European Journal of Taxonomy 452: 1–36 ISSN 2118-9773 https://doi.org/10.5852/ejt.2018.452 www.europeanjournaloftaxonomy.eu 2018 · Stec D. et al. This work is licensed under a Creative Commons Attribution 3.0 License. Research article urn:lsid:zoobank.org:pub:76171A51-C284-455F-9504-C4CCEFE42D9D STEC D. et al., Tardigrades from the Canary Islands European Journal of Taxonomy 452: 1–36 (2018) 3 1 2 Integrative description of Macrobiotus canaricus sp. nov. with notes on M. recens (Eutardigrada: Macrobiotidae) Daniel STEC 1,*, Łukasz KRZYWAŃSKI 2 & Łukasz MICHALCZYK 3 1,2,3 Department of Entomology, Institute of Zoology and Biomedical Research, Jagiellonian University, Gronostajowa 9, 30-387 Kraków, Poland. * Corresponding author: daniel_stec@interia.eu 2 Email: lukasz.s.krzywanski@gmail.com 3 Email: LM@tardigrada.net 1 urn:lsid:zoobank.org:author:C148C15D-9F12-4312-9702-B1E1513C63CC 2 urn:lsid:zoobank.org:author:0B32C511-BC10-4946-8191-7623AF798C65 3 urn:lsid:zoobank.org:author:6592787E-AAB1-4717-90C9-B967488F06F4 Abstract. In this paper we describe Macrobiotus canaricus sp. nov., a new tardigrade species of the Macrobiotus hufelandi group from the Canary Islands. Moreover, with the use of DNA sequencing, we confirm that Macrobiotus recens Cuénot, 1932 represents the hufelandi group, even though eggs laid by this species do not exhibit the typical hufelandi group morphology. Our study is based on both classical taxonomic methods that include morphological and morphometric analyses conducted with the use of light and scanning electron microscopy, and on the analysis of nucleotide sequences of four molecular markers (three nuclear: 18S rRNA, 28S rRNA, ITS-2, and one mitochondrial: COI). Our analyses revealed that M. canaricus sp. nov. is most similar to Macrobiotus almadai Fontoura et al., 2008 from the Archipelago of the Azores, from which it differs by the absence of granulation patches on the external and internal surfaces of legs I–III as well as by the absence of a cuticular pore in the centre of the external patch on legs I–III. Molecular sequences allowed us to pinpoint the phylogenetic positions of M. canaricus sp. nov. and M. recens within the M. hufelandi group. Keywords. Canary Islands, COI, Macrobiotus hufelandi group, phylogeny, tardigrades. Stec D., Krzywański Ł. & Michalczyk Ł. 2018. Integrative description of Macrobiotus canaricus sp. nov. with notes on M. recens (Eutardigrada: Macrobiotidae). European Journal of Taxonomy 452: 1–36. https://doi.org/10.5852/ejt.2018.452 Introduction Tardigrades are a phylum of globally distributed microinvertebrates which inhabit terrestrial and marine habitats (Nelson et al. 2015). However, the majority of the over 1200 known species were found and described from terrestrial mosses and lichens (Guidetti & Bertolani 2005; Degma & Guidetti 2007; Degma et al. 2009–2017). Although tardigrade faunistic studies have been conducted for more than two centuries, many regions are still very poorly known in terms of tardigrade species composition. One such place is the Canary Islands (Spain), where only two studies on tardigrades have been performed until now (Heinis 1908; Guil & Guidetti 2005). In total, 13 taxa identified to species level have been reported from the Canary Islands: Echiniscus arctomys Ehrenberg, 1853, E. canadensis Murray, 1910, E. quadrispinosus Richters, 1902, E. mediantus Marcus, 1930, E. trisetosus Cuénot, 1932, Isohypsibius tuberculatus (Plate, 1888), Macrobiotus echinogenitus Richters, 1904, M. furcatus Ehrenberg, 1859, M. hufelandi C.A.S. Schultze, 1834, M. occidentalis Murray, 1910, Milnesium tardigradum Doyère, 1840, Minibiotus intermedius (Plate, 1888) and M. gumersindoi Guil & Guidetti, 2005. Moreover, both studies reported undetermined species of the Macrobiotus hufelandi group and Heinis (1908) noted an undetermined species of Echiniscus C.A.S. Schultze, 1840. However, not all of the thirteen species identifications can be considered certain according to modern taxonomic standards. For example, records of M. hufelandi and M. tardigradum predate the redescriptions of these species (Bertolani & Rebecchi 1993 and Michalczyk et al. 2012, respectively), and thus they need to be verified against modern literature. We examined moss and lichen samples from the island of Gran Canaria. In two of them, we found two species of the Macrobiotus hufelandi group, one new to science and the other conforming to the description of Macrobiotus recens Cuénot, 1932. For both these populations, we provide taxonomic descriptions based on morphological and morphometric data acquired with phase contrast light microscopy (PCM) as well as from scanning electron microscopy (SEM). Additionally, we sequenced four DNA markers, three nuclear (18S rRNA, 28S rRNA, ITS-2) and one mitochondrial (COI), that allowed us to pinpoint the phylogenetic positions of the two species. Specimens of M. cf. recens conform to the diagnosis of the hufelandi group, but they lay eggs that depart from the typical hufelandi group egg ornamentation, i.e., instead of inverted goblet-shaped processes, the eggs are equipped with conical processes. Thus, the species either has not (Biserov 1990a, 1990b) or has (Bertolani & Rebecchi 1993; Kaczmarek & Michalczyk 2017b) been classified within the hufelandi group. Therefore, the first DNA sequences for M. recens allowed us to verify whether the species represents the hufelandi group. Material and methods Sample processing and tardigrade culturing The moss and lichen samples containing the new species and M. cf. recens, respectively, were collected from two localities in Gran Canaria by Marta Kapała on 16 February 2017 (see Table 1 for details). Samples were examined for terrestrial tardigrades using standard methods described by Dastych (1980) with modification by Stec et al. (2015). A total of 21 individuals of the new species and 29 individuals of M. cf. recens were extracted from the first and the second sample, respectively. Live animals were placed in a culture to obtain more individuals and eggs required for integrative analyses (see Table 1 for details). Animals were reared on plastic Petri dishes according to the protocol in Stec et al. (2015). Microscopy and imaging Specimens for light microscopy were mounted on microscope slides in a small drop of Hoyer’s medium and secured with a cover slip, following the protocol in Morek et al. (2016b). Slides were then dried for five to seven days at 60 °C. Dried slides were sealed with a transparent nail polish and examined under a Nikon Eclipse 50i phase contrast light microscope (PCM) associated with a Nikon Digital Sight DS-L2 digital camera. In order to obtain clean and extended specimens for SEM, tardigrades were processed according to the protocol in Stec et al. (2015). In short, specimens were first subjected to a 60 °C water bath for 30 min to obtain fully extended animals, followed by a water/ethanol and an ethanol/acetone series, and then CO2 critical point drying and finally sputter coated with a thin layer of gold. Specimens were examined under high vacuum in a Versa 3D DualBeam Scanning Electron Microscope at the ATOMIN facility of the Jagiellonian University, Kraków, Poland. Both studied populations were also examined for the presence of males with aceto-orcein staining (Bertolani 1971) in accordance with Stec et al. (2016a). All figures were assembled in Corel Photo-Paint X6, v. 16.4.1.1281. For structures that could not be satisfactorily focused in a single photograph, a stack of 2–8 images was taken with an equidistance of ca 0.2 μm and assembled manually into a single deep-focus image. Morphometrics and morphological nomenclature All measurements are given in micrometres (μm). Sample size was adjusted following recommendations by Stec et al. (2016b). Structures were measured only if their orientation was suitable. Body length was measured from the anterior extremity to the end of the body, excluding the hind legs. The terminology used to describe oral cavity armature and egg shell morphology follows Michalczyk & Kaczmarek (2003) and Kaczmarek & Michalczyk (2017b). Macroplacoid length sequence is given according to Kaczmarek et al. (2014). Buccal tube length and the level of the stylet support insertion point were measured according to Pilato (1981). The pt index is the ratio of the length of a given structure to the length of the buccal tube expressed as a percentage (Pilato 1981). All other measurements and nomenclature are given in accordance with Kaczmarek & Michalczyk (2017b). In brief, the buccal tube width was measured as the external and internal diameter at the level of the stylet support insertion point. Lengths of the claw branches were measured from the base of the claw (i.e., excluding the lunula) to the top of the branch, including accessory points. Distance between egg processes was measured as the shortest line connecting base edges of the two closest processes. Morphometric data were handled using the ‘Parachela’ v. 1.3 template available from the Tardigrada Register (Michalczyk & Kaczmarek 2013). Tardigrade taxonomy follows Bertolani et al. (2014). Comparative material The taxonomic key for the hufelandi group provided by Kaczmarek & Michalczyk (2017b) was used to determine whether the isolated species had previously been described. After one species could not be identified with the key, we compared it with the original description of the most similar species of the hufelandi group, Macrobiotus almadai Fontoura et al., 2008. Additionally, we also compared our new species with three paratypes and one egg of M. almadai kindly loaned to us by Professor Paulo Fontoura. Moreover, thanks to the courtesy of Matteo Vecchi, we analysed photomicrographs of animals and eggs identified as ‘M. recens’ by Maucci (1979) from several Portuguese populations deposited in the Maucci collection. Although our population conformed to the original description of M. recens, the description is outdated and, in our opinion, an accurate identification of the species is not possible until a modern redescription is available (see Discussion for more details). Therefore, we identified our population as M. cf. recens. Nevertheless, given that our population fits the description of M. recens, it has to represent at least a closely related species and, as such, it can be used to estimate the phylogenetic position of M. recens s.str. Genotyping The DNA was extracted from individual animals following the Chelex® 100 resin (Bio-Rad) extraction method of Casquet et al. (2012) with modifications described in detail in Stec et al. (2015). We sequenced four DNA fragments, three nuclear (18S rRNA, 28S rRNA, ITS-2) and one mitochondrial (COI). All fragments were amplified and sequenced according to the protocols described in Stec et al. (2015); primers and original references for specific PCR programs are listed in Table 2. Sequencing products were read with the ABI 3130xl sequencer at the Molecular Ecology Lab, Institute of Environmental Sciences of the Jagiellonian University, Kraków, Poland. Sequences were processed in BioEdit v. 7.2.5 (Hall 1999) and submitted to GenBank. Comparative molecular analysis For molecular comparisons, all published sequences of the four above-mentioned markers for species of the hufelandi complex were downloaded from GenBank (listed in Table 3). Additionally, we also sequenced the four DNA fragments for a new population of Macrobiotus macrocalix Bertolani & Rebecchi, 1993 collected in Łękawica, southern Poland by DS in October 2015 (see Table 1 for sample details). The sequences were aligned using the default settings (in the case of COI) and the Q-INS-I method (in the case of ribosomal markers: 18S rRNA, 28S rRNA and ITS-2) of MAFFT v. 7 (Katoh et al. 2002; Katoh & Toh 2008) and manually checked against non-conservative alignments in BioEdit. Then, the aligned sequences were trimmed to 763 (18S rRNA), 712 (28S rRNA), 317 (ITS-2) and 618 (COI), bp. All COI sequences were translated into protein sequences in MEGA7 v. 7.0 (Kumar et al. 2016) to check against pseudogenes. According to the recommendation by Srivathsan & Meier (2012), uncorrected pairwise distances were calculated using MEGA7 instead of genetic distances corrected by Kimura 2 parameter model (K2P). Data deposition Raw morphometric measurements underlying the description of Macrobiotus canaricus sp. nov. are given in Supplementary Materials (SM.1) and are deposited in the Tardigrada Register (Michalczyk & Kaczmarek 2013) under www.tardigrada.net/register, whereas raw morphometric data underlying the description of the population of M. cf. recens are given in Supplementary Materials (SM.2). The DNA sequences for the type population are deposited in GenBank (https://www.ncbi.nlm.nih.gov/genbank). Uncorrected pairwise distances are given in Supplementary Materials (SM.3). Phylogenetic analysis To verify the phylogenetic positions of the new species and M. cf. recens, phylogenetic trees were constructed using (1) all macrobiotid 18S rRNA sequences available from GenBank, (2) concatenated 18S rRNA+28S rRNA+ITS-2+COI sequences of macrobiotid species for which at least three of these markers were sequenced, and (3) all published M. hufelandi group COI sequences. In addition to the sequences of the hufelandi group listed in Table 3, we also used sequences of other species of the family Macrobiotidae published so far: Sands et al. (2008), Guidetti et al. (2009), Guil & Giribet (2012), Bertolani et al. (2014), Mapalo et al. (2016), Vecchi et al. (2016), Zawierucha et al. (2016), Mapalo et al. (2017), Stec & Kristensen (2017), Stec et al. (2018b). The sequences of Milnesium variefidum Morek et al., 2016a and Mi. berladnicorum Ciobanu et al., 2014 from Morek et al. (2016a) were used as the outgroup in the first two analyses, whereas four species of Mesobiotus served as the outgroup in the third analysis: Me. ethiopicus Stec & Kristensen, 2017, Me. hilariae Vecchi et al., 2016, Me. insanis Mapalo et al., 2017 and Me. philippinicus Mapalo et al., 2016. The sequences were aligned using the default settings (in the case of COI) and with the Q-INS-I method (in the case of ribosomal markers: 18S rRNA, 28S rRNA and ITS-2) of MAFFT v. 7 (Katoh et al. 2002; Katoh & Toh 2008) and then edited and checked manually in BioEdit. The alignments of 18S rRNA and COI sequences were trimmed to 739 bp and 618 bp, respectively, whereas the aligned sequences that were used to construct the concatenated data matrix were trimmed to: 728 bp (18S rRNA), 754 bp (28S rRNA), 570 bp (ITS-2) and 621 bp (COI). The sequences were concatenated in SequenceMatrix (Vaidya et al. 2011). The concatenated data matrix comprises species for which at least three of the aforementioned molecular markers are available. This resulted in only two gaps within the matrix: for the 18S rRNA sequence of Me. insanis (sequence too short) and the ITS-2 sequence of Me. ethiopicus (sequence not available). Using PartitionFinder v. 2.1.1 (Lanfear et al. 2016) under the Bayesian Information Criterion (BIC), the best scheme of partitioning and substitution models were chosen for posterior phylogenetic analysis. We ran the analysis to test all possible models implemented in the program. As the COI is a protein coding gene, before partitioning, we divided our alignments of this marker into 3 data blocks constituting separated three codon positions. As best-fit partitioning scheme, PartitionFinder always suggested the retention of all predefined partitions separately. See Table 4 for specific substitution models suggested for all tested data sets and partitions. Bayesian inference (BI) marginal posterior probabilities were calculated for both data sets using MrBayes v. 3.2 (Ronquist & Huelsenbeck 2003). Random starting trees were used and the analysis was run for ten million generations, sampling the Markov chain every 1000 generations. An average standard deviation of split frequencies of < 0.01 was used as a guide to ensure the two independent analyses had converged. The program Tracer v. 1.7 (Rambaut et al. 2018) was then used to ensure Markov chains had reached stationarity and to determine the correct ‘burn-in’ for the analysis, which was the first 10% of generations. The ESS values were greater than 200 and a consensus tree was obtained after summarising the resulting topologies and discarding the ‘burn-in’. All final consensus trees were viewed and visualised by FigTree v. 1.4.3, available from http://tree.bio.ed.ac.uk/software/figtree. Abbreviations PCM = phase contrast light microscopy SEM = scanning electron microscopy IZiBB = Institute of Zoology and Biomedical Research, Jagiellonian University, Kraków, Poland Results Taxonomic account Phylum Tardigrada Doyère, 1840 Class Eutardigrada Richters, 1926 Order Parachela Schuster et al., 1980 Superfamily Macrobiotoidea Thulin, 1928 (in Marley et al. 2011) Family Macrobiotidae Thulin, 1928 Genus Macrobiotus C.A.S. Schultze, 1834 Macrobiotus canaricus sp. nov. urn:lsid:zoobank.org:act:AE3AAEA9-D20E-4917-8ECD-12B30DB514B6 Figs 1–7, Tables 5–6 Etymology The specific epithet refers to the Canary Islands, the place where the new species was found. Material examined (162 animals, 57 eggs) Specimens mounted on microscope slides in Hoyer’s medium (120 animals + 42 eggs), fixed on SEM stubs (20 + 15), processed for DNA sequencing (7 animals), and aceto-orcein staining (15 animals). Holotype SPAIN: Canary Islands, Gran Canaria, Fagajesto, 28°03′05″ N, 15°38′21″ W, moss on a tree trunk in a pine forest (slide IZiBB ES.004.04). Paratypes SPAIN: 127 specimens, same data as for holotype (slides IZiBB ES.004.01–24); 42 eggs, same data as for holotype (slides IZiBB ES.004.25–33). Description Animals (measurements and statistics in Table 5) Body white in adults, after fixation in Hoyer’s medium transparent (Fig. 1A). Eyes present both in live animals and in specimens mounted in Hoyer’s medium. Round and oval pores (0.4–0.7 μm in diameter), visible under PCM and SEM, scattered randomly on entire body cuticle (Fig. 2A–F), including external and internal surface of all legs (Fig. 2A–F). Extremely fine body granulation (granules 0.06–0.09 μm in diameter), visible only under SEM, present on the dorso-posterior cuticle (Fig. 2E–F). Granulation patches on external surface of legs I–III clearly visible both under PCM and SEM (Fig. 3A–B). Granulation patches on internal surface of legs I–III weakly visible under PCM but clearly visible under SEM (Fig. 3C–D, empty indented arrowheads). Single, large, oval pore present at centre of each external patch on legs I–III (Fig. 3A–B, filled flat arrowheads). Cuticular bulge, resembling pulvinus, present on internal surface of legs I–III (Fig 3C–D, filled indented arrowheads). This structure is visible only if legs are fully extended and correctly oriented on slide. Cuticular granulation on legs IV present and always clearly visible both under PCM and SEM (Fig. 3E–F). Mouth antero-ventral. Bucco-pharyngeal apparatus of the Macrobiotus type, with the ventral lamina and ten small peribuccal lamellae followed by six buccal sensory lobes (Fig. 4A–C). Under PCM, the oral cavity armature is of the maculatus type, i.e., only the third band of teeth is visible (Fig. 4A). Under SEM, the oral cavity is always composed of three bands of teeth (Fig. 4B–C). The first band of teeth is composed of numerous extremely small cones arranged in one or two rows, situated anteriorly in the oral cavity, on the basal part of the peribuccal lamellae (Fig. 4B–C, filled arrowhead). The second band of teeth is situated between the ring fold and the third band of teeth and consists of cones, clearly larger than those of the first band (Fig. 4B–C, empty arrowhead). The teeth of the third band are located within the posterior portion of the oral cavity, between the second band of teeth and the buccal tube opening (Fig. 4B–C). The third band of teeth is discontinuous and divided into a dorsal and a ventral portion. Under PCM, the dorsal teeth form a transversal ridge weakly divided into three teeth, whereas the ventral teeth appear as two separate lateral transverse ridges between which a roundish median tooth is visible (Fig. 4A). Under SEM, the dorsal teeth are divided into three separate teeth: one median and two lateral, the median tooth has a slightly serrated edge (Fig. 4B). The ventral teeth are also separated into one median and two lateral teeth (Fig. 4C). The medio-ventral tooth is much smaller than the medio-dorsal tooth (Fig. 4B–C). Pharyngeal bulb spherical, with triangular apophyses, two rod-shaped macroplacoids and a small microplacoid (Fig. 4A). The first and the second macroplacoids have a fine central and a subterminal constriction, respectively. The macroplacoid length sequence is 2 < 1. Claws Y-shaped, of the hufelandi type (Fig. 5A–D). Primary branches with distinct accessory points and with an evident stalk connecting the claw to the lunula (Fig. 5A–D). Lunulae under all claws smooth (Fig. 5A–D). Cuticular bars under claws absent but muscle attachments are visible under claws I–III (Fig. 5A, C, filled arrowhead). Eggs (measurements and statistics in Table 6) Laid freely, white, spherical or slightly oval (Figs 6A–B, 7A). The surface between processes of the hufelandi type, i.e., covered by a reticulum with very thin walls (Figs 6D–E, 7A–F). Peribasal meshes slightly larger and with slightly thicker walls compared to interbasal meshes (Figs 6D–E, 7B–F). The mesh diameter is always larger then mesh walls and nodes/knots (Figs 6D–E, 7B–F). The meshes are 0.3–1.0 μm in diameter, polygonal but with rounded edges. Under SEM, meshes deep and empty inside (Fig. 7C–F). Processes in the shape of inverted goblets with concave conical trunks and well-defined terminal discs (Figs 6C–F, 7A–F). Terminal discs strongly serrated, with a concave central area (Figs 6C–F, 7B–F). Sparse ultragranulation on the edges of terminal discs visible only under SEM (Fig. 7E–F). Three to five microgranules (0.25–0.30 μm in diameter), covered with ultragranulation, present in the centre of the terminal disc (visible only under SEM; Fig. 7B–F, empty arrowheads). Reproductive mode The examined population is dioecious (gonochoristic). Males were identified using aceto-orcein staining, which revealed testicles filled with spermatozoa. However, no morphological secondary sexual dimorphism, such as gibbosities on hind legs in males, was identified. DNA sequences We obtained sequences for all four of the above-mentioned molecular markers. The two conservative nuclear markers (18S rRNA, 28S rRNA) were represented by single haplotypes, whereas ITS-2 and COI exhibited three and two haplotypes, respectively. The p-genetic distance between the ITS-2 haplotypes ranged from 0.5 to 1.1% and between COI haplotypes it was equal to 1.3%. The 18S rRNA sequence (GenBank: MH063925) was 1033 bp long. The 28S rRNA sequence (GenBank: MH063934) was 721 bp long. The ITS-2 haplotypes 1–3 were 413 bp long (GenBank: MH063928, MH063929 and MH063930, respectively). The COI haplotypes 1–2 were 658 bp long (GenBank: MH057765 and MH057766, respectively). Phenotypic differential diagnosis By the oral cavity armature of the maculatus type and hufelandi type of egg shell ornamentation, smooth lunules under claws of all legs and granulation at least on legs IV, the new species is similar to M. almadai Fontoura et al., 2008, M. humilis Binda & Pilato, 2001, and M. rawsoni Horning et al., 1978, but can be differentiated specifically from: Macrobiotus almadai, known only from the Azores (Fontoura et al. 2008), by the presence of the external and the internal patch of granulation on legs I–III (legs I–III smooth in M. almadai) and by the presence of a single large pore in the centre of the external patch on legs I–III (occasionally, regular cuticular pores may be present on some legs, but such pores are small and never present on all legs in the same place in M. almadai). Macrobiotus humilis, reported only from its type locality in Sri Lanka (Binda & Pilato 2001), by the presence of three separated dorsal teeth of the third band (dorsal teeth fused into a single transversal ridge in M. humilis), the presence of a subterminal constriction in the second macroplacoid (second macroplacoid without constrictions in M. humilis), more posteriorly inserted stylet supports (pt = 74.3–76.8 in the new species vs pt = 71.1–71.3 in M. humilis), slightly higher pt of the second macroplacoid length (pt=14.6–19.6 in the new species vs pt = 12.5–14.4 in M. humilis) and by irregularly serrated edges of the terminal discs on egg processes (edges of terminal discs regularly indented in M. humilis). Macrobiotus rawsoni, known only from its type locality in New Zealand (Horning et al. 1978; Kaczmarek & Michalczyk 2017a), by the presence of granulation on all legs (granulation present only on legs IV in M. rawsoni), the presence of a subterminal constriction in the second macroplacoid (second macroplacoid without constrictions in M. rawsoni), the absence of cuticular bars under the claws on legs I–III (thin paired bars present in M. rawsoni), more anteriorly inserted stylet supports (pt = 74.3–76.8 in the new species vs pt = 77.0–77.1 in M. rawsoni), a different morphology of reticulation on the egg surface between processes (several lines of mesh between neighbouring egg processes in the new species vs two lines of mesh between neighbouring egg processes in M. rawsoni) and by a smaller mesh size in the chorion reticulum (0.3–1.0 μm in diameter in the new species vs 1.8–2.5 μm in diameter in M. rawsoni). Genotypic differential diagnosis The ranges of uncorrected genetic p-distances between the new species and species of the Macrobiotus hufelandi complex, for which sequences are available from GenBank, are as follows: • 18S rRNA: 0.5–3.7% (2.0% on average), being most similar to two undetermined species of the M. hufelandi group from Spain (FJ435738–9) and to M. macrocalix from Poland (MH063926) and the least similar to M. polypiformis Roszkowska et al., 2017 from Ecuador (KX810008) • 28S rRNA: 1.9–13.2% (6.1% on average), being most similar to three undetermined species of the M. hufelandi complex from Spain (FJ435751 and FJ435754–5) and the least similar to M. polypiformis from Ecuador (KX810009) • ITS-2: 5.3–27.8% (17.0% on average), with the most similar being M. macrocalix from Poland (MH063931) and the least similar being M. polypiformis from Ecuador (KX810010) • COI: 17.2–24.7% (19.2% on average), with the most similar being M. hannae Nowak & Stec, 2018 from Poland (MH057764) and the least similar being M. papei Stec et al., 2018 from Tanzania (MH057763) Macrobiotus cf. recens Cuénot, 1932 Figs 8–14, Tables 7–8 Material examined (114 animals, 48 eggs) SPAIN: Canary Islands, Gran Canaria, Presa de Lugarejos, 28°02′38″ N, 15°40′22″ W, 885 m a.s.l., lichen on a stone wall. Specimens mounted on microscope slides in Hoyer’s medium (87 animals + 38 eggs), fixed on SEM stubs (8+10), processed for DNA sequencing (4 animals) and used for aceto-orcein staining (15 animals). Slide depositories: 87 animals (slides: ES.006.*, where the asterisk can be substituted by the following numbers 1–4, 10–12, 14–17 ) and 38 eggs (slides: ES.006.*: 5–9, 13) (IZiBB). Description of the population from Gran Canaria Animals (measurements and statistics in Table 7) Body white in juveniles and slightly yellowish in adults, after fixation in Hoyer’s medium transparent (Fig. 8A). Eyes present in live animals and in specimens mounted in Hoyer’s medium. Elliptical and sometimes roundish pores (1.0–1.8 μm in diameter), visible under PCM and SEM, scattered randomly on entire body cuticle (Fig. 8B–E), including the external and internal surface of all legs (Fig. 9A–I). Inside pores several granules, visible only under SEM, always present (Fig. 8E). Granulation patch on external and internal surfaces of legs I–III present (Fig. 9A–E). Single pore present at centre of each external granulation patch (Fig. 9A–C). Granulation patch on external surface larger and more distinct than the one on internal surface (Fig. 9A–E). Faint cuticular fold present on external surface of legs I–III just above claws (Fig. 9A–B, empty arrowhead), whereas on internal surface of legs I–III there is a cuticular bulge resembling pulvinus (Fig. 9D–E, filled arrowhead). Both external fold and internal bulge visible only if legs are fully extended and correctly oriented on slide (particularly cuticular fold above claws). Granulation on legs IV always clearly visible and consists of two granulation patches: the distal patch with densely distributed granules situated just above claws and the proximal patch being wider with more sparsely distributed granules located immediately above distal patch (Fig. 9G–I). Mouth antero-ventral. Bucco-pharyngeal apparatus of the Macrobiotus type (Fig. 10A–C), with ventral lamina and ten small peribuccal lamellae followed by six buccal sensory lobes. Under PCM, oral cavity armature of the hufelandi type, i.e., with all three bands of teeth always visible (Fig. 10B–C). First band of teeth composed of numerous very small cones arranged in four to six rows situated anteriorly in oral cavity, just behind bases of peribuccal lamellae (Figs 10B–C, 11A–B, filled arrowhead). Second band of teeth situated between ring fold and third band of teeth and comprises 4–5 rows of small cones, slightly larger than those of first band (Figs 10B–C, 11A–B, empty arrowhead). Teeth of the third band located within posterior portion of oral cavity, between the second band of teeth and buccal tube opening (Figs 10B–C, 11A–B). Third band of teeth discontinuous and divided into dorsal and ventral portions. Under PCM, dorsal teeth seen as three distinct transversal ridges, whereas ventral teeth appear as two separate lateral transversal ridges and a roundish median tooth (Fig. 10B–C). Under SEM, both dorsal and ventral teeth clearly distinct (Fig.11A–B). Medio-ventral tooth rarely divided into two or three smaller teeth (Fig. 11B). Under SEM, margins of dorsal and latero-ventral teeth slightly serrated (Fig. 11A–B). Pharyngeal bulb spherical, with triangular apophyses, two rod-shaped macroplacoids and one big rod-shaped microplacoid (Fig. 10D–E). Macroplacoid length sequence 2 < 1. First and second macroplacoid with the central and the subterminal constriction, respectively (Fig. 10D–E). Claws Y shaped, of the hufelandi type (Fig. 12A–D). Primary branches with distinct accessory points and with an evident stalk connecting claw to lunula (Fig. 12A–D). Lunulae I–III smooth (Fig. 12A, C), whereas lunulae IV slightly crenulated (Fig. 12B, D). Faint cuticular bars under claws I–III present, more visible in larger specimens (Fig. 12A, C). Horseshoe-shaped structure connects anterior and posterior lunules (Fig. 12B). Eggs (measurements and statistics in Table 8) Laid freely, white/light yellow, spherical or slightly oval (Figs 13A–B, 14A). Surface between processes of the hufelandi type, i.e., chorion surface between processes covered by reticulum with very small meshes (Figs 13C–D, 14B–E). Under PCM, surface between processes seems to be covered by dark dots (Fig. 13C) and only sometimes a clear reticulation is visible (Fig. 13D). Several rows of meshes between egg processes (usually 7–8). Mesh borders and nodes/knots thick and sometimes wider than mesh diameter (Fig. 14B–E). Meshes circular or slightly oval (0.4–0.9 μm in diameter) and under SEM all pores empty inside (Fig. 14C–D). Mesh diameter decreases gradually from peribasal to interbasal meshes (Fig. 14C–D). Short thickenings radiating from process bases are often visible under PCM and always visible under SEM; thickenings may create a crown around process bases (Figs 13C, 14C–D, filled arrowheads). Processes in the shape of high and thin cones with straight conical trunks, devoid of terminal discs and sometimes bifurcated (Figs 13A–F, 14A–F). Trunks of processes slightly undulated. Undulations covered by numerous granules composed of microgranule aggregations. Undulations and granules poorly visible under PCM (Fig. 13C–F). Undulations, granules and microgranules always clearly visible under SEM (Fig. 14B, E–F). Reproductive mode The examined population is dioecious (gonochoristic). Males were identified using aceto-orcein staining, which revealed testicles filled with spermatozoa. However, no morphological secondary sexual dimorphism, such as gibbosities on hind legs in males, was identified. DNA sequences We obtained sequences for all four of the above-mentioned molecular markers. The two conservative nuclear markers (18S rRNA, 28S rRNA) were represented by single haplotypes, whereas both ITS-2 and COI exhibited two haplotypes. The p-genetic distance between the ITS-2 as well as between the COI haplotypes was 1.1%. The 18S rRNA sequence (GenBank: MH063927) was 1033 bp long, the 28S rRNA sequence (GenBank: MH063936) was 725 bp long, the ITS-2 haplotype 1 and 2 sequences (GenBank: MH063932 and MH063933, respectively) were 420 bp long; the COI haplotype 1 and 2 sequences (GenBank: MH057768 and MH057769, respectively) were 658 bp long. Genotypic differential diagnosis The ranges of uncorrected genetic p-distances between the new species and species of the Macrobiotus hufelandi complex, for which sequences are available from GenBank, are as follows: • 18S rRNA: 0.4–4.0% (2.0% on average), with the most similar being M. macrocalix from Poland (MH063926) and the least similar being M. polypiformis from Ecuador (KX810008) • 28S rRNA: 1.3–13.6% (6.5% on average), with the most similar being M. macrocalix from Poland (MH063935) and the least similar being M. polypiformis from Ecuador (KX810009) • ITS-2: 3.7–26.7% (16.9% on average), with the most similar being M. macrocalix from Poland (MH063931) and the least similar being M. polypiformis from Ecuador (KX810010) • COI: 16.4–24.7% (19.1% on average), with the most similar being M. macrocalix from Italy (FJ176203–7 and FJ176213–7) and the least similar being M. papei from Tanzania (MH057763) Molecular phylogeny Phylogenetic analyses conducted on macrobiotid 18S rRNA sequences as well as on the concatenated macrobiotid data set unambiguously confirmed that the two studied species represent the M. hufelandi group (Figs 15–16). The phylogeny based on the COI sequences of the hufelandi group also corroborated these results, since none of the two species were recovered external to the species of the hufelandi group (Fig. 17). In all analyses, two clades within the hufelandi group were present, although the species composition varied slightly between phylogenies based on different markers. One clade grouped exclusively species that exhibit modified egg processes (M. paulinae Stec et al., 2015, M. polypiformis, M. papei, M. shonaicus Stec et al., 2018a, M. scoticus Stec et al., 2017b and M. kristenseni Guidetti, Peluffo, Rocha, Cesari & Moly de Peluffo, 2013); ‘the kristenseni clade’ henceforth. The other clade comprised mostly species with typical inverted goblet-shaped egg processes (‘the hufelandi clade’ hereafter). In contrast to our predictions, M. cf. recens, with its atypical egg processes, was always embedded within the hufelandi clade. The two clades were well supported in phylogenies based on the concatenated data set and on COI sequences, but weakly supported in the 18S rRNA tree (Figs 15–17). Moreover, in the 18S rRNA analysis the kristenseni clade, in addition to the majority of species with modified egg processes, comprised M. sapiens Binda & Pilato, 1984 (DQ839601) and undetermined species of the M. hufelandi group (HQ604971), of which at least the first species exhibits the typical egg morphology. In contrast to other analyses, the 18S rRNA phylogeny recovered a clade, with X. pseudohufelandi (Iharos, 1966) and M. polonicus Pilato, Kaczmarek, Michalczyk & Lisi, 2003, that was in a sister relationship to all other species of the hufelandi group, suggesting that the hufelandi group is polyphyletic or that Xerobiotus belongs to the hufelandi group (Fig. 15). Discussion Thanks to the detailed morphological and molecular analyses, we were able to describe a new species M. canaricus sp. nov. and characterise a population of M. cf. recens, both collected on the Canary Islands. Moreover, by the use of phylogenetic inference, we confirmed the affinity of both studied species with the M. hufelandi group. Macrobiotus recens was described in 1932 by Lucien Cuénot, but soon after, some researchers started questioning the status of this species and classified it as ‘M. hufelandi forma recens’ (Marcus 1936; Ramazzotti 1945, 1962, 1972; Rudescu 1946; Grigarick et al. 1973). However, Maucci (1979) analysed several Portuguese populations he identified as M. recens morphologically and confirmed that the taxon is indeed a ‘bona species’. However, it should be noted that the locus typicus for M. recens is La Tardière, a commune in the Vendée department, in the Pays de la Loire region in western France. Thus, similarly to our study, the comparison presented by Maucci (1979) has to be treated with caution, as it may be based on a related species rather than M. recens s.str. Since the original description is outdated and incomplete, a confident identification of M. recens is currently not possible. Although Pilato & Bertolani (2004) supported the identification of the Portuguese populations studied by Maucci (1979) as M. recens, we think that without an integrative redescription of the species it is not possible to test whether M. recens is a single species or a complex of morphologically similar taxa. Thus, instead of designating the Canary Islands population as M. recens, we treat it as M. cf. recens, at the same time highlighting the need for a redescription based on new material from the locus typicus. It could be hypothesised that our Canarian population, as well as previous records of M. recens outside the type locality, i.e., from Romania (Rudescu 1946), Italy (Ramazzotti 1945), Greece (Marcus 1936), Portugal (Maucci 1979) and China (Rahm 1937; Bartoš 1963), may represent distinct taxa that could become identifiable only after a modern redescription of M. recens. This would not be surprising, as such a pattern has been observed before, for example, when nominal taxa in other groups were described: Macrobiotus hufelandi (redescribed by Bertolani & Rebecchi 1993 and Bertolani et al. 2011a), Milnesium tardigradum (redescribed by Michalczyk et al. 2012), Mesocrista spitsbergensis (redescribed by Gąsiorek et al. 2016), Hypsibius dujardini (redescribed by Gąsiorek et al. 2018) or Ramazzottius oberhaeuseri (redescribed by Stec et al. 2018c). Thus, in our opinion, all records of M. recens outside its type locality should be considered as uncertain identifications until a redescription of M. recens is available and at least some of the reports can be verified. Following this logic, if the record of M. cf. recens from the Canary Islands turns out to represent M. recens s.str., then this study will be an addition to our poor knowledge of the intraspecific variability in tardigrades. If, however, the current record represents a new species, then, with the data already presented in our study, only a new name will have to be proposed to erect a new species. Recently, Stec et al. (2018a) showed two well supported COI lineages within the M. hufelandi group: one clade grouping species with hufelandi-type egg processes in the shape of inverted goblets, and the other with modified egg process (conical processes or processes with filaments growing out of terminal discs). Thus, M. cf. recens, with its conical egg processes, should be expected to cluster with species with modified egg processes. However, in contrast to this prediction, M. cf. recens is embedded within the clade with species exhibiting typical egg morphology in our COI analysis (Fig. 17). Moreover, the same position of the species was recovered in the 18S rRNA phylogeny (Fig. 15) and in the analysis based on four concatenated markers (Fig. 16). Also, M. sapiens, with typical egg processes, should be present in the hufelandi clade, but in our analyses it clustered with species exhibiting mostly modified processes (Fig. 15). Importantly, however, the 18S rRNA sequence identified as M. sapiens (DQ839601; Schill & Steinbrück 2007) comes from Croatia, whereas the type locality of the species is in Sicily (Binda & Pilato 1984). Thus, given that there are no type/neotype sequences for M. sapiens and Schill & Steinbrück (2007) did not provide SEM photomicrographs of Croatian eggs, it is not possible to verify the identity of the DQ839601 sequence. In other words, the sequence may represent a similar species of the hufelandi group that may exhibit terminal disc filaments. Nevertheless, regardless of the phylogenetic position of M. sapiens, the affinity of M. cf. recens with species exhibiting typical egg processes shows that the morphological criterion proposed by Stec et al. (2018a) to distinguish the two clades is not universal. Therefore, more species representing the two clades need to be sequenced to elucidate the taxonomic status of the two clades. Another species whose phylogenetic position diverges from the predicted is M. polonicus. This species, with typical egg processes, should be embedded within the hufelandi clade. However, in the 18S rRNA phylogeny, not only does it not cluster within the hufelandi clade, but it clusters with Xerobiotus pseudohufelandi in a clade that is in a sister relationship to the entire hufelandi group. The M. polonicus + X. pseudohufelandi clade was also found to be a sister group to all other hufelandi group species by Bertolani et al. (2014). The genus Xerobiotus Bertolani & Biserov, 1996 shares a similar morphology of the buccal apparatus, egg shell and spermatozoa with species of the M. hufelandi group, but it differs from them by having strongly reduced claws. Given that the M. polonicus + X. pseudohufelandi clade contains only a single hufelandi group sequence, the clade could be a statistical artefact or possibly the 18S rRNA marker is too conservative to solve the relationships between the hufelandi group and the genus Xerobiotus. Therefore, it is crucial to analyse more populations of M. polonicus, other species of the persimilis subgroup (sensu Kaczmarek & Michalczyk 2017b) and species of the genus Xerobiotus, both in terms of the 18S rRNA marker and additional, more variable DNA fragments. If, however, more species of the hufelandi group turn out to cluster with the genus Xerobiotus, then the taxonomic status of the genus and of the hufelandi group should be reconsidered. Although our multilocus phylogeny supports the presence of two distinct evolutionary lineages within the hufelandi group, the taxon sample size is still very low and more precise conclusions concerning the phylogeny of the group cannot currently be made. Thus, to provide more reliable conclusions, much more effort should be made to obtain multilocus molecular data linked to morphology for a larger number of species and populations. Particularly, it would be beneficial to increase the sample size, both in terms of markers as well as species, for the M. polonicus + X. pseudohufelandi clade to test whether the genus Xerobiotus and the hufelandi group are monophyletic. Another clade which would benefit from an increased sample size is the clade that comprises species with modified egg processes. Currently, the clade consists of species with two very different process morphotypes, i.e., species having processes with flexible filaments on the terminal discs and species having conical processes devoid of terminal discs. Thus, it might be possible that increased sampling could reveal the presence of two further clades that differ by the egg process morphology. Nevertheless, our findings support the previous results that egg morphology seems to be evolving faster than animal morphology, which underlines the usefulness of chorion ornamentation in the delineation of closely related species (Guidetti et al. 2013; Stec et al. 2016a, 2017a, 2018a). Acknowledgments We are grateful to Marta Kapała for collecting the samples on the Canary Islands. We would also like to thank Professor Paulo Fontoura (University of Porto, Portugal) for lending us slides with M. almadai and to Matteo Vecchi (University in Modena, Italy), who kindly sent us the photos of specimens and eggs from Portugal identified by Maucci (1979) as M. recens and deposited in the Maucci collection. We are also grateful to Matteo Vecchi and an anonymous reviewer, whose comments helped to improve our work. We would like to thank the Aquatic Ecosystems Team from the Institute of Environmental Sciences (Jagiellonian University, Kraków) for providing us with the rotifers. 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Manuscript received: 10 April 2018 Manuscript accepted: 25 June 2018 Published on: 24 July 2018 Topic editor: Rudy Jocqué Desk editor: Kristiaan Hoedemakers Printed versions of all papers are also deposited in the libraries of the institutes that are members of the EJT consortium: Muséum national d’Histoire naturelle, Paris, France; Botanic Garden Meise, Belgium; Royal Museum for Central Africa, Tervuren, Belgium; Natural History Museum, London, United Kingdom; Royal Belgian Institute of Natural Sciences, Brussels, Belgium; Natural History Museum of Denmark, Copenhagen, Denmark; Naturalis Biodiversity Center, Leiden, the Netherlands; Museo Nacional de Ciencias Naturales-CSIC, Madrid, Spain; Real Jardín Botánico de Madrid CSIC, Spain; Zoological Research Museum Alexander Koenig, Bonn, Germany. Table 1. Sample collection details and numbers of individuals and eggs used for integrative analyses of M. canaricus sp. nov. and M. cf. recens Cuénot, 1932. (PCM = phase contrast microscopy; SEM = scanning electron microscopy; DNA = DNA sequencing; ORC = orcein staining) *Eggs were taken from the culture and incubated individually until hatching, then the juveniles were used for DNA sequencing whereas egg chorions (isogenophores) were mounted on microscope slides in Hoyer’s medium. Table 2. Primers and references for PCR protocols for amplification of the four DNA fragments sequenced in the study. Table 3. Sequences of species of the Macrobiotus hufelandi group used for molecular comparisons and the phylogenetic analyses of Macrobiotus canaricus sp. nov. and M. cf. recens Cuénot, 1932. Underlined GenBank accession numbers indicate type or neotype sequences, whereas bolded numbers indicate new sequences obtained in this study. Table 4. The best scheme of partitioning and substitution models chosen for posterior phylogenetic analysis using PartitionFinder v. 2.1.1 under the Bayesian Information Criterion (BIC). The analyses were run to test all possible models implemented in the program. Table 5. Measurements (in µm) of selected morphological structures of individuals of Macrobiotus canaricus sp. nov. mounted in Hoyer’s medium (N = number of specimens / structures measured; Range = the smallest and the largest structure among all measured specimens; SD = standard deviation) Table 6. Measurements (in µm) of selected morphological structures of the eggs of Macrobiotus canaricus sp. nov. mounted in Hoyer’s medium. (N = number of eggs / structures measured; Range = the smallest and the largest structure among all measured specimens; SD = standard deviation) Table 7. Measurements (in µm) of selected morphological structures of individuals of Macrobiotus cf. recens Cuénot, 1932 from the Canary Islands mounted in Hoyer’s medium. (N = number of specimens / structures measured; Range = the smallest and the largest structure among all measured specimens; SD = standard deviation) Table 8. Measurements (in µm) of selected morphological structures of the eggs of Macrobiotus cf. recens Cuénot, 1932 from the Canary Islands mounted in Hoyer’s medium. (N = number of eggs /structures measured; Range = the smallest and the largest structure among all measured specimens; SD = standard deviation) Species Sample code Coordinates; altitude Sample type, habitat PCM SEM DNA ORC M. canaricus sp. nov. ES.004 28°03′05″ N, 15°38′21″ W; 1050 m a.s.l. moss on a tree trunk in a pine forest 120 ind. 42 eggs 20 ind. 15 eggs 4 ind. 3 eggs* 15 ind. 0 eggs M. cf. recens ES.006 28°02′38″ N, 15°40′22″ W; 885 m a.s.l. lichen on a stone wall 87 ind. 38 eggs 8 ind. 10 eggs 0 ind. 4 eggs* 15 ind. 0 eggs M. macrocalix PL.110 49°31′27″ N, 21°02′23″ E; 254 m a.s.l. moss on a roof 60 ind. 72 eggs 0 ind. 0 eggs 4 ind. 0 eggs 0 ind. 0 eggs DNA fragment Primer name Primer direction Primer sequence (5’-3’) Primer source PCR programme 18S rRNA 18S_Tar_Ff1 forward AGGCGAAACCGCGAATGGCTC Stec et al. (2017a) Zeller (2010) 18S_Tar_Rr1 reverse GCCGCAGGCTCCACTCCTGG 28S rRNA 28SF0001 forward ACCCVCYNAATTTAAGCATAT Mironov et al. (2012) Mironov et al. (2012) 28SR0990 reverse CCTTGGTCCGTGTTTCAAGAC ITS-2 ITS2_Eutar_Ff forward CGTAACGTGAATTGCAGGAC Stec et al. (2018c) Stec et al. (2018c) ITS2_Eutar_Rr reverse TCCTCCGCTTATTGATATGC COI LCO1490 forward GGTCAACAAATCATAAAGATATTGG Folmer et al. (1994) Michalczyk et al. (2012) HCO2198 reverse TAAACTTCAGGGTGACCAAAAAATCA DNA marker Species Accession number Source 18S rRNA “M. hufelandi” Schultze, 1834 GQ849024 Giribet et al. (1996) M. hufelandi group species HQ604971, FJ435738–40 Bertolani et al. (2014), Guil & Giribet (2012) M. hannae Nowak & Stec, 2018 MH063922 Nowak & Stec (2018) “M. joannae” Pilato & Binda, 1983 [= M. hannae Nowak & Stec, 2018] HQ604974–5 Bertolani et al. (2014) M. kristenseni Guidetti et al., 2013 KC193577 Guidetti et al. (2013) M. macrocalix Bertolani & Rebecchi, 1993 HQ604976, MH063926 Bertolani et al. (2014), this study M. papei Stec et al., 2018 MH063881 Stec et al. (2018d) M. paulinae Stec et al., 2015 KT935502 Stec et al. (2015) M. polypiformis Roszkowska et al., 2017 KX810008 Roszkowska et al. (2017) M. polonicus Pilato et al., 2003 HM187580 Wełnicz et al. (2011) M. sapiens Binda & Pilato, 1984 DQ839601 Bertolani et al. (2014) M. scoticus Stec et al., 2017 KY797265 Stec et al. (2017b) M. shonaicus Stec et al., 2018 MG757132 Stec et al. (2018a) 28S rRNA M. hannae Nowak & Stec, 2018 MH063924 Nowak & Stec (2018) M. hufelandi group species FJ435751, FJ435754–5 Guil & Giribet (2012) M. macrocalix Bertolani & Rebecchi, 1993 MH063935 this study M. papei Stec et al., 2018 MH063880 Stec et al. (2018d) M. paulinae Stec et al., 2015 KT935501 Stec et al. (2015) M. polypiformis Roszkowska et al., 2017 KX810009 Roszkowska et al. (2017) M. scoticus Stec et al., 2017 KY797266 Stec et al. (2017b) M. shonaicus Stec et al., 2018 MG757133 Stec et al. (2018a) ITS-2 M. hannae Nowak & Stec, 2018 MH063923 Nowak & Stec (2018) M. macrocalix Bertolani & Rebecchi, 1993 MH063931 this study M. papei Stec et al., 2018 MH063921 Stec et al. (2018d) M. paulinae Stec et al., 2015 KT935500 Stec et al. (2015) M. polonicus Pilato et al., 2003 HM150647 Wełnicz et al. (2011) M. polypiformis Roszkowska et al., 2017 KX810010 Roszkowska et al. (2017) M. sapiens Binda & Pilato, 1984 GQ403680 Schill et al. (2010) M. scoticus Stec et al., 2017 KY797268 Stec et al. (2017b) M. shonaicus Stec et al., 2018 MG757134–5 Stec et al. (2018a) COI M. hannae Nowak & Stec, 2018 MH057764 Nowak & Stec (2018) M.cf. hufelandi, Schultze, 1834 HQ876589–94, HQ876596 Bertolani et al. (2011a) M. hufelandi, Schultze, 1834 HQ876584, HQ876586–8 Bertolani et al. (2011a) M. kristenseni Guidetti et al., 2013 KC193575–6 Guidetti et al. (2013) M. macrocalix Bertolani & Rebecchi, 1993 FJ176203–07, FJ176208–17, HQ876571, MH057767 Cesari et al. (2009), Bertolani et al. (2011a) this study M. papei Stec et al., 2018 MH057763 Stec et al. (2018d) M. paulinae Stec et al., 2015 KT951668 Stec et al. (2015) M. polypiformis Roszkowska et al., 2017 KX810011–2 Roszkowska et al. (2017) M. sandrae Bertolani & Rebecchi, 1993 HQ876566–67, HQ876569–70, HQ876562–73, HQ876574–83 Bertolani et al. (2011a) M. scoticus Stec et al., 2017 KY797267 Stec et al. (2017b) M. shonaicus Stec et al., 2018 MG757136–7 Stec et al. (2018a) M. terminalis Bertolani & Rebecchi, 1993 JN673960, AY598775 Cesari et al. (2011), Guidetti et al. (2005) M. vladimiri Bertolani et al., 2011 HM136931–2, HM136933–4, HQ876568 Bertolani et al. (2011a, 2011b) Data set Substitution model for the given partition 18S 28S ITS-2 COI-1st COI-2nd COI-3rd 18S SYM+I+G – – – – – COI – – – SYM+I+G GTR+I+G TRN+G 18S+28S+ ITS-2+COI SYM+G GTR+G SYM+G GTR+I+G GTR+I+G GTR+G Character N Range Mean SD Holotype µm pt µm pt µm pt µm pt Body length 30 306–431 902–1206 368 1094 30 72 359 1088 Buccopharyngeal tube           Buccal tube length 30 29.5–37.6 –   33.7 – 1.8 – 33.0 – Stylet support insertion point 30 22.4–28.1 74.3–76.8 25.4 75.4 1.3 0.7 24.6 74.5 Buccal tube external width 30 3.2–5.4 10.3–15.5 4.3 12.8 0.4 1.1 4.3 13.0 Buccal tube internal width 30 2.1–3.3 6.6–9.5 2.7 7.9 0.3 0.8 2.5 7.6 Ventral lamina length 28 16.2–24.4 53.4–68.2 20.5 60.8 1.5 3.7 19.5 59.1 Placoid lengths           Macroplacoid 1 30 5.5–9.5 17.7–28.4 7.3 21.5 1.0 2.4 8.0 24.2 Macroplacoid 2 30 4.6–7.1 14.6–20.5 5.8 17.2 0.6 1.4 6.0 18.2 Microplacoid 30 1.6–2.7 4.7–7.8 2.0 6.0 0.3 0.8 2.1 6.4 Macroplacoid row 30 11.5–17.5 36.8–52.0 14.1 41.8 1.4 3.1 14.4 43.6 Placoid row 30 14.0–19.4 42.6–55.3 16.6 49.2 1.5 3.0 17.0 51.5 Claw 1 lengths           External primary branch 30 8.3–11.3 25.1–33.5 9.5 28.3 0.7 2.0 8.3 25.2 External secondary branch 30 6.2–9.3 19.0–26.2 7.4 22.1 0.7 1.9 6.5 19.7 Internal primary branch 29 7.5–10.6 23.8–32.0 9.2 27.4 0.6 1.9 8.6 26.1 Internal secondary branch 29 6.0–8.4 17.4–23.8 7.0 20.7 0.6 1.7 6.6 20.0 Claw 2 lengths           External primary branch 30 8.6–11.8 25.3–35.6 9.8 29.3 0.7 2.2 9.0 27.3 External secondary branch 30 5.8–8.7 18.4–26.0 7.4 22.1 0.7 2.2 6.3 19.1 Internal primary branch 29 8.2–10.8 24.2–31.2 9.4 28.0 0.6 1.8 8.2 24.8 Internal secondary branch 29 5.4–8.6 15.9–25.1 7.2 21.5 0.6 2.0 7.4 22.4 Claw 3 lengths           External primary branch 28 8.9–11.0 25.8–32.6 10.0 29.8 0.6 1.9 9.2 27.9 External secondary branch 28 6.4–9.7 18.8–28.4 7.7 22.8 0.7 2.4 7.2 21.8 Internal primary branch 28 5.6–11.3 16.5–31.6 9.5 28.1 1.0 3.0 8.1 24.5 Internal secondary branch 28 6.0–8.9 16.8–25.7 7.3 21.7 0.7 1.9 6.9 20.9 Claw 4 lengths           Anterior primary branch 27 8.6–20.5 28.5–57.1 11.2 33.1 2.0 5.2 10.3 31.2 Anterior secondary branch 25 6.7–9.7 18.5–28.8 8.1 24.1 0.8 2.3 6.9 20.9 Posterior primary branch 26 9.6–13.2 28.3–37.6 11.2 33.2 0.8 2.7 10.5 31.8 Posterior secondary branch 23 6.6–9.5 19.4–27.3 8.1 24.0 0.9 2.3 7.4 22.4 Fig. 1. Macrobiotus canaricus sp. nov., holotype, habitus. A. Dorso-ventral projection (Hoyer’s medium, PCM). B. Dorsal view under SEM. Scale bars in μm. Fig. 2. Macrobiotus canaricus sp. nov., paratypes, body cuticle. A–B. Pores on the dorsal and ventral cuticle, respectively (PCM). C–D. Pores on the dorsal and ventral cuticle, respectively (SEM). E. Pores and fine granulation on the dorso-posterior cuticle (SEM). F. Close-up of a pore and granulation on the dorso-posterior cuticle (SEM). Scale bars in μm. Fig. 3. Macrobiotus canaricus sp. nov., paratypes, cuticular structures on legs. A–B. The patch of granulation on the external surface of leg III with a large oval cuticular pore at the centre of the patch (PCM and SEM, respectively). C–D. The patch of granulation and the cuticular bulge (pulvinus) on the internal surface of leg II (PCM and SEM, respectively). E–F. Granulation on leg IV (PCM and SEM, respectively). Filled flat arrowheads indicate the large pore in the centre of the external granulation patch, filled indented arrowheads indicate the cuticular fold (pulvinus) on the internal leg surface, and empty indented arrowheads indicate granulation on the internal leg surface. Scale bars in μm. Fig. 4. Macrobiotus canaricus sp. nov., paratypes, buccal apparatus and the oral cavity armature seen under PCM and SEM. A. Dorsal projection of the entire buccal apparatus; the lower insert shows a ventral view of the oral cavity armature, the upper insert shows macroplacoid morphology (PCM). B–C. The oral cavity armature of a single paratype seen under SEM from different angles. Filled arrowheads indicate the teeth of the first band, empty arrowheads indicate the teeth of the second band, median teeth are marked with M, andlateral teeth are marked with L. Scale bars in μm. Fig. 5. Macrobiotus canaricus sp. nov., paratypes, claws. A–B. Claws III and IV seen under PCM. C–D. Claws I and IV seen under SEM. Arrowheads indicate faint muscle attachments under claws; Figs A and B assembled from several photos. Scale bars in μm. Fig. 6. Macrobiotus canaricus sp. nov., egg seen under PCM. A. Midsection under 400 × magnification. B. Surface under 400 × magnification. C. Midsection under 1000 × magnification. D–E. Surface under 1000 × magnification. F. Outlines of terminal discs of egg processes under 1000 × magnification. Scale bars in μm. Fig. 7. Macrobiotus canaricus sp. nov., egg chorion morphology seen under SEM. A. Entire egg. B. Close up of the egg surface and processes. C–D. Details of the egg surface. E–F. Details of the terminal discs. Arrowheads indicate granules in the centre of the terminal discs. Scale bars in μm. Fig. 8. Macrobiotus cf. recens Cuénot, 1932, Gran Canaria, habitus and cuticular pores. A. Dorso-ventral projection (PCM). B–C. Pores on the dorsal and ventral cuticle, respectively (PCM). D. Pores on the dorsal cuticle (SEM). E. Granulation inside a single pore (SEM). Scale bars in μm. Fig. 9. Macrobiotus cf. recens Cuénot, 1932, Gran Canaria, structures on forelegs seen under PCM and under SEM. A–C. The patch of granulation on the external surface of leg II, with the cuticular pore at the centre of the patch and the cuticular fold above the claws. D–F. The patch of granulation and the cuticular bulge (pulvinus) on the internal surface of leg III. G–I. Granulation on leg IV. Filled flat arrowheads indicate the large pore at the centre of the external granulation patch, empty flat arrowheads indicate the cuticular fold above the claws on the external leg surface, filled indented arrowheads indicate the cuticular fold (pulvinus) on the internal leg surface, and empty indented arrowheads indicate granulation on the internal leg surface. Scale bars in μm. Fig. 10. Macrobiotus cf. recens Cuénot, 1932, Gran Canaria, buccal apparatus and the oral cavity armature seen under PCM. A. Dorsal projection of the entire buccal apparatus. B–C. Dorsal and ventral view of the oral cavity armature, respectively. D–E. Placoid morphology. Scale bars in μm. Fig. 11. Macrobiotus cf. recens Cuénot, 1932, Gran Canaria, oral cavity armature seen under SEM from different angles. A. View of the dorsal portion. B. View of the ventral portion. Filled arrowheads indicate the teeth of the first band, empty arrowheads indicate the teeth of the second band, median teeth are marked with M, and lateral teeth are marked with L. Scale bars in μm. Fig. 12. Macrobiotus cf. recens Cuénot, 1932, Gran Canaria, claws. A–B. Claws III and IV seen under PCM, with smooth and weakly crenulated lunules, respectively. C–D. Claws II and IV seen under SEM, with smooth and weakly crenulated lunules, respectively. A and B assembled from several photos. Filled arrowheads indicate faint cuticular bars under claws whereas empty arrowhead indicates a horseshoe-shaped structure. Scale bars in μm. Fig. 13. Macrobiotus cf. recens Cuénot, 1932, Gran Canaria, egg seen under PCM. A. Midsection under 400 × magnification. B. Surface under 400 × magnification. C–D. Surface under 1000 × magnification. E–F. Midsection under 1000 × magnification. Filled arrowheads indicate short thickenings radiating from the processes bases. Scale bars in μm. Fig. 14. Macrobiotus cf. recens Cuénot, 1932, Gran Canaria, egg chorion morphology seen under SEM. A. Entire egg. B. Close up of the egg surface and processes. C–D. Details of the egg surface. E–F. Details of process morphology. Filled arrowheads indicate short thickenings radiating from the process bases. Scale bars in μm. Fig. 15. The Bayesian Inference (BI) phylogeny constructed from 18S rRNA sequences of the family Macrobiotidae. Numbers at nodes indicate the Bayesian posterior probability. Species of the hufelandi group with typical and atypical egg processes are indicated by blue and red fonts, respectively. The X. pseudohufelandi + M. polonicus clade, which is in a sister relationship to all other species of the hufelandi group, is indicated by green branches. See Table 2 and the Phylogenetic analysis subsection in Material and methods for details on species sequences used in the analysis. The remaining macrobiotids are marked with black and the outgroup is marked with grey. Scale bar represents substitutions per position. Fig. 16. The Bayesian Inference (BI) phylogeny constructed from concatenated sequences (18S rRNA + 28S rRNA + ITS-2 + COI) of the family Macrobiotidae. Numbers at nodes indicate the Bayesian posterior probability. Species of the hufelandi group with typical and atypical egg processes are indicated by blue and red fonts, respectively. See Table 2 and the Phylogenetic analysis subsection in Material and methods for details on the species sequences used in the analysis. The remaining macrobiotids are marked with black and the outgroup is marked with grey. The scale bar represents substitutions per position. Fig. 17. The Bayesian Inference (BI) phylogeny constructed from COI sequences of the species of the Macrobiotus hufelandi group. Numbers at nodes indicate the Bayesian posterior probability. Species of the hufelandi group with typical and atypical egg processes are indicated by blue and red fonts, respectively. See Table 2 for details on the species sequences used in the analysis. The outgroup is marked with grey. The scale bar represents substitutions per position. Character N Range Mean SD Egg bare diameter 30 59.9–81.6 68.7 5.0 Egg full diameter 30 70.2–96.2 80.8 5.4 Process height 90 3.9–8.2 5.5 1.0 Process base width 90 3.3–7.5 5.3 0.8 Process base/height ratio 90 60%–160% 99% 19% Terminal disc width 90 3.4–7.5 4.8 0.8 Inter-process distance 90 1.5–5.5 3.4 0.8 Number of processes on the egg circumference 30 23–31 25.5 2.0 Character N Range Mean SD µm pt µm pt µm pt Body length 30 353–731 684–1174 576 1042 66 90 Buccopharyngeal tube           Buccal tube length 30 48.5–70.3 –   55.4 – 5.8 – Stylet support insertion point 30 39.3–57.2 79.1–83.5 44.7 80.8 4.9 1.0 Buccal tube external width 30 8.0–15.6 15.4–22.8 9.8 17.6 1.6 1.4 Buccal tube internal width 30 6.1–12.2 11.6–17.8 7.5 13.4 1.3 1.3 Ventral lamina length 30 24.9–42.3 47.3–63.8 32.9 59.6 3.2 4.2 Placoid lengths           Macroplacoid 1 30 9.4–19.4 18.1–30.5 13.3 24.0 2.3 2.6 Macroplacoid 2 30 8.2–17.0 15.9–24.8 10.6 19.0 2.0 1.8 Microplacoid 30 3.1–8.4 5.8–12.3 4.7 8.5 1.1 1.3 Macroplacoid row 30 12.5–39.1 24.1–57.1 25.4 45.7 5.1 6.2 Placoid row 30 17.3–52.6 33.4–76.8 32.7 58.7 6.1 6.3 Claw 1 lengths           External primary branch 30 12.3–18.0 23.1–32.7 14.6 26.6 1.5 2.4 External secondary branch 29 9.6–14.6 17.9–23.8 11.7 21.1 1.2 1.6 Internal primary branch 30 12.0–17.0 22.7–29.1 14.1 25.6 1.2 1.9 Internal secondary branch 30 8.2–13.7 15.7–24.5 11.1 20.1 1.3 2.4 Claw 2 lengths           External primary branch 30 12.8–18.3 23.2–31.6 15.0 27.2 1.3 2.3 External secondary branch 30 8.0–14.6 16.5–26.4 11.7 21.2 1.5 2.5 Internal primary branch 30 12.0–20.5 22.2–39.7 14.7 26.6 1.8 3.3 Internal secondary branch 30 8.5–14.9 15.5–25.6 11.2 20.4 1.6 2.7 Claw 3 lengths           External primary branch 30 13.3–18.2 23.9–33.7 15.2 27.6 1.2 2.4 External secondary branch 30 8.9–16.2 17.2–26.1 11.7 21.2 1.5 2.0 Internal primary branch 30 11.1–17.5 20.4–32.7 14.5 26.3 1.4 2.5 Internal secondary branch 30 9.1–15.1 16.7–29.2 11.5 20.8 1.3 2.5 Claw 4 lengths           Anterior primary branch 29 11.8–22.2 22.6–36.7 17.3 31.3 1.9 3.2 Anterior secondary branch 28 11.1–17.2 20.6–29.0 13.1 23.7 1.5 2.0 Posterior primary branch 27 14.5–23.4 28.0–38.9 18.1 32.7 1.6 2.5 Posterior secondary branch 26 11.1–17.4 20.2–32.0 13.7 24.6 1.6 3.1 Character N Range Mean SD Egg bare diameter 30 86.1–113.7 102.1 8.0 Egg full diameter 30 113.6–151.4 137.6 9.3 Process height 90 13.4–26.3 20.0 2.7 Process base width 90 3.2–10.7 6.3 1.4 Process base/height ratio 90 16%–45% 32% 6% Inter-process distance 90 3.7–74.0 6.6 7.3 Number of processes on the egg circumference 30 21–27 24.6 1.5