High lineage diversity and host sharing of malarial parasites in a local avian assemblage

Bird populations often have high prevalences of the haemosporidians Haemoproteus spp. and Plasmodium spp., but the extent of host sharing and host switching among these parasite lineages and their avian hosts is not well known. While sampling within a small geographic region in which host individuals are likely to have been exposed to the same potential parasite lineages, we surveyed highly variable mitochondrial DNA from haemosporidians isolated from 14 host taxa representing 4 avian families (Hirundinidae, Parulidae, Emberizidae, and Fringillidae). Analyses of cytochrome b sequences from 83 independent infections identified 29 unique haplotypes, representing 2 well-differentiated Haemoproteus spp. lineages and 6 differentiated Plasmodium spp. lineages. A phylogenetic reconstruction of relationships among these lineages provided evidence against host specificity at the species and family levels, as all haemosporidian lineages recovered from 2 or more host individuals (2 Haemoproteus and 3 Plasmodium lineages) were found in at least 2 host families. We detected a similar high level of host sharing; the 3 most intensively sampled host species each harbored 4 highly differentiated haemosporidian lineages. These results indicate that some Haemoproteus spp. and Plasmodium spp. lineages exhibit a low degree of host specificity, a phenomenon with implications for ecological and evolutionary interactions among these parasites and their hosts.     

Prevalence and differential host-specificity of two avian blood parasite genera in the Australo-Papuan region

The degree to which widespread avian blood parasites in the genera Plasmodium and Haemoproteus pose a threat to novel hosts depends in part on the degree to which they are constrained to a particular host or host family. We examined the host distribution and host-specificity of these parasites in birds from two relatively understudied and isolated locations: Australia and Papua New Guinea. Using polymerase chain reaction (PCR), we detected infection in 69 of 105 species, representing 44% of individuals surveyed (n = 428). Across host families, prevalence of Haemoproteus ranged from 13% (Acanthizidae) to 56% (Petroicidae) while prevalence of Plasmodium ranged from 3% (Petroicidae) to 47% (Ptilonorhynchidae). We recovered 78 unique mitochondrial lineages from 155 sequences. Related lineages of Haemoproteus were more likely to derive from the same host family than predicted by chance at shallow (average LogDet genetic distance = 0, n = 12, P = 0.001) and greater depths (average distance = 0.014, n = 11, P < 0.001) within the parasite phylogeny. Within two major Haemoproteus subclades identified in a maximum likelihood phylogeny, host-specificity was evident up to parasite genetic distances of 0.029 and 0.007 based on logistic regression. We found no significant host relationship among lineages of Plasmodium by any method of analysis. These results support previous evidence of strong host-family specificity in Haemoproteus and suggest that lineages of Plasmodium are more likely to form evolutionarily-stable associations with novel hosts.     

Cross-species infection of blood parasites between resident and migratory songbirds in Africa

We studied the phylogeny of avian haemosporidian parasites, Haemoproteus and Plasmodium, in a number of African resident and European migratory songbird species sampled during spring and autumn in northern Nigeria. The phylogeny of the parasites was constructed through sequencing part of their mitochondrial cytochrome b gene. We found eight parasite lineages, five Haemoproteus and three Plasmodium, infecting multiple host species. Thus, 44% of the 18 haemospiridian lineages found in this study were detected in more than one host species, indicating that host sharing is a more common feature than previously thought. Furthermore, one of the Plasmodium lineages infected species from different host families, Sylviidae and Ploceidae, expressing exceptionally large host range. We mapped transmission events, e.g. the occurrence of the parasite lineages in resident bird species in Europe or Africa, onto a phylogenetic tree. This yielded three clades, two Plasmodium and one Haemoproteus, in which transmission seems to occur solely in Africa. One Plasmodium clade showed European transmission, whereas the remaining two Haemoproteus clades contained mixes of lineages of African, European or unknown transmission. The mix of areas of transmission in several branches of the phylogenetic tree suggests that transmission of haemosporidian parasites to songbirds has arisen repeatedly in Africa and Europe. Blood parasites could be viewed as a cost of migration, as migratory species in several cases were infected with parasite lineages from African resident species. This cost of migration could have considerable impact on the evolution of migration and patterns of winter distribution in migrating birds.     

Avian haematozoan parasites and their associations with mosquitoes across Southwest Pacific Islands

The degree to which haematozoan parasites can exploit a range of vectors and hosts has both ecological and evolutionary implications for their transmission and biogeography. Here we explore the extent to which closely related mosquito species share the same or closely related haematozoan parasites, and examine the overlap in parasite lineages with those isolated from avian hosts, Zosterops species, sampled across the same study sites. Mosquito samples were collected and analysed (14 species, n = 804) from four islands in Vanuatu and the main island of New Caledonia. Using polymerase chain reaction, 15.5% (14/90) of pooled mosquito (thoracic) samples showed positive amplifications. Subsequent phylogenetic analysis of the cytochrome b gene identified four genetically distinct Plasmodium and two Haemoproteus lineages from these samples, five of which were identical to parasite lineages (n = 21) retrieved from the avian hosts. We found that three Plasmodium lineages differing by a maximum of 0.9% sequence divergence were recovered from different species and genera of mosquitoes and two Haemoproteus lineages differing by 4.6% sequence divergence were carried by 10 distantly related (11-21% divergent) mosquito species. These data suggest a lack of both cospeciation and invertebrate host conservatism. Without experimental demonstration of the transmission cycle, it is not possible to establish whether these mosquitoes are the biological vectors of isolated parasite lineages, reflecting a limitation of a purely polymerase chain reaction-based approach. Nonetheless, our results raise the possibility of a new transmission pathway and highlight extensive invertebrate host shifts in an insular mosquito-parasite system.     

Nonspecific patterns of vector, host and avian malaria parasite associations in a central African rainforest

Malaria parasites use vertebrate hosts for asexual multiplication and Culicidae mosquitoes for sexual and asexual development, yet the literature on avian malaria remains biased towards examining the asexual stages of the life cycle in birds. To fully understand parasite evolution and mechanism of malaria transmission, knowledge of all three components of the vector-host-parasite system is essential. Little is known about avian parasite-vector associations in African rainforests where numerous species of birds are infected with avian haemosporidians of the genera Plasmodium and Haemoproteus. Here we applied high resolution melt qPCR-based techniques and nested PCR to examine the occurrence and diversity of mitochondrial cytochrome b gene sequences of haemosporidian parasites in wild-caught mosquitoes sampled across 12 sites in Cameroon. In all, 3134 mosquitoes representing 27 species were screened. Mosquitoes belonging to four genera (Aedes, Coquillettidia, Culex and Mansonia) were infected with twenty-two parasite lineages (18 Plasmodium spp. and 4 Haemoproteus spp.). Presence of Plasmodium sporozoites in salivary glands of Coquillettidia aurites further established these mosquitoes as likely vectors. Occurrence of parasite lineages differed significantly among genera, as well as their probability of being infected with malaria across species and sites. Approximately one-third of these lineages were previously detected in other avian host species from the region, indicating that vertebrate host sharing is a common feature and that avian Plasmodium spp. vector breadth does not always accompany vertebrate-host breadth. This study suggests extensive invertebrate host shifts in mosquito-parasite interactions and that avian Plasmodium species are most likely not tightly coevolved with vector species.     

Low haemosporidian diversity and one key-host species in a bird malaria community on a mid-Atlantic island (São Miguel, Azores)

When host species colonize new areas, the parasite assemblage infecting the hosts might change, with some parasite species being lost and others newly acquired. These changes would likely lead to novel selective forces on both host and its parasites. We investigated the avian blood parasites in the passerine bird community on the mid-Atlantic island of S?o Miguel, Azores, a bird community originating from continental Europe. The presence of haemosporidian blood parasites belonging to the genera Haemoproteus, Plasmodium, and Leucocytozoon was assessed using polymerase chain reaction. We found two Plasmodium lineages and two Leucocytozoon lineages in 11 bird species (84% of all breeding passerine species) on the island. These lineages were unevenly distributed across bird species. The Eurasian Blackbird (Turdus merula) was the key-host species (total parasite prevalence of 57%), harboring the main proportion of parasite infections. Except for Eurasian Blackbirds, all bird species had significantly lower prevalence and parasite diversity compared to their continental populations. We propose that in evolutionary novel bird communities, single species may act as key hosts by harboring the main part of the parasite fauna from which parasites "leak" into the other species. This would create very different host-parasite associations in areas recently colonized by hosts as compared to in their source populations.     

A new PCR assay for simultaneous studies of Leucocytozoon, Plasmodium, and Haemoproteus from avian blood

Many bird species host several lineages of apicomplexan blood parasites (Protista spp., Haemosporida spp.), some of which are shared across different host species. To understand such complex systems, it is essential to consider the fact that different lineages, species, and families of parasites can occur in the same population, as well as in the same individual bird, and that these parasites may compete or interact with each other. In this study, we present a new polymerase chain reaction (PCR) protocol that, for the first time, enables simultaneous typing of species from the 3 most common avian blood parasite genera (Haemoproteus, Plasmodium, and Leucocytozoon). By combining the high detection rate of a nested PCR with another PCR step to separate species of Plasmodium and Haemoproteus from Leucocytozoon, this procedure provides an easy, rapid, and accurate method to separate and investigate these parasites within a blood sample. We have applied this method to bird species with known infections of Leucocytozoon spp., Plasmodium spp., and Haemoproteus spp. To obtain a higher number of parasite lineages and to test the repeatability of the method, we also applied it to blood samples from bluethroats (Luscinia svecica), for which we had no prior knowledge regarding the blood parasite infections. Although only a small number of different bird species were investigated (6 passerine species), we found 22 different parasite species lineages (4 Haemoproteus, 8 Plasmodium, and 10 Leucocytozoon).     

Big bang in the evolution of extant malaria parasites

Malaria parasites (genus Plasmodium) infect all classes of terrestrial vertebrates and display host specificity in their infections. It is therefore assumed that malaria parasites coevolved intimately with their hosts. Here, we propose a novel scenario of malaria parasite-host coevolution. A phylogenetic tree constructed using the malaria parasite mitochondrial genome reveals that the extant primate, rodent, bird, and reptile parasite lineages rapidly diverged from a common ancestor during an evolutionary short time period. This rapid diversification occurred long after the establishment of the primate, rodent, bird, and reptile host lineages, which implies that host-switch events contributed to the rapid diversification of extant malaria parasite lineages. Interestingly, the rapid diversification coincides with the radiation of the mammalian genera, suggesting that adaptive radiation to new mammalian hosts triggered the rapid diversification of extant malaria parasite lineages.     

Diversity, distribution and exchange of blood parasites meeting at an avian moving contact zone

Research on contact zones has paid relatively little attention to host-parasite interactions, although these situations have important but different implications depending on whether one considers the host or the parasite's perspective. We investigated both the role of a host contact zone in parasite expansion and whether parasites could influence contact zone dynamics. We studied the diversity and the patterns of parasite exchange (genera Haemoproteus and Plasmodium) infecting two parapatric sibling passerines meeting at a moving contact zone in western Europe. We amplified and sequenced a fragment of the parasite cytochrome b gene. The expanding host harboured more diverse parasites, which might indicate a superior ability to face a diverse parasite fauna than the receding host. Prevalence was very high in both hosts, due to the frequent occurrence of two sister Haemoproteus lineages. Despite the recent movement of the contact zone, these two parasites fitted almost perfectly to the geographic range of their main host species. Yet, we found several cases of cross-species infection in sympatric areas and evidences of asymmetrical spreading of parasites from the expanding host towards the receding host. Altogether, our results suggest that the host contact zone mainly acts as a barrier to parasite expansion even if recurrent host shifts are observed. Besides, they also support the idea that parasite-mediated competition might contribute to the displacement of hosts' contact zones, thereby emphasizing the role of parasitism on the population dynamics of sympatric species.     

Temporal dynamics and diversity of avian malaria parasites in a single host species

1. We have used molecular methods to unravel a remarkable diversity of parasite lineages in a long-term population study of great reed warblers Acrocephalus arundinaceus that was not foreseen from traditional microscopic examination of blood smears. This diversity includes eight Haemoproteus and 10 Plasmodium lineages of which most probably represent good biological species. 2. Contrary to expectation, the relative frequency of parasite lineages seemed not to change over the 17-year study period and we found no effects of the parasites on a male secondary sexual ornament (song repertoire size) and two measures of fitness (adult survival and production of recruited offspring). 3. We discuss whether the absence of fitness consequences of the parasites might relate to the fact that we have studied the host at the breeding sites in Europe, whereas the transmission seems to take place at the wintering sites in Africa, where the na?ve birds encounter the parasites for the first time and the resulting primary infections likely make them sicker than during the chronic phase of the infection. 4. The prevalence of the three most common lineages appeared to fluctuate in parallel with a periodicity of approximately 3-4 years. Theoretical models based on intrinsic interactions between parasite antigen and host immune genes cannot explain such dynamics, suggesting that knowledge of extrinsic parameters such as vector distribution and alternative hosts are required to understand these patterns.     

Parasite diversity declines with host evolutionary distinctiveness: A global analysis of carnivores

Evolutionarily distinctive host lineages might harbor fewer parasite species because they have fewer opportunities for parasite sharing than hosts having extant close relatives, or because diverse parasite assemblages promote host diversification. We evaluate these hypotheses using data from 930 species of parasites reported to infect free‐living carnivores. We applied nonparametric richness estimators to estimate parasite diversity among well‐sampled carnivore species and assessed how well host evolutionary distinctiveness, relative to other biological and environmental factors, explained variation in estimated parasite diversity. Species richness estimates indicate that the current published literature captures less than 50% of the true parasite diversity for most carnivores. Parasite species richness declined with evolutionary distinctiveness of carnivore hosts (i.e., length of terminal ranches of the phylogeny) and increased with host species body mass and geographic range area. We found no support for the hypothesis that hosts from more diverse lineages support a higher number of generalist parasites, but we did find evidence that parasite assemblages might have driven host lineage diversification through mechanisms linked to sexual selection. Collectively, this work provides strong support for host evolutionary history being an essential predictor of parasite diversity, and offers a simple model for predicting parasite diversity in understudied carnivore species.     

LINKAGE BETWEEN NUCLEAR AND MITOCHONDRIAL DNA SEQUENCES IN AVIAN MALARIA PARASITES: MULTIPLE CASES OF CRYPTIC SPECIATION?

Analyses of mitochondrial cytochrome b diversity among avian blood parasites of the genera Haemoproteus and Plasmodium suggest that there might be as many lineages of parasites as there are species of birds. This is in sharp contrast to the approximately 175 parasite species described by traditional methods based on morphology using light microscopy. Until now it has not been clear to what extent parasite mitochondrial DNA lineage diversity reflects intra- or interspecific variation. We have sequenced part of a fast-evolving nuclear gene, dihydrofolate reductase-thymidylate synthase (DHFR-TS), and demonstrate that most of the parasite mitochondrial DNA lineages are associated with unique gene copies at this locus. Although these parasite lineages sometimes coexist in the same host individual, they apparently do not recombine and could therefore be considered as functionally distinct evolutionary entities, with independent evolutionary potential. Studies examining parasite virulence and host immune systems must consider this remarkable diversity of avian malaria parasites.     

Multiple cryptic species of sympatric generalists within the avian blood parasite Haemoproteus majoris

The avian haemosporidian parasite Haemoproteus majoris has been reported to infect a wide range of passerine birds throughout the Holarctic ecozone. Five cytochrome b (cyt b) lineages have been described as belonging to the morphological species H. majoris, and these form a tight phylogenetic cluster together with 13 undescribed lineages that differ from each other by < 1.2% in sequence divergence. Records in a database (MalAvi) that contains global findings of haemosporidian lineages generated by universal primers suggest that these lineages vary substantially in host distribution. We confirm this pattern in a data set collected at Lake Kvismaren, Sweden, where three of the generalist lineages have local transmission. However, whether these lineages represent intraspecific mitochondrial diversity or clusters of cryptic species has previously not been examined. In this study, we developed novel molecular markers to amplify the partial segments of four nuclear genes to determine the level of genetic diversity and gene phylogenies among the five morphologically described cyt b lineages of H. majoris. All five cyt b lineages were strongly associated with unique nuclear alleles at all four nuclear loci, indicating that each mitochondrial lineage represents a distinct biological species. Within lineages, there was no apparent association between nuclear alleles and host species, indicating that they form genetically unstructured populations across multiple host species.     

Variation in host specificity between species of avian hemosporidian parasites: evidence from parasite morphology and cytochrome B gene sequences

A parasite's shift to a new host may have serious evolutionary consequences, since host switching usually is associated with a change in virulence and may lead to the evolution of emerging diseases. This phenomenon remains insufficiently studied in wildlife. Here, we combine microscopic examination of blood films and PCR-based methods to investigate the natural host specificity of Haemoproteus and Plasmodium spp. in birds of 4 families of the Passeriformes within a small geographic area. The material was collected on the Curonian Spit in the Baltic Sea between May and July in 2003-2004. A nested-PCR protocol was used for amplifying and sequencing a fragment of 480 nucleotides of the cytochrome b gene of the mtDNA of these parasites. Blood samples from 282 birds, which were positive both by microscopic examination of blood films and mtDNA amplification, were used in this study. We found that Haemoproteus majoris (lineages hPARUS1, hCCF5, hWW2, and hPHSIB1), Haemoproteus sp. (hWW1), Plasmodium (Haemamoeba) sp. (pSGS1), and Plasmodium (Haemamoeba) sp. (pGRW11) are capable of infecting birds belonging to different families of passeriform birds. Some species of Haemoproteus are less specific than have been traditionally believed. Haemoproteus majoris appears to have a genetic predisposition to have a broad host range. The level of host specificity varies markedly among different species of hemosporidian parasites of birds. The natural host range is thus not a reliable taxonomic character in the systematics of these parasites in the form in which it is still accepted in some recent taxonomic studies.     

Diversity and distribution of avian haematozoan parasites in the western Indian Ocean region: a molecular survey

The genetic diversity of haematozoan parasites in island avifauna has only recently begun to be explored, despite the potential insight that these data can provide into the history of association between hosts and parasites and the possible threat posed to island endemics. We used mitochondrial DNA sequencing to characterize the diversity of 2 genera of vector-mediated parasites (Plasmodium and Haemoproteus) in avian blood samples from the western Indian Ocean region and explored their relationship with parasites from continental Africa. We detected infections in 68 out of 150 (45·3%) individuals and cytochrome b sequences identified 9 genetically distinct lineages of Plasmodium spp. and 7 lineages of Haemoproteus spp. We found considerable heterogeneity in parasite lineage composition across islands, although limited sampling may, in part, be responsible for perceived differences. Two lineages of Plasmodium spp. and 2 lineages of Haemoproteus spp. were shared by hosts in the Indian Ocean and also on mainland Africa, suggesting that these lineages may have arrived relatively recently. Polyphyly of island parasites indicated that these parasites were unlikely to constitute an endemic radiation and instead probably represent multiple colonization events. This study represents the first molecular survey of vector-mediated parasites in the western Indian Ocean, and has uncovered a diversity of parasites. Full understanding of parasite community composition and possible threats to endemic avian hosts will require comprehensive surveys across the avifauna of this region.     

Host specificity in avian blood parasites: a study of Plasmodium and Haemoproteus mitochondrial DNA amplified from birds

A fragment of the mitochondrial cytochrome b gene of avian malaria (genera Haemoproteus and Plasmodium) was amplified from blood samples of 12 species of passerine birds from the genera Acrocephalus, Phylloscopus and Parus. By sequencing 478 nucleotides of the obtained fragments, we found 17 different mitochondrial haplotypes of Haemoproteus or Plasmodium among the 12 bird species investigated. Only one out of the 17 haplotypes was found in more than one host species, this exception being a haplotype detected in both blue tits (Parus caeruleus) and great tits (Parus major). The phylogenetic tree which was constructed grouped the sequences into two clades, most probably representing Haemoproteus and Plasmodium, respectively. We found two to four different parasite mitochondrial DNA (mtDNA) haplotypes in four bird species. The phylogenetic tree obtained from the mtDNA of the parasites matched the phylogenetic tree of the bird hosts poorly. For example, the two tit species and the willow warbler (Phylloscopus trochilus) carried parasites differing by only 0.6% sequence divergence, suggesting that Haemoproteus shift both between species within the same genus and also between species in different families. Hence, host shifts seem to have occurred repeatedly in this parasite host system. We discuss this in terms of the possible evolutionary consequences for these bird species.     

Prevalence and diversity of avian hematozoan parasites in Asia: a regional survey

Tissue samples from 699 birds from three regions of Asia (Myanmar, India, and South Korea) were screened for evidence of infection by avian parasites in the genera Plasmodium and Haemoproteus. Samples were collected from November 1994 to October 2004. We identified 241 infected birds (34.0%). Base-on-sequence data for the cytochrome b gene from 221 positive samples, 34 distinct lineages of Plasmodium, and 41 of Haemoproteus were detected. Parasite diversity was highest in Myanmar followed by India and South Korea. Parasite prevalence differed among regions but not among host families. There were four lineages of Plasmodium and one of Haemoproteus shared between Myanmar and India and only one lineage of Plasmodium shared between Myanmar and South Korea. No lineages were shared between India and South Korea, although an equal number of distinct lineages were recovered from each region. Migratory birds in South Korea and India originate from two different migratory flyways; therefore cross-transmission of parasite lineages may be less likely. India and Myanmar shared more host species and habitat types compared to South Korea. Comparison between low-elevation habitat in India and Myanmar showed a difference in prevalence of haematozoans.     

A restriction enzyme-based assay to distinguish between avian hemosporidians

We describe a reliable and relatively inexpensive method for detecting and differentiating between the commonly studied avian blood parasite genera Haemoproteus, Plasmodium, and Leucocytozoon. The assay takes advantage of a Haemoproteus-specific restriction site identified by sequencing full mitochondrial genomes from two Haemoproteus and three Plasmodium lineages and an adjacent, genus-specific restriction site identified in Leucocytozoon spp. The assay was sensitive to simulated parasitemias of approximately 8 x 10(-6) per erythrocyte and was 100% accurate in differentiating between parasite genera isolated from a broad geographical and taxonomic sampling of infected hosts.     

MalAvi: a public database of malaria parasites and related haemosporidians in avian hosts based on mitochondrial cytochrome b lineages

Research in avian blood parasites has seen a remarkable increase since the introduction of polymerase chain reaction-based methods for parasite identification. New data are revealing complex multihost-multiparasite systems which are difficult to understand without good knowledge of the host range and geographical distribution of the parasite lineages. However, such information is currently difficult to obtain from the literature, or from general repositories such as GenBank, mainly because (i) different research groups use different parasite lineage names, (ii) GenBank entries frequently refer only to the first host and locality at which each parasite was sampled, and (iii) different researchers use different gene fragments to identify parasite lineages. We propose a unified database of avian blood parasites of the genera Plasmodium, Haemoproteus and Leucocytozoon identified by a partial region of their cytochrome b sequences. The database uses a standardized nomenclature to remove synonymy, and concentrates all available information about each parasite in a public reference site, thereby facilitating access to all researchers. Initial data include a list of host species and localities, as well as genetic markers that can be used for phylogenetical analyses. The database is free to download and will be regularly updated by the authors. Prior to publication of new lineages, we encourage researchers to assign names to match the existing database. We anticipate that the value of the database as a source for determining host range and geographical distribution of the parasites will grow with its size and substantially enhance the understanding of this remarkably diverse group of parasites.     

A molecular phylogeny of malarial parasites recovered from cytochrome b gene sequences

A phylogeny of haemosporidian parasites (phylum Apicomplexa, family Plasmodiidae) was recovered using mitochondrial cytochrome b gene sequences from 52 species in 4 genera (Plasmodium, Hepatocystis, Haemoproteus, and Leucocytozoon), including parasite species infecting mammals, birds, and reptiles from over a wide geographic range. Leucocytozoon species emerged as an appropriate out-group for the other malarial parasites. Both parsimony and maximum-likelihood analyses produced similar phylogenetic trees. Life-history traits and parasite morphology, traditionally used as taxonomic characters, are largely phylogenetically uninformative. The Plasmodium and Hepatocystis species of mammalian hosts form 1 well-supported clade, and the Plasmodium and Haemoproteus species of birds and lizards form a second. Within this second clade, the relationships between taxa are more complex. Although jackknife support is weak, the Plasmodium of birds may form 1 clade and the Haemoproteus of birds another clade, but the parasites of lizards fall into several clusters, suggesting a more ancient and complex evolutionary history. The parasites currently placed within the genus Haemoproteus may not be monophyletic. Plasmodium falciparum of humans was not derived from an avian malarial ancestor and, except for its close sister species, P. reichenowi, is only distantly related to haemospordian parasites of all other mammals. Plasmodium is paraphyletic with respect to 2 other genera of malarial parasites, Haemoproteus and Hepatocystis. Explicit hypothesis testing supported these conclusions.