Emilia, Modena, Italy b Faculty of Natural and Exact Sciences, National University of La. Pampa, Santa Rosa, Argentina Published online: 27 Jun 2013.

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1 This article was downloaded by: [Universita degli Studi di Modena e Reggio Emilia ] On: 15 July 2013, At: 01:04 Publisher: Taylor & Francis Informa Ltd Registered in England and Wales Registered Number: Registered office: Mortimer House, Mortimer Street, London W1T 3JH, UK Journal of Natural History Publication details, including instructions for authors and subscription information: 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) Roberto Guidetti a, Julio Ricardo Peluffo b, Alejandra Mariana Rocha b, Michele Cesari a & María Cristina Moly de Peluffo b a Department of Life Sciences, University of Modena and Reggio Emilia, Modena, Italy b Faculty of Natural and Exact Sciences, National University of La Pampa, Santa Rosa, Argentina Published online: 27 Jun To cite this article: Journal of Natural History (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, DOI: / To link to this article: PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the Content ) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content.

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3 Journal of Natural History, 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) Roberto Guidetti a *, Julio Ricardo Peluffo b, Alejandra Mariana Rocha b, Michele Cesari a and María Cristina Moly de Peluffo b a Department of Life Sciences, University of Modena and Reggio Emilia, Modena, Italy; b Faculty of Natural and Exact Sciences, National University of La Pampa, Santa Rosa, Argentina (Received 14 December 2012; final version received 19 April 2013) Worldwide knowledge of tardigrade fauna is still limited, and many areas such as South America are not well studied. The collection of new substrates in Argentinean urban areas provided an opportunity to describe the new tardigrade species Macrobiotus kristenseni sp. nov. This species has been studied with an integrative taxonomic approach, analysing its morphology by light and scanning electron microscopy, and considering two genes (cox1 and 18S rrna) for DNA barcoding and phylogenetic purposes. The species belongs to the Macrobiotus hufelandi group of species, and it is characterized by egg processes in the shape of elongated cones with truncated and enlarged apexes, and by a high genetic distance with respect to closely related species (cox1: %). Morphological and molecular data showthatthe Macrobiotus hufelandi group of species has to be considered a true biological entity; one of the more widespread tardigrade lineages in continental environments. CE499D97B0F1 Keywords: DNA barcoding; Macrobiotus kristenseni sp. nov.; phylogeny; Eutardigrada Introduction Worldwide knowledge of tardigrade fauna is still very limited, and many geographic areas are not well studied. One of these areas is South America, for which we have inadequate knowledge of its tardigrade species, and the number of recorded tardigrade taxa is small when compared with that of other areas such as Europe (McInnes 1994; Pilato et al. 2003, 2004). Specifically, only 108 species have been recorded in Argentina (Claps et al. 2008) from a total of more than one thousand recorded worldwide (Guidetti and Bertolani 2005, 2011). Among studies that have contributed to the knowledge of tardigrade fauna in Argentina are those of Claps and Rossi (1981, 1984, 1988), Rossi and Claps (1980, 1983, 1989, 1991), Rossi et al. (2009); Maucci (1988); Moly de Peluffo and Peluffo (1995); Moly de Peluffo et al. (2006); Peluffo et al. (2002a, 2002b, 2007) and Rocha et al. (2007). Among all terrestrial environments, a poorly analysed habitat in tardigrade research is the urban environment: at a worldwide scale only 16 species of *Corresponding author. roberto.guidetti@unimore.it 2013 Taylor & Francis

4 2 R. Guidetti et al. tardigrades have been recorded in urban areas (Moly de Peluffo et al. 2006). Of these species, five have been recorded in cities located in central Argentina (Moly de Peluffo et al. 2006; Peluffo et al. 2007). We consider it important to study the tardigrade fauna of the urban areas of Argentina to evaluate the effects of urbanization on these organisms. The collection of new substrates in the city of Santa Rosa (La Pampa, Argentina) gave us the opportunity to describe a new species, which was referred to as Macrobiotus sp. in previous studies (Moly de Peluffo et al. 2006; Peluffo et al. 2007), and to perform molecular taxonomic and phylogenetic studies on the Macrobiotus hufelandi group of species. Material and methods The new species was found in lichen communities growing on different tree species, in two cities (Santa Rosa and General Pico) and one natural site (Natural Reserve Parque Luro) in La Pampa Province (Argentina). Sampling took place between 1999 and For separation and fixation of the tardigrades we used the method described in Moly de Peluffo et al. (2006). Morphological analysis Specimens were studied using light microscopy (LM) and scanning electron microscopy (SEM). For LM observations, specimens were mounted in Faure Berlese medium or in polyvinyl-lactophenol mounting medium. These specimens mounted on permanent slides can be considered as paragenophores (i.e. voucher specimens obtained from animals extracted from the same sample as the animal used for molecular analysis, Pleijel et al. 2008). For SEM observations, the animals were fixed in neutral buffered formalin, then dehydrated by critical-point drying, whereas eggs were fixed and dehydrated in ethanol and finally all specimens were coated in gold. The SEM photographs of animals were taken with a JEOL JSM35CF SEM at the SEM Laboratory of the Centro Regional de Investigaciones Básicas y Aplicadas de Bahía Blanca (CRIBABB, Regional Centre for Basic and Applied Research at Bahía Blanca, Argentina), while those of eggs were taken with an SEM XL 40 (Fei Company-Oxford Instruments) available at the Centro Interdipartimentale Grandi Strumenti of the University of Modena and Reggio Emilia, Italy. Morphometric data were obtained using an optical micrometer. Animal length was measured excluding the fourth leg pair. Percentage ratios between the length of the structure considered and the buccal tube (pt) were calculated following Pilato (1981). Molecular analysis Before molecular analysis, each animal was observed in vivo with LM and identified. To do this, each specimen was mounted in a drop of water under a cover slip on a slide and examined with a Leitz DM RB using the 40 objective and sometimes up to 100 oil objective. The specimen was recollected from the slide by adding water at the edge of the cover slip, and then gently removing the cover slip with a needle. Genomic DNA was extracted from single adult animals (Table 1) following the protocol described by Cesari et al. (2009). Two individuals were also photographed before DNA extraction following the protocol described in Cesari et al. (2011) in order to obtain hologenophore voucher specimens (i.e. voucher specimens obtained from the

5 Journal of Natural History 3 Table 1. Specimens used as source of DNA extraction and GenBank accession numbers of the new analysed sequences and of those used in phylogenetic analyses. Species Specimen Source Voucher specimen GenBank Accession Number cox1 18S rrna Macrobiotus kristenseni sp. nov. C3291-A01 V1 Adult hologenophore KC NA Macrobiotus kristenseni sp. nov. C3291-A02 V2 Adult hologenophore KC KC Macrobiotus kristenseni sp. nov. C3291-A03 Adult KC NA Macrobiotus kristenseni sp. nov. C3291-A04 Adult KC NA Macrobiotus kristenseni sp. nov. C3291-A05 Adult KC NA Species No. of specimens Macrobiotus terminalis 5 JN Macrobiotus cf. terminalis 1 AY Macrobiotus vladimiri 5 HM , HQ FJ Macrobiotus sandrae 16 HQ , HQ , HQ Macrobiotus hufelandi 5 HQ GQ Macrobiotus gr. hufelandi sp FJ , HQ Macrobiotus macrocalix 20 FJ , HQ876571, JN , AY Macrobiotus sapiens 1 DQ Macrobiotus polonicus 1 HM Macrobiotus pallarii 1 FJ Macrobiotus furciger 3 EU Macrobiotus sp. 6 EU266926, EU , U49912, U32393 Paramacrobiotus richtersi 1 DQ Paramacrobiotus gr. richtersi 2 FJ Paramacrobiotus fairbanksi 1 EU (Continued)

6 4 R. Guidetti et al. Table 1. (Continued). Species Specimen Source Voucher specimen GenBank Accession Number cox1 18S rrna Paramacrobiotus kenianus 2 EU Paramacrobiotus tonollii 1 DQ Paramacrobiotus areolatus 1 DQ Paramacrobiotus sp. 1 X81442 Minibiotus furcatus 1 FJ Minibiotus gumersindoi 1 FJ Minibiotus sp. 3 EU Richtersius coronifer 4 EU , AY582123, DQ Isohypsibius papillifer 1 EU Voucher specimens as defined by Pleijel et al. (2008). Letters followed by numbers indicate code in voucher specimen collection (C: collected sample, A: adult animals, V: in vivo). NA: not available.

7 Journal of Natural History 5 same animal used for molecular analysis, Pleijel et al. 2008). Polymerase chain reaction (PCR) amplification of a portion of the mitochondrial cox1 gene was carried out following the Cesari et al. (2009) protocol, using LCO-1490 (5 -GGT CAA CAA ATC ATA AAG ATA TTG G-3 ; Folmer et al. 1994) and HCO-2198 (5 -TAA ACT TCA GGG TGA CCA AAA AAT CA-3 ; Folmer et al. 1994) primers. A phylogenetic investigation was also carried out using a fragment of the nuclear 18S ribosomal RNA (rrna) gene that was amplified using primers SSU F04 (5 -GCT TGT CTC AAA GAT TAA GCC-3 ; Kiehl et al. 2007) and SSU R26 (3 -CAT TCT TGG CAA ATG CTT TCG-5 ; Kiehl et al. 2007), and the following PCR protocol: 35 cycles with 1 min at 94 C, 90 seconds at 52 C and 2 min at 72 C, with a final elongation step at 72 C for 10 min. The amplified products were gel purified using the Wizard Gel and PCR cleaning kit (Promega, Madison, WI, USA). Both strands were subjected to sequencing reaction using the Big Dye Terminator 1.1 kit (Applied Biosystems, Foster City, CA, USA) and sequenced using an ABI Prism 3100 sequencer (Applied Biosystems). Chromatograms obtained were checked for presence of ambiguous bases. For cox1 gene analysis, nucleotide sequences were aligned with the CLUSTAL algorithm implemented in MEGA5 (Tamura et al. 2011; pairwise and multiple alignment parameters: Gap opening penalty 15, Gap extend penalty 6.66) and checked by eye. The cox1 sequences were translated to amino acids by using the invertebrate mitochondrial code implemented in MEGA to check for the presence of stop codons and therefore of pseudogenes. Nucleotide sequences of the newly analysed specimens were submitted to GenBank (Table 1). For appropriate molecular comparisons, we included in our analysis cox1 sequences from GenBank pertaining to other species of the Macrobiotus hufelandi group and of Macrobiotoidea (Table 1). Intraspecific, interspecific, and overall mean Kimura two-parameter (K2P) distances between scored haplotypes were determined using MEGA5. An unrooted neighbour-joining dendrogram was computed on K2P distances using MEGA5; bootstrap values were obtained after 2000 replicates. For 18S rrna gene analysis, nucleotide sequences were aligned with the MUSCLE algorithm, using default parameters implemented in MEGA5. A sequence identified as Isohypsibius papillifer (GenBank A.N.: EU266925) was used as the outgroup. Other Macrobiotoidea sequences from GenBank were also included in the analysis for appropriate comparisons (Table 1). A Bayesian inference dendrogram was computed using the program MRBAYES 3.2 (Ronquist et al. 2012). Best fitting model evaluations were performed taking into account the Akaike Information Criterion and Bayes Information Criterion (JMODELTEST 0.0.1; Posada 2008) which identified the GTR + G model as the most suitable. Two independent runs, each of four Metropolis-coupled Markov chain Monte Carlo method, were launched for generations, trees were sampled every 100 generations and the first 17,500 were discarded. The analyses were run three times, all of which resulted in identical topologies. Results Species description Macrobiotus kristenseni sp. nov. (Figures 1 5 and Table 2)

8 6 R. Guidetti et al. Table 2. Summary of morphometric data (in µm, except pt) formacrobiotus kristenseni sp. nov. Character Holotype Mean SD Min Max pt mean pt s.d. pt min pt max pt c.v. n pt bl btl btd vl ssi plr pl pl mpl pb sb pb sb pb sb pb sb Notes: bl = body length; btl = buccal tube length; btd = diameter of the buccal tube; vl = ventral lamina length; ssi = insertion of stylet supports; plr = macroplacoid row length; 1 pl = first macroplacoid length; 2 pl = second macroplacoid length; mpl = microplacoid length; pb1-4 = length of primary branch of the claw on first-fourth leg pair; sb1-4 length of secondary the claw on first-fourth leg pair; pt = index pt (length of a structure/buccal tube length 100; Pilato, 1981); min = minimum; max = maximum; SD = standard deviation; c.v. = coefficient of variation; n = number of considered specimens.

9 Journal of Natural History 7 Diagnosis Size medium. Cuticle with pearls (pores). Eye-spots present. Buccal tube of average width. Buccal armature with two bands of teeth and transverse ridges. Bulb with two macroplacoids and small microplacoid. Long Y-shaped claws with two short accessory points on the main branch, lunule smooth or weakly crenate in the hind legs. Free eggs with processes in shape of elongated cones with truncated and enlarged apexes. Limnoterrestrial. Description Size range: µm long. Colourless, yellowish, greyish or light brown, sometimes with transverse bands with brown pigment. Eye-spots present in all specimens observed (Figure 1), eye-spot colour not detected. Cuticle smooth under LM, except small granulations on outer part of legs. Cuticular pores rounded or ovate approximately 1 µm in diameter (Figure 2), generally with thickened edges, irregularly distributed on the body surface (dorsally and ventrally). Near the anterior end, a ring of pores with evident thickened edges and placed in a regular manner. Ten small peribuccal lamellae surround the mouth opening, followed by six buccal sensory lobes (Figure 3). Buccal armature, poorly distinguishable under LM, formed by an anterior band of small teeth of variable thickness at the base of the buccal lamellae, sometimes reduced to a single row of teeth (Figures 1 and 3); a posterior band of larger teeth, sometimes in a single row, followed by three dorsal and three ventral fine transverse ridges (Figures 1 and 3). Ventral transversal ridges clearly smaller than the dorsal, lateral ridges larger than the middle ones (Figure 1). Long ventral strengthening bar, about 65% of buccal tube total length, with an anterior crest on its ventral margin (Figure 1). Stylet support insertion point posteriorly placed, at about 75% the distance of the buccal tube from the anterior margin of the stylet sheaths. Anterior end of the piercing stylet with external edge straight and serrate, and internal edge curved and smooth (Figure 3). Pharyngeal bulb oval to circular, with well-developed pharyngeal apophyses. Two elongate macroplacoids and a small elongate microplacoid close to the second macroplacoid. First macroplacoid the longest with a small medial constriction, second macroplacoid quadrangular or circular (Figure 1). Slanting pharyngeal bars present between the apophyses and the first macroplacoids (Figure 1). Long double-claws. Main branch long with two fine accessory points, not reaching the distal bend of the main branch, and not clearly visible at LM (Figure 2). Secondary branch long and evidently bended. Short and large common tract, without distal part, and with an evident stalk connecting the claw to the lunule (Figure 2). Hind claws larger than the others. Lunules smooth in legs I III, larger and with weakly crenate edges in the hind legs (Figure 2). Measurements of different animal cuticular structures, pt values and a descriptive statistical analysis are given in Table 2. Ornamented round egg laid free (Figures 4 and 5). Egg ornamentation formed by processes in the shape of elongated empty cones (height µm, diameter of their base µm) with truncated and enlarged apexes (diameter less than 2 µm) (Figures 4, 5). Several processes with a long thick filament developing from the distal disk (Figures 4, 5). Ornamentations with smooth surface frequently ringed. Diameter of eggs without processes µm, diameter of eggs with processes

10 8 R. Guidetti et al. Figure 1. Macrobiotus kristenseni sp. nov. (A) Habitus; (B) anterior portion of the animal; (C) bucco-pharyngeal apparatus in ventral view; (D) dorsal and (E) ventral transversal crests of the buccal armature; (F) buccal pharyngeal apparatus in lateral view. (A C) holotype (phase contrast); (D F) paratypes (differential interference contrast). Scale bars: A = 50 µm; B, C, F, = 20 µm; D, E = 5 µm µm. Number of processes present on a diameter Chorion smooth; in some eggs a fine granulation and very small scattered pores in between processes visible by LM (Figure 4). Three embryonated eggs were found (Figure 4). In addition to the typical eggs described above, few eggs present slightly higher and more flexible processes internally septated and with a smaller base (Figures 4, 5). Type locality South America, Argentina, La Pampa Province, city of Santa Rosa (36 39 S, W) at 177 m above sea level, epiphytic lichens.

11 Journal of Natural History 9 Figure 2. Macrobiotus kristenseni sp. nov. (A) Claws of the hind pair of legs; (B) claws of the hind legs and cuticular dots on the external surface of the leg (arrow); (C) cuticular pores (arrow) in the body cuticle; (D) drawing of the claws of the hind legs; (E) claws of the second pair of legs; (F) claws of a hind leg. A C: scanning electron micrographs; E, F: holotype (phase contrast). Scale bars: A C = 5 µm; E, F = 10 µm. Type material Holotype (MLP-Oi 3726), four paratypes (MLP-Oi 3727) and three eggs are housed at the Museum of Natural Sciences, National University of La Plata (Argentina), and 32 paratypes are housed in J. Peluffo s personal collection in the Department of Natural Sciences at the National University of La Pampa (Argentina). The voucher specimens (two pictures of the hologenophore animals, 16 cryoconserved paragenophore animals, eight paratypes/paragenophore animals and eggs) and a fragment of the lichen are deposited in the MOD:BRT collection of the Department of Life Sciences (University of Modena and Reggio Emilia, Italy) registered in the Registry of Biological Repositories (

12 10 R. Guidetti et al. Figure 3. Macrobiotus kristenseni sp. nov. (A) Most anterior end of the animal bearing the mouth opening, regular ring of pores (arrow), buccal sensory lobes (L), peribuccal lamellae (asterisk); (B) mouth opening with buccal lamellae (asterisk) and buccal armature: first band of teeth (arrow head), second band of teeth (arrow); (C) mouth opening with buccal lamellae (asterisk) and buccal armature: second band of teeth (arrow head), transversal crests (arrow); (D) mouth opening with buccal lamellae (asterisk) and protruding piercing stylets (ps). A D: scanning electron micrographs. Scale bars: A, D = 5 µm; B, C = 2 µm. Additional material South America, Argentina, La Pampa Province, city of General Pico at 143 m above sea level (125 km northeast of Santa Rosa) and Natural Reserve Parque Luro (35 km south of Santa Rosa, caldén forest), in all cases on epiphytic lichens. Etymology The name is dedicated in honour of Prof. Reinhardt M. Kristensen, of the Invertebrate Department of The Natural History Museum of Denmark of the University of Copenhagen. Remarks Under SEM, the general surface of the cuticle appears slightly wrinkled (Figure 2). It should be verified whether this character is an effect of SEM preparation. An egg

13 Journal of Natural History 11 Figure 4. Macrobiotus kristenseni sp. nov. (A) Embryonated eggs; (B) egg surface with small rounded meshes (arrow) bearing elongated conical processes; (C) egg with embryo buccopharyngeal apparatus in development (arrow); (D) egg processes with filamentous matter developing from their tips (arrow). A D: phase contrast. Scale bars: A C = 20 µm; D = 5 µm. observed by SEM had processes with filamentous strips on their surface (Figure 5). These strips seem to be present on the same side of all processes: it is not clear whether they are artefacts or real morphological structures. Where M. kristenseni sp. nov. appears in a sample, it is either the only or the dominant tardigrade species. In the latter case, it is accompanied by one or more of the following species: Echiniscus rufoviridis du Bois Raymond-Marcus, 1944; Milnesium cf. tardigradum; Ramazzottius cf.oberhaeuseri. The phorophyte of the lichen communities in which it was recorded more frequently is the autochthonous tree species Prosopis caldenia (Burk.). Taxonomic remarks. Macrobiotus kristenseni sp. nov. is similar to species of the M. hufelandi group. Among these species, Macrobiotus sandrae Bertolani and Rebecchi, 1993 is the most similar, sharing a weak buccal armature, reduced ventral

14 12 R. Guidetti et al. Figure 5. Macrobiotus kristenseni sp. nov. (A) Egg; (B) egg processes with filamentous matter developing from their tips (arrows); (C, D) egg processes with annulated surface (arrow) and filamentous matter developing from their tips (arrow head). A D: scanning electron micrographs. Scale bars: A = 10 µm; B D = 2 µm. transversal crests, and indented lunule only on the fourth leg pair, but it differs from M. kristenseni sp. nov. by longer claw accessory points and larger dorsal transversal crests. Macrobiotus kristenseni sp. nov. differs from all the M. hufelandi group of species in that the shape of the egg processes is different from any other egg of the species group. The most similar eggs are those of Macrobiotus recens Cuénot, 1932, which share similar thin, long, conical processes with a truncated apex but in M. recens an evident series of small indentations around the base of the egg processes is present (Pilato and Bertolani 2004). Molecular results The molecular phylogeny based on the 18S rrna gene (897 bp) indicates that M. kristenseni sp. nov. does not share its 18S rrna sequence with any other Macrobiotus species sequenced so far (Figure 6), but it is in a sister-group relationship with Macrobiotus sapiens Binda and Pilato, 1984 within a well-supported clade grouping all the M. hufelandi group of species (i.e. M. sapiens, Macrobiotus hufelandi C.A.S. Schultze, 1834, Macrobiotus polonicus Pilato, Kaczmarek, Michalczyk and Lisi, 2003, Macrobiorus vladimiri Bertolani, Biserov, Rebecchi and Cesari, 2011).

15 Journal of Natural History 13 Figure 6. Unrooted neighbour joining dendrogram computed on Macrobiotus hufelandi group cox1 sequences. Bootstrap percentages computed after 2000 replicates are shown above branches. Macrobiotus kristenseni sp. nov. specimens are shown in bold. Acronyms as in Table 1.

16 14 R. Guidetti et al. Table 3. Kimura two-parameter distances computed among all newly analysed specimens (below the diagonal), and their standard error (above the diagonal). Specimen C3291 A01-V C3291 A02-V C3291 A C3291 A C3291 A Acronyms as in Table 1. Figure 7. Bayesian inference dendrogram computed on 18S rrna sequences. Numbers near nodes indicate posterior probability. Macrobiotus kristenseni sp. nov. specimen is shown in bold (C3291 A02-V2). Molecular analysis of the mitochondrial DNA cox1 gene was carried out on sequences of bp. Two different sequences (i.e. two haplotypes) are present in the five specimens analysed: three specimens are of one haplotype, two specimens are of the other (Table 3; Figure 7). The two haplotypes are very similar and the intraspecific genetic distance among M. kristenseni sp. nov. specimens is low ( %; Table 3). Mean K2P distances between specimens pertaining to different species are very high

17 Journal of Natural History 15 Table 4. Mean Kimura two-parameter distances computed among different species pertaining to the Macrobiotus hufelandi group (below the diagonal), and their standard errors (above the diagonal), and within each species (column D). Species D 1 M. kristenseni ± M. terminalis ± M. vladimiri ± M. sandrae ± M. hufelandi ± M. cf. hufelandi sp ± M. macrocalix ± M. cf. terminalis NP Theanalysis was carriedout ona 624-base-pair dataset, comprisingallm. hufelandi group cox1 sequences available (NP: not possible, only one sequence was present). ( %; Table 4). In particular, among the considered species, M. kristenseni sp. nov. has the greatest genetic difference with respect to the other M. hufelandi group of species ( %), while the difference among the considered species excluding M. kristenseni sp. nov. is %. The neighbour-joining dendrogram based on cox1 sequences confirms these differences between the two M. kristenseni sp.nov.haplotypes, and the differences among this species and the other species of M. hufelandi group (Figure 7). Discussion The integrative taxonomic approach used to define the species M. kristenseni sp. nov. demonstrated that this species has good support from both morphological (peculiar egg shape) and molecular (genetic distance) points of view. The presence of a standardized defined sequence for this species, together with the presence of voucher specimens, allowed the barcoding of the species for better future taxonomic identifications (Taylor and Harris 2012). Macrobiotus kristenseni sp. nov. belongs to the hufelandi group of species from both molecular and morphological points of view. The animals of M. kristenseni sp. nov. exhibit the characteristic features of the hufelandi group: two bands of small teeth in the buccal armature, two macroplacoids and a microplacoid positioned close to the second macroplacoid, and pearls (pores) on the cuticular surface. However, the egg morphology is quite peculiar: generally species of the hufelandi group have eggs with processes in the shape of inverted goblets, while M. kristenseni sp. nov. has long thin cones with truncated and enlarged apexes. This process shape deviates significantly from the classical goblet-like form, but it could be viewed as a derivation of a very elongated inverted goblet-like process, e.g. a step further of the egg process shape of Macrobiotus diversus Biserov, 1990 (species belonging to the hufelandi group). The large genetic distances (cox1 sequences) found between M. kristenseni sp. nov. and the other M. hufelandi group of species could be explained by the fact that all of these species have been collected in Europe, whereas M. kristenseni sp. nov. represents the only analysed species that probably developed in a different continent (South America).

18 16 R. Guidetti et al. From the molecular and morphological results of this study and previous research (Bertolani et al. 2011; Cesari et al. 2011; Guil and Giribet 2012), the M. hufelandi group of species has to be considered not just a taxonomic group created for convenience but a true biological entity, supported by morphological and molecular data. During the evolution of the hufelandi group phylogenetic line the general shape of the animals seems to have remained very conserved, while the egg shape may have been more subject to morphological variation, as testified for example by the different egg morphologies recorded among M. kristenseni sp. nov., M. hufelandi (type species of the genus and of the group), M. recens, M. diversus, Macrobiotus dariae Pilato and Bertolani, 2004, Macrobiotus denticulus Dastych, 2002, and Macrobiotus kazmierskii Kaczmarek and Michalczyk, In fact, the egg shape (e.g. diameter, process shapes and sizes, chorion surface) is a fundamental character for taxonomic identification because it is the most variable character among the hufelandi group of species, as already pointed out by Bertolani and Rebecchi (1993) in their revision of the group. According to our findings the M. hufelandi group of species should be defined as a group of species phylogenetically related, and characterized by animals with two bands of small teeth in the buccal armature, with two macroplacoids and a microplacoid positioned close to the second macroplacoid within the pharynx, and pearls (pores) on the cuticular surface. To further define the M. hufelandi group more in-depth molecular analyses should be devoted to the barcoding of the species whose animals exhibit these characters. The results of this study are particularly important because they define a new animal species and underline the biological validity of a well-defined evolutionary line of tardigrades (M. hufelandi group of species) that can be considered one of the more widespread lineages in continental environments. Moreover, the results of this study are particularly significant because they are aimed at a habitat (the urban environment) and at a region (South America) that are under-represented in tardigrade studies. Acknowledgements Special thanks to Prof. Paul Bartels (Warren Wilson College, Swannanoa, NC, USA) for the English revision of the manuscript, and to the Laboratory of Sequencing Labgen (University of Modena and Reggio Emilia, Italy) for the sequencing service. We want also to thank Mrs Irene Doma who provided the material from Parque Luro. This work was partially supported by funds from the National University of La Pampa, Argentina. References Bertolani R, Rebecchi L A revision of the Macrobiotus hufelandi group (Tardigrada, Macrobiotidae), with some observations on the taxonomic characters of eutardigrades. Zool. Scripta. 22: Bertolani R, Rebecchi L, Giovannini I, Cesari M 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: Cesari M, Bertolani R, Rebecchi L, Guidetti R DNA barcoding in Tardigrada: the first case study on Macrobiotus macrocalix Bertolani & Rebecchi 1993 (Eutardigrada, Macrobiotidae). Mol. Ecol. Res. 9:

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