DNA Barcodes Applied to a Rapid Baseline Construction in Biodiversity Monitoring for the Conservation of Aquatic Ecosystems in the Sian Ka’an Reserve (Mexico) and Adjacent Areas

Round 1

Reviewer 1 Report

The manuscript presents an outline of a eDNA and barcode library generating project with the emphasis on biomonitoring. The premise and general aims are of interest to readers and the wider field. There needs to be some careful attention to improve the structure of the introduction and discussion to more clearly present the aims and objectives and to outline the key findings, with regards to the data presented. Additional information regarding the methods used, and principally a completion of the statistical analyses, are needed prior to publication.

Introduction: there are many odd sentences to start paragraphs that could be modified to better link paragraphs together. The current format makes it difficult to follow the train of logic the authors are making to support their study aims and objectives.

31-35: expand the opening paragraph or integrate the two sentences into the following paragraph.

36: consider dropping ‘therefore’ from the start of the sentence.

62-65: incomplete/partial paragraph. This is an interesting topic, but it needs to be integrated with the introduction. Consider where you want to lead the reader and what points need to be made here to engage the reader towards the aims and objectives of the study.

95-121: These are all fragments of paragraphs and need to be put into a structure with the rest of the introduction.

122-126: This is a bit unclear. Is the study reanalyzing previous data obtained in previous publications or are the findings new?

If the aim is to assess eDNA sampling for use in biomonitoring it would be good to introduce the topic to the readers see

Seymour, M. et al. (2020) ‘Executing multi-taxa eDNA ecological assessment via traditional metrics and interactive networks’, Science of The Total Environment, 729, p. 138801. doi: 10.1016/j.scitotenv.2020.138801.

Pereira-da-Conceicoa, L. et al. (2021) ‘Metabarcoding unsorted kick-samples facilitates macroinvertebrate-based biomonitoring with increased taxonomic resolution, while outperforming environmental DNA’, Environmental DNA. John Wiley & Sons, Ltd, 3(2), pp. 353–371. doi: https://doi.org/10.1002/edn3.116.

153-154: is there a reference guide that can be cited for the taxonomic assignments?

166-184: specify that these steps are to generate individual sequences for the barcode database. I would also suggest specifying that these extracts are individual specimens.

212: add space after employing

194-242: I would argue that this is metabarcoding and not metagenomics since amplicons and not genomes are used to generate the data.

219-220: Were the paired end tags unique pairs, or were the paired ends unique tag combinations (across the libraries)?

245-250: betapart is also generally used to generate the complementary simpson index and the corresponding nestedness value from the simspon and Sorenson metrics. Is there a reason why Sorenson was used that can be provided here?

Results and discussion – there should be separate sections for 1) sequencing results 2) metabarcoding data 3) community statistical analyses. Presently discussion is missing regarding the inference of the molecular derived communities from this study to biomonitoring suggested policy, as suggested in the introduction. See

Seymour, M. et al. (2021) ‘Environmental DNA provides higher resolution assessment of riverine biodiversity and ecosystem function via spatio-temporal nestedness and turnover partitioning’, Communications Biology, 4(1), p. 512. doi: 10.1038/s42003-021-02031-2.

Additional information regarding the sequencing output should be provided, including number of reads, chimera detection, control sample results/contamination checking, etc.

It is unclear what the phyologentic trees are illustrating with regards to the main theme of the paper (biomonitoring). It may be more informative to relocate some of the extra figures to an appendix and consider including additional illustrations to show the spatial distribution of the species and community biodiversity.

255- again these are not genomes, but amplicons. Specific primers targeting fragements of genes were used to generate metabarcoding libraries (e.g. amplicons). Genomics would require different molecular methods, such as shotgun sequencing to capture the genomic information.

258-261: please outline what the suggestions were and what methods were used in the present study.

314-322 as well as throughout the results: replace qualitative statements such as “less represented” with quantitative statements to avoid ambiguity and to make the interpretations more convincing.

428-445: These statements should be verified with some statistical test. A glm may suffice, for example, but a mixed effect model may be needed to account for spatial variation or unequal sampling effort between samples.

484-502: There should be a direct link between the data generated in this study to biomonitoring interpretation. Calling for more data and investment is standard rhetoric at this stage. What major findings from the data can be used to inform managers wanting to manage your study area?

Figures/Tables

Figure 1: indicate the location in the legend.

Table 2: indicate the 5’ to 3’ direction of the primers as written in the table

Figure 3: provide the support values for the node

Figure 4: provide the support values for the node

Figure 5: provide the support values for the node

Figure 6: provide the support values for the node

Consider moving figure 3-6 & 7to an appendix

Table 3: These are the number of reads per species, correct? It would be more informative to report the number of reads per sample, perhaps. It may be also useful to show instead a figure with relative read counts per species across the locations.

Figure 7: provide the support values for the nodes.

Figure 8: clarify what dissimilarity measure is shown in the legend. Adjust the color to show the full range of color in the table to reflect the color in the legend. At present, there are not blue squares.

 

Author Response

Replies to Referee 1

The manuscript presents an outline of a eDNA and barcode library generating project with the emphasis on biomonitoring. The premise and general aims are of interest to readers and the wider field. There needs to be some careful attention to improve the structure of the introduction and discussion to more clearly present the aims and objectives and to outline the key findings, with regards to the data presented. Additional information regarding the methods used, and principally a completion of the statistical analyses, are needed prior to publication.

R: We corrected the introduction and discussion accordingly. Methods were explained in more detail, and we give boostrap support after 500 replications to our trees, altohough this not usual in ID trees (our trees are not phylogenetic in the strict sense). See Elías-Gutiérrez et al. (2018) or Montes-Ortiz and Elías-Gutiérrez (2020)

Introduction: there are many odd sentences to start paragraphs that could be modified to better link paragraphs together. The current format makes it difficult to follow the train of logic the authors are making to support their study aims and objectives.

R: We prepared a new version following a more logical connection between paragraphs.

31-35: expand the opening paragraph or integrate the two sentences into the following paragraph.

R: Done

36: consider dropping ‘therefore’ from the start of the sentence.

R: Done

62-65: incomplete/partial paragraph. This is an interesting topic, but it needs to be integrated with the introduction. Consider where you want to lead the reader and what points need to be made here to engage the reader towards the aims and objectives of the study.

R: We changed the sequence of the introduction. This point was included later.

95-121: These are all fragments of paragraphs and need to be put into a structure with the rest of the introduction.

R: Corrected

122-126: This is a bit unclear. Is the study reanalyzing previous data obtained in previous publications or are the findings new?

R: We corrected the goals of this study.

If the aim is to assess eDNA sampling for use in biomonitoring it would be good to introduce the topic to the readers see

Seymour, M. et al. (2020) ‘Executing multi-taxa eDNA ecological assessment via traditional metrics and interactive networks’, Science of The Total Environment, 729, p. 138801. doi: 10.1016/j.scitotenv.2020.138801.

Pereira-da-Conceicoa, L. et al. (2021) ‘Metabarcoding unsorted kick-samples facilitates macroinvertebrate-based biomonitoring with increased taxonomic resolution, while outperforming environmental DNA’, Environmental DNA. John Wiley & Sons, Ltd, 3(2), pp. 353–371. doi: https://doi.org/10.1002/edn3.116.

R: We have seen both studies, and our personal experience, and some published studies do not agree at all with the results presented mainly in the first study. For example, see Karanovic et al. (2020) for experience with 18s. We follow the route given by Valdez-Moreno et al. (2019), trying to go to species level, not to genera. About the second, it is quite limited in scope. Their database has only 249 specimens. Just for comparison our database for the paper of Valdez-Moreno et al. (2019) and this one, we used a database (DS-EBACALAR) with 3534 specimens among others, as explained in the manuscript. 

153-154: is there a reference guide that can be cited for the taxonomic assignments?

R: We added some citations and examples how we identified the material

166-184: specify that these steps are to generate individual sequences for the barcode database. I would also suggest specifying that these extracts are individual specimens.

R: It is clearly stated in the title of the section

212: add space after employing

R: Done

194-242: I would argue that this is metabarcoding and not metagenomics since amplicons and not genomes are used to generate the data.

R: To avoid discussions we used DNA barcoding and metabarcoding.

219-220: Were the paired end tags unique pairs, or were the paired ends unique tag combinations (across the libraries)?

R: Right now it is explained in the text

245-250: betapart is also generally used to generate the complementary simpson index and the corresponding nestedness value from the simspon and Sorenson metrics. Is there a reason why Sorenson was used that can be provided here?

R: Yes, thank you for your observation. We used Sorensen index since data are absence-presence. In text we change line 249. 

 Results and discussion – there should be separate sections for 1) sequencing results 2) metabarcoding data 3) community statistical analyses. Presently discussion is missing regarding the inference of the molecular derived communities from this study to biomonitoring suggested policy, as suggested in the introduction. See

Seymour, M. et al. (2021) ‘Environmental DNA provides higher resolution assessment of riverine biodiversity and ecosystem function via spatio-temporal nestedness and turnover partitioning’, Communications Biology, 4(1), p. 512. doi: 10.1038/s42003-021-02031-2.

R: The sections are clearly identified. The suggested study has a different focus from ours. Temperate systems have low diversity with high abundance. The opposite is in tropical systems: high diversity with low abundance, mostly in systemas as ours, characterized by their oligotrophic nature.

Additional information regarding the sequencing output should be provided, including number of reads, chimera detection, control sample results/contamination checking, etc.

R: Done

It is unclear what the phyologentic trees are illustrating with regards to the main theme of the paper (biomonitoring). It may be more informative to relocate some of the extra figures to an appendix and consider including additional illustrations to show the spatial distribution of the species and community biodiversity.

R: The focus is not biomonitoring, the focus is creating the baselines for biomonitoring. It is clearly stated in the goal of the study. Please see Hajibabaei et al. (2016); Moriniere et al. (2019). Additionally, we must emphazise: our trees are ID, not really phylogenetic, although they show a limited phylogenetic signal. This is a common confussion: Please read explanation in Quiroz-Vazquez and Elías-Gutiérrez (2009), and notice that we never used the word phylogenetic in these papers: Andrade-Sossa et al. (2020); Gutiérrez-Aguirre et al. (2020); Montes-Ortiz and Elías-Gutiérrez (2020). That is why in ample comparisons we do not provide support to the branches, they do not provide additional information. At the end, we discuss the future application for biomonitoring.

255- again these are not genomes, but amplicons. Specific primers targeting fragements of genes were used to generate metabarcoding libraries (e.g. amplicons). Genomics would require different molecular methods, such as shotgun sequencing to capture the genomic information.

R: We changed the term.

258-261: please outline what the suggestions were and what methods were used in the present study.

R: Done

314-322 as well as throughout the results: replace qualitative statements such as “less represented” with quantitative statements to avoid ambiguity and to make the interpretations more convincing.

R: Done

428-445: These statements should be verified with some statistical test. A glm may suffice, for example, but a mixed effect model may be needed to account for spatial variation or unequal sampling effort between samples.

R: Thank you for your suggestion. But in our case the sampling effort was standardized (please look for M&M). Additionally, as we comment previously, data used were absence-presence (0-1), Moreover, absence-presence data were generated, not for a typical sample organism separation, data about species presence were based on BINs. Then, the possible bias, in sample comparisons based on Sorensen index is negligible.

484-502: There should be a direct link between the data generated in this study to biomonitoring interpretation. Calling for more data and investment is standard rhetoric at this stage. What major findings from the data can be used to inform managers wanting to manage your study area?

R: We clarified this point at the end of this paragraph

Figures/Tables

Figure 1: indicate the location in the legend.

R: Done

Table 2: indicate the 5’ to 3’ direction of the primers as written in the table

R: Done

Figure 3: provide the support values for the node

Figure 4: provide the support values for the node

Figure 5: provide the support values for the node

Figure 6: provide the support values for the node

Consider moving figure 3-6 & 7to an appendix

R: We provided support. We do not agree to put in appendix these figures, because they are the main result for the baseline.

Table 3: These are the number of reads per species, correct? It would be more informative to report the number of reads per sample, perhaps. It may be also useful to show instead a figure with relative read counts per species across the locations.

R: Each location is represented by one sample, the reads correspond to the samples.

Figure 7: provide the support values for the nodes.

R: Figure title was wrong, in really it is not a cladogram it is a similarity dendrogram

Title was changed to:

Figure 7. Similarity dendrogram of the localities sampled. Abbreviations are the same as Fig. 8.

Then, values of branches are in the plot axis.

Figure 8: clarify what dissimilarity measure is shown in the legend. Adjust the color to show the full range of color in the table to reflect the color in the legend. At present, there are not blue squares.

R: Legend was changed to:

Figure 8. Sorensen dissimilarities among sampling aquatic systems. Abbreviations: Tres Reyes sinkhole 2, Rey2; Chunyaxche 2, Chunya 2; Pucte 2 sinkhole, Pucte; Chancah Veracruz, Chancah; Santa Teresa sinkhole, Teresa; Laguna Muyil 1, Muyil 1; Pucte Cafetal sinkhole, Cafetal; Tres Reyes sinkhole 1, Rey1; Minicenote sinkhole, Mini; Sijil Noh Há sinkhole, NohHha; Chunyaxche 1, Chunya1; Km 48, Km48; El Toro sinkhole, Toro; Laguna Muyil 2, Muyil 2.

Colors were changed

References cited

Andrade-Sossa, C., L. Buitron-Caicedo & M. Elías-Gutiérrez, 2020. A new species of Scapholeberis Schoedler, 1858 (Anomopoda: Daphniidae: Scapholeberinae) from the Colombian Amazon basin highlighted by DNA barcodes and morphology. Peerj 8 doi:10.7717/peerj.9989.

Elías-Gutiérrez, M., M. Valdez-Moreno, J. Topan, M. R. Young & J. A. Cohuo-Colli, 2018. Improved protocols to accelerate the assembly of DNA barcode reference libraries for freshwater zooplankton. Ecology and Evolution 8(5):3002-3018 doi:10.1002/ece3.3742.

Gutiérrez-Aguirre, M. A., A. Cervantes-Martinez, M. Elías-Gutiérrez & A. Lugo-Vazquez, 2020. Remarks on Mastigodiaptomus (Calanoida: Diaptomidae) from Mexico using integrative taxonomy, with a key of identification and three new species. Peerj 8:e8416 doi:10.7717/peerj.8416.

Hajibabaei, M., D. J. Baird, N. A. Fahner, R. Beiko & G. B. Golding, 2016. A new way to contemplate Darwin's tangled bank: how DNA barcodes are reconnecting biodiversity science and biomonitoring. Philosophical Transactions of the Royal Society B-Biological Sciences 371(1702) doi:10.1098/rstb.2015.0330.

Karanovic, I., P. T. M. Huyen, H. Yoo, Y. Nakao & A. Tsukagoshi, 2020. Shell and appendages variability in two allopatric ostracod species seen through the light of molecular data. Contributions to Zoology 89(3):247-269 doi:10.1163/18759866-20191423.

Montes-Ortiz, L. & M. Elías-Gutiérrez, 2020. Water Mite Diversity (Acariformes: Prostigmata: Parasitengonina: Hydrachnidiae) from Karst Ecosystems in Southern of Mexico: A Barcoding Approach. Diversity-Basel 12(9) doi:10.3390/d12090329.

Moriniere, J., M. Balke, D. Doczkal, M. F. Geiger, L. A. Hardulak, G. Haszprunar, A. Hausmann, L. Hendrich, L. Regalado, B. Rulik, S. Schmidt, J. W. Wagele & P. D. N. Hebert, 2019. A DNA barcode library for 5,200 German flies and midges (Insecta: Diptera) and its implications for metabarcoding-based biomonitoring. Molecular Ecology Resources 19(4):900-928 doi:10.1111/1755-0998.13022.

Quiroz-Vazquez, P. & M. Elías-Gutiérrez, 2009. A New Species of the Freshwater Cladoceran Genus Scapholeberis Schoedler, 1858 (Cladocera: Anomopoda) from the Semidesert Northern Mexico, Highlighted by DNA Barcoding. Zootaxa 2236:50-64.

Valdez-Moreno, M., N. V. Ivanova, M. Elías-Gutiérrez, S. L. Pedersen, K. Bessonov & P. D. N. Hebert, 2019. Using eDNA to biomonitor the fish community in a tropical oligotrophic lake. Plos One 14(4) doi:10.1371/journal.pone.0215505.

 

Reviewer 2 Report

The manuscript presents DNA barcodes as a powerful tool for the rapid assessment of most animal taxa inhabiting any freshwater system. The authors analyzed different freshwater ecosystems on the Yucatan Peninsula and they detected 167 Molecular Operational Taxonomic Units (MOTU), from which they identified 43 species. This study gives new insight biodiversity of freshwater habitats in Mexico. However, the authors concluded that most other zooplankton MOTU are a possible new species for the sciences (lines 26-27). This does not have to be true because many of the zooplankton species (even quite common) do not have barcodes in databases (BOLD, GenBank). The other problem is the level of taxonomical identification of deposited sequences (especially for copepods). Therefore, barcodes and eDNA are very valuable tools for discovering biodiversity, however, this method needs reference databases with nucleotide sequences for accurately identified species which are critically needed to allow taxonomic identification of MOTU. Nevertheless, in recent years the level of taxonomic identification in developed countries has been dramatically reduced due to the well-known ‘global taxonomy crisis’. This should be at least discussed.

The other weakness of the manuscript is the sequencing data analysis which was made only via BOLD tools.

I noticed grammatical errors. So, I suggest reviewing the text by the native in the language.

 

Minor comments:

Line 96: ‘It’ – should be ‘it’

Lines 105-107: This paragraph could more emphasize the lack of proper taxonomical identification due to the ‘global taxonomy crisis’ which are constraints in discover biodiversity. Nowadays, we have discovered many new barcodes but we do not know what is it.

Line 140: remove dot after ‘polygon’

Line 150: ‘processed in Canada’ - provide the same information as in line 196-197

Line 153-154: ‘Stereo Microscope’ change to ‘stereo microscope’

Line 252: ‘All aquatic ecosystems studied here are of transparent blue water’ - one was eutrophic? line 84, and photography.

Line 323-331: domination of Ceriodaphnia is common in highly eutrophic systems, while higher share Calanoida is typical for oligotrophic lakes

Line 329: ‘Ceriodaphnia cf. rigaudi’ – italics species name

Figure 4: why Mesocyclops edax is separated from other Cyclopoida species? e.g. M. longisetus

Lines 337-338: ‘All other taxa registered here are restricted to Yucatan or the southern of Mexico’ - not only, there are at least few species with a more global range like Tropocyclops prasinus (also in groundwater), Macrocyclops albidus.

Line 355: ‘When calanoids were present, usually they were the most common group in the samples’ - maybe due to oligotrophic?

Lines 455, 464: correct species names

Lines 484-485: ‘As it was said previously, there is not any group or lab in the world with the ability to identify all species found in any aquatic ecosystem’ - but for sure there are specialists from some groups like Cladocera, Cyclopoida, Calanoida, Ostracoda, etc.

Author Response

Replies to referee 2

The manuscript presents DNA barcodes as a powerful tool for the rapid assessment of most animal taxa inhabiting any freshwater system. The authors analyzed different freshwater ecosystems on the Yucatan Peninsula and they detected 167 Molecular Operational Taxonomic Units (MOTU), from which they identified 43 species. This study gives new insight biodiversity of freshwater habitats in Mexico. However, the authors concluded that most other zooplankton MOTU are a possible new species for the sciences (lines 26-27). This does not have to be true because many of the zooplankton species (even quite common) do not have barcodes in databases (BOLD, GenBank). The other problem is the level of taxonomical identification of deposited sequences (especially for copepods).

R: Mexico has one of the most complete public databases of freshwater zooplankton. Just in BOLD is the second with more Branchiopoda (2196 specimens, representing at least 113 species),the third in Copepoda (2536 specimens, with 54 species) and the first on Rotifera (644 specimens, 95 species). Most of the OTUs we can not identify have been new to science, and we are publishing them. However, to avoid discussions we deleted this part. We do not understand the sense of the level of identification in copepods. We are making progress with them, because we keep the vouchers (see the paper by Elías-Gutiérrez et al., 2018).

Therefore, barcodes and eDNA are very valuable tools for discovering biodiversity, however, this method needs reference databases with nucleotide sequences for accurately identified species which are critically needed to allow taxonomic identification of MOTU. Nevertheless, in recent years the level of taxonomic identification in developed countries has been dramatically reduced due to the well-known ‘global taxonomy crisis’. This should be at least discussed.

R:  The goal in this paper is not to go deep in the discussion of the taxonomic impediment. We just pretend the implementation of a baseline of sequencies and demonstrate how it works with one of the most studied groups with DNA barcodes, the fishes. However, we included some citation about this problem.

The other weakness of the manuscript is the sequencing data analysis which was made only via BOLD tools.

R: We do not understand why it is a weakness, it has been demonstrated that simple analyses do not change the final conclusions and the Barcode Index Name has been widely adapted (see Mercado-Salas et al., 2018; Miller et al., 2016; Moriniere et al., 2017) for examples. We applied a bootstrap method with 500 replications in this new version, and the main results did not change. If the scope of the paper was to contrast species delimitation, we will use different methods as ABGD or coalescence, as we did in Gutiérrez-Aguirre et al. (2020) but this was not the goal for this study.

I noticed grammatical errors. So, I suggest reviewing the text by the native in the language.

 R: We double checked the English.

Minor comments:

Line 96: ‘It’ – should be ‘it’

R: We did not find in this line this observation:

Lines 105-107: This paragraph could more emphasize the lack of proper taxonomical identification due to the ‘global taxonomy crisis’ which are constraints in discover biodiversity. Nowadays, we have discovered many new barcodes, but we do not know what is it.

R: We changed the introduction, and we are aware about this problem. It was mentioned but is not the scope of this study to discuss it.

Line 140: remove dot after ‘polygon’

R: Done

Line 150: ‘processed in Canada’ - provide the same information as in line 196-197

R: It was changed

Line 153-154: ‘Stereo Microscope’ change to ‘stereo microscope’

R: It was changed

Line 252: ‘All aquatic ecosystems studied here are of transparent blue water’ - one was eutrophic? line 84, and photography.

R: We corrected the paragraph.

Line 323-331: domination of Ceriodaphnia is common in highly eutrophic systems, while higher share Calanoida is typical for oligotrophic lakes

R: Not the case here, we found it in a blue water system.

Line 329: ‘Ceriodaphnia cf. rigaudi’ – italics species name

R: It was changed

Figure 4: why Mesocyclops edax is separated from other Cyclopoida species? e.g. M. longisetus

R: It is corrected.

Lines 337-338: ‘All other taxa registered here are restricted to Yucatan or the southern of Mexico’ - not only, there are at least few species with a more global range like Tropocyclops prasinus (also in groundwater), Macrocyclops albidus.

R: We added an explanation

Line 355: ‘When calanoids were present, usually they were the most common group in the samples’ - maybe due to oligotrophic?

R: This is not the case, for example in oligotrophic lake Bacalar Arctodiaptomus dorsalis and Pseudodiaptomus marshi are the dominants (see Elías-Gutiérrez et al., 2018). As well in the oligotrophic Cenote Azul, a form of A. dorsalis is also dominant (see (Montes-Ortiz and Elías-Gutiérrez, 2018)

Lines 455, 464: correct species names

R: Corrected

Lines 484-485: ‘As it was said previously, there is not any group or lab in the world with the ability to identify all species found in any aquatic ecosystem’ - but for sure there are specialists from some groups like Cladocera, Cyclopoida, Calanoida, Ostracoda, etc.

R: Yes, but they are specialized in biotas from different regions. In our experience, even for them is difficult to identify the Mexican material.

 

References

Gutiérrez-Aguirre, M. A., A. Cervantes-Martinez, M. Elías-Gutiérrez & A. Lugo-Vazquez, 2020. Remarks on Mastigodiaptomus (Calanoida: Diaptomidae) from Mexico using integrative taxonomy, with a key of identification and three new species. Peerj 8:e8416 doi:10.7717/peerj.8416.

Mercado-Salas, N. F., S. Khodami, T. C. Kihara, M. Elías-Gutiérrez & P. M. Arbizu, 2018. Genetic structure and distributional patterns of the genus Mastigodiaptomus (Copepoda) in Mexico, with the description of a new species from the Yucatan Peninsula. Arthropod Systematics & Phylogeny 76(3):487-507.

Miller, S. E., A. Hausmann, W. Hallwachs & D. H. Janzen, 2016. Advancing taxonomy and bioinventories with DNA barcodes. Philosophical Transactions of the Royal Society B-Biological Sciences 371(1702) doi:10.1098/rstb.2015.0339.

Montes-Ortiz, L. & M. Elías-Gutiérrez, 2018. Faunistic survey of the zooplankton community in an oligotrophic sinkhole, Cenote Azul (Quintana Roo, Mexico), using different sampling methods, and documented with DNA barcodes. Journal of Limnology 77(3):428-440 doi:10.4081/jlimnol.2018.1746.

Moriniere, J., L. Hendrich, M. Balke, A. J. Beermann, T. Konig, M. Hess, S. Koch, R. Muller, F. Leese, P. D. N. Hebert, A. Hausmann, C. D. Schubart & G. Haszprunar, 2017. A DNA barcode library for Germanys mayflies, stoneflies and caddisflies (Ephemeroptera, Plecoptera and Trichoptera). Molecular Ecology Resources 17(6):1293-1307 doi:10.1111/1755-0998.12683.