Experimental Evolutionary Ecology (Stelzer Lab)
Our research focusses on genome size evolution in Eukaryotic organisms, and for this we are mainly using rotifers (Fig. 1) as model organisms. Rotifers are short-lived (1-2 weeks), small aquatic metazoans, only a few hundred micrometers in size, common in fresh and brackish water habitats throughout the world. They are an important component of aquatic food webs as consumers of microalgae and heterotrophic microorganisms on the one hand, but also as prey for higher trophic levels like crustaceans or fish larvae. In our research we capitalize on the ease of culturing large populations of rotifers in the lab, their fast generation times, and their interesting biological properties (more on that, below).
Fig. 1 The rotifer Brachionus asplanchnoidis. a Female rotifer with two attached (asexually produced) eggs. b Clonal rotifer cultures in our lab, which currently encompasses >100 genotypes sampled from the wild, experimental crosses, and various selfed lines. c Our main field site and the origin of many of our rotifers is Obere Halbjochlacke, a small, shallow soda lake in East Austria, close to the Hungarian border.
Why are Eukaryotic genomes so variable in size and structure?
This is the main question driving our research. A genome contains the complete set of instructions to build, run and maintain an organism. However, genome organization in Eukaryotes is neither straightforward, streamlined, nor efficient. Surprisingly, most eukaryotic genomes contain lots of sequence repetitions and huge amounts of DNA of presumably low or no informational value. Eukaryotic genomes also display an enormous size variation, ranging from 2.3 million base pairs in the microsporidian Encephalitozoon intestinalis (i.e., less than 1/1000th of the human genome) to 152 billion base pairs in the monocot plant Paris japonica (i.e., 50 times the human genome). Genome sequencing projects of the last two decades have established the presence of vast amounts of repetitive DNA in Eukaryotic genomes, for example transposable elements (“jumping genes”), satellite DNA (a form of repeat in which short motives are repeated tandemly thousands of times), or pseudogenes, which are the inactive remainders of formerly duplicated genes. However, despite ever-increasing additions of newly sequenced genomes, it is still unclear how this repetitive proportion of the genome evolves in short and long-term, why it is so large, how it affects other parts of the genome and, ultimately, the phenotype of its carriers. We are fascinated by such questions and study them by focusing on one end of the genome size diversity-spectrum: on genome size variation among members of a population.
Our model system
The rotifer Brachionus asplanchnoidis (Fig. 1) is one of the few well-documented cases of within-population genome size variation. We have previously shown that natural populations harbor significant variation in genome size, generally 1.2-1.3-fold, with exceptions of up to approximately twofold (Riss et al. 2017, Stelzer et al. 2019). Genome size is highly heritable, and the mean genome size of a population will increase if only the largest (by GS) individuals interbreed with each other. Genome size variation in B. asplanchnoidis is mediated by large genomic elements, up to a few megabases in size, which segregate independently from each other during meiosis. These elements consist largely of noncoding DNA, predominantly of satellite DNA, with only few genes or other interspersed DNA sequences (Stelzer et al. 2021a), although transposable elements do significantly contribute to total genome size in this species (Blommaert et al. 2019). We have also found that the differences in DNA content among B. asplanchnoidis individuals affect their phenotype, with individuals of large genome size having slightly larger body or egg sizes, and longer embryonic development times (Stelzer et al. 2021b).
Riss S., Arthofer W., Steiner F.M., Schlick-Steiner B.C., Pichler M., Stadler P., Stelzer C.-P. (2017). Do genome size differences within Brachionus asplanchnoidis (Rotifera, Monogononta) cause reproductive barriers among geographic populations? Hydrobiologia, 796(1): 59-75.
Stelzer C.-P., Pichler M., Stadler P., Hatheuer A., Riss S. (2019): Within-population genome size variation is mediated by multiple genomic elements that segregate independently during meiosis. Genome Biology and Evolution, evz253.
Blommaert J., Riss S., Hecox-Lea B., Mark Welch D.B., Stelzer C.-P. (2019). Small, but surprisingly repetitive genomes: transposon expansion and not polyploidy has driven a doubling in genome size in a metazoan species complex. BMC Genomics 20: 466
Stelzer C.P., J. Blommaert, A.M. Waldvogel, M. Pichler, B. Hecox-Lea, D.B. Mark Welch (2021a) Genome structure of Brachionus asplanchnoidis, a Eukaryote with intrapopulation variation in genome size. bioRxiv 2021.03.09.434534
Stelzer C.P., Pichler M., Hatheuer, A. (2021b). Linking genome size variation to population phenotypic variation within the rotifer, Brachionus asplanchnoidis. Communications Biology 4:596.
Current research
The overall goal of our research is to disentangle the contributions of two genome fractions to the phenotype of B. asplanchnoidis: the contribution of genetic variation in coding regions vs. the contribution of predominantly structural elements (presence/absence of large sections of non-coding, repetitive DNA). We are interested both in the causes and consequences of these two types of genomic variation. Using flow cytometry (Fig. 2), we measure differences in total genome size among individuals, and it allows us to identify and size particularly large genomic elements that are present in some individuals. Using whole-genome sequencing and various PCR-based techniques (Fig. 3), we characterize at a finer scale the genomic differences and structural variation among individuals of the population. Informed by these data, we identify and track CNVs (copy number variants) that contribute to variation in genome size in the population. At a whole-organism level, we are interested in the phenotypic consequences of this genomic variation, e.g. the effects on body size/shape, developmental rates, growth patterns and metabolic efficiency - both on the individual and on the population level. To this end, we use laboratory experiments (Fig. 4), including long-term experimental evolution in continuous cultures. Much of this work is done in combination with automated image analysis (Fig. 4), which allows us to track phenotypic changes (e.g. body size) in the evolved populations. Thus, we are trying to address all the major hypotheses about genome size change, i.e. selection-based hypotheses (“optimal DNA”), mutational, and drift-based hypotheses.
Fig. 2 Flow cytometry. a Peter Stadler performing flow cytometry measurements to estimate genome size of rotifers. b Flow cytometry allows accurate quantification of the DNA content of thousands of nuclei within a few minutes c Intraspecific genome size variation in the rotifer Brachionus asplanchnoidis. d Example measurements of three rotifer clones (differing in genome size) and the internal control, the fruit fly Drosophila melanogaster (pink).
Fig. 3 Genome sequencing and DNA-based analyses. a Maria Pichler extracting DNA for whole-genome sequencing. b PCR-based approaches allow us to detect presence/absence of specific genomic regions c Droplet-digital PCR is used to estimate copy numbers of such regions d Example of gene, repeat annotations and depth-of-coverage of a genomic region containing a CNV (copy-number variable) region.
Fig. 4 Laboratory experiments. a Continuous cultures (chemostats) in which rotifers evolve for several weeks to months under defined conditions. b All our continuous cultures are monitored by an automated sampling and counting device. c Claus-Peter Stelzer ensuring proper function of the ABESM system, our in-house developed system for large-scale Automated Body Egg Size Measurements using a 96-well plate format. d Example of body size measurement with the ABESM system.