Remarks on the diversity of Hysteromorpha Lutz 1931 (Digenea, Diplostomidae), with erection of a new species from South America

Syst Parasitol (2025) 102:28 https://doi.org/10.1007/s11230-025-10224-0 Remarks on the diversity of Hysteromorpha Lutz 1931 (Digenea, Diplostomidae), with erection of a new species from South America Tyler J. Achatz · Sean A. Locke · Florencia Arrascaeta · Martin M. Montes · Jorge Barneche · Alan Fecchio · Jeffrey A. Bell · Pablo Oyarzún‑Ruiz · Geza T. R. Souza · Ricardo M. Takemoto · Vasyl V. Tkach Received: 31 December 2024 / Accepted: 7 March 2025 / Published online: 29 March 2025 © The Author(s) 2025 Abstract Hysteromorpha Lutz, 1931 is a small but broadly distributed genus of diplostomoidean digeneans parasitic as adults primarily in cormorants, but also reported from some other fish-eating birds. Their metacercariae were found in a variety of freshwater fishes as second intermediate hosts. Prior to this study, the genus included only 3 nominal species, 2 of them distributed in the Old World and 1 in the New World. We obtained sequences of partial mitochondrial cox1 gene and nuclear rDNA operon from new specimens collected in Europe, North and South America and used them for species comparisons and phylogenetic analysis. We also examined Supplementary Information The online version contains supplementary material available at https://doi. org/10.1007/s11230-025-10224-0. T. J. Achatz Department of Natural Sciences, Middle Georgia State University, Macon, GA, USA S. A. Locke Department of Biology, University of Puerto Rico at Mayagüez, Mayagüez, PR, USA F. Arrascaeta · M. M. Montes · J. Barneche Centro de Estudios Parasitológicos y Vectores (CEPAVE), Consejo Nacional de Investigaciones Científicas y Técnicas, Universidad Nacional de La Plata (CCT, CONICET-UNLP), La Plata, Buenos Aires, Argentina A. Fecchio Department of Ornithology, Academy of Natural Sciences of Drexel University, Philadelphia, PA, USA morphology of our newly collected specimens as well as museum specimens. Our analysis has demonstrated that at least 2 (likely 3) species of Hysteromorpha are distributed in the Americas. Hysteromorpha sp. previously sequenced from larval stages, clearly represents a new species named herein Hysteromorpha ostrowskiae n. sp. Achatz, Locke et Tkach. Morphology of adults of the new species was earlier described in sufficient detail under the name Hysteromorpha triloba (Rudolphi, 1819). Our analyses also suggest the presence of another unknown Hysteromorpha sp. in North America represented by cox1 sequence from J. A. Bell · V. V. Tkach (*) Department of Biology, University of North Dakota, Grand Forks, ND, USA e-mail: P. Oyarzún‑Ruiz Departamento de Microbiología, Facultad de Ciencias Biológicas, Universidad de Concepción, Concepción, Chile G. T. R. Souza Federal Institute of Education, Science and Technology of São Paulo, Salto, São Paulo, Brazil R. M. Takemoto Postgraduate Program in Ecology of Continental Aquatic Environments (PEA), Limnology, Ichthyology and Aquaculture Research Center (NUPELIA), State University of Maringá, Maringá, Paraná, Brazil Vol.: (0123456789) 28 Page 2 of 10 southeastern Canada (GenBank JF769473), but no morphological vouchers are available for this species. Introduction Hysteromorpha Lutz, 1931 is a small genus of diplostomids (Digenea Carus, 1863: Diplostomidae Poirier, 1886) that has a broad geographic distribution (Australia, Asia, Europe, North and South America) (Drago et al., 2011; Dubois, 1970a; González-Acuña et al., 2020; Heneberg et al., 2020; Locke et al., 2018; Sereno-Uribe et al., 2019; Sudarikov, 1960). Although Hysteromorpha spp. have been reported from various fish-eating birds, cormorants are their preferred definitive hosts (Dubois, 1970a; Forrester & Spalding, 2003; González-Acuña et al., 2020; Heneberg et al., 2020; Locke et al., 2018; Sereno-Uribe et al., 2019; Sudarikov, 1960). Members of this genus utilize a diversity of freshwater fishes as second intermediate hosts (Dubois, 1970a; Locke et al., 2018; Sudarikov, 1960; Sereno-Uribe et al., 2019). At present, the genus includes 3 nominal species: Hysteromorpha triloba (Rudolphi, 1819), Hysteromorpha plataleae Dubinin et Dubinina, 1940 and Hysteromorpha corti (Hughes, 1929); Hysteromorpha triloba and H. plataleae are distributed in the Old World, while H. corti is distributed in the New World (Dubois, 1970a; Locke et al., 2018; Sudarikov, 1960). López-Hernández et al. (2019) described cercariae and metacercariae of an additional species-level genetic lineage of Hysteromorpha collected in Brazil, but no adults of this lineage have been previously sequenced. Several previous studies have reported adults identified as H. triloba in various parts of South America (Drago et al., 2011; GonzálezAcuña et al., 2020; Lutz, 1931). In the present study, we re-evaluate the diversity of Hysteromorpha. We generated sequences of the internal transcribed spacer region (ITS1 + 5.8S + ITS2) and partial large ribosomal subunit gene (28S) of the nuclear ribosomal DNA operon and cytochrome c oxidase subunit 1 (cox1) mtDNA gene for 3 species of Hysteromorpha spp. collected in Europe, North America and South America. Due to its higher variability, cox1 sequences were used for distinguishing among species and phylogenetic analysis of the interrelationships among Hysteromorpha spp. Vol:. (1234567890) Syst Parasitol (2025) 102:28 Materials and methods Morphological study Adult specimens of Hysteromorpha spp. were collected from intestines of various cormorant species and common redshank Tringa totanus (L.) (Scolopacidae Rafinesque) in the USA (Minnesota), Brazil (State of Mato Grosso), Chile (Diguillín Province) and Ukraine (Kherson Oblast). Metacercariae of Hysteromorpha spp. were collected from black bullhead catfish Ameiurus melas Rafinesque and white sucker Catostomus commersonii (Lacépède) in the USA (North Dakota) and peppered cory Hoplisoma paleatum (Jenyns) in Argentina (Buenos Aires Province). Live digeneans were heat-killed and preserved in 80% ethanol; specimens for light microscopy were stained with aqueous alum carmine, permanently mounted following Lutz et al. (2017) and examined using an O lympus© BX53 microscope (Olympus America, Pennsylvania, USA) equipped with DIC optics. Type specimens of the new species are deposited by Ostrowski de Núñez (1970) in the Museo Argentino de Ciencias Naturales (MACN). Our voucher specimens are deposited in the collection of the Harold W. Manter Laboratory (HWML), University of Nebraska State Museum, Lincoln, Nebraska and Helminthological Collection of the Museo de La Plata, Argentina (MLP-He) (Table 1). Molecular study Extraction of genomic DNA from whole specimens was performed according to Tkach & Pawlowski (1999) and amplification by polymerase chain reactions (PCR) occurred in a T100TM thermal cycler (Bio-Rad, California, USA). The forward primer digL2 (5′-AAG CAT ATC ACT AAG CGG-3′) and reverse primer 1500R (5′-GCT ATC CTG AGG GAA ACT TCG-3′) were used to amplify 28S (Tkach et al., 2003). The ITS region was amplified using forw (...truncated)