Arctic and Antarctic cryoconite holes harbour distinct bacterial and eukaryotic communities
The structure of bacterial and eukaryotic cryoconite communities was found to vary according to their geographical location, as shown by 16S and 18S rRNA gene T-RFLP analysis (Fig. 2). Whilst prior research had demonstrated local (glacier to glacier) variation within Svalbard cryoconite microbial communities (Edwards et al., 2011), our research has demonstrated variability across a global scale. The communities from cryoconite holes in individual glacier locations often clustered together, suggesting localized sources of organisms and/or similar environmental selection pressures within a number of glacial locations. The most pronounced differences were found upon comparison between Arctic and Antarctic cryoconite communities, which may be a consequence of bipolar variation in mean total carbon content (Table 1).
Within the Arctic, location-specific clustering was more commonly found with respect to bacterial than eukaryotic communities. These findings reflect earlier microscopy-based studies that showed cyanobacterial, algal and invertebrate composition to be similar between cryoconite holes from five different glaciers within Taylor Valley, Antarctica (Porazinska et al., 2004), whilst communities present within an Arctic glacier and an Antarctic glacier were found to be different (Mueller & Pollard, 2004).
Aeolian transport has previously been proposed to account for the similarities across cryoconite communities over short distances (Porazinska et al., 2004). However, more recent molecular characterization of bacterial cryoconite communities present in three Svalbard glaciers (< 10 km apart) suggests that more subtle abiotic variability (e.g. in temperature, pCO2 and cryoconite inorganic content) leads to local variation in bacterial community structure (Edwards et al., 2011). If the microbial communities within cryoconite holes are seeded in ways similar to their original formation, as described by Gajda (1958), then aeolian transportation of local terrestrial or aquatic microorganisms onto the glacial surface will be an important contributor to the communities that establish.
In this study, Cercozoa-related sequences were the most abundant and diverse eukaryotic lineage within the cryoconite holes. Note we recognize that identification of the most abundant sequences from eukaryotic communities that comprise both unicellular and multicellular communities can be biased by multiple copies of rRNA genes being amplified from DNA from different cells from the same organism, that is, the relative abundance of multicellular organisms may be overestimated in such communities. Typically, Cercozoa are found in high abundances and are important within soil, freshwater and marine ecosystems (Bass & Cavalier-Smith, 2004). The high abundance of diverse Cercozoa within cryoconite communities would support their aeolian transport from other ecosystems onto these glacial surfaces. Similarly, Chlorophyta have been found to be transported from soil onto snow surfaces (Stibal & Elster, 2005), and terrestrial bacteria, including cyanobacteria, have been proposed to colonize permanent lake ice (Gordon et al., 2000). Cryoconite communities have also previously been noted for their similarities to local permanent lake ice communities and microbial mat communities (Christner et al., 2003).
As the geographical location of a cryoconite hole varies, so will the composition of microbial communities within the local environment that are available to seed them (Mueller et al., 2001). As a consequence, unique communities that are characteristic to each region may develop. It is, however, possible that biological inputs are not solely restricted to the neighbouring environment but that material can be transported over far greater distances, on trade winds (Rousseau et al., 2005; Price, 2009). Whilst local winds (Lyons et al., 2003) and polar easterlies and westerlies might cause community mixing within either Arctic or Antarctic ecosystems, foreign biological material could also travel from temperate regions to be deposited onto polar ice surfaces (Bovallius et al., 1978). However, the extent of this biological input and the ability of these foreign organisms to tolerate and survive the cryosphere's extreme conditions are limited, although studies of bacteria isolated from Antarctic ice cores (e.g. Christner et al., 2000) suggest that survival is possible.
Taxonomic composition of cryoconite bacterial and eukaryotic communities
Sequencing of rRNA genes was used to study the composition and diversity of cryoconite microbial communities that were representative of different geographical locations and of T-RFLP-derived clusters (Fig. 2). With the proviso that all PCR-based assessments of microbial community diversity are subject to potential biases (von Wintzingerode et al., 1997; Sipos et al., 2007), these cryoconite communities were found to contain members of between six and eight bacterial phyla, five and eight eukaryotic first-rank taxa and, at the two Antarctic locations only, representatives of two archaeal phyla. Interestingly, sequences related to both bacterial (Cyanobacteria and/or Chloroflexi) and eukaryotic (Archaeplastida and/or Stramenopiles) photoautotrophs were identified within every location studied, which together will provide a source of organic carbon to these environments.
All of the communities also contained organisms related to both bacterial heterotrophs and eukaryotic grazers, suggesting a self-contained multi-level trophic web. Metazoa (tardigrades) as top grazers were found in Arctic cryoconite holes (Fig. 3 and Fig. S2), but were not identified at the two Antarctic locations, in contrast to Christner et al. (2003), who found tardigrade-related sequences together with those from nematodes and rotifers within a cryoconite community in Taylor Valley, Antarctica. Additionally, the previous observation and identification of viruses within cryoconite holes (Säwström et al., 2002; Anesio et al., 2007) will contribute further to carbon cycling within cryoconite trophic webs (Säwström et al., 2006).
We identified representatives of all of the bacterial phyla and classes that were previously identified, using molecular analysis, within Antarctic (Christner et al., 2003) and Arctic (Svalbard) cryoconite holes (Edwards et al., 2011) were also found in this study. In addition, Deltaproteobacteria and Firmicutes were found in the majority of holes from both the Arctic and Antarctic that were sampled, whilst Epsilonproteobacteria were also present in cryoconite communities in Svalbard. Perhaps, the most striking discovery within this study is the ubiquity, high relative abundance (26–35% of clones sequenced) and diversity of Rhizaria-related sequences in all of the Arctic and Antarctic locations studied. In contrast, no Rhizaria-related sequences were identified by Christner et al. (2003), perhaps due to differences in specificity of the primers used in their study. Moreover, members of the first-rank lineages Haptophyta, Choanomonada and Centroheliozoa have also been found for the first time within cryoconite communities in our study.
Despite the extreme conditions of the cryosphere, taxon diversity within these cryoconite communities was surprisingly high. Taxon richness estimates (at an OTU threshold of 97% similarity) indicate that up to approximately 46% of the total estimated number of bacterial species and 67% of the total estimated number of eukaryotic species within these cryoconite communities were identified within this study. The overall estimated taxon richness (i.e. 449 bacterial and 139 eukaryotic OTUs) across these communities was comparable to that in bacterial communities found in more hospitable climates within arable and grassland soils (Øvreås, 2000; Hughes et al., 2001), although substantially lower than global estimates of bacterial species in soil (4 × 106) and in the oceans (8000) (Curtis et al., 2002). Bacterial taxon richness was not estimated in the two prior studies of cryoconite holes (Christner et al., 2003; Edwards et al., 2011), in which clone libraries were smaller in size (18 and 36 per hole, respectively), in contrast to the larger libraries generated within our current study (mean of 112 clones per hole; Table S1).
The reasons for the relatively high taxon diversity observed within cryoconite holes are unclear. Cryoconite holes are recognized as being important hydrological and biological systems, providing a harbour from extreme polar conditions (MacDonell & Fitzsimons, 2008), and provide a refugia for biota from physical factors, such as wind, desiccation, freezing, glacial flush-out (Fountain et al., 2004, 2008) and high UV intensities (Vincent et al., 2004). Moreover, the development of cryoconite holes helps to enhance localized water and nutrient availability (Takeuchi et al., 2001b; Säwström et al., 2002; Mueller & Pollard, 2004; Hodson et al., 2005; Stibal et al., 2006). Such resulting physicochemical conditions are biologically favourable and thus may help to support elevated taxon richness.
Taxon diversity was particularly high within the Comamondaceae (Betaproteobacteria) and the Sphingobacteriales (Bacteroidetes), although the majority of such sequences were only found at a single cryoconite location (data not shown). Within the Cyanobacteria, members of two orders, Oscillatoriales and Nostocales, were identified (Fig. S1). However, sequences related to members of the order Chroococcales were not identified within any of the six cryoconite communities, despite descriptions of these organisms within other cryoconite studies (Mueller et al., 2001; Christner et al., 2003; Porazinska et al., 2004 and Mueller & Pollard, 2004). Many of the closest relatives of the bacteria present within the cryoconite communities were related at genus level to sequences within the microbial mat communities of Lake Fryxell, Antarctica (Brambilla et al., 2001).
Identification of archaea within Antarctic cryoconite holes
Archaea were identified within the Antarctic (but not the Arctic) cryoconite communities. To our knowledge, there have been no other reports of archaea within cryoconite holes; archaea were not detected by PCR-based approaches in a recent molecular investigation of cryoconite holes on three Svalbard glaciers (Edwards et al., 2011). However, archaea have previously been identified on Alpine glaciers (Battin et al., 2001) and have been found frequently within many cold environments (DeLong, 1998; Cavicchioli, 2006), including Antarctic rock structures (Smith et al., 2000; de la Torre et al., 2003), within polar oceans (DeLong et al., 1994) and in Arctic rivers and the Arctic Ocean (Galand et al., 2006).
Representatives of two archaeal phyla were found (Fig. 4), with the majority identified as Thaumarchaeota, related to sequences from a range of environments, including 49 of the 63 sequences that were most closely related to an archaeon found in the cold sulphur-rich springs of Lake Erie (FJ968078; Chaudhary et al., 2009). Other Thaumarchaeota sequences were related to an endolithic archaeon from the pores of European Alpine dolomite rock (Horath & Bachofen, 2009). A further 11 clones from Signy Island, Antarctica, were most closely related to archaea within the rhizosphere of macrophytes within freshwater sediments that were thought to contribute towards elevated nitrification via ammonia oxidation (Herrmann et al., 2008). It is therefore possible that some of the Thaumarchaeota, identified within this cryoconite system, are similarly contributing towards ammonia oxidation and nitrification, facilitating nitrogen cycling within these environments, as is further evidenced via PCR amplification of Thaumarchaeota-related amoA genes from this site (Cameron, Hodson and Osborn; in preparation).
Members of the methanogenic Euryarchaeota classes Methanobacteriaceae and Methanomicrobia (Woese, 1987; Boone et al., 1993) were also identified within the Antarctic cryoconite holes. Methanomicrobiaceae have been previously found within the microbial mats of Antarctica's Lake Fryxell (Brambilla et al., 2001).