Macrobiotus cf. recens Cuénot, 1932

Stec, Daniel, Krzywański, Łukasz & Michalczyk, Łukasz, 2018, Integrative description of Macrobiotus canaricus sp. nov. with notes on M. recens (Eutardigrada: Macrobiotidae), European Journal of Taxonomy 452, pp. 1-36 : 17-26

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 acetoorcein 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. 8 B–E), including the external and internal surface of all legs ( Fig. 9 A–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. 9 A–E). Single pore present at centre of each external granulation patch ( Fig. 9 A–C). Granulation patch on external surface larger and more distinct than the one on internal surface ( Fig. 9 A–E). Faint cuticular fold present on external surface of legs I– III just above claws ( Fig. 9 A–B, empty arrowhead), whereas on internal surface of legs I–III there is a cuticular bulge resembling pulvinus ( Fig. 9 D–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. 9 G–I).

Mouth antero-ventral. Bucco-pharyngeal apparatus of the Macrobiotus type ( Fig. 10 A–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. 10 B–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 10 B–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 10 B–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 10 B–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. 10 B–C). Under SEM, both dorsal and ventral teeth clearly distinct ( Fig.11 A–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. 11 A–B). Pharyngeal bulb spherical, with triangular apophyses, two rod-shaped macroplacoids and one big rod-shaped microplacoid ( Fig. 10 D–E). Macroplacoid length sequence 2 <1. First and second macroplacoid with the central and the subterminal constriction, respectively ( Fig. 10 D–E).

Claws Y shaped, of the hufelandi type ( Fig. 12 A–D). Primary branches with distinct accessory points and with an evident stalk connecting claw to lunula ( Fig. 12 A–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 13 A–B, 14A). Surface between processes of the hufelandi type, i.e., chorion surface between processes covered by reticulum with very small meshes ( Figs 13 C–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. 14 B–E). Meshes circular or slightly oval (0.4–0.9 μm in diameter) and under SEM all pores empty inside ( Fig. 14 C–D). Mesh diameter decreases gradually from peribasal to interbasal meshes ( Fig. 14 C–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 , 14 C–D, filled arrowheads). Processes in the shape of high and thin cones with straight conical trunks, devoid of terminal discs and sometimes bifurcated ( Figs 13 A–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. 13 C–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 ).

Figures

Fig. 8. Macrobiotus cf. recens Cuénot, 1932, Gran Canaria, habitus and cuticular pores. A. Dorsoventral 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 horseshoeshaped 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.