The effects of human leukemia inhibitory factor (hLIF) and culture medium on in vitro differentiation of cultured porcine inner cell mass (pICM)

Summary Isolation and maintenance of porcine embryonic stem (pES) cells have been hindered by the inability to inhibit differentiation of the porcine inner cell mass (pICM) in vitro. Culture conditions currently in use have been developed from mouse ES cell culture and are not effective for maintaining the pICM. Optimizing culture conditions for the pICM is essential. We have developed a grading system to detect changes in the differentiation status of in vitro cultured pICM. Porcine ICMs (Day 7) were isolated by immunosurgery and cultured for 4 d in either Dulbecco’s modified Eagle’s medium (DMEM)-based medium (D medium) or DMEM/Ham’s F-10 (1:1)-based medium (D/H medium) with or without human Leukemia Inhibitory Factor (hLIF, 1000 iu/ml). Colonies were photographed daily for morphological analysis. pICMs were categorized into one of two types based on their morphological profile: type A, nonepithelial or type B, epithelial-like. Eight investigators evaluated pICM differentiation using standardized differentiation profiles. Each pICM series was graded on a scale of 1 (fully undifferentiated) to 5 (fully differentiated) for each time point. Differentiation was verified by alkaline phosphatase activity, cytokeratin staining, and scanning electron microscopy. Neither hLIF nor culture medium delayed differentiation of pICMs (P=0.08 and P=0.25, respectively). The grading system employed was an effective tool for detecting treatment effects on differentiation of the developing pICM. These results demonstrate that hLIF cannot significantly inhibit differentiation of the pICM, and is unlikely to assist in porcine ES cell isolation. Future experiments utilizing homologous cytokines may prove more beneficial.     

The variability of the hypericin content in the regenerants of Hypericum perforatum

The hypericin content of in vitro regenerated plants of Hypericum perforatum L. was determined by spectrophotometry. A significant variability of some of the morphological characters, of the biomass production and the hypericin formation was found within the somaclones of the same genetic origin and among the regerants of different genotypes. The concentration of BAP which promoted the shoot differentiation did not affect the hypericin content and the gland density. New information on the ultrastructure of hypericin-containing multicellular glands is presented here.     

Analysis of meristic and mitochondrial DNA variation in Syngnathus abaster (Teleostea: Syngnathidae) from two western Mediterranean lagoons

Syngnathus abaster is a euryhaline pipefish distributed in the Mediterranean, Black Sea, and the north-eastern Atlantic. Although its populations are characterised by high morphological plasticity, neither congruent information about the morphological differentiation of S. abaster populations from the Mediterranean lagoons is available, nor population genetic surveys have been so far performed. In this context, the aims of our study were as follow: i) to describe the variation at nine meristic characters of S. abaster from two western Mediterranean brackish-water areas: the Tunis north lagoon (Tunisia) and the Mauguio lagoon (France); ii) to analyse sequences of four mitochondrial DNA regions in order to evaluate the occurrence of genetic variation between the two areas, if any. The morphological survey revealed a subdivision into two distinct groups: the first included the Tunisian specimens, the second the French ones. Genetic analysis evidenced the occurrence of a sharp genetic structuring with high levels of genetic differentiation between Tunisian and French S. abaster populations. Results suggest that the evolutionary forces driven by the different biogeographical and ecological conditions of the two Mediterranean brackish-water areas have boosted the morphological and genetic divergence of the populations here analysed. The scarce potential of long-distance dispersal of S. abaster may have also enhanced the divergence retrieved.     

Symposium 10: Differentiation Plasticity of Stem Cells. Direct differentiation of radial glial cells from embryonic stem cells

The major role of radial glial cells in neuronal development is to provide support and guidance for neuronal migration. In vitro, neurons, astrocytes and oligodendrocytes have also been generated from neural stem cells and embryonic stem cells, but the generation of radial glial cells in vitro has not yet been reported. Since radial glial cells can lead to neurons and astrocytes during brain development, neurogenesis and gliogenesis of stem cells in vitro may at least in part also utilize the same mechanisms. To test this hypothesis, we utilized five different clones of embryonic (ES) and embryonal carcinoma (EC) stem cell lines to investigate the differentiation of radial glial cells during in vitro neural differentiation. Here, we demonstrate that radial glial cells can be generated from ES/EC cell lines. These ES/EC cell‐derived radial glial cells are similar in morphology to radial glial cells in vivo. They also express several cytoskeletal markers that are characteristics of radial glial cells in vivo. The processes of these in vitro‐generated radial glial cells are organized into scaffolds that appear to support the migration of newly generated neurons in culture. Like radial glial cells in vivo, they appear to differentiate subsequently into astrocytes. Differentiation of radial glial cells may be a common pathway during in vitro neural differentiation of ES cells. This novel in vitro model system may facilitate the investigation of regulation of radial glial cell differentiation and its biological function. Acknowledgements: Supported by USPHS Grant NS11853 and a grant from the Children's Medical Research Foundation.     

Expression of developmentally relevant proteins by rodent embryo CNS cells in vivo and in vitro: Proto-oncogene pp60c-src and high molecular weight neurofilament protein

The expression of proteins that play a role in neuronal differentiation was examined in central nervous system (CNS) micromass embryo cell cultures and compared to expression at comparable developmental stages in vivo. The protein product of the src proto-oncogene (pp60^c-src) has been postulated to have a specific role in development because, although it is expressed in many tissues, marked increases in amount and activity of pp60^c-src occur in neurons at the time of differentiation. Another protein of interest, high molecular weight neurofilament (NF) protein, is found in differentiated neurons. In the present study, changes over time in the expression of these two proteins in vitro and in vivo were examined. In the micromass cell cultures, primary cells from day 12 rat embryo CNS are plated at high density and differentiate into neurons during five days in culture. Tissues from embryos grown in vivo were assessed at 12 and 17 days post-coitum. Proteins were quantified by PAGE separation of equal amounts of total protein followed by transfer to membranes, immunoblotting, and densitometric scanning of blots. Increases in the amount of both proteins with neuronal differentiation was shown. Protein kinase activity of immunoprecipitated pp60^c-src also increased in cell cultures and in embryos. Similarity in patterns of expression between in vitro and in vivo tissue samples provides further evidence that the cultures closely simulate in vivo differentiation and are a useful system for examining expression of developmental genes in vitro.Abbreviations BCIP 5-bromo-4-chloro-3-indolylphosphate p-toluidine salt - CNS central nervous system - DPBS Dulbecco's phosphate-buffered saline - GAM-AP goat anti-mouse IgG alkaline phosphatase conjugate - LB limb bud - NF high molecular weight neurofilament protein - NBT nitroblue tetrazolium chloride - SDS-PAGE polyacrylamide gel electrophoresis - PVDF polyvinylidene difluoride - RIPA radioimmunoprecipitation - TBS Tris-buffered saline - TTBS TBS with 0.05% Tween-20 Presented in part at the 1989 and 1990 Teratology Society Meetings and the 1990 Society of Toxicology Meetings.     

The role of neurotrophins in the developing nervous system

Neurotrophins were originally identified by their ability to promote the survival of developing neurons. However, recent work on these proteins indicates that they may also influence the proliferation and differentiation of neuron progenitor cells and regular several differentiated traits of neurons throughout life. Moreover, the effects of neurotrophins on survival have turned out to be more complex than originally thought. Some neurons switch their survival requirements from one set of neurotrophins to another during development, and several neurotrophins may be involved in regulating the survival of a population of neurons at any one time. Much of our understanding of the developmental physiology of neurotrophins has come from studying neurons of the peripheral nervous system. Because these neurons and their progenitors are segregated into anatomically discrete sites, it has been possible to obtain these cell for in vitro experimental studies from the earliest stage of their development. The recent generation of mice having null mutations in the neurotrophin and neurotrophin receptor genes has opened up an unparalleled opportunity to assess the physiological relevance of the wealth of data obtained from these in vitro studies. Here I provide a chronological account of the effects of members of the NGF family of neurotrophins on cells of the neural lineage with special reference to the peripheral nervous system. 1994 John Wiley & Sons, Inc.     

Excitotoxin-induced changes in transglutaminase during differentiation of cerebellar granule cells

Summary. Excitotoxicity induced by NMDA receptor stimulation is able to increase the activity of many enzymes involved in neuronal cell death. Primary cultures of rat cerebellar granule cells were used to elucidate the role of transglutaminase reaction in the excitotoxic cell response, and to evaluate the role of glutamate receptors in cell survival and degeneration. Granule neurons, maintained in vitro for two weeks, were exposed to NMDA at different stages of differentiation. Following NMDA receptor activation, increases in transglutaminase activity were observed in cell cultures. The levels of enzyme activity were higher in cells at 5 days in vitro than in those at 8–9 or 13–14 days in vitro. Moreover, NMDA exposure up-regulated tTG expression in neurons as young as 5 days in vitro. These cultures also exhibited morphological changes with clear apoptotic features. Results obtained demonstrate that susceptibility of granule cells to excitotoxicity depends on the developmental stage of neurons.     

Multivariate morphometric analysis of the Potamogeton compressus group (Potamogetonaceae)

The Potamogeton compressus group is a complex of three to five closely related species with a circumpolar distribution in the Northern Hemisphere. Multivariate morphometric analyses (principal component analysis, cluster analysis, canonical and classificatory discriminant analyses) were used to elucidate the patterns of variation within this group and to test the morphological differentiation of the species recognized in the current literature. From the entire distribution range, 156 specimens of the group were included in the numerical methods. Results from morphological comparison are discussed in relation to molecular data, reproductive behaviour and geographical distribution. Morphometric analyses provided evidence that this complex can be clearly divided into three groups, one of which was subdivided mainly on the basis of allopatric occurrence and genetic differentiation. These groups correspond to four species accepted here: P. acutifolius (temperate regions of Europe), P. compressus (boreal and temperate regions of Europe and Asia), P. manchuriensis (northeastern China and Russian Far East) and P. zosteriformis (boreal and temperate regions of North America). Two species, P. acutifolius and P. compressus, are partly sympatric, but clearly differentiated morphologically and genetically, and effectively isolated reproductively. Endemic P. manchuriensis is characterized by a unique combination of characters and an occurrence in a limited geographical area. Allopatric P. zosteriformis is weakly differentiated morphologically from P. compressus, but differs markedly in molecular markers correlated with geographical differentiation. It may represent a cryptic species. In contrast, a recently suggested concept of southern Siberian P. henningii was not supported by our analyses. Plants so named are considered here as slender phenotypes of the widespread and variable P. compressus. © 2012 The Linnean Society of London, Botanical Journal of the Linnean Society, 2012, 170 , 112–130.     

Optimizing neuronal differentiation of human pluripotent NT2 stem cells in monolayer cultures

Human pluripotent embryonal carcinoma (NT2) cells are increasingly considered as a suitable model for in vitro developmental toxicity and neurotoxicity (DT/DNT) studies as they undergo neuronal differentiation upon stimulation with retinoic acid (RA) and allow toxicity testing at different stages of maturation. However, differentiation of NT2 cells is not straightforward. There are different protocols available in the literature reporting varying results with regard to differentiation efficiency, expression of neuronal markers and morphological characteristics of differentiated cells. Yet, the efficiency of available protocols has not been systematically compared. To address this question, we quantified the number and size of cell cluster formed during differentiation using published and modified protocols and analyzed the abundance of neuronal and non‐neuronal expression markers using immunocytochemistry. In the course of the experiments we observed that differentiation results strongly depend on the cell density at differentiation‐initiation as well as on the type of used cell culture plastic ware. Based on those observations and the results from our comparative analysis, we created our own optimized and robust protocol that reproducibly reveals differentiated cells with high yield. We conclude that our method may be superior to differentiation of NT2 cells for systematic in vitro‐based primary screening for developmental toxicants and neurotoxicants at different stages of maturation over previous protocols used. Our approach will also contribute to reduce animal testing in the context of the 3Rs.     

The preparation,in vitro screening and molecular docking of symmetrical bisquaternary cholinesterase inhibitors containing a but-(2E)-en-1,4-diyl connecting linkage

Carbamate inhibitors (e.g. pyridostigmine bromide) are used as a pre-treatment for the prevention of organophosphorus poisoning. They work by blocking the native function of acetylcholinesterases (AChE) and thus protect AChE against irreversible inhibition by organophosphorus compounds. However, carbamate inhibitors are known for their many undesirable side effects related to the carbamylation of AChE. In this paper, we describe 17 novel bisquaternary compounds and have analysed their effect on AChE inhibition. The newly prepared compounds were evaluated in vitro using both human erythrocyte AChE and human plasmatic butyrylcholinesterase. Their inhibitory ability was expressed as the half maximal inhibitory concentration (IC₅₀) and then compared to the standard carbamate drugs and two AChE reactivators. One of these novel compounds showed promising AChE inhibition in vitro (nM range) and was better than the currently used standards. Additionally, a kinetic assay confirmed the non-competitive inhibition of hAChE by this novel compound. Consequently, the docking results confirmed the apparent π-π or π-cationic interactions with the key amino acid residues of hAChE and the binding of the chosen compound at the enzyme catalytic site.     

Pollen morphology of Astragalus section Hymenostegis (Fabaceae) and evaluation of its systematic implications

Astragalus is with nearly 3000 described species the largest genus of flowering plants. So far analyses of pollen characters have only been conducted for a few species of the groups within the genus. Here we analyse pollen grains of 22 species representative for Astragalus section Hymenostegis using scanning electron microscopy. We found the basic shape of the pollen grains to be oblate-spheroidal and apertures to be tricolpate as for other eudicots. The sculpturing pattern of the exine is micro-reticulate. Pollen grains show low morphological variation among different species of this section, but differences occur between sections of the genus. We conclude that the vast morphological differentiation that occurred during the rapid radiation of section Hymenostegis was not accompanied by comparable differentiation in pollen morphology.     

Glutamate receptor properties of human mesencephalic neural progenitor cells: NMDA enhances dopaminergic neurogenesis in vitro

Human midbrain‐derived neural progenitor cells (NPCs) may serve as a continuous source of dopaminergic neurons for the development of novel regenerative therapies in Parkinson’s disease. However, the molecular and functional characteristics of glutamate receptors in human NPCs are largely unknown. Here, we show that differentiated human mesencepahlic NPCs display a distinct pattern of glutamate receptors. In whole‐cell patch‐clamp recordings, l ‐glutamate and NMDA elicited currents in 93% of NPCs after 3 weeks of differentiation in vitro. The concentration‐response plots of differentiated NPCs yielded an EC₅₀ of 2.2 μM for glutamate and an EC₅₀ of 36 μM for NMDA. Glutamate‐induced currents were markedly inhibited by memantine in contrast to 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione (CNQX) suggesting a higher density of functional NMDA than alpha‐amino‐3‐hydroxy‐5‐methylisoxazole‐4‐propionate (AMPA)/kainate receptors. NMDA‐evoked currents and calcium signals were blocked by the NR2B‐subunit specific antagonist ifenprodil indicating functional expression of NMDA receptors containing subunits NR1 and NR2B. In calcium imaging experiments, the blockade of voltage‐gated calcium channels by verapamil abolished AMPA‐induced calcium responses but only partially reduced NMDA‐evoked transients suggesting the expression of calcium‐impermeable, GluR2‐containing AMPA receptors. Quantitative real‐time PCR showed a predominant expression of subunits NR2A and NR2B (NMDA), GluR2 (AMPA), GluR7 (kainate), and mGluR3 (metabotropic glutamate receptor). Treatment of NPCs with 100 μM NMDA in vitro during proliferation (2 weeks) and differentiation (1 week) increased the amount of tyrosine hydroxylase‐immunopositive cells significantly, which was reversed by addition of memantine. These data suggest that NMDA receptors in differentiating human mesencephalic NPCs are important regulators of dopaminergic neurogenesis in vitro.     

Ultrastructural differentiation during drosophila neurogenesis in vitro

Drosophila melanogaster neuroblasts differentiate in vitro, and each gives rise to a cluster of about 18 daughter neurons. Electron microscopic observations of single clusters show that axons from daughter neurons form a neuropile within the cluster of cell bodies. The neuropile increases in size and complexity for several hours, during which time chemical, and probably electrotonic, synapses form between neurites. Clear vesicles with diameters of about 35 nm and dense core vesicles with diameters of about 60 and 160 nm were detected. The development of the neuropile indicates that the prerequisite cell recognition phenomena were manifested during differentiation in vitro, and the complexity of the neuropile suggests it may have attained the capacity to process information.     

Morphological and genetic variation of Fucus in the eastern Gulf of Bothnia,northern Baltic Sea

Fucus vesiculosus and F. radicans (Phaeophyceae) are important habitat-formers on rocky shores in the Bothnian Sea. While both species occur sympatrically along the entire western Bothnian Sea coast, F. radicans has been found only in the northern part of the eastern coast. According to previous studies, the two species can be distinguished based on morphology, F. radicans having narrower thalli and a bushier appearance. However, marine mapping in the eastern Bothnian Sea has revealed that high morphological variation in Fucus, partly caused by gradients in salinity and exposure, makes differentiation between the two species difficult. We studied morphological and genetic variation to find out whether the two Fucus species can be differentiated in the south-eastern Bothnian Sea, and if F. radicans occurs in the area. The study was carried out in six subareas including 350 km of coast, with a salinity gradient of 3.5–6.5 PSU, and varying wave exposure. We found a gradual change towards smaller and narrower thalli and a higher number of holdfasts in Fucus populations when moving northwards to lower salinities. Distinct Fucus morphs were often found within the study sites but the morphs were genetically differentiated only at one study site in the Skaftung subarea, suggesting the occurrence of both species. However, in the Vasa subarea the sample size for analysing genetic differentiation was low due to high clonality. In the Luvia subarea south of Skaftung, Fucus morphology corresponded to that of F. radicans in earlier studies but the population was genetically more similar to F. vesiculosus in the southern subareas. We conclude that by using only morphological characteristics it is not possible to differentiate between the two species in central and northern parts of the eastern Bothnian Sea. Based on genetic analyses, the southernmost known occurrence of F. radicans in the eastern Bothnian Sea is in Skaftung.     

Clonal analysis of vertebrate myogenesis. VI. Acetylcholinesterase and acetylcholine receptor in myogenic and nonmyogenic clones from chick embryo leg cells.

Myogenic clones grown in vitro from cells of 4-, 6-, and 12-day chick embryo leg buds demonstrate reproducible stage-specific characteristics of morphology, extent of myotube formation, and culture medium requirements for differentiation, suggesting heterogeneity in the myogenic cell populations of the developing limb. To determine whether there is heterogeneity in the cytodifferentiation of different muscle colony types, clones have been examined for the appearance of two muscle-specific gene products—acetylcholinesterase (AChE) and acetylcholine receptor (AChR). AChE (detected by cytochemical reaction) and AChR (detected by autoradiography of [¹²⁵I]α-bungarotoxin binding) appeared in myotubes of all muscle colony types, and also appeared in about 5% of the mononucleated cells of all muscle colonies; but neither were detectable in cells of nonfused clones (colonies containing no myotubes). The results suggest that all muscle colony-forming cell types have equivalent capacities to elaborate muscle-specific gene products once the process of differentiation is initiated. However, when putative muscle colony-forming cells are grown under certain conditions that do not permit cell fusion (e.g., conditioned medium-requiring clones grown in fresh medium), mononucleated cells do not accumulate AChE or AChR. Conditioned medium-dependent differentiation thus differs from the fusion-specific processes affected by Ca²⁺ deprivation and phospholipase C treatment, since in these cases mononucleated cells exhibit differentiated functions. The apparent cytodifferentiation (without fusion) of some mononucleated cells within muscle colonies in which most mononucleated cells continue to proliferate raises questions concerning the control of myoblast differentiation and its relationship to the cell cycle and to fusion.     

Guidance of regenerating motor axons in vivo by gradients of diffusible peripheral nerve‐derived factors

During development of the central nervous system, neurons rely on target‐derived factors to guide their outgrowing processes. Several CNS target‐derived chemoattractive and repellant factors have been isolated and characterized, and their mechanism of action determined. For the peripheral nervous system, the results from numerous experiments suggest that during regeneration axons also respond to concentration gradients of target‐derived factors leading to an oriented outgrowth up the gradient to the denervated target in vivo. The results from in vitro experiments have shown that diffusible concentration gradients of factors released from a length of denervated peripheral nerve, composed predominantly of Schwann cells, direct the outgrowth of sensory and motor neuron growth cones over distances of several hundred microns. However, a conclusive demonstration of a chemoattractive influence of diffusible concentration gradients on regenerating adult motor axons in vivo has remained elusive. The present experiments show that concentration gradients of denervated peripheral nerve‐released factors direct the regeneration of adult motor axons in vivo, and that these gradients are effective over distances of more than 6.5 mm. Nonconditioned medium exerted no influence on the regenerating axons. Thus, results from in vivo experiments parallel those from in vitro experiments and indicate that isolated peripheral nerve‐released factors that are effective in vitro will play a similar role on sensory and motor axons in vivo. Finally, the results show that diffusible concentration gradients of target‐derived factors direct axon outgrowth both during both development and regeneration, as well as in vivo and in vitro. © 2000 John Wiley & Sons, Inc. J Neurobiol 42: 212–219, 2000     

mTOR kinase is needed for the development and stabilization of dendritic arbors in newly born olfactory bulb neurons

Neurogenesis is the process of neuron generation, which occurs not only during embryonic development but also in restricted niches postnatally. One such region is called the subventricular zone (SVZ), which gives rise to new neurons in the olfactory bulb (OB). Neurons that are born postnatally migrate through more complex territories and integrate into fully functional circuits. Therefore, differences in the differentiation of embryonic and postnatally born neurons may exist. Dendritogenesis is an important process for the proper formation of future neuronal circuits. Dendritogenesis in embryonic neurons cultured in vitro was shown to depend on the mammalian target of rapamycin (mTOR). Still unknown, however, is whether mTOR could regulate the dendritic arbor morphology of SVZ‐derived postnatal OB neurons under physiological conditions in vivo. The present study used in vitro cultured and differentiated SVZ‐derived neural progenitors and found that both mTOR complex 1 and mTOR complex 2 were required for the dendritogenesis of SVZ‐derived neurons. Furthermore, using a combination of in vivo electroporation of neural stem cells in the SVZ and genetic and pharmacological inhibition of mTOR, it was found that mTOR was crucial for the growth of basal and apical dendrites in postnatally born OB neurons under physiological conditions and contributed to the stabilization of their basal dendrites. © 2016 Wiley Periodicals, Inc. Develop Neurobiol 76: 1308–1327, 2016     

Fibrillar β‐amyloid 1‐42 alters cytokine secretion,cholinergic signalling and neuronal differentiation

Adult neurogenesis is impaired by inflammatory processes, which are linked to altered cholinergic signalling and cognitive decline in Alzheimer's disease. In this study, we investigated how amyloid beta (Aβ)‐evoked inflammatory responses affect the generation of new neurons from human embryonic stem (hES) cells and the role of cholinergic signalling in regulating this process. The hES were cultured as neurospheres and exposed to fibrillar and oligomeric Aβ_1‐42 (Aβf, AβO) or to conditioned medium from human primary microglia activated with either Aβ_1‐42 or lipopolysaccharide. The neurospheres were differentiated for 29 days in vitro and the resulting neuronal or glial phenotypes were thereafter assessed. Secretion of cytokines and the enzymes acetylcholinesterase (AChE), butyrylcholinesterase (BuChE) and choline acetyltransferase (ChAT) involved in cholinergic signalling was measured in medium throughout the differentiation. We report that differentiating neurospheres released various cytokines, and exposure to Aβf, but not AβO, increased the secretion of IL‐6, IL‐1β and IL‐2. Aβf also influenced the levels of AChE, BuChE and ChAT in favour of a low level of acetylcholine. These changes were linked to an altered secretion pattern of cytokines. A different pattern was observed in microglia activated by Aβf, demonstrating decreased secretion of TNF‐α, IL‐1β and IL‐2 relative to untreated cells. Subsequent exposure of differentiating neurospheres to Aβf or to microglia‐conditioned medium decreased neuronal differentiation and increased glial differentiation. We suggest that a basal physiological secretion of cytokines is involved in shaping the differentiation of neurospheres and that Aβf decreases neurogenesis by promoting a microenvironment favouring hypo‐cholinergic signalling and gliogenesis.     

ADAPTIVE RADIATION AND GENETIC DIFFERENTIATION IN HAWAIIAN BIDENS

The genus Bidens (Asteraceae) has undergone extensive adaptive radiation on the Hawaiian Islands. The 19 species and eight subspecies endemic to Hawaii exhibit much more morphological and ecological differentiation than the continental members of the genus. However, the Hawaiian taxa have the same chromosome number and retain the capacity to interbreed in all possible combinations. Twenty-two populations of 15 Hawaiian taxa and four populations of American taxa were compared at 21 loci controlling eight enzyme systems. Populations of Hawaiian taxa are highly polymorphic. However, little genetic differentiation has occurred among taxa in spite of the high levels of genetic variability. Genetic identities calculated for pairs of populations show that populations of the same taxon are genetically more similar than are populations belonging to different taxa, but all values are high. The level of genetic differentiation that has occurred among the species of Hawaiian Bidens is comparable to the level of genetic differences found among populations within single continental plant species. Moreover, there is no correlation between the isozyme data and morphological data. No groups of taxa are evident in the genetic data, although morphological groups exist. Genetic differentiation at isozyme loci has not occurred at the same rate as the acquisition of presumably adaptive morphological and ecological characters in Hawaiian Bidens. Adaptive radiation may be limited to a few genes controlling morphological and ecological characters.     

Host environment and local genetic adaptation determine phenotype in parasitic Rhinanthus angustifolius

Plants have a remarkable capacity to adapt to local environmental conditions, which can result in ecotypic differentiation. Patterns of differentiation can, however, also be influenced by the extensive phenotypic plasticity exhibited by many plant species. In this study, we evaluated the distinctness of two putative ecotypes of the parasitic herb Rhinanthus angustifolius. We compared population means of characters commonly used to distinguish between the putative ecotypes after growing individuals of R. angustifolius with a variety of host species in a common garden. Resulting data were also pooled over environments to study how phenotypic plasticity affects the distinctness of ecotypes and individual populations. Except for node number, most of the characters were plastic. The pattern of differentiation was consistent with the existence of two, or possibly three, habitat‐related groups of populations; however, we observed considerable overlap in flowering time and morphological characters after pooling data across host environments. The results show that the complex phenological and morphological variation in R. angustifolius is caused by a combination of genetically determined ecotypic differentiation and plastic responses to the host environment and other factors. © 2015 The Linnean Society of London, Botanical Journal of the Linnean Society, 2016, 180 , 89–103.