Mitochondrial DNA sequences in three genera of Italian lampreys

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Mitochondrial DNA sequences in three genera of Italian lampreys a

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James Tagliavini , Rubens Tizzi , Franco Conterio , Paolo Mariottini & Gilberto Gandolfi

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Dipartimento di Biologia evolutiva , Università di Parma , viale delle Scienze, Parma, I‐43100, Italy b

Istituto di Scienze biochimiche , Università di Parma , viale delle Scienze, Parma, I‐43100, Italy c

Dipartimento di Biologia e Fisiologia generali , Università di Parma , viale delle Scienze, Parma, I‐43100, Italy Published online: 28 Jan 2009.

To cite this article: James Tagliavini , Rubens Tizzi , Franco Conterio , Paolo Mariottini & Gilberto Gandolfi (1994) Mitochondrial DNA sequences in three genera of Italian lampreys, Bolletino di zoologia, 61:4, 331-333, DOI: 10.1080/11250009409355903 To link to this article: http://dx.doi.org/10.1080/11250009409355903

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Boll. Zool. 61: 331-333 (1994)

Mitochondrial DNA sequences in three genera of Italian lampreys JAMES TAGLIAVINI RUBENS TIZZI FRANCO CONTERIO Dipartimento di Biologia evolutiva, Università di Parma, viale delle Scienze, I-43100 Parma (Italy)

PAOLO MARIOTTINI Istituto di Scienze biochimiche, Università di Parma, viale delle Scienze, I-43100 Parma (Italy)

GILBERTO GANDOLFI

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Dipartimento di Biologia e Fisiologia generali, Università di Parma, viale delle Scienze, I-43100 Parma (Italy)

INTRODUCTION The Agnatha, and in particular the species of the Holarctic family Petromyzontidae, are very interesting organisms for their basic divergence in the vertebrate phylogenetic tree. The four Italian lampreys are actually assigned to three genera, i.e., Petromyzon Linnaeus (P. marinus), Lethenteron Creaser & Hubbs (I. zanandreai), and Lampetra Bonnaterre (L. fluviatilis and L. planeri). In this study, we have considered one species for each genera. Two of them are freshwater and non-parasitic species, with allopatric distribution: L. planeri (Bloch), whose range extends through the river systems draining toward the North Sea and Atlantic coastlines of France and the British Isles, and is also found on the western slopes of the Apennines within the Mediterranean area; L. zanandreai (Vladykov), which is endemic on the Italian Alpine watershed to the left side of the River Po and in the Venetian region from the Adige to the Isonzo river drainages. Notwithstanding the great morphological and ecological likeness of these two species, on the basis of some differences in their patterns of dentition they have been assigned to different genera by Vladykov & Follett (1967), Vladykov & Kott (1979) and Holcik"(1986, footnote on p. 238), although other authors recognize Lethenteron as a subgenus of Lampetra (Creaser & Hubbs, 1922; Hubbs & Potter, 1971; Bailey, 1980; Potter, 1980; Nelson, 1984). The third species, P. marinus Linnaeus, is anadromous and parasitic, and widely distributed on both sides of the North Atlantic; in the Mediterranean, it occurs in the western part and extends to the Ionian and Adriatic Seas.

KEYWORDS: Petromyzontidae - Lampreys - Mitochondrial DNA - Cytochrome b - tRNAGlu.

On the basis of morphological characteristics, the phylogenetic derivation of both Lampetra and Lethenteron from Petromyzon is a well-stated fact (Hubbs & Potter, 1971; Potter, 1980; Hardisty, 1986). In the northern hemisphere, the widely distributed monotypic species P. marinus could represent the possible way by which genes spread in the other Italian species until relatively recent times during some particular geological events, e. g. the last glaciation. To verify this hypothesis, and to study the genetic diversity among the three genera, we have sequenced and compared two generegions of the mitochondrial genome of L. planeri and L. zanandreai, i.e., the fast evolving tRNAGlu gene and a partial sequence of the evolutionarily conserved and slowly evolving cytochrome b (cyt-b) gene. We have also sequenced and analysed the same cyt-b region of P. marinus.

ACKNOWLEDGEMENTS

MATERIALS AND METHODS

ABSTRACT The genetic diversity obtained by comparing mitochondrial DNA regions (the slowly evolving protein-coding gene cytochrome b and the tRNAGlu gene) of three lamprey species belonging to different genera, Petromyzon marinus, Lethenteron zanandreai, and Lampetra planeri, is reported. The low degree of intergeneric variability detected, as compared with differences described for other fish taxa, supports the view of a recent evolutive separation of both Lethenteron and Lampetra from Petromyzon.

This study was partially supported by CNR grant No. 94.026l6.CT04. The authors would like to thank Prof. P. Melotti, Dr P. Turin and Dr S. Zerunian for their assistance in supplying lamprey specimens. (Received 10 May 1994 - Accepted 14 July 1994)

Samples were collected from frozen or ethanol conserved animals captured in three separate localities: adult P. marinus from the Po River (northern Italy), ammocoetes and adult L. zanandreai from tributaries of the Brenta River (northwestern Italy), ammocoetes and adult L. planeri from the Aniene River (Tiber drainage, central Italy). Muscle samples (0.5 g wet weight) were washed twice with 20 vol

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of cold 25 mM Tris-HCl (pH 8), 10 mM EDTA, 50 mM Glucose (GTE buffer) and homogenized on ice by a glass tissue homogenizer (Wheaton, pestle B, 10 strokes). Mitochondrial DNAs (mt-DNA) were isolated from homogenates by alkaline lysis (Csaikl & Csaikl, 1988) and purified by conventional methods (Sambrook et al., 1989). The mt-DNAs were Hindlll-EcoRI digested and cloned into a PUC18 vector. On the basis of their sequence homologies, some cloned fragments proved to contain part of the cyt-b and tRNA°'u genes. Sanger's sequencing was carried out by Ml3 universal primers for tRNAG1" sequences and by Kocher's specific primers for the cyt-b gene region (Kocher et al., 1989). Nucleotidic and derived protein sequences were analysed by PCGene software (Intelligenetics) and compared with DNA and protein sequence collections (GenBank, Swiss-Prot).

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EMBL accession numbers of analyzed lamprey sequences: X78804 Lampetra planeri tRNAGl11; X78806 Lethenteron zanandreai tRNAGl11; X79109 Lethenteron zanandreai cyt-b partial CDS; X79110 Lampetra planeri cyt-b partial CDS; X791H Petromyzon marinus cyt-b partial CDS.

RESULTS AND DISCUSSION Comparison between the 71 bp long tRNAGlu genes of L. planeri and L. zanandreai shows 100% identicity. Multiple alignment adding the same gene sequences, available in the EMBL data collection for two osteichthyes, Crossostoma lacustre (Tzeng et al., 1992) and Cyprinus carpio (Huang, unpublished), and for the amphibian Xenopus laevis (Roe et al., 1985) reveals identities of 77.5%, 74.6%, and 74.6%, respectively (Fig- 1). Comparison between a 270 bp long region of the cyt-b genes of L. planeri and L. zanandreai shows 97.8% identities, with five nucleotide transitions and one transversion in their sequences. Cyt-b DNA fragments of P. marinus and L. planeri show identical sequences (Fig. 2 A). The alignment of the derived aminoacidic sequences of the cyt-b proves the existence of 100% identicity be-

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tween L. planeri and P. marinus, and 98.9% identicity between these two species and L. zanandreai, due to a single aminoacidic substitution (Fig. 2B). On the basis of divergence of mt-DNA sequence data and fossil records, Martin et al. (1992) have described some relationships between point-mutation occurrence and time of divergence in sharks. In particular, comparing the cyt-b sequences of two individuals from Sphyrna tiburo populations separated by the Isthmus of Panama about 3.5 million years ago, they suggested a maximum divergence rate of 2.3% per million years. Among osteichthyes, considering 239 bp sequences of the same cyt-b portion of gene, the Cichlidae Cicblasoma citrinellum and Julidochromis regani show 83.7% identicity, due to 27 transitions and 12 transversions (Kocher étal., 1989; Meyer, 1993). Comparing 270 bp of the cyt-b gene in Crossostoma lacustre (Tzeng et al., 1992) and Cyprinus carpio (Huang, unpublished, X61010), both Cypriniformes belonging to the families Homalopteridae and Cyprinidae, we obtain 79-3% identicity, due to 30 transitions and 26 transversions. The last reported genera have been reproductively separated for approximatively 100 million years. In all cases, the relatively high similarity observed in the derived aminoacidic sequences proves that a large amount of nucleotidic diversity is evolutionarily neutral. ~ On the basis of the molecular homologies observed among the cyt-b sequencies of the three lamprey genera, and assuming for heterotermic vertebrates an almost constant rate of nucleotidic substitutions in the same gene (Kocher et al, 1989; Thomas & Beckenbach, 1989), we can estimate a recent evolutive origin of L. zanandreai and L. planeri from P. marinus, probably not exceeding one million years. Moreover, the rapid evolutionary sequence changes described for the family of tRNA genes (Brown et al., 1982) and the observed 100% homology between the tRNAGlu genes of L. zanandreai and L.

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similarity G T T C T T A T A G T T O AATTCTA CAAC G G T G G T TTTTCAA GTCGC TAGT TTAAG TTAAAGT CTTAA TGAGAATG

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Fig. 1 - Nucleotide sequences for tRNAG'u genes. The functional regions are indicated above the sequences. The anticodon is underlined; a dot represents an identical nucleotide. On the right side the percentage of similarity is given with reference to the Lampetra planeri (L. p.) sequence. Lethenteron zanandreai (L. z.); Cyprinus carpio (C. c , Huang, unpublished); Crossostoma lacustre (C. 1., Tzeng et al., 1992); Xenopus laevis (X. 1., Roe et al., 1985).

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MITOCHONDRIAL DNA SEQUENCES OF ITALIAN LAMPREYS

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L. p . P. m. Ï..

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p . m.

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L. p . P. m. L.

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Fig. 2 - Sequence comparisons of the analysed cytochrome b region in the three lamprey species: Lethenteron zanandreai (L. z.), Petromyzon marinus (P. m.) and Lampetra planeri (L. p.); A, nucleotidic sequences alignment; B, deduced alignment of aminoacid sequences from residue 36 to residue 125.

planeri, confirm their recent reproductive isolation. It is worth pointing out that also their minor morphological differences are in agreement with the mitochondrial molecular data. Our results support neither a Pannonic (Middle Miocene) nor a Messinian (Lower Miocene) penetration of Lethenteron in northern Adriatic drainages, as hypothesized by Bianco (1987). The origin of both Lethenteron and Lampetra can most probably be ascribed to Pleistocenic events. Their close genetic likeness, which results in mtDNA regions, seems not to support their assignment to different genera. It is also known that other species of Lethenteron (L. japonicum) and Lampetra (L. fluviatilis) have identical aminoacidic composition of their hemoglobins and show minor differences from the hemoglobin V of P. marinus (Hardisty, 1979). An analysis of other mitochondrial gene regions and an extension of the study to Caspiomyzon and Eudontomyzon, which are supposed to have evolved independently and prior to the Lethenteron-Lampetra complex (Hubbs & Potter, 1971; Potter, 1980; Hardisty, 1986), would probably clarify the problem of the evolution of European lampreys.

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