Molecular phylogeny of Diplostomum, Tylodelphys, Austrodiplostomum and Paralaria (Digenea: Diplostomidae) necessitates systematic changes and reveals a history of evolutionary host switching events

Abstract

The Diplostomidae Poirier, 1886 is a large, globally distributed family of digeneans parasitic in intestines of their definitive hosts. Diplostomum and Tylodelphys spp. are broadly distributed, commonly reported, and the most often sequenced diplostomid genera. The majority of published DNA sequences from these genera originated from larval stages only, which typically cannot be identified to the species level based on morphology alone. We generated partial large ribosomal subunit (28S) rRNA and cytochrome c oxidase subunit 1 (cox1) mtDNA gene sequences from 14 species/species-level lineages of Diplostomum, six species/species-level lineages of Tylodelphys, two species/species-level lineages of Austrodiplostomum, one species previously assigned to Paralaria, two species/species-level lineages of Dolichorchis and one unknown diplostomid. Our DNA sequences of 11 species/species-level lineages of Diplostomum (all identified to species), four species/species-level lineages of Tylodelphys (all identified to species), Austrodiplostomum compactum, Paralaria alarioides and Dolichorchis lacombeensis originated from adult specimens. 28S sequences were used for phylogenetic inference to demonstrate the position of Paralaria alarioides and Dolichorchis spp. within the Diplostomoidea and study the interrelationships of Diplostomum, Tylodelphys and Austrodiplostomum. Our results demonstrate that two diplostomids from the North American river otter (P. alarioides and a likely undescribed taxon) belong within Diplostomum. Further, our results demonstrate the non-monophyly of Tylodelphys due to the position of Austrodiplostomum spp., based on our phylogenetic analyses and morphology. Furthermore, the results of phylogenetic analysis of 28S confirmed the status of Dolichorchis as a separate genus. The phylogenies suggest multiple definitive host-switching events (birds to otters and among major avian groups) and a New World origin of Diplostomum and Tylodelphys spp. Our DNA sequences from adult digeneans revealed identities of 10 previously published lineages of Diplostomum and Tylodelphys, which were previously identified to genus only. The novel DNA data from this work provide opportunities for future comparisons of larval diplostomines collected in ecological studies.

Keywords: 28S; Cox1; Diplostomidae; Diplostomum; Molecular phylogeny; Tylodelphys.

Fig. 1.

Phylogenetic interrelationships among 43 diplostomoidean taxa including 13 members of Diplostomum, Tylodelphys and Austrodiplostomum (including a former Paralaria sp.), two species-level lineages of Dolichorchis and an unknown diplostomid based on Bayesian inference analysis of partial 28S rRNA gene sequences. Members of Diplostomum + Tylodelphys are indicated by the shaded rectangle. Bayesian inference posterior probability values lower than 80% are not shown. The new sequences generated in this study are indicated in bold. The scale bar indicates the number of substitutions per site. GenBank accession numbers are provided after the names of species. The previously accepted/published names are provided in parentheses after GenBank accession numbers.

Fig. 2.

Phylogenetic interrelationships among 21 taxa of Diplostomum (including a former Paralaria sp.) based on Bayesian inference analysis of partial 28S rRNA gene sequences. Bayesian inference posterior probability values lower than 80% are not shown. The new sequences generated in this study are indicated in bold. The scale bar indicates the number of substitutions per site. GenBank accession numbers are provided after the names of species. References to origins of species numbering/naming systems are provided in parentheses after GenBank accession numbers followed by the biogeographical realms where specimens were collected, life stages of isolates and families of definitive hosts (for adult isolates and larvae molecularly matched to adult forms). Abbreviations for references to the original designations of species-level lineages: H, Hoogendoorn et al. (2020); L, Locke et al. (2010a, b; 2015); N, Nakao and Sasaki, (2021). The previously accepted/published names are provided in parentheses after GenBank accession numbers. Abbreviations for biogeographical realms: Afr, Afrotropical realm; Nea, Nearctic realm; Neo, Neotropical realm; Pal, Palaearctic realm. Abbreviations for life stage: Adu, adult; Cer, cercaria; Met, metacercaria. Abbreviations for family of definitive host: Ana, Anatidae; Ard, Ardeidae; Gav, Gaviidae; Lar, Laridae; Mus, Mustelidae; Rec, Recurvirostridae; Sco, Scolopacidae.

Fig. 3.

Phylogenetic interrelationships among 13 taxa of Tylodelphys and Austrodiplostomum spp. based on Bayesian inference analysis of partial 28S rRNA gene sequences. Bayesian inference posterior probability values lower than 80% are not shown. The new sequences generated in this study are indicated in bold. The scale bar indicates the number of substitutions per site. GenBank accession numbers are provided after the names of species. References to origins of species numbering/naming systems are provided in parentheses after GenBank accession numbers followed by the biogeographical realms where specimens were collected, life stages of isolate and families of definitive hosts (for adult isolates and larvae molecularly matched to adult forms). Abbreviations for references to the original designations of species-level lineages: L, Locke et al. (2010a, b; 2015); R, Rosser et al. (2016a). The previously accepted/published names are provided in parentheses after GenBank accession numbers. Abbreviations for biogeographical realms: Nea, Nearctic realm; Neo, Neotropical realm; Pal, Palaearctic realm. Abbreviations for life stage: Adu, adult; Cer, cercaria; Met, metacercaria. Abbreviations for families of definitive hosts: Cic, Ciconiidae; Gav, Gaviidae; Pel, Pelecanidae; Pha, Phalacrocoracidae; Pod, Podicipedidae.

Fig. 4.

Phylogenetic interrelationships among 27 taxa of Tylodelphys and Austrodiplostomum spp. based on Bayesian inference analysis of partial cox1 mtDNA gene sequences. Bayesian inference posterior probability values lower than 80% are not shown. The new sequences generated in this study are indicated in bold. The scale bar indicates the number of substitutions per site. GenBank accession numbers are provided after the names of species. References to origins of species numbering/naming systems are provided in parentheses after GenBank accession numbers followed by the biogeographical realms where specimens were collected, life stages of isolate and families of definitive hosts (for adult isolates and larvae molecularly matched to adult forms). Abbreviations for references to the original designations of species-level lineages: C, Chibwana et al. (2013); Ch, Chaudhary et al. (unpublished); Go, Gordy and Hanington (2019); L, Locke et al. (2010a, b; 2015); P, Pelegrini et al. (2019); R, Rosser et al. (2016a); Se, Sereno-Uribe et al. (2019a); So, Soldánová et al. (2017). Abbreviations for biogeographical realms: Afr, Afrotropical realm; Aus, Australasian realm; Ind, Indomalayan realm; Nea, Nearctic realm; Neo, Neotropical realm; Pal, Palaearctic realm. Abbreviations for life stages: Adu, adult; Cer, cercaria; Met, metacercaria. Abbreviations for families of definitive hosts: Ard, Ardeidae; Cic, Ciconiidae; Gav, Gaviidae; Pel, Pelecanidae; Pha, Phalacrocoracidae; Pod, Podicipedidae.