Despite the presence of well-documented changes in vegetation and faunal communities at the Pleistocene-Holocene transition, it is unclear whether similar shifts occurred in soil microbes. Recent studies do not show a clear connection between soil parameters and community structure, suggesting permafrost microbiome-climate studies may be unreliable. However, the majority of the permafrost microbial ecological studies have been performed only in either Holocene-or Pleistocene-aged sediments and not on permafrost that formed across the dramatic ecosystem reorganization at the Pleistocene-Holocene transition. In our study, we used permafrost recovered in proximity to the Pleistocene-Holocene transition subsampled under strict sterile conditions developed for ancient DNA studies. Our ordination analyses of microbial community composition based on 16S RNA genes and chemical composition of the soil samples resulted into two distinct clusters based on whether they were of late Pleistocene or Holocene age, while samples within an epoch were more similar than those across the boundary and did not result in age based separation. Between epochs, there was a statistically significant correlation between changes in OTU composition and soil chemical properties, but only Ca and Mn were correlated to OTU composition within Holocene aged samples; furthermore, no chemical parameters were correlated to OTU composition within Pleistocene aged samples. Thus, the results indicate that both soil chemical and microbial parameters are fairly stable until a threshold, driven by climate change in our study, is crossed, after which there is a shift to a new steady state. Modern anthropogenic climate change may lead to similar transitions in state for soil biogeochemical systems and microbial communities in Arctic regions.

25This study aims to act as a methodological guide for contamination monitoring, 26 decontamination, and DNA extraction for peaty and silty permafrost samples with low 27 biomass or difficult to extract DNA. We applied a biological tracer, either only in the 28 field or both in the field and in the lab, via either spraying or painting. Spraying in the 29 field followed by painting in the lab resulted in a uniform layer of the tracer on the core 30 sections. A combination of bleaching, washing, and scraping resulted in complete 31 removal of the tracer leaving sufficient material for DNA extraction, while other widely 32 used decontamination methods did not remove all detectable tracer. In addition, of four 33 widely used commercially available DNA extraction kits, only a modified 34ZymoBIOMICS TM DNA Microprep kit was able to acquire PCR amplifiable DNA. 35Permafrost chemical parameters, age, and soil texture did not have an effect on 36 decontamination efficacy; however, the permafrost type did influence DNA extraction. 37Based on these findings, we developed recommendations for permafrost microbiologists 38 to acquire contaminant-free DNA from permafrost with low biomass. 39 IMPORTANCE: 40Permafrost has the capacity to preserve microbial and non-microbial genomic material for 41 millennia; however, major challenges are associated with permafrost samples, including 42 decontamination of samples and acquiring pure DNA. Contamination of samples during 43 coring and post coring handling and processing could affect downstream analyses and 44 interpretations. Despite the use of multiple different decontamination and DNA extraction 45 methods in studies of permafrost, the efficacy of these methods is not well known. We 46 used a biological tracer to test the efficacy of previously published decontamination 47 methods, as well as a bleach-based method we devised, on two chemically and 48 structurally different permafrost core sections. Our method was the only one that 49 removed all detectable tracer. In addition, we tested multiple DNA extraction kits and 50 modified one that is able to acquire pure, PCR amplifiable DNA from silty, and to some 51 extent from peaty, permafrost samples. 52 53 54 55

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Arbuscular mycorrhizal fungal diversity can be altered by intercropping plant species, as well as N fertilizer applications. This study examined the effects of oat-pea intercropping and N fertilizer addition on the richness and diversity of mycorrhizal species, as well as identified the most common arbuscular mycorrhizal fungi (AMF) genera recruited for oats and peas in two growing seasons (2019 and 2020). The AMF diversity was higher in an intercropped system compared to their respective monocropping system. Under drier conditions in 2019, arbuscular mycorrhizal richness decreased with N fertilizer addition in sole peas and increased with N fertilizer addition in sole oats, but no significant change in richness was observed in oat-pea intercropping. During the wetter growing season 2020, arbuscular mycorrhizal diversity increased when oat and pea were intercropped, compared to either sole oat or sole pea. Diversispora in sole pea was a significant indicator differentiating the root associated AMF community from sole oat. Claroideoglomus richness increased in peas in 2020, thus this genus could be moisture dependent. Paraglomus richness in oat-pea intercropping was similar to sole oat in 2019, and similar to sole pea in 2020. This can suggest that Paraglomus is an indicator of plant stress under intercropping, as based on the premise that stressed plants release more exudates, and the subsequent mycorrhizal associations favor these plants with higher exudation. Future investigations can further reveal the functions and benefits of these mycorrhizal genera in annual monocrop and intercropping systems.

this study aims to act as a methodological guide for contamination monitoring, decontamination, and DNA extraction for peaty and silty permafrost samples with low biomass or difficult to extract DNA. We applied a biological tracer, either only in the field or both in the field and in the lab, via either spraying or painting. Spraying in the field followed by painting in the lab resulted in a uniform layer of the tracer on the core sections. A combination of bleaching, washing, and scraping resulted in complete removal of the tracer leaving sufficient material for DNA extraction, while other widely used decontamination methods did not remove all detectable tracer. in addition, of four widely used commercially available DNA extraction kits, only a modified ZymoBIOMICS DNA Microprep kit was able to acquire PCR amplifiable DNA. Permafrost chemical parameters, age, and soil texture did not have an effect on decontamination efficacy; however, the permafrost type did influence DNA extraction. Based on these findings, we developed recommendations for permafrost researchers to acquire contaminant-free DnA from permafrost with low biomass. Permafrost, i.e. Earth materials below 0 °C for at least two years and up to millions of years, acts as an archive of past environments and ecosystems, preserving biological material as a result of its isolation from atmospheric inputs, low temperatures, and low water activity 1. Ancient DNA derived from long-dead organisms is an important example of such material and has been used for a variety of purposes, ranging from reconstructing human migration patterns to reconstituting the genomes of extinct organisms such as the woolly mammoth and North American horses 2-6. Furthermore, permafrost-dwelling microbes may also play important roles in carbon cycling by conversion of permafrost organic carbon to methane and carbon dioxide, both important greenhouse gasses 7-10. The use of high-throughput sequencing technologies has enriched our understanding of microbial communities in permafrost and ancient DNA. However, these technologies require the extraction of high yields of DNA devoid of contaminants 11. External contamination is particularly problematic in DNA-based approaches due to the high sensitivity in detecting, amplifying and sequencing of DNA. The nature of these contaminants are primarily DNA from humans and background or exogenous DNA of microorganisms 12,13. Obtaining DNA devoid of contaminants from environmental samples, especially from those with low biomass such as permafrost, is often challenging. Such samples are prone to external contamination during drilling and collection in the field and handling in the laboratory, which could lead to misinterpretation of microbial diversity, activity, or ancient DNA studies 14-16. Several methods have been used for permafrost decontamination, such as scraping the outer surface of cores, fracturing of cores followed by clean subsampling from the interior of the core sections (i.e. "disk sampling"), or washing the cores with DNase (e.g. 17...