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Santiago Alarcon Et Al2010PhylogeneticRelationshipsColumbiformHaemosporidia

Santiago Alarcon Et Al2010PhylogeneticRelationshipsColumbiformHaemosporidia

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Se estudiaron las relaciones filogenéticas y la colonización de los parásitos Haemosporida a las Islas Galápagos en Columbiformes o palomas.
Se estudiaron las relaciones filogenéticas y la colonización de los parásitos Haemosporida a las Islas Galápagos en Columbiformes o palomas.

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Published by: Diego Santiago Alarcon on Dec 08, 2011
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This article appeared in a journal published by Elsevier. The attachedcopy is furnished to the author for internal non-commercial researchand education use, including for instruction at the authors institutionand sharing with colleagues.Other uses, including reproduction and distribution, or selling orlicensing copies, or posting to personal, institutional or third partywebsites are prohibited.In most cases authors are permitted to post their version of thearticle (e.g. in Word or Tex form) to their personal website orinstitutional repository. Authors requiring further informationregarding Elsevier’s archiving and manuscript policies areencouraged to visit:http://www.elsevier.com/copyright
 
Author's personal copy
Phylogenetic relationships of haemosporidian parasites in New WorldColumbiformes, with emphasis on the endemic Galapagos dove
q
Diego Santiago-Alarcon
*
, Diana C. Outlaw
1
, Robert E. Ricklefs, Patricia G. Parker
University of Missouri – St. Louis, Department of Biology, One University Boulevard, Saint Louis, MO 63121, USA
a r t i c l e i n f o
 Article history:
Received 24 August 2009Received in revised form 25 September2009Accepted 2 October 2009
Keywords:Haemoproteus
(
Haemoproteus
)ColumbiformesAvian HaemosporidaGalapagos
 Zenaida galapagoensis
a b s t r a c t
DNA-sequence analyses of avian haemosporidian parasites, primarily of passerine birds, have describedthe phylogenetic relationships of major groups of these parasites, which are in general agreement withmorphological taxonomy. However, lessattentionhasbeenpaidtohaemosporidianparasitesofnon-pas-serine birds despite morphological and DNA-sequence evidence for unique clades of parasites in thesebirds. Detection of haemosporidian parasites in the Galapagos archipelago has raised conservation con-cerns and prompted us to characterise the origins and diversity of these parasites in the Galapagos dove(
 Zenaida galapagoensis
). We used partial mitochondrial cytochrome
b
(cyt
b
) and apicoplast caseinolyticprotease C (ClpC) genes to develop a phylogenetic hypothesis of relationships of haemosporidian para-sites infecting NewWorld Columbiformes, paying special attention to those parasites infecting the ende-mic Galapagos dove. We identified a well-supported and diverse monophyletic clade of haemosporidianparasites uniquetoColumbiformes, whichbelongtothesub-genus
Haemoproteus
(
Haemoproteus
). Thisisasister clade toall the
Haemoproteus
(
Parahaemoproteus
) and
Plasmodium
parasites so far identifiedfrombirdsaswellasthe
Plasmodium
parasitesofmammalsandreptiles.Ourdatasuggestthatthediverse
Hae-moproteus
parasites observedinGalapagos doves are not endemic tothearchipelago andlikelyrepresentmultiple recent introductions.
Ó
2009 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.
1. Introduction
Haemosporidianparasitesarevector-bornparasitesintheorderHaemosporida (PhylumApicomplexa) that are commonlyfound inreptiles, birds and mammals (Valki
unas, 2005). Avian haemospo-ridian parasites have a cosmopolitan distribution and are dividedintofourgenera:
Plasmodium
,
Haemoproteus
,
Fallisia
and
Leucocyto- zoon
(Atkinson and van Riper, 1991; Atkinson, 1991; Valki
unas,2005). Only recently have evolutionary biologists and ecologistsapplied molecular approaches to study this group of parasites infree-ranging hosts (e.g., seeSchall and Marghoob, 1995; Schalland Pearson, 2000; Salkeld and Schwarzkopf, 2005for lizards;andBensch et al., 2000; Ricklefs and Fallon, 2002; Fallon et al.,2003, 2004, 2005; Pérez-Tris and Bensch, 2005; Valki
unas, 2005;Hellgren et al., 2007afor birds). These studies have revealed thatthe diversity of parasite DNA lineages greatly surpasses the num-ber of named species based on morphological characters (Ricklefsand Fallon, 2002; Krizˇ anauskiene˙et al., 2006; Martinsen et al.,2006; Hellgren et al., 2007a).Phylogenetic studies of avian haemosporidian parasites, mostlybased on mitochondrial (mtDNA) cytochrome
b
(cyt
b
), have iden-tifiedmanyparasitelineagesofthegenera
Plasmodium
and
Haemo- proteus
infectingpasserinebirds(PerkinsandSchall, 2002;Ricklefsand Fallon, 2002; Martinsen et al., 2008); however, few studieshave specifically addressed the haemosporidian parasites of non-passerine birds. Traditional taxonomy based on morphologicalcharacters places
Haemoproteus
parasites that infect Columbifor-mes in the sub-genus
Haemoproteus
, whereas all other
Haemopro-teus
parasites belong to the sub-genus
Parahaemoproteus
(Valki
unas, 2005). Sequence (mtDNA cyt
b
and cytochrome
oxi-dase subunit I (COI), apicoplast caseinolytic protease C (ClpC) andnuclear adenylosuccinate lyase (asl), genes) have verified the phy-logenetic position of the sub-genus
Haemoproteus
(
Haemoproteus
)clade as sister to the sub-genus
Haemoproteus
(
Parahaemoproteus
)and the genus
Plasmodium
(Martinsen et al., 2008); however, thatstudy only included three samples from domestic pigeons (
Colum-ba livia
),which represent one morphological species of parasite(
Haemopr oteus columbae
). Thus, samplingof haemosporidian para-sites from pigeons and doves, as well as from other non-passerinebirds, has been rather limited in phylogenetic analyses. Wide geo-graphicalsamplingofbloodparasitesinfectingnon-passerinebirds
0020-7519/$36.00
Ó
2009 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.doi:10.1016/j.ijpara.2009.10.003
q
Note:
Nucleotide sequence data reported in this paper are available in theGenBank
TM
database under the accession numbers FJ462649–FJ462685 andFJ467560–FJ467608.
*
Corresponding author. Present address: University of Freiburg, Biology I.Hauptstraße 1, Freiburg D-79104, Germany. Tel.: +49 761 203 2545; fax: +49 761203 2544.
E-mail address:
onca77@yahoo.com(D. Santiago-Alarcon).
1
Present address: Mississippi State University, Department of Biological Sciences,Mississippi State, MS 39762, USA.
International Journal for Parasitology 40 (2010) 463–470
Contents lists available atScienceDirect
International Journal for Parasitology
journal homepage: www.elsevier.com/locate/ijpara
 
Author's personal copy
will certainly provide a refinement of the taxonomy and systemat-ics of the order Haemosporida.Traditional taxonomy recognises 19 morphological species of haemosporidian parasites infecting Columbiformes (Bennett andPeirce, 1990; Valki
unas, 2005). Of these, six belong to the genus
Haemoproteus
,11to
Plasmodium
,oneto
Fallisia
andoneto
Leucocy-tozoon
(Valki
unas, 2005).
H. columbae
,
H. sacharovi
,
H. palumbis
,
H.turtur 
,
Plasmodium gabaldoni
,
P. columbae
,
Fallisia neotropicalis
and
Leucocytozoon marchouxi
have been described from bird species of the family Columbidae (Valki
unas, 2005). The other two
Hemopro-teus
parasites (
H. krylovi
and
H. pteroclis
) that infect pigeons anddoves have been described from the bird family Pteroclididae.Theremainingparasitesinfectingpigeonsanddoveshavebeende-scribed from birds belonging to the families Passeridae, Turdidae,Hirundinidae, Phasianidae, Mimidae and Psittacidae (Valki
unas,2005). Thevertebratehostsofthepresentstudybelongtothefam-ily Columbidae, sub-family Columbinae, with approximately 60species recognised across the American continent (Baptista et al.,1997). The endemic Galapagos dove (
 Zenaida galapagoensis
) be-longs to a genus comprising seven species, within which its sisterspecies is the widespread South American eared dove (
 Z. auricula-ta
) ( Johnson and Clayton, 2000).The study of avian haemosporidian parasites has contributedtounderstanding emerging infectious diseases in novel hosts (e.g.,Mackenzie et al., 2004; Kilpatrick et al., 2006; Jourdain et al.,2007). Because millions of migratory birds travel enormous dis-tances, they potentially can transmit parasites between distantgeographical locations, evenbetweencontinents (e.g.,Waldeströmet al., 2002; Ricklefs et al., 2005; Pérez-Tris and Bensch, 2005; Fal-lon et al., 2006; Svensson et al., 2007; but seeHellgren et al.,2007b). In addition, many avian haemosporidian parasites are ableto infect species from different bird families (Ricklefs and Fallon,2002; Ricklefs et al., 2004; Krizˇ anauskiene˙et al., 2006; but seeIezhova et al., 2005).Introduced avian diseases are a concern for the conservation of endemic species in the Galapagos archipelago (Padilla et al., 2004;Parker et al., 2006). Recently, we have detected high prevalence(>80%) and infection intensities of 
Haemoproteus
spp. in endemicGalápagos doves from eight different islands of the archipelago(Padilla et al., 2004; Santiago-Alarcon et al., 2008). We suspectedthat these haemosporidian parasites might have arrived in theGalapagos recently, perhaps via introduced domestic pigeons,which were repeatedly brought to the islands during the last cen-tury (Harmon et al., 1987; Padilla et al., 2004). An exterminationprogram removed the last remaining domestic pigeons from theGalapagosin2002,leavingtheendemicGalapagosdoveastheonlycolumbiform species inhabiting the archipelago.Until now, no attempt has been made to characterise the diver-sity of columbiform haemosporidian parasites using molecularmethods. In this study, we provide a comprehensive phylogeneticanalysis of columbiform haemosporidian parasites using partialmtDNA cyt
b
and ClpC genes. We also characterise the haplotypediversity of haemosporidian parasites of Galapagos doves in rela-tiontoparasite lineagesobtainedfromdoves across LatinAmerica.
2. Materials and methods
 2.1. General field and laboratory methods
Pigeons and doves were captured using mist nets and handnets. Our sample comprised 439blood samplesfromdoves and pi-geons of North and South America and the West Indies. We col-lected 166 blood samples from Galapagos doves on eight islands(Santiago, Santa Cruz, Santa Fe, Española, San Cristobal, Genovesa,Darwin and Wolf) between 2002 and 2005. In 2002, we also ob-tained samples from 14 domestic pigeons on San Cristobal Island.We obtained blood samples from New World Columbiformes (17species from seven genera) in the United States (two samples),Mexico (seven), Caribbean islands (10), Venezuela (126), Peru(29), Uruguay (two), Ecuador (73) and Guatemala (10). Samplesfrom Ecuador (Galapagos and mainland), Peru, USA and CaribbeanIslands were collected by the authors. Samples from other locali-ties were provided to us by colleagues. To develop a broader phy-logenetic perspective, we also used published DNA sequencesavailable in GenBank fromMartinsen et al. (2008).Wecollected
$
50
l
l of bloodfromeachcapturedbirdvia punc-tureofthewingvein.Sampleswerepreservedinlysisbuffer(Long-mire et al., 1988). DNA was extracted by phenol–chloroformfollowed by dialysis in 1
Â
TNE
2
(Sambrook and Russell, 2001).Infection was determined by visual examination of blood smearsand by PCR amplification of parasite gene sequences (Fallonet al., 2003) in endemic Galapagos doves, and by PCR only in allother columbiform samples. We prepared two thin blood smearsfrom each sampled Galapagos dove. Smears were air dried, fixedin methyl alcohol and stained with Giemsa’s stain. We scannedfrom 100 to 200 fields at 100
Â
magnification to visually identifyinfected individuals (Valki
unas et al., 2006). We sequenced a frag-ment of 
$
600bp of mtDNA cyt
b
and
$
550bp of the apicoplastgene ClpC using published primers that are specific for haemospo-ridian parasites (Perkins and Schall, 2002; Martinsen et al., 2008).PCR was conducted in 50
l
l reactions containing 5
l
l 10
Â
Ex Taqbuffer, 4
l
l of 25mM MgCl
2
, 4
l
l of dNTP mixture (2.5mM each),0.25U of Takara Ex Taq polymerase, 1
l
l of 10mM primers, 1
l
lof stock DNA, and 1
l
l of BSA only for mtDNA cyt
b
reactions.We conducted nested PCRs. For mtDNA cyt
b
we used primerpairs DW2–DW4 for the outer reaction and DW1–HaemoR forthenestedreaction(PerkinsandSchall,2002)andforClpCweusedprimer pairs ClpC/outer and ClpC/nested (Martinsen et al., 2008).When several bands were present in the amplified products, weoptimisedthePCRtosuppressmostorallunspecificproduct.Then,we purified the targeted fragment by using a QIAquick gel extrac-tion kit (QIAGEN). When a single band was obtained, we directlycleaned the PCR product using Antarctic phosphatase and Exonu-clease I (# M0289S and # M0293S, respectively, New England BioLabs, Inc.). We used sequence chromatograms to determine sam-ples with possible mixed infections. When we detected doublepeaksinsequencechromatograms,wesequencedthesampleagainand adjusted conditions to verify that it was not an artefact of thesequencing reaction. If we again obtained a chromatogram withdouble peaks we removed the sample from analyses. We eitherused an ABI 3100 microcapillary genetic analyzer to sequenceDNA products or sent the samples for sequencing at Macrogen,Inc. (Korea). DNA sequences were compared with sequences of the GenBank database by using the BLAST algorithm to verify theamplification of the correct gene. All sequences have been depos-ited in GenBank (Accession Nos.: FJ462649–FJ462685 andFJ467560–FJ467608).
 2.2. Phylogenetic analyses
Sequences were edited in SeqManII version 4 (1989–1999,DNASTAR, Inc.) and aligned by eye in Se-Al version 2.0a11(1996–2002,http://tree.bio.ed.ac.uk/software/seal/). We con-structed phylogenetic hypotheses using maximum parsimony,maximumlikelihoodandBayesianmethods.Phylogeneticanalyseswere performed in PAUP
Ã
version 4.0b10 (Swofford, D., 2002.Paup
Ã
version 4.0b10. Massachusetts: Sunderland.) and Mr. Bayes2.01 (Huelsenbeck and Ronquist, 2001), and the best-fit model of DNA evolution was determined using MODELTEST (Posada andCrandall, 1998). All analyses were executed in the Beowulf com-puting cluster of the University of Missouri St. Louis, USA
464
D. Santiago-Alarcon et al./International Journal for Parasitology 40 (2010) 463–470

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