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ABSTRACT

  1. Top of page
  2. ABSTRACT
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDGMENTS
  7. LITERATURE CITED

Satellite DNAs represent a large portion of all high eukaryotic genomes. They consist of numerous very similar repeated sequences, tandemly arranged in large clusters up to 100 million base pairs in length, usually located in the heterochromatic parts of chromosomes. The biological significance of satDNAs is still under discussion, but most of their proposed functions are related to heterochromatin and/or centromere formation and function. Because information about the structure of reptilian satDNA is far from exhaustive, we present a molecular and cytogenetic characterization of two satDNA families in four lacertid species. Two families of tandemly repeated DNAs, namely TaqI and HindIII satDNAs, have been cloned and sequenced from four species belonging to the genus Iberolacerta. These satDNAs are characterized by a monomer length of 171–188 and 170–172 bp, and by an AT content of 60.5% and 58.1%, respectively. FISH experiments with TaqI satDNA probe produced bright signals in pericentromeric regions of a subset of chromosomes whereas all the centromeres were marked by HindIII probe. The results obtained in this study suggest that chromosome location and abundance of satDNAs influence the evolution of these elements, with centromeric families evolving tenfold faster than interstitial/pericentromeric ones. Such different rates render different satellites useful for phylogenetic investigation at different taxonomic ranks. J. Exp. Zool. (Mol. Dev. Evol.) 322B: 13–26, 2014. © 2013 Wiley Periodicals, Inc.

Satellite DNAs (satDNAs) form a substantial part of eukaryotic genomes and consist of tandemly repeated DNA sequences typically arranged in large clusters of hundreds or thousands of copies usually located in the heterochromatic regions of chromosomes, mainly in the regions close to the centromeres and telomeres. The biological significance of satDNAs remains intriguing and challenging. The sequence conservation of some satellites over long evolutionary times, the presence of differentially expressed transcripts in several species and interactions with centromeric-specific proteins (e.g., the histone H3 variant CENH3) suggest a biological role for some satellites, although this is not fully understood (see Plohl et al., 2008; Plohl, 2010).

A satDNA family could arise in a phylogenetically short period by explosive amplification (Bachmann and Sperlich, 1993) and afterwards its repeats could follow a gradual mode of sequence evolution during a long evolutionary time (Bachmann and Sperlich, 1993). The processes by which satDNA families arise are not well known. A set of molecular-exchange mechanisms has been proposed to account for its origin by amplification of a tandem array of multi-copy sequences. These mechanisms include unequal crossing-over (Smith, 1976), transposition (Miller et al., 2000), or extrachromosomal rolling-circle replication and reintegration of tandem arrays into the genome (Feliciello et al., 2006). A recently originated tandem array is initially homogeneous in sequence because of the multi-copy amplification of the same repeat. In the course of time, random mutations would accumulate and the repeats would diverge. However, the nonallelic repeats of a satDNA family do not evolve independently, but concertedly leading to near homogeneity for species-specific mutations (Bachmann and Sperlich, 1993; Rudd et al., 2006). This phenomenon, known as concerted evolution, is achieved by a number of genomic mechanisms, mainly unequal crossing-over, biased gene conversion, slippage replication, and amplification by rolling-circle (Dover, 1982; Walsh, 1987; Charlesworth et al., 1994). However, the rates of sequence change (homogenization and fixation) vary for each satDNA family or even for the same satDNA family within different lineages. Levels of sequence variation among repeats would depend on factors such as mutation rate, inter- and intrachromosomal recombination rates, copy number, array size and structure, chromosomal distribution, chromosomal structure, population size, divergence time, and reproductive mode; it is also subject to random genetic drift and possibly natural selection (Strachan et al., 1985; Stephan and Cho, 1994; Luchetti et al., 2003; Navajas-Pérez et al., 2005; Dawe and Henikoff, 2006; Kuhn et al., 2007). The relative importance of each factor remains controversial.

In this context, very little information exists on satDNA array size, composition and long-range organization, especially in reptiles (see Giovannotti et al., 2009). An exception is represented by Lacertidae, a species rich family of squamate reptiles, widespread in the Palaearctic region (Sindaco and Jeremcenko, 2008). This family comprises the subfamilies Gallotiinae and Lacertinae, with the latter comprising two monophyletic tribes, the Eremiadini of Africa and arid southwest and central Asia, and the Lacertini of Europe (Arnold et al., 2007). So far, five satDNA families have been described for the genome of the Lacertinae subfamily: the pLCS (190 bp in length) is shared by the genera Algyroides, Teira, Lacerta, and Podarcis (Capriglione et al., 1989, 1991; Capriglione, 2000); the pLHS (140 bp) is specific for Podarcis only (Capriglione et al., 1994; Capriglione, 2000); the pGPS (185 bp) is present in the genome of Podarcis and in species belonging to the genera Archaeolacerta, Algyroides, Lacerta, and Zootoca (Capriglione et al., 1998), so that its appearance would precede the divergence within the Lacertinae subfamily; the CLsat family is described for the Caucasian genus Darevskia (145–147 bp, Ciobanu et al., 2003; Grechko et al., 2006); the Agi160 is restricted to the genus Lacerta (138–184 bp, Ciobanu et al., 2004; Grechko et al., 2005). These satDNA families revealed several common features, such as the same range of monomer lengths (140–190 bp), AT content (tendency toward AT enrichment 50–65%) and homopolymeric (A3–4 and T3–4) stretches (Capriglione et al., 1991; Ciobanu et al., 2001, 2004). All these features were also found in other nonreptilian satDNAs (see King and Cummings, 1997).

The genus Iberolacerta (see Arribas, 1999) has a disjunct range in mountain areas of western Europe: a portion comprises central Portugal, central and northern Spain and Pyrenees; another part embraces western Alps and northern Dinaric chain. Until recently the rock-lizard populations endemic to the Iberian Peninsula were considered to represent a single species, Lacerta monticola Boulenger, 1905 (see Salvador, 1985), that has recently been split into the following taxa: Iberolacerta aranica, I. aurelioi, and I. bonnali restricted to the Pyrenees and I. cyreni, I. galani, I. martinezricai, and I. monticola, in the central-western parts of Iberian Peninsula (see Arribas et al., 2006). An additional species is represented by the east-Alpine and Dynaric species I. horvathi. This classification was based on (i) morphological (biometry, scalation), ostelogical, and karyological data; (ii) on the use of molecular tools, namely nuclear (c-mos) and mitochondrial DNA (12S and cytochrome b), and (iii) on the construction of phylogenetic trees ranking the different allopatric populations based on the degree of genetic divergence, with I. horvathi as the most basal species (for a revision see Arribas et al., 2006). Another conceptual framework influencing the species subdivision of these largely allopatric lizards is the phylogenetic species concept, according to which species are segments of a phylogenetic lineage beyond nodes, irrespective of the degree of reproductive isolation (for a criticism see Mace, 2004). Considering the well-known usefulness of satDNAs in facing phylogenetic issues (i.e., Martinsen et al., 2009), the aim of the present paper was to isolate and characterise satDNA in some lacertid species in order to (i) increase the knowledge of this genomic elements in an important amniote group for which data on occurrence, genomic distribution, and evolutionary rates are limited to a handful of species; (ii) use the satDNAs isolated to verify the robustness of the proposed phylogenetic reconstruction for some Iberolacerta taxa on the light of independent molecular markers.

MATERIALS AND METHODS

  1. Top of page
  2. ABSTRACT
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDGMENTS
  7. LITERATURE CITED

Samples

Two males and two females of Iberolacerta monticola (from Fragas do Eume, A Capela, Galicia, Spain) and two males and two females of I. galani (from A Ponte, Pena Trevinca, A Veiga, Galicia, Spain) were used to make metaphase chromosomes and to extract genomic DNA. In addition, genomic DNA was extracted from seven ethanol preserved specimens of I. cyreni from three different Iberian locations (Navacerrada, Sierra de Guadarrama, Segovia-Madrid, Spain; Pico Zapatero, Sierra de la Paramera, Ávila; Puerto de Peña Negra, Sierra de Villafranca, Ávila, Spain) and one of I. martinezricai (Puerto El Portillo, Salamanca, Spain). Permissions for field work and experimental procedures were issued by the competent Spanish authorities: Xunta de Galicia (for I. monticola and I. galani) (permission number 79/2008) and Junta de Castilla y León (for I. cyreni and I. martinezricai) (permission numbers: 20051630007003/2005, 20061630024599/2006, 2007167004130/2007, 20081630020386/2008, 20092390004760/2009). Finally, genomic DNA of Lacerta bilineata, Podarcis muralis, P. siculus, and Timon lepidus, was extracted from ethanol preserved tissues of voucher specimens belonging to one of the authors (Vincenzo Caputo Barucchi).

Isolation and Characterization of Satellite DNAs

Genomic DNA was extracted from whole blood, using standard protocols with proteinase K digestion followed by phenol/chloroform extraction (see Sambrook et al., 1989). Fifteen restriction endonucleases (AluI, ApaI, AvaII, BamHI, BcnI, BglI, BglII, DraI, EcoRV, HindIII, MspI, RsaI, SmaI, TaqI, XbaI) (Fermentas International, Inc., Burlington, ON, USA) were screened and about 8 µg of I. monticola and I. galani purified genomic DNA were utilized for each digestion. Electrophoresis on 2% agarose gel of the digested DNA revealed a band of about 170 bp for HindIII and 190 bp for TaqI, corresponding to the monomeric unit of repetitive DNA (Fig. 1A), whereas no clear bands were produced by the remaining 13 endonucleases. The 170 and 190 bp fragments were excised from agarose gel, purified with Pure Link Quick Gel Extraction Kit (Invitrogen, Carlsabad, CA, USA) and cloned in the pCR®-blunt vector with Zero Blunt PCR Cloning Kit (Invitrogen) following the manufacturer's recommendations. Ten clones of each I. monticola satellite DNAs (HindIII and TaqI satDNAs henceforth) and 13 (HindIII) and 16 (TaqI) of I. galani satDNAs were sequenced on an ABI PRISM 3730XL (Applied Biosystems, Foster City, CA, USA) automatic sequencer.

image

Figure 1. Comparisons of consensus sequences of HindIII (a) and TaqI (b) between the four Iberolacerta species analysed. Repeated motifs are highlighted. sfI: HindIII satDNA subfamily I; sfII: HindIII satDNA subfamily II. Dots refer to nucleotide identity and dashes indicate indels.

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Digoxigenin-labeled probes were produced by PCR amplification of single clones and used in Southern hybridization experiments to verify that the elements isolated were tandemly arranged, as expected for satDNAs. In these experiments, HindIII and TaqI digested genomic DNAs from I. monticola and other lizards (I. cyreni, I. galani, I. martinezricai, Lacerta bilineata, Podarcis muralis, P. siculus, Timon lepidus) were used in order to assess the presence of these repetitive elements in other genera of this family. The hybridization with the digoxigenin-labeled satDNA probes was performed at 50°C overnight with the Sure Blot CHEMI Hybridization and Detection Kit (EMD Millipore Co., Billerica, MA, USA) following the manufacturer's recommendations. The hybridization was detected with the same kit.

The genomic abundance of satDNAs was estimated by quantitative dot blot analysis. Dilutions of genomic DNA and clones containing HindIII and TaqI satDNAs used as a standard were blotted onto a nylon membrane with BIO-DOT® microfiltration apparatus (Bio-Rad Laboratories, Hercules, CA, USA), following manufacturer's recommendations. In order to avoid errors due to the differences in the hybridization kinetics, sonicated salmon sperm DNA was used as a carrier and added to each sample up to a final amount of 0.5 µg DNA/sample (see Cafasso et al., 2003). Hybridization was performed overnight at 45°C. The same clones as those used as a standard were employed to produce digoxigenin-labeled probes. The detection protocol was carried out with the same protocol as the one used for Southern hybridization.

From the sequences of the monomeres of I. monticola and I. galani, HindIII and TaqI satDNAs two pairs of primers (HindIII-F: 5′-TGAGTGTTTTACAGTTGAAAAGCT-3′; HindIII-R: 5′-CATTGTGTTATTTGAGCGCAA-3′; TaqI-F: 5′-ATTCTGACCCTGGGGGTTAG-3′; TaqI-R: 5′-CATATTTAAAGAAATCAGGCCTCG-3′) were designed and used for isolation of these satellites from the genomes of the other two Iberolacerta species. PCR products from the amplification of Iberolacerta genomic DNAs with above primers were run on 2% agarose gel, the band corresponding to the amplified monomers excised from the gel, purified with Pure Link Quick Gel Extraction Kit (Invitrogen) and cloned in the pCR®-blunt vector with Zero Blunt PCR Cloning Kit (Invitrogen) following manufacturer's recommendations. Clones of HindIII and TaqI satDNAs were sequenced on an ABI PRISM 3730XL (Applied Biosystems) automatic sequencer. These sequences were then aligned in CLUSTAL W (Larkin et al., 2007), using default parameters. The visual inspection of sequence alignments was carried out to check for the presence of shared nucleotide changes, which could serve as diagnostic positions to define subsets (subfamilies) within each satDNA family. A GenBank search was performed in order to compare HindIII and TaqI satDNAs with other satDNAs in the database.

Maximum parsimony (MP), neighbor joining (NJ), maximum likelihood (ML), and Bayesian analyses (BA) were used to infer the phylogenetic relationships among sequences of each satDNA. MP consensus trees (50% majority rule) were constructed with PAUP* version 4.0b10 (Swofford, 2002) using the heuristic search method with 1,000 random-addition-sequence replicates, tree-bisection-reconnection (TBR) branch swapping and holding 100 trees at each cycle of the stepwise-addition procedure. To increase the number of informative characters, gaps were coded as binary (presence/absence) characters.

NJ analyses were performed in MEGA version 5 (Tamura et al., 2011). The NJ trees were based on distances obtained by the maximum composite likelihood method, with pairwise deletion and 1,000 bootstrap replicates. ML analyses were conducted in MetaPIGA v.2.1.3 (http://www.metapiga.org) (Helaers and Milinkovitch, 2010) using the metapopulation genetic algorithm (metaGA) with probability consensus pruning among four populations of four individuals each. The best-fitting nucleotide substitution models [Jukes–Cantor (JC) for HindIII satDNA and Hasegawa–Kishino–Yano plus Gamma (HKY + G) for TaqI satDNA] were selected based on the Likelihood Ratio Test implemented in this software. Branch support values that approximate the posterior probability distribution of the corresponding branches were estimated by performing a minimum of 100 replicated metaGA searches that were stopped when the mean relative error (MRE) among 10 consecutive consensus trees remained below 5%. BA were carried out using the software MrBayes v.3.2.1 (Ronquist and Huelsenbeck, 2003). As in the MP analyses, gaps were coded as binary characters and included as a separate data partition in the matrix. A binary model (lset coding = variable) was applied to the coded gaps, whereas the previously selected models of sequence evolution, JC and HKY + G, were applied to the DNA partitions of HindIII and TaqI satDNAs, respectively. The analyses included two separate concurrent Monte Carlo Markov Chain (MCMC) runs, each composed of four chains (one cold, three heated). Each Markov chain was started from a random tree and run for up to 106 generations, sampling every 500 generations. Stationarity was assessed using the software Tracer v.1.5 (Rambaut and Drummond, 2009). Samples obtained during the first 25% generations were discarded as burn-in, and the remaining data were used to generate a majority-rule consensus tree where the percentage of samples recovering any particular clade of the consensus tree represented the clade's posterior probability.

Intraspecific nucleotide diversity (π) was estimated using DnaSP v. 5 (Librado and Rozas, 2009). Net average genetic distances between groups were calculated under the appropriate substitution model for each satDNA family (see above) with MEGA v. 5. Rates of HindIII and TaqI satDNAs evolution were determined according to the divergence times estimated for the four Iberolacerta species here investigated by Arribas et al. (2006).

The occurrence of genetic differentiation between the four species analyzed was assessed with the analysis of molecular variance (AMOVA) (Excoffier et al., 1992) calculating Φ-statistics. This test was performed at two hierarchical levels to test how satDNAs sequence variability was distributed within species and among species, for both HindIII and TaqI satDNAs. The test was based on pair wise genetic distances between clones and performed as implemented in ARLEQUIN 2.000 (Schneider et al., 2000), using 1,000 permutations.

The repeats of the analyzed species were compared using satDNA Analyzer version 1.2 (Navajas-Pérez et al., 2007). This program allows the discrimination between shared and nonshared polymorphic sites. The program identifies polymorphic sites shared between two species when the same polymorphism is found in both species. When this occurs, we assume that these are ancestral sites that appeared before the split between the two species (Navajas-Pérez et al., 2005). By contrast, nonshared polymorphic sites are autapomorphies, representing different transitional stages in the process of intraspecific sequence homogenization and interspecific divergence. Under the assumption that concerted evolution is a time dependent process, the expected stages of transition during the spread of a variant repeat unit toward its fixation can be defined according to the model of Strachan et al. (1985). This is a method of partitioning the variation by analyzing the patterns of variation at each nucleotide site considered independently among all the repeats of a repetitive family when comparing a pair of species (Strachan et al., 1985; Navajas-Pérez et al., 2007). This method examines the distribution of nucleotide sites among six stages (Classes I–VI) in the spread of variant repeats through the family and the species. Briefly, the Class I site represents complete homogeneity across all repeat units sampled from a pair of species, whereas Classes II, III, and IV represent intermediate stages in which one of the species shows a polymorphism. The frequency of the new nucleotide variant at the site considered is low in Class II and intermediate in Class III, while Class IV represents sites in which a mutation has replaced the progenitor base in most members of the repetitive family in the other species. Class V represents diagnostic sites in which a new variant is fully homogenized and fixed in all the members of one of the species while the other species retains the progenitor nucleotide. A Class VI site represents an additional step over the stage of Class V (new variants appear in some of the members of the repetitive family at a site fully divergent between the two species). The statistical significance (P-value) of the variation in the relative proportions of Strachan transitions stages among different interspecific comparisons was evaluated using chi-square heterogeneity tests that were performed in the interactive online calculator available at http://www.quantpsy.org/chisq/chisq.htm (Preacher, 2001).

Chromosome Analysis

For metaphase preparations, about 50 µl of blood were taken from I. monticola and I. galani individuals with a sterile heparinized syringe and cultured in CO2 incubators using the culture conditions indicated by Ezaz et al. (2005). Metaphase preparations were obtained by exposing cell cultures to 75 ng/ml of Demecolcine (Sigma-Aldrich Co., St Louis, MO, USA) for 4 hr before harvesting (Ezaz et al., 2005). Cells were hypotonized in KCl 0.75 M for 30 min at 37°C, prefixed by adding several drops of freshly prepared methanol:acetic acid fixative (3:1), then fixed through three changes of fixative. Suspensions of fixed cells were dropped onto microscope slides and air dried at room temperature.

Fluorescence in situ hybridization (FISH) experiments were performed on metaphase preparations using (i) a telomeric probe (TTAGGG)n produced by PCR according to Ijdo et al. (1991), and (ii) the probes obtained by PCR amplification of TaqI and HindIII satDNA clones. Telomeric and TaqI probes were also used in two-color FISH experiments. The probes were labeled by PCR either with biotin-16-dUTP (Roche) or digoxigenin-11-dUTP (Roche Diagnostics GmbH, Mannheim, Germany). Slide pretreatment, denaturation, hybridization, post-hybridization washes, and detection were performed according to Schwarzacher and Heslop-Harrison (2000). The HindIII satDNA and telomeric probes were evidenced with fluorescein iso-thyocianate (FITC) and tetramethyl rhodamine iso-thyocianate (TRITC), respectively. Chromosomes were observed with a Nikon Eclipse 800 epifluorescence microscope and the images were captured and processed with a Leica CytoVision version 7.2 system.

In order to define the relationships between satDNAs and the constitutive heterochromatin, C-banding was performed on metaphase plates following Sumner (1972). The relations between AT-rich heterochromatic regions and satDNAs were determined by staining C-banded metaphases with 4′,6-diamidino-2-phenylindole (DAPI) (Schweizer, 1976).

RESULTS

  1. Top of page
  2. ABSTRACT
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDGMENTS
  7. LITERATURE CITED

Isolation and Characterization of Satellite DNAs

The digestion of I. monticola and I. galani genomic DNA with HindIII and TaqI restriction enzymes revealed bands corresponding to a monomer of a repetitive element of about 170 and 190 bp, respectively (not shown). PCR amplification using primers designed by aligning I. monticola and I. galani sequences of both satDNAs was successful in individuals representing the other two lineages of Iberolacerta recognized as distinct species (I. martinezricai, I. cyreni). The length of the 45 clones sequenced for HindIII ranged between 170 and 172 bp, whereas the length of the 42 clones sequenced for TaqI ranged between 171 and 188 bp (Table 1). Sequences of both satDNAs were deposited in GenBank (HindIII accession numbers: from KF453637 to KF453681; TaqI accession numbers: from KF453682 to KF453723). When HindIII and TaqI satDNA sequences were subjected to a BLASTN search, no significant similarities with sequences deposited in databases were found.

Table 1. Summary of repeat features and π values
SpeciesHindIIITaqI
n%ATRepeat lengthπn%ATRepeat lengthNucleotide diversity (π)
  1. Number of monomeric repeats sequenced (n), nucleotide composition of repeats (AT), length of repeats (expressed in base pairs), and nucleotide diversity (π) ± SE for both satDNAs for each Iberolacerta species investigated. sfI: HindIII satDNA subfamily I; sfII: HindIII satDNA subfamily II

I. cyreni1157.01710.0055 ± 0.0022960.2186–1870.0384 ± 0.0058
I. galani1358.9170–1710.0358 ± 0.00331660.1186–1870.0475 ± 0.0070
I. galani (sfI)659.41710.0175 ± 0.0031    
I. galani (sfI)758.51700.0101 ± 0.0020    
I. monticola1059.0170–1710.0187 ± 0.00351060.8171–1880.0569 ± 0.0062
I. monticola (sfI)959.01710.0062 ± 0.0019    
I. monticola (sfI)158.8170    
I. martinezricai1058.7171–1720.0105 ± 0.0052760.1187–1880.0428 ± 0.0114

Southern blot analysis revealed hybridization of both satDNA probes onto Iberolacerta monticola digested genomic DNA with a ladder-like pattern, indicating the tandem arrangement of repeating units which is typical of satDNAs. A strong hybridization signal was also produced on the other three Iberolacerta species whit both HindIII and TaqI probes; this latter probe also produced a clear signal on the other lizards tested, whereas no signal appeared when HindIII probe was hybridized on representatives of the genera Lacerta, Podarcis, and Timon (not shown).

Quantitative dot blot analysis revealed that HindIII satDNA represents around 10% of I. monticola and I. galani, and 5% of I. cyreni and I. martinezricai genomes. TaqI satDNA represents 5% of I. cyreni, I. galani, and I. monticola genomes, and 2.5% in I. martinezricai (data not shown). The estimation of the number of repeats was not possible because the genome size of these lizards is not known.

The consensus sequences of the two satDNAs were very similar in the four Iberolacerta species, with an AT average content of 58.4% for HindIII and 60.3% for TaqI, indicating an enrichment in AT (Table 1). Both satellites repeats are characterized by the occurrence of short motifs such A and T stretches and dinucleotides steps TG and CA, with more numerous and longer A (T) stretches in TaqI satDNA (Fig. 1), as expected from its higher AT content. Within HindIII satDNA, two monomer variants or subfamilies (I and II) were detected in I. galani and I. monticola (Fig. 1A). The consensus sequences of subfamily I in both species were virtually identical to the consensus of I. martinezricai, whereas subfamily II showed several (nine) randomly distributed diagnostic nucleotide substitutions, as well as three exclusive indels located in the terminal region of the monomer. Both monomer variants were present in similar proportions in the sequence data set of I. galani, but only one out of ten sequences in I. monticola belonged to subfamily II (Table 1).

The phylogenetic tree obtained from the Bayesian analysis of HindIII satDNA is shown in Figure 2. The four different phylogenetic analyses (NJ, MP, ML, and BA) yielded very similar topologies, with some minor incongruences. Two major clades were recovered with maximum support, one harbouring I. cyreni clones and the other the sequences of the remaining three Iberolacerta species. Within this second cluster, monomers of subfamily II constitute a well-supported clade sister to that formed by sequences belonging to subfamily I, [with the exception of two clones from I. galani (IGA_32 and IGA_39) that share some private nucleotide substitutions]. Within subfamily I, relationships between most monomers were poorly resolved and they were not grouped according to the species of origin.

image

Figure 2. Bayesian phylogenetic tree depicting the the phylogenetic relationships between the 45 monomeric units of HindIII satDNA sequenced. Support values obtained by four different methods of analysis are shown at each node; from left to right: Bayes posterior probability (100×), metaGA branch support values (100×), NJ-bootstrap (%), and equally MP trees (%). A hyphen was inserted whenever a particular method did not support the Bayesian topology. Numbers after the species names are experimental number for clone identification. ICY: Iberolacerta cyreni; IGA: Iberolacerta galani; IMO: Iberolacerta monticola; IMR: Iberolacerta martinezricai.

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The Bayesian tree constructed using the sequences of TaqI satDNA was largely unresolved, regardless of the phylogenetic method employed, showing that this satellite cannot discriminate effectively the four Iberolacerta species here investigated (Fig. 3). Even though several well-supported subclusters including conspecific monomers were recognized, the number of diagnostic mutations shared by these sequences was too low to be considered species-specific TaqI satDNA subfamilies (not shown).

image

Figure 3. Bayesian phylogenetic tree depicting the the phylogenetic relationships between the 42 monomeric units of TaqI satDNA sequenced. Support values obtained by four different methods of analysis are shown at each node; from left to right: Bayes posterior probability (100×), metaGA branch support values (100×), NJ-bootstrap (%) and equally MP trees (%). A hyphen was inserted whenever a particular method did not support the Bayesian topology. Numbers after the species names are experimental number for clone identification. ICY: Iberolacerta cyreni; IGA: Iberolacerta galani; IMO: Iberolacerta monticola; IMR: Iberolacerta martinezricai.

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The π values indicated that intraspecific sequence heterogeneity is higher for TaqI satDNA (from 3.84% in I. cyreni to 5.69% in I. monticola) than for HindIII satDNA (from 0.55% in I. cyreni to 3.58% in I. galani) (Table 1). Interspecific mean net distances are low and similar for both satellites when I. cyreni is excluded from the analysis of HindIII satDNA (from 0.04% between I. monticola subfamily I and I. martinezricai to 5.60% between I. galani subfamily II and I. martinezricai for HindIII, and from 0.90% between I. galani and I. martinezricai to 1.30% between I. monticola and I. galani for TaqI satDNA) (Tables 2 and 3). Pair wise comparisons of HindIII satDNA involving I. cyreni and the other Iberolacerta analyzed, showed distance values substantially higher, between 8.40% and 13.90% (Table 2).

Table 2. Interspecific and intersubfamily comparative analysis of HindIII repeats
Species comparisonSP (%)Strachan sites II–III (%)Strachan sites IV–VI (%)Genetic distance
  1. The table reports number and percentage of shared polymorphic sites (SP); variable nucleotide sites classified according to Strachan et al. (1985); net genetic distances (Jukes–Cantor method) in pair wise comparisons of species. sfI: HindIII satDNA subfamily I; sfII: HindIII satDNA subfamily II.

HindIII
I. cyreni versus I. galani (sfI)0 (0%)4 (2.3%)15 (8.8%)0.0838 ± 0.0232
I. cyreni versus I. galani (sfII)0 (0%)5 (2.9%)21 (12.3%)0.1388 ± 0.0326
I. cyreni versus I. monticola (sfI)0 (0%)5 (2.9%)16 (9.4%)0.1025 ± 0.0265
I. cyreni versus I. martinezricai0 (0%)5 (2.9%)15 (8.8%)0.0996 ± 0.0258
I. galani (sfI) versus I. monticola (sfI)1 (0.59%)7 (4.1%)1 (0.59%)0.0038 ± 0.0025
I. galani (sfI) versus I. martinezricai2 (1.2%)6 (3.5%)1 (0.59%)0.0034 ± 0.0026
I. monticola (sfI) versus I. martinezricai2 (1.2%)4 (2.3%)0 (0%)0.0004 ± 0.0005
I. galani (sfII) versus I. monticola (sfI)0 (0%)8 (4.7%)9 (5.3%)0.0545 ± 0.0190
I. galani (sfII) versus I. martinezricai0 (0%)8 (4.7%)9 (5.3%)0.0555 ± 0.0192
I. galani (sfI) versus I. galani (sfII)0 (0%)6 (3.5%)10 (5.8%)0.0447 ± 0.0160
Table 3. Interspecific comparative analysis of TaqI repeats
Species comparisonSP (%)Strachan sites II–III (%)Strachan sites IV–VI (%)Genetic distance
  1. The table reports number and percentage of shared polymorphic sites (SP); variable nucleotide sites classified according to Strachan et al. (1985); net genetic distances (maximum composite likelihood method) in pair wise comparisons of species.

TaqI
I. cyreni versus I. galani8 (4.3%)51 (27.3%)3 (1.6%)0.0099 ± 0.0040
I. cyreni versus I. monticola9 (4.8%)25 (13.4%)1 (0.5%)0.0113 ± 0.0040
I. cyreni versus I. martinezricai7 (3.7%)25 (13.4%)2 (1.1%)0.0109 ± 0.0039
I. galani versus I. monticola10 (5.3%)43 (23%)1 (0.5%)0.0130 ± 0.0057
I. galani versus I. martinezricai5 (2.7%)57 (30.5%)3 (1.6%)0.0089 ± 0.0037
I. monticola versus I. martinezricai7 (3.7%)31 (16.6%)2 (1.1%)0.0114 ± 0.0040

In addition, higher levels of sequence divergence were obtained in the comparisons between subfamilies I and II of HindIII satDNA in I. galani (4.5%) than in the comparisons between monomeric repeats belonging to subfamily I in different species (from 0.04% to 0.4%) (Table 2).

The evolutionary rate of these two satellites was then calculated based on sequence divergence between I. cyreni and the other three species, that were considered as a single taxonomic unit not being discriminated by either satellite. The values found are 1.2% for HindIII and 0.14% for TaqI, indicating an evolutionary rate almost 10-fold faster for the former.

The poor phylogenetic differentiation of these species based on the sequences of the satDNAs here isolated was confirmed by AMOVA analysis. When this test was performed on the HindIII sequences, most of the percentage of the molecular variation was distributed among species (69.60%; ΦST 0.69596, P < 0.0001) whereas the percentage of variation within species was much lower, but still significant (30.40%; ΦST 0.69596, P < 0.001) (Table 4). The variance among species became much lower (32.07%; ΦST 0.32072, P < 0.001) and the one within populations became the preponderant variance component (67.93%; ΦST 0.32072, P < 0.001) when the sequences of I. cyreni were excluded from the analysis (Table 4). This result can be explained by the fact that I. cyreni was recovered as a distinct cluster with a high support in the phylogeny based on HindIII sequences, whereas the other three cannot be discriminated by this molecular marker. The AMOVA test carried out on TaqI satDNA sequences produced results very similar to those obtained with HindIII sequences after excluding I. cyreni, with a preponderant variance component distributed within species (82.69%; ΦST 0.17314, P < 0.001), confirming that this satDNA cannot effectively discriminate between these Iberolacerta species (Table 4). These results emerged also by analyzing the pattern of variation at each nucleotide position considered independently among all HindIII repeats (Table 2). Indeed, when comparing I. cyreni with the other species, a high percentage of Strachan sites belonging to the categories IV, V, and VI were found (average = 9.9%), while 5.1% of sites per repeat were Strachan transition stages (II + III), and no shared polymorphic sites were observed. Conversely, for TaqI satDNA sites of the classes IV–VI were very few (average = 0.5%) in all the comparison, while 20.7% of the sites represented Strachan stages II–III and an average of 4.1% were polymorphic sites (Table 3). According to the chi-square heterogeneity test, these differences in the relative proportions of Strachan transition stages between HindIII and TaqI satDNAs are highly significant (P < 0.001).

Table 4. AMOVA analysis
Source of variationVariance componentsPercentage of variation
  1. The test was carried on HindIII satDNA sequences including the four species selected for this study (first line of each hierarchical level), and removing Iberolacerta cyreni from the analysis (second line of each hierarchical level). The test on TaqI satDNA sequences included all four of the Iberolacerta investigated (third line of each hierarchical level). Φ-statistics were highly significant in all comparisons (P < 0.001).

Among species4.2127569.60
 1.0771932.07
 1.1421817.31
Within species1.8403530.40
 2.2815267.93
 5.4548782.69

The relatively high degree of genetic differentiation detected in the analysis of sequence divergence between HindIII subfamily II and subfamilies I from I. galani, I. monticola, and I. martinezricai was also evident in the comparisons of Strachan transition stages among these groups (Table 2). No shared polymorphisms were found and the number of sites falling in classes IV and V (between 5% and 6%) was significantly larger (P < 0.001) than the average frequency of these “differentiated sites” in the comparisons among subfamilies I in different species.

Chromosome Analysis

FISH experiments with HindIII satDNA probe on metaphase chromosomes of I. galani and I. monticola revealed that this repetitive element is widespread in the genome of these species, occurring at centromeres of all the 36 chromosomes of the diploid complement (Fig. 4A,B), with no differences between males and females. The occurrence of “bouquet” figures where chromosomes are linked together at the level of centromeres seems to indicate that this satDNA is involved in the interchromosome connection during mitosis (Fig. 4B). FISH with TaqI satDNA probe produced bright signals in interstitial position in a subset of 18 chromosomes in I. galani and 20 in I. monticola. No differences between males and females were detected with this probe either (Fig. 4C,D). Results of FISH experiments are consistent with the genomic abundance of HindIII and TaqI satDNAs as showed by quantitative dot blot analysis for these two species, with the former around twofold more abundant than the latter.

image

Figure 4. FISH with HindIII probe onto metaphases from females of Iberolacerta galani (A) and I. monticola (B). FISH with TaqI probe onto a metaphase of I. galani female (C). Two-color FISH with telomeric (red) and TaqI (green) probes on a metaphase of I. monticola female (D). C-banding on I. monticola male (E) and I. galani female (F) metaphases. The W chromosome of I. galani is indicated by an arrow.

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FISH with a telomeric probe (TTAGGG)n produced a fluorescent signal at telomeres of all the chromosomes. Besides telomeric signals, also interstitial telomeric sites (ITS) were marked in about five chromosome pairs. When a two-color FISH with both telomeric and TaqI satellite probes were performed, the fluorescent signals of ITS resulted distally located to the satellite ones (Fig. 4D).

C-banding, performed in order to assess the relationships between the isolated satellites and constitutive heterochromatin, revealed that in Iberolacerta the chromosomal distribution of HindIII satDNA overlaps the centromeric heterochromatic blocks, whereas TaqI probe colocalizes with pericentromeric heterochromatin (Fig. 4E,F).

DISCUSSION

  1. Top of page
  2. ABSTRACT
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDGMENTS
  7. LITERATURE CITED

Satellite DNAs represent rapidly evolving genomic elements, and therefore, even among most closely related species, they usually differ in nucleotide sequence, copy number, and/or composition of satellite families (Csink and Henikoff, 1998). However, some satDNA families evolve more slowly than others and occur in several closely related species with different degrees of sequence similarity (Bachmann and Sperlich, 1993; Mantovani et al., 1997; Watabe et al., 1997). Some satDNAs seem to be rather ancient and are widely distributed among higher taxa (Modi et al., 2004; Robles et al., 2004). Consequently, some satDNAs may be valuable taxonomic identification tools while others might be useful for phylogenetic analyses at higher taxonomic levels. In the present study, we compared sequences of two different satDNA families (HindIII and TaqI) in four closely related lacertid species, allopatrically distributed in mountain areas of the Iberian Peninsula. These satDNAs seem to evolve at different rates in the studied lizards, with HindIII showing a 10-fold faster evolutionary rate than TaqI. Indeed, Southern blot analysis using Iberolacerta satellite probes revealed a clear hybridization pattern also in other lizard genera (namely, Lacerta, Podarcis, and Timon) only for TaqI repeats, whereas HindIII seems to be restricted to the genus Iberolacerta. However, a significant level of genetic divergence was detected only in comparisons involving I. cyreni when HindIII satDNA was considered. For this satDNA, analysis of turnover dynamics indicate the effectiveness of the molecular drive process, after species split, in the spreading of new sequence variants leading to intraspecific homogeneity (0.56% of sequence variation within I. cyreni) and interspecific divergence (around 9% of sequence divergence between I. cyreni and the other species), an evolutionary pattern known as concerted evolution (Dover, 1982). The fact that the other species are scarcely differentiated at HindIII repeats can be interpreted in two alternative ways: (i) it may represent the outcome of the relatively recent (approximately 2 mya, Arribas et al., 2006) and rapid succession of speciation events within this group. In fact, previous molecular analyses based on nuclear and mitochondrial markers also failed to resolve the phylogenetic relationships or even track lineage splitting at this taxonomic level (Mayer and Arribas, 2003; Carranza et al., 2004; Crochet et al., 2004; Arribas et al., 2006; Arnold et al., 2007); (ii) the specific status for these three taxa might not have been reached yet. Indeed, estimation of divergence times among these three Iberolacerta species are similar to those recorded for different populations of the lizard Podarcis muralis that diverged genetically in separate refuges during glaciations, currently not showing evidence for reproductive isolation (Giovannotti et al., 2010).

The deep divergence observed between I. cyreni and the other Iberolacerta species here investigated with HindIII satellite is in good accordance with the molecular phylogenies published so far (Mayer and Arribas, 2003; Carranza et al., 2004; Crochet et al., 2004; Arribas et al., 2006; Arnold et al., 2007). This analysis showed that this species was the most diverged clade of the tree, with an estimated splitting time of about 7.5 million years. The relatively scarce representation of transitional stages (only 5% of the nucleotide positions) might suggest that the concerted evolution mechanisms have led to sequence differentiation between I. cyreni and the other species, probably due to the efficiency of the molecular-exchange homogenizing mechanisms among chromosomes.

The occurrence of two different types of monomeric variants or subfamilies was described for HindIII satDNA sequences. These subfamilies were defined according to a set of particular nucleotide substitutions or indels, in two of the four species examined. However, given the almost simultaneous speciation processes between I. monticola, I. galani, and I. martinezricai, it seems unlikely that subfamily II constitutes a specific variant of I. monticola and I. galani. An interspecific analysis of the pattern of nucleotide change was not possible for subfamily II due to the lack of a representative number of sequences in I. monticola or I. martinezricai. Even so, our results show that both subfamilies are presumably evolving independently, as indicated by the substantially high percentage of transitions stages IV and V between the monomers of subfamily II (I. galani) and the sequences of subfamily I, either belonging to I. galani, I. monticola, or I. martinezricai. The coexistence and divergent evolution of satellite subfamilies in the genomes of these species could be in agreement with the Nijman and Lenstra model (2001), in which mutations inhibiting the interactions of repeat units in a satellite family would lead to sequence diversification and the independent amplification or contraction of concurrent sequence variants. Nevertheless, a more extensive survey of HindIII satDNA will be the subject of further studies, in order to assess the presence and abundance of both monomeric variants in other Iberolacerta species, as well as to elucidate the processes driving the evolution of this satellite family.

Conversely to HindIII sequences, the tandem arrays of TaqI show a low sequence change rate when comparing I. cyreni with the other Iberolacerta. In fact, we detected a low rate of sequence change (0.1% per Myr), a rate 10-fold lower than that estimated for HindIII sequences (about 1.2% per Myr) and only 1.1% of Strachan stages IV–VI compared to 18% of II–III stages. In addition, we also observed some shared polymorphic sites and a comparatively higher intraspecific heterogeneity, suggesting that most of the intraspecific variability in each species is ancestral, originated prior to the separation of these lineages; moreover, the high number of transitional stages of differentiation (Strachan stages II–III) suggest that after the allopatric isolation, processes of concerted evolution were less efficient than in the HindIII repeats. In addition, contrarily to HindIII, Southern hybridization with TaqI probe produced a clear signal also in other lacertid genera, like Lacerta, Podarcis, and Timon, also suggesting a strong conservation of this satellite DNA family.

Various factors were invoked to explain different evolutionary turnover rates between satDNA families, like interchromosomal and intrachromosomal recombination rates, copy number, array size and structure, chromosomal distribution, chromosomal structure, population size, divergence time and reproductive mode. Moreover, evolutionary conservation of satDNA repeats might be a likely indication of functional constraints and natural selection (see Plohl et al., 2008). Unfortunately, very few examples are found in the literature with both fast-evolving and slow-evolving satDNAs found within the same species. For instance, in the genus Dolichopoda, a comparison among three satDNA families showed a trend of sequence variability and copy number being positively correlated, and a trend of sequence variability and length of repeats being negatively correlated (Martinsen et al., 2009). Like in Dolichopoda, it seems that also in the studied lizards an increase in copy number is linked to a trend of sequence homogenization. In fact, it was observed that HindIII repeats represent between 5% and 10% of the Iberolacerta genome, while TaqI satDNA between 2.5% and 5%. The different chromosome localization of the two satellites may also play a role in the different rate of sequence homogenization recorded for the two satDNA families. First of all, it should be noted that HindIII repeats are centromerically located on all the acrocentric chromosomes of I. galani and I. monticola karyotypes. In fact, it is reported that satellite DNAs at centromeres of acrocentric chromosomes show greater homology and a higher rate of homogenization than in noncentromeric locations or nonacrocentric chromosomes (Jantsch et al., 1990; Bandyopadhyay et al., 2001). It has been hypothesized that homogenization occurs through physical association and crossing-over between nonhomologous chromosomes (Ohno et al., 1961). Indeed, acrocentric chromosomes associate at the heterochromatic regions during meiotic prophase and somatic interphase (Schmid et al., 1983; Tuck-Muller et al., 1984; Kuznetsova et al., 2007) and we also observed typical “bouquet” figures, where chromosomes are linked together at the level of centromeres (Fig. 4B). This process may be the most important mechanisms for spontaneous chromosomal mutation, concerted evolution, and homogenization of satellite subfamilies of DNA among acrocentric chromosomes (Maeda and Smithies, 1986).

Conversely, TaqI repeats are pericentromerically located on a lower number of chromosomes (10 pairs in I. monticola and 9 in I. galani). In this case, we could explain the low homogenization rate within single species in terms of primary rate of the homogenization process. That is, it is possible that the exchange between nonhomologous chromosomes having TaqI sequences is limited. The TaqI repeats are indeed restricted to a subset of chromosomes in these species and located in a pericentromeric position less prone to physical association: this could reduce interchromosomal exchange and homogenization, thus determining a lower rate of interspecific divergence and a higher degree of intraspecific repeat heterogeneity. Similar considerations were reported for satDNAs of Rumex, where repeats in nonrecombining Y chromosomes show low rates of concerted evolution and intraspecific variability increase with no interspecific divergence (Navajas-Pérez et al., 2009; see also Kuhn et al., 2008), and to explain the lower mutation rate of satDNAs in sturgeons as compared to sparids. In fact, the more symmetrical karyotypes of these latter fishes would represent no physical barrier to interchromosomal exchange (de la Herrán et al., 2001a, 2001b). However, also these AT-rich pericentromeric repeats could represent chromosome sites favoring spontaneous rearrangements. Indeed, we observed that the majority of the TaqI repeats are flanked by interstitial telomeric sequences that would insert in these chromosome points during the repair of double strand breaks (see Bolzán and Bianchi, 2006). These unstable sequences might explain the high rate of Robertsonian translocation observed in Pyrenean Iberolacerta (Odierna et al., 1996).

In conclusion, our study suggests the effect of differential location and repeat copy number in the evolution of satDNAs, revealing features that could also improve the use of this genomic component as a molecular marker in phylogenetic analyses. Moreover, these results indicate that some molecular markers should be used cautiously in species identification when divergence times are shallow among the taxa compared.

ACKNOWLEDGMENTS

  1. Top of page
  2. ABSTRACT
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDGMENTS
  7. LITERATURE CITED

Special thanks are given to Pedro Galán for providing the samples of Iberolacerta monticola and I. galani used in this study. All authors declare that no conflict of interests exists for this manuscript.

LITERATURE CITED

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  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDGMENTS
  7. LITERATURE CITED
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