Immature Stages of Genus Hexatoma (Diptera, Limoniidae) in the Korean Peninsula

Round 1

Reviewer 1 Report

The manuscript uses the most appropriate methodology (DNA barcoding and morphology) to detect and describe unknown larvae of an important insect group in aquatic environment. Larvae taxonomy is still a neglected field of entomology, but with growing interest by incorporating new taxonomy methods, like DNA data.

Limoniidae, where the target group, Hexatoma belongs are a taxonomically challenged group, especially larvae having important gaps in species level identification. The manuscript is clearly formulated, relevant for the field with and excellent iconography and important new data.

Based on the novelty of data we highly recommend for publication in this journal.

Comments for author File: Comments.pdf

Author Response

We agree with all comments and suggestions and made changes in manuscript.

 

Introduction

 

 

- “a brief history on taxonomy data on adults Hexatoma, especially Eriocera, as well as larvae. Some problems are highlighted, but not detailed. I highly recommend more information on taxonomic criteria (or problems) for delimiting the subgenus Eriocera among Hexatoma larvae” -  required information is provided.

 

- “more detailed information on morphological groups established among Eriocera larvae

(ex. in paragraph in line 47)” - required information is provided.

 

-“The introduction part is mixed with methods (lines 52-55, as well as lines 65-68) and discussions (lines 56-64). Please be more rigorous on delimiting different parts of the manuscript” – we excluded lines 52-55 and 65-68, but we decided to leave lines 56-64 in introduction part. In our opinion, this information is appropriate for the Introducyion section.

 

- “line 29 – please reformulate the phrase Instead of this sentence: Hexatoma adults are a common group in aquatic habitats on the Korean peninsula….this should be written: Hexatoma larvae are common in aquatic habitats on the Korean peninsula” – changes are made.

 

 

Results

 

- “we highly recommend to present here the most important morphological data supporting the molecular results before presenting the Key to the last instar larvae” -  required information is provided.

 

Discussion

 

-“We highly recommend to introduce in the end of Discussions a short presentation of the habitat of identified larvae, which will be a valuable information for further investigation in biological assessment of freshwaters with a more general audience. Please comment if there are some differences in ecological requirements (water-type) of taxonomically different larvae. – there are no differences in ecological requirements of Hexatoma larvae” – We have provided a note on the ecological requirements for larvae. They were collected in similar types of water bodies in very similar habitats.

 

We would like to thank the reviewer for his valuable comments and remarks, which improved the quality of the paper.

 

Reviewer 2 Report

This is an excellent paper setting the standard for further studies of immature stages of Nematocera, particularly in the Tipuloidea.  This paper fully implements a technique of using DNA barcoding for assessing species identification in multiple species of the genus Hexatoma, along with some traditional rearing methods.  The photographic images are of the highest quality and again sets the standard for future papers describing larval and pupae in Tipuloidea.  This genus is important in biotic analysis of water quality as it is common in macroinvertebrate water quality samples and this paper and its identification key, like the first author's previous paper on the genus, provides important tools for benthological scientists.  The paper is well written, the descriptions are detailed and excellent, and the introduction and discussion appropriate.

I have a few suggestions:

I would make sure the comments in line 750-753 are also included in the Abstract.  The authors should point out in the abstract the importance of this work.

I was a bit unclear on exactly which species of Korean (or broadly) eastern Palaeactic/Asian Hexatoma have been barcoded from adult specimens.  I was trying to evaluate how many species in the region might possibly be matched to Hexatoma sp. 1 and 2 if the sequences are available.  Are there no sequences of sakhalinensis or ussurienses available?  Are any other Hexatoma in the region (broader than Korea - including Russia, Mongolia, China) that have been sequenced and could be included in Fig. 1?  It might help elucidate what Hexatoma sp. 1 or 2 might be?  Are the thoughts that additional species of Hexamoa on the Korean peninsula remain to be discovered?

Table 2 - what do the colors mean?  Please put this info somewhere in the legend.

Line 169. Might add an arrow in Fig 3A and B showing the "median labrum lobe"

Line 164. Why isnt sakahleniensis included in the key - is the larva and pupa unknown?

line 172 - scarcely, instead of scarcy

Line 261 - might note in the figure legend of Fig 4  that the longer hairs from the spiracular lobes have been cut off in the image.

line 282 - maybe an arrow showing the sensory structures

line 493 - the mandible teeth structure seem overall in all the species to be useful distinguishing characteristics but are not used in the diagnoses or the key for the species.  Why not?  is it because they can be hard to see without dissection, or do they show wear patterns among individuals?

Author Response

We agree with almost all of the comments and suggestions and made changes in manuscript.

Few comments:

“I would make sure the comments in line 750-753 are also included in the Abstract.  The authors should point out in the abstract the importance of this work”

- we include this information into Abstracts.

“I was a bit unclear on exactly which species of Korean (or broadly) eastern Palaeactic/Asian Hexatoma have been barcoded from adult specimens.  I was trying to evaluate how many species in the region might possibly be matched to Hexatoma sp. 1 and 2 if the sequences are available.  Are there no sequences of sachalinensis or ussurienses available?  Are any other Hexatoma in the region (broader than Korea - including Russia, Mongolia, China) that have been sequenced and could be included in Fig. 1?  It might help elucidate what Hexatoma sp. 1 or 2 might be?  Are the thoughts that additional species of Hexatoma on the Korean peninsula remain to be discovered?” –

- Only Hexatoma (Eriocera)spinosa (USA), Hexatoma (Eriocera) gressittiana (China), Hexatoma (Eriocera) nudivena (China), Hexatoma (Hexatoma) fuscipennis (Finland), Hexatoma (Hexatoma) obscura (Portugal), Hexatoma (Hexatoma) nigra (Portugal), and Hexatoma (Eriocera) longicornis (USA) species have been found with reliable sequences.  We compared the sequences of Hexatoma sp.1 and Hexatoma sp.2 and they did not match any of the deposited sequences (neither with nor without the species name). We are sure that these are not ussuriensis and sachaliniensis, as the senior author has described the larvae of these species in previous studies. A small percentage of species overlap with China, but a large percentage overlap with Japan (22 species) and the Russian Far East (12 species, some overlapping with Japan). Korea is also known to be highly endemic, so there is a high probability (but not 100%) that these are species new to science. We also only had old specimens of Hexatoma masaki and could not successfully extract DNA from them. This species was collected between 1950 and 1960, but this does not mean that the species is extinct. So, given all this information, we have decided not to go into further discussion.

“Table 2 - what do the colors mean?  Please put this info somewhere in the legend.”

 – colors mean nothing. We change into regular black color.

 

“Line 169. Might add an arrow in Fig 3A and B showing the "median labrum lobe"

– 3A has arrow marked as “lal” showing median labrum lobe.

 

“Line 164. Why isnt sachaliniensis included in the key - is the larva and pupa unknown?”

 – sachalinensis included in the key

“Line 172 - scarcely, instead of scarcy”

– we made changes

 

“Line 261 - might note in the figure legend of Fig 4 that the longer hairs from the spiracular lobes have been cut off in the image”

– we made a note

 

“line 282 - maybe an arrow showing the sensory structures”

 – we made 2 arrows showing sensory structures

 

“line 493 - the mandible teeth structure seems overall in all the species to be useful distinguishing characteristics but are not used in the diagnoses or the key for the species.  Why not?  is it because they can be hard to see without dissection, or do they show wear patterns among individuals?”

 – It is very hard to see basal teeth of mandible without high magnification. We have plenty of more obvious characters which are much easier to see.

 

 

We would like to thank the reviewer for his valuable comments and remarks, which improved the quality of the paper.

 

Reviewer 3 Report

This article uses molecular methods to identify three Hexatoma larvae species from South Korea and matches the larval stage with the adult stage. The larval stages of these species also be described and illustrated.

The larvae of two species that are not associated with any adult are also described and their COI gene fragment sequences are presented. The study is  meaningful for understanding the larval stage of Hexatoma. In addition, the figures is very good in quality.

However, there are still many small errors in the text that need to be carefully corrected, especially the format of references. Other comments are in the uploaded manuscript.

 

Comments for author File: Comments.pdf

Minor editing of English language required.

Author Response

Responses to the second reviewer's comments and suggestions:

 

We agree with almost all of the comments and suggestions and made changes in manuscript.

Few comments:

“According to Catalogue of the Craneflies of the World, there are currently 610 species and subspecies” – 610 species and subspecies, but just 599 species. We decided to use just number of species.

“Have sp 1 and sp 2 been identified in the database to determine if they are unknown species? “– yes, we didn’t find matching sequences in any data bases.

 

We would like to thank the reviewer for his valuable comments and remarks, which improved the quality of the paper.