Y-Chromosome Variability in Four Native American Populations from Panama

Home , Haplogroup, Haplogroup R (Y-DNA)

1 Y-Chromosome Variability in Four Native American Populations 2 from Panama 3 4 5 marina s. ascunce,1,2* angelica gonzález-oliver,1,3* and connie j. 6 mulligan1 7 8 9 Abstract The allele and haplotype frequencies for 13 Y-chromosome short tandem repeats (STRs) [nine STR loci of the minimal Y-chromosome haplo- 10 type (DYS19 -DYS385a -DYS385b -DYS389I -DYS389II -DYS390 -DYS391 - 11 DYS392 -DYS393) plus four additional loci (DYS388, DYS426, DYS439, 12 DXYS156)] were determined in 99 males from 4 Panamanian native American [First Page] 13 populations, including the Chibcha-speaking Ngöbé and Kuna and the Chocó- 14 speaking Emberá and Wounan. Fifty haplotypes were identified, of which 48 [287], (1) 15 (96%) were specific to a single population and 29 (63%) were found in only 16 a single individual. Gene diversity per locus per population ranged from 0 17 to 0.814, with the highest gene diversity present at the DYS389II locus in the Lines: 0 to 45 Emberá. The haplotypic discrimination capacity was low, ranging from 42.3% 18 ——— 19 in the Kuna to 63.1% in the Wounan. The four tribes showed a high degree of differentiation both at the Y chromosome and in the mitochondrial genome, -0.32797pt PgVar 20 ——— highlighting the importance of genetic structure even in geographically prox- 21 imate and linguistically related populations. Custom Page (6.0pt) 22 PgEnds: TEX 23 24 Many studies have pointed out the importance of language, cultural differences, 25 and geographic distance as barriers to gene flow between neighboring populations. [287], (1) 26 In particular, the sex-specific modes of inheritance of the mitochondrial genome 27 (maternal lineage) and the Y chromosome (paternal lineage) allow the descrip- 28 tion of female and male demographic patterns, respectively, which may be af- 29 fected by different behaviors such as marriage practice. Seventy percent of human 30 populations practice patrilocality customs, in which newly married women move 31 into the natal household of their husbands (Murdock 1981). On the other hand, in 32 matrilocal populations the women stay in their birthplace and the men move. In 33 comparisons of mitochondrial and Y-chromosome data, some investigators have 34 35 1Department of Anthropology, University of Florida, 1376 Mowry Road, Gainesville, FL 32610. 2Current address: USDA-ARS Center for Medical, Agricultural, and Veterinary Entomology, 1600 SW 23rd 36 Drive, Gainesville, FL 32608. 37 3Departamento de Biología, Facultad de Ciencias, Universidad Nacional Autónoma de México, Mexico 38 City 04510, Mexico. *These authors contributed equally to this work. 39 40 Human Biology, June 2008, v. 80, no. 3, pp. 287–302. 41 Copyright © 2008 Wayne State University Press, Detroit, Michigan 48201-1309 42 key words: y chromosome, short tandem repeats (strs), haplotypes, 43 native americans, ngöbé, kuna, emberá, wounan, forensics, lower cen- 44 tral america, panama, colombia, venezuela, DYS19, DYS385, DYS388, DYS389, DYS390, DYS391, DYS392, DYS393, DYS426, DYS439, DXYS156.

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1 observed low differentiation at the mitochondrial DNA (mtDNA) level and high 2 differentiation for the Y chromosome, suggesting that there is genetic evidence for 3 a higher global female than male migration rate in humans through patrilocality 4 (Seielstad et al. 1998). Other studies have proposed that patrilocality effects are 5 evident only on the local and regional scale (Stoneking 1998; Hammer et al. 2001; 6 Oota et al. 2001). 7 Over the last 20 years, the evolutionary history of New World peoples has 8 been the subject of considerable research to understand the colonization of the 9 Americas (Wallace et al. 1985; Schurr et al. 1990; Schurr and Sherry 2004; Torroni 10 et al. 1993; Kolman et al. 1996). The presence of four major founder American 11 mitochondrial DNA haplogroups (A, B, C, and D) was originally interpreted as 12 indicating more than one migratory wave during the initial colonization of the 13 Americas (Horai et al. 1993; Torroni et al. 1993). However, other mtDNA studies 14 have proposed a single migration to the continent (Kolman et al. 1996; Merri- [288], (2) 15 wether and Ferrell 1996; Bonatto and Salzano 1997; Silva et al. 2002). In the last 16 10 years, Y-chromosome evidence has supported the occurrence of one (Pena et 17 al. 1995; Santos et al. 1996; Underhill et al. 1996; Zegura et al. 2004) or two Lines: 45 to 59 18 major male migrations (Karafet et al. 1999; Lell et al. 2002; Bortolini et al. 2003). ——— 19 Two groups of investigators have evaluated sex-biased migration patterns in some 0.0pt PgVar 20 native American groups (Mesa et al. 2000; Bortolini et al. 2002). Neither group ——— 21 found a different migration rate between sexes in the native American populations Normal Page 22 analyzed, but a north to south gradient of increasing genetic drift in the Americas PgEnds: TEX 23 has been suggested by other investigators (Cavalli-Sforza et al. 1994). 24 Because of Panama’s unique geographic position, as the land bridge between 25 North and South America, several investigators have suggested that this area was a [288], (2) 26 dynamic migration corridor through which Paleo-Indians traveled repeatedly dur- 27 ing colonization of the New World (Bartlett and Barghoorn 1973; Linares 1977; 28 Piperno 1988). These studies led to the hypothesis that native Americans from 29 lower Central America would exhibit high genetic diversity. Chibcha-speaking 30 tribes are distributed along lower Central America, extending from eastern Hon- 31 duras to northern South America, reaching east of Lake Maracaibo in Venezuela 32 (Hoopes and Fonseca 2003). Genetic studies using both protein polymorphisms 33 (Barrantes et al. 1990; Thompson et al. 1992) and mtDNA evidence (Batista et al. 34 1995; Kolman et al. 1995; Kolman and Bermingham 1997; Melton et al. 2007) 35 have found that the Chibcha present low genetic diversity and a high level of 36 differentiation, reflecting an isolated long-term presence in lower Central Amer- 37 ica. However, based on the analysis of five Y-chromosome markers, Kolman and 38 Bermingham (1997) did not find a significant genetic differentiation at the Y- 39 chromosome level when comparing four tribes from Panama. Recently, a study 40 of five Chibchan tribes, four from Costa Rica and one from Panama, indicated 41 a genetic diversity structure on the basis of nine markers on the Y chromosome 42 (Ruiz-Narváez et al. 2005). 43 In the present study, we describe the genetic variability at 13 Y-chromosome 44 STR loci in four native American populations from Panama: the Chibcha-speaking

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 [289], (3) Figure 1. Distributions in Panama of the four native American tribes analyzed. The geographic range 15 of each population is indicated by lines encircling the letters, and letters represent collec- 16 tion sites. N, Ngöbé; K, Kuna; E, Emberá; W, Wounan. 17 Lines: 59 to 64 18 ——— 19 Ngöbé and Kuna and the Chocó-speaking Emberá and Wounan. The Y-chromo- -0.19199pt PgVar 20 some STR loci analyzed in this study consist of the nine STR loci of the min- ——— 21 imal Y-chromosome haplotype deﬁned by the Y Chromosome Haplotype Ref- Normal Page 22 erence Database (available at http://www.yhrd.org/) (DYS19-DYS385a-DYS385b- PgEnds: TEX 23 DYS389I-DYS389II-DYS390-DYS391-DYS392-DYS393) plus four additional loci 24 (DYS388, DYS426, DYS439, DXYS156). The new Y-chromosome data, which in- 25 [289], (3) clude a larger number of individuals and more Y-chromosome markers than in pre- 26 vious publications (Kolman and Bermingham 1997; Karafet et al. 1999; Zegura et 27 al. 2004), allow us to evaluate the genetic differentiation among these Panamanian 28 populations by comparing Y-chromosome and previously reported mitochondrial 29 data. We also incorporate previously published Y-chromosome data of other native 30 American populations for a comprehensive study of the lower Central American 31 and northern South American region in terms of male genetic structure. We ob- 32 served different patterns of genetic differentiation, highlighting the importance 33 of generating a regional Y-chromosome database for evolutionary and forensic 34 purposes. 35 36 37 Materials and Methods 38 39 Samples. Blood samples were collected from individuals from four Amer- 40 indian groups of Panama: Ngöbé and Kuna, belonging to the Chibcha linguis- 41 tic family; and Emberá and Wounan, representing the Chocó linguistic family 42 (Figure 1). DNA was isolated from blood samples using proteinase K digestion 43 of leukocytes followed by organic extraction and ethanol precipitation (Kolman 44 and Bermingham 1997). Ninety-nine males were included in the Y-chromosome

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1 microsatellite analysis: 32 Ngöbé, 26 Kuna, 22 Emberá, and 19 Wounan. Pre- 2 viously published mitochondrial control region I sequences from 46 Ngöbé, 63 3 Kuna, 44 Emberá, and 31 Wounan (Batista et al. 1995; Kolman et al. 1995; Kol- 4 man and Bermingham 1997) were incorporated into the study. RFLP analysis was 5 performed on the sequenced individuals to define mtDNA haplogroups following 6 the haplogroup definition by Torroni et al. (1993). Six sets of primers were used 7 in balanced PCR reactions to screen for seven polymorphic sites located outside 8 mtDNA control region I, including HaeIII (bp 663), AluI (bp 5176), COII/tRNALys 9 deletion (bps 8272–8289), DdeI (bp 10394) and AluI (bp 10397), AluI (bp 13262), 10 and HaeIII (bp 16517) (Batista et al. 1995; Kolman et al. 1995; Kolman and 11 Bermingham 1997). 12 13 Y-Chromosome STR Analysis. Eight Y-chromosome STR loci were ampli- 14 fied for the 99 Panamanian males using previously reported primer pairs (Kayser [290], (4) 15 et al. 1997; Thomas et al. 1999; Bosch et al. 2002). Six STRs (DYS19, DYS388, 16 DYS390, DYS391, DYS392, and DYS393) were amplified in one multiplex reac- 17 tion, which was slightly modified from Thomas et al. (1999) following Bosch et Lines: 64 to 70 18 al. (2002), and two STRs (DYS389I and DYS389II) were amplified separately fol- ——— 19 lowing Bosch et al. (2002). PCR reactions were carried out in 10-μl volumes con- -0.47998pt PgVar 20 taining 200 μM each dNTP, 0.02 ng/ml BSA, 2.2 mM MgCl2, 0.08 unit AmpliTaq ——— 21 Gold DNA polymerase (Applied Biosystems, Foster City, California), 1× PCR Normal Page 22 Gold buffer, 2 μl of DNA template, and 1 μlof10× mix primers. Amplifications PgEnds: TEX 23 were performed in a GeneAmp PCR System 9700 (Applied Biosystems) using the 24 PCR parameters described by Bosch et al. (2002). Aliquots (0.5 μl) of the PCR 25 products were run on a CEQ 8000 Genetic Analysis System (Beckman Coulter, [290], (4) 26 Fullerton, California) using the program Fragment 3 with default conditions. Al- 27 lele sizes were determined automatically using the Fragment Analysis program 28 of the CEQ 8000 Genetic Analysis software. Y-chromosome STR alleles were 29 labeled according to the number of repeat units, which was established using ref- 30 erence DNA samples provided by Mark Thomas. Previously published data on five 31 Y-chromosome markers (DYS385*A, DYS385*B, DYS426, DYS439, DXYS156; 32 Kolman and Bermingham 1997; Karafet et al. 1999; Zegura et al. 2004), assayed in 33 a subset of the samples analyzed (17 Ngöbé, 9 Kuna, 10 Emberá, and 14 Wounan), 34 were also incorporated into the current study. Haplogroups were named according 35 to the proposals of the Y Chromosome Consortium (2002). 36 37 Analysis of Data. Y-chromosome allele frequencies, number of polymorphic 38 loci, and haplotypic diversity based on Nei (1987) were calculated using Arlequin, 39 version 2.00 (Schneider et al. 2000). To determine the power of our sample sizes to 40 detect differences in allele and haplotype frequencies, we followed Allendorf and 41 Luikart (2007), using the product rule probability to evaluate the minimum sample 42 size necessary to detect rare alleles or genotypes. The probability of not detecting 43 an allele at frequency p = 0.1 in a sample size of x is x(1 − p). Therefore the 44 sample size required to have a 95% chance of sampling an allele with a frequency

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1 of 0.10 is 29 haploid individuals. The Ngöbé presented an adequate sample size 2 and the Kuna presented a borderline adequate sample size, but both the Emberá 3 and Wounan did not, indicating the need for further studies including more samples 4 from these populations to confirm the results. 5 We measured the distribution of Y-chromosome diversity using an analy- 6 sis of molecular variance (AMOVA) (Excoffier et al. 1992) based on the sum of 7 squared differences (RST), as implemented in Arlequin, version 2.00 (Schneider et 8 al. 2000). The statistical significance of this test was evaluated using 1,023 random 9 permutations. Gene diversity based on Nei (1987), number of alleles sampled, and 10 allele richness were calculated using FSTAT (Goudet 2001). Haplotype discrim- 11 ination capacity was calculated as DC = H/N, where H is the total number of 12 different haplotypes and N is the total number of individuals in the sample (Kayser 13 et al. 1997). Y-chromosome haplotypes were compared with the worldwide Y 14 Chromosome Haplotype Reference Database to evaluate European and African [291], (5) 15 admixture. Comparative analysis of genetic structure of Y-chromosome diversity 16 was performed using previously published data from native American populations 17 from northern South America distributed in northern Colombia and Venezuela Lines: 70 to 163 18 (Ruiz-Linares et al. 1999; Mesa et al. 2000; Bortolini et al. 2003). These groups ——— 19 were assigned to the following linguistic families, as described by Bortolini et al. -0.15599pt PgVar 20 (2003): the Chibcha-Paenzan, Bari (n = 12) and Warao (n = 12); the equatorial ——— 21 Tucano, Wayuu (n = 15); the Ge-Pano-Carib, Zenu (n = 12); and with unknown Normal Page 22 linguistic affiliation, Yukpa (n = 11) (Ruiz-Linares et al. 1999; Mesa et al. 2000; PgEnds: TEX 23 Bortolini et al. 2003). 24 25 Results and Discussion [291], (5) 26 27 We examined 13 Y-chromosome STR loci in four Native American pop- 28 ulations from Panama: the Chibcha-speaking Ngöbé and Kuna and the Chocó- 29 speaking Emberá and Wounan. Allele frequencies within populations ranged from 30 0.031 to 1 (data not shown). Five unique alleles were found in the Ngöbé, three 31 in the Kuna, and two each in the Emberá and Wounan. The highest power of ex- 32 clusion (gene diversity) was found at locus DYS389II in all populations (Table 1). 33 DYS388 and DYS426 exhibited zero gene diversity in some populations, whereas 34 DYSX156-Y was monomorphic in all individuals (Table 1). When pooling all pop- 35 ulations, gene diversity values tended to increase relative to gene diversity in a 36 single population, demonstrating the risk of overestimating the exclusion capacity 37 when ethnic composition is not taken into consideration (Table 1). 38 Fifty haplotypes were identified, of which 48 (96%) were specific to a single 39 population and 29 (63%) were found in only a single individual (Table 2). Only 40 two haplotypes were shared, and they may reflect shared ancestry. The apparent 41 absence of gene flow among these populations, as indicated by the low number 42 of shared haplotypes, is striking, considering their geographic proximity, related 43 languages, and shared cultural practices (Constenla-Umaña 1991). This lack of 44 shared haplotypes is even stronger than the pattern observed among Chibchan

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1 Table 1. Gene Diversity, Number of Alleles, and Allelic Richness per Locus in 13 2 Y-Chromosome Short Tandem Repeats (STRs) in Four Panamanian Native American 3 Populations 4 All Ngöbé Kuna Emberá Wounan 5 Locus Measure (N = 99) (N = 32) (N = 26) (N = 22) (N = 19) 6 DYS19 Gene diversity 0.333 0.063 0.551 0.091 0.281 7 Number of alleles 32322 8 Allelic richness 2.577 1.839 2.931 1.984 2.000 9 DYS388 Gene diversity 0.060 0.063 0.157 0.000 0.000 10 Number of alleles 32311 Allelic richness 1.932 1.839 2.892 1.000 1.000 11 DYS389I Gene diversity 0.596 0.280 0.280 0.495 0.602 12 Number of alleles 33333 13 Allelic richness 2.999 2.838 2.931 2.993 3.000 14 DYS389II Gene diversity 0.809 0.778 0.638 0.814 0.743 [292], (6) 15 Number of alleles 97555 Allelic richness 6.426 6.516 4.919 5.000 5.000 16 DYS390 Gene diversity 0.576 0.466 0.618 0.455 0.105 17 Number of alleles 42422Lines: 163 to 163 18 Allelic richness 3.570 2.000 3.928 2.000 2.000 ——— DYS391 Gene diversity 0.241 0.417 0.080 0.173 0.199 19 * 37.80138pt PgVar 20 Number of alleles 33222 Allelic richness 2.863 2.997 1.946 2.000 2.000 ——— 21 DYS392 Gene diversity 0.592 0.458 0.630 0.255 0.444 Normal Page 22 Number of alleles 64433PgEnds: TEX 23 Allelic richness 4.403 3.815 3.944 2.984 3.000 24 DYS393 Gene diversity 0.241 0.333 0.077 0.000 0.509 25 Number of alleles 43213[292], (6) Allelic richness 3.408 2.994 1.931 1.000 3.000 26 27 All Ngöbé Kuna Emberá Wounan = = = = = 28 (N 50) (N 17) (N 9) (N 10) (N 14) 29 DYS385*A Gene diversity 0.6048 0.6851 0.1975 0.4200 0.5612 30 Number of alleles 55223 Allelic richness 3.909 4.533 2.000 2.000 2.881 31 DYS385*B Gene diversity 0.6176 0.4567 0.4938 0.6400 0.6224 32 Number of alleles 63244 33 Allelic richness 4.087 2.955 2.000 3.989 3.762 34 DYS426 Gene diversity 0.0392 0 0 0.1800 0 35 Number of alleles 21121 Allelic richness 1.329 1.000 1.000 1.995 1.000 36 DYS439 Gene diversity 0.6216 0.5260 0.6420 0.5800 0.6122 37 Number of alleles 33333 38 Allelic richness 2.981 2.786 3.000 2.995 2.990 39 DYSX156-Y Gene diversity 00000 40 Number of alleles 11111 Allelic richness 1.000 1.000 1.000 1.000 1.000 41 42 43 44

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1 tribes studied by Ruiz-Narváez et al. (2005), where from 39 Y-chromosome hap- 2 lotypes, 6 were shared among the populations, although in this previous study only 3 9 markers were surveyed (Ruiz-Narváez et al. 2005). 4 Most of the Chibchan groups, including the Kuna, practice matrilocal mar- 5 riage customs (the husband moves to the wife’s home), whereas the Chocoan Em- 6 berá and Wounan and the Chibchan Ngöbé practice patrilocal marriage traditions. 7 In terms of genetic diversity, in patrilocal groups one would expect increased 8 mitochondrial diversity and decreased Y-chromosome variation. Y-chromosome 9 haplotype diversity and Y-chromosome haplotypic discrimination capacity were 10 lowest in the Kuna and slightly higher in the Ngöbé, Emberá, and Wounan (Table 11 3). However, when the standard deviations of the haplotype diversities were used 12 to calculate confidence intervals, the Y-chromosome haplotype diversities were 13 not significantly different among the populations (Figure 2). Mitochondrial DNA 14 haplotype diversity was lowest in the matrilocal Kuna (0.59) and slightly higher [293], (7) 15 in the patrilocal Ngöbé (0.76), Wounan (0.91), and Emberá (0.94). In contrast to 16 the Y-chromosome diversity, when the standard errors of the diversity measures 17 were taken into account, the mitochondrial haplotype diversity was significantly Lines: 163 to 178 18 different between the Kuna and all other groups and between the Ngöbé and all ——— 19 other groups (Table 3; Figure 2). Among Panamanian groups, we could not detect 0.0pt PgVar 20 a correlation between diversity values (Y chromosome and mitochondrial) and ——— 21 marriage traditions (matrilocal vs. patrilocal; Table 3). This may be a consequence Normal Page 22 of the historical demography of these groups, which included founder events as- * PgEnds: Eject 23 sociated with the colonization of the Americas as well as bottlenecks in relation 24 to the tribes’ ethnogenesis. 25 Two main Y-chromosome founding haplogroups have been identified for [293], (7) 26 the Americas: haplogroups C and Q. In particular, founder lineages Q-M3 and Q- 27 M242 are restricted to the Americas or Asia (Bortolini et al. 2003). Among the 28 four Panamanian populations for which haplogroup classifications are available, 29 all populations exhibit haplogroup Q-M3. In addition, the Ngöbé, Emberá, and 30 Wounan present haplogroup Q-P36, and two Wounan men have haplogroup R-P25 31 (Table 2). Haplogroup R is also found in North and Central American indigenous 32 populations, but it is thought to have been introduced by European populations, 33 where it is the most frequent haplogroup (Bortolini et al. 2003; Zegura et al. 2004). 34 All haplotypes reported in Table 2 were compared with haplotypes reported 35 in the Y Chromosome Haplotype Reference Database. The first search was con- 36 ducted based on ethnic affiliation. The comparisons included 877 haplotypes from 37 worldwide populations in a set of 14 populations. Only haplotypes H22 (Kuna), 38 H24 (Kuna), and H36 (Emberá), which contain no data for loci DYS385 and 39 DYS439, present neighbor haplotypes (one allele difference in one locus) with 40 European and Eurasian populations. Haplotype H22 matches three Eurasian hap- 41 lotypes, H24 matches five Eurasian haplotypes and one Asian haplotype, and 42 H36 matches seven Eurasian haplotypes. The same results were obtained when 43 populations were defined by geographic affiliation (this comparison involved a 44 worldwide population sample of 22,093 haplotypes) instead of ethnic affiliation.

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1 19)

2 = 3 N

4 22) (

5 = 6 N

7 26) ( = 2690

8 . 9 N

10 32) ( = 2500 11 . Ngöbé Kuna Emberá Wounan N 12 ( 13 14 [294], (8) 15 16 17 Lines: 178 to 243 18 ——— 19 * 516.0pt PgVar 20 ——— 21 Normal Page 22 * PgEnds: PageBreak 23 24 25 [294], (8) 26 27

28 Allele at 29 30 31 32 33 34 35 36 DYS388 DYS389I DYS389II DYS390 DYS391 DYS392 DYS393 DYS426 DYS439

37 c 38 39 40 41 DYS19 DYS385

42 Y-Chromosome Haplotypes Identiﬁed by 12 STR Loci and Their Frequencies in b 43 p 44 H a t H H20 Q-M3H21 Q-M3H22 14H23 14 Q-M3 14–17H24 15–17H25 13 12H26 14 Q-M3 12 14–15H27 Q-M3 14 13 14 12 14 14 14 14–15 14–17 32 14 12 12 31 12 11 24 31 28 14 12 24 14 10 14 23 22 32 10 14 31 14 10 15 10 23 32 24 13 14 13 11 10 24 24 10 12 13 12 13 14 10 15 12 12 13 13 15 13 11 12 13 12 0.0384 13 12 0.1920 13 0.0770 0.0770 0.0384 0.0384 0.1530 0.0384 Table 2. Four Panamanian Native American Tribes H01 Q-P36H02 Q-M3 13H03 Q-M3H04 13 Q-M3 15–16H05 13 Q-M3 14–17H06 13 12 Q-P36 14–17H07 13 12 14–17 13H08 Q-M3 12 16–19H09 13 Q-P36 15–16 12H10 13 13 12 15H11 13 13 Q-M3 12 14–16 28 13 13–19 13 30 13 12 13 30 12 25 12 13–17 30 24 30 14 12 12 24 27 14 6 24 11 24 31 10 13 14 25 10 14 31 10 15 10 15 24 32 32 13 6 14 15 24 13 17 13 11 12 25 25 14 13 10 33 12 14 12 14 11 10 10 13 12 10 12 25 12 11 0.1250 12 15 15 12 0.0312 12 14 10 0.0625 11 12 0.0312 13 12 11 12 15 0.0625 12 0.0312 12 12 13 0.0312 0.0312 0.0454 12 0.0526 0 0.0312 0.0312 H12 Q-M3H13 Q-M3H14 13 Q-M3H15 13 Q-M3 12–17H16 13 14–17H17 13 Q-M3 12 14–17H18 Q-M3 12 13–17H19 13 Q-M3 12 13 13 13 12 13–17 13 13 13–17 13 14–15 12 32 13 12 31 12 34 13 12 24 31 13 24 14 24 32 10 13 25 30 10 32 11 15 25 31 10 15 25 15 23 13 10 15 25 13 10 13 10 12 15 13 10 12 15 12 14 12 13 12 15 12 13 0.0312 12 13 12 0.0312 12 12 12 0.0312 12 0.0312 11 13 11 0.0625 0.0625 0.0312 0

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1 1050 2110

2 . . 3 4 2730 0 5 . 6 7 8 9 10 11 12 13 14 [295], (9) 15 16 17 Lines: 243 to 298 18 ——— 19 * 516.0pt PgVar 20 ——— 21 Normal Page 22 was ampliﬁed * PgEnds: PageBreak 23

24 DYS385 25 [295], (9) 26 27 28 29 30 31 32 33 34 35 36 37 38 39

40 and that the observed fragments should be treated as genotypes with the alleles 41 42 43 is characterized by the amplification of two fragments. In this study DYS385 loci 44 marked in boldface. Karafet et al. (1999), and Zegura et al. (2004). by a single pair ofterm primers. In those cases, the Y Chromosome Consortium (2002) recommends the separated by a hyphen. DYS385 H33 Q-M3H34 Q-M3H35 13H36 13 14–17H37 Q-M3 13–15H38 12H39 13 13 Q-M3 12H40 14 Q-M3 14–15H41 13 12 Q-P36H42 13 13 12 Q-M3 12 14–20H43 13 14–15H44 29 14 Q-M3 14–16 12 12H45 29 13 12 12 14–17H46 13 12 24H47 13 12 13 Q-M3 24 12 12 13–17H48 32 13 13 R-P25H49 13 13 10 14 Q-M3 12H50 14 10 29 13 29 12 13 Q-M3 25 14–16 29 30 13 14 14–15 30 13 14 14 24 12 24 12 14–14 31 29 10 12 24 24 13–15 13 24 12 12 13 30 11 10 13 13 14 24 12 24 12 11 10 13 12 10 12 13 14 14 15 24 13 31 30 10 10 13 12 13 14 13 30 14 11 13 13 31 32 10 13 14 14 24 24 13 13 29 30 24 13 12 14 13 24 23 13 13 10 10 12 24 24 11 12 12 14 10 10 12 14 14 13 10 10 13 11 12 14 14 12 0.0910 13 14 15 14 13 12 11 13 12 11 13 12 0.0910 0 12 12 12 11 0.0454 13 0.0454 0.0454 0.0454 11 0.0454 0.0454 0.1050 0.1050 0.0526 0.1050 0.0526 0.0526 0.0526 0.0526 0.0526 H28 Q-M3H29H30 13 Q-M3H31 14–17H32 13 Q-P36 15 12 13 14–15 13 12–17 12 13 12 13 12 29 13 12 31 12 25 28 12 25 26 10 24 30 10 15 24 10 14 25 14 11 14 13 10 12Empty 13 cells denote that data 13 are not 12a. available. 14 Haplotype 12 The defined 13 as most each frequent distinctb. Haplogroup Y haplotype chromosome definition for identified 12 by based each Y-chromosome STRs. on population 13 is biallelic 13 markersc. as given by 12 Kolman and Bermingham (1997), 0.0384 0.1360 0.0384 0 0.0910

BOOKCOMP, Inc. — Wayne State University Press / Page 295 / ﬁnal proof / Human Biology 80-3 / June 2008 296 / ascunce et al. ) 0 1 0 ± 2 ± 3 4 Patrilocal 5 6 7 )( 0 0.96 8 0 0.91 ± 9 ± 10 Patrilocal 11 12 13 14 [296], (10) )( 0 0.90 15 0 0.94 ± ± 16

17 Patrilocal Lines: 298 to 346 18 ——— 19 * 516.0pt PgVar 20 , and ———

21 )( Normal Page 0 0.90 0 0.76 22 Chibcha-Speaking Chocó-Speaking DYS392 ± ± * PgEnds: PageBreak 23 ,

24 Matrilocal (

25 DYS391 [296], (10) , 26 27 DYS390 28 , 29 0 0.85 30 ± DYS389II ,

31 0.96 56 7 (4/3/0/0) 15 (8/7/0/0) 20 (5/9/6/0) 14 (5/4/4/1) 32

33 DYS389I 34 ,

35 b DYS388 36 , 37 ) NR 0.009 0.012 0.017 0.019 DYS19

38 φ 39 a 40

41 Y-Chromosome STRs and Mitochondrial Genetic Diversity in Chibchan- 42 ). 43 Number of polymorphic lociNumber of unique allelesSample sizeNumber of haplotypes (A/B/C/D) 12 12 184 11 3 63 11 5 10 46 2 10 44 2 31 Number of unique haplotypesHaplotype diversityNucleotide diversity ( 30 NR 5 0.59 4 12 9 Number of segregating sites NR 10 12 23 29 Sample sizeNumber of haplotypesDiscrimination capacity (%)Number of unique haplotypesHaplotype diversity 50.50 48 50 99 42.30 10 11 26 56.25 17 18 32 54.54 10 12 22 63.15 12 9 19 lineages A, B, C, and DBermingham (Batista1997). et al. 1995; Kolman et al. 1995; Kolman and 44 Y-chromosome STRs ( DYS393 mtDNA and Chocó-Speaking Populations from Panama Table 3. Linguistic Familyand Diversity IndexY-chromosome STRs All NR, not reported. a. Values of haplotype diversity were Kuna estimated including all the individuals and based on eight Ngöbé Emberá Wounan b. A/B/C/D indicates the four mitochondrial founder lineages described for the Americas:

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 [297], (11) 15 16 Figure 2. Mean and standard deviation for haplotype diversity of Y-chromosome STRs (open bars) and mitochondrial DNA (hatched bars) in the matrilocal Kuna population (gray bars) and 17 the patrilocal tribes Ngöbé, Emberá, and Wounan. Lines: 346 to 365 18 ——— 19 1.144pt PgVar 20 ——— 21 Furthermore, H48 (Wounan), which belongs to haplogroup R-P25 and is character- Normal Page 22 istic of European populations, matches one haplotype from an admixed population PgEnds: TEX 23 from Colombia. In sum, these results suggest that the four Panamanian populations 24 have experienced little European admixture. 25 The Y-chromosome AMOVA results indicate that the major component of [297], (11) 26 variation corresponds to intrapopulation variation (80.19%) and that differences 27 among populations also account for a significant amount of variation (φST = 28 0.198, P < 0.00001), indicating a high degree of differentiation among these pop- 29 ulations. Using the sum of squared differences among Y-chromosome haplotypes, 30 pairwise RST among populations indicate that the Emberá and Wounan popu- 31 lations are not significantly differentiated based on Y-chromosome data. Con- 32 versely, mitochondrial data indicate a significant level of differentiation (Kolman 33 and Bermingham 1997). The pattern of high genetic structure at the Y-chromo- 34 some level has been found in other groups from Central America (Ruiz-Narváez 35 et al. 2005) and South America (Mesa et al. 2000; Bortolini et al. 2002), suggesting 36 that genetic drift has played a major role in the colonization of the Americas. One 37 of the main observations in our study is the apparent absence of gene flow, as indi- 38 cated by the low number of shared Y-chromosome haplotypes (only 2 haplotypes 39 out of 50). This significant genetic differentiation both at the Y-chromosome and 40 the mitochondrial level is more likely to be the result of fragmentation of ancestral 41 populations into separate tribal groups in agreement with the idea of a continuous 42 presence of Amerindian groups in the isthmian region since their arrival (Cooke 43 2005). In this scenario, cultural transitions in lower Central America were the 44 result of cultural adaptation by endogenous populations rather than replacement

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1 or introgression (Kolman and Bermingham 1997). Archeological records indicate 2 that native Americans in lower Central America had strong social interactions 3 among neighboring groups (Cooke 2005), suggesting that genetic barriers existed 4 among these populations despite their geographic proximity, related languages, 5 and social interactions. 6 To further evaluate the role of the Panama area during the colonization 7 of South America, in terms of pattern of genetic diversity, we compared the Y- 8 chromosome variability of populations of Panama and northern South America. 9 These comparisons consisted of our Panamanian populations and previously an- 10 alyzed Colombian and Venezuelan populations and included six STRs (DYS19, 11 DYS388, DYS390, DYS391, DYS392, DYS393) (Ruiz-Linares et al. 1999; Mesa et 12 al. 2000; Bortolini et al. 2003). DYS389 (I and II) was not included because of 13 a lack of information for populations from Colombia and Venezuela. DYS389II 14 exhibits the highest level of gene diversity in Panamanians (see Table 1); thus [298], (12) 15 some differentiation is lost with the absence of this marker. With this set of six 16 STRs, 43 haplotypes were described among 161 males. Thirty-two haplotypes 17 were present in only 1 tribe, whereas 11 were shared among tribes. Three haplo- Lines: 365 to 414 18 types were shared among the Panamanian groups, four among the Colombian and ——— 19 Venezuelan populations, and four among populations from Panama, Colombia, -0.12999pt PgVar 20 and Venezuela (data not shown). The Y-chromosome AMOVA results indicate ——— 21 that the major component of variation corresponds to intrapopulation variation Normal Page 22 (75.32%) and that differences among populations also account for a significant PgEnds: TEX 23 amount of variation (φST = 0.246, P < 0.00001), indicating a high degree of differ- 24 entiation among these populations. When groups are compared pairwise, among 25 the Panamanian groups the Emberá and Wounan are not significantly differenti- [298], (12) 26 ated as well as the Emberá and Kuna (Table 4). The loss of genetic differentiation 27 between the Kuna and Emberá when only 6 STR loci are assayed points out the 28 need to include more markers to further evaluate the Y-chromosome differentia- 29 tion among these tribes. Among the populations from Colombia and Venezuela, 30 only the Warao and Yukpa are not significantly differentiated (Table 4). Compar- 31 isons between Panama and Colombia/Venezuela show that the Wounan are not 32 significantly differentiated from the Warao (Colombia) and that the Emberá are 33 not significantly differentiated from the Zenu (Colombia), Warao (Venezuela), or 34 Yukpa (Venezuela). One of the outstanding results is the constant differentiation 35 of the Ngöbé group from the remaining tribes and to a certain degree the Kuna, 36 both groups with the largest sample sizes. The uniqueness of these groups from 37 Panama might be the result of a population bottleneck that was associated with 38 Chibchan ethnogenesis (Kolman and Bermingham 1997). Thus genetic drift might 39 have played a strong role in Chibchan groups because of particular demographic 40 events. 41 42 Concluding Remarks. Our study provides new Y-chromosome data and in- 43 cludes a larger number of individuals than in previous studies (Kolman and Berm- 44 ingham 1997; Karafet et al. 1999; Zegura et al. 2004). Panamanian Y-chromosome

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1 2 3 4 5 6 a 7 8 9 10 11 0.39934 0.13058 12 13 14 [299], (13) 01446 . 15 0 16

17 ) Lines: 414 to 414 18 ——— 0.34715 0.0270 0.0360 0.3513 0.0000 0.0991 0.0000 0.0000 0.0000 19 DYS393 , * 516.0pt PgVar 20 ——— 21 Normal Page DYS392 22 , 08094 . PgEnds: TEX 0.403160.20977 0.27028 0.44998 0.19143 0.28009 0.0270 23 0.9909 0.0180 0.7567 0.0000 0.1712 0.2522 DYS391 24 Values , 25 P [299], (13) ST

26 R 07429 01535 03463 0 04271 DYS390 . . . . , 0 0 –0 27 –0 28 a 29 DYS388 Panama Colombia and Venezuela , 30 DYS19 31 0185 . 32 0 33

34 (Below the Diagonal) and ST

35 R 36 Ngöbé Kuna Emberá Wounan Bari Warao Wayuu Zenu Yukpa 37

38 b 39 values are marked in bold.

40 ST R

41 Population Pairwise 42 43 WayuuZenu 0.59261 0.39992 0.42725 0.34832 0.20899 0.39122 0.22789 0.22251 0.0000 0.0090 Yukpa 0.26233 0.13458 NgöbéKunaEmberáBariWarao 0.31813 0.11659 0.0000 0.56133 0.23593 0.379 0.11604 0.0000 0.2612 0.0000 0.11134 0.0180 0.0000 0.44105 0.0000 0.0000 0.0450 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 0.0450 0.0000 0.0000 Wounan 0.24817 0.09608 Mesa et al. (2000), and Bortolini et al. (2003). 44 from groups from Panama and northern South America. b. Haplotype data for Colombia and Venezuela were reported by Ruiz-Linares et al. (1999), a. Comparisons are based on data from six STRs ( Table 4. Panama Nonsigniﬁcant (Above the Diagonal) (Signiﬁcant at the 0.05 Level) Colombia and Venezuela

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1 data are important for future evolutionary genetic studies of native Americans. The 2 forensic utility of the assayed Y-chromosomemarkers is limited because of the low 3 diversity in these populations, which is most likely a result of population bottle- 4 necks associated with colonization of the Americas, ethnogenesis, and European 5 contact (Kolman and Bermingham 1997). The four Panamanian native American 6 populations show a high degree of differentiation both at the Y-chromosome and 7 the mitochondrial level, highlighting the importance of population structure even 8 in geographically proximate and linguistically related populations. It is significant 9 that these populations show virtually no gene flow among themselves or with non- 10 indigenous groups, suggesting that these populations have remained genetically 11 isolated since their ethnogenesis. 12 13 Acknowledgments We acknowledge the participation of the Ngöbé, Kuna, Emberá, and 14 [300], (14) Wounan people in our study. We thank Eldredge Bermingham, who supported the initial 15 stages of this project, and Mark Thomas, who provided control DNAs for calibration of the 16 microsatellite alleles. We are grateful to A. Ruiz-Linares, M. C. Bortolini, and T. Karafet for 17 providing the haplotype data published in their studies. We thank G. Clark (Interdisciplinary Lines: 414 to 446 18 Center for Biotechnology Research, University of Florida) and D. Shoemaker (U.S. De- ——— 19 partment of Agriculture, Agricultural Research Service, Center for Medical, Agricultural, 0.16899pt PgVar 20 and Veterinary Entomology, Gainesville, Florida) for comments on the manuscript. The ——— 21 Dirección General de Asuntos del Personal Académico (DGAPA), Universidad Nacional Normal Page 22 Autónoma de México, provided support to Angelica González-Oliver. PgEnds: TEX 23 24 Received 29 November 2007; revision received 1 March 2008. 25 [300], (14) 26 27 Literature Cited 28 Allendorf, F. W., and G. Luikart. 2007. Conservation and the Genetics of Populations. Malden, MA: 29 Blackwell. 30 Barrantes, R., P. E. Smouse, H. W. Mohrenweiser et al. 1990. Microevolution in lower Central Amer- 31 ica: Genetic characterization of the Chibcha-speaking groups of Costa Rica and Panama and a consensus taxonomy based on genetic and linguistic affinity. Am. J. Hum. Genet. 46:63–84. 32 Bartlett, A. S., and S. Barghoorn. 1973. Phytogeographic history of the isthmus of Panama during 33 the past 12,000 years: A history of vegetation, climate, and sea-level change. In Vegetation and 34 Vegetational History of Northern South America, A. Graham, ed. New York: Elsevier, 233–247. 35 Batista, O., C. J. Kolman, and E. Bermingham. 1995. Mitochondrial DNA diversity in the Kuna 36 Amerinds of Panama. Hum. Mol. Genet. 5:921–929. Bonatto, S. L., and F. M. Salzano. 1997. A single and early migration for the peopling of the Americas 37 supported by mitochondrial DNA sequence data. Proc. Natl. Acad. Sci. USA 94:1866–1871. 38 Bortolini, M. C., F. M. Salzano, C. H. D. Bau et al. 2002. Y-chromosome biallelic polymorphisms and 39 native American population structure. Am. J. Hum. Genet. 66:255–259. 40 Bortolini, M. C., F. M. Salzano, M. G. Thomas et al. 2003. Y-chromosome evidence for differing 41 ancient demographic histories in the Americas. Am. J. Hum. Genet. 73:524–539. Bosch, E., A. C. Lee, F. Calafell et al. 2002. High resolution Y-chromosome typing: 19 STRs amplified 42 in three multiplex reactions. Forensic Sci. Int. 125:42–51. 43 Cavalli-Sforza, L. L., P. Menozzi, and A. Piazza. 1994. The History and Geography of Human Genes. 44 Princeton, NJ: Princeton University Press.

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