Integrative description of a new Tunisian tardigrade species, Macrobiotus azzunae sp. nov. (Eutardigrada, Macrobiotidae, hufelandi group)

Keywords: Tardigrada, Ain Soltan Forest, Tunisia, Macrobiotus azzunae, new species


In this paper a new tardigrade species, Macrobiotus azzunae sp. nov., from Tunisia, is described. An integrative taxonomic approach was applied by combining morphological, morphometric and molecular data. In particular, light and scanning electron microscopy observations, and four genetic markers, three nuclear (18S rRNA, 28S rRNA and ITS-2) and one mitochondrial (COI) were used. The analysis showed that M. azzunae sp. nov. belongs to the Macrobiotus hufelandi group and is most similar to Macrobiotus sandrae Bertolani & Rebecchi, 1993. It differs from M. sandrae by a more pronounced constriction of the first macroplacoid (hardly visible in M. sandrae) and for the eggshell shape, with thinner wires of the reticulum and meshes around the processes larger than the inter-process meshes in M. azzunae sp. nov., while all meshes are similar in size in M. sandrae. The species is gonochoristic. With this discovery, there are 33 species of tardigrades identified in Tunisia, all non-marine. This result, compared with nearby Sicily, where more research has been conducted, indicates that there is a considerable potential for identification of new species. Further research will be most informative if multiple habitats are explored and if carried out with an integrated approach as done in this present work.


Bertolani R., Biserov V., Rebecchi L. & Cesari M. 2011a. Taxonomy and biogeography of tardigrades using an integrated approach: new results on species of the Macrobiotus hufelandi group. Invertebrate Zoology 8 (1): 23–26.

Bertolani R., Rebecchi L., Giovannini I. & Cesari M. 2011b. DNA barcoding and integrative taxonomy of Macrobiotus hufelandi C.A.S. Schultze 1834, the first tardigrade species to be described, and some related species. Zootaxa 2997: 19–36.

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: 110–126.

Binda M.G. & Pilato G. 1987. Tardigradi dell’Africa. V: Notizie sui Tardigradi del Nordafrica e descrizione della nuova specie Macrobiotus diffusus. Animalia 14: 177–191.

Biserov V.I. 1990. On the revision of the Macrobiotus genus. The subgenus Macrobiotus s. str. is a new taxonomic status of the hufelandi group (Tardigrada, Macrobiotidae). Communication 2. Zoologicheskij Zhurnal 69: 38–50. [In Russian.]

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.

Cesari M., Giovannini I., Bertolani R. & Rebecchi L. 2011. An example of problems associated with DNA barcoding in tardigrades: a novel method for obtaining voucher specimens. Zootaxa 3104: 42–51.

Cesari M., Vecchi M., Palmer A., Bertolani R., Pilato G., Rebecchi L. & Guidetti R. 2016. What if the claws are reduced? Morphological and molecular phylogenetic relationships of the genus Haplomacrobiotus May. 1948 (Eutardigrada, Parachela). Zoological Journal of the Linnean Society 178 (4): 819–827.

Cesari M., Montanari M., Kristensen R.M., Bertolani R., Guidetti R. & Rebecchi L. 2020. An integrated study of the biodiversity within the Pseudechiniscus suillus-facettalis group (Heterotardigrada: Echiniscidae). Zoological Journal of the Linnean Society 188 (3): 717–732.

Clement M., Posada D. & Crandall K. 2000. TCS: a computer program to estimate gene genealogies. Molecular Ecology 9: 1657–1660.

Coughlan K. & Stec D. 2019. Two new species of the Macrobiotus hufelandi complex (Tardigrada: Eutardigrada: Macrobiotidae) from Australia and India, with notes on their phylogenetic position. European Journal of Taxonomy 573: 1–38.

Coughlan K., Michalczyk Ł. & Stec D. 2019. Macrobiotus caelestis sp. nov., a new tardigrade species (Macrobiotidae: hufelandi group) from the Tien Shan Mountains (Kyrgyzstan). Annales Zoologici 69 (3): 499–513.

Darriba D., Taboada G.L., Doallo R. & Posada D. 2012. jModelTest 2: more models, new heuristics and parallel computing. Nature Methods 9 (8): 772.

Degma P., Bertolani R. & Guidetti R. 2020. Actual checklist of Tardigrada. 36th Edition: 8–07–2020. Available from [accessed 28 Jun. 2021].

Gąsiorek P., Stec D., Morek W., Marnissi J. & Michalczyk Ł. 2017. The tardigrade fauna of Tunisia, with an integrative description of Bryodelphax maculatus sp. nov. (Heterotardigrada: Echiniscidae). African Zoology 52 (2): 77–89.

Guidetti R. & Bertolani R. 2005. Tardigrade taxonomy: an updated checklist of the taxa and a list of characters for their identification. Zootaxa 845 (1): 1–46.

Guidetti R., Gandolfi A., Rossi V. & Bertolani R. 2005. Phylogenetic analysis of Macrobiotidae (Eutardigrada,Parachela): a combined morphological and molecular approach. Zoologica Scripta 34: 235–244.

Guidetti R., Altiero T., Marchioro T., Sarzi Amadè L., Avdonina A.M., Bertolani R. & Rebecchi L. 2012. Form and function of the feeding apparatus in Eutardigrada (Tardigrada). Zoomorphology 131: 127–148.

Guidetti R., Peluffo J.R., Rocha A.M., Cesari M. & Moly de Peluffo M.C. 2013. The morphological and molecular analyses of a new South American urban tardigrade offer new insights on the biological meaning of the Macrobiotus hufelandi group of species (Tardigrada: Macrobiotidae). Journal of Natural History 47: 2409–2426.

Guil N. & Giribet G. 2012. A comprehensive molecular phylogeny of tardigrades — adding genes and taxa to a poorly resolved phylum-level phylogeny. Cladistics 27: 1–29.

Guindon S. & Gascuel O. 2003. A simple, fast and accurate method to estimate large phylogenies by maximum-likelihood. Systematic Biology 52: 696–704.

Hart M.W. & Sunday J. 2007. Things fall apart: biological species form unconnected parsimony networks. Biology Letters 3: 509–512.

Huelsenbeck J.P. & Ronquist F. 2001. MRBAYES: Bayesian inference of phylogenetic trees. Bioinformatics 17: 754–755.

Iharos G. 1978. Data to the knowledge of the Tardigrada fauna of Tunisia. Folia Entomologica Hungarica 31: 161–164.

Jørgensen A., Faurby S., Hansen J.G., Møbjerg N. & Kristensen R.M. 2010. Molecular phylogeny of Arthrotardigrada (Tardigrada). Molecular Phylogenetics and Evolution 54: 1006–1015.

Kaczmarek Ł. & Michalczyk Ł. 2017a. The Macrobiotus hufelandi (Tardigrada) group revisited. Zootaxa 4363: 101–123.

Kaczmarek Ł. & Michalczyk Ł. 2017b. A description of Macrobiotus horningi sp. nov. and redescriptions of M. maculatus comb. nov. Iharos, 1973 and M. rawsoni Horning et al., 1978 (Tardigrada: Eutardigrada: Macrobiotidae: hufelandi group). Zootaxa 4363: 79–100.

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: 3059–3066.

Katoh K., Rozewicki J. & Yamada K.D. 2017. MAFFT online service: multiple sequence alignment, interactive sequence choice and visualization. Briefings in Bioinformatics 20 (4): 1160–1166.

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.

Kumar A., Sharma P., Gomar Alba M., Shcheprova Z., Daulny A., Sanmartín T., Matucci I., Funaya C., Beato M. & Mendoza M. 2018. Daughter-cell-specific modulation of nuclear pore complexes controls cell cycle entry during asymmetric division. Nature Cell Biology 20 (4):432–442.

McInnes S.J. 1994. Zoogeographic distribution of terrestrial/freshwater tardigrades from current literature. Journal of the Natural History 28: 257–352.

McInnes S.J., Michalczyk Ł. & Kaczmarek Ł. 2017. Annotated zoogeography of non-marine Tardigrada. Part IV: Africa. Zootaxa 4284: 1–71.

Michalczyk Ł. & Kaczmarek Ł. 2003. A description of the new tardigrade Macrobiotus reinhardti (Eutardigrada, Macrobiotidae, harmsworthi group) with some remarks on the oral cavity armature within the genus Macrobiotus Schultze. Zootaxa 331: 1–24.

Miller M.A., Pfeiffer W. & Schwartz T. 2010. Creating the CIPRES Science Gateway for inference of large phylogenetic trees. 2010 Gateway Computing Environments Workshop (GCE): 1–8.

Múrias dos Santos A., Cabezas M.P., Tavares A.I., Xavier R. & Branco M. 2016. tcsBU: a tool to extend TCS network layout and visualization. Bioinformatics 32 (4): 627–628.

Nelson D.R., Guidetti R. & Rebecchi L. 2015. Phylum Tardigrada. In: Thorp J. & Rogers D.C. (eds) Ecology and General Biology: Thorp and Covich’s Freshwater Invertebrates: 347–380. Academic Press, London.

Nowak B. & Stec D. 2018 An integrative description of Macrobiotus hannae sp. nov. (Tardigrada: Eutardigrada: Macrobiotidae: hufelandi group) from Poland. Turkish Journal of Zoology 42: 269–286.

Nylander J.A.A. 2004. MrModeltest v2. Program distributed by the author. Evolutionary Biology Centre, Uppsala University.

Pilato G. 1981. Analisi di nuovi caratteri nello studio degli Eutardigradi. Animalia 8: 51–57.

Pilato G. & Binda M.G. 2010. Definition of families, subfamilies, genera and subgenera of the Eutardigrada, and keys to their identification. Zootaxa 2404: 1–54.

Pilato G. & Lisi O. 2009. Tardigrades of the Seychelles Islands, with the description of three new species. Zootaxa 2124: 1–20.

Pilato G., Binda M.G. & Azzaro M. 1990. Tardigradi di Terra del Fuoco e Magallanes. III. Macrobiotus punctillus, nuova specie di Macrobiotidae del gruppo hufelandi. Animalia 17: 123–129.

Pilato G., Binda M.G. & Catanzaro R. 1991. Remarks on some tardigrades of the African fauna with the description of three new species of Macrobiotus Schultze 1834. Tropical Zoology 4: 167–178.

Pilato G., Kiosya Ye., Lisi O. & Sabella G. 2012. New records of Eutardigrada from Belarus with the description of three new species. Zootaxa 3179: 39–60.

Pilato G., Sabella G., D’Urso V. & Lisi O. 2017. Macrobiotus nebrodensis and Adropion vexatum, two new species of Eutardigrada (Tardigrada, Parachela) from Sicily. Zootaxa 4362 (2): 267–279.

Pilato G., D’Urso V., Sabella G. & Lisi O. 2019. A new species of isohypsibiid (Eutardigrada) from Sicily. Zootaxa 4608 (3): 543–554.

Puillandre N., Lambert A., Brouillet S. & Achaz G. 2012. ABGD, Automatic Barcode Gap Discovery for primary species delimitation. Molecular Ecology 21: 1864–1877.

Ramazzotti G. & Maucci W. 1983. Il Phylum Tardigrada. Memorie dell’Istituto Italiano di Idrobiologia, Pallanza 41: 1–1012.

Rambaut A., Drummond A.J., Xie D., Baele G. & Suchard M.A. 2018. Posterior summarisation in Bayesian phylogenetics using Tracer 1.7. Systematic Biology 67 (5): 901–904.

Rebecchi L. & Bertolani R. 1988. New cases of parthenogenesis and polyploidy in the genus Ramazzottius (Tardigrada, Hypsibiidae) and a hypothesis concerning their origin. International Journal of Invertebrate Reproduction and Development 14: 187–196.

Richters F. 1926. Tardigrada. In: Kukenthal W. & Krumbach T. (eds) Handbuch der Zoologie, Vol. III: 58–61. Walter de Gruyter & Co, Berlin and Leipzig.

Ronquist F. & Huelsenbeck J.P. 2003. MRBAYES 3: Bayesian phylogenetic inference under mixed models. Bioinformatics 19: 1572–1574.

Roszkowska M., Ostrowska M., Stec D., Janko K. & Kaczmarek Ł. 2017. Macrobiotus polypiformis sp. nov., a new tardigrade (Macrobiotidae; hufelandi group) from the Ecuadorian Pacific coast, with remarks on the claw abnormalities in eutardigrades. European Journal of Taxonomy 327: 1–19.

Schill R.O. (ed.) 2018. Water Bears: The Biology of Tardigrades. Vol. 419. Springer, Cham, Switzerland.

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 and Evolution 10: 287–296.

Schultze C.A.S. 1834. Macrobiotus Hufelandii animal e crustaceorum classe novum, reviviscendi post diuturnam asphyxia et ariditaten potens, etc. C. Curths, Berlin.

Schuster R.O, Nelson D.R., Grigarick A.A. & Christenberry D. 1980. Systematic criteria of the Eutardigrada. Transaction of the American Microscopical Society 99: 284–303.

Stamatakis A. 2006. RAxML-VI-HPC: maximum likelihood-based phylogenetic analyses with thousands of taxa and mixed models. Bioinformatics 22: 2688–2690.

Stamatakis A., Hoover P. & Rougemont J. 2008. A rapid bootstrap algorithm for the RAxML web servers. Systematic Biology 57: 758–771.

Stambouli-Essassi S., Roche E. & Bouzid S. 2007. Evolution of vegetation and climatic changes in North-Western Tunisia during the last 40 millennia. Geo-Eco-Trop 31: 171–214.

Stec D., Smolak R., Kaczmarek Ł. & Michalczyk Ł. 2015. An integrative description of Macrobiotus paulinae sp. nov. (Tardigrada: Eutardigrada: Macrobiotidae: hufelandi group) from Kenya. Zootaxa 4052 (2): 501–526.

Stec D., Morek W., Gąsiorek P., Blagden B. & Michalczyk Ł. 2017. Description of Macrobiotus scoticus sp. nov. (Tardigrada: Macrobiotidae: hufelandi Group) from Scotland by means of integrative taxonomy. Annales Zoologici 67 (2): 181–197.

Stec D., Arakawa K. & Michalczyk Ł. 2018a. An integrative description of Macrobiotus shonaicus sp. nov. (Tardigrada: Macrobiotidae) from Japan with notes on its phylogenetic position within the hufelandi group. PLoS ONE 13 (2): e0192210.

Stec D., Kristensen R.M. & Michalczyk Ł. 2018b. Integrative taxonomy identifies Macrobiotus papei, a new tardigrade species of the Macrobiotus hufelandi complex (Eutardigrada: Macrobiotidae) from the Udzungwa Mountains National Park (Tanzania). Zootaxa 4446 (2): 273–291.

Stec D., Krzywański Ł. & Michalczyk Ł. 2018c. Integrative description of Macrobiotus canaricus sp. nov. with notes on M. recens (Eutardigrada: Macrobiotidae). European Journal of Taxonomy 452: 1–36.

Stec D., Morek W., Gąsiorek P. & Michalczyk Ł. 2018d. Unmasking hidden species diversity within the Ramazzottius oberhaeuseri complex, with an integrative redescription of the nominal species for the family Ramazzottiidae (Tardigrada: Eutardigrada: Parachela). Systematics and Biodiversity 16 (4): 357–376.

Stec D., Dudziak M. & Michalczyk Ł. 2020a. Integrative descriptions of two new Macrobiotidae species (Tardigrada: Eutardigrada: Macrobiotoidea) from French Guiana and Malaysian Borneo. Zoological Studies 59 (23):1–25.

Stec D., Tumanov D.V. & Kristensen R.M. 2020b. Integrative taxonomy identifies two new tardigrade species (Eutardigrada: Macrobiotidae) from Greenland. European Journal of Taxonomy 614: 1–40.

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.

Sugiura K., Arakawa K. & Matsumoto M. 2020. Distribution of Macrobiotus shonaicus Stec, Arakawa & Michalczyk, 2018 (Tardigrada: Eutardigrada: Macrobiotidae) in Japan. Zootaxa 4767 (1): 56–70.

Tang C.Q., Humphreys A.M., Fontaneto D. & Barraclough T.G. 2014. Effects of phylogenetic reconstruction method of the robustness of species delimitation using single-locus data. Methods in Ecology and Evolution 5: 1086–1094.

Templeton A.R., Crandall K.A. & Sing C.F. 1992. A cladistic analysis of phenotypic association with haplotypes inferred from restriction endonuclease mapping and DNA sequence data. III. Cladogram estimation. Genetics 132 (2): 619–633.

Thulin G. 1928. Über die Phylogenie und das System der Tardigraden. Hereditas 11 (2–3): 207–266.

Vecchi M., Cesari M., Bertolani R., Jönsson K.I., Rebecchi L. & Guidetti R. 2016. Integrative systematic studies on tardigrades from Antarctica identify new genera and new species within Macrobiotoidea and Echiniscoidea. Invertebrate Systematics 30: 303–322.

Vicente F., Cesari M., Serrano A. & Bertolani R. 2013. The impact of fire on terrestrial tardigrade biodiversity: a first case-study from Portugal. Journal of Limnology 72 (s1): 152–159.

Wełnicz W., Grohme M.A., Kaczmarek Ł., Schill R.O. & Frohme L. 2011. ITS-2 and 18S rRNA data from Macrobiotus polonicus and Milnesium tardigradum (Eutardigrada, Tardigrada). Journal of Systematics and Evolutionary Research 49 (s1): 34–39.

Zhang J., Kapli P., Pavlidis P. & Stamatakis A. 2013. A general species delimitation method with applications to phylogenetic placements. Bioinformatics 29: 2869–2876.

How to Cite
Ben Marnissi, J., Cesari, M., Rebecchi, L., & Bertolani, R. (2021). Integrative description of a new Tunisian tardigrade species, Macrobiotus azzunae sp. nov. (Eutardigrada, Macrobiotidae, hufelandi group). European Journal of Taxonomy, 758(1), 122–146.
Research article