NewKaczmarek Zealand & Journal Michalczyk— of Zoology, 2007, krzysztofiVol. 34: 297–302, new from Costa Rica 297 0301–4223/07/3404–0297 © The Royal Society of New Zealand 2007

A new species of Tardigrada (Eutardigrada: ): Milnesium krzysztofi from Costa Rica (Central America)

Łukasz Kaczmarek 1990 (Chile), M. dujianensis Yang 2003 (China), Department of & Ecology M. eurystomum Maucci, 1991 (Greenland), M. ka- Institute of Environmental Biology tarzynae Kaczmarek et al., 2004 (China), M. longiun- A. Mickiewicz University gue Tumanov, 2006 (India), M. reductum, Tumanov Szamarzewskiego 91 a 2006 (Kirghizia), M. reticulatum Pilato et al., 2002 60-569 Poznań, Poland (Seychelles), M. slovenskyi Bertolani & Grimaldi, [email protected] 2000 (known only from amber), M. tardigradum Doyère, 1840 (described from France, Łukasz Michalczyk until recently thought to be cosmopolitan) and M. Centre for Ecology tetralamellatum Pilato & Binda, 1991 (Tanzania). Evolution and Conservation All species except for M. tardigradum are known School of Biological Sciences only from their type localities (Binda & Pilato 1990; University of East Anglia Maucci 1991; Pilato & Binda, 1991, 2001; McInnes Norwich NR4 7TJ, UK 1994; Bertolani & Grimaldi 2000; Pilato et al. 2002; [email protected] Yang 2003; Kaczmarek et al. 2004; Tumanov 2006). A new species, the fourteenth in the Milne- sium, is described and figured in this paper. Abstract A new species Milnesium krzysztofi sp. nov. is described from Costa Rica. M. krzysztofi sp. nov. differs from the most similar Milnesium katarzynae Kaczmarek et al., 2004 main- Material and Methods ly by the presence of spurs on internal claws I–III Thirteen specimens of M. krzysztofi sp. nov. were and on external claw IV. found in a lichen sample from an agricultural area near the road from Volcano Irazu to Cartago, 21.0 km Keywords Costa Rica; Milnesiidae; Milnesium to Cartago (Costa Rica). Ten specimens were mount- krzysztofi sp. nov., M. katarzynae; Tardigrada ed on microscopic slides in Hoyer’s medium and three remaining specimens were prepared for the scanning electron microscope (SEM). Introduction All measurements are given in micrometres (µm). Body length was measured from the mouth to the Until now only 13 species of the genus Milnesium end of the body including the hind legs. Buccal Doyére have been known: Milnesium almatyense tube length and level of the stylet support insertion Tumanov, 2006 (Kazakhstan), M. antarcticum Tu- point were measured from anterior margin of stylet manov, 2006 (Antarctica), M. asiaticum Tumanov, sheaths. Buccal tube widths were measured as the 2006 (Kirghizia), M. brachyungue Binda & Pilato external diameters at the level of the stylet support insertion point. The pt ratio is the ratio of the length of a given structure to the length of the buccal tube expressed as a percentage (Pilato 1981). In the de- scription of the holotype, the pt is given after the µm value (in square brackets and italic). Terminology Z07024; Online publication date 14 September 2007 describing the claws and measurements of claws are Received 4 June 2007; 23 August 2007 given according to Tumanov (2006). 298 New Zealand Journal of Zoology, 2007, Vol. 34

Fig. 1–3 Milnesium krzysztofi sp. nov.—habitus: 1, Ventro-lateral view (PCM); 2, lateral view (SEM); 3, dorsal view (SEM).

Taxonomic accounts retraction of the buccal tube. Buccal tube 26.7 long and 9.4 [35.2] wide (Fig. 12). Stylet supports in- Milnesium krzysztofi sp. nov. Fig. (1–15) serted on buccal tube at 17.4 [65.2]. ­Pharyngeal bulb description (measurements of the holotype): Body elongated and pear-shaped. ­Placoids and septula in length 380.4 (Fig. 1–3). Body white/transparent, pharyngeal bulb absent. Claws of Milnesium type eyes absent. Dorsal cuticle arranged in “segments” (Fig. 13–15). Main branch on all legs with small ac- (Fig. 2–3, 5, 9–10). Dorsal side of the body with cessory points. Internal secondary branches on legs fine reticular design (diameter of polygons 0.5– I–III with three points, external with two points (Fig. 1.5), better developed in the caudal end (Fig. 8–11). 13–14). External secondary branches on legs IV with On the dorsal side, on one of the last dorsal segments three points, internal with two points (Fig. 15). Claw a distinct irregular depression present (Fig. 4–7). lengths: leg I (internal): primary (= main) branch: Cuticle sculpturing present also on all legs (Fig. 14). 11.9 [44.6], claw base + secondary branch 9.5 [35.6]; Six peribuccal and two lateral papillae present. Peri­ leg II (internal): pb 12.1 [45.3]; leg III (internal): buccal lamellae invisible in mounted due to pb 13.0 [48.7], cb+sb. 9.4 [35.2]; leg IV (external/ Kaczmarek & Michalczyk—Milnesium krzysztofi, new tardigrade from Costa Rica 299

Fig. 4–7 Milnesium krzysztofi sp. nov. 4–5, Dorso-caudal cuticle with irregular depressions; 6–7, irregular ­depressions. 4, 6—PCM; 5, 7—SEM. anterior): pb 16.6 [62.2], cb+sb 10.4 [39.0], spur Taxonomy and Ecology, A. Mickiewicz University, 2.1 [10.1]. Due to unfavourable orientation of the Umultowska 89, Poznań, Poland. holotype the rest of claws were not measured. Long e t y m o l o g y : We dedicate the new species to Mr transversal, cuticular bars on first three pairs of legs Krzysztof Kaczmarek, the first author’s father. present (Fig. 14). Eggs white/transparent, deposited in exuvium. differential d i a g n o s i s : The species within the genus Milnesium can be divided into two clear groups with r e m a r k s : In some specimens irregular cuticular respect to the cuticle sculpturing. The majority of depressions may be present on more than one dorsal Milnesium species have smooth cuticle (Milnesium “segments”, however the most caudal one is always almatyense, M. antarcticum, M. asiaticum, the biggest (Fig. 4–5). Results of simple statistical M. brachyungue, M. dujianensis, M. eurystomum, analysis of measurements and pt values of selected M. longiungue, M. reductum, M. slovenskyi, morphological structures for six specimens are given M. tardigradum and M. tetralamellatum), however in Table 1. No males were found. a small group of species has a distinct reticular t y p e l o c a l i t y : Central America, Costa Rica, Cartago ­design on the dorsal cuticle (M. katarzynae, M. re- Province, agricultural area, lichen from tree near the ticulatum, and the new species). Because M. road from Volcano Irazu to Cartago, 21.0 km before ­krzysztofi sp. nov. is readily distinguishable from all Cartago, 17.12.2002, leg. Ł. Kaczmarek. species with a smooth cuticle, we differentiate it only t y p e repositories : Holotype (No. CR467/1) and 8 from the species that also have the reticular design paratypes are preserved at the Department of Animal on the dorsal cuticle. 300 New Zealand Journal of Zoology, 2007, Vol. 34

Fig. 8–11 Milnesium krzysztofi sp. nov.—dorsal cuticle surface. 8—PCM; 9–11—SEM.

Table 1 measurements (in µm) and pt values of selected morphological structures of specimens of Milnesium krzysztofi sp. nov. mounted in Hoyer’s medium (range refers to the smallest and the largest structure found among all measured specimens; N, number of specimens/structures measured; SD, standard deviation.

Range Mean SD Character N (µm) (pt) (µm) (pt) (µm) (pt) Body 6 320.5–536.8 1261.8–1424.7 412.43 1362.5 79.9 56.9 Buccal tube 6 24.7–40.1 – 30.28 – 5.9 – Stylet support insertion point 6 16.6–26.2 63.3–67.3 19.78 65.5 3.6 1.6 Buccal tube external width 6 8.4–15.4 33.1–38.4 10.72 35.1 2.7 1.8 Internal claw I: primary (main) branch 5 10.3–14.3 40.4–45.3 12.08 42.6 1.8 2.2 Internal claw I: base + secondary branch 4 9.0–10.8 34.2–36.4 9.60 35.5 0.8 1.0 Internal claw I: spur 1 2.5–2.5 10.1–10.1 2.50 10.1 ? ? Internal claw II: primary (main) branch 4 9.5–14.9 37.4–47.2 12.85 43.7 2.6 4.3 Internal claw II: base + secondary branch 3 9.0–12.0 34.3–38.0 10.80 35.9 1.6 1.9 Internal claw II: spur 2 2.5–2.8 0.0–9.8 2.65 6.2 0.2 5.4 Internal claw III: primary (main) branch 4 10.9–16.0 42.9–50.6 13.78 47.0 2.3 3.4 Internal claw III: base + secondary branch 3 8.1–11.3 31.9–35.8 9.60 34.3 1.6 2.1 Internal claw III: spur 1 3.4–3.4 10.8–10.8 3.40 10.8 – – External claw IV: primary (main) branch 4 16.6–22.8 56.9–66.5 20.33 62.1 2.6 4.0 External claw IV: base + secondary branch 4 10.4–16.0 39.0–43.7 13.40 40.7 2.3 2.1 External claw IV: spur 4 2.7–3.2 8.0–10.1 2.95 9.1 0.2 0.9 Kaczmarek & Michalczyk—Milnesium krzysztofi, new tardigrade from Costa Rica 301

Fig. 12–15 Milnesium krzysztofisp. nov. 12, Buccal tube; 13, legs II (note cuticle sculpture above claws); 14, claws III with the cuticular bar; 15, claws IV. 12 and 15—PCM; 13 and 14—SEM.

M. krzysztofi sp. nov. is the most similar to M. Ministry of Environment and Energy (MINAE) with funds katarzynae but differs from it by: the presence of from the Global Environment Facility (GEF) through the spurs on the internal secondary branches of claws World Bank and project ALAS with funds from National Science Foundation (grant no. DEB-0072702), the State I–III and on external/anterior secondary branches of Committee for Scientific Research (KBN) (grant no. claw IV, more visible reticular design on the dorsal 2P04C 07729: “Niesporczaki (Tardigrada) Kostaryki— side of the body, larger body size (320.5–536.8 in the przenikanie się fauny Nearktycznej i Neotropikalnej”) new species and 285.0–294.5 in M. katarzynae), a and a grant from the Foundation for Polish Science lower pt of the stylet supports (63.3–67.3 in the new (FNP) to the first author and by a grant from theE uropean species and 73.3–78.3 in M. katarzynae), a wider Commission’s programme “Transnational Access to Major buccal tube (8.4–15.4 in the new species and 6.6–7.6 Research Infrastructures” to SYNTHESYS (grant no. DK- TAF-2576) to both authors. in M. katarzynae) and a higher pt of the buccal tube width (33.1–38.4 in the new species and 21.7–26.6 in M. katarzynae). The new species differs also from M. reticulatum References by the absence of the dorsal gibbosities and eyes. Bertolani R, Grimaldi D 2000. A new eutardigrade (Tardigrada: Milnesiidae) in amber from the Upper Cretaceous () of New Jersey. Acknowledgments In: Grimaldi D ed. Studies on fossils in amber, with particular reference of the cretaceous of The research was partially supported by grants from New Jersey. Leiden, The Netherlands, Backhuys the Instituto Nacional de Biodiversidad (INBio) and the Publishers. Pp. 103–110. 302 New Zealand Journal of Zoology, 2007, Vol. 34

Binda MG, Pilato G 1990. Tardigradi di terra del fuoco Pilato G, Binda MG 1991. Milnesium tetralamellatum new e Magallanes. Milnesium brachyungue, nuova species of Milnesiidae from Africa (Eutardigrada). specie di Tardigrado Milnesidae. Animalia 17: Tropical Zoology 4: 103–106. 105–110. Pilato G, Binda MG 2001. Biogeography and limno- Kaczmarek Ł, Michalczyk Ł, Beasley CW 2004. Mil- terrestrial : are they truly incompatible nesium katarzynae sp. nov., a new species of binomials? Zoologischer Anzeiger 240: 511–516. eutardigrade (Milnesiidae) from China. Zootaxa Pilato G, Binda MG, Lisi O 2002. Notes on tardigrades of 743: 1–5. the Seychelles with the description of two new spe- cies. Bollettino dell’Accademia Gioenia Scienze Maucci W 1991. Tre nouve specie di Eutardigradi Naturali 35: 503–517. della Groenlandia Meridionale. Bollettino del Museo Civico di Storia Naturale di Verona 15: Tumanov DV 2006. Five new species of the genus Mil- 279–289. nesium (Tardigrada, Eutardigrada, Milnesiidae). Zootaxa 1122: 1–23. McInnes SJ 1994. Zoogeographic distribution of terres- Yang T 2003. Two new species and three new records trial/freshwater tardigrades from current literature. of the Tardigrada (Heterotardigrada: Echinisci- Journal of Natural History 28: 257–352. dae; Eutardigrada: Milnesiidae, Macrobiotidae, Pilato G 1981. Analisi di nuovi caratteri nello studio degli Hypsibiidae). Acta Zootaxonomica Sinica 28(2): Eutardigradi. Animalia 8: 51–57. 6–11.