery died...stuck at home, have no money to get new one
2 out of 3 kids have gastro bug...now me too
Its only the 4th
I desire to have a Happy 2013. hugs to pp
Posted 04 January 2013 - 01:51 PM
No real dramas so far, but I am hoping with all fingers and toes crossed that 13 is lucky for us.
Posted 04 January 2013 - 01:54 PM
Sadly a friend's husband died on New Year's Day so that was pretty awful. Otherwise though it has been pretty laid back.
Posted 04 January 2013 - 02:00 PM
So far I'd class it as HOT.
Posted 04 January 2013 - 02:06 PM
It can only improve from here.
Posted 04 January 2013 - 02:06 PM
Not sure yet. However I did have a dream 2 nights ago (how very timely!) about moving to a brand-new house, that's supposed to be an omen for new beginnings in your life so let's hope it's true!
Posted 04 January 2013 - 02:11 PM
Crap. Too hot, my anxiety is hitting me full force and some expectations I had from a certain person show no sign of being fulfilled any time soon. Sick of going over and over the same conversation, getting the same promise at the end and no follow through.
Posted 04 January 2013 - 02:17 PM
I've spent the start of the year waking up slowly while someone else plays with the baby, changing into bathers and walking to the river for a swim, coming back for someone to pass me a G&T or two or three at the Scrabble board while taking in the view of the ocean between turns.
It's going to be hard to keep up with that level of luxury for the rest of the year but it's certainly a good start.
Posted 04 January 2013 - 02:19 PM
Edited by bye, 29 March 2013 - 02:57 PM.
Posted 04 January 2013 - 02:26 PM
Can I curl up in a corner somewhere and sleep out the rest of 2013??
In all seriousness... it can only get better, right?
Posted 04 January 2013 - 02:26 PM
I have pretty much cracked it, the kids are getting on my nerves in the first bit of 2013 and I find I am just losing it slowly !
maryanne - I am sorry your DH is being a bit of a douche lately, sometimes it is hard staying the course and saying no is seems that you're negative all the time, it sucks, I hope you can find some peace and soon. hugs.
Posted 04 January 2013 - 02:35 PM
Spent the first day of 2013 in A&E with a nasty rash which has since been diagnosed as shingles. Local weather forecast for today is 46.
Posted 04 January 2013 - 02:55 PM
Crap, been stuck at home with half my face swollen and in bad pain from a tooth. Finally got into the dentist today and got told I need root canal. I don't have the money Hoping the antibiotics today will clear up the infection and swelling. And it's the tooth next to my top front tooth so no way I am getting it pulled.
Posted 04 January 2013 - 03:04 PM
ive been on the couch un able to move with bad sciatica down my left leg..
so great that it is numb from my ham string down the bottom of foot....
i have a 9month old who is starting to crawl.. bugga i cant bend over to pick him up....
Posted 04 January 2013 - 03:09 PM
2012 was a very tough year for us, and ended with my best friend's husband having an accident causing brain trauma only a month after their wedding.
2013 has to be better.
Posted 04 January 2013 - 03:23 PM
Well considering I went to the hospital at 2am yesterday and got diagnosed with a kidney infection (again) I would say it is not off to the best start...
but trying to look at the positive in that at least I caught it early and it shouldn't go septic or anything like the last time and I was able to be discharged in the afternoon.
DD has a low grade fever too at the moment, but she is still bright and bubbly so hoping it gets no further.
This is meant to be my 'good' year...they used to be good years when odd, and bad years when even. I think they got mixed up though and last year was a good year
Posted 04 January 2013 - 03:26 PM
Mine has been fine. Pretty regular everyday life. Had a nice lead up although a quiet NYE as worked New Year's Day. Just waiting on settlement for our house in March so I need to start getting organized for the move. I'm very excited about moving, so I suspect 2013 to be a bit busy but a good year.
Posted 04 January 2013 - 03:26 PM
Sorry and hugs for everyone who's having a bad time. 2013 is good so far here - hot but manageable, DH and I are sharing the kids well, no real dramas. I do know that from February on our work is going to be very busy, so we need to keep smiling and living healthily through that.
Posted 04 January 2013 - 03:34 PM
Pretty effed up.
Posted 04 January 2013 - 03:55 PM
So far, better than 2012.
I'm determined to be stronger in 2013!
So far, so good.
The kids are with me. Ex and I are getting along. I'm on holidays for another 3 days.
And it's cooler after a week of 40+ days.
Posted 04 January 2013 - 04:10 PM
I have had an awful 18 months with lots of life changing drama happening in my life. 2013 has shown some side effects from that still, with rumors being spread like wild fire about me. It's hard, I have lost a lot over the last 18 months, my world has been turned upside down and most 'friends' have now turned their backs on me because of rumors from one vicious family whom are in leadership at my ex church, so of course everyone believes them. The reason they are doing this is because I reported to other leaders about what this family had done to us (bullying and threatening) and they are trying to discredit us. There is nothing I can do about it.
But I got through worse crap in the last 18 months I can get through this. Gotta keep looking upwards and forwards.
Posted 04 January 2013 - 04:12 PM
Really good 2012 was crap.
I'm happy, kids are happy and DH is happy.
I feel for those that have had a rocky start to 2013.
Posted 04 January 2013 - 04:28 PM
I feel for all those that have had a crap start. It is awesome here.
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Employee benefits
Both the number of employers providing benefits for employees who adopt (including leave when a child arrives in the home, reimbursement of adoption expenses, and assistance with adoption information and referral services) and the dollar amount of these benefits are growing. The average expense reimbursement now approaches $4,000, with some employers providing up to $10,000. For help asking your employer for this benefit, see the National Adoption Center's adoption and workplace initiative: www.adopt.org/workplace. Also read Elizabeth Mair's article "How to Lobby Your Employer for Adoption Benefits" online: www.adoptivefamilies.com/clip.php.
Adoption grants and loans
A number of adoption agencies and organizations offer adoption grants and/or low-cost loans. Some are limited to certain types of children or adoptions, while others are available to any prospective parents who meet the organization's criteria. Examples of adoption agencies with such grant or loan programs: Holt International, World Association for Children and Parents (WACAP), Dillon International (for contact information, see the adoption program listings in this Adoption Guide). Two organizations offering adoption grants are the Gift of Adoption Fund: www.giftofadoption.org, and A Child Waits, www.achildwaits.org (Eastern European adopters). For more granting organizations, search the Web for "adoption grants," but be wary of demands for application or other fees.
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Cabool High School senior Madison Carder, right, was crowned the 2016 Hoop Queen last Friday night by last year's queen, Heather Goforth, left. Carder was chosen by a vote of the CHS student body and crowned during ceremonies held between the junior varsity and varsity boys' basketball games with Houston at the CHS gym.
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District > District News >


text: On Wednesday, April 5th, the Shawnee Clinic, as well as the rest of the health system, will close at the noon hour for health's first of the month administrative duties. Shawnee Clinic will not re-open at 5:00 P.M. The Extended Hours service will be closed on this particular evening and will re-open the following business day. However, if you need to be seen for an acute illness or injury on the evening of the April 5th, you can be seen at the PlusCare Clinic, 15702 East State Highway 9, Norman, OK 73026, or call (405)447-0477. They will re-open at 5:00 P.M. and remain open until 9:00 P.M. with the last patient accepted at 8:30 P.M.


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Alright, my first Ouran High School Host Club fanfic! This story is going to be based somewhat around the concept of "A Midsummer Night's Dream." Also, since I haven't really read the manga, this fic is going to be based in the anime universe. However, I may borrow some things from the manga from time to time. I'm also using some Japanese words, so here they are with their translation:
Konnichi wa – Good afternoon
Ano – ummm
Neesan – big sister
Okaasan – mother
Otoosan – father
I will now turn it over to my muse who will give the disclamer.
Alfador: SquiggleSmurf does not own any part of Ouran High School Host Club. She is merely a fan of the anime and is writing this story for fun. She does however own the three original characters who appear in this fic: Naoki, Usagi, and Mizuki. Enjoy!
'Ah, I'm out. Finally,' Haruhi Fujioka quickly slipped out of the doors of Ouran Academy. Classes had ended about two hours ago, but Host Club activities kept her late. As much as she loved the Host Club, things could get pretty hectic, with Tamaki being his usual dramatic self, Hunny eating all those cakes, Hikaru and Kaoru acting mischievous, and all those screaming girls. But Friday had come at last, and now Haruhi could take a break from the activities for the weekend.
'I just hope they don't make a surprise visit,' Haruhi thought, making her way out of the gates of the school. 'I have to study for that English test on Monday.'
As she turned to walk down the sidewalk, she saw a limousine and a male student sitting on the sidewalk, working on a Sudoku puzzle book.
Haruhi looked in confusion. 'Why is that student sitting there instead of getting on his limo and going home? Isn't his limo driver getting annoyed?'
Looking closer, Haruhi recognized the student. He was Naoki Kozu, a somewhat short boy with short, dark brown hair styled neatly and hazel eyes. Though he was only fifteen years old, he was in the same class as Haruhi, as he had started school a year early. Curious as to why he was waiting outside by himself, Haruhi approached him. "Naoki-kun?"
The fifteen-year-old brunette looked up from his puzzle and turned towards Haruhi. Upon seeing his classmate, Naoki smiled and replied, "Konnichi wa, Haruhi-kun!" He pushed up his round glasses, put his puzzle book back in his school bag, and got up to face Haruhi.
Haruhi gave a small wave. "Konnichi wa. Ano, Naoki-kun, what are you doing here so late?"
"Oh, I'm waiting for my Neesan," he explained, a little annoyed. "You didn't happen to see Mizuki at the Host Club, did you?"
"Hmmm, Mizuki-sempai…" Haruhi tried to remember if she saw the black-haired second year. "No, I don't think I remember seeing her. She's one of Tamaki-sempai's clients, right?"
"Yeah, and she's also a fan of twinest," Naoki sighed. "Where could she be? She's never this late…"
Haruhi glanced at the limousine. "That's yours, right? Has your limo driver been waiting all this time?"
Naoki nodded. "Usagi-san is always scheduled to pick me and Neesan up after school."
"He must be really annoyed, having to wait all this time…"
"No, it's okay. He's paid to wait for us. That's his job! Right, Usagi-san?"
Usagi gave a nod from inside the limousine. Naoki grinned, and Haruhi gave a weak smile. 'Rich bastards.'
Looking back towards Naoki, Haruhi asked, a bit concerned, "Do you have any idea of why she might be late?"
"Hmmmm," Naoki thought deeply. "Well, we did have a little fight two days ago, but I don't think-"
Just then, a seventeen-year old girl with long black hair in a ponytail came running out of the gate. Frantically, she ran to Naoki (knocking down Haruhi in the process), grabbed him, shoved him into the limousine before jumping in herself, and screamed, "DRIVE USAGI-SAN! DRIVE NOW! DRIVE LIKE YOU NEVER DRIVEN BEFORE!"
With that, the limo took off, leaving Haruhi lying face down on the sidewalk. She got up, twitching slightly. 'Rich bastards…'
"Neesan! Why did you do that?" Naoki scolded his sister, looking out the back window of the limo. "I think you hurt Haruhi-kun!"
"Forget Haruhi-kun! I have something much more important than any cute boy!" Mizuki whipped out a small black glass bottle from her purse, eyeing it admirably with her dark brown eyes.
Naoki gulped. "Neesan, what exactly is that?" he asked carefully.
Mizuki smirked. "This, little brother, is the answer to certain doubts I've been having recently."
"Which ones?"
Mizuki glared annoyingly at the brunette. "Show some respect for your Neesan! This is partly your fault anyway!"
"Don't act like you've forgotten that horrible thing you said two days ago!"
"… you're still thinking about that?" Naoki sighed.
"Naoki, why can't you understand how deeply you hurt me with that language!?"
"I didn't say anything bad!" Naoki raised his voice, getting mad.
"What do you mean you didn't say anything bad!? You said things worse than any curse the Dark Magic Club could conjure up! You are obviously a heartless beast with no remorse!"
"I was just telling the truth!"
"The truth!? THE TRUTH!? Let me tell you something: Hikaru and Kaoru are so deeply in love! IT IS NOT AN ACT LIKE YOU ACCUSE IT OF BEING!"
Naoki groaned. "The Host Club only wants to bring joy to woman. They only act incestuous to-"
There was an uneasy silence in the limo for the next several minutes. Mizuki, her knees to her chest, stroked the bottle while Naoki stared out the window, dumbfounded.
'Why does my sister have to be so crazy?' he thought hopelessly. 'How is twincest so hot anyway?'
Naoki decided to break the silence. "Neesan, how exactly is that bottle supposed to help you?"
Mizuki smirked, straightened herself out, and looked at her brother. "Although I refuse to believe any word you say, creature, you made me doubt the Hitachiin twins' relationship. But this little bottle will straightened everything out."
Naoki prepared himself for the worst. "So what is it?"
Mizuki took a deep breath and held up the bottle. "This, is a love potion."
Naoki stared at his sister. And stared. And gaped. But mostly stared.
"Yes, yes, I know what you're thinking," Mizuki said, rolling her eyes. "'Love potions don't work.' But I know this one is real!"
"And how do you know that?" Naoki asked, afraid of the answer.
Mizuki beamed. "I got it from Nekozawa-san!"
The younger teen narrowed his hazel eyes. "Nekozawa-san gave you a love potion."
"Well, not exactly," Mizuki admitted. "I had to sneak into his dark magic room."
"You WHAT!?"
"Well, what did you expect me to do!?" Mizuki huffed. "Ask Nekozawa himself and risk getting cursed or just forget about the whole thing?"
"Actually, the latter doesn't sound like a bad-"
"Shut up Naoki!" Mizuki shouted. She quickly lowered her voice and went back to her explanation. "Now, like I was saying, I had to sneak into his room. And I'll tell you: it wasn't easy. Do you know how hard it is to wait for a door that may or may not appear? And then I had to look through that room. Now that was creepy. There were all those candles and magic circles. Seriously, one wrong turn and I could have been turned into a toad or something! But I finally found this little bottle: Love Potion #69."
"Love Potion #69?" Naoki sweat dropped.
His older sister shrugged. "That's what the bottle said."
"Well, I suppose no harm's been done as long as you didn't get caught."
Mizuki glanced around nervously.
"Neesan?" Naoki started to get very serious. "No one saw you, right?"
"Ummm…. not exactly," Mizuki admitted, sheepishly. "I mean, I think he may have seen me, but it's taken care of."
Naoki moaned. "Neesan, I don't want our family name to be ruined because you had to steal for a stupid reason."
Mizuki twitched at the word 'stupid' but attempted to comfort her brother. "No Naoki, listen to me. You see, I was almost to the door when Nekzsawa-san came in."
The boy moaned louder.
"No, Naoki! It's okay! Listen! I kind of panicked when I saw him. So, I saw a container on a nearby shelf. Since I didn't know what else to do, I took it and opened it. It contained this dust-like substance. With no other better plan, I took some of the dust and blew it at him."
Naoki moaned something about assault charges.
"No, I didn't hurt him! It just put him to sleep. See? It's sleep dust," Mizuki pulled the container out of her bag.
Her younger brother looked up at her and moaned again. "You stole that too?"
"Hey, it might be useful," Mizuki shrugged.
The brunette hung his head and groaned, "Just tell me you didn't take anything else."
"Well, there was one other thing…"
Naoki shot up. "Neesan! What else!?"
The black-haired teen bit her lip and pulled out…
"Beelzenef!? You stole Nekozawa's Beelzenef!?"
"Look, just in case Nekozawa-san saw me, I need some leverage against him. Plus, he can't curse me without his little puppet here."
Naoki covered his face with his hands. "Okaasan and Otoosan are going to kill us!"
Mizuki rolled her eyes. "It really isn't going to matter. After he fell asleep, I put some of the love potion on Nekozawa-san. When he wakes up, he will probably be too busy being in love to even think about his lost puppet."
"Neesan, why don't you understand? That love potion isn't going to work!"
"Silence!" Mizuki commanded. "I've had enough of you and your idiotic logic! This love potion is real and it is going to work!"
"Has anyone told you that you're insane?"
"Oh sure, make fun of me now but you'll see," Mizuki once again eyed and stroked the bottle in awe. "Come Monday afternoon, Hikaru and Kaoru will be bound forever by an irresistible love!"
Naoki's right eyebrow began to itch.
And so it begins. Stay tuned for Chapter 2 and drop a review if you have the time!


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Becker cuddles up to partner watching tennisPosted on: April 1, 2010 20:08:43
Three-time Wimbledon champion Boris Becker decided to take his partner and son Elias to watch a spot of tennis in Florida.
Becker, who became the youngest winner of Wimbledon as a 17-year-old in 1985, was joined by his wife Sharley Kerssenberg and ten-year-old son Elias to watch Roger Federer in action at the Sony Ericsson Open.
The 42-year-old married Dutch model Sharley, also known as Lilly, in a ceremony in Switzerland last year.
However, it hasn't always been plain sailing for the tennis ace.
They originally split up in 2008 and Boris turned his attentions to Sandy Meyer-Wolden, the daughter of his late manger Axel, the Daily Mail reports.
After a change of heart, he announced his relationship with Lilly was back on and earlier this year his 33-year-old partner gave birth to his son Amadeus.
Elias and his 16-year-old brother Noah Gabriel are the product of Becker's marriage to Barbara Feltus. He also has a nine-year-old daughter from an infamous dalliance at a London restaurant with a Russian model.
Looking for love again like Boris? Maybe you could consider European dating?
Related European Dating News
Jenny Frost and Vicente Juan Spiteri wedPosted on: August 16, 2011 12:23:47
Lonely hearts looking for love using online dating websites could be pleased to hear that Jenny Frost has married boyfriend Vicente Juan Spiteri Photographs from the big day are set...
(read more)
Selma Blair spotted with new babyPosted on: August 11, 2011 16:57:30
UK singles looking for a new partner to start a perfect family with could receive encouragement from recent pictures of Selma Blair Published in the Daily Mail the photographs show...
(read more)
Snooki single again?Posted on: June 8, 2011 16:51:07
Lonely hearts looking for a new partner using online dating websites could be sad to hear rumours that Snooki has split up with her boyfriend The Jersey Shore star who...
(read more)
Cristiano Ronaldo and Irina Shayk spotted in PortugalPosted on: May 26, 2011 17:08:15
Lonely hearts looking for a new partner using online dating sites could be happy to hear that things appear to be going well for Cristiano Ronaldo and Irina Shayk The...
(read more)
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|Day 1
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|Day 2
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Philipp Geist | Videogeist
Philipp Geist 2005–2011 Light Installations/ Video Mapping
HQ (1080p) youtu.be/Xdo9qMGxlLs
videogeist.com / philipp-geist.de
The Berlin artist Philipp Geist (1976) works internationally as a mulitmedia artist in the mediums of video- / light installation, photography and painting. In 2012 he showed his installation "time drifts - words of berlin" during the Festival of Lights at the Potsdamer Platz in Berlin, the installation "time drifts" was also shown in Frankfurt at the Luminale 2012 and earlier in 2011 during the 125th anniversary of the city of Vancouver, and in 2010, 'Time Drifts' could be experienced by the visitors of the Montreal Mutek festival for new media. In 2011, he has shown abstract photo works in a solo exhibition in the "Museum gegenstandsfreier Kunst Otterndorf, including the video mapping show 'Construction|Deconstruction' on the museum facade and the installation "Liquid Time" on the historical Water Tower in Cuxhaven. In December 2009, Geist showed a 4D mapping installation on the facade of the royal throne in Bangkok on the occasion of king Bhumibol's 82nd birthday. The one-hour-show was seen by 2,5 million of visitors. At the Glow Festival 2009 in Eindhoven he showed the light installation 'Timing'. In 2008, during the 'Long Night of the Museums', he exhibited his video installation 'Time Fades' at the Kulturforum. In 2007, he realized the video installation 'Time Lines' on the entire front of the Palazzo delle Espozioni in Rome. In 2006, he opened the Salon Noir within the context of the exhibition 'Melancholie, Genie und Wahnsinn' ('Melancholy, Genius and Insanity') in the Berlin Neue National Galerie. Together with the symphony orchestra OBC Barcelona and the Finnish sound ensemble Pan Sonic, Geist opened the Sonar Festival in Barcelona in 2004. He has exhibited his art work and audio-visual performances internationally at various renowned festivals and art spaces. Geist´s projects are characterized by their complexity and the integration of the location, the sound and moving images. In his video mapping installations, he avoids using canvasses and turns diverse architectures in moving, painterly light sculptures, which challenge the onlookers' perception of two- and threedimensionality.