Hidden heterochromatin: Characterization in the Rodentia species Cricetus cricetus, Peromyscus eremicus (Cricetidae) and Praomys tullbergi (Muridae)

The use of in situ restriction endonuclease (RE) (which cleaves DNA at specific sequences) digestion has proven to be a useful technique in improving the dissection of constitutive heterochromatin (CH), and in the understanding of the CH evolution in different genomes. In the present work we describe in detail the CH of the three Rodentia species, Cricetus cricetus, Peromyscus eremicus (family Cricetidae) and Praomys tullbergi (family Muridae) using a panel of seven REs followed by C-banding. Comparison of the amount, distribution and molecular nature of C-positive heterochromatin revealed molecular heterogeneity in the heterochromatin of the three species. The large number of subclasses of CH identified in Praomys tullbergi chromosomes indicated that the karyotype of this species is the more derived when compared with the other two genomes analyzed, probably originated by a great number of complex chromosomal rearrangements. The high level of sequence heterogeneity identified in the CH of the three genomes suggests the coexistence of different satellite DNA families, or variants of these families in these genomes.     

Cytogenetics of the Brazilian Bolitoglossa paraensis (Unterstein, 1930) salamanders (Caudata,Plethodontidae)

Plethodontid salamanders of genus Bolitoglossa constitute the largest and most diverse group of salamanders, including around 20% of living caudate species. Recent studies have indicated the occurrence of five recognized species in the Brazilian Amazon Rainforest. We present here the first cytogenetic data of a Brazilian salamander, which may prove to be a useful by contribution to the cytotaxonomy of the genus. Specimens were collected near the “type” locality (Utinga, Belém, PA, Brazil). Chromosomal preparations from duodenal epithelial cells and testes were subjected to Giemsa staining, C-banding and DAPI/CMA₃ fluorochrome staining. All specimens showed a karyotype with 13 bi-armed chromosome pairs (2n = 26). Nucleolar Organizer Regions, evidenced by CMA₃, were located distally on the long arm of pair 7 (7q). DAPI+ heterochromatin was predominantly centromeric, with some small pericentromeric bands. Although the C-banding patterns of other Bolitoglossa species are so far unknown, cytogenetic studies conducted in other Plethodontid salamanders have demonstrated that pericentromeric heterochromatin is a useful cytological marker for identifying interspecific homeologies. Species diversification is usually accompanied by chromosomal changes. Therefore, the cytogenetic characterization of Bolitoglossa populations from the middle and western Brazilian Amazon Basin could identify differences which may lead to the identification of new species.     

Karyotype differentiation in three species of Tripogandra Raf. (Commelinaceae) with different ploidy levels

Most species of the genus Tripogandra (Commelinaceae) are taxonomically poorly circumscribed, in spite of having a relatively stable basic number x = 8. Aiming to estimate the cytological variation among Tripogandra species carrying this base number, several structural karyotypic characters were investigated in the diploid T. glandulosa, the hexaploid T. serrulata, and the octoploid T. diuretica. A careful evaluation of chromosome size and morphology did not reveal clear chromosome homeologies among karyotypes. The mean chromosome size was strongly reduced in the octoploid species, but not in the hexaploid species. They also differed largely in the CMA(+) banding pattern and in the number of 5S and 45S rDNA sites per monoploid chromosome complement. All three species showed proximal DAPI (+) heterochromatin, although in T. serrulata this kind of heterochromatin was only visible after FISH. Further, the meiosis in T. serrulata was highly irregular, suggesting that this species has a hybrid origin. The data indicate that, in spite of the conservation of the base number, these species are karyologically quite different from each other.     

Effect of an Ice-Nucleating Activity Agent on Subzero Survival of Nematode Juveniles

Juveniles of five species of nematodes, Caenorhabditis elegans, Panagrellus redivivus, Pratylenchus agilis, Pristionchus pacificus, and Distolabrellus veechi, were added to solutions with (treatment) and without (control) a commercial ice-nucleating activity (INA) agent. Ten-microliter droplets of the solutions containing the juveniles were placed on glass microscope slides and transferred to a temperaturecontrolled freeze plate where the temperature was reduced to -6 to -8 °C. At this temperature, the droplets containing the INA agent froze while those without the agent remained liquid. After 2 minutes, the temperature of the plate was raised to 24 °C, and the slides were examined with a light microscope to determine the viability of the juveniles. The results showed that usually most juveniles (43% to 88%, depending on species) in solutions that did not contain the INA agent (controls) were active, indicating that the juveniles were capable of supercooling and were thereby protected from the subzero temperatures. Alternatively, less than 10% of the juveniles that had frozen for 2 minutes in solutions containing the INA agent remained viable, indicating that inoculative freezing of the solution was lethal to the supercooled juveniles. Our results suggest that, in geographical areas where winter temperatures may not be sufficiently low or sustained to freeze soil, the addition of an INA agent may help induce ice nucleation and thereby reduce the populations of nematode species that are unable to survive when the soil solution is frozen.     

The karyotype of Adenomera diptyx (Boettger 1885) (Anura, Leptodactylidae) from northeastern Argentina

In this work we analyzed the karyotype of five populations of Adenomera diptyx from Argentina after conventional staining, Ag-NOR and C-banding. All specimens presented 2n = 26 and FN = 34. The karyotype was formed by three submetacentric, one metacentric and nine telocentric pairs. Silver staining revealed that the NOR was located on a secondary constriction in pair 7. C- banding evidenced constitutive heterochromatin at the pericentromeric region of all chromosomes. The karyotype of A. diptyx was similar to that of A. hylaedactyla (2n = 26, FN = 34) and different from that of A. andreae (2n = 26, FN = 40) in the fundamental number and secondary constriction position. It also differed from the karyotypes of A. marmorata (2n = 24, FN = 34 and 36) and of A. aff. bokermanni (2n = 23, FN = 34) in diploid number. Until a comprehensive cytogenetic analysis of all the species of the genus is performed, their chromosome evolution will remain poorly understood.     

Subtropical mouse-tailed bats use geothermally heated caves for winter hibernation

We report that two species of mouse-tailed bats (Rhinopoma microphyllum and R. cystops) hibernate for five months during winter in geothermally heated caves with stable high temperature (20°C). While hibernating, these bats do not feed or drink, even on warm nights when other bat species are active. We used thermo-sensitive transmitters to measure the bats’ skin temperature in the natural hibernacula and open flow respirometry to measure torpid metabolic rate at different ambient temperatures (T_a, 16–35°C) and evaporative water loss (EWL) in the laboratory. Bats average skin temperature at the natural hibernacula was 21.7 ± 0.8°C, and no arousals were recorded. Both species reached the lowest metabolic rates around natural hibernacula temperatures (20°C, average of 0.14 ± 0.01 and 0.16 ± 0.04 ml O₂ g⁻¹ h⁻¹ for R. microphyllum and R. cystops, respectively) and aroused from torpor when T_a fell below 16°C. During torpor the bats performed long apnoeas (14 ± 1.6 and 16 ± 1.5 min, respectively) and had a very low EWL. We hypothesize that the particular diet of these bats is an adaptation to hibernation at high temperatures and that caves featuring high temperature and humidity during winter enable these species to survive this season on the northern edge of their world distribution.     

Cytogenetic analysis of three species of Pseudacteon (Diptera, Phoridae) parasitoids of the fire ants using standard and molecular techniques

Pseudacteon flies, parasitoids of worker ants, are being intensively studied as potentially effective agents in the biological control of the invasive pest fire ant genus Solenopsis (Hymenoptera: Formicidae). This is the first attempt to describe the karyotype of P. curvatus Borgmeier, P. nocens Borgmeier and P. tricuspis Borgmeier. The three species possess 2n = 6; chromosomes I and II were metacentric in the three species, but chromosome pair III was subtelocentric in P. curvatus and P. tricuspis, and telocentric in P. nocens. All three species possess a C positive band in chromosome II, lack C positive heterochromatin on chromosome I, and are mostly differentiated with respect to chromosome III. P. curvatus and P. tricuspis possess a C positive band, but at different locations, whereas this band is absent in P. nocens. Heterochromatic bands are neither AT nor GC rich as revealed by fluorescent banding. In situ hybridization with an 18S rDNA probe revealed a signal on chromosome II in a similar location to the C positive band in the three species. The apparent lack of morphologically distinct sex chromosomes is consistent with proposals of environmental sex determination in the genus. Small differences detected in chromosome length and morphology suggests that chromosomes have been highly conserved during the evolutionary radiation of Pseudacteon. Possible mechanisms of karyotype evolution in the three species are suggested.     

Does the intensive grazing and aridity change the relations between the dominant shrub Artemisia kopetdaghensis and plants under its canopies?

The interspecific plant interactions along grazing and aridity stress gradients represent a major research issue in plant ecology. However, the combined effects of these two factors on plant–plant interactions have been poorly studied in the northeast of Iran. To fill this knowledge gap, 144 plots were established in 12 study sites with different grazing intensities (high vs. low) and climatic characteristics (arid vs. semiarid) in northeastern Iran. A dominant shrub, Artemisia kopetdaghensis, was selected as the model species. Further, we studied changes in plant life strategies along the combined grazing and aridity stress gradients. In this study, we used relative interaction indices calculated for species richness, Shannon diversity, and species cover to determine plant–plant interactions using linear mixed‐effect models (LMM). The indicator species analysis was used to identify the indicator species for the undercanopy of shrub and for the adjacent open areas. The combined effects of grazing and aridity affected the plant–plant interactions and plant life strategies (CSR) of indicator species. A. kopetdaghensis showed the highest facilitation effect under high stress conditions (high grazing, high aridity), which turned into competition under the low stress conditions (low grazing, low aridity). In the arid region, the canopy of the shrub protected ruderals, annual forbs, and grasses in both high and low grazing intensities. In the semiarid region and high grazing intensity (low aridity/high grazing), the shrubs protected mostly perennial forbs with C‐strategy. Our findings highlight the importance of context‐dependent shrub management to restore the vegetation damaged by the intensive grazing.     

Karyotype differentiation of four Cestrum species (Solanaceae) revealed by fluorescent chromosome banding and FISH

The karyotypes of four South American species of Cestrum (C. capsulare,C. corymbosum,C. laevigatum and C. megalophylum) were studied using conventional staining, C-CMA/DAPI chromosome banding and FISH with 45S and 5S rDNA probes. The karyotypes showed a chromosome number of 2n = 2x = 16, with metacentric chromosomes, except for the eighth submeta- to acrocentric pair. Several types of heterochromatin were detected, which varied in size, number, distribution and base composition. The C-CMA(+) bands and 45S rDNA were located predominantly in terminal regions. The C-CMA (+) /DAPI (+) bands appeared in interstitial and terminal regions, and the C-DAPI (+) bands were found in all chromosome regions. The 5S rDNA sites were observed on the long arm of pair 8 in all species except C. capsulare, where they were found in the paracentromeric region of the long arm of pair 4. The differences in band patterns among the species studied here, along with data from other nine species reported in the literature, suggest that the bands are dispersed in an equilocal and non-equilocal manner and that structural rearrangements can be responsible for internal karyotype diversification. However, it is important to point out that the structural changes involving repetitive segments did not culminate in substantial changes in the general karyotype structure concerning chromosome size and morphology.     

Preferential Breakpoints in the Recovery of Broken Dicentric Chromosomes in Drosophila melanogaster

We designed a system to determine whether dicentric chromosomes in Drosophila melanogaster break at random or at preferred sites. Sister chromatid exchange in a Ring-X chromosome produced dicentric chromosomes with two bridging arms connecting segregating centromeres as cells divide. This double bridge can break in mitosis. A genetic screen recovered chromosomes that were linearized by breakage in the male germline. Because the screen required viability of males with this X chromosome, the breakpoints in each arm of the double bridge must be closely matched to produce a nearly euploid chromosome. We expected that most linear chromosomes would be broken in heterochromatin because there are no vital genes in heterochromatin, and breakpoint distribution would be relatively unconstrained. Surprisingly, approximately half the breakpoints are found in euchromatin, and the breakpoints are clustered in just a few regions of the chromosome that closely match regions identified as intercalary heterochromatin. The results support the Laird hypothesis that intercalary heterochromatin can explain fragile sites in mitotic chromosomes, including fragile X. Opened rings also were recovered after male larvae were exposed to X-rays. This method was much less efficient and produced chromosomes with a strikingly different array of breakpoints, with almost all located in heterochromatin. A series of circularly permuted linear X chromosomes was generated that may be useful for investigating aspects of chromosome behavior, such as crossover distribution and interference in meiosis, or questions of nuclear organization and function.     

Conditional cold avoidance drives between-population variation in germination behaviour in Calluna vulgaris

Background and Aims

Across their range, widely distributed species are exposed to a variety of climatic and other environmental conditions, and accordingly may display variation in life history strategies. For seed germination in cold climates, two contrasting responses to variation in winter temperature have been documented: first, an increased ability to germinate at low temperatures (cold tolerance) as winter temperatures decrease, and secondly a reduced ability to germinate at low temperatures (cold avoidance) that concentrates germination towards the warmer parts of the season.

Methods

Germination responses were tested for Calluna vulgaris, the dominant species of European heathlands, from ten populations collected along broad-scale bioclimatic gradients (latitude, altitude) in Norway, covering a substantial fraction of the species'' climatic range. Incubation treatments varied from 10 to 25 °C, and germination performance across populations was analysed in relation to temperature conditions at the seed collection locations.

Key Results

Seeds from all populations germinated rapidly and to high final percentages under the warmer incubation temperatures. Under low incubation temperatures, cold-climate populations had significantly lower germination rates and percentages than warm-climate populations. While germination rates and percentages also increased with seed mass, seed mass did not vary along the climatic gradients, and therefore did not explain the variation in germination responses.

Conclusions

Variation in germination responses among Calluna populations was consistent with increased temperature requirements for germination towards colder climates, indicating a cold-avoidance germination strategy conditional on the temperature at the seeds'' origin. Along a gradient of increasing temperatures this suggests a shift in selection pressures on germination from climatic adversity (i.e. low temperatures and potential frost risk in early or late season) to competitive performance and better exploitation of the entire growing season.     

The fission yeast homologue of CENP-B, Abp1, regulates directionality of mating-type switching

In fission yeast, mating-type switching involves replacing genetic information contained at the expressed mat1 locus by that of either the mat2P or mat3M donor loci. Donor selection is nonrandom, as mat1P cells preferentially use mat3M for switching, whereas mat1M cells use mat2P. Switching directionality is determined by the cell-type-specific distribution of the Swi2-Swi5 complex that, in mat1P cells, localises to mat3M and, only in mat1M cells, spreads to mat2P in a heterochromatin-dependent manner. Mechanisms regulating spreading of Swi2-Swi5 across heterochromatin are not fully understood. Here, we show that the fission yeast homologue of CENP-B, Abp1, binds to the silent domain of the mating-type locus and regulates directionality of switching. Deletion of abp1 prevents utilisation of mat2P, as when heterochromatin is disrupted and spreading of Swi2-Swi5 is impaired. Our results show that, indeed, deletion of abp1 abolishes spreading of Swi2-Swi5 to mat2P. However, in abp1Delta cells, heterochromatin organisation at the mating-type locus is preserved, indicating that Abp1 is actually required for efficient spreading of Swi2-Swi5 through heterochromatin. Cbh1 and Cbh2, which are also homologous to CENP-B, have only a minor contribution to the regulation of directionality of switching, which is in contrast with the strong effects observed for Abp1.     

Karyology of the Atlantic forest rodent Juliomys (Cricetidae): A new karyotype from southern Brazil

Juliomys is a small rodent from the family Cricetidae which inhabits the Atlantic forest and forests from Argentina to eastern Brazil. The three species recognized so far have different karyotypes. In this paper, we describe a new karyotype with 2n = 32, FN = 48 found in Juliomys specimens from a high-altitude area in the Atlantic forest of southern Brazil. The karyotype was analyzed after G- and C-banding and silver staining of the nucleolus organizer regions (Ag-NOR) and its G-banding patterns were compared with those of the newly described species Juliomys ossitenuis (2n = 20, FN = 36). The 2n = 32 karyomorph presented peculiar features and was very different from those of the other species of the genus: J. pictipes (2n = 36, FN = 34), J. rimofrons (2n = 20, FN = 34) and J. ossitenuis (2n = 20, FN = 36). Differences were mostly due to centric and tandem fusions, pericentric inversion and loss of heterochromatin. The karyotype represents a powerful tool to differentiate Juliomys species and our data suggest that the karyotype described herein belongs to a new species.     

Molecular organization of heterochromatin in malaria mosquitoes of the Anopheles maculipennis subgroup

Although heterochromatin makes up a significant portion of the malaria mosquito genome, its organization, function, and evolution are poorly understood. Sibling species of the Anopheles maculipennis subgroup, the European malaria mosquitoes, are characterized by striking differences in the morphology of pericentric heterochromatin; however, the molecular basis for the rapid evolutionary transformation of heterochromatin is not known. This study reports an initial survey of the molecular organization of the pericentric heterochromatin in nonmodel species from the A. maculipennis subgroup. Molecular identity and chromosomal localization were established for short DNA fragments obtained by microdissection from the pericentric diffuse β-heterochromatin of A. atroparvus. Among 102 sequenced clones of the Atr2R library, twenty had sequence similarity to transposable elements (TEs) from the Anopheles gambiae and Aedes aegypti genomes. At least six protein-coding single-copy genes from A. gambiae and four single-copy genes from Drosophila melanogaster were homologous to eight clones from the library. Most of these conserved genes were heterochromatic in A. gambiae but euchromatic in D. melanogaster. The remaining 74 clones were characterized as noncoding repetitive DNA. Comparative chromosome mapping of twelve clones in the sibling species A. atroparvus and A. messeae demonstrated that the noncoding repetitive sequences and the TEs have undergone independent chromosome-specific and species-specific gains and losses in the morphologically different pericentric heterochromatic regions, in accordance with the “library model.”     

The karyotype of Nothoscordum arenarium Herter (Gilliesioideae, Alliaceae): A populational and cytomolecular analysis

The genus Nothoscordum Kunth comprises approximately 20 species native to South America. Karyologically, the genus is remarkable for its large chromosomes and Robertsonian translocations. Variation in chromosome number has been recorded in a few polyploid species and it is unknown among diploids. This study presents the chromosome number and morphology of 53 individuals of seven populations of N. arenarium Herter (2n = 10). In addition, karyotype analyses after C-banding, staining with CMA and DAPI, and in situ hybridization with 5S and 45S rDNA probes were performed in six individuals from one population. All individuals exhibited 2n = 10 (6M + 4A), except for one tetraploid (2n = 20, 12M + 8A) and one triploid (2n = 15, 9M + 6A) plant. C-banding revealed the presence of CMA(+) /DAPI (-) heterochromatin in the short arm and in the proximal region of the long arm of all acrocentric chromosomes. The 45S rDNA sites co-localized with the CMA (+) regions of the acrocentrics short arms, while the 5S rDNA probe only hybridized with the subterminal region of a pair of metacentric chromosomes. A change in the pattern of CMA bands and rDNA sites was observed in only one individual bearing a reciprocal translocation involving the long arm of a metacentric and the long arm of an acrocentric chromosome. These data suggest that, despite isolated cases of polyploidy and translocation, the karyotype of N. arenarium is very stable and the karyotypic instability described for other species may be associated with their polyploid condition.     

Genomic population structure of freshwater‐resident and anadromous ide (Leuciscus idus) in north‐western Europe

Climate change experts largely agree that future climate change and associated rises in oceanic water levels over the upcoming decades, will affect marine salinity levels. The subsequent effects on fish communities in estuarine ecosystems however, are less clear. One species that is likely to become increasingly affected by changes in salinity is the ide (Leuciscus idus). The ide is a stenohaline freshwater fish that primarily inhabits rivers, with frequent anadromous behavior when sea salinity does not exceed 15%. Unlike most other anadromous Baltic Sea fish species, the ide has yet to be subjected to large‐scale stocking programs, and thus provides an excellent opportunity for studying the natural population structure across the current salinity gradient in the Danish Belts. To explore this, we used Genotyping‐by‐Sequencing to determine genomic population structure of both freshwater resident and anadromous ide populations in the western Baltic Sea region, and relate the results to the current salinity gradient and the demographic history of ide in the region. The sample sites separate into four clusters, with all anadromous populations in one cluster and the freshwater resident populations in the remaining three. Results demonstrate high level of differentiation between sites hosting freshwater resident populations, but little differentiation among anadromous populations. Thus ide exhibit the genomic population structure of both a typical freshwater species, and a typical anadromous species. In addition to providing a first insight into the population structure of north‐western European ide, our data also (1) provide indications of a single illegal introduction by man; (2) suggest limited genetic effects of heavy pollution in the past; and (3) indicate possible historical anadromous behavior in a now isolated freshwater population.     

Sympatric species of Hibbertia (Dilleniaceae) vary in dormancy break and germination requirements: implications for classifying morphophysiological dormancy in Mediterranean biomes

Background and Aims

Several ecologically important plant families in Mediterranean biomes have seeds with morphophysiological dormancy (MPD) but have been poorly studied. The aim of this study was to understand the seed ecology of these species by focusing on the prominent, yet intractably dormant Australian genus Hibbertia. It was hypothesized that the slow germination in species of this genus is caused by a requirement for embryo growth inside the seed before germination, and that initiation of embryo growth is reliant upon a complex sequence of environmental cues including seasonal fluctuations in temperature and moisture, and an interplay with light and smoke. Using the results, the classification of the MPD level in species of Hibbertia is considered.

Methods

Four species of Hibbertia in winter rainfall south-western Australia were selected. These species, whilst differing in geographic distributions, are variously sympatric, and all are important understorey components of plant communities. The following aspects related to dormancy break, embryo growth and germination were investigated: temperature and moisture requirements; effects of karrikinolide, gibberellic acid and aerosol smoke; and phenology.

Key Results

Following exposure to wet/dry cycles at low or high temperatures, embryo growth and germination occurred, albeit slowly in all species at low temperatures when moisture was unlimited, corresponding to winter in south-west Australia. Photo regime influenced germination only in H. racemosa. Aerosol smoke triggered substantial germination during the 1st germination season in H. huegelii and H. hypericoides.

Conclusions

Although the study species are con-generic, sympatric and produce seeds of identical morphology, they possessed different dormancy-break and germination requirements. The physiological component of MPD was non-deep in H. racemosa but varied in the other three species where more deeply dormant seeds required >1 summer to overcome dormancy and, thus, germination was spread over time. Embryos grew during winter, but future studies need to resolve the role of cold versus warm stratification by using constant temperature regimes. To include Mediterranean species with MPD, some modifications to the current seed-dormancy classification system may need consideration: (a) wet/dry conditions for warm stratification and (b) a relatively long period for warm stratification. These outcomes have important implications for improving experimental approaches to resolve the effective use of broadcast seed for ecological restoration.