New tardigrade records for the Baltic states with a description of Minibiotus formosus sp. n. (Eutardigrada, Macrobiotidae).

In sixteen moss, lichen and mixed (moss/lichen) samples, collected from Estonia, Latvia and Lithuania, 291 specimens, 48 simplexes, including one exuvium with 6 eggs, and 8 free-laid eggs of eutardigrades were found. In total, 17 species, together with one new to science, were identified (all are new records for the Baltic states): Astatumen bartosi, Diphascon (Adropion) prorsirostre, D. (Diphascon) bullatum, D. (D.) pingue pingue, D. (D.) recamieri, D. (D.) rugosum, Hypsibius convergens, H. dujardini, H. cf. scabropygus, Isohypsibius ronsisvallei, I. sattleri, Macrobiotus harmsworthi harmsworthi, M. hufelandi hufelandi, Milnesium asiaticum, Milnesium tardigradum tardigradum, Minibiotus formosus sp. n. and Paramacrobiotus richtersi. The new species is most similar to Minibiotus gumersindoi, but differs from it mainly by the presence of two types of cuticular pores, the absence of a triangular or pentagonal arrangement of pores above a single large pore on legs, the presence of granulation on all legs and a different macroplacoid length sequence. In this paper we also provide photographs and morphometrics of H. cf. scabropygus.

Introduction

The Baltic States, i.e. Estonia, Latvia and Lithuania, are located on the eastern coast of the Baltic Sea, and fall within the Palearctic ecozone (Holt et al. 2012). The topography of the three countries is dominated by lowlands with the highest peaks at ca. 300 m asl. The temperate climate is intermediate between maritime and continental. Even though the phylum Tardigrada is cosmopolitan and currently comprises ca. 1,200 species (Degma et al. 2013), so far only six tardigrade taxa have been reported from the Baltic States. Specifically, two from Estonia: (Binda, 1969) and cf. marcellinoi (Binda & Pilato, 1971), two from Latvia: group and group and two from Lithuania: sp. and sp. (Šatkauskienė and Vosyliūtė 2010, Zawierucha and Kaźmierski 2012, Ziemelis et al. 2012).

In this study we report seventeen tardigrade species, which are all new records for the Baltic States. Moreover, one of these species is also new to science. The new species belongs to the genus R.O. Schuster, 1980, that until 1988 contained only a single species, (Plate, 1888). In 1988 Pilato and Claxton (1988) described , and within the last decade fourteen new species have been described. Several species have also been transferred to from the genus based on characters defined by Claxton (1998) and later supplemented by Guidetti et al. (2007) (Michalczyk and Kaczmarek 2003a, Pilato et al. 2003, Michalczyk and Kaczmarek 2004, Guil and Guidetti 2005, Michalczyk et al. 2005, Pilato and Lisi 2006b, Li et al. 2008, Fontoura et al. 2009a, b, Rossi et al. 2009, Meyer and Hinton 2009, Meyer and Domingue 2011, Meyer et al. 2011 Meyer 2012). Currently, the total number of species amounts to as many as forty seven.

In addition to the description of the new species, we also provide morphometric data and photographs of cf. scabropygus, a rare species that belongs to a large group of hypsibiids with at least partially sculptured dorsal cuticle and pharynx with two macroplacoids and without the microplacoid.

Material and methods

Sixteen moss, lichen and mixed (moss/lichen) samples from trees, soil and stones were collected from 15 localities in Estonia, Latvia and Lithuania between the 29 April and the 5 May 2012 by the third author (more details below). Samples were collected and examined for tardigrades using standard methods (see Dastych 1980). After extraction, PageBreakanimals were mounted on microscope slides in Hoyer’s medium. All specimens were examined measured and photographed using Phase Contrast Microscopy (PCM) or Scanning Electron Microscopy (SEM). In total 358 specimens (including 47 simplexes), one exuvium with 6 eggs, and 8 free-laid eggs were examined.

All measurements are given in micrometers [μm]. Structures were measured only if their orientation was appropriate. Body length was measured from the anterior extremity to the end of the body, excluding the hind legs. Buccal tube length and the level of the stylet support insertion point were measured according to Pilato (1981). Buccal tube width was measured as the external diameter at the level of the stylet support insertion point. Lengths of the claw branches were measured from the base of the claw to the top of the branch including accessory points for and according to Beasley et al. (2008) for . 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). Macroplacoid length sequence is given according to Kaczmarek et al. (2014b), i.e. macroplacoids are listed from the shortest to the longest and their relative sizes are denoted with appropriate inequality, approximation and/or equality signs (<, ≤, ≈, =). Morphometric data were handled using the ‘Macrobiotoidea’ ver. 1.1 template available from the Tardigrada Register (www.tardigrada.net/register, Michalczyk and Kaczmarek 2013).

For species identification and differentiation, keys in Claxton (1998), Fontoura and Pilato (2007), Kaczmarek et al. (2011), Michalczyk et al. 2012a, b and Ramazzotti and Maucci (1983), and original descriptions and redescriptions (Ehrenberg 1859, Ramazzotti 1959, 1962, Horning et al. 1978, Bertolani and Rebecchi 1993, Dastych 1988, 1990, Binda and Pilato 1992, Michalczyk and Kaczmarek 2004, Michalczyk et al. 2005, Miller et al. 2005, Fontoura et al. 2009a, b, Meyer and Hinton 2009, Meyer et al. 2011) as well as for insertion of the stylet muscles Pilato (2013) were used. Tardigrade taxonomy is presented according to Marley et al. (2011). Only specimens determined to species level are provided in the list of species (we omitted all specimens determined only to the species group level, e.g. the hufelandi group or the oberhaeuseri group). In the species list Roman numbers indicate sample codes (see sampling localities) and Arabic numbers indicate the number of specimens, exuvia/simplexes and eggs.

Raw data underlying the description of sp. n. are deposited in the Tardigrada Register (Michalczyk and Kaczmarek 2013) under www.tardigrada.net/register/0012.htm.

56°03'08"N, 24°24'10"E, ca. 33 m asl: Lithuania, Panevėžys county, Pasvalys district municipality, along the road E67, 0.5 km before the turning to Pasvalys, moss from tree and soil (slide code: LT 2422), date: 29.04.2012.

55°25'59"N, 24°13'32"E, ca. 59 m asl: Lithuania, Kaunas county, Kėdainiai district municipality, Truskava city, near a church, lichens from tree and moss from soil (slide code: LT 2423), date: 29.04.2012.

55°17'12"N, 23°58'57"E, ca. 30 m asl: Lithuania, Kaunas county, Kėdainiai district municipality, Kėdainiai city, Kranto II street; moss from wall (slide code: LT 2424), date: 29.04.2012.

55°17'13"N, 23°58'56"E, ca. 30 m asl: Lithuania, Kaunas county, Kėdainiai district municipality, Kėdainiai city, Paeismilgio street; moss from stone (slide code: LT 2425), date: 29.04.2012.

55°43'35"N, 24°21'30"E, ca. 62 m asl: Lithuania, Panevėžys county, Panevėžys district municipality, Panevėžys city, Garden Street near Holy Trinity Rector; moss from tree (slide code: LT 2440), date: 05.05.2012.

56°38'53"N, 23°43'18"E, ca. 7 m asl: Latvia, Zemgale region, Jelgava municipality, Jelgava city, City Park; moss from soil (slide code: ŁO 2426), date: 29.04.2012.

57°10'33"N, 24°50'32"E, ca. 45 m asl: Latvia, Vidzeme region, Sigulda municipality, Gutmana Cave in the Gauja National Park; moss from rocks (slide code: ŁO 2427), date: 30.04.2012.

56°23'55"N, 24°07'33"E, ca. 25 m asl: Latvia, Zemgale region, Bauska municipality, along Road No P103, 0.5 km from Saulaine; lichens from tree (slide code: ŁO 2428) date: 29.04.2012.

57°09'55"N, 24°51'03"E, ca. 73 m asl: Latvia, Vidzeme region, Sigulda municipality, Turaida city, Turaida Castle; moss from stone (slide code: ŁO 2430), date: 30.04.2012.

56°54'32"N, 24°08'45"E, ca. 10 m asl: Latvia, Riga Region, boundary of Ķekava municipality, along road no A2; moss from tree (slide code: ŁO 2431), date: 30.04.2012.

57°09'59"N, 24°50'59"E, ca. 91 m asl: Latvia, Vidzeme region, Sigulda municipality, Sigulda city, Sigulda Castle; moss from stone (slide code: ŁO 2432), date: 30.04.2012.

56°41'22"N, 23°47'43"E, ca. 4 m asl: Latvia, Zemgale region, Ozolnieki municipality, Ozolnieki city, about 100 m from the Ozolnieki Lake; moss from soil (slide code: ŁO 2433), date: 29.04.2012.

57°45'43"N, 24°20'59"E, ca. 3 m asl: Latvia, Vidzeme region, Salacgriva municipality, Salacgriva city; moss from soil, near the beach (slide code: ŁO 2434), date: 01.05.2012.

59°10'44"N, 24°30'06"E, ca. 59 m asl: Republic of Estonia, Harju county, Kernu Parish municipality, Road No 4, moss from tree (slide code: ES 2420), date: 04.05.2012.

59°10'44"N, 24°30'06"E, ca. 59 m asl: Republic of Estonia, Harju county, Kernu Parish municipality, Road No 4, moss from tree (slide code: ES 2421), date: 04.05.2012.

58°48'47"N, 24°24'46"E, ca. 32 m asl: Republic of Estonia, Rapla County, Märjamaa municipality, forest near Konuvere village, moss from tree (slide codes: ES 2487), date: 29.04.2012.

Results

Taxonomic accounts of species found in the study

Phylum: Tardigrada (Spallanzani, 1777)

Class: Eutardigrada Richters, 1926

Order: Apochela Schuster, Nelson, Grigarick and Christenberry, 1980

Family: Milnesiidae Ramazzotti, 1962

Genus: Doyère, 1840

Tumanov, 2006

Localities and specimen numbers.

XV: 1 specimen.

Remarks.

Our specimen corresponds perfectly to the original description. was originally described from Kirghizstan and subsequently found in the Svalbard archipelago (Tumanov 2006, Kaczmarek et al. 2012).

Doyère, 1840

VIII: 31 specimens (including 6 simplexes) + 1 exuvium with 6 eggs.

Specimens correspond perfectly with the redescription by Michalczyk et al. (2012a, b). This species was reported from many localities throughout the World, however records prior to Michalczyk et al. (2012a, b) need to be verified. So far, all confirmed localities are exclusively European (Michalczyk et al. 2012a, b).

Order: Parachela Schuster, Nelson, Grigarick & Christensen, 1980

Superfamily: Hypsibioidea Pilato, 1969 (in Marley et al. 2011)

Family: Hypsibiidae Pilato, 1969 Subfamily: Diphasconinae Dastych, 1992 Genus: Plate, 1888 Subgenus: Pilato, 1987

Murray, 1905

Localities and specimen numbers.

XIII: 1 specimen.

Remarks.

Specimens correspond well with the limited original description (Murray 1905) and also with later descriptions (Argue 1974 and Dastych 1980, 1988). This species is very similar to (Binda & Pilato, 1971) and, as suggested by Dastych (1988), these two species could be synonymous. Thus, to clarify the taxonomic status of , a re-description of is necessary based on material from locus typicus in Scotland (the type material probably does not exist) (Dastych 1988).

(Marcus, 1936)

XV: 11 specimens.

Although we have found only 11 specimens, we were confident in identifying them to because they corresponded perfectly to the partial redescriptions by Pilato and Binda (1997/1998, 1999) and we also identified them with the key by Fontoura and Pilato (2007). The species belongs to the pingue group and has been previously recorded from numerous localities throughout the World, however the majority of records should be verified based on the modern taxonomy (Pilato and Binda 1997/1998, 1999). Currently, exclusively verified localities of this species are only from Europe and North America (Pilato and Binda 1997/1998).

Richters, 1911

XV: 1 specimen.

The species has previously been found in many localities, mostly in the Holarctic (McInnes 1994).

(Bartoš, 1935)

II: 3 specimens.

The species has previously been found in many localities in the Holarctic (McInnes 1994).

Genus: Pilato, 1987

Thulin, 1928

Localities and specimen numbers.

XIV: 2 specimens, XV: 2 specimens.

Remarks.

The species has been previously found in many localities, mostly in the Holarctic (McInnes 1994).

Subfamily: Hypsibiinae Pilato, 1969 Genus: Ehrenberg, 1848

(Urbanowicz, 1925)

IX: 6 specimens (including 2 simplexes).

Belonging to the cosmopolitan PageBreakconvergens-dujardini complex of species (McInnes 1994, Miller et al. 2005, Kaczmarek and Michalczyk 2009a, Kaczmarek et al. 2014a), used to be considered cosmopolitan, but it is most likely a complex of very similar (possibly also cryptic) species found throughout the world. The original description no longer conforms to modern standards and therefore a redescription is required. Nevertheless, the examined specimens correspond perfectly with the original description and the characteristics reviewed by Miller et al. (2005).

(Doyère, 1840)

XIV: 2 specimens (including 1 simplex), XV: 5 specimens.

belongs to the cosmopolitan convergens-dujardini complex of species (McInnes 1994, Miller et al. 2005, Kaczmarek and Michalczyk 2009a, Kaczmarek et al. 2014a), and used to be considered cosmopolitan, but it is most likely a complex of similar (possibly cryptic) species found throughout the world. Due to the limited original description, needs a modern redescription. Nevertheless, the examined specimens correspond perfectly with the original description and the characteristics reviewed by Miller et al. (2005).

Cuénot, 1929

Table 1 , Figs 1 –7

Table 1.

Measurements and pt values of selected morphological structures of cf. scabropygus Cuénot, 1929 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	14	183	–	293	808	–	1132	235	949	33	97
Buccopharyngeal tube
Buccal tube length	15	22.0	–	28.2		–		24.6	–	2.0	–
Stylet support insertion point	15	12.0	–	15.9	51.3	–	57.1	13.5	54.9	1.1	1.6
Buccal tube external width	15	1.5	–	2.0	6.5	–	8.0	1.8	7.2	0.2	0.5
Buccal tube internal width	15	0.6	–	0.9	2.1	–	3.4	0.7	2.7	0.1	0.4
Placoid lengths
Macroplacoid 1	15	1.8	–	3.2	7.7	–	12.3	2.4	9.6	0.4	1.3
Macroplacoid 2	15	1.7	–	2.7	7.2	–	11.3	2.2	8.9	0.3	1.1
Macroplacoid row	15	4.7	–	6.3	19.1	–	26.5	5.3	21.7	0.5	1.8
Claw 1 lengths
External base	14	2.2	–	4.5	9.1	–	17.2	3.5	14.1	0.7	2.1
External primary branch	14	4.0	–	8.8	18.2	–	36.1	6.9	28.1	1.5	5.0
External secondary branch	14	2.3	–	5.9	10.5	–	24.4	4.4	17.9	1.0	3.9
Internal base	12	2.1	–	4.1	9.3	–	15.5	3.3	13.4	0.6	1.8
Internal primary branch	12	3.8	–	5.9	16.0	–	23.5	4.8	19.3	0.6	2.0
Internal secondary branch	12	2.4	–	4.3	9.9	–	16.5	3.3	13.3	0.6	2.0
Claw 2 lengths
External base	11	3.0	–	5.2	12.4	–	19.9	4.1	16.7	0.7	2.2
External primary branch	13	6.7	–	10.4	29.7	–	43.7	8.5	34.7	1.3	4.4
External secondary branch	13	4.3	–	6.7	19.0	–	27.2	5.4	21.9	0.7	2.6
Internal base	10	2.4	–	4.5	10.9	–	18.9	3.6	14.6	0.7	2.2
Internal primary branch	12	4.0	–	6.7	17.7	–	27.2	5.4	22.0	0.9	2.9
Internal secondary branch	12	2.6	–	5.4	11.8	–	22.0	4.1	16.7	0.9	3.0
Claw 3 lengths
External base	9	2.7	–	6.2	11.9	–	23.8	4.3	17.3	1.0	3.5
External primary branch	9	7.2	–	10.4	29.3	–	43.7	8.8	35.7	1.1	4.4
External secondary branch	9	3.6	–	6.5	12.8	–	27.3	5.2	21.0	1.0	4.3
Internal base	11	2.3	–	4.1	10.5	–	17.2	3.4	13.9	0.6	1.9
Internal primary branch	13	3.8	–	6.5	17.3	–	27.3	5.4	21.8	0.9	3.1
Internal secondary branch	12	2.7	–	6.1	12.2	–	24.8	3.9	16.0	0.9	3.5
Claw 4 lengths
Anterior base	13	3.3	–	5.6	12.8	–	20.1	4.1	16.8	0.6	2.2
Anterior primary branch	13	4.4	–	7.5	19.5	–	31.1	5.9	24.2	1.1	3.9
Anterior secondary branch	11	3.1	–	13.2	13.0	–	47.3	4.8	18.9	2.9	9.7
Posterior base	12	2.7	–	5.4	12.3	–	21.5	4.5	18.2	0.9	3.1
Posterior primary branch	12	4.9	–	14.9	22.0	–	60.6	10.3	41.9	2.9	11.2
Posterior secondary branch	12	4.0	–	6.5	15.4	–	25.6	5.2	21.2	0.9	3.4

Figures 1–5.

cf. scabropygus Cuénot, 1929: 1 habitus (dorso-lateral view) 2–4 caudo-dorsal cuticle with distinct sculpturing – tubercles and tubercles merged into platelets 5 a single caudo-dorsal platelet. (1–3: PCM, 4–5: SEM).

Figures 6–7.

cf. scabropygus Cuénot, 1929: 6 bucco-pharyngeal apparatus (dorso-ventral projection, ventral placoids in the insert) 7 claws IV (arrow indicates a small cuticular bar near the posterior claw). (Both PCM).

XI: 1 simplex, XII: 1 specimen, XIV: 34 specimens (including 4 simplexes), XV: 24 specimens (including 6 simplexes).

Description

(measurements in Body transparent/white (after preparation), eyes present in 6 of 15 (40%) specimens mounted in Hoyer’s medium (Fig. 1). Dorsal cuticle sculptured: from head to legs II without tubercles but clearly thickened, from legs II to the caudal end of the body (including legs IV) with irregular tubercles and platelets. Tubercles increasing in size from the anterior to the posterior part of the body, reaching maximum dimensions between legs III and IV, where tubercles sometimes merge and form irregular platelets (Figs 2–5). Irregular tubercles 1.0–6.0 μm in diameter. Ventral cuticle smooth (i.e. without sculpturing). Gibbosities and cuticular pores absent.

Bucco-pharyngeal apparatus of the type, without the ventral lamina, and with forked apophyses for stylet muscles (Fig. 6). Peribuccal lamellae absent. Teeth in the oral cavity armature absent or not visible under PCM. Pharyngeal bulb with apophyses and with two granular macroplacoids (both, without constrictions). Macroplacoid length sequence 2<1. Microplacoid and septulum absent.

Claws of the type, internal claws much smaller and of a different shape than the external claws (Fig. 7). All main branches with large accessory points. Smooth, indistinct areoles under claws usually visible only on posterior claws IV. Cuticular bars under claws I-III absent but a small bar is present near the posterior claw IV (Fig. 7, arrow).

Eggs. Unknown.

has been recorded from many localities, mostly in the Holarctic (McInnes 1994). In general, our specimens correspond to the original description by Cuénot (1929, 1932) and later descriptions by Marcus (1930) (= spec. nov.), and by Ramazzotti and Maucci (1983). However, importantly, none of the above mentioned descriptions reported a bar between anterior and posterior claws IV, which is present in all our specimens. Given the bar is small, it is possible that it was overlooked by Cuénot and later authors. If, however, does not have the bar, then our specimens should probably be classified as a new species. Thus, until is redescribed, our Latvian and Estonian records should be regarded as cf. scabropygus. As there is a possibility of our specimens belonging to a new species, we provide standard morphometrics (Table 1) and photographs (Figs 1–7).

Subfamily: Itaquasconinae Rudescu, 1964 Genus: Pilato, 1997

(Węglarska, 1959)

XIV: 1 specimen.

Our specimen corresponds perfectly with characters of proposed by Dastych (1988) with the main difference between and being the absence/presence of cuticular bars on legs II–III. Due to the notorious difficulties in differentiating the two species, the actual distribution of cannot currently be described with confidence. McInnes (1994) cited this species from several localities in Europe and from single African, Asian and South American sites.

Superfamily: Isohypsibioidea Marley, McInnes & Sands, 2011

Family: Isohypsibiidae Marley, McInnes & Sands, 2011 Genus: Thulin, 1928

Binda & Pilato, 1969

Localities and specimen numbers.

III: 1 specimen.

Remarks.

The species has previously been reported from several, mostly European, localities in the Holarctic (McInnes 1994).

(Richters, 1902)

IX: 1 specimen, XI: 8 specimens (including 5 simplexes), XIV: 3 specimens, XV: 5 specimens, XVI: 1 specimen.

The species has previously been reported from many localities throughout the World, thus it is considered cosmopolitan (McInnes 1994, Kaczmarek et al. 2014a).

Superfamily: Macrobiotoidea Thulin, 1928 in Marley et al. 2011

Family: Macrobiotidae Thulin, 1928 Genus: C.A.S. Schultze, 1834

Murray, 1907

XI: 1 egg, XIV: 8 specimens, 1 egg.

The species belongs to the harmsworthi group which is widely distributed across a broad range of ecosystems throughout the world (McInnes 1994, Kaczmarek et al. 2014a). In the last decade many new species within this group were described from a variety of localities (Michalczyk and Kaczmarek 2003b, Pilato et al. 2004, Tumanov 2005a, Pilato and Lisi 2006a, b, Pilato et al. 2006a, Kaczmarek et al. 2007, Kaczmarek and Michalczyk 2009b, Pilato and Lisi 2009a, Rossi et al. 2009, see also Kaczmarek et al. 2011 for the diagnostic key to the group). Due to many uncertain reports of , especially in older literature, the distribution of the species is currently unknown. Specimens found in the present study correspond well to the characters presented in Pilato et al. (2000) and were successfully identified with the key by Kaczmarek et al. (2011).

C.A.S. Schultze, 1833

XIV: 4 specimens, 1 egg.

The species belongs to the hufelandi group which is widely distributed across a broad range of ecosystems throughout the world (McInnes 1994, Kaczmarek et al. 2014a). In the last decade new species belonging to this group have been described from various localities (Pilato et al. 2003, Kaczmarek and Michalczyk 2004, Dastych 2002, 2005, Fontoura et al. 2008, Bartels et al. 2009, Kaczmarek and Michalczyk 2009b, Pilato and Lisi 2009b, Bertolani et al. 2011, Biserov et al. 2011, Pilato et al. 2012, Guidetti et al. 2013, see also Bertolani and Rebecchi 1993 for the diagnostic key to the group). Due to many uncertain reports of , especially in older literature, the distribution of the species is currently unknown. Specimens found in the present study correspond well with the redescription by Bertolani and Rebecchi (1993).

Genus: R.O. Schuster, 1980 sp. n.

http://zoobank.org/BDBE49B7-84CF-4FE2-BE55-A399A537DE77

http://species-id.net/wiki/Minibiotus_formosus

http://www.tardigrada.net/register/0012.htm

Tables 2 –3 , Figs 8 –15

Table 2.

Measurements and pt values of selected morphological structures of sp. n. 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, ? – trait oriented unsuitably for measurement).

CHARACTER	N	RANGE						MEAN		SD		Holotype
		µm			pt			µm	pt	µm	pt	µm	pt
Body length	7	113	–	236	636	–	1034	184	901	39	139	194	848
Buccal tube
Length	9	17.7	–	22.9		–		20.3	–	1.7	–	22.9	–
Stylet support insertion point	9	9.5	–	12.2	49.5	–	56.2	10.9	53.8	0.9	2.0	12.2	53.3
External width	9	1.3	–	2.0	7.0	–	9.9	1.6	7.8	0.2	0.8	1.7	7.4
Internal width	9	0.5	–	0.7	2.4	–	3.4	0.6	2.8	0.1	0.3	0.7	3.1
Placoid lengths
Macroplacoid 1	9	1.3	–	1.9	6.9	–	8.3	1.5	7.6	0.2	0.4	1.8	7.9
Macroplacoid 2	9	1.2	–	1.7	6.8	–	8.3	1.5	7.5	0.2	0.4	1.7	7.4
Macroplacoid 3	9	1.4	–	2.2	7.4	–	9.6	1.7	8.2	0.3	0.6	1.9	8.3
Microplacoid	8	0.5	–	0.9	2.4	–	4.7	0.7	3.3	0.1	0.8	0.8	3.5
Macroplacoid row	9	4.5	–	6.8	24.3	–	33.7	5.5	26.9	0.7	3.0	5.9	25.8
Placoid row	8	5.2	–	7.9	27.1	–	39.1	6.4	31.0	0.9	3.9	6.7	29.3
Claw 1 lengths
External primary branch	4	4.7	–	6.5	22.8	–	31.6	5.8	27.7	0.8	3.7	6.2	27.1
External secondary branch	3	3.6	–	4.3	17.5	–	20.9	3.9	19.4	0.4	1.7	?	?
Internal primary branch	6	4.7	–	6.6	24.3	–	30.4	5.7	27.4	0.7	2.4	6.3	27.5
Internal secondary branch	5	3.1	–	4.6	15.0	–	20.4	4.0	18.7	0.5	2.3	4.6	20.1
Claw 2 lengths
External primary branch	5	5.2	–	6.9	25.2	–	34.0	5.9	28.6	0.6	3.5	5.9	25.8
External secondary branch	4	3.3	–	4.7	16.0	–	23.2	3.9	19.4	0.6	2.9	?	?
Internal primary branch	5	5.2	–	6.4	27.1	–	31.9	6.0	29.7	0.5	2.3	6.2	27.1
Internal secondary branch	3	3.5	–	4.1	18.5	–	20.9	3.9	19.9	0.3	1.3	?	?
Claw 3 lengths
External primary branch	5	5.1	–	6.9	27.0	–	33.2	6.3	30.6	0.7	2.4	6.9	30.1
External secondary branch	6	3.6	–	4.9	19.0	–	24.1	4.6	22.1	0.5	1.8	4.9	21.4
Internal primary branch	7	5.1	–	6.6	25.7	–	32.5	5.9	29.2	0.6	2.5	6.4	27.9
Internal secondary branch	4	4.1	–	4.6	20.9	–	22.8	4.3	21.6	0.2	0.8	?	?
Claw 4 lengths
Anterior primary branch	5	6.0	–	8.0	31.6	–	39.6	7.0	34.2	0.8	3.5	?	?
Anterior secondary branch	4	4.0	–	6.0	21.2	–	29.7	5.0	24.1	0.9	3.9	?	?
Posterior primary branch	6	6.0	–	7.8	30.7	–	38.4	7.0	33.9	0.7	3.1	?	?
Posterior secondary branch	5	3.9	–	5.5	20.6	–	27.1	4.8	23.8	0.6	2.6	?	?

Table 3.

Measurements of selected morphological structures of sp. n. eggs mounted in Hoyer’s medium.

CHARACTER	egg 1	egg 2	egg 3
Diameter of egg without processes	45.7	44.1	?
Diameter of egg with processes	55.6	55.1	?
Process height	4.5–5.2	4.8–5.2	4.6–5.3
Process base width	2.8–3.4	2.8–3.1	2.4–2.6
Process base/height ratio	57%–69%	54%–65%	47%–57%
Distance between processes	2.0–2.5	1.9–3.9	1.8–2.0
Number of processes on the egg circumference	30	29	30

Figures 8–11.

sp. n.: 8 habitus (holotype, ventral view) 9 dorsal cuticle with pores (holotype) 10–11 bucco-pharyngeal apparatus (10 dorso-ventral projection, paratype 11 lateral view, paratype). All PCM.

Figures 12–15.

sp. n.: 12 leg II with claws, granulation (arrowhead) and a single large pore (arrow) (holotype) 13 claws IV (paratype) 14 egg (mid-section) 15 egg surface with processes. All PCM.

Type material.

Holotype and 23 paratypes, 24 specimens (including 2 simplexes) and 3 unembryonated eggs).

Type locality.

57°10'33"N, 24°50'32"E, ca. 45 m asl: Latvia, Vidzeme region, Sigulda municipality, Gutmana Cave in the Gauja National Park; moss from rock (1 sample, slide codes: ŁO 2427/*, where the asterisk can be substituted by any of the following numbers: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12).

Description

(measurements in Body white/colourless (Fig. 8). Eyes present in 18 of 24 (75%) specimens mounted in Hoyer’s medium. Entire cuticle covered with small (0.4–1.1 μm) and large (1.9–2.5 μm) round or oval pores (Fig. 9). Pores arranged in 9–10 poorly defined transverse bands. Pores on the dorsal cuticle arranged more densely than on the ventral cuticle. A single large pore (diameter: 2.1–2.9 μm) present on external side of legs I–III (Fig. 12, arrow). A ring of pores around the mouth opening absent. Cuticle without granulation, except for legs which are all covered with fine and regular granulation (better developed on legs IV) visible only in larger specimens (Fig. 12, arrowhead).

Mouth antero-ventral. Ten peribuccal papulae present. Bucco-pharyngeal apparatus of the type (Figs 10–11). Oral cavity armature absent or not visible under PCM. Buccal tube with a poorly visible ventral lamina and with an anterior and a posterior bend (both visible in lateral view only, Fig. 11). Buccal tube walls thickened just below the stylet supports insertion point. Pharyngeal apophyses triangular, very near to the first macroplacoid. Three granular macroplacoids and a minute microplacoid present in the pharyngeal bulb. All macroplacoids of similar but not identical sizes, the macroplacoid length sequence: 2<1<3. Septulum absent.

Claws of the type (Figs 12–13). Primary branches of claws with thin, but obvious accessory points detaching at the apogee of the primary branch curve. Smooth lunules present on all legs, distinctly larger under external and posterior claws. Bars and other cuticular thickenings on legs absent.

Eggs (measurements in White/transparent, laid freely (Fig. 14). Spherical, without areolation. Processes in the shape of short, smooth, slightly flexible cones (Fig. 15). Processes are distributed on the surface of the egg close one to another but never in contact. Surface between processes smooth under PCM (Fig. 15).

Since ventral lamina is very poorly visible, the measurements of this structure are not included in Table 2. Three unembryonated eggs have been found alongside the described specimens. Given that PageBreak sp. n. was the only species in the sample and because no Binda & Pilato, 1986 was found in the samples, we assumed that these eggs belong to the new species.

Etymology.

Given that we found the composition of small and large pores in the new species beautiful, we decided to name the animal after this impression (in Latin ‘formosus’ means ‘beautiful’).

Type depositories.

Holotype 23 paratypes and 3 eggs are deposited in the Department of Animal Taxonomy and Ecology at the Adam Mickiewicz University (Poznań, Poland).

Differential diagnosis

The new species is most similar to Guil & Guidetti, 2005, but it differs from it by: the presence of two types of cuticular pores (small and large) in the new species vs pores of a uniform size in , the absence of a triangular or a pentagonal arrangement of pores placed above a single large pore on legs, the presence of granulation on legs, a different macroplacoid length sequence (2<1<3 in the new species vs 1=2=3 in ), and by slightly larger macroplacoids (I: 1.3–1.9 μm; II: 1.2–1.7 μm III: 1.4–2.2 μm in the new species vs 1.0 μm in ).

Other species to which sp. n. is similar by some characteristics of adult and/or egg morphology (e.g. pores in transverse bands, eggs with conical processes), include species listed below. The new species differs specifically from:

(Horning et al. 1978) by: the absence of trilobed and star-shaped pores (although their presence was not mentioned in the original description, they are clearly visible in Fig. 114 in Horning et al. (1978), and by stylet supports inserted in a more anterior position (pt=49.5–56.2 in the new species vs pt≈60.3 in (according to Claxton 1998)).

Michalczyk & Kaczmarek, 2004 by: a different arrangement of pores on the dorsal cuticle (9–10 transverse bands in the new species vs 8 bands in ), the absence of star-shaped pores, the absence of four pores around the mouth opening, the presence of a single large pore on lateral sides of legs I-III, slightly shorter buccal tube (17.7–22.9 μm in the new species vs 24.7–34.2 μm in ), stylet supports inserted in more anterior position (9.5–12.2 μm [pt=49.5–56.2] in the new species vs 16.2–23.8 μm [pt=65.4–70.6] in ), a different macroplacoid sequence (2<1<3 in the new species vs 2<3<1 μm in ), slightly shorter placoids, and by slightly smaller external claws I–IV (compare Table 2 below and Table 1 in Michalczyk and Kaczmarek 2004 for exact differences in dimensions of placoids and claws).

(Ehrenberg, 1859) (according to Binda and Pilato 1992) by: the absence of tri- and quadrilobed cuticular pores, the presence of two types of cuticular pores (small and large in the new species vs uniformly small pores present in ), the presence of a single large pore on each of legs I–III, the presence of granulation on legs, the absence of the oral cavity armature, stylet supports inserted in a more anterior position (pt=49.5–56.2 in the new species vs pt≈68.4 in ), a different macroplacoid length sequence (2<1<3 in the new species vs 2<3<1 in ), and by egg processes without an obvious flexible portion (and never bifurcated).

Meyer & Hinton, 2009 by: the absence of tri- and quadrilobed cuticular pores, the presence of two types of pores (small and large) over the entire cuticle in the new species vs small pores present only in the anterior part of the body and large pores present only in the posterior part of the body in , the presence of a single large pore on each of legs I–III, stylet supports inserted in a more anterior position (pt=49.5–56.2 in the new species vs pt=61.4–67.6 in ), a different macroplacoid length sequence (2<1<3 in the new species vs 2≤3<1 μm in ), a different shape of egg processes (short, single-tipped cones in the new species vs elongated, tapering cones with bulbous bases in ), a smaller diameter of eggs without and with processes (44.1–45.7 μm and 55.1–55.6 μm in the new species vs 66.1–80.0 μm and 78.2–101.9 μm in ), a slightly lower number of processes on egg circumference (29–30 in the new species vs 32–41 in ), and by smaller egg processes (4.5–5.3 μm in the new species vs 7.6–12.8 μm in ).

Meyer et al., 2011 by: the absence of trilobed and polygonal pores, the presence of two types of cuticular pores (small and large) in the new species vs small pores present only in the anterior part of the body, intermediate in size in the middle of the body and large pores in the posterior part of the body in ), the presence of a single large pore on each of legs I-III, the presence of granulation on all legs, a slightly shorter buccal tube (17.7–22.9 μm in the new species vs 24.4–29.6 μm in ), stylet supports inserted in a more anterior position (pt=49.5–56.2 in the new species vs pt=63.0–65.6 in ), a slightly smaller external diameter of the buccal tube (1.3–2.0 μm [pt=7.0-9.9] in the new species vs 2.1–2.6 μm [pt=7.4–10.7] in ), a different macroplacoid length sequence (2<1<3 in the new species vs 1<2<3 in ), slightly shorter macroplacoids II and III (II: 1.2–1.7 μm [pt=6.8–8.6]; III: 1.4–2.2 μm [pt=7.4–9.6] in the new species vs II: 1.9–2.3 μm [pt=7.1–8.8]; III: 2.4–2.6 μm [pt=8.4–9.9] in ), a slightly shorter macroplacoid row (4.5–6.8 μm [24.3–33.7] in the new species vs 7.0–8.4 μm [pt=27.0–34.4] in ), the presence of a microplacoid, and by slightly shorter primary and secondary branches of external claws I–IV (compare Table 2 below and Table 2 in Meyer et al. 2011).

Claxton, 1998 by: the lack of red pigment granules, the presence of two types of cuticular pores (small and large) in the new species vs pores uniform in size (ca. 1.0 μm) in ), the presence of a single large pore on each of legs I-III, a slightly shorter buccal tube (17.7–22.9 μm in the new species vs 24.9–28.4 μm in ), stylet supports inserted in a more anterior position (pt=49.5–56.2 in the new species vs pt≈60.6 in ), a different macroplacoid length sequence (2<1<3 in the new species vs 2=3<1 in ), a slightly shorter macroplacoid row (4.5–6.8 μm in the new species vs 7.0–7.6 μm in ), the lack of a membrane around egg processes, a smaller diameter of eggs with processes (55.1–55.6 μm in the new species vs 65.0–85.0 μm in ), a larger number of processes on egg circumference (29–30 in the new species vs ca. 11 in ), smaller egg processes (4.5–5.3 μm in the new species vs 11.0–16.0 μm in ), narrower egg processes bases (2.4–3.4 μm in the new species vs 9.0–12.0 μm in ), and by slightly smaller distances between egg processes (1.8–3.9 μm in the new species vs 4.0–6.0 μm in ).

Fontoura et al., 2009 by: cuticular pores arranged in 9–10 transverse bands (11 transverse bands present in ), the absence of tri- and quadrilobed cuticular pores, the presence of two types of pores (small and large) in the new species vs all pores of similar size in , the presence of a single large pore on each of legs I–III, the presence of granulation on all legs, stylet supports inserted in a more anterior position (pt=49.5–56.2 in the new species vs pt=66.5–67.8 in ), a different shape of egg processes (short, single tip cones without a membrane in the new species vs screw-like processes with a membrane and six areoles in ), a slightly larger number of processes on egg circumference (29–30 in the new species vs ca. 24 in ), and by smaller distances between egg processes (1.8–3.9 μm in the new species vs 6.4–6.9 μm in ).

Claxton, 1998 by: cuticular pores arranged in 9–10 transverse bands (8 transverse bands in ), the presence of two types of pores (small and large) in the new species vs uniform pore size in ), the presence of a single large pore on each of legs I–III, stylet supports inserted in a more anterior position (pt=49.5–56.2 in the new species vs pt≈59.5 in ), a different macroplacoid length sequence (2<1<3 in the new species vs 2=3<1 in ), a different shape of egg processes (short, single-tipped cones without a membrane in the new species vs. screw-like processes within a membrane in ), the absence of granulation on egg shell, a larger number of processes on egg circumference (29–30 in the new species vs 18–20 in ), slightly smaller egg processes (4.5–5.3 μm in the new species vs 6.5–7.0 μm in ), and by smaller distances between egg processes (1.8–3.9 μm in the new species vs 6.0–8.0 μm in ).

(Ramazzotti, 1959) by: the absence of triangular and polygonal pores, the presence of two types of cuticular pores (small and large) in the new species vs small pores present only in the anterior part of the body, intermediate in size in the middle of the body and the large pores in the posterior part of the body in ), the presence of a single large pore on each of legs I–III and, egg processes without a filiform bristle.

Binda & Pilato, 1992 by: pores arranged in bands, the presence of two types of pores (small and large) in the new species vs universal pores size in ), the presence of a single large pore on each of legs I–III, the absence of the oral cavity armature, stylet supports inserted in a more anterior position (pt=49.5–56.2 in the new species vs pt=68.2–68.3 in ), a different macroplacoid length sequence (2<1<3 in the new species vs 3<2<1 in ), and by a lower number of processes on egg circumference (29–30 in the new species vs ca. 34–41 in ).

(Ramazzotti, 1962) by the presence of cuticular pores, the presence of granulation on all legs, and by fully developed lunules (only small open lunules present in ).

Binda & Pilato, 1992 by: pores arranged in bands, the absence of tri- and quadrilobed pores, the presence of two types of pores (small and large) in the new species vs uniform pore size in ), the presence of a single large pore on each of legs I–III, the absence of the oral cavity armature, a larger mean body size (184 μm in the new species vs 380 μm in ), stylet supports inserted in a more anterior position (pt=49.5–56.2 in the new species vs pt=66.1–68.7 in ), a different macroplacoid length sequence (2<1<3 in the new species vs 2<3<1 μm in ), a smaller diameter of eggs without and with processes (44.1–45.7 μm and 55.1–55.6 μm in the new species vs ca. 76.4 μm and ca. 88.0 μm in ), a slightly larger number of processes on egg circumference (29–30 in the new species vs ca. 26 in ), egg processes without flexible filaments, smaller egg processes (4.5–5.3 μm in the new species vs ca. 8.2 μm in ), and by narrower bases of egg processes (2.4–3.4 μm in the new species vs ca. 5.0 μm in ).

Michalczyk et al., 2005 by: the absence of bi-, trilobed and star-shaped pores, the presence of two types of pores (small and large) in the new species vs uniform pore size in ), the absence of 3–5 large triangular or irregularly shaped pores on the caudo-dorsal cuticle above hind legs, the presence of a single large pore on each of legs I-III, a different shape of egg processes (short, single tip cones without a membrane in the new species vs. screw-like processes within a membrane in ), a slightly larger number of processes on egg circumference (29–30 in the new species vs ca. 24 in ), and by slightly wider bases of egg processes (2.4–3.4 μm in the new species vs 1.6–2.0 μm in ).

Fontoura et al., 2009 by: the absence of trilobed pores, the presence of two types of pores (small and large) in the new species vs all pores of similar size in ), the presence of a single large pore on each of legs I–III, the presence of granulation on all legs, a smaller body size (113–236 μm in the new species vs 275–410 μm in ), stylet supports inserted in a more anterior position (pt=49.5–56.2 in the new species vs pt=66.1–67.9 in ), a different macroplacoid length sequence (2<1<3 in the new species vs 2<3<1 in ). shorter macroplacoids (I: 1.3–1.9 μm [pt=6.9–8.3]; II: 1.2–1.7 μm [pt=6.8–8.6] III: 1.4–2.2 μm [pt=7.4–9.6] in the new species vs I: 3.6–4.5 μm [12.7–13.8]; II: 2.9–3.6 μm [10.3–11.1] III: 3.0–3.9 μm [pt=10.9–11.9] in ), a shorter microplacoid (0.5–0.9 μm [pt=2.4–4.7] in the new species vs 1.5–2.0 [5.0–6.2] in ), a shorter macroplacoid row (4.5–6.8 μm [pt=24.3–33.7] in the new species vs 9.8–12.6 μm [pt=35.6–38.5] in ), a shorter placoid row (5.2–7.9 μm [pt=27.1–39.1] in the new species vs 10.9–13.9 μm [39.6–43.3] in ), a different shape of egg processes (short, single-tipped cones in the new species vs long cones with bi- or multi-tipped tips in ), egg shell and processes without granulation, a smaller diameter of eggs without and with processes (44.1–45.7 μm and 55.1–55.6 μm in the new species vs 56.0–79.0 μm and 80.0–99.2 μm in ), a larger number of processes on egg circumference (29–30 in the new species vs 20–23 in ), smaller egg processes (4.5–5.3 μm in the new species vs 10.6–19.0 μm in ), and by slightly narrower bases of egg processes (2.4–3.4 μm in the new species vs 3.7–6.6 μm in ).

Genus: Guidetti, Schill, Bertolani, Dandekar & Wolf, 2009

(Murray, 1911)

Localities and specimen numbers.

XI: 2 specimens (including 1 simplex) and 1 egg.

Remarks.

species (until recently a collection of species within ) can be divided into three groups: areolatus, huziori and richtersi, with respect to the combination of two traits: the presence/absence of the microplacoid in the pharynx and the type of egg areolation. , considered cosmopolitan, is recognised as the nominal species for a group of very similar taxa that require careful taxonomic examination of adults and egg morphology for correct identification. In the last decade many new species of this group have been described from various localities (e.g. Pilato et al. 2004, Kaczmarek et al. 2005, Tumanov 2005b, Michalczyk and Kaczmarek 2006a, b, Michalczyk et al. 2006, Pilato et al. 2006a, b, Degma et al. 2008, Bartels et al. 2009, Pilato et al. 2012), with more recent additions including molecular data (Guidetti et al. 2009, Schill et al. 2010).