Timber Tracking of Jacaranda copaia from the Amazon Forest Using DNA Fingerprinting
, Celine Blanc-Jolivet 2, Niklas Tysklind 3, Stephen Cavers 4, Malte Mader 2, Barbara Rocha Venancio Meyer-Sand 1, Kathelyn Paredes-Villanueva 5, Eurídice Nora Honorio Conorado 6, Carmen Rosa García-Dávila 7, Valérie Troispoux 3, Adline Delcamp 8 and Alexandre Magno Sebbenn 1,9,*Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsThis is an interesting paper addressing an important application of molecular genetic approaches to timber identification in the context of illegal logging. The authors demonstrate convincing genetic structure in Amazonian Jacaranda copaia providing a basis by which timber samples could be assigned to countries and regions of origin.
A number of deficiencies mar the work as currently presented. These need to be corrected.
1. Although the introduction provided to laws and regulations in various countries is relevant and necessary it does not seem to be entirely accurate, and the references provided do not always seem to provide sufficient basis for the claims made. For example, three references are cited to support the statement that "[A] Great part of trade timber harvest around the world from natural forest has illegal origin", but all three are papers similar to the present work and not directly about the great part of trade timber harvest and its legality. Another example is reference made to "... Australia (prohibition bill)..." which I can only assume is meant to refer the reader to the Australian Illegal Logging Prohibition Act 2012. Note that in this context the word Bill usually applies to a piece of legislation during its progress through a parliament and the word Act to pieces of legislation once passed. This part of the introduction needs to be carefully checked and possibly reduced.
2. The analyses combining cytoplasmic (plastid and mitochondrial markers) and nuclear markers is not sufficiently justified since it seems markers from (presumeably) haploid and diploid genomes are being analysed together as if all were diploid. As a result the reader is presented with heterozygosity measures for haploid markers - a situation which makes very little sense.
3. Unless I missed it, no evidence is presented that the markers used in this study can actually be assayed from logged or otherwise processed timber since leaf and cambium tissue preserved on silica gel was used. I think this issue needs to be addressed.
In addition, the paper could be improved by addressing these less critical points.
Communication around "group and individual self-assignment" and exactly what the numbers mean is unclear. This might be clear to other readers but certainly isn't to me.
The phrase "reference data" appears numerous times but it is never explained exactly what this means in relation to data analysed. Is all the sample data reference data?
The figures in the review version are not legible, both when printed and viewed electronically.
I am surely not unique in being unfamiliar with the MassARRAY® iPLEXTM technology. Some brief description of it and its advantages over other assay methods (for example, amplicon sequencing) in the context of timber tracking would be helpful. I would be interested in things like rates of genotyping error which would would be important in a legal/regulatory context.
Although there is no doubt a good association between genetic and geographic distance, there are some pairwise comparisons between geographically distant populations with very low Fst values. Some comment on this would seem appropriate.
Comments on the Quality of English LanguageGrammar and spelling are poor throughout, and in places makes the writing difficult to interpret. Other English language conventions are not followed, for example in the use of commas and decimal points in numbers.
Author Response
Reviewer 1
This is an interesting paper addressing an important application of molecular genetic approaches to timber identification in the context of illegal logging. The authors demonstrate convincing genetic structure in Amazonian Jacaranda copaia providing a basis by which timber samples could be assigned to countries and regions of origin.
A number of deficiencies mar the work as currently presented. These need to be corrected.
Answer: Thank you by your constructive criticism.
1.Although the introduction provided to laws and regulations in various countries is relevant and necessary it does not seem to be entirely accurate, and the references provided do not always seem to provide sufficient basis for the claims made. For example, three references are cited to support the statement that "[A] Great part of trade timber harvest around the world from natural forest has illegal origin", but all three are papers similar to the present work and not directly about the great part of trade timber harvest and its legality. Another example is reference made to "... Australia (prohibition bill)..." which I can only assume is meant to refer the reader to the Australian Illegal Logging Prohibition Act 2012. Note that in this context the word Bill usually applies to a piece of legislation during its progress through a parliament and the word Act to pieces of legislation once passed. This part of the introduction needs to be carefully checked and possibly reduced.
Answer: This part of the introduction has been corrected and reduced.
- The analyses combining cytoplasmic (plastid and mitochondrial markers) and nuclear markers is not sufficiently justified since it seems markers from (presumeably) haploid and diploid genomes are being analysed together as if all were diploid. As a result the reader is presented with heterozygosity measures for haploid markers - a situation which makes very little sense.
Answer: Thank you for bringing attention to He estimation for plastid and mitochondrial markers. It really doesn't make sense to make such an estimate. However, the combination of nuclear, plastid and mitochondrial loci is possible for Fst estimates and sel-assignenmet tests, as they are estimates based on allele frequency and not on genotypic frequency. Furthermore, cytoplasmic and mitochondrial markers show greater genetic differentiation between populations and therefore help in determining the origins of populations. Therefore, we exclude He estimates for haplotypes.
- Unless I missed it, no evidence is presented that the markers used in this study can actually be assayed from logged or otherwise processed timber since leaf and cambium tissue preserved on silica gel was used. I think this issue needs to be addressed. In addition, the paper could be improved by addressing these less critical points.
Answer: Has been implemented. I agree that it is important to clarify that we used DNA samples obtained from population trees as examples of samples that could have been collected from wood along the chain of custody of commercialized wood (for example, where wood is stored in timber companies, sawn wood, instruments musicals, furniture, etc). Thus, the following sentence has been added to M&M, Assignment test. „Although no wood DNA samples were collected, tests to determine individuals for their populations or country of origin can represent a simulation of DNA collected from wood stored in timber companies, sawn wood, instruments musicals, furniture, etc. The same procedure has been used in other studies fort he same purpose [1-4].“
Communication around "group and individual self-assignment" and exactly what the numbers mean is unclear. This might be clear to other readers but certainly isn't to me.
Answer: Has been clarified in 2.5. Genetic Assignenment... used to assign group (grouping of individual from each population)…
Answer: Has been clarified in 2.5. Genetic Assignenment (Here the samples of individuals from the original populations (reference data).... The reference data means samples of individuals from the original populations.
The figures in the review version are not legible, both when printed and viewed electronically.
Answer: Has been implemented.
I am surely not unique in being unfamiliar with the MassARRAY® iPLEXTM technology. Some brief description of it and its advantages over other assay methods (for example, amplicon sequencing) in the context of timber tracking would be helpful. I would be interested in things like rates of genotyping error which would would be important in a legal/regulatory context.
Answer: Genotyping error has been implemented in Table 2 and a new Table S1 (for individual loci).
Although there is no doubt a good association between genetic and geographic distance, there are some pairwise comparisons between geographically distant populations with very low Fst values. Some comment on this would seem appropriate.
Answer: These Fst values in Figure 3A lower than 0.2, between pairs of population samples located at distances ranging from 1,500 to 2,000 km involved pairs of Brazilian population samples with fewer than 15 sampled individuals (i.e. 7BW-Ja, 8BW-Jr, 10BW-Cu, 12BE-Av, 14BE-Ar, , 15BE-Te, and 31PS-Ca). So, wem ade an new figure including only populations with sampel saize > 16 individuals.
Comments on the Quality of English Language
Grammar and spelling are poor throughout, and in places makes the writing difficult to interpret. Other English language conventions are not followed, for example in the use of commas and decimal points in numbers.
Answer: Has been implemented. The manuscript was corrected by one of the authors fluent in English grammar.
Answer: Thank you by your constructive criticism.
Reviewer 2 Report
Comments and Suggestions for AuthorsTimber tracking of Jacaranda copaia from Amazon Forest using DNA fingerprint
Comments and suggestions
The DNA fingerprinting markers developed for the Neotropical tree, Jacaranda copaia are crucial for combating illegal logging. However, the manuscript requires significant improvements in writing quality and language proficiency. Additionally, numerous spelling errors need to be corrected. Please revise the manuscript to enhance its clarity and accuracy. In addition, specific points need attention (see below).
Abstract
P1: Please revise the abstract to improve its clarity.
P1, L26: Power refers to “Power of discrimination”?
P1, L27: Please provide the full term of CpSNP, MtSNP, CpMtSNP, etc., rather than the abbreviation.
P1, L29: Correct “specialy” with especially; “isolation by distance pathern” with pattern.
P1, L30: Improve sentence: e.g. … we were able to determine the country and population origin of all samples with 100% accuracy.
Introduction
P1, L47: Correct “many international lows”, with laws; “de” with the
P2, L49: Spell in full: United States (Lacey Act), European Union (The EU Timber Regulation), Australia (Illegal Logging Prohibition Act)
P2, L57-71: Improve and shorten the write-up for laws and policies used in Brazil.
P3, L111: Improve the sentence as “Reproduction is primarily mediated by outcrossing, with rates ranging from 0.942 to 0.993”.
P3, L120: Rewrite: “geographic location of origin of wood of the species” to geographic origin of wood
Materials and Methods
P4, L124: 2.1 Sampling: The sentence is incomplete: “Were collected cambium…”
P4, L147: The sentence is repeated twice in the same paragraph. “After sampling, all collected plant material was stored and dried in silica gel”.
P4, L128-149: Improve the write-up for samples collected from various countries, the paragraph is too lengthy. Correct the names: “Institut National de la Recherche.Agronomique INRA”
P6: Correct “French Guyana” with French Guiana
Fig. 1 & Fig. 3: The resolution of the maps should be improved.
Table 1: Improve the table’s layout. When the sample size (n) appeared in 2 columns, it seemed confusing to the authors.
P6: L163-167: Include the detailed method for MassARRAY® iPLEXTM genotyping. Please clarify the meaning of “all selected for genetic tracking analysis due to show a minimum amplification rate of 95%”? Spell in full: insertions/deletions (InDels)
P6, L165: Correct the typo error for the “cloroplastidial”. Please confirm the usage of chloroplastidial or chloroplast?
P6, L180: To improve clarification, please mention in the text that nuclear, chloroplast, and mitochondrial SNP markers will hereafter be referred to as nCpMtSNP markers.
P7, L197-199: Correct SNPs “makers” with markers; change 10.000 to 10,000
Results
P8, L251: Table 2: polymorphic; To improve clarity, prepare P%(128) in a separate column under the individual heading of nCpMtSNPs.
P12, L315: The STRUCTURE analysis based on nSSR? Ensure consistency of the markers used in the study.
P12, L331-332: Please clarify which result this sentence refers to: “The assignment test for individuals for was able to self-assignment among 56.4–98.6% (mean rate of 98.7%)?
Table 4: Please clarify the meaning of the term "Rate Total."
Discussion
The discussion section requires enhancement and there is room for a more comprehensive exploration of results.
P14, L363-364: Correct the typo error for the “population origing”, with origin; “chain of custodia”, with custody
P14, L368: Please include a citation for genetic diversity reported for other Neotropical trees, as a comparison.
P14, L372: Discuss more the observation of low genetic diversity of J. copaia in some North Peru populations.
P14, L391: There are inconsistencies between the abbreviation used, e.g. SNPs, nSNPs, and CpMtSNps.
Please include the discussion for 𝐹𝑠𝑡 among all populations and countries was higher for CpMtSNPs than for all nCpMtSNPs (0.484 and 0.295, respectively) and nSNPs (0.415 and 0.233, respectively).
P15, L396: “Within countries for all SNPs markers, 𝐹𝑠𝑡 was highest among the Brazilian and Peru population. These results indicate strong potential for assigning timber origins between populations and countries”. Referring to this paragraph, please include the percentage from the self-assignment test as support.
Italicize the scientific names throughout the manuscript.
Comments on the Quality of English Language
The manuscript requires significant improvements in writing quality and language proficiency. Additionally, numerous spelling errors need to be corrected. Please revise the manuscript to enhance its clarity and accuracy.
Author Response
Reviewer 2
Comments and suggestions
The DNA fingerprinting markers developed for the Neotropical tree, Jacaranda copaia are crucial for combating illegal logging. However, the manuscript requires significant improvements in writing quality and language proficiency. Additionally, numerous spelling errors need to be corrected. Please revise the manuscript to enhance its clarity and accuracy. In addition, specific points need attention (see below).
Answer: Thank you by your constructive criticism.
Abstract
P1: Please revise the abstract to improve its clarity.
Answer: Has been implemented.
P1, L26: Power refers to “Power of discrimination”?
Answer: Yes, has been corrected.
P1, L27: Please provide the full term of CpSNP, MtSNP, CpMtSNP, etc., rather than the abbreviation.
Answer: Has been implemented.
P1, L29: Correct “specialy” with especially; “isolation by distance pathern” with pattern.
Answer: Has been corrected.
P1, L30: Improve sentence: e.g. … we were able to determine the country and population origin of all samples with 100% accuracy.
Answer: Has been corrected.
Introduction
P1, L47: Correct “many international lows”, with laws; “de” with the
Answer: Has been corrected.
P2, L49: Spell in full: United States (Lacey Act), European Union (The EU Timber Regulation), Australia (Illegal Logging Prohibition Act)
Answer: Has been corrected.
P2, L57-71: Improve and shorten the write-up for laws and policies used in Brazil.
Answer: Has been implemented.
P3, L111: Improve the sentence as “Reproduction is primarily mediated by outcrossing, with rates ranging from 0.942 to 0.993”.
Answer: Has been implemented.
P3, L120: Rewrite: “geographic location of origin of wood of the species” to geographic origin of wood
Answer: Has been implemented.
Materials and Methods
P4, L124: 2.1 Sampling: The sentence is incomplete: “Were collected cambium…”
Answer: Has been corrected.
P4, L147: The sentence is repeated twice in the same paragraph. “After sampling, all collected plant material was stored and dried in silica gel”.
Answer: Has been corrected.
P4, L128-149: Improve the write-up for samples collected from various countries, the paragraph is too lengthy. Correct the names: “Institut National de la Recherche.Agronomique INRA”
Answer: Has been implemented.
P6: Correct “French Guyana” with French Guiana
Answer: Has been implemented.
Fig. 1 & Fig. 3: The resolution of the maps should be improved.
Answer: Has been implemented.
Table 1: Improve the table’s layout. When the sample size (n) appeared in 2 columns, it seemed confusing to the authors.
Answer: Has been implemented.
P6: L163-167: Include the detailed method for MassARRAY® iPLEXTM genotyping. Please clarify the meaning of “all selected for genetic tracking analysis due to show a minimum amplification rate of 95%”? Spell in full: insertions/deletions (InDels)
Answer: Has been implemented.
P6, L165: Correct the typo error for the “cloroplastidial”. Please confirm the usage of chloroplastidial or chloroplast?
Answer: We chouse to use chloroplast.
P6, L180: To improve clarification, please mention in the text that nuclear, chloroplast, and mitochondrial SNP markers will hereafter be referred to as nCpMtSNP markers.
Answer: Has been implemented.
P7, L197-199: Correct SNPs “makers” with markers; change 10.000 to 10,000
Answer: Has been corrected.
Results
P8, L251: Table 2: polymorphic; To improve clarity, prepare P%(128) in a separate column under the individual heading of nCpMtSNPs.
Answer: Has been implemented.
P12, L315: The STRUCTURE analysis based on nSSR? Ensure consistency of the markers used in the study.
Answer: No, for all nCpMtSNP markers.
P12, L331-332: Please clarify which result this sentence refers to: “The assignment test for individuals for was able to self-assignment among 56.4–98.6% (mean rate of 98.7%)?
Answer: Has been corrected.
Table 4: Please clarify the meaning of the term "Rate Total."
Answer: Has been corrected. The total rate is the rate of individuals assigned to the correct source population, regardless of the score value; Rates > 80 and > 95% are the rates of individuals assigned to the correct source population, with a score value ≥ than 80 and 95%.
Discussion
The discussion section requires enhancement and there is room for a more comprehensive exploration of results.
Answer: It has been implemented. The results were better discussed, following the suggestions of both reviewers.
P14, L363-364: Correct the typo error for the “population origing”, with origin; “chain of custodia”, with custody
Answer: Has been corrected.
P14, L368: Please include a citation for genetic diversity reported for other Neotropical trees, as a comparison.
Answer: has been implemented.
P14, L372: Discuss more the observation of low genetic diversity of J. copaia in some North Peru populations.
Answer: Has been implemented.
P14, L391: There are inconsistencies between the abbreviation used, e.g. SNPs, nSNPs, and CpMtSNps.
Answer: Has been corrected.
Please include the discussion for ??? among all populations and countries was higher for CpMtSNPs than for all nCpMtSNPs (0.484 and 0.295, respectively) and nSNPs (0.415 and 0.233, respectively).
Answer: |Has been implemented.
P15, L396: “Within countries for all SNPs markers, ??? was highest among the Brazilian and Peru population. These results indicate strong potential for assigning timber origins between populations and countries”. Referring to this paragraph, please include the percentage from the self-assignment test as support.
Answer: Has been implemented.
Italicize the scientific names throughout the manuscript.
Answer: Has been correted.
Answer: Thank you by your constructive criticism.
Round 2
Reviewer 1 Report
Comments and Suggestions for AuthorsThis manuscript is much improved.
Some remaining issues mostly with language.
Line 58. Insert 'it' after 'declare'
Line 86. Delete 'been'.
Line 106. Replace 'individuals' with 'individual'.
Line 113. Replace 'was' with 'were'.
Line 121. Replace 'collected in' with 'collected at'.
Line 226. Replace 'the estimated' with 'estimating'.
Line 343. Replace 'number the' with 'number of'.
Line 540. Population is miss-spelt.
The resolution of the figures is still not adequate. It might be good to lay them out landscape to increase the sized. The green shades used in fig. 3B and 3C are very similar to my eyes, this could be improved.
Comments on the Quality of English Language
see above
Author Response
Dear Reviewer,
All your corrections were accepted.
I would also like to thank you for the previous suggestions and corrections, which were fundamental to improving the quality of the manuscript.
Best regards,
Alexandre
This manuscript is much improved.
Some remaining issues mostly with language.
Line 58. Insert 'it' after 'declare'
Answer: Has been implemented.
Line 86. Delete 'been'.
Answer: Has been corrected.
Line 106. Replace 'individuals' with 'individual'.
Answer: It was corrected to ….one tree per hectare….
Line 113. Replace 'was' with 'were'.
Answer: Has been corrected.
Line 121. Replace 'collected in' with 'collected at'.
Answer: Has been corrected.
Line 226. Replace 'the estimated' with 'estimating'.
Answer: Has been corrected.
Line 343. Replace 'number the' with 'number of'.
Answer: Has been corrected.
Line 540. Population is miss-spelt.
Answer: Has been corrected.
The resolution of the figures is still not adequate. It might be good to lay them out landscape to increase the sized. The green shades used in fig. 3B and 3C are very similar to my eyes, this could be improved.
Answer: Has been implemented.
