Uncovering further diversity of Ochoterenatrema Caballero, 1943 (Digenea: Lecithodendriidae) in South American bats

Syst Parasitol (2024) 101:43 https://doi.org/10.1007/s11230-024-10165-0 Uncovering further diversity of Ochoterenatrema Caballero, 1943 (Digenea: Lecithodendriidae) in South American bats Vasyl V. Tkach · Roxanne Gasperetti · Thayane F. Fernandes Carlos A. Carrión‑Bonilla · Joseph A. Cook · Tyler J. Achatz · Received: 13 January 2024 / Accepted: 15 April 2024 © The Author(s) 2024 Abstract Ochoterenatrema Caballero, 1943 is a genus of lecithodendriid digeneans that prior to this study included 8 species parasitic in bats in the Western Hemisphere. Species of Ochoterenatrema possess a unique morphological feature in form of the pseudogonotyl on the sinistral side of the ventral sucker. In this study, we describe 2 new species of Ochoterenatrema from bats in Ecuador. The new species V. V. Tkach (*) Department of Biology, University of North Dakota, 10 Cornell Street, Grand Forks, ND 58202, USA e-mail: R. Gasperetti Department of Biology and Museum of Natural History, University of Wisconsin-Stevens Point, Stevens Point, WI 54481, USA T. F. Fernandes Faculdade Facimp Wyden, Imperatriz, Maranhão 65914‑335, Brazil C. A. Carrión‑Bonilla Museo de Zoología QCAZ, Facultad de Ciencias Biológicas, Pontificia Universidad Católica del Ecuador, Quito, Ecuador J. A. Cook Museum of Southwestern Biology and Department of Biology, University of New Mexico, Albuquerque, NM 87131, USA T. J. Achatz Department of Natural Sciences, Middle Georgia State University, Macon, GA 31206, USA are readily differentiated from their congeners by a combination of morphological characters, including the distribution of vitelline follicles, length of oesophagus, sucker ratio and the body shape, among other features. We have generated partial nuclear 28S rDNA and mitochondrial cox1 gene DNA sequences from both new species. The newly obtained sequences were used to differentiate among species and study the phylogenetic interrelationships among Ochoterenatrema spp. The internal topology of the clade was weakly supported, although the cox1 tree was much better resolved than the 28S tree. Comparison of sequences revealed 0–1.2% interspecific divergence in 28S and 3.3–20.5% interspecific divergence in cox1 among Ochoterenatrema spp. The new findings demonstrate that bats in South America likely harbor multiple additional undescribed species of Ochoterenatrema. More extensive sampling from broader geographic and host ranges, especially in North America, should allow for a better understanding of the evolution of host associations and morphological traits of this lineage of lecithodendriid digeneans. Introduction Ochoterenatrema Caballero, 1943 is a relatively small genus of digeneans belonging to the highly diverse family Lecithodendriidae Lühe, 1901. Members of Ochoterenatrema are parasitic exclusively in bats in the Western Hemisphere. They are characterized by Vol.: (0123456789) 13 43 Page 2 of 13 the presence of a unique morphological feature among lecithodendriids, namely the pseudogonotyl which is formed by thickened tegument on the sinistral side of the ventral sucker (Lotz & Font, 2008). Some authors have mistakenly referred to this structure as the genital pore and its function remains unknown (see references in Fernandes et al., 2022). Until recently, molecular data on Ochoterenatrema were lacking and its phylogenetic affinities were unknown. Fernandes et al. (2022) provided the first DNA sequence data for 4 species of Ochoterenatrema from South and North America. Their phylogenetic analysis confirmed the monophyly of Ochoterenatrema and demonstrated its position as the closest taxon to the genus Lecithodendrium. In addition, Fernandes et al. (2022) described 2 new species, Ochoterenatrema sphaerula Fernandes, Melo, Santos, Achatz et Tkach, 2022 and Ochoterenatrema gracilis Fernandes, Achatz, McAllister et Tkach, 2022, and provided quality morphological descriptions and illustrations of Ochoterenatrema diminutum (Chandler, 1938), Ochoterenatrema fraternum Freitas et Ibañez, 1963 and Ochoterenatrema cf. labda Caballero, 1943. Eight species of Ochoterenatrema have been described, 3 of them from the Nearctic and 5 from the Neotropics (original descriptions in Macy, 1936, 1938; Chandler, 1938; Caballero, 1943; Freitas, 1957; Freitas & Ibañez, 1963; Fernandes et al., 2022). Fernandes et al. (2022) hypothesized that Ochoterenatrema spp. may be more diverse in Neotropics than currently known. In the course of parasitological examination of bats in Ecuador, we discovered 2 previously unknown species of Ochoterenatrema. Herein, we provide morphological descriptions of the new species accompanied by molecular differentiation and phylogenetic analysis based on partial sequences of the nuclear large ribosomal subunit (28S) gene and the mitochondrial cytochrome c oxidase 1 (cox1) DNA gene. Materials and methods Morphological data Specimens of 2 new species of Ochoterenatrema were collected from the intestines of bats in Yasuni National Park, Orellana Province and the Centro Científico Rio Palenque, Los Ríos Province, and Luis Vargas Torres, community of Playa de Oro, Vol:. (1234567890) 13 Syst Parasitol (2024) 101:43 Esmeraldas province, Ecuador in 2016 and 2017. Bats were trapped using mist nets. Digeneans were collected live, rinsed in saline, heat-killed with hot water, and immediately fixed in 70% ethanol for morphological and molecular studies. Specimens for light microscopy were stained with aqueous alum carmine, dehydrated in an ethanol series of ascending concentrations, cleared in clove oil and mounted permanently in Damar gum (Lutz et al., 2017). The identification, measurements, and drawings of the specimens were made using an Olympus BX51 microscope (Olympus Corporation, Tokyo, Japan) equipped with DIC and digital imaging system. Drawings were made with the aid of a drawing tube. All measurements are in micrometers; forebody was measured as distance from anterior end of the body to the center of ventral sucker. The CV is a percentage value of the ratio of the standard deviation to the mean of a particular metric character. Characters with lower CV have values that are more stable around the mean than those with higher CV. The type specimens are deposited in the collection of the Museo de Zoología QCAZ, Facultad de Ciencias Biológicas, Pontificia Universidad Católica del Ecuador (QCAZI), Quito, Ecuador. Museum accession numbers are provided in the descriptions below. Molecular data Genomic DNA was extracted according to the protocol described by Tkach and Pawlowski (1999). An approximately 1,300 bp long fragment at the 5′ end of 28S was amplified by polymerase chain reactions (PCR) on a T100™ thermal cycler (Bio-Rad, Hercules, California, USA) using forward primer digL2 (5′–AAG CAT ATC ACT AAG CGG – 3′) and the reverse primer 1500R (5′–GCT ATC CTG AGG GAA ACT TCG–3′) (Tkach et al., 2003). A 396 bp long (upon trimming of primers) fragment of the cox1 gene was amplified using forward primer (...truncated)