Culture collection-
To confirm that our isolates cultured from fresh and historic nodules were not the result of contamination from the herbarium or the lab, we compared our media blanks and herbarium leaf controls to growth on plates that were streaked with either fresh or historic crushed nodules. We didn’t find any growth on our negative controls (Fig. 2A) or historic leaf samples (Fig. 2B), compared to ~ 50% probability of growth on plates streaked with nodules from historic and contemporary sources (X2 =177.6, df=2,P >0.0001). The probability of growth was almost three times higher for contemporary nodules compared to historic nodules (contemporary probability= 0.968, historic probability= 0.359,X2 =82.82, df=1, P >0.0001, Fig. 2). We also found that collection year (historic nodules only) and media type interacted to determine the probability of growth (X2 =21.17, df=12, P =0.04795, Fig. 2D, G, H). Unfortunately, due to small samples for each media type, planned contrasts could not detect differences between media types, however it seems that isolates from 2004 did markedly worse on LB media, whereas those from 1950 had a slight preference for tryptone yeast.