Supplementary Materials for

A 6000-year-long genomic transect from the Bogotá Altiplano reveals multiple

genetic shifts in the demographic history of Colombia

Kim-Louise Krettek et al.

Corresponding author: Kim-Louise Krettek, [email protected];

Andrea Casas-Vargas, [email protected]; Cosimo Posth, [email protected]

Sci. Adv. 11, eads6284 (2025)

DOI: 10.1126/sciadv.ads6284

The PDF file includes:

Supplementary Text
Figs. S1 to S21
Legends for tables S1 to S8
References

Other Supplementary Material for this manuscript includes the following:

Tables S1 to S8
Supplementary Text

Anthropological and archaeological context

The reconstruction of the peopling of South America has raised different hypotheses on the
origin of the first settlers. Two of the main theories for interpreting biological variation in
Native American populations, which have exhibited heterogeneous traits in their cranial and
dental morphology, are based on archeological and anthropological evidence. The oldest
skeletons differ morphologically from those of late Native Americans. The first, so-called
“Paleoamericans”, are characterized by having longer, narrower and smaller skulls
(Dolichocephalic) with larger teeth (Sundadont), while later populations, so-called
“Amerindians”, tend to have a more rounded skull and a wider cranial vault (Brachycephalic)
with smaller teeth (Sinodont) (fig. S1-S5) (63). These characteristics have led to different
hypotheses about the possible origin of these settlers: 1) Migratory hypothesis, which
suggests that variation among South American groups was the result of multiple waves of
migration and 2) Microevolutionary hypothesis, which explains the emergence of biological
diversity as consequence of evolutionary processes (e.g. genetic drift, natural selection), so
that all Indigenous groups would be descendants of the same ancestral population (64, 65,
66).
Given its geographical location, Colombia was a mandatory steppingstone for entering (and
leaving) South America. Several studies have been carried out from both genetic and
archeological perspectives, to elucidate possible routes into Colombia and the subcontinent,
with no definite conclusions (21, 67).
According to archaeological data, one region that has shed light on the first human
settlements of Colombia is the Eastern Andes, since this region allows to test several
hypotheses regarding their origins. The pre-colonial phase is commonly divided into three
periods in which human populations occupied this mountain range: Preceramic (~12,000 to
3,000 BP), Formative (~3,000 BP to 1,000 BP) and Muisca (~1,000 BP to 400 BP) (68, 69).
Populations from these periods possibly had different origins and were the product of several
migration waves, displacing or integrating predecessor groups (70, 71). Bio-anthropological
studies have suggested that the Muisca settlements can be traced back to the Formative
period without major migratory processes of foreign peoples, but rather through
microevolutionary processes (68).

Description of analyzed individuals and archeological sites

In the current study we analyzed a total of 21 ancient individuals from five sites associated
with the Preceramic period (Checua), Formative period (Laguna de La Herrera) and Muisca
period (Las Delicias, Soacha, Purnia). A description of the archeological sites and analyzed
specimens is provided below and in table S1.

Checua (PREC), Nemocon, Bogotá ~6,000 BP

Coordinates: 5.0674157 Latitude, -73.8849739 Longitude

• PREC001 (93-CHII-03): no collagen preserved, MAMS-54219

• PREC002 (CHII-08): 3,951-3,789 calBC, MAMS-54221
• PREC003 (CHII-06)
 • PREC004 (CHII-07): 4,699-4,545 calBC, MAMS-5418
• PREC005 (CHII-04): no collagen preserved, MAMS-52227
• PREC006 (CHII02A)
• PREC007 (CHII-03A): 3,951-3,788 calBC, MAMS-54220

The Checua site in Nemocón, Bogotá is an open-air site extending over the top of a hill, rising
about 15 feet above the surrounding area. It was named after the subdivision of the
municipality containing the site, a region of subtropical lower-montane dry forest with a 2,600
m.a.s.l. average altitude, harboring the Checua river and the Neusa river (26, 72-74). The site
has produced archeological evidence for hunter-gatherer presence between 8,500 and 3,000
BP (radiocarbon dated) with two extensive excavation campaigns subdividing the site into
eight (first excavation) and seven (more recent excavation) stratigraphic layers (26, 72-74).
The first excavation, covering 300 m2, conducted by A.M. Groot and colleagues in 1991,
revealed eight stratigraphic levels with two human occupation zones, with zone one being
dated to 9,500-8,700 BP and zone two to 9,100-8,300 BP (26, 75). Human remains found at
the site were identified as Indigenous people based on anthropological assessment (26, 76).
Lithic tools, faunal remains and bone fragments linked the site to hunter-gatherer activities
(26, 72-74), painting a picture of complex funerary processes and cultural practices.

Laguna de La Herrera (FORM), Madrid, Bogotá ~2,000 BP

Coordinates: 4.7333069 Latitude, -74.2827857 Longitude

• FORM001 (Madrid 2-41 individuo 8)

• FORM002 (Madrid Ind 1): 36 cal BC-117 cal AD, MAMS-52224
• FORM003 (Madrid Ind 5)
• FORM004 (Madrid 2-41)
• FORM005 (Madrid 2-41 Indv 6)
• FORM006 (Madrid 2-41 Indv 2)
• FORM007 (Madrid 2-41 Indv 14)
• FORM008 (Madrid 2-41 Indv 15 corte 0)
• FORM009 (Madrid 2-41 corte 8 cráneo infantile)

Madrid in Cundinamarca is a collective group of burials belonging to the Early Formative

period (3,000 to 2,000 BP) and located on the edge of the ancient Laguna de La Herrera. The
remains were accompanied by foreign pottery from the Magdalena River valley, animal bones
and ochre. Their physical features display a Paleoamerican morphology, and some individuals
showed osteological signs that they suffered from infections of treponematosis origin. A
remarkable feature of the site is an astronomical observatory, which was built during the Late
Formative period (2,000 BP to 1,000 BP), consisting of inverted pyramids dug into the ground,
cones and canals aligned 24˚ NW.

Las Delicias (EMUI), Bogotá ~1,200BP

Coordinates: 4.6486259 Latitude, -74.2478931 Longitude

• EMUI001 (Delicias Marzo 14-90): 664-773 cal BC, MAMS-52223

• EMUI002 (Delicias RS-1)
The site Las Delicias, within the neighborhood of Las Delicias in Bogotá, was discovered
during construction work. The site has been severely destroyed by looting as well as
environmental processes. Initial assessment of suitability for becoming an archeological
study site was conducted by the Colombian Institute of Anthropology in collaboration with Dr.
Francisco Guacheta. The site comprises a residential area and a burial area located on an
alluvial terrace of the Tunjuelito River and was inhabited between 1010 ± 60 BP and 1180 ±
70 BP according to radiocarbon dates obtained from charcoal as well as archeological
material found at the site. Within the site, houses containing artifacts as well as occasional
burials without funerary goods were found. Based on archeological evidence, the inhabitants
consumed flora and fauna that is currently extinct in the area, as well as maize. Furthermore,
the archeological material was associated with the Muisca culture (77). During an excavation
conducted by Enciso, the remains of 19 human individuals were found within the boundaries
of the burial area.

Soacha (MUIS), Soacha, Bogotá ~520 BP

Coordinates: 4.5754900 Latitude, -74.2312017 Longitude

• MUIS001 (Soacha 1987 T50): 1,413-1,447 calAD, MAMS-52226

At the Portalegre site of Soacha, Cundinamarca, Álvaro Botiva (1988) excavated a total of 130
tombs and four floors of houses dated to 900 to 700 BP. Most of the tombs are simple
rectangular shallow pits of which 10 % were covered with slabs. The bodies were in the
extended dorsal decubitus position, predominantly facing south and east. The grave goods
consisted of moccasins, bowls, cups, jars, two-handled globular pots, seashell beads and
some lithic artifacts (spindle whorls, grinding hands, metates and an axe). Shuttle hooks and
bone needles seem to be associated with male individuals, while spindle whorls seem to be
associated with female individuals.

Purnia (GUAN), Mesa de los Santos, Santander ~530 BP

Coordinates: 6.9425442 Latitude, -73.0445416 Longitude

• GUAN001 (Purnia 19): 1,402-1,436 calAD, MAMS-52225

• GUAN002 (Purnia 01)

Purnia refers to an archeological site located within the La Mesa de los Santos district in
Santander, Colombia. It encompasses two rock shelters near the El Salto del Duende
waterfall, which were reportedly used by people associated with the Guane as a burial
ground. During a campaign in 1988, led by Professor Arturo Cifuentes from the Universidad
Industrial de Santander, students found multiple human remains and artifacts within the
caves (78). The human remains in question entailed a mummified human body of a possibly
high-rank pre-Guane individual inhumated with various grave goods like several rolled
blankets, a loom, a baton stick, several ceramic pieces, necklaces, food, and surrounded by
scattered human skeletons (35). While the mummified body, dating back to 900-1000 BP,
showed severe cephalic deformation, only one other skeleton showed paleopathological
signs, possibly congenital syphilis (35, 78).
Sample selection and sequencing strategy

We screened 21 individuals from five different archeological sites and four different time
periods from the Altiplano for ancient DNA preservation. Our sample set encompasses
seven individuals from the preceramic site Checua, with an average age for the radiocarbon
dated individuals of 6,000 BP, nine individuals form the site Laguna de la Herrera dated to
2,000 BP, two individuals from the early Muisca site Las Delicias dated to 1,200 BP, two
individuals from the Guane-associated site Purnia dated to 530 BP, and one individual from
the late Muisca site Soacha dated to 520 BP (table S1).

For each individual we constructed double-stranded, double-indexed Illumina libraries (51)

and evaluated endogenous DNA content via shotgun sequencing. We considered a human
endogenous DNA content above 0.1 % in combination with the presence of the
characteristic damage pattern for ancient DNA (55), as necessary preservation features for
in-solution capture (56). All screened individuals met these criteria with preservation of
human endogenous DNA between 0.31 % and 48 %. Therefore, we proceeded for all
individuals with both nuclear and mitochondrial DNA in-solution captures. For individuals
with less than 1 % human DNA, we performed two rounds of capture, while for individuals
with above 1 %, we performed one round of capture. We reached an average capture
efficiency of 25 and 228 for one and two rounds of capture, respectively. Additionally, we
sequenced each library multiple times to maximize the number of available SNPs for each
individual. With this strategy, we were able to obtain usable genome-wide data for 19
individuals from the Bogotá plateau and two individuals from the Los Curos area, both on the
Atiplano Cundiboyacense.
We integrated the genomic data with previously published ancient and modern genomes
from the Allen Ancient Genome Diversity project version v.54.1 genotyped on ~1,24M SNPs
(table S8.A) (79), the Human Origins panel genotyped on ~600K SNPs (table S8.B,C) (37)
and the Native American Illumina panel genotyped on ~365K SNPs (table S8.C,D) (23),
resulting in three different datasets to obtain the highest overlapping SNP coverage for
different analyses.

Uniparental markers and biological kinship

We observed substantially different mtDNA haplogroup frequencies across time in our

dataset corresponding to the observation of a complete genetic replacement of preceramic
individuals by an incoming ancestry sometime after 6,000 BP. In the preceramic group, the
most prevalent mtDNA haplogroup is A2 and its subgroups, with haplogroup C being present
in only one individual (table S1). Even though we were able to establish second- and third-
degree kinship between preceramic individuals from Colombia_Checua_6000BP, they do
not share the same mtDNA haplogroup, excluding a connection through the maternal line.
Instead, we do observe some patterns of Y-haplogroup sharing. Nevertheless, our Checua
samples represent a limited subset of the individuals buried at the site, which limits our
ability to assess the social structure of the population in greater detail.
Interestingly, individuals from Colombia_LagunadelaHerrera_2000BP show a prevalence of
mtDNA haplogroup B2d, with haplogroup C1c being present in two individuals and D1 in one
(table S1). This suggests a turnover in mtDNA haplogroup diversity, which is in line with the
genetic replacement observed at the nuclear DNA level. In addition, haplogroups B2d and
A2w were previously found in pre-Hispanic individuals from Panama, confirming the genetic
link between the post-2,000 BP individuals from the Altiplano and ancient populations from
Lower Central America.
We were able to reconstruct a parent-offspring relationship between two female individuals
(FORM004 and FORM006) and a second-degree kinship between the female individuals
FORM004 and FORM008. Based on our analysis, these females share the same mtDNA
haplogroup, namely B2d, which might indicate that they derive from a shared maternal
lineage. From 1,200 BP onwards, we observe the survival of mtDNA haplogroup B2d, in
agreement with large-scale genetic continuity between ~2,000 and 500 BP in the Altiplano.

Population structure and effective population size

Archeological and anthropological evidence has been used to derive population size
dynamics of human groups on the Altiplano. It was suggested that preceramic hunter-
gatherer populations lived in relatively small groups and relied on a foraging lifestyle that
transitioned towards sedentism through time (27, 80, 81). Populations associated with the
Herrera ceramics are instead considered to have had larger group size and to be mainly
based on a farming subsistence (80, 81). With the beginning of the Muisca period, roughly
around 1,200 BP, population size increased even further, until it declined again due to
demographic impact of the Spanish colonization (82, 83).
We investigated the changes in effective population size, as well as mating patterns, by
analyzing runs of homozygosity (ROH) and Identity by decent (IBD) using hapROH (41). The
analysis of the Colombia_Checua_6000BP group shows a large proportion of short (4-8 cM)
ROHs in all individuals with a few medium (8-20 cM) and even fewer long (>20 cM) ROHs
(fig. S9). This pattern is indicative of a small effective population size rather than a close kin
mating pattern. To further expand on this observation, we inferred IBD for male individuals
as implemented in HapROH. We were able to detect several short IBD stretches, again
indicative of a small effective population size but with limited close kin mating (fig S8).
In individuals from the Colombia_LagunadelaHerrera_2000BP group, ROHs of short and
medium size decrease significantly compared to Checua (fig. S9). However, a substantial
amount of long ROHs is present in FORM006 and FORM009, inferring a higher level of
consanguinity than the other individuals analyzed from the same archeological site. The
male individuals from Laguna de la Herrera do not share any IBDs (fig S7), supporting the
scenario of a larger effective population size in this site than in Checua.
In all other individuals we observe a general decline in short and medium ROHs through
time, which is consistent with the described population size increase from the early Muisca
period onwards. Finally, we report a potential case of close-kin mating offspring for
GUAN002. The pattern of ROHs is the closest resembling that of two second cousins mating
(fig S9).

Principal Component Analysis

Since Principal Component Analysis (PCA) is significantly influenced by missing data, we

created three different datasets to build PCAs by merging our generated genomes with the
1240K SNP panel downloaded from the Allen Ancient Genome diversity project (79) (table
S8.A), two datasets of modern-day Native American individuals genotyped on the Human
Origins panel (table S8.B) (20, 60) and the unmasked, unadmixed version of the Illumina
Native American panel (table S8.D) (23). We analyzed these datasets separately to retain as
many SNPs as possible while minimizing missing data.
We first built a PCA using present-day genomes from the SGDP panel (37) and projected
the ancient individuals on this variation to verify their placements within a worldwide genetic
diversity map as a visual indicator of potential non-Native American admixture or
contamination (fig S6). As expected from their pre-colonial age and the low-level levels of
contamination estimated with schmutzi and ANGSD (57, 58), all analyzed ancient individuals
fall within the genetic diversity of modern Native Americans. This confirms no signs of recent
admixture with non-Native American ancestries nor modern-day DNA contamination (table
S1 and fig S11).
We further explored the relationship of the generated ancient genomes from Colombia and
previously published ancient genome from Panama and Venezuela in relation to the
unadmixed and unmasked Native American populations from a dataset published by Reich
et al. (23). We restricted the number of individuals used to build the PCA by selecting
populations from the Isthmo-Colombian area and projected the ancient individuals on this
modern-day genetic variation (fig. S12). In addition, we projected Ceramic-age and Archaic-
age individuals from the Caribbean (44, 45) due to general affinities with individuals from the
Isthmo-Colombian area revealed through f-statistics (table S4.D). In this PCA we find a
pattern where ancient Colombians from the Altiplano fall in-between East Colombian
populations like Piapoco and Guahibo, and Panamanian and Costa Rican populations like
Guna, Cabécar, Teribe and Bribri, as well as ancient Panamanians. Interestingly, when we
project Ceramic-age Venezuelans into the same PCA space, these individuals fall on a
widespread cline with some being more drawn towards ancient and present-day Isthmus,
while others more towards eastern Colombians. As expected from previous studies (44, 45).
Ceramic-Age Caribbeans are the closest groups to Piapoco, while Archaic-age Caribbeans
fall in an intermediate position between Ceramic-Age Caribbeans on one side, and
Venezuelans and Colombians on the other. Interestingly, present-day populations from
Northern Colombia like Waunana and Embera, but also some Costa Rican populations like
Maleku and Guaymi are removed from the main cline and extend along PC2. Lastly, all
individuals from the Colombia_Checua_6000BP plot with Archaic- and Ceramic-age
individuals from the Caribbean, which is interpreted as an artifact due to the equal genetic
distance of this group from all present-day Indigenous populations from South Americans
observed in f-statistics.
Finally, our analyses showed a distinct affinity of ancient individuals younger than 2,000 BP
from the Bogotá plateau to ancient and modern individuals form the Isthmus. Thus, we used
previously published genotypes of present-day individuals from Panama (20) to build a third
PCA and project ancient individuals on this variation (fig. S13). As previously reported by
Capodiferro and colleagues, Guna is a highly drifted population, as also visible in our PCA.
Instead, the other modern Panamanian populations form two distinct clines, one mainly
comprised of Ngabe and Naso individuals, and the other of Embera individuals. Ancient
individuals from Panama, Colombia and Venezuela plot close to the center of this triangle,
with post-2,000 BP Colombians largely overlapping and ancient Venezuelans placed along a
gradient of higher-to-lower affinity to ancient Panamanians. Therefore, this PCA built with a
Human Origins dataset confirms the main findings observed in the previous PCA built with
an Illumina dataset (fig. S12).
ADMIXTURE

To obtain a general overview of the clustering pattern in different genetic ancestry

components of ancient and modern Native American individuals we used ADMIXTURE
(Version 1.3.0). We created a dataset comprising the generated ancient genomes from
Colombia, previously published ancient Panamanians and Ceramic-age Venezuelans (20,
44), as well a subset of masked individuals from the Illumina dataset (23) (fig. S11). We ran
ADMIXTURE in unsupervised mode with the number of ancestral populations K ranging from
3 to 15. The calculation of the cross-validation error for each K reveals that K=6 has the
lowest value, with a clear increase from K=7 onwards.
Starting from K=3, ancient Colombians as well as Panamanians and Venezuelans are
largely comprised of a component shared with Cabecar, one with Quechua/Pima, and a third
component virtually shared with all Native South Americans. In the run of K=6 (Fig. 3A), we
observe different proportions of a component maximized with Cabecar in ancient
Colombians younger than 2,000 BP, Panamanians and Venezuelans, but it is absent in
individuals from Checua. Nevertheless, we observed a component shared between all these
ancient groups, including Checua, which is maximized in Quechua and Aymara. While
ADMIXTURE analysis suggests a shared genetic ancestry component between these
ancient and present-day populations, we were unable to confirm this with f-statistics (see
below). Therefore, we interpret this shared component as the results of an appropriate
modeling of the genetic ancestry profile of ancient Colombians.

F-statistics

We conducted multiple f-statistics by creating three separate datasets, one containing only
ancient and present-day genome-wide data overlapping up to 1.24M SNPs (79), one with
the masked individuals from the Illumina dataset (23) and one with the Human Origins
genotypes for present-day Colombian individuals (60).
We initially tested whether the individuals of one site could be grouped by conducting a f3-
outgroup statistic in the form of f3(Mbuti, Individual 1; Individual 2). We ran this statistic both
within and among the sites to exclude the possibility of closer inter-site affinities. We were
able to show that there is intra-site homogeneity, and we grouped individuals by site (tables
S1 and S2.A-B).
Additionally, we performed a f3-outgroup statistic of the form f3(Mbuti, X; Ancient South
America), where X stands for our reported individuals and ‘Ancient South America’ for major
genetic ancestry lineages of the sub-continent. We then built a genetic dissimilarity matrix for
multidimensional scaling (MDS) through 1-f3-outgroup statistics (Fig. 3B). To confirm the
patterns observed with the f3-outgroup statistics, we performed f4-statistics in the form of
f4(Mbuti, X; Ancient Americans, Ancient Americans), with X representing the sequenced
ancient Colombian groups (tables S3.C and S4.H).
We then tested for genetic affinities of ancient Colombian individuals with available modern
Native American populations through f3(Mbuti; Ancient Colombia, X), with X being individuals
from Reich et al. (23). We found a strong similarity between ancient Colombians younger
than 2,000 BP and modern-day populations from the Isthmus, significantly less affinity to
northern Colombian populations, and no direct links to other neighboring populations within
and outside of Colombia (Fig. 4A). However, we also observed a generalized affinity of
Colombia_Checua_6000BP to all available Central and South Americans, consistent with its
placement as a deeply divergent lineage (Fig. 2B). Additionally, despite the limited SNP
overlap between the Illumina and Human Origins panels (~78,000 SNPs), we run f3(Mbuti; X,
Ancient Colombia) combining the Reich et al., (23) and Arias et al. (60) datasets to maximize
the number of present-day individuals from the Isthmo-Colombia area. We observe the same
pattern of generalized affinity of Colombia_Checua_6000BP with populations from the
Isthmus and South America (fig. S20A). Individuals from the Altiplano younger than 2,000
BP confirm a specific affinity to Isthmian populations to the exception of the highly drifted
Chorotega group (fig. S20B-E). We further confirmed the differential affinities highlighted
with f3-outgroup statistics in corresponding f4-outgroup statistics of the form f4(Mbuti, Ancient
Colombia; Modern Native Americans, Modern Native Americans) (tables S3.D and S4.C).
Moreover, we conducted a series of f4-statistics to specifically test for distinct genetic links to
Anzick-1- and California Channel Island-related ancestries with f4(Mbuti, Anzick-1; Chile_Los
Rieles_12000BP/Peru_Lauricocha_8600BP, X) and f4(Mbuti, California Channel Island;
Peru_Cuncaicha_4200BP/Peru_Lauricocha_8600BP, X), where X is our generated ancient
Colombian data (table S3.A-B).
We then conducted the following f4-statistics f4(Mbuti, Ancient Colombia; Modern Northern
Colombia, Modern Isthmus) (table S7.K). These reveal a significant affinity of ancient
Colombians younger than 2,000 BP to modern-day as well as ancient populations of the
Isthmus, Ceramic-age Caribbean, and Ceramic-age Venezuelans with an absence of
significant affinity towards modern northern Colombian populations.
Furthermore, we tested whether other modern-day Colombians provide a better proxy for
ancient Colombian than either modern Northern Colombians or modern Isthmus populations
by performing two f4-statistics of the form f4(Mbuti, Ancient Colombians; Modern Colombians,
Modern Northern Colombians) and f4(Mbuti, Ancient Colombians; Modern Colombians,
Modern Isthmus) (table S7.D).
Lastly, we tested whether ancient and modern-day populations from the Isthmus are equally
related to ancient Colombians by conducting the following f4-outgroup statistic f4(Mbuti,
Ancient Colombians; Ancient Isthmus, Modern Isthmus). By extension we also tested
whether ancient Colombians and Isthmian populations exhibit differential affinity to the
modern-day Isthmus groups with f4(Mbuti, Modern Isthmus; Ancient Colombians, Ancient
Isthmus) (table S7A-J).
The affinity to Ceramic-age Venezuela was tested in multiple ways, firstly in relation to
populations from the Isthmus and the Ceramic-age Caribbeans. We tested whether ancient
and modern-day Isthmus populations remain the best proxy compared to Ceramic-age
Venezuelans with f4(Mbuti, Ancient Colombians; Ancient Isthmus, Ceramic-age Venezuela)
and f4(Mbuti, Modern Americans; Ancient Colombians, Ceramic-age Venezuela) (table
S4.G-H). In addition, we investigated the Ceramic-age Caribbean affinity with the following
f4-statistics f4(Mbuti, Ancient Colombians; Ceramic-age Venezuela, Ceramic-age Caribbean)
and f4(Mbuti, Ancient Colombians; Ancient Isthmus, Ceramic-age Caribbean) (table S4.I-J).

qpGraph

The f-statistics performed on our reported ancient Colombian individuals from

Colombia_Checua_6000BP showed a generalized genetic affinity to all previously
sequenced ancient and modern-day Native South Americans, and lack of Anzick-1 and
California Channel Island-related ancestries. We therefore hypothesize these individuals
being part of the initial South American radiation event. In fact, a previous study (8) has
proposed that the ancestral population of South Americans likely experienced a rapid
diversification process giving rise to multiple South American lineages. However, there was
no genome-wide data available for hunter-gatherer individuals from northern South America,
making it difficult to assess where this process took place. Here, we attempted to model
Colombia_Checua_6000BP within this previously defined admixture graph topology using
the Admixtools package qpGraph (v7.0.2).
We tested multiple placements of the Colombia_Checua_6000BP group, placing it as: 1) a
lineage splitting within the North American variation; 2) as part of the radiation event; 3)
splitting off from each lineage leading to distinct South American ancestries (fig. S10).
Placing Colombia_Checua_6000BP as a lineage splitting from a North American source,
yields a worst Z-score that deviates significantly from zero (Z=-5.1), indicating a non-viable
model (fig. S10J). Instead, when positioning Colombia_Checua_6000BP as deriving from a
basal split before the South American radiation we obtain the best available Z-score of 3.2
but with an internal edge length of 0 (fig. S10A). This Z-score value is maintained when
modeling Colombia_Checua_6000BP as deriving from the radiation (fig. S10B-D, S10G).
Instead, worst Z-score values are obtained when Colombia_Checua_6000BP is placed as
derived within one of the South American lineages (fig. S10E-F, S10H). We therefore
consider as the most viable model the one where Colombia_Checua_6000BP derive from
the initial radiation event into South America, suggesting that this process took place latest
as human groups reached the southern sub-continent.

TreeMix

We used TreeMix v.1.13 to better understand the relative relationships between ancient
Colombians, Panamanians and Venezuelans in our dataset, in comparison to Ceramic-and
Archaic-age Caribbeans. Previous analyses provide evidence for a significant genetic
heterogeneity within the Ceramic-age Venezuelan group, where three individuals show a
higher attraction to ancient Panamanians compared to the other five individuals from the
same archeological site and similar date (table S4.G). We therefore subdivided this group
into two clusters, Venezuela_LasLocas_Ceramic_1 and Venezuela_LasLocas_Ceramic_2,
with the latter including the three individuals with more Panamanian-related ancestry.
For each Treemix analysis we included an outgroup, Ceramic- and Archaic-age individuals
from the Caribbean, ancient Panamanians plus one ancient Colombian group and one
Venezuelan group at the time to exclude the possibility of distorting the analysis by intra-site
genetic attraction (fig. S14-S18). We used USA_Ancient_Beringian (2) at the outgroup, due
to its equal genetic distance to most ancient and modern American populations. Earlier
publications have established Archaic-age and Ceramic-age populations from the Caribbean
to be largely homogenous populations with little to no admixture between them (44, 45). To
keep complexity to a minimum, we included only one group for each Caribbean-related
ancestry. In f4-outgroup statistics, we have observed Cuba_PlayadelMango_Archaic to
reveal some affinity with ancient Venezuelan and Colombian groups, and thus we
specifically chose this population as a representative for the Archaic-age populations (table
S4). To represent the Ceramic-age Caribbean group, we chose the best covered site in
terms of number of individuals available (Dominican_LaCaleta_Ceramic). For each tree
composition we inferred admixture edges from 0 to 5.
When performing the analysis including Colombia_Checua_6000BP as the ancient
Colombian group, all trees reveal it as an outgroup to the exclusion off all other populations
(except USA_Ancient_Beringian). This confirms the modeled placement of
Colombia_Checua_6000BP in qpGraph. Additionally, some admixture edges suggest the
contribution of an ancient source into Venezuela_LasLocas_Ceramic_1 and
Colombia_Checua_6000BP. However, we were unable to confirm such link with f-statistics
or ADMIXTURE analysis (fig. S14).
The group Colombia_LagunadelaHerrera_2000BP exhibits a differential behavior when
modelled in relation to both ancient Venezuelan clusters. When analyzed with cluster 1,
individuals from Laguna de la Herrera form a sister group with ancient Panamanians.
Instead, when plotted with cluster 2, ancient Venezuelans form a siter group with ancient
Panamanians. Admixture edges infer gene flow between ancient Venezuelans,
Panamanians and Colombia_LagunadelaHerrera_2000BP. However, we were unable to
infer directionality or direct admixture proportions with other analyses. This observed pattern
extents to Colombians between 1,200 BP and 520 BP (fig. S15-S18).
We interpret this pattern as a cline in the proportion of Chibchan-related ancestry in ancient
Venezuelans and post-2,000 BP ancient Colombians. This suggests that Chibchan-related
genetic contributions in Venezuela and Colombia underwent different demographic
processes.
Figure S1: Photographs taken of the archeological site Checua and excavated
human remains. Pictures of the Checua archeological site (left) and of a largely complete
skull of a hunter-gatherer individual excavated at the site (right), Photo Credit: Jose-Vicente
Rodriguez Cuenca, Universidad Nacional de Colombia (84).

Figure S2: Excavation photograph of Madrid and excavated human remains in situ.
Pictures of Madrid 2-41 in the Laguna de La Herrera archeological site (left) and of an
almost complete skeleton (UE1-F6) excavated at the site (right), Photo Credit: Jose-
Vicente Rodriguez Cuenca, Universidad Nacional de Colombia (84).
Figure S3: Human remains in situ excavated at Madrid. Burial 18 of an incomplete
skeleton (above) and infant burial (below) at Madrid 2-41, Cundinamarca, Photo Credit:
Jose-Vicente Rodriguez Cuenca, Universidad Nacional de Colombia (84).

Figure S4: Picture of an almost complete skull. The individual was excavated at the
Soacha site in Colombia and is associated with the Muisca culture, Photo Credit: Jose-
Vicente Rodriguez Cuenca, Universidad Nacional de Colombia (84).
Figure S5: Picture of a skull without mandible. This individual was excavated at the
Purnia site in the Los Curos area and was associated with the Guane culture, Photo Credit:
Jose-Vicente Rodriguez Cuenca, Universidad Nacional de Colombia (84).
 0.005
0.000
−0.005
PC2

−0.010
−0.015
−0.020

−0.015 −0.010 −0.005 0.000 0.005 0.010

PC1
Adygei.DG Eskimo_Chaplin.DG Lahu.DG Sherpa.DG
Albanian.DG Eskimo_Naukan.DG Lemande.DG Sindhi.DG
Aleut.DG Eskimo_Sireniki.DG Lezgin.DG Somali.DG
Altaian.DG Estonian.DG Luhya.DG Spanish.DG
Altai.DG Even.DG Luo.DG Surui.DG
Altais.DG Finnish.DG Madiga.DG Tajik.DG
Ami.DG French.DG Makrani.DG Tajiks.DG
Armenian.DG Gambian.DG Mala.DG Teleuts.DG
Atayal.DG Georgian.DG Mandenka.DG Thai.DG
Australian.DG Greek.DG Mansi.DG Tibetan.DG
Balochi.DG Han.DG Maori.DG Tlingit.DG
BantuHerero.DG Hawaiian.DG Masai.DG TomskTatars.DG
BantuKenya.DG Hazara.DG Mayan.DG Tubalar.DG
BantuTswana.DG Hazaras.DG Mbuti.DG Tu.DG
Bashkirs.DG Hezhen.DG Mende.DG Tujia.DG
Basque.DG Hungarian.DG Mexico_Zapotec.DG Turkish.DG
BedouinB.DG Icelandic.DG Miao.DG Turkmens.DG
Bengali.DG Igbo.DG Mixe.DG Tuscan.DG
Bergamo.DG Igorot.DG Mixtec.DG Ulchi.DG
Biaka.DG Iranian.DG Mongola.DG Uyghurs.DG
Bougainville.DG Iraqi_Jew.DG Mozabite.DG Uygur.DG
Brahmin.DG Irula.DG Nahua.DG Uzbeks.DG
Brahui.DG Itelman.DG Naxi.DG Vindija.DG
Bulgarian.DG Japanese.DG Onge.DG VolgaTatars.DG
Burmese.DG Jordanian.DG Orcadian.DG Xibo.DG
Burusho.DG Ju_hoan_North.DG Oroqen.DG Yadava.DG
Buryats.DG Kalash.DG Palestinian.DG Yakut.DG
Cambodian.DG Kalmyks.DG Papuan.DG Yemenite_Jew.DG
Chane.DG Kapu.DG Pathan.DG Yi.DG
Chechen.DG Karakalpaks.DG Piapoco.DG Yoruba.DG
Chipewyan.DG Karitiana.DG Pima.DG Zapotec.DG
Chukchi.DG Kashmiri_Pandit.DG Polish.DG Greenland_Saqqaq.SG
Cree.DG Kazakhs.DG Punjabi.DG Panama_IsthmoColombian_PreColonial.SG
Crete.DG Kazkahs.DG Quechua.DG Venezuela_LasLocas_Ceramic
Czech.DG Kharia.DG Relli.DG Venezuela_LasLocas_Ceramic2
Dai.DG Khomani_San.DG Russia_Abkhasian.DG Colombia_Soacha_520BP
Daur.DG Khonda_Dora.DG Russian.DG Colombia_Purnia_530BP
Dinka.DG Kinh.DG Russia_North_Ossetian.DG Colombia_LasDelicias_1200BP
Druze.DG Kongo.DG Saami.DG Colombia_LagunadelaHerrera_2000BP
Dungan.DG Korean.DG Saharawi.DG Colombia_Checua_6000BP
Dusun.DG Kurumba.DG Samaritan.DG
English.DG Kusunda.DG Sardinian.DG
Esan.DG Kyrgyz.DG She.DG

Figure S6: Principal Component Analysis built on worldwide present-day genetic diversity
Mallick et al. (37). Ancient individuals were projected. Native American populations are depicted in
yellow, other present-day individuals in black and ancient individuals in other colors.
Figure S7: Identity by Descent (IBD) assigned with hapROH Ringbauer et al. (41). Assignment
was based on the X-Chromosome for pairs of male individuals within the
Colombia_LagunadelaHerrera_2000BP group.
Figure S8: Identity by Descent (IBD) assigned with hapROH Ringbauer et al. (41). Assignment
was based on the X-Chromosome for pairs of male individuals within the Colombia_Checua_6000BP
group.
Figure S9: Runs of Homozygosity (ROH) assigned with hapROH for every individual,
Ringbauer et al. (41). Individual IDs are indicated in Supplementary Table 1. Color code refers to
ROH lengths as follows: dark blue (4-8 cM), light blue (8-12 cM), yellow (12-20 cM), red (20-300
cM).
A Han CA_ Arg ASO -0.005567 -0.003764 0.001803 0.000571 3.157 B R
Han CA_ Arg ASO -0.005567 -0.003764 0.001803 0.000571 3.157 C Han

R
CA_ Arg ASO -0.005567 -0.003764 0.001803 0.000571 3.157
D Han CA_ Arg ASO -0.005567 -0.003764 0.001803 0.000571 3.157
R R

33 33
33 33 33 33 33 33

Mbuti nonAfrica Mbuti nonAfrica

Mbuti nonAfrica Mbuti nonAfrica

9 9
9 9

EastEurasia 35 EastEurasia 35
EastEurasia 35 EastEurasia 35

7 31 7 31
7 31 7 31

EastAsia Onge_DG WestEurasia EastAsia Onge_DG WestEurasia

EastAsia Onge_DG WestEurasia EastAsia Onge_DG WestEurasia

6 2 1 67 6 2 1 67
6 2 1 67 6 2 1 67

EastAsia2 EastAsia3 E_HG2 EastAsia2 EastAsia3 E_HG2

EastAsia2 EastAsia3 E_HG2 EastAsia2 EastAsia3 E_HG2

1 4 16% E_HG3 84% 1 4 16% E_HG3 84%

1 4 16% E_HG3 84% 1 4 16% E_HG3 84%

Han EastAsia4 33% ANE Han EastAsia4 33% ANE

Han EastAsia4 33% ANE
Han EastAsia4 33% ANE
67% 95 67% 95
67% 95
67% 95
FA Russia_MA1_HG_SG
FA Russia_MA1_HG_SG
FA Russia_MA1_HG_SG
FA Russia_MA1_HG_SG
10
10
10
10
Beringia
Beringia
Beringia
Beringia
142 8
142 8
142 8
142 8 USA_Ancient_Beringian NA
USA_Ancient_Beringian NA
USA_Ancient_Beringian NA
USA_Ancient_Beringian NA 117 6
117 6
117 6
117 6 ASO_SG NA2
ASO_SG NA2
ASO_SG NA2
ASO_SG NA2 81 1
81 1
81 1
81 1 CA_Islands CA
CA_Islands CA
CA_Islands CA
CA_Islands CA 1
1
1
1 SA
SA
SA
0 0
Source
2 0
2 0
Source SA3
72 0
SA2 Source
SA2 SA3
72 2 39 2
PREC_6000BP SA
124 79 72 0
124 79 40 0
PREC_6000BP SA2 Brazil_LapaDoSanto_9600BP SA4
2 0
Peru_Cuncaicha_9000BP Lauricocha_8600BP PREC_6000BP SA3
124 79 59 124 Peru_Cuncaicha_9000BP Lauricocha_8600BP Brazil_LapaDoSanto_9600BP Source
SA2 SA3
39 2
Peru_Cuncaicha_9000BP Lauricocha_8600BP Argentina_ArroyoSeco2_7700BP Chile_LosRieles_5100BP 71 2
124 79 39 2
Brazil_LapaDoSanto_9600BP SA4
PREC_6000BP SA4
Peru_Cuncaicha_9000BP Lauricocha_8600BP Brazil_LapaDoSanto_9600BP SA4
59 124
59 124
59 124
Argentina_ArroyoSeco2_7700BP Chile_LosRieles_5100BP
Argentina_ArroyoSeco2_7700BP Chile_LosRieles_5100BP

Argentina_ArroyoSeco2_7700BP Chile_LosRieles_5100BP

E R
Per PRE Lau PRE 0.071077 0.073936 0.002859 0.000798 3.582
F R
Per PRE Lau PRE 0.071077 0.073936 0.002859 0.000798 3.582

G R
Han CA_ Arg ASO -0.005567 -0.003764 0.001803 0.000571 3.157

33 33 33 33
33 33

Mbuti nonAfrica Mbuti nonAfrica

Mbuti nonAfrica

9 9 9

EastEurasia 35 EastEurasia 35 EastEurasia 35

7 31 7 31 7 31

EastAsia Onge_DG WestEurasia EastAsia Onge_DG WestEurasia EastAsia Onge_DG WestEurasia

6 3 1 67 6 3 1 67 6 2 1 67

EastAsia2 EastAsia3 E_HG2 EastAsia2 EastAsia3 E_HG2

EastAsia2 EastAsia3 E_HG2

1 4 16% E_HG3 84%

1 4 16% E_HG3 84% 1 4 16% E_HG3 84%

Han EastAsia4 33% ANE

Han EastAsia4 33% ANE Han EastAsia4 33% ANE
67% 95
67% 95 67% 95
FA Russia_MA1_HG_SG
FA Russia_MA1_HG_SG FA Russia_MA1_HG_SG
10
10 10
Beringia
Beringia Beringia
142 8
142 8 142 8
USA_Ancient_Beringian NA

USA_Ancient_Beringian NA USA_Ancient_Beringian NA
117 6

117 6 117 6
ASO_SG NA2

ASO_SG NA2 ASO_SG NA2 81 1

81 1 81 1 CA_Islands CA

CA_Islands CA CA_Islands CA 1

1 1 SA

SA SA 2 0

1 0 SA2 SA3
1 0

124 79 0 2
SA2 SA3 SA2 SA3

Peru_Cuncaicha_9000BP Lauricocha_8600BP Source SA4

81 0 39 2 125 0 39 2

39 72 59 124
Lauricocha_8600BP Source Brazil_LapaDoSanto_9600BP SA4 Peru_Cuncaicha_9000BP Source Brazil_LapaDoSanto_9600BP SA4
Brazil_LapaDoSanto_9600BP PREC_6000BP Argentina_ArroyoSeco2_7700BP Chile_LosRieles_5100BP
125 71 59 124 81 71 59 124

Peru_Cuncaicha_9000BP PREC_6000BP Argentina_ArroyoSeco2_7700BP Chile_LosRieles_5100BP Lauricocha_8600BP PREC_6000BP Argentina_ArroyoSeco2_7700BP Chile_LosRieles_5100BP

H R
Ong Arg Chi PRE 0.000000 -0.002725 -0.002725 0.000628 -4.336
I R
Ong Arg Chi PRE 0.000000 -0.002725 -0.002725 0.000628 -4.336
J R
Arg ASO CA_ PRE -0.000000 -0.003138 -0.003138 0.000618 -5.081

33 33 33 33 33 33

Mbuti nonAfrica Mbuti nonAfrica Mbuti nonAfrica

9 9 9

EastEurasia 35 EastEurasia 35 EastEurasia 35

7 31 7 31 7 31

EastAsia Onge_DG WestEurasia EastAsia Onge_DG WestEurasia EastAsia Onge_DG WestEurasia

6 2 1 67 6 2 1 67 6 2 1 67

EastAsia2 EastAsia3 E_HG2 EastAsia2 EastAsia3 E_HG2 EastAsia2 EastAsia3 E_HG2

1 4 16% E_HG3 84% 1 4 16% E_HG3 84% 1 4 16% E_HG3 84%

Han EastAsia4 33% ANE Han EastAsia4 33% ANE Han EastAsia4 33% ANE

67% 95 67% 95 67% 94

FA Russia_MA1_HG_SG FA Russia_MA1_HG_SG FA Russia_MA1_HG_SG

10 10 10

Beringia Beringia Beringia

142 8 142 8 142 8

USA_Ancient_Beringian NA USA_Ancient_Beringian NA USA_Ancient_Beringian NA

117 6 117 6 117 6

ASO_SG NA2 ASO_SG NA2 ASO_SG NA2

81 1 81 1 73 0

CA_Islands CA CA_Islands CA PREC_6000BP NA3

1 1 81 1

SA SA CA_Islands SA

2 0 2 0 2 0

SA2 SA3 SA2 SA3 SA2 SA3

124 79 40 1 124 79 40 1 124 79 39 2

Peru_Cuncaicha_9000BP Lauricocha_8600BP Brazil_LapaDoSanto_9600BP SA4 Peru_Cuncaicha_9000BP Lauricocha_8600BP Brazil_LapaDoSanto_9600BP SA4 Peru_Cuncaicha_9000BP Lauricocha_8600BP Brazil_LapaDoSanto_9600BP SA4

125 0 60 0 59 124

Chile_LosRieles_5100BP Source Argentina_ArroyoSeco2_7700BP Source Argentina_ArroyoSeco2_7700BP Chile_LosRieles_5100BP

60 71 125 71

Argentina_ArroyoSeco2_7700BP PREC_6000BP Chile_LosRieles_5100BP PREC_6000BP

Figure S10: Admixture graph of Colombia_Checua_6000BP in multiple positions among the

modelled North and South American genetic lineages. Colombia_Checua_6000BP is here indicated
as “PREC_6000BP”. Each panel (A-J) shows a different branching of the Checua group from a fixed tree
scaffold topology.
 K=6
K=5

K=8
K=7
K=4
K=3
Karitiana Karitiana Karitiana Karitiana Karitiana Karitiana Karitiana

Piapoco Piapoco Piapoco Piapoco Piapoco Piapoco

Piapoco
Chono Chono Chono Chono Chono Chono
Chono

Kaqchikel Kaqchikel Kaqchikel Kaqchikel Kaqchikel Kaqchikel

Kaqchikel

Mixe Mixe Mixe Mixe Mixe Mixe Mixe

Hulliche Hulliche Hulliche Hulliche Hulliche Hulliche

Hulliche

Zapotec1 Zapotec1 Zapotec1 Zapotec1 Zapotec1 Zapotec1

Zapotec1

Jamamadi Jamamadi Jamamadi Jamamadi Jamamadi Jamamadi

Jamamadi Parakana Parakana Parakana Parakana Parakana Parakana

Parakana Kaingang Kaingang Kaingang Kaingang Kaingang Kaingang

Kaingang

Aymara Aymara Aymara Aymara Aymara Aymara Aymara

Chilote Chilote Chilote Chilote Chilote Chilote Chilote

Inga
Inga Inga Inga Inga Inga Inga

Ojibwa
Ojibwa Ojibwa Ojibwa Ojibwa Ojibwa Ojibwa

Huetar
Huetar Huetar Huetar Huetar Huetar Huetar

Cabecar Cabecar Cabecar Cabecar Cabecar Cabecar

Cabecar

Bribri Bribri Bribri Bribri Bribri Bribri

Bribri
Yaghan Yaghan Yaghan Yaghan Yaghan Yaghan Yaghan

Teribe Teribe Teribe Teribe Teribe Teribe Teribe

Yaqui Yaqui Yaqui Yaqui Yaqui Yaqui

Yaqui
Diaguita Diaguita Diaguita Diaguita Diaguita Diaguita
Diaguita
Maleku Maleku Maleku Maleku Maleku Maleku
Maleku
Wayuu
Wayuu Wayuu Wayuu Wayuu Wayuu Wayuu

Arhuaco Arhuaco Arhuaco Arhuaco Arhuaco Arhuaco

Arhuaco
Guaymi Guaymi Guaymi Guaymi Guaymi Guaymi Guaymi

Waunana Waunana Waunana Waunana Waunana Waunana

Waunana
Chorotega Chorotega Chorotega Chorotega Chorotega Chorotega

Chorotega Palikur Palikur Palikur Palikur Palikur Palikur

Palikur Ticuna Ticuna Ticuna Ticuna Ticuna Ticuna

Ticuna
Embera Embera Embera Embera Embera Embera

Venezuela on the 1240K. Analyses are ranging from K=3 to K=8.

Embera
Mixtec Mixtec Mixtec Mixtec Mixtec Mixtec Mixtec

Arara Arara Arara Arara Arara Arara

Arara Toba Toba Toba Toba Toba Toba

Toba
Guarani Guarani Guarani Guarani Guarani Guarani
Guarani
Kogi Kogi Kogi Kogi Kogi Kogi Kogi

Chane Chane Chane Chane Chane Chane

Chane
Wichi Wichi Wichi Wichi Wichi Wichi
Wichi
Algonquin Algonquin Algonquin Algonquin Algonquin Algonquin Algonquin

Purepecha Purepecha Purepecha Purepecha Purepecha Purepecha

Purepecha
Guahibo Guahibo Guahibo Guahibo Guahibo Guahibo Guahibo

Pima Pima Pima Pima Pima Pima

Pima

Quechua Quechua Quechua Quechua Quechua Quechua

Quechua

SuruiSurui Surui Surui Surui Surui Surui

Zapotec2 Zapotec2 Zapotec2 Zapotec2 Zapotec2 Zapotec2

Zapotec2

Maya1 Maya1 Maya1 Maya1 Maya1 Maya1 Maya1

Figure S11: Unsupervised ADMIXTURE analysis including present-day Native American populations. Modern
popualtions were genotyped on the Illumina panel, Reich et al. (23), and ancient individuals from Panama, Colombia and

Maya2 Maya2 Maya2 Maya2 Maya2 Maya2

Maya2

Tepehuano Tepehuano Tepehuano Tepehuano Tepehuano Tepehuano

Tepehuano

Panama_IsthmoColombian Panama_IsthmoColombian_PreColonial.SG Panama_IsthmoColombian_PreColonial.SG Panama_IsthmoColombian_PreColonial.SG Panama_IsthmoColombian_PreColonial.SG Panama_IsthmoColombian_PreColonial.SG Panama_IsthmoColombian_PreColonial.SG

_Precolonial
Venezuela_LasLocas_Ceramic Venezuela_LasLocas_Ceramic Venezuela_LasLocas_Ceramic Venezuela_LasLocas_Ceramic Venezuela_LasLocas_Ceramic Venezuela_LasLocas_Ceramic
Venezuela_LasLocas_Ceramic
Colombia_LasDelicias_1200BP Colombia_LasDelicias_1200BP Colombia_LasDelicias_1200BP Colombia_LasDelicias_1200BP Colombia_LasDelicias_1200BP Colombia_LasDelicias_1200BP
Colombia_LasDelicias_1200BP
Colombia_LagunadelaHerrera Colombia_LagunadelaHerrera_2000BP Colombia_LagunadelaHerrera_2000BP Colombia_LagunadelaHerrera_2000BP Colombia_LagunadelaHerrera_2000BP Colombia_LagunadelaHerrera_2000BP Colombia_LagunadelaHerrera_2000BP

_2000BP
Colombia_Purnia_530BP Colombia_Purnia_530BP Colombia_Purnia_530BP Colombia_Purnia_530BP Colombia_Purnia_530BP Colombia_Purnia_530BP
Colombia_Purnia_530BP Colombia_Soacha_520BP Colombia_Soacha_520BP Colombia_Soacha_520BP Colombia_Soacha_520BP Colombia_Soacha_520BP Colombia_Soacha_520BP

Colombia_Soacha_520BP
Colombia_Checua_6000BP Colombia_Checua_6000BP Colombia_Checua_6000BP Colombia_Checua_6000BP Colombia_Checua_6000BP Colombia_Checua_6000BP

Colombia_Checua_6000BP
 0.15
0.10
0.05
PC2

0.00
−0.05
−0.10

−0.005 0.000 0.005

PC1

Bribri Bahamas_LongIsl_Ceramic PuertoRico_LosIndios_Ceramic

Cabecar Bahamas_SouthAndros_Ceramic PuertoRico_Monserrate_Ceramic
Embera Cuba_CuevaEsqueletos_Ceramic PuertoRico_PasodelIndio_Ceramic
Guahibo Cuba_ElMorrillo_Ceramic PuertoRico_PuntaCandelero_Ceramic
Guaymi Dominican_Andres_Ceramic PuertoRico_SantaElena_Ceramic
Maleku Dominican_Atajadizo_Ceramic PuertoRico_Tibes_Ceramic
Piapoco Dominican_CuevaJuana_Ceramic StLucia_Lavoutte_Ceramic
Teribe Dominican_EdilioCruz_Ceramic Curacao_deSavaan_Ceramic
Waunana Dominican_ElFrances_Ceramic Curacao_SantaCruz_Ceramic
Wayuu Dominican_ElSoco_Ceramic Guadeloupe_AnseGourde_Ceramic
Panama_IsthmoColombian_PreColonial.SG Dominican_JuanDolio_Ceramic Cuba_CanimarAbajo_Archaic
Venezuela_LasLocas_Ceramic Dominican_LaCaleta_Ceramic Cuba_CuevaCalero_Archaic
Colombia_Soacha_520BP Dominican_LaUnion_Ceramic Cuba_CuevaPerico_Archaic
Colombia_Purnia_530BP Dominican_LomaPerenal_Ceramic Cuba_GuayaboBlanco_Archaic
Colombia_LasDelicias_1200BP Dominican_LosCorniel_Ceramic Cuba_LasCarolinas_Archaic
Colombia_LagunadelaHerrera_2000BP Dominican_LosMuertos_Ceramic Cuba_Manuelito_Archaic
Colombia_Checua_6000BP Dominican_Macao_Ceramic Cuba_PlayadelMango_Archaic
Bahamas_AbacoIsl_Ceramic PuertoRico_CaboRojo11_Ceramic Dominican_Andres_Archaic
Bahamas_CrookedIsl_Ceramic PuertoRico_CanasColloresMonserrate_Ceramic Dominican_CuevaRoja_Archaic
Bahamas_EleutheraIsl_Ceramic PuertoRico_Collores_Ceramic

Figure S12: Principal Component Analysis built with the genetic variation of a subset of
modern-day individuals from the unmasked and unadmixed Illumina dataset, Reich et al. (23).
Ancient individuals were projected onto the PCA.
 0.010
0.005
PC2

0.000
−0.005
−0.010

−0.015 −0.010 −0.005 0.000 0.005 0.010 0.015

PC1

PaNASO PaEMBERA Colombia_Purnia_530BP Venezuela_LasLocas_Ceramic

PaBRIBRI PaGUNA Colombia_Soacha_520BP Colombia_LasDelicias_1200BP
PaNGABE Colombia_LagunadelaHerrera_2000BP Colombia_Checua_6000BP Panama_IsthmoColombian_PreColonial.SG

Figure S13: Principal Component Analysis built with the genetic variation of unadmixed
modern-day Panamanian individuals genotyped on the Human Origins panel, Capodiferro et
al. (20). Ancient individuals were projected onto the built PCA.
 USA_Ancient_Beringian

USA_Ancient_Beringian

Dominican_LaCaleta_Ceramic

Venezuela_LasLocas_Ceramic_1

Panama_IsthmoColombian

Panama Migration
IsthmoColombian
weight

Migration 0.5

weight
Venezuela_LasLocas_Ceramic_2
0.5

Dominican_LaCaleta_Ceramic

0 Cuba_PlayadelMango_Archaic
0 Colombia_Checua_6000BP

Colombia_Checua_6000BP
10 s.e.
Cuba_PlayadelMango_Archaic
10 s.e.

0.00 0.02 0.04 0.06 0.08 0.00 0.02 0.04 0.06 0.08

Drift parameter Drift parameter

USA_Ancient_Beringian
Panama_IsthmoColombian

Cuba_PlayadelMango_Archaic
Venezuela_LasLocas_Ceramic_1

Dominican_LaCaleta_Ceramic
Dominican_LaCaleta_Ceramic
Migration

Migration weight

weight 0.5

0.5
Panama_IsthmoColombian
Cuba_PlayadelMango_Archaic

0 Venezuela_LasLocas_Ceramic_2
0 Colombia_Checua_6000BP

USA_Ancient_Beringian Colombia_Checua_6000BP
10 s.e. 10 s.e.

0.00 0.02 0.04 0.06 0.08 0.00 0.02 0.04 0.06 0.08

Drift parameter Drift parameter

Panama_IsthmoColombian
USA_Ancient_Beringian

Dominican_LaCaleta_Ceramic
Cuba_PlayadelMango
_Archaic

Panama_IsthmoColombian Venezuela_LasLocas_Ceramic_2

Migration
Migration

weight weight

0.5 0.5

Venezuela_LasLocas_Ceramic_1 Cuba_PlayadelMango_Archaic

0 Dominican_LaCaleta_Ceramic 0 Colombia_Checua_6000BP

Colombia_Checua
10 s.e. _6000BP USA_Ancient_Beringian
10 s.e.
0.00 0.02 0.04 0.06 0.08 0.10
0.00 0.02 0.04 0.06 0.08
Drift parameter
Drift parameter
USA_Ancient_Beringian
Panama_IsthmoColombian

Venezuela_LasLocas_Ceramic_2
Venezuela_LasLocas_Ceramic_1

Panama_IsthmoColombian
Dominican_LaCaleta_Ceramic
Migration

Migration
weight

weight
0.5
0.5
Dominican_LaCaleta_Ceramic
Cuba_PlayadelMango_Archaic

0 Colombia_Checua_6000BP
0 Cuba_PlayadelMango_Archaic

USA_Ancient_Beringian Colombia_Checua_6000BP
10 s.e. 10 s.e.

0.00 0.02 0.04 0.06 0.08 0.00 0.02 0.04 0.06 0.08

Drift parameter Drift parameter

Figure S14: Treemix analysis of the Colombia_Checua_6000BP group, including ancient

individuals from Panama and Ceramic-age Venezuela. Trees have been constructed using USR1
as an outgroup and one representative of Ceramic-age and Archaic-age Caribbean populations. Due to
their heterogenous genetic profile, Ceramic-age Venezuelan individuals were subdivided into two
groups, namely Venezuela_LasLocas_Ceramic_1 (left) and Venezuela_LasLocas_Ceramic_2 (right).
Trees are reported from 0 to 3 admixture edges (from top to bottom).
 USA_Ancient_Beringian
USA_Ancient_Beringian

Cuba_PlayadelMango_Archaic
Venezuela_LasLocas_Ceramic_1

Dominican_LaCaleta_Ceramic
Colombia_LagunadelaHerrera_2000BP
Migration
Migration
weight
weight
0.5
0.5
Panama_IsthmoColombian Colombia_LagunadelaHerrera_2000BP

0 Venezuela_LasLocas_Ceramic_2
0 Cuba_PlayadelMango_Archaic

Panama_IsthmoColombian
Dominican_LaCaleta_Ceramic 10 s.e.
10 s.e.
0.00 0.02 0.04 0.06 0.08 0.10
0.00 0.02 0.04 0.06 0.08 0.10
Drift parameter
Drift parameter

Dominican_LaCaleta_Ceramic USA_Ancient_Beringian

Colombia_LagunadelaHerrera_2000BP
Venezuela_LasLocas_Ceramic_1

Panama_IsthmoColombian Panama_IsthmoColombian

Migration Migration

weight weight

0.5 0.5

Colombia_LagunadelaHerrera_2000BP Venezuela_LasLocas_Ceramic_2

0 Cuba_PlayadelMango_Archaic 0 Cuba_PlayadelMango_Archaic

USA_Ancient_Beringian Dominican_LaCaleta_Ceramic
10 s.e. 10 s.e.

0.00 0.02 0.04 0.06 0.08 0.10 0.00 0.02 0.04 0.06 0.08 0.10

Drift parameter Drift parameter

USA_Ancient_Beringian
USA_Ancient_Beringian

Colombia_LagunadelaHerrera_2000BP Dominican_LaCaleta_Ceramic

Panama_IsthmoColombian Venezuela_LasLocas_Ceramic_2

Migration Migration

weight weight

0.5
0.5
Panama_IsthmoColombian
Venezuela_LasLocas_Ceramic_1

0 Cuba_PlayadelMango_Archaic 0 Colombia_LagunadelaHerrera_2000BP

Dominican_LaCaleta_Ceramic Cuba_PlayadelMango_Archaic
10 s.e. 10 s.e.

0.00 0.02 0.04 0.06 0.08 0.10 0.00 0.02 0.04 0.06 0.08 0.10 0.12

Drift parameter Drift parameter

Venezuela_LasLocas_Ceramic_2

Colombia_LagunadelaHerrera_2000BP
Panama_IsthmoColombian

Panama_IsthmoColombian

Cuba_PlayadelMango_Archaic

Venezuela_LasLocas_Ceramic_1 Migration

weight

Migration 1

weight
Dominican_LaCaleta_Ceramic
0.5

Dominican_LaCaleta_Ceramic

0
Colombia_LagunadelaHerrera_2000BP
0 Cuba_PlayadelMango_Archaic

USA_Ancient_Beringian
USA_Ancient_Beringian
10 s.e.
10 s.e.

0.00 0.02 0.04 0.06 0.08 0.10 0.00 0.02 0.04 0.06 0.08 0.10

Drift parameter Drift parameter

Figure S15: Treemix analysis of the Colombia_LagunadelaHerrera_2000BP group, including

ancient individuals from Panama and Ceramic-age Venezuela. Trees have been constructed using
USR1 as an outgroup and one representative of Ceramic-age and Archaic-age Caribbean populations.
Due to their heterogenous genetic profile, Ceramic-age Venezuelan individuals were subdivided into
two groups, namely Venezuela_LasLocas_Ceramic_1 (left) and Venezuela_LasLocas_Ceramic_2
(right). Trees are reported from 0 to 3 admixture edges (from top to bottom).
 USA_Ancient_Beringian USA_Ancient_Beringian

Cuba_PlayadelMango_Archaic Colombia_LasDelicias_1200BP

Dominican_LaCaleta_Ceramic Dominican_LaCaleta_Ceramic

Migration Migration

weight weight

0.5 0.5

Venezuela_LasLocas_Ceramic_1 Cuba_PlayadelMango_Archaic

Panama_IsthmoColombian 0
Panama_IsthmoColombian
0

Colombia_LasDelicias_1200BP Venezuela_LasLocas_Ceramic_2
10 s.e. 10 s.e.

0.00 0.02 0.04 0.06 0.08 0.10 0.00 0.02 0.04 0.06 0.08 0.10

Drift parameter Drift parameter

Dominican_LaCaleta_Ceramic USA_Ancient_Beringian

Colombia_LasDelicias_1200BP Colombia_LasDelicias_1200BP

Panama_IsthmoColombian
Cuba_PlayadelMango_Archaic

Migration
Migration
weight
weight

0.5
0.5

Venezuela_LasLocas_Ceramic_1 Venezuela_LasLocas_Ceramic_2

Panama_IsthmoColombian 0
Dominican_LaCaleta_Ceramic
0

USA_Ancient_Beringian Cuba_PlayadelMango_Archaic
10 s.e. 10 s.e.

0.00 0.02 0.04 0.06 0.08 0.10 0.00 0.02 0.04 0.06 0.08 0.10

Drift parameter Drift parameter

Cuba_PlayadelMango_Archaic
Dominican_LaCaleta_Ceramic

Dominican_LaCaleta_Ceramic
Cuba_PlayadelMango_Archaic

Colombia_LasDelicias_1200BP
Panama_IsthmoColombian
Migration

Migration weight

weight 1

0.5
USA_Ancient_Beringian
Colombia_LasDelicias_1200BP

0
Panama_IsthmoColombian
0
Venezuela_LasLocas_Ceramic_1

USA_Ancient_Beringian Venezuela_LasLocas_Ceramic_2
10 s.e. 10 s.e.

0.00 0.02 0.04 0.06 0.08 0.10 0.00 0.02 0.04 0.06 0.08

Drift parameter Drift parameter

USA_Ancient_Beringian USA_Ancient_Beringian

Panama_IsthmoColombian
Colombia_LasDelicias_1200BP

Panama_IsthmoColombian
Colombia_LasDelicias_1200BP

Migration Migration

weight weight

0.5 0.5

Dominican_LaCaleta_Ceramic Venezuela_LasLocas_Ceramic_2

0 Cuba_PlayadelMango_Archaic 0
Dominican_LaCaleta_Ceramic

Venezuela_LasLocas_Ceramic_1 Cuba_PlayadelMango_Archaic
10 s.e. 10 s.e.

0.00 0.02 0.04 0.06 0.08 0.10 0.00 0.02 0.04 0.06 0.08 0.10

Drift parameter Drift parameter

Figure S16: Treemix analysis of the Colombia_LasDelicias_1200BP group, including ancient

individuals from Panama and Ceramic-age Venezuela. Trees have been constructed using USR1
as an outgroup and one representative of Ceramic-age and Archaic-age Caribbean populations. Due to
their heterogenous genetic profile, Ceramic-age Venezuelan individuals were subdivided into two
groups, namely Venezuela_LasLocas_Ceramic_1 (left) and Venezuela_LasLocas_Ceramic_2 (right).
Trees are reported from 0 to 3 admixture edges (from top to bottom).
 USA_Ancient_Beringian USA_Ancient_Beringian

Dominican_LaCaleta_Ceramic Cuba_PlayadelMango_Archaic

Cuba_PlayadelMango_Archaic Venezuela_LasLocas_Ceramic_2

Migration Migration

weight weight

0.5 0.5

Venezuela_LasLocas_Ceramic_1 Panama_IsthmoColombian

0
Panama_IsthmoColombian 0 Colombia_Purnia_530BP

Colombia_Purnia_530BP Dominican_LaCaleta_Ceramic
10 s.e. 10 s.e.

0.00 0.02 0.04 0.06 0.08 0.00 0.02 0.04 0.06 0.08

Drift parameter Drift parameter

USA_Ancient_Beringian USA_Ancient_Beringian

Dominican_LaCaleta_Ceramic Colombia_Purnia_530BP

Cuba_PlayadelMango_Archaic Panama_IsthmoColombian

Migration
Migration

weight
weight

0.5
0.5

Panama_IsthmoColombia Venezuela_LasLocas_Ceramic_2

0 Colombia_Purnia_530BP 0
Dominican_LaCaleta_Ceramic

Venezuela_LasLocas_Ceramic_1 Cuba_PlayadelMango_Archaic
10 s.e.
10 s.e.

0.00 0.02 0.04 0.06 0.08 0.00 0.02 0.04 0.06 0.08

Drift parameter Drift parameter

USA_Ancient_Beringian Dominican_LaCaleta_Ceramic

Panama_IsthmoColombian Colombia_Purnia_530BP

Colombia_Purnia_530BP Cuba_PlayadelMango_Archaic

Migration Migration

weight weight

0.5 1

Dominican_LaCaleta_Ceramic USA_Ancient_Beringian

0 Cuba_PlayadelMango_Archaic 0 Panama_IsthmoColombian

Venezuela_LasLocas_Ceramic_1 Venezuela_LasLocas_Ceramic_2
10 s.e. 10 s.e.

0.00 0.02 0.04 0.06 0.08 0.00 0.02 0.04 0.06 0.08

Drift parameter Drift parameter

Panama_IsthmoColombian USA_Ancient_Beringian

Colombia_Purnia_530BP Venezuela_LasLocas_Ceramic_2

Dominican_LaCaleta_Ceramic Panama_IsthmoColombian

Migration Migration

weight weight

0.5 0.5

Venezuela_LasLocas_Ceramic_1 Colombia_Purnia_530BP

0 Cuba_PlayadelMango_Archaic 0 Cuba_PlayadelMango_Archaic

USA_Ancient_Beringian Dominican_LaCaleta_Ceramic
10 s.e. 10 s.e.

0.00 0.02 0.04 0.06 0.08 0.00 0.02 0.04 0.06 0.08

Drift parameter Drift parameter

Figure S17: Treemix analysis of the Colombia_Purnia_530BP group, including ancient

individuals from Panama and Ceramic-age Venezuela. Trees have been constructed using USR1
as an outgroup and one representative of Ceramic-age and Archaic-age Caribbean populations. Due to
their heterogenous genetic profile, Ceramic-age Venezuelan individuals were subdivided into two
groups, namely Venezuela_LasLocas_Ceramic_1 (left) and Venezuela_LasLocas_Ceramic_2 (right).
Trees are reported from 0 to 3 admixture edges (from top to bottom).
 USA_Ancient_Beringian USA_Ancient_Beringian

Venezuela_LasLocas_Ceramic Cuba_PlayadelMango_Archaic

Cuba_PlayadelMango_Archaic Dominican_LaCaleta_Ceramic

Migration Migration

weight weight

0.5 0.5
Colombia_Soacha_520BP
Dominican_LaCaleta_Ceramic

Colombia_Soacha_520BP
0 0 Panama_IsthmoColombian

Panama_IsthmoColombian Venezuela_LasLocas_Ceramic_2
10 s.e. 10 s.e.

0.00 0.02 0.04 0.06 0.08 0.10 0.12 0.00 0.02 0.04 0.06 0.08 0.10 0.12

Drift parameter Drift parameter

USA_Ancient_Beringian

USA_Ancient_Beringian

Panama_IsthmoColombian

Dominican_LaCaleta_Ceramic

Venezuela_LasLocas_Ceramic_1

Migration
Cuba_PlayadelMango_Archaic
weight

Migration
0.5
weight
Cuba_PlayadelMango_Archaic
0.5
Colombia_Soacha_520BP

0 Dominican_LaCaleta_Ceramic
0
Venezuela_LasLocas_Ceramic_2

Colombia_Soacha_520BP
10 s.e.

0.00 0.02 0.04 0.06 0.08 0.10 0.12

Panama_IsthmoColombian
10 s.e.

Drift parameter 0.00 0.02 0.04 0.06 0.08 0.10 0.12

Drift parameter

Dominican_LaCaleta_Ceramic Dominican_LaCaleta_Ceramic

Colombia_Soacha_520BP
Colombia_Soacha_520BP

Cuba_PlayadelMango_Archaic Venezuela_LasLocas_Ceramic_2

Migration Migration

weight weight

0.5 0.5

Panama_IsthmoColombian Panama_IsthmoColombian_PreColonial

0
Venezuela_LasLocas_Ceramic_1 0
Cuba_PlayadelMango_Archaic

USA_Ancient_Beringian USA_Ancient_Beringian
10 s.e. 10 s.e.

0.00 0.02 0.04 0.06 0.08 0.10 0.12 0.00 0.02 0.04 0.06 0.08

Drift parameter Drift parameter

Venezuela_LasLocas_Ceramic_1 Cuba_PlayadelMango_Archaic

Panama_IsthmoColombian Venezuela_LasLocas_Ceramic_2

Dominican_LaCaleta_Ceramic Panama_IsthmoColombian

Migration
Migration

weight
weight

0.5
0.5 Colombia_Soacha_520BP
Colombia_Soacha_520BP

0 Cuba_PlayadelMango_Archaic
0
Dominican_LaCaleta_Ceramic

USA_Ancient_Beringian USA_Ancient_Beringian
10 s.e.
10 s.e.

0.00 0.02 0.04 0.06 0.08

0.00 0.02 0.04 0.06 0.08
Drift parameter
Drift parameter

Figure S18: Treemix analysis of Colombia_Soacha_520BP, including ancient individuals from

Panama and Ceramic-age Venezuela. Trees have been constructed using USR1 as an outgroup and
one representative of Ceramic-age and Archaic-age Caribbean populations. Due to their heterogenous
genetic profile, Ceramic-age Venezuelan individuals were subdivided into two groups, namely
Venezuela_LasLocas_Ceramic_1 (left) and Venezuela_LasLocas_Ceramic_2 (right). Trees are
reported from 0 to 3 admixture edges (from top to bottom).
 Panam a_Isthm ocolom bi
an_Precolonial

Panam a_Isthm ocolom bian_Precolonial

Colom bia_Soacha_520BP

Colom bia_Soacha_520BP

Colom bia_Purnia_530BP

Panam a_Isthm ocolom bi Panam a_Isthm ocolom bian_Precolonial

an_Precolonial

Colom bia_Purnia_530BP

Colom bia_LasDelicias_1200BP

Venezuelas_LasLocas_Ceram ic2

Venezuelas_LasLocas_Ceram ic2 Panam a_Isthm ocolom bian_Precolonial

Panam a_Isthm ocolom bian

_Precolonial

Colom bia_LasDelicias_1200BP

Panam a_Isthm ocolom bian_

Precolonial

Venezuelas_LasLocas_Ceram ic2

Panam a_Isthm ocolom bian_

Precolonial

Venezuelas_LasLocas_Ceram ic2

Figure S19: Treemix analysis of ancient Colombians, including ancient individuals from
Panama and both Ceramic-age Venezuela groups (Venezuela_LasLocas_Ceramic_1 and
Venezuela_LasLocas_Ceramic_2). Trees have been constructed using USR1 as an outgroup and one
representative of Ceramic-age and Archaic-age Caribbean populations. Trees are reported for all four
post-2000BP ancient Colombian groups, and from 0 to 1 admixture edges (left and right panels,
respectively).
 A
f3(Mbuti; X, Colombia_Checua_6000BP)
B f3(Mbuti; X, Colombia_LagunadelaHerrera_2000BP) C f3(Mbuti; X, Colombia_LasDelicias_1200BP)
10

10
10
0.395 0.406 0.407
5

5
5
Latitude

Latitude
Latitude
0

0
0
0.331 0.331 0.331
−5

−5
−5

−85 −80 −75 −70 −85 −80 −75 −70 −85 −80 −75 −70

Longitude Longitude Longitude

f3(Mbuti; X, Colombia_Purnia_530BP) f3(Mbuti; X, Colombia_Soacha_520BP)

D E
10

10

0.41 0.41
5

5
Latitude

Latitude
0

0.332 0.332
−5

−5

−85 −80 −75 −70 −85 −80 −75 −70

Longitude Longitude

Figure S20: Map with f3-outgroup statistics measuring the shared genetic drift of ancient
Colombian individuals with present-day Indigenous populations from Panama and Costa
Rica. The Costa Rican populations were genotyped on the Illumina panel, Reich et al. (23), and the
Panamanian and Colombian populations were genotyped on the Human Origins panel, Capodiferro
et al. (20) and Arias et al. (60). Each panel (A-E) reports the f3-outgroup statistics of present-day
populations (indicated with colored circles) with different ancient groups analyzed in this study
(indicated with a black triangle).
 f4(Mbuti;Colombia_Checua_6000BP, Cabecar, X) f4(Mbuti;Colombia_LasDelicias_1200BP, Cabecar, X)
Algonquin Algonquin
Ojibwa Ojibwa
Pima Yaqui
Yaqui Pima
Purepecha Purepecha
Tepehuano Tepehuano
Zapotec1 Mixe
Mixe Zapotec2
Zapotec2 Mixtec
Mixtec Zapotec1
Arhuaco Maya2
Kogi Maya1
Maya1 Chilote
Maya2 Yaghan
Ticuna Jamamadi
Kaqchikel Hulliche
Embera Quechua
Huetar Aymara
Waunana Kaqchikel
Bribri Chono
Guaymi Piapoco
Maleku Parakana
Piapoco Guarani
X

X
Chorotega Diaguita
Kaingang Chane
Parakana Karitiana
Teribe Toba
Jamamadi Wichi
Inga Palikur
Wayuu Surui
Karitiana Kaingang
Toba Ticuna
Chilote Guahibo
Yaghan Arhuaco
Guarani Kogi
Hulliche Arara
Guahibo Wayuu
Diaguita Embera
Palikur Inga
Quechua Waunana
Surui Chorotega
Chono Huetar
Aymara Bribri
Wichi Maleku
Chane Guaymi
Arara Teribe
−0.008 −0.006 −0.004 −0.002 0.000 0.002 −0.012 −0.008 −0.004 0.000
f4 f4

f4(Mbuti;Colombia_Purnia_530BP, Cabecar, X) f4(Mbuti;Colombia_Soacha_520BP, Cabecar, X)

Algonquin Algonquin
Ojibwa Ojibwa
Pima Pima
Purepecha Yaqui
Tepehuano Purepecha
Zapotec1 Tepehuano
Mixe Mixtec
Zapotec2 Zapotec1
Yaqui Zapotec2
Mixtec Mixe
Kaingang Maya2
Yaghan Hulliche
Hulliche Maya1
Maya1 Yaghan
Maya2 Chilote
Parakana Chane
Diaguita Kaqchikel
Chane Palikur
Karitiana Parakana
Aymara Jamamadi
Kaqchikel Chono
Guahibo Guarani
Quechua Quechua
X

Toba Aymara
Piapoco Diaguita
Chilote Guahibo
Guarani Karitiana
Palikur Piapoco
Arara Ticuna
Chono Wichi
Wichi Kaingang
Ticuna Toba
Inga Surui
Surui Arara
Jamamadi Inga
Arhuaco Embera
Chorotega Waunana
Embera Kogi
Wayuu Chorotega
Waunana Arhuaco
Kogi Wayuu
Huetar Huetar
Maleku Maleku
Guaymi Guaymi
Bribri Teribe
Teribe Bribri
−0.010 −0.005 0.000 −0.012 −0.008 −0.004 0.000
f4 f4

Figure S21: f4-statistics to test the relative affinity of ancient Colombian individuals
compared to Cabecar and other present-day Native American populations. All populations
were genotyped on the Illumina dataset, Reich et al. (23). Tests are reported with 1 SE and red
symbols indicate Z-scores below -3.
Captions for Supplementary tables

Table S1: Colombian dataset. Meta data of ancient Colombian individuals analyzed in this
study.

Table S2.A: f4-outgroup-statistics exploring potential within-group asymmetric

relationships. Ancient Colombian populations are compared with ancient populations
available in the 1240K.

Table S2.B: f4-outgroup-statistics exploring potential asymmetric relationships within

ancient Colombian groups. Ancient Colombian populations are compared with present-day
Indigenous populations available in the Illumina dataset Reich et al. (23).

Table S3.A: f4-outgroup statistic describing the affinity of Colombia_Checua_6000BP

to Anzick-1. This table shows the results of testing the allele sharing between
Colombia_Checua_6000BP and the Anzick-1 individual.

Table S3.B: f4-outgroup statistic describing the affinity of Colombia_Checua_6000BP

to the California Channel Islands. Results showcase the allele sharing between both
populations.

Table S3.C: f4-outgroup statistic exploring Colombia_Checua's relationship with

ancient individuals available in the 1240K panel. This table contains comparisons with all
available Native American populations without prior pre-selection.

Table S3.D: f4-statistic describing Colombia_Checua's relationship with modern-day

Indigenous individuals. Populations were not pre-selected and are available in the masked
Illumina dataset, Reich et al (23).

Table S3.E: f4-outgroup-statistics exploring potential excess affinity of

Colombia_Checua_6000BP to Indigenous individuals. All populations are available in the
1240K v54.

Tabel S3.F: f4-outgroup-statistics describing the relationship between

Colombia_checua_6000BP and post-2000BP ancient Colombians. Individuals have
been grouped by site.

Tabel S3.G: f4-outgroup-statistics exploring the relationship between post-2000BP

ancient Colombians, Colombia_Checua_6000BP and Indigenous populations availabe
in the 1240K. Results show the allele sharing between these populations. No pre-selection
of 1240K populations took place.

Tabel S3.H: f4-outgroup-statistics exploring the relationship between

Colombia_Checua_6000BP, post-2000BP ancient Colombians and present-day
Indigenous populations. Within all populations European and/or African ancestry has been
masked. Populations are available in the Illumina dataset, Reich et al. (23).

Table S4.A: f4-outgroup-statistics exploring the relationship of ancient Colombians

with present-day Indigenous populations of the Americas. Non-Indigenous ancestry as
been masked and populations are available in the Illumina dataset (Reich et al. (23)).
Table S4.B: f4-outgroup-statistics exploring the relationship between ancient
Colombian populations and present-day Indigenous populations (Reich et al. (23)). All
populations are available in the masked version of the Illumina dataset.

Table S4.C: f4-outgroup-statistics showing the relationship between ancient

Colombian populations, present-day Indigenous Colombians (Arias et al. (60)), and
present-day Indigenous populations in the Illumina dataset (Reich et al 2012 (23)). All
non-Native American ancestry was masked by the respective authors prior to publication.

Table S4.D: f4-outgroup-statistics exploring the relationship of ancient Colombians

and ancient Indigenous populations of the Americas. Results show asymmetric allele
sharing of ancient Colombian populations through time.

Table S4.E: f4-outgroup-statistics describing the relationship between ancient

Panamanians and ancient Colombians compared to Indigenous populations of the
1240K. Populations from the 1240K have not been preselected and results suggest affinity
of post-2000BP Colombians to ancient Isthmians.

Table S4.F: f4-outgroup-statistics describing the relationship between ceramic-age

Venezuelans and ancient Colombians compared to ancient Indigenous populations of
the 1240K. Ceramic-age Venezuelans have been subdevided into two groups by their
respective affinities to ancient Panamanians with Venezuela_LasLocas_Ceramic2 exhibiting
higher affinity to ancient Panamanians.

Table S4.G: f4-outgroup-statistics describing the relationship between ancient

Indigenous populations of Colombia, ceramic-age Venezuelans, and ancient
Panamanians. The test has been conducted for two set ups of ceramic-age Venezuelan
grouping; 1) no sub-division, 2) subdivision based on Panamanian affinity.

Table S4.H: f4-outgroup-statistics highlighting the relationship between ancient

Indigenous populations of Colombia, ceramic-age Venezuelans, and modern Isthmian
populations. Within modern populations non-Native American ancestry has been masked.
Venezuelan populations have been subdivided based on their affinity to ancient Isthmian
populations.

Table S4.I: f4-outgroup-statistics showcasing the relatinship between ancient

Indigenous populations of Colombia, ceramic-age Venezuelans, and ancient
Indigenous Caribbeans. Venezuelans have been subdivided into their respective groups
based on affinity to ancient Panamanians. This test uses all available ceramic and archaic
Caribbean populations.

Table S4.J: f4-outgroup-statistics describing the allele sharing between ancient

Indigenous populations of Colombia, ancient Panamanians, and ancient Indigenous
Caribbeans. This test utilizes all available ceramic and archaic Caribbean populations of the
1240K v54.

Table S5.A: qpWave analysis. This analysis is testing whether Colombia_Checua_6000BP

and Colombia_LagunadelaHerrera_2000BP can be modeled as one ancestry wave.

Table S5.B: qpWave analysis. This test is exploring whether Colombia_Checua_6000BP

and Colombia_LasDelicias_1200BP can be modeled as one ancestry wave.

Table S5.C: qpWave analysis. This analysis is testing whether Colombia_Checua_6000BP

and Colombia_Purnia_530BP can be modeled as coming from the same ancestry wave.
Table S5.D: qpWave analysis. This test is rejecting H0 for Colombia_Checua_6000BP and
Colombia_Soacha_520BP.

Table S5.E: qpWave analysis. These results are proving that H0 can not be rejected for
Colombia_LagunadelaHerrera_2000BP and Colombia_LasDelicias_1200BP.

Table S5.F: qpWave analysis. For Colombia_LagunadelaHerrera_2000BP and

Colombia_Purnia_530BP a one wave model cannot be rejected.

Table S5.G: qpWave analysis. qpWave is suggesting a potential one-wave model for
Colombia_LagunadelaHerrera_2000BP and Colombia_Soacha_520BP.

Table S5.H: qpWave analysis. For Colombia_LasDelicias_1200BP and

Colombia_Purnia_530BP we are assuming a one wave model.

Table S5.I: qpWave analysis. We are testing whether Colombia_LasDelicias_1200BP and

Colombia_Soacha_520BP can be fitted within a one wave model.

Table S5.J: qpWave analysis. We are testing whether Colombia_Purnia_530BP and

Colombia_Soacha_520BP can be fit withing the validity of a one wave model.

Table S6.A: f4-outgroup-statistics describing the relationship between ceramic-age

Venezuelans and ancient Colombians. Ancient Venezuelans have been subdivided into
Venezuela_LasLocas_Ceramic and Venezuela_LasLocas_Ceramic2 in regards to their
affinity to ancient Panamanians with the Venezuela_LasLocas_Ceramic2 showing more
allele sharing with Panama.

Table S6.B: f4-outgroup-statistics exploring the asymmetrical relationship of ceramic-

age Venezuelans to ancient Panamanians. Individuals of ceramic age Venezuela have
been numbered from one to eight as a unique identifier. The results show that Venezuela 6,
5, and 8 share more affinity to ancient Panamanians.

Table S6.C: f4-outgroup-statistics exploring the asymmetrical allele sharing of

ceramic-age Venezuelans and ancient Indigenous populations within the 1240K.
Populations within the 1240K have not been pre-selected to avoid bias in analysis.

Table S6.D: f4-outgroup-statistics exploring the asymmetrical relationship of grouped

ceramic-age Venezuelans to ancient Panamanians. The three Venezuelans that show
more affinity to ancient Panamanians have been grouped into
Venezuela_LasLocas_Ceramic2. Results confirm their excess allele sharing with Panama
compared to Venezuela_LasLocas_Ceramic.

Table S6.E: f4-outgroup-statistics showing the relationship of ceramic-age

Venezuelans, ancient Colombians and Indigenous populations of the 1240K. This
analysis utilizes an ancient African captured outgroup, which was chosen based on number
of available individuals to yield the highest coverage possible. This analysis inquires whether
there is a sequencing protocol bias and/or 1240K batch effect.

Table S6.F: f4-outgroup-statistics describing the relationship of two ceramic-age

Venezuelan groups and Indigenous individuals of the 1240K. We are mplementing a
captured ancient African outgroup to correct for sequencing protocols and 1240K batch
effect.
Table S6.G: f4-outgroup-statistics exploring the asymmetrical relationship of ceramic-
age Venezuelans to ancient Panamanians. Using a captured ancient African outgroup
corrcts for sequencing protocol biases.

Table S6.H: f4-outgroup-statistics exploring the relationship of ancient Panamanians,

ancient Colombians and Indigenous populations of the 1240K. With a captured ancient
African outgroup we account for sequencing procotol biases.

Table S6.I: f4-outgroup-statistics highlighting the relationship of ancient Colombians

and Indigenous populations of the 1240K. Through implementing a captured ancient
African outgroup we correct for sequencing protocol biases and 1240K batch effect.

Table S7.A: f4-outgroup-statistics describing the relationship between ancient

Colombians, present-day and ancient populations from the Isthmus. Present-day
populations have been selected from the masked Illumina dataset from Reich et al. (23).

Table S7.B: f4-outgroup-statistic exploring the allele sharing between different ancient
Colombians to modern-day Indigenous Native Americans. All populations are available
in the Illumina dataset without pre-selection and non_Native ancestry has been masked by
Reich et al. (23).

Table S7.C: f4-outgroup-statistic exploring the allele sharing between ancient

Colombians and modern-day Indigenous Native Americans from the whole of
Colombia (Arias et al. (60)). Modern-day Colombians have been genotpyed on the HO and
non-Native American ancestry has been masked prior to publication.

Table S7.D: f4-outgroup-statistic exploring the relationship of ancient Colombians,

present-day Indigenous people (Reich et al. (23)) and modern-day Indigenous Native
Americans from Colombia (Arias et al. (60)). All non-Native ancestry was maksed in the
repsective presentday datasets.

Table S7.E: f4-outgroup-statistics describing the allele sharing between present-day

people from Costa Rica and ancient Colombians. Costa Ricans are part of the masked
Illumina dataset from Reich et al. (23).

Table S7.F: f4-outgroup-statistic showing the allele sharing between different ancient
Colombians and modern-day Indigenous Native Americans available in the Illumina
dataset (Reich et al. (23)). Non-native ancestry was masked and no pre-selection pf
specific populations took place.

Table S7.G: f4-outgroup-statistic exploring the relationship between ancient

Colombians and present-day Indigenous peoples from Colombia (Arias et al. (60)) and
the Isthmus (Reich et al. (23)). Modern-day individuals form Colombia were genotyped on
the HO, Isthmian populations on the Illumina panel. Non-Native ancestry was masked.

Table S7.H: f4-outgroup-statistics comparing ancient Colombians with present-day

Indigenous Colombians available in the Illumina dataset (Reich et al. (23)). Present-day
Colombians were taken from the masked Illumina panel.

Table S7.I: f4-outgoup-statistics comparing post-2000BP Colombians to Cabecar and

present-day Indigenous populations (Reich et al. (23)). Cabecar was chosen based on
previous f4-statistic showing the highest allele sharing with ancient Colombians.
Table S7.J: f4-outgroup-statistics comparing Isthmian populations to present-day
Indigenous populations (Reich et al. (23)). All populations are from the masked Illumina
dataset.

Table S7.K: f4-outgroup-statistics comparing North Colombian Indigenous

populations and Isthmian populations (Reich et al. (23)) against ancient Colombains.
All present-day Indigenous populations had their non-Native ancestry masked.

Table S8.A: 1240K meta dataset used in this study. The dataset, version v54, has been
restricted to the individuals and populations used in this study.

Table S.8.B: Meta data of present-day Indigenous Panamanians genotyped on the HO.
The table was adapted from its respective publication. Data was restricted to individuals
used in this study’s analyses.

Table S.8.C: Meta data of present-day Indigenous Americans in the Illumina dataset.
Native American ancestry has been masked prior to publication by Reich et al. (23). The
data table has been adapted from Reich et al. (23) and Capodiferro et al. (20).

Table S.8.D: Meta data of unadmixed present-day Indigenous Americans in the

Illumina dataset. This dataset only contains individuals without non-Native ancestry.

Table S.8.E: Meta data of present-day Indigenous Colombians genotyped on the HO.
Data has been provided by Arias et al. (60).
Auxiliary Files:
Supplementary Table S1: Meta data table for the generated ancient Colombian genomes.
Supplementary Table S2: f4-outgroup statistics for inter- and intra-site affinities
Supplementary Table S3: f4-outgroup statistics for Colombia_Checua_6000BP
Supplementary Table S4: f4-outgroup statistics for post-2000P ancient Colombians
Supplementary Table S5: qpWave-analyses of ancient Colombians
Supplementary Table S6: Comparative f4-outgroup statistics for
Venezuela_LasLocas_Ceramic and ancient Colombians
Supplementary Table S7: f4-outgroup statistics between ancient Colombians and present-
day Indigenous populations
Supplementary Table S8: Comparative meta data used in this study
 REFERENCES AND NOTES

1. M. Raghavan, M. Steinrücken, K. Harris, S. Schiffels, S. Rasmussen, M. De Giorgio, A.

Albrechtsen, C. Valdiosera, M. C. Ávila-­Arcos, A.-­S. Malaspinas, A. Eriksson, I. Moltke, M.
Metspalu, J. R. Homburger, J. Wall, O. E. Cornejo, J. V. Moreno-­Mayar, T. S. Korneliussen, T.
Pierre, M. Rasmussen, P. F. Campos, P. de Barros Damgaard, M. E. Allentoft, J. Lindo, E.
Metspalu, R. Rodríguez-­Varela, J. Mansilla, C. Henrickson, A. Seguin-­Orlando, H.
Malmström, T. Stafford Jr., S. S. Shringarpure, A. Moreno-­Estrada, M. Karmin, K. Tambets, A.
Bergström, Y. Xue, V. Warmuth, A. D. Friend, J. Singarayer, P. Valdes, F. Balloux, I.
Leboreiro, J. L. Vera, H. Rangel-­Villalobos, D. Pettener, D. Luiselli, L. G. Davis, E. Heyer, C.
P. E. Zollikofer, M. S. Ponce de León, C. I. Smith, V. Grimes, K.-­A. Pike, M. Deal, B. T.
Fuller, B. Arriaza, V. Standen, M. F. Luz, F. Ricaut, N. Guidon, L. Osipova, M. I. Voevoda, O.
L. Posukh, O. Balanovsky, M. Lavryashina, Y. Bogunov, E. Khusnutdinova, M. Gubina, E.
Balanovska, S. Fedorova, S. Litvinov, B. Malyarchuk, M. Derenko, M. J. Mosher, D. Archer,
J. Cybulski, B. Petzelt, J. Mitchell, R. Worl, P. J. Norman, P. Parham, B. M. Kemp, T. Kivisild,
C. Tyler-­Smith, M. S. Sandhu, M. Crawford, R. Villems, D. G. Smith, M. R. Waters, T.
Goebel, J. R. Johnson, R. S. Malhi, M. Jakobsson, D. J. Meltzer, A. Manica, R. Durbin, C. D.
Bustamante, Y. S. Song, R. Nielsen, E. Willerslev, Genomic evidence for the Pleistocene and
recent population history of Native Americans. Science 349, aab3884 (2015).

2. J. V. Moreno-­Mayar, B. A. Potter, L. Vinner, M. Steinrücken, S. Rasmussen, J. Terhorst, J. A.

Kamm, A. Albrechtsen, A.-­S. Malaspinas, M. Sikora, J. D. Reuther, J. D. Irish, R. S. Malhi, L.
Orlando, Y. S. Song, R. Nielsen, D. J. Meltzer, E. Willerslev, Terminal Pleistocene Alaskan
genome reveals first founding population of Native Americans. Nature 553, 203–207 (2018).

3. M. Sikora, V. V. Pitulko, V. C. Sousa, M. E. Allentoft, L. Vinner, S. Rasmussen, A. Margaryan,

P. de Barros Damgaard, C. de la Fuente, G. Renaud, M. A. Yang, Q. Fu, I. Dupanloup, K.
Giampoudakis, D. Nogués-­Bravo, C. Rahbek, G. Kroonen, M. Peyrot, H. McColl, S. V.
Vasilyev, E. Veselovskaya, M. Gerasimova, E. Y. Pavlova, V. G. Chasnyk, P. A. Nikolskiy, A.
V. Gromov, V. I. Khartanovich, V. Moiseyev, P. S. Grebenyuk, A. Y. Fedorchenko, A. I.
Lebedintsev, S. B. Slobodin, B. A. Malyarchuk, R. Martiniano, M. Meldgaard, L. Arppe, J. U.
Palo, T. Sundell, K. Mannermaa, M. Putkonen, V. Alexandersen, C. Primeau, N. Baimukhanov,
R. S. Malhi, K.-­G. Sjögren, K. Kristiansen, A. Wessman, A. Sajantila, M. M. Lahr, R. Durbin,
 R. Nielsen, D. J. Meltzer, L. Excoffier, E. Willerslev, The population history of northeastern
Siberia since the Pleistocene. Nature 570, 182–188 (2019).

4. H. Yu, M. A. Spyrou, M. Karapetian, S. Shnaider, R. Radzevičiūtė, K. Nägele, G. U.

Neumann, S. Penske, J. Zech, M. Lucas, P. LeRoux, P. Roberts, G. Pavlenok, A. Buzhilova, C.
Posth, C. Jeong, J. Krause, Paleolithic to Bronze Age Siberians reveal connections with first
Americans and across Eurasia. Cell 181, 1232–1245.e20 (2020).

5. J. V. Moreno-­Mayar, L. Vinner, P. de Barros Damgaard, C. de la Fuente, J. Chan, J. P. Spence,

M. E. Allentoft, T. Vimala, F. Racimo, T. Pinotti, S. Rasmussen, A. Margaryan, M. Iraeta
Orbegozo, D. Mylopotamitaki, M. Wooller, C. Bataille, L. Becerra-­Valdivia, D. Chivall, D.
Comeskey, T. Devièse, D. K. Grayson, L. George, H. Harry, V. Alexandersen, C. Primeau, J.
Erlandson, C. Rodrigues-­Carvalho, S. Reis, M. Q. R. Bastos, J. Cybulski, C. Vullo, F. Morello,
M. Vilar, S. Wells, K. Gregersen, K. L. Hansen, N. Lynnerup, M. Mirazón Lahr, K. Kjær, A.
Strauss, M. Alfonso-­Durruty, A. Salas, H. Schroeder, T. Higham, R. S. Malhi, J. T. Rasic, L.
Souza, F. R. Santos, A.-­S. Malaspinas, M. Sikora, R. Nielsen, Y. S. Song, D. J. Meltzer, E.
Willerslev, Early human dispersals within the Americas. Science 362, eaav2621 (2018).

6. C. L. Scheib, H. Li, T. Desai, V. Link, C. Kendall, G. Dewar, P. W. Griffith, A. Mörseburg, J.

R. Johnson, A. Potter, S. L. Kerr, P. Endicott, J. Lindo, M. Haber, Y. Xue, C. Tyler-­Smith, M.
S. Sandhu, J. G. Lorenz, T. D. Randall, Z. Faltyskova, L. Pagani, P. Danecek, T. C. O’Connell,
P. Martz, A. S. Boraas, B. F. Byrd, A. Leventhal, R. Cambra, R. Williamson, L. Lesage, B.
Holguin, E. Ygnacio-­de Soto, J. T. Rosas, M. Metspalu, J. T. Stock, A. Manica, A. Scally, D.
Wegmann, R. S. Malhi, T. Kivisild, Ancient human parallel lineages within North America
contributed to a coastal expansion. Science 360, 1024–1027 (2018).

7. M. Rasmussen, S. L. Anzick, M. R. Waters, P. Skoglund, M. DeGiorgio, T. W. Stafford Jr., S.

Rasmussen, I. Moltke, A. Albrechtsen, S. M. Doyle, G. D. Poznik, V. Gudmundsdottir, R.
Yadav, A. S. Malaspinas, S. S. W. V, M. E. Allentoft, O. E. Cornejo, K. Tambets, A. Eriksson,
P. D. Heintzman, M. Karmin, T. S. Korneliussen, D. J. Meltzer, T. L. Pierre, J. Stenderup, L.
Saag, V. M. Warmuth, M. C. Lopes, R. S. Malhi, S. Brunak, T. Sicheritz-­Ponten, I. Barnes, M.
Collins, L. Orlando, F. Balloux, A. Manica, R. Gupta, M. Metspalu, C. D. Bustamante, M.
 Jakobsson, R. Nielsen, E. Willerslev, The genome of a Late Pleistocene human from a Clovis
burial site in western Montana. Nature 506, 225–229 (2014).

8. C. Posth, N. Nakatsuka, I. Lazaridis, P. Skoglund, S. Mallick, T. C. Lamnidis, N. Rohland, K.

Nägele, N. Adamski, E. Bertolini, N. Broomandkhoshbacht, A. Cooper, B. J. Culleton, T.
Ferraz, M. Ferry, A. Furtwängler, W. Haak, K. Harkins, T. K. Harper, T. Hünemeier, A. M.
Lawson, B. Llamas, M. Michel, E. Nelson, J. Oppenheimer, N. Patterson, S. Schiffels, J.
Sedig, K. Stewardson, S. Talamo, C.-­C. Wang, J.-­J. Hublin, M. Hubbe, K. Harvati, A. N.
Delaunay, J. Beier, M. Francken, P. Kaulicke, H. Reyes-­Centeno, K. Rademaker, W. R. Trask,
M. Robinson, S. M. Gutierrez, K. M. Prufer, D. C. Salazar-­García, E. N. Chim, L. M. P.
Gomes, M. L. Alves, A. Liryo, M. Inglez, R. E. Oliveira, D. V. Bernardo, A. Barioni, V.
Wesolowski, N. A. Scheifler, M. A. Rivera, C. R. Plens, P. G. Messineo, L. Figuti, D. Corach,
C. Scabuzzo, S. Eggers, P. De Blasis, M. Reindel, C. Méndez, G. Politis, E. Tomasto-­Cagigao,
D. J. Kennett, A. Strauss, L. Fehren-­Schmitz, J. Krause, D. Reich, Reconstructing the deep
population history of Central and South America. Cell 175, 1185–1197.e22 (2018).

9. N. Nakatsuka, B. Holguin, J. Sedig, P. E. Langenwalter, J. Carpenter, B. J. Culleton, C. García-­

Moreno, T. K. Harper, D. Martin, J. Martínez-­Ramírez, A. Porcayo-­Michelini, V. Tiesler, M. E.
Villapando-­Canchola, A. Valdes Herrera, K. Callan, E. Curtis, A. Kearns, L. Iliev, A. M.
Lawson, M. Mah, S. Mallick, A. Micco, M. Michel, J. N. Workman, J. Oppenheimer, L. Qiu,
F. Zalzala, N. Rohland, J. L. Punzo Diaz, J. R. Johnson, D. Reich, Genetic continuity and
change among the Indigenous peoples of California. Nature 624, 122–129 (2023).

10. T. Ferraz, X. S. Villagran, K. Nägele, R. Radzevičiūtė, R. B. Lemes, D. C. Salazar-­García, V.

Wesolowski, M. L. Alves, M. Bastos, A. R. Py-­Daniel, H. P. Lima, J. M. Cardoso, R.
Estevam, A. Liryo, G. M. Guimarães, L. Figuti, S. Eggers, C. R. Plens, D. M. A. Erler, H. A.
V. Costa, I. da Silva Erler, E. Koole, G. Henriques, A. Solari, G. Martin, S. F. S. M. da Silva,
R. Kipnis, L. M. Müller, M. Ferreira, J. C. Resende, E. Chim, C. A. da Silva, A. C. Borella, T.
Tomé, L. M. P. Gomes, D. B. Fonseca, C. S. da Rosa, J. D. de Moura Saldanha, L. C. Leite,
C. M. S. Cunha, S. A. Viana, F. O. Almeida, D. Klokler, H. L. A. Fernandes, S. Talamo, P. De
Blasis, S. M. de Souza, C. de Paula Moraes, R. E. Oliveira, T. Hünemeier, A. Strauss, C.
Posth, Genomic history of coastal societies from eastern South America. Nat. Ecol. Evol. 7,
1315–1330 (2023).
11. N. Nakatsuka, I. Lazaridis, C. Barbieri, P. Skoglund, N. Rohland, S. Mallick, C. Posth, K.
Harkins-­Kinkaid, M. Ferry, É. Harney, M. Michel, K. Stewardson, J. Novak-­Forst, J. M.
Capriles, M. A. Durruty, K. A. Álvarez, D. Beresford-­Jones, R. Burger, L. Cadwallader, R.
Fujita, J. Isla, G. Lau, C. L. Aguirre, S. LeBlanc, S. C. Maldonado, F. Meddens, P. G.
Messineo, B. J. Culleton, T. K. Harper, J. Quilter, G. Politis, K. Rademaker, M. Reindel, M.
Rivera, L. Salazar, J. R. Sandoval, C. M. Santoro, N. Scheifler, V. Standen, M. I. Barreto, I. F.
Espinoza, E. Tomasto-­Cagigao, G. Valverde, D. J. Kennett, A. Cooper, J. Krause, W. Haak, B.
Llamas, D. Reich, L. Fehren-­Schmitz, A paleogenomic reconstruction of the deep population
history of the Andes. Cell 181, 1131–1145.e21 (2020).

12. J. W. Hoopes, O. Fonseca, “Goldwork and Chibchan identity: Endogenous change and
diffuse unity in the Isthmo-­Colombian area,” in Gold and Power in Ancient Costa Rica,
Panama, and Colombia (Dumbarton Oaks, 2003), pp. 49–89.

13. J. C. Niño Vargas, S. Beckerman, “Universo chibcha, universos chibchas: introducción a la
unidad y la diversidad del área istmocolombiana,” in Universos chibchas: Nuevas
aproximaciones a la unidad y la diversidad humana del área istmocolombiana. J. C. Nino
Vargas, S. Beckerman eds. (Universidad de los Andes, 2024), pp. 1–59.

14. M. Pache, “Contribution to Chibchan historical linguistics,” thesis, University Leiden (2018).

15. M. Pache, Tracing sound change in Nasa Yuwe (western Colombia): Evidence from Andaqui
(western Colombia) and Misumalpan languages (Central America). LIAMES: Línguas
Indígenas Americanas 24, e024003 (2024).

16. M. Urban, Language classification, language contact and Andean prehistory: The North.
Lang. Linguist. Compass 15, e12414 (2021).

17. A. Constenla Umaña, L. Campbell, V. Grondona, “Chibchan languages,” in The Indigenous
Languages of South America: A Comprehensive Guide, V. G. L. Campbell, Ed. (De Gruyter
Mouton, 2012), pp. 391–439.
18. A. Constenla Umaña, Sobre el estudio diacrónico de las lenguas chibchenses y su
contribución al conocimiento del pasado de sus hablantes. Boletín Museo del Oro 3839, 1356
(1995).

19. L. Casas Vargas, L. M. Romero, W. Usaquén, S. Zea, M. Silva, I. Briceño, A. Gomez, J. V.
Rodríguez, Mitochondrial DNA diversity in Prehispanic bone remains on the Eastern
Colombian Andes. Biomedica 37, 548–560 (2017).

20. M. R. Capodiferro, B. Aram, A. Raveane, N. Rambaldi Migliore, G. Colombo, L. Ongaro, J.

Rivera, T. Mendizábal, I. Hernández-­Mora, M. Tribaldos, U. A. Perego, H. Li, C. L. Scheib,
A. Modi, A. Gòmez-­Carballa, V. Grugni, G. Lombardo, G. Hellenthal, J. M. Pascale, F.
Bertolini, G. S. Grieco, C. Cereda, M. Lari, D. Caramelli, L. Pagani, M. Metspalu, R.
Friedrich, C. Knipper, A. Olivieri, A. Salas, R. Cooke, F. Montinaro, J. Motta, A. Torroni, J.
G. Martín, O. Semino, R. S. Malhi, A. Achilli, Archaeogenomic distinctiveness of the
Isthmo-­Colombian area. Cell 184, 1706–1723.e24 (2021).

21. P. E. Melton, I. Briceño, A. Gómez, E. J. Devor, J. E. Bernal, M. H. Crawford, Biological

relationship between central and South American Chibchan speaking populations: Evidence
from mtDNA. Am. J. Phys. Anthropol. 133, 753–770 (2007).

22. M. C. Noguera-­Santamaría, C. E. Anderson, D. Uricoechea, C. Durán, I. Briceño-­Balcázar, J.

Bernal Villegas, Mitochondrial DNA analysis suggests a Chibchan migration into Colombia.
Universitas Scientiarum 20, 261–278 (2015).

23. D. Reich, N. Patterson, D. Campbell, A. Tandon, S. Mazieres, N. Ray, M. V. Parra, W. Rojas,
C. Duque, N. Mesa, L. F. García, O. Triana, S. Blair, A. Maestre, J. C. Dib, C. M. Bravi, G.
Bailliet, D. Corach, T. Hünemeier, M. C. Bortolini, F. M. Salzano, M. L. Petzl-­Erler, V.
Acuña-­Alonzo, C. Aguilar-­Salinas, S. Canizales-­Quinteros, T. Tusié-­Luna, L. Riba, M.
Rodríguez-­Cruz, M. Lopez-­Alarcón, R. Coral-­Vazquez, T. Canto-­Cetina, I. Silva-­Zolezzi, J.
C. Fernandez-­Lopez, A. V. Contreras, G. Jimenez-­Sanchez, M. J. Gómez-­Vázquez, J. Molina,
A. Carracedo, A. Salas, C. Gallo, G. Poletti, D. B. Witonsky, G. Alkorta-­Aranburu, R. I.
Sukernik, L. Osipova, S. A. Fedorova, R. Vasquez, M. Villena, C. Moreau, R. Barrantes, D.
Pauls, L. Excoffier, G. Bedoya, F. Rothhammer, J.-­M. Dugoujon, G. Larrouy, W. Klitz, D.
 Labuda, J. Kidd, K. Kidd, A. D. Rienzo, N. B. Freimer, A. L. Price, A. Ruiz-­Linares,
Reconstructing Native American population history. Nature 488, 370–374 (2012).

24. A. Moreno-­Estrada, S. Gravel, F. Zakharia, J. L. McCauley, J. K. Byrnes, C. R. Gignoux, P.

A. Ortiz-­Tello, R. J. Martínez, D. J. Hedges, R. W. Morris, C. Eng, K. Sandoval, S. Acevedo-­
Acevedo, P. J. Norman, Z. Layrisse, P. Parham, J. C. Martínez-­Cruzado, E. G. Burchard, M.
L. Cuccaro, E. R. Martin, C. D. Bustamante, Reconstructing the population genetic history of
the Caribbean. PLOS Genet. 9, e1003925 (2013).

25. G. Correal Urrego, Aguazuque. Evidencia de cazadores, recolectores y plantadores en la

altiplanicie de la cordillera oriental (FIAN, 1990).

26. A. M. Groot, Checua: Una secuencia cultural entre 8 500 y 3 000 años antes del presente
(FIAN, 1992).

27. J. V. Rodríguez, Tras las huellas de los chibchas de los Andes Orientales de Colombia
(ICANH, 2024).

28. A. Gómez, J. C. Berrio, H. Henry, M. Becerra, R. Marchant, A Holocene pollen record of
vegetation change and human impact from Pantano de Vargas, an intra-­Andean basin of
Duitama, Colombia. Rev. Palaeobot. Palynol. 145, 143–157 (2007).

29. S. M. Broadbent, Reconocimiento arqueológico de la laguna de La Herrera. Revista

colombiana de antropología 15, 173–191 (1970).

30. M. E. Delgado Burbano, Mid and Late Holocene population changes at the Sabana de Bogotá
(Northern South America) inferred from skeletal morphology and radiocarbon chronology.
Quat. Int. 256, 2–11 (2012).

31. M. Delgado, F. Rodríguez, K. Kassadjikova, L. Fehren-­Schmitz, A paleogenetic perspective

of the Sabana de Bogotá (Northern South America) population history over the Holocene
(9000–550 cal BP). Quat. Int. 578, 73–86 (2021).

32. A. M. Boada Rivas, The Evolution of Social Hierarchy in a Muisca Chiefdom of the Northern
Andes of Colombia (University of Pittsburgh,Universidad de los Andes, 2007).
33. S
 . Archila, A. M. Groot, J. P. Ospina, M. Mejía, C. Zorro, Dwelling the hill: Traces of
increasing sedentism in hunter-­gatherers societies at Checua site, Colombia (9500-­5052
cal BP). Quat. Int. 578, 102–119 (2021).

34. C. H. Langebaek, Regional Archaeology in the Muisca territory: A Study of the Fúquene and
Susa Valleys (University of Pittsburgh, Universidad de los Andes, 1995).

35. A. Casas-­Vargas, A. Gómez, I. Briceño, M. Díaz-­Matallana, J. E. Bernal, J. V. Rodríguez,

High genetic diversity on a sample of pre-­Columbian bone remains from Guane territories in
northwestern Colombia. Am. J. Phys. Anthropol. 146, 637–649 (2011).

36. M. Díaz-­Matallana, A. Gómez, I. Briceño, J. V. Rodríguez, Genetic analysis of Paleo-­

Colombians from Nemocón, Cundinamarca provides insights on the early peopling of
northwestern South America. Revista Acad. Colomb. Ci. Exact. 40, 461–483 (2016).

37. S. Mallick, H. Li, M. Lipson, I. Mathieson, M. Gymrek, F. Racimo, M. Zhao, N. Chennagiri,
S. Nordenfelt, A. Tandon, P. Skoglund, I. Lazaridis, S. Sankararaman, Q. Fu, N. Rohland, G.
Renaud, Y. Erlich, T. Willems, C. Gallo, J. P. Spence, Y. S. Song, G. Poletti, F. Balloux, G.
van Driem, P. de Knijff, I. G. Romero, A. R. Jha, D. M. Behar, C. M. Bravi, C. Capelli, T.
Hervig, A. Moreno-­Estrada, O. L. Posukh, E. Balanovska, O. Balanovsky, S. Karachanak-­
Yankova, H. Sahakyan, D. Toncheva, L. Yepiskoposyan, C. Tyler-­Smith, Y. Xue, M. S.
Abdullah, A. Ruiz-­Linares, C. M. Beall, A. di Rienzo, C. Jeong, E. B. Starikovskaya, E.
Metspalu, J. Parik, R. Villems, B. M. Henn, U. Hodoglugil, R. Mahley, A. Sajantila, G.
Stamatoyannopoulos, J. T. S. Wee, R. Khusainova, E. Khusnutdinova, S. Litvinov, G. Ayodo,
D. Comas, M. F. Hammer, T. Kivisild, W. Klitz, C. A. Winkler, D. Labuda, M. Bamshad, L.
B. Jorde, S. A. Tishkoff, W. S. Watkins, M. Metspalu, S. Dryomov, R. Sukernik, L. Singh, K.
Thangaraj, S. Pääbo, J. Kelso, N. Patterson, D. Reich, The Simons genome diversity project:
300 genomes from 142 diverse populations. Nature 538, 201–206 (2016).

38. Y.-­Z. Huang, H. Pamjav, P. Flegontov, V. Stenzl, S.-­Q. Wen, X.-­Z. Tong, C.-­C. Wang, L.-­X.
Wang, L.-­H. Wei, J.-­Y. Gao, L. Jin, H. Li, Dispersals of the Siberian Y-­chromosome
haplogroup Q in Eurasia. Mol. Genet. Genomics 293, 107–117 (2018).
39. V. Grugni, A. Raveane, L. Ongaro, V. Battaglia, B. Trombetta, G. Colombo, M. R.
Capodiferro, A. Olivieri, A. Achilli, U. A. Perego, J. Motta, M. Tribaldos, S. R. Woodward,
L. Ferretti, F. Cruciani, A. Torroni, O. Semino, Analysis of the human Y-­chromosome
haplogroup Q characterizes ancient population movements in Eurasia and the Americas.
BMC Biol. 17, 3 (2019).

40. D. Popli, S. Peyrégne, B. M. Peter, KIN: A method to infer relatedness from low-­coverage
ancient DNA. Genome Biol. 24, 10 (2023).

41. H. Ringbauer, J. Novembre, M. Steinrücken, Parental relatedness through time revealed
by runs of homozygosity in ancient DNA. Nat. Commun. 12, 5425 (2021).

42. J. L. Garcia, “The foods and crops of the Muisca: A dietary reconstruction of the intermediate
chiefdoms of Bogotá (Bacatá) and Tunja (Hunza), Colombia,” thesis, University of Central
Florida (2012).

43. F. M. Olivares, J. M. Madero, A. Casas-­Vargas, S. Z. Montoya, D. S. Medellín, L. Gusmão,

W. Usaquén, Contrasting the ancestry patterns of three distinct population groups from the
northernmost region of South America. Am. J. Phys. Anthropol. 173, 437–447 (2020).

44. D. M. Fernandes, K. A. Sirak, H. Ringbauer, J. Sedig, N. Rohland, O. Cheronet, M. Mah, S.

Mallick, I. Olalde, B. J. Culleton, N. Adamski, R. Bernardos, G. Bravo, N.
Broomandkhoshbacht, K. Callan, F. Candilio, L. Demetz, K. S. D. Carlson, L. Eccles, S.
Freilich, R. J. George, A. M. Lawson, K. Mandl, F. Marzaioli, W. C. McCool, J.
Oppenheimer, K. T. Özdogan, C. Schattke, R. Schmidt, K. Stewardson, F. Terrasi, F. Zalzala,
C. A. Antúnez, E. V. Canosa, R. Colten, A. Cucina, F. Genchi, C. Kraan, F. La Pastina, M.
Lucci, M. V. Maggiolo, B. Marcheco-­Teruel, C. T. Maria, C. Martínez, I. París, M. Pateman,
T. M. Simms, C. G. Sivoli, M. Vilar, D. J. Kennett, W. F. Keegan, A. Coppa, M. Lipson, R.
Pinhasi, D. Reich, A genetic history of the pre-­contact Caribbean. Nature 590, 103–110
(2021).

45. K. Nägele, C. Posth, M. Iraeta Orbegozo, Y. Chinique de Armas, S. T. Hernández Godoy, U.
M. González Herrera, M. A. Nieves-­Colón, M. Sandoval-­Velasco, D. Mylopotamitaki, R.
Radzeviciute, J. Laffoon, W. J. Pestle, J. Ramos-­Madrigal, T. C. Lamnidis, W. C. Schaffer, R.
 S. Carr, J. S. Day, C. Arredondo Antúnez, A. Rangel Rivero, A. J. Martínez-­Fuentes, E.
Crespo-­Torres, I. Roksandic, A. C. Stone, C. Lalueza-­Fox, M. Hoogland, M. Roksandic, C.
L. Hofman, J. Krause, H. Schroeder, Genomic insights into the early peopling of the
Caribbean. Science 369, 456–460 (2020).

46. J. W. Hoopes, The emergence of social complexity in the Chibchan world of southern Central
America and northern Colombia, AD 300–600. J. Archaeol. Res. 13, 1–47 (2005).

47. J. V. Rodríguez, C. Vargas Vargas, Evolución y tamaño dental en poblaciones humanas de
Colombia. Revista Acad. Colomb. Ci. Exact. 34, 423–439 (2010).

48. M. Delgado, Stable isotope evidence for dietary and cultural change over the Holocene at the
Sabana de Bogotá region, Northern South America. Archaeol. Anthropol. Sci. 10, 817–832
(2018).

49. D. J. Kennett, M. Lipson, K. M. Prufer, D. Mora-­Marín, R. J. George, N. Rohland, M.

Robinson, W. R. Trask, H. H. J. Edgar, E. C. Hill, E. E. Ray, P. Lynch, E. Moes, L.
O’Donnell, T. K. Harper, E. J. Kate, J. Ramos, J. Morris, S. M. Gutierrez, T. M. Ryan, B. J.
Culleton, J. J. Awe, D. Reich, South-­to-­north migration preceded the advent of intensive
farming in the Maya region. Nat. Commun. 13, 1530 (2022).

50. J. Dabney, M. Knapp, I. Glocke, M.-T. Gansauge, A. Weihmann, B. Nickel, C. Valdiosera, N.
García, S. Pääbo, J.-L. Arsuaga, M. Meyer, Complete mitochondrial genome sequence of a
Middle Pleistocene cave bear reconstructed from ultrashort DNA fragments. Proc. Natl.
Acad. Sci. U.S.A. 110, 15758–15763 (2013).

51. N. Rohland, E. Harney, S. Mallick, S. Nordenfelt, D. Reich, Partial uracil–DNA–glycosylase

treatment for screening of ancient DNA. Philos. Trans. R Soc. Lond. B Biol. Sci. 370,
20130624 (2015).

52. A. Peltzer, G. Jäger, A. Herbig, A. Seitz, C. Kniep, J. Krause, K. Nieselt, EAGER: Efficient
ancient genome reconstruction. Genome Biol. 17, 60 (2016).
53. M. Schubert, S. Lindgreen, L. Orlando, AdapterRemoval v2: Rapid adapter trimming,
identification, and read merging. BMC. Res. Notes 9, 88 (2016).

54. H. Li, R. Durbin, Fast and accurate short read alignment with Burrows-­Wheeler transform.
Bioinformatics 25, 1754–1760 (2009).

55. H. Jónsson, A. Ginolhac, M. Schubert, P. L. F. Johnson, L. Orlando, mapDamage2.0: Fast

approximate Bayesian estimates of ancient DNA damage parameters. Bioinformatics 29,
1682–1684 (2013).

56. Q. Fu, M. Meyer, X. Gao, U. Stenzel, H. A. Burbano, J. Kelso, S. Pääbo, DNA analysis of an
early modern human from Tianyuan Cave, China. Proc. Natl. Acad. Sci. U.S.A. 110, 2223–
2227 (2013).

57. G. Renaud, V. Slon, A. T. Duggan, J. Kelso, Schmutzi: Estimation of contamination and
endogenous mitochondrial consensus calling for ancient DNA. Genome Biol. 16, 224 (2015).

58. T. S. Korneliussen, A. Albrechtsen, R. Nielsen, ANGSD: Analysis of next generation

sequencing data. BMC Bioinformatics 15, 356 (2014).

59. N. Patterson, A. L. Price, D. Reich, Population structure and eigenanalysis. PLOS Genet. 2,
e190 (2006).

60. L. Arias, N. Q. Emlen, S. Norder, N. Julmi, M. Lemus Serrano, T. Chacon, J. Wiegertjes, A.
Howard, M. C. B. C. Azevedo, A. Caine, S. Dunn, M. Stoneking, R. Van Gijn, Interpreting
mismatches between linguistic and genetic patterns among speakers of Tanimuka (Eastern
Tukanoan) and Yukuna (Arawakan). Interface Focus 13, 20220056 (2023).

61. N. Patterson, P. Moorjani, Y. Luo, S. Mallick, N. Rohland, Y. Zhan, T. Genschoreck, T.

Webster, D. Reich, Ancient admixture in human history. Genetics 192, 1065–1093 (2012).

62. M. Stuiver, H. A. Polach, Discussion reporting of 14C data. Radiocarbon 19, 355–363 (1977).
63. S. I. Perez, V. Bernal, P. N. Gonzalez, M. Sardi, G. G. Politis, Discrepancy between cranial
and DNA data of early Americans: Implications for American peopling. PLOS ONE 4, e5746
(2009).

64. M. Hubbe, W. A. Neves, K. Harvati, Testing evolutionary and dispersion scenarios for the
settlement of the new world. PLOS ONE 5, e11105 (2010).

65. S. de Azevedo, A. Nocera, C. Paschetta, L. Castillo, M. González, R. González-­José,

Evaluating microevolutionary models for the early settlement of the New World: The
importance of recurrent gene flow with Asia. Am. J. Phys. Anthropol. 146, 539–552 (2011).

66. R. González-­José, M. C. Bortolini, F. R. Santos, S. L. Bonatto, The peopling of America:

Craniofacial shape variation on a continental scale and its interpretation from an
interdisciplinary view. Am. J. Phys. Anthropol. 137, 175–187 (2008).

67. G. Keyeux, C. Rodas, N. Gelvez, D. Carter, Possible migration routes into South America
deduced from mitochondrial DNA studies in Colombian Amerindian populations. Hum. Biol.
74, 211–233 (2002).

68. J. V. Rodríguez Cuenca, La identificación humana en Colombia. Avances y perspectivas

(Universidad Nacional de Colombia, 2011).

69. J. V. Rodríguez Cuenca, El Parque Arqueológico de Facatativá (CAR, Universidad Nacional,
2015).

70. C. H. Langebaek, in Mercados, poblamiento e integración étnica entre los muiscas (Banco de
la República, 1986).

71. R. Lleras, Los Muiscas en la literatura histórica y antropológica. Bol. Hist. Antig. 92, 307–
338 (2005).

72. A. M. Groot, Arqueología y patrimonio: Conocimiento y apropiación social. Revista Acad.
Colomb. Ci. Exact. 10.18257/raccefyn.30(114).2006.2210 , (2006).
73. A. M. Groot, “Checua: Un aporte para el conocimiento del precerámico de la sabana de
Bogotá,” in Ámbito y Ocupaciones Tempranas de la América Tropical (Instituto Colombiano
de Antropología, 1995), pp. 45–58.

74. A. Minelli, M. Cozzolino, A. Di Nucci, S. Guglielmi, M. Giannantonio, D. D'Amore, E.

Pittoni, A. M. Groot, The prehistory of the Colombian territory: The result of the Italian
archaeological investigation on the Checua Site (Municipality of Nemocòn, Cundinamarca
Department). J. Biol. Res.-­Boll. Soc. Ital. Biol. Sper. 85, 10.4081/jbr.2012.4073 (2012).

75. W. A. Neves, M. Hubbe, G. Correal, Human skeletal remains from Sabana de Bogotá,
Colombia: A case of Paleoamerican morphology late survival in South America? Am. J. Phys.
Anthropol. 133, 1080–1098 (2007).

76. H. M. Pucciarelli, S. I. Perez, G. G. Politis, Early Holocene human remains from the
Argentinean Pampas: Additional evidence for distinctive cranial morphology of early South
Americans. Am. J. Phys. Anthropol. 143, 298–305 (2010).

77. E. E. R. Braida, Arqueología de rescate, en el barrio las delicias (Bogotá). Rev. Colomb.
Antropol. 28, 156–160 (1991).

78. A. Cifuentes Toro, Reseña de un sitio arqueológico en la Mesa de los Santos (Santander).
Bol. Arqueol. FIAN 4, 33–40 (2014).

79. S. Mallick, A. Micco, M. Mah, H. Ringbauer, I. Lazaridis, I. Olalde, N. Patterson, D. Reich,
The Allen Ancient DNA Resource (AADR) a curated compendium of ancient human
genomes. Sci. Data 11, 182 (2024).

80. M. H. Kruschek, “The evolution of the Bogotá chiefdom: A household view,” thesis,
University of Pittsburgh (2003).

81. S. Rivas, D. Calderón, C. Marulanda, L. F. Mendoza, G. R. Scott, S. R. Poulson, M. Delgado,

Stable isotopes and paleodiet of the ancient inhabitants of Nueva Esperanza: A late Holocene
site from Sabana de Bogotá (Colombia). Int. J. Osteoarchaeol. 34, e3244 (2024).
82. J. P. Quintero-­Guzmán, El Dorado offerings in Lake Guatavita: A muisca ritual
archaeological site. Latin Am. Antiq. 35, 483–499 (2024).

83. P. A. Sánchez-­Castañeda, Memory in sacred places: The revitalization process of the Muisca
community. Urban Plan. 5, 263–273 (2020).

84. J. V. Rodríguez, Los chibchas: Hijos del sol, la luna y los Andes. Orígenes de su diversidad
(Universidad Nacional de Colombia, 2011).