The Old World Sparrows (Genus Passer) Phylogeography and Their RelativeAbundance of Nuclear mtDNA Pseudogenes
Luis M. Allende, Isabel Rubio, Valentin Ruız-del-Valle, Jesus Guille´n, Jorge Martınez-Laso, Ernesto Lowy,Pilar Varela, Jorge Zamora, Antonio Arnaiz-Villena
Department of Immunology and Molecular Biology, Hospital 12 de Octubre, Universidad Complutense, Madrid, 28041 Spain
Received: 29 November 2000 / Accepted: 22 March 2001
Abstract. The phylogenetic relationships of genusPasser(Old World sparrows) have been studied withspecies covering their complete world living range. Mi-tochondrial (mt) cyt b genes and pseudogenes have beenanalyzed, the latter being strikingly abundant in genusPassercompared with other studied songbirds. The sig-nificance of thesePasserpseudogenes is presently un-clear. The mechanisms by which mt cyt b genes becomepseudogenes after nuclear translocation are discussed to-gether with their mode of evolution, i.e., transition/transversion mitochondrial ratio is decreased in thenucleus, as is the constraint for variability at the threecodon positions. However, the skewed base compositionaccording to codon position (in 1st position the percent-age is very similar for the four bases, in 2nd position thereare fewer percentage of A and G and more percentage ofT, and in 3rd codon position fewer percentage of G andT and is very rich in A and C) is maintained in thetranslocated nuclear pseudogenes. Different nuclear in-ternal mechanisms and/or selective pressures must existfor explaining this nuclear/mitochondrial differentialDNA base evolutive variability. Also, the phylogeneticusefulness of pseudogenes for defining relationships be-tween closely related lineages is stressed. The analysessuggest that the primitive genus Passer species comesfrom Africa, the Cape sparrow being the oldest:P.hispaniolensis italiaeis more likely conspecific toP.domesticusthan toP. hispaniolensis.Also, Passerspe-
cies are not included within weavers orEstrildinae orEmberizinae,as previously suggested. European andAmerican Emberizinaesparrows are closely related toeach other and seem to be the earliest species that radi-ated among the studied songbirds (all in the MioceneEpoch).
The Old World sparrows (genus Passer) are probably themost familiar group of bird species, since their world-wide distribution reflects that of humans. House sparrow(Passer domesticus), Spanish sparrow (P. hispaniolen-sis), tree sparrow (P. montanus), and others can be re-garded as human commensals; also, mostPasserspeciesnest in human-made constructions. The relationshipsamong the sparrow species within genus Passer and toother finches (Passeridaeand New World sparrows)have not been fully resolved. On the other hand, it iswidely believed that Pleistocene temperature variations(glaciations) and subsequent isolation are the most im-portant factors provoking the appearance of new extantbird species (Gill 1995). Recent contradictory evidencesuggests that speciation of some genera and orders mayhave already occurred a long time ago (Chiappe 1995;Feduccia 1995; Hackett 1996; Hedges et al. 1996; Ha¨rlidet al. 1997), particularly inPasserines(Klicka and Zink1997) and inCarduelinae(Marten and Johnson 1986;Fehrer 1996; Arnaiz-Villena et al. 1998, 1999a). In the
present work, we have collectedPasserand other relatedspecies samples from around the world (Passeridae;Sib-ley and Monroe 1990) in order to sequence an ortholo-gous gene from each of them: the mt cyt b (924 bp).Mitochondrial DNA has proven to be helpful for defin-ing the evolutionary relationships among relatively dis-tant and closely related birds (Arnason and Gullberg1994; Seutin et al. 1994); thus, we have also aimed tostudy the relatedness of these bird species in the contextof the paleogeography and molecular clock timing, inorder to get an overall picture of the evolution and therelative time of origin of these birds (Paturi 1991, pp.284–496; Smith et al. 1994; pp. 24–39; Cox and Moore1995, pp. 134–276). In addition, the genetic relationshipsbetween some of thePasseridaesubfamilies (Sibley andMonroe 1990; i.e.,Passerinae, Ploceinae, Estrildinae)have been studied. New and Old WorldEmberizinaesparrow-like finches are also used for determining thecorresponding phylogenetic relationships. Finally, the
very striking observation that many mitochondrial cyt b(probably nuclear) pseudogenes were only found at veryhigh frequency ingenus Passerspecies among thePas-serinebirds is discussed in the light of the evolutionarysignificance and phylogenetic usefulness of this obser-vation.
Materials and Methods
Bird samples come from species and places that are described in Table1. GenBank sequence accession numbers are also given. Blood fromliving birds was drawn after photographing and cutting the claws lo-cally anesthetized with a lidocaine ointment. Blood was collected inEDTA cooled at 4°C and frozen until use. DNA was obtained andmitochondrial cytochrome b gene (mt cyt b) (924 DNA bases) wasamplified with primers L14841 58-AAAAAGCTTCCATCCAA-CATCTC AGCATGATGAAA-38 and H15767 58-ATGAAGGGAT-GTTCTACTGGTTG-38 as detailed by Edwards et al. (1991). At leasttwo birds per species were sequenced in order to discard any variation
Table 1. List of species, origin, mt cytochrome b, and nuclear pseudogenes sequence identification
SpeciesMt cyt bsequence
Nuclear pseudogenessequence Sample region
1 House Sparrow(Passer domesticus domesticus) AF230906 AF230916 Madrid, Spain
All specimens studied are males and belong to the FamiliesPasseridaeor Fringilidae. Passeridae:Old World sparrows (Passerinae) Genus Passer+Rock-sparrows (Passerinae), #African Estrildine (Estrildinae). *Weaver (Ploecinae), Fringillidae: $(Fringilinae), &Old World Emberizid.CNewWorld Emberizid (Sibley and Monroe 1990).
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among individuals. Polymerase chain reaction (PCR), cloning, and au-tomatic DNA sequencing were performed as previously described (Ed-wards et al. 1991; Arnaiz-Villena et al. 1992). At least four clones fromeach of two different PCRs were sequenced from each species in orderto assess both PCR and DNA sequencing quality. For comparing thespecific existence of mitochondrial nuclear pseudogenes ingenusPasser,120 other species belonging toOrder Passeriformes(including20 differentgenera), and the following families/subfamilies:Corvidae,Dicrurinae, Vangini, Turdinae, Muscicapinae, Sturnidae, Poliopti-nilae, Paridae, Alaudidae, Passerinae, Ploceinae, Estrildinae, andFringillidae were tested (Arnaiz-Villena et al. 2001). Eight mt cyt bclones were analyzed for each bird and an average of five clones perindividual were suitable to be fully analyzed in the automatic sequencerbecause an average of one to three clones showed PCR mistakes andwere discarded. Three different phylogenetic tree-constructing meth-odologies were used to confirm independently the robustness of thetopologies (Ha¨rlid et al. 1997): unweighted parsimony, neighbor join-ing (NJ), and unweighted pair group with arithmetic mean (UPGMA).The matrix of genetic distances for the NJ tree was obtained by themaximum-likelihood method, and Kimura two-parameter distanceswere used for the UPGMA dendrogram. The UPGMA tree was alsoobtained for estimating relative coalescence times from known out-groups’ divergence times (Vincek et al. 1997). Times of species diver-gence are only a rough and relative estimate. Maximum parsimony(MP), NJ, and UPGMA dendrograms and their corresponding bipara-metric distance matrices were obtained with the PAUP*4.0b2 program,kindly provided by D.L. Swofford (1996). The following statisticalcalculations were carried out: (1) number of substitutions of the twogroup of sequences, mitochondrial and nuclear, (2) their transitions andtransversions ratios, and (3) the base composition according to codonposition in both genes and pseudogenes. Bootstrap values were calcu-lated to test the topology robustness of trees (Felsenstein 1985), andlow-bootstrap value branches are shown because a similar tree branchtopology is obtained by at least two different tree construction meth-odologies, as suggested by Edwards 1995. The LINTRE test was alsoperformed in order to test the molecular clock homogeneity among alllineages used in our analyses (Takezaki et al. 1995); thus, linearizedtrees were obtained, which reestimate the branch lengths under theassumption of a constant rate of evolution. The computer software usedfor these calculations can be obtained from the web site: ftp://ftp.bio.indiana.edu/molbio/evolve/lintr/ or http://cib.nig.ac.jp/dda/ntakezak.html.
The universal criteriun for defining aPasserpseudogene as such isthe existence of a stop codon at position 77 (nuclear reading code). Inaddition, different anomalies occur, such as deletions (see below).
In order to show the evolutionary difference between mt andnuclear pseudogenes, pairwise comparisons of species represented byboth the mt sequence and the nuclear counterpart were done (Table 2).The mean sequence divergence was also calculated for each data par-
tition by adding the respective pairwise divergences (%) and divided bythe total number of comparisons done (see Table 2).
Results and Discussion
Patterns of Base Substitution
Saturation was considered to have occurred in any of thedata partitions if the scatter of points showed a levellingoff of mutations as sequence divergence increased. Satu-ration plots for cyt b DNA (Fig. 1) indicated that onlythird position transitions showed a clear levelling offassociated with saturation; this occurred at about 13%uncorrected total sequence divergence (Passer/Petronia)and at sequence divergences of more than 13% in thecomparisons ofPasser/Gallus(see Fig. 1). Assessmentof saturation gave similar results by using Kimura’s twoparameter distances. Also, the number of variable andphylogenetically informative sites (403 and 278, respec-tively, out of 924 mt cyt b DNA bases), between the birdsdescribed in Table 1, was appropriate to establish soundphylogenetic comparisons (Hillis et al. 1994).
The nucleotide distribution pattern of the mt cyt bgene of thePasserbirds under study was similar to thepattern found in a previous analysis of this gene in birdsand mammals (Kornegay et al. 1993). At the first codonpositions, the four bases were equally distributed; at thesecond position, fewer G residues and a higher amount ofT were seen. At the third codon position, the bias againstG and T was strong, as previously found by others (Ed-wards et al. 1991; Kornegay et al. 1993). This bias inbase composition was similar in all species studied (re-sults not shown). Thus, our parsimony methodologyseems to be adequate for all species studied (Lockhart etal. 1994). Our cyt b gene variability was theoreticallysufficient to establish phylogenetic relationships accord-ing to the number of phylogenetically informative sites(Hillis et al. 1994); most of the differences were silentsubstitutions, as expected for a protein-coding gene, par-ticularly for close relatives, such as species within asingle genus,i.e., Passer,(Kocher et al. 1989). Withinspecies variability of the mt cyt b sequence was very lowin thePasserspecies tested (between 0.0 and 0.1%). Ourdata are concordant with those already published (Aviseand Ball 1991). Therefore, within species variability wasnot likely to interfere with interspecific comparisonswhich were in a higher range (see Fig. 2).
As expected for gene evolving relatively rapidly un-der strong functional contraints, most (53%) of the thirdcodon positions amongPasserspecies, in which substi-tutions are often silent, were variable. By contrast, rela-tively few of the first and second positions, (9% and 1%,respectively) were variable. Therefore, more than three-quarters (84%) of the variable sites occurred in thirdpositions of codons. A similar evolution rate for the mtcyt b DNA was found in the subfamiliesEstrildinae,Ploceinae,andEmberizinae(56% of variable sites in thethird position of codons, and 15% and 6% of the first and
Table 2. Matrix of pairwise Kimura two-parameter distances (%) incyt b genes (below diagonal) and cyt b nuclear pseudogenes (abovediagonal)
Speciesa 1 2 6 7 9 10
House sparrow(1) 3.81 9.34 0.98 4.30 4.16Sudan golden sparrow(2) 7.76 9.61 3.92 3.72 3.81Spanish sparrow
a Numbers in parentheses after species names correspond to columnnumbers to the right.
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second positions, respectively, were variable; results notshown).
Also, an attempt to estimate the relative tempo ofevolution ofPasserand other sparrows and finches wasundertaken: in order to estimate the tempo of evolution,the calculations done by Takahata, Klein, and ourselves(Vincek et al. 1997, Arnaiz-Villena et al. 1998, 1999a) toassess the time of appearance of Darwin’s finches, ca-naries, and goldfinches were followed. An UPGMA den-drogram (Fig. 2) was constructed because this type ofphylogenetic tree is more suitable for estimating coales-cence times than other methods, particularly when a mo-lecular clock exists (shown in this case by the LINTREtest, see below) (Nei 1987). The UPGMA tree was ob-tained by comparing the mt cyt b DNA sequences of thepheasant (Wittzell et al. 1994) and the chicken (Zoorobet al. 1990), two species that have diverged around 18MYA (Helm-Bychowski and Wilson 1986), not shownin Fig. 2. The mt cyt b comparison ofgenus Passeryields
an evolutionary rate (per lineage) of 0.68 × 10−9 non-synonymous substitutions per nonsynonymous site peryear and 1.41 × 10−8 synonymous substitutions per syn-onymous site per year, so that the overall rate is 4.0 ×10−9 ± 0.26 (Vincek et al. 1997). This results in a sub-stitution rate of 0.4% per million years, which leads to arough approximation to the 4% of nucleotide substitutionper lineage between the most distantPasserspecies.Taking into account that thePassermt DNA substitutionrate was found between the most distant species (ap-proximates to 4%), thePasserradiation seems to havestarted shortly before that ofSerinusandCarduelisgen-era (Arnaiz-Villena et al. 1999a and unpublished), about11 MYA (not shown in Fig. 2). However, the substitutionrate found by us (0.4% per million years) differs from thestandard 2% per million years. Also, cranes show a fasterrate of nucleotide substitution, closer to our results foundin Carduelis, Serinus,andPasser;(about 1%) (Krajew-ski and King 1996).
Fig. 1. Saturation plots for the cytochrome b gene that relate uncorrected sequence divergence to changes due to transitions (top) and transversions(bottom) at first, second, and third codon positions.
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Notwithstanding, the calculation of species diver-gence times needs to be confirmed by other methodolo-gies and with additional species in this particular studyand others (i.e., Ha¨rlid et al. 1997). In the later study,Passerinesare found to be older than paleognathousbirds; also, the relative close-to-Passer Carduelini(Car-duelis, Serinus,and others) species are considered nearlyas old asPasser(Marten and Johnson 1986; Fehrer 1996;Arnaiz-Villena et al. 1998, 1999a, 2001).
The use of either Chaffinch or chicken and pheasantas outgroups would seem to be correct, because onlythird position transitions appear to be saturated (Fig. 1).Notwithstanding, the LINTRE computer program(Takezaki et al. 1995) was used to assess constancy ofevolutionary rates among the used bird lineages. The“two cluster test” shows that the pseudogene cluster(with the exception ofP. hispaniolensispseudogenewhich clusters with the genes, see Fig. 2 and Fig. 3)evolves with a significantly different rate (>1%). Otheranalyzed sequences do not show a significant heteroge-neity of evolutionary rate. Two linearized trees were ob-
tained (one for the genes and one for the pseudogenes) byalso using the LINTRE software; these trees do notshown significant differences from our UPGMA tree inbranch length (Fig. 2) and their topology was also iden-tical. This supports the fact that a constant evolutionaryrate there exists among different gene sequences and thevalidity of our time calculations.
Phylogeography ofPasserspecies
In the present work, the complete geographical range ofOld World sparrows distribution is covered with thestudied species (i.e., Africa and Eurasia) (Table 1). TheAfrican grey-headed (P. griseus) and black-headed(P. melanurus) sparrows together with the Saxaul spar-row (P. ammodendri) seem to form a different clade inrelation to the other sparrows in all the trees (Figs. 2, 3a,3b). This clade seems to have originated earlier (Fig. 2)and this grouping suggests that (1) Saxaul sparrow mayalso have had its living range in the African desserts at
Fig. 2. Relative time of appearance ofgenus Passerand other spar-rows lineages. UPGMA methodology tends to perform poorly if theassumption of equal rate of cytb evolution among species does notreflect their actual evolution; however, LINTRE test shows that a mo-lecular clock exists (see text). Also, it seems to perform correctly in theclosely related bird species used for this work since groups of taxa are
similar to those obtained in NJ and parsimony dendrograms (see Fig.3). Bird species used are detailed in Table 1; Ps: note that theP. h.hispaniolensispseudogene cluster with the genes (see text). The abso-lute timing is uncertain; however, our data support the Miocene/Pliocene species appearance.
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one time (Sahara, Arabian Peninsula) and (2) Old Worldsparrows originated in Africa. The five species of Afri-can grey sparrows may all belong to one species (Sum-mers-Smith 1988). The species studied by us (P. griseus)is related to the Cape Sparrow (P. melanurus) (Figs. 2,3a, 3b), the latter living only in the southern SaharaAfrican range where grey sparrows do not thrive (exceptP. diffusus).
The oldestgenus Passerspecies seems to be the SouthAfrican P. melanurus(Cape sparrow) (Fig. 2); it alsoclusters together with the African speciesP. griseus(grey-headed sparrow). The latter is a representative ofthe grey-headed sparrows group, which thrive from theSouth Sahara down to South Africa. OtherPasserspe-cies should also be tested, particularly African ones (al-though our study includes representatives for each Afri-can group, i.e., grey-headed, yellow ones).P. luteuslivesin Africa in a land band just bellow the Sahara Desert(Sahel) from the East to the West Coast (Summers-Smith1988); its position in the phylogenetic trees is ambigous.
It may or may not be closely related to the other Africansparrows. However, it is clear that the claims thatP.luteusdoes not belong togenus Passeris not supportedby our genetic data. It may have originated in Sudan andmight have expanded from West to East following thehunter-gathered Cushitic people emigration (McEvedy1980; Summers-Smith 1988).P. ammodendri(the singlesparrow species, other thanP. melanurus,that has ablack nape) is tightly linked toP. melanurus;this couldhave given rise to mutation(s) that diluted melanin atvarious parts of the head and body and could have orig-inated the grey-headed sparrows, including theP. griseusgroup—P. ammodendriandP. domesticus(grey-patchedhead).P. ammodendriis now restricted to the Asiandeserts but may have also been originated in Africa andinhabited its deserts in the past, likeP. simplex(withwhich it shares a common living range in Asia) (Sum-mers-Smith 1988).P. ammodendrimay have been dis-placed by a recent introduction ofP. simplexin Africa.
The origin ofgenus Passeris likely to be confined to
Fig. 3. Maximum unweighted parsimony tree. Heuristic search wasused (PAUP). Only one tree was the best parsimonious. Consistencyand retention indexes were 0.57 and 0.57, respectively. The majorityrule bootstrap consensus tree of mt cyt b genes and nuclear pseudo-genes is shown. First, second, and third codon bases were used un-weighted. Parsimony was used unweighted because weighting is onlyrecommended for greater amounts of evolutionary divergence, whichare not expected among the relatively closely related species analyzed(Hillis et al. 1994). Parsimony bootstrap analysis was done with 1,000replications, and values (in percent) shown above branches. The num-
ber of events is shown underlined below branches. Bird species usedare detailed in Table 1. Ps: Note that theP. h. hispaniolensispseudo-gene clusters with the genes (see text). b. Neighbor-joining bootstraptree (1,000 replications) based on 924 bases of cyt b genes and nuclearpseudogenes. Bootstrap values are shown above branches; branchlengths (× 1,000) are shown underlined below branches. The evolu-tionary model used was “the minimum evolution.” Distance matriceswere calculated based on maximum likelihood analysis (Swofford1996). Bird species used are detailed in Table 1. Note that theP. h.hispaniolensispseudogene clusters with the genes (see text).
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Africa because of the highest number of extant specieson this continent (Summers-Smith 1988). Our resultsagree with this assertion,P. melanurusbeing the oldestextant species: it would have been the origin of grey-headed African sparrows (includingP. domesticus) andalso yellow and other Paleartic bab-sparrows. It has beenpostulated that the bab-sparrows have probably arisen atthe Nile or Rift Valley and followed the human expan-sion towards Eurasia, giving rise to all Eurasian bab-species (Summers-Smith 1988). That this could havehappened would be more feasible if the human expan-sion had occurred much earlier than thought (5 millionyears ago. Not shown; see Fig. 2).P. rutilans (Asia)seems to be the oldest of the Eurasian sparrows althoughits phylogenetic placing within thegenusis undefined(Figs. 2, 3a, 3b). Thegenus Passeris strongly associatedwith man, since most of species nest on man-made build-ings.
Passer domesticusand P. hispaniolensiscannot bedistinguished osteologically and their ancestor may haveappeared 4 MYA (Fig. 2 and not shown). Fossil evidencefor this precursor is found 350,000 years ago in Palestine(Summers-Smith 1988). However, the Pleistocene originfor sparrows (this paper) and otherPasserines(includingSerinusand Carduelis; Klicka and Zink 1997; Arnaiz-Villena et al. 1998, 1999a) could be placed much furtherback into the Miocene or Pliocene Epochs, Pleistocenebeing more important in subspeciation. Our Fig. 2 sug-gests thatP. domesticusandP. hispaniolensissubspecia-tion may have occurred in the Pleistocene. However,Passerines appearance timing are still much debated anda Pleistocene origin for species is also suggested (Aviseand Walker 1998).
The following conclusions about the much debatedsystematic uncertainties within Old World sparrows(Summers-Smith 1988) have been reached in the presentstudy:
1) The origin ofgenus Passerseems to be African be-cause the highest number of extant species is Africanand our phylogenetic results are also suggestive (Figs.2, 3a, 3b); this is in accordance with Summers-Smith1988. P. melanurusor other related extinct speciesmight be the parental one.
2) P. melanurus(Cape sparrow) is found to be related tothe grey-headed African sparrow and also toP. am-modendri(Saxaul sparrow) (Figs. 2, 3a, 3b). All threespecies males have melanic pigmentation in the headcrown and nape (Figs. 2, 3a, 3b). Also,P. domesticusmay be included in the grey-headed sparrows becauseof its grey head crown (Fig. 2, picture), although noprecise placement is found in our phylogenetic trees,probably because more African species need to bestudied.
3) P. hispaniolensis italiaeis probably aP. domesticus(grey head crown) subspecies according to the re-
spective branch lengths observed in NJ and MP den-drograms (Fig. 3a and 3b) and not aP. hispaniolensissubspecies (brown head). This is in accordance withthe classical view, but not with more recent opinions(Summers-Smith 1988).P.h. italiae (brown head)may have arisen fromP. domesticus(grey headcrown) by hybridization or divergence/speciation.
On the other hand, the relationships of Old Worldsparrows (G. Passer) to otherPasseridae:Old and NewWorld Emberizinaesparrows, weaver birds (Ploceinae),andEstrildinaehave been studied. Rock sparrow (genusPetronia) is the closest relative studied to Old Worldsparrows within the SubfamilyPasserinae; Petroniaismore robust in appearance and has specialized habitatrequirements (drier and more arid areas). Its distributionis African and Euroasiatic and according to Figs. 2, 3a,and 3b, seems to be the sister group togenus Passerbutstands as a separate lineage (Clement et al. 1993, pp.442–468). AfricanPasseridae, Lonchura cucullata(Es-trildinae), andQuelea cardinalis(a weaver-bird,Plocei-nae) clade together corroborating their relatedness; how-ever,genus Passeris not included within thePloceinae(Figs. 2, 3a, 3b) in contrast to the suggestion of previousstudies in which they were included within weavers bysome authors based mainly on skeletal characters, nestbuilding, and DNA hybridization (Summers-Smith1988). Emberizinaeare also separate fromPasser inspite of suggestions to join both families according toegg-white protein studies (Summers-Smith 1988).Frin-gilla coelebs(the cropless Chaffinch) is closer toLonchuraandQueleathan to theEmberizinaein the UPGMA andNJ dendrograms; however, it stands as a separate lineagein the parsimony tree. It is clear from all the trees thatOld and New worldEmberizinaesparrows represent aseparate (and single) radiation compared to bothPasse-ridae family and Chaffinches (Fringillidae). This is con-cordant with the fact that otherFringillidae (Goldfinchesand Canaries, Arnaiz-Villena et al. 1998, 1999a) do notcluster with any finch of thePasseridaefamily (Sibleyand Monroe 1990), described in this paper (Arnaiz-Villena et al. 2001, and unpublished results). Also, theAmerican speciesSpizella passerinamay represent theoldest split within theEmberizinae(Fig. 2); this maysuggest thatEmberizinaefinches originated in America,although the study of more species is needed.
Abundance of Nuclear Mitochondrial cyt bPseudogenes ingenus Passerand Their CharacteristicMode of Evolution
Mitochondrial DNA fragments have been found in thenuclear DNA of yeast, locust,Podospora,sea urchin,maize, rat, human (Fukuda et al. 1985), and birds(Arctander 1995, for review see Blanchard and Lynch2000). Although the mtDNA fragments seem to have
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been continuously integrated into nuclear DNA duringevolution, the evolutionary significance and mechanismsof such phenomena are unclear. The present study de-scribes for the first time the relative abundance of inte-grated mt cyt b pseudogenes among species of a singlegenus(Passer). The different types of alterations foundin Passermt cyt b pseudogenes are depicted in Fig. 4.About 610Passerinemt cyt b DNA clones have beensequenced and studied in our laboratory during the lastfew years (see Materials and Methods) and we onlyfound pseudogenes withingenus Passerspecies. Noteven a single pseudogene has been detected in otherPasserine(or non-Passerine) species (Arnaiz-Villena etal. 2001), includingCarduelis, Serinus(Arnaiz-Villenaet al. 1998, 1999a), and in any other related-to-Passergenus,like the ones studied in the present paper. Also, aFischer test was performed: 0 pseudogenes found in 610clones belonging to non-Passerfinches were comparedwith 39 pseudogenes found in 65 clones belonging to
Passerspecies;P < 0.00001 was obtained, supportingthe fact that the pseudogenes finding inPasserspecieswas not due to chance. In fact, the number of pseudogeneand gene clones obtained were, respectively:P. domes-ticus (4 and 5),P. luteus(4 and 5),P. h. hispaniolensis(1 and 3),P. griseus(3 and 6),P. h. italiae(3 and 3), andP. ammodendri(24 and 4).
Our phylogeny suggests that the most ancientanomaly (Fig. 4) in pseudogenes is a stop signal at codon77 seen inP. griseus, P. ammodendri, P. luteus, P. do-mesticus, and P. hispaniolensis italiae.This stop codononly appeared because of the mitochondrial-to-nucleartranslocation itself in the first three species: it is a stopcodon in the nuclear code, but not in the mitochondrialone (see Fig. 4 caption). It may have first occurred inP. griseus(or any of the African grey-headed group)after separation of theP. melanuruslineage or theirextinct ancestor(s). Fourteen clones have been studied inP. melanurusand no pseudogene has been found, but
Fig. 4. Mitochondrial cyt b protein. Black circles refer to the speciesnumbered in Table 1, i.e., 14 Passer domesticus,2 4 Passer luteus,6 4 Passer hispaniolensis hispaniolensis,7 4 Passer hispaniolensisitaliae, 9 4 Passer griseus griseus,10 4 Passer ammodendri ammo-dendri. First stop codons (red square) and deletions (blue square) ofnuclear pseudogenes are depicted in a cytochrome b molecule. Morestop codons were found along the nuclear pseudogenes. The mecha-nism that generates the first stop codons is simply a translocation ofmitochondrial genes to the nucleus (tr), and true base change (tc) mayalso occur; normal mt cyt b gene was used for comparisons. The typeof observed stop codons and their order are the following: (a) InP.
domesticus,tr: 113, 141, 163, 165, 272 and tc: 77, 89, 107, 223, aftercodon 284 (deletion of 7 bases) the open reading frame was lost. (b) InP. luteus,tr: 77, 113, 141, 163, 165, 272 and no tc, after codon 284(deletion of 7 bases), the open reading frame was lost. (c) InP. h.hispaniolensis,tr: 113, 135, 141, 163, 165, 272 and no tc, after codon284 (deletion of 7 bases) the open reading frame was lost. (d) InP. h.italiae, tr: 113, 141, 163, 165, 272 and tc: 77 and 89; after codon 284(deletion of 7 bases) the open reading frame was lost. (e) InP. griseus,tr: 77 and 113 and no tc, after codon 114 (deletion of 3 bases) the openreading frame was lost. (f) InP. ammodendri,tr: 77, and tc: 104, aftercodon 105 (deletion of 1 base) the open reading frame was lost.
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there is still the possibility that pseudogenes are presentin all Passerlineages and they have not been found by usin some species by chance. The fact that bothP. hispani-olensis italiaeand P. domesticushave this first stopcodon because of a true base change (TGG→TGA, seeFig. 4 caption) further corroborates that their relatednessis closer than that ofP. hispaniolensis hispaniolensisandP. hispaniolensis italiaebecause the former has a differ-ent stop codon (Fig. 4). Thus, evolutive pressures atcodon 77 exist to render it as a stop signal in both ofthese species. Position 77 is placed on the extramito-chondrial region of the molecule (Fig. 4) and it shows alow change rate and probably is an integral part of the Qoredox core; most of the outer surface is implicated in theQo redox center and this appears to be a major contribu-tor to the reduced evolutionary rate for the outer surface(Howell 1989). Deletions may be secondary to the ap-pearance of this 77 stop codon. A number of stop codonsmay only be generated because of the putative mitochon-drial/nuclear translocation (Fig. 4, caption), once thecodon 77 is read as “stop,” the translocated DNA mayescape then to the translation and transcription gene ma-chinery control.
The fact thatP. griseusandP. ammodendrishow thehighest number of anomalies in pseudogenes also sup-ports the fact that they belong to the oldest group ofsparrows (Fig. 4). A stop codon at 113 has been found intheP. h. hispaniolensispseudogene (Canary Islands) andcodon 77 was not a stop codon either in mitochondrial ornuclear reading code in this particular bird. This pseu-dogene clusters with the normal mt cyt b genes in thedendrograms (Figs. 2, 3a, 3b), suggesting that the trans-location has occurred independently and more recently inthe relatively isolated Canary Islands sparrow popula-tions and that the pseudogene is in the process of accu-mulating more differences with respect to its mitochon-drial gene. This pseudogene has been detected in threeunrelated birds.
Pseudogenes may also be useful to ascertain phylog-enies in birds; because the nuclear copy has a slowersubstitution rate, it is more likely to be more similar tothe ancestral form (Fukuda et al. 1985; Arctander 1995).In fact, original African sparrows (P. griseusandP. am-modendri) cluster together and are separated fromP.domesticusandP. h. italiae,the latter being a subspeciesof the former (Figs. 2, 3a, 3b).P. luteus is probablylinked to the Mediterranean sparrows (in its origin)(Summers-Smith 1988), i. e., toP. domesticuswhichprobably thrived close to the first urban nucleus aroundthe once fertile Sahara (Arnaiz-Villena et al. 1999b) and/or the Nile Valley (Summers-Smith 1988). Also, higherbootstrap values are found in the pseudogene tree (aspreviously described by Arctander 1995); this may helpto define phylogenetic relationships between closely re-lated species, as in our case.
Evolutive constrictions has also been observed in the
Passernuclear pseudogenes. First and second codon po-sition substitutions in mitochondrial genes of six speciesamounted to 137, whereas third codon position substitu-tions amounted to 756 (ratio 756/1374 5.5). In the caseof 6 nuclear pseudogenes (each belonging to a differentspecies), first and second codon position substitutionswere 323 and third codon position substitutions were 381(ratio 381/3234 1.18). A constriction for third codonposition substitution was clearly found in the nuclearmitochondrial pseudogenes and also a higher constric-tion at the first and second codon positions was observed,when compared with the mt genes. Arctander (1995) didnot distinguish the substitution rate according to codonposition because his data set did not contain several spe-cies within a singlegenus.In addition, and in order togain a more complete picture for the mode of evolutionof mitochondrial pseudogenes in birds, the nucleotidedistribution patterns of the mt cyt b genes and pseudo-genes were analyzed and found to be similar in bothnuclear pseudogenes and mitochondrial genes to the pat-terns found by others for mt cyt b in birds and mammals(Kornegay et al. 1993; Hackett 1996; Arnaiz-Villena etal. 1998; 1999a). At the first codon positions, the fourbases were equally distributed; at the second position,fewer G residues and a higher amount of T were seen. Atthe most variable third codon position, the bias against Gand T was strong, as previously found by others (notshown, Edwards et al. 1991; Kornegay et al. 1993). Thus,it may be postulated that the evolutive mechanisms thatmaintain general constraints for both functional DNAand pseudogenes variation and reduce the DNA basechange rate are different from those that preserve thespecific skewed base variation; the latter, and not theformer, are universally maintained (both in nucleus andmitochondria) in mammals and birds. This is confirmedby our results inPasserspecies (not shown).
The ts(498)/tv(232) ratio in six Passer pseudogeneswas 2.14 and the same ts(738)/tv(155) ratio in six Passergenes was 4.80; this relative constriction for transitions(or the lack of bias against transversion) that occurs inthe bird nucleus for a mt gene was also found by others(Arctander 1995).
The evolutionary difference (% mean sequence diver-gence) is lower in pseudogenes (5.6%) than in genes(6.9%) (Table 2); however, the pseudogene mean differ-ence is much lower (3.6%) ifP. h. hispaniolensispseu-dogene is left out. This pseudogene clusters with genes inthe dendrograms (Figs. 2 and 3) and is supposed to haverecently been translocated into the nucleus, thus showingonly a few anomalies (see above).
The evidences suggesting that we are dealing withnuclear pseudogenes are shown in Fig. 4. This is alsoshown by the distinctive mode of mitochondrial andnuclear gene evolution, i.e., ts/tv ratio, nuclear con-straints for DNA base variability, and phylogenetic stud-ies that show how they cluster together in dendrograms
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(Figs. 2, 3a, 3b, Arctander 1995). Therefore, it may bepostulated that translocation of mitochondrial genes tothe nucleus is a common phenomenon in nature (Fukudaet al. 1985; Collura and Stewart, 1995; Blanchard andLynch 2000), and may occur together with the generationof generaand species i.e., in ourgenus Passerit is notfound in the postulated ancestor species (Petronia) andother species. The pathway of mtDNA integration intonuclear DNA is mediated by mechanisms similar tothose seen in the case of Simian Virus 40 integration tonuclear DNAs, but not to those observed in retroviralintegration (Fukuda et al. 1985). By studying closelyrelated species likegenus Passer,a possible sequence ofevents may be hypothesised for the evolutionary changeof nuclear mt cyt b DNA:
1) The nuclear reading code detects stop codons (eithercaused only by a mt-to-nucleus translocation or byindependent true base changes).
2) The new mtDNA integrated into the nucleus escapesthe control of normal nuclear genes repairing machin-ery.
3) Scattered deletions are established.
It is possible that the mitochondrial genes may alreadyarrive to the nucleus with promoter anomalies, but thepossibility that the priming sites (for PCR amplification)in genus Passerare different from otherPasserinesmaybe ruled out since our primers attach to the mt cyt bcoding DNA (Arnaiz-Villena et al. 1998, 1999a).
A more general inference may be drawn from thepresent work: mt pseudogenes bird phylogenies are morerobust with pseudogenes (also found by Arctander 1995)and the conjoint evolutive analysis of genes and pseudo-genes may help to unveil the mechanisms by which mtpseudogenes come into the nucleus and to follow theirevolution thereafter.
Finally, it is not clear why genus Passer accumulatesa high number of nuclear mtDNA pseudogenes and otherclosely related bird species do not (Arnaiz-Villena et al.1998, 1999a, 2001). The only general characteristic ofgenus Passer is that they have become human commen-sals. This may have rendered these finches more exposedto xenobiotics. It might be hypothesized that popullantsmay have driven the mtDNA integration to the nucleusthrough processes that damaged mitochondria in the firstplace. Indeed, the amount of mtDNA in human genomeis high (Fukuda et al. 1985).
Acknowledgments. We are indebted to the following Spanish orni-thologists: Bernardino Yebes, Gloria Gardo´, Francisco Mira Chin-chilla, Arturo Ferna´ndez Cagiao, and Alvaro Guille´n. This work wassupported in part by Ministerio de Educacio´n grants (PM95-57, PM96-21 and PM99-23) and Comunidad de Madrid (06/70/97 and 8.3/14/98).L. Allende and I. Rubio contributed equally to this paper.
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