Integrative approach reveals new species of water bears (Pilatobius, Grevenius, and Acutuncus) from Arctic cryoconite holes, with the discovery of hidden lineages of Hypsibius

Abstract

A crucial step in understanding glacial ecosystems is to elucidate their biodiversity. Although tardigrades (water bears) are one of the most common and abundant metazoans inhabiting glaciers, knowledge of their taxonomic diversity on these ice masses remains scarce. The fragmentary data about the diversity of metazoans in glacial ecosystems is a warning sign in the era of global warming that seriously threatens cold-adapted specialists. Based on morphological, morphometric and DNA analyses (18S rRNA, 28S rRNA, COI), we identified tardigrade taxa from water-filled reservoirs in the glacier surface (cryoconite holes) in the Arctic (Svalbard and Greenland). Here we describe three species new to science: Pilatobius glacialis sp. nov., Acutuncus mariae sp. nov. and Grevenius cryophilus sp. nov. In addition to their morphological and genetic differences, these species are easily distinguished from their close relatives by their thermally stable niche, cryoconite holes, in which water temperature at the bottom oscillates around 0.1 °C during the polar summer. Moreover, we found the genus Acutuncus, considered a pan-Antarctic taxon, in cryoconite holes on Svalbard. Additionally, we discovered three new species of the Hypsibius dujardini complex by DNA analysis. Their delimitation is currently based on genetic differences and further morphometric studies may aid in distinguishing them. Our study revealed a new view on the biogeography of glacier tardigrades and the genus Acutuncus, as well as emphasized the uniqueness of glacier metazoans. Tardigrade species inhabiting cryoconite holes may be either morphologically the same (P. glacialis sp. nov.), or very similar to species inhabiting other ecosystems (H. dujardini complex), but they differ in DNA. On other hands, others are unique in both morphology and DNA (G. cryophilus sp. nov.) indicating that the properties of specific habitats shape speciation and, as in cryoconite holes, the reason might be their thermal regime. Therefore, we suggest using the ecological background as well as habitat characteristics in taxonomic descriptions and the differential diagnosis of new tardigrade species.

Introduction

Recent climate changes and increasing warming of polar and high mountain ecosystems are the main threats to psychrophiles. Consequently, due to a rapid change in environmental conditions, these organisms might completely disappear or hide in cold refugia (Fountain et al. 2012; Milner et al. 2017; Zawierucha & Shain 2019). Considering that the world is being subjected to the so-called sixth mass extinction, description and documentation of the current status of biodiversity is highly needed (Eisenhauer et al. 2019). Therefore, studies on the biodiversity in cryospheric habitats have gained special attention in recent years (Cauvy-Fraunie & Dangles 2019; Zawierucha & Shain 2019; Stibal et al. 2020). Glaciers and ice sheets form one of the coldest ecosystems, with the surface of a glacier (called the supraglacial zone), being the most biologically active and productive (Hodson et al. 2008; Cook et al. 2016b). The supraglacial zone consists of various habitats and forms myriads of niches for unicellular and multicellular organisms (Takeuchi et al. 2000; Stibal et al. 2006; Hodson et al. 2008; Franzetti et al. 2017; Zawierucha et al. 2019a). The most species-rich glacial ecosystems are cryoconite holes - extreme water-filled reservoirs embodied directly in the ice (Takeuchi et al. 2000; Hodson et al. 2008; Sommers et al. 2019a, b). The dark colour of cryoconite sediment reduces albedo of the glacier surface and absorbs solar radiation, which influences the formation of these water pools (Takeuchi et al. 2001; Cook et al. 2016b). Cryoconite holes are inhabited by primary producers like cyanobacteria and algae, heterotrophic bacteria, protozoans and invertebrates (Porazinska et al. 2004; Zawierucha et al. 2016a, 2018a; Pittino et al. 2018) represented by tardigrades and rotifers in Arctic cryoconite holes (Zawierucha et al. 2016a, 2020). Due to strong environmental selection pressure in cryoconite holes, organism assemblages inhabiting these ecosystems are mostly unique and different from those found in tundra or in other freshwater Arctic ponds. Instead of being embedded in soils or rocks, water and sediments in cryoconite holes are surrounded by ice that keeps the temperatures at near freezing even during summer days. For instance, the average temperature at the bottom of cryoconite holes on Arctic glacier is 0.08 °C with the maximum of 0.22 °C (Zawierucha et al. 2019b). In addition to low temperatures, Arctic cryoconite holes on small valley glaciers are dynamic and characterised by periodic freezing, removal of sediments from the bottom, and/or collapsing during stochastic weather events (e.g. rain and foehn winds) (Mueller et al. 2001; Zawierucha et al. 2019b). Such abiotic conditions most probably favour specialised, cold-adapted stenothermic ice inhabitants (Zawierucha et al. 2019a; Stibal et al. 2015). Recent biological studies on cryoconite holes are mostly focused on bacterial and photoautotroph physiology and diversity (e.g., Williamson et al. 2018; Pittino et al. 2018; Sommers et al. 2019a, b). Invertebrates, regrettably, although they are apex consumers in such extreme ecosystems, were not in the main scope of most of these studies (for review of the literature on the topic see Hodson et al. 2008; Cook et al. 2016b; Kaczmarek et al. 2016).

The fragmentary data on the diversity of metazoans in glacial ecosystems is a warning sign that the biodiversity of glacial habitats is still largely overlooked and underestimated. This is especially worrisome given the magnitude of glacial habitat loss anticipated this century (e.g. WGMS 2020; Zawierucha & Shain 2019). Indeed, the diversity of invertebrates inhabiting ice, although poor, is considered unique and often endemic (Zawierucha et al. 2016a, 2018c, 2019a), therefore a description of such endangered extremophiles is highly needed.

Tardigrada, also known as water bears, are one of the most common and abundant invertebrates inhabiting cryoconite holes worldwide, with densities in the holes being similar to other freshwater pools or limno-terrestrial ecosystems (Porazinska et al. 2004; Zawierucha et al. 2019a,b). So far, over 1300 tardigrade species have been described, mostly from bryophytes and lichens (Nelson et al. 2015; McInnes & Pugh 2018; Degma et al. 2020). As a result of their evolutionary history, tolerance to unfavourable conditions, small size and probable long-range distance dispersal by wind, tardigrades inhabit a variety of environments, from limno-terrestrial (soil, bryophytes, lichens) to aquatic (sediments, plants), including cryoconite holes (e.g., Nelson et al. 2015; McInnes et al. 2017; Zawierucha et al. 2019a). Tardigrades play an important role in trophic webs at a multitrophic level as carnivorous, omnivorous, herbivorous and microbivorous species (Hallas & Yeates 1972; Guidetti et al. 2012; Guil & Sanchez-Moreno 2013). As apex consumers in cryoconite holes due to grazing, they may potentially influence the diversity and density of other organisms inhabiting cryoconite holes ( T. Jaromerska pers. observ.), thus, they might be an important part of cryoconite ecosystems. Tardigrades seem to be well adapted to cryoconite hole environments due to intrinsic physiological features favouring life on ice, e.g. ability of tardigrades to enter cryobiosis (Møbjerg et al. 2011; Zawierucha et al. 2018c). Even though taxonomic studies on tardigrade biodiversity have been conducted in high mountain and polar regions (Cesari et al. 2016; Kaczmarek et al. 2018; Guidetti et al. 2019; Zawierucha et al. 2018b, 2019c; Dueñas-Cedillo et al. 2020; Stec et al. 2020), they focused mostly on bryophytes or lichens, and other glacial or glacier-adjacent environments have rarely been investigated (Grøngaard 1994; Zawierucha et al. 2019a, b). Studies on tardigrades in cryoconite holes have a long tradition, but published records are fragmentary and rare. To the best of our knowledge, tardigrades along with rotifers were the first animals described from cryoconite holes (Von Drygalski 1897; Jensen 1928), and studies on tardigrade fauna have continued since then (e.g. Dastych 1985, 2019; Grøngaard 1994; Grøngaard et al. 1992, 1999; Dabert et al. 2015; Zawierucha et al. 2018a, 2019a, b).

Recently, two genera of tardigrades new for science have been described from cryoconite. Zawierucha et al. (2018c) erected a new genus Cryoconicus from glaciers in central Asia. Molecular data placed Cryoconicus in the family Ramazzottiidae as a sister clade to the cosmopolitan genus Ramazzottius. The classical erection of a genus based on morphology was supported by the dark pigmentation unusual for limno-terrestrial tardigrades, the morphology of ventral apophyses for the insertion of the stylet muscles (AISM), the absence of accessory points on the claw branches and the presence of cuticular bars under claws I-III (Guidetti et al. 2019; Zawierucha et al. 2018c). Dastych (2019) erected a new glacier-dwelling genus Cryobiotus that for years was classified as Hypsibius (currently comprising four cryoconite obligatory species, see also Dastych et al. 2003). The erection of Cryobiotus was based on morphological characters: pigmentation, big eyes, the morphology of claws and AISM (Dastych 2019). Recent descriptions of new tardigrade genera from glaciers is a robust indicator that tardigrades in glacial ecosystems still await discovery.

Tardigrade species richness in extreme glacial habitats is known to be lower than that of tundra or high mountain ecosystems, potentially as a consequence of the strong selection pressure of the cryosphere. However, our knowledge of diversity of glacier-dwelling tardigrades are underestimated due to lack of taxonomic focus on these habitats and there is an increasing evidence that some tardigrade species in cryoconite holes are unique obligatory glacier-dwelling animals (e.g. Dastych et al. 2003; Zawierucha et al. 2016a, 2019a).

In order to fully understand glacial ecosystems, elucidating their biodiversity is a crucial step. Zawierucha et al. (2016a) described the diversity of tardigrades from Arctic cryoconite holes and emphasised, to avoid taxonomic confusion, that species should be described as new taxa only if additional fresh material with individuals for exact comparison is available. Since that time, several contributions have been published and taxonomic obstacles removed (Cesari et al. 2016; Dastych 2018; Gąsiorek et al. 2017, 2018, 2019). Therefore, here we describe three new species of tardigrades and reveal new genetic lineages of likely cryptic taxa from the High Arctic cryoconite holes in Svalbard and Greenland.