Phlebotomus argentipes is the vector of Leishmania donovani which causes the disease leishmaniasis, a neglected tropical disease and a growing health problem in Sri Lanka. A proper understanding of the population genetic structure of sand fly vectors is considered important prior to planning and implementation of a successful vector control program. Thus, the present study was conducted to determine the population genetic structure of sand fly vectors in Sri Lanka. Two mitochondrial genes namely Cytochrome c oxidase subunit 1 (Cox 1) and Cytochrome b (Cytb), and the internal transcribed spacer 2 (ITS2) region from the nuclear ribosomal DNA were used for molecular characterization. Analyses included maximum likelihood method, network analysis and DNA polymorphisms. The outcome revealed unique sequences of all genomic regions studied except the cox 1 gene had a relationship with sand flies isolated previously from Sri Lanka, India and Israel and cytb gene of 4 sand flies that aligned with those isolated earlier from Sri Lanka and 3 from Madagascar. Furthermore, cox 1 gene and ITS 2 region analyses based on FST values indicated a possible gene flow between the study sites whereas cytb gene analysis favoured the existence of genetically distinct populations of P. argentipes in each of the study sites. Poor population differentiation of P. argentipes, a possible consequence of a gene flow, is indeed of concern due to the risk imposed by promoting the spread of functionally important phenotypes such as insecticide resistance across the country, making future vector control efforts challenging. Leishmania. Phlebotomus argentipes (Diptera: Psycodidae) is the known vector of Leishmania donovani, the causal agent of the clinical form VL which is considered the second parasitic disease cause of a high number of deaths, in India, Nepal and Bangladesh  ATT CAA CC[A/T]GAATGATA-3 ′; N1N-PDR: 5 ′ - GGTA [C/T] [A/T] TTGC CTCGA [T/A] TTCG [T/A]TATGA– 3′  and, Neutrality tests (Tajima’s D and Fu’s Fs) were determined using DNA Sequences Polymorphism software (dnaSP) (version 5.1.10). Pairwise differences and population structures of each species were evaluated by analysis of molecular variance (AMOVA) in Arlequin software (version 3.11) (cmpg.unibe.ch/software/arlequin3/) and significance was evaluated based on 1000 permutations. Median joining (MJ) network analysis was conducted based on the number of nucleotide differences and haplotype networks of these three regions, and haplotype networks were constructed using Network software 5.0.0.1 (http://www.fluxus-engineering.com) to determine the interrelationship between haplotypes.Trace files of DNA sequences were manually edited and aligned using ClustalW in BioEdit 7.2.5 software. The finalized sequences were subjected to a BLAST search, on the National Center for Biotechnology information (NCBI) database GenBank , cytb (~550bp) genes and rDNA ITS2 ( ~350bp) region of P. argentipes specimens collected from each locality were used in the phylogenetic analysis. Alignment of cox 1, cytb and ITS2 sequences with those available in public domain in Genbank confirmed their identification as P. argentipes    Tables.cox 1, cytb and ITS2 sequences of P. argentipes are shown in The evolutionary analysis by maximum likelihood method respectively for cox 1, cytb genes and ITS2 region were separated in to 2 different clades except SLITSI42 sample from Thalawa population (cox 1 gene (A and B) comprised 21 individuals  (cytb gene (X and Y) comprised 35 and 7 individuals   and the ividuals  in each ividuals .cox 1 gene clustered with P. argentipes previously reported in Sri Lanka and 2 individual sequences from Mirigama and Thalawa from the same clade A grouped with P. argentipes found in Kerala, India. Clade B of the cox 1 gene clustered with P. argentipes found in Israel  of n Israel . Sequenc in NCBI . None of in NCBI .cox 1 (~650bp), cytb (~550bp) genes and rDNA ITS2 ( ~350bp) region respectively. The total number of polymorphic sites were 263, 105 and 83 respectively for cox 1, cytb genes and ITS2 region. The haplotype diversities (Hd) and nucleotide diversities (Pi) were similarly high for all species in each study site for cox 1 gene. P. argentipes from Mamadala population had the lowest haplotype diversity (0.182) and nucleotide diversity (0.007) with haplotype diversities ranging from 0.182 to 0.733 and nucleotide diversities from 0.007 to 0.276 when compared with data from other populations for cytb gene. Similar haplotype diversities values were observed for cytb gene and ITS2 region in both Mamadala and Thalawa  of 0.878, 0.513 and 0.324 for mtDNA  Thalawa .cox 1 gene, both Tajima’s D and Fu’s Fs values were significant in Mamadala (P<0.02), Thalawa (P<0.05) and Mirigama (P<0.05) populations whereas these values were not significant in Pannala (P>0.1) population  in Mirigama population whereas significant in other populations (P<0.05) where the population may have experienced a bottleneck event in the past (Tajima’s D index was positive (+) for all study sites of the past . When cothe past .ST values obtained for three different genes of P. argentipes in this study are presented in ST values of P. argentipes between different study sites were not significant (P>0.1) with an absence of genetic differentiation between populations. Interestingly, the pairwise FST values between Mirigama and Pannala (P = 0.01) and between Mamadala and Pannala (P = 0.00) for cytb gene were significant favouring existence of genetic differentiation between populations (The pairwise Fulations .ST (Fixation index) values were 0.0579, 0.4221 and 0.0607 for cox 1, cytb genes and ITS2 region respectively for P. argentipes in this study (cox 1 gene and ITS2 region whereas significant for (P<0.05) cytb gene among studied localities  and cox 1 (8 haplotypes) genes of P. argentipes. The most dominant haplotypes were haplotype 5 and 6 for cox 1 and cytb genes respectively , and haplotype 2  in ITS2 region  were observed while 66.67% haplotypes sharing were shown by both cytb gene  and ITS2 (haplotypes 1 and 2). Out of shared haplotypes, only haplotype 5 and 6 respectively for cox 1 gene and cytb gene shared among 4 localities while other haplotypes shared among 2 or 3 study localities (The lowest number of haplotypes (n = 3) were shown by ITS2 region when compared to the 2 region . The lowcalities . Howevercalities .cox 1 gene of P. argentipes, haplotype 6, 7 and 8 respectively had a relationship with sand flies isolated previously from Sri Lanka, India and Israel  in all localities i.e. Thalawa, Mirigama and Mamadala,  that demonstrates the presence of high variations that exist within study sites with observed average pairwise differences (π) being higher than the expected average pairwise differences (ϴ). Thus the natural selection may have led to a low variation, which might result in a bottleneck event. This effect was further confirmed using ITS2 region analysis, which resulted in a positive (+) Tajima’s D value in Pannala.Tajima’s D is the difference between observed average pairwise difference (π) and expected average pairwise difference (ϴ) . Tajima’cytb gene was minus (-) for Thalawa and Mamadala indicating a low genetic variation within the study sites and therefore, the π is lower than ϴ. This might indicate a bottleneck event in the past in these sites. Such an effect could be confirmed in Mamadala also based on ITS2 region analysis.Tajima’s D of ST value of 0.4221 with cytb gene analysis in this study suggest genetically distinct populations between the sites (P<0.02). However, that assumption does not hold true when FST values from cox 1 gene and ITS2 region were considered (P>0.05). Interestingly, the pairwise FST values between Mirigama and Pannala and between Mamadala and Pannala for cytb gene were different (P<0.02), which may demonstrate genetic differentiation between populations.High FST values derived through the analysis of molecular variance) using cox 1 and ITS2 markers may indicate a possible gene flow between regions . Sri Lanka is an island with a relatively small land mass and there are no major geographical barriers between the studied localities. Hence, regardless of the geographic distance the likelihood of a gene flow between the study sites could be considerably high. The presence of a known genetic mutation associated with insecticide resistance in a notable proportion of flies in these study locations was previously reported .Line 70: change weren´t to were notLine 78, 83, 103: delete this spaceLine 86: Colombia is not an Old world country, please make the correction. Additionally there are more barcode studies for species from the new world, such as USA, Mexico, Brazil, please keep it in mindLine 99: change phylogeograpy to phylogeographyLine 61-77: Although the information is relevant is very long, please be more specific.Line 93-102: I suggest that only provide the information regarding the relevance of the use of the cox1, cytb and ITS2 genes for the genetic characterization of Phlebotomus argentipes. And do not include information that is not relevant in your research. For example: Line 94-96:In South America the phylogenetic analysis of cox 1 sequences of Verrucarum species of New World sand flies of the genus Lutzomyia was investigated [20].Line:97-98: Mitochondrial introgression in the Lutzomyia townsendi in Colombia was studied using cytb gene [12].Line 48-107: In general, the introduction is very long, and contains information that is not very relevance. I kindly suggest that it be summarized a bit to make it more clear and precise.Line106-107: “to study the effect of evolutionary forces that may spread resistance genes from one population to another”.I have a question. The genes to be amplified are not necessarily genes that are related to insecticide resistance. How do you plan to evaluate and/or justify that the genetic variability that you may find are related to resistance to insecticides?It is not very clear to me. Please in the part of your introduction, where you talk about insecticides could explain what genes are useful for do this objective.Material and MethodLine 111-116: A total of 126 of adult sand flies were collected, this sentences is a result not is part of the method. Please move this information to the next section. Also specify how many days you collected in each of those locations.ResultsLine:189-190: A total of 126 of adult sand flies were collected and were identified as Phlebotomus argentipes .Please, also specify how many specimens you collected in each one of the four locations that you studied. Why you did not collect other sand fly species and only obtain specimens of Phlebotomus argentipes, this is very common in other studies? There are not other sand fly species in your country? How many males and females did you collect? Did you analysis 126 specimens by PCR or how many?Line193: how did you select those 10 specimens of the best quality?Line 214, 217,305,306,308: Change flies or fly to sand flies or sand fly according to the case in the entire document.Line 217: correct P. argentipesLine 237: In the table, please include the number of specimens that you analyzed, for each gene and locations.DiscussionLine 322: two cladesLine 325: network analysisLine 326: P. argentipes were closely related to specimens of the same species collected in Sri Lanka, India and Israel.Line 327: of three sand fliesLine 327-328: cytb is a marker that shows variability at the intraespecific level, different from cox1 that shows differences at the interespecific level. So, why is relevant that some specimens are similar to specimens from Madagascar? Explain more about it, although you have a small sample, it is interesting that you have haplotypes similar to a locality that is considerably distant from your collection point.Line 329: did notLine 330: Add a sentence highlighting that your work is one of the first sequences for this gene and for this species. I think it is very important that you highlight this contribution of your work.Line 345: does not.Line 352-353: put the name of the area of study not numbers, because you did not listed before.Line 354-360: Although it is an interesting hypothesis, I think it is very risky to assert something of this style, given that the number of specimens that you analyzed is very small. I do not consider that ten specimens are enough to think that there is gene flow in all communities of sand flies and those possible resistance genes are being inherited. Additionally, you did not analyze genetic markers for resistance.Line 363-373: This information is not informative for this article, I suggest that you focus on the group of sand flies and compare your results with those of other researchers. I suggest you read the article by:https://doi.org/10.1016/j.meegid.2013.02.0041. Pech-May et al. 2016. Genetic structure and divergence in populations of Lutzomyia cruciata, a phlebotomine sand fly (Diptera: Psychodidae) vector of Leishmania mexicana in southeastern Mexico https://scholarworks.uttyler.edu/biology_grad/53/2. Wolkoff, 2018. Population structure, demographic history, and environmental niche of the sand fly disease vector Lutzomyia shannoni (Dyar) (Diptera: Psychodidae) in the U.S., Mexico, and Colombia Line 362-382: I suggest that you change these paragraphs as follows and fill them in with additional information, so that you can keep comparing your results.The molecular markers have been extensively used for genotyping and study of evolutionary history of mosquitoes . The analysis using cytb gene has demonstrated been useful to detect the lack of population differentiation and the presence of gene flow in some species of dipterans in South-east Asia [32]. The gene flow between species of insect vectors may lead to alterations in the disease patterns [35]. Low genetic structure variation in some sand fly species as a result of geographical isolation and restricted gene flow may have led to reduced flight abilities and formation of cryptic species that ultimately influence the capacity to transmit parasites such as Leishmania in Latin America [36].Line 384-391: again, I do not agree with these statements, I suggest that you approach it in another way for greater support, or that you only suggest it as a possible hypothesis in a more subtle way.ReferencesPlease check the structure of the citations again, to make it homogeneous. Scientific and gene names are also missing in italics, and there are spelling mistakes **********what does this mean?). If published, this will include your full peer review and any attached files.6. PLOS authors have the option to publish the peer review history of their article  digital diagnostic tool,  3 Jul 2021Author responses to editor’s and reviewers comments:Additional Editor’s Comments:Q: it is pivotal to modify the ms. according to comments of both reviewers and focusing the population genetic analysis on microsatellite loci; otherwise, you have to limit the study only on the molecular typing of sand fly species examined.R: The current study was designed to determine the population genetic structure of sand flies using two mitochondrial markers cox 1, cytb and a ribosomal region ITS2. However, no microsatellite loci were used for genotyping due their unavailability of such markers in P. argentipes. While we as a group, continue our efforts in developing such robust markers for P. argentipes, the authors believe the selected antigenic loci of P. argentipes are good enough for the said purpose due to the following reasons: Previous studies have successfully used cox 1and cyb as the genetic marker in analysing the population genetic structure of insects including sand flies. https://doi.org/10.1186/s13071-016-1603-5.Ebrahimi, et al. Genetic dynamics in the sand fly (Diptera: Psychodidae) nuclear and mitochondrial genotypes: evidence for vector adaptation at the border of Iran with Iraq. Parasites Vectors 9, 319 (2016). Flanley, et al. Population genetics analysis of Phlebotomus papatasi sand flies from Egypt and Jordan based on mitochondrial cytochrome b haplotypes. Parasites Vectors. 2018; 11, 214, doi:10.1186/s13071-018-2785-9. Guernaoui, et al. Population Genetics of Phlebotomus papatasi from Endemic and Nonendemic Areas for Zoonotic Cutaneous Leishmaniasis in Morocco, as Revealed by Cytochrome Oxidase Gene Subunit I Sequencing. Microorganisms. 2020; 8(7): 1010.Arrivillaga et al. Phylogeography of the neotropical sand fly Lutzomyia longipalpis inferred from mitochondrial DNA sequences. Infect Genet Evol. 2002; 2: 83-95.Latrofaa et al. Multilocus molecular and phylogenetic analysis of phlebotomine sand flies ( Diptera : Psychodidae ) from southern Italy. Acta trop. 2011;119:91–8. doi:10.1016/j.actatropica.2011.04.013.Cohnstaedt et al. Phylogenetics of the phlebotomine sand fly group Verrucarum (Diptera: Psychodidae: Lutzomyia). Am J Trop Med Hyg. 2011;84(6):913–22. https://doi.org/10.1186/s12936-018-2419-x.Weeraratne et al. Genetic diversity and population structure of malaria vector mosquitoes Anopheles subpictus, Anopheles peditaeniatus, and Anopheles vagus in five districts of Sri Lanka. Malar. J. 2018; 17: 271. Gene flow also successfully determined using these markers in following studies.Marcondes et al. Introgression between Lutzomyia intermedia and both Lu. neivai and Lu. whitmani, and their roles as vectors of Leishmania braziliensis. Trans R Soc Trop Med Hyg. 1997; 91(6): 725-6. https://doi.org/10.1111/j.1948-7134.2011.00110.x.Belen et al. Genetic structures of sand fly (Diptera: Psychodidae) populations in a leishmaniasis endemic region of Turkey. Journal of vector biology. 2011; 36 (Supplement 1): 32-48. Studies appear below have proven that ITS regions could successfully determine the population genetic structure of many organisms including parasites and mosquitoes. Further, Manni et al (2015) have used microsatellites and ITS2 as the genetic markers and have proven that both markers are equally successful in determining the population genetic structure.https://doi.org/10.1186/s13071-015-0794-5Manni, M., Gomulski, L.M., Aketarawong, N. et al. Molecular markers for analyses of intraspecific genetic diversity in the Asian Tiger mosquito, Aedes albopictus . Parasites Vectors 8, 188 (2015). https://doi.org/10.1186/s13071-014-0461-2. Villalobos, G., Orozco-Mosqueda, G.E., Lopez-Perez, M. et al. Suitability of internal transcribed spacers (ITS) as markers for the population genetic structure of Blastocystis spp. Parasites Vectors 7, 461 (2014). Latrofaa MS, Dantas-Torresa F, Weigla S, Taralloa VD, Parisib A, Traversac D, et al. Multilocus molecular and phylogenetic analysis of phlebotomine sand flies ( Diptera : Psychodidae ) from southern Italy. Acta trop. 2011;119:91–8. doi:10.1016/j.actatropica.2011.04.013.Journal Requirements:1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. 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Please reply and provide explicit written permission to publish XXX under a CC BY license and complete the attached form.”“I request permission for the open-access journal PLOS ONE to publish XXX under the Creative Commons Attribution License (CCAL) CC BY 4.0  under Materials and Method section in the manuscript. Reviewer Comments:Reviewer 1:Q1: 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: NoReviewer #2: PartlyR1: A minimum of 10 sequences of each marker from each study site was used in the analysis. Previous published work on population genetic structure analysis have used sample numbers between 6-10 from a single study location and have obtained successful results. I suggest adding one statement to the discussion in line 342-343 and changing the following sentence in the conclusion in line 370-373, 375-377– ‘Gene flow was evident with lack of population differentiation even between geographically distant populations of P. argentipes perhaps due to absence of geographic barriers and the continuity of habitats.’ ‘Results also validated the use of cox 1, cytb genes and ITS2 region as a tool in understanding the population genetic structure of Phlebotominae sand flies in Sri Lanka.’As, Both cox 1 and ITS2 indicated a gene flow with lack of population differentiation even between geographically distant populations of P. argentipes perhaps due to absence of geographic barriers and the continuity of habitats.Results showed the possible use of cox 1, cytb genes and ITS2 region as markers in determining the population genetic structure of Phlebotominae sand flies in Sri Lanka.Q2. Has the statistical analysis been performed appropriately and rigorously?Reviewer #1: NoReviewer #2: Yes R2: Standard statistical software and analysis were used in the study by referring to previous published work. Q3: The markers used in this paper are not suitable for assessing population genetic structure and gene flow among populations. At this aim, microsatellite or SNPs markers should be used. Consequently the inferred conclusions are not supported by genetic data.The genetic analyses carried out in this paper revealed unique sequences of “all genomic regions studied except the cox 1 gene in 21 flies that aligned with those from Kerala, India and cytb gene of 4 flies that aligned with those isolated earlier from Sri Lanka and 3 from Madagascar”.The authors say that Cox 1 gene and ITS 2 region analyses revealed gene flow between the study sites”. However, the lack of differentiation among populations is due to incomplete lineage sorting and the lack of power of these markers for intra-specific studies.R3: The current study was designed to determine the population genetic structure of sand flies using two mitochondrial markers cox 1, cytb and a ribosomal region ITS2. However, no microsatellite loci were used for genotyping due their unavailability of such markers in P. argentipes. While we as a group, continue our efforts in developing such robust markers for P. argentipes, the authors believe the selected antigenic loci of P. argentipes are good enough for the said purpose due to the following reasons: Most of the studies have been conducted to investigate population genetic analysis using these markers . Gene flow also successfully determined using these markers in following studies.. Further, several studies have conducted using one or two of the markers used in the present study and microsattelites and have proved that both could be used sucessfully in population genetic structure analysis . we suggest changing the following sentence in the conclusion in line 370-373, 375-377– ‘Gene flow was evident with lack of population differentiation even between geographically distant populations of P. argentipes perhaps due to absence of geographic barriers and the continuity of habitats.’‘Results also validated the use of cox 1, cytb genes and ITS2 region as a tool in understanding the population genetic structure of Phlebotominae sand flies in Sri Lanka.’As, Both cox 1 and ITS2 indicated a gene flow with lack of population differentiation even between geographically distant populations of P. argentipes perhaps due to absence of geographic barriers and the continuity of habitats.Results showed the possible use of cox 1, cytb genes and ITS2 region as markers in determining the population genetic structure of Phlebotominae sand flies in Sri Lanka.Reviewer 2: Q1: 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: NoReviewer #2: PartlyR1: A minimum of 10 sequences of each marker from each study site was used in the analysis. Previous published work on population genetic structure analysis have used sample numbers between 6-10 from a single study location and have obtained successful results. I suggest changing the following sentence in the discussion and conclusion in line 342-343, 370-373, 375-377– ‘Gene flow was evident with lack of population differentiation even between geographically distant populations of P. argentipes perhaps due to absence of geographic barriers and the continuity of habitats.’ ‘Results also validated the use of cox 1, cytb genes and ITS2 region as a tool in understanding the population genetic structure of Phlebotominae sand flies in Sri Lanka.’As, Both cox 1 and ITS2 indicated a gene flow with lack of population differentiation even between geographically distant populations of P. argentipes perhaps due to absence of geographic barriers and the continuity of habitats.Results showed the possible use of cox 1, cytb genes and ITS2 region as markers in determining the population genetic structure of Phlebotominae sand flies in Sri Lanka.Q2: Is the manuscript presented in an intelligible fashion and written in standard English?Reviewer #1: YesReviewer #2: NoR2: The typographical and grammatical errors suggested by the reviewers were corrected and changes are highlighted in the manuscript. AbstractQ3: Line 30: Two mitochondrial genes viz. Please correct this sentence, I consider the word “viz.” it is misused.R3: Agreed and changed the sentence in line 30.Q4: Line 31: please delete oxidase, just leave Cytochrome b (CytB)R4: Agreed and deleted it in line 31.Q5: Line 30-31: parentheses are not italicizedR5: Agreed and done.Q6: Line 33: Change Network to networkR6: Done it in line 33. Q7: Line 35: change flies to sand fliesR7: Done it in line 35.Q8: Line 38-42: Please restructure the sentences, because it is a bit confusing.R8: Changed the sentence in line 38-42.IntroductionQ9: Line 51-54: I suggest change the paragraph this way:Phlebotomus argentipes (Diptera: Psycodidae) is the known vector of Leishmania donovani, the causal agent of the clinical form VL which is considered the second parasitic disease cause of a high number of deaths, in India, Nepal and Bangladesh [4].R9: Agreed and done the changes to line 50-52.Q10: Line 70: change weren´t to were notR10: Agreed and done in line 65.Q11: Line 78, 83, 103: delete this spaceR11: Deleted spaces.Q12: Line 86: Colombia is not an Old world country, please make the correction. Additionally there are more barcode studies for species from the new world, such as USA, Mexico, Brazil, please keep it in mind.Q12: Done the changes to the sentence in line 75-77.Q13: Line 99: change phylogeograpy to phylogeographyR13: Changed as ‘phylogeography’ in line 85.Q14: Line 61-77: Although the information is relevant is very long, please be more specific.R14: Agreed and done in line 59-70.Q15: Line 93-102: I suggest that only provide the information regarding the relevance of the use of the cox1, cytb and ITS2 genes for the genetic characterization of Phlebotomus argentipes. And do not include information that is not relevant in your research. For example: Line 94-96:In South America the phylogenetic analysis of cox 1 sequences of Verrucarum species of New World sand flies of the genus Lutzomyia was investigated [20].Line:97-98: Mitochondrial introgression in the Lutzomyia townsendi in Colombia was studied using cytb gene [12].R15: Due to restricted studies in P. argentipes corrected statement in line 83-85.Q16: Line 48-107: In general, the introduction is very long, and contains information that is not very relevance. I kindly suggest that it be summarized a bit to make it more clear and precise.R16: The changes were made according to the comments suggested by the reviewers to the introduction to be summarized. Q17: Line106-107: “to study the effect of evolutionary forces that may spread resistance genes from one population to another”.I have a question. The genes to be amplified are not necessarily genes that are related to insecticide resistance. How do you plan to evaluate and/or justify that the genetic variability that you may find are related to resistance to insecticides?R17: There is no link between the genes used in the study and the insecticide resistance genes. This statement was made as there is a high chance of entering such genes from one populations to another population in the absence of population genetic structure variation. Q18: It is not very clear to me. Please in the part of your introduction, where you talk about insecticides could explain what genes are useful for do this objective.R18: Explained in line 67-69.Material and MethodQ19: Line 111-116: A total of 126 of adult sand flies were collected, this sentences is a result not is part of the method. Please move this information to the next section. Also specify how many days you collected in each of those locations.R19: Moved it to the result section and added information to the material and method in line 92-97. ResultsQ20: Line:189-190: A total of 126 of adult sand flies were collected and were identified as Phlebotomus argentipes .R20: Added it to line 171.Q21: Please, also specify how many specimens you collected in each one of the four locations that you studied. Why you did not collect other sand fly species and only obtain specimens of Phlebotomus argentipes, this is very common in other studies? There are not other sand fly species in your country? How many males and females did you collect? Did you analysis 126 specimens by PCR or how many?R21: About 42 specimens were collected in each one of the four locations with a total of 52 females and 74 males. There are Sergentomyia spp. in the country. However, trapping method was basically effective for cattle-baited net trap. Thus, Phlebotomus argetipes were found apart from one or two Sergentomyia spp. specimens collected from light traps in some days, not routinely. Due to absence of sufficient numbers of samples, Sergentomyia spp. was not included in the study. Due to the study were based on the vector species, Phlebotomus argentipes, a total of 126 species were analysed by PCR followed by the analysis and interpretation were based on vector control strategies.Q22: Line193: how did you select those 10 specimens of the best quality?R22: The chromatograms of each and every sequence was checked manually and only the good quality sequences were used in the study.Q23: Line 214, 217,305,306,308: Change flies or fly to sand flies or sand fly according to the : case in the entire document.R23: Agreed and corrected.Q24: Line 217: correct P. argentipesR24: Corrected it line 199.Q25: Line 237: In the table, please include the number of specimens that you analyzed, for each gene and locations.R25: Added to the Table 1.DiscussionQ26: Line 322: two cladesR26: Agreed and done it in line 302.Q27: Line 325: network analysisR27: Done it in line 305.Q28: Line 326: P. argentipes were closely related to specimens of the same species collected in Sri Lanka, India and Israel.R28: Corrected it in line 306-307.Q29: Line 327: of three sand fliesR29: Corrected it in line 307.Q30: Line 327-328: cytb is a marker that shows variability at the intraespecific level, different from cox1 that shows differences at the interespecific level. So, why is relevant that some specimens are similar to specimens from Madagascar? Explain more about it, although you have a small sample, it is interesting that you have haplotypes similar to a locality that is considerably distant from your collection point.R30: The larger sample will be used for further studies to confirm this specimens exactly from Madagascar or not. If so, there is a genetic exchange may apply in between those specimens.Q31: Line 329: did notR31: Corrected it in line 309.Q32: Line 330: Add a sentence highlighting that your work is one of the first sequences for this gene and for this species. I think it is very important that you highlight this contribution of your work.R32: Done it in line 311-312.Q33: Line 345: does not.R33: Corrected it in line 327.Q34: Line 352-353: put the name of the area of study not numbers, because you did not listed before.R34: Agreed it and corrected in line 334-335.Q35: Line 354-360: Although it is an interesting hypothesis, I think it is very risky to assert something of this style, given that the number of specimens that you analyzed is very small. I do not consider that ten specimens are enough to think that there is gene flow in all communities of sand flies and those possible resistance genes are being inherited. Additionally, you did not analyze genetic markers for resistance.R35: Previously published work on population genetic structure analysis have used sample numbers between 6-10 from a single study location and have obtained successful results. This study used a minimum of 10 sequences from each marker from each locality. However, as the sample size is not very high it was stated as “possible” gene flow. Insecticide resistance genes was taken as an example gene that could be introduced into other populations due to gene flow. The sentence, “The possible gene flow revealed in the current study demonstrate the risk of dispersal of such resistance genes between populations with the potential for the spread of insecticide resistance and implications for future vector control” could be changed as “The possible gene flow revealed in the current study demonstrate the risk of dispersal of genes such as insecticide resistance genes between populations with the potential for the spread of insecticide resistance and implications for future vector control” in line 339-342 in the discussion.Q36: Line 363-373: This information is not informative for this article, I suggest that you focus on the group of sand flies and compare your results with those of other researchers. I suggest you read the article by:https://doi.org/10.1016/j.meegid.2013.02.0041. Pech-May et al. 2016. Genetic structure and divergence in populations of Lutzomyia cruciata, a phlebotomine sand fly (Diptera: Psychodidae) vector of Leishmania mexicana in southeastern Mexico https://scholarworks.uttyler.edu/biology_grad/53/2. Wolkoff, 2018. Population structure, demographic history, and environmental niche of the sand fly disease vector Lutzomyia shannoni (Dyar) (Diptera: Psychodidae) in the U.S., Mexico, and Colombia R36: Added information to line 351-358.Q37: Line 362-382: I suggest that you change these paragraphs as follows and fill them in with additional information, so that you can keep comparing your results.The molecular markers have been extensively used for genotyping and study of evolutionary history of mosquitoes . The analysis using cytb gene has demonstrated been useful to detect the lack of population differentiation and the presence of gene flow in some species of dipterans in South-east Asia [32]. The gene flow between species of insect vectors may lead to alterations in the disease patterns [35]. Low genetic structure variation in some sand fly species as a result of geographical isolation and restricted gene flow may have led to reduced flight abilities and formation of cryptic species that ultimately influence the capacity to transmit parasites such as Leishmania in Latin America [36].R37: Added information to line 344-351.Q38: Line 384-391: again, I do not agree with these statements, I suggest that you approach it in another way for greater support, or that you only suggest it as a possible hypothesis in a more subtle way.R38: Corrected it in line 360-366.ReferencesQ39: Please check the structure of the citations again, to make it homogeneous. Scientific and gene names are also missing in italics, and there are spelling mistakes R39: Agreed and done it.AttachmentResponse to Reviewers.docxSubmitted filename: Click here for additional data file. 4 Aug 2021PONE-D-21-16188R1Genetic diversity and population structure of Phlebotomus argentipes: vector of leishmaniasis in Sri Lanka.PLOS ONEDear Dr. Karunaweera,Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.Please submit your revised manuscript by 16th August 2021. 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Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.http://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.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: We look forward to receiving your revised manuscript.Kind regards,Maria Stefania LatrofaAcademic EditorPLOS ONEJournal Requirements:Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.Additional Editor Comments (if provided):I agree with the authors on that the target genes analyzed are good for the specimen’s identification and for the analyses of the genetic divergence within and among phlebotomine sand fly populations, but not on the gene flow. Indeed, Weeraratne and colleagues only hypothesized the presence of the gene flow using the cox1, whilst Manni et al., define that SSR markers are, as expected, more informative than ITS2 in revealing the slight genetic diversity between native and derived populations both in terms of variability and differentiation. The authors have to be more cautious when talk on the gene flow. I suggest, for example, putting your considerations as a hypothesis of the “existence of a potential gene flow” based on the results obtained of the genes analyzed, and that the gene flow have to be further investigated or supported by the analysis of microsatellites.See also the article Prudhomme et al., “Altitude and hillside orientation shapes the population structure of the Leishmania infantum vector Phlebotomus ariasi.” Scientific Reports, 2020 DOI: 10.1038/s41598-020-71319-w.Change the title: since sand flies are vector of pathogen and not of diseasesLines 36-37: I suggest changing “revealed gene flow” into “may indicate a potential gene flow”, due to the motivation above, and throughout the ms.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.While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool,  16 Aug 2021Author responses to journal requirements and additional editor’s comments:Journal Requirements:Q: Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.R: Reviewed the reference list and it was completed and corrected. Following changes were made in the reference list.• Journal name abbreviation was corrected in line 428, 443.• Page numbers were added in line 467.• Journal name and page numbers were added in line 473.• Journal name, issue, page numbers and DOI number were added in line 475-476.Following reference was removed from the reference list as it contains unpublished work. Thus, the statement in line 353-358 referred to as “unpublished work” in line 358. Wolkoff. Population structure, demographic history, and environmental niche of the sand fly disease vector Lutzomyia shannoni (Dyar) (Diptera: Psychodidae) in the U.S., Mexico, and Colombia. University of Texas at Tyler; 2018.Additional Editor’s Comments:Q1: I agree with the authors on that the target genes analyzed are good for the specimen’s identification and for the analyses of the genetic divergence within and among phlebotomine sand fly populations, but not on the gene flow. Indeed, Weeraratne and colleagues only hypothesized the presence of the gene flow using the cox1, whilst Manni et al., define that SSR markers are, as expected, more informative than ITS2 in revealing the slight genetic diversity between native and derived populations both in terms of variability and differentiation. The authors have to be more cautious when talk on the gene flow. I suggest, for example, putting your considerations as a hypothesis of the “existence of a potential gene flow” based on the results obtained of the genes analyzed, and that the gene flow have to be further investigated or supported by the analysis of microsatellites.See also the article Prudhomme et al., “Altitude and hillside orientation shapes the population structure of the Leishmania infantum vector Phlebotomus ariasi.” Scientific Reports, 2020 DOI: 10.1038/s41598-020-71319-w.R1: Authors agreed and suggested changes have been added in the abstract section in line 32-42 and in the conclusion section in line 369-373 and 374-385.“Therefore, there is a high risk of transferring genes of functional significance, such as those that confer insecticide resistance from one population to another with the risk of eventually spreading across the country. However, confirmation of such an assumption would require further investigations and supportive evidence probably with the use of more neutral genotyping markers such as microsatellites, which have been used in other Phlebotomus sp. for similar investigations including for those related to the influence of geographic distance and altitude . The study also confirms the possible use of cox 1, cytb genes and ITS2 region as genotyping markers for the determination of the population genetic structure of Phlebotominae sand flies in Sri Lanka. Thus, the findings will pave the way for more extensive investigations, probably with the use of more genotyping markers, to aid a vector control strategy that may need to be adopted within a future national leishmaniasis control programme.”Q2: Change the title: since sand flies are vector of pathogen and not of diseasesR2: The title was changed into ‘Genetic diversity and population structure of Phlebotomus argentipes: vector of Leishmania donovani in Sri Lanka.’Q3. Lines 36-37: I suggest changing “revealed gene flow” into “may indicate a potential gene flow”, due to the motivation above, and throughout the ms.R3: Changed as ‘may indicate a possible gene flow’ in line 37, 39-40, 333-334, 337, 339-343, 363-364, 372.Authors suggested to acknowledge funding source in the Acknowledgements section in line 391-394.AttachmentResponse to Reviewers.docxSubmitted filename: Click here for additional data file. 17 Aug 2021Genetic diversity and population structure of Phlebotomus argentipes: vector of Leishmania donovani in Sri Lanka.PONE-D-21-16188R2Dear Dr. Karunaweera,We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at onepress@plos.org.If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact Kind regards,Maria Stefania LatrofaAcademic EditorPLOS ONE 3 Sep 2021PONE-D-21-16188R2 Phlebotomus argentipes: vector of Leishmania donovani in Sri Lanka. Genetic diversity and population structure of Dear Dr. Karunaweera: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. onepress@plos.org.If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact plosone@plos.org. If we can help with anything else, please email us at Thank you for submitting your work to PLOS ONE and supporting open access. 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One way in which the wine sector is reacting to the challenge of climate change is to develop plant material that is adapted to the new conditions. Such a strategy will allow the continuation of quality viticulture in traditional winemaking areas that will otherwise be abandoned. The objective of this study was to characterize the anthocyanin composition and content of selected intraspecific hybrids of Monastrell with two other varieties (Syrah and Cabernet Sauvignon). The experiment was carried out over three successive seasons, and the polyphenolic quality of the grapes and wines was assessed along with the adaptation of the hybrids to the high temperatures which will inevitably affect our area (south-eastern Spain). The results showed that, compared with grapes of the Monastrell variety and the wines made from them, most of the hybrids  had a higher total anthocyanin concentration and overall content of acylated anthocyanins, depending on the year studied. Grapevine is one of the most important fruit crops worldwide and constitutes a socio-economic activity of great significance for wine-producing countries . Spain hOne of fighting back in this new scenario is to develop breeding programmes that will hopefully produce new varieties better adapted to the new conditions. However, two things must be borne in mind in this respect: the need to develop new varieties that are more resilient to climate change while maintaining the principal features cultivars, and increasing consumer demand for wines whose production is respectful of the environment. At the present time, crossbreeding and bud mutation are still regarded as the best ways for developing new grape cultivars .Many different breeding programs are being developed around the world, and the progenies obtained as a result of these programs are still under evaluation. Several new cultivars have been generated by IMIDA , among them, ten red vinification grape crosses (five of them in awaiting registration), which are still being characterized for their quality and yield. The ten crosses are well-adapted to hot, dry climatic conditions, which should mean that will be suited to the forthcoming climatic scenario of the Mediterranean area.Current data suggest that climate change may adversely affect grape and wine quality by affecting color, an important indicator for evaluating the quality of red wine and one of the most influential factors when consumers all over the world choose a wine. As is well-known, the color of red wine is mainly determined by the composition and concentration of anthocyanins, which are responsible for the slight red to dark purple color of wine. When studying grapes for winemaking, it is not only the quantity of anthocyanins that is important but also their anthocyanidin profile. The hydroxylation pattern of the B-ring is one of the main structural features of flavonoids and is an important determinant of their coloration, stability and antioxidant capacity .Many factors can influence the concentration and profile of anthocyanins in grapes, which is primarily decided by genetic factors, with some modifications due to climate. Previous studies have shown the effect of temperature on the anthocyanin content of grape berries e.g., –7], and,, and,7)]To the best of our knowledge, there have been few reports on the individual anthocyanin composition of new Monastrell varieties, and only two reports , 13 in wVitis vinifera L. cv Monastrell was planted in 1997, and the intraspecific crosses were planted between 2007 and 2012. The vegetal material was obtained from intra-varietal crosses between Monastrell and Syrah (S) and Cabernet Sauvignon (C). There are 20 plants of each of these new varieties that are grafted on R110 and the planting density was 3,200 vines ha−1 (2.50 m between rows and 1.25 m between vines). The training system was a bilateral cordon trellised to a three-wire vertical system and drip irrigation was applied. Grapes were harvested at optimum technological maturity. Subsequently, grapes were transported to the winery (Estación Enológica de Jumilla), where wines were elaborated in accordance with a traditional vinification protocol in 50-L steel tanks.The present study was carried out over three consecutive seasons (2017–2019) in an experimental field in Bullas . 2/100 kg). Total acidity was corrected to 5.5 g/L with tartaric acid, and selected yeasts were added . All vinifications were conducted at 25 ± 1°C. Throughout the fermentation pomace contact period (10 days), the cap was punched down twice a day, and the temperature and must density were recorded. At the end of this period, wines were pressed at 1.5 bars in a 75 L tank membrane press. Free-run and press wines were combined and stored at room temperature. The analyses were carried out at the end of alcoholic fermentation in triplicate.All vinifications were made using 50 kg of grapes, which were destemmed, crushed and sulfited  were analyzed using an Abbé-type refractrometer (Atago RX-5000) and titratable acidity and pH were measured using an automatic titrator  with 0.1 N NaOH.Grapes were peeled with a scalpel, and the skins were stored at −20°C until analysis. Samples (2 g) were immersed in methanol (40 mL) in hermetically closed tubes and placed on a stirring plate at 150 r.p.m. and 25°C. After 4 h, the methanolic extracts were filtered (0.45 μm) and analyzed by high-performance liquid chromatography (HPLC) injecting wines samples directly into the chromatograph.1. The compounds were identified according to Gil-Muñoz et al. , equipped with a Waters 996 diode array, and a Primisep B2 SIELC column , 25 × 0.4 cm, 5 μm particle size and a CORTECS® Shield RP18 column , 150 × 0.46 mm, 2.7 μm particle size were used to analyse the grapes and wines, respectively. In both analyses type were used as solvents 4.5% formic acid solution (solvent A) and HPLC grade acetonitrile (solvent B) at a flow rate of 0.9 mL minz et al. . CompounAll the analyses were performed with the statistical package Statgraphics 5.1. The data were analyzed using analysis of variance (ANOVA) and a two-way analysis of variance (MANOVA) procedures, and means were separated by Duncan's multiple range test in order to highlight similarities and/or differences within samples (grapes and wines). Finally, discriminant analysis was used to identify the most discriminant variables.The physical data of the progenies and of the Monastrell parental berries are presented in There were differences in terms of the yield obtained for each variety and among years. The parental (Monastrell) plants had their highest yield in 2019 and the lowest in 2017. With regards to the crosses, in general, hybrids attained the lowest production level in 2018 and the highest in 2019. The hybrids with the lowest production were MC80 and MS34, while those with the highest production were MC94 and MS104, the last one reaching an exceptionally high level (5.4 kg/vine) in 2019. This may have been because in 2019 the pruning system was changed to pruning at the two bud site which resulted in an increase in the yield of all plants, although the climatological conditions may also have affected the results -so 2018 was the year with the lowest temperatures, and 2019 the year with the maximum radiation .Berry size provides information about the skin surface/berry volume ratio, which can influence the winemaking process. Monastrell cultivar had significantly higher berry weights in the three seasons than the grapes of all the progenies. Only two of the crosses, obtained higher values during the two first seasons: MC98 and MS104. In the last season, the hybrids with the highest berry weight were MC80 and MS10. These data reflect the findings of Ortega-Régules et al. , who fouTotal acidity also varied among the different cultivars analyzed during the 3 years studied. The highest value obtained in 2017 was that of MS10, in 2018 that of MC85 and in 2019 of MC18. During the 3 years the lowest values were obtained by the parental (Monastrell) although MC4 had similar values in the first season. Also the pH values varied during these 3 years, although in general the highest values were found in Monastrell and the lowest in the MC18 variety. These values suggest that the crosses are the well-adapted the new climatic scenario. Lower acidity levels are also frequently correlated with higher grape pH, although the relationship is affected by potassium accumulation, which is dependent on temperature . It has The average soluble solids content of the studied grapes ranged from 20.0 (MC4) to 24.6 (MS34) °Brix, in the first season; 21.3 (MC4) to 27.4 (MS34) in the second season and from 17.5 (MS104) to 23.0 (MC111) in the third season. In 2019, the grapes were harvested 1 week earlier than in 2018. As mentioned above, climatological conditions could have influenced the results obtained, and, in general, °Brix values were higher in 2018, when the highest maximum temperatures were reached in August and the lowest rainfall was recorded . Also, tThe total anthocyanin concentrations found in grapes and wines from Monastrell and its hybrids are shown in The concentrations in grapes (expressed as μg/g skin) were higher in the hybrids than in Monastrell during the 3 years of the study, except in the case of the cross MC4 in 2018 and 2019. The appearance of a large number of hybrids in which the anthocyanin concentration is outside the range of concentrations found in their parental phenotypes is called transgressive segregation, and is a frequent occurrence in intraspecific crosses and in domesticated populations . Song etAs can be observed in As regards the hybrids, the highest concentration of total anthocyanins in 2017 was obtained in MS10, as it was in 2018, when it had similar values to MS34; in 2019 the highest levels were recorded for MC111, followed by MC85 and MS34. Of note is the fact that MS10 is one of the earlier varieties, since it is harvested at the end of August, when temperatures are highest (~35–40°C) and there is limited contrast between day and night temperatures. However, several authors, such as Shinomiya et al.  or YamanAs regards the wines at the end of alcoholic fermentation , the lowThe concentration of total anthocyanins in the wines made with the hybrids varied with the year, and differed from the concentrations in grapes. This can be explained by the inherited properties of each of the crosses in terms of their cell walls, which may hinder or facilitate the extraction of anthocyanins during the wine-making process. However, other aspects also need to be taken into account; for example, Medina-Plaza et al.  reportedThe anthocyanin profile in grapes differed among the cultivars. They were present in non-acylated and acylated form as well as in combination with p-coumaric and caffeic acids. The category, the proportion and amount of anthocyanins in red grapes largely depend on the grape variety and the growing conditions, such as viticulture practices and the weather . It is gAs can be observed in In general, hybrids had a higher percentage of tri-hydroxylate forms  than di-hydroxylate forms , although Monastrell grapes had the highest percentage of cyanidin (6.7–8.0%) and peonidin (9.6–12.6%). Goméz-Plaza et al.  found thO-glucoside in grape berry increases during the early stages of berry ripening to reach a maximum value during this time, and subsequently decreases until harvest due to its conversion into petunidin-3-Oglucoside and malvidin-3-O-glucoside through the action of methyl transferases.The different percentages of tri-hydroxylated monoglucosides obtained by the varieties studied are also shown in O glucoside had the highest percentage among the non-acylated anthocyanins in both Monastrell and Cabernet Sauvignon grapes, a finding echoed by Kyraleou et al. (O-glucoside is the main anthocyanin in both grapes and wines of many European red vine varieties (With respect to the percentage of petunidin (Pet) only in 2017, the following crosses provided higher percentages than Monastrell: MC111, MC85, and MS34. In a study made over two seasons (2015 and 2016), Gil-Muñoz et al.  also fouu et al.  in some arieties , 33. CarVitis vinifera the presence of acylated anthocyanins is to be expected, but the quantity of this type of anthocyanin can vary among cultivars, as reported by several authors. For example, Ortega-Regules et al.  and 14% for MS34. Zhang et al.  reportedFinally, coumaryl anthocyanin levels varied significantly among the cultivars and the values obtained were both higher and lower than those obtained by Monastrell, which showed a 3 year average of 15%. The hybrids that exceeded this percentage were the MC4 cross , MS104 (26%), MS34 (23%) and MS10 with 16% (profile more similar to Syrah). The values for the rest of the hybrids were lower values, ranging between 5% (MC94) and 13% (MC98). These results are important because coumaroylglucoside derivatives may also be more reactive and, consequently, more involved in the formation of derived pigments than other anthocyanins. Their higher reactivity has previously been observed during fermentation, when the rate of p-coumaroylvitisin formation was estimated to be higher than that of non-acylated vitisins .Finally, Vitis vinifera varieties  are more stable in wines than di-hydroxylated ones (cyanidin and peonidin-3-glucosides) . The % DRegarding acylated anthocyanins in wines, as can be observed in As for the percentage of acetates and coumarates, as occurred in grapes, once again the percentage of acetates in the wines made with crosses was always much higher than that obtained by the wines made with the Monastrell variety, which had an average of 7% for the 3 years studied. The values obtained by the wines made with the hybrids were always two-fold, and in some cases three-fold or even four-fold the percentage obtained for the Monastrell wines. These values ranged from 16% for the wines made with MS34 to 23% for the wines made with MC4. Gil-Muñoz et al.  also fouAs regards the coumarates, as occurred with the grapes, both higher and lower percentages than those obtained in the Monastrell wines were found for the wines made with the crosses. The wines made with Monastrell grapes had an average of 11% coumarates in the three vintages studied. The wines made from the hybrids that had higher percentages than the Monastrell wines were MS10, MS104, and MS34, all of them with 15% coumarates, followed by the wine made with MC4 (11%) and the wines made with the crosses MC11, MC18, and MC98 (12% coumarates). Only the wines made with the MC85 and MC94 crosses had lower percentages than the wines made with the Monastrell variety.Finally, Finally, the interactions between the two factors (variety and year) were not statistically significant. This means that the anthocyanin profile, which differed among varieties, can be taken as an individual characteristic of each one, regardless of the interannual differences that may exist.A discriminant analysis was conducted using the following variables: % Dp, % Cy, % Pt; % Pn, % Mv, % acetates and % coumarates as independent variables to obtain the coefficients. The first two discriminant functions explained 82% of the variance. The first canonical function accounted for 60% of the variability and the second 30%.The standardized coefficients of the discriminant functions  obtainedTherefore, according to the groups of cultivars depicted in the plot, it may be said that the variables were able to discriminate varieties according to their anthocyanin profiles.Vitis vinifera from Monastrell variety growing in the southeast of Spain were analyzed.The anthocyanin composition of 10 intraspecific red hybrids of In general, both the grapes and the wines of the new varieties showed a higher concentration of total anthocyanins, tri-hydroxylated forms and acylated compounds than the Monastrell variety. Based on the results obtained, it can be concluded that most of the hybrids selected have an anthocyanin profile far from that of Monastrell variety, and closer to that of the other parentals (Syrah or Cabernet Sauvignon). While there were interannual differences in terms of the concentration of the different types of anthocyanin, the profile was well-defined for each of these new varieties and their wines.In summary, these results are indicative of the anthocyanin richness of the obtained hybrids compared with Monastrell and show their potential for producing quality red wines. This suggests that the ten selected hybrids may be useful for alleviating the effect of climate change, since, despite being harvested earlier than Monastrell, they contain higher concentrations of anthocyanins, suggesting they will adapt well to expected climatic conditions.The original contributions presented in the study are included in the article/JM-O, MG-B, DP-Q, and AC-P: formal analysis. JM-O: data curation. JF-F: investigation. JF-F, RG-M, and MG-B: methodology. JB-S: conceptualization. RG-M: writing—original draft, funding acquisition, project administration, and resources. All authors: have read and agreed to the published version of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Rhododendron, 26 SSR primers were designed and validated in three species.Microsatellites  are co-dominant nuclear markers that are widely used in population genetic studies. Population genetic parameters from different studies might be significantly influenced by differences in marker number. In our study, 265 sequences with polymorphic microsatellites were obtained from SLAF-seq data. Then, subpopulations containing different numbers  of markers were genotyped 10 times to investigate the impact of marker numbers on population genetic diversity results. Our results show that genotyping with less than 11 or 12 microsatellite markers lead to significant deviations in the population genetic diversity or genetic structure results. In order to provide markers for population genetic and conservation studies for  Although single nucleotide polymorphic loci (SNPs) have been used in a variety of genetic studies2 recently, they still have not replaced SSRs completely as microsatellites are highly informative, codominant, exhibit high specificity, are transferable among related species and have relatively low costs. As such, microsatellites continue to be widely used, especially for wild species, particularly when sample sizes are large. The number of loci used for genotyping is one of the key issues concerning the use of microsatellites. Koskinen et al. show a substantial decrease in standard deviation estimates of the interpopulation genetic distances by increasing the number of loci from six to seventeen. They indicate that the stability of commonly used genetic distances and phylograms is determined by the number of microsatellites investigated3. In addition, the results obtained from 205 red deer indicate significant effects on population genetic parameter if the number of microsatellite loci smaller than six4. However, in experimental studies of Drosophila nigrosparsa, Arthofer et al. demonstrate that a large proportion of individuals can still be correctly assigned to population of origin when using eight loci, and the population structure is still retained when using only two highly polymorphic loci5.Microsatellites  have been the most frequently used genetic marker in population genetics over the past 20 years4. Compared with the total number of microsatellites in genome, 16 or 17 microsatellite loci are too low and may be inconsistent with actual experimental design. This is limited by previous de novo SSR development, which can be a tedious and costly process6. More recently, next generation sequencing (NGS) technology has facilitated the development of hundreds microsatellite loci based on sequence data with reduced cost and effort10, offering the possibility of resampling different numbers of microsatellites from genome.Unfortunately, to investigate the impact of different numbers of loci on population genetic results, the subpopulations containing different loci were generated from the total set of 16 or 17 microsatellite loci in previous studiesRhododendron, R. dauricum, and R. mucronulatum, distributed in northeastern China, using 664,406 SNPs based on specific-locus amplified fragment sequencing (SLAF-seq), a recently developed, high-resolution strategy for the discovery of large-scale de novo genotyping of SNPs12.In our previous study, we estimated the population genetic structure and demographic history of two closely related species of R. dauricum and R. mucronulatum. Rhododendron species are widely distributed around the world ranging from tropical to polar climates, and used as valuable horticultural plants due to their beautiful vegetative forms and remarkably bright-colored flowers13. The microsatellite markers developed in this study will aid genetic diversity studies of Rhododendron.Here, using SSR sequences from this dataset, we evaluated the impact of using different numbers of SSR loci, generated from the whole genome, on the stability of population genetic results. Furthermore, we used the SLAF-seq data to develop polymorphic microsatellite markers for R. dauricum and R. mucronulatum (accession number: PRJNA589346), we removed some populations that contain only one individual and merged some populations were close to each other geographically. Our dataset for this part consisted of 38 R. dauricum and 25 R. mucronulatum samples, and the sample vouchers have been deposited at the Northeast Normal University Herbarium . The lowest threshold of repeats for dinucleotides was set to six, while all others were set to five. Two SSRs motifs with the maximum interruption less than 100 bp were considered as one compound microsatellite.Microsatellite motifs from di-nucleotides to hexa-nucleotides were identified from the polymorphic SLAF tag sequences in MISA-web 16 with our previously used parameters11. Raw INDELs were filtered using our custom Perl scripts with the cutoff “mapping quality (MQ) > 30, read depth (DP) > 3.” Moreover, PLINK 217 was used to further filter with the minor allele frequency (MAF) of 0.04 and maximum missing rate of 0.1. Finally, microsatellite genotypes for each individual were determined based on the sequence length of core motifs.Additionally, insertions/deletions (INDELs) were called using the program SAMtools p-values, which indicate whether markers or populations deviate from Hardy–Weinberg equilibrium, were tested by 1000 random permutations using FSTAT version 2.9.3.218. When FIS values for a locus deviated significantly from zero (p < 0.01), loci were excluded from further analyses. In addition, number of alleles (NA), allelic richness (Ar) and genetic diversity (Hs)19 were calculated for each species using FSTAT software. The population genetic structure was analyzed using the Bayesian clustering program STRUCTURE version 2.3.320. The admixture model with correlated allele frequencies was chosen, as recommended for faint population structures. The number of clusters (K) assumed was set to , and each value of K was run 10 times. Each run was performed with 20,000 MCMC iterations and an initial burn-in of 180,000. The final posterior probability of K, ln p(K), and Delta K (ΔK) was calculated using STRUCTURE HARVESTER21 to determine the most likely K value.Inbreeding coefficients (FIS) and corresponding t-test with the IBM-SPSS package version 24.To assess the effect of the number of microsatellites on the stability of the genetic diversity and genetic structure, population genetic analysis was assessed by the following procedure: data files consisting of 5, 6, 7,…, 15, 20, 25, 30, 35, 40 microsatellite loci were created by resampling from the complete data set randomly using a python script and repeated 10 times for each subset of microsatellite loci. Genetic diversity parameters and genetic structure analysis were constructed for each replicated data set as described above. Statistical analysis was done used one sample http://bioinfo.ut.ee/primer3-0.4.0/). The primer size ranged from 18 to 22 bp with the optimal size of 20 bp. The optimum GC content was 50%, the optimum melting temperature was 60 °C (ranged from 50 to 65 °C), and the maximum acceptable difference between the melting temperatures of the forward and reverse primers was 5 °C.Polymorphism is one of the important criteria for judging the usability of microsatellite markers. To determine if SLAF data of populations can be used for highly polymorphic microsatellite marker development, we selected 66 loci with both highly polymorphic microsatellite motif  and at most one individual missing data for primer design, and finally only 40 pairs of primers were synthesized since their flanking regions were long enough. All primers were designed by the program Primer v3 , 10 samples of population JC (R. mucronulatum), moreover, and 12 individuals of R. aureum following a modified CTAB procedure22 and verified by electrophoresis on 1% agarose gel. PCR amplifications were performed in 20 μL reactions containing 50 ng genomic DNA, 1 × PCR buffer (plus Mg2+), 0.2 mM of dNTPs, and 0.5 μM of each primer, with each forward primer labeled with fluorescent dye  (Invitrogen) and 1 unit (U) of Taq polymerase (Takara). Thermal cycling began with an initial denaturation step at 95 °C for 5 min, followed by 35 cycles of 30 s at 94 °C, 30 s at an optimal annealing temperature  contained more than one microsatellite motif. Di-nucleotides were the most abundant repeat motif , followed by tri-nucleotides (11.69%), tetra-nucleotides (1.38), penta-nucleotides (0.48%) and hexa-nucleotides (0.34%). In addition, the highest number of repeats per locus was 6  , so they were excluded from subsequent analyses. Among the remaining 265 loci, the number of alleles per locus was 7, 6, 5, 4, 3 and 2 for 91, 48, 39, 18, 25 and 44 loci, respectively. For the remaining 265 loci, allelic richness (Ar) and the genetic diversity (Hs) measured 1.508 and 0.511 for R. dauricum with 5 to 10 markers were from 1.553 to 1.745  are given in Fig. R. mucronulatum. Compared with the full set of 265 loci, with fewer than twelve loci, at least one incorrect cluster was detected in all datasets. Remarkably, when data of only five or six loci were used, the error rate of populations clustering reached 50%.STRUCTURE analysis based all SSR markers (265 loci) showed clear differentiation between species Fig. , similar ΔK Fig. . The resAmong the 265 polymorphic microsatellites, a total of 40 primer pairs were designed for evaluating PCR amplification efficiency and polymorphism in congeneric species. Of the primers tested, 14 primers were excluded from further analysis because these primers did not generate clear microsatellite peaks or failed to be amplified. The remaining 26 primers exhibited high amplification success and were screened in 40 samples from three species. Of the 26 polymorphic SSR loci, 23 were dinucleotides, two were trinucleotides and one was a tetranucleotide.R. dauricum, R. mucronulatum and R. aureum, respectively  ranging from 4 to 19. The Ar values ranged from 3.023 to 9.339, with an average of 6.082, while the HS varied from 0.297 to 0.917, 0 to 0.929 and 0 to 0.968 in 24. Large number of papers published in the last few years have involved the use of microsatellites. However, as shown here and elsewhere, the low number of loci used may lead to erroneous conclusions when comparing populations3.Simple sequence repeats (SSRs) are co-dominant nuclear markers that are widely used in population genetic studies, which provide insights and guidelines for preserving the genetic diversity of populationsR. dauricum was significantly different from the Hs based 265 loci (0.511) when compared to analyses of less than 10 microsatellites  when used 25 microsatellite loci , one of the most reported measures of genetic variation, is also referred to as allelic diversity or mean number of alleles per locus. And in FSTAT software, the Ar is corrected for sample size, thus compared with Ar, Hs is more sensitive to the sample size.For example, in our study, significant deviations in Ar were found when using less than 11 loci, compared to the value from the full dataset of 265 loci. Moreover, the genetic diversity parameter Hs of 5, which were much larger than the average Ar of microsatellite sites in the other study27. Thus, it is difficult to de novo develop microsatellite primers with such high polymorphism. Therefore, two microsatellite loci are far from enough in actual genetic structure research. Moreover, our previous study illustrated R. dauricum and R. mucronulatum clustered into distinct groups and showed majority populations collected from the Changbai Mountains  of R. dauricum with some admixture from R. mucronulatum11. However, in our study, the differentiation between species was not obvious with low microsatellite loci used. Remarkably, the incorrect assignment was still possible with fewer than 12 loci. With decreasing number of microsatellites, a reliable comparison between species cannot be achieved, even if Bayesian methods are used.These effects could also be confirmed for the population genetic structure, which was investigated by STRUCTURE. Arthofer et al. demonstrate that the population structure was still retained, though about a quarter of a individuals cannot be correctly assigned, when using only two loci with the Arwere 12.94 and 13.54, , respectivelyR. dauricum, R. mucronulatum and R. aureum. The levels of diversity observed at these microsatellite loci, measured as allelic richness (Ar) and genetic diversity (Hs), were similar to those in previous studies11. Rhododendron is a familiar ornamental plant worldwide, ranging from tropical to polar climates13. This study provides a potentially highly polymorphic SSR markers library for the research of Rhododendron subgen. Rhodorastrum, which will facilitate the further study of the genetics of Rhododendron subgen. Rhodorastrum, even Rhododendron. Furthermore, we explored a simple route to develop polymorphic SSR markers from non-model species based on SLAF-seq, which is well suited for polymorphic SSR marker discovery in non-model organisms.In addition, 26 polymorphic microsatellite loci were validated and characterized for individuals of Rhododendron species will be important for investigating population genetic diversity and genetic structure, and these results in turn will provide crucial information for conservation and management of Rhododendron species.Previous studies showed considerable differences of genetic diversity and genetic structure with regard to the number of microsatellite loci. Our results indicated significant effects on population genetic parameters if the number of microsatellite loci was less than 12. With decreasing marker numbers, the accuracy of population genetic of and the genetic structure decreases. Fortunately, the SLAF-seq data of populations offers an effective approach to develop polymorphic microsatellite markers for non-model species. The 26 polymorphic microsatellite markers we developed for Supplementary Information.