Wednesday, May 28, 2008

Dillon and Frankis 2004

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.

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.

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.

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.

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.

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.

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.

Krishan 1990

Krishan A. 1990. Rapid DNA content analysis by the Propidium iodide-hypotonic citrate method. Methods in Cell Biology 33: 121-125.

This author describes a set of protocols for staining nuclear DNA with Propidium iodide for use in flow cytometry. The basic protocol is similar to the methods we use in our lab currently, though this author claims that prepared stain solution can be stored at room temperature in a light-proof bottle in a large volume (e.g. 1 L), and that stained specimens can be stored up to 24 hours on ice without noticeable impact on measurements. Trypsin and heparin, frequently used in cell dissociation or blood storage procedures, will interfere with the binding of Propidium iodide to nuclear DNA.

Cerra et al. 1990

Cerra R, Zarbo RJ, Crissman JD. Dissociation of cells from solid tumours. Methods in Cell Biology 33: 1-12.

These authors describe methods to produce cell suspensions from a range of cancer tumour types collectively described as “solid”. Many tumours are relatively easy to extract individual cells in suspension; this paper describes methods suitable for tumours in which the cells are bound to each other and to extracellular materials. Existing methods for flow cytometry analysis of these tumours have focused on enucleation techniques, which avoid the problems associated with cell membrane bonds to other cells by rupturing cell membranes. This causes the loss of potentially informative cell-membrane-bound and cytoplasmic materials, and these authors state they are not convinced enucleation is the best option.

Dissociating cells can be accomplished by a vast range of mechanical and enzymatic techniques, many of which can be used in combination, such that the optimum technique to use on a given tissue must be determined empirically for each case. Mechanical dissociation techniques typically start with cells in the early stages of cell culture. The authors recommend the use of two scalpel blades rather than scissors to slice tissues because scissors tend to mash the tissue rather than cut. The resulting semi-dissociated tissue should be sieved through something called a “100-mesh cell sieve”, whatever that is. I think this step removes clumps of cells and large pieces of extracellular debris. Other mechanical dissociation methods include the use of tissue attached to a wine cork and vibrated with a mechanical engraver to release cells (Eade et al. 1981).

Enzymatic dissociation methods tend to focus either on the use of proteases to disrupt intercellular desmosomes, or collagenases to disrupt the extracellular matrix. Each category of enzymes may damage or remove cell-membrane features that may be useful in diagnosis during flow cytometry. In all cell dissociation techniques, DNAases are routinely used to remove the very sticky free DNA released by dead cells. Liotta et al. (1982) provide a review of the use of collagenases in cell dissociation.

Enucleation techniques can be applied to a wider range of tissues than can cell dissociation techniques, including fixed and frozen tissues. I get the impression that fixed and / or frozen tissues are routinely used in cancer flow cytometry with a range of human tissues. The authors caution that enucleation typically causes high levels of background debris compared to cell dissociation. Thornthwaite et al. (1980) and Vindeløv et al. (1983) provide protocols for one-step isolation and staining of nuclei; these authors provide a more complicated method for the use of paraffin-embedded tissues.

Singh 1998

Singh NP. 1998. A rapid method for the preparation of single-cell suspensions from solid tissues. Cytometry 31: 229-232.

This author evaluated the use of a tissue press (BioSpec Product, Inc.) to produce cell suspensions suitable for examination of DNA double-stranded breaks in rat brain tissue. The use of manual cell dissociation with forceps and scalpels was compared to the device, and the device was found to be highly effective and efficient at producing cell suspensions.

This device is not expensive (approx. $30) and looks like it may be useful for my own work with preparing cell suspensions for flow cytometry.

Brockhoff et al. 1999

Brockhoff G, Fleischmann S, Meier A, Wachs F-P, Hofstaedter F, Knuechel R. 1999. Use of a mechanical dissociation device to improve standardization of flow cytometric cytokeratin DNA measurements of colon carcinomas. Cytometry (Communications in Clinical Cytometry) 38: 184-191.

These authors evaluated the use of a Medimachine (DAKO Diagnostica GmbH) to dissociate cells from specimens of colon tumours in a large number of patients. They compared flow cytometry results from specimens sliced by the Medimachine’s “little crossed blades” to manual dissociation using scalpels. In general, the Medimachine produced a higher fraction of useable cells in flow cytometry, and was more consistent in an interlaboratory comparison.

Saturday, May 17, 2008

Rodríguez-Juiz et al. 1996

Rodríguez-Juiz AM, Torrado M, Méndez J. 1996. Genome-size variation in bivalve molluscs determined by flow cytometry. Marine Biology 126: 489-497.

These authors measured nuclear DNA contents in 10 species of bivalves of commercial importance. Genome size variation in plants and poikilothermal animals had previously been associated with life-history and ecological traits, suggesting links between genome size variation and speciation events. There had been few previous studies of DNA content in molluscs. Early examples of such studies include Mirsky and Ris (1951), Hinegardner (1973, 1976), and Cavalier-Smith (1978); several earlier papers on molluscs and other poikilotherms related genome size to “specialization”, particularly the work of Hinegardner and colleagues.

Unlike most studies of DNA content, these authors included relatively large samples of each species, using 20 individuals in each species. All individuals were purchased from commercial shellfish sellers, either in Spain (9 species) or the Netherlands (Mytilus edulis), and maintained alive in the laboratory until dissection of gill tissue; the assumption that gill tissue is diploid is never stated explicitly but was used in the calculations of genome sizes. Tissue was placed in filtered, autoclaved seawater and subjected to mechanical shaking for 30 minutes; these authors do not describe in detail this shaking, is there a standard rate and magnitude of mechanical shaking of mollusc tissue? The presence of isolated cells was verified using a microscope, and cell preparations were strained through 15mm mesh and sonicated for two minutes to remove cell membranes. The nuclei were then centrifuged, resuspended in buffer that appears similar to Galbraith’s buffer (Galbraith et al. 1983), and fixed with 0.1% formaldehyde on ice. Finally, aggregations of nuclei were disrupted using a 26-gauge needle, pumped three times.

Two internal standards were employed: Capsicum annuum and chicken red blood cells (CRBCs). Isolated nuclei from these species were added to bivalve nuclei suspensions before staining, thus standards and specimens here were co-stained rather than co-prepared. The CRBCs produced a peak in the flow cytometry histograms overlapping seven of the 10 bivalve species, thus the introduction of the plant nuclei. C. annuum nuclei were employed after checking for consistent measurements with the three species of bivalves that did not overlap in peak area with CRBCs, and comparison between C. annuum and CRBCs to determine a C. annuum diploid nuclear content of 8.4 pg, larger than any bivalve measured in this study. 10 000 nuclei were measured per histogram, presumably this means total events recorded above the debris cut-off, and each specimen was measured three times.

These authors report significant intraspecific genome size variation in all 10 bivalve species. Interspecific (and between higher taxa) was much greater than intraspecific variation, but the intraspecific variation was statistically significant under 2-way ANOVA and “GSD” calculations based on the work of Gold and Amemiya (1987) and Alvarez-Fuster et al. (1991).

The discovered and possibly unexpected intraspecific variation is used to bolster an argument made in the discussion that large samples are necessary for accurate determination of genome size and genome size variation in species. This explains the difference between these results and the no-intraspecific-variation results of some previous authors that did not use large samples per species.

Following this is a discussion of Hinegardner’s (several papers in the 1970s) “specialization” assumption / hypothesis. It is described as one, then the other. These data do not support this hypothesis, which is not surprising considering how vague and taxon-specific the terms “specialized” and “generalized” are, and their underlying assumptions about species and lineage ages and rates of evolution.

Healy and Rota 1992

Healy B, Rota E. 1992. Methods for collecting Enchytraeidae during expeditions. Soil Biology and Biochemistry 24: 1279-1281.

These authors succinctly describe methods for collecting, extracting from soil and other materials, sorting, maintaining alive, and fixing and preserving enchytraeid worms. Enchytraeids are found in all moist soils, and other materials such as tide debris and forest litter. In this study, 0.5 kg soil samples were kept in plastic bags for up to five weeks before extraction of worms.

The methods described here were developed during an expedition by the authors to north Africa; Ireland, their home, does not allow import of soil samples so they were forced to extract worms in the field. The basic extraction method is a modification of O’Connor’s (1957) “wet funnel”, which is much more clearly described and illustrated here than in that older work. Figure 1. of this paper shows a wet funnel. Worms move down away from the source of light and heat (a light bulb) into a funnel full of water connected to a bottle. After an undescribed period, probably at least three hours, the water in the bottle is dumped to Petri dishes and worms sorted. The authors describe sorting using the naked eye, but recommend a magnifying glass with attached light source for smaller specimens. Living enchytraeids can be maintained in culture with soil agar, made from 2% agar and a 1:1 mixture of soil and distilled water. Soil and debris added with the worms will ruin sterility, but provides food for the worms. Water should be added periodically to keep everything moist, about every five days. Worms in this study were narcotized with soda water, and the authors describe dilute beer as an acceptable substitute. Fixation and preservation can use “any of the usual fixatives”, though contraction of specimens can make it difficult to distinguish taxonomically-important internal organs.

Briones et al. 2007

Briones MJI, Ineson P, Heinemeyer A. 2007. Predicting potential impacts of climate change on the geographical distribution of enchytraeids: a meta-analysis approach. Global Change Biology 13: 2252-2269.

These authors conducted a meta-analysis of all studies describing population abundances of enchytraeids. This meta-analysis required certain standards of error reporting and sample sizes for the analysis, thus many papers were not included. The authors seem inordinately enthusiastic about their meta-analysis, going to great lengths to describe both meta-analyses in general, and their own approach.

These authors focused on enchytraeids because they are often the dominant-biomass organisms of organic soils. Organic soils are not well defined in this paper, but are apparently those with very high carbon contents, thus these soils are important in the context of global climate change because changes to these systems could result in large changes in these soils’ roles as either carbon sinks or sources. Biomass of enchytraeids in organic soils can exceed 50% of all animal biomass in the soil, often dominated by one or a few species, feeding primarily on bacteria and detritus.

As an additional layer of analysis, these authors focused on one species of enchytraeid, Cognettia sphagnetorum, commonly found in European organic soils such as marshlands. The majority of studies analysed were situated in Europe, principally the UK and other parts of north-western Europe. The authors repeatedly describe this geographic bias, but do not seem otherwise concerned.

In general, high population sizes of enchytraeids were associated with Hungary (one site), alpine meadows, tropical grasslands, tropical rainforests, moorlands, moder and brown-earth soils, slightly acidic soils (pH 4 – 6), temperate rainy climates with moisture all year, and regions with moderate or cold summers. Mean annual temperature (I think that’s what the undefined acronym “MAT” stands for) higher than 16°C was strongly associated with reduced population sizes, and the loss of the focal species C. sphagnetorum. MAT higher than 10°C appears to be an inflection point, with reduced population sizes above that limit. Additionally, small population sizes were associated with warm dry summer climates (e.g. Mediterranean) and cold snowy tundra climates.

The authors present a confusing and possibly meaningless discussion of the results of their geographic analysis. They describe the range of population densities in their studies (more than 500 000 m-2 down to less than 5 000 m-2), then state these differences in mean densities were not significant under the Wilcoxon test. If the means are not different, they’re not different, so why bother to report them, except to describe the error associated with comparing across ecosystems? They ran a regression analysis using these population densities, after stating the data were not normally distributed; I do not recall how sensitive to departures from normality regression analysis may be.

The authors state that there was no association between enchytraeid population density and depth in soil, then go on to rather confusingly describe the most enchytraeid-rich soil depth horizons. Apparently, enchytraeids are generally concentrated in the top 3 to 4 cm, with very few individuals found deeper than 12 cm. Enchytraeids generally seem to require permanent high moisture levels.

The focal species apparently reproduces asexually by fragmentation; many of the studies included in the meta-analysis describe numbers of individuals that are “whole” or “regenerating”. This curious and surprising life-history trait is never referenced in this paper, seemingly treated as common knowledge among enchytraeidologists. I know of few animals that habitually reproduce this way.

Overall, I found this a confusing and disappointing paper, though I admire their attempt to reconcile a highly heterogeneous dataset. The reference list contains probably the majority of available papers on Enchytraeidae, and may be very useful in that context.

Tuesday, May 13, 2008

Dillon 2006

Dillon RT. 2006. The classification of the Lymnaeidae. Letter to the FWGNA project group, December 2006. http://www.cofc.edu/~fwgna/archive/28Dec06.html.

This article describes the current taxonomic uncertainty of the freshwater gastropod family Lymnaeidae, with reference to the major monographs and taxonomic works of the 20th century.

Baker (1911) describes 113 species and subspecies of Lymnaeidae in North America, including seven genera (one of which is only known from fossils). Later work, including aspects of the Modern Synthesis emphasized the considerable intraspecific morphological variation of this and other families of freshwater snails, leading to a major revision of the family by Hubendick (1951), a monograph that this author refers to as a “masterpiece”.

Hubendick (1951) reduced the family to two genera, with most species placed in Lymnaea; only the “weirdo” limpet-like species of genus Lanx remain separate. Approximately 12 species were recognized for North America, either as endemics or holarctic.

Later work, for example by Burch (1980, 1982), did not adopt Hubendick’s (1951) scheme, resulting in a compromise taxonomic scheme somewhere between that of Hubendick (1951) and Baker (1911). This author proposes a modified scheme for current workers, listing two genera (as Hubendick, 1951), and reducing most of Baker’s (1911) genera to subgenus level, to preserve the utility of such terms as “fossarine” and “stagnicoline” as descriptors of groups of species.

I have assembled a taxonomy of the Lymnaeidae for North America from this letter and from Clarke (1973).

Leslie et al. 1997

Leslie AJ, Crisman TL, Prenger JP, Ewel KC. 1997. Benthic macroinvertebrates of small Florida pondcypress swamps and the influence of dry periods. Wetlands 17: 447-455.

These authors examined the faunas of three pondcypress swamps in northeastern Florida, over about 18 months from 1993 to 1995. Pondcypress swamps are wetlands with unpredictably-fluctuating water levels and a canopy formed by pondcypress trees (Taxodium distichum var. nutans). These swamps may remain wet for years or dry repeatedly within a single year, presenting a very challenging abiotic environment to aquatic organisms. Prior to this study, there had been few papers describing the biodiversity of pondcypress swamps, particularly during dry periods referred to as “drawdowns”.

The three ponds differed from each other in area, depth, and most dramatically emergent macrophyte vegetation. All three contained large abundances of Sphagnum mosses, and responded to local weather conditions similarly, tending to be driest at the same time, in June 1994. All ponds were highly acidic, with pH ranging between 3.4 and 4.4, and changing over time.

Benthic macroinvertebrates were collected by cores in a random fashion, to allow calculation of species richness and individual density per unit area, in this case reported as per square meter. Collected animals were identified to genus when possible using a range of identification keys from the literature (e.g. Pennak 1978). The beetle families Dytiscidae and Hydrophilidae and the fly family Chironomidae contributed large numbers of genera, and two genera of chironomids and one amphipod genus (Crangonyx) contributed the majority of individuals. Up to 52% of total individuals collected were specimens of Crangonyx.

Total species richness varied between the three pools, with the pool with least emergent vegetation having the lowest richness. Species richnesses were higher than those reported for other wetland habitats in the same region.

The authors report some surprise at the levels of biodiversity maintained even during dry periods. There is some speculation based on Barlocher et al. (1978) that drawdown periods are accompanied by an increase in bacterial and fungal activity, with a net increase in protein levels and therefore food quality in the detritus that forms the bulk of the food of most of the collected animals. Crangonyx and many of the other species found are generalists, and can cope with unpredictable dry periods by burrowing into wet soil or escaping to other habitats, while many of the species excluded during dry periods are obligate aquatics or have restricted life cycles and require a certain minimum period of wetness to complete one or more stages of the life cycle.

The high species richness of Dytiscidae and high population densities of Crangonyx are good indications for my project, as I expect to visit such habitats during the summer of 2008.

Sunday, May 11, 2008

Miller et al. 2007

Miller KB, Alarie Y, Whiting MF. 2007. Description of the larva of Notaticus fasciatus (Coleoptera: Dytiscidae) associated with adults using DNA sequence data. Annals of the Entomological Society of America 100: 787-797.

These authors were able to associate some larvae collected in French Guiana to a species widespread in lowland South America that did not previously have described larvae. This represents the first description of larvae of the tribe Aubehydrini, though this tribe contains only one described genus with two possibly synonymous species. This is the last tribe in the subfamily Dytiscinae to be so described.

The general methods closely follow those of Miller et al. (2005), though there is little in the way of phylogenetic discussion because, as stated by the authors, this work forms part of the basis of ongoing and future projects to examine dytiscine phylogeny in detail.

The larvae were clearly identified as belonging to the genus Notaticus, and are most likely members of the species N. fasciatus, with sequence differences well below the usual 2% threshold relative to adults of that species. However, the adults used in the comparison came from geographically distant populations in Bolivia, and the sequences of the larvae are sister to the sequences of the adults, rather than nested within in the cladogram. Thus, it is possible though unlikely that these larvae are actually members of a different species within the genus, either N. confusus or some as-yet-undescribed species. In addition, the adults that have been described of the two species of Notaticus are morphologically very similar, and may actually represent members of one species.

Miller et al. 2005

Miller KB, Alarie Y, Wolfe GW, Whiting MF. 2005. Association of insect life stages using DNA sequences: the larvae of Philodytes umbrinus (Motschulsky) (Coleoptera: Dytsicidae). Systematic Entomology 30: 499-509.

These authors used DNA sequence data in the form of 806 bp of mtCOI, to associate unidentified larvae to a described species. The insects were collected from pools in the desert region of the Skeleton Coast of northern Namibia. The larvae were morphologically associated with one tribe of dytiscids, and were collected with adults and larvae of two other species in the tribe in the genus Laccophilus, but were larger-bodied than known larvae of that genus.

Between species differences in sequence ranged between 1.9 and 19.9%, similar to values reported for other insect species. Within species differences were between 0 and 0.82%, and the differences in sequences between the larvae and the adults of their assigned species was 0 to 0.14%, providing clear evidence of the association.

The authors provide a very detailed list of morphological characters that are diagnostic for larvae of this genus (Philodytes), and a longer and even more detailed list of characters diagnostic for the species (P. umbrinus). They state near the end of the paper that their goal was not to provide molecular diagnostic features, but to use DNA sequence data to work backwards to find diagnostic morphological features which had not previously been described. There is some additional discussion of the uses, abuses, and limits of DNA barcoding in a taxonomic context, with a final point that taxonomy without morphology would not be as interesting.

Friday, May 9, 2008

Bajer et al. 1961

Bajer A, Hansen-Melander E, Melander Y, Molè-Bajer J. 1961. Meiosis in Cepaea nemoralis studied by microcinematography. Chromosoma (Berl.) 12: 374-381.

These authors studied spermatogenesis in the helicid land snail Cepaea nemoralis, an hermaphrodite with accessible meiotic cells containing 28 bivalents. The stated aim of this paper is to contribute to knowledge of basic meiotic processes in animals, and compare them to meiosis in plants. This was accomplished through the use of an apparently cutting-edge-technology phase-contrast microscope, and for at least this paper, 16mm film.

Most of this paper is a description of the changes in shape and size of the nucleus of meiotic cells, and the movements of the chromosomes in the minutes immediately before and after the disappearance of the nuclear membrane. The nucleus is apparently somewhat unstable during prophase, changing position in the cell in a random fashion and changing shape rapidly. The chromosomes inside the intact nucleus also seem to move randomly, in a manner that does not suggest pulling by microtubules or other cytoskeletal components. Just prior to the disappearance of the nuclear membrane, the nucleus increases in size; this phenomenon is not explained even by speculation in this paper.

Previous work by these authors, using the same microscope, included measurements of nucleus mass by interference microscopy. How mass is measured is not clearly explained, but this paper is the first reference to this measurement I have seen.