J Mol Evol (2001) 53:144–154 DOI: 10.1007/s002390010202 © Springer-Verlag New York Inc. 2001 The Old World Sparrows (Genus Passer) Phylogeography and Their Relative Abundance of Nuclear mtDNA Pseudogenes Luis M. Allende, Isabel Rubio, Valentin Ruı´z-del-Valle, Jesus Guille´n, Jorge Martı´nez-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 genus cies are not included within weavers or Estrildinae or Passer (Old World sparrows) have been studied with Emberizinae, as previously suggested. European and species covering their complete world living range. Mi- American Emberizinae sparrows are closely related to tochondrial (mt) cyt b genes and pseudogenes have been each other and seem to be the earliest species that radi- analyzed, the latter being strikingly abundant in genus ated among the studied songbirds (all in the Miocene Passer compared with other studied songbirds. The sig- Epoch). nificance of these Passer pseudogenes is presently un- clear. The mechanisms by which mt cyt b genes become Key words: Mitochondrial DNA — Nuclear pseudo- pseudogenes after nuclear translocation are discussed to- genes — Sparrow — Passerines — Emberiza — Petronia gether with their mode of evolution, i.e., transition/ transversion mitochondrial ratio is decreased in the nucleus, as is the constraint for variability at the three Introduction codon positions. However, the skewed base composition The Old World sparrows (genus Passer) are probably the according to codon position (in 1st position the percent- most familiar group of bird species, since their world- age is very similar for the four bases, in 2nd position there wide distribution reflects that of humans. House sparrow are fewer percentage of A and G and more percentage of (Passer domesticus), Spanish sparrow (P. hispaniolen- T, and in 3rd codon position fewer percentage of G and sis), tree sparrow (P. montanus), and others can be re- T and is very rich in A and C) is maintained in the garded as human commensals; also, most Passer species translocated nuclear pseudogenes. Different nuclear in- nest in human-made constructions. The relationships ternal mechanisms and/or selective pressures must exist among the sparrow species within genus Passer and to for explaining this nuclear/mitochondrial differential other finches (Passeridae and New World sparrows) DNA base evolutive variability. Also, the phylogenetic have not been fully resolved. On the other hand, it is usefulness of pseudogenes for defining relationships be- widely believed that Pleistocene temperature variations tween closely related lineages is stressed. The analyses (glaciations) and subsequent isolation are the most im- suggest that the primitive genus Passer species comes portant factors provoking the appearance of new extant from Africa, the Cape sparrow being the oldest: P. bird species (Gill 1995). Recent contradictory evidence hispaniolensis italiae is more likely conspecific to P. suggests that speciation of some genera and orders may domesticus than to P. hispaniolensis. Also, Passer spe- have already occurred a long time ago (Chiappe 1995; Feduccia 1995; Hackett 1996; Hedges et al. 1996; Ha¨rlid et al. 1997), particularly in Passerines (Klicka and Zink Correspondence to: A. Arnaiz-Villena; e-mail: aarnaiz@eucmax. 1997) and in Carduelinae (Marten and Johnson 1986; sim.ucm.es. website: http://chopo.pntic.mec.es/∼biolmol Fehrer 1996; Arnaiz-Villena et al. 1998, 1999a). In the 145 Table 1. List of species, origin, mt cytochrome b, and nuclear pseudogenes sequence identification Mt cyt b Nuclear pseudogenes Species sequence sequence Sample region 1 House Sparrow (Passer domesticus domesticus) AF230906 AF230916 Madrid, Spain 2 Sudan Golden-Sparrow (Passer luteus) L76714 AF230919 Dakar, Senegal 3 Eurasian Tree Sparrow (Passer montanus montanus) AF230911 — Madrid, Spain 4 Pegu Sparrow (Passer flaveolus) AF230907 — Ho-Chi-Minh city, Vietnam 5 Cape Sparrow (Passer melanurus melanurus) AF230910 — Capetown, South Africa 6 Spanish Sparrow (Passer hispaniolensis hispaniolensis) AF230909 AF230918 Gran Canaria, Canary Islands, Spain 7 Spanish Sparrow (Passer hispaniolensis italiae) AF255708 AF255707 Piacenza, Italy 8 Russet Sparrow (Passer rutilans rutilans) AF230912 — Beijing, China 9 Grey headed Sparrow (Passer griseus griseus) AF230908 AF230917 Dakar, Senegal 10 Saxaul Sparrow (Passer ammodendri ammodendri) AF230905 AF230915 Almaty (Kazakhstan), Russia 11 Rock Sparrow+ (Petronia petronia petronia) AF230914 — Madrid, Spain 12 Bronze Mannikin# (Lonchura cucullata cucullata) AF255705 — Kimshasa, Zaire 13 Cardinal Quelea*# (Quelea cardinalis) AF255709 — Kimshasa, Zaire 14 Chaffinch$ (Fringilla coelebs coelebs) L76609 — Madrid, Spain 15 Corn Bunting& (Miliaria calandra calandra) AF255706 — Madrid, Spain 16 Lark SparrowC¸ (Chondestes grammacus grammacus) AF255704 — Mexico DF, Mexico 17 Chipping SparrowC¸ (Spizella passerina passerina) AF255710 — Mexico DF, Mexico All specimens studied are males and belong to the Families Passeridae or Fringilidae. Passeridae: Old World sparrows (Passerinae) Genus Passer +Rock-sparrows (Passerinae), #African Estrildine (Estrildinae). *Weaver (Ploecinae), Fringillidae: $(Fringilinae), &Old World Emberizid. C¸ New World Emberizid (Sibley and Monroe 1990). present work, we have collected Passer and other related very striking observation that many mitochondrial cyt b species samples from around the world (Passeridae; Sib- (probably nuclear) pseudogenes were only found at very ley and Monroe 1990) in order to sequence an ortholo- high frequency in genus Passer species among the Pas- gous gene from each of them: the mt cyt b (924 bp). serine birds is discussed in the light of the evolutionary Mitochondrial DNA has proven to be helpful for defin- significance and phylogenetic usefulness of this obser- ing the evolutionary relationships among relatively dis- vation. tant and closely related birds (Arnason and Gullberg 1994; Seutin et al. 1994); thus, we have also aimed to study the relatedness of these bird species in the context Materials and Methods of the paleogeography and molecular clock timing, in order to get an overall picture of the evolution and the Bird samples come from species and places that are described in Table relative time of origin of these birds (Paturi 1991, pp. 1. GenBank sequence accession numbers are also given. Blood from 284–496; Smith et al. 1994; pp. 24–39; Cox and Moore living birds was drawn after photographing and cutting the claws lo- 1995, pp. 134–276). In addition, the genetic relationships cally anesthetized with a lidocaine ointment. Blood was collected in between some of the Passeridae subfamilies (Sibley and EDTA cooled at 4°C and frozen until use. DNA was obtained and Monroe 1990; i.e., Passerinae, Ploceinae, Estrildinae) mitochondrial cytochrome b gene (mt cyt b) (924 DNA bases) was amplified with primers L14841 5Ј-AAAAAGCTTCCATCCAA- have been studied. New and Old World Emberizinae CATCTC AGCATGATGAAA-3Ј and H15767 5Ј-ATGAAGGGAT- sparrow-like finches are also used for determining the GTTCTACTGGTTG-3Ј as detailed by Edwards et al. (1991). At least corresponding phylogenetic relationships. Finally, the two birds per species were sequenced in order to discard any variation 146 Table 2. Matrix of pairwise Kimura two-parameter distances (%) in tition by adding the respective pairwise divergences (%) and divided by cyt b genes (below diagonal) and cyt b nuclear pseudogenes (above the total number of comparisons done (see Table 2). diagonal) Speciesa 1267910 Results and Discussion House sparrow(1) 3.81 9.34 0.98 4.30 4.16 Patterns of Base Substitution Sudan golden sparrow(2) 7.76 9.61 3.92 3.72 3.81 Spanish sparrow Saturation was considered to have occurred in any of the “hispaniolensis”(6) 3.01 6.76 9.46 9.67 9.48 data partitions if the scatter of points showed a levelling Spanish sparrow off of mutations as sequence divergence increased. Satu- “italiae”(7) 0.54 7.63 2.66 4.41 4.27 ration plots for cyt b DNA (Fig. 1) indicated that only Grey headed sparrow(9) 8.61 7.74 8.34 8.61 3.02 third position transitions showed a clear levelling off Saxaul sparrow(10) 8.74 8.48 7.97 8.48 7.85 associated with saturation; this occurred at about 13% a Numbers in parentheses after species names correspond to column uncorrected total sequence divergence (Passer/Petronia) numbers to the right. and at sequence divergences of more than 13% in the comparisons of Passer/Gallus (see Fig. 1). Assessment of saturation gave similar results by using Kimura’s two among individuals. Polymerase chain reaction (PCR), cloning, and au- parameter distances. Also, the number of variable and tomatic DNA sequencing were performed as previously described (Ed- phylogenetically informative sites (403 and 278, respec- wards et al. 1991; Arnaiz-Villena et al. 1992). At least four clones from tively, out of 924 mt cyt b DNA bases), between the birds each of two different PCRs were sequenced from each species in order to assess both PCR and DNA sequencing quality. For comparing the described in Table 1, was appropriate to establish sound specific existence of mitochondrial nuclear pseudogenes in genus phylogenetic comparisons (Hillis et al. 1994). Passer, 120 other species belonging to Order Passeriformes (including The nucleotide distribution pattern of the mt cyt b 20 different