Does high intraspecific variability of two genome fragments indicate a recent speciation process of Paramecium dodecaurelia (P. aurelia species complex,Ciliophora, Protozoa)?

Species of the Paramecium aurelia complex show different levels of intraspecific polymorphism, with P. dodecaurelia revealing a high level of intraspecific variation. Paramecium dodecaurelia strains originating from distant localities in the Palaearctic, North America (USA), and Oceania (Hawaii) were studied in terms of intraspecific differentiation and the degree of speciation. Sequences of genes encoding the ITS1–5.8S-ITS2–5’ end of LSU rDNA (1063–1097 bp) and cytochrome c oxidase subunit I mtDNA (638–644 bp) were obtained from 33 strains of P. dodecaurelia, other P. aurelia species, and another species of the genus Paramecium, with Tetrahymena sp. used as an outgroup. In phylograms, the majority of P. dodecaurelia strains from the Palaearctic appear in one cluster, while strains from Japan, Hawaii and the USA are grouped in another cluster, together with some strains from Italy and representatives of the P. aurelia species complex. Our results tend to support the hypothesis that P. dodecaurelia seems to be a polyphyletic species with several haplotypes similar to or even shared with other members of the P. aurelia species complex. However, it is still an open question whether the revealed intraspecific differences within P. dodecaurelia are the result of ongoing speciation, or perhaps they just indicate genetic differentiation within a species that has a wide geographic distribution.     

Distribution of the Paramecium aurelia Species Complex (Protozoa,Ciliophora) in the Carpathian Chain of Poland*

Four species of the Paramecium aurelia complex were found in the Carpathian chain: P. primaurelia, P. biaurelia, P. tetraurelia and P. novaurelia. However, variation in the frequency of settlement of these species in the region was observed; P. biaurelia and P. novaurelia appeared with the greatest frequency, P. Primaurelia occurred less frequently. while P. tetraurelia was rather rare being found only in the Tatras.     

Comparison of the Esterases and Acid Phosphatases in Paramecium multimicronucleatum,Syngens 1–5, P. jenningsi,P. caudatum,and the P. aurelia Complex1

ABSTRACT. Forty-eight stocks in Paramecium jenningsi, syngens 1–5 of P. multimicronucleatum, P. caudatum, P. primaurelia, P. biaurelia, and P. tetraurelia were grown axenically and tested for their esterases and acid phosphatases using starch gel electrophoresis. The five esterases and the acid phosphatases previously characterized in species of the P. aurelia complex were also found in P. jenningsi, and three to four of the esterases and the acid phosphatases were found in the P. multimicronucleatum species complex and in P. caudatum. Additional subtypes were observed for each of the enzyme phenotypes in these new (though here unnamed) species of Paramecium. Two of the new acid phosphatase subtypes, which depart radically in mobility and in pattern, were found in syngen 3 of P. multimicronucleatum and in P. caudatum. Except for syngens 1 and 5 in P. multimicronucleatum, the degree of similarity between syngens 1, 5 and 2, 3, and 4 appears to be very low—perhaps even lower than that seen for species in the aurelia complex. More realistically, the syngens of P. multimicronucleatum should be considered as separate species although they are not here given separate taxonomic names. Limited sharing of subtypes occurred between species in different species complexes. This observation suggests that the molecular distances between species complexes may be even greater than between species within a complex.     

Worldwide sampling reveals low genetic variability in populations of the freshwater ciliate <Emphasis Type="Italic">Paramecium biaurelia</Emphasis> (<Emphasis Type="Italic">P. aurelia</Emphasis> species complex,Ciliophora, Protozoa)

Species (or cryptic species) identification in microbial eukaryotes often requires a combined morphological and molecular approach, and if possible, mating reaction tests that confirm, for example, that distant populations are in fact one species. We used P. biaurelia (one of the 15 cryptic species of the P. aurelia complex) collected worldwide from 92 sampling points over 62 years and analyzed with the three above mentioned approaches as a model for testing protistan biogeography hypotheses. Our results indicated that despite the large distance between them, most of the studied populations of P. biaurelia do not differ from each other (rDNA fragment), or differ only slightly (COI mtDNA fragment). These results could suggest that in the past, the predecessors of the present P. biaurelia population experienced a bottleneck event, and that its current distribution is the result of recent dispersal by natural or anthropogenic factors. Another possible explanation for the low level of genetic diversity despite the huge distances between the collecting sites could be a slow rate of mutation of the studied DNA fragments, as has been found in some other species of the P. aurelia complex. COI haplotypes determined from samples obtained during field research conducted in 2015–2016 in 28 locations/374 sampling points in southern Poland were shared with other, often distant P. biaurelia populations. In the Kraków area, we found 5 of the 11 currently known COI P. biaurelia haplotypes. In 5 of 7 reservoirs from which P. biaurelia was obtained, two different COI haplotypes were identified.     

Polymorphism of Paramecium pentaurelia (Ciliophora, Oligohymenophorea) strains revealed by rDNA and mtDNA sequences

Paramecium pentaurelia is one of 15 known sibling species of the Paramecium aurelia complex. It is recognized as a species showing no intra-specific differentiation on the basis of molecular fingerprint analyses, whereas the majority of other species are polymorphic. This study aimed at assessing genetic polymorphism within P. pentaurelia including new strains recently found in Poland (originating from two water bodies, different years, seasons, and clones of one strain) as well as strains collected from distant habitats (USA, Europe, Asia), and strains representing other species of the complex. We compared two DNA fragments: partial sequences (349 bp) of the LSU rDNA and partial sequences (618 bp) of cytochrome B gene. A correlation between the geographical origin of the strains and the genetic characteristics of their genotypes was not observed. Different genotypes were found in Kraków in two types of water bodies (Opatkowice—natural pond; Jordan's Park—artificial pond). Haplotype diversity within a single water body was not recorded. Likewise, seasonal haplotype differences between the strains within the artificial water body, as well as differences between clones originating from one strain, were not detected. The clustering of some strains belonging to different species was observed in the phylogenies.     

Intraspecies Variability in the Esterases and Acid Phosphatases of Paramecium jenningsi and Paramecium multimicronucleatum: Assignment of Unidentified Paramecia; Comparison with the P. aurelia Complex1

ABSTRACT. Enzyme electrophoresis was exploited to identify stocks of paramecia previously not identified to particular species. Stocks collected in India and one from Panama belong to Paramecium jenningsi, while others collected in Panama or in Brazil are assignable to syngen 2 of P. multimicronucleatum on the basis of similarity of their esterase and acid phosphatase phenotypes. Inclusion of these doubled the numbers of stocks available in the two species, thereby facilitating examination of intraspecies variation and comparison of particular features of intraspecies variation found for the P. aurelia complex. Variant stocks were observed in P. jenningsi and in syngens 2, 3, and 4 of P. multimicronucleatum. In some cases the variant lacked the enzyme; in others, a change in mobility of the enzyme occurred that resulted in an electrophoretic form similar to one common in another species. Unique phenotypes were displayed by the variants of syngen 2 in P. multimicronucleatum. Hypervariability for Esterase B was observed in this syngen, where, in addition, several subtypes were seen for three other esterases. Unique phenotypes and hypervariability were also noted in P. biaurelia. Clustered variations were observed in these species and in the P. aurelia species. Unlike the situation for members of the aurelia complex, where lack of geographical differentiation between stocks in the same species is a unique feature, some such differentiation does occur in P. multimicronucleatum-2. The frequency of variant stocks in P. jenningsi was similar to that observed in the aurelia sibling species. In contrast, a significantly higher frequency of variant stocks was found in syngens 2, 3, and 4 of P. multimicronucleatum.     

Esterase Variants in Four Species of the Paramecium aurelia Complex1

One hundred eighty-eight stocks of Paramecium primaurelia, P. biaurelia, P. tetraurelia, and P. octaurelia were grown axenically and tested for five esterases, visualized by starch gel electrophoresis, in a search for variant stocks. The five esterases can be distinguished on the bases of their substrate specificity, sensitivity to an inhibitor, and response to different growth conditions. This paper addresses the nature of the electrophoretic change in mobility of the variant stocks in order that species relationships can be more accurately assessed. Crosses carried out in all four species show that single genes determine the differences in mobility between variant and common subtypes. Extracts of variant stocks that gave similar patterns were run against each other, tested for their sensitivity to the inhibitor, and the pattern was compared to that found in extracts of stocks with variant and common subtypes in other species. The majority of the variants in P. primaurelia, P. tetraurelia, and P. octaurelia show an electrophoretic mobility characteristic of a common subtype, or a variant, in another species. The same proportion of variant subtypes as common subtypes have mobilities similar to esterase subtypes found in other species. Of the four species examined in this paper, P. tetraurelia and P. octaurelia appear to be most closely related on the basis of shared esterase subtypes. In P. biaurelia the mobilities of most of the variants are unique, as are the common esterase subtypes in this species. P. biaurelia stands out as having the greatest number of esterase subtypes, with very few of them homologous to subtypes found in other species. This observation supports the idea of greater diversification of stocks within P. biaurelia than for the other three species.     

Effect of Acetate on Esterase C Activity During the Growth Cycle of Paramecium*

SYNOPSIS Enhanced esterase C activity could be demonstrated by starch gel electrophoresis in various stocks of Paramecium spp. (P. primaurelia stocks 90 and 540, P. biaurelia stock 93, P. tetraurelia stock 29. P. pentaurelia stock 87, P. octaurelia stocks 31 and 300, and P. multimicronucleatum species 3, stock 8 MO) grown in Adaptation Medium. This esterase, however, was barely detectable when they were cultivated in Axenic Medium. Addition of trypticase to Adaptation Medium resulted in reduction of esterase C in the ciliates. This effect is ascribable to Na acetate present in trypticase. Since esterase C increased with the decrease in acetate concentration (as estimated by gas-liquid chromatography) during growth of Paramecium, acetate appears to be utilized by the cells. Sensitivity of esterase C to acetate occurs in all 6 species of Paramecium examined. Different stocks within a species may have different levels of sensitivity; in one case this is genetically determined. The results emphasize the importance of controlling and manipulating growth conditions for the assessment of inter- and intraspecies variations in the isozymes of Paramecium.     

Interspecies Relationships in the Paramecium aurelia Complex: Acid Phosphatase Variation1

ABSTRACT. Up to five zones of acid phosphatase activity appear in gels after electrophoresis of detergent-treated extracts from 13 of the 14 species of the Paramecium aurelia complex. The overall pattern is somewhat similar for all species: differences in intensity and mobility of individual zones permit the grouping of these sibling species into eight groups. All 14 species can be identified using the procedure of enzyme electrophoresis, although two of them are more similar than is usually the case. Problems of misclassification are discussed in terms of the nature and frequency of variants. With the judicious choice of enzymes used to screen new stocks, these problems can be circumvented. Species relationships are updated using 11 enzymes. A dendrogram constructed from the matrix of genetic distances shows four clusters of species: (i) P. biaurelia, P. triaurelia; (ii) P. primaurelia, P. pentaurelia, P. sexaurelia, P. novaurelia; (iii) P. septaurelia, P. undecaurelia, P. tredecaurelia, P. quadecaurelia; and (iv) P. tetraurelia, P. octaurelia, P. decaurelia, P. dodecaurelia. Distances between the species are large, on the order of the differences between Drosophila species. The species are characterized by an extraordinary lack of geographical differentiation and great morphological similarity, which contrasts strongly with the molecular differentiation.     

Insights into Three Whole-Genome Duplications Gleaned from the Paramecium caudatum Genome Sequence

Paramecium has long been a model eukaryote. The sequence of the Paramecium tetraurelia genome reveals a history of three successive whole-genome duplications (WGDs), and the sequences of P. biaurelia and P. sexaurelia suggest that these WGDs are shared by all members of the aurelia species complex. Here, we present the genome sequence of P. caudatum, a species closely related to the P. aurelia species group. P. caudatum shares only the most ancient of the three WGDs with the aurelia complex. We found that P. caudatum maintains twice as many paralogs from this early event as the P. aurelia species, suggesting that post-WGD gene retention is influenced by subsequent WGDs and supporting the importance of selection for dosage in gene retention. The availability of P. caudatum as an outgroup allows an expanded analysis of the aurelia intermediate and recent WGD events. Both the Guanine+Cytosine (GC) content and the expression level of preduplication genes are significant predictors of duplicate retention. We find widespread asymmetrical evolution among aurelia paralogs, which is likely caused by gradual pseudogenization rather than by neofunctionalization. Finally, cases of divergent resolution of intermediate WGD duplicates between aurelia species implicate this process acts as an ongoing reinforcement mechanism of reproductive isolation long after a WGD event.     

Intraspecies Variability in the Esterases and Acid Phosphatases of Four Species of the Paramecium aurelia Complex1

ABSTRACT. One hundred eighty-eight stocks of Paramecium primaurelia. P. biaurelia, P. tetraurelia. and P. octaurelia were grown axenically and screened for variation in four different esterases and acid phosphatase using starch gel electrophoresis. Major observations: frequency of intraspecies variation for these enzymes is much lower in these four species than in other organisms; hypervariability for two esterases occurs in P. biaurelia both in isolates from worldwide locales and in a restricted locale; clustering of variations occurs in a high proportion of variant stocks in all four species; frequency of intraspecies variation is highest in Central and South America for all four species; and geographical differentiation is lacking between stocks in the same species both for common as well as variant phenotypes despite the cosmopolitan distribution of these species. These results are not correlated with adaptations that favor inbreeding over outbreeding. nor is the possession of bacterial endosymbionts strongly correlated with enzyme variation. When the riequency of intraspecies variation was examined for the aurelia complex of species as a whole for 13 enzymes, mitochondrial DNA, and ribosomal DNA, differences between enzymes in frequency of variation could be seen, ranging from less than 2% for seven enzymes to 12.4% for glucosephosphate isomerase, a value similar to that observed for malic dehydrogenase, mitochondrial DNA, and ribosomal DNA in P. tetraurelia. The percentage of polymorphic enzyme loci in the complex as a whole was found to be much lower than that observed for other organisms. For the species more intensely studied in this paper the level of genetic polymorphism was also much lower, although P. biaurelia showed greater variability for two of the enzymes.     

The Spatial and Temporal Distribution of Paramecium bursaria in the Littoral Zone1

The spatial and seasonal distribution of Paramecium bursaria in two small Indiana ponds was studied using a sampling grid. Very small (5.0 ml) samples were taken so that the individual microhabitats could be studied. The results were evaluated in comparison to the data collected for the P. aurelia complex collected in the same manner and at the same sites. It was found that P. bursaria exist in a clumped distribution, but that the distribution was not very different from random. Paramecium bursaria also exist at the surface and at the mud-water interface. Temperature does not seem to play a statistically significant role in determining population size. The breeding system of P. bursaria is optimized for an outbreeding population of low density. In comparison, the species of the P. aurelia complex exist in a very clumped distribution, are found only at the mud-water interface, and are inbreeders. The evolutionary strategies of the two types of paramecia are discussed.     

Toxic effects of antimalarial drugs in Paramecium : role of calcium channels

The antimalarial drugs, quinacrine, quinine and mefloquine, as well as the structurally-similar compound, W-7, inhibit calcium-dependent backward swimming and calcium currents in Paramecium calkinsi. These drugs are also toxic to paramecia at high concentrations. Therefore, one site of toxic action of the drugs may be the calcium channel. To test this hypothesis, the toxicity of the antimalarials and W-7 was compared in paramecia with and without calcium channels. Since calcium channels are located on the cilia, calcium channels were removed from the paramecia by deciliating the cells. Deciliated cells were found to be less susceptible to the lethal effects of the antimalarials and W-7 than their ciliated counterparts. Moreover, Pawns, mutants of P. tetraurelia that possess cilia but lack functional calcium channels, were also less susceptible to the antimalarials than wild-type cells. Thus, calcium channels may be one site of toxic action of the antimalarial drugs in paramecia and perhaps in other protists. Accepted: 27 December 1996     

New Paramecium quadecaurelia strains (P. aurelia spp. complex,Ciliophora) identified by molecular markers (rDNA and mtDNA)

Paramecium quadecaurelia is a rare species (previously known only from two locations) belonging to the P. aurelia species complex. In the present paper, fragments of an rDNA gene (ITS1-5.8S-ITS2-5′ rDNA) and mtDNA genes (cytochrome oxidase subunit I and cytochrome b regions) were employed to assist in the identification and characterization of three new strains collected from Ecuador and Thailand. Molecular data were confirmed by mating reactions. In rDNA and mtDNA trees constructed for species of the P. aurelia complex, all P. quadecaurelia strains, including the three new strains discussed in this study and two known previously from Australia and Africa, form a monophyletic but differentiated clade. The present study shows that genetic differentiation among the strains of P. quadecaurelia is equal to or even greater than the distances between some other P. aurelia species, e.g., P. primaurelia and P. pentaurelia. Such great intra-specific differentiation may indicate a future splitting of the P. quadecaurelia species into reproductively isolated lines.     

The Toxic Symbiont Caedibacter caryophila in the Cytoplasm of Paramecium novaurelia

Endosymbiotic bacteria were observed to inhabit the cytoplasm of the freshwater ciliateParamecium novaurelia. Transmission electron microscopy and toxicity tests with sensitive paramecia showed that the endosymbionts belong to the genusCaedibacter. The bacteria conferred a killer trait to their host paramecia. The production of a proteinaceous inclusion body (“R-body”) in the bacterial cell makes them toxic to other paramecia after they become enclosed in food vacuoles. R-bodies ofCaedibacter sp were associated with phages, which are known in most otherCaedibacter species to code for the R-body proteins. The killer-effect ofP. novaurelia on sensitiveP. caudatum strains was of the “paralysis” type, which is a characteristic of the symbiont speciesCaedibacter caryophila. Until nowC. caryophila was known to inhabit the macronucleus ofParamecium caudatum only. Sequencing of the 16S rRNA-gene proved thatCaedibacter sp from the cytoplasm ofP. novaurelia belongs to the speciesC. caryophila as well. The rDNA-sequence of 1695 bp length differed in a total of only 1 bp from the corresponding gene inC. caryophila from the macronucleus ofP. caudatum. The results indicate that the infection of specific host cell compartments may depend on host genes, but not on different traits of the infecting symbiont species. The occurrence of killer and sensitive paramecia strains together in one pond is discussed with respect to the competitive advantage of the killer trait.     

Identifying conservation priority ponds of an endangered minnow, <Emphasis Type="Italic">Pseudorasbora pumila</Emphasis>, in the area invaded by <Emphasis Type="Italic">Pseudorasbora parva</Emphasis>

We investigated the occurrence pattern of the pond-living endangered cyprinid, Pseudorasbora pumila, and also compared its habitat characteristics with those of the congeneric invasive species, Pseudorasbora parva, in the contact zone. Comparison of 16 environmental variables among the P. pumila habitats, P. parva habitats, and unoccupied ponds revealed that conductivity was a common limitation factor of distribution of both species. We found that emergent vegetation occupancy along the pond bank was the most important factor determining P. pumila occurrence and that ponds with steep banks may have a low probability of containing P. parva. We constructed a logistic regression model to predict the establishment risk of P. parva in ponds occupied by P. pumila. The model demonstrated that more than half of the ponds exhibited a high establishment risk of P. parva. Principal component analysis using six parameters selected from stepwise logistic regression analysis revealed that seven unoccupied ponds had the potential to sustain P. pumila, suggesting that our study site is capable of supporting more P. pumila populations and expanding the current range.     

Spatial distribution patterns of Littoraria species in Hong Kong mangroves

Variation in the abundance and distribution of two species of mangrove littorinid was investigated using a nested sampling design at different spatial scales, in the two dominant seasons, in Hong Kong. The abundance of Littoraria melanostoma, which was less abundant than L. ardouiniana, showed large-scale spatial variation whilst abundance of L. ardouiniana varied on both large and smaller spatial scales and interacted with seasons, indicating that the abundance of this species varied between mangroves in summer and winter. Small-scale variation suggested a patchy distribution of littorinids within a mangrove, whilst the large-scale variation might reflect changes in physical factors possibly associated with habitat fragmentation. Investigation of the vertical distribution of the littorinids on the mangrove trees revealed that L. melanostoma were located at a lower overall level than L. ardouiniana. This zonation pattern may be a result of the morphological differences of the two species, as L. melanostoma has a thick shell, whilst L. ardouiniana has a thinner, but colour polymorphic shell. Both species of mangrove littorinid showed patchy distributions at a variety of scales and these patterns highlight the importance of using a hierarchical sampling approach when investigating spatially fragmented habitats.     

Response of littoral vegetation on climate warming in the boreal zone; an experimental simulation

The impact of climate warming on the littoral zone of a boreal lake ecosystem was studied experimentally for three growing seasons in two artificial ponds (10×27 m) and in replicated chamber experiments. One pond was enclosed in a plastic greenhouse and another untreated pond served as a reference system. During the growing seasons temperature in the greenhouse was maintained at levels 2–3 °C higher than ambient with a computer-controlled ventilation system. One growing season prior to initiation of the experiment, a vegetated littoral zone with equal densities of water horsetail (Equisetum fluviatile) was established in both ponds. Although changes occurred in the species dominance (E. fluviatile - Alisma plantago-aquatica - Sparganium erectum spp. microcarpum - Elodea canadensis) within the three years of the study, the emergent macrophytes emerged earlier and grew better in the warmer conditions of the greenhouse pond compared with those in the reference pond. The difference in above-ground biomass throughout the growing seasons was >2 fold and after three experimental growing seasons the difference in below-ground biomass of macrophytes was 2.5-fold between the ponds. In replicated chamber experiments the biomass growth of E. fluviatile was also significantly higher in a 2–3 °C higher temperature than under ambient conditions. An ecosystem-scale induced change, characterized by a heavy growth of filamentous algae (mainly chlorophytes) was evident in the vegetated littoral zone of the greenhouse pond. A hypothesis that macrophyte rhizomes function as `phosphorus pumps' from the sediment and thus accelerate eutrophication in a warmer climate should be further studied.     

不同水深条件下刺参养殖池塘大型底栖动物群落结构季节性变化特征

于2012年7月至2013年4月调查了荣成靖海湾3个不同水深的刺参(Apostichopus japonicus)养殖池塘内大型底栖动物的构成,以了解不同水深对刺参养殖池塘环境条件的影响以及由此引起的大型底栖生物群落结构的改变。结果表明:3个不同水深梯度池塘(1#浅水位、2#正常水位和3#高水位)底部光照强度、叶绿素a(Chla)和总有机物(TOM)含量存在显著差异,各池塘水温差异不显著。光强、Chla和TOM含量在夏季、冬季和春季均表现为1#池塘显著高于3#池塘;秋季各池塘间光强和TOM含量差异不显著,Chla含量则表现为3#池塘显著高于1#池塘。各季节3个池塘间大型底栖动物在种类组成、丰度、生物量和多样性指数上均存在显著性差异。大型底栖动物丰度和生物量夏季均表现为1#池塘显著高于3#池塘,秋季和冬季则相反;春季1#池塘丰度显著高于3#池塘,生物量则差异不显著。这些差异主要与其各自优势种及其优势度指数大小有关。大型底栖动物多样性指数夏季和秋季均表现为1#池塘高于3#池塘,春季则相反,冬季各池塘间多样性指数差异不显著。单因子相似性分析(ANOSIM)表明,各季节3个池塘间大型底栖动物群落结构均存在显著差异,表明水深梯度对刺参养殖池塘大型底栖动物群落结构造成显著性影响。相似性百分比分析(SIMPER)显示,各季节对3个池塘间大型底栖动物群落差异起主要作用的物种为各个池塘的优势种。典范对应分析(CCA)表明,水深、Chla和TOM含量为影响大型底栖动物群落的主要环境因子。     

Effects of Actinomycin D on Generation Time and Morphogenesis in Paramecium*

SYNOPSIS. The sensitivity of Paramecium tetraurelia (=P. aurelia syngen 4) cells to pulse treatments with various doses of Actinomycin D (AMD) was estimated by comparing the generation times of treated and untreated sister cells. It was found that the delay of division in treated cells depended on the concentration of AMD, on their “age” at the time of the pulse treatment, and on their individual sensitivity. Sensitivity of Paramecium to AMD changes during the cell cycle in a predictable way. About 3 1/2 hr before the normally expected cell fission (total generation time ～ 5 1/2 hr) there is a decrease of sensitivity. Thereafter, the cell enters a new stage with a progressive increase of sensitivity. This 2nd phase ends at the “transition point” (～ 2 hr before cell division), when sensitivity drops abruptly. The division process itself may be altered and slowed down by high concentrations of AMD, even if the drug is applied after the transition point, but this process can never be completely annulled. The impairment of the division mechanism may lead to morphologic anomalies in the offspring. Resorption of oral anlagen in P. tetraurelia probably never occurs during the cell cycle after AMD treatment. The reason for individual variability of the cells, mechanisms controlling development, and the question of an obligate sequence of gene action in each cell cycle are discussed.