Evolutionary Dynamics of Satellite DNA Family PIM357 in Species of the Genus Pimelia (Tenebrionidae, Coleoptera)
Joan Pons*
,1,
Eduard Petitpierre*
and
Carlos Juan*
*Laboratori de Genètica, Departament de Biologia, Universitat de les Illes Balears, Palma de Mallorca, Balearic Islands, Spain;
Division of Insect Biology, ESPM, University of California;
Departament de Recursos Naturals, Institut Mediterrani d'Estudis Avançats (CSIC-UIB), C/. Miquel Marqués, Esporles, Balearic Islands, Spain
 |
Abstract
|
---|
A large number of repeats of a satellite DNA (stDNA) family have been cloned and sequenced from species and populations of the genus Pimelia (Tenebrionidae, Coleoptera). The beetles were collected in the Canary Islands, Morocco, the Iberian Peninsula, and the Balearic Islands in order to analyze the evolutionary forces and processes acting on abundant stDNAs conserved at the genus level. This repetitive family is composed of an abundant A-T-rich stDNA, with basic units of 357 bp. All the sequences obtained showed similarity to the 22 repeat units of the PIM357 stDNA family described previously for six Iberian Pimelia species (Pons et al. 1997
). An analysis based on similarity shows the presence of three different groups of sequences clearly in accordance with their geographical origin. One is composed of satellite sequences from Iberian and Balearic species, a second group from the Moroccan taxa, whereas the third one is from the Pimelia species endemic to the Canary Islands. The latter group shows higher nucleotide diversities for their stDNA sequences and a lack of relationship between transition stages to fixation and sequence divergence. Phylogeographic data of Canarian Pimelia show that the PIM357 stDNA family has persisted for more than 8 Myr and could probably be traced to the origin of the lineage. The data suggest that distinct demographic and phylogenetic patterns related to the colonization of the volcanic Canarian island chain account for particular evolutionary dynamics of the repeat DNA family in this group.
 |
Introduction
|
---|
Satellite DNA (stDNA) is a genomic component almost universally present in eukaryotic genomes with a characteristic tandemly repeated sequence up to the level of megabase DNA clusters and typically associated with heterochromatic blocks (Elder and Turner 1995
). Tandemly repeated units are thought to spread horizontally within the repeat family by means of unequal crossing-over, transposition, and gene conversion, although the relative contribution of each process is not clear (Charlesworth, Sniegowski, and Stephan 1994
). These mechanisms usually lead to a high intraspecific similarity of array repeats and low or undetectable interspecific similarity, making many stDNAs species-specific after fixation in the population by inbreeding, random genetic drift, or selection. This pattern of nonindependent evolution of the repetitive units within a family is known as concerted evolution (Smith 1976
) and has been explained by molecular drive (Dover 1982
). Unequal recombination and segregation seem to be the main factors acting at stDNA evolution in many cases, as shown in the comparison of repetitive sequences from bisexual and automictic parthenogenetic species of stick insects of the genus Bacillus (Mantovani et al. 1997
). The evolutionary dynamics of tandemly repetitive DNAs in sexual organisms leads to a gradual and cohesive spreading of a new repeat unit produced by mutation through a particular stDNA family (known as homogenization) and through the whole population (fixation). The probability and time necessary for homogenization and fixation are dependent on population size, stDNA copy number, and rates and biases of nonreciprocal transfers (Dover 1982
; Ohta and Dover 1984
).
Darkling beetles (family Tenebrionidae) show the presence of significant amounts of stDNA, and their highly repetitive sequences have been studied in detail in species of the genera Tribolium, Tenebrio, and Palorus (see Ugarkovic et al. 1995
for a review). Although the usual pattern in these taxa is the presence of species-specific stDNAs, six species of Pimelia (also a genus in the same family Tenebrionidae) share a highly conserved stDNA (named PIM357, Pons et al. 1997
). This repetitive family is composed of an abundant A-T-rich stDNA, with basic units with an average size of 357 bp. The analysis of 22 cloned repeats showed high intra- and interspecific sequence similarity among repeats, grouped into two clusters of related sequences showing no particular species-specific mutations with the exception of one of the species (P. integra). One group of stDNA sequences was obtained from the species P. interjecta, P. criba, and P. elevata, whereas the other was present in P. baetica, P. variolosa, and P. integra. These species-groups show disjunct geographical distributions: in the Northeast Iberian Peninsula plus Balearic Islands and in the Southeast of the Iberian Peninsula, respectively. There are other instances in which a major satellite family is conserved and spread among diverse taxonomic groups of different evolutionary ages (e.g., Fanning et al. 1988
; Arnason, Gretarsdottir, and Widegren 1992
; Garrido-Ramos et al. 1995
; Modi, Gallagher, and Womack 1996
; King and Cummings 1997
). However, there are few experimental data showing the gradual stages of spreading of particular repeat variants in a phylogenetic framework (see for example Strachan, Webb, and Dover 1985
; Mantovani et al. 1997
; Mantovani 1998
).
In this article we have extended the study on the PIM357 stDNA to 20 additional congeneric species and populations of Pimelia collected from the Canary Islands, Morocco, the Iberian Peninsula, and the Balearic Islands to study the taxonomic extent and molecular evolution of the repeated family in deeper detail.
 |
Material and Methods
|
---|
Sampling and DNA Isolation
Beetles were collected at localities of the Canary Islands, the Iberian Peninsula, the Balearic Islands, and Morocco (table 1 ). DNA was isolated from adults by standard phenol extraction and ethanol precipitation procedures (Sambrook, Fritsch, and Maniatis 1989
, pp. 1619).
View this table:
[in this window]
[in a new window]
|
Table 1 List of Specimens (Pimelia), Collection Localities, Percentages of stDNA, and Characteristics for Cloned Repeat Units Studied
|
|
Isolation, Cloning, and Sequencing of stDNA
Digestions of genomic DNA with restriction enzymes were performed according to the instructions of the manufacturer (Roche), and the fragments were separated by electrophoresis on 1.5% agarose gels. The DNA bands corresponding to monomers or oligomer sequences were cut from the agarose gel and purified with the Gene Clean Kit (Bio 101 Inc.), ligated into the EcoRI or the SmaI site of the plasmid pUC18 vector (Amersham), and used to transform Escherichia coli DH5
. The clones were screened using the ß-galactosidase gene blue-white color system (Sambrook, Fritsch, and Maniatis 1989
, pp. 8587). Recombinant clones were digested with EcoRI or EcoRI plus HindIII and subsequently separated by electrophoresis on 1.5% agarose gel to check the size of the insert. The positive clones were sequenced on both strands by the dideoxy sequencing method (Sanger, Nicklen, and Coulson 1977
) using the Dig Taq DNA Sequencing Kit for Cycle Sequencing (Boehringer Mannheim) and the semiautomatic sequencing system GATC 1500-System Direct Blotting Electrophoresis (Boehringer Mannheim). For Southern analysis, 5 µg of genomic DNA from each species was digested with different restriction enzymes and blotted on nylon membranes. Satellite clones labeled with digoxigenin were used as hybridization probes under high and low stringency conditions. The relative amount of stDNA was determined from digestion of genomic DNA (EcoRI or HindIII) electrophoresed on an agarose gel. The digitalization and densitometric measurements from the gel photographs were performed with the help of the Sun View program (Pharmacia).
PCR Amplification of Cytochrome Oxidase I (COI)
DNA from single individuals was extracted using the standard procedure outlined earlier. The two oligonucleotide primers used for PCR amplifications were 5'-CCAACAGGAATTAAAATTTTTAGATGATTAGC-3' and 5'-TCCAATGCACTAATCTGCCATATTA-3' (Juan, Oromí, and Hewitt 1995
). The amplification conditions for each of 35 cycles were: denaturation at 94°C for 1 min, annealing at 50°C for 1 min, and primer extension at 72°C for 2 min. PCR products were sequenced directly using the same primers.
Sequence Analysis
Multiple alignment was performed using Clustal W v. 1.7 (Higgins, Thompson, and Gibson 1996
) available on-line (http://bioweb.pasteur.fr/seqanal/interfaces/clustalw.html). Sequence divergences were calculated according to the two-parameter method of Kimura (1980)
and distance trees produced by the neighbor-joining method (NJ, Saitou and Nei 1987
) using PAUP* v. 4.0 (Swofford 1999
). Details of the nucleotide composition,
2 homogeneity test of base frequency across sequences, and patterns of nucleotide substitution in the repetitive units were estimated using the PAUP* package. Nucleotide diversity, pairwise sequence divergences, and putative gene conversion events were estimated using the DnaSP v. 2.52 package (Rozas and Rozas 1997
). The divergence was calculated as the average of nucleotide substitution per site between species (Dxy value from DnaSP 2.52, Nei 1987
, pp. 221226). Search for A or T
three tracts, their expected frequencies in a random sequence of the same A+T content, and analysis of retarded mobility of stDNA sequences on nondenatured polyacrylamide gel electrophoresis were performed as described in Barceló et al. (1997)
. We have followed the method proposed by Strachan, Webb, and Dover (1985)
to analyze the pattern of variation at each nucleotide position of a stDNA shared by two species. This allows us to deduce the transition stages of the concerted evolution of a tandem repeated family. This method compares the sequences obtained from two species at individual nucleotide positions considered independently, each position being classified in one of six different homogenization stages (classes 16). A detailed explanation of different nucleotide position classes is presented in figure 1
.

View larger version (25K):
[in this window]
[in a new window]
|
Fig. 1.Graphic representation of transition stages during the spread of new mutations (modified from Strachan, Webb, and Dover 1985
). Classes 16 represent the patterns of distributions of mutations at individual nucleotide positions across clones af in two species, A and B. Class 1 represents complete homogeneity across all clones randomly sampled from a pair of species (white circles). Classes 2, 3, and 4 would represent intermediate stages where a new mutation (black circle) is gradually spread throughout the stDNA family in one species, whereas the other species remains homogeneous for the progenitor base in the corresponding position. Class 2 represents mutation in a single clone or low levels of subsequent spread, resulting in the appearance of a minority of clones. Class 3 includes those cases where no decision can be made between minority and majority frequencies, in that mutation and progenitor base are in equal frequencies. Class 4 covers those positions in which a mutation has replaced the progenitor base in the majority of members in the other species. Class 5 (diagnostic positions) represents positions where the two species are internally homogeneous for bases (one species shows the progenitor bases and the other the new one). All subsequent mutations (speckled circle) beyond this point are represented by the pattern shown in class 6
|
|
 |
Results
|
---|
Digestion of genomic DNA from most Pimelia taxa with the restriction enzyme EcoRI showed the presence of characteristic ladders of stDNAs (table 1
and fig. 2
). Similar results were obtained for P. lutaria and some of the Moroccan taxa using the restriction enzyme HaeIII instead of EcoRI. A prominent band of about 360 bp was common to all digestions and was the repeating unit used for subsequent cloning experiments. Cloned fragments were used as hybridization probes after Southern transfer of a panel of Pimelia genomic DNAs digested with EcoRI and HaeIII, at stringency conditions allowing annealing of sequences with 60% or higher similarity (representative digestion and Southern hybridizations are shown in fig. 2
). This revealed the presence of the characteristic ladders of stDNA in all the species. Densitometric quantification of the stDNAs was either performed from EcoRI genomic DNA digestions run in agarose gels (from which high molecular nondigested DNA and digested bands corresponding to stDNA could be compared directly) or from dot-blot experiments in the case of species with stDNA recognized by HaeIII (not shown) using a cloned repeat as standard. These estimations suggested that there is a high proportion of the stDNA sequences in Pimelia species' genomes, ranging from 27.1% to 43.6% (table 1
), which given the DNA content of the screened species of the genus roughly corresponds to an average of 4.5 x 105 copies per haploid genome.

View larger version (81K):
[in this window]
[in a new window]
|
Fig. 2.Agarose gel electrophoresis of P. sparsa sparsa and P. atlantis atlantis genomic DNAs digested with EcoRI (lane 1) and HaeIII (lane 3). Lane 2 corresponds to a DNA molecular standard (Marker VI from Roche) with bands ranging from 2,100 to 300 bp. The right panel corresponds to a Southern blot of the same gel hybridized with the cloned repeat unit pSSP40 from P. sparsa sparsa as probe under high stringency conditions
|
|
A total of 156 random clones obtained from the different taxa of Pimelia (table 1
) was sequenced. All the sequences obtained showed similarity to the 22 repeat units of the PIM357 stDNA family described previously for six Iberian Pimelia species which showed a consensus size unit of 357 bp (Pons et al. 1997
). The alignment of all 178 repeat units is deposited in the EMBL/GeneBank database under accession number DS45063. The sequences of P. lutaria were the most divergent with respect to the consensus, which can be explained both by nucleotide substitutions and two internal autopomorphic duplications of 10 and three nucleotides, respectively. These duplications make it the longest unit in the family (362365 bp). The Pimelia satellite sequences proved to be homogeneous in their nucleotide composition (average of 69% A+T rich) as suggested by the
2 homogeneity test of base frequency across sequences and taxa (P < 0.001). Moreover, the obtained sequences show a higher frequency of tracts of A or T
3 than the expected for a random sequence of the same length and A+T composition (Barceló et al. 1997
). These A-T rich motifs seem to be arranged in a phase which is relatively conserved throughout the repeating sequences of the different species analyzed (fig. 3
). Several stDNA studies have related the presence of periodical A or T tracts
3 (with an average of 10 nucleotides along the sequence) with the induction of DNA helix bending (Koo, Wu, and Crothers 1986
; Martínez-Balbás et al. 1990
). Nondenaturing polyacrylamide gels to test for DNA bending demonstrated retarded mobility on PIM357 sequences, and this was also supported by computational analyses using three different algorithms (Barceló et al. 1997
; Pons et al. 1997
).

View larger version (62K):
[in this window]
[in a new window]
|
Fig. 3.Nucleotide sequence alignment of a sample of cloned PIM357 repeat units of Pimelia species from the Canary Islands, Morocco, the Iberian Peninsula and the Balearic Islands. Dashes denote gaps. The A or T tracts 3 (shadowed) are phase distributed throughout the repeat, and this pattern seems relatively conserved in all the studied repeats
|
|
The nucleotide sequences of PIM357 stDNA clones were analyzed to retrieve potential subrepeat structures. A further search for direct and inverted repeats revealed a low number of short perfect subrepeats up to the size of pentanucleotides. However, the distribution pattern of these repeats was not conserved across stDNA units, suggesting that the A+T richness of the sequences could account for their appearance.
stDNA Sequence Divergence
Nucleotide substitutions are the major cause of sequence variability if we do not consider the two indels in the P. lutaria lineage. These substitutions are randomly distributed, as revealed by the
statistical test (Sneath 1995
), and they are able to discriminate whether sites differing in a pair of aligned sequences are significantly clustered. A phylogenetic analysis of all sequences based on their similarity was performed using the divergences corrected according to the two-parameter model of Kimura (1980)
and the NJ tree building method (Saitou and Nei 1987
). Figure 4
shows a plot relating the proportion of transitional changes as a function of pairwise uncorrected divergences in Pimelia PIM357 stDNA. As can be seen, there is evidence of saturation in the stDNA data set because the ts/tv ratio drops to less than one for interspecific divergence values higher than
0.15. The phylogenetic analysis showed the presence of three different groups of sequences clearly in accordance with their geographical origin (fig. 5
).

View larger version (12K):
[in this window]
[in a new window]
|
Fig. 4.Graphical representation of the transitions-transversions ratio as a function of uncorrected divergence in PIM357 stDNA sequences. Rhombs and dots represent intraspecific and interspecific comparisons, respectively.
|
|
We have produced an independent rooted phylogeny of the examined species based on partial COI mitochondrial sequences, adding to the Canary Island taxa COI data set reported elsewhere (Juan, Oromí, and Hewitt 1995
) sequences obtained from all the species studied in the present article. Unfortunately this mitochondrial marker seems to be too variable to robustly infer the basal evolutionary relationships of the major Pimelia species groups (fig. 6
). Low bootstrap values were obtained for most basal nodes using different tree-building algorithms or molecular evolutionary assumptions and hence, no firm conclusions could be drawn about monophyly of the Moroccan and the Iberian-Balearic Island groups, their hypothetical sister relationship, or basality with respect to the Canarian clade. In the stDNA unrooted phylogeny, the basal relationships cannot be determined but the Moroccan and Canary Islands form monophyletic clusters. The average divergence between any stDNA sequence from the Iberian-Balearic group and one from the African group is 0.20, whereas the average divergence between a stDNA sequence from these clusters and the Canarian group is 0.30 (see additional information at MBE website: www.molbiolevol.org).

View larger version (25K):
[in this window]
[in a new window]
|
Fig. 6.The NJ dendrogram of 26 Pimelia taxa based on Kimura's two-parameter distances from the mitochondrial COI gene sequences (Accession Numbers X97209X97222 and AJ248198AJ2481209). Tenebrio molitor is included as outgroup (Accession Number X88966). Bar represents genetic distance d = 0.02. Numbers at each node indicate the percentage of trees presenting the particular node out of 1,000 bootstrap replicates for Maximum Parsimony (first), NJ using K2p distances (second) and in an analysis using Maximum-Likelihood distances (third). The bootstrap values are indicated only for nodes for which at least one of the three mentioned analyses gave percentages higher than 50
|
|
The Iberian, Balearic, and Moroccan species showed low nucleotide diversities and relatively short branches in the NJ tree (fig. 5 ). Generally, their stDNA sequences are clustered species-specifically with no sharing of repeats among species, but few of the species show diagnostic nucleotide substitutions (only in the cases of P. integra, P. costata, P. maura, and P. boyeri). The Iberian-Balearic group shows two monophyletic clusters of stDNA sequences (CRI-ELE-INJ and BAE-VAR-ING-COS) and two or maybe three groups probably occur in the Moroccan cluster. Pimelia maura is distributed over Northwest Africa and reaches the south of the Iberian Peninsula, from where the samples sequenced in this study were collected. However, the analyzed stDNA sequences from this species are clearly close to its African relatives. The divergences (between sequences of two different species) are higher than their respective nucleotide diversities (within sequences of the same species), but some pairwise comparisons from the same cluster, such as INJ-CRI, BAE-VAR, or BOY-MAB, show similar levels of nucleotide diversities and divergences (see additional information).
In contrast, the tree topology for the stDNA sequences from the Canarian taxa shows that the species distribution of the repeats is less species-specific. These taxa showed higher nucleotide diversities for their stDNA sequences and relatively longer branches in the NJ tree (fig. 5
) than the Iberian, Balearic, and African counterparts. In fact, nucleotide diversities within the Canarian species (0.100.20) are in many cases similar to the values for the pairwise species comparisons. Three general groups of sequences can be deduced in the sample of Canarian taxa; one group is formed by P. lutaria sequences (LUT, a species exclusive to Fuerteventura and Lanzarote Islands), a group that is clearly monophyletic. The two other groups with moderately low bootstrap support are not arranged species-specifically and reflect their geographical provenance only in part. One includes sequences from the Tenerife endemics (RRA, RGR, RAS, and CAN sequences) and from P. laevigata (La Gomera, La Palma, and El Hierro islands, LLA, LVA, and LCO sequences). The sequences from P. laevigata proved to be very close to the ones from Tenerife, although they show some diagnostic nucleotide substitutions with respect to the latter. Finally, a third group of very divergent sequences is mainly composed of sequences from Gran Canaria taxa (note the significantly longer branch lengths in the tree in fig. 5
for SSP, SSE, SAH, GRA, and EST sequences). The sequences from P. fernandezlopezi (FER, a species endemic to La Gomera) are more related to the Gran Canarian sequences (P. granulicollis, GRA and P. estevezi, EST). Moreover, many of the Canarian species show the coexistence of two divergent subfamilies of repeats in their genomes. Stretches of mutations shared between two or more sequences can indicate the effect of gene conversion (Strachan, Webb, and Dover 1985
; Drouin and Dover 1990
). Possible gene conversion events were detected in some Canarian species having these two subfamilies. For example, the monomer pGRA1a69 from P. granulicollis showed a tract of 10 nucleotides (positions 1221) identical to the sequence of another subfamily (fig. 7a
). Longer gene conversion tracts have been detected too, such as the 145-bp tract (positions 8152) from pLVA7 which is recognized by 17 diagnostic nucleotides (fig. 7
b) or the one found in the repeats pCAN11 and pCAN13 (positions 851). In addition, unequal crossing-over between repeats and subsequent amplification of the recombination products has probably led to some of the observed subfamilies of stDNA. These are the sequences isolated from P. fernandezlopezi, P. laevigata laevigata, and a common sequence found in P. canariensis and P. radula ascendens satellites (Accession Numbers AJ247418, AJ247419, and AJ247420-3, respectively). The latter is highly conserved in length (383 bp) and sequence (94% similarity among repeats), being possibly the result of an unequal crossing-over between two PIM357 sequences, one of which had already a long internal inversion (fig. 8
).

View larger version (75K):
[in this window]
[in a new window]
|
Fig. 7.Nucleotide sequence alignment of two subfamilies of cloned PIM357 repeat units isolated from P. granulicollis (a) and P. laevigata validipes (b). Dots denote nucleotides that are the same as the first sequence; dashes denote gaps; and question marks denote any nucleotide (A, C, G, or T). The monomer pGRA1a69 shows a tract of 10 nucleotides (a) and pLVA7 a tract of 145 nucleotides (b) probably caused by gene conversion events (shadowed)
|
|

View larger version (23K):
[in this window]
[in a new window]
|
Fig. 8.Rearranged sequence of 383 bp isolated from P. canariensis and P. radula ascendens showing an internal inverted repeat (77% similarity). The positions 1288 are identical to the corresponding ones of the canonical PIM357 sequence
|
|
Transition Stages in Pimelia stDNA Evolution
The results of studying the extent of mutations in the satellite arrays by the method proposed by Strachan, Webb, and Dover (1985)
, comparing positions between pairs of related species are shown in table 2
for the species in which five or more repeats have been sequenced. Again, quite different results were obtained from the comparisons between closely related North African, Iberian, and Balearic species on the one hand, and between the Canarian species on the other hand. In the first group of sequences, the majority of nucleotide positions (>95%) can be classified into one of the six transitional stages, indicating the existence of a major characteristic sequence in each species. The presence of classes 1 and 2 only, indicates that two species share the same major monomeric variant (class 1) and the presence of rare mutations or low levels of subsequent spread of these (class 2). On the other hand, the predominance of classes 3 and 4 would indicate the gradual spreading of new species-specific variants and the subsequent divergence of the stDNA families of the two species. In the first steps of the process, the sequence variant is not completely fixed (presence of classes 1, 2, 3, and 4 but absence of classes 5 and 6, e.g., CRI-ELE). Whereas during the later steps, when the divergence becomes higher, the six classes should be present, showing a more extensive spreading of variant repeats (classes 5 and 6 or diagnostic positions, e.g., CRI-ING and AAT-ING). There is a clear linear relationship between transition stages to fixation and sequence divergence (table 2
and fig. 9
) among non-Canarian sequences. On the contrary, the Canarian sequences show a significant higher number of nucleotide positions falling in stage two and many positions showing rare mutations unclassifiable in any of the six transitional classes.
View this table:
[in this window]
[in a new window]
|
Table 2 List of stDNA Divergence, Percentage of Nucleotide Positions Falling into One of the Six Classes of Transition Stages Proposed by Strachan, Webb and Dover (1985), and the Percentage of Classes 16 in Pairwise Comparisons. Taxa with Less than Five Repeat Unit Sequences Analyzed Have Not Been Included in this Study Because this Method of Analysis of the Spread of Mutations in the Satellite Arrays Needs a Minimum Number of stDNA Sequences to Accurately Classify Each Nucleotide Position into One of the Six Transition Stages. The Code Species-sequence is as Indicated In Table 1
|
|

View larger version (14K):
[in this window]
[in a new window]
|
Fig. 9.Linear regression of the percentage of nucleotide positions completely homogenized and fixed (classes 5 plus 6) versus the nucleotide divergence from the six pairwise comparisons, including Iberian, Balearic, and African taxa (or both) (see table 2
)
|
|
 |
Discussion
|
---|
The evolution of satellite tandem repeats is one of the central topics of genome organization and evolution, but the usual large sizes of their clusters and the difficulties inherent in obtaining population data make them one of the less understood existing tandem repeats (Charlesworth, Sniegowski, and Stephan 1994
). The species of the genus Pimelia show an abundant tandemly repeated DNA family conserved across all the species examined (Pons et al. 1997
; this article). In a separate study of Palorus species (also of the family Tenebrionidae), major species-specific stDNAs were found without significant sequence similarities between them, even in the two species P. ratzeburgii and P. genalis, which are the most closely related having comparable COI mtDNA sequence divergences to some of the Pimelia species studied here (Mestrovic et al. 2000
). Our sample of Pimelia taxa includes all the endemic species present in the Canary Islands and 12 congeneric taxa distributed over the Iberian Peninsula, Morocco, and the Balearic Islands. A mitochondrial phylogeny of the Canarian taxa reported elsewhere was used to deduce colonization patterns. A stepwise colonization sequence from older to younger islands in the volcanic chain with three main lineages was proposed (Juan, Oromí, and Hewitt 1995
; Juan et al. 2000
). The mtDNA molecular clock calibrated at about 2%/Myr suggested a range of 68 Myr for the oldest inter-island migration, with Fuerteventura Island being the first to be colonized from North Africa (Juan, Oromí, and Hewitt 1995
). These phylogeographic data suggest that the PIM357 stDNA family has persisted for more than 8 Myr and could probably be traced to the origin of the lineage as suggested by its presence in all the taxa studied. Satellite sequences in other animal groups have been inferred as surviving for large evolutionary time periods; 10 Myr in Triturus (Varley, MacGregor, and Barnett 1990
), 20 Myr in species of the Drosophila virilis group (Heikkinen et al. 1995
), and at least 40 Myr in the Cetacea stDNA (Arnason, Gretarsdottir, and Widegren 1992
). Tandem repetitive sequences have been used both as phylogenetic and taxonomic markers in cases where related taxa share the same family (Arnason 1990
; Busche van den et al. 1993
; Wijers, Zijlstra, and Lenstra 1993
; Garrido-Ramos et al. 1999
). In the case of Pimelia, the NJ tree shows three main clusters of related PIM357 sequences partly concordant with the three main possible mtDNA phylogenetic lineages (although the inferences based in this mtDNA marker are not robust): (1) Iberian Peninsula-Balearic Islands taxa, (2) North Africa, and (3) Canary Islands species. From pairwise comparisons of sequenced clones obtained from related species, we can deduce that this repeated family evolves in a gradual manner, and the different stages of concerted evolution fit quite well with the mtDNA-deduced divergences. The stDNA from Iberian, Balearic, and Morocco Pimelia species show a high intraspecific conservation of the repeat units, suggesting that the mechanisms producing concerted evolution have been efficient in these taxa. In addition, comparing individual nucleotide positions between related species shows a paucity in the spreading of variants in the family with stDNA divergence, an indication of a constant rate of homogenization of the repeated cluster. This trend is absent in the comparisons of stDNA sequences from Canarian taxa. In these endemic taxa there is often sharing of stDNA repeats among species, and different repeat subfamilies in the same genome coexist in some cases. These facts point to weaker homogenization processes in the stDNA of Pimelia from these Atlantic islands and perhaps different rates of homologous to nonhomologous recombination which would explain the incipient genome compartmentalization of the repeats.
Furthermore, the Canarian taxa allow us to compare the phylogenetic signal contained in their stDNA sequences with the mtDNA-deduced phylogenetic patterns. Signatures of the main island colonizations and subsequent radiations are clearly present at the stDNA sequences. However, only P. lutaria, endemic to Fuerteventura and Lanzarote forms a distinct monophyletic group of PIM357 sequences which appears as being ancestral to the sequences obtained from the taxa of the remaining islands. There is also a certain phylogenetic structuring of the Tenerife-La Gomera-La Palma-El Hierro PIM357 stDNA sequences on the one hand, and Gran Canaria on the other hand, similar to the results shown by the mtDNA, reflecting independent colonization events (Juan, Oromí, and Hewitt 1995
). The stDNA and COI data also suggest the presence of two repeat subfamilies widely spread in the genome of the ancestor of Tenerife-La Gomera-La Palma-El Hierro lineage and another two different subfamilies in the ancestor of the P. granulicollis-P. estevezi-P. fernandezlopezi lineage.
Theoretical studies have demonstrated that tandem repeated sequences can be generated by computer simulations allowing for different values of unequal sister-chromatid exchange and slippage replication (which can be extended to homologous unequal exchange and rolling circle replication of extrachromosomal copies followed by reinsertion) (Smith 1976
; Stephan 1989
; Stephan and Cho 1994
). In these models, very low recombination rate values and sequence-dependant amplification processes such as slippage replication spontaneously forms satellite-like DNA arrays, i.e., high copy number long-DNA stretches. It has been shown that in a finite population in which there is no selection against high copy numbers, the equilibrium distribution of the number of repeats in an array depends on unequal exchange and random genetic drift only, processes causing both expansion and contraction of the DNA array (Charlesworth, Sniegowski, and Stephan 1994
). The time to reach a single copy per chromosome (the effective disappearance of the repeated family because unequal exchange cannot generate new copies) is inversely proportional to the rate of unequal exchange
, if 4Ne
<< 1. However, if amplification processes regenerate the repeated sequences from time to time, the repeated size will be highest in regions of low recombination. Thus, the evolutionary persistence of large tandem arrays depends on the ratio between amplification and unequal exchange rates (Charlesworth, Sniegowski, and Stephan 1994
; Stephan and Cho 1994
). Chromosomal regions with very low recombination and weak selective constraints on array length and episodic, even if rare, amplification bursts of the repeats, should be the ones where stDNAs expand and persist over evolutionary time. The PIM357 stDNA family is located in the large centromeric heterochromatic blocks of all chromosomes as evidenced by in situ hybridization with cloned repeats (unpublished data). The centromere area is a chromosomal region were recombination is supposed to be very rare, which would explain the persistence and abundance of stDNA in these regions. Amplification or the sudden increase in array length has been proposed to be caused by replication slippage, rolling circle replication, or by conversion-like mechanisms (Charlesworth, Sniegowski, and Stephan 1994
). Slippage, although probably of importance in the initial stages for the formation of many stDNAs, is an unlikely cause of amplification in the large repeats of the PIM357 family. Rolling circle replication of extrachromosomal copies of an array followed by reinsertion is a possibility, but it has been demonstrated in very few cases (Okumura, Kiyama, and Oishi 1987
). However, there are some indications from sequence comparisons among PIM357 repeats that gene conversion is acting in this family. The same mechanism has been deduced in other tenebrionid stDNAs like the stDNAs of Alphitobius diaperinus (Plohl and Ugarkovic 1994
) and Tribolium madens (Ugarkovic, Durajlija, and Plohl 1996
).
Stephan and Cho (1994)
have incorporated selection on array length (and not on the sequence per se) in the theoretical models of tandem repetitive DNA evolution. From this model, two main conclusions arise: (1) the interrepeat variability should decrease with increasing rates of the relative unequal crossing-over (unequal crossing-over with respect to the mutation rate
/u) and (2) the repeat length should decrease with the rate of relative unequal crossing-over. Regions of low recombination seem to maintain the interrepeat variability at low levels by formation of the longer multimeric repeats often found in stDNAs (Stephan and Cho 1994
). Because the repeated length in the PIM357 stDNA family is highly conserved and there is no evidence of higher order repeats, this would reflect a similar rate of relatively low recombination in the different taxa and hence equivalent rates of unequal exchanges in all species-populations of Pimelia. However, the data show that the interrepeat variability is significantly higher in the insular Canarian species than in their continental relatives (including the Balearic islands species), a fact that could be explained by different ratios of unequal exchange to the random mutation rate in the two species groups (
/u in the Stephan and Cho [1994]
model) because of higher fixation probabilities for neutral mutations in the Canarian taxa.
In summary, the persistence of an abundant tandemly repeated DNA family evolving in a gradual manner in the radiation of Pimelia traces the evolutionary history of the genus; so it can be used as a phylogenetic marker, it being a poor taxonomic indicator. The stDNA sequence evolution in Canarian Pimelia endemics appears to be different compared with the one in the two other congeneric groups, maybe reflecting particular demographic and phylogenetic patterns.
 |
Acknowledgements
|
---|
We are very grateful to Drs. P. Oromí, J. Gomez-Zurita, M. Palmer, C. Garin, and Lucas Riera who provided many of the Pimelia samples. Dr. Oromí and Dr. Gomez-Zurita also helped in the taxonomic determination. We also thank Drs. R. Zardoya, M Plohl, D. Ugarkovic, and L. Haste for valuable suggestions. The comments and suggestions of two anonymous referees substantially improved the article. This work was supported by the Spanish Research Funds REN2000-0282/GLO to C.J. and BOS2000-0822 to E.P. J.P. was supported by a postdoctoral fellowship of the Obra Social i Cultural de "Sa Nostra" de Balears (Spain).
 |
Footnotes
|
---|
Axel Meyer, Reviewing Editor
1 Present address: Entomology Department, The National History Museum, Cromwell Road, London SW7 5BD 
Keywords: satellite DNA
concerted evolution
Pimelia
Tenebrionidae
Coleoptera 
Address for correspondence and reprints: Joan Pons, Entomology Department, The National History Museum, Cromwell Road, London SW7 5BD. joap{at}nhm.ac.uk 
 |
References
|
---|
Arnason U., 1990 Phylogeny of marine mammals: evidence from chromosomes and DNA Chromosomes Today 10:267-278
Arnason U., S. Gretarsdottir, B. Widegren, 1992 Mysticetae (baleen whale) relationships based upon the sequence of the common cetacean DNA satellite Mol. Biol. Evol 9:1018-1028[Abstract]
Barceló F., J. Pons, E. Petitpierre, I. Barjau, J. Portugal, 1997 Polymorphic curvature of satellite DNA in three subspecies of the beetle Pimelia sparsa Eur. J. Biochem 244:318-324[Abstract]
Busche van den R. A., R. J. Baker, H. A. Wichman, M. J. Hamilton, 1993 Molecular phylogenetics of Stenodermatini bat genera: congruence of data from nuclear and mitochondrial DNA Mol. Biol. Evol 10:944-959[Abstract]
Charlesworth B., P. Sniegowski, W. Stephan, 1994 The evolutionary dynamics of repetitive DNA in eukaryotes Nature 371:215-220[ISI][Medline]
Dover G., 1982 Molecular drive: a cohesive mode of species evolution Nature 299:111-117[ISI][Medline]
Drouin G., G. A. Dover, 1990 Independent gene evolution in the potato actin gene family demonstrated by phylogenetic procedures for resolving gene conversion and the phylogeny of angiosperm genes J. Mol. Evol 31:132-150[ISI][Medline]
Elder J. F., B. J. Turner, 1995 Concerted evolution of repetitive DNA sequences in eukaryotes Q. Rev. Biol 70:297-323[ISI][Medline]
Fanning T. G., W. S. Modi, P. K. Wayne, S. J. O'brien, 1988 Evolution of heterochromatin-associated satellite DNA loci in felids and canids (Carnivora) Cytogenet. Cell Genet 48:214-219[ISI][Medline]
Garrido-Ramos M. A., R. De La Herrán, M. Jamilena, R. Lozano, C. Ruiz Rejón, M. Ruiz Rejón, 1999 Evolution of centromeric satellite DNA and its use in phylogenetic studies of the Sparidae family (Pisces, Perciformes) Mol. Phylogenet. Evol 12:200-204[ISI][Medline]
Garrido-Ramos M. A., M. Jamilena, R. Lozano, R. Cárdenas, C. R. Ruiz-Rejón, M. R. Ruiz-Rejón, 1995 Phylogenetic relationships of the Sparidae family (Pisces, Perciformes) inferred from satellite DNA Hereditas 122:1-6[ISI]
Heikkinen E., V. Launonen, E. Muller, L. Bachmann, 1995 The pvB370 BamHI satellite DNA family of the Drosophila virilis group and its evolutionary relation to mobile dispersed genetic pDv elements J. Mol. Evol 41:604-614[ISI][Medline]
Higgins D. G., J. D. Thompson, T. J. Gibson, 1996 Using clustal for multiple sequence alignments Methods Enzymol 266:383-402[ISI][Medline]
Juan C., B. C. Emerson, P. Oromí, G. M. Hewitt, 2000 Colonization and diversification: towards a phylogeographic synthesis for the Canary Islands Trends Ecol. Evol 15:104-108[ISI][Medline]
Juan C., P. Oromí, G. M. Hewitt, 1995 Mitochondrial DNA phylogeny and sequential colonization of Canary Islands by darkling beetles of the genus Pimelia (Tenebrionidae) Proc. R. Soc. B 261:173-180[ISI][Medline]
Kimura M., 1980 A simple method for estimating evolutionary rate of base substitution through comparative studies of nucleotide sequences J. Mol. Biol 16:111-120
King L. M., M. P. Cummings, 1997 Satellite DNA repeat sequence variation is low in three species of burying beetles in the genus Nicrophorus (Coleoptera, Silphidae) Mol. Biol. Evol 14:1088-1095[Abstract]
Koo H., H. Wu, D. M. Croters, 1986 DNA bending at adenine-thymine tracts Nature 320:501-506[ISI][Medline]
Mantovani B., 1998 Satellite sequence turnover in parthenogenetic systems: the apomictic triploid hybrid Bacillus lynceorum (Insecta, Phasmatodea) Mol. Biol. Evol 15:1288-1297[Abstract/Free Full Text]
Mantovani B., F. Tinti, L. Bachmann, V. Scalli, 1997 The Bag 320 satellite DNA family in Bacillus stick insects (Phasmatodea): different rates of molecular evolution of highly repetitive DNA in bisexual and parthenogenetic taxa Mol. Biol. Evol 14:1197-1205[Abstract]
Martínez-Balbás A., A. Rodríguez-Campos, M. García-Ramírez, J. Sainz, P. Carrera, J. Aymani, F. Azorín, 1990 Satellite DNAs contain sequences that induce curvature Biochemistry 29:2342-2348.[ISI][Medline]
Mestrovic N., B. Mravinac, C. Juan, D. Ugarkovic, M. Plohl, 2000 Comparative study of satellite sequences and phylogeny of five species from the genus Palorus (Insecta, Coleoptera) Genome 43:776-785[ISI][Medline]
Modi W. S., D. S. Gallagher, J. E. Womack, 1996 Evolutionary histories of highly repeated DNA families among the Artiodactyla (Mammalia) J. Mol. Evol 42:337-349[ISI][Medline]
Nei M., 1987 Molecular evolutionary genetics Columbia University Press, New York
Ohta T., G. Dover, 1984 The cohesive population genetics of molecular drive Genetics 108:501-521[Abstract/Free Full Text]
Okumura K., R. Kiyama, M. Oishi, 1987 Sequence analysis of extrachromosomal Sau3A and related family DNA: analysis of recombination in the excision event Nucleic Acid Res 15:7477-7489[Abstract]
Plohl M., D. Ugarkovic, 1994 Analysis of divergence of Alphitobius diaperinus satellite DNA: roles of recombination, replication slippage and gene conversion Mol. Gen. Genet 242:297-304[ISI][Medline]
Pons J., B. Bruvo, C. Juan, E. Petitpierre, M. Plohl, D. Ugarkovic, 1997 Conservation of satellite DNA in species of the genus Pimelia (Tenebrionidae, Coleoptera) Gene 255:183-190
Rozas J., R. Rozas, 1997 DnaSP version 2.0 a novel software package for extensive molecular population genetic analysis Comput. Appl. Biosci 13:307-311[Abstract]
Saitou N., M. Nei, 1987 The Neighbor-Joining method: a new method for reconstructing phylogenetic trees Mol. Biol. Evol 4:406-425[Abstract]
Sambrook J., E. F. Fritsch, T. Maniatis, 1989 Molecular cloning: a laboratory manual. 2nd edition Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York
Sanger F., S. Nicklen, A. R. Coulson, 1977 DNA sequencing with chain terminating inhibitors Proc. Natl. Acad. Sci 74:5463-5467[Abstract]
Smith G. P., 1976 Evolution of repeated DNA sequences by unequal crossover Science 191:528-535[ISI][Medline]
Sneath P. H. A., 1995 The distribution of the random division of a molecular sequence Binary 7:148-152[ISI]
Stephan W., 1989 Tandem-repetitive noncoding DNA: forms and forces Mol. Biol. Evol 6:198-212[Abstract]
Stephan W., S. Cho, 1994 Possible role of natural selection in formation of tandem-repetitive noncoding DNA Genetics 136:333-341[Abstract/Free Full Text]
Strachan T., D. Webb, G. Dover, 1985 Transition stages of molecular drive in multiple-copy DNA families in Drosophila EMBO J 4:1701-1708[ISI]
Swofford D. L., 1999 PAUP*: phylogenetics analysis using parsimony (*and other methods). Version 4 Sinauer Associates, Sunderland, Mass
Ugarkovic D., S. Durajlija, M. Plohl, 1996 Evolution of Tribolium madens (Insecta, Coleoptera) satellite DNA through DNA inversion and insertion J. Mol. Evol 42:350-358[ISI][Medline]
Ugarkovic D., E. Petitpierre, C. Juan, M. Plohl, 1995 Satellite DNAs in tenebrionid species: structure, organization and evolution Croat. Chem. Acta 68:627-638[ISI]
Varley J. M., H. C. MacGregor, L. Barnett, 1990 Characterization of a short, highly repeated and centromerically localized DNA sequence in crested marbled newts of the genus Triturus Chromosoma 100:15-31[ISI][Medline]
Wijers E. R., C. Zijlstra, J. A. Lenstra, 1993 Rapid evolution of horse satellite DNA Genomics 18:113-117[ISI][Medline]
Accepted for publication April 12, 2002.