Macrobiotus wandae Kayastha & Berdi & Mioduchowska & Gawlak & Łukasiewicz & Gołdyn & Kaczmarek 2020, sp. nov.

Macrobiotus wandae sp. nov.

(Tables 2–3, Figs. 1–18)

Type material examined. 88 adults and 8 eggs (holotype and 95 paratypes) on microscope slides in Hoyer’s medium.

Description of the new species. Adults (measurements and statistics in Table 2). Body transparent after fixation in Hoyer’s medium, eyes present in 90% of fixed specimens (Fig. 1). Entire cuticle covered with conspicuous round and lenticular pores (1.0– 3.6 µm in diameter) distributed randomly, but larger pores present on dorsal side in the anterior and posterior part of the body (Figs 3–4). Bucco-pharyngeal apparatus of the Macrobiotus type, with ventral lamina and ten peribuccal lamellae (Fig. 5). Mouth antero-ventral. Oral cavity armature of the maculatus type, with only third bands of teeth visible under LM (Fig. 6). The third band of teeth is composed of three dorsal and three ventral teeth, which appear under LM in the shape of transverse ridges (Fig. 6, arrow). Pharyngeal bulb spherical with triangular apophyses, two rod-shaped macroplacoids and a triangular microplacoid. Macroplacoid length configuration 2<1 (Fig. 5). The first macroplacoid possesses a central constriction (Fig. 5, the empty arrowhead). The second macroplacoid with sub-terminal constriction (Fig. 5, the filled arrowhead). Claws of the hufelandi type, stout (Figs 7–8, 11–12). Primary branches with distinct accessory points. Lunules under all claws smooth (Figs 7, 11). Thin and paired cuticular bars under claws I–III present. Easily visible granulation present on legs I–IV (Figs 7, 9–11). Other cuticular thickenings on legs absent.

Eggs (measurements and statistics in Table 3). Eggs spherical, ornamented and laid freely (Figs 13–18), with a chorion surface of the maculatus type with peribasal pores around each processes (Figs 15–16, empty arrowheads). Peribasal pores (ca. 15 around each process) elongated 1.4–1.7×0.7–1.0 in diameter. In general, it is one row of pores around each process but some additional pores could be also present. Processes in the shape of concave cones with terminal discs. Terminal discs slightly concave or flat with serrated margins or with irregular teeth (Figs 17–18, filled arrowheads). In SEM on the surface of the discs small granules present (Fig. 18, filled arrowheads).

Etymology. We dedicate this species to Wanda Rutkiewicz, who was an outstanding Polish mountain climber. She was the first European woman to reach the summit of Mount Everest and the first woman who reached K2. In total she summited eight eight-thousanders. She died in Himalaya, climbing the summit of Kangchenjunga in May 1992.

Type locality. 27°58’48’’N, 86°49’43’’E, 5,170 m a.s.l., Nepal, Koshi /Purbanchal, Gorak Shep, moss on stone, 02 October 2018, coll. Aleksandra Łukasiewicz and Bartłomiej Gołdyn.

Type depositories. Holotype and 87 paratypes (including 5 exuviae) (slide: 5.1, 5.2, 5.3) and eight eggs (slide: 5.4) are deposited at the Department of Animal Taxonomy and Ecology, Institute of Environmental Biology, Adam Mickiewicz University in Poznań, Uniwersytetu Poznańskiego 6, 61-614 Poznań, Poland.

Differential diagnosis. Based on egg processes morphology (egg shell between processes with rings of peribasal pores – maculatus type), the new species is most similar to Mac. biserovi Bertolani, Guidi and Rebecchi 1996, Mac. denticulus Dastych 2002, Mac. diversus Biserov 1990a, Mac. glebkai Biserov 1990b, Mac. macrocalix Bertolani and Rebecchi 1993, Mac. maculatus Iharos 1973 and Mac. ramoli Dastych 2005 but differs specifically from:

Mac. biserovi, known only from the type locality in Italy (Bertolani et al. 1996), by: a different oral cavity armature type (maculatus type in new species vs hufelandi type in Mac. biserovi), smooth lunules

under claws IV (slightly dentate in Mac. biserovi), different egg shell sculpture (large oval pores situated regularly around the egg processes bases in new species vs small round pores distributed randomly on entire egg shell in Mac. biserovi), shorter egg processes (4.1–6.0 µm in new species vs 7.7–12.2 µm in Mac. biserovi), narrower egg processes bases (4.8–6.3 µm in new species vs 8.3–9.2 µm in Mac. biserovi) and larger pores around egg processes (1.4–1.7×0.7–1.0 µm in diameter in new species vs 0.3–0.6 µm in diameter in Mac. biserovi).

Mac. denticulus, known from Kerguelen Islands (Dastych 2002), by: absence of additional tooth situat- ed at the level of stylet sheath, presence of granulation on legs I–III, higher pt of stylet support insertion point (77.2–79.8 in new species vs 71.2–76.7 in Mac. denticulus), higher number of processes on the egg circumference (33–37 in new species vs 22–29 in Mac. denticulus), shorter egg processes (4.1–6.0 µm in new species vs 8.1–12.6 µm in Mac. denticulus) and narrower egg processes bases (4.8–6.3 µm in new species vs 8.1–12.0 µm in Mac. denticulus).

Mac. diversus, known only from Russia (Biserov 1990a, 1991), by: presence of three dorsal ridges in oral cavity (only one present in Mac. diversus) smooth lunules under claws IV (dentated in Mac. diversus), different egg shell sculpture (large oval pores situated regularly around the egg processes bases in new species vs small round pores distributed randomly on entire egg shell in Mac. diversus), shorter egg processes (4.1–6.0 µm in new species vs 6.0–10.0 µm in Mac. diversus) and narrower egg processes bases (4.8–6.3 µm in new species vs 8.3–10.5 µm in Mac. diversus).

Mac. glebkai, known only from Russia (Biserov 1990b, 1991), different egg processes (typical stout inverted goblets vs cones with very long and slender endings and poorly visible terminal discs in Mac. glebkai), a higher number of processes on the egg circumference

(33–37 in new species vs 12–14 in Mac. glebkai), shorter egg processes (4.1–6.0 µm in new species vs 15.0– 22.5 µm in Mac. glebkai) and narrower egg processes bases (4.8–6.3 µm in new species vs 15.0–18.0 µm in Mac. glebkai).

Mac. macrocalix, recorded from several localities in Europe and the Seychelles (Bertolani and Rebecchi 1996, Vargha 1998, Kaczmarek and Michalczyk 2002, Pilato et al. 2002, Jönsson 2007, Guil 2008), by: a different oral cavity armature type (maculatus type in

new species vs hufelandi type in Mac. macrocalix), a higher number of processes on the egg circumference (33–37 in new species vs ca. 26 in Mac. macrocalix), shorter egg processes (4.1–6.0 µm in new species vs 7.7–12.0 µm in Mac. macrocalix), narrower egg processes bases (4.8–6.3 µm in new species vs 8.6–12.1 µm in Mac. macrocalix), smaller diameter of terminal discs on the egg processes (2.7–3.5 µm in new species vs 7.2–10.9 µm in Mac. macrocalix) and larger pores around egg processes (1.4–1.7×0.7–1.0 µm in diameter in new species vs up to 0.6 µm in diameter in Mac. macrocalix).

Mac. maculatus, known only from the type locality in New Guinea (Iharos 1973) and later redescription (Kaczmarek and Michalczyk 2017b), by: the presence of sub-terminal constriction in second macroplacoid, smaller maximum size of cuticular pores (up to 3.6 µm in new species vs pores up to 9.5 µm in Mac. maculatus), shorter macroplacoids and claws (for details see Table 2 in this paper and Table 2 in Kaczmarek and Michalczyk (2017b)), more stout egg processes (very slender in Mac. maculatus), different terminal discs of egg processes (serrated margins or with irregular teeth on their margins in the new species vs smooth in

Mac. maculatus), shorter egg process (4.1–6.0 µm in new species vs 7.1–9.5 µm in Mac. maculatus) and higher processes base/height ratio (100–137% in new species vs 71–83% in Mac. maculatus).

Mac. ramoli, known only from Austria (Dastych 2005), by: smooth lunules under claws IV (strongly dentated in Mac. ramoli) and different egg shell sculpture (oval large: 1.4–1.7×0.7–1.0 µm in diameter pores in new species vs small: 0.2–0.5 µm in diameter round pores in Mac. ramoli).

Genotypic differential diagnosis

All the obtained DNA barcode sequences of Mac. wandae sp. nov. were unique and distinct from those deposited in GenBank (see Appendix 1 and 2). The COI sequences (GenBank accession numbers: MN482681 – MN482687, seven specimens) were 619–644 bp-long and two distinct haplotypes were found. No insertions, deletions or stop codons were identified. The translation was successfully carried out with the invertebrate mitochondrial codon table and the -3 th reading frame. The p-distance between Mac. wandae sp. nov. haplotypes (H01 and H02) was 0.16%. The p-distances between COI haplotypes of all species of the hufelandi group available in GenBank ranged from the most similar 17.21%, for Mac. sandrae Bertolani and Rebecchi 1993 (haplotype H23 / GenBank accession numbers: HQ876569 –73, HQ876566 –67 and haplotype H24 / Gen- Bank accession numbers: HQ876574, HQ876577 –79, HQ876581), to least similar 28.36% for Mac. polypiformis Roszkowska, Ostrowska, Stec, Janko and Kaczmarek, 2017 (haplotype H20 / GenBank accession number: KX810012), with an average distance of 20.19%.

The 18S rRNA sequences (GenBank accession numbers: MN435109 – MN435113, five specimens) were 990–997 bp-long and no genetic differences between the studied specimens were found. The ranges of uncorrected genetic p-distances were 0.13% between the most similar species of Mac. hufelandi group (GenBank accession number: FJ435738 –39) and 3.87% between the least similar Mac. polypiformis (Gen- Bank accession number: KX810008), with an average distance of 1.7%.

The 28S rRNA sequences (GenBank accession numbers: MN435114 – MN435119, six specimens) were 777–793 bp-long. No genetic differences in conservative 28S rRNA gene fragment was observed. The p-distances between Mac. wandae sp. nov. and species of the hufelandi group, for which sequences of this molecular marker are available in GenBank, are as follows: species of the Mac. hufelandi group (Gen- Bank accession numbers: FJ435751, FJ435754 –55) – 0.45% and Mac. polypiformis (GenBank accession number: KX810009) – 12.24%, with an average distance of 6.13%.

The ITS2 sequences (GenBank accession numbers: MN435120 – MN435122, three specimens) were 424 bplong and no genetic differences were found. The ranges of uncorrected genetic p-distances were 8.76% between the new species described and the most similar Mac. macrocalix (GenBank accession number: MH063931), the least similar was Mac. polypiformis (GenBank accession number: KX810010) – 30.41%, with an average distance of 19.59%.

Phylogenetic analyses

For the mtDNA data, 77 sequences (32 haplotypes; Appendix 1) of 17 taxa of the hufelandi group were compared. The alignment of the COI gene produced 611 characters, 273 of which were variable and 257 parsimony-informative. The phylogenetic analysis showed that the new species belongs to the hufelandi group of species. The topology of the ML tree revealed an evident two highly supported clades (Fig. 19, colour lines). Macrobiotus wandae sp. nov. was clustered together with Mac. sandrae, Mac. terminalis Bertolani and Rebecchi 1993, Mac. vladimiri Bertolani, Biserov, Rebecchi and Cesari 2011a and Mac. macrocalix. The same was earlier shown and discussed by Stec et al. (2018a, b) and possibly connected with morphology of adults and eggs. However, taking into consideration, taxonomic position of some sequences deposited in GenBank, described as Mac. hufelandi group still needs further verification.