Integrating coalescent species delimitation with analysis of host specificity reveals extensive cryptic diversity despite minimal mitochondrial divergence in the malaria parasite genus Leucocytozoon

Abstract

BackgroundCoalescent methods that use multi-locus sequence data are powerful tools for identifying putatively reproductively isolated lineages, though this approach has rarely been used for the study of microbial groups that are likely to harbor many unrecognized species. Among microbial symbionts, integrating genetic species delimitation methods with trait data that could indicate reproductive isolation, such as host specificity data, has rarely been used despite its potential to inform species limits. Here we test the ability of an integrative approach combining genetic and host specificity data to delimit species within the avian malaria parasite genus Leucocytozoon in central Alaska.ResultsWe sequenced seven nuclear loci for 69 Leucocytozoon samples and used multiple species delimitation methods (GMYC and BPP models), tested for differences in host infection patterns among putative species based on 406 individual infections, and characterized parasite morphology. We found that cryptic morphology has masked a highly diverse Leucocytozoon assemblage, with most species delimitation methods recovering support for at least 21 separate species that occur sympatrically and have divergent host infection patterns. Reproductive isolation among putative species appears to have evolved despite low mtDNA divergence, and in one instance two Leucocytozoon cytb haplotypes that differed by a single base pair (~ 0.2% divergence) were supported as separate species. However, there was no consistent association between mtDNA divergence and species limits. Among cytb haplotypes that differed by one to three base pairs we observed idiosyncratic patterns of nuclear and ecological divergence, with cytb haplotype pairs found to be either conspecific, reproductively isolated with no divergence in host specificity, or reproductively isolated with divergent patterns of host specialization.ConclusionIntegrating multi-locus genetic species delimitation methods and non-traditional ecological data types such as host specificity provide a novel view of the diversity of avian malaria parasites that has been missed previously using morphology and mtDNA barcodes. Species delimitation methods show that Leucocytozoon is highly species-rich in Alaska, and the genus is likely to harbor extraordinary species-level diversity worldwide. Integrating genetic and ecological data will be an important approach for understanding the diversity and evolutionary history of microbial symbionts moving forward.Electronic supplementary materialThe online version of this article (10.1186/s12862-018-1242-x) contains supplementary material, which is available to authorized users.

An inverse latitudinal gradient in infection probability and phylogenetic diversity for Leucocytozoon blood parasites in New World birds

“…Experimental infections under laboratory conditions will be necessary to confirm whether high temperature is a limiting factor for Leucocytozoon parasite development within their vectors, which ultimately affects prevalence and distributions among their avian hosts, thus constraining lineage diversity across biogeographical regions. Nevertheless, higher prevalence of Leucocytozoon in boreal (Galen et al, ; Oakgrove et al, ; this study) and temperate regions (Merino et al, , this study) supports the idea that Leucocytozoon parasites (and perhaps their vectors) may be better adapted for development and transmission in colder environments (Valkiūnas, ).…”

Genomic sequence capture of haemosporidian parasites: Methods and prospects for enhanced study of host–parasite evolution

“…Some lineages differing by a single nucleotide in the cytb barcode region are considered to be reproductively isolated, biological species (Nilsson et al, ), while others are considered to represent intraspecific variants (Hellgren et al, ; Outlaw & Ricklefs, ). Galen, Nunes, et al () used seven nuclear loci to show that both phenomena occur in Leucocytozoon , confirming the poor resolution of mtDNA for inferring species limits. Second, developing large, multilocus nuclear DNA sequence data sets is needed to advance the study of haemosporidian evolutionary dynamics.…”

“…Relatively few studies thus far have included multilocus nuclear data of avian haemosporidian parasites (Figure ). Studies demonstrate that the cytb barcode provides limited resolution; a single cytb haplotype can include multiple cryptic species (Falk, Glor, & Perkins, ; Galen, Nunes, Sweet, & Perkins, ), and phylogenies estimated from multiple nuclear loci substantially improve inferences of evolutionary relationships (Borner et al, ; Galen, Borner, et al, ). Several challenges, however, have previously prevented any large‐scale efforts to obtain genomic data from avian haemosporidians.…”

Population structure of avian malaria parasites

“…The avian malaria parasite lineages assessed here have been defined based on their similarity at the partial cytochrome b gene, but whether they represent good phylogenetic species has not been determined. Broad host ranges, propensity for host switching, and difficulty in linking morphological species and genetic lineages have all contributed to the problem of delimiting species in this group (Galen, Nunes, Sweet, & Perkins, ; Martinsen, Perkins, & Schall, ; Outlaw & Ricklefs, ). Moreover, a cut‐off based on percentage of divergence is not a useful way to define these species because it has been demonstrated through combined geographic range, host range, and corroboration with multiple loci that good phylogenetic species can exhibit low divergence at CYTB (Nilsson et al., ; Outlaw & Ricklefs, ).…”

“…The avian malaria parasite lineages assessed here have been defined based on their similarity at the partial cytochrome b gene, but whether they represent good phylogenetic species has not been de- Outlaw . In the absence of morphological data, species delimitation is thought to be best addressed by comparing multiple gene trees (Bensch, Pérez-Tris, Waldenström, & Hellgren, 2004;Galen et al, 2018;.…”