Analysis of the earliest complete mtDNA genome of a Caribbean colonial horse (Equus caballus) from 16th-century Haiti.

Unlike other European domesticates introduced in the Americas after the European invasion, equids (Equidae) were previously in the Western Hemisphere but were extinct by the late Holocene era. The return of equids to the Americas through the introduction of the domestic horse (Equus caballus) is documented in the historical literature but is not explored fully either archaeologically or genetically. Historical documents suggest that the first domestic horses were brought from the Iberian Peninsula to the Caribbean in the late 15th century CE, but archaeological remains of these early introductions are rare. This paper presents the mitochondrial genome of a late 16th century horse from the Spanish colonial site of Puerto Real (northern Haiti). It represents the earliest complete mitogenome of a post-Columbian domestic horse in the Western Hemisphere offering a unique opportunity to clarify the phylogeographic history of this species in the Americas. Our data supports the hypothesis of an Iberian origin for this early translocated individual and clarifies its phylogenetic relationship with modern breeds in the Americas.

Introduction

Despite considerable speculation, the history of the domestic horse in North America remains unclear. We reconstructed the complete mitochondrial genome of one of the earliest specimens of domestic horse (Equus caballus) in the Americas in order to better understand the origins and trajectory of the first horses introduced by the Europeans after 1500. The specimen is from a 16th-century archaeological deposit at the site of Puerto Real, Haiti. Puerto Real was one of the first towns founded by the Spanish in the Americas and served as an important port. Horses, cows (Bos taurus), and other domestic animals of Eurasian origin were vital to the town [1, 2] and according to early colonial chronicles, all of these animals were brought to Hispaniola from the Iberian Peninsula via the Canary islands [3]. Other than the documentary evidence, little is known about source populations and the spread of domestic horse throughout the Americas. Here we use mitogenomics to place our sample in a phylogeny composed of global horse samples to understand the phylogeographic history of Western Hemisphere horses. Although sampling is limited to a single specimen, it is the oldest horse mitochondrial genome of the post-Columbian era sequenced to date, and of significance for understanding of the introduction of domestic horses in the Western Hemisphere.

A brief history of horses in the Americas

The Equus genus first appeared on the North American continent during the Pliocene era [4] and spread to and across Eurasia beginning around 2.5 million years ago [5]. All equids disappeared from the Western Hemisphere during the megafauna extinction event at the end of the Pleistocene and the last glacial period [6]. It was not until the late 15th-century and the arrival of European explorers in the Caribbean that equids returned to the Americas, this time as Equus caballus, the domestic horse.

The introduction of the domestic horse began with the second voyage of Columbus. In 1493, a royal ordinance from the Castilian monarchs ordered Fernando de Zafra to hire Andalusian horsemen to accompany Columbus on his second exploration [7]. Oviedo y Valdés [3] reports that, after Columbus boarded these horses on ships in the Canary Islands, they arrived on Hispaniola, in the town of La Isabela (Dominican Republic), on November 28, 1493.

Over the next decade, the horse population on Hispaniola grew rapidly from a few dozen individuals to large herds, thanks to favorable environmental conditions and continued importation of animals from the Iberian Peninsula. Eventually, the governor of the West Indies, Nicolás de Ovando, decreed there was no need to import more horses because the population size was sufficient on the island [7]. In this same decade, horses were taken to other Caribbean islands as Spanish interests expanded. By the 1520s horses had reached the Mesoamerican mainland and by 1538, the northern coast of the Gulf of Mexico in what is now Florida (USA) [8, 9]. A permanent Spanish presence was established on the North American coast in 1565. One of the most iconic episodes of equid introduction in the Americas is the arrival, in 1519, of Hernan Cortés’ 16 horses on the Mexican Gulf coast, contributing to the military conquest of Mexico. These animals were so essential that Spanish chronicler Bernal Diaz del Castillo gave a detailed description of each animal, with its name and appearance [10].

In later years, horses from other regions in Europe were brought to the Americas. For example, in the 1620s, in the British-sponsored colonies of New England, horses were brought from northern Europe, mostly from England and the Netherlands [11]. These later imports were of diverse breeds, some of them larger and better suited to labor than were the earlier Spanish imports [12]. Horse breeding rapidly became an important activity in some British colonies. By the 18th century, New England horses were a major export to the Caribbean where the sugarcane industry was in critical need of draft animals [13].

Horses were not only pivotal to the European military expeditions in the Americas, they also held a crucial role in the implementation of post-Columbian industries such as ranching in which horses were used to manage the wide-ranging cattle [14-16]. With the rapid growth of cattle herds, many horses became feral. By the 1650s they were widespread from Mexico to the Great Plains of North America, where they were tamed and re-domesticated by Native American communities of the Great Plains [15, 17]. In many regions horses deeply impacted Native cultures by providing greater mobility through an equestrian lifestyle [17, 18].

Horse lineages and mtDNA

The complete mitochondrial genome of the modern horse, which matrilineal lineages, was sequenced for the first time in 1994 [19]. Other modern horse mitogenome studies have targeted small portions of the mitochondrion, focusing particularly on clarifying the early phylogenetic history of horse domestication [20-23]. These studies revealed a weak geographic structure among mitochondrial haplogroups, the highest diversity of which are found in Asian horses, and also show that European and Middle Eastern horses lack genetic representation of the most ancestral lineages, suggesting an Asian origin [23]. More recently, ancient DNA analyses of archaeological horse specimens focusing on the whole genome (i.e. including the nuclear genome) of ancient horses confirmed the genetic homeland of modern horses as central Asia, and revealed that modern domestic horses probably originated around 4,200 years ago on the steppes near the Volga and Don rivers, now part of Russia, before spreading across Eurasia, ultimately replacing all pre-existing horse lineages [24].

The mitochondrial data from Modern horses suggest that domestic horses emerged via several distinct domestication events from populations with high maternal haplotypic diversity, and a later mixing of these different haplotypes during the early stages of domestication [23]. This contrasts with the evolutionary history of all the other Near Eastern domestic ungulates which arose from a limited number of animals domesticated in a small number of places around 10–8 ky BP [25, 26]. In contrast, studies of ancient horse lineages from the Y chromosome of modern individuals show almost no diversity of past male lineages, which contrasts with the great diversity of matrilineal lineages [27]. Together, these studies suggest that the domestication of horses involved a few closely related males and a very diverse population of females [27].

In the only study of the population genetics of present-day horses of the Western Hemisphere, an analysis of the control region of the D-loop in modern individuals of Iberian and American horse breeds reveals that Iberian animals were more diverse than any American horse breeds, and that many Western hemisphere breeds present a high frequency of haplotypes that can be traced back to the Iberian Peninsula, indicating that most have Iberian ancestry [9]. It also showed that among the Western Hemisphere breeds, diversity is highest in the Caribbean (based only on the Puerto Rican Paso Fino breed), followed by the South American breeds, and then the North American breeds which have the lowest genetic diversity.

Relatively little is known about the introduction of horses to the Americas from their archaeological remains, in part because early colonial horse remains may be mistakenly identified as either pre-extinction equids or Historic/modern intrusions [28]. Horse remains are rare in most 16th-century vertebrate assemblages of the Americas. For example, they represent only 2.3% of the faunal remains in early colonial deposits at the site of Ek’ Balam in Yucatan, Mexico [29], 1.75% of faunal remains from an Indigenous midden the site of El Japón in Mexico City [30], and 0.23% of a Spanish elite midden at the site of Justo Sierra in Mexico City [31]. This relative lack of specimens also could be related to the composition of archaeological faunal deposits and the status of horses in colonial societies. Many archaeological deposits are garbage dumps primarily containing food waste and debris from artisan activities. In colonial Hispanic society, horses were highly valued animals used in a multitude of different contexts, e.g. agriculture, transportation, or warfare, but they were not considered appropriate for human consumption, and are unlikely to be recovered amongst food waste [32]. Ancient DNA studies of archaeological horse remains in the Americas are equally rare. While several studies analyzed the extinct lineages of pre-Holocene horses [6, 33] only one published study contains genomic data (no mtDNA) of an historical horse specimen [28]. Thus, the Puerto Real specimen and its ancient DNA presented here is critical to understanding the history of domestic horses in the Americas.

Our study focuses on a single archaeological horse specimen recovered from excavations at the post-Columbian site of Puerto Real founded by Spain in 1503, and the final resting spot of some of the earliest colonial horses. While limited to a single specimen, this study is of significance to our understanding of the introduction and phylogeography of domestic horses in the Western Hemisphere as it constitutes the earliest complete equid mitochondrial genome sequenced to date for the post-Columbian era. Our approach focuses on the whole mitochondrial sequence, as earlier studies have demonstrated that the analysis of shorter sections of mtDNA do not provide sufficient detail to allow an assessment of the phylogeny of a species in all its complexity [23]. This paper characterizes this genome by comparing it with published equid sequences, situating it in the horse phylogeographic history. Our study provides a unique opportunity to address the question of the origins of the first colonial horses and their relationships to European breeds of the time, and also reveals genetic affinities between colonial horses from the Caribbean and modern populations from North America. This archaeogenomic evidence discusses the early colonial horse in the broader context of 16th-century Eurasian domesticates in the Americas, and contributes to our understanding of the history of the Spanish colonization, highlighting the long-term presence of horses alongside Spanish explorers and colonists on the Atlantic coast of North America.

Study area

Puerto Real was an early sixteenth-century town in what is now Haiti (Fig 1) [2, 34]. It was founded in 1503, one of the first 15 towns founded on Hispaniola. Nearby Puerto Plata was officially the last port of call for ships sailing from the Americas to Spain until 1515, when Havana, Cuba, was founded [35]. The site was home to a diverse population of Europeans, Taino Natives, and Africans. The economy of Puerto Real primarily was based on mineral resources (copper) and cattle. Colonists exploited cattle hides, which were more valued than meat, and exported them and other cattle products to Europe [1, 36]. Much of this trade was with Portuguese, Dutch, French, and English ships that called at Spanish ports on the northern coast of Hispaniola [36]. This was a violation of Spanish mercantile policies that gave the Spanish Casa de Contratación a monopoly on the American trade. Repeated efforts to counteract pirates and corsairs, limit the extensive illegal trade with foreign vessels, and exert more control over commerce on Hispaniola failed. Accordingly, the Crown ordered Puerto Real and other northern ports to be abandoned in 1578 and Puerto Real was destroyed by Spanish officials in 1579, though a few people remained in temporary housing at the site as squatters. The area was abandoned by the remaining Spanish, African, and Native residents in 1605, when the colonial authorities ordered the entire population of the western part of Hispaniola to relocate. After that date, most people on the northern coast were buccaneers, sailors licensed by colonial powers to prey on Spanish colonies, who hunted the herds of feral cattle left behind when Spain abandoned the coast [31].

Fig 1

Location of the site of Puerto Real and other locations mentioned in the study (original map by N. Delsol, cartographic data from USGS EROS http://eros.usgs.gov/, source: NASA/METI/AIST/Japan Spacesystems and U.S./Japan ASTER Science Team).

Excavations at Puerto Real were conducted by Dr. Kathleen Deagan, Curator Emerita of the Florida Museum Historical Archaeology Division, between 1979 and 1990. The 16th-century town was arranged in a 500 m x 400 m rectangle [37]. The excavations revealed several buildings including the church and its cemetery, two elite households and a middle social status residence [2].

Material

The zooarchaeological collection from Puerto Real represents an invaluable source of data on animal use and human-animal relationship in the colonial Caribbean. The collection was originally identified and analyzed by several researchers including E. Reitz, B. McEwan, and R.A. Marrinan [1, 38]. The Puerto Real faunal materials are curated by the Environmental Archaeology Program of the Florida Museum (the horse specimen is curated in EAP Accession number 0295) and cultural materials are curated by the Historical Archaeology program (cultural materials associated with the horse specimen are curated in ANT 92–002). Of the over 127,000 vertebrate specimens identified from Puerto Real thus far, only eight were attributed to horse when this study began [38]. Horse equipage was recovered from the town, including harness and tack artifacts. C.R. Ewen reports 31 items in this category from Locus 19 [39], McEwan reports three from Locus 33/35 [40], and Locus 39 contained a horseshoe and six harness/tack fragments [38].

The horse specimen used in this study (FLMNH Environmental Archaeology catalog number 02951527, see Fig 2) is a small tooth fragment initially identified as a cow (Bos taurus) by the lead author and included in a genetic study of early colonial cattle. Its identification as a horse came about during the genetic analysis itself. The specimen is a lower third molar from the left side. Its occlusal surface presents moderate wear, which implies the individual was an adult horse. The specimen was recovered during Deagan’s 1980 fieldwork campaign in an area originally called “Building A” (EAP Accession #0295), later interpreted as the church of the Spanish town [41]. The tooth is from Unit 770N-764E located just north of the church. Stratigraphically, it is from a level deposited during the initial abandonment of the town in 1578 and sealed by a sterile layer above it (i.e. a layer containing no archaeological materials). This archaeological context contains exclusively 16th century materials. Historical and archaeological contexts and material culture permit the excavators to reliably date the horse tooth to the last quarter of the 16th century. The deposit, therefore, represents activities of remnant squatters at the site between 1578 and 1605, after which the town was burned and remaining residents removed. Historical and archaeological contexts and material culture permit the excavators to reliably date the horse tooth to the last quarter of the 16th century.

Fig 2

Picture of the horse specimen R0121-03 (FM-EAP Catalog Number 02951527).

Methods

Data acquisition and permits

All necessary permits were obtained for the described study, which complied with all relevant regulations. Permission for the excavation, import, and curation of the materials were provided by the Haitian government to the Florida Museum in 1980. Permission for use of the specimen in this research (FM-EAP Catalog Number 02951527) was provided following evaluation by the curators and collection managers of the Florida Museum Divisions of Historical Archaeology (Curator Charles Cobb, and Collection Manager Gifford Waters) and Environmental Archaeology (Curator Kitty Emery and Collection Manager Nicole Cannarozzi)in accordance with the Florida Museum Destructive Analysis Policy (https://www.floridamuseum.ufl.edu/wp-content/uploads/sites/32/2017/03/Destructive_Sampling_Policy.pdf)

DNA extraction and sequencing

All DNA extraction work was conducted in the Florida Museum Ancient DNA laboratory that includes a clean room facility specifically dedicated to the extraction of ancient and historical DNA samples. We first isolated a fragment of the molar weighing about 500 mg. This sample was then frozen using liquid nitrogen and pulverized with a stainless-steel mortar and pestle. Following Yang and colleagues’ protocol [42] as implemented by Oswald et al. [43], the bone powder was combined with an extraction buffer composed of 949 μl of 0.5 M ethylenediaminetetraacetic acid (EDTA), 25 μl 20 mg/ml proteinase K, 21 μl of 10 mg/ml 1,4-dithiothreitol (DTT), and 5 ul of 10% sodium dodecyl sulfate (SDS). We incubated the sample at 60°C for 24 h, centrigugated it,and then intermittently vortexed and then concentrated it with two Amicon® Ultra-4 Centrifugal Filter Unit and purified using a Qiagen QiaQuick MinElute Kit and eluted in 48 μl of elution buffer according to the manufacturer’s directions. A negative control was included alongside each extraction to monitor contamination. DNA extraction of the sample and the negative control were quantified with a Qubit® 2.0 Fluorometer.

After the extraction, we sent the sample to Rapid Genomics (Gainesville, Florida) for library preparation, mtDNA enrichment, and sequencing. Rapid Genomics is a private company dedicated to the processing of genomic samples and sequencing. They followed a strict protocol adapted to the processing of ancient and degraded samples. This protocol was designed in collaboration with the authors. After initial quantification, the libraries were built using Swift Methyl Seq Kits. This kit uses a uracil-tolerant polymerase and performs well with degraded and low-yield samples. Bisulfite conversion was not performed on the horse sample. To retain lower molecular weight fragments and improve libraries with degraded DNA, SPRI bead cleanup ratios were modified as follows: post-extension SPRI ratio 1.8; post-ligation SPRI ratio 1.6; post-PCR SPRI ratio 1.6. Otherwise, the protocol was followed per manufacturers’ instructions. For the enrichments, Rapid Genomics designed RNA bait kits using 12,000 probes based on the domestic cow (Bos taurus) mitogenome (cattle mitochondrial reference genome, NC_006853.1). Capture reactions were performed on the library using all the library product or up to 500 ng following Rapid Genomics customized workflow. The hybridization was performed at 60°C for 48 hours. After hybridization, enrichment PCRs were performed for 15 cycles. The sample was sequenced on an Illumina MiSeq sequencer (2x150 paired-end sequencing).

NGS reads processing

The quality of the reads from the sequencing was first assessed using FASTQC [44]. Adapter removal (i5 and i7 Illumina primers) and quality trimming [28] was performed with the program adapterremoval [45]. The forward and reverse reads were then merged into a single read and duplicates were removed in Geneious Prime (https://www.geneious.com/,version 2021.1). The sequence was attributed to a horse after a BLAST search on the reads with the blastn command line application in Geneious using the nt database and the default options (E-value 1e-03). We mapped the reads to the horse mitochondrial reference genome (Genbank NC_001640.1) using the Geneious mapper algorithm set on “Custom sensitivity” with a minimum mapping quality of 30, allowing for only 5% mismatch between reads and not allowing gaps. The Geneious algorithm maps reads to the reference up to 5 times. A contiguous mitochondrial genome sequence was generated by Geneious with a threshold of 75% and quality set on “Highest”. The consensus generated with Geneious out of contig pileups represents a mean coverage of 5622.9X across the horse reference mitochondrion. The damage patterns of the mitogenome assembly were assessed using MapDamage [46]. The DNA damage patterns for the mapped mitogenome of the Puerto Real horse are consistent with those of ancient DNA, with elevated C-T transitions occurring on the 5’ and 3’ ends (S1 Fig). This confirms a lack of contamination from modern DNA. Following analysis, the raw reads were uploaded on the Sequence Read Archive (SRA, accession number PRJNA817535) and the assembled sequence on Genbank (accession number ON168403).

Phylogeny and haplotype map

The mitochondrial genome of the Puerto Real horse was aligned with the 83 mtDNA sequences of modern horses published by Achilli and colleagues [23] and obtained from GenBank to establish the closest relatives of this individual (S1 Table). This operation was performed using Geneious alignment algorithm with a gap open penalty of 12 and a gap extension penalty of 3. We processed the FASTA alignment file through GBlocks to remove ambiguously aligned sequences [47] resulting in 16,652 bp in the final alignment. To identify the Puerto Real horse sequence and its relationship to known matrilineal haplogroups, we applied a maximum likelihood (ML) model using RAxML [48] with the GTRCAT model of rate heterogeneity (S2 Fig) and 10,000 bootstrap replicates (option “-f a”). The data were partitioned based on the annotations imported from the reference sequence into coding (CDS), non-coding (introns), rRNA, and tRNA regions to produce a phylogeny using different estimates for each of these regions. We used the reference mitogenome of the donkey (Equus asinus, Genbank RefSeq: NC_001788.1) as an outgroup.

The relationships of the Puerto Real horse to the 83 other mitogenomes also was visualized using a median-joining network. This approach permits the reconstruction of the networks illustrating the relationships between organisms based on Kruskal’s algorithm for generating minimum spanning trees and Farris’s maximum-parsimony (MP) heuristic algorithm [49]. The network was realized using the PopART (Population Analysis with Reticulate Trees) software [50]. This visualization provides results similar to the maximum likelihood phylogenetic analysis (Fig 3).

Fig 3

Median joining network of the 85 equid specimens (including Equus asinus as an outgroup and the Puerto Real horse).

Numbers on the branches indicate the number of mutations separating each mitogenome.

Results

This study instead explicitly focuses on the whole mitochondrial sequence following more recent horse phylogeographic analyses showing that the short segments of the mtDNA used in earlier studies were often accompanied by high levels of recurrent mutations, thus blurring the structure of the phylogenetic analyses and rendering some clades more difficult to distinguish from one another [23]. Thus, most complete mitogenomes published on Genbank stem from the Achilli and colleagues study mentioned above. A GenBank query using the terms “horse mitochondrion” returned a total of 511 complete mtDNA sequences (~16.6 kbp). None of the returned sequences were individuals from Iberia and all the specimens from the Americas found using this query were included in our study. The study by Achilli and colleagues [23] identified a total of 667 SNPs, including 549 sites in the coding region and 118 in the non-coding region, mostly represented by the D-loop (113 out of 960 nucleotides) and with these data identified 18 haplogroups (S1 Table). The Puerto Real horse falls into the haplogroup A (Figs 3 and 4; S2 Fig). This equine branch is defined by a specific mutation at nucleotide position 15,720. According to Achilli et al. [23], individuals of the equine haplogroup A mostly are found in Central Asia (46.7% of the modern specimens), Southern Europe (37.3%), and the Middle East (10.0%). Horses belonging to this haplogroup also were identified in Bronze Age archaeological contexts in the Iberian Peninsula [23, 51]. As with most of the other mitochondrial lineages, members of haplogroup A are characterized by a wide geographical range including, for example, the Maremmano (Italy), Akhal Teke (Turkmenistan, central Asia), Caspian ponies (Central Asia), and several breeds from the Middle East. However, this equine group does not include any Northern European breeds, indicating that the Puerto Real horse is least likely to have been bred from horses in that region. The phylogeny and haplotype maps indicate that the Puerto Real horse is most closely related to a modern specimen of the American Chincoteague pony breed (JN398377) (Fig 3), a finding backed by 86% bootstrap support (Fig 4). These two taxa differ by six mutations (three transitions at positions 6792, 16298, and 16404, and three transversions at positions 12,735, 16,343, and 16,403).

Fig 4

Detail of the haplogroup A phylogeny.

The complete phylogeny of all 85 sampled individuals can be found in S2 Fig.

Discussion

The introduction of domestic horses into the Western Hemisphere was part of the massive exchange of organisms between both sides of the Atlantic Ocean that began with the European invasion of the Americas [52]. Eurasian domesticates such as cows, pigs (Sus scrofa), sheep (Ovis aries), and chicken (Gallus gallus) had been central to the economic, social, and symbolic life of European and African societies since the Neolithic. They were brought to the Americas by colonists whose lifestyles and economies relied on these animals. In this context, horses played a central role in colonial life, and were crucial symbols of social status for Spanish colonists. The ownership of horses often characterized high-status individuals such as hidalgos (members of the Iberian nobility) and access to them by Native people in the Americas was restricted [53]. Besides being symbols of elite social status, horse labor was vital in some colonial economies and conditioned colonists use of American environments. Horses also facilitated the implementation of other animal-related industries, such as ranching, that fundamentally reshaped much of the American landscape [16, 54].

Historical scholarship on the sources of Spanish colonial horses suggests they were brought directly from Castile (central Spain) via the Canary Islands. Previous DNA studies on modern breeds from the Americas also suggest a Spanish origin for early colonial horses [9, 55]. The attribution of the Puerto Real horse to the haplogroup A is consistent with an Iberian origin for horses in the Caribbean after 1492. This maternal lineage extends from Central Asia to southern Europe and has been present in the Iberian Peninsula since at least the Bronze Age.

The specimen that presents the closest affinities with the Puerto Real horse is an individual belonging to the Chincoteague pony breed. Chincoteague ponies are a feral equine breed found on the islands of Chincoteague and Assateague, off the Maryland and Virginia coasts (United States) [56]. This small population (28 individuals in 1968, 175 in 2001) has been extensively studied from a conservation and ecological perspective to assess their status as an American heritage breed [56, 57]. The origin of the Chincoteague ponies is popularized by a local oral history retold in the mid-20th century children’s novel “Misty of Chincoteague” [58]. According to this story, Chincoteague ponies descend from a small herd of horses that escaped the shipwreck of a Spanish galleon during the colonial era. The galleon is said to have set sail from the Caribbean but was caught in a storm and shipwrecked close to Chincoteague Island. This narrative has generated some opposition, some authors arguing that the ponies did not precede the arrival of British settlers on the island because early British documents on the island did not describe feral horse population [59], or that it was not likely for horses to survive a shipwreck by swimming to the shore [60]. Other authors argue that the presence of a 1750 Spanish shipwreck close to the island lends veracity to the story [61, 62]. An earlier study, based on the comparative analysis of 10 protein loci revealed genetic affinities between the Chincoteague ponies and specimens of the Paso Fino breed from the Caribbean region [57].

Beyond folk stories, affinities between early Caribbean horse breeds and the Chincoteague ponies may reflect Spanish efforts to colonize the Atlantic coast of North America. These colonizing activities persisted throughout the 16th-century, reaching as far north as Chesapeake Bay. Most of these efforts originated on Hispaniola, Cuba, and Puerto Rico. Colonization, trading, and raiding among European interests in the Caribbean and along the Atlantic coast into the 1800s offered many opportunities for Spanish horses to reach the mid-Atlantic coast. By revealing these genetic affinities, this study contributes a new perspective on colonization’s impact on domestication, colonial economies, and its environmental consequences.

Conclusion

While limited to a single mitogenome, this study has a particular significance as it constitutes the first archaeogenomic evidence of post-Columbian horses in the Western Hemisphere. Our analysis suggests that the Puerto Real horse belongs to the equine haplogroup A, a maternal branch commonly found in Central Asia and Southern Europe. These results are consistent with an Iberian origin of this horse, as specimens of this haplogroup are found as early as the Bronze Age in the Iberian Peninsula. This supports early colonial accounts that suggest Spanish Andalusia was the source of the first horses brought to the Caribbean. We also found that the Puerto Real horse is closely related to another breed currently found in the Americas, the feral population of Chincoteague ponies. Further work on feral horse populations along the Atlantic coast of the United States, and continued horse archaeogenomic sampling promises to further clarify our understanding of how horses colonized the Western Hemisphere.

MapDamage [37] fragment misincorporation plot for the Puerto Real horse sample.

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Partitioned RAxML (39) phylogeny.

Bootstrap support is on the nodes (10,000 iterations). Letters on the right correspond to the equine haplogroups defined by Achilli et al. [22].

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Samples from GenBank that were included in the phylogeographic analysis.

Except for NC001788 (Equus asinus) all are published in Achilli et al. [22].

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1 Mar 2022

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Maps at the CIA (public domain): https://www.cia.gov/library/publications/the-world- factbook/index.html and https://www.cia.gov/library/publications/cia-maps-publications/index.html

NASA Earth Observatory (public domain): http://earthobservatory.nasa.gov/

Landsat: http://landsat.visibleearth.nasa.gov/

USGS EROS (Earth Resources Observatory and Science (EROS) Center) (public domain): http://eros.usgs.gov/#

5. Thank you for stating the following in the Acknowledgments Section of your manuscript:

“This research was funded by the National Science Foundation to Emery and Delsol (DDIG 1930628), the Fulbright program (to Delsol), and additional support from the Florida Museum Ancient DNA Laboratory (from Guralnick). Permission for excavation and export of the faunal materials for curation at the Florida Museum was provided to Deagan by the government of Haiti, and permission for analysis was provided by the curators of the Historical Archaeology (Cobb) and Environmental Archaeology (Emery) programs of the Florida Museum.”

We note that you have provided additional information within the Acknowledgements Section that is not currently declared in your Funding Statement. Please note that funding information should not appear in the Acknowledgments section or other areas of your manuscript. We will only publish funding information present in the Funding Statement section of the online submission form.

Please remove any funding-related text from the manuscript and let us know how you would like to update your Funding Statement. Currently, your Funding Statement reads as follows:

“KFE and ND received support from NSF DDRIG grant (#1930628) for this research (https://beta.nsf.gov/funding/opportunities/archaeology-and-archaeometry-0). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.”

Please include your amended statements within your cover letter; we will change the online submission form on your behalf.

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Partly

Reviewer #4: Yes

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: N/A

Reviewer #4: Yes

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: No

Reviewer #3: No

Reviewer #4: Yes

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: It is an interesting research. Author briefed an interesting history about horses in the Americas and take us an overview about the origin of early translocated individual and its phylogenetic relationship with modern breeds in the Americas.

Reviewer #2: The manuscript reads very well and presents a nice story that fits with the historical records for the introduction of the horse into the Americas in the 16th century. The only minor issue with the results presented is that it is only based on a single specimen.

While the authors indicate that the sequence is publicly available at the NCBI, no accession number is provided. The accession number needs to be added before the paper can be published.

For the rest, I only have a few minor comments:

Line 73: "By the 1650s century" delete "century"

Lines 167-175. It would have been nice if the authors would have added a map of the archeological site in the supplementary material.

Lines 232-237: I assume the medium-joining network mentioned in this paragraph is the one shown in figure 3?

Line 259: "Figure S1" should be "Figure S2"

Reviewer #3: The paper reports assembly of the whole mt genome of a single horse dating back to 16th century. It is well written, and although only one sample was analyzed I think it adds to current knowledge.

However, I have several suggestions to improve the paper before publication on PLOS ONE, mainly related to reproducibility and data availability.

In particular, authors declared that they made the data publicly available, but I wasn't able to find any link pointing to them.

I list my concerns below regarding Data availability and Methods (and reproducibility)

Data availability issues:

Raw reads should be deposited on SRA. The assembled genome should be deposited in GenBank. Authors declared that the data are available, but they are not.

Methods and reproducibility issues:

First of all, I think authors performed all the analysis (mapping, assembling, phylogenetic tree construction) using the Geneious commercial software. This should be clearly declared.

Most importantly, for each analysis step, all the parameters and options used should be clarified so that a user buying Genious and having access to the data would be able to repeat the experiment. I think we are still far from this. I give some suggestions below, but I invite the authors to carefully check the methods and identify more missing details.

Line 210: “The 3’ and 5’ reads were imported, paired, and deduplicated in Geneious (https://www.geneious.com/).” This description should be more detailed. 1) What does it mean that read were “imported, paired”? Reads are already provided as paired by the sequencing instrument. Did the authors mean that the tried to merge paired reads in a single read? 2) What does it mean that reads were “deduplicated”? Did authors remove duplicates? 3) I assume 3’ and 5’ reads are the so-called forward and reverse reads. I think it is more common to name them forward and reverse, but this is not an issue.

Line 211: “The sequence was attributed to a horse after a BLAST search on the reads.” Did the authors use the blast online service? If so, they should provide the link and/or a reference to BLAST publications. Did the authors use default parameters? Did the authors blast against nt or specifically against the horse genome? Which threshold did they set in order to accept the hits as horse?

Lines 211-213: Which software was used for mapping? Which parameters were used to ensure that the maximum proportion of mismatches was 5%?

Lines 213-214: It is not clear how authors went from mapping reads to assemble the mitochondrial genome. This should be described.

Lines 216-218: Which software used for building the consensus? How were contigs generated? Which requisites were required for a contig to be used in the assembly? (e.g. contig length, contig coverage). Which overlap between contigs was required to assemble them into a scaffold? “We built the consensus sequence of the whole mitochondrion of the Puerto Real horse with a threshold of 75% and a quality set on “Highest”. The consensus was generated out of contig pileups that represented a mean coverage of 5622.9X of the horse reference mitochondrion.”

Lines 229-230: “The mitochondrial data was partitioned into coding, non-coding, and RNA regions to produce a phylogeny using different model estimates for each of these regions.” What are the differences between the model estimates? Are they implemented in Geneious? What are their names?

Reviewer #4: This study focused on a single ancient horse sample from 16th century /Caribbean colonial horse/. Complete mitochondrial sequencing of this single sample was performed. According to the authors, this mitochondrial genotype is associated with group A, and is the most closely related to a modern specimen of the American Chincoteague pony breed from North America (JN398377). I did not find a deposit of your genomic sequence and Genbank. Why?

The article is well discussed historically, but in contrast, relatively genetic research in this region is lacking. It would be good to make a comparative analysis of the available genetic data on the biodiversity of mitochondrial lines (modern and ancient) for America and the Iberian Peninsula. I would recommend adding data on modern local horses from the study region.

**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

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Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

Reviewer #4: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

11 Apr 2022

We would like to thank the academic editor and the reviewers for their thoughtful comments which have greatly helped improve our manuscript. Please find here our response to the points they raised. For clarity purposes, the editor and reviewers’ initial comments are inserted before our responses.

A. Editor’s comment:

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and

https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

We made sure to implement PLOS ONE’s style template in the main manuscript and changed the figures file names to follow the file naming template.

2. In your manuscript, please provide additional information regarding the specimens used in your study. Ensure that you have reported specimen numbers and complete repository information, including museum name and geographic location.

If permits were required, please ensure that you have provided details for all permits that were obtained, including the full name of the issuing authority, and add the following statement:

'All necessary permits were obtained for the described study, which complied with all relevant regulations.'

If no permits were required, please include the following statement:

'No permits were required for the described study, which complied with all relevant regulations.'

For more information on PLOS ONE's requirements for paleontology and archaeology research, see https://journals.plos.org/plosone/s/submission-guidelines#loc-paleontology-and-archaeology-research.

We added a Methods subsection that details additional information on the permits and curation institution, including the statement 'All necessary permits were obtained for the described study, which complied with all relevant regulations.'

3. Please include a complete copy of PLOS’ questionnaire on inclusiveness in global research in your revised manuscript. Our policy for research in this area aims to improve transparency in the reporting of research performed outside of researchers’ own country or community. The policy applies to researchers who have traveled to a different country to conduct research, research with Indigenous populations or their lands, and research on cultural artifacts. The questionnaire can also be requested at the journal’s discretion for any other submissions, even if these conditions are not met. Please find more information on the policy and a link to download a blank copy of the questionnaire here: https://journals.plos.org/plosone/s/best-practices-in-research-reporting. Please upload a completed version of your questionnaire as Supporting Information when you resubmit your manuscript.

The questionnaire on inclusiveness in global research will be uploaded in the SI section together with the revised documents.

4. We note that Figure 1 in your submission contain [map/satellite] images which may be copyrighted. All PLOS content is published under the Creative Commons Attribution License (CC BY 4.0), which means that the manuscript, images, and Supporting Information files will be freely available online, and any third party is permitted to access, download, copy, distribute, and use these materials in any way, even commercially, with proper attribution. For these reasons, we cannot publish previously copyrighted maps or satellite images created using proprietary data, such as Google software (Google Maps, Street View, and Earth). For more information, see our copyright guidelines: http://journals.plos.org/plosone/s/licenses-and-copyright.

We require you to either (1) present written permission from the copyright holder to publish these figures specifically under the CC BY 4.0 license, or (2) remove the figures from your submission:

1. You may seek permission from the original copyright holder of Figures 1 to publish the content specifically under the CC BY 4.0 license.

We recommend that you contact the original copyright holder with the Content Permission Form (http://journals.plos.org/plosone/s/file?id=7c09/content-permission-form.pdf) and the following text:

“I request permission for the open-access journal PLOS ONE to publish XXX under the Creative Commons Attribution License (CCAL) CC BY 4.0 (http://creativecommons.org/licenses/by/4.0/). Please be aware that this license allows unrestricted use and distribution, even commercially, by third parties. Please reply and provide explicit written permission to publish XXX under a CC BY license and complete the attached form.”

Please upload the completed Content Permission Form or other proof of granted permissions as an "Other" file with your submission.

In the figure caption of the copyrighted figure, please include the following text: “Reprinted from [ref] under a CC BY license, with permission from [name of publisher], original copyright [original copyright year].”

2. If you are unable to obtain permission from the original copyright holder to publish these figures under the CC BY 4.0 license or if the copyright holder’s requirements are incompatible with the CC BY 4.0 license, please either i) remove the figure or ii) supply a replacement figure that complies with the CC BY 4.0 license. Please check copyright information on all replacement figures and update the figure caption with source information. If applicable, please specify in the figure caption text when a figure is similar but not identical to the original image and is therefore for illustrative purposes only.

The following resources for replacing copyrighted map figures may be helpful:

USGS National Map Viewer (public domain): http://viewer.nationalmap.gov/viewer/

The Gateway to Astronaut Photography of Earth (public domain): http://eol.jsc.nasa.gov/sseop/clickmap/

Maps at the CIA (public domain): https://www.cia.gov/library/publications/the-world- factbook/index.html and https://www.cia.gov/library/publications/cia-maps-publications/index.html

NASA Earth Observatory (public domain): http://earthobservatory.nasa.gov/

Landsat: http://landsat.visibleearth.nasa.gov/

USGS EROS (Earth Resources Observatory and Science (EROS) Center) (public domain): http://eros.usgs.gov/#

Figure 1 is a map that was designed by the lead author (Nicolas Delsol) using the open-source GIS software QGIS (version 3.24.1). The rasters used for the design of this map come from public and non-copyright cartographic resources generated by the NASA and and Japan’s Ministry of Economy, Trade, and Industry (METI). These resources were retrieved at the url http://eros.usgs.gov/# and were recommended by the Academic Editor in his comments. In the caption of the figure we mention the source of this raw cartographic data.

5. Thank you for stating the following in the Acknowledgments Section of your manuscript:

“This research was funded by the National Science Foundation to Emery and Delsol (DDIG 1930628), the Fulbright program (to Delsol), and additional support from the Florida Museum Ancient DNA Laboratory (from Guralnick). Permission for excavation and export of the faunal materials for curation at the Florida Museum was provided to Deagan by the government of Haiti, and permission for analysis was provided by the curators of the Historical Archaeology (Cobb) and Environmental Archaeology (Emery) programs of the Florida Museum.”

We note that you have provided additional information within the Acknowledgements Section that is not currently declared in your Funding Statement. Please note that funding information should not appear in the Acknowledgments section or other areas of your manuscript. We will only publish funding information present in the Funding Statement section of the online submission form.

Please remove any funding-related text from the manuscript and let us know how you would like to update your Funding Statement. Currently, your Funding Statement reads as follows:

“KFE and ND received support from NSF DDRIG grant (#1930628) for this research (https://beta.nsf.gov/funding/opportunities/archaeology-and-archaeometry-0). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.”

Please include your amended statements within your cover letter; we will change the online submission form on your behalf.

The Acknowledgement section has been changed to reflect the Editor’s requests. We also removed funding-related text from the manuscript.

In addition, we ask the Academic Editor to change the funding statement in the following way:

“KFE and ND received support from NSF DDRIG grant (#1930628) for this research (https://beta.nsf.gov/funding/opportunities/archaeology-and-archaeometry-0). ND’s initial research was also funded by a Fulbright scholarship. The field research in Haiti was financed through contributions of the Organization of American States, the National Endowment for the Humanities, Dr. William Goya, the Wentworth Foundation, the University of Florida Division of Sponsored Research, and the Florida Museum of Natural History. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.”

B. Reviewers’ comments:

- Reviewers #2, #3, and #4 expressed some concern about the availability of the consensus sequence and the raw reads on public databases. The raw reads have been deposited on the NCBI SRA repository (BioProject# PRJNA817535, SRA# SRR18395639). The annotated mitochondrial consensus sequence has been deposited on Genbank under the reference # ON168403. Both datasets are set to be publicly released upon publication of the article.

- Reviewer #2:

The manuscript reads very well and presents a nice story that fits with the historical records for the introduction of the horse into the Americas in the 16th century. The only minor issue with the results presented is that it is only based on a single specimen.

While the authors indicate that the sequence is publicly available at the NCBI, no accession number is provided. The accession number needs to be added before the paper can be published.

For the rest, I only have a few minor comments:

Line 73: "By the 1650s century" delete "century"

Lines 167-175. It would have been nice if the authors would have added a map of the archeological site in the supplementary material.

Lines 232-237: I assume the medium-joining network mentioned in this paragraph is the one shown in figure 3?

Line 259: "Figure S1" should be "Figure S2"

We made the minor revisions asked for on lines 73 and 259. We also added a reference to Fig 3 to refer to the median-joining network. About the site map, we did not have access to a high-quality reproduction of a map of the site, given that most publications on this site are almost 30 years old. The syntheses of the site that were cited (Deagan 1995, 1996) provide some cartographic data.

- Reviewer #3:

The paper reports assembly of the whole mt genome of a single horse dating back to 16th century. It is well written, and although only one sample was analyzed I think it adds to current knowledge.

However, I have several suggestions to improve the paper before publication on PLOS ONE, mainly related to reproducibility and data availability.

In particular, authors declared that they made the data publicly available, but I wasn't able to find any link pointing to them.

I list my concerns below regarding Data availability and Methods (and reproducibility)

Data availability issues:

Raw reads should be deposited on SRA. The assembled genome should be deposited in GenBank. Authors declared that the data are available, but they are not.

Methods and reproducibility issues:

First of all, I think authors performed all the analysis (mapping, assembling, phylogenetic tree construction) using the Geneious commercial software. This should be clearly declared.

Most importantly, for each analysis step, all the parameters and options used should be clarified so that a user buying Genious and having access to the data would be able to repeat the experiment. I think we are still far from this. I give some suggestions below, but I invite the authors to carefully check the methods and identify more missing details.

Line 210: “The 3’ and 5’ reads were imported, paired, and deduplicated in Geneious (https://www.geneious.com/).” This description should be more detailed. 1) What does it mean that read were “imported, paired”? Reads are already provided as paired by the sequencing instrument. Did the authors mean that the tried to merge paired reads in a single read? 2) What does it mean that reads were “deduplicated”? Did authors remove duplicates? 3) I assume 3’ and 5’ reads are the so-called forward and reverse reads. I think it is more common to name them forward and reverse, but this is not an issue.

Line 211: “The sequence was attributed to a horse after a BLAST search on the reads.” Did the authors use the blast online service? If so, they should provide the link and/or a reference to BLAST publications. Did the authors use default parameters? Did the authors blast against nt or specifically against the horse genome? Which threshold did they set in order to accept the hits as horse?

Lines 211-213: Which software was used for mapping? Which parameters were used to ensure that the maximum proportion of mismatches was 5%?

Lines 213-214: It is not clear how authors went from mapping reads to assemble the mitochondrial genome. This should be described.

Lines 216-218: Which software used for building the consensus? How were contigs generated? Which requisites were required for a contig to be used in the assembly? (e.g. contig length, contig coverage). Which overlap between contigs was required to assemble them into a scaffold? “We built the consensus sequence of the whole mitochondrion of the Puerto Real horse with a threshold of 75% and a quality set on “Highest”. The consensus was generated out of contig pileups that represented a mean coverage of 5622.9X of the horse reference mitochondrion.”

Lines 229-230: “The mitochondrial data was partitioned into coding, non-coding, and RNA regions to produce a phylogeny using different model estimates for each of these regions.” What are the differences between the model estimates? Are they implemented in Geneious? What are their names?

Reviewer #3 had several recommendations to improve the paper, particularly in terms of reproducibility and data availability. We already addressed the question of data accessibility (see above). In the methods section, we added some clarifications as requested by the reviewer as shown in the new version of the manuscript. More specifically, we added additional details on how the reads were assembled using the commercial software Geneious (lines 210-218).

- Reviewer #4:

This study focused on a single ancient horse sample from 16th century /Caribbean colonial horse/. Complete mitochondrial sequencing of this single sample was performed. According to the authors, this mitochondrial genotype is associated with group A, and is the most closely related to a modern specimen of the American Chincoteague pony breed from North America (JN398377). I did not find a deposit of your genomic sequence and Genbank. Why?

The article is well discussed historically, but in contrast, relatively genetic research in this region is lacking. It would be good to make a comparative analysis of the available genetic data on the biodiversity of mitochondrial lines (modern and ancient) for America and the Iberian Peninsula. I would recommend adding data on modern local horses from the study region.

Reviewer #4 also expressed some concern about the accessibility of the sequence and the reads (see above for the response). We agree with their suggestion that adding more mitochondrial genomes of horses from the Caribbean would be helpful. However, there are few published and available mitogenomes from the Caribbean on public repositories such as Genbank or the ENA, and we used all available, relevant mitogenomes in our analysis. We hope that further work on horse heritage breeds from across the Caribbean will provide more data to fill this gap.

Again, we thank the academic editor and the reviewers for their comments and suggestions. We sincerely hope the changes in this revised version of the manuscript reflects these recommendations.

Sincerely,

Submitted filename: PLOS1 rebuttal letter_Delsol_et_al_final.docx

Click here for additional data file.

17 May 2022

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.

A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.

An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Tzen-Yuh Chiang

Academic Editor

PLOS ONE

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #3: All comments have been addressed

Reviewer #4: (No Response)

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #3: Yes

Reviewer #4: Partly

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #3: N/A

Reviewer #4: No

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #3: Yes

Reviewer #4: No

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #3: Yes

Reviewer #4: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: (No Response)

Reviewer #3: All my comments and concerns have been addressed, and I think that the paper is now suitable for publication. Authors provided the required details for reproducing the work using Geneious and the other software they used. Authors also provided accession numbers for the raw reads and the genome assembly. The data will be made available after publication.

Reviewer #4: The authors of the article did not comment on my two remarks.

- I did not find a deposit of your genomic sequence and Genbank?

- It would be good to make a comparative analysis of the available

genetic data on the biodiversity of mitochondrial lines (modern and ancient) for

America and the Iberian Peninsula. I would recommend adding data on modern local

horses from the study region.

In addition, the article is not written according to the requirements of PlosOne. As an example, too short a description of the results and the discussion. The abstract section is not formatted according to the requirements of the journal.

The Materials and Methods section describes the isolation of ancient DNA that does not conform to the protocols of good practice for working with ancient DNA. Тhere are no control reactions for DNA contamination as example.

I cannot believe that DNA isolation is possible in the described way to dissolve 500 mg of dry bone material in 1 ml of buffer.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #3: No

Reviewer #4: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

6 Jun 2022

We would like to renew our thanks to the academic editor and the reviewers for their thoughtful comments which have greatly helped improve our manuscript. We were happy to see that two of the three reviewers found our prior submission satisfactory. Please find here our responses to the points raised by Reviewer #4 in this version.

I did not find a deposit of your genomic sequence and Genbank?

As stated in the letter attached to our first resubmission, the assembly of the horse mitogenome has been submitted successfully to Genbank (accession number ON168403) and the raw reads were submitted to the Sequence Read Archive (accession number PRJNA817535). These have been explicitly called out in the latest revisions to this article (lines 245-247). As customary, we have embargoed the release of these data until after the publication of the manuscript. As a proof of these submissions, we provide the confirmation emails we received from both repositories.

It would be good to make a comparative analysis of the available genetic data on the biodiversity of mitochondrial lines (modern and ancient) for America and the Iberian Peninsula. I would recommend adding data on modern local horses from the study region.

We agree with Reviewer 4 that such a comparative analysis of this 16th-century mitogenome with modern and ancient mtDNA from archaeological and modern horses from Iberia and the Americas would be extremely interesting. In the introduction section, we mention an older study that outlined some genetic similarities between American and Iberian horses using short regions of the D-loop between 360- to 442-bp long (Luís C, Bastos-Silveira C, Cothran EG, Oom M do M. Iberian Origins of New World Horse Breeds. J Hered. 2006 Mar 1;97(2):107–13).

To address Reviewer 4’s concerns, we have further clarified our approach and have explicitly explained the value of using complete mtDNA only (lines 126-128 of the revised manuscript). We also added the following paragraph at the beginning of the Results section (lines 268-275).

“This study instead explicitly focuses on the whole mitochondrial sequence following more recent horse phylogeographic analyses showing that the short segments of the mtDNA used in earlier studies were often accompanied by high levels of recurrent mutations, thus blurring the structure of the phylogenetic analyses and rendering some clades more difficult to distinguish from one another (23). Thus, most complete mitogenomes published on Genbank stem from the Achilli and colleagues study mentioned above. A GenBank query using the terms “horse mitochondrion” returned a total of 511 complete mtDNA sequences (~16.6 kbp). None of the returned sequences were individuals from Iberia and all the specimens from the Americas found using this query were included in our study.”

In addition, the article is not written according to the requirements of PlosOne. As an example, too short a description of the results and the discussion. The abstract section is not formatted according to the requirements of the journal.

Thank you for pointing out the formatting requirements for this journal. In this revision we have made every effort to ensure our formatting falls within the guidelines found on https://journals.plos.org/plosone/s/submission-guidelines. We have also refined the text in several places. We have expanded the results section, but feel that the discussion provides sufficient content to contextualize our findings.

The Materials and Methods section describes the isolation of ancient DNA that does not conform to the protocols of good practice for working with ancient DNA.

Тhere are no control reactions for DNA contamination as example.

I cannot believe that DNA isolation is possible in the described way to dissolve 500 mg of dry bone material in 1 ml of buffer.

Our methods for ancient DNA work have been outlined in a number of publications (Oswald et al, 2019, 2020, 2021). We have further described the Florida Museum Ancient DNA Laboratory and its protocols. As stated in our earlier MS and clarified in this version, this study did include negative controls. As per the Florida Museum aDNA laboratory, all extractions (for this study and others) include negative controls. As shown by our results in these several studies, our ratio of sample to buffer is appropriate and effective. The protocols used for DNA extraction are based not only on our work but also on published studies such as those of Yang et al. 1998, 2008 and others.

1. Yang, D. Y., Eng, B., Waye, J. S., Dudar, J. C. & Saunders, S. R. Technical note: Improved DNA extraction from ancient bones using silica-based spin columns. Am. J. Phys. Anthropol. 105, 539–43 (1998).

2. Yang, D. Y., Liu, L., Chen, X. & Speller, C. F. Wild or domesticated: DNA analysis of ancient water buffalo remains from north China. J. Archaeol. Sci. 35, 2778–2785 (2008).

To clarify our protocols and further describe the measures we used to limit cross-contamination, we have added details about our negative control reactions during the extraction process (lines 205-207).

Again, we thank the academic editor and the reviewers for their comments and suggestions. We hope the changes we have made in this revised version of the manuscript effectively addresses these recommendations.

Sincerely,

Nicolas Delsol

Corresponding author

Submitted filename: PLOS1 rebuttal letter#2_ND_2nd resubmission.docx

Click here for additional data file.

14 Jun 2022

Analysis of the earliest complete mtDNA genome of a Caribbean colonial horse (Equus caballus) from 16th-century Haiti.

PONE-D-21-38977R2

Dear Dr. Delsol ,

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Reviewer #3: All comments have been addressed

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30 Jun 2022

PONE-D-21-38977R2

Analysis of the earliest complete mtDNA genome of a Caribbean colonial horse (Equus caballus) from 16th-century Haiti

Dear Dr. Delsol:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

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