In this study, we revealed that tardigrades and rotifers were concentrated on seasonal snow patches in beech forests in Japan, particularly in green snow. Our observation suggests that snow algae blooming is truly benefitable for tardigrades and rotifers where they have stable and abundant food source. According morphology, we found two tardigrade taxa represented by genus Hypsibius and bdelloid rotifers on snow (Fig. 3). The high densities of microinvertebrates, their presence through more than one seasons and various size classes (Fig. 4, 6, 7) suggests that tardigrades and rotifers living on the seasonal snow patches and adapt to snow surface environment. Although snow algae blooming serves a good habitat for tardigrades, not all taxa found in mosses have been found on snow. Under laboratory conditions, tardigrades represented by tardigrade genus Macrobiotus which was found in mosses from trees at the study site are well known to feed on algae32, but was absent in snow. We expect that some physiological or dispersal constrains might limits their appearance on seasonal snow patches. Most probably it is a temperature regime, which seems shape tardigrade communities on glaciers26. Regrettably, we cannot say clearly where Hypsibius species on snow came from, whether mosses, lichens growing on ground or in tree canopies.
Most probably, microinvertebrates on snow origin from passive dispersal by wind. Hypothetically, if they would be passively transported to snow from surrounding mosses, more taxa should be found during sampling campaigns than only two Hypsibius. This observation suggests that even some tardigrades might appear on snow, they are not adapted enough to grow and reproduce in these ecosystems. In fact, previous work conducted in Akashibo snow patches in Japan, reported tardigrades and other microinvertebrates (e.g. Nematoda, Oligochaeta) in snow layer under the snow patches, which probably migrated from soil, however these microinvertebrates did not seem to be active in snow such as growing and reproduction33. Although the harsh conditions (low temperature and periodic freezing) occurred on seasonal snow patches in Mt. Gassan, due to presence of snow algae, these ephemeral ecosystems supports growth and reproduction of microinvertebrates. Tardigrades and rotifers found in green snow seems to not only be cold lovers, but taking into account high biomass and densities they must benefits from being on snow. From the above, a simple, hypothetical estimation of the microinvertebrates life cycle is as follow (I): dispersal to snow surface by wind from moss growing on tree trunks or canopies (April), (II): grow and reproduce by eating green snow algae (May), (III): passive transport with melting snow to mosses and spend their life in there (no snowy seasons).
There were no differences of concentration of chlorophyll a between colored snow in 2018 (Fig. 6), however, it seems that green snow form the most favorable conditions for growth and reproduction of tardigrades and rotifers in the study area. In green snow, exuvia of tardigrades and rotifers were found (supplementary Fig. S3), body length of tardigrades in May 2018 were bigger than in April 2018 (Fig. 4) which suggest successful growth. At the same time concentration of chlorophyll a in the samples also increased in time, which means that along with available food sources tardigrades increased their size and numbers. Although seasonal variability may result in growth of tardigrades body size, such effect has not been widely accepted.
Microinvertebrates were found with the highest population density in green snow, none or low in red, yellow and white snow (Fig. 6, 7 and Table 1). Tardigrades in green snow are typical algae feeders32,34 and indeed their intestine were full of algae cells (Fig. 3a and supplementary Fig. S1). At this moment it is hardly to say whether tardigrades and rotifers utilize the same food source on snow, or they use different food; tardigrades eat algae and rotifers might eat some of the associated suspension bacteria or yeasts. Although, the same two dominant algae genera on snow were found in Canada5 (Chloromonas spp. or Sanguina spp.). Authors presented tardigrades, rotifers, mites and springtails in red snow, and showed that both tardigrades and rotifers have the same red intestine content. Regrettably, authors did not presented densities of animals, hence we cannot compare data. In Mt. Gassan, it is known that snow algae which compose green and red snow are mix of several species of genus Chloromonas, yellow snow are Ochromonas species10,11,35,36. Even though green and red snow may consist of same genus Chloromonas, still they are different species, the difference in their color is probably due to the difference in life cycle of snow algae. In green snow, algal cells were mostly motile vegetative cells, which have chloroplasts without any secondary pigment, and were actively swimming using flagella. They can asexually reproduce in the melting snow surface and form visible green snow. In contrast, algal cells were mostly cysts with ribbed and thick cell walls, they contained orange or red colored secondary carotenoids, which are probably astaxanthin. Such secondary pigments are usually produced when snow algae are stimulated by light, for minimizing the amount of light available36,37. Tardigrades belong to Hypsibius also found in snow are considered to eat algae by sucking34, so this thick cell walls make it difficult for tardigrades to suck algae. Moreover, tardigrades in laboratory cultures feed on Chlorella vulgaris, Chlorella sp.,38,39 and green algae on snow have similar size ( 2-10 µm for C. vulgaris40. It was mentioned that size of algae is not important for their dietary preferences34, by far we cannot say whether size of snow algae affect their dietary preferences. For yellow snow algae, their cell walls and size do not seem to interfere with the tardigrade’s dietary so we can assume that tardigrades get something beneficial from green snow algae pigments not from yellow snow algae. However, these relations required exact testing in the future.
Due to simplicity of snow ecosystems, low organic matter content, few primary producers and few consumers, snow ecosystems may facilitate recognition and understanding of ecological processes in the cryosphere. In cryoconite holes which are cylindrical water filled reservoirs contained cyanobacteria, dust, organic matter and mineral particles on glacial ice41,42, although some study showed correlation between tardigrades and rotifers30,43,44, the significant relation between tardigrades or rotifers and any organisms as a typical source of food in cryoconite have not been found29,43. There are two potential scenarios which might explain this unrecognized pattern in cryoconite holes. In comparison with areas of snow algae blooming, cryoconite holes host very diverse biota and rich organic matter, thus diet of invertebrates might be complex which may constrain understanding of food preferences45. Secondly, dynamic nature of cryoconite holes in Arctic and alpine regions like rapid ablation, inter-hole water-sediment mixing and stochastic weather events disturb or sometimes destroy habitats on surface of ice like cryoconite holes30,46, hence in dynamic habitats findings of any ecological relations are problematic.
In the seasonal snow patches, meltwater is considered the only way causing removal of biotic components including microinvertebrates. In this case, snow surface was mostly flat and during field works we could not see meltwater flowing on the surface. Therefore animals might migrate along with the movement of snow algae, and maintain stable until they are supported by snow (as a liquid water) and algae (as a food). Nevertheless, population density of microinvertebrates in the seasonal snow patches was lower than that of in the cryoconite holes with note that snow patches and cryoconite holes have different conditions of water (solid or liquid) so the densities cannot be simply compared; in Greenland, average was approx. (looks as same as snow but reached up approx. for tardigrades, approx. for rotifers29, in European Alps, approx. (reached up approx. ) for tardigrades26, in Svalbard, reached up approx. for tardigrades, approx. for rotifers47, in Antarctica, reached up approx. for tardigrades, approx. for rotifers43. In other cold environments, tardigrades were found in mosses in Antarctica with higher population density and biomass (reached up to approx. ) than that of in the seasonal snow patches48. Rotifers were found in weathered snow/ice in accumulation zone with lower population density (reached up to ) than that of in the seasonal snow patches49.
The need for holistic studies on snow and biosphere including microbial activity which change the chemical composition of snow is well acknowledged7. Many taxa inhabit the seasonal snow5,6,16, however in many cases it is hard to infer which faunal elements are true snow ecosystems element and which randomly feed on snow algae. Herein we showed that tardigrades and rotifers are abundant where snow algae blooming occurred with preferences towards green snow (Chloromonas sp.) over the two sampling seasons in Japanese mountain forests. Intestine contents which contained algae cells, various instars and size classes of animals suggests that microinvertebrates are virtual faunal element in snow blooming while snow patches serve as a new arena for studies on biodiversity and ecology of these ubiquitous microinvertebrates. Further studies on the consumption rate by snow algae consumers may bring a new data on how invertebrates suppress algae blooming and how these algae respond on abundant invertebrates. These studies also have possibility to contribute to understanding material cycle such as carbon and nitrogen in snow which related to microbial activity on the snow surface50,51. While it was also mentioned carbon and nitrogen cycle in snow cover environments and emphasized impact of presence of seasonal snow cover for such cycle in soil ecosystem under the snow52, microbial activity in and on snow were not considered. Understanding the relationship between snow and biosphere including microinvertebrates is important in anticipation future changes and biogeocycles in snow ecosystems.