A demographic approach to study effects of climate change in desert plants

Desert species respond strongly to infrequent, intense pulses of precipitation. Consequently, indigenous flora has developed a rich repertoire of life-history strategies to deal with fluctuations in resource availability. Examinations of how future climate change will affect the biota often forecast negative impacts, but these—usually correlative—approaches overlook precipitation variation because they are based on averages. Here, we provide an overview of how variable precipitation affects perennial and annual desert plants, and then implement an innovative, mechanistic approach to examine the effects of precipitation on populations of two desert plant species. This approach couples robust climatic projections, including variable precipitation, with stochastic, stage-structured models constructed from long-term demographic datasets of the short-lived Cryptantha flava in the Colorado Plateau Desert (USA) and the annual Carrichtera annua in the Negev Desert (Israel). Our results highlight these populations'' potential to buffer future stochastic precipitation. Population growth rates in both species increased under future conditions: wetter, longer growing seasons for Cryptantha and drier years for Carrichtera. We determined that such changes are primarily due to survival and size changes for Cryptantha and the role of seed bank for Carrichtera. Our work suggests that desert plants, and thus the resources they provide, might be more resilient to climate change than previously thought.     

Projected effects of climate change on tick phenology and fitness of pathogens transmitted by the North American tick Ixodes scapularis

Ixodes scapularis is the principal tick vector of the Lyme borreliosis agent Borrelia burgdorferi and other tick-borne zoonoses in northeastern North America. The degree of seasonal synchrony of nymphal and larval ticks may be important in influencing the basic reproductive number of the pathogens transmitted by I. scapularis. Because the seasonal phenology of tick vectors is partly controlled by ambient temperature, climate and climate change could shape the population biology of tick-borne pathogens. We used projected monthly normal temperatures, obtained from the second version of the Canadian Coupled Global Climate Model (CGCM2) under emissions scenario A2 of the Intergovernmental Panel on Climate Change for a site in southern Ontario, Canada, to simulate the phenology of I. scapularis in a mathematical model. The simulated seasonal abundance of ticks then determined transmission of three candidate pathogens amongst a population of white-footed mice (Peromyscus leucopus) using a susceptible-infected-recovered (SIR) model. Fitness of the different pathogens, in terms of resilience to changes in tick and rodent mortality, minima for infection duration, transmission efficiency and particularly any additional mortality of rodents specifically associated with infection, varied according to the seasonal pattern of immature tick activity, which was different under the temperature conditions projected for the 2020s, 2050s and 2080s. In each case, pathogens that were long-lived, highly transmissible and had little impact on rodent mortality rates were the fittest. However, under the seasonal tick activity patterns projected for the 2020s and 2050s, the fitness of pathogens that are shorter-lived, less efficiently transmitted, and more pathogenic to their natural hosts, increased. Therefore, climate change may affect the frequency and distribution of I. scapularis-borne pathogens and alter their evolutionary trajectories.     

Land, lake, and fish: Investigation of fish remains from Gesher Benot Ya'aqov (paleo-Lake Hula)

The question of whether or not pre-modern hominins were responsible for the accumulation of fish remains is discussed through analyses of remains recovered from two lacustrine facies (I-4 and I-5) from Area A of the Acheulian site of Gesher Benot Ya‘aqov (GBY) in the Jordan Rift Valley, Israel. The fish remains provide the first glimpse into the naturally accumulated fish assemblage from the fluctuating shores of a lake that had been continually exploited by early hominins some 780,000 years ago. Preliminary analysis of the remains show that thirteen of the seventeen species native to Lake Hula were identified at GBY. These represent three of the five freshwater fish families native to the lake: Cyprinidae (carps), Cichlidae (tilapini, St. Peter's fish), and Clariidae (catfish). From a taphonomical perspective, a significant difference is found between the two lithofacies (Layers I-4 and I-5) in terms of species composition, richness, diversity, and skeleton completeness. It appears that the fish remains recovered from Layer I-4 (clay) are better preserved than those from Layer I-5 (coquina). In both lithofacies, Cyprinidae are highly abundant while Cichlidae and Clariidae are rare and under-represented, especially when compared to the Lake Hula fishery report from the 1950s. All of these identified species may have contributed significantly to the diet of GBY hominins.     

Macroalgal-mediated transfers of water column nitrogen to intertidal sediments and salt marsh plants

In many temperate estuaries, mats of opportunistic macroalgae accumulate on intertidal flats and in lower elevations of salt marshes, perhaps playing a role in linking water column nitrogen (N) supply to these benthic habitats. Using a flow-through seawater system and tidal simulator, we varied densities (equivalent to 0, 1, 2, or 3 kg m⁻² wet mass) of ¹⁵N-labelled macroalgae (Enteromorpha intestinalis) on estuarine sediments in microcosms with/without pickleweed (Salicornia virginica) to assess N transfers from algae. In the 6-week experiment, macroalgal biomass increased from initial levels in the lower density treatments but all algae lost N mass, probably through both leakage and decomposition. With all densities of algae added, sediments and pickleweed became enriched in ¹⁵N. With increasing mat density, losses of algal N mass increased, resulting in stepwise increases in ¹⁵N labeling of the deeper sediments and pickleweed. While we did not detect a growth response in pickleweed with macroalgal addition during the experiment, N losses from algal mats that persist over many months and/or recur each year could be important to the mineral nutrition of N-limited marsh plants. We conclude that N dynamics of intertidal sediments and lower salt marsh vegetation are linked to the N pools of co-occurring macroalgae and that further study is needed to assess the magnitude and importance of N transfers.     

Evidence for a new lineage of primary ambrosia fungi in Geosmithia Pitt (Ascomycota: Hypocreales)

Geosmithia is a genus of mitosporic filamentous fungi typically associated with phloeophagous bark beetles world-wide. During this study, the fungal associates of ambrosia beetles Cnesinus lecontei, Eupagiocerus dentipes, and Microcorthylus sp. from Costa Rica, were studied using morphology and DNA sequences. Fungal associates belonged to four undescribed Geosmithia species. Geosmithia eupagioceri sp. nov. and G. microcorthyli sp. nov. are evidently primary ambrosia fungi of their respective vectors E. dentipes and Microcorthylus species. They both have convergently evolved distinct morphological adaptations including the production of large, solitary and globose conidia, and yeast-like cells. Tunnels of C. lecontei contained an undescribed Geosmithia species, but its nutritional importance for its vector is unclear. An auxiliary ambrosia fungus, Geosmithia rufescens sp. nov., was found associated with both G. eupagioceri and the Geosmithia species associated with C. lecontei. G. microcorthyli is genetically quite similar to the phloem-associated Geosmithia sp. 8 from Europe. Large differences in morphology between these two species suggest the rapid co-evolution resulting from the close symbiosis of the former with its beetle host. The ITS rDNA sequences of G. microcorthyli and Geosmithia sp. 8 were not diagnostic, suggesting that alternative markers such as EF-1α, IGS rDNA or β-tubulin should be used, together with morphological and ecological data, for species delimitation in this genus. The primary ambrosia fungi described here are derived from phloem-associated ancestors, and represent two independent lineages of ambrosia fungi in the Hypocreales and a new ecological strategy within Geosmithia.     

Side steps: the erratic pattern of hominin postcranial change through time

The hominin fossil record reveals brain-size expansion, canine reduction, premolar metaconid development, and numerous other craniodental features that become more human-like through time. In general, the postcranial skeleton also gets more human-like through time, but in some respects it does not. This is particularly apparent in the overall morphology of one of the most frequently preserved elements, the distal humerus. Some of the earliest hominins display quite human-like morphologies, whereas later specimens are quite unusual among extant species of Hominoidea: when described metrically and subjected to multivariate discriminant analyses in the context of large samples of extant hominoid humeri, the shapes of the earliest hominin fossils are more human-like than many of the later specimens. The Mahalanobis distances between many of the 1.5-2Ma hominin humeri and Homo sapiens are remarkably large. Many of the less well-represented postcranial specimens do not follow a linear path through time of increasing hominization either. This is particularly noticeable in the fore-to-hind limb joint-size proportions, ulnar morphology, and pelvic architecture. The hominin postcranial fossil record reveals many side-steps: there appears to be no simple march toward our human bodies, but a pattern better explained as adaptations to proximate conditions and constrained by ontogeny and history.     

Local adaptation in brown trout early life-history traits: implications for climate change adaptability

Knowledge of local adaptation and adaptive potential of natural populations is becoming increasingly relevant due to anthropogenic changes in the environment, such as climate change. The concern is that populations will be negatively affected by increasing temperatures without the capacity to adapt. Temperature-related adaptability in traits related to phenology and early life history are expected to be particularly important in salmonid fishes. We focused on the latter and investigated whether four populations of brown trout (Salmo trutta) are locally adapted in early life-history traits. These populations spawn in rivers that experience different temperature conditions during the time of incubation of eggs and embryos. They were reared in a common-garden experiment at three different temperatures. Quantitative genetic differentiation (QST) exceeded neutral molecular differentiation (FST) for two traits, indicating local adaptation. A temperature effect was observed for three traits. However, this effect varied among populations due to locally adapted reaction norms, corresponding to the temperature regimes experienced by the populations in their native environments. Additive genetic variance and heritable variation in phenotypic plasticity suggest that although increasing temperatures are likely to affect some populations negatively, they may have the potential to adapt to changing temperature regimes.     

Phosphoproteins analysis in plants: a proteomic approach

The study of phosphoproteome on a global scale represents one of the challenges in the post-genomic era. Here, we propose an integrated procedure starting from the crude protein extract, that consists of sequential purification steps, and ending up in the identification of phosphorylation sites. This involves (i) an enrichment in phosphoproteins with a commercially available chromatography matrix, (ii) a 2-D gel analysis of the enriched fraction followed by the selective staining with the phosphospecific fluorescent dye Pro-Q Diamond, (iii) a phosphopeptide capture, from the tryptic lysate of 2-D spots, using IMAC micro-columns. In the end, the identification of the phosphoproteins and their corresponding phosphorylation sites were achieved by MALDI-TOF-TOF spectrometry. The method was applied to contrasting samples prepared from cell suspension cultures of Arabidopsis thaliana and roots of Medicago truncatula. The results obtained, demonstrated the robustness of the combination of two enrichment stages, sequentially at the protein and at the peptide levels, to analyse phosphoproteins in plants.     

Coexistence of Abies alba (Mill.) - Fagus sylvatica (L.) and climate change impact in the Iberian Peninsula: A climatic-niche perspective approach

Abies alba Mill. (European silver fir) and Fagus sylvatica L. (beech) are Eurosiberian species dispersed over the Iberian Peninsula. Climate change predictions indicate a rise in temperature and a decrease in precipitation in this region, threatening the future existence of these species. In the present study we analyzed the future topo-climatic suitability of Abies alba and Fagus sylvatica and the mixed forests of these two species, using the General Linear Models technique and data from the third National Forest Inventory (Ministerio de Agricultura PyA, 2007). We considered two modeling approaches based on niche theory: modeling community (Abieti-Fagetum) and overlapping individual species models. General trends showed an overall decrease in both species’ topo-climatic suitability and indicated that the Pyrenees will play a crucial role as a climatic refuge. The modeling approaches markedly differed, however, in their current and future spatial agreement. Despite good accuracy results, community modeling through co-occurrence does not encompass the environmental space of individual species prejudicing future assessments in new environmental situations, suggesting a need for future studies in community modeling.     

Evidence for mitochondrial-derived alternative oxidase in the apicomplexan parasite Cryptosporidium parvum: a potential anti-microbial agent target

The observation that Plasmodium falciparum possesses cyanide insensitive respiration that can be inhibited by salicylhydroxamic acid (SHAM) and propyl gallate is consistent with the presence of an alternative oxidase (AOX). However, the completion and annotation of the P. falciparum genome project did not identify any protein with convincing similarity to the previously described AOXs from plants, fungi or protozoa. We undertook a survey of the available apicomplexan genome projects in an attempt to address this anomaly. Putative AOX sequences were identified and sequenced from both type 1 and 2 strains of Cryptosporidium parvum. The gene encodes a polypeptide of 336 amino acids and has a predicted N-terminal transit sequence similar to that found in proteins targeted to the mitochondria of other species. The potential of AOX as a target for new anti-microbial agents for C. parvum is evident by the ability of SHAM and 8-hydroxyquinoline to inhibit in vitro growth of C. parvum. In spite of the lack of a good candidate for AOX in either the P. falciparum or Toxoplasma gondii genome projects, SHAM and 8-hydroxyquinoline were found to inhibit the growth of these parasites. Phylogenetic analysis suggests that AOX and the related protein immutans are derived from gene transfers from the mitochondrial endosymbiont and the chloroplast endosymbiont, respectively. These data are consistent with the functional localisation studies conducted thus far, which demonstrate mitochondrial localisation for some AOX and chloroplastidic localization for immutans. The presence of a mitochondrial compartment is further supported by the prediction of a mitochondrial targeting sequence at the N-terminus of the protein and MitoTracker staining of a subcellular compartment in trophozoite and meront stages. These results give insight into the evolution of AOX and demonstrate the potential of targeting the alternative pathway of respiration in apicomplexans.     

Lutzomyia vectors for cutaneous leishmaniasis in Southern Brazil: ecological niche models,predicted geographic distributions,and climate change effects

Geographic and ecological distributions of three Lutzomyia sand flies that are cutaneous leishmaniasis vectors in South America were analysed using ecological niche modelling. This new tool provides a large-scale perspective on species' geographic distributions, ecological and historical factors determining them, and their potential for change with expected environmental changes. As a first step, the ability of this technique to predict geographic distributions of the three species was tested statistically using two subsampling techniques: a random-selection technique that simulates 50% data density, and a quadrant-based technique that challenges the method to predict into broad unsampled regions. Predictivity under both test schemes was highly statistically significant. Visualisation of ecological niches provided insights into the ecological basis for distributional differences among species. Projections of potential geographic distributions across scenarios of global climate change suggested that only Lutzomyia whitmani is likely to be experiencing dramatic improvements in conditions in south-eastern Brazil, where cutaneous leishmaniasis appears to be re-emerging; Lutzomyia intermedia and Lutzomyia migonei may be seeing more subtle improvements in climatic conditions, but the implications are not straightforward. More generally, this technique offers the possibility of new views into the distributional ecology of disease, vector, and reservoir species.     

Evidence for multiple hybrid groups in Trypanosoma cruzi

A role for parasite genetic variability in the spectrum of Chagas disease is emerging but not yet evident, in part due to an incomplete understanding of the population structure of Trypanosoma cruzi. To investigate further the observed genotypic variation at the sequence and chromosomal levels in strains of standard and field-isolated T. cruzi we have undertaken a comparative analysis of 10 regions of the genome from two isolates representing T. cruzi I (Dm28c and Silvio X10) and two from T. cruzi II (CL Brener and Esmeraldo). Amplified regions contained intergenic (non-coding) sequences from tandemly repeated genes. Multiple nucleotide polymorphisms correlated with the T. cruzi I/T. cruzi II classification. Two intergenic regions had useful polymorphisms for the design of classification probes to test on genomic DNA from other known isolates. Two adjacent nucleotide polymorphisms in HSP 60 correlated with the T. cruzi I and T. cruzi II distinction. 1F8 nucleotide polymorphisms revealed multiple subdivisions of T. cruzi II: subgroups IIa and IIc displayed the T. cruzi I pattern; subgroups IId and IIe possessed both the I and II patterns. Furthermore, isolates from subgroups IId and IIe contained the 1F8 polymorphic markers on different chromosome bands supporting a genetic exchange event that resulted in chromosomes V and IX of T. cruzi strain CL Brener. Based on these analyses, T. cruzi I and subgroup IIb appear to be pure lines, while subgroups IIa/IIc and IId/IIe are hybrid lines. These data demonstrate for the first time that IIa/IIc are hybrid, consistent with the hypothesis that genetic recombination has occurred more than once within the T. cruzi lines.     

Comparison of two methods characterising the seed bank of amphibious plants in submerged sediments

We analysed the submerged soil seed bank of three fishponds in the Waldviertel region in Austria. We aimed our study at comparing the efficiency of two methods in detecting seeds quantitatively from soil samples of four characteristic mud-flat species, i.e. Carex bohemica, Coleanthus subtilis, Elatine hexandra, and Eleocharis ovata: the seedling-emergence and the rinsing method. Additionally, the actual vegetation was described based on 65 phytosociological relevés. Soil seed bank analyses were based on 31 samples representing the vegetation zonation. Mixed soil samples were portioned into 62 subsamples further treated with the two methods in parallel.     

Investigating early hominin dispersal patterns: developing a framework for climate data integration

An investigation using the Stepping Out model of early hominin dispersal out of Africa is presented here. The late arrival of early hominins into Europe, as deduced from the fossil record, is shown to be consistent with poor ability of these hominins to survive in the Eurasian landscape. The present study also extends the understanding of modelling results from the original study by Mithen and Reed (2002. Stepping out: a computer simulation of hominid dispersal from Africa. J. Hum. Evol. 43, 433-462). The representation of climate and vegetation patterns has been improved through the use of climate model output. This study demonstrates that interpretative confidence may be strengthened, and new insights gained when climate models and hominin dispersal models are integrated.     

Hominid environments at Hadar from paleosol studies in a framework of Ethiopian climate change

The amount and seasonal distribution of paleo-rainfall is a major concern of paleoanthropology because they determine the nature of the vegetation and the structure of the ecosystem, particularly in eastern Africa. The δ¹⁸O and δ¹³C of paleosol carbonates are quantitative proxies of these critical features of the paleoenvironment. The Afar region of Ethiopia lies between the African and Indian summer monsoons, and is prone to profound climate change. In the western Afar, the dominant paleoenvironment of the Hadar Formation during the late Pliocene was a major meandering river's distal low, flat floodplain, on which muds accreted that were continuously transformed into vegetated soils with Bk horizons rich in CaCO₃. The mean δ¹³C of paleosols throughout the Hadar Formation translates to an average vegetative cover across the extensive floodplain of about 30% of the C₄ grasses and 70% of unspecified C₃ plants. The character of the paleosols, such as the one at Locality 333, and their δ¹⁸O_Carbonate argue for a highly seasonal rainfall of about twice today's amount, implying that the C₃ plants were mostly sizeable trees and that the biome for Australopithecus afarensis was a grassy woodland. The amount of grasses abruptly increased in the lower Busidima Formation with its early Homo and artifacts to a more open grassy woodland of ca. 50% grasses. However, this transition in δ¹³C is not mirrored in the δ¹⁸O, which persists at a quite negative average value of −6.4‰ over the entire >2-Myr duration of both formations. This value for the carbonate means that the paleosoil water was a quite negative −4.1‰, a significant 5‰ more negative than our estimate of modern rain at Hadar. We put the negative δ¹⁸O of paleo-Hadar's rainfall into an isotopic framework of the dynamic history of climate change in sub-Saharan northern Africa. There have been two end-member climate regimes: (1) an earlier persistently pluvial Pliocene regime, with its strong summer monsoon, as registered in the Hadar Formation; and (2) the modern cyclical, mostly arid regime that began ca. 1 Myr ago, which has been punctuated by about ten cyclically predictable brief millennia-long pluvial episodes. The best known pluvial of the latter regime is the latest one, the African Humid Period (AHP), just 9.0-6.5 kyr ago, whose δ¹⁸O_Rainfall matches that for paleo-Hadar. The known climatological factors that brought on the AHP are probably the same ones that were persistently present for the Afar of the Pliocene. This dynamic rainfall history undoubtedly has influenced hominid occupation of the keystone Afar area at the gateway out of, and into, Africa.     

Carnivory in plants as a beneficial trait in wetlands

A new hypothesis for the benefit of carnivory in plants (i.e., an alternative to aerenchyma for avoiding hypoxia) is evaluated. Root porosity and root depth were quantified in eight carnivorous plant species and 48 non-carnivorous species within a nutrient-poor wet pine savanna in south Mississippi, USA. Carnivorous and non-carnivorous plant species were contrasted with respect to their indication of wetlands, open habitats, and habitats with nutrient-poor soils. We used path analysis, multiplicative regression, and a field experiment to test hypotheses of the effects of soil moisture/hypoxia on the abundance of carnivorous and non-carnivorous plants. All carnivorous plant species produced non-porous roots (or no roots), which were shallower than the average for non-carnivorous plants (6.9 ± 0.95 cm vs. 11.9 ± 0.96 cm), even after correcting for plant size. Root porosity in non-carnivorous species (mean = 22%) was positively correlated with root depth (r = 0.6). Despite lacking porous roots, carnivorous plants were four times more indicative of wetland habitats than were the non-carnivorous species encountered in the wetland studied here. Carnivorous plants, along with non-carnivorous plants with well-developed aerenchyma, were positively associated with the wettest microsites and were more negatively affected by elevating the substrate than were non-carnivorous plants with low-porosity roots. Non-carnivorous plants with shallow roots, while less indicative of wetlands and less abundant in wet microsites of the wet pine savanna than were carnivorous plants, were no less indicative of nutrient-poor soils than were carnivorous plants. Results supported the hypothesis that carnivory is advantageous in wet soils and disadvantageous in drier (including mesic) soils and are more indicative of wetland conditions than of low soil fertility.     

Evidence for an immune function of lepidopteran silk proteins

Hemolymph coagulation stops bleeding and protects against infection. Clotting factors include both proteins that are conserved during evolution as well as more divergent proteins in different species. Here we show that several silk proteins also appear in the clot of the greater wax moth Galleria mellonella. RT-PCR analysis reveals that silk proteins are expressed in immune tissues and induced upon wounding in both Galleria and Ephestia kuehniella, a second pyralid moth. Our results support the idea that silk proteins were co-opted for immunity and coagulation during evolution.     

Evidence for common machinery utilized by the early and late RNA localization pathways in Xenopus oocytes

In Xenopus, an early and a late pathway exist for the selective localization of RNAs to the vegetal cortex during oogenesis. Previous work has suggested that distinct cellular mechanisms mediate localization during these pathways. Here, we provide several independent lines of evidence supporting the existence of common machinery for RNA localization during the early and late pathways. Data from RNA microinjection assays show that early and late pathway RNAs compete for common localization factors in vivo, and that the same short RNA sequence motifs are required for localization during both pathways. In addition, quantitative filter binding assays demonstrate that the late localization factor Vg RBP/Vera binds specifically to several early pathway RNA localization elements. Finally, confocal imaging shows that early pathway RNAs associate with a perinuclear microtubule network that connects to the mitochondrial cloud of stage I oocytes suggesting that motor driven transport plays a role during the early pathway as it does during the late pathway. Taken together, our data indicate that common machinery functions during the early and late pathways. Thus, RNA localization to the vegetal cortex may be a regulated process such that differential interactions with basal factors determine when distinct RNAs are localized during oogenesis.     

Composition and anti-plasmodial activities of essential oils from some Cameroonian medicinal plants

In a search for new plant-derived biologically active compounds against malaria parasites, five essential oils extracted from the Cameroonian plants Xylopia phloiodora, Pachypodanthium confine, Antidesma laciniatum, Xylopia aethiopica, and Hexalobus crispiflorus were evaluated in regard to their anti-plasmodial activity against the W2 strain of Plasmodium falciparum. The oils were obtained from the plants with 0.12, 0.13, 0.18, 0.6 and 0.1% yields (relatively to dried material weight) respectively. Analysis by gas chromatography and mass spectrometry identified mainly terpenoids, among which alpha-copaene, gamma-cadinene, delta-cadinene, alpha-cadinol, spathulenol and caryophyllene oxide were most commonly found. The five oils were active against Plasmodium falciparum in culture. The most effective was the oil of Hexalobus crispiflorus, with an IC50 of 2 microg/ml.     

Evidence for structural integrity in the undecameric c-rings isolated from sodium ATP synthases

The Na(+)-translocating ATP synthases from Ilyobacter tartaricus and Propionigenium modestum contain undecameric c subunit rings of unusual stability. These c(11) rings have been isolated from both ATP synthases and crystallized in two dimensions. Cryo-transmission electron microscopy projection maps of the c-rings from both organisms were identical at 7A resolution. Different crystal contacts were induced after treatment of the crystals with dicyclohexylcarbodiimide (DCCD), which is consistent with the binding of the inhibitor to glutamate 65 in the C-terminal helix on the outside of the ring. The c subunits of the isolated c(11) ring of I.tartaricus were modified specifically by incubation with DCCD with kinetics that were indistinguishable from those of the F(1)F(o) holoenzyme. The reaction rate increased with decreasing pH but was lower in the presence of Na(+). From the pH profile of the second-order rate constants, the pK of glutamate 65 was deduced to be 6.6 or 6.2 in the absence or presence of 0.5mM NaCl, respectively. These pK values are identical with those determined for the F(1)F(o) complex. The results indicate that the isolated c-ring retains its native structure, and that the glutamate 65, including binding sites near the middle of the membrane, are accessible to Na(+) from the cytoplasm through access channels within the c-ring itself.