MSc Student
Advisor: Luise Kruckenhauser
Unit for Integrative Zoology, Department of Evolutionary Biology
University of Vienna &
Natural History Museum Vienna
Abstract
There is ample evidence that global species extinctions continue to increase, leading to a global biodiversity crisis (Cowie et al. 2022). Therefore, the importance and urgency of describing new species before they become extinct is increasing. Likewise, it is important to document changes in distribution and occurrence so that disappearances and extinctions of species can be detected. Hence, identifying known species and describing newly discovered species represent important goals of any biological research in the 21st century.
Isopoda are a group of crustaceans with more than 10.000 species distributed worldwide, showing a great ecological diversity (Boyko et al. 2008). Throughout time many synonyms and subspecies have been proposed (Schmalfuss 2003). Which leads me to the importance of natural history collections, that provide a valuable contribution to taxonomic research. The Natural History Museum of Vienna owns a remarkable isopod collection comprising 5383 lots collected in Austria alone, including about 120 different species and subspecies (some of which may no longer be valid). These specimens were almost entirely determined by the first director of the Natural History Museum, Hans Strouhal (* October 2, 1897 in Vienna, † January 25, 1969), an isopod expert, who himself described some of these species.
Morphology-based identifications, however, require a high level of expertise. To this end various molecular methods have been developed, such as DNA barcoding, that provide taxonomy with useful tools. Compared to morphological species identification, DNA barcoding is a rapid alternative that allows the identification of individuals that cannot be determined from morphological characteristics, such as juveniles, species with strong sexual dimorphism, or damaged specimens. In this process, a short gene sequence of a standardized part of the genome, namely a DNA barcode, can be used as an identifier for different taxa. For most animal phyla the cytochrome c oxidase I gene (COI) is commonly used. The DNA barcode sequence of the unknown specimen is then compared with a digital DNA barcode database (eg. BOLD Barcode of Life Database; Hebert et al. 2002). Currently, 66 species of terrestrial isopods are listed for Austria. Earlier listings give an additional 13 subspecies, which have since been synonymized (Schmalfuss 2003; Schmölzer 1965) . A considerable part these Austrian species and subspecies has not yet received an entry in the database.
In 2021 Raupach et al. presented the first DNA barcode library for German Isopods. However, DNA barcodes have been supplied for only some of the German species. Moreover, note that Germany has a smaller number of isopod species than Austria. Their results show very high levels of intraspecific genetic differentiation for COI sequences of numerous species, including common and well-known species such as Porcellio scaber and Trachelipus rathkii. While the authors discuss possible reasons for these high distances, the possibility of cryptic species is not considered.
The main goal of my Master's thesis will therefore be the creation of a DNA Barcoding database for terrestrial isopods that includes sampling sites in all Austrian provinces. For this purpose, DNA will be extracted from approximately 200 collected samples and a fragment of the COI region will be amplified by polymerase chain reaction (PCR). PCR products will then be sequenced by Sanger sequencing and subsequently edited and compared.
In addition to sequencing data from fresh material, I will try to gather data from museum specimens. Unfortunately, working with historical DNA is difficult due to high fragmentation of the DNA. Sanger sequencing is not sufficient to obtain sequence data from such historical samples. However, next-generation sequencing (NGS) makes it possible to obtain full-length COI sequences from samples with highly degraded DNA by the use of genome skimming. This involves sequencing small fragments of the whole genome with the aim of covering the entire COI region.
Standard methods for genome skimming require double-stranded DNA. However, historical samples have a high proportion of single-stranded DNA, which should not simply be discarded given such small amounts of DNA per sample. Therefore, with a number of tests, I will try to find a method that amplifies complementary DNA from single-stranded fragments.
The objectives of my Master's thesis will thus be the following:
- In order to gain new insights for taxonomy through molecular analyses of the COI region, I will attempt to create a DNA barcoding database for terrestrial isopod species in Austria.
- By applying phylogeny-based methods to these sequences, I will get an estimation on how many species might occur in Austria, but also get a better indication of the presence of cryptic species.
- Furthermore, I will compare COI sequences with morphological species identifications of historical specimens identified by previous taxonomic experts. Thus, creating a direct link from the physical voucher specimen to the DNA barcode.
References
Boyko CB, Bruce NL, Hadfield KA, Merrin KL, Ota Y, Poore GCB, Taiti S (Eds) (2008 onwards). World Marine, Freshwater and Terrestrial Isopod Crustaceans database. Accessed at https://www.marinespecies.org/isopoda on 2023-03-08. doi:10.14284/365 [Accessed 2023-03-08]
Cowie RH, Bouchet P, Fontaine B (2022) The Sixth Mass Extinction: fact, fiction or speculation?. Biol Rev, 97: 640-663. https://doi.org/10.1111/brv.12816
Hebert PDN, Cywinska A, Ball AL, deWaard JR (2002): Biological identifications through DNA barcodes. The Royal Society 270: 313-321
Raupach MJ, Rulik B, Spelda J (2022) Surprisingly high genetic divergence of the mitochondrial DNA barcode fragment (COI) within Central European woodlice species (Crustacea, Isopoda, Oniscidea). ZooKeys 1082: 103-125. https://doi.org/10.3897/zookeys.1082.69851
Schmalfuss H (2003): World catalog of terrestrial isopods (Isopoda: Oniscidea). Stuttgarter Beiträge zur Naturkunde, Serie A, Nr. 654: 341 pp. [Updated by Schmalfuss in 2004]
Schmölzer K (1965): Ordnung Isopoda (Landasseln). Bestimmungsbücher zur Bodenfauna Europas. Akademie-Verlag GmbH, 108 Berlin, Leipziger Straße 3-4
