Chaetognath transcriptome reveals ancestral and unique features among bilaterians

Abstract

Background: The chaetognaths (arrow worms) have puzzled zoologists for years because of their astonishing morphological and developmental characteristics. Despite their deuterostome-like development, phylogenomic studies recently positioned the chaetognath phylum in protostomes, most likely in an early branching. This key phylogenetic position and the peculiar characteristics of chaetognaths prompted further investigation of their genomic features.

Results: Transcriptomic and genomic data were collected from the chaetognath Spadella cephaloptera through the sequencing of expressed sequence tags and genomic bacterial artificial chromosome clones. Transcript comparisons at various taxonomic scales emphasized the conservation of a core gene set and phylogenomic analysis confirmed the basal position of chaetognaths among protostomes. A detailed survey of transcript diversity and individual genotyping revealed a past genome duplication event in the chaetognath lineage, which was, surprisingly, followed by a high retention rate of duplicated genes. Moreover, striking genetic heterogeneity was detected within the sampled population at the nuclear and mitochondrial levels but cannot be explained by cryptic speciation. Finally, we found evidence for trans-splicing maturation of transcripts through splice-leader addition in the chaetognath phylum and we further report that this processing is associated with operonic transcription.

Conclusion: These findings reveal both shared ancestral and unique derived characteristics of the chaetognath genome, which suggests that this genome is likely the product of a very original evolutionary history. These features promote chaetognaths as a pivotal model for comparative genomics, which could provide new clues for the investigation of the evolution of animal genomes.

Figure 1

Overall composition of the EST collection. The annotation of transcripts is based on SwissProt (score >150) and led to identification of mitochondrial genes. The conceptual translation of ESTs allowed detection of those that include coding sequences. The large portion of non-coding polyadenylate nuclear transcripts and RPs among nuclear transcripts is the most prominent aspect of this distribution as well as the unexpected presence of mitochondrial rRNAs (12 and 16S) related to their polyadenine stretches.

Figure 2

Visualization of relative similarity between the transcriptome of S. cephaloptera and (a) selected species or (b) corresponding clades: H. sapiens as a deuterostome, D. melanogaster as an ecdsyzoan and L. rubellus as a lophotrochozoan. The graphs are based on whole transcriptome Blast comparisons and the plotting of respective Blast scores was performed using Simitri [77] (cut-off score 150). Genes at the center of the plot are equally related to the three databases and hence represent valuable phylogenetic markers, whereas genes attracted by a node share a greater similarity with the corresponding database. Genes on the edge do not have a match in the database from the opposite vertex and those on the vertex only have a match in the corresponding database; these two types of genes constitute candidates for signature genes that have possibly been lost in a peculiar lineage. The color scale indicates the relevancy of scores.

Figure 3

RP minimization of missing data in EST-based phylogenomic datasets. Dataset completeness was estimated for datasets composed of 78 RPs (red) or 115 other genes (green) retrieved from EST collections of a large range of sizes.

Figure 4

The basal-protostome branching of chætognaths is confirmed through improved inference methods and expanded taxon sampling. A RP alignment of 11,730 positions (after GBlock filtration; see Additional data file 4) was analyzed using two classes of models. (a) Site-homogeneous model (WAG) implemented in a maximum-likelihood framework (PhyML [80] and Treefinder [81]). Similar topology and maximal posterior probabilities were obtained with Bayesian analyses using the same model (MrBayes). (b) Site-heterogeneous model (CAT) implemented in a bayesian framework (Phylobayes [79]). Plain colored circles denote nodes for which significant support values were obtained (likelihood ratio statistics based on expected-likelihood weights (LR-ELW) >0.95 for site-homogenous and PP >0.95 for site-heterogenous). Support values are indicated for selected nodes: LR-ELW statistics and bootstrap (bold type) for maximum likelihood (ML) using the WAG model and posterior probabilities for Bayesian inference using the CAT model.

Figure 5

Alternative forms of selected markers amplified by PCR in order to assess the origin of polymorphism. (a) Localization of sperm within sperm receptacles (SR) and sperm ducts (SD) in the body of chætognath S. cephaloptera along with ovaries (Ov) and testis (Te). The double arrow indicates that head and body of individuals were split to perform independent PCR amplifications with the purpose of detecting possible contamination from the sperm genome. (b) Paralogous copies of nuclear genes RP L36 and L40 with their intron positions and average lengths, which are distinct in both cases (Additional data files 5 and 6). The names and positions of primers used for the amplification are also specified (Table S3 in Additional data file 2). (c) Relationships between alternative copies of Cytb retrieved within the ESTs with the three different forms detected by the designed primers (Additional data file 8). Boostrap proportions are indicated for selected nodes.

Figure 6

Relationships between haplotypes of nine individuals, including distinct head and body sequences for four marker genes, including two pairs of paralogous sequences: (a) L36 form 1; (b) L36 form 2; (c) L40 form 1; (d) L40 form 2. The indels are plotted onto the branches (green lines). Noticeably, some individuals display mixed sequences from different haplotypes, which is explained by recombination events between alleles (purple star). The substitutions occurring between copies of the same allele in the head and body of individuals (blue branches) are assumed to be somatic mutations. These neighbor-joining trees were inferred assuming kimura 2 parameter distances from Additional data files 7-10. Boostrap proportions are indicated for selected nodes.

Figure 7

Identified S. cephaloptera operon within the BAC 35YA21. (a) Structure of the 158 kb BAC 35A21, including the predicted genes and the mapping of ESTs that bear SLs (purple). Detailed EST/BAC alignments are provided as Additional data files 15-17. (b) Detailed structure of the identified chætognath operon with RP S14 and PCNA genes and the corresponding ESTs (purple) that exhibit SL sequences. UTR (genomic, light orange; EST, light purple) and coding sequences (genomic, orange; EST, purple). (c) Alignments of selected regions of the operon, beginning and end of genes showing genomic DNA (orange) and transcripts with their alternative SL forms. The very short distance encountered between the end and beginning of the two genes argues for polycistronic transcription.

Figure 8

Categories of trans-spliced transcripts for chætognath (a) S. cephaloptera and (b) nematode C. elegans. The presence of a SL sequence is related to coding properties and homologous matches in SwissProt (score >150) of the sequences. C. elegans exhibits less non-coding transcripts than S. cephaloptera.