Macrobiotus canaricus, Stec & Krzywański & Michalczyk, 2018

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 : 7-17

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. 2 A–F), including external and internal surface of all legs ( Fig. 2 A–F). Extremely fine body granulation (granules 0.06–0.09 μm in diameter), visible only under SEM, present on the dorso-posterior cuticle ( Fig. 2 E–F). Granulation patches on external surface of legs I–III clearly visible both under PCM and SEM ( Fig. 3 A–B). Granulation patches on internal surface of legs I–III weakly visible under PCM but clearly visible under SEM ( Fig. 3 C–D, empty indented arrowheads). Single, large, oval pore present at centre of each external patch on legs I–III ( Fig. 3 A–B, filled flat arrowheads). Cuticular bulge, resembling pulvinus, present on internal surface of legs I–III ( Fig 3 C–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. 3 E–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. 4 A–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. 4 B–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. 4 B–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. 4 B–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. 4 B–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. 4 B–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. 5 A–D). Primary branches with distinct accessory points and with an evident stalk connecting the claw to the lunula ( Fig. 5 A–D). Lunulae under all claws smooth ( Fig. 5 A–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 6 A–B, 7A). The surface between processes of the hufelandi type, i.e., covered by a reticulum with very thin walls ( Figs 6 D–E, 7A–F). Peribasal meshes slightly larger and with slightly thicker walls compared to interbasal meshes ( Figs 6 D–E, 7B–F). The mesh diameter is always larger then mesh walls and nodes/knots ( Figs 6 D–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. 7 C–F). Processes in the shape of inverted goblets with concave conical trunks and well-defined terminal discs ( Figs 6 C–F, 7A–F). Terminal discs strongly serrated, with a concave central area ( Figs 6 C– F, 7B–F). Sparse ultragranulation on the edges of terminal discs visible only under SEM ( Fig. 7 E–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. 7 B–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 )

Figures

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.