- Email: [email protected]
Mitochondrial phylogeny of hedgehogs and monophyly of Eulipotyphla
MOLECULAR PHYLOGENETICS AND EVOLUTION Molecular Phylogenetics and Evolution 28 (2003) 276–284 www.elsevier.com/locate/ympev
Mitochondrial phylogeny of hedgehogs and monophyly of Eulipotyphlaq Masato Nikaido,a Ying Cao,b Masashi Harada,c Norihiro Okada,a,* and Masami Hasegawab,d a
Department of Biological Sciences, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, Yokohama, Japan b Institute of Statistical Mathematics, Tokyo, Japan c Osaka City University Graduate School of Medicine, Osaka, Japan d Department of Biosystems Science, Graduate University for Advanced Studies, Hayama, Japan Received 4 October 2002; revised 19 January 2003
Abstract We sequenced the complete mitochondrial (mt) genomes of three insectivores: the long-eared hedgehog Hemiechinus auritus, the Japanese mole Mogera wogura, and the greater Japanese shrew-mole Urotrichus talpoides. These mtDNA data together with other previously sequenced mtDNAs were analyzed using a maximum likelihood method to infer their phylogenetic relationships among eutherians. Previous mitochondrial protein analyses used a simple model that did not consider site-heterogeneity, and Erinaceoidea (hedgehogs and moonrats) was placed at the basal eutherian position that is separated from Soricoidea (shrews) and Talpoidea (moles), suggesting the exclusion of the Erinaceoidea–Eulipotyphla tree. By including the new mtDNA sequences and introducing site-heterogeneity into the model, the Erinaceoidea–Eulipotyphla tree emerges as the best tree or as a tree with a log-likelihood score indistinguishable from that of the best tree. However, this conclusion depends on species sampling in Erinaceoidea, demonstrating the importance of both species sampling and use of an appropriate substitution model when inferring phylogenetic relationships. Ó 2003 Elsevier Science (USA). All rights reserved.
1. Introduction One of the most important discoveries in mammalian phylogeny was the grouping Afrotheria, which united endemic African mammals. This grouping was determined by successive molecular analyses since the 1980s (de Jong et al., 1981; Madsen et al., 2001; Murphy et al., 2001a,b; Springer et al., 1997; Stanhope et al., 1998a). Afrotheria includes two insectivorous mammals, Chrysochloridae (golden moles), and Tenrecidae (tenrecs), which have been traditionally thought to be closely related to moles, shrews, and hedgehogs. Thus, monophyly of the order Insectivora was excluded by the afrotherian status, and it was divided into the two q
The nucleotide sequences reported in this paper have been submitted to GenBank and have been assigned Accession Nos. AB099481, AB099482, and AB099483. * Corresponding author. Fax: +81-45-924-5835. E-mail address: [email protected] (N. Okada).
groups Eulipotyphla (moles, shrews, and hedgehogs) and Afrosoricida (golden moles and tenrecs) (Stanhope et al., 1998b; van Dijk et al., 2001; Waddell et al., 1999a). Although the paraphyly of insectivores has already been accepted, the grouping of Eulipotyphla has not been confirmed by the mitochondrial (mt) genome analyses. Krettek et al. (1995) determined the complete mt genome of the hedgehog, and suggested that hedgehogs be placed at the basal position of Eutheria. Later, by sequencing complete genome of mole, Mouchaty et al. (2000) suggested Eulipotyphla paraphyly; i.e., mole is closely related to Fereuungulata, while hedgehog represents the basal eutherian lineage. This conclusion is inconsistent with morphological and nuclear DNA analyses that group hedgehogs with moles and shrews (Butler, 1988; Douady et al., 2002; MacPhee and Novacek, 1993; Madsen et al., 2001; McKenna and Bell, 1997; Murphy et al., 2001a,b). There is a slight difference among the conclusions drawn from several
1055-7903/03/$ - see front matter Ó 2003 Elsevier Science (USA). All rights reserved. doi:10.1016/S1055-7903(03)00120-9
M. Nikaido et al. / Molecular Phylogenetics and Evolution 28 (2003) 276–284
morphological analyses and nuclear DNA analyses; i.e., McKenna and BellÕs (1997) morphological analysis suggested that moles and hedgehogs are more closely related to each other than either is to shrew, and ButlerÕs (1988) morphological analysis suggested closer relationship between shrews and moles than to hedgehogs, while Murphy et al.Õs (2001a,b) molecular analyses based mainly on nuclear DNA suggeted a hedgehog/ shrew clade excluding mole as an outgroup. However, all these studies support the monophyly of Eulipotyphla. Given that the analyses based on hedgehog mtDNA sequences result in a different conclusion than analyses of morphology and nuclear DNA, particular attention should be paid to the hedgehog mt-genome data in inferring the position of hedgehogs among Eutheria, and also inferring the phylogeny of eutherians themselves. Recently, Nikaido et al. (2001) used a complete mtgenome to examine the phylogenetic position of the hedgehog, taking into account site-heterogeneity. The results showed that the Eulipotyphla monophyly was not excluded even in mitochondrial phylogeny. The phylogenetic position of hedgehogs is still unresolved, representing apparent discrepancies between a nuclear DNA tree and a mtDNA tree. Here, we reexamined this issue by increasing the number of species of relevant taxa, including the long-eared hedgehog, the Japanese mole, the greater Japanese shrew-mole (all of which were sequenced in this work) the moonrat, the Formosan shrew (sequenced by Lin et al., 2002), and others. The phylogenetic relationships of broad mammalian taxa were also extensively analyzed by the maximum likelihood method, in which mt-genome data of hedgehogs and their relatives were included or excluded to test whether changes in species sampling and/ or substitution model affects the results.
2. Materials and methods 2.1. Genomic DNA samples Fresh liver samples were obtained from the Japanese mole, greater Japanese shrew-mole, and long-eared hedgehog and preserved in 99% ethanol. Total genomic DNA was isolated from each sample using phenol and chloroform extraction and ethanol precipitation (Blin and Stafford, 1976) and stored at 4 °C. 2.2. Sequencing We determined the complete mt-genome sequence of three insectivores using primer walking and/or shotgun sequencing as described in our previous studies (primer walking, Nikaido et al., 2000, 2001; shotgun sequencing, Murata et al., 2002). Shotgun sequencing is more rapid and cost effective, in that there is no need to design and
277
order lots of primers for ‘‘walking’’ unknown region of the mitochondrial genomes for each animal. 2.3. Sequence data Complete mitochondrial genome data used in this study were from the 42 species used in Nikaido et al. (2001); Artibeus jamaicensis (Jamaican fruit-eating bat; AF061340), Pteropus dasymallus (Ryukyu flying fox; AB042770), Rhinolophus pumilus (horseshoe bat; AB061526), Pipistrellus abramus (Japanese pipistrelle; AB061528), Talpa europaea (European mole; Y19192), Sorex unguiculatus (long-clawed shrew; AB061527), Lama pacos (alpaca; Y19184), Sus scrofa (pig; AJ002189), Bos taurus (cow; V00654), Ovis aries (sheep; AF010406), Hippopotamus amphibius (hippopotamus; AJ010957), Balaenoptera physalus (fin whale; X61145), Balaenoptera musculus (blue whale; X72204); Physeter macrocephalus (sperm whale; AJ277029), Phoca vitulina (harbor seal; X63726), Halichoerus grypus (grey seal; X72004), Canis familiaris (dog; U96639), Felis catus (cat; U20753), Equus caballus (horse; X79547), Equus asinus (donkey; X97337), Rhinoceros unicornis (Indian rhinoceros; X97336), Ceratotherium simum (white rhinoceros; Y07726), Dasypus novemcinctus (nine-banded armadillo; Y11832), Loxodonta africana (African elephant; AJ224821), Orycteropus afer (aardvark; Y18475), Oryctolagus cuniculus (rabbit; AJ001588), Glis glis (fat dormouse; AJ001562), Cavia procellus (guinea pig; AJ222767), Mus musculus (mouse; V00711), Rattus norvegicus (rat; X14848), Homo sapiens (human; D38112), Pan troglodytes (chimpanzee; D38113), Pan paniscus (bonobo; D38116), Gorilla gorilla (gorilla; D38114), Pongo pygmaeus p. (Bornean orangutan, D38115), Pongo pygmaeus abelii (Sumatran orangutan; X97707), Hylobates lar (common gibbon; X99256), Papio hamadryas (baboon; Y18001), Erinaceus europaeus (hedgehog; X88898), Didelphis virginiana (Virginia opossum; Z29573), Macropus robustus (wallaroo; Y10524), Ornithorhynchus anatinus (platypus; X83427), and the following 26 species: Mogera wogura (Japanese mole; AB099482), Urotrichus talpoides (greater Japanese shrew-mole; AB099483), Hemiechinus auritus (longeared hedgehog; AB099481), Echinosorex gymnura (greater moonrat; AF348079), Soriculus fumidus (Formosan shrew; AF348081), Pteropus scapulatus (little red flying fox; AF321050), Chalinolobus tuberculatus (longtailed bat; AF321051), Rhinolophus monoceros (Formosan lesser horseshoe bat; AF406806), Ursus maritimus (polar bear; AF303111), Macaca sylvanus (Barbary macaque; AJ309865), Cebus albifrons (white-fronted capuchin; AJ309866), Nycticebus coucang (slow loris; AJ309867), Tupaia belangeri (northern tree shrew; AF217811), Ochotona princeps (American pika; AF348080), Thryonomys swinderianus (greater cane rat; AJ301644), Volemys kikuchii (vole; AF348082), Dugong
278
M. Nikaido et al. / Molecular Phylogenetics and Evolution 28 (2003) 276–284
dugon (dugong; AJ421723), Echinops telfairi 1 (lesser hedgehog tenrec; AJ400734), Echinops telfairi 2 (lesser hedgehog tenrec; AB099484), Chrysochloris asiatica (cape golden mole; AB096866), Elephantulus sp. (elephant shrew; AB096867), Procavia capensis (cape hyrax; AB096865), Trichosurus vulpecula (silver-gray brushtail possum; AF357238), Isoodon macrourus (northern brown bandicoot; AF358864), Vombatus ursinus (common wombat; AJ304826), Tachyglossus aculeatus (Australian echidna; AJ303116). 2.4. Phylogenetic analysis The deduced amino acid sequences of 12 proteins encoded on the same strand of mtDNA were prepared and aligned as in Cao et al. (2000) and Nikaido et al. (2001), and 3392 amino acid sequences thus obtained were used in the analyses. The ProtML program in the MOLPHY package (ver. 2.3) (Adachi and Hasegawa, 1996) and the CodeML program in the PAML package (Yang, 1997) with the mtREV-F model (Adachi and Hasegawa, 1996) were used for the analyses. When using CodeML, the discrete C-distribution model (with eight categories) for site-heterogeneity was used. The Kishino and Hasegawa (1989) (KH) test, the weighted Shimodaira and Hasegawa (1999) (WSH) test, and the approximately unbiased (AU) test (Shimodaira, 2002) were executed with the CONSEL program (Shimodaira and Hasegawa, 2001).
3. Results and discussion Using a combination of primer walking and shotgun sequencing, we determined the complete mtDNA sequence of three insectivorous mammals, namely the Japanese mole (16,814 bp), the Japanese greater shrewmole (16,904 bp), and the long-eared hedgehog (17,283 bp). Due to heteroplasmy caused by differences in the number of repeated motifs in the control region, mtDNA sometimes varies in length, and therefore the number of nucleotides reported here may vary from that of other specimens. The content and arrangement of the mt-genes determined in this study are consistent with those of other mammals. Likewise, the initiation codons are usually ATG, but sometimes varies to ATA, ATT, or GTG, the termination codons are not always TAA but are sometimes incomplete, and there are overlapping regions and spacers between each gene. All of these phenomena had also been observed to the other mitochondrial genomes of mammals. The length of the control region of the long-eared hedgehog is relatively long (1813 bp) compared with those of other eutherians (1000 bp), although the control region of the European hedgehog is also long (1988 bp).
A neighbor-joining (NJ) tree of the 69 mt-protein sequences was first analyzed. Based on this tree, a ProtML tree was obtained with local rearrangements in which marsupials and monotremes were used as outgroups of the eutherian tree (Fig. 1). The quartet-puzzling method (Strimmer and von Haeseler, 1996) with mtREV + C model was also applied to the data, and a consistent tree was obtained, although several branchings remained unresolved (data not shown). Several portions of the tree, which remained ambiguous by the previous analyses (Cao et al., 2000; Murata et al., 2003; Nikaido et al., 2001, 2003) were left as multifurcations. In this tree, Erinaceoidea (hedgehog and moonrat) represents the first branching lineage in eutherians. Subsequently, Myomorpha branched before Sciuromorpha and Hystricomorpha diverged from the remaining eutherians, consistent with the rodent-polyphyly hypothesis proposed by mt-protein analyses (DÕErchia et al., 1996; Reyes et al., 2000). The basal position of Erinaceoidea in Fig. 1 is also consistent with the conclusion of Krettek et al. (1995) from mt-genome analysis. However, these placements are in sharp contradiction with both traditional morphology (McKenna and Bell, 1997; Novacek, 1992) and the analyses of nuclear sequences in that both of these analyses support the rodent monophyly and place the hedgehog in Eulipotyphla (core insectivores) together with Soricoidea (shrews) and Talpoidea (moles) (Madsen et al., 2001; Murphy et al., 2001a,b). When we apply a more realistic model of amino acid substitutions that accounts for site-heterogeneity with the C model, the mt-protein analysis supports the rodent-monophyly tree rather than rodent-polyphyly. The apparent polyphyly of rodents may be attributable to an insufficient tree topology search and to model misspecification (Cao et al., 2000; Nikaido et al., 2001; Sullivan and Swofford, 1997). The hedgehog mt-genome has a high evolutionary rate and a strong base composition bias, and hence an amino acid composition bias (Penny et al., 1999; Sullivan and Swofford, 1997; Waddell et al., 1999b). Thus, the phylogenetic placement of the hedgehog in the basal position of eutherians based on the mt-genome data must be viewed with caution. Indeed, Sullivan and Swofford (1997) demonstrated that the hedgehog can be placed in any of the eutherian lineages with only a minor difference in log-likelihood score (although their analysis was at the nucleotide level using only the first and second codon positions of mt-protein genes). More recently, using 12 mt-protein sequences and two mt-rRNA sequences, Nikaido et al. (2001) showed that the log-likelihood difference between the hedgehogEulipotyphla tree and the best hedgehog-basal tree is insignificant when site-heterogeneity is accounted for by the C-distribution model. However, ML analysis without considering site-heterogeneity strongly rejects the hedgehog-Eulipotyphla tree.
M. Nikaido et al. / Molecular Phylogenetics and Evolution 28 (2003) 276–284
279
Fig. 1. Tree-1 of the 69 mt-protein sequences obtained by the local rearrangement option of the ProtML program starting from a NJ tree without considering site-heterogeneity. The horizontal length of each branch is proportional to the number of amino acid substitutions estimated by the ProtML. Arrows show the successive generation of numbered trees using Tree-1 as the starting point.
280
M. Nikaido et al. / Molecular Phylogenetics and Evolution 28 (2003) 276–284
The v2 test was used to compare the amino acid composition of each sequence to the frequency distribution assumed in the maximum likelihood model (with TREE-PUZZLE; Strimmer and von Haeseler, 1996). Among the 69 concatenated amino acid sequences of mt-proteins, only data from three species fail to pass the v2 test at the 5% level (baboon, 3.3%; and the two hedgehog species, the long-eared hedgehog, 4.8%, and European hedgehog, 2.5%). These results suggest the presence of strong amino acid biases in these three species. In contrast with hedgehogs, the moonrat has a very similar amino acid composition to other eutherian species (P value of the test for moonrat was as high as 85.8%), although it is grouped morphologically along with hedgehogs within Erinaceoidea. Although Lin et al. (2002) noted an unusual C/T nucleotide ratio common to the hedgehog and moonrat but different from other eutherians, we observed unusual amino acid composition only for hedgehog but not for moonrat. To examine the impact of both the bias of amino acid composition and species sampling on the phylogenetic inference, we prepared three datasets: (1) 69 OTUs including two hedgehogs and the moonrat, (2) 68 OTUs including two hedgehogs but excluding the moonrat, and (3) 67 OTUs including the moonrat but excluding two hedgehogs. Tree-1 is represented in Fig. 1. Tree-2 was generated by combining the Sciuromorpha/Hystricomorpha clade with Myomorpha (resulting in rodent-monophyly) as indicated by arrow 2. Tree-3 was then generated from Tree-2 by moving Scandentia (tree shrew) to the Primates lineage (Euarchonta). Trees-4, -5, and -6 were obtained by successively moving the monophyletic Rodentia to Lagomorpha (Glires) in Tree-3, Erinaceoidea to the Soricoides/Talpoidea lineage forming the Eulipotyphla clade in Tree-4, and the Xenarthra/Afrotheria to the basal eutherian position in Tree-5, respectively. These successive movements were performed to fit the tree supported by the nuclear gene analysis (Murphy et al., 2001b), and Tree-6 presented in Fig. 2 fits Murphy et al.Õs tree except for the relationships among moles, shrews, and hedgehogs as discussed before and for the relationships among Afrotheria, Xenarthra, and the remaining eutherians. Trees-7 and -8 were generated from Tree-6 by moving Erinaceoidea to the Talpoidea and Soricoidea lineages, respectively. Trees-9 and -10 were generated from Tree-6 by moving Afrotheria and Xenarthra, respectively, to a more basal position as indicated by arrows. As discussed below, we examined whether the mt-protein data fit this improved tree topology, which is based on the tree of Murphy et al. (2001b). When site-heterogeneity was not taken into account (without C analysis), an Erinaceoidea-basal tree was favored and the Erinaceoidea–Eulipotyphla trees (Tree numbers > 5) were rejected by the KH and AU tests at the 5% level of significance (Table 1). However, de-
pending on the dataset, some Erinaceoidea–Eulipotyphla trees were not rejected by the WSH test, which is more conservative than the KH and AU tests. For the 68-OTU data, which excludes the moonrat, all the Erinaceoidea–Eulipotyphla trees were rejected by the WSH test with P < 0:015. However, for the 67-OTU data, which excludes two hedgehogs, P values for Trees6 and -10 were as large as 0.131 and 0.193, and these trees could not be rejected significantly at the 5% level. The 69-OTU data yielded an intermediate result, with P values of 0.053 and 0.105 for Trees-6 and -10, respectively. Given an Erinaceoidea–Eulipotyphla tree as suggested by the nuclear DNA analyses (Murphy et al., 2001b), this dependence of species sampling from Erinaceoidea demonstrates that the unusual amino acid compositions of the two hedgehogs contributed to the construction of an erroneous tree. However, this explanation alone is not sufficient since we obtained a putatively incorrect tree even when including the moonrat (which has a normal amino acid composition) and excluding the hedgehogs. However, the Erinaceoidea–Eulipotyphla trees could not be rejected by the WSH test in this case. The LogDet transformation (Lake, 1994; Lockhart et al., 1994) could not compensate for the above anomaly, as the hedgehog was still placed with the basal eutherians (Penny et al., 1999; Waddell et al., 1999b). Even though the new data for the moonrat, another Erinaceoidea species, does not show anomalous amino acid composition, this animal was placed at the basal position of eutherians with a conventional analysis (Lin et al., 2002). Therefore, a factor other than amino acid composition may underlie this unorthodox placement. The scenario changed significantly when site-heterogeneity was taken into account with the C model. Although the Erinaceoidea-basal trees could not be excluded, more reasonable trees of Erinaceoidea– Eulipotyphla were favored when only the moonrat was used from Erinaceoidea (Table 1c). Thus, the apparent strong support for the basal position of Erinaceoidea (Krettek et al., 1995) is attributable to model misspecification; that is, use of an oversimplified model that did not consider site-heterogeneity. Tree-4 was the ML tree when the 69-OTU data was used (Table 1a), but a more reasonable Tree-6 became the ML tree when the 67OTU data was used (Table 1c). In the nuclear sequence analysis, hedgehog was closer to shrew than to mole in the monophyletic Eulipotyphla (Murphy et al., 2001b). In our analysis with C, log-likelihood scores were almost indistinguishable among Trees-6, -7, and -8, and therefore the relationships within Eulipotyphla could not be resolved by the mt-protein data. Traditionally, morphologists have long regarded Xenarthra as a sister-group to all other eutherians called Epitheria (Gregory, 1910; McKenna and Bell, 1997; Novacek, 1992). Tree-9 represents this hypothesis, and
M. Nikaido et al. / Molecular Phylogenetics and Evolution 28 (2003) 276–284
281
Fig. 2. Tree-6 of the 69 mt-protein sequences. The horizontal length of each branch is proportional to the number of amino acid substitutions estimated by CodeML with the C model. Arrows show the successive generation of numbered trees using Tree-6 as the starting point. Nomenclature for higher order taxa are from Waddell et al. (1999a).
282
M. Nikaido et al. / Molecular Phylogenetics and Evolution 28 (2003) 276–284
Table 1 Differences in log-likelihood scores of alternative trees from the highest likelihood tree, and P values of the Kishino–Hasegawa (KH) and the weighted Shimodaira–Hasegawa (WSH) tests The log-likelihood scores of the highest likelihood trees are given in brackets. Tree topologies: 1 (((((Pri,(Sca,Lag)),((Scr,(Sor,Tal)),(Xen,Afr))),SciHys),Myo),Eri) 2 ((((Pri,(Sca,Lag)),((Scr,(Sor,Tal)),(Xen,Afr))),(SciHys,Myo)),Eri) 3 (((((Pri,Sca),Lag),((Scr,(Sor,Tal)),(Xen,Afr))),(SciHys,Myo)),Eri) 4 ((((Pri,Sca),(Lag,(SciHys,Myo))),((Scr,(Sor,Tal)),(Xen,Afr))),Eri) 5 (((Pri,Sca),(Lag,(SciHys,Myo))),((Scr,((Sor,Tal),Eri)),(Xen,Afr))) 6 ((((Pri,Sca),(Lag,(SciHys,Myo))),(Scr,((Sor,Tal),Eri))),(Xen,Afr)) 7 ((((Pri,Sca),(Lag,(SciHys,Myo))),(Scr,(Sor,(Tal,Eri)))),(Xen,Afr)) 8 ((((Pri,Sca),(Lag,(SciHys,Myo))),(Scr,((Sor,Eri),Tal))),(Xen,Afr)) 9 (((((Pri,Sca),(Lag,(SciHys,Myo))),(Scr,((Sor,Tal),Eri))),Xen),Afr) 10 (((((Pri,Sca),(Lag,(SciHys,Myo))),(Scr,((Sor,Tal),Eri))),Afr),Xen) Abbreviations. Pri, Primates; Sca, Scandentia; Scr, Scrotifera (Carnivora + Perissodactyla + Cetartiodactyla + Chiroptera); Xen, Xenarthra; Afr, Afrotheria; Myo, Myomorpha; SciHys, Sciuromorpha + Hystricomorpha; Eri, Erinaceoidea; Sor, Soricoidea; Tal, Talpoidea. Without C With C KH
WSH
(a) 69 OTUs including two hedgehogs and moon rat 1 h)117320.7i 0.999 0.995 2 )64.7 19.1 0.001 0.002 3 )72.6 25.4 0.003 0.011 4 )68.8 41.1 0.047 0.177 5 )145.7 57.1 0.005 0.036 6 )135.6 56.6 0.007 0.053 7 )167.8 58.9 0.002 0.013 8 )160.3 58.3 0.003 0.020 9 )147.5 57.4 0.005 0.031 10 )118.0 59.2 0.023 0.105
AU
KH
WSH
AU
0.965 0.000 0.003 0.084 0.004 0.019 0.003 0.001 0.001 0.036
)13.3 26.7 )35.7 22.5 )39.3 20.5 h)104619.2i )4.1 22.1 )1.0 22.3 )19.1 26.0 )14.8 25.9 )8.2 23.5 )3.4 23.7
0.306 0.056 0.030 0.508 0.432 0.492 0.233 0.283 0.367 0.449
0.575 0.108 0.111 0.883 0.647 0.978 0.182 0.360 0.464 0.820
0.338 0.001 0.002 0.620 0.299 0.750 0.071 0.184 0.143 0.512
(b) 68 OTUs including two hedgehogs and excluding moon rat 1 h)114711.0i 0.894 0.983 0.900 2 )57.8 18.8 0.002 0.005 0.000 3 )70.5 25.3 0.004 0.014 0.002 4 )51.4 41.1 0.106 0.346 0.121 5 )176.6 56.5 0.001 0.000 0.000 6 )167.3 56.0 0.001 0.001 0.002 7 )199.9 58.4 0.000 0.000 0.000 8 )188.0 57.7 0.001 0.001 0.000 9 )177.2 56.8 0.001 0.001 0.000 10 )148.9 58.2 0.005 0.015 0.001
)12.4 25.6 )33.5 21.2 )37.4 18.9 h)102377.7i )30.2 18.6 )26.7 18.8 )44.5 22.0 )45.3 21.7 )32.6 20.1 )28.0 20.2
0.318 0.056 0.025 0.682 0.054 0.079 0.022 0.018 0.053 0.084
0.575 0.139 0.098 0.965 0.244 0.392 0.094 0.074 0.236 0.319
0.360 0.008 0.002 0.813 0.069 0.233 0.016 0.012 0.064 0.174
(c) 67 OTUs including moon rat and excluding two hedgehogs 1 h)112383.5i 0.999 0.994 0.960 2 )57.9 17.7 0.001 0.003 0.000 3 )66.9 24.3 0.004 0.016 0.010 4 )62.4 39.5 0.059 0.210 0.099 5 )122.1 54.2 0.013 0.076 0.005 6 )110.2 54.0 0.022 0.131 0.036 7 )133.6 55.5 0.009 0.053 0.004 8 )108.3 55.9 0.026 0.120 0.034 9 )123.2 55.1 0.014 0.076 0.004 10 )95.5 57.2 0.050 0.193 0.083
)19.3 33.9 )40.7 29.8 )46.0 28.5 )7.9 19.6 )3.6 4.1 h)100109.9i )12.5 8.5 )2.8 10.3 )6.8 6.4 )2.1 7.3
0.290 0.087 0.054 0.339 0.188 0.620 0.076 0.390 0.145 0.380
0.551 0.088 0.094 0.730 0.622 0.996 0.286 0.782 0.511 0.825
0.309 0.007 0.000 0.450 0.328 0.763 0.068 0.511 0.185 0.531
its log-likelihood score is almost indistinguishable with those of Trees 6 and 10 from either of the three data sets when the C model was taken into account with respect to the relationships among Xenarthra, Afrotheria, and other eutherians. As indicated by the horizontal branch lengths in Fig. 2 (estimated numbers of substitutions with the C model) multiple substitutions in a site must be abundant in the mt-proteins to infer the order of deep
eutherian branches, and therefore these branches may be difficult to resolve. Nevertheless, the monophyly of all eutherians other than Xenarthra and Afrotheria seems quite likely from our analyses, which is consistent with nuclear DNA analysis (Murphy et al., 2001b). Our work demonstrates the importance of using an appropriate substitution model when inferring phylogenetic relationships, and the significance of species
M. Nikaido et al. / Molecular Phylogenetics and Evolution 28 (2003) 276–284
sampling with regard to avoiding species with unusual nucleotide or amino acid compositions.
Acknowledgments This work was supported by Research Grants from the Ministry of Education, Culture, Sports, Science and Technology of Japan (to N.O. and M.H.), from JSPS (to M.N., N.O., and M.H.), and from the Graduate University for Advanced Studies (Group Research to M.H.).
References Adachi, J., Hasegawa, M., 1996. ‘‘MOLPHY: Programs for Molecular Phylogenetics, ver.2.3,’’ Institute of Statistical Mathematics, Tokyo. Blin, N., Stafford, D.W., 1976. A general method for isolation of high molecular weight DNA from eukaryotes. Nucleic Acids Res. 3, 2303–2308. Butler, P.M., 1988. Phylogeny of the insectivores. In: The Phylogeny and Classification of the Tetrapods. In: Benton, M.J. (Ed.),. Mammals, vol. 2. Clarendon, Oxford, pp. 117–141. Cao, Y., Fujiwara, M., Nikaido, M., Okada, N., Hasegawa, M., 2000. Interordinal relationships and time-scale of eutherian evolution as inferred from mitochondrial genome data. Gene 259, 149–158. DÕErchia, A.M., Gissi, C., Pesole, G., Saccone, C., Arnason, U., 1996. The guinea-pig is not a rodent. Nature 381, 597–600. de Jong, W.W., Zweers, A., Goodman, M., 1981. Relationship of aardvark to elephants, hyraxes and sea cows from a-crystallin sequences. Nature 292, 538–540. Douady, C.J., Chatelier, P.I., Madsen, O., de Jong, W.W., Catzeflis, F., Springer, M.S., Stanhope, M.J., 2002. Molecular phylogenetic evidence confirming the Eulipotyphla concept and in support of hedgehogs as the sister group to shrews. Mol. Phylogenet. Evol. 25, 200–209. Gregory, W.K., 1910. The orders of mammals. Amer. Mus. Nat. Hist. Bull. 27, 1–524. Kishino, H., Hasegawa, M., 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, 170–179. Krettek, A., Gullberg, A., Arnason, U., 1995. Sequence analysis of the complete mitochondrial DNA molecule of the hedgehog, Erinaceus europaeus, and the phylogenetic position of the Lipotyphla. J. Mol. Evol. 41, 952–957. Lake, J.A., 1994. Reconstructing evolutionary trees from DNA and protein sequences: paralinear distances. Proc. Natl. Acad. Sci. USA 91, 1455–1459. Lin, Y.H., McLenachan, P.A., Gore, A.R., Phillips, M.J., Ota, R., Hendy, M.D., Penny, D., 2002. Four new mitochondrial genomes and the increased stability of evolutionary trees of mammals from improved taxon sampling. Mol. Biol. Evol. 19, 2060–2070. Lockhart, P.J., Steel, M.A., Hendy, M.D., Penny, D., 1994. Recovering evolutionary trees under a more realistic model of sequence evolution. Mol. Biol. Evol. 11, 605–612. MacPhee, R.D.E., Novacek, M.J., 1993. Definition and relationships of Lipotyphla. In: Szalay, F.S., Novacek, M.J., McKenna, M.C. (Eds.), Mammal Phylogeny: Placentals. Springer, New York, pp. 13–31. Madsen, O., Scally, M., Douady, C.J., Kao, D.J., DeBry, R.W., Adkins, R., Amrine, H.M., Stanhope, M.J., de Jong, W.W.,
283
Springer, M.S., 2001. Parallel adaptive radiations in two major clades of placental mammals. Nature 409, 610–614. McKenna, M.C., Bell, S.K., 1997. Classification of Mammals: Above the Species Level. Columbia University Press, New York. Mouchaty, S.K., Gullberg, A., Janke, A., Arnason, U., 2000. The phylogenetic position of the Talpidae within Eutheria based on analysis of complete mitochondrial sequences. Mol. Biol. Evol. 17, 60–67. Murata, Y., Nikaido, M., Sasaki, T., Fukumoto, Y., Hasegawa, M., Okada, N., 2003. Afrotherian phylogeny as inferred from complete mitochondrial genomes. Mol. Phylogenet. Evol. (in press). Murphy, W.J., Eizirik, E., Johnson, W.E., Zhang, Y.P., Ryder, O.A., OÕBrien, S.J., 2001a. Molecular phylogenetics and the origins of placental mammals. Nature 409, 614–618. Murphy, W.J., Eizirik, E., OÕBrien, S.J., Madsen, O., Scally, M., Douady, C.J., Teeling, E., Ryder, O.A., Stanhope, M.J., de Jong, W.W., Springer, M.S., 2001b. Resolution of the early placental mammal radiation using Bayesian phylogenetics. Science 294, 2348–2351. Nikaido, M., Cao, Y., Okada, N., Hasegawa, M., 2003. The phylogenetic relationships of insectivores with special reference to the lesser hedgehog tenrecs as inferred from the complete sequence of their mitochondrial genome. Genes Genet. Syst. 78, 107– 111. Nikaido, M., Harada, M., Cao, Y., Hasegawa, M., Okada, N., 2000. Monophyletic origin of the order Chiroptera and its phylogenetic position among Mammalia inferred from the complete mitochondrial DNA sequence of a Japanese megabat, Ryukyu flying fox (Pteropus dasymallus). J. Mol. Evol. 51, 318– 328. Nikaido, M., Kawai, K., Cao, Y., Harada, M., Tomita, S., Okada, N., Hasegawa, M., 2001. Maximum likelihood analysis of the complete mitochondrial genomes of eutherians and a reevaluation of the phylogeny of bats and insectivores. J. Mol. Evol. 53, 508–516. Novacek, M.J., 1992. Mammalian phylogeny: shaking the tree. Nature 356, 121–125. Penny, D., Hasegawa, M., Waddell, P.J., Hendy, M.D., 1999. Mammalian evolution: timing and implications from using the LogDeterminant transform for proteins of differing amino acid composition. Syst. Biol. 48, 76–93. Reyes, A., Gissi, C., Pesole, G., Catzeflis, F.M., Saccone, C., 2000. Where do rodents fit? Evidence from the complete mitochondrial genome of Sciurus vulgaris. Mol. Biol. Evol. 17, 979–983. Shimodaira, H., 2002. An approximately unbiased test of phylogenetic tree selection. Syst. Biol. 51, 492–508. Shimodaira, H., Hasegawa, M., 1999. Multiple comparisons of loglikelihoods with applications to phylogenetic inference. Mol. Biol. Evol. 16, 1114–1116. Shimodaira, H., Hasegawa, M., 2001. CONSEL: for assessing the confidence of phylogenetic tree selection. Bioinformatics 17, 1246– 1247. Springer, M.S., Cleven, G.C., Madsen, O., de Jong, W.W., Waddell, V.G., Amrine, H.M., Stanhope, M.J., 1997. Endemic African mammals shake the phylogenetic tree. Nature 388, 61–64. Stanhope, M.J., Madsen, O., Waddell, V.G., Cleven, G.C., de Jong, W.W., Springer, M.S., 1998a. Highly congruent molecular support for a diverse superordinal clade of endemic African mammals. Mol. Phylogenet. Evol. 9, 501–508. Stanhope, M.J., Waddell, V.G., Madsen, O., de Jong, W.W., Hedges, S.B., Cleven, G.C., Kao, D., Springer, M.S., 1998b. Molecular evidence for multiple origins of insectivora and for a new order of endemic African insectivore mammals. Proc. Natl. Acad. Sci. USA 95, 9967–9972. Strimmer, K., von Haeseler, A., 1996. Quartet puzzling: a quartet maximum-likelihood method for reconstructing tree topologies. Mol. Biol. Evol. 13, 964–969.
284
M. Nikaido et al. / Molecular Phylogenetics and Evolution 28 (2003) 276–284
Sullivan, J., Swofford, D.L., 1997. Are guinea pigs rodents? The importance of adequate models in molecular phylogenetics. J. Mammal. Evol. 4, 77–86. van Dijk, M.A.M., Madsen, O., Catzeflis, F., Stanhope, M.J., de Jong, W.W., Pagel, M., 2001. Protein sequence signatures support the African clade of mammals. Proc. Natl. Acad. Sci. USA 98, 188–193. Waddell, P.J., Okada, N., Hasegawa, M., 1999a. Towards resolving the interordinal relationships of placental mammals. Syst. Biol. 48, 1–5.
Waddell, P.J., Cao, Y., Hauf, J., Hasegawa, M., 1999b. Using novel phylogenetic methods to evaluate mammalian mtDNA, including amino acid-invariant sites-LogDet plus site stripping, to detect internal conflicts in the data, with special reference to the positions of hedgehog, armadillo, and elephant. Syst. Biol. 48, 31–53. Yang, Z., 1997. PAML: a program package for phylogenetic analysis by maximum likelihood. CABIOS 13, 555–556.
Mitochondrial phylogeny of hedgehogs and monophyly of Eulipotyphlaq Masato Nikaido,a Ying Cao,b Masashi Harada,c Norihiro Okada,a,* and Masami Hasegawab,d a
Department of Biological Sciences, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, Yokohama, Japan b Institute of Statistical Mathematics, Tokyo, Japan c Osaka City University Graduate School of Medicine, Osaka, Japan d Department of Biosystems Science, Graduate University for Advanced Studies, Hayama, Japan Received 4 October 2002; revised 19 January 2003
Abstract We sequenced the complete mitochondrial (mt) genomes of three insectivores: the long-eared hedgehog Hemiechinus auritus, the Japanese mole Mogera wogura, and the greater Japanese shrew-mole Urotrichus talpoides. These mtDNA data together with other previously sequenced mtDNAs were analyzed using a maximum likelihood method to infer their phylogenetic relationships among eutherians. Previous mitochondrial protein analyses used a simple model that did not consider site-heterogeneity, and Erinaceoidea (hedgehogs and moonrats) was placed at the basal eutherian position that is separated from Soricoidea (shrews) and Talpoidea (moles), suggesting the exclusion of the Erinaceoidea–Eulipotyphla tree. By including the new mtDNA sequences and introducing site-heterogeneity into the model, the Erinaceoidea–Eulipotyphla tree emerges as the best tree or as a tree with a log-likelihood score indistinguishable from that of the best tree. However, this conclusion depends on species sampling in Erinaceoidea, demonstrating the importance of both species sampling and use of an appropriate substitution model when inferring phylogenetic relationships. Ó 2003 Elsevier Science (USA). All rights reserved.
1. Introduction One of the most important discoveries in mammalian phylogeny was the grouping Afrotheria, which united endemic African mammals. This grouping was determined by successive molecular analyses since the 1980s (de Jong et al., 1981; Madsen et al., 2001; Murphy et al., 2001a,b; Springer et al., 1997; Stanhope et al., 1998a). Afrotheria includes two insectivorous mammals, Chrysochloridae (golden moles), and Tenrecidae (tenrecs), which have been traditionally thought to be closely related to moles, shrews, and hedgehogs. Thus, monophyly of the order Insectivora was excluded by the afrotherian status, and it was divided into the two q
The nucleotide sequences reported in this paper have been submitted to GenBank and have been assigned Accession Nos. AB099481, AB099482, and AB099483. * Corresponding author. Fax: +81-45-924-5835. E-mail address: [email protected] (N. Okada).
groups Eulipotyphla (moles, shrews, and hedgehogs) and Afrosoricida (golden moles and tenrecs) (Stanhope et al., 1998b; van Dijk et al., 2001; Waddell et al., 1999a). Although the paraphyly of insectivores has already been accepted, the grouping of Eulipotyphla has not been confirmed by the mitochondrial (mt) genome analyses. Krettek et al. (1995) determined the complete mt genome of the hedgehog, and suggested that hedgehogs be placed at the basal position of Eutheria. Later, by sequencing complete genome of mole, Mouchaty et al. (2000) suggested Eulipotyphla paraphyly; i.e., mole is closely related to Fereuungulata, while hedgehog represents the basal eutherian lineage. This conclusion is inconsistent with morphological and nuclear DNA analyses that group hedgehogs with moles and shrews (Butler, 1988; Douady et al., 2002; MacPhee and Novacek, 1993; Madsen et al., 2001; McKenna and Bell, 1997; Murphy et al., 2001a,b). There is a slight difference among the conclusions drawn from several
1055-7903/03/$ - see front matter Ó 2003 Elsevier Science (USA). All rights reserved. doi:10.1016/S1055-7903(03)00120-9
M. Nikaido et al. / Molecular Phylogenetics and Evolution 28 (2003) 276–284
morphological analyses and nuclear DNA analyses; i.e., McKenna and BellÕs (1997) morphological analysis suggested that moles and hedgehogs are more closely related to each other than either is to shrew, and ButlerÕs (1988) morphological analysis suggested closer relationship between shrews and moles than to hedgehogs, while Murphy et al.Õs (2001a,b) molecular analyses based mainly on nuclear DNA suggeted a hedgehog/ shrew clade excluding mole as an outgroup. However, all these studies support the monophyly of Eulipotyphla. Given that the analyses based on hedgehog mtDNA sequences result in a different conclusion than analyses of morphology and nuclear DNA, particular attention should be paid to the hedgehog mt-genome data in inferring the position of hedgehogs among Eutheria, and also inferring the phylogeny of eutherians themselves. Recently, Nikaido et al. (2001) used a complete mtgenome to examine the phylogenetic position of the hedgehog, taking into account site-heterogeneity. The results showed that the Eulipotyphla monophyly was not excluded even in mitochondrial phylogeny. The phylogenetic position of hedgehogs is still unresolved, representing apparent discrepancies between a nuclear DNA tree and a mtDNA tree. Here, we reexamined this issue by increasing the number of species of relevant taxa, including the long-eared hedgehog, the Japanese mole, the greater Japanese shrew-mole (all of which were sequenced in this work) the moonrat, the Formosan shrew (sequenced by Lin et al., 2002), and others. The phylogenetic relationships of broad mammalian taxa were also extensively analyzed by the maximum likelihood method, in which mt-genome data of hedgehogs and their relatives were included or excluded to test whether changes in species sampling and/ or substitution model affects the results.
2. Materials and methods 2.1. Genomic DNA samples Fresh liver samples were obtained from the Japanese mole, greater Japanese shrew-mole, and long-eared hedgehog and preserved in 99% ethanol. Total genomic DNA was isolated from each sample using phenol and chloroform extraction and ethanol precipitation (Blin and Stafford, 1976) and stored at 4 °C. 2.2. Sequencing We determined the complete mt-genome sequence of three insectivores using primer walking and/or shotgun sequencing as described in our previous studies (primer walking, Nikaido et al., 2000, 2001; shotgun sequencing, Murata et al., 2002). Shotgun sequencing is more rapid and cost effective, in that there is no need to design and
277
order lots of primers for ‘‘walking’’ unknown region of the mitochondrial genomes for each animal. 2.3. Sequence data Complete mitochondrial genome data used in this study were from the 42 species used in Nikaido et al. (2001); Artibeus jamaicensis (Jamaican fruit-eating bat; AF061340), Pteropus dasymallus (Ryukyu flying fox; AB042770), Rhinolophus pumilus (horseshoe bat; AB061526), Pipistrellus abramus (Japanese pipistrelle; AB061528), Talpa europaea (European mole; Y19192), Sorex unguiculatus (long-clawed shrew; AB061527), Lama pacos (alpaca; Y19184), Sus scrofa (pig; AJ002189), Bos taurus (cow; V00654), Ovis aries (sheep; AF010406), Hippopotamus amphibius (hippopotamus; AJ010957), Balaenoptera physalus (fin whale; X61145), Balaenoptera musculus (blue whale; X72204); Physeter macrocephalus (sperm whale; AJ277029), Phoca vitulina (harbor seal; X63726), Halichoerus grypus (grey seal; X72004), Canis familiaris (dog; U96639), Felis catus (cat; U20753), Equus caballus (horse; X79547), Equus asinus (donkey; X97337), Rhinoceros unicornis (Indian rhinoceros; X97336), Ceratotherium simum (white rhinoceros; Y07726), Dasypus novemcinctus (nine-banded armadillo; Y11832), Loxodonta africana (African elephant; AJ224821), Orycteropus afer (aardvark; Y18475), Oryctolagus cuniculus (rabbit; AJ001588), Glis glis (fat dormouse; AJ001562), Cavia procellus (guinea pig; AJ222767), Mus musculus (mouse; V00711), Rattus norvegicus (rat; X14848), Homo sapiens (human; D38112), Pan troglodytes (chimpanzee; D38113), Pan paniscus (bonobo; D38116), Gorilla gorilla (gorilla; D38114), Pongo pygmaeus p. (Bornean orangutan, D38115), Pongo pygmaeus abelii (Sumatran orangutan; X97707), Hylobates lar (common gibbon; X99256), Papio hamadryas (baboon; Y18001), Erinaceus europaeus (hedgehog; X88898), Didelphis virginiana (Virginia opossum; Z29573), Macropus robustus (wallaroo; Y10524), Ornithorhynchus anatinus (platypus; X83427), and the following 26 species: Mogera wogura (Japanese mole; AB099482), Urotrichus talpoides (greater Japanese shrew-mole; AB099483), Hemiechinus auritus (longeared hedgehog; AB099481), Echinosorex gymnura (greater moonrat; AF348079), Soriculus fumidus (Formosan shrew; AF348081), Pteropus scapulatus (little red flying fox; AF321050), Chalinolobus tuberculatus (longtailed bat; AF321051), Rhinolophus monoceros (Formosan lesser horseshoe bat; AF406806), Ursus maritimus (polar bear; AF303111), Macaca sylvanus (Barbary macaque; AJ309865), Cebus albifrons (white-fronted capuchin; AJ309866), Nycticebus coucang (slow loris; AJ309867), Tupaia belangeri (northern tree shrew; AF217811), Ochotona princeps (American pika; AF348080), Thryonomys swinderianus (greater cane rat; AJ301644), Volemys kikuchii (vole; AF348082), Dugong
278
M. Nikaido et al. / Molecular Phylogenetics and Evolution 28 (2003) 276–284
dugon (dugong; AJ421723), Echinops telfairi 1 (lesser hedgehog tenrec; AJ400734), Echinops telfairi 2 (lesser hedgehog tenrec; AB099484), Chrysochloris asiatica (cape golden mole; AB096866), Elephantulus sp. (elephant shrew; AB096867), Procavia capensis (cape hyrax; AB096865), Trichosurus vulpecula (silver-gray brushtail possum; AF357238), Isoodon macrourus (northern brown bandicoot; AF358864), Vombatus ursinus (common wombat; AJ304826), Tachyglossus aculeatus (Australian echidna; AJ303116). 2.4. Phylogenetic analysis The deduced amino acid sequences of 12 proteins encoded on the same strand of mtDNA were prepared and aligned as in Cao et al. (2000) and Nikaido et al. (2001), and 3392 amino acid sequences thus obtained were used in the analyses. The ProtML program in the MOLPHY package (ver. 2.3) (Adachi and Hasegawa, 1996) and the CodeML program in the PAML package (Yang, 1997) with the mtREV-F model (Adachi and Hasegawa, 1996) were used for the analyses. When using CodeML, the discrete C-distribution model (with eight categories) for site-heterogeneity was used. The Kishino and Hasegawa (1989) (KH) test, the weighted Shimodaira and Hasegawa (1999) (WSH) test, and the approximately unbiased (AU) test (Shimodaira, 2002) were executed with the CONSEL program (Shimodaira and Hasegawa, 2001).
3. Results and discussion Using a combination of primer walking and shotgun sequencing, we determined the complete mtDNA sequence of three insectivorous mammals, namely the Japanese mole (16,814 bp), the Japanese greater shrewmole (16,904 bp), and the long-eared hedgehog (17,283 bp). Due to heteroplasmy caused by differences in the number of repeated motifs in the control region, mtDNA sometimes varies in length, and therefore the number of nucleotides reported here may vary from that of other specimens. The content and arrangement of the mt-genes determined in this study are consistent with those of other mammals. Likewise, the initiation codons are usually ATG, but sometimes varies to ATA, ATT, or GTG, the termination codons are not always TAA but are sometimes incomplete, and there are overlapping regions and spacers between each gene. All of these phenomena had also been observed to the other mitochondrial genomes of mammals. The length of the control region of the long-eared hedgehog is relatively long (1813 bp) compared with those of other eutherians (1000 bp), although the control region of the European hedgehog is also long (1988 bp).
A neighbor-joining (NJ) tree of the 69 mt-protein sequences was first analyzed. Based on this tree, a ProtML tree was obtained with local rearrangements in which marsupials and monotremes were used as outgroups of the eutherian tree (Fig. 1). The quartet-puzzling method (Strimmer and von Haeseler, 1996) with mtREV + C model was also applied to the data, and a consistent tree was obtained, although several branchings remained unresolved (data not shown). Several portions of the tree, which remained ambiguous by the previous analyses (Cao et al., 2000; Murata et al., 2003; Nikaido et al., 2001, 2003) were left as multifurcations. In this tree, Erinaceoidea (hedgehog and moonrat) represents the first branching lineage in eutherians. Subsequently, Myomorpha branched before Sciuromorpha and Hystricomorpha diverged from the remaining eutherians, consistent with the rodent-polyphyly hypothesis proposed by mt-protein analyses (DÕErchia et al., 1996; Reyes et al., 2000). The basal position of Erinaceoidea in Fig. 1 is also consistent with the conclusion of Krettek et al. (1995) from mt-genome analysis. However, these placements are in sharp contradiction with both traditional morphology (McKenna and Bell, 1997; Novacek, 1992) and the analyses of nuclear sequences in that both of these analyses support the rodent monophyly and place the hedgehog in Eulipotyphla (core insectivores) together with Soricoidea (shrews) and Talpoidea (moles) (Madsen et al., 2001; Murphy et al., 2001a,b). When we apply a more realistic model of amino acid substitutions that accounts for site-heterogeneity with the C model, the mt-protein analysis supports the rodent-monophyly tree rather than rodent-polyphyly. The apparent polyphyly of rodents may be attributable to an insufficient tree topology search and to model misspecification (Cao et al., 2000; Nikaido et al., 2001; Sullivan and Swofford, 1997). The hedgehog mt-genome has a high evolutionary rate and a strong base composition bias, and hence an amino acid composition bias (Penny et al., 1999; Sullivan and Swofford, 1997; Waddell et al., 1999b). Thus, the phylogenetic placement of the hedgehog in the basal position of eutherians based on the mt-genome data must be viewed with caution. Indeed, Sullivan and Swofford (1997) demonstrated that the hedgehog can be placed in any of the eutherian lineages with only a minor difference in log-likelihood score (although their analysis was at the nucleotide level using only the first and second codon positions of mt-protein genes). More recently, using 12 mt-protein sequences and two mt-rRNA sequences, Nikaido et al. (2001) showed that the log-likelihood difference between the hedgehogEulipotyphla tree and the best hedgehog-basal tree is insignificant when site-heterogeneity is accounted for by the C-distribution model. However, ML analysis without considering site-heterogeneity strongly rejects the hedgehog-Eulipotyphla tree.
M. Nikaido et al. / Molecular Phylogenetics and Evolution 28 (2003) 276–284
279
Fig. 1. Tree-1 of the 69 mt-protein sequences obtained by the local rearrangement option of the ProtML program starting from a NJ tree without considering site-heterogeneity. The horizontal length of each branch is proportional to the number of amino acid substitutions estimated by the ProtML. Arrows show the successive generation of numbered trees using Tree-1 as the starting point.
280
M. Nikaido et al. / Molecular Phylogenetics and Evolution 28 (2003) 276–284
The v2 test was used to compare the amino acid composition of each sequence to the frequency distribution assumed in the maximum likelihood model (with TREE-PUZZLE; Strimmer and von Haeseler, 1996). Among the 69 concatenated amino acid sequences of mt-proteins, only data from three species fail to pass the v2 test at the 5% level (baboon, 3.3%; and the two hedgehog species, the long-eared hedgehog, 4.8%, and European hedgehog, 2.5%). These results suggest the presence of strong amino acid biases in these three species. In contrast with hedgehogs, the moonrat has a very similar amino acid composition to other eutherian species (P value of the test for moonrat was as high as 85.8%), although it is grouped morphologically along with hedgehogs within Erinaceoidea. Although Lin et al. (2002) noted an unusual C/T nucleotide ratio common to the hedgehog and moonrat but different from other eutherians, we observed unusual amino acid composition only for hedgehog but not for moonrat. To examine the impact of both the bias of amino acid composition and species sampling on the phylogenetic inference, we prepared three datasets: (1) 69 OTUs including two hedgehogs and the moonrat, (2) 68 OTUs including two hedgehogs but excluding the moonrat, and (3) 67 OTUs including the moonrat but excluding two hedgehogs. Tree-1 is represented in Fig. 1. Tree-2 was generated by combining the Sciuromorpha/Hystricomorpha clade with Myomorpha (resulting in rodent-monophyly) as indicated by arrow 2. Tree-3 was then generated from Tree-2 by moving Scandentia (tree shrew) to the Primates lineage (Euarchonta). Trees-4, -5, and -6 were obtained by successively moving the monophyletic Rodentia to Lagomorpha (Glires) in Tree-3, Erinaceoidea to the Soricoides/Talpoidea lineage forming the Eulipotyphla clade in Tree-4, and the Xenarthra/Afrotheria to the basal eutherian position in Tree-5, respectively. These successive movements were performed to fit the tree supported by the nuclear gene analysis (Murphy et al., 2001b), and Tree-6 presented in Fig. 2 fits Murphy et al.Õs tree except for the relationships among moles, shrews, and hedgehogs as discussed before and for the relationships among Afrotheria, Xenarthra, and the remaining eutherians. Trees-7 and -8 were generated from Tree-6 by moving Erinaceoidea to the Talpoidea and Soricoidea lineages, respectively. Trees-9 and -10 were generated from Tree-6 by moving Afrotheria and Xenarthra, respectively, to a more basal position as indicated by arrows. As discussed below, we examined whether the mt-protein data fit this improved tree topology, which is based on the tree of Murphy et al. (2001b). When site-heterogeneity was not taken into account (without C analysis), an Erinaceoidea-basal tree was favored and the Erinaceoidea–Eulipotyphla trees (Tree numbers > 5) were rejected by the KH and AU tests at the 5% level of significance (Table 1). However, de-
pending on the dataset, some Erinaceoidea–Eulipotyphla trees were not rejected by the WSH test, which is more conservative than the KH and AU tests. For the 68-OTU data, which excludes the moonrat, all the Erinaceoidea–Eulipotyphla trees were rejected by the WSH test with P < 0:015. However, for the 67-OTU data, which excludes two hedgehogs, P values for Trees6 and -10 were as large as 0.131 and 0.193, and these trees could not be rejected significantly at the 5% level. The 69-OTU data yielded an intermediate result, with P values of 0.053 and 0.105 for Trees-6 and -10, respectively. Given an Erinaceoidea–Eulipotyphla tree as suggested by the nuclear DNA analyses (Murphy et al., 2001b), this dependence of species sampling from Erinaceoidea demonstrates that the unusual amino acid compositions of the two hedgehogs contributed to the construction of an erroneous tree. However, this explanation alone is not sufficient since we obtained a putatively incorrect tree even when including the moonrat (which has a normal amino acid composition) and excluding the hedgehogs. However, the Erinaceoidea–Eulipotyphla trees could not be rejected by the WSH test in this case. The LogDet transformation (Lake, 1994; Lockhart et al., 1994) could not compensate for the above anomaly, as the hedgehog was still placed with the basal eutherians (Penny et al., 1999; Waddell et al., 1999b). Even though the new data for the moonrat, another Erinaceoidea species, does not show anomalous amino acid composition, this animal was placed at the basal position of eutherians with a conventional analysis (Lin et al., 2002). Therefore, a factor other than amino acid composition may underlie this unorthodox placement. The scenario changed significantly when site-heterogeneity was taken into account with the C model. Although the Erinaceoidea-basal trees could not be excluded, more reasonable trees of Erinaceoidea– Eulipotyphla were favored when only the moonrat was used from Erinaceoidea (Table 1c). Thus, the apparent strong support for the basal position of Erinaceoidea (Krettek et al., 1995) is attributable to model misspecification; that is, use of an oversimplified model that did not consider site-heterogeneity. Tree-4 was the ML tree when the 69-OTU data was used (Table 1a), but a more reasonable Tree-6 became the ML tree when the 67OTU data was used (Table 1c). In the nuclear sequence analysis, hedgehog was closer to shrew than to mole in the monophyletic Eulipotyphla (Murphy et al., 2001b). In our analysis with C, log-likelihood scores were almost indistinguishable among Trees-6, -7, and -8, and therefore the relationships within Eulipotyphla could not be resolved by the mt-protein data. Traditionally, morphologists have long regarded Xenarthra as a sister-group to all other eutherians called Epitheria (Gregory, 1910; McKenna and Bell, 1997; Novacek, 1992). Tree-9 represents this hypothesis, and
M. Nikaido et al. / Molecular Phylogenetics and Evolution 28 (2003) 276–284
281
Fig. 2. Tree-6 of the 69 mt-protein sequences. The horizontal length of each branch is proportional to the number of amino acid substitutions estimated by CodeML with the C model. Arrows show the successive generation of numbered trees using Tree-6 as the starting point. Nomenclature for higher order taxa are from Waddell et al. (1999a).
282
M. Nikaido et al. / Molecular Phylogenetics and Evolution 28 (2003) 276–284
Table 1 Differences in log-likelihood scores of alternative trees from the highest likelihood tree, and P values of the Kishino–Hasegawa (KH) and the weighted Shimodaira–Hasegawa (WSH) tests The log-likelihood scores of the highest likelihood trees are given in brackets. Tree topologies: 1 (((((Pri,(Sca,Lag)),((Scr,(Sor,Tal)),(Xen,Afr))),SciHys),Myo),Eri) 2 ((((Pri,(Sca,Lag)),((Scr,(Sor,Tal)),(Xen,Afr))),(SciHys,Myo)),Eri) 3 (((((Pri,Sca),Lag),((Scr,(Sor,Tal)),(Xen,Afr))),(SciHys,Myo)),Eri) 4 ((((Pri,Sca),(Lag,(SciHys,Myo))),((Scr,(Sor,Tal)),(Xen,Afr))),Eri) 5 (((Pri,Sca),(Lag,(SciHys,Myo))),((Scr,((Sor,Tal),Eri)),(Xen,Afr))) 6 ((((Pri,Sca),(Lag,(SciHys,Myo))),(Scr,((Sor,Tal),Eri))),(Xen,Afr)) 7 ((((Pri,Sca),(Lag,(SciHys,Myo))),(Scr,(Sor,(Tal,Eri)))),(Xen,Afr)) 8 ((((Pri,Sca),(Lag,(SciHys,Myo))),(Scr,((Sor,Eri),Tal))),(Xen,Afr)) 9 (((((Pri,Sca),(Lag,(SciHys,Myo))),(Scr,((Sor,Tal),Eri))),Xen),Afr) 10 (((((Pri,Sca),(Lag,(SciHys,Myo))),(Scr,((Sor,Tal),Eri))),Afr),Xen) Abbreviations. Pri, Primates; Sca, Scandentia; Scr, Scrotifera (Carnivora + Perissodactyla + Cetartiodactyla + Chiroptera); Xen, Xenarthra; Afr, Afrotheria; Myo, Myomorpha; SciHys, Sciuromorpha + Hystricomorpha; Eri, Erinaceoidea; Sor, Soricoidea; Tal, Talpoidea. Without C With C KH
WSH
(a) 69 OTUs including two hedgehogs and moon rat 1 h)117320.7i 0.999 0.995 2 )64.7 19.1 0.001 0.002 3 )72.6 25.4 0.003 0.011 4 )68.8 41.1 0.047 0.177 5 )145.7 57.1 0.005 0.036 6 )135.6 56.6 0.007 0.053 7 )167.8 58.9 0.002 0.013 8 )160.3 58.3 0.003 0.020 9 )147.5 57.4 0.005 0.031 10 )118.0 59.2 0.023 0.105
AU
KH
WSH
AU
0.965 0.000 0.003 0.084 0.004 0.019 0.003 0.001 0.001 0.036
)13.3 26.7 )35.7 22.5 )39.3 20.5 h)104619.2i )4.1 22.1 )1.0 22.3 )19.1 26.0 )14.8 25.9 )8.2 23.5 )3.4 23.7
0.306 0.056 0.030 0.508 0.432 0.492 0.233 0.283 0.367 0.449
0.575 0.108 0.111 0.883 0.647 0.978 0.182 0.360 0.464 0.820
0.338 0.001 0.002 0.620 0.299 0.750 0.071 0.184 0.143 0.512
(b) 68 OTUs including two hedgehogs and excluding moon rat 1 h)114711.0i 0.894 0.983 0.900 2 )57.8 18.8 0.002 0.005 0.000 3 )70.5 25.3 0.004 0.014 0.002 4 )51.4 41.1 0.106 0.346 0.121 5 )176.6 56.5 0.001 0.000 0.000 6 )167.3 56.0 0.001 0.001 0.002 7 )199.9 58.4 0.000 0.000 0.000 8 )188.0 57.7 0.001 0.001 0.000 9 )177.2 56.8 0.001 0.001 0.000 10 )148.9 58.2 0.005 0.015 0.001
)12.4 25.6 )33.5 21.2 )37.4 18.9 h)102377.7i )30.2 18.6 )26.7 18.8 )44.5 22.0 )45.3 21.7 )32.6 20.1 )28.0 20.2
0.318 0.056 0.025 0.682 0.054 0.079 0.022 0.018 0.053 0.084
0.575 0.139 0.098 0.965 0.244 0.392 0.094 0.074 0.236 0.319
0.360 0.008 0.002 0.813 0.069 0.233 0.016 0.012 0.064 0.174
(c) 67 OTUs including moon rat and excluding two hedgehogs 1 h)112383.5i 0.999 0.994 0.960 2 )57.9 17.7 0.001 0.003 0.000 3 )66.9 24.3 0.004 0.016 0.010 4 )62.4 39.5 0.059 0.210 0.099 5 )122.1 54.2 0.013 0.076 0.005 6 )110.2 54.0 0.022 0.131 0.036 7 )133.6 55.5 0.009 0.053 0.004 8 )108.3 55.9 0.026 0.120 0.034 9 )123.2 55.1 0.014 0.076 0.004 10 )95.5 57.2 0.050 0.193 0.083
)19.3 33.9 )40.7 29.8 )46.0 28.5 )7.9 19.6 )3.6 4.1 h)100109.9i )12.5 8.5 )2.8 10.3 )6.8 6.4 )2.1 7.3
0.290 0.087 0.054 0.339 0.188 0.620 0.076 0.390 0.145 0.380
0.551 0.088 0.094 0.730 0.622 0.996 0.286 0.782 0.511 0.825
0.309 0.007 0.000 0.450 0.328 0.763 0.068 0.511 0.185 0.531
its log-likelihood score is almost indistinguishable with those of Trees 6 and 10 from either of the three data sets when the C model was taken into account with respect to the relationships among Xenarthra, Afrotheria, and other eutherians. As indicated by the horizontal branch lengths in Fig. 2 (estimated numbers of substitutions with the C model) multiple substitutions in a site must be abundant in the mt-proteins to infer the order of deep
eutherian branches, and therefore these branches may be difficult to resolve. Nevertheless, the monophyly of all eutherians other than Xenarthra and Afrotheria seems quite likely from our analyses, which is consistent with nuclear DNA analysis (Murphy et al., 2001b). Our work demonstrates the importance of using an appropriate substitution model when inferring phylogenetic relationships, and the significance of species
M. Nikaido et al. / Molecular Phylogenetics and Evolution 28 (2003) 276–284
sampling with regard to avoiding species with unusual nucleotide or amino acid compositions.
Acknowledgments This work was supported by Research Grants from the Ministry of Education, Culture, Sports, Science and Technology of Japan (to N.O. and M.H.), from JSPS (to M.N., N.O., and M.H.), and from the Graduate University for Advanced Studies (Group Research to M.H.).
References Adachi, J., Hasegawa, M., 1996. ‘‘MOLPHY: Programs for Molecular Phylogenetics, ver.2.3,’’ Institute of Statistical Mathematics, Tokyo. Blin, N., Stafford, D.W., 1976. A general method for isolation of high molecular weight DNA from eukaryotes. Nucleic Acids Res. 3, 2303–2308. Butler, P.M., 1988. Phylogeny of the insectivores. In: The Phylogeny and Classification of the Tetrapods. In: Benton, M.J. (Ed.),. Mammals, vol. 2. Clarendon, Oxford, pp. 117–141. Cao, Y., Fujiwara, M., Nikaido, M., Okada, N., Hasegawa, M., 2000. Interordinal relationships and time-scale of eutherian evolution as inferred from mitochondrial genome data. Gene 259, 149–158. DÕErchia, A.M., Gissi, C., Pesole, G., Saccone, C., Arnason, U., 1996. The guinea-pig is not a rodent. Nature 381, 597–600. de Jong, W.W., Zweers, A., Goodman, M., 1981. Relationship of aardvark to elephants, hyraxes and sea cows from a-crystallin sequences. Nature 292, 538–540. Douady, C.J., Chatelier, P.I., Madsen, O., de Jong, W.W., Catzeflis, F., Springer, M.S., Stanhope, M.J., 2002. Molecular phylogenetic evidence confirming the Eulipotyphla concept and in support of hedgehogs as the sister group to shrews. Mol. Phylogenet. Evol. 25, 200–209. Gregory, W.K., 1910. The orders of mammals. Amer. Mus. Nat. Hist. Bull. 27, 1–524. Kishino, H., Hasegawa, M., 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, 170–179. Krettek, A., Gullberg, A., Arnason, U., 1995. Sequence analysis of the complete mitochondrial DNA molecule of the hedgehog, Erinaceus europaeus, and the phylogenetic position of the Lipotyphla. J. Mol. Evol. 41, 952–957. Lake, J.A., 1994. Reconstructing evolutionary trees from DNA and protein sequences: paralinear distances. Proc. Natl. Acad. Sci. USA 91, 1455–1459. Lin, Y.H., McLenachan, P.A., Gore, A.R., Phillips, M.J., Ota, R., Hendy, M.D., Penny, D., 2002. Four new mitochondrial genomes and the increased stability of evolutionary trees of mammals from improved taxon sampling. Mol. Biol. Evol. 19, 2060–2070. Lockhart, P.J., Steel, M.A., Hendy, M.D., Penny, D., 1994. Recovering evolutionary trees under a more realistic model of sequence evolution. Mol. Biol. Evol. 11, 605–612. MacPhee, R.D.E., Novacek, M.J., 1993. Definition and relationships of Lipotyphla. In: Szalay, F.S., Novacek, M.J., McKenna, M.C. (Eds.), Mammal Phylogeny: Placentals. Springer, New York, pp. 13–31. Madsen, O., Scally, M., Douady, C.J., Kao, D.J., DeBry, R.W., Adkins, R., Amrine, H.M., Stanhope, M.J., de Jong, W.W.,
283
Springer, M.S., 2001. Parallel adaptive radiations in two major clades of placental mammals. Nature 409, 610–614. McKenna, M.C., Bell, S.K., 1997. Classification of Mammals: Above the Species Level. Columbia University Press, New York. Mouchaty, S.K., Gullberg, A., Janke, A., Arnason, U., 2000. The phylogenetic position of the Talpidae within Eutheria based on analysis of complete mitochondrial sequences. Mol. Biol. Evol. 17, 60–67. Murata, Y., Nikaido, M., Sasaki, T., Fukumoto, Y., Hasegawa, M., Okada, N., 2003. Afrotherian phylogeny as inferred from complete mitochondrial genomes. Mol. Phylogenet. Evol. (in press). Murphy, W.J., Eizirik, E., Johnson, W.E., Zhang, Y.P., Ryder, O.A., OÕBrien, S.J., 2001a. Molecular phylogenetics and the origins of placental mammals. Nature 409, 614–618. Murphy, W.J., Eizirik, E., OÕBrien, S.J., Madsen, O., Scally, M., Douady, C.J., Teeling, E., Ryder, O.A., Stanhope, M.J., de Jong, W.W., Springer, M.S., 2001b. Resolution of the early placental mammal radiation using Bayesian phylogenetics. Science 294, 2348–2351. Nikaido, M., Cao, Y., Okada, N., Hasegawa, M., 2003. The phylogenetic relationships of insectivores with special reference to the lesser hedgehog tenrecs as inferred from the complete sequence of their mitochondrial genome. Genes Genet. Syst. 78, 107– 111. Nikaido, M., Harada, M., Cao, Y., Hasegawa, M., Okada, N., 2000. Monophyletic origin of the order Chiroptera and its phylogenetic position among Mammalia inferred from the complete mitochondrial DNA sequence of a Japanese megabat, Ryukyu flying fox (Pteropus dasymallus). J. Mol. Evol. 51, 318– 328. Nikaido, M., Kawai, K., Cao, Y., Harada, M., Tomita, S., Okada, N., Hasegawa, M., 2001. Maximum likelihood analysis of the complete mitochondrial genomes of eutherians and a reevaluation of the phylogeny of bats and insectivores. J. Mol. Evol. 53, 508–516. Novacek, M.J., 1992. Mammalian phylogeny: shaking the tree. Nature 356, 121–125. Penny, D., Hasegawa, M., Waddell, P.J., Hendy, M.D., 1999. Mammalian evolution: timing and implications from using the LogDeterminant transform for proteins of differing amino acid composition. Syst. Biol. 48, 76–93. Reyes, A., Gissi, C., Pesole, G., Catzeflis, F.M., Saccone, C., 2000. Where do rodents fit? Evidence from the complete mitochondrial genome of Sciurus vulgaris. Mol. Biol. Evol. 17, 979–983. Shimodaira, H., 2002. An approximately unbiased test of phylogenetic tree selection. Syst. Biol. 51, 492–508. Shimodaira, H., Hasegawa, M., 1999. Multiple comparisons of loglikelihoods with applications to phylogenetic inference. Mol. Biol. Evol. 16, 1114–1116. Shimodaira, H., Hasegawa, M., 2001. CONSEL: for assessing the confidence of phylogenetic tree selection. Bioinformatics 17, 1246– 1247. Springer, M.S., Cleven, G.C., Madsen, O., de Jong, W.W., Waddell, V.G., Amrine, H.M., Stanhope, M.J., 1997. Endemic African mammals shake the phylogenetic tree. Nature 388, 61–64. Stanhope, M.J., Madsen, O., Waddell, V.G., Cleven, G.C., de Jong, W.W., Springer, M.S., 1998a. Highly congruent molecular support for a diverse superordinal clade of endemic African mammals. Mol. Phylogenet. Evol. 9, 501–508. Stanhope, M.J., Waddell, V.G., Madsen, O., de Jong, W.W., Hedges, S.B., Cleven, G.C., Kao, D., Springer, M.S., 1998b. Molecular evidence for multiple origins of insectivora and for a new order of endemic African insectivore mammals. Proc. Natl. Acad. Sci. USA 95, 9967–9972. Strimmer, K., von Haeseler, A., 1996. Quartet puzzling: a quartet maximum-likelihood method for reconstructing tree topologies. Mol. Biol. Evol. 13, 964–969.
284
M. Nikaido et al. / Molecular Phylogenetics and Evolution 28 (2003) 276–284
Sullivan, J., Swofford, D.L., 1997. Are guinea pigs rodents? The importance of adequate models in molecular phylogenetics. J. Mammal. Evol. 4, 77–86. van Dijk, M.A.M., Madsen, O., Catzeflis, F., Stanhope, M.J., de Jong, W.W., Pagel, M., 2001. Protein sequence signatures support the African clade of mammals. Proc. Natl. Acad. Sci. USA 98, 188–193. Waddell, P.J., Okada, N., Hasegawa, M., 1999a. Towards resolving the interordinal relationships of placental mammals. Syst. Biol. 48, 1–5.
Waddell, P.J., Cao, Y., Hauf, J., Hasegawa, M., 1999b. Using novel phylogenetic methods to evaluate mammalian mtDNA, including amino acid-invariant sites-LogDet plus site stripping, to detect internal conflicts in the data, with special reference to the positions of hedgehog, armadillo, and elephant. Syst. Biol. 48, 31–53. Yang, Z., 1997. PAML: a program package for phylogenetic analysis by maximum likelihood. CABIOS 13, 555–556.