Effects of point-source PCB contamination on breeding performance and post-fledging survival in the dipper Cinclus cinclus

Despite widespread information on the incidence and biochemical effects of polychlorinated biphenyls (PCBs) in birds, field studies of effects on population processes are still scarce. This is particularly so in passerines. We therefore assessed breeding performance and post-fledging survival in dippers, Cinclus cinclus, breeding along the Afon Mule, a Welsh river where previous work indicated locally elevated PCB concentrations. We hypothesised that marked toxicological effects, including endocrine disruption, would be detectable as altered clutch-size, hatching success, nest success, brood-size, post-fledging survival or recruitment to the breeding population. Congener-specific analysis of PCBs in failed and deserted dipper eggs during 1990–93 revealed that geometric mean concentrations were four to 20 times higher along the Mule than elsewhere in Wales, UK. PCB signatures also varied highly significantly from other locations, with congeners 138, 153 and 180 dominant at the contaminated site, but 118 or 170 more frequent in eggs elsewhere. Subsequent analysis of surface runoff confirmed a small breaker's yard as a point source. Neither laying dates, clutch-sizes, brood-sizes, nest failure nor hatching failure along the contaminated Mule (n=46 breeding attempts and 218 eggs) differed significantly from dippers along adjacent reference rivers (n=82 breeding attempts and 315 eggs) or Welsh rivers as a whole (n=332 breeding attempts and 1534 eggs). Subsequent recaptures of breeding dippers ringed initially as nestlings were nearly identical between the Mule (7.7% of 182 nestlings), the reference set (7.5% of 323 nestlings) and all other Welsh rivers (7.3% of 2821 nestlings). These data illustrate the value of congener-specific analysis of bird's eggs in indicating local PCB sources. The data show also that PCBs can occur in dipper eggs at total concentrations of 0.49 (geometric mean)–1.29 (upper quartile) μg g⁻¹ wet mass without effects on breeding performance and survival. These values are below those at which biochemical or reproductive effects on other passerines have been detected, but above current concentrations in any other population of European dippers for which PCB data are available. Population effects by PCBs on European dippers are therefore unlikely. Survivors from locally contaminated sites like that in our study might allow field assessments of second-generation effects on breeding performance in wild birds that have developed under PCB exposure.     

Egg mass and shell thickness in dippers Cinclus cinclus in relation to stream acidity in Wales and Scotland

The shell thickness and mass of eggs of the dipper (Cinclus cinclus) collected on streams of different pH in Wales and Scotland were measured. The aluminium, phosphorus and calcium content of their invertebrate prey were also measured. In a regression analysis, significant at p<0.05, stream pH accounted for up to 7% of the variance in shell thickness, with shells decreasing by 2.5% of the overall mean with each unit of pH decline. In the Welsh sample, differences in shell thickness due to pH were small compared with differences between years across all sites. In data pooled between Scotland and Wales, pH accounted for 17% of the variance in egg mass, but a greater proportion (25%) in Scottish eggs alone. Aluminium concentrations in invertebrates showed no relationship with stream pH, but calcium levels in two insect orders increased significantly with pH. Calcium rich prey, such as Gammarus, were found only in circumneutral streams. The importance of calcium in the diet of dippers before and during egg formation is discussed. No evidence that aluminium in prey adversely affected dipper eggs was found.     

Relationships between stable isotopes and metal contaminants in feathers are spurious and biologically uninformative

Stable isotopes of carbon (δ¹³C) and nitrogen (δ¹⁵N) are used frequently in ecotoxicological investigations of birds to relate contaminant levels to trophic position (δ¹⁵N) or foraging location (δ¹³C) and many researchers using avian feathers in such investigations use δ¹³C or δ¹⁵N as a predictor of contaminant concentrations. Contaminants, especially mercury, however, are integrated into feathers over different time periods than are stable isotopes, resulting in spurious relationships that have no biological meaning. I show the fundamental principles behind the conclusion that relating δ¹³C or δ¹⁵N to contaminant concentrations in feathers is not appropriate in light of the number of recent studies that have employed this approach, and make recommendations for those wishing to investigate the relationship between contaminants and stable isotope ratios.     

Long-range air pollution and its impact on heavy metal accumulation in dippers Cinclus cinclus in Norway

Heavy metals such as cadmium, lead and zinc are deposited in Norway as long-range transported air pollution, while copper deposition is of mainly local origin. This study examined whether the deposition influences the metal concentrations in dippers Cinclus cinclus. The lead concentration in dippers reflected the deposition pattern of long-range air pollution, with higher concentrations in Southern Norway than Central Norway and a significant decrease (>/=40%) between 1987 and 1994. For cadmium, copper and zinc there were no geographical or temporal variations in metal concentrations. For zinc, this lack of geographic and temporal variation probably reflects the fact that it is a homeostatically regulated metal. For both cadmium and copper, local sources are probably more important than long-range sources. Zinc and copper was at higher level in livers of females than in males during the egg laying period. The concentrations of zinc and copper in livers were positively correlated.     

Effects of point-source PCB contamination on breeding performance and post-fledging survival in the dipper Cinclus cinclus

Despite widespread information on the incidence and biochemical effects of polychlorinated biphenyls (PCBs) in birds, field studies of effects on population processes are still scarce. This is particularly so in passerines. We therefore assessed breeding performance and post-fledging survival in dippers, Cinclus cinclus, breeding along the Afon Mule, a Welsh river where previous work indicated locally elevated PCB concentrations. We hypothesised that marked toxicological effects, including endocrine disruption, would be detectable as altered clutch-size, hatching success, nest success, brood-size, post-fledging survival or recruitment to the breeding population. Congener-specific analysis of PCBs in failed and deserted dipper eggs during 1990-93 revealed that geometric mean concentrations were four to 20 times higher along the Mule than elsewhere in Wales, UK. PCB signatures also varied highly significantly from other locations, with congeners 138, 153 and 180 dominant at the contaminated site, but 118 or 170 more frequent in eggs elsewhere. Subsequent analysis of surface runoff confirmed a small breaker's yard as a point source. Neither laying dates, clutch-sizes, brood-sizes, nest failure nor hatching failure along the contaminated Mule (n=46 breeding attempts and 218 eggs) differed significantly from dippers along adjacent reference rivers (n=82 breeding attempts and 315 eggs) or Welsh rivers as a whole (n=332 breeding attempts and 1534 eggs). Subsequent recaptures of breeding dippers ringed initially as nestlings were nearly identical between the Mule (7.7% of 182 nestlings), the reference set (7.5% of 323 nestlings) and all other Welsh rivers (7.3% of 2821 nestlings). These data illustrate the value of congener-specific analysis of bird's eggs in indicating local PCB sources. The data show also that PCBs can occur in dipper eggs at total concentrations of 0.49 (geometric mean)-1.29 (upper quartile) microg g(-1) wet mass without effects on breeding performance and survival. These values are below those at which biochemical or reproductive effects on other passerines have been detected, but above current concentrations in any other population of European dippers for which PCB data are available. Population effects by PCBs on European dippers are therefore unlikely. Survivors from locally contaminated sites like that in our study might allow field assessments of second-generation effects on breeding performance in wild birds that have developed under PCB exposure.     

A review of the likely causal pathways relating the reduced density of breeding dippers Cinclus cinclus to the acidification of upland streams

Previous work has shown that the breeding density of a bird characteristic of upland streams, the dipper Cinclus cinclus, is markedly reduced at low pH in both Wales and Scotland. Populations also declined when streams became more acidic. Evidence of causal explanation for these relationships is that: (1) Food quantity is reduced in acidic streams, and important prey, including those rich in calcium, are scarce; (2) Blood chemistry in pre-breeding birds differs between acid and circumneutral streams, with plasma calcium reduced in those breeding at low pH. Skeletal sources of calcium are probably limited; (3) The time spent foraging by pre-breeding birds on acidic streams is markedly increased, even though overall energy costs on acidic and circumneutral streams are similar. Body condition is inferior to birds on circumneutral streams; (4) Egg laying is significantly delayed on acidic streams irrespective of an effect on laying of altitude, and clutch and brood sizes are significantly reduced; (5) Eggs are lighter and shells thinner at low pH; (6) Chick growth is reduced at low pH; (7) Contamination by heavy metals and persistent organochlorines is low in the populations on acidic streams and cannot explain the impaired breeding performance. None of these features can exclude the possibilities that acidic streams either hold populations of poor quality birds, which show the above features, or that acidity affects the breeding ecology of all dippers that attempt to breed at low pH. The qualitative outcome of these two alternatives is identical.     

Element patterns in albatrosses and petrels: Influence of trophic position, foraging range, and prey type

We investigated the concentrations of 22 essential and non-essential elements among a community of Procellariiformes (and their prey) to identify the extent to which trophic position and foraging range governed element accumulation. Stable isotope analysis (SIA) was used to characterise trophic (δ¹⁵N) and spatial patterns (δ¹³C) among species. Few consistent patterns were observed in element distributions among species and diet appeared to be highly influential in some instances. Arsenic levels in seabird red blood cells correlated with δ¹⁵N and δ¹³C, demonstrating the importance of trophic position and foraging range for arsenic distribution. Arsenic concentrations in prey varied significantly across taxa, and in the strength of association with δ¹⁵N values (trophic level). In most instances, element patterns in Procellariiformes showed the clearest separation among species, indicating that a combination of prey selection and other complex species-specific characteristics (e.g. moult patterns) were generally more important determining factors than trophic level per se.     

Organochlorines, brominated flame retardants and mercury levels in six seabird species from the Gulf of St. Lawrence (Canada): Relationships with feeding ecology, migration and molt

Concentrations of organochlorines (OCs), brominated flame retardants (BFRs) and mercury (Hg) were measured in eggs of six seabird species breeding in the Gulf of St. Lawrence, Canada. Stable nitrogen (δ¹⁵N) and carbon (δ¹³C) isotopes were used as ecological tracers to measure trophic level and connectivity with benthos, respectively. Concentrations, patterns as well as ecological tracers varied significantly between species. The sum of polychlorinated biphenyls (ΣPCBs) was the most important group measured in all seabird species based on concentration followed generally by the sum of chlorinated pesticides (ΣCPs), the sum of brominated flame retardants (ΣBFRs) and finally total Hg (THg). ΣPCBs, ΣCPs and ΣBFRs increased with trophic level, whereas THg did not. Only ΣBFRs increased with a higher connectivity with the benthos. Seabird species resident to the Great Lakes-St. Lawrence ecosystem showed higher Hg and BFR levels than migratory species. Molt patterns were used to explain variations of contaminant levels.     

Tissue-specific accumulation and lactational transfer of polychlorinated biphenyls, chlorinated pesticides, and brominated flame retardants in hooded seals (Cistophora cristata) from the Gulf of St. Lawrence: applications for monitoring

Accumulation and mother-pup transfer of halogenated organic contaminants was studied in hooded seal tissues from eastern Canada. Blubber polychlorinated biphenyl (PCB) and total pesticide concentrations were relatively high, possibly due to their high trophic level and demersal feeding habits. Blood plasma showed the lowest contaminant concentrations compared to blubber and liver, possibly due to a lower affinity of these compounds to lipoproteins in blood plasma. Total contaminant body burden correlated well with blubber, liver, and milk contaminants, but not with blood plasma contaminants, indicating that blood plasma might be less suitable to monitor contaminants in hooded seals. Lactational transfer favored less lipophilic contaminants and was associated with relatively high blood plasma PCB and polybrominated diphenyl ether concentrations in females. Despite lactational transfer, females did not show significantly lower blubber contaminant concentrations or burdens than males. This might be caused by their low blubber, and thus contaminant, loss during lactation compared to other species.     

Trophodynamics and distribution of silver in a Patagonia mountain lake

Silver (Ag) ions are among the most toxic metallic ions to aquatic biota. In southern Argentina, fish from Patagonian lakes have liver Ag concentrations [Ag] among the highest ever reported globally. Silver concentration in phytoplankton from Lake Moreno (1.82 ± 3.00 μg g⁻¹ dry weight, DW) was found to be significantly higher than [Ag] in zooplankton (0.25 ± 0.13 μg g⁻¹). Values in snails and decapods (0.60 ± 0.28 μg g⁻¹ and 0.47 ± 0.03 μg g⁻¹ respectively), were higher than in insect larvae (0.28 ± 0.39 μg g⁻¹ for Trichoptera). We examined trophic transfer of Ag in the biota using stable nitrogen and carbon isotopes ratios (δ¹⁵N and δ¹³C respectively). Silver concentrations in the biota of Lake Moreno were not associated with any particular C source, as assessed by δ¹³C. Hepatic [Ag] significantly increased with trophic position, as measured by δ¹⁵N, within the brook trout sample set. Biodilution of Ag was observed between primary producers and small forage fish when whole body [Ag] was analyzed. Nevertheless, when considering whole food web biomagnification and hepatic [Ag] of top predator fish, a significant positive regression was found between [Ag] and trophic position, as measured by δ¹⁵N. The importance of species-specific and tissue-specific considerations to obtain more information on Ag trophodynamics than that usually presented in the literature is shown. To the best of our knowledge, this is the first study in assessing Ag trophodynamics and tissue-specific biomagnification in a whole freshwater food web.     

Trace elements and stable isotopes (delta13C and delta15N) in shallow and deep-water organisms from the East China Sea

Trace elements (22) and stable isotope ratios (δ¹⁵N and δ¹³C) were analyzed in marine organisms from shallow (SW) and deep-water (DW) of the East China Sea to understand biomagnification and prey source of trace elements. In the benthic marine organisms from DW, δ¹⁵N values were negatively correlated with Ba, Cu, Ag, Mo, Sr, As, and Co concentrations. This may be due to the specific accumulation in lower trophic animals and/or the biodilution through the food web in DW. Relationships between δ¹⁵N and concentrations of Co, Cr, Bi, and Tl in fish and Ag, Bi, V, Hg, and Tl in crustaceans showed positive correlations, suggesting that trophic position was affecting the concentrations of those elements in phyla, with higher trophic animals retaining higher concentrations than the lower trophic animals. Positive correlations between δ¹³C and Rb were observed in marine organisms. Therefore, Rb may be a possible substitute of δ¹³C as tracer of prey source in the East China Sea although further investigation is required.     

Below-ground carbon allocation in mature beech and spruce trees following long-term, experimentally enhanced O3 exposure in Southern Germany

Canopies of adult European beech (Fagus sylvatica) and Norway spruce (Picea abies) were labeled with CO₂ depleted in ¹³C to evaluate carbon allocation belowground. One-half the trees were exposed to elevated O₃ for 6 yrs prior to and during the experiment. Soil-gas sampling wells were placed at 8 and 15 cm and soil CO₂ was sampled during labeling in mid-late August, 2006. In beech, δ¹³CO₂ at both depths decreased approximately 50 h after labeling, reflecting rapid translocation of fixed C to roots and release through respiration. In spruce, label was detected in fine-root tissue, but there was no evidence of label in δ¹³CO₂. The results show that C fixed in the canopy rapidly reaches respiratory pools in beech roots, and suggest that spruce may allocate very little of recently-fixed carbon into root respiration during late summer. A change in carbon allocation belowground due to long-term O₃ exposure was not observed.     

Oxygen and carbon isotopic signatures reveal a long-term effect of free-air ozone enrichment on leaf conductance in semi-natural grassland

The effect of ozone on leaf gas diffusion was investigated by analyzing the stable oxygen isotopic signatures (δ¹⁸O) in leaves of Holcus lanatus L., Plantago lanceolata L., Ranunculus friesianus (Jord.), and Trifolium pratense L. grown in temperate, semi-natural grassland. Dried material from plants exposed to ambient or elevated ozone levels in a long-term free-air experiment was sampled in 2002 and 2003. A general increase in δ¹⁸O in elevated ozone indicated increased limitation to gas diffusion, which was strongest during the driest and warmest period in 2003. In three out of four species, the increase in δ¹⁸O paralleled an increase in δ¹³C measured earlier in the same samples, meaning that the dominant effect of ozone was on gas diffusion and not on CO₂ fixation. Only in R. friesianus, ozone affected both processes simultaneously. It is concluded that elevated ozone not only affects productivity, but also the water status of important component species of grassland communities.     

Dietary exposure to technical hexabromocyclododecane (HBCD) alters courtship, incubation and parental behaviors in American kestrels (Falco sparverius)

Hexabromocyclododecane (HBCD) is a high production volume brominated flame retardant that has been detected in the environment and wildlife at increasing concentrations. This study was designed to determine potential effects of dietary exposure to environmentally relevant levels of HBCD on behavior during reproduction in captive American kestrels. Twenty kestrel pairs were exposed to 0.51 μg technical HBCD g⁻¹ kestrel d⁻¹ from 4 weeks prior to pairing until chicks hatched (∼75 d). Ten pairs of controls received the safflower oil vehicle only and were used for comparison. During the courtship period the chitter-calls were reduced in both sexes (p = 0.038) and females performed fewer bonding displays (p = 0.053). Both sexes showed a propensity to be less active than controls during courtship. The reduction in male courtship behavior was correlated with reduced courtship behaviors of females (p ? 0.008) as well as reduced egg mass (p = 0.019). During incubation, nest temperatures of treatment pairs were lower at mid-incubation (p = 0.038). HBCD-exposed males performed fewer key parental behaviors when rearing nestlings, including entering the nest-box, pair-bonding displays and food-retrievals. HBCD-exposed females appeared to compensate for the reduced parental behavior of their mates by performing these same behaviors more frequently than controls (p = 0.004, p = 0.027, p = 0.025, respectively). This study demonstrates that HBCD affects breeding behavior in American kestrels throughout the reproductive season and behavioral alterations were linked to reproductive changes (egg size). This is the first study to report HBCD effects on reproductive behavior in any animal model.     

Food web of a confined and anthropogenically affected coastal basin (the Mar Piccolo of Taranto) revealed by carbon and nitrogen stable isotopes analyses

Carbon and nitrogen stable isotope analysis was used to examine the food web of the Mar Piccolo of Taranto, a coastal basin experiencing several anthropogenic impacts. Main food sources (algal detritus, seaweeds, particulate organic matter (POM) and sediment organic matter (SOM)) and benthic and pelagic consumers were collected during two contrasting seasons (June and April), at four sites distributed over two inlets, and characterized by different level of confinements, anthropogenic inputs and the presence of mussels farming. δ¹³C values of organic sources revealed an important contribution of POM to both planktonic and benthic pathways, as well as the influence of terrigenous inputs within both inlets, probably due to high seasonal land runoff. Although δ¹³C of both sources and consumers varied little between sampling sites and dates, δ¹⁵N spatial variability was higher and clearly reflected the organic enrichment in the second inlet as well as the uptake of anthropogenically derived material by benthic consumers. On the other hand, within the first inlet, the isotopic composition of consumers did not change in response to chemical contamination. However, the impact of polluted sediments near the Navy Arsenal in the first inlet was detectable at the level of the macrobenthic trophic structure, showing high dominance of motile, upper level consumers capable to face transient conditions and the reduction of the more resident deposit feeders. We therefore underline the great potential of matching stable isotope analysis with quantitative studies of community structure to assess the effects of multiple anthropogenic stressors.     

Anthropogenic NOx emissions alter the intrinsic water-use efficiency (WUEi) for Quercus cerris stands under Mediterranean climate conditions

We investigated the effect of N deposition (Ndep) on intrinsic water-use efficiency (WUE_i), the ratio of photosynthesis (A) to stomatal conductance (g_s), for two Quercus cerris stands at different distances to an oil refinery in Southern Italy. We used δ¹³C in tree rings for assessing changes in WUE_i; while the influence of climate and NO_x emission was explored through δ¹⁸O and δ¹⁵N, respectively. Differences in WUE_i between the two sites were significant, with trees exposed to different degrees of NO_x emissions showing an abrupt increase with the onset of pollution. Assuming similar g_s at the two sites, as inferred through δ¹⁸O, the higher N availability at the polluted site caused the shift of the A/g_s ratio in favour of A. Overall, our result suggests that an increase of Ndep may enhance tree WUE under a scenario of reduction of precipitation predicted for Mediterranean area.     

A freshwater amphipod toxicity test based on postexposure feeding and the population consumption inhibitory concentration

Consumption inhibition in natural populations, namely due to contaminants, may have direct and immediate effects on ecosystems, by hampering ecosystem key functions (e.g., organic matter decomposition, grazing), long before effects at the individual level (e.g., reproduction, growth, emergence) have time-delayed consequences at successively higher levels of biological organization. The present study aimed at developing a cost-effective (short and easy) toxicity test based on the postexposure feeding of a freshwater amphipod present in the Iberian Peninsula and at evaluating the immediate impact of contamination on the population-level consumption rate. First, methodologies to quantify postexposure feeding were developed and optimized, the most important criterion being a feeding period as short as possible to minimize physiological recovery from the contaminant exposure. Second, the sensitivities of 48-h postexposure feeding and 48-h lethality tests were compared, using a reference chemical - copper. Third, the latter responses were integrated in a single parameter, the median population consumption inhibitory concentration. When Echinogammarus meridionalis males were fed on 100 defrosted Artemia franciscananauplii during 30 min in darkness at 19-21 °C, the eaten proportion was approximately 80%, without truncated data distributions. The 48-h median effective copper concentration for postexposure feeding was approximately two times lower than the 48-h LC50 - 91 versus 198 μg L⁻¹, respectively. Two techniques were used to quantify the median population consumption inhibitory copper concentration, both leading to similar values (75 and 68 μg L⁻¹). In conclusion, when contaminant concentrations elicit both lethality and feeding depression, the integration of both responses can provide an ecologically relevant evaluation on the potency of a contaminant to immediately disrupt ecosystem functioning.     

Bioaccumulation and tissue distribution of organochlorine pesticides (OCPs) in freshwater fishes: a case study performed in Poyang Lake,China’s largest lake

Concentrations and tissue distribution of organochlorine pesticides (OCPs) in different tissues of freshwater fish, silver carp (Hypophthalmichthys molitrix) and bighead carp (Aristichthys nobilis), collected from Poyang Lake, China’s largest shallow lake, and were studied. OCPs were detected with the observed concentrations ranging from 280.67 to 1,006.58 ng/g wet weight (ww) for bighead carp and from 67.28 to 930.06 ng/g ww for silver carp. Composition analysis demonstrated OCPs in both fish were from the same polluted environment, and then, the species-specific bioaccumulation might be mainly due to the different fish age as well as the different feeding habits elucidating from the stable carbon (δ¹³C) and nitrogen (δ¹⁵N) isotope analysis. Tissue distribution indicated that dietary intake was the major exposure route of OCPs for both fish and higher accumulation potency of OCPs by the hepatobiliary-related tissues (such as liver, kidney, bile, and heart). The higher metabolic activities of these tissues elucidating from the higher values of δ¹⁵N might be the potential-determined factor responsible for the tissue-specific accumulation.     

Tree-ring stable isotopes and historical perspectives on pollution - An overview

Hydrogen (δ²H), carbon (δ¹³C), oxygen (δ¹⁸O) and nitrogen (δ¹⁵N) isotopes of tree rings growing in field conditions can be indicative of past pollution effects. The characteristic δ¹³C trend is a positive shift generally explained by invoking closure of stomata, but experimental studies suggest that increased rates of carboxylation could also generate such trends. In many cases the δ¹⁸O and δ²H values decrease in trees exposed to pollution and exhibit inverse coinciding long-term trends with δ¹³C values. However, some trees exposed to diffuse pollution and experimental conditions can show an increase or no δ¹⁸O change even if δ¹³C values increase. These diverse responses depend on how stress conditions modify physiological functions such as stomatal conductance, carboxylation, respiration, and perhaps water assimilation by the root system. Recent studies suggest that δ¹⁵N changes in trees can be caused by soil acidification and accumulation of anthropogenic emissions with isotopic signals deviating from natural N.     

Origin and vertical variation of the bound fatty acids in core sediments of Lake Dianchi in Southwest China

Based on the molecular distribution of bound fatty acid (BFA) compound classes in core sediments of Lake Dianchi combined with the compound-specific δ¹³C values of the straight-chain BFAs, origin and vertical changes of organic matters in the sediments were investigated. The results indicated a significant change of BFA sources over the past 700 years. Contrast to the low concentrations of the terrestrial BFAs, the abundance of BFAs derived from the plankton/bacteria in the top sections (1944–recent) was more than 80 %. The increasing proportions of the branched and unsaturated BFAs in total fatty acids were closely correlated with the heavy eutrophication and the frequent algal blooms in the decades. Furthermore, the positive shift of δ ¹³C of C16 and C18 (~2?‰) in the upper section might be an indicator of the excess phytoplankton productivity. However, it was found that the plankton/bacteria-derived BFAs were more easily degraded during the early diagenetic process. The special compound carbon isotopic compositions of the long straight-chain BFAs (C24 and C26) in the sediments showed a depletion of heavier δ ¹³C values (ca. ?30?‰) in the midsections (1559–1787), reflecting a relatively growing contribution of C3 plants to C4 plants or that C4 plant growth was inhibited in cold and arid climates during the period.