East of the Andes: The genetic profile of the Peruvian

J_ID: Customer A_ID: AJPA23209 Cadmus Art: AJPA23209 Ed. Ref. No.: AJPA-2016-00184.R1 Date: 10-March-17

Received: 31 May 2016

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Revised: 8 November 2016

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Accepted: 28 February 2017

DOI 10.1002/ajpa.23209

RESEARCH ARTICLE

East of the Andes: The genetic profile of the Peruvian Amazon populations T. Di Corcia1 | C. Sanchez Mellado2 | T. J. Davila Francia2 | G. Ferri4 | S. Sarno3 | D. Luiselli3 | O. Rickards1 1 Department of Biology, University of Rome “Tor Vergata,”, Via della Ricerca Scientifica n. 1, Roma 00173, Italy

Abstract Objectives: Assuming that the differences between the Andes and the Amazon rainforest at envi-

2

Faculty of Intercultural Education and Humanity, National Intercultural University of Amazon, Yarinacocha, Coronel Portillo, Ucayali 25000, Peru

ronmental and historical levels have influenced the distribution patterns of genes, languages, and cultures, the maternal and paternal genetic reconstruction of the Peruvian Amazon populations was used to test the relationships within and between these two extreme environments.

3

Department of Biological, Geological, and Environmental Sciences, University of Bologna, Bologna 40126, Italy 4 Department of Diagnostic and Clinical Medicine and Public Health, University of Modena and Reggio Emilia, Modena 41124, Italy

Correspondence T. Di Corcia, O. Rickards, Department of Biology, University of Rome “Tor Vergata,” Via della Ricerca Scientifica n. 1, Roma 00173, Italy. Email: [email protected]; O. Rickards, Email: [email protected] Funding information The European Research Council, Grant Number ERC-2011-AdG 295733 grant (Langelin)

Materials and Methods: We analyzed four Peruvian Amazon communities (Ashaninka, Huambisa, Cashibo, and Shipibo) for both Y chromosome (17 STRs and 8 SNPs) and mtDNA data (control region sequences, two diagnostic sites of the coding region, and one INDEL), and we studied their variability against the rest of South America. Results: We detected a high degree of genetic diversity in the Peruvian Amazon people, both for mtDNA than for Y chromosome, excepting for Cashibo people, who seem to have had no exchanges with their neighbors, in contrast with the others communities. The genetic structure follows the divide between the Andes and the Amazon, but we found a certain degree of gene flow between these two environments, as particularly emerged with the Y chromosome descent cluster’s (DCs) analysis. Discussion: The Peruvian Amazon is home to an array of populations with differential rates of genetic exchanges with their neighbors and with the Andean people, depending on their peculiar demographic histories. We highlighted some successful Y chromosome lineages expansions originated in Peru during the pre-Columbian history which involved both Andeans and Amazon Arawak people, showing that at least a part of the Amazon rainforest did not remain isolated from those exchanges. KEYWORDS

mtDNA, Peru, South America, STR, Y chromosome

1 | INTRODUCTION

different vertical levels of exploitability. In northern and central Andes, although the beginnings of food production began earlier, the

The Peruvian territory spans vast latitude. This distance, in combina-

first domestication of the potato was achieved in the highland Andes

tion with other geographical factors, contributes to the extreme

at least as early as 2,500 BCE, along with others staples (Dillehay,

diversity of the territory’s ecosystem and has impacted the history of

2011).

its peoples and cultures. The whole landscape is divided into three

Later, the rapid spread of maize crops from south-central Mexico

zones: the coast, the mountain (including the Andes, with an average

to lowland and highland South America by at least 1,800 BCE marked

height of 4,000 m.a.s.l.) and the Amazon rainforest. The forest covers

the beginning of the full-scale agriculture and resulted in a shift in

60% of the territory, while the mountain range and the coast respec-

social strategies and the establishing of larger settlements (Fagan &

tively cover 28 and 12%. In mountainous areas the altitude delineates

Durrani, 2016).

Am J Phys Anthropol. 2017;1–11

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In the Andean regions, much before the age of states and empires,

lighted some ancient demographic flows across the Andes/Amazon

a certain degree of social complexity was reached, for instance, with

divide using the mitochondrial DNA (mtDNA) and Y chromosome data

the early urbanism of Caral site that date to 3,100–1,800 BCE (Solís,

of the Peruvian Amazon people called the Yanesha. Similarly, other

2008), or with the Chavín de Huant ar culture in the central highlands

scholars have shown (using noncoding unlinked autosomal loci) that

dated back to 1,200–400 BCE (Rick et al., 2009).

the genetic diversity of some inhabitants of the Peruvian Amazon

Later, the age of empires saw the arise of the Wari culture in the

stems from a subset of the Central-Andean Quechuas (Scliar et al.,

Central Andes which began at about 550–600 CE and continued until

2014). Here, we generated novel genetic data from the Peruvian peo-

about 1,000 CE, and Tiwanaku on the southern shores of Lake Titicaca

ple settled in the rainforest, comparing their genetic variability against

(400–1,100 CE) which emerged as a contemporary rival of the Wari

the South American genetic background. All of the communities

state (Moore, 2014), only to cite some of the most important examples.

sampled live today in different ecosystems ranging from Amazon rivers

Between approximately 1,400 and 1,535 CE, the largest state of the

basins to the eastern slopes of the Andes, where the mountain range

pre-Columbian Americas, the Inca empire, arose from the highlands of

meets the Amazon River headwaters. Three of these communities (the

Peru and spread across a large portion of western South America along

Ashaninka, Cashibo, and Shipibo) occupy the Ucayali river basin near

the Andean mountain ranges, including large parts of Ecuador, western

the city of Pucallpa, while the Huambisa tribe lives in the upper

and south-central Bolivia, northwest Argentina, north and central Chile,

~o n river, near the city of Iquitos. Here we briefly report upon the Maran

and a small part of southern Colombia. In contrast, the Amazonian

four populations sampled.

archaeology is much less known, and poorly studied.

The Ash aninka belong to the large Arawak linguistic family and live

But cultural and commercial relations occurring within the last mil-

today in the rainforests of Peru and in the State of Acre (Brazil). Their

lennia among the people living in the Andes and the Amazon regions

ancestral territories are located in the forests of Junín, Pasco, Huanuco,

are archaeologically documented. The Wari, along with many of the

and part of Ucayali (Rojas Zolezzi, 1994), and their population is esti-

Inca, submitted several tribes living in the foothills, especially in the

mated to include about 88,000 people (INEI, 2009). Santos (1992)

southern Andes (Renard-Casevitz, Saignes, & Taylor, 1988). People

describes that the Ashaninka people maintained trade relations with

inhabiting the lowlands likely had to contend with strong expansionist

Andean populations long before the birth of the Inca empire. The Shipibo

policies enacted by those trying to conquer territory essential for the

live in the central region of the Rio Ucayali, with Pucallpa as the geo-

survival of the empire. Moreover, they likely responded by adopting

graphical center of the whole area (Bergman, 1980); the documented

one or more of the following strategies: partnership, submission, raids,

number of Shipibo people is variable, with estimates of up to 20,000 indi-

or war (Mc Neish, 1977). To clarify the demographic histories in South

viduals (INEI, 2009). The population settled in Ucayali between the sev-

America (Cabana et al., 2014; Fuselli et al., 2003; Guerra Amorim et al.,

enth and ninth centuries CE, and there is evidence that the Shipibo have

2013; Ramallo et al., 2013; Roewer et al., 2013), many scholars have

been in contact—at least commercially—with the Incas (Lathrap, 1970).

focused on the current distribution of language families and genes,

The Cashibo tribes currently live along the rivers of the Aguaytía, San Ale-

finding that the genetic profile of highland and lowland peoples reflects

jandro, Shamboyacu, and Sungaroyacu, as well as the tributaries of the

a disparity between the Andes and the Amazon akin to their languages

Pachitea, in the Ucayali region (Ribeiro & Wise, 1978). Along with the

distribution: the Amazon languages are numerous, diverse, and inter-

Shipibo, they belong to the Pano linguistic group (Campbell, 1997). They

spersed, while the Andean languages are less numerous but are spoken

are known for their hostility towards their neighbors and European mis-

by larger groups of people. Many of these studies take into account

sionaries and their population comprises about 1,880 individuals (INEI,

the uniparental genetic markers of the Native American populations,

2009). The Huambisa live in settlements that cover about five thousand

which retain traces of the bottleneck resulting from the first human

square kilometers along the eastern buttresses of the Andes and share a

migrations from Siberia to the American continent, which were caused

common history with the Achuar and the Aguaruna, belonging to the

by a strong founder effect (Bortolini et al., 2003; Lewis et al., 2007;

same linguistic family: the Jivaros (Guallard-Martínez, 1990). They occupy

Torroni et al., 1993). Indeed, only the macro-haplogroups A, B, C, D

about thirty-five communities along the Santiago River and its streams

and X (the latter restricted to North American populations) are found

and twenty communities along the Morona River, and about 6,000 total

in the mitochondrial DNA of these populations (Achilli et al., 2008; Per-

individuals have been documented (INEI, 2009).

ego et al., 2009; Schurr, 2004), and only two lineages are present in

We analyzed uniparental markers in 162 samples, providing

the Y chromosome: the macro-haplogroups Q and C (Underhill, Jin,

mtDNA data and Y chromosome data to assign the haplogroups and

Zemans, Oefner, & Cavalli-Sforza, 1996; Zegura, Karafet, Zhivotovsky,

explore the general variability. We built different datasets, some includ-

& Hammer, 2004). The current knowledge about the western South

ing only Peruvian profiles, others including data from all around South

American populations is mostly based on archaeological, cultural, and

America. Ultimately, we sought to: (a) characterize the genetic diversity

molecular studies on the Andean people. However, because of their

of each population sampled, (b) investigate the genetic relationships

peculiar geographical position in South America, the Peruvian Amazon

between the Peruvian Amazon populations and their Western Andean

populations could represent an interesting reservoir of genetic diver-

neighbors, (c) estimate whether sex-biased demographic processes

sity. At present, few molecular studies have been conducted on the

occurred, and (d) connect the pre-Columbian histories of all the Peru-

Peruvian Amazon tribes. Recently, however, Barbieri et al. (2014) high-

vian Amazon peoples.



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2 | MATERIALS AND METHODS

3

Cashibo, Shipibo, Ashaninka, and Huambisa communities (Figure 1).

F1

The subjects collected are representative of the total unrelated popula-

2.1 | Data collection and DNA extraction Two fieldwork expeditions were carried out in the Peruvian Amazon (Ucayali and Loreto departments) between the years 2012 and 2015. Buccal swabs were collected from 162 unrelated individuals from the

tion residing in the communities (Table 1). Informed consent about the purpose of the study and the anonymous use of their data was obtained from all sampled subjects. This study and the collection of samples were approved by the representative of the regional

Map with the Amazon villages sampled. In (A), the location of Huambisa village in northern Amazonia is shown; while in (B), the locations of Shipibo, Cashibo, and Ashaninka villages in central Amazonia are shown

FIGURE 1


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Details of the populations sampled

Pop name

Pop census

Number of mtDNA

Number of Y chr

Sampling location

Longitude (X)

Latitude (Y)

H (mtDNA)

H (Y chr)

Shipibo

300

60

22

Yarinacocha

274.576

28.353

0.9347

0.9004

Cashibo

277

60

28

Padre Abad

275.533

28.733

0.6622

0.8598

Ashaninka

200

20

8

Iparia

274.443

29.307

0.9105

0.9643

Huambisa

150

22

16

Morona

273.311

23.710

0.9333

0.9917

The haplotype diversity indices (H) for both markers are shown.

organization of Ucayali (AIDESEP), and a formal agreement was signed

sub-branch Q1a3a* was the only present in the sample. The Y-SNP

by the president of COSHIKOX (Consejo Shipibo Conibo Xetebo). The

haplogroup nomenclature assignment was completed in accordance

project was also approved by the Ethic Committee of the University of

with Karafet et al. (2008).

Rome Tor Vergata. DNA was extracted in accordance with the standard procedures (Miller, Dykes, & Polesky, 1988).

2.4 | Data analysis Diversity indices for mtDNA and Y chromosome, AMOVA, and uST dis-

2.2 | mtDNA genotyping

tance matrices were computed through the software Arlequin ver 3.11

All 162 individuals were amplified through PCR for the hypervariable

(Excoffier & Heckel, 2006); for mtDNA, the HVSI sequences collected

segment I (HVSI) with the primers L15996 and H16401 and the hyper-

in the database were used to calculate all the parameters in the

variable segment II (HVSII) with the primers L29 and H408. They were

sampled communities and in the other South American samples. The

then sequenced using the 3.1 BigDye Terminator protocol of Applied

program PAST ver. 2.16 (Hammer, 2011) was used for computations of

BiosystemsTM on an ABI PRISM 3130 genetic analyzer. The sequences

nonmetric multidimensional scaling (nMDS), and the data were repre-

were aligned by the software Bioedit v7.2.5, using the reference

sented in a two-dimensional plot. Two comparative datasets of

sequence RSRS (Behar et al., 2012) to find the polymorphic sites. Sub-

sequences from throughout South America were prepared: for the

sequently, the haplotypes were subjected to the prediction on Haplo-

mtDNA HVS-I sequences, collections were taken from 1,712 individu-

grep (haplogrep.uibk.ac.at/) according to the nomenclature of the latest

als from 50 native populations; we considered only the haplotypes

version of Phylotree Build 16 (phylotree.org, van Oven & Kayser,

belonging to the A, B, C, and D native macro-haplogroups. For the Y

2009). Only haplotypes belonging to haplogroup B—when the classifi-

chromosome, STR haplotypes for 15 loci were collected from 1, 217

cation based on the HVSI and HVSII regions—could not be resolved by

individuals from 47 populations, and we only considered the haplo-

Haplogrep’s rank values; two properly selected diagnostic sites for

types belonging to the native haplogroup Q (we excluded DYS385a

mtDNA phylogeny were analyzed in the mtDNA coding region (primers

and DYS385b because they were absent in many samples included in

L4770 and H5193 for the first fragment and primers L11141 and

the dataset, due to instability as loci). As a general rule, all the popula-

H11271 for the second), along with the INDEL 8281–8289d specific

tions taken from the literature and included in the dataset contained at

to haplogroup B.

least 10 individuals. Another dataset of frequencies was prepared only for mtDNA data (Table S2). The populations were divided into two

2.3 | Y chromosome genotyping

geographical macro-groups: Andean and Amazon, taking into account

All the males in our sample were genotyped for Y chromosomes (74

also the language and the linguistic family of each population.

individuals). Y-STRs were analyzed using the AmpFISTR YfilerTM

Median Joining (MJ) network analyses, performed with Network

(Applied Biosystems) following the manufacturer’s instructions. Once

4.1 software (Fluxus Technology Ltd., Clare, Suffolk, UK) were carried

amplified, we used an ABI PRISM 3130 Genetic Analyzer (Applied

out on HVSI, HVSII of mtDNA, and Y chromosome STRs profiles. In

Biosystems) to detect the products, following the recommended

mtDNA MJ network, different weights were assigned to the polymor-

sequencing kit protocols. Hence, we analysed the products with Gene

phic sites according to the transition/transversion rates previously

MapperR ID Software v3.2, which identifies an allele for each STR

described (Meyer, Weiss, & von Haeseler, 1999) and to the recurrence

locus. The 74 individuals were successfully genotyped for the 17 loci

of mutations to minimize homoplasy. For the Y chromosome networks,

of the Y-filer kit; all the haplotypes are listed in Table S1. All the indi-

15 Y-STRs were used (excluding DYS385a and DYS385b) and were

viduals assigned to the Native American Q haplogroup through their Y-

weighted in a proportion twice the inverse of the square root variance.

STR profiles (Bayesian inference by http://www.hprg.com/hapest5/)

For the Y chromosome STR data, we performed a cluster analysis

were used for the following analyses. To assign each individual to a

to identify past episodes of Y lineage success over generations. This

sub-lineage of Q, we tested eight SNPs with a SNaPshotTM Multiplex

analysis was conducted using the software Star Cluster Generator, one

kit (Applied Biosystems) following the protocol described by Sevini

of two pieces of software designed by Balaresque et al. (2015) to recall

et al. (2013). We aimed to observe whether the common Amerindian

clusters centered on frequent haplotypes. The first (the map generator)

R



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5

generates an n-dimensional map for a given population of individuals,

We also tested the variability at the sequence level for each com-

in which each dimension corresponds to a marker. Based on this map,

munity; the number of haplotypes and the diversity indices (based on

the second piece of software (the cluster generator) builds a descent

HVSI) were calculated to assess the mtDNA diversity, and our samples

cluster that corresponds to a given haplotype and a given haplogroup.

were included in a dataset of 77 Amerindian populations to compare

We estimated the TMRCA using Batwing software (Wilson, Weale, &

them at a sub-continental level (Table S3). The standard diversity indi-

Balding, 2003) with an exponential growth model, adopting a genera-

ces showed that the Ashaninka, Shipibo, and Huambisa have a high

tion time of 30 years, which is compatible with population-based esti-

heterogeneity at the mtDNA level, while the same indices in the Cash-

mates for males (Fenner, 2005). We also implemented a mutation rate

ibo have considerably lower values; we expected as much on the basis

l corresponding to a mean rate of 0.022 per locus per generation for

of their recent history. Indeed, the Cashibo remained more isolated

the microsatellites (Kayser et al., 2000). The Batwing outfiles were then

from their neighbors refuging in the rainforest in small groups during

postprocessed with R (http://cran.r-project.org) to calculate the means

the last centuries (Morin, 1998) unlike the others three communities.

for all the parameters values, along with the mean values of TMRCA.

Pairwise genetic distances (uST) were then used to build an nMDS plot (Figure 2): the four samples of the present study were included in a dataset of 50 Amerindian populations to compare HVSI haplotypes. We

3 | RESULTS

distinguished Amazon from Andean peoples using white and black colors,

3.1 | mtDNA

respectively. The linguistic affiliation was specified using symbols to

Every one of the 162 samples shows Amerindian ancestry, each belong-

the member status of a specific linguistic macro-group. Along the second

ing to one of the pan-American native macro-haplogroups: A, B, C, and

coordinate one can distinguish the Andean group from the Amazon one.

D. For the Cashibo and Shipibo (both affiliated with the Panoan lan-

In the upper part of the plot, all Quechua and Aymara populations from

guage), the most represented macro-haplogroup is C, with frequencies

Peru and Bolivia are grouped together: the only “outsiders” who fall

of 70% and 45%, respectively. D appears less frequently, except in the

within the Andeans are the Huambisa. The Ashaninka and Shipibo lie

Ashaninka (35%). And B prevails in the Huambisa, with a percentage of

within a group mainly composed of Amazon populations from Brazil,

63% (Table S2). The PCA analysis (Figure S1) summarizes this trend,

eastern Bolivia, and eastern Peru. The Cashibo remain the most isolated

with Andeans grouping together along the B or D component and Ama-

in the center of the plot, along with the Xavante from Mato Grosso (Bra-

zons scattering in the plot since they have a more variable distribution

zil). There does not appear to be any clustering of linguistic affiliation

of frequencies. The Huambisa fall closer to the Central Andean cluster,

among the Amazon populations. Instead, the big Andean linguistic fami-

together with the other Peruvian Amazons: the Yanesha.

lies tend to group together: the Quechua and Aymara from Peru and

hypothetically visualize relationships between populations depending on

nMDS plot based on mtDNA Fst distances (stress value 5 0.08). The black, white, and gray symbols indicate Andeans, Amazon, and Central American (or Northern South American) peoples, respectively. The symbols indicate the linguistic family: circles for Andean languages (Aymara, Quechua and Araucarian), squares for Ge, trapezes for Jivaroan, crosses for Arawak, triangles for Chibchan, stars for Tupi, and rhombus for Panoan. The white circles indicate unknown (or mixed) languages

FIGURE 2


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nMDS plot based on Y-STRs Rst distances (stress value 5 0.162). The black, white, and gray symbols indicate Andeans, Amazon, and Central American (or Northern South American) peoples, respectively. The symbols indicate the linguistic family: circles for Andeans (Aymara, Quechua, and Araucarian), squares for Ge, trapezes for Jivaroan, crosses for Arawaks, triangles for Chibchan, stars for Tupi, and rhombus for Panoan. The white circles indicate unknown (or mixed) languages

FIGURE 3

Bolivia (the black circles on the top), the Araucarian from Chile and Argentina (the black circles on the right).

The nMDS plot (Figure 3) based on Rst distances between Y-STRs profiles suggests a pattern of distribution of diversity across South

In the network built on the HVSI and HVSII sequences (Figure S2)

America that differs from that of the mtDNA data dramatically. Along

we found two shared nodes between the Peruvian Andeans (both

the first coordinate, an area of more dispersed populations (mostly

Quechua and Aymara) and the Huambisa among the B2 haplotypes;

Amazon) could be visualized on the left, while along the second coordi-

moreover, we found two shared nodes between the Quechua (and

nate an Amazon (below) and Andean group (above) could be distin-

Aymara) and Ashaninka among the B2 and D1 haplotypes.

guished; an area of overlap lies between the two groups in the middle

AMOVA analysis was performed with 15 Peruvian populations

of the plot. The Huambisa appear more isolated than in the previous

present in the dataset (Table S4). In total, 452 mtDNA HVSI haplotypes

plot, based on the mtDNA data, while the Shipibo and Cashibo remain

were extracted from the dataset and processed. We classified the pop-

in a remote position in the left part of the plot within other populations

ulations on the basis of their geographical distribution (Andes or Ama-

of Brazilian Amazon. Ashaninka, on the other side, falls in the middle of

zon). The AMOVA tests indicated that those living in the Peruvian

the plot lying closer to the Andean group, which includes the two

Andes have a lower variance between populations (9.37%) than the

others Amazon Arawak present in the dataset: Yanesha (Peru) and

Peruvian Amazon people (12.86%), as expected by previous data (Call-

Wayuu (Colombia).

egari-Jacques et al., 2011; Yang et al., 2010), and that a relevant percentage of variance between the two groups exists.

This trend seems to be confirmed by the AMOVA results (Table S4) which indicate that Andes have a smaller percentage of variance between populations than Amazon (9.09 vs. 12.93%) as happens for mtDNA, but the variation observed between Andes and Amazon is

3.2 | Y chromosome

lower for Y chromosome than for mtDNA (3.37 vs. 9.59%). The prevailing haplogroup found in the samples on the basis of the Y-

The networks built on Y-STRs haplotypes (Figures S3 and S4),

STRs profiles (Table S1) is the native haplogroup Q. When we analyzed

unlike the mtDNA network, indicate that all the nodes shared between

the SNPs to investigate the sub-haplogroups of Q, we found that most

Peruvian Amazonian and Andean people include only Yanesha (Ama-

of the samples belong to sub-branch Q1a3a*. Interestingly, in Huam-

zon Arawak) and Aymara or Quechua (Andean), while Cashibo and Shi-

bisa sample we found five individuals displaying the basal M346–

pibo samples show a reduced distribution, having no sharing with

Q1a3* mutation without the downstream M3–Q1a3a*, a less frequent

anyone, and Ashaninka have one haplotype shared with the Peruvian

combination in South America (Bailliet et al., 2009).

Arawak Yanesha.

The standard diversity indices calculated for the Y chromosome

We also searched for signals of transmission of Y lineages success

data shown in Table S3 indicate that the Cashibo comprise the most

over generations by analyzing the Y descent clusters (DCs) recently

homogeneous sample, as they did for the mtDNA.

published by Balaresque et al. (2015) to detect recent episodes of male


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Features of the three descent clusters (DCs): number of microsatellite profiles per DC (N), haplogroup (HG), maximum number of mutational steps from core, countries where the DCs have expanded, and maximum values of frequency and variance found for each DC

T A B LE 2

DCs

N

HG

Max steps from core

Countries

Max frequency

Max variance

DC1

70

Q1a3a

5

Peru–Bolivia

81% (Peru)

0.078 (Peru)

DC2

82

Q1a3a

7

Mexico–Colombia–Venezuela–Brazil–Peru– Bolivia–Argentina

54% (Argentina)

0.25 (Mexico)

DC3

26

Q1a3a

3

Peru–Argentina

77% (Argentina)

0.07 (Peru)

lineage expansion across the continent. To achieve this goal, our novel

decrease going south, but the maximum frequency was found in popu-

Peruvian Y-microsatellites data were combined with other 1,440 pub-

lations of the Argentine Pampas (54%). The DC3 is smaller and includes

lished data points in order to search for the most frequent haplotypes

prevalently Argentine haplotypes from the Salta province and Peruvian

and their associated DCs. In total, 1,514 Y chromosome microsatellite

Andeans, with the maximum variance found in the Peruvians (0.07).

profiles belonging to 53 populations distributed from Mexico to Argen-

Figure S5 shows the distribution of frequencies for the three clusters

tina were analyzed (only Q haplogroups were taken into account). We

(DC1, DC2, and DC3). TMRCA was estimated for all the three descent

ranked the haplotypes by frequency, because particularly frequent hap-

clusters using BATWING with an exponential growth model. Table 3

lotypes should represent potential “cores” for the descent clusters, indi-

illustrates the resulting TMRCA estimates for all three DCs. DC2 is the

cating past expansions of Y lineages. The entire dataset contained 853

oldest cluster, having originated during American prehistory; DC1 and

haplotypes (gleaned from a total of 1,514), 639 of which (75%) were

DC3 are younger, having originated around 400 and 176 years BCE,

unique. And 17 haplotypes (1.9%) were present >10 times in the

respectively, in Peru.

dataset. Among these 17 haplotypes, those found within the same population or within the same restricted geographical area were systematically discarded. The three resulting haplotypes that remained after these filtering procedures were used as “core haplotypes” for the

4 | DISCUSSION 4.1 | Genetic landscape of the Peruvian Amazon

cluster analysis, shown in Table S5. Each of these three frequent haplo-

T2

types was used to define a DC centered upon them, using the script

The four native communities analyzed in this study are strategically

“Cluster Generator,” which extracts the DCs from the database. Table

important because of their location in the Peruvian rainforest, a poorly

S6 illustrates for each “core haplotype” all the haplotypes that the

studied but wide ecoregion that spans the foothills of the Andes to the

script has included in the DC and their distance from the core (1, 2, 3,

vast Amazon lowlands. These peoples still live in a state of isolation

and more mutational steps). The resulting descent clusters are called

within their Amazon settlements at different latitudes, very likely pre-

DC1, DC2, and DC3. Their most important features are illustrated in

serving their genetic pool from admixtures, and are ideal candidates to

Table 2: the number of haplotypes per DC (N), the haplogroup, the

clarify whether genetic exchanges with Andean regions could have

maximum number of mutational steps from the core haplotype, and

occurred during the precolonial times. The genetic data collected from

the countries in which the DC has been found. We also calculated the

these communities were first analyzed to assess the degree of genetic

frequency and the variance of the DCs for each country; the frequency

diversity in the Peruvian Amazon native populations.

can be used to identify the place where the maximum expansion of

As in most Peruvian and Bolivian Andeans, in the Huambisa we

DCs occurred, while the variance could suggest the most likely place of

observed a high percentage of mitochondrial haplogroup B2 (Table S2),

origin for each cluster. In the DC1, the Andean haplotypes prevailed,

while in the Cashibo and Shipibo the most represented group was C1,

with Quechua and Aymara populations dominating the cluster, with

a haplogroup previously observed along the northwestern portion of

some Amazon haplotypes from the Arawak Machiguenga and Yanesha

the continent (especially in Peru), with additional high spots in southern

populations. The maximum value of the microsatellite variance for DC1

Brazil, northern Argentina, and Chile. The Ashaninka sample shows a

is found in Peru (0.078), while the lowest is found in Bolivia (0.005). In

higher incidence of haplogroup D, typical of the contemporary Amazo-

the DC2, the Argentine haplotypes from Chaco province prevailed, fol-

nian populations (Bisso-Machado, Bortolini, & Salzano, 2012). Regard-

lowed by the Bolivian and Peruvian haplotypes (Andeans and Yanesha);

ing chromosome Y, only two sub-branches of the Q haplogroup were

the maximum variance of DC2 was found in Mexico (0.25) with a

found: Q1a3a, defined by the Y-SNP M3, and Q1a3, defined by M346

T A B LE 3

TMRCA estimates, growth rate for generation (alpha) and maximum variance value

DCs

TMRCA (95% CI)

Alpha

Max variance (pop)

Period

DC1

2,415 (727–5,520)

0.0369

0.078 (Peru)

400 BCE

DC2

5,637 (1,801–12,375)

0.0214

0.25 (Mexico)

3,600 BCE

DC3

2,191 (425–6,395)

0.0249

0.07 (Peru)

176 BCE


T3


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(Table S1). The former dominates in all samples, while the latter has

mtDNA and Y chromosome). At the same time, Andean populations

been found only in the Huambisa. Most of the South American natives

are genetically more heterogeneous than Amazonian ones, as found in

possess the founder Y chromosome defined by M3 and commonly

previous researches that evidenced contrasting patterns of genetic

classified as Q1a3a, while the upstream paragroup Q1a3 has been

variation in Western (mostly Andean) versus Eastern South America,

observed with a lower frequency in South America. Many studies have

characterized by lower within-population diversity (and greater differ-

shown that the distribution of the Q1a3a lineage encompasses North,

entiation) in the East relative to the West (Bodner et al., 2012;

Central, and South America in a star-like shape phylogeny (Battaglia

Callegari-Jacques et al., 2011; Yang et al., 2010). Also within the Peru-

et al., 2013) indicative of a first general north-to-south diffusion, fol-

vian territory, we found that genetic structure follows the contrasts

lowed by local expansions in Canada, Mexico, and the Andean region.

between the two environments: Andes and Amazon, being greater for

In contrast, Q1a3 (M346) is distributed mainly along the northwest

mtDNA than for Y chromosome as stated by AMOVA (Table S4) and

border of South America, and it was previously described only in

previously reported by other authors (Luiselli, Simoni, Tarazona-Santos,

Argentine and Bolivian populations (Bailliet et al., 2009).

Pastor, & Pettener, 2000; Tarazona-Santos et al. 2001). This is probably

The lowest values of genetic diversity for both markers have been

due to different population histories in these distinct geographical

found in the Cashibo community (Table S3), the least contacted and

areas, with a lower genetic drift effect in the Andes than in the rest of

smallest community, while the others three samples (Shipibo, Asha-

the country.

ninka, and Huambisa) exhibit levels of diversity more similar to the

Excepting the Cashibo sample, all the other samples analyzed in

Andean populations, especially in mtDNA indices, that is unusual for

the present study fall within the typical variability of the contemporary

Amazon people. We know from the historical sources that Cashibo

Western populations. On the basis of the haplogroup frequencies, it

probably moved to Ucayali from eastern Bolivia between the third and

can be argued that a noticeable effect of the genetic drift is particularly

sixth centuries CE together with Shipibo (Noble, 1965) and they likely

evident in the Amazonian populations. According to the PCA (Figure

began warring with the other Pano groups (also Shipibo) in the

S1), only the Huambisa sample stayed quite close to the Quechua and

Pampa/Sacramento area soon after. They did not cease warring until

Aymara from the Central Andes, together with the populations inhabit-

the twentieth century; this further contributed to their actual state of

ing the highest areas of the Peruvian Amazon (the Yanesha and Machi-

isolation. The Cashibo widely practiced the sororal polygyny (the old-

guenga). A clean-cut genetic ‘divide’ between the Andes and Amazon

est daughter of a family gets married and her sisters become her co-

emerged in the nMDS analysis, based on HVSI of mtDNA (Figure 2). In

wives), a practice that is evidenced by the lower values of their

the nMDS, based on Y-STRs, this partition is less clear (Figure 3), prob-

mtDNA diversity indices. Shipibo, on the other hand, joined to Conibo

ably because Y chromosome STRs are more informative for recent con-

and Setebos in the last centuries to drive away the missionaries from

tact effects. On the maternal side, only the Huambisa people clearly

Ucayali, forming a group that is considered today as a unique ethnic-

point towards the Andean variability, likely because they belonged in

ity: the Shipibo-Conibo. During this period started the fusion Shipibo Setebos - Conibo, a process ended in the mid-XX century CE (Lathrap, 1970), which led to their actual rate of diversity. Also the Ashaninka, the larger population of the Peruvian Amazon, and the Huambisa shared their territory with others populations, and experimented exchanges for commercial reasons (the salt trade, for instance), especially in the last decades (Mayor Aparicio & Bodmer, 2009), so that we can assume a higher degree of gene flow or a higher effective population size if compared with the other inhabitants of the Amazon basin. If we look at mtDNA network (Figure S2), Cashibo do not seem to have had genetic exchanges with the other Peruvian Amazon communities analyzed, while there is some maternal gene flow among the Shipibo and their neighbors, Ashaninka. The unique haplotype shared between the Ashaninka and Yanesha in the Y chromosome Q1a3a network (Figure S4), together with the low Rst index, could suggest a recent flow of paternal lineages or a common origin for these Arawak populations. Generally, the high percentage of genetic variation observed between the Amazon populations of Peru, as measured via AMOVA (Table S4) indicates that there is a greater heterogeneity in these populations than in the Andean ones.

4.2 | Relationships between Andes and Amazon

ancient times to a conjunct of ethnicities of the northern Andes, known as the Palta-Jivaro, that has since been disrupted (Taylor, 1991). As such, they were originally more closely related to the Andean peoples. However, on the paternal side, they could have lost this heredity with successive events of admixture. The Ashaninka and Yanesha (both affiliated with the Arawak linguistic group) fall closer to the variability of the Andean Quechua and Aymara, if we look at their male contributions. Previous studies have highlighted a genetic signature of Andeans in the Arawak peoples of the Peruvian Amazon (Barbieri et al., 2014; Sandoval el al., 2013), and high-frequency Y microsatellite haplotypes can signal past episodes of high reproductive success of one or more men and their patrilineal descendants (Xue et al., 2005). Accordingly, we performed a cluster analysis using the software Cluster Generator designed by Balaresque et al. (2015) to investigate whether population expansions of Y lineages across the entire South American continent also involved populations from the Amazon rainforest. Indeed, only the Amazonian people of Ecuador, Peru, and Bolivia could have been involved in the population expansions from the Andean regions, as reported by other authors (Scliar et al., 2014). As shown in Figure S5, we found one cluster (DC2) spanning from Mexico to Argentina that originated in 3,600 BCE according to its TMRCA (Table 3) and probably

At a continental level, the Peruvian and Bolivian populations have the

representing one of the late prehistorical migrations from Mesoamerica

highest rates of diversity, while the Brazilians have the lowest (for both

towards the southern part of the continent, since the maximum



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variance was found in Mexico. Moreover, we found two descent clusters (DC1 and DC3) that expanded across the western area of the continent and likely originated in Peru around 400 and 176 years BCE, respectively, according to an estimate by TMRCA (Table 3). This period preceded the age of the great empires in the Central Andes, and was characterized by various emergent complex societies that built peculiar types of public architecture focused on religion and ceremonies, as we observe at the site of Kotosh (2,500 BCE–100 CE) and Chavín de Huantar (1,200 BCE–400 BCE). Particularly, the historical development of Chavín de Huantar is seen as a cultural ‘horizon’, due to its wide artistical and religious influence in the Peruvian Andes that suggest some form of cultural integration (Moore, 2014). The two successful male lineages clusters DC1 and DC3 originated in that period, also called Formative Period, even if we cannot exclude that they underwent further and later expansions during the following ages of States and Empires. Interestingly, each of these two clusters includes a certain number of Peruvian Amazon Arawak (mostly Yanesha) whose place of origin is still debated, confirming the existence of a male-mediated gene flow among the Andean people and the Amazonian Arawak. We do not know how and when the Arawak went to the eastern buttresses of the Andes, but, according to the historical sources, it is possible that their east-west expansion (Aikhenvald, 1999), which was further prompted by the migrations of the Pano people, led them to these heights. Hence, although the current database does not include populations from all over the Peruvian Amazon, from the actual data we can nevertheless hypothesize that the AndesAmazon divide has not been always a sharp obstacle to the population exchanges.

ACKNOWLE DGMENTS This study was supported by the Peruvian NGO “Sin razas, sin fronteras”. DL and SS are supported by the European Research Council ERC-2011-AdG 295733 grant (Langelin). We are grateful to all the Shipibo, Cashibo, Ashaninka, and Huambisa people who participated in this project, and we thank Ornella Maggiulli for assisting in the

9

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S UPPORTING IN FORMATION

TABLE S3 Diversity indices based on mitochondrial data (HVSI) and

Additional Supporting Information may be found online in the sup-

Y chromosome data (15 STRs)

porting information tab for this article.

TABLE S4 AMOVA

FIGURE S1 PCA analysis performed on mtDNA haplogroups

TABLE S5 Core haplotypes launched in “Star Cluster Generator”

frequencies

software

FIGURE S2 Median joining network based on HVSI and HVSII poly-

TABLE S6 Descent clusters 1, 2, 3 (DC1, DC2, DC3)

morphisms and some coding region SNPs FIGURE S3 Median joining network based on Q1a3* haplotypes FIGURE S4 Median joining network based on Q1a3a* haplotypes

How to cite this article: Di Corcia T, Sanchez Mellado C, Davila

FIGURE S5 The distribution of frequencies for DC1 (A), DC2 (B) and

TJ, et al. East of the Andes: The genetic profile of the Peruvian

DC3 (C)

Amazon populations. Am J Phys Anthropol. 2017;00:1–11.

TABLE S1 The eight SNPs tested for Q phylogeny

https://doi.org/10.1002/ajpa.23209

TABLE S2 Frequency of the haplogroups A, B, C, and D in Native South American populations
