Ramazzottius claudii, Vecchi & Stec, 2025

Vecchi, Matteo & Stec, Daniel, 2025, Mitogenome of a new Ramazzottius species (Tardigrada: Eutardigrada: Ramazzottiidae) discovered in rock pools along with its temperature and desiccation-related proteins repertoire, Organisms Diversity & Evolution 25 (1), pp. 119-135 : 126-131

Ramazzottius claudii sp. nov. Vecchi & Stec, 2024

Zoobank registration: urn:lsid:zoobank.org:act:

Ramazzottius sp. A in ( Vecchi et al., 2022)

Ramazzottius sp. Italy in ( Zawierucha et al., 2023)

Type locality. Rock pool sediment from vicinity of Sella del Marmagna , Corniglio, Parma, Italy ( 44°23′45.8″N 10°00′15.7″E) .

Material examined. Holotype ( Slide IT.158.7 ), 23 paratypes (Slides IT.158.4 – IT.158.10 ) and 20 eggs (Slides IT.158.01 – IT.158.03 ) mounted on slides in Hoyer’s medium. 4 animals (Stub TAR.2.06) and 5 eggs (Stub TAR.2.07) mounted on stubs for SEM. 3 animals used for DNA sequencing (Ram. sp.IT.158.01, Ram.sp.IT.158.03 and Ram_IT.158_WGA_1) .

Material repository. Tardigrada collection of the Institute of Systematics and Evolution of Animals (Polish Academy of Sciences), Sławkowska 17, 31–016, Kraków, Poland.

Etymology. This species is dedicated to Claudio Ferrari (University of Parma, Italy), to acknowledge his constant support and help in exploring the Italian Northern Apennines in search of rock pools.

( Tables 4 and 5, Figs. 3 , 4 , 5 , 6 , 7 , 8 , and 9, Online resources 01).

Description. Animals (morphometrics in Table 4 , raw measurements in Online resources 01): Eyes absent in live individuals. Pigmentation of the cuticle red-brown, distributed in one ring around the mouth, a dorso-caudal continuous band, and 8 lateral-ventral bands ( Fig. 3 ). Cuticle with faint sculpture, more visible in the dorso-caudal part of the body ( Figs. 4 and 5 ). A pair of sensory organs present on the head ( Fig. 5A ). The sculpture is composed by rounded polygonal granules; however, in some individuals (in particular in the caudal portion of bigger individuals), the granules have an elongated “spindle” shape ( Figs. 4 and 5 ).

Bucco-pharyngeal apparatus of the Ramazzottius - type ( Fig. 6 ). Mouth opening antero-ventral. Oral cavity armature visible under PCM ( Fig. 6B and C ). The armature is composed of one band of teeth, located in the posterior oral cavity ( Fig. 6B and C ). The band composed of a single row of large and regularly spaced granular teeth. In ventral view, the teeth are less visible and seem connected to the posterior edge of the oral cavity ( Fig. 6C ). Under PCM, other structures within oral cavity not visible ( Fig. 6B and C ). Apophyses for the insertion of stylet muscles (AISM) in the shape of blunt hooks and asymmetrical in size and shape with respect to the frontal plane ( Fig. 6D ). Stylet furcae with rounded ends. Buccal tube with a posterior bend and thickened walls posteriorly from the stylet support insertion point. Pharyngeal bulb (bulbus) almost oval, with apophyses and two clearly separated macroplacoids. Pharyngeal apophyses triangular, smaller than macroplacoids ( Fig. 6E ). Macroplacoid length configuration 2 <1. Microplacoid and septulum absent ( Fig. 6E ). Macroplacoids are usually roundish; however, in bigger specimens, they look slightly more elongated. Constrictions in the macroplacoids are present. In the first macroplacoid a central constriction is present, whereas in the second macroplacoids a constriction in central-posterior position is present.

Claws of the Ramazzottius - type. Primary branches of external and posterior claws long and thin. A non-sclerotized light refracting unit (LRU) is present between the claw base and the primary branch, which are connected by a couple of thin cuticular filaments ( Fig. 7A, B ).

Internal and anterior claws much smaller and of a different shape than external claws ( Fig. 7 ). Claws with smooth pseudolunules ( Fig. 7B ). Accessory points on primary branches of all claws present ( Fig. 7 D-E). Bars and other cuticular thickenings on legs, absent. A papilla is present on the external side of legs IV, but not always visible depending on the animal positioning ( Fig. 7C ).

Eggs (morphometrics in Table 5, raw measurements in Online resources 01): Laid freely, white and spherical, covered in processes with shape ranging from spike-like to filamentous ( Fig. 8 ). The processes exhibit extreme diversity in size, with most of them being within the range of 4.6–14.4 μm, but with some of them reaching up to 27.9 μm ( Fig. 8C ). Egg processes and surface between processes dotted under PCM, smooth under SEM ( Fig. 8I ).

Males Individuals with gonad filled with developing spermatids were found, indicating the presence of males ( Fig. 9 ). DNA sequences.

SSU (18S): PQ108467 – 8, PQ108470;

LSU (28S): PQ108475 – PQ108477;

COI: MW306832 – MW306836 , PQ109084 – 5;

ITS-2: PQ110584 – 6.

Differential diagnosis

By having eggs with dotted chorion and elongated processes, R. claudii sp. nov. is similar to six other species, however it differs from:

• Ramazzottius anomalus ( Ramazzotti, 1962) : by having a thinner buccal tube [external diameter pt 11.5 in R anomalus vs. 7.1 – 9.8 μm in R. claudii sp. nov.] and by the shape of the egg processes [long cone/aculeus in R. anomalus spines/filaments in R. claudii sp. nov.].

• Ramazzottius groenlandensis Kihm et al., 2023 : by having smaller eggs [bare diameter 80.6 3 μm in R. groenlandensis vs. 55.4 – 65.7 μm in R. claudii sp. nov.] and by the processes shape [mostly cone shaped in R. groenlandensis vs. elongated spikes and filamentous in R. claudii sp. nov.].

• Ramazzottius horningi Binda & Pilato, 1994 : by having longer external I-III claws [52.26 – 55.73 pt in R. horningi vs. 58.3 – 85.8 pt in R. claudii sp. nov.], and by the absence of a crown of dots around the base of the egg processes [crown of dots presents in R. horningi vs. absent in R. claudii sp. nov.].

• Ramazzottius theroni Dastych, 1993 : by the absence of eyes [eyes present in R. theroni vs. absent in R. claudii sp. nov.] and a weakly visible cuticle sculpturing [very evident in R. theroni vs. weakly visible in R. claudii sp. nov.]

• Ramazzottius valaamis Biserov & Tumanov, 1993 : by the shape of the egg processes [very thin filaments in R. valaamis vs. spines/filaments in R. claudii sp. nov.]

• Ramazzottius varieornatus Bertolani & Kinchin, 1993 : by having smaller eggs [bare diameter 69.9 – 73.3 μm in R. varieornatus vs. 55.4 – 65.7 μm in R. claudii sp. nov.], with generally longer processes [up to 10.2 μm in R. varieornatus vs. up to 27.9 μm in R. claudii sp. nov.], a shorter external claws II-III [58.2 – 68.6 pt in R. varieornatus vs. 61.8 – 85.8 pt in R. claudii sp. nov.], and by the absence of males [absent in R. varieornatus vs. present in R. claudii sp. nov.].

Additionally, for five other Ramazottius species, the eggs are unknown; however, the new species differs from them by:

• Ramazzottius belubellus Bartels et al., 2011 : by the absence of spines on the dorsal cuticle [present in R. belubellus vs. absent in R. claudii sp. nov.].

• Ramazzottius baumanni ( Ramazzotti, 1962) : by the absence of stronged sculptured cuticle with elevated polygons [present in R. baumanni vs. absent in R. claudii sp. nov.].

• Ramazzottius montivagus ( Dastych, 1983) : by having a sculptured cuticle [smooth in R. montivagus vs. sculptured in R. claudii sp. nov.] and by more slender primary branch of external claws (morphometric data for R. montivagus not available, but see Figs. 5 , 6 , and 7 in Dastych, 1983).

• Ramazzottius saltensis ( Claps & Rossi, 1984) : by the absence of stronged sculptured cuticle with elevated polygons [present in R. saltensis vs. absent in R. claudii sp. nov.].

• Ramazzottius szeptyckii ( Dastych, 1980) : by the absence of dorsolateral gibbosities [present in R. szeptycki vs. absent in R. claudii sp. nov.].

Figures

Fig.3 Ramazzottius claudii sp. nov. in toto. A In vivo animal under transmitted light showing pigmentation bands. B Holotype under PCM. Indented arrowheads: dorso-lateral pigmentation bands. Indented arrowhead:pigmentation ring around the mouth. Scale bars 100 μm

Fig. 4 Ramazzottius claudii sp. nov. cuticle under PCM. A Anterior dorsal cuticle. B Median dorsal cuticle. C Caudal dorsal cuticle. D Anterior dorsal cuticle in cross section. E Median dorsal cuticle in cross section. F Caudal dorsal cuticle in cross section. G Cuticle of a large individual showing “spin- dle” shaped granulation, more pronounced on the caudal side (on the right side of the image). White arrowhead: spindle- shaped granulation. Scale bars 20 μm

Fig. 5 Ramazzottius claudii sp. nov. cuticle under SEM. A Dorsal cephalic region show- ing sensory organs. B Dorsal caudal cuticle showing granular sculpture. C Dorsal caudal cuticle showing “spindle” like granulation. Arrowheads: cephalic sensory organs. White arrowhead: spindle-shaped granulation. Scale bars A, C 10 μm; B 2 μm

Fig. 6 Ramazzottius claudii sp. nov. buccal apparatus under PCM. A Buccal apparatus in toto in dorsal view. B OCA in dorsal view. C Oral cavity armature (OCA) in ventral view. D AISM in lateral view. E Placoids in ventral view. Arrowheads: third band of OCA. Indented arrowhead: dorsal hook of AISM. Empty indented arrowhead: ventral hook of AISM. Empty arrow: pharyngeal apophyses. White arrows: macroplacoids constric- tions. Scale bars 10 μm

Fig. 7 Ramazzottius claudii sp. nov. legs associated structures. A Claws III under PCM. B Claws IV under PCM. C Papilla on leg IV under PCM. D Claws III under SEM. E Claws IV under SEM. Black arrow- heads: pseudolunules. White arrowheads: claws accessory points. White arrows: LRU with cuticular filaments. Indented arrowhead: Papilla on leg IV. Scale bars 10 μm

Fig.8 Ramazzottius claudii sp. nov. eggs. A–H Eggs under PCM. I Egg under SEM. Scale bars 20 μm

Fig. 9 Ramazzottius claudii sp. nov. males under PCM. A–B Males posterior part. Arrow- heads: gonads with developing spermatids. Scale bars 100 μm

Tables

Table 5 Summary of eggs morphometrics of Ramazzottius claudii sp. nov

Character	N	Range						Mean		SD		Holotype
		μm			pt			μm	pt	μm	pt	μm	pt
Claw 4 lengths
Anterior base	13	6.3	–	10.0	15.3	–	26.5	8.2	22.2	1.3	3.1	8.5	25.2
Anterior primary branch	13	9.0	–	13.6	26.1	–	35.8	11.4	30.7	1.4	2.9	12.1	35.8
Anterior secondary branch	9	6.2	–	12.8	18.7	–	31.5	9.7	25.9	2.1	4.2	9.5	28.2
Anterior cbt ratio	12	58.8	–	82.5		–		71.6	–	7.6	–	70.3	–
Anterior br ratio	9	61.3	–	99.1				81.9		12.1
Anterior total	12	13.5	–	17.9	37.1	–	45.3	15.5	41.1	1.6	2.7	15.1	44.8
Posterior base	16	0.4	–	14.6	1.1	–	36.1	10.3	27.8	3.1	7.7	10.2	30.0
Posterior primary branch	19	13.3	–	22.2	44.0	–	56.9	18.5	50.3	2.5	3.7	17.2	50.8
Posterior secondary branch	12	6.5	–	10.6	19.7	–	27.9	9.0	23.9	1.3	2.8	8.1	24.1
Posterior cbt ratio	15	47.7	–	70.2		–		58.7	–	6.6	–	59.0	–
Posterior br ratio	11	41.8	–	56.9				48.1		5.1
Posterior total	14	24.2	–	37.3	72.6	–	92.1	29.4	79.1	4.3	5.9	24.9	73.5