Introduction
The process of species identification is widely acknowledged as one of the most essential requirements for understanding the diversity, ecology, and evolution of the living world (e.g., Elphick, Reference Elphick2008; Farnsworth et al., Reference Farnsworth, Chu, Kress, Neill, Best, Pickering, Stevenson, Courtney, VanDyk and Ellison2013; Kürzel et al., Reference Kürzel, Kaiser, Lörz, Rossel, Paulus, Peters, Schwentner, Martinez Arbizu, Coleman, Svavarsson and Brix2022). In recent decades, DNA sequencing technologies have become integral to all branches of zoology, enabling precise delimitation and characterization of specific species, as well as establishing their taxonomic positions. Importantly, in the contemporary taxonomy of Digenea, DNA technologies have not only facilitated accurate species delimitation and valuable phylogenetic reconstructions but have also provided a unique means of identifying and differentiating all life stages of trematodes. This capability has greatly enhanced the understanding of trematode life cycles and helped recognize cryptic diversity (e.g., Georgieva et al., Reference Georgieva, Selbach, Faltýnková, Soldánová, Sures, Skírnisson and Kostadinova2013a, Reference Georgieva, Soldánová, Pérez-Del-Olmo, Dangel, Sitko, Sures and Kostadinova2013b; Bray et al., Reference Bray, Cutmore and Cribb2022; Pyrka et al., Reference Pyrka, Kanarek, Gabrysiak, Jeżewski, Cichy, Stanicka, Żbikowska, Zaleśny and Hildebrand2022; Valadão et al., Reference Valadão, Alves, López-Hernández, Assis, Coelho, Geiger and Pinto2023). Given these advancements, molecular data have become a ‘must-have’ tool for nearly all ecological and evolutionary research, large-scale biodiversity surveys, and species identification and delineation. However, a significant limitation in using DNA sequences for the taxonomy of Digenea – and zoology in general – is the necessity for precise taxonomic identification of sequenced specimens using conventional morphology-based analysis. This step is crucial to ensure that the obtained sequences are correctly linked to their taxonomic identities. Therefore, molecular techniques should always be combined with high-quality morphological studies to achieve reliable results (e.g., Blasco-Costa et al., Reference Blasco-Costa, Cutmore, Miller and Nolan2016; Schwelm et al., Reference Schwelm, Georgieva, Grabner, Kostadinova and Sures2021; Faltýnková et al., Reference Faltýnková, Kudlai, Pantoja, Yakovleva and Lebedeva2022). Additionally, the increasing problem of taxonomic misidentification in public DNA databases, which arises from improperly identified isolates (due to issues such as invalid labelling, poor quality, lack of voucher specimens for further comparison, or the use of non-informative DNA markers in constructed phylogenies), has been widely discussed in contemporary literature (Locke et al., Reference Locke, Al-Nasiri, Caffara, Drago, Kalbe, Lapierre, McLaughlin, Nie, Overstreet, Souza, Takemoto and Marcogliese2015; Kanarek et al., Reference Kanarek, Zaleśny, Sitko and Blanco2016; Steinegger and Salzberg, Reference Steinegger and Salzberg2020; Tang, Reference Tang2020; Bensch et al., Reference Bensch, Inumaru, Sato, Lee Cruz, Cunningham, Goodman, Levin, Parker, Casanueva, Hernández, Moreno-Rueda and Rojo2021; Achatz et al., Reference Achatz, Martens, Kostadinova, Pulis, Orlofske, Bell, Fecchio, Oyarzún-Ruiz, Syrota and Tkach2022; Coca-de-la-Iglesia et al., Reference Coca-de-la-Iglesia, Gallego-Narbón, Alonso and Valcárcel2024).
Despite certain challenges, the understanding of the genus Cotylurus (Szidat, Reference Szidat1928) (Diplostomoidea: Strigeidae) has evolved significantly in recent years. This change has been largely due to the application of molecular methods and an integrative taxonomy approach. Cotylurus comprises a relatively small group of highly specialized species that inhabit the intestines and the bursa of Fabricius of water and wading birds (for further details, see Pyrka et al., Reference Pyrka, Kanarek, Zaleśny and Hildebrand2021, and references therein). Established by Szidat in 1928, the genus Cotylurus includes strigeid trematodes from avian hosts, characterized by vitellaria that are limited to the opisthosoma and a well-developed genital bulb. Cotylurus operates on a 3-host life cycle closely associated with the freshwater environment. The first intermediate hosts, in which the larval stages multiply asexually, are typically pulmonate snails. A variety of pulmonate and prosobranch water snails, as well as leeches, serve as the second intermediate hosts (for further details, see Pyrka et al., Reference Pyrka, Kanarek, Zaleśny and Hildebrand2021, Reference Pyrka, Kanarek, Gabrysiak, Jeżewski, Cichy, Stanicka, Żbikowska, Zaleśny and Hildebrand2022, and references therein). The metacercariae of the tetracotyle type are transmitted to the avian definitive hosts when they ingest the second intermediate host (Sudarikov et al., Reference Sudarikov, Shigin, Kurochkin, Lomakin, Stenko and Yurlova2002; Cribb et al., Reference Cribb, Bray, Olson and Littlewood2003; Blasco-Costa and Locke, Reference Blasco-Costa and Locke2017).
Cotylurus is widely recognized as a valid genus (Niewiadomska, Reference Niewiadomska, Gibson, Jones and Bray2002; Heneberg et al., Reference Heneberg, Sitko, Těšínský, Rząd and Bizos2018; Pyrka et al., Reference Pyrka, Kanarek, Zaleśny and Hildebrand2021); however, its species composition remains controversial. The absence of easily visible morphological features and the high level of morphological variability observed in adult forms of species have led to numerous taxonomic decisions of questionable validity by various authors (e.g., Dubois, Reference Dubois1968; Sudarikov, Reference Sudarikov1984). Consequently, this group has been the focus of extensive discussion and ongoing taxonomic changes over time. As a result, the legitimacy of certain species within the genus Cotylurus and the true diversity of this genus remain ambiguous and require further confirmation. Among the many species of Cotylurus, one with a particularly complicated and still unclear taxonomic status is Cotylurus brandivitellatus (Belogurov et al., Reference Belogurov, Maksimova and Tolkacheva1966). This species was originally described based on trematode specimens collected from the intestines of a wide range of ducks (including the northern shoveler [Spatula clypeata], garganey [Spatula querquedula], greater scaup [Aythya marila], and long-tailed duck ([langula hyemalis]) sampled in the lower reaches of the Yenisei River, along the coast of the Sea of Okhotsk, and in Kazakhstan (Belogurov et al., Reference Belogurov, Maksimova and Tolkacheva1966).
Initially, Belogurov et al. (Reference Belogurov, Maksimova and Tolkacheva1966) placed his species within the genus Cotylurostrigea established by Sudarikov (Reference Sudarikov1961) for strigeid trematodes found in Anatid birds. Cotylurostigea is characterized by a unique combination of morphological features: the presence of vitellaria in both the prosoma and opisthosoma (a trait typical for the genus Strigea) and a well-developed genital bulb (typical for the genus Cotylurus). Sudarikov (Reference Sudarikov1961) initially included only one species in this new genus – Cotylurostrigea raabei (Bezubik, Reference Bezubik1958) – and later added another taxon, Cotylurostrigea strigeoides (Dubois, Reference Dubois1958) (e.g., Sudarikov et al., Reference Sudarikov, Shigin, Kurochkin, Lomakin, Stenko and Yurlova2002). Over the years, the validity of Cotylurostrigea has been widely debated. Dubois (Reference Dubois1968) and Odening (Reference Odening1969) considered it a synonym of Cotylurus, while Yamaguti (Reference Yamaguti1971) assigned it a subgeneric status within Strigea. However, Sudarikov (Reference Sudarikov1984) still regarded Cotylurostrigea as a valid taxon. A cladistic analysis based on morphological and ecological features by Zazornova and Sysoev (Reference Zazornova and Sysoev1993) recognized Cotylurostrigea as a synonym of Cotylurus, a view later supported by Niewiadomska (Reference Niewiadomska, Gibson, Jones and Bray2002). Recent molecular studies have further corroborated these conclusions (Pyrka et al., Reference Pyrka, Kanarek, Zaleśny and Hildebrand2021, Reference Pyrka, Kanarek, Gabrysiak, Jeżewski, Cichy, Stanicka, Żbikowska, Zaleśny and Hildebrand2022). Regardless of the varying generic status of Cotylurostrigea, the validity and taxonomic position of C. brandivitellatus have also been the subject of extensive discussion over the years and remain unresolved. Odening (Reference Odening1969) and Sudarikov (Reference Sudarikov1984) treated this species as valid, while McDonald (Reference McDonald1981) and Niewiadomska (Reference Niewiadomska2010) suggested that it be considered a synonym of C. strigeoides. Thus, the validity and taxonomic position of C. brandivitellatus remain contentious and require further detailed studies.
In this study, we present comprehensive morphological and molecular characterization of adults of C. brandivitellatus, sourced from the intestines of naturally infected mute swans (Cygnus olor) in Gdańsk Pomerania. Our analysis adheres to the recommended ‘best practices’ established by Blasco-Costa et al. (Reference Blasco-Costa, Cutmore, Miller and Nolan2016) for molecular approaches in trematode systematics. Based on our findings, we assert new insights regarding the validity and position of C. brandivitellatus, as well as its implications for the structure of the genus Cotylurus. We firmly believe that this study contributes significantly to clarifying the species spectrum within the Cotylurus genus through an integrative taxonomy approach, supported by sequences that correlate with the well-documented morphology of adult specimens.
Materials and methods
Host sampling, parasite recovery protocol and morphological analysis
Adult trematodes were collected from the intestinal tract of a juvenile mute swan (Cygnus olor) specimen weighing approximately 2.5 kg. This swan was received deceased with permission (RDOŚ-Gd-WZG.6401.64.2023.AB.3) from the Pomeranian Wildlife Rehabilitation Centre ‘Ostoja’. The research material consisted of bird specimens that had been euthanized due to severe injuries while providing necessary veterinary assistance to sick and weakened birds from the Gdańsk Pomerania area. The avian hosts were transported to the laboratory immediately after death and then frozen at −25°C for further necropsy. Specimens of Cotylurus sp. were identified in the intestine, washed in tap water and preserved in hot 70% ethanol for further morphological and molecular studies. The collected trematodes were initially identified under a stereomicroscope following the original description provided by Belogurov et al. (Reference Belogurov, Maksimova and Tolkacheva1966). Some selected trematodes were designated for further molecular studies and were vouchered according to the protocols outlined by Pleijel et al. (Reference Pleijel, Jondelius, Norlinder, Nygren, Oxelman, Schander, Sundberg and Thollesson2008) and Blasco-Costa et al. (Reference Blasco-Costa, Cutmore, Miller and Nolan2016). A series of microphotographs were taken using a digital camera. For DNA extraction, a small fragment of the opisthosoma was excised from the selected specimens. The remaining voucher specimen (hologenophore) and other trematode specimens identified as C. brandivitellatus, collected from the same hosts (paragenophores), were stained with iron acetocarmine, dehydrated in ethanol, cleared in clove oil, and mounted in Canada balsam for detailed morphological studies. All measurements were taken from the paragenophores stained and mounted in Canada balsam using NIS-Elements D image analysis software. The voucher specimens were deposited in the Natural History Museum of Geneva under number MHNG-PLAT-0159729.
DNA processing and phylogenetic analyses
DNA was extracted from small piece of hologenophores and selected single alcohol-fixed paragenophores using a commercial kit (DNeasy Blood and Tissue kit; Qiagen, Hilden, Germany), according to the manufacturer’s protocol.
PCR amplification of nuclear 28S and the mitochondrial gene encoding CO1 was carried out using the KAPA2G Robust HotStart ReadyMix (Sigma-Aldrich, St. Louis, MO. USA). Reaction conditions and primers were selected based on the literature and our previous study, i.e., LSU5, digl2 and 1500 R for 28S rDNA, and JB3 and CO1_Rtrema for CO1 (Pyrka et al., Reference Pyrka, Kanarek, Zaleśny and Hildebrand2021). The PCR products were visualised following electrophoresis in a 1% agarose gel and purified using the Exo-BAP kit (EURx) and sequenced directly in both directions using the PCR primers. Contiguous sequences were assembled using Geneious software (Geneious 9.1.8; https://www.geneious.com).
The alignments including newly obtained sequences and closely related representatives of Strigeidae currently available in GenBank were prepared using ClustalW multiple alignment implemented in MegaX (Kumar et al., Reference Kumar, Stecher, Li, Knyaz and Tamura2018). Sequences of the partial 28S rDNA gene (1042 bp) and the CO1 fragment (307 bp) were aligned in one concatenated dataset. Phylogenetic analyses were conducted using Bayesian inference criteria as implemented in MrBayes ver. 3.2.7 software (Ronquist et al., Reference Ronquist, Teslenko, van der Mark, Ayres, Darling, Höhna, Larget, Liu, Suchard and Huelsenbeck2012). The general time-reversible model with estimates of invariant sites and gamma distributed among-site variation (GTR + I + G) was identified as the best-fitting nucleotide substitution model for each marker independently and the concatenated dataset. The consensus trees were visualized in FigTree ver. 1.4.4 software (Rambaut, ) and annotated in CorelDraw® (Corel Corp., Ottawa, ON, Canada)
Results
Morphological description of the voucher material
Family: Strigeidae Railliet, 1919
Subfamily: Strigeinae Railliet, 1919
Genus: Cotylurus (Szidat, Reference Szidat1928)
• Cotylurus brandivitellatus (Belogurov et al., Reference Belogurov, Maksimova and Tolkacheva1966; Odening, Reference Odening1969)
• Host: Cygnus olor Gmelin, 1789, one individual infected with 42 specimens
• Locality: Chmielno, Gdańsk Pomerania, northern Poland (54°19′34″ N, 18°6′2″E)
• Site of the infection: ileum
• Material: 3 hologenophores and 27 paragenophores
• Representative DNA sequences: PX047982 (28S rDNA), PX056574 (CO1 mtDNA)
Adult specimen of Cotylurus brandivitellatus (host: Cygnus olor, locality: Chmielno, Gdańsk Pomerania, Northern Poland).
Comparison of selected morphometric characteristics of C. brandivitellatus, C. magniactebulus, C. lutzi and related species
* Min–max (mean ± SD).
** Min–max (mean).
*** Min–max.
**** Ratio calculated on base of literature data.
The following information are based on 27 paragenophores stained with iron acetocarmine and mounted on slides in Canada balsam. All measurements are expressed in micrometres (min-max; mean ± SD) unless otherwise specified. The specimens are mounted laterally, ensuring that the prosoma and opisthosoma organ widths match their dorso-ventral diameters.
Body distinctly bipartite, slightly curved, total length 2024.65–2404.03 (2207.99 ± 91.34). Tegument smooth. Prosoma cupuliform, hemispherical to spheroidal 736.56–870.40 (795.88 ± 37.35) length and 699.66–869.26 (769.68 ± 46.91) width, maximum width at the level of the ventral sucker. Prosoma well separated from subcylindrical and slightly arched opisthosoma, 1308.00–1576.57 (1406.88 ± 77.67) length and 490.55–644.63 (574.48 ± 40.15) width. The length ratio of the prosoma to the opisthosoma is 1:1.53–2.05 (1.77 ± 0.11). Oral sucker muscular, terminal and elongate-oval, 136.42–159.02 (148.94 ± 5.99) length and 120.25–153.66 (135.54 ± 9.76) width. Ventral sucker muscular and elongate-oval, larger than the oral sucker, usually positioned at the mid-level of the prosoma, 186.21–241.49 (207.59 ± 13.78) length and 153.01–229.41 (191.39 ± 17.80) width. The oral to ventral sucker length ratio is 1:1.17–1.68 (1.40 ± 0.12). No prepharynx observed. The pharynx muscular weakly and slightly oval, 75.23–110.08 (94.13 ± 8.63) length and 71.18–92.20 (80.82 ± 5.61) width. The oesophagus, intestinal bifurcation, and caeca in the prosoma not observed. The caeca in the opisthosoma poorly visible and asymmetrical. The holdfast organ consists of 2 large lobes with a deep slit. The ovary oval to reniform, pretesticular, 120.10–199.25 (147.79 ± 17.87) length and 104.67–169.45 (138.85 ± 17.07) width, positioned 212.91–339.44 (268.83 ± 28.91) from the anterior end of the opisthosoma. The pre-ovary distance in the opisthosoma relatively large, occupying 15.90–22.67 (19.10 ± 1.60)% of the total opisthosoma length. Laurer’s canal and Mehlis gland not visible. Two testes, post-ovarian, large, tandem and extended-oval, usually bilobed with asymmetrical lobes. Slightly larger anterior testis 193.32–263.70 (224.87 ± 22.03) length and 109.73–235.97 (186.18 ± 37.75) at the widest point. The posterior testis 169.63–272.86 (212.87 ± 29.00) length and 125.77–223.22 (163.86 ± 27.54) width. The length ratio of the anterior to posterior testis is 1:0.75–1.39 (0.94 ± 0.18). The post-testicular region of the opisthosoma measures 327.04–467.40 (397.9 ± 35.35), covering 24.25–31.96 (28.39 ± 2.12)% of the opisthosoma length. The seminal vesicle not observed. The vitellaria dense and follicular, containing numerous vitelline follicles, present in both the prosoma and opisthosoma, with follicles in the prosoma extending anteriorly into the distal parts of the lobes of the holdfast organ at various lengths, typically asymmetrically. Vitellaria absent in the ‘neck’ region and do not occur in the parenchyma tissue between the prosoma and opisthosoma. In the opisthosoma, the vitelline fields extend from the pre-ovarian region to the mid-level of the genital bulb. The genital bulb well-expressed, clearly delimited from the surrounding parenchyma. The excretory vesicle and excretory pore not observed. The uterus extends anterior to the ovary, ventral to the gonads, containing 15–32 (25.11 ± 4.86) eggs. The eggs oval, 70.94–96.52 (86.09 ± 7.50) length, 46.07–62.95 (55.11 ± 4.47) width.
Remarks
The analysed material aligns with the diagnosis of the genus Cotylurus, as described by Niewiadomska (Reference Niewiadomska, Gibson, Jones and Bray2002); moreover, the morphology of the presented materials corresponds well with the brief description of Cotylurostrigea brandivitellata as provided by Belogurov et al. (Reference Belogurov, Maksimova and Tolkacheva1966). This includes an elongate, subcylindrical, slightly curved opisthosoma, a cupuliform, hemispherical to spheroidal prosoma with 2 lobes of the holdfast organ, as well as well-developed suckers and pharynx. The large lobed testes and, importantly, the vitelline follicles located mainly in the opisthosoma – which characteristically extend anteriorly into 2 lobes of the holdfast organ in the prosoma (Figs. 1 and 2) – are also significant features. From a morphometric perspective, most mean body dimensions of the recently analyzed material from a mute swan are slightly larger compared to the originally described specimens of C. brandivitellatus from a wide range of duck species. However, the width of the opisthosoma, dimensions of the acetabulum and pharynx, as well as the measurements of the testes, are slightly larger in the original description (Table 1). Notably, the range of variability in measurements from the originally described trematodes is significantly broader than that of the recently studied specimens (Table 1). Additionally, the mean ratio of sucker lengths in the description by Belogurov et al. (Reference Belogurov, Maksimova and Tolkacheva1966) is lower than that of the analyzed material (1:1.09 vs. 1:1.40 – Table 1). Despite these differences, we believe that the generally well-corresponding morphology – especially the structure of vitellaria in the prosoma – along with the similarity in dimensions of the body, internal organs, and certain morphological indices (such as the ratio of prosoma to opisthosoma length) supports the conclusion that our material represents C. brandivitellatus.
Comparison of vitellaria in prosoma of 3 Cotylurus species from anatid avian hosts: (A) – Cotylurus raabei; (B) – Cotylurus strigeoides; (C) – Cotylurus brandivitellatus. Arrows indicate the distribution of the vitelline follicles in the prosoma.
The morphology of C. brandivitellatus is characterized by a unique structure of vitelline follicles in the prosoma, which closely resembles 2 other species of Cotylurus, i.e. C. lutzi (Basch, Reference Basch1969) and C. magniacetabulus (Dubois and Angel, Reference Dubois and Angel1972). C. lutzi was described based on trematode specimens obtained experimentally from tetracotyle, which were collected from the naturally infected snail, Biomphalaria glabrata, in Bahia, Brazil and fed to atypical avian passeriform hosts: the Sunda zebra finch (Taeniopygia guttata), black-rumped waxbill (Estrilda troglodytes) and white-rumped munia (Lonchura striata) (Basch, Reference Basch1969).
The adult trematodes obtained in this study, although similar in the structure of vitellaria in the opisthosoma and sucker length ratio (1.35–1.74 in C. lutzi vs. 1.17-1.68 in C. brandivitellatus), differ significantly from C. brandivitellatus specimens in several aspects: smaller body size and dimensions of all internal organs, round (not elongated) testes, and a significantly larger prosoma to opisthosoma length ratio (1:2.19–2.45 vs. 1:1.53–2.05) (Table 1). Notably, adult specimens of C. lutzi have not been recorded in definitive avian hosts since their description, leaving their true morphological and morphometric variability unknown. The other species, C. magniacetabulus, was characterized based on several trematode specimens collected from the ‘lower intestine’ of a naturally infected black swan (Cygnus atratus) in Australia (Dubois and Angel, Reference Dubois and Angel1972) Besides the evident similarity in the structure of vitellaria in the prosoma, C. magniacetabulus shows similarities in general morphology with the recently analysed specimens of C. brandivitellatus. However, the 2 species differ in a few body dimensions (Table 1). The main differences between the trematode specimens analysed and the original description of C. magniacetabulus include larger total body length, shorter prosoma and longer opisthosoma in C. magniacetabulus (resulting in a slightly higher prosoma to opisthosoma length ratio: 1:1.53–2.05 vs. 1:2–2.6), larger testes, wider ovary and larger egg width (Table 1). Interestingly, since the description, Cotylurus specimens identified on the basis of morphology as C. magniacetabulus have been recorded in Anatidae in South America (Padilla-Aguilar et al., Reference Padilla-Aguilar, Romero-Callejas, Osorio-Sarabia, Pérez–ponce de León and Alcalá-Canto2020a, Reference Padilla-Aguilar, Romero-Callejas, Ramírez-Lezama, Osorio-Sarabia, García-Prieto, Manterola, García-Márquez and Zarza2020b), a region typical for C. lutzi. Despite these differences, we believe that the morphology of C. brandivitellatus, particularly the arrangement of vitelline follicles in the prosoma, is uniquely consistent within the genus Cotylurus, aligning with further descriptions of C. lutzi and C. magniacetabulus. The existence or absence of vitelline follicles in the prosoma, along with their structure, is widely regarded as one of the most important and relatively constant morphological features crucial for the precise identification of particular species within Cotylurus (e.g., Dubois, Reference Dubois1968; Sudarikov, Reference Sudarikov1984). Therefore, it seems unlikely that the identical structure of vitellaria in the prosoma of these 3 species is merely coincidental. When considering the broader implications, the observed differences in morphology and morphometry among C. brandivitellatus, C. lutzi and C. magniacetabulus are, in our opinion, not significant and may be results of the origins of the voucher specimens (especially the experimental infections of non-specific passeriform hosts in the case of C. lutzi) and geographic variations. Literature extensively describes phenotypic variations in Digenea caused by the identity of the host (‘host-induced variation’) (e.g., Stunkard, Reference Stunkard1957; Nolan and Cribb, Reference Nolan and Cribb2005; Hildebrand et al., Reference Hildebrand, Adamczyk, Laskowski and Zaleśny2015). Notably, the original descriptions of C. lutzi and C. magniacetabulus (Basch, Reference Basch1969; Dubois and Angel, Reference Dubois and Angel1972) do not include a differential diagnosis with C. brandivitellatus. Given these observations, we are convinced that the 3 taxa are conspecific; however, final confirmation of this hypothesis requires morphological and molecular analyses of Cotylurus specimens from Australia and South America.
Phylogenetic results
Phylogenetic analysis supports the hypothesis of independent taxonomic position and validity of C. brandivitellatus. In the case of 28S rDNA, C. brandivitellatus forms an independent lineage sister to C. strigeoides (Figure 3) with a sequence similarity of 99.4% (8 nucleotide difference). Phylogenetic analysis based on mtDNA COI even more clearly shows the independent status of C. brandivitellatus, also with C. strigeoides as a sister lineage to C. brandivitellatus (Figure 3). The genetic similarity of the sequences is at the level of 90%. Obtained results also confirmed separate position of C. raabei within genus Cotylurus (Figure 3).
The phylogenetic reconstruction of C. brandivitellatus based on the concatenated COI mtDNA and 28S rDNA markers. The analysis was performed by the use of Bayesian inference, square symbol indicates posterior probability greater than 90%. AD – adult; LV – larvae.
Discussion
Despite extensive efforts in recent years, the actual diversity, phylogenetic relationships and species composition of the genus Cotylurus are still not fully established. Recent study, which combine morphological and molecular analyses of adult Cotylurus collected from avian hosts in Central Europe, have revealed an unexpectedly high level of molecular diversity within morphologically well-defined species. This includes the unclear status of the typical species of the genus Cotylurus, such as C. cornutus (Rudolphi, Reference Rudolphi1808; Szidat, Reference Szidat1928), the polyphyletic nature of C. syrius Dubois, Reference Dubois1934 and the distinct species status of C. raabei (Bezubik, Reference Bezubik1958; Pyrka et al., Reference Pyrka, Kanarek, Zaleśny and Hildebrand2021). Further research focusing on molecular analyses of tetracotyle metacercariae of Cotylurus, collected from snail second intermediate hosts, has again shown high molecular diversity and clear evidence of cryptic taxa. Several putative novel species lineages have also been identified. These findings reaffirm the polyphyletic nature of C. syrius (with 3 separate molecular species-level lineages) and C. cornutus (with 4 separate molecular species-level lineages). This strongly suggests that these taxa, in fact, consist of a complex of species (Pyrka et al., Reference Pyrka, Kanarek, Gabrysiak, Jeżewski, Cichy, Stanicka, Żbikowska, Zaleśny and Hildebrand2022). Notably, 2 divergent phylogenetic and ecological lineages within Cotylurus were demonstrated – one utilizing leeches and the other freshwater snails as second intermediate hosts, which differ significantly in their life history strategies (Pyrka et al., Reference Pyrka, Kanarek, Zaleśny and Hildebrand2021, Reference Pyrka, Kanarek, Gabrysiak, Jeżewski, Cichy, Stanicka, Żbikowska, Zaleśny and Hildebrand2022). Despite these advancements in understanding the diversity, morphological and molecular variability, and phylogeny of Cotylurus, several issues regarding the taxonomy, biology, and ecology of these trematodes remain unresolved.
Our results obtained from an integrative taxonomy approach shed new light on the current understanding of the phylogeny and diversity of the genus Cotylurus, leading to the confirmation of the validity and phylogenetic affiliations of C. brandivitellatus. In our opinion, the new material of adult C. brandivitellatus, despite some morphometric differences, corresponds well with the original description. The unique morphology of the analyzed trematode specimens – especially the structure of vitelline follicles in the prosoma, as well as the body and internal organ measurements – generally aligns with the original description and the body dimensions provided by Belogurov et al. (Reference Belogurov, Maksimova and Tolkacheva1966). The observed variability in specific morphometric features between the recently collected material and the original description is likely related to geographical differences (Europe and Asia), the phylogenetic distance, and the body dimensions of the avian hosts. It is noteworthy that since the description of C. brandivitellatus, it has rarely been recorded in the former Soviet Union across various anatid avian hosts, including the red-throated loon (Gavia stellata) and the short-billed dowitcher (Limnodromus griseus) (Sudarikov, Reference Sudarikov1984), as well as in Poland in mute swans (Sulgostowska, Reference Sulgostowska1972). The recent findings of this trematode species in a juvenile, flightless specimen of mute swan clearly indicate a local origin of the infection, suggesting that the life cycle of C. brandivitellatus is completed within the aquatic ecosystems of Central Europe.
The phylogenetic analysis confirms that C. brandivitellatus is a valid species within the genus Cotylurus, refuting earlier claims by McDonald (Reference McDonald1981) and Niewiadomska (Reference Niewiadomska2010) of it being conspecific with C. strigeoides. The analysis also highlights close affinities between these 2 taxa. Historically, C. brandivitellatus and C. strigeoides, along with C. raabei, were classified as Cotylurostrigea by Sudarikov (Reference Sudarikov1961, Reference Sudarikov1984). He argued that their unique features warranted a separate genus. However, recent molecular studies indicate that Cotylurostrigea is a junior synonym of Cotylurus (Pyrka et al., Reference Pyrka, Kanarek, Zaleśny and Hildebrand2021). Our findings support this matter, highlighting the evolutionary significance of the distribution of vitelline follicles in the prosoma. Additionally, C. raabei is confirmed as distinct from C. brandivitellatus and C. strigeoides (Figure 3), contributing to a clearer understanding of Cotylurus phylogeny. Niewiadomska’s (Reference Niewiadomska1971) hypothesis posits that early divergent Cotylurus forms had vitelline follicles in both the prosoma and opisthosoma, while evolutionary derived forms exhibit them only in the opisthosoma. This aligns with the observation that C. raabei is the earliest divergent morphological form, with distinct vitellaria extending from the opisthosoma to the prosoma. Moreover, C. lutzi and C. magniacetabulus exhibit similar vitelline follicles patterns to C. brandivitellatus, suggesting potential conspecific status that deserves further investigation. A thorough study combining morphological, molecular, and ecological data, especially with a focus on non-European specimens, is needed. We believe this will yield valuable insights into the diversity and relationships within the Cotylurus genus.
Author contributions
J.H., G.K. and G.Z. conceived and designed the study, B.R. preserved avian host, G.K. and J.G. performed necropsy of avian host, processed material for further research and identified morphologically collected trematodes, J.H., J.G., S.W. and G.Z. performed molecular analyses. J.H., G.K., J.G. and G.Z. drafted the manuscript. All authors read and approved final version of the manuscript.
Financial support
This research received no specific grant from any funding agency, commercial or not-for-profit sectors.
Competing interests
The authors declare that there are no conflicts of interest.
Ethical standards
Not applicable.
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