Back in time: a new systematic proposal for the Bilateria

Conventional wisdom suggests that bilateral organisms arose from ancestors that were radially, rather than bilaterally, symmetrical and, therefore, had a single body axis and no mesoderm. The two main hypotheses on how this transformation took place consider either a simple organism akin to the planula larva of extant cnidarians or the acoel Platyhelminthes (planuloid-acoeloid theory), or a rather complex organism bearing several or most features of advanced coelomate bilaterians (archicoelomate theory). We report phylogenetic analyses of bilaterian metazoans using quantitative (ribosomal, nuclear and expressed sequence tag sequences) and qualitative (HOX cluster genes and microRNA sets) markers. The phylogenetic trees obtained corroborate the position of acoel and nemertodermatid flatworms as the earliest branching extant members of the Bilateria. Moreover, some acoelomate and pseudocoelomate clades appear as early branching lophotrochozoans and deuterostomes. These results strengthen the view that stem bilaterians were small, acoelomate/pseudocoelomate, benthic organisms derived from planuloid-like organisms. Because morphological and recent gene expression data suggest that cnidarians are actually bilateral, the origin of the last common bilaterian ancestor has to be put back in time earlier than the cnidarian-bilaterian split in the form of a planuloid animal. A new systematic scheme for the Bilateria that includes the Cnidaria is suggested and its main implications discussed.     

The fine structure of protonephridia in Gnathostomulida and their comparison within Bilateria

Summary The fine structure of the protonephridia of Haplognathia rosea (Filospermoidea) and Gnathostomula paradoxa (Bursovaginoidea) is described. Each protonephridium consists of three different cells: (1) a monociliated terminal cell which constitutes the filtration area, (2) a nonciliated canal cell showing a special protonephridial outlet system, and (3) an intraepidermal cell — the nephroporus cell — constituting the nephroporus. The protonephridia are arranged serially. There is no canal system connecting the protonephridial units.Protonephridial characters in other Bilateria are considered. The pattern of characters in the protonephridia in the last common gnathostomulid stem species and presumed apomorphies in the protonephridia of the Gnathostomulida investigated are discussed.Abbreviations used in figures ac acessory centriole - AC additional epidermal cell - bb basal body - bl basal lamina - bm bundle of microvilli - c cilium - cc cilium duct cell - cd cilium duct - cr ciliary rootlet - crs structures resembling ciliary rootlets - di diplosome - ds desmosome - dy dictyosome - f filtration area - g granules - m mitochondrium - mv microvillus - n nucleus - NC nephroporus cell - np nephroporus - oc outlet canal - TC terminal cell - tl tubules of lacunar system     

T-box and homeobox genes from the ctenophore Pleurobrachia pileus: comparison of Brachyury, Tbx2/3 and Tlx in basal metazoans and bilaterians

Most animals are classified as Bilateria and only four phyla are still extant as outgroups, namely Porifera, Placozoa, Cnidaria and Ctenophora. These non-bilaterians were not considered to have a mesoderm and hence mesoderm-specific genes. However, the T-box gene Brachyury could be isolated from sponges, placozoans and cnidarians. Here, we describe the first Brachyury and a Tbx2/3 homologue from a ctenophore. In addition, analysing T-box and homeobox genes under comparable conditions in all four basal phyla lead to the discovery of novel T-box genes in sponges and cnidarians and a Tlx homeobox gene in the ctenophore Pleurobrachia pileus. The conservation of the T-box and the homeobox genes suggest that distinct subfamilies with different roles in bilaterians were already split in non-bilaterians.     

Evidence for a Diverse Cys-Loop Ligand-Gated Ion Channel Superfamily in Early Bilateria

The genome sequences of Caenorhabditis elegans and Drosophila melanogaster reveal a diversity of cysteine-loop ligand-gated ion channels (Cys-loop LGICs) not found in vertebrates. To better understand the evolution of this gene superfamily, I compared all Cys-loop LGICs from rat, the primitive chordate Ciona intestinalis, Drosophila, and C. elegans. There are two clades of GABA receptor subunits that include both verterbate and invertebrate orthologues. In addition, I identified nine clades of anion channel subunits found only in invertebrates, including three that are specific to C. elegans and two found only in Drosophila. One well-defined clade of vertebrate cation channel subunits, the α7 nicotinic acetylcholine receptor subunits (nAChR), includes invertebrate orthologues. There are two clades of invertebrate nAChRs, one of α-type subunits and one of non-α subunits, that are most similar to the two clades of vertebrate neuronal and muscle α and non-α subunits. There is a large group of divergent C. elegans nAChR-like subunits partially resolved into clades but no orthologues of 5HT3-type serotonin receptors in the invertebrates. The topology of the trees suggests that most of the invertebrate-specific Cys-loop LGIC clades were present in the common ancestor of chordates and ecdysozoa. Many of these disappeared from the chordates. Subsequently, selected subunit genes expanded to form large subfamilies. Electronic Supplementary Material Electronic Supplementary material is available for this article at and accessible for authorised users. [Reviewing Editor: Dr. Rafael Zardoya]     

Changes in the eruption order of the first permanent tooth and their relation to season of birth in Japan

In order to examine the eruption order of the first two permanent teeth, kindergarten children 5 to 6 years old were examined in Hakone, Japan. Among a total of 817 children examined from 1976 to 1984, 349 were determined as I-type children, whose mandibular first incisor erupted earlier than the mandibular first molar, and 183 were as M-type children, whose first molar erupted earlier in the mandible. The mandibular I-type rate, i.e., the proportion of the I-type among a total of I- and M-type children, was 66% (349/532). In 1983-1984, the I-type rate was 70% among boys and 62% among girls, but the overall I-type rate did not differ significantly by the sex of the subject or by the year of examination. The I-type rate varied significantly with the season of subject's birth. Those born in October or November showed a significantly lower I-type rate (33%) than the other subjects (P less than 0.001), in spite of a shift of birth season with low I-type rate toward winter in 1983-1984. The difference of the I-type rate according to birth season suggests that the causes responsible for this change are primarily environmental and act at the prenatal or perinatal stage of life.     

Sympatric speciation and radiative evolution of socially parasitic ants - Heretic hypotheses and their factual background

According to current hypotheses the main types of social parasitism among ants, namely slavery, temporary parasitism, and inquilinism, arose from such features as predation on other ants, or territorial behavior, both presumed precursors of slavemaking, and polygyny, a presumed precursor of temporary parasitism and inquilinism. The latter is believed also to represent a final instar in several evolutionary pathways leading from slavery, temporary parasitism, and xenobiosis to this permanently parasitic, workerless condition. Speciation, the origin of parasitic species from their usually closely related host species, is suggested to occur due to temporary geographic isolation and subsequent transition of one of the newly formed daughter species to parasitism in the nests of the other. Evidence is presented suggesting that the main types of social parasitism originated independently of each other. 15 ant genera are parasitized exclusively by inquilines, Eve other genera exclusively by temporary parasites. Only four groups of non-parasitic ant species (Formica, Tet-ramorium, Leptothorax subgenera Leptothorax and Myrafant) have parasites of several types each. Within these roups, however, there is little evidence of evolutionary transitions from one type to another. The few exceptions, mainly workerless species of the genera Epimyrma and Chalepoxenus, represent parasites which clearly derive from slave-making congeners, but differ from ordinary inquilines in that they eliminate the host colony queens like their actively dulotic ancestors. The new hypothesis suggests that all forms of interspecific true social parasitism (excluding xenobiosis) orginated from a common “preparasitic” stage, a subpopulation of reproductives in polygynous colonies and species, with diverging sexual behavior (near-nest mating vs. swarming) and caste ratios (production of more sexuals vs. workers). Arguments for sympatric speciation are compiled. Various features of the ancestral, and then host species (colony sizes, population density and structure, transition from polygyny to monoyny, etc.), and of the “preparasite” (production of few, or no workers, etc.) may shape the developing parasite to become a slave-maker, inquiline, or temporary parasite. These features usually leave open only one, or in a few genera, several options. The different types of parasitism within one host species group thus may have developed in a radiative manner from the common, preparasitic stage, which explains that independent colony foundation is a common feature of all true social parasites among ants.     

Spermatogenesis and the structure of the testes in Isodiametra pulchra (Isodiametridae,Acoela)

Boone M., Willems M., Claeys M. and Artois, T. 2011. Spermatogenesis and the structure of the testes in Isodiametra pulchra (Isodiametridae, Acoela). —Acta Zoologica (Stockholm) 92 : 101–108. Spermatogenesis and the structure of the testes were studied ultrastructurally in Isodiametra pulchra (Smith and Bush, Transactions of the American Microscopical Society 1991; 110: 12; Hooge and Tyler, Journal of zoological systematics and evolutionary research 2005; 43: 100). The testes are paired, compact, non‐follicular and lie dorsally and dorso‐laterally to the paired ovaries, partially enfolding them. All stages of spermatogenesis, including spermiogenesis, are described at the ultrastructural level and their spatial organization within the testes is discussed. The cells at the early stages of spermatogenesis (spermatogonia and spermatocytes) are located on the dorsal and dorso‐lateral sides of the testes, while the late stages (spermatids and filiform spermatozoa with 9+2 axonemes) lie at the ventral and inner periphery of the testes, adjacent to ovaries. All the cell types can be found both at the anterior and the posterior end of the testes. The value of the structure of the testes as a phylogenetic marker is addressed.     

Water crystallization within rat precision-cut liver slices in relation to their viability

This study examined whether tissue vitrification, promoted by partitioning within the tissue, could be the mechanism explaining the high viability of rat liver slices, rapidly frozen after preincubation with 18% Me2SO or VS4 (a 7.5 M mixture of Me2SO, 1,2-propanediol, and formamide with weight ratio 21.5:15:2.4). To achieve this, we first determined the extent to which crystallization or vitrification occurred in cryoprotectant solutions (Me2SO and VS4) and within liver slices impregnated with these solutions. Second, we determined how these events were related to survival of slices after thawing. Water crystallization was evaluated by differential scanning calorimetry and viability was determined by histomorphological examination of the slices after culturing at 37 degrees C for 4 h. VS4-preincubated liver slices indeed behaved differently from bulk VS4 solution, because, when vitrified, they had a lower tendency to devitrify. Vitrified VS4-preincubated slices that were warmed sufficiently rapid to prevent devitrification had a high viability. When VS4 was diluted (to 75%) or if warming was not fast enough to prevent ice formation, slices had a low viability. With 45% Me2SO, low viability of cryopreserved slices was caused by cryoprotectant toxicity. Surprisingly, liver slices preincubated with 18% Me2SO or 50% VS4 had a high viability despite the formation of ice within the slice. In conclusion, tissue vitrification provides a mechanism that explains the high viability of VS4-preincubated slices after ultrarapid freezing and thawing (>800 degrees C/min). Slices that are preincubated with moderately concentrated cryoprotectant solutions (18% Me2SO, 50% VS4) and cooled rapidly (100 degrees C/min) survive cryopreservation despite the formation of ice crystals within the slice.     

Evidence for a microRNA expansion in the bilaterian ancestor

Understanding how animal complexity has arisen and identifying the key genetic components of this process is a central goal of evolutionary developmental biology. The discovery of microRNAs (miRNAs) as key regulators of development has identified a new set of candidates for this role. microRNAs are small noncoding RNAs that regulate tissue-specific or temporal gene expression through base pairing with target mRNAs. The full extent of the evolutionary distribution of miRNAs is being revealed as more genomes are scrutinized. To explore the evolutionary origins of metazoan miRNAs, we searched the genomes of diverse animals occupying key phylogenetic positions for homologs of experimentally verified human, fly, and worm miRNAs. We identify 30 miRNAs conserved across bilaterians, almost double the previous estimate. We hypothesize that this larger than previously realized core set of miRNAs was already present in the ancestor of all Bilateria and likely had key roles in allowing the evolution of diverse specialist cell types, tissues, and complex morphology. In agreement with this hypothesis, we found only three, conserved miRNA families in the genome of the sea anemone Nematostella vectensis and no convincing family members in the genome of the demosponge Reniera sp. The dramatic expansion of the miRNA repertoire in bilaterians relative to sponges and cnidarians suggests that increased miRNA-mediated gene regulation accompanied the emergence of triploblastic organ-containing body plans. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

VSP417-6, a variant-specific surface protein encoded at a sixth locus within the vsp417 gene subfamily of Giardia intestinalis

A sixth locus (vsp417-6) belonging to the vsp417 gene subfamily, a subset of the family of genes that encodes 'variant-specific' surface proteins (VSP) in Giardia, is described. The sequence of vsp417-6(A-I), the ortholog representing the vsp417-6 locus in isolates of the type A-I (Assemblage A, Group I) genotype of Giardia intestinalis, was determined from a cloned 5.5-kb Hind III fragment of genomic DNA derived from isolate Ad-1/C1. The gene encodes a 704 residue polypeptide (VSP417-6(A-I), Mr 71,674) that has 75% identity (92% similarity) over a 718 residue overlap with the prototype of the VSP417 subfamily, VSP417-1(A-I)-encoded by the vsp417-1 (syn. tsa417) locus in type A-I isolates. Alignment of VSP417-6(A-I) with the deduced sequences of other known members of this subfamily identified one polypeptide, encoded by a gene found in type A-II (Assemblage A, Group II) isolates, whose homology with VSP417-6(A-I) (91% identity, 98% similarity over 713-residues) indicated that it was VSP417-6(A-II), the VSP417-6 ortholog in type A-II isolates. Sequence-based phylogenetic analyses of known VSP417 subfamily members defined several loci that predate the emergence of the A-I and A-II sublineages of G. intestinalis. Related sequences that may correspond to additional, uncharacterised vsp417 subfamily genes were identified in genomic DNA by Southern hybridisation using subfamily- and locus-specific probes. Variant-specific expression of vsp417-1 and vsp417-6 within axenic cultures of G. intestinalis was detected by in situ mRNA hybridization, indicating that these genes are functional and that they are expressed in an alternative fashion with other vsp genes in these organisms.     

Improvement of molecular phylogenetic inference and the phylogeny of Bilateria

Inferring the relationships among Bilateria has been an active and controversial research area since Haeckel. The lack of a sufficient number of phylogenetically reliable characters was the main limitation of traditional phylogenies based on morphology. With the advent of molecular data, this problem has been replaced by another one, statistical inconsistency, which stems from an erroneous interpretation of convergences induced by multiple changes. The analysis of alignments rich in both genes and species, combined with a probabilistic method (maximum likelihood or Bayesian) using sophisticated models of sequence evolution, should alleviate these two major limitations. We applied this approach to a dataset of 94 genes and 79 species using CAT, a previously developed model accounting for site-specific amino acid replacement patterns. The resulting tree is in good agreement with current knowledge: the monophyly of most major groups (e.g. Chordata, Arthropoda, Lophotrochozoa, Ecdysozoa, Protostomia) was recovered with high support. Two results are surprising and are discussed in an evo-devo framework: the sister-group relationship of Platyhelminthes and Annelida to the exclusion of Mollusca, contradicting the Neotrochozoa hypothesis, and, with a lower statistical support, the paraphyly of Deuterostomia. These results, in particular the status of deuterostomes, need further confirmation, both through increased taxonomic sampling, and future improvements of probabilistic models.     

Distinguishing geology from biology in the Ediacaran Doushantuo biota relaxes constraints on the timing of the origin of bilaterians

The Ediacaran Doushantuo biota has yielded fossils that include the oldest widely accepted record of the animal evolutionary lineage, as well as specimens with alleged bilaterian affinity. However, these systematic interpretations are contingent on the presence of key biological structures that have been reinterpreted by some workers as artefacts of diagenetic mineralization. On the basis of chemistry and crystallographic fabric, we characterize and discriminate phases of mineralization that reflect: (i) replication of original biological structure, and (ii) void-filling diagenetic mineralization. The results indicate that all fossils from the Doushantuo assemblage preserve a complex mélange of mineral phases, even where subcellular anatomy appears to be preserved. The findings allow these phases to be distinguished in more controversial fossils, facilitating a critical re-evaluation of the Doushantuo fossil assemblage and its implications as an archive of Ediacaran animal diversity. We find that putative subcellular structures exhibit fabrics consistent with preservation of original morphology. Cells in later developmental stages are not in original configuration and are therefore uninformative concerning gastrulation. Key structures used to identify Doushantuo bilaterians can be dismissed as late diagenetic artefacts. Therefore, when diagenetic mineralization is considered, there is no convincing evidence for bilaterians in the Doushantuo assemblage.     

Egg size of skippers (Lepidoptera: Hesperiidae) in relation to their host specificity and to leaf toughness of host plants

The adaptive significance of egg size of skippers (Lepidoptera; Hesperiidae) in Japan was evaluated in relation to the leaf toughness of their major host grasses. The hesperids that fed on tougher grasses laid larger eggs. Hesperids that laid larger eggs were larger in body size, but lower in fecundity. They also had a wider host range. Thus, despite the lower fecundity, hesperids may benefit from large eggs by having a wider host range of larvae. Grass feeders had wider range of host plants than broadleaf feeders.     

Gestation length variation in domesticated horses and its relation to breed and body size diversity

The domestication process and selective breeding reportedly alter some life history variables. In horses, it has been claimed that gestation length is particularly variable. Some of the factors influencing gestation length are already known and can be grouped into environmental and genetic factors, but the effects of breed and body size have rarely been evaluated. In this study we tested the influence of breed and body size on gestation length for 25 horse breeds from Central Europe. The mean gestation length for all breeds was 342.3 ± 10.2 days and we found significant differences among breeds with a variation of up to 11 days. Body size did not show a significant correlation with gestation length. Our data suggest that breed affiliation explains part of the large variability of gestation length in horses.     

Ultrastructure of the protonephridial system in Neodasys chaetonotoideus (Gastrotricha: Chaetonotida) and in the ground pattern of Gastrotricha

The taxon Neodasys has a basal position within Gastrotricha. This makes it very interesting for phylogenetic considerations in this group. To complete the reconstruction of the nephridial system in the stem species of Gastrotricha started earlier, we have studied the whole protonephridial system of Neodasys chaetonotoideus by means of complete sets of ultrathin sections and TEM. In many characters, protonephridia of N. chaetonotoideus resemble those of macrodasyidan gastrotrich species. For example, each of the six protonephridia, arranged in three pairs, consists of three distinct cells that constitute the continuous protonephridial lumen. Especially, the terminal cell of the protonephridia of N. chaetonotoideus shows a striking pattern: The perforation of the filter region is a meandering cleft that is continuous with the seam of the enfolded lumen of that cell. With the results presented here and that of former TEM studies, we give a comprehensive idea of the excretory organs in the ground pattern of Gastrotricha. Moreover, we can elaborate on the hypothesized protonephridial system in the stem species of Bilateria. We suggest that a meandering filtration cleft is a feature of the ground pattern of the Bilateria.     

Body-plan evolution in the Bilateria: early antero-posterior patterning and the deuterostome-protostome dichotomy

Recent molecular analyses reveal common themes in early antero-posterior patterning in the four major groups of invertebrate deuterostomes and vertebrates in spite of large differences in the mode of gastrulation. Comparisons with Drosophila and Cnidarians suggest a scheme for evolution of the Bilaterian body plan and emphasize the pressing need for similar studies in a wider variety of organisms, especially more basal protostomes.     

The Origin of Patterning Systems in Bilateria——Insights from the Hox and ParaHox Genes in Acoelomorpha

Hox and ParaHox genes constitute two families of developmental regulators that pattern the Anterior-Posterior body axis in all bilaterians.The members of these two groups of genes are usually arranged in genomic clusters and work in a coordinated fashion,both in space and in time. While the mechanistic aspects of their action are relatively well known,it is still unclear how these systems evolved. For instance,we still need a proper model of how the Hox and ParaHox clusters were assembled over time.This problem is due to the shortage of information on gene complements for many taxa (mainly basal metazoans) and the lack of a consensus phylogenetic model of animal relationships to which we can relate our new findings.Recently, several studies have shown that the Acoelomorpha most probably represent the first offshoot of the Bilateria. This finding has prompted us,and others, to study the Hox and ParaHox complements in these animals,as well as their activity during development.In this review,we analyze how the current knowledge of Hox and ParaHox genes in the Acoelomorpha is shaping our view of bilaterian evolution.     

Systematics and taxonomy of the Spanish Anchitheriinae, and their relationship with regional climate changes: a comment on Eronen et al

In a recent paper, Eronen et al. (2010; hereafter EEFJ) observe differences in occlusal morphology, tooth crown height, and mesowear pattern between populations of the Miocene tridactyl equid Anchitherium from Spain and Germany, proposing that Spanish Anchitherium underwent adaptive evolution to local or regional arid conditions. However, these authors do not take into account the actual diversity of Iberian representatives of Anchitherium, or the fact that the Spanish fossils cover a wider temporal and geographical range than those from Germany. For these reasons, we suggest that their subsequent statistical work should be reconsidered.