DNA Barcode Library of Megadiverse Lepidoptera in an Alpine Nature Park (Italy) Reveals Unexpected Species Diversity

Round 1

Reviewer 1 Report

The study reports the species diversity of Lepidoptera in a well-studied protected area of Italy (the Cottian Alps) through molecular analysis. The authors originally performed a DNA-based inventory (including ~1,200 species) of such a species-rich group, demonstrating the successfulness of barcode sequences as a proxy of species identification (1,206 BINs belonging to 1,214 named species). The data also revealed cryptic diversity, with new records for Italy and potential undescribed species, increasing the regional faunistic knowledge.

The analytical methods employed are adequate and the field collection effort is relevant, leading the results to be well-supported, and of broad interest, as the taxonomic approach of the BIN versus Linnean systems.  

The barcode gap analysis pointed out a mean distance of 7% to the nearest neighbor species, with a maximum of 16%, likely indicating the existence of some taxonomic issues (common pattern of microlepidoptera).

The authors raised the necessity of further integrative analysis on these specimens with 2-3% of divergence that was assigned to new BINs. I strongly recommend going ahead with the descriptions! A species without a name cannot be remembered/protected, valid also for tiny moths.

In general, I am satisfied with the data and conclusions of the study, including the usefulness of the barcode library to further studies and/or improvement of delineating conservation strategies. The moderate diversity (comparative to the tropics) of European Lepidoptera, coupled with the great scientific effort throughout years of collecting, describing, and cataloging specimens in reliable institutions on the continent, allow the construction of nearly complete regional DNA barcode libraries. This is a good example of the return of taxonomic description efforts performed by experts in the past.

Minor issues regarding the overall presentation are pointed out:

It was not clear when (year and season) fieldwork was performed. 

Along the same line, the sampling was performed using light traps and a butterfly net. So, how about the immature stages? Maybe part of the species diversity is not in the stage (adult) sampled.

Figure 1 - it is hard to identify in Europe map the exact location of the study. Maybe including the geographic coordinates and/or individual identification of collection sites will help.

Table 1 - it is not intuitive. I do not have a better idea, but I can say that it is not clear. May be give more importance to the BIN column.

Author Response

Response to reviewer 1

Many thanks for your careful review!

The study reports the species diversity of Lepidoptera in a well-studied protected area of Italy (the Cottian Alps) through molecular analysis. The authors originally performed a DNA-based inventory (including ~1,200 species) of such a species-rich group, demonstrating the successfulness of barcode sequences as a proxy of species identification (1,206 BINs belonging to 1,214 named species). The data also revealed cryptic diversity, with new records for Italy and potential undescribed species, increasing the regional faunistic knowledge.

The analytical methods employed are adequate and the field collection effort is relevant, leading the results to be well-supported, and of broad interest, as the taxonomic approach of the BIN versus Linnean systems.  

The barcode gap analysis pointed out a mean distance of 7% to the nearest neighbor species, with a maximum of 16%, likely indicating the existence of some taxonomic issues (common pattern of microlepidoptera).

The authors raised the necessity of further integrative analysis on these specimens with 2-3% of divergence that was assigned to new BINs. I strongly recommend going ahead with the descriptions! A species without a name cannot be remembered/protected, valid also for tiny moths.

In general, I am satisfied with the data and conclusions of the study, including the usefulness of the barcode library to further studies and/or improvement of delineating conservation strategies. The moderate diversity (comparative to the tropics) of European Lepidoptera, coupled with the great scientific effort throughout years of collecting, describing, and cataloging specimens in reliable institutions on the continent, allow the construction of nearly complete regional DNA barcode libraries. This is a good example of the return of taxonomic description efforts performed by experts in the past.

Minor issues regarding the overall presentation are pointed out:

It was not clear when (year and season) fieldwork was performed. 

All data are documented in the public dataset. However, we are grateful that you spotted this problem and we now added detailed data and alsoexpanded the abstract accordingly.

Along the same line, the sampling was performed using light traps and a butterfly net. So, how about the immature stages? Maybe part of the species diversity is not in the stage (adult) sampled.

We added a clarifying phrase.

Figure 1 - it is hard to identify in Europe map the exact location of the study. Maybe including the geographic coordinates and/or individual identification of collection sites will help.

We added geographic coordinates to the main text as well as to Figure 1 and furthermore added numbers of the 4 major study areas in text and Figure 1.

Table 1 - it is not intuitive. I do not have a better idea, but I can say that it is not clear. May be give more importance to the BIN column.

Many thanks for discussing this issue. Obviously we tried to include too much information in this table. We therefore now deleted the last column with numbers of barcodes/BIN from the study area which is not crucial and furthermore split the table into one dealing with BIN sharing species and the second with barcode sharing species.

Reviewer 2 Report

The article "DNA Barcode Library of Megadiverse Lepidoptera in an Alpine Nature Park (Italy) Reveals Unexpected Species Diversity" is devoted to the precise identification of butterfly species in a nature reserve and to study of the diversity of their DNA. The authors collected a large amount of material, characterized the specimens by morphological features, and thus carried out a preliminary identification to the species level. They then selected several representatives of each morphotype and sent samples for sequencing of a standard mtDNA fragment to a Canadian research center. Based on the results obtained, the authors identify species, intraspecific variability of the studied DNA fragment, genetic distances between taxa, and draw conclusions about the importance of barcoding natural specimens. The authors have extensive experience with Lepidoptera species in this area. Therefore, some data from previous works are also presented in this paper. The article fits the content of the special issue Frontiers in DNA Barcoding and Implications for Entomology.

 After reading the article, I have some comments:

The introduction can be extended. For example, to talk about known species for this region (in a narrow or broad sense) and what is known about their barcoding or other genetic studies. Although this is partly described in the Discussion, some literature data can also be given in the Introduction.

In the Materials and Methods section, a link to the sequencing center is given. However, it would be useful to indicate in the text which primers were used for PCR and sequencing in this particular cases. Readers who wish to study the same species might not need to look for additional information.

It is difficult to keep track of the numbers of the results obtained. In line 128 authors wrote  about 1,214 Linnean species found. In the next lines - 927 species are represented by a unique sequence from the study area, whereas the remaining 298 species... So, this is 1225 species in total. I think it's a mistake.

Lines 148-153 show the results of finding identical DNA sequences in different species. It is clear from the text of the article that the morphological differences in these groups of taxa are also not significant. But, before closing the species, they must be studied deeply. In such cases, it is necessary to examine another DNA region, for example, the nuclear genes of ribosomal RNA. This region is also often investigated and serves as a barcode for nuclear genes. Is there any such information for the species discussed in the article?

The fact that the obtained sequences for the vast majority of species coincide with those previously published for the same species from other studies indicates the stability of the COI analysis.

The method made it possible to identify 22 new taxa, whose sequences differ from closely related ones by more than 2% , i.e. at the interspecies level. In these cases, it is also possible to prove the existence of new taxa not only by careful morphological examination, but also by studying other genes in candidate species.

Therefore, in some cases, in addition to the well-established COI barcode, it is necessary to study another DNA fragment. This fragment must also be standardized in order to be able to compare samples. It is the large database of COI that makes this barcode method indispensable for the accurate identification of taxa.

Author Response

Reviewer 2. response

Many thanks for your careful review!

The article "DNA Barcode Library of Megadiverse Lepidoptera in an Alpine Nature Park (Italy) Reveals Unexpected Species Diversity" is devoted to the precise identification of butterfly species in a nature reserve and to study of the diversity of their DNA. The authors collected a large amount of material, characterized the specimens by morphological features, and thus carried out a preliminary identification to the species level. They then selected several representatives of each morphotype and sent samples for sequencing of a standard mtDNA fragment to a Canadian research center. Based on the results obtained, the authors identify species, intraspecific variability of the studied DNA fragment, genetic distances between taxa, and draw conclusions about the importance of barcoding natural specimens. The authors have extensive experience with Lepidoptera species in this area. Therefore, some data from previous works are also presented in this paper. The article fits the content of the special issue Frontiers in DNA Barcoding and Implications for Entomology.

 After reading the article, I have some comments:

The introduction can be extended. For example, to talk about known species for this region (in a narrow or broad sense) and what is known about their barcoding or other genetic studies. Although this is partly described in the Discussion, some literature data can also be given in the Introduction.

We expanded  parts of the introduction and discussion.

In the Materials and Methods section, a link to the sequencing center is given. However, it would be useful to indicate in the text which primers were used for PCR and sequencing in this particular cases. Readers who wish to study the same species might not need to look for additional information.

We added relevant primers to MM

It is difficult to keep track of the numbers of the results obtained. In line 128 authors wrote  about 1,214 Linnean species found. In the next lines - 927 species are represented by a unique sequence from the study area, whereas the remaining 298 species... So, this is 1225 species in total. I think it's a mistake.

Grateful that you spotted this error! In fact we now have undergone a re-calculations in EXCEL and have corrected all errors.

Lines 148-153 show the results of finding identical DNA sequences in different species. It is clear from the text of the article that the morphological differences in these groups of taxa are also not significant. But, before closing the species, they must be studied deeply. In such cases, it is necessary to examine another DNA region, for example, the nuclear genes of ribosomal RNA. This region is also often investigated and serves as a barcode for nuclear genes. Is there any such information for the species discussed in the article?

Rephrased and slightly expanded

The fact that the obtained sequences for the vast majority of species coincide with those previously published for the same species from other studies indicates the stability of the COI analysis.

The method made it possible to identify 22 new taxa, whose sequences differ from closely related ones by more than 2% , i.e. at the interspecies level. In these cases, it is also possible to prove the existence of new taxa not only by careful morphological examination, but also by studying other genes in candidate species.

We slightly adapted the Discussion

Therefore, in some cases, in addition to the well-established COI barcode, it is necessary to study another DNA fragment. This fragment must also be standardized in order to be able to compare samples. It is the large database of COI that makes this barcode method indispensable for the accurate identification of taxa.

We added the necessity of integrating nuclear markers into discussion and expanded literature