The interstitial environment of marine sandy bottoms is a nutrient-rich, sheltered habitat whilst at the same time also often a turbulent, space-limited, and ecologically challenging environment dominated by meiofauna. The interstitial fauna is one of the most diverse on earth and accommodates miniaturized representatives from many macrofaunal groups as well as several exclusively meiofaunal phyla. The colonization process of this environment, with the restrictions imposed by limited space and low Reynolds numbers, has selected for great morphological and behavioral changes as well as new life history strategies.Here we describe a new enteropneust species inhabiting the interstices among sand grains in shallow tropical waters of the West Atlantic. With a maximum body length of 0.6 mm, it is the first microscopic adult enteropneust known, a group otherwise ranging from 2 cm to 250 cm in adult size. Asexual reproduction by paratomy has been observed in this new species, a reproductive mode not previously reported among enteropneusts. Morphologically, Meioglossus psammophilus gen. et sp. nov. shows closest resemblance to an early juvenile stage of the acorn worm family Harrimaniidae, a result congruent with its phylogenetic placement based on molecular data. Its position, clearly nested within the larger macrofaunal hemichordates, suggests that this represents an extreme case of miniaturization. The evolutionary pathway to this simple or juvenile appearance, as chiefly demonstrated by its small size, dense ciliation, and single pair of gill pores, may be explained by progenesis. The finding of M. psammophilus gen. et sp. nov. underscores the notion that meiofauna may constitute a rich source of undiscovered metazoan diversity, possibly disguised as juveniles of other species.
Many recent studies on invertebrates have shown how morphology not always captures the true diversity of taxa, with cryptic speciation often being discussed in this context. Here, we show how diversification patterns can be very different in two clades of closely related earthworms in the genus Hormogaster stressing the risk of using nonspecific substitution rate values across taxa. On the one hand, the Hormogaster elisae species complex, endemic to the central Iberian Peninsula, shows morphological stasis. On the other hand, a clade of Hormogaster from the NE Iberian Peninsula shows an enormous morphological variability, with 15 described morphospecies. The H. elisae complex, however, evolves faster genetically, and this could be explained by the harsher environmental conditions to which it is confined-as detected in this study, that is, sandier and slightly poorer soils with lower pH values than those of the other species in the family. These extreme conditions could be at the same time limiting morphological evolution and thus be responsible for the observed morphological stasis in this clade. Contrarily, Hormogaster species from the NE Iberian Peninsula, although still inhabiting harsher milieu than other earthworm groups, have had the opportunity to evolve into a greater morphological disparity. An attempt to delimit species within this group following the recently proposed general mixed Yule-coalescent method showed a higher number of entities than expected under the morphospecies concept, most probably due to the low vagility of these animals, which considerably limits gene flow between distant conspecific populations, but also because of the decoupling between morphological and genetic evolution in the H. elisae complex.
We used 16S ribosomal RNA (rRNA) and cytochrome c oxidase subunit I (COI) sequence data to investigate the population structure in the centipede Craterostigmus tasmanianus Pocock, 1902 (Chilopoda: Craterostigmomorpha: Craterostigmidae) and to look for possible barriers to gene flow on the island of Tasmania, where C. tasmanianus is a widespread endemic. We first confirmed a molecular diagnostic character in 28S rRNA separating Tasmanian Craterostigmus from its sister species Craterostigmus crabilli (Edgecombe and Giribet 2008) in New Zealand and found no shared polymorphism in this marker for the 2 species. In Tasmania, analysis of molecular variance analysis showed little variation at the 16S rRNA and COI loci within populations (6% and 13%, respectively), but substantial variation (56% and 48%, respectively) among populations divided geographically into groups. We found no clear evidence of isolation by distance using a Mantel test. Bayesian clustering and gene network analysis both group the C. tasmanianus populations in patterns which are broadly concordant with previously known biogeographical divisions within Tasmania, but we did not find that genetic distance varied in a simple way across cluster boundaries. The coarse-scale geographical sampling on which this study was based should be followed in the future by sampling at a finer spatial scale and to investigate genetic structure within clusters and across cluster boundaries.
INTRODUCTION: Traditionally, genomic or transcriptomic data have been restricted to a few model or emerging model organisms, and to a handful of species of medical and/or environmental importance. Next-generation sequencing techniques have the capability of yielding massive amounts of gene sequence data for virtually any species at a modest cost. Here we provide a comparative analysis of de novo assembled transcriptomic data for ten non-model species of previously understudied animal taxa. RESULTS: cDNA libraries of ten species belonging to five animal phyla (2 Annelida [including Sipuncula], 2 Arthropoda, 2 Mollusca, 2 Nemertea, and 2 Porifera) were sequenced in different batches with an Illumina Genome Analyzer II (read length 100 or 150 bp), rendering between ca. 25 and 52 million reads per species. Read thinning, trimming, and de novo assembly were performed under different parameters to optimize output. Between 67,423 and 207,559 contigs were obtained across the ten species, post-optimization. Of those, 9,069 to 25,681 contigs retrieved blast hits against the NCBI non-redundant database, and approximately 50% of these were assigned with Gene Ontology terms, covering all major categories, and with similar percentages in all species. Local blasts against our datasets, using selected genes from major signaling pathways and housekeeping genes, revealed high efficiency in gene recovery compared to available genomes of closely related species. Intriguingly, our transcriptomic datasets detected multiple paralogues in all phyla and in nearly all gene pathways, including housekeeping genes that are traditionally used in phylogenetic applications for their purported single-copy nature. CONCLUSIONS: We generated the first study of comparative transcriptomics across multiple animal phyla (comparing two species per phylum in most cases), established the first Illumina-based transcriptomic datasets for sponge, nemertean, and sipunculan species, and generated a tractable catalogue of annotated genes (or gene fragments) and protein families for ten newly sequenced non-model organisms, some of commercial importance (i.e., Octopus vulgaris). These comprehensive sets of genes can be readily used for phylogenetic analysis, gene expression profiling, developmental analysis, and can also be a powerful resource for gene discovery. The characterization of the transcriptomes of such a diverse array of animal species permitted the comparison of sequencing depth, functional annotation, and efficiency of genomic sampling using the same pipelines, which proved to be similar for all considered species. In addition, the datasets revealed their potential as a resource for paralogue detection, a recurrent concern in various aspects of biological inquiry, including phylogenetics, molecular evolution, development, and cellular biochemistry.
The proximo-distal axis of the arthropod leg is patterned by mutually antagonistic developmental expression domains of the genes extradenticle, homothorax, dachshund, and Distal-less. In the deutocerebral appendages (the antennae) of insects and crustaceans, the expression domain of dachshund is frequently either absent or, if present, is not required to pattern medial segments. By contrast, the dachshund domain is entirely absent in the deutocerebral appendages of spiders, the chelicerae. It is unknown whether absence of dachshund expression in the spider chelicera is associated with the two-segmented morphology of this appendage, or whether all chelicerates lack the dachshund domain in their chelicerae. We investigated gene expression in the harvestman Phalangium opilio, which bears the plesiomorphic three-segmented chelicera observed in "primitive" chelicerate orders. Consistent with patterns reported in spiders, in the harvestman chelicera homothorax, extradenticle, and Distal-less have broadly overlapping developmental domains, in contrast with mutually exclusive domains in the legs and pedipalps. However, unlike in spiders, the harvestman chelicera bears a distinct expression domain of dachshund in the proximal segment, the podomere that is putatively lost in derived arachnids. These data suggest that a tripartite proximo-distal domain structure is ancestral to all arthropod appendages, including deutocerebral appendages. As a corollary, these data also provide an intriguing putative genetic mechanism for the diversity of arachnid chelicerae: loss of developmental domains along the proximo-distal axis.
Among chelicerates, Hox gene expression has only been investigated in representatives of two arachnid orders to date: Acari (mites and ticks) and Araneae (spiders). Limited data are available for the "primitive" arachnid orders, such as Scorpiones (scorpions) and Opiliones (harvestmen). Here, we present the first data on Hox gene expression in the harvestman Phalangium opilio. Ten Hox genes of this species were obtained from a de novo assembled developmental transcriptome using the Illumina GAII platform. All 10 genes are expressed in characteristic Hox-like expression patterns, and the expression of the anterior and central Hox genes is similar to those of other chelicerates. However, intriguingly, the three posteriormost genes-Ultrabithorax, abdominal-A, and Abdominal-B-share an identical anterior expression boundary in the second opisthosomal segment, and their expression domains extend through the opisthosoma to the posterior growth zone. The overlap in expression domains of the posterior Hox genes is correlated with the absence of opisthosomal organs posterior to the tubular tracheae, which occur on the second opisthosomal segment. Together with the staggered profile of posterior Hox genes in spiders, these data suggest the involvement of abdominal-A and Abdominal-B in the evolution of heteronomous patterning of the chelicerate opisthosoma, providing a mechanism that helps explain the morphological diversity of chelicerates.
Transcriptome sequencing with next-generation sequencing technologies has the potential for addressing many long-standing questions about the biology of sponges. Transcriptome sequence quality depends on good cDNA libraries, which requires high-quality mRNA. Standard protocols for preserving and isolating mRNA often require optimization for unusual tissue types. Our aim was assessing the efficiency of two preservation modes, (i) flash freezing with liquid nitrogen (LN(2)) and (ii) immersion in RNAlater, for the recovery of high-quality mRNA from sponge tissues. We also tested whether the long-term storage of samples at -80 degrees C affects the quantity and quality of mRNA. We extracted mRNA from nine sponge species and analysed the quantity and quality (A260/230 and A260/280 ratios) of mRNA according to preservation method, storage time, and taxonomy. The quantity and quality of mRNA depended significantly on the preservation method used (LN(2)) outperforming RNAlater), the sponge species, and the interaction between them. When the preservation was analysed in combination with either storage time or species, the quantity and A260/230 ratio were both significantly higher for LN(2)-preserved samples. Interestingly, individual comparisons for each preservation method over time indicated that both methods performed equally efficiently during the first month, but RNAlater lost efficiency in storage times longer than 2 months compared with flash-frozen samples. In summary, we find that for long-term preservation of samples, flash freezing is the preferred method. If LN(2) is not available, RNAlater can be used, but mRNA extraction during the first month of storage is advised.
The origins of tropical southwest Pacific diversity are traditionally attributed to southeast Asia or Australia. Oceanic and fragment islands are typically colonized by lineages from adjacent continental margins, resulting in attrition of diversity with distance from the mainland. Here, we show that an exceptional tropical family of harvestmen with a trans-Pacific disjunct distribution has its origin in the Neotropics. We found in a multi-locus phylogenetic analysis that the opilionid family Zalmoxidae, which is distributed in tropical forests on both sides of the Pacific, is a monophyletic entity with basal lineages endemic to Amazonia and Mesoamerica. Indo-Pacific Zalmoxidae constitute a nested clade, indicating a single colonization event. Lineages endemic to putative source regions, including Australia and New Guinea, constitute derived groups. Divergence time estimates and probabilistic ancestral area reconstructions support a Neotropical origin of the group, and a Late Cretaceous (ca 82 Ma) colonization of Australasia out of the Fiji Islands and/or Borneo, which are consistent with a transoceanic dispersal event. Our results suggest that the endemic diversity within traditionally defined zoogeographic boundaries might have more complex evolutionary origins than previously envisioned.
Revived interest in molluscan phylogeny has resulted in a torrent of molecular sequence data from phylogenetic, mitogenomic, and phylogenomic studies. Despite recent progress, basal relationships of the class Bivalvia remain contentious, owing to conflicting morphological and molecular hypotheses. Marked incongruity of phylogenetic signal in datasets heavily represented by nuclear ribosomal genes versus mitochondrial genes has also impeded consensus on the type of molecular data best suited for investigating bivalve relationships. To arbitrate conflicting phylogenetic hypotheses, we evaluated the utility of four nuclear protein-encoding genes-ATP synthase beta, elongation factor-1alpha, myosin heavy chain type II, and RNA polymerase II-for resolving the basal relationships of Bivalvia. We sampled all five major lineages of bivalves (Archiheterodonta, Euheterodonta [including Anomalodesmata], Palaeoheterodonta, Protobranchia, and Pteriomorphia) and inferred relationships using maximum likelihood and Bayesian approaches. To investigate the robustness of the phylogenetic signal embedded in the data, we implemented additional datasets wherein length variability and/or third codon positions were eliminated. Results obtained include (a) the clade (Nuculanida+Opponobranchia), i.e., the traditionally defined Protobranchia; (b) the monophyly of Pteriomorphia; (c) the clade (Archiheterodonta+Palaeoheterodonta); (d) the monophyly of the traditionally defined Euheterodonta (including Anomalodesmata); and (e) the monophyly of Heteroconchia, i.e., (Palaeoheterodonta+Archiheterodonta+Euheterodonta). The stability of the basal tree topology to dataset manipulation is indicative of signal robustness in these four genes. The inferred tree topology corresponds closely to those obtained by datasets dominated by nuclear ribosomal genes (18S rRNA and 28S rRNA), controverting recent taxonomic actions based solely upon mitochondrial gene phylogenies.
Arthropods are the most diverse group of animals and have been so since the Cambrian radiation. They belong to the protostome clade Ecdysozoa, with Onychophora (velvet worms) as their most likely sister group and tardigrades (water bears) the next closest relative. The arthropod tree of life can be interpreted as a five-taxon network, containing Pycnogonida, Euchelicerata, Myriapoda, Crustacea, and Hexapoda, the last two forming the clade Tetraconata or Pancrustacea. The unrooted relationship of Tetraconata to the three other lineages is well established, but of three possible rooting positions the Mandibulata hypothesis receives the most support. Novel approaches to studying anatomy with noninvasive three-dimensional reconstruction techniques, the application of these techniques to new and old fossils, and the so-called next-generation sequencing techniques are at the forefront of understanding arthropod relationships. Cambrian fossils assigned to the arthropod stem group inform on the origin of arthropod characters from a lobopodian ancestry. Monophyly of Pycnogonida, Euchelicerata, Myriapoda, Tetraconata, and Hexapoda is well supported, but the interrelationships of arachnid orders and the details of crustacean paraphyly with respect to Hexapoda remain the major unsolved phylogenetic problems.
In order to study the tempo and the mode of spider orb web evolution and diversification, we conducted a phylogenetic analysis using six genetic markers along with a comprehensive taxon sample. The present analyses are the first to recover the monophyly of orb-weaving spiders based solely on DNA sequence data and an extensive taxon sample. We present the first dated orb weaver phylogeny. Our results suggest that orb weavers appeared by the Middle Triassic and underwent a rapid diversification during the end of the Triassic and Early Jurassic. By the second half of the Jurassic, most of the extant orb-weaving families and web designs were already present. The processes that may have given origin to this diversification of lineages and web architectures are discussed. A combination of biotic factors, such as key innovations in web design and silk composition, as well as abiotic environmental changes, may have played important roles in the diversification of orb weavers. Our analyses also show that increased taxon sampling density in both ingroups and outgroups greatly improves phylogenetic accuracy even when extensive data are missing. This effect is particularly important when addition of character data improves gene overlap.
BACKGROUND: Seamount-associated faunas are often considered highly endemic but isolation and diversification processes leading to such endemism have been poorly documented at those depths. Likewise, species delimitation and phylogenetic studies in deep-sea organisms remain scarce, due to the difficulty in obtaining samples, and sometimes controversial. The phylogenetic relationships within the precious coral family Coralliidae remain largely unexplored and the monophyly of its two constituent genera, Corallium Cuvier and Paracorallium Bayer & Cairns, has not been resolved. As traditionally recognized, the diversity of colonial forms among the various species correlates with the diversity in shape of their supporting axis, but the phylogenetic significance of these characters remains to be tested. We thus used mitochondrial sequence data to evaluate the monophyly of Corallium and Paracorallium and the species boundaries for nearly all named taxa in the family. Species from across the coralliid range, including material from Antarctica, Hawaii, Japan, New Zealand, Taiwan, Tasmania, the eastern Pacific and the western Atlantic were examined. RESULTS: The concatenated analysis of five mitochondrial regions (COI, 16S rRNA, ND2, and ND3-ND6) recovered two major coralliid clades. One clade is composed of two subgroups, the first including Corallium rubrum, the type species of the genus, together with a small group of Paracorallium species (P. japonicum and P. tortuosum) and C. medea (clade I-A); the other subgroup includes a poorly-resolved assemblage of six Corallium species (C. abyssale, C. ducale, C. imperiale, C. laauense, C. niobe, and C. sulcatum; clade I-B). The second major clade is well resolved and includes species of Corallium and Paracorallium (C. elatius, C. kishinouyei, C. konojoi, C. niveum, C. secundum, Corallium sp., Paracorallium nix, Paracorallium thrinax and Paracorallium spp.). A traditional taxonomic study of this clade delineated 11 morphospecies that were congruent with the general mixed Yule-coalescent (GMYC) model. A multilocus species-tree approach also identified the same two well-supported clades, being Clade I-B more recent in the species tree (18.0-15.9 mya) than in the gene tree (35.2-15.9 mya). In contrast, the diversification times for Clade II were more ancient in the species tree (136.4-41.7 mya) than in the gene tree (66.3-16.9 mya). CONCLUSIONS: Our results provide no support for the taxonomic status of the two currently recognized genera in the family Coralliidae. Given that Paracorallium species were all nested within Corallium, we recognize the coralliid genus Corallium, which includes the type species of the family, and thus consider Paracorallium a junior synonym of Corallium. We propose the use of the genus Hemicorallium Gray for clade I-B (species with long rod sclerites, cylindrical autozooids and smooth axis). Species delimitation in clade I-B remains unclear and the molecular resolution for Coralliidae species is inconsistent in the two main clades. Some species have wide distributions, recent diversification times and low mtDNA divergence whereas other species exhibit narrower allopatric distributions, older diversification times and greater levels of mtDNA resolution.
