Tenuibiotus voronkovi ( Tumanov, 2007 )

Zawierucha, Krzysztof, Kolicka, Małgorzata & Kaczmarek, Łukasz, 2016, Re-description of the Arctic tardigrade Tenuibiotus voronkovi (Tumanov, 2007) (Eutardigrada; Macrobiotidea), with the first molecular data for the genus, Zootaxa 4196 (4), pp. 498-510 : 501-506

Tenuibiotus voronkovi ( Tumanov, 2007)

( Figs. 1–18 , Tables 1–3 )

Macrobiotus voronkovi in Tumanov (2007)

Material examined: Type material: Paratype from Spitsbergen, Kongsfjorden (1 adult, 1 egg). Additional material: Spitsbergen: 1 specimen, 1 egg, Nordaustlandet: 23 specimens and 51 eggs, Edgeøya: 22 specimens and 18 eggs.

Description: Adults. Body transparent or light yellow in living specimens and transparent after fixation in Hoyer’s medium ( Figs 1–2 ). Faint granulation visible on leg I–III and obvious granulation present on legs IV ( Figs 7–8 ). Dorsal and ventral cuticle without gibbosities, papillae, spines or sculpturing.

Bucco–pharyngeal apparatus of the Macrobiotus type ( Figs 3–6 ), with ventral lamina and poorly visible peribuccal lamellae. Mouth antero–ventral. First and second row of teeth in the oral cavity armature, absent (or not visible under PCM). Third row of teeth composed dorsally of “three transverse ridges (medial ridge arched with the convexity turned backwards and very thin lateral ridges” ( Fig. 3 ) and ventrally of a “single medial ridge arched with the convexity turned backwards and a pair of lateral ridges fragmented into granules” Tumanov (2007) ( Fig. 4 ). Pharyngeal bulb with apophyses, two rod–shaped macroplacoids and a triangular microplacoid. Macroplacoid length sequence 2<1. The first macroplacoid with a central constriction ( Figs 3, 5 ).

Claws of the Tenuibiotus type ( Figs 7–8 ). Primary branches with distinct accessory points ( Figs 7–8 ). Lunules under claws on legs I–III smooth and dentate on legs IV ( Figs 7–8 ). Other cuticular structures on legs absent.

Eggs. Spherical and ornamented, with conical processes. Processes have blunt or attenuate apices with reticulation. Egg shell between processes with poorly visible short irregular ridges.

Complementary description. Eyes are present in some individuals preserved on the slides. Claws on leg IV of the Tenuibiotus type ( Figs 7–8 ). Primary branches of claw IV with distinct accessory points ( Fig. 7 ). Less clear constriction is present prior to the caudal end of the second macroplacoid. Eggs with short or long conical processes ( Figs 9–12 ).

Remarks. Some of our observed live individuals were yellowish. Additional measurements of paratype (adult): internal buccal width, ventral lamina length, and measurements of primary and secondary branches of external and internal claws I, and II are given in Table 1 . Supplementary measurement of egg (paratype) are: 11.2, 11.3, 10.3 for processes length (height), 13.6, 11.4, 11.4 for process base diameters, 3.2, 2.2, 1.8 for egg shell distances between processes. Supplementary measurements of all important taxonomic traits for specimens and eggs of T. voronkovi from Nordaustlandet and Edgeøya are presented in Tables 2 and 3 .

We have complemented the original description of T. voronkovi with claw IV description, variability of egg processes ( Figs 9–12 , 13–18 ) and other morphometric traits of adults (internal width of buccal tube, ventral lamina, claws II and IV). Our measurements increase the morphometric range (min, max) in all measured traits of T. voronkovi .

No significant differences in body length were found between individuals from Edgeøya and Nordaustlandet (Mann–Whitney test, z = -0.582, p = 0,560). We therefore present the morphometric data in a single table ( Table 1 ). Statistical analysis of morphometric traits of the eggs collected from Edgeøya and Nordaustlandet showed no significant differences in process height between the eggs (T–test, t =-0.061, p<0.951), but there was a statistical difference in process base diameter (Mann–Whitney test, z =-2.61, p = 0.008) and the egg shell distances between processes (T–test, t =-5.44, p<0.005). However, as only five eggs were measured from Edgeøya, and the raw measurements from both islands overlapped, we decided to put all eggs measurements in a single table ( Table 3 ).

Type depositories: The holotype (Slide No. 205 (1)) is preserved at the Zoological Institute of the Russian Academy of Sciences, St. Petersburg, Russia . The paratype (adult and egg) has been given to the tardigrade collection at the Department of Animal Taxonomy and Ecology , Institute of Environmental Biology , Adam Mickiewicz University in Poznań (Slide No. 205 (2)). Additional material from Spitsbergen (slide code: 102.8/1), Nordaustlandet (slide codes: 103.1/1, 2, 4, 5, 7, 9, 103.2/1, 3, 5, 6, 7, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42), and Edgeøya (slide codes: 104/2, 113.1/2, 3, 4, 118.1/2, 4, 5, 7, 8, 9, 10) are preserved in the tardigrade collection at the Department of Animal Taxonomy and Ecology , Institute of Environmental Biology, Adam Mickiewicz University in Poznań.

All sequences for T. voronkovi obtained in this study were unique and distinct from all other sequences deposited in GenBank. The 28S rRNA alignment comprised 1004–bp with two haplotypes (two sequences from two adults) that differed in one nucleotide position (the uncorrected p-distances = 0.2%) (sequences deposited in GenBank under accession numbers: KX810049 –50 respectively). The 18S rRNA comprised 1702–bp (one adult, sequence deposited in GenBank under accession number: KX810045 ). The COI alignment comprised 432–bp with three haplotypes (three sequences from two adult and one egg) that differed in eight nucleotide positions (the uncorrected p-distances varied from 0.9% to 1.9%) (sequences deposited in GenBank under accession numbers: KX810042 –44 respectively). The ITS2 alignment comprised 1044-bp with three haplotypes (three sequences from three adult) that differed in six nucleotide positions (the uncorrected p-distances varied from 0.1% to 0.6%) (sequences deposited in GenBank under accession numbers: KX810046 –48 respectively).

The low genetic distance between the COI and ITS–2 haplotypes indicate that, at the molecular level, all individuals and egg should be classified as a single species.

Nucleotide blast searches using T. voronkovi 28S rRNA and 18S rRNA sequences, suggested that this taxon is most closely related to Macrobiotus furcatus Ehrenberg, 1859 , Paramacrobiotus richtersi ( Murray, 1911) , M. pallarii Maucci, 1954 (28S rRNA), P. tonollii ( Ramazzotti, 1956) and P. richtersi ( Murray, 1911) (18S rRNA). These results placed the genus Tenuibiotus in the family Macrobiotidae . The analysis of 28S rRNA involved four nucleotide sequences. All positions containing gaps and missing data were eliminated. There were a total of 1052 positions in the final dataset. The p-distances for 28S rRNA between T. voronkovi (GenBank accession number: KX810049 –50), M. furcatus (FJ435760.1), P. richtersi (FJ435757.1) and M. pallarii (FJ435756.1) varied from 6.7% to 7%. The analysis of 18S rRNA involved three nucleotide sequences. As before, all positions containing gaps and missing data were eliminated, providing a total of 1699 positions in the final dataset. The p-distances for 18S rRNA between T. voronkovi (GenBank accession number: KX810045 ), P. tonollii (DQ839605.1) and P. richtersii (EU038078.1) varied from 1.3% to 2.1%.

Figures

FIGURES 1 – 2. Tenuibiotus voronkovi — habitus: 1 - dorso-ventral projection, exoskeleton after DNA extraction (PCM); 2 - dorso-ventral projection (DIC).

FIGURES 3 – 6. Tenuibiotus voronkovi — buccal apparatus: 3 — buccal apparatus, dorso-ventral projection (PCM); 4 — ventral view of the buccal armature, arrowhead indicate row of teeth (PCM); 5 — buccal apparatus, dorso-ventral projection (DIC); 4 — dorsal view of the buccal armature, arrowhead indicate single teeth (PCM).

FIGURES 7 – 8. Tenuibiotus voronkovi — claws of leg IV seen in PCM: 7 — arrowhead indicate accessory points; 8 — dentate lunules and granulation.

FIGURES 9 – 12. Tenuibiotus voronkovi — eggs and juveniles: 9 — egg midsection (DIC); 10 — egg midsection with embryo (PCM); 11, 12 — juveniles and eggs.

FIGURES 13 – 18. Tenuibiotus voronkovi — egg process details, different shape of processes seen in PCM.

Tables

TABLE 1. Measurements [in µm] of selected morphological structures of holotype and paratype of T. voronkovi from Tumanov (2007). New measurements not included in the original description are bolded and italicized.

CHARACTER	µm	pt	µm	pt
	Holotype		Paratype
Body length	532	876
Buccal tube length	60.7	-	63.6	-
Stylet support insertion point		74.4		76.7
Buccal tube external width	6.7	11.0	7.4	11.6
Buccal tube internal width			5.1	8.0
Ventral lamina length			46.4	73.0
Macroplacoid 1	14.1	23.2	16.3	25.6
Macroplacoid 2	8.1	13.3	14.1	22.2
Microplacoid	2.2	3.6	3.3	5.2
Macroplacoid row	22.2	36.6	28.1	44.2
Placoid row	25.2	41.5	32.2	50.6
Claw 1 lengths
External primary branch			17.1	26.9
External secondary branch			12.3	19.3
Internal primary branch			16.9	26.6
Internal secondary branch			12.3	19.3
Claw 2 lengths
External primary branch			18.2	28.6
External secondary branch			14.6	23.0
Internal primary branch			18.0	28.3
Internal secondary branch			13.5	21.2

TABLE 2. Measurements [in µm] of selected morphological structures of individuals of T. voronkovi 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	23	407–769 871–1534	646 1241	111 131
Buccopharyngeal tube
Buccal tube length	27	33.5–56.5 -	50.5 –	6.0 –
Stylet support insertion point	27	24.2–42.6 72.2–77.1	37.9 75.0	4.6 1.4
Buccal tube external width	27	4.5–7.2 11.0–14.7	6.3 12.6	0.7 0.9
Buccal tube internal width	27	2.2–4.7 5.6–9.2	3.6 7.1	0.7 0.9
Ventral lamina length	19	18.0–32.7 50.2–64.1	27.4 56.0	3.9 3.9
Placoid lengths
Macroplacoid 1	27	8.8–17.6 21.9–33.2	14.0 27.7	2.6 3.1
Macroplacoid 2	27	4.9–9.2 12.5–18.7	7.9 15.6	1.6 1.7
Microplacoid	27	1.5–3.6 4.2–8.2	3.0 5.8	0.7 1.0
Macroplacoid row	27	14.3–27.7 35.7–53.5	23.9 47.0	4.3 4.7
Placoid row	26	17.1–32.8 42.6–63.5	27.9 54.8	5.2 6.0
Claw 1 lengths
External primary branch	13	12.2–17.5 25.3–36.1	15.5 29.9	2.0 3.1
External secondary branch	13	8.5–12.5 18.2–26.1	11.2 21.6	1.4 2.3
Internal primary branch	14	11.5–17.0 25.4–33.4	15.0 29.2	2.5 2.6
Internal secondary branch	14	9.0–12.4 17.1–26.0	11.2 22.0	1.4 2.1
Claw 2 lengths
External primary branch	23	9.5–18.5 23.6–34.8	15.6 30.9	2.6 2.7
External secondary branch	23	8.6–16.9 21.4–32.7	12.7 25.2	2.0 2.4
Internal primary branch	19	12.5–18.5 26.9–36.4	15.9 31.0	2.0 2.7
Internal secondary branch	19	9.2–14.2 19.7–29.2	12.4 24.3	1.7 2.4
Claw 3 lengths
External primary branch	21	11.8–20.0 26.9–41.2	16.8 32.6	3.1 3.4
External secondary branch	20	9.0–14.2 19.2–27.3	12.7 24.8	1.9 2.2
Internal primary branch	23	12.4–21.0 26.5–38.5	16.3 31.5	2.3 3.5
Internal secondary branch	23	9.1–17.0 19.4–37.5	13.1 25.2	2.9 3.7
Claw 4 lengths
Anterior primary branch	21	9.7–21.7 28.8–41.3	17.1 34.1	3.3 3.4
Anterior secondary branch	21	7.6–15.9 21.1–31.3	12.9 25.8	2.3 2.6
Posterior primary branch	19	7.1–20.0 21.2–40.9	16.4 33.3	3.3 4.2
Posterior secondary branch	19	8.5–14.9 20.1–30.7	12.5 25.6	2.3 3.4
Sequences data

TABLE 3. Measurements [in µm] of selected morphological structures of 18 eggs of T. voronkovi from Nordaustlandet (13 eggs) and Edgeøya (5 eggs) mounted in Hoyer’s medium (N—number of eggs / structures measured, RANGE refers to the smallest and the largest structure among all measured traits; SD—standard deviation).

CHARACTER	N	RANGE	MEAN	SD
Diameter of egg without processes	3	86.6–123.2	104.0	18.4
Diameter of egg with processes	2	137.0–182.0	159.5	31.8
Process height	49	12.3–37.3	21.8	5.7
Process base width	53	9.7–25.4	15.6	3.5
Process base/height ratio	49	44%–124%	75%	18%
Distance between processes	49	0.6–4.2	1.8	0.8
Number of processes on the egg circumference	2	17–22	19.5	3.5