Macrobiotus siderophilus, Bertolani & Cesari & Giovannini & Rebecchi & Guidetti & Kaczmarek & Pilato, 2023

Bertolani, Roberto, Cesari, Michele, Giovannini, Ilaria, Rebecchi, Lorena, Guidetti, Roberto, Kaczmarek, Łukasz & Pilato, Giovanni, 2023, The Macrobiotus persimilis-polonicus complex (Eutardigrada, Macrobiotidae), another example of problematic species identification, with the description of four new species, Organisms Diversity & Evolution (New York, N. Y.) 23 (2), pp. 329-368 : 357-359

Macrobiotus siderophilus sp. nov. differs from:

Macrobiotus persimilis by: having a shorter placoid length (see Tables 4 and 6) and longer, flexible and gradually thinner egg processes, without distinct neck at the end.

Macrobiotus hyperboreus by: the presence of eyes, more anterior insertion of the stylet supports, longer claws (see Tables 4 and 6), the presence of indented lunules of the hind legs, the presence of males with a lateral gibbosity on each hind leg, absence of dots around the egg processes bases, flexible distal part of the egg processes and absence of plates on the distal discs.

Macrobiotus polonicus by: having longer placoids and claws (see Tables 4 and 6) and different egg processes (that are taller, slender and tapered, with distal discs clearly smaller than the bases and with a small depression).

Macrobiotus marlenae by: the presence of longer placoids (mean pt 57.0 ± 2.4 in M. siderophilus sp. nov. vs 50.8 ± 3.3 in M. marlenae ), longer claws (mean pt of the posterior claw of the hind legs: 37.2 ± 1.4 in M. siderophilus sp. nov. vs 24.4 ± 2.0 in M. marlenae ), presence of gibbosities on the hind legs in males, higher number of processes on the egg circumference (26–28 in M. siderophilus sp. nov. vs ca. 16 in M. marlenae ) and shorter egg processes (5.7–7.3 μm in M. siderophilus sp. nov. vs ca. 8.6 μm in M. marlenae ).

Macrobiotus trunovae by: having much smaller eggs without processes (69–77 µm in diameter in M. siderophilus sp. nov. vs ca. 134.3 µm in diameter in M. trunovae ), smaller body length (up to 420 µm in length in M. siderophilus sp. nov. vs up to 639 µm in length in M. trunovae ), stylet supports inserted in a more anterior position (pt 75.8–78. 2 in M. siderophilus sp. nov. vs 81.6–82.4 in M. trunovae ), longer claws (pt range of the posterior claw of the hind legs 35.8–38.7 in M. siderophilus sp. nov. vs 27.9–29.3 in M. trunovae ) and shorter egg processes (up to 7.3 µm high in in M. siderophilus sp. nov. vs up to 10.9 µm in M. trunovae ).

Macrobiotus anemone by: the presence of eyes, presence of the posterior band of teeth in the buccal armature, indented lunules on the hind legs and egg processes flexible and with indented distal discs and without tentacular arms.

Macrobiotus halophilus by: the clearly separated dorsal ridges of the buccal armature (almost fused in M. halophilus ) and by having taller, more slender and flexible egg processes (5.4–7.3 µm high in M. siderophilus sp. nov. vs 3.9–5.9 µm in M. halophilus ), and with distal discs smaller than the processes bases.

Macrobiotus dolosus sp. nov. by having higher and more slender egg processes (see Table 5) that are flexible and become gradually thinner distally and by the presence of distal discs with a small depression. There is also a large difference at the molecular level: cox1 p-distance between the two species ranges from 16.7 to 18.3% ( Table 3).

Macrobiotus fontourai sp. nov. ( Figs. 6a , 16 and 17; Tables 5 and 6).

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Type locality: Italy, Osimo (Ancona Province), 43° 29.249′ N, 13° 29.140′ E, 223 m asl .

Type depositories: Holotype C2861-S 9 ♀ D, one egg C2861-S 14 in Faure-Berlese deposited in the Civic Museum

◂ Fig. 16 Macrobiotus fontourai sp. nov. ( Osimo , Italy). a Habitus; b dorsal buccal armature; c ventral buccal armature; d buccal-pharyngeal apparatus with microplacoid in focus; e the same apparatus as in d at another level of focus; f claws on the third pair of legs and weak bar (arrowhead); g claws on the hind legs with large and indented lunules (arrowheads). a–g Holotype (PhC)

of Natural History of Verona ( Italy), 18 paratypes and two eggs deposited in the Bertolani collection at the Department of Life Sciences , University of Modena and Reggio Emilia, Modena, Italy ; three paratypes and two eggs in Faure-Berlese deposited in the Binda & Pilato collection, Museum of the Department of Biological, Geological and Environmental Sciences, University of Catania, Italy ; one paratype and one egg in Faure-Berlese deposited at the Department of Animal Taxonomy and Ecology, Faculty of Biology, Adam Mickiewicz University, Poznań, Poland .

Description of the holotype (slide C2861-S 9 ♀ D; Osimo A)

Female (detailed measurements in Table 7). Body 409.0 µm in length (Fig. 16a); cuticle smooth, with roundish pores, often barely visible with PhC; eye spots present, even after permanent mounting in Faure-Berlese. Mouth surrounded by 10 peribuccal lamellae; anterior band of teeth of the buccal armature poorly visible, posterior band with clearly visible oval teeth in 3–4 rows. System of ridges in the posterior part of the mouth cavity dorsally formed by three long rectangular crests of similar length, ventrally by three crests, the two lateral ones being triangular, the median one rectangular (Fig. 16b, c). Pharyngeal bulb with well-developed apophyses in shape of truncated cones with the apex facing backwards, two rod-shaped macroplacoids and microplacoid (Fig. 16d). First macroplacoid with evident, but not very deep central constriction (Fig. 16e). Lunules small and smooth on the first three pairs of legs, larger and with tiny teeth on the hind legs (Fig. 16g). Weak transverse cuticular bars visible only under some claws on the first three pairs of legs (Fig. 16f, arrowhead). Granulation on the legs not visible in LM.

Other type material. Paratypes similar to the holotype. In two specimens, granulation visible on all the legs, but in another specimen granulation just visible on the hind legs. There are males with a lateral gibbosity on each hind leg and females with seminal receptacle. The spermatozoon head is similar to that of M. dolosus sp. nov. Six bivalents were observed in the oocytes I (Fig. 17a–c).

Since two species were present in the same sample, the relationship between the morphology of the animal and that of the egg was ascertained using females and eggs laid by them.

Eggs (measurements in Table 5). Egg processes ( Figs. 6a and 17d–g) relatively close to each other, with a distal part rigid and thinner than the basal part, without a neck, topped by a clearly dentate distal disc (teeth longer than 1 µm) that is a little smaller than the egg process base and with a wide depression (Fig. 17f, g). In LM, process bases surrounded by very weak dots.

Other non-type material. Animals found in Nardò, named Nardò A and attributed to M. fontourai sp. nov. were similar to those of the type locality, including the presence of a shallow constriction of the first macroplacoid ( Fig. 18b ). Animals with either many or few pores in the cuticle, uniformly arranged ( Fig. 18a ); one lateral gibbosity on each hind leg of the males ( Fig. 18c ) and a seminal receptacle in the females. Only one specimen measured ( Table 7). Corresponding eggs ( Fig. 18d–f ) without a visible neck (or a barely visible neck), a wide depression on the distal disc. In LM, surface of the eggs slightly rough and without dots; in SEM the egg surface appears the same, i.e. rough and without pits. Although, unfortunately, there is no molecular confirmation (for this material no animals attributable to M. fontourai sp. nov. were successfully sequenced), we propose that due to the morphological characteristics, some of the animals and eggs found in Nardò (Lecce, Italy) are attributed to this species, while other animals and eggs from the same sample (some of them sequenced) should be attributed to Macrobiotus cf. muralis (see below). For both species, the certain relationship between animal and egg morphology was obtained by observing females and eggs laid by them when isolated.

Etymology: The species is named in honor of the Portuguese tardigradologist and our friend dr. Paulo Fontoura.

Figures

Fig.6 Eggs of the populations of the Macrobiotus persimilis- polonicus complex. a Osimo A, Italy (PhC); b Osimo B, Italy (SEM); c Paris, France (DIC); d egg process bases with dots (arrowheads) (near Poznań, Poland, PhC); e Paris, France (SEM); f egg process bases with dots (arrowheads) (Nardò B, Italy, PhC); egg’s mother was sequenced (see Table III); g Novellara, Italy (SEM)

Fig.18 Macrobiotus fontourai sp. nov. (Nardò A) (a–f) and Macrobiotus cf. muralis (Nardò B) (g–i). a Cuticular pores (orcein,PhC);b buccal-pharyngeal apparatus with middle constric- tion in the first macroplacoid (orcein, PhC); c gibbosities (arrowheads) on the hind legs of a male (fresh, DIC). d Distal discs on the egg processes (PhC); e process bases and egg surface (PhC); f egg processes and egg surface (SEM); g distal discs on the egg processes (PhC); h egg processes in profile (PhC); i processes and egg surface (SEM). Egg in g–i from the female A (its sister egg sequenced, see in Fig. 10 and Table 5)