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OPEN Enclaves of genetic diversity

resisted Inca impacts on population
history
Received: 5 October 2017 Chiara Barbieri 1, Jos R. Sandoval 2, Jairo Valqui 3, Aviva Shimelman1, Stefan
Accepted: 29 November 2017 Ziemendorff4, Roland Schrder5, Maria Geppert6, Lutz Roewer6, Russell Gray1, Mark
Published: xx xx xxxx Stoneking5, Ricardo Fujita2 & Paul Heggarty1
The Inca Empire is claimed to have driven massive population movements in western South America,
and to have spread Quechua, the most widely-spoken language family of the indigenous Americas.
A test-case is the Chachapoyas region of northern Peru, reported as a focal point of Inca population
displacements. Chachapoyas also spans the environmental, cultural and demographic divides between
Amazonia and the Andes, and stands along the lowest-altitude corridor from the rainforest to the
Pacific coast. Following a sampling strategy informed by linguistic data, we collected 119 samples,
analysed for full mtDNA genomes and Y-chromosome STRs. We report a high indigenous component,
which stands apart from the network of intense genetic exchange in the core central zone of Andean
civilization, and is also distinct from neighbouring populations. This unique genetic profile challenges
the routine assumption of large-scale population relocations by the Incas. Furthermore, speakers of
Chachapoyas Quechua are found to share no particular genetic similarity or gene-flow with Quechua
speakers elsewhere, suggesting that here the language spread primarily by cultural diffusion, not
migration. Our results demonstrate how population genetics, when fully guided by the archaeological,
historical and linguistic records, can inform multiple disciplines within anthropology.

Genetic studies have begun to contribute significantly to our understanding of the pre-colonial history of the
Americas, and are able to fill in some of the gaps in the archaeological and historical records. Archaeology faces
preservation biases between the diverse environments of the desert along the Pacific coast, the Andean highlands
and the Amazonian rainforest. Written history in the Andes begins only with the Spanish conquest in the1530s,
and these first chronicles are fragmentary and contradictory.
Genetic results have returned important insights particularly on the macro-scale: the initial colonization
events13 and the broad patterns of diversity, such as the genetic contrast between the ecological domains of the
Andes and Amazonia4,5. Studies focused on sub-regions, however, face significant limitations when attempting
to evaluate past population movements and contacts on a much finer scale, often due to the low resolution of
the genetic markers selected, or to poor sample coverage. This study therefore explicitly selects high-resolution
markers, and to improve sampling follows a strategy fully informed by historical, archaeological and linguistic
contexts.
Our focus is on northern Peru, selected as a case-study for its significance in both environmental and his-
torical terms. The Andes here are at their lowest elevation, and thus serve as a preferential corridor between
Amazonia and the Pacific6. Towards the eastern slopes, where the environmental transition to Amazonia begins,
is the cloud forest region of Chachapoyas7. The archaeological record here attests to a rich diversity of regional
cultures over time, up until Inca conquest in the 1470s. Chachapoya culture serves as a collective term for a series
of political entities, independent but sharing common architecture, art (ceramic style) and iconography8,9. In the

1
Department of Linguistic and Cultural Evolution, Max Planck Institute for the Science of Human History, D-
07745, Jena, Germany. 2Centro de Investigacin de Gentica y Biologa Molecular (CIGBM) Universidad de San
Martn de Porres, Lima, Peru. 3Departamento de Lingstica, Universidad Nacional Mayor de San Marcos, Lima,
Peru. 4Independent researcher, Chachapoyas (city), Chachapoyas, Peru. 5Department of Evolutionary Genetics,
Max Planck Institute for Evolutionary Anthropology, Leipzig, Germany. 6Institute of Legal Medicine and Forensic
Sciences, Department of Forensic Genetics, CharitUniversittsmedizin Berlin, Berlin, Germany. Correspondence
and requests for materials should be addressed to C.B. (email: [email protected]) or P.H. (email: paul.
[email protected])

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Figure 1. Map of sampling locations and approximate distribution of sub-branches of the Quechua language
family, as traditionally classified (Adapted from13). Red dot 1 marks the sampling locations in the Amazonas
region (Chachapoyas City, Luya, Huancas, Utcubamba South, La Jalca); red dot 2 marks that in the San Martn
region (Lamas, Wayku neighbourhood). The inset zooms in on the sampling locations in Amazonas. Map
generated in R - version 3.3.0 www.R-project.org/62. Raster physical map adapted from www.naturalearthdata.
com/ (public domain).

earliest historical accounts, Spanish chronicles make extensive mention of Chachapoyas for its long resistance to
Inca conquest and then as a clear example of the Inca state policy of forced resettlements. Chachapoyas is taken
as one of few regions where the recalcitrant local population was essentially completely removed and replaced10
making this a test-case for the ability of population genetics to challenge or confirm the (proto-)historical record,
of questionable veracity here10 (91118).
The presumed native language of Chachapoya culture (referred to as Chacha) is extinct, although a few of its
characteristics can be inferred from surviving placenames and surnames11,12. Quechua, meanwhile, never seems
to have been dominantly established across northern Peru, and is spoken, in diverse local forms, only in sporadic
pockets scattered across the region (Fig.1). One of these pockets is Chachapoyas, where a small proportion of
the present-day population, in a handful of small communities, still speaks a now moribund variety of Quechua
that is difficult to classify within the familys phylogeny. Another pocket of Quechua, also covered in this study, is
the town of Lamas in San Martn province, in the Andean foothills at the edge of Amazonia. Both these forms of
Quechua have usually been assigned to a putative Quechua IIb branch, together with others spoken elsewhere
in the Amazonian lowlands of northern Peru (in Loreto province), the Inga spoken in southern Colombia, and
the widely spoken Ecuadoran Kichwa13. Quechua did once serve as lingua franca for the Inca Empire, but that
can explain only some of its diversity and distribution through the Andes. Other potential drivers include the
forced population movements under the Incas, and the cultural and/or demographic impacts of earlier complex
societies in the Andes, and of the Spanish colonial regime14,15. For further archaeological, historical and linguistic
contextualization, see the Supplementary Text.
To introduce a genetic perspective on these scenarios from archaeology, history and linguistics, this study
focuses on uniparental markers: i.e. mtDNA and the non-recombinant portion of the Y chromosome.
For mitochondrial DNA, in the Americas data are relatively abundant for the hypervariable region only. At a
broad level, it is possible to identify variation in the frequencies of the four haplogroups most heavily represented
in Native Americans (A2, B2, C1 and D1: for an overview see16). Beyond that, however, sequencing the hyper-
variable region alone is often insufficient to disentangle population relationships. This is a result of the Americas
being settled only relatively recently, and through a bottleneck in Beringia1,2. Sequencing full mtDNA genomes
gives an important gain in resolution, as demonstrated by recent studies thatgenerate native mitogenomes for
some parts of the Americas1721. So by sequencing new sets of full mtDNA genomes, from regions of the continent
selected for their distinctive local population prehistories, this study adds to our database for the Americas, and
uncovers new patterns of genetic variation that bear directly on population prehistory here.
For the Y chromosome, standardized Short Tandem Repeat (STR) sets have been successfully applied to
explore genetic diversity in the paternal line in South America2225. The most widely used STR sets cover 12 or
17 markers, but newly available sets for 23 markers give more fine-grained results. Data from the Andean region
reveal a nucleus of homogeneity across the central highlands, from central Peru to northern Bolivia, in popu-
lations speaking languages of the Quechua or Aymara families, which both likely originated within this broad

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Figure 2. CA plot of mtDNA haplogroup frequencies.

region13. This pattern is confirmed in the mtDNA data23,26, and has typically been associated with the demo-
graphic impacts of the expansion and resettlement policies of the Inca Empire, and perhaps also of their most
significant predecessor polities, Wari and Tiwanaku in the Middle Horizon period (c. 5001050 AD)24.
Beyond this nucleus of homogeneity in the Andes, occasional populations in the Amazonian lowlands also
speak the highland language Quechua. Genetically, however, these populations do not show any particular rela-
tionship to the central highlands that would parallel this linguistic link. Quechua speakers from Lamas likely
acquired the language by processes of cultural diffusion, not by significant migration from the highlands25. Finally,
a recent study of 23 Y-chromosome STRs and the mtDNA HVSI has reported high genetic diversity in popula-
tions from Chachapoyas and surrounding areas27. It remains to be clarified, however, what the relationships are
between Chachapoyas and other parts of the Andes, which we test here using more and higher-resolution genetic
data from Chachapoyas.
For this study, we generated 23-STR profiles and full mtDNA genomes from Quechua-speaking populations
of northern Peru, with the aim of exploring the impacts of the Inca period, the possible genetic inheritance from
pre-Inca periods, and the dynamics behind the diffusion and differentiation of the multiple Quechua varieties
scattered across the region. We sampled 119 individuals, with an explicit strategy targeting firstly the surviving
Quechua-speaking pockets in Chachapoyas and Lamas. Secondly, we focused on surnames characteristic of puta-
tive Chacha linguistic origin (i.e. neither Spanish nor Quechua), to try to trace back to a possible genetic legacy
of the Chachapoya population from before the Inca conquest (Supplementary Text and Supplementary TableS1).
We uncover an enclave of diversity on a micro-geographical scale in both Y-chromosome and mtDNA, inde-
pendent of the main network of intense genetic (and linguistic) interaction in the south-central Andes. Our
findings have multiple implications for Andean (pre)history. Firstly, we find evidence to challenge the presump-
tion of major Inca impacts and resettlements affecting the Chachapoyas population. Secondly, we find no direct
demographic connections between populations that speak varieties of the disputed QIIb clade of Quechua, and
support a cultural rather than demographic model for its spread in northern Peru.

Results
mtDNA. Full mitochondrial genomes were successfully sequenced for 116 individuals (data deposited in
GenBank under accession numbers MG571104 - MG571221). Of these, 113 could be assigned (given the local ori-
gins of their parents and grandparents) to one of six broadly-defined geographical groups (Fig.1, Supplementary
TableS1). Sample sizes range from eight individuals in the Chachapoyas city group to 36 in the Luya group. All
except two individuals have Native American haplogroups: most frequent in our sample is B2 (35%), followed by
A2 (26%), D (D1+D4h3 23%) and C1 (16%). Differences in haplogroup compositions are summarized in a CA
plot (Fig.2). Haplogroup frequencies were also compared to those of populations from across South America in a
broader CA plot (Supplementary Fig.S1). Each group is more similar in haplogroup composition to other groups
outside the region than to any other group within Chachapoyas itself.
Full mtDNA genomes were first screened for diversity within each group, comparing nucleotide diversity
and haplotype diversity (Supplementary TableS2). For both values of diversity, the same trend emerges: Luya,
Chachapoyas and Utcubamba South are the most diverse, Huancas the least diverse. Between populations, ST
genetic distances (Supplementary TableS2) indicate that La Jalca has a highly distinctive genetic make-up, show-
ing the highest distance to other groups, in particular to Huancas, Wayku and Utcubamba South.
We constructed Networks (Supplementary Fig.S2, Supplementary Text) to compare the diversity of our sam-
ple with other full mtDNA sequences available from the literature. Our samples either stand alone, or group in
branches mostly with each other. Only rarely do they fall together with samples drawn from the literature, in a
shared branch. This effect can be in principle explained by our target populations having remained relatively iso-
lated from other sampling localities in our database, although it may also reflect lack of adequate sampling in the
literature, and/or extinction of more closelyrelated populations in the post-colonial era20.

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Figure 3.(a) BSP for mtDNA sequences for the whole sample. (b) BSP for mtDNA sequences for populations
with sample size > 14.

BEAST was used to construct a tree genealogy and Bayesian Skyline Plots (BSPs). The BSP for all the sequences
generated in this study displays a steep curve indicative of an expansion starting ~17,000 years ago (kya): the High
Probability Density (HPD) ranges from 15 to 22kya (Fig.3a), in line with mainstream views on the timing of the
first settlement of the Americas1,20. The increase in population size across our whole sample is more than 33-fold:
for a generation time of 28 years28, this corresponds to an increase in effective population size (Ne) from 1000 to
35,000 individuals. BSPs for the individual population groups (Fig.3b) do reveal some differences: La Jalca and
Wayku show a fairly stable population size, with but a faint indication of increase through time. Luya instead
clearly reproduces the increase in population size observed for the whole dataset.
The annotated tree from the BEAST runs (Fig.4) gives age estimates for the sequence of major branching
events, and mirrors the signal of expansion in the BSP, and its time-depth, with a strong effect of branch diver-
gence between 15 and 20kya. Some cases of more recent divergence are found at the tips of the tree, in closely
related sequences, clustered together in a triangle span for illustrative purposes. The divergence time for these
sequences is estimated at roughly within the last 1000 years. One notable aspect of the results, even if not unex-
pected when sequencing mitogenomes from an understudied region of the Americas, is that a large proportion
of the branches we report here represent new sub-haplogroups not yet identified in previous research (verified
against Phylotree build 1729 and a recent review19). Within A2, B2 and D1 we find 27 previously unreported
branches with divergence times 15,000 years ago, i.e. possibly shortly after first human entry to the Americas.
Only within C1 could all the lineages be assigned to already known sub-haplogroups: C1b, C1c and C1d.

Y chromosome. Male individuals were first genotyped for haplogroup assignment; only those belonging
to haplogroup Q, the characteristic Native American marker of interest for our study, were then typed for STR
haplotypes and included in the intra- and inter-population analyses. The other Native American haplogroup,
C2-M217, was not found in our sample. The remaining samples belong to broad macro-haplogroups other than
Q, as detailed in the Supplementary Text. Haplogroup frequencies for each population are included in TableS2.
The highest frequency of non-Native haplogroups is in La Jalca (40%), the lowest in Luya (20%).
Between population comparisons at a regional scale are based on the 23-loci dataset, which includes the sam-
ple from Guevara et al.27. The haplotype sharing plot (Supplementary Fig.S3) and the Median Joining network
(Supplementary Fig.S4) show that sharing is limited (139 unique haplotypes over 175 individuals, of which only
13 are shared between two or more populations) and that several branches are highly localized (Supplementary
Text). These local branches correspond in some cases to samples marked for non-Spanish surnames (of Chacha
or Quechua origin Supplementary Fig.S5).
After controlling for non-significant RST values, amount of haplotype sharing and compatible diversity values,
we merged some of our smallest samples with other related ones, to allow for continent-wide comparisons. The
Chachapoya sample from Guevara et al. was merged with our small Chachapoyas citygroup, the Huancas sample
from Guevara et al. with our Huancas, and finally the Quechua Lamas sample from Sandoval et al.25 (only 17 loci)
with our Wayku.
Between population comparisons at a continental scale are based on 17 or 15 loci. Supplementary TableS3
lists the 90 populations included and their diversity values. The least diverse of our samples (partially merged
with Guevara et al. and Sandoval et al.) are those from Huancas, La Jalca and Wayku: their low variance is similar
to that found in populations predominantly from Amazonia. Higher values for within population variance (but
close to the average for the continent) are found for the Chachapoyas city, Luya and Utcubamba South groups.
Haplotype diversity values follow the same trend.
To identify close connections between populations, identical haplotypes were annotated with two meth-
ods: first by a heatplot of sharing between relevant populations from Ecuador and Peru, based on 17 loci
(Supplementary Fig.S6), then in a map to display the full amount of sharing throughout the continent, based on
15 stable loci, weighted for their mutation rates (Fig.5). The patterns of sharing do not support any recent connec-
tion to speakers of other Quechua IIb varieties. The Wayku do show affinities to some other Quechua speakers:
those in north-east Peruvian Amazonia (see Supplementary Text for details).

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Figure 4. Annotated tree of mtDNA sequences generated with BEAST. Branches are named after Phylotree
v.1729; branches in italics correspond to the nomenclature recently proposed by19. Individual samples are colour-
coded according to the sub-population affiliation.

The continental sharing map helps visualize connections between the Chachapoyas region and its close and
distant neighbours. The two maps in Fig.5 show shared identical haplotypes (Fig.5a), and shared similar haplo-
types, i.e. allowing for one stepwise mutation in the more rapidly mutating loci (Fig.5b). Supplementary Fig.S7
zooms in on the region of interest, and shows a limited amount of sharing involving the target populations near
Chachapoyas (in blue, four cases of direct sharing which involve Utcubamba South). This stands in stark contrast
to the nucleus of homogeneity in the highlands, across the southern half of Peru and Bolivia, where rates of iden-
tical and similar haplotypes are remarkably high, creating a dense network of exchange (see Supplementary Text
for details on haplotype sharing).
Supplementary Fig.S8 visualizes the correlation between the frequency of shared haplotypes and the sample
size of each population. The sample from Chachapoyas City shares relatively few haplotypes for its relatively large
sample size, and is more isolated than average Amazonian populations when sharing between close neighbours
is ruled out (Supplementary Fig.S8b). Sharing inChachapoyas is lower than in the Andes but higher than in
Amazonia when sharing with close neighbours is not excluded (Supplementary Fig.S8a). The sharing pattern is
thendecomposed to theregional level, bydividing the South American samples into10 groups (Supplementary

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Figure 5. Map depicting patterns of Y chromosome haplotype sharing at a continental scale. Thin yellow
lines indicate the lowest levels of exchange (from just a single pair of individuals sharing an identical or similar
haplotype); thick red lines the highest (up to a maximum of 154 identical and 310 similar haplotypes shared
between the Toba and Pilaga samples). (a) Sharing of identical haplotypes. (b) Sharing of similar haplotypes
(allowing 1 step mutation in loci with a high mutation rate see Methods). Map generated in R - version 3.3.0
(www.R-project.org/)62.

Fig.S9a) and focusing on five linguistic groups of interest for the Andean region (Supplementary Fig.S9b). The
highest level of sharing involves the Central South Andes, and the speakers of Quechua IIc: the two groups include
almost the same samples (Supplementary Fig.S9c,d). Finally, coalescent simulations were applied to estimate the
incidence of haplotype sharing between two populations with varying Ne, time of divergence, and symmetric
migration rate. The results reveal that the target case of no haplotype sharing, as observed between Chachapoyas
city and the other published samples, is detectable (>5% of cases) until 10 generations split time and Ne>1000.
For more recent split times and Ne1000, instead, haplotype sharing would be predominant (>95% of cases; Fig.
S10). When migration between the two populations is considered, no haplotype sharing could still be detected
with appreciable frequency only for large Ne (3000)(Fig.S10d), while for smaller Ne no haplotype sharing is
detectable for a maximum migration rate of 0.015 (Ne=1000, Fig.S10c) or 0.005 (Ne=500, Fig.S10b).

Discussion
First we look at a continental scale, and the position of the population of Chachapoyas and Lamas (Wayku)
within the Americas. On this broadest level, our high-resolution analysis of uniparental markers reveals a pro-
nounced (high frequency of Native American haplogroups) and distinctive (unique to the region, in particular
in Chachapoyas) native component. Our dataset of 114 Native American mtDNA genomes corresponds to a new
hotspot of diversity, and suggests that other regions of South America may likewise harbour pockets of diversity
that have so far remained undetected.
Recent publications of mtDNA genomes have mostly focused on specific lineages of interest, misrepresenting
the diversity at the population level at the fullest resolution for this (maternally inherited) marker. Our new
dataset has not only been able to distinguish characteristic lineages for the population groups involved, but has
also uncovered notable diversity within the four major Native American mtDNA haplogroups. In the BEAST tree
reconstruction (Fig.4), 65 of these new sequences within A2, B2 and D are sub-haplogroups newly identified
here, i.e. they cannot be assigned to any of those already defined in previous studies (Phylotree v. 1729 and the
latest publication by Brandini et al.19).
This result entails two significant findings: a) Previous studies have underestimated the extent of diversity and
geographical differentiation within Native American mtDNA lineages; and b) Despite the generally low diver-
sity caused by the strong founding bottleneck in Beringia, the significantly higher resolution available from full
mitogenome analysis over the HVSI does make it possible to reveal demographic dynamics at a regional scale.
As illustrated in Fig.4, each of the six groups contains exclusive sub-branches in the network, a result compatible
with residence patterns localised here over time.

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Secondly, we compared our results against competing hypotheses on the origins of the Chachapoyas popu-
lation: an allochthonous origin (either from the lowlands, the highlands or from several localities, as a result of
the Inca resettlement policies) or an autochtonous one (see Supplementary Text). To explore these hypotheses,
the present-day genetic data can be interrogated for any signals to support connections to other regions, and to
assess whether the genetic make-up of Chachapoyas has or has not been relatively stable through time. Since
mitogenome data from the Andes are still too scarce, we considered the Y-chromosome STR dataset (of 90 pop-
ulations), with the caveat that it reflects only paternal population history. In principle, the high internal diversity
of our sample could be compatible with populations relocated here from multiple, distinct and far-flung regions
of the Inca empire or indeed from earlier highland polities. Our sample from the Chachapoyas region turns out
not to be genetically close, however, to the core Andean exchange network in the highlands of southern Peru
and Bolivia, sharing only four haplotypes with Utcubamba South (Fig.5, Supplementary FigsS6,S7,S8,S9c). This
result is unexpected for what is considered a strategic crossroads region, a corridor from Amazonia to the Andes6,
and particularly in comparison to the conspicuous amount of sharing found in other populations, for example
the Yanesha of central Peru, supposedly more isolated both geographically and culturally24. The high-altitude
Yanesha, with a sample size of 55 individuals, share haplotypes with 20 other populations (the highest number in
our continental dataset). While occasional other populations in the database display a unique genetic profile with
no haplotype sharing (Fig.S8), the general sharing trend is higher in all neighbouring regions (Fig.S9).
A recent allochthonous origin would entail some level of haplotype exchange, and our sample size is large
enough that any such exchange should be detected but we do not find this (Supplementary Fig.S8). It is highly
improbable that noshared haplotypes would be reportedfor a population split within the past 20 generations
(~600 years), as oursimulations confirm for a compatibly small Ne of 500 or 1000 (Fig.S10). This time constraint
arguesagainst the scenario of a complete relocation forced by the Incas or by subsequent Spanishcolonial poli-
cies. The hypothesis most directly and parsimoniously compatible with the distinctiveness of our Chachapoyas
sample seems to be that of autochthonous development and continuity over thegenerations (see Supplementary
Text for further discussion). Nevertheless, more ancient connections to the lowlands and/or the highlands can-
not be ruled out, given the fast mutation rate of the Y chromosome and the overall low diversity of the Native
American genetic component. The ultimate tool to test and refine these interpretations would be the analysis of
ancient DNA from both before and after Inca conquest.
The support for local continuity over at least the last 20 generations (Fig.S10) allows us to link the current
genetic makeup of the region back to the immediately pre-Inca period, and explore microgeographical diversity
patterns. Our six sub-regions display a considerable degree of population structure for such a small sample area:
this is reflected by the presence of characteristic lineages for both Y chromosome and mtDNA (see networks in
FigsS2 and S4). In mtDNA the samples from Luya and La Jalca (which have the largest population sizes) are dis-
tinct in haplogroup frequencies (Fig.2, Supplementary Fig.S1), in internal diversity (Supplementary TableS2),
in projected population sizes (Fig.3b) and in admixture histories (see Supplementary text). This fine population
structure within the provinces of Chachapoyas and Luya appears consistent with the archaeological record, which
points to a collection of societies forming a shared Chachapoyas culture, but without a single over-arching polit-
ical unit8. Obviously, drift has exacerbated differences in haplogroup frequencies, especially through the bottle-
neck after European contact, when widespread epidemics led to a dramatic loss of native lineages throughout the
Americas20.
Thirdly, our data can also test between hypotheses on the dispersal and phylogeny of Quechua. In sampling,
we targeted localities in Chachapoyas identified as recently or still currently Quechua-speaking (Supplementary
Table1, Supplementary Text). Our results can thus provide a new perspective on which demographic processes
may connect the various Quechua-speaking populations across northern Peru and beyond, particularly those
traditionally classed within Quechua IIb. We focused on haplotype-sharing patterns, to restrict the time-depth of
our analysis to population connections within historical times here (see above), adjusting for the mutation rates
of the markers used (see Methods).
A primary finding from our linguistically-targeted sampling is that the samples from Chachapoyas and Lamas
are not genetically connected, and that no major connections appear between speakers of the different varieties
of the putative Quechua IIb branch overall (Supplementary Figs6 and 7). Despite our large sample size from
Chachapoyas and adjacent provinces, the level of haplotypesharing with other populations is nil, even with their
supposedly close linguistic relatives the only exception is Utcubamba South, which does share haplotypes with
three populations outside the province. Rather, the speakers from Wayku share a network of connections with
other lowland populations (Supplementary FigsS7,S9c). Nor does our Chachapoyas sample share haplotypes
with Quechua-speakers in the Ecuadorian lowlands (QIIb) who instead share haplotypes with other lowland
populations, and have a typically Amazonian genetic profile24,25 (Fig.S9c,d).
This rules out any strong demographic connection between speakers of the disparate varieties of Quechua
all traditionally classified into the IIb clade, and bears on the open question of when and how Quechua came to
be spoken in northern Peru, Ecuador and southern Colombia. In the case of Wayku, historical sources report
how Jesuit and Franciscan missionaries from Quito purposely introduced Quechua as a lingua franca in the
Reducciones (mission stations) that they established among various tribes of the Maran, Napo and Amazon
rivers30,31. This finding is compatible with recent genetic analyses of the Wayku population as predominantly local
Amazonian25. For Chachapoyas, however, there is little parallel documentary evidence for a missionary diffusion
model, and the alternative explanation remains that Quechua arrived here with at least some population reset-
tled here by the Incas, from some other part(s) of their Empire. But in either scenario, the genetic data offer no
support for the existence of any putative single Proto-QIIb language, whose speakers could have been a common
migration source for the populations that today speak the Quechua varieties of Ecuador, Colombia, Chachapoyas
and Wayku.

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Zooming out to the rest of northern Peru, there are only two other Quechua-speaking groups in the region,
both traditionally classified within a hotly disputed QIIa clade. Only for one of these Cajamarca does the
genetic literature include samples that may represent speakers of the local Quechua (or their descendants). Again,
however, our Chachapoyas sample shows no connections to the Cajamarca sample, which itself shows no con-
nections to other Quechua-speakers in northern Peru (Fig.S9d), but only to the (linguistically unrelated) Jvaro
from Amazonia.
Finally, zooming out further still, the origin of the Quechua language lineage as a whole is generally set in the
south-central highlands of Peru13. That linguistic homeland falls within the network of very high homogeneity in
the gene pool and high levels of haplotype sharing across the highlands of southern Peru and Bolivia, as already
reported in the literature23,24,26,32 (Fig.S9d). This is the core territory of the best known form of Quechua, with by
far the largest number of speakers: Southern Quechua, traditionally classified as a QIIc clade. The genetic net-
work also includes all speakers of Aymara in this region, and is consistent, furthermore, with the intense contact,
shift and convergence between the distinct Quechua and Aymara language lineages33.
Although the uniparental markers are analysed here to high resolution, even so they still constitute only a lim-
ited perspective on population history, which thus may underrate the amount of contact in the region of interest.
It is conceivable that high-throughput genomic data may reveal more connections that bear on both the origin of
Chachapoyas populations and any relationships among Quechua-speaking groups across northern Peru. This would
entail extensive and targeted sampling coverage, however, from both these regions and the broader Andean context.
In conclusion, with a targeted sampling strategy focused on tracing autochthonous surnames and local
Quechua-speaking survivals, we have been able to uncover new genetic variation, and a potential signature of
continuity through multiple layers of history. High-resolution uniparental markers prove able to disentangle
relationships not just over the Andean region as a whole, but also at a local scale, despite the relatively low overall
genetic diversity so often regarded as an obstacle to recovering fine-grained population history in the Americas.
The long and complex (pre)history of Andean civilization has left traces that can be recovered not only from a
rich archaeological record and a complex linguistic panorama, but also from the details of the genetic makeup of
Andean populations living there today. This precision also helps clarify models of the diffusion of the largest sur-
viving language family of the entire Americas, Quechua. Clearly, those models need to combine two contrasting
mechanisms: significant demographic exchange in the south, and language shift by predominantly cultural forces
in the north. To further test this double scenario, and to gain a full insight into the genetic ancestry of the region,
this study should ideally be complemented by autosomal genomic data. Finally, the full mtDNA genome dataset
released here will stand as a valuable resource for broader, continent-wide comparisons, and for ancient DNA
studies for which the case-study region of Chachapoyas holds out particular potential.

Methods
Sample design and strategy: genealogical and linguistic characterization. This work is the result
of a fieldwork expedition conducted in 2015 to the regions of Amazonas and San Martn, with the support of local
political authorities and cultural representatives. Our sampling strategy was guided by linguistic indicators that
parallel, and can help tease apart, the three main layers of history in the region: the Chachapoya autochthonous
substrate; the brief period of Inca influence and then dominance, from the 1470s to the 1530s, which presumably
fostered the use of Quechua; and finally Spanish rule immediately thereafter.
Linguistic research directed our sampling in two ways: firstly it enabled us to focus on the Native American
genetic component, and secondly, within the native component, it allowed us to further distinguish between
Chacha and Quechua, most plausibly corresponding to periods before and after Inca impacts here. The presence
of Quechua, surviving in just a few villages scattered across Chachapoyas, enabled us to target sampling on those
locations. In each one, we conducted a survey to assess how far Quechua was (or still is) spoken in each partici-
pants family, and to identify the birthplaces of parents and grandparents. We also conducted extensive archival
work on historical patterns in local surname origins, following not just Quechua markers but trace indicators of
the now extinct Chacha language, too. Supplementary TableS1 shows our results by presence of Quechua and by
surname origin, with further details on linguistic and surname characterization given in the Supplementary Text.

The genetic sample. Saliva samples were collected from healthy volunteers. Each participant signed a writ-
ten consent form, after being fully informed of the purpose of the study, with the opportunity to ask questions
for further clarification. The project and the informed consent were approved by the Ethics Committee of the
University of San Martn de Porres, Lima (Comit Institucional de tica en Investigacin de la Universidad de
San Martn de Porres Clnica Cada Mujer, Ofcio No. 579-2015-CIEI-USMP-CCM, 12/05/2015) and by the
Ethics Committee of the University of Jena (Ethik-Kommission des Universittsklinikums Jena, Bearbeitungs Nr.
4840-06/16). All methods were performed in accordance with the relevant guidelines and regulations. The sample
analysed in this study represents only a small fraction of the population living in the provinces of Chachapoyas
and Luya, and in Wayku, and so is only partially representative of the complex demographic history of these
regions and of their inhabitants ancestors.
For Y-chromosome and mitochondrial analysis, individuals were assigned to different regional groups accord-
ing to their self-identified paternal or maternal ancestry (up to two generations): Huancas, La Jalca, Chachapoyas
city, Utcubamba South, Luya. These locations are all in the Amazonas region; our final sample is from the region
of San Martn, specifically from the Wayku neighbourhood in Lamas. (For further details on sample locations,
see the map in Fig.1 and the Supplementary Text). Information on the 119 individuals sampled is given in
Supplementary Table1. The precise identification of their home villages is not given, however, in order to guaran-
tee anonymity. Individuals marked OUT have maternal or paternal ancestry from other regions of Peru, outside
the provinces that are the focus of this study, and were excluded from the group comparisons.
DNA was extracted with QIAamp DNA Mini Kits (Qiagen) according to the manufacturers protocol.

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mtDNA. Libraries were prepared with a multiplex protocol for the Illumina Genome Analyzer platform, and
enriched for mtDNA with in-solution capture following standard protocols as performed in previous studies34,35.
Libraries were pooled and sequenced in Illumina HiSeq. 2500 on two Rapid lanes with 100+7+100+7 cycles.
The Kit for the chemistry was V2.
Base-calling was performed using freeIbis36, and reads were processed as follows37,38: read adaptors were
trimmed, and reads were filtered for at most 5 bases with a quality score <15, and indexes for no bases with
quality scores <10. Sequences were manually checked with Bioedit (www.mbio.nc-su.edu/BioEdit/bioedit.html).
Average coverage was ~3000X, with a minimum of 58X and a maximum of 12,093X.
The two poly-C regions (np 303315, 1618316194), prone to sequencing errors, were trimmed from the
final alignment used in the analysis. Sequence alignments, including the RSRS sequence39, were assembled with
MAFFT v7.123b40.
Haplogroup assignment was performed with Haplofind41 and Haplogrep 242, and manually confirmed by
checking diagnostic positions as described in Phylotree v.1729. Haplogrep was also used to list the polymorphisms
in each sample, as included in TableS1.
Two sets of comparative data were collected from the literature: a dataset of haplogroup frequencies from
across the Americas, adapted from24 (see original publication for population labels), and a dataset of full genome
mtDNA sequences2,1719,39,4354. The comparative sequence dataset includes a total of 725 sequences from hap-
logroups A, B, C and D, from North America, Mesoamerica and South America, and was screened to minimize
the number of missing sites. The maximum number of missing sites allowed per sequence was 2. The list of
sequences included, with references to the corresponding publications, is available in TableS4.

Y chromosome. Eighty-eight male individuals were typed with a basal SNaPshot multiplex, as described
in55. The Q samples were also tested for markers downstream of M3: Y14998418(MG2), PV2, SA01, M19,

M557 55,56. Samples belonging to haplogroup Q were typed with the PowerPlex Y23 System (Promega,

Mannheim, Germany) as previously described57. The data were analyzed with GeneMapper ID-X1.1.1. (Life
Technologies, Darmstadt, Germany). Haplogroup nomenclature follows van Oven et al.58 where possible, with
the name of the diagnostic marker following the conventional name of the haplogroup. Haplogroup affiliations
and STR profiles are reported in Supplementary Table1.
Intra-population comparisons were performed on a set of 23 or 17 loci. Comparisons with 23 loci were per-
formed with the analogous dataset from27, which included other individuals from Chachapoyas (some from La
Jalca and some from the province of Luya) and Huancas, as well as neighbouring populations from Cajamarca in
the highlands, and the Jvaro in Amazonia. The dataset from Guevara et al. was filtered for individuals assigned
to haplogroup Q with more than 90% accuracy from the haplogroup predictor. The resulting dataset for 23 loci
included a total of 177 individuals. Continent-wide comparisons were performed with a subset of 17 loci, to
include further data available from the literature, for a total of 90 populations2225,32,5961.

Computational analysis. Values of diversity, ST, haplogroup and haplotype comparisons, haplotype shar-
ing and correlations between features of the dataset were calculated and plotted in R62 using the packages Pegas63,
factoextra64, MASS65, vegan66, ape67, maps68, geosphere69, ggplot270and ggmap71. Median-joining networks were
calculated with Network 4.6.1.3 (Fluxus Technology, http://www.fluxus-engineering.com) and plotted with
Network Publisher. In the Network analysis of the STR haplotypes, weights were assigned to each individual
STR locus in inverse proportion to the variance observed in our dataset. Individuals from the published dataset
who were missing values for one or more loci were excluded from this analysis. Direct haplotype sharing was
performed with the 23 and 17 loci datasets and visualized in heatplot maps. The maps showing amounts of shared
identical and similar haplotypes were calculated for a subset of 15 loci (excluding unstable loci DSY385a and b),
adjusted for the mutation rate for each locus (mutation rates from https://yhrd.org/). The similar profiles allow up
to one mutation in one of five loci DYS389II, DYS439, DYS456, DYS458 and DYS635 with mutation rates higher
than 0.003 mutations per year per generation.
Phylogenetic trees and Bayesian Skyline Plots (BSPs) were generated with BEAST v1.872. BEAST runs were
performed with full mtDNA genomes, for the entire dataset as well as for individual populations. The dataset
was partitioned into the coding region, to which we assigned a rate of 1.708108 substitutions per nucleotide
per year, and the non-coding region, to which we assigned a rate of 9.883108 substitutions per nucleotide per
year73. The best substitution model for each partition and for each population subset was determined using jMod-
elTest v2.1.774. In order to determine the best clock model and tree model, different runs were performed with
BEAST and evaluated by a Bayes Factor (BF) analysis75. For the clock model, we compared a strict clock model, a
relaxed exponential, an uncorrelated relaxed lognormal (ULN) clock model and a random clock.
The best substitution models determined by jModelTest for the whole dataset were TNI+Invariant Sites for
the coding part of the alignment, and HKI+Invariant Sites+gamma for the non-coding part. Maximum like-
lihood estimates for the different combinations of clock models were evaluated by BF analysis76, and showed
decisive support for a ULN clock for the coding region and a random clock for the D-Loop. The Bayesian Skyline
tree model was chosen in order todisplay theBSP (given the strong expansion shown by the Native American
sequences, a constant size model was not considered). A total of 50 million chains were executed for the entire
sequence set, to ensure reliable ESS values. For the single populations set, we performed 1020 million chains.
Multiple runs were performed on each dataset, and combined using logCombiner. The maximum clade credibil-
ity was determined using TreeAnnotator and visualized with FigTree (http://tree.bio.ed.ac.uk/software/figtree/).
Coalescent simulations for 15 Y chromosome STRs were performed with Simcoal v.2.1.2 (http://cmpg.
unibe.ch/software/simcoal2/), with the mutation rates used above, a stepwise mutation model with a geometric
parameter of 0.1, and a simple population history of two populations (pop1 sample size 70, corresponding to
Chachapoyas, and pop2 sample size 20, chosen as a rounding down of the average sample size of 23) coalescing at

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a given split time T1 with no growth rate. We performed a total of 800 runs of 100 simulations each with varying
conditions: T1 between 10 and 200 generations, Ne for pop1 and pop2 between 500 and 5000, and symmetric
mutation rate between pop1 and pop2 between 0 and 0.1. For each simulation, we recorded the presence of hap-
lotype sharing events, to give a percentage of sharing events for each simulated condition.

Data availability. The STR and mtDNA haplotypes generated during this study are included in the
Supplementary Information files. The whole mtDNA sequences are available in GenBank under accession num-
bers MG571104 - MG571221.

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Acknowledgements
We are grateful to all volunteer participants, and to all those who supported our research in the provinces of
Chachapoyas, Luya and Lamas. In particular, we wish to thank Napoleon Culqui, Antenor Chuquizuta and
Segundo Felipe Cachique Amasifuen for their insights into the linguistic and historical background of their
home regions. We thank Pierina Danos and Anna Paschall for their assistance with sample preparation and DNA
extraction, and Fabrizio Mafessoni for support in bash scripting. We finally thank Bernard Comrie for supporting
the genetic and linguistic fieldwork with funds from the Max Planck Society.

Author Contributions
Project design was by P.H. and C.B.; community engagement, ethics and sampling by R.F., J.R.S., J.V., A.S. and
C.B.; linguistic and historical contextualization by P.H., J.V. and S.Z.; laboratory support by L.R., M.G., M.S. and
R.G.; data generation by C.B. and R.S.; data processing and analysis by C.B.; data interpretation by C.B., J.R.S.,
R.F., S.Z., J.V., M.S. and P.H.; manuscript writing by C.B. and P.H. with inputs from all co-authors.

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Additional Information
Supplementary information accompanies this paper at https://doi.org/10.1038/s41598-017-17728-w.
Competing Interests: The authors declare that they have no competing interests.
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The Author(s) 2017

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