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Published in final edited form as: Zool Scr. 2019 May 29;48(4):545–556. doi: 10.1111/zsc.12360

Molecular phylogeny of the Cyathocotylidae (Digenea, Diplostomoidea) necessitates systematic changes and reveals a history of host and environment switches

Tyler J Achatz 1, Eric E Pulis 2, Kerstin Junker 3, Tran Thi Binh 4, Scott D Snyder 5, Vasyl V Tkach 6
PMCID: PMC6959977  NIHMSID: NIHMS1028137  PMID: 31937984

Abstract

The Cyathocotylidae is a globally distributed family of digeneans parasitic as adults in fish, reptiles, birds, and mammals in both freshwater and marine environments. Molecular phylogenetic analysis of interrelationships among cyathocotylids is lacking with only a few species included in previous studies. We used sequences of the nuclear 28S rRNA gene to examine phylogenetic affinities of 11 newly sequenced taxa of cyathocotylids and the closely related family Brauninidae collected from fish, reptiles, birds, and dolphins from Australia, Southeast Asia, Europe, North America and South America. This is the first study to provide sequence data from adult cyathocotylids parasitic in fish and reptiles. Our analyses demonstrated that the members of the genus Braunina (family Brauninidae) belong to the Cyathocotylidae, placing the Brauninidae into synonymy with the Cyathocotylidae. In addition, our DNA sequences supported the presence of a second species in the currently monotypic Braunina. Our phylogeny revealed that Cyathocotyle spp. from crocodilians belong to a separate genus (Suchocyathocotyle, previously proposed as a subgenus) and subfamily (Suchocyathocotylinae subfam. n.). Morphological study of Gogatea serpentum indicum supported its elevation to species as Gogatea mehri. The phylogeny did not support Holostephanoides within the subfamily Cyathocotylinae; instead, Holostephanoides formed a strongly supported clade with members of the subfamily Szidatiinae (Gogatea and Neogogatea). Therefore, we transfer Holostephanoides into the Szidatiinae. DNA sequence data revealed the potential presence of cryptic species reported under the name Mesostephanus microbursa. Our phylogeny indicated at least two major host switching events in the evolutionary history of the subfamily Szidatiinae which likely resulted in the transition of these parasites from birds to fish and snakes. Likewise, the transition to dolphins by Braunina represents another major host switching event among the Cyathocotylidae. In addition, our phylogeny revealed more than a single transition between freshwater and marine environments demonstrated in our dataset by Braunina and some Mesostephanus.

Keywords: Cyathocotylidae, molecular phylogeny, systematics, Suchocyathocotylinae subfam. n., host switching

Introduction

The Cyathocotylidae Mühling, 1896 is a small, globally distributed family of diplostomoidean (superfamily Diplostomoidea Poirier, 1886) digeneans that are parasitic as adults in the intestine of birds, reptiles and, rarely, mammals and fishes. Unlike most other diplostomoideans, cyathocotylids usually have an undivided body, a cirrus-sac enclosing a cirrus and a seminal vesicle and, sometimes, a small caudal appendix (Niewiadomska, 2002). Some members of this family are of economic and conservation concern; for instance, Cyathocotyle bushiensis Khan, 1962 has been associated with massive die-offs of aquatic birds in the Midwestern United States (Gibson et al., 1972; Hoeve & Scott, 1988; Herrmann & Sorensen, 2009). Mühling (1896) initially established the subfamily Cyathocotyleae Mühling, 1896 for his newly described genus Cyathocotyle Mühling, 1896 with Cyathocotyle prussica Mühling, 1896 as the type-species. Later, Poche (1926) elevated the status of the group to family level. The most recent revision of the Cyathocotylidae by Niewiadomska (2002) recognized five subfamilies: Cyathocotylinae Mühling, 1896 (four genera), Muhlinginae Mehra, 1950 (one genus), Prohemistominae Lutz, 1935 (five genera), Prosostephaninae Szidat, 1936 (three genera) and Szidatiinae Dubois, 1938 (three genera).

The current systematics and taxonomy of the Cyathocotylidae is based entirely on morphological characters. While members of the family parasitize fishes, reptiles, birds and mammals worldwide, the lack of a robust (or any) phylogeny of the group prevented examination of intriguing questions regarding patterns of their current and past geographic distribution, host associations and environmental switches. In fact, no molecular phylogenetic study of the family has been carried out. Monophyly of the family as a whole and its constituent subfamilies and genera have not been tested yet using molecular data. Likewise, the interrelationships among the genera within the Cyathocotylidae remain completely unknown. Currently, mostly non-comparable DNA sequences are available from the adult forms of only three species belonging to the genera, Mesostephanus Lutz, 1935 and Holostephanus Szidat, 1936 (Dzikowski et al., 2004; Hernández-Mena et al., 2017; El-Bahy et al., 2017); moreover, all three species are from avian hosts, thus preventing evolutionary analysis of the patterns of host associations among cyathocotylids.

Other studies have generated DNA sequence data from other life cycle stages, mostly cercariae and metacercariae (Locke et al., 2010; Karamian et al., 2011; Ciparis et al., 2013; Blasco-Costa & Locke, 2017; Locke et al., 2018); however, the limited sequence data from adult forms prevent accurate species- or genus-level diagnoses of the sequenced life cycle stages. While recent molecular phylogenetic studies (Blasco-Costa & Locke, 2017; Hernández-Mena et al., 2017; Locke et al., 2018) have indicated the general position of the Cyathocotylidae among other diplostomoidean families, the very limited number of taxa used in these analyses did not allow exploration of questions of evolution and systematics related to geographic distribution, host associations and environmental switches of the group. For instance, no molecular data have been published on any of the genera of cyathocotylids parasitizing non-avian reptiles and fish as adults. From a geographic point of view, all available cyathocotylid DNA sequences so far come from Europe and North America, thus leaving their relationships with members of the family from other regions completely unknown.

Similarly, the phylogenetic affinities of another diplostomoidean family, the Brauninidae Wolf, 1903, have always been unclear. At present, the Brauninidae only includes the monotypic genus Braunina Heider, 1900 that parasitize cetaceans as adults. Braunina shares atypical diplostomoidean morphological characters with the Cyathocotylidae (e.g. a cirrus-sac enclosing a cirrus and a seminal vesicle) (Niewiadomska, 2002). The validity and systematic position of the Brauninidae in relation to the Cyathocotylidae has been recently called into question based on the results of molecular phylogenetic studies of the group (Fraija-Fernández et al. 2015; Blasco-Costa & Locke, 2017; Hernández-Mena et al., 2017) which placed Braunina in the clade with members of the Cyathocotylidae. However, the Brauninidae currently remains a separate family due to insufficient data and low diversity of cyathocotylids included in previous phylogenetic analyses.

Herein, we examine the phylogenetic interrelationships and host associations of the Cyathocotylidae and re-evaluate the taxonomic status of its constituent lineages as well as the family Brauninidae using 28S rRNA sequences from quality specimens of cyathocotylids newly collected from fish, reptiles, birds and mammals in Europe, Asia, Australia, Africa and North America. In addition, we used mitochondrial cytochrome c oxidase subunit 1 (CO1) gene sequences for comparison between samples of Braunina originating from different hosts and geographic regions as well as between two genetically different forms identified as Mesostephanus microbursa Caballero, Grocott & Zerecero, 1953.

Methods

Morphological data

Specimens belonging to the families Cyathocotylidae and Brauninidae were collected from the intestines of fish, snakes, crocodilians, birds and dolphins in Australia, Southeast Asia, Europe and North America between 2003 and 2016 (Table 1). In most cases, live digeneans removed from the hosts were briefly rinsed in saline, killed with hot water and fixed in 70% ethanol. The digeneans from the Nile crocodile Crocodylus niloticus Laurenti were killed in hot saline, fixed in 10% formalin and transferred to 70% ethanol. Specimens of Braunina were collected from stranded common bottlenose dolphins Tursiops truncatus (Montagu) which were already dead at the time of necropsy and thus were placed directly in 70% ethanol. Specimens for light microscopy were stained with aqueous alum carmine, dehydrated in an ethanol series of ascending concentration, cleared in clove oil, mounted permanently in Damar gum and identified using a DIC-equipped Olympus BX40 compound microscope (Tokyo, Japan) with digital imaging system. Morphological vouchers are deposited in the collection of the Harold W. Manter, University of Nebraska State Museum, Lincoln, NE, U.S.A.

Table 1.

List of cyathocotylid species used in our phylogenetic analysis of 28S rRNA including their host species, geographical origin of material, morphological voucher numbers and GenBank accession numbers.

Digenean taxa Host species Country Museum No. Accession numbers
 Reference
rRNA cox1
Braunina cordiformis Wolf, 1903 Delphinus delphis Argentina − KM258670 MF124272 Fraija-Fernandez et al., 2015; Blasco-Costa & Locke, 2017
Braunina sp. Tursiops truncatus U.S.A. HWML-110857 MK650438, MK650439 MK645805 Present study
Cyathocotyle bushiensis Khan, 1962 Aythya affinis U.S.A. HWML-139967 MK650440 − Present study
Cyathocotyle prussica Mühling, 1896 Gasterosteus aculeatus Germany − MH521249 − Locke et al., 2018
Cyathocotylidae sp. Clypeomorus batillariaeformis Australia − MH257776 − Huston et al., 2018
Gogatea mehri Mehra, 1947 Acrochordus granulatus Vietnam − MK650441 − Present study
Gogatea sp. Acrochordus javanicus Thailand − MK650442 − Present study
Holostephanoides ictaluri Vernberg, 1952 Ameiurus sp. U.S.A. _ MK650443 − Present study
Holostephanus dubinini Vojtek & Vojtkova, 1968 Phalacrocorax carbo Ukraine HWML-139968 MK650444 − Present study
Mesostephanus cubaensis Alegret, 1941 Morus bassanus U.S.A. HWML-139969 MK650445 − Present study
Mesostephanus microbursa Caballero, Grocott, & Zerecero, 1953 Mo. bassanus U.S.A. HWML-139970 MK650446 MK645806 Present study
Me. microbursa Sula nebouxii Mexico − MF398325 MF398316 Hernández-Mena et al., 2017
Neogogatea sp. Lophodytes cucullatus U.S.A. HWML-139971 MK650447–MK650449 − Present study
Suchocyathocotyle crocodili (Yamaguti, 1954) Crocodylus johnsoni Australia HWML-139972 MK650450, MK650451 − Present study
Suchocyathocotyle fraterna (Odhner, 1902) Crocodylus niloticus South Africa HWML-139373 MK650452 − Present study
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Molecular data

Genomic DNA was extracted according to the protocol described by Tkach & Pawlowski (1999). An approximately 1,300 bp long fragment at the 5’ end of the 28S rRNA was amplified by polymerase chain reactions (PCR) on a T100™ thermal cycler (Bio-Rad, Hercules, CA, U.S.A.) using forward primer digL2 (5’−AAG CAT ATC ACT AAG CGG−3’) and reverse primer 1500R (5’−GCT ATC CTG AGG GAA ACT TCG−3’) (Tkach et al., 2003). For our specimens of Braunina and Me. microbursa, we amplified a fragment of the CO1 gene using the forward primers Plat-diploCOX1F (5’−CGT TTR AAT TAT ACG GAT CC−3’) and Cox1_Schist_5’ (5’−TCT TTR GAT CAT AAG CG−3’) and reverse primers Plat-diploCOX1R (5’−AGC ATA GTA ATM GCA GCA GC−3’) and JB5 (5’−AGC ACC TAA ACT TAA AAC ATA ATG AAA ATG−3’) (Lockyer et al., 2003; Derycke et al., 2005; Moszczynska et al., 2009). PCRs were performed in a total volume of 25 μl using New England Biolabs (Ipswich, MA, U.S.A.) One-Taq quick load PCR mix according to the manufacturer’s protocol and using an annealing temperature of 53oC for nuclear rRNA and 45oC for CO1.

PCR products were purified using ExoSap PCR clean-up enzymatic kit from Affimetrix (Santa Clara, CA, U.S.A.) following the manufacturer’s protocol. PCR products were cycle-sequenced directly using ABI BigDyeTM 3.1 (Applied Biosystems, Foster City, California, U.S.A.) chemistry, alcohol precipitated, and run on an ABI 3130 automated capillary sequencer (Life Technologies, Grand Island, New York, U.S.A.). PCR primers and additional internal forward primer DPL600F (5’−CGG AGT GGT CAC CAC GAC CG−3’) and reverse primer DPL700R (5’−CAG CTG ATT ACA CCC AAA G−3’) designed for this study by TJA and VVT were used in sequencing reactions. Contiguous sequences were assembled using Sequencher ver. 4.2 (GeneCodes Corp., Ann Arbor, Michigan, U.S.A.) and submitted to GenBank (for accession numbers see Table 1). Representative sequences are deposited in the National Institutes of Health genetic sequence database (GenBank).

Sequences were initially aligned using ClustalW implemented in Mega7 (Kumar et al., 2016). The alignment was then trimmed to the length of the shortest sequences. Clinostomum tataxumui Sereno-Uribe, Pinacho-Pinacho, Garcia-Varela & Pérez-Ponce de León, 2013 was. used as outgroup based on the phylogeny recently published by Hernández-Mena et al. (2017). The alignment included newly obtained sequences of one specimen of Braunina sp. and 10 cyathocotylid taxa, previously published sequences of Braunina coridiformis, three cyathocotylid taxa, 13 representatives of the Diplostomidae Poirier, 1886, one species of the Proterodiplostomidae Dubois, 1936 and 10 taxa of the Strigeidae Railliet, 1919 in order to test the interrelationships among all these digenean families.

Phylogenetic analyses were conducted using Bayesian inference (BI) as implemented in MrBayes Ver. 3.2.6 software (Ronquist & Huelsenbeck, 2003) and Maximum Likelihood (ML) as implemented in Mega7 (Kumar et al., 2016). 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 using jModelTest 2 software (Darriba et al., 2012). Nodal support of ML analysis was estimated by performing 1000 bootstrap pseudoreplicates. BI analysis was performed using MrBayes software as follows: Markov chain Monte Carlo (MCMC) chains were run for 3,000,000 generations with sample frequency set at 100. Log-likelihood scores were plotted and only the final 75% of trees were used to produce the consensus trees by setting the “burn-in” parameter at 7500. This number of generations was considered sufficient because the SD dropped below 0.01. The trees were visualized in FigTree ver. 1.4 software (Rambaut, 2016) and annotated in Adobe Illustrator®.

Results

The 28S rRNA alignment was 1,083 bp long. In the phylogenetic trees resulting from the BI and ML analyses, all members of the Cyathocotylidae and Brauninidae formed a very highly supported (100% in BI and 94% in ML), monophyletic clade (Fig. 1). Within this clade, Cyathocotyle (Suchocyathocotyle) crocodili from the freshwater crocodile Crocodylus johnsoni Krefft collected in Australia, and Cyathocotyle (Suchocyathocotyle) fraterna collected from the Nile crocodile Crocodylus niloticus in South Africa formed a very highly supported clade (100% in BI and 82% in ML) as the sister-group to the remaining cyathocotylid taxa. All other cyathocotylids included in our analysis formed the second, very highly supported clade (100% in BI and 99% in ML) with overall highly supported internal topology. One of the subclades included strongly supported clusters of Cyathocotyle + Holostephanus (100% in BI and ML) and Gogatea Lutz, 1935 + Neogogatea Chandler & Rausch, 1947 + Holostephanoides Dubois, 1983 (100% in BI and 73% in ML). Both sequenced forms of Braunina were nested within the Cyathocotylidae clade and formed a strongly supported clade with the Mesostephanus spp. and an unidentified cyathocotylid cercaria from Australia (98% in BI and 80% in ML). All three adult forms of Mesostephanus formed a strongly supported clade (100% in BI and 97% in ML) (Fig. 1).

Fig. 1.

Fig. 1

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Phylogenetic interrelationships among 38 diplostomoidean taxa based on Bayesian inference (BI) and Maximum Likelihood (ML) analyses of partial 28S rRNA sequences. Topology from BI analysis provided. Bayesian inference posterior probability values and Maximum Likelihood bootstrap values associated with the branches are shown as BI/ML; support values lower than 90% (BI) and 50% (ML) are not shown. New sequences obtained in this study are in bold. Branch length scale bar indicates number of substitutions per site. GenBank accession numbers are provided after the names of species. Biogeographical realms, definitive host groups and types of environment, and subfamilies are indicated for the members of the Cyathocotylidae only. Abbreviations for biogeographical realms: AF – Afrotropical realm, AUS – Australasian realm, IM – Indo-Malayan realm, NA – Nearctic realm, NT –Neotropical realm, PA – Palaearctic realm. Abbreviations for cyathocotylid subfamilies: Bra – Braunininae, Cya – Cyathocotylinae, Pro – Prohemistominae, Suc – Suchocyathocotylinae, Szi –Szidatiinae. Abbreviations for environment type of definitive host: FW –freshwater, SW – saltwater. *Previously a member of subfamily Cyathocotylinae. **The definitive host of C. prussica is a member of Anatidae, but the sequence was obtained from a metacercaria collected from fish. ***Cyathocotyle bushiensis is a Palaearctic species only recently introduced to the Nearctic.

At the species level, our DNA sequences obtained from a Braunina specimen collected from Tu. truncatus in the Gulf of Mexico differed by two bases (0.16%) in 28S and by 41 bases (8.9%) in CO1 from the comparable sequences of Br. cordiformis from Delphinus delphis Linnaeus available in GenBank (KM258670, MF124272). The phylogenetic analysis indicated that our 28S sequence of Me. microbursa does not form a monophyletic group with the sequence of Me. microbursa from GenBank (MF398325). However, Hernández-Mena et al. (2017) have also published a CO1 sequence from the same specimen (Genbank MF398316). We, therefore, also obtained CO1 sequence from our sample for comparison. The two forms differed by 30 nucleotide positions (2.7%) in 28S sequences and 80 nucleotide positions (16.4%) in CO1 sequences.

Other accepted families of the superfamily Diplostomoidea formed a strongly supported (100% in BI and 97% in ML) clade (Fig. 1). This clade was, however, poorly resolved internally with multiple taxa of the Diplostomidae, Proterodiplostomidae and Strigeidae forming a polytomy. The Strigeidae as currently accepted was polyphyletic with two distinct clades. The first clade was strongly supported (100% in both BI and ML) and included members of Apatemon Szidat, 1928, Australapatemon Sudarikov, 1959 and a well-supported group of Strigea Abildgaard, 1790 + Apharyngostrigea Ciurea, 1927 + Parastrigea Szidat, 1928. The second supported clade (97% in BI and 62% in ML) united Cardiocephaloides Sudarikov, 1959 and a strongly supported clade of Cotylurus Szidat, 1928 + Ichthyocotylurus Odening, 1969 (100% in BI and 99% in ML).

The Diplostomidae were also found to be paraphyletic and formed seven distinct clades: 1) Posthodiplostomum Dubois, 1936 + Ornithodiplostomum Dubois, 1936 (100% in both BI and ML), 2) Bolbophorus Dubois, 1935, 3) Uvulifer Yamaguti, 1934, 4) Diplostomum von Nordmann, 1832 + Austrodiplostomum Szidat & Nani, 1951 + Tylodelphys Diesing, 1850 (100% in BI and 91% in ML), 5) Alaria Schrank, 1788, 6) Neodiplostomum Railliet, 1919 and 7) Hysteromorpha Lutz, 1931.

Discussion

The Cyathocotylidae is a relatively small digenean group, therefore, even a limited set of taxa allowed us to answer some questions about their systematics and reveal important trends in the evolution of their morphological traits, host associations and geographic distribution. We have for the first time obtained and combined in one study sequences of cyathocotylids from five continents and all main host groups (fishes, snakes, crocodilians, birds and mammals). The results of our phylogenetic analysis challenge the current morphology-based systematic framework of the Cyathocotylidae in several ways. While the molecular phylogeny supported the monophyly of some of the existing subfamilies (Fig. 1 and discussion below), the paraphyletic nature of the type-genus of the family as well as the position of Holostephanoides outside of the Cyathocotylinae and particularly the inclusion of Braunina within the Cyathocotylidae, necessitates taxonomic and systematic changes. Below we discuss the main findings of this study focusing on well-supported topologies only.

Braunina and the amended diagnosis of the Cyathocotylidae

The genus Braunina is a unique group of diplostomoideans parasitizing cetaceans as adults and is the only genus of the family Brauninidae (Niewiadomska, 2002). Braunina was first included in a molecular phylogenetic analysis by Fraija-Fernández et al. (2015), however, no cyathocotylids were included in their analysis. Recent studies by Hernández-Mena et al. (2017) and Blasco-Costa & Locke (2017) called the systematic position of the Brauninidae in relation to the Cyathocotylidae into question. Blasco-Costa & Locke (2017) demonstrated that Br. cordiformis forms a clade with a metacercaria of unknown species of Cyathocotyle (the name was later changed to Cy. prussica) and Mesostephanus based on CO1 mtDNA and ITS1–5.8S-ITS2 rRNA sequences. Both Hernández-Mena et al. (2017) and Blasco-Costa & Locke (2017) have noted the strong morphological similarities between Braunina and cyathocotylids and their differences from other diplostomoideans (e.g. presence of a cirrus-sac in Braunina and cyathocotylids vs absence in all other diplostomoideans). While Blasco-Costa & Locke (2017) indicated that the Cyathocotylidae would be monophyletic upon inclusion of the Brauninidae, no definitive taxonomic conclusion was drawn. Those authors stated that knowledge of the morphology of the larval stages of Braunina would eventually strengthen the argument for its transfer to the Cyathocotylidae. In our opinion, larval morphology is desirable, but not required for this taxonomic action. Our molecular phylogenetic results as well as the details of adult morphology provide sufficient grounds for synonymization. Therefore, we transfer Braunina into the family Cyathocotylidae and the Brauninidae becomes a junior synonym of the Cyathocotylidae. It is noteworthy that the Brauninidae had already been considered a synonym of the Cyathocotylidae in the past (Wolf, 1903; La Rue, 1957; Yamaguti, 1971). At the same time, based on its unique morphology and the results of our phylogenetic analysis, we retain this taxon as subfamily Braunininae Wolf, 1903 within the Cyathocotylidae, with diagnosis of the type- and currently only genus Braunina. The amended diagnosis of the Cyathocotylidae is provided below.

Cyathocotylidae Mühling, 1896. Diagnosis: Diplostomoidea, with body generally undivided, oval, pyriform, linguiform, or cordiform, with small, conical, truncate, or sometimes elongate caudal appendix. Holdfast organ round or oval, sometimes large and overlaid by ventral fold forming deep cavity (in Braunina). Oral sucker present or absent; when absent, subterminal oral opening leads directly to pharynx. Ventral sucker present or absent. Pseudosuckers absent. Oesophagus short; caeca usually reaching close to posterior end of body, rarely sinuous. Position of testes and ovary variable. Cirrus-sac present, occasionally rudimentary, enclosing seminal vesicle, pars prostatica, and cirrus. Genital pore terminal. Eggs typically large, not numerous. Vitellarium follicular, variable in extent. Life-cycle with longifurcate furcocercous cercaria having excretory system composed of four stems united anteriorly, two lateral and two median joined anterior to ventral sucker. Metacercaria of ‘prohemistomulum’ type, with sucker-like holdfast organ and crown-like reserve bladder. Parasites of reptiles, birds, and mammals. Mother- and daughter-sporocysts in gastropods (Prosobranchia); metacercariae in fishes, amphibians, and aquatic invertebrates. Type-genus Cyathocotyle Mühling, 1896.

Braunina contains the only valid species, Br. Cordiformis, originally described from the short-beaked common dolphin De. delphis collected in the Adriatic Sea (Wolf, 1903). Previously published sequences of Br. cordiformis (GenBank KM258670, MF124272) also came from material obtained from a De. delphis, but collected off the Atlantic coast of Argentina (Fraija-Fernandez et al., 2015). Our DNA sequences obtained from a Braunina species collected from Tu. truncatus in the Gulf of Mexico differ by 8.9% in CO1 from the matching sequence of Br. cordiformis in GenBank, thus strongly indicating the presence of a second species in the genus. At the same time, relationships of either of these species with the form originally described from the Adriatic Sea remain unknown. Molecular data for Br. cordiformis collected close to the type-locality, are needed to verify the identity of currently available sequences.

It is possible that Br. cordiformis is distributed in both the Old and New World, however, it may also be a complex of more than two morphologically similar species. While our specimens appear in relatively fair condition, they are not ideal for descriptive work, especially considering their size and shape. Due to the protected status of dolphins, opportunities to obtain fresh material from them are very rare and chances of obtaining live digeneans that can be properly fixed are extremely low. Until quality specimens associated with DNA sequences become available, knowledge of the diversity of the genus will mostly rely on molecular data.

Composition of the Cyathocotylinae Mühling 1896

The most recent revision of the Cyathocotylinae by Niewiadomska (2002) recognized four genera: Cyathocotyle, Holostephanoides, Pseudhemistomum Szidat, 1936 and Holostephanus. Our analysis included three of these genera (Cyathocotyle, Holostephanus and Holostephanoides). According to the most recent revision of Cyathocotyle by Dubois (1984), this genus contains seventeen species distributed on all continents except for Antarctica and Australia. Fourteen species are parasites of birds as adults, while the remaining three species have been described from crocodilians. No Cyathocotyle species is restricted to North America or was originally described from the continent, however, the European species Cy. bushiensis was reported from the Midwestern United States. Often, these reports were associated with massive die-offs of waterfowl (Gibson et al., 1972; Hoeve & Scott, 1988; Herrmann & Sorensen, 2009). Surprisingly, no DNA sequences of this rather infamous species were available until now.

Originally, Cy. bushiensis was described in the United Kingdom based on digeneans experimentally grown in laboratory ducklings from metacercariae obtained from naturally infected prosobranch snails belonging to Bithynia tentaculata Linnaeus (Khan, 1962). The first study of Cy. bushiensis in North America compared the morphology of North American and European specimens and noted several morphological differences between them including egg length, relative cirrus length, shape and relative size of testes (Gibson et al., 1972). Our specimens collected from Lake Winnibigoshish, Minnesota very closely correspond to those described by Khan (1962).

Currently the genus Cyathocotyle is split into the subgenera Cyathocotyle Mühling, 1896 and Suchocyathocotyle Dubois, 1984 on the basis of testes orientation (opposite testes in Cyathocotyle and tandem in Suchocyathocotyle), cirrus-sac length (reaching or extending beyond the middle of the body in Cyathocotyle and never extending beyond the middle of the body in Suchocyathocotyle), egg size (smaller in Cyathocotyle and larger in Suchocyathocotyle), and definitive hosts (birds in Cyathocotyle and crocodilians in Suchocyathocotyle). It should be noted that the relative length of the cirrus-sac cannot be used for reliable differentiation between these taxa because at least some species of Cyathocotyle (Cyathocotyle) have a short cirrus-sac not fitting Dubois’ (1984) diagnosis. On the other hand, one species of Cyathocotyle (Cyathocotyle), namely Cyathocotyle (Cyathocotyle) fulicae Ginetzinskaja, 1952, was described as having tandem testes. However, it is evident from the provided illustration (Ginetzinskaja, 1952) that the specimen was very strongly flattened at the time of fixation which likely caused a shift in the position of internal organs.

Suchocyathocotyle was named after the crocodilian hosts of three of the four included species: Cy. (S.) crocodili (type-species) was described from the saltwater crocodile Crocodylus porosus Schneider in Indonesia, Cyathocotyle (Suchocyathocotyle) brasiliensis Ruiz & Leão, 1943 from the spectacled caiman Caiman crocodilus Linnaeus (=Caiman sclerops) in Brazil, Cy. (S.) fraterna from Cr. niloticus (=Champse vulgaris) in Egypt (Odhner, 1902; Ruiz & Leão, 1943; Yamaguti, 1954; Dubois, 1984). The fourth species, Cyathocotyle (Suchocyathocotyle) szidatiana Faust & Tang, 1938, was described from a mallard duck Anas platyrhynchos Linnaeus (=Anas boschas) in China (Faust & Tang, 1938). Dubois (1984) argued that the duck infection with Cy. (S.) szidatiana may have been accidental and the true definitive host is a crocodilian, presumably the Chinese alligator Alligator sinensis Fauvel.

Our phylogenetic analysis places the type-species Cy. (S.) crocodili and Cy. (S.) fraterna as the basal branch within the Cyathocotylidae, genetically distant from Cyathocotyle parasitic in birds. Based on the results of our molecular phylogenetic analysis combined with the morphological characters used by Dubois (1984) to separate the subgenera of Cyathocotyle, we elevate these two subgenera to genus status. Therefore, Cy. (S.) crocodili (type-species), Cy. (S.) brasiliensis, Cy. (S.) fraterna and Cy. (S.) szidatiana are transferred to Suchocyathocotyle Dubois, 1984. However, we place the latter species in Suchocyathocotyle with caution. Although it has tandem testes, the description of some important morphological characters in this species is vague. In general, multiple cases of poor descriptions and/or poor fixation of specimens used for descriptions in the literature present a major hindrance for a full revision of the group. Collection of fresh, properly fixed specimens is desirable for the majority of species. For instance, the cirrus-sac of S. szidatiana as described by Faust & Tang (1938) was strongly contracted, which causes uncertainty and places it between Cyathocotyle and Suchocyathocotyle. Thus, the systematic position of S. szidatiana may eventually change. Due to the fact that the diagnoses of the subgenera Cyathocotyle and Suchocyathocotyle by Dubois (1984) were very brief and Niewiadomska’s (2002) diagnosis combined features of both Dubois’ subgenera, there is an obvious need for amended diagnoses of Cyathocotyle and Suchocyathocotyle. They are provided below. We would like to emphasize that only properly mounted, not flattened specimens are likely to fit these diagnoses. Also, the only species of Cyathocotyle (Cyathocotyle) with eggs larger than 113 µm is Cyathocotyle (Cyathocotyle) melanittae Yamaguti, 1934, in which the position of testes is uncertain, appearing nearly tandem in a flattened, laterally mounted specimen (Yamaguti, 1934).

Cyathocotyle Mühling, 1896. Diagnosis (after Dubois, 1984 and Niewiadomska, 2002, with changes). Body massive, oval, pyriform or fusiform. Holdfast organ large, round, with aperture of variable shape, elevated above ventral surface. Oral sucker and pharynx well-developed; ventral sucker small, near intestinal bifurcation, in some species absent or not visible, covered by holdfast organ. Oesophagus very short or absent. Testes round or elongate, opposite or somewhat oblique. Cirrus-sac well-developed, claviform, with large seminal vesicle occupying proximal part of cirrus-sac. Genital pore subterminal. Ovary round, small, variable in position and ventral to testes. Eggs small to medium sized (57−127μm). Vitellarium in form of coarse follicles surrounding holdfast organ in peripheral part of body and overlying caeca, usually does not extend into organ. In different groups of birds. Europe, Asia, North America. Metacercariae of ‘prohemistomulum’ type, in fishes or leeches; one species in Bithynia Leach. Cercariae, with flame-cell formula 2[(3 + 3) + (3 + [3])] = 24, developing in Prosobranchia (Bithynia, Bellamya Jousseaume) or Pulmonata (Bulinus Müller). Type-species Cy. prussica Mühling, 1896. Other species: Cy. anhinga Vidyarthi, 1948, Cy. bithyniae Sudarikov, 1974, Cy. bushiensis Khan, 1962, Cy. fulicae Ginetzinskaja, 1952, Cy. indica Mehra, 1943, Cy. japonica Kurisu, 1931, Cy. malayi Palmieri, Krishnasamy & Sullivan, 1979, Cy. melanittae, Cy. opaca (Wisniewski, 1934) Vojtek, 1971, Cy. orientalis Faust, 1922, Cy. oviformis Szidat, 1936, Cy. skrjabini Petrov & Sudarikov, 1963.

Suchocyathocotyle Dubois, 1984. Diagnosis (after Dubois, 1984, with changes). Body massive, oval, pyriform with or without caudal appendix. Holdfast organ large, round, with aperture of variable shape, elevated above ventral surface. Oral sucker and pharynx well-developed; ventral sucker weakly developed, near intestinal bifurcation. Prepharynx and oesophagus very short. Ceca reaching to second or last third of body. Testes oval, tandem. Cirrus-sac claviform, thin-walled, typically short, never extending beyond equator of body, containing elongated seminal vesicle, terminating with muscular ejaculatory duct. Genital pore subterminal. Ovary round, small, either at the level of the anterior testis or inter-testicular. Uterus with few coils, metraterm short. Laurer’s canal absent. Eggs large sized (117 to 144 μm). Vitellarium in form of large coarse follicles, filling most of body. Excretory pore ventral to genital pore; dorso-lateral stems reach as far as anterior extremity. Parasites of crocodilians in Africa, Asia, Australia and South America. Type-species: S. crocodili (Yamaguti, 1954). Other species: S. fraterna (Odhner, 1902), S. brasiliensis (Ruiz & Leão, 1943), S. szidatiana (Faust & Tang, 1938).

Based on the results of the phylogenetic analysis demonstrating that Suchocyathocotyle forms a clade clearly separated from Cyathocotylinae and the remaining cyathocotylid subfamilies included in our analysis, we establish herein a subfamily Suchocyathocotylinae subfam. n. with Suchocyathocotyle as the type- and currently only genus. The diagnosis of the subfamily is the same as that of Suchocyathocotyle.

The two Suchocyathocotyle sequences included in our analysis are characterized by long branches which likely reflects the long evolutionary and geographic separation between these parasites and their hosts. While it is possible that the African species S. fraterna may deserve to be separated into its own genus, the morphological study of our specimens as well as descriptions in the literature did not yield convincing evidence to warrant creation of a new genus.

Composition of the Szidatiinae Dubois, 1938

The most recent revision of the subfamily Szidatiinae recognised the three genera: Szidatia Dubois, 1938, Gogatea and Neogogatea (Niewiadomska, 2002). Adult Gogatea and Szidatia parasitize snakes in Africa and Asia (Joyeux & Bear, 1934; Gogate, 1932), whereas Neogogatea parasitizes birds in North America and Asia (Chandler & Rausch, 1947; Zazornova, 1995). No member of the subfamily has been used in any molecular study to date. Our analysis used two of the genera (Gogatea and Neogogatea) and confirmed their close relationships and separation from other main cyathocotylid linages (Fig. 1).

Dubois (1989) recognized Gogatea serpentum (Gogate, 1932) as the only member of Gogatea with two subspecies, Gogatea serpentum serpentum (Gogate, 1932) and Gogatea. serpentum indicum Mehra, 1947. The latter form had been elevated to species level by Dwivedi & Chauhan (1969), based on several morphological characteristics (including the relative length of cirrus-sac and position of testes), and named Gogatea mehri Mehra, 1947. Dubois (1975; 1980) rejected this change and synonymized G. mehri as well as three other species, with G. serpentum indicum. Our, high quality specimens, freshly collected in Vietnam, fully corresponded morphologically to the description of G. mehri, but not to the original description of G. serpentum. Therefore, we consider G. mehri a separate species. The taxonomic status of other species synonymized by Dubois (1975; 1980) requires a revision using quality specimens.

The present work is the first molecular systematic study to include sequences of any member of either Gogatea or Neogogatea. Our specimens of Neogogatea sp. (deposited as HWML-139971), collected from the hooded merganser Lophodytes cucullatus (Linnaeus) in Mississippi, U.S.A., were not fully mature (lacked eggs), but still had traits of Neogogatea (e.g. lack of ventral sucker and vitellarium in form of horseshoe).

Somewhat surprisingly, Holostephanoides ictaluri Vernberg, 1952, the type-species of Holostephanoides (previously a member of the Cyathocotylinae), appeared in the phylogenetic tree within a strongly supported clade with members of Szidatiinae with Neogogatea as its closest relative. While the general morphology of Holostephanoides (a digenean with rounded appearance), Gogatea and Neogogatea (both include digeneans with an enlarged anterior part of the body and elongated posterior part) differs substantially, the phylogenetic analysis suggests that these differences are likely a result of recent adaptation. Other than the body shape, these genera do not have other dramatic morphological differences. Moreover, available data on the excretory system including the protonephridial formulas support relatedness between Holostephanoides and Neogogatea. Both genera have the same excretory formula 2[(3+3+3)+(3+3+[3])]=36 (Cable, 1938; Hoffman & Dunbar, 1963; Stang & Cable, 1966). On the other hand, Cyathocotyle and Holostephanus belonging to the Cyathocotylinae have different protonephridial formulas, 2[(3+3)+3)3+[3])]=24 in Cyathocotyle and 2[(2+2+2)+(2+2+[2])]=24 in Holostephanus (Dubois, 1983; Dubois, 1984). The excretory formula of Pseudhemistomum, the other genus belonging to subfamily Cyathocotylinae, is currently unknown. Due to the fact that our analysis included the type-species of the genus, we transfer Holostephanoides into the Szidatiinae. However, the monophyly of Holostephanoides needs to be tested using DNA sequences and data on the excretory system of its only other member, Holostephanoides hoeppliana (Tang & Tang, 1989) from Eurasian curlew Numenius arquata (Linnaeus) in China.

Notes on the Prohemistominae Lutz, 1935

The most recent revision of subfamily Prohemistominae by Niewiadomska (2002) recognises five genera: Mesostephanoides Dubois, 1951, Mesostephanus, Prohemistomum Odhner, 1913, Linstowiella Szidat, 1933 and Paracoenogonimus Katsurada, 1914. Our analyses are limited to Mesostephanus, a cosmopolitan genus of cyathocotylids that parasitize birds and mammals as adults. Our analysis shows strong support for the Mesostephanus clade.

We compared our sequences of Me. microbursa collected from the Northern gannet Morus bassanus (Linnaeus) off the coast of Mississippi with the sequence of Me. microbursa (GenBank MF398316, MF398325) collected from the blue-footed booby Sula nebouxii Milne-Edwards in Nayarit, Mexico (Hernández-Mena et al., 2017). These samples differed by 2.7% in the 28S gene and by 16.4% in the CO1 gene. This divergence level clearly indicates that our specimens and those sequenced by Hernández-Mena et al. (2017) represent different species. Neither the specimens sequenced by Hernández-Mena et al. (2017), nor our specimens came from the type-host, the brown pelican Pelecanus occidentalis Linnaeus. Our material was in excellent condition and morphologically corresponded very well to the original description. It is unclear whether the identification of specimens used in the two studies as conspecifics stems from the specimen quality or indicates the existence of cryptic species.

Definitive host associations and environmental switches

Based on the broad, essentially cosmopolitan distribution, great diversity of definitive host groups ranging from fishes to mammals, and clear phylogenetic separation from the remaining diplostomoideans, the Cyathocotylidae is undoubtedly a very ancient digenean lineage. Based on the presence of cyathocotylids in crocodilians in Australia, Southeast Asia, Africa and South America, combined with the strong separation of Suchocyathocotyle from the remaining members of the family, cyathocotylids likely already existed as a separate lineage in the late Cretaceous (ca. 65–70 mya). The family could be more ancient, but the available data are insufficient for a confident conclusion on this matter.

Our analysis revealed some strongly supported cyathocotylid clades associated with certain groups of definitive hosts. Although cyathocotylids are not particularly diverse in crocodilian hosts, the basal position and strong separation of Suchocyathocotyle (Suchocyathocotylinae subfam. n.) from other members of the family likely reflects the ancient nature of their host association rather than a secondary host switching event.

The subfamily Szidatiinae is represented in our tree by Gogatea (parasites of snakes) and Neogogatea (parasites of birds) and Holostephanoides (parasites of fishes) forming a 100% supported clade in BI and 73% in ML. The placement of Holostephanoides ictaluri, a parasite of freshwater fishes, in the Szidatiinae represents a significant secondary host switching event, in this case from tetrapods to fish. While this is certainly a very rare event, it has occurred in a variety of digenean groups. For example, a few microphallid species have transitioned to parasitism in fishes (Gibson, 1996), while members of Caballerotrema Prudhoe, 1960 are the only echinostomatoidean digeneans that secondarily switched to parasitism of freshwater fishes (Tkach et al., 2016). Thus, the evolutionary history of the Szidatiinae sub-clade has included at least two major host switching events. Based on the fact that almost all other members of the Cyathocotylinae and Szidatiinae that appeared as sister groups in our analysis (Fig. 1) are parasitic in birds, it is somewhat plausible to hypothesize the general direction of host switching from avian hosts to other vertebrates. However, we abstain here from any definitive conclusions until more taxa of the Szidatiinae can be included in phylogenetic analysis.

Braunininae also form a very strongly supported clade that includes cyathocotylids that transitioned to parasitism of marine mammals, namely dolphins. This shift was accompanied by significant morphological changes; the phylogenetic position and systematic history of Braunina was discussed in detail above.

Lastly, the 100% BI and 99% ML supported clade of Mesostephanus represents the subfamily Prohemistominae in our analysis. Species included in this study are all parasitic in water birds, but some members of the subfamily are known from other vertebrates such as reptiles. It is difficult to speculate on the exact nature of these associations until a more detailed phylogeny of the Cyathocotylidae becomes available. The systematic position and definitive hosts of Cyathocotylidae sp. (GenBank MH257776) sequenced from a cercaria is presently unknown.

Along with the multiple definitive host switches that occurred in the evolutionary history of the family, cyathocotylids have also transitioned more than once between freshwater and marine environments. While what is currently known about their geographic distribution as well as definitive and first intermediate hosts strongly supports freshwater life cycles among the ancestral cyathocotylids, members of the former family Brauninidae and some of the prohemistomatine taxa have switched to marine life cycles.

Finally, mapping the geographic distribution of the taxa used in our analysis onto the phylogenetic tree did not reveal clades strongly associated with distinct biogeographical realms (Fig. 1). The mosaic nature of the geographic distribution of cyathocotylid taxa across the phylogenetic tree provides support for a likely ancient origin of the group as a whole.

To conclude, this is the first molecular phylogenetic analysis of the Cyathocotylidae that includes a broad variety of taxa from different continents and a wide range of host groups. Importantly, almost all taxa used in our analysis, were represented by adult digeneans, most of them well-fixed and morphologically identifiable. This is the first study to report DNA sequence data for several cyathocotylid taxa and the first to provide molecular data from representatives of the family parasitic in reptiles. Our phylogenetic analysis provided grounds for revisions in the system of the Cyathocotylidae that include transfer of the former Brauninidae into the Cyathocotylidae as a subfamily and erection of the Suchocyathocotylinae subfam. n. Future molecular phylogenetic studies will need to include a higher number of cyathocotylid taxa, including members of the not yet sequenced Muhlinginae and Prosostephaninae, in order to test the monophyly and interrelationships of the currently accepted subfamilies as well as further explore the evolution of their host associations.

Acknowledgements

We are grateful to Dr. Danny Govender and Prof. Joop Boomker (both at the University of Pretoria, South Africa) and the stuff of SANParks, South Africa for facilitating and assisting in parasite collections from Nile crocodiles; the help of Mr. Frans Masubelle and Mr. Daniel Chipana, ARC-Onderstepoort Veterinary Institute, in parasite recovery from crocodiles is greatly appreciated. We also sincerely thank Dr. Nguyen Van Ha (Institute of Ecology and Biological Resources, Hanoi, Vietnam) for the help with collecting of specimens in Vietnam. Collecting and processing of the specimens from Southeast Asia, North America and Europe were supported in part by grant R15AI092622 from the National Institutes of Health, U.S.A. and grant DEB1021431 from the National Science Foundation to V. V. Tkach and E. W. H. Wheeler Award from the Department of Biology, University of North Dakota to T. J. Achatz.

Contributor Information

Tyler J. Achatz, Department of Biology, University of North Dakota, 10 Cornell Street, Grand Forks, North Dakota 58202, U.S.A.

Eric E. Pulis, Department of Science and Mathematics, Northern State University, Aberdeen, South Dakota 57401, U.S.A.

Kerstin Junker, Epidemiology, Parasites and Vectors, ARC-Onderstepoort Veterinary Institute, Private Bag X05, Onderstepoort, 0110 South Africa.

Tran Thi Binh, Department of Parasitology, Institute of Ecology and Biological Resources, Vietnam Academy of Science and Technology, Nghiado, Caugiay, Hanoi, Vietnam.

Scott D. Snyder, College of Science and Engineering, Idaho State University, 921 S 8th Ave, Pocatello, ID, 83209 U.S.A.

Vasyl V. Tkach, Department of Biology, University of North Dakota, 10 Cornell Street, Grand Forks, North Dakota 58202, U.S.A..

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