Marine snow is a ubiquitous feature of the ocean and an important agent in the transport of energy and nutrients through marine ecosystems. Diatom aggregates, which form during blooms and, to a lesser extent, by the resuspension of benthic biofilms, are a primary source of marine snow. Genera commonly found in diatom aggregates are: Nitzschia, Chaetoceros, Rhizosolenia, Leptocylindricus, Skeletonema and Thalassionema. Most fieldwork has been restricted to a limited number of locations in the Northern Hemisphere. To quantify the global impact of diatom aggregation there is a need to conduct fieldwork in a wider range of areas, particularly in the Southern Hemisphere. Aggregates form when particles collide and stick together. Collisions in the water column are affected by turbulence, differential settlement and animal feeding, whereas diatom stickiness is affected by extracellular polymeric substances (EPS). Laboratory experiments have demonstrated that diatoms produce more EPS under nutrient limitation, although little is known about how limitation by different nutrients affects the quantity and composition of EPS and subsequent stickiness. EPS form three pools in the environment: cell coatings, soluble EPS and transparent exopolymeric particles (TEP). There is a need to investigate the dynamics of conversion between the pools of EPS by both abiotic and biological processes and how these conversions affect aggregate concentration and structure. Processes governing disaggregation have been largely overlooked, although they are as important as aggregation in determining the dynamics of aggregate concentrations in the water column. The biogeochemical significance of diatom aggregates as a means of transporting carbon and other nutrients from the euphotic zone to the seabed is well established. However, the internal biogeochemistry of aggregates is not well understood. Aggregates contain anaerobic microsites and further work is required to establish whether aggregates are significant sinks for nitrogen in the water column through anaerobic denitrification. Several hypotheses have been proposed to explain diatom aggregation in the field, but many of these are flawed because the mechanisms and adaptive explanations proposed require natural selection to operate at the level of populations rather than genes or individuals.

A compilation of data on the elemental composition of marine phytoplankton from published studies was used to determine the range of C[ratio ]N[ratio ]P. The N[ratio ]P ratio of algae and cyanobacteria is very plastic in nutrient-limited cells, ranging from <5 mol N[ratio ]mol P when phosphate is available greatly in excess of nitrate or ammonium to >100 mol N[ratio ]mol P when inorganic N is present greatly in excess of P. Under optimal nutrient-replete growth conditions, the cellular N[ratio ]P ratio is somewhat more constrained, ranging from 5 to 19 mol N[ratio ]mol P, with most observations below the Redfield ratio of 16. Limited data indicate that the critical N[ratio ]P that marks the transition between N- and P-limitation of phytoplankton growth lies in the range 20–50 mol N[ratio ]mol P, considerably in excess of the Redfield ratio. Biochemical composition can be used to constrain the critical N[ratio ]P. Although the biochemical data do not preclude the critical N[ratio ]P from being as high as 50, the typical biochemical composition of nutrient-replete algae and cyanobacteria suggests that the critical N[ratio ]P is more likely to lie in the range between 15 and 30. Despite the observation that the overall average N[ratio ]P composition of marine particulate matter closely approximates the Redfield ratio of 16, there are significant local variations with a range from 5 to 34. Consistent with the culture studies, lowest values of N[ratio ]P are associated with nitrate- and phosphate-replete conditions. The highest values of N[ratio ]P are observed in oligotrophic waters and are within the range of critical N[ratio ]P observed in cultures, but are not so high as to necessarily invoke P-limitation. The C[ratio ]N ratio is also plastic. The average C[ratio ]N ratios of nutrient-replete phytoplankton cultures, oceanic particulate matter and inorganic N and C draw-down are slightly greater than the Redfield ratio of 6·6. Neither the analysis of laboratory C[ratio ]N[ratio ]P data nor a more theoretical approach based on the relative abundance of the major biochemical molecules in the phytoplankton can support the contention that the Redfield N[ratio ]P reflects a physiological or biochemical constraint on the elemental composition of primary production.

The plastid organelle of plants, eukaryotic algae and apicomplexan protists contains its own DNA genome (the ‘plastome’). In higher plants, at least two RNA polymerases have been shown to participate in the transcription of plastid genes. The multisubunit ‘plastid-encoded plastid RNA polymerase’ (PEP) is derived from the cyanobacterial ancestor of the organelle and is structurally similar to eubacterial RNA polymerases. The core subunits of PEP are encoded by genes on the plastome, whilst sigma-like transcription factors and other regulatory factors are encoded in the nucleus. In contrast, the ‘nuclear-encoded plastid RNA polymerase’ (NEP) is a single-subunit enzyme related to the T3/T7 bacteriophage and mitochondrial RNA polymerases that is believed to have arisen by duplication of the nuclear gene for the mitochondrial enzyme. The situation in algal plastids is less well understood. All algal groups, apart from possibly the dinoflagellates, possess a PEP but it is far from clear whether any possess additional plastid RNA polymerases. In this article we review what is known about the transcription apparatus of algal plastids, and examine the evidence for and against the presence of NEPs.

Microphytobenthos are the major intertidal primary producers in many estuarine systems and assessment of their biomass is essential in ecological studies. Algal biomass can be assessed by the use of proxy measurements, e.g. chlorophyll a (Chl a), using a lengthy and destructive extraction procedure. Fluorescence is a non-destructive and in situ alternative, recently applied to microphytobenthos, using the minimum fluorescence signal (Fo) as a measurement of Chl a. In this study, Fo after 15 min of dark adaptation (Fo15) showed a significant positive correlation with Chl a (r = 0.84, p<0.001, n = 24). The relationship between Fo15 and Chl a extracted from sediment samples of increasing sample depth (0.2–2 mm) became less significant as sediment depth increased. The strongest relationship between Fo15 and Chl a, expressed as concentration (mg m−2) and content (mg kg−1), was found when all data were combined, but nearly comparable levels of association were found for in situ measurements using sediment cores up to 2.0 mm deep. Results suggested that full dark adaptation was not always achieved because a significant negative correlation was observed between Fv/Fm and ambient irradiance for seasonal data. In addition, Fo15 showed a more significant correlation with Chl a than with Fm15, suggesting some residual level of non-photochemical quenching after 15 min in the dark. Despite the incomplete dark adaptation, the highly significant relationships between Fo15 and both Chl a concentration and content suggest that the use of Fo15 is a suitable in situ proxy method of estimating algal biomass.

The utility of absorbance and fluorescence-emission spectra for discriminating among microalgal phylogenetic groups, selected species, and phycobilin- and non-phycobilin-containing algae was examined using laboratory cultures. A similarity index algorithm, in conjunction with fourth-derivative transformation of absorbance spectra, provided discrimination among the chlorophyll [Chl] a/phycobilin (cyanobacteria), Chl a/Chl c/phycobilin (cryptophytes), Chl a/Chl b (chlorophytes, euglenophytes, prasinophytes), Chl a/Chl c/fucoxanthin (diatoms, chrysophytes, raphidophytes) and Chl a/Chl c/peridinin (dinoflagellates) spectral classes, and often between/among closely related phylogenetic groups within a class. Spectra for phylogenetic groups within the Chl a/Chl c/fucoxanthin, Chl a/Chl c/peridinin, Chl a/phycobilins and Chl a/Chl c/phycobilin classes were most distinguishable from spectra for groups within the Chl a/Chl b spectral class. Chrysophytes/diatoms/raphidophytes and dinoflagellates (groups within the comparable spectral classes, Chl a/Chl c/fucoxanthin and Chl a/Chl c/peridinin, respectively) displayed the greatest similarity between/among groups. Spectra for phylogenetic groups within the Chl a/Chl c classes displayed limited similarity with spectra for groups within the Chl/phycobilin classes. Among the cyanobacteria and chlorophytes surveyed, absorbance spectra of species possessing dissimilar cell morphologies were discriminated, with the greatest range of differentiation occurring among cyanobacteria. Among the cyanobacteria, spectra for selected problematic species were easily discriminated from spectra from each other and from other cyanobacteria. Fluorescence-emission spectra were distinct among spectral classes and the similarity comparisons involving fourth-derivative transformation of spectra discriminated the increasing contribution of distinct cyanobacterial species and between phycobilin- and non-phycobilin-containing species within a hypothetical mixed assemblage. These results were used to elucidate the application for in situ moored instrumentation incorporating such approaches in water quality monitoring programmes, particularly those targeting problematic cyanobacterial blooms.

The crude phlorotannins from the brown alga Eisenia bicyclis showed inhibitory activity against 10 of 13 kinds of glycosidases present in the viscera of the turban shell Turbo cornutus. Phloroglucinol and its oligomers – eckol (a trimer), phlorofucofuroeckol A (a pentamer), dieckol and 8,8′-bieckol (hexamers), and an unidentified tetramer – were isolated from the crude phlorotannins by column and thin-layer chromatography. Phlorofucofuroeckol A, dieckol and 8,8′-bieckol inhibited α-fucosidase, β-galactosidase and β-mannosidase partially purified from T. cornutus, while phloroglucinol, eckol and the unidentified tetramer were weakly active. Dieckol was a competitive inhibitor of α-fucosidase with an inhibition constant (Ki) of 0.12 mM. The amounts of phlorotannins released after the immersion of freshly collected E. bicyclis in seawater or deionized water were estimated by high-performance liquid chromatography. Nearly all the phlorotannins were exuded into the medium following the death of the algae, whereas no phlorotannins were detected in the medium of living algae. These findings indicate that the phlorotannins deter the feeding of marine herbivorous gastropods by inhibiting the glycosidases.

Fourier-transform infrared (FTIR) spectroscopy was carried out on single colonies of Pediastrum duplex present in air-dried preparations of mixed phytoplankton samples isolated from a eutrophic freshwater lake. FTIR absorption spectra had 12 distinct bands over the wavenumber range 3300–900 cm−1 which were tentatively assigned to a range of chemical groups, including -OH (residual water, wavenumber 3299 cm−1), -CH2 (lipid, 2924), -C=O (cellulose, 1739), amide (protein, 1650 and 1542), >P=O (nucleic acid, 1077) and -C-O (starch, 1151 and 1077). Measurement of band areas identified residual water, protein and starch as the major detectable constituents. Areas of single bands and combined bands of -CH2, -C-O and >P=O species normalized to protein (to correct for differences in specimen hydration and thickness) showed wide variation between colonies, indicating environmental heterogeneity. Correlation analysis demonstrated close statistical associations between different molecular species. Particularly high levels of correlation between bands 3/4 (CH2), 6/7 (amide) and 8/9 (-CH3) was consistent with their joint origin from the same molecular species. The isolation of bands 11 and 12 in the correlation pattern was confirmed by factor analysis, suggesting that variation in the level of starch is statistically unrelated to other macromolecules being monitored. The use of FTIR spectroscopy to characterize an algal micro-population within mixed phytoplankton has potential for future studies on biodiversity and environmental interactions at the species level.

We report here the complete sequence of the mitochondrial genome of the brown alga Laminaria digitata (Hudson) J.V. Lamouroux. L. digitata mtDNA is a circular molecule of 38,007 bp (64·9% A+T), encoding 63 genes and 3 ORFs and with only 6·7% of non-coding sequences. Based on gene content and order, its overall organization is very similar to that of the mitochondrial genome of Pylaiella littoralis, another brown alga belonging to a different sublineage of the Phaeophyceae. In particular, the two genomes share unusual features, which hence could be unique to brown algae among the heterokont lineage, namely the presence of a rn5 gene, a short nad11 gene, a cox2 gene with a large in-frame insertion and α-proteobacterial-like promoter sequences. On the other hand, L. digitata lacks the sequences which are thought to have been transmitted horizontally to the P. littoralis genome, that is, the group-II introns in the rnl and cox1 genes, and it features only traces of an ancestral T7-like RNA polymerase. Distance phylogenetic trees inferred from concatenated mitochondrial genes confirm that speciation of brown algae occurred recently compared to other heterokonts.

Nutrient manipulation experiments were conducted on a natural planktonic community in outdoor mesocosms. Inorganic nitrogen (N) and silicon (Si) were added to achieve N[ratio ]Si ratios of 1[ratio ]1 and 4[ratio ]1. Total particulate carbon (PC) biomass of the microbial assemblage was determined by elemental analysis. Cell volume measurements by microscope on individual components of the community (bacteria, diatoms, photosynthetic nanoflagellates, heterotrophic nanoflagellates, dinoflagellates and ciliates) were also made. We applied published C[ratio ]volume relationships to determine the volume estimated C content (CBV) of these microbial groups and hence of the total assemblage. The total CBV and total PC were compared to test the applicability of C[ratio ]volume relationships under different nutrient regimes both before and after nutrient exhaustion. For initial N[ratio ]Si ratios of 1[ratio ]1, prior to nutrient exhaustion, the relationship between CBV and PC was linear with a gradient of approximately 1, (0.99±0.06), indicating that the published C[ratio ]volume relationships accurately predicted the C content of the microbial assemblage. For N[ratio ]Si ratios of 4[ratio ]1, a linear relationship was again evident between CBV and PC (slope: 1.36±0.08). However, statistical comparison using a general linear model indicated that the gradient of this relationship differed significantly from that when the N[ratio ]Si ratio was 1[ratio ]1, and hence CBV overestimated elemental C. For both N[ratio ]Si ratios, subsequent to nutrient exhaustion (N or Si), and hence when the diatom fraction of the microbial assemblage was in yield-limited post-exponential phase, the two measures of biomass were not well correlated. This indicated that measured cytoplasmic cell volume was a poor indicator of C biomass within the microbial assemblage in nutrient-deplete conditions.

The population of Stephanodiscus rotula in a temperate eutrophic lake was studied over a 2 month period (January to early March). Although during the study period nutrient concentrations in the lake water remained far in excess of phytoplankton requirements, no notable increase in chlorophyll levels was recorded. This suggests that algae were limited by shortage of light and low temperatures. Frequency distribution analysis of cell diameters showed a dramatic size reduction at the end of winter, followed by restoration of higher values by early spring. Electron probe X-ray microanalysis spectra from Stephanodiscus cells routinely showed peaks of Ca, K, Si, P, S, Fe, Al, Mn, Mg, Na and Cl, with substantial variation in elemental concentrations both between and within samples. End-of-winter reduction in the cell size coincided with a considerable depletion of intracellular chemical levels of Si, P, Cl and K and could be related to the concurrent decrease in dissolved organic C and increase in intracellular Al. Correlation and factor analysis of intracellular elemental concentrations showed that statistical elemental associations within Stephanodiscus cells were mainly determined by three factors, with P, Cl, Si and K showing higher loadings on the first, Ca, Mn and S on the second, and Fe, Ca and Al on the third factor. Significant correlations among the elements of the first association may indicate the importance of P (ATP), K (through involvement in P metabolism) and Cl (possibly charge balance) in the active Si uptake during the study period.

The reproductive stages of Dermocarpella gardneri and D. stellata, which have been reported only once, are described. Formation of baeocytes occurs by cellular divisions that are parallel to the substratum, followed by a series of anticlinal radial divisions. In some cases in D. gardneri, the superior cell, resulting from the first division parallel to the substratum, is liberated prior to radial divisions, and these probably represent the ‘macrogonidia’ originally described by Lemmermann for D. hemisphaerica. The baeocytes are released through a circular apical pore, which develops after the formation of a papilla that eventually dissolves to form a pore.

Patterns of nuclear and organelle inheritance among artificial hybrids of the seaweeds Fucus serratus and F. evanescens were detected using single-strand conformation polymorphism (SSCP). Three alleles were identified in the 231 bp rDNA-ITS1 gene (nuclear): two in F. serratus and one in F. evanescens. Alleles differed by 1–2 bp and all hybrids possessed one allele from each parent. Two haplotypes were present in the 288 bp Rubisco spacer (chloroplast), differentiated by a 33 bp indel. Two haplotypes differing by a single nucleotide were found in a 135 bp region of nad11 gene (mitochondrion). Both organelles are maternally inherited, as all hybrids contained the haplotypes of the parent contributing the egg. Although laboratory hybrids among Fucus spp. have been produced previously, this is the first time that both nuclear and cytoplasmic genetic markers have been used to document inheritance patterns. SSCPs analysed on an automated sequencer offer a rapid and powerful approach for identifying suspected hybrids from field samples, as well as a screen for intraspecific and intra-individual variation in DNA regions prior to confirmation of variations by sequencing.

The apex of the tetrasporangial branches of Osmundea spectabilis var. spectabilis (= Laurencia spectabilis var. spectabilis) exhibits cavities in which tufts of multicellular trichoblasts occur. Trichoblast development in Osmundea spectabilis var. spectabilis begins with the differentiation of an epidermal cell within the crypt. This cell differentiates into a trichoblast mother cell (TMC). The TMC divides to form a two-celled incipient trichoblast. Successive periclinal divisions of the apical cell of the young trichoblast result in the formation of a multicellular developing trichoblast. With the exception of the apical cell all trichoblast cells are at the same developmental stage. They possess a large nucleus, abundant plastids with peripheral and some internal thylakoids and dictyosomes. Daughter chloroplasts result from one constriction or multiple fission of a single chloroplast. Dictyosomal cisternae and mucilage sacs contribute material to wall formation. Each differentiating trichoblast cell is surrounded by a bi-layered wall. The outer wall layer represents the trichoblast mother cell wall and the inner wall layer is the trichoblast cell wall. Mature trichoblast cells have thin walls, probably as a consequence of mucilage extrusion, the most likely function of trichoblasts in Osmundea.

Time-lapse videomicroscopy was used to observe the effects of various cytoskeletal inhibitors on three important fertilization events in Bostrychia moritziana: spermatial mitosis, gamete fusion (formation of a fertilization pore) and nuclear migration along the trichogyne. The microtubule inhibitor oryzalin disrupted spermatial mitosis but had no other effect on fertilization. The actin inhibitors, jasplakinolide, cytochalasin B, latrunculin A and B and mycalolide B inhibited gamete fusion while BDM, a myosin-disrupting drug, inhibited all three major fertilization events. FL-Phallacidin was used to stain actin filaments in spermatia and trichogynes while microtubules were labelled with antibodies at appropriate stages of fertilization. Microtubules were only evident during spermatial nuclear division. Actin filaments were present in both trichogynes and spermatia throughout fertilization; they formed a discrete ring around the fertilization pore and ensheathed male nuclei as the latter migrated into and along the trichogyne. These results suggest that the actin/myosin system plays a role in the events of fertilization.

The excretion of extracellular polymeric substances (EPS) by an axenic culture of the benthic diatom Cylindrotheca closterium was investigated. Two sequential extraction steps proved to be sufficient to remove the bulk of the EPS present. Soluble EPS was recovered by a simple centrifugation step and represented a fraction that was not or was only loosely associated with diatom cells. For the extraction of bound EPS, different procedures were compared. The best results were obtained using distilled water as extraction solvent (1 h, 30 °C). The sugars that were recovered using this procedure were typically associated with aggregates of diatoms. In addition to the distinct differences in localization of the different types of EPS, their temporal dynamics differed in relation to the light–dark cycle. Soluble EPS were continuously released into the medium at a rate of 1·6 pg cell−1 day−1. In contrast, the production of bound EPS was highly light-dependent. In the dark, this bound EPS rapidly disappeared, probably as the result of its utilization by the diatoms.

Crosses were performed to identify the sources of variation in zygote production (via cystocarp production) in Gracilaria gracilis, a red haploid-diploid seaweed. First, because male gametes are short-lived (<6 h), the rate of gamete encounters was evaluated in a time-course experiment. Second, the effect of water motion on gamete encounters was assessed by introducing turbulent eddies in the crossing tank and by comparing fertilization rates with and without this added turbulence. Third, variation due to individual performance was explored by performing multiple-donor crosses using 12 males and 12 females from three populations. Paternity of cystocarps produced in these crosses was determined using microsatellite markers. The results show that cystocarp yield increased with exposure time: fertilization occurred in as little as 15 min after the introduction of male branches into the crossing tank and maximum cystocarp production values were observed at 6 h. There were no significant differences in cystocarp production between the two turbulence levels. On the other hand, cystocarp production was highly influenced by male and female parental identities and to a lesser degree by an interaction between the male and female parents. The variation in cystocarp production according to male and female identity was not due to population origin as there was no difference between intra- and inter-population crosses. Thus non-random mating occurs in controlled conditions and arises from differential performance in G. gracilis. There was a strong deviation from equality of male performance, implicating post-adhesion events and/or male gamete production as important in generating non-random mating. Consequently, non-random mating may play a role in the evolution of mating patterns in G. gracilis.

Viable diatom and dinoflagellate resting stages were recovered from sediments in Koljö Fjord on the west coast of Sweden. To determine the maximum survival time of buried resting stages, samples from sediment depths down to 50 cm were incubated at temperatures of 3, 10 and 18 °C. Sediment cores were dated by 210Pb and the age of samples containing viable resting stages was determined using the constant rate of supply model. Dilution cultures of surface sediments allowed semiquantitative estimates of the potential seed bank. Dinoflagellate cysts from species such as Diplopsalis sp., Gymnodinium nolleri, Oblea rotunda and Protoceratium reticulatum were viable down to 15 cm depth, or 37 years old. Spores and resting cells of the diatoms Chaetoceros spp., Detonula confervacea and Skeletonema costatum were viable to over 40 cm depth, and may have been buried for many decades. The seed bank of living resting stages in surficial sediments was found to be rich (c. 57000 diatom resting stages g−1 wet weight and c. 200 dinoflagellate cysts g−1 wet weight), and the percentage of viable resting stages was higher for spore- and cyst-forming species. The oxygen-deficient sediments in Koljö Fjord appear to be a natural conservator of cell viability, a condition not easily simulated in laboratory studies. These results are ecologically important since spores and cysts are a repository of genetic material able to repopulate waters if resuspended and exposed to suitable light, temperature and nutrients.

Field emission scanning electron microscopy (FESEM) preparation techniques have been successfully adapted for visualization of the internal and external ultrastructure of Mougeotia filaments and protoplasts. FESEM of the innermost layer of cell wall in Mougeotia filaments revealed that microfibrils are deposited parallel to each other in an interconnected mesh and are oriented perpendicular to the direction of elongation. For the first time, the surface of protoplasts at different stages of regeneration has been observed using FESEM. Nascent microfibril deposition occurs between 1 and 2 h after isolation and arrangement of these microfibrils is random for at least 8 h. Observation of the inner surface of the plasma membrane in burst protoplasts showed that microtubules are not strongly attached for at least 3 h after protoplast isolation.

The presence of intracellular bacteria in the dinoflagellate Gyrodinium instriatum Freudenthal & Lee has previously been described but the bacterial flora associated with this species has not been characterized. In this study, new results of transmission electron microscopy (TEM) and in situ hybridization using several bacterial group-specific oligonucleotide probes are presented. The long-term association of endocytoplasmic and endonuclear bacteria with G. instriatum has been confirmed. All endonuclear and most of the endocytoplasmic bacteria labelled were identified as belonging to the betaproteobacteria. Large clusters of Cytophaga-Flavobacterium-Bacteroides (CFB) were labelled and observed in the cytoplasm of the dinoflagellate cells, but were absent from the nucleus. Gammaproteobacteria were only observed outside the dinoflagellates. No alphaproteobacteria were detected either free-living or intracellular. Empirical observation of intracellular CFB reflected a degradation process of moribund dinoflagellate cells, whereas the systematic colonization of dinoflagellate nucleoplasm by betaproteobacteria suggested a true symbiotic relationship. Natural colonization may have occurred, perpetuated by vertical transmission of intracellular bacteria to the dinoflagellate daughter cells, via a pool of bacteria sequestered within the nucleus. Dividing bacteria were observed in the nucleus and equilibrium may be maintained by release of endonuclear bacteria to the cytoplasm through nuclear envelope constrictions.

Differentiation in temperature responses (survival and growth) was investigated among isolates of two tropical to temperate green algal lineages: the Cladophora vagabunda complex and the C. albida/sericea complex. The results were analysed in relation to published data on 18S rRNA and ITS sequence divergence, which have shown that the overall degree of genetic divergence is similar in the two lineages but that very different patterns of radiation have occurred. In the C. vagabunda complex, the two main clades in the well-resolved phylogenetic tree differed mainly in their tolerance to low temperatures. Within-clade variation was no stronger in the Atlantic/Pacific than in the all-Pacific clade. In the C. albida/sericea complex, six distinctive ITS types indicated early radiation. Although distinctive differences were found between some of these types, the thermal responses of others were very similar, indicating physiological stasis. In both lineages there was evidence for some adaptation to local temperature regimes but phylogenetic constraints were generally more important. Isolates with the same ITS sequences showed similar temperature responses even though collected from different climate zones. Evidence was found for a physiological trade-off between growth at high and at low temperatures in the C. albida/sericea complex, whereas, in the C. vagabunda complex, one clade showed more eurythermal growth responses than the other. In the C. vagabunda complex, which is the ancestral lineage of the C. albida/sericea complex, major differentiation was found in cold tolerance but not in heat tolerance, whereas the reverse pattern was found in the derived C. albida/sericea complex. These findings suggest that an acquisition of cold tolerance preceded the loss of heat tolerance during adaptation to colder climates.