The xerophilic genera Xerobiotus and Pseudohexapodibius (Macrobiotidae; Tardigrada): biodiversity, biogeography and phylogeny Author Vincenzi, Joel Department of Life Sciences, University of Modena and Reggio Emilia, Modena, Italy joel.vincenzi@unimore.it Author Cesari, Michele Department of Life Sciences, University of Modena and Reggio Emilia, Modena, Italy & NBFC, National Biodiversity Future Center, Palermo, Italy Author Kaczmarek, Łukasz Department of Animal Taxonomy and Ecology, Faculty of Biology, Adam Mickiewicz University, Poznań, Poland Author Roszkowska, Milena Department of Animal Taxonomy and Ecology, Faculty of Biology, Adam Mickiewicz University, Poznań, Poland Author Mioduchowska, Monika Department of Evolutionary Genetics and Biosystematics, Faculty of Biology, University of Gdansk, Poland Author Rebecchi, Lorena Department of Life Sciences, University of Modena and Reggio Emilia, Modena, Italy & NBFC, National Biodiversity Future Center, Palermo, Italy Author Guideưi, Yevgen Kiosya and Roberto text Zoological Journal of the Linnean Society 2024 2023-10-10 200 1 111 141 http://dx.doi.org/10.1093/zoolinnean/zlad129 journal article 10.1093/zoolinnean/zlad129 0024-4082 10470587 Xerobiotus litus sp.nov. ( Figs 10 , 11 ; measurements and statistics are in Tables 2 and 3 ; Supporting Information, Table S3 ). ZooBank: urn:lsid:zoobank.org:act: C27CCE1F-3672-459A- A2FD-13185397B9A8 Type locality: Praia do Martinhal , Sagres , Portugal ( 37°1ʹ14.736N , 8°55ʹ31.5192E ), moss on sandy dunes, coll. R . Guideưi . Material examined: Thirty-two adults and three eggs . Type repositories: The holotype ( 4383s2b ), 10 paratypes and two eggs deposited in Bertolani’s Collection of UNIMORE ; two paratypes and one egg deposited in the Museum of Natural History of Verona ( Italy ) . Three paratypes and two eggs were mounted on stubs for SEM observations . Etymology: The name of the species means beach ( litus in Latin), for the type of environment in which the species was found. Description: Body whitish; eye-spots (orange) present in live and fixed specimens ( Fig. 10A ). Very small, scaưered pores (~0.5 µm in diameter, visible only with SEM) on the dorsolateral cuticle and legs ( Fig. 11C ). Very small single granules (visible only with SEM), distributed almost regularly, present on the entire cuticle ( Fig. 11B ). Legs of the first pair clearly smaller than those of the second and third pairs. The area of the leg cuticle surrounding the claws with a swelling (forming a furbelow-like structure; Fig. 10G ) covered with microdigitations (clearly visible only with SEM; Fig. 11D ). Buccopharyngeal apparatus of Macrobiotus type , with anteroventral mouth and 10 small peribuccal lamellae. Oral cavity armature of the maculatus type (with LM), with only third band of teeth and three dorsal transversal crests (in contact), two ventral lateral crests and the medioventral crest split in two granules ( Fig. 10C, D ). Typically shaped stylet furcae, with oval condyles supported by short branches provided with rounded apophyses. In the pharynx ( Fig. 10B ): large and triangular pharyngeal apophyses overlapping the first macroplacoid; two rod-shaped macroplacoids, length sequence 2 <1 (in lateral view), and evident drop-shaped microplacoid. In frontal view, the first macroplacoid is in the shape of a drop with a medial slight constriction longer than the second, the second rectangular with rounded corners and with a small terminal slight constriction. Figure 10. Xerobiotus litus . A, animal in toto . B, buccopharyngeal apparatus. C, buccal armature (dorsal view). D, buccal armature (ventral view). E, claw II. F, claw IV (arrowheads indicate thickenings under claw base). G, legs I and II, with the swellings of the furbelow-like structure (arrowheads). H, egg. I, egg surface and processes. J, sperm in the gonad (stained with Orcein). A, C–G, PhC; B, H–J, DIC. Scale bars: 20 µm in A, G–I; 10 µm in B–F, J. Figure 11. Xerobiotus litus . A, animal in toto . B, crybrose area and pores of the posterolateral sensory field (black arrowhead); white arrowheads indicate granules on the cuticle. C, legs II–III, with furbelow-like structures; arrowhead indicates pore. D, claws II. E, egg processes. F, egg surface and processes. A–F, SEM. Scale bars: 50 µm in A; 3 µm in B; 10 µm in C, E; 5 µm in D, F. Claws I–III of Xerobiotus type , with thick base (without lunules), a triangular common tract and a right angle between the two claw branches ( Figs 10E , 11C, D ). Claw IV with a longer common tract, small and short claw branches, proximal portion laterally enlarged to form a concavity (lunules absent), and a cuticular thickening in the shape of a ring with irregular margins present under the claw base (and connected with it) ( Fig. 10F ). Main branches of all claws with small accessory points. Eggs spherical, laid freely ( Figs 10H , 11E ). Surface between processes of hufelandi type , i.e. covered with a reticulum formed by a mesh of small, densely distributed pores, uniform in size and evenly distributed ( Figs 10I , 11F ). Processes of the hufelandi type , relatively large and with a straight trunk and a large and convex terminal disc. The edge of the terminal discs indented; indentations arranged regularly to form a crenated circumference and ornamented with granules (visible only with SEM; Fig. 11F ). Egg with an embryo not found; the eggs were aưributed to this species for the morphological affinities of their morphologies to the other eggs of the genus. Reproduction: Gonochoristic amphimictic species; females and male present. Molecular characterization: Four haplotypes for cox1 (p-distance 0.2%–0.9%), two haplotypes for ITS2 (p-distance 0.2%), two haplotypesfor 18S, andonehaplotypefor 28Sgenes (GenBankaccession numbers in Supporting Information, Table S1 ; p-distances in Supporting Information, Table S8). The more similar sequences of X. litus belong:for cox1 , to Xerobiotus arenosum and to Xerobiotus n. sp. 1, with p-distances of 9.3%; for ITS2, to X. gretae and Xerobiotus n. sp. 1, with p-distances of 0.4%–0.7% and 0.2%–2.0%, respectively (Supporting Information, Table S8). Differential diagnosis: Xerobiotus litus differs from all other Xerobiotus species by a common tract in claw IV, with proximal portion laterally enlarged, generating a concavity, and the largest terminal disc of the egg processes among all the other species of the genus (diameter 4.5–7.0 µm). Moreover, X. litus differs from: X. reductus by the shape of claws I–III (with wider and more triangular common tract) and claws IV (with proximal portion laterally enlarged, forming a concavity and a cuticular thickening under the claw base), and convex terminal discs of egg processes; X. euxinus by the absence of small plates at the base of claws I–III (visible with LM as semilunar thickening), the absence of lunules in claws IV, and convex terminal discs of egg processes; X. gretae by the shape of claws I–III (more stumpy, with triangular common tract and a clear right angle between the two claw branches), the absence of lunules in claws IV, and convex terminal discs of egg processes; X. naginae by the shape of the claw I–III (with triangular common tract and right angle between the two claw branches), and convex terminal discs of egg processes; X. xerophilus by the absence of cuticular plates (visible only with SEM) at the base of all claws ( Dastych and Alberti 1990 ) and the shape of egg processes (without a funnel-like depression).