*Centro Studio Mitocondri e Metabolismo Energetico, Consiglio Nazionale delle Ricerche, Bari, Italy;
Dipartimento di Biochimica e Biologia Molecolare, Università di Bari, Bari, Italy;
Dipartimento di Fisiologia e Biochimica Generali, Università di Milano, Milano, Italy;
§Laboratoire de Paléontologie, Paléobiologie et Phylogénie, Institut des Sciences de l'Evolution, Centre National de la Recherche Scientifique, Université Montpellier II, Montpellier, France
Nowadays, the order Rodentia represents almost half of all living mammalian species, classified into 3033 families (Hartenberger 1998
), and shows high levels of variability in morphology, habitat utilization, behavior, life history strategy, and geographic distribution (Eisenberg 1981
; Wilson and Reeder 1993
).
Morphological classifications have considered the order Rodentia a monophyletic group on account of dental, cranial, postcranial, and soft anatomical attributes (Luckett and Hartenberger 1993
; Hartenberger 1998
). Nevertheless, this view was challenged in different molecular surveys at the beginning of this decade (Graur, Hide, and Li 1991
; Graur et al. 1992
; Li et al. 1992
). More recent studies have relied on the analysis of complete mitochondrial genomes of four rodent species, namely, rat, mouse, guinea pig, and dormouse, encompassing three major lineages: Muridae, Caviidae, and Gliridae (D'Erchia et al. 1996
; Reyes, Pesole, and Saccone 1998
). Irrespective of the methodological approach, the results obtained in these studies showed the existence of two well-supported rodent clades, one including murid rodents (rat and mouse) and the other including nonmurid rodents (guinea pig and dormouse). These surveys would support rodent paraphyly; however, some authors claim rodent monophyly based on different mitochondrial data sets or methodological approaches (Cao, Okada, and Hasegawa 1997
; Philippe 1997
; Sullivan and Swofford 1997
; Philippe and Laurent 1998
). On the whole, molecular studies are still scarce and do not resolve the relationships among the major lineages of this order (see Huchon, Catzeflis, and Douzery [1999]
and references therein), let alone between rodent families. Thus, the relationships among rodents and the issue of rodent monophyly versus paraphyly/polyphyly are far from being settled, and only additional data could help solve them. In this context, we have sequenced, according to standard procedures described elsewhere (e.g., Reyes, Pesole, and Saccone 1998
), the complete mitochondrial (mt) genome of the European red squirrel, Sciurus vulgaris (specimen number V-784 of F. Catzeflis), a representative of the Sciuridae family which represents one of the major rodent lineages.
The mtDNA sequence of the squirrel S. vulgaris has been deposited in the EMBL database under accession number AJ238588. The control region is 1,059 nt long and shows some peculiar features, such as the lack of repeated motifs, the lack of two out of the three conserved sequence blocks (CSBs) (namely, CSB2 and CSB3), and the existence of a 58-bp region next to the tRNA-Pro which shows high similarity (average value 83.5%) to the corresponding region of rabbit, dormouse, and guinea pig but not with that of rat and mouse.
Phylogenetic analyses were carried out on the complete mammalian mtDNA sequences available in the EMBL database (release 61): human (Homo sapiens, V00662), common chimpanzee (Pan troglodytes, D38116), pigmy chimpanzee (Pan paniscus, D38113), gorilla (Gorilla gorilla, D38114), orangutan (Pongo pygmaeus, D38115), gibbon (Hylobates lar, X99256), baboon (Papio hamadryas, Y18001), horse (Equus caballus, X79547), donkey (Equus asinus, X97337), Indian rhinoceros (Rhinoceros unicornis, X97336), white rhinoceros (Ceratotherium simum, Y07726), harbor seal (Phoca vitulina, X63726), gray seal (Halichoerus grypus, X72004), cat (Felis catus, U20753), dog (Canis familiaris, U96639), fin whale (Balenoptera physalus, X61145), blue whale (Balenoptera musculus, X72204), cow (Bos taurus, V00654), sheep (Ovis aries, AF010406), pig (Sus scrofa, AJ002189), hippopotamus (Hippopotamus amphibius, AJ010957), Neotropical fruit bat (Artibeus jamaicensis, AF061340), African elephant (Loxodonta africana, AJ224821), aardvark (Orycteropus afer, Y18475), armadillo (Dasypus novemcintus, Y11832), rabbit (Oryctolagus cuniculus, AJ001588), guinea pig (Cavia porcellus, AJ222767), fat dormouse (Glis glis, AJ001562), rat (Rattus norvegicus, X14848), mouse (Mus musculus, V00711), hedgehog (Erinaceus europaeus, X88898), opossum (Didelphis virginiana, Z29573), wallaroo (Macropus robustus, Y10524), and platypus (Ornithorhyncus anatinus, X83427). Noneutherian sequences were always used as mammalian outgroups.
Concatenated supergenes for ribosomal 12S and 16S RNA genes and for protein genes coded by the H-strand were used for phylogenetic analyses. For protein genes, only first and second codon positions (P12) were considered, due to the substitution saturation and base composition heterogeneity observed in third codon positions (data not shown). Phylogenetic analyses were conducted using different approaches: the Markov model (Saccone et al. 1990
; also called general time reversible [GTR] in PAUP*) for the calculation of genetic distances, and the minimum-evolution (ME; Rzhetsky and Nei 1992
) or neighbor-joining (NJ; Saitou and Nei 1987
) method for tree reconstruction. The GTR analyses were also performed on the two supergenes assuming a gamma distribution for substitution rate across sites, where the parameter
(Yang 1994
) and the proportion of invariant sites (I) were estimated with the maximum-likelihood method assuming the GTR-ME phylogeny using PAUP*. Maximum-parsimony (MP) and maximum likelihood with the mtREV24-F model were used and a heuristic search (PROTML) was conducted on concatenated H-strand encoded amino acid sequences using the PAUP* (Swofford 1998
) and MOLPHY (Adachi and Hasegawa 1996
) packages, respectively. Bootstrap values were based on 1,000 replicates, except in the case of PROTML, where only 100 replicates were performed. Furthermore, log-likelihood ratio tests (Kishino and Hasegawa 1989
) were performed on both protein-coding and ribosomal genes in order to determine the degree of support for alternative hypotheses of relationships among rodents and other mammals.
To avoid a compositional bias in the analysis, the hedgehog sequence was excluded from the phylogenetic reconstruction, because it deviates significantly from the mean nucleotide frequency on the P12 sites (2 = 74.18, df = 3, P < 0.001). Figures 1 and 2 show the tree obtained by means of PROTML and GTR-ME on amino acid and protein-coding genes, respectively. In both cases, relationships within ferungulates (carnivores, perissodactyls, cetaceans, artiodactyls) and their clustering with Chiroptera are supported by high bootstrap values, in agreement with previous studies (e.g., Pumo et al. 1998
; Reyes, Pesole, and Saccone 1998
; Ursing and Arnason 1998
). Elephants and aardvarks, both members of the African clade (Springer et al. 1997
), are closely related to one another (bootstrap values 91% and 93%), Armadillo clusters with the African clade, when resolved, with a high bootstrap value (92%). With regard to rodent species, two different clades supported by high bootstrap values were observed: one containing rat and mouse (100%) and the other containing guinea pig, squirrel, and dormouse (86% and 90%). Within the latter group, squirrel is most closely related to dormouse (bootstrap values 96% and 83%). Rabbit was found to cluster with nonmurid rodents, leading to a "quasi-Glires" clade, even though bootstrap support is poor (52% and 66%). The main difference between figures 1 and 2 is that the former suggests rodent polyphyly and the latter suggests rodent paraphyly if the position of rabbit is not considered. The phylogenetic trees obtained with the other methods and data sets (i.e., GTR-ME and GTR-NJ either assuming constant rates of evolution or with
= 0.77, I = 0.42 for CDS and
= 0.64, I = 0.24 for rRNA supergenes, and MP on the protein supergene) show the same general topology (data not shown). Such trees exclude rodent monophyly, but they do not discriminate between paraphyly and polyphyly. Furthermore, log-likelihood ratio tests for different rodent tree topologies revealed that monophyly is significantly rejected in all cases and that the best tree corresponds to the one showing rodent polyphyly and the clustering of nonmurids with rabbit (fig. 1
). However, the alternative tree, assuming rodent paraphyly and rabbit clustering with nonmurids (fig. 2
), is not significantly different (best tree in fig. 1
shows a -ln L of 83,193.8, while the corresponding value for the alternative tree shown in fig. 2
is -ln L = 83,203.2, based on P12 sites).
|
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It is noteworthy that using the largest available mitochondrial data set (34 mammalian species, of which 5 are rodents) and different methodological approaches (homogeneous- and heterogeneous-site rate models), we never found support for rodent monophyly, in contrast to what is reported in previous studies (Cao, Okada, and Hasegawa 1997
; Philippe 1997
; Sullivan and Swofford 1997
; Philippe and Laurent 1998
). Finally, it has also been suggested that rodent monophyly is obscured by the higher evolutionary rate of murids and subsequent long-branch attraction with noneutherian species at the basal position of the tree (Philippe and Laurent 1998
). Nevertheless, we confirmed by relative-rate test using the CODRATES program (Muse and Gaut 1994
) that, except for squirrels, all rodent species considered here show the same or similar evolutionary rates on ribosomal and P12 sites. Thus, long-branch attraction should affect both murid and nonmurid species in the same way (with the exception of squirrel) and would not explain why murids remain at a basal position while nonmurids are placed as a sister group of the nonprimate eutherians.
All in all, the position of rodents in the mammalian tree remains an open question, as data are in agreement with both paraphyly and polyphyly of rodents. The confirmation of one of these hypotheses or even a completely different picture may arise from the analysis of new rodent sequences. In particular, the sampling of murid-related taxa, such as Spalacidae, Rhizomyidae, and Dipodidae, or of representatives of other rodent lineages (e.g., Ctenodactylidae, Bathyergidae, and Anomaluridae) would be of great interest.
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Footnotes |
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1 Keywords: Sciurus vulgaris,
mitochondrial DNA
rodent phylogeny
mammalian evolution
rodent paraphyly
rodent polyphyly
2 Address for correspondence and reprints: Cecilia Saccone, Dipartimento di Biochimica e Biologia Molecolare. Università di Bari, Via Orabona 4/A, 70125 Bari, Italy. E- mail: saccone{at}area.ba.cnr.it
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Adachi, J., and M. Hasegawa. 1996. MOLPHY version 2.3: programs for molecular phylogenetics based on maximum likelihood. Comput. Sci. Monogr. 28:1150.
Bugge, J. 1985. Systematic value of the carotid arterial pattern in rodents. Pp. 333354 in W. P. Luckett and J.-L. Hartenberger, eds. Evolutionary relationships among rodents. A multidisciplary analysis. Plenum Press, New York and London.
Cao, Y., N. Okada, and M. Hasegawa. 1997. Phylogenetic position of guinea pigs revisited. Mol. Biol. Evol. 14:461464.
D'Erchia, A. M., C. Gissi, G. Pesole, C. Saccone, and U. Arnason. 1996. The guinea-pig is not a rodent. Nature 381:597599.
Eisenberg, J. F. 1981. The mammalian radiations. An analysis of trends in evolution, adaptation, and behavior. University of Chicago Press, Chicago.
Graur, D., W. A. Hide, and H.-W. Li. 1991. Is the guinea-pig a rodent? Nature 351:649652.
Graur, D., A. Zarkikh, W. A. Hide, and H.-W. Li. 1992. The biochemical phylogeny of guinea pigs and gundies and the paraphyly of the order Rodentia. Comp. Biochem. Physiol. B 101:495498.
Hartenberger, J.-L. 1985. The order Rodentia: major questions on their evolutionary origin, relationships and suprafamilial systematics. Pp. 132 in W. P. Luckett and J.-L. Hartenberger, eds. Evolutionary relationships among rodents. A multidisciplinary analysis. Plenum Press, New York.
Hartenberger, J.-L. 1996. Les débuts de la radiation adaptive des Rodentia (Mammalia). C. R. Acad. Sci. Paris 323:631637.
Hartenberger, J.-L. 1998. Description de la radiation des Rodentia (Mammalia) du Paléocène supérieur au Miocène; incidences phylogénétiques. C. R. Acad. Sci. Paris 326:439444.
Huchon, D., F. Catzeflis, and E. Douzery. 1999. Molecular evolution of the nuclear von Willebrand factor gene in mammals and the phylogeny of rodents. Mol. Biol. Evol. 16:577589.[Abstract]
Kishino, H., and M. Hasegawa. 1989. Evaluation of the maximum likelihood estimate of the evolutionary tree topologies from DNA sequence data, and the branching order in Hominoidea. J. Mol. Evol. 29:170179.[ISI][Medline]
Kramerov, D., N. Vassetzky, and I. Serdobova. 1999. The evolutionary position of dormice (Gliridae) in Rodentia determined by a novel short retroposon. Mol. Biol. Evol. 16:715717.
Lavocat, R., and J.-P. Parent. 1985. Phylogenetic analysis of middle ear features in fossil and living rodents. Pp. 333354 in W. P. Luckett and J.-L. Hartenberger, eds. Evolutionary relationships among rodents. A multidisciplinary analysis. Plenum Press, New York and London.
Li, W.-H., W. A. Hide, A. Zharkika, D. P. Ma, and D. Graur. 1992. The molecular taxonomy and evolution of the guinea pig. J. Hered. 83:174181.[ISI][Medline]
Luckett, W. P., and J.-L. Hartenberger. 1993. Monophyly or polyphyly of the order Rodentia: possible conflict between morphological and molecular interpretations. J. Mamm. Evol. 1:127147.
McKenna, M. C., and S. K. Bell. 1997. Classification of mammals above the species level. Columbia University Press, New York.
Muse, S. V., and B. S. Gaut. 1994. A likelihood approach for comparing synonymous and nonsynonymous nucleotide substitution rates, with application to the chloroplast genome. Mol. Biol. Evol. 11:715724.
Nebdal, M. A., R. L. Honeycutt, and D. A. Schlitter. 1996. Higher-level systematics of rodents (Mammalia, Rodentia): evidence from the mitochondrial 12S rRNA gene. J. Mamm. Evol 3:201237.
Philippe, H. 1997. Rodent monophyly: pitfalls of molecular phylogenies. J. Mol. Evol. 45:712715.[ISI][Medline]
Philippe, H., and J. Laurent. 1998. How good are deep phylogenetic trees? Curr. Opin. Genet. Dev. 8:616623.[ISI][Medline]
Pumo, D. E., P. S. Finamore, W. R. Franek, C. J. Phillips, S. Tarzami, and D. Balzarano. 1998. Complete mitochondrial genome of a neotropical fruit bat, Artibeus jamaicensis, and a new hypothesis of the relationships of bats to other eutherian mammals. J. Mol. Evol. 47:709717.[ISI][Medline]
Reyes, A., G. Pesole, and C. Saccone. 1998. Complete mitochondrial DNA sequence of the fat dormouse, Glis glis: further evidence of rodent paraphyly. Mol. Biol. Evol. 15:499505.[Abstract]
Rzhetsky, A., and M. Nei. 1992. A simple method for estimating and testing minimum evolution trees. Mol. Biol. Evol. 9:945967.
Saccone, C., C. Lanave, G. Pesole, and G. Preparata. 1990. Influence of base composition on quantitative estimates of gene evolution. Methods Enzymol. 183:570583.[ISI][Medline]
Saitou, N., and M. Nei. 1987. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol. Biol. Evol. 4:406425.[Abstract]
Springer, M. S., G. C. Cleven, O. Madsen, W. de Jong, V. G. Waddell, H. M. Amrine, and M. J. Stanhope. 1997. Endemic African mammals shake the phylogenetic tree. Nature 388:6164.
Sullivan, J., and D. L. Swofford. 1997. Are guinea pigs rodents? The importance of adequate models in molecular phylogenetics. J. Mamm. Evol. 4:7786.
Swofford, D. L. 1998. PAUP* (developmental version). Sinauer, Sunderland, Mass.
Ursing, B. M., and U. Arnason. 1998. Analyses of mitochondrial genomes strongly support a hippopotamus-whale clade. Proc. R. Soc. Lond. B Biol. Sci. 265:22512255.[ISI][Medline]
Wilson, D. E., and D. M. Reeder. 1993. Mammal species of the world. A taxonomic and geographic reference. Smithsonian Institution Press, Washington, D.C., and London.
Yang, Z. 1994. Maximum likelihood phylogenetic estimation from DNA sequences with variable rates over sites: approximate methods. J. Mol. Evol. 39:306314.[ISI][Medline]