A landmark-free analysis of the pelvic girdle in Sulawesi ricefishes (Adrianichthyidae): How 2D and 3D geometric morphometrics can complement each other in the analysis of a complex structure.

Geometric morphometrics (GM) enable the quantification of morphological variation on various scales. Recent technical advances allow analyzing complex three-dimensional shapes also in cases where landmark-based approaches are not appropriate. Pelvic girdle bones (basipterygia) of Sulawesi ricefishes are 3D structures that challenge traditional morphometrics. We hypothesize that the pelvic girdle of ricefishes experienced sex-biased selection pressures in species where females provide brood care by carrying fertilized eggs supported by elongated pelvic fins ("pelvic brooding"). We test this by comparing pelvic bone shapes of both sexes in species exhibiting pelvic brooding and the more common reproductive strategy "transfer brooding," by using landmark-free 2D and 3D GM, as well as qualitative shape descriptions. Both landmark-free approaches revealed significant interspecific pelvic bone variation in the lateral process, medial facing side of the pelvic bone, and overall external and internal wing shape. Within pelvic brooders, the three analyzed species are clearly distinct, while pelvic bones of the genus Adrianichthys are more similar to transfer brooding Oryzias. Female pelvic brooding Oryzias exhibit prominent, medially pointing tips extending from the internal wing and basipterygial plate that are reduced or absent in conspecific males, Adrianichthys and transfer brooding Oryzias, supporting our hypothesis that selection pressures affecting pelvic girdle shape are sex-biased in Sulawesi ricefishes. Furthermore, both sexes of pelvic brooding Oryzias have overall larger pelvic bones than other investigated ricefishes. Based on these differences, we characterized two reproductive strategy- and sex-dependent pelvic girdle types for Sulawesi ricefishes. Morphological differences between the investigated pelvic brooding genera Adrianichthys and Oryzias provide additional evidence for two independent origins of pelvic brooding. Overall, our findings add to a better understanding on traits related to pelvic brooding in ricefishes and provide a basis for upcoming studies on pelvic girdle function and morphology.

Potential Contribution of Ancient Introgression to the Evolution of a Derived Reproductive Strategy in Ricefishes.

Transitions from no parental care to extensive care are costly and involve major changes in life history, behavior, and morphology. Nevertheless, in Sulawesi ricefishes, pelvic brooding evolved from transfer brooding in two distantly related lineages within the genera Adrianichthys and Oryzias, respectively. Females of pelvic brooding species carry their eggs attached to their belly until the fry hatches. Despite their phylogenetic distance, both pelvic brooding lineages share a set of external morphological traits. A recent study found no direct gene flow between pelvic brooding lineages, suggesting independent evolution of the derived reproductive strategy. Convergent evolution can, however, also rely on repeated sorting of preexisting variation of an admixed ancestral population, especially when subjected to similar external selection pressures. We thus used a multispecies coalescent model and D-statistics to identify gene-tree-species-tree incongruencies, to evaluate the evolution of pelvic brooding with respect to interspecific gene flow not only between pelvic brooding lineages but also between pelvic brooding lineages and other Sulawesi ricefish lineages. We found a general network-like evolution in Sulawesi ricefishes, and as previously reported, we detected no gene flow between the pelvic brooding lineages. Instead, we found hybridization between the ancestor of pelvic brooding Oryzias and the common ancestor of the Oryzias species from the Lake Poso area. We further detected signs of introgression within the confidence interval of a quantitative trait locus associated with pelvic brooding in O. eversi. Our results hint toward a contribution of ancient standing genetic variation to the evolution of pelvic brooding in Oryzias.

Integrating gene annotation with orthology inference at scale.

Annotating coding genes and inferring orthologs are two classical challenges in genomics and evolutionary biology that have traditionally been approached separately, limiting scalability. We present TOGA (Tool to infer Orthologs from Genome Alignments), a method that integrates structural gene annotation and orthology inference. TOGA implements a different paradigm to infer orthologous loci, improves ortholog detection and annotation of conserved genes compared with state-of-the-art methods, and handles even highly fragmented assemblies. TOGA scales to hundreds of genomes, which we demonstrate by applying it to 488 placental mammal and 501 bird assemblies, creating the largest comparative gene resources so far. Additionally, TOGA detects gene losses, enables selection screens, and automatically provides a superior measure of mammalian genome quality. TOGA is a powerful and scalable method to annotate and compare genes in the genomic era.

How to stay attached-Formation of the ricefish plug and changes of internal reproductive structures in the pelvic brooding ricefish, Oryzias eversi Herder et al. (2012) (Beloniformes: Adrianichthyidae).

Teleost fishes show an enormous diversity of parental care, ranging from no care to viviparity with maternal provisioning of embryos. External brooders carry their developing eggs attached to their bodies. This requires the formation of novel morphological structures to support attachment. The pelvic brooding ricefish Oryzias eversi evolved such a structure, called the "plug." The plug anchors attaching filaments from the fertilized eggs inside the female reproductive system, allowing the female to carry the embryos until hatching. Using histological sections and µ-computed tomography scanning, we show that the plug is formed by several types of interstitial cells, blood capillaries, and collagen fibrils that encapsulate the end of the attaching filaments in the anterior part of the gonoduct. Even 15 days after the loss of the protruding attaching filaments, the plug remains. In addition, the developed plug contains multinucleated giant cells that are derived from fusing macrophages. We thus hypothesize that the ricefish plug, which is vital for egg attachment in O. eversi, evolved due to an inflammatory reaction. We assume that it forms similar to a foreign body granuloma, as a reaction to irritation or injury of the gonoduct epithelium by the attaching filaments. Our study further corroborates that pelvic brooding entails a complex set of adaptations to prolonged egg-carrying in the female reproductive system. During brooding, for instance, ovulation in the ovary is suppressed and the anterior part of the gonoduct is characterized by an intricate, recessed folding.

Evolutionary Divergence and Radula Diversification in Two Ecomorphs from an Adaptive Radiation of Freshwater Snails.

(1) Background: Adaptive diversification of complex traits plays a pivotal role in the evolution of organismal diversity. In the freshwater snail genus Tylomelania, adaptive radiations were likely promoted by trophic specialization via diversification of their key foraging organ, the radula. (2) Methods: To investigate the molecular basis of radula diversification and its contribution to lineage divergence, we used tissue-specific transcriptomes of two sympatric Tylomelania sarasinorum ecomorphs. (3) Results: We show that ecomorphs are genetically divergent lineages with habitat-correlated abundances. Sequence divergence and the proportion of highly differentially expressed genes are significantly higher between radula transcriptomes compared to the mantle and foot. However, the same is not true when all differentially expressed genes or only non-synonymous SNPs are considered. Finally, putative homologs of some candidate genes for radula diversification (hh, arx, gbb) were also found to contribute to trophic specialization in cichlids and Darwin's finches. (4) Conclusions: Our results are in line with diversifying selection on the radula driving Tylomelania ecomorph divergence and indicate that some molecular pathways may be especially prone to adaptive diversification, even across phylogenetically distant animal groups.

The genetic basis of a novel reproductive strategy in Sulawesi ricefishes: How modularity and a low number of loci shape pelvic brooding.

The evolution of complex phenotypes like reproductive strategies is challenging to understand, as they often depend on multiple adaptations that only jointly result in a specific functionality. Sulawesi ricefishes (Adrianichthyidae) evolved a reproductive strategy termed as pelvic brooding. In contrast to the more common transfer brooding, female pelvic brooders carry an egg bundle connected to their body for weeks until the fry hatches. To examine the genetic architecture of pelvic brooding, we crossed the pelvic brooding Oryzias eversi and the transfer brooding Oryzias nigrimas (species divergence time: ∼3.6 my). We hypothesize, that a low number of loci and modularity have facilitated the rapid evolution of pelvic brooding. Traits associated to pelvic brooding, like rib length, pelvic fin length, and morphology of the genital papilla, were correlated in the parental species but correlations were reduced or lost in their F1 and F2 hybrids. Using the Castle-Wright estimator, we found that generally few loci underlie the studied traits. Further, both parental species showed modularity in their body plans. In conclusion, morphological traits related to pelvic brooding were based on a few loci and the mid-body region likely could evolve independently from the remaining body parts. Both factors presumably facilitated the evolution of pelvic brooding.

Inflammation and convergent placenta gene co-option contributed to a novel reproductive tissue.

The evolution of pregnancy exposes parental tissues to new, potentially stressful conditions, which can trigger inflammation.1 Inflammation is costly2,3 and can induce embryo rejection, which constrains the evolution of pregnancy.1 In contrast, inflammation can also promote morphological innovation at the maternal-embryonic interface as exemplified by co-option of pro-inflammatory signaling for eutherian embryo implantation.1,4,5 Given its dual function, inflammation could be a key process explaining how innovations such as pregnancy and placentation evolved many times convergently. Pelvic brooding ricefishes evolved a novel "plug" tissue,6,7 which forms inside the female gonoduct after spawning, anchors egg-attaching filaments, and enables pelvic brooders to carry eggs externally until hatching.6,8 Compared to pregnancy, i.e., internal bearing of embryos, external bearing should alleviate constraints on inflammation in the reproductive tract. We thus hypothesized that an ancestral inflammation triggered by the retention of attaching filaments gave rise to pathways orchestrating plug formation. In line with our hypothesis, histological sections of the developing plug revealed signs of gonoduct injuries by egg-attaching filaments in the pelvic brooding ricefish Oryzias eversi. Tissue-specific transcriptomes showed that inflammatory signaling dominates the plug transcriptome and inflammation-induced genes controlling vital processes for plug development such as tissue growth and angiogenesis were overexpressed in the plug. Finally, mammalian placenta genes were enriched in the plug transcriptome, indicating convergent gene co-option for building, attaching, and sustaining a transient tissue in the female reproductive tract. This study highlights the role of gene co-option and suggests that recruiting inflammatory signaling into physiological processes provides a fast-track to evolutionary innovation.

Complex sexually dimorphic traits shape the parallel evolution of a novel reproductive strategy in Sulawesi ricefishes (Adrianichthyidae).

BACKGROUND: Pelvic brooding is a form of uni-parental care, and likely evolved in parallel in two lineages of Sulawesi ricefishes. Contrary to all other ricefishes, females of pelvic brooding species do not deposit eggs at a substrate (transfer brooding), but carry them until the fry hatches. We assume that modifications reducing the costs of egg carrying are beneficial for pelvic brooding females, but likely disadvantageous in conspecific males, which might be resolved by the evolution of sexual dimorphism via sexual antagonistic selection. Thus we hypothesize that the evolution of pelvic brooding gave rise to female-specific skeletal adaptations that are shared by both pelvic brooding lineages, but are absent in conspecific males and transfer brooding species. To tackle this, we combine 3D-imaging and morphometrics to analyze skeletal adaptations to pelvic brooding. RESULTS: The morphology of skeletal traits correlated with sex and brooding strategy across seven ricefish species. Pelvic brooding females have short ribs caudal of the pelvic girdle forming a ventral concavity and clearly elongated and thickened pelvic fins compared to both sexes of transfer brooding species. The ventral concavity limits the body cavity volume in female pelvic brooders. Thus body volumes are smaller compared to males in pelvic brooding species, a pattern sharply contrasted by transfer brooding species. CONCLUSIONS: We showed in a comparative framework that highly similar, sexually dimorphic traits evolved in parallel in both lineages of pelvic brooding ricefish species. Key traits, present in all pelvic brooding females, were absent or much less pronounced in conspecific males and both sexes of transfer brooding species, indicating that they are non-beneficial or even maladaptive for ricefishes not providing extended care. We assume that the combination of ventral concavity and robust, elongated fins reduces drag of brooding females and provides protection and stability to the egg cluster. Thus ricefishes are one of the rare examples where environmental factors rather than sexual selection shaped the evolution of sexually dimorphic skeletal adaptations.

The untapped potential of medaka and its wild relatives.

The medaka is a fish that has served as a model organism for over a century, yet there is still much to learn about its life in the wild. Here we summarize the current knowledge, highlight recent progress and outline remaining gaps in our understanding of the natural history of medaka. It has also become clear over time that rather than being a single species, medaka comprises an entire species complex, so disentangling the species boundaries is an important goal for future research. Moreover, medaka and other ricefishes exhibit striking functional diversity, little of which has been investigated to date. As such, there are opportunities to use the resources developed for medaka to study other ricefishes, and to learn more about medaka itself in an evolutionary context.

Cryptic species in a well-known habitat: applying taxonomics to the amphipod genus Epimeria (Crustacea, Peracarida).

Taxonomy plays a central role in biological sciences. It provides a communication system for scientists as it aims to enable correct identification of the studied organisms. As a consequence, species descriptions should seek to include as much available information as possible at species level to follow an integrative concept of 'taxonomics'. Here, we describe the cryptic species Epimeria frankei sp. nov. from the North Sea, and also redescribe its sister species, Epimeria cornigera. The morphological information obtained is substantiated by DNA barcodes and complete nuclear 18S rRNA gene sequences. In addition, we provide, for the first time, full mitochondrial genome data as part of a metazoan species description for a holotype, as well as the neotype. This study represents the first successful implementation of the recently proposed concept of taxonomics, using data from high-throughput technologies for integrative taxonomic studies, allowing the highest level of confidence for both biodiversity and ecological research.

Novel Genes, Ancient Genes, and Gene Co-Option Contributed to the Genetic Basis of the Radula, a Molluscan Innovation.

The radula is the central foraging organ and apomorphy of the Mollusca. However, in contrast to other innovations, including the mollusk shell, genetic underpinnings of radula formation remain virtually unknown. Here, we present the first radula formative tissue transcriptome using the viviparous freshwater snail Tylomelania sarasinorum and compare it to foot tissue and the shell-building mantle of the same species. We combine differential expression, functional enrichment, and phylostratigraphic analyses to identify both specific and shared genetic underpinnings of the three tissues as well as their dominant functions and evolutionary origins. Gene expression of radula formative tissue is very distinct, but nevertheless more similar to mantle than to foot. Generally, the genetic bases of both radula and shell formation were shaped by novel orchestration of preexisting genes and continuous evolution of novel genes. A significantly increased proportion of radula-specific genes originated since the origin of stem-mollusks, indicating that novel genes were especially important for radula evolution. Genes with radula-specific expression in our study are frequently also expressed during the formation of other lophotrochozoan hard structures, like chaetae (hes1, arx), spicules (gbx), and shells of mollusks (gbx, heph) and brachiopods (heph), suggesting gene co-option for hard structure formation. Finally, a Lophotrochozoa-specific chitin synthase with a myosin motor domain (CS-MD), which is expressed during mollusk and brachiopod shell formation, had radula-specific expression in our study. CS-MD potentially facilitated the construction of complex chitinous structures and points at the potential of molecular novelties to promote the evolution of different morphological innovations.

The complete mitochondrial transcript of the red tail loach Yasuhikotakia modesta as assembled from RNAseq (Teleostei: Botiidae).

Here, we present the first complete mitochondrial genome of the Red Tailed Loach Yasuhikotakia modesta (Teleostei: Botiidae) from Thailand, assembled from next-generation transcriptome sequencing data. The assembled transcript corresponds to the full length mitochondrial genome of Y. modesta, which measured 16,865 bp in length, and contained 13 protein-coding genes, 2 ribosomal RNA genes, and 22 transfer RNA genes. A slight A + T bias was observed in the mitogenome of Y. modesta with an overall base composition of 32.2% A, 25.8% T, 26.4% C, and 15.4% G, and a GC content of 41.8%. The gene arrangement was identical to that of previously described loach mitogenomes.

The complete mitochondrial genome of the viviparous freshwater snail Tylomelania sarasinorum (Caenogastropoda: Cerithioidea).

Here, we present the first complete mitochondrial genome within the gastropod family Pachychilidae, using the viviparous freshwater snail Tylomelania sarasinorum. This species is a representative member of the lacustrine Tylomelania radiations of the Malili-Lakes-System (Sulawesi, Indonesia). The mitochondrial genome was 16,632 bp long and contained 13 protein-coding genes, 2 rRNA genes and 22 tRNA genes. A pronounced A + T bias was observed with an overall base composition of 29.5% A, 35.7% T, 18.3% G and 16.6% C. Tylomelania sarasinorum exhibited a novel mitochondrial gene arrangement, differing from all Caenogastropoda mitochondrial genomes published to date.

Localization of copper and copper transporters in the human brain.

Disturbances in brain copper result in rare and severe neurological disorders and may play a role in the pathogenesis and progression of multiple neurodegenerative diseases. Our current understanding of mammalian brain copper transport is based on model systems outside the central nervous system and no data are available regarding copper transport systems in the human brain. To address this deficit, we quantified regional copper concentrations and examined the distribution and cellular localization of the copper transport proteins Copper transporter 1, Atox1, ATP7A, and ATP7B in multiple regions of the human brain using inductively coupled plasma-mass spectrometry, Western blot and immunohistochemistry. We identified significant relationships between copper transporter levels and brain copper concentrations, supporting a role for these proteins in copper transport in the human brain. Interestingly, the substantia nigra contained twice as much copper than that in other brain regions, suggesting an important role for copper in this brain region. Furthermore, ATP7A levels were significantly greater in the cerebellum, compared with other brain regions, supporting an important role for ATP7A in cerebellar neuronal health. This study provides novel data regarding copper regulation in the human brain, critical to understand the mechanisms by which brain copper levels can be altered, leading to neurological disease.