High-Density Genetic Linkage Map of the Southern Blue-ringed Octopus (Octopodidae: Hapalochlaena maculosa)
, Peter Morse 1, Jan M. Strugnell 1,2, Ira R. Cooke 3,4 and Kyall Zenger 1
Michael Kuba
Round 1
Reviewer 1 Report
Summary:In the present work, Whitelaw et al., provide a high-density linkage map for the southern blue-ringed octopus Hapalochlaena maculosa, which is the first for any cephalopod species. According to the linkage map, the markers (including 2166 SNPs and 2,455 presence/absence variants) were distributed into 47 linkage groups. This information was used to improve a highly-fragmented genome published previously for this octopus species. First, scaffold orientation and re-scaffolding were performed based on the linkage maps, then scaffolds were arranged into pseudo-chromosomes based on the 47 linkage groups. Using this data, authors were able to identify and locate six essential developmental genes of the HOX family, showing a cluster of three HOX genes in the same scaffold. The high-density linkage map obtained in this study is presented as a promising tool for the development of quantitative trait loci (QTL) in related species with aquaculture potential; besides, the authors suggested its use for improving other fragmented genomes from different octopus species.
General comments:
My general concern is that the manuscript should be reoriented to be in line with the aims and scope of the MDPI Diversity journal. For instance, in the introduction, authors are encouraged to make more emphasis on how their work contributes to preserving the genetic diversity of the populations, to understand the evolution of octopus genomes, or how it impacts fields like conservation genomics or evo-devo studies. Moreover, according to the journal’s aim “to encourage scientists to publish their experimental and theoretical results in as much detail as possible” and that “Full experimental details must be provided so that the results can be reproduced”, the methods section requires further details that will be specified in the paragraphs below. Although the results contribute greatly to the field of cephalopod research, I consider that the manuscript is not acceptable for publication in its current form; however, it could be publishable after revision.
Specific comments:
Abstract
Lines 14-15: There is no relationship between the first sentence and the rest of the abstract. Please remove the first sentence.
Introduction
Lines 30-41: This paragraph is out of the context of the whole manuscript; it seems unnecessary. After reading the paragraph, I was expecting to find some analysis focused on genes encoding neurotoxins but that was not the case, so I recommend removing this paragraph. Instead of such a paragraph, you may write another mentioning the importance of developing this kind of molecular tools for fields like population diversity, conservation genomics, evolutionary genomics, and cephalopod research.
Materials and Methods
All the protocols must be approved by an Ethics or Bioethics committee from a scientific or academic institution. Protocols must accomplish the guidelines and good practices for the welfare and care of octopuses in experimental procedures (see Fiorito et al., 2015; Winlow et al., 2018).
Lines 108-124: According to the journal guidelines “Full experimental details must be provided so that the results can be reproduced”. Please provide details of the experimental design including the number of females and males captured and used in each group during the experimental procedure. Please mention and cite the protocols used for DNA purification, quality check, restriction enzyme digestion, library preparation (specifying the library type, fragment size and expected read size), sequencing strategy and technology, and quality control of the sequencing data. If authors performed any of these processes, please provide the names of all instruments and reagents utilized.
Line 150: Since both female and male octopuses were obtained from the wild (nobody knows if they mated previously there), and both sexes are known to be polygamous, I highly recommend running Colony again, activating the polygamy option for both males and females.
Results
Line 232: the term “sex averaged” should be sex-averaged? On the other hand, the number of males and females used to obtain the average was not specified. This is important since in the experimental design, apparently (not specified), you used 24 males and 12 females. If you used these numbers of octopuses to calculate the sex average, the result could be biased since the number of males and females is unequal.
Line 236: Please use italics for all the scientific names.
Line 254: Table 1. The same comment of line 232 regarding a possible bias in the sex average calculation.
Line 281: Table 3. Please, write numbers in a consistent format. In the “number of Scaffolds” row, remove decimals and ad commas to separate large numbers as in the “N count” and “Gaps” rows.
Line 301: Figure 2. Try to represent the scaffolds of the three different genomes at the same scale, you may transform scaffold lengths into an appropriate logarithmic scale to improve this representation. It is confusing in its current form.
For all the Tables (including all the Supplementary Tables): Make sure that all abbreviations used for variables like LOD, Ge1, Ge2, Rec, etc., are fully described at the bottom of the table (table description).
Discussion
Line 351: “carrying eggs within their web” should be “within their mantle cavity”? or what do you mean by “web”?
Line 405: Since the construction of the pseudo-chromosomes integrated only one-third of the total genome, I feel that the sentence “the genome was reassembled” is not appropriate. I suggest changing it to “the genome assembly was reorganized into 47 pseudo-chromosomes”.
References suggested
Fiorito G, Affuso A, Basil J, Cole A, de Girolamo P, D’angelo L, Dickel L, Gestal C, Grasso F, Kuba M, Mark F, Melillo D, Osorio D, Perkins K, Ponte G, Shashar N, Smith D, Smith J, Andrews P lr. 2015. Guidelines for the Care and Welfare of Cephalopods in Research –A consensus based on an initiative by CephRes, FELASA and the Boyd Group. Laboratory Animals 49:1–90. DOI: 10.1177/0023677215580006.
Winlow W, Polese G, Moghadam H-F, Ahmed IA, Di Cosmo A. 2018. Sense and Insensibility - An Appraisal of the Effects of Clinical Anesthetics on Gastropod and Cephalopod Molluscs as a Step to Improved Welfare of Cephalopods. Frontiers in physiology 9:1147. DOI: 10.3389/fphys.2018.01147.
Author Response
Reviewer 1
Summary:
In the present work, Whitelaw et al., provide a high-density linkage map for the southern blue-ringed octopus Hapalochlaena maculosa, which is the first for any cephalopod species. According to the linkage map, the markers (including 2166 SNPs and 2,455 presence/absence variants) were distributed into 47 linkage groups. This information was used to improve a highly-fragmented genome published previously for this octopus species. First, scaffold orientation and re-scaffolding were performed based on the linkage maps, then scaffolds were arranged into pseudo-chromosomes based on the 47 linkage groups. Using this data, authors were able to identify and locate six essential developmental genes of the HOX family, showing a cluster of three HOX genes in the same scaffold. The high-density linkage map obtained in this study is presented as a promising tool for the development of quantitative trait loci (QTL) in related species with aquaculture potential; besides, the authors suggested its use for improving other fragmented genomes from different octopus species.
General comments:
My general concern is that the manuscript should be reoriented to be in line with the aims and scope of the MDPI Diversity journal. For instance, in the introduction, authors are encouraged to make more emphasis on how their work contributes to preserving the genetic diversity of the populations, to understand the evolution of octopus genomes, or how it impacts fields like conservation genomics or evo-devo studies. Moreover, according to the journal’s aim “to encourage scientists to publish their experimental and theoretical results in as much detail as possible” and that “Full experimental details must be provided so that the results can be reproduced”, the methods section requires further details that will be specified in the paragraphs below. Although the results contribute greatly to the field of cephalopod research, I consider that the manuscript is not acceptable for publication in its current form; however, it could be publishable after revision.
I would like to thank the reviewer for taking the time to provide feedback on this manuscript. All comments have been carefully taken into account and addressed.
Specific comments:
Abstract
Lines 14-15: There is no relationship between the first sentence and the rest of the abstract. Please remove the first sentence.
Response: We agree with the reviewer and the sentence has been removed. Line 14
Introduction
Lines 30-41: This paragraph is out of the context of the whole manuscript; it seems unnecessary. After reading the paragraph, I was expecting to find some analysis focused on genes encoding neurotoxins but that was not the case, so I recommend removing this paragraph. Instead of such a paragraph, you may write another mentioning the importance of developing this kind of molecular tools for fields like population diversity, conservation genomics, evolutionary genomics, and cephalopod research.
Response:
The paragraph mentioned serves to provide some context as to why this map was generated, in this case for a unique species (Southern blue-ringed octopus) within a genus which exhibits traits specific to the genus (aposematic colouration and sequestration of a potent neurotoxin). I understand the reviewers point that information focusing on the utility of the molecular tool generated should be included in the introduction. The lines 58-98 serve to address and justify the generation of a linkage map for cephalopods relating to utility in an evolutionary context.
Materials and Methods
All the protocols must be approved by an Ethics or Bioethics committee from a scientific or academic institution. Protocols must accomplish the guidelines and good practices for the welfare and care of octopuses in experimental procedures (see Fiorito et al., 2015; Winlow et al., 2018).
Response:
Details for housing and care are stated in the published manuscript “Mating behaviour and postcopulatory fertilization patterns in the southern blue-ringed octopus, Hapalochlaena maculosa” by Morse et al 2018 and are referenced in text. Line 112
I have included the appropriate excerpt here from Morse et al 2018 below for your reference:
“Animal Acquisition and Maintenance
False-shelter traps were used to source 12 female and 24 male wild H. maculosa from Cockburn Sound, Western Australia over September and October 2015 under Western Australia DPaW permit: SF010531 and Fisheries exemption: 26367. False-shelter traps consisted of 20 cm lengths of PVC pipe 20–25 mm in diameter with a cement plug in the centre. Cable ties were used to connect these traps along 20 m lengths of rope that were held down with cement blocks, and these trap lines were marked and checked weekly using GPS. It was ensured that all captured female animals weighed at least 5 g, as this was the minimum size at which females of this species consistently accept copulation attempts from males (Morse et al., 2015).
Animals were brought to the Fremantle Octopus facilities in O'Conner, WA, Australia where they were housed in individual 1-litre plastic containers connected to a closed flow-through system in a 1000-litre sump. Water parameters were continuously maintained at 22 °C and 34–35 ppt salinity. Each animal received an appropriately sized shell to use as a shelter, and fed ad libitum with sections of thawed frozen bait prawn. Animals were given a 14 h daylight cycle using ReefOne biOrb intelligent LED aquarium lights. As there was no means to ensure that females had not already mated in the wild, they were maintained in the laboratory for a minimum of 48 h prior to being introduced to candidate males. This was done to ensure that the time between laboratory pairings would have been at least as long as the time since the female might have mated with another male in the wild. All use and treatment of animals was approved by the James Cook University Animal Ethics Committee (Approval No. A1850). ”
Lines 108-124: According to the journal guidelines “Full experimental details must be provided so that the results can be reproduced”. Please provide details of the experimental design including the number of females and males captured and used in each group during the experimental procedure. Please mention and cite the protocols used for DNA purification, quality check, restriction enzyme digestion, library preparation (specifying the library type, fragment size and expected read size), sequencing strategy and technology, and quality control of the sequencing data. If authors performed any of these processes, please provide the names of all instruments and reagents utilized.
Response:
Details for the sequencing, library preparation and SNP calling have been added. Lines :120-130
“Muscle tissue samples were taken from the arm tip of adults and the body of embryos. DNA was extracted from samples using the CTAB protocol followed by purification with a Sephadex G-50 (GE Healthcare Life Sciences 2000). Quality and quantity of genomic DNA was assessed through visualization on a 0.8% agrose gel. SNP discovery was conduced by Diversity Arrays Technology PL, Canberra ACT, Australia using a restriction digest method, specifically enzymes Pstl and Hpall in conjunction with proprietary barcodes. Samples exhibiting uneven digestion patterns were excluded from library preparation. Pooled libraries were sequenced using a HiSeq2500 and resulting reads were filtered for quality (Q<25) [29]. SNPs were called on the subsequent data set using the KDcompute pipeline (DArT) [30]. “
Line 150: Since both female and male octopuses were obtained from the wild (nobody knows if they mated previously there), and both sexes are known to be polygamous, I highly recommend running Colony again, activating the polygamy option for both males and females.
Response:
Multiple paternity was assessed using colony and cervus which detected matings with unknown males in the wild prior to capture. Offspring produced from these matings were relatively few and were not included in the generation of the linkage map. Full details for the families generated in the study including the number of males to females, number of offspring per family and which families contained unknown males not captured in this study are found in the referenced supplementary table S1
Results
Line 232: the term “sex averaged” should be sex-averaged? On the other hand, the number of males and females used to obtain the average was not specified. This is important since in the experimental design, apparently (not specified), you used 24 males and 12 females. If you used these numbers of octopuses to calculate the sex average, the result could be biased since the number of males and females is unequal.
Response:
Full details for the families generated in the study including the number of males to females, number of offspring per family and which families were included are found in the referenced supplementary table S1. I would like to clarify that sex average maps are the result of informative meiosis events irrespective of whether the event is informative in the maternal or paternal parent. This means that the number of males to females does not introduce a bias. Additionally, the sex specific maps use informative events in all offspring regardless their gender. The number of informative meiosis changes depending on whether the male or female parent genotype for that SNP is informative or not. However, I agree that the number of females to males included should be mentioned in the text and this has been added.
L: 254
Line 236: Please use italics for all the scientific names.
Response:
We agree with the reviewer and the change has been implemented. Line 262
Line 254: Table 1. The same comment of line 232 regarding a possible bias in the sex average calculation.
Response:
As mentioned above the methods has been corrected to specify the number of females and males.
Line 254
Line 281: Table 3. Please, write numbers in a consistent format. In the “number of Scaffolds” row, remove decimals and ad commas to separate large numbers as in the “N count” and “Gaps” rows.
Response:
We agree with the reviewer and the change has been implemented.
Line 301: Figure 2. Try to represent the scaffolds of the three different genomes at the same scale, you may transform scaffold lengths into an appropriate logarithmic scale to improve this representation. It is confusing in its current form.
Response:
The figure aims to provide a context for the position of genes with relative sizes comparable using the text provided with each scaffold. I agree that a logarithmic scale would aid with clarity and this has been added
Figure 2. Comparison of HOX gene arrangement in the a) owl limpet (Lottia gigantea), b) California two spot octopus (Octopus bimaculoides) and the southern blue-ringed octopus (Hapalochlaena maculosa). Genes are coloured consistently between species. Scaffold lengths for b) follow a logarithmic scale.
For all the Tables (including all the Supplementary Tables): Make sure that all abbreviations used for variables like LOD, Ge1, Ge2, Rec, etc., are fully described at the bottom of the table (table description).
Response:
We agree with the reviewer and the requested changes have been implemented.
Manuscript: Lines 283 and 284. Supp materials: Tables 2,5,7 and 9
Discussion
Line 351: “carrying eggs within their web” should be “within their mantle cavity”? or what do you mean by “web”?
Response:
The web refers to the thin tissue joining each arm to the adjacent ones. H. maculosa are known to “cradle” their eggs within their web in order to carry and protect them. In response to another comment this line was removed to improve flow.
Line 405: Since the construction of the pseudo-chromosomes integrated only one-third of the total genome, I feel that the sentence “the genome was reassembled” is not appropriate. I suggest changing it to “the genome assembly was reorganized into 47 pseudo-chromosomes”.
Response:
We agree with the reviewer and the change has been implemented. Lines 463-465
References suggested
Fiorito G, Affuso A, Basil J, Cole A, de Girolamo P, D’angelo L, Dickel L, Gestal C, Grasso F, Kuba M, Mark F, Melillo D, Osorio D, Perkins K, Ponte G, Shashar N, Smith D, Smith J, Andrews P lr. 2015. Guidelines for the Care and Welfare of Cephalopods in Research –A consensus based on an initiative by CephRes, FELASA and the Boyd Group. Laboratory Animals 49:1–90. DOI: 10.1177/0023677215580006.
Winlow W, Polese G, Moghadam H-F, Ahmed IA, Di Cosmo A. 2018. Sense and Insensibility - An Appraisal of the Effects of Clinical Anesthetics on Gastropod and Cephalopod Molluscs as a Step to Improved Welfare of Cephalopods. Frontiers in physiology 9:1147. DOI: 10.3389/fphys.2018.01147.
Author Response File: 
Author Response.docx
Reviewer 2 Report
I enjoyed reading the manuscript and i think that it is a great next stepp to better understand cephalopods. As these animals might be potential model animals i would ask the authors to give more details about the keeping system. Was there any enrichment for the animals? What type of food was provided for the animals?
Thanks a lot
Author Response
Reviewer 2
Comments and Suggestions for Authors
I enjoyed reading the manuscript and i think that it is a great next step to better understand cephalopods. As these animals might be potential model animals i would ask the authors to give more details about the keeping system. Was there any enrichment for the animals? What type of food was provided for the animals?
I would like to thank the reviewer for taking the time to provide feedback on this manuscript. All comments have been carefully taken into account and addressed.
Response:
Details for housing and care are stated in the published manuscript “Mating behaviour and postcopulatory fertilization patterns in the southern blue-ringed octopus, Hapalochlaena maculosa” by Morse et al 2018 and are referenced in text at Line 112
I have included the appropriate excerpt from Morse et al 2018 here for your reference:
“Animal Acquisition and Maintenance
False-shelter traps were used to source 12 female and 24 male wild H. maculosa from Cockburn Sound, Western Australia over September and October 2015 under Western Australia DPaW permit: SF010531 and Fisheries exemption: 26367. False-shelter traps consisted of 20 cm lengths of PVC pipe 20–25 mm in diameter with a cement plug in the centre. Cable ties were used to connect these traps along 20 m lengths of rope that were held down with cement blocks, and these trap lines were marked and checked weekly using GPS. It was ensured that all captured female animals weighed at least 5 g, as this was the minimum size at which females of this species consistently accept copulation attempts from males (Morse et al., 2015).
Animals were brought to the Fremantle Octopus facilities in O'Conner, WA, Australia where they were housed in individual 1-litre plastic containers connected to a closed flow-through system in a 1000-litre sump. Water parameters were continuously maintained at 22 °C and 34–35 ppt salinity. Each animal received an appropriately sized shell to use as a shelter, and fed ad libitum with sections of thawed frozen bait prawn. Animals were given a 14 h daylight cycle using ReefOne biOrb intelligent LED aquarium lights. As there was no means to ensure that females had not already mated in the wild, they were maintained in the laboratory for a minimum of 48 h prior to being introduced to candidate males. This was done to ensure that the time between laboratory pairings would have been at least as long as the time since the female might have mated with another male in the wild. All use and treatment of animals was approved by the James Cook University Animal Ethics Committee (Approval No. A1850). ”
Author Response File: Author Response.docx
Reviewer 3 Report
The ms presents results from a fairly technical study to generate linkage maps from the blue ringed octopus. As such it appears to be well designed and conducted as a study, with suitable detail and analyses in the Methods and Results. The discussion present some interesting observations and suggestions for future studies. A solid and well presented study, worthy of publication after consideration of a few minor issues noted below.
Good Introduction - excellent, concise review of the background and uses of linkage maps, and justification of the study.
Methods section generally adequate, giving suitable levels of information and detail, but some details need addressing – see specific notes below.
Results section is well structured, detailed and informative, with suitable support from Figures and Tables. All Figures useful. Perhaps Table 3 could be moved to supplementary?
Discussion is appropriate for the results presented, containing some useful discussion of points for future consideration. Several sections could be shortened, as noted in detailed comments below, as they read like direct transpositions from a thesis – perhaps not so much detail, referencing and postulation needed given the amount of data/results presented? But overall well written and compelling.
Detailed comments:
l.65 - QTL = Quantitative trait loci?
l.100-103 - this ms obviously developed from a thesis chapter, but the references to chapters should be removed (and text edited appropriately). Likewise see lines 109-10 - this may have been appropriate for a thesis chapter, but not for a paper - just refer to previous publication.
l.118-123 - again, this is thesis language (especially as the people named are authors) - re-write and give a little more reference to methods used.
l.126 - what is SNP? Give abbreviations in full at first use (like you do for PAV on l.135).
l.145-149 - text isn't clear - rewrite, and give reference for programme Cervus
l.296 - 5 species, but lists 6 names?
l.327-8 - sentence not clear -edit
l.339 - 363 - these two paragraphs are a bit repetitive and could be condensed and combined - the issue is potential effect of small family sizes, which doesn't need dwelling on at this length as it's just a note for future improvement?
l.367-9 - clarity of second part of this sentence falls apart - needs editing.
l.385-391 - is this info on other cephalopods needed? The point made is about chromosome number - for this simple point, only the 1st sentence needed as reference?
Author Response
Reviewer 3
Comments and Suggestions for Authors
The ms presents results from a fairly technical study to generate linkage maps from the blue ringed octopus. As such it appears to be well designed and conducted as a study, with suitable detail and analyses in the Methods and Results. The discussion present some interesting observations and suggestions for future studies. A solid and well presented study, worthy of publication after consideration of a few minor issues noted below.
Good Introduction - excellent, concise review of the background and uses of linkage maps, and justification of the study.
Methods section generally adequate, giving suitable levels of information and detail, but some details need addressing – see specific notes below.
Results section is well structured, detailed and informative, with suitable support from Figures and Tables. All Figures useful. Perhaps Table 3 could be moved to supplementary?
Discussion is appropriate for the results presented, containing some useful discussion of points for future consideration. Several sections could be shortened, as noted in detailed comments below, as they read like direct transpositions from a thesis – perhaps not so much detail, referencing and postulation needed given the amount of data/results presented? But overall well written and compelling.
I would like to thank the reviewer for taking the time to provide feedback on this manuscript. All comments have been carefully taken into account and addressed.
Detailed comments:
l.65 - QTL = Quantitative trait loci?
Response:
We agree with the reviewer and the change has been implemented. Line 67
l.100-103 - this ms obviously developed from a thesis chapter, but the references to chapters should be removed (and text edited appropriately). Likewise see lines 109-10 - this may have been appropriate for a thesis chapter, but not for a paper - just refer to previous publication.
Response:
We agree with the reviewer and the text has been modified to make it more appropriate for a research paper publication. Lines 99-107
Original:
“Generation of a linkage map for cephalopods has not previously been attempted, in part due to the absence of genome assemblies and genetic marker resources prior to 2015 [8], but also due the difficulty in keeping and breeding cephalopods in captivity. This latter requirement is essential to track pedigree information and attain the large family sizes, which are required for robust linkage analysis [27]. This chapter presents the first linkage map of any cephalopod, the southern blue-ringed octopus (Hapalochlaena maculosa) constructed using a combination of single nucleotide polymorphism (SNP) and presence absence variant (PAV) markers. The assembly generated in chapter 2 is incorporated here with the linkage map to produce an improved assembly leading to insights in HOX gene placement within H. maculosa.”
Corrected
“Generation of a linkage map for cephalopods has not previously been attempted, in part due to the absence of genome assemblies and genetic marker resources prior to 2015 [8], but also due the difficulty in keeping and breeding cephalopods in captivity. This latter requirement is essential to track pedigree information and attain the large family sizes, which are required for robust linkage analysis [27]. This study presents the first linkage map of any cephalopod, the southern blue-ringed octopus (Hapalochlaena maculosa) constructed using a combination of single nucleotide polymorphism (SNP) and presence absence variant (PAV) markers.”
*l.118-123 - again, this is thesis language (especially as the people named are authors) - re-write and give a little more reference to methods used.
Response:
We agree with the reviewer and the text has been modified to make it more appropriate for a research paper publication. Lines 111-118
Corrected:
“Detailed descriptions of sample collection, mating structure and housing are available in Morse et al. 2018 . Briefly, specimens of H. maculosa were collected from Cockburn Sound, Western Australia using false shelter traps. Animals were individually housed at the Fremantle Octopus facility in 1 litre flow-through aquaria within a 1,000 L sump. Collected animals (36 in total) were divided into 12 groups with each female being paired with one of the two males sequentially. Animals were paired in a larger separate 30L aquarium overnight and interactions recorded. Resulting families' clutch sizes are located in table S1.”
l.126 - what is SNP? Give abbreviations in full at first use (like you do for PAV on l.135).
Response:
We agree with the reviewer and the change has been implemented. Line 133
l.145-149 - text isn't clear - rewrite, and give reference for programme Cervus
Response:
We agree with the reviewer and the specific program version and reference has been added. Parameters listed have been clarified as best as possible with the understanding that they are technical and specific to this analysis. Lines: 166-171
Original:
“Cervus was used to calculate allele frequencies across all samples, simulate parentage and assign parent to offspring as follows (parent pairs with known sexes, 1,600 offspring, 15 candidate mothers, probability mother sampled 0.95, 20 candidate fathers, probability father sampled 0.95, probability of loci typed 0.95, probability of loci mistyped 0.01, and minimum loci typed 200).”
Corrected:
“Cervus v3.0.7 [34] was used to calculate allele frequencies across all samples, simulate parentage and assign parent to offspring parameters used were as follows (parent pairs with known sexes, 1,600 offspring, 15 candidate mothers, probability of mother sampled 0.95, 20 candidate fathers, probability of father sampled 0.95, probability of loci typed 0.95, probability of loci mistyped 0.01, and minimum loci typed 200).”
l.296 - 5 species, but lists 6 names?
Response:
Six species is the correct number and has been corrected in text. Line 324
l.327-8 - sentence not clear -edit
Response: We agree with the reviewer and the sentence has been clarified. Lines 366-367
Original:
“The reduced family sizes present in this result in subsequently fewer recombination events observed between loci”
Corrected:
“The relatively smaller family sizes present in this study results in fewer recombination events observed between loci”
l.339 - 363 - these two paragraphs are a bit repetitive and could be condensed and combined - the issue is potential effect of small family sizes, which doesn't need dwelling on at this length as it's just a note for future improvement?
Response: We agree with the reviewer and the paragraphs have been merged and condensed. Lines 388-392
Original:
“This study utilised data generated from a complimentary multiple paternity study on H. maculosa with parental samples sourced from the wild (Cockburn Sound, Western Australia) [28], while this was the most successful captive breeding attempt to date in this genus and rendered our current work possible, the data were not without limitations for the purposes of the current study. Before capture, and during the multiple paternity study, females were exposed to multiple males resulting in observed cases of multiple paternity. All females exhibited multiple paternity, including five unknown males (presumably from matings in the wild prior to the females capture). As a result, family sizes were reduced as female clutches were divided among the multiple males, which resulted in the loss of nine families, each containing < 10 individuals, in addition to the five families containing unknown paternity.
Hapalochlaena maculosa are semelparous and produce a single relatively small clutch (30-170) of large eggs (6-7mm) [28,49], which they carry within their web. On hatching offspring are well-developed and assume a benthic lifestyle immediately. Other members of the genus such as H. lunulata exhibit a merobenthic lifestyle and produce a larger quantity (270) of smaller eggs (3.5mm) [50,51], which may make them more suitable for linkage mapping purposes. Due to the smaller family sizes used in this study resulting from limitations in clutch size and multiple paternity, a linkage mapping algorithm was selected, that was able to use a combined dataset integrating all families to maximise power in linkage group determination and marker order. Lep-Map3 was employed in this study because it allows not only for the integration of all families as one dataset, but also takes into account sibship between offspring [35]. This was of particular importance for this study since all H. maculosa females produced clutches with multiple paternity. To ensure quality of families analysed, paternity was verified using multiple methods and loci screened for Mendelian errors prior to map construction. ”
Corrected:
“This study utilised data generated from a complimentary multiple paternity study on H. maculosa with parental samples sourced from the wild (Cockburn Sound, Western Australia) [28], while this was the most successful captive breeding attempt to date in this genus and rendered our current work possible, the data were not without limitations for the purposes of the current study. Before capture, and during the multiple paternity study, females were exposed to multiple males resulting in observed cases of multiple paternity. All females exhibited multiple paternity, including five unknown males (presumably from matings in the wild prior to the females capture). As a result, family sizes were reduced as female clutches were divided among the multiple males, which resulted in the loss of nine families, each containing < 10 individuals, in addition to the five families containing unknown paternity. To address there challenges a linkage mapping algorithm was selected, that was able to use a combined dataset integrating all families to maximise power in linkage group determination and marker order. Lep-Map3 was employed in this study because it allows not only for the integration of all families as one dataset, but also takes into account sibship between offspring [36].”
l.367-9 - clarity of second part of this sentence falls apart - needs editing.
Response:
We agree with the reviewer and the sentence has been clarified. Lines 394-398
Original:
“Map length in H. maculosa is larger than many other published molluscan linkage maps including of the bivalve Pinctada maxima (831.7cM) [52] and the gastropod Biomphalaria glabrata (746.7cM) [53] in line with their relatively smaller genomes 290Mb (Neomenia permagna) - 2.21Gb (Bathymodiolus plantifrons) [54] compared to H. maculosa (4Gb)”
Corrected:
“Map length in H. maculosa is larger than many other published molluscan linkage maps including of the bivalve Pinctada maxima (831.7cM) [53] and the gastropod Biomphalaria glabrata (746.7cM) [54], which have relatively smaller genomes 290Mb (Neomenia permagna) - 2.21Gb (Bathymodiolus plantifrons) [55] compared to H. maculosa (4Gb).”
l.385-391 - is this info on other cephalopods needed? The point made is about chromosome number - for this simple point, only the 1st sentence needed as reference?
Response: We agree with the reviewer and the paragraph has been modified. Lines: 446-454
Original:
“Prior to this study, no genetic linkage maps have been produced for any cephalopod, however, karyological studies conducted on a subset of 11 cephalopods estimate chromosome numbers for octopodiformes (n30), decapodiformes (n46) and nautiloids (n26) [12,13,60]. Structural variation was present between octopod karyotypes with the most divergent being Amphioctopus fangsiao (d’Obigny, 1839-1841), which exhibited no sub-telocentric chromosomes [13]. In composition, metacentric, submetacentric, teleocentric and sub-teleocentric chromosomes displayed slight variation between Callistoctopus minor (Sasaki, 1920) and Cistopus chinensis (Zheng et al., 2012). Karyograms revealed evolutionary distances between species to be congruent with molecular phylogenies conducted on the three octopods, C. minor, A. fangsiao and C. chinensis [13]. Linkage groups generated in this study do not correspond 1:1 with the expected chromosome number for H. maculosa suggesting the need for either a larger number of families, a larger number of progeny per family, or a larger set of informative markers to allow for chromosome level resolution and improved contiguity. ”
Corrected
“Prior to this study, no genetic linkage maps have been produced for any cephalopod, however, karyological studies conducted on a subset of 11 cephalopods estimate chromosome numbers for octopodiformes (n30), decapodiformes (n46) and nautiloids (n26) [12,13,61]. Karyograms revealed evolutionary distances between species to be congruent with molecular phylogenies conducted on the three octopods, C. minor, A. fangsiao and C. chinensis [13]. Linkage groups generated in this study do not correspond 1:1 with the expected chromosome number for H. maculosa suggesting the need for either a larger number of families, a larger number of progeny per family, or a larger set of informative markers to allow for chromosome level resolution and improved contiguity.”
Author Response File: Author Response.docx
