Unraveling the Genetic Threads of History: mtDNA HVS-I Analysis Reveals the Ancient Past of the Aburra Valley

This article presents a comprehensive genetic study focused on pre-Hispanic individuals who inhabited the Aburrá Valley in Antioquia, Colombia, between the tenth and seventeenth centuries AD. Employing a genetic approach, the study analyzed maternal lineages using DNA samples obtained from skeletal remains. The results illuminate a remarkable degree of biological diversity within these populations and provide insights into their genetic connections with other ancient and indigenous groups across the American continent. The findings strongly support the widely accepted hypothesis that the migration of the first American settlers occurred through Beringia, a land bridge connecting Siberia to North America during the last Ice Age. Subsequently, these early settlers journeyed southward, crossing the North American ice cap. Of particular note, the study unveils the presence of ancestral lineages from Asian populations, which played a pivotal role in populating the Americas. The implications of these results extend beyond delineating migratory routes and settlement patterns of ancient populations. They also enrich our understanding of the genetic diversity inherent in indigenous populations of the region. By revealing the genetic heritage of pre-Hispanic individuals from the Aburrá Valley, this study offers valuable insights into the history of human migration and settlement in the Americas. Furthermore, it enhances our comprehension of the intricate genetic tapestry that characterizes indigenous communities in the area.


Introduction
The Antioquia region has played a pivotal role in the pre-Hispanic history of South America, serving as a gateway for the initial human migrations to the continent and as a hub for the development of a sophisticated culture deeply rooted in the utilization of natural resources and advanced technologies [1].Colombia boasts a multitude of archaeological sites that have enabled the tracking of the earliest human arrivals, dating back to approximately sixteen thousand four hundred (16,400) ± four hundred twenty (420) A.P. [2], as determined through radiocarbon dating of artifacts linked to ancient human activities.Among these sites, Pubenza, nestled in the lowlands of the middle-valley of the Magdalena River [3], and El Jordán in the municipality of Roncesvalles (Tolima), stand out Genes 2023, 14, 2036 3 of 23 tombs investigated in the three hills of the Aburrá Valley, where the city of Medellín is located, belong to this latter period.
Molecular biology, leveraging DNA analysis, has played a pivotal role in shedding light on hypotheses concerning the origins of human populations.DNA, the repository of hereditary information and essential protein-coding data, was first structurally elucidated in 1953 by Watson, Crick, and Franklin.Subsequently, Sanger's sequencing method paved the way for reading and decoding genetic information.Recent advancements in data storage, analytical technologies, and informatics have catalyzed breakthroughs across multiple disciplines, including archaeology.Despite the inevitable degradation of DNA post-mortem, it can adhere to hydroxyapatite in bones, maintaining sufficient quality for reconstructing ancient genomes.
Mitochondrial DNA (mtDNA) analysis, particularly in tracing maternal lineages, has emerged as a powerful tool for unraveling population history and genetic ancestry.The exclusive maternal transmission of mtDNA enables the tracking of maternal lineages across generations, facilitating the reconstruction of ancient population movements and migrations.
The high copy-number of mtDNA within cells empowers the recovery and examination of genetic information from ancient remains, thereby aiding our comprehension of genetic diversity and population dynamics over time.Maternal lineages, a focal point of mtDNA analysis, are identified through the detection of characteristic mutations that serve as markers for distinct population groups.These findings have significantly contributed to the development of theories regarding the settlement of pre-Hispanic communities in Colombia.
Ancient DNA studies have sought to unravel the genetic structure of pre-Colombian populations and establish genetic links between historical and contemporary population groups.These investigations are instrumental in supporting models related to population replacements, migrations, and genetic admixtures.As of now, genetic ancestry data in the Aburrá Valley are scarce.
To gain a more comprehensive understanding of population dynamics, migration patterns, and genetic relationships within the region, future studies should consider incorporating complete nuclear and mitochondrial genome analysis.This advanced approach will enable a more detailed and precise assessment of the genetic history of the pre-Hispanic inhabitants in the Aburrá Valley; thereby, enriching our comprehension of the complex tapestry of human settlement and evolution in this pivotal region.

Sample Collection
This study was centered on the analysis of mitochondrial DNA HVS-I extracted from ten individuals originating from the Aburrá Valley in Antioquia.These individuals were associated with pit tombs characterized by lateral chambers.Gender determination was based on the anthropological examination of the skeletal remains.
We conducted research at three distinct archaeological sites: Cerro del Volador, which yielded one individual (bone); Alto de las Flores in Envigado, where we analyzed one individual (bone) in Medellín; and La Colinita, where we investigated eight individuals (molars) in Medellín (Figure 1).The samples selected for analysis date back to the late pre-Hispanic period and the contact period, specifically spanning from the eleventh to the eighteenth centuries AD [7].

Ancient DNA Extraction
The genetic analysis of individuals from bone remains was conducted at the Human Genetics Laboratory within the specialized area dedicated to archaic DNA procedures at the University of Sabana.Stringent authenticity criteria, designed to prevent contamination of ancient DNA as proposed by Cooper, Poinar, and Gilbert [8], were strictly adhered to during the entire process.Mitochondrial DNA sequences from the HVS-I region were detected through the polymerase chain reaction (PCR) amplification method.Subsequently, these sequences were aligned with the revised Cambridge Reference Sequence (rCRS) for detailed analysis [9].
To ensure the accuracy and reliability of the findings, the procedures were independently replicated by two researchers on separate occasions.Furthermore, a subset of the samples was sent to the Department of Genetic Epidemiology and Bioinformatics at the University of Southampton in England.

PCR Amplification and Sequencing
To determine the mitochondrial haplotypes of the individuals in this study, we focused on a 388 bp fragment of HVS-I, specifically nucleotide positions 16024-16408.For amplification, we employed four pairs of overlapping PCR primers that were specifically developed for pre-Hispanic American populations [10]: H15995: GCT AAG ATT CTA ATT TAA ACT ATT CT (26 bp); L16174: GGA TTG GGT TTT TAT GTA CTA C (22 bp); H16105: TGC CAG CCA CCA TGA ATA TTG TAC (24 bp); L16256: GCT TTG GAG TTG CAG TTG ATG TGT (24 bp); H16194: ATG CTT ACA AGC AAG TAC AGC AA (23 bp); L16360: GAG AAG GGA TTT GAC TGT AAT GTG (24 bp); H16261: CCT CAC CCA CTA GGA TAC CAA CAA (24 bp); L16429: CCT CAC CCA CTA GGA TAC CAA CAA (21 bp).For the amplification of the HVS-I in human bone and molar remains containing ancient DNA, a standardized process was employed.This process involved a final volume of 10 µL and PCR amplification utilizing a hot start Polymerase and dNTPs.The resulting PCR product was verified using a 2% agarose gel, and the most suitable bands were selected for Sanger sequencing.Stringent alignment and analysis was performed using the QIAGEN CLC Genomics Workbench 20.0.4 program.Variants were independently and repeatedly identified by three investigators; each was assigned a GenBank accession number within the

Ancient DNA Extraction
The genetic analysis of individuals from bone remains was conducted at the Human Genetics Laboratory within the specialized area dedicated to archaic DNA procedures at the University of Sabana.Stringent authenticity criteria, designed to prevent contamination of ancient DNA as proposed by Cooper, Poinar, and Gilbert [8], were strictly adhered to during the entire process.Mitochondrial DNA sequences from the HVS-I region were detected through the polymerase chain reaction (PCR) amplification method.Subsequently, these sequences were aligned with the revised Cambridge Reference Sequence (rCRS) for detailed analysis [9].
To ensure the accuracy and reliability of the findings, the procedures were independently replicated by two researchers on separate occasions.Furthermore, a subset of the samples was sent to the Department of Genetic Epidemiology and Bioinformatics at the University of Southampton in England.

PCR Amplification and Sequencing
To determine the mitochondrial haplotypes of the individuals in this study, we focused on a 388 bp fragment of HVS-I, specifically nucleotide positions 16024-16408.For amplification, we employed four pairs of overlapping PCR primers that were specifically developed for pre-Hispanic American populations [10]: H15995: GCT AAG ATT CTA ATT TAA ACT ATT CT (26 bp); L16174: GGA TTG GGT TTT TAT GTA CTA C (22 bp); H16105: TGC CAG CCA CCA TGA ATA TTG TAC (24 bp); L16256: GCT TTG GAG TTG CAG TTG ATG TGT (24 bp); H16194: ATG CTT ACA AGC AAG TAC AGC AA (23 bp); L16360: GAG AAG GGA TTT GAC TGT AAT GTG (24 bp); H16261: CCT CAC CCA CTA GGA TAC CAA CAA (24 bp); L16429: CCT CAC CCA CTA GGA TAC CAA CAA (21 bp).For the amplification of the HVS-I in human bone and molar remains containing ancient DNA, a standardized process was employed.This process involved a final volume of 10 µL and PCR amplification utilizing a hot start Polymerase and dNTPs.The resulting PCR product was verified using a 2% agarose gel, and the most suitable bands were selected for Sanger sequencing.Stringent alignment and analysis was performed using the QIAGEN CLC Genomics Workbench 20.0.4 program.Variants were independently and repeatedly identified by three investigators; each was assigned a GenBank accession number within the range BankIt 2736055: OR478541-OR478622.Haplogroups and haplotypes were determined using the HaploGrep2 program.Variations with a quality score exceeding 60% present in at least three duplicate samples were documented to ensure the reliability of our findings.

Statistical Analyses
To conduct individual analyses, we employed specific haplotypes and constructed phylogenetic trees using unique polymorphisms for each haplogroup.We determined common evolutionary relationships through several approaches, including neighbor-joining algorithms, UPGMA, direct networks using the NETWORK 10 program, and the HaploGrep 2 program [11].
Our study also encompassed intra-and inter-population analyses, performed with the Arlequin 3.5 program.We calculated various indices to discern population trends, and mean pairwise F ST genetic distances were computed between populations based on their mitochondrial haplotype frequencies.
In this analysis, we considered twenty present-day populations from Colombia and seventy-seven pre-Hispanic American populations.These were categorized into eight groups based on previous research.We obtained distance matrices and visualized them using the MDS ALSCAL algorithm within the SPSS program.This method provides a balanced approach, addressing concerns regarding false positives and statistical power, as opposed to relying solely on phylogenetic trees [12][13][14][15].

Results
In our study, we identified a total of ten individuals, collectively representing the four founding haplogroups: A (60%), B (10%), C (20%), and D (10%) (Table 2).These haplotypes were defined by seventeen-point polymorphic sites, revealing thirty-one T↔C and thirteen A↔G transitions.It is worth noting that each individual possessed a unique haplotype, indicating the absence of a direct matrilineal relationship among the individuals under analysis.To classify the mitochondrial haplogroups, we followed the Phylotree hierarchy [16], which begins with the common ancestor R, followed by N, M, and A. Among the individuals with the C16290T polymorphism, six were categorized into haplogroup A, with four of them belonging to the A2 subgroup due to distinct variants.Individual Abu10 was assigned to haplogroup B2s but also exhibited a variant characteristic of B4.Individuals Abu7 and Abu9 were designated as haplogroup C and shared specific variants, while the individual with the C16223T polymorphism was placed in haplogroup D, with individual Abu6 specifically categorized as D4j1a based on another unique variant.
The phylogenetic tree (Figure 2) illustrates the presence of various haplogroups and their corresponding samples.In the tree, we observe samples classified under haplogroup A, including A2, A2af1b1b, A, A2i, A2+(64)+ @16111, and A2ah.Additionally, there are samples classified under haplogroup C, namely C and C1c4.The tree also shows a sample categorized under haplogroup D, specifically D4j1a.Lastly, there is a sample denoted as B2s under haplogroup B2.These findings provide valuable insights into the genetic diversity and relationships among the studied population.To classify the mitochondrial haplogroups, we followed the Phylotree hierarchy [16], which begins with the common ancestor R, followed by N, M, and A. Among the individuals with the C16290T polymorphism, six were categorized into haplogroup A, with four of them belonging to the A2 subgroup due to distinct variants.Individual Abu10 was assigned to haplogroup B2s but also exhibited a variant characteristic of B4.Individuals Abu7 and Abu9 were designated as haplogroup C and shared specific variants, while the individual with the C16223T polymorphism was placed in haplogroup D, with individual Abu6 specifically categorized as D4j1a based on another unique variant.
The phylogenetic tree (Figure 2) illustrates the presence of various haplogroups and their corresponding samples.In the tree, we observe samples classified under haplogroup A, including A2, A2af1b1b, A, A2i, A2+(64)+ @16111, and A2ah.Additionally, there are samples classified under haplogroup C, namely C and C1c4.The tree also shows a sample categorized under haplogroup D, specifically D4j1a.Lastly, there is a sample denoted as B2s under haplogroup B2.These findings provide valuable insights into the genetic diversity and relationships among the studied population.
Figure 2. Phylogenetic tree (samples classified in haplogroups and haplotypes).All sorted samples are combined into a resulting tree that includes all rCRS-related polymorphisms.The polymorphisms marked in blue are private polymorphisms for this group, already known by Phylotree.Individuals are marked in green.Based on Phylotree [16], the phylogenetic tree was constructed using the neighbor-joining algorithm.The samples names have been abbreviated for clarity.
Median-joining (MJ) analysis was employed to explore maternal relationships between individuals in family graves discovered in the Aburrá Valley (Figure 3).The analysis did not reveal direct matrilineal relationships; however, it did show an ancestral connection between an Abu1 node and individual Abu9.
Our study's results revealed the presence of ten individuals from the Aburrá Valley, representing four founding haplogroups (A, B, C, and D).Each individual exhibited a unique haplotype, indicating the absence of direct matrilineal relationships among them.Haplogroup assignments were made based on specific variants and polymorphisms identified in their sequences.

D4
Figure 2. Phylogenetic tree (samples classified in haplogroups and haplotypes).All sorted samples are combined into a resulting tree that includes all rCRS-related polymorphisms.The polymorphisms marked in blue are private polymorphisms for this group, already known by Phylotree.Individuals are marked in green.Based on Phylotree [16], the phylogenetic tree was constructed using the neighbor-joining algorithm.The samples names have been abbreviated for clarity.
Median-joining (MJ) analysis was employed to explore maternal relationships between individuals in family graves discovered in the Aburrá Valley (Figure 3).The analysis did not reveal direct matrilineal relationships; however, it did show an ancestral connection between an Abu1 node and individual Abu9.
Our study's results revealed the presence of ten individuals from the Aburrá Valley, representing four founding haplogroups (A, B, C, and D).Each individual exhibited a unique haplotype, indicating the absence of direct matrilineal relationships among them.Haplogroup assignments were made based on specific variants and polymorphisms identified in their sequences.
The haplotype network offers valuable insights into the genealogical relationships among haplotypes.It allows us to assess the degree of divergence between different lineages and estimate the distance to their most recent common ancestor.It is important to note, though, that the haplotype network does not directly establish maternal relationships between specific individuals.Instead, it primarily serves as a tool for understanding the genealogical connections and ancestral origins of different haplotypes.Its relevance lies in elucidating the historical relationships and evolutionary patterns within a population rather than establishing direct relationships between specific individuals.The haplotype network offers valuable insights into the genealogical relationships among haplotypes.It allows us to assess the degree of divergence between different lineages and estimate the distance to their most recent common ancestor.It is important to note, though, that the haplotype network does not directly establish maternal relationships between specific individuals.Instead, it primarily serves as a tool for understanding the genealogical connections and ancestral origins of different haplotypes.Its relevance lies in elucidating the historical relationships and evolutionary patterns within a population rather than establishing direct relationships between specific individuals.
Furthermore, the results of population diversity in the Aburrá Valley are presented in Table 3, with 'n' representing ten individuals and 'K' denoting ten sequences analyzed.The nucleotide diversity (π) for the study population is calculated as 0.012.Analysis of the Tajima index (-D) during pre-Hispanic periods indicates population expansion, with a result of −0.72937 and a p-value greater than 0.05; which suggests no significant differences between the analyzed samples [14].In line with the research conducted by Raff [15], a multiscale analysis comparing seventy-eight ancient populations across the Americas with twenty contemporary indigenous Colombian populations highlights the highest genetic similarity in the Aburráes population (Figure 4).Furthermore, the results of population diversity in the Aburrá Valley are presented in Table 3, with 'n' representing ten individuals and 'K' denoting ten sequences analyzed.The nucleotide diversity (π) for the study population is calculated as 0.012.Analysis of the Tajima index (-D) during pre-Hispanic periods indicates population expansion, with a result of −0.72937 and a p-value greater than 0.05; which suggests no significant differences between the analyzed samples [14].In line with the research conducted by Raff [15], a multiscale analysis comparing seventy-eight ancient populations across the Americas with twenty contemporary indigenous Colombian populations highlights the highest genetic similarity in the Aburráes population (Figure 4).This similarity is particularly striking with pre-Hispanic populations in Mesoamerica, as well as in the Arctic and Sub-Arctic regions.The study reveals the presence of haplogroups A, C, and D in both current and ancient populations throughout the Americas.Notably, haplogroup B is absent in the Caribbean, while haplogroup X is reported to be in low proportions in the northern part of the continent.Haplogroup M, on the other hand, is exclusively identified in an ancient population in the northwestern United States and not in current populations (Table 4).This similarity is particularly striking with pre-Hispanic populations in Mesoamerica, as well as in the Arctic and Sub-Arctic regions.The study reveals the presence of haplogroups A, C, and D in both current and ancient populations throughout the Americas.Notably, haplogroup B is absent in the Caribbean, while haplogroup X is reported to be in low proportions in the northern part of the continent.Haplogroup M, on the other hand, is exclusively identified in an ancient population in the northwestern United States and not in current populations (Table 4).The dataset analyzed includes archaeological sites categorized into various geographic zones, namely the Arctic/Subarctic, northwestern United States, Great Basin/southwestern United States, northeastern United States, Mesoamerica, Caribbean, and South America.This regional categorization, as presented in Table 4, is primarily based on the work of Raff [15].However, it has been refined and expanded upon through recent research, with a particular focus on the inclusion of Colombia.The geographic distribution of these analyzed zones across the Americas is visually represented in Figure 5. Which employs a color scheme for easy identification of geographical locations and highlights the diverse array of regions covered in our study.
For more comprehensive information, including the haplogroup frequencies used for the multidimensional scaling (MDS) analysis, please consult Table 4.The specific matrix of F ST values can be found in Table S1.
The distribution of mitochondrial haplogroups among the individuals revealed several patterns.Five individuals with the C16290T polymorphism were assigned to haplogroup A, with four of them specifically identified as A2.Notably, individual Abu10 was assigned to haplogroup B2s but also exhibited a variant typical of B4.Meanwhile, Individuals Abu4 and Abu7 were assigned to haplogroup C and shared specific variants.On the other hand, the individual with the C16223T polymorphism was placed in haplogroup D, with individual Abu6 classified as D4j1a, based on another specific variant (Table 5).These findings provide insights into the diversity of mitochondrial haplogroups in the studied population.
Basin/southwestern United States, northeastern United States, Mesoamerica, Caribbean, and South America.This regional categorization, as presented in Table 4, is primarily based on the work of Raff [15].However, it has been refined and expanded upon through recent research, with a particular focus on the inclusion of Colombia.The geographic distribution of these analyzed zones across the Americas is visually represented in Figure 5. Which employs a color scheme for easy identification of geographical locations and highlights the diverse array of regions covered in our study.For more comprehensive information, including the haplogroup frequencies used for the multidimensional scaling (MDS) analysis, please consult Table 4.The specific matrix of FST values can be found in Table S1.
The distribution of mitochondrial haplogroups among the individuals revealed several patterns.Five individuals with the C16290T polymorphism were assigned to haplogroup A, with four of them specifically identified as A2.Notably, individual Abu10 was assigned to haplogroup B2s but also exhibited a variant typical of B4.Meanwhile, Individuals Abu4 and Abu7 were assigned to haplogroup C and shared specific variants.On the other hand, the individual with the C16223T polymorphism was placed in haplogroup

Discussion
The findings of this study offer valuable genetic insights into the population history of pre-Hispanic inhabitants in the Aburrá Valley region.This research primarily focused on the analysis of mitochondrial DNA (mtDNA), which has provided significant information about migratory patterns, interactions, and genetic relationships among ancient American populations.
Studying ancient populations is a multidisciplinary endeavor that combines genetics and archaeology to unravel the complex tapestry of human history.Haplogroups, which are genetic markers passed down through generations, serve as invaluable tools for understanding ancient population dynamics.In this research, we present a comprehensive table featuring archaeological sites across distinct geographic zones, each holding a wealth of genetic information in the form of haplogroups A, B, C, D, X, M, and H.
Variations in haplogroup distribution are evident across these geographic zones, indicating that different regions were inhabited by populations with unique genetic profiles.By examining these archaeological periods, we can observe how haplogroup distributions evolved over millennia; shedding light on genetic adaptations and the ebb and flow of ancient populations.Comparative analysis of haplogroups allows us to reconstruct population movements, migrations, and genetic relationships, providing insights into the interactions between ancient human groups.However, it is crucial to consider sample sizes, as they impact the statistical robustness of genetic findings.Larger sample sizes lead to more reliable insights.
Regarding genetic diversity, the Aburráes population in Colombia exhibited a genetic diversity level of 0.644.When compared to various populations from different geographic regions and archaeological periods, this falls within the range of genetic diversity observed across these populations.Some populations displayed higher genetic-diversity values, while others had lower values.Thus, reflecting the intricate tapestry of human genetic variation across different time periods and geographical locations.It is important to note that, in general, a larger number of samples provide a more accurate reflection of the true diversity of the population or community being studied.Therefore, to obtain reliable and representative results, it is recommended to use a significant number of samples in the diversity analysis.
It is important to emphasize that the haplotype network does not directly establish maternal relationships between specific individuals.Instead, its primary function is to elucidate genealogical connections and ancestral origins among various haplotypes.Its significance lies in unveiling historical relationships and evolutionary patterns within a population rather than confirming direct relationships between specific individuals.
Regarding the Colinita tomb, it's worth noting that the eight adult individuals found within it are not from the same family.This information holds significant value for archaeologists and anthropologists as it provides insights into burial rituals and helps determine whether the tombs are related to a single family or not.
The meticulous examination of various haplogroups, including A2, B2, C1, and D4j1a, has provided valuable insights into the intricate demographic processes that shaped the region.Notably, Haplogroup A2, renowned for its prevalence in the high latitudes of the Americas and its historical ties to Siberian and Eskimo tribes, has emerged as a pivotal contributor to the initial waves of migration into the continent [16,74].Moreover, the identification of three distinct sub-haplogroups within A2 among pre-Hispanic Antioquia populations signifies local genetic diversification, underscoring the region's role as a genetic melting-pot.
This research delves deeply into the dynamics of these populations.The discovery of A2af1b1b within contemporary populations in Panama and Costa Rica carries profound implications, pointing to significant demographic events approximately ten thousand years ago.While this specific haplogroup is presently observed in only two individuals-one from Chiriqui, Panama, and the other from Costa Rica-it originated 4700 years before the present, with a time to most recent common ancestor (TMRCA) of 1000 years before the present [78].
Most intriguingly, the study identifies the presence of the A2af1b1b sub-haplogroup in an individual from the Valle de Aburra.This finding suggests that A2af1b1b has deep historical roots in the region, indicating its presence within ancient populations.Furthermore, the fact that this haplogroup is still present in contemporary populations emphasizes its enduring legacy [78].This underscores the notion that A2af1b1b is not just a relic of the past but has persisted through generations and is still part of the genetic makeup of current populations in the region.
The enduring presence of Haplogroup A2ah for over five millennia across various regions offers a compelling narrative of historical population movements.This relatively rare lineage among Native Americans, discerned through comprehensive analyses of complete mitochondrial genomes, is characterized by the uncommon combination of tandem transitions, T16097C and A16098G.Which is rarely encountered in mitochondrial DNA databases [94].
Within the larger Haplogroup A2, A2ah emerges as a distinct branch within the Native American mitochondrial family.This research marks a significant milestone in the field of genetics.Notably, for the first time, researchers have unveiled a mitochondrial lineage in the Andean region that was previously believed to be exclusive to the South American lowlands.Specifically, they have identified the A2ah lineage in the Andean area, even within a pre-Hispanic individual.The implications of this discovery are profound, raising intriguing questions about the potential gene flow between the Andean and lowland regions before the Spanish arrival [95].
Archaeological evidence indicates interactions and trade among diverse populations across various eco-regions during pre-Hispanic times.The presence of trade networks, such as those connected to the Quebrada de La Cueva ravine, which housed the Pukara de La Cueva, suggests extensive pre-Hispanic connectivity.Yet, whether these networks also facilitated gene-flow remains uncertain.This underscores the significance of ancient DNA studies in shedding light on this aspect of pre-Hispanic population dynamics [96].
If indeed gene-flow was a regular occurrence between highland and lowland pre-Hispanic populations, it becomes plausible that certain genetic variants from one region could be found in the other.The identification of an infrequent mitochondrial haplotype from the South American lowlands in the Andean area in this study lends support to this hypothesis.To further investigate this proposition, the researchers recommend expanding genetic analyses to encompass other individuals retrieved from Pukara de La Cueva at the population level.Additionally, isotopic analysis could offer insights into the region where the analyzed individual resided during their early years [97].
The research suggests an approximate origin dating back to 5200 years ago, with some within-clade variations.Geographically, this lineage appears to be primarily concentrated in Bolivia and its adjacent regions with a limited distribution [98][99][100].
The discovery of the A2ah mitochondrial lineage in the Andean region, as previously discussed, presents an intriguing parallel to the genetic findings in the Valle de Aburra.In both instances, researchers have uncovered the existence of uncommon mitochondrial haplogroups in regions that defied earlier expectations.
This shared discovery of sub-haplogroup A2ah among both ancient and contemporary Native American populations emphasizes its profound historical importance and its enduring legacy within the tapestry of mitochondrial DNA.The presence of this lineage in diverse temporal contexts underscores its resilience and persistence through time.
By delving deeper into the study of this unique lineage, researchers can unlock invaluable insights into the historical narratives, migration patterns, and genetic tapestry of Native American communities.This connection between seemingly distant regions challenges preconceived notions and beckons for further exploration into the intricate web of human genetic heritage across the Americas.
The genetic narrative of the A2i haplogroup, explored in Achilli et al.'s 2008 study within the Valle de Aburra [75], finds resonance in the genetic tapestry of Alaska.Recent studies involving mitogenomes from Alaska have brought to light a range of haplogroups, including A2, A2a, and A2i.The intriguing presence of A2i in both locales speaks to the remarkable genetic continuity and adaptation across distinct regions and epochs [85][86][87].
Within the complex genetic framework of lineage C1, a thorough network analysis has unveiled a compelling historical narrative.This analysis, which considered sequences from diverse regions such as the Amur River, Japan, and the Americas, lends support to the notion that the foundational haplotype for Native American C1 finds its roots in the Amur River region, echoing previous hypotheses proposed by Starikovskaya [86].
The C1c subclade, an exclusive genetic branch within haplogroup C1 exclusive to Native American populations, stands as a testament to the deep ancestral ties between indigenous peoples and the Americas.C1c plays a pivotal role in genetic and phylogenetic investigations, offering a unique perspective into the evolutionary history and migration patterns of specific groups.This subclade provides invaluable insights into the genetic diversity of Native American populations and their ancestral journeys [87].
The research also delved into broader colonization patterns.The presence of haplogroup B2 across diverse Native American populations adds weight to the idea of an early expansion from North to South America.Additionally, the widespread distribution of haplogroup C1 and its subclades suggests a unified migratory wave that played a crucial role in populating the Americas.The identification of variant 16298C in Abu4 within haplogroup C underscores the distinctive genetic legacy of pre-Hispanic Native American settlers.
Moreover, the presence of Haplogroup C1c, with its older coalescent age and evidence of intra-haplogroup variation, in both Mexican Americans and ancient Valle de Aburra populations hints at a captivating connection.This shared genetic heritage suggests that the genetic diversity within Haplogroup C1c, as well as its sub-branches, was firmly established among ancestral populations before their migration and expansion into the Americas.The exploration of this haplogroup's distribution and variation across populations promises profound insights into historical movements and genetic relationships among these groups, ultimately unraveling the intricate history of Native American origins and migrations.
Within the realm of Native American haplogroups, B2 emerges as a distinct lineage exclusive to indigenous populations of the Americas.Setting it apart from the other major founding haplogroups (A2, C1, D1, and X2a), the basal B2 lineage lacks defining variants in the control region (CR).This unique feature presents a challenge in distinguishing B2 from closely related haplogroups, a critical task for distinguishing Native American from Asian haplotypes [91].
Compelling evidence suggests that the initial wave of people entering what is now Mexican territory carried with them the A2 and B2 haplogroups around 15-18-thousand years ago.The distribution of individuals with higher frequencies of B2 or C1 compared to A2 indicates a specific migration pattern.A2, B2, and C1 appear to have been primarily dispersed along a Pacific coast route, spanning approximately 800 km along the Neovolcanic axis region.Subsequently, they made their way southward along the Pacific coast route, eventually reaching the Yucatan Peninsula via the Gulf coast route.
It is noteworthy that the Neovolcanic axis region, characterized by the presence of significant rivers like Lerma and Balsas, acts as a natural barrier against adverse Pacific weather conditions.This geographical feature likely played a pivotal role in facilitating the spread of these haplogroups [86,91].
Sub-haplotype D4j1a stands out from the norm when it comes to substitutions observed in mitochondrial DNA clusters associated with the southern distribution of aboriginal Siberian peoples.These substitutions are typically linked to major mitochondrial DNA haplogroups prevalent in East and South Asia.The intriguing presence of sub-haplotype D4j1a among both aboriginal Siberian populations and the ancient inhabitants of the Valle de Aburra unveils a fascinating genetic connection spanning vast geographical distances.Importantly, this sub-haplotype diverges from the usual mitochondrial DNA clusters found in aboriginal Siberian populations, aligning instead with major mitochondrial DNA haplogroups prevalent in East and South Asia [87][88][89][90][91][92][93].
The genetic connection between Siberia and the Valle de Aburra holds the potential to illuminate ancient migration patterns and the interactions between diverse populations.To gain a deeper understanding of the historical context and the implications of this shared genetic marker, further genetic and archaeological investigations are imperative (Figure 5).
Understanding the genetic connection between Siberia and Valle de Aburra offers a glimpse into the intricate journey of ancient human migrations.It suggests that populations from distant regions might have come into contact, possibly through migratory movements, trade routes, or cultural exchanges.
To provide a meaningful context for this genetic link, it is crucial to highlight the fundamental role of archaeological investigations, which, in combination with genetics, offer a more accurate representation of ancient life.The genetic relationship between the Valle de Aburra and Siberia, along with its proximity to Mesoamerica, suggests the diffusion of genetic markers across populations.Mesoamerica's genetic diversity, a result of its complex history, can provide insights into ancient cultural exchanges and trade routes.A multidisciplinary approach, encompassing genetics and archaeological excavations, is essential for a comprehensive understanding of the history and implications of this shared genetic marker.
The revelation of a genetic connection weaving through Siberia, Mesoamerica, and Valle de Aburra is nothing short of enthralling.This revelation acts as a beckoning call, compelling us to embark on an expedition of profound historical significance.Expanding our investigation into Mesoamerica opens doors to unraveling ancient ties, migratory routes, and the intricate web of interactions that wove together diverse populations.In the realm of collaborative genetic and archaeological exploration, we stand on the precipice of unearthing the narratives of our forebears, shedding brilliant light upon the enigmatic pathways of our shared past.
Comparisons with other populations across the Americas revealed that the Aburráes population showed the highest genetic similarity with pre-Hispanic populations in America.The results presented in our study contribute to the understanding of the genetic diversity and ancestral connections of the Aburráes population.These findings have implications for the population history and migration patterns in the Americas.
Furthermore, the study sheds light on population relationships across regions.The genetic affinity between the Aburráes and Mesoamerican and Arctic-sub-Arctic populations showcase intricate gene flow and migratory dynamics.These findings align with the hypothesis of a coastal migration route and potential interactions between the indigenous Chibchas population and the Mayan population.
The primary limitation of this study arises from its exclusive focus on the HVS-I of ancient mtDNA.This choice was necessitated by the inherent challenges in working with degraded and ancient DNA samples.While the results obtained are indeed intriguing and informative, it is imperative to recognize and acknowledge this limitation.
Likewise, it is advisable to approach the study's conclusions with a degree of caution.This caution is particularly warranted due to the relatively small sample size, consisting of only 10 individuals, and the restriction of the analysis to a single mitochondrial region (HVS-I).
Despite these challenges, it is noteworthy that the study was successful in elucidating important haplogroups.These findings make a significant contribution to our understanding of the genetic history of the ancient population under investigation.

Conclusions
In conclusion, the present study has provided valuable insights into the genetic diversity and population history of pre-Hispanic inhabitants in the Aburrá Valley region.The identification of various mitochondrial haplogroups, particularly A2 and its sub-lineages (A2af1b1b, A2ah, and A2i), Haplogroup B2, Haplogroup C1, and Haplogroup D4j1a, has shed light on the complex migratory patterns and interactions of ancient populations in the Americas.
One of the most striking findings of this research is the presence of distinct subhaplogroups within the larger haplogroups, such as A2af1b1b, A2ah, and A2i.These subhaplogroups not only reveal local genetic diversification but also highlight the enduring legacy of certain genetic lineages in the region.
While the genetic data presented in this study contribute valuable insights, it represents only a starting point in the investigation of the region's population history.The pursuit of more extensive and refined research will undoubtedly bring us closer to a deeper appreciation of the rich and complex human history that unfolded in the Aburrá Valley throughout the ages.
Ongoing research efforts are vital to fully unravel the region's population history.By continuously exploring and integrating diverse data sources, we can achieve a more nuanced understanding of the region's past, its connections to broader human migrations in the Americas, and the factors shaping its distinctive genetic landscape.

Figure 1 .
Figure 1.Geographic overview of excavation sites for pre-Hispanic human remains in the department of Antioquia, municipalities of Medellín, and Envigado.

Figure 1 .
Figure 1.Geographic overview of excavation sites for pre-Hispanic human remains in the department of Antioquia, municipalities of Medellín, and Envigado.

Figure 3 .
Figure 3. Network analysis of ten HVS-I sequences was performed using the reduced median algorithm.The star contraction option was applied to condense extensive data by identifying phylogenetic clusters into a single ancestral type.

Figure 3 .
Figure 3. Network analysis of ten HVS-I sequences was performed using the reduced median algorithm.The star contraction option was applied to condense extensive data by identifying phylogenetic clusters into a single ancestral type.

Figure 4 .
Figure 4. MDS analysis comparing haplotype frequencies of pre-Hispanic populations.AP: ancient population; CP: current population (the stress value for the matrix is 0.05841, with an RSQ of 0.98776).

Figure 4 .
Figure 4. MDS analysis comparing haplotype frequencies of pre-Hispanic populations.AP: ancient population; CP: current population (the stress value for the matrix is 0.05841, with an RSQ of 0.98776).

Figure 5 .
Figure 5. Geographic locations of analyzed populations in the Americas.These zones are colorcoded to simplify identification: blue: Arctic and Subarctic; green: northwestern United States; orange: Great Basin; yellow: southwestern United States; purple: Mesoamerican regions; gray: Caribbean; dark blue: Colombia and light green: South America.

Figure 5 .Table 5 .
Figure 5. Geographic locations of analyzed populations in the Americas.These zones are color-coded to simplify identification: blue: Arctic and Subarctic; green: northwestern United States; orange: Great Basin; yellow: southwestern United States; purple: Mesoamerican regions; gray: Caribbean; dark blue: Colombia and light green: South America.Table 5. Distribution of mitochondrial haplogroups in the sampled population.Offering an insightful breakdown of the genetic haplogroups assigned to each individual in the study.This comprehensive presentation aids in the categorization and enhances our understanding of the genetic diversity within the sampled population.

Table 2 .
Results of haplogroups and haplotypes identification based on the HVS-I sequence for each of the analyzed samples with the gender assignment, the origin of the excavation site and type of tissue used for DNA extraction.

Table 3 .
Measures of diversity in the Aburráes population from the mitochondrial sequences of the HVS-I.Where k number of different sequences; H, haplotype/genetic diversity; π, nucleotide diversity and Tajima's index (-D) and p value.

Table 3 .
Measures of diversity in the Aburráes population from the mitochondrial sequences of the HVS-I.Where k number of different sequences; H, haplotype/genetic diversity; π, nucleotide diversity and Tajima's index (-D) and p value.

Table 4 .
Table of haplogroups, showing genetic diversity across ancient and contemporary populations throughout the American continent.The genetic diversity (h) for various archaeological sites in different geographic zones, including the Aburráes region in Colombia.Sites with "N.C." values indicate that there is no genetic diversity data available for those sites.AP = 0 for modern indigenous populations.

Table 4 .
Table of haplogroups, showing genetic diversity across ancient and contemporary populations throughout the American continent.The genetic diversity (h) for various archaeological sites in different geographic zones, including the Aburráes region in Colombia.Sites with "N.C." values indicate that there is no genetic diversity data available for those sites.AP = 0 for modern indigenous populations.