Phylogenetic relationships, expanded diversity and distribution of Crassiphiala spp. (Digenea, Diplostomidae), agents of black spot disease in fish

Abstract

Crassiphiala is a monotypic genus of diplostomid digeneans and is the type genus of the subfamily Crassiphialinae. The type species Crassiphiala bulboglossa parasitizes kingfishers in the Nearctic and has a Neascus-type metacercaria that encysts on fish intermediate hosts, often causing black spot disease. While recent molecular phylogenetic studies included some members of the Crassiphialinae, no DNA sequence data of Crassiphiala is currently available. Our molecular and morphological study of adult and larval crassiphialines from the Americas revealed the presence of at least three lineages of Crassiphiala from the Nearctic and two lineages from the Neotropics. This is the first record of Crassiphiala from the Neotropics. Herein, we provide the first molecular phylogeny of the Diplostomoidea that includes Crassiphiala. Our data revealed 0.2-2.4% divergence among 28S sequences and 11-19.8% among CO1 sequences of lineages of Crassiphiala. The results of our analyses did not support the monophyly of Crassiphialinae. Our results clearly demonstrated that the diversity of Crassiphiala has been underestimated.

Keywords: Black spot disease; Crassiphiala; Diplostomidae; Diversity; Molecular phylogeny.

Fig. 1

Phylogenetic interrelationships among 38 diplostomoidean taxa including Crassiphiala based on Bayesian Inference (BI) analysis of partial 28S rRNA gene sequences. Members of the subfamily Crassiphialinae as currently recognized are indicated by the shaded rectangles. Bayesian Inference posterior probability values lower than 80% are not shown. New sequences obtained in this study are marked by an asterisk. Scale bar indicates number of substitutions per site. Abbreviations: BR – Brazil, USA – United States of America.

Fig. 2

Phylogenetic interrelationships among Crassiphiala lineages based on Bayesian Inference (BI) analysis of partial CO1 mtDNA sequences. Bayesian Inference posterior probability values lower than 80% are not shown. All sequences of Crassiphiala included in our analysis are new and marked by an asterisk. Scale bar indicates number of substitutions per site. Abbreviations: BR – Brazil, USA – United States of America.

Fig. 3

Adult specimens of Crassiphiala from Megaceryle torquata from Pantanal, Brazil. (a) Ventral view of whole mount of Crassiphiala lineage 4. (b) Ventral view of the prosoma of Crassiphiala lineage 4. (c) Lateral view of posterior body end of Crassiphiala lineage 4. (d) Ventral view of the prosoma of Crassiphiala lineage 5. (e) Ventral view of whole mount of Crassiphiala lineage 5. Scale bars: (a) = 500 μm; (b) = 100 μm; (c) = 100 μm; (d) = 100 μm; (e) = 300 μm.