Publication: Bringing together approaches to reporting on within species genetic diversity

In the new article in the Journal of Applied Ecology, our pilot study for a monitoring of genetic diversity has been compared with and related to other approaches to reporting on within species genetic diversity. The publication shows the international importance of a monitoring of genetic diversity. The publication is available via this link in the Journal of Applied Ecology.

Supplementary sampling 2022

After the largely successful sampling of all five selected species in 2021, the missing false heath fritillary, Melitaea diamina, populations will be sampled this year. In addition, 200 contemporary populations of Hare’s-tail cottongrass, Eriophorum vaginatum, will be sampled for retrospective monitoring of genetic diversity. Where possible, these will be collected from similar places as the 200 historical samples from collections and museums. The over 200 historic samples of Eriophorum and 200 samples of Melitaea are currently being processed in the clean-lab and will soon be sequenced.

As a consequence of the rainy and cool weather in spring and summer 2021, some populations of the false heath fritillary could not be successfully sampled. Therefore, 10-15 populations of the false heath fritillary will be resampled this year, mostly in eastern Switzerland. The collection points were chosen more specifically to increase the chance of successful sampling in the second trial. Since the false heath fritillary is also studied retrospectively, in addition to the recent specimens, specimens from museums the same place of origin, if possible, are also studied. The collection points were slightly optimised for this purpose without compromising our proportionally stratified rantom sampling strategy. This is because it is important that the location of the historical and present-day material coincide as closely as possible, as otherwise geographical rather than temporal differences in genetic diversity are inferred.

Sampling locations for the false heath fritillary. Green dots: Successful sampling 2021, red dots: Resampling 2022

For the retrospective analysis of Hare’s-tail cottongrass (Eriophorum vaginatum), about 200 populations are sampled. The aim is to sample a population of each selected herbarium record, which are between 70 and 150 years old, as close as possible to the historical site. The difficulty is to match the approx. 100 year old field descriptions on the labels of the herbarium specimens with today’s field descriptions on the maps and to find potential sites where there is a chance to find and sample the species today. This is especially difficult when the landscape has changed considerably due to human activity. With the populations sampled this year and the approximately 100 years of herbarium specimens, it should be possible to carry out analyses that provide information on whether certain locally adapted plants have come under particular pressure. This is particularly relevant with regard to the massive loss of Hare’s-tail cottongrass habitats (predominantly raised bogs), which has mainly taken place on the Swiss Central Plateau in recent centuries. In addition, it should be possible to determine whether genetic diversity in this species has changed over the time-period studied.

For each herbarium specimen of the Hare’s-tail cottongrass, the most accurate possible location on today’s maps was searched for the given field name. In this example, the field name “Prantin” could be found on today’s maps.
For each herbarium specimen of the Hare’s-tail cottongrass, the most accurate possible location on today’s maps was searched for the given field name. In this example, the field name “Prantin” could be found on today’s maps.

Results sampling 2021

Karte mit den Sammelpunkten der fünf Arten der Pilotstudie

Sampling of the five species was fairly successful despite a wet and cold spring and a gloomy summer.

The various collection sites of the carthusian pink (Dianthus carhusianorum), yellowhammer (Emberiza citrinella), natterjack toad (Epidalea calamita), hare’s-tail cottongrass (Eriophorum vaginatum), and false heath fritillary (Melitaea diamina) are shown.

The various collection sites of the carthusian pink (Dianthus carhusianorum), yellowhammer (Emberiza citrinella), natterjack toad (Epidalea calamita), hare’s-tail cottongrass (Eriophorum vaginatum), and false heath fritillary (Melitaea diamina) are shown.

At the beginning of April 2021, the teams of the Swiss Ornithological Institute started sampling the Yellowhammer (Emberiza citrinella). They sampled about 120 bird over a two-month period. The anticipated number of 150 samples was not reached because bird trapping was hampered by the cold and wet spring. Nevertheless, birds were sampled in 23 of the 30 planned collection quadrats.

Between early April and late July 2021, experts commissioned by the Coordination Center for the protection of Amphibians and Reptiles (karch) were able to collect a total of about 150 samples in the form of embryos or tadpoles of the natterjack toad (Epidalea calamita) in 28 of the 30 planned quadrats. It was often necessary to find replacement quadrats because the natterjack toad, as a pioneer species, often changes its habitat, causing occurrences to disappear and emerge elsewhere. In addition, it is also one of the amphibian species in Switzerland whose populations are in sharp decline.

Just over 220 false heat fritillaries (Melitaea diamina) were collected by various environmental agencies between mid-June and mid-August 2021 in 22 of the 30 planned sampling quadrats. Rainy weather and hailstorms that severely impacted vegetation in some study areas hampered the collectors’ work and limited the number of sunny days on which butterflies were flying and could be sampled.

From early June to early August 2021, environmental agencies successfully sampled 30 populations and 300 individuals of hare’s tail cottongrass (Eriophorum vaginatum). A replacement collection quadrat was needed twice because the species was either no longer present or there were too few individuals at the actual designated location.

Sampling of the Carthusian pink (Dianthus carthusianorum) by environmental agencies was also successful. 31 populations and 330 individuals were sampled between late May and early July 2021. Six replacement collection quadrats were needed, often in regions where Carthusian pink was artificially seeded.

The random stratified proportional sampling strategy proved to be efficient in the field and we covered the climatic space of each species well, as it can be seen in the figure below with the hare’s tail cottongrass as an example. The randomly sampled individuals and populations (red dots) cover the climate space of the species in Switzerland (gray dots) well. The climate space covers the range from low to high mean annual temperature (e.g. low and high elevation regions) on the X-axis, and high to low rainfall regions can be seen on the Y-axis.

Climate space of Eriophorum vaginatum (grey dots) with allocation of the sampled populations (red dots)

The randomly sampled individuals and populations (Eriophorum vaginatum), (red dots) cover very well the expected climate space (gray dots) of the species in Switzerland. The X-axis shows low to high mean annual temperature (e.g. low and high elevation regions) on the X-axis, and high to low rainfall regions can be seen on the Y-axis.

The work on retrospective analyses using museum and herbarium samples is currently underway. DNA from museum and herbarium samples that are older than 40 years is highly fragmented, usually with fragments shorter than 70 base pairs. It is also more damaged compared to DNA from freshly collected samples, making it highly susceptible to contamination by new and fresh DNA from humans or other samples processed in the laboratory. These properties necessitate the use of state-of-the-art clean lab, such as those available at the Institute of Evolutionary Medicine (IEM) at the University of Zurich. This clean lab has all the necessary equipment for the various stages of ancient DNA analysis, including a positive pressure system, UV sterilization, and several isolated workstations where research personnel work in full-body protective gear. Only thanks to this careful approach, the recovery and sequencing of DNA from voucher specimens is possible in a reliable manner. Currently, about 200 voucher specimens each of the hare’s tail cottongrass and the false heat fritillaries from Swiss collections are processed in the clean lab.

For the retrospective analysis, samples of the false heat fritillaries and the hare’s tail cottongrass from collections are processed in the Clean lab. This is the only way to obtain and sequence DNA in a reliable way and justifies the use of the valuable herbarium or collection specimens.
For the retrospective analysis, samples of the false heat fritillaries and the hare’s tail cottongrass from collections are processed in the Clean lab. This is the only way to obtain and sequence DNA in a reliable way and justifies the use of the valuable herbarium or collection specimens.

The preparation of the reference genomes of the five species is well advanced and thanks to new sequencing technologies (PacBio HiFi Reads and OMNI-C Scaffolding) the quality of the reference genomes have exceeded our expectations. Most genomes were assembled almost to chromosome level. This is even true for the natterjack toad, which has the largest genome of the five species and with 3.8 billion base pairs, it is even larger than the human genome.

Great work awaits us this year as well. The DNA of the 1,120 collected samples will be extracted, sequenced and analyzed. In addition, work continues on the museum samples in the clean lab. After the extraction and sequencing of the DNA, these sequences will be analyzed and compared with the modern samples.

Retrospective Analyses

False heath fritillary, Melitaea diamina, in the Entomological Collection Zurich.

For the retrospective analysis, more than 700 voucher specimens were found for the Hare’s-tail cottongrass and more than 1,000 for the False heath fritillary in collections in Switzerland – from Geneva to Basel. Where not already done, each specimen was entered into a database and the location was recorded and georeferenced using the original label. In a next step, a selection of the vouchers will now be made. A report on part of our research on collection specimens of the False heath fritillary has just been written and published in the ETH journal GLOBE: https://ethz.ch/en/news-and-events/eth-news/news/2021/06/silent-witnesses.html

False heath fritillary, Melitaea diamina, in the Entomological Collection Zurich.
False heath fritillary, Melitaea diamina, from the Entomological Collection Zurich. © Martin C. Fischer

Sampling has started

Pond where natterjack toad spawn was found. Foto: Jérôme Pellet

The fieldwork season began this spring. The teams of the Swiss Ornithological Institute started earliest at the beginning of April in Ticino and Graubünden. Since then, 117 yellowhammers have been caught, sampled and released throughout Switzerland. Breeding calls of natterjack toads began at the end of April, a sign for which the staff of the karch had been waiting to begin sampling. However, the search for natterjack toad populations at the planned collection sites have not always been successful and often the collection sites planned as reserves needed to be used. This was due to characteristics of the natterjack toad’s pioneer habitat, which is composed primarily of temporary pools in gravel pits and floodplains, which are not stable long-term. The input of the karch staff, with their knowledge of local natterjack toad spawning areas and ecology, has been invaluable. Thanks to them, spawning areas that are no longer active could be removed from the list of collection sites in advance, and new collection sites could be recorded where necessary. Sampling of the populations of the Carthusian pink and the Hare’s-tail cottongrass has also started. Lastly, depending on the weather, sampling of the False heath fritillary will soon begin. We are curious to see what challenges this particular sampling holds for our experts.

Pond where natterjack toad spawn was found. Foto: Jérôme Pellet
In this pond on the site of a gravel pit, the search for natterjack toad spawn was successful. This illustrates the very special pioneer habitats of the natterjack toad. (Picture provided by Jérôme Pellet)

Assembly of reference genomes

High-molecular DNA is extracted from samples of a single individual of each of the five species, and the entire genome is sequenced using the latest “long read sequencing method” (PacBio Sequel II; HiFi reads). These long DNA fragments are necessary so that the whole genome can be assembled as accurately and completely as possible. The newly assembled genomes for each species then serve as reference genomes and are the basis for further genetic analyses. They represent a kind of template, whereas the extracted and sequenced DNA segments of each individual are compared and genetic variations are detected. As the assembly of the reference genomes is a complicated and complex process, various specialists are involved.

Update May 2021, Retrospective Analyses

Historical genetic diversity will be investigated for the Hare’s-tail cottongrass (Eriophorum vaginatum) and the Valerian Fritillary (Melitaea diamina). For this purpose, we have reached out to numerous museums and herbaria about whether their collections contain specimens of these two species that are at least 50 years old. We found 767 vouchers of the Hare’s-tail cottongrass and about 1’400 vouchers of the Valerian Fritillary that are eligible for our investigation, which requires the removal of a small piece of tissue. Important in the selection of individuals to be sequenced from the collections is now where the two species were collected in the past and at which frequency. In order to obtain the relevant geographical information, these specimens have to be georeferenced using old handwritten labels (see pictures). A polygon is defined where the specimens were originally collected, based on the locations on the labels. The coordinates are archived in a database, which serves as a basis for sample selection. The example of Eriophorum from 1907 shows how much the landscape has changed and the habitats had to give way to settlements.

Beleg Eriophorum 1907
Voucher and enlarged label of a Hare’s-tail cottongrass from 1907 (United Herbarium Z + ZT).
Collection sites of a specimen of Hare’s-tail cottongrass from 1907 marked on the map of then and now.

Update December 2020

Testing DNA extraction from tissues of the study types

The tissue samples for creating the reference genomes of the five species have arrived in the laboratory. The reference genome contains the entire hereditary information of an individual (DNA). It is needed to later visualise the genetic differences between different individuals of a species. In the laboratory, different extraction methods are currently being tested for the natterjack toad, the valerian fritillary and the yellowhammer. To create a genome with as few gaps as possible, DNA fragments of maximum length are needed.

Testing DNA extraction from tissues of the study types
Various tests are made to determine the best extraction method of DNA for each study species.

A strategy for collecting samples from the five study species will be designed. A random selection of collection sites is aimed at in order to ensure comparability between species. Using information on the distribution of the species from the InfoSpecies databases and other sources, the distribution throughout Switzerland is outlined. From this, the collection points can be randomly selected, for example.

Exemple for choosing sampling points
Map with possible distribution of collection points: Distribution of hare’s-tail cottongrass, Eriophorum vaginatum, in Switzerland. Modelled with data from InfoSpecies and arranged in a 5×5 km grid with 35 randomly drawn sampling points.