Macrobiotus azzunae, Marnissi & Cesari & Rebecchi & Bertolani, 2021

Marnissi, Jamila Ben, Cesari, Michele, Rebecchi, Lorena & Bertolani, Roberto, 2021, Integrative description of a new Tunisian tardigrade species, Macrobiotus azzunae sp. nov. (Eutardigrada, Macrobiotidae, hufelandi group), European Journal of Taxonomy 758 (1), pp. 122-146 : 126-138

Macrobiotus azzunae sp. nov.

urn:lsid:zoobank.org:act:933CCC06-F69D-49E2-AF4F-0C042D8F5C99

Figs 1–4 , 5A, C , 7

Etymology

The new species is dedicated in honor of Atf Azzouna, professor in the Faculty of Mathematical, Physical and Natural Sciences of Tunis and supervisor of the PhD thesis of Jamila Ben Marnissi.

Type material

Holotype TUNISIA • spec. of unidentified sex; North-West Tunisia, Kroumiri Mountains, Ain Soltan forest , Jendouba; 36º31′21.788″ N, 8º19′57.741″ E; 893 m a.s.l.; Apr. 2017; Marnissi leg.; moss on trunk of Quercus canariensis ; UNIMORE, slide code C4218–S32.

Paratypes

TUNISIA • 17 specs, sex unidentified; same collection data as for holotype; UNIMORE, slide codes C4218–S2 to C4218–S7, C4218–S9, C4218–S17, C4218–S30, C4218–S31, C4218–S33 to C4218– S35 • 3 eggs; same collection data as for holotype; UNIMORE, slide codes C4218–S10, C4218–S11, C4218–S25 .

Type depositories

The holotype (slide: C 4218– S 32), 17 paratypes (slides C 4218– S 2 to C 4218– S 7, C 4218– S 17, C 4128– S 30, C 4218– S 31, C 4218– S 33 to C 4218- S 35), 3 eggs (slides: C 4218– S 10/11/25) and two vouchers (slides SP 04 and SP 07, corresponding to specimens C 4218 V 4 and C 4218 V 7, respectively) mounted in Faure-Berlese fluid, are deposited in the Bertolani collection at the Department of Life Science, UNIMORE, Modena, Italy.

Type locality

NW Tunisia, Kroumrie mountains , Ain Soltan forest , Jendouba, 36º31′21.788″ N, 8º19′57.741″ E. Altitude 893 m a.s.l .

Description

Adult specimens

Body white, transparent after mounting in Faure-Berlese, from 162.2 to 410.3 µm in length ( Fig. 2A , Table 3 ; structures measured only in the animals more than 200 µm in length). Eye spots present, even after mounting. Cuticle smooth but with small round or oval pores, 1–1.5 µm in diameter ( Fig. 2B ), better visible after fixation in Carnoy and orcein staining ( Fig. 3C ), scattered randomly on the entire cuticle, including the dorsal surface of all legs. With SEM, pores look oval or in the shape of a seed ( Fig. 3A, D ) with the largest diameter of 0.7–0.8 µm. Weak cuticular granulation also present on the lateral surface of all legs and specially on legs IV ( Fig. 2B , arrow). Only with SEM is it possible to define the shape of the granulation on the legs, which looks as a regular disposition of star-shaped protuberances (about 0.3 µm; Fig. 3F ). Six buccal sensory lobes around the mouth, well recognizable with SEM. Mouth antero-ventral; buccal-pharyngeal apparatus of the Macrobiotus type (sensu Pilato & Binda 2010), with ventral lamina and ten small peribuccal lamellae (in the holotype, after mounting, separated from the mouth). Buccal armature, corresponding to oral cavity armature, OCA, according to Michalczyk & Kaczmarek (2003), without an anterior band of teeth visible, corresponding to the first band of teeth according to Michalczyk & Kaczmarek (2003), and to the anterior band of the buccal ring according to Guidetti et al. (2012); posterior band of teeth poorly visible, corresponding to second band of teeth, according to Michalczyk & Kaczmarek (2003), followed by three dorsal and three ventral crests, corresponding to third band of teeth according to Michalczyk & Kaczmarek (2003); the dorsal crests ( Fig. 2D ) are distinct transverse ridges, whereas the ventral crests ( Fig. 2E ) appear as two separate lateral transverse ridges and a roundish median tooth. The posterior band of teeth and the transverse ridges are part of the buccal tube, according to Guidetti et al. (2012). Buccal tube narrow; pharyngeal bulb spherical with triangular apophyses, two rod-shaped macroplacoids, relatively short, the first longer than the second and evidently but not deeply narrowed at its middle ( Fig. 2C ), the second with a not particularly evident subterminal constriction. Microplacoid present. Slender claws of the hufelandi type (sensu Pilato & Binda 2010); the external claw longer than the internal one and the posterior longer than

the anterior. Primary branches of each claw with distinct accessory points ( Fig. 2F ), a common tract of medium length (about a third of the total claw length) and an evident stalk connecting the claw to the lunule. Lunules under all claws, smooth, larger on the hind legs ( Figs 2G , 3E ). Cuticular bars under claws absent.

The population is dioecious (gonochoristic). Males were recognized using orcein staining, which revealed that the testis is filled with spermatozoa with a coiled head ( Fig. 3G ) and spermatids. No morphological secondary sexual dimorphism, such as gibbosities on legs IV in males, was identified.

Eggs

Eggs are laid freely, and are white, spherical or slightly oval. One egg containing a fully developed embryo showed the shape of the buccal-pharyngeal apparatus ( Fig. 4A ). Processes of the eggshell are in the shape of inverted goblets ( Fig. 4B ) with conical trunks and well-defined distal discs as large as the process bases (for measurements see Table 4 ). Distal discs concave, with a median small protuberance and, using PhC, with border often smooth, or sometimes slightly jagged, or slightly ragged ( Fig. 4C ),

but never clearly jagged, serrated or dentate. Surface among processes of the hufelandi type (sensu Kaczmarek & Michalczyk 2017a), i.e., covered by a very thin grid ( Fig. 4D ). Meshes around the process bases slightly larger and with slightly thicker wires compared with interbasal meshes. Mesh diameter around 0.5 µm.

Comparisons

Macrobiotus azzunae sp. nov. has eggs with processes as inverted goblets and a reticulate eggshell between the processes. Consequently, a comparison must be done with the Macrobiotus species listed by Kaczmarek & Michalczyk (2017a) with hufelandi type eggshells, excluding the species with processes that are not like inverted goblets, and adding the species with hufelandi type chorion eggs described after that publication. The species with hufelandi type chorion eggs that do not have processes as inverted goblets are Macrobiotus acadianus (Meyer & Domingue, 2011) , M. dariae Pilato & Bertolani, 2004 , M. lissostomus Durante Pasa & Maucci, 1979 , M. santoroi Pilato & D’Urso, 1976 , and M. scoticus Stec, Morek, Gąsiorek, Blagden & Michalczyk, 2017 . Moreover, M. azzunae sp. nov. has egg processes with distal discs with a smooth or slightly jagged border, therefore it differs from all the species that have clearly indented, serrated or clearly jagged distal discs, such as: Macrobiotus canaricus Stec, Krzywański & Michalczyk, 2018 , M. crustulus Stec, Dudziak & Michalczyk, 2020 , M. hannae Nowak & Stec, 2018 (whose egg surface is more cribriform than reticulate), M. hibiscus de Barros, 1942 , M. horningi Kaczmarek & Michalczyk, 2017b (which also has very high processes), M. hufelandi C.A.S. Schultze, 1834 , M. humilis Binda & Pilato, 2001 , M. iharosi Pilato, Binda & Catanzaro 1991 , M. joannae Pilato & Binda, 1983 , M. julianae (Meyer, 2012) , M. kamilae Coughlan & Stec, 2019 , M. modestus Pilato & Lisi, 2009 , M. noonragi s Coughlan & Stec, 2019, M. papei Stec, Kristensen & Michalczyk, 2018 (with particularly long filaments starting from the distal disc), M. paulinae Stec, Smolak, Kaczmarek & Michalczyk, 2015 , M. polypiformis Roszkowska, Ostrowska, Stec, Janko & Kaczmarek, 2017 (even with cog-teeth extended to form a long, thin, hair-like and flexible filament), M. punctillus Pilato, Binda & Azzaro, 1990 , M. sapiens Binda & Pilato, 1984 , M. sottilei Pilato, Kiosya, Lisi & Sabella, 2012 .

For the shape of the egg Macrobiotus azzunae sp. nov. differs from M. rawsoni Horning, Schuster & Grigarick, 1978 because this species has only one strip of meshes around each egg process (see Kaczmarek & Michalczyk 2017b); it differs from M. serratus Bertolani, Guidi & Rebecchi, 1996 because in this species the egg surface is porous more than reticulated, with pores small and spaced from each other, and its egg processes have a large, often square, distal disc; it differs from M. seychellensis Biserov, 1994 because the distal disc of the egg processes of this species, even though not dentate, has long and very developed lobes.

The remaining nine species of the hufelandi group should be compared singularly.

Macrobiotus almadai Fontoura, Pilato & Lisi, 2008

Macrobiotus azzunae sp. nov. differs from M. almadai in having a posterior band of teeth in the buccal cavity visible with LM (not visible in M. almadai ), and distal disc with a jagged margin instead of very small teeth as in M. almadai .

Macrobiotus canaricus Stec, Krzywański & Michalczyk, 2018

With LM the margin of the distal disc of M. azzunae sp. nov., never dentate in this species, looks similar to that of M. canaricus , but the SEM images of the eggs of the latter species evidence the presence of an almost dentate disc. Moreover, the peribasal meshes of the eggshell are larger than interbasal ones in the new species while they do not differ from the interbasal ones in M. canaricus ; regarding the animals there are differences in the buccal armature: in M. azzunae sp. nov. the posterior band of teeth is visible with LM (even though poorly) and the three dorsal crests are distinct transverse ridges, while in M. canaricus the posterior band of teeth is visible only with SEM and with LM the dorsal teeth form a transversal ridge weakly divided into three teeth.

Macrobiotus madegassus Maucci, 1993

The new species differs from M. madegassus by the presence of the eye spots (absent in M. madegassus ), pores on the cuticle (absent in M. madegassus ), presence in the buccal armature of posterior band of teeth, even though weak (fully absent in M. madegassus ), buccal tube much larger (pt of the holotypes 15.9 vs 7), insertion of the stylet supports on the buccal tube much more posterior (pt of the holotypes 76.1 vs 68), first and second macroplacoid longer (pt of the holotypes 25.5 and 18.1 vs 21.3 and 12.0), lunules on the hind legs without kerning (crenate in M. madegassus ), eggshell processes with distal disc as large as the base (similar range 3.2–5.2 for both measurements) with respect to that of M. madegassus (disc vs base: 4.3–5.4 vs 2.3–2.6).

Macrobiotus martini Bartels, Pilato, Lisi & Nelson, 2009

The cuticular pores in M. azzunae sp. nov. are much smaller than those of M. martini ; the distal disc of the egg processes in M. azzunae sp. nov. has a diameter similar to that of the process base, while in M. martini the distal disc is much narrower than the base.

Macrobiotus nebrodensis Pilato, Sabella, D’Urso & Lisi, 2017

Macrobiotus azzunae sp. nov. differs from M. nebrodensis by the absence of the cuticular bar near the lunules on the first three pairs of legs (a faint bar is present in M. nebrodensis ). The egg processes of M. azzuane sp. nov. are in higher number on the circumference (29–33) with respect to those of M. nebrodensis (18). In the latter species there are some egg processes very high (up to 20.6 µm), while in the new species process height and shape are more uniform. The difference in the eggshell between meshes around the process base and the others is much less evident in M. azzunae sp. nov. than in M. nebrodensis .

Macrobiotus personatus Biserov, 1990

The new species differs from M. personatus by the posterior band of the buccal armature less evident, the presence of a clear constriction in the first macroplacoid ( Fig. 5A ), in the paratype of M. personatus examined by us barely identifiable ( Fig. 6A ) and, according to Biserov (1990) usually absent in the type material of that species. The pores on the cuticle of M. azzunae sp. nov. are small, approximately 1 µm in diameter, while in M. personatus they are strongly elliptic and about 3 µm in length ( Fig. 6B ). Lunules on leg IV are always smooth in M. azzunae sp. nov., sometimes indented in M personatus . With respect to the eggs of M. personatus ( Fig. 6C–D ), the egg processes of M. azzunae sp. nov. ( Figs 4C–D , 5C ) are clearly shorter, 5.4 ± 0.6 vs 9.5 ± 0.5 (range 4.2–6.4 vs 9–10.5) and with a narrower base and distal disc (both 3.2–5.2 vs 7–10.5 and 7–9 respectively). Males are present in the new species, while in M. personatus only females were found ( Biserov 1990), suggesting parthenogenesis in that species.

Macrobiotus sandrae Bertolani & Rebecchi, 1993

The new species differs from M. sandrae for the eggshell shape, with thinner wires of the reticulum and meshes around the processes larger than the inter-process meshes in M. azzunae sp. nov. ( Fig. 5C ), all meshes similar in size in M. sandrae ( Fig. 5D ). Figure 5C–D also show a difference in the process base diameter, narrower in M. azzunae sp. nov. With regard to the animals, M. azzunae sp. nov. differs from M. sandrae by a constriction of the first macroplacoid much more pronounced ( Fig. 5A ; it is hardly visible in M. sandrae ; Fig. 5B ). Moreover, in animals of similar size the posterior band of the buccal armature is just less evident in the new species, and lunules on the hind legs are without hint of teeth (but teeth, present in the holotype of M. sandrae , are often difficult to identify in other specimens of that species).

Macrobiotus terminalis Bertolani & Rebecchi, 1993

Macrobiotus azzunae sp. nov. differs from M. terminalis for the absence of granulation on the cuticle (noted only in the redescription of M. terminalis ; see Cesari et al. 2011), for the absence of teeth on the lunules, especially evident on the hind legs of M. terminalis , and for the presence of males, absent in M. terminalis (see redescription by Cesari et al. 2011).

Macrobiotus vladimiri Bertolani, Biserov, Rebecchi & Cesari, 2011

With respect to M. vladimiri , animals of M. azzunae sp. nov. reach a shorter length (up to 410.3 µm vs 515.1 µm), in M. azzunae sp. nov. the posterior band of teeth of the buccal armature is less evident and the lunules on the hind legs are not indented. In M. azzunae sp. nov. the egg diameter without processes (64.7–80.6 µm) is less than that of the eggs of M. vladimiri (89.9–92.0 µm); the processes are shorter (4.2–6.4 µm in the new species vs 6.5–8 µm in M. vladimiri ). In the new species the base process diameter is narrower (3.2–5.2 µm) than in M. vladimiri (5.1–7.3 µm), the distal disc is weakly or not jagged (clearly jagged in M. terminalis ). In M. azzunae sp. nov. males are present, while they are absent in M. vladimiri .

Genetic distances

The ranges of uncorrected genetic p-distances between M. azzunae sp. nov. and the other species of the M. hufelandi group (Supp. file 7, Supp. file 8, Supp. file 9, Supp. file 10), are as follows:

18S 0.1–5.6%, with the most similar being M. sandrae from Germany (present paper)

28S 0.1%, with the only available M. vladimiri from Spain ( FJ435751 –5)

ITS-2 7.7–32.2%, with the most similar being Macrobiotus vladimiri ( MN888347 ) from Finland

COI 6.3–25.6%, with the most similar being Macrobiotus sandrae ( HQ876574 , HQ876577 , HQ876578 , HQ876579 , HQ876581 ) from Germany

The COI dataset is the most complete and informative for species delimitation investigation. Both phylogenetic reconstructions on the COI dataset resulted in the same topology, and thus the ML tree was utilized for the PTP analysis ( Fig. 7 , left), which shows 14 putative species clusters: M. crustulus , M.hannae , M. cf. recens , M.canaricus , M.hufelandi , M. cf. hufelandi sp.1, M. terminalis , M. cf. terminalis , M. wandae , M. macrocalix , M. cf. macrocalix , M. vladimiri , M. sandrae and M. azzunae sp. nov. This subdivision is further validated by both the ABGD and the haplotype network analysis ( Fig. 7 , centre and right). Present molecular data therefore confirms the validity of the erection of M. azzunae sp. nov.

Figures

Fig. 1. Map of the sampling locality (circle) in the North-West of Tunisia.

Fig. 2. Macrobiotus azzunae sp. nov., holotype (UNIMORE, slide C4218–S32). A. In toto animal. B. Cuticular pores and leg granulation (arrow) on the hind legs. C. Buccal-pharyngeal apparatus. D. Buccal armature: dorsal crests (arrow). E. Buccal armature: ventral crests (arrow). F. Claw and lunulae of the third pair of legs. G. Claw and lunulae of the fourth pair of legs. A–G: PhC.

Fig. 3. Macrobiotus azzunae sp. nov., paratypes. A. In toto animal. B–D. Cuticular pores. E. Fourth pair of legs with smooth lunules and peculiar granulation on the legs. F. Granulation on the legs with a starshaped organization. G. Male with testis full of mature spermatozoa with elongate, helicoidal nucleus. A, D–F: SEM (stub-C4218); B: in vivo DIC; C, G: orcein (not permanent slide TN02–04) PhC.

Fig. 4. Egg of Macrobiotus azzunae sp. nov., paratype (UNIMORE, slide C4218–S11). A. In toto egg with buccal-pharyngeal apparatus of its embryo at the end of development. B. Processes of the eggshell (midsection). C. Distal discs of the eggshell processes. D. Surface of the eggshell between processes. A, C–D: PhC; B: DIC.

Fig. 5. Comparison betweenM. azzunae sp. nov. andM. sandrae Bertolani & Rebecchi, 1993.A. Placoids in M. azzunae sp. nov., paratype (UNIMORE, slide C4218–S30).B. Placoids inM. sandrae (UNIMORE, slide C442–S79); arrowheads evidence the different constriction depth of the first macroplacoid. C. Eggshell in M. azzunae sp. nov., paratype (UNIMORE, slide C4218–S4). D. Eggshell in M. sandrae (UNIMORE, slide C2346–S2); in M. azzunae sp. nov. there are smaller processes and reticulation with thinner wires and larger net around the processes than in M. sandrae. A–D: PhC.

Fig. 7. Left: tree resulting from both the maximum likelihood analysis and the Bayesian inference of cytochrome c oxidase subunit I (COI) in M. azzunae sp. nov. specimens and sequences from GenBank. Values above branches point out bootstrap values, while values under branches represent posterior probability values. Results of the Poisson tree process analysis are provided using differently coloured branches: putative species are indicated using transitions from blue-coloured branches to red-coloured branches. Newly scored haplotypes are in bold. The scale bar shows the number of substitutions per nucleotide position. Centre: haplotype network of COI gene in M. hufelandi complex. Circles represent haplotypes, while circle surface denotes haplotype frequency. Networks falling below the value of the 95% connection limit are disconnected. Right: rectangles denote specimens grouped by ABGD analysis.

Fig. 6. Macrobiotus personatus Biserov, 1990, paratypes (Civic Museum of Natural History of Verona, Italy, CT14701). A. Buccal-pharyngeal apparatus with macroplacoids. B. Pores on the cuticle. C. Eggshell reticulation and egg processes. D. Egg processes. A–D: PhC.

Tables

Table 3. Measurements (in μm) and pt of selected morphological structures of individuals ofMacrobiotus azzunae sp. nov. mounted in Faure-Berlese. Abbreviations: Nr = number of specimens/structures measured; Range = smallest–largest value; SD = standard deviation. * Three animals below 200 μm in length were not considered in the analysis. Two were not measurable, while data of the only measurable animal below 200 μm is available in the supplementary material (Supp. file 1).

Character	Nr	Range µm	Mean ± SD	Holotype µm
Body length	10 *	246.0–410.3		374.0
Buccal tube length	13	29.4–38.5		36.6
		pt		µm	pt
Buccal tube external width	13	11.6–15.9	13.3 ± 1.2	5.8	15.9
Stylet support insertion point	13	75.1–80.5	76.7 ± 1.5	27.9	76.1
Placoid row	13	50.1–57.7	51.8 ± 2.4	18.9	51.5
Macroplacoid row	13	37.7–48.7	44.6 ± 3.1	15.7	42.7
First macroplacoid	13	24.3–29.7	26.6 ± 2.2	9.3	25.5
Second macroplacoid	13	16.0–20.7	18.3 ± 1.5	6.6	18.1
Microplacoid	13	5.9–8.6	6.9 ± 1.0	2.6	7.0
External Claws III main branch	13	27.1–31.3	29.8 ± 1.3	11.5	31.3
External Claws III secondary branch	13	20.1–25.8	22.5 ± 1.6	8.1	22.1
Internal Claws III main branch	13	24.9–30.2	27.7 ± 1.6	10.2	27.9
Internal Claws III secondary branch	13	18.8–24.2	21.4 ± 1.7	7.2	19.7
Posterior Claws IV main branch	9	29.1–34.6	32.7 ± 1.8	12.2	33.2
Posterior Claws IV secondary branch	9	21.6–24.9	23.8 ± 1.6	9.1	24.9
Anterior Claws IV main branch	10	28.2–32.5	30.1 ± 1.9	11.1	30.3
Anterior Claws IV secondary branch	10	20.4–26.2	23.2 ± 1. 7	8.8	24.0

Table 4. Measurements (in μm) of selected morphological structures of the eggs of Macrobiotus azzunae sp. nov. mounted in Faure-Berlese. Abbreviations: Nr = number of eggs/structures measured; Range = smallest–largest value; SD = standard deviation.

Character	Nr	Range	Mean ± SD
Egg diameter without processes	2	64.7–80.6	–
Egg diameter with processes	2	72.4–89.2	–
Process nr on egg circumference	2	29–33	–
Process height	10	4.2–6.4	5.4 ± 0.6
Process base width	10	3.2–5.2	4.2 ± 0.5
Distal disc width	10	3.2–5.2	4.4 ± 0.6
Inter process distance	10	2.7–4.8	3.5 ± 0.5