Jochen BW Wolf

Abstract: Our understanding of how cooperation can arise in a population of selfish individuals has been greatly advanced by theory. More than one approach has been used to explore the effect of population structure. Inclusive fitness theory uses genetic relatedness r to express the role of population structure. Evolutionary graph theory models the evolution of cooperation on network structures and focuses on the number of interacting partners k as a quantity of interest. Here we use empirical data from a hierarchically structured animal contact network to examine the interplay between independent, measurable proxies for these key parameters. We find strong inverse correlations between estimates of r and k over three levels of social organization, suggesting that genetic relatedness and social contact structure capture similar structural information in a real population.

Abstract: Development implies a change in allocation of resources from somatic growth to reproduction. In a highly variable environment, growth can vary from year to year thereby in�uencing the long-term life history perspective. The Galapagos sea lion (Zalophus wollebaeki) lives in a highly unpredictable marine environment in � which food abundance varies not only seasonally, but also annually due to El Nino. Galapagos sea lions are restricted to a patch of cold upwelling waters surrounding the archipelago and are closely tied to land as nursing females alternate between foraging at sea and nursing ashore. Therefore, their offspring are especially vulnerable to ocean warming causing reduced food abundance. We found a signi�cant correlation between sea surface temperature (SST) and early growth: Both mass at birth and linear growth within the �rst 2 mo of life correlated negatively with SST. Absolute mass gain was higher for males, but both sexes gained equally 1.9% of birth mass per day. Until the age of 3 yr male and female juveniles showed similar growth to an asymptotic mass of 40 and 35 kg, respectively. As a consequence of the highly variable environment, the plasticity in growth strategy of Galapagos sea lion juveniles appears wider than that of all other sea lions allowing them to cope with poor conditions.

Abstract: Background Species with heteromorphic sex chromosomes face the challenge of large-scale imbalance in gene dose. Microarray-based studies in several independent male heterogametic XX/XY systems suggest that dosage compensation mechanisms are in place to mitigate the detrimental effects of gene dose differences. However, recent genomic research on female heterogametic ZZ/ZW systems has generated surprising results. In two bird species and one lepidopteran no evidence for a global dosage compensating mechanism has been found. The recent advent of massively parallel RNA sequencing now opens up the possibility to gauge the generality of this observation with a broader phylogenetic sampling. It further allows assessing the validity of microarray-based inference on dosage compensation with a novel technology. Results We here expemplify this approach using massively parallel sequencing on barcoded individuals of a bird species, the European crow (Corvus corone), where previously no genetic resources were available. Testing for Z-linkage with quantitative PCR (qPCR,) we first establish that orthology with distantly related species (chicken, zebra finch) can be used as a good predictor for chromosomal affiliation of a gene. We then use a digital measure of gene expression (RNA-seq) on brain transcriptome and confirm a global lack of dosage compensation on the Z chromosome. RNA-seq estimates of male-to-female (m:f) expression difference on the Z compare well to previous microarray-based estimates in birds and lepidopterans. The data further lends support that an up-regulation of female Z-linked genes conveys partial compensation and suggest a relationship between sex-bias and absolute expression level of a gene. Correlation of sex-biased gene expression on the Z chromosome across all three bird species further suggests that the degree of compensation has been partly conserved across 100 million years of avian evolution. Conclusions This work demonstrates that the study of dosage compensation has become amenable to species where previously no genetic resources were available. Massively parallel transcriptome sequencing allows re-assessing the degree of dosage compensation with a novel tool in well-studies species and, in addition, gain valuable insights into the generality of mechanisms across independent taxonomic group for both the XX/XY and ZZ/ZW system.

Abstract: Changes in expression of genes are thought to contribute significantly to evolutionary divergence. To study the relative role of selection and neutrality in shaping expression changes, we analyzed 24 genes in three different tissues of the house mouse (Mus musculus). Samples from two natural populations of the subspecies M. m. domesticus and M. m. musculus were investigated using quantitative PCR assays and sequencing of the upstream region. We have developed an approach to quantify expression polymorphism within such populations and to disentangle technical from biological variation in the data. We found a correlation between expression polymorphism within populations and divergence between populations. Furthermore, we found a correlation between expression polymorphism and sequence polymorphism of the respective genes. These data are most easily interpreted within a framework of a predominantly neutral model of gene expression change, where only a fraction of the changes may have been driven by positive selection. Although most genes investigated were expressed in all three tissues analyzed, significant changes of expression levels occurred predominantly in a single tissue only. This adds to the notion that enhancer-specific effects or transregulatory effects can modulate the evolution of gene expression in a tissue-specific way.

Abstract: Local variation in neutral substitution rate across mammalian genomes is governed by several factors, including sequence context variables and structural variables. In addition, the interplay of replication and transcription, known to induce a strand bias in mutation rate, gives rise to variation in substitutional strand asymmetries. Here, we address the conservation of variation in mutation rate and substitutional strand asymmetries using primate- and rodent-specific repeat elements located within the introns of protein-coding genes. We find significant but weak conservation of local mutation rates between human and mouse orthologs. Likewise, substitutional strand asymmetries are conserved between human and mouse, where substitution rate asymmetries show a higher degree of conservation than mutation rate. Moreover, we provide evidence that replication and transcription are correlated to the strength of substitutional asymmetries. The effect of transcription is particularly visible for genes with highly conserved gene expression. In comparison with replication and transcription, mutation rate influences the strength of substitutional asymmetries only marginally.

Abstract: Next-generation sequencing technology provides an attractive means to obtain large-scale sequence data necessary for comparative genomic analysis. To test the feasibility of this approach for studies within a divergent group of non-model organisms, we performed brain transcriptome sequencing using Roche 454 technology of 10 different avian species. Contigs from de novo assemblies were aligned to the two available avian reference genomes, chicken and zebra finch. In total, we identified 6,499 different genes across all 10 species, with approximately 1,000 genes found in each full run per species. We found evidence for a higher mutation rate of the Z chromosome than of autosomes (male-biased mutation) and a negative correlation between the neutral substitution rate (dS) and chromosome size. Analyses of the mean dN/dS ratio (�) of genes across chromosomes supported the Hill-Robertson effect and point at stochastic problems with � as an independent measure of selection. Overall, this study demonstrates the usefulness of next-generation sequencing for obtaining genomic resources for comparative genomic analysis of non-model organisms.

Abstract: Recent advances in sequencing technology promise to provide new strategies for studying population differentiation and speciation phenomena in their earliest phases. We focus here on the black carrion crow (Corvus [corone] corone) which forms a zone of hybridization and overlap with the grey coated hooded crow (Corvus [corone] cornix). However, although these semispecies are taxonomically distinct, previous analyses based on several types of genetic markers did not reveal significant molecular differentiation between them. We here corroborate this result with sequence data obtained from a set of 25 nuclear intronic loci. Thus, the system represents a case of a very early phase of species divergence that requires new molecular approaches for its description. We have therefore generated RNA-Seq expression profiles using barcoded massively parallel pyrosequencing of brain mRNA from six individuals of the carrion crow and five individuals from a hybrid zone with the hooded crow. We obtained 856,675 reads from two runs, with average read length of 270 nt and coverage of 8.44. Reads were assembled de novo into 19,552 contigs, 70% of which could be assigned to annotated genes in chicken and zebra finch. This resulted in a total of 7,637 orthologous genes and a core set of 1,301 genes that could be compared across all individuals. We find a clear clustering of expression profiles for the pure carrion crow animals and disperse profiles for the animals from the hybrid zone. These results suggest that gene expression differences may indeed be a sensitive indicator of initial species divergence.

Abstract: The view of species as entities subjected to natural selection and amenable to change put forth by Charles Darwin and Alfred Wallace laid the conceptual foundation for understanding speciation. Initially marred by a rudimental understanding of hereditary principles, evolutionists gained appreciation of the mechanistic underpinnings of speciation following the merger of Mendelian genetic principles with Darwinian evolution. Only recently have we entered an era where deciphering the molecular basis of speciation is within reach. Much focus has been devoted to the genetic basis of intrinsic postzygotic isolation in model organisms and several hybrid incompatibility genes have been successfully identified. However, concomitant with recent technological advancements in genome analysis and a newfound interest in the role of ecology in the differentiation process, speciation genetic research is becoming increasingly open to non-model organisms. This development will expand speciation research beyond the traditional boundaries and unveil the genetic basis of speciation from manifold perspectives and at various stages of the splitting process. This review aims at providing an extensive overview of speciation genetics. Starting from key historical developments and core concepts of speciation genetics, we focus much of our attention on evolving approaches and introduce promising methodological approaches for future research venues.

Abstract: Background Obtaining a draft genome sequence of the zebra finch (Taeniopygia guttata), the second bird genome to be sequenced, provides the necessary resource for whole-genome comparative analysis of gene sequence evolution in a non-mammalian vertebrate lineage. To analyze basic molecular evolutionary processes during avian evolution, and to contrast these with the situation in mammals, we aligned the protein-coding sequences of 8,384 1:1 orthologs of chicken, zebra finch, a lizard and three mammalian species. Results We found clear differences in the substitution rate at fourfold degenerate sites, being lowest in the ancestral bird lineage, intermediate in the chicken lineage and highest in the zebra finch lineage, possibly reflecting differences in generation time. We identified positively selected and/or rapidly evolving genes in avian lineages and found an over-representation of several functional classes, including anion transporter activity, calcium ion binding, cell adhesion and microtubule cytoskeleton. Conclusions Focusing specifically on genes of neurological interest and genes differentially expressed in the unique vocal control nuclei of the songbird brain, we find a number of positively selected genes, including synaptic receptors. We found no evidence that selection for beneficial alleles is more efficient in regions of high recombination; in fact, there was a weak yet significant negative correlation between � and recombination rate, which is in the direction predicted by the Hill-Robertson effect if slightly deleterious mutations contribute to protein evolution. These findings set the stage for studies of functional genetics of avian genes.

Abstract: Sexual selection theory predicts competitive males and choosy females. Nevertheless, since the advent of molecular marker based studies paternity outside the expected mating patterns has increasingly been described. Even in highly polygynous systems, where paternal share is expected to be strongly skewed towards large dominant males, alternative mating tactics have been suggested. We examined reproductive success in the polygynous Galápagos sea lion (Zalophus wollebaeki). Semi-aquatic territoriality allows females to move freely and may lower the degree of polygyny otherwise suggested by territorial behaviour and strong sexual dimorphism. We assigned paternities with 22 microsatellites and analysed how male reproductive success related to size, dominance status, intra-sexual agonistic behaviour, proximity to females, and attendance in the colony. Male behaviour was consistent across two seasons for all parameters under consideration. Attendance was by far the most important determinant of paternal success. Skew in reproductive success towards large, dominant males was weak and dominance status played no role. This appears to be caused by an extremely drawn out reproductive season lasting for five or more months making it difficult for any male to monopolize receptive females. Females seem to choose displaying males that were present in the colony for a long time rather than dominance per se. Sexual dimorphism in Galápagos sea lions may thus be more influenced by selection for fasting than fighting ability. Our data further point towards the possibility of alternative mating tactics, as several males gained relatively high paternity success despite short attendance and hardly any involvement in agonistic interactions.

Abstract: Selection modulates gene sequence evolution in different ways by constraining potential changes of amino acid sequences (purifying selection) or by favoring new and adaptive genetic variants (positive selection). The number of nonsynonymous differences in a pair of protein-coding sequences can be used to quantify the mode and strength of selection. To control for regional variation in substitution rates, the proportionate number of nonsynonymous differences (dN) is divided by the proportionate number of synonymous differences (dS). The resulting ratio (dN/dS) is a widely used indicator for functional divergence to identify particular genes that underwent positive selection. With the ever-growing amount of genome data, summary statistics like mean dN/dS allow gathering information on the mode of evolution for entire species. Both applications hinge on the assumption that dS and mean dS ([~]branch length) are neutral and adequately control for variation in substitution rates across genes and across organisms, respectively. We here explore the validity of this assumption using empirical data based on whole-genome protein sequence alignments between human and 15 other vertebrate species and several simulation approaches. We find that dN/dS does not appropriately reflect the action of selection as it is strongly influenced by its denominator (dS). Particularly for closely related taxa, such as human and chimpanzee, dN/dS can be misleading and is not an unadulterated indicator of selection. Instead, we suggest that inconsistencies in the behavior of dN/dS are to be expected and highlight the idea that this behavior may be inherent to taking the ratio of two randomly distributed variables that are nonlinearly correlated. New null hypotheses will be needed to adequately handle these nonlinear dynamics.

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Abstract: Parent-offspring conflict theory is well supported by theoretical arguments. However, empirical observations are often difficult to interpret and have contradicted one of its most appealing predictions that parent and offspring should disagree over killing of nest or littermates. We present the first examples of deadly conflict between siblings of different cohorts. In Galapagos fur seals (Arctocephalus galapagoensis) and sea lions (Zalophus wollebaeki), mothers often wean their single offspring at 2 years. This leads to a situation where up to 23% of all pups are born while the older sibling is still being nursed. Younger siblings are disadvantaged by being born lighter than neonates without older still dependent siblings. Pups born while an older sib is still dependent grow less in early life (fur seal) and suffer increased early mortality (both species) through direct aggression or scramble competition with the older sibling. This effect is much stronger in years of high sea surface temperature (El Nino) indicating low marine productivity and if the older offspring is a male. In both species, mothers interfere aggressively in this conflict by defending the younger offspring. In years of El Nino, intense resistance to maternal aggression by the older offspring happens frequently in the fur seal. Such resistance against weaning can induce maternal neglect of the newborn. Given substantial year to year variation in offspring growth, maternal aggression forces weaning in the older sibling only if it has reached sufficient size to support itself by foraging. In Galapagos fur seals, pups with older siblings can either represent insurance against loss of older offspring or extra reproductive value.

Abstract: Male vocal displays play an important role in sexual selection through both male-male competition and female choice. While this is supported by numerous studies in birds, much less attention has been paid to the role of such displays in mammals. We investigated the function of vocal displays in a polygynous mammal, the Galapagos sea lion (Zalophus wollebaeki). In our study population a large proportion of mature males are unable to establish a territory, providing the opportunity to compare the vocal behaviour between territorial and non-territorial males. We examined how seasonal patterns of vocalizations differed between territorial and non-territorial males and how the number of females present in a territory influenced behaviour of territorial males. We found that territorial males vocalized at higher rates than non-territorial males, and territorial males vocalized more at the onset than towards the end of territory tenure. During the onset of territory-tenure vocalizations of territorial males were directed more often towards other males than females. Furthermore, we found that vocalizations of territorial males were not only given in male-male interactions, but were also actively directed towards females. Territorial males vocalized at higher rates when more females were present in their territory. Our results suggest that vocalizations are important in male-male interactions, are relevant in territorial disputes and are used as a proxy for females to assess a male's quality.

Abstract: In mammals, hematocrit (Hct) is optimized between the divergent requirements of blood flow characteristics and oxygen transport and storage capacity. This trade-off plays a particularly major role in marine mammals, in which oxygen demand during sustained diving requires high Hct levels. Galapagos sea lions (Zalophus wollebaeki) need a long time after birth to develop from terrestrial life to the state of an independent forager at sea. We here show that pups were born with high Hct of 45%, then reduced Hct during the first 40 d of life to 31% while they remain constantly ashore, and increased Hct again until the adult level (57%) is reached at 1 yr of age when they begin to dive for foraging at sea. A similar, but much more rapidly changing pattern is seen in Weddell seal pups, but not in northern elephant seals, where no reduction in Hct is seen after birth. These and similarly changing patterns in terrestrial mammals likely reflect species specific functional adjustments during development due to a trade-off between the costs of circulation and the changing need to store and transport oxygen.

Abstract: Population substructuring is a fundamental aspect of animal societies. A growing number of theoretical studies recognize that who-meets-whom is not random, but rather determined by spatial relationships or illustrated by social networks. Structural properties of large highly dynamic social systems are notoriously difficult to unravel. Network approaches provide powerful ways to analyse the intricate relationships between social behaviour, dispersal strategies and genetic structure. Applying network analytical tools to a colony of the highly gregarious Galapagos sea lion (Zalophus wollebaeki), we find several genetic clusters that correspond to spatially determined 'network communities'. Overall relatedness was low, and genetic structure in the network can be interpreted as an emergent property of philopatry and seems not to be primarily driven by targeted interactions among highly related individuals in family groups. Nevertheless, social relationships between directly adjacent individuals in the network were stronger among genetically more similar individuals. Taken together, these results suggest that even small differences in the degree of relatedness can influence behavioural decisions. This raises the fascinating prospect that kin selection may also apply to low levels of relatedness within densely packed animal groups where less obvious co-operative interactions such as increased tolerance and stress reduction are important.

Abstract: Background: Oceans are high gene flow environments that are traditionally believed to hamper the build-up of genetic divergence. Despite this, divergence appears to occur occasionally at surprisingly small scales. The Galapagos archipelago provides an ideal opportunity to examine the evolutionary processes of local divergence in an isolated marine environment. Galapagos sea lions (Zalophus wollebaeki) are top predators in this unique setting and have an essentially unlimited dispersal capacity across the entire species range. In theory, this should oppose any genetic differentiation. Results: We find significant ecological, morphological and genetic divergence between the western colonies and colonies from the central region of the archipelago that are exposed to different ecological conditions. Stable isotope analyses indicate that western animals use different food sources than those from the central area. This is likely due to niche partitioning with the second Galapagos eared seal species, the Galapagos fur seal (Arctocephalus galapagoensis) that exclusively dwells in the west. Stable isotope patterns correlate with significant differences in foraging-related skull morphology. Analyses of mitochondrial sequences as well as microsatellites reveal signs of initial genetic differentiation. Conclusion: Our results suggest a key role of intra- as well as inter-specific niche segregation in the evolution of genetic structure among populations of a highly mobile species under conditions of free movement. Given the monophyletic arrival of the sea lions on the archipelago, our study challenges the view that geographical barriers are strictly needed for the build-up of genetic divergence. The study further raises the interesting prospect that in social, colonially breeding mammals additional forces, such as social structure or feeding traditions, might bear on the genetic partitioning of populations.

Abstract: The processes governing social interactions and the resulting relationships among members of colonially organized animals are largely unknown. Using network analytical tools we investigated fundamental components of social structure in a highly fluid fission-fusion society. We constructed a social network and studied the relative importance of sex and age class, fine-scale site fidelity and male territory distribution in a breeding colony of the Galapagos sealion, Zalophus wollebaeki. The social system was partitioned into a hierarchy with at least three levels. The outermost level of organization was given by a single social network, where all individuals of the population were interconnected. This level split into communities and further into cliques. Sex and age class influenced social structure at the population level, whereas fine-scale site fidelity explained most of the structure found at the community level. None of these assortment variables could account for clique structure, which might therefore be explained in terms of individual preferences, genetic relatedness or a combination of both. Male territories did not form the basic unit of social structure, but appeared simply to be superimposed on the structural backbone formed by females and young. (C) 2007 The Association for the Study of Animal Behaviour. Published by Elsevier Ltd. All rights reserved.

Abstract: Under sperm competition, paternity is apportioned by polyandrous females according to the order of matings and the genetic quality of the inseminating males. In order to distinguish between these two effects, we sequentially paired 12 female smooth newts (Lissotriton vulgaris) with each of two males and, where possible, repeated the same procedure in reverse order of the identical males after assumed sperm depletion. For a total of 578 offspring, amplified fragment length polymorphisms genetic markers revealed multiple paternities in all matings, without significant first- or second-male sperm precedence. The paternity share of individual males was transitive across the two trials with male order switch, and successful males had a significantly higher genetic dissimilarity to the female than expected by chance. We argue that patterns of paternity in natural newt populations are determined through a combination of good genes and relatedness.

Abstract: Background Accurate formal taxonomic designations are thought to be of critical importance for the conservation of endangered taxa. The Galápagos sea lion (GSL), being appreciated as a key element of the Galápagos marine ecosystem, has lately been listed as 'vulnerable' by the IUCN. To date there is, however, hardly any scientific evidence, whether it constitutes a separate entity from its abundant Californian neighbour (CSL). In this paper, we delineate the taxonomic relationships within the genus Zalophus being comprised of the Galápagos sea lion, the Californian sea lion and the already extinct Japanese sea lion (JSL). Results Using a set of different phylogenetic reconstruction approaches, we find support for monophyly of all three taxa without evidence of reticulation events. Molecular clock estimates place time to common ancestry of the Galápagos sea lion and the Californian sea lion at about 2.3 ± 0.5 mya. Genetic separation is further suggested by diagnostic SNPs in the mitochondrial and nuclear genome. Microsatellite markers confirm this trend, showing numerous private alleles at most of the 25 investigated loci. Microsatellite-based estimates of genetic differentiation between the Galápagos sea lion and the Californian sea lion indicate significant genetic differentiation. Gene diversity is 14% lower in the Galápagos sea lion than in the Californian sea lion, but there is no evidence for recent bottleneck events in the Galápagos sea lion. Conclusion Based on molecular evidence we build a case for classifying the Galápagos sea lion (Zalophus wollebaeki), the Californian sea lion (Zalophus californianus) and the Japanese sea lion (Zalophus japonicus) as true species. As morphological characters do not necessarily fully reflect the rapid divergence on the molecular level, the study can be considered as a test case for deriving species status from molecular evidence. We further use the results to discuss the role of genetics in conservation policy for an organism that already is under the general protection of the habitat it lives in.

Abstract: We isolated and characterized 10 novel dinucleotide microsatellite loci from the Galapagos sea lion (Zalophus californianus wollebaeki) and tested their amplification utility in four further otariid species (Zalophus californianus californianus, Arctocephalus gazella, Arctocephalus australis and Eumetopias jubatus) and three phocid species (Hydrurga leptonyx, Halichoerus grypus and Phoca vitulina). All of the loci amplified polymorphic polymerase chain reaction (PCR) products in at least three species other than the Galapagos sea lion. These markers will be useful for studies of pinniped mating systems, genetic structure and genetic diversity.

Abstract: Site fidelity has been widely discussed, but rarely been related explicitly to a species' social context. This is surprising, as fine-scale site fidelity constitutes an important structural component in animal societies by setting limits to an individual's social interaction space. The study of fine-scale site fidelity is complicated by the fact that it is inextricably linked to patterns of habitat use. We here document fine-scale site fidelity in the Galapagos sea lion (Zalophus wollebaeki) striving to disentangle these two aspects of spatial behaviour. Regardless of sex and age, all individuals used small, cohesive home ranges, which were stable in size across the reproductive and non-reproductive season. Home ranges showed a large individual component and did not primarily reflect age- or sex-specific habitat requirements. Site specificity could be illustrated up to a resolution of several metres. Long-term site fidelity was indicated by home range persistence over 3 years and the degree of site fidelity was unaffected by habitat, but showed seasonal differences: it was lower between reproductive and non-reproductive periods than between reproductive seasons. We further examined static and social interaction within mother-offspring pairs, which constitute a central social unit in most mammalian societies. Regardless of the occupied habitat type, adult females with offspring had smaller home range sizes than non-breeding females, demonstrating the importance of spatial predictability for mother-offspring pairs that recurrently have to reunite after females' foraging sojourns. While social interaction with the mother dropped to naught in both sexes after weaning, analysis of static interaction suggested female-biased home range inheritance. Dispersal decisions were apparently not based on habitat quality, but determined by the offspring's sex. We discuss the implication of observed fine-scale site fidelity patterns on habitat use, dispersal decisions and social structure in colonial breeding pinnipeds.

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Abstract: In many sexually dimorphic species adult sexes tend to segregate socially, spatially, or in habitat use. Several hypotheses have been formulated regarding underlying mechanisms. We investigated terrestrial habitat use and sexual segregation in a tropical otariid, the Galapagos sea lion (Zalophus californianus wollebaeki), where most of the hypotheses can be ruled out a priori. Factors relating to thermoregulation and costs of locomotion were of prime importance for habitat use. Habitats directly adjacent to the sea, with simple structured flat surfaces, shade, and tide pools were most frequented, but sexes and age classes differed in their usage patterns. Sexual segregation, both spatial and by habitat was pronounced in the reproductive period (RP), but remained high during the nonreproductive period (NRP). A GLM model of habitat use showed that in both seasons adult males frequented habitat types that adult females and other age classes used much less. Males were most abundant in suboptimal inland habitats, which offered only shade for cooling. Females with newborns differed in habitat use from females with older offspring and lone females. Spatial and habitat segregation are explained most parsimoniously as by-products of social processes, primarily intrasexual competition and female avoidance of male harassment, linked to the polygynous mating system.

Abstract: DNA activation tagging was used to generate tobacco mutants with increased tolerance to antimicrotubular herbicides and chilling stress. After transformation, protoplast-derived calli were screened for tolerance to treatments that affect microtubule assembly. In one screen mutants with tolerance to aryl carbamates ( a blocker of microtubule assembly) were selected, the second screen was targeted to chilling-tolerant mutants that could survive for several months at 3degreesC, a third screen combined both factors. The resistance of these mutants to aryl carbamates or chilling was accompanied by resistance of microtubules to these factors. The carbamate tolerant mutants were cross-resistant to chilling stress. This was mirrored by an adaptive reorganization of microtubules and a reduction of microtubule dynamics in response to chilling. The analysis of these mutants suggests ( 1) that microtubule dynamics limit the tolerance to chilling and EPC, and ( 2) that the cold sensitivity of microtubules limits chilling tolerance in tobacco.