Macrobiotus kirghizicus, TUMANOV, 2005

Stec, Daniel, Vončina, Katarzyna, Kristensen, Reinhardt Møbjerg & Michalczyk, Łukasz, 2022, The Macrobiotus ariekammensis species complex provides evidence for parallel evolution of claw elongation in macrobiotid tardigrades, Zoological Journal of the Linnean Society 195, pp. 1067-1099 : 1076-1090

publication ID	https://doi.org/ 10.1093/zoolinnean/zlab101
publication LSID	lsid:zoobank.org:pub:B66AA0F0-04D1-4CEE-833A-1C9EE623166E
DOI	https://doi.org/10.5281/zenodo.6994548
persistent identifier	https://treatment.plazi.org/id/03A1D418-FF90-5A17-FCFB-FB4FFC02FE1A
treatment provided by	Plazi
scientific name	Macrobiotus kirghizicus
status

Treatment

MACROBIOTUS KIRGHIZICUS TUMANOV, 2005 View in CoL

( TABLES 6 View Table 6 , 7 View Table 7 ; FIGS 12–18 View Figure 12 View Figure 13 View Figure 14 View Figure 15 View Figure 16 View Figure 17 View Figure 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 GoogleMaps .

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 View Table 6 ): Body whitish in adults and colourless in smaller individuals, after fixation in Hoyer’s medium transparent ( Fig. 12A View Figure 12 ). 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 View Figure 12 ), scattered randomly on the entire body cuticle, including the external and internal surface of all legs ( Fig. 13A–F View Figure 13 ). Extremely fine body granulation (c. 60 nm in diameter), visible only in SEM, present on the entire dorsocaudal cuticle ( Fig. 12C View Figure 12 ). 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 View Figure 13 ). A cuticular bulge, resembling a pulvinus, is present on the internal surfaces of legs I–III ( Fig. 13C, D View Figure 13 ). Cuticular granulation on legs IV present and always clearly visible both in PCM and SEM ( Fig. 13E, F View Figure 13 ).

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 View Figure 14 ). Primary branches with indistinct accessory points, barely visible in PCM, but clearly visible in SEM ( Fig. 14A, B, D, E View Figure 14 ). Lunulae I–III smooth ( Fig. 14A, D View Figure 14 ), whereas lunulae IV with clear dentation ( Fig. 14B, E View Figure 14 ).

Mouth anteroventral with ten peribuccal lamellae ( Fig. 16A, B View Figure 16 ). Bucco-pharyngeal apparatus of the Macrobiotus - type ( Fig. 15A View Figure 15 ). Under PCM, only the second and third band of teeth visible, with the second band being faintly marked ( Fig. 15B, C View Figure 15 ). 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 View Figure 16 ). 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 View Figure 15 , 16B View Figure 16 ; note: in Fig 16B View Figure 16 , 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 View Figure 15 , 16A, B View Figure 16 ). 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 View Figure 15 ). 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 View Figure 16 ). Pharyngeal bulb spherical, with triangular apophyses, cuticular spikes, two rodshaped macroplacoids (macroplacoid sequence: 2 <1) and a triangular small microplacoid ( Fig. 16A, E View Figure 16 ). The first macroplacoid exhibits weak central constriction, whereas the second macroplacoid is sub-terminally and weakly constricted ( Figs 15E View Figure 15 ).

Eggs (measurements and statistics in Table 7 View Table 7 ): Eggs laid freely, whitish, spherical or slightly oval ( Figs 17A, B View Figure 17 , 18A View Figure 18 ). 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 View Figure 17 , 18B–D View Figure 18 ). Egg processes single-walled (without reticulation caused by labyrinthine layer) with domeshaped basal part and rigid spine-like distal part ( Figs 17A–F View Figure 17 , 18A–F View Figure 18 ). In PCM, the basal and distal portions are clearly separated from with single internal septum ( Fig 17C–F View Figure 17 ). 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 View Figure 17 , 18B–F View Figure 18 ). 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 View Figure 17 ). The apical part of the processes is devoid of terminal discs and is covered with short, thin and flexible filaments ( Figs 17C–F View Figure 17 , 18A–F View Figure 18 ).

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 View Materials , MZ463666 View Materials , MZ463667 View Materials . 28S rRNA: MZ463671 View Materials , MZ463672 View Materials , MZ463673 View Materials . ITS2: MZ463659 View Materials , MZ463660 View Materials , MZ463661 View Materials .

COI: MZ461002 View Materials , MZ461003 View Materials , MZ461004 View Materials .

PHYLOGENY

The phylogenetic reconstruction ( Fig. 19 View Figure 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 View Figure 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 View Figure 19 ). However, the Macrobiotus persimilis complex, which was monophyletic in the two earlier studies, appears to be paraphyletic in the current analysis ( Fig. 19 View Figure 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.

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.

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.