Andrew D. Calcino

Intestinal inflammation continues in a subset of patients with celiac disease despite a gluten-free diet. Here, by applying multi-omic single-cell analysis to duodenal biopsies, we found that low-grade malignancies with lymphoma driver mutations in patients with refractory celiac disease type 2 (RCD2) are comprised by surface CD3-negative (sCD3 ⁻ ) lymphocytes stalled at an innate lymphoid cell (ILC)–progenitor T cell stage undergoing extensive TRA , TRB , and TRD TCR recombination. In people with refractory celiac disease type 1 (RCD1), a disease currently lacking explanation, we identified sCD3 ⁺ T cells with lymphoma driver mutations in 6 of 10 individuals with RCD1 and in one of the patients with active, recently diagnosed celiac disease. Furthermore, the mutant T cells formed large TCRαβ clones and displayed inflammatory and cytotoxic molecular profiles. Thus, accumulation of lymphoma driver–mutated T cells and sCD3 ⁻ progenitors may contribute to chronic, nonresponsive celiac disease.

Bryozoans are mostly sessile colonial invertebrates that inhabit all kinds of aquatic ecosystems. Extant bryozoan species fall into two clades with one of them, Phylactolaemata, being the only exclusively freshwater clade. Phylogenetic relationships within the class Phylactolaemata have long been controversial owing to their limited distinguishable characteristics that reflect evolutionary relationships. Here, we present the first phylogenomic analysis of Phylactolaemata using transcriptomic data combined with dense taxon sampling of six families to better resolve the interrelationships and to estimate divergence time. Using maximum-likelihood and Bayesian inference approaches, we recovered a robust phylogeny for Phylactolaemata in which the interfamilial relationships are fully resolved. We show Stephanellidae is the sister taxon of all other phylactolaemates and confirm that Lophopodidae represents the second offshoot within the phylactolaemate tree.Plumatella fruticosaclearly falls outside Plumatellidae as previous investigations have suggested, and instead clusters with Pectinatellidae and Cristatellidae as the sister taxon of Fredericellidae. Our results demonstrate that cryptic speciation is very likely inF. sultanaand in two species ofPlumatella(P. repensandP. casmiana). Divergence time estimates show that Phylactolaemata appeared at the end of the Ediacaran and started to diverge in the Silurian, although confidence intervals were large for most nodes. The radiation of most extant phylactolaemate families occurred mainly in the Palaeogene and Neogene highlighting post-extinction diversification.

The mesoderm gives rise to several key morphological features of bilaterian animals including endoskeletal elements and the musculature. A number of regulatory genes involved in mesoderm and/or muscle formation (e.g., Brachyury (Bra), even-skipped (eve), Mox, myosin II heavy chain (mhc)) have been identified chiefly from chordates and the ecdysozoans Drosophila and Caenorhabditis elegans, but data for non-model protostomes, especially those belonging to the ecdysozoan sister clade, Lophotrochozoa (e.g., flatworms, annelids, mollusks), are only beginning to emerge. Within the lophotrochozoans, Mollusca constitutes the most speciose and diverse phylum. Interestingly, however, information on the morphological and molecular underpinnings of key ontogenetic processes such as mesoderm formation and myogenesis remains scarce even for prominent molluscan sublineages such as the bivalves. Here, we investigated myogenesis and developmental expression of Bra, eve, Mox, and mhc in the quagga mussel Dreissena rostriformis, an invasive freshwater bivalve and an emerging model in invertebrate evodevo. We found that all four genes are expressed during mesoderm formation, but some show additional, individual sites of expression during ontogeny. While Mox and mhc are involved in early myogenesis, eve is also expressed in the embryonic shell field and Bra is additionally present in the foregut. Comparative analysis suggests that Mox has an ancestral role in mesoderm and possibly muscle formation in bilaterians, while Bra and eve are conserved regulators of mesoderm development of nephrozoans (protostomes and deuterostomes). The fully developed Dreissena veliger larva shows a highly complex muscular architecture, supporting a muscular ground pattern of autobranch bivalve larvae that includes at least a velum muscle ring, three or four pairs of velum retractors, one or two pairs of larval retractors, two pairs of foot retractors, a pedal plexus, possibly two pairs of mantle retractors, and the muscles of the pallial line, as well as an anterior and a posterior adductor. As is typical for their molluscan kin, remodelling and loss of prominent larval features such as the velum musculature and various retractor systems appear to be also common in bivalves.

As complete genomes become easier to attain, even from previously difficult-to-sequence species, and as genomic resequencing becomes more routine, it is becoming obvious that genomic structural variation is more widespread than originally thought and plays an important role in maintaining genetic variation in populations. Structural variants (SVs) and associated gene presence–absence variation (PAV) can be important players in local adaptation, allowing the maintenance of genetic variation and taking part in other evolutionarily relevant phenomena. While recent studies have highlighted the importance of structural variation in Mollusca, the prevalence of this phenomenon in the broader context of marine organisms remains to be fully investigated. Here, we describe a straightforward and broadly applicable method for the identification of SVs in fully assembled diploid genomes, leveraging the same reads used for assembly. We also explain a gene PAV analysis protocol, which could be broadly applied to any species with a fully sequenced reference genome available. Although the strength of these approaches have been tested and proven in marine invertebrates, which tend to have high levels of heterozygosity, possibly due to their lifestyle traits, they are also applicable to other species across the tree of life, providing a ready means to begin investigations into this potentially widespread phenomena. Key words Hemizygosity Genome Presence–Absence Variation Structural Variation Pangenome

The mesoderm is considered the youngest of the three germ layers. Although its morphogenesis has been studied in some metazoans, the molecular components underlying this process remain obscure for numerous phyla including the highly diverse Mollusca. Here, expression of Hairy and enhancer of split (HES), Mox, and myosin heavy chain (MHC) was investigated in Acanthochitona fascicularis, a representative of Polyplacophora with putative ancestral molluscan features. While AfaMHC is expressed throughout myogenesis, AfaMox1 is only expressed during early stages of mesodermal band formation and in the ventrolateral muscle, an autapomorphy of the polyplacophoran trochophore. Comparing our findings to previously published data across Metazoa reveals Mox expression in the mesoderm in numerous bilaterians including gastropods, polychaetes, and brachiopods. It is also involved in myogenesis in molluscs, annelids, tunicates, and craniates, suggesting a dual role of Mox in mesoderm and muscle formation in the last common bilaterian ancestor. AfaHESC2 is expressed in the ectoderm of the polyplacophoran gastrula and later in the mesodermal bands and in putative neural tissue, whereas AfaHESC7 is expressed in the trochoblasts of the gastrula and during foregut formation. This confirms the high developmental variability of HES gene expression and demonstrates that Mox and HES genes are pleiotropic.

Ecdysis-related neuropeptides (ERNs), including eclosion hormone, crustacean cardioactive peptide, myoinhibitory peptide, bursicon alpha, and bursicon beta regulate molting in insects and crustaceans. Recent evidence further revealed that ERNs likely play an ancestral role in invertebrate life cycle transitions, but their tempo-spatial expression patterns have not been investigated outside Arthropoda. Using RNA-seq and in situ hybridization, we show that ERNs are broadly expressed in the developing nervous system of a mollusk, the polyplacophoran Acanthochitona fascicularis. While some ERN-expressing neurons persist from larval to juvenile stages, others are only present during settlement and metamorphosis. These transient neurons belong to the “ampullary system,” a polyplacophoran-specific larval sensory structure. Surprisingly, however, ERN expression is absent from the apical organ, another larval sensory structure that degenerates before settlement is completed in A. fascicularis. Our findings thus support a role of ERNs in A. fascicularis metamorphosis but contradict the common notion that the apical organ-like structures shared by various aquatic invertebrates (i.e., cnidarians, annelids, mollusks, echinoderms) are of general importance for this process.

The advent of complete genomic sequencing has opened a window into genomic phenomena obscured by fragmented assemblies. A good example of these is the existence of hemizygous regions of autosomal chromosomes, which can result in marked differences in gene content between individuals within species. While these hemizygous regions, and presence/absence variation of genes that can result, are well known in plants, firm evidence has only recently emerged for their existence in metazoans. Here, we use recently published, complete genomes from wild-caught molluscs to investigate the prevalence of hemizygosity across a well-known and ecologically important clade. We show that hemizygous regions are widespread in mollusc genomes, not clustered in individual chromosomes, and often contain genes linked to transposition, DNA repair and stress response. With targeted investigations of HSP70-12 and C1qDC, we also show how individual gene families are distributed within pan-genomes. This work suggests that extensive pan-genomes are widespread across the conchiferan Mollusca, and represent useful tools for genomic evolution, allowing the maintenance of additional genetic diversity within the population. As genomic sequencing and re-sequencing becomes more routine, the prevalence of hemizygosity, and its impact on selection and adaptation, are key targets for research across the tree of life. This article is part of the Theo Murphy meeting issue ‘Molluscan genomics: broad insights and future directions for a neglected phylum’.

Background: Animal mitochondrial genomes are typically circular, 14-20 kb in length, maternally inherited, contain 13 coding genes, two ribosomal genes and are homoplasmic. In contrast, plant mitogenomes display frequent gene rearrangements, often contain greatly expanded repetitive regions, encode various open reading frames of unknown function and may be heteroplasmic due to differential repeat expansions between molecules. Error correction by recombination is common in plant mitochondria and has been proposed as the driver behind the rearrangements and repeat expansions that are often observed. In contrast, most animal mitochondria never or only very seldomly recombine and their utilisation of other repair mechanisms for mitochondrial genome error correction is a potential driver of their non-coding DNA reduction. Results: Using PacBio long reads for genome assembly and structural variant detection, we identify evidence of heteroplasmy in the form of variable repeat lengths within two blocks of repetitive DNA within the expanded 46 kb mitochondrial genome of the bivalve mollusc, quagga mussel, Dreissena rostriformis . The quagga mussel also has a greatly expanded repertoire of coding genes in comparison to most animals which includes an additional nine open reading frames (ORFs) encoding predicted transmembrane peptides of unknown orthology. Conclusions: The genome size, repeat content and coding gene repertoire of the quagga mussel mitogenome closely resemble those of plants and the identification of repeat-associated heteroplasmy is consistent with the utilisation of plant-like recombination-based error correction mechanisms. Given the frequency of mitochondrial repeat expansions within the Bivalvia, recombination may be an underappreciated mechanism for mitogenomic error correction within this and other animal lineages. Significance Statement: Unlike most animals, the mitochondrial genomes of many bivalve molluscs are often greatly expanded and contain large non-coding regions and additional predicted genes of unknown function. While these features are uncommon in other animal groups, they are common features of plant mitochondrial genomes. Here we show that the mitochondrial genome of the bivalve mollusc, the quagga mussel, displays many plant-like features and additionally, shows evidence of variability in the repeat lengths between mitochondrial molecules within an individual mussel. We propose that similar error correction mechanisms in plants and bivalves may play a role in these observed commonalities.

The advent of complete genomic sequencing has opened a window into genomic phenomena obscured by fragmented assemblies. A good example of these is the existence of hemizygous regions of autosomal chromosomes, which can result in marked differences in gene content between individuals within species. While these hemizygous regions, and presence/absence variation of genes that can result, are well known in plants, firm evidence has only recently emerged for their existence in metazoans. Here we use recently published, complete genomes from wild-caught molluscs to investigate the prevalence of hemizygosity and pan-genomes across a well-known and ecologically important clade. We show that hemizygous regions are widespread in mollusc genomes, not clustered in individual chromosomes, and often contain genes linked to transposition, DNA repair and stress response. With targeted investigations of HSP70-12 and C1qDC , we also show how individual gene families are distributed within pan-genomes. This work suggests that pan-genomes are widespread across the conchiferan Mollusca, and represent useful tools for genomic evolution, allowing the maintenance of additional genetic diversity within the population. As genomic sequencing and re-sequencing becomes more routine, the prevalence of hemizygosity, and its impact on selection and adaptation, are key targets for research across the tree of life.