Neurological symptoms in tricolor sharkminnow Balantiocheilos melanopterus associated with Myxobolus balantiocheili n. sp. infecting the central nervous system

A new species, Myxobolus balantiocheili, is described from the central nervous system of wild and cultured tricolor sharkminnow Balantiocheilos melanopterus from Thailand. The infection was associated with emaciation and severe neurological symptoms, including sudden uncoordinated darting, rolling and pitching. Whitish cysts of varying shape, measuring 70 to 500 microm, were located in the brain, spinal cord and in the meninx, and were sometimes accompanied by haemorrhages. In the brain, most cysts were located in the medulla oblongata and the mesencephalon. Cysts with a segmental distribution were observed in the meninx of the spinal cord. Single di- to hexasporic pansporoblasts, and free mature spores, were frequently seen in both the white and grey matter of the brain and spinal cord, mostly associated with blood vessels. Mature spores varied in shape from oval to ellipsoid, measuring 12.3 +/- 0.59 (range 10.6 to 13.3) x 10.0 +/- 0.47 (9.2 to 10.9) x 6.9 +/- 0.20 (6.7 to 7.1) microm. Spore size and morphology, in addition to fish-host species and apparently narrow tissue tropism, clearly distinguishes this new Myxobolus. No marked inflammatory response or gliosis was associated with the lesions, but compression and degeneration of the nervous tissue surrounding large cysts was observed. We suggest that the high temperature of the fish hosts' tropical habitat may have increased the rate of development and the virulence of M. balantiocheili.     

Multidimensional on-line sample preparation of verapamil and its metabolites by a molecularly imprinted polymer coupled to liquid chromatography-mass spectrometry

A new molecularly imprinted polymer (MIP) material was synthesized selective for verapamil and utilized for on-line metabolic screening of this common calcium antagonist in biological samples. Since some metabolites of verapamil have also shown pharmacological properties, a selective and sensitive sample preparation approach that provides a metabolic profile in biologically relevant samples is important. The MIP material was coupled on-line to a restricted access material (RAM) precolumn. The multidimensional nature of this set-up removed large matrix interferents such as proteins from the sample, while the selectivity of the MIP enabled further cleanup of the smaller analytes. The selectivity and extraction efficiency of the MIP for verapamil and its metabolites was evaluated in various biological matrices, such as cell cultures and urine. The experimental set-up with the developed method enabled the direct injection of biological samples for the selective isolation, preconcentration, identification and analysis of verapamil and its phase I metabolites by LC-MS(n). This multidimensional approach provided much qualitative information about the metabolic profile of verapamil in various biological matrices. An analytical method was developed for the quantification of verapamil and gallopamil in urine, plasma and cell culture. Acceptable linearity (R(2)=0.9996, 0.9982 and 0.9762) with an average injection repeatability (n=3) of 10, 25 and 15% R.S.D. was determined for urine, plasma and cell culture, respectively. This is the first application of the procedure for the selective metabolic screening of verapamil in biological samples.     

Pleistocene speciation in the genus Populus (salicaceae)

The macroevolutionary consequences of recent climate change remain controversial, and there is little paleobotanical or morphological evidence that Pleistocene (1.8-0.12 Ma) glacial cycles acted as drivers of speciation, especially among lineages with long generation times, such as trees. We combined genetic and ecogeographic data from 2 closely related North American tree species, Populus balsamifera and P. trichocarpa (Salicacaeae), to determine if their divergence coincided with and was possibly caused by Pleistocene climatic events. We analyzed 32 nuclear loci from individuals of P. balsamifera and P. trichocarpa to produce coalescent-based estimates of the divergence time between the 2 species. We coupled the coalescent analyses with paleodistribution models to assess the influence of climate change on species' range. Furthermore, measures of niche overlap were used to investigate patterns of ecological differentiation between species. We estimated the divergence date of P. balsamifera and P. trichocarpa at approximately 75 Ka, which corresponds closely with the onset of Marine Isotope Stage 4 (～76 Ka) and a rapid increase in global ice volume. Significance tests of niche overlap, in conjunction with genetic estimates of migration, suggested that speciation occurred in allopatry, possibly resulting from the environmental effects of Pleistocene glacial cycles. Our results indicate that the divergence of keystone tree species, which have shaped community diversity in northern North American ecosystems, was recent and may have been a consequence of Pleistocene-era glaciation and climate change.     

Provision of nestboxes raises the breeding density of Great Tits Parus major equally in coniferous and deciduous woodland

Nestbox provision is a technique used to increase nest-site availability for secondary cavity-nesting birds. However, little is known about the demographic consequences of nestbox provision in different habitat types. To assess how nestbox provision affects the density of hole-nesting birds simultaneously in two contrasting habitats, we compared the breeding density of Great Tits along transects without nestboxes with that in transects where nestboxes were provided. Although the initial density of breeders was considerably higher in the deciduous habitat than in the coniferous habitat, provision of nestboxes increased density by a similar number of additional pairs in each habitat type. Thus, the provision of nestboxes in managed coniferous forests may be as effective in increasing the breeding opportunities of cavity nesters as in deciduous stands. Moreover, previous research showed that pairs in deciduous habitat with nestboxes have consistently lower breeding success than those in coniferous habitat with nestboxes. It is possible that the addition of nestboxes in the preferred habitat increased density to such an extent that density-dependent effects became apparent.     

The development and morphogenesis of Camallanus cotti Fujita, 1927 (Nematoda: Camallanidae), with notes on its phylogeny and definitive host range

The freshwater fish nematode Camallanus cotti Fujita, 1927 (Spirurida: Camallanidae) is naturally distributed in East, South and Southeast Asia. During the past three or four decades the species has been disseminated to Europe, North America, Australia and Hawaii, mainly due to the extensive ornamental fish trade. In the present study, its development and larval morphogenesis is described. Newborn first-stage larvae are ingested by copepods and within hours reach the haemocoel, where the worms moult twice. At 22°C ambient temperature, fully-developed third-stage larvae appear on day 11 p.i. After transmission into the fish intestine two further moults occur before the adult stage is reached. At 23°C water temperature, the final moult is initiated around day 33 p.i. in males and between 34 and 42 days p.i. in females. Morphologically the young and older adults are similar, but the buccal capsule of both young males and young females is not yet fully sclerotised. Based on the close similarity in the development and adult morphology of the buccal capsule in C. cotti and three congeners from Europe and North America, we suggest, in general agreement with earlier workers, that the Camallanus Railliet & Henry, 1915 originated in tropical Asia and subsequently radiated to species when adapting to new climatic and/or host-related conditions in the Old World and the New World. C. cotti may thus represent a lineage of speciation which did not apparently involve extensive changes in relation to their natural geographical distribution or definitive host range.     

Phylogenetics and diversification of Cotyledon (Crassulaceae) inferred from nuclear and chloroplast DNA sequence data

Crassulaceae includes approximately 35 genera and 1500 species of leaf and stem succulent flowering plants. The family is nearly cosmopolitan in distribution, but is particularly diverse in southern Africa, where five genera comprising approximately 325 species are found. One of these genera, Cotyledon, includes 10 species that are largely confined to South Africa, where they are commonly found on rocky hillsides, coastal flats, and cliff faces. Species of Cotyledon are characterized by five-parted, pendulous, sympetalous flowers, but the genus is highly diverse in growth form, flower color and size, and leaf morphology. One particularly variable species, C. orbiculata, has been divided into five varieties based on leaf morphology and biogeography; however, the monophyly of this species as well as the relationships among the varieties have not previously been investigated. Parsimony analyses of a combined data set of DNA sequences from chloroplast and nuclear genome provided the first estimate of phylogeny for Cotyledon, and resulted in two minimum-length trees and a fully resolved phylogeny for the genus. Results indicate that C. orbiculata is not monophyletic and suggest the need for additional studies and a revised classification within the genus.     

Local selection across a latitudinal gradient shapes nucleotide diversity in balsam poplar, Populus balsamifera L

Molecular studies of adaptive evolution often focus on detecting selective sweeps driven by positive selection on a species-wide scale; however, much adaptation is local, particularly of ecologically important traits. Here, we look for evidence of range-wide and local adaptation at candidate genes for adaptive phenology in balsam poplar, Populus balsamifera, a widespread forest tree whose range extends across environmental gradients of photoperiod and growing season length. We examined nucleotide diversity of 27 poplar homologs of the flowering-time network-a group of genes that control plant developmental phenology through interactions with environmental cues such as photoperiod and temperature. Only one gene, ZTL2, showed evidence of reduced diversity and an excess of fixed replacement sites, consistent with a species-wide selective sweep. Two other genes, LFY and FRI, harbored high levels of nucleotide diversity and exhibited elevated differentiation between northern and southern accessions, suggesting local adaptation along a latitudinal gradient. Interestingly, FRI has also been identified as a target of local selection between northern and southern accessions of Arabidopsis thaliana, indicating that this gene may be commonly involved in ecological adaptation in distantly related species. Our findings suggest an important role for local selection shaping molecular diversity and reveal limitations of inferring molecular adaptation from analyses designed only to detect species-wide selective sweeps.