A New Species of Bisexual Milnesium (Eutardigrada: Apochela) Having Aberrant Claws from Innhovde, Dronning Maud Land, East Antarctica

There have been several records in the last 60 years for East Antarctica for Milnesium tardigradum Doyère, 1840 sensu lato, now considered a species complex. During the 56th Japanese Antarctic Research Expedition summer operation (2014–2015), a new tardigrade species in the genus Milnesium Doyère, 1840 was found in an ice-free Innhovde area along Lützow-Holm Bay, Dronning Maud Land, East Antarctica. The new species has aberrant claws with four to seven points on each secondary claw branch, which distinguishes it from other Milnesium species. A male specimen was found in the population and evidence showed that an isolated adult female moulted twice without oviposition. This strongly suggested bisexual reproduction for this population. The new species, Milnesium rastrum sp. nov., is described with its phylogenetic position and a discussion on the reproductive strategies for the harsh environments.


INTRODUCTION
The genus Milnesium Doyère, 1840 was established with the description of Milnesium tardigradum Doyère, 1840 from Saint-Maur near Paris. This tardigrade was characterised by four claws on each leg, with two long primary claws and two short secondary claws, with the anterior secondary claw divided into three branches and the posterior with two branches (Doyère, 1840). The second species, Milnesium alpigenum Ehrenberg, 1853, was distinguished by having three branches on all secondary claws (Ehrenberg, 1853;illustrated in Ehrenberg, 1854). Despite the differing number of branches (or points) on the secondary claws, this character was regarded as merely intraspecific variation by early researchers (Richters, 1902(Richters, , 1904(Richters, , 1908Marcus, 1936). Later, Michalczyk et al. (2012) emphasized the taxonomic importance of the secondary claws and proposed a notation system to describe the claw configuration (CC). Among more than 40 described species in this genus, almost all species have two or three branches (reviewed by Suzuki, 2022) but M. quadrifidum Nederström, 1919 has four branches on each secondary claw. A more recent exception, M. wrightae Kaczmarek et al., 2019, has a very small fourth point near the base of the secondary claws of leg IV. The significant exceptions in publications were of specimens from East Antarctica, which were identified as M. tardigradum in spite of having five or more branches on the secondary claws (Sudzuki, 1964;Dastych, 1984).
During the 56th Japanese Antarctic Research Expedition (JARE-56) summer operation (2014)(2015), field investigation and monitoring of terrestrial ecosystems were carried out by three of the authors (ACS, MT, and RN) in several areas along Lützow-Holm Bay, Dronning Maud Land, East Antarctica. The main aim of the investigation was to examine and characterize diversity and distribution of the meiofauna and the microbiome in the ice-free areas in this region. In this context, re-examination of Milnesium sp. mentioned above was planned, and microscopic examinations were performed as much as possible during the short field stay. Although Milnesium specimens described by Sudzuki (1964) were collected from the Langhovde area (69°13′S, 39°45′E) during JARE-5 (1960JARE-5 ( -1962, this time we found this tardigrade during the first biological survey of the Innhovde (69°51′S, 37°06′E) region.

Sample collection, rearing conditions, and microscopy
The ice-free area of Innhovde is situated at ca. 120 km southwest of Syowa Station (Figs. 1,2). Along the north-eastern slope near the coast are exposed rocks, about 3 km long from the NW end (ca. 10 m above sea level) toward the SE end (160 m a.s.l.). We conducted a field study in the Innhovde area on 11 January 2015. Limited vegetation occurred in the northern quarter of the area; in contrast, the rest of the southern area was relatively dry with little vegetation, and lakes were still frozen at the time of our visit. Mosses and lichens from the northern area were collected in paper bags and kept dried in cold storage, at 4°C during the expedition, then later at − 20°C in a cold room at National Institute of Polar Research (NIPR), Tokyo.
Several samples were examined during the expedition in our field laboratory at Kizahashi-hama, Skarvsnes (69°28′22″S, 39°36′06″E). The samples were divided into several portions, one of which was immediately soaked in Milli Q water and the small animals that were liberated were picked out under a stereo microscope (Wild M3C, Leica). Two adult females and a juvenile of the Milnesium species were found in the moss Ceratodon purpureus. The adult specimens were mounted on glass slides in gum-chloral solution, and the juvenile was fixed and stored in ethanol for DNA analysis. Images of the slide specimens were taken at the field laboratory with a digital camera (NEX5-N, Sony) on a compound microscope (Nikon L-Ke) with differential interference contrast (DIC), and again later using another microscope (BX50, Olympus) with DIC. The rest of the samples were returned to Japan, where the remainder of the moss sample that had yielded the Milnesium specimens at the field laboratory was examined. Additional specimens comprising an exuvia with three eggs, an adult female, a premature simplex individual, and a moulting male were extracted. The premature simplex specimen was fixed with ethanol for DNA analysis, while the moulting male and three hatchlings from the eggs were mounted on microslides. The adult female, which was presumed to be the mother of the three hatchlings, was reared in a plastic dish and the life history was observed under a stereo microscope (Wild/Leica M-10). The rearing conditions were as described in Suzuki (2003) with a slight modification, using rotifers Lecane inermis (Bryce, 1892) as a food source, and at 4 or 10°C under 16L:8D photoperiod. After this female individual died, the specimen was processed for scanning electron microscopy (SEM). It was fixed in 10% formalin, post-fixed in 1% OsO4 in 0.1 M sodium cacodylate, pH 7.2 for 1 hr at room temperature, dehydrated through a  graded series of ethanol and 100% t-butyl alcohol, frozen at 4°C overnight, and lyophilized using a JFD-320 (JEOL). The specimen was mounted on an aluminium stub, sputter-coated with gold, and observed with a JSM 6510 (JEOL) at 20 kV.
Specimens for morphometry were observed under an Olympus BX-50 DIC microscope with a digital camera, and measurements were taken using ImageJ (https://imagej.nih.gov/ij/). Structures were measured when their orientation was suitable. Body length was measured from the anterior extremity to the end of the body excluding the hind legs. Buccal tube length and stylet support insertion point were measured according to Tumanov (2006). Buccal tube width (diameter) was measured at the anterior, standard, and posterior positions . The pt index (Pilato, 1981) was used for comparison, i.e., the percentage of the length of each structure relative to the buccal tube length, expressed in italics. Illustrations were made using a drawing tube attached to a BX-50 microscope and finished with Inkscape software (https:// inkscape.org/). The terminology for the Milnesium claw system generally followed Camarda et al. (2022), i.e., we now replace the terms "primary/secondary branches" in the previous works (reviewed in Suzuki, 2022) by "primary/secondary claws", and instead we call the hooks, or points, on the secondary claws "branches". Following Suzuki (2022), we avoid using the directional terms internal/external for leg I-III, and describe the CC by the revised notation system (Suzuki, 2022) with a modification, i.e., the number of "branches" on the "secondary claws" expressed in brackets as {anterior/posterior} in order through all legs. The position of legs was indicated by a superscript Roman numeral behind the brackets, if necessary. By this notation, the adult CC of M. tardigradum is expressed simply as {3-2}, or more precisely {3-2} I-IV . Specimens of the new species in the present study have a different number of branches, even on the left/right legs in one individual, so that every CC was described for each leg.
Other samples of vegetation collected from Antarctica during JARE-56 have been kept at NIPR for future investigation.

DNA extraction and sequencing
DNA extraction was performed as described by Kagoshima et al. (2013). Each specimen was transferred individually to 20 μL of 0.25 N NaOH in a 0.2 mL tube and kept at room temperature for 12 hr. The tube was heated for 3 min at 95°C, and 4 μL of 1 M HCl and 10 μL of 0.5 M Tris-HCl (pH 8.0) were added to neutralize the base followed by 1 μL of 2% Triton X-100. The lysate was heated for a further 3 min at 95°C and stored at − 20°C. Polymerase chain reaction (PCR) amplification was performed with primers as follows: for the 18S rRNA gene we used the primers SSU04F and SSU81R (Blaxter et al., 1998), for the 28S rRNA gene the primers were 28S_Eutar_F (Gąsiorek et al., 2017) and 28SR0990 (Mironov et al., 2012), for ITS-2 they were ITS3 and ITS4 (White et al., 1990), and for COI the primers were LCO1490 and HCO2198 (Folmer et al., 1994). PCR conditions for the 18S rRNA gene and COI were: 94°C for 2 min, followed by 40 cycles of 94°C for 10 sec, 52°C for 30 sec, and 72°C for 10 min. Conditions for the 28S rRNA genes and ITS-2 were: 94°C for 2 min, 40 cycles of denaturing at 94°C for 30 sec, annealing at 50°C for 30 sec, extension at 68°C for 75 sec, and final extension for 7 min. Sequencing reactions were performed with Big-Dye terminator cycle sequencing kits and run on the ABI 3130xl Genetic Analyzer (Applied Biosystems, Foster City, CA, USA) for 18S rRNA gene and COI, and sequencing for the other two regions was performed by Eurofins Genomics (Japan). The sequences were checked and trimmed with SnapGene Viewer, a software from Insightful Science (snapgene.com).

Phylogenetic analyses
For phylogenetic analyses, we used the publicly available Milnesium spp. sequences of 18S rRNA, 28S rRNA, ITS-2, and COI in the DDBJ/ENA/GenBank databases as shown in Supplementary   Table S1. Antarctic taxa and some species with published sequences were added to the list. The sequences were aligned using MAFFT v7.222 (Katoh et al., 2002;Katoh and Toh, 2008) with the default setting, then obtained alignments were checked by using MEGA7 (Kumar et al., 2016) and trimmed manually to 193 bp in 18S rRNA, 564 bp in 28S rRNA, 104 bp in ITS-2, and 478 bp in COI. The p-distances were calculated with MEGA7.
Phylogenetic analyses were run with COI and 18S + 28S + ITS-2 + COI concatenated sequences. The concatenated sequences included only the taxa with the COI sequence and at least one nuclear DNA sequence. Four Antarctic COI sequences: KJ857001, KJ857002, KP013598, and KP013613, were added for the concatenated sequences even though the taxa with only COI sequence was available. The best-fit evolution model and Maximum Likelihood (ML) topologies were calculated with IQ-TREE v1.6.12 (Minh et al., 2020) by 10,000 rapid bootstrap replicates. TVM + F + G4, GTR + F + I + G4, K3Pu + F + G4 and HKY+ F + R7 were estimated to ITS-2, 1st, 2nd and 3rd codon of COI, respectively. In addition, K2P + G4 and T2P + R5 were estimated to the respective partitions for 18S and 28S of the concatenated sequence. Bayesian Inference (BI) probabilities were calculated with MrBayes v3.2.6 (Ronquist and Huelsenbeck, 2003;Ronquist et al., 2012) under GTR + I + G model with "nst = 6 rates = invgamma" option, and the analyses were run for over 5,000,000 generations, sampling the Markov chain every 1,000 generations. Moreover, an average standard deviation of split frequencies of < 0.01 was confirmed. The trees were visualised in FigTree v1.4.3 (http://tree.bio.ed.ac.uk/software/figtree).

Diagnosis.
Milnesium with four-seven branches on each secondary claws. Live adult body orange in color under stereomicroscopy. Eyes present. Male present, with modified claw on leg I. The presence of a male in the population suggests sexual reproduction.
Materials examined. Holotype: Adult female (slide No. NSMT-Tg-334), collected by the first author on 11 January 2015, mounted in gum-chloral solution. Type locality: Innhovde (69°51′13.8″S, 37°06′12.9″E), Dronning Maud Land, East Antarctica. All other materials examined from the same locality. Paratypes: A male at the stage of moult between juvenile and adult with newly formed adult claws (NSMT-Tg-335) and three hatchlings (NSMT-Tg-336), all mounted in gum-chloral solution. Deposited in National Museum of Nature and Science, Tsukuba, Japan. Another adult female (designated as female-2 in this study) was regrettably lost during an unsuccessful attempt at restoration after the mounting media deteriorated. However, we were left with good photographs of the specimen and measurements shown in this article.
Other materials: An exuviae (NSMT-Tg-337) with three 100 µm 100 µm egg shells that produced the three paratype hatchlings. An adult female (designated as female-3 in this study) was reared for 2 months, during which time she moulted once without oviposition (exuviae mounted in gum-choral, NSMT-Tg-338), and a second time but died in the process. Female-3 was prepared and mounted on an aluminium stub for SEM (NSMT-Tg-339). Two further specimens were used for DNA analyses.
Etymology. The specific name rastrum means a rake in Latin, and especially a 5-pointed writing implement to draw lines for sheet music; representing the shape of the secondary claws with many branches.
Description. All measurements shown in Table 1.

Observation of a solitary adult female in culture
When the exuviae including three eggs was found, an adult female (female-3) was also retrieved from the same sample. This female, which might have been the mother of the above three eggs in spite of no firm evidence, was temporarily dried and kept in a refrigerator. After a week it was rehydrated ( Fig. 3A-D). The dried body was 244 μm long and expanded to 707 μm after rehydration. Once rehydrated it was put into a 35-mm dish filled with water and provided with rotifers as the food source. Table 2 shows the summary of the observations of female-3 for more than 3 months. The movement of the female was very slow, and its eating behavior was not directly observed. However, its gut sometimes looked full of some grey substance.
At day 19 after rehydration, the female lost her buccal apparatus going into simplex (i.e., the moulting stage), but no distinct eggs were discerned in her ovary. It took about 10 days at 10°C to form new buccal apparatus (day 29). Two days later (day 31), the locomotive activity ceased, and apolysis, i.e., detachment of the old cuticle from the newly formed epidermis, was observed. It took another week to finish the ecdysis (day 38). The remains of a rotifer trophi in the exuviae between the 3rd-leg pair (in the old cuticle of the hind gut) provided evidence that this female had been eating the rotifers (Fig. 9).
The new-instar female continued her slow locomotion for a month until day 68, and then the next moulting began, indicated by the loss of the buccal tube again. The ovary, as before, had no large oocytes. It took 14 days to recognize the beginning of the buccal tube regeneration. At day 86, the observation was interrupted for 10 days at 4°C (caused by a conference trip of the first author). By day 96, when the animal was returned to 10°C, the new buccal tube had already formed. However, she did not complete ecdysis and apparently died at day 103. The body was fixed and processed for SEM observation (Fig. 6D). Overall during this observation, this female moulted twice without egg laying.

Phylogeny
The phylogenetic trees based on the COI and concatenated sequences are shown in Fig. 10 and Supplementary Figure S1, respectively; the supported values of BI trees were usually low, thus the phylogenetic trees based on the ML trees are shown. The COI ML tree indicates that M. rastrum sp. nov. is a separate species with 100% probability by both maximum likelihood and Bayesian supported solutions, and the new species is included in a clade with Antarctic specimens (Milnesium spp. Miln06 224, MilnC 025, Ta47-01, and Ta46-01).

Differential diagnosis
Milnesium rastrum sp. nov. is clearly distinguished from all other congeners by having four-seven branches on the secondary claws, which CC is probably expressed as {(4-7)-(4-7)}. Milnesium quadrifidum has {4-4} CC, M. wrightae has {3-3}{4-4} IV with a minute 4th branch near the base of the secondary claw, and all other reported species have either CC of {3-3}, {3-2}, or {2-2}, or a mixture of these combinations (Suzuki, 2022 Milnesium antarcticum by having a shorter buccal tube for a similar body length (range: 62.5-64.1 μm in the new species and 67.0-74.7 μm in M. antarcticum); the adult body length for M. antarcticum was reported to exceed 800 μm (Tumanov, 2006) and thus may be similar to that of the new species; and by having a more cephalic position of stylet Milnesium validum, by having a shorter buccal tube with respect to the body length (percent ratio 6.9-7.9 in the new species and 10.4-11.5 in M. validum).

Other records of Antarctic Milnesium specimens
The distribution of Milnesium spp. recorded so far in and around Antarctica is shown in Fig. 11. The morphological trait of multiple branches observed in the new species from Innhovde has already been reported from other areas within the East Antarctic region. The specimens from Langhovde (Sudzuki, 1964) showed two-five branches on the secondary claws. Although the quality of the illustrations was far from perfect, this description suggested that the samples possibly included specimens with up to five-branched secondary claws. Moreover, although this report did not men-tion any males, one of the illustrations clearly showed the modified secondary claw of a male (see fig. 12 in Sudzuki, 1964). This indicates that a dioecious Milnesium population also occurred at Langhovde, and it is possible these belong to the new species. Co-localization of other Milnesium sp. with {3-3} CC has been suggested by the literature that described M. tardigradum sensu lato from Molodeznaya, in the vicinity of Lützow-Holm Bay (Utsugi and Ohyama, 1991). Dastych (1984)  That branch is single in typical specimens […], in mentioned form it is divided into 2-5 teeth, mostly 3-4; […]. A number of teeth is different within this species, often even on one leg."  Dastych's (1984) description of the 'typical' three-branched specimens from King George Island would correspond with the more recent description of M. antarcticum, also from King George Island (Tumanov, 2006). The Continental Antarctic 'form' that Dastych (1984) described with four-seven branches on the secondary claws, mostly five-six branches, would match our description for M. rastrum sp. nov.
The COI sequence of Milnesium sp. Ta46-01 was obtained from a specimen collected from the Stornes Penisula, Larsemann Hills (Velasco-Castrillón et al., 2015). Our unpublished data indicates there is a population of Milnesium in the Larsemann Hills with multiple secondary claw branches. Another study noted the distribution of Milnesium sp. from the inland nunataks at the base of the Antarctic Peninsula, Palmer, and Ellsworth Land, and suggested that morphology indicated, "there may be distinct 'forms' or speciation occurring at the different sites within the continental, maritime, and sub-Antarctic" (Convery and McInnes, 2005). The details of morphological differences were not stated. However, the COI sequence of Milnesium sp. Miln06-224 (in Velasco-Castrillón et al., 2015) was obtained from Ellsworth Land, the region under discussion in Convery and McInnes (2005). There is currently no information about CC of Ta47-01 from Queen Maud Mountain.

Phylogeography
In the ML phylogenetic tree constructed from Milnesium COI, M. rastrum sp. nov. appeared in a clade with Antarctic strains: Milnesium sp. Ta47 Afrotropical strains are also found among many other clades in the phylogenetic trees. In comparison, the Nearctic/Palaearctic strains appeared in a more derived position, after the seventh or eighth node (Fig. 10B, C), and are supported by more studies from these regions. This view of the tree accords with the Gondwana origin hypothesis of several lines of tardigrades (McInnes and Pugh, 1998;Guidetti et al., 2017).
It is interesting that Marion Island harbored the most basal Milnesium strain. This island is the tip of an active oceanic intraplate volcano, considered to be less than one million years old and with an earliest subaerial eruption estimated by K-Ar dating at ca. 450,000 years ago (McDougall et al., 2001). On the other hand, the split between Parachela and Apochela is estimated to be at 432 million years ago (Mya) (range, 323-540 Mya) during the Palaeozoic, and the most recent common ancestor of Milnesium was estimated to appear 162 Mya (range, 116-207 Mya) (Morek et al., 2021), which would coincide with the onset of the Gondwana breakup about 164 Mya (Mueller and Jokat, 2019). Thus, the island is quite young in comparison with Milnesium diversification. However, Marion Island is on the Marion Hotspot that has been stationary since 88 Mya and played an active role in the breakup of Madagascar and India (Storey et al., 1995;Torsvik et al., 1998). According to this geographic view, Madagascar would have migrated North, with its Milnesium ancestor, away from the Marion Hotspot about 88 Mya. This Milnesium population would have evolved in response to various environments, with the extant Madagascan species M. matheusi and M. wrightae, as the modern representatives. However, this does not explain how Milnesium spp. arrived on Marion Island. We cannot say where the ancestral Milnesium species existed until we get more spatiotemporal data and hopefully some fossil records. Nevertheless, with the molecular results of Figs. 10 and 11, it is possible to
It is unknown whether any species in the above list have facultative parthenogenesis. Although M. inceptum always reproduces without males, male individuals emerged at a very low frequency even in such a thelytokous population (Suzuki, 2008); the reason for this has not yet been elucidated. The first author has observed that individuals of M. inceptum, since 2000, have laid at least one egg during the moulting period, even if the nutritional condition was not good. On the other hand, the solitary female of M. rastrum sp. nov. did two cycles of moult without oviposition, suggesting an obligate mode of bisexual reproduction. In dioecious tardigrades, females isolated from males never lay eggs (Lemloh et al., 2011;Bingemer et al., 2016). The same condition was apparently observed in M. eurystomum GB.005, in which males were found, and described as follows: "Since mature females extracted from the GB.005 sample did not lay additional eggs, the culture was terminated after a short period of time" (Morek et al., 2020b).
While parthenogenesis is considered more common in harsh environments (Nelson et al., 2015), it is interesting to note that an isolated ice-free area like Innhovde contains the presumed non-parthenogenetic species M. rastrum sp. nov. This may account for the rarity of Milnesium over the East Antarctica area, and suggests that M. rastrum sp. nov. had retained the plesiomorphic trait of bisexual reproduction since the isolation of Antarctica.
Tardigrade dispersal mechanisms are relatively poorly studied and the exact means are unknown. In the genus Ramazzottius Binda and Pilato, 1987, which has both reproductive modes, the diploid bisexual populations were found mostly in large moss or lichen encrustations on rocky outcrops, while the triploid or tetraploid parthenogenetic populations were from newly formed patches of moss or lichen on tree trunks (Bertolani et al., 1990). This observation suggests a theory that parthenogenesis with multiploidy originated somewhere, and then settled and propagated in harsh and isolated habitats. However, this theory cannot be applied to the genus Milnesium because parthenogenetic populations of M. tardigradum, M. pacificum Sugiura et al., 2020, and M. inceptum all showed diploid chromosomes (Sugiura et al., 2020). So, parthenogenetic Milnesium flourish in harsh environments regardless of the ploidy.
Antarctica was a warm and well-vegetated land mass before becoming isolated and frozen about 30 Mya. Tardigrades, able to survive the harsher environment, would have survived, staying in the remaining vegetation but becoming more isolated by glacier formations. This, and the more plesiomorphic bisexual reproduction as the ancestral mode may account for the isolated pockets of Antarctic dioecious tardigrades, e.g., M. rastrum sp. nov., Mopsechiniscus franciscae Guidetti et al., 2014, (Victoria Land), and Echiniscus corrugicaudatus McInnes, 2010 (inland nunataks in Ellsworth Land). While these Antarctic environments are extremely isolated, they have also provided very stable and rich habitats as refugia for tiny bisexual creatures for several tens of millions of years. However, this also makes these ecosystems very fragile against settlement of more fertile parthenogenetic animals as well as contemporary invaders (see Hughes et al., 2020) or possible future destruction of the natural barrier around the Antarctic Continent (Convey and Peck, 2019).