Morfologická a molekulární diverzita volně žijících zástupců čeledi Metopidae a objev nové linie anaerobních nálevníků

Anoxické sedimenty slouží jako životní prostředí mnohým nálevníkům. Přestože je známo, že anaerobióza vznikla nezávisle v několika liniích z 11 hlavních linií Ciliophora, a anaerobové byli nalezeni v nejméně osmi liniích nálevníků, diverzita anaerobních nálevníků třídy Armophorea je podstatně přehlížena. Podobně, jen velmi málo je známo o jejich hydrogenosomech. Abychom prohloubili vědomosti o diverzitě volně žijících zástupců třídy Armophorea, kultivovali jsme více než 100 armophoridních kmenů ze sladkovodních, brakických, i mořských anoxických sedimentů z celého světa. Stanovili jsme jejich SSU rDNA sekvence, provedli barvení protargolovou technikou, a studovali jejich morfologii ve světelné mikroskopii. Kromě toho jsme použili transmisní elektronovou mikroskopii pro posouzení ultrastruktury některých kmenů. Identifikovali jsme několik nových linií, které pravděpodobně představují nové druhy. Významnou součástí práce je objevení nové hluboké linie mořských anaerobních nálevníků - Muránů. Podle analýzy SSU rDNA se větví mezi třídami skupiny SAL (Spirotrichea, Armophorea a Litostomatea), ale tvoří samostatnou hlubokou linii, případně novou třídu. Došli jsme k závěru, že anoxické sedimenty ukrývají velkou diverzitu dosud nepopsaných anaerobních nálevníků. Klíčová slova: Anerobióza, Armophorea,...Anoxic sediments host a wide variety of ciliates. Although it is known that anaerobiosis has independently arisen in several lineages of the main 11 lineages of Ciliophora and anaerobes have been found in at least eight lineages of ciliates, the diversity of anaerobic ciliates of the class Armophorea is severely understudied. Similarly, not much is known about their hydrogenosomes. To deepen our knowledge about the diversity of Armophorea, we have cultivated more than 100 armophorid strains from fresh water, brackish, and marine anoxic sediments worldwide. We determined their SSU rDNA sequences, performed protargol staining techniques, and studied light-microscopic morphology. In addition, we used transmission electron microscopy to assess the ultrastructure of some of the strains. Several novel clades of metopids, the free-living anaerobic ciliates of the class Armophorea, were identified. Importantly, a new deep lineage of marine anaerobic ciliates, muranes, was discovered. According to the SSU rDNA analysis, it is related to SAL group (Spirotrichea, Armophorea, and Litostomatea) with Cariacotrichea, but forms a separate lineage, possibly a novel class. We conclude that anoxic sediments harbour a high diversity of undescribed anaerobic ciliates. Key words: Anaerobiosis, Armophorea, ciliates,...Department of ZoologyKatedra zoologiePřírodovědecká fakultaFaculty of Scienc

Morfologická a molekulární diverzita volně žijících zástupců čeledi Metopidae a objev nové linie anaerobních nálevníků

5 ABSTRAKT Anoxické sedimenty slouží jako životní prostředí mnohým nálevníkům. Přestože je známo, že anaerobióza vznikla nezávisle v několika liniích z 11 hlavních linií Ciliophora, a anaerobové byli nalezeni v nejméně osmi liniích nálevníků, diverzita anaerobních nálevníků třídy Armophorea je podstatně přehlížena. Podobně, jen velmi málo je známo o jejich hydrogenosomech. Abychom prohloubili vědomosti o diverzitě volně žijících zástupců třídy Armophorea, kultivovali jsme více než 100 armophoridních kmenů ze sladkovodních, brakických, i mořských anoxických sedimentů z celého světa. Stanovili jsme jejich SSU rDNA sekvence, provedli barvení protargolovou technikou, a studovali jejich morfologii ve světelné mikroskopii. Kromě toho jsme použili transmisní elektronovou mikroskopii pro posouzení ultrastruktury některých kmenů. Identifikovali jsme několik nových linií, které pravděpodobně představují nové druhy. Významnou součástí práce je objevení nové hluboké linie mořských anaerobních nálevníků - Muránů. Podle analýzy SSU rDNA se větví mezi třídami skupiny SAL (Spirotrichea, Armophorea a Litostomatea), ale tvoří samostatnou hlubokou linii, případně novou třídu. Došli jsme k závěru, že anoxické sedimenty ukrývají velkou diverzitu dosud nepopsaných anaerobních nálevníků. Klíčová slova: Anerobióza, Armophorea,...6 ABSTRACT Anoxic sediments host a wide variety of ciliates. Although it is known that anaerobiosis has independently arisen in several lineages of the main 11 lineages of Ciliophora and anaerobes have been found in at least eight lineages of ciliates, the diversity of anaerobic ciliates of the class Armophorea is severely understudied. Similarly, not much is known about their hydrogenosomes. To deepen our knowledge about the diversity of Armophorea, we have cultivated more than 100 armophorid strains from fresh water, brackish, and marine anoxic sediments worldwide. We determined their SSU rDNA sequences, performed protargol staining techniques, and studied light-microscopic morphology. In addition, we used transmission electron microscopy to assess the ultrastructure of some of the strains. Several novel clades of metopids, the free-living anaerobic ciliates of the class Armophorea, were identified. Importantly, a new deep lineage of marine anaerobic ciliates, muranes, was discovered. According to the SSU rDNA analysis, it is related to SAL group (Spirotrichea, Armophorea, and Litostomatea) with Cariacotrichea, but forms a separate lineage, possibly a novel class. We conclude that anoxic sediments harbour a high diversity of undescribed anaerobic ciliates. Key words: Anaerobiosis, Armophorea, ciliates,...Katedra zoologieDepartment of ZoologyFaculty of SciencePřírodovědecká fakult

Morphological and molecular diversity of the free-living representatives of the family Metopidae and the discovery of a new lineage of anaerobic ciliates

Anoxic sediments host a wide variety of ciliates. Although it is known that anaerobiosis has independently arisen in several lineages of the main 11 lineages of Ciliophora and anaerobes have been found in at least eight lineages of ciliates, the diversity of anaerobic ciliates of the class Armophorea is severely understudied. Similarly, not much is known about their hydrogenosomes. To deepen our knowledge about the diversity of Armophorea, we have cultivated more than 100 armophorid strains from fresh water, brackish, and marine anoxic sediments worldwide. We determined their SSU rDNA sequences, performed protargol staining techniques, and studied light-microscopic morphology. In addition, we used transmission electron microscopy to assess the ultrastructure of some of the strains. Several novel clades of metopids, the free-living anaerobic ciliates of the class Armophorea, were identified. Importantly, a new deep lineage of marine anaerobic ciliates, muranes, was discovered. According to the SSU rDNA analysis, it is related to SAL group (Spirotrichea, Armophorea, and Litostomatea) with Cariacotrichea, but forms a separate lineage, possibly a novel class. We conclude that anoxic sediments harbour a high diversity of undescribed anaerobic ciliates. Key words: Anaerobiosis, Armophorea, ciliates,..

The method of DNA barcoding and its use in protists

DNA barcoding is a molecular method of species identification based on short regions of DNA sequences. These DNA barcodes should be unique for each species. The sequences within species should be identical or very much alike, while between species they should display a significant amount of differences. The sequences of an undetermined specimen can be compared with sequences vouchered to a particular species in a global DNA barcode database. The database was developed with the aim to simplify traditional taxonomy, eliminate its problems and accelerate its approaches. However, DNA barcoding has its own limitations that may hinder achieving its original goals and a compliance with the concept. The aim of this bachelor thesis consists of explaning the evolution of DNA barcoding through time and assessing the importance of this method at present, particularly in protists. Species identification may be very difficult in many protist groups, which can be explained by several reasons. To appraise this method completely is a hard task, because creation of the database has not yet been finished and official DNA barcodes have not been chosed for many groups. We can only find out in the future, therefore i will only focus on factors that influence the success of DNA barcoding at present

The method of DNA barcoding and its use in protists

DNA barcoding is a molecular method of species identification based on short regions of DNA sequences. These DNA barcodes should be unique for each species. The sequences within species should be identical or very much alike, while between species they should display a significant amount of differences. The sequences of an undetermined specimen can be compared with sequences vouchered to a particular species in a global DNA barcode database. The database was developed with the aim to simplify traditional taxonomy, eliminate its problems and accelerate its approaches. However, DNA barcoding has its own limitations that may hinder achieving its original goals and a compliance with the concept. The aim of this bachelor thesis consists of explaning the evolution of DNA barcoding through time and assessing the importance of this method at present, particularly in protists. Species identification may be very difficult in many protist groups, which can be explained by several reasons. To appraise this method completely is a hard task, because creation of the database has not yet been finished and official DNA barcodes have not been chosed for many groups. We can only find out in the future, therefore i will only focus on factors that influence the success of DNA barcoding at present

Anaerobic ciliates as a model group for studying the biodiversity and symbioses in anoxic environments

Ciliates are also of the most extensively studied and diverse groups of unicellular eukaryotes, and yet, their anaerobic representatives have been largely neglected; in part due to culturing difficulties. Although all main ciliate lineages contain anaerobes, their diversity and evolution of anaerobiosis are especially poorly understood and just starting to gain attention. In fact, Ciliophora is an excellent model group to study adaptations to life in anoxia, since it, apart from the aerobic majority, includes free-living and endobiotic obligately anaerobic lineages, facultative anaerobes, microaerophiles, and microaerotolerant species. The diversity of Metopida, the free-living order of obligately anaerobic class Armophorea, has been partially revised and significantly broadened during the past years, including numerous redescriptions using modern methods, as well as the description of novel families Tropidoatractidae and Apometopidae, several genera, and multiple species. Oxygen plays a crucial role in ATP production via oxidative phosphorylation that takes place in the mitochondrion in most known eukaryotes. Nevertheless, anaerobic ciliates, among many other eukaryotes that have adapted to low oxygen concentrations or even its absence, have modified their mitochondria and energetic metabolism to..

Morphological and molecular diversity of the free-living representatives of the family Metopidae and the discovery of a new lineage of anaerobic ciliates

6 ABSTRACT Anoxic sediments host a wide variety of ciliates. Although it is known that anaerobiosis has independently arisen in several lineages of the main 11 lineages of Ciliophora and anaerobes have been found in at least eight lineages of ciliates, the diversity of anaerobic ciliates of the class Armophorea is severely understudied. Similarly, not much is known about their hydrogenosomes. To deepen our knowledge about the diversity of Armophorea, we have cultivated more than 100 armophorid strains from fresh water, brackish, and marine anoxic sediments worldwide. We determined their SSU rDNA sequences, performed protargol staining techniques, and studied light-microscopic morphology. In addition, we used transmission electron microscopy to assess the ultrastructure of some of the strains. Several novel clades of metopids, the free-living anaerobic ciliates of the class Armophorea, were identified. Importantly, a new deep lineage of marine anaerobic ciliates, muranes, was discovered. According to the SSU rDNA analysis, it is related to SAL group (Spirotrichea, Armophorea, and Litostomatea) with Cariacotrichea, but forms a separate lineage, possibly a novel class. We conclude that anoxic sediments harbour a high diversity of undescribed anaerobic ciliates. Key words: Anaerobiosis, Armophorea, ciliates,..

Morphological and molecular diversity of the free-living representatives of the family Metopidae and the discovery of a new lineage of anaerobic ciliates

6 ABSTRACT Anoxic sediments host a wide variety of ciliates. Although it is known that anaerobiosis has independently arisen in several lineages of the main 11 lineages of Ciliophora and anaerobes have been found in at least eight lineages of ciliates, the diversity of anaerobic ciliates of the class Armophorea is severely understudied. Similarly, not much is known about their hydrogenosomes. To deepen our knowledge about the diversity of Armophorea, we have cultivated more than 100 armophorid strains from fresh water, brackish, and marine anoxic sediments worldwide. We determined their SSU rDNA sequences, performed protargol staining techniques, and studied light-microscopic morphology. In addition, we used transmission electron microscopy to assess the ultrastructure of some of the strains. Several novel clades of metopids, the free-living anaerobic ciliates of the class Armophorea, were identified. Importantly, a new deep lineage of marine anaerobic ciliates, muranes, was discovered. According to the SSU rDNA analysis, it is related to SAL group (Spirotrichea, Armophorea, and Litostomatea) with Cariacotrichea, but forms a separate lineage, possibly a novel class. We conclude that anoxic sediments harbour a high diversity of undescribed anaerobic ciliates. Key words: Anaerobiosis, Armophorea, ciliates,..

Anaerobní nálevníci jako modelová skupina pro studiu biodiverzity a symbióz v anoxických prostředích

Ciliates are also of the most extensively studied and diverse groups of unicellular eukaryotes, and yet, their anaerobic representatives have been largely neglected; in part due to culturing difficulties. Although all main ciliate lineages contain anaerobes, their diversity and evolution of anaerobiosis are especially poorly understood and just starting to gain attention. In fact, Ciliophora is an excellent model group to study adaptations to life in anoxia, since it, apart from the aerobic majority, includes free-living and endobiotic obligately anaerobic lineages, facultative anaerobes, microaerophiles, and microaerotolerant species. The diversity of Metopida, the free-living order of obligately anaerobic class Armophorea, has been partially revised and significantly broadened during the past years, including numerous redescriptions using modern methods, as well as the description of novel families Tropidoatractidae and Apometopidae, several genera, and multiple species. Oxygen plays a crucial role in ATP production via oxidative phosphorylation that takes place in the mitochondrion in most known eukaryotes. Nevertheless, anaerobic ciliates, among many other eukaryotes that have adapted to low oxygen concentrations or even its absence, have modified their mitochondria and energetic metabolism to...6 ABSTRAKT Nálevníci patří k nejrozsáhlejším a nejrozmanitějším skupinám jednobuněčných eukaryot, avšak jejich anaerobní zástupci byli do značné míry zanedbáváni; částečně kvůli kultivačním obtížím. Přestože všechny hlavní linie nálevníků obsahují anaeroby, jejich diverzita a evoluce anaerobiózy jsou zvláště málo studovány a teprve začínají získávat pozornost. Ve skutečnosti je kmen Ciliophora vynikající modelovou skupinou pro studium adaptací na život v anoxii, protože kromě aerobní většiny zahrnuje volně žijící a endobiotické obligátně anaerobní linie, fakultativní anaeroby, mikroaerofily i mikroaerotolerantní druhy. Diverzita řádu Metopida, volně žijících nálevníků ze striktně anaerobní třídy Armophorea, byla v posledních letech částečně revidována a výrazně rozšířena, včetně četných redeskripcí pomocí moderních metod, jakož i popisu nových čeledí Tropidoatractidae a Apometopidae, několika rodů a druhů. Kyslík hraje klíčovou roli při produkci ATP oxidační fosforylací, která se vyskytuje v mitochondrii ve většině známých eukaryot. Nicméně anaerobní nálevníci, mezi mnoha dalšími eukaryoty, kteří se přizpůsobili nízkým koncentracím kyslíku nebo dokonce jeho nepřítomnosti, modifikovali své mitochondrie a energetický metabolismus tak, aby byli schopní produkovat energii i při anoxii. Objev dvou nových tříd...Department of ZoologyKatedra zoologiePřírodovědecká fakultaFaculty of Scienc

Tropidoatractidae fam. nov., a Deep Branching Lineage of Metopida (Armophorea, Ciliophora) Found in Diverse Habitats and Possessing Prokaryotic Symbionts

We report a discovery of a novel family of anaerobic ciliates, Tropidoatractidae fam. nov. Phylogenetic analyses based on the 18S rRNA gene show that the family Tropidoatractidae corresponds to the previously reported clade of environmental sequences closely related to the lineage consisting of orders Metopida and Clevelandellida. The family comprises two genera, Tropidoatractus and Palmarella, and five species, two of which are newly described herein. Tropidoatractidae are cosmopolitan Metopida with sparse somatic and oral ciliature, deep, cup-like buccal cavity, and hyaline cortex with interkinetal ridges. Moreover, all species occur in two morphotypes, slender and stout. They inhabit microoxic or anoxic freshwater, brackish, and marine sediments and possess anaerobic mitochondrion-related organelles and various prokaryotic symbionts. The discovery of Tropidoatractidae provides valuable information about the evolution of Armophorea and gives us insights to the diversity and ecological preferences of anaerobic ciliates in general