Paramacrobiotus metropolitanus Sugiura & Matsumoto & Kunieda 2022, sp. nov

Species Paramacrobiotus metropolitanus sp. nov. Sugiura, Matsumoto & Kunieda, 2022 (Figs 1–7, Tables 2–3) Paramacrobiotus sp. TYO in: Sugiura et al. (2019), Hara et al. (2021), Sugiura & Matsumoto (2021), and Sugiura et al. (2022). Material examined Forty animals (NMST-Tg-281–310, NHMD-918038–918047, holotype: NMST-Tg-299) and 33 eggs (NMST-Tg-311–333, NHMD-918048–918057) were examined under light microscopes and were deposited in National Museum of Nature and Science (NMST), Tsukuba, Japan, and Natural History Museum (NHMD), Copenhagen, Denmark, respectively. In addition, over 30 animals and eggs were observed with the scanning electron microscope and stored in the laboratory of Keio University. Description Measurements and statistics of animals are given in Table 2. The Holotype is illustrated in Fig. 1A and a paratype observed with SEM in Figs 1B–C. Adult P. metropolitanus sp. nov. specimens whitish under dark-field illumination, the juvenile transparent (data not shown). Eyes absent in live animals. Mouth anteroventral (Figs 2A–C). Buccopharyngeal apparatus the Macrobiotus type, with ten peribuccal lamellae around the mouth (Fig. 2A). Numerous conical teeth present, forming the first band of teeth posterior to the peribuccal lamellae. Conical teeth present at just behind the ring fold, forming the second band, and ridges parallel to the main axis of the buccal tube as the third band (Figs 2A–C). Approximately 20–30 small and sharp ridges on the second band, and two to four larger ridges on the third band (Fig. 2A–C). Three macroplacoids present in the pharynx, the length sequence 2hufelandi type (Y-shaped, Figs 3–4). Smooth cuticle with a cuticular bulge on the inner side of legs I–III (Fig. 3), whereas granulation on the inner surface of leg IV visible under both PCM and SEM (Figs 4A–B). Primary branches with two distinct accessory points (Fig. 4C). A long common tract with a stalk connects the claw to the lunule. Lunules smooth (Fig. 4C). Smooth body cuticle without pores, sculpturing or granulation (Figs 1 and 5). Muscle attachments (apodemes/cribriform areas) clearly visible on the cuticle, with two holes, each with densely packed pores (Fig. 5). Eggs laid freely, yellow yolk seen internally and transparent conical processes on the surface present in PCM (Figs 6A–B). Reticulated pattern with annual rings present on the surface of the processes, with slightly rough tops (Figs 6C–D). Approximately 10 areolae around each process on the surface of the egg (Fig. 6B). Smooth surface of areoles (Fig. 6E). Morphometric data are available in Table 3. All morphometric data are listed in Supporting Materials SM.01. Type locality 35.742983°N, 139.549757°E; 55 m asl: Japan, Honshu mainland, Kanto region, Tokyo, Nishi-Tokyo city, Tozenji temple; urban cemetery; bamboo leaf litter. The locality described in Sugiura et al. (2019) was corrected in Hara et al. (2021). Etymology The species name of “ metropolitanus ” refers to the Tokyo Metropolitan Area (Tokyo Cho-mei mushi in Japanese), the region where the species was collected. Sequence data We confirmed 100% matches between the sequence of PCR-amplicon and the sequence retrieved from the published genome of the species for 18S rDNA, 28S rDNA and ITS-2 sequences. As for COI sequence, one mismatched base was found between the sequence of 658 bp PCR-amplicon and the sequence retrieved from the genome. The sequences were deposited in GenBank with IDs: LC637243 (18S rDNA), LC649795 (28S rDNA of the PCR amplicon using primer-set of 28S_Eutar_F and 28SR0090), LC649797 (28S rDNA of the PCR amplicon using primer-set of 28SF0001 and 28SR0990), LC649794 (ITS-2), LC637242 (COI sequence deduced from the genome), and LC649796 (COI sequence of PCR-amplicon). Phenotypic differential diagnosis The new species P. metropolitanus sp. nov. with microplacoid, smooth lunules, and its egg without pored surface on the areolae is similar to the following seven species but differs specifically from (data from original descriptions of the compared species): • P. danielae Pilato, Binda, Napolitano & Moncada 2001, known from Ecuador and Peru, by the lack of eyes, and the lack of body granulation, larger body length (200–390 μm in P. danielae vs 393–711 μm in the new species). • P. experimentalis, known only from Madagascar, by the lack of wrinkled surface on the egg areolae, the presence of cuticular bulge on inner surface of claws I–III and granulation on inner surface of leg IV. • P. garynahi Kaczmarek, Michalczyk & Diduszko, 2005, known only from Russia, by the lack of oval pores on the cuticle, the lack of granulation in the first three pairs of legs, the lack of a cap-like structure on the top of the egg process, smaller egg bare diameter (96.0–132.0 μm in P. garynahi vs 58.1–90.1 μm in the new species), and by a smaller egg diameter with processes (142.0–180.0 μm in P. garynahi vs 81.7–112.1 μm in the new species). • P. hapukuensis Pilato, Bind & Lisi, 2006, known only from New Zealand, by the lack of finger-shaped apices of egg processes, and the presence of leg granulation. • P. peteri Pilato, Claxton & Binda, 1989, known only from Australia and Indonesia (Pilato et al. 1989; Pilato and Binda, 1990; Mackness, 2003), by the lack of a cuticular thickening near the lunules I–III, the lack of subdivided apices of egg processes, a lower number of processes on the egg circumference (16–18 in P. peteri vs 10–15 in the new species), and by a higher number of areolae around egg processes (6–7 in P. peteri, approximately 10 in the new species). • P. rioplatensis Claps & Rossi, 1997, known only from Uruguay, by the lack of eyespots, a larger body (227– 372 μm in P. rioplatensis vs 392.5–710.8 μm in the new species), a longer buccal tube (38 μm in P. rioplatensis, over 40–58.4 μm in the new species), a larger egg full diameter (68–79 μm in P. rioplatensis vs 81.7–112.1 μm in the new species), the lack of the a filament-shaped apex of egg processes, higher egg processes (4.6 μm height in P. rioplatensis vs over 7.4 μm in the new species), and by a lower number of processes on the egg circumference (17–19 in P. rioplatensis vs 10–15 in the new species). • P. savai Binda & Pilato, 2001, known from only Sri Lanka, by the lack of eyespots, the lack of a cuticular thickening near the lunules of the first three legs, a different egg process shapes (blunt in P. savai vs conical in the new species), and by a slightly smaller egg full diameter (115 μm in P. savai vs 81.7–112.1 μm in the new species). Genetic differential diagnosis Below is a summary of genetic p-distances, whereas detailed matrices are available in Supporting Material SM.02. • 18S rDNA: 0.5%–3.6%, with the most similar species being P. experimentalis from Madagascar (MN073467 –8 and MH664938) and the least similar being P. areolatus NO. 385 from Svalbard (MH664931). • 28S rDNA: 0.98–7.5%, with the most similar species being P.experimentalis (MN073465 –6and MH664956) and the least being Paramacrobiotus tonollii Ramazzotti, 1956 strain US.035 from USA (MH664963). • ITS-2: 9.9–33.9%, with the most similar species being P. experimentalis (MH073463 –4 and MH666087) and the least similar species being P. tonollii (GQ403679, MH666096). • COI: 21.2–32.6%, with the most similar species being Paramacrobiotus sp. strain TZ.018 from Tanzania (MH676017) and with the least similar species being Paramacrobiotus depressus Guidetti, Cesari, Bertolani, Altiero & Rebecchi, 2019 from Italy (MK041013). Phylogeny The supported bootstrap values of ML trees were usually low (P. kenianus, P. experimentalis, and an undescribed species from Tanzania) and the Neotropics (two unidentified species from Brazil). Genetic species delineation Both, the ITS-2 and the COI BI trees indicated that the cluster of P. metropolitanus sp. nov. is a separate species with>93% probabilities by both maximum likelihood and Bayesian supported solutions. (A) SEM image of the postero-dorsal surface of P. metropolitanus sp. nov. (B) Magnified image of dorsal cuticle apodemes corresponding to the white dashed box in A. (C) Magnified image of the left apodemes in B. White-filled indented arrowheads indicate the apodemes. Scale bars = μm.