Paramacrobiotus bengalensis, Basu & Babu & Siddique & Purushothaman, 2023

Basu, Subhrangshu, Babu, Rahul, Siddique, Alfisa & Purushothaman, Jasmine, 2023, Integrative description of Paramacrobiotus bengalensis sp. nov. (Tardigrada: Eutardigrada: Macrobiotidae), a new limno-terrestrial tardigrade species from the state of West Bengal, India, European Journal of Taxonomy 890 (1), pp. 23-48 : 30-38

Paramacrobiotus bengalensis sp. nov.

urn:lsid:zoobank.org:act:19ABAA28-D46D-4045-A126-FC628526C114

Figs 2–7 , Tables 3–4

Etymology

The specific toponymic bengalensis refers to the State of West Bengal, India, where the type locality of this new species, ‘Acharya Jagadish Chandra Bose Indian Botanic Garden’, Shibpur, is situated.

Material examined

32 specimens (one holotype, 28 paratypes, three voucher specimens) and five eggs were mounted on microscopic slides in Hoyer’s medium. Three specimens and two eggs were fixed for SEM preparation. Three specimens were processed for genotyping.

Holotype INDIA • West Bengal, Howrah, Shibpur, Acharya Jagadish Chandra Bose Indian Botanic Garden ( Fig. 1 ); 22°33′18.1′′ N, 88°17′30.7′′ E; Rahul Babu and Subhrangshu Basu leg.; moss from a tree (Sample Code: BSI/M 8 ); ZSI-HQ/GNC/T1/1 .

Paratypes INDIA • 3 specs (slides); same collection data as for holotype; ZSI-HQ/GNC/T2/1 , ZSI-HQ/GNC/ T3/1 , ZSI-HQ/GNC/T4/1 National Zoological Collection, Zoological Survey of India, Kolkata, India • 24 specs (slides); same collection data as for holotype; ZSI/TAR_IND/WB/001 to ZSI/TAR_IND/ WB/024 National Zoological Collection, Zoological Survey of India, Kolkata, India • 3 voucher specs; same collection data as for holotype; ZSI/TAR_IND/WB/V001 to ZSI/TAR_IND/WB/V003 • 5 eggs; same collection data as for holotype; ZSI/TAR_IND/WB/E001 Protozoology Section, Zoological Survey of India HQ, New Alipore, M Block, Kolkata, India .

Description

The body is almost transparent in juveniles, white in adults, and transparent after mounting in Hoyer’s medium ( Fig. 2 ; measurements and statistics provided in Table 3 ). Eyes are present in live specimens but dissolve after mounting in Hoyer’s medium.

Mouth anteroventral, bucco-pharyngeal apparatus of the Macrobiotus type ( Fig. 3 ) with 10 peribuccal lamellae and ventral lamina. The oral cavity armature is well developed and composed of three bands of teeth ( Fig. 3B–C ). The teeth in the first band are granular in shape and smaller than those in the other two bands. The first band of teeth is situated in the anterior portion of the oral cavity behind the bases of the peribuccal lamellae. The second band, situated between the ring fold and the third band ( Fig. 3B–C ) is intermediate in size, continuous, and arranged in a row that runs around the oral cavity wall. The second band comprises cone shaped teeth which are parallel to the main axis of the buccal tube. Teeth of the second band are uniform and regular and are not joined to each other. The third band is located at the rear end of the oral cavity between the second band teeth and the buccal tube opening. The third band is divided into two parts: dorsal and ventral, with three ventral and three dorsal teeth each (two lateral and one median, which is always slightly shorter than the lateral ones). The dorsal and ventral portions are visible under the PCM as one median ridge and two lateral transverse ridges. The medioventral tooth of the third band of teeth is subdivided into two to three smaller teeth ( Fig. 3B ). Additional granular teeth are absent between the second and third band of teeth on the ventral side. The pharyngeal bulb is spherically shaped with triangular apophyses. Three macroplacoids and rod-shaped microplacoids are present and distinctly visible under PCM ( Fig. 3A, D ). The macroplacoid sequence is 2<1 ≤3, and the first macroplacoid is anteriorly thinner and arrow-shaped. The second macroplacoid is bar-shaped without constriction, whereas the third macroplacoid has a distinct sub-terminal constriction ( Fig. 3D ).

The claws are Y-shaped and of the hufelandi type. The primary claw branches have distinct accessory points, a common tract, and a stalk that connects the claw and lunula ( Fig. 4A ). The lunulae under all the claws on all the legs are smooth ( Fig. 4 ). Leg cuticle is smooth, without any granulations present in legs I–III. Granulation is present on the hind legs but only faintly visible ( Fig. 4D ). Cuticular bars under the claws absent. In PCM, muscle attachments under claws I to III are visible ( Fig. 4B–C ).

Eggs laid freely, white/colourless with 12–14 cone-shaped processes on the circumference ( Fig. 5 ; measurements and statistics provided in Table 4 ). The space between processes is areolated with 8 to 10 areolas present around each process ( Fig. 6 ). The surface of the areoles is without pores but sculptured with wrinkles. Processes trunk cone shaped with a cap-like structure on the top ( Fig. 7 ), with fine villilike protrusions. Under PCM egg processes walls have fine reticulation which is caused by the internal labyrinthine layer within the chorion ( Fig. 7A–B ).

DNA sequences

We obtained sequences for two DNA markers. Out of these two successfully sequenced markers, 18S rRNA was represented by two haplotypes, whereas a single haplotype was found for COI: the 18S rRNA haplotype 1 sequence (GenBank: ON923868), 1017 bp long; the 18S rRNA haplotype 2 sequence (GenBank: ON923866) 1014 bp long; the COI haplotype 1 sequence (GenBank: OP531839), 658 bp long.

Phylogenetic analysis

The phylogenetic reconstruction performed with the BI and ML methods on the concatenated dataset of the two DNA markers showed almost identical topologies, with lower support values for the ML tree ( Fig. 8A ). Our analysis revealed that the Paramacrobiotus richtersi morphogroup forms a monophyletic clade, whereas the Paramacrobiotus areolatus morphogroup was recovered as a paraphyletic group ( Fig. 8A–B ), which was consistent with the results presented by Stec et al. (2020c). Phylogenetic analysis supported the discovery of Paramacrobiotus bengalensis sp. nov. The new species is strongly supported in the monophyletic clade of Paramacrobiotus richtersi in both the BI and ML phylogenetic trees, establishing that it does indeed belong to the Paramacrobiotus richtersi morphogroup.

Ecological information

The moss (species unknown) was collected from the mango tree Mangifera indica L., at a height of approximately 2 m from the ground. The altitude of the type locality is 12 m above sea level. The type locality is situated on the banks of River Ganges.

Species delimitation

ASAP and mPTP analyses of 81 COI sequences (Supp. File 4) identified 24 partitioned subsets (asapscore = 6.0) and 30 delimited species. Both analyses revealed Paramacrobiotus bengalensis sp. nov. as a putative new species.

Figures

Fig. 2. Paramacrobiotus bengalensis sp. nov. A. Holotype (ZSI-HQ/GNC/T1/1), habitus, dorso-ventral projection. B–C. Paratypes (ZSI-HQ/GNC/T2/1). B. Habitus dorsal projection. C. Scanning electron microphotograph of a whole specimen. Scale bars: A, C = 100 μm; B = 200 μm.

Fig. 3. Paramacrobiotus bengalensis sp. nov., paratypes. A, D. Specimen ZSI-HQ/GNC/T3/1. B–C. Specimen ZSI/TAR_IND/WB/024. A. Buccal apparatus, dorso-ventral projection with dorsal teeth in focus. B–C. Oral cavity armature (B, ventral teeth; C, dorsal teeth). D. Macroplacoids. White unfilled arrow indicate the second band of teeth, unfilled checkered arrow indicate the first band of teeth, white unfilled chevron arrow indicate the third band of teeth, white filled chevron arrow indicates medioventral teeth, a white filled arrow indicates the subterminal constriction in the third macroplacoid. All PCM. Scale bars: A = 10 μm; B–D = 5 μm.

Fig. 4. Paramacrobiotus bengalensis sp. nov., claws. A. Holotype (ZSI-HQ/GNC/T1/1), claws I with small smooth lunulae. B–C. Paratype (ZSI/TAR_IND/WB/024). B. Claws II with small smooth lunulae. C. Claws III with small smooth lunulae. D. Paratype (ZSI-HQ/GNC/T2/1), claws IV with smooth lunulae, (black filled arrow indicates smooth lunuale for A–D). E–F. Claws II and III without any visible granulation the legs (black filled checkered arrow). Figures assembled from several photos. A–D = PCM; E–F = SEM. Scale bars: A–C = 5 μm; D = 10 μm; E–F = 0.4 μm.

Fig. 5. Paramacrobiotus bengalensis sp. nov., paratype (ZSI/TAR_IND/WB/E001). A. Entire egg under SEM. B–F. Eggs seen in PCM under 1000× magnification. B. Midsection. D–F. Surfaces. Scale bars: A = 3 μm; B–F = 10 μm.

Fig. 6. Paramacrobiotus bengalensis sp. nov., paratype (ZSI/TAR_IND/WB/E001), egg chorion details. A–B. Zoom on egg surface between the processes with clearly visible areolae and cap like structures on processes (SEM), white empty chevron arrows indicate wrinkles inside the areolae. Scale bars: A = 5 μm; B = 2 μm.

Fig. 7. Paramacrobiotus bengalensis sp. nov., paratype (ZSI/TAR_IND/WB/E001), egg process details. A–B. An egg process under PCM and SEM with weakly visible reticulum. Black empty arrows indicate the cap like structure on the top of the process, black empty checkered arrows indicate the fine reticulation on the process. Scale bars: A = 10 μm; B = 1 μm.

Fig. 1. Type locality of the new taxa (marked with red square).

Fig. 8. Phylogeny of Paramacrobiotus Guidetti, Schill, Bertolani, Dandekar & Wolf, 2009 based on the nucleotide sequences of two markers (18S rRNA + COI). A. ML tree. B. BI tree. The numbers at nodes represent Bayesian posterior probability and bootstrap support values. The blue coloured clade represents species from the Paramacrobiotus richtersi morphogroup, while the red coloured clade represents species from the Paramacrobiotus areolatus morphogroup. Both trees have a red colour for the new species. The outgroup is denoted by black triangles. The scale bar reflects the number of substitutions per position.

Tables

Table 3. Measurements (in µm) of selected morphological structures of individuals of Paramacrobiotus bengalensis sp. nov. mounted in Hoyer’s medium.Abbreviations: 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	29	158–412	780–1117	295	899	85	94	193	925
Buccal tube
Buccal tube length	29	19.2–47.0	–	32.8	–	9.2	–	20.9	–
Stylet support insertion point	29	13.5–37.1	70.3–79.6	25.1	76.3	7.3	2.2	16.3	77.9
Buccal tube external width	29	3.6–8.7	17.2–22.1	6.4	19.4	1.8	1.2	3.6	17.2
Buccal tube internal width	29	2.8–7.8	13.1–16.6	5.1	15.4	1.6	1.1	2.8	13.2
Ventral lamina length	29	9.9–25.6	49.2–54.5	17.2	52.4	4.9	1.6	10.6	50.8
Placoid lengths
Macroplacoid 1	29	2.8–7.1	14.2–15.5	4.8	14.7	1.4	0.4	3.0	14.2
Macroplacoid 2	29	1.9–5.0	9.7–11.8	3.5	10.8	1.0	0.5	2.4	11.3
Macroplacoid 3	29	3.0–7.0	14.1–16.7	5.0	15.5	1.3	0.7	3.3	15.6
Microplacoid	29	1.0–2.7	4.4–7.3	1.8	5.5	0.5	0.7	1.1	5.5
Macroplacoid row	29	7.7–18.2	36.6–42.8	13.3	40.7	3.7	1.4	8.7	41.9
Placoid row	29	10.3–24.5	48.0–55.0	17.0	51.9	4.7	1.6	11.5	55.0
Claw I heights
External primary branch	26	3.0–7.7	14.9–18.5	5.3	16.1	1.4	0.7	3.9	18.5
External secondary branch	26	2.3–6.0	10.3–13.0	4.1	12.3	1.1	0.6	2.7	12.8
Internal primary branch	25	3.0–7.7	15.2–18.4	5.3	16.2	1.4	0.7	3.9	18.4
Internal secondary branch	24	2.3–6.0	11.0–13.3	4.0	12.3	1.2	0.5	2.8	13.3
Claw II heights
External primary branch	27	3.1–7.8	15.0–18.4	5.3	16.1	1.4	0.6	3.8	18.4
External secondary branch	27	2.2–6.0	10.2–13.4	4.0	12.2	1.2	0.7	2.5	12.0
Internal primary branch	26	3.2–7.8	14.9–17.6	5.4	16.1	1.4	0.6	3.7	17.6
Internal secondary branch	25	2.3–5.9	10.9–13.0	4.1	12.2	1.1	0.6	2.4	11.4
Claw III heights
External primary branch	27	3.1–7.7	14.8–18.1	5.3	16.2	1.5	0.6	3.8	18.1
External secondary branch	27	2.2–5.9	10.0–13.6	4.0	12.2	1.2	0.8	2.4	11.4
Internal primary branch	23	3.2–7.2	14.8–17.6	5.3	16.1	1.4	0.6	3.7	17.6
Internal secondary branch	22	2.3–5.4	10.6–13.2	3.9	12.2	1.1	0.6	2.3	11.0
Claw IV heights
Anterior primary branch	25	4.5–9.7	20.0–25.5	7.3	22.2	1.7	1.3	5.3	25.5
Anterior secondary branch	25	3.4–8.5	16.5–19.5	6.0	18.2	1.6	0.8	3.9	18.4
Posterior primary branch	22	4.7–9.7	20.1–25.6	7.1	22.5	1.8	1.3	5.4	25.6
Posterior secondary branch	20	3.5–8.5	16.8–19.7	5.8	18.3	1.7	0.8	3.9	16.8