Metal-induced stress in survivor plants following the end-Permian collapse of land ecosystems

The Permian-Triassic mass extinction (PTME) had a catastrophic impact on both marine and terrestrial ecosystems and was the most severe crisis of the Phanerozoic (e.g., Wignall, 2015). The contemporaneous eruption of the Siberian Traps large igneous province is thought to have been the main trigger of the biological crisis, with the release of vast quantities of volatiles such as CO₂, SO₂, organohalogens, and metals to the atmosphere resulting in the collapse of the biosphere (Beerling et al., 2007; Bond and Wignall, 2014; Burgess et al., 2017; Broadley et al., 2018). Work in Australia and southwestern China showed that collapse of the terrestrial ecosystems occurred prior to the marine mass extinction (Fielding et al., 2019; Chu et al., 2020). The loss of terrestrial ecosystems is evidenced by the sudden disappearance of the Gigantopteris flora and coal swamps, an abrupt drop to very low total organic carbon (TOC) content of coastal sediments, and frequent wildfires in terrestrial sections of southwestern China (Chu et al., 2020; Dal Corso et al., 2020). Some vegetation survived the collapse of the Permian terrestrial ecosystems in the tropics, as seen in southwestern China, where the luxuriant Permian Gigantopteris-dominated rainforests were replaced by herblands comprising almost monospecific lycophyte populations (Feng et al., 2020).

Spore tetrads and pollen malformations have been observed globally in sediments deposited during the PTME and are commonly reported in relatively high abundances in some quantitative studies (e.g., Looy et al., 2001, 2005; Visscher et al., 2004; Hochuli et al., 2017; Fig. 1). These malformations have been attributed to the effects on plants of extreme environmental conditions (Visscher et al., 2004; Foster and Afonin, 2005; Hochuli et al., 2017; Benca et al., 2018). However, no consensus exists on the actual stressor. Increased levels of ultraviolet-B (UV-B) radiation associated with ozone-layer depletion has been invoked as a possible mutating agent (e.g., Visscher et al., 2004), but no independent evidence has yet been found. Pollution of the atmosphere by volcanic volatiles, in particular Hg, has been postulated as a possible cause of malformed plant spores (Hochuli et al., 2017). Similarly, Lindström et al. (2019) suggested that a correlation between teratological sporomorphs and Hg enrichments across the end-Triassic mass extinction indicates mutagenic effects of emissions from the Central Atlantic magmatic province. Phytotoxic volcanic gas emissions can cause abnormalities in plants and their spores or pollen, including stunted growth, lesions, malformed spores and pollen, and tetrads (e.g., Lindström et al., 2019).

A marked increase of Hg concentrations, as well as of other heavy metals like Cu, has been found in many Permian-Triassic boundary marine and terrestrial successions (Grasby et al., 2015, 2019; Shen et al., 2019; Wang et al., 2019; Chu et al., 2020). Improved chronology of the terrestrial records of the PTME (e.g., Chu et al., 2020), where floral changes are better documented, allows exploration of the cause-and-effect relationship between the collapse of ecosystems on land, the geochemical changes observed, and the occurrence of mutated spores and pollen. Hence, we investigated phytotoxic heavy metal concentrations and stress responses of terrestrial plants, specifically in the record of spore tetrad abundances, in a well-calibrated terrestrial Permian-Triassic boundary section in southwestern China.

We studied the ZK4703 borehole from the South China block (drilled 15 km south of Fuyuan at the Dahe Mine [25.54151°N, 104.28994°E]). It records paralic conditions at equatorial paleolatitudes during the Permian-Triassic transition (Fig. 1). The borehole encompasses, in ascending order, the Chang-hsingian upper Xuanwei, Permian-Triassic transitional Kayitou, and earliest Triassic Dongchuan Formations (Fig. 2). Mudrock samples were processed using standard palynological acid maceration techniques involving digestion of 40 g of mudstone in hydro-chloric and hydrofluoric acid, then dilution with ultrapure water to reach neutral pH. The residue was sieved over a 100 μm mesh screen, and both size fractions were collected. The organic and inorganic residues in the <100 μm size fractions were separated using ZnCl₂. Ten dry slides were sealed by neutral gum for each sample. To assess the percentage of the lycopsid spore tetrads (herein referred to as tetrads), all normal and tetrad specimens of spores were counted separately within the dry slides from each sample using a Zeiss Axio Scope A1 microscope until ≥100 specimens were counted or ten entire slides were scanned. Cu and Al contents in shales and mudstones were measured using an Agilent 7700e inductively coupled plasma–mass spectrometer at Wuhan Sample-Solution Analytical Technology Co., Ltd. (Wuhan, China). Data for organic δ¹³C (δ¹³C_org), TOC content, and Hg concentrations are from our previous work (Chu et al., 2020).

The palynological and geochemical data are provided in the Supplemental Material¹ and in Figure 2. Cu concentrations are plotted along with values normalized to Al, to account for potential lithologic changes, and to TOC, given that Cu bonds with organic matter in terrestrial plants and soil. Cu values are relatively constant from the Xuanwei Formation to the base of the Kayitou Formation but increase in concentration (and also Cu/Al and Cu/TOC) above this, reaching a peak Cu value of 417 ppm (Fig. 2D). Hg and Hg/TOC rise above background levels following the drop in TOC and immediately above the onset of a negative carbon isotope excursion (CIE) (Fig. 2; Chu et al., 2020). High Hg concentrations are observed in a ∼2 m interval of the lower part of Kayitou Formation where Hg increased >10× above background levels (as high as 324 ppb) (Fig. 2C). Other heavy minerals, e.g., Ni, Pb, Cr, and Zn, show very low concentrations and no changes across the section (see Table S1 in the Supplemental Material).

Temporal trends of tetrad abundances for the ZK4703 core were examined by calculating their frequency in the total number of spores. Spore tetrads are rare and their frequency never exceed 1% of all spores from the Xuanwei Formation to the base of the Kayitou Formation (Fig. 2E). A distinct peak of tetrads is then observed in the Kayitou Formation where Hg and Cu enrichments occur. At this level, as much as 19% of all spores are tetrads (Fig. 2E). The peak in tetrad abundance is precisely synchronous with the Cu peak (Figs. 2D and 2E). All the tetrads and normal spores showed the same taphonomic features and thermal alteration stage throughout the succession, indicated by the uniform phytoclast composition and palynomorph color (Fig. 3; Fig. S1; Chu et al., 2020), ruling out selective recycling. Tetrads are essentially absent below and above their interval of peak abundance. The lycopsid tetrads belong to Aratrisporites yunnanensis and Lapposisporites echinatus (e.g., Ouyang and Norris, 1999).

In southwestern China, an increase in lycopsid tetrads occurred after the collapse of the terrestrial ecosystem, which is marked by a sharp decline in TOC to almost 0%; it coincides with an interval of increased Cu and Hg concentrations and occurs within the negative CIE (Fig. 2). Correlation of the δ¹³C_org and Hg records indicates that the tetrad spike is synchronous with the marine mass extinction (Chu et al., 2020).

In both extant and fossil assemblages, malformed sporomorphs >5% of the total population indicates environmental stress (e.g., Lindström et al., 1997; Foster and Afonin, 2005), and modern studies show that an increased abundance of teratologic sporomorphs is an indicator of local environmental disturbance (e.g., Mičieta and Murín, 1996). The peak frequencies of tetrads in our study (6%–19%) therefore indicate considerable environmental stress. However, the increase in lycopsid tetrads occurs immediately after the floral extinctions and last coal beds, indicating ecological pressure was focused on the surviving lycopsids. Intriguingly, lycopsids do not display this stress indicator at the time of extinction amongst the Permian flora. Similar increases in immature tetrads of survivor lycophytes have been found in Greenland and Norway and have been explained as the possible effect of prolonged UV-B radiation on plants or atmospheric pollution by toxic metals (Visscher et al., 2004; Hochuli et al., 2017). Lycopsids were widespread during the PTME interval and are considered stress-tolerant opportunistic plants, capable of surviving the terrestrial catastrophe (e.g., Looy et al., 2001; Visscher et al., 2004). However, even these hardy plants were clearly suffering high levels of stress in the extinction aftermath.

Data normalized for TOC and Al contents show that the Hg and Cu peaks are not due to changes in organic carbon contents nor lithology but are instead the result of enhanced metal loading. To date, a sharp Hg spike has been widely observed in numerous marine PTME intervals (e.g., Grasby et al., 2015, 2019; Wang et al., 2019) as well as in some terrestrial records (Shen et al., 2019; Chu et al., 2020). The sources of Hg at the Permian-Triassic transition are multiple (e.g., Grasby et al., 2019, 2020; Dal Corso et al., 2020, and references therein). Modeling suggests oxidation of terrestrial bio-mass, and increased soil erosion related to the collapse of the terrestrial ecosystems, can explain the sharp spike in Hg, which is observed in both terrestrial and marine depositional environments, and the coeval negative Δ¹⁹⁹Hg and δ¹³C values recorded at the marine PTME stratigraphic level, while volcanism could account for a longer-term increase in Hg loading (Grasby, et al., 2020; Dal Corso et al., 2020). The release of thermogenic Hg produced from organic-rich sediments in contact with Siberian Traps intrusions could have been an additional source of Hg (Sanei et al., 2012). Cu could also have derived from multiple sources, although fewer data are available to fully understand its behavior at the Permian-Triassic boundary. Cu spikes have been observed in Spitsbergen (Norway) and have been explained as related to higher loading rates from the volcanic ash of Siberian Traps eruptions (Grasby et al., 2015). Cu is also present in high amounts in soil, and a large part of it (∼36%) is bound to organic matter (e.g., Adriano, 1986); Cu is an essential element during photosynthesis by higher plants (Nagajyoti et al., 2010). Therefore, a mechanism of Cu release from organic-matter oxidation and soil erosion, similar to that proposed for Hg (Dal Corso et al., 2020), can be also hypothesized.

The coincidence between metal enrichment and relatively abundant spore tetrads that we observe (Fig. 2) suggests that Hg and Cu may have acted as phytotoxic pollutants and represented a stressor on terrestrial ecosystems after the terrestrial mass extinction. Metal toxicity is known to induce oxidative stress by triggering the generation of reactive oxygen species (ROSs), which is linked to oxidation of proteins and membrane lipids and to alterations of DNA (e.g., Nagajyoti et al., 2010; Lindström et al., 2019). ROSs can damage the DNA strand or interact with proteins used to repair the DNA, eventually causing mutagenesis; damaged DNA can then be transferred to the next generation (e.g., Kültz, 2005). Hg is well known as one of the most toxic metals and could have caused inhibition of plant growth and photosynthesis, oxidative stress, and damage of proteins and DNA (Azevedo et al., 2018). Cu is a very important micronutrient and essential component of proteins for plants; however, excess Cu also plays a cytotoxic role (Angelé-Martínez et al., 2017; Nagajyoti et al., 2010). Just like Hg-induced genotoxicity, Cu toxicity can also generate oxidative stress with ROSs, inducing growth inhibition and lipid peroxidation (İşeri et al., 2011). We therefore propose that metal genotoxicity most likely caused the observed malformations in the survivor lycophytes at the PTME, as also invoked for the marine extinction (Grasby et al., 2015, 2020), but was not observed during the terrestrial extinction. Ozone depletion during the PTME is unproven; its evaluation awaits the development of an independent test for ancient ozone levels. However, given the global distribution of mutated sporomorphs, UV-B radiation as a mutagenic mechanism remains contentious because ozone can be rapidly replenished in the atmosphere, making global ozone destruction difficult (Black et al., 2014).

In summary, the terrestrial successions of southwestern China offer a detailed picture of terrestrial events: vegetation first entered into crisis, Permian flora declined, and wildfire activity increased (Chu et al., 2020); then, the terrestrial ecosystems suddenly collapsed (at the level of the onset of the marine PTME), as evidenced by the disappearance of the Gigantopteris forests and coal deposits, causing sediment TOC to fall to ∼0%. This was immediately followed by increased Hg and Cu loading, causing a major increase in the abundance of undeveloped spore tetrads (Fig. 2). The reason for the extinction of Gigantopteris flora remains unknown because sporomorphs' mutagenesis, in southwestern China, was only observed in the survivor lycophytes, and no or <1% unseparated spore tetrads are found before this interval, when the decline of the Permian forests and increased wildfire activity are recorded (Fig. 2).

The record of spore tetrad abundances and metal concentrations during the Permian-Triassic mass extinction in a well-preserved core (ZK4703) encompassing the terrestrial record of southwestern China provides insight into the terrestrial ecosystem collapse. A significant peak of lycophyte tetrads occurs in the immediate aftermath of a sharp TOC drop, which is associated with the loss of the Permian Gigantopteris flora and coal swamps. Both Hg and Cu concentrations show a large peak during the tetrad acme. The close temporal relationship between extinction, metal concentrations, and spore teratology suggests that survivor lycophyte plants were under huge stress after the loss of the Permian Gigantopteris forests. The mutagenesis of the reproductive organs was likely driven by environmental pollution linked to a sudden rise of Hg and Cu loading into the system that followed. Importantly, this toxic stress postdates the terrestrial extinction and indicates some other, as yet undiagnosed, mechanism caused the losses.

We are grateful to reviewers D. Bond, S. Lindström, and C. Fielding for their constructive comments. This study was supported by the National Natural Science Foundation of China (grants 42072025 and 41821001), and the UK Natural Environment Research Council's Eco-PT project (grant NE/P01377224/1), which is a part of the Biosphere Evolution, Transitions and Resilience (BETR) Program.