Od czasu wynalezienia mikroskopu świetlnego przez Antoniego van Leeuwenhoek’a w XVII wieku, człowieka fascynuje świat mikroorganizmów. Już ponad 300 lat badacze z mozołem i determinacją opisują i próbują poznać te powszechnie otaczające nas organizmy, od których w ogromnym stopniu jest zależna nasza egzystencja. Tymczasem współczesne projekty naukowe prowadzone na dużą skalę takie jak TARA lub BIOMARKS pokazują, że nasza wiedza o ich bogactwie,zróżnicowaniu gatunkowymi ekologii jest wciąż bardzo ograniczona. Jedną ze najsłabiej poznanych pod tym względem grup mikroorganizmów eukariotycznych są eugleniny zielone (jednokomórkowe glony), które pojawiły się na ziemi ponad miliard lat temu. Wszystkie są miksotrofami (pomimo zdolności do prowadzenia fotosyntezy odżywiają się również na drodze osmotrofii) dlatego masowo występują w wodach eutroficznych i hiper eutroficznych (m.in.stawach rybnych, sadzawkach wiejskich, miejskich sadzawkach parkowych oraz zbiornikach śródpolnych), i chociaż ich rola nie jest do końca rozpoznana to są dowody na ich udział w samooczyszczaniu się wód. W ostatnich latach pojawiły się doniesienia o gatunku toksycznym (Euglena sanguinea), który produkując ichtiotoksyny doprowadził do masowej śmierci ryb w stawach hodowlanych w Północnej Karolinie i spowodował dotkliwe straty materialne. Badania paramylonu u Euglena gracilis (węglowodanu, materiału zapasowego) wykazały jego aktywność przeciwnowotworową oraz regeneracyjną komórek wątroby i skóry u zwierząt eksperymentalnych. W komórkach innego gatunku (Euglena viridis) wykazano obecność aktywnych związków przeciwbakteryjnych. Estry woskowe,wytwarzane podczas fermentacji beztlenowej, są doskonałym surowcem do produkcji biopaliw. Gromadzone w komórkach duże ilości nutraceutyków i kosmeceutyków sprawiają, ze biomasa euglenin ma dużą wartość odżywczą. Podane przykłady tylko w niewielkim stopniu pokazują duże możliwości tych niezwykłych organizmów, które mógłby wykorzystać człowiek. Niestety, lepsze ich poznanie było dotąd bardzo trudne, praco-i czasochłonne z uwagi na dużą liczbę opisanych gatunków (ponad tysiąc), kłopoty z identyfikacją i praktycznie brak wiarygodnych informacji na temat ich ekologii czy zróżnicowania gatunkowego. Bezprecedensową okazją zmiany tej sytuacji jest pojawienie się wydajnych technik molekularnych (sekwencjonowania nowej generacji) oraz zaawansowanych narzędzi bioinformatycznych. Współczesne badania różnorodności i bogactwa gatunkowego w środowiskach wodnych i lądowych prowadzone tymi metodami pokazują, że mikroorganizmy eukariotyczne są znacznie bardziej zróżnicowane niż dotychczas sądzono, a prawdziwa skala tej różnorodności, rozmieszczenie gatunków, dynamika ich pojawów oraz znaczenie ekologiczne, są praktycznie nieznane. Zdecydowanie najmniej wiadomo o małych, eutroficznych i hipereutroficznych zbiornikach słodkowodnych, mimo ich dużego rozpowszechnienia i znaczenia ekologicznego. Ponadto niektóre grupy mikroorganizmów eukariotycznych, w tym także autotroficzne eugleniny, dominujące w tych siedliskach, nie są odpowiednio reprezentowane w sekwencjonowaniu środowiskowym. Jest to prawdopodobnie związane z tym, że wykorzystywany powszechnie w tzw. barkodingu środowiskowym (ang. metabarcoding) fragment DNA (rejon V4 nSSU rDNA; rodzaj „kodu kreskowego”) przy identyfikacji molekularnej gatunków, jest wyjątkowo długi i zmienny u euglenin, co może generować problemy. Dlatego w planowanym projekcie zostanie użyty inny fragment (V2 nSSU rDNA), który wytypowaliśmy na kod kreskowy DNA, w celu oszacowania składu gatunkowego oraz różnorodności biologicznej autotroficznych euglenin występujących w czterech wybranych typach charakterystycznych dla nich siedlisk eutroficznych (stawach rybnych, sadzawkach wiejskich i miejskich oraz zbiornikach śródpolnych), różniących się zarówno parametrami fizyczno-chemicznymi wody jak i lokalizacją. W tym celu z dwunastu zbiorników zlokalizowanych na terenie Polski (na Mazowszu, Mazurach i w Wielkopolsce) zostaną wielokrotnie pobrane próby planktonu w okresie kolejnych trzech sezonów wegetacyjnych i zmierzone wybrane parametry fizyczno-chemiczne wody. Później, stosując metody klasyczne (mikroskopowe),molekularne nowej generacji i zaawansowane narzędzia bioinformatyczne zostanie przeprowadzona identyfikacja gatunków we wszystkich pobranych próbach. Ostatnim krokiem będą analizy statystyczne. Spodziewamy się, że planowane badania (1) pozwolą lepiej i szybciej ocenić bogactwo i zróżnicowanie taksonomiczne zielonych euglenin występujących w siedliskach eutroficznych, wskazać taksony dominujące, ustalić dynamikę pojawów i czynniki ją kształtujące. Spodziewamy się również (2) wskazać gatunki wskaźnikowe, które mogą być wykorzystane w biomonitoriungu takich ekstremalnych ekosystemów, (3) odkryć nowe dla nauki i Polski gatunki, (4) poznać zróżnicowanie genetyczne w poszczególnych kladach drzewa filogenetycznego euglenin, zwłaszcza wewnątrzgatunkowe (obecnie wiele kladów jest reprezentowanych przez pojedyncze szczepy), (5) odkryć nowe linie filogenetyczne (rodzaje). Badania będą prowadzone wyłącznie na terenie Polski, ale zważywszy na fakt, że zielone eugleniny są kosmopolitami (podobnie jak większość mikroorganizmów), to uzyskane wyniki będą miały charakter uniwersalny.
Publikacje
2022
Chaber, Katarzyna; Łukomska-Kowalczyk, Maja; Fells, Alicja; Milanowski, Rafał; Zakryś, Bożena
Toward the robust resolution of taxonomic ambiguity within Lepocinclis (Euglenida) based on DNA sequencing and morphology Journal Article
In: Journal of Phycology, vol. 58, no. 1, pp. 105-120, 2022.
@article{https://doi.org/10.1111/jpy.13220,
title = {Toward the robust resolution of taxonomic ambiguity within Lepocinclis (Euglenida) based on DNA sequencing and morphology},
author = {Katarzyna Chaber and Maja Łukomska-Kowalczyk and Alicja Fells and Rafał Milanowski and Bożena Zakryś},
url = {https://onlinelibrary.wiley.com/doi/abs/10.1111/jpy.13220},
doi = {https://doi.org/10.1111/jpy.13220},
year = {2022},
date = {2022-01-01},
journal = {Journal of Phycology},
volume = {58},
number = {1},
pages = {105-120},
abstract = {DNA sequences were analyzed for three groups of species from the Lepocinclis genus (L. acus-like, L. oxyuris-like, and L. tripteris-like) along with cellular morphology. Phylogenetic analyses were based on nuclear SSU rDNA, LSU rDNA, and plastid-encoded LSU rDNA. DNA sequences were obtained from species available in culture collections (L. acus SAG 1224-1a and UTEX 1316) and those isolated directly from the environment in Poland (48 isolates), resulting in 79 new sequences. The obtained phylogenetic tree of Lepocinclis included 27 taxa, five of which are presented for the first time (L. convoluta, L. gracillimoides, L. longissima, L. pseudospiroides, and L. torta) and nine taxonomically verified and described. Based on morphology, literature data, and phylogenetic analyses, the following species were distinguished: in the L. acus-like group, L. longissima and L. acus; in the L. tripteris-like group, L. pseudospiroides, L. torta, and L. tripteris; in the L. oxyuris-like group, L. gracillimoides, L. oxyuris var. oxyuris, and L. oxyuris var. helicoidea. For all verified species, diagnostic descriptions were emended, nomenclatural adjustments were made, and epitypes were designated.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
DNA sequences were analyzed for three groups of species from the Lepocinclis genus (L. acus-like, L. oxyuris-like, and L. tripteris-like) along with cellular morphology. Phylogenetic analyses were based on nuclear SSU rDNA, LSU rDNA, and plastid-encoded LSU rDNA. DNA sequences were obtained from species available in culture collections (L. acus SAG 1224-1a and UTEX 1316) and those isolated directly from the environment in Poland (48 isolates), resulting in 79 new sequences. The obtained phylogenetic tree of Lepocinclis included 27 taxa, five of which are presented for the first time (L. convoluta, L. gracillimoides, L. longissima, L. pseudospiroides, and L. torta) and nine taxonomically verified and described. Based on morphology, literature data, and phylogenetic analyses, the following species were distinguished: in the L. acus-like group, L. longissima and L. acus; in the L. tripteris-like group, L. pseudospiroides, L. torta, and L. tripteris; in the L. oxyuris-like group, L. gracillimoides, L. oxyuris var. oxyuris, and L. oxyuris var. helicoidea. For all verified species, diagnostic descriptions were emended, nomenclatural adjustments were made, and epitypes were designated.
2021
Gumińska, Natalia; Łukomska-Kowalczyk, Maja; Chaber, Katarzyna; Zakryś, Bożena; Milanowski, Rafał
Evaluation of V2 18S rDNA barcode marker and assessment of sample collection and DNA extraction methods for metabarcoding of autotrophic euglenids Journal Article
In: Environmental Microbiology, vol. 23, no. 6, pp. 2992–3008 , 2021.
@article{https://doi.org/10.1111/1462-2920.15495,
title = {Evaluation of V2 18S rDNA barcode marker and assessment of sample collection and DNA extraction methods for metabarcoding of autotrophic euglenids},
author = {Natalia Gumińska and Maja Łukomska-Kowalczyk and Katarzyna Chaber and Bożena Zakryś and Rafał Milanowski},
url = {https://sfamjournals.onlinelibrary.wiley.com/doi/abs/10.1111/1462-2920.15495},
doi = {https://doi.org/10.1111/1462-2920.15495},
year = {2021},
date = {2021-04-08},
journal = {Environmental Microbiology},
volume = {23},
number = {6},
pages = {2992–3008 },
abstract = {Summary Even though the interest in metabarcoding in environmental studies is growing, euglenids are still underrepresented in both sea and freshwater bodies researches. The reason for this situation could be the unsuitability of universal eukaryotic DNA barcodes and primers as well as the lack of a verified protocol, suitable to assess euglenid diversity. In this study, using specific primers for the V2 hypervariable region of 18S rDNA for metabarcoding resulted in obtaining a high fraction (85%) of euglenid reads and species-level identification of almost 90% of them. Fifty species were detected by the metabarcoding method, including almost all species observed using a light microscope. We investigated three biomass harvesting methods (filtering, centrifugation and scraping the side of a collection vessel) and determined that centrifugation and filtration outperformed scrapes, but the choice between them is not crucial for the reliability of the analysis. In addition, eight DNA extraction methods were evaluated. We compared five commercially available DNA isolation kits, two CTAB-based protocols and a chelating resin. For this purpose, the efficiency of extraction, quality of obtained DNA, preparation time and generated costs were taken into consideration. After examination of the aforementioned criteria, we chose the GeneMATRIX Soil DNA Purification Kit as the most suitable for DNA isolation.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Summary Even though the interest in metabarcoding in environmental studies is growing, euglenids are still underrepresented in both sea and freshwater bodies researches. The reason for this situation could be the unsuitability of universal eukaryotic DNA barcodes and primers as well as the lack of a verified protocol, suitable to assess euglenid diversity. In this study, using specific primers for the V2 hypervariable region of 18S rDNA for metabarcoding resulted in obtaining a high fraction (85%) of euglenid reads and species-level identification of almost 90% of them. Fifty species were detected by the metabarcoding method, including almost all species observed using a light microscope. We investigated three biomass harvesting methods (filtering, centrifugation and scraping the side of a collection vessel) and determined that centrifugation and filtration outperformed scrapes, but the choice between them is not crucial for the reliability of the analysis. In addition, eight DNA extraction methods were evaluated. We compared five commercially available DNA isolation kits, two CTAB-based protocols and a chelating resin. For this purpose, the efficiency of extraction, quality of obtained DNA, preparation time and generated costs were taken into consideration. After examination of the aforementioned criteria, we chose the GeneMATRIX Soil DNA Purification Kit as the most suitable for DNA isolation.
Łukomska-Kowalczyk, Maja; Chaber, Katarzyna; Fells, Alicja; Milanowski, Rafał; Zakryś, Bożena
Description of Flexiglena gen. nov. and new members of Discoplastis and Euglenaformis (Euglenida) Journal Article
In: Journal of Phycology, vol. 57, no. 3, pp. 766-779, 2021.
@article{https://doi.org/10.1111/jpy.13107,
title = {Description of Flexiglena gen. nov. and new members of Discoplastis and Euglenaformis (Euglenida)},
author = {Maja Łukomska-Kowalczyk and Katarzyna Chaber and Alicja Fells and Rafał Milanowski and Bożena Zakryś},
url = {https://onlinelibrary.wiley.com/doi/abs/10.1111/jpy.13107},
doi = {https://doi.org/10.1111/jpy.13107},
year = {2021},
date = {2021-01-01},
journal = {Journal of Phycology},
volume = {57},
number = {3},
pages = {766-779},
abstract = {Environmental sampling in Poland and the United States and phylogenetic analyses based on 567 sequences of four genes (155 sequences of nuclear SSU rDNA, 139 of nuclear LSU rDNA, 135 of plastid-encoded SSU rDNA, and 138 of plastid-encoded LSU rDNA) resulted in description of the new genus Flexiglena, which has been erected by accommodating Euglena variabilis, and enriching the Discoplastis and Euglenaformis genera with five new species. Four of them have joined the Discoplastis genus, currently consisting of six representatives: D. adunca, D. angusta (=Euglena angusta), D. constricta (=Lepocinclis constricta), D. excavata (=E. excavata), D. gasterosteus (=E. gasterosteus), and D. spathirhyncha. One of them has enriched the Euglenaformis genus, currently represented by two species: Euf. chlorophoenicea (= E. chlorophoenicea) and Euf. proxima. For most studied species, the diagnostic descriptions have been emended and epitypes were designated. Furthermore, the emending of Discoplastis and Euglenaformis diagnoses was performed.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Environmental sampling in Poland and the United States and phylogenetic analyses based on 567 sequences of four genes (155 sequences of nuclear SSU rDNA, 139 of nuclear LSU rDNA, 135 of plastid-encoded SSU rDNA, and 138 of plastid-encoded LSU rDNA) resulted in description of the new genus Flexiglena, which has been erected by accommodating Euglena variabilis, and enriching the Discoplastis and Euglenaformis genera with five new species. Four of them have joined the Discoplastis genus, currently consisting of six representatives: D. adunca, D. angusta (=Euglena angusta), D. constricta (=Lepocinclis constricta), D. excavata (=E. excavata), D. gasterosteus (=E. gasterosteus), and D. spathirhyncha. One of them has enriched the Euglenaformis genus, currently represented by two species: Euf. chlorophoenicea (= E. chlorophoenicea) and Euf. proxima. For most studied species, the diagnostic descriptions have been emended and epitypes were designated. Furthermore, the emending of Discoplastis and Euglenaformis diagnoses was performed.
2020
Łukomska-Kowalczyk, Maja; Fells, Alicja; Chaber, Katarzyna; Milanowski, Rafał; Zakryś, Bożena
Taxon‐rich phylogeny and taxonomy of the genus Phacus (Euglenida) based on morphological and molecular data Journal Article
In: Journal of Phycology, vol. 56, no. 5, pp. 1135-1156, 2020.
@article{https://doi.org/10.1111/jpy.13028,
title = {Taxon‐rich phylogeny and taxonomy of the genus Phacus (Euglenida) based on morphological and molecular data },
author = {Maja Łukomska-Kowalczyk and Alicja Fells and Katarzyna Chaber and Rafał Milanowski and Bożena Zakryś},
url = {https://onlinelibrary.wiley.com/doi/10.1111/jpy.13028},
doi = { https://doi.org/10.1111/jpy.13028},
year = {2020},
date = {2020-05-19},
journal = {Journal of Phycology},
volume = {56},
number = {5},
pages = {1135-1156},
abstract = {Morphological and molecular features were analyzed for a species of Phacus to better understand the phylogenetic relationships among them and establish the taxonomy. Phylogenetic analyses were based on nSSU rDNA and the research resulted in 55 new sequences. The study included species available in algal collections and those isolated directly from the environment in Poland and the Czech Republic. As a result, the obtained phylogenetic tree of Phacus includes 50 species, out of which 7 are represented on a tree for the first time (Phacus anacoelus, P. anomalus, P. curvicauda, P. elegans, P. lismorensis, P. minutus and P. stokesii) and many have been taxonomically verified. For all verified species, diagnostic descriptions were amended, the naming was reordered and epitypes were designated.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Morphological and molecular features were analyzed for a species of Phacus to better understand the phylogenetic relationships among them and establish the taxonomy. Phylogenetic analyses were based on nSSU rDNA and the research resulted in 55 new sequences. The study included species available in algal collections and those isolated directly from the environment in Poland and the Czech Republic. As a result, the obtained phylogenetic tree of Phacus includes 50 species, out of which 7 are represented on a tree for the first time (Phacus anacoelus, P. anomalus, P. curvicauda, P. elegans, P. lismorensis, P. minutus and P. stokesii) and many have been taxonomically verified. For all verified species, diagnostic descriptions were amended, the naming was reordered and epitypes were designated.
Łukomska-Kowalczyk, Maja; Chaber, Katarzyna; Fells, Alicja; Milanowski, Rafał; Zakryś, Bożena
Molecular and Morphological Delimitation of Species in the Group of Lepocinclis Ovum-like taxa (Euglenida) Journal Article
In: Journal of Phycology, vol. 56, no. 2, pp. 283-299, 2020.
@article{https://doi.org/10.1111/jpy.12949,
title = {Molecular and Morphological Delimitation of Species in the Group of Lepocinclis Ovum-like taxa (Euglenida)},
author = {Maja Łukomska-Kowalczyk and Katarzyna Chaber and Alicja Fells and Rafał Milanowski and Bożena Zakryś},
url = {https://onlinelibrary.wiley.com/doi/abs/10.1111/jpy.12949},
doi = {https://doi.org/10.1111/jpy.12949},
year = {2020},
date = {2020-01-01},
journal = {Journal of Phycology},
volume = {56},
number = {2},
pages = {283-299},
abstract = {Although Lepocinclis ovum is recognized as a cosmopolitan and common species, and Lepocinclis globulus is the type species of the genus Lepocinclis, their correct identification is nearly impossible. The reason is that over 30 morphologically similar taxa appear in the literature, but no good diagnostic features exist to distinguish amongst them. Using environmental sampling and nuclear SSU rDNA sequencing, we delimited species within the group of Lepocinclis ovum-like taxa. Morphological and molecular features were analyzed for taxa isolated from Poland and six cultured strains from algal collections. In the case of environmental sampling, DNA was obtained from a small number of cells (20–400) isolated with a micropipette without setting up laboratory cultures (52 isolates), and phylogenetic analyses were based on the variation in nSSU rDNA. Apart from L. ovum and L. globulus, 13 other species were distinguished and four taxa (Lepocinclis conica comb. nov., L. fominii comb. nov., L. gracilicauda comb. nov., and L. pseudofominii nom. nov.) had their taxonomic ranks changed. For all verified species, diagnostic descriptions were emended and epitypes designated. The only exception was L. ovum, for which the epitype was questioned and thus, a new candidate for the epitype was suggested for future adoption.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Although Lepocinclis ovum is recognized as a cosmopolitan and common species, and Lepocinclis globulus is the type species of the genus Lepocinclis, their correct identification is nearly impossible. The reason is that over 30 morphologically similar taxa appear in the literature, but no good diagnostic features exist to distinguish amongst them. Using environmental sampling and nuclear SSU rDNA sequencing, we delimited species within the group of Lepocinclis ovum-like taxa. Morphological and molecular features were analyzed for taxa isolated from Poland and six cultured strains from algal collections. In the case of environmental sampling, DNA was obtained from a small number of cells (20–400) isolated with a micropipette without setting up laboratory cultures (52 isolates), and phylogenetic analyses were based on the variation in nSSU rDNA. Apart from L. ovum and L. globulus, 13 other species were distinguished and four taxa (Lepocinclis conica comb. nov., L. fominii comb. nov., L. gracilicauda comb. nov., and L. pseudofominii nom. nov.) had their taxonomic ranks changed. For all verified species, diagnostic descriptions were emended and epitypes designated. The only exception was L. ovum, for which the epitype was questioned and thus, a new candidate for the epitype was suggested for future adoption.
