Stage-dependent remodeling of the nuclear envelope and lamina during rabbit early embryonic development.

Utilizing 3D structured illumination microscopy, we investigated the quality and quantity of nuclear invaginations and the distribution of nuclear pores during rabbit early embryonic development and identified the exact time point of nucleoporin 153 (NUP153) association with chromatin during mitosis. Contrary to bovine early embryonic nuclei, featuring almost exclusively nuclear invaginations containing a small volume of cytoplasm, nuclei in rabbit early embryonic stages show additionally numerous invaginations containing a large volume of cytoplasm. Small-volume invaginations frequently emanated from large-volume nuclear invaginations but not vice versa, indicating a different underlying mechanism. Large- and small-volume nuclear envelope invaginations required the presence of chromatin, as they were restricted to chromatin-positive areas. The chromatin-free contact areas between nucleolar precursor bodies (NPBs) and large-volume invaginations were free of nuclear pores. Small-volume invaginations were not in contact with NPBs. The number of invaginations and isolated intranuclear vesicles per nucleus peaked at the 4-cell stage. At this stage, the nuclear surface showed highly concentrated clusters of nuclear pores surrounded by areas free of nuclear pores. Isolated intranuclear lamina vesicles were usually NUP153 negative. Cytoplasmic, randomly distributed NUP153-positive clusters were highly abundant at the zygote stage and decreased in number until they were almost absent at the 8-cell stage and later. These large NUP153 clusters may represent a maternally provided NUP153 deposit, but they were not visible as clusters during mitosis. Major genome activation at the 8- to 16-cell stage may mark the switch from a necessity for a deposit to on-demand production. NUP153 association with chromatin is initiated during metaphase before the initiation of the regeneration of the lamina. To our knowledge, the present study demonstrates for the first time major remodeling of the nuclear envelope and its underlying lamina during rabbit preimplantation development.

Vertebrate time-tree elucidates the biogeographic pattern of a major biotic change around the K-T boundary in Madagascar.

The geographic and temporal origins of Madagascar's biota have long been in the center of debate. We reconstructed a time-tree including nearly all native nonflying and nonmarine vertebrate clades present on the island, from DNA sequences of two single-copy protein-coding nuclear genes (BDNF and RAG1) and a set of congruent time constraints. Reconstructions calculated with autocorrelated or independent substitution rates over clades agreed in placing the origins of the 31 included clades in Cretaceous to Cenozoic times. The two clades with sister groups in South America were the oldest, followed by those of a putative Asian ancestry that were significantly older than the prevalent clades of African ancestry. No colonizations from Asia occurred after the Eocene, suggesting that dispersal and vicariance of Asian/Indian groups were favored over a comparatively short period during, and shortly after, the separation of India and Madagascar. Species richness of clades correlates with their age but those clades that have a large proportion of species diversity in rainforests are significantly more species-rich. This finding suggests an underlying pattern of continuous speciation through time in Madagascar's vertebrates, with accelerated episodes of adaptive diversification in those clades that succeeded radiating into the rainforests.

RNA-seq pinpoints a Xanthomonas TAL-effector activated resistance gene in a large-crop genome.

Transcription activator-like effector (TALE) proteins of the plant pathogenic bacterial genus Xanthomonas bind to and transcriptionally activate host susceptibility genes, promoting disease. Plant immune systems have taken advantage of this mechanism by evolving TALE binding sites upstream of resistance (R) genes. For example, the pepper Bs3 and rice Xa27 genes are hypersensitive reaction plant R genes that are transcriptionally activated by corresponding TALEs. Both R genes have a hallmark expression pattern in which their transcripts are detectable only in the presence and not the absence of the corresponding TALE. By transcriptome profiling using next-generation sequencing (RNA-seq), we tested whether we could avoid laborious positional cloning for the isolation of TALE-induced R genes. In a proof-of-principle experiment, RNA-seq was used to identify a candidate for Bs4C, an R gene from pepper that mediates recognition of the Xanthomonas TALE protein AvrBs4. We identified one major Bs4C candidate transcript by RNA-seq that was expressed exclusively in the presence of AvrBs4. Complementation studies confirmed that the candidate corresponds to the Bs4C gene and that an AvrBs4 binding site in the Bs4C promoter directs its transcriptional activation. Comparison of Bs4C with a nonfunctional allele that is unable to recognize AvrBs4 revealed a 2-bp polymorphism within the TALE binding site of the Bs4C promoter. Bs4C encodes a structurally unique R protein and Bs4C-like genes that are present in many solanaceous genomes seem to be as tightly regulated as pepper Bs4C. These findings demonstrate that TALE-specific R genes can be cloned from large-genome crops with a highly efficient RNA-seq approach.

Acoustic underwater signals with a probable function during competitive feeding in a tadpole.

Acoustic communication is widespread among adult stages of terrestrial animals and fish and has also been observed in insect larvae. We report underwater acoustic communication in the larvae of a frog, Gephyromantis azzurrae, from Isalo, a sandstone massif in western Madagascar. According to our field data, these tadpoles live in streams and prefer habitats characterized by comparatively low temperatures, shallow water depth, and a relatively fast current. Feeding experiments indicated that the tadpoles are carnivorous and macrophagous. They consumed insect larvae and, to a lesser extent, small shrimps, and conspecific as well as heterospecific tadpoles. Calls of these tadpoles consisted either of single click notes or of irregular series of various clicks. Some complex calls have a pulsed structure with three to nine indistinct energy pulses. Production of the pulses coincided with rapid closure of the jaw sheaths and often with an upward movement of the body. Calls were emitted while attacking prey and occurred significantly more often when attacking conspecifics. Tadpoles that had not been fed for some time emitted sounds more frequently than those that had been regularly fed. The spectral frequency of the calls differed in tadpole groups of different size and was higher in groups of smaller tadpoles, suggesting that spectral frequency carries some information about tadpole size which might be important during competitive feeding to assess size and strength of competitors. This report differs from those for the larvae of South American horned frogs, Ceratophrys ornata. These are the only other tadpoles for which sound production has reliably been reported but the calls of Ceratophrys tadpoles occur mainly in a defensive context.

The world's richest tadpole communities show functional redundancy and low functional diversity: ecological data on Madagascar's stream-dwelling amphibian larvae.

BACKGROUND: Functional diversity illustrates the range of ecological functions in a community. It allows revealing the appearance of functional redundancy in communities and processes of community assembly. Functional redundancy illustrates the overlap in ecological functions of community members which may be an indicator of community resilience. We evaluated patterns of species richness, functional diversity and functional redundancy on tadpole communities in rainforest streams in Madagascar. This habitat harbours the world's most species-rich stream tadpole communities which are due to their occurrence in primary habitat of particular interest for functional diversity studies. RESULTS: Species richness of tadpole communities is largely determined by characteristics of the larval habitat (stream structure), not by adult habitat (forest structure). Species richness is positively correlated with a size-velocity gradient of the streams, i.e. communities follow a classical species-area relationship. While widely observed for other taxa, this is an unusual pattern for anuran larvae which usually is expected to be hump-shaped. Along the species richness gradient, we quantified functional diversity of all communities considering the similarity and dissimilarity of species in 18 traits related to habitat use and foraging. Especially species-rich communities were characterised by an overlap of species function, i.e. by functional redundancy. By comparing the functional diversity of the observed communities with functional diversity of random assemblages, we found no differences at low species richness level, whereas observed species-rich communities have lower functional diversity than respective random assemblages. CONCLUSIONS: We found functional redundancy being a feature of communities also in primary habitat, what has not been shown before using such a continuous measure. The observed species richness dependent pattern of low functional diversity indicates that communities with low species richness accumulate functional traits randomly, whereas species in species-rich communities are more similar to each other than predicted by random assemblages and therefore exhibit an accumulation of stream-specific functional traits. Beyond a certain species richness level, therefore, stream-specific environmental filters exert influence whereas interspecific competition between species does not influence trait assemblage at any species richness level.

Opposing Patterns of Seasonal Change in Functional and Phylogenetic Diversity of Tadpole Assemblages.

Assemblages that are exposed to recurring temporal environmental changes can show changes in their ecological properties. These can be expressed by differences in diversity and assembly rules. Both can be identified using two measures of diversity: functional (FD) and phylogenetic diversity (PD). Frog communities are understudied in this regard, especially during the tadpole life stage. We utilised tadpole assemblages from Madagascan rainforest streams to test predictions of seasonal changes on diversity and assemblage composition and on diversity measures. From the warm-wet to the cool-dry season, species richness (SR) of tadpole assemblages decreased. Also FD and PD decreased, but FD less and PD more than expected by chance. During the dry season, tadpole assemblages were characterised by functional redundancy (among assemblages-with increasing SR), high FD (compared to a null model), and low PD (phylogenetic clustering; compared to a null model). Although mutually contradictory at first glance, these results indicate competition as tadpole community assembly driving force. This is true during the limiting cool-dry season but not during the more suitable warm-wet season. We thereby show that assembly rules can strongly depend on season, that comparing FD and PD can reveal such forces, that FD and PD are not interchangeable, and that conclusions on assembly rules based on FD alone are critical.

Remodeling of the Nuclear Envelope and Lamina during Bovine Preimplantation Development and Its Functional Implications.

The present study demonstrates a major remodeling of the nuclear envelope and its underlying lamina during bovine preimplantation development. Up to the onset of major embryonic genome activation (MGA) at the 8-cell stage nuclei showed a non-uniform distribution of nuclear pore complexes (NPCs). NPCs were exclusively present at sites where DNA contacted the nuclear lamina. Extended regions of the lamina, which were not contacted by DNA, lacked NPCs. In post-MGA nuclei the whole lamina was contacted rather uniformly by DNA. Accordingly, NPCs became uniformly distributed throughout the entire nuclear envelope. These findings shed new light on the conditions which control the integration of NPCs into the nuclear envelope. The switch from maternal to embryonic production of mRNAs was accompanied by multiple invaginations covered with NPCs, which may serve the increased demands of mRNA export and protein import. Other invaginations, as well as interior nuclear segments and vesicles without contact to the nuclear envelope, were exclusively positive for lamin B. Since the abundance of these invaginations and vesicles increased in concert with a massive nuclear volume reduction, we suggest that they reflect a mechanism for fitting the nuclear envelope and its lamina to a shrinking nuclear size during bovine preimplantation development. In addition, a deposit of extranuclear clusters of NUP153 (a marker for NPCs) without associated lamin B was frequently observed from the zygote stage up to MGA. Corresponding RNA-Seq data revealed deposits of spliced, maternally provided NUP153 mRNA and little unspliced, newly synthesized RNA prior to MGA, which increased strongly at the initiation of embryonic expression of NUP153 at MGA.

Reprogramming of fibroblast nuclei in cloned bovine embryos involves major structural remodeling with both striking similarities and differences to nuclear phenotypes of in vitro fertilized embryos.

Nuclear landscapes were studied during preimplantation development of bovine embryos, generated either by in vitro fertilization (IVF), or generated as cloned embryos by somatic cell nuclear transfer (SCNT) of bovine fetal fibroblasts, using 3-dimensional confocal laser scanning microscopy (3D-CLSM) and structured illumination microscopy (3D-SIM). Nuclear landscapes of IVF and SCNT embryonic nuclei were compared with each other and with fibroblast nuclei. We demonstrate that reprogramming of fibroblast nuclei in cloned embryos requires changes of their landscapes similar to nuclei of IVF embryos. On the way toward the 8-cell stage, where major genome activation occurs, a major lacuna, enriched with splicing factors, was formed in the nuclear interior and chromosome territories (CTs) were shifted toward the nuclear periphery. During further development the major lacuna disappeared and CTs were redistributed throughout the nuclear interior forming a contiguous higher order chromatin network. At all stages of development CTs of IVF and SCNT embryonic nuclei were built up from chromatin domain clusters (CDCs) pervaded by interchromatin compartment (IC) channels. Quantitative analyses revealed a highly significant enrichment of RNA polymerase II and H3K4me3, a marker for transcriptionally competent chromatin, at the periphery of CDCs. In contrast, H3K9me3, a marker for silent chromatin, was enriched in the more compacted interior of CDCs. Despite these striking similarities, we also detected major differences between nuclear landscapes of IVF and cloned embryos. Possible implications of these differences for the developmental potential of cloned animals remain to be investigated. We present a model, which integrates generally applicable structural and functional features of the nuclear landscape.

Diversity of the strongly rheophilous tadpoles of Malagasy tree frogs, genus Boophis (Anura, Mantellidae), and identification of new candidate species via larval DNA sequence and morphology.

This study provides detailed morphological descriptions of previously unknown tadpoles of the treefrog genus Boophis Tschudi and analyses of habitat preferences of several of these tadpoles in Ranomafana National Park. A total of twenty-two tadpoles determined via DNA barcoding are characterized morphologically herein, fourteen of them for the first time. Twelve of these tadpoles belong to taxonomically undescribed candidate species which in several cases are so far only known from their larval stages. Our data show that the larvae of some of these candidate species occur syntopically yet maintaining a clearly correlated genetic and morphological identity, suggesting that they indeed are true biological and evolutionary species. Tadpoles considered to belong to the "adherent" ecomorphological guild inhabit fast-running waters and their oral disc is commonly to continuously attached to the rocky substrate, supposedly to keep their position in the water current. Some of these species are characterized by the presence of a dorsal gap of papillae and the absence of an upper jaw sheath. This guild includes the tadpoles of the Boophis albipuncatus group (Boophis ankaratra, Boophis schuboeae, Boophis albipunctatus, Boophis sibilans, Boophis luciae), and of the Boophis mandraka group (Boophis sambirano and six candidate species related to this species and to Boophis mandraka). Tadpoles considered belonging to the "suctorial" guild inhabit fast-running waters where they use frequently their oral disc to attach to the substrate. They have an enlarged oral disc without any dorsal gap, including two nominal species (Boophis marojezensis, Boophis vittatus), and five candidate species related to Boophis marojezensis. An ecological analysis of the tadpoles of Boophis luciae, Boophis schuboeae and Boophis marojezensis [Ca51 JQ518198] from Ranomafana National Park did not provide evidence for a clear preference of these tadpoles to the fast flowing microhabitat sections of the stream, although the tadpoles discussed in this study are typically caught in this habitat.