Phylogeny of the supertribe Nebriitae (Coleoptera, Carabidae) based on analyses of DNA sequence data.

The phylogeny of the carabid beetle supertribe Nebriitae is inferred from analyses of DNA sequence data from eight gene fragments including one nuclear ribosomal gene (28S), four nuclear-protein coding genes (CAD, topoisomerase 1, PEPCK, and wingless), and three mitochondrial gene fragments (16S + tRNA-Leu + ND1, COI ("barcode" region) and COI ("Pat/Jer" region)). Our taxon sample included 264 exemplars representing 241 species and subspecies (25% of the known nebriite fauna), 39 of 41 currently accepted genera and subgenera (all except Notiokasis and Archileistobrius), and eight outgroup taxa. Separate maximum likelihood (ML) analyses of individual genes, combined ML analyses of nuclear, nuclear protein-coding, and mitochondrial genes, and combined ML and Bayesian analyses of the eight-gene-fragment matrix resulted in a well-resolved phylogeny of the supertribe, with most nodes in the tree strongly supported. Within Nebriitae, 167 internal nodes of the tree (out of the maximum possible 255) are supported by maximum-likelihood bootstrap values of 90% or more. The tribes Notiophilini, Opisthiini, Pelophilini, and Nebriini are well supported as monophyletic but relationships among these are not well resolved. Nippononebria is a distinct genus more closely related to Leistus than Nebria. Archastes, Oreonebria, Spelaeonebria, and Eurynebria, previously treated as distinct genera by some authors, are all nested within a monophyletic genus Nebria. Within Nebria, four major clades are recognized: (1) the Oreonebria Series, including eight subgenera arrayed in two subgeneric complexes (the Eonebria and Oreonebria Complexes); (2) the Nebriola Series, including only subgenus Nebriola; (3) the Nebria Series, including ten subgenera arrayed in two subgeneric complexes, the Boreonebria and Nebria Complexes, with the latter further subdivided into three subgeneric subcomplexes (the Nebria, Epinebriola, and Eunebria Subcomplexes)); and (4) the Catonebria Series, including seven subgenera arrayed in two subgeneric complexes (the Reductonebria and Catonebria Complexes). A strong concordance of biogeography with the inferred phylogeny is noted and some evident vicariance patterns are highlighted. A revised classification, mainly within the Nebriini, is proposed to reflect the inferred phylogeny. Three genus-group taxa (Nippononebria, Vancouveria and Archastes) are given revised status and seven are recognized as new synonymies (Nebriorites Jeannel, 1941 and Marggia Huber, 2014 = Oreonebria Daniel, 1903; Pseudonebriola Ledoux & Roux, 1989 = Boreonebria Jeannel, 1937; Patrobonebria Bänninger, 1923, Paranebria Jeannel, 1937 and Barbonebriola Huber & Schmidt, 2017 = Epinebriola Daniel & Daniel, 1904; and Asionebria Shilenkov, 1982 = Psilonebria Andrewes, 1923). Six new subgenera are proposed and described for newly recognized clades: Parepinebriola Kavanaugh subgen. nov. (type species: Nebria delicata Huber & Schmidt, 2017), Insulanebria Kavanaugh subgen. nov. (type species: Nebria carbonaria Eschscholtz, 1829), Erwinebria Kavanaugh subgen. nov. (type species Nebria sahlbergii Fischer von Waldheim, 1828), Nivalonebria Kavanaugh subgen. nov. (type species: Nebria paradisi Darlington, 1931), Neaptenonebria Kavanaugh subgen. nov. (type species: Nebria ovipennis LeConte, 1878), and Palaptenonebria Kavanaugh subgen. nov. (type species: Nebria mellyi Gebler, 1847). Future efforts to better understand relationships within the supertribe should aim to expand the taxon sampling of DNA sequence data, particularly within subgenera Leistus and Evanoleistus of genus Leistus and the Nebria Complex of genus Nebria.

Adaptation via pleiotropy and linkage: Association mapping reveals a complex genetic architecture within the stickleback Eda locus.

Genomic mapping of the loci associated with phenotypic evolution has revealed genomic "hotspots," or regions of the genome that control multiple phenotypic traits. This clustering of loci has important implications for the speed and maintenance of adaptation and could be due to pleiotropic effects of a single mutation or tight genetic linkage of multiple causative mutations affecting different traits. The threespine stickleback (Gasterosteus aculeatus) is a powerful model for the study of adaptive evolution because the marine ecotype has repeatedly adapted to freshwater environments across the northern hemisphere in the last 12,000 years. Freshwater ecotypes have repeatedly fixed a 16 kilobase haplotype on chromosome IV that contains Ectodysplasin (Eda), a gene known to affect multiple traits, including defensive armor plates, lateral line sensory hair cells, and schooling behavior. Many additional traits have previously been mapped to a larger region of chromosome IV that encompasses the Eda freshwater haplotype. To identify which of these traits specifically map to this adaptive haplotype, we made crosses of rare marine fish heterozygous for the freshwater haplotype in an otherwise marine genetic background. Further, we performed fine-scale association mapping in a fully interbreeding, polymorphic population of freshwater stickleback to disentangle the effects of pleiotropy and linkage on the phenotypes affected by this haplotype. Although we find evidence that linked mutations have small effects on a few phenotypes, a small 1.4-kb region within the first intron of Eda has large effects on three phenotypic traits: lateral plate count, and both the number and patterning of the posterior lateral line neuromasts. Thus, the Eda haplotype is a hotspot of adaptation in stickleback due to both a small, pleiotropic region affecting multiple traits as well as multiple linked mutations affecting additional traits.

Phosphorus limitation does not drive loss of bony lateral plates in freshwater stickleback (Gasterosteus aculeatus).

Connecting the selective forces that drive the evolution of phenotypes to their underlying genotypes is key to understanding adaptation, but such connections are rarely tested experimentally. Threespine stickleback (Gasterosteus aculeatus) are a powerful model for such tests because genotypes that underlie putatively adaptive traits have been identified. For example, a regulatory mutation in the Ectodysplasin (Eda) gene causes a reduction in the number of bony armor plates, which occurs rapidly and repeatedly when marine sticklebacks invade freshwater. However, the source of selection on plate loss in freshwater is unknown. Here, we tested whether dietary reduction of phosphorus can account for selection on plate loss due to a growth advantage of low-plated fish in freshwater. We crossed marine fish heterozygous for the 16 kilobase freshwater Eda haplotype and compared the growth of offspring with different genotypes under contrasting levels of dietary phosphorus in both saltwater and freshwater. Eda genotype was not associated with growth differences in any treatment, or with mechanisms that could mitigate the impacts of phosphorus limitation, such as differential phosphorus deposition, phosphorus excretion, or intestine length. This study highlights the importance of experimentally testing the putative selective forces acting on phenotypes and their underlying genotypes in the wild.

Divergent Fine-Scale Recombination Landscapes between a Freshwater and Marine Population of Threespine Stickleback Fish.

Meiotic recombination is a highly conserved process that has profound effects on genome evolution. At a fine-scale, recombination rates can vary drastically across genomes, often localized into small recombination "hotspots" with highly elevated rates, surrounded by regions with little recombination. In most species studied, the location of hotspots within genomes is highly conserved across broad evolutionary timescales. The main exception to this pattern is in mammals, where hotspot location can evolve rapidly among closely related species and even among populations within a species. Hotspot position in mammals is controlled by the gene, Prdm9, whereas in species with conserved hotspots, a functional Prdm9 is typically absent. Due to a limited number of species where recombination rates have been estimated at a fine-scale, it remains unclear whether hotspot conservation is always associated with the absence of a functional Prdm9. Threespine stickleback fish (Gasterosteus aculeatus) are an excellent model to examine the evolution of recombination over short evolutionary timescales. Using a linkage disequilibrium-based approach, we found recombination rates indeed varied at a fine-scale across the genome, with many regions organized into narrow hotspots. Hotspots had highly divergent landscapes between stickleback populations, where only ∼15% of these hotspots were shared. Our results indicate that fine-scale recombination rates may be diverging between closely related populations of threespine stickleback fish. Interestingly, we found only a weak association of a PRDM9 binding motif within hotspots, which suggests that threespine stickleback fish may possess a novel mechanism for targeting recombination hotspots at a fine-scale.

Two Drosophilids exhibit distinct EGF pathway patterns in oogenesis.

Deciphering the evolution of morphological structures is a remaining challenge in the field of developmental biology. The respiratory structures of insect eggshells, called the dorsal appendages, provide an outstanding system for exploring these processes since considerable information is known about their patterning and morphogenesis in Drosophila melanogaster and dorsal appendage number and morphology vary widely across Drosophilid species. We investigated the patterning differences that might facilitate morphogenetic differences between D. melanogaster, which produces two oar-like structures first by wrapping and then elongating the tubes via cell intercalation and cell crawling, and Scaptodrosophila lebanonensis, which produces a variable number of appendages simply by cell intercalation and crawling. Analyses of BMP pathway components thickveins and P-Mad demonstrate that anterior patterning is conserved between these species. In contrast, EGF signaling exhibits significant differences. Transcripts for the ligand encoded by gurken localize similarly in the two species, but this morphogen creates a single dorsolateral primordium in S. lebanonensis as defined by activated MAP kinase and the downstream marker broad. Expression patterns of pointed, argos, and Capicua, early steps in the EGF pathway, exhibit a heterochronic shift in S. lebanonensis relative to those seen in D. melanogaster. We demonstrate that the S. lebanonensis Gurken homolog is active in D. melanogaster but is insufficient to alter downstream patterning responses, indicating that Gurken-EGF receptor interactions do not distinguish the two species' patterning. Altogether, these results differentiate EGF signaling patterns between species and shed light on how changes to the regulation of patterning genes may contribute to different tube-forming mechanisms.

Evolution of Schooling Behavior in Threespine Sticklebacks Is Shaped by the Eda Gene.

Despite longstanding interest in the genetic mechanisms that underlie behavioral evolution, very few genes that underlie naturally occurring variation in behavior between individuals or species are known, particularly in vertebrates. Here, we build on our previous forward genetic mapping experiments and use transgenic approaches to identify Ectodysplasin as a gene that causes differences in schooling behavior between wild populations of threespine stickleback (Gasterosteus aculeatus) fish. This work provides rare insight into the proximate mechanisms that have shaped the evolution of vertebrate behavior.

The genomic landscape of juvenile myelomonocytic leukemia.

Juvenile myelomonocytic leukemia (JMML) is a myeloproliferative neoplasm (MPN) of childhood with a poor prognosis. Mutations in NF1, NRAS, KRAS, PTPN11 or CBL occur in 85% of patients, yet there are currently no risk stratification algorithms capable of predicting which patients will be refractory to conventional treatment and could therefore be candidates for experimental therapies. In addition, few molecular pathways aside from the RAS-MAPK pathway have been identified that could serve as the basis for such novel therapeutic strategies. We therefore sought to genomically characterize serial samples from patients at diagnosis through relapse and transformation to acute myeloid leukemia to expand knowledge of the mutational spectrum in JMML. We identified recurrent mutations in genes involved in signal transduction, splicing, Polycomb repressive complex 2 (PRC2) and transcription. Notably, the number of somatic alterations present at diagnosis appears to be the major determinant of outcome.

Multiple paternity is a shared reproductive strategy in the live-bearing surfperches (Embiotocidae) that may be associated with female fitness.

According to Bateman's principle, female fecundity is limited relative to males, setting the expectation that males should be promiscuous, while females should be choosy and select fewer mates. However, several surfperches (Embiotocidae) exhibit multiple paternity within broods indicating that females mate with multiple males throughout the mating season. Previous studies found no correlation between mating success and reproductive success (i.e., a Bateman gradient). However, by including samples from a broader range of reproductive size classes, we found evidence of a Bateman gradient in two surfperch species from distinct embiotocid clades. Using microsatellite analyses, we found that 100% of the spotfin surfperch families sampled exhibit multiple paternity (Hyperprosopon anale, the basal taxon from the only clade that has not previously been investigated) indicating that this tactic is a shared reproductive strategy among surfperches. Further, we detected evidence for a Bateman gradient in H. anale; however, this result was not significant after correction for biases. Similarly, we found evidence for multiple paternity in 83% of the shiner surfperch families (Cymatogaster aggregata) sampled. When we combine these data with a previous study on the same species, representing a larger range of reproductive size classes and associated brood sizes, we detect a Bateman gradient in shiner surfperch for the first time that remains significant after several conservative tests for bias correction. These results indicate that sexual selection is likely complex in this system, with the potential for conflicting optima between sexes, and imply a positive shift in fertility (i.e., increasing number) and reproductive tactic with respect to the mating system and number of sires throughout the reproductive life history of females. We argue that the complex reproductive natural history of surfperches is characterized by several traits that may be associated with cryptic female choice, including protracted oogenesis, uterine sac complexity, and sperm storage.

HoxA and HoxD expression in a variety of vertebrate body plan features reveals an ancient origin for the distal Hox program.

BACKGROUND: Hox genes are master regulatory genes that specify positional identities during axial development in animals. Discoveries regarding their concerted expression patterns have commanded intense interest due to their complex regulation and specification of body plan features in jawed vertebrates. For example, the posterior HoxD genes switch to an inverted collinear expression pattern in the mouse autopod where HoxD13 switches from a more restricted to a less restricted domain relative to its neighboring gene on the cluster. We refer to this program as the 'distal phase' (DP) expression pattern because it occurs in distal regions of paired fins and limbs, and is regulated independently by elements in the 5' region upstream of the HoxD cluster. However, few taxa have been evaluated with respect to this pattern, and most studies have focused on pectoral fin morphogenesis, which occurs relatively early in development. RESULTS: Here, we demonstrate for the first time that the DP expression pattern occurs with the posterior HoxA genes, and is therefore not solely associated with the HoxD gene cluster. Further, DP Hox expression is not confined to paired fins and limbs, but occurs in a variety of body plan features, including paddlefish barbels - sensory adornments that develop from the first mandibular arch (the former 'Hox-free zone), and the vent (a medial structure that is analogous to a urethra). We found DP expression of HoxD13 and HoxD12 in the paddlefish barbel; and we present the first evidence for DP expression of the HoxA genes in the hindgut and vent of three ray-finned fishes. The HoxA DP expression pattern is predicted by the recent finding of a shared 5' regulatory architecture in both the HoxA and HoxD clusters, but has not been previously observed in any body plan feature. CONCLUSIONS: The Hox DP expression pattern appears to be an ancient module that has been co-opted in a variety of structures adorning the vertebrate bauplan. This module provides a shared genetic program that implies deep homology of a variety of distally elongated structures that has played a significant role in the evolution of morphological diversity in vertebrates.

A re-consideration of the taxonomic status of Nebria lacustris Casey (Coleoptera, Carabidae, Nebriini) based on multiple datasets - a single species or a species complex?

This study gathered evidence from principal component analysis (PCA) of morphometric data and molecular analyses of nucleotide sequence data for four nuclear genes (28S, TpI, CAD1, and Wg) and two mitochondrial genes (COI and 16S), using parsimony, maximum likelihood, and Bayesian methods. This evidence was combined with morphological and chorological data to re-evaluate the taxonomic status of Nebria lacustris Casey sensu lato. PCA demonstrated that both body size and one conspicuous aspect of pronotal shape vary simultaneously with elevation, latitude, and longitude and served to distinguish populations from the southern Appalachian highlands, south of the French Broad, from all other populations. Molecular analyses revealed surprisingly low overall genetic diversity within Nebria lacustris sensu lato, with only 0.39% of 4605 bp varied in the concatenated dataset. Evaluation of patterns observed in morphological and genetic variation and distribution led to the following taxonomic conclusions: (1) Nebria lacustris Casey and Nebria bellorum Kavanaugh should be considered distinct species, which is a NEW STATUS for Nebria bellorum. (2) No other distinct taxonomic subunits could be distinguished with the evidence at hand, but samples from northeastern Iowa, in part of the region known as the "Driftless Zone", have unique genetic markers for two genes that hint at descent from a local population surviving at least the last glacial advance. (3) No morphometric or molecular evidence supports taxonomic distinction between lowland populations on the shores of Lake Champlain and upland populations in the adjacent Green Mountains of Vermont, despite evident size and pronotal shape differences between many of their members.

Germline CBL mutations cause developmental abnormalities and predispose to juvenile myelomonocytic leukemia.

CBL encodes a member of the Cbl family of proteins, which functions as an E3 ubiquitin ligase. We describe a dominant developmental disorder resulting from germline missense CBL mutations, which is characterized by impaired growth, developmental delay, cryptorchidism and a predisposition to juvenile myelomonocytic leukemia (JMML). Some individuals experienced spontaneous regression of their JMML but developed vasculitis later in life. Importantly, JMML specimens from affected children show loss of the normal CBL allele through acquired isodisomy. Consistent with these genetic data, the common p.371Y>H altered Cbl protein induces cytokine-independent growth and constitutive phosphorylation of ERK, AKT and S6 only in hematopoietic cells in which normal Cbl expression is reduced by RNA interference. We conclude that germline CBL mutations have developmental, tumorigenic and functional consequences that resemble disorders that are caused by hyperactive Ras/Raf/MEK/ERK signaling and include neurofibromatosis type 1, Noonan syndrome, Costello syndrome, cardiofaciocutaneous syndrome and Legius syndrome.

Mutations in CBL occur frequently in juvenile myelomonocytic leukemia.

Juvenile myelomonocytic leukemia is an aggressive myeloproliferative disorder characterized by malignant transformation in the hematopoietic stem cell compartment with proliferation of differentiated progeny. Seventy-five percent of patients harbor mutations in the NF1, NRAS, KRAS, or PTPN11 genes, which encode components of Ras signaling networks. Using single nucleotide polymorphism arrays, we identified a region of 11q isodisomy that contains the CBL gene in several JMML samples, and subsequently identified CBL mutations in 27 of 159 JMML samples. Thirteen of these mutations alter codon Y371. In this report, we also demonstrate that CBL and RAS/PTPN11 mutations were mutually exclusive in these patients. Moreover, the exclusivity of CBL mutations with respect to other Ras pathway-associated mutations indicates that CBL may have a role in deregulating this key pathway in JMML.

Single-cell profiling identifies aberrant STAT5 activation in myeloid malignancies with specific clinical and biologic correlates.

Progress in understanding the molecular pathogenesis of human myeloproliferative disorders (MPDs) has led to guidelines incorporating genetic assays with histopathology during diagnosis. Advances in flow cytometry have made it possible to simultaneously measure cell type and signaling abnormalities arising as a consequence of genetic pathologies. Using flow cytometry, we observed a specific evoked STAT5 signaling signature in a subset of samples from patients suspected of having juvenile myelomonocytic leukemia (JMML), an aggressive MPD with a challenging clinical presentation during active disease. This signature was a specific feature involving JAK-STAT signaling, suggesting a critical role of this pathway in the biological mechanism of this disorder and indicating potential targets for future therapies.

JAK2 V617F positive polycythemia Vera in a child with neurofibromatosis type I.

We report a child with polycythemia vera (PV). This patient demonstrates the acquired somatic JAK2 V617F mutation and also has neurofibromatosis type I (NF1). NF1, while not previously associated with PV, is associated with another childhood MPD, juvenile myelomonocytic leukemia (JMML). Thus we examined a number of genetic abnormalities identified in JMML patients, but found no association in this case. Neurofibromin sequencing failed to identify a causative mutation. An unknown genetic abnormality resulting in NF1 may have predisposed this young child to acquiring the common JAK2 mutation.

Development of an allele-specific minimal residual disease assay for patients with juvenile myelomonocytic leukemia.

Juvenile myelomonocytic leukemia is an aggressive and frequently lethal myeloproliferative disorder of childhood. Somatic mutations in NRAS, KRAS, or PTPN11 occur in 60% of cases. Monitoring disease status is difficult because of the lack of characteristic leukemic blasts at diagnosis. We designed a fluorescently based, allele-specific polymerase chain reaction assay called TaqMAMA to detect the most common RAS or PTPN11 mutations. We analyzed peripheral blood and/or bone marrow of 25 patients for levels of mutant alleles over time. Analysis of pre-hematopoietic stem-cell transplantation, samples revealed a broad distribution of the quantity of the mutant alleles. After hematopoietic stem-cell transplantation, the level of the mutant allele rose rapidly in patients who relapsed and correlated well with falling donor chimerism. Simultaneously analyzed peripheral blood and bone marrow samples demonstrate that blood can be monitored for residual disease. Importantly, these assays provide a sensitive strategy to evaluate molecular responses to new therapeutic strategies.