Macrobiotus polypiformis, Roszkowska & Ostrowska & Stec & Janko & Kaczmarek, 2017

Roszkowska, Milena, Ostrowska, Marta, Stec, Daniel, Janko, Karel & Kaczmarek, Łukasz, 2017, Macrobiotus polypiformis sp. nov., a new tardigrade (Macrobiotidae; hufelandi group) from the Ecuadorian Pacific coast, with remarks on the claw abnormalities in eutardigrades, European Journal of Taxonomy 327, pp. 1-19 : 6-12

Macrobiotus polypiformis sp. nov.

urn:lsid:zoobank.org:act:3858645A-2909-4DB9-82CD-CF2B4D7051C2

Figs 1–29 ; Tables 3–4

Etymology

The specific epithet ‘ polypiformis ’ refers to the similarity of the egg processes to the polyp form found in the phylum Cnidaria.

Material examined

Specimens mounted on microscope slides in Hoyer’s medium, fixed on SEM stubs or processed for DNA sequencing.

Holotype

ECUADOR: slide 1215/ 19, 3 Jan. 2015, Milena Roszkowska and Łukasz Kaczmarek leg. ( MECN).

Paratypes

ECUADOR: 135 animals (including 10 simplex), one exuvia and 44 eggs (including two with developed embryos), same data as holotype ( MECN, slide 1215 /19 (with holotype), 4 paratypes (slides: 1215 /*, where the asterisk can be substituted by any of the following numbers: 23, 25) and two eggs (slides: 1215 /*: 8, 9); DATE, 92 paratypes (slides: 1215 /*: 1, 2, 3, 10, 11, 12, 13, 14, 15, 16, 19, 20, 22, 24) and 24 eggs (slides: 1215 /*: 1, 4, 5, 7, 26, 27); ZMUC, 5 paratypes (slides 1215 /*: 17, 21) and 3 eggs (slide 1215 /6)).

Type locality

ECUADOR: Manabí Province, 1°04′06″ S, 89°52′18″ W; 370 m asl, moss sample from a concrete wall, next to E15 road, ca 3.5 km W from San Lorenzo, tropical rainforest.

Description

Animals (measurements and statistics in Table 3 )

Body white in juveniles and adults, transparent after fixation in Hoyer’s medium ( Figs 1–2 ). Eyes present (in 93% of measured specimens). Dorsal and ventral cuticle smooth under LM. Additionally, small oval and round pores (0.9–1.2 μm in diameter), sometimes difficult to observe under LM, are scattered randomly on the entire cuticle ( Figs 3–4 ). A ring of pores, difficult to observe under LM, is present around the mouth opening, below the peribuccal sensory lobes. One patch of fine and dense granulation above claws on legs I–IV present ( Figs 16–19 ).

Mouth antero-ventral. Bucco-pharyngeal apparatus of the Macrobiotus type ( Figs 5–6 ), with the ventral lamina and ten small peribuccal lamellae ( Figs 6 , 9 ) followed by six buccal sensory lobes. Oral cavity armature composed of three bands of teeth, of which only the third band is visible under LM ( Fig. 7 , empty arrowhead). SEM is required to reveal the first and the second bands of teeth (see Figs 8–9 ). The first band of teeth comprises extremely small cones arranged in 3–4 rows situated at the anterior portion of the oral cavity, at the base of the peribuccal lamellae ( Figs 8–9 , filled arrowheads). The second band of teeth is composed of 4–5 rows of small cones (but larger than those on the first band), positioned towards the rear of the oral cavity, between the ring fold and the third band of teeth ( Figs 8–9 , arrows). The teeth of the third band are positioned at the rear of the oral cavity, between the second band of teeth and the buccal tube opening ( Figs 7–9 , empty arrowheads).

Under LM, the teeth of the third band appear as a single, thin, transversal ridge both ventrally and dorsally ( Fig. 7 , empty arrowhead). Although SEM reveals that both ventral and dorsal teeth do indeed form continuous ridges, there are evident median (M) and lateral (L) peaks corresponding to the median and lateral teeth found in species with better developed oral cavity armatures ( Figs 8–9 ). Median teeth are smaller than the lateral teeth ( Fig. 8 ). In addition, there are a number of smaller accessory teeth (a) placed laterally to the lateral teeth. These accessory teeth are better developed ventrally than dorsally ( Fig. 8 ). Pharyngeal bulb spherical, with triangular apophyses, two rod-shaped macroplacoids and a triangular microplacoid ( Figs 5–6 ). The macroplacoid length sequence 1>2. The first macroplacoid with a central constriction ( Figs 5–6 , the filled arrowhead).

Claws small and slender of the hufelandi type ( Figs 10–14 ). Primary branches with distinct accessory points. Lunules on legs I–III smooth ( Figs 10, 13 ), those on legs IV dentate ( Figs 12, 14 ). Bars under claws absent but paired muscle attachments below claws I–III present (hardly visible under LM and only slightly more visible under SEM).

Eggs (measurements and statistics in Table 4 )

Laid freely, white to light yellow, spherical and with a hufelandi type chorion ornamentation ( Figs 20–28 ). The surface between processes is covered with a dense regular reticulum (mesh diameter 0.5– 0.8 μm) ( Figs 22 , 25–28 ). Processes in the shape of inverted goblets with slightly concave, conical, micro-granulated trunks and well-defined terminal discs ( Figs 22–23 , 25–26, 28 ). When observed under SEM, some process trunks have 3–5 faint, annular ring undulations ( Figs 26, 28 ), whereas in LM these undulations are not visible. Terminal discs are cog-shaped, with each of the 8–10 ‘cog-teeth’ extended to form a long, thin, hair-like and flexible filament that probably serves to enhance the adhesive function of egg processes. Under SEM, small rounded granules or aggregations of granules, 0.06–0.15 μm in diameter, are visible on the filaments ( Fig. 29 ). Central area of the terminal disc with sparse, randomly distributed, small granules ( Figs 25, 28 ). Moreover, the area between the granules on the filaments, the surface of the terminal disk and the trunk of the processes appear, under SEM, to be micro-granulated ( Figs 28–29 ).

DNA sequences

Initially, four molecular markers obtained from four of the eight individuals were sequenced. The sequences from 18S rRNA, 28S rRNA, ITS-2 exhibited a lack of polymorphism, whereas for COI two distinct haplotypes were obtained. Knowing that ITS-2 and COI have relatively high mutation rates, these fragments were sequenced for the remaining four specimens. The eight individuals were found to have identical ITS-2 sequences while COI revealed two haplotypes (frequency 1:1) differing by 20 substitutions. One consensus sequence for each nuclear marker and one consensus sequence for each COI haplotype were deposited in GenBank: 18S rRNA sequence, 1726 bp long (GenBank accession number: KX810008 ), 28S rRNA sequence, 725 bp long ( KX810009 ), COI sequence for haplotype 1, 658 bp long ( KX810011 ), COI haplotype 2, 658 bp long ( KX810012 ), and ITS-2 sequence, 425 bp long ( KX810010 ).

Remark

Among the 136 individuals of the new species, one specimen had abnormal claws, i.e., internal and external claw II and III on one leg had additional secondary branches ( Figs 15 , 19 ). Additionally, on the

claws of the IV pair of legs, unique, upwardly turned spurs were present near the lower half of the claw ( Fig. 19 ).

Differential diagnosis

Morphological

Macrobiotus polypiformis sp. nov., by the presence of long, flexible filaments on the egg processes and faint annular undulations on their trunks, is most similar to Ma. paulinae Stec et al., 2015 , but differs from it by possessing larger pores on the cuticle (0.9–1.2 μm in Ma. polypiformis sp. nov. vs 0.3–0.5 μm in Ma. pauliane ), smooth cuticle (seven dorso-lateral patches of sparse granulation arranged symmetrically on both sides of the body in Ma. paulinae ), one patch of granulation on legs I–IV (two distinct patches of granulation, fine and dense granulation above claws and a more robust and sparse granulation in the middle of each leg in Ma. paulinae ), a different number of rows of teeth in the first band of teeth in the oral cavity (3–4 rows in Ma. polypiformis sp. nov. vs a single row in Ma. paulinae ), larger reticulum mesh diameter covering the egg surface (0.5–0.8 μm in Ma. polypiformis sp. nov. vs 0.05–0.2 μm in Ma. paulinae ), trunks of the egg processes with faint annular undulations (distinct and clearly visible in Ma. paulinae ), different morphology of the terminal disc margins (8–10 long, hair-like, flexible filaments in Ma. polypiformis sp. nov. vs small, irregular teeth, instead of filaments, in Ma. paulinae , with only some processes having one to a few flexible filaments), lower number of processes on the egg circumference (19–23 in Ma. polypiformis sp. nov. vs 24–32 in Ma. paulinae ).

Genotypic

It was confirmed, using Basic Local Alignment Search Tool (BLAST; Altschul et al. 1990), that no sequences deposited in GenBank were identical with sequences obtained from the type population of Ma. polypiformis sp. nov. The p-distances calculated for ITS-2 between the new species and Ma. polonicus Pilato, Kaczmarek, Michalczyk & Lisi, 2003 , Ma. spaiens Binda & Pilato, 1984 and Ma. paulinae are 39.4%, 25.6% and 31.1%, respectively. In the case of COI, genetic distances between the two haplotypes of Ma. polypiformis sp. nov. is 3.1%, which is above the threshold for species delineation proposed by Cesari et al. (2009). However, sequences from haplotype 1 and 2 are clearly distant from sequences of each species used in the analysis. For haplotype 1, p-distance range from 20.6% to 25.8%; haplotype 2 from 21.0% to 25.9%. In each case the most similar to the new species is Ma. paulinae ( KT951668 ) and the most distinct is Ma. macrocalix Bertolani & Rebecchi, 1993 ( HQ876571 , FJ176208 -12). Given there is no polymorphism in the ITS-2 sequences coming from the type population and the morphologically most similar species Ma. paulinae differs from the new species by a genetic distance of nearly 21%, it can be claimed that Ma. polypiformis sp. nov. is a valid new species exhibiting two COI haplotypes. P-distances for more conservative DNA fragments (28S rRNA and 18S rRNA) are, as was expected, lower than for more variable markers (COI and ITS-2). The range for 28S rRNA is 7.3%–12.9% with the most similar species Ma. paulinae ( KT935501 ) and for 18S rRNA 3.0%–6.2% where the closest related species are Ma. paulinae ( KT935502 ) and Ma. sapiens ( DQ839601 ).

Figures

Figs 1–2. Macrobiotus polypiformis sp. nov., holotype, habitus, dorso-ventral projection. 1. Seen in PCM. 2. Seen in DIC. Scale bars in μm.

Figs 3–6. Macrobiotus polypiformis sp. nov. 3. Cuticular pores visible in PCM (paratype). 4. Cuticular pores visible in SEM (paratype). 5–6. Bucco-pharyngeal apparatus (dorso–ventral projection), the filled arrowhead indicates the first macroplacoid with central constriction (Fig. 5 = paratype in PCM; Fig. 6 = holotype in DIC). Scale bars in μm.

Figs 7–9. Macrobiotus polypiformis sp. nov., paratypes. 7. Third band of teeth visible under PCM (empty arrowhead). 8–9. Oral cavity armature (SEM), the filled arrowheads indicate teeth of the first band, arrows indicate teeth of the second band, the empty arrowheads indicate teeth of the third band, M = median teeth, L = lateral teeth, a = accessory teeth, rhombi = ring folds, asterisks = ventral side.

Figs 10–15. Claws. 10. Claw II with smooth lunules (PCM, paratype). 11. Claw IV (PCM, holotype). 12. Indented lunules on claw IV (PCM, paratype). 13. Claw I with smooth lunules (SEM, paratype). 14. Claw IV with indented lunules (SEM, paratype). 15. Aberrant claw IV (SEM, paratype). Numbers 1–2 indicate normally developed claw branches/spurs, whereas 3–4 indicate aberrant claw branches/ spurs. Scale bars in μm.

Figs 16–19. Macrobiotus polypiformis sp. nov., paratypes. 16. Granulation on the II pair of legs, arrow (PCM). 17. Granulation on IV pair of legs, arrow (PCM). 18. Granulation on II pair of legs (SEM). 19. Modified claw IV (arrowheads) and granulation on IV pair of legs, arrow (SEM). Scale bars in μm.

Figs 20–23. Macrobiotus polypiformis sp. nov. 20. Chorion. 21. Long, hair-like flexible filaments on terminal discs. 22. Egg processes and the surface between egg processes with reticular design. 23. Egg processes with filaments. All in PCM. Scale bars in μm.

Figs 24–29. Macrobiotus polypiformis sp. nov. 24. Chorion. 25. Long, hair-like flexible filaments on terminal discs. 26. Egg processes with faint annular undulations. 27. Surface between egg processes with reticular design. 28. Terminal discs with small, randomly arranged granules. 29. Small granules on filaments. All in SEM. Scale bars in μm.