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(Chironomidae) in Heavy Metal Polluted Ponds

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(Chironomidae) in Heavy Metal Polluted Ponds DNA-BARCODING OF NON-BITING MIDGE LARVAE (CHIRONOMIDAE) IN HEAVY METAL POLLUTED PONDS By Valeria Manfredi Canterbury Christ Church University Thesis submitted for the degree of MSc by Research 2019 Table of Contents List of Figures and Tables ................................................................................................................ 4 Abstract .............................................................................................................................................. 6 Introduction ....................................................................................................................................... 7 Water quality assessment ............................................................................................................ 7 Environmental Pollution ............................................................................................................. 8 Parys Mountain and pollution .................................................................................................. 10 Heavy metals in the sediments ................................................................................................. 12 Benthic invertebrates ................................................................................................................ 13 Chironomidae ........................................................................................................................... 14 Molecular ecology .................................................................................................................... 16 DNA-barcoding as a molecular tool ......................................................................................... 20 Cytochrome Oxidase Subunit 1 (CO1) ..................................................................................... 21 Aims and Objectives ........................................................................................................................ 22 Material and Methods ..................................................................................................................... 23 Sampling sites ............................................................................................................................ 23 Chironomid larvae, sediment and water samplings ................................................................... 25 Sediment analysis ..................................................................................................................... 25 Statistical analysis ..................................................................................................................... 28 Molecular analysis ..................................................................................................................... 29 Phylogenetic analysis ................................................................................................................ 30 Assessment of genetic and community structure ....................................................................... 31 Community structure .................................................................................................................. 31 Results ............................................................................................................................................... 32 Environmental conditions and sediment analysis ..................................................................... 32 Magnetic properties ................................................................................................................... 35 DNA sequence and BLAST ...................................................................................................... 40 Phylogenetic analysis ................................................................................................................. 41 DNA barcoding and Community composition ........................................................................... 44 DNA sequence polymorphism ................................................................................................... 47 Discussion ......................................................................................................................................... 52 DNA-barcoding… ...................................................................................................................... 52 Community composition ............................................................................................................ 54 Environmental conditions ............................................................................................................. 56 Future directions ............................................................................................................................ 58 Conclusions ...................................................................................................................................... 59 Acknowledgments ........................................................................................................................... 61 References......................................................................................................................................... 62 Glossary............................................................................................................................................. 74 Appendix .......................................................................................................................................... 77 List of Figure and Tables Figure 1. Chironomids larvae. Figure 2. Map of the sampling area. Table 1. Protocol used in the Microwave Digester. Table 2. Mann-Whitney-U test results. Figure 3. Kruskal- Wallis test on the six environmental conditions Table 3. Concentrations in calibration units of Cu, Fe, Pb and Zn in sediment samples from Wales. Table 4. Concentrations in calibration units of Cu, Fe, Pb and Zn in water samples from Wales. Figure 4. Magnetic susceptibility among the seven ponds in Wales Table 5. Eigenvectors of the Principal Components Analysis. Only values above 0.3 were taken (first four Principal Components). Figure 5. Scree plot of the Eigenvalue across the sixteen components. Table 6. Average GenBank identity (using BLAST) of the chironomid larvae from Wales per site and per genus. Table 7. Population genetics statistics for global Chironomidae family OTUs (GenBank and Wales). Figure 6. Score plot of the autoscalated data. Figure 7. Phylogenetic tree. Figure 8. Phylogenetic networks for genera Table 8. Population genetics statistics for global Chironomida family OTUs in all the seven examined ponds present in Wales. Table 9. Pairwise Fst values of Chironomidae larvae among the seven ponds in Wales 4 Table 10. Simpson’s index across the seven ponds in Wales. Figure 9. Haplotype network of Chironomidae family based on variation of COI. Figure 10. Number (abundance) of chironomid genera for each collection site in Wales. Figure 11. Bray-Curtis similarity among collection sites. Figure S1. Three sampling sites within the seven ponds in Wales. Figure S2. Calibration curve for Cu,Fe,Pb and Zn. Figure S3. Outlier plot for Principal Component Analysis. Table S1. GPS coordinates of sampling sites. Table S2. Eigenanalysis of the Correlation Matrix Table S3. Overall correct classification in Discriminant analysis Table S4. Summary of Classification of the seven ponds in Wales Table S5. Squared distance between groups in discriminant analysis. Figure S4A-D. Zoomed in section of the phylogenetic tree. 5 Abstract Chironomids, or non-biting midges (Chironomidae), belong to the order of true flies (Diptera), and are frequently used as bio-indicators of water quality, where higher diversity levels reflect less polluted habitats. However, the identification of chironomids (as well as other freshwater invertebrates) is done using morphological characters, a process that can be difficult and time- consuming. With the use of the taxonomic identification tool DNA barcoding, it is possible to identify the specimens to genus or species level without the use of morphological identifications which often difficult and time-consuming. In this study, DNA barcoding using the mitochondrial (mt) DNA the cytochrome oxidase 1 gene (CO1) was used to identify chironomid larvae from seven different ponds in Wales in relationship to sediment and water environmental characteristics. Chironomid larvae, sediment and water samples were taken from five ponds in Parys Mountain, an old copper mine in northern Wales, and from two ponds in Cwm-Idwal in Snowdonia Natural Park. Because of the history of the mining site, four ponds from Parys Mountain were suspected to be polluted. Environmental conditions (pH, dissolved oxygen, conductivity, salinity and temperature), magnetic susceptibility (MS) and isothermal remanent magnetisation (IRM), and presence of heavy metals were analysed to characterize the sites and to understand the effect on chironomid larvae diversity and community structure. Chironomid larvae were DNA-barcoded and the genetic and community structure for each pond were calculated and analysed in relationship with environmental conditions, magnetic characteristics and presence of heavy metals. A total of 15 genera of chironomids were found among the seven ponds. The results indicated a significant difference between suspected non- polluted ponds (WAPM1,WACI1,WACI2) and most of the suspected polluted ponds (WAPM2,WAPM3,WAPM4,WAPM5);
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