Uniwersytet im. Adama Mickiewicza w Poznaniu Wydział Biologii Zakład Taksonomii i Ekologii Zwierząt ZTiEZ, Poznań Rodzaj Paramacrobiotus (Tardigrada): taksonomia integratywna, biogeografia i oraz wpływ czynników stresowych na wybrane gatunki. Uniwersytet im. Adama Mickiewicza w Poznaniu Pushpalata Kayastha Rozprawa doktorska Adam Mickiewicz University in Poznan Faculty of Biology Department of Animal Taxonomy and Ecology ZTiEZ, Poznań The genus Paramacrobiotus (Tardigrada): integrative taxonomy, biogeography and effects of stress factors on the selected species. Pushpalata Kayastha Doctoral thesis Promotor: Dr hab. Prof. UAM Łukasz Kaczmarek Promotor pomocniczy: Dr Monika Mioduchowska Poznan, Poland 2023 The genus Paramacrobiotus (Tardigrada): integrative taxonomy, biogeography and effects of stress factors on the selected species. Pushpalata Kayastha ACKNOWLEDGEMENTS My time spent as a PhD student at the Department of Animal Taxonomy and Ecology was like a roller coaster ride. Firstly, I would like to thank my parents (Birendra Prasad Lal Karna and Lalita Karna) and my siblings (Sanandan Kayastha and Yogita Kayastha) for always cheering, supporting and being there for me. Next, I thank myself for being able to go through this journey, survive and come out victorious. I would also like to thank my supervisor, prof. UAM dr. hab. Łukasz Kaczmarek, for giving me the opportunity to be his student and to experience a truly exceptional atmosphere of both hard work and great joy in making scientific discoveries; for his outstanding support, understanding and inspiration to excel. My co-supervisor Dr. Monika Mioduchowska for helping and guiding me at every step alongside my supervisor. The head of the Department of Animal Taxonomy and Ecology prof. UAM dr. hab. Wojciech Ł. Magowski for outstanding support during my PhD. Professor Hanna Kmita for providing the laboratory workspace to carry out the molecular biology part of the integrative taxonomy approach. Moreover, I would like to thank all my friends, colleagues, co-workers, professors, and administration support staff (especially Dr. Karolina Cerbin & Ms. Arleta Kucz) from the Adam Mickiewicz University in Poznań, and a huge thanks to the friends from my laboratory (Milena Roszkowska, Tomasz Bartylak, Amit Nagwani, Magdalena Górna and Michalina Krakowiak) for their invaluable help, scientific, as well as social support, encouragement, and inspiration. Nonetheless, I would like to thank all my friends (Swapnil Gaware, Megha Gusain, Saurabh Gaur, Neha Kudumula, Parathamesh Dhanpalwar, Neyaz Khan, Shejin T, Rahiman Faiyaz, Krupa Vyas, Prerit Berman, Omkar Apshankar, Alisha, Akansha Tripathi, Deepti Varshney, Anand Maurya, Kishor Gawade, Dheeraj Sharkar, Nishita Mandal, Aravind Selvaram and Amit K. Nagwani) for supporting me and tolerating me during the highs and lows of this PhD. All members of our collaborator’s group of Dr. hab. Izabela Poprawa at the University of Katowice and a group of Prof. Marek Michalak at the University of Alberta, Canada. Rafał Gryka and his family (Paweł Gryka, Dorota Gryka and Emilia Gryka) in Poznań played a pivotal role in my integration into the Polish culture and made me feel at home in a foreign land. The genus Paramacrobiotus (Tardigrada): integrative taxonomy, biogeography and effects of stress factors on the selected species. Pushpalata Kayastha Dedicated to both my late grandmothers, whom I lost during the period of my PhD. The genus Paramacrobiotus (Tardigrada): integrative taxonomy, biogeography and effects of stress factors on the selected species. Pushpalata Kayastha TABLE OF CONTENTS Sr. no Title Page no. 1 Streszczenie 5 2 Abstract 8 3 List of scientific papers included in the dissertation 11 4 Other publications published during PhD 12 5 Funding 14 6 Scientific collaboration 14 7 Summary 15 8 References 30 9 Publication 1: Integrative taxonomy reveals new, widely distributed tardigrade species of the genus Paramacrobiotus (Eutardigrada: Macrobiotidae). 41 10 Publication 2: Morphological and genetic variability in cosmopolitan tardigrade species - Paramacrobiotus fairbanksi Schill, Förster, Dandekar & Wolf, 2010 80 11 Publication 3: Tolerance against exposure to solution of magnesium perchlorate in microinvertebrates 188 12 Publication 4: Elevated external temperature affect cell ultrastructure and heat shock protein (HSP) in Paramacrobiotus experimentalis Kaczmarek, Mioduchowska, Poprawa & Roszkowska, 2020 208 13 Publication 5: A Review on the Genus Paramacrobiotus (Tardigrada) with a New Diagnostic Key. 233 14 Author statements 257 15 Co-author statements 263 The genus Paramacrobiotus (Tardigrada): integrative taxonomy, biogeography and effects of stress factors on the selected species. Pushpalata Kayastha 5 STRESZCZENIE Paramacrobiotus stanowi jeden z rodzajów należących do typu Tardigrada (grupy zwierząt potocznie nazywanej niesporczakami lub misiami wodnymi). Rodzaj ten został utworzony ponad dekadę temu. Poprzednio, przedstawiciele tego rodzaju byli zaliczani do kompleksu richtersi-areolatus wewnątrz rodzaju Macrobiotus, by w końcu, przy pomocy technik analizy molekularnej i filogenetycznej zostać wyodrębnionymi jako nowy rodzaj. Jak sugeruje tytuł, celem niniejszej rozprawy były badania rodzaju Paramacrobiotus, z uwzględnieniem zastosowania taksonomii integratywnej w opisie nowych dla wiedzy gatunków, ustalenie potencjalnych zasięgów występowania gatunków partenogenetycznych oraz sprawdzenie zasadności hipotezy „wszystko jest wszędzie” względem badanych gatunków, zbadanie wpływu czynników stresowych na gatunek Pam. experimentalis, badania nad rozmieszczeniem poszczególnych gatunków, badania mikrobiomu, a także przyjrzenie się historiom życiowym poszczególnych gatunków z uwzględnieniem zdolności do anhydrobiozy. Ponadto sporządzony został nowy klucz diagnostyczny dla rodzaju Paramacrobiotus wykorzystujący cechy morfologiczne i morfometryczne osobników dorosłych i jaj. Wykorzystując techniki taksonomii integratywnej (klasyczna morfologia i morfometria oraz badania genetyczne), nowy gatunek, Pam. gadabouti, został opisany na podstawie materiału pochodzącego z Ribeiro Frio na Maderze. Ponadto, eksperymentalnie zbadano sposób rozmnażania się tego gatunku, co potwierdziło wzajemne powiązania między szerokim rozprzestrzenieniem a partenogenezą. W kolejnej pracy przeanalizowano rozmieszczenie oraz zróżnicowanie genetyczne dwóch partenogenetycznych gatunków z rodzaju Paramacrobiotus, tj. Pam. gadabouti oraz Pam. fairbanksi potwierdzając prawdziwość hipotezy „wszystko jest wszędzie” przynajmniej dla niektórych gatunków niesporczaków. Modelowanie niszy środowiskowych wykonane przy użyciu narzędzia MaxEnt, potwierdza szeroki zasięg obydwu partenogenetycznych gatunków. W kolejnej publikacji zbadano przeżywalność niesporczaków z gatunku Pam. experimentalis, wystawianych na działanie różnych stężeń (0,25%; 0,50% oraz 1,00%) nadchloranu magnezu (uwzględniając stężenia zaobserwowane w marsjańskim regolicie) w dwóch różnych przedziałach czasowych (24h i 72h). W próbach, w których osobniki zostały poddane 24-godzinnej ekspozycji, kolejno 33,3%; 16,7% oraz 0% osobników pozostało aktywnych w stężeniach 0,25%; 0,50% oraz 1,00%. Jednakże, ponad 75% z nich powróciło do The genus Paramacrobiotus (Tardigrada): integrative taxonomy, biogeography and effects of stress factors on the selected species. Pushpalata Kayastha 6 stanu aktywnego po przeniesieniu ich do medium hodowlanego (93,3%; 76,7% oraz 86,7% osobników w stężeniach 0,25%; 0,50% oraz 1,00%). W próbach, w których osobniki zostały poddane 72-godzinnej ekspozycji, kolejno 30,0%; 26,7% oraz 0% osobników pozostało aktywnych w stężeniach 0,25%; 0,50% oraz 1,00%. Po przeniesieniu ich do medium hodowlanego kolejno 83,3%; 86,7% oraz 10,0% osobników w stężeniach 0,25%; 0,50% oraz 1,00% powróciło do stanu aktywnego. W następnej pracy zbadano zmiany w ultrastrukturze komórek spichrzowych zarówno u aktywnych osobników, jak i u osobników w anhydrobiozie. Zbadano też poziom syntezy białek szoku cieplnego (Hsp27, Hsp60 oraz Hsp70) u aktywnych osobników Pam. experimentalis wystawionych na podwyższoną temperaturę (35 °C, 37 °C, 40 °C i 42 °C) przez pięć godzin, w porównaniu do optymalnej temperatury hodowlanej (20 °C). Pojedyncze komórki spichrzowe z grupy kontrolnej, znajdowały się w jamie ciała, pośród narządów wewnętrznych, przyjmując kuliste i ameboidalne kształty. Niewielkie, choć zauważalne zmiany w mitochondriach zostały zaobserwowane u osobników aktywnych wystawionych na temperaturę 35 °C. Znaczące zmiany w ultrastrukturze komórek spichrzowych zaobserwowane zostały u osobników poddanych temperaturze 37 °C. Zaobserwowano u nich liczne, uszkodzone mitochondria z zauważalnym zanikiem grzebieni oraz pojawieniem się struktur autofagicznych. Jeszcze więcej zmian zaobserwowano w temperaturze 40 °C, objawiających się nieregularnym kształtem komórek spichrzowych, uszkodzeniami organelli komórkowych oraz zwiększoną obecnością ciał autofagicznych i autolizosomów w mitochondriach. Jednakże najbardziej drastyczne zmiany zaobserwowano w 42 °C, gdzie nastąpiła pełna degradacja komórek i organelli, wskazująca na wystąpienie martwicy, do poziomu utrudniającego rozpoznanie komórek. Jednocześnie, osobniki w anhydrobiozie, poddane działaniu podwyższonej temperatury, nie wykazywały jakichkolwiek negatywnych zmian w komórkach spichrzowych na poziomie ultrastrukturalnym. Spośród pięciu zastosowanych w badaniu temperatur, trzy uznane za najważniejsze zostały wybrane (20 °C – optymalna temperatura do hodowli, 35 °C – najwyższa temperatura, po której nastąpił powrót osobników do aktywności oraz 42 °C – gdzie zaobserwowana została pełna martwica) i wykorzystane do określenia poziomów białek szoku cieplnego (Hsp27, Hsp60 oraz Hsp70) w aktywnych osobnikach. Wszystkie próbki wskazywały na wyraźny wzrost poziomu ekspresji białek wraz ze wzrostem temperatury. The genus Paramacrobiotus (Tardigrada): integrative taxonomy, biogeography and effects of stress factors on the selected species. Pushpalata Kayastha 7 W ostatniej pracy składającej się na rozprawę doktorską zebrano całkowitą dostępną wiedzę na temat rodzaju Paramacrobiotus. Podsumowując więc, do rodzaju Paramacrobiotus należy obecnie 45 gatunków (wliczając w to Pam. gadabouti dodany w ramach niniejszej pracy), które można znaleźć na całym świecie, popierając tezę, że rodzaj ten jest kosmopolityczny. W rodzaju występują zarówno partenogenetyczne, jak i obupłciowe gatunki, wykazujące zarówno krótką jak i długą długość życia. Gatunki w tym rodzaju mogą być wszystkożerne (jednak z przewagą drapieżników), a odżywiają się między innymi, cyjanobakteriami, algami, grzybami, wrotkami, nicieniami oraz niesporczakami. Gatunki z tego rodzaju charakteryzują się dość dobrą zdolnością do kryptobiozy, dzięki czemu często stanowią obiekt badań w pracach nad anhydrobiozą. Slowa kluczowe: Tardigrada, taksonomia integratywna, hipotezy „wszystko jest wszędzie”, kosmopolityczny, misiami wodnymi The genus Paramacrobiotus (Tardigrada): integrative taxonomy, biogeography and effects of stress factors on the selected species. Pushpalata Kayastha 8 ABSTRACT Paramacrobiotus is one of the genera of the phylum Tardigrada (commonly referred as water bears). This genus was erected more than a decade ago. Previously all representatives of this genus were included in the richtersi-areolatus complex within the genus Macrobiotus, but with the help of molecular and phylogenetic analysis, this new genus was identified and established. As the title of thesis suggests, the goal of this dissertation was the overall study of the genus Paramacrobiotus, including: incorporating integrative taxonomy in new species descriptions, working out the distribution patterns of parthenogenetic Pramacrobiotus species, testing if the ‘everything is everywhere’ hypothesis is true for them, testing the influence of different types of stressors on the species Pam. experimentalis, assessing biogeography, distribution, microbiome, reproduction, feeding behaviour, life history, Wolbachia endosymbiont identification and cryptobiotic abilities of the species from the genus Pramacrobiotus and providing a new diagnostic key for the genus using morphological and morphometric characters of adults and eggs. Using an integrative taxonomy approach (classical morphology and morphometry, as well as, genotypic using DNA barcodes and phylogenetic tree), a new species: Pam. gadabouti was described from a moss sample collected in Ribeiro Frio, Madeira. Furthermore, the mode of reproduction of this species was studied experimentally, which corroborates the interrelatedness between wide distribution and parthenogenesis. In the subsequent paper constituting the doctoral dissertation, two parthenogenetic species of the Paramacrobiotus, i.e., Pam. gadabouti and Pam. fairbanksi were analysed to study the distribution, as well as genetic variability which showed that the ‘everything is everywhere’ hypothesis is true for, at least, some tardigrade species. Environmental niche modelling performed using MaxEnt supports the wide distribution of these two parthenogenetic species. In the next publication, survivability of the Pam. experimentalis was tested at various concentrations (0.25%, 0.50% and 1.00%) of magnesium perchlorates (in range with the concentration present in Martian regolith) for two different time points (24h and 72h). In experiments where specimens were exposed to 24h time period, 33.3%, 16.7% and 0% were active in 0.25%, 0.50% and 1.00% solutions, respectively. However, more than 75% returned to activity after transferring them to the culture medium (93.3%, 76.7% and 86.7% of The genus Paramacrobiotus (Tardigrada): integrative taxonomy, biogeography and effects of stress factors on the selected species. Pushpalata Kayastha 9 specimens exposed to 0.25%, 0.50% and 1.00% solutions, respectively). In experiments where specimens were exposed to 72h time period, 30.0%, 26.7% and 0% were active in 0.25%, 0.50% and 1.00% solutions, respectively and after transferring them to the culture medium, 83.3%, 86.7% and 10.0% of specimens exposed to 0.25%, 0.50% and 1.00% solutions, respectively, returned to activity. In the following paper constituting the doctoral dissertation, changes in ultrastructure for both active animals and desiccated tuns, as well as levels of heat shock proteins (Hsp27, Hsp60 and Hsp70) in active animals of Pam. experimentalis were studied when exposed at higher temperatures (35 °C, 37 °C, 40 °C, and 42 °C) for 5 hours, compared to optimal temperature (20 °C). Isolated storage cells from the control group persisted in the body cavity among internal organs in an amoeboid or spherical shape. Small, but visible changes were observed in specimens exposed to 35°C in the form of alterations in the mitochondria. Significant ultrastructural changes were observed in storage cells of specimens exposed to 37 °C. There were multiple deteriorating mitochondria with the loss of its cristae and there was a presence of autophagic structures. The level of changes increased at 40°C, with the irregular and shrunken shape of storage cells, deteriorated cell organelles and mitochondria with a higher number of autophagosomes and autolysosomes. Most drastic changes were observed at 42 °C, with full degeneration of cells and organelles showing signs of necrosis, making even cell identification difficult. However, when exposed to higher temperatures, tuns exhibited absolutely no differences from the control group. Out of five temperatures tested, the three deemed most important were selected (20 °C – optimum temperature, 35 °C – the highest temperature from which the return to activity was observed and 42 °C – where full necrosis was observed) and used for quantification of heat shock proteins (Hsp27, Hsp60 and Hsp70) in active specimens. All of them showed significant upregulation with the increase of the temperature. In the last paper, all available information on the genus Paramacrobiotus were summarized. Thus, in summary, the genus Paramacrobiotus currently includes 45 species (including Pam. gadabouti added as a part of the present thesis). The species of this genus can be found everywhere throughout the globe, supporting the statement that the genus is cosmopolitan. Both dioecious and unisexual species are present in the genus, with both long and short lifespan. The species in this genus are generally carnivorous with their food preference, including certain rotifers, nematodes and juvenile tardigrades. However, it was The genus Paramacrobiotus (Tardigrada): integrative taxonomy, biogeography and effects of stress factors on the selected species. Pushpalata Kayastha 10 reported that they could also feed on cyanobacteria, algae, and fungi. The species generally have a good affinity for cryptobiosis, which is why multiple studies involving anhydrobiosis have been performed to date using specimens of various species of the Paramacrobiotus. Keywords: Tardigrada, integrative taxonomy, Everything is Everywhere hypothesis, cosmopolitan, water bear The genus Paramacrobiotus (Tardigrada): integrative taxonomy, biogeography and effects of stress factors on the selected species. Pushpalata Kayastha 11 LIST OF SCIENTIFIC WORKS INCLUDED IN THE DISSERTATION The results of the experimental works are described in the following papers: 1. Kayastha, P., Stec, D., Sługocki, Ł., Gawlak, M., Mioduchowska, M., & Kaczmarek, Ł. (2023). Integrative taxonomy reveals new, widely distributed tardigrade species of the genus Paramacrobiotus (Eutardigrada: Macrobiotidae). Scientific Reports, 13(1), 2196. https://doi.org/10.1038/s41598-023-28714-w, IF (2021): 4.997; MEiN points: 140. 2. Kayastha, P., Szydło, W., Mioduchowska, M. & Kaczmarek, Ł. (after review). Morphological and genetic variability in cosmopolitan tardigrade species - Paramacrobiotus fairbanksi Schill, Förster, Dandekar & Wolf, 2010. Scientific Reports. https://doi.org/10.21203/rs.3.rs-2736709/v2, IF (2021): 4.997; MEiN points: 140. 3. Kayastha, P., Rzymski, P., Gołdyn, B., Nagwani, A.K., Fiałkowska, E., Pajdak-Stós, A., Sobkowiak, R., Robotnikowski, G. & Kaczmarek, Ł. (2023). Tolerance against exposure to solution of magnesium perchlorate in microinvertebrates. Zoological Journal of the Linnean Society, (online first). https://doi.org/10.1093/zoolinnean/zlad060. IF (2021): 3.838; MEiN points: 140. 4. Kayastha, P., Wieczorkiewicz, F., Pujol, M., Robinson, A., Michalak, M., Kaczmarek, Ł. & Poprawa, I. (in review). Elevated external temperature affects cell ultrastructure and heat shock proteins (HSPs) in Paramacrobiotus experimentalis Kaczmarek, Mioduchowska, Poprawa, & Roszkowska, 2020, Scientific Reports. IF (2021): 4.997; MEiN points: 140. In addition, the following review paper containing an overview of the genus Paramacrobiotus was published. 5. Kayastha, P., Mioduchowska, M., Warguła, J., Kaczmarek, Ł. (2023). A Review on the Genus Paramacrobiotus (Tardigrada) with a New Diagnostic Key. Diversity. 15(9):977. https://doi.org/10.3390/d15090977. IF (2021): 3.031; MEiN points: 70 https://doi.org/10.1038/s41598-023-28714-w https://doi.org/10.21203/rs.3.rs-2736709/v2 https://doi.org/10.21203/rs.3.rs-2736709/v1 https://doi.org/10.3390/d15090977 The genus Paramacrobiotus (Tardigrada): integrative taxonomy, biogeography and effects of stress factors on the selected species. Pushpalata Kayastha 12 OTHER PUBLICATIONS DURING MY PHD WHICH ARE NOT INCLUDED IN THESIS 1. Polishchuk, A., Kayastha, P., Kovalenko, P., Parnikoza, I. & Kaczmarek, Ł. (2023). New records of tardigrades from the Danco and Graham Coasts, the maritime Antarctic. Annales Zoologici, 73: 17–28. https://doi.org/10.3161/00034541ANZ2023.73.1.002, IF (2022): 1.1; MEiN points: 100 2. Kaczmarek, Ł., Rutkowski, T., Zacharyasiewicz, M., Surmacki, A., Osiejuk, T. & Kayastha, P. (2023). New species of Macrobiotidae (Eutardigrada) from Cameroon (Africa), characteristics of Macrobiotus morpho-groups and a key to the nelsonae group. Annales Zoologici, 73: 1–15, https://doi.org/10.3161/00034541ANZ2023.73.1.001, IF (2022): 1.1; MEiN points: 100 3. Kayastha, P., Mioduchowska, M., Gawlak, M., Sługocki, Ł., Araújo, R., Silva, J.J.G. & Kaczmarek, Ł. (2023). Integrative description of Macrobiotus kosmali sp. nov. (hufelandi group) from the Island of Madeira (Portugal). The European Zoological Journal, 90(1): 126–38. https://doi.org/10.1080/24750263.2022.2163312, IF (2022): 2.1; MEiN points: 140 4. Kaczmarek, Ł., Kayastha, P., Roszkowska, M., Gawlak, M. & Mioduchowska, M. (2022). Integrative redescription of the Minibiotus intermedius (Plate, 1888)—The type species of the genus Minibiotus R.O. Schuster, 1980. Diversity, 14(5): 356. https://doi.org/10.3390/d14050356, IF (2022): 2.4; MEiN points: 70 5. Kaczmarek, Ł., Kayastha, P., Gawlak, M., Mioduchowska, M. & Roszkowska, M. (2022). An integrative redescription of Echiniscus quadrispinosus quadrispinosus Richters, 1902 (Heterotardigrada; Echiniscidae) from the terra typica in Taunus Mountain Range (Europe; Germany). European Journal of Taxonomy, 823: 102–124. https://doi.org/10.5852/ejt.2022.823.1819, IF (2022): 2.0; MEiN points: 70 6. Bartylak, T., Kayastha, P., Roszkowska, M., Kepel, A., Kepel, M. & Kaczmarek, Ł., (2022). Tardigrades of the Ivohibory forest (south-central Madagascar) with a description of a new Bryodelphax species. The European Zoological Journal, 89(1): 423–436. https://doi.org/10.1080/24750263.2022.2042404, IF(2022): 2.1; MEiN points: 140 https://doi.org/10.3161/00034541ANZ2023.73.1.002 https://doi.org/10.3161/00034541ANZ2023.73.1.001 https://doi.org/10.1080/24750263.2022.2163312 https://doi.org/10.3390/d14050356 https://doi.org/10.5852/ejt.2022.823.1819 https://doi.org/10.1080/24750263.2022.2042404 The genus Paramacrobiotus (Tardigrada): integrative taxonomy, biogeography and effects of stress factors on the selected species. Pushpalata Kayastha 13 7. Kaczmarek, Ł., Kayastha, P., Gawlak, M., Mioduchowska, M. & Roszkowska, M. (2021). An integrative description of a Diploechiniscus oihonnae (Richters, 1903) population from near the original type locality in Merok. Zootaxa, 4964(1): 83–102. https://doi.org/10.11646/zootaxa.4964.1.4, IF (2022): 1.026; MEiN points: 70 8. Kayastha, P., Wiśniewska, J., Kuzdrowska, K. & Kaczmarek, Ł. (2021). Aquatic tardigrades in Poland – a review. Limnological Review, 21(3): 147–154. https://doi.org/10.2478/limre-2021-0013, IF (2022): 0.69; MEiN points: 70 9. Kayastha, P., Roszkowska, M., Mioduchowska, M., Gawlak, M. & Kaczmarek, Ł. (2021) Integrative descriptions of two new tardigrade species along with the new record of Mesobiotus skorackii Kaczmarek et al., 2018 from Canada. Diversit, 13(8): 394. https://doi.org/10.3390/d13080394, IF (2022): 2.4; MEiN points: 70 10. Kayastha, P., Berdi, D., Mioduchowska, M., Gawlak, M., Łukasiewicz, A., Gołdyn, B., Jędrzejewski, S. & Kaczmarek, Ł. (2020). Description and molecular characterization of Richtersius ziemowiti sp. nov. (Richtersiidae) from Nepal (Asia) with evidence of heterozygous point mutation events in the 28S rRNA. Annales Zoologici, 70(3): 381–396. https://doi.org/10.3161/00034541ANZ2020.70.3.010, IF (2022): 1.1; MEiN points: 100 11. Kayastha, P., Bartylak, T., Gawlak, M. & Kaczmarek, Ł. (2020). Integrative description of Pseudechiniscus lalitae sp. nov. (Tardigrada: Heterotardigrada: Echiniscidae) from the Azores Archipelago (Portugal). Annales Zoologici, 70(4): 487– 505. https://doi.org/10.3161/00034541ANZ2020.70.4.002, IF (2022): 1.1; MEiN points: 100 12. Kayastha, P., Berdi, D., Mioduchowska, M., Gawlak, M., Łukasiewicz, A., Gołdyn, B. & Kaczmarek, Ł. (2020). Some tardigrades from Nepal (Asia) with integrative description of Macrobiotus wandae sp. nov. (Macrobiotidae: hufelandi group). Annales Zoologici, 70(1): 121–142. https://doi.org/10.3161/00034541ANZ2020.70.1.007, IF (2022): 1.1; MEiN points: 100 https://doi.org/10.11646/zootaxa.4964.1.4 https://doi.org/10.2478/limre-2021-0013 https://doi.org/10.3390/d13080394 https://doi.org/10.3161/00034541ANZ2020.70.3.010 https://doi.org/10.3161/00034541ANZ2020.70.4.002 https://doi.org/10.3161/00034541ANZ2020.70.1.007 The genus Paramacrobiotus (Tardigrada): integrative taxonomy, biogeography and effects of stress factors on the selected species. Pushpalata Kayastha 14 FUNDING This work was supported by the following sources: • 'Support for publications in prestigious research journals'-Uczelnia Badawcza' No 085/08/POB1/0001 • 'Initiative of Excellence - Research University'- Uczelnia Badawcza' No 071/06/POB1/0022. • 'Excellence Initiative - Research University'- Uczelnia Badawcza' No 070/13/UAM/0032 • Second edition of the "MINIGRANTY" competition as part of the Passport to the Future Project - Interdisciplinary Studies doctoral studies at the Faculty of Biology, AMU POWR.03.02.00-00-I006 / 17. No: POWER8/2021/2ed • UNIWERSYTET JUTRA - zintegrowany program rozwoju Uniwersytetu im. Adama Mickiewicza w Poznaniu”, No POWR.03.05.00-00-Z303/17 • Dean’s Grant for PhD students at Faculty of Biology, Uniwersytetu im. Adama Mickiewicza w Poznaniu: No 506000/604/4102000000/BW002020 (GDWB-08/2020) SCIENTIFIC COLLABORATION All the experiments which are part of my PhD thesis were performed at the Department of Animal Taxonomy and Ecology, Faculty of Biology, Adam Mickiewicz University in Poznan, Poland, but also with collaboration with: • Dr. hab. Izabela Poprawa Team (University of Silesia in Katowice, Poland) • Prof. Marek Michalak (Michalak Lab, Department of Biochemistry, University of Alberta, Canada) The genus Paramacrobiotus (Tardigrada): integrative taxonomy, biogeography and effects of stress factors on the selected species. Pushpalata Kayastha 15 SUMMARY Background Tardigrades are invertebrates from the phylum Tardigrada, also known as water bears or moss piglets. There are ca. 1,500 species and subspecies in this phylum till date (Degma & Guidetti, 2009-2023). Within the phylum are currently 33 families, 159 genera, 1464 species, and 21 more subspecies (Nelson et al., 2020) (Figure 1). Tardigrades are widespread throughout the world's biomes and can be found in freshwater, marine, and terrestrial environments, despite their understudied status (Nelson et al., 2018) (Figure 2). Marine tardigrades have been reported from the intertidal and subtidal zones to the deepest depths of the sea (Kristensen & Sterrer, 1985). Freshwater tardigrades inhabit various running and standing water sources and underground habitats. Terrestrial species live in a wide variety of habitats, such as mosses, lichens, and liverworts on rocks, soil, tree trunks, leaf litter, and soil, but to be active they need to be surrounded by a film of water (Ramazzotti & Maucci, 1983). Limno-terrestrial species inhabit both freshwater and terrestrial environments. Tardigrades have adapted to their environment in many ways. The most common ones that they undergo are cryptobiosis which are anhydrobiosis (lack of water), anoxybiosis (lack of oxygen), chemobiosis (harsh chemical condition), cryobiosis (very low temperature) and osmobiosis (higher salt concentration) (Keilin, 1959). Furthermore, our understanding of tardigrade biodiversity and biogeography is fast changing due to constant discoveries of new species and the development of integrative taxonomy employing combined morphological and genetic data, which is currently deemed vital for future tardigrade studies (Nelson et al., 2010, 2015). The genus Paramacrobiotus (Tardigrada): integrative taxonomy, biogeography and effects of stress factors on the selected species. Pushpalata Kayastha 16 Figure 1. Taxonomic position of genus Paramacrobiotus within phylum Tardigrada based on Degma & Guidetti (2009) (but, not all genera and subgenera are listed). Phylum: TARDIGRADA Class: HETEROTARDIGRADA Order: ARTHROTARDIGRADA Family: 1. Anisonychidae 2. Archechiniscidae 3. Batillipedidae 4. Coronarctidae 5. Halechiniscidae 6. Neoarctidae 7. Neostygarctidae 8. Renaudarctidae 9. Stygarctidae 10. Styraconyxidae 11. Tanarctidae . Order: ECHINISCOIDEA Family: 1. Echiniscoididae 2. Carphanidae 3. Oreellidae 4. Echiniscidae Class: MESOTARDIGRADA Order: THERMOZODIA Family: Thermozodiidae Class: EUTARDIGRADA Order: APOCHELA Family: Milnesiidae Order: PARACHELA Family: 1. Eohypsibiidae 2. Calohypsibiidae 3. Hypsibiidae 4. Microhypsibiidae 5. Ramazzottiidae 6. Doryphoribiidae 7. Halobiotidae 8. Hexapodibiidae 9. Isohypsibiidae 10. Adorybiotidae 11. Macrobiotidae 12. Murrayidae 13. Richtersiusidae 14. Beornidae 15. Necopinatidae Genus: 1. Biserovus 2. Calcarobiotus 3. Famelobiotus 4. Insuetifurca 5. Macrobiotus 6. Mesobiotus 7. Minibiotus 8. Minilentus 10. Pseudodiphascon 11. Pseudohexapodibius 12. Schusterius 13. Sisubiotus 14. Tenuibiotus 15. Xerobiotus 9. Paramacrobiotus The genus Paramacrobiotus (Tardigrada): integrative taxonomy, biogeography and effects of stress factors on the selected species. Pushpalata Kayastha 17 Figure 2. Habitats where tardigrades can be found (Created using BioRender.com). Integrative taxonomy In the past, species were described using a traditional taxonomy approach which was solely based on morphology and morphometrics and therefore came with its limits (Dayrat, 2005). For this reason, taxonomists have embraced the integrative taxonomy approach (Figure 3), which is based on both morphological and morphometric data, as well as, phylogenetic analysis using DNA barcodes (Figure 4). In the case of tardigrades, four DNA markers, three nuclear (18S rRNA, 28S rRNA, ITS-2) and one mitochondrial (COI) are used to characterize the species genetically. The mitochondrial marker COI alone can be enough to identify and distinguish taxa at the species level (Cesari et al., 2009), but for higher taxonomic units, more conserved 18S rRNA and 28S rRNA markers concatenated with both COI and ITS or either one of them are more accurate. For morphology and morphometrics, Phase Contrast or Nomarski Contrast Microscopy and Scanning Electron Microscopy are used to study the morphology of both eggs and adults, as well as, to measure various characteristics of both eggs and adults. The genus Paramacrobiotus (Tardigrada): integrative taxonomy, biogeography and effects of stress factors on the selected species. Pushpalata Kayastha 18 Figure 3. Schematic representation of the integrative taxonomy approach. Morphology Morphometry Genetic analysis DNA barcodes Phylogenetic trees Traditional taxonomy Molecular taxonomy Integrative taxonomy Legend The genus Paramacrobiotus (Tardigrada): integrative taxonomy, biogeography and effects of stress factors on the selected species. Pushpalata Kayastha 19 Figure 4. Schematic representation of the DNA barcoding methodology for tardigrades (Created using BioRender.com). “Everything is Everywhere” (EiE) hypothesis Finlay (2002) proposed in the 'everything is everywhere' concept that species below 1μm have the potential to be distributed everywhere and that the environment will determine if they can survive. Although some morpho-species (species determined by morphological criteria only) may be found throughout the world, their gene pools may or may not be disjunct (Baas-Becking, 1934; Beijerinck, 1913; Finlay, 2002; Finlay & Clarke, 1999). Many researchers proposed or hypothesized tardigrade cosmopolitism (e.g. Ramazzotti & Maucci, 1983; ,Nielsen, 2012). However, few previous investigations, based exclusively on classical taxonomy and indirectly testing the ‘EiE’ hypothesis (Guil, 2011; Guil et al., 2009; Pilato and Binda 2001), did not support the cosmopolitan distribution of tardigrades. Furthermore, recent genetic results suggested that certain tardigrade species' ranges may be significantly smaller than previously thought. These findings show that previous conclusions on the distribution of tardigrades, which were all based entirely on classical taxonomy, were most likely incorrect. However, as of today, there are four tardigrade species known from more than one The genus Paramacrobiotus (Tardigrada): integrative taxonomy, biogeography and effects of stress factors on the selected species. Pushpalata Kayastha 20 zoogeographic realm namely Echiniscus testudo (Doyère, 1840), Milnesium inceptum (Morek , Suzuki, Schill, Georgiev, Yankova, Marley & Michalczyk, 2019), Pam. fairbanksi (Schill, Förster, Dandekar & Wolf, 2010) and Pam. gadabouti (Kayastha , Stec, Mioduchowska and Kaczmarek, 2023). Genetic findings confirmed the distribution of these species in more than one zoogeographic realm. So, cosmopolitan distribution might not generally be true for all tardigrades, but it is true at least for some tardigrade species. Environmental niche modelling (ENM) The notion of ecological niches was developed by Joseph Grinnell (Grinnell, 1917), and he was the first who investigated links between ecological niches and species distribution. His concept, which was later adapted into contemporary jargon, was that a species' ecological niche is the set of conditions in which the species may sustain generations despite requiring immigration from other localities (Grinnell, 1917). The ENMs are similar to species distribution models and are experimental or analytical estimates of a species' ecological niche (Sillero et al., 2021). They are statistical approaches or theoretically generated response surfaces that describe and predict species distributions by relating physiological or distribution data to environmental variables (Franklin, 2014; Guisan et al., 2017; Peterson et al., 2011; Sillero, 2011; Sillero et al., 2021). The ENMs are generated using a variety of methods, using georeferenced occurrence databases (i.e. sample localities that include latitude and longitude data) and data pertaining to the environment in the form of GIS (Geographic Information System) datasets (Elith et al., 2006). Some ENM-specific modelling algorithms, such as Maximum Entropy (Phillips et al., 2006, 2017) or Ecological Niche Factor Analysis (ENFA) (Hirzel et al., 2002), are only available in dedicated statistical software. They are increasingly used in studying bioinvasions, conservation biology, bioresponse to climate change, disease transmission in space, and various aspects of ecology and evolutionary studies (GengPing et al., 2013). There has been a significant increase in the use of ENM in recent years. The ENMs are used to explain populations' or species' ecological tolerances. Furthermore, the simultaneous advancements in phylogeography resulting from the increased spatial data accessibility and methodological breakthroughs in species distribution modelling, led to widening the area of problems explored and delivering of unique insights (Alvarado-Serrano & Knowles, 2014). The ENMs also help predict the invasion and cosmopolitanism or wide distribution of various species (Ba et al., 2010; Iniesta et al., 2020). Only two studies using The genus Paramacrobiotus (Tardigrada): integrative taxonomy, biogeography and effects of stress factors on the selected species. Pushpalata Kayastha 21 ENM have been performed using tardigrades to date. Gąsiorek et al. ( 2019) used ENM for the first time to model statistical predictions of tardigrades’ geographical distribution. The other study that used ENM to provide proof of cosmopolitanism for two widely distributed Paramacrobiotus species was published by Kayastha et al., 2023). Stressors Stress is a complex force that is essential to life’s evolution. Organisms change in response to stress, and stressful environments can constitute selection limits (Kültz, 2020). Species are frequently subjected to an array of stresses, both natural and generated by human (Sih et al., 2004). Increase in pollution as a consequence of anthropogenic activities are causing biodiversity loss, affected ecosystems, and habitat degradation (Ojekunle & Sodipe, 2020). Various pollutants including toxic heavy metals and chemicals are released into the environment resulting in environmental problems. Nonetheless, temperature is another stressor used extensively to understand the effects of global climate change on various biological systems (e.g. Doney et al., 2012; Morón Lugo et al., 2020). Invertebrates are at risk when exposed to both toxic chemicals and higher temperatures; see, e.g., the effect of temperature on metabolic energy balance in marine invertebrates (Newell & Branch, 1980), temperature rises affect invertebrate population and slowing down decomposition (Figueroa et al., 2021), a large-scale ecotoxicology/health stressor trial for mussel embryos (Young et al., 2023), toxicology of sodium chloride-based road salt formulations in juvenile aquatic invertebrates (Harwood et al., 2023) and so on. Tardigrades are known for their ability to undergo cryptobiosis and were subjected to different kinds of stressors, and have also been used as a model system to study the effect of temperature, both high-temperature tolerance and freeze tolerance (e.g. Hengherr, Worland, Reuner, Brümmer, et al., 2009; Hengherr, Worland, Reuner, Brummer, et al., 2009; Neves et al., 2020, 2022; Rebecchi et al., 2009). Furthermore, the effects of toxic chemicals have also been studied on the tardigrades (Czerneková et al., 2017; Hygum et al., 2017; Ojekunle & Sodipe, 2020; Wieczorkiewicz et al., 2023). One of the studies performed was to check the tolerance of magnesium perchlorate on various invertebrates including tardigrades (Kayastha et al., 2023) since presence of different concentrations of perchlorates (ClO4 −), reaching a mean of 0.6 wt% in the Martian regolith (a blanket of unconsolidated, loose, heterogeneous surface deposits overlaying solid rock), are regarded as a severe obstacle for terrestrial life forms (Glavin et al., 2013; Hecht et al., 2009; The genus Paramacrobiotus (Tardigrada): integrative taxonomy, biogeography and effects of stress factors on the selected species. Pushpalata Kayastha 22 Kounaves et al., 2010, 2014; Leshin et al., 2013; Martin et al., 2020; Ming et al., 2014; Sutter et al., 2017). On Earth, perchlorate is produced both naturally and artificially (Brown & Gu, 2006; Isobe et al., 2013; Vega et al., 2018) and is a pollutant that can remain in groundwater and soil and is regularly identified at human-health-relevant quantities in various ecosystems (Acevedo-Barrios et al., 2022). However, perchlorate concentrations on Mars much exceed those observed on Earth (Calderón et al., 2014; Ericksen, 1983) which is why the study was performed using range of magnesium perchlorate solutions higher than that found in Martian regolith. The main aims of my PhD thesis are: 1. To describe new Paramacrobiotus species using an integrative taxonomy approach. 2. To study distribution patterns of parthenogenetic Paramacrobiotus species and test the ’everything is everywhere’ hypothesis. 3. To study the influence of different stress factors on the ultrastructure, survivability and heat shock proteins expression in the species of the genus Paramacrobiotus. 4. To verify the available data on barcodes and construct a phylogeny, biogeography, distribution, microbiome, reproduction, feeding behaviour, life history and cryptobiotic ability of the species from the genus Paramacrobiotus. 5. To prepare a new diagnostic key for Paramacrobiotus based on the morphological and morphometric characters of adults and eggs. The first aim of my thesis is accomplished in studies presented in publication 1. Further, the second aim is fulfilled in the investigation performed and reported in publication 2. Similarly, the third aim of the thesis is achieved in the results produced in publications 3 and 4. Lastly, the fourth and fifth aim of the thesis is fulfilled in publication 5. In the first paper (Kayastha et al., 2023), which is part of my PhD thesis, I am presenting the description of a species of tardigrade from the genus Paramacrobiotus new to science, identified using integrative taxonomy – Pam. gadabouti Kayastha, Stec, Mioduchowska and Kaczmarek 2023. The new species belongs to the Pam. richtersi group due to the presence of microplacoid in the pharynx and richtersi-type of eggs. Paramacrobiotus. gadabouti shares the most similarity with Pam. alekseevi (Tumanov, 2005) (Tumanov 2005), Pam. filipi The genus Paramacrobiotus (Tardigrada): integrative taxonomy, biogeography and effects of stress factors on the selected species. Pushpalata Kayastha 23 Dudziak, Stec and Michalczyk 2020 (Stec et al., 2020) and Pam. garynahi (Kaczmarek, Michalczyk and Diduszko 2005) (Kaczmarek et al., 2005), but differs from them mostly in egg morphology and adult morphometrics (Table 1). For genotyping, four barcodes were sequenced i.e. three nuclear (18S rRNA, 28S rRNA, ITS-2) and one mitochondrial (COI). The description of the new species increased the number of known Paramacrobiotus species to forty-five. Additionally, this new species' reproduction mode was determined to be parthenogenetic (Figure 5). This fact supports that parthenogenetic species of Paramacrobiotus are widespread (Paper 1). Table 1. Shortened differential diagnosis of Pam. gadabouti based on morphometrical characters. Characters Pam. gadabouti Pam. alekseevi Pores inside egg areoles Present Absent pt value of second macroplacoid Higher Lower Microplacoid length Longer Shorter Characters Pam. gadabouti Pam. filipi Dorsal cuticle granulation Absent Present Second macroplacoid Longer Shorter pt value of macroplacoid row Higher Lower Placoid row Longer Shorter Full egg diameter Larger Smaller Characters Pam. gadabouti Pam. garynahi Medioventral tooth in the third band of teeth in the oral cavity Divided Not divided Eggs chorion ornamentation richtersi type areolatus type pt value of macroplacoid and placoid rows Higher Lower Eggs bare and full diameter Smaller Larger The genus Paramacrobiotus (Tardigrada): integrative taxonomy, biogeography and effects of stress factors on the selected species. Pushpalata Kayastha 24 Figure 5. Workflow used to determine of the parthenogenetic reproduction mode in Pam. gadabouti (Created using BioRender.com). In the second paper, part of my PhD thesis, I am presenting the distribution patterns of two parthenogenetic Pramacrobiotus species i.e. Pam. fairbanksi and Pam. gadabouti. I analysed nine populations of the Pam. fairbanksi from various localities. Five known from literature and new finding from Albania, Canada, Madeira and Mongolia (Kayastha et al., 2023). The distribution pattern obtained using ecological niche modelling strengthens the claim for the cosmopolitanism of the species. Ecological niche modelling using maximum entropy approach was performed using MaxEnt. MERRAclim dataset (Vega et al., 2017) was used for bioclimatic data as it contains dataset for Antarctica, including one of the localities where Pam. fairbanksi was found. Similarly, five populations of the Pam. gadabouti has been analysed and the distribution pattern obtained using the ecological niche model shows its wide distribution globally, favouring the areas with Mediterranean climates. The wide distribution of these two parthenogenetic Paramacrobiotus species confirms that the hypothesis 'everything is everywhere' is correct, at least for some tardigrades. Furthermore, the morphological and genetic variability of Pam. fairbanksi was studied. Analysis of variance (ANOVA) test with The genus Paramacrobiotus (Tardigrada): integrative taxonomy, biogeography and effects of stress factors on the selected species. Pushpalata Kayastha 25 post hoc comparison of pairs of measurements, applying Bonferroni correction was used to statistically analyse single characters and R script provided in Stec et al. (2021) was used to execute Principal Component Analysis (PCA). Statistically significant morphometric differences were observed in the specimens from the populations from different localities. Furthermore, the analysed species showed higher haplotype diversity in COI compared to other barcodes, but the overall variability remains very low (Paper 2). In the third paper which is part of my PhD thesis, I tested the influence of magnesium perchlorate on the survivability in the Pam. experimentalis Kaczmarek, Mioduchowska, Poprawa and Roszkowska, 2020 (Kaczmarek et al., 2020). A three different solutions of magnesium perchlorate were used, i.e. 0.25%, 0.50% and 1.00% (mean of 0.6 wt % found in Martian regolith) for two different time periods, i.e. 24 and 72h (Figure 6). The study showed that 33.3% of the tardigrades were active after 24h in 0.25% solution, 16.7% after 24h in 0.50% solution and 0% after 24h in 1.00% solution. However, 93.3%, 76.7% and 86.7% of specimens exposed to 0.25%, 0.50% and 1.00% solutions returned to activity when placed back in culture medium for 24h. Furthermore, 30.0% of the specimens were active after 72h in 0.25% solution, 26.7% after 72h in 0.50% solution and 0.00% after 72h in 1.00% solution. Later it was found that 83.3%, 86.7% and 10.0% of specimens exposed to 0.25%, 0.50% and 1.00% solutions returned to activity when placed back in culture medium for 24h. Additionally, median deactivation time (i.e. >50% of the specimens showed no activity) was calculated. The median deactivation time was the same for specimens exposed to 0.25% and 0.50% magnesium perchlorate solutions (13.5-24h) and significantly lower for specimens exposed to 1.00% magnesium perchlorate solutions (1.5-2.5h) (Paper 3). The genus Paramacrobiotus (Tardigrada): integrative taxonomy, biogeography and effects of stress factors on the selected species. Pushpalata Kayastha 26 Figure 6. Protocol for testing Paramacrobiotus experimentalis tolerance against exposure to magnesium perchlorate solution (Created using BioRender.com). In the fourth paper, part of my PhD thesis, I tested how increased temperature affects storage cells ultrastructure and heat shock proteins levels in active specimens and in anhydrobiotic tuns of Pam. experimentalis. All active specimens’ experiments were carried out in 1.5 ml Eppendorf tubes with 10 specimens placed in the culture medium. For 5 hours, the Eppendorf tubes were placed on a heat block with an open cover set to 20 °C, 35 °C, 37 °C, 40 °C, and 42 °C. Specimens were then transferred to Petri dishes and subjected to ultrastructural and biochemical investigations (Figure 7). For anhydrobiotic tuns, specimens were first subjected to anhydrobiosis and then exposed to 20 °C, 35 °C, 37 °C, 40 °C, and 42 °C for 5 hours and then subjected to ultrastructural investigations (Figure 8). The genus Paramacrobiotus (Tardigrada): integrative taxonomy, biogeography and effects of stress factors on the selected species. Pushpalata Kayastha 27 Figure 7. Protocol for testing effects of heat stress on active specimens of the Paramacrobiotus experimentalis (Created using BioRender.com). The genus Paramacrobiotus (Tardigrada): integrative taxonomy, biogeography and effects of stress factors on the selected species. Pushpalata Kayastha 28 Figure 8. Protocol for anhydrobiosis and testing effects of heat stress on anhydrobiotic tuns of the Paramacrobiotus experimentalis (Created using BioRender.com) The genus Paramacrobiotus (Tardigrada): integrative taxonomy, biogeography and effects of stress factors on the selected species. Pushpalata Kayastha 29 When active animals were exposed to 35 °C, 37 °C, 40 °C, and 42 °C, various changes were observed (see below for details). Active specimens incubated at 35 °C already showed the first modifications in the ultrastructure of storage cells. Mitochondria showed signs of degradation and lost their cristae. Most of the mitochondria in the storage cells of active animals that were exposed to 37 °C deteriorated and lost their crests. Numerous autophagic structures also emerged. Individuals incubated at 40 °C showed similar, but significantly more pronounced changes. Additionally, at 42 °C, the cytoplasm of the storage cells became electron-lucent, the cell membrane ruptured, and necrotic symptoms were visible in degenerated cells and organs. In anhydrobiotic tuns, there were no differences in the ultrastructure of storage cells between the control group (20 °C) and the cells treated to 35 °C, 37 °C, 40 °C. However, in the tuns storage cells incubated at 42 °C, the karyolymph grew denser and the mitochondrial electron-dense substance accumulated. Furthermore, the levels of three distinct heat shock proteins (Hsp27 (sHsp), Hsp60, and Hsp70) were measured in active specimens of Pam. experimentalis exposed to 20 °C, 37 °C, and 42 °C. It was found that heat stress leads to upregulation of expression of all studied HSPs (Paper 4). In the fifth paper, part of my PhD thesis, all the information regarding the genus Pramacrobiotus till date was reviewed using available literature. It was deemed necessary to compile all the data, such as the geographical distribution of all species, feeding behaviour, life history, microbiome community, Wolbachia endosymbiont identification, reproduction, phylogeny, morphological and molecular taxonomy and cryptobiotic ability, to give them a proper overview. The genus Paramacrobiotus consists of 45 species till date, among which 13 belong to the areolatus group and 32 to the richtersi group, and the species are both bisexual and unisexual. The genus is truly cosmopolitan, as species are present throughout the world. The species are sometimes omnivorous (but most often carnivorous), consuming cyanobacteria, algae, fungi, rotifers and tardigrades. In our analysis of COI barcode sequences, speciation events that resulted in polytomies within the phylogeny of the genus Paramacrobiotus were observed (Kayastha et al., 2023). Furthermore, only few species’ lifespan is known till date including Pam. fairbanski, Pam. kenianus (Schill, Förster, Dandekar & Wolf, 2010), Pam. metropolitanus (Sugiura Matsumoto & Kunieda, 2022), Pam. richtersi (Murray, 1911) and Pam. tonollii (Ramazzotti, 1956). Also, only Pam. metropolitanus whole genome is available among species of the genus Paramacrobiotus. Similarly, the microbiomes of a few species have been studied to date. Proteobacteria and Bacteroidetes were found in the The genus Paramacrobiotus (Tardigrada): integrative taxonomy, biogeography and effects of stress factors on the selected species. Pushpalata Kayastha 30 microbial community of Pam. areolatus (Murray, 1907). Two unique patterns in the diversity detected between tardigrades and their substrates demonstrate that tardigrades had much less microbial variety than their substrates (Vecchi et al., 2018). Also, microbiome analysis on two populations of Pam. experimentalis from Madagascar and their laboratory culture environment were conducted where Proteobacteria, Firmicutes and Bacteroides were the most abundant phyla (Kaczmarek et al., 2020). Also, Rickettsiales endosymbionts were identified as possible endosymbionts. Moreover, Wolbachia endosymbiont identification was performed by Mioduchowska et al., 2021. Proteobacteria, Firmicutes, and Actinobacteria were most common, but the purpose was to study Wolbachia endosymbiont and both Rickettsiales and Wolbachia were detected in adult Paramacrobiotus sp. Moreover, few studies regarding cryptobiosis in the species of Paramacrobiotus have also been conducted. To better understand the energy aspect of anhydrobiosis, Reuner et al., 2010 studied how starvation and anhydrobiosis alter the size and number of storage cells in Pam. tonollii. Antioxidant defence (the ability to combat reactive oxygen species (ROS)) in Pam. richtersi in both active and dehydrated stages was studied by Rizzo et al. (2010). Giovannini et al., 2022 studied the formation of reactive oxygen species and the participation of bioprotectants during anhydrobiosis in Pam. spatialis Guidetti, Cesari, Bertolani, Altiero & Rebecchi, 2019 where they concluded that ROS production corresponds to the time spent in anhydrobiosis. Furthermore, Roszkowska et al. (2023) have investigated how long several tardigrades, including Pam. experimentalis, can survive in anhydrobiotic conditions. All such data were summarized in my review. Additionally, a new diagnostic key to the genus Paramacrobiotus is provided based on the morphological and morphometric characters of adults and eggs (Paper 5). References: Acevedo-Barrios, R., Rubiano-Labrador, C., Navarro-Narvaez, D., Escobar-Galarza, J., González, D., Mira, S., Moreno, D., Contreras, A., & Miranda-Castro, W. (2022). Perchlorate-reducing bacteria from Antarctic marine sediments. Environmental Monitoring and Assessment, 194(9), 654. https://doi.org/10.1007/s10661-022-10328-w Alvarado-Serrano, D. F., & Knowles, L. L. (2014). Ecological niche models in phylogeographic studies: Applications, advances and precautions. Molecular Ecology Resources, 14(2), 233–248. https://doi.org/10.1111/1755-0998.12184 The genus Paramacrobiotus (Tardigrada): integrative taxonomy, biogeography and effects of stress factors on the selected species. Pushpalata Kayastha 31 Ba, J., Hou, Z., Platvoet, D., Zhu, L., & Li, S. (2010). Is Gammarus tigrinus (Crustacea, Amphipoda) becoming cosmopolitan through shipping? Predicting its potential invasive range using ecological niche modeling. Hydrobiologia, 649(1), 183–194. https://doi.org/10.1007/s10750-010-0244-5 Baas-Becking, L. G. M. (1934). Geobiologie; of inleiding tot de milieukunde. WP Van Stockum & Zoon NV. Beijerinck, M. W. (1913). De infusies en de ontdekking der bakterien. Johannes Müller. Brown, G. M., & Gu, B. (2006). The chemistry of perchlorate in the environment. In B. Gu & J. D. Coates (Eds.), Perchlorate (pp. 17–47). Kluwer Academic Publishers. https://doi.org/10.1007/0-387-31113-0_2 C. Vega, G., Pertierra, L. R., & Olalla-Tárraga, M. Á. (2017). MERRAclim, a high-resolution global dataset of remotely sensed bioclimatic variables for ecological modelling. Scientific Data, 4(1), 170078. https://doi.org/10.1038/sdata.2017.78 Calderón, R., Palma, P., Parker, D., Molina, M., Godoy, F. A., & Escudey, M. (2014). Perchlorate levels in soil and waters from the Atacama desert. Archives of Environmental Contamination and Toxicology, 66(2), 155–161. https://doi.org/10.1007/s00244-013-9960-y Cesari, M., Bertolani, R., Rebecchi, L., & Guidetti, R. (2009). DNA barcoding in Tardigrada: The first case study on Macrobiotus macrocalix Bertolani & Rebecchi 1993 (Eutardigrada, Macrobiotidae). Molecular Ecology Resources, 9(3), 699–706. https://doi.org/10.1111/j.1755-0998.2009.02538.x Czerneková, M., Jönsson, K. I., Chajec, L., Student, S., & Poprawa, I. (2017). The structure of the desiccated Richtersius coronifer (Richters, 1903). Protoplasma, 254(3), 1367– 1377. https://doi.org/10.1007/s00709-016-1027-2 Stec, D., Dudziak, M., & Michalczyk, Ł. (2020). Integrative descriptions of two new Macrobiotidae species (Tardigrada: Eutardigrada: Macrobiotoidea) from French Guiana and Malaysian Borneo. Zoological Studies, (59) e23. https://doi.org/10.6620/ZS.2020.59-23 Dayrat, B. (2005). Towards integrative taxonomy. Biological Journal of the Linnean Society, 85(3), 407–415. https://doi.org/10.1111/j.1095-8312.2005.00503.x Degma, P., & Guidetti, R. (2009-2023). Actual checklist of Tardigrada species https://doi.org/10.25431/11380_1178608 The genus Paramacrobiotus (Tardigrada): integrative taxonomy, biogeography and effects of stress factors on the selected species. Pushpalata Kayastha 32 Doney, S. C., Ruckelshaus, M., Emmett Duffy, J., Barry, J. P., Chan, F., English, C. A., Galindo, H. M., Grebmeier, J. M., Hollowed, A. B., Knowlton, N., Polovina, J., Rabalais, N. N., Sydeman, W. J., & Talley, L. D. (2012). Climate change impacts on marine ecosystems. Annual Review of Marine Science, 4(1), 11–37. https://doi.org/10.1146/annurev-marine-041911-111611 Doyère, L. (1840). Memoire sur les Tardigrades. I. (Annales des Sciences Naturelles Paris Series 2), 14, 269-362. Elith, J., H. Graham, C., P. Anderson, R., Dudík, M., Ferrier, S., Guisan, A., J. Hijmans, R., Huettmann, F., R. Leathwick, J., Lehmann, A., Li, J., G. Lohmann, L., A. Loiselle, B., Manion, G., Moritz, C., Nakamura, M., Nakazawa, Y., McC. M. Overton, J., Townsend Peterson, A., … E. Zimmermann, N. (2006). Novel methods improve prediction of species’ distributions from occurrence data. Ecography, 29(2), 129–151. https://doi.org/10.1111/j.2006.0906-7590.04596.x Ericksen, G. E. (1983). The Chilean nitrate deposits. American Scientist, 71(4), 366–374. USGS Publications Warehouse. Figueroa, L. L., Maran, A., & Pelini, S. L. (2021). Increasing temperatures reduce invertebrate abundance and slow decomposition. Plos One, 16(11), e0259045. https://doi.org/10.1371/journal.pone.0259045 Finlay, B. J. (2002). Global dispersal of free-living microbial eukaryote species. Science, 296(5570), 1061–1063. https://doi.org/10.1126/science.1070710 Finlay, B. J., & Clarke, K. J. (1999). Ubiquitous dispersal of microbial species. Nature, 400(6747), 828–828. https://doi.org/10.1038/23616 Franklin, J. (2014). Summary for policymakers. In Climate Change 2013 – The Physical Science Basis (1st ed., pp. 1–30). Cambridge University Press. https://doi.org/10.1017/CBO9781107415324.004 Gąsiorek, P., Jackson, K. J., Meyer, H. A., Zając, K., Nelson, D. R., Kristensen, R. M., & Michalczyk, Ł. (2019). Echiniscus virginicus complex: The first case of pseudocryptic allopatry and pantropical distribution in tardigrades. Biological Journal of the Linnean Society, blz147. https://doi.org/10.1093/biolinnean/blz147 GengPing, Z., GuoQing, L., WenJun, B., & YuBao, G. (2013). Ecological niche modeling and its applications in biodiversity conservation. Biodiversity Science, 21(1), 90–98. Giovannini, I., Boothby, T. C., Cesari, M., Goldstein, B., Guidetti, R., & Rebecchi, L. (2022). The genus Paramacrobiotus (Tardigrada): integrative taxonomy, biogeography and effects of stress factors on the selected species. Pushpalata Kayastha 33 Production of reactive oxygen species and involvement of bioprotectants during anhydrobiosis in the tardigrade Paramacrobiotus spatialis. Scientific Reports, 12(1), 1938. https://doi.org/10.1038/s41598-022-05734-6 Glavin, D. P., Freissinet, C., Miller, K. E., Eigenbrode, J. L., Brunner, A. E., Buch, A., Sutter, B., Archer, P. D., Atreya, S. K., Brinckerhoff, W. B., Cabane, M., Coll, P., Conrad, P. G., Coscia, D., Dworkin, J. P., Franz, H. B., Grotzinger, J. P., Leshin, L. A., Martin, M. G., … Mahaffy, P. R. (2013). Evidence for perchlorates and the origin of chlorinated hydrocarbons detected by SAM at the Rocknest aeolian deposit in Gale Crater: Evidence for perchlorates at Rocknest. Journal of Geophysical Research: Planets, 118(10), 1955–1973. https://doi.org/10.1002/jgre.20144 Grinnell, J. (1917). Field tests of theories concerning distributional control. The American Naturalist, 51(602), 115–128. https://doi.org/10.1086/279591 Guidetti, R., Cesari, M., Bertolani, R., Altiero, T., & Rebecchi, L. (2019). High diversity in species, reproductive modes and distribution within the Paramacrobiotus richtersi complex (Eutardigrada, Macrobiotidae). Zoological Letters 5, 1–28. https:// doi. org/ 10. 1186/ s40851- 018- 0113-z. Guil, N. (2011). Molecular approach to micrometazoans. Are they here, there and everywhere? In D. Fontaneto (Ed.), Biogeography of Microscopic Organisms (1st ed., pp. 284–306). Cambridge University Press. https://doi.org/10.1017/CBO9780511974878.015 Guil, N., Sánchez‐Moreno, S., & Machordom, A. (2009). Local biodiversity patterns in micrometazoans: Are tardigrades everywhere? Systematics and Biodiversity, 7(3), 259– 268. https://doi.org/10.1017/S1477200009003016 Guisan, A., Thuiller, W., & Zimmermann, N. E. (2017). Habitat suitability and distribution models: With applications in R. Cambridge University Press. Harwood, A., Wilson, H., St John, L., & Centurione, I. (2023). Acute toxicity of sodium chloride-based road salt formulations to juvenile aquatic invertebrates. Available at SSRN 4508985. Hecht, M. H., Kounaves, S. P., Quinn, R. C., West, S. J., Young, S. M. M., Ming, D. W., Catling, D. C., Clark, B. C., Boynton, W. V., Hoffman, J., DeFlores, L. P., Gospodinova, K., Kapit, J., & Smith, P. H. (2009). Detection of perchlorate and the soluble chemistry of martian soil at the phoenix lander site. Science, 325(5936), 64–67. https://doi.org/10.1126/science.1172466 The genus Paramacrobiotus (Tardigrada): integrative taxonomy, biogeography and effects of stress factors on the selected species. Pushpalata Kayastha 34 Hengherr, S., Worland, M. R., Reuner, A., Brummer, F., & Schill, R. (2009). Freeze tolerance, supercooling points and ice formation: Comparative studies on the subzero temperature survival of limno-terrestrial tardigrades. Journal of Experimental Biology, 212(6), 802– 807. Hengherr, S., Worland, M. R., Reuner, A., Brümmer, F., & Schill, R. O. (2009). High‐ temperature tolerance in anhydrobiotic tardigrades is limited by glass transition. Physiological and Biochemical Zoology, 82(6), 749–755. https://doi.org/10.1086/605954 Hirzel, A. H., Hausser, J., Chessel, D., & Perrin, N. (2002). Ecological‐niche factor analysis: How to compute habitat‐suitability maps without absence data? Ecology, 83(7), 2027– 2036. Hygum, T. L., Fobian, D., Kamilari, M., Jørgensen, A., Schiøtt, M., Grosell, M., & Møbjerg, N. (2017). Comparative investigation of copper tolerance and identification of putative tolerance related genes in tardigrades. Frontiers in Physiology, 8, 95. Iniesta, L. F. M., Bouzan, R. S., Rodrigues, P. E. S., Almeida, T. M., Ott, R., & Brescovit, A. D. (2020). Ecological niche modeling predicting the potential invasion of the non- native millipede Oxidus gracilis (C. L. Koch, 1847) (Polydesmida: Paradoxosomatidae) in Brazilian Atlantic Forest. Annales de La Société Entomologique de France (N.S.), 56(5), 387–394. https://doi.org/10.1080/00379271.2020.1834873 Isobe, T., Ogawa, S. P., Sugimoto, R., Ramu, K., Sudaryanto, A., Malarvannan, G., Devanathan, G., Ramaswamy, B. R., Munuswamy, N., Ganesh, D. S., Sivakumar, J., Sethuraman, A., Parthasarathy, V., Subramanian, A., Field, J., & Tanabe, S. (2013). Perchlorate contamination of groundwater from fireworks manufacturing area in South India. Environmental Monitoring and Assessment, 185(7), 5627–5637. https://doi.org/10.1007/s10661-012-2972-7 Kaczmarek, Ł., Michalczyk, Ł., & Diduszko, D. (2005). Some tardigrades from Siberia (Russia, Baikal region) with a description of Macrobiotus garynahi sp. nov. (Eutardigrada: Macrobiotidae: richtersi group). Zootaxa, 1053(1), 35. https://doi.org/10.11646/zootaxa.1053.1.3 Kaczmarek, Ł., Roszkowska, M., Poprawa, I., Janelt, K., Kmita, H., Gawlak, M., Fiałkowska, E., & Mioduchowska, M. (2020). Integrative description of bisexual Paramacrobiotus experimentalis sp. Nov. (Macrobiotidae) from republic of Madagascar (Africa) with The genus Paramacrobiotus (Tardigrada): integrative taxonomy, biogeography and effects of stress factors on the selected species. Pushpalata Kayastha 35 microbiome analysis. Molecular Phylogenetics and Evolution, 145, 106730. https://doi.org/10.1016/j.ympev.2019.106730 Kayastha, P., Mioduchowska, M., Warguła, J., Kaczmarek, Ł. (2023). A review on the genus Paramacrobiotus (Tardigrada) with a new diagnostic key. Diversity. 15(9):977. https://doi.org/10.3390/d15090977. Kayastha, P., Rzymski, P., Gołdyn, B., Nagwani, A. K., Fiałkowska, E., Pajdak-Stós, A., Sobkowiak, R., Robotnikowski, G., & Kaczmarek, Ł. (2023). Tolerance against exposure to solution of magnesium perchlorate in microinvertebrates. Zoological Journal of the Linnean Society, zlad060. https://doi.org/10.1093/zoolinnean/zlad060 Kayastha, P., Stec, D., Sługocki, Ł., Gawlak, M., Mioduchowska, M., & Kaczmarek, Ł. (2023). Integrative taxonomy reveals new, widely distributed tardigrade species of the genus Paramacrobiotus (Eutardigrada: Macrobiotidae). Scientific Reports, 13(1), 2196. https://doi.org/10.1038/s41598-023-28714-w Kayastha, P., Szydło, W., Mioduchowska, M., & Kaczmarek, Ł. (2023). Morphological and genetic variability in cosmopolitan tardigrade species—Paramacrobiotus fairbanksi Schill, Förster, Dandekar & Wolf, 2010 [Preprint]. In Review Scientific Reports. https://doi.org/10.21203/rs.3.rs-2736709/v1 Keilin, D. (1959). The Leeuwenhoek Lecture - The problem of anabiosis or latent life: History and current concept. Proceedings of the Royal Society of London. Series B - Biological Sciences, 150(939), 149–191. https://doi.org/10.1098/rspb.1959.0013 Kounaves, S. P., Chaniotakis, N. A., Chevrier, V. F., Carrier, B. L., Folds, K. E., Hansen, V. M., McElhoney, K. M., O’Neil, G. D., & Weber, A. W. (2014). Identification of the perchlorate parent salts at the Phoenix Mars landing site and possible implications. Icarus, 232, 226–231. https://doi.org/10.1016/j.icarus.2014.01.016 Kounaves, S. P., Hecht, M. H., Kapit, J., Gospodinova, K., DeFlores, L., Quinn, R. C., Boynton, W. V., Clark, B. C., Catling, D. C., Hredzak, P., Ming, D. W., Moore, Q., Shusterman, J., Stroble, S., West, S. J., & Young, S. M. M. (2010). Wet Chemistry experiments on the 2007 Phoenix Mars Scout Lander mission: Data analysis and results. Journal of Geophysical Research, 115, E00E10. https://doi.org/10.1029/2009JE003424 Kristensen, R., & Sterrer, W. (1985). Phylum Tardigrada (water bears). Sterrer, W.(Ed.), Marine Flora and Fauna of Bermuda, 265–268. The genus Paramacrobiotus (Tardigrada): integrative taxonomy, biogeography and effects of stress factors on the selected species. Pushpalata Kayastha 36 Kültz, D. (2020). Defining biological stress and stress responses based on principles of physics. Journal of Experimental Zoology Part A: Ecological and Integrative Physiology, 333(6), 350–358. https://doi.org/10.1002/jez.2340 Leshin, L. A., Mahaffy, P. R., Webster, C. R., Cabane, M., Coll, P., Conrad, P. G., Archer, P. D., Atreya, S. K., Brunner, A. E., Buch, A., Eigenbrode, J. L., Flesch, G. J., Franz, H. B., Freissinet, C., Glavin, D. P., McAdam, A. C., Miller, K. E., Ming, D. W., Morris, R. V., … Moores, J. E. (2013). Volatile, isotope, and organic analysis of Martian Fines with the Mars Curiosity Rover. Science, 341(6153), 1238937. https://doi.org/10.1126/science.1238937 Martin, P. E., Farley, K. A., Douglas Archer, P., Hogancamp, J. V., Siebach, K. L., Grotzinger, J. P., & McLennan, S. M. (2020). Reevaluation of perchlorate in Gale Crater Rocks suggests geologically recent perchlorate addition. Journal of Geophysical Research: Planets, 125(2). https://doi.org/10.1029/2019JE006156 Ming, D. W., Archer, P. D., Glavin, D. P., Eigenbrode, J. L., Franz, H. B., Sutter, B., Brunner, A. E., Stern, J. C., Freissinet, C., McAdam, A. C., Mahaffy, P. R., Cabane, M., Coll, P., Campbell, J. L., Atreya, S. K., Niles, P. B., Bell, J. F., Bish, D. L., Brinckerhoff, W. B., … Moores, J. E. (2014). Volatile and organic compositions of sedimentary rocks in Yellowknife Bay, Gale Crater, Mars. Science, 343(6169), 1245267. https://doi.org/10.1126/science.1245267 Mioduchowska, M., Nitkiewicz, B., Roszkowska, M., Kačarević, U., Madanecki, P., Pinceel, T., Namiotko, T., Gołdyn, B., & Kaczmarek, Ł. (2021). Taxonomic classification of the bacterial endosymbiont Wolbachia based on next-generation sequencing: Is there molecular evidence for its presence in tardigrades? Genome, 64(10), 951–958. https://doi.org/10.1139/gen-2020-0036 Morek, W., Suzuki, A. C., Schill, R. O., Georgiev, D., Yankova, M., Marley, N. J., & Michalczyk, Ł. (2019). Redescription of Milnesium alpigenum Ehrenberg, 1853 (Tardigrada: Apochela) and a description of Milnesium inceptum sp. nov., a tardigrade laboratory model. Zootaxa, 4586(1). https://doi.org/10.11646/zootaxa.4586.1.2 Morón Lugo, S. C., Baumeister, M., Nour, O. M., Wolf, F., Stumpp, M., & Pansch, C. (2020). Warming and temperature variability determine the performance of two invertebrate predators. Scientific Reports, 10(1), 6780. https://doi.org/10.1038/s41598-020-63679- 0 The genus Paramacrobiotus (Tardigrada): integrative taxonomy, biogeography and effects of stress factors on the selected species. Pushpalata Kayastha 37 Murray, J. (1907). XXV.—Arctic Tardigrada, collected by Wm. S. Bruce. Earth and Environmental Science Transactions of The Royal Society of Edinburgh, 45(3), 669– 681. Cambridge Core. https://doi.org/10.1017/S0080456800011789 Murray, J. (1911). Scottish Tardigrada. A review of our present knowledge. Annals of Scottish Natural History, 78, 88–95. Nelson, D. R., Bartels, P. J., & Guil, N. (2018). Tardigrade Ecology. In R. O. Schill (Ed.), Water Bears: The Biology of Tardigrades (Vol. 2, pp. 163–210). Springer International Publishing. https://doi.org/10.1007/978-3-319-95702-9_7 Nelson, D. R., Guidetti, R., & Rebecchi, L. (2010). Chapter 14: Tardigrada. In Ecology and classification of North American freshwater invertebrates (pp. 455–484). https://scholar.google.com/scholar_lookup?&title=Chapter%2014%3A%20Tardigrad a&pages=455- 484&publication_year=2010&author=Nelson%2CDR&author=Guidetti%2CR&autho r=Rebecchi%2CL Nelson, D. R., Guidetti, R., & Rebecchi, L. (2015). Phylum Tardigrada. In Thorp and Covich’s Freshwater Invertebrates (pp. 347–380). Elsevier. https://doi.org/10.1016/B978-0-12- 385026-3.00017-6 Nelson, D. R., Guidetti, R., Rebecchi, L., Kaczmarek, Ł., & McInnes, S. (2020). Phylum Tardigrada. In Thorp and Covich’s Freshwater Invertebrates (pp. 505–522). Elsevier. https://doi.org/10.1016/B978-0-12-804225-0.00015-0 Neves, R. C., Hvidepil, L. K. B., Sørensen-Hygum, T. L., Stuart, R. M., & Møbjerg, N. (2020). Thermotolerance experiments on active and desiccated states of Ramazzottius varieornatus emphasize that tardigrades are sensitive to high temperatures. Scientific Reports, 10(1), 94. https://doi.org/10.1038/s41598-019-56965-z Neves, R. C., Møbjerg, A., Kodama, M., Ramos-Madrigal, J., Gilbert, M. T. P., & Møbjerg, N. (2022). Differential expression profiling of heat stressed tardigrades reveals major shift in the transcriptome. Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology, 267, 111169. Newell, R. C., & Branch, G. M. (1980). The influence of temperature on the maintenance of metabolic energy balance in marine invertebrates. In Advances in Marine Biology (Vol. 17, pp. 329–396). Elsevier. https://doi.org/10.1016/S0065-2881(08)60304-1 Nielsen, C. (2012). Animal evolution: Interrelationships of the living phyla. Oxford University The genus Paramacrobiotus (Tardigrada): integrative taxonomy, biogeography and effects of stress factors on the selected species. Pushpalata Kayastha 38 Press. Ojekunle, O. O., & Sodipe, A. (2020). Antioxidative effect of selenium in cadmium-exposed tardigrade (H. exemplaris). Water, Air, & Soil Pollution, 231, 1–11. Peterson, A. T., Soberón, J., Pearson, R. G., Anderson, R. P., Martínez-Meyer, E., Nakamura, M., & Araújo, M. B. (2011). Ecological niches and geographic distributions (MPB-49). In Ecological Niches and Geographic Distributions (MPB-49). Princeton University Press. Phillips, S. J., Anderson, R. P., Dudík, M., Schapire, R. E., & Blair, M. E. (2017). Opening the black box: An open-source release of Maxent. Ecography, 40(7), 887–893. https://doi.org/10.1111/ecog.03049 Phillips, S. J., Anderson, R. P., & Schapire, R. E. (2006). Maximum entropy modeling of species geographic distributions. Ecological Modelling, 190(3–4), 231–259. Pilato, G., & Binda, M.G. (2001) Biogeography and Limno-terrestrial Tardigrades: Are They Truly Incompatible Binomials? Zoologischer Anzeiger, 240(3-4), 511–516. Ramazzotti, G. (1956). Tre nouve specie di Tardigradi ed altre specie poco comuni. 10, 284– 291. Ramazzotti, G., & Maucci, W. (1983). II Phylum Tardigrada. III. edizione riveduta e aggiornata.(II Phylum Tardigrada. (3rd ed.) 41, pp. 1-1012. Rebecchi, L., Boschini, D., Cesari, M., Lencioni, V., Bertolani, R., & Guidetti, R. (2009). Stress response of a boreo-alpine species of tardigrade, Borealibius zetlandicus (Eutardigrada, Hypsibiidae). Journal of Limnology, 68(1), 64. https://doi.org/10.4081/jlimnol.2009.64 Reuner, A., Hengherr, S., Brümmer, F., & Schill, R. O. (2010). Comparative studies on storage cells in tardigrades during starvation and anhydrobiosis. Current Zoology, 56(2), 259– 263. https://doi.org/10.1093/czoolo/56.2.259 Rizzo, A. M., Negroni, M., Altiero, T., Montorfano, G., Corsetto, P., Berselli, P., Berra, B., Guidetti, R., & Rebecchi, L. (2010). Antioxidant defences in hydrated and desiccated states of the tardigrade Paramacrobiotus richtersi. Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology, 156(2), 115–121. https://doi.org/10.1016/j.cbpb.2010.02.009 Roszkowska, M., Gołdyn, B., Wojciechowska, D., Księżkiewicz, Z., Fiałkowska, E., Pluskota, M., Kmita, H., & Kaczmarek, Ł. (2023). How long can tardigrades survive in the The genus Paramacrobiotus (Tardigrada): integrative taxonomy, biogeography and effects of stress factors on the selected species. Pushpalata Kayastha 39 anhydrobiotic state? A search for tardigrade anhydrobiosis patterns. PLOS ONE, 18(1), e0270386. https://doi.org/10.1371/journal.pone.0270386 Schill, R. O., Förster, F., Dandekar, T., & Wolf, M. (2010). Using compensatory base change analysis of internal transcribed spacer 2 secondary structures to identify three new species in Paramacrobiotus (Tardigrada). Organisms Diversity & Evolution, 10(4), 287–296. https://doi.org/10.1007/s13127-010-0025-z Sih, A., Bell, A. M., & Kerby, J. L. (2004). Two stressors are far deadlier than one. Trends in Ecology & Evolution, 19(6), 274–276. https://doi.org/10.1016/j.tree.2004.02.010 Sillero, N. (2011). What does ecological modelling model? A proposed classification of ecological niche models based on their underlying methods. Ecological Modelling, 222(8), 1343–1346. https://doi.org/10.1016/j.ecolmodel.2011.01.018 Sillero, N., Arenas-Castro, S., Enriquez‐Urzelai, U., Vale, C. G., Sousa-Guedes, D., Martínez- Freiría, F., Real, R., & Barbosa, A. M. (2021). Want to model a species niche? A step- by-step guideline on correlative ecological niche modelling. Ecological Modelling, 456, 109671. https://doi.org/10.1016/j.ecolmodel.2021.109671 Stec, D., Vecchi, M., Dudziak, M., Bartels, P. J., Calhim, S., & Michalczyk, Ł. (2021). Integrative taxonomy resolves species identities within the Macrobiotus pallarii complex (Eutardigrada: Macrobiotidae). Zoological Letters, 7(1), 9. https://doi.org/10.1186/s40851-021-00176-w Sugiura, K., Matsumoto, M., & Kunieda, T. (2022). Description of a model tardigrade Paramacrobiotus metropolitanus sp. Nov. (Eutardigrada) from Japan with a summary of its life history, reproduction and genomics. Zootaxa, 5134(1), 92–112. https://doi.org/10.11646/zootaxa.5134.1.4 Sutter, B., McAdam, A. C., Mahaffy, P. R., Ming, D. W., Edgett, K. S., Rampe, E. B., Eigenbrode, J. L., Franz, H. B., Freissinet, C., Grotzinger, J. P., Steele, A., House, C. H., Archer, P. D., Malespin, C. A., Navarro-González, R., Stern, J. C., Bell, J. F., Calef, F. J., Gellert, R., … Yen, A. S. (2017). Evolved gas analyses of sedimentary rocks and eolian sediment in Gale Crater, Mars: Results of the Curiosity rover’s sample analysis at Mars instrument from Yellowknife Bay to the Namib Dune: SAM-Evolved Gas Analysis at Gale Crater. Journal of Geophysical Research: Planets, 122(12), 2574– 2609. https://doi.org/10.1002/2016JE005225 Tumanov, D. V. (2005). Notes on the Tardigrada of Thailand, with a description of The genus Paramacrobiotus (Tardigrada): integrative taxonomy, biogeography and effects of stress factors on the selected species. Pushpalata Kayastha 40 Macrobiotus alekseevi sp. Nov. (Eutardigrada, Macrobiotidae). Zootaxa, 999(1), 1. https://doi.org/10.11646/zootaxa.999.1.1 Vecchi, M., Newton, I. L. G., Cesari, M., Rebecchi, L., & Guidetti, R. (2018). The microbial community of tardigrades: Environmental influence and species specificity of microbiome structure and composition. Microbial Ecology, 76(2), 467–481. https://doi.org/10.1007/s00248-017-1134-4 Vega, M., Nerenberg, R., & Vargas, I. T. (2018). Perchlorate contamination in Chile: Legacy, challenges, and potential solutions. Environmental Research, 164, 316–326. https://doi.org/10.1016/j.envres.2018.02.034 Wieczorkiewicz, F., Sojka, J., & Poprawa, I. (2023). Effect of paracetamol on the storage cells of Hypsibius exemplaris – ultrastructural analysis. https://doi.org/10.1093/zoolinnean/zlad051 Young, T., Gale, S. L., Ragg, N. L., Sander, S. G., Burritt, D. J., Benedict, B., Le, D. V., Villas- Bôas, S. G., & Alfaro, A. C. (2023). Metabolic regulation of copper toxicity during marine mussel embryogenesis. Metabolites, 13(7), 838. The genus Paramacrobiotus (Tardigrada): integrative taxonomy, biogeography and effects of stress factors on the selected species. Pushpalata Kayastha 41 Paper I Kayastha, P., Stec, D., Sługocki, Ł. et al. Integrative taxonomy reveals new, widely distributed tardigrade species of the genus Paramacrobiotus (Eutardigrada: Macrobiotidae). Sci Rep 13, 2196 (2023). https://doi.org/10.1038/s41598-023-28714-w Link: https://www.nature.com/articles/s41598- 023-28714-w https://www.nature.com/articles/s41598-023-28714-w https://www.nature.com/articles/s41598-023-28714-w The genus Paramacrobiotus (Tardigrada): integrative taxonomy, biogeography and effects of stress factors on the selected species. Pushpalata Kayastha 42 Paper II Kayastha, P., Szydło, W., Mioduchowska, M. and Kaczmarek, Ł. Morphological and genetic variability in cosmopolitan tardigrade species - Paramacrobiotus fairbanksi Schill, Förster, Dandekar & Wolf, 2010. (In review Scientific Reports) https://doi.org/10.21203/rs.3.rs- 2736709/v2 Link: https://www.researchsquare.com/article/rs- 2736709/v2 https://doi.org/10.21203/rs.3.rs-2736709/v2 https://doi.org/10.21203/rs.3.rs-2736709/v2 https://www.researchsquare.com/article/rs-2736709/v2 https://www.researchsquare.com/article/rs-2736709/v2 The genus Paramacrobiotus (Tardigrada): integrative taxonomy, biogeography and effects of stress factors on the selected species. Pushpalata Kayastha 43 Paper III Kayastha, P., Rzymski, P., Gołdyn, B., Nagwani, A.K., Fiałkowska, E., Pajdak-Stós, A., Sobkowiak, R., Robotnikowski, G. and Kaczmarek, Ł. Tolerance against exposure to solution of magnesium perchlorate in microinvertebrates, Zool J Linn Soc, zlad060 (2023) https://doi.org/10.1093/zoolinnean/zlad060 Link: https://academic.oup.com/zoolinnean/advance- article/doi/10.1093/zoolinnean/zlad060/7229189?u tm_source=authortollfreelink&utm_campaign=zoo linnean&utm_medium=email&guestAccessKey=a 6bc45c0-a647-4866-916b-e4e521a4a43f https://doi.org/10.1093/zoolinnean/zlad060 https://academic.oup.com/zoolinnean/advance-article/doi/10.1093/zoolinnean/zlad060/7229189?utm_source=authortollfreelink&utm_campaign=zoolinnean&utm_medium=email&guestAccessKey=a6bc45c0-a647-4866-916b-e4e521a4a43f https://academic.oup.com/zoolinnean/advance-article/doi/10.1093/zoolinnean/zlad060/7229189?utm_source=authortollfreelink&utm_campaign=zoolinnean&utm_medium=email&guestAccessKey=a6bc45c0-a647-4866-916b-e4e521a4a43f https://academic.oup.com/zoolinnean/advance-article/doi/10.1093/zoolinnean/zlad060/7229189?utm_source=authortollfreelink&utm_campaign=zoolinnean&utm_medium=email&guestAccessKey=a6bc45c0-a647-4866-916b-e4e521a4a43f https://academic.oup.com/zoolinnean/advance-article/doi/10.1093/zoolinnean/zlad060/7229189?utm_source=authortollfreelink&utm_campaign=zoolinnean&utm_medium=email&guestAccessKey=a6bc45c0-a647-4866-916b-e4e521a4a43f https://academic.oup.com/zoolinnean/advance-article/doi/10.1093/zoolinnean/zlad060/7229189?utm_source=authortollfreelink&utm_campaign=zoolinnean&utm_medium=email&guestAccessKey=a6bc45c0-a647-4866-916b-e4e521a4a43f The genus Paramacrobiotus (Tardigrada): integrative taxonomy, biogeography and effects of stress factors on the selected species. Pushpalata Kayastha 44 Paper IV Kayastha, P., Wieczorkiewicz, F., Pujol, M., Robinson, A., Michalak, M., Kaczmarek, Ł. and Poprawa, I. Elevated external temperature affect cell ultrastructure and heat shock protein (HSP) in Paramacrobiotus experimentalis Kaczmarek, Mioduchowska, Poprawa & Roszkowska, 2020’ https://doi.org/10.21203/rs.3.rs-3202172/v1 Link: https://www.researchsquare.com/article/rs- 3202172/v1 https://doi.org/10.21203/rs.3.rs-3202172/v1 https://www.researchsquare.com/article/rs-3202172/v1 https://www.researchsquare.com/article/rs-3202172/v1 The genus Paramacrobiotus (Tardigrada): integrative taxonomy, biogeography and effects of stress factors on the selected species. Pushpalata Kayastha 45 Paper V Kayastha, P., Mioduchowska, M., Warguła, J., Kaczmarek, Ł. A Review on the Genus Paramacrobiotus (Tardigrada) with a New Diagnostic Key. Diversity 15(9):977 (2023). https://doi.org/10.3390/d15090977 Link: https://www.mdpi.com/1424-2818/15/9/977 https://doi.org/10.3390/d15090977 The genus Paramacrobiotus (Tardigrada): integrative taxonomy, biogeography and effects of stress factors on the selected species. Pushpalata Kayastha 46 Authorship Statements of the PhD candidate The genus Paramacrobiotus (Tardigrada): integrative taxonomy, biogeography and effects of stress factors on the selected species. Pushpalata Kayastha 47 AUTHOR STATEMENT for the research article ‘Integrative taxonomy reveals new, widely distributed tardigrade species of the genus Paramacrobiotus (Eutardigrada: Macrobiotidae). Sci Rep 13, 2196 (2023).’ I declare that the research article ‘Kayastha et al. Integrative taxonomy reveals new, widely distributed tardigrade species of the genus Paramacrobiotus (Eutardigrada: Macrobiotidae). Sci Rep 13, 2196 (2023). https://doi.org/10.1038/s41598-023-28714-w is part of my PhD dissertation. I, Pushpalata Kayastha along with my supervisor Łukasz Kaczmarek conceptualized the idea, curated the data, performed formal analysis, investigated the research study, prepared tables and figures, wrote the original drafts and performed all editorial work. Date: 03.07.2023 Name: Pushpalata Kayastha (P.K.) Signature: https://doi.org/10.1038/s41598-023-28714-w https://doi.org/10.1038/s41598-023-28714-w https://doi.org/10.1038/s41598-023-28714-w https://doi.org/10.1038/s41598-023-28714-w https://doi.org/10.1038/s41598-023-28714-w https://doi.org/10.1038/s41598-023-28714-w https://doi.org/10.1038/s41598-023-28714-w https://doi.org/10.1038/s41598-023-28714-w The genus Paramacrobiotus (Tardigrada): integrative taxonomy, biogeography and effects of stress factors on the selected species. Pushpalata Kayastha 48 AUTHOR STATEMENT for the research article ‘Morphological and genetic variability in cosmopolitan tardigrade species - Paramacrobiotus fairbanksi Schill, Förster, Dandekar & Wolf, 2010’ I declare that the research article ‘Kayastha et al. Morphological and genetic variability in cosmopolitan tardigrade species - Paramacrobiotus fairbanksi Schill, Förster, Dandekar & Wolf, 2010’ https://doi.org/10.21203/rs.3.rs-2736709/v2 is part of my PhD dissertation. I, Pushpalata Kayastha along with my supervisor Łukasz Kaczmarek conceptualized the idea, curated the data, performed formal analysis, investigated the research study, prepared tables and figures, wrote the original drafts and performed all editorial work. Date: 30.07.2023 Name: Pushpalata Kayastha (P.K.) Signature: https://doi.org/10.21203/rs.3.rs-2736709/v1 The genus Paramacrobiotus (Tardigrada): integrative taxonomy, biogeography and effects of stress factors on the selected species. Pushpalata Kayastha 49 AUTHOR STATEMENT for the research article ‘Tolerance against exposure to solution of magnesium perchlorate in microinvertebrates’ I declare that the research article ‘Kayastha et al. Tolerance against exposure to solution of magnesium perchlorate in microinvertebrates. Zoological Journal of the Linnean Society, zlad060. https://doi.org/10.1093/zoolinnean/zlad060’ is part of my PhD dissertation. I, Pushpalata Kayastha along with my supervisor Łukasz Kaczmarek and Dr. Piotr Rzymski conceptualized the idea, performed all the experiments for tardigrade and nematodes, performed statistical analysis, prepared all the figures, wrote the original drafts and performed all editorial work. Date: 30.07.2023 Name: Pushpalata Kayastha (P.K.) Signature: https://doi.org/10.21203/rs.3.rs-2736709/v1 The genus Paramacrobiotus (Tardigrada): integrative taxonomy, biogeography and effects of stress factors on the selected species. Pushpalata Kayastha 50 AUTHOR STATEMENT for the research article ‘Elevated external temperature affects cell ultrastructure and heat shock protein (HSP) in Paramacrobiotus experimentalis Kaczmarek, Mioduchowska, Poprawa & Roszkowska, 2020.’ I declare that the research article ‘Kayastha et al. Elevated external temperature affect cell ultrastructure and heat shock protein (HSP) in Paramacrobiotus experimentalis Kaczmarek, Mioduchowska, Poprawa & Roszkowska, 2020’ https://doi.org/10.21203/rs.3.rs-3202172/v1 is part of my PhD dissertation. I, Pushpalata Kayastha along with my supervisor Łukasz Kaczmarek conceptualized the idea, performed all the experiments for heat stress, performed statistical analysis, prepared all the figures, wrote the original drafts and performed all editorial work. Date: 30.07.2023 Name: Pushpalata Kayastha (P.K.) Signature: https://doi.org/10.21203/rs.3.rs-3202172/v1 The genus Paramacrobiotus (Tardigrada): integrative taxonomy, biogeography and effects of stress factors on the selected species. Pushpalata Kayastha 51 AUTHOR STATEMENT for the review article ‘A review on genus Paramacrobiotus’ I declare that I am aware of that the work in the research article ‘Kayastha et al. A review on genus Paramacrobiotus. Preprints.org 2023, 2023071250. https://doi.org/10.20944/preprints202307.1250.v1’ of which I am co-author is a part to PhD dissertation by Pushpalata Kayastha. Conceptualization, P.K. and Ł.K.; methodology, P.K. and M.M.; formal analysis, P.K. and M.M.; investigation, P.K.; data curation, P.K.; writing—original draft preparation, P.K.; writing—review and editing, P.K., M.M. and Ł.K; visualization, P.K. and M.M.; supervision, Ł.K. All authors have read and agreed to the published version of the manuscript. Date: 30.07.2023 Name: Pushpalata Kayastha (P.K.) Signature: https://doi.org/10.20944/preprints202307.1250.v1 The genus Paramacrobiotus (Tardigrada): integrative taxonomy, biogeography and effects of stress factors on the selected species. Pushpalata Kayastha 52 Co-authors Statements The genus Paramacrobiotus (Tardigrada): integrative taxonomy, biogeography and effects of stress factors on the selected species. Pushpalata Kayastha 53 CO-AUTHOR STATEMENT for the research article 'Integrative taxonomy reveals new, widely distributed tardigrade species of the genus Paramacrobiotus (Eutardigrada: Macrobiotidae)' I declare that I am aware that the work in the research article 'Kayastha et al. 'Integrative taxonomy reveals new, widely distributed tardigrade species of the genus Paramacrobiotus (Eutardigrada: Macrobiotidae). Sci Rep 13, 2196 (2023). https://doi.org/10.1038/s41598-023-28714-w of which I am co-author is a part to PhD dissertation by Pushpalata Kayastha. Conceptualization, P.K. and Ł.K.; data curation, P.K.; sample collection, Ł.S.; formal analysis, P.K., D.S., M.M. and Ł.K.; investigation, P.K., D.S., Ł.S., M.M., M.G. and Ł.K.; methodology, P.K., D.S., M.M. and Ł.K.; supervision, Ł.K.; validation, P.K., D.S., M.M. and Ł.K.; visualization, P.K. and M.G.; writing—original draft, P.K., D.S., M.M. and Ł.K.; writing—review and editing, All authors reviewed the manuscript. All authors have read and agreed to the published version of the manuscript. Date: 01.06.2023 Name: Daniel Stec (D.S.) Address: Institute of Systematics and Evolution of Animals, Polish Academy of Sciences, Slawkowska 17, 31-016, Krak6w, Poland Signature• https://doi.org/10.1038/s41598-023-28714-w The genus Paramacrobiotus (Tardigrada): integrative taxonomy, biogeography and effects of stress factors on the selected species. Pushpalata Kayastha 54 CO-AUTHOR STATEMENT for the research article 'Integrative taxonomy reveals new, widely distributed tardigrade species of the genus Paramacrobiotus (Eutardigrada: Macrobiotidae)' I declare that I am aware that the work in the research article 'Kayastha et al. 'Integrative taxonomy reveals new, widely distributed tardigrade species of the genus Paramacrobiotus (Eutardigrada: Macrobiotidae). Sci Rep 13, 2196 (2023). https://doi.org/10.1038/s41598-023-28714-w of which I am co-author is a part to PhD dissertation by Pushpalata Kayastha. Conceptualization, P.K. and Ł.K.; data curation, P.K.; sample collection, Ł.S.; formal analysis, P.K., D.S., M.M. and Ł.K.; investigation, P.K., D.S., Ł.S., M.M., M.G. and Ł.K.; methodology, P.K., D.S., M.M. and Ł.K.; supervision, Ł.K.; validation, P.K., D.S., M.M. and Ł.K.; visualization, P.K. and M.G.; writing—original draft, P.K., D.S., M.M. and Ł.K.; writing—review and editing, All authors reviewed the manuscript. All authors have read and agreed to the published version of the manuscript. Date: 01.06.2023 Name: Łukasz Sługocki (Ł.S.) Address: Department of Hydrobiology, Institute of Biology, University of Szczecin, Szczecin, Poland Signature:……………………. https://doi.org/10.1038/s41598-023-28714-w The genus Paramacrobiotus (Tardigrada): integrative taxonomy, biogeography and effects of stress factors on the selected species. Pushpalata Kayastha 55 CO-AUTHOR STATEMENT for the research article 'Integrative taxonomy reveals new, widely distributed tardigrade species of the genus Paramacrobiotus (Eutardigrada: Macrobiotidae)' I declare that I am aware that the work in the research article 'Kayastha et al. 'Integrative taxonomy reveals new, widely distributed tardigrade species of the genus Paramacrobiotus (Eutardigrada: Macrobiotidae). Sci Rep 13, 2196 (2023). https://doi.org/10.1038/s41598-023-28714-w of which I am co-author is a part to PhD dissertation by Pushpalata Kayastha. Conceptualization, P.K. and Ł.K.; data curation, P.K.; sample collection, Ł.S.; formal analysis, P.K., D.S., M.M. and Ł.K.; investigation, P.K., D.S., Ł.S., M.M., M.G. and Ł.K.; methodology, P.K., D.S., M.M. and Ł.K.; supervision, Ł.K.; validation, P.K., D.S., M.M. and Ł.K.; visualization, P.K. and M.G.; writing—original draft, P.K., D.S., M.M. and Ł.K.; writing—review and editing, All authors reviewed the manuscript. All authors have read and agreed to the published version of the manuscript. Date: 01.06.2023 Name: Magdalena Gawlak (M.G.) Address: The Institute of Plant ProtectionNational Research Institute, Wegorka 20, 60-318, Poznan, Poland Signature: https://doi.org/10.1038/s41598-023-28714-w The genus Paramacrobiotus (Tardigrada): integrative taxonomy, biogeography and effects of stress factors on the selected species. Pushpalata Kayastha 56 CO-AUTHOR STATEMENT for the research article 'Integrative taxonomy reveals new, widely distributed tardigrade species of the genus Paramacrobiotus (Eutardigrada: Macrobiotidae)' I declare that I am aware that the work in the research article 'Kayastha et al. 'Integrative taxonomy reveals new, widely distributed tardigrade species of the genus Paramacrobiotus (Eutardigrada: Macrobiotidae). Sci Rep 13, 2196 (2023). https://doi.org/10.1038/s41598-023-28714-w of which I am co-author is a part to PhD dissertation by Pushpalata Kayastha. Conceptualization, P.K. and Ł.K.; data curation, P.K.; sample collection, Ł.S.; formal analysis, P.K., D.S., M.M. and Ł.K.; investigation, P.K., D.S., Ł.S., M.M., M.G. and Ł.K.; methodology, P.K., D.S., M.M. and Ł.K.; supervision, Ł.K.; validation, P.K., D.S., M.M. and Ł.K.; visualization, P.K. and M.G.; writing—original draft, P.K., D.S., M.M. and Ł.K.; writing—review and editing, All authors reviewed the manuscript. All authors have read and agreed to the published version of the manuscript. Date: 18.07.2023 Name: Monika Mioduchowska (M.M.) Address: Department of Evolutionary Genetics and Biosystematics, Faculty of Biology, University of Gdańsk, Gdańsk, Wita Stwosza 59, 80-308, Gdańsk, Poland Signature:……………………. https://doi.org/10.1038/s41598-023-28714-w The genus Paramacrobiotus (Tardigrada): integrative taxonomy, biogeography and effects of stress factors on the selected species. Pushpalata Kayastha 57 CO-AUTHOR STATEMENT for the research article 'Integrative taxonomy reveals new, widely distributed tardigrade species of the genus Paramacrobiotus (Eutardigrada: Macrobiotidae)' I declare that I am aware that the work in the research article 'Kayastha et al. 'Integrative taxonomy reveals new, widely distributed tardigrade species of the genus Paramacrobiotus (Eutardigrada: Macrobiotidae). Sci Rep 13, 2196 (2023). https://doi.org/10.1038/s41598-023-28714-w of which I am co-author is a part to PhD dissertation by Pushpalata Kayastha. Conceptualization, P.K. and Ł.K.; data curation, P.K.; sample collection, Ł.S.; formal analysis, P.K., D.S., M.M. and Ł.K.; investigation, P.K., D.S., Ł.S., M.M., M.G. and Ł.K.; methodology, P.K., D.S., M.M. and Ł.K.; supervision, Ł.K.; validation, P.K., D.S., M.M. and Ł.K.; visualization, P.K. and M.G.; writing—original draft, P.K., D.S., M.M. and Ł.K.; writing—review and editing, All authors reviewed the manuscript. All authors have read and agreed to the published version of the manuscript. Date: 31.07.2023 Name: Łukasz Kaczmarek (Ł.K.) Address: Department of Animal Taxonomy and Ecology, Faculty of Biology, Adam Mickiewicz University in Poznań, Poznań, Uniwersytetu Poznańskiego 6, 6 1-614, Poznań, Poland Signature:……………………. https://doi.org/10.1038/s41598-023-28714-w The genus Paramacrobiotus (Tardigrada): integrative taxonomy, biogeography and effects of stress factors on the selected species. Pushpalata Kayastha 58 CO-AUTHOR STATEMENT for the research article 'Morphological and genetic variability in cosmopolitan tardigrade species - Paramacrobiotusfairbanksi Schill, Förster, Dandekar & Wolf, 2010' I declare that I am aware of that the work in the research article 'Kayastha et al. 'Morphological and genetic variability in cosmopolitan tardigrade species Paramacrobiotus fairbanksi Schill, Förster, Dandekar & Wolf, 2010 https://doi.org/10.21203/rs.3.rs- 2736709/v2 of which I am co-author is a part to PhD dissertation by Pushpalata Kayastha. Conceptualization, P.K. and L.K.; data curation, P.K.; formal analysis, PK.; investigation, P.K., M.M., and L.K.; methodology, P.K., M.M. and L.K.; supervision, L.K.; validation, P.K., W.S. M.M. and L.K.; visualization, P.K.; writing—original draft, P.K., M.M. and L.K.; writing— review and editing, P.K., W.S., M.M. and L.K. Date: 18.07.2023 Name: Wiktoria Szydlo (W.S.) Address: Center for Advanced Technology, Adam Mickiewicz University in Poznafi, Uniwersytetu Poznafiskiego 10, 61-614 Poznafi, Poland Signature:. https://doi.org/10.21203/rs.3.rs-2736709/v2 https://doi.org/10.21203/rs.3.rs-2736709/v2 The genus Paramacrobiotus (Tardigrada): integrative taxonomy, biogeography and effects of stress factors on the selected species. Pushpalata Kayastha 59 CO-AUTHOR STATEMENT for the research article ‘Morphological and genetic variability in cosmopolitan tardigrade species - Paramacrobiotus fairbanksi Schill, Förster, Dandekar & Wolf, 2010’ I declare that I am aware of that the work in the research article ‘Kayastha et al. ‘Morphological and genetic variability in cosmopolitan tardigrade species - Paramacrobiotus fairbanksi Schill, Förster, Dandekar & Wolf, 2010 https://doi.org/10.21203/rs.3.rs-2736709/v2 of which I am co-author is a part to PhD dissertation by Pushpalata Kayastha. Conceptualization, P.K. and Ł.K.; data curation, P.K.; formal analysis, P.K.; investigation, P.K., M.M., and Ł.K.; methodology, P.K., M.M. and Ł.K.; supervision, Ł.K.; validation, P.K., W.S., M.M. and Ł.K.; visualization, P.K.; writing—o