Table 2. Overview of the research questions and general approaches in the study of rare microbes.

	Research questions	Method	Critical issues	Options	References
Synthetic communities
	Order of arrival	Vary order of arrival to test priority effects	Cultivation dependent	Selective/spatially structured media	Fukami and Morin, 2003
	Density-dependent effects	Vary abundances to test effects of rare species	Cultivation dependent	Cell separation via microfluidic or flow cytometry	Zhang et al., 2009
Manipulation of natural communities
Removal	Consequence of rare species loss	Dilution-to-extinction	Equal biomass in all treatments	Incubation period for recovery of biomass	Philippot et al., 2013; Mallon et al., 2015; Hol et al., 2015a,2015b; Delgado-Baquerizo et al., 2016
Enrichment	Responders to changing conditions	Salinity, dry–rewet, predation, pollution, nutrient amendments	Molecular methods for composition (DNA) and activity (RNA)	DNA normalization; improve coverage rare biosphere via single-cell genomics	Giebler et al., 2013; Aanderud et al., 2015
In situ
	Microbial population dynamics	Time series	Availability of data sets	Increase sampling	Shade et al., 2014
	Genome recovery of rare species; predict metabolic pathways	Single-cell genomics	Selection of target	Labeling via FISH	Podar et al., 2007; Freilich et al., 2011
	Function of rare species	SIP; Nano-SIMS	Low throughput	Combine with single-cell analysis	Musat et al., 2008; Pester et al., 2010

Abbreviations: FISH, fluorescence in situ hybridization; SIMS, secondary ion mass spectrometry; SIP, stable isotope probing.