Genetic diversity and population structure of the rockpool shrimp Palaemon elegans based on microsatellites: evidence for a cryptic species and differentiation across the Atlantic-Mediterranean transition.

The rockpool shrimp Palaemon elegans is an ecologically important crustacean species within the European coastline fauna. In the present study, genetic diversity and population structure and connectivity were assessed by examining 21 polymorphic microsatellite loci at 13 sampling sites located along the natural distribution range. All localities showed similar levels of genetic variability. Significant deficits of heterozygosity were recorded, most likely due to the presence of null alleles. Genetic structure analyses revealed two clearly genetically distinct groups within P. elegans but without following any geographical or oceanographic basis. Thus, our results provided nuclear evidence for the existence of a Mediterranean cryptic species within P. elegans, highlighting the need to revise its taxonomic status. Regarding P. elegans sensu stricto, population structuring was reported across the Atlantic-Mediterranean transition area, where the Almería-Orán Front restricts the gene flow between the Atlantic and the Mediterranean population. Moreover, while population connectivity was suggested between all Mediterranean localities, some substructure was found within the Atlantic group. Canary Islands exhibited a weak but significant genetic differentiation from all Atlantic mainland localities, consistent with the isolation-by-distance pattern detected throughout the Atlantic population. Overall, all these findings provided new insights into the population biology of P. elegans complex.

Isolation and characterization of 21 polymorphic microsatellite loci for the rockpool shrimp Palaemon elegans using Illumina MiSeq sequencing.

The rockpool shrimp Palaemon elegans is considered an important crustacean species within the European coastline fauna. This species is experiencing an ongoing geographical expansion beyond its native distribution range due to unintentional human introductions. A better knowledge of the genetic diversity, geographic structure and connectivity of its populations is necessary. In the present study, microsatellite loci were isolated using the Illumina MiSeq platform. The microsatellite-enriched library sequencing produced 3.9 million raw reads. Reads were processed and primer pairs were designed for microsatellite sequences amplification. Ninety-six microsatellite loci were preliminary screened in individuals from Atlantic and Mediterranean localities. From them, 21 loci exhibited reliable polymorphism and were thoroughly characterized in 30 individuals from a Cantabrian locality (Spain). No linkage disequilibrium between pairs of loci was detected. Number of alleles per locus ranged from 2 to 12. Observed and expected heterozygosities ranged from 0.033 to 0.833 and from 0.033 to 0.869 respectively. No significant departure from the Hardy-Weinberg equilibrium was detected in most of loci. This is the first time that microsatellite markers have been developed for P. elegans. This characterized microsatellite suite provides new suitable tools for further analyses, facilitating the understanding of population genetics both in natural and introduced populations.

Comparative cytogenetic analysis of marine Palaemon species reveals a X1X1X2X2/X1X2Y sex chromosome system in Palaemon elegans.

BACKGROUND: The maintenance of species and the promotion of speciation are closely related to chromosomal rearrangements throughout evolution. Decapoda represents the most species-rich order among crustaceans and, despite its ecological and economic importance, little is known about decapod karyology. We aim at cytogenetically characterizing two sympatric prawn species. RESULTS: Analysis of mitotic metaphases and meiotic diakinesis of the common prawn Palaemon serratus and the rockpool prawn P. elegans, revealed considerable differences between their karyotypes including chromosome numbers and sex determination systems. The cytogenetic data for P. serratus showed a diploid number of 56 and the putative absence of heteromorphic sex chromosomes. However, the diploid chromosome number in P. elegans was 90 for females and 89 for males. The karyotype of the females consisted of the three largest acrocentric pairs and 42 submetacentric and metacentric pairs, while the karyotype of the males comprised a clearly identifiable large metacentric chromosome and two acrocentric pairs as well as the smaller 42 pairs. These results highlight the presence of the X1X1X2X2/X1X2Y multiple sex chromosome system in P. elegans, which constitute the only sexual system for Decapoda reported cytogenetically using modern techniques. The origin of this sex chromosome system is discussed. We hypothesize that the chromosome evolution within the genus could involve several fusion events giving rise to a reduction on the chromosome number in P. serratus. In both species, the major ribosomal genes were located in two chromosome pairs and hybridization signals of the telomeric sequences (TTAGGG)n were visualized at the telomeres of all chromosomes. C-banding revealed that, when present, constitutive heterochromatin had a predominantly telomeric distribution and no centromeric constitutive heterochromatin was observed. CONCLUSIONS: Although more comparative cytogenetic analyses are needed to clarify our hypotheses, the findings of this work indicate that the prawns of the genus Palaemon represent a promising model among Decapoda representatives to investigate the karyotype evolution and the patterns of sex chromosome differentiation.

De novo transcriptome assembly of shrimp Palaemon serratus.

The shrimp Palaemon serratus is a coastal decapod crustacean with a high commercial value. It is harvested for human consumption. In this study, we used Illumina sequencing technology (HiSeq 2000) to sequence, assemble and annotate the transcriptome of P. serratus. RNA was isolated from muscle of adults individuals and, from a pool of larvae. A total number of 4 cDNA libraries were constructed, using the TruSeq RNA Sample Preparation Kit v2. The raw data in this study was deposited in NCBI SRA database with study accession number of SRP090769. The obtained data were subjected to de novo transcriptome assembly using Trinity software, and coding regions were predicted by TransDecoder. We used Blastp and Sma3s to annotate the identified proteins. The transcriptome data could provide some insight into the understanding of genes involved in the larval development and metamorphosis. SPECIFICATIONS: [Table: see text].

Evolutionary dynamics of two satellite DNA families in rock lizards of the genus Iberolacerta (Squamata, Lacertidae): different histories but common traits.

Satellite DNAs compose a large portion of all higher eukaryotic genomes. The turnover of these highly repetitive sequences is an important element in genome organization and evolution. However, information about the structure and dynamics of reptilian satellite DNA is still scarce. Two satellite DNA families, HindIII and TaqI, have been previously characterized in four species of the genus Iberolacerta. These families showed different chromosomal locations, abundances, and evolutionary rates. Here, we extend the study of both satellite DNAs (satDNAs) to the remaining Iberolacerta species, with the aim to investigate the patterns of variability and factors influencing the evolution of these repetitive sequences. Our results revealed disparate patterns but also common traits in the evolutionary histories of these satellite families: (i) each satellite DNA is made up of a library of monomer variants or subfamilies shared by related species; (ii) species-specific profiles of satellite repeats are shaped by expansions and/or contractions of different variants from the library; (iii) different turnover rates, even among closely related species, result in great differences in overall sequence homogeneity and in concerted or non-concerted evolution patterns, which may not reflect the phylogenetic relationships among taxa. Contrasting turnover rates are possibly related to genomic constraints such as karyotype architecture and the interspersed organization of diverging repeat variants in satellite arrays. Moreover, rapid changes in copy number, especially in the centromeric HindIII satDNA, may have been associated with chromosomal rearrangements and even contributed to speciation within Iberolacerta.

Molecular organization and phylogenetic analysis of 5S rDNA in crustaceans of the genus Pollicipes reveal birth-and-death evolution and strong purifying selection.

BACKGROUND: The 5S ribosomal DNA (5S rDNA) is organized in tandem arrays with repeat units that consist of a transcribing region (5S) and a variable nontranscribed spacer (NTS), in higher eukaryotes. Until recently the 5S rDNA was thought to be subject to concerted evolution, however, in several taxa, sequence divergence levels between the 5S and the NTS were found higher than expected under this model. So, many studies have shown that birth-and-death processes and selection can drive the evolution of 5S rDNA. In analyses of 5S rDNA evolution is found several 5S rDNA types in the genome, with low levels of nucleotide variation in the 5S and a spacer region highly divergent. Molecular organization and nucleotide sequence of the 5S ribosomal DNA multigene family (5S rDNA) were investigated in three Pollicipes species in an evolutionary context. RESULTS: The nucleotide sequence variation revealed that several 5S rDNA variants occur in Pollicipes genomes. They are clustered in up to seven different types based on differences in their nontranscribed spacers (NTS). Five different units of 5S rDNA were characterized in P. pollicipes and two different units in P. elegans and P. polymerus. Analysis of these sequences showed that identical types were shared among species and that two pseudogenes were present. We predicted the secondary structure and characterized the upstream and downstream conserved elements. Phylogenetic analysis showed an among-species clustering pattern of 5S rDNA types. CONCLUSIONS: These results suggest that the evolution of Pollicipes 5S rDNA is driven by birth-and-death processes with strong purifying selection.

The 5S rDNA gene family in mollusks: characterization of transcriptional regulatory regions, prediction of secondary structures, and long-term evolution, with special attention to Mytilidae mussels.

Several reports on the characterization of 5S ribosomal DNA (5S rDNA) in various animal groups have been published to date, but there is a lack of studies analyzing this gene family in a much broader context. Here, we have studied 5S rDNA variation in several molluskan species, including bivalves, gastropods, and cephalopods. The degree of conservation of transcriptional regulatory regions was analyzed in these lineages, revealing a conserved TATA-like box in the upstream region. The evolution of the 120 bp coding region (5S) was also studied, suggesting the occurrence of paralogue groups in razor clams, clams, and cockles. In addition, 5S rDNA sequences from 11 species and 7 genus of Mytilidae Rafinesque, 1815 mussels were sampled and studied in detail. Four different 5S rDNA types, based on the nontranscribed spacer region were identified. The phylogenetic analyses performed within each type showed a between-species gene clustering pattern, suggesting ancestral polymorphism. Moreover, some putative pseudogenized 5S copies were also identified. Our report, together with previous studies that found high degree of intragenomic divergence in bivalve species, suggests that birth-and-death evolution may be the main force driving the evolution of 5S rDNA in these animals, even at the genus level.

Analysis of ITS1 and ITS2 sequences in Ensis razor shells: suitability as molecular markers at the population and species levels, and evolution of these ribosomal DNA spacers.

Internal transcribed spacer 1 and 2 (ITS1 and ITS2) sequences were analysed in Ensis razor shells (Mollusca: Bivalvia: Pharidae). We aimed to (1) test ITS1 and ITS2 as molecular markers at the population level in the successful alien E. directus (Conrad, 1843); (2) test these spacers at the species level in E. directus and three other Ensis species, E. siliqua (L., 1758), E. macha (Molina, 1782), and E. magnus (Schumacher, 1817); and (3) analyse the evolutionary processes that may be shaping Ensis ITS1 and ITS2 extant variation. In E. directus, despite the intragenomic divergence detected, ITS1 and ITS2 were informative in differentiating the geographic areas considered (Denmark and Canada) by means of both the insertion-deletion polymorphism and the nucleotide polymorphism. In this species, the 5.8S ribosomal gene (5.8S) showed scarce polymorphism. At the species level, maximum parsimony and maximum likelihood analyses revealed that ITS1 and ITS2 may be suitable to reconstruct Ensis phylogenetic relationships. Finally, the evolutionary models that best fit the long-term evolution of Ensis ITS1-5.8S-ITS2 are discussed. A mixed process of concerted evolution, birth-and-death evolution, and selection is chosen as an option that may reconcile the long-term evolution of Ensis ITS1-5.8S-ITS2 and 5S ribosomal DNA.

Long-term evolution of 5S ribosomal DNA seems to be driven by birth-and-death processes and selection in Ensis razor shells (Mollusca: Bivalvia).

A study of nucleotide sequence variation of 5S ribosomal DNA from six Ensis species revealed that several 5S ribosomal DNA variants, based on differences in their nontranscribed spacers (NTS), occur in Ensis genomes. The 5S rRNA gene was not very polymorphic, compared with the NTS region. The phylogenetic analyses performed showed a between-species clustering of 5S ribosomal DNA variants. Sequence divergence levels between variants were very large, revealing a lack of sequence homogenization. These results strongly suggest that the long-term evolution of Ensis 5S ribosomal DNA is driven by birth-and-death processes and selection.

Common evolutionary origin and birth-and-death process in the replication-independent histone H1 isoforms from vertebrate and invertebrate genomes.

The H1 histone multigene family shows the greatest diversity of isoforms among the five histone gene families, including replication-dependent (RD) and replication-independent (RI) genes, according to their expression patterns along the cell cycle and their genomic organization. Although the molecular characterization of the RI isoforms has been well documented in vertebrates, similar information is lacking in invertebrates. In this work we provide evidence for a polyadenylation signature in the Mytilus "orphon" H1 genes similar to the polyadenylation characteristic of RI H1 genes. These mussel genes, together with the sea urchin H1delta genes, are part of a lineage of invertebrate "orphon" H1 genes that share several control elements with vertebrate RI H1 genes. These control elements include the UCE element, H1-box and H4-box. We provide evidence for a functional evolution of vertebrate and invertebrate RI H1 genes, which exhibit a clustering pattern by type instead of by species, with a marked difference from the somatic variants. In addition, these genes display an extensive silent divergence at the nucleotide level which is always significantly larger than the nonsilent. It thus appears that RI and RD H1 isoforms display similar long-term evolutionary patterns, best described by the birth-and-death model of evolution. Notably, this observation is in contrast with the theoretical belief that clustered RD H1 genes evolve in a concerted manner. The split of the RI group from the main RD group must therefore have occurred before the divergence between vertebrates and invertebrates about 815 million years ago. This was the result of the transposition of H1 genes to solitary locations in the genome.

Birth-and-death evolution with strong purifying selection in the histone H1 multigene family and the origin of orphon H1 genes.

Histones are small basic nuclear proteins with critical structural and functional roles in eukaryotic genomes. The H1 multigene family constitutes a very interesting histone class gathering the greatest number of isoforms, with many different arrangements in the genome, including clustered and solitary genes, and showing replication-dependent (RD) or replication-independent (RI) expression patterns. The evolution of H1 histones has been classically explained by concerted evolution through a rapid process of interlocus recombination or gene conversion. Given such intriguing features, we have analyzed the long-term evolutionary pattern of the H1 multigene family through the evaluation of the relative importance of gene conversion, point mutation, and selection in generating and maintaining the different H1 subtypes. We have found the presence of an extensive silent nucleotide divergence, both within and between species, which is always significantly greater than the nonsilent variation, indicating that purifying selection is the major factor maintaining H1 protein homogeneity. The results obtained from phylogenetic analysis reveal that different H1 subtypes are no more closely related within than between species, as they cluster by type in the topologies, and that both RD and RI H1 variants follow the same evolutionary pattern. These findings suggest that H1 histones have not been subject to any significant effect of interlocus recombination or concerted evolution. However, the diversification of the H1 isoforms seems to be enhanced primarily by mutation and selection, where genes are subject to birth-and-death evolution with strong purifying selection at the protein level. This model is able to explain not only the generation and diversification of RD H1 isoforms but also the origin and long-term persistence of orphon RI H1 subtypes in the genome, something that is still unclear, assuming concerted evolution.

Molecular evolutionary characterization of the mussel Mytilus histone multigene family: first record of a tandemly repeated unit of five histone genes containing an H1 subtype with "orphon" features.

The present work represents the first characterization of a clustered histone repetitive unit containing an H1 gene in a bivalve mollusk. To complete the knowledge on the evolutionary history of the histone multigene family in invertebrates, we undertake its characterization in five mussel Mytilus species, as an extension of our previous work on the H1 gene family. We report the quintet H4-H2B-H2A-H3-H1 as the major organization unit in the genome of Mytilus galloprovincialis with two 5S rRNA genes with interspersed nontranscribed spacer segments linked to the unit, which is not justified by their cotranscription with histone genes. Surprisingly, 3' UTR regions of histone genes show two different mRNA termination signals, a stem-loop and a polyadenylation signal, both related to the evolution of histone gene expression patterns throughout the cell cycle. The clustered H1 histones characterized share essential features with "orphon" H1 genes, suggesting a common evolutionary origin for both histone subtypes which is supported by the reconstructed phylogeny for H1 genes. The characterization of histone genes in four additional Mytilus species revealed the presence of strong purifying selection acting among the members of the family. The chromosomal location of most of the core histone genes studied was identified by FISH close to telomeric regions in M. galloprovincialis. Further analysis on nucleotide variation would be necessary to assess if H1 proteins evolve according to the birth-and-death model of evolution and if the effect of the strong purifying selection maintaining protein homogeneity could account for the homologies detected between clustered and "orphon" variants.

Molecular and evolutionary analysis of mussel histone genes (Mytilus spp.): possible evidence of an "orphon origin" for H1 histone genes.

Linker histones are a divergent group of histone proteins with an independent evolutionary history in which, besides somatic subtypes, tissue- and differentiation-specific subtypes are included. In the present work H1 histone coding and noncoding segments from five Mytilus mussel species (Mollusca: Bivalvia) widely distributed throughout the world have been determined and characterized. Analysis of promoter regions shows clear homologies among Mytilus H1 genes, sea urchin H1 genes, and vertebrate differentiation-specific H1 subtypes (H5 and H1(o)), all having an H4 box motif in common. The amino acid sequence of the H1 protein central conserved domain is also closely related to that previously defined for the vertebrate divergent subtypes. A phylogenetic tree reconstructed from different H1 genes from several species strengthens the hypothesis of an "orphon" origin for the Mytilus H1 genes, as well as for the H1(o)/H5 genes from vertebrates and the H1D gene from the sea urchin Strongylocentrotus purpuratus, is suggested. As additional data, the average copy number of the H1 genes in the species analyzed was estimated as being 100 to 110 copies per haploid genome, where FISH revealed telomeric chromosomal location for several H1 copies in M. galloprovincialis. The contribution of such proximity to heterochromatic regions over the amount of codon bias detected for H1 genes is discussed.