Phylogenomic reconstruction of Solenogastres (Mollusca, Aplacophora) informs hypotheses on body size evolution.

Body size is a fundamental characteristic of animals that impacts every aspect of their biology from anatomical complexity to ecology. In Mollusca, Solenogastres has been considered important to understanding the group's early evolution as most morphology-based phylogenetic reconstructions placed it as an early branching molluscan lineage. Under this scenario, molluscs were thought to have evolved from a small, turbellarian-like ancestor and small (i.e., macrofaunal) body size was inferred to be plesiomorphic for Solenogastres. More recently, phylogenomic studies have shown that aplacophorans (Solenogastres + Caudofoveata) form a clade with chitons (Polyplacophora), which is sister to all other molluscs, suggesting a relatively large-bodied (i.e., megafaunal) ancestor for Mollusca. Meanwhile, recent investigations into aplacophoran phylogeny have called the assumption that the last common ancestor of Solenogastres was small-bodied into question, but sampling of meiofaunal species was limited, biasing these studies towards large-bodied taxa and leaving fundamental questions about solenogaster body size evolution unanswered. Here, we supplemented available data with transcriptomes from eight diverse meiofaunal species of Solenogastres and conducted phylogenomic analyses on datasets of up to 949 genes. Maximum likelihood analyses support the meiofaunal family Meiomeniidae as the sister group to all other solenogasters, congruent with earlier ideas of a small-bodied ancestor of Solenogastres. In contrast, Bayesian Inference analyses support the large-bodied family Amphimeniidae as the sister group to all other solenogasters. Investigation of phylogenetic signal by comparing site-wise likelihood scores for the two competing hypotheses support the Meiomeniidae-first topology. In light of these results, we performed ancestral character state reconstruction to explore the implications of both hypotheses on understanding of Solenogaster evolution and review previous hypotheses about body size evolution and its potential consequences for solenogaster biology. Both hypotheses imply that body size evolution has been highly dynamic over the course of solenogaster evolution and that their relatively static body plan has successfully allowed for evolutionary transitions between meio-, macro- and megafaunal size ranges.

Radula and Shell Microstructure Variations are Congruent with a Molecular Estimate of Shallow-Water Japanese Chitons.

Variations of the radula and shell microstructures in 33 species of Japanese chiton were investigated along with molecular phylogenetic trees. The molecular phylogenetic trees indicated that Chitonida was composed of four clades, of which two clades formed Acanthochitonina and corresponded to Mopalioidea and Cryptoplacoidea, respectively, and the other clades formed Chitonina. In the radula, the shapes of the central and centro-lateral teeth and the petaloid process varied greatly among species or genera and were useful for the identification of particular species or genera. The presence of accessory and petaloid processes and the cusp shape were relatively conserved and useful for recognizing particular genera or even suborders. In the valves, four to six shell layers were found at the section, but the ventral mesostracum was not observed in Acanthochitonina. The shell microstructures in the ventral sublayer of the tegmentum varied at suborder, but those in the other layers were almost constant. The megalaesthete chamber type varied at superfamily and was helpful to identify particular families or superfamilies. The characteristics of the shell layers and shell microstructures appear to be a synapomorphy shared by the members of Acanthochitonina. The classification within Chitonina needs to be reexamined because the variations of the cusp shape and megalaesthete chamber type were relatively large and did not correspond to the current classification. Callochiton formed a sister group with Chitonida and would be equally closely related to Chitonina and Acanthochitonina because of possessing a mosaic of characteristics from both.

Genome of the lepidopleurid chiton Hanleya hanleyi (Mollusca, Polyplacophora).

Mollusca is the second most species-rich phylum and includes animals as disparate as octopuses, clams, and chitons. Dozens of molluscan genomes are available, but only one representative of the subphylum Aculifera, the sister taxon to all other molluscs, has been sequenced to date, hindering comparative and evolutionary studies. To facilitate evolutionary studies across Mollusca, we sequenced the genome of a second aculiferan mollusc, the lepidopleurid chiton Hanleya hanleyi (Bean 1844), using a hybrid approach combining Oxford Nanopore and Illumina reads. After purging redundant haplotigs and removing contamination from this 1.3% heterozygous genome, we produced a 2.5 Gbp haploid assembly (>4X the size of the other chiton genome sequenced to date) with an N50 of 65.0 Kbp. Despite a fragmented assembly, the genome is rather complete (92.0% of BUSCOs detected; 79.4% complete plus 12.6% fragmented). Remarkably, the genome has the highest repeat content of any molluscan genome reported to date (>66%). Our gene annotation pipeline predicted 69,284 gene models (92.9% of BUSCOs detected; 81.8% complete plus 11.1% fragmented) of which 35,362 were supported by transcriptome and/or protein evidence. Phylogenomic analysis recovered Polyplacophora sister to all other sampled molluscs with maximal support. The Hanleya genome will be a valuable resource for studies of molluscan biology with diverse potential applications ranging from evolutionary and comparative genomics to molecular ecology.

HES and Mox genes are expressed during early mesoderm formation in a mollusk with putative ancestral features.

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.

The Iron-Responsive Genome of the Chiton Acanthopleura granulata.

Molluscs biomineralize structures that vary in composition, form, and function, prompting questions about the genetic mechanisms responsible for their production and the evolution of these mechanisms. Chitons (Mollusca, Polyplacophora) are a promising system for studies of biomineralization because they build a range of calcified structures including shell plates and spine- or scale-like sclerites. Chitons also harden the calcified teeth of their rasp-like radula with a coat of iron (as magnetite). Here we present the genome of the West Indian fuzzy chiton Acanthopleura granulata, the first from any aculiferan mollusc. The A. granulata genome contains homologs of many genes associated with biomineralization in conchiferan molluscs. We expected chitons to lack genes previously identified from pathways conchiferans use to make biominerals like calcite and nacre because chitons do not use these materials in their shells. Surprisingly, the A. granulata genome has homologs of many of these genes, suggesting that the ancestral mollusc may have had a more diverse biomineralization toolkit than expected. The A. granulata genome has features that may be specialized for iron biomineralization, including a higher proportion of genes regulated directly by iron than other molluscs. A. granulata also produces two isoforms of soma-like ferritin: one is regulated by iron and similar in sequence to the soma-like ferritins of other molluscs, and the other is constitutively translated and is not found in other molluscs. The A. granulata genome is a resource for future studies of molluscan evolution and biomineralization.

A mitogenomic phylogeny of chitons (Mollusca: Polyplacophora).

BACKGROUND: Polyplacophora, or chitons, have long fascinated malacologists for their distinct and rather conserved morphology and lifestyle compared to other mollusk classes. However, key aspects of their phylogeny and evolution remain unclear due to the few morphological, molecular, or combined phylogenetic analyses, particularly those addressing the relationships among the major chiton lineages. RESULTS: Here, we present a mitogenomic phylogeny of chitons based on 13 newly sequenced mitochondrial genomes along with eight available ones and RNAseq-derived mitochondrial sequences from four additional species. Reconstructed phylogenies largely agreed with the latest advances in chiton systematics and integrative taxonomy but we identified some conflicts that call for taxonomic revisions. Despite an overall conserved gene order in chiton mitogenomes, we described three new rearrangements that might have taxonomic utility and reconstructed the most likely scenario of gene order change in this group. Our phylogeny was time-calibrated using various fossils and relaxed molecular clocks, and the robustness of these analyses was assessed with several sensitivity analyses. The inferred ages largely agreed with previous molecular clock estimates and the fossil record, but we also noted that the ambiguities inherent to the chiton fossil record might confound molecular clock analyses. CONCLUSIONS: In light of the reconstructed time-calibrated framework, we discuss the evolution of key morphological features and call for a continued effort towards clarifying the phylogeny and evolution of chitons.

Dorsoventral decoupling of Hox gene expression underpins the diversification of molluscs.

In contrast to the Hox genes in arthropods and vertebrates, those in molluscs show diverse expression patterns with differences reported among lineages. Here, we investigate 2 phylogenetically distant molluscs, a gastropod and a polyplacophoran, and show that the Hox expression in both species can be divided into 2 categories. The Hox expression in the ventral ectoderm generally shows a canonical staggered pattern comparable to the patterns of other bilaterians and likely contributes to ventral patterning, such as neurogenesis. The other category of Hox expression on the dorsal side is strongly correlated with shell formation and exhibits lineage-specific characteristics in each class of mollusc. This generalized model of decoupled dorsoventral Hox expression is compatible with known Hox expression data from other molluscan lineages and may represent a key characteristic of molluscan Hox expression. These results support the concept of widespread staggered Hox expression in Mollusca and reveal aspects that may be related to the evolutionary diversification of molluscs. We propose that dorsoventral decoupling of Hox expression allowed lineage-specific dorsal and ventral patterning, which may have facilitated the evolution of diverse body plans in different molluscan lineages.

Phylogenomics of Aplacophora (Mollusca, Aculifera) and a solenogaster without a foot.

Recent molecular phylogenetic investigations strongly supported the placement of the shell-less, worm-shaped aplacophoran molluscs (Solenogastres and Caudofoveata) and chitons (Polyplacophora) in a clade called Aculifera, which is the sister taxon of all other molluscs. Thus, understanding the evolutionary history of aculiferan molluscs is important for understanding early molluscan evolution. In particular, fundamental questions about evolutionary relationships within Aplacophora have long been unanswered. Here, we supplemented the paucity of available data with transcriptomes from 25 aculiferans and conducted phylogenomic analyses on datasets with up to 525 genes and 75 914 amino acid positions. Our results indicate that aplacophoran taxonomy requires revision as several traditionally recognized groups are non-monophyletic. Most notably, Cavibelonia, the solenogaster taxon defined by hollow sclerites, is polyphyletic, suggesting parallel evolution of hollow sclerites in multiple lineages. Moreover, we describe Apodomenia enigmatica sp. nov. , a bizarre new species that appears to be a morphological intermediate between Solenogastres and Caudofoveata. This animal is not a missing link, however; molecular and morphological studies show that it is a derived solenogaster that lacks a foot, mantle cavity and radula. Taken together, these results shed light on the evolutionary history of Aplacophora and reveal a surprising degree of morphological plasticity within the group.

Phylogenetic Relationships Among Japanese Species of the Genus Ischnochiton (Polyplacophora: Ischnochitonidae), Including a New Species.

Seven (including one new) species of the polyplacophoran genus Ischnochiton (Ischnochitonidae) from the Pacific coast of Japan, namely, I. boninensis, I. comptus, I. manazuruensis, I. hakodadensis, I. hayamii sp. nov., I. paululus, and I. poppei, were investigated on the basis of DNA sequence analyses of COI, 16S rRNA, 18S rRNA, and 28S rRNA gene regions. For the latter four species, SEM observations were simultaneously carried out. A molecular phylogenetic tree based on the four gene regions for 18 chiton species indicated that the seven Japanese Ischnochiton species are polyphyletic and originated from two different clades. A haplotype network based on the COI gene region for the six Japanese Ischnochiton species, except I. hakodadensis, showed that the genetic distances among them were large. The SEM observations revealed that the denticles of the major lateral teeth in the seven Japanese Ischnochiton species were bicuspid, and an accessory process was only observed in the minor lateral teeth of I. hakodadensis. Ischnochiton hayamii sp. nov. cooccurs with I. boninensis, I. comptus, and I. manazuruensis at the two investigated localities, and was difficult to distinguish from other, similar species by naked eyes. However, these can be discriminated based on a combination of adult body size, girdle scales, and valve sculpturing in the lateral and central areas.

Mitochondrial gene order evolution in Mollusca: Inference of the ancestral state from the mtDNA of Chaetopleura apiculata (Polyplacophora, Chaetopleuridae).

The mitochondrial genome architecture of polyplacophorans has been usually regarded as being very ancient in comparison to all mollusks. However, even if some complete chiton mtDNAs have been recently sequenced, thorough studies of their evolution are lacking. To further expand the set of complete chiton mtDNAs and perform such analysis, we sequenced the mitochondrial genome of the Eastern beaded chiton Chaetopleura apiculata (Chaetopleuridae) using next-generation sequencing. With mitochondrial sequences from all available chiton mtDNAs, we also built a phylogeny on which we reconstructed the evolution of gene arrangement in this class. The arrangement of C. apiculata proved to be the most primitive known so far for polyplacophorans. Comparing this gene order to those of other molluscan classes, we found that it most probably is the original gene order of the last common ancestor to all extant Mollusca. The ancient mitochondrial genome organization of C. apiculata is an important information that may help reconstructing the phylogeny of Mollusca and their relationship with other lophotrochozoans.

The complete mitochondrial genomes of two chiton species (Sypharochiton pelliserpentis and Sypharochiton sinclairi) obtained using Illumina next generation sequencing.

Using an Illumina platform, we shot-gun sequenced the complete mitochondrial genomes of two sister chiton species (Sypharochiton pelliserpentis and Sypharochiton sinclairi) to an average coverage of 172× and 60×, respectively. We performed a de novo assembly using SOAPdenovo2 and determined the total mitogenome lengths to be 15,048 and 15,028 bps, respectively. The gene organization was similar to that of other chitons, with 13 protein-coding genes, 24 transfer RNAs and 2 ribosomal RNAs. These data will contribute for resolving the taxonomy and population genetic structures of these species.

Lepidochitona pseudoliozonis, una nueva especie de quitón (Polyplacophora: Ischnochitonidae) del norte del Caribe / Lepidochitona pseudoliozonis, a new species of chiton (Polyplacophora: Ischnochitonidae) from the North of the Caribbean

The genus Lepidochitona (Gray 1821) contains relatively small chitons with a distinctive girdle, dorsally clothed with non-overlapping calcareous corpuscles. In the Caribbean, it is represented by four species: L. liozonis (Dall, & Simpson, 1901), L. rosea Kaas, 1972, L. rufoi García-Ríos, 2010 and L. bullocki García-Ríos, 2011. A rutinary morphological inspection of 10 specimens of a Lepidochitona species from the Florida Keys was concordant with L. liozonis (the only species of the genus informed for Florida). They did not show many morphological differences that could justify its separation from the specimens from Puerto Rico (the type locality). However, the comparison of sequences of the mitochondrial gene coding for cytochrome oxidase I (COI) of L. liozonis from Puerto Rico and the Florida specimens showed a divergence of 14%. This divergence is incompatible with a reproductively connected species. In addition to their genetic differences, the new species differs from L. liozonis in having bigger size, longer marginal spicules and a postmucronal slope very concave. Rev. Biol. Trop. 63 (2): 369-384. Epub 2015 June 01.

El género Lepidochitona (Gray, 1821) agrupa a quitones relativamente pequeños con un cinturón distintivo, por estar cubierto dorsalmente por corpúsculos calcáreos no solapados. Esta representado en el Caribe por cuatro especies: L. liozonis (Dall, & Simpson, 1901), L. rosea Kaas, 1972, L. rufoi García-Ríos, 2010 y L. bullocki García-Ríos, 2011. La inspección de una muestra de ejemplares de los cayos de la Florida permite distinguir ejemplares de lepidoquitones que podrían clasificarse como L. liozonis (la única especie del género informada para la Florida) por no presentar grandes diferencias morfológicas que justifiquen su separación de los de Puerto Rico (localidad del tipo). Sin embargo, la comparación de secuencias del gen mitocondrial que codifica para el citocromo oxidasa I (COI) de los ejemplares de la Florida con ejemplares de L. liozonis de Puerto Rico evidencia una divergencia de 14%. Esta divergencia es incompatible con especies reproductivamente conectadas. Además de sus diferencias genéticas, la nueva especie se puede distinguir de su especie gemela por ser de mayor tamaño, tener espículas marginales más largas y una pendiente posmucronal muy cóncava.

Novel repeated DNAs in the antarctic polyplacophoran Nuttallochiton mirandus (Thiele, 1906).

Within the scope of a project on the characterization of satellite DNAs in polar mollusks, the Antarctic chiton Nuttallochitonmirandus (Thiele, 1906) was analyzed. Two novel families of tandemly repeated DNAs, namely NmH and NmP, are described in their structure and chromosomal localization, and, furthermore, their presence was analyzed in related species. Data reported here display a particular variability in the structural organization of DNA satellites within this species. Processes driving satellite evolution, which are likely responsible for the intriguing variability of the identified satellite DNAs, are discussed.

The continuing debate on deep molluscan phylogeny: evidence for Serialia (Mollusca, Monoplacophora + Polyplacophora).

Molluscs are a diverse animal phylum with a formidable fossil record. Although there is little doubt about the monophyly of the eight extant classes, relationships between these groups are controversial. We analysed a comprehensive multilocus molecular data set for molluscs, the first to include multiple species from all classes, including five monoplacophorans in both extant families. Our analyses of five markers resolve two major clades: the first includes gastropods and bivalves sister to Serialia (monoplacophorans and chitons), and the second comprises scaphopods sister to aplacophorans and cephalopods. Traditional groupings such as Testaria, Aculifera, and Conchifera are rejected by our data with significant Approximately Unbiased (AU) test values. A new molecular clock indicates that molluscs had a terminal Precambrian origin with rapid divergence of all eight extant classes in the Cambrian. The recovery of Serialia as a derived, Late Cambrian clade is potentially in line with the stratigraphic chronology of morphologically heterogeneous early mollusc fossils. Serialia is in conflict with traditional molluscan classifications and recent phylogenomic data. Yet our hypothesis, as others from molecular data, implies frequent molluscan shell and body transformations by heterochronic shifts in development and multiple convergent adaptations, leading to the variable shells and body plans in extant lineages.

Evolutionary consequences of microhabitat: population-genetic structuring in kelp- vs. rock-associated chitons.

Rafting has long been invoked as a key marine dispersal mechanism, but biologists have thus far produced little genetic evidence to support this hypothesis. We hypothesize that coastal species associated with buoyant seaweeds should experience enhanced population connectivity owing to rafting. In particular, invertebrates strongly associated with the buoyant bull-kelp Durvillaea antarctica might be expected to have lower levels of population-genetic differentiation than taxa mainly exploiting nonbuoyant substrates. We undertook a comparative genetic study of two codistributed, congeneric chiton species, assessing population connectivity at scales of 61-516 km, using ≥ 186 polymorphic AFLP loci per species. Consistent with predictions, population-genetic differentiation was weaker in the kelp-associated Sypharochiton sinclairi than in the rock-associated S. pelliserpentis. Additionally, while we found a significant positive correlation between genetic and oceanographic distances in both chiton species, the correlation was stronger in S. pelliserpentis (R(2) = 0.28) than in S. sinclairi (R(2) = 0.18). These data support the hypothesis that epifaunal taxa can experience enhanced population-genetic connectivity as a result of their rafting ability.

Molecular and cytogenetic characterization of repetitive DNA in the Antarctic polyplacophoran Nuttallochiton mirandus.

Two highly repeated DNAs, designated NmE1/NmE2 and NmE5, were identified by EcoRV digestion in the chiton Nuttallochiton mirandus (Mollusca: Polyplacophora). The comparison of the sequences obtained showed high similarity in 5' and 3' regions and the NmE5 sequence displayed an inserted sequence that might arise from a transposable element. Southern blotting analyses suggested a tandem organization of both satellite DNA families identified. Moreover, dot blot analyses, performed on several molluscan species, revealed a different degree of conservation of the repeated DNAs. Fluorescence in-situ hybridizations (FISH) on metaphase chromosomes showed that both satellite DNAs are located at centromeric regions.

Karyology of the Antarctic chiton Nuttallochiton mirandus (Thiele, 1906) (Mollusca: Polyplacophora) with some considerations on chromosome evolution in chitons.

We describe the karyotype, location of nucleolus-organizing regions (NORs) and heterochromatin distribution and composition in the Antarctic chiton Nuttallochiton mirandus. Specimens had a karyotype of 2n = 32 chromosomes, of which two were microchromosomes. Among macrochromosomes, the elements of the first and fourth pair were bi-armed, the others were telocentric. At least six NOR sites were detected with NOR-FISH, but only four were Ag-NOR-banding-positive. The two microchromosomes were essentially euchromatic, while all macrochromosomes exhibited clear pericentromeric C bands that were found to be AT-rich (being quinacrine- and DAPI-positive) and resistant to digestion with AluI and HaeIII. N. mirandus has the largest number of chromosomes (2n = 32) and telocentric elements (26) of all the chiton species studied to date. The karyological results of our study agree with previous molecular data indicating N. mirandus as a sister taxon of Acanthochitona crinita. The karyotypes of the two species could be related as a result of Robertsonian rearrangements. According to the more parsimonious hypothesis, the former would be the primitive karyotype, although other evolutionary events cannot be ruled out.

Hox genes in the antarctic polyplacophoran Nuttallochiton mirandus.

Hox genes are conserved across all bilaterians and encode transcription factors involved in the formation of the anteroposterior axis during embryo development. Differences in homeotic gene evolution have been observed not only between deuterostomes and protostomes, but also between the two large protostome clades, Ecdysozoa and Lophotrochozoa.Among lophotrochozoans, the phylum Mollusca displays high diversity of body plans, ranging from the wormlike appearance of aplacophorans to the complex body plan of cephalopods. Using a PCR-based method, we were able to identify eight Hox genes in the polyplacophoran Nuttallochiton mirandus, two orthologous to the anterior class (lab, pb), four to the central class (Scr, Lox5, Antp, Lox2) and two to the posterior class (Post-1, Post-2). Comparison with the results obtained in other molluscs seems to confirm the conservation of Hox genes in this phylum in terms of both presence and characteristics.

Southern hospitality: a latitudinal gradient in gene flow in the marine environment.

In recent years population genetics and phylogeographic studies have become increasingly valuable tools for inferring both historical and present-day genetic patterns within marine species. Here, we take a comparative approach to population-level study, analyzing original mitochondrial DNA data from 969 individuals representing 28 chiton (Mollusca: Polyplacophora) species to uncover large-scale genetic patterns along the Pacific coast of North America. The data reveal a distinct latitudinal connectivity gradient among chitons: species that exist at lower latitudes tend to have more isolated populations. This trend appears to be a product of between-species differences; within species, no significant gradient in connectivity is observed. Lower average annual sea surface temperatures are hypothesized to contribute to longer larval duration (and by extension, greater connectivity) among lecithotrophic species, providing a mechanism for the observed positive correlation between gene flow and latitude. Because increased isolation among populations may lead to speciation, a latitudinal trend in gene flow may contribute to the increased species diversity observed at lower latitudes.

Evidence for a clade composed of molluscs with serially repeated structures: monoplacophorans are related to chitons.

Monoplacophorans are among the rarest members of the phylum Mollusca. Previously only known from fossils since the Cambrian, the first living monoplacophoran was discovered during the famous second Galathea deep-sea expedition. The anatomy of these molluscs shocked the zoological community for presenting serially repeated gills, nephridia, and eight sets of dorsoventral pedal retractor muscles. Seriality of organs in supposedly independent molluscan lineages, i.e., in chitons and the deep-sea living fossil monoplacophorans, was assumed to be a relic of ancestral molluscan segmentation and was commonly accepted to support a direct relationship with annelids. We were able to obtain one specimen of a monoplacophoran Antarctic deep-sea species for molecular study. The first molecular data on monoplacophorans, analyzed together with the largest data set of molluscs ever assembled, clearly illustrate that monoplacophorans and chitons form a clade. This "Serialia" concept may revolutionize molluscan systematics and may have important implications for metazoan evolution as it allows for new interpretations for primitive segmentation in molluscs.