Two new tardigrade species from Romania (Eutardigrada: Milnesiidae, Macrobiotidae), with some remarks on secondary sex characters in Milnesium dornensis sp. nov. Author Roszkowska, Daniel Adrian Ciobanu Milena Author Kaczmarek, Łukasz text Zootaxa 2015 3941 4 542 564 journal article 41596 10.11646/zootaxa.3941.4.4 0e136add-c127-45be-bfe9-0987b523fb87 1175-5326 233659 66179A2D-14A6-4C5F-91F7-331E7922D1B0 Milnesium dornensis sp. nov. http://www.tardigrada.net/register/0018.htm ( Figs 1 14 , Tables 1 2 ) Material examined: Holotype (female), 46 paratypes ( 36 females and 10 males ) and five exuvia with 18 smooth eggs. Description of the adults ( Figures 1 2 ) (measurements in µm, pt ratios 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 granulatum group ( 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, 9 and 11 13 ). Primary claws on all legs are simple, unbranched with small accessory points detaching from the branch at its greatest curvature ( Figs 10 and 14 ). Secondary claws on all legs with rounded basal thickenings (lunules) (sometimes barely visible) ( Figs 9 and 13 ). Secondary claws on all legs with three branches (claw configuration: [3-3]-[3-3]) ( Figs 7, 9 and 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 7 and 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 Milnesium species. Locus typicus : 47°20'13.7''N , 25°19'38.8''E ; 968 m asl: Romania , Suceava County, Vatra Dornei town, lichen ( Usnea sp .) from tree ( Picea abies L., H. Karst.). Etymology: Milnesium dornensis is named after Vatra Dornei, the town, where the species was found. Type depositories: Holotype (female; slide: VD-7), 36 paratypes ( 29 females and 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 pt values 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 pt values 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 granulatum group (Michalczyk et al. 2012a,b). The new species is most similar to M. alabamae Wallendorf & Miller, 2009 , M. beasleyi Kaczmarek et al ., 2012 a, M. granulatum ( Ramazzotti, 1962 ) , M. katarzynae Kaczmarek et al ., 2004 , M. krzysztofi Kaczmarek & Michalczyk, 2007 , M. lagniappe Meyer et al ., 2013 and M. reticulatum Pilato et al ., 2002 , but it differs from: M. alabamae by: different dorsal sculpture (pseudopores not arranged in bands, sparsely distributed and not forming a reticular design in M. dornensis sp. nov. vs pseudopores 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 pt of the body length ([ 1,496–1,986 ] in M. dornensis sp. nov. vs [ 821–1,388 ] in M . alabamae ). M. beasleyi by: 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 pt of peribuccal papillae length ([ 22.0–28.3 ] in M. dornensis sp. nov. vs [ 19.6–21.3 ] in M . beasleyi ), larger pt of the anterior buccal tube width ([ 42.5–54.5 ] in M. dornensis sp. nov. vs [ 35.3–41.8 ] in M . beasleyi ) larger pt of 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. granulatum by: a different dorsal sculpture (pseudopores not arranged in bands, sparsely distributed and not forming reticular design in M. dornensis sp. nov. vs pseudopores densely distributed and forming a reticular design in M. granulatum ) different lengths of claws on legs IV (see Table 1 below and Table 2 in 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. katarzynae by: 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. vs pseudopores densely distributed and forming a reticular design in M. katarzynae ), a larger body size (384 874 µm in M. dornensis sp. nov. vs 285.0 294.5 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. vs 6.6–7.6 Μm in M. katarzynae ) and a different pt of buccal tube standard width ([ 37.8–51.6 ] in M. dornensis sp. nov. vs [ 21.7–26.6 ] in M. katarzynae ). M. krzysztofi by: 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. vs pseudopores densely distributed and forming a reticular design in M. krzysztofi ), larger pt of 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. vs I: 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. lagniappe by: 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. vs nine 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 pt of 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. vs 63% 78% in M. lagniappe ) and a smaller pt of 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. reticulatum by: 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. vs pseudopores 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 1 below and Table 2 in Pilato et al. (2002) for the exact differences in dimensions of the claws).