The samples were genotyped for five Y‐SNPs found in the South American natives, M130, M242, M346, L54 and M3 (Karafet et al., 2008; Jota et al., 2011), using TaqMan assays (ABI) and a 7900HT Fast Real‐Time PCR System (ABI). Next, the Q‐M3 and Q‐M346* lineages were genotyped with 17 Y‐chromosome short tandem repeats (Y‐STRs) using Y‐filer Kit (ABI). The PCR reactions were performed following Sandoval et al. (2013b) and their products were subjected to capillary electrophoresis using the ABI 3130XL Genetic Analyzer (Applied Biosystems), with the alleles being visualised by the GeneMapper ID v3.2 software (Applied Biosystems, Foster City, California, USA). The DYS389b allele scoring was done by subtracting DYS389I from DYS389II (Zerjal et al., 1997) and the DYS385 marker was not included in the statistical analyses.
The complete mtDNA control region (1122 bp, 16024–576) according to the revised Cambridge Reference Sequence (rCRS; Andrews et al., 1999) was amplified and sequenced following Sandoval et al. (2013b), using a 3130XL Genetic Analyzer (ABI) and Big Dye Terminator v. 3.1. protocol. The sequence alignments were performed in relation to rCRS through SeqScape 2.6 software (Applied Biosystems), and major haplogroup assignment was obtained by MitoTool (Fan & Yao, 2011) or haplogroup prediction tool from the Genographic Project (Behar et al., 2007). Due to phylogenetic uncertainty and alignment controversy, the alignments at nucleotide positions 309.1C, 315.1C, indels at 515–522, 16182C, 16183C, 16193.1C and 16519 were not taken into account in the statistical analyses.

Y‐Chromosome Results
After SNP and STR genotyping in all 15 Peruvian and Ecuadorian populations, we considered only Q‐M3 Y‐chromosome haplotypes (n = 277) for the analyses because they represent the dominant autochthonous lineage that appeared in all populations. The list of 17 Y‐STR haplotypes of Q‐M3 and Q‐M346* lineages obtained for the studied populations is found in Tables S1a and S1b.

To reveal the genetic relationships among the Y‐STR haplotypes at the individual level, we used them to generate median joining networks (Bandelt et al., 1999). Thus, in Figure Figure3,3, we observe shared haplotypes between some Quechua‐Lamistas and individuals from different Amazonian ethnic groups (Achuar, Shipibo, Yine and Andoas locality), including one Cocama‐speaking individual from Iquitos as well as from Loreto, San Martin and Chachapoyas (Fig. S1). In some branches, the Quechua‐Lamistas were clustered according to their clan “relatives” (differentiated by one or two mutation steps), which were identified by patrilineal connections, and a family surname. There were five recognisable male lineage clusters (see also, Table S1a), here named from W1 to W5. Clan W1 (haplotype code = 125, n = 8) is connected to Machiguenga‐speaking individuals (Fig. S2). Another clan was W2 (haplotype code = 26, 27 and 29, n = 4) is derived from haplotypes appearing in Kichwa and Shawi individuals. A third clan was W3 (haplotype codes = 143 and 174, n = 5), which shared a haplotype with Andoas, and was connected by one mutation step to another Quechua‐Lamista, and by four mutation steps to an individual from Andoas. In Figure S1, we show also that the W3 clan shared a haplotype with an individual from the San Martin Department. A fourth clan was W4 (haplotype code = 47 and 48, n = 3), which was linked by one mutation step to a Quechua‐speaking individual from Ecuador (Fig. (Fig.3).3). Also, haplotype 48, which belongs to the same W4 clan, shared a haplotype with an individual from Chachapoyas (Fig. S1). In addition, we observe a shared haplotype (code = 49) between Quechua‐Lamistas (n = 2) and Andoas (n = 2), which is connected by three mutation steps to a Yine‐speaking individual. In another branch, connected to the W4 clan, we observe a shared haplotype between Yine (n = 1) and Quechua‐Lamista (n = 1; Fig. Fig.3,3, and haplotype code = 79). Clan W5 (haplotype code = 55, n = 2) shared a haplotype with one Achuar‐speaking individual from Rio Tigre (Loreto), a lineage located in a separated branch. In another branch, we observe a shared haplotype (code = 73) between Quechua‐Lamista (n = 1) and a Shipibo (n = 1), a Cocama, and two individuals from Loreto Department (Fig. S1). Finally, haplotype 73 is connected by two mutation steps to a shared haplotype between Achuar and Andoas (Fig. (Fig.3;3; haplotype code 78 in Table S1a). Despite the fact that other haplotypes from Quechua‐Lamistas are heterogeneous and differentiated by several mutations from the five main clans, most of them appeared to be more connected to individuals from Amazonia than to Andean people.

We identified 174 mtDNA CR haplotypes (GenBank accession numbers for 168 new sequences: KT997553‐KT997720). At the individual level, we conducted a phylogenetic analysis of mtDNA CR sequences using the median joining algorithm. The genetic relationship of the mtDNA control region haplotypes among the 404 individuals from 15 populations is shown in Figure Figure5.5. There are two interesting features about the Quechua‐Lamistas’ maternal lineages. First, independently of the mtDNA haplogroup, 37 out of 40 individuals from Lamas do not share haplotypes with individuals from other ethnic groups, which could suggest a separation of several generations from other related Amazonians. Another feature is that there is much less genetic differentiation between Quechua‐Lamistas and Amazonians, than between Quechua‐Lamistas and Andeans. The complete set of polymorphic sites of mtDNA sequences or haplotypes relative to rCRS is listed in Table S2a and the most common haplogroup found among the Quechua‐Lamistas was B2. Regarding this lineage, we observed many haplotypes shared among Quechua‐Lamistas, mostly from Wayku and Pamashto, but also from Chumbakiwi and other nearby localities (haplotype code = 157, in Table S2a). In addition, common mtDNA haplotypes were correlated with the self‐declared clans identified by “surname” (W1, W3, W4), appeared only among Quechua‐Lamistas (code = 157). In another branch, two Chopccas and two Chankas shared haplotypes (code = 113), and few other haplotypes were shared between individuals speaking different languages. For example, there was a shared haplotype (code = 50) between Chopcca (n = 1), Jivaro (Awajun, n = 3), Quechuas from Ecuador (Quichua, n = 2; Karanki, n = 2) and Andoas (n = 1). Among other haplotypes, the Jivaroan (n = 6) communities and Andoas (n = 1) shared a haplotype (code = 63); haplotype 34 was also shared between Andoas (n = 10), Huambisa (n = 1) and Kichwa_LO‐PTZ (n = 1); haplotype 163 was shared between Jivaro‐speaking groups (Huambisa, n = 4 and Awajun, n = 2); haplotype 170 was shared among Jivaro (Awajun, n = 1), Arawak (Yine, n = 1) and Andoas (n = 1); haplotype 111 was shared between Kichwa_LO (n = 4) and Jivaro (Achuar, n = 1); and haplotype 58 was shared between Andoas (n = 3) and Awajun (n = 1).

In A2 and D1 haplogroups, we observed shared haplotypes among Kichwa_LO, Jivaro (Achuar) and Quechua‐Lamistas as well as among the Kichwa_LO, Kichwa_PTZ, Andoas, Jivaro (Achuar) and Quechua‐Lamistas, respectively (see also Table S2a). Furthermore, in these haplogroups, most of the shared haplotypes were observed among Quechuas from Pastaza (Ecuador) and Loreto (Peru). However, in the C1 (with haplotype code = 85), one individual (a woman from the Tabalosos locality) from Quechua‐Lamistas (n = 1) shared a haplotype with Kichwa (n = 1), Andoas (n = 2), Jivaro (Achuar, n = 1; Huambisa, n = 1), a Quichua (n = 1) and a Chanka (n = 1). In B2 (haplotype code = 57) and C1 (haplotype code = 12), some haplotypes were shared by Quichua and Karanki from Ecuador, while in the D1, Quechuas and Chopccas from Huancavelica shared a haplotype (haplotype code = 131).

To characterise the genetic variability among the 15 Peruvian and Ecuadorian indigenous communities, we carried out the AMOVA analyses taking into account the pairwise haplotype differences according to Sandoval et al. (2013b) There was considerable variability within populations (Fis = 0.891) and an intermediate level of differentiation among them (Fst = 0.109). When ethnic groups were divided into two regional categories (Andes vs Amazonia), higher interpopulation differentiation was observed among Amazonian subpopulations (Fst = 0.1397, p = 0.000) than among Andeans (Fst = 0.0073, p = 0.229; Table S2b). The haplotype diversity indices and neutrality tests (Tajima's D and Fu's Fs statistics) showed high intrapopulation genetic diversity among Andean populations (Quichua and Karanki, Quechua_Chopcca‐HVC and Chanka) in contrast to Amazonians which had lower values, but with the exceptions of Andoas, Jivaro (Achuar) and Kichwa_LO (Table S2c). The nucleotide diversity indices (π) among the Quechua‐Lamistas were similar to those of Awajun and Huambisa populations. The distribution of mtDNA haplogroup frequencies among the 15 Peruvian and Ecuadorian communities (n = 404) is shown in Table S2d.
The MDS plot based on the Reynolds’ linearised distances of mtDNA's Fst estimates shows that Quechua‐speaking populations from Peru and Ecuador were grouped in a compact cluster in comparison to the Amazonian communities (Fig. (Fig.6),6), and in agreement with previous observations. In addition, the Jivaro populations (Awajun, Huambisa) were associated with each other as expected due to their higher B2 haplotype frequencies, as well as to the Quechua‐Lamista population. However, the Achuar population was located at the opposite side of the two‐dimensional space because of the high frequency of A2 haplotypes. Notwithstanding this configuration in the MDS, the Awajun, Huambisa and Achuar populations belonged to the same Jivaroan language family. Furthermore, there was a close relationship between the Achuar and Quechua speakers from the Loreto and Pastaza River (Kichwa_LO; Kichwa_PTZ), such as Pano (Shipibo) and Cahuapana (Shawi).

In addition, we included the control region mtDNA (16024‐16365 bp) haplotypes of several individuals (n = 51) from lowland Amazonia‐Yurimaguas (Justice et al., 2012) and Chachapoyas (n = 14, from Amazonia‐Andes) in the network analysis. The phylogenetic tree showed that Quechua‐Lamistas and Chankas shared some B2, A2 and C1 haplotypes amongst them and with other individuals from different Amazonian groups (Fig. S4). In the D1 lineage, one Quechua‐Lamista shared a haplotype with the Quechua speakers from Pastaza and Loreto as well as Andoas, Jivaro (Achuar) and Yurimaguas. At the population level, analyses using A2, B2, C1 and D1 haplogroup frequencies, populations of Yurimaguas, Chachapoyas and Andoas showed intermediate levels of genetic diversity relative to Andean and Amazonian populations. Besides, the set of mtDNA haplogroup frequencies among Quechua‐Lamistas ranged between that of Quichua‐Karanki from Ecuador and those of the Huambisa‐Awajun populations (Fig. S5).

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