Taxonomic reanalysis of the genus Richtersius (Tardigrada; Eutardigrada), with description of two new species from Italy and Sweden
Abstract
The tardigrade genus Richtersius Pilato & Binda, 1989 has been considered monotypic for more than 30 years since its establishment and is frequently used in experimental studies on physiological adaptations to stress. Only recently, integrative taxonomy has allowed us to disentangle and describe different but similar species. In this study, we provide a taxonomic reanalysis of the genus Richtersius with an integrative description of two new species based on light and scanning electron microscopy as well as DNA sequencing of four markers (18S rDNA, 28S rDNA, ITS-2, and COI). Richtersius nicolai sp. nov. and Richtersius ingemari sp. nov. are distinguished from congeneric species based on a combination of pore density in newborn’s dorsal cuticle, egg diameters, placoid sizes and reproductive modes. This reanalysis of the genus Richtersius will facilitate the future descriptions of new species and provides a solid taxonomic background for the identification of the species used in experimental research.
References
Bertolani R., Guidetti R., Marchioro T., Altiero T., Rebecchi L. & Cesari M. 2014. Phylogeny of Eutardigrada: New molecular data and their morphological support lead to the identification of new evolutionary lineages. Molecular Phylogenetics and Evolution 76 (1): 110–126. https://doi.org/10.1016/j.ympev.2014.03.006
Bortolus A. 2008. Error cascades in the biological sciences: The unwanted consequences of using bad taxonomy in ecology. AMBIO: A Journal of the Human Environment 37 (2): 114–118. https://doi.org/10.1579/0044-7447(2008)37[114:ECITBS]2.0.CO;2
Camarda D., Massa E., Guidetti R. & Lisi O. 2023. A new, simplified, drying protocol to prepare tardigrades for scanning electron microscopy. Microscopy Research and Technique 87 (4): 716–726. https://doi.org/10.1002/jemt.24460
Caputi L., Andreakis N., Mastrototaro F., Cirino P., Vassillo M. & Sordino P. 2007. Cryptic speciation in a model invertebrate chordate. Proceedings of the National Academy of Sciences 104 (22): 9364–9369. https://doi.org/10.1073/pnas.0610158104
Casquet J.T., Thebaud C. & Gillespie R.G. 2012. Chelex without boiling, a rapid and easy technique to obtain stable amplifiable DNA from small amounts of ethanol-stored spiders. Molecular Ecology Resources 12 (1): 136–141. https://doi.org/10.1111/j.1755-0998.2011.03073.x
Czernekova M. & Jönsson K.I. 2016. Experimentally induced repeated anhydrobiosis in the eutardigrade Richtersius coronifer. PLoS ONE 11 (11): e0164062. https://doi.org/10.1371/journal.pone.0164062
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
Czerneková M., Janelt K., Student S., Jönsson K.I. & Poprawa I. 2018. A comparative ultrastructure study of storage cells in the eutardigrade Richtersius coronifer in the hydrated state and after desiccation and heating stress. PLoS ONE 13 (8): e0201430. https://doi.org/10.1371/journal.pone.0201430
Dunn C.W., Hejnol A., Matus D.Q., Pang K., Browne W.E., Smith S.A., Seaver E., Rouse G.W., Obst M., Edgecombe G.D., Sørensen M.V., Haddock S.H.D., Schmidt-Rhaesa A., Okusu A., Kristensen R.M., Wheeler W.C., Martindale M.Q. & Giribet G. 2008. Broad phylogenomic sampling improves resolution of the animal tree of life. Nature 452 (7188): 745–749. https://doi.org/10.1038/nature06614
Edgar R.C. 2004. MUSCLE: Multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Research 32 (5): 1792–1797. https://doi.org/10.1093/nar/gkh340
Faurby S., Jönsson K.I., Rebecchi L. & Funch P. 2008. Variation in anhydrobiotic survival of two eutardigrade morphospecies: A story of cryptic species and their dispersal. Journal of Zoology 275 (2): 139–145. https://doi.org/10.1111/j.1469-7998.2008.00420.x
Guidetti R., Gandolfi A., Rossi V. & Bertolani R. 2005. Phylogenetic analysis of Macrobiotidae (Eutardigrada, Parachela): A combined morphological and molecular approach. Zoologica Scripta 34 (3): 235–244. https://doi.org/10.1111/j.1463-6409.2005.00193.x
Guidetti R., Rebecchi L., Bertolani R., Jönsson K.I., Kristensen R.M. & Cesari M. 2016. Morphological and molecular analyses on Richtersius (Eutardigrada) diversity reveal its new systematic position and lead to the establishment of a new genus and a new family within Macrobiotoidea. Zoological Journal of the Linnean Society 178 (4): 834–845. https://doi.org/10.1111/zoj.12428
Guidetti R., Vecchi M., Ferrari A., Newton I.L., Cesari M. & Rebecchi L. 2019. Further insights in the Tardigrada microbiome: Phylogenetic position and prevalence of infection of four new Alphaproteobacteria putative endosymbionts. Zoological Journal of the Linnean Society 188 (3): 925–937. https://doi.org/10.1093/zoolinnean/zlz128
Guidetti R., Schill R.O., Giovannini I., Massa E., Goldoni S.E., Ebel C., Förschler M.I., Rebecchi L. & Cesari M. 2021. When DNA sequence data and morphological results fit together: Phylogenetic position of Crenubiotus within Macrobiotoidea (Eutardigrada) with description of Crenubiotus ruhesteini sp. nov. Journal of Zoological Systematics and Evolutionary Research 59 (3): 576–587. https://doi.org/10.1111/jzs.12449
Hagelbäck P. & Jönsson K.I. 2023. An experimental study on tolerance to hypoxia in tardigrades. Frontiers in Physiology 14: 1249773. https://doi.org/10.3389/fphys.2023.1249773
Halberg K.A., Larsen K.W., Jørgensen A., Ramløv H. & Møbjerg N. 2012. Inorganic ion composition in Tardigrada: Cryptobionts contain large fraction of unidentified organic solutes. Journal of Experimental Biology 216 (7): 1235–1243. https://doi.org/10.1242/jeb.075531
Halberg K.A., Jørgensen A. & Møbjerg N. 2013. Desiccation tolerance in the tardigrade Richtersius coronifer relies on muscle mediated structural reorganization. PLoS ONE 8 (12): e85091. https://doi.org/10.1371/journal.pone.0085091
Hindborg Mortensen L., Lynge Nilsson L., Nyby C. & Vilstrup A. 2010. Quantifying 8-hydroxy-2′-deoxyguanosine with Enzyme-linked Immunosorbent Assay (ELISA) as a Measure of DNA Damage in the Eutardigrade Richtersius coronifer. Report, Roskilde University, Roskilde. Available from https://rucforsk.ruc.dk/ws/portalfiles/portal/57743684/samlet.pdf [accessed 14 Feb. 2025].
Ivarsson H. & Jönsson K.I. 2004. Aggregation effects on anhydrobiotic survival in the tardigrade Richtersius coronifer. Journal of Experimental Zoology Part A: Comparative Experimental Biology 301A (2): 195–199. https://doi.org/10.1002/jez.a.20018
Jönsson K.I. 2007. Long-term experimental manipulation of moisture conditions and its impact on moss-living tardigrades. Journal of Limnology 66 (s1): 119–125. https://doi.org/10.4081/jlimnol.2007.s1.119
Jönsson K.I. & Guidetti R. 2001. Effects of methyl bromide fumigation on anhydrobiotic micrometa-zoans. Ecotoxicology and Environmental Safety 50 (1): 72–75. https://doi.org/10.1006/eesa.2001.2090
Jönsson K.I. & Rebecchi L. 2002. Experimentally induced anhydrobiosis in the tardigrade Richtersius coronifer: Phenotypic factors affecting survival. Journal of Experimental Zoology 293 (6): 578–584. https://doi.org/10.1002/jez.10186
Jönsson K.I. & Schill R.O. 2007. Induction of Hsp70 by desiccation, ionising radiation and heat-shock in the eutardigrade Richtersius coronifer. Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology 146 (4): 456–460. https://doi.org/10.1016/j.cbpb.2006.10.111
Jönsson K.I., Borsari S. & Rebecchi L. 2001. Anhydrobiotic survival in populations of the tardigrades Richtersius coronifer and Ramazzottius oberhaeuseri from Italy and Sweden. Zoologischer Anzeiger 240 (3–4): 419–423. https://doi.org/10.1078/0044-5231-00050
Jönsson K.I., Harms-Ringdahl M. & Torudd J. 2005. Radiation tolerance in the eutardigrade Richtersius coronifer. International Journal of Radiation Biology 81 (9): 649–656. https://doi.org/10.1080/09553000500368453
Jørgensen A. & Kristensen R.M. 2004. Molecular phylogeny of Tardigrada—investigation of the monophyly of Heterotardigrada. Molecular Phylogenetics and Evolution 32 (2): 666–670. https://doi.org/10.1016/j.ympev.2004.04.017
Kamilari M., Jørgensen A., Schiøtt M. & Møbjerg N. 2019. Comparative transcriptomics suggest unique molecular adaptations within tardigrade lineages. BMC Genomics 20 (1): 607. https://doi.org/10.1186/s12864-019-5912-x
Katoh K. & Toh H. 2008. Recent developments in the MAFFT multiple sequence alignment program. Briefings in Bioinformatics 9 (4): 286–298. https://doi.org/10.1093/bib/bbn013
Katoh K., Misawa K., Kuma K.I. & Miyata T. 2002. MAFFT: A novel method for rapid multiple sequence alignment based on fast Fourier transform. Nucleic Acids Research 30 (14): 3059–3066. https://doi.org/10.1093/nar/gkf436
Kayastha P., Berdi D., Mioduchowska M., Gawlak M., Łukasiewicz A., Gołdyn B., Jȩdrzejewski 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
Kiosya Y. & Stec D. 2022. New species of the genus Richtersius Pilato & Binda, 1989 (Tardigrada: Eutardigrada: Richtersiusidae) from Uzbekistan. Folia Biologica 70 (4): 141–150. https://doi.org/10.3409/fb_70-4.18
Kiosya Y., Pogwizd J., Matsko Y., Vecchi M. & Stec D. 2021. Phylogenetic position of two Macrobiotus species with a revisional note on Macrobiotus sottilei Pilato, Kiosya, Lisi & Sabella, 2012 (Tardigrada: Eutardigrada: Macrobiotidae). Zootaxa 4933 (1): 113–135. https://doi.org/10.11646/zootaxa.4933.1.5
Lanfear R., Frandsen P.B., Wright A.M., Senfeld T. & Calcott B. 2017. PartitionFinder 2: New methods for selecting partitioned models of evolution for molecular and morphological phylogenetic analyses. Molecular Biology and Evolution 34 (3): 772–773. https://doi.org/10.1093/molbev/msw260
Lisi O., Londoño R. & Quiroga S. 2020. Description of a new genus and species (Eutardigrada: Richtersiidae) from Colombia, with comments on the family Richtersiidae. Zootaxa 4822 (4): 531–550. https://doi.org/10.11646/zootaxa.4822.4.4
Massa E., Vecchi M., Calhim S. & Choong H. 2024. First records of limnoterrestrial tardigrades (Tardigrada) from Haida Gwaii, British Columbia, Canada. The European Zoological Journal 91 (1): 1–20. https://doi.org/10.1080/24750263.2023.2288824
Michalczyk Ł. & Kaczmarek Ł. 2013. The Tardigrada Register: A comprehensive online data repository for tardigrade taxonomy. Journal of Limnology 72: 175–181. https://doi.org/10.4081/jlimnol.2013.s1.e22
Mutterer J. & Zinck E. 2013. Quick-and-clean article figures with FigureJ. Journal of Microscopy 252 (1): 89–91. https://doi.org/10.1111/jmi.12069
Nilsson E.J.C., Ingemar Jönsson K. & Pallon J. 2010. Tolerance to proton irradiation in the eutardigrade Richtersius coronifer a nuclear microprobe study. International Journal of Radiation Biology 86 (5): 420–427. https://doi.org/10.3109/09553000903568001
Padial J M., Miralles A., De la Riva I. & Vences M. 2010. The integrative future of taxonomy. Frontiers in Zoology 7(1): 16. https://doi.org/10.1186/1742-9994-7-16
Pedersen B.H., Malte H., Ramløv H. & Finster K. 2020. A method for studying the metabolic activity of individual tardigrades by measuring oxygen uptake using micro-respirometry. Journal of Experimental Biology 223 (22): jeb233072. https://doi.org/10.1242/jeb.233072
Pedersen B.H., Malte H., Finster K. & Ramløv H. 2021. Respiration measurements of individual tardigrades of the species Richtersius cf. coronifer as a function of temperature and salinity and termination of anhydrobiosis. Astrobiology 21 (7): 853–865. https://doi.org/10.1089/ast.2020.2371
Persson D.K., Halberg K.A., Jørgensen A., Ricci C., Møbjerg N. & Kristensen R.M. 2011. Extreme stress tolerance in tardigrades: Surviving space conditions in low earth orbit. Journal of Zoological Systematics and Evolutionary Research 49 (s1): 90–97. https://doi.org/10.1111/j.1439-0469.2010.00605.x
Pilato G. 1981. Analisi di nuovi caratteri nello studio degli Eutardigradi. Animalia 8: 51–57.
Pilato G. & Binda M.G. 1987. Richtersia, nuovo genere di Macrobiotidae, e nuova definizione di Adorybiotus Maucci e Ramazzotti. 1981 (Eutardigrada). Animalia 14 (1): 147–152.
Pilato G. & Binda M.G. 1989. Richtersius, nuovo nome genetico in sostituzione di Richtersia Pilato e Binda, 1987 (Eutardigrada). Animalia 16: 147–148.
Pilato G. & Binda M.G. 2010. Definition of families, subfamilies, genera and subgenera of the Eutardigrada, and keys to their identification. Zootaxa 2404 (1): 1–54. https://doi.org/10.11646/zootaxa.2404.1.1
Pogwizd J. & Stec D. 2022. An integrative description of a new Richtersius species from Greece (Tardigrada: Eutardigrada: Richtersiusidae). Acta Zoologica Academiae Scientiarum Hungaricae 68 (1): 1–21. https://doi.org/10.17109/AZH.68.1.1.2022
Puillandre N., Brouillet S. & Achaz G. 2021. ASAP: Assemble Species by Automatic Partitioning. Molecular Ecology Resources 21 (2): 609–620. https://doi.org/10.1111/1755-0998.13281
Ramløv H. & Westh P. 1992. Survival of the cryptobiotic Eutardigrade Adorybiotus coronifer during cooling to -196°C: Effect of cooling rate, trehalose level, and short term acclimation. Cryobiology 29 (1): 125–130. https://doi.org/10.1016/0011-2240(92)90012-Q
Ramløv H. & Westh P. 2001. Cryptobiosis in the eutardigrade Adorybiotus (Richtersius) coronifer: Tolerance to alcohols, temperature and de novo protein synthesis. Zoologischer Anzeiger 240 (3–4): 517–523. https://doi.org/10.1078/0044-5231-00062
Rebecchi L., Rossi V., Altiero T., Bertolani R. & Menozzi P. 2003. Reproductive modes and genetic polymorphism in the tardigrade Richtersius coronifer (Eutardigrada, Macrobiotidae). Invertebrate Biology 122 (1): 19–27. https://doi.org/10.1111/j.1744-7410.2003.tb00069.x
Richters F. 1903. Nordische Tardigraden. Zoologischer Anzeiger 27: 168–172.
Ronquist F., Teslenko M., Van Der Mark P., Ayres D.L., Darling A., Höhna, S., Larget B., Liu L., Suchard M.A. & Huelsenbeck J.P. 2012. MrBayes 3.2: Efficient Bayesian phylogenetic inference and model choice across a large model space. Systematic Biology 61 (3): 539–542. https://doi.org/10.1093/sysbio/sys029
Stec D. & Michalczyk Ł. 2020. Macrobiotus coronifer Richters, 1903 (type species for Richtersius Pilato & Binda, 1989): Designating a new neotype from the original type locality described within the integrative taxonomy framework. Zootaxa 4858 (2): 292–294. https://doi.org/10.11646/zootaxa.4858.2.10
Stec D. & Morek W. 2022. Reaching the monophyly: Re-evaluation of the enigmatic species Tenuibiotus hyperonyx (Maucci, 1983) and the genus Tenuibiotus (Eutardigrada). Animals 12 (3): 404. https://doi.org/10.3390/ani12030404
Stec D., Kristensen R.M. & Michalczyk Ł. 2020a. An integrative description of Minibiotus ioculator sp. nov. from the Republic of South Africa with notes on Minibiotus pentannulatus Londoño et al., 2017 (Tardigrada: Macrobiotidae). Zoologischer Anzeiger 286: 117–134. https://doi.org/10.1016/j.jcz.2020.03.007
Stec D., Krzywański Ł., Arakawa K. & Michalczyk Ł. 2020b. A new redescription of Richtersius coronifer, supported by transcriptome, provides resources for describing concealed species diversity within the monotypic genus Richtersius (Eutardigrada). Zoological Letters 6 (1): 2. https://doi.org/10.1186/s40851-020-0154-y
Stec D., Vecchi M., Maciejowski W. & Michalczyk Ł. 2020c. Resolving the systematics of Richtersiidae by multilocus phylogeny and an integrative redescription of the nominal species for the genus Crenubiotus (Tardigrada). Scientific Reports 10 (1): 19418. https://doi.org/10.1038/s41598-020-75962-1
Stec D., Vecchi M., Calhim S. & Michalczyk Ł. 2021. New multilocus phylogeny reorganises the family Macrobiotidae (Eutardigrada) and unveils complex morphological evolution of the Macrobiotus hufelandi group. Molecular Phylogenetics and Evolution 160: 106987. https://doi.org/10.1016/j.ympev.2020.106987
Vecchi M. & Bruneaux M. 2021. concatipede: An R package to concatenate fasta sequences easily. http://doi.org/10.5281/zenodo.5130604
Vecchi M., Newton I.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
Westh P. & Kristensen R.M. 1992. Ice formation in the freeze-tolerant eutardigrades Adorybiotus coronifer and Amphibolus nebulosus studied by differential scanning calorimetry. Polar Biology 12 (8): 693–699. https://doi.org/10.1007/BF00238869
Westh P. & Ramløv H. 1991. Trehalose accumulation in the tardigrade Adorybiotus coronifer during anhydrobiosis. Journal of Experimental Zoology 258 (3): 303–311. https://doi.org/10.1002/jez.1402580305
Zawierucha K., Vecchi M., Takeuchi N., Ono M. & Calhim S. 2023. Negative impact of freeze–thaw cycles on the survival of tardigrades. Ecological Indicators 154: 110460. https://doi.org/10.1016/j.ecolind.2023.110460
Copyright (c) 2025 Matteo Vecchi, Jakub Godziek, Reinhardt M. Kristensen, Lucia Piemontese, Sara Calhim, Daniel Stec
This work is licensed under a Creative Commons Attribution 4.0 International License.
Creative Commons Copyright Notices
Authors who publish with this journal agree to the following terms:
- Authors retain copyright and grant the journal right of first publication with the work simultaneously licensed under a Creative Commons Attribution License (CC BY 4.0) that allows others to share the work with an acknowledgement of the work's authorship and initial publication in this journal.
- Authors are able to enter into separate, additional contractual arrangements for the non-exclusive distribution of the journal's published version of the work (e.g., post it to an institutional repository or publish it in a book), with an acknowledgement of its initial publication in this journal.
- Authors are NOT PERMITTED to post their submitted work online (e.g., in institutional repositories or on personal websites) prior to publication, as it may lead to nomenclatural issues.
