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Molecular Phylogeny and Classification of Filamentous Microalgae Belonging to the Family Kornmanniaceae

南波, 紀昭 筑波大学

2021.12.02

概要

The class Ulvophyceae is one of the groups of the phylum Chlorophyta. This group includes microalgae and macroalgae like seaweeds. Ulvophycean algae have various morphologies such as unicellular, colonial, filamentous, blade-like, and siphonous. Although most species of the Ulvophyceae grow in marine environment, species that grow in freshwater and terrestrial environment also exist. Moreover, some species have symbiotic relationships with other organisms. Microalgae are less studied taxonomically than macroalgae. In particular, the taxonomic research on branched filamentous microalgae has not made much progress. Although these branched filamentous algal taxonomy were performed based on thallus and cell morphology or reproductive mode, their morphologies are easily changed by cultivate conditions and the fluctuation of morphology is large. Moreover, there also have similar morphology among phylogenetically different species so that this makes difficult their taxonomy. However, these algae are very common in marine, freshwater and terrestrial environments. Accordingly, the development of the taxonomy on branched filamentous algae is important to establish the classification system of green algae. In these backgrounds, Darienko & Pröschold (2017) performed the large-scale taxonomic study on some ulvophycean species including branched filamentous species. They reclassified branched filamentous algae that are confused taxonomically using freshwater and marine strains maintained in public culture collections according to information of 18S rDNA and ITS-2 phylogeny, CBC approach and morphology. Although the framework of taxonomical foundation was established by this study, it had some problems, because they used only strains in culture collection and they mainly focused only on freshwater species, while branched filamentous green algae are found universally in marine and terrestrial environments.

I have been studied the taxonomy of branched filamentous ulvophycean microalgae growing in marine and terrestrial environments with aim to reclassify and get the robust taxonomy of them. For this aim, I collected and isolated branched filamentous microalgae from several locations, and observed their morphology and performed preliminary phylogenetic analyses. The results showed that many branched filamentous microalgae were the members of the Kornmanniaceae. Therefore, in this study, I aimed to clarify and discuss the diversity, phylogeny, taxonomy and evolution of the Kornmanniaceae. I wish that this study leads to understanding the diversity and evolution of whole Ulvophyceae in the future.

I collected various samples (e.g., water, water sample of washed leaves and rocks, sand, and green shells of door snails) from several locations in Japan and Republic of Palau. These samples were primary cultured using liquid medium of BBM for freshwater samples, or ESM and IMK for seawater samples, and 1.5 % agar BBM or ESM medium containing anti biotics. After 2-3 weeks, unialgal colony was picked up and established unialgal strains. These strains were observed morphological features by light microscope and some strains were also observed ultrastructure by scanning electron microscope and transmission electron microscope. The total DNA was extracted from these strains, and the 18S rDNA and ITS region were amplified by polymerase chain reaction. Amplified products were sequenced and analyzed to construct phylogenetic trees by ML analysis and Bayesian analysis. The sequences of ITS-2 were also analyzed to compare the secondary structure among closely related strains.

In this study, about 85 strains of branched filamentous green algae from marine, freshwater and door snail shells were established. In preliminary phylogenetic analysis, it was suggested that many strains belonged to the Kornmanniaceae, so that these strains were studied in phylogenetic analyses and morphology in detail. Most of these strains were collected from marine environment but some strains were collected from freshwater and terrestrial environment (include on the shell surface of door snails). It could be said that many branched filamentous microalgae growing marine are belonged to the Kornmanniaceae by this study. These strains have very similar each other in morphology such as the thallus composed of aggregated cell and peripheral radiating filaments. The size and morphology of cells have large fluctuation. Most of cells have one parietal chloroplast, with one pyrenoid. A part of strains was observed reproduction by quadriflagellate zoospores. These characters were similar to Pseudendoclonium, Paulbroadya, Lithotrichon belonging to the Kornmanniaceae and some genera transferred from the Ulvales to Ulotrichales by Darienko & Pröschold (2017). However, the number of branching, length, ratio of cylindrical cell and elliptical cell composing filaments was different among strains. In the result of detailed phylogenetic analysis, these strains were separated at least about 7 linages (it might be corresponded to genus revel). 4 of them were novel linages that never been reported in previous studies. There were some strains having fast evolving sequence of 18S rDNA in Pseudendoclonium. Many strains were closely related to Lithotrichon. Another 6 strains possessed the similar sequences to the environmental sequence of the photobiont of marine lichen collected from Ascension Island. This photobiont was not examined taxonomically and present study showed that this group was sister to the shell-attached algae discovered in this study (see below). In this study, some shell-attached algal strains were established from several places and several clausiliid species. Interestingly, they showed no CBCs in their ITS-2 secondary structure, so that it was suggested that these strains are the same species. Based on this study I described a new genus and new species, Annulotesta cochlephila for the shell-attached alga. This study revealed the hidden diversity of the Kornmanniaceae that was unknown previously. Although the Kornmanniaceae includes some macroalgae such as Kornmania, most members of this family are typically branched filamentous microalgae. Moreover, present study indicates that the habitats of the Kornmanniaceae were very diverse, such as marine, freshwater, land, lichen photobiont and surface of clausillid shell. The phylogenetic relationships among these algae suggest that they were originally marine algae and colonized freshwater independently several times. Interestingly, some lineages of the Kornmanniaceae include lichen photobionts, and these lichen symbioses might be the intermediate stages in the evolution of these frequently change of habitats.

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