Milnesium berladnicorum sp. n. (Eutardigrada, Apochela, Milnesiidae), a new species of water bear from Romania

Abstract In a lichen sample collected from a tree in Bârlad town (Vaslui County, Romania), a new tardigrade species belonging to the genus Milnesium (granulatum group) was found. Milnesium berladnicorum sp. n. is most similar (in the type of dorsal sculpture) to Milnesium beasleyi Kaczmarek et al., 2012 but differs from it mainly by having a different claw configuration and some morphometric characters. Additionally, the new species differs from other congeners of the granulatum group by the different type of dorsal sculpture, claw configuration and some morphometric characters.


Introduction
In Romania, studies on tardigrades were rather fragmentary and with a significant discontinuity in time (last 40 years). Even though during this period more than 150 taxa (species and subspecies) were reported for this region (Rudescu 1964), many of them are now considered as non-valid, have been synonymized, or require confirmation. In fact, in the light of modern taxonomy, only 127 tardigrades species are consider to be present in Romania (in 26 of 41 Romanian counties) (Ciobanu et al. 2014). Species from the genus Milnesium Doyère, 1840 are known from many localities throughout the world, from the Antarctic through tropical and temperate zones to the Arctic regions (e.g. Tumanov 2006;Kaczmarek et al. 2012a, b;Michalczyk et al. 2012a). Since the genus was recently re-described (Michalczyk et al. 2012a, b) new records and species have been reported from various localities (e.g. Kaczmarek et al. 2012a;Meyer et al. 2013;Zawierucha et al. 2014;Ciobanu et al. 2014). Taking into consideration that some morphological characters were omitted in older records of Milnesium specimens, all such records should be verified (Michalczyk et al. 2012a, b). Until now in Romania only three Milnesium species have been reported: Milnesium tardigradum sensu lato Doyère, 1840, M. granulatum (Ramazzotti, 1962) and M. asiaticum Tumanov, 2006, but all early records of the first species should be verified (Michalczyk et al. 2012a, b;Ciobanu et al. 2014). In this paper a new species of the genus Milnesium is described and illustrated.

Materials and methods
In a lichen sample collected by the first author in Bârlad town in July, 2013, 53 individuals and two exuvia (with 16 eggs) of the new species were found. Additionally, 55 specimens of Ramazzottius oberhaeuseri (Doyère, 1840) were found in the same sample, including 9 specimens in simplex stage and 9 eggs.
All specimens were extracted according to Dastych (1980Dastych ( , 1985 and mounted on microscope slides in Hoyer's medium. Observations, measurements and photomicrographs were taken using Phase Contrast Microscopy (PCM) (Olympus BX41 with digital camera ARTCAM-300Mi). All measurements (determined with QuickPhoto Camera 2.3) are given in micrometers [μm].
Body length was measured from the mouth to the end of the body excluding the hind legs. The buccal tube and claws characteristics were measured according to Tumanov (2006) and Michalczyk et al. (2012a). Subsequently, claw configuration is described according to Michalczyk et al. (2012a, b). Other morphometric data were calculated using the pt ratio: the ratio of the length of a given structure to the length of the buccal tube, expressed as a percentage (Pilato 1981). The pt values are always provided in italics in order to differentiate them from length values.
Characteristics and measurements of the species used in the differential diagnosis are given according to the original descriptions (Ramazzotti 1962;Pilato et al. 2002;Kaczmarek et al. 2004;Tumanov 2006;Kaczmarek and Michalczyk 2007;Wallendorf and Miller 2009;Kaczmarek et al. 2012a;Meyer et al. 2013) or are based on direct examination of type material (holotype and paratypes of Milnesium beasleyi Kaczmarek et al. 2012a). Ramazzottius specimens were verified and identified using the key to the World Tardigrada (Ramazzotti and Maucci 1983), a more modern key to the genus Ramazzottius (Biserov 1998), and remarks discussed by Pilato et al. (2013).
Description (measurements and statistics in Table 1). Body brownish (in live specimens) or transparent (in fixed specimens) with eyes (visible before and after mounting in Hoyer's medium -90% of fixed specimens had eyes). Six peribuccal papillae (ventral papilla smallest) and six peribuccal lamellae (of equal size) around the mouth opening present. Two cephalic papillae positioned laterally. The cuticle is covered with numerous tiny, shallow and rounded depressions (pseudopores) . Under PCM these pseudopores are visible as light spots, placing the species within the granulatum group. Bucco-pharyngeal apparatus of the Milnesium type ( Fig. 6). Buccal tube funnelshaped, wider anteriorly (on average the posterior diameter is 73% of the anterior di-    Etymology. This new species is named after the Berladnici, an ancient population with a controversial origin (most probably Slavs) who previously lived in the area of the present Bârlad town.

Differential diagnosis
Due to the sculptured cuticle, Milnesium berladnicorum sp. n. belongs to the granulatum group (Michalczyk et al. 2012a, b (Ramazzotti, 1962) Tumanov, 2006 but differs by having a sculptured dorsal cuticle and by presence of eyes.

Discussion
Until 1990, the genus Milnesium Doyère, 1840 was considered as monotypic with only one described cosmopolitan species, Milnesium tardigradum Doyère, 1840. In 1990, Binda and Pilato described a second species, Milnesium brachyungue from Chile. Later, additional species in the genus Milnesium were described sporadically up to 2006 when Tumanov published the first, but partial, revision of the genus Milnesium and described five new species (Tumanov 2006). In 2012, the genus Milnesium was redescribed in more detail by Michalczyk et al. (2012 a, b), and the nominal species M. tardigradum tardigradum sensu stricto Doyère,1840 obtained a clear and definitive diagnosis.
Including the new species described here, the total number of valid tardigrade taxa recorded in Romania is 128, with three valid Milnesium species (not including M. tardigradum tardigradum sensu stricto, which requires confirmation of presence in Romania).