Polychromophilus cytb

Arnuphapprasert, Apinya, Riana, Elizabeth, Ngamprasertwong, Thongchai, Wangthongchaicharoen, Monsicha, Soisook, Pipat, Thanee, Suchansa, Bhodhibundit, Phanaschakorn & Kaewthamasorn, Morakot, 2020, First molecular investigation of haemosporidian parasites in Thai bat species, International Journal for Parasitology: Parasites and Wildlife 13, pp. 51-61 : 56-57

3.3. Bat haemosporidian phylogenetic analysis based upon cytb nucleotide sequence

We obtained a total of 62 partial cytb nucleotide sequences, comprised of 50 sequences of Hepatocysti s, 10 of Nycteria , and two of Polychromophilus . The residual 378 bp fragment of cytb was subjected to the sequence analyses. The sequence located corresponding to nucleotide positions 463 to 840 (amino acid positions 155 to 280) of the cytb gene in Nycteria heischi (accession no. KX090648). The percentage of trees in which the associated taxa clustered together is shown next to the branches. Among the 50 sequences of Hepatocysti s, 19 different haplotypes were detected. Haplotype numbers 1 of Hepatocysti s (originated from H. larvatus ; n = 19 and H. armiger ; n = 1); and 6 ( H. larvatus ; n = 9) were the dominant haplotypes (Supplementary Table S3). Haplotype numbers 3 ( R. pearsonii ; n = 2), 4 ( C. thonglongyai and R. pearsonii ; n = 2), 7 ( H. larvatus ; n = 3), and 12 ( H. larvatus ; n = 2) were less abundant. The remaining haplotypes were comprised of only one sequence each. Representatives of each haplotype were included in the phylogenetic analysis. Pairwise nucleotide identity among Hepatocystis in the present study ranged from 86.7 to 99.7%.

The cytb sequences of Hepatocysti s were divided into 19 haplotypes and clustered into three subclades (1a, 1b, and 2) ( Fig. 5 ). Almost all the Hepatocysti s in this study were placed in the same subclade (1a) as the Hepatocysti s previously reported in H. larvatus from Cambodia ( Duval et al., 2007). This subclade was comprised of Hepatocysti s isolated from

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H. larvatus , H. armiger , R. pearsonii , and C. thonglongyai . Another subclade (1b) was unique to the Hepatocysti s in Australasian small flying fox in the family Pteropodidae and none of our Hepatocysti s was placed in this subclade. Only one Hepatocysti s from Cr. brachyotis in the current study was placed in the same clade (2) with Hepatocysti s isolated from the same frugivorous bat species in Malaysia with strong BS support.

We detected Nycteria in M. spasma , C. thonglongyai , T. melanopogon , and E. spelaea bats that originated from the Phetchaburi, Ratchaburi, and Kanchanaburi provinces. A high nucleotide similarity among the sequences of Nycteria was found (97.6–100%). The Nycteria observed in our study were placed in the same clade as the Nycteria previously reported in M. spasma from Cambodia ( Duval et al., 2007), but in a distinct clade from the Nycteria observed in bats in the family Rhinolophidae from Africa (clade 1) ( Fig. 6 ). Nycteria in M. spasma was split into three separate subclades (subclades 2a, 2c, and 2d). Nycteria in C. thonglongyai , E. spelaea , and T. melanopogon was placed in separate subclades (2b, 2c, and 2d, respectively). Nycteria in T. melanopogon from Phetchaburi was closely related to Nycteria isolated from M. spasma in Kanchanaburi. Nycteria in E. spelaea from Ratchaburi was closely related to Nycteria isolated from M. spasma in Kanchanaburi and Cambodia. It is important to note that BS support only in subclades 2c and 2d were greater than 50.

Polychromophilus was detected in T. melanopogon and My. siligorensis bats in Kanchanaburi province. In the present study, the nucleotide similarity between the two sequences of Polychromophilus was 97.8%. The BLASTn results of Polychromophilus revealed two distinct species comprised of P. murinus and P. melanipherus (with percent identity 99.47 and 99.74, respectively). The P. melanipherus originating from T. melanopogon was clustered in clade 1 together with Polychromophilus seen in the African insectivorous bats in the family Miniopteridae ( Duval et al., 2012) ( Fig. 7 ), while P. murinus from My. siligorensis was placed in clade 2 with the P. murinus found in bats in the family Vespertilionidae in Switzerland ( Megali et al., 2011).

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Figures

Fig. 5. Phylogenetic (ML) relationship of the Hepatocystis cytb gene fragment (378 bp) in the present study (taxa with red circles) and the other global isolates from the GenBank™ database. Clade (number) and subclade (letter) are indicated. Haplotype 10 of Hepatocystis in the current study originated from the frugivorous bat Cy. brachyotis shares the same clade with Malaysian isolate (in clade 2). The percentage of trees in which the associated taxa clustered together is shown next to the branches. BS values greater than 50% are shown in the figure. The other clades, consisting of Nycteria, Polychromophilus, Plasmodium, and outgroup are collapsed. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 6. Phylogenetic (ML) relationship of the Nycteria cytb gene fragment (378 bp) obtained in the present study (taxa with blue circles) and the other isolates previously deposited in GenBank™ database. Nycteria from Asian bats clustered in clade 2 and a separate clade from the African bat isolates (in clade 1). BS values greater than 50% are shown in the figure. The other clades, consisting of Hepatocystis, Polychromophilus, Plasmodium, and outgroup are collapsed. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 7. Phylogenetic (ML) relationship of the Polychromophilus cytb gene fragment (378 bp) found in this study (taxa with green circles) and the other global isolates. Polychromophilus isolated from T. melanopogon is placed in the same clade with African bat isolates of P. melanipherus in clade 1. Polychromophilus isolated from My. siligorensis shares the same clade with European bat isolates of P. murinus in clade 2. BS values greater than 50% are shown in the figure. The other clades, consisting of Hepatocystis, Nycteria, Plasmodium, and outgroup are collapsed. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)