Florida Museum of Natural History

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Florida Museum of Natural History Powell Hall, 3215 Hull Rd.

McGuire Center for Lepidoptera and Biodiversity Gainesville, FL 32611-2710

Tel: 352-392-3516

Fax: 352-392-4400

Dr Thomas W. Sappington

Editor, Environmental Entomology

January 07, 2015
Dear Editor:
We are resubmitting the manuscript EN-14-216 titled “Genetic record for a recent invasion of the mealybug Phenacoccus solenopsis in Asia” as an article to the Environmental Entomology. We have revised the MS substantially according to reviewer s’ suggestions. We have acknowledged the problem of misrepresentation of genetic groups as species. We agree that data currently does not support the species level delimitation however, the date does support the genetic group level identities. Since reviewer#2 thinks genetic group may sound like too strong of a word and might be misleading to species level boundaries, we have replaced the term “genetic group” with “group”. The two groups, which were previously called genetic groups, are also based on geographical boundaries and are representative of Asia and America. We have performed additional species delimitation analysis suggested by reviewer#1 to reconfirm that these genetic groups are not species.
We have responded to all comments of both reviewers one by one following the reviewer s’ comment in blue text.


The authors should test if these genetic groups are species by using species delimitation software. See:Boykin, L.M., K.F. Armstrong, L. Kubatko, and P. De Barro. 2012. Species Delimitation and Global Biosecurity. Evolutionary Bioinformatics 8: 1-37 (and references therein).

  • Response 1.1: We have used PTP method (Tang et al. 2014) similar to other methods described in Boykin et al 2012. For PTP method, we have used tree both rooted (with Phenacoccus solni) and unrooted with values of 100000 MCMC, Burnin 0.1 and seed 123. We did not get significant support for species delimitation between Asia and NewWorld genetic groups. In addition we did not get any significant support for species delimitation among haplotypes H1-H9 in Asia and H1-H3 in NewWorld. We have clarified it in MS and replaced the term of genetic group with group.

    Tang CQ, Humphreys AM, Fontaneto D, Barraclough TG. 2014. Effects of phylogenetic reconstruction method on the robustness of species delimitation using single-locus data. Methods in Ecology and Evolution 5:1086-1094.


Also, how did you determine the model of molecular evolution? K2P might not be the best fitting model (as assumed in the ms).

  • Response1.2: We have used partition finder software and based on lowest BIC (Bayesian Information Criterion) value; we have selected the K2P model as best model. We did describe it in previous version of MS in the line 177-178; “The best model and partitioning scheme was chosen using the Bayesian Information Criterion (BIC) in partition finder v1.0.1 (Lanfear et al. 2012)

    Lanfear R, Calcott B, Ho SYW, Guindon S. 2012. PartitionFinder: Combined Selection of Partitioning Schemes and Substitution Models for Phylogenetic Analyses. Molecular Biology and Evolution 29:1695-1701.

The authors have appeared to overstated the results. I don't see evidence for the congruent spread of the mealybug and parasitoid. You would need to collect microsat or SNP data to make those conclusions.

  • Response1.3: We have toned down this and discussed that further analysis using more refined molecular maker will be needed to clarify this in the future.

The figures are very good.
Reviewer 2

A lot of work went into this manuscript, but there is unclarity in the data presented. I think

the authors need to shorten the introduction and discussion. It is written in a disorganized

manner. In many of the places sentences are repeated. Unrelated and wrong references are

given. Authors should be careful and serious about writing the MS in a comprehensible

manner with the relevant data. In addition, the text suffers from numerous problems of

grammar, syntax and use of tenses. Before any publication, the text should be carefully

revised for the English writing

  • Response2.1: We have shortened the introduction and discussion. We also taken out less important references and added the more important ones. In addition, we also revised the language.

Moreover, don’t jumb into a conclusion of making genetic groups. That can mislead the

followers. Putative genetic groups are named on the basis that lack morphological distinction but genetically distinct populations. In case of P. solenopsis there is clear morphological separation regarding multilocular disc pores between Asian and American populations. Also in case of other insects like Bemisia tabaci genetic groups are defined after a series of studies on its biology, host range, virus transmission, allozyme pattern, esterase pattern and RAPD. So very problematic is the use of genetic groups. Genetic distance between the groups should be 3.5% or above. So there is some criteria for making into groups/ species complex. It appears you have the data to start to develop this framework, which I would strongly suggest prior to resubmission.

  • Response2.2: We think, this is a misunderstanding; using the term genetic groups here was not meant to mean species in our MS. We did mention this in previous draft for example in line 333-334, “Given criteria of putative species split by a 95% TCS network, it seems Asian and American populations are likely one species.”, We have clarified that these genetic groups are not species. However, it appears that the term genetic group has already used to represent the species level boundaries in others cases like Bemisia species complex. Therefore, we have replaced the term “genetic group” with “group” to avoid the confusion. We have explained it above. In addition, we also performed additional analysis suggested by reviewer#1 to reconfirm that these genetic groups are not species. We also agreed with reviewer that additional data is required to declare them species. We have acknowledged the fact that these are not different species but genetically different populations of Phenacoccus solenopsis, which are found in various geographical regions.

Lines 2. Author name should be given after species name. Phenacoccus solenopsis Tinsley

  • Response2.3: We have incorporated suggested changes.

Line 13. Suggestion..Recent invasion of cotton mealybug

  • Response2.4: We have incorporated suggested changes.

Line 28-29. In this study, we performed the field surveys during 2010-2012 and sequenced

mtCOI from across China and Pakistan then compared them with already available mtCOI

sequences from other Asian and American countries. SENTENCE SHOULD BE IN THIRD


field surveys were performed during 2010-1012 and analysed mtCOI sequences across China and Pakistan and compared with the sequences available in the NCBI database.

  • Response2.5: Editor suggested that it is not necessary to change the style to 3rd person.

Line 30-32. Repetition. Our genetic analysis of samples collected from China and Pakistan

along with already published samples from India, Vietnam and USA classified P. solenopsis into two putative genetic groups. BE FOCUSSED. Suggestion Analysis of samples from different geographical regions revealed genetic differences among the populations. AVOID THE USE OF PUTATIVE GENTIC GROUPS.

  • Response2.6: We have deleted the repetition and also replaced the “putative genetic groups” with “groups”.

Lines 44-48. Give Relevant References only. Major Reference is lacking Ben Dov et al 2003.

  • Response2.7: We could not find the reference of Ben Dov et al 2003 which reviewer suggested in his/her comment. However, we assumed that reviewers wanted to suggested the references of Ben Dov 2005 which we have included in our MS.

Line 53-57. Give Relevant References only. Mention only scalenet Reference.

  • Response2.8: We have deleted unnecessary references.

Line 94-96 Recently twelve more species of Planococcus were characterized as pests, including the morphological similar species Planococcus citri and Planococcus minor (Miller et al. 2002).


Line 96-99. I don’t understand this

  • Response2.9: We have incorporated suggested changes.



  • Response2.10: We have taken out unnecessary material from introduction and made it more precise.

Materials And Methods



  • Response2.11: Editor suggested that it is not necessary to change the style to 3rd person.

Line 123-125 Phenacoccus solenopsis were identified according to the morphological

characteristics such as living female adults usually with pairs of dark spots and/or stripes dorsally suggestion “ P. solenopsis can be distinguished by the presence of paired dark stripes on the

dorsal region”

  • Response2.12: We have incorporated suggested changes.

Line 127-128. There were no specific collection permits required to collect P. solenopsis

samples at these locations. ……Need to mention this?

  • Response2.13: We have deleted this line.

Line 129-132. Parasitized P. solenopsis nymph were also collected and kept in Petri dishes until they emerged from nymph bodies. suggestion to change “Parasitized specimens were kept in petri dishes for emergence and the emerged ones were preserved in 95% alcohol for molecular studies.

  • Response2.14: We have incorporated necessary changes.

Line 134. The total genomic DNA from either P. solenopsis or its parasitoids using the whole body of one individual. We used Promega Wizard Genomic DNA purification Kit (Cat# A1120) according to protocol for animal tissues. Suggestion. “The total genomic DNA was extracted from individual specimens of P. solenopsis and its parasitoids using Promega Wizard Genomic DNA purification Kit.

  • Response2.15: We have incorporated suggested changes.



  • Response2.16: We have incorporated suggested changes.



It is thus difficult to provide a general feeling about this manuscript. I would first recommend to perform major modifications not only of form but also to justify the relevance of the work and the importance of the results, given the context described in the introduction

  • Response2.17: In comment#1, reviewer suggested to shorten the discussion. We have shortened the discussion and also clarified the significance of work according to the description mentioned in introduction.


Muhammad Z Ahmed

Postdoctoral Associate

Florida Museum of Natural History

University of Florida

Powell Hall, 3215 Hull Road

Gainesville, FL 32611-2710, USA

Tel: 352.283.0173

Email: zaheerento@gmail.com


Ma Jun


Plant Quarantine Laboratory

Guangdong Inspection and Quarantine Technology Center

No. 66 Huacheng Ave., Guangzhou 510623, P.R.China

Tel: 86-20-38290936 / 86-20 38290951

Emial: majmail@163.com

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