Increase In Thermal Tolerance Research Articles

Emergent increase in coral thermal tolerance reduces mass bleaching under climate change

Recurrent mass bleaching events threaten the future of coral reefs. To persist under climate change, corals will need to endure progressively more intense and frequent marine heatwaves, yet it remains unknown whether their thermal tolerance can keep pace with warming. Here, we reveal an emergent increase in the thermal tolerance of coral assemblages at a rate of 0.1 °C/decade for a remote Pacific coral reef system. This led to less severe bleaching impacts than would have been predicted otherwise, indicating adaptation, acclimatisation or shifts in community structure. Using future climate projections, we show that if thermal tolerance continues to rise over the coming century at the most-likely historic rate, substantial reductions in bleaching trajectories are possible. High-frequency bleaching can be fully mitigated at some reefs under low-to-middle emissions scenarios, yet can only be delayed under high emissions scenarios. Collectively, our results indicate a potential ecological resilience to climate change, but still highlight the need for reducing carbon emissions in line with Paris Agreement commitments to preserve coral reefs.

Rapid heat hardening in embryos of the lizard Anolis sagrei.

Adaptive thermal tolerance plasticity can dampen the negative effects of warming. However, our knowledge of tolerance plasticity is lacking for embryonic stages that are relatively immobile and may benefit the most from an adaptive plastic response. We tested for heat hardening capacity (a rapid increase in thermal tolerance that manifests in minutes to hours) in embryos of the lizard Anolis sagrei. We compared the survival of a lethal temperature exposure between embryos that either did (hardened) or did not (not hardened) receive a high but non-lethal temperature pre-treatment. We also measured heart rates (HRs) at common garden temperatures before and after heat exposures to assess metabolic consequences. 'Hardened' embryos had significantly greater survival after lethal heat exposure relative to 'not hardened' embryos. That said, heat pre-treatment led to a subsequent increase in embryo HR that did not occur in embryos that did not receive pre-treatment, indicative of an energetic cost of mounting the heat hardening response. Our results are not only consistent with adaptive thermal tolerance plasticity in these embryos (greater heat survival after heat exposure), but also highlight associated costs. Thermal tolerance plasticity may be an important mechanism by which embryos respond to warming that warrants greater consideration.

Evolution of thermal tolerance and phenotypic plasticity under rapid and slow temperature fluctuations.

Global warming is associated with an increase in sea surface temperature and its variability. The consequences of evolving in variable, fluctuating environments are explored by a large body of theory: when populations evolve in fluctuating environments the frequency of fluctuations determines the shapes of tolerance curves (indicative of habitats that organisms can inhabit) and trait reaction norms (the phenotypes that organisms display across these environments). Despite this well-established theoretical backbone, predicting how trait and tolerance curves will evolve in organisms at the foundation of marine ecosystems remains a challenge. Here, we used a globally distributed phytoplankton, Thalassiosira pseudonana, and show that fluctuations in temperature on scales of 3–4 generations rapidly selected for populations with enhanced trait plasticity and elevated thermal tolerance. Fluctuations spanning 30–40 generations selected for the formation of two stable, genetically and physiologically distinct populations, one evolving high trait plasticity and enhanced thermal tolerance, and the other, akin to samples evolved under constant warming, with lower trait plasticity and a smaller increase in thermal tolerance.

Gene expression plasticity, genetic variation and fatty acid remodelling in divergent populations of a tropical bivalve species.

Ocean warming challenges marine organisms' resilience, especially for species experiencing temperatures close to their upper thermal limits. A potential increase in thermal tolerance might significantly reduce the risk of population decline, which is intrinsically linked to variability in local habitat temperatures. Our goal was to assess the plastic and genetic potential of response to elevated temperatures in a tropical bivalve model, Pinctada margaritifera. We benefit from two ecotypes for which local environmental conditions are characterized by either large diurnal variations in the tide pools (Marquesas archipelago) or low mean temperature with stable to moderate seasonal variations (Gambier archipelago). We explored the physiological basis of individual responses to elevated temperature, genetic divergence as well as plasticity and acclimation by combining lipidomic and transcriptomic approaches. We show that P. margaritifera has certain capacities to adjust to long-term elevated temperatures that was thus far largely underestimated. Genetic variation across populations overlaps with gene expression and involves the mitochondrial respiration machinery, a central physiological process that contributes to species thermal sensitivity and their distribution ranges. Our results present evidence for acclimation potential in P. margaritifera and urge for longer term studies to assess populations resilience in the face of climate change.

Repeatability of Voluntary Thermal Maximum and Covariance with Water Loss Reveal Potential for Adaptation to Changing Climates.

AbstractAlthough climate warming poses a grave threat to amphibians, little is known about the capacity of this group to evolve in response to warming. The capacity of key traits to evolve depends on the presence of genetic variation on which selection can act. Here, we use repeatability estimates to estimate the potential upper bounds of heritable genetic variation in voluntary and critical thermal maxima of gray-cheeked salamanders (Plethodon metcalfi). Increases in thermal tolerance may also require concordant increases in resistance to water loss because hotter temperatures incur greater evaporative risk. Therefore, we also tested for a correlation between voluntary thermal maxima and resistance to water loss and conducted an acclimation study to test for covariation between these traits in response to warming. Voluntary thermal maxima exhibited low to moderate levels of repeatability (, ), while critical thermal maxima exhibited no statistically significant repeatability (, ). Voluntary thermal maxima also correlated positively with resistance to water loss (, ) but only when controlling for body mass. Voluntary thermal maxima and resistance to water loss also exhibited different acclimatory responses across control (12°C-18°C) and warm (18°C-24°C) temperature regimes, indicating a potential decoupling of traits in different thermal environments. By addressing the repeatability of thermal tolerance and the potential for covariation with resistance to water loss, we begin to address some of the key requirements of amphibians to evolve in warming climates.

Negative relationship between thermal tolerance and plasticity in tolerance emerges during experimental evolution in a widespread marine invertebrate.

Whether populations can adapt to predicted climate change conditions, and how rapidly, are critical questions for the management of natural systems. Experimental evolution has become an important tool to answer these questions. In order to provide useful, realistic insights into the adaptive response of populations to climate change, there needs to be careful consideration of how genetic differentiation and phenotypic plasticity interact to generate observed phenotypic changes. We exposed three populations of the widespread copepod Acartia tonsa (Crustacea) to chronic, sublethal temperature selection for 15 generations. We generated thermal survivorship curves at regular intervals both during and after this period of selection to track the evolution of thermal tolerance. Using reciprocal transplants between ambient and warming conditions, we also tracked changes in the strength of phenotypic plasticity in thermal tolerance. We observed significant increases in thermal tolerance in the Warming lineages, while plasticity in thermal tolerance was strongly reduced. We suggest these changes are driven by a negative relationship between thermal tolerance and plasticity in thermal tolerance. Our results indicate that adaptation to warming through an increase in thermal tolerance might not reduce vulnerability to climate change if the increase comes at the expense of tolerance plasticity. These results illustrate the importance of considering changes in both a trait of interest and the trait plasticity during experimental evolution.

Salinity-Conveyed Thermotolerance in the Coral Model Aiptasia Is Accompanied by Distinct Changes of the Bacterial Microbiome

Coral bleaching, i.e., the loss of photosynthetic algal endosymbionts, caused by ocean warming is now among the main factors driving global reef decline, making the elucidation of factors that contribute to thermotolerance important. Recent studies implicate high salinity as a contributing factor in cnidarians, potentially explaining the high thermotolerance of corals from the Arabian Seas. Here we characterized bacterial community composition under heat stress at different salinities using the coral model Aiptasia. Exposure of two Aiptasia host-algal symbiont pairings (H2-SSB01 and CC7-SSA01) to ambient (25°C) and heat stress (34°C) temperatures at low (36 PSU), intermediate (39 PSU), and high (42 PSU) salinities showed that bacterial community composition at high salinity was significantly different, concomitant with reduced bleaching susceptibility in H2-SSB01, not observed in CC7-SSA01. Elucidation of bacteria that showed increased relative abundance at high salinity, irrespective of heat stress, revealed candidate taxa that could potentially contribute to the observed increased thermotolerance. We identified 4 (H2-SSB01) and 3 (CC7-SSA01) bacterial taxa belonging to the orders Alteromonadales (1 OTU), Oligoflexales (1 OTU), Rhizobiales (2 OTUs), and Rhodobacterales (2 OTUs), suggesting that only few bacterial taxa are potential contributors to an increase in thermal tolerance at high salinities. These taxa have previously been implicated in nitrogen and DMSP cycling, processes that are considered to affect thermotolerance. Our study demonstrates microbiome restructuring in symbiotic cnidarians under heat stress at different salinities. As such, it underlines how host-associated bacterial communities adapt to prevailing environmental conditions with putative consequences for the environmental stress tolerance of the emergent metaorganism.

The effects of repeat acute thermal stress on the critical thermal maximum (CTmax) and physiology of juvenile shortnose sturgeon (Acipenser brevirostrum)

The shortnose sturgeon (Acipenser brevirostrum Lesueur, 1818) is a species of special concern in Canada, but little is known about their thermal biology. Information on the upper thermal tolerance of shortnose sturgeon becomes valuable for predicting future survival particularly with climate change and improving species management. Using a modified critical thermal maximum (CTmax) methodology, the objective is to determine whether previous thermal stress affects the thermal tolerance of juvenile shortnose sturgeon when exposed to a second thermal stress event. Prior exposure to thermal stress (CTmax1) did not affect the thermal tolerance (CTmax2) of juvenile shortnose sturgeon when a 24 h recovery period was allotted between tests. However, a significant increase in thermal tolerance occurred when the recovery time between the two thermal challenges was 1 h. Plasma glucose, lactate, and osmolality were all significantly affected by thermal stress, but values returned to control levels within 24 h. Hematocrit and plasma chloride concentrations were not significantly affected by thermal stress. All fish survived the CTmax testing. The data indicate that the thermal tolerance of juvenile shortnose sturgeon is modified when multiple thermal stresses occur closer together (1 h) but not if separated by a longer time period (24 h).

Impact of Changing Temperature on Critical Thermal Maximum and Metabolic Rate of Uca perplexa and Uca crassipes

This study quantifies thermal tolerance and metabolic rates for two fiddler crab species ( Uca perplexa and Uca crassipes ) exposed to increasing temperatures. Uca perplexa prefers sun-exposed mangal zones, while U. crassipes inhabits shaded as well as sun-exposed areas. A total forty crabs (20 crabs from each species) were collected from the Ambeua mangrove on Kaledupa Island, and transported to the Hoga Island Research Laboratory for testing. Fifteen crabs of each species were used for CTmax trials, and five crabs were used in metabolic trials. Crabs were acclimated for 10 days at 26, 28, or 30°C prior to experimentation. Critical Thermal Maximum (CTmax) was measured by increasing the temperature by 0.3°C per minute until loss of righting responses was observed. A Gilson differential respirometer was used to determine oxygen uptake at 26 and 31°C for each species, and the results used to calculate temperature quotient (Q 10 ) values. While both crab species showed an increase in thermal tolerance with increasing acclimation temperature, U. crassipes was more heat tolerant (CTmax = 42.21°C), than U. perplexa (CTmax = 41.95°C). Likewise, U. crassipes was less metabolically sensitive to temperature increase (Q 10 = 1.33) than U. perplexa , (Q 10 = 2.38) suggesting that U. crassipes is better adapted to high environmental temperature conditions.

Long-Term Acclimation to Different Thermal Regimes Affects Molecular Responses to Heat Stress in a Freshwater Clam Corbicula Fluminea.

Global climate change (GCC) can negatively affect freshwater ecosystems. However, the degree to which freshwater populations can acclimate to long-term warming and the underlying molecular mechanisms are not yet fully understood. We used the cooling water discharge (CWD) area of a power plant as a model for long-term warming. Survival and molecular stress responses (expression of molecular chaperones, antioxidants, bioenergetic and protein synthesis biomarkers) to experimental warming (20–41 °C, +1.5 °C per day) were assessed in invasive clams Corbicula fluminea from two pristine populations and a CWD population. CWD clams had considerably higher (by ~8–12 °C) lethal temperature thresholds than clams from the pristine areas. High thermal tolerance of CWD clams was associated with overexpression of heat shock proteins HSP70, HSP90 and HSP60 and activation of protein synthesis at 38 °C. Heat shock response was prioritized over the oxidative stress response resulting in accumulation of oxidative lesions and ubiquitinated proteins during heat stress in CWD clams. Future studies should determine whether the increase in thermal tolerance in CWD clams are due to genetic adaptation and/or phenotypic plasticity. Overall, our findings indicate that C. fluminea has potential to survive and increase its invasive range during warming such as expected during GCC.

Role of sequential low-tide-period conditions on the thermal physiology of summer and winter laboratory-acclimated fingered limpets, Lottia digitalis

The rocky intertidal zone is among one of the most highly variable environments on Earth, with rapid and unpredictable changes in temperature on a daily basis. Numerous studies have investigated the thermal physiology of intertidal animals; however, few have focused on an organism’s capacity to withstand repeated heat stress during the emersion of low-tide periods and how previous exposures to sublethal increases in temperature may modulate the capacity to withstand severe heat stress. Lottia digitalis, a species of limpet ubiquitous along the Pacific coast of North America, were acclimated under ambient ocean conditions in the laboratory during the summer and winter months. Limpets were aerially exposed with or without preliminary heat exposures of varying magnitudes (15–35 °C for 2 h) the day before being challenged to a lethal temperature increase to investigate how sequential low-tide-period conditions affect upper thermal tolerance and temperature sensitivity. Limpets with a preliminary aerial exposure of 25–35 °C (summer) and 20 °C (winter) had greater upper critical thermal limits of cardiac performance as determined by final breakpoint temperature (~1.5–6 °C increase) and flatline temperature (~1–2 °C increase) than limpets with no previous exposure. The magnitude of temperature increase that conferred significant increases in thermal tolerance differed in summer and winter, reflecting seasonal differences in the thermal environment in nature. Fingered limpets’ upper thermal tolerance is plastic and likely modulated by the previous day’s low-tide exposure, demonstrating the importance of incorporating the repeated nature of stress into thermal physiology research in intertidal organisms.

Rapid evolution of thermal tolerance in the water flea Daphnia

A manipulation experiment, combined with reconstructed evolutionary history from a dormant egg bank found in recent lake sediments, reveals that water fleas (Daphnia) can exhibit a rapid increase in their capacity to tolerate higher temperatures. Global climate is changing rapidly, and the degree to which natural populations respond genetically to these changes is key to predicting ecological responses1,2,3. So far, no study has documented evolutionary changes in the thermal tolerance of natural populations as a response to recent temperature increase. Here, we demonstrate genetic change in the capacity of the water flea Daphnia to tolerate higher temperatures using both a selection experiment and the reconstruction of evolution over a period of forty years derived from a layered dormant egg bank. We observed a genetic increase in thermal tolerance in response to a two-year ambient +4 °C selection treatment and in the genotypes of natural populations from the 1960s and 2000s hatched from lake sediments. This demonstrates that natural populations have evolved increased tolerance to higher temperatures, probably associated with the increased frequency of heat waves over the past decades, and possess the capacity to evolve increased tolerance to future warming.

Thermal stability engineering of Glomerella cingulata cutinase

Cutinase has been ascertained as a biocatalyst for biotechnological and industrial bioprocesses. The Glomerella cingulata cutinase was genetically modified to enhance its enzymatic performance to fulfill industrial requirements. Two sites were selected for mutagenesis with the aim of altering the surface electrostatics as well as removing a potentially deamidation-prone asparagine residue. The N177D cutinase variant was affirmed to be more resilient to temperature increase with a 2.7-fold increase in half-life at 50°C as compared with wild-type enzyme, while, the activity at 25°C is not compromised. Furthermore, the increase in thermal tolerance of this variant is accompanied by an increase in optimal temperature. Another variant, the L172K, however, exhibited higher enzymatic performance towards phenyl ester substrates of longer carbon chain length, yet its thermal stability is inversely affected. In order to restore the thermal stability of L172K, we constructed a L172K/N177D double variant and showed that these two mutations yield an improved variant with enhanced activity towards phenyl ester substrates and enhanced thermal stability. Taken together, our study may provide valuable information for enhancing catalytic performance and thermal stability in future engineering endeavors.

Differential thermal tolerance and energetic trajectories during ontogeny in porcelain crabs, genus Petrolisthes

Thermal tolerance limits of marine intertidal zone organisms are elevated compared to subtidal species, but are typically just slightly higher than maximal habitat temperatures. The small thermal safety margins maintained by intertidal zone organisms suggest that high thermal tolerance is associated with a physiological cost. If true, we hypothesize that species that transition between intertidal zone and planktonic habitats during ontogeny, will adjust their thermal tolerance accordingly to capitalize upon potential energy savings while in a thermally benign habitat. We tested this hypothesis in porcelain crabs that transition between the thermally stressful, intertidal zone as embryos, to the thermally benign pelagic zone as larvae, and back at settlement. We found the more thermally tolerant, mid-intertidal zone species, Petrolisthes cinctipes, and the less thermally tolerant, subtidal zone species, Petrolisthes manimacilis, exhibited reduced thermal tolerance (LT50) in the transition from embryos to larvae. This was associated with an increased oxygen consumption rate in both species, though P. cinctipes exhibited a significantly greater increase in oxygen consumption. P. cinctipes also showed an increase in thermal tolerance in settled juveniles compared to pelagic zoea I larvae, resulting in an overall V-shaped thermal tolerance relationship during ontogeny, while in P. manimaculis thermal tolerance was significantly lower in juveniles compared to zoea I. In neither species were these changes (zoea I to juvenile) associated with a significant change in metabolism. While embryos and juveniles of P. cinctipes have thermal tolerance limits near intertidal habitat thermal maxima (∼32.5°C), all three life-history stages in P. manimaculis (especially embryos and larvae) exhibit considerable thermal safety margins. The mechanisms underlying this “excess” thermal tolerance in P. manimacilis embryos are unknown, but suggest that patterns of thermal tolerance in early life history stages are species-specific.

Limited potential for adaptation to climate change in a broadly distributed marine crustacean.

The extent to which acclimation and genetic adaptation might buffer natural populations against climate change is largely unknown. Most models predicting biological responses to environmental change assume that species' climatic envelopes are homogeneous both in space and time. Although recent discussions have questioned this assumption, few empirical studies have characterized intraspecific patterns of genetic variation in traits directly related to environmental tolerance limits. We test the extent of such variation in the broadly distributed tidepool copepod Tigriopus californicus using laboratory rearing and selection experiments to quantify thermal tolerance and scope for adaptation in eight populations spanning more than 17° of latitude. Tigriopus californicus exhibit striking local adaptation to temperature, with less than 1 per cent of the total quantitative variance for thermal tolerance partitioned within populations. Moreover, heat-tolerant phenotypes observed in low-latitude populations cannot be achieved in high-latitude populations, either through acclimation or 10 generations of strong selection. Finally, in four populations there was no increase in thermal tolerance between generations 5 and 10 of selection, suggesting that standing variation had already been depleted. Thus, plasticity and adaptation appear to have limited capacity to buffer these isolated populations against further increases in temperature. Our results suggest that models assuming a uniform climatic envelope may greatly underestimate extinction risk in species with strong local adaptation.

Major bleaching events can lead to increased thermal tolerance in corals

Climate change is a major threat to coral reef ecosystems worldwide. A key determinant of the fate of reef corals in a warming climate is their capacity to tolerate increasing thermal stress. Here, an increase in thermal tolerance is demonstrated for three major coral genera (Acropora, Pocillopora and Porites) following the exten- sive mass bleaching event that occurred on the Great Barrier Reef (Australia) in 1998. During the subsequent and more severe thermal stress event in 2002, bleaching severity was 30-100% lower than predicted from the relationship between severity and thermal stress in 1998, despite higher solar irradiances during the 2002 thermal event. Coral genera most susceptible to thermal stress (Pocillopora and Acropora) showed the greatest increase in tolerance. Although bleaching was severe in 1998, whole- colony mortality was low at most study sites. Therefore, observed increases in thermal tolerance cannot be explained by selective mortality alone, suggesting a capacity for acclimatization or adaptation. Although the vulnerability of coral reefs remains largely dependent on the rate and extent of climate change, such increase in thermal tolerance may delay the onset of mass coral mor- talities in time for the implementation of low-emission scenarios and effective management.

Inducible heat tolerance in Antarctic notothenioid fishes

Significant increases in heat tolerance (time of survival at 14� C) were observed for some, but not all, species of notothenioid fishes collected from McMurdo Sound, Antarctica (77� 51¢S) following acclimation to 4� C. The increase in thermal tolerance was rapid in Trematomus bernacchii, developing within 1-2 days of acclimation to 4� C. Long-term (6-8 weeks) acclimation to 4� C led to greater heat tolerance in Trematomus pennellii than in T. bernacchii. Unlike its demersal congeners, the cryopelagic notothenioid Pa- gothenia borchgrevinki did not increase heat tolerance during warm acclimation. A deep-living zoarcid fish, Lycodichthys dearborni, also failed to increase heat tolerance, but survived significantly (> threefold) longer at 14� C than the notothenioids.

Global assessment of coral bleaching and required rates of adaptation under climate change.

Elevated ocean temperatures can cause coral bleaching, the loss of colour from reef-building corals because of a breakdown of the symbiosis with the dinoflagellate Symbiodinium. Recent studies have warned that global climate change could increase the frequency of coral bleaching and threaten the long-term viability of coral reefs. These assertions are based on projecting the coarse output from atmosphere-ocean general circulation models (GCMs) to the local conditions around representative coral reefs. Here, we conduct the first comprehensive global assessment of coral bleaching under climate change by adapting the NOAA Coral Reef Watch bleaching prediction method to the output of a low- and high-climate sensitivity GCM. First, we develop and test algorithms for predicting mass coral bleaching with GCM-resolution sea surface temperatures for thousands of coral reefs, using a global coral reef map and 1985-2002 bleaching prediction data. We then use the algorithms to determine the frequency of coral bleaching and required thermal adaptation by corals and their endosymbionts under two different emissions scenarios. The results indicate that bleaching could become an annual or biannual event for the vast majority of the world's coral reefs in the next 30-50 years without an increase in thermal tolerance of 0.2-1.0°C per decade. The geographic variability in required thermal adaptation found in each model and emissions scenario suggests that coral reefs in some regions, like Micronesia and western Polynesia, may be particularly vulnerable to climate change. Advances in modelling and monitoring will refine the forecast for individual reefs, but this assessment concludes that the global prognosis is unlikely to change without an accelerated effort to stabilize atmospheric greenhouse gas concentrations.

Thermal tolerance and heat shock proteins in encysted embryos of Artemia from widely different thermal habitats

Encysted embryos (cysts) of the brine shrimp, Artemia provide an excellent model system for the study of biochemical adaptation to environmental extremes. Here, we describe an experiment in which cysts of A. franciscana from the San Francisco Bay (SFB), California, U.S.A., were inoculated into experimental ponds in the Mekong Delta region of Vietnam where water temperatures are much higher than the SFB. Cysts produced in each of three successive growing seasons (1996–1998) were collected and examined in the laboratory for resistance to high temperature and relative contents of three stress proteins (Hsp-70, artemin and p26). Thermal adaptation took place rapidly, during the first growing season. The increase in thermal tolerance was reflected in an overall increase in stress protein content, compared to SFB cysts used for the initial inoculation. Also examined were cysts of A. tibetiana collected from a lake on the high plateau of Tibet, PR China, almost 4.5 km above sea level. These cysts were very sensitive to high temperatures, and contained much lower levels of all stress proteins examined, compared to A. franciscana cysts from SFB and Vietnam. Cysts of A. sinica, collected from a hypersaline lake in Inner Mongolia, PR China, were examined in the same fashion and found to be similar to SFB cysts in terms of thermal resistance and stress protein content. The harsh environments in which Artemia are found, and the great diversity of its habitats, world-wide, provide excellent opportunities to relate the ecological setting of an organism to the underlying physiological and biochemical processes enabling its survival.

Heat hardening in response to two types of heat shock in the lungless salamanders Eurycea bislineata and Desmognathus ochrophaeus

1. 1. The induction and decay kinetics of rapid increases in thermal tolerance (heat hardening) in Eurycea bislineata following exposure to either Critical Thermal Maximum (CTM) or 1 h at 2° below CTM were similar. Thermal tolerance increased to a maximum within 1 1 2 h post-heat treatment of either type, and returned to near the original level within 24 h. However, the one hour heat treatment resulted in a larger amount of heat hardening (0.9 vs 0.6°C at optimum development). 2. 2. Most Desmognathus ochrophaeus did not heat harden in response to either CTM or a one hour sub-CTM exposure. 3. 3. CTM was significantly higher in D. ochrophaeus (33.9°C) than in E. bislineata (33.2°C), whereas certain sub-CTM changes in behavior/coordination were lower in D. ochrophaeus. The importance of these sub-CTM changes is discussed.