Identification and validation of X- and Y-linked genomic scaffolds.

<p><b>(A)</b> Plot of the fold difference in coverage of each genomic scaffold between two female individuals (blue) or a male and a female individual (red), in genome re-sequencing data. Fold difference for each scaffold is shown as a logarithm to base 2. The plot reveals a number of scaffolds with different patterns of abundance in the two sexes. The regions of the plot containing scaffolds over- or underrepresented in males are indicated with brackets on the right. Only scaffolds 10 kb or more in length are shown. The scaffolds are ordered by length along the horizontal axis, with largest scaffolds on the left. Scaffolds independently validated as being located on the X or Y chromosome are indicated with arrowheads or arrows, respectively. <b>(B)</b> Quantitative PCR confirms that three tested genomic scaffolds are present at approximately twice the copy number in females as in males. The Y-axis is the mean ratio of C_T value of the candidate scaffold to the C_T value of the autosomal reference scaffold (for scaffolds 1–3: males n = 12, females n = 9; for scaffold 4: males n = 9, females n = 8). Error bars represent the standard error of the mean (SEM). Student’s t test * <i>P</i><0.05; ** <i>P</i><0.001; n.s. = not significant. Scaffolds 1–3 are scf7180001248200, scf7180001248049 and scf7180001247190, respectively; the control scaffold is scf7180001247533. <b>(C)</b> Identification of six male-specific genomic scaffolds. Primers designed against these 6 scaffolds only amplify a PCR product from male genomic DNA samples. In contrast, a PCR against an autosomal scaffold amplifies a product in both sexes (control). None of the primer sets amplified a product with water as the template (NTC = no template control). Scaffolds 1–6 are scf7180001247258, scf7180001245067, scf7180001243011, scf7180001247286, scf7180001247324 and scf7180001247297, respectively; the control scaffold is scf7180001247533. Original, uncropped gels are provided in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0150292#pone.0150292.s002" target="_blank">S2 Fig</a>. The raw data for this figure are provided in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0150292#pone.0150292.s004" target="_blank">S2 Table</a>.</p

Identification of the X chromosome in the <i>Strigamia</i> karyotype by FISH with a set of X-chromosome derived DNA probes.

<p><b>(A)</b> The relative positions and sizes of the five PCR fragments, distributed on two different X-linked scaffolds, which were used to produce the X-probes. Two of the fragments are located on scf7180001248200 and three on scf7180001248049. The genomic distance between these scaffolds is not known. The black line depicts the scaffold. The numbers above the line indicate the number of bases, starting at 1 on the left hand side. Green boxes represent the length and relative position of the PCR products, numbered arbitrarily from 1 to 5. <b>(B)</b> A mitotic metaphase chromosome spread prepared from a single embryo. Hybridization signals of the X-probes identify a middle-sized element in the <i>Strigamia</i> karyotype as the X chromosome. As there are two chromosomes with the X-probe signals, we infer that this chromosome spread is derived from a female embryo (XX). <b>(C)</b> Two <i>Strigamia</i> karyotypes constructed from the mitotic metaphases of embryonic cells. They are derived from different embryos. Upper panel: karyotype derived from the female metaphase shown in (B). Lower panel: karyotype derived from an inverted image of a DAPI-stained metaphase of unknown sex. It is the same as that shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0150292#pone.0150292.g001" target="_blank">Fig 1A</a>. We infer that the pair of sex chromosomes represents the 4^th pair of chromosomes by size (asterisks). (D, E, F) Meiotic chromosome spreads, prepared from sub-adult male testes. <b>(D)</b> Late zygotene complement showing a clump of incompletely paired bivalents. The X-probes label the longer chromosome of a partially paired bivalent, as schematically illustrated in (D’). We thus infer that this is the X chromosome, and that the other shorter chromosome, without hybridization signals, is the Y chromosome. The X and Y chromosomes are only paired at the distal part of the X chromosome, with a large proximal part unpaired. <b>(E)</b> A particularly clear and well-spread XY bivalent at a similar stage to (D). It shows hybridization signals of X-probes on the unpaired proximal part of the X chromosome, while the Y chromosome is completely paired except for the DAPI-highlighted centromere (see schematic drawing below the XY bivalent). <b>(F)</b> Pachytene complement showing 8 bivalents, each with DAPI-highlighted centromeric chromatin. X-probe hybridization signals are visible on the unpaired segment of the longer chromosome, near the centromere (see schematic drawing on the right-hand side). The X and Y chromosomes now appear almost equal in length in the bivalent. Scale bar is equal to 5 μm in (B) and 10 μm in (D, E, F). Chromosomes were counterstained with DAPI (blue). Arrowheads indicate hybridization signals of the digoxigenin-labelled X-probes (green); arrows indicate a pair of the largest chromosomes (B) or the largest bivalent (F).</p

Ancestral protein kinases are extensively lost during arthropod evolution.

<p><i>S. maritima</i> is an exception and retains the largest number of ancestral kinases. Numbers of kinase subfamilies in selected species are shown in parentheses after species names. The gains, losses, and inferred content of common ancestors are listed on internal branches. Kinases found in at least two species from human, <i>C. elegans</i> and <i>Nematostella vectenesis</i> were used as an outgroup.</p

Expansion of chemosensory receptor families.

<p>(A) Phylogenetic relationships among <i>S. maritima</i> (Smar), <i>I. scapularis</i> (Isca), <i>D. pulex</i> (Dpul), and a few insect GRs that encode for sugar, fructose, and carbon dioxide receptors (Dmel, <i>D. melanogaster</i>, and Amel, <i>A. mellifera</i>). (B) Phylogenetic relationships among <i>S. maritima</i>, <i>I. scapularis</i>, and a few <i>D. melanogaster</i> IRs and IgluR genes (the suffix at the end of the protein names indicates: i, incomplete and p, pseudogene).</p

Presence and absence of immunity genes in different arthropods.

<p>Counts of immune genes are shown for <i>S. maritima</i>, <i>D. pulex</i><a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002005#pbio.1002005-McTaggart1" target="_blank">[131]</a>, <i>A. mellifera</i><a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002005#pbio.1002005-Evans1" target="_blank">[86]</a>, <i>T. castaneum</i>, <i>Anopheles gambiae</i>, and <i>D. melanogaster</i><a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002005#pbio.1002005-Dasmahapatra1" target="_blank">[132]</a>. ∼, identity of the gene is uncertain; -, not investigated.</p

Homeobox gene clusters.

<p>(A) The Hox gene cluster of <i>S. maritima</i> compared to the cluster that can be deduced for the ancestral arthropod. <i>S. maritima</i> provides the first instance of an arthropod Hox cluster with tight linkage of an <i>Even-skipped (Eve)</i> gene (see text). Hox3 is the only gene missing from the <i>S. maritima</i> Hox cluster, but may be present elsewhere in the genome on a separate scaffold (see main text and <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002005#pbio.1002005.s072" target="_blank">Text S1</a> for details). The <i>S. maritima</i> cluster is drawn approximately to scale and spans 457 kb from the start codon of <i>labial (lab)</i> to the start codon of <i>Eve-b</i>. Arrows denote the transcriptional orientation. (B) Remains of clustering and linkage of ANTP class genes in <i>S. maritima</i>. The blue boxes are genes belonging to the ANTP class. The brown box is a gene belonging to the HNF class. The orange box is a gene belonging to the SINE class. The intergenic distances are indicated in kb. (C) Clusters of non-ANTP class homeobox genes in <i>S. maritima</i>. The green boxes are genes belonging to the TALE class. The red boxes are genes belonging to the PRD class. The intergenic distances are indicated in kb, except in the case of Rx-Hbn as these genes are overlapping but with opposite transcriptional orientations. All scaffold numbers are indicated in brackets.</p

Plot showing that DNA from a male individual contains a distinct fraction of scaffolds that is underrepresented (black arrow), and presumably derives from heterogametic sex chromosomes.

<p>No such fraction is present in the sequenced DNA of two individual females. The data underlying this plot is presented in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002005#pbio.1002005.s068" target="_blank">File S4</a>.</p

Conserved macro synteny signal between <i>S. maritima</i> and the chordate lancelet <i>B. floridae</i> clustered into ancestral linkage groups.

<p>Each dot represents a pair of genes, one in <i>B. floridae</i>, one in <i>S. maritima</i>, assigned to the same gene family by our orthology analysis. The ancestral linkage group identifiers refer to groups of scaffolds from the <i>S. maritima</i> (SmALG) or <i>B. floridae</i> (BfALG) assemblies, as detailed in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002005#pbio.1002005.s066" target="_blank">File S2</a>. The identification of ALGs is described in the SI. Note that two <i>S. maritima</i> scaffolds were divided across ALGs, and so appear multiple times in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002005#pbio.1002005.s066" target="_blank">File S2</a>.</p

Arthropod phylogenetic tree (with nematode outgroup) showing selected events of gene loss, gene gain, and gene family expansions.

<p>Main taxa are listed on the tips, with representative species for which there is a fully sequenced genome listed below. Major nodes are also named. Data from the genome of <i>S. maritima</i> allow us to map when in arthropod evolution these events occurred, even when these events did not occur on the centipede lineage. A plausible node for the occurrence of each event is marked and colour-coded, with the possible range marked with a thin line of the same colour. The events, listed from left to right are: (1) Dscam alternative splicing as a strategy for increasing immune diversity is known from <i>D. melanogaster</i>, as well as the crustacean <i>D. pulex</i>, and thus probably evolved in the lineage leading to pancrustacea, after the split from centipedes. (2) Several wnt genes have been lost in holometabolous insects, leaving only seven of the 13 ancestral families. This loss occurred gradually over arthropod evolution, but reached its peak at the base of the Holometabola. (3) Selenoproteins are rare in insects. The presence of a large number of selenoproteins in <i>S. maritima</i> as well as in other non-insect arthropods suggests that the loss of many selenoproteins occurred at the base of the Insecta. (4) Expansion of chemosensory gene families occurred independently in different arthropod lineages as they underwent terrestrialisation. The OR family is expanded in insects only. (5) Chemosensory genes of the GR and IR genes have undergone a lineage specific expansion in the genome of <i>S. maritima</i>. As these are probably also linked with terrestrialisation we suggest that this expansion happened at the base of the Chilopoda, but it could have also occurred later in the lineage leading to <i>S. maritima</i>. (6) Cuticular proteins of the RR-1 family are numerous in the <i>S. maritima</i> genome. They are found in other arthropods, but not in chelicerates nor in any non-arthropod species. This suggests that the RR-1 subfamily evolved at the base of the Mandibulata. (7) The genome of <i>S. maritima</i> has a large complement of wnt genes, but is missing <i>wnt8</i>. Since this gene is found in the Diplopod <i>G. marginata</i> (a species without a fully sequenced genome), the loss most likely occurred at the base of the Chilopoda. (8) Unlike the situation in <i>D. melanogaster</i>, immune diversity in the <i>S. maritima</i> genome is achieved through multiple copies of the Dscam gene. This expansion of the family could have happened at any time after the split between Myriapoda and Pancrustacea. (9) Both circadian rhythm genes and many light receptors are missing in <i>S. maritima</i>. These losses are most likely due to the subterranean life style of geophilomorph centipedes and are probably specific to this group. However, we cannot rule out the possibility that they were lost somewhere in the lineage leading to myriapods. (10) The existence of JH signalling in <i>S. maritima</i> as well as in all other arthropods studied to date strengthens the idea that this signalling system evolved with the exoskeleton of arthropods, though its origins could be even more ancient and date back to the origin of moulting at the base of the Ecdysozoa.</p

Dscam diversity caused either by gene and/or exon duplication in different Metazoa.

<p>^aOnly canonical Dscam paralogues were considered. ^bIn <i>D. melanogaster</i> and <i>D. pulex</i> the paralogue Dscam-L2 has two Ig7 alternative coding exons. ^cPotential number of Dscam isoforms, circulating in one individual, produced by mutually exclusive alternative splicing of duplicated exons.</p