Periodic Reporting for period 1 - PALECOEVOL (Paleo-ecogenomics in Daphnia: developing a model for studying evolutionary impacts of climate and environmental change)
Reporting period: 2015-06-01 to 2017-05-31
Global climate change and anthropogenic pollution are among the most prominent forces that affect biodiversity. Numerous recent examples demonstrate microevolutionary changes in response to an increase in temperature and added indirect effects of anthropogenic stressors, magnifying the negative effects on biological systems. Temperature changes have been especially pronounced in the polar regions, with the Arctic warming faster than any other region on earth. In Greenland, air temperature has repeatedly gone through cycles of warm and cold periods over the past 2000 years. In order to study the response of organisms and their populations, historical datasets across centuries are especially useful to document genomic alterations related to such environmental changes.
In this project, paleogenetic and paleogenomic methods were applied to reconstruct ancient populations and their individual genomes. The overarching goal of this project was to study the genomic responses of the ecological and genomic model organism Daphnia, a small aquatic crustacean that is central to aquatic foodwebs, to repeated shifts in temperature and related environmental parameters.
A new method was refined for amplifying ancient DNA of eggs that lay dormant in the sediment of lakes. This DNA amplification is necessary to allow sequencing of the minute amount of DNA material present in these eggs. The successful amplification of DNA of 300 eggs collected from sediment (10 to 300 years old) was achieved, and the resulting genomic libraries are now being prepared for sequencing. In the next step, this will enable the association of genetic and genomic changes with detailed information on the environmental history on a long-term temporal scale to uncover genomic mechanisms and adaptation to rapidly changing environments. A preliminary population genetic analysis detected identical microsatellite genotypes across hundreds of years, and suggests genetic stability despite rapid environmental change.
In this project, paleogenetic and paleogenomic methods were applied to reconstruct ancient populations and their individual genomes. The overarching goal of this project was to study the genomic responses of the ecological and genomic model organism Daphnia, a small aquatic crustacean that is central to aquatic foodwebs, to repeated shifts in temperature and related environmental parameters.
A new method was refined for amplifying ancient DNA of eggs that lay dormant in the sediment of lakes. This DNA amplification is necessary to allow sequencing of the minute amount of DNA material present in these eggs. The successful amplification of DNA of 300 eggs collected from sediment (10 to 300 years old) was achieved, and the resulting genomic libraries are now being prepared for sequencing. In the next step, this will enable the association of genetic and genomic changes with detailed information on the environmental history on a long-term temporal scale to uncover genomic mechanisms and adaptation to rapidly changing environments. A preliminary population genetic analysis detected identical microsatellite genotypes across hundreds of years, and suggests genetic stability despite rapid environmental change.
The first task of the project was concerned with obtaining the biological material from Greenland. An expedition to West Greenland provided replicate sediment samples from 4 lakes. Upon returning the material to the UK, sediment was processed to quantify ephippia with dormant eggs for DNA extraction or whole genome amplification. All lakes contained Daphnia pulex ephippia at least throughout the past several hundred years, however, only 2 lakes contained sufficient egg numbers for further analysis. Eggs were used for initial genotyping (microsatellite marker) from 3 lakes covering a period of several hundred years. Loss-On-Ignition (LOI) profiles were constructed of selected sediment cores using sediment subsamples along the entire core for correlation with previous core collections, providing a comprehensive archive of historical environmental data from previous work.
Ephippia were collected and quantified from Lake SS4 which contained the best-preserved eggs for DNA analysis. Eggs (for observation of genomic changes over time) were collected at a high temporal resolution of 30-year intervals, covering a total of ~300 years. From 10 time periods, 30 eggs were collected and frozen at -80 C until further analysis, yielding a total of 300 isolates.
The success of the project depended heavily upon successful methodological optimisation for processing of isolated Daphnia eggs. This required rigorous optimisation through a series of steps: (1) pretreatment (cleaning) of eggs to avoid external contamination, (2) egg homogenization and subsequent whole genome amplification, (3) quantification and quality control of amplified DNA, (4) genomic library construction. Further, (5) a sequencing test was run to provide information for the optimal strategy of the final sequencing of 300 egg isolates (whole genome amplified). Finally (6) bioinformatic analysis of test samples was carried out to identify the fraction of foreign DNA (a common problem in ancient DNA samples) and the resulting calculation of the targeted depth-of-coverage for the final sequencing of 300 samples.
A preliminary study was performed, using microsatellite analysis of individual eggs isolated from the sediment. This included egg from three lakes, across 50 - 300 years. Ten microsatellite markers were used to genotype individual eggs, revealing low clonal diversity in the lakes at all time periods tested, detecting distinct genotypes unique to each lakes despite their geographic proximity. Further, the Comet Assay, a method by which DNA damage can be quantified, was optimised for its use on individuals eggs and will be a useful tool for future studies.
Results of this study were partly included in the reports of 3 student projects supervised by Dr Frisch: (1) O'Grady, C. The development and optimization of whole genome and whole transcriptome amplification in historic Daphnia magna and Daphnia pulex diapause eggs. University of Birmingham MIBTP Rotation project, 2016; (2) Dane, M. Stability of spatio-temporal genetic variation across the centuries: a case study of arctic asexual Daphnia pulex populations from three West Greenland lakes. University of Birmingham. Third Year Practical Project Report, 2016; (3) Salimraj, R. Optimization of the Comet Assay to assess environmental impact on DNA damage in diapause eggs of Daphnia. University of Birmingham. Third Year Practical Project Report, 2017. Based on these project outcomes, three manuscripts are currently in advanced preparation, to be submitted to journals included in the science citation index. Additional publications are expected as soon as the current sequencing experiment is finalised.
Ephippia were collected and quantified from Lake SS4 which contained the best-preserved eggs for DNA analysis. Eggs (for observation of genomic changes over time) were collected at a high temporal resolution of 30-year intervals, covering a total of ~300 years. From 10 time periods, 30 eggs were collected and frozen at -80 C until further analysis, yielding a total of 300 isolates.
The success of the project depended heavily upon successful methodological optimisation for processing of isolated Daphnia eggs. This required rigorous optimisation through a series of steps: (1) pretreatment (cleaning) of eggs to avoid external contamination, (2) egg homogenization and subsequent whole genome amplification, (3) quantification and quality control of amplified DNA, (4) genomic library construction. Further, (5) a sequencing test was run to provide information for the optimal strategy of the final sequencing of 300 egg isolates (whole genome amplified). Finally (6) bioinformatic analysis of test samples was carried out to identify the fraction of foreign DNA (a common problem in ancient DNA samples) and the resulting calculation of the targeted depth-of-coverage for the final sequencing of 300 samples.
A preliminary study was performed, using microsatellite analysis of individual eggs isolated from the sediment. This included egg from three lakes, across 50 - 300 years. Ten microsatellite markers were used to genotype individual eggs, revealing low clonal diversity in the lakes at all time periods tested, detecting distinct genotypes unique to each lakes despite their geographic proximity. Further, the Comet Assay, a method by which DNA damage can be quantified, was optimised for its use on individuals eggs and will be a useful tool for future studies.
Results of this study were partly included in the reports of 3 student projects supervised by Dr Frisch: (1) O'Grady, C. The development and optimization of whole genome and whole transcriptome amplification in historic Daphnia magna and Daphnia pulex diapause eggs. University of Birmingham MIBTP Rotation project, 2016; (2) Dane, M. Stability of spatio-temporal genetic variation across the centuries: a case study of arctic asexual Daphnia pulex populations from three West Greenland lakes. University of Birmingham. Third Year Practical Project Report, 2016; (3) Salimraj, R. Optimization of the Comet Assay to assess environmental impact on DNA damage in diapause eggs of Daphnia. University of Birmingham. Third Year Practical Project Report, 2017. Based on these project outcomes, three manuscripts are currently in advanced preparation, to be submitted to journals included in the science citation index. Additional publications are expected as soon as the current sequencing experiment is finalised.
Obtaining high quality DNA for whole genome sequencing is a key obstacle in evolutionary studies of small organisms such as Daphnia, and in particular their dormant eggs. This project has provided the crucial achievement of optimising this methodological aspect that will allow the scientific community to move significantly beyond the current knowledge and exploit the historic ressources of zooplankton eggbanks. The project provided further an unmatched collection of historic DNA of a single metazoan population across several centuries during which individuals were exposed to an unprecedented change in environmental conditions, including shifts in climate. Aside from the methodological progress, the expected DNA sequence material of these Daphnia populations will allow an in-depth understanding of the genomic processes involved in evolutionary adaptation to environmental change.
The expertise of the fellow Dr. Frisch within the research field of environmental paleogenomics has been well-received during the time of her fellowship, leading to invitations to deliver five departmental seminars in European academic institutions, to co-organise a special symposium on evolutionary ecology, to co-edit a special issue in the journal Evolutionary Applications, and an invitation to present her work in a European workshop on ancient DNA. Dr Frisch was further invited to join a research expedition on Arctic zooplankton in and to participate in sequencing project of economically important zooplankton. Lastly, the methodology developed here led to Dr. Frisch's successful application for funding of a pilot project (Natural Environment Research Council, UK) on the epigenetic memory of environmental stress in Daphnia.
The expertise of the fellow Dr. Frisch within the research field of environmental paleogenomics has been well-received during the time of her fellowship, leading to invitations to deliver five departmental seminars in European academic institutions, to co-organise a special symposium on evolutionary ecology, to co-edit a special issue in the journal Evolutionary Applications, and an invitation to present her work in a European workshop on ancient DNA. Dr Frisch was further invited to join a research expedition on Arctic zooplankton in and to participate in sequencing project of economically important zooplankton. Lastly, the methodology developed here led to Dr. Frisch's successful application for funding of a pilot project (Natural Environment Research Council, UK) on the epigenetic memory of environmental stress in Daphnia.