Steinemann M, Steinemann S. 1998. Enigma of Y chromosome degeneration: Neo-Y and Neo-X chromosomes of Drosophila miranda a model for sex chromosome evolution. Genetica 102/103: 409-420.
These authors examined the Neo-Y chromosome and its meiotic-pairing partner X2 in Drosophila miranda, a species in the Drosophila psuedoobscura species subgroup, about 25 million years removed from D. melanogaster.
These sex chromosomes are unique to D. miranda, and represent an early stage in the evolution of degenerate Y chromosomes. Two major changes distinguish degenerate Y chromosomes from X chromosomes: loss of functional genes by psuedogenization and conversion of euchromatin to heterochromatin.
Muller’s ratchet and the accumulation of point mutations on a non-recombining Y chromosome can account for the first difference, but does not explain the heterochromatinization of the Neo-Y chromosome. A block of repeats homologous to telomere sequences was found in the interior region of the Neo-Y, suggesting an end-to-end chromosome fusion event as the origin of the Neo-Y. These authors examined a region of the Neo-Y associated with a clustered family of genes. On the Neo-Y but not on the X2 they found a “massive accumulation of transposable elements” in this region, associated with the silencing of two of the genes in the examined family.
Finally, the authors suggest that transposable elements are directly involved in the formation of heterochromatin, though the mechanism by which this occurs is not described. The high level of TE insertion in the Neo-Y of D. miranda is strong evidence for chromosome degeneration as in Y chromosomes driven at least partly by the activities of TEs.
Monday, January 28, 2008
Wednesday, January 23, 2008
Blumenstiel et al. 2002
Blumenstiel JP, Hartl Dl, Lozovsky ER. 2002. Patterns of insertion and deletion in contrasting chromatin domains. Molecular Biology and Evolution 19: 2211-2225.
These authors examined “dead-on-arrival” (DOA) transposable elements (TEs) in the genome of Drosophila melanogaster. They divided the genome into three categories for their analysis: heterochromatin, euchromatin of low recombination frequency, and euchromatin of high recombination frequency. They also calculated local rates of DNA gain or loss by small indels.
TEs were more abundant in heterochromatin than in either type of euchromatin, and more abundant in low-recombination euchromatin than in high-recombination euchromatin. Young TEs (more recent insertions) were more evenly distributed while older “mature” TEs were heavily biased towards heterochromatin. The average rate of DNA loss over the entire genome examined was 5.6 bp per nucleotide substition, a rate of spontaneous DNA loss much faster than found in mammals (Petrov and Hartl, 1998).
The authors propose four potential mechanisms to explain the observed distribution of TEs. The first two, that TEs insert preferentially in heterochromatin and that the rate of spontaneous deletion is lowest in heterochromatin, are each rejected. The examined TEs do not insert preferentially anywhere in the genome, as evidenced by the even distribution of the youngest TEs. Spontaneous deletion rates calculated in this study did not vary across regions of the genome, and were insensitive to local recombination rates. Small deletions in TEs should be favoured (and commonly detected) if the deletion rate varies in such an adaptive manner.
The remaining explanatory mechanisms both involve selection, either relaxed selection against TEs in heterochromatin relative to in euchromatin, or positive selection on TEs in heterochromatin for some reason. This analysis suggested the relaxed selection hypothesis is more likely, though it is neutral with respect to the relative contributions to that selection of avoiding ectopic recombination and avoidance of interference with gene function.
The authors suggest that while they have no evidence that small deletions are favoured any more in one region than another, they claim to have some evidence that large deletions, greater than 400 bp, may be favoured. However, this statement is based on a difference in mean deletion sizes between regions that is not statistically significant; they seem to be claiming a trend that does not exist! Perhaps I misread that section of the Discussion.
These authors examined “dead-on-arrival” (DOA) transposable elements (TEs) in the genome of Drosophila melanogaster. They divided the genome into three categories for their analysis: heterochromatin, euchromatin of low recombination frequency, and euchromatin of high recombination frequency. They also calculated local rates of DNA gain or loss by small indels.
TEs were more abundant in heterochromatin than in either type of euchromatin, and more abundant in low-recombination euchromatin than in high-recombination euchromatin. Young TEs (more recent insertions) were more evenly distributed while older “mature” TEs were heavily biased towards heterochromatin. The average rate of DNA loss over the entire genome examined was 5.6 bp per nucleotide substition, a rate of spontaneous DNA loss much faster than found in mammals (Petrov and Hartl, 1998).
The authors propose four potential mechanisms to explain the observed distribution of TEs. The first two, that TEs insert preferentially in heterochromatin and that the rate of spontaneous deletion is lowest in heterochromatin, are each rejected. The examined TEs do not insert preferentially anywhere in the genome, as evidenced by the even distribution of the youngest TEs. Spontaneous deletion rates calculated in this study did not vary across regions of the genome, and were insensitive to local recombination rates. Small deletions in TEs should be favoured (and commonly detected) if the deletion rate varies in such an adaptive manner.
The remaining explanatory mechanisms both involve selection, either relaxed selection against TEs in heterochromatin relative to in euchromatin, or positive selection on TEs in heterochromatin for some reason. This analysis suggested the relaxed selection hypothesis is more likely, though it is neutral with respect to the relative contributions to that selection of avoiding ectopic recombination and avoidance of interference with gene function.
The authors suggest that while they have no evidence that small deletions are favoured any more in one region than another, they claim to have some evidence that large deletions, greater than 400 bp, may be favoured. However, this statement is based on a difference in mean deletion sizes between regions that is not statistically significant; they seem to be claiming a trend that does not exist! Perhaps I misread that section of the Discussion.
Tuesday, January 22, 2008
Nikaido et al. 2003
Nikaido M, Nishihara H, Hukumoto Y, Okada N. 2003. Ancient SINEs from African endemic mammals. Molecular Biology and Evolution 20: 522-527.
These authors discovered a family of Short Interspersed Nuclear Elements (SINEs) restricted to the genomes of Afrotherian mammals. Afrotheria is clade of mammals that originated in Africa during the 60 million years or so it was isolated from the other continents, from approximately 100 to 40 mya, and includes elephants, hyraxes, aardvarks and other mammals. The monophyly of the afrotherian clade is a recent hypothesis, which these authors sought to test by a molecular phylogenetic analysis.
SINEs are not expected to experience horizontal transmission, in contrast to some other retrotransposons, and endemic SINE families have been found in other mammal clades. For example, the Alu family of SINEs are only found in primates.
The main result of this study was the discovery of this family of SINEs and its restriction to animals included in the Afrotheria clade by previous authors based on other data. This result supports the monophyly hypothesis for Afrotheria, and thus helps to connect phylogeny with plate tectonics.
These authors discovered a family of Short Interspersed Nuclear Elements (SINEs) restricted to the genomes of Afrotherian mammals. Afrotheria is clade of mammals that originated in Africa during the 60 million years or so it was isolated from the other continents, from approximately 100 to 40 mya, and includes elephants, hyraxes, aardvarks and other mammals. The monophyly of the afrotherian clade is a recent hypothesis, which these authors sought to test by a molecular phylogenetic analysis.
SINEs are not expected to experience horizontal transmission, in contrast to some other retrotransposons, and endemic SINE families have been found in other mammal clades. For example, the Alu family of SINEs are only found in primates.
The main result of this study was the discovery of this family of SINEs and its restriction to animals included in the Afrotheria clade by previous authors based on other data. This result supports the monophyly hypothesis for Afrotheria, and thus helps to connect phylogeny with plate tectonics.
Monday, January 21, 2008
Palestis et al. 2004
Palestis BG, Burt A, Jones RN, Trivers R. 2004. B chromosomes are more frequent in mammals with acrocentric karyotypes: support for the theory of centromeric drive. Proceedings of the Royal Society of London B (Supplement) 271: S22-S24.
These authors tested the predictions of the theory of centromeric drive (apparently proposed by Pardo-Manuel de Villena and Sapienza, 2001) using a dataset of cytological and taxonomic information for mammals.
The theory of centromeric drive explains the distribution of B chromosomes among species by asymmetries during female meiosis. Female meiosis involves the production of only one gamete from a diploid progenitor cell, compared with the four gametes from one diploid progenitor in males. This unbalance in female meiosis offers an opportunity for selfish genetic elements like B chromosomes to achieve meiotic drive by exploiting differences in spindle capabilities between eggs and polar bodies.
A “strong” spindle in the egg compared with the polar body will capture more centromeres during meiotic division when there are odd numbers, as in the case of a centromeric fission or fusion translocation event that leads to meiotic pairing of one metacentric with two acrocentric chromosomes. This stronger spindle will also tend to capture the centromeres of any B chromosomes, as well. Conversely, if the polar body has the stronger spindle, B chromosomes will be lost from the population more often than chance.
This study found support for the theory of centromeric drive, in that most mammals with Bs had predominantly acrocentric A chromosomes, while acrocentrics were relatively rare among species without Bs. These data support both the proliferation of Bs in predominantly-acrocentric genomes and the typical failure of Bs to establish in predominantly-metacentric genomes.
Two alternative hypotheses for these data are presented, but no other evidence consistent with either acrocentric chromosomes being prone to generating Bs nor with other selection pressures on centromeres are described, apparently because such evidence has not been found. Additionally, although mammal Bs are poorly studied, two cases of strong female meiotic drive in rat Bs are known, associated with weak or absent drive in males, and two other species in which nothing is known about female meiotic drive but mitotic instability in males has been found do not contradict the theory of centromeric drive.
These authors tested the predictions of the theory of centromeric drive (apparently proposed by Pardo-Manuel de Villena and Sapienza, 2001) using a dataset of cytological and taxonomic information for mammals.
The theory of centromeric drive explains the distribution of B chromosomes among species by asymmetries during female meiosis. Female meiosis involves the production of only one gamete from a diploid progenitor cell, compared with the four gametes from one diploid progenitor in males. This unbalance in female meiosis offers an opportunity for selfish genetic elements like B chromosomes to achieve meiotic drive by exploiting differences in spindle capabilities between eggs and polar bodies.
A “strong” spindle in the egg compared with the polar body will capture more centromeres during meiotic division when there are odd numbers, as in the case of a centromeric fission or fusion translocation event that leads to meiotic pairing of one metacentric with two acrocentric chromosomes. This stronger spindle will also tend to capture the centromeres of any B chromosomes, as well. Conversely, if the polar body has the stronger spindle, B chromosomes will be lost from the population more often than chance.
This study found support for the theory of centromeric drive, in that most mammals with Bs had predominantly acrocentric A chromosomes, while acrocentrics were relatively rare among species without Bs. These data support both the proliferation of Bs in predominantly-acrocentric genomes and the typical failure of Bs to establish in predominantly-metacentric genomes.
Two alternative hypotheses for these data are presented, but no other evidence consistent with either acrocentric chromosomes being prone to generating Bs nor with other selection pressures on centromeres are described, apparently because such evidence has not been found. Additionally, although mammal Bs are poorly studied, two cases of strong female meiotic drive in rat Bs are known, associated with weak or absent drive in males, and two other species in which nothing is known about female meiotic drive but mitotic instability in males has been found do not contradict the theory of centromeric drive.
SanMiguel et al. 1998
SanMiguel P, Gaut BS, Tikhonov A, Nakajima Y, Bennetzen JL. 1998. The paleontology of intergene retrotransposons of maize. Nature Genetics 20: 43-45.
These authors dated the insertions of 16 retrotransposons inserted in the 240kb region of the maize genome near the gene adh1. The retrotransposons in this region encompass approximately 160kb; three protein-coding genes have been described from this region.
The dating method was based on divergence of the Long Terminal Repeats (LTR) of this family of retrotransposons. The two repeats per retrotransposon would have been identical in sequence when the elements inserted, but have diverged by neutral substitution since that time. If a good estimate of neutral substitution is known, then the divergence in sequence between LTR can provide an estimate of time since insertion.
Many of the retrotransposons inserted into existing retrotransposons in a hierarchical manner, leading to a prediction that LTR divergences of retros-within-retros should be lower (i.e. more recent) than retrotransposons inserted directly into the maize genome. This prediction was correct for 10 of 11 examined retros-in-retros; in the eleventh, the divergences of the LTRs in the two retrotransposons were similar, suggesting similar dates of insertion. This is at least not inconsistent with the prediction.
When the authors compared the maize adh1 region to the orthologous region in sorghum, no retrotransposons were found in sorghum. These authors estimated divergence between the two plant species at 17.4 million years ago, consistent with a previously-published estimate of 16 mya. Thus, all 16 retrotransposon insertions most likely occurred in the last 16 million years or so. The dating based on LTR divergences suggest that almost all of these insertions occurred in the last 6 million years.
The authors discuss and are able to cautiously disregard several potential sources of error, such as gene converstion altering LTR sequences and RNA recombination among distantly-related transcripts.
Their main conclusion, which is well-supported by their data, is that the 240-kb region of the adh1 gene has experienced intense retrotransposon insertion activity over the last 6 million years, greatly increasing the size of this local region. From this, and from four published LTR retrotransposon sequences in other parts of the maize genome, they extrapolate that the maize genome doubled in size from 1200 Mb to 2400 Mb over roughly the same period, a conclusion I find consistent with their results but not particularly well-supported. They end with a request for greater study of this and related phenomena in plant genomes.
These authors dated the insertions of 16 retrotransposons inserted in the 240kb region of the maize genome near the gene adh1. The retrotransposons in this region encompass approximately 160kb; three protein-coding genes have been described from this region.
The dating method was based on divergence of the Long Terminal Repeats (LTR) of this family of retrotransposons. The two repeats per retrotransposon would have been identical in sequence when the elements inserted, but have diverged by neutral substitution since that time. If a good estimate of neutral substitution is known, then the divergence in sequence between LTR can provide an estimate of time since insertion.
Many of the retrotransposons inserted into existing retrotransposons in a hierarchical manner, leading to a prediction that LTR divergences of retros-within-retros should be lower (i.e. more recent) than retrotransposons inserted directly into the maize genome. This prediction was correct for 10 of 11 examined retros-in-retros; in the eleventh, the divergences of the LTRs in the two retrotransposons were similar, suggesting similar dates of insertion. This is at least not inconsistent with the prediction.
When the authors compared the maize adh1 region to the orthologous region in sorghum, no retrotransposons were found in sorghum. These authors estimated divergence between the two plant species at 17.4 million years ago, consistent with a previously-published estimate of 16 mya. Thus, all 16 retrotransposon insertions most likely occurred in the last 16 million years or so. The dating based on LTR divergences suggest that almost all of these insertions occurred in the last 6 million years.
The authors discuss and are able to cautiously disregard several potential sources of error, such as gene converstion altering LTR sequences and RNA recombination among distantly-related transcripts.
Their main conclusion, which is well-supported by their data, is that the 240-kb region of the adh1 gene has experienced intense retrotransposon insertion activity over the last 6 million years, greatly increasing the size of this local region. From this, and from four published LTR retrotransposon sequences in other parts of the maize genome, they extrapolate that the maize genome doubled in size from 1200 Mb to 2400 Mb over roughly the same period, a conclusion I find consistent with their results but not particularly well-supported. They end with a request for greater study of this and related phenomena in plant genomes.
Friday, January 18, 2008
Watanabe et al. 1999
Watanabe K, Yahara T, Denda T, Kosuge K. 1999. Chromosomal evolution in the genus Brachyscome (Asteraceae, Astereae): Statistical tests regarding correlation between changes in karyotype and habit using phylogenetic information. Journal of Plant Research 112: 145-161.
These authors used most of the described species in a large genus of Australasian flowering shrubs to test hypotheses regarding chromosome evolution and life-history. Brachyscome contains about 80 species; these authors examined 73, plus 8 other species in related genera. The genus includes a wide range of karyotypes, from 2n=4 up to 2n=36, with a mode of 2n=18. The species are distributed across a strong environmental gradient, from high-raingfall coasts and mountains to variable and semi-arid areas in central Australia. Lower chromosome numbers have been previously reported for species occuring in dryer areas, though there is a strong phylogenetic signal associated with those data.
The authors state in the Introduction that the main purpose of this study was to find “some evolutionary trends” in karyotypes and to test the correlations between changes in chromosome number, karyotype, and life-history habit using independent contrasts and a phylogenetic tree. They did this by asking three main questions:
1. Has the reduction in chromosome number occurred in correlation with the evolution of annuality?
2. Has the reduction in total chromosome length occurred in correlation with the reduction in chromosome number?
3. Have karyotypic asymmetry and length heterogeneity increased in correlation with the reduction in chromosome number?
Their phylogenetic tree was based on Denda et al. (in press), who used 47 taxa in this genus and the matK sequence. They resolved polytomies with morphological, anatomical, and cytological data, being careful not to make any assumptions about the directionality of chromosome evolution to “avoid a circular argument”.
Their main finding was that reductions in chromosome number and total chromosome length (i.e. genome size) were indeed associated with a shift from wet habitats to drier and more variable habitats, and strongly associated with a shift from perennial to annual life-history. These data support the existing hypothesis that annual life-history contributes to selection for smaller genomes.
An interesting section in the Discussion maintains that even if smaller genomes are selected for in annuals, it is still necessary to explain why large genomes would be maintained in perennials, in which it would also seem advantageous to have small genomes and rapid growth and early reproduction. The hypothesis proposed here involves population size: perennials (at least in this genus) have much larger effective population sizes than annuals, apparently due to the ephemeral and variable nature of the more xeric habitats typical of annual species. The neutral or mildly advantageous translocations involved in chromosome reduction and loss would not be as likely to become fixed in the larger effective populations of perennial species.
The model of chromosome reduction used in this paper involves translocation of chromosome segments, followed by loss of the centromere on the donor chromosome and associated loss of remaining segments. They found clear evidence supporting a history of large translocations in this genus, which is also consistent with a model of genome dynamics characterized by frequent small increases in genome size by proliferation of transposable elements and larger, rarer losses by translocation and centromere loss.
These authors used most of the described species in a large genus of Australasian flowering shrubs to test hypotheses regarding chromosome evolution and life-history. Brachyscome contains about 80 species; these authors examined 73, plus 8 other species in related genera. The genus includes a wide range of karyotypes, from 2n=4 up to 2n=36, with a mode of 2n=18. The species are distributed across a strong environmental gradient, from high-raingfall coasts and mountains to variable and semi-arid areas in central Australia. Lower chromosome numbers have been previously reported for species occuring in dryer areas, though there is a strong phylogenetic signal associated with those data.
The authors state in the Introduction that the main purpose of this study was to find “some evolutionary trends” in karyotypes and to test the correlations between changes in chromosome number, karyotype, and life-history habit using independent contrasts and a phylogenetic tree. They did this by asking three main questions:
1. Has the reduction in chromosome number occurred in correlation with the evolution of annuality?
2. Has the reduction in total chromosome length occurred in correlation with the reduction in chromosome number?
3. Have karyotypic asymmetry and length heterogeneity increased in correlation with the reduction in chromosome number?
Their phylogenetic tree was based on Denda et al. (in press), who used 47 taxa in this genus and the matK sequence. They resolved polytomies with morphological, anatomical, and cytological data, being careful not to make any assumptions about the directionality of chromosome evolution to “avoid a circular argument”.
Their main finding was that reductions in chromosome number and total chromosome length (i.e. genome size) were indeed associated with a shift from wet habitats to drier and more variable habitats, and strongly associated with a shift from perennial to annual life-history. These data support the existing hypothesis that annual life-history contributes to selection for smaller genomes.
An interesting section in the Discussion maintains that even if smaller genomes are selected for in annuals, it is still necessary to explain why large genomes would be maintained in perennials, in which it would also seem advantageous to have small genomes and rapid growth and early reproduction. The hypothesis proposed here involves population size: perennials (at least in this genus) have much larger effective population sizes than annuals, apparently due to the ephemeral and variable nature of the more xeric habitats typical of annual species. The neutral or mildly advantageous translocations involved in chromosome reduction and loss would not be as likely to become fixed in the larger effective populations of perennial species.
The model of chromosome reduction used in this paper involves translocation of chromosome segments, followed by loss of the centromere on the donor chromosome and associated loss of remaining segments. They found clear evidence supporting a history of large translocations in this genus, which is also consistent with a model of genome dynamics characterized by frequent small increases in genome size by proliferation of transposable elements and larger, rarer losses by translocation and centromere loss.
Thursday, January 17, 2008
Matlock and Dornfeld 1981
Matlock DB, Dornfeld EJ. 1981. Somatic polyploidy in the marine isopod Idothea wasnesenskii. Comparative Biochemistry and Physiology 69A: 777-781.
These authors examined polyploid somatic tissues in an intertidal isopod with a fun name, using a combination of Feuglen cytophotometry (DNA contents) and Autoradiography (DNA synthesis). They were able to examine three tissues (hepatic cecum, midgut, testis sheath) in adult, juvenile, and “emerger” isopods, except testis tissue in emergers.
Endopolyploidy was found to start first in hepatic cecum, as emergers had very few polyploid cells in their midguts. The pattern of cell ploidy distribution varied between tissues: midgut and cecum cells showed a continuous series of larger cells up to 256C, while testis sheath cells did not exceed 32C, and were distributed discontinuously.
Four hypotheses regarding the intercell synchronisation and duration of DNA synthesis were proposed in the Discussion section, though none of the four could be conclusively rejected based on these data. However, the authors suggest that hypothesis 2, that DNA synthesis is confined to a definite S phase in each cell, but there is no synchrony between cells, is probably correct for the studied tissues in this species. Finally, the authors tentatively suggest that moulting hormones may play a role in stimulating DNA synthesis, a phenomenon that has been partly demonstrated in other contexts in arthropods.
These authors examined polyploid somatic tissues in an intertidal isopod with a fun name, using a combination of Feuglen cytophotometry (DNA contents) and Autoradiography (DNA synthesis). They were able to examine three tissues (hepatic cecum, midgut, testis sheath) in adult, juvenile, and “emerger” isopods, except testis tissue in emergers.
Endopolyploidy was found to start first in hepatic cecum, as emergers had very few polyploid cells in their midguts. The pattern of cell ploidy distribution varied between tissues: midgut and cecum cells showed a continuous series of larger cells up to 256C, while testis sheath cells did not exceed 32C, and were distributed discontinuously.
Four hypotheses regarding the intercell synchronisation and duration of DNA synthesis were proposed in the Discussion section, though none of the four could be conclusively rejected based on these data. However, the authors suggest that hypothesis 2, that DNA synthesis is confined to a definite S phase in each cell, but there is no synchrony between cells, is probably correct for the studied tissues in this species. Finally, the authors tentatively suggest that moulting hormones may play a role in stimulating DNA synthesis, a phenomenon that has been partly demonstrated in other contexts in arthropods.
Wednesday, January 16, 2008
Doležel et al. 1998
Doležel J, Greilhuber J, Lucretti S, Meister A, Lysák MA, Nardi L, Obermayer R. 1998. Plant genome size estimation by flow cytometry: Inter-laboratory comparison. Annals of Botany 82 (Supplement A): 17-26.
These authors compared pairwise ratios of measured genome sizes of nine species of plants covering a genome size range of 0.3-30pg, between four laboratories in central Europe (Austria, Czech Republic, Germany, Italy), using flow cytometry. The purpose of this “exercise” was to test the reliability and variability of flow cytometry for genome-size measurement in plants. Each lab used its own method of nuclei isolation and staining; one lab also measured these plant species using Feulgen densitometry. Two labs used laser-based flow cytometers, two used mercury arc-lamp-based flow cytometers. All labs used propidium iodide (PI), a DNA-intercalating agent that is not biased by sequence; one of each type of machine was also used with DAPI, which preferentially binds to AT-rich regions of chromosomes.
Their primary findings were 1. intra-lab variation was very low, 2. inter-lab variation was low but statistically significant (partly because intra-lab variation was so low), and 3. a consistent difference was found between mercury arc-lamp and laser-based flow cytometers. Additionally, they found that DAPI was not suitable for genome size estimation because of differences between plant species in GC contents. Variation in nuclei-isolating buffers did not have measureable effects on the results.
They make two key recommendations for future research into plant genome size variation using flow cytometry:
1. All samples should be analysed in one lab using one instrument. Replication of one lab’s findings by another should analyse whole sets of samples for reliable ratio comparisons.
2. Inter-lab and machine-type differences should be considered when evaluating published reports of genome size variation in plants.
Both of these recommendations underscore the need for detailed, clear Methods & Materials sections in scientific publications, including the need to identify staining protocols, instrument types, and similar fine details.
Finally, they urge a broad agreement on reference standards (i.e. species used as standards during measurement) and their calibration, to allow higher precision in estimated genome size variation among and within plant species.
These authors compared pairwise ratios of measured genome sizes of nine species of plants covering a genome size range of 0.3-30pg, between four laboratories in central Europe (Austria, Czech Republic, Germany, Italy), using flow cytometry. The purpose of this “exercise” was to test the reliability and variability of flow cytometry for genome-size measurement in plants. Each lab used its own method of nuclei isolation and staining; one lab also measured these plant species using Feulgen densitometry. Two labs used laser-based flow cytometers, two used mercury arc-lamp-based flow cytometers. All labs used propidium iodide (PI), a DNA-intercalating agent that is not biased by sequence; one of each type of machine was also used with DAPI, which preferentially binds to AT-rich regions of chromosomes.
Their primary findings were 1. intra-lab variation was very low, 2. inter-lab variation was low but statistically significant (partly because intra-lab variation was so low), and 3. a consistent difference was found between mercury arc-lamp and laser-based flow cytometers. Additionally, they found that DAPI was not suitable for genome size estimation because of differences between plant species in GC contents. Variation in nuclei-isolating buffers did not have measureable effects on the results.
They make two key recommendations for future research into plant genome size variation using flow cytometry:
1. All samples should be analysed in one lab using one instrument. Replication of one lab’s findings by another should analyse whole sets of samples for reliable ratio comparisons.
2. Inter-lab and machine-type differences should be considered when evaluating published reports of genome size variation in plants.
Both of these recommendations underscore the need for detailed, clear Methods & Materials sections in scientific publications, including the need to identify staining protocols, instrument types, and similar fine details.
Finally, they urge a broad agreement on reference standards (i.e. species used as standards during measurement) and their calibration, to allow higher precision in estimated genome size variation among and within plant species.
Marescalchi et al. 1990
Marescalchi O, Scali V, Zuccotti M. 1990. Genome size in parental and hybrid species of Bacillus (Insecta, Phasmatodea) from southeastern Sicily: a flow cytometric analysis. Genome 33: 789-793.
These authors present the first flow cytometry analysis of genome size in the insect order Phasmatodea (stick insects); an earlier paper by other authors reported the genome size of the leaf-insect Extatosoma tiaratum.
Two of the five species studied are diploid bisexuals; one is a double allotriploid (three parental species), one is a diploid thelytokous parthenogenetic species, and one is a diploid thelytokous parthenogenetic hybrid between the two diploid bisexual species. In the two bisexual diploids, male genome sizes were significantly smaller than female, by about 15% in both cases; this difference exceeds that attributable to the absent X chromosome in these XO males, the X chromosome accounts for about 4% of the total haploid C-value. The authors suggest that male midgut cells (the measured tissue in all cases) may have higher DNA packing densities, a suggestion indirectly supported by their report that male karyotypes appear somewhat more compact than female.
The genome size data collected were used to support the hypotheses regarding the origins of the hybrid diploid and triploid species; non-significances in Student’s t-tests are argued to support intermediate genome size values.
The methods used here appear similar to the ‘best practices’ suggested by DeSalle et al. (2005): 50ug/mL of Propidium Iodide, incubated with the sample and co-prepared standard for at least 20 minutes. Interestingly, their narcotizing agent of choice was chloroform, as we use in our lab.
Tuesday, January 8, 2008
Marescalchi et al. 1998
Marescalchi O, Scali V, Zuccotti M. 1998. Flow-cytometric analyses of intraspecific genome size variations in Bacillus atticus (Insecta, Phasmatodea). Genome 41: 629-635.
These authors measured nuclear DNA contents of 12 populations of a walking-stick insect distributed around the shores of the eastern Mediterranean sea. This species is thelytokous (see Boivin and Candau, 2007), and includes both diploid and triploid populations. Chromosome number is mostly conserved across populations of the same ploidy level.
Their flow cytometry methodology is more fully described in an earlier paper (Marescalchi et al. 1990), but seems fairly good (at this level of detail) as they describe copreparation of a standard, mouse thymocytes in all samples.
A clear correlation of genome size and longitude was found, with the smallest genomes in Sardinia at the species’ western extent. As one moves west, genomes in this species become gradually smaller, a situation that the authors argue can be explained by the relative ages of the populations. The high diversity within this species among and within eastern populations suggests that the eastern Mediterranean houses the oldest populations. The authors argue for a pattern of reduced genome size with younger populations, though the details of what type of DNA has changed or been lost is not known, no mechanism linking colonization versus nondispersal and genome size evolution is proposed, and the theory discussed includes apparently long-since-discounted ideas about ecological specialization.
These authors measured nuclear DNA contents of 12 populations of a walking-stick insect distributed around the shores of the eastern Mediterranean sea. This species is thelytokous (see Boivin and Candau, 2007), and includes both diploid and triploid populations. Chromosome number is mostly conserved across populations of the same ploidy level.
Their flow cytometry methodology is more fully described in an earlier paper (Marescalchi et al. 1990), but seems fairly good (at this level of detail) as they describe copreparation of a standard, mouse thymocytes in all samples.
A clear correlation of genome size and longitude was found, with the smallest genomes in Sardinia at the species’ western extent. As one moves west, genomes in this species become gradually smaller, a situation that the authors argue can be explained by the relative ages of the populations. The high diversity within this species among and within eastern populations suggests that the eastern Mediterranean houses the oldest populations. The authors argue for a pattern of reduced genome size with younger populations, though the details of what type of DNA has changed or been lost is not known, no mechanism linking colonization versus nondispersal and genome size evolution is proposed, and the theory discussed includes apparently long-since-discounted ideas about ecological specialization.
Boivin and Candau 2007
Boivin T, Candau J-N. 2007. Flow cytometric analysis of ploidy levels in two seed-infesting Megastigmus species: applications to sex ratio and species determination at the larval stage. Entomologia Experimentalis et Applicata 124: 125-131.
These authors tested the utility of flow cytometry for both species and sex discrimination in the larvae of two congeneric species of pest wasps. These wasps are recently-introduced pests of cedar trees in southern France, and cannot be identified to either species or sex by morphology during their larval stages. In this genus, most species have an obligate larval diapause that can last more than 5 years. Thus, a method for determining sex ratios (for population-dynamics studies) and species (for invasion dynamics) was required.
Their method of flow cytometry involved a commercially-available staining solution containing DAPI, and no mention of known standards appears. They do not report absolute or relative genome sizes, rather they report ratios of cells of various ploidy levels.
Flow cytometry was shown to be effective for identifying both sex and species in this system. Like other hymenoptera, these species have haplodiploid sex determination, though they differ in the details: Megastigmus pinsapinis are thelytokous, M. schimitscheki are arrhenotokous. Species can be discriminated in female larvae, because M. schimitscheki female larvae include large numbers of haploid cells. The failure of species discrimination among male larvae is not critical, as the sex ratio of M. pinsapinus is severly female-biased, with males occuring at the rate of about 1 in 4000.
The discovery of haploid cells in the abdomens of female M. schimitscheki lead to a brief discussion and some speculation regarding haploidization and gametogenesis. However, I am under the impression that universally among animals, female mieosis does not complete until after fertilization, and thus eggs are never actually haploid. A reference in this section, Pannebakker et al. (2004), may shed light on this issue.
Glossary of Terms:
Arrhenotoky: females and males result from fertilized and unfertilized eggs, respectively.
Thelytoky: parthenogenetic diploid females develop from unfertilized eggs, males are absent or rare.
These authors tested the utility of flow cytometry for both species and sex discrimination in the larvae of two congeneric species of pest wasps. These wasps are recently-introduced pests of cedar trees in southern France, and cannot be identified to either species or sex by morphology during their larval stages. In this genus, most species have an obligate larval diapause that can last more than 5 years. Thus, a method for determining sex ratios (for population-dynamics studies) and species (for invasion dynamics) was required.
Their method of flow cytometry involved a commercially-available staining solution containing DAPI, and no mention of known standards appears. They do not report absolute or relative genome sizes, rather they report ratios of cells of various ploidy levels.
Flow cytometry was shown to be effective for identifying both sex and species in this system. Like other hymenoptera, these species have haplodiploid sex determination, though they differ in the details: Megastigmus pinsapinis are thelytokous, M. schimitscheki are arrhenotokous. Species can be discriminated in female larvae, because M. schimitscheki female larvae include large numbers of haploid cells. The failure of species discrimination among male larvae is not critical, as the sex ratio of M. pinsapinus is severly female-biased, with males occuring at the rate of about 1 in 4000.
The discovery of haploid cells in the abdomens of female M. schimitscheki lead to a brief discussion and some speculation regarding haploidization and gametogenesis. However, I am under the impression that universally among animals, female mieosis does not complete until after fertilization, and thus eggs are never actually haploid. A reference in this section, Pannebakker et al. (2004), may shed light on this issue.
Glossary of Terms:
Arrhenotoky: females and males result from fertilized and unfertilized eggs, respectively.
Thelytoky: parthenogenetic diploid females develop from unfertilized eggs, males are absent or rare.
DeSalle et al. 2005
DeSalle R, Gregory TR, Johnston JS. 2005. Preparation of samples for comparative studies of arthropod chromosomes: visualization, In Situ hybridization, and genome size estimation. Methods in Enzymology 395: 460-488.
This paper appears to be formatted like a book chapter, and covers a broad range of topics in arthropod cytogenetics. These authors describe the fundamental concepts of arthropod chromosome research and genome size, focusing primarily on general techniques and best-practices recommendations.
The section concerning chromosome manipulation includes general instructions for preparing chromosome squashes and descriptions of some of the most widely-used stains. Most of the stains described are useful for studying GC content, though silver stains are mentioned for studies of Nucleolus Organizer Regions (NORs), and Flourescent In-Situ Hybridization (FISH) is also described. A table lists books and websites for more detailed protocols.
The section on genome size estimation is obviously of greater direct relevance for my work. Besides strong justification for the work I do, this part of this paper provides some interesting factoids about the range of genome sizes in various arthropod groups, though I know some more recent additions to the database extend these ranges. Genome size estimation protocols, for both Feulgen Image Analysis Densitometry and Flow Cytometry are also provided, including a detailed FC protocol in Box III. Interestingly, the FC protocol includes recommendations for both very long stain incubations (up to 24 hours, but always at least 1h) and very long sampling runs (up to 20 minutes), the latter due to the generally low cell density of insect preparations. I have copied Box III to a MS-Word document for inclusion in my lab notebook.
This paper appears to be formatted like a book chapter, and covers a broad range of topics in arthropod cytogenetics. These authors describe the fundamental concepts of arthropod chromosome research and genome size, focusing primarily on general techniques and best-practices recommendations.
The section concerning chromosome manipulation includes general instructions for preparing chromosome squashes and descriptions of some of the most widely-used stains. Most of the stains described are useful for studying GC content, though silver stains are mentioned for studies of Nucleolus Organizer Regions (NORs), and Flourescent In-Situ Hybridization (FISH) is also described. A table lists books and websites for more detailed protocols.
The section on genome size estimation is obviously of greater direct relevance for my work. Besides strong justification for the work I do, this part of this paper provides some interesting factoids about the range of genome sizes in various arthropod groups, though I know some more recent additions to the database extend these ranges. Genome size estimation protocols, for both Feulgen Image Analysis Densitometry and Flow Cytometry are also provided, including a detailed FC protocol in Box III. Interestingly, the FC protocol includes recommendations for both very long stain incubations (up to 24 hours, but always at least 1h) and very long sampling runs (up to 20 minutes), the latter due to the generally low cell density of insect preparations. I have copied Box III to a MS-Word document for inclusion in my lab notebook.
Thursday, January 3, 2008
Nardon et al. 2005
Nardon C, Deceliere G, Lœvenbruck, Weiss M, Vieira C, Biémont C. 2005. Is genome size influence by colonization of new environments in dipteran species? Molecular Ecology 14: 869-878.
These authors examined the genome sizes of many populations around the world of each of 6 species of drosophilid flies. Most species did not show differences between continents in genome size as measured by flow cytometry, except that populations of Drosophila melanogaster from east Africa, where this species is thought to have originated, had smaller genomes.
The methods of this paper are substantially the same as the basic protocol described in Nardon et al. (2003), apparently using the “best practices” (my own terminology) discovered in that earlier work.
I quote here the most important, in my mind, sentence in this paper:
These authors examined the genome sizes of many populations around the world of each of 6 species of drosophilid flies. Most species did not show differences between continents in genome size as measured by flow cytometry, except that populations of Drosophila melanogaster from east Africa, where this species is thought to have originated, had smaller genomes.
The methods of this paper are substantially the same as the basic protocol described in Nardon et al. (2003), apparently using the “best practices” (my own terminology) discovered in that earlier work.
I quote here the most important, in my mind, sentence in this paper:
“Because D. melanogaster is thought to have colonized the entire globe a long time ago, whereas D. simulans is thought to have dispersed more recently, this suggests that the time since colonization occurred may matter, and that change in genome size, when it happens, is a very slow process.”
This paper is not cited by me in my PhD proposal, an oversight that seems obvious now that I have read this paper. The note after the literature cited section, describing this paper in the context of a larger project on genome size evolution, suggests I should periodically search for new papers by these authors on this and related topics.
Nardon et al. 2003
Nardon C, Weiss M, Vieira C, Biémont C. 2003. Variation of the genome size estimate with environmental conditions in Drosophila melanogaster. Cytometry Part A 55A: 43-49.
These authors examined the effects of various temperatures and humidities during development on measures of genome size by flow cytometry in Drosophila melanogaster male adult brains. They also looked at the effects of varying temperatures of prepared solutions after extraction of cell nuclei, and of the age of the flies when they were killed.
Previous studies by various authors have found a range of factors that can affect genome size estimation, such as age of the individual or temperature. The results reported here are consistent with those earlier reports.
Unfortunately, I am rather suspicious of these results because of some odd phrases in this paper. There are some probably trivial and meaningless mistakes, such as refering to eclosion of adult flies from pupae as “hatching”, but other putative mistakes are more worrisome, such as the confusing discussion of the statistical tests employed. Five of seven experiments in temperature (i.e. five of seven comparisons between 17°C and 25°C) are reported to show differences in measured stain flourescence intensity, but the next clause says that three of those five were significant differences. If it is not significant, it is not different, no?
Their data seem to show some noise, which is of course to be expected in any experiment. They interpret this noise in a somewhat confusing manner, never refering to it as “noise”. I am not certain I understood the discussion section properly.
The most important finding in this paper to me is probably the warning that prepared samples of extracted nuclei must be maintained at low temperatures (i.e. on ice) for consistent results.
These authors examined the effects of various temperatures and humidities during development on measures of genome size by flow cytometry in Drosophila melanogaster male adult brains. They also looked at the effects of varying temperatures of prepared solutions after extraction of cell nuclei, and of the age of the flies when they were killed.
Previous studies by various authors have found a range of factors that can affect genome size estimation, such as age of the individual or temperature. The results reported here are consistent with those earlier reports.
Unfortunately, I am rather suspicious of these results because of some odd phrases in this paper. There are some probably trivial and meaningless mistakes, such as refering to eclosion of adult flies from pupae as “hatching”, but other putative mistakes are more worrisome, such as the confusing discussion of the statistical tests employed. Five of seven experiments in temperature (i.e. five of seven comparisons between 17°C and 25°C) are reported to show differences in measured stain flourescence intensity, but the next clause says that three of those five were significant differences. If it is not significant, it is not different, no?
Their data seem to show some noise, which is of course to be expected in any experiment. They interpret this noise in a somewhat confusing manner, never refering to it as “noise”. I am not certain I understood the discussion section properly.
The most important finding in this paper to me is probably the warning that prepared samples of extracted nuclei must be maintained at low temperatures (i.e. on ice) for consistent results.
Wednesday, January 2, 2008
Husband 2000
Husband BC. 2000. Constraints on polyploid evolution: a test of the minority cytotype exclusion principle. Proceedings: Biological Sciences 267: 217-223.
This author looked for assortative mating and frequency-dependent selection on tetraploids and diploids in laboratory fireweed populations. Fireweed occurs throughout North America, with populations of different ploidy usually geographically separated, though sympatric and parapatric populations occur in a zone of contact along the Rocky Mountains. In contrast to other plants, fireweed diploids occur more often at higher latitudes and at higher altitudes.
Frequency-dependent selection against rare genotypes (or cytotypes) is generally expected because in the absence of assortative mating, the rare type will suffer a greater proportion of failed matings (4x X 2x = 3x failure). Assortative mating driven by any of a large number of factors could therefore explain the apparent importance of polyploidy in evolution.
In contrast to expectations, tetraploid absolute fitness did not vary with frequency, but diploid absolute fitness did, leading to changes in relative fitness. While this specific result was unexpected, it is consistent with the general expectation of reduced relative fitness in rare types.
This author found that the threshold of frequency of equal relative fitness was approximately 0.61 tetraploid, suggesting a difference in absolute fitness favouring diploids. In this study, diploid plants had approximately twice as many flowers as tetraploids, and the tetraploid flowering period was about 10 days shorter (out of 53 days) than the diploid.
Two factors examined in this study could drive assortative mating among tetraploids, thus improving their odds of establishment (and “instant speciation”).
1. Tetraploid flowering time completely encompasses and extends beyond diploid. However, in this study, the opposite occurred.
2. Pollinator preferences drive a higher-than-expected frequency of 4 to 4 pollinator trips. When looking between individual plants, the opposite occurred; however, when visits between flowers within individual plants were considered, a very high proportion of trips were within tetraploid individuals, a potential source of assortative mating.
The author concludes that other, unexamined, factors may be driving assortative mating and polyploid evolution in this and other plants.
This author looked for assortative mating and frequency-dependent selection on tetraploids and diploids in laboratory fireweed populations. Fireweed occurs throughout North America, with populations of different ploidy usually geographically separated, though sympatric and parapatric populations occur in a zone of contact along the Rocky Mountains. In contrast to other plants, fireweed diploids occur more often at higher latitudes and at higher altitudes.
Frequency-dependent selection against rare genotypes (or cytotypes) is generally expected because in the absence of assortative mating, the rare type will suffer a greater proportion of failed matings (4x X 2x = 3x failure). Assortative mating driven by any of a large number of factors could therefore explain the apparent importance of polyploidy in evolution.
In contrast to expectations, tetraploid absolute fitness did not vary with frequency, but diploid absolute fitness did, leading to changes in relative fitness. While this specific result was unexpected, it is consistent with the general expectation of reduced relative fitness in rare types.
This author found that the threshold of frequency of equal relative fitness was approximately 0.61 tetraploid, suggesting a difference in absolute fitness favouring diploids. In this study, diploid plants had approximately twice as many flowers as tetraploids, and the tetraploid flowering period was about 10 days shorter (out of 53 days) than the diploid.
Two factors examined in this study could drive assortative mating among tetraploids, thus improving their odds of establishment (and “instant speciation”).
1. Tetraploid flowering time completely encompasses and extends beyond diploid. However, in this study, the opposite occurred.
2. Pollinator preferences drive a higher-than-expected frequency of 4 to 4 pollinator trips. When looking between individual plants, the opposite occurred; however, when visits between flowers within individual plants were considered, a very high proportion of trips were within tetraploid individuals, a potential source of assortative mating.
The author concludes that other, unexamined, factors may be driving assortative mating and polyploid evolution in this and other plants.
BrummellBlog JournalClub
Welcome to Martin Brummell's new blog, my Journal Club. This does not replace BrummellBlog, this provides a space for me to post about the peer-reviewed scientific articles I'm reading, for two purposes.
1. So that others can comment. Really, if you've read the same paper as I, or (hope springs eternal) are an author of one of these papers, or if you just take issue with something I've said, please comment. I find I get a great benefit from discussing the papers I've read, and this blog exists to foster that kind of discussion.
2. So I can organize my thoughts about the literature I'm reading in a manner distinct from that of a traditional bibliography. I plan to take advantage of Blogger's software features such as labels to help with this. While my on-harddrive annotated bibliography is useful to me as is, in its alphabetical-by-author-name format in MS Word, I think the features of a blog format will allow some interesting re-examination of the papers I read.
Most of the posts here will simply be copy-pasted from my annotated bibliography as I make entries there. Since I started my annotated bibliography a little less than a year ago, I've entered 167 papers with my commentary. I've always written it in complete-sentences, semi-formal style, so that's what will appear here. Not everything I read will appear here, and I don't think I'll be pulling much from the back library; this will be almost exclusively for stuff I read from now on.
1. So that others can comment. Really, if you've read the same paper as I, or (hope springs eternal) are an author of one of these papers, or if you just take issue with something I've said, please comment. I find I get a great benefit from discussing the papers I've read, and this blog exists to foster that kind of discussion.
2. So I can organize my thoughts about the literature I'm reading in a manner distinct from that of a traditional bibliography. I plan to take advantage of Blogger's software features such as labels to help with this. While my on-harddrive annotated bibliography is useful to me as is, in its alphabetical-by-author-name format in MS Word, I think the features of a blog format will allow some interesting re-examination of the papers I read.
Most of the posts here will simply be copy-pasted from my annotated bibliography as I make entries there. Since I started my annotated bibliography a little less than a year ago, I've entered 167 papers with my commentary. I've always written it in complete-sentences, semi-formal style, so that's what will appear here. Not everything I read will appear here, and I don't think I'll be pulling much from the back library; this will be almost exclusively for stuff I read from now on.
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