Molecular characterization and morphological description of cryptic haemoproteids in the laughingthrushes (Leiothrichidae) in the western and eastern Himalaya, India

Abstract

Background: Laughingthrushes (family: Leiothrichidae) consists of diverse and widespread species found in the Indian subcontinent but there is a lack of information on their avian haemosporidians. Methods: We sampled 231 laughingthrushes of 8 species in the western and eastern Himalaya in India. Using parasite morphology and cytochrome b sequences we describe 2 new Haemoproteus species harbored in 3 species of laughingthrushes and report a case of cryptic speciation. Results: First Haemoproteus lineage TROERY01 (GenBank: KY623720) found in Trochalopteron erythrocephalum (27.47%) and Trochalopteron variegatum (2.9%) in mid to high altitude tropical forests in the western and eastern Himalaya, was described as Haemoproteus (Parahaemoproteus) leiothrichus n. sp. (Haemosporida: Haemoproteidae). Second Haemoproteus lineage TROERY02 (GenBank: KY623721) described as Haemoproteus (Parahaemoproteus) homoleiothrichus n. sp. (Haemosporida: Haemoproteidae) was found in T. erythrocephalum (2.19%) and Trochalopteron lineatum (3.84%), albeit in low intensity, only in the western Himalaya. Both H. homoleiothrichus n. sp. and H. leiothrichus n. sp. showed no significant difference in morphological features in blood stages. A genetic divergence of 4.4% along with distinct phylogenetic position indicates that these 2 lineages represent cryptic species. Previously, T. erythrocephalum has been described as an additional host for a morphologically described Haemoproteus timalus in the oriental region. Our described species have several morphological features that are absent in H. timalus. These are, the presence of dumbbell-like shaped mature gametocytes, 'arm' like extensions of gametocytes and lateral displacement of nuclei of infected erythrocytes. Illustrations of blood stages of the new species are given, and phylogenetic analysis with morphologically described Haemoproteus species identifies parasites closely related to the 2 described parasites. Conclusions: The lineages described here have been recorded only in the laughingthrushes so far. These are the first parasites to be described with T. erythrocephalum as a type host from the western and eastern Himalaya in India.

Keywords: Haemoproteus; India; Laughingthrushes; cryptic species.

Figure 1.. Study sites for laughingthrush sampling in India.

Figure 2.. Gametocytes of Haemoproteus leiothrichus n. sp.

( A– C) Growing gametocyte, ( D– H) mature macrogametocyte and ( I– L) mature microgametocytes. Scale bar = 10 µm. Long arrow points to the nucleus of the parasite; short arrow - indicates unfilled spaces between gametocytes and envelope of infected erythrocyte; arrow head shows pigment granules; long narrow arrow shows volutin granules.

Figure 3.. Gametocytes of Haemoproteus homoleiothrichus n. sp.

( A– C, F) Growing gametocyte, ( D, E) mature macrogametocyte and ( G– I) mature microgametocytes. Scale bar = 10 µm. Long arrow points to the nucleus of the parasite; short arrow - indicates unfilled spaces between gametocytes and envelope of infected erythrocyte; arrow head shows pigment granules; long narrow arrow shows volutin granules.

Figure 4.. Bayesian phylogeny of 40 Haemoproteus spp. lineages described morphologically together with Haemoproteus leiothrichus sp. nov.

(TROERY01) and Haemoproteus homoleiothrichus sp. nov. (TROERY02), the lineages recorded in the Trochalopteron erythrocephalum based on mitochondrial cytochrome b gene. Leucocytozoon majoris (GenBank Accession no: AY393804) has been used as an outgroup. Bootstrap values with greater than 60% support have been mentioned on the branches. Names of new species are given in bold.

Figure 5.. Gametocytes of Haemoproteus timalus (blood of the Garrulax mitratus; accession numbers 4062, in the collection of the International Reference Centre for Avian Haematozoa at the Queensland Museum, Australia); 5.

( A) growing gametocyte, ( B, C) macrogametocytes and ( D) microgametocyte. Scale bar = 10 µm. Long arrow points to the nucleus of the parasite; short arrow - indicates unfilled spaces between gametocytes and envelop of infected erythrocyte; arrow head shows pigment granules; long narrow arrow shows volutin granules.