Genome size estimation of an endoparasitoid wasp,Cotesia plutellae,using quantitative real-time polymerase chain reaction

An endoparasitoid wasp, Cotesia plutellae, is a natural enemy against the diamondback moth, Plutella xylostella, which is the most destructive insect pest of cruciferous crop plants. The wasp genome contains genetic information of several parasitic factors, such as its symbiotic virus (C. plutellae bracovirus), venom, teratocyte as well as the parasitoid itself. These parasitic factors interfere with physiological processes of the immature stages of P. xylostella and need to be analyzed concerning their genetic components. A full genome sequence of C. plutellae would be highly informative to determine functional genes associated with these parasitic factors. Before a full genome sequence analysis of C. plutellae can be undertaken, an estimate of genome size is needed. In this study, we used a strategy using a quantitative real-time polymerase chain reaction (qPCR) to measure the wasp genome size. qPCR determined the number of a single copy gene hexokinase in a total mass of genomic DNA. The resulting molecular weight of the DNA sample was used to calculate the genome size in base pair (bp). This estimation approach indicated a genome size of C. plutellae of 186.06 ± 1.21 Mb.     

Controlling cell volume for efficient PHB production by Halomonas

Bacterial morphology is decided by cytoskeleton protein MreB and cell division protein FtsZ encoded by essential genes mreB and ftsZ, respectively. Inactivating mreB and ftsZ lead to increasing cell sizes and cell lengths, respectively, yet seriously reduce cell growth ability. Here we develop a temperature-responsible plasmid expression system for compensated expression of relevant gene(s) in mreB or ftsZ disrupted recombinants H. campaniensis LS21, allowing mreB or ftsZ disrupted recombinants to grow normally at 30 °C in a bioreactor for 12 h so that a certain cell density can be reached, followed by 36 h cell size expansions or cell shape elongations at elevated 37 °C at which the mreB and ftsZ encoded plasmid pTKmf failed to replicate in the recombinants and thus lost themselves. Finally, 80% PHB yield increase was achieved via controllable morphology manipulated H. campaniensis LS21. It is concluded that controllable expanding cell volumes (widths or lengths) provides more spaces for accumulating more inclusion body polyhydroxybutyrate (PHB) and the resulting cell gravity precipitation benefits the final separation of cells and product during downstream.     

Genome size variation and polyploidy in the resurrection plant genus Ramonda: Cytogeography of living fossils

The genus Ramonda includes three preglacial paleoendemic species surviving as the rare resurrection angiosperms of the Northern hemisphere in refugia habitats in the Balkan (Ramonda nathaliae and Ramonda serbica) and Iberian Peninsulas (Ramonda myconi). This study focuses on: assessing genome size and base composition, determining chromosome number and ploidy level in several populations, evaluating inter- and intra-specific variations in DNA content and chromosome number as well as looking for the possible hybridization in the sympatric zones of Balkan species. R. nathaliae and R. myconi are diploid species (2n = 2x = 48) while R. serbica is hexaploid (2n = 6x = 144). The mean 2C DNA values ranged from 2.30 pg for R. nathaliae to 2.59 pg for R. myconi compared to 7.91 pg for R. serbica. The base composition for R. nathaliae was 42.1% GC, for R. myconi 39.9% and for R. serbica 41.2%. In one population of R. serbica the DNA content ranged from 2C = 7.65 to 11.82 pg, revealing different ploidy levels among its individuals. In sympatric populations genome size was intermediary (～5 pg) between the diploid and hexaploid classes which indicates the hybridization ability between R. serbica and R. nathaliae. It appears that polyploidization is the major evolutionary mechanism in the genus Ramonda.     

Reproductive isolation and differentiation between five populations of Cotesia plutellae (Hymenoptera: Braconidae), parasitoid of Plutella xylostella (Lepidoptera: Plutellidae)

Many taxa used in biological control may be composed of several population units reproductively isolated to various degrees. Yet, the introductions of non-compatible populations may result in a depression of intended biological control. Cotesia plutellae has been widely used as a biological control agent of the diamondback moth, Plutella xylostella. However, not all introductions and augmentations were successful. With the aim to develop markers of compatibility for this species, we studied the reproductive compatibility between five populations (South Africa, Benin, Martinique, Reunion, and Taiwan) and analyzed its statistical relationship with three morphological characters (antennae and body length and antennae/body ratio), one molecular character (sequence of a cytochrome b fragment) and the Wolbachia infection status. This study shows that C. plutellae populations formed two interbreeding groups. The antenna/body length ratio of females, the ratio between male and female body length, one non-silent base pair substitution observed in cytochrome b sequences, and Wolbachia infection status differentiate these groups and were all good predictors of reproductive compatibility. Our results suggest also that genetic factors independent from Wolbachia are needed to fully explain reproductive isolation between C. plutellae populations. We conclude that C. plutellae is a single species composed of at least two partially incompatible population aggregates. Diagnostic markers were proposed for the prediction of the level of reproductive compatibility between populations which can be used for further biocontrol introductions.     

Comparison of the thermal performance between a population of the olive fruit fly and its co-adapted parasitoids

Long-term separation of a host from its native parasitoids may result in divergent thermal adaptation between host and parasitoid. The olive fruit fly, Bactrocera oleae (Rossi), most likely originated from Sub-Saharan Africa, but has since had a long invasion history in cultivated olives that spans geographical barriers and continents. This study compared three major thermal performance profiles (development, survival, and reproduction) across a wide range of temperatures (10–34 °C) among a Californian population of the olive fruit fly and two African parasitoids, Psyttalia lounsburyi (Silvestri) and Psyttalia humilis (Silvestri), believed to have co-adapted with the fruit fly in its native range. Temperature ranges for the development and survival were 10–30 °C for the fly, 10–28 °C for P. lounsburyi, and 14–32 °C for P. humilis. There was no difference in any thermal performance measured between two P. humilis populations (Kenya and Namibia) tested. The most suitable temperature ranges for reproduction were 22–30 °C for the fly, 18–32 °C for P. humilis, and 18–26 °C for P. lounsburyi. The results showed slight differences in the thermal profiles among olive fruit fly and both parasitoids species, with P. humilis being more heat tolerant whereas P. lounsburyi was less heat tolerant than the fruit fly. The results are discussed with respect to thermal co-adaptation and classical biological control of the olive fruit fly.     

Dispersal behavior of the parasitic wasp Cotesia urabae (Hymenoptera: Braconidae): A recently introduced biocontrol agent for the control of Uraba lugens (Lepidoptera: Nolidae) in New Zealand

Estimates of the dispersal range of a recently introduced biocontrol agent in its new environment are vital to understanding its relative searching capacity, and to foresee the maximum area that could be covered in a release event. In New Zealand, the solitary endoparasitoid Cotesia urabae Austin and Allen (Hymenoptera: Braconidae) was first released in January 2011 as a biological control agent for the gum leaf skeletoniser, Uraba lugens Walker (Lepidoptera: Nolidae). The objective of this study was to utilize an experimental approach to quantify the dispersal behavior of one generation of C. urabae. In our experiment, which used sentinel larvae as target hosts, parasitoids dispersed up to 20 m away from the release point but parasitism was highest within 5 m of the release site. A high level of parasitism was observed at the release tree (87.6%) which suggests that most of the females released may have stayed there. According to the dispersal model developed from the data collected, Cotesia would be able to disperse up to 53 m in one release event. In addition, significant differences were found between the different directions tested for dispersal, showing a clear downwind effect on dispersal suggesting that wind has a direct effect on the dispersal behavior of C. urabae in the field.     

Chloroplast DNA variation and phylogeographic patterns in the Chinese endemic marsh herb Sagittaria potamogetifolia

We examined chloroplast DNA (cpDNA) atpB–rbcL intergenic spacer sequences variation within Sagittaria potamogetifolia, an endangered and endemic marsh herb in China. Sequence data were obtained from 54 individuals in six extant populations of the species. Sequences appeared to evolve neutral (Tajima's criterion D = −1.59826, 0.1 > P > 0.05 and Fu and Li's tests D* = −1.44484, P > 0.1; F* = −1.83446, P > 0.1). Eleven haplotypes were identified in S. potamogetifolia. A relatively high level of haplotype diversity (h = 0.0.699) and low level of nucleotide diversity (pi = 0.0035 ± 0.0020) were detected in S. potamogetifolia. Pairwise comparisons of F_st and Nm deduced from cpDNA variation suggested no significant genetic differentiation between populations of S. potamogetifolia excepted for the WY-1 population. Low genetic differentiation among populations and also among regions was consistently indicated by both hierarchical analyses of molecular variance (AMOVA) and the structure of a neighbor-joining tree. Lack of population differentiation between populations or between regions in cpDNA sequences may be due to effects of lower substitution rates or lineage sorting. In the minimum spanning network, all tip haplotypes except for the haplotype J were unique to a particular population, while the interior nodes except for the haplotype E were widespread (haplotype A). From nested clade analysis (NCA), the evolutionary events such as restricted gene flow with isolation by distance and allopatric fragmentation were inferred to responsible for the current distribution of S. potamogetifolia populations, as well as their genetic diversity.     

Longicorn beetle that vectors pinewood nematode carries many Wolbachia genes on an autosome

Monochamus alternatus is the longicorn beetle notorious as a vector of the pinewood nematode that causes the pine wilt disease. When two populations of M. alternatus were subjected to diagnostic polymerase chain reaction (PCR) detection of four Wolbachia genes, only the ftsZ gene was detected from one of the populations. The Wolbachia ftsZ gene persisted even after larvae were fed with a tetracycline-containing diet for six weeks. The inheritance of the ftsZ gene was not maternal but biparental, exhibiting a typical Mendelian pattern. The ftsZ gene titres in homozygotic ftsZ⁺ insects were nearly twice as high as those in heterozygotic ftsZ⁺ insects. Exhaustive PCR surveys revealed that 31 and 30 of 214 Wolbachia genes examined were detected from the two insect populations, respectively. Many of these Wolbachia genes contained stop codon(s) and/or frame shift(s). Fluorescent in situ hybridization confirmed the location of the Wolbachia genes on an autosome. On the basis of these results, we conclude that a large Wolbachia genomic region has been transferred to and located on an autosome of M. alternatus. The discovery of massive gene transfer from Wolbachia to M. alternatus would provide further insights into the evolution and fate of laterally transferred endosymbiont genes in multicellular host organisms.     

Development of primary cell cultures from the adult xylem-feeding leafhopper,Kolla paulula,as a tool for studying Wolbachia biology

Xylophagous leafhoppers are vectors of xylem-limited plant pathogens such as citrus variegated chlorosis (CVC) and Pierce's disease, which cause Xylella diseases. Currently, no cure for Xylella diseases exists. The objective of endosymbiont control using Wolbachia pipientis is to reduce the populations of insect vectors, potentially preventing the expansion of Xylella diseases. The purpose of this study was to establish primary cell cultures from adult xylem-feeding leafhoppers to study Wolbachia biology. Cells from adult male and female Kolla paulula (Walker) (Hemiptera: Membracoidea: Cicadellinae) were successfully cultured in Dulbecco's Modified Eagle Medium containing 20% fetal bovine serum and were maintained for more than 6 months. Cells of both male and female adults are round and semi-attached. The doubling times for male and female K. paulula cells are approximately 8 and 10 days, respectively. The presence of Wolbachia in K. paulula cell cultures was detected by polymerase chain reaction (PCR) amplification of Wolbachia surface protein (wsp) gene. The cell cultures developed in this study may be useful in studying interactions between Wolbachia and its hosts of different genders.     

Molecular distinction of C × R hybrid (Coffea congensis × Coffea canephora) from morphologically resembling male parent using rbcL and matK gene sequences

Interspecific C × R hybrid (Coffea congensis × Coffea canephora) in India is cultivated as mixed population with male parent C. canephora as this species is an efficient pollen donor for enhanced yield. But distinction of C × R hybrid from C. canephora in old plantation is difficult due to varying plant sizes of C × R hybrid and often resembles with C. canephora. C × R hybrid cultivated under different agroclimatic conditions show distinct vegetative growth pattern with varying yields. Thus development of DNA marker for identification of C × R hybrid is important for clonal propagation and seed preparation from selective individuals. In this study, two DNA bar coding loci of chloroplast genome (rbcL and matK) of parents, F1 hybrid and its back cross progeny were partially sequenced to identify SNPs as DNA marker for distinction of C × R hybrid from C. canephora. Seven SNPs in the matK gene sequence and three nucleotides in the rbcL gene sequence were identified as DNA markers for the genetic identity of C. congensis. These SNPs were found in F1 and advanced progenies of C × R hybrid due to maternal inheritance. Large number of samples of C × R hybrids with varying morphological features revealed no polymorphism among C × R hybrid and C. congensis. Thus, the SNPs in C. congensis can be used as DNA markers for precise identification of C × R hybrid for production of clones besides tagging the chloroplast inheritance in advanced progenies.     

Tree species diversity and community composition in a human-dominated tropical forest of Western Ghats biodiversity hotspot,India

The structure, function, and ecosystem services of tropical forest depend on its species richness, diversity, dominance, and the patterns of changes in the assemblages of tree populations over time. Long-term data from permanent vegetation plots have yielded a wealth of data on the species diversity and dynamics of tree populations, but such studies have only rarely been undertaken in tropical forest landscapes that support large human populations. Thus, anthropogenic drivers and their impacts on species diversity and community structure of tropical forests are not well understood. Here we present data on species diversity, community composition, and regeneration status of tropical forests in a human-dominated landscape in the Western Ghats of southern India. Enumeration of 40 plots (50 m × 20 m) results a total of 106 species of trees, 76 species of saplings and 79 species of seedlings. Detrended Correspondence Analysis ordination of the tree populations yielded five dominant groups, along disturbance and altitudinal gradients on the first and second axes respectively. Abundant species of the area such as Albizia amara, Nothopegia racemosa and Pleiospermum alatum had relatively few individuals in recruiting size classes. Our data indicate probable replacement of rare, localized, and old-growth ‘specialists’ by disturbance-adapted generalists, if the degradation is continuing at the present scale.     

Cyclestheria hislopi (Crustacea: Branchiopoda): A group of morphologically cryptic species with origins in the Cretaceous

Cyclestheria hislopi is thought to be the only extant species of Cyclestherida. It is the sister taxon of all Cladocera and displays morphological characteristics intermediate of Spinicaudata and Cladocera. Using one mitochondrial (COI) and two nuclear (EF1α and 28S rRNA) markers, we tested the hypothesis that C. hislopi represents a single circumtropic species. South American (French Guiana), Asian (India, Indonesia, Singapore) and several Australian populations were included in our investigation. Phylogenetic and genetic distance analyses revealed remarkable intercontinental genetic differentiation (uncorrected p-distances COI > 13%, EF1α > 3% and 28S > 4%). Each continent was found to have at least one distinct Cyclestheria species, with Australia boasting four distinct main lineages which may be attributed to two to three species. The divergence of these species (constituting crown group Cyclestherida) was, on the basis of phylogenetic analyses of COI and EF1α combined with molecular clock estimates using several fossil branchiopod calibration points or a COI substitution rate of 1.4% per million years, dated to the Cretaceous. This was when the South American lineage split from the Asian–Australian lineage, with the latter diverging further in the Paleogene. Today’s circumtropic distribution of Cyclestheria may be best explained by a combination of Gondwana vicariance and later dispersal across Asia and Australia when the tectonic plates of the two continents drew closer in the early Miocene. The lack of morphological differentiation that has taken place in this taxon over such a long evolutionary period contrasts with the high level of differentiation and diversification observed in its sister taxon the Cladocera. Further insights into the evolution of Cyclestheria may help us to understand the evolutionary success of the Cladocera.     

Population genetic structure and conservation implications of Ceriops decandra in Malay Peninsula and North Australia

Comprehensive information of mangrove genetic resources is requisite for developing strategies for their effective conservation and sustainable use. Genetic diversity within and among populations of a widespread viviparous mangrove Ceriops decandra was determined using inter-simple sequence repeat (ISSR). Ten natural populations were collected from Malay Peninsula and North Australia. At the species level, high genetic variation was detected (P = 72%, H_E = 0.253, and I = 0.379). The estimate of G_ST was 0.882, indicating a high level of genetic differentiation among populations. When populations were grouped according to geographic regions, i.e., East Malaya, West Malaya, Southmost Malaya, and North Australia, AMOVA suggested that most of the total variation (87%) was accounted for by differentiation between regions, with only 4% accounting for variation among populations within regions, and a further 9% partitioned among individuals within a population. A UPGMA dendrogram based on genetic distance revealed a deep split between populations from the eastern Indian Ocean and all others from the western Pacific Ocean, which may result from the historical lowering of sea level at these regions during the recent Pleistocene glaciations. An understanding of the genetic structure of C. decandra provides insight for the conservation and management of this species.     

Cytology and embryology of the pompom weed,Campuloclinium macrocephalum (Eupatorieae,Asteraceae)

Campuloclinium macrocephalum (Less.) DC. is a perennial herb widely distributed in the New World, but introduced to South Africa, where it is commonly called “pompom weed”. This species is considered one of the most important weeds from Brazil and it has been included among the plant invaders of South Africa. Results of the meiotic and embryological analyses of six populations of C. macrocephalum are reported in this paper. The microsporogenesis analysis revealed five triploid (2n = 3x = 30) and one diploid (2n = 2x = 20) populations with a basic chromosome number x = 10. The diploid specimens showed regular meiotic behavior, but the triploid plants presented irregular chromosome pairing which result in the formation of univalents, bivalents and trivalents at diakinesis. In consequence, laggard chromosomes, unbalanced nuclei and micronuclei were observed in subsequent phases of meiosis. The embryological analysis showed that the triploid specimens of C. macrocephalum have embryo sac development from a nucellar cell (apospory), which indicates that these specimens are apomictic. Almost all cases of apomixis found in tribe Eupatorieae are diplosporous apomixis. Campuloclinium macrocephalum constitutes the second species of the tribe and the first of the genus with apospory as reproductive system. The aposporous apomixis combined with the presence of xylopodium would be two important factors responsible for the invasiveness of C. macrocephalum.     

Response to heat stress of populations of two Sphagnum species from alpine bogs at different altitudes

Sphagnum mosses are a fundamental component of bog vegetation in northern regions, where these plants play a major role in controlling important ecosystem processes. As heat waves are expected to become increasingly intense and frequent, especially in cold territories, it is important to improve our knowledge of heat resistance in Sphagnum species. We investigated the response to heat stress of S. fuscum and S. magellanicum. Three populations of the two species collected at different altitudes (1090 m, 1870 m and 2100 m) were grown at three daytime temperature levels: 25 °C (AT); 36 °C (MT); 43 °C (HT). The HT treatment decreased concentrations of chlorophyll and nitrogen in the plant tissues, which resulted in lower net CO₂ exchange rates and quantum yield of PS_II. The plants recovered significantly within six days, probably because temperature in the living tissue did not reach lethal thresholds because of the high water content in the plant tissues. Contrary to our main hypothesis, that S. magellanicum had greater resistance to high temperatures because of its more southern distribution, the two species showed much the same response patterns to heat stress. Supporting our second hypothesis, populations of both species originating from the highest site suffered somewhat stronger, although still reversible, damage when grown at HT. Heat stress brought about by heat waves will unlikely have differential effects on these two Sphagnum species. We also conclude that heat waves are unlikely to exert irreversible damage to the Sphagnum layer in bog ecosystems if high temperatures are not coupled with drought.     

Dispersal pattern of airborne emissions from an aluminium smelter in Ouro Preto,Brazil, as expressed by foliar fluoride accumulation in eight plant species

In order to evaluate the dispersal pattern of airborne fluoride emissions, from a single source in the city of Ouro Preto, Brazil, the fluoride impact on some herbaceous plant species was studied using the plants as passive bioindicators. Foliar fluoride contents of eight species collected at different distances from an aluminium smelter were analyzed. The plant species were: Baccaharis dracunculifolia, Bidens pilosa, Borreria verticillata, Calopogonium mucunoides, Erigeron bonariensis, Hedychium coronarium, Ipomoea purpurea and Ipomoea cairica. In all species the fluoride accumulation decreased exponentially with the distance from the emission source. There was specific and distinct variation in fluoride accumulation among the species, a group of high-accumulator species (B. dracunculifolia and Bidens pilosa) and a group of low-accumulator species (I. cairica, H. coronarium and Borreria verticillata). C. mucunoides and E. bonariensis occupied an intermediate position. There was a pattern of plant contamination response during the periods analyzed. The plants nearest to the emission source, between 0.4 km northwest and 1.1 km east, showed fluoride contamination traits in leaves reaching values between 100 and 500 μg g⁻¹. Moreover, fluoride contents higher than 1000 μg g⁻¹ were found in these plants. At the most distant stations, situated 2.9 km northwest and 6 km east from the factory, the fluoride content of the dry matter was less than 10 μg g⁻¹ showing that plants at those distances were submitted to minimum contamination. There were different patterns of tolerance among the species analyzed. While B. dracunculifolia accumulated fluoride up to 1500 μg g⁻¹ in dry matter without any signs of injury, Borreria verticillata showed severe necrosis in leaves, but the fluoride content found was not higher than 120 μg g⁻¹.     

Population genetics and conservation of the Azorean tree Picconia azorica

Picconia azorica (Tutin) Knobl. (Oleaceae) is an endangered species, endemic to the Azores. Samples from 31 populations in 8 islands were genotyped using 8 newly developed nuclear microsatellite markers. From the amplified loci, 81% were polymorphic across all populations and the species showed a relatively high total genetic diversity (H_T = 0.7). Several populations were close to Hardy–Weinberg equilibrium while others presented positive F_IS values (0.02–0.2). The largest proportion of genetic variation (98%) occurred within populations and the level of differentiation between populations, was generally low, although 27% of the population pairwise comparisons showed relatively high differentiation values (0.25 ≤ R_ST ≤ 0.65). Relatively high levels of gene flow were also found among most populations. Using the Bayesian clustering method implemented in STRUCTURE we found a particular genetic pattern in Corvo samples, and also similarities between Santa Maria, São Miguel and Flores populations. Considerable levels of genetic admixture within P. azorica populations might have resulted from: (i) fruit dispersal by native birds; and/or (ii) human mediated dispersal between islands. Our results revealed the existence of some genetically depauperate populations needing specific conservation measures, and indicate that arbitrary translocation of individuals between islands should be avoided.     

Phylogenetic relationships of Cochlodinium polykrikoides Margalef (Gymnodiniales,Dinophyceae) from the Mediterranean Sea and the implications of its global biogeography

Although the diversity of dinoflagellates has been intensively studied in several locations in the Mediterranean Sea since the 1950s, it is only during the last two decades that the morphotype of the toxic unarmoured dinoflagellate Cochlodinium polykrikoides Margalef has been detected, coinciding with its apparent worldwide expansion in marine coastal waters. In this study, vegetative cells of C. polykrikoides morphotype from the Catalan coast (NW Mediterranean Sea) were detected and isolated, and the DNA from collected cells was sequenced. While in the Mediterranean Sea, detections are scarce and C. polykrikoides is consistently present at low concentrations, we reported exceptional blooms of this species, in which the maximum abundance reached 2 × 10⁴ cells L⁻¹. Partial LSU rDNA region sequences showed that most C. polykrikoides populations from the Catalan coast formed a new differentiated ribotype, but others were included within the ‘Philippines’ ribotype, demonstrating their coexistence in the Mediterranean Sea. Thus, the current biogeographic nomenclature of the ribotypes is likely to be invalid with respect to the available information from populations comprising the ‘Philippines’ ribotype. The phylogeny suggests the existence of cryptic species that should be evaluated for species-level status. Accordingly, the ribotype determination must be carefully evaluated for all detections and bloom events, since accurate characterization of the morphology, ecophysiology and distribution of the ribotypes are not well resolved.     

Geometric morphometrics on Greek house mouse populations (Mus musculus domesticus) with Robertsonian and all-acrocentric chromosomal arrangements

This work aims to give the first comprehensive morphometric analysis of intraspecific variation for the different populations of the western house mouse (Mus musculus domesticus), in the Robertsonian (Rb) system of the N-NW Peloponnisos. Furthermore, we study all-acrocentric karyotype populations (2n = 40) of the species coming from several localities of Greece. We apply 2D shape analysis, i.e. landmark analysis and Elliptic Fourier Analysis, on the dorsal and ventral side of skull and the occlusal view of the first upper molar (M¹), respectively. Although significant genetic divergence between typical and Rb populations and even ongoing speciation processes have been reported for this species, this was not the case for the Greek populations studied. However, our analyses herein reveal morphologically differentiated chromosome groups in N-NW Peloponnisos Rb system and a clear geographical discrimination of the all-acrocentric (2n = 40) populations for all characters studied. We suggest that in all-acrocentric (2n = 40) karyotype mice the geographical distance drives their differentiation while within the Rb system of N-NW Peloponnisos, karyotype is the key factor that acts on their phenotypic variation.