Journal of Heredity Advance Access originally published online on February 24, 2005
Journal of Heredity 2005 96(4):318-328; doi:10.1093/jhered/esi037
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Structure of the Mitochondrial Control Region of the Eurasian Otter (Lutra lutra; Carnivora, Mustelidae): Patterns of Genetic Heterogeneity and Implications for Conservation of the Species in Italy
From the Dipartimento di Biologia Animale e dell'Uomo, Università di Roma "La Sapienza," V.le dell'Università 32, I-00185 Rome, Italy. V. Ketmaier is currently at the Unit of Evolutionary Biology/Systematic Zoology, Institute of Biochemistry and Biology, University of Potsdam, D–14476, Potsdam, Germany
Address correspondence to Valerio Ketmaier at the address above, or e-mail: valerio.ketmaier{at}uniroma1.it.
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Abstract |
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In this study we determined the complete sequence of the mitochondrial DNA (mtDNA) control region of the Eurasian otter (Lutra lutra). We then compared these new sequences with orthologues of nine carnivores belonging to six families (Mustelidae, Mephitidae, Canidae, Hyaenidae, Ursidae, and Felidae). The comparative analyses identified all the conserved regions previously found in mammals. The Eurasian otter and seven other species have a single location with tandem repeats in the right domain, while the spotted hyena (Hyaenidae) and the tiger (Felidae) have repeated sequences in both the right and left domains. To assess the degree of genetic heterogeneity of the Eurasian otter in Italy we sequenced two fragments of the gene and analyzed length polymorphisms of repeated sequences and heteroplasmy in 32 specimens. The study includes 23 museum specimens collected in northern, central, and southern Italy; most of these specimens are from extinct populations, while the southern Italian samples belong to the sole extant Italian population of the Eurasian otter. The study also includes all the captive-reared animals living in the colony "Centro Lontra, Caramanico Terme" (Pescara, central Italy). The colony is maintained for reintroduction of the species. We found a low level of genetic polymorphism; a single haplotype is dominant, but our data indicate the presence in central and southern Italy of two slightly divergent haplotypes. One haplotype belongs to an extinct population, the other is present in the single extant Italian population. Analyses of length polymorphisms and heteroplasmy indicate that the autochthonous Italian samples are characterized by a distinct array of repeated sequences from captive-reared animals.
The control region (CR) is the main noncoding region of mitochondrial DNA (mtDNA). In mammals, the CR is flanked by the transfer RNA genes t-RNAPro on the 5' side of the light (L) strand and t-RNAPhe on the 3' side of the heavy (H) strand. In comparative studies, Douzery and Randi (1997) and Saccone et al. (1991) demonstrated that the CR is a highly structured gene formed by a conserved central region (CCR) and two peripheral domains. The CCR region is conserved even among divergent taxa, probably because of strong selective constraints related to its role in the cleaving of the L-strand transcript. In contrast, the two peripheral domains are rapidly evolving regions that show a high rate of both nucleotide substitutions and variation in copy number of tandem repeats. This variation in copy number of tandemly repeated sequences has been regarded as the major source of mtDNA length variation in animals (Brown et al. 1986).
In this study we used two levels of investigation. First, we sequenced the entire CR in the Eurasian otter (Lutra lutra) in order to describe its general organization and to compare it with CRs of other carnivore species. Comparisons were made with increasingly divergent carnivore taxa, according to the phylogeny of the order based on the cytochrome b gene (Ledje and Arnason 1996). The entire CR has been sequenced in nine carnivores belonging to six families (Table 1). This allowed us to explore patterns of organization of the different regions of the gene from the species to the family level.
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We also wanted to explore the utility of the left and right domains and the repetitive sequences in the conservation genetics of the Eurasian otter in Italy. This species was once widespread throughout wetland areas of Europe, but from the end of the 19th century, the species started declining sharply because of habitat loss, water pollution, and direct killing (Ansorge et al. 1997; Kruuk 1995, MacDonald and Mason 1994). At the time of writing, vital populations of the species are still present in several parts of Europe, but their distribution is highly fragmented (Randi et al. 2003). In Italy, the species is presently limited to a small area in the southern part of the country (several thousand kilometers from the closest viable population). This extant population is reduced to approximately 100 individuals (Cagnolaro et al. 1975; Prigioni 1991). The decline of the Eurasian otter along the entire peninsula was quite rapid if one considers that it was still common and widespread during the first two decades of the 20th century (Cagnolaro et al. 1975; Ghigi 1911). Following international efforts devoted to the conservation and reintroduction of the species (Foster-Turley et al. 1990), a colony of captive-bred otters was recently established in central Italy (Centro Lontra, Caramanico Terme) with the aim of reintroducing the species where extinct and/or to maintain the declining populations; the founders descend from the colony in the Norfolk Wildlife Centre (Norfolk, U.K.). This reintroduction program lacks genetic information, even though this aspect is of paramount importance to avoid the potential negative effects of genetic incompatibility (Drew et al. 2003).
Previous studies demonstrated a lack of genetic variability in the CR of populations of L. lutra in Denmark and Germany (Cassens et al. 2000; Effenberger and Suchentrunk 1999; Mucci et al. 1999; Pertoldi et al. 2001). Randi et al. (2003), using microsatellite loci, showed that this lack of polymorphism is not related to recent human-induced bottlenecks, but is due to a recent recolonization of the areas following the withdrawal of the Quaternary glaciers. We used sequence variation of the 5' and 3' domains of the CR and we analyzed length polymorphisms and heteroplasmy of the region with repetitive sequences to determine the degree of genetic differentiation between two samples of the extant Italian population (one museum and one contemporary) and the animals living in the Caramanico colony. We also evaluated the historical pattern of genetic heterogeneity of the species in Italy by comparing 22 museum specimens collected throughout the Italian peninsula in localities where the Eurasian otter is now extinct.
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Materials and Methods |
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Sampling and DNA Extraction
We obtained samples from 32 Eurasian otters. Details on the geographic origins of the samples are given in Table 2. The age of museum samples ranged from 74 to 132 years. Genomic DNA from contemporary samples was extracted from hairs using the DNeasy tissue kit from Qiagen (Venlo, Netherlands). Before using the kit, samples were cut into small pieces and then placed in 200 µl of a solution containing 10 mM Tris-HCl (pH 8.0), 10 mM EDTA, 100 mM NaCl, 40 mM DTT, 2% SDS, and 250 µg/ml proteinase K and incubated at 55°C overnight. DNA from museum samples was extracted from both hair and bone. Extractions from hair and bone were performed with the same protocol adopted for contemporary samples. Bones were crushed into small fragments and ground to a fine powder; the powder was decalcified with 0.5 M EDTA (pH 7.5). Decalcification was repeated five times. All museum extractions were accompanied by blank extractions to aid in the detection of contamination and were carried out under a hood separate from extractions of contemporary samples.
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Polymerase Chain Reaction Amplification and Sequencing
We amplified the entire CR of all the contemporary captive-bred samples using the primers L-Pro (5'-CGT CAG TCT CAC CAT CAA CCC CCA AAG C-3') and H-Phe (5'-GGG AGA CTC ATC TAG GCA TTT TCA GTG-3') (Mucci et al. 1999), which bind to the flanking tRNAPro and tRNAPhe genes, respectively. Polymerase chain reaction (PCR) amplifications were carried out in 25 µl total volume with the following recipe: 10 mM Tris-HCl (pH 8.8), 50 mM KCl, 1.5 mM MgCl2, 0.8 µM each dNTP, 2–50 nM each of primers, 1 U of AmpliTaq polymerase (PE Applied Biosystems, Foster City, CA) and 10–1000 ng of genomic DNA. PCR conditions were a first denaturation step at 94°C for 2 min followed by 30 cycles consisting of 94°C for 15 s, 50°C for 15 s, and 72°C for 15 s. Cycling was followed by an elongation step at 72°C for 5 min. PCR products were ligated in pt-AdvVector, linearized, and transformed in TOP10F' Escherichia coli competent cells using the Advantage PCR cloning kit (Clontech Laboratories, Palo Alto, CA). Positive clones were sequenced (both directions) using the 310 autosequencer with ABI BigDye chemistry (PE Applied Biosystems) according to the manufacturers' protocols. We sequenced four clones for each of the contemporary samples.
We amplified a fragment of the 5' region from the museum specimens and from LLU with the primers L-Pro and 363rev (5'-CCA TTC GAG ATG TCC CAT TTG-3'); a fragment of the 3' region of the gene was amplified with primers Ott-LD2 (5'-CCT ATA TTG TCC TGC CAA ACC C-3') and Ott-HD1 (5'-TAA CAA GTG GTG GGA GAG AGA AGC-3') (Mucci et al. 1999); the region with repetitive sequences was amplified with primers 363for (the reverse of 363rev) and Ott-D2rev (the reverse of Ott-LD2). We designed the primer 363rev on the basis of an alignment of CR sequences of the Eurasian otter retrieved from GenBank. PCR conditions for these regions were the same as described above. To avoid contamination, PCR mixtures with museum DNA were prepared under a dedicated hood where extractions of DNA from contemporary samples (or manipulations of PCR products) had never occurred. Aerosol-resistant tips were used whenever possible. Blank extractions were tested for amplifications and a negative control containing no DNA was added to each PCR experiment. All PCR products were cleaned with the GenElute PCR DNA purification kit (Sigma-Aldrich, St. Louis, MO) and sequenced in both directions. The size of the region with repetitive sequences was first deduced by running PCR products on a 2% agarose gel stained with ethidium bromide and by comparing them with a standard molecular weight marker. To further check the size of this region, we amplified it using the 363for primer labeled with fluorescent phosphoramidites (6-Fam). PCR products were analyzed with the 310 autosequencer. The size of the bands was calculated with GeneScan software based on an internal standard DNA (Rox 1000). Each band scored on an agarose gel was excised from the gel, purified with the GenElute PCR DNA purification kit and sequenced with the primers used for PCR amplification of the region. All the sequences obtained in this study have been submitted to GenBank (accession no. AY860320–AY860386).
Data Analysis
Sequences were edited and aligned with Sequencher 4.2.2 (Gene Codes Corp., Ann Arbor, MI); alignment was also checked by eye. The organization of the entire CR of the Eurasian otter was compared with those of other carnivore taxa (six families, including Mustelidae; see Table 1) for which data were available from GenBank. We searched for repeats in the CR of these species using the Tandem Repeats Finder program (Benson 1999). This program implements an algorithm that has several advantages: it uses the k-tuple matching method (no full-scale alignment matrix computations needed); it does not require a priori knowledge of the pattern, pattern size, and number of copy repeats; there are no restrictions on the size of the repeat; and substitutions and indels are treated separately. The secondary structure and thermal stability of regions with repeats were predicted using the MFOLD 3.0 server (available at http://mfold.burnet.edu.au/).
Phylogenetic analyses were carried out on two datasets. In the first dataset, we combined sequences (5' + 3' regions of the CR) of all the contemporary and museum samples included in the study plus a sequence from a population from Denmark (DK; data from Mucci et al. 1999; not available in the National Center for Biotechnology Information [NCBI] and European Molecular Biology Laboratory [EMBL] databases). In the second dataset, all the above sequences of the 5' region were combined with homologous sequences of Eurasian otter populations from central and northern Europe available in GenBank (data from Cassens et al. 2000; GenBank accession no. AJ006174–AJ006178; clone LUT1–LUT5). For both datasets we carried out the analyses by considering gaps either as a fifth base or as missing data. We used PAUP*4.0b10 (Swofford 2003) to calculate the number and pattern of nucleotide substitutions and to perform maximum parsimony (MP; heuristic searches, ACCTRAN character-state optimization, 100 random stepwise additions, TBR branch-swapping algorithm) (Farris 1970; Hendy and Penny 1982) and neighbor-joining (NJ) (Saitou and Nei 1987) analyses. Robustness of the phylogenetic hypotheses generated by the MP and NJ searches was tested by 1000 bootstrap replicates (Felsenstein 1985). Lontra canadensis was used as an outgroup in the phylogenetic analyses. We also performed a network analysis to estimate gene genealogies using the TCS program (Clement et al. 2000), which implements the Templeton et al. (1992) statistical parsimony procedure. This program collapses sequences into haplotypes and produces a network linking different haplotypes only if they have a 95% probability of being justified by the parsimony criterion. Input data were individual sequences.
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Organization of the CR in the Eurasian Otter
The CRs we cloned and sequenced from the captive-bred otters were either 918 or 1116 bp long; this difference is due to the variation in the number of tandem repeats (see below). A schematic diagram of the organization of the gene is presented in Figure 1A. Figure 1A and the following results refer to the 1116 bp variant. At position 151 we found a motif with homology to the termination-associated sequences (TAS-A) of other mammals. A few nucleotides upstream we found a GCCCC motif associated with the termination of the displacement loop structure (D-loop) of the CR. We identified all eight putative conserved sequence blocks (CSB1-3 and B-F) previously found in mammals. A TATA motif, the putative promoter of light strand transcription (LSP), starts at position 957. The putative promoter of the heavy strand transcription (HSP; same TATA motif) maps between nucleotides 1069 and 1072.
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We found only a single region with tandem repeats (called RS3 according to Douzery and Randi [1997]); this region is located between CSB-1 and CSB-2, as in the vast majority of mammals investigated so far (Fumagalli et al. 1996; Hoelzel et al. 1994). The length of a single repeat is 22 bp and there are 10 of them. These repeats are flanked at the 3' end by a short motif that is 6 bp long. The 10 repeats share the same core motif ACGT. Repeats 1 and 2 show a T-C transition at position 22, while repeats 8, 9, and 10 show a C-G transversion at the same position. These repeats also show a T-C transition at position 9. Among the clones we sequenced, we found an alternative array of repeats. In this case there were only six repeats that were 8 bp long, based on the core motif ACAT and flanked at the 3' end by a motif 5 bp long. The core motif of these repeats shows a G-A transition at position 3 in repeats 2 and 4, a T-C transition at position 6 in repeat 2, and a G-A transition at position 4 in repeat 5. PCR amplifications with primers flanking the RS3 domain produced two bands of about 600 bp and 390 bp for the captive-bred animals and two bands of about 600 bp and 470 bp for the museum samples. Direct sequencing revealed that the 600 bp band is identical between museum and contemporary samples. The 470 bp band contains five repeats 22 bp long; the motif of the repeat is identical to the one found in the 600 bp variant.
All but one of the captive-bred specimens were heteroplasmic; the sole homoplasmic individual carried the 600 bp array. Sixty percent of the museum samples were heteroplasmic; we found homoplasmic individuals for both variants, but those carrying the 600 bp one were more common (75% of the homoplasmic individuals). The sequence of repeated motifs and their array and distribution among specimens included in the study are presented in Table 3.
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GeneScan analyses revealed a single peak for all the homoplasmic individuals, which gave a single PCR band on the agarose gel. In contrast, for heteroplasmic individuals (two PCR bands), the analysis showed an apparent continuum of low peaks differing in size by approximately 20 bp between the two most intense ones (600 bp and 390 bp in contemporary samples; 600 bp and 470 bp in museum samples). These bands of intermediate size were not visible on agarose gels; this could imply that the two arrays visible on agarose gels are far more common in the mitochondria than the faint ones and that the repeated element 22 bp long is the one that varies in copy number.
The three main arrays could form stable secondary structures (not shown); the free energy was –7.2 kcal/mol for the 390 bp variant, –16.9 kcal/mol for the 470 bp variant, and –35.4 kcal/mol for the 600 bp variant. Our results agree with those of Fumagalli et al. (1996), who showed that repeated units are capable of folding into secondary structures and that the associated free energy increases linearly with repeat copy number.
Comparison with Other Carnivore Taxa
Figure 1B summarizes the information on the conserved regions of the CR among carnivore taxa for which the entire gene has been sequenced. The D-loop termination motif is perfectly conserved among Mustelidae, Mephitidae, and Canidae; this is the only part of the CR that does not vary across different families. CSB-F, C, and 2 are identical in the eastern and West-South American hog-nosed skunk; CSB-1 and 3 are perfectly conserved among the three Canidae species included in the study. After combining the 10 conserved regions shown in Figure 1B, we found 9.4 ± 2.2% of sequence divergence among different families; at this taxonomic level, 47.5% of all substitutions are transitions. There are two levels of sequence divergence at the intrafamily level; we found a similar level of divergence within Mustelidae (6.7%) and Canidae (5.3 ± 1.2%), while sequence divergence was substantially lower within Mephitidae (2.1%). In all these cases, most of the detected substitutions were transitions (from 71.8% in Canidae to 81.2% in Mustelidae).
Table 4 reports the repeated motifs and their array and localization in the nine carnivore taxa included for comparison in the study. Most of the species show tandem repeats between CSB-1 and CSB-2; Tandem Repeats Finder (Benson 1999) found repeated motifs in the left domain of the CR only in the spotted hyena and the tiger. The repeated elements in the left domain of the gene are generally longer than those located between CSB-1 and CSB-2 (75 bp and 80 bp in Crocuta crocuta and Panthera tigris, respectively). Repeats located between CSB-1 and CSB-2 are always based on the core motif ACGT. Different arrays are based on the repetition of variants of a single element; nucleotide substitutions among different variants are insertion/deletion and transitions, while we did not observe any transversions between variants of a given element. Repeated sequences between CSB-1 and CSB-2 are arranged in a single array in most of the species; only the eastern hog-nosed skunk has two different arrays in this region of the gene. The repeated element of the river otter is 22 bp long, as in the case of the 600 and 470 bp arrays of the Eurasian otter, but the respective sequences are very different. The domestic dog, wolf, and coyote share an element 10 bp long; the sequence of the element is identical in the domestic dog and coyote.
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Phylogeography of the Eurasian Otter in Italy
We found very low levels of genetic variation in both the datasets analyzed phylogenetically. Aligned datasets (5' and 3' domains) were 249 bp and 134 bp long, respectively. We identified four and five variable characters that defined four and seven haplotypes, respectively. Detected substitutions were almost exclusively transitions; there was only a C-G transversion between LUT1-2-3-5 and the remaining samples. Captive samples differed from samples from the provinces of Florence and Naples by two single A-G transitions. In our alignment, transition at position 60 identifies four samples from central Italy (Tuscany, province of Florence, specimens 6845, 11825, 11829, 12407), while substitution at position 237 discriminates southern Italian samples (PEP, LLU, Campania, province of Naples). All the remaining native Italian samples shared the same haplotype with the captive ones. MP and NJ searches carried out on the two datasets produced largely unresolved trees (not shown); the outcome of the analyses did not change when we considered gaps either as a fifth base or as missing data. We always recovered two resolved clades in all phylogenetic searches. The first clade groups the four individuals from the province of Florence, while the second one contains PEP, LLU, and DK (Denmark) placed basal. These clades received moderate bootstrap support (from 60% to 65%) in MP and NJ analyses.
Figure 2 shows the relationships among all the haplotypes obtained from the network analyses of the two datasets. Treating gaps as a fifth base or as a missing character did not change the result of the analyses. A single haplotype shared among all the captive-bred individuals and 18 of the 23 museum samples is dominant across the samples included in the study; TCS analyses gave support to MP and NJ searches in identifying two groups of individuals from central and southern Italy. When sequences of the 5' and 3' regions of the gene are combined, LLU and PEP are characterized by their own haplotype; these individuals share the same haplotype with DK and LUT4 if the analysis is limited to the 5' region. In this case there is also a higher level of homoplasy in the data, as indicated by the reticulation (loop) among haplotypes.
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Conserved and Repeated Domains of the CR
The CR of the Eurasian otter shows all the main functional sites already identified in a variety of vertebrates (e.g., Gemmell et al. 1996). These regions can easily be aligned in all the carnivore families included in the study and even with some Cervidae taxa (data not shown; Douzery and Randi 1997); this implies that these regions of the gene were conserved throughout more than 65 million years of evolution, corresponding to the emergence of Carnivora and Artiodactyla (Ledje and Arnason 1996). The D-loop termination motif is the only one conserved across different carnivore families; furthermore, the same GCCCC putative stop point has already been identified in a variety of mammalian taxa (Doda et al. 1981; Douzery and Randi 1997). In contrast, the TAS-A region shows a relatively high level of nucleotide substitutions, even between recently divergent species (i.e., wolf and domestic dog). The D-loop termination motif and TAS-A have great functional importance because they are both involved in the termination of the H-strand replication and in the displacement of the original H-strand to create a three-stranded structure usually called the D-loop. Our comparisons demonstrate that, despite its functional importance, the TAS-A region evolved rapidly, giving rise to heterogeneity in both length and base composition.
Three of the eight CSBs (CSB-F, CSB-C, and CSB-2) are identical in the two Mephitidae taxa, while CSB-1 and CSB-3 are perfectly conserved among wolf, domestic dog, and coyote. None of the CSBs is identical between the Eurasian otter and the river otter. The patterns of nucleotide substitutions in the CSBs between and within Mephitidae, Mustelidae, and Canidae agree with previous molecular phylogenies of carnivores. Dragoo and Honeycutt (1997) and Ledje and Arnason (1996) recommended considering the previous subfamily Mephitinae as a family (Mephitidae) distinct from Mustelidae. Phylogenetic analyses of mitochondrial and nuclear genes (Koepfli and Wayne 1998, 2003) supported the suggestion of van Zyll de Jong (1972, 1987), based on morphology, to place the Eurasian otter and the river otter in different genera (Lutra and Lontra, respectively). Finally, Ledje and Arnason (1996) showed that the genus Canis underwent a recent diversification.
Despite the different patterns of nucleotide substitutions across CSB sequences, they are still quite similar among all the carnivore taxa included in this study and, even more important, these regions of the CR are relatively similar in a variety of vertebrates (Gemmell et al. 1996). Several studies focused on the possible role played by CSBs in the transcription and replication of mtDNA in the D-loop containing region (Brown et al. 1986; Saccone et al. 1987). Our finding of relative uniformity of sequences among CSBs might be considered a further indication of the functional importance of these regions.
The size of the mitochondrial genome can vary extensively among animal taxa; this phenomenon is rarely associated with large amplifications or deletions in the protein-coding portions of the molecule (Moritz and Brown 1987; Zevering et al. 1992). Rather, the apparently more common mechanism responsible of mtDNA length polymorphism is variation in the copy number of tandemly repeated sequences of the CR. Length polymorphism can also occur among different mitochondria of the same individual (heteroplasmy). The CR of mammals typically has two potential locations with tandem repeats; in the left and right domains of the gene, respectively (Wilkinson et al. 1997). Repeated sequences in the left domain of the CR are usually about 80 bp long and include TAS elements. Repeated sequences in the right domain of the CR (RS3 in this study) are located between CSB-1 and CSB-2. This location has only been described in mammals (Fumagalli et al. 1996; Hoelzel et al. 1994). These repeated sequences are usually shorter than those in the left domain of the gene (typically between 6 and 30 bp) and exhibit length variation similar to that described for nuclear microsatellite loci. Because of their location upstream of the origin of H-strand replication, their variation does not influence D-loop size and probably has no impact on the rate of mtDNA transcription and replication.
Among the nine carnivore taxa used for comparison in this study, two (the spotted hyena and tiger) showed repeated sequences both in the left domain and between CSB-1 and CSB-2; in all the other species, repeated sequences were only located between CSB-1 and CSB-2. Interestingly, while the tiger has only two 80 bp long repeated sequences with TAS elements in the left domain of the CR, the spotted hyena has two blocks of three and four repeats (9 bp and 75 bp long, respectively) separated by 176 bp of a nonrepeated sequence. In this species we identified TAS elements only in the 75 bp long repeats.
Concerning RS3, all the species included in this study except the eastern hog-nosed skunk have a single array of repeated sequences based on or derived from the core motif ACGT (Hoelzel et al. 1994). The eastern hog-nosed skunk has two contiguous arrays of repeats 6 bp long (28 and 14 repeats, respectively). This could be an exceptional example of the short single "imperfect" copy of the tandem repeats at the 3' end of the RS3 generated by slippage-mispairing events (Fumagalli et al. 1996). Indeed, this is what we observed in all the carnivore taxa included in the study (see Table 4). In all the species, we found at least one substitution and/or insertion/deletion among the repeats that make up the RS3 array. This agrees with previous findings on the same region in a variety of mammals (e.g., Fumagalli et al. 1996; Wilkinson et al. 1997). However, sequence variations of repeats and/or variations in the array of the repeats among different species do not seem to be informative for phylogenetic reconstruction above the species level.
Phylogeography of the Eurasian Otter in Italy
The phylogenetic and network analyses (Figure 2) revealed the existence of three different groups of haplotypes in Italy. Four museum specimens from the province of Florence (Tuscany) and two individuals from the province of Naples (Campania) (one museum and one contemporary) have distinct haplotypes, which are one mutational step from the most common haplotype. This common haplotype is shared by the animals living in the Caramanico colony and 18 of the 23 historical samples from northern and central Italy. The two individuals (PEP, LLU) from southern Italy, the only part of the country where the Eurasian otter is still present, have a distinct haplotype when the 5' and 3' fragments of the CR are combined. Furthermore, a geographic component is evident if we analyze the partitioning of RS3 arrays; the 470 bp array is present only in historical specimens sampled in Italy, while the 390 bp variant only appears in the captive-bred individuals. It is important to note that the independent variation we found between sequence variation of the 5' and 3' fragments of the gene and length polymorphism of the RS3 region tends to exclude contamination between contemporary and historical samples, a risk frequently associated with studies that simultaneously analyze DNA extracted from fresh and old/poorly preserved specimens.
The captive-bred otters do not possess the haplotype LUT1, which Cassens et al. (2000) found to be the most abundant and widespread in populations sampled over a broad geographic area from Austria to Russia. However, only incomplete information is available about the origin of most of the founders of European captive populations of the Eurasian otter. Vogt (1995) reported that these populations probably originated from wild-caught otters from Scotland. Unfortunately, complete pedigrees are available only for a small number of individuals. This obviously leaves many questions open and does not allow us to place our results in a well-defined framework.
We generally found little structuring by geographic origin of the samples, with the exception of individuals from the provinces of Florence and Naples. The network analyses reported in Figure 2 revealed a pattern of relationships among haplotypes based on a single abundant (ancestral?) haplotype and a few derived, locally restricted ones. This pattern could indicate a bottleneck followed by a population expansion (Stanley et al. 1996). Cassens et al. (2000), Pertoldi et al. (2001), and Randi et al. (2003) identified the Quaternary glaciations as the main events responsible for a drastic decline of the Eurasian otter populations in Europe; the species presumably survived in a few refuge areas and dispersed northward after the glaciations. Our findings agree with this hypothesis. Nevertheless, we also have to consider the possible role of anthropogenic disturbance in altering the geographic distribution of molecular markers. Small extant isolated populations of threatened species can often be diagnosed on the basis of a few base pair substitutions.
Recently Goldstein and De Salle (2003) and Palkovacs et al. (2004) reported examples of how the diagnosability of such populations might be the result of human disturbance that altered the original distribution of species; extinction of many contiguous populations and the reduced size of the extant ones can make such populations appear more genetically divergent than in the past. Therefore it is possible that our results on the distinctiveness of the two geographic groups of haplotypes we identified in Italy might at least partly reflect population fragmentation due to human disturbance. It should be noted, however, that the nucleotide substitutions that diagnose these haplotypes are fixed, even in comparisons with specimens sampled over a geographically contiguous area (especially in the case of Tuscany); this supports the hypothesis that the pattern of genetic heterogeneity we detected reflects the true (past) phylogeography of the species.
Despite the past efforts, a robust phylogeography of the Eurasian otter is still not available, even though it is needed to plan adequate and scientifically sound conservation measures. Neither mtDNA CRs nor microsatellites have proved decisive in reconstructing evolutionary relationships among populations of the species (Cassens et al. 2000; Dallas et al. 1999, 2002, 2003; Effenberger and Suchentrunk 1999; Mucci et al. 1999; Randi et al. 2003). Our results on RS3 suggest that this region of the CR could be a valuable tool when used in conjunction with other markers. RS3 seems to have several advantages. First, characterization of its variation is relatively easy and inexpensive; second, it seems to be variable enough (especially if one considers the presence of the many peaks revealed by GeneScan analysis) to describe population structure and to complement microsatellite analyses from an exclusively maternal perspective; third, it is likely that this region of the CR varies freely (and therefore neutrally), since it is not subject to any of the functional constraints acting on other regions of the gene.
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In this study we have characterized for the first time the entire mtDNA CR of the Eurasian otter and have compared it with the CR of selected carnivore species. We have also explored the potential utility of different regions of the gene in tracing evolutionary relationships among populations of this vulnerable species; our data, based also on museum specimens, suggest an apparent uniqueness of two Italian populations. One of these populations is now extinct, but the other is the sole natural population of the species still present in Italy. We are aware that our results must be considered cautiously due to the small number of individuals analyzed. Nevertheless, given the difficulty in obtaining samples of this elusive and rare species, these data can serve, at a minimum, as a starting point for further research aimed at a detailed characterization of the genetic traits of the Eurasian otter in Italy. Since the present and previous studies have revealed a certain degree of genetic heterogeneity among Eurasian otter populations, but have failed to clearly define the phylogeography of the species, the reintroduction of individuals from captive-bred stocks should be avoided for the moment.
Although the genetic characterization of populations of declining species is important, it is by itself inadequate when the causes of the decline are habitat loss and fragmentation of suitable environments, pollution, and direct persecution. The Eurasian otter has demonstrated the ability to recover when habitat conditions are adequately restored and corridors are made available to reconnect scattered populations (MacDonald and Mason 1994). These should undoubtedly be the primary conservation goals.
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Acknowledgments |
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We would like to thank Leonardo Latella (Museo Civico di Storia Naturale, Verona), Paolo Agnelli (Museo Zoologico "La Specola," Florence), Gloria Svampa (Museo Civico di Zoologia, Rome), Fernando Di Fabrizio (Museo Naturalistico Lago di Penne, Pescara), Livia Mattei (Corpo Forestale dello Stato, Pescara), Ettore Randi (Istituto Nazionale per la Fauna Selvatica, Bologna), and all the people working in the Centro Lontra of Caramanico Terme (Pescara) for their invaluable assistance in obtaining samples. Thanks to Federica Venanzetti for technical assistance with GeneScan analyses. We are also thankful to Elvira De Matthaeis, Spartaco Gippoliti, Brian Bowen, and two reviewers for their comments on an earlier draft of this article. This research was supported by Ministry for Higher Education, Training and Research (MIUR) funds "Progetto Giovani Ricercatori" (to V.K.).
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Footnotes |
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Corresponding Editor: Brian Bowen
Received June 28, 2004
Accepted December 15, 2004
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