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Genetic Diversity and Population Structure of Alpacas (Vicugna pacos) in Peru: A Microsatellite Analysis

Diversity 2025, 17(5), 353; https://doi.org/10.3390/d17050353

by Wilber Peralta¹, Agustin Nestares¹, Julyssa Gamarra¹, Miler Rojas¹, Juan Sullca²

and Richard Estrada^2,*

Reviewer 1: Anonymous

Reviewer 2: Anonymous

Reviewer 3:

Alexander Balakirev

Diversity 2025, 17(5), 353; https://doi.org/10.3390/d17050353

Submission received: 19 March 2025 / Revised: 7 May 2025 / Accepted: 12 May 2025 / Published: 16 May 2025

(This article belongs to the Special Issue 15th Anniversary of Diversity—Biodiversity, Conservation and Ecology of Animals, Plants and Microorganisms)

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Main observations

Populations naming: Authors have to clearly state that Suitucancha and Quimsachata alpacas populations belongs to INIA´s experimental centers and the Huayre population belongs to a Community of Huayre. So, alpacas from Suitucancha are not genetically managed as Huayre population and they not belong to the Paccha community. It is not clear the differences in genetic management between the two experimental centers. It was reported that an Alpacas Germplasm Bank was installed in Quimsachata (Huanca et al 2007, https://repositorio.inia.gob.pe/server/api/core/bitstreams/767334b4-7150-4b09-9aca-f8ece25d01c3/content)

Description of the management: It is not clear what the authors want to describe. Do they refer to open or close populations, type of mating systems (single-sire or group sire mating), selection or conservation goals?

Limitations of the research was not discussed.

See other comments in the attached file.

Comments for author File: Comments.pdf

Author Response

Main observations

Thank you for your comment. We have revised the manuscript to clearly state that the alpacas from Suitucancha belong to the Santa Ana Experimental Station (EEA Santa Ana) of INIA and are part of a genetically managed herd under institutional supervision, while the alpacas from Huayre are owned and bred by members of the local rural community without formal genetic management. We have removed the reference to the Paccha community, as it was incorrect, and we have clarified the distinction in breeding practices between the two populations. These differences have been highlighted in the revised version to more accurately reflect the origin and genetic context of each group.

“The study was conducted in two distinct locations in the department of Junín, Peru: the Suitucancha estate, where the Santa Ana Experimental Station (EEA Santa Ana) of INIA is located, and the rural community of Huayre. The Suitucancha population, managed by INIA’s experimental program, constitutes a semi-closed nucleus under a controlled genetic improvement scheme that employs group-sire mating and selection for fiber quality and reproductive traits. In contrast, the Huayre population is a community-managed, open population, with natural multiple-sire mating systems and no structured selection or conservation strategy in place..”

Thank you for your comment. We have added a description of the management practices for both alpaca populations. The Suitucancha herd (EEA Santa Ana – INIA) is a semi-closed population managed under a controlled genetic improvement program with group-sire mating and selection for fiber quality and reproductive traits. In contrast, the Huayre herd is an open, community-managed population with natural mating practices, typically involving multiple sires per group, and without structured selection or conservation objectives.

“The Suitucancha population, managed by INIA’s Santa Ana Experimental Station, constitutes a semi-closed nucleus under a controlled genetic improvement program that employs group-sire mating and selection for fiber quality and reproductive traits. In contrast, the Huayre population is a community-managed, open population, with natural multiple-sire mating systems and no structured selection or conservation strategy in place.”

Limitations of the research was not discussed.

Thank you for your comments. A paragraph has been added to the discussion to address methodological considerations without undermining the value of the findings. It clarifies the scope of the analysis and emphasizes the reliability and relevance of the observed patterns. The paragraph reads:

“While this study provides valuable insights into the genetic structure of alpacas under contrasting management systems, certain methodological considerations should be acknowledged. The analysis was based on specific microsatellite markers and targeted local populations, which, while representative of distinct reproductive contexts, may not reflect the full genetic panorama of alpacas across the Andean region. Additionally, the integration of external data required careful standardization to ensure comparability across datasets. Despite these considerations, the patterns observed in this study are consistent and informative, reinforcing the relevance of reproductive management practices in shaping genetic diversity at the population level.”

Summary

Need to be updated according to the suggested changes in the following sections.

Lines 14-15 What is an open breeding system?. Be more specific.

Thank you for your comment. The expression "open breeding system" has been clarified in the revised abstract. We now specify that the Huayre population corresponds to a community-based herd where natural, unregulated mating practices are common. The sentence has been revised as follows:

"…the genetically managed herd of INIA’s Santa Ana Experimental Station (Suitucancha) and the community-based herd of Huayre, where natural, unregulated mating practices are common."

Line 20 Be more specific about the breeding practices and genetic flow.

Thank you for your comment. The sentence has been revised to provide a clearer description of the breeding practices and gene flow. We now specify that the observed differentiation is likely influenced by the use of selected males under controlled mating in Suitucancha, versus natural, unregulated group mating in Huayre, which facilitates broader gene flow. The revised sentence reads as follows:

“…indicated moderate genetic differentiation between Suitucancha and Huayre, likely influenced by the use of selected males under controlled mating in Suitucancha versus natural, unregulated group mating in Huayre, which facilitates broader gene flow.”

Line 21. Be more specific about management strategies

Thank you for your suggestion. We have revised the sentence to clarify that the genetic distinctiveness of the Quimsachata group is likely due to its role as a germplasm conservation nucleus managed under a closed reproductive scheme. The updated sentence reads:

“The Quimsachata group displayed distinct genetic characteristics, likely reflecting limited gene flow due to its role as a germplasm conservation nucleus under closed reproductive management.”

Line 22-23 Erase the statement, because it is not a result of this study.

Thank you for your observation. The sentence has been reformulated to avoid general recommendations and now focuses strictly on the observed relationship between reproductive management and population structure. The revised sentence reads:

“These results reflect how differences in reproductive management may influence population structure in alpacas.”

Line 25 What does “to enhance conservation and breeding practices” means?, please be

more specific.

Thank you for your comment. The original sentence has been reformulated to avoid general recommendations and now focuses solely on the observed link between reproductive management and population structure. The phrase “to enhance conservation and breeding practices” has been removed for clarity.

Lines 27-28 I suggest not include Andean Region, Gene Flow and Conservation genetics,

Thank you for your suggestion. The keywords have been revised accordingly, and the terms “Andean Region”, “Gene flow”, and “Conservation genetics” have been removed to better reflect the specific scope of the study.

Introduction

Lines 41-42 The sentence is not clear.

Thank you for your observation. The sentence has been revised for clarity and now reads:

“One of the main challenges in the genetic improvement of South American camelids is to assess productive traits, develop innovative strategies for managing reproductive males, and preserve the genetic variability available in these species [6].”

Line 47-51. The sentence is not clear. Please be more specific. Take in account that most farmers seek genetic improvement by selecting animals for specific traits, and that selection and conservation have different goals and opposite genetic consequences.

Thank you for your insightful comment. The sentence has been revised to clarify the distinction between selective breeding and genetic conservation. We now specify that the referenced studies provide a foundation for breeding programs focused on productive traits, while also emphasizing the need to manage genetic variability to prevent erosion. The updated version reads:

“Based on previous genetic characterization studies [7–9], which include analyses using microsatellite and mitochondrial DNA markers, significant differences have been identified in genetic diversity and population structure between wild and domestic South American camelids. These studies also revealed substantial genetic variability within alpaca populations, providing a reference for selective breeding programs focused on productive traits, while underscoring the importance of managing this diversity to avoid unintended genetic erosion.”

Line 55. The sentence is right, but the authors did not carry out a gene flow analysis. Do they have to do that?

Thank you for your observation. Since this statement appears in the introduction, it was intended as general background information rather than a reflection of our own analyses. However, the sentence has been revised to clarify that it refers to theoretical insights about gene flow rather than results derived from this study. The updated version reads:

“Genetic diversity within a population can provide general insights into evolutionary processes such as gene flow, drift, and selection, as it reflects the distribution and frequency of genetic variants over time.”

Line 61. Add a literature reference for alpaca

Thank you for your suggestion. In addition to the existing reference [12], we have included a specific study on alpacas (Yalta et al., 2014) to further support the statement regarding the informativeness of microsatellite markers in this species.

Line 64-66. Add a literature reference.

Thank you for your suggestion. A specific reference has been added to support the statement regarding the role of genetic markers in identifying populations with conservation value. The updated sentence reads:

“The information these markers provide is key to the conservation of alpacas and llamas, as they detect populations with sufficient variability to serve as genetic reservoirs (Vallejo Trujillo et al. 2012).”

Line 67. Genetic improvement by using selection does not prevent allelic diversity loss.

Thank you for your observation. We agree that selection alone does not prevent allelic diversity loss and may, in fact, reduce genetic variability if not properly managed. To address this, the sentence has been revised for accuracy and now reads:
“Furthermore, they are valuable in genetic improvement, guiding breeding programs that aim to enhance desirable traits while implementing strategies to monitor and preserve genetic diversity.”

Please correct the statement. Microsatellites are still used for parentage verification, which is important for genetic value prediction.

Thank you for your observation. We agree and have clarified this point in the introduction. A sentence has been added to highlight that microsatellites remain essential tools for parentage verification in alpacas and llamas, contributing to accurate pedigree reconstruction and supporting breeding value estimation. The updated text now reads:

“Additionally, microsatellites remain essential for parentage verification in alpacas and llamas, providing accurate pedigree information that supports breeding value prediction and genetic management.”

Line 69. The Suitucancha population seems not belongs the Paccha Community but it belongs to the INIA experimental Center called “Santa Ana”. Please make the appropriate correction in the objectives statement. What is a microsatellite analysis?, … maybe it is better to write … by using microsatellite molecular markers.

Thank you for your observation. The objectives statement has been revised to accurately indicate that the Suitucancha population corresponds to alpacas managed by INIA’s Santa Ana Experimental Station, rather than the Paccha Community. In addition, the expression “microsatellite analysis” has been replaced with “by using microsatellite molecular markers” for greater clarity and precision. The updated sentence now reads:

“Therefore, this study aims to evaluate the genetic diversity and population structure of alpacas from INIA’s Santa Ana Experimental Station (located in the Suitucancha area) and from the rural community of Huayre, by using microsatellite molecular markers.”

Materials and methods

Line 84-86. Please erase the word “State of the”. Please give information about the sex and age of the alpacas selected by random sampling. Please, clarify if the Huayre population came from different herds. Be more specific in the methodology used to assure the adequate representation of both populations.

Line 92. Are you sure that you carried out DNA extraction from the “sera” or “serum”?

Thank you for your observation. You are correct—DNA extraction was not performed from serum. The text has been corrected to accurately reflect that, after centrifugation, the leukocyte layer (buffy coat) was recovered and stored under cold conditions for subsequent DNA extraction. The revised sentence now reads:

“Blood samples were collected from these alpacas through direct puncture of the caudal artery of the thigh, using sterile vacutainers with anticoagulant and 22 x 1½ gauge needles. Subsequently, the samples were placed in racks at a 45° inclination and transported under controlled conditions to the Molecular Biology and Genomics Laboratory of INIA – Lima, where they were centrifuged, and the leukocyte layer (buffy coat) was recovered and stored under cold conditions for subsequent DNA extraction.”

Line 92-93. The sentence must be moved to DNA extraction section.

Thank you for your observation. The sentence describing the centrifugation of the blood samples and the recovery of the leukocyte layer (buffy coat) has been relocated to the DNA extraction section (2.3), as it more appropriately corresponds to the description of the extraction protocol. The revised text now reads:

“Upon arrival at the laboratory, the blood samples were centrifuged, and the leukocyte layer (buffy coat) was recovered and stored under cold conditions for subsequent DNA extraction.”

Line 95-96 The sentence is not clear.

Thank you for your comment. The sentence has been clarified to indicate that the extraction followed the manufacturer’s protocol with minor adaptations for the sample type. The revised version reads:

“DNA extraction was performed using the PureLink® Genomic DNA Kit (Invitrogen), following the manufacturer’s instructions with minor modifications adapted to the sample type.”

Line 100. Please add the accepted range for the DNA sample.

Thank you for your observation. The sentence has been revised to include the accepted absorbance ratio ranges for assessing DNA purity. The updated version reads:

“ensuring absorption ratios (A260/A280 between 1.8–2.0 and A260/A230 between 2.0–2.2), which are within acceptable ranges for genetic analyses.”

Line 111. Please add information about sex, color or age of the alpacas sampled from Quimsachata.

Thank you for your comment. The Quimsachata population corresponds to external data obtained from a previously published study [15]. Unfortunately, the original source does not specify the sex, age, or color of the individuals included in that dataset. Therefore, this information could not be incorporated into the present analysis.

Line 117-120. The sentence is no clear. Please give a better explanation.

Thank you for your comment. The sentence has been revised for clarity. We now specify the type of statistics estimated by AMOVA and their purpose. The updated version reads:

“To assess the genetic structure of the alpaca populations, an Analysis of Molecular Variance (AMOVA) was performed using Arlequin v3.5. This analysis estimated F-statistic coefficients, including overall F<sub>ST</sub> for population differentiation and F<sub>IS</sub> for intra-population inbreeding. The significance of the variance components was tested under the assumption of Hardy-Weinberg equilibrium.”

Line 126. I think the right term is Principal Component Analysis. Please make the appropriate correction.

Thank you for your comment. However, the analysis conducted was a Principal Coordinates Analysis (PCoA), which is the correct terminology when using GenAlEx v6.5, as it is based on a genetic distance matrix rather than a variance-covariance matrix. Therefore, no change was made to this term.

Line 150. Merge Table 1 and 2

Thank you for your suggestion. Tables 1 and 2 have been merged into a single table.

Line 167 Erase to avoid redundancy

Thank you for your observation. The sentence mentioning negative Fis values in the Huayre population has been revised and integrated into the previous statement to avoid redundancy while maintaining the relevant information. The updated text now reads:

“The overall average Fis for the Huayre population was approximately 0.064, reflecting a mild tendency toward heterozygote deficiency, although negative values were also detected at loci LCA24, LCA37, and LCA66.”

Line 169 Add below the table NS= pvalue > 0.05

Thank you for your suggestion. The explanation “NS = p-value > 0.05” has been added below the table as requested.

Line 172. How you get 16.6% from the table 4?. Please check it.

Thank you for your observation. To improve clarity and accuracy, we have revised the description to report the exact variance components (Sigma²) obtained from the AMOVA, rather than converting them to percentages. This adjustment avoids rounding inconsistencies and presents the original outputs directly from the analysis software.

Line 175 Correct the population name

Thank you for your observations. The name of the Quimsachata population has been corrected to match the terminology used throughout the manuscript.

Line 174-176 Erase to avoid redundancy

Thank you for your comment. The paragraph has been revised to eliminate redundancy while preserving the key interpretation of the AMOVA results.

Line 176. How you get 32.5% from the table 5?. Please check it.

Thank you for your observation. To improve clarity and accuracy, we have revised the description to report the exact variance components (Sigma²) obtained from the AMOVA, rather than converting them to percentages. This adjustment avoids rounding inconsistencies and presents the original outputs directly from the analysis software.

Line 177 replace “between” by “among”

Thank you for your observation. The term “between” has been replaced with “among” to reflect the comparison of more than two populations, as suggested.

Line 180 tables 4 and 5: Place the full value of the p-value. Move df to the second column

Thank you for your suggestions. The full p-values have been added to Tables 4 and 5, and the “df” column has been moved to the second position as requested.

Line 201. Figura 1. Replace p1,p2, p3 for P1, P2 and P3 respectively.

Thank you for your observation. The labels in Figure 1 have been corrected, and “p1”, “p2”, and “p3” have been replaced with “P1”, “P2”, and “P3” respectively.

Line 223. Genetic Complexity?. Be more specific.

Thank you for your comment. The phrase “genetic complexity” has been revised to “greater levels of genetic admixture,” which more accurately describes the presence of multiple genetic clusters inferred by the STRUCTURE analysis.

Line 248. Huancayo? or Junin?

Thank you for your observation. The correct reference is Huancayo, which is the province where the Suitucancha population is located. Junín refers to the department and is not the appropriate administrative level in this context. The text has been reviewed to ensure consistency.

Line 254 Figure 2: dark or brown?. Please correct

Thank you for your observation. The label has been corrected to “brown” in Figure 2, as this more accurately represents the color category referenced

Discussion

Line 258 More information about the genetic program (selection, conservation, etc), mating system (single-sire mating or grouped sire mating) is needed for all populations.

Thank you for your suggestion. A sentence has been added to describe each population's mating systems and genetic management strategies. The updated text now reads:

“This study examined the genetic diversity and relationships among the alpaca populations of Suitucancha and Huayre in Junín, along with an external group from Quimsachata. Suitucancha is managed by INIA’s Santa Ana Experimental Station as a genetic improvement nucleus, applying a grouped-sire mating system and selection focused on fiber quality and reproductive traits. This population exhibits low levels of genetic variation and a high frequency of shared genotypes, likely due to the long-term application of controlled breeding practices. In contrast, Huayre is a community-managed herd with natural multiple-sire mating and no structured selection or conservation program, which likely contributes to greater genetic heterogeneity. Meanwhile, the Quimsachata population is part of INIA’s Germplasm Bank and is managed under a conservation-oriented program with controlled reproduction and limited external gene flow. The analysis using microsatellite markers provides insights into the evolutionary processes and management strategies that have shaped the genetic structure of these populations, guiding future breeding and conservation efforts.”

Line 259. Homogeneus gene pool?. Be more specific.

Thank you for your comment. The phrase “homogeneous gene pool” has been replaced with a more precise description. The revised text now specifies that the Suitucancha population exhibits low levels of genetic variation and a high frequency of shared genotypes, likely resulting from long-term controlled breeding practices.

Line 273-275 it is not true for all cases

Thank you for your comment. The sentence has been revised to reflect that the increase in heterozygosity is a potential outcome, but not a universal one. The updated version now reads:

“Additionally, research by La Manna et al. (2011) [25] and Figueroa et al. (2023) [15] suggests that the reproductive exchange and introduction of novel alleles can contribute to increased heterozygosity, which may help explain the relatively high expected heterozygosity values observed in Huayre.”

Line 280. The sentence is not clear.

Thank you for your observation. The sentence has been revised for clarity. The updated version now reads:

“From a scientific perspective, the negative Fis values observed in Suitucancha may reflect reduced inbreeding and a tendency toward heterozygote excess, likely resulting from controlled breeding practices that encourage genetic mixing.”

Line 283. Please revise the sentence. There is a contradiction with line 268.

Thank you for your observation. The paragraph has been revised to clarify that the positive Fis values and Hardy–Weinberg deviations observed in Huayre do not necessarily contradict its high genetic diversity. The updated version specifies that these patterns may result from internal substructure or localized inbreeding due to reproductive isolation within family groups, rather than from population-wide breeding restrictions. The revised paragraph now reads:

“On the other hand, the Suitucancha population showed a trend toward an excess of heterozygotes, as evidenced by negative Fis indices, which may reflect reduced inbreeding resulting from controlled breeding practices that promote genetic mixing. In contrast, the Huayre population exhibited positive fixation indices and significant deviations from Hardy–Weinberg equilibrium in some loci. These patterns may indicate the presence of internal substructure or localized inbreeding within family groups, rather than generalized breeding restrictions. Although Huayre displays high overall genetic diversity, reproductive events may occur in semi-isolated herds, which can lead to deviations from equilibrium expectations. Similar patterns have been reported in comparable studies [13,26], emphasizing the importance of implementing management strategies that enhance genetic exchange to maintain diversity and reduce inbreeding in alpaca populations.”

Line 288. Reproductive management and gene flow?. Please add an explanation of their effects.

Thank you for your suggestion. The paragraph has been revised to include a brief explanation of the effects of reproductive management and gene flow on genetic structure. The updated version now reads:

“Controlled reproductive management, as implemented in Suitucancha and Quimsachata, can lead to reduced variability due to selection and limited mating among selected individuals. In contrast, gene flow—more common in open or community-managed systems like Huayre—introduces new alleles and increases intra-population variability, while reducing genetic differentiation between populations.”

Line 291-296. The message is not clear. What is community-based management systems? Managing genetic resources?.

Thank you for your comment. The paragraph has been revised to clarify that community-based management systems refer to local breeding practices where herds are managed independently by farmers, usually without structured or centralized mating strategies. It now specifies that the exchange of animals among these independently managed herds can contribute to maintaining genetic diversity and resilience. The updated text now reads:

“This phenomenon suggests that in community-based breeding systems—where each herd is managed independently by local farmers, often without coordinated mating plans—the exchange of animals between herds helps preserve a broad genetic pool, enhancing adaptability and resilience in the species.”

Line 298. Include reference about Quimsachata (Huanca et al 2007)

Thank you for your suggestion. The reference to Huanca et al. (2007) has been included in the revised manuscript to support the description of the Quimsachata population and its management context.

Line 317. The term management strategies is too general. Be more specific

Thank you for your comment. The sentence has been revised to specify the type of genetic management practices recommended, including rotational exchange of breeding males, pedigree tracking, and avoidance of consanguineous matings. The revised sentence now reads:

“…highlights the need for targeted genetic management practices that consider the particularities of each group—such as rotational exchange of breeding males, maintenance of pedigree records, and avoidance of close-relative mating—to preserve both overall diversity and the unique characteristics of each genetic lineage.”

Line 319. Limitations of the research have to be mentioned and discussed.

Thank you for your comments. A paragraph has been added to the discussion to address methodological considerations without undermining the value of the findings. It clarifies the scope of the analysis and emphasizes the reliability and relevance of the observed patterns. The paragraph reads:

Conclusions

Must be updated according to changes made in the other sections.

Thank you for your observation. The conclusions have been revised to reflect the updated interpretation of the results and the clarifications made throughout the manuscript. The revised version now reads:

“The genetic differentiation observed between the Suitucancha and Huayre populations reflects the influence of distinct reproductive management practices on their population structure. The inclusion of the Quimsachata population as an external reference revealed a more divergent genetic profile, likely shaped by its conservation-oriented management and relative reproductive isolation. These results highlight the importance of breeding context in shaping genetic variability and suggest that coordinated practices—such as promoting reproductive exchange and minimizing substructuring—can support the preservation of genetic diversity in alpaca herds. Future research should incorporate higher-resolution genomic tools and broader geographic sampling to enhance population structure assessments and inform conservation and breeding strategiess.”

References

See the authors guidelines and correct when it corresponds.

Thank you for your observation. The reference section has been reviewed and formatted according to the journal’s author guidelines.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript entitled ‘Genetic Diversity and Population Structure of Alpacas in Peru: A Microsatellite Analysis’ explores the genetic diversity and population structure of alpacas bred in Peru. Alpacas from two locations in the Junín department were analyzed, with one outgroup included, using 12 microsatellite markers. This study is particularly important for breeding programs and the management of alpaca herds in Peru. The research is valuable and timely, given the growing global interest in alpaca breeding. The manuscript is well-structured and clearly written. However, I have some comments.

L83-L93: Is the number of 20 individuals adequate to determine genetic diversity and perform calculations of genetic parameters? Please explain.

L102: You write twelve markers, you give eleven, please correct it.

L111: This sentence needs to be improved, I mean the brackets and quoting

In Table 1 and Table 2 at locus LCA99 there is some error in Ho. How is it possible that there is such a difference between the Ho and He value? I suspect that there may have been incorrect genotyping of the alleles.

In table 3 LCA99 Fis=0,591 and 0,573???

Why is the error value so large in Tables 4 and 5?

My main question is what data did you use from the Quimsachata population? Were they ready-made DNA profiles or did you use other data?

Comments for author File: Comments.pdf

Author Response

Thank you very much for your positive evaluation of our manuscript and for highlighting its relevance to alpaca breeding and herd management in Peru. We greatly appreciate your thoughtful comments and constructive feedback, which have been carefully addressed in the revised version.

L83-L93: Is the number of 20 individuals adequate to determine genetic diversity and perform calculations of genetic parameters? Please explain.

Thank you for your observation. While we recognize that larger sample sizes are generally preferred, several peer-reviewed studies have successfully used 20 or fewer individuals per population in microsatellite-based analyses. For example, Barreta et al. (2012) analyzed genetic diversity and population structure in Bolivian alpacas using microsatellite markers with similar sample sizes per group, yielding robust and biologically meaningful results. Likewise, Väli et al. (2008) demonstrated that microsatellite data from as few as 20 individuals per population can reliably reflect genome-wide genetic diversity patterns in natural populations. These studies support the methodological validity of using small but representative samples, provided the genetic markers are informative and the results are interpreted with appropriate caution.

Väli, Ü., Einarsson, A., Waits, L., & Ellegren, H. (2008). To what extent do microsatellite markers reflect genome‐wide genetic diversity in natural populations?. Molecular ecology, 17(17), 3808-3817.

Barreta, J., Iñiguez, V., Saavedra, V., Romero, F., Callisaya, A. M., Echalar, J., ... & Arranz, J. J. (2012). Genetic diversity and population structure of Bolivian alpacas. Small Ruminant Research, 105(1-3), 97-104.

L102: You write twelve markers, you give eleven, please correct it.

Thank you for your observation. The text has been corrected to indicate that eleven microsatellite markers were used in the final analysis.

L111: This sentence needs to be improved, I mean the brackets and quoting

Thank you for the suggestion. The sentence has been revised for clarity and proper formatting as follows: “Allele designation was carried out using GeneMapper v4.0 (Applied Biosystems) software.”

Thank you for your observation. The issue at locus LCA99, where an anomalously low observed heterozygosity (Ho) was reported despite a high expected heterozygosity (He), was due to a high proportion of missing genotype data (coded as 0) likely caused by amplification failure. As a result, locus LCA99 was removed from the final analysis in both the Suitucancha and Huayre populations, and the summary tables have been updated accordingly to reflect only loci with reliable genotyping.

In table 3 LCA99 Fis=0,591 and 0,573???

Thank you for your comment. Locus LCA99 has been removed from Table 3 due to inconsistent genotyping results and a high proportion of missing data, which affected the reliability of the Fis estimates.

Why is the error value so large in Tables 4 and 5?

Thank you for your comment. Tables 4 and 5 have been updated following the removal of two samples with missing data. The observed within-population variance components (error terms) remain high, which is consistent with previous studies on alpacas and other South American camelids. This pattern has been widely reported and reflects the high genetic diversity and low population structure characteristic of these species, where over 90% of the total genetic variation typically resides within populations (Paredes et al. 2020; Casey et al. 2018; Barreta et al. 2012; MacHugh & Bradley, 2001).

MacHugh, D. E., & Bradley, D. G. (2001). Livestock genetic origins: goats buck the trend. Proceedings of the National Academy of Sciences, 98(10), 5382-5384.

Casey, C. S., Orozco-terWengel, P., Yaya, K., Kadwell, M., Fernández, M., Marín, J. C., ... & Bruford, M. W. (2018). Comparing genetic diversity and demographic history in co-distributed wild South American camelids. Heredity, 121(4), 387-400.

Paredes, G. F., Yalta-Macedo, C. E., Gutierrez, G. A., & Veli-Rivera, E. A. (2020). Genetic diversity and population structure of llamas (Lama glama) from the camelid germplasm bank—quimsachata. Genes, 11(5), 541.

My main question is what data did you use from the Quimsachata population? Were they ready-made DNA profiles or did you use other data?

Thank you for your question. The data from the Quimsachata population consisted of genotypes previously generated by the same INIA laboratory and technical personnel who conducted the present study. The microsatellite profiles were obtained under comparable laboratory conditions and protocols, ensuring consistency in allele scoring and marker interpretation across datasets. To address this, we have revised the manuscript to clarify that only the microsatellite loci overlapping be tween our dataset and the external dataset (Figueroa et al., 2023) were used in the analysis (LCA08, LCA19, LCA24, LCA37, LCA56, LCA65, LCA66, LCA94, LGU49, YWLL29, and YWLL40). This practice of combining external genotype data based on shared loci has been widely validated in population genetics studies across livestock and wildlife species, including sheep (Paiva et al., 2011), cattle (Freeman et al., 2006), and arctic foxes (Hasselgren et al., 2018), where authors emphasize the importance of using common markers and calibrating allele sizes where necessary. By ensuring methodological compatibility through only matching loci, our analysis remains robust and consistent with established approaches in the field. This clarification has been incorporated into the revised manuscript.
Paiva, S. R., Mariante, A. D. S., & Blackburn, H. D. (2011). Combining US and Brazilian microsatellite data for a meta-analysis of sheep (Ovis aries) breed diversity: facilitating the FAO global plan of action for conserving animal genetic resources. Journal of Heredity, 102(6), 697-704.
Freeman, A. R., Bradley, D. G., Nagda, S., Gibson, J. P., & Hanotte, O. (2006). Combination of multiple microsatellite data sets to investigate genetic diversity and admixture of domestic cattle. Animal Genetics, 37(1), 1-9.
Hasselgren, M., Angerbjörn, A., Eide, N. E., Erlandsson, R., Flagstad, Ø., Landa, A., ... & Norén, K. (2018). Genetic rescue in an inbred Arctic fox (Vulpes lagopus) population. Proceedings of the Royal Society B: Biological Sciences, 285(1875), 20172814.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

Review

General notes

I have read the manuscript sent and must note that, overall, the study was carried out with serious methodological errors on an extremely scarce amount of original material, which together makes most of the authors' conclusions untenable. Also, it is necessary to note the rather chaotic language and low quality of illustrations.

Notes at the text

Header

It would be desirable if the Latin name of the animal that served as the object of study was mentioned at least once in the text of the article or in keywords…

Abstract

A detailed description of the research methodology and software is usually not included in the abstract. The abstract needs to be rewritten.

Line 31 Peru has more than 3,700,000 alpaca specimens within its territory,…

The introduction should clearly indicate whether the study is about wild populations or about farm animals, livestock. Mandatory

Also, in the Introduction, it is necessary to briefly explain to the reader the specific position of the studied species within the natural ecosystems and agricultural systems of Peru.

Line 47

What is this? What type of analyses do you talk about?

Line 56-57 … it, offering information on the number of genes that have originated in the

population and estimating their contribution to the current population [11].

This phrase makes one doubt whether the authors understand the meaning of the word gene in its biological sense...

Materials and Methods

Lines 76-80

I think it would be more convenient to provide a map indicating the location of the research points. In addition, it is not entirely clear why the authors indicate in detail the climatic parameters of the research sites.

Lines 85-87

It should be noted that a sample of 20 individuals and only 2 populations being studied cannot be considered sufficient for population studies.

Here it is necessary to provide a reference to the study in which this set of microsatellites was first proposed and tested. It is also necessary to provide a table with the basic data of these microsatellites and the primers used for their amplification. MANDATORY

Line 112

I must say that this is a rather strange study design. As far as I can see, a different set of microsatellite markers was used in this work (Figueroa et al., 2023). Therefore, combining these data is methodologically incorrect, making all further manipulations by the authors meaningless.

Lines 245-251 The phylogenetic analysis using the Neighbor-Joining (NJ) method revealed the genetic relationships among the Suitucancha (blue), Huayre (pink), and the external Quimsachata (black) populations (Figure 5). The Suitucancha and Huayre individuals clustered into distinct clades in the upper part of the tree, reflecting genetic proximity between these two populations. However, the Quimsachata individuals (black) formed scattered clades around the circular dendrogram, indicating high genetic diversity and a clear differentiation from the local Huancayo populations (Suitucancha and Huayre).

This part of the analysis should be recognized as absolutely methodologically incorrect, because, firstly, it is impossible to compare different sets of microsatellites in one data set, and secondly, the tree shows that the groups under consideration are not monophyletic, and most of the tree nodes are not reliable.

Figure 5

The choice of tree representation (concentric) is extremely unfortunate. In addition, it is obvious that most of the branching nodes are not reliable. In this regard, the point of using this tree as an illustration becomes unclear.

Comments for author File: Comments.pdf

Comments on the Quality of English Language

Not too strong...

Author Response

General notes

Thank you for your feedback. We have revised the manuscript to improve clarity, language, and figure quality, and we believe the methodology is sound and appropriate for the study objectives.

Notes at the text

Header

It would be desirable if the Latin name of the animal that served as the object of study was mentioned at least once in the text of the article or in keywords…

Thank you for your suggestion. The Latin name of the species (Vicugna pacos) has been added to the keywords section of the manuscript.

Abstract

A detailed description of the research methodology and software is usually not included in the abstract. The abstract needs to be rewritten.

Thank you for your observation. The abstract has been rewritten to remove excessive methodological detail and now provides a concise summary focused on the study’s objectives, main findings, and relevance.

“This study evaluated the genetic diversity and population structure of Vicugna pacos (Huacaya alpacas) from two contrasting breeding contexts in Junín, Peru: the genetically managed herd of INIA’s Santa Ana Experimental Station (Suitucancha) and the community-based herd of Huayre. An external reference population from Quimsachata was also included. Genetic diversity parameters revealed high allelic richness and heterozygosity within all populations. Analyses of molecular variance (AMOVA), principal coordinate analysis (PCoA), Bayesian clustering, and phylogenetic reconstruction indicated moderate genetic differentiation between Suitucancha and Huayre, influenced by breeding practices and gene flow. The Quimsachata group displayed distinct genetic characteristics, suggesting relative reproductive isolation. These results highlight the relevance of breeding system context in shaping genetic diversity and underscore the importance of implementing tailored genetic management strategies to conserve local alpaca resources and guide future breeding programs in the Andean region.”

Line 31 Peru has more than 3,700,000 alpaca specimens within its territory,…

Thank you for your comment. The sentence has been revised to clarify that the study refers to domesticated alpacas (Vicugna pacos), raised as livestock within high-Andean agricultural systems in Peru.

The introduction should clearly indicate whether the study is about wild populations or about farm animals, livestock. Mandatory

Thank you for your suggestion. The paragraph has been revised to clarify that the study focuses on domesticated alpacas (Vicugna pacos), and their role within Peruvian highland agricultural systems. It now specifies their economic and ecological importance as fiber-producing livestock managed under traditional systems in puna ecosystems. The revised paragraph reads:

“Peru has more than 3,700,000 alpaca specimens within its territory, representing 87% of the world's population, and it is the leading global producer of fiber from this high-Andean camelid [1]. Alpacas (Vicugna pacos) are fully domesticated livestock species, managed mainly for their high-quality fiber under traditional and semi-intensive systems in the Andean highlands. They are integral to local agricultural systems and adapted to puna ecosystems, where they contribute to both the cultural identity and economic sustainability of rural communities…”

Also, in the Introduction, it is necessary to briefly explain to the reader the specific position of the studied species within the natural ecosystems and agricultural systems of Peru.

Thank you for your observation. The introduction has been revised to clarify the position of alpacas as domesticated livestock species within high-Andean agricultural systems. Their role in puna ecosystems and their economic and cultural relevance to rural communities in Peru is now explicitly stated.

Line 47 What is this? What type of analyses do you talk about?

Thank you for your comment. The sentence has been clarified in the revised version to specify that the cited references refer to genetic characterization studies based on microsatellite and mitochondrial DNA analyses, which assess levels of genetic diversity, structure, and differentiation among wild and domestic South American camelids.

“Based on previous genetic characterization studies [7–9], which include analyses using microsatellite and mitochondrial DNA markers, significant differences have been identified in genetic diversity and population structure between wild and domestic South American camelids. These studies also revealed substantial genetic variability within alpaca populations, offering a valuable baseline for the development of medium- and long-term genetic improvement programs by alpaca producers.”

Line 56-57 … it, offering information on the number of genes that have originated in the population and estimating their contribution to the current population [11]. This phrase makes one doubt whether the authors understand the meaning of the word gene in its biological sense…

Thank you for your observation. The term “genes” was imprecise in this context and has been replaced with “alleles” or “genetic variants” to accurately reflect the intended meaning and ensure correct biological usage.

“A population's genetic diversity provides insight into how gene flow has occurred within it, offering information on the genetic variants that have emerged and their frequencies within the population [11].”

Materials and Methods

Lines 76-80 I think it would be more convenient to provide a map indicating the location of the research points. In addition, it is not entirely clear why the authors indicate in detail the climatic parameters of the research sites.

Thank you for your comment. The climatic data and geographic coordinates have been removed from the text for clarity and will instead be incorporated into a map illustrating the study sites in the revised manuscript (Figure S1).

Lines 85-87 It should be noted that a sample of 20 individuals and only 2 populations being studied cannot be considered sufficient for population studies.

Thank you for your comment. While we recognize that larger sample sizes are generally preferred, several peer-reviewed studies have successfully used 20 or fewer individuals per population in microsatellite-based analyses. For example, Barreta et al. (2012) analyzed genetic diversity and population structure in Bolivian alpacas using microsatellite markers with similar sample sizes per group, yielding robust and biologically meaningful results. Likewise, Väli et al. (2008) demonstrated that microsatellite data from as few as 20 individuals per population can reliably reflect genome-wide genetic diversity patterns in natural populations. These studies support the methodological validity of using small but representative samples, provided the genetic markers are informative and the results are interpreted with appropriate caution.

Lines 102-104 For microsatellite allele determination, twelve microsatellite loci described for alpacas and llamas were used, specifically LCA08, LCA19, LCA24, LCA37, LCA56, LCA65, LCA66, LCA94, LGU49, YWLL29, and YWLL40. Here it is nec essary to provide a reference to the study in which this set of microsatellites was first proposed and tested. It is also necessary to provide a table with the basic data of these microsatellites and the primers used for their amplification. MANDATORY

-Thank you for your observation. In response to your suggestion, we have included the requested information regarding the twelve microsatellite loci (LCA08, LCA19, LCA24, LCA37, LCA56, LCA65, LCA66, LCA94, LGU49, YWLL29, and YWLL40) in Supplementary Table 2. This table provides the reference to the original study where these markers were first described and validated.

Line 112. I must say that this is a rather strange study design. As far as I can see, a different set of microsatellite markers was used in this work (Figueroa et al., 2023). Therefore, combining these data is methodologically incorrect, making all further manipulations by the authors meaningless.

Thank you for your comment. We agree that combining datasets with different microsatellite markers without harmonization would be methodologically inappropriate. To address this, we have revised the manuscript to clarify that only the microsatellite loci overlapping between our dataset and the external dataset (Figueroa et al., 2023) were used in the analysis (LCA08, LCA19, LCA24, LCA37, LCA56, LCA65, LCA66, LCA94, LGU49, YWLL29, and YWLL40). This practice of combining external genotype data based on shared loci has been widely validated in population genetics studies across livestock and wildlife species, including sheep (Paiva et al., 2011), cattle (Freeman et al., 2006), and arctic foxes (Hasselgren et al., 2018), where authors emphasize the importance of using common markers and calibrating allele sizes where necessary. By ensuring methodological compatibility through only matching loci, our analysis remains robust and consistent with established approaches in the field. This clarification has been incorporated into the revised manuscript.

Paiva, S. R., Mariante, A. D. S., & Blackburn, H. D. (2011). Combining US and Brazilian microsatellite data for a meta-analysis of sheep (Ovis aries) breed diversity: facilitating the FAO global plan of action for conserving animal genetic resources. Journal of Heredity, 102(6), 697-704.

Freeman, A. R., Bradley, D. G., Nagda, S., Gibson, J. P., & Hanotte, O. (2006). Combination of multiple microsatellite data sets to investigate genetic diversity and admixture of domestic cattle. Animal Genetics, 37(1), 1-9.

Hasselgren, M., Angerbjörn, A., Eide, N. E., Erlandsson, R., Flagstad, Ø., Landa, A., ... & Norén, K. (2018). Genetic rescue in an inbred Arctic fox (Vulpes lagopus) population. Proceedings of the Royal Society B: Biological Sciences, 285(1875), 20172814.

Besides, we emphasize that the data from the Quimsachata population consisted of genotypes previously generated by the same INIA laboratory and technical personnel who conducted the present study. The microsatellite profiles were obtained under comparable laboratory conditions and protocols, ensuring consistency in allele scoring and marker interpretation across datasets

Lines 245-251. The phylogenetic analysis using the Neighbor-Joining (NJ) method revealed the genetic relationships among the Suitucancha (blue), Huayre (pink), and the external Quimsachata (black) populations (Figure 5). The Suitucancha and Huayre individuals clustered into distinct clades in the upper part of the tree, reflecting genetic proximity between these two populations. However, the Quimsachata individuals (black) formed scattered clades around the circular dendrogram, indicating high genetic diversity and a clear differentiation from the local Huancayo populations (Suitucancha and Huayre).

Thank you for your comments. First, we confirm that only the microsatellite loci common to both our dataset and the external dataset (LCA08, LCA19, LCA24, LCA37, LCA56, LCA65, LCA66, LCA94, LGU49, YWLL29, and YWLL40) were used in the joint analysis. This clarification has been added to the revised version of the manuscript to avoid misunderstanding.

“External data from 116 alpacas was included [15], considering only the microsatellite loci that overlapped with those used in the present study (LCA08, LCA19, LCA24, LCA37, LCA56, LCA65, LCA66, LCA94, LGU49, YWLL29, and YWLL40).”

Second, the issue at locus LCA99, where an anomalously low observed heterozygosity (Ho) was reported despite a high expected heterozygosity (He), was due to a high proportion of missing genotype data (coded as 0) likely caused by amplification failure. As a result, locus LCA99 was removed from the all analysis in both the Suitucancha and Huayre populations, and the tree has been updated accordingly to reflect only loci with reliable genotyping. As shown in the new tree (Figure 4), we obtained notably higher bootstrap values, particularly for the clades grouping individuals from Suitucancha and Huayre , which significantly strengthens the phylogenetic signal and provides greater confidence in the inferred clustering patterns. These results confirm a more reliable monophyletic grouping of the local populations,

Figure 4. Neighbor-joining cladogram showing the genetic relationship among individuals from Suitucancha (blue) and Huayre (pink).

Figure 5 The choice of tree representation (concentric) is extremely unfortunate. In addition, it is obvious that most of the branching nodes are not reliable. In this regard, the point of using this tree as an illustration becomes unclear.

Thank you for your comment. The phylogenetic tree has been modified from its original circular layout to a radial representation to improve visual clarity and facilitate the interpretation of sample relationships across clusters. However, bootstrap values for the Quimsachata (black) population did not improve significantly in the updated analysis. We hypothesize that, since all populations belong to the same species, they likely share a substantial proportion of alleles, resulting in limited genetic structuring. This reduced divergence may hinder phylogenetic resolution and lead to poorly supported internal nodes within the tree (Guichoux et.al, 2011, Selkoe et.al, 2006)

-Guichoux, E., Lagache, L., Wagner, S., Chaumeil, P., Léger, P., Lepais, O., ... & Petit, R. J. (2011). Current trends in microsatellite genotyping. Molecular Ecology Resources, 11(4), 591-611. https://doi.org/10.1111/j.1755-0998.2011.03014.x

-Selkoe, K. A., & Toonen, R. J. (2006). Microsatellites for ecologists: a practical guide to using and evaluating microsatellite markers. Ecology Letters, 9(5), 615-629. https://doi.org/10.1111/j.1461-0248.2006.00889.x

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Authors could find suggestions to improve their manuscript in the attached file.

Comments for author File: Comments.pdf

Comments on the Quality of English Language

Writing style need to be improved

Author Response

Authors could find suggestions to improve their manuscript in the attached file.

-Thank you for your message. I appreciate your time and feedback. I will carefully review the attached file and consider all the suggestions provided to improve the manuscript.

Comment: “The sentence is not clear. Add reference.”

Response: Thank you for your observation. The sentence regarding the role of alpacas in local systems was clarified and supported with appropriate references.

Revision:

They play a fundamental role in Andean agropastoral systems and are well adapted to the harsh puna ecosystems. In these regions, alpacas are not only a key source of income through fiber commercialization, but also hold deep cultural significance for rural communities, reinforcing traditional livelihoods and contributing to their economic resilience (Wheeler et al., 1995; FAO, 2007).

Comment: “This is true, but also the number and distribution of the used microsattelites along the genome is important.”

Response: Thank you for your valuable suggestion. A clarification was added to indicate that the number and genomic distribution of markers affect the resolution of genetic structure.

Revision:

However, the effectiveness of these analyses depends not only on the inherent variability of the markers, but also on the number and genomic distribution of the microsatellite loci used, as inadequate coverage can limit the detection of genetic patterns.

Comment: Please, clarify if the Huayre population came from different familiar herds or for one communal herd

Response: Thank you for your comment. We have clarified in the text that the alpacas sampled from Huayre belonged to different family herds within the community.

Revision:

“…and 2019 from different family-managed herds within the rural community of Huayre…”

Comment: “Mating and sub-structure is at population level. Please be clear.”

Response: Thank you for the comment. The sentence was revised to reflect that mating patterns and substructure occur at the population level.

Revision:

...suggesting the presence of non-random mating or population substructure that may be reflected at these loci.

Comment: “Add below the table: NS= Not significant (pvalue>0.05)...”

Response: Thank you for your observation. The requested legend was added below the table for clarity.

Revision:

NS = Not significant (p-value > 0.05); * = p-value < 0.05; ** = p-value < 0.01

Comment: “The values that are in the text, are different from the table.”

Response: Thank you for noticing. The text was corrected to match the exact numerical values presented in the tables.

Revision:

...with a variance component of 3.7071 attributed to differences among populations and 19.72 to variation within populations...

...a variance component of 4.1259 among populations and 10.4463 within populations...

Comment: “Not clear, erase or rephrased.” (referring to “indicating a significant population structure”)

Response: Thank you for the suggestion. The sentence was rephrased to clearly convey population-level genetic divergence.

Revision:

...suggesting population-level genetic divergence between Suitucancha and Huayre.

Comment: “I expect to find higher genetic variability within Quimsachata and Suitucancha than Huayre... Please discuss this.”

Response: Thank you for the comment. A paragraph was added to discuss the expected high variability in conservation-managed populations.

Revision:

Although this study found higher genetic dispersion in the Huayre population, it is important to note that conservation-managed herds like Quimsachata are generally expected to harbor high genetic variability due to structured breeding practices aimed at preserving diversity. Indeed, Figueroa et al. (2023) reported that the Quimsachata alpaca population maintained high allelic richness and expected heterozygosity, comparable to or exceeding that of community herds, confirming the effectiveness of conservation-oriented management in sustaining broad genetic pools.

Comment: “I suggest to merge these paragraph with the previous one or erase it to avoid redundancy.”

Response: Thank you for your suggestion. The two paragraphs were merged to eliminate redundancy and improve narrative cohesion.

Revision:

The more homogeneous clustering of Suitucancha and the greater dispersion in Huayre suggest differences in internal genetic connectivity, likely influenced by variations in reproductive management and breeding frequency, as observed in Andean studies [13,28]. Simultaneously, the inclusion of the external Quimsachata population revealed an increase in genetic variation explained between groups (Huanca et al., 2007), suggesting that this population has differentiated due to relative isolation or specific management practices. The clear genetic separation of Quimsachata in the dendrogram supports this interpretation, indicating that factors such as geographic isolation or controlled reproductive strategies have generated a distinct gene pool [15,30]. This situation has been documented in recent studies, which indicate that even subtle restrictions on gene flow or reproductive exchange can lead to the formation of genetic substructures [27–29]. This differentiation, corroborated by comparative analyses in both Peruvian and global contexts [31,32], emphasizes the importance of targeted genetic management strategies—such as rotational exchange of breeding males, maintenance of pedigree records, and avoidance of close-relative mating—to mitigate inbreeding accumulation and preserve the genetic diversity essential for alpaca improvement and long-term sustainability.

Comment: “Limitations of the research have to be mentioned and discussed in these section.”

Response: Thank you for your recommendation. A concise limitations paragraph was added at the end of the discussion and supported with recent literature.

Revision:

Despite the valuable insights provided, this study has certain limitations. The use of a limited number of molecular markers and a modest sampling effort may constrain the resolution of genetic patterns and lead to an underestimation of allelic diversity. Previous research in South American camelids has highlighted how small marker sets or restricted sample sizes can reduce the accuracy and representativeness of diversity estimates (Figueroa et al., 2023; Mamani et al., 2021; Paredes et al., 2020). Future studies incorporating larger datasets and higher-density genomic tools are recommended to enhance genetic assessments and inform long-term conservation strategies.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

Dear authors, I have no more comments after the corrections.

Author Response

Thank you for your confirmation. We appreciate your valuable feedback throughout the review process.

Reviewer 3 Report

Comments and Suggestions for Authors

Dear colleagues.

I have read the corrected version of the article and must say that the new version is definitely better than the original one. The authors have corrected and clarified most of the points that I drew attention to earlier. Of course, I still have to note that the sample size is clearly insufficient for broad generalizations, but overall, I must agree with the authors' conclusions. And although due to the limited amount of data, their value cannot be called high, I still believe that the article can be published. I just want to recommend adding an illustration to the article with a map indicating the collection sites at the main text instead of Supplementary.

With respect

Author Response

Thank you very much for your detailed review and kind comments. We sincerely appreciated your positive evaluation of the revised version of the manuscript and were pleased to know that the changes addressed your previous concerns.

We acknowledged the limitation regarding the sample size and agreed that it constrained the potential for broad generalizations. Regarding your recommendation to include a map indicating the collection sites in the main text instead of the Supplementary Materials, we made this adjustment in the final version of the manuscript.

Once again, we were grateful for your valuable feedback and your support for the publication of the article.

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Genetic Diversity and Population Structure of Alpacas (Vicugna pacos) in Peru: A Microsatellite Analysis

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