Methodological/ethical considerations
We controlled for environmental contamination by rinsing nodules and including leaf controls, which presumably were exposed to the same sources of environmental contamination. However, post-harvest plants have significantly different microbiomes than actively growing plants both in taxonomic diversity and function. This “post-mortem” microbiome was found to be a significant portion of the microbes identified on herbarium specimens (Alternaria alternata comprised up to 7% of the total reads, Bieker et al. 2020). However, authors found that non-bacterial microbes were the largest contributors of the leaf post-mortem microbiome (Bieker et al. 2020). Potentially, some of the microbes that we cultured are constituents of Medicago’spost-mortem microbiome. We did isolate genera that are known constituents of the Medicago nodule microbiome includingMicromonospora and Paenibaccillus (Martínez-Hidalgo et al. 2014, Lai et al. 2015, Hansen et al. 2020). Further work should compare historic and contemporary nodule microbiomes of Medicago lupulina to determine occurrence of taxa found in our collection in contemporary populations.
General ethical issues that occur when using herbarium specimens are also relevant for type of work. We took a highly conservative sampling approach because our approach requires destructively sampling specimens. We chose a common species that is abundant in herbaria across the country. It would be impractical and unethical to work with rare specimens that occur sporadically in herbaria. Fortunately, with the digitization of collections all over the country and world access to suitable specimens this kind of work is increasingly possible. Finally, it is important to acknowledge that herbaria, like any biological collection, benefitted from the period of overt colonialism spanning the 15th to mid-20th centuries. The majority of plant specimens are held in the global north, despite being collected globally, a pattern that has created an inverse relationship between global biodiversity and where collections are housed (Park et al. 2023). This trend in collections highlights not only a trend for the colonizing countries to exploit foreign resources, but also wrests the control of knowledge and biological resources away from countries of origin, depriving resident explorers/scientists/researchers from local resources and discoveries.
CONCLUSIONS : Herbarium collections contain specimens that represent taxa from a wide range of locations collected over a timeframe that is well beyond that of modern research. Our work demonstrates that not only are herbarium specimens a source of genetic/genomic/metagenomic information about plant species and their associated microorganisms, but they are also the source of historic microbes. While microbial diversity has been examined extensively, microbial function is only now being explored. A source of data for understanding microbiome function are growing culture collections. Enhancing culture collections through herbarium collections will not only expand the source of culture collection material but might help identify novel functions that are relevant to pressing problems such as climate change (Rudgers et al. 2020). Additionally, herbarium microbial culture collections will allow us to test hypotheses about plant-microbe interactions over larger temporal and spatial scales. For example, previous work has suggested that increasing soil nitrogen availability could cause the erosion of interactions between nitrogen fixing microbial bacteria and plant hosts, Although some support exists for this idea (Weese et al. 2015) , herbarium cultures could further the temporal window to test these ideas.