We investigated Zn and Cd tolerance in North American S. brevipes and S. tomentosus and found pronounced inter- and intraspecific metal tolerance variation. S. brevipes was overall more tolerant to Zn, while S. tomentosus was more tolerant to Cd, even though isolates from both species showed a wide range of Cd and Zn tolerance. Surprisingly, we did not find a correlation between soil contamination and isolate metal tolerance or sporocarp metal accumulation.
Our findings of inter- and intraspecific variation in metal tolerance are consistent with previous work on Suillus and highlight the diversity of metal tolerance within the genus. Suillus is known to be tolerant to Zn and Cd and to show marked differences across species and isolates. For example, S. bovinus displays Zn tolerance intraspecific variation (Colpaert and Vanassche 1992), while S. luteus, S. granulatus and S. variegatus isolates show differences in Zn and Cd tolerance (S. granulatus is more Cd tolerant, and S. variegatus is more Zn tolerant ; (Colpaert et al. 2004; Blaudez et al. 2000; Hartley et al. 1997). Our results expand the already documented widespread Suillus metal tolerance variation to American species. Geography aside, there are consistent metal tolerance differences across and within Suillus species, with closely related isolates showing very different abilities to withstand metal toxicity.
Previous research in Belgian S. luteus found that isolates from geographically distinct but relatively close sites (~ 20 km apart) display marked differences in their ability to tolerate soil metal contamination, generally correlating with metal contamination at the site of origin (Colpaert et al. 2000). Interestingly, even though these tolerant and sensitive isolates show genetic differentiation at genes involved in metal homeostasis, whole genome analyses indicated that they all belong to the same population, with differences in metal tolerance thought to be a result of standing genetic variation (Bazzicalupo et al. 2020). On top of genomic variation, transcriptomic control of tolerance mechanisms such as efflux pumps, chelators, and ROS detoxification systems seems to play an important role in S. luteus Zn tolerance. Specifically, metal tolerant isolates show very different gene expression patterns compared to sensitive isolates and are likely constitutively metal tolerant because of differential gene expression (Smith et al. 2023). It is yet unclear whether the Suillus isolates from Colorado follow a similar pattern of population structure, with one population of S. brevipes and another of S. tomentosus including sensitive and tolerant isolates. Further research will shed light on this possibility.
Our Colorado isolates appear to be much less tolerant to Cd and Zn than other species in the genus. Specifically, the highest detected S. brevipes and S. tomentosus Cd EC50 values are less than half of those seen in S. luteus from Europe (Krznaric et al. 2009; Colpaert et al. 2000). In addition, the EC50 values of our most Cd tolerant isolates match those of the most sensitive European isolates. Furthermore, our S. brevipes and S. tomentosus Zn sensitive isolates are much more sensitive to Zn than Zn sensitive European Suillus isolates (Colpaert et al. 2000). These findings corroborate the differences across the genus Suillus, with some species showing much higher tolerance than others. These differences can stem from distinct soil contamination levels, as the Colorado sites display much lower Zn contamination levels compared to previously studied European sites (our most contaminated site contained 260 ppm Zn compared to up to 1750 ppm in Belgium (Colpaert et al. 2000)). However low soil contamination does not explain the differences in Cd tolerance, as our sites were much more heavily contaminated with Cd (our most contaminated site had 45 ppm Cd, compared to 14 ppm in Belgium (Colpaert et al. 2000)).
We were surprised by the lack of correlation between soil contamination and isolate metal tolerance. This comes in contrast with other Suillus studies, that document the majority of isolates from metal-contaminated sites showing higher metal tolerance (Colpaert et al. 2004; Colpaert et al. 2000; Colpaert and Vanassche 1992; Colpaert and van Assche 1987). However, these reports also detected metal sensitive Suillus isolates inhabiting contaminated soils, as was seen in our data, possibly due to soil heterogeneity and the potential existence of pockets with little to no contamination in polluted sites that allow metal sensitive isolates to persist (Fomina et al. 2005; Colpaert et al. 2004; Blaudez et al. 2000; Adriaensen et al. 2005). In addition, metal tolerance has been suggested to have little to no fitness costs in uncontaminated environments, which could explain the presence of tolerant isolates in non-contaminated soils (Bazzicalupo et al. 2020; Colpaert et al. 2004).
We found Zn and Cd tolerance to be positively correlated in S. brevipes and negatively correlated not in S. tomentosus. Neither of these correlations were statistically significant, but tests returned p values of 0.05 and 0.053 respectively. Even if not strictly significant, the positive correlation between Zn and Cd tolerance detected in S. brevipes is a novel finding that warrants further investigation. Previous research showed no correlation between Zn and Cd tolerance in S. luteus (Colpaert et al. 2000). One possible explanation is that tolerance to the two metals in S. brevipes is achieved through mechanisms involving the same genes and pathways and/or that the genes involved in tolerance to each metal are linked and inherited together. Follow up genomic and genetic investigations will contribute to clarify this pattern.
We found that faster growing S. tomentosus isolates are more sensitive to Cd toxicity. This suggests that cellular metal load may be a limiting factor for tolerance. It is likely that in the absence of effective exclusion mechanisms, isolates growing more quickly may be taking up more metal from the environment, increasing cell toxicity as Cd serves no known nutritional function and is toxic at low levels (De Oliveira and Tibbett 2018).
We found no correlation between sporocarp metal content across species, tolerance level, or soil contamination. In S. luteus, sporocarp Zn is also not correlated with soil Zn contamination, but sporocarp Cd matches soil Cd levels (Colpaert et al. 2000). This indicates that while there may be differences in mycelial tolerance strategies across species and metals, the transport of Zn into the sporocarps seems to be tightly regulated internally. However, we found that S. tomentosus sporocarps contained significantly more Cd than S. brevipes sporocarps, showing interspecific variation in the ability to either control Cd uptake from the soil, or in the ability to sequester and manipulate Cd mobility intracellularly. Potential reasons for these differences include the presence and high efficiency of Cd efflux pumps or more stringent uptake mechanisms. Again, deeper investigation into the genomics, transcriptomics and internal metal mobility of each species could help us understand the basis for these interspecific differences in metal tolerance.
When we examined mycelium metal uptake, we found that, for Zn, there was a negative correlation between uptake and tolerance for both S. brevipes and S. tomentosus, however we did not see any correlation for Cd uptake. The Zn trend appears to match previously documented results in S. luteus, where it was seen that tolerant isolates take up less metal than sensitive isolates when grown on the same concentration of metal (Colpaert et al. 2005). In the case of Cd, however, the lack of correlation between tolerance and uptake in both S. brevipes and S. tomentosus appears to be a novel finding, as previous research in S. luteus demonstrated that Cd tolerant isolates take up less Cd than sensitive isolates (Krznaric et al. 2009).
It is likely that S. brevipes and S. tomentosus rely on distinct mechanisms to achieve Cd tolerance compared to S. luteus, perhaps depending more on internal metal sequestration and/or detoxification instead of exclusion to persist in high metal environments. It is also possible that Cd tolerant S. brevipes and S. tomentosus have efficient extra-cellular chelation, rendering metals immobilized in the outer cell wall. Given we used ICP-MS to quantify the total mycelial metal content, we are not able to assess whether Zn and Cd are sequestrated within the cell or on the cell wall. Further work focusing on metal cell localization in S. tomentosus and S. brevipes is needed to explain our results.
In summary, our investigation into Zn and Cd tolerance in North American Suillus brevipes and S. tomentosus unveils substantial diversity in metal tolerance within the genus. The observed inter- and intraspecific metal tolerance variation, coupled with comparisons to the better studied Suillus luteus, emphasize the complexity of metal stress responses and indicate the existence of diverse metal tolerance mechanisms within the genus. The lack of relationship between site contamination and metal tolerance challenges previous ideas of environmental adaptation at the population level and warrants deeper investigation. The possible correlation between Zn and Cd tolerance in S. brevipes hints at the existence of shared genetic pathways for tolerance to multiple metals, while the lack of correlation in S. tomentosus highlights the possibility of a divergence of pathways in this organism. Moving forward, exploring the genetic basis of these correlations and alternative tolerance strategies, such as internal metal sequestration and metal detoxification, will be essential. These insights contribute not only to our understanding of Suillus ecology but also have broader implications for predicting fungal contributions to forest ecosystems, especially in metal contaminated sites in North America.