Andrew Winston Liu

Genome structural variations within species are rare. How selective constraints preserve gene order and chromosome structure is a central question in evolutionary biology that remains unsolved. Our sequencing of several genomes of the appendicularian tunicateOikopleura dioicaaround the globe reveals extreme genome scrambling caused by thousands of chromosomal rearrangements, although showing no obvious morphological differences between these animals. The breakpoint accumulation rate is an order of magnitude higher than in ascidian tunicates, nematodes,Drosophila,or mammals. Chromosome arms and sex-specific regions appear to be the primary unit of macrosynteny conservation. At the microsyntenic level, scrambling did not preserve operon structures, suggesting an absence of selective pressure to maintain them. The uncoupling of the genome scrambling with morphological conservation inO. dioicasuggests the presence of previously unnoticed cryptic species and provides a new biological system that challenges our previous vision of speciation in which similar animals always share similar genome structures.

SUMMARY: Genes are not randomly distributed throughout chromosomes. How gene order evolves and how selective constraints act to preserve or vary gene order, both at the macrosyntenic level of whole chromosomes or microsyntenic level of gene blocks, are central questions of evolutionary biology and genomics that remain largely unsolved. Here, after sequencing several genomes of the appendicularian tunicate Oikopleura dioica from different locations around the globe, we show an unprecedented amount of genome scrambling in animals with no obvious morphological differences, consistent with cryptic speciation. Our assemblies suggest that all members of this clade possess a common 3-chromosome karyotype, and that different species largely preserve gene content, despite the presence of thousands of rearrangements in gene order. The movements of genes are largely restricted to chromosome arms and sex-specific regions, which appear to be the primary unit of macrosynteny conservation, and examples of these within-arm movements can be seen in the Hox and Fgf gene families. Our approach employing whole-genome alignments demonstrates that segments containing protein-coding elements tend to be preserved at the microsyntenic scale, consistent with strong purifying selection, with appreciably less preservation of non-coding elements. Unexpectedly, scrambling did not preserve operon structure across species, suggesting an absence of selective pressure to maintain operon structure. As well, genome scrambling does not occur uniformly across all chromosomes, as short chromosome arms possess shorter genes, smaller operons, more breakpoints, and elevated dN/dS values compared to long chromosome arms. Estimation of divergence times among the cryptic O. dioica lineages yielded an estimated breakpoint accumulation rate of 6 to 25 breakpoints per megabase per million years, which is an order of magnitude higher than the rates for other ascidian tunicates or Drosophila species. Therefore, O. dioica appears to be an attractive animal system to unravel the mechanisms that underlie gene order and synteny conservation, as well as exploring the limits of genome scrambling without an apparent impact on phenotypic evolution.

Appendicularian tunicates are some of the most abundant mesozooplankton organisms with key roles in marine trophic webs and global carbon flux. Like most appendicularians with cosmopolitan distributions, Oikopleura dioica Fol, 1872 is considered a single species worldwide based on morphological features that distinguish them from other appendicularians. Despite their abundance, however, there are still only ~ 70 described appendicularian species, compared to over 2800 ascidian tunicates. Here we perform a molecular phylogenetic, morphological, and reproductive assessment of O. dioica specimens collected from the Ryukyu Archipelago, mainland Japan, and Europe. The specimens are morphologically very similar, with only detailed examination of the oikoplastic epithelium and quantitative measurements revealing minor distinguishing characteristics. Phylogenetic analyses of the ribosomal gene loci and mitochondrial cytochrome oxidase I (COI) gene strongly indicate that they form three separate genetic clades despite their morphological similarities. Finally, in vitro crosses between the Ryukyu and mainland Japanese specimens show total prezygotic reproductive isolation. Our results reveal that the current taxonomic O. dioica classification likely hides multiple cryptic species, highlighting the genetic diversity and complexity of their population structures. Cryptic organisms are often hidden under a single species name because their morphological similarities make them difficult to distinguish and their correct identification is fundamental to understanding Earth’s biodiversity. O. dioica is an attractive model to understand how morphological conservation can be maintained despite pronounced genetic divergence.

Appendicularian tunicates are some of the most abundant mesozooplankton organisms with key roles in marine trophic webs and global carbon flux. Like most appendicularians with cosmopolitan distributions, Oikopleura dioica Fol, 1872 is considered a single species worldwide based on morphological features that distinguish them from other appendicularians. Despite their abundance however, there are still only ∼70 described appendicularian species, compared with over 2,800 ascidian tunicates. Here we perform a molecular phylogenetic, morphological, and reproductive assessment of O. dioica specimens collected from the Ryukyu Archipelago, mainland Japan, and Europe. The specimens are morphologically very similar, with only detailed examination of the oikoplastic epithelium and quantitative measurements revealing minor distinguishing characteristics. Phylogenetic analyses of the ribosomal gene loci and mitochondrial cytochrome oxidase I (COI) gene strongly indicate that they form three separate genetic clades despite their morphological similarities. Finally, in vitro crosses between the Ryukyu and mainland Japanese specimens show total prezygotic reproductive isolation. Our results reveal that the current taxonomic O. dioic a classification likely hides multiple cryptic species, highlighting the genetic diversity and complexity of their population structures. Cryptic organisms are often hidden under a single species name because their morphological similarities make them difficult to disinguish and their correct identification is fundamental to understanding Earth’s biodiversity. O. dioica is an attractive model to understand how morphological conservation can be maintained despite pronounced genetic divergence.

Background: Automation has increasingly become more commonplace in the research laboratory workspace. The introduction of articulated robotic arms allows the researcher more flexibility in the tasks a single piece of automated machinery can perform. We set out to incorporate automation in processing of genomic DNA organic extractions to increase throughput and limit researchers to the exposure of organic solvents. Methods: In order to automate the genome sequencing pipeline in our laboratory, we programmed a dual-arm anthropomorphic robot, the Robotic Biology Institute's Maholo LabDroid, to perform organic solvent-based genomic DNA extraction from cell lysates. To the best of our knowledge, this is the first time that automation of phenol-chloroform extraction has been reported. Results: We achieved routine extraction of high molecular weight genomic DNA (>100 kb) from diverse biological samples including algae cultured in sea water, bacteria, whole insects, and human cell lines. The results of pulse-field electrophoresis size analysis and the N50 sequencing metrics of reads obtained from Nanopore MinION runs verified the presence of intact DNA suitable for direct sequencing. Conclusions: We present the workflow that can be used to program similar robots and discuss the problems and solutions we encountered in developing the workflow. The protocol can be adapted to analogous methods such as RNA extraction, and there is ongoing work to incorporate further post-extraction steps such as library construction. This work shows the potential for automated robotic workflows to free molecular biological researchers from manual interventions in routine experimental work. A time-lapse movie of the entire automated run is included in this report.

In order to automate the genome sequencing pipeline in our laboratory, we programmed a dual-arm anthropomorphic robot, the Robotic Biology Institute’s Maholo LabDroid, to perform organic solvent-based genomic DNA extraction from cell lysates. To the best of our knowledge, this is the first time that automation of phenol-chloroform extraction has been reported. We achieved routine extraction of high molecular weight genomic DNA (>100 kb) from diverse biological samples including algae cultured in sea water, bacteria, whole insects, and human cell lines. The results of pulse-field electrophoresis size analysis and the N50 sequencing metrics of reads obtained from Nanopore MinION runs verified the presence of intact DNA suitable for direct sequencing. We present the workflow that can be used to program similar robots and discuss the problems and solutions we encountered in developing the workflow. The protocol can be adapted to analogous methods such as RNA extraction, and there is ongoing work to incorporate further post-extraction steps such as library construction. This work shows the potential for automated robotic workflows to free molecular biological researchers from manual interventions in routine experimental work. A time-lapse movie of the entire automated run can be seen at http://doi.org/10.6084/m9.figshare.17097065

Eusociality represents a major evolutionary transition that arose independently in at least 12 insect lineages. Despite this prevalence, there remains considerable uncertainty surrounding the catalysing event and underlying genomic changes that enable such modifications. Commonly associated with this evolutionary transition is establishing and maintaining the reproductive division of labour (e.g. a reproductive queen and no-reproductive workers). This division is, at least in part, induced and maintained by highly species-specific pheromones. However, genomic analysis remains conflicted on the role of pheromones in this evolutionary transition. Specifically, if there was co-option of a common pheromone-sensitive genetic pathway present in all progenitor species or strong lineage-specific selection converging on similar transcriptomic signatures. Using a solitary insect model, we sought to determine if various species-specific pheromones induced similar transcriptomic responses, thus activating similar pathways. We measured the transcriptomic and physiological response of a solitary insect, Drosophila melanogaster , to pheromones from bumblebees, honey bees, and termites. Each treatment induced the same strong physiological response - a decreased ovary size. However, employing several methods of transcriptomic analysis, we did not observe conservation in pheromone-mediated gene/pathway regulation. Thus, despite a conserved phenotypic response, the underpinning transcriptome was vastly different. This suggests that pheromone-mediated eusociality is the result of convergent evolution. We propose that mechanisms maintaining eusociality (i.e. proto-pheromone) in early stages of eusocial evolution in each group, thus, acting as a primer for eusociality. This early state is then refined through strong selective pressure, resulting in a converging eusocial phenotype. Visual Figure 1.

Oikopleura dioica is a ubiquitous marine zooplankton of biological interest owing to features that include dioecious reproduction, a short life cycle, conserved chordate body plan, and a compact genome. It is an important tunicate model for evolutionary and developmental research, as well as investigations into marine ecosystems. The genome of north Atlantic O. dioica comprises three chromosomes. However, comparisons with the genomes of O. dioica sampled from mainland and southern Japan revealed extensive sequence differences. Moreover, historical studies have reported widely varying chromosome counts. We recently initiated a project to study the genomes of O. dioica individuals collected from the coastline of the Ryukyu (Okinawa) Islands in southern Japan. Given the potentially large extent of genomic diversity, we employed karyological techniques to count individual animals’ chromosomes in situ using centromere-specific antibodies directed against H3S28P, a prophase-metaphase cell cycle-specific marker of histone H3. Epifluorescence and confocal images were obtained of embryos and oocytes stained with two commercial anti-H3S28P antibodies (Abcam ab10543 and Thermo Fisher 07-145). The data lead us to conclude that diploid cells from Okinawan O. dioica contain three pairs of chromosomes, in line with the north Atlantic populations. The finding facilitates the telomere-to-telomere assembly of Okinawan O. dioica genome sequences and gives insight into the genomic diversity of O. dioica from different geographical locations. The data deposited in the EBI BioImage Archive provide representative images of the antibodies’ staining properties for use in epifluorescent and confocal based fluorescent microscopy.

Background: The larvacean Oikopleura dioica is an abundant tunicate plankton with the smallest (65-70 Mbp) non-parasitic, non-extremophile animal genome identified to date. Currently, there are two genomes available for the Bergen (OdB3) and Osaka (OSKA2016) O. dioica laboratory strains. Both assemblies have full genome coverage and high sequence accuracy. However, a chromosome-scale assembly has not yet been achieved. Results: Here, we present a chromosome-scale genome assembly (OKI2018_I69) of the Okinawan O. dioica produced using long-read Nanopore and short-read Illumina sequencing data from a single male, combined with Hi-C chromosomal conformation capture data for scaffolding. The OKI2018_I69 assembly has a total length of 64.3 Mbp distributed among 19 scaffolds. 99% of the assembly is in five megabase-scale scaffolds. We found telomeres on both ends of the two largest scaffolds, which represent assemblies of two fully contiguous autosomal chromosomes. Each of the other three large scaffolds have telomeres at one end only and we propose that they correspond to sex chromosomes split into a pseudo-autosomal region and X-specific or Y-specific regions. Indeed, these five scaffolds mostly correspond to equivalent linkage groups of OdB3, suggesting overall agreement in chromosomal organization between the two populations. At a more detailed level, the OKI2018_I69 assembly possesses similar genomic features in gene content and repetitive elements reported for OdB3. The Hi-C map suggests few reciprocal interactions between chromosome arms. At the sequence level, multiple genomic features such as GC content and repetitive elements are distributed differently along the short and long arms of the same chromosome. Conclusions: We show that a hybrid approach of integrating multiple sequencing technologies with chromosome conformation information results in an accurate de novo chromosome-scale assembly of O. dioica ’s highly polymorphic genome. This assembly will be a useful resource for genome-wide comparative studies between O. dioica and other species, as well as studies of chromosomal evolution in this lineage.

Oikopleura dioica is a ubiquitous marine tunicate of biological interest due to features that include dioecious reproduction, short life cycle, and vertebrate-like dorsal notochord while possessing a relatively compact genome. The use of tunicates as model organisms, particularly with these characteristics, offers the advantage of facilitating studies in evolutionary development and furthering understanding of enduring attributes found in the more complex vertebrates. At present, we are undertaking an initiative to sequence the genomes of Oikopleura individuals in populations found among the seas surrounding the Ryukyu Islands in southern Japan. To facilitate and validate genome assemblies, karyotyping was employed to count individual animals’ chromosomes in situ using centromere-specific antibodies directed against H3S28P, a prophase-metaphase cell cycle-specific marker of histone H3. New imaging data of embryos and oocytes stained with two different antibodies were obtained; interpretation of these data lead us to conclude that the Okinawan Oikopleura dioica has three pairs of chromosomes, akin to previous results from genomic assemblies in Atlantic populations. The imaging data have been deposited to the open-access EBI BioImage Archive for reuse while additionally providing representative images of two commercially available anti-H3S28P antibodies’ staining properties for use in epifluorescent and confocal based fluorescent microscopy.