Dillon RT, Frankis RC. 2004. High levels of mitochondrial DNA sequence divergence in isolated populations of freshwater snails of the genus Goniobasis Lea, 1862. American Malacological Bulletin 19: 69-77.
These authors examined sequence divergence within and between populations and species of “prosobranch” freshwater gastropods in the south-eastern USA. These species are relatively well studied, and previous work by other authors had suggested very high levels of mtDNA heterogeneity, between populations and between species.
Part of the justification for this work is as a way of calibrating a new measurement tool: DNA sequences may be useful for systematic assignment, but if levels of sequence divergence are to be used to distinguish species, first the levels of divergence between species well-established by other means (e.g. interspecific hybridization trials) must be determined. These snails had previously been well studied for traits associated with concepts of species such as pre- and post-mating isolation (e.g. Dillon 1986).
The other major reason for this paper was to test the hypothesis that freshwater snail populations may be so old, so large, and so isolated that intrapopulation mtDNA sequence divergence is likely to swamp interpopulation (and interspecies) mtDNA divergence. Under this hypothesis, within-population levels of divergence will overlap with between-population levels. Populations studied here are extremely isolated, with no freshwater connections between them; this area was not inundated nor was it ice-bound by the Pleistocene glaciations, suggesting that some populations may have been isolated for millions of years.
The levels of divergence found in COI and 16S mtDNA sequences were extremely high, especially among populations of one species, Goniobasis proxima. In one population, individual conspecific snails collected from adjacent rocks may have more divergent mtDNA than individual snails collected from populations separated by over 400km of (impassable) land. In that same highly heterogeneous-mtDNA population, morphology and seven nuclear markers (allozymes) were essentially homogeneous.
The authors end with a caution that systematic inference must be made with care when faced with high levels of intrapopulation divergences. This paper appears to provide an example situation in mtDNA phylogeography that does not show the “barcode gap”, i.e. a clear distinction between intra- and interpopulation divergences.
Wednesday, May 28, 2008
Saturday, May 24, 2008
Richardson and Goff 2001
Richardson MS, Goff ML. 2001. Effects of temperature and intraspecific interaction on the development of Dermestes maculatus Coleoptera: Dermestidae). Journal of Medical Entomology 38: 347-357.
These authors measured survivorship and development rate in Dermestes maculatus from Hawaii, and compared these variables between beetles raised at a range of temperatures between 15°C and 35°C, and larval densities between one and 120 individuals per container, with modifications to prevent cannibalism by larvae of eggs.
Beetles were maintained in large plastic boxes (see also Hefti et al. 1980) lined with paper towels, and fed on Lighthouse brand (60% protein) fish meal in Petri dishes, with distilled water provided in separate Petri dishes. Humidity was not controlled, but varied between 60% and 80% relative.
Development times (egg to adult) varied from about three months at 20°C to a little longer than one month at 35°C. Females are generally slightly larger, and this species (or at least this population) shows a reversed size-temperature rule, with larger adult body sizes at higher temperatures. High density impairs larval survival, though there were few differences between intermediate-density treatments.
The authors suggest that optimal growth and survival is probably close to 30°C, and at intermediate population densities. Fish meal seems to be an appropriate and useful growth medium.
These authors measured survivorship and development rate in Dermestes maculatus from Hawaii, and compared these variables between beetles raised at a range of temperatures between 15°C and 35°C, and larval densities between one and 120 individuals per container, with modifications to prevent cannibalism by larvae of eggs.
Beetles were maintained in large plastic boxes (see also Hefti et al. 1980) lined with paper towels, and fed on Lighthouse brand (60% protein) fish meal in Petri dishes, with distilled water provided in separate Petri dishes. Humidity was not controlled, but varied between 60% and 80% relative.
Development times (egg to adult) varied from about three months at 20°C to a little longer than one month at 35°C. Females are generally slightly larger, and this species (or at least this population) shows a reversed size-temperature rule, with larger adult body sizes at higher temperatures. High density impairs larval survival, though there were few differences between intermediate-density treatments.
The authors suggest that optimal growth and survival is probably close to 30°C, and at intermediate population densities. Fish meal seems to be an appropriate and useful growth medium.
Hefti et al. 1980
Hefti E, Trechsel U, Rufenacht H, Fleisch H. 1980. Use of dermestid beetles for cleaning bones. Calcified Tissue International 31: 45-47.
These authors evaluated the quantitative effects on a range of bone measures of carcass cleaning by Dermestes maculatus vs. manually. Standard Wistar rats, healthy and osteoporetic, were cleaned by either a beetle colony or by hand, a time-consuming and tedious process. Hand-cleaning invariably leaves behind small pieces of soft tissue, while the beetles are more thorough but may consume bone when the food supply of softer tissue is depleted. The beetles did not significantly reduce the measured aspects of the bones compared to manual dissection, as long as the rat carcasses were not exposed to the beetles for too long.
The lab beetle colony was maintained in a metal box, because the larvae can “burn through” some types of plastic. When not engaged in cleaning rat carcasses, the beetles were fed on “greaves”, the remainders of industrial fat production.
These authors evaluated the quantitative effects on a range of bone measures of carcass cleaning by Dermestes maculatus vs. manually. Standard Wistar rats, healthy and osteoporetic, were cleaned by either a beetle colony or by hand, a time-consuming and tedious process. Hand-cleaning invariably leaves behind small pieces of soft tissue, while the beetles are more thorough but may consume bone when the food supply of softer tissue is depleted. The beetles did not significantly reduce the measured aspects of the bones compared to manual dissection, as long as the rat carcasses were not exposed to the beetles for too long.
The lab beetle colony was maintained in a metal box, because the larvae can “burn through” some types of plastic. When not engaged in cleaning rat carcasses, the beetles were fed on “greaves”, the remainders of industrial fat production.
Sunday, May 18, 2008
Gill and Cain 1980
Gill JJB, Cain AJ. 1980. The karyotype of Cepaea sylvatica (Pulmonata: Helicidae) and its relationship to those of C. hortensis and C. nemoralis. Biological Journal of the Linnean Society 14: 293-301.
These authors describe the karyotypes of three species of congeneric land snails found in Europe, and discuss the evolutionary implications of their findings in the context of previous work on the genetics of shell colour patterns, particularly in the relatively well-studied Cepaea nemoralis. This paper is an explicit attempt to unite genetic and cytogenetic studies of these snails.
Previous karyotype examinations in this genus had been performed only on meiotic tissue, which these authors consider unsuitable. Mitotic cells for karyotype analysis are difficult to obtain, however, so these authors used embryos from 5-day-old eggs, following the method of Page (1978). Shell pattern variation had previously been associated with between seven and nine tightly linked loci, calculated by Cook (1969) to be most likely located together (as a “supergene”) on one arm of the largest chromosome pair in C. nemoralis.
Among the three studied species, two (C. nemoralis and C. hortensis) have similar karyotypes, with 2n = 44 and one “conspicuously large” pair of chromosomes, while the third species (C. sylvatica) has 2n = 50 and a large pair of chromosomes. A fourth species (C. vindobonensis), possibly restricted to Russia and not studied by these authors, also has 2n = 50 but probably lacks the conspicuous and large chromosomes (Baltzer 1913).
These authors were not able to apply standard banding and u.v. fluorochrome techniques to the karyotypes, rendering most of the chromomes in all three species indistinguishable. The only chromosomes that could be reliably distinguished were the two largest pairs (by their sizes) and a third pair with a clear constriction on one arm. The other chromosomes showed approximately continuous variation in size and no other morphological characteristics.
The large pair of chromosomes in C. sylvatica do not resemble the large chromosomes in the other two species and are therefore probably not homologous. A scenario of chromosome fusions is briefly discussed, but is dismissed as unlikely to generate the patterns of genetic linkage observed. There is also a rather strange evolutionary path described, in which the karyotypes of extant species are taken as indicative of ancestral states, with gradual evolution of the “derived” karyotype of C. nemoralis and C. hortensis from “ancestral” karyotypes represented by C. vindobonensis and C. sylvatica. The authors end with a statement that other species in the subfamily Helicinae have also shown conspicuous large chromosomes and probable high degrees of karytotype stability, though they do not describe shell colour patterns in those other species, nor do they provide references for these observations.
These authors describe the karyotypes of three species of congeneric land snails found in Europe, and discuss the evolutionary implications of their findings in the context of previous work on the genetics of shell colour patterns, particularly in the relatively well-studied Cepaea nemoralis. This paper is an explicit attempt to unite genetic and cytogenetic studies of these snails.
Previous karyotype examinations in this genus had been performed only on meiotic tissue, which these authors consider unsuitable. Mitotic cells for karyotype analysis are difficult to obtain, however, so these authors used embryos from 5-day-old eggs, following the method of Page (1978). Shell pattern variation had previously been associated with between seven and nine tightly linked loci, calculated by Cook (1969) to be most likely located together (as a “supergene”) on one arm of the largest chromosome pair in C. nemoralis.
Among the three studied species, two (C. nemoralis and C. hortensis) have similar karyotypes, with 2n = 44 and one “conspicuously large” pair of chromosomes, while the third species (C. sylvatica) has 2n = 50 and a large pair of chromosomes. A fourth species (C. vindobonensis), possibly restricted to Russia and not studied by these authors, also has 2n = 50 but probably lacks the conspicuous and large chromosomes (Baltzer 1913).
These authors were not able to apply standard banding and u.v. fluorochrome techniques to the karyotypes, rendering most of the chromomes in all three species indistinguishable. The only chromosomes that could be reliably distinguished were the two largest pairs (by their sizes) and a third pair with a clear constriction on one arm. The other chromosomes showed approximately continuous variation in size and no other morphological characteristics.
The large pair of chromosomes in C. sylvatica do not resemble the large chromosomes in the other two species and are therefore probably not homologous. A scenario of chromosome fusions is briefly discussed, but is dismissed as unlikely to generate the patterns of genetic linkage observed. There is also a rather strange evolutionary path described, in which the karyotypes of extant species are taken as indicative of ancestral states, with gradual evolution of the “derived” karyotype of C. nemoralis and C. hortensis from “ancestral” karyotypes represented by C. vindobonensis and C. sylvatica. The authors end with a statement that other species in the subfamily Helicinae have also shown conspicuous large chromosomes and probable high degrees of karytotype stability, though they do not describe shell colour patterns in those other species, nor do they provide references for these observations.
Baršiene et al. 1996
Baršiene J, Tapia G, Barsyte D. 1996. Chromosomes of molluscs inhabiting some mountain springs of eastern Spain. Journal of Molluscan Studies 62: 539-543.
These authors report karyotypes for four species of molluscs (three gastropods and one bivalve) collected from small, high-altitude freshwater habitats in Spain. These habitats are quite diverse, with some very small springs occupying only 3-4 m^2 of surface area. Previous studies had associated small and isolated populations with polyploidy and its effects on tolerance of environmental stress and avoiding inbreeding depression.
Collected animals were injected with (large body) or immersed in (small body) a colchicine solution before dissection. The authors do not clearly describe what tissues from which species and populations were used, though they do describe “gonal and somatic cells”; presumably the gonadal cells were sperm, and the somatic cells are loosely described as “soft tissues” that were dissociated in 45% acetic acid by pipetting.
The high fraction of “hypodiploid” cells found in some individuals of Lymnaea peregra and some instances of apparent cell degeneration was attributed to “ecological stress”, though no supporting evidence or further discussion appears. They also state that the bivalve in their study, Pisidium casertanum, was almost certainly diploid, despite different populations having chromsome numbers ranging from 150 to 180, and other, uncited studies that found high and idiosyncratic levels of polyploidy in this family (Pisidiidae) and the closely related family Sphaeriidae (e.g. Burch and Huber 1966). As a final negative criticism of this paper, I found the frequent reference of the authors’ own unpublished data in support of presumed trends to be rather annoying.
These authors report karyotypes for four species of molluscs (three gastropods and one bivalve) collected from small, high-altitude freshwater habitats in Spain. These habitats are quite diverse, with some very small springs occupying only 3-4 m^2 of surface area. Previous studies had associated small and isolated populations with polyploidy and its effects on tolerance of environmental stress and avoiding inbreeding depression.
Collected animals were injected with (large body) or immersed in (small body) a colchicine solution before dissection. The authors do not clearly describe what tissues from which species and populations were used, though they do describe “gonal and somatic cells”; presumably the gonadal cells were sperm, and the somatic cells are loosely described as “soft tissues” that were dissociated in 45% acetic acid by pipetting.
The high fraction of “hypodiploid” cells found in some individuals of Lymnaea peregra and some instances of apparent cell degeneration was attributed to “ecological stress”, though no supporting evidence or further discussion appears. They also state that the bivalve in their study, Pisidium casertanum, was almost certainly diploid, despite different populations having chromsome numbers ranging from 150 to 180, and other, uncited studies that found high and idiosyncratic levels of polyploidy in this family (Pisidiidae) and the closely related family Sphaeriidae (e.g. Burch and Huber 1966). As a final negative criticism of this paper, I found the frequent reference of the authors’ own unpublished data in support of presumed trends to be rather annoying.
Dressler 1990
Dressler LG. 1990. Controls, standards, and histogram interpretation in DNA flow cytometry. Methods in Cell Biology 33: 157-171.
This author describes and discusses various procedural components and concerns involved in using flow cytometry to measure nuclear DNA contents, in the context of human cancer research and diagnosis.
Among controls, one important control described by this author is the use of cytologic or histologic examination, for example by staining a subsample of a specimen and examining under a compound microscope. In my work, this can be accomplished through Feulgen image analysis densitometry, though I think this author had in mind a less involved technique with the intention of verifying cell types and cell densities. This author also reiterates that the use of internal (ideally co-prepared) standards is critical for flow cytometry.
This paper is divided into procedures for first, fresh and frozen tissues and second, formalin-fixed, paraffin-embedded tissues. For fresh and frozen tissues, the use of duplicates, where one duplicate is the specimen alone, the other the specimen plus the standard, is urged. This fits well with what we have already been doing in our lab, using the unknown specimen alone to determine the approximate range of the position of the peak in the histogram, and the specimen coprepared with a known standard to actually estimate genome size. For fixed tissue, additional procedures are described for removing nuclei from the paraffin matrix, and the DNA index (Vindelov and Christensen 1990) is clearly defined; it is essentially the same as our calculation of genome size by comparison to a known standard.
This author also includes a recipe for freezing medium for use at -70°C, and advice on clarifying peaks in the histogram.
This author describes and discusses various procedural components and concerns involved in using flow cytometry to measure nuclear DNA contents, in the context of human cancer research and diagnosis.
Among controls, one important control described by this author is the use of cytologic or histologic examination, for example by staining a subsample of a specimen and examining under a compound microscope. In my work, this can be accomplished through Feulgen image analysis densitometry, though I think this author had in mind a less involved technique with the intention of verifying cell types and cell densities. This author also reiterates that the use of internal (ideally co-prepared) standards is critical for flow cytometry.
This paper is divided into procedures for first, fresh and frozen tissues and second, formalin-fixed, paraffin-embedded tissues. For fresh and frozen tissues, the use of duplicates, where one duplicate is the specimen alone, the other the specimen plus the standard, is urged. This fits well with what we have already been doing in our lab, using the unknown specimen alone to determine the approximate range of the position of the peak in the histogram, and the specimen coprepared with a known standard to actually estimate genome size. For fixed tissue, additional procedures are described for removing nuclei from the paraffin matrix, and the DNA index (Vindelov and Christensen 1990) is clearly defined; it is essentially the same as our calculation of genome size by comparison to a known standard.
This author also includes a recipe for freezing medium for use at -70°C, and advice on clarifying peaks in the histogram.
Vindeløv and Christensen 1990
Vindeløv L, Christensen IJ. 1990. An integrated set of methods for routine flow cytometric DNA analysis. Methods in Cell Biology 33: 127-137.
These authors present a set of protocols for standard flow cytometry in a clinical setting. The variables that can be obtained from flow cytometry are the number of subpopulations of cells with different DNA contents in a specimen, the relative sizes of these subpopulations, the “DNA index (DI)”, and the fractions of cells in G1, S, and G2 + M phases. DNA index is used here in a human-cancer context, and is roughly equivalent to nuclear DNA content per nucleus. In running flow cytometry, there are six major problems to overcome. These are sample acquisition, storage, standardization, staining, flow cytometry itself, and statistical analysis, also referred to here as “deconvolution”.
Optimal methods for sample acquisition vary with specimen type; these authors were apparently concerned primarily with the differences between soft and solid tumours from humans, but the point applies across eukaryotes in my opinion. These authors recommend storage of specimens at -80°C with DMSO, but caution that specimens should be frozen and thawed at most once. I am not certain that DMSO is a good idea. Standardization is achieved through the use of internal standards; these authors recommend erythrocytes (blood) from chicken (Gallus gallus domesticus) and trout (Onchorynchus mykiss). These authors describe a staining protocol in three steps, that includes the use of trypsin during cell dissociation and a trypsin inhibitor in a later step to prevent interaction with Propidium iodide (see also Krishan 1990). The chicken blood is described as being mixed with heparin at the time of collection, but no mention is made here of the potential interactions between heparin and Propidium iodide (Krishan 1990).
During flow cytometry, the positions of the peaks in the histogram corresponding to the standards (chicken and trout) are used to determine the DNA index of the measured specimen. These histogram heights can be made equal by mixing the standards together and with the specimen in a manner described by these authors; essentially, cell concentrations are measured and the known fluorescence characteristics of the standards are taken into account. Agitation of specimens will tend to increase the presence of debris and the occurrence of nuclear clumping.
These authors report satisfactory results from these protocols with 17 000 samples for all (human) tissues except sperm.
These authors present a set of protocols for standard flow cytometry in a clinical setting. The variables that can be obtained from flow cytometry are the number of subpopulations of cells with different DNA contents in a specimen, the relative sizes of these subpopulations, the “DNA index (DI)”, and the fractions of cells in G1, S, and G2 + M phases. DNA index is used here in a human-cancer context, and is roughly equivalent to nuclear DNA content per nucleus. In running flow cytometry, there are six major problems to overcome. These are sample acquisition, storage, standardization, staining, flow cytometry itself, and statistical analysis, also referred to here as “deconvolution”.
Optimal methods for sample acquisition vary with specimen type; these authors were apparently concerned primarily with the differences between soft and solid tumours from humans, but the point applies across eukaryotes in my opinion. These authors recommend storage of specimens at -80°C with DMSO, but caution that specimens should be frozen and thawed at most once. I am not certain that DMSO is a good idea. Standardization is achieved through the use of internal standards; these authors recommend erythrocytes (blood) from chicken (Gallus gallus domesticus) and trout (Onchorynchus mykiss). These authors describe a staining protocol in three steps, that includes the use of trypsin during cell dissociation and a trypsin inhibitor in a later step to prevent interaction with Propidium iodide (see also Krishan 1990). The chicken blood is described as being mixed with heparin at the time of collection, but no mention is made here of the potential interactions between heparin and Propidium iodide (Krishan 1990).
During flow cytometry, the positions of the peaks in the histogram corresponding to the standards (chicken and trout) are used to determine the DNA index of the measured specimen. These histogram heights can be made equal by mixing the standards together and with the specimen in a manner described by these authors; essentially, cell concentrations are measured and the known fluorescence characteristics of the standards are taken into account. Agitation of specimens will tend to increase the presence of debris and the occurrence of nuclear clumping.
These authors report satisfactory results from these protocols with 17 000 samples for all (human) tissues except sperm.
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