Our results demonstrate subtle differences in the host strategies of Haemoproteus
spp. and Plasmodium
spp. and corroborate patterns emerging from previous regional surveys that have revealed broad variability of host-parasitism strategies employed by both genera of avian haematozoa (Beadell et al., 2004
; Ricklefs et al., 2004
; Fallon et al., 2005
; Krizanauskiene et al., 2006
). Application of methods similar to those described here to a haematozoan parasite fauna from Australia and Papua New Guinea indicated that lineages of Haemoproteus
generally appeared to be more constrained at the family level than lineages of Plasmodium
(Beadell et al., 2004
). In the present study we demonstrated that, on average, lineages of Haemoproteus
were more constrained at the level of host species than were lineages of Plasmodium
. This difference in host specificity, however, was not evident following removal of the three lineages of Plasmodium
with the broadest host distribution (WA20, WA9 and WA15). Furthermore, the difference in specificity did not extend to the level of host family. In fact, probing host-parasite associations across the parasite phylogeny using logistic regression suggested that at least one large collection of Plasmodium
lineages (clade PA) exhibited a signal of host specificity at the host family level (0.026) that was equivalent to the signal obtained for a well-defined group of Australo-Papuan Haemoproteus
lineages (0.029) and stronger than any signal recovered for groups of West African Haemoproteus
(range = 0.013 to 0.017). Thus, at least some lineages of Plasmodium
appear to be constrained to certain host groups to the same extent as lineages of Haemoproteus
Because of the extreme variability in observed host-parasitism strategies, particularly in certain lineages of Plasmodium
, comparing average strategies among haematozoan genera may not be valuable. We recovered individual lineages of Plasmodium
from between one and 27 different avian host species. The extreme diversity of hosts observed for a single lineage is in keeping with the 39 species of host infected by lineage GRW4 worldwide (Beadell et al., 2006
) and the 27 hosts infected by lineage PA in the Antilles (Fallon et al., 2005
). We recovered lineages of Haemoproteus
from a maximum of three host species (three families), but other regional surveys have detected certain lineages of Haemoproteus
in up to seven (Krizanauskiene et al., 2006
) and even 26 (Fallon et al., 2005
) host species, suggesting that some Haemoproteus
lineages may exhibit similarly broad host distributions. In contrast to these generalist lineages, we also identified at least several individual lineages that exhibited significant host constraint at both the host species and host family level. More intensive and thorough sampling of the West African avifauna will undoubtedly expand the host ranges of many of the apparent specialist haematozoan lineages, but the signals of host specificity extending deeper within the Haemoproteus
phylogeny suggest that many of these lineages are likely to be true specialists. Thus, both Haemoproteus
appear to harbor lineages with strongly divergent host-parasitism strategies.
Why do related parasites exhibit such striking difference in host specificity? As outlined previously, specialists presumably benefit from relatively high fitness in the limited number of hosts that they utilize and may be able to evolve more quickly in response to changes in host defense or physiology. Generalists, on the other hand, may be less prone to extinction because they maintain larger populations distributed over a greater number of hosts. Thus, the strategy adopted by a parasite represents a fine balance between the selective pressures favoring either specialist or generalist strategies (Woolhouse et al., 2001
). Consequently, host-parasitism strategies may shift rapidly so that even closely related parasites may exhibit very different host ranges. Alternatively, it is possible that the generalist strategy does not exist, or that the generalist strategy represents an unstable and ephemeral transition state (Stireman, 2005
). Large observed haematozoan host ranges may reflect the spillover of parasites into hosts in which the full transmission cycle may never be completed. Or, they may simply reflect our inability to distinguish between cryptic parasite species. This phenomenon could explain the extreme lack of specialization observed in Plasmodium
lineage WA9, detected in 27 different species representing nine host families, and lineage WA15 which was detected in 15 species also representing nine different host families. We cannot rule out the possibility that these lineages actually represent a group of species that are so young that mutations have not yet accumulated to allow differentiation of the numerous specialists that may compose the group.
If mitochondrial lineage diversity is generated too slowly to reflect host specialization, one might question whether mitochondrial lineages are a useful taxonomic unit for investigating host specificity. Certainly, we may be limited in our ability to distinguish recently evolved parasite species. In this case, the broad host ranges of parasite lineages such as WA9 or WA15 may place a downward bias on our estimates of host-specificity. But, at the other end of the spectrum, we may ask whether mitochondrial lineages are valid proxies for long-established reproductively-isolated parasite species. If not, how does this affect estimates of host specificity?
Evidence from two nuclear genes suggests that mitochondrial lineages do provide a reasonable taxonomic metric, at least at the level of mitochondrial differentiation examined here. Although we cannot determine whether lineages that share a given mitochondrial haplotype are currently reproductively-isolated, sequencing of fragments of the DHFR-TS and transferase genes provided no evidence to refute the common ancestry of parasites sharing a single mitochondrial signature. In other words, the apparent generalism attributed to WA9, for example, did not appear to be the consequence of a single mitochondrial lineage having introgressed into multiple evolutionarily distinct parasites. Nuclear sequences from individuals possessing the WA9 mitochondrial signatures were either identical (transferase) or differed by a single nucleotide (DHFR), suggesting that the parasites sharing this mitochondrial lineage also share a similar nuclear genome.
Evaluated more broadly across other Plasmodium lineages, the correspondence between mitochondrial haplotype and nuclear sequences provided support for the use of mitochondrial lineages as the taxonomic basis for evaluating host associations. Mitochondrial lineages tended to be associated with distinct nuclear genotypes; however, the correspondence was not perfect. In at least one case (WA20), a single mitochondrial lineage encompassed three distinct nuclear signatures, evident in both transferase and DHFR. If this mitochondrial lineage is really composed of three reproductively-isolated species, then the host range of this seemingly generalist lineage could be inflated. In several other cases, we identified distinct mitochondrial lineages that shared at least one nuclear haplotype (e.g., WA37 and WA38 (transferase), WA19 and WA20 (DHFR and transferase), WA20 and WA21 (DHFR)). This sharing may represent instances of incomplete lineage sorting in which mitochondrial lineages do actually represent reproductively-isolated species. Alternatively, this sharing of nuclear haplotypes could indicate that the associated mitochondrial lineages simply represent intraspecific diversity. In this case, artificially separating these lineages would inflate estimates of host specificity. For lineages WA37 and WA38, the distinction is irrelevant given that both lineages were found in the same host species. Similarly, changes in host distributions arising from the genetic associations of lineages WA19, WA20 and WA21 would not dramatically alter the signal of host generalism arising from these lineages.
The apparent validity of using mitochondrial lineages as a foundation for investigating host-parasite associations is due in part to the resolution provided by our opportunistic sampling. Among the lineages that we sampled, the average genetic distance between pairs of most closely-related Plasmodium
lineages (first-step nodes) was about 1.4%. For Haemoproteus
lineages, the average minimum divergence was about 1.9%. As points of reference, the well-defined and closely-related species Plasmodium falciparum
and Plasmodium reichenowi
exhibit a divergence of about 2.3% across the mitochondrial genome (Joy et al., 2003), while morphospecies of Haemoproteus
can exhibit as little as 0.7% divergence (Hellgren et al., 2007
). Mean intra-morphospecies divergence can be substantially higher but at least some of this divergence may represent differentiation among cryptic biological species that share a similar morphology (Beadell et al., 2006
; Hellgren et al., 2007
). Therefore these data, combined with the strong correspondence between mitochondrial and nuclear haplotypes, suggest that the majority of the parasite lineages in our sample represent species-level taxonomic units. Further integration of genetic and morphological studies (Martinsen et al., 2006; Hellgren et al., 2007
), combined with experimental studies of parasite transmission (Iezhova et al., 2005
), should help to resolve the species limits of avian blood parasites and lend context to the lineage-level host ranges provided by regional surveys.
Our data suggest that host-parasitism strategies within the genera Haemoproteus
are variable and can show extreme differences even among closely-related lineages. While we demonstrated that at least some parasites within both genera have been constrained at the level of host-family and even host-species over their evolutionary history, we found evidence of apparent broad host generalism, particularly in certain lineages of Plasmodium
. Importantly, wide variability in host specificity among lineages of avian haematozoa may be linked to wide variation in virulence (Garamszegi, 2006
). Although several studies have demonstrated negative consequences of haematozoan infection for survival, clutch size, incubation period, fledging success, motor activity and fat accumulation (Bennett et al., 1993; Gustafsson et al., 1994
; Nordling et al., 1998
; Merino, 2000
; Valkiunas, 2005
), few have accounted for possible differences in the virulence of different lineages infecting a particular host population (but see Zehtindjiev et al., 2008
). Variability in host specificity, and therefore virulence, of closely related parasite lineages should be accounted for when estimating their impact on host fitness, immunity or life history. Regional surveys have uncovered numerous parasite lineages with extremely divergent host-parasitism strategies; these would now make good candidates for experimentally testing the assumed linkage between host-specificity and virulence.