Mobilization of retrotransposons as a cause of chromosomal diversification and rapid speciation: the case for the Antarctic teleost genus Trematomus.

BACKGROUND: The importance of transposable elements (TEs) in the genomic remodeling and chromosomal rearrangements that accompany lineage diversification in vertebrates remains the subject of debate. The major impediment to understanding the roles of TEs in genome evolution is the lack of comparative and integrative analyses on complete taxonomic groups. To help overcome this problem, we have focused on the Antarctic teleost genus Trematomus (Notothenioidei: Nototheniidae), as they experienced rapid speciation accompanied by dramatic chromosomal diversity. Here we apply a multi-strategy approach to determine the role of large-scale TE mobilization in chromosomal diversification within Trematomus species. RESULTS: Despite the extensive chromosomal rearrangements observed in Trematomus species, our measurements revealed strong interspecific genome size conservation. After identifying the DIRS1, Gypsy and Copia retrotransposon superfamilies in genomes of 13 nototheniid species, we evaluated their diversity, abundance (copy numbers) and chromosomal distribution. Four families of DIRS1, nine of Gypsy, and two of Copia were highly conserved in these genomes; DIRS1 being the most represented within Trematomus genomes. Fluorescence in situ hybridization mapping showed preferential accumulation of DIRS1 in centromeric and pericentromeric regions, both in Trematomus and other nototheniid species, but not in outgroups: species of the Sub-Antarctic notothenioid families Bovichtidae and Eleginopsidae, and the non-notothenioid family Percidae. CONCLUSIONS: In contrast to the outgroups, High-Antarctic notothenioid species, including the genus Trematomus, were subjected to strong environmental stresses involving repeated bouts of warming above the freezing point of seawater and cooling to sub-zero temperatures on the Antarctic continental shelf during the past 40 millions of years (My). As a consequence of these repetitive environmental changes, including thermal shocks; a breakdown of epigenetic regulation that normally represses TE activity may have led to sequential waves of TE activation within their genomes. The predominance of DIRS1 in Trematomus species, their transposition mechanism, and their strategic location in "hot spots" of insertion on chromosomes are likely to have facilitated nonhomologous recombination, thereby increasing genomic rearrangements. The resulting centric and tandem fusions and fissions would favor the rapid lineage diversification, characteristic of the nototheniid adaptive radiation.

New nuclear markers and exploration of the relationships among Serraniformes (Acanthomorpha, Teleostei): the importance of working at multiple scales.

We explore the relationships within Serraniformes (Li et al., 2009) using a dense taxon sampling and seven nuclear markers. Six had already used been for teleost phylogeny (IRBP, MC1R, MLL4, Pkd1, Rhodopsin, and RNF213) at other scales, and one (MLL2) is new. The results corroborate the composition of Serraniformes described in previous publications (some Gasterosteiformes, Perciformes and Scorpaeniformes). Within the clade, Notothenioidei and Zoarcoidei are each monophyletic. Cottoidei was not monophyletic due to placement of the genus Ebinania (Psychrolutidae). Our independent data confirm the sister-group relationship of Percophidae and Notothenioidei as well as the division of Platycephaloidei in four different groups (Bembridae, Platycephalidae, Hoplichthyidae and Peristediidae with Triglidae). Within Cottoidei, Liparidae and Cyclopteridae formed a clade associated with Cottidae, the genus Cottunculus (Psychrolutidae), and Agonidae. Serranidae and Scorpaenidae are not monophyletic, with the Serranidae divided in two clades (Serraninae and Epinephelinae/Anthiinae) and Scorpaenidae including Caracanthidae and the genus Ebinania (Psychrolutidae). We discuss some morphological characters supporting clades within the Scorpaenidae.

Tracking the elusive monophyly of nototheniid fishes (Teleostei) with multiple mitochondrial and nuclear markers.

Since the first molecular study of the suborder Notothenioidei in 1994, many phylogenetic studies have been published. Among these, those with a sufficient number of taxa have all suggested that the Nototheniidae, as currently defined, is monophyletic only with the inclusion of the Channichthyidae, Artedidraconidae, Bathydraconidae and Harpagiferidae. This is corroborated by more recent studies including more taxa, but in these studies either the number of nuclear markers or the number of taxa included remained low. We obtained sequences for a large sampling covering most nototheniid genera for five markers described previously for other samplings (COI, Rhodopsin retrogene, Pkd1, HECW2, and SSRP1) and one nuclear marker never used before in phylogenetic inference (PPM1d). The topology for the combined analysis of the nuclear coding genes, as well as the topology for SSRP1 (non-coding) and the combined analysis for all markers all support the paraphyly of Nototheniidae, the genus Notothenia (including Paranotothenia) is the sister group of the clade Channichthyidae, Artedidraconidae, Bathydraconidae and Harpagiferidae, and genus Gobionotothen is a sister group to both. As in previous studies, Trematomus, Lepidonotothen and Patagonotothen form a clade that also includes Indonotothenia cyanobrancha. The position of Pleuragramma antarctica, Dissostichus species and Aethotaxis mitopteryx remains unstable and dependant on markers and analyses. We therefore propose the inclusion of the four families of the High Antarctic clade in the Nototheniidae, and their transformation into subfamilies. We transfer Paranotothenia magellanica to the genus Notothenia, as Notothenia magellanica.

Phylogenetic footprints of an Antarctic radiation: the Trematominae (Notothenioidei, Teleostei).

The teleost suborder Notothenioidei is restricted to the Southern Ocean and has been described as a species flock spanning the whole of it. Within the suborder, the subfamily Trematominae is important for coastal Antarctic ecosystems. The eleven Trematomus species occupy a large range of ecological niches. The genus is monophyletic if the genus Pagothenia (two additional species) and Cryothenia amphitreta, also nested within it, are included. Although the Trematominae have received much interest, the relationships among these fourteen species are still unclear. Several recent studies have tried to resolve these interrelationships; however no complete and clear picture has emerged, probably because of the use of a low number of insufficiently variable markers. The only common results places T. scotti as the sister-group of the rest of the subfamily and T. loennbergi close to T. lepidorhinus. We use here more variable markers. Four nuclear markers, two of which are new, and a mitochondrial marker for the biggest trematomine sampling ever gathered (14 species, 78 specimens). We found that several nuclear haplotypes are shared by several species (mostly in very closely related species). The haplotype patterns coupled with the cytogenetics of the subfamily suggest that a phenomenon of incomplete lineage sorting (ILS) is likely to be at play. Using a calibration linked to fossil evidence, we evaluate the relative ages of each clade within the Trematominae to assess the proximity of the speciation events to one another. The main trematomine diversification was recent and sudden.

Genetics of sex determination in tilapiine species.

We identified DNA markers linked to sex determining genes in six closely related species of tilapiine fishes. The mode of sex determination differed among species. In Oreochromis karongae and Tilapia mariae the sex-determining locus is on linkage group (LG) 3 and the female is heterogametic (WZ-ZZ system). In O. niloticus and T. zillii the sex-determining locus is on LG1 and the male is heterogametic (XX-XY system). A more complex pattern was observed in O. aureus and O. mossambicus, in which markers on both LG1 and LG3 were associated with sex. We found evidence for sex-linked lethal effects on LG1, as well as interactions between loci in the two linkage groups. Comparison of genetic and physical maps demonstrated a broad region of recombination suppression harboring the sex-determining locus on LG3. Sex-specific recombination suppression was found in the female heterogametic sex. Sequence analysis showed the accumulation of repetitive elements in this region. Phylogenetic analysis suggests that at least two transitions in the mode of sex determination have occurred in this clade. This variation in sex determination mechanisms among closely related species makes tilapias an excellent model system for studying the evolution of sex chromosomes in vertebrates.

Mapping of sox2 and sox14 in tilapia (Oreochromis spp.).

Sox genes encode transcription factors that are involved in a variety of embryonic developmental pathways. Sox2 and Sox14 are located on the same chromosomal arm in several mammalian and bird species and on the basis of comparative maps were suggested as candidate genes for the major sex-determining locus on tilapia LG3. We have sequenced the sox2 and sox14 genes in four tilapia species and mapped them to different chromosomes, LG17 and LG23 respectively. Although excluded as being one of the major sex-determining genes so far mapped in tilapia, sox14 did fall within a QTL region for growth, stress response, embryonic mortality and a minor effect on sex determination.

Diversity and clustered distribution of retrotransposable elements in the compact genome of the pufferfish Tetraodon nigroviridis.

We report the characterization and chromosomal distribution of retroelements in the compact genome of the pufferfish Tetraodon nigroviridis. We have reconstructed partial/complete retroelement sequences, established their phylogenetic relationship to other known eukaryotic retrotransposons, and performed double-color FISH analyses to gain new insights into their patterns of chromosomal distribution. We could identify 43 different reverse transcriptase retrotransposons belonging to the three major known subclasses (14 non-LTR retrotransposons from seven clades, 25 LTR retrotransposons representing the five major known groups, and four Penelope-like elements), and well as two SINEs (non-autonomous retroelements). Such a diversity of retrotransposable elements, which seems to be relatively common in fish but not in mammals, is astonishing in such a compact genome. The total number of retroelements was approximately 3000, roughly representing only 2.6% of the genome of T. nigroviridis. This is much less than in other vertebrate genomes, reflecting the compact nature of the genome of this pufferfish. Major differences in copy number were observed between different clades, indicating differential success in invading and persisting in the genome. Some retroelements displayed evidence of recent activity. Finally, FISH analysis showed that retrotransposable elements preferentially accumulate in specific heterochromatic regions of the genome of T. nigroviridis, revealing a degree of genomic compartmentalization not observed in the human genome.

Lack of correspondence between CMA3-, Ag-positive signals and 28S rDNA loci in two Iberian minnows (Teleostei, Cyprinidae) evidenced by sequential banding.

Despite the growing outcome of results that put doubt upon the reliability of silver (Ag) staining and chromomycin A3 (CMA3) fluorescent banding in the detection of major ribosomal gene sites (NORs), these methods have been widely used, especially in fishes. In order to clarify the previous patterns obtained with those techniques, we performed fluorescence in situ hybridisation (FISH) with 28S rDNA probe followed by sequential CMA3 and Ag staining in diploid non-hybrid males of the Squalius alburnoides complex and in Squalius pyrenaicus. The results from all the studied specimens revealed a lack of correlation between classical and molecular techniques. Not just some other regions besides NORs were stained with CMA3 and Ag, but also the majority of the 28S rDNA sites were not detected. Care should then be taken in considering CMA3- and Ag-stained sites as NORs since their accuracy for that purpose may not always correspond to the expectations.

Viability of X-autosome translocations in mammals: an epigenomic hypothesis from a rodent case-study.

X-autosome translocations are highly deleterious chromosomal rearrangements due to meiotic disruption, the effects of X-inactivation on the autosome, and the necessity of maintaining different replication timing patterns between the two segments. In spite of this, X-autosome translocations are not uncommon. We here focus on the genus Taterillus (Rodentia, Gerbillinae) which provides two sister lineages differing by two autosome-gonosome translocations. Despite the recent and dramatic chromosomal repatterning characterising these lineages, the X-autosome translocated species all display intercalary heterochromatic blocks (IHBs) between the autosomal and the ancestral sexual segments. These blocks, composed of highly amplified telomeric repeats and rDNA clusters, are not observed on the chromosomes of the non-translocated species, nor the Y1 and Y2 of the translocated species. Such IHBs are found in all mammals documented for X-autosome translocation. We propose an epigenomic hypothesis which explains the viability of X-autosome translocations in mammals. This posits that constitutive heterochromatin is probably selected for in X-autosome translocations since it may (1) prevent facultative heterochromatinization of the inactivated X from spreading to the autosomal part, and (2) allow for the independent regulation of replication timing of the sex and autosomal segments.

Comparative cytogenetics and chromosomal characteristics of ribosomal DNA in the fish genus Vimba (Cyprinidae).

Karyotypic and cytogenetic characteristics of Vimba vimba and V. elongata were investigated using differential staining techniques (sequential C-banding, Ag- and CMA3-staining) and fluorescent in situ hybridization (FISH) with 28S rDNA probe. The diploid chromosome number in both species was 2n = 50 with 8 pairs of metacentrics, 14 pairs of submetacentrics to subtelocentrics and 3 pairs of subtelo- to acrocentrics. The largest chromosome pair of the complements was characteristically subtelo- to acrocentric. The nucleolar organizer regions (NORs) in both species were detected in the telomeres of a single, middle-sized subtelocentric chromosome pair, a pattern common in a number of other Leuciscinae. FISH with rDNA probe produced consistently positive hybridization signals detected in the same regions indicated by Ag-staining and CMA3-fluorescence. The distribution of C-positive heterochromatin was identical in both species, including a conspicuous size polymorphism of heterochromatic blocks in the largest metacentric and subtelo- to acrocentric chromosomal pairs. No heteromorphic sex chromosomes were detected. A single analyzed individual of V. melanops possessed the same karyotype and NOR phenotype as V. vimba and V. elongata. The apparent karyotype homogeneity and chromosomal characteristics of ribosomal DNA in all three species of the genus Vimba is consistent to that found in most other representatives of the European leuciscine cyprinid fishes.

Evolution of rRNA gene clusters and telomeric repeats during explosive genome repatterning in TATERILLUS X (Rodentia, Gerbillinae).

A survey of 28S and 5S rRNA gene clusters, and telomeric repeats was performed using single and double FISH in the Taterillus genus (Rodentia, Muridae, Gerbillinae). Taterillus was previously demonstrated to have undergone a very recent and extensive chromosomal evolution. Our FISH results demonstrate that rRNA genes can vary in location and number irrespective of the phylogenetic relationships. Telomeric repeats were detected in pericentromeric and interstitial regions of several chromosomes, thus providing nonambiguous evolutionary footprints of Robertsonian and tandem translocation events. These footprints are discussed in reference to the molecular process of these karyotypical changes. Also, examples of colocation of rDNA clusters and telomeric repeats lend support to their possible involvement in nucleolus formation. Finally, the presence of rRNA genes, and the extensive amplification of telomeric repeats at specific loci within a double X-autosome translocated element which were not observed on the homologous Y1 and Y2, served as basis for an epigenomic hypothesis on X-autosome translocation viability in mammals.

Conservation of the 5S-bearing chromosome pair and co-localization with major rDNA clusters in five species of Astyanax (Pisces, Characidae).

Major and 5S ribosomal genes have been localized in chromosomes from five fish species, genus Astyanax, using in situ hybridization (FISH) with 28S and 5S rDNA probes. In situ signals for the major rDNA co-localized with the 5S rDNA clusters in the pericentromeric region of one marker chromosome in all five species analyzed. The conserved localization of these two rDNA clusters in the five related Astyanax species was considered as indicative of a close relationship among them. The use of these molecular markers for elucidating evolutionary relationships among closely related taxa is discussed.

"Ag-NORs" are not always true NORs: new evidence in mammals.

In spite of uncertainty about the biochemical processes involved, silver staining is a widely used technique for assessing the locations of active NORs in eukaryotic genomes in general, and in mammalian genomes in particular. However, following a previous study of hedgehog chromosomes, we present here a second example from two gerbil species (Rodentia, Muridae), which have several clear Ag-positive signals that do not correspond to 28S rDNA clusters. Although this pattern may be characteristic of particular genomes displaying unusual heterochromatic features, our study casts doubt upon the reliability and universality of Ag-staining for detecting active NORs.

Extensive polymorphism and chromosomal characteristics of ribosomal DNA in a loach fish, Cobitis vardarensis (Ostariophysi, Cobitidae) detected by different banding techniques and fluorescence in situ hybridization (FISH).

When surveying the karyotype diversity of European loaches of the genus Cobitis to identify species involved in hybrid polyploid complexes, an extensive polymorphism in number and location of NORs was discovered in C. vardarensis using Ag-staining, C-banding, CMA3-fluorescence and fluorescence in situ hybridization (FISH). This species had 2n = 50, the karyotype contained 13 pairs of metacentric, 10 pairs of submetacentric and two pairs of subtelocentric chromosomes. The NOR-bearing chromosomes included one medium-sized metacentric pair with a large CMA3-positive heterochromatic pericentromeric block, one small metacentric as well as one large submetacentric pairs. Ribosomal sites were always located in telomeres of these chromosomes. Each of the pair of NOR-bearing chromosomes occurred in three variants - (1) presence and/or (2) absence of NORs on both homologues and (3) heterozygous combination where only one of the homologues bears NORs. Altogether, 10 different NOR cytotypes from 27 theoretically possible ones were discovered among 20 indviduals examined. The number of NORs ranged from two to five per specimen. The results regarding the number and locations of NORs as revealed by banding techniques were confirmed using FISH with rDNA probe. NOR sites were of CMA3-positive, suggesting that ribosomal sites are associated with GC-rich DNA. Very similar structural polymorphism with multiple NORs is expressed in the Danubian loach C. elongatoides indicating a close relationship between both species.

Characterization and repeat analysis of the compact genome of the freshwater pufferfish Tetraodon nigroviridis.

Tetraodon nigroviridis is a freshwater pufferfish 20-30 million years distant from Fugu rubripes. The genome of both tetraodontiforms is compact, mostly because intergenic and intronic sequences are reduced in size compared to other vertebrate genomes. The previously uncharacterized Tetraodon genome is described here together with a detailed analysis of its repeat content and organization. We report the sequencing of 46 megabases of bacterial artificial chromosome (BAC) end sequences, which represents a random DNA sample equivalent to 13% of the genome. The sequence and location of rRNA gene clusters, centromeric and subtelocentric satellite sequences have been determined. Minisatellites and microsatellites have been cataloged and notable differences were observed in comparison with microsatellites from Fugu. The genome contains homologies to all known families of transposable elements, including Ty3-gypsy, Ty1-copia, Line retrotransposons, DNA transposons, and retroviruses, although their overall abundance is <1%. This structural analysis is an important prerequisite to sequencing the Tetraodon genome.

Karyotype and chromosome location of characteristic tandem repeats in the pufferfish Tetraodon nigroviridis.

Karyotype analysis of Tetraodon nigroviridis, a pufferfish of the family Tetraodontidae with a small compact genome (385 Mb) which is currently being investigated in our laboratory, indicates that this species has 2n = 42 chromosomes. The small chromosome size (the largest pair measuring less than 3 microm) has complicated accurate chromosome pairing based on morphology alone. DAPI staining, however, provides a banding-like pattern. Because of quantitative variations of some heterochromatin classes, the chromosome formula can not be established precisely, but is estimated to include approximately 20 meta- or submetacentric chromosomes and 22 subtelocentric chromosomes. A centromeric satellite, telomeric repeats, and the major and minor rRNA clusters have been localized unequivocally by FISH. As a result, the 28S and 5S rDNA sequences can be used as chromosome-specific probes.

Ribosomal RNA location in the Antarctic fish Champsocephalus gunnari (Notothenioidei, Channichthyidae) using banding and fluorescence in situ hybridization.

A biotinylated 28S rDNA probe was prepared from the genomic DNA of the Antarctic ice-fish Champsocephalus gunnari and hybridized to metaphase chromosomes of the same species by fluorescence in situ hybridization (FISH). The hybridization signal appeared over the whole heterochromatic arm of the submetacentric chromosomes bearing the nucleolar organizer regions. The results of rDNA/FISH are compared with those coming from classical cytogenetic (C, Q, Ag-NOR, chromomycin A3) banding techniques. The in situ detection of a specific DNA sequence offers a new more precise perspective for understanding the evolving process in chromosomes of Antarctic fish and will provide an interesting contribution to comparative cytogenetics of lower vertebrates.