Evolution of ancient satellite DNAs in extant alligators and caimans (Crocodylia, Reptilia).

BACKGROUND: Crocodilians are one of the oldest extant vertebrate lineages, exhibiting a combination of evolutionary success and morphological resilience that has persisted throughout the history of life on Earth. This ability to endure over such a long geological time span is of great evolutionary importance. Here, we have utilized the combination of genomic and chromosomal data to identify and compare the full catalogs of satellite DNA families (satDNAs, i.e., the satellitomes) of 5 out of the 8 extant Alligatoridae species. As crocodilian genomes reveal ancestral patterns of evolution, by employing this multispecies data collection, we can investigate and assess how satDNA families evolve over time. RESULTS: Alligators and caimans displayed a small number of satDNA families, ranging from 3 to 13 satDNAs in A. sinensis and C. latirostris, respectively. Together with little variation both within and between species it highlighted long-term conservation of satDNA elements throughout evolution. Furthermore, we traced the origin of the ancestral forms of all satDNAs belonging to the common ancestor of Caimaninae and Alligatorinae. Fluorescence in situ experiments showed distinct hybridization patterns for identical orthologous satDNAs, indicating their dynamic genomic placement. CONCLUSIONS: Alligators and caimans possess one of the smallest satDNA libraries ever reported, comprising only four sets of satDNAs that are shared by all species. Besides, our findings indicated limited intraspecific variation in satellite DNA, suggesting that the majority of new satellite sequences likely evolved from pre-existing ones.

Bread wheat satellitome: a complex scenario in a huge genome.

In bread wheat (Triticum aestivum L.), chromosome associations during meiosis are extremely regulated and initiate at the telomeres and subtelomeres, which are enriched in satellite DNA (satDNA). We present the study and characterization of the bread wheat satellitome to shed light on the molecular organization of wheat subtelomeres. Our results revealed that the 2.53% of bread wheat genome is composed by satDNA and subtelomeres are particularly enriched in such DNA sequences. Thirty-four satellite DNA (21 for the first time in this work) have been identified, analyzed and cytogenetically validated. Many of the satDNAs were specifically found at particular subtelomeric chromosome regions revealing the asymmetry in subtelomere organisation among the wheat subgenomes, which might play a role in proper homologous recognition and pairing during meiosis. An integrated physical map of the wheat satellitome was also constructed. To the best of our knowledge, our results show that the combination of both cytogenetics and genome research allowed the first comprehensive analysis of the wheat satellitome, shedding light on the complex wheat genome organization, especially on the polymorphic nature of subtelomeres and their putative implication in chromosome recognition and pairing during meiosis.

Comparison between the Gametophyte and the Sporophyte Transcriptomes of the Endangered Fern Vandenboschia speciosa.

Genomic resources are essential to understanding the evolution and functional biology of organisms. Nevertheless, generating genomic resources from endangered species may be challenging due to the scarcity of available specimens and sampling difficulties. In this study, we compare the transcriptomes of the sporophyte and the gametophyte of the endangered fern Vandenboschia speciosa. After Illumina sequencing and de novo transcriptome assembly of the gametophyte, annotation proved the existence of cross-species contamination in the gametophyte sample. Thus, we developed an in silico decontamination step for the gametophyte sequences. Once the quality check of the decontaminated reads passed, we produced a de novo assembly with the decontaminated gametophyte reads (with 43,139 contigs) and another combining the sporophyte and in silico decontaminated gametophyte reads (with 42,918 contigs). A comparison of the enriched GO terms from the top 1000 most expressed transcripts from both tissues showed that the gametophyte GO term set was enriched in sequences involved in development, response to stress, and plastid organization, while the sporophyte GO term set had a larger representation of more general metabolic functions. This study complements the available genomic resources on the life cycle of the endangered fern Vandenboschia speciosa.

Satellitome comparison of two oedipodine grasshoppers highlights the contingent nature of satellite DNA evolution.

BACKGROUND: The full catalog of satellite DNA (satDNA) within a same genome constitutes the satellitome. The Library Hypothesis predicts that satDNA in relative species reflects that in their common ancestor, but the evolutionary mechanisms and pathways of satDNA evolution have never been analyzed for full satellitomes. We compare here the satellitomes of two Oedipodine grasshoppers (Locusta migratoria and Oedaleus decorus) which shared their most recent common ancestor about 22.8 Ma ago. RESULTS: We found that about one third of their satDNA families (near 60 in every species) showed sequence homology and were grouped into 12 orthologous superfamilies. The turnover rate of consensus sequences was extremely variable among the 20 orthologous family pairs analyzed in both species. The satDNAs shared by both species showed poor association with sequence signatures and motives frequently argued as functional, except for short inverted repeats allowing short dyad symmetries and non-B DNA conformations. Orthologous satDNAs frequently showed different FISH patterns at both intra- and interspecific levels. We defined indices of homogenization and degeneration and quantified the level of incomplete library sorting between species. CONCLUSIONS: Our analyses revealed that satDNA degenerates through point mutation and homogenizes through partial turnovers caused by massive tandem duplications (the so-called satDNA amplification). Remarkably, satDNA amplification increases homogenization, at intragenomic level, and diversification between species, thus constituting the basis for concerted evolution. We suggest a model of satDNA evolution by means of recursive cycles of amplification and degeneration, leading to mostly contingent evolutionary pathways where concerted evolution emerges promptly after lineages split.

Transposable element landscapes illuminate past evolutionary events in the endangered fern Vandenboschia speciosa.

Vandenboschia speciosa is an endangered tetraploid fern species with a large genome (10.5 Gb). Its geographical distribution is characterized by disjoined tertiary flora refuges, with relict populations that survived past climate crises. Here, we analyzed the transposable elements (TEs) and found that they comprise approximately 76% of the V. speciosa genome, thus being the most abundant type of DNA sequence in this gigantic genome. The V. speciosa genome is composed of 51% and 5.6% of Class I and Class II elements, respectively. LTR retrotransposons were the most abundant TEs in this species (at least 42% of the genome), followed by non-LTR retrotransposons, which constituted at least 8.7% of the genome of this species. We introduce an additional analysis to identify the nature of non-annotated elements (19% of the genome). A BLAST search of the non-annotated contigs against the V. speciosa TE database allowed for the identification of almost half of them, which were most likely diverged sequence variants of the annotated TEs. In general, the TE composition in V. speciosa resembles the TE composition in seed plants. In addition, repeat landscapes revealed three episodes of amplification for all TEs, most likely due to demographic changes associated with past climate crises.

The Genomics of Plant Satellite DNA.

The twenty-first century began with a certain indifference to the research of satellite DNA (satDNA). Neither genome sequencing projects were able to accurately encompass the study of satDNA nor classic methodologies were able to go further in undertaking a better comprehensive study of the whole set of satDNA sequences of a genome. Nonetheless, knowledge of satDNA has progressively advanced during this century with the advent of new analytical techniques. The enormous advantages that genome-wide approaches have brought to its analysis have now stimulated a renewed interest in the study of satDNA. At this point, we can look back and try to assess more accurately many of the key questions that were left unsolved in the past about this enigmatic and important component of the genome. I review here the understanding gathered on plant satDNAs over the last few decades with an eye on the near future.

Expanding the Search for Sperm Transmission Elements in the Mitochondrial Genomes of Bivalve Mollusks.

Doubly uniparental inheritance (DUI) of mitochondrial DNA (mtDNA) in bivalve mollusks is one of the most notable departures from the paradigm of strict maternal inheritance of mtDNA among metazoans. Recently, work on the Mediterranean mussel Mytilus galloprovincialis suggested that a nucleotide motif in the control region of this species, known as the sperm transmission element (STE), helps protect male-transmitted mitochondria from destruction during spermatogenesis. Subsequent studies found similar, yet divergent, STE motifs in other marine mussels. Here, we extend the in silico search for mtDNA signatures resembling known STEs. This search is carried out for the large unassigned regions of 157 complete mitochondrial genomes from within the Mytiloida, Veneroida, Unionoida, and Ostreoida bivalve orders. Based on a sliding window approach, we present evidence that there are additional putative STE signatures in the large unassigned regions of several marine clams and freshwater mussels with DUI. We discuss the implications of this finding for interpreting the origin of doubly uniparental inheritance in ancestral bivalve mollusks, as well as potential future in vitro and in silico studies that could further refine our understanding of the early evolution of this unusual system of mtDNA inheritance.

De Novo Sporophyte Transcriptome Assembly and Functional Annotation in the Endangered Fern Species Vandenboschia speciosa (Willd.) G. Kunkel.

We sequenced the sporophyte transcriptome of Killarney fern (Vandenboschia speciosa (Willd.) G. Kunkel). In addition to being a rare endangered Macaronesian-European endemism, this species has a huge genome (10.52 Gb) as well as particular biological features and extreme ecological requirements. These characteristics, together with the systematic position of ferns among vascular plants, make it of high interest for evolutionary, conservation and functional genomics studies. The transcriptome was constructed de novo and contained 36,430 transcripts, of which 17,706 had valid BLAST hits. A total of 19,539 transcripts showed at least one of the 7362 GO terms assigned to the transcriptome, whereas 6547 transcripts showed at least one of the 1359 KEGG assigned terms. A prospective analysis of functional annotation results provided relevant insights on genes involved in important functions such as growth and development as well as physiological adaptations. In this context, a catalogue of genes involved in the genetic control of plant development, during the vegetative to reproductive transition, in stress response as well as genes coding for transcription factors is given. Altogether, this study provides a first step towards understanding the gene expression of a significant fern species and the in silico functional and comparative analyses reported here provide important data and insights for further comparative evolutionary studies in ferns and land plants in general.

Full plastome sequence of the fern Vandenboschia speciosa (Hymenophyllales): structural singularities and evolutionary insights.

We provide here the first full chloroplast genome sequence, i.e., the plastome, for a species belonging to the fern order Hymenophyllales. The phylogenetic position of this order within leptosporangiate ferns, together with the general scarcity of information about fern plastomes, places this research as a valuable study on the analysis of the diversity of plastomes throughout fern evolution. Gene content of V. speciosa plastome was similar to that in most ferns, although there were some characteristic gene losses and lineage-specific differences. In addition, an important number of genes required U to C RNA editing for proper protein translation and two genes showed start codons alternative to the canonical AUG (AUA). Concerning gene order, V. speciosa shared the specific 30-kb inversion of euphyllophytes plastomes and the 3.3-kb inversion of fern plastomes, keeping the ancestral gene order shared by eusporangiate and early leptosporangiate ferns. Conversely, V. speciosa has expanded IR regions comprising the rps7, rps12, ndhB and trnL genes in addition to rRNA and other tRNA genes, a condition shared with several eusporangiate ferns, lycophytes and hornworts, as well as most seed plants.

Long-term monitoring of B-chromosome invasion and neutralization in a population of Prospero autumnale (Asparagaceae).

B chromosomes have been reported in about 15% of eukaryotes, but long-term dynamics of B chromosomes in a single natural population has rarely been analyzed. Prospero autumnale plants collected in 1981 and 1983 at Cuesta de La Palma population had shown the presence of B chromosomes. We analyze here seven additional samples collected between 1987 and 2015, and show that B frequency increased significantly during the 1980s and showed minor fluctuations between 2005 and 2015. A mother-offspring analysis of B chromosome transmission, at population level, showed significant drive on the male side (kB  = 0.65) and significant drag on the female side (kB  = 0.33), with average B transmission rate being very close to the Mendelian rate (0.5). No significant effects of B chromosomes were observed on a number of vigor and fertility-related traits. Within a parasite/host framework, these results suggest that B chromosomes' drive on the male side is the main pathway for B chromosome invasion, whereas B chromosome drag on the female side might be the main manifestation of host genome resistance in this species. Prospero autumnale thus illuminates a novel evolutionary pathway for B chromosome neutralization by means of a decrease in B transmission through the nondriving sex.

Identification and Characterization of TALE Homeobox Genes in the Endangered Fern Vandenboschia speciosa.

We report and discuss the results of a quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis of the expression patterns of seven three amino acid loop extension (TALE) homeobox genes (four KNOTTED-like homeobox (KNOX) and three BEL1-like homeobox (BELL) genes) identified after next generation sequencing (NGS) and assembly of the sporophyte and gametophyte transcriptomes of the endangered fern species Vandenboschia speciosa. Among the four KNOX genes, two belonged to the KNOX1 class and the other two belonged to the KNOX2 class. Analysis of the deduced amino acid sequences supported the typical domain structure of both types of TALE proteins, and the homology to TALE proteins of mosses, lycophytes, and seed plant species. The expression analyses demonstrate that these homeodomain proteins appear to have a key role in the establishment and development of the gametophyte and sporophyte phases of V. speciosa lifecycle, as well as in the control of the transition between both phases. Vandenboschia speciosa VsKNAT3 (a KNOX2 class protein) as well as VsBELL4 and VsBELL10 proteins have higher expression levels during the sporophyte program. On the contrary, one V. speciosa KNOX1 protein (VsKNAT6) and one KNOX2 protein (VsKNAT4) seem important during the development of the gametophyte phase. TALE homeobox genes might be among the key regulators in the gametophyte-to-sporophyte developmental transition in regular populations that show alternation of generations, since some of the genes analyzed here (VsKNAT3, VsKNAT6, VsBELL4, and VsBELL6) are upregulated in a non-alternating population in which only independent gametophytes are found (they grow by vegetative reproduction outside of the range of sporophyte distribution). Thus, these four genes might trigger the vegetative propagation of the gametophyte and the repression of the sexual development in populations composed of independent gametophytes. This study represents a comprehensive identification and characterization of TALE homeobox genes in V. speciosa, and gives novel insights about the role of these genes in fern development.

Satellite DNA: An Evolving Topic.

Satellite DNA represents one of the most fascinating parts of the repetitive fraction of the eukaryotic genome. Since the discovery of highly repetitive tandem DNA in the 1960s, a lot of literature has extensively covered various topics related to the structure, organization, function, and evolution of such sequences. Today, with the advent of genomic tools, the study of satellite DNA has regained a great interest. Thus, Next-Generation Sequencing (NGS), together with high-throughput in silico analysis of the information contained in NGS reads, has revolutionized the analysis of the repetitive fraction of the eukaryotic genomes. The whole of the historical and current approaches to the topic gives us a broad view of the function and evolution of satellite DNA and its role in chromosomal evolution. Currently, we have extensive information on the molecular, chromosomal, biological, and population factors that affect the evolutionary fate of satellite DNA, knowledge that gives rise to a series of hypotheses that get on well with each other about the origin, spreading, and evolution of satellite DNA. In this paper, I review these hypotheses from a methodological, conceptual, and historical perspective and frame them in the context of chromosomal organization and evolution.

Satellite DNA in Plants: More than Just Rubbish.

For decades, satellite DNAs have been the hidden part of genomes. Initially considered as junk DNA, there is currently an increasing appreciation of the functional significance of satellite DNA repeats and of their sequences. Satellite DNA families accumulate in the heterochromatin in different parts of the eukaryotic chromosomes, mainly in pericentromeric and subtelomeric regions, but they also span the functional centromere. Tandem repeat sequences may spread from subtelomeric to interstitial loci, leading to the formation of chromosome-specific loci or to the accumulation in equilocal sites in different chromosomes. They also appear as the main components of the heterochromatin in the sex-specific region of sex chromosomes. Satellite DNA, required for chromosome organization, also plays a role in pairing and segregation. Some satellite repeats are transcribed and can participate in the formation and maintenance of heterochromatin structure and in the modulation of gene expression. In addition to the identification of the different satellite DNA families, their characteristics and location, we are interested in determining their impact on the genomes, by identifying the mechanisms leading to their appearance and amplification as well as in understanding how they change over time, the factors affecting these changes, and the influence exerted by the evolutionary history of the organisms. On the other hand, satellite DNA sequences are rapidly evolving sequences that may cause reproductive barriers between organisms and promote speciation. The accumulation of experimental data collected in recent years and the emergence of new approaches based on next-generation sequencing and high-throughput genome analysis are opening new perspectives that are changing our understanding of satellite DNA. This review examines recent data to provide a timely update on the overall information gathered about this part of the genome, focusing on the advances in the knowledge of its origin, its evolution, and its potential functional roles.

Satellite-DNA diversification and the evolution of major lineages in Cardueae (Carduoideae Asteraceae).

In a previous work, we characterized the HinfI satellite DNA family in the subtribe Centaureinae (Cardueae) demonstrating that a "library" of eight HinfI subfamilies would exist in the common ancestor of all Centaureinae, which were differentially amplified in different lineages. Now, we extend our study by analyzing a total of 219 additional repeats from fifteen species belonging to Carlininae, Echinopsinae and Carduinae, and comparing them to those of Centaureinae. Most HinfI sequences belonged to the subfamily II, although a few sequences of other subfamilies were detected in some species. Additionally, a new subfamily characteristic of several Carduinae species was discovered. Although phylogenetic trees grouped sequences by subfamily affinity instead of species provenance, when comparing repeats of the same subfamily, the degree of divergence between any pair of sequences was related to the evolutionary distance between the species compared in most cases. Exceptions were in comparisons between sequences of some Centaureinae species, and between sequences of some Carduinae species and those of Centaureinae. Our results demonstrate that: (1) At least nine HinfI subfamilies would exist in the common ancestor of Cardueae, each one differentially amplified in different lineages; (2) After differential spreading, sequences of each subfamily evolved concertedly through molecular drive, resulting in the gradual divergence of repeats between different species; (3) The rate to which concerted evolution occurred was different between lineages according to the evolutionary history of each one.

Differential spreading of HinfI satellite DNA variants during radiation in Centaureinae.

BACKGROUND AND AIMS: Subtribe Centaureinae appears to be an excellent model group in which to analyse satellite DNA and assess the influence that the biology and/or the evolution of different lineages have had on the evolution of this class of repetitive DNA. Phylogenetic analyses of Centaureinae support two main phases of radiation, leading to two major groups of genera of different ages. Furthermore, different modes of evolution are observed in different lineages, reflected by morphology and DNA sequences. METHODS: The sequences of 502 repeat units of the HinfI satellite DNA family from 38 species belonging to ten genera of Centaureinae were isolated and compared. A phylogenetic reconstruction was carried out by maximum likelihood and Bayesian inference. KEY RESULTS: Up to eight different HinfI subfamilies were found, based on the presence of a set of diagnostic positions given by a specific mutation shared by all the sequences of one group. Subfamilies V-VIII were mostly found in older genera (first phase of radiation in the subtribe, late Oligocene-Miocene), although some copies of these types of repeats were also found in some species of the derived genera. Subfamilies I-IV spread mostly in species of the derived clade (second phase of radiation, Pliocene to Pleistocene), although repeats of these subfamilies exist in older species. Phylogenetic trees did not group the repeats by taxonomic affinity, but sequences were grouped by subfamily provenance. Concerted evolution was observed in HinfI subfamilies spread in older genera, whereas no genetic differentiation was found between species, and several subfamilies even coexist within the same species, in recently radiated groups or in groups with a history of recurrent hybridization of lineages. CONCLUSIONS: The results suggest that the eight HinfI subfamilies were present in the common ancestor of Centaureinae and that each spread differentially in different genera during the two main phases of radiation following the library model of satellite DNA evolution. Additionally, differential speciation pathways gave rise to differential patterns of sequence evolution in different lineages. Thus, the evolutionary history of each group of Centaureinae is reflected in HinfI satellite DNA evolution. The data reinforce the value of satellite DNA sequences as markers of evolutionary processes.

Concerted evolution of satellite DNA in Sarcocapnos: a matter of time.

SarkOne is a genus-specific satellite-DNA family, isolated from the genomes of the species of the genus Sarcocapnos. This satellite DNA is composed of repeats with a consensus length of 855 bp and a mean G+C content of 52.5%. We have sequenced a total of 189 SarkOne monomeric repeats belonging to a total of seven species of the genus Sarcocapnos. The comparative analysis of these sequences both at the intraspecific and the interspecific levels have revealed divergence patterns between species are proportional to between-species divergence according to the phylogeny of the genus. Our study demonstrates that the molecular drive leading to the concerted-evolution pattern of this satellite DNA is a time-dependent process by which new mutations are spreading through genomes and populations at a gradual pace. However, time is a limiting factor in the observation of concerted evolution in some pairwise comparisons. Thus, pairwise comparisons of species sharing a recent common ancestor did not reveal nucleotide sites in transitional stages higher than stage III according to the Strachan's model. By contrast, there was a gradation in the percentage of upper transition stages (IV, V, VI) the more phylogenetically distant the species were. In addition, closely related species shared a high number of polymorphic sites, but these types of sites were not common when comparing more distant species. All these data are discussed in the light of current life-cycle models of satellite-DNA evolution.

Molecular cytogenetic characterization of Rumex papillaris, a dioecious plant with an XX/XY(1)Y (2) sex chromosome system.

Rumex papillaris Boiss, & Reut., an Iberian endemic, belongs to the section Acetosa of the genus Rumex whose main representative is R. acetosa L., a species intensively studied in relation to sex-chromosome evolution. Here, we characterize cytogenetically the chromosomal complement of R. papillaris in an effort to enhance future comparative genomic approaches and to better our understanding of sex chromosome structure in plants. Rumex papillaris, as is common in this group, is a dioecious species characterized by the presence of a multiple sex chromosome system (with females 2n = 12 + XX and males 2n = 12 + XY(1)Y(2)). Except for the X chromosome both Y chromosomes are the longest in the karyotype and appear heterochromatic due to the accumulation of at least two satellite DNA families, RAE180 and RAYSI. Each chromosome of pair VI has an additional major heterochromatin block at the distal region of the short arm. These supernumerary heterochromatic blocks are occupied by RAE730 satellite DNA family. The Y-related RAE180 family is also present in an additional minor autosomal locus. Our comparative study of the chromosomal organization of the different satellite-DNA sequences in XX/XY and XX/XY(1)Y(2) Rumex species demonstrates that of active mechanisms of heterochromatin amplification occurred and were accompanied by chromosomal rearrangements giving rise to the multiple XX/XY(1)Y(2) chromosome systems observed in Rumex. Additionally, Y(1) and Y(2) chromosomes have undergone further rearrangements leading to differential patterns of Y-heterochromatin distribution between Rumex species with multiple sex chromosome systems.

Detection of Marteilia refringens using nested PCR and in situ hybridisation in Chamelea gallina from the Balearic Islands (Spain).

In the course of a histopathological survey performed to discover the cause of mass mortality of the striped clam Chamelea gallina in the Balearic Islands (Spain, Mediterranean Sea), we detected a Marteilia-like parasite in 3 clams. Molecular methods were applied to identify the parasite. DNA extracted from a paraffin block was used to carry out a PCR assay for Marteilia refringens detection based on a rDNA sequence of the parasite (the intergenic spacer of ribosomal genes, IGS). The nucleotide sequence of the IGS amplified fragment and the positive signal obtained by in situ hybridisation analysis with a M. refringens-specific probe allowed us to confirm the presence of this parasite in the digestive gland tissue of C. gallina.

Identification of Marteilia refringens infecting the razor clam Solen marginatus by PCR and in situ hybridization.

Marteilia refringens is a protozoan parasite recognized as a significant pathogen of the European flat oyster Ostrea edulis. It is believed to have a complex life-cycle involving several hosts. In this study, we applied molecular approaches to identify this parasite in samples of the razor clam Solen marginatus from the south west coast of Spain. We used a PCR assay to amplify a fragment of the IGS rDNA region. PCR products were sequenced and the phylogenetic affinity of the sequences was determined. In situ hybridization analysis showed tissue distribution and presence of different developmental stages of the parasite in the digestive diverticula epithelium, which suggested a true parasitism in these individuals. This is the first report of the occurrence of M. refringens in the razor clam S. marginatus in the south Atlantic. The methodology described herein may be useful for accurate identification of the parasite strain in different hosts and thus provide valuable information for marteiliosis control programmes.