Sympatric speciation—the divergence of populations into new species in absence of geographic barriers to hybridization—is the most debated mode of diversification of life forms. Parasitic organisms are prominent models for sympatric speciation, because they may colonise new hosts within the same geographic area and diverge through host specialization. However, it has been argued that this mode of parasite divergence is not strict sympatric speciation, because host shifts likely cause the sudden effective isolation of parasites, particularly if these are transmitted by vectors and therefore cannot select their hosts. Strict sympatric speciation would involve parasite lineages diverging within a single host species, without any population subdivision.
Here we report a case of extraordinary divergence of sympatric, ecologically distinct, and reproductively isolated malaria parasites within a single avian host species, which apparently occurred without historical or extant subdivision of parasite or host populations.
This discovery of within-host speciation changes our current view on the diversification potential of malaria parasites, because neither geographic isolation of host populations nor colonization of new host species are any longer necessary conditions to the formation of new parasite species.
Citation: Pérez-Tris J, Hellgren O, Križanauskienė A, Waldenström J, Secondi J, Bonneaud C, et al. (2007) Within-Host Speciation of Malaria Parasites. PLoS ONE2(2): e235. https://doi.org/10.1371/journal.pone.0000235
Academic Editor: Angus Buckling, Oxford University, United Kingdom
Received: November 8, 2006; Accepted: February 1, 2007; Published: February 21, 2007
Copyright: © 2007 Perez-Tris et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: The study was funded by EU Marie Curie grants (to J.P. and J.S.), the Spanish Ramón y Cajal programme (J.P.), the Swedish Research Council and Carl Trygger Foundation (D.H. and S.B.), and the Ministère de la Recherche (C.B.). None of these funding bodies played any role in the experimental design, execution or writing of the paper.
Competing interests: The authors have declared that no competing interests exist.
Malaria parasites comprise a diverse group of protozoans that infect reptiles, mammals and birds, and that are transmitted through the bite of different families of blood-feeding insect vectors , . They encompass two closely related genera, Plasmodium and Haemoproteus , which contain about 170 and 150 morphologically distinct species, respectively , and many more cryptic species as revealed by DNA sequencing of mitochondrial and nuclear gene fragments . Genetic techniques have substantially improved our understanding of both the diversity and the modes of lineage divergence in non-human malaria parasites . Speciation in malaria parasites is now known to follow from the subdivision of parasite populations in discrete habitats, such as different geographic regions in which host populations become isolated, or different host species accidentally colonised with the assistance of vectors , . Such findings fit to the expectations of usual modes of allopatric and sympatric speciation, respectively, which have been proposed for parasitic organisms –. In contrast, theoretical expectations of strict sympatric, within-host speciation involve the evolution of reproductive isolation among the members of an interbreeding population of parasites within a single host species . Such a mode of diversification can be inferred from phylogenetic relationships and host distributions of extant parasite species, the critical observation being a fully sympatric community of reproductively isolated sister parasite lineages within a geographically unstructured host species , .
Here we describe a case of great diversification of Haemoproteus parasite lineages that has occurred within a single bird host species, the blackcap Sylvia atricapilla. To illustrate such occurrence, we (i) determine the distribution of parasite lineages among 47 passerine species that are sympatric to the blackcap, showing that most blackcap parasites are exclusive to this species, (ii) determine the evolutionary relationships of parasites found in blackcaps and its closest relatives, demonstrating that many blackcap parasites are included in a monophyletic group that apparently diverged within the blackcap, (iii) analyse whether such group of parasites are reproductively isolated entities, and (iv) analyse the geographic structuring of genetic variation of blackcap parasites, showing that the probability that this group of parasites evolved in allopatry is very slim.
We sequenced part of the mitochondrial cytochrome b gene of malaria parasites found in European passerine birds to broadly investigate the phylogenetic diversity of this group of organisms. Our survey included 4470 individual birds of 47 European species, and 1911 parasite infections, each one involving one to four parasite lineages distinguished through their DNA sequences . We were able to identify whether parasites belonged to the Plasmodium (45 lineages) or the Haemoproteus (92 lineages) genera by comparing their DNA sequences with those of parasites identified by microscopy.
Parasite richness in the blackcap
One warbler species, the blackcap, was exceptionally rich in parasites, harbouring 19% of all parasites found among the 47 screened European bird species. This pattern remained when we restricted the analysis to widely sampled bird species, for which we had scored at least 25 parasite infections. For these species, we could estimate intraspecific parasite richness independent of sampling effort as the number of parasite lineages accumulated in 25 scored infections (an index we named R25). This index of parasite richness was higher in blackcaps (R25 = 9.6±0.06) than in any other widely sampled bird species (14 species with more than 25 scored infections: R25 = 6.0±0.58, Fig. 1). Most importantly, blackcaps had the largest proportion of exclusive parasites observed in any species studied (73.1%), with other widely sampled birds harbouring a significantly lower number of exclusive lineages (33.1%±4.8, higher 95% confidence limit = 68.4%, P<0.05, Table 1). Sampling bias did not explain these results, as blackcaps showed higher parasite diversity than other bird species for which the sampling effort was often higher (Fig. 1).
Variation in richness of malaria parasites among different bird host species. The curves represent parasite cumulative richness in 15 passerine species with more than 25 scored parasite infections, belonging to 10 bird genera (represented in different colours, see details in Table 1). The curves provide a standard estimate of parasite richness (R25) for each species, given by the number of parasites accumulated after analysing 25 infected individuals. The small graph shows the curves of parasite accumulation including all infections scored in each species (n = 26-353), crossed by a dotted line at R25. Blackcaps show the highest richness of malaria parasites in both graphs.
Endemism of blackcap parasites
Seventeen parasites found only in blackcaps, summing up 18.5% of all Haemoproteus parasites found in 47 European host species, made the bulk of a monophyletic group in the genus Haemoproteus (Fig. 2). The microscopic investigation of various parasites from this group places them within the morphological species H. parabelopolskyi . These blackcap parasites are not shared with African birds species either, according to an extensive survey (over 5800 individuals) of European and African birds, including more than 100 African passerine species , which strongly supports endemism of this group of parasites in blackcaps. A search in GenBank (run in January 23, 2007) failed to find any parasite sequence within this monophyletic group that had been retrieved from any bird species other than the blackcap. However, the search identified some of the sequences found in our study as blackcap parasites independently found in places not included in our survey. Given that GenBank includes hundreds of cytochrome b sequences of Haemoproteus parasites retrieved from hundreds of bird species sampled worldwide, the result of our search further supports endemism of blackcap parasites.
The blackcap parasite flock placed in a phylogenetic context. The tree A shows the phylogenetic position of parasites found in blackcaps (red), garden warblers (blue) and African hill babblers (purple), among 143 parasite lineages found in 48 bird species. Monophyletic parasite groups apparently exclusive to some species are encircled in orange (species names are mentioned and the number of parasites in each group is shown in brackets). The tree B shows the parasite flock (represented by a triangle in the tree A). Numbers represent >75% support to internal branches, based on bootstrap replicates (above) or posterior probabilities (below). For six parasites in this cluster, the figure shows the match between phylogenies based on mitochondrial cytochrome b and nuclear DHFR-TS genes (the number of cases with each association of haplotypes is indicated, and bootstrap support is reported if >75%).
This phylogenetic “flock of parasites” also included three parasites infecting only garden warblers Sylvia borin, the closest extant relative species to the blackcap. However, garden warbler parasites were never found infecting blackcaps, or vice versa (n = 179 blackcaps and 54 garden warblers infected by parasites of this group; Fisher exact P<0.0001), despite of the fact that both species are sympatric over most of their ranges and were often trapped at the same sites (Fig. 3), supporting high host specificity in this group of closely related parasites. Within our sample, other bird species often harboured nearly as many parasites as the blackcap (Table 1). However, the blackcap was unique in that the majority of its parasites were sister lineages not found in other species. Other species never had more than five parasites forming monophyletic groups, even if the parasites of several closely related bird species were considered together (Fig. 2).
The geographic distribution of the parasite flock. The map shows the location of our sampling sites within the range of blackcaps, garden warblers and African hill babblers, which are shadowed in different colours according to the key below the map. Blackcaps and garden warblers are sympatric (S) in wide areas of Europe and Africa, during the breeding season and in winter, respectively. African hill babblers are year-round residents. Many blackcaps and garden warblers from Europe spend the winter in the range of African hill babblers, where the three species occur in the same habitat. For each sampling site, the squares indicate the number of parasite lineages from the flock that were found in each species (blackcap parasites in red, garden warbler parasites in blue, and hill babbler parasites in purple).
Given that blackcaps harboured many more of these monophyletic parasites than garden warblers, we suspected that parasites might have diversified in blackcaps after both bird species diverged from each other, and then some parasite lineages switched host from blackcaps to garden warblers (Fig. 2). To further examine this possibility, we analysed a sample of African hill babblers (Sylvia [Pseudoalcippe] abyssinica), which is the closest extant relative to the species pair formed by blackcaps and garden warblers . Among 43 screened hill babblers, we found 16 infections involving 7 parasite lineages. Three of these parasites were included in the monophyletic group infecting blackcaps and garden warblers (one of them shared with blackcaps), and another two formed an outgroup to the above parasite cluster, supporting the possibility that parasites cospeciated with their host species when blackcaps and garden warblers diverged from African hill babblers (Fig. 2). We obtained good statistical support for the monophyly of the parasite group shared by all three species (93–100%), as well as for the early divergence of two African hill babbler parasites in the evolutionary history of the group (100%), despite the minor mtDNA sequence differences among these parasites and their closest relatives (Fig. 2). Because of the latter reason, however, the evolutionary relationships between the three host species and the parasites in the flock were difficult to establish. Frequent host switching might drive parasite divergence in this system, but such scenario is unlikely because it implies newly diverged parasites nearly always switching host to blackcaps and becoming extinct in the other two host species, although the three host species are sympatric. In our view, the three bird species likely acquired their distinct parasites from a common ancestor, and later on such parasites went through substantial diversification in blackcaps, dramatically more than in garden warblers or in African hill babblers. At this point, we do not have a plausible explanation that can explain why parasite diversification may have been so much stronger in the blackcap than in the other investigated passerines.
Reproductive isolation of blackcap parasites
To evaluate whether sister blackcap parasites are reproductively isolated, we sequenced part of the nuclear DHFR-TS gene of parasites in 13 infections involving six members of the aforementioned Haemoproteus parasite flock. Both cytochrome b and DHFR-TS genes produced identical phylogenies, although two mitochondrial lineages shared identical nuclear sequences (Fig. 2). The latter case might signify intraspecific polymorphism, or conservation of the nuclear sequence between reproductively isolated parasite lineages. In either case, random effects are unlikely to produce identical topologies for two independent trees with six lineages (P = 0.0095). Random effects are also unlikely to explain linkage between nuclear and mitochondrial haplotypes determined in single infections of the same parasite lineages (four cases with mtDNA SYAT01 and five with mtDNA SYAT02, obtained from blackcaps captured in different geographical areas and times, were never observed to interchange nuclear sequences, Fisher exact P = 0.0079, Fig. 2). Therefore, we concluded that these parasites remain reproductively isolated, both during evolutionary time and at present. Arguably, such pattern could be due to long-term selfing in isolated populations leading to genetically “clonal” diversification of various strains of the same parasite species . However, two observations argue against this possibility. First, we often observed two or three members of this parasite flock co-infecting individual blackcaps, with 82% of the flock members sometimes co-infecting individual blackcaps together with other flock members, and many apparently reproductively isolated lineages participated in such mixtures (Fig. 4). Recurrent coexistence of parasite lineages in vectors' bloodmeals should therefore bring about frequent interbreeding opportunities . Second, parasite transmission rate was very high, as demonstrated by 92% of young blackcaps becoming infected soon after leaving the nest , which should prevent the long-term maintenance of “clonal” lineages resulting from selfing . Note also that Haemoproteus parabelopolskyi parasites of blackcaps undergo normal sexual reproduction in the vector , and strict clonality resulting from asexual reproduction has never been demonstrated for Plasmodium parasites . Given these facts, reproductive isolation between coexisting lineages is the most likely explanation for the observed linkage between parasite mitochondrial and nuclear haplotypes , , although the isolating mechanisms involved remain to be uncovered.
Co-occurrence of sister blackcap parasites (see tree in Fig. 2B) in mixed infections of individual hosts. Numbers indicate the frequency of occurrence of each parasite combination. Combinations in red involve parasites for which frequent hybridisation is unlikely, according to linkage between mitochondrial and nuclear haplotypes.
Testing for host and parasite population subdivision
Blackcaps were sampled in seven breeding populations distributed between southern Spain and Sweden, but there was no geographic structure in the genetic variation contained in the parasite flock infecting this species (AMOVA: ΦST = −0.01; d.f. = 6,121; P = 0.60, estimated genetic variance among populations = 0). This result supports the hypothesis that the diversification of blackcap malaria parasites occurred without long-term population subdivision.
In addition, blackcap populations show an extremely weak neutral genetic structure across their geographical range . Therefore, we conclude that blackcap parasites are very unlikely to have diverged during historical periods of host population isolation. Supporting this view, various European passerines that show strong evidence of population fragmentation in the past , and pairs of sister species which apparently diverged in different glacial refugia , did not show the diversity of monophyletic parasites found in blackcaps, even if phylogenetic analyses support a long-term association between these species and their parasites , .
Our results imply that the blackcap parasite flock consists of sympatric biological species , which have diverged within a single avian host species without population subdivision. It is important to note that some of these parasites show clear ecological differences, such as different times of transmission (seasonal or year-round), dispersal rates, or local prevalence . If such diverse life histories mean that these parasites exploit different adaptive optima within the same host species, natural selection for ecological specialization could trigger parasite speciation without host switching . In addition, the fact that parasites undergo sexual reproduction in the vector's gut make us anticipate that intricate associations between blackcap parasites and Culicoides impunctatus biting midges (the vector of Haemoproteus parabelopolskyi in blackcaps ) may play a relevant role as causes of both parasite reproductive isolation and restricted host range of this group of parasites.
Our results are the first to show that sympatric, within-host divergence possibly occurs in malaria parasites. This divergence mode has important implications because diverse but closely related parasites infecting the same host species may establish complex ecological interactions , . Such scenarios may intensify disease impacts on populations, because a diverse parasite community can greatly constrain a host's life history strategies and ultimately fitness by imposing a larger antigenic variation for the host's immune system , and perhaps by boosting up virulence as a consequence of within-host competition among closely related parasites . Malaria parasites are economically and socially important , . Therefore, understanding the origins of diversity in these parasites transcends the interest of evolutionary biologists , such knowledge being essential for disease control and wildlife conservation .
Materials and Methods
Blood samples of wild birds were obtained at different European localities from southern Spain to Sweden, at migration stopover sites, and in European and African wintering areas. We screened 4513 wild birds in total, corresponding to 48 passerine species (47 European species plus the African hill babbler; Table 1).
We detected malaria infections by amplification of 479 bases of the parasite cytochrome b gene using DNA extracted from bird blood and highly efficient polymerase chain reaction (PCR) methods , . Different malaria lineages were distinguished by one or more nucleotide differences . Multiple infections revealed by mixed sequences were resolved by TA-cloning . In total, we scored 1927 infected birds, the average sample size being 40 infected individuals per species (median = 9.5, range 1–357; Table 1). We found 143 distinct parasite lineages, each one being found in 1.9 species on average (range 1–22 species). In 13 blackcap cases (from distantly located sites or different years), we also amplified 220 bases of the nuclear DHFR-TS gene as described previously . All these sequences were retrieved from singly infected blackcaps, so that the association between parasite nuclear and mitochondrial DNA sequences could be unambiguously determined. We could not retrieve the sequence of this gene from all blackcap parasites because many occurred in mixed infections or were not amplified using our PCR . The DNA sequences used in this study have been deposited in GenBank (Table 1).
We used PAUP  to construct a maximum likelihood phylogenetic tree based on parasite cytochrome b sequences (Fig. 2), using a General Time Reversible model of nucleotide substitution with gamma parameter α = 0.623, and assumed proportion of invariable sites = 0.427. This was the best of 56 models according to the Akaike information criterion implemented in Modeltest . Support to internal branches was estimated by bootstrap analyses (1000 replicates) . We confirmed the tree by repeating the analysis using Bayesian methods as implemented in mrBayes 3.0 , under the same model of nucleotide substitution. This method produced the same tree topology, and similar or even stronger support for internal branches, as evaluated by posterior probabilities derived from trees sampled every 500 generations from a 10-million generations Markov Chain Monte Carlo series, with a burn-in time of 250000 generations that removed any trees generated before convergence had been reached. To construct the DHFR-TS tree, we used PAUP and a Kimura 3-parameters model with unequal base frequencies , . The exact probability of obtaining identical topologies for trees based on nuclear and mitochondrial genes was calculated by generating all possible trees with six leaves using COMPONENT 2.00a .
We analysed the genetic structure of the blackcap parasite flock using an analysis of molecular variance (AMOVA) , comparing seven breeding populations covering most of the species' range, from southern Spain to Sweden (Fig. 3). The analysis used Kimura two-parameter distances under a gamma distribution with α = 0.12, as estimated from the data. The significance of the fixation index (ΦST) was tested by 5000 permutations of parasite haplotypes among populations .
Aside from blackcaps, we extensively sampled 14 other species (n>40 birds and >25 scored infections), which were used to estimate intraspecific parasite richness (Fig. 1, Table 1). To avoid sampling effects, curves of cumulative lineage richness (addition of new parasite lineages as new infected hosts are inspected) were constructed, and the number of parasite lineages found after scoring 25 infections was used as a standard estimate of parasite richness (R25). Average curves and R25 values (±S.E.) were derived from 1000 richness curves constructed by randomly changing the order in which individual hosts were screened. While the number of parasite lineages found in one species depended on the number of infections scored (r2 = 0.58, n = 48, P<0.0001), R25 was independent of sample size (r2 = 0.04, n = 15, P = 0.43).
This paper greatly benefited from inspiring ideas and discussion with A. J. Helbig, and we dedicate it to his memory.
All parasite sequences used in this study can be found in GenBank, with accession numbers (parasite names cited in Fig. 2 are in brackets): AF069609 (P. falciparum); AF254962 (GRW2); AF254963; AF254967; AF254968; AF254969; AF254971; AF254975; AF254977 (PARUS1); AF495547; AF495550; AF495551; AF495552; AF495556; AF495558; AF495559; AF495560; AF495563; AF495564; AF495565; AF495566; AF495568 (RTSR1); AF495569; AF495570; AF495571 (SGS1); AF495572; AF495573; AF495574; AF495575 (SYBOR1); AF495576 (TURDUS1); AF495577; AF495578 (WW4); AF495579; AF495580; AY099040 (GRW1); AY393792; AY393793; AY393794; AY393805 (CWT4); AY393806; AY393807; AY831747 (COLL1); AY831748; AY831749 (SYAT24); AY831750 (SYAT01); AY831751 (SYAT02); AY831752 (SYAT03); AY831753 (SYAT04); AY831754 (SYAT07); AY831755; AY831756 (SYAT09); AY831757 (SYAT10); AY831758 (SYAT11); AY831759 (SYAT12); AY831760 (SYAT13); AY831761 (SYAT14); AY831762 (SYAT16); AY831763 (SYAT17); AY831764 (SYAT18); AY831765 (SYAT19); AY831766 (SYAT21); AY831767 (SYAT26); AY831768 (SYAT28); AY831769 (SYAT29); DQ000320; DQ000321; DQ000322; DQ000323; DQ000324; DQ000325; DQ058611; DQ058612; DQ058613; DQ058614; DQ060764; DQ060765; DQ060766; DQ060767; DQ060768; DQ060769; DQ060770; DQ060771; DQ060772; DQ060773; DQ063577; DQ063578; DQ067581; DQ368339; DQ368340; DQ368341; DQ368342 (CWT2); DQ368343; DQ368344; DQ368345; DQ368346; DQ368347; DQ368348; DQ368349; DQ368350; DQ368351; DQ368352 (PABY01); DQ368353 (PABY02); DQ368354 (PABY03); DQ368355 (PABY04); DQ368356 (PABY06); DQ368357; DQ368358; DQ368359; DQ368360; DQ368361; DQ368362; DQ368363; DQ368364; DQ368365 (SYBOR3); DQ368366 (SYBOR4); DQ368367; DQ368368; DQ368369; DQ368370; DQ368371; DQ368372; DQ368373; DQ368374; DQ368375; DQ368376; DQ368377; DQ368378; DQ368379; DQ368380; DQ368381; DQ368382; DQ368383; DQ368384; DQ368385; DQ368386; DQ368387 (PABY05); DQ368388; DQ368389; DQ368390 (SYBOR10); DQ368391 (SYBOR11); DQ368392 (SYBOR2); DQ368393 (SYBOR5); DQ368394 (SYBOR9); DQ368395.
Conceived and designed the experiments: SB JP. Performed the experiments: JP OH AK JW. Analyzed the data: JP OH. Contributed reagents/materials/analysis tools: SB DH JP OH AK JW JS CB JF. Wrote the paper: JP. Other: Improved the paper with comments: AK JW JS CB JF DH SB OH.
- 1. Kreier JP (1994) Parasitic protozoa. 2nd edition. New York: Academic Press. JP Kreier1994Parasitic protozoa. 2nd edition.New YorkAcademic Press
- 2. Pérez-Tris J, Hasselquist D, Hellgren O, Krizanauskiene A, Waldenström J, Bensch S (2005) What are malaria parasites? Trends Parasitol 21: 209–211.J. Pérez-TrisD. HasselquistO. HellgrenA. KrizanauskieneJ. WaldenströmS. Bensch2005What are malaria parasites?Trends Parasitol21209211
- 3. Bensch S, Pérez-Tris J, Waldenström J, Hellgren O (2004) Linkage between nuclear and mitochondrial DNA sequences in avian malaria parasites – multiple cases of cryptic speciation? Evolution 58: 1617–1621.S. BenschJ. Pérez-TrisJ. WaldenströmO. Hellgren2004Linkage between nuclear and mitochondrial DNA sequences in avian malaria parasites – multiple cases of cryptic speciation?Evolution5816171621
- 4. Ricklefs RE, Fallon S, Bermingham E (2004) Evolutionary relationships, cospeciation, and host switching in avian malaria parasites. Syst Biol 53: 111–119.RE RicklefsS. FallonE. Bermingham2004Evolutionary relationships, cospeciation, and host switching in avian malaria parasites.Syst Biol53111119
- 5. Waldenström J, Bensch S, Kiboi S, Hasselquist D, Ottosson U (2002) Cross-species infection of blood parasites between resident and migratory songbirds in Africa. Mol Ecol 11: 1545–1554.J. WaldenströmS. BenschS. KiboiD. HasselquistU. Ottosson2002Cross-species infection of blood parasites between resident and migratory songbirds in Africa.Mol Ecol1115451554
- 6. de Meeûs T, Michalakis Y, Renaud F (1998) Santa Rosalia revisited: or why are there so many kinds of parasites in ‘The Garden of Earthly Delights’? Parasitol Today 14: 10–13.T. de MeeûsY. MichalakisF. Renaud1998Santa Rosalia revisited: or why are there so many kinds of parasites in ‘The Garden of Earthly Delights’?Parasitol Today141013
- 7. Filchak KE, Roethele JB, Feder JL (2000) Natural selection and sympatric divergence in the apple maggot Rhagoletis pomonella. Nature 407: 739–742.KE FilchakJB RoetheleJL Feder2000Natural selection and sympatric divergence in the apple maggot Rhagoletis pomonella.Nature407739742
- 8. McKoy KD (2003) Sympatric speciation in parasites – what is sympatry? Trends Parasitol 19: 400–404.KD McKoy2003Sympatric speciation in parasites – what is sympatry?Trends Parasitol19400404
- 9. Coyne JA, Orr HA (2004) Speciation. Sunderland: Sinauer Associates. JA CoyneHA Orr2004Speciation.SunderlandSinauer Associates
- 10. Valkiūnas G, Križanauskienė A, Iezhova TA, Hellgren O, Bensch S (2007) Molecular phylogenetic analysis of circumnuclear hemoproteids (Haemosporida, Haemoprotidae) of Sylviid birds, with description of Haemoproteus parabelopolskyi, nov. J Parasitol.. G. ValkiūnasA. KrižanauskienėTA IezhovaO. HellgrenS. Bensch2007Molecular phylogenetic analysis of circumnuclear hemoproteids (Haemosporida, Haemoprotidae) of Sylviid birds, with description of Haemoproteus parabelopolskyi, nov.J Parasitol.In press. In press.
- 11. Hellgren O, Waldenström J, Pérez-Tris J, Szöllősi E, Hasselquist D, et al. (2007) Detecting shifts of transmission areas in avian blood parasites - a phylogenetic approach. Mol Ecol.. O. HellgrenJ. WaldenströmJ. Pérez-TrisE. SzöllősiD. Hasselquist2007Detecting shifts of transmission areas in avian blood parasites - a phylogenetic approach.Mol Ecol.In press. In press.
- 12. Böhning-Gaese K, Schuda MD, Helbig AJ (2003) Weak phylogenetic effects on ecological niches of Sylvia warblers. J Evol Biol 16: 956–965.K. Böhning-GaeseMD SchudaAJ Helbig2003Weak phylogenetic effects on ecological niches of Sylvia warblers.J Evol Biol16956965
- 13. Razakandrainibe FG, Durand P, Koella JC, de Meeûs T, Rousset F, et al. (2005) “Clonal” population structure of the malaria agent Plasmodium falciparum in high-infection regions. Proc Natl Acad Sci USA 102: 17388–17393.FG RazakandrainibeP. DurandJC KoellaT. de MeeûsF. Rousset2005“Clonal” population structure of the malaria agent Plasmodium falciparum in high-infection regions.Proc Natl Acad Sci USA1021738817393
- 14. Pérez-Tris J, Bensch S (2005) Dispersal increases local transmission of avian malarial parasites. Ecol Lett 8: 838–845.J. Pérez-TrisS. Bensch2005Dispersal increases local transmission of avian malarial parasites.Ecol Lett8838845
- 15. Valkiūnas G, Iezova T (2004) The transmission of Haemoproteus belopolskyi (Haemosporida: Haemoproteidae) of Blackcap by Culicoides impunctatus (Diptera: Ceratopogonidae). J Parasitol 90: 196–198.G. ValkiūnasT. Iezova2004The transmission of Haemoproteus belopolskyi (Haemosporida: Haemoproteidae) of Blackcap by Culicoides impunctatus (Diptera: Ceratopogonidae).J Parasitol90196198
- 16. Pérez-Tris J, Bensch S, Carbonell R, Helbig AJ, Tellería JL (2004) Historical diversification of migration patterns in a passerine bird. Evolution 58: 1819–1832.J. Pérez-TrisS. BenschR. CarbonellAJ HelbigJL Tellería2004Historical diversification of migration patterns in a passerine bird.Evolution5818191832
- 17. Bensch S, Hasselquist D (1999) Phylogeographic population structure of great reed warblers: an analysis of mtDNA control region sequences. Biol J Linn Soc 66: 171–185.S. BenschD. Hasselquist1999Phylogeographic population structure of great reed warblers: an analysis of mtDNA control region sequences.Biol J Linn Soc66171185
- 18. Reullier J, Pérez-Tris J, Bensch S, Secondi J (2006) Diversity, distribution and exchange of blood parasites meeting at an avian moving contact zone. Mol Ecol 15: 753–763.J. ReullierJ. Pérez-TrisS. BenschJ. Secondi2006Diversity, distribution and exchange of blood parasites meeting at an avian moving contact zone.Mol Ecol15753763
- 19. Schluter D (2000) The ecology of adaptive radiation. Oxford: Oxford University Press. D. Schluter2000The ecology of adaptive radiation.OxfordOxford University Press
- 20. Rohani P, Green CJ, Mantilla-Beniers NB, Grenfell BT (2003) Ecological interference between fatal diseases. Nature 422: 885–888.P. RohaniCJ GreenNB Mantilla-BeniersBT Grenfell2003Ecological interference between fatal diseases.Nature422885888
- 21. de Roode JC, Read AF (2003) Evolution and ecology, after the malaria genomes. Trends Ecol Evol 18: 60–61.JC de RoodeAF Read2003Evolution and ecology, after the malaria genomes.Trends Ecol Evol186061
- 22. de Roode JC, Pansini R, Cheesman SJ, Helinski MEH, Huijben S, et al. (2005) Virulence and competitive ability in genetically diverse malaria infections. Proc Natl Acad Sci USA 102: 7624–7628.JC de RoodeR. PansiniSJ CheesmanMEH HelinskiS. Huijben2005Virulence and competitive ability in genetically diverse malaria infections.Proc Natl Acad Sci USA10276247628
- 23. Sachs J, Malaney P (2002) The economic and social burden of malaria. Nature 415: 680–685.J. SachsP. Malaney2002The economic and social burden of malaria.Nature415680685
- 24. Daszak P, Cunningham AA, Hyatt AD (2000) Emerging infectious diseases of wildlife - threats to biodiversity and human health. Science 287: 443–449.P. DaszakAA CunninghamAD Hyatt2000Emerging infectious diseases of wildlife - threats to biodiversity and human health.Science287443449
- 25. Waldenström J, Bensch S, Hasselquist D, Östman Ö (2004) A new nested PCR method very efficient in detecting Plasmodium and Haemoproteus infections from avian blood. J Parasitol 90: 191–194.J. WaldenströmS. BenschD. HasselquistÖ. Östman2004A new nested PCR method very efficient in detecting Plasmodium and Haemoproteus infections from avian blood.J Parasitol90191194
- 26. Hellgren O, Waldenström J, Bensch S (2004) A new PCR assay for simultaneous studies of Leucocytozoon spp., Plasmodium spp. and Haemoproteus spp. from avian blood. J Parasitol 90: 797–802.O. HellgrenJ. WaldenströmS. Bensch2004A new PCR assay for simultaneous studies of Leucocytozoon spp., Plasmodium spp. and Haemoproteus spp. from avian blood.J Parasitol90797802
- 27. Pérez-Tris J, Bensch S (2005) Diagnosing genetically diverse avian malarial infections using mixed-sequence analysis and TA-cloning. Parasitology 131: 15–23.J. Pérez-TrisS. Bensch2005Diagnosing genetically diverse avian malarial infections using mixed-sequence analysis and TA-cloning.Parasitology1311523
- 28. Swofford DL (2003) PAUP*. Phylogenetic Analysis Using Parsimony (*and Other Methods). Version 4.0b 10. Sunderland: Sinauer Associates. DL Swofford2003PAUP*. Phylogenetic Analysis Using Parsimony (*and Other Methods). Version 4.0b 10.SunderlandSinauer Associates
- 29. Posada D, Crandall KA (1998) Modeltest: testing the model of DNA substitution. Bioinformatics 14: 817–818.D. PosadaKA Crandall1998Modeltest: testing the model of DNA substitution.Bioinformatics14817818
- 30. Ronquist F, Huelsenbeck JP (2003) MrBayes 3: Bayesian phylogenetic inference under mixed models. Bioinformatics 19: 1572–1574.F. RonquistJP Huelsenbeck2003MrBayes 3: Bayesian phylogenetic inference under mixed models.Bioinformatics1915721574
- 31. Page RDM (1993) COMPONENT. Ver 2.00a. London: The Natural History Museum. RDM Page1993COMPONENT. Ver 2.00a.LondonThe Natural History Museum
- 32. Schneider S, Roessli D, Excoffier L (2000) Arlequin ver. 2000: A software for population genetics data analysis. Geneva: Genetics and Biometry Laboratory, University of Geneva. S. SchneiderD. RoessliL. Excoffier2000Arlequin ver. 2000: A software for population genetics data analysis.GenevaGenetics and Biometry Laboratory, University of Geneva