Community change of microorganisms in the Muztagata and Dunde glacier 1 and climatic and environmental implications 2

Microorganisms are continuously blown onto the glacier snow, and thus the glacial depth 10 profiles provide excellent geographic archives of the microbial communities. However, it is uncertain 11 about how the microbial communities respond to the climatic and environmental changes over the glacier 12 ice. In the present study, the live microbial density, stable isotopic ratios, 18 O/ 16 O in the precipitation, and 13 mineral particle concentrations along the glacial depth profiles were collected from ice cores from the 14 Muztagata glacier and the Dunde ice cap. Six bacterial 16S rRNA gene clone libraries were established 15 from the Dunde ice core. The Muztagata ice core presented seasonal response patterns for both live and 16 total cell density with high cell density occurring in the warming spring and summer. Both ice core data 17 showed a frequent association of dust and microorganisms in the ice. Genera Polaromas sp., Pedobacter 18 sp, Flavobacterium sp., Cryobacteriium sp., and Propionibacterium/Blastococcus sp. frequently appeared 19 at the six tested ice layers, and constituted the dominant species endemic to the Dunde ice cap, whereas 20 some genera such as Rhodoferax sp., Variovorax sp., Sphingobacterium. sp., Cyanobacterium sp., 21 Knoellia sp., and Luteolibacter sp. rarely presented in the ice. In conclusion, data present a discrete 22 increase of microbial cell density in the warming seasons and biogeography of the microbial communities 23 associated with the predominance of a few endemic groups in the local glacial regions. This reinforces our 24 hypothesis of dust-borne and post-deposition being the main agents interactively controlling microbial 25 load in the glacier ice. 26


Introduction.
Microorganisms are continuously blown onto the glacier snow, and thus the glacial depth profiles provide good geographic archives of the microbial communities during the course of global climatic and environmental processes.However it is unclear how the microbial communities respond to the climatic and environmental changes over the glacier ice.Recently, microbiological data have been collected from ice cores extracted from the geographically different glaciers such as the Vostok Station ice core, Antarctica (Abyzov et al., 1998;Christner et al., 2006;Priscu et al., 2008), the Malan Glacier (Yao et al., 2006) and the Guoqu Glacier in the Mount Geladaindong on the central Tibetan Plateau (Yao et al., 2008).
All results of the ice cores have showed a high microbial abundance corresponds with a high concentration of particles, which suggests a strong effect of aeolian activities on the influx of dust born microorganisms in the glacier ice.
However, the obvious transition of microbial diversity structures between the surface and subsurface snow suggests an importance of the post-deposition mechanisms on the microbial community succession in the glacier ice.Cyanobacteria were dominant across the surface snow slope in the Kuytun 51 Glacier, but rarely in the subsurface snow layers (Xiang et al., 2009b); Red Chlamydomonas were frequently observed at the pink to red surface snow, sometimes 15 cm below the snow surface in New Zealand and on the Harding icefield, Alaska (Thomas and Broady, 1997;Takeuchi et al., 2006).The visible community transitions are good indications of cold-adapted bacterial growth and colonization in the ice, and thus strengthen the important role of post-deposition on the biogeographically development of microbial communities in the glaciers.
Previous DNA sequence analysis have showed a significant difference in bacterial communities, and δ 18 O data were previously described by Tian et al. (2006).
For microbial analysis, the Muztagata ice core sections were cut into 156 samples, while the Dunde ice sections were cut into 37 in intervals of 12-30 cm using a band saw within walk-in freezers (-18 to -24° C).
The ice samples were cut between the visible dust layers, and ice layers were collected separately.The outside layers of the ice core sections were moved out, and the inner sections were slowly melted at 4C by following the protocols previously described by Yao et al. (2006).The freshly melted water (10 ml) from the Muztagata and Dunde ice cores was diluted 10 fold. 100 µl of diluted sample was added to the known concentration of fluorescent-dyed bead solution Trucount (Becton Dickinson) mixture with cell sorting marker carboxyfluorescein diacetate (cFDA) and propidium iodide (PI).Three groups of bacteria could be identified based on the difference of the bound probes: cFDA-stained, cFDA/PI-double-stained and PI-stained group, indicating viable, injured, and dead cells, respectively (Xiang et al., 2009b).The cFDA and PI staining was separately prepared by following the method of Amor et al. (2002), except for the cell suspensions which were incubated for 15 min in the dark at the room temperature (25°C) for cell staining.The live and total cell numbers in the melt-water were determined with a FACSCalibur flow cytometer (Becton Dickinson Immunocytometry Systems, San Jose, California, U.S.A.) by following manufacturer's instruction.
For DNA analysis, six clone libraries of the bacterial 16S rRNA genes were collected from the Dunde ice cap.Approximately 400 ml of ice core melt-water was used for DNA extraction.DNA extraction and further clone library establishment procedure were conducted by following the same protocols as previously used in a microbial analysis of the Kuytun 51 Glacier samples (Xiang et al., 2009b (5′-AGAGTTTGATCATGGCTCAG) and 1492R (5′-CGGTTACCTTGTTACGACTT) (Lane 1991;Weisenburg et al., 1991).To avoid possible bias, the three PCR products were pooled and used to establish clone library from each ice column.A total of 137 clones were selected for sequencing by HaeIII-based ARDRA (amplified rRNA restriction analysis) out of the 406 clones from the Dunde ice core.
Each sequence was named using the initial of Dunde ice cap (DD1, noted for one out of the 5 ice cores drilled in October 2002, Wu et al., 2009), along with the ice depth (D84, D107, D238, D324, D386, and D466: 84, 107, 238, 324, 386, and 466 cm below the snow surface) followed by the clone number (1 to 163).For example, clones DD1D84-9, DD1D107-55, and DD1D466-123 were the clone representatives of the ice core DD1 at the depth 84, 107, and 466 cm below the snow surface.The accession numbers of the cloned sequences obtained from the Dunde ice core in GenBank are: KU060881 -KU061017.
All 137 sequences from the Dunde ice cap were checked by DECIPHER (Wright et al., 2012, sequence chemera check tool available: http://decipher.cee.wisc.edu/FindChimerasOutputs.html), and aligned with the Blast references (Altschul et al., 1990) by using ClustalX (Thompson et al., 1997).A Neighbor-Joining phylogeny for the aligned sequences was constructed using MEGA 6.0 ( similarity with reported species, and thus were designated separately.

Changes in physical-chemical and biological records in the Muztagata ice cores
There was a strong influence of aeolian activities on the physical and biological records along the ice core extracted at 7010 m ASL of the Muztagata Glacier (Fig. 2).An apparent seasonal temperature change was indicated by the proxy value of the stable isotopic ratios, 18 O/ 16 O (δ 18 O) with a low value in winter and high value in summer (Fig. 2b).The live cell density was greatly variable at a range from 6.5  10 2 to 2.1  10 4 cells/ml during 1964 to 2000 (Fig. 2a), the total cell density varied from 4.4 10 4 to 8.7  10 5 cells/ml (Fig. 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 9 events, A1 to A9 (open triangles in Fig. 2a), respectively, while cell density peaks were found during the winter-spring (filled triangles in Fig. 2a).This ice core also had an increased density of the total microorganisms in summer in 1978, 1988 and 1993 (open triangles B1, B2, and B3 in Fig. 2c), and in spring of 1995 and 2000 (B4 and B5 in Fig. 2c), respectively, which was consistent with the live cell density patterns (Fig. 2a).The high microbial cell density significantly correlated with the peaks of mineral particle concentrations and possessed a high R 2 value of 0.7 (Fig. 3).
Approximately here, Fig. 2 Bacterial cell density, mineral particles and δ 18 O in the Muztagata ice core.It was not successful for the seasonal analysis of Dunde ice core because of the limitation of sample resolution (Fig. 4).Oxygen isotope ratios of the melt-water samples from the Dunde ice core showed a temperature change from -10.78‰ to -8.24‰ (temperature proxy 18 O/ 16 O, Fig. 4d), while microbial cell density varied from 1.2 10 3 to 9.1  10 4 cells/ml (Fig. 4b) and 1.3 10 5 to 1.9  10 6 cells/ml (Fig. 4c) for live and total cell density, respectively.Three peaks C2, C3 and C4 of the total cell density were found in 1988-1989, 1992, and 2000, only one peak C1 in 1985, respectively (Fig. 4c, respectively (Fig. 4c).The live cell density response pattern was consistent with the total cell density tendency (the dash lines in Figs.4b and 4c).
Abundance of microbial cells frequently occurred at the dirty ice layers (Cell density peaks C1, C3, and C4 at the dust layers labeled as the dash lines in Fig. 4), rarely presented at the clean ice layer (small density peak C2 at the A1 layer in Fig. 4).
Approximately here, Fig. 4 Bacterial cell density, mineral particles and δ 18 O in the Dunde ice core.

Phylogenetic analysis of bacterial 16S rRNA gene amplified from the Dunde ice core
The dominant bacteria in six ice layers of the Dunde ice core were investigated by 16S rRNA gene clone library, as well as sequencing techniques, and BLAST and phylogenetic tools.A total of 24 bacterial genera were identified in the Dunde ice core.

Changes in proportion of the main bacterial genera along the Dunde ice core profile
There was a great difference in proportion of the main phylogenetic groups along the Dunde

Discussion
Our previous studies have documented a zonal distribution of microbial community at the surface snow, indicating the spatial biogeography of microorganisms across the western mountain glaciers in China (Xiang et al., 2009b(Xiang et al., , 2010)).Similar zonal phenomena have also been found in the glacier depth profiles

Dust deposition and microbial distribution along the glacial depth profiles
Ice core data from the Muztagata and Dunde glacier showed a frequent association of microbial abundance with high concentrations of particles (Fig. 3, and Fig. 4), which was consistent with previous data from the Antarctic Glacier (Abysov 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 trace elements Tb, Sr, Th, and U, and rare earth elements (REE) including light REE La, Ce, Pr, Nd, Sm, and Eu, and heavy REE Gd, Tb, Dy, Ho, Er, Tm, Yb, and Lu were extracted from the same series of ice core section (Wu et al., 2009).The 4a . This indicates that microbial loading onto the glacier surface does not always associate with the dust deposits or "dirty" wind, may transport with "clean" wind or snow, which implies influences of the processes like aerosol, and snow deposition (microbial deposition with snow, wet-deposition), and post-deposition and other factors.

Changes in glacial microbial density at variable temperatures
The present data sets from the Muztagata glacier revealed clear seasonal patterns with high microbial cell density occurring in the warming spring (filled triangles in Fig. 2) and summer (open triangles Fig. 2), which indicated the positive temperature effects on the microbial density patterns.The direct evidences of positive temperature effect on the microbial growth at the snow were reported on the red Chlamydomonas growth at the surface snow in New Zealand and on the Harding icefield, Alaska in late spring and summer (Thomas and Broady, 1997;Takeuchi et al., 2006).Thus it is not surprise for the high live cell density in summer as a result of microbial growing in the surface snow.This is consistent with another independent microbial investigation on the Muztagata glacier (Liu et al., 2013).Uetake et al. (2006) 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, so did Price and Bay (2012).The positive relationship between microbial abundance and temperature was very evident in the Guoqu Glacier in the Geladaindong mountain regions (Yao et al., 2008).In addition to those cell density peaks in summer, there were many density peaks in spring from 1963-2000 (filled triangles in Figs.2a and 2c).The growth of red Chlamydomonas were also observed on the late spring snow (Thomas and Broady, 1997).So, the yearly discrete increasing pattern of microbial density presenting along the Dunde ice core profile could be

Temporal and spatial biogeography of microbial community in the glacier ice
Under the same project guideline for microorganisms in glacier ice and the relation to climatic and environmental changes, microbiological data have been collected from the ice core depth profiles from the geographically different glaciers in western China over the last decade.The same methodological system has been used for the investigation on the labelled glaciers in Figure 1, which makes it possible to explore the geographical features of microorganisms in the glacier ice across western China.We here will initially discuss the temporal and spatial biogeography of microbial community in western China.
Our previous sequence data showed a clear phylogenetic distance with only 87% similarity among the bacteria Polaromonas sp. from the different geographically glaciers (An et al., 2010), although Polaromonas sp. were identified from all of the ice layers from the glaciers Dunde, and Muztagata, and Kuytun 51, Qiangyong, and Puruogangri (An et al., 2010;Xiang et al., 2009aXiang et al., , 2009bXiang et al., , 2010)).Statistical analyses showed that the genetic distances among 43 unique glacier Polaromonas sequences were positively correlated with geographic distance among the glacier sites (Franzetti et al., 2013).6b), while Polaromonas sp., and Flexibacter sp. were found at all of three tested ice columns of Puruogangri glacier from 1600 -1920 (Zhang et al., 2009;An et al., 2010).These results clearly indicate that the biogeography of microbial communities associates with the presence/absence of several dominant genus groups within the specific glacier region.
The changing composition of dominant groups endemic to the local glacier regions could be attributed to the climatic and environmental differences in the different geographical glacier regions.As shown in Figure 1, the precipitations over the Muztagata glacier is mostly influenced by the westerly depressions, while the precipitation over the Dunde ice cap and Puruogangri ice cap is mainly driven by the westerly depressions in winter and Indian monsoon in summer (Wake et al., 1990;Davis et al., 2005;Dregne, 1968;Murakami, 1987).The dramatic change of climatic and environmental processes across the Tibetan Plateau mountainous glaciers may lead to differences in the microbial communities uploaded onto the snow.Moreover, the heterogeneity of local conditions such as temperature, light intensity, meltwater availability and nutrient concentrations in the ice may drive the temporal and spatial patterning of were frequently found at the four tested ice depths of Muztagata glacier.This study presented a discrete seasonal increase pattern of microbial cell density, and community transition of dominant endemic bacterial community among the different geographically glaciers.This strengthens the importance of post-deposition, and reinforces our hypothesis of dust-borne and post-deposition being the main agents interactively controlling microbial load in the glacier ice.The tree was generated by the Neighbour-Joining method after sequence alignment, and rooted with two Methanosaeta strains (accession no.AY817738 and NR102903).Bootstrap values (100 replications) were specified for each Node.Cut-off value for the condensed tree was 55%.Numbers of the obtained snow-ice clones (had the same ARDRA pattern to the sequenced representatives listed on the tree) and relative sequence affiliations corresponding to GenBank accession number were provided in parentheses.The sequences discussed in this study were noted bold.See a detailed description for the assigned sequence references and numbers in materials and methods.

Fig. 5b
Phylogenetic analysis of the 16S rRNA genes for the Actinobacteria, Cyanobacteria, Verrucomicrobia, and Firmicutes clones from the Dunde ice core and the closest relatives.The tree was constructed by following the protocol as described in Fig. 5a.

Fig. 5c
Phylogenetic analysis of the 16S rRNA genes for the Bacteroidetes and TM7 candidate clones from the four geographically isolated glaciers and the closest relatives.The tree was constructed by following the protocol as described in Fig. 5a.

Fig. 6
Proportion of the main phylogenetic groups in the Dunde and Muztagata ice cores.Muztagata ice core dada was adapted from our previous report (An et al., 2010).

Fig. 3
Fig. 3 Correlation between mineral particle concentration and total cell density in the Muztagata ice Fig.5aPhylogenetic analysis of the 16S rRNA genes for Alphaproteobacteria, Betaproteobacteria,

Fig. 5c
Fig. 5c Phylogenetic analysis of the 16S rRNA genes for Bacteroidetes and TM7 candidate clones

Fig. 6
Fig. 6 Proportion of the main phylogenetic groups in the Dunde and Muztagata ice cores.
from the Dunde ice core and Muztagata ice cores.However, the current data have presented a change of the dominant endemic community composition, indicating an association of the microbial spatial patterning with the presence/absence of the dominant species within the specific glaciers.The new data have also presented seasonal response patterns of cell density in the Muztagata ice core.All results reinforce the concept of interactive mechanisms, aeolian-and post-deposition of microorganisms on the glacier surface.
trace element and REE analyses revealed 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, which implies the long range of transportation of the Dunde dust and dust-borne Biogeosciences Discuss., doi:10.5194/bg-2015-637,2016 Manuscript under review for journal Biogeosciences Published: 18 January 2016 c Author(s) 2016.CC-BY 3.0 License.microorganisms from the western desert.These results suggest an aeolian driving effect on both dust and microbial deposition in the ice.One small cell density peak C2 presented at the clean ice layer A1 in Fig.
Biogeosciences Discuss., doi:10.5194/bg-2015-637,2016 Manuscript under review for journal Biogeosciences Published: 18 January 2016 c Author(s) 2016.CC-BY 3.0 License.attributed to microbial growth followed by the new snow cover during the spring-summer months.All results suggest the fundamental contribution of dry-aeolian and wet-deposition (with snow) to the basic population pool size of microorganisms, and the cascade effect of post-deposition of microorganisms by microbial growth, enhanced metabolic activity and increased population density in spring and summer.
These results indicate an evident biogeography of Polaromonas sp.Bacteria Cryobacteria more frequently presented in the Dunde ice cap than in the Muztagata glacier, while Enterobacter sp.appeared throughout the four tested ice layers of Muztagata glacier, but rarely in the Dunde ice cap (Figs 6a and 6b).The presence or absence of the dominant species indicates a clear spatial patterning of bacterial group endemic to the special glacier regions.Biogeosciences Discuss., doi:10.5194/bg-2015-637,2016 Manuscript under review for journal Biogeosciences Published: 18 January 2016 c Author(s) 2016.CC-BY 3.0 License.Several bacterial genus groups were frequently identified along the Dunde ice core depth, and became the dominant groups endemic to the local glacier regions (labeled as the dashed lines in Fig. 6b BiogeosciencesDiscuss., doi:10.5194/bg-2015Discuss., doi:10.5194/bg--637, 2016     Manuscript under review for journal Biogeosciences Published: 18 January 2016 c Author(s) 2016.CC-BY 3.0 License.microbial community by the effects on successful colonization and primary succession of the endemic species dominant in the ice.More data on the meteorologic, physical and chemical characteristics of the ice core will be helpful for better understanding the biogeography of microorganisms in the ice.5 ConclusionsPhysical-chemical and microbiological data sets from the Muztagata glacier showed a seasonal pattern during the rapidly changing temperature phases.The cell density peaks are frequently associated with high concentration of particles in the warming spring-summer.These suggest the importance of aeolian and post-deposition on the microbial upload on to the glacier snow.Sequence analyses of 16S rRNA gene clone libraries from the Dunde ice core showed an obvious difference in composition of the dominant genus groups between the two glaciers Muztagata and Dunde.Five bacterial dominant genus groups Polaromonas, Pedobacter sp., Flavobacterium sp., Propionibacterium/Blastococcus sp., and Cryobacterium sp.frequently appeared at the six tested ice layers, constituting the dominant species endemic to the Dunde ice cap, while Seven genus groups Polaromonas sp., Enterobacter sp., Acinetobacter sp., Flexibacter sp., Thermus sp., Propionibacteria/Luteococcus sp., and Flavisolibacter sp.

Fig. 1
Fig. 1 Map illustrating the location of glaciers discussed in this study.

Fig. 3
Fig. 3 Correlation between mineral particle concentration and total cell density in the Muztagata ice core.

Fig. 4
Fig. 4 Bacterial cell density, mineral particles and δ 18 O in the Dunde ice core.(a) Mineral particle