There was an obvious seasonal effect on temperature and biological parameters along the ice core extracted at 7010 m ASL of the Muztagata Glacier (Figure 2). An apparent seasonal temperature change was indicated by the proxy value of the stable isotopic ratios, ¹⁸O/¹⁶O (δ¹⁸O), with a low value in winter and a high value in summer (Figure 2B). The live cell density was greatly variable and ranged from 6.5 × 10² to 2.1 × 10⁴ cells/ml between 1964 and 2000 (Figure 2A). The total cell density varied from 4.4 × 10⁴ to 8.7 × 10⁵ cells/ml (Figure 2C). Several live cell density peaks were formed during the summer seasons in 1969, 1970, 1973, 1979, 1982, 1983, 1988, 1990, and 1993 for a total of nine events, a1 to a9 (open triangles in Figure 2A), while cell density peaks were found in spring (filled triangles in Figure 2A). This ice core also had an increased density of the total number of microorganisms in the summer of 1978, 1988, and 1993 (open triangles c1, c2, and c3 in Figure 2C), and in the spring of 1995 and 2000 (c4 and c5 in Figure 2C), which was consistent with the live cell density patterns (Figure 2A). The microbial cell density correlated with the concentrations of mineral particles and possessed a high R² value of 0.68 only from 1994 to 2000 (from ice core depth 2.5 to 9.3 m, Figures 3A,B), but did not correlate with mineral particle concentrations from 1990 to 1993 (from ice core depth 9.3 to 12.5 m, with R² < 0.1, Figure 3B).

Seasonal analysis of the Dunde ice core was not successful due to the limitations of sample resolution (Figure 4). Oxygen isotope ratios of the melt-water samples from the Dunde ice core showed a change range from –10.78‰ to –8.24‰ (temperature proxy ¹⁸O/¹⁶O, Figure 4D), while microbial cell density varied from 1.2 × 10³ to 9.1 × 10⁴ cells/ml (Figure 4B) and 1.3 × 10⁵ to 1.9 × 10⁶ cells/ml (Figure 4C) for live and total cell density, respectively. Three peaks c2, c3, and c4 of the total cell density were found in the spring of 1988–1989, 1992, and 2000, only one peak, c1, was found in the summer of 1985 (Figure 4C). The live cell density response pattern was consistent with the total cell density tendency (the dash lines in Figures 4B,C). An abundance of microbial cells frequently occurred at the dirty ice layers (Cell density peaks c1, c3, and c4 at the dust layers labeled as a1, a3, and a4 at the dash lines in Figures 4A,C), but were rarely found at the clean ice layer (small density peak c2 at the a1 ice layer in Figure 4).

The dominant bacteria in six ice layers of the Dunde ice core were investigated using 16S rRNA gene clone library, sequencing techniques, BLAST and phylogenetic tools. A total of 24 bacterial genera were identified in the Dunde ice core. They belonged to genera Polaromonas sp., Rhodoferax sp., Variovorax sp., Burkholderiales, Herbaspirillum sp., Xanthomonadaceae, Ketogulonicigenium sp., Devosia sp., Bacteriovorax sp., Hymenobacter sp., Pedobacter sp., Flavobacterium sp., Flectobacillus sp., Cytophagales, Sphingobacteriaceae, Cryobacterium-related, Propionibacterium/Blastococcus sp., Salinibacterium/Frigoribacterium sp., Knoellia sp., Cyanobacteria, Luteolibacter sp., Paenibacillus sp., Anoxybacillus sp., and TM7 candidates (Figures 5–7). Three genus groups Cryobacterium-related, Salinibacterium/Frigoribacterium sp., and Propionibacterium/Blastococcus sp. were clustered with 65–76% similarity to the known species but grouped with genus Knoellia sp. with 95% similarity in the family members of Actinobacteria (Figure 6). Only one clone DD1D107-100 was 100% similar to the uncultured Bacteroidetes clone AKYG1686 (Figure 7). All tested bacterial clones in the ice fell into members of bacteria phyla Alpha, Beta, Gamma, and Deltaproteobacteria, Actinobacteria, Bacteroidetes, Firmicutes, Verrucomicrobia, and TM7 candidates.

There was a large difference in the proportion of the main phylogenetic groups along the Dunde glacier depth profile, which indicated the seasonal changes of microbial communities in the glacier (Figures 8A1–A6). The bacterial clones were comprised of five dominant genus groups, Polaromonas sp., Pedobacter sp., Flavobacterium sp., Propionibacterium/Blastococcus sp., and Cryobacterium-related, which accounted for more than 55% of the total 406 clones and frequently appeared in the six tested ice layers from 1990 to 2000 (dashed lines in Figures 8A1–A6). Nine genus groups such as Rhodoferax sp., Variovorax sp., Burkholderiales, Flectobacillus sp., Cytophagales, Sphingobacteriaceae, Knoellia sp., Cyanobacteria rarely occurred in the ice. Other opportunistic bacterial clones occasionally appeared in the ice.

Contamination of the DNA samples (from the inner core columns) used in this study is unlikely because the outer surfaces of ice core and reagents for DNA analysis were cautiously decontaminated, and all the procedures were performed within a sterile, positive pressure laminar flow hood. Only small DNA fragments (<100 bp) were detected from the ice column control of (autoclaved sterile water), which were not considered for further sequence analysis in this study. It should be noted that Herbaspirillum sequences, also found in this study, have previously been identified as potential contaminants in glacier debris and ice samples (Cameron et al., 2016). However, the experimental procedures used by Cameron were completely different from ours in the present study. Their protocols and procedures were used for glacier cryoconite debris and surface ice samples. Herbaspirillum sp. found in this study, are well-known plant root-associated nitrogen-fixing (Baldani et al., 1986) and non-nitrogen-fixing environmental species (Ding and Yokota, 2004; Dobritsa et al., 2010). They were also reported in the Alaska Gulkana glacier, an Antarctica glacier forefield and the Antarctica Lake Vida brine (Segawa et al., 2011; Bajerski et al., 2013; Kuhn et al., 2014).

Various molecular techniques, CFM with cell stains cFDA, PI, and SYTOX have been used to investigate viable bacteria (Amor et al., 2002; Schumann et al., 2003; Xiang et al., 2009). These tools helped us to examine the abundance of live cells and the potential metabolic activities of microorganisms in an environment. However, the CFM approach has certain limitations because of interference from dust particles or spurious abiotic autofluorescence and underestimation of the accurate cell counts under the typical CFM parameters (Stibal et al., 2015). Despite the limitations, the background noise can be counterweighed by data series from the ice core profiles. In this study, the apparent seasonal tendency suggests that our analyses were based on a substantial fraction of bacteria.

For the phylogenetic analysis of bacteria, more than 600 clones were picked and sequenced. A total of 406 valid bacterial clones were obtained from the Dunde ice core after vector, and chimeric checking. The rarefaction curves of six clone libraries from the ice core were approaching asymptotes (dada not shown). Data also showed the prevalence of a few dominant genus-related groups at the different ice core depths (Figures 8A1–A6). This indicated that the identified clones were based on the dominant bacterial taxa.

The present data sets from the Muztagata glacier at 7010 m ASL (38°17′N, 75°04′E) revealed a high correlation between dust and microbial abundance from 1994 to 2000, which indicated a strong influence of aeolian activities on the microbial deposition in the glacier snow (Figures 3A,B). This was also consistent with another independent microbial investigation on the Muztagata glacier at 6300 m above sea level (Liu et al., 2013). The Dunde ice core also presented a frequent association of microbial cell density peaks with high concentrations of mineral particles (a1, a3, and a4, verse c1, c3, and c4 in Figures 4A,C). The strong association of microorganisms with dust was also found in previous data from the Antarctic glacier (Abyzov et al., 1998; Priscu et al., 2008), the Malan glacier (Yao et al., 2006), and the Guoqu glacier on the Tibetan Plateau (Yao et al., 2008). The analyses of trace and rare earth elements extracted from the same series of Dunde ice core sections showed that the fine fractions in the Dunde dust were more similar to those in the western Qaidam Basin and Tarim Taklimakan Desert than those in the Badain Juran and Tengger Desert (Wu et al., 2009). The Nd-Sr isotopic composition of mineral particles in the Dunde ice core is also similar to that in desert sand from Qaidam and Tarim Taklimakan (Wu et al., 2010). All results revealed that the Qaidam Basin and the Tarim Taklimakan Desert was the main source of dust in the Dunder glacier, implying the transportation of dust-borne microorganisms from the western desert Tarim Taklimakan and adjacent Qaidam to the Dunde glacier.

However, the Muztagata ice core data showed independence of microbial load with dust deposition from ice core depth 9.3 to 12.5 m (Figure 3B). The Dunde ice core data also showed one small cell density peak c2 appearing at the clean ice layer a2 in Figure 4. These results indicate that microbial deposition in the glacier snow does not always associate with the dust deposits or “dirty” wind and may in fact be transported by “clean” wind or snow, which implies influences of the processes such as aerosol and precipitation deposition, along with other factors (Bottos et al., 2014; Pearce et al., 2016).

The present data sets from the Muztagata glacier at 7010 m ASL (38°17′N, 75°04′E), revealed clear seasonal patterns with high microbial cell density occurring in the warming summer months (open triangles Figure 2), which indicated positive temperature effects on the microbial density patterns. This was consistent with another independent microbial investigation on the Muztagata glacier at 6300 m ASL (38°17′N, 75°06′E, Liu et al., 2013). The high repeatability of both ice cores from the Muztagata glacier confirmed the reliability of the data sets discussed here. Evidence for a positive temperature effect includes the algae growth of red Chlamydomonas at the surface snow in New Zealand and on the Alaska Harding icefield and on Greenland and Iceland’s glaciers in late spring and summer (Thomas and Broady, 1997; Takeuchi et al., 2006; Yallop et al., 2012; Lutz et al., 2015). Further temperature effects on bacterial growth, colonization and community transition were reported on Kuytun 51 Glacier, where bacterial Cyanobacteria were dominant across the surface snow slope in the warming spring-summer, but rarely in the subsurface, winter-snow-layers (Xiang et al., 2009). As expected, the live cell density during the summer was high as a result of microbial growth in the surface snow. Other groups, Uetake et al. (2006), Yao et al. (2008), and Price and Bay (2012) also found that high microbial abundance was present in the warming spring-summer seasons in the Sofiyskiy glacier in the south Chuyskiy range of the Russian Altai, the Guoqu glacier in the Geladaindong mountain regions, and the deep Arctic and Antarctic ice cores, respectively. The obvious seasonal patterns of bacterial populations with a high cell density in summer strengthen the post-deposition effect on the microbial populations in glaciers.

In addition to those cell density peaks during the summer (open triangles in Figures 2A,C), there were also many density peaks in the spring from 1963 to 2000 (filled triangles in Figures 2A,C). The seasonal pattern of bacterial density was generally consistent with the dynamic mineral particle deposition with frequently dust outbreaks in spring and summer (Figures 3 and 4 in this study; Wu et al., 2008; Liu et al., 2015). This indicated an important influence of dust deposition on the microbial communities in glaciers. All of the results suggest the fundamental contribution of dust-microbe deposition to the basic population pool size and the intensifying effect of post-deposition by microbial growth in the warming seasons.

The present data showed that the Polaromonas sp. from the Dunde ice core clustered together more closely than those from other environments (Figure 5). The phenomena of Polaromonas sp. from the same location readily grouping together was also found in the Muztagata and Puruogangri glaciers (An et al., 2010; Xiang et al., 2010). Although Polaromonas sp. were widely distributed across the geographically different glaciers, statistical analyses demonstrated a large genetic distance among 43 unique glacier Polaromonas sequences, which positively associated with geographic distance (Franzetti et al., 2013). Similar geographic phenomenon of individual microorganisms was also found in the deep ice core. Bacteria Alternaria sp. were common in the deep ice cores of Greenland GISP2D and Antarctic Vostok 5G and Byrd, but their DNA sequences were phylogenetically different between the two polar regions (Knowlton et al., 2013). The geographic differences of Polaromonas sp. and Alternaria sp. across the isolated glaciers suggests that the mountain “barriers” to the microbial transportation can be surmounted by suitable adaptations, which leads to the geographic patterns of individual microorganisms.

Geographic differences are not only evident for Polaromonas sp. and Alternaria sp. but also for the taxonomic groups. It is obvious that there is a geographic distinction of taxonomic groups at the cryoconite habitats on three High-Arctic glaciers from the associated moraines and adjacent tundra on the Brøggerhalvøya peninsula, Svalbard (Edwards et al., 2013, 2014). Significant differences in the composition of dominant taxonomic groups are also found between alpine and Arctic cryoconite habitats (Edwards et al., 2014). The present data from the Dunde ice core showed that similar taxonomic groups frequently appeared along the ice core profiles as historical events (Figures 8A1–A6). Bacterial genus groups Cryobacterium-related, Flavobacterium sp., Pedobacter sp., Polaromonas sp., and Propionibacterium/Blastococcus sp. were frequently found at the six tested ice layers of Dunde glacier from 1990 to 2000 (Figures 8A1–A6). Another example of similar group members sharing the related ice core layers can be found in the recently reported Dunde ice. Genera Polaromonas sp. and Flavobacterium sp. commonly found between 1990 to 2000 were also identified from the Dunde ice column AD 1780–1830 (Zhang et al., 2009). Although the dominant genus-related groups are similar in the related ice cores, the composition of the main genus-related groups is largely different across the geographically different glaciers. The bacteria Cryobacterium-related was more abundant in the Dunde ice cap than in the Muztagata glacier, while Enterobacter sp. appeared throughout the four tested ice layers of the Muztagata glacier but rarely in the Dunde ice cap (Figures 8A1–A6,B1–B4). Seven genus groups Polaromonas sp., Enterobacter sp., Acinetobacter sp., Flexibacter sp., Thermus sp., Propionibacteria/Luteococcus sp., and Flavisolibacter sp. were frequently identified in the four tested ice layers of Muztagata glacier from 1970–1988 (labeled as the dashed lines in Figures 8B1–B4), while Polaromonas sp. and Flexibacter sp. were found at all three tested ice columns of Puruogangri glacier from 1600 to 1920 (Zhang et al., 2009; An et al., 2010). All results clearly show that a few genus-related groups are dominant in the mountain ice cores and constitute the main taxonomic groups endemic to the local glacier regions. The difference of taxonomic group members across the geographically different glaciers suggests intermingling of the bacterial taxonomic groups to the point of geographic separation. More data of microorganisms in the deep ice are necessary for our better understanding of the biogeography of microorganisms in glaciers.

The geographic pattern of bacterial taxonomic groups could be attributed to the influence of the moisture and dust source area, which might vary across the mountain glaciers on the Tibetan Plateau (Figure 1; Table 1). Precipitation over the Muztagata glacier is mostly influenced by the westerly depressions, while precipitation over the Dunde ice cap and Puruogangri ice cap is mainly driven by the westerly depressions in winter and Indian monsoon in summer (Murakami, 1987; Wake et al., 1990; Davis et al., 2005). Dust in the mountain glacier Muztagata is mainly derived from deserts including Sary-Ishykotrau, Muyun Kum, Kyzyl Kum and Kara Kum, Taklimakan, and Gurbantunggut (Figure 1; Wake et al., 1993), while the Dunde ice cap is very close to the Gobi Desert, Qaidam Basin (Figure 1) and thus its dust components are more likely strongly affected by local dust storms and dominated by mineral particles from the two deserts Qaidam Basin and Tarim Taklimakan (Wu et al., 2009, 2010). The dramatic changes of the moisture sources and dust pathways across the mountainous glaciers may lead to differences in the microbial communities deposited in the glacier snow. Moreover, the heterogeneity of local conditions such as temperature, light intensity, melt-water availability, and nutrient concentrations in the snow may drive the spatial patterning of the microbial community by influencing the colonization of the dominant endemic species in the snow. Concerns on how surface communities are incorporated into the cores, how much they change after burial, and how the post processes contribute the geographic differences of microbial communities are still open questions. More data on the microbiological, meteorological, and physical and chemical characteristics of the glacier surface and subsurface snow and ice cores will be helpful for a better understanding of the biogeography of microorganisms in glaciers.