Towards unravelling the real biodiversity of terrestrial green algae

• Thesis title in Czech: Výzkum reálné diverzity půdních zelených řas
• Thesis title in English: Towards unravelling the real biodiversity of terrestrial green algae
• Academic year of topic announcement: 2021/2022
• Thesis type: dissertation
• Thesis language: angličtina
• Department: Department of Botany (31-120)
• Supervisor: doc. Mgr. Pavel Škaloud, Ph.D.
• Author: hidden - assigned and confirmed by the Study Dept.
• Date of registration: 08.03.2022
• Date of assignment: 09.03.2022
• Confirmed by Study dept. on: 09.03.2022

Preliminary scope of work

Background
Microalgae are photosynthetic microorganisms that are responsible for almost half of the total global carbon dioxide fixation and thus play a pivotal role as primary producers in the world's ecosystems. As photoautotrophs, microalgae play a key role in land ecosystems. In fact, they constitute the basis of soil food webs and take part in its processes to favor fertility. Moreover, some of them are good bioindicators of the environment. Terrestrial green microalgae belonging to the Chlorophyta and Streptophyta groups represent a major component of the microbial flora, and they occurring on exposed aerial surfaces and in almost every habitat (Leliaert et al. 2012). Microalgae in terrestrial habitats exhibit morphological convergence, their morphology has narrow to a few types: unicellular, uniseriate filamentous, sarcinoid colony (Rindi 2011). Terrestrial green algae represent a polyphyletic group; molecular data have demonstrated that the morphology of subaerial green algae is highly homoplasious. To date, the knowledge of the diversity and distribution of free-living aero-terrestrial green algae, including the lichen symbiotic partners, is still very limited. Much of our knowledge comes from the traditional microscopic investigations of enriched cultures followed by the development of pure cultures or from sequencing of clone libraries derived of environmental samples (Lukešová & Hoffmann 1996; Hoffmann et al., 2007; Škaloud 2009; Neustupa & Škaloud 2010). However, both approaches uncover only a tiny fraction of overall species diversity (Rippin.et al. 2018). Since the identification of terrestrial green algae based on the morphology is impossible or only tentative because it offers very few characters, it will be necessary to define individual species and the taxonomy of the main groups of these organisms, and subsequently, clarify their systematics and biogeography using molecular markers. In fact, the molecular data are reshaping species delimitations in terrestrial green algae and, indirectly, basic concepts about their biogeography. My priority will be to increase the number of sequences and to expand substantially the taxon sampling. New molecular data based on environmental sampling will improve the characterization of algal biodiversity. Moreover, a combination of multi-tool field-based observations with novel experimental approaches allows me to get insight into the diversity of free-living terrestrial green algae.
Main questions
1) Do soil algal communities undergo major temporal fluctuations throughout the year?
2) What is the spatial heterogeneity of free-living algae at a micro-scale (cm to m)?
3) What is the proportion of uncultivable green algae?
4) How large is the inter- and intraspecific variability of this communities between different sites?
5) How do artificial changes of environmental conditions influence the pools of terrestrial algae?
Methodology
A total of two long-term study sites will be established. The study sites will be sampled repeatedly to monitor temporal dynamics. In addition, the single sampling will be performed to investigate the spatial heterogeneity. The soil will be collected from the upper horizon (0.5 cm) into separate sterilized containers, immediately frozen, and processed for chemical analyses and DNA metabarcoding. There, the entire green algal community will be characterized by both metabarcoding and Sanger sequencing of isolated algal cells. To answer the questions specified above, a number of experiments will be performed on these plots:
ad 1,2) I plan to sample four times per year. A sample mixed soil and sample mixed rock collected around the spots previously select inside of our sampling sites, will be inoculated on several Petri agar plates with BBM + Carbendozim 0.1 mg/mL. After a few weeks, I will transfer the colonies from the Petri plates into 24-well multiwall plates. Finally, the colonies will be molecularly characterized by Sanger sequencing.
ad 3) From the same soil and rock samples, I will isolate free-living algae separated from soil particles using Percoll gradient centrifugation. The single cells will be frozen and after then molecularly characterized by Sanger sequencing. I will also perform DNA metabarcoding approache to genetically characterize the entire community of green algae.
ad 4,5) Artificial exposure to elevated temperatures simulated by open-top chambers and translocation experiments will be performed to simulate changes of major environmental conditions in situ.