Genetic Diversity Evaluation and Population Structure Analysis of Red Swamp Crayfish (Procambarus clarkii) from Lakes and Rice Fields by SSR Markers
Anne Schrimpf
Round 1
Reviewer 1 Report
Abstract: Crayfish is not a “shrimp”
“Genetic diversity was slightly higher in the cultured populations that” replace “that” by “then”
21-22: “Genetic differentiation revealed a slight degree of genetic differentiation” rephrase
24: “(PCoA) ” no abbreviations in the Abstract.
26-27: It soulds like you would discuss a management strategy in this manuscript, but this is not discussed.
49-50: I don’t agree with the definition of genetic diversity.
67-68: SSR cannot be detected by PCR, PCR only amplifies a DNA fragment. Rephrase.
87-88: This is true, but the discussion about conservation and improved breeding of the studied populations is missing in the manuscript.
General comment for all headlines: The headlines are very short and do not fully explain what can be seen in the tables.
103: replace “Type” by “code”. Later you mention the name of the city of the populations. This is confusing because the reader do not know which city belongs to which abbreviation. Please add these names to table 1.
114: what is “appropriate ddH2O”?
115-117: no space between number and °C 94 °C should be 94°C
131-132: Where is the result of this part (absent, present)?
148: Usually the burn-in is smaller then the number of MCMC.
154: For uniformity, also add the number of mono-nucleotide repeats.
Table 4: explain the abbreviations in the headline of the table. Delete the .00 in the second column. The 2 “Mean” are confusing in the table. Explain this in the headline.
185 and following: which values are significant? You could make these values bold.
212: In the AMOVA I would chose “Within populations”, “Among populations” and “Among groups of Populations” (wild – hatchery). Within individual is not common.
Table 7: more information about the abbreviation in the headline. The F-statistics in the middle of the table below d.f. are confusing.
Figure 3: What do the circles indicate. Explain.
227-229: Repetition from the method section. Delete here.
Figure 4b: it would be important to sort the graph by the populations and not by the structure-groups to see which populations share which genetic structure-groups.
Figure 4c can be deleted, or it needs to be sorted by populations as well.
Paragraph 3.4: The diversity values were presented at the beginning of the results section. This part should be moved to the other diversity values.
245-246: Here I read the first time about the 3 generations. So this is the same hatchery in 3 different years. This should be explained in the method section. It is also very important for the genetic distance values. This must be discussed.
252: What does “mixed state” means?
280 and 303: What about this previous study? You write about it as if each reader should know about it. Please explain this study and why it is important for your manuscript.
General comment at the end: P. clarkii is an invasive species in China. Therefore the wild populations also experiences a founder effect and can not only be explained be “wild population. Since when is the species in China? So how old are the wild populations roughly?
Author Response
Response to Reviewer 1 Comments
Point 1: Abstract: Crayfish is not a “shrimp”
Response 1: Thank you for the suggestion. We have made this modification in line 16.
Point 2: “Genetic diversity was slightly higher in the cultured populations that” replace “that” by “than”
Response 2: Thank you for the suggestion. We have made this modification in line 20.
Point 3: 21-22: “Genetic differentiation revealed a slight degree of genetic differentiation” rephrase
Response 3: Thank you for the suggestion. We have rewritten this sentence in line 21.
Point 4: 24: “(PCoA) ” no abbreviations in the Abstract.
Response 4: Thank you for the suggestion. We have made this modification.
Point 5: 26-27: It soulds like you would discuss a management strategy in this manuscript, but this is not discussed.
Response 5: Thank you for the suggestion. We have added the description in line 314-316.
Point 6: 49-50: I don’t agree with the definition of genetic diversity.
Response 6: Thank you for the suggestion. We have made modifications in line 52-53.
Point 7: 67-68: SSR cannot be detected by PCR, PCR only amplifies a DNA fragment. Rephrase.
Response 7: Thank you for the suggestion. We have rewritten the sentence in line 71-72.
Point 8: 87-88: This is true, but the discussion about conservation and improved breeding of the studied populations is missing in the manuscript.
Response 8: Thank you for the suggestion. We have added the description in line 346-348.
Point 9: General comment for all headlines: The headlines are very short and do not fully explain what can be seen in the tables.
Response 9: Thank you for the suggestion. We have added the details.
Point 10: 103: replace “Type” by “code”. Later you mention the name of the city of the populations. This is confusing because the reader do not know which city belongs to which abbreviation. Please add these names to table 1.
Response 10: Thank you for the suggestion. We have made modifications.
Point 11: 114: what is “appropriate ddH2O”?
Response 11: Thank you for the suggestion. We have made this modification.
Point 12: 115-117: no space between number and °C 94 °C should be 94°C
Response 12: Thank you for the suggestion. We have made this modification.
Point 13: 131-132: Where is the result of this part (absent, present)?
Response 13: Thank you for the suggestion. We have added the result in Table S1.
Point 14: 148: Usually the burn-in is smaller then the number of MCMC.
Response 14: Thank you for the suggestion. There is a writing error here, we have made this modification.
Point 15: 154: For uniformity, also add the number of mono-nucleotide repeats.
Response 15: Thank you for the suggestion. We have added the description in line 162-163.
Point 16: Table 4: explain the abbreviations in the headline of the table. Delete the .00 in the second column. The 2 “Mean” are confusing in the table. Explain this in the headline.
Response 16: Thank you for the suggestion. We have made modification.
Point 17: 185 and following: which values are significant? You could make these values bold.
Response 17: Thank you for the suggestion. We have made modification in line 210-211.
Point 18: 212: In the AMOVA I would chose “Within populations”, “Among populations” and “Among groups of Populations” (wild – hatchery). Within individual is not common.
Response 18: Thank you for the suggestion. Many articles are chosen for these three indexes.
Point 19: Table 7: more information about the abbreviation in the headline. The F-statistics in the middle of the table below d.f. are confusing.
Response 19: Thank you for the suggestion. We have made modifications.
Point 20: Figure 3: What do the circles indicate. Explain.
Response 20: Thank you for the suggestion. We have added explanations in line 270-274.
Point 21: 227-229: Repetition from the method section. Delete here.
Response 21: Thank you for the suggestion. We have made the modifications.
Point 22: Figure 4b: it would be important to sort the graph by the populations and not by the structure-groups to see which populations share which genetic structure-groups.
Response 22: Thank you for the suggestion. Because of the low degree of genetic differentiation between populations, these individuals are very dispersed and not clustered together by population.
Point 23: Figure 4c can be deleted, or it needs to be sorted by populations as well.
Response 23: Thank you for the suggestion. We have deleted the Figure 4c.
Point 24: Paragraph 3.4: The diversity values were presented at the beginning of the results section. This part should be moved to the other diversity values.
Response 24: Thank you for the suggestion. We have made modifications and this part is moved to 3.3.
Point 25: 245-246: Here I read the first time about the 3 generations. So this is the same hatchery in 3 different years. This should be explained in the method section. It is also very important for the genetic distance values. This must be discussed.
Response 25: Thank you for the suggestion. We have added the description in line 99-100 and 231-233.
Point 26: 252: What does “mixed state” means?
Response 26: Thank you for the suggestion. We have added the description in line 214-216.
Point 27: 280 and 303: What about this previous study? You write about it as if each reader should know about it. Please explain this study and why it is important for your manuscript.
Response 27: Thank you for the suggestion. We have added references in line 306-307.
Point 28: General comment at the end: P. clarkii is an invasive species in China. Therefore the wild populations also experiences a founder effect and can not only be explained be “wild population. Since when is the species in China? So how old are the wild populations roughly?
Response 28: Thank you for the suggestion. We consider the red swamp crayfish populations collected from wild lakes as wild populations. The crayfish were introduced to Nanjing, China in 1929, about 90 years ago.
Author Response File: 
Author Response.docx
Reviewer 2 Report
The paper presents a nice population genetic study using microsatellites, comparing population of the red swamp crayfish in cultured and wild populations. The study is well performed, only some minor errors are evidenced in the attached revised pdf.
My main concern is not with the study itself, but with the introduction and conclusion of the paper. Reading it, it seems that the only purpose of Chinese scientists is to improve genetic diversity of this species given its high importance for aquaculture and protect wild populations. However, this is in contrast with a wider ecological and conservation biology point of view. I suggest the author to read and cite in the introduction an important paper published in Scientific Reports: Yi, S., Li, Y., Shi, L. et al. Characterization of Population Genetic Structure of red swamp crayfish, Procambarus clarkii, in China. Sci Rep 8, 5586 (2018). https://doi.org/10.1038/s41598-018-23986-z
Apart the interest of this paper as a background for the analysis reported in the present manuscript, it includes important inputs. I worked a lot to eradicate wild populations of this crayfish in Europe, and in the cited paper it is clearly written that: "The red swamp crayfish (Procambarus clarkii) is one of the most economically important farmed aquatic species in China. However, it is also a famous invasive species in the world. This invasive species was dispersed most via human activities including intentional or unintentional carry in China".
Well, the interest in this paper towards protection of populations of this crayfish in the wild is against common sense and a serious management policy of natural ecosystem. I understand that the paper is focused on aquaculture, but I strongly suggest authors to report the negative consequences of the presence of this species outside breeding sites, its invasiveness and the damage to natural populations, especially fish and most macroinvertebrates. It is not needed they rewrite the paper, but they should be aware of this, state this in the introduction, and 'mitigate' in some way the protection purpose of wild populations which is a serious threat to freshwater ecosystems.
Comments for author File: 
Comments.pdf
Author Response
Response to Reviewer 2 Comments
The paper presents a nice population genetic study using microsatellites, comparing population of the red swamp crayfish in cultured and wild populations. The study is well performed, only some minor errors are evidenced in the attached revised pdf.
My main concern is not with the study itself, but with the introduction and conclusion of the paper. Reading it, it seems that the only purpose of Chinese scientists is to improve genetic diversity of this species given its high importance for aquaculture and protect wild populations. However, this is in contrast with a wider ecological and conservation biology point of view. I suggest the author to read and cite in the introduction an important paper published in Scientific Reports: Yi, S., Li, Y., Shi, L. et al. Characterization of Population Genetic Structure of red swamp crayfish, Procambarus clarkii, in China. Sci Rep 8, 5586 (2018). https://doi.org/10.1038/s41598-018-23986-z
Apart the interest of this paper as a background for the analysis reported in the present manuscript, it includes important inputs. I worked a lot to eradicate wild populations of this crayfish in Europe, and in the cited paper it is clearly written that: "The red swamp crayfish (Procambarus clarkii) is one of the most economically important farmed aquatic species in China. However, it is also a famous invasive species in the world. This invasive species was dispersed most via human activities including intentional or unintentional carry in China".
Well, the interest in this paper towards protection of populations of this crayfish in the wild is against common sense and a serious management policy of natural ecosystem. I understand that the paper is focused on aquaculture, but I strongly suggest authors to report the negative consequences of the presence of this species outside breeding sites, its invasiveness and the damage to natural populations, especially fish and most macroinvertebrates. It is not needed they rewrite the paper, but they should be aware of this, state this in the introduction, and 'mitigate' in some way the protection purpose of wild populations which is a serious threat to freshwater ecosystems.
Point 1: 16: “Red” should be “The red”.
Response 1: Thank you for the suggestion. We have changed “Red” into “The red” in line 16.
Point 2: 33: “values” should be “value”
Response 2: Thank you for the suggestion. We have changed “values” into “value” in line 35.
Point 3: 33: “delicious” This is a matter of taste; better to say 'edible' or similar
Response 3: Thank you for the suggestion. We have changed “delicious” into “edible” in line 35.
Point 4: 34: “nutrition” should be “nutritional power”.
Response 4: Thank you for the suggestion. We have changed “nutrition” into “nutritional power” in line 36.
Point 5: 34: “red” should be “the red”.
Response 5: Thank you for the suggestion. We have made modifications.
Point 6: 39: “in” should be “of”.
Response 6: Thank you for the suggestion. We have changed “in” into “of” in line 42.
Point 7: 50: “validated” should be “supposed”.
Response 7: Thank you for the suggestion. We have changed “validated” into “supposed” in line 53.
Point 8: 51: “for” should be “, because”.
Response 8: Thank you for the suggestion. We have changed “for” into “, because” in line 54.
Point 9: 115-118: please delete the space between numerals and °C - and in this way you avoid carriage return in the middle of the datum.
Response 9: Thank you for the suggestion. We have made modification.
Point 10: 127-129: same as above.
Response 10: Thank you for the suggestion. We have made modifications.
Point 11: 134: “following” should be “the following”.
Response 11: Thank you for the suggestion. We have changed “following” into “the following” in line 142.
Point 12: 140: Coordinates is most widely used, but just a suggestion.
Response 12: Thank you for the suggestion. We have changed “Co-ordinates” into “Coordinates” in line 148.
Point 13: 147: “twenty” should be “20”.
Response 13: Thank you for the suggestion. We have changed “twenty” into “20” in line 155.
Point 14: 153: “tetra” should be “tetra-“.
Response 14: Thank you for the suggestion. We have changed “tetra” into “tetra-“ in line 161.
Point 15: 213: Do you mean the first two axes of PCoA, I suppose.
Response 15: Thank you for the suggestion. We have made modification in line 258-259.
Point 16: 214: These values are very small and indicate after all a failure, from a statistical point of view, of the analysis to represent well your dataset. This is of course due to the high within individuals variability encountered in your study. May you spend a sentence to remark this point?
Response 16: Thank you for the suggestion. We have added explanations in line 260.
Point 17: 280: “declining” should be “decline”.
Response 17: Thank you for the suggestion. We have changed “declining” into “decline” line 308.
Point 18: 305: “the” should be “of the”.
Response 18: Thank you for the suggestion. We have changed “the” into “of the” in line 355.
Point 19: 334: Why do you have to protect wild genetic resources of this alien species???
Response 19: Thank you for the suggestion. We have added references about the hazards of the crayfish as an invasive species in line 33-40 in the introduction, but the benefits of the crayfish in China currently outweigh the drawbacks and it is very popular aquatic animals. Our study of genetic diversity in wild populations can provide germplasm resources for cultured populations and better parents for captive breeding and genetic breeding.
Author Response File: Author Response.docx
