EGU2020-19272
https://doi.org/10.5194/egusphere-egu2020-19272
EGU General Assembly 2020
© Author(s) 2021. This work is distributed under
the Creative Commons Attribution 4.0 License.

Shotgun DNA, pollen and biological multi-proxy analysis of Lateglacial lake sediments from Monticchio, Italy

Laura Parducci1, Kevin Nota1, Willy Tinner3, Jacqueline van Leeuwen3, Pim van der Knaap3, Dirk Sachse4, Zuobing Liang4, Achim Brauer5, Markus J. Schwab5, Xuery Zhao5, Aldo Marchetto6, Andrea Lami6, and Sabine Wulf7
Laura Parducci et al.
  • 1Uppsala University, Ecology and Genetics, Plant Ecology and Evolution, Uppsala, Sweden (laura.parducci@ebc.uu.se)
  • 3Institute of Plant Sciences, University of Bern, Switzerland
  • 4Section Geomorphology, GFZ German Research Centre for Geosciences, Potsdam, Germany
  • 5Section Climate Dynamics and Landscape Evolution, GFZ German Research Centre for Geosciences, Potsdam, Germany
  • 6National Research Council of Italy, Water Research Institute (CNR-IRSA) Verbania Pallanza, Italy
  • 7School of the Environment, Geography and Geosciences, University of Portsmouth, United Kingdom

We used shotgun DNA sequencing of the full metagenome preserved in varved lake sediments from southern Italy (Lago Grande di Monticchio) to investigate the whole diversity of taxonomic groups present. We combine sedimentary aDNA and pollen data as well as other biological multi-proxy data and tested if it was possible to correlate the relative abundances of plants and other biological communities to distinct climatic shifts that occurred between the Late Glacial and Holocene. In addition, we used the metabarcoding technique to compare the two sequencing approaches specifically for plants.

Our studies showed that the inhibition of DNA replication was almost absent in older (full glacial) sediment samples while it increased substantially in more recent samples. DNA provides a strong signal of plant community changes and a large number of new plant taxa were recorded. A comparison between sequencing approaches and proxies highlights differences and similarities and supports earlier findings that plants growing close to or within a lake are often recorded by DNA and that DNA provides important complementary information to that collected from palaeoecological analyses. Nevertheless, increasing DNA reference libraries and enrichment strategies prior to sequencing are necessary to improve the potential and accuracy of plant identification using the metagenomic approach.

How to cite: Parducci, L., Nota, K., Tinner, W., van Leeuwen, J., van der Knaap, P., Sachse, D., Liang, Z., Brauer, A., Schwab, M. J., Zhao, X., Marchetto, A., Lami, A., and Wulf, S.: Shotgun DNA, pollen and biological multi-proxy analysis of Lateglacial lake sediments from Monticchio, Italy, EGU General Assembly 2020, Online, 4–8 May 2020, EGU2020-19272, https://doi.org/10.5194/egusphere-egu2020-19272, 2020.

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Display material version 2 – uploaded on 04 May 2020
Version description: I added a missing label with colours for the DNA diagram in the third slide and add more information on the[...]
  • CC1: Comment on EGU2020-19272, Charline Giguet-Covex, 04 May 2020

    Dear Laura,

    Do you have any idea of the cause(s) of the inhibition? In the middle of the record we cannot see specific lithology but maybe you have information from XRF data or organic matter analyses?

    It is a little out of the topic, but I was asking me if there are studies on the effects of volcanic ash on the aquatic life (biodiversity, primary productivity)?

    Charline

    • AC2: Reply to CC1, Laura Parducci, 04 May 2020

      Hi Charline, there seems to be some but weak correlation with organic in the core. It increase from 14.5 but it decreases again afterwards and at the same time we have diatoms that dominate. 

      • AC5: Reply to AC2, Kevin Nota, 04 May 2020

        To add to Laura's comment. We did correlate TOC from a section of the core (~11-22 kyr. BP, not published), but the correlation was weak and not significant. 

    • AC3: Reply to CC1, Laura Parducci, 04 May 2020

      Charline, I dont know much about ash and impact aquatic life unfortunately.

  • AC1: Comment on EGU2020-19272, Sabine Wulf, 04 May 2020

    Dear Charlene,

    regarding your question on the effect of volcanic ash on the aquatic life we have not done systematic analyses at this point. We noticed, however, that the amount of planktonic diatoms is strongly increased after the deposition of thick (several centimeter) tephra layers. I agree it would be worth to look into much more detail on the diatom quantities and species change, especially since there are so many tephra events to investigate!

    Kind regards,

    Sabine 

    • AC4: Reply to AC1, Laura Parducci, 04 May 2020

      Thank you Sabine! 

      • CC2: Reply to AC4, Charline Giguet-Covex, 04 May 2020

        Thank you Laura and Sabine for your precisions. I would be happy to see more results from this very good lake sediment record :)

  • CC3: Comment on EGU2020-19272, Sandra Gomes, 05 May 2020

    Dear Laura Parducci and co-authors,

    Thank you for sharing the information about Shotgun DNA and pollen. I would like to ask when question because I find this approach very interesting, sophisticated and hopefully with future. 

    As the shotgun DNA or metabarcoding (or another similar technique) been applied to pollen from marine sediments? 

    • AC7: Reply to CC3, Laura Parducci, 05 May 2020

      Hi and nice to meet you. We did not have shotgun on pollen, which can be done however. We did shotgun on sedimentary DNA. We do work with pollen only however and we did metabarcoding on bulk pollen but fresh and not ancient; it works. It can certainly also work with shotgun seqeuncing. What exactly is your material?

      • CC6: Reply to AC7, Sandra Gomes, 05 May 2020

        Hemipelagic clays mostly from a IODP core.

  • AC6: Comment on EGU2020-19272, Laura Parducci, 05 May 2020

    Hi and nice to meet you. We did not have shotgun on pollen, which can be done however. We did shotgun on sedimentary DNA. We do work with pollen only however and we did metabarcoding on bulk pollen but fresh and not ancient; it works. It can certainly also work with shotgun seqeuncing. What exactly is your material?

  • CC4: Comment on EGU2020-19272, Kathleen Stoof-Leichsenring, 05 May 2020

    Dear Laura,

    I would like to learn more about your approch to identify inhibition in the samples and how this effects diversity in your samples. You use qPCR and different dilution sof DNA extracts. How does this affect the diversity in your samples? Is a lower concentration of DNA better? How many cycles to you typically run in teh metabarcoding PCR? Do you think diversity id more affected by PCR cycling number or inhibition of PCR?

    Thanks Kathleen 

    • AC8: Reply to CC4, Kevin Nota, 06 May 2020

      Hi Kathleen, 

      For the qPCR inhibition testing, we spike the qPCR reactions with a known synthetic oligo that we designed, which we then amplify. We dilute the DNA extract down until we see an amplification that is nearly identical to our positive control, this gives us an indication of the level of inhibition in our sample. The only problem with this is that the polymerase we use for metabarcoding is less sensitive to inhibitors than our qPCR Taq-polymerase. So when we set up the metabarcoding we didn't correct for PCR-inhibitors (We were under time pressure and didn't sufficiently check the amplification efficiency in inhibited samples). When we got the sequences back we saw a clear decrease in reads acquired per sample which is quite highly correlated with the inhibition that we found with qPCR. In the presentation we excluded any sample that showed very low read counts due to inhibition, so we haven't really looked into how the diversity in our samples changed between inhibited samples and not inhibited samples, we did however made a quick PCA (unfiltered, dereplicated reads) and it looks like inhibited samples group closer with PCR-negatives. I think the amplification in our inhibited samples becomes more random, so it will affect diversity, but how and how much we can't say at this point. We will look into this soon:) For the PCR we use typically 50 PCR cycles, with Platinum II hot-start Taq.   

      We did our extraction with the PowerSoil kit but didn't use any other kits to remove PCR inhibitors. Someone mentioned that Zymospin PCR inhibitor removal columns are pretty good, so we will probably check those out.

      Kevin

      • CC7: Reply to AC8, Kathleen Stoof-Leichsenring, 07 May 2020

        Dear Kevin,

        thanks for the detailed reply. We typically use a clean up kit after extraction with PowerSoil, which purifies and concentrates the DNA extracts. With this we have relatively, constantly nice PCR products, IN oiur recent projects we also shifted to 40 cycles in the PCR, because we got a lot of critisms in reviews when doing 50 cycles (especially for modern/historis lake sediment samples, so not really ancient). We already saw that diversity increased in our PCR products, because we have more bands of different, but expected, length in the PCRs with 40 cycles compared to 50 cycle PCR products, but we will be sure, when we have sequenced these products. Concerning qPCR, which Kit (polymerase) do you use and are you using the typically g/h primers or another plant marker? 

        Kind regards

        Kathleen

        • AC10: Reply to CC7, Kevin Nota, 08 May 2020

          Thank you so much for your reply Kathleen. What clean-up kits do you usually use? We are planning to soon send new samples from the Monticchio core for sequencing and also trying some capture. Getting rid of the inhibitors would be amazing! It will also be great to compare the sequence results before and after the clean up to see what the effect of the inhibition is. Using fewer PCR cycles would be nice to test as well on our ancient samples, I think the fewer cycles the better, it will probably also reduce contamination.

          The qPCR kit we use is TATAA SYBR GtandMaster mix (Taq). For metabarcoding, we use g/h primers, but we will be testing ITS primers for Asterales and Poales as well. For testing inhibition, we have primer set specific for the spike in template.

          Kevin

  • CC5: Comment on EGU2020-19272, Charline Giguet-Covex, 05 May 2020

    Laura, to follow on discussion on inhibition. Did you try to use commercial kits to remove them. My experience with that was on soil samples. The kit allows to remove a significant part (probably humic substances) of inhibitors but probably not all of them.

    • AC9: Reply to CC5, Kevin Nota, 06 May 2020

      Hi Charline, I answered your question in the reply to Kathleen.

      Kevin

      • CC8: Reply to AC9, Charline Giguet-Covex, 12 May 2020

        thanks for your reply Kevin.

        I don't know if it is the best, but for the inhibitors in my soil samples, I used the OneStepTM PCR Inhibitor Removal Kit.

        best

        Charline

         

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