Using tardigrades from a single moss sample as a case study,
we propose a new method for tardigrade species identification, which is
often problematic, due to the low number of morphological characters.
Identification at generic level was carried out on adults, while morphological
analyses were performed on animals (LM) and eggs (LM and SEM), including
hologenophores, vouchers used also for molecular analysis of COI mtDNA.
This multi-approach method revealed the presence of three species of the
“Macrobiotus hufelandi group” instead of the two species identified in a
previous study. The validity of the method is shown, indicating that it could be
applied to studies of problematic meiofauna taxa

Bertolani, Roberto

Cesari, Michele

Rebecchi, Lorena

Using tardigrades from a single moss sample as a case study,we propose a new method for tardigrade species identification, which is often problematic, due to the low number of orphological characters. Identification at generic level was carried out on adults, while morphological analyses were performed on animals (LM) and eggs (LM and SEM), including hologenophores, vouchers used also for molecular analysis of COI mtDNA. This multi-approach method revealed the presence of three species of the“Macrobiotus hufelandi group” instead of the two species identified in a previous study. The validity of the method is shown, indicating that it could be applied to studies of problematic meiofauna taxa

BERTOLANI, Roberto

REBECCHI, Lorena

CESARI, Michele

Archivio istituzionale della ricerca - Università di Modena e Reggio Emilia

333A model study for tardigrade identificationRoberto Bertolani, Lorena Rebecchi, Michele CesariAbstract — Using tardigrades from a single moss sample as a case study, we propose a new method for tardigrade species identification, which is often problematic, due to the low number of morphological characters. Identification at generic level was carried out on adults, while morphological analyses were performed on animals (LM) and eggs (LM and SEM), including hologenophores, vouchers used also for molecular analysis of COI mtDNA. This multi-approach method revealed the presence of three species of the “Macrobiotus hufelandi group” instead of the two species identified in a previous study. The validity of the method is shown, indicating that it could be applied to studies of problematic meiofauna taxa.Index Terms — COI, DNA barcoding, morphology, Tardigrades, taxonomy.——————————   u   ——————————1 introductionTardigrades consist of more than 1,000 described species [1], [2] colonizing marine, limnic and terrestrial environments, including “hostile to life” and unpredictable habitats. In the seventies, a new evaluation of the intraspecific variability and new morphological characters for species identification were proposed [3], [4], [5], which led the number of tardigrade species to increase from less than 500 species described to that date to the current number. An example of this improvement in identifying species can be found in Macrobiotus hufelandi, the first described [6] and most commonly identified tardigrade species. What was considered a single species is currently represented by more than 25 species. Nonetheless, tardigrade identification at the species level is often problematic due to the low number of taxonomic characters. During our work it was not rare to find in the same moss sample more than one tardigrade species, not only in the same genus but also in the same species group, creating problems of species identification. For this reason we have begun to identify species by coupling a detailed evaluation of animal and egg shell morphology with DNA barcoding [7]. Using one moss sample as a case study, we propose a new method for tardigrade species identification and, in general, for identification of meiofaunal taxa whose morphological ————————————————The authors are with the Department of Biology, University of Modena and Reggio Emilia, 41125 Modena, Italy. E-mail: roberto.bertolani@unimore.it; lorena.rebecchi@unimore.it; michele.cesari@unimore.it.Nimis P. L., Vignes Lebbe R. (eds.) Tools for Identifying Biodiversity: Progress and Problems – pp. 333-339.ISBN 978-88-8303-295-0. EUT, 2010.334characters are often very limited.2 material and methodSThe moss sample was collected at Andalo (Central Alps; province of Trento, Italy, 46°N 10.133, 011°E 00.017, 1050 m) on a rock already examined by us (Fig. 1) [7], [8]. Several tardigrade species were present in the sample, belonging to different genera of eutardigrades (Macrobiotus, Minibiotus, Ramazzottius, Milnesium) and heterotardigrades (Echiniscus) but only the specimens belonging to the so-called “Macrobiotus hufelandi group” have been used in this study. Two species in this group, Macrobiotus macrocalix Bertolani & Rebecchi, 1983 (amphimictic) and M. cf. terminalis (parthenogenetic), were already found in the previous collections [7], [8].Fig. 1 – The rock located in Andalo (Italy), with the moss patch that was used in the study.Morphological analyses of paragenophore voucher specimens (sensu Pleijel et al. [9]) were carried out by mounting animals and eggs (that have species-specific shell processes) in Faure-Berlese fluid for light microscopy (LM) observations. Other eggs were fixed and dehydrated for scanning electron microscopy (SEM) analysis. Additional animals were stained with acetic lactic orcein for gender identification.For molecular analysis, DNA was extracted from single entire animals. Some of these specimens were newborns hatched from isolated eggs, whose shells were mounted in Faure-Berlese fluid, obtaining hologenophore voucher specimens (sensu Pleijel et al. [9]). Amplification and sequencing of 684 bp of the COI mtDNA gene were carried out, following the procedures described in Cesari et al. [7]. Kimura 2-parameters distances between haplotypes were scored by using MEGA4 [10], while neighbor joining and maximum parsimony dendrograms were computed using PAUP* 4.01b10 [11], also using sequences retrieved from GenBank (EU244599; FJ435804-7; AY598773-5; FJ176203-17). For possible further investigations, a fragment of the moss sample was stored at -80°C. 3353 reSultSObservations of animals stained with acetic lactic orcein confirmed the presence of males and females morphologically attributable to M. macrocalix by the presence of a strong buccal armature, with thick crests and large bands of evident teeth and with a relatively wide buccal tube, also observed in mounted specimens (Fig. 2a). In addition, males were also found among the specimens characterized by a weaker buccal armature and narrower buccal tube (Fig. 2b). Fig. 2 – Buccal-pharyngeal apparatuses (Faure-Berlese fluid, phase contrast). a: Strong buccal armature and wide buccal tube. b: Weak buccal armature and narrow buccal tube. Scale bars = 10 µm.Molecular analysis of specimens and eggs belonging to the “Macrobiotus hufelandi group” revealed three clearly distinct haplogroups (a-c, Fig. 3), with very high genetic distances among them (Tab. 1). Tab. 1 – Kimura 2-parameters distances computed among (under the diagonal) and inside (column d) haplogroups.A detailed analysis of egg shell morphology both of the hologenophores and of other voucher specimens (paragenophores) showed the presence of three types of eggs, all bearing processes as inverted goblets on the shell (Fig. 4). One type of egg exhibited high (9.6-10.7 µm) and wide (7.9-8.6 µm) processes with large (7.0-8.0 µm) smooth distal discs and very large pits located only around the process bases (typical of M. macrocalix) (Fig. 4a, d). The second type of egg was characterized by clearly smaller processes (7.4-8.4 µm) than 1 2 3 d1 Haplogroup a 0.0052 Haplogroup b 0.193 0.0003 Haplogroup c 0.169 0.181 0.001336those of M. macrocalix and having an irregularly edged distal disc (6.3-7.0 µm in diameter) and a non-uniform reticulated egg shell with a thick meshwork (Fig. 4b, e). The third type of egg had small processes (5.0-5.3 µm in height) with a slightly irregular edge on the distal disc (4.7-5.2 µm in diameter), and a very uniform reticulated egg shell with a very thin meshwork (Fig. 4c, f).Fig. 3 – Dendrogram combining neighbor joining (NJ, ME score: 0.731) and maximum parsimony (MP, consistency index: 0.743; retention index: 0.920; rescaled consistency index: 0.684) analyses. Numbers above branches indicate mutational steps, while numbers in parentheses show bootstrap values computed after 2000 replicates (above branches: MP; below branches: NJ). a-c denote different haplogroups, while H denotes individuals for which hologenophore voucher specimens are available. Names in bold indicate specimens pertaining to the studied moss.337Fig. 4 – Voucher specimens consisting of egg shells. a-c: Faure-Berlese fluid (LM, phase contrast). a: Macrobiotus macrocalix, haplogroup a (hologenophore H1). b: M. cf. terminalis, haplogroup b (paragenophore). c: M. sandrae, haplogroup c (paragenophore). d-f: SEM (paragenophores). d: M. macrocalix. e: M. cf. terminalis. f: M. sandrae. Scale bars = 5 µm.4 diScuSSion The sex ratio analysis of the tardigrades belonging to the “Macrobiotus hufelandi group” in the moss sample revealed a much more complicated situation than that known from the literature [7], [8]. Nevertheless, by comparing the results of a detailed morphological analysis with those obtained by DNA barcoding, and in particular by sequencing the newborns’ DNA and linking their sequences to the related egg shell shapes (hologenophores), the problem can finally be solved.The distance values among the three different haplogroups are very high, far exceeding the 3% threshold and the 10x rule proposed by Hebert et al. [12], [13], [14], thus supporting the specific rank of the three haplogroups. Two species, M. macrocalix and M. cf. terminalis (currently being described as a new species), morphologically correspond to what was previously found on the same rock at Andalo [7], [8]. With regards to the third species, the animals look similar to the specimens of M. cf. terminalis (even through probably smaller), but the eggs are quite distinguishable and allow us to attribute them to Macrobiotus sandrae Bertolani & Rebecchi, 1983. This species is known to be amphimictic 338[8], a situation consistent with the presence of males among the animals with a weaker buccal armature and narrower buccal tube. 5 concluSionSThe methods described here allow us to solve intricate tardigrade identification problems, validating our new approach based on linking morphological and molecular data. The use of voucher specimens, and in particular of the hologenophores, is critical for obtaining a correct species diagnosis. A hologenophore can also be obtained by culturing an isolated female until oviposition, mounting it as voucher and using its developing eggs either for molecular analysis and/or as further vouchers. Further information important for identification can also be obtained from other tardigrades, which can be photographed in vivo up to maximum magnification (100x objective) before being used in molecular investigations. In our opinion, our multi-approach method for tardigrade identification can be easily applied to other meiofaunal taxa, whose few morphological characters can generate problems in species identification.acknowledgementThe authors wish to thank Diane R. Nelson, East Tennessee State University, U.S.A, for her help in the English revision and for her suggestions. This work was supported by a grant from the Fondazione Cassa di Risparmio di Modena (Italy) and the University of Modena and Reggio Emilia (Italy): “MoDNA project (Morphology and DNA): DNA barcoding and phylogeny of tardigrades, basic research and applications”.referenceS[1] R. Guidetti and R. Bertolani, “Tardigrade taxonomy: an updated check list of the taxa and a list of characters for their identification”, Zootaxa, vol. 845, pp. 1-46, 2005.[2] D. R. Nelson, R. Guidetti and L. Rebecchi, “Tardigrada”. In: J. H. Thorp. A. P. Covich (eds.), Ecology and Classification of North American Freshwater Invertebrates, Elsevier Inc., Amsterdam, The Netherlands, pp. 455-484, 2010.[3] G. Pilato, “Structure, intraspecific variability and systematic value of the buccal armature of eutardigrades”, Z. f. Zool. Systematik u. Evolutionforschung, vol. 10, pp. 65-78, 1972.[4] G. Pilato, “Redescription of Haplomacrobiotus hermosillensis May, 1948, and consideration of the genus Haplomacrobiotus (Eutardigrada)”, Z. f. Zool. Systematik u. Evolutionforschung, vol. 11, pp. 283-286, 1973.[5] G. Pilato, “On the taxonomic criteria of the Eutardigrada”, Mem. Ist. Ital. Idrobiol., vol. 32, Suppl., pp. 277-303, 1975.[6] C. A. S. Schultze, Macrobiotus Hufelandii animal e crustaceorum classe novum, reviviscendi post diuturnam asphyxiam et ariditatem potens, C. Curths, Berlin, 6 pp. 1 tab., 1834.[7] M. Cesari, R. Bertolani, L. Rebecchi and R. Guidetti, “DNA barcoding in Tardigrada: the first case study on Macrobiotus macrocalix Bertolani & Rebecchi 1993 (Eutardigrada, Macrobiotidae)”, Mol. Ecol. Resour., vol. 9, pp. 699-706, 2009.[8] R. Bertolani and L. Rebecchi, “A revision of the Macrobiotus hufelandi group (Tardigrada, Macrobiotidae), with some observations on the taxonomic characters of eutardigrades”, Zool. Scripta, vol. 22, pp.127-152, 1993.[9] F. Pleijel, U. Jondelius, E. Norlinder, A. Nygren, B. Oxelman, C. Schander, P. Sundberg and M. Thollesson, “Phylogenies without roots? A plea for the use of vouchers in molecular phylogenetic studies”, Mol. Phylogenet. Evol., vol. 48, pp. 369-371, 2008.339[10] K. Tamura, J. Dudley, M. Nei and S. Kumar, “MEGA4: Molecular Evolutionary Genetics Analysis (MEGA), software version 4.0”, Mol. Bio. Evol., vol. 24, pp. 1596-1599, 2007.[11] D. L. Swofford, “PAUP* Phylogenetic analysis using parsimony (*and other methods)”, Version 4.0b10 win32”, Sinauer Associates, Sunderland, USA, 2002.[12] P. D. N. Hebert, A. Cywinska, S. L. Ball and J. R. deWaard, “Biological identifications through DNA barcodes”, P. Roy. Soc. Lond. B Biol., vol. 270, pp. 313-321, 2003.[13] P. D. N. Hebert, S. Ratnasingham and J. R. deWaard, “Barcoding animal life: cytochrome c oxidase subunit 1 divergences among closely related species”, P. Roy. Soc. Lond. B Bio., vol. 270, pp. 596-599, 2003.[14] P. D. N. Hebert, M. Y. Stoeckle, T. S. Zemlak and C. M. Francis, “Identification of birds through DNA barcodes”, PLoS Biol., vol. 2, e312, 2004.

A model study for tardigrade identification

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A model study for tardigrade identification

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