PCR primers designed in this study
In 1 of 13 PCR products amplified using the previously reported Haemoproteus primer set, HaemF, and HaemR2, we identified Leucocytozoon sp. rather than Haemoproteus sp. by sequencing a part of cob (Bird ID S9 in Supplementary Table 2). This result led us to infer that some Leucocytozoon lineages exist that are not amplified using HaemFL and HaemR2L (Hellgren et al. 2004). We thus confirmed whether the cob regions of Leucocytozoon that can be annealed by the HaemFL/HaemR2L primers are conserved. To do this, we aligned cob sequences from 12 Leucocytozoon lineages reported by Pacheco et al. (2018) (Fig. 1). We focused on conservation of cob regions that anneal with the HaemFL and HaemR2L primers. We found several mutations in this region, so we designed new primers (HaemFLn and HaemRLn) to amplify minor-lineage sequences that would be difficult to amplify with the conventional primers (Fig. 1) (see Supplementary Table 1 for their nucleotide sequences). Our new primers were designed as follows: i) at sites with mutations in more than one lineage, the minority base was used at the new primer sequence (Fig. 1) (T219C, C228T, T234C, T723A); ii) the highly conserved position at 715 (C) was chosen as the 3' terminal of HaemR2Ln the primer since the 3'-terminal region is critical for PCR (that of HaemR2L primer is not well conserved).
Prevalence of haemosporidian parasites
Samples were taken from a total of 32 individual birds, 19 S. orientalis, and 13 C. livia, for PCR analysis. In the Haemoproteus-specific PCR assay, 12 out of 32 birds were positive. These birds were all S. orientalis and were co-infected with Leucocytozoon, as described in Table 1 and Supplementary Table 2. In the Leucocytozoon-specific assay, 16 out of 32 birds were positive using the conventinal primers (HeamFL/HaemR2L), and 15 were positive using our primers (HeamFLn/HaemR2Ln). The infection ratio was 79% (15/19) and 8% (1/13) in S. orientalis and C. livia, respectively. All the samples with positive amplifications were sequenced, and multiple infections were detected by visualizing the double-base calling in sequence electropherograms. PCR with Haemoproteus-specific primers did not detect any multiple infections (data not shown). PCR with the HeamFL/HaemR2 primers detected multiple infections in 16% of the samples (5/32), whereas our primers HeamFLn/HaemR2n detected multiple infections in 44% (14/32) (Fig. 2), indicating that our primers are more suitable for detection of multiple infection.
Identification of haemosporidian lineages in single and multiple infection
Of the 12 samples that tested positive for Haemoproteus, 11 were identified as STRORI01 lineage, and the remaining one (Bird ID S10 in Supplementary Table 2) demonstrated an SNP at position 430 of cob (Supplementary Table 2). The PCR products that were identified as “multiple infections” by their sequencing electropherograms were cloned, and 10 clones from each PCR product were sequenced. SNPs were sequenced more than three times for confirmation. Three different sequences were found from Bird ID S3, six from S4, two from S13, two from S23, and four from S29 (Fig. 3a). We registered these as newly discovered lineages (STRORI06 to STRORI15). However, no novel lineages were identified from PCRs using the conventional primers. All of the 16-Leucocytozoon-PCR-positive specimens amplified using the conventional primer set belonged to one of the four previously reported lineages (Fig. 3a). In addition to the three lineages (AMO02, COLIV04, and STRORI05) that were reported, ten novel lineages were identified using our primers, HaemFLn/HaemRLn (Fig. 3b), which could detect all lineages detected by the conventional primers except for one (STRORI02) (Fig. 3). All the novel lineages were found in samples indicated to be multiple infections. Of the ten novel lineages, eight demonstrated the same sequence found in more than two individual birds. In Bird ID S10, a STRORI05 lineage was detected with the conventional primer set, while the AEMO02 and COLIV04 lineages were detected using our primer set, indicating that detection of a number of lineages does depend on the primers used (Fig. 3).
Lineage diversity and phylogenetic analyses
To confirm the phylogenetic diversity of the lineages found in this study, a phylogenetic analysis was performed. Nine of the ten novel lineages were closely related and belong to the same phylogenetic group (Fig. 4). The new primers detected a wide range of Leucocytozoon compared with those detected using the conventional primers (Fig. 4). To show the variety of SNPs from all lineages found in this study, we compared them to AEMO02, which is the most widely infected lineage. Consequently, we identified 59 SNPs (Fig. 5). To see if unknown SNPs only found in novel lineages were present, we compared ten novel lineages with four known ones and determined that 13 new mutations were found only in the novel lineages. (Fig. 5).