Control, Suppression, and Monitoring of Infectious Laryngotracheitis in a Multiage Commercial Layer Pullet Farm in Canada.

A multiage commercial layer pullet operation with a history of chicken embryo-origin (CEO) modified live infectious laryngotracheitis (ILT) virus vaccination suffered severe ILT outbreaks in 2017. The initial sequencing revealed that the circulating virus was of vaccine origin. Changes to the timing and dosage of CEO ILT vaccine failed to control the outbreak. The clinical resolution of the outbreak occurred with the transition to a turkey herpesvirus vector vaccine given in-hatchery, followed by a tissue culture-origin vaccine given on the farm. The circulating ILT viruses were monitored periodically by next-generation sequencing. This site became free of ILT virus within 1 yr after implementing the new vaccination program.

Reporte de caso­Control, supresión y seguimiento de la laringotraqueítis infecciosa en una granja de pollitas de postura comerciales de edades múltiples en Canadá Una operación de pollitas de postura comerciales de edades múltiples con antecedentes de inmunización contra el virus de la laringotraqueítis infecciosa con vacuna viva modificada con origen en embriones de pollo (CEO) sufrió brotes severos de laringotraqueítis infecciosa en el año 2017. La secuenciación inicial reveló que el virus circulante era de origen vacunal. Los cambios en el momento y la dosis de la vacunación con vacuna viva modificada con origen en embriones de pollo para laringotraqueítis no lograron controlar el brote. La resolución clínica del brote se produjo con la transición a una vacuna con vector de herpesvirus de pavo administrada en la incubadora, seguida de una vacuna de origen en cultivo de tejidos administrada en la granja. Los virus de laringotraqueítis circulantes se controlaron periódicamente mediante secuenciación de próxima generación. Este sitio quedó libre del virus de la laringotraqueítis después de un año después de implementar el nuevo programa de vacunación.

Gene-rich UV sex chromosomes harbor conserved regulators of sexual development.

Nonrecombining sex chromosomes, like the mammalian Y, often lose genes and accumulate transposable elements, a process termed degeneration. The correlation between suppressed recombination and degeneration is clear in animal XY systems, but the absence of recombination is confounded with other asymmetries between the X and Y. In contrast, UV sex chromosomes, like those found in bryophytes, experience symmetrical population genetic conditions. Here, we generate nearly gapless female and male chromosome-scale reference genomes of the moss Ceratodon purpureus to test for degeneration in the bryophyte UV sex chromosomes. We show that the moss sex chromosomes evolved over 300 million years ago and expanded via two chromosomal fusions. Although the sex chromosomes exhibit weaker purifying selection than autosomes, we find that suppressed recombination alone is insufficient to drive degeneration. Instead, the U and V sex chromosomes harbor thousands of broadly expressed genes, including numerous key regulators of sexual development across land plants.

Area from image analyses accurately estimates dry-weight biomass of juvenile tissue from the moss Ceratodon purpureus.

PREMISE: Mosses have long served as models for studying many areas of plant biology. Investigators have used two-dimensional measurements of juvenile growth from photographs as a surrogate for dry-weight biomass. The relationship between area and biomass, however, has not been critically evaluated. METHODS: Here we grew axenic tissue cultures of 10 Ceratodon purpureus isolates to study the relationship between these parameters. We measured area and biomass on replicate cultures with two distinct starting inoculum sizes each week for three weeks. We then examined the correlation between area and biomass as well as the influence of variation in inoculum size on both parameters. RESULTS: We found a strong correlation between area and biomass after two weeks of growth. Furthermore, we found inoculum size affected biomass during the first week of growth but not in subsequent weeks and inoculum size had no detectable effect on area. DISCUSSION: These analyses provide experimental confirmation that area is a suitable proxy for biomass and provide clear guidelines for when inoculum size variation may affect downstream growth estimates.

Microarthropod contributions to fitness variation in the common moss Ceratodon purpureus.

The evolution of sustained plant-animal interactions depends critically upon genetic variation in the fitness benefits from the interaction. Genetic analyses of such interactions are limited to a few model systems, in part because genetic variation may be absent or the interacting species may be experimentally intractable. Here, we examine the role of sperm-dispersing microarthropods in shaping reproduction and genetic variation in mosses. We established experimental mesocosms with known moss genotypes and inferred the parents of progeny from mesocosms with and without microarthropods, using a pooled sequencing approach. Moss reproductive rates increased fivefold in the presence of microarthropods, relative to control mesocosms. Furthermore, the presence of microarthropods increased the total number of reproducing moss genotypes, and changed the rank-order of fitness of male and female moss genotypes. Interestingly, the genotypes that reproduced most frequently did not produce sporophytes with the most spores, highlighting the challenge of defining fitness in mosses. These results demonstrate that microarthropods provide a fitness benefit for mosses, and highlight the potential for biotic dispersal agents to alter fitness among moss genotypes.

A pseudomolecule-scale genome assembly of the liverwort Marchantia polymorpha.

Marchantia polymorpha has recently become a prime model for cellular, evo-devo, synthetic biological, and evolutionary investigations. We present a pseudomolecule-scale assembly of the M. polymorpha genome, making comparative genome structure analysis and classical genetic mapping approaches feasible. We anchored 88% of the M. polymorpha draft genome to a high-density linkage map resulting in eight pseudomolecules. We found that the overall genome structure of M. polymorpha is in some respects different from that of the model moss Physcomitrella patens. Specifically, genome collinearity between the two bryophyte genomes and vascular plants is limited, suggesting extensive rearrangements since divergence. Furthermore, recombination rates are greatest in the middle of the chromosome arms in M. polymorpha like in most vascular plant genomes, which is in contrast with P. patens where recombination rates are evenly distributed along the chromosomes. Nevertheless, some other properties of the genome are shared with P. patens. As in P. patens, DNA methylation in M. polymorpha is spread evenly along the chromosomes, which is in stark contrast with the angiosperm model Arabidopsis thaliana, where DNA methylation is strongly enriched at the centromeres. Nevertheless, DNA methylation and recombination rate are anticorrelated in all three species. Finally, M. polymorpha and P. patens centromeres are of similar structure and marked by high abundance of retroelements unlike in vascular plants. Taken together, the highly contiguous genome assembly we present opens unexplored avenues for M. polymorpha research by linking the physical and genetic maps, making novel genomic and genetic analyses, including map-based cloning, feasible.

Hitchhiking the high seas: Global genomics of rafting crabs.

Population differentiation and diversification depend in large part on the ability and propensity of organisms to successfully disperse. However, our understanding of these processes in organisms with high dispersal ability is biased by the limited genetic resolution offered by traditional genotypic markers. Many neustonic animals disperse not only as pelagic larvae, but also as juveniles and adults while drifting or rafting at the surface of the open ocean. In theory, the heightened dispersal ability of these animals should limit opportunities for species diversification and population differentiation. To test these predictions, we used next-generation sequencing of genomewide restriction-site-associated DNA tags (RADseq) and traditional mitochondrial DNA sequencing, to investigate the species-level relationships and global population structure of Planes crabs collected from oceanic flotsam and sea turtles. Our results indicate that species diversity in this clade is low-likely three closely related species-with no evidence of cryptic or undescribed species. Moreover, our results indicate weak population differentiation among widely separated aggregations with genetic indices showing only subtle genetic discontinuities across all oceans of the world (RADseq F ST = 0.08-0.16). The results of this study provide unprecedented resolution of the systematics and global biogeography of this group and contribute valuable information to our understanding of how theoretical dispersal potential relates to actual population differentiation and diversification among marine organisms. Moreover, these results demonstrate the limitations of single gene analyses and the value of genomic-level resolution for estimating contemporary population structure in organisms with large, highly connected populations.

Terrestrial species adapted to sea dispersal: Differences in propagule dispersal of two Caribbean mangroves.

A central goal of comparative phylogeography is to understand how species-specific traits interact with geomorphological history to govern the geographic distribution of genetic variation within species. One key biotic trait with an immense impact on the spatial patterns of intraspecific genetic differentiation is dispersal. Here, we quantify how species-specific traits directly related to dispersal affect genetic variation in terrestrial organisms with adaptations for dispersal by sea, not land-the mangroves of the Caribbean. We investigate the phylogeography of white mangroves (Laguncularia racemosa, Combretaceae) and red mangroves (Rhizophora mangle, Rhizophoraceae) using chloroplast genomes and nuclear markers (thousands of RAD-Seq loci) from individuals throughout the Caribbean. Both coastal tree species have viviparous propagules that can float in salt water for months, meaning they are capable of dispersing long distances. Spatially explicit tests of the role of ocean currents on patterning genetic diversity revealed that ocean currents act as a mechanism for facilitating dispersal, but other means of moving genetic material are also important. We measured pollen- vs. propagule-mediated gene flow and discovered that in white mangroves, seeds were more important for promoting genetic connectivity between populations, but in red mangroves, the opposite was true: pollen contributed more. This result challenges our concept of the importance of both proximity to ocean currents for moving mangrove seeds and the extent of long-distance pollen dispersal. This study also highlights the importance of spatially explicit quantification of both abiotic (ocean currents) and biotic (dispersal) factors contributing to gene flow to understand fully the phylogeographic histories of species.

Adding loci improves phylogeographic resolution in red mangroves despite increased missing data: comparing microsatellites and RAD-Seq and investigating loci filtering.

The widespread adoption of RAD-Seq data in phylogeography means genealogical relationships previously evaluated using relatively few genetic markers can now be addressed with thousands of loci. One challenge, however, is that RAD-Seq generates complete genotypes for only a small subset of loci or individuals. Simulations indicate that loci with missing data can produce biased estimates of key population genetic parameters, although the influence of such biases in empirical studies is not well understood. Here we compare microsatellite data (8 loci) and RAD-Seq data (six datasets ranging from 239 to 25,198 loci) from red mangroves (Rhizophora mangle) in Florida to evaluate how different levels of data filtering influence phylogeographic inferences. For all datasets, we calculated population genetic statistics and evaluated population structure, and for RAD-Seq datasets, we additionally examined population structure using coalescence. We found higher F ST using microsatellites, but that RAD-Seq-based estimates approached those based on microsatellites as more loci with more missing data were included. Analyses of RAD-Seq datasets resolved the classic Gulf-Atlantic coastal phylogeographic break, which was not significant in the microsatellite analyses. Applying multiple levels of filtering to RAD-Seq datasets can provide a more complete picture of potential biases in the data and elucidate subtle phylogeographic patterns.

Multiple factors influence population sex ratios in the Mojave Desert moss Syntrichia caninervis.

PREMISE OF RESEARCH: Natural populations of many mosses appear highly female-biased based on the presence of reproductive structures. This bias could be caused by increased male mortality, lower male growth rate, or a higher threshold for achieving sexual maturity in males. Here we test these hypotheses using samples from two populations of the Mojave Desert moss Syntrichia caninervis. METHODS: We used double-digest restriction-site associated DNA (RAD) sequencing to identify candidate sex-associated loci in a panel of sex-expressing plants. Next, we used putative sex-associated markers to identify the sex of individuals without sex structures. KEY RESULTS: We found a 17:1 patch-level phenotypic female to male sex ratio in the higher elevation site (Wrightwood) and no sex expression at the low elevation site (Phelan). In contrast, on the basis of genetic data, we found a 2:1 female bias at the Wrightwood site and only females at the Phelan site. The relative area occupied by male and female genets was indistinguishable, but males were less genetically diverse. CONCLUSIONS: Our data suggest that both male-biased mortality and sexual dimorphism in thresholds for sex expression could explain genetic and phenotypic sex ratio biases and that phenotypic sex expression alone over-estimates the extent of actual sex ratio bias present in these two populations of S. caninervis.

A method for eliminating bacterial contamination from in vitro moss cultures.

PREMISE OF THE STUDY: Bacterial contamination is a major problem in plant tissue culture, resulting in loss of experimental strains or preventing use of field-collected isolates. Here we evaluated an agar embedding method for eliminating bacteria from experimental cultures of the mosses Ceratodon purpureus and Physcomitrella patens. • METHODS AND RESULTS: We blended moss protonema that had been inoculated with bacteria and embedded the cell fragments in antibiotic-containing, low-concentration agar. The plants were placed in a growth chamber and allowed to grow until the moss grew out of the media. The plants were then transferred to new plates and observed for contamination. The embedding method consistently outperformed standard procedures. • CONCLUSIONS: The embedding method places moss in direct contact with antibiotics, arresting bacterial replication and allowing moss to outgrow contamination. We anticipate this method will prove valuable for other plants capable of clonal propagation by blending.

Meiotic sex ratio variation in natural populations of Ceratodon purpureus (Ditrichaceae).

UNLABELLED: • PREMISE OF STUDY: Sex ratio variation is a common but often unexplained phenomenon in species across the tree of life. Here we evaluate the hypothesis that meiotic sex ratio variation can contribute to the biased sex ratios found in natural populations of the moss Ceratodon purpureus.• METHODS: We obtained sporophytes from several populations of C. purpureus from eastern North America. From each sporophyte, we estimated the mean spore viability by germinating replicate samples on agar plates. We estimated the meiotic sex ratio of each sporophyte by inferring the sex of a random sample of germinated spores (mean = 77) using a PCR-RFLP test. We tested for among-sporophyte variation in viability using an ANOVA and for deviations from 1:1 sex ratio using a χ(2)-test and evaluated the relationship between these quantities using a linear regression.• KEY RESULTS: We found among-sporophyte variation in spore viability and meiotic sex ratio, suggesting that genetic variants that contribute to variation in both of these traits segregate within populations of this species. However, we found no relationship between these quantities, suggesting that factors other than sex ratio distorters contribute to variation in spore viability within populations.• CONCLUSIONS: These results demonstrate that sex ratio distortion may partially explain the population sex ratio variation seen in C. purpureus, but more generally that genetic conflict over meiotic segregation may contribute to fitness variation in this species. Overall, this study lays the groundwork for future studies on the genetic basis of meiotic sex ratio variation.

Recent gene-capture on the UV sex chromosomes of the moss Ceratodon purpureus.

Sex chromosomes evolve from ordinary autosomes through the expansion and subsequent degeneration of a region of suppressed recombination that is inherited through one sex. Here we investigate the relative timing of these processes in the UV sex chromosomes of the moss Ceratodon purpureus using molecular population genetic analyses of eight newly discovered sex-linked loci. In this system, recombination is suppressed on both the female-transmitted (U) sex chromosome and the male-transmitted (V) chromosome. Genes on both chromosomes therefore should show the deleterious effects of suppressed recombination and sex-limited transmission, while purifying selection should maintain homologs of genes essential for both sexes on both sex chromosomes. Based on analyses of eight sex-linked loci, we show that the nonrecombining portions of the U and V chromosomes expanded in at least two events (~0.6-1.3 MYA and ~2.8-3.5 MYA), after the divergence of C. purpureus from its dioecious sister species, Trichodon cylindricus and Cheilothela chloropus. Both U- and V-linked copies showed reduced nucleotide diversity and limited population structure, compared to autosomal loci, suggesting that the sex chromosomes experienced more recent selective sweeps that the autosomes. Collectively these results highlight the dynamic nature of gene composition and molecular evolution on nonrecombining portions of the U and V sex chromosomes.

Estimating the nucleotide diversity in Ceratodon purpureus (Ditrichaceae) from 218 conserved exon-primed, intron-spanning nuclear loci.

PREMISE OF THE STUDY: We developed and tested primers for 218 nuclear loci for studying population genetics, phylogeography, and genome evolution in bryophytes. • METHODS AND RESULTS: We aligned expressed sequence tags (ESTs) from Ceratodon purpureus to the Physcomitrella patens genome sequence, and designed primers that are homologous to conserved exons but span introns in the P. patens genome. We tested these primers on four isolates from New York, USA; Otavalo, Ecuador; and two laboratory isolates from Austria (WT4 and GG1). The median genome-wide nucleotide diversity was 0.008 substitutions/site, but the range was large (0-0.14), illustrating the among-locus heterogeneity in the species. • CONCLUSIONS: These loci provide a valuable resource for finely resolved, genome-wide population genetic and species-level phylogenetic analyses of C. purpureus and its relatives.