MBE Advance Access originally published online on September 18, 2006
Molecular Biology and Evolution 2006 23(12):2316-2325; doi:10.1093/molbev/msl119
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Research Articles |
Interplay of Selective Pressure and Stochastic Events Directs Evolution of the MEL172 Satellite DNA Library in Root-Knot Nematodes
trovi
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* Department of Molecular Biology, Ru
er Bokovi Institute, Zagreb, Croatia
Interactions Plantes-Microorganismes et Santé Végétale, INRA/UNSA/CNRS, UMR1064, Sophia Antipolis, France
E-mail: plohl{at}irb.hr.
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Abstract |
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According to the library model, related species can have in common satellite DNA (satDNA) families amplified in differing abundances, but reasons for persistence of particular sequences in the library during long periods of time are poorly understood. In this paper, we characterize 3 related satDNAs coexisting in the form of a library in mitotic parthenogenetic root-knot nematodes of the genus Meloidogyne. Due to sequence similarity and conserved monomer length of 172 bp, this group of satDNAs is named MEL172. Analysis of sequence variability patterns among monomers of the 3 MEL172 satellites revealed 2 low-variable (LV) domains highly reluctant to sequence changes, 2 moderately variable (MV) domains characterized by limited number of mutations, and 1 highly variable (HV) domain. The latter domain is prone to rapid spread and homogenization of changes. Comparison of the 3 MEL172 consensus sequences shows that the LV domains have 6% changed nucleotide positions, the MV domains have 48%, whereas 78% divergence is concentrated in the HV domain. Conserved distribution of intersatellite variability might indicate a complex pattern of interactions in heterochromatin, which limits the range and phasing of allowed changes, implying a possible selection imposed on monomer sequences. The lack of fixed species-diagnostic mutations in each of the examined MEL172 satellites suggests that they existed in unaltered form in a common ancestor of extant species. Consequently, the evolution of these satellites seems to be driven by interplay of selective constraints and stochastic events. We propose that new satellites were derived from an ancestral progenitor sequence by nonrandom accumulation of mutations due to selective pressure on particular sequence segments. In the library of particular taxa, established satellites might be subject to differential amplification at chance due to stochastic mechanisms of concerted evolution.
Key Words: satellite DNA library • sequence variability • concerted evolution • selective pressure • parthenogenesis • root-knot nematode
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Introduction |
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TOPAbstractIntroductionMaterials and MethodsResultsDiscussionSupplementary MaterialAcknowledgementsReferences |
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Satellite DNA (satDNA) sequences are highly reiterated genomic components located in heterochromatic compartments including functional centromeres. They often differ in copy number, nucleotide sequence, and composition, making entire profile of satDNA sequences species specific. However, specificity of satellite profiles can only be the result of rapid fluctuations in copy number within a set of satDNAs, without changes in nucleotide sequences (reviewed in Ugarkovi
and Plohl 2002
). According to the library model, an ancestor of closely related species contains a set of satDNAs within its genome, and as new species emerge, one or few satDNAs become predominant whereas other repeats remain present as low–copy number sequences (Fry and Salser 1977; Metrovi et al. 1998). Differential amplification/deletion of (peri)centromeric satellites from the library can be linked with speciation processes (Metrovi et al. 1998). However, the library model does not predict if the choice of diverse satellite families able to enter and persist in a library is driven solely by random processes or the selection of satellite families is constrained.
SatDNA sequences evolve according to principles of concerted evolution, in which mutations are homogenized throughout a repetitive family and fixed within a group of reproductively linked organisms in a stochastic process of molecular drive (Dover 1986). However, study of sequence variability in Arabidopsis centromeric satellite and in human 
-satellite (Hall et al. 2003) showed nonrandom patterns of evolution within their monomer sequences. This study highlighted the hypothesis that the number and distribution of changes in satDNA monomers are the result of a balance between forces that generate and spread sequence changes and the selective pressure(s) acting on satellite sequences. The hypothesis is supported by recent observations that satDNAs may be involved in various functional interactions in heterochromatin. For example, satDNAs located in a region of functional centromere are proposed to interact specifically with centromere-specific, histonelike variants and drive their evolution (Talbert et al. 2004; Henikoff and Dalal 2005, and references therein). Recent analyses showed that transcripts of satDNAs can be functional in the form of small interfering RNAs, which act as signals in heterochromatin formation and maintenance (Martienssen 2003).
Root-knot nematodes (RKNs) of the genus Meloidogyne are agriculturally important, widely distributed plant-parasitic nematodes, with species that mostly reproduce by parthenogenesis (mitotic or meiotic), whereas amphimixis is encountered less frequently (Castagnone-Sereno 2006). In our previous study, we characterized 2 high–copy number satDNAs in the mitotic nematodes Meloidogyne javanica and Meloidogyne paranaensis (Metrovi et al. 2005). The satellite repeats from M. javanica could not be resolved from those described earlier in Meloidogyne arenaria (Castagnone-Sereno et al. 2000) and were therefore indicated as a single satDNA family named MARJA. In addition, this satDNA showed 65% similarity in comparison with the MPA1 satDNA characterized in M. paranaensis (Metrovi et al. 2005). Intra- and intervariability analysis of MARJA and MPA1 satDNAs disclosed one domain of high and one of low variability in segments of monomer sequences, equilocated within and between the 2 satellite families.
In order to study sequence dynamics of satDNAs in congeneric Meloidogyne species, in this paper, we characterize additional MARJA and MPA1 monomers from Meloidogyne incognita and Meloidogyne hapla B and a new related class of satDNA, AJL, identified in M. arenaria, M. javanica, and M. incognita. New monomer sequences were analyzed together with those obtained previously, MARJA form M. arenaria and M. javanica and MPA1 from M. paranaensis (Castagnone-Sereno et al. 2000; Metrovi et al. 2005) by phylogenetic approach, by analysis of distribution of polymorphic nucleotide positions and by the study of transition stages in the process of sequence homogenization. To our knowledge, for the first time, analysis of spread and homogenization of mutated nucleotides along the monomer sequences of MEL172 satDNAs disclosed domains with 3 different levels of variability conserved across the set of related satDNAs.
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Materials and Methods |
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TOPAbstractIntroductionMaterials and MethodsResultsDiscussionSupplementary MaterialAcknowledgementsReferences |
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Sampling and DNA Isolation
Five parthenogenetic Meloidogyne species were used in this study: M. javanica (Brazil), M. paranaensis (Brazil), M. incognita (France), M. arenaria (France), and M. hapla strains A and B (France). Nematodes were maintained on tomatoes (Lycopersicon esculentum cv. Saint Pierre) grown at 20 °C in a greenhouse. They were specifically identified morphologically and according to their isoesterase electrophoretic pattern (Carneiro et al. 2000
). Eggs were collected from infested roots, according to the procedure described earlier (Castagnone-Sereno et al. 2000). For each nematode isolate, total genomic DNA was purified from 100 to 200 µl eggs using the DNeasy Tissue Kit (Qiagen, Courtaboeuf, France) according to the manufacturer's instructions.
Detection and Cloning of Satellite Sequences
Satellite sequences were amplified in Meloidogyne species with specific primers designed on consensus sequences derived from MARJA and MPA1 satellites analyzed previously (Metrovi et al. 2005) and from the AJL satDNA characterized in this study. The following primer pairs were used to amplify sequences homologous to MARJA (aj 5'-ACGAT AAACTTTGTGGGGTAA-3' and aj1 5'-GGGTACAATTCGTCTTGGTAA-3'), MPA1 (p1 5'-TGATTTTCTGGATGGTGATACG-3' and p2 5'-GATTGGGTACAGGTCGTGGT-3'), and AJL (ajL1 5'-AATGACGTTTAAATTGTTTTAATATCG-3' and ajL2 5'-TTTATGACTGGCACTATTCAAAG-3') satDNAs. Positions of primers are indicated in figure 5. Amplifications were performed with HotMaster Taq DNA polymerase (Eppendorf, Hamburg, Germany) using annealing temperature of 55 °C and 1-min extension at 65 °C for 35 cycles. Southern hybridization analysis of polymerase chain reaction (PCR) products was performed with cloned satellite monomers (MARJA, MPA1, and AJL) as probes. Hybridization was done using the DIG DNA Labeling and Detection kit (Roche Biochemical, Mannheim, Germany) according to the manufacturer's instructions under high stringency conditions (hybridization temperature: 68 °C; washing: 0.1 x SSC, 1% sodium dodecyl sulfate (SDS) at 66 °C). Under relaxed conditions, PCR amplification (annealing at 52 °C) and Southern hybridization (60 °C; washing: 0.1 x SSC, 1% SDS at 58 °C) revealed the same pattern (data not shown). The control of PCR reaction was done with internal transcribed spacer (ITS) primers (Zijlstra 1997). The possibility of contamination with nonspecific DNA was excluded through reactions with sequence-characterized amplified region (SCAR) primers specific for M. javanica, M. arenaria, and M. paranaensis. SCAR primers produce randomly amplified polymorphic DNA fragments that are specific for 1 species only (Randig et al. 2002). The amount of satDNAs in Meloidogyne species was estimated by quantitative dot-blot analysis, as described previously (Metrovi et al. 2005).
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The selected products amplified by PCR with primer pair specific for MPA1, MARJA, and AJL satellites were ligated into a pGEM T-vector System I (Promega, Madison, WI) and transformed in Escherichia coli DH5
-competent cells. Recombinant clones with dimers and trimers of satDNA were sequenced on both strands by Genome Express (France). The sequences were trimmed to the monomer length avoiding primer-binding sites to prevent any effect on the analysis of sequence variability. Monomer sequences obtained in this work were deposited in European Molecular Biology Laboratory databank under accession numbers: DQ435688–DQ435766 and DQ834644–DQ834669.
Sequence Analysis
Multiple sequence alignments were done using ClustalX. Phylogenetic analyses were performed using the parsimony criterion in PAUP*, version 4b10 (Swofford 1998). All nucleotide positions were considered equivalent and weighted equally, using gaps as missing data. A parsimony tree was obtained after 500 random searches with Tree Bisection-Reconnection branch swapping and the Multrees option. Bootstrap values were calculated on 500 replicates. The nucleotide diversity was estimated using DnaSP program v.3.99 (Rozas J and Rozas R 1999). The nucleotide diversity Pi is defined as the average number of nucleotide differences per site between any 2 DNA sequences chosen randomly from the sample population. The conserved and variable regions in satDNAs were defined by sliding window analysis using a window length of 15 bp and a step size of 2 bp. Windows exhibiting variability Pi 
(average + 2 standard deviation) or Pi 
(average – 2 standard deviation) were considered significant.
The variation pattern at each nucleotide position of monomer sequences shared between 2 groups of sequences was analyzed by the method of Strachan et al. (1985), recently reintroduced in analysis of tandemly repeated sequences (Pons et al. 2002). This method enables comparisons between 2 sets of sequences. Each nucleotide position is classified into 1 of the 6 homogenization classes (Strachan et al. 1985), which we further grouped into 3 transition stages: homogenized (class 1), intermediate (classes 2–4), and advanced (classes 5 and 6). The homogenized stage represents complete homogeneity of a nucleotide position across all monomers from 2 satDNAs. The intermediate stage includes gradual spreading of a new mutation in 1 satDNA family, whereas the other family remains homogeneous for a progenitor base. The advanced stage includes positions where the 2 satDNAs are completely homogeneous for different nucleotides (class 5) and positions in which homogenized mutation in one set of sequences is subsequently replaced with a new one (class 6).
Detection of internal repeats was performed on the consensus sequence through the online program OligoRep (http://www.mgs.bionet.nsc.ru/mgs/programs/oligorep/). Tertiary structure was studied by constructing 3-dimensional models of DNA helix axis by the online program model.it (http://www.icgeb.trieste.it/dna/).
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Results |
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TOPAbstractIntroductionMaterials and MethodsResultsDiscussionSupplementary MaterialAcknowledgementsReferences |
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Distribution of MARJA and MPA1 SatDNAs in Meloidogyne spp.
PCR amplification with primers specific for MARJA (Me
trovi et al. 2005) and the corresponding Southern hybridization analysis revealed a well-defined ladder of bands with expected fragment length in M. incognita, M. paranaensis, and M. hapla B but not in meiotic M. hapla A (fig. 1a and b). Genomic DNAs from M. arenaria and M. javanica were not included in this analysis because they harbor already characterized high-copy MARJA repeats (Castagnone-Sereno et al. 2000; Metrovi et al. 2005). Results of PCR amplification of M. arenaria, M. javanica, M. incognita, and M. hapla A and B genomic DNAs with primers specific for MPA1 satDNA and subsequent Southern hybridization analysis of amplified fragments are presented in figure 1c and d. Again, the source of previously determined high-copy MPA1 repeats, M. paranaensis, was not included in the PCR screen (Metrovi et al. 2005). Bands of expected size were obtained in the amplification of M. incognita and M. hapla A and B genomic DNAs, although in the latter signals were of very low intensity. After amplification of M. arenaria and M. javanica genomic DNAs, bands of expected size appeared masked within intensive smear of fragments of various size. Because primer construction was limited by monomer length, high A + T content (>70%), and sequence similarity, the smeared signal might be a consequence of nonspecific annealing of p1 to MARJA satellite (see fig. 5), which is present in high–copy number in these species.
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Characterization of Satellite Repeats
PCR products obtained after amplification of genomic DNA from M. incognita and M. hapla B with primers specific for MARJA (fig. 1a) and for MPA1 (fig. 1c) were cloned and sequenced. MARJAi and MARJAhB monomers were obtained after amplification of M. incognita and M. hapla B genomic DNA with MARJA primers, respectively. Nucleotide diversity within MARJAi (Pi = 0.050) and MARJAhB (Pi = 0.056) monomers is slightly higher with respect to the diversity of original MARJA monomers (Pi = 0.040) from M. arenaria and M. javanica determined previously (Me
trovi et al. 2005), whereas the overall nucleotide diversity is 0.048 (table 1). Alignment of all MARJA monomers did not reveal any species-diagnostic mutations, and the whole set is therefore indicated as MARJAall (Supplement 1, Supplementary Materials online).
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MPA1 homologs from M. incognita and M. hapla B were cloned and sequenced. Monomers from M. hapla B (MPA1hB), with internal variability of 0.045, did not show any specific features compared with the original MPA1 satDNA determined previously (Me
trovi et al. 2005). On the contrary, sequence analysis of monomers obtained after amplification of M. incognita genomic DNA with primers specific for MPA1 disclosed 2 groups of repeating units. One group of monomers (MPA1i) falls into the range of internal variability (Pi = 0.050) of original MPA1 monomer variants. All MPA1 variants determined from examined taxa are indicated as MPA1all (see Supplement 1, Supplementary Materials online). Interestingly, the second group of highly homogenous set of monomers (AJL1i) shows lower divergence when compared with MARJAall than with MPA1all repeats, despite the fact that AJL1i monomers were obtained after amplification with primers specific for MPA1 (fig. 5). To reduce a potential bias induced by nonspecific annealing of MPA1 primers, new primers specific for AJL1i monomers (fig. 5) were designed with changed 3' end with respect to the MPA1 and MARJA satDNA sequences. To check for the presence of AJL1i monomers in Meloidogyne species, we performed PCR with specific primers and subsequent hybridization analysis. The results revealed a regular ladder-like pattern in M. arenaria, M. javanica, and M. incognita, but it was not possible to detect any band in M. paranaensis and in both strains of M. hapla (fig. 1e and f). Phylogenetic analysis (fig. 2) of all AJL monomers from M. incognita (AJL1i and AJL2i), M. arenaria (AJL2a), and M. javanica (AJL2j) does not show any subclustering. This new homogeneous group of repeats is indicated as AJLall.
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Sequence alignment of all monomer variants analyzed above with the original MARJA and MPA1 monomers described previously (Me
trovi et al. 2005) showed that the divergence is mainly due to nucleotide substitutions, with 120 variable sites out of 172 nt positions (see Supplement 1, Supplementary Materials online). The phylogenetic analysis (fig. 2) showed clustering of monomers into 3 groups (MARJAall, MPA1all, and AJLall) in the maximum parsimony tree, as described above. The 3 groups of sequences are resolved with high confidence, showing clearly that they belong to the 3 homogeneous satDNAs. Within each satDNA, monomer sequences could not be grouped according to the species of origin. Due to similarities in nucleotide sequence and monomer length, examined satellites are indicated as MEL172 group. Sequence analysis in order to detect substructure of satDNA monomers based on direct or inverted repeats as well as analysis of tertiary structure induced by bent DNA helix axis did not reveal any prominent feature (not shown). The relative contribution of MARJA, MPA1, and AJL, assessed from dot-blot experiments, show 2 distinctive levels of abundance in congeneric Meloidogyne species (table 2): the high–copy number (>10,000 copies per genome) and the low–copy number (between 100 and 1,000 copies per genome). Under conditions used in performed experiments, it was not possible to detect particular satDNAs in some species, either in dot-blot or in PCR analyses. However, level of sensitivity could not definitively exclude presence of residual copies of examined satDNAs in those species. It can be concluded that all 3 related satDNAs described above are differentially amplified in the set of congeneric Meloidogyne species, making a library of conserved satellite sequences.
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Distribution of Variability among MEL172 SatDNAs
The study of the distribution of nucleotide diversity among MEL172 satDNAs was performed by sliding window analysis. Sets of satDNA monomers were already defined in the phylogenetic analysis as MARJAall, AJLall, and MPA1all satDNAs (fig. 2). First, we performed pairwise analysis of examined satDNAs (fig. 3a–c) and then, using the same criteria, analysis on all 3 sequence sets together (fig. 3d). The analysis revealed 2 low-variable (LV) (LV1 and LV2), 2 moderately variable (MV) (MV1 and MV2), and 1 highly variable (HV) domain. It is difficult to define precisely the range of particular domains in monomer sequences, and approximate limits of each domain were determined as interception of the curve in figure 3d and the corresponding Pi ± 2 standard deviation value. Both LV domains remained unaffected irrespectively of the nucleotide divergence between compared satDNAs taken in the variability analysis. Between them, the most conserved sequence segment is the 19 bp long LV2 domain, with only 1 homogenized mutation (see fig. 5). MV domains showed a similar level of variability irrespectively of the sequence set taken in the analysis (fig. 3). In the HV domain, the variability increases in parallel with overall nucleotide divergence between satDNAs included in the analysis (fig. 3). However, overall intrasatellite variability is low in all examined satDNAs (table 1), and mutations are in general uniformly distributed along the monomer (not shown). The only exceptions are 2 segments in MARJA and MPA1 satDNAs in which intrasatellite variability roughly corresponds to intersatellite distribution of mutations (Me
trovi et al. 2005).
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In order to study dynamics of nucleotide changes in the process of sequence homogenization in domains defined above, we analyzed transition stages by the method proposed by Strachan et al. (1985)
, modified as described in Materials and Methods. Each position in the pairwise analysis of above-defined sets of satDNA monomers was classified into 1 of the 3 transitional stages: homogenized, intermediate, and advanced stage. The results of the analysis are presented in figure 4a for LV domains LV1 and LV2 together, in figure 4b for both MV domains MV1 and MV2, and in figure 4c for the HV domain. Limits of domains were as in sliding window analysis (fig. 3) and are also shown in figure 5. Most positions (>70%) in LV domains in all comparisons (fig. 4a, columns I–III; divergence Pi over the entire monomer length is indicated at the top) belong to the homogenized stage, and the rest of positions can be mostly classified in the intermediate stage (18–22% of mutated positions). In MV domains (fig. 4b, columns I–III), the number of sites in homogenized stage decreases, whereas the number of sites in intermediate stage increases in parallel with divergence between sets of satDNAs. Interestingly, the portion of mutations in advanced stage (from 21% to 25%) remains similar regardless of the divergence between sets of satDNAs, indicating a limited homogenization of new mutations in the MV domain. On the contrary, the number of sites falling in the advanced stage increases dramatically in the HV domain (from 20% to 60%; fig. 4c, columns I–III) in parallel with increased sequence divergence.
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The comparison among the consensus sequences of each MEL172 satDNA (fig. 5) shows that sequence divergence is concentrated in the HV domain, in which 29 nt positions out of 37 are changed (78%). In MV domains, 42 positions out of 88 (48%) are changed. However, only 3 positions are changed in 48 nt long segments of LV domains (6%).
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Discussion |
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TOPAbstractIntroductionMaterials and MethodsResultsDiscussionSupplementary MaterialAcknowledgementsReferences |
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The satDNAs analyzed here in RKNs of the genus Meloidogyne have 2 important features concerning evolution of satDNA sequences. First, variability analysis revealed nonrandom accumulation of mutations along monomer sequences of the 3 related MEL172 satDNAs, showing domains with different variability levels preserved across the set of satDNAs. Second, related satDNAs analyzed in this work coexist in the majority of examined parthenogenetic taxa in differing abundances, thus forming a library of conserved repeats.
Intersatellite comparisons show that the entire monomer sequence of MEL172 satDNAs can be subdivided into 2 LV domains, 2 MV domains, and 1 HV domain. The LW domains are highly homogeneous between satDNAs. Whereas the MV domains are characterized by the limited number of mutations homogenized within each satDNA family, the number of homogenized changes in the HV domain increases in parallel with sequence divergence between MEL172 satDNAs. This observation supports the idea about extensive homogenization processes in the fixation of changes on "allowed" positions in limited sequence segments of MEL172 satDNAs. Domains with different variability levels alternating in the monomer sequence of MEL172 satDNAs suggest a complex pattern of interactions that might be involved, for example, in nucleosome phasing and heterochromatin formation. In that case, LV domains might represent the yet uncharacterized DNA sequence motifs involved in DNA–protein interactions. Unfortunately, the small size of nematode chromosomes (<0.5 µm in metaphase) and their holocentric nature (Triantaphyllou 1985) impose serious limitations on the precise localization of characterized satDNAs, and in addition, nothing is known about the heterochromatin structure and histone variants in holocentric RKN chromosomes (reviewed in Castagnone-Sereno 2006). Polymorphism observed recently in Arabidopsis thaliana centromeric satDNA and in human -satellite was interpreted as an indication of constraints imposed on particular segments of monomer sequence (Hall et al. 2003). It was suggested that the effect may be caused by functional interactions between the satDNA and various protein components in heterochromatin and/or in the functional centromere, but direct experimental evidence for this hypothesis is still lacking. Alternatively, the lack of conservation of LV domains in satDNAs from different Arabidopsis species led to an assumption that regions with reduced polymorphism may serve as sites that promote recombination within repeats (Hall et al. 2005). A similar alternative function of LV domains might be important in the spread of MEL172 satDNAs along holocentric chromosomes. Structural characteristics of satDNAs (e.g., dyad structures and intrinsically bent helix axis) might represent additional features involved in tight packing of DNA in heterochromatin (Ugarkovi and Plohl 2002). The fact that no prominent secondary or tertiary structure was detected in any of MEL172 satDNAs reinforces the hypothetical significance of nucleotide sequence variability pattern. The repeat length could also be an important determinant required for uniform nucleosome phasing and heterochromatin propagation (Henikoff et al. 2001). The repeat length of MEL172 satellite monomers is well conserved and equals that of human -satellite (Durfy and Willard 1990) and A. thaliana satellite monomers (Hall et al. 2003).
Based on presented results, we propose 2 phases in the evolution of MEL172 satDNAs, formation of satDNAs, and persistence of satDNAs in the library. Constitution of the library must be a process that occurred in ancestral organism(s), before the onset of extant species. Molecular studies based on mitochondrial DNA markers have shown that mitotic parthenogenetic RKN species share a common lineage and that they diverged from amphimictic/meiotic species (Hugall et al. 1997). A set of divergent satDNAs could be formed from an ancestral repetitive sequence by amplification of mutated monomer variants nascent in regions of restricted sequence homogenization. In accord with Dover's (1986) hypothesis, it has been suggested that limited sequence homogenization between distantly located arrays can explain origin of divergence among subsets of Arabidopsis centromeric satellites (Hall et al. 2005). Similarly, the source of novel (sub)families can be in aberrant satDNA repeats accumulated at array ends due to low homogenization efficiency in bordering regions (Schueler et al. 2005). In our case, we propose that mutated monomers of the ancestral MEL172 sequence can be amplified and constitute the library of satDNAs only if they have sequence characteristics necessary for putative interactions in heterochromatin. Therefore, the hypothesis could be raised that monomers of MEL172 satDNAs were initially shaped and spread under selective pressure due to functional interactions. Once established, these satDNAs might be subject to differential amplification in particular taxa at chance, due to dynamics of recombinational mechanisms in the process of concerted evolution. Consistent with the library hypothesis, the whole set can be in distant species replaced with satDNA families of unrelated origin, what might be followed by adaptive coevolution of DNA-binding components. Results indicating adaptive coevolution of centromere-specific, histone-like variants and satDNAs in several animal and plant experimental systems (Henikoff and Dalal 2005) add to the reliability of the proposed scenario.
Based on the hypothesis of ancient origin of M. hapla, dated about 43 MYA (Hugall et al. 1997), we propose a long-term conservation of MEL172 satDNAs. Paradoxically, MEL172 satDNAs in the library are interspecifically conserved in a way that their constitutive monomers remain unaltered in the entire length, even in domains characterized as HV. This would indicate that homogenization mechanisms act on the satellite monomer as a whole, favoring persistence of the ancestral monomer sequence. The theory of concerted evolution predicts that a small bias in favor of some set of monomer variants can result in extreme sequence conservation, preventing the fixation of new sequence variants at the species level (Ohta and Dover 1984; Strachan et al. 1985). It is possible that this effect of concerted evolution can be enhanced by functional constraints imposed on the sequence. Alternatively, conservation of satellite sequences in sturgeons for more than 100 Myr is explained as an effect of slow rate of both sequence changes and concerted evolution (Robles et al. 2004). Long-term conservation of satDNAs among RKN species presented in this paper and in recent analysis (Metrovi et al. 2006) is comparable to that found in highly conserved satDNAs in sexual species (Mravinac et al. 2002, 2005). In accord with this observation, satellite monomer variants in parthenogenetic Bacillus taxa constitute an ancestral library, conserved during significant evolutionary periods (Cesari et al. 2003). Evolution of MEL172 satDNAs does not show any specificities that could be linked directly with the parthenogenetic mode of reproduction, similar to that observed in comparative analysis of Bag320 in bisexual and unisexual Bacillus taxa (Luchetti et al. 2003). These results indicate that turnover mechanisms in mitotic parthenogens might be sufficient to preserve some "optimal" set of monomer variants for long periods of time.
Phylogenetic studies suggested that the ancestor of meiotic/mitotic M. hapla separated first from the other examined species and represents the most divergent species in the group (Hugall et al. 1997). M. incognita, M. javanica, and M. arenaria form a sister species cluster, whereas M. paranaensis is more distant (De Ley et al. 2002; Tenente et al. 2004). An additional hypothesis based on analysis of ITS fragments indicates hybridization and subsequent polyploidization during evolution of mitotic lineages (Hugall et al. 1999). Nevertheless, besides deep ancestry of M. hapla, exact dating and sequence of events in Meloidogyne is only partially understood (reviewed in Castagnone-Sereno 2006). It has been observed that gradual accumulation of divergences among satDNA sequences of related species or populations can generate useful molecular marker on different taxonomic levels, depending on the rate of accumulation of nucleotide substitutions (reviewed in Ugarkovi and Plohl 2002). In contrast to that, sequence comparison of related satDNAs in the MEL172 library has no relevance to the phylogeny of Meloidogyne species. In that context, presence/absence of satDNAs in particular Meloidogyne species can be more informative because taxonomic relationships might be mirrored by the time of satDNA variant birth or elimination in course of genomic turnover processes. For example, the AJL satDNA is distributed within the sister cluster M. arenaria, M. javanica, and M. incognita, whereas it could not be detected in more distant M. paranaensis and in both races of M. hapla. Inability to detect most, if not all, MEL172 satDNAs in M. hapla A may be related to a higher level of sequence dynamics due to meiotic reproductive lifestyle and/or specific species/taxon traits (Luchetti et al. 2003).
In conclusion, we suggest that interplay of stochastic events and selective pressure can explain constitution and persistence of related satDNAs in the library. The sequence features might represent a limiting factor in the formation and survival of new satDNA families in the library, although their ultimate sequence dynamics will depend on stochastic turnover processes in a particular species. In addition to the examined MEL172 satellites, presence of other satellites of this group could not be excluded in Meloidogyne species. In the future, the characterization of nucleosomal phasing and DNA–protein interactions in heterochromatin compartments of RKN species could represent a convenient experimental system to test constraints that limit changes in satellite monomers and possible role of these satDNAs in centromeric function of holocentric chromosomes.
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Supplementary Material |
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TOPAbstractIntroductionMaterials and MethodsResultsDiscussionSupplementary MaterialAcknowledgementsReferences |
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Supplement 1 is available at Molecular Biology and Evolution online (http://www.mbe.oxfordjournals.org/).
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Acknowledgements |
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TOPAbstractIntroductionMaterials and MethodsResultsDiscussionSupplementary MaterialAcknowledgementsReferences |
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This work has been supported by the Research Fund of Republic of Croatia and French Institut National de la Recherche Agronomique. Short exchange visits were funded through the Croatian–French bilateral project "COGITO" #06410ZL. We would like to thank Daphne Preuss and Joan Pons for critical reading and useful suggestions during preparation of the manuscript, as well as to 2 anonymous referees, whose comments helped to significantly improve the manuscript.
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Footnotes |
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Aoife McLysaght, Associate Editor
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References |
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