Distinguishing Long-Discussed Cryptic Species of the Epibiotic Goose-Neck Barnacle of the Genus Conchoderma (Thoracicalcarea: Lepadidae) with Integrative Taxonomy
Round 1
Reviewer 1 Report
The manuscript provides an integrated analysis of morphological and molecular data for the discrimination of two species of the genus Conchoderma: C. virgatum and C. hunteri. These two species have a long history of misidentification/synonymizing/ and being re-erected to species always based only on morphological data/observation (since Darwin). The authors concluded that the two species are indeed different both morphologically and molecularly.
The manuscript is well written, the introduction explains properly the convoluted taxonomic situation, the morphological description is accurate, and the morphological pictures are really well done. However, the molecular data were not properly explored and analyzed.
The authors used only distance-based methodologies for the species delimitation: ASAP and K2P distances. Nevertheless, the latter is not a delimitation method and only shows the average distance between morphospecies. Also, for the ASAP analysis, would also be better to use more substitution models and not only the default as it would provide more information and/or support to the analyses. Further, in the results the authors mentioned the intraspecific distance that can also be shown in the table 2 in diagonal.
Moreover, for molecular species delimitation, what is usually done is to take into account different methodologies to avoid the single possible bias. Analyses based on the topology of phylogeny are advised like: GMYC (https://species.h-its.org/gmyc/), mPTP (https://mptp.h-its.org/#/tree), bPTP (https://species.h-its.org/ptp/). The first requires an ultrametric tree that can be obtained from MrBayes, the other two use any kind of phylogeny, and is also possible to exclude outgroups.
I also suggest the authors to include the bootstrap values of the ML in the BI phylogeny (or the opposite) in order to provide more informative and less pictures.
I also saw that the data were not uploaded to BOLD systems (http://v4.boldsystems.org/), and any alignment was provided now during the submission, I’d prefer to see the alignments before any acceptance, as they should be freely available later, but also for the reviewing process. From BOLD, the authors will also be able to run other analyses like Cluster sequences (MOTU delimitation), which will also complement their results.
At least one further nuclear gene would integrate well these analyses providing stronger results and support, however, I understand that this can be an issue and I leave it to the authors.
For these reasons, I suggest major revision to provide some time to run the new analyses and submit the new sequences to BOLD.
I have a couple of minor comments for the text:
The title: “The epibiotic striped gooseneck barnacle Conchoderma hunteri (Thoracicalcarea: Lepadidae) is a valid species but not sub-species, variety or growth form of Conchoderma virgatum” is a bit wordy. I suggest rephrasing it avoiding the second part (e.g., The epibiotic striped gooseneck barnacle Conchoderma hunteri (Thoracicalcarea: Lepadidae) is a valid species), or changing it completely through something like: Distinguishing long-discussed cryptic species of the genus Conchoderma with integrative taxonomy. I leave it to the authors.
Figure 1 (line 61): Please include in the caption the abbreviations’ explanation.
Line 61: Uruguay.
Author Response
Reviewer 1:
Comment: The manuscript provides an integrated analysis of morphological and molecular data for the discrimination of two species of the genus Conchoderma: C. virgatum and C. hunteri. These two species have a long history of misidentification/synonymizing/ and being re-erected to species always based only on morphological data/observation (since Darwin). The authors concluded that the two species are indeed different both morphologically and molecularly.
The manuscript is well written, the introduction explains properly the convoluted taxonomic situation, the morphological description is accurate, and the morphological pictures are really well done. However, the molecular data were not properly explored and analyzed.
Response: Thanks for the appreciation. We have further conducted the molecular analysis and detailed responses are as below.
Comment: The authors used only distance-based methodologies for the species delimitation: ASAP and K2P distances. Nevertheless, the latter is not a delimitation method and only shows the average distance between morphospecies. Also, for the ASAP analysis, would also be better to use more substitution models and not only the default as it would provide more information and/or support to the analyses. Further, in the results the authors mentioned the intraspecific distance that can also be shown in the table 2 in diagonal.
Moreover, for molecular species delimitation, what is usually done is to take into account different methodologies to avoid the single possible bias. Analyses based on the topology of phylogeny are advised like: GMYC (https://species.h-its.org/gmyc/), mPTP (https://mptp.h-its.org/#/tree), bPTP (https://species.h-its.org/ptp/). The first requires an ultrametric tree that can be obtained from MrBayes, the other two use any kind of phylogeny, and is also possible to exclude outgroups.
I also suggest the authors to include the bootstrap values of the ML in the BI phylogeny (or the opposite) in order to provide more informative and less pictures.
Response: We have used the ASAP analysis and use 3 models used for analysis, 1: Jukes-Cantor model (JC69), 2: Kimura 2-parameter model (K2P) and 3: Simple Distance (p-distances). We also conducted additional PTP analysis based on Bayesian Poisson tree processes (bPTP) and Maximum likelihood solution. We also conducted GMYC analysis. All these results are overlapped in new Figure 13 (Please see the revised molecular methods in page 3, revised molecular results in page 18, 19 and the new figure 13.
Comment: I also saw that the data were not uploaded to BOLD systems (http://v4.boldsystems.org/), and any alignment was provided now during the submission, I’d prefer to see the alignments before any acceptance, as they should be freely available later, but also for the reviewing process. From BOLD, the authors will also be able to run other analyses like Cluster sequences (MOTU delimitation), which will also complement their results.
Response: We have now uploaded the sequences in BOLD (please see last paragraph of section 2.3)
Comment: At least one further nuclear gene would integrate well these analyses providing stronger results and support, however, I understand that this can be an issue and I leave it to the authors.
Response: We understand nuclear gene can provide further evidence. Since the two species we compared have distinct divergences in mitochondria COI marker and with diagnostic morphological differences. I think the molecular and morphological evidence is strong enough to discriminate these two species.
Comment: For these reasons, I suggest major revision to provide some time to run the new analyses and submit the new sequences to BOLD.
Response: We have added sequences in BOLD.
Comment: I have a couple of minor comments for the text:
The title: “The epibiotic striped gooseneck barnacle Conchoderma hunteri (Thoracicalcarea: Lepadidae) is a valid species but not sub-species, variety or growth form of Conchoderma virgatum” is a bit wordy. I suggest rephrasing it avoiding the second part (e.g., The epibiotic striped gooseneck barnacle Conchoderma hunteri (Thoracicalcarea: Lepadidae) is a valid species), or changing it completely through something like: Distinguishing long-discussed cryptic species of the genus Conchoderma with integrative taxonomy. I leave it to the authors.
Response: We have revised the title as suggested.
Comment: Figure 1 (line 61): Please include in the caption the abbreviations’ explanation.
Response: Addressed.
Comment: Line 61: Uruguay.
Response: Addressed.
Reviewer 2 Report
The manuscript in general is good, but it lacks of serious components, first in introduction and methodology for any taxonomic and systematics works it MUST cite WORMS (www.marinespecies.org), it is a valid internet source for get the current and right taxonomic status, you must cite it in methodology and results, and verify your results in according to the indications of this site
The discussion is very poor, I think that must be improved with more ecological and biogeographical components that support the results of molecular analysis. The molecular analysis provide a results, but you must explain the possibles causes of this results, and confirm or reject your hypothesis in according to the molecular analysis and taxonomical evidence.
Author Response
Comment: The manuscript in general is good, but it lacks of serious components, first in introduction and methodology for any taxonomic and systematics works it MUST cite WORMS (www.marinespecies.org), it is a valid internet source for get the current and right taxonomic status, you must cite it in methodology and results, and verify your results in according to the indications of this site
Response: We have cited WoRMS in reference 14, and in the first paragraph of the manuscript.
Comment: The discussion is very poor, I think that must be improved with more ecological and biogeographical components that support the results of molecular analysis. The molecular analysis provide a results, but you must explain the possible causes of this results, and confirm or reject your hypothesis in according to the molecular analysis and taxonomical evidence.
Response: We have revised the discussion. In the first two sentences we stated we rejected the hypothesis that C. virgatum and C. hunteri are conspecific “Our results did not support the hypothesis in Hiro (1937) and Zevina (1982) that C. virgatum and C. hunteri are conspecific.” We also added in second paragraph that these two species formed sister relationship in the molecular phylogenetic tree, due to their similar habitats and morphology “Based on our molecular analysis, Conchoderma virgatum and C. hunteri form sister clades with high support, indicating that the two species are closely related and these two species also share similar epibiotic habitats [2-6] and similar morphology (cirri and filamentary appendages on somatic bodies).”
Round 2
Reviewer 1 Report
I would like to compliment the authors that answered and complied with all my previous suggestions.
I believe the manuscript definitely improved and it is ready to be published after some minor comments which are easily amendable.
Line 108: “with 1 × 106 generations of 5”, 106 is a typo and probably exponential: 106
Lines 346 to 352: “The species delimitation results of ASAP with three models and GMYC method areall suggested 3 Conchoderma groups which is coincide with the phylogeny and morphology result. The results of PTP in highest Bayesian supported solution and maximum likelihood solution divided C. hunter and C. virgatum into 2 groups respectively, which consisted with other results. However, C. virgatum were further designated into 2 groups in two solutions. Moreover, C. auritum were also divided into 2 groups, which shows 5 groups in Conchoderma spp..” I’d rewrite the whole paragraph as it is wordy and redundant: Generally, the three morphospecies of Conchoderma were always in different MOTUs (Molecular Operational Taxonomic Units). However, PTP analyses further split C. hunter and C. virgatum in two MOTUs each.
Lines 371-372: “The present study revealed that Conchoderma virgatum and C. hunteri are different species based on morphological and molecular evidence. Our results did not support the hypothesis in Hiro (1937) and Zevina (1982) that C. virgatum and C. hunteri are conspecific.”, I’d rephrase the second sentence: …. morphological and molecular evidence, rejecting the hypothesis of their conspecificity proposed by Hiro (1937) and Zevina (1982).
Author Response
We thanks the reviewer for further editing of our manuscript. All of the editing comments were addressed and highlighted in yellow in the new revised MS. We acknowledged the two anonymous reviewers for their comments in the acknowledgement of the manuscript.
