Introduction
The digenetic trematode Cathaemasia hians (Rudolphi, Reference Rudolphi1809) was initially described from the black stork Ciconia nigra. Later, C. hians has been repeatedly shown to be hosted by both stork species that nest in Central Europe, Ciconia ciconia and Ci. nigra (C. nigra: Viborg Reference Viborg1795; Rudolphi Reference Rudolphi1809, Reference Rudolphi1819; Nathusius Reference Nathusius1837; Dujardin Reference Dujardin1845; von Willemoes Suhm Reference Von Willemoes Suhm1873; Müller Reference Müller1897; Mühling Reference Mühling1898; Yoshida and Toyoda Reference Yoshida and Toyoda1930; Szidat Reference Szidat1940a; Macko Reference Macko1960b; Van den Broek Reference Van den Broek1963; Gundlach Reference Gundlach1969; Merino et al. Reference Merino, Martínez, Lanzarot, Cano, Fernández-García and Rodríguez-Caabeiro2001; Saad Reference Saad2009; Liptovszky et al. Reference Liptovszky, Majoros and Perge2012; Hampl and Sitko Reference Hampl and Sitko2013; Königová et al. Reference Königová, Hrčková, Molnár, Major and Várady2015; Sitko and Heneberg Reference Sitko and Heneberg2015; Ramilo et al. Reference Ramilo, Caetano, Brazio, Mira, Antunes, Pereira da Fonseca and Cardoso2021; C. ciconia: Gurlt Reference Gurlt1845; Baird Reference Baird1853; van Beneden Reference Van Beneden1868; Mühling Reference Mühling1897; Van den Broek Reference Van den Broek1960, Reference Van den Broek1963; Mettrick Reference Mettrick1963; Grünberg and Kutzer Reference Grünberg and Kutzer1964; Gundlach Reference Gundlach1969; Schuster et al. Reference Schuster, Schaffer and Shimalov2002; Sitko and Heneberg Reference Sitko and Heneberg2015; Michalczyk et al. Reference Michalczyk, Sokół, Gesek, Mączyński and Będzłowicz2020; Sitko and Heneberg Reference Sitko and Heneberg2021). These stork species differ substantially in diet. The black stork Ci. nigra feeds predominantly on fish and, to a lesser extent, on amphibians and mollusks and hunts for them in wetlands, particularly slow-flowing waters (Merino et al. Reference Merino, Martínez, Lanzarot, Cano, Fernández-García and Rodríguez-Caabeiro2001; Liptovszky et al. Reference Liptovszky, Majoros and Perge2012). In contrast, Czech populations of the white stork Ci. ciconia feed mainly on mammals and earthworms, with amphibians present in the diet in the past but only rarely in recent years (Reif et al. Reference Reif, Voříšek, Šťastný and Bejček2006; Voříšek Reference Voříšek2006); fish are absent from the common prey types of this bird species. The dominant diet types may differ with respect to the landscape context and can be seasonal. The main feeding habitats of Ci. ciconia include arable fields, dry pastures, and rubbish dumps (Alonso et al. Reference Alonso, Alonso and Carrascal1991; Carrascal et al. Reference Carrascal, Bautista and Lázaro1993). The diet change in Czech populations of Ci. ciconia was hypothesized to be associated with the recent decline in C. hians prevalence in Ci. ciconia of Czech origin (Sitko and Heneberg Reference Sitko and Heneberg2021). Although the findings of C. hians from other host species are known, only a recent report of C. hians from Aquila heliaca (Aves: Accipitridae) (Juhásová et al. Reference Juhásová, Königová, Molnár, Major, Králová-Hromadová and Čisovská Bazsalovicsová2023) represents a correctly identified specimen, whereas the findings of C. hians in Ardea cinerea, Ardea purpurea, and Nycticorax nycticorax (all Aves: Ardeidae) by Parona (Reference Parona1899) represented misidentifications (The only individuals from A. heliaca were not fixed on slides but lysed for DNA isolation. The first author of the cited study, Ľ. Juhásová, refused to share the deposited DNA or to analyze the species identity of this individual (Ľ. Juhásová, in litt.); thus, the species identity of this finding remains unclear.) Other possible satellite hosts include Ardea cinerea (Stossich Reference Stossich1891), Ardea goliath (Dollfus Reference Dollfus1950), and Hydrocoloeus minutus (Condorelli-Francaviglia Reference Condorelli-Francaviglia1897).
We hypothesized that the Cathaemasia hians isolates ex Ci. nigra and Ci. ciconia represent two independent species. Therefore, in the present study, we performed the first molecular analyses of C. hians individuals that were consistent with the diagnosis of C. hians hians (ex Ci. nigra; Table 2) and C. hians longivitellata (ex Ci. ciconia; Table 3), providing integrative evidence to support the reclassification of C. hians longivitellata as a standalone species.
Table 1. New sequences of Cathaemasia spp. that were collected from Czechia and generated throughout the course of the present study (NCBI GenBank accession numbers are indicated)
Table 2. Measurements of Cathaemasia hians sensu stricto based on adult individuals ex Ciconia nigra (data are shown as a range [mean ± standard deviation]; measurements are shown in μm)
1 Some of the measurements provided by Macko (Reference Macko1960a) are valid for individuals that were alive at the time of the measurement; these measurements are shown in square brackets.
2 We double-checked and measured the material deposited on slides by Macko (Reference Macko1960a) and provide measurements of the eight individuals identified by Macko (Reference Macko1960a) as C. hians hians (including only individuals with eggs). The differences between measurements of live (published by Macko Reference Macko1960a) and pressure-fixed individuals (materials collected by Macko Reference Macko1960a measured in the present study) are caused by pressure fixation and by the application of a series of 96% ethanol, carboxylol, and xylol in the course of the fixation of the latter individuals.
3 The data from Braun (Reference Braun1901) were reported originally for Cathaemasia fodicans ex Sterna nigra (the host identity was not confirmed by Braun himself, but it was retrieved from the Vienna Museum label; Braun only measured the archived specimen). Later authors, including Odhner (Reference Odhner1926), Yoshida and Toyoda (Reference Yoshida and Toyoda1930), and Szidat (Reference Szidat1939), suggested that the examined specimen represented C. hians and that the label of the host species was probably erroneous and should be Ciconia nigra.
4 The measurements provided by Ramilo et al. (Reference Ramilo, Caetano, Brazio, Mira, Antunes, Pereira da Fonseca and Cardoso2021) likely contained an erroneously positioned decimal point in measurements of oral and ventral suckers, which were claimed to be one order of magnitude larger than commonly observed values; the data provided in this table include the correction of this obvious error.
Table 3. Measurements of Cathaemasia longivitellata sensu Macko (Reference Macko1960a) based on adult individuals ex Ciconia ciconia (data are shown as a range [mean ± standard deviation]; measurements are shown in μm)
1 Some of the measurements provided by Macko (Reference Macko1960a) are valid for individuals that were alive at the time of the measurement; these measurements are shown in square brackets.
2 We double-checked and measured the material deposited on slides by Macko (Reference Macko1960a) and provide measurements of the holotype and six paratypes (including only individuals with eggs). The differences between measurements of live (published by Macko Reference Macko1960a) and pressure-fixed individuals (materials collected by Macko Reference Macko1960a measured in the present study) are caused by pressure fixation and by the application of a series of 96% ethanol, carboxylol, and xylol in the course of the fixation of the latter individuals.
Materials and methods
Sampling
We examined helminths from carcasses of storks provided dead for deposition in the Comenius Museum in Přerov. The birds died from various causes at various sites in the Czech Republic (48°39′N–50°59′N, 12°19′E–18°29′E), Central Europe. We examined the carcasses immediately or froze them and examined them within two months of receipt. For the phylogenetic analyses, we fixed representative individuals of helminths in 96% ethanol from May 2011 to May 2022 for further analyses. A complete list of the sequenced individuals is provided in Table 1. For the comparative morphological analyses, we stained another set of 45 C. hians sensu lato individuals (30 ex Ci. nigra and 15 ex Ci. ciconia) in Semichon’s carmine, followed by dehydration through an alcohol series, and we then mounted the helminths in Canada balsam. For the analyses of egg length, we measured the longest egg present within each examined adult individual. The body measurements are shown as the range (mean ± standard deviation) and are presented in μm unless otherwise specified.
We also measured the material deposited on slides by Macko (Reference Macko1960a) and provide measurements of the holotype and seven paratypes diagnosed by Macko as C. hians longivitellata (ex Ci. ciconia) and eight adult individuals diagnosed by Macko as C. hians hians (ex Ci. nigra). We included only individuals with eggs in these measurements. These materials are currently deposited at the Institute of Parasitology, Czech Academy of Sciences in České Budějovice, Czech Republic.
DNA extraction, amplification, and sequencing
We extracted, amplified, and sequenced the DNA using primers that targeted nuclear ribosomal DNA (partial 18S rDNA and ITS2) and mitochondrial (CO1 and ND1) loci as described by Heneberg et al. (Reference Heneberg, Casero, Waap, Sitko, Azevedo, Těšínský and Literák2018). We submitted the resulting visually checked sequences to NCBI GenBank under accession numbers OR533419 (18S rDNA), OR533496-OR533499 (ITS2), OR536618-OR536623 (CO1), OR544075 (ND1), PP157883-PP157886 (ND1), PP177534-PP177536 (18S rDNA), and PP177542-PP177543 (ITS2) (Table 1).
Alignments and phylogenetic analyses
We aligned the obtained sequences and publicly available sequences of closely related species retrieved from NCBI GenBank as of February 8, 2024, along with sequences of corresponding outgroups (selected as sequences of species with the highest similarity of the sequence of the respective locus to the sequences of the same locus obtained from Cathaemasia spp. and publicly available in NCBI GenBank at a time when the analyses were performed) using ClustalW (with the following parameters: gap opening penalty 7 and gap extension penalty 2 for both pairwise and multiple alignments, DNA weight matrix IUB, and transition weight 0.1). We manually corrected any inconsistencies in alignments and trimmed the alignments to the length of the shortest sequence. The trimmed 18S rDNA locus (partial SSU rRNA coding sequence) corresponded to nt. 63-1741 (1679 bp) of Petasiger phalacrocoracis (Echinostomatidae) AY245709.1. The trimmed ITS2 locus (partial 5.8S ribosomal RNA, full-length ITS2, and partial 28S ribosomal RNA sequences) corresponded to nt. 2478-3196 (719 bp) of Isthmiophora hortensis (Echinostomatidae) AB189982.1. The trimmed CO1 locus (partial CO1 coding sequence) corresponded to nt. 7626-7955 (330 bp) of Fasciolopsis buski (Fasciolidae) NC_030528.1. The trimmed ND1 locus (partial ND1 coding sequence) corresponded to nt. 13-365 (352 bp) of Echinochasmus coaxatus (Echinochasmidae) MN720147.1.
We calculated the maximum likelihood fits of the 24 nucleotide substitution models for each locus. We employed a bootstrap procedure with 1,000 replicates and nearest-neighbor interchange as the maximum likelihood heuristic method of choice for tree inference when we generated the initial tree using a neighbor-joining algorithm. We then determined the best substitution model based on the lowest Bayesian Information Criterion scores and used best-fit models for the maximum likelihood phylogenetic analyses. The models used to construct the maximum likelihood phylogenetic trees were the Kimura 2-parameter model with gamma-distributed rates among sites (18S rDNA and ITS2) and the Hasegawa-Kishino-Yano model with gamma-distributed rates among sites (five discrete gamma categories) (CO1 and ND1). We also used these models to estimate the evolutionary divergence between sequences. We conducted all the maximum likelihood analyses in MEGA5.
To validate the maximum likelihood analysis data, we employed Bayesian inference. We converted the ClustalW alignments generated in MEGA5 to the Nexus format in Mesquite 3.04. We then performed the Bayesian analysis using the mixed model of nucleotide substitution in MrBayes 3.2.5. We used four Monte Carlo Markov chains for 10,000,000 generations and trees sampled every 1,000th generation, with the average standard deviation of split frequencies not exceeding 0.0030. We discarded the first 25% of samples as burn-in. We used the remaining dataset to generate a 50% majority-consensus tree with the posterior probabilities of branches indicated and visualized the resulting trees in FigTree 1.4.2. We obtained the following summary statistics for analyses performed: average standard deviation of split frequencies 0.006–0.025, maximum standard deviation of split frequencies 0.017–0.090, average potential scale reduction factor 1.000–1.005, and maximum potential scale reduction factor 1.000–1.007.
Results
Molecular phylogenetics
We sequenced and analyzed differences between C. hians hians (ex Ci. nigra) and C. hians longivitellata (ex Ci. ciconia) using three DNA loci representing hypervariable DNA regions (CO1, ND1, and ITS2). We also sequenced partial 18S rDNA, but we were able to amplify this locus for only one of the subspecies. Molecular phylogenetic analyses of the three hypervariable regions provided clear support for the elevation of C. hians longivitellata (ex Ci. ciconia) to the species level (Fig. 1). Of particular interest were the analyses of CO1 (Fig. 1A) and ITS2 (Fig. 1E), which illustrate well the genetic distance between the two proposed species. The conclusions from maximum likelihood analyses were confirmed using the Bayesian approach (Fig. S1).
Figure 1. Maximum likelihood analyses of the sequences of the mitochondrial and nuclear DNA loci of Cathaemasia spp. (A) CO1, (B) ND1, (C) ITS2, and (D) 18S rDNA. The bars indicate the number of substitutions per nucleotide. The numbers above the internodes indicate the percentage of trees in which the associated taxa clustered together.
The genetic distance between the CO1 loci of C. hians hians (ex Ci. nigra) and C. hians longivitellata (ex Ci. ciconia) was 12.8%. There was no intraspecific genetic variability among the sequences of the respective Cathaemasia spp. The genetic distance between the ITS2 loci of C. hians hians (ex Ci. nigra) and C. hians longivitellata (ex Ci. ciconia) was 4.2%-4.5%. The intraspecific variability among C. hians hians (ex Ci. nigra) was 0.0% to 0.1%, and the variability was 0.3% among isolates of C. hians longivitellata (ex Ci. ciconia).
Species descriptions
Cathaemasia longivitellata Macko sp. n.
Synonym: Cathaemasia hians longivitellata Macko, 1960
Host: Ciconia ciconia (Aves: Ciconiiformes) (prevalence 4.1 %; intensity of infection 1–14 individuals).
Location in host: Esophagus, muscular stomach.
Locality: Czech Republic: Strachotín (48.90°N, 16.65°E).
Other localities: Czech Republic: Bartošovice (49.66°N, 18.05°E), Záhlinice (49.29°N, 17.48°E).
Examined specimens: Type specimen and six paratype specimens D534/2, all collected by J. K. Macko; currently in the collection of the Institute of Parasitology, Czech Academy of Sciences, České Budějovice, Czech Republic. Additional 15 specimens P-P-1865/1, all in the collection of Comenius Museum, Přerov, Czech Republic. All represent adult individuals with eggs present. DNA samples are deposited at the Charles University, Third Faculty of Medicine, Prague, Czech Republic (marked as 3LF-4426 and 3LF-4427).
Zoobank accession: The Life Science Identifier for Cathaemasia longivitellata sp. n. is urn:lsid:zoobank.org:act:D6DCBD4B-B1FA-4600-84BE-A1C1DC5A2CC5.
DNA sequences: ITS2: PP177542 and PP177543; CO1: OR536622 and OR536623; ND1: PP157886 and OR544075.
Etymology: The specific epithet longivitellata is identical to the name previously proposed by Macko (Reference Macko1960a), who was the first to recognize the morphologic distinctness of this species and propose its status as a subspecies Cathaemasia hians longivitellata. The species name refers to a characteristic identification feature of this species: the prominent length of vitellaria relative to the total body length.
Description (15 specimens ex Ciconia ciconia) (Fig. 2A-B): Body medium to large, elongately oval, with rounded extremities and maximum width at mid hindbody, 10,000–15,710 × 3,430–5,290 (12,189 × 4,396), body length/width ratio 1: 2.33–3.58 (2.79) (body width equals to 42%–53 % of body length). Forebody length 2,657–4,860 (3,693), hindbody length 6,290–9,140 (7,346), forebody/hindbody length ratio 1: 1.72–2.5 (2); forebody occupies 29% to 36 % of the body length. Post-testicular field length 714 to 1,571; post-testicular field occupies 7% to 12% of body length. Tegument wrinkled, thickness 220 to 480, greater in median portions than lateral margins. Dorsal and ventral thicknesses of tegument similar. Cercariae have collar with 47 spines (Bykhovskaya-Pavlovskaya and Kulakova Reference Bykhovskaya-Pavlovskaya and Kulakova1977); in adult digeneas collar rudimentary, with only 20 to 36 (31) small, sharply pointed spines, 24 to 70 × 13–27, in two lateral groups. Body covered with scales, approximately one third smaller in juveniles than adults. Scales in area of suckers round shaped, on anterior body edge larger than on lateral body edges, median edge of oral sucker 34 × 34, lateral margin 24 × 24, scales of ventral sucker median edge 48 × 48, and lateral margin 48 × 42. Scales oval-shaped on remaining parts of body: scales from esophagus and intestinal bifurcation 45 × 42, scales of ovary and testes medially 64 × 71, and lateral margin 54 × 64, behind posterior testes 62 × 62. Oral sucker spherical, small 685 to 1,120 × 629 to 1,143 (847 × 916). Ventral sucker spherical, longer than oral sucker, in second quarter of body 971 to 1,486 × 1,000 to 1,486 (1,244 × 1,210), oral/ventral suckers length ratio 1: 0.53 to 1.67 (1.42), width ratio 1: 1.12 to 2.01 (1.36). Prepharynx short, 57 to 200 (141). Pharynx globular, 400 to 596 × 429 to 714 (490 × 530). Esophagus moderately long, 600 to 1.143 (890), without lateral diverticula. Intestinal bifurcation approximately halfway between pharynx and ventral sucker. Ceca sinuous, with short outer lateral diverticula. Testes large, contiguous, deeply lobed to branched, in posterior quarter of body. Anterior testis 1,143 to 3,000 × 1,426 to 3,430 (1,682 × 2,077), always slightly larger than posterior 1,120 to 2,143 × 1,286 to 2,428 (1,420 × 1,907). Cirrus pouch elongately oval, entirely anterior to ventral sucker 522 to 1,371 × 536 to 1,371 (763 × 845). Internal seminal vesicle large, saccate. Prostatic pars short. Genital pore median, approximately halfway between intestinal bifurcation and ventral sucker. Ovary small, elongate or round, submedian, postequatorial, 429 to 571 × 457 to 514 (500 × 486). Mehlis´ gland diffuse, contiguous with ovary, 134 to 429 × 209 to 457 (338 × 344). Vitellarium in two compact lateral small follicles, from pharynx or halfway between pharynx and ventral sucker up to posterior body extremity, left branch 7,430 to 12,860 (9,839), right branch 8,000 to 12,860 (10,181). Vitellarium occupies 68% to 98% (85%) of body length. Stem of excretory vesicle may bear lateral diverticula, pore terminal. Uterus long 4,860 to 6,543 (uterus occupies 38%–48% [40%] of body length), loops numerous between ovary and ventral sucker, may overlap ceca. Metraterm indistinct. Eggs numerous, relatively small 87 to 104 × 54 to 62 (99 × 59), contain fully developed miracidium with distinct eyespots.
Figure 2. Representative photographs of C. longivitellata sp. n. (A, B) and C. hians sensu stricto (C, D). (A) Cathaemasia longivitellata sp. n. ex Ciconia ciconia, female, May 1, 1967, Napajedla, district Zlín, Czech Republic, site: esophagus. (B) Cathaemasia longivitellata sp. n. ex Ciconia ciconia, male, July 27, 1999, Nošovice, district Frýdek-Místek, Czech Republic, site: esophagus. (C-D) Cathaemasia hians ex Ciconia nigra, female, May 28, 1976, Šišma, district Přerov, Czech Republic, site: esophagus.
Remarks: Specialized parasite of white storks (Ciconia ciconia) in Europe and Africa. The two newly proposed Cathaemasia spp. differ mainly in the length of their vitellaria. However, as the total body length is highly variable in both of these species, we propose using the vitellaria length ratio to the total body length. In C. hians sensu stricto, the vitellaria/body length ratio is 48% to 64% (57%), whereas it is 67% to 97% (82%) of the total body length in C. longivitellata sp. n. The genetic distance between the CO1 loci of C. hians sensu stricto and C. longivitellata sp. n. was 0.128 base substitutions per site. An example of characteristic C. longiviellata sp. n. sequence is GGGTTTGGATGTTC (CO1 locus; position 153–166 in OR536622), whereas the sequence of this locus in C. hians sensu stricto is AGGTTTAGATGTAC.
Note: Pressure-fixed holotype and six paratypes collected by Macko (Reference Macko1960a) were re-examined; the measurements are provided in Table 3, and photograph of the holotype is provided in Fig. 3A. Some of the individuals deposited by Macko were subadults and did not have developed eggs. We measured only those with eggs, which caused differences in the lower ranges of some of the measurements.
Figure 3. Photographs of the C. longivitellata sp. n. holotype (A, ex Ciconia ciconia, 1957, Senné, Slovakia, site: esophagus) and representative individual of C. hians sensu stricto (B, ex Ciconia nigra, undisclosed date, Košický region, Slovakia, site: esophagus), both collected and prepared by Macko (Reference Macko1960a). Photographs were merged from two images each. Specimens in the collection by J. K. Macko were not numbered individually, only the holotype was labeled.
Figure 4. Drawings of C. longivitellata sp. n. (A) and C. hians sensu stricto (B).
Cathaemasia hians (Rudolphi, Reference Rudolphi1809) Looss, 1899
Synonym: Distoma hians Rudolphi, Reference Rudolphi1809; Cathaemasia hians hians Macko, 1960
Host: Ciconia nigra (Aves: Ciconiiformes) (prevalence 41.2 %; intensity of infection 1–32 individuals).
Location in host: Esophagus, muscular stomach.
Localities: Czech Republic: Bartošovice (49.66°N, 18.05°E), Hrabyně (49.87°N, 18.03°E), Huslenky (49.29°N, 18.09°E), Komorní Lhotka (49.66°N, 18.49°E), Přerov (49.45°N, 17.46°E), Záhlinice (49.29°N, 17.48°E).
Examined specimens: Specimens D534/1, all collected by J. K. Macko; currently in the collection of the Institute of Parasitology, Czech Academy of Sciences, České Budějovice, Czech Republic. Additional 30 specimens P-P-1865/1, all in the collection of the Comenius Museum, Přerov, Czech Republic. All represent adult or subadult individuals with eggs present. DNA samples were deposited at Charles University, Third Faculty of Medicine, Prague, Czech Republic (marked as 3LF-2331, 3LF-2332, 3LF-2333, and 3LF-3989).
DNA sequences: 18S rDNA: PP177534-6, OR533419; ITS2: OR533496-9; CO1: OR536618-21; ND1: PP157883-5.
Description (30 specimens ex Ciconia nigra) (Fig. 2C-D): Digenea pink to freshly red in color. Body medium to large, elongately oval, with rounded extremities and maximum width at mid-hindbody, 7,436 to 16,159 × 2,814 to 5,710 (10,986 × 3,801), body length/width ratio 1: 2.14 to 3.56 (3.02) (body width equals to 28%–47% of body length). Forebody length 2,414 to 3,140 (2,776), hindbody length 4,420 to 5,430 (5,058), forebody/hindbody length ratio 1: 1.52 to 2.24 (1.84); forebody occupies 27% to 34% of the body length. Post-testicular field length 571 to 1,286; post-testicular field occupies 6% to 15% of body length. Tegument wrinkled, thickness 220 to 480, greater in median portions than lateral margins. Dorsal and ventral thickness of tegument similar. Cercariae likely have collars with 47 spines (Bykhovskaya-Pavlovskaya and Kulakova Reference Bykhovskaya-Pavlovskaya and Kulakova1977), in adult digeneas collar rudimentary, with only 24 to 36 (34) small, sharply pointed spines 40 to 75 × 19 to 27 in two lateral groups. Body covered with scales, approximately one third smaller in juveniles than in adults. Scales in area of suckers round shaped, on anterior edge larger than on lateral edges, median edge of oral sucker 31 × 26, lateral margin 16 × 26, scales of ventral sucker: median edge 48 × 48, and lateral margin 42 × 48. Scales oval-shaped on remaining parts of the body: scales from esophagus and intestinal bifurcation 32 × 54, scales of ovary and testes medially 48 to 51 × 64 to 71, lateral margin 48 × 48, behind posterior testes 64 × 64. Oral sucker subglobular 542 to 1,143 × 626 to 1,143 (749 × 816). Ventral sucker spherical, longer than oral sucker, in second quarter of body, 904 to 1,514 × 940 to 1,486 (1,109 × 1,145). Oral/ventral suckers length ratio 1: 1.32 to 1.67 (1.36), width ratio 1: 1.3 to 1.5 (1.4). Prepharynx short, 312 to 415 (410). Pharynx long oval, 422 to 602 × 443 to 771 (527 × 541). Esophagus short, 216 to 714 (440), without lateral diverticula. Intestinal bifurcation approximately halfway between pharynx and ventral sucker. Ceca sinuous, with short outer lateral diverticula, long, reaches up to middle distance of sucker. Testes large, contiguous, deeply lobed to branched, in posterior quarter of body. Anterior testis 714 to 2,571 × 1,200 to 3,718 (1,374 × 1,877), always slightly larger than posterior 572 to 2,220 × 686 to 3,146 (1,277 × 1,466). Cirrus pouch elongate oval, entirely anterior to ventral sucker 482 to 1,429 × 361 to 1,429 (762 × 635). Internal seminal vesicle large, saccate. Prostatic pars short. Cirrus tubular, unarmed. Genital pore median, approximately halfway between intestinal bifurcation and ventral sucker. Ovary small transversely elongate or round, submedian, postequatorial 216 to 571 × 180 to 514 (293 × 333). Mehlis´ gland diffuse, contiguous with ovary 241 to 361 × 265 to 578 (321 × 422). Vitellarium nonconfluent, in two compact laterals extra cecal fields of small follicles composed of individual follicles, reach from rear edge of ventral sucker up to posterior body extremity, left branch 2,860 to 5,710 (4,423) and right branch 2,823 to 5,600 (4,205). Vitellarium occupies 48% to 64% (55%) of body length. Stem of excretory vesicle may bear lateral diverticula, pore terminal. Uterus long 2,571 to 6,061 (uterus occupies 33%–43% of body length), loops numerous, between ovary and ventral sucker, may overlap ceca. Metraterm indistinct. Eggs numerous, relatively small 97 to 108 × 54 to 62 (105 × 59), contain fully developed miracidium with distinct eyespots.
Remarks: Specialized parasite of black storks (Ciconia nigra) in Europe and Africa.
Note: The largest individuals of both species (C. hians and C. longivitellata, sp. n.) were of similar size. Eight pressure-fixed individuals collected by Macko (Reference Macko1960a) were re-examined; the measurements are provided in Table 2, and a photograph of the representative slide is provided in Fig. 3B. Some of the individuals deposited by Macko were subadults and did not have developed eggs. We measured only those with eggs, which caused differences in the lower ranges of some of the measurements.
Discussion
Several previous studies have noted morphological differences between the proposed species. First, Macko (Reference Macko1960a) proposed the existence of two subspecies, C. hians hians and C. hians longivitellata. A year later, Feizullaev (Reference Feizullaev1961) described a new species, Cathaemasia skrjabini ex Ciconia ciconia from Azerbaijan. Feizullaev noted that the vitellaria of C. skrjabini extend anteriorly to the level of the genital bursa. The same author also proposed an alternative explanation in his follow-up study (Feizullaev Reference Feizullaev1962), claiming that the reported morphological differences might result from development in different intermediate hosts. That Feizullaev (Reference Feizullaev1961) described the new species ex Ci. ciconia based on material from the Transcaucasian region (Azerbaijan) caused a somewhat chaotic situation when some Western European parasitologists, such as Van den Broek (Reference Van den Broek1963), continued to recognize the materials from European Ci. nigra and Ci. ciconia as C. hians but accepted the materials from Azerbaijani Ci. ciconia as C. skrjabini sensu Feizullaev (Reference Feizullaev1961). Other authors, including Gundlach (Reference Gundlach1969), recognized both subspecies, confirming their strict host specificity.
Another issue associated with descriptions of C. hians by previous authors stems from the absence of mentions of host species in some of the descriptions or from the mixing of data from both host species. For example, Szidat (Reference Szidat1940b) published a drawing of C. longivitellata sp. n. ex Ci. ciconia. However, the text of his study does not recognize C. longivitellata sp. n. and proposes that previously suggested C. fodicans (here synonymized with C. longivitellata sp. n.) ex Chlidonias niger is identical to C. hians and that the host published by Braun (Reference Braun1901) was in fact Ci. nigra (which is most likely an accurate claim). Later, Chiriac and Udrescu (Reference Chiriac and Udrescu1973) reprinted the C. longivitellata sp. n. drawing from Szidat (Reference Szidat1940b) but claimed that it was hosted by Ci. nigra.
Several researchers provided measurements that were identical to those published by other authors earlier but mentioned themselves as the authors of the measurements. This applies, for example, to the descriptions of C. hians from Hungary by Edelényi (Reference Edelényi1974), who provided identical measurements as Lühe (Reference Lühe and Brauer1909). However, the descriptions by Lühe (Reference Lühe and Brauer1909) were also not original because they were identical to those provided by Braun (Reference Braun1902). Surprisingly, this does not correspond to the provided illustrative drawings because Edelényi (Reference Edelényi1974) published a drawing of C. longivitellata sp. n., whereas Lühe (Reference Lühe and Brauer1909) published a drawing of C. hians. Additionally, Bykhovskaya-Pavlovskaya and Kulakova (Reference Bykhovskaya-Pavlovskaya and Kulakova1977) provided C. hians measurements, but these were identical to those published by Macko (Reference Macko1960a), who remained uncited by Bykhovskaya-Pavlovskaya and Kulakova (Reference Bykhovskaya-Pavlovskaya and Kulakova1977).
Some previous host-parasite records were erroneous. Ardea cinerea, Ardea purpurea, and Nycticorax nycticorax were reported as C. hians hosts in Italy by Parona (Reference Parona1899), but these records represented erroneously identified trematodes of the genus Clinostomum.
In addition to the above-named species, the C. hians species complex also contains the African species Cathaemasia variabilis. This species was described from Sphenorhynchus abdimii in Africa and was recognized as valid by Van den Broek (Reference Van den Broek1963) but was synonymized with C. hians as C. hians variabilis by Bykhovskaya-Pavlovskaya and Kulakova (Reference Bykhovskaya-Pavlovskaya and Kulakova1977). The extent of vitellaria is identical to that of C. hians. Therefore, without subsequent DNA analyses, it is impossible to draw conclusions regarding the systematics of members of the C. hians complex in tropical and subtropical regions outside Europe.
In our view, valid descriptions of individuals of C. hians sensu stricto were published by Macko (Reference Macko1960a), Yoshida and Tomoda (Reference Yoshida and Toyoda1930), and Braun (Reference Braun1901). We provide a comparative table of measurements provided by these authors in Table 2. Note that the drawing and description in Braun (Reference Braun1901) is consistent with the C. hians sensu stricto diagnosis. Nevertheless, the author claimed that the host was Chlidonias niger (identified as Sterna niger according to the taxonomy valid at a time of the description). However, it is unlikely that the black tern was infected by the species strictly specialized to Ci. nigra, and we assume that the host was recorded erroneously. The valid descriptions of individuals of C. longivitellata sp. n. were provided by Macko (Reference Macko1960a), Feizullaev (Reference Feizullaev1961), and Iskova (Reference Iskova1985), and these descriptions are compared in Table 3.
The intensity of infection by C. hians sensu stricto was greater than that by C. longivitellata sp. n., which contributes to the apparently smaller size of individuals of this species. The largest individuals of both Cathaemasia spp. are of similar size. Because of the dietary changes of both examined stork species, particularly from the nearly complete dietary change of the Czech population of C. ciconia from amphibians to small mammals and other types of dietary items (Sitko and Heneberg Reference Sitko and Heneberg2021), both Cathaemasia spp. have become rare in recent years. These species were dominant among the trematodes of both stork species, but recently, we had to examine more than 100 stork individuals to find them only once in the past 10 years.
Species identity of C. hians sensu lato findings from intermediate hosts remains to be elucidated. The first intermediate hosts of C. hians sensu lato are snails; the repeatedly reported hosts are Planorbis planorbis (Baršiené Reference Baršiené1991; Zhytova and Korol Reference Zhytova and Korol2012; Tkach et al. Reference Tkach, Kudlai and Kostadinova2016) and Lymnaea stagnalis (Grabda-Kazubska et al. Reference Grabda-Kazubska, Bayssade-Dufour and Kiseliene1990; Baršiené Reference Baršiené1990; Faltýnková et al. Reference Faltýnková, Našincová and Kablásková2008). The presence of C. hians sensu lato in the first of these two species was also confirmed by molecular analysis (Tkach et al. Reference Tkach, Kudlai and Kostadinova2016). Other Planorbiidae and Lymnaeidae are also hypothesized to be permissive intermediate hosts (Szidat Reference Szidat1939; Zhytova & Korol Reference Zhytova and Korol2012); for example, infections of Planorbis and Anisus spp. were reported by Zdun (Reference Zdun1961). Notably, the karyotypes of C. hians sensu lato isolated from P. planorbis and L. stagnalis differed from one another (Baršiené Reference Baršiené1991). It is unclear whether these isolates of C. hians sensu lato represented different species or which of the isolates should be assigned to C. hians sensu stricto. The second intermediate hosts of C. hians sensu lato are amphibian tadpoles, including those of Bombina bombina, Pelophylax ridibundus, Pelophylax esculentus, and Ranidae spp. (Volgar-Pastukhova Reference Volgar-Partukhova1959; Vojtková Reference Vojtková1982; Grabda-Kazubska and Lewin Reference Grabda-Kazubska and Lewin1989). It is unclear whether other vertebrate species may also serve as second intermediate hosts. Merino et al. (Reference Merino, Martínez, Lanzarot, Cano, Fernández-García and Rodríguez-Caabeiro2001) proposed that C. hians sensu lato requires a warm climate to complete its life cycle. However, the authors mentioned above provided multiple pieces of evidence of the presence of infected snails and amphibians locally (Poland, Czech Republic, Lithuania, and Ukraine – Sandner Reference Sandner1949; Zdun Reference Zdun1961; Vojtková and Křivanec Reference Vojtková and Křivanec1970; Balúsek and Vojtek Reference Balúsek and Vojtek1973; Baršiené Reference Baršiené1990, Reference Baršiené1991; Grabda-Kazubska et al. 1998; Faltýnková et al. Reference Faltýnková, Našincová and Kablásková2008; Zhytova and Korol Reference Zhytova and Korol2012). Vojtková (Reference Vojtková1982) examined 1536 amphibian tadpoles from 82 sampling sites across the Czech Republic and Slovakia, reporting differences in C. hians sensu lato prevalence from zero up to 35% (Pelophylax esculentus tadpoles from Palkovičovo (recently termed Sap), Slovakia). Studies of adult amphibians often conclude the absence of C. hians sensu lato metacercariae in adult frogs (Kozák Reference Kozák1973) at localities where highly prevalent C. hians sensu lato infections of storks are known (Macko Reference Macko1961). Supporting the completion of the life cycle locally, there is also evidence of infection of juvenile C. nigra, which was approximately 76 days old, in the Czech Republic (Hampl and Sitko Reference Hampl and Sitko2013), and infection of C. nigra nestlings in Spain was reported by Merino et al. (Reference Merino, Martínez, Lanzarot, Cano, Fernández-García and Rodríguez-Caabeiro2001). The second author of the present study also found two flightless nestlings at sampling sites Strachotín and Napajedla (both Czech Republic), which were positive for C. hians sensu lato (J. Sitko, pers. obs.).
In conclusion, combined molecular and comparative morphological analyses of central European Cathaemasia individuals ex Ci. nigra and Ci. ciconia led to the proposal of a split of C. hians into C. hians sensu stricto (formerly C. hians hians sensu Macko Reference Macko1960a) and C. longivitellata sp. n. (formerly C. hians longivitellata sensu Macko Reference Macko1960a). Morphological analyses confirmed that the length of the vitellaria was the key identification feature of the two abovementioned species. Both Cathaemasia spp. have strict host specificity, which might be related to differences in food preferences of the two stork species, and they substantially differ at the molecular level.
Supplementary material
The supplementary material for this article can be found at http://doi.org/10.1017/S0022149X24000622.
Declaration
Ethical approval
Not applicable. All the host birds were obtained dead and therefore no ethics permit was required by Czech law. The research on bird helminths was authorized by the Ministry of the Environment of the Czech Republic; the most recent permit was issued on August 3, 2009, under No. 11171/ENV/09-747/620/09-ZS 25.
Availability of data and materials
Representative specimens of the helminths analysed in this study are available in the collections of the Comenius Museum in Přerov. All data are available in the main text or the supplementary materials.
Competing interest
On behalf of both authors, the corresponding author states that there is no conflict of interest.
Author contribution
P.H. performed the molecular and phylogenetic analyses and wrote the manuscript; J.S. conceived the study, examined the host birds, and performed the morphological analyses. Both authors revised the manuscript and agreed on its final version.
Funding
The study was supported by the Ministry of Culture of the Czech Republic project DE07P04OMG007.
Acknowledgements
We thank the governmental and local authorities for providing the necessary permissions to conduct this long-term research. We also thank the landlords, gamekeepers, and the staff of local rescue stations for providing us with carcasses of untreatable birds. We thank Tomáš Scholz and Blanka Škoríková (Institute of Parasitology, Czech Academy of Sciences, České Budějovice, Czech Republic) for providing access to the specimens from the collection of J. K. Macko.
Open access
