Two new tardigrade species from Romania (Eutardigrada: Milnesiidae, Macrobiotidae), with some remarks on secondary sex characters in Milnesium dornensis sp. nov. Roszkowska, Daniel Adrian Ciobanu Milena Kaczmarek, Łukasz Zootaxa 2015 3941 4 542 564 [151,405,801,827] Polychaeta Aphroditidae Milnesium Animalia Phyllodocida 2 544 Annelida species dornensis sp. nov.   Material examined: Holotype(female), 46 paratypes( 36 femalesand 10 males) and five exuvia with 18 smooth eggs.   Description of the adults ( Figures 1 –2) (measurements in µm, ptratios and statistics in Tables 1 –2):Body brownish (in live specimens) or transparent (after fixation in Hoyer’s medium). Eyes present or absent (visible before and after mounting; 68% of fixed specimens had eyes). Cuticle sculptured with pseudopores (0.5–0.7 Μm in diameter in females and 0.3–0.5 Μm in males), not arranged in bands, sparsely distributed and not forming a reticular design. Under PCM these pseudopores are visible as light spots, placing the species within the  granulatumgroup ( Figs 4 and 6). On the dorsal side, in the caudal region, an area similar to plate structures is visible ( Fig. 8). These plates are visible only in a few females and at present it is hard to evaluate their taxonomic significance. Six peribuccal papillae and six peribuccal lamellae around the mouth opening present ( Figs 3 and 5). Two cephalic papillae, positioned laterally. Bucco-pharyngeal apparatus of the  Milnesium type( Figs 3 and 5). Buccal tube slightly funnel-shaped, wider anteriorly (posterior diameter on average 90% of the anterior diameter). Pharyngeal bulb elongated, pear-shaped and without placoids or septulum. Claws of the  Milnesium type, slender ( Figs 7, 9and 11 –13). Primary claws on all legs are simple, unbranched with small accessory points detaching from the branch at its greatest curvature ( Figs 10and 14). Secondary claws on all legs with rounded basal thickenings (lunules) (sometimes barely visible) ( Figs 9and 13). Secondary claws on all legs with three branches (claw configuration: [3-3]-[3-3]) ( Figs 7, 9and 12–13), with the exception of the sexually dimorphic modified claws of the males of first pair of legs ( Fig. 11). Single, long and transverse cuticular bars under claws I –III present ( Figs 7and 12). In males, the cuticular bars on first pair of legs distinctly wider than those on legs II–III.  Eggs:Oval, smooth and deposited in exuvium as in all other known  Milnesiumspecies.   Locus typicus: 47°20'13.7''N, 25°19'38.8''E; 968 masl: Romania, Suceava County, Vatra Dornei town, lichen (  Usneasp.) from tree (  Picea abiesL., H. Karst.).   Etymology:  Milnesium dornensisis named after Vatra Dornei, the town, where the species was found.    Typedepositories: Holotype(female; slide: VD-7), 36 paratypes( 29 femalesand seven males; slides VD1, VD2, VD10, VD11, VD13, VD14) and one exuvium with eggs (slide VD10) are preserved at the Department of Animal Taxonomy and Ecology, Adam Mickiewicz University in Poznań, Umultowska 89, 61–614 Poznań, Poland. Additionally, three paratypes(females) (slides: VD3, VD4, VD5) are deposited at the Natural History Museum of “Alexandru Ioan Cuza” University from Iaşi, Romania(Bd. Independenţei No.16, 700101) and eight paratypes(five females and three males; slides: VD6, VD8, VD9, VD12) are deposited at the Department of Zoology, Comenius University, Bratislava, Slovakia.   TABLE 1. Measurements and ptvalues of selected morphological structures of fifteen females from the type population of  Milnesium dornensis  sp. nov.mounted in Hoyer’s medium (N—number of specimens/ structures measured, RANGE refers to the smallest and the largest structure among all measured specimens; SD—standard deviation).    CHARACTER N RANGE MEAN SD Holotype  µm pt µm pt µm pt µm pt  Body length 15 384–874 1496–1986 679 1730 152 144 837 1816  Peribuccal papillae length 11 5.7–12.0 22.0–28.3 9.9 25.2 1.9 1.9 11.5 24.9  Lateral papillae length 14 4.2–9.2 15.0–23.7 7.2 18.7 1.2 2.4 6.9 15.0  Buccal tube  Length 15 24.5–46.1 - 39.0 – 6.7 – 46.1 –  Stylet support insertion point 14 16.2–31.1 64.3–68.1 26.1 66.0 4.3 1.3 31.1 67.5  Anterior width 15 10.7–24.0 42.5–54.5 18.1 46.3 3.8 3.3 22.9 49.7  Standard width 15 9.4–22.7 37.8–51.6 17.3 44.1 3.8 3.9 22.7 49.2  Posterior width 15 9.0–22.8 34.8–51.8 16.3 41.6 3.7 4.5 22.0 47.7  Standard width/length ratio 15 38%–52% - 44% – 4% – 49% –  Posterior/anterior width ratio 15 80%–98% - 90% – 6% – 96% –  Claw 1 lengths  External primary branch 14 11.6–22.0 40.1–50.4 18.1 46.7 3.5 3.1 20.8 45.1  External base + secondary branch 14 9.2–18.3 34.2–40.9 15.3 38.5 2.8 2.3 17.7 38.4  External spur 8 3.1–5.3 8.4–11.8 4.1 9.6 0.6 1.0 4.4 9.5  Internal primary branch 14 10.8–21.0 36.9–47.7 16.7 43.0 3.4 3.0 20.0 43.4  Internal base + secondary branch 14 8.8–17.2 33.0–38.6 14.4 36.3 2.5 1.8 16.6 36.0  Internal spur 10 4.3–6.3 10.6–14.4 5.3 12.7 0.7 1.5 4.9 10.6  Claw 2 lengths  External primary branch 15 12.0–22.0 44.7–52.4 18.9 48.5 3.5 1.9 21.7 47.1  External base + secondary branch 14 9.8–18.2 35.3–40.8 15.0 38.3 2.5 1.7 18.2 39.5  External spur 7 2.9–5.8 7.6–14.1 4.3 10.6 1.1 2.1 4.7 10.2  Internal primary branch 15 11.5–22.0 41.3–51.2 17.6 45.2 3.2 2.7 20.4 44.3  Internal base + secondary branch 14 9.4–16.5 33.8–39.1 14.1 36.0 2.1 1.9 16.3 35.4  Internal spur 9 4.1–6.5 10.3–14.6 5.4 13.1 0.9 1.5 6.5 14.1  Claw 3 lengths  External primary branch 15 13.2–23.7 47.0–54.4 19.6 50.4 3.4 2.6 23.0 49.9  External base + secondary branch 13 10.0–17.8 36.7–43.7 15.2 39.5 2.3 2.2 17.0 36.9  External spur 10 2.6–6.0 8.9–13.6 4.1 10.7 0.9 1.7 4.5 9.8  Internal primary branch 14 12.4–21.8 42.2–50.6 18.3 47.3 3.3 2.3 21.1 45.8  Internal base + secondary branch 13 9.0–17.3 33.3–41.7 14.3 37.1 2.3 2.6 16.6 36.0  Internal spur 9 3.5–6.2 11.3–14.3 5.1 13.1 0.9 1.2 5.2 11. 3  Claw 4 lengths  Anterior primary branch 13 14.8–30.7 57.9–67.0 25.2 64.1 4.8 3.1 30.7 66.6  Anterior base + secondary branch 12 10.3–21.4 40.9–50.3 18.0 45.5 3.0 3.3 21.2 46.0  Anterior spur 12 3.5–6.6 12.1–17.9 5.6 14.2 0.9 1.5 5.6 12.1  Posterior primary branch 13 16.1–30.4 58.0–69.1 24.9 63.7 4.2 3.5 29.0 62.9  Posterior base + secondary branch 13 11.5–22.2 44.7–52.6 19.0 48.6 3.0 2.7 21.1 45.8  Posterior spur 11 3.1–6.0 8.2–14.1 4.5 11.4 0.9 2.1 4.5 9.8   TABLE 2. Measurements and ptvalues of selected morphological structures of ten males from the type population of  Milnesium dornensis  sp. nov.mounted in Hoyer’s medium (N—number of specimens/ structures measured, RANGE refers to the smallest and the largest structure among all measured specimens; SD—standard deviation).    CHARACTER N RANGE MEAN SD  µm pt µm pt µm pt  Body length 10 448–498 1500–1938 474 1647 18 121  Peribuccal papillae length 10 4.1–4.9 12.5–19.1 4.5 15.8 0.3 1.9  Lateral papillae length 10 5.1–5.9 16.5–21.5 5.6 19.5 0.2 1.7  Buccal tube  Length 10 25.7–32.7 - 28.9 – 2.1 –  Stylet support insertion point 10 15.8–21.0 61.5–64.7 18.3 63.4 1.5 1.1  Anterior width 10 10.6–12.9 36.5–44.7 11.3 39.2 0.8 2.4  Standard width 10 9.0–10.4 30.6–38.9 9.8 33.9 0.5 2.5  Posterior width 10 8.7–10.8 30.4–37.0 9.7 33.6 0.6 1.8  Standard width/length ratio 10 31%–39% - 34% – 3% –  Posterior/anterior width ratio 10 80%–90% - 86% – 3% –  Claw 1 lengths  External primary branch 4 15.0–18.1 53.6–69.3 17.1 62.5 1.4 6.5  External base + secondary branch 5 15.1–18.2 53.9–63.3 16.7 59.2 1.3 3.4  External spur 0 ? ? ? ? ? ?  Internal primary branch 5 14.1–20.4 50.4–65.8 17.1 60.6 2.4 5.9  Internal base + secondary branch 6 15.0–19.0 53.6–68.1 17.3 61.5 1.7 5.4  Internal spur 0 ? ? ? ? ? ?  Claw 2 lengths  External primary branch 10 17.7–20.0 60.3–71.2 19.2 66.5 0.7 3.7  External base + secondary branch 10 13.1–15.8 47.1–56.0 15.0 51.9 0.7 2.5  External spur 6 3.1–4.0 11.3–15.6 3.6 12.5 0.4 1.7  Internal primary branch 9 16.6–19.4 51.7–66.5 18.1 62.0 0.9 4.7  Internal base + secondary branch 10 11.9–15.3 45.6–54.4 14.3 49.4 1.0 3.1  Internal spur 7 4.1–4.9 13.2–17.1 4.5 15.2 0.3 1.6  Claw 3 lengths  External primary branch 9 17.3–20.0 61.2–72.0 19.0 65.6 0.9 3.7  External base + secondary branch 8 13.1–15.4 47.1–53.2 14.6 50.1 0.8 1.9  External spur 2 2.6–2.8 9.0–9.3 2.7 9.1 0.1 0.2  Internal primary branch 9 16.5–19.3 55.0–66.4 17.9 61.8 1.0 3.9  Internal base + secondary branch 8 13.1–15.1 45.9–54.5 14.3 49.2 0.7 2.7  Internal spur 6 4.0–5.7 12.9–21.4 4.8 16.4 0.7 3.5  Claw 4 lengths  Anterior primary branch 10 20.9–24.4 71.0–88.7 22.7 78.9 1.1 6.1  Anterior base + secondary branch 10 14.4–17.0 50.6–61.5 15.7 54.5 0.8 3.6  Anterior spur 9 3.7–5.1 11.9–18.2 4.4 15.3 0.6 2.7  Posterior primary branch 9 21.3–25.0 76.1–89.5 23.2 80.4 1.1 4.7  Posterior base + secondary branch 9 15.0–16.6 51.3–61.1 15.8 55.6 0.5 3.1  Posterior spur 8 2.9–3.4 9.7–12.2 3.1 11.0 0.2 0.9   FIGURES 1–2.  Milnesium dornensis  sp. nov.habitus: 1—female (ventral view); 2—male (ventral view).   FIGURES 3–6.  Milnesium dornensis  sp. nov.female: 3—buccal apparatus (ventral view); 4—sculpture on dorsal cuticle; male: 5—buccal apparatus; 6—sculpture on dorsal cuticle (ventral view).   FIGURES 7–10.  Milnesium dornensis  sp. nov.female: 7—claws I; 8—dorsal plates on caudal end; 9—claws IV; 10—accessory point on main branch of claw.  Differential diagnosis (for adult females).Based on having a sculptured dorsal cuticle,  Milnesium dornensis  sp. nov.belongs to the  granulatumgroup (Michalczyk et al.2012a,b). The new species is most similar to  M. alabamae Wallendorf & Miller, 2009,  M. beasleyiKaczmarek et al., 2012a,  M. granulatum( Ramazzotti, 1962),  M. katarzynaeKaczmarek et al., 2004,  M. krzysztofi Kaczmarek & Michalczyk, 2007,  M. lagniappe Meyer et al., 2013and  M. reticulatum Pilato et al., 2002, but it differs from:   M. alabamaeby: different dorsal sculpture (pseudopores not arranged in bands, sparsely distributed and not forming a reticular design in  M. dornensis  sp. nov. vspseudopores arranged in bands (especially in caudal region), densely distributed and forming a reticular design in  M. alabamae), the presence of accessory points on primary branches of claws and a larger ptof the body length ([ 1,496–1,986] in  M. dornensis  sp. nov. vs[ 821–1,388] in  M. alabamae).   M. beasleyiby: a different claw configuration ([3-3]-[3-3] in  M. dornensis  sp. nov. vs[2-3]-[3-2] in  M. beasleyi), the presence of rounded basal thickenings under secondary branches of claws (sometimes poorly visible), larger ptof peribuccal papillae length ([ 22.0–28.3] in  M. dornensis  sp. nov. vs[ 19.6–21.3] in  M. beasleyi), larger ptof the anterior buccal tube width ([ 42.5–54.5] in  M. dornensis  sp. nov. vs[ 35.3–41.8] in  M. beasleyi) larger ptof the anterior primary branch of the claw IV ([ 57.9–67.0] in  M. dornensis  sp. nov. vs[ 53.1–57.4] in  M. beasleyi).   M. granulatumby: a different dorsal sculpture (pseudopores not arranged in bands, sparsely distributed and not forming reticular design in  M. dornensis  sp. nov. vspseudopores densely distributed and forming a reticular design in  M. granulatum) different lengths of claws on legs IV (see Table 1below and Table 2in Bartels et al.(2014)for the exact differences in dimensions of the claws).   FIGURES 11–14.  Milnesium dornensis  sp. nov.male: 11—modified claws I; 12—claws III; 13—claws IV; 14—accessory point on main branch of claw.   M. katarzynaeby: a different claw configuration ([3-3]-[3-3] in  M. dornensis  sp. nov. vs[2-2]-[2-2] in  M. katarzynae), different dorsal sculpture (pseudopores not arranged in bands, sparsely distributed and not forming a reticular design in  M. dornensis  sp. nov. vspseudopores densely distributed and forming a reticular design in  M. katarzynae), a larger body size (384 –874 µm in  M. dornensis  sp. nov. vs285.0 –294.5 µm in  M. katarzynae), the stylet supports inserted in a more anterior position ([ 64.3–68.1] in  M. dornensis  sp. nov. vs[ 73.3–78.3] in  M. katarzynae), a different buccal tube standard width (9.4–22.7 Μm in  M. dornensis  sp. nov. vs6.6–7.6 Μm in  M. katarzynae) and a different ptof buccal tube standard width ([ 37.8–51.6] in  M. dornensis  sp. nov. vs[ 21.7–26.6] in  M. katarzynae).   M. krzysztofiby: a different claw configuration ([3-3]-[3-3] in  M. dornensis  sp. nov. vs[2-3]-[3-2] in  M. krzysztofi), a different dorsal sculpture (pseudopores not arranged in bands, sparsely distributed and not forming a reticular design in  M. dornensis  sp. nov. vspseudopores densely distributed and forming a reticular design in  M. krzysztofi), larger ptof the body length ([ 1,496–1,968] in  M. dornensis  sp. nov. vs[ 1,262–1,425] in  M. krzysztofi) larger internal/anterior spurs of claws I –IV (I: 4.3 –6.3 Μm [ 10.6–14.4]; II: 4.1 –6.5 Μm [ 10.3–14.6]; III: 3.5 –6.2 Μm [ 11.3–14.3]; IV: 3.5 –6.6 Μm claws [ 12.1–17.9] in  M. dornensis  sp. nov. vsI: 2.5 –2.5Μm [ 10.1–10.1]; II: 2.5 –2.8 Μm [? –9.8]; ca.III: 3.4 Μm [ ca. 10.8]; IV: 2.7–3.2 Μm [ 8.0–10.1] in  M. krzysztofi).   M. lagniappeby: a different claw configuration ([3-3]-[3-3] in  M. dornensis  sp. nov. vs[2-3]-[3-2] in  M. lagniappe), a different dorsal sculpture (pseudopores not arranged in bands, sparsely distributed and not forming a reticular design in  M. dornensis  sp. nov. vsnine dorsal and lateral sculptured bands bearing a reticulated pattern of polygons in  M. lagniappe), having six peribuccal lamellae (four in  M. lagniappe), the stylet supports inserted in a more anterior position ([ 64.3–68.1] in  M. dornensis  sp. nov. vs[ 69.7–73.4] in  M. lagniappe), smaller ptof width of the buccal tube ([ 42.5–54.5], [ 37.8–51.6], [ 34.8–51.8] anterior, standard and posterior respectively in  M. dornensis  sp. nov. vs[ 68.0–77.5], [ 63.4–77.9], [ 61.8–70.8] anterior, standard and posterior respectively in  M. lagniappe), a smaller buccal tube standard width/length ratio (38% –52% in  M. dornensis  sp. nov. vs63% –78% in  M. lagniappe) and a smaller ptof the external base+secondary branch of claws I ([ 34.2–40.9] in  M. dornensis  sp. nov. vs[ 41.6–51.0] in  M. lagniappe).   M. reticulatumby: a different claw configuration ([3-3]-[3-3] in  M. dornensis  sp. nov. vs[2-3]-[3-2] in  M. reticulatum), a different dorsal sculpture (pseudopores not arranged in bands, sparsely distributed and not forming a reticular design in  M. dornensis  sp. nov. vspseudopores arranged in nine sculptured bands, forming a reticular design in  M. reticulatum), absence of cuticular gibbosities, having six peribuccal lamellae (four in  M. reticulatum), the stylet supports inserted in a more anterior position ([ 64.3–68.1] in  M. dornensis  sp. nov. vs[ 68.5–69.8] in  M. reticulatum) and larger claws I –IV (see Table 1below and Table 2in Pilato et al.(2002)for the exact differences in dimensions of the claws).