Mitochondrial DNA points to Lanius meridionalis as a polyphyletic species

Molecular Phylogenetics and Evolution 47 (2008) 1227–1231

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Molecular Phylogenetics and Evolution journal homepage: www.elsevier.com/locate/ympev

Short Communication

Mitochondrial DNA points to Lanius meridionalis as a polyphyletic species Tilman E. Klassert a, M. Ángeles Hernández b, Francisco Campos c, Octavio Infante d, Teresa Almeida a, Nicolás M. Suárez e, José Pestano e, Mariano Hernández a,* a

University Institute of Tropical Diseases of Canary Islands, Genetics, University of La Laguna, Avda. Astrofisico Fco. Sanchez S/N, E-38071 La Laguna, Tenerife, Spain Department of Zoology and Ecology, Faculty of Sciences, University of Navarre, E-31080 Pamplona, Spain c European University Miguel de Cervantes, Padre Julio Chevalier 2, E-47012 Valladolid, Spain d SEO/BirdLife Conservation Unit, C/Melquiades Biencinto 34, E-28053 Madrid, Spain e Department of Genetics, Faculty of Medicine, University of Las Palmas de Gran Canaria, E-35080 Las Palmas, Spain b

a r t i c l e

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Article history: Received 28 February 2007 Revised 18 January 2008 Accepted 6 March 2008 Available online 16 March 2008

1. Introduction The family Laniidae (Order Passeriformes) includes, among others, the genus Lanius. Some species of this genus are the Great Grey Shrike (Lanius excubitor), the Southern Grey Shrike (Lanius meridionalis), the Loggerhead Shrike (Lanius ludovicianus) and the Chinese Grey Shrike (Lanius sphenocercus), all of them with similar sizes and morphological characteristics and clearly differentiated from other shrike species. According to Lefranc and Worfolk (1997), Lanius meridionalis encompasses ten subspecies distributed along the southern and eastern Palearctic region and the North of the Afrotropical region (meridionalis, algeriensis, koenigi, elegans, leucopygos, aucheri, lathora, pallidirostris, buryi and uncinatus). Lanius excubitor includes seven subspecies in the northern Palearctic region (excubitor, homeyeri, leucopterus, sibiricus, bianchii, mollis and funereus) and two in the Nearctic region (borealis and invictus). Lanius ludovicianus is comprised of eight subspecies, two of them (mearnsi and anthonyi) in the islands off the coast of California and the other six (ludovicianus, migrans, mexicanus, excubitorides, miamensis and grinnelli) in North America. Lanius sphenocercus includes two subspecies (giganteus and sphenocercus), both from eastern Asia (Russia, China). The taxonomy of Lanius meridionalis has been under debate for several decades. Vaurie (1959) suggested that there is only one polytypic species (Lanius excubitor), of which L. e. meridionalis is a subspecies. However, Lanius meridionalis has been proposed and accepted at international level as a separate species (British Ornithologists’ Union, 1997). Furthermore, Harris and Franklin

* Corresponding author. Fax: +34 9223 18311. E-mail address: [email protected] (M. Hernández). 1055-7903/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.ympev.2008.03.012

(2000) and Hernández et al. (2004) have suggested the existence of three species (L. excubitor, L. meridionalis and L. pallidirostris). To date, there have been few works published showing phylogenetic relationships among these shrike species. Mundy and Helbig (2004), studying the mtDNA control region, separated the shrikes into two groups according to whether or not they had tandem repeats. Hernández et al. (2004) proposed three groups of species and subspecies for the shrikes according to the number of these tandem repeats: (1) L. e. excubitor in the North of Europe and Asia; (2) L. m. pallidirostris and L. m aucheri in the Central and South-eastern Asia and North of Africa, and (3) L. m. meridionalis and L. m. koenigi in the South of Europe and the Canary Islands. Despite these works, a broader study is required to jointly analyze different shrike species to reveal their phylogenetic relationships. In the present work, four shrike species are studied: L. excubitor, L. meridionalis, L. sphenocercus and L. ludovicianus. L. excubitor includes two subspecies (excubitor and invictus) and L. meridionalis includes five subspecies (algeriensis, meridionalis, aucheri, pallidirostris and koenigi) (Fig. 1). Sequences for L. ludovicianus and L. e. invictus were taken from the GenBank. 2. Materials and methods 2.1. Samples for molecular analyses Seventy individuals were analyzed representing eight species/ subspecies of the Lanius genus. In addition, our analysis includes representative individuals from other three species based on nucleotide sequences deposited in the GenBank (one of them, Rook Corvus frugileus, has been used as outgroup). Species names and collecting areas are reported in Table 1.

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Fig. 1. Origin of the samples analyzed in this work.

Samples were obtained by venipuncture of the ulnar vein. A total of 50 lL of blood was then preserved in a citrate buffer (0.129 M sodium citrate) or in a FTA card (Whatman International Ltd.). 2.2. DNA extraction, PCR amplification and sequencing From the samples conserved in citrate buffer, total genomic DNA was extracted using a standard phenol/chloroform extraction procedure (Sambrook et al., 1989). DNA from FTA cards was obtained following the method proposed by Gutiérrez-Corchero et al. (2002). A fragment of the mitochondrial cytochrome b gene (cytb) was amplified with the primers L-15035 (50 -gCCTAATTgTgCAAATTAC CAC-30 ) and H-15985 (50 -CTTgCgATAgggAATAggAC-30 ) specifically designed for this study. Numbers in the primer names refer to the 30 base positions of the primers as referenced to the chicken mtDNA sequence (Desjardins and Morais, 1990). PCR amplifications were performed in a total volume of 50 lL, including 1 buffer (Ecogen, Barcelona), 150 lM of each dNTP, 0.2 lM of each primer, 0.6 U Ecotaq polymerase (Ecogen, Barcelona), 2.5 mM MgCl2 and 20 ng of total genomic DNA. PCR conditions were as follows: 2 min at 94 °C followed by 35 cycles of denaturation at 94 °C for 10 s, annealing at 54 °C for 20 s, and extension at 72 °C for 30 s, with a final extra extension step at 72 °C for 5 min. PCR products were purified using the wizard purification systems (Promega, Madison, WI). Sequencing reactions were performed for both strands and sequenced on an ABI PRISM 3770 DNA Analyzer (Applied Biosystems) following the manufacturer’s instructions. 2.3. Sequence alignment and phylogenetic analyses DNA sequences were edited and assembled using MEGA v. 3.1 (Kumar et al., 2004). Sequence alignment was performed using CLUSTAL W (Thompson et al., 1994) as implemented in MEGA. Site saturation was investigated by plotting the numbers of observed transitions and transversions against genetic distance for each sequence pair. Variation among sequences in base pair composition was tested using a Chi-square (v2) analysis as implemented in PAUP (v4.0b10; Swofford, 2002). Neutrality of the data was tested using the Tajima’s and Fu and Li’s test included in the DnaSP package v4.10 (Rozas et al., 2003). Phylogenetic relationships were constructed by maximum parsimony (MP), maximum likelihood (ML) and Bayesian inference (BI) using unique haplotypes. MP analyses were performed in PAUP (v4.0b10; Swofford, 2002), under the heuristic search option, using the tree-bisection

and reconnection (TBR) branch-swapping algorithm, with 100 random additions of taxa. Branch supports were evaluated using 1000 bootstrap replicates. For the ML phylogenetics analysis, nucleotide substitution model parameters were determined using MODELTEST v. 3.7 (Posada and Crandall, 1998), while for the BI analysis the MrMODELTEST 2.2 (Nylander, 2004) was used. Based on arguments presented by Posada and Buckley (2004), we used the Akaike Information Criterion (Akaike, 1974) to select best-fit models. For both phylogenetic analyses, the general time reversible model with invariant sites proportion (GTR + I) was the appropriate substitutional model for the dataset (ln L = 2593.2227, AIC = 5204.4453). Maximum likelihood tree was reconstructed using TREEFINDER (Jobb, 2006) under the above-mentioned model with P = 0.6972 as the invariant site proportion. BI analysis was conducted with MrBayes v3.1.2 (Ronquist and Huelsenbeck, 2003), using the previously determined model of nucleotide evolution (GTR + I). Analysis was run for 107 generations with a sampling frequency of 102 generations. Of the resulting trees, the first 10,001 trees were discarded as burn in after checking for stationarity with TRACER v1.3 (Rambaut and Drummond, 2003), and the following 90,000 trees were used to estimate topology and tree parameters. The percentage of times a node occurred within those 90,000 trees was interpreted as the posterior probability of the node.

3. Results No evidence of heteroplasmy or nuclear mitochondrial insertions (Numts) was found in the analyzed sequences. The electropherograms presented only one specific band without length mutations, and no stop codons were observed along the sequence. Among the 828 nucleotide sites utilized for the phylogenetic analyses, 204 were variable and 97 were phylogenetically informative. Chi-square tests indicated homogeneity of nucleotide frequencies across taxa (v2 = 9.48; d.f. = 219; P = 1.0). Base composition analyses showed that the sequences were comparable to cytb sequences from other vertebrates with 28.5% A, 31.6% C, 26.2% T and 13.7% G (Kornegay et al., 1993). First, second and third codon positions for all taxa showed dissimilar percentages of nucleotides, especially at third positions with a lack of G (3.4%) and an abundance of A and C (41.1 and 39.2%, respectively). Second positions had an overabundance of T (40.6%) and a low percentage of G (13.4%). First positions had similar percentages for all four bases (A: 24.0%, C: 29.8%, T: 21.6%, G: 24.5%).

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T.E. Klassert et al. / Molecular Phylogenetics and Evolution 47 (2008) 1227–1231 Table 1 List of specimens examined in the present study remarking their provenance localities and haplotype codes according to Fig. 2 Species/subspecies

Haplotype

Locality

N

GenBank Accession

01 02 03 04 05 06 07 08 09 10

Canary Canary Canary Canary Canary Canary Canary Canary Canary Canary

1 1 1 4 2 8 1 10 4 2

AM494415 AM494416 AM494417 AM494418 AM494419 AM494420 AM494421 AM494422 AM494423 AM494424

L. m. algeriensis

Algeria

1

AM494425

L. m. aucheri 01 L. m. aucheri 02

Israel Israel

1 2

AM494426 AM494427

L. e. excubitor 01 L. e. excubitor 02 L. e. excubitor 03

Poland and Hungary Hungary Poland

5 1 2

AM494428 AM494429 AM494430

L. m. pallidirostris

Kazakhstan

2

AM494431

Cáceres, Navarra (Spain) Salamanca (Spain) Cáceres (Spain) Cáceres (Spain) Salamanca (Spain) Navarra (Spain) Navarra (Spain) Navarra (Spain) Navarra (Spain) Cáceres (Spain) Navarra (Spain) Navarra (Spain) Navarra (Spain)

6 1 2 1 1 1 2 1 1 1 1 1 1

AM494432 AM494433 AM494434 AM494435 AM494436 AM494437 AM494438 AM494439 AM494440 AM494441 AM494442 AM494443 AM494444

L. e. invictus

Minnesota (USA)

1

AY030106a

L. s. sphenocercus

Eastern Russia

1

AM494445

L. ludovicianus 01 L. ludovicianus 02

California (USA) USA

1 1

AY030105a X74259a

L. senator

Navarra (Spain)

1

AM494446

Lanius meridionalis koenigi L. L. L. L. L. L. L. L. L. L.

m. m. m. m. m. m. m. m. m. m.

koenigi koenigi koenigi koenigi koenigi koenigi koenigi koenigi koenigi koenigi

Islands Islands Islands Islands Islands Islands Islands Islands Islands Islands

Lanius meridionalis algeriensis Lanius meridionalis aucheri

Lanius excubitor excubitor

Lanius meridionalis pallidirostris Lanius meridionalis meridionalis L. L. L. L. L. L. L. L. L. L. L. L. L.

m. m. m. m. m. m. m. m. m. m. m. m. m.

meridionalis meridionalis meridionalis meridionalis meridionalis meridionalis meridionalis meridionalis meridionalis meridionalis meridionalis meridionalis meridionalis

01 02 03 04 05 06 07 08 09 10 11 12 13

Lanius excubitor invictus Lanius sphenocercus Lanius ludovicianus

Lanius senator Corvus frugilegus Corvus

Y18522a

N, sample size. a Taken from GenBank.

The saturation plots for cytb showed that transitions and transversions were roughly linearly correlated, with the uncorrected pairwise distances with no obvious tendency to level off (data not shown). None of the tests for neutrality of the dataset was significant (Tajima’s D = 0.1779, P > 0.10, Fu and Li’s D = 0.8390 P > 0.10), providing no evidence for selection. 3.1. Phylogenetic trees A single likelihood tree was produced from the Bayesian analysis (Fig. 2). Most nodes within the topology are supported by high posterior probabilities. The likelihood analysis strongly supports (100%) L. m. koenigi as a sister to the L. m. algeriensis subspecies. In addition, the close relationship among L. m. aucheri, L. m. pallidirostris and L. e. excubitor seems to be clear. All of them shared a recent common ancestor (99% posterior support) although the relationships among them are not resolved.

The clades koenigi-algeriensis and aucheri-pallidirostris-excubitor show a closer relationship between them (98% of posterior probability) than with the other clade, which includes among others, L. m. meridionalis specimens from the Iberian Peninsula. Our results indicate that this species (L. m. meridionalis) is more related to L. e. invictus from North America and to L. ludovicianus and L. s. sphenocercus than to the other meridionalis subspecies. The last clade including L. m. meridionalis-L. e. invictus-L. ludovicianus-L. s. sphenocercus and the other species mentioned above constitutes a monophyletic group. An unweighted maximum parsimony analysis of the cytb sequences resulted in 127 most parsimonious trees with step lengths of 287 (CI = 0.7021, RI = 0.6545, RC = 0.7341). The maximum parsimony bootstrap consensus tree showed an identical topology to that of the Bayesian tree with similar node support values. Maximum likelihood analysis gave the same tree and branch support values similar to those found for the Bayesian and parsimony

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Fig. 2. Bayesian tree of the taxa analyzed in this work. Numbers match with those in Table 1. On nodes are represented: posterior probability of Bayesian inference and bootstrap values of the maximum likelihood, and parsimony analyses, respectively. In brackets, the numbers of analyzed specimens for each taxon.

analysis. We have added the percentage of bootstraps obtained by both methods to the Bayesian tree (see Fig. 2). The log likelihood value of the ML tree (GTR + I) was compared with that of the same tree constructed under molecular clock assumptions. The results showed no significant difference between the likelihoods of the two trees (2 log D = 70.48, which approximates to v2 distribution with 70 d.f.; P = 0.52), which allowed us to use the sequences to estimate dates. In this sense, we calibrated our tree using a previously established rate for evolution of this gene in passerines (1.9% substitutions/million years, Fleischer et al., 1998; Barker et al., 2004).

4. Discussion The results of this work show a unique cluster encompassing the four shrike species herein analyzed. This is in agreement with the findings of Harris and Franklin (2000), who studied the taxon-

omy of the Lanius genus based on species mate recognition. Our phylogenetic tree is divided in two clades: (a) Clade 1 including shrikes from northern Africa, Canary Islands, Central Asia (Kazakhstan) and Central Europe (Poland and Hungary). Moreover, two additional groups are differentiated, one with the African subspecies (L. m. algeriensis and L. m. koenigi) and the other one with the Asian subspecies (L. m. pallidirostris and L. m. aucheri) and one European subspecies (L. e. excubitor). Surprisingly, L. e. excubitor is linked to the subspecies currently included in the group meridionalis-lathora suggested by Panov (1995). (b) Clade 2 includes shrikes from North America, Iberia and eastern Asia (Russia, China). The great similarity between L. e. invictus and L. m. meridionalis, two subspecies geographically distant at present, deserves special mention (Fig. 1). This finding is also in agreement with Mundy and Helbig (2004) who studied the evolution of tandem repeats in the mitochondrial DNA control region. These authors proposed a common origin of these two subspecies and L. ludovicianus, which are clearly separated from L. e. excubitor.

T.E. Klassert et al. / Molecular Phylogenetics and Evolution 47 (2008) 1227–1231

The shrike species analyzed in this work and the other species of the genus Lanius belong to the Core Corvoidea sensu (Sibley and Ahlquist, 1990), also admitted by other authors (e.g. Ericson et al., 2003; Barker et al., 2004). According to Barker et al. (2004) the first group of the Core Corvoidea spread from Australia about 34–40 million years ago (MYA). Later, at the end of the Eocene period, a second expansion of other Corvoidea (including the Lanius genus) occurred. These ancestors from Australia arrived to Asia (Briggs, 1987), from where they could spread to America (by the Bering Land Bridge, Ericson et al., 2003), Africa (connected to Eurasia during the Miocene period) and Europe. Applying the substitution rates previously mentioned, our data point to a common ancestor for the shrike species studied in this work around 1.7–1.8 MYA. This date coincides with the beginning of the Pleistocene period, when glaciations in the northern hemisphere began (Haug et al., 2005). The species and subspecies of the above-mentioned clade 1 could have derived from this ancestor. It seems likely that the glaciations forced the species to seek refuge in at least two areas: a Mediterranean one harboring the ancestors of extant subspecies koenigi and algeriensis, and an Asian refuge from where the rest of European and Asian extant species of this clade emerged (0.8–0.9 MYA). The species included in clade 2 could also have derived from the first ancestor. In such case, two branches derived from it 1.1–1.2 MYA, one with L. ludovicianus and the other one with L. s. sphenocercus, L. m. meridionalis and L. e. invictus. However, the origin of these last two subspecies is not clear. All methods of analysis showed polyphyly in the species meridionalis and excubitor (Fig. 2). For the meridionalis species, the phylogenetic tree does not reflect any direct relationship among the meridionalis subspecies from the Iberian Peninsula with either the African, the European, or the western and central Asian subspecies, but with those in North America and eastern Asia. On the contrary, L. e. invictus is more closely related to other North American species than to Paleartic subspecies of L. excubitor. However, these results should be taken with caution since not all species/subspecies of this shrike group have been studied. In any case, the need for a thorough revision of the taxonomy of the shrikes analyzed has become apparent, since our results contradict the current taxonomy. Specifically, we suggest an update of the following issues: (1) Lanius meridionalis meridionalis should be elevated to species status. Our data indicate that it is most closely related to the North American form (invictus) of excubitor and not a member of an otherwise well-supported ‘‘meridionalis” clade. (2) The New World and Old World forms of L. excubitor are not each other’s closest relatives. Each of these clades is deserving of species status. (3) L. m. pallidirostris, L. m. algeriensis, L. m. aucheri and L. m. koenigi, currently included in the species Lanius meridionalis, should be reviewed and assigned to different species. According to these remarks, it is likely that the Lanius [excubitor] superspecies proposed by Lefranc (1993) and Panov (1995) should also be modified. It is clear that more phylogenetic analyses are needed, including other subspecies of L. excubitor and L. meridionalis from the Holarctic region and Africa to elucidate the taxonomy of these shrikes and their phylogenetic relationships.

Acknowledgments Our thanks to Francisco Gutiérrez-Corchero (Spain), Amber E. Budden (Israel), Martin Hromada (Slovakia), Boris Gubin (Kazakhstan), Alexei P. Kryukov (Russia) and Hunor Török (Hungary) for providing the blood samples. Other samples belong to the BIOTAgenes project. This work was partially financed by the Fundación Universitaria de Navarra and for the Consejería de Medio Ambiente y Política Territorial del Gobierno de Canarias. Thanks to Javier

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Hernández and Claire Graham for their assistance. We thank the anonymous referees for helpful and thorough comments on the manuscript.

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