The Macrobiotus ariekammensis species complex provides evidence for parallel evolution of claw elongation in macrobiotid tardigrades Author Stec, Daniel Author Vončina, Katarzyna Author Kristensen, Reinhardt Møbjerg Author Michalczyk, Łukasz text Zoological Journal of the Linnean Society 2022 2022-02-05 195 1067 1099 journal article 121762 10.1093/zoolinnean/zlab101 e67af1dc-6b12-44cf-9a1d-27aad0e6f39a 0024-4082 6994499 B66AA0F0-04D1-4CEE-833A-1C9EE623166E MACROBIOTUS KIRGHIZICUS TUMANOV, 2005 ( TABLES 6 , 7 ; FIGS 12–18 ) Material examined: Altogether 66 animals, and 15 eggs . Specimens mounted on microscope slides in Hoyer’s medium (53 animals + ten eggs ), fixed on SEM stubs (ten + five), processed for DNA sequencing (three animals) . Population locality : 41°32’37.98’’N , 75°10’2.28’’E ; 2288 m a.s.l. : Kyrgyzstan , Chui , Kegeti , moss on rock . Specimens depositories: Altogether 53 animals (slides: KG.062.006. 1, 9–14, SEM stub: 18.07) and ten eggs (slides: KG.062. 5–8, SEM stub: 18.07) are deposited at the Institute of Zoology and Biomedical Research , Jagiellonian University , Gronostajowa 9, 30-387, Kraków , Poland . Description of the Kyrgyz Republic population Animals (measurements and statistics in Table 6 ): Body whitish in adults and colourless in smaller individuals, after fixation in Hoyer’s medium transparent ( Fig. 12A ). After mounting in Hoyer’s medium eyes present in all specimens. Small, oval pores (0.5–0.8 µm in diameter), visible under PCM and SEM ( Fig. 12B, C ), scattered randomly on the entire body cuticle, including the external and internal surface of all legs ( Fig. 13A–F ). Extremely fine body granulation ( c . 60 nm in diameter), visible only in SEM, present on the entire dorsocaudal cuticle ( Fig. 12C ). Patches of dense granulation present on the internal and external surfaces of all legs I–III and clearly visible both in PCM and SEM ( Fig. 13A–D ). A cuticular bulge, resembling a pulvinus, is present on the internal surfaces of legs I–III ( Fig. 13C, D ). Cuticular granulation on legs IV present and always clearly visible both in PCM and SEM ( Fig. 13E, F ). Table 4. Measurements (in µm) and pt values (italics) of selected morphological structures of the holotype and the paratypes of Macrobiotus ariekammensis groenlandicus subsp. nov. mounted in Hoyer’s medium ( N , number of specimen/ structures measured, Ranges refer to the smallest and the largest structure among all measured specimens; SD, standard deviation)

Character	N	Ranges			Mean	SD		Holotype
µm	pt	µm	pt	µm	pt	µm	pt
Body length	22	377	–	888	950	–	1734	633	1324	148	187	811	1483
Buccal tube
Buccal tube length	22	37.2	–	56.5	–	47.3	–	5.7	–	54.7	–
Stylet support insertion point	22	27.1	–	41.1	71.0	–	73.2	34.1	72.2	4.1	0.8	40.0	73.1
Buccal tube external width	22	4.5	–	9.6	11.0	–	18.4	6.1	12.9	1.3	1.7	7.5	13.7
Buccal tube internal width	22	3.0	–	5.5	6.8	–	10.5	3.9	8.2	0.7	0.9	5.5	10.1
Ventral lamina length	22	22.1	–	35.8	53.6	–	63.7	28.3	59.9	3.4	2.3	33.7	61.6
Placoid lengths
Macroplacoid 1	22	7.2	–	15.9	18.1	–	31.4	11.2	23.5	2.5	3.2	13.4	24.5
Macroplacoid 2	22	4.5	–	9.8	11.3	–	18.3	6.9	14.4	1.6	1.9	8.9	16.3
Microplacoid	22	1.7	–	5.2	4.3	–	10.4	2.9	6.1	0.8	1.4	3.3	6.0
Macroplacoid row	22	13.6	–	26.5	34.3	–	52.3	19.8	41.6	4.0	4.6	24.6	45.0
Placoid row	22	15.1	–	30.2	37.3	–	59.2	23.4	49.0	5.0	5.8	29.2	53.4
Claw I heights
External primary branch	20	13.2	–	25.6	33.2	–	54.6	21.1	44.0	4.1	5.2	25.6	46.8
External secondary branch	15	8.9	–	18.2	22.4	–	36.1	14.6	30.4	3.1	3.9	?	?
Internal primary branch	18	13.4	–	23.4	35.3	–	49.8	19.6	41.2	3.5	4.2	?	?
Internal secondary branch	17	8.9	–	16.6	23.9	–	33.9	13.8	29.1	2.5	2.6	?	?
Claw II heights
External primary branch	22	14.7	–	27.5	37.5	–	54.9	21.9	46.1	4.1	4.8	27.3	49.9
External secondary branch	15	9.9	–	21.4	26.6	–	42.6	16.2	33.8	3.3	4.0	19.0	34.7
Internal primary branch	21	12.6	–	26.2	31.7	–	53.7	20.6	43.2	4.4	5.8	24.0	43.9
Internal secondary branch	18	9.3	–	18.6	25.0	–	37.1	14.8	31.2	2.8	3.3	?	?
Claw III heights
External primary branch	21	14.4	–	28.0	36.5	–	57.8	22.8	48.0	4.5	5.9	27.8	50.8
External secondary branch	19	9.9	–	20.5	24.9	–	42.3	16.5	34.5	3.5	4.5	20.5	37.5
Internal primary branch	21	13.7	–	28.0	34.5	–	57.4	21.6	45.5	4.7	6.3	26.9	49.2
Internal secondary branch	14	9.1	–	19.1	23.7	–	38.7	15.2	31.5	3.5	4.2	19.1	34.9
Claw IV heights
Anterior primary branch	16	20.1	–	39.9	50.6	–	73.3	30.1	63.3	6.0	6.3	39.9	72.9
Anterior secondary branch	13	11.4	–	21.9	30.6	–	46.1	17.6	37.5	3.2	4.1	21.0	38.4
Posterior primary branch	21	21.0	–	41.0	52.9	–	82.9	32.0	67.7	6.2	7.2	40.7	74.4
Posterior secondary branch	19	11.6	–	22.9	30.2	–	46.9	18.6	39.6	3.6	4.7	21.7	39.7

Table 5. Measurements (in µm) of selected morphological structures of the eggs of Macrobiotus ariekammensis groenlandicus subsp. nov. mounted in Hoyer’s medium ( N , number of eggs/structures measured, Range refers to the smallest and the largest structure among all measured specimens; SD, standard deviation)

Character	N	Range		Mean	SD
Egg bare diameter	15	95.9	–	118.1	103.5	7.0
Egg full diameter	15	122.9	–	150.9	134.9	8.4
Process height	60	9.3	–	19.4	15.3	2.3
Process base width	60	10.0	–	18.8	14.0	2.2
Process base/height ratio	60	62%	–	130%	92%	15%
Process distal portion width	60	1.4	–	5.0	3.1	0.8
Inter-process distance	57	1.8	–	4.7	3.0	0.6
Number of processes on the egg circumference	14	20	–	25	23.1	1.5

Figure 5. Macrobiotus ariekammensis groenlandicus subsp. nov. A, habitus, dorsoventral projection (holotype, Hoyer’s medium, PCM); B, C, well-visible granulation on the dorsal (B) and ventral (C) parts of the body seen in PCM; D, less-visible granulation on the dorsal part of the body seen in PCM; E, F, granulation on the dorsal part of the body seen in SEM; G, magnification on the cuticular pore and granulation on the dorsal part of the body in SEM. Filled flat arrowheads indicate the granules of granulation seen in SEM. Scale bars in µm. Figure 6. Macrobiotus ariekammensis groenlandicus subsp. nov. – cuticular structures on legs: A–C, granulation on the external surface of legs III seen in PCM (A) and SEM (B, C), respectively; C, shows a magnification of leg granulation above the dense granulation patch; D–F, granulation on the internal surface of legs III seen in PCM (D) and SEM (E, F), respectively; F, shows a magnification of leg granulation above the dense granulation patch; G–I, granulation on the dorsal and dorsolateral surface of leg IV seen in PCM (G) and SEM (H, I); I, shows a magnification of leg granulation above the dense granulation patch. Filled flat arrowheads indicate the dense granulation patch on the external leg surface, empty indented arrowhead indicates the cuticular bulge (pulvini), empty flat arrowheads indicate the dense granulation patch on the internal leg surface, filled indented arrowhead indicates cuticular bar under the claws. Scale bars in µm. Claws slender, with flat and wide common tract, beginning with an evident stalk that connects the claws to the wide lunulae and ending with extremely elongated branches (especially the primary branch; Fig. 14A, B, D, E ). Primary branches with indistinct accessory points, barely visible in PCM, but clearly visible in SEM ( Fig. 14A, B, D, E ). Lunulae I–III smooth ( Fig. 14A, D ), whereas lunulae IV with clear dentation ( Fig. 14B, E ). Mouth anteroventral with ten peribuccal lamellae ( Fig. 16A, B ). Bucco-pharyngeal apparatus of the Macrobiotus - type ( Fig. 15A ). Under PCM, only the second and third band of teeth visible, with the second band being faintly marked ( Fig. 15B, C ). However, under SEM, all of the three bands of teeth are visible, with the first band being situated at the base of peribuccal lamellae and composed of several irregular rows of small granular teeth surrounding the oral cavity ( Fig. 16A, C ). The second band of teeth is situated between the ring fold and the third band of teeth, and is comprises of small cones, barely visible in PCM ( Figs 15B , 16B ; note: in Fig 16B , only distal portion of these teeth are visible from behind the ring fold; due to unsuitable positioned specimen it was impossible to get better image in SEM). The teeth of the third band are located within the posterior portion of the oral cavity, between the second band of teeth and the buccal tube opening ( Figs 15B–D , 16A, B ). The third band of teeth is discontinuous and divided into a dorsal and a ventral portion. Under PCM, the dorsal teeth form a transversal ridge weakly divided into two granular teeth, whereas the ventral teeth are smaller and faintly visible as two separate lateral transverse ridges with granular/roundish thickening at their medial extremities ( Fig 15B–D ). In SEM, both the dorsal and the ventral portion of the third band of teeth are visible as one fused ridge with two evident teeth extending from the medial portion of the ridge ( Fig 16A, B ). Pharyngeal bulb spherical, with triangular apophyses, cuticular spikes, two rodshaped macroplacoids (macroplacoid sequence: 2 <1) and a triangular small microplacoid ( Fig. 16A, E ). The first macroplacoid exhibits weak central constriction, whereas the second macroplacoid is sub-terminally and weakly constricted ( Figs 15E ). Figure 7. Macrobiotus ariekammensis groenlandicus subsp. nov. – claws: A, B, claws III and IV seen in PCM, respectively; C–E, claws I, III and IV seen in SEM, respectively. Filled flat arrowheads indicate double muscles attachments under the claws, filled indented arrowhead indicates cuticular bar under the claws. Scale bars in µm. Eggs (measurements and statistics in Table 7 ): Eggs laid freely, whitish, spherical or slightly oval ( Figs 17A, B , 18A ). Although the spaces between processes are small, the surface between processes is of the persimilis - type , i.e. with the continuous smooth chorion, with few, randomly distributed pores ( Figs 17A, B , 18B–D ). Egg processes single-walled (without reticulation caused by labyrinthine layer) with domeshaped basal part and rigid spine-like distal part ( Figs 17A–F , 18A–F ). In PCM, the basal and distal portions are clearly separated from with single internal septum ( Fig 17C–F ). The bases of egg processes are pierced with pores of uniform size (0.3–0.7 µm in diameter), distributed evenly around the base and most often arranged in two rows ( Figs 17A, B , 18B–F ). In PCM, short, dark thickenings are sometimes visible around the process bases below or at the same level as the lower ring of pores ( Fig 17A, B ). The apical part of the processes is devoid of terminal discs and is covered with short, thin and flexible filaments ( Figs 17C–F , 18A–F ). Figure 8. Macrobiotus ariekammensis groenlandicus subsp. nov. – buccal apparatus and the oral cavity armature seen in PCM: A, dorsoventral projection of the entire buccal apparatus; B, C, oral cavity armature visible from dorsal (B) and ventral (C) view, respectively; D, oral cavity armature visible from lateral view; E, macroplacoid morphology. Filled flat arrowheads indicate a single tooth in dorsal portion of the third band of teeth in the oral cavity, empty arrows indicate dorsal cuticular spikes, empty indented arrowheads indicate central constrictions in first macroplacoids and subterminal constriction in second macroplacoid. Scale bars in µm. Reproduction: The population is dioecious (the examination of specimens freshly mounted in Hoyer’s medium revealed testes filled with spermatozoa), but no secondary sexual dimorphism has been observed. DNA sequences: All obtained DNA sequences were represented by a single haplotype per each marker: 18S rRNA: MZ463665 , MZ463666 , MZ463667 . 28S rRNA: MZ463671 , MZ463672 , MZ463673 . ITS2: MZ463659 , MZ463660 , MZ463661 . COI : MZ461002 , MZ461003 , MZ461004 . Figure 9. Macrobiotus ariekammensis groenlandicus subsp. nov. – buccal apparatus and oral cavity armature seen in SEM: A, entire buccal apparatus; B, ventral view of the buccal crown; C, D, placoids morphology; E, magnification of the middle part of the buccal apparatus with stylet support insertion points; F, the oral cavity armature. Filled arrows indicate two globular protuberances on the ventral side of the buccal crown and buccal tube, empty arrows indicate dorsal cuticular spikes, empty indented arrowheads indicate central constrictions in first macroplacoid and subterminal constriction in second macroplacoid, filled flat arrowhead indicates a single tooth in dorsal portion of the third band of teeth in the oral cavity. Scale bars in µm. Figure 10. Macrobiotus ariekammensis groenlandicus subsp. nov. – egg chorion morphology seen in PCM: A–D, egg surface under 1000× magnification; E–P, egg processes midsections under 1000× magnification. Filled flat arrowheads indicate a crown of dark thickenings and pores arranged alternately around egg process bases, filled indented arrowheads indicate light-refracting dots in the egg surface between the processes, empty flat arrowheads indicate septum between the basal and distal portion of egg process. Scale bars in µm. PHYLOGENY The phylogenetic reconstruction ( Fig. 19 ) shows three well-supported distinct lineages constituting three separate genera within superclade I ( sensu Stec et al. , 2021a ) of the family Macrobiotidae : the clade comprising Macrobiotus species , and further two monophyletic groups: one corresponding to the genus Mesobiotus Vecchi et al. , 2016 , and the other representing Sisubiotus Stec et al. , 2021a ( Fig. 19 ). Macrobiotus is divided into three well-supported subclades: A, B and C, sensu Stec et al. (2021a) . All of the three newly found populations investigated in this study, M. a. ariekammensis , M. a. groenlandicus and M. kirghizicus , are nested in subclade A, which contains species of the Macrobiotus hufelandi morphogroup sensu Stec et al. (2021a) and Macrobiotus basiatus Nelson et al. , 2020 , which exhibits unique egg morphology. Subclade B comprises three species complexes delineated by Stec et al. (2021a) . As in Stec et al. (2021a) and Vecchi & Stec (2021) , the Macrobiotus pallari complex and the Macrobiotus pseudohufelandi complex are monophyletic also in the present study ( Fig. 19 ). However, the Macrobiotus persimilis complex, which was monophyletic in the two earlier studies, appears to be paraphyletic in the current analysis ( Fig. 19 ). Thus, further studies are needed to clarify the phyletic character of the latter species complex. Subclade C comprises species of the Macrobiotus hufelandi morphogroup. Figure 11. Macrobiotus ariekammensis groenlandicus subsp. nov. – egg chorion morphology seen in SEM: A, entire egg; B, magnification of the egg surface; C, D, details of the egg processes; E, F, details of the terminal portion of egg processes. Filled flat arrowheads indicate pores within the basal portion of the process wall. Scale bars in µm. Table 6. Measurements (in µm) and pt values (italics) of selected morphological structures of the paratypes of Macrobiotus kirghizicus from the Kyrgyz Republic mounted in Hoyer’s medium ( N , number of specimen/structures measured, Ranges refer to the smallest and the largest structure among all measured specimens; SD, standard deviation)

Character	N	Ranges		Mean	SD
µm	pt	µm	pt	µm	pt
Body length	21	453	–	738	926	–	1549	582	1207	85	134
Buccal tube
Buccal tube length	21	38.8	–	55.4	–	48.2	–	3.9	–
Stylet support insertion point	21	28.3	–	40.3	71.4	–	73.4	35.0	72.5	2.8	0.6
Buccal tube external width	21	5.1	–	8.2	11.7	–	15.3	6.3	13.1	0.8	0.9
Buccal tube internal width	21	3.4	–	4.8	7.0	–	9.1	4.0	8.3	0.4	0.6
Ventral lamina length	21	23.2	–	34.1	57.7	–	64.2	29.7	61.6	2.7	1.7
Placoid lengths
Macroplacoid 1	21	9.0	–	15.6	23.2	–	30.5	13.0	26.8	1.6	1.9
Macroplacoid 2	21	6.4	–	9.4	14.1	–	18.7	7.9	16.3	0.8	1.2
Microplacoid	21	2.6	–	3.9	5.3	–	8.0	3.3	6.8	0.4	0.7
Macroplacoid row	21	16.0	–	26.7	40.7	–	53.4	22.4	46.3	2.6	3.0
Placoid row	21	19.4	–	31.3	49.5	–	65.2	26.8	55.6	2.9	3.3
Claw I heights
External primary branch	17	16.2	–	25.4	40.8	–	46.9	21.4	44.0	2.1	2.1
External secondary branch	12	11.8	–	18.6	30.4	–	38.9	15.9	33.3	1.9	2.4
Internal primary branch	16	15.4	–	23.0	35.3	–	46.7	20.2	42.1	1.9	3.1
Internal secondary branch	13	11.7	–	18.0	28.1	–	35.6	15.3	32.1	1.9	2.1
Claw II heights
External primary branch	19	17.3	–	26.6	44.6	–	53.3	23.1	48.2	2.4	2.6
External secondary branch	11	15.6	–	21.1	33.5	–	38.8	17.3	35.6	1.7	1.6
Internal primary branch	18	17.0	–	25.3	41.5	–	51.2	21.9	45.7	2.1	2.5
Internal secondary branch	15	13.1	–	19.1	29.4	–	40.1	16.2	33.5	1.8	3.0
Claw III heights
External primary branch	16	18.5	–	29.6	46.7	–	58.5	24.2	50.7	2.9	3.4
External secondary branch	10	13.2	–	23.7	33.3	–	46.8	18.1	38.3	3.0	4.3
Internal primary branch	17	17.4	–	27.6	44.8	–	53.6	23.3	48.5	2.7	2.7
Internal secondary branch	9	14.8	–	22.1	30.3	–	43.7	17.9	36.7	2.1	3.6
Claw IV heights
Anterior primary branch	12	27.6	–	42.6	71.1	–	85.8	36.7	75.8	3.9	4.9
Anterior secondary branch	8	17.2	–	28.0	44.3	–	56.5	24.0	49.3	3.7	3.9
Posterior primary branch	17	28.6	–	42.9	73.2	–	87.9	38.6	79.7	3.3	4.4
Posterior secondary branch	14	18.7	–	30.6	48.2	–	59.9	26.4	54.2	2.9	3.0

Table 7. Measurements (in µm) of selected morphological structures of the eggs of Macrobiotus kirghizicus from the Kyrgyz Republic mounted in Hoyer’s medium ( N , number of eggs/structures measured, Range refers to the smallest and the largest structure among all measured specimens; SD, standard deviation)

Character	N	Range	Mean	SD
Egg bare diameter	5	93.3	–	114.3	103.3	8.0
Egg full diameter	5	134.1	–	186.3	161.6	18.6
Process height	27	19.9	–	40.8	31.3	5.7
Process base width	27	9.2	–	17.5	13.2	1.9
Process base/height ratio	27	34%	–	57%	43%	7%
Inter-process distance	18	2.1	–	4.1	3.1	0.6
Number of processes on the egg circumference	5	21	–	24	22.2	1.3

Figure 12. Macrobiotus kirghizicus from the Kyrgyz Republic: A, habitus, dorsoventral projection (Hoyer’s medium, PCM); B, C, cuticular pores on the dorsocaudal part of the body seen in PCM and SEM, respectively. Filled flat arrowheads indicate faint body granulation. Scale bars in µm. SPECIES DELIMITATION AND GENETIC DISTANCES The PTP analysis identified 49 and 55 putative species in ML and BI approach, respectively. The ASAP analysis, on the other hand, identified 48 putative species. These results are in line with the general inspection of the tree terminals and the morphological information that would suggest also 48 species among the ingroup taxa. However, for two out of the three newly found populations analysed in this study, both PTP approaches were not congruent with ASAP results. The PTP approaches indicated that M. a. ariekammensis and M. a. groenlandicus constitute a single species, whereas the ASAP analysis identified them as separate entities. Uncorrected pairwise distances between the three newly found populations analysed in this study are as follows: • 18S rRNA: 0.2% for M. a. ariekammensis and M. a. groenlandicus ; 0.1% for M. a. ariekammensis and M. kirghizicus ; 0.3% for M. a. groenlandicus and M. kirghizicus . • 28S rRNA: 0.1% for M. a. ariekammensis and M. a. groenlandicus ; 0.3% for M. a. ariekammensis in PCM (E) and SEM (F). Filled flat arrowheads indicate granulation patch on the external leg surface, empty indented arrowheads indicate cuticular bulge (pulvini), filled indented arrowhead indicates cuticular bar, empty flat arrowheads indicate granulation patch on the internal leg surface. Scale bars in µm. Figure 13. Macrobiotus kirghizicus from the Kyrgyz Republic – cuticular structures on legs: A, B, granulation on the external surface of legs III seen in PCM (A) and SEM (B), respectively; C, D, granulation on the internal surface of legs III and II seen in PCM (C) and SEM (D), respectively; E–F, granulation on the dorsal and dorsolateral surface of leg IV seen Figure 14. Macrobiotus kirghizicus from the Kyrgyz Republic – claws (paratypes): A, B, claws II and IV seen in PCM, respectively; C, single continuous cuticular bar and double muscle attachments on leg III seen in PCM; D, E, claws II and IV seen in SEM, respectively. Empty flat arrowheads indicate accessory points, filled flat arrowheads indicate double muscles attachments under the claws, filled indented arrowhead indicates cuticular bar. Scale bars in µm. Figure 15. Macrobiotus kirghizicus from the Kyrgyz Republic – buccal apparatus and the oral cavity armature seen in PCM: A, dorsoventral projection of the entire buccal apparatus; B, C, oral cavity armature visible from dorsal (B) and ventral (C) view, respectively; D, oral cavity armature visible from lateral view; E, macroplacoid morphology. Filled flat arrowheads indicate the third band of teeth in the oral cavity, empty arrow indicates dorsal cuticular spike, empty flat arrowhead indicates the second band of teeth in the oral cavity, empty indented arrowheads indicate central constrictions in first macroplacoid and subterminal constriction in second macroplacoid. Scale bars in µm. and M. kirghizicus ; 0.1% for M. a. groenlandicus and M. kirghizicus . • ITS2: 0.3% to 0.8% for M. a. ariekammensis and M. a. groenlandicus ; 6.1% for M. a. ariekammensis and M. kirghizicus ; 6.3% for M. a. groenlandicus and M. kirghizicus . • COI : 3.3% for M. a. ariekammensis and M. a. groenlandicus ; 16.3% for M. a. ariekammensis and M. kirghizicus ; 16.4% for M. a. groenlandicus and M. kirghizicus . Given the discrepancies between the PTP and ASAP species delineation results, shallow genetic divergence and low p -distances in COI and ITS2 between M. a. ariekammensis and M. a. groenlandicus , we interpreted the morphological differences between the two taxa as intraspecific variability, hence the later taxon is described here as a subspecies rather than a separate species.