Molecular Phylogenetics and Evolution 54 (2010) 984–994

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Molecular Phylogenetics and Evolution

journal homepage: www.elsevier .com/locate /ympev

The evolutionary diversification of parrots supports a taxon pulse modelwith multiple trans-oceanic dispersal events and local radiations

Manuel Schweizer a,*, Ole Seehausen b,c, Marcel Güntert a, Stefan T. Hertwig a

a Naturhistorisches Museum der Burgergemeinde Bern, Bernastrasse 15, CH 3005 Bern, Switzerlandb Aquatic Ecology and Macroevolution, Institute of Ecology & Evolution, University of Bern, Baltzerstrasse 6, CH 3012 Bern, Switzerlandc Fish Ecology and Evolution, EAWAG, Seestrasse 79, CH 6047 Kastanienbaum, Switzerland

a r t i c l e i n f o a b s t r a c t

Article history:Received 12 June 2009Accepted 17 August 2009Available online 21 August 2009

Keywords:ParrotsBiogeographyDispersalVicarianceNuclear genesTaxon pulse model

1055-7903/$ - see front matter � 2009 Elsevier Inc. Adoi:10.1016/j.ympev.2009.08.021

* Corresponding author. Fax: +41 31 350 74 99.E-mail address: manuel.schweizer@nmbe.ch (M. S

Vicariance is thought to have played a major role in the evolution of modern parrots. However, as therelationships especially of the African taxa remained mostly unresolved, it has been difficult to draw firmconclusions about the roles of dispersal and vicariance. Our analyses using the broadest taxon samplingof old world parrots ever based on 3219 bp of three nuclear genes revealed well-resolved and congruentphylogenetic hypotheses. Agapornis of Africa and Madagascar was found to be the sister group to Loriculusof Australasia and Indo-Malayasia and together they clustered with the Australasian Loriinae, Cyclo-psittacini and Melopsittacus. Poicephalus and Psittacus from mainland Africa formed the sister group ofthe Neotropical Arini and Coracopsis from Madagascar and adjacent islands may be the closest relativeof Psittrichas from New Guinea. These biogeographic relationships are best explained by independent col-onization of the African continent via trans-oceanic dispersal from Australasia and Antarctica in thePaleogene following what may have been vicariance events in the late Cretaceous and/or early Paleogene.Our data support a taxon pulse model for the diversification of parrots whereby trans-oceanic dispersalplayed a more important role than previously thought and was the prerequisite for range expansion intonew continents.

� 2009 Elsevier Inc. All rights reserved.

1. Introduction

Species richness is highly unevenly distributed among taxo-nomic groups and studying the most diverse clades is often associ-ated with major systematic challenges (Soltis, 2007). As speciesrichness is influenced by both the biological traits of the organismsthemselves and their environment (Newton, 2003), the study ofspecies-rich groups can provide insights into the mechanisms ofspeciation and accumulation of species diversity, as well as intohistorical biogeography. For a long time, vicariance approacheshave dominated historical biogeography; recent works suggesthowever, that dispersal is an important process in speciation andthe build-up of regional fauna and that the importance of oceanicdispersal has been strongly underestimated (de Queiroz, 2005;Cowie and Holland, 2006; McGlone, 2005; Yoder and Nowak,2006).

With 353 species currently recognized, parrots represent one ofthe most species-rich groups of birds (Collar, 1998; Rowley, 1998).They have radiated extensively in the Neotropical and Australasianregion and to a smaller extent in the Afrotropical and Indo-Mala-yan region (Collar, 1998; Cracraft, 2001; Rowley, 1998; Smith,

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chweizer).

1975; Wright et al., 2008). Recent molecular phylogenetic studiesagree that the New Zealand taxa Kea (Nestor notabilis) and Kakapo(Strigops habroptilus) are the sister group of all other parrots (deKloet and de Kloet, 2005; Tavares et al., 2006; Tokita et al.,2007; Wright et al., 2008). The mainly Australasian Cacatuidae isthe taxon that branches off next (Tokita et al., 2007; Wrightet al., 2008) and the monophyly of the new world parrots is wellsupported (de Kloet and de Kloet, 2005; Tavares et al., 2006; Tokitaet al., 2007; Wright et al., 2008). These phylogenetic and biogeo-graphic relationships have been interpreted as supporting avicariance speciation model and as modern parrots are mostlynon-migratory (Collar, 1998), it has been suggested that theirdiversification pattern and evolutionary history may be less influ-enced by dispersal than in other avian groups. This has been takenin turn to suggest that vicariance was a major force in parrot diver-sification following the break-up of Gondwana (cf. Wright et al.,2008). There is still some controversy about the timescale of theevolution of parrots similar to other groups of modern birds withthe fossil record suggesting a Cenozoic origin for most lineageswhereas molecular genetic approaches date the origin of the samelineages in the Cretaceous before the K-Pg boundary (e.g. Brownet al., 2007, 2008; Cooper and Penny, 1997; Cracraft, 2001; Ericsonet al., 2006; Pratt et al., 2009). Although no representative of crowngroup Psittaciformes is known from Paleogene fossil deposits

M. Schweizer et al. / Molecular Phylogenetics and Evolution 54 (2010) 984–994 985

(Mayr, 2009), molecular dating analyses calibrated with externaldating points and/or biogeographic approaches suggest a Gondwa-nan origin of the parrots during the late Cretaceous (de Kloet andde Kloet, 2005; Tavares et al., 2006; Wright et al., 2008). However,a recent molecular dating of the origin of the major Neoaves lin-eages based on complete mitochondrial genomes and using twofossil calibration points suggested that the Kakapo split from theremaining parrots only after the K-Pg boundary (Pratt et al.,2009). Wright et al. (2008) compared two internal calibrationpoints to estimate the divergence times within the parrots. First,the separation of New Zealand from Gondwana 82–85 MYA assum-ing that this date coincides with the separation of the New ZealandKea and Kakapo from all other Parrots. This calibration point wasalso used by other authors (Ribas et al., 2007; Tavares et al.,2006). Second, a minimum age of 50 MYA for the same initial splitbased on fossil records of stem parrots in Europe accounting for thehypothesis of a radiation of modern bird orders during the Paleo-gene. Wright et al. (2008) considered the latter scenario less likelyas it would have required oversea dispersal to New Zealand, Mad-agascar and South America from Australia.

However, to assess the likelihood of possible dispersal or colo-nization routes and vicariance events, we need to answer a fewcritical phylogenetic questions. Especially the phylogenetic andbiogeographic history of the recent African parrots remains contro-versial. African parrots include four endemic genera, Coracopsis (2species) from Madagascar, the Comoro Islands and the Seychelles,the African and Malagasy Agapornis (9 species) as well as the Afri-can genera Poicephalus (9 species) and Psittacus (1 species). Further,Psittacula echo occurs on Mauritius and Africa has an endemic sub-species of the otherwise Asian Psittacula krameri (Collar, 1998).Various phylogenetic relationships have been proposed for Africanparrots and it was unclear how the different groups reached thiscontinent (de Kloet and de Kloet, 2005).

The aim of the present study is therefore to investigate the phy-logenetic and biogeographic history of parrots to infer the sistergroup relationship especially of African parrots in order to testthe role of dispersal vs. vicariance in their diversification. Com-pared to previous studies, we increased the taxon sampling andchose a different set of markers consisting of the three nuclearexons c-mos, Rag-1 and Zenk. Rag-1 is a single copy nuclear exoninvolved in recombination and has been widely applied to phylo-genetic studies at genus level within avian orders and families(e.g. Groth and Barrowclough, 1999; Paton et al., 2003; Barkeret al., 2004; Griffiths et al., 2004; Tavares et al., 2006; Pereiraet al., 2007; Treplin et al., 2008). Zenk is a single copy nuclear tran-scription factor that has utility to resolve divergences among(Chubb, 2004a; Long and Salbaum, 1998) as well as within avianorders (Chubb, 2004b; Treplin et al., 2008). Finally, the single copynuclear proto oncogene c-mos has been used successfully forresolving phylogenetic relationships among intermediate andmore distantly related species of birds (Lovette and Bermingham,2000; Overton and Rhoads, 2004).

2. Materials and methods

2.1. Taxon sampling and character sampling

We concentrated on sampling parrots from the old world as themonophyly of the new world taxa is well supported (see above).We sampled 60 old world species plus one Neotropical species.We analyzed between two and five species from those genera thatwere of particular interest because their sister group relationshipscould not be resolved in previous studies (five species of Agapornis,four of Neophema, three of Loriculus and two each of Micropsitta,Coracopsis and Poicephalus). A phylogenomic study by Hackett

et al. (2008) revealed a sister group relationship between the Psit-taciformes and the Passeriformes with both forming a clade to-gether with the Falconidae. We selected therefore two suboscinePasseriformes (Pipra and Pitta), two oscine Passeriformes (Corvusand Picathartes) as well as one Falco as outgroups and rooted treeswith the latter taxon (Table 1).

2.2. Laboratory methods

Frozen tissues of birds collected in the wild or from captivity aswell as feathers sampled from captive birds were used for the iso-lation of genomic DNA (Table 1). Total genomic DNA was isolatedusing peqGOLD tissue DNA Mini Kit following manufacturer’s rule(Peqlab). Partial sequences of the three nuclear genes c-mos, RAG-1and Zenk (second exon) were amplified with Polymerase chainreaction (PCR) using different sets of published primer sequences(Table 2). PCR reaction volumes were 20 ll containing 10 ll PCR-Master-Mix S (c-mos) or PCR-Master-Mix Y (RAG1, ZENK) (Peqlab),2–3 ll genomic DNA, 2 ll of each primer with a concentration of10 lM and 3–4 ll ddH2O. PCR was performed on a Techne TC-512 thermo-cycler. Amplifications of c-mos was performed withthe following parameters: initial denaturation of 94 �C for 2 minfollowed by 33 cycles of denaturation at 90 �C for 30 s, annealingat 55 �C for 30 s, and extension at 72 �C for 1 min, with a finalextension at 72 �C for 5 min. The PCR reaction profile publishedby Groth and Barrowclough (1999) was used for RAG1 with the ini-tial denaturation step reduced to 2 min. For Zenk, the PCR profile ofChubb (2004a) was used with the annealing temperature set to53.5 �C. PCR products were examined by gel electrophoresis toconfirm the amplification of the target fragment. PCR productswere either excised from gels and cleaned using the Wizard� SVGel and PCR Clean-UP System (Promega) or directly purified withthe above mentioned kit or with the peqGOLD MicroSpin Cycle-Pure Kit (Peqlab). To increase the quantity of DNA for problematicsamples, the products of two independent PCR runs were put to-gether before the cleaning or a second PCR was performed afterthe cleaning. Sequencing was carried out with Microsynth AG(Balgach, Switzerland) using the same primers as for amplification.All three genes were sequenced from both sides leading to com-plete overlapping fragments for Rag-1 and c-mos and an overlap-ping fragment of about 600 bp for Zenk. Sequencing files werechecked with Chromas (Technelysium Pty., Ltd.) and ambiguitieswere assigned standard IUB codes. The Alignment of the sequenceswas done manually with BioEdit 7.0.5.2 (Hall, 1999). We checkedindividual Sequences and the whole alignment further for qualityby searching for apparent stop codons after the translation of se-quences into amino acids and for indels that were not a multipleof three bases.

2.3. Sequence and phylogenetic analyses

A Chi-square test of homogeneity of base frequencies acrosstaxa as implemented in PAUP* (Swafford, 2001) was used to testthe variation of base frequencies between taxa for each gene. Datasets of the different genes were tested pairwise for heterogeneityusing the incongruent length difference test (ILD) (Farris et al.,1995) implemented in PAUP* to asses combinability of the differ-ent data sets with taxa missing from either data set excluded (heu-ristic search, 1000 replicates, number of max trees limited to1000). Additionally, we tested the potential loss of information inthe third codon position of all three genes due to substitutionsaturation estimating the index of substitution saturation (Iss) byXia et al. (2003) with DAMBE (Xia and Xie, 2001). The phylogeneticinformativeness per site was calculated as the quotient of the num-ber of parsimony-informative sites and the sequence length to

Table 1Species sampled, Museum and collection number, GenBank Accession numbers for the three genes analyzed and sample type.

Species Museum/collection Collection nb. c-mos Rag-1 Zenk type

Agapornis canus NMBE 1056201 GQ505083 GQ505191 GQ505138 TissueAgapornis fischeri NMBE 1056202 GQ505084 GQ505192 GQ505139 TissueAgapornis lilianae NMBE 1056205 GQ505087 — — TissueAgapornis nigrigenis NMBE 1056203 GQ505085 GQ505193 GQ505140 TissueAgapornis roseicollis NMBE 1056204 GQ505086 GQ505194 GQ505141 TissueAlisterus chloropterus NMBE 1056207 GQ505091 GQ505199 GQ505145 TissueAlisterus scapularis NMBE 1056206 GQ505090 GQ505198 GQ505144 TissueAprosmictus jonquillaceus NMBE 1056208 GQ505092 GQ505200 GQ505146 TissueBarnardius zonarius NMBE 1056210 GQ505095 GQ505203 GQ505149 TissueCacatua galerita fitzroyi NMBE 1056235 GQ505120 GQ505231 GQ505173 TissueCacatua moluccensis NMBE 1056236 GQ505121 GQ505232 GQ505174 TissueCalyptorhynchus funereus NMBE 1056233 GQ505118 GQ505229 – TissueCalyptorhynchus latirostris NMBE 1056234 GQ505119 GQ505230 GQ505172 TissueCharmosyna pulchella NMBE 1056241 GQ505126 GQ505237 GQ505179 TissueCoracopsis nigra GQ505114 GQ505224 — FeathersCoracopsis vasa UWBM 85986/2004-001 GQ505113 GQ505223 GQ505167 TissueCyanoramphus auriceps NMBE 1056221 GQ505104 GQ505213 GQ505158 TissueCyanoramphus novaezelandiae NMBE 1056220 GQ505103 GQ505212 GQ505157 TissueCyclopsitta diophthalma GQ505130 — — FeathersEclectus roratus NMBE 1056248 GQ505135 GQ505244 GQ505187 TissueEos cyanogenia NMBE 1056237 GQ505122 GQ505233 GQ505175 TissueEunymphicus cornutus cornutus NMBE 1056223 GQ505106 GQ505215 GQ505159 TissueEunymphicus cornutus uvaeensis NMBE 1056224 GQ505107 GQ505216 GQ505160 TissueLathamus discolor NMBE 1056219 GQ505102 GQ505211 GQ505156 TissueLoriculus catamene ZMUC 115584 GQ505088 GQ505195 GQ505142 TissueLoriculus galgulus UWBM 73841/2002-006 GQ505089 GQ505196 — TissueLoriculus philippensis ZMUC 130608 – GQ505197 GQ505143 TissueLorius garrulus NMBE 1056240 GQ505125 GQ505236 GQ505178 TissueMelopsittacus undulatus UWBM 60748/1998-068 – GQ505222 GQ505166 TissueMicropsitta finschii tristrami UWBM 66040/2000-022 GQ505128 GQ505240 GQ505182 TissueMicropsitta pusio UWBM 67905/2001-054 GQ505129 GQ505241 GQ505183 TissueNeophema chrysogaster NMBE 1056227 GQ505110 GQ505219 GQ505163 TissueNeophema chrysostoma NMBE 1056228 GQ505111 GQ505220 GQ505164 TissueNeophema pulchella NMBE 1056226 GQ505109 GQ505218 GQ505162 TissueNeophema splendida NMBE 1056225 GQ505108 GQ505217 GQ505161 TissueNeopsephotos bourkii UWBM 57542/1996-109 GQ505112 GQ505221 GQ505165 TissueNestor notabilis NMBE 1056242 — GQ505238 GQ505180 TissuePlatycercus caledonicus NMBE 1056212 GQ505097 GQ505205 GQ505151 TissuePlatycercus eximius NMBE 1056213 — GQ505206 GQ505152 TissuePlatycercus flaveolus NMBE 1056215 GQ505099 GQ505208 — TissuePlatycercus venustus NMBE 1056214 GQ505098 GQ505207 GQ505153 TissuePoicephalus rufiventris NMBE 1056230 GQ505116 GQ505226 GQ505169 TissuePoicephalus senegalus NMBE 1056231 — GQ505227 GQ505170 TissuePolytelis alexandrae NMBE 1056209 GQ505093 GQ505201 GQ505147 TissuePolytelis anthopeplus NMBE 1056657 GQ505094 GQ505202 GQ505148 TissuePrioniturus discurus NMBE 1056246 GQ505133 — GQ505186 TissuePrioniturus luconensis NMBE 1056247 GQ505134 — — TissueProsopeia tabuensis NMBE 1056252 GQ505105 GQ505214 — TissuePsephotus dissimilis NMBE 1056218 GQ505101 GQ505210 GQ505155 TissuePsephotus varius NMBE 1056217 GQ505100 GQ505209 GQ505154 TissuePsittacula eupatria NMBE 1056250 GQ505137 GQ505246 GQ505189 TissuePsittaculirostris desmarestii NMBE 1056244 GQ505131 GQ505242 GQ505184 TissuePsittaculirostris edwardsii NMBE 1056245 GQ505132 GQ505243 GQ505185 TissuePsittacus erithacus erithacus NMBE 1056229 GQ505115 GQ505225 GQ505168 TissuePsitteuteles goldiei NMBE 1056239 GQ505124 GQ505235 GQ505177 TissuePsittinus cyanurus NMBE 1056251 — GQ505247 GQ505190 TissuePsittrichas fulgidus NMBE 1056243 GQ505127 GQ505239 GQ505181 TissuePurpureicephalus spurius NMBE 1056211 GQ505096 GQ505204 GQ505150 TissueTanygnathus megalorhynchus NMBE 1056249 GQ505136 GQ505245 GQ505188 TissueTrichoglossus johnstoniae NMBE 1056238 GQ505123 GQ505234 GQ505176 TissueTriclaria malachitacea NMBE 1056232 GQ505117 GQ505228 GQ505171 TissuePipra coronata AY056951 AY057020 AF492518Pitta AY056952 AY057021 EF568299Corvus corone AY056918 AY056989 EF568306Picathartes gymnocephalus AY056950 AY057019 EF568314Falco AY447974 AY461399 AF490155

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account for the different number of characters sampled from eachgene (Treplin et al., 2008).

Phylogenetic analyses were conducted using model based ap-proaches (Bayesian inference BI and maximum likelihood ML)and maximum parsimony analyses (MP). BI of the phylogeneticrelationships was done with MrBayes 3.1 (Huelsenbeck andRonquist, 2001; Ronquist and Huelsenbeck, 2003). We chose a

mixed-model approach and evaluated the significance of six differ-ent biologically relevant ways to partition our data into gene and/or codon positions. Separate models for the sequences of the differ-ent data partitions were evaluated with Mr. Modeltest 2.3 (Nyland-er, 2004) using the Akaike information criteria (Akaike, 1974). Therelevance of the different partitions was evaluated with the BayesFactor (BF) (Brown and Lemmon, 2007; Kass and Raftery, 1995) and

Table 2Primers used for the amplification of the three genes analyzed in this study.

Gene Primer name References

c-mosF944 Cooper and Penny (1997)R1550or05 Overton and Rhoads (2004)R1550hb99 Hughes and Baker (1999)

Rag-1R8 Groth and Barrowclough (1999)R11B Groth and Barrowclough (1999)R17 Groth and Barrowclough (1999)R18 Groth and Barrowclough (1999)

ZenkZ1F Chubb (2004a)Z9R Chubb (2004a)

M. Schweizer et al. / Molecular Phylogenetics and Evolution 54 (2010) 984–994 987

the harmonic mean calculated by MrBayes was used as an estima-tion of the marginal likelihood of the data (Kass and Raftery,1995). A more complicated model (i.e. more partitions) was favoredcompared to a simpler model if 2lnBF was greater than 10 (Brownand Lemmon, 2007). Base frequencies, rate matrix, shape parameterand proportion of invariable sites were allowed to vary between par-titions. For each data partition, we ran two independent runs ofMetropolis-coupled Markov chain Monte Carlo analyses each con-sisting of one cold chain and three heated chains with default tem-perature of 0.2. The chains were run for 5 million generations withsampling every 100 generations and the first 25% of samples werediscarded as burn-in (12,500 trees) and we checked that the averagestandard deviation of split frequencies converged towards zero.

For the maximum likelihood search, we used RAxML 7.0.4 (Sta-matakis, 2006) running the program on the Web-server with 100rapid bootstrap inferences with all free model parameters esti-mated by the software (Stamatakis et al., 2008). We partitionedour alignment into three genes with splitting each gene into threecodon positions (9 partitions) as this partition was chosen as thebest model in our BI (see below) and allowed RAxML to estimatean individual model of nucleotide substitution for each partition.

Analyses using the maximum parsimony criterion were con-ducted in PAUP* (heuristic search, 1000 random taxon-additionreplicates, TBR branch swapping). Gaps were treated as fifth char-acter state. Nodal support was estimated with a maximum parsi-mony bootstrap analysis (1000 pseudo-replicates, heuristic

Table 3Sequence characteristics and model parameters for the three genes and their different par

c-cmos Rag-1

1pos

1&2pos

2pos

3pos

1pos

1&2pos

2pos

Size (bp) 603 1461%A 0.24 0.26 0.28 0.17 0.32 0.33 0.34 0.35%C 0.24 0.22 0.23 0.29 0.20 0.21 0.20 0.19%G 0.30 0.37 0.30 0.29 0.22 0.27 0.24 0.20%T 0.22 0.16 0.20 0.25 0.25 0.19 0.23 0.27Model GTR HKY HKY K80 HKY GTR GTR GTR GTRTi/Tv ratio 3.06 4.09 5.85 3.74A–C 0.07 0.07 0.13 0.09 0.04A–G 0.31 0.34 0.29 0.33 0.36A–T 0.04 0.04 0.06 0.04 0.04C–G 0.05 0.07 0.08 0.10 0.13C–T 0.49 0.43 0.40 0.40 0.39G–T 0.04 0.04 0.05 0.04 0.04Prop Inv 0.42 0.72 0 0 0 0.00 0.00 0.00 0.48Gamma 1.03 — 0.10 0.11 1.26 0.44 0.45 0.27 0.62ParsInf 128 304inormativeness

per site0.21 0.21

Resolved nodesin MP search (nb)

102 108

search, 10 random taxon-addition replicates, TBR branch swap-ping, number of max trees limited to 100). MP search was con-ducted for the combined data set and each gene separately.Clades were considered as supported by our analyses when boot-strap values were P70% (Hillis and Bull, 1993) and clade credibilityvalues for the BI P 0.95 (Huelsenbeck and Ronquist, 2001).

2.4. Reconstruction of ancestral area states

We choose a maximum likelihood approach to infer the ancestralareas where the different groups of parrots have originated based onthe tree topology obtained with the BI and ML analysis. We inferredfor each node the area assignment that maximizes the probability ofarriving at the observed areas in the terminal taxa given a stochasticMarkov model of evolution (Lewis, 2001) as implemented in Mes-quite 2.5 (build j77) (Maddison and Maddison, 2006, 2008). The cur-rent distribution areas of the different species were categorized infive discrete character states based on the following biogeographicrealms (Newton, 2003): Afrotropical, Australasian, Indo-Malayanand Neotropical. We further included Madagascar and adjacent is-lands as a discrete character state. The distribution areas assignedto the different species follow Collar (1998) and Rowley (1998). Lori-culus catamene occurs on Sangihe in Wallacea, the transition zonebetween the Indo-Malayan and the Australasian realms. However,it is part of a superspecies complex which contains taxa of New Gui-nea, the Bismarck Archipelago, Halmahera, Sulawesi and adjacentisland and was therefore assigned to the Australasian region. Ourspecies sampling covered the whole distribution areas of the differ-ent groups with the exception that several species of the Loriinae oc-cur additionally in the Oceanian region, one Cockatoo species entersthe Indo-Malayan region and that within the Psittaculini, Psittaculakrameri has a subspecies occurring in Africa and Psittacula echo oc-curs on Mauritius.

3. Results

3.1. Sequence characteristics

The final alignment was 3219 bp in length, consisting of 603 bpfor c-mos, 1461 bp for Rag-1 and 1155 bp for Zenk (Table 3). It con-tained one indel of four amino acids for c-mos, one indel of threeamino acids and one indel of one amino acid for Rag-1 as well as

titions analyzed. Base substitution rates are the mean values obtained with MrBayes.

Zenk comb. data set

3pos

1pos

1&2pos

2pos

3pos

1pos

1&2pos

2pos

3pos

1155 32190.29 0.24 0.30 0.27 0.25 0.17 0.28 0.31 0.31 0.30 0.240.21 0.35 0.27 0.35 0.43 0.38 0.27 0.24 0.26 0.28 0.270.20 0.20 0.20 0.18 0.15 0.23 0.22 0.26 0.23 0.19 0.230.30 0.20 0.23 0.21 0.17 0.23 0.23 0.19 0.21 0.23 0.26HKY HKY HKY GTR GTR GTR HKY HKY GTR GTR HKY4.16 3.32 2.14 3.57 2.32 3.91

0.07 0.07 0.07 0.08 0.040.48 0.43 0.52 0.39 0.410.04 0.06 0.05 0.04 0.040.09 0.18 0.04 0.08 0.100.21 0.21 0.28 0.36 0.370.10 0.05 0.04 0.05 0.03

0.00 0.00 0.00 0.76 0.86 0.00 0.38 0.45 0.58 0.66 0.001.23 0.31 0.23 — — 1.62 0.97 0.87 0.79 0.64 1.03

174 6060.15 0.19

93 123

988 M. Schweizer et al. / Molecular Phylogenetics and Evolution 54 (2010) 984–994

four indels of one amino acid and each one indel of three and twoamino acids for Zenk. Due to missing data at the end of some se-quences, their length ranged from 502 to 603 bp (88%>580 bp)for c-mos, 701–1461 bp (79%>1300 bp) for Rag-1 and 763–1155 bp (95%>1075 bp) for Zenk. The translation into amino acidsdid not reveal any unexpected stop codons and the indels were amultiple of three bases. Both the Chi-square test of homogeneityof base frequencies and the pairwise incongruence length differ-ence test revealed no significant heterogeneity. Substantial satura-tion at the third codon position of each gene did not influence ourphylogenetic inference as tested by Iss statistics (all P < 0.05).

3.2. Phylogenetic analysis

The model based approaches and the MP analysis yielded highlycongruent trees and the sister group relationships of most African

Fig. 1. 50% Majority-rule consenus tree of the Bayesian inference with clade credibilityclade credibility values of 1 or bootstrap values of 100. The notations for the different clatree.

taxa could be resolved with large confidence. Different models ofnucleotide evolution were selected for the different partitions ofour data set (Table 3). The six partitions tested with BI yielded al-most identical trees with only small changes in the value of theclade credibility intervals. The only topological difference con-cerned the placement of the clade of Coracopsis and Psittrichas inthe 50% majority rule consensus tree of the concatenated dataset compared to the other partitions. After calculation of the BayesFactors, the partitioning into three genes with splitting each geneinto three codon positions (nine partitions) was chosen as thebest-fit model. The 50% majority rule consensus tree of this analy-sis was identical in topology to the best-scoring maximum likeli-hood tree (Fig. 1).

As no significant heterogeneity between the three genes was re-vealed with the pairwise incongruence length difference test, MPanalysis was performed with the concatenated data set where

values and bootstrap values of the ML likelihood tree indicated at nodes. *Indicatedes used in the text based on traditional taxonomy are indicated to the right of the

M. Schweizer et al. / Molecular Phylogenetics and Evolution 54 (2010) 984–994 989

120 trees were equally parsimonious with 123 resolved nodes inthe strict consensus tree (Fig. 2). Apart from different numbers ofresolved nodes, there were no conflicts among the topologies ofthe strict consensus trees for the single markers and of the com-bined data set (Table 3) and the latter did not show any conflictto the BI and ML tree.

As expected, the sister group position of Nestor to the remainingparrots was highly supported overall (we did not include Strigopsin our sampling). The next group branching off were the Cacatui-dae. A clade consisting of ((Poicephalus + Psittacus) + Triclaria) re-ceived robust support throughout, thus revealing a sister grouprelationship between two of the endemic African genera and theNeotropical parrots. A clade containing all Platycercini, the Lorii-nae, the Cyclopsittacini as well as Loriculus and Agapornis was sup-ported by the BI and ML analysis. Within this clade, the monophylyof the analyzed Platycercini group A (cf. Fig. 1) was supported over-all, as was a clade containing Agapornis, Loriculus, the Loriinae, theCyclopsittacini and Melopsittacus. Within the latter clade, the clus-ter of the African Agapornis and the Indo-Malayan/AustralasianLoriculus was the sister group of an Australasian clade consisting

Fig. 2. Strict consensus tree of the maximum parsimony analysis of the

of ((Loriinae + Melopsittacus) + Cyclopsittacini). Coracopsis fromMadagascar and adjacent islands clustered with Psittrichas fromNew Guinea in the BI and ML analysis, but this clade was not ro-bustly supported. The sister group position of the Micropsittini tothe Psittaculini group A was well supported in the model basedapproaches.

3.3. Reconstruction of ancestral area states

Our ancestral area reconstruction revealed that several majortrans-oceanic dispersal events must be considered to explain thecurrent distribution patterns of parrots (see below). The Markov-ML reconstruction of ancestral areas indicated that the commonancestor of all extant parrots occurred in Australasia (Fig. 3). Thecommon ancestor of Psittrichas and Malagassy Coracopsis as wellas of the ‘‘Loricoloriinae” lived in Australasia, and so did the com-mon ancestor of Loriculus and the African and Malagassy Agapornis.However, the ancestral areas of the common ancestor of Agapornisand of that of the clade containing the Arini, Poicephalus and Psitta-cus could not be unambiguously resolved.

concatenated data set with bootstrap values indicated at each node.

Fig. 3. Cladogram with Markov-ML reconstruction of ancestral areas. Pie charts at each node represent the proportion of the total likelihood received by each biogeographicregion as the ancestral area of a given clade. The present distribution of the taxa is indicated by the color of the circles at the tips of the tree. Inferred dispersal events areindicated at the clades concerned. The colonization of Africa from Indo-Malaysia by Psittacula krameri is also indicated, although this species is not included in our sampling.

990 M. Schweizer et al. / Molecular Phylogenetics and Evolution 54 (2010) 984–994

4. Discussion

4.1. Utility of marker system

Our analyses based on 3219 bp of three nuclear exons with thebroadest taxon sampling of old world parrots analyzed so farrecovered with high support the phylogenetic relationship of somecontroversial taxa. We revealed exactly the same topology thatHackett et al. (2008) recovered with about 10 times more bp whencomparing the taxa sampled in both studies (Hackett et al. (2008)included only 7 parrot species in their study). Hence, we can havesome confidence that the relationships among all other taxa sam-pled are reasonably reliably resolved. A recent simulation studyprovided evidence that introns have a greater potential than exons

to reconstruct phylogenies at least for regions in the tree with clo-sely-spaced branching events like at the base of Neoaves (Choj-nowski et al., 2008). However, in our analyses using exons, treeresolution and nodal support were higher for some clades withinand among the different old world parrot taxa compared to a re-cent phylogenetic study of parrots based on nuclear introns andmitochondrial genes (Wright et al., 2008). This difference couldbe due to our broader taxon sampling (e.g. Poe and Swofford,1999; Tavares et al., 2006; Zwickl and Hillis, 2002). We sampledmore old world taxa than Wright et al. (2008) and included twoto more representatives of controversial taxa. Our results supportthe utility of nuclear genes for the reconstruction of phylogeneticrelationships within avian orders consistent with recent data forthe Passeriformes (Treplin et al., 2008).

M. Schweizer et al. / Molecular Phylogenetics and Evolution 54 (2010) 984–994 991

In the MP analyses, Zenk showed the lowest informativenessper site and the lowest amount of resolved nodes in the MP strictconsensus tree compared to Rag-1 and c-mos. This is in contrast tothe study of Treplin et al. (2008) where the informativeness persite was similar between Zenk and Rag-1 and the number of re-solved nodes in the strict consensus tree of the MP analyses washigher for Zenk than for Rag-1. However, we sequenced only thesecond exon of Zenk, whereas Treplin et al. (2008) included 5020

bp of the 30 UTR of Zenk additional to 1149 bp of the second exon.The second exon of Zenk together with the 30 UTR has the greatestutility in resolving splits among deep lineages within major aviangroups which occurred roughly between 60 and 10 MYA (Chubb,2004b), but was also able to resolve even older splits within thePasseriformes based on a broader taxon sampling (Treplin et al.,2008). In our data set, the second exon of Zenk failed to resolvedeep splits in the Psittaciformes similar to c-mos, while Rag-1 per-formed best throughout. Since at least the deep splits within thePsittaciformes were thought of as having similar ages as withinthe Passeriformes (Barker et al., 2004; Wright et al., 2008) a similartree resolution of Zenk compared to Rag-1 would have been ex-pected for these two groups. The 30 UTR of Zenk could thereforebe more useful to resolve such deep splits than its second exon.However, alternatively, highly variable substitution rates amongavian lineages may account for these differences (Hackett et al.,2008).

4.2. Phylogenetic relationships within the parrots

We succeeded to resolve the phylogenetic relationships of sev-eral controversial taxa with high nodal support and confirmed re-cent results of molecular phylogenies which were in conflict withtraditional views of the evolutionary history of the parrots (Collar,1998). Previous molecular studies could not resolve the position ofthe African taxa Poicephalus and Psittacus, however, we identifiedthe Neotropical Arini as their sister group. Psittacus and Poicephalushave been grouped together with Coracopsis from Madagascar andadjacent islands as Psittacini in part because their relationships toother groups were not clear (Collar, 1998). However, de Kloet andde Kloet (2005) hypothesized a close relationship of Coracopsiswith Psittrichas fulgidus from New Guinea. Our study recovered thisclade but without robust support, which leaves the affinities ofthese taxa still not unambiguously resolved.

In congruence with Wright et al. (2008), we found a clade ofAfrican, Australasian and Indo-Malayan taxa containing the Platy-cercini group A and the Loricoloriinae. Melopsittacus was tradition-ally included within the Platycercini (Collar, 1998), however, wefound it to belong to a clade consisting of the Loriinae and theCyclopsittacini. This is congruent with other molecular studies(de Kloet and de Kloet, 2005; Wright et al., 2008). The sister groupof this clade could not be unambiguously resolved by Wright et al.(2008). We revealed it to be a clade consisting of Agapornis fromAfrica and Madagascar and the Indo-Malayan/Australasian Loricu-lus. A close relationship between the latter two genera had alreadybeen suggested earlier (cf. Smith, 1975), and was found recentlyalso by Wright et al. (2008). Within the Agapornis species analyzed,A. canus was the sister taxon to the remaining species. A. canus isthe only species of this genus occurring exclusively on Madagascar,while all others are confined to Africa. Within Loriculus, we foundthat the Australasian L. catamene is the sister species of the Indo-Malayan species pair L. philippensis and L. galgulus. Agapornis andLoriculus were traditionally treated as Psittaculini, but our findingscorroborate the proposition of Mayr (2008) based on hypotarsalmorphology that these two genera are closely related to the Lorii-nae, the Cyclopsittacini and Melopsittacus. Mayr (2008) proposedthe name Loricoloriinae for this clade. However, he included Micro-psitta (Micropsittini) in the Loricoloriinae which we found to be the

sister group of the Psittaculini group A. The Australasian Platycer-cini group A were revealed to be monophyletic in contrast toWright et al. (2008), where the position of Neopsephotus and Neop-hema was not consistently resolved. The latter were found to bethe sister group to the remaining Platycercini group A. The mono-typic genus Lathamus was for the first time analyzed with molecu-lar data here. It has been variously treated as a member of theLoriinae, but our data confirm its placement within the Platycercini(cf. Collar, 1998).

4.3. Multiple events of long distance trans-oceanic dispersal

Our data suggest Australasia is where a common ancestor of allextant parrots lived. This was also suggested recently by Wrightet al. (2008). To explain the current distribution patterns of the dif-ferent groups based on our robustly supported phylogenetichypothesis, several long distance dispersal and colonization eventshave to be invoked regardless if modern parrots initially split be-fore or after the K-Pg boundary, and especially the African conti-nent must have been colonized independently more than once(Fig. 3).

The origin of the Psittacini of Africa (Poicephalus, Psittacus,excluding Coracopsis) was dated around the K-Pg boundary byWright et al. (2008) when they used a Gondwana calibration point.African was already separated from the southern continents in thelate Cretaceous no matter which of several different models for thebreak-up of Gondwana is assumed (Upchurch, 2008). The origin ofthe Psittacini can therefore not be explained by vicariant evolutionas a consequence of the separation of Africa from Gondwana. Wefound the Psittacini to be the sister group of the Neotropical Arini,but the ancestral distribution area of their common ancestor couldnot be unambiguously resolved. We hypothesize that a commonancestor of the Arini and the Psittacini became separated fromthe Australasian lineages on Antarctica through vicariant evolutioncoinciding with the beginning of seafloor spreading between Aus-tralia and Antarctica in the late Cretaceous and their final separa-tion about 40 MYA (Li and Powell, 2001). At the beginning of thePaleogene, Antarctica was ice free, warmer and wetter than todayand separated into West and East Antarctica by a seaway (Lawverand Gahagan, 2003). We hypothesize that the common ancestor ofthe Psittacini and that of the Arini diverged between East and WestAntarctica and that the major lineages of the Arini identified byTavares et al. (2006) evolved subsequently on West Antarctica,the southern cone of South America and the rest of this continent.When climate change began during the Eocene with a trend to-wards cooler conditions, continental ice-sheets expanded rapidlyon Antarctica in the earliest Oligocene (Zachos et al., 2001). Parrotsprobably dispersed out of Antarctica as was proposed for the Ariniby Tavares et al. (2006). The ancestors of the Psittacini may havereached Africa by trans-oceanic dispersal from East Antarctica viathe Kerguelen Plateau (de Kloet and de Kloet, 2005; Fig. 4, seebelow).

The genus Agapornis is restricted to Africa and Madagascar,whereas its sister genus Loriculus is distributed in the Indo-Mala-yan and Australasian region with eight of 13 species occurringexclusively in Australasia when the Wallace’s line is taken as aboundary between the Australasian and Indo-Malayan region (Col-lar, 1998). We identified Australasia as the ancestral area for acommon ancestor of Agapornis and Loriculus. The ancestor of Aga-pornis, hence, must have reached Africa from Australasia. The splitbetween these genera is clearly younger than the separation ofGondwana, and thus requires another event of trans-oceanic dis-persal. We hypothesize that the common ancestor of Agapornis dis-persed from Australasia to Madagascar and finally, Agaporniscolonized the African continent from Madagascar (Fig. 4).

Fig. 4. Inferred trans-oceanic dispersal routes of parrots. Emergent continentsabove sea level today are shaded in grey and continental shelves are indicated withblack lines (redrawn from Li and Powell (2001)), corresponding to the middleEocene, when the dispersal of Agapornis, the Psittacini and Coracopsis could haveoccurred. Following Fjeldså and Bowie (2008), the Broken Ridge and the Kerguelenarchipelago are indicated where volcanic islands could have served as steppingstones. The dispersal of Loriculus, Psittacus and Psittinus probably occurred lateraround 20–25 MYA.

992 M. Schweizer et al. / Molecular Phylogenetics and Evolution 54 (2010) 984–994

A third case of trans-oceanic dispersal to the African region, andspecifically again to Madagascar, is indicated by the relationshipbetween Psittrichas fulgidus and Coracopsis (see above). The Mala-gasy avifauna is supposed to be strictly comprised of Cenozoictrans-oceanic dispersers and most groups are thought to have orig-inated from African ancestors (Yoder and Nowak, 2006). However,trans-oceanic dispersal from Australasia to Madagascar or Mauri-tius was also suggested for Alectroenas pigeons (Shapiro et al.,2002), Anas dabbling ducks (Johnson and Sorenson, 1999) andcuckoo-shrikes, whose genus Coracina further colonized Africavia Madagascar (Fuchs et al., 2007; Jonsson et al., 2008). Around40 MYA, dispersal from Australasia to Madagascar and Africa couldhave been facilitated by volcanic islands in the southern IndianOcean possibly serving as stepping stones when the Broken Ridgeof Western Australia was connected with the Kerguelen archipel-ago (Fjeldså and Bowie, 2008; Fuchs et al., 2006a; Fig. 4).

Indo-Malayasia was probably colonized by Psittacula, Psittinusand Loriculus in three independent colonization events when Aus-tralasia approached Southeast Asia and reached its present posi-tion around 20–25 MYA (Li and Powell, 2001). Parrots of thegenus Psittacula then further colonized Africa. This fourth case ofsuccessfully colonizing Africa may have happened as recently asthe Plio-Pleistocene boundary (Groombridge et al., 2004). Finally,Prioniturus and Tanygnathus may have dispersed to Indo-Malaysiafrom Australasia as they include species from both regions.

Wright et al. (2008) considered a Paleogene origin of the Psit-taciformes less likely than a Cretaceous origin because the formerrequires several oversea dispersal events. Importantly, our datasuggests that trans-oceanic dispersal has to be invoked no matterif parrots originated in the Paleogene or in the Cretaceous. Vicari-ance had long been considered more plausible than dispersal to ex-plain major distribution patterns, but recent work suggests thattrans-oceanic dispersal may be more important than previously

thought (de Queiroz, 2005; Cowie and Holland, 2006; McGlone,2005; Yoder and Nowak, 2006). Vicariance is usually consideredthe null hypothesis that is rejected if phylogenetic divergencedates are incongruent with potential vicariance events, leaving dis-persal as the most likely alternative (e.g. Pramuk et al., 2008;Trenel et al., 2007). Trans-oceanic dispersal has recently been in-voked as a plausible mechanism to explain distribution patternsof several bird taxa, e.g. Turdus thrushes (Voelker et al., 2009),cuckoo-shrikes and allies (Campephagidae) (Fuchs et al., 2007;Jonsson et al., 2008), Columbiformes (Pereira et al., 2007; Shapiroet al., 2002), vangas, bushshrikes and allies (Fuchs et al., 2006b).Trans-oceanic dispersal is thought to have played a role in thediversification of other terrestrial vertebrates too, including cha-meleons (Raxworthy et al., 2002), lizards (Vicario et al., 2003),frogs (Heinicke et al., 2007; Vences et al., 2003), Carnivora and le-murs (Yoder et al., 2003) as well as monkeys (Schrago and Russo,2003).

Our data suggest that trans-oceanic dispersal may have alsoplayed a major role in the spread and diversification of parrots.Their ability to disperse long distances over water is shown bytheir colonization of distant islands in the Pacific (Collar, 1998).We suggest multiple dispersal events between the Afrotropical,Indo-Malayan, Neotropical and Australasian regions as well as Ant-arctica during the Paleogene, at similar rate and timescale as hasbeen recently proposed for the Columbiformes (Pereira et al.,2007). Halas et al. (2005) proposed the null hypothesis for histor-ical biogeography may have to be changed from vicariance to taxonpulses. A taxon pulse model of diversification may indeed best ex-plain the global spread and radiation of parrots because initialvicariance events within the center of parrot diversity were fol-lowed by episodes of dispersal to and in-situ radiation on all othertropical continents. In fact both Africa and the Indo-Malaysia werecolonized by multiple lineages at different times, and more oftenthan not these lineages subsequently radiated, resulting in a bioge-ographically reticulated history (cf. Erwin, 1981; Halas et al., 2005).Tavares et al. (2006) proposed an important role for ecological spe-ciation via niche diversification in combination with the coloniza-tion of open-dry habitats for a clade of Neotropical parrots.Contrasting with conclusions of a recent study on the radiationof plant lineages in the Southern Hemisphere, which found biomeconservatism prevalent (Crisp et al., 2009), relatively frequentbiome shifts through dispersal followed by local adaptive radiationinto new habitats seem responsible for much of the diversificationof parrots. The newly colonized, previously parrot-free areas mayhave presented under-utilized adaptive zones with several ecolog-ical opportunities facilitating diversification in comparable ways aswhen newly emerged or remote and ecologically under-utilized is-lands are colonized (cf. Losos and Ricklefs, 2009).

Acknowledgments

We especially thank the Silva Casa Foundation for financialsupport of this project. We are grateful to S. Birks (University ofWashington, Burke Museum), Dr. R. Burkhard, Dr. A. Fer-genbauer-Kimmel, J. Fjeldså and J.-B. Kristensen (Zoological Mu-seum, University of Copenhagen), H. Gygax, Dr. P. Sandmeier, T.and P. Walser and Dr. G. Weiss for kindly providing us with tissueor feather samples. We further thank the following people for valu-able support: B. Blöchlinger, R. Burri, H. Frick, M. Hohn, S. Klopf-stein, S. Lauper, L. Lepperhof, M. Maan, M. Rieger, T. Roth and C.Sherry.

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