Abstract
Background
The Antarctic continent harbors unique microfauna that evolved independently under extreme conditions. The tardigrade Milnesium rastrum, discovered in East Antarctica, is characterized by atypical, aberrant claw configurations (CC) and pronounced sexual dimorphism. However, its detailed morphology and ecological roles have remained obscure due to an extreme scarcity of specimens. During the 67th Japanese Antarctic Research Expedition (JARE67, 2025–2026), we discovered a moss colony where M. rastrum was abundant near Langhovde, providing a rare opportunity for comprehensive morphometric and ecological analyses.
Results
Out of 33 collected individuals, 17 were males, 14 were adult females, and two were juveniles, indicating a balanced sex ratio of approximately 1:1. Significant sexual dimorphism was observed: females were larger (up to 742 μm) than males (up to 549 μm), and males exhibited a more slender posterior body. On leg I, males possessed enlarged, hook-like secondary branches. Males consistently exhibited a {4–4} CC, with a {2–2} pattern on leg I, whereas female body length showed a positive correlation with the number of points on the secondary branches, with larger individuals showing as many as seven points. Intestine content analysis revealed remnants of Hypsibiidae claws and Diphascon-like long buccal tubes. This provides direct evidence that M. rastrum preys on the sympatric tardigrade Diphascon langhovdense in its natural habitat.
Conclusions
This study provides the first comprehensive characterization of sexual dimorphism, growth dynamics, and the wild diet of M. rastrum. Our findings clarify distinct morphological variations between sexes and suggest different developmental strategies regarding molting. Additionally, confirming D. langhovdense as a natural prey source clarifies the specific ecological role of M. rastrum as a predator in the Langhovde microfauna community. Overall, these insights fundamentally advance our understanding of the morphological adaptations and ecological interactions of endemic tardigrades in extreme Antarctic environments.
Supplementary Information
The online version contains supplementary material available at https://doi.org/10.1186/s40851-026-00270-x.
Keywords: Tardigrades, Antarctica, Milnesium rastrum, Sexual dimorphism, Diet
Background
The Antarctic continent is surrounded by ocean and exposed to one of the harshest natural environments on Earth. Consequently, many species endemic to Antarctica have become established and evolved independently there. This includes the microfauna [1, 2], with endemic species having been discovered among rotifers, nematodes and tardigrades (e.g. the rotifer Philodina gregaria Murray, 1910; the nematode Plectus murrayi Yeates, 1970; the tardigrade Acutuncus antarcticus (Richters, 1904)). However, fundamental questions remain regarding the extent of diversity within these animal groups and the ecological characteristics they exhibit in such harsh conditions.
Tardigrades are organisms found across a wide range of habitats on Earth and of the more than 1,500 species within the phylum Tardigrada [3, 4], 42 species have been described from Antarctica alone [5]. Among species of the genus Milnesium, claw configuration (CC [6]), which indicates the number of points on the secondary branches, is typically consistent and used as a taxonomic key. Also, males in 16 species in the genus Milnesium exhibit sexual dimorphism (reviewed in [7, 8]), robust hook-shaped claws on the first pair of legs. It has also been reported that males tend to have a slimmer body than females e.g. [9].
In contrast, Milnesium rastrum Suzuki, Sugiura, Tsujimoto & McInnes, 2023, which is found only in scattered locations near Lützow-Holm Bay in Dronning Maud Land, East Antarctica, exhibits a unique CC. The number of points on the secondary branches vary irregularly from four to seven among individuals [10]. Although males have also been confirmed, only a single specimen has been found, in the process of molting [10]. Laboratory observations have indicated that this species feeds on rotifers. However, these findings are based on a very small number of specimens. Therefore, critical aspects of its biology remain poorly understood, including sexual dimorphism, allometric morphological changes in CC, and its actual diet in the wild.
From late 2025, we participated in the 67th Japanese Antarctic Research Expedition (JARE67) and collected moss samples near Lützow-Holm Bay. We discovered a colony of M. rastrum near Yukidori Valley Hut in Langhovde (Lützow-Holm Bay, Dronning Maud Land, East Antarctica) and conducted a detailed examination of the specimens.
Methods
Sample collection
Moss samples were collected in Langhovde, Lützow-Holm Bay, Dronning Maud Land, East Antarctica (69°14’38.58–68“S 39°43’09.49–54“E, ca.10 a.s.l.) between January 8th to 11th, 2026 (Fig. 1). Moss samples from the surrounding area were also collected. The samples were soaked in water for one day, and were then observed under a stereomicroscope. All Milnesium species found were picked, mounted with Hoyer’s medium on glass slides, and sealed with coverslips.
Fig. 1.
Sampling site in Langhovde, East Antarctica. Maps and pictures of the sampling site in Antarctica. (A) Maps showing the sampling site in the Antarctica (top left) and Lützow-Holm Bay. The triangle, star and circle indicate the location of the Syowa Station, the sampling site and Innhovde (the type locality of M. rastrum). B: a picture of the sampling site. The star indicates the sampling site. C: sampled mosses
Morphology observation and morphometrics
The Milnesium specimens were observed using phase-contrast microscopy. The structures of the specimens were measured using Apochela v1.4 [11]. The notation for CC followed [12]. The pt index (the ratio of measured values to the buccal tube) was used for comparison [13]. Body width was measured at each of the 10 transverse folds (see Fig. 2, abbreviated as F1 to F10). To compare the samples, a score was calculated by dividing body width by body length. To compare CC, the average number of points per leg across each secondary branch was calculated. Measurements were performed using ImageJ 1.53k (https://imagej.nih.gov/ij/) based on photographs. A Wilcoxon rank-sum test was used to statistically compare the claw configurations of males and females, while a Student’s t-test was used to compare body length, secondary branch length and body width on R v.3.6.1 [14].
Fig. 2.
Female and male of Milnesium rastrum. specimens of female and male M. rastrum. Left: whole body. Center: leg I. Right: leg IV. An and Po are abbreviations of anterior secondary branch and posterior secondary branch, respectively. Arrows indicate body folds of F1 to F10 (‘F’ stands for ‘fold’). Arrowheads indicate points of secondary branches. Scales = μm
Sexual dimorphism was quantified using the two-step ratio Sexual dimorphism index (SDI [15]). For each morphological trait, the SDI was calculated for every possible female and male pair. The resulting SDI values were then averaged to obtain a single estimate for the trait, in accordance with the procedure outlined for Echiniscidae tardigrades [16]. Positive SDI values indicate female-biased dimorphism, whereas negative values indicate male-biased dimorphism; the absolute value represents the magnitude of dimorphism. Statistical significance was assessed using a permutation test in which sex labels were randomly reassigned among individuals while preserving the original sample sizes (500 permutations). The observed SDI was then compared with the null distribution to obtain a two-tailed, permutation-based p-value. As multiple morphological traits were analyzed, the permutation-derived p-values were adjusted using the Benjamini–Hochberg false discovery rate (FDR) procedure. Traits with an FDR-adjusted permutation p-value < 0.05 were considered to exhibit significant sexual dimorphism. R v3.6.1 was used for comparisons [14]. The code used in this study was uploaded on FigShare (10.6084/m9.figshare.32843480).
Results
Sexual dimorphism of Milnesium rastrum
At Langhovde, near the Yukidori Valley Hut, we discovered that a moss colony located directly behind a glacial erratic serves as a colony where M. rastrum lives in abundance (Fig. 1). All 33 individuals collected during field observations were mounted as slide specimens (Fig. 2). Of these, 17 were males, 14 were adult females, and two were juveniles. Morphometrical data were on Tables 1, 2, and Table S1. The specimens of M. rastrum possessed secondary branches with up to seven points (Fig. 2, see next section). While the males measured up to 549 μm, some females were found to be as large as 742 μm (Tables 1 and 2, Table S1). Females were significantly longer in body length (p < 0.01 in a Student’s t-test, Fig. 3A). In male specimens, the secondary branch of leg I displayed the robust hook shape and, when compared using pt values, was significantly larger than that of females (Fig. 3B). Furthermore, when comparing the sizes of the anterior and posterior secondary branches of the male’s leg I, the anterior secondary branch was significantly larger (Fig. 3B). In addition, we measured the body width at 10 body folds and divided these values by body length to determine the slenderness of the body relative to its length (see Fig. 2). A comparison between males and females revealed that, in the region posterior to the sixth to tenth body fold (roughly posterior to the third leg), males were more slender than females (Fig. 3C, p < 0.05). Analysis using SDI revealed sex differences in buccal tube width (both actual measurements and pt values) and the lengths of several claws (pt values), in addition to the traits mentioned above (Table S2). Specifically, females had wider buccal tubes, while males often had longer secondary branches.
Table 1.
Morphometrics of female Milnesium rastrum
| Character | N | Range | Mean | SD | |||||||
|---|---|---|---|---|---|---|---|---|---|---|---|
| µm | pt | µm | pt | µm | pt | ||||||
| Body length | 14 | 533.4 | – | 741.6 | 993.2 | – | 1419.8 | 627.1 | 1239.6 | 50.6 | 115.4 |
| Peribuccal papillae length | 6 | 2.4 | – | 6.2 | 5.2 | – | 10.9 | 3.9 | 7.2 | 1.4 | 2.2 |
| Lateral papillae length | 6 | 3.7 | – | 7.8 | 8.5 | – | 13.6 | 5.1 | 10.3 | 1.4 | 1.8 |
| Buccal tube | |||||||||||
| Length | 14 | 41.7 | – | 65.3 | – | 51.2 | – | 7.6 | – | ||
| Stylet support insertion point | 14 | 23.2 | – | 45.8 | 53.6 | – | 73.5 | 30.5 | 59.5 | 6.2 | 5.3 |
| Anterior width | 14 | 11.1 | – | 23.7 | 22.5 | – | 36.3 | 14.8 | 28.9 | 3.2 | 4.0 |
| Standard width | 14 | 10.3 | – | 18.8 | 20.4 | – | 37.5 | 14.0 | 27.4 | 3.1 | 4.9 |
| Posterior width | 14 | 6.2 | – | 17.1 | 14.8 | – | 34.4 | 12.0 | 23.3 | 3.1 | 4.9 |
| Posterior/anterior width ratio | 14 | 0.5 | – | 1.1 | – | 0.8 | – | 0.2 | – | ||
| Claw I heights | |||||||||||
| Posterior (Posterior (External)) primary branch | 14 | 13.8 | – | 22.6 | 27.9 | – | 45.0 | 17.7 | 34.9 | 2.6 | 4.4 |
| Posterior (External) base + secondary branch | 14 | 9.9 | – | 15.8 | 21.2 | – | 31.1 | 13.0 | 25.6 | 2.0 | 2.7 |
| Posterior (External) spur | 14 | 1.3 | – | 3.8 | 3.2 | – | 5.9 | 2.2 | 4.3 | 0.7 | 0.9 |
| Posterior (External) branches length ratio | 14 | 0.6 | – | 0.9 | – | 0.7 | – | 0.1 | – | ||
| Anterior (Internal) primary branch | 14 | 10.7 | – | 25.8 | 22.7 | – | 45.2 | 16.8 | 33.1 | 3.7 | 6.8 |
| Anterior (Internal) base + secondary branch | 13 | 10.3 | – | 15.7 | 20.8 | – | 31.6 | 13.3 | 26.6 | 1.7 | 3.1 |
| Anterior (Internal) spur | 13 | 1.2 | – | 4.4 | 2.5 | – | 10.5 | 2.5 | 5.1 | 1.1 | 2.2 |
| Anterior (Internal) branches length ratio | 13 | 0.5 | – | 1.2 | – | 0.8 | – | 0.2 | – | ||
| Claw II heights | |||||||||||
| Posterior (External) primary branch | 14 | 15.6 | – | 26.4 | 25.3 | – | 50.8 | 20.1 | 39.7 | 3.9 | 7.1 |
| Posterior (External) base + secondary branch | 13 | 9.8 | – | 15.1 | 17.7 | – | 30.6 | 12.8 | 25.7 | 1.6 | 3.8 |
| Posterior (External) spur | 13 | 1.0 | – | 3.9 | 2.5 | – | 7.8 | 2.4 | 4.8 | 0.9 | 1.7 |
| Posterior (External) branches length ratio | 13 | 0.5 | – | 0.8 | – | 0.6 | – | 0.1 | – | ||
| Anterior (Internal) primary branch | 14 | 14.0 | – | 29.1 | 27.4 | – | 50.9 | 18.9 | 37.2 | 4.2 | 7.1 |
| Anterior (Internal) base + secondary branch | 14 | 9.6 | – | 15.7 | 19.1 | – | 29.7 | 12.5 | 24.7 | 1.6 | 3.5 |
| Anterior (Internal) spur | 14 | 1.0 | – | 4.5 | 2.5 | – | 8.6 | 2.9 | 5.8 | 0.9 | 1.9 |
| Anterior (Internal) branches length ratio | 14 | 0.5 | – | 0.8 | – | 0.7 | – | 0.1 | – | ||
| Claw III heights | |||||||||||
| Posterior (External) primary branch | 14 | 12.9 | – | 25.4 | 31.0 | – | 50.6 | 21.0 | 41.3 | 3.2 | 6.1 |
| Posterior (External) base + secondary branch | 14 | 10.0 | – | 15.7 | 23.0 | – | 30.8 | 13.7 | 26.9 | 1.7 | 2.1 |
| Posterior (External) spur | 14 | 1.6 | – | 4.0 | 3.4 | – | 6.9 | 2.6 | 5.1 | 0.7 | 1.1 |
| Posterior (External) branches length ratio | 14 | 0.5 | – | 0.9 | – | 0.7 | – | 0.1 | – | ||
| Anterior (Internal) primary branch | 14 | 14.8 | – | 23.7 | 31.1 | – | 50.4 | 19.5 | 38.3 | 3.0 | 5.5 |
| Anterior (Internal) base + secondary branch | 13 | 10.3 | – | 15.6 | 22.0 | – | 31.0 | 13.3 | 26.7 | 1.6 | 2.8 |
| Anterior (Internal) spur | 13 | 1.5 | – | 5.9 | 3.2 | – | 10.4 | 3.2 | 6.2 | 1.2 | 1.9 |
| Anterior (Internal) branches length ratio | 13 | 0.6 | – | 0.9 | – | 0.7 | – | 0.1 | – | ||
| Claw IV heights | |||||||||||
| Anterior primary branch | 14 | 18.3 | – | 31.0 | 37.2 | – | 54.6 | 23.1 | 45.4 | 3.7 | 5.4 |
| Anterior base + secondary branch | 14 | 9.3 | – | 17.6 | 18.8 | – | 32.1 | 14.0 | 27.5 | 2.4 | 3.6 |
| Anterior spur | 14 | 1.2 | – | 5.4 | 2.6 | – | 11.4 | 2.7 | 5.2 | 1.2 | 2.4 |
| Anterior branches length ratio | 14 | 0.5 | – | 0.8 | – | 0.6 | – | 0.1 | – | ||
| Posterior primary branch | 14 | 15.8 | – | 29.5 | 32.0 | – | 54.3 | 21.9 | 43.0 | 4.2 | 6.7 |
| Posterior base + secondary branch | 14 | 11.8 | – | 18.3 | 26.1 | – | 37.4 | 15.1 | 29.6 | 2.2 | 3.4 |
| Posterior spur | 14 | 1.0 | – | 4.6 | 2.3 | – | 9.7 | 3.3 | 6.4 | 1.1 | 1.9 |
| Posterior branches length ratio | 14 | 0.5 | – | 0.9 | – | 0.7 | – | 0.1 | – | ||
Table 2.
Morphometrics in male Milnesium rastrum
| Character | N | Range | Mean | SD | |||||||
|---|---|---|---|---|---|---|---|---|---|---|---|
| µm | pt | µm | pt | µm | pt | ||||||
| Body length | 17 | 408.9 | – | 548.5 | 914.1 | – | 1581.8 | 492.5 | 1176.8 | 42.1 | 180.7 |
| Peribuccal papillae length | 12 | 2.5 | – | 3.4 | 5.6 | – | 8.3 | 23.8 | 52.8 | 72.4 | 159.2 |
| Lateral papillae length | 15 | 2.4 | – | 5.2 | 5.3 | – | 15.0 | 4.1 | 9.9 | 2.4 | 5.3 |
| Buccal tube | |||||||||||
| Length | 17 | 33.3 | – | 48.7 | – | 42.4 | – | 4.7 | – | ||
| Stylet support insertion point | 17 | 19.2 | – | 36.0 | 42.1 | – | 77.0 | 23.9 | 56.6 | 5.2 | 10.7 |
| Anterior width | 17 | 9.0 | – | 13.5 | 19.7 | – | 30.9 | 10.3 | 24.6 | 1.2 | 3.7 |
| Standard width | 17 | 7.6 | – | 11.4 | 17.3 | – | 25.7 | 8.7 | 20.8 | 1.0 | 3.0 |
| Posterior width | 17 | 6.6 | – | 9.9 | 14.8 | – | 24.0 | 8.0 | 19.1 | 0.9 | 2.2 |
| Posterior/anterior width ratio | 16 | 0.6 | – | 1.1 | – | 0.8 | – | 0.1 | – | ||
| Claw I heights | |||||||||||
| Posterior (Posterior (External)) primary branch | 17 | 9.3 | – | 18.4 | 26.8 | – | 46.3 | 15.4 | 36.4 | 2.0 | 5.2 |
| Posterior (External) base + secondary branch | 17 | 8.9 | – | 16.7 | 23.7 | – | 36.8 | 12.6 | 29.9 | 2.0 | 4.3 |
| Posterior (External) spur | 17 | 1.0 | – | 3.1 | 2.5 | – | 8.3 | 1.9 | 4.6 | 0.6 | 1.5 |
| Posterior (External) branches length ratio | 16 | 0.6 | – | 1.0 | – | 0.8 | – | 0.1 | – | ||
| Anterior (Internal) primary branch | 17 | 13.0 | – | 18.6 | 28.8 | – | 47.4 | 16.0 | 38.1 | 1.6 | 5.6 |
| Anterior (Internal) base + secondary branch | 17 | 13.0 | – | 19.0 | 29.4 | – | 49.2 | 15.6 | 37.4 | 1.7 | 6.0 |
| Anterior (Internal) spur | 17 | 1.2 | – | 2.9 | 2.9 | – | 8.8 | 1.8 | 4.3 | 0.4 | 1.3 |
| Anterior (Internal) branches length ratio | 16 | 0.8 | – | 1.1 | – | 1.0 | – | 0.1 | – | ||
| Claw II heights | |||||||||||
| Posterior (External) primary branch | 17 | 13.8 | – | 23.8 | 36.7 | – | 66.7 | 19.7 | 47.0 | 2.5 | 8.1 |
| Posterior (External) base + secondary branch | 16 | 8.2 | – | 13.8 | 18.1 | – | 37.1 | 11.8 | 28.2 | 1.3 | 4.9 |
| Posterior (External) spur | 16 | 1.2 | – | 3.2 | 2.9 | – | 7.5 | 2.2 | 5.3 | 0.6 | 1.4 |
| Posterior (External) branches length ratio | 15 | 0.5 | – | 0.8 | – | 0.6 | – | 0.1 | – | ||
| Anterior (Internal) primary branch | 17 | 14.6 | – | 23.5 | 33.7 | – | 65.7 | 19.6 | 46.7 | 2.6 | 8.4 |
| Anterior (Internal) base + secondary branch | 17 | 10.4 | – | 14.9 | 23.3 | – | 38.6 | 12.5 | 29.8 | 1.3 | 4.3 |
| Anterior (Internal) spur | 17 | 1.2 | – | 4.2 | 2.7 | – | 9.4 | 2.8 | 6.5 | 0.9 | 2.0 |
| Anterior (Internal) branches length ratio | 16 | 0.5 | – | 0.8 | – | 0.6 | – | 0.1 | – | ||
| Claw III heights | |||||||||||
| Posterior (External) primary branch | 17 | 16.7 | – | 23.7 | 38.2 | – | 59.6 | 20.4 | 48.5 | 1.9 | 5.4 |
| Posterior (External) base + secondary branch | 16 | 7.9 | – | 14.3 | 22.8 | – | 37.9 | 12.2 | 29.0 | 1.6 | 4.3 |
| Posterior (External) spur | 16 | 1.2 | – | 2.7 | 2.6 | – | 6.4 | 2.0 | 4.8 | 0.4 | 1.1 |
| Posterior (External) branches length ratio | 15 | 0.5 | – | 0.7 | – | 0.6 | – | 0.1 | – | ||
| Anterior (Internal) primary branch | 17 | 15.5 | – | 23.6 | 34.6 | – | 60.3 | 19.6 | 46.6 | 2.6 | 6.7 |
| Anterior (Internal) base + secondary branch | 17 | 9.4 | – | 14.6 | 20.7 | – | 38.1 | 12.2 | 29.2 | 1.6 | 4.7 |
| Anterior (Internal) spur | 17 | 1.3 | – | 3.8 | 3.1 | – | 8.2 | 2.5 | 5.8 | 0.7 | 1.5 |
| Anterior (Internal) branches length ratio | 16 | 0.5 | – | 0.8 | – | 0.6 | – | 0.1 | – | ||
| Claw IV heights | |||||||||||
| Anterior primary branch | 17 | 15.1 | – | 24.8 | 33.7 | – | 69.4 | 21.0 | 50.3 | 2.6 | 9.7 |
| Anterior base + secondary branch | 16 | 2.5 | – | 15.3 | 5.4 | – | 40.7 | 12.7 | 30.5 | 3.1 | 8.3 |
| Anterior spur | 16 | 1.2 | – | 20.4 | 3.0 | – | 43.6 | 3.9 | 8.9 | 4.5 | 9.4 |
| Anterior branches length ratio | 15 | 0.1 | – | 0.8 | – | 0.6 | – | 0.1 | – | ||
| Posterior primary branch | 17 | 11.4 | – | 25.7 | 25.4 | – | 69.0 | 21.4 | 51.2 | 3.5 | 10.7 |
| Posterior base + secondary branch | 16 | 9.9 | – | 15.7 | 22.2 | – | 43.5 | 13.6 | 32.4 | 1.5 | 5.1 |
| Posterior spur | 16 | 1.6 | – | 4.4 | 3.6 | – | 11.6 | 2.8 | 6.7 | 0.8 | 2.0 |
| Posterior branches length ratio | 16 | 0.5 | – | 0.9 | – | 0.6 | – | 0.1 | – | ||
Fig. 3.
Morphometrical comparison between female and male. A: body length (μm). p-value is indicated. B: anterior and posterior secondary branch length (pt index) of leg I. p-values with significant differences are indicated (Student’s t-test). C: body width (%body length). ‘F’ stands for ‘fold’. 25th to 75th percentiles with a line at the median. Whiskers extend to 1.5 times the interquartile range. Individual dots indicate respective values. *: p < 0.05 (Student’s t-test)
Claw configuration aberration of Milnesium rastrum
At the base of the male’s secondary branches on leg I of M. rastrum, there were spurs similar to those found on the other secondary branches (Fig. 4A). Therefore, we determined that the CC of leg I was {2–2}. All males exhibited the {2–2} pattern on leg I. We counted the number of points on the secondary branches of both males and females and calculated the average number of points per secondary branch per leg (Fig. 4B). Leg I in males was consistently limited to two points, whilst legs 2–4 generally had four points. In contrast, the median number of points for females across all legs was 4–5, and some individuals were observed to have as many as seven. Including data from the paper by Suzuki et al. [10], we examined the correlation between body length and the average number of points per secondary branch per leg (Fig. 4C). In the dataset encompassing hatchlings, juveniles, males, and females, the correlation coefficient was 0.46, indicating a weak correlation. In contrast, the correlation coefficient for the data comprising only females was 0.84, indicating a strong correlation.
Fig. 4.
Claw configuration of female and male. A: spurs of male secondary branch. Scale = μm. B: averaged number of points on secondary branches. Individual dots indicate respective values. p-values with significant differences are indicated (Wilcoxon rank-sum test). C: correlation between average number of points per secondary branch per leg and body length. Data includes described in Suzuki et al. [10]. “All specimens” includes morphometrical data of hatchlings, juveniles, males, and females. Correlation coefficients R2 values are indicated.
Diet of Milnesium rastrum
We examined the intestinal contents of the specimens (Fig. 5), and frequently found the placoids, buccal tubes, and claws of other tardigrades (Fig. 5B), as well as trophi of bdelloid rotifer mastaces (Fig. 5C). It was not uncommon to find fragments of multiple tardigrades within both female and male individuals, indicating that M. rastrum preys on other tardigrades in its wild habitat. The tardigrade fragments found in the intestine had claws with a Hypsibiidae-type, and traces resembling four placoids associated with pharynx were also observed (Fig. 5B). Furthermore, Diphascon-like long buccal tubes attached four placoids were found in the intestine (Fig. 5B).
Fig. 5.
Intestine of Milnesium rastrum. A: intestine of M. rastrum specimen. An and Po indicate anterior and posterior, respectively. Arrowheads indicate gastric contents. Dotted lines indicate the outline of the intestine. Expanded images of labeled B and C region are shown in lower panels. B: tardigrades in the intestine. Arrowheads indicate claws, buccal tubes, or placoids. C: a trophi of rotifer mastax in the intestine. Scales = μm
Discussion
Geographical distribution of Milnesium rastrum
This report marks the fourth record of a species believed to be M. rastrum. Sudzuki reported a single specimen from Langhovde in 1964 [17], followed by Dastych in 1984 [18], and finally, Suzuki et al. described the species in 2023 [10]. Although the exact location within Langhovde where Sudzuki collected his specimen is unclear, it is certain that it was not far from the site of the present collection [17]. Dastych’s report states that the specimen was collected near Molodeznaya [18], while Suzuki et al. designated Innhovde as the type locality for this species [10]. Molodeznaya, Langhovde, and Innhovde are approximately 300–450 km apart, and the species is likely scattered throughout this region, at least.
Sexual differences of Milnesium rastrum
The original description [10] was based on a limited number of specimens, and the only male specimen was an individual undergoing the molt to adulthood. In this study, the discovery of an area where numerous M. rastrum specimens could be collected enabled access to more detailed morphological data. Statistically, it was first confirmed that males are smaller and more slender than females. One possible explanation is that females accommodate developing ovaries. SDI analysis indicated that the buccal tube is thicker in females. On the other hand, fragments of tardigrades and trophi of rotifers were found in both males and females; therefore, based on the current data, it is not possible to assess whether differences in feeding habits are related to buccal tube thickness in M. rastrum. Furthermore, this study confirmed a positive correlation between female body size and the number of points on the secondary branches. If M. rastrum tends to increase in body size with each molt and the number of points also increase, this would be consistent with the observations of this study: that females have more points than males. The CC of males prior to sexual maturity is of the typical “juvenile type” [10]. It is possible that, in males, the number of points and the body length do not change after the molting into adulthood.
Among the 58 currently described species in the genus Milnesium [4], males have been reported in 16 species (Table 3), and in all of them, the secondary branch of leg I is highly developed in a hook-like manner. If this structure were necessary for walking, it would not be developed exclusively in males; nor, given the shortness of the legs themselves, is it likely used in combat between males. Pseudobiotus megalonyx Thulin, 1928, which, like the milnesiids, belongs to the class Eutardigrada, lays its eggs in molted exuvia, similar to the milnesiids, and the claws on the males’ forelegs differ from those of the females (reviewed in [7, 8]. Pseudobiotus megalonyx reproduces sexually, and there are descriptions of males boring holes into the molted exuvia where females are laying eggs in order to ejaculate (reviewed in [35]). Based on these findings, we hypothesize that these hook-like claws are used to restrain the female during mating or to create holes for ejaculation. Although no observations of mating behavior in milnesiids have been reported to date, we look forward to future research in this area.
Table 3.
Sex ratio of Milnesium species including specimens of Suzuki et al., 2023 [10]
| Male | Female | %Male | References | |
|---|---|---|---|---|
| M. inceptum | Very few | Parthenogenetic | - | [9] |
| Milnesium sp. | 1 | 25 | 3.8 | [19] |
| Milnesium sp. | 2 | 36 | 5.3 | [20] |
| M. milenae | 1 | 8 | 11.1 | [21] |
| M. grandicupula | 3 | 22 | 12.0 | [22] |
| M. tetralamellatum | 1 | 6 | 14.3 | [23] |
| M. beasleyi | 1 | 5 | 16.7 | [24] |
| M. eurystomum | 17 | 84 | 16.8 | [25] |
| M. lagniappe | 3 | 12 | 20.0 | [26] |
| M. warrenwilsonensis | 5 | 20 | 20.0 | [27] |
| M. dornensis | 10 | 37 | 21.3 | [28] |
| M. fridae | 5 | 16 | 23.8 | [29] |
| Milnesium sp. | 18 | 57 | 24.0 | [20] |
| M. matheusi | 3 | 9 | 25.0 | [30] |
| M. myriae | 1 | 3 | 25.0 | [21] |
| Milnesium sp. MilnC | 1 | 2 | 33.3 | [31] |
| Milnesium sp. | 9 | 17 | 34.6 | [20] |
| Milnesium sp. | 29 | 47 | 38.2 | [20] |
| M. decorum | 5 | 5 | 50.0 | [32] |
| M. swansoni | 5 | 5 | 50.0 | [33] |
| M. rastrum | 18 | 17 | 51.4 | [10]; This study |
| M. burgessi | ? | ? | - | [34] |
Sex ratio of the genus Milnesium
Furthermore, this study revealed that males are present in M. rastrum, and the sex ratio is approximately 1:1. Of the 21 dioecious species or populations in the genus Milnesium, 18 have a higher proportion of females (see Table 3). The remaining three species—Milnesium decorum Morek, Wałach & Michalczyk, 2022 [32], Milnesium swansoni Young, Chappell, Miller & Lowman, 2016 [33], and M. rastrum—show a sex ratio close to 1:1. On the other hand, a major issue is the small number of specimens. As with Milnesium eurystomum Maucci, 1901, a survey involving at least 100 specimens would be necessary [25]. Imbalances in sex ratios have been reported in many tardigrade species outside the milnesiids as well (e.g. [36–39]). To date, no sex chromosomes have been found in tardigrades, and it has been reported that no sex-specific regions can be detected even through comparisons of male and female genomes [40]. Elucidating the sex-determination system in tardigrades is desirable in order to understand the causes of sex ratio variation.
Ecological relation between Milnesium rastrum and Diphascon langhovdense
Although Suzuki et al. reported finding a rotifer trophi within the intestine of M. rastrum [10], these were fed to the organisms under laboratory conditions. Instances of milnesiids preying on other tardigrades have been reported in e.g. Milnesium sp. [41], Milnesium burgessi Schlabach, Donaldson, Hobelman, Miller & Lowman, 2018 [34], Milnesium lagniappe Meyer, Hinton & Dupré, 2013 [26, 42], and M. eurystomum [25], and this phenomenon is by no means rare. In particular, McInnes et al. reported the discovery of Diphascon sp. with a long buccal tube in the intestine of a milnesiid from Antarctica [41]. Given the presence of Hypsibiidae claws, the four placoids (three macroplacoids and one microplacoid), and the shape of buccal tube remnants in the intestine of M. rastrum is reasonable to conclude that they belong to Diphascon langhovdense Sudzuki, 1964, which was also found with M. rastrum in Langhovde [17]. This observation indicated that M. rastrum actively preys on D. langhovdense in the natural environment. Acutuncus antarcticus is found across a wide area of Antarctica, including Langhovde [43]. In the specimens examined in this study, no short buccal tubes resembling those of A. antarcticus were found within the intestines. One factor worth noting may be the body size of the prey. Adult A. antarcticus exceeds 300 µm [44], whereas D. langhovdense is just over 100 µm [17]. Milnesium rastrum is probably capable of swallowing at least small prey whole. Detailed examinations of the intestines of M. rastrum collected from another location would likely clarify whether M. rastrum preferentially preys on D. langhovdense or whether it feeds indiscriminately on other species, such as A. antarcticus.
Conclusions
In conclusion, our investigation of the M. rastrum colony at Langhovde significantly advances the morphological and ecological understanding of this endemic Antarctic tardigrade. We successfully documented pronounced sexual dimorphism, establishing that males are smaller and more slender than females, and possess highly specialized, enlarged robust hook-like claws on their first pair of legs. The strong positive correlation between female body length and the number of points on the secondary branches suggests that CC may change with body growth in females. Furthermore, it was suggested that the sex ratio of M. rastrum in the wild is approximately 1:1. Finally, the identification of D. langhovdense remnants within the intestine confirms that M. rastrum is a predator of other tardigrades.
Electronic supplementary material
Below is the link to the electronic supplementary material.
Acknowledgements
This survey was conducted as part of the 67th JARE projects. We thank Yumi Nakayama, Yukiko Fukuhama, and the member of JARE67 and Japan Marine Self-Defense Force (JMSDF) for supporting our field work. We also thank Drs. Atsushi C. Suzuki, James F. Fleming, Katsuyuki Eguchi, and Sakae Kudoh for their advice on improvements. We are grateful to the two anonymous reviewers for their constructive comments, which greatly improved our manuscript. The samples were used with the permission of the minister of Ministry of the Environment and Ministry of Agriculture, Forestry and Fisheries based on the Plant Protection Act.
Author contributions
KS, NT, TW performed sampling and improved the manuscript. KS examined all observations and analyses.
Funding
This work was supported by the Japanese Antarctic Research Expedition (AP1011 to TW), Grant-In-Aid KAKENHI Early-Career Scientists (26JP21465 to TW), Grant-In-Aid KAKENHI Scientific Research (B) (25K02323 to KS).
Data availability
Draft data of this study is uploaded on FigShare (DOI: 10.6084/m9.figshare.32843480).
Declarations
Ethics approval and consent to participate
Not applicable.
Consent of publication
Not applicable.
Competing interests
The authors declare that they have no competing interests.
Footnotes
Publisher’s Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
References
- 1.Pugh PJA, Convey P. Surviving out in the cold: antarctic endemic invertebrates and their refugia. J Biogeogr. 2008;35(12):2176–86. 10.1111/j.1365-2699.2008.01953.x. [DOI] [Google Scholar]
- 2.Wada T, Kudoh S, Koyama H, Iakovenko N, Elster J, Kvíderová J, et al. Abundance and biomass of Bdelloid rotifers in the microbial mats from East Antarctica: the ecological relations between microscopic phototrophs and invertebrates. Ecol Res. 2023;38(2):317–30. 10.1111/1440-1703.12368. [DOI] [Google Scholar]
- 3.Morek W, Surmacz B, Lopez-Lopez A, Michalczyk Ł. “Everything is not everywhere”: time-calibrated phylogeography of the genus Milnesium (tardigrada). Mol Ecol. 2021;30(14):3590–609. 10.1111/mec.15951. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 4.Degma P, Guidetti R. Actual checklist of Tardigrada species. 45th. 2026. 10.5281/zenodo.20746849. [DOI] [Google Scholar]
- 5.Vecchi M, Brandoli S, Trokhymets VM. Community analysis reveals biogeographical patterns and biodiversity shortfalls in Antarctic tardigrades. J Biogeogr. 2025;52(3):735–49. 10.1111/jbi.15063. [DOI] [Google Scholar]
- 6.Morek W, Stec D, Gąsiorek P, Surmacz B, Michalczyk Ł. Milnesium tardigradum Doyère, 1840: the first integrative study of interpopulation variability in a tardigrade species. J Zool Syst Evol Res. 2018;57(1):1–23. 10.1111/jzs.12233. [DOI] [Google Scholar]
- 7.Nelson DR, Guidetti R, Rebecchi L. Tardigrada. In: Thorp JH, Covich AP, editors. Ecology and classification of North American freshwater invertebrates. 3rd. San Diego: Academic Press; 2010. p. 455–84. [Google Scholar]
- 8.Nelson DR, Guidetti R, Rebecchi L. Phylum Tardigrada. In: Thorp JH, Covich AP, editors. Thorp and Covich’s freshwater invertebrates. 4th. Vol. 1. Academic Press; 2015. p. 347–80. [Google Scholar]
- 9.Suzuki AC. Appearance of males in a thelytokous strain of Milnesium cf. tardigradum (Tardigrada). Zoological Sci. 2008;25(8):849–53. 10.2108/zsj.25.849. [DOI] [PubMed] [Google Scholar]
- 10.Suzuki AC, Sugiura K, Tsujimoto M, Nakai R, McInnes SJ, Kagoshima H, et al. A new species of bisexual Milnesium (Eutardigrada: Apochela) having aberrant claws from Innhovde, Dronning Maud Land, East Antarctica. Zoological Sci. 2023;40(3):246–61. 10.2108/zs220085. [DOI] [PubMed] [Google Scholar]
- 11.Michalczyk Ł, Kaczmarek Ł. The tardigrada register: a comprehensive online data repository for tardigrade taxonomy. J Limnol. 2013;72(1s):175–77. 10.4081/jlimnol.2013.s1.e22. [DOI] [Google Scholar]
- 12.Suzuki AC. Beautiful claws of a tiny water bear: a review and proposal for claw configuration. Zoological Sci. 2022;39(2):167–75. 10.2108/zs210087. [DOI] [PubMed] [Google Scholar]
- 13.Pilato G. Analisi di nuovi caratteri nello studio degli eutardigrad. Animalia. 1981;8:51–57. [Google Scholar]
- 14.R Core Team. R: a language and environment for statistical computing. Vienna: R Foundation for Statistical Computing; 2016.
- 15.Lovich JE, Gibbons JW. A review of techniques for quantifying sexual size dimorphism. Growth Dev Aging. 1992;56(4):269–81. [PubMed] [Google Scholar]
- 16.Vecchi M, Calhim S. Patterns of sexual dimorphism in the armoured tardigrades. Biol Lett. 2024;20(9):20240301. 10.1098/rsbl.2024.0301. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 17.Sudzuki M. On the microfauna of the Antarctic region I. Moss-water community at Langhovde. JARE 1956–1962 Sci Rep Ser E. 1964;19:1–41. [Google Scholar]
- 18.Dastych H. The tardigrada from Antarctic with descriptions of several new species. Acta Zool Cracov. 1984;27(19):377–436. [Google Scholar]
- 19.Marcus E. Tardigrada. Berlin: Walter de Gruyter; 1936. [Google Scholar]
- 20.Rebecchi L, Nelson DR. Evaluation of a secondary sex character in eutardigrades. Invertebr Biol. 1998;117(3):194–98. 10.2307/3226985. [DOI] [Google Scholar]
- 21.Kayastha P, Warguła J, Bartylak T, Mioduchowska M, Gawlak M, Kaczmarek Ł. Integrative description of two new Milnesium species (Apochela: Milnesiidae) from Canada and Nepal. Eur Zool J. 2025;92(1):1358–82. 10.1080/24750263.2025.2562907. [DOI] [Google Scholar]
- 22.Kihm JH, Zawierucha K, Rho HS, Park T-YS. Greenlandic water bears reveal a new morphological trait of external head sensory organs. Sci Rep. 2025;15(1):22556. 10.1038/s41598-025-06766-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 23.Pilato G, Binda MG. Milnesium tetralamellatum, new species of Milnesiidae from Africa (Eutardigrada). Trop Zool. 1991;4(1):103–06. 10.1080/03946975.1991.10539480. [DOI] [Google Scholar]
- 24.Kaczmarek Ł, Jakubowska N, Michalczyk Ł. Current knowledge on Turkish tardigrades with a description of Milnesium beasleyi sp. nov. (Eutardigrada: Apochela: Milnesiidae, the granulatum group). Zootaxa. 2012;3589(1):29–49. 10.11646/zootaxa.3589.1.3. [DOI] [Google Scholar]
- 25.Morek W, Blagden B, Kristensen RM, Michalczyk Ł. The analysis of inter- and intrapopulation variability of Milnesium eurystomum maucci, 1991 reveals high genetic divergence and a novel type of ontogenetic variation in the order Apochela. Syst Biodivers. 2020;18(6):614–32. 10.1080/14772000.2020.1771469. [DOI] [Google Scholar]
- 26.Meyer HA, Hinton JG, Dupré MC. Milnesium lagniappe, a new species of water bear (Tardigrada, Eutardigrada, Apochela, Milnesiidae) from the Southern United States. West N Am Naturalist. 2013;73(3):295–301. 10.3398/064.073.0305. [DOI] [Google Scholar]
- 27.Bartels PJ, Kayastha P, Bartylak T, Mioduchowska M, Dmuchowska W, Kaczmarek Ł. A new species in the genus Milnesium (Eutardigrada) from USA (North Carolina). Zootaxa. 2025;5661(2):201–20. 10.11646/zootaxa.5661.2.3. [DOI] [PubMed] [Google Scholar]
- 28.Ciobanu DA, Roszkowska M, Kaczmarek Ł. Two new tardigrade species from Romania (Eutardigrada: Milnesiidae, Macrobiotidae), with some remarks on secondary sex characters in Milnesium dornensis sp. nov. Zootaxa. 2015;3941(4):542–64. 10.11646/zootaxa.3941.4.4. [DOI] [PubMed] [Google Scholar]
- 29.Moreno-Talamantes A, León-Espinosa GA, García-Aranda MA, Flores-Maldonado JJ, Kaczmarek Ł. The genus Milnesium Doyère, 1840 in Mexico with description of a new species. Ann Zoologici. 2020;70(4):489–98. 10.3161/00034541ANZ2020.70.4.001. [DOI] [Google Scholar]
- 30.Kaczmarek Ł, Grobys D, Kulpa A, Bartylak T, Kmita H, Kepel M, et al. Two new species of the genus Milnesium Doyère, 1840 (Tardigrada, Apochela, Milnesiidae) from Madagascar. ZK. 2019;884:1–22. 10.3897/zookeys.884.29469. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 31.Sands CJ, Convey P, Linse K, McInnes SJ. Assessing meiofaunal variation among individuals utilising morphological and molecular approaches: an example using the Tardigrada. BMC Ecol. 2008;8(1):7. 10.1186/1472-6785-8-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 32.Morek W, Wałach K, Michalczyk Ł. Rough backs: taxonomic value of epicuticular sculpturing in the genus Milnesium Doyère, 1840 (Tardigrada: Apochela). Sci Rep. 2022;12(1):9857. 10.1038/s41598-022-10758-z. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 33.Young A, Chappell B, Miller W, Lowman M. Tardigrades of the tree canopy: Milnesium swansoni sp. nov. (Eutardigrada: Apochela: Milnesiidae) a new species from Kansas, U.S.A. Zootaxa. 2016;4072(5):559–68. . 10.11646/zootaxa.4072.5.3. [DOI] [PubMed] [Google Scholar]
- 34.Schlabach S, Donaldson E, Hobelman K, Miller WR, Lowman MD. Tardigrades of the canopy: Milnesium burgessi nov. sp. (Eutardigrada: Apochela: Milnesiidae) a new species from Kansas, U.S.A. Trans Kansas Acad Sci. 2018;121(1–2):39–48. . 10.1660/062.121.0204. [DOI] [Google Scholar]
- 35.Sugiura K, Matsumoto M. Sexual reproductive behaviours of tardigrades: a review. Invertebr Reproduction Devel. 2021;65(4):279–87. 10.1080/07924259.2021.1990142. [DOI] [Google Scholar]
- 36.Nagwani AK, Melosik I, Kaczmarek Ł, Kmita H. Recovery from anhydrobiosis in the tardigrade Paramacrobiotus experimentalis: better to be young than old and in a group than alone. Heliyon. 2024;10(5):e26807. 10.1016/j.heliyon.2024.e26807. [DOI] [PMC free article] [PubMed]
- 37.Rebecchi L, Bertolani R. Maturative pattern of ovary and testis in eutardigrades of freshwater and terrestrial habitats. Invertebr Reproduction Devel. 1994;26(2):107–17. 10.1080/07924259.1994.9672407. [DOI] [Google Scholar]
- 38.Rebecchi L, Rossi V, Altiero T, Bertolani R, Menozzi P. Reproductive modes and genetic polymorphism in the tardigrade Richtersius coronifer (Eutardigrada, Macrobiotidae). Invertebr Biol. 2003;122(1):19–27. 10.1111/j.1744-7410.2003.tb00069.x. [DOI] [Google Scholar]
- 39.Sugiura K, Minato H, Suzuki AC, Arakawa K, Kunieda T, Matsumoto M. Comparison of sexual reproductive behaviors in two species of Macrobiotidae (Tardigrada: Eutardigrada). Zoological Sci. 2019;36(2):120–27. 10.2108/zs180103. [DOI] [PubMed] [Google Scholar]
- 40.Sugiura K, Yoshida Y, Hayashi K, Arakawa K, Kunieda T, Matsumoto M. Sexual dimorphism in the tardigrade Paramacrobiotus metropolitanus transcriptome. Zoological Lett. 2024;10(1):11. 10.1186/s40851-024-00233-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 41.McInnes SJ, Chown SL, Dartnall HJG, Pugh PJA. Milnesium cfr. tardigradum (Milnesiidae, Apochela, Tardigrada): a monitor of high altitude meiofauna on sub-antarctic Marion Island. Zoologischer Anz - J Comp Zool. 2001;240(3–4):461–65. 10.1078/0044-5231-00054. [DOI] [Google Scholar]
- 42.Meyer HA, Larsen HE, Akobi NO, Broussard G. Predator and prey detection in two species of water bear (Tardigrada). Zoological J The Linn Soc. 2020;188(3):860–64. 10.1093/zoolinnean/zlz141. [DOI] [Google Scholar]
- 43.Tsujimoto M, Kagoshima H, Kanda H, Watanabe K, Imura S. Reproductive performance of the Antarctic tardigrades, Acutuncus antarcticus (Eutardigrada: Hypsibiidae), revived after being frozen for over 30 years and of their offspring. Zoological J The Linn Soc. 2020;188(3):839–47. 10.1093/zoolinnean/zlz137. [DOI] [Google Scholar]
- 44.Tsujimoto M, Imura S, Kanda H. Recovery and reproduction of an Antarctic tardigrade retrieved from a moss sample frozen for over 30 years. Cryobiology. 2016;72(1):78–81. 10.1016/j.cryobiol.2015.12.003. [DOI] [PubMed] [Google Scholar]
Associated Data
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Data Availability Statement
Draft data of this study is uploaded on FigShare (DOI: 10.6084/m9.figshare.32843480).