Integrative taxonomy and conservation status of amphibians in western Panama with an emphasis on the highlands of the Cordillera Central
- Amphibians have existed on the planet for over 300 million years and are today one of the most diverse vertebrate classes in the world with over 7000 known species and still many more to be discovered. However, several studies assume that approximately one third of the world´s known living amphibians are directly threatened with extinction, making it the most endangered vertebrate class. In relation to the relatively small land mass that is occupied by the state of Panama, it supports one of the most diverse amphibian faunas. However, in many cases the ecological role of single species in a wider context and their habitat preferences are still poorly understood and subject to ongoing research. Modern taxonomic approaches in other tropical regions have shown that former assumptions of amphibian diversity were distinct underestimations of the actual species diversity; a situation that is probably also true for Panama. Concurrently, the collection of amphibian diversity data and the description of new species is a race against time. The amphibian fauna of the world and that of Panama in particular, has suffered from an unprecedented loss of diversity over the last 30 years. The reasons are manifold and include destruction, alteration, and fragmentation of their natural habitats as the main causes, but also the deadly amphibian disease chytridiomycosis caused by the fungal pathogen Batrachochytrium dendrobatidis (Bd). In Panama and Costa Rica, this Emerging Infectious Disease (EID) spread in a wave-like manner from west to east causing mass die-offs and reduced amphibian diversity even in well-preserved habitats. The disease has primarily affected stream-associated highland species. The last large-scale evaluation of the conservation status of Panama´s amphibians through the IUCN Red List of Threatened Species in 2004 concluded that approximately 30% of the known species are acutely threatened with extinction. Furthermore, around 17% of the amphibian species that have been known back then lacked adequate data to be assessed. In view of Panama´s already overwhelming amphibian diversity, as well as the variety of habitats and the large number of sites that have not been examined with regard to amphibians before, I started this study with the conviction that the inventory of Panama´s amphibian diversity is far from being completed. Furthermore, when I started this study, it was uncertain if there would be any surviving amphibian species in areas where chytridiomycosis had emerged. The loss of whole amphibian communities in upland western Panama following Bd arrival led to a shift of amphibian research to lowland sites in central and eastern Panama aiming primarily on pathogen arrival and the documentation of epizootic outbreak and subsequent population decline. The situation of amphibian communities in areas post-decline was therefore largely unknown. Accordingly, the main goals of my study were to add to the taxonomic inventory of amphibians in Panama and to assess the situation of amphibian populations in habitats where chytrid-driven declines have been observed. To address these tasks I conducted fieldwork in western Panama with a focus on mountainous elevations between 1000 and 3475 m asl. Additionally, I visited different lowland sites between sea level and 1000 m asl to collect comparative material. In the period between 2008 and 2013, I conducted five collection trips to Panama that add up to a total of approximately 13 months in the field. I have sampled nine regions in western Panama and collected 767 specimens together with student collaborators, 531 of which were collected under my personal field number. Additional data obtained from those specimens include 68 male anuran call recordings, 102 standardized color descriptions of specimens in life, and 259 tissue samples that to date yielded 185 16S mtDNA sequences. This comprises the most comprehensive data set for amphibians of Panama and the first large-scale DNA barcoding approach for western Panama to date. After a preliminary DNA barcoding and subsequent comparative examination of morphological und bioacoustic data of all specimens collected, the number of taxonomic problems that needed to be addressed was higher than I previously anticipated. For most genetic lineages deeper taxonomic analyses were required to reach conclusive results. A selection had to be made with which lineages to proceed in the analyses, in view of the substantial financial and time expenditure that would be needed for a complete taxonomic revision. Therefore, I chose to run deeper analyses on one genus from each of the three amphibian orders in Panama. The genera selection depended largely on the availability of sufficient material and the scientific relevance of the respective genus.
I selected the genus Diasporus from the order Anura. These small frogs are omnipresent in many habitats and thus relatively easy to find. In addition, the genus is underrepresented in taxonomic studies. This is the first taxonomic study on the genus Diasporus to include a molecular phylogeny and the first comparison of advertisement calls between several populations from western Panama. In total, I collected 67 Diasporus specimens throughout western Panama and compared them morphologically with 49 additional specimens from Central America in collections, including the primary types of D. diasporus and D. hylaeformis. Additional comparative data were taken from literature. The DNA barcoding analysis of a fragment of the 16S rRNA gene included 43 own sequences that were complemented with 15 relevant GenBank sequences. In addition, I compared the advertisement calls of 26 male individuals among each other and with call descriptions from the literature. The DNA barcoding approach revealed several unnamed genetic lineages, but in some cases also resulted in the lumping of morphologically and bioacoustically distinct specimens. Generally, the morphological examination of the collected material revealed almost no specific characters that could be used to distinguish between genetic lineages. However, it was possible to identify species using a combination of several morphological characteristics. Which ones are relevant in the individual case depends on the respective species. My extensive collection of call recordings made it possible to test for the first time the intraspecific call variation of D. hylaeformis in dependency of various parameters. This analysis showed that the dominant frequency depends significantly on the body size of the calling male; the smaller the calling male, the higher the frequency of the call. A similar relationship was observed between the call rate and temperature: the lower the temperature during calling, the lower the call rate. I suppose that these general patterns, which have already been observed in other anuran genera, are also true in other Diasporus species that could not be tested in this study. Taking into account the intraspecific variation of Diasporus advertisement calls, I consider comparative call analyses to be the best way to distinguish between species. This is especially true in syntopic species. Integration of the three lines of evidence (i.e., morphology, DNA barcoding, and bioacoustics) led to the identification of four new species, two of which (i.e., D. citrinobapheus and D. igneus) colleagues and I have already formally described.
I conducted an integrative taxonomic analysis of the western Panamanian representatives of the genus Bolitoglossa from the order Caudata, the larger of the two Panamanian salamander genera. Bolitoglossa is very species-rich with a centre of diversification in the high mountains of Costa Rica and western Panama. I collected 53 Bolitoglossa specimens and compared them to twelve specimens in collection, including the holotype and one paratype of B. gomezi. The dataset was complemented with information from the literature. Among the sampled specimens were two species considered to be endangered that have not been collected or observed for several decades; B. magnifica has not been seen for 34 years and B. anthracina has not been seen for 22 years. Further, I collected salamanders at several new locations. To date, my 16S mtDNA barcoding analysis represents the densest taxon sampling for Panamanian Bolitoglossa composed of 21 own sequences that were combined in the final alignment with 47 GenBank sequences. Even though the molecular phylogeny is based only on a single marker, the received trees largely coincide with previous studies and the nodes received high statistical support. In these trees, I retrieve all previously defined subgenera and species groups. On the basis of this molecular phylogeny, I placed B. anthracina, here sequenced for the first time, in the B. subpalmata species group. Due to the fact that B. anthracina is a large and dark colored species it had previously been placed by implication in the B. schizodactyla species group along with other large black salamanders of the B. nigrescens species complex. Moreover, I found deep divergent genetic lineages among geographically separated populations of B. minutula. However, until now there were no additional morphological characteristics detectable to distinguish between these lineages. Additionally, my colleagues and I described a new deep divergent lineage in the B. robinsoni species group as B. jugivagans, a species new to science. In contrast, I found only minor genetic differences between specimens of B. sombra and B. nigrescens. After combining morphometric data and tooth counts from literature of both species with additional data from specimens of B. sombra that I collected near the type locality, the distinguishing features blurred. In particular, including much larger specimens of B. sombra, not yet known at the time of its description, showed that the tooth count difference is dependent on the size and age of the specimen examined. Larger specimens have more maxillary and vomerine teeth. Based on this evidence I regard B. sombra as a junior synonym of B. nigrescens. Further, I revised the Panamanian distribution of the two relatively common lowland salamanders, B. colonnea and B. lignicolor. Besides filling the gaps in the fragmentary known distributions of these species, I assessed the molecular and morphological variation of both species among populations in Panama. While there was little variation in B. lignicolor, I found divergent genetic lineages among geographically distinct populations of B. colonnea that require further taxonomic examination.
Caecilians (order Gymnophiona) are among the least investigated terrestrial vertebrates. After I received a first specimen of the predominantly South American genus Oscaecilia (family Caeciliidae) in western Panama, I started to work more extensively on the taxonomy of Caeciliidae in Central America. The specimens from western Panama were not readily assignable to a single described species, but shared characters with O. elongata and O. osae. While O. osae was only known from the holotype, the type material of O. elongata was destroyed during World War II. On the basis of the original description, the unique feature in O. elongata within Oscaecilia is the absence of subdermal scales in the posterior part of the body. In a referred specimen of O. elongata mentioned in the original description from eastern Panama, this characteristic cannot be examined as it consists of head and neck only. Therefore, I used non-destructive high-resolution, synchrotron-based X-ray micro CT imaging (HRμCT) to examine cranial characters in the specimens in question and took normal radiographs to count vertebrae and to make subdermal scales visible. I found that the fragmented specimen from eastern Panama likely belongs to the well-sampled species O. ochrocephala and has not much in common with O. osae or the specimens from western Panama. Contrarily, O. osae and the specimens from western Panama share many morphological characters, but also show some differences. Genetic barcoding revealed that both species are close relatives, but the genetic distance could not be finally resolved, because 16S sequences obtained from blood samples of living O. osae were of poor quality. Thus, I compare the Oscaecilia from western Panama to O. osae in this study, but postpone a taxonomic decision until further material becomes available. Further, I designate O. elongata a nomen dubium, because the type material is lost, the type locality is not defined in more detail than “Panama”, and the original description does not allow for a definite assignment. Since previous molecular studies only considered O. ochrocephala, the monophyly of Oscaecilia was never tested before. So far, the genus Oscaecilia is based largely on a single cranial character, the eyes covered with bone. Here, I combined two 16S mtDNA sequences of O. osae from Costa Rica and two sequences from O. sp. from western Panama with two sequences of O. ochrocephala and ten sequences of four species of the genus Caecilia, the sister genus of Oscaecilia. The resulted phylogeny contains two well-supported clades, one clade containing two species of Caecilia, one from Panama and one from western Ecuador and all species of Oscaecilia tested. The other clade consists of two species of Caecilia from the Amazon basin. I therefore assume that the split in both clades is due to the rise of the Andes, what led to today’s cis-trans-Andean distribution of the two clades. For now, to restore monophyly, I suggest to place Oscaecilia within the synonymy of Caecilia until more taxa have been tested. When assessing the conservation status of the amphibian species in mountainous western Panama, I first compiled a list of known species that I potentially could have found during my fieldwork. Using the IUCN categories, I analyzed how many of the endangered species I actually found and how these are distributed over families and species groups. Surprisingly, my rediscoveries of lost species were not equally distributed among the four families that comprise most endangered amphibian species (i.e., Bufonidae, Craugastoridae, Hylidae, and Plethodontidae). While I discovered ten of eleven endangered hylids and six of nine endangered plethodontids, I found only one of four endangered bufonids and none of the nine endangered craugastorids. I assume that the secretive living plethodontids, for which no Bd related declines have been documented, were just overlooked in the past decades. In contrast, I propose that hylids, in which Bd related population decline is well documented, developed distinct evolutionary solutions permitting coexistence with the pathogen. The situation is obviously different in bufonids and craugastorids, where I found no signs of population recoveries at present. So far, the only surviving populations of species from these families exist in climatic or physiographic niches that have probably shielded them from Bd. My data confirm the current view that the risk for naïve amphibian populations to decline during Bd epizootics is predicted by ecological traits (e.g., aquatic index, vertical distribution) and not dependent on taxonomic affiliation. However, I propose that only certain amphibian families (e.g., hylids and centrolenids) have the ability to acquire immunity solutions to coexist with the pathogen during enzootic stages. This is a very new perspective on the worst infectious disease in amphibians worldwide, allowing for new research approaches to understand the host-pathogen dynamics. Moreover, I examined where the share of surviving endangered amphibian species is particularly high in mountainous western Panama. As was to be expected, most of the endangered species are found within the boundaries of protected areas. One exception is the unprotected Cerro Colorado region in the Comarca Ngöbe-Buglé that provides habitat for a wide variety of endangered and undiscovered amphibian species. Nonetheless, planned open pit mining would destroy the forests in a large part of the area. This demonstrates once again that human activities are the biggest threat to amphibians in Panama and elsewhere.
Spatio-temporal evolution of Cedrela (Meliaceae) : climatic niche dynamics, phylogeography and taxonomy
Anna Valerie Köcke
- Understanding major causes of biodiversity and range dynamics requires research on evolutionary processes under consideration of environmental changes. In my thesis, I investigated the spatio-temporal evolution of the Neotropical tree genus Cedrela from the Meliaceae family by studying its genetic diversity, taxonomy, colonization history, climatic niche changes and dynamics of species distributions. My results show that climatic and geological changes are major drivers of biological diversification in Cedrela.
Long-term observation of arabidopsis thaliana root growth under close-to-natural conditions using light sheet-based fluorescence microscopy
Daniel Wangenheim von
- In the interest of understanding the development of a multicellular organism, subcellular events must be seen in the context of the entire three-dimensional tissue. In addition, events that occur within a short period of time can be of great importance for the relatively long developmental process of the organ. Thus, it is required to capture subcellular events in a larger spatio-temporal scale context, which has been up to now a technical challenge. In developmental biology, light microscopy has always been an important tool. The dilemma of light microscopy, in particular fluorescence microscopy, is that molecules receive high light intensities that might change the conformation of molecules, which can have signaling or toxic effects. In Light Sheet-based Fluorescence Microscopy (LSFM), the energy required for a single recording is reduced by several orders of magnitude compared to other fluorescence microscopy techniques. During the last ten years, LSFM has emerged as a preferred tool to capture all cells during embryogenesis of the zebrafish Danio rerio, the fruit fly Drosophila melanogaster or recently the red flour beetle Tribolium castaneum for a period of several days. The motivation of this work was to gain new insights in developmental related processes of plant organs. The aim of this work was to establish a protocol for imaging plant growth over a long period of time using LSFM and perform comprehensive analyses at the cellular level. Plants have to cope with a variety of environmental conditions, therefore the conditions inside the microscope chamber had to be brought under control. The sample preparation methods and the standardized conditions at a physiological level allowed the study of gravity response, day-night rhythms, organ shape development as well as the intracellular dynamic events of the cytoskeleton and endosomal compartments in an unprecedented manner. Several of these projects were successfully published in collaborations with Prof. Jozef Šamaj (Palacký University Olomouc, Czech Republic), Prof. Niko Geldner (University of Lausanne, Switzerland), Prof. Malcom Bennett (University of Nottingham, UK) and Dr. Jürgen Kleine-Vehn (University of Natural Resources and Life Sciences, Austria). The main part of my work focused on the formation of lateral roots in Arabidopsis thaliana and was conducted in close collaboration with Dr. Alexis Maizel (University of Heidelberg, Germany). Previously, most experiments that describe lateral root formation have been performed on a small number of cells and for short periods of time. Capturing the complete process of lateral roots is an ambitious goal, because first, the primordium of a lateral root is located deep inside the primary root and imaging quality is impaired due to scattering of the overlaying tissue. Second, the process takes about 48 h, i.e. the plant has to be kept healthy for the whole period. Third, the amount of excitation light required for the spatio-temporal might have phototoxic effects that lead to a stop of growth at least in conventional microscopic techniques. In Arabidopsis embryogenesis, the sequence of cell divisions is relatively invariant. However, whether lateral root organogenesis follows particular cell division patterns has been unknown. The complete process of lateral root formation was captured from the first cell division until after the emergence from the main root. Images of a nuclei marker and a plasmamembrane marker were recorded every 5 min for a time period of up to 64 h. The positions and cell divisions of all cells were tracked manually. In collaboration with Alexander Schmitz (Goethe University Frankfurt am Main, Germany) and Dr. Jens Fangerau (University of Heidelberg, Germany), comprehensive analyses of the data were performed. A lateral root forms from initially 8-15 founder cells, arranged in a patch of 5-8 parallel files. The occurrence of new cell layers by periclinal divisions, as well as the sequence of layer generation was conserved and resembles the sequence suggested by Malamy and Benfey in 1997. Besides this stereotyped occurrence of periclinal divisions, radial divisions were found to appear stochastically, following no particular pattern. A large variability was also found in the contribution of founder cells and cell files to the final lateral root. In summary, the results suggest that a stereotyped pattern of cell divisions at particular developmental stages and a dynamically adapted control of cell divisions exist in parallel. Both properties allow a controlled but flexible development of the organ according to variations in cell topology and mechanical properties of the surrounding tissue. This work shows that LSFM, the sample preparation methods and controlled environmental conditions allow to capture and analyse the development of plants over several days at high resolution in an unprecedented manner.
A revised infrageneric classification and synopsis of the Afro-Eurasian genus Moraea (Iridaceae: Irideae)
John C. Manning
- Molecular phylogenetic studies of Moraea Mill. and the inclusion of Barnardiella Goldblatt, Galaxia Thunb., Gynandriris Parl., Hexaglottis Vent., Homeria Vent. and Roggeveldia Goldblatt in the genus have rendered the existing infrageneric classification, dating from 1976, in need of substantial revision. In particular, subg. Moraea and subg. Vieusseuxia have been shown to be paraphyletic. We propose a new infrageneric classification, based, as far as current data permit, on phylogenetic principles. Monophyletic subgenera and sections are circumscribed based on molecular phylogenies alone or in combination with morphological considerations. We recognize 11 subgenera, 15 sections and three series, arranged as follows in phylogenetic sequence: Plumarieae; Visciramosae (with sect. Multifoliae and sect. Visciramosae); Moraea (with sect. Moraea and sect. Polyphyllae); Galaxia (with ser. Unguiculatae, ser. Eurystigma and ser. Galaxia); Monocephalae; Acaules; Polyanthes (with sect. Serpentinae, sect. Deserticola, sect. Hexaglottis, sect. Gynandriris, sect. Polyanthes and sect. Pseudospicatae); Grandifl orae; Vieusseuxia (with sect. Integres, sect. Vieusseuxia and sect. Villosae); and Homeria (with sect. Stipanthera, sect. Flexuosae, sect. Homeria and sect. Conantherae). Most are moderately to well circumscribed at the morphological level either by floral or vegetative characters, except subg. Moraea, which includes a small number of unspecialized species apparently not linked by any apomorphic features. With over 27 new species described in the past 25 years and another 60 transferred to the genus, Moraea now includes 214 species. We provide a full taxonomic synopsis of the genus.
Physiology and mechanics of insect hearing organs
- Tympanal hearing organs of insects emit distortion-product otoacoustic emissions (DPOAEs) which are indicative of nonlinear mechanical sound processing. General characteristics of insect DPOAEs are comparable to those measured in vertebrates, despite distinct differences in ear anatomy. DPOAEs appear during simultaneous stimulation with two pure tones (f1<f2) as additional spectral peaks at frequencies of nf1-(n-1)f2 and nf2-(n-1)f1, with the 2f1-f2 emission being the most prominent one. Insect DPOAEs are highly vulnerable to manipulations that interfere with the animal's physiological state and disappear after death. First evidence from locusts suggested that scolopidial mechanoreceptors might play a role in frequency-specific DPOAE generation (Möckel et al. 2007). The overall aim of this thesis was to determine the source of sensitive, nonlinear hearing at high frequencies and of DPOAE generation in tympanal organs of insects.
The first project of the present thesis involved general characteristics of DPOAE generation in the bushcricket Mecopoda elongata and the selective exclusion of the scolopidial mechanoreceptors using the neuroactive insectizide pymetrozine (Möckel et al. 2011). Pymetrozin appears to act highly effective and selectively on chordotonal organs, without affecting other sensory organs that lack scolopidial receptors. Pymetrozine solutions were applied as closely as possible to the scolopidia via a cuticle opening in the tibia, distally to the organ. Applications at concentrations between 10-3 and 10-7 M led to a pronounced and irreversible decrease of DPOAE amplitudes. Both this study on bushcrickets (Möckel et al. 2011) and an earlier one on locusts (Möckel et al. 2007) hence indicate the involvement of scolopidia in DPOAE generation in insects, by using complementary methods (pharmacological versus mechanical manipulation) and different animal models.
The second project of the present thesis investigated the temperature-dependence of DPOAEs in the locust Locusta migratoria (Möckel et al. 2012). The suggested biological origin of acoustic two-tone distortions in insects should involve metabolic processes, whose temperature-dependence would directly affect the DPOAE generation. Body temperature shifts resulted in reversible, level- and frequency-dependent effects on the 2f1–f2 emission. Using low f2 frequencies of up to 10 kHz, a body temperature increase (median +8–9°C) led to an upward shift of DPOAE amplitudes of approximately +10 dB, whereas a temperature decrease (median –7°C) was followed by a reduction of DPOAE amplitudes by 3 to 5 dB. Both effects were only present in the range of the low-level component of DPOAE growth functions below f2 stimulus levels of approximately 30-40 dB SPL. Emissions induced by higher stimulus levels and frequencies (e.g. 12 and 18 kHz) remained unaffected by any temperature shifts. The Arrhenius activation energy of the underlying cellular component amounted to 34 and 41 kJmol-1 (for growth functions measured with 8 and 10 kHz as f2, respectively). Such activation energy values provide a hint that an intact dynein-tubulin system within the scolopidial receptors could play an essential part in the DPOAE generation in tympanal organs.
The third project of this thesis demonstrated mechanical DPOAE analogs in the tympanum's vibration pattern during two-tone stimulation in the locust Schistocerca gregaria, using laser Doppler vibrometry (Möckel et al. 2014). DPOAE generation crucially relies on the integrity of the scolopidial mechanoreceptors (Möckel et al. 2007, 2011), which in locusts, directly attach to the tympanal membrane. During two-tone stimulation, DPOAEs were shown to mechanically emerge at the tympanum region where the auditory mechanoreceptors are attached. Those emission-coupled vibrations differed remarkably from tympanum waves evoked by external pure tones of the same frequency, in terms of wave propagation, energy distribution, and location of amplitude maxima. In contrast to traveling wave-like characteristics of externally evoked vibrations, intrinsically generated waves were locally restricted to the region around the high frequency receptors’ attachment position. The mechanical gradient of the tympanal membrane that leads to direction-dependent properties probably avoids the spreading of these locally evoked waves, which are then reflected and occur only in restricted areas as standing waves. Selective inactivation of mechanoreceptors by mechanical lesions did not affect the tympanum's response to external pure tones, but abolished the emission's displacement amplitude peak. These findings provide evidence that tympanal auditory receptors, comparable to the situation in mammals, comprise the required nonlinear response characteristics, which during two-tone stimulation lead to additional, highly localized deflections of the tympanum.
Direct observation of mobility state transitions in RNA trajectories by sensitive single molecule feedback tracking
Tim P. Kaminski
Jennifer S. Rinne
- Observation and tracking of fluorescently labeled molecules and particles in living cells reveals detailed information about intracellular processes on the molecular level. Whereas light microscopic particle observation is usually limited to two-dimensional projections of short trajectory segments, we report here image-based real-time three-dimensional single particle tracking in an active feedback loop with single molecule sensitivity. We tracked particles carrying only 1-3 fluorophores deep inside living tissue with high spatio-temporal resolution. Using this approach, we succeeded to acquire trajectories containing several hundred localizations. We present statistical methods to find significant deviations from random Brownian motion in such trajectories. The analysis allowed us to directly observe transitions in the mobility of ribosomal (r)RNA and Balbiani ring (BR) messenger (m)RNA particles in living Chironomus tentans salivary gland cell nuclei. We found that BR mRNA particles displayed phases of reduced mobility, while rRNA particles showed distinct binding events in and near nucleoli.
Phenotypic variation of 38 European Ambrosia artemisiifolia populations measured in a common garden experiment
Marion Carmen Leiblein-Wild
- The knowledge of phenotypic variation in the European range of the highly allergenic Ambrosia artemisiifolia L. (common ragweed) is not entirely complete, even though it is an invasive species of utmost concern. We hypothesized the prevalence of phenotypic differentiations between common ragweed populations in the introduced range, and we assumed that those differentiations were related to environmental conditions at the points of origin. Using a common garden experiment, we investigated biomass allocation, growth rates, and flowering phenology of 38 European common ragweed populations originating from a major geographical gradient. We observed considerable phenotypic variation in growth parameters and flowering phenology, e.g. mean aboveground biomass varied from 23.3 to 47.3 g between the populations. We were able to relate most measured traits with environmental parameters prevailing at the points of origin. For example, early growth of ruderal populations was highly correlated with temperature and precipitation at the point of origin. Late growth and flowering phenology were highly correlated with latitude, i.e. individuals from northern populations grew smaller and flowered and dispersed their pollen and seeds up to 5 weeks earlier than individuals from southern populations. We also found a longitudinal gradient in flowering phenology which has not yet been described. The existence of such a high variability in the introduced range may facilitate further range expansion. We suggest that the correlation with environmental variables rests upon genetic variation possibly due to adaptations to the respective environment. To clarify if such adaptation results from multiple events of introduction or as evolutionary response after introduction, genetic investigations are needed.
Characterization of an invertebrate-type dopamine receptor of the American cockroach, Periplaneta americana
- We have isolated a cDNA coding for a putative invertebrate-type dopamine receptor (Peadop2) from P. americana brain by using a PCR-based strategy. The mRNA is present in samples from brain and salivary glands. We analyzed the distribution of the PeaDOP2 receptor protein with specific affinity-purified polyclonal antibodies. On Western blots, PeaDOP2 was detected in protein samples from brain, subesophageal ganglion, thoracic ganglia, and salivary glands. In immunocytochemical experiments, we detected PeaDOP2 in neurons with their somata being located at the anterior edge of the medulla bilaterally innervating the optic lobes and projecting to the ventro-lateral protocerebrum. In order to determine the functional and pharmacological properties of the cloned receptor, we generated a cell line constitutively expressing PeaDOP2. Activation of PeaDOP2-expressing cells with dopamine induced an increase in intracellular cAMP. In contrast, a C-terminally truncated splice variant of this receptor did not exhibit any functional property by itself. The molecular and pharmacological characterization of the first dopamine receptor from P. americana provides the basis for forthcoming studies focusing on the significance of the dopaminergic system in cockroach behavior and physiology.
The genome of the basal agaricomycete Xanthophyllomyces dendrorhous provides insights into the organization of its acetyl-CoA derived pathways and the evolution of Agaricomycotina
- Background: Xanthophyllomyces dendrorhous is a basal agaricomycete with uncertain taxonomic placement, known for its unique ability to produce astaxanthin, a carotenoid with antioxidant properties. It was the aim of this study to elucidate the organization of its CoA-derived pathways and to use the genomic information of X. dendrorhous for a phylogenomic investigation of the Basidiomycota.
Results: The genome assembly of a haploid strain of Xanthophyllomyces dendrorhous revealed a genome of 19.50 Megabases with 6385 protein coding genes. Phylogenetic analyses were conducted including 48 fungal genomes. These revealed Ustilaginomycotina and Agaricomycotina as sister groups. In the latter a well-supported sister-group relationship of two major orders, Polyporales and Russulales, was inferred. Wallemia occupies a basal position within the Agaricomycotina and X. dendrorhous represents the basal lineage of the Tremellomycetes, highlighting that the typical tremelloid parenthesomes have either convergently evolved in Wallemia and the Tremellomycetes, or were lost in the Cystofilobasidiales lineage. A detailed characterization of the CoA-related pathways was done and all genes for fatty acid, sterol and carotenoid synthesis have been assigned.
Conclusions: The current study ascertains that Wallemia with tremelloid parenthesomes is the most basal agaricomycotinous lineage and that Cystofilobasidiales without tremelloid parenthesomes are deeply rooted within Tremellomycetes, suggesting that parenthesomes at septal pores might be the core synapomorphy for the Agaricomycotina. Apart from evolutionary insights the genome sequence of X. dendrorhous will facilitate genetic pathway engineering for optimized astaxanthin or oxidative alcohol production.
Massive analysis of cDNA Ends (MACE) and miRNA expression profiling identifies proatherogenic pathways in chronic kidney disease
Adam M. Zawada
Kyrill S. Rogacev
Gunnar H. Heine
- Epigenetic dysregulation contributes to the high cardiovascular disease burden in chronic kidney disease (CKD) patients. Although microRNAs (miRNAs) are central epigenetic regulators, which substantially affect the development and progression of cardiovascular disease (CVD), no data on miRNA dysregulation in CKD-associated CVD are available until now. We now performed high-throughput miRNA sequencing of peripheral blood mononuclear cells from ten clinically stable hemodialysis (HD) patients and ten healthy controls, which allowed us to identify 182 differentially expressed miRNAs (e.g., miR-21, miR-26b, miR-146b, miR-155). To test biological relevance, we aimed to connect miRNA dysregulation to differential gene expression. Genome-wide gene expression profiling by MACE (Massive Analysis of cDNA Ends) identified 80 genes to be differentially expressed between HD patients and controls, which could be linked to cardiovascular disease (e.g., KLF6, DUSP6, KLF4), to infection / immune disease (e.g., ZFP36, SOCS3, JUND), and to distinct proatherogenic pathways such as the Toll-like receptor signaling pathway (e.g., IL1B, MYD88, TICAM2), the MAPK signaling pathway (e.g., DUSP1, FOS, HSPA1A), and the chemokine signaling pathway (e.g., RHOA, PAK1, CXCL5). Formal interaction network analysis proved biological relevance of miRNA dysregulation, as 68 differentially expressed miRNAs could be connected to 47 reciprocally expressed target genes. Our study is the first comprehensive miRNA analysis in CKD that links dysregulated miRNA expression with differential expression of genes connected to inflammation and CVD. After recent animal data suggested that targeting miRNAs is beneficial in experimental CVD, our data may now spur further research in the field of CKD-associated human CVD.