Extreme genetic depauperation and differentiation of both populations and species in Eurasian feather grasses (Stipa)

A highly selfing breeding system affects gene flow, which may have consequences for patterns of genetic variation and differentiation on both the population and species level. Feather grasses (Stipa spp.) are dominant elements of Eurasian steppes that persist in Central Europe in scattered isolated populations that are of great conservation interest. Cleistogamy is common in the Stipa pennata group, the phylogeny of which is largely unresolved. Intraspecific patterns of genetic variation can be characterised by lack of gene flow due to selfing, but also by large-scale historical migrations and long-term isolation. We analysed both 5 species within the S. pennata group and 33 populations of Stipa pulcherrima sampled across a large part of its range. Using AFLP markers we assessed phylogenetic relationships of the S. pennata group and patterns of genetic variation within and among populations. The S. pennata group formed a consistent clade separated from S. capillata. Stipa pulcherrima was sister to S. eriocaulis, but the relationships among S. pennata s. str., S. borysthenica., and S. tirsa remained unresolved. Within-population genetic variation was extremely low in all species of the S. pennata group (H _e = 0.0–0.013). In S. pulcherrima, genetic variation was consistently relatively high in the east (Romania, Russia) and declined toward western populations, with many populations at the western range edge lacking genetic variation entirely. Populations were strongly differentiated (F _ST = 0.762), and this differentiation did not follow a classical pattern of isolation by distance. Bayesian cluster analysis revealed nine gene pools in S. pulcherrima, which were mostly geographically clustered. Overall the results suggest that S. pulcherrima and species of the S. pennata group are characterised by a cleistogamous breeding system leading to extremely low levels of genetic variation and high levels of population differentiation at both the population and species level. Postglacial colonisation, current population isolation, and population bottlenecks at the western range periphery have further reduced genetic variation and obviated gene exchange. Thus, genetic variation can only be preserved by the conservation of multiple populations.     

Systematic parameter optimization of a Me(2)SO- and serum-free cryopreservation protocol for human mesenchymal stem cells

Human mesenchymal stem cells (hMSCs) have great potential for clinical therapy and regenerative medicine. One major challenge concerning their application is the development of an efficient cryopreservation protocol since current methods result in a poor viability and high differentiation rates. A high survival rate of cryopreserved cells requires an optimal cooling rate and the presence of cryoprotective agents (CPA) in sufficient concentrations. The most widely used CPA, dimethylsulfoxide (Me₂SO), is toxic at high concentrations at temperatures >4 °C and has harmful effects on the biological functionality of stem cell as well as on treated patients.Thus, this study investigates different combinations of non-cytotoxic biocompatible substances, such as ectoin and proline, as potential CPAs in a systematic parametric optimization study in comparison to Me₂SO as control and a commercial freezing medium (Biofreeze®, Biochrom). Using a freezing medium containing a low proline (1%, w/v) and higher ectoin (10%, w/v) amount revealed promising results although the highest survival rate was achieved with the Biofreeze® medium. Cryomicroscopic experiments of hMSCs revealed nucleation temperatures ranging from −16 to −25 °C. The CPAs, beside Me₂SO, did not affect the nucleation temperature. In most cases, cryomicroscopy revealed intracellular ice formation (IIF) during the cryopreservation cycle for all cryoprotocols. The occurence of IIF during thawing increased with the cooling rate. In case of hMSC there was no correlation between the rate of IIF and the post-thaw cell survival. After thawing adipogenic differentiation of the stem cells demonstrated cell functionality.     

The Rediscovery of a Long Described Species Reveals Additional Complexity in Speciation Patterns of Poeciliid Fishes in Sulfide Springs

The process of ecological speciation drives the evolution of locally adapted and reproductively isolated populations in response to divergent natural selection. In Southern Mexico, several lineages of the freshwater fish species of the genus Poecilia have independently colonized toxic, hydrogen sulfide-rich springs. Even though ecological speciation processes are increasingly well understood in this system, aligning the taxonomy of these fish with evolutionary processes has lagged behind. While some sulfide spring populations are classified as ecotypes of Poecilia mexicana, others, like P. sulphuraria, have been described as highly endemic species. Our study particularly focused on elucidating the taxonomy of the long described sulfide spring endemic, Poecilia thermalis Steindachner 1863, and investigates if similar evolutionary patterns of phenotypic trait divergence and reproductive isolation are present as observed in other sulfidic species of Poecilia. We applied a geometric morphometric approach to assess body shape similarity to other sulfidic and non-sulfidic fish of the genus Poecilia. We also conducted phylogenetic and population genetic analyses to establish the phylogenetic relationships of P. thermalis and used a population genetic approach to determine levels of gene flow among Poecilia from sulfidic and non-sulfidic sites. Our results indicate that P. thermalis'' body shape has evolved in convergence with other sulfide spring populations in the genus. Phylogenetic analyses placed P. thermalis as most closely related to one population of P. sulphuraria, and population genetic analyses demonstrated that P. thermalis is genetically isolated from both P. mexicana ecotypes and P. sulphuraria. Based on these findings, we make taxonomic recommendations for P. thermalis. Overall, our study verifies the role of hydrogen sulfide as a main factor shaping convergent, phenotypic evolution and the emergence of reproductive isolation between Poecilia populations residing in adjacent sulfidic and non-sulfidic environments.     

A cost of being large: genetically large yellow dung flies lose out in intra-specific food competition

Most life history traits are positively influenced by body size, while disadvantages of large size are poorly documented. To investigate presumed intrinsic costs of large body size in yellow dung flies (Scathophaga stercoraria; Diptera: Scathophagidae), we allowed larvae from replicate lines artificially selected for small and large body size for 21 generations to compete directly with each other at 20°C (benign) and 25°C (stressful) and low and high food (dung) availability. Greater mortality of large line flies was evident at low food independent of temperature, suggesting a cost of fast growth and/or long development for genetically large flies during larval scramble competition under food limitation. Our results are congruent with a previous study assessing mortality when competing within body size lines, so no additional mechanisms affecting scramble or contest behavior of larvae need be invoked to explain the results obtained beyond the costs of longer development and faster growth. Thus, artificial selection producing larger yellow dung flies than occur in nature revealed some, albeit weak mortality costs of large body size that otherwise might have remained cryptic. We conclude, however, that these costs are insufficient to explain the evolutionary limits of large body size in this species given persistently strong fecundity and sexual selection favoring large size in both sexes.     

Air–liquid interface cultures enhance the oxygen supply and trigger the structural and functional differentiation of intestinal porcine epithelial cells (IPEC)

The specific function of the epithelium as critical barrier between the intestinal lumen and the organism’s internal microenvironment is reflected by permanent maintenance of intercellular junctions and cellular polarity. The intestinal epithelial cells are responsible for absorption of nutritional components, facing mechanical stress and a changing oxygen supplementation via blood stream. Oxygen itself can regulate the barrier and the absorptive function of the epithelium. Therefore, we compared the dish cell culture, the transwell-like membrane culture and the oxygen enriched air–liquid interface (ALI) culture. We demonstrated strong influence of the different culture conditions on morphology and function of intestinal porcine epithelial cell lines in vitro. ALI culture resulted in a significant increase in cell number, epithelial cell layer thickness and expression as well as apical localisation of the microvilli-associated protein villin. Remarkable similarities regarding the morphological parameters were observed between ALI cultures and intestinal epithelial cells in vivo. Furthermore, the functional analysis of protein uptake and degradation by the epithelial cells demonstrated the necessity of sufficient oxygen supply as achieved in ALI cultures. Our study is the first report providing marked evidence that optimised oxygen supply using ALI cultures directly affects the morphological differentiation and functional properties of intestinal epithelial cells in vitro.     

The effects of polymorphisms in genes from the renin–angiotensin, bradykinin, and fibrinolytic systems on plasma t-PA and PAI-1 levels are dependent on environmental context

Thrombosis is a key factor in the pathophysiology of cardiovascular disease. Important biochemical constituents of the fibrinolytic system, affecting thrombosis, include tissue-type plasminogen activator (t-PA) and plasminogen activator inhibitor-1 (PAI-1). Both t-PA and PAI-1 are determined by multiple genetic and environmental factors. We aimed to investigate whether the effects of polymorphism in genes from the renin–angiotensin, bradykinin, and fibrinolytic systems on t-PA or PAI-1 levels are dependent on environmental factors in a large population-based sample from the PREVEND study in Groningen, The Netherlands (n = 2,527). We found strong evidence (P ≤ 0.02) for interaction effects of polymorphisms from the bradykinin receptor (BDKRB2) gene and alcohol consumption on t-PA in females and males and on PAI-1 in males. Only suggestive evidence (P ≤ 0.10) was present for an interaction effect of the BDKRB2 gene and alcohol consumption on PAI-1 levels in females. Another consistent finding was evidence for an interaction between bradykinin receptor (BDKRB2) gene polymorphisms and body size as measured by body mass index and/or waist–hip-ratio. For each gender and for both t-PA and PAI-1 there was at least one BDKRB2–body size combination that exhibited suggestive (P ≤ 0.10), significant (P ≤ 0.04) and/or strong evidence (P ≤ 0.02) for interaction. In conclusion, the genetic architecture of t-PA and PAI-1 is dependent on the environmental context such as body size and alcohol use. The present study emphasizes the importance of including environmental factors in genetic analyses to fully comprehend the genetic architecture of a specific trait. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.