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Research Article
Molecular phylogeny, including a new species of Anindobothrium (Cestoda, Rhinebothriidea) from the Southern eagle ray Myliobatis goodei, finally solves the taxonomic enigma of Phyllobothrium myliobatidis
expand article infoGuillermina García Facal‡, Sebastián Franzese‡, Martín Miguel Montes§, Adriana Menoret‡
‡ Universidad de Buenos Aires, Buenos Aires, Argentina
§ Universidad Nacional de La Plata, La Plata, Argentina

Corresponding author: Guillermina García Facal ( guillefacal@gmail.com )

Academic editor: Tom Artois
© 2024 Guillermina García Facal, Sebastián Franzese, Martín Miguel Montes, Adriana Menoret.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation: García Facal G, Franzese S, Montes MM, Menoret A (2024) Molecular phylogeny, including a new species of Anindobothrium (Cestoda, Rhinebothriidea) from the Southern eagle ray Myliobatis goodei, finally solves the taxonomic enigma of Phyllobothrium myliobatidis. Zoosystematics and Evolution 100(4): 1401-1417. https://doi.org/10.3897/zse.100.131971
ZooBank: urn:lsid:zoobank.org:pub:50A363FC-F02F-4B6A-A0A8-963EC8D5A65C
Open Access

Abstract

During a parasitological survey of tapeworms from Myliobatis goodei Garman, 1885 (Myliobatiformes: Myliobatidae) in coastal waters off Argentina in the Southwestern Atlantic, a new rhinebothriidean cestode species, Anindobothrium danielae sp. nov., is described using morphological and molecular techniques. This species differs from its congeners by a particular combination of features, including the configuration of the bothridia, the number of marginal loculi, and the number and distribution of testes. Additionally, Anindobothrium myliobatidis comb. nov. is proposed based on several morphological traits, including the presence of stalked bothridia with marginal loculi and an apical sucker, euapolytic strobila, and postvaginal testes. The diagnosis of the genus Anindobothrium Marques, Brooks & Lasso, 2001 is amended to include the features exhibited by these two species; two species subsets are suggested based on the configuration of the bothridia. The presence of A. danielae sp. nov. and A. myliobatidis comb. nov. in the studied area not only increases the number of cestodes in M. goodei here from eight to ten but also represents the first report of a rhinebothriidean cestode parasitizing stingrays of the family Myliobatidae in the Southwestern Atlantic.

Key Words

Anindobothriidae, description, molecular identification, morphology, Myliobatiformes, Southwest Atlantic, tapeworms

Introduction

The rhinebothriidean cestode genus Anindobothrium Marques, Brooks & Lasso, 2001 currently consists of four valid species: A. anacolum (Brooks, 1977), A. carrioni Trevisan, Primon & Marques, 2017, A. inexpectatum Trevisan, Primon & Marques, 2017, and A. lisae Marques, Brooks & Lasso, 2001. According to Trevisan et al. (2017), these species are principally characterized by a scolex composed of four stalked bothridia typically longer than wide, with or without longitudinal septa, with apical sucker and marginal loculi, with or without facial loculi; testes numerous arranged in two lateral columns; the presence of postporal testes; a genital pore in the anterior half of the proglottid; and a vitellarium interrupted by terminal genitalia and partial or totally interrupted by ovary.

Members of Anindobothrium were found in stingrays of the family Potamotrygonidae (Myliobatiformes) in marine and freshwater environments. Three species were found in potamotrygonids of the marine genus Styracura Carvalho, Loboda & da Silva, 2016, with A. anacolum and A. inexpectatum from several localities in the Caribbean Sea in Central and South America, and A. carrioni from the Tropical Eastern Pacific Ocean in Central America (Trevisan et al. 2017). To date, A. lisae Marques, Brooks & Lasso, 2001 was exclusively found in freshwater stingrays of the genus Potamotrygon Garman, 1877, in rivers of South America (Marques et al. 2001; Trevisan et al. 2017). Recently, Trevisan et al. (2017), based on the phylogeny of Anindobothrium, suggested that the diversification of the genus follows the colonization by their hosts, the marine-derived freshwater stingrays, with an ancestor in rivers of South America after marine incursions in northern latitudes (Fontenelle et al. 2021), which consequently explains the presence of Anindobothrium in both environments.

During field trips off the coastal waters off Argentina in the Southwestern Atlantic (SWA), eagle rays of the genus Myliobatis Cuvier, 1816 were examined for cestodes. Preliminary studies on the collected specimens based mainly on the morphology of the scolex and the proglottids indicated that these were similar to those exhibited by A. lisae. However, an exhaustive morphological study based on entire mature worms, fine histology, and scanning electron microscopy (SEM), in addition to molecular analyses, allowed us to identify a new species of Anindobothrium.

Two eagle rays of the genus Myliobatis (i.e., Myliobatis goodei Garman, 1885 and Myliobatis ridens Ruocco, Lucifora, Díaz de Astarloa, Mabragaña & Delpiani, 2012) have been previously reported as hosts for several species of tapeworms in the SWA, being parasitized by nine species of cestodes of the orders Diphyllidea, Lecanicephalidea, “Tetraphyllidea,” and Trypanorhyncha, and one of uncertain placement, Phyllobothrium myliobatidis Brooks, Mayes & Thorson, 1981 (see Franzese et al. 2023). Furthermore, prior to this study, there are no other records of Myliobatis being infected by rhinebothriideans in the SWA.

Of particular interest is P. myliobatidis, originally described as a member of the family Phyllobothriidae, included in the polyphyletic order “Tetraphyllidea,” parasitizing the Southern eagle ray, M. goodei, near the Río de la Plata estuary. Phyllobothrium myliobatidis exhibits, among other morphological features, a scolex composed of four bothridia with marginal loculi, a cephalic peduncle, and a genital pore located in the anterior third of proglottid (Brooks et al. 1981). Subsequently, Ruhnke (2011) suggested that this species should be placed in the order Rhinebothriidea since, like Anindobothrium, its members also possess bothridial stalks, a diagnostic morphological criterion for the order. Similarities in morphology and host association between P. myliobatidis and the specimens recently collected along the SWA cast doubts about its actual generic and ordinal placement. Consequently, in addition to the new species herein described, a new combination is proposed based on morphological characters, and P. myliobatidis is transferred to the genus Anindobothrium in anticipation of eventual new material to be included in future molecular phylogenetic studies.

Materials and methods

Tapeworms and host collection

Cestodes examined in this study were collected from the spiral intestines of seven individuals of M. goodei (Myliobatiformes: Myliobatidae) caught from different localities along the coast of the Argentine Sea. Two specimens were caught off Puerto Quequén, Buenos Aires Province, at 38°53.00'S, 58°27.00'W in July 2001 (assigned unique host number VIPQ-052) and January 2018 (GGPQ-115), and two specimens were caught off Balneario San Cayetano at 38°54.01'S, 59°12.02'W, Buenos Aires Province, in February 2018 (AGPQ-001, AGPQ-024), all by commercial trawlers (Fig. 1). One specimen was caught in San Matías Gulf, Río Negro Province, at 41°11.00'S, 64°03.26'W in March 2011 (PD3-155) and two were caught off Bahía San Blas, Buenos Aires Province, at 40°42.92'S, 62°00.58'W in April 2013 (PD7-512, PD7-514) with bottom trawls on board the RV Puerto Deseado (CONICET). Additional five specimens of M. goodei were also examined, but no rhinebothriidean cestodes were recovered. Three were caught off Balneario San Cayetano, Buenos Aires Province, at 38°54.01'S, 59°12.02'W in February 2018, and two specimens were caught off Puerto Quequén, Buenos Aires Province, at 38°53.00'S, 58°27.00'W in December 2019 also by commercial trawlers. Cestode specimens included in this study were recovered from the spiral intestines using a stereomicroscope. All tapeworms were removed from the spiral intestine of their respective host and relaxed in seawater. A subset of specimens was fixed in 10% formalin and transferred to 70% ethanol for long-term storage; a total of two specimens were fixed in ethanol and stored at -20 ºC.

Figure 1. 

Sampling sites of the southern eagle rays, Myliobatis goodei, examined during the present study. Ad, Anindobothrium danielae sp. nov.; Am, Anindobothrium myliobatidis comb. nov. Red circles, hosts examined in this study; black circles, hosts examined in Brooks et al. (1981).

Morphological examination of cestodes

The specimens prepared for light microscopy (permanent mounts) were hydrated in a graded ethanol series, stained with Harris’ haematoxylin, dehydrated in a graded ethanol series, cleared in methyl salicylate, and mounted in Canada balsam. A single bothridium was removed from the scolex of five tapeworms, observed in glycerine (non-permanent mount), and posteriorly included in the permanent mount.

One bothridium and the terminal portion of three strobilae were embedded in paraffin and serially cross-sectioned at a thickness of 7 micrometers (μm). Histological sections were stained with Harris' haematoxylin, counterstained with eosin, and mounted in Canada balsam. Whole mounts, non-permanent mounts, and histological sections were measured using an Olympus BX 51 compound microscope. Drawings were made with the aid of a drawing tube attached to the Olympus BX 51 compound microscope. All measurements of reproductive structures were taken from mature proglottids in which the vas deferens was not sperm-filled. Measurements are expressed as the range, followed in parentheses by the mean, standard deviation (when n ≥ 3), and the number of worms from which the measurements were taken (n). All measurements are in µm unless otherwise stated.

Worms prepared for scanning electron microscopy (SEM) were hydrated in a graded ethanol series, post-fixed in 1% osmium tetroxide overnight at room temperature, dehydrated in a graded ethanol series, and dried using hexamethyldisilazane. After drying, the specimens were mounted on stubs with carbon tape, coated with c. 40 nm of gold/palladium in a Thermo VG Scientific Polaron SC 7630, and examined in either a Zeiss GeminiSEM 360 or a Carl Zeiss NTS-SUPRA 40 scanning electron microscope.

Molecular methods and phylogenetic analysis

Total genomic DNA was extracted from two cestode specimens using PURO-Genomic DNA produced by PB-L (Productos Bio-Lógicos, Argentina), according to the manufacturer’s protocol. The D1–D3 region of the nuclear large subunit ribosomal gene (28S rDNA) was amplified by PCR using the forward primer LSU-5 (5′-TAG GTC GAC CCG CTG AAYTTA AGC A-3′) and the reverse primer 1500R (5′-GCT ATC CTG AGG GAA ACT TCG-3′) (Olson et al. 2003). The reactions were carried out with a Mastercycler thermocycler (Eppendorf) in a 50 μL reaction mixture containing 25 μL of PB-L master mix (Productos Bio-Lógicos, Argentina), 0.4 μM of each forward and reverse primer, and 6 μL of the template DNA. The PCR cycling conditions were: 3 min denaturing at 94 °C; 45 cycles of 30 s at 94 °C, 30 s at 50 °C, 1 min 45 s at 72 °C; followed by a final extension period of 2 min at 72 °C.

Sample sequencing was carried out in a specialized laboratory (Macrogen, Korea). Sequences were assembled using the platform Geneious 5.0.4. In addition to the new sequences reported in the present study, the phylogenetic analysis included known sequences of 29 species of Rhinebothriidea obtained from GenBank, which are representatives of the six families currently included in the order (Ruhnke et al. 2015; Trevisan et al. 2017; Herzog et al. 2023) (Table 1). Additional sequences obtained from GenBank of nine phyllobothriidean species with scolex morphology similar to specimens collected from M. goodei (flat or foliose bothridia with marginal loculi) were also included in the phylogenetic analysis (Table 1). Two outgroup species of the orders Litobothriidea and Trypanorhyncha were also included (Table 1). Taxa names, GenBank accession numbers for 28S, voucher accession numbers, host, and sources are given for the 40 species in Table 1.

Table 1.
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Cestodes included in the phylogenetic analysis.

Cestode species Host Order Rhinebothriidean family No. GenBank 28S No. voucher Source
Dollfusiella tenuispinis (Linton, 1890)* Hypanus sabinus TR NA DQ642796 BMNH 2008.5.21.2 Olson et al. (2010)
Litobothrium amplifica (Kurochkin and Slankis, 1973)* Alopias pelagicus LI NA KF685906 LRP8279 Caira et al. (2014)
Anindobothrium anacolum (Brooks, 1977) Styracura schmardae RH Anindobothriidae MF920345 MZUSP 7778 Trevisan et al. (2017)
Anindobothrium carrioni Trevisan, Primon & Marques, 2017 Styracura pacifica RH Anindobothriidae MF920342 MZUSP 7785 Trevisan et al. (2017)
Anindobothrium danielae sp. nov. Myliobatis goodei RH Anindobothriidae PQ346666 MACN-Pa 801/1 Present study
PQ346665 MACN-Pa 801/2
Anindobothrium inexpectatum Trevisan, Primon & Marques, 2017 Styracura schmardae RH Anindobothriidae MF920353 MZUSP 7767 Trevisan et al. (2017)
Anindobothrium lisae Marques, Brooks & Lasso, 2001 Potamotrygon orbignyi RH Anindobothriidae MF920362 MZUSP 7782 Trevisan et al. (2017)
Anthocephalum alicae Ruhnke, 1994 Hypanus americanus RH Anthocephaliidae KM658205 LRP 8508 Ruhnke et al. (2015)
Anthocephalum healyae Ruhnke, Caira & Cox, 2015 Neotrygon kuhlii RH Anthocephaliidae KM658200 LRP 8512 Ruhnke et al. (2015)
Barbeaucestus ralickiae Caira, Healy, Marques & Jensen, 2017 Taenyura lima RH Anthocephaliidae FJ177108 LRP3922 (CH35) Healy et al. (2009)
Barbeaucestus jockuschae Caira, Healy, Marques & Jensen, 2017 Neotrygon kuhlii RH Anthocephaliidae FJ177109 LRP3894 (CH3) Healy et al. (2009)
Divaricobothrium tribelum Caira, Healy, Marques & Jensen, 2017 Maculabatis cf. gerrardi RH Anthocephaliidae FJ177107 LRP3902 (CH11) Healy et al. (2009)
Echeneibothrium sp. 1 Rostroraja velezi RH Echeneibothriidae FJ177098 LRP4217 (TE94) Healy et al. (2009)
Echeneibothrium sp. 2 Raja miraletus RH Echeneibothriidae KF685876 LRP8312 Caira et al. (2014)
Escherbothrium cielochae Bueno, Trevisan & Caira, 2024 Urotrygon rogersi RH Escherbothriidae KM658197 LRP 8519 Ruhnke et al. (2015)
Mixobothrium healyae Herzog, Caira & Jensen, 2023 Pristis clavata RH Mixobothriidae FJ177119 LRP4220 (CH26) Healy et al. (2009)
Mixobothrium carinesmarinei Herzog, Caira & Jensen, 2023 Pristis pristis RH Mixobothriidae OQ429320 LRP10963 Herzog et al. (2023)
Mixobothrium bengalense Herzog, Caira & Jensen, 2023 Glaucostegus obtusus RH Mixobothriidae OQ429316 LRP10970 Herzog et al. (2023)
Pseudanthobothrium sp. Leucoraja erinacea RH Echeneibothriidae KF685750 LRP8324 Caira et al. (2014)
Rhabdotobothrium anterophallum Campbell, 1975 Mobula hypostoma RH Rhinebothriidae AF286961 BMNH 2001.1.31.3-4 Olson et al. (2001)
Rhinebothrium sp. Maculabatis pastinacoides RH Rhinebothriidae FJ177121 LRP3903 (CH12) Healy et al. (2009)
Rhinebothrium megacanthophallus Healy, 2006 Urogymnus polylepis RH Rhinebothriidae FJ177120 LRP3901 (CH10) Healy et al. (2009)
Rhinebothroides sp. Potamotrygon wallacei RH Rhinebothriidae MF920365 MZUSP 7792 Trevisan et al. (2017)
Rhodobothrium paucitesticulare Mayes & Brooks, 1981 Rhinoptera bonasus RH Rhinebothriidae FJ177100 LRP4216 (TE61) Healy et al. (2009)
Scalithrium sp. Hypanus longus RH Rhinebothriidae KF685878 LRP8333 Caira et al. (2014)
Semiorbiseptum yakiae (Franzese, Montes, Shumabukuro & Arredondo, 2024) Sympterygia bonapartii RH Escherbothriidae OR791403 MACN-Pa 785/4 Franzese et al. (2024)
Spongiobothrium sp. Rhynchobatus cf. australiae RH Rhinebothriidae FJ177134 LRP3919 (CH32) Healy et al. (2009)
Stillabothrium amuletum (Butler, 1987) Glaucostegus typus RH Escherbothriidae FJ177117 LRP 3917 (CH-30) Healy et al. (2009)
Stillabothrium cadenati (Euzet, 1954) Zanobatus schoenleinii RH Escherbothriidae FJ177110 LRP3924 (CH37) Healy et al. (2009)
Stillabothrium davidcynthiaorum Daigler & Reyda, 2016 Brevitrygon walga RH Escherbothriidae FJ177116 LRP3926 (CH45) Healy et al. (2009)
Sungaicestus kinabatanganensis (Healy, 2006) Urogymnus polylepis RH Anthocephaliidae FJ177118 LRP3900 (CH9) Healy et al. (2009)
Chimaerocestos sp. Rhinochimaera pacifica PH NA KF685758 LRP8303 Caira et al. (2014)
KF685882 LRP8348
Rockacestus carvajali Caira, Bueno & Jensen, 2021 Dipturus chilensis PH NA MW419973 LRP8913 Caira et al. (2021)
Rockacestus conchai Caira, Bueno & Jensen, 2021 Bathyraja albomaculata PH NA MW419959 LRP10324 Caira et al. (2021)
Scyphophyllidium guariticus (Marques, Brooks & Lasso, 2001) Paratrygon aiereba PH NA KF685888 LRP8286 Caira et al. (2014)
Scyphophyllidium janineae (Ruhnke, Healy & Shapero, 2006) Hemipristis elongata PH NA HQ680625 QM G 231309–14 Cutmore et al. (2011)
Scyphophyllidium kirstenae (Ruhnke, Healy & Shapero, 2006) Hemigaleus microstoma PH NA KC505626 LRP7962 Ruhnke and Workman (2013)
Scyphophyllidium orectolobi (Butler, 1987) Orectolobus maculatus PH NA MG008940 – Cutmore et al. (2017)
Scyphophyllidium randyi (Ruhnke, Caira & Carpenter, 2006) Chiloscyllium hasseltii PH NA KF685767 LRP8318 Caira et al. (2014)
Scyphophyllidium tyleri (Ruhnke, Caira & Carpenter, 2006) Chiloscyllium punctatum PH NA KF685890 LRP8315 Caira et al. (2014)

Outgroup species: LI, Litobothriidea; NA, not apply; PH, Phyllobothriidea; RH, Rhinebothriidea; TR, Trypanorhyncha.

Sequences were aligned using the online version of MAFFT v.7 (Katoh et al. 2019). Ambiguously aligned and hypervariable regions in the 28S dataset were removed with Gblocks online version 0.91b (Talavera and Castresana 2007), according to a secondary structure model, with the parameter settings of a less stringent selection (allowing smaller final blocks, gap positions within the final blocks, and less strict flanking positions).

The substitution model was chosen under the Bayesian Information Criterion (BIC) in Jmodeltest 2.1 (Darriba et al. 2012). The appropriate nucleotide substitution model implemented for the 28S rDNA matrix was GTR+I+G.

The phylogenetic reconstruction was performed using Bayesian Inference (BI) through MrBayes v. 3.2.6 (Ronquist et al. 2012). Phylogenetic trees were constructed using two parallel analyses of Metropolis-Coupled Markov Chain Monte Carlo (MCMC) for 10 million generations each to estimate the posterior probability (PP) distribution. Topologies were sampled every 1,000 generations, and the average standard deviation of split frequencies was observed to be less than 0.01 at the end of the run, as suggested by Ronquist et al. (2012). The robustness of clades was assessed using Bayesian posterior probability (PP), where PP > 0.95 was considered strongly supported. A majority consensus tree with branch lengths was reconstructed for each run after discarding the first 25% of trees sampled as “burn-in.” The consensus tree for the 28S gene was visualized in FigTree 1.4.3 (Rambaut, 2014). Additionally, uncorrected p-distances were calculated using MEGA X (Kumar et al. 2018) with the bootstrap method (1000 replicates) and nucleotide substitution (transition + transversions). A uniform rate was applied, and gaps/missing data were considered as complete deletions. The newly generated sequences were submitted to GenBank.

Mapping and geographic sites

Geographic coordinates of type and additional localities of species of Anindobothrium are expressed in degrees and minutes. Estimated coordinates were assigned to records of specimens of Brooks et al. (1981) (Fig. 1). The geographic distribution of the Anindobothrium species was charted using the PANMAP software v.0.9.6 (Diepenbroek et al. 2002).

Terminology for microtriches, sources of valid zoological names, marine regionalization, and abbreviations of zoological names

Terminology for the morphology of microtriches follows Chervy (2009). Valid cestode names follow Caira et al. (2022). Valid host names follow Froese and Pauly (2024). Marine regionalization follows Spalding et al. (2007). Zoological abbreviations include A. for Anindobothrium and An. for Anthocephalum.

Material examined and museum abbreviations

The museum material examined includes light micrographs of the holotype (USNM No. 1371266) and three paratypes (USNM No. 1371267) of P. myliobatidis provided by Anna Phillips from the Smithsonian National Museum of Natural History–Invertebrate Zoology Collection, Washington, D.C., USA. Museum abbreviations are as follows: BMNH, History Museum, London, United Kingdom; LRP, Lawrence R. Penner Parasitology Collection, Department of Ecology and Evolutionary Biology, University of Connecticut, Storrs, Connecticut, USA; MACN-Pa, Museo Argentino de Ciencias Naturales, Colección Parasitológica, Buenos Aires, Argentina; MLP-He, Museo de La Plata, Colección de Invertebrados, La Plata, Argentina; MZUSP, Museu de Zoologia da Universidade de São Paulo, São Paulo, Brazil; QM G, Queensland Museum, Brisbane, Australia.

Results

Order Rhinebothriidea Healy, Caira, Jensen, Webster & Littlewood, 2009

Genus Anindobothrium Marques, Brooks & Lasso, 2001

Anindobothrium danielae sp. nov.

https://zoobank.org/68F38AF4-AF06-4F51-BFE4-1B35734CC36F
Figs 2, 3, 4, 5

Type material

Holotype • an entire, mature worm, off Bahía San Blas, Buenos Aires Province, Argentina (40°42.92'S, 62°00.58'W), 23 m, 2 Apr 2013, A. Menoret leg., MACN-Pa No. 798.

Paratypes • Three entire, mature worms, cross-section of 2 attached mature proglottids, same data as preceding, MACN-Pa No. 799/1–5 • Two entire, mature worms, same data as preceding, MLP-He No. 8136 • Six entire, mature worms, off Puerto Quequén, Buenos Aires Province (38°53.00'S, 58°27.00'W), 20 Jul 2001, V. A. Ivanov leg., MACN-Pa Nos. 795/1–2 and 800/1–4 • Two entire, mature worms, same data as preceding, MLP-He No. 8135 • One entire, mature worm, off Puerto Quequén, Buenos Aires Province, Argentina (38°53.00'S, 58°27.00'W), 22 Jan 2018, G. García Facal leg., MACN-Pa No. 796 • Three entire, mature worms, off Balneario San Cayetano, Buenos Aires Province, Argentina (38°54.01'S, 59°12.02'W), 37 m, 20 Feb 2018, G. García Facal & A. Menoret leg., MACN-Pa No. 797/1–3 • Cross-section of 1 scolex, off San Matías Gulf, Rio Negro Province, Argentina (41°11.00'S, 64°03.26'W), 73 m, 20 Mar 2011, A. Menoret & V. A. Ivanov leg., MACN-Pa No. 800.

Description

Based on 23 specimens in total: 18 entire, mature worms, cross-sections of 2 terminal mature proglottids, cross-sections of 1 scolex, and SEM of 2 worms. Worms euapolytic, 9.1–21.5 (14.2 ± 4.1, n = 17) mm long, 22–49 (32 ± 9, n = 17) acraspedote proglottids (Fig. 2A). Scolex 510–1,150 (775 ± 176, n = 17) long, 600–1,625 (1011 ± 309, n = 17) wide, composed of four stalked bothridia (Figs 2B, 3A). Bothridia orbicular-shaped, 272–850 (561 ± 213, n = 7) long, 272–1,000 (729 ± 296, n = 7) wide, with 101–121 (115 ± 10, n = 5) marginal loculi and anterior apical sucker. Apical sucker 40–90 (64 ± 19, n = 6) long, 48–100 (66 ± 21, n = 6) wide (Figs 2B, C, 3A–C). Transverse and longitudinal septa absent. Distal portion of marginal septa formed by marginal muscle bundles; proximal portion formed by radial muscles with proximal fibers ending adjacent to each other (Fig. 2D). Cephalic peduncle 410–1,400 (856 ± 292, n = 18) long, 100–300 (192 ± 63, n = 18) wide.

Figure 2. 

Light micrographs of Anindobothrium danielae sp. nov. from Myliobatis goodei. A. Entire, mature worm (holotype MACN-Pa No. 798); B. Scolex (paratype MACN-Pa No. 799/2); C. Bothridium (paratype MACN-Pa No. 799/1); D. Longitudinal section of bothridium (paratype MACN-Pa No. 800). Abbreviations: l - loculus; mmb - marginal muscle bundles; ms - marginal septum; rm - radial muscle. Arrowhead indicates the apical sucker.

Figure 3. 

Scanning electron micrographs of Anindobothrium danielae sp. nov. from Myliobatis goodei. A. Scolex, small letters indicate locations of detail shown in B–G; B. Bothridial apical sucker; C. Bothridial marginal loculi; D. Detail of apex surface with acicular filitriches; E. Detail of distal bothridial surface with capilliform filitriches and coniform spinitriches; F. Detail of proximal bothridial surface of marginal loculi with acicular filitriches; arrowheads indicate cilia; G. Detail of cephalic peduncle surface with capilliform filitriches.

Apex of scolex proper covered with acicular filitriches (Fig. 3D). Proximal bothridial surface covered with acicular filitriches, cilia present in marginal loculi (Fig. 3F). Distal surface covered with capilliform filitriches and coniform spinitriches (Fig. 3E). Surface of bothridial stalks covered with acicular filitriches. Cephalic peduncle and anterior portion of strobila covered with capilliform filitriches (Fig. 3G).

Immature proglottid, initially wider than long, becoming longer than wide with maturity. Most terminal proglottids and some subterminal proglottids with sperm-filled vas deferens. Mature proglottids without sperm-filled vas deferens 1,080–2,150 (1,491 ± 269, n = 18) long, 315–600 (423 ± 103, n = 18) wide, length to width ratio 2.3–5.3 (3.7 ± 0.9, n = 18): 1 (Figs 2A, 4A). Terminal mature proglottids with sperm-filled vas deferens 1,525–2,675 (2,013 ± 312, n = 15) long, 300–620 (427 ± 107, n = 15) wide, length to width ratio 3.1–6.8 (4.9 ± 1.0, n = 15): 1 (Figs 2A, 4B), mature proglottids 2–7 (4 ± 1, n = 18) in number per worm (Fig. 2A). Gravid proglottids not observed.

Figure 4. 

Line drawings of Anindobothrium danielae sp. nov. from Myliobatis goodei. A. Subterminal mature proglottid (holotype MACN-Pa No. 798); B. Terminal mature proglottid with sperm-filled vas deferens (holotype MACN-Pa No. 798); C. Detail of terminal genitalia of subterminal proglottid, ventral view, (paratype MACN-Pa No. 795/4).

Testes round to oval, 52–83 (68 ± 8, n = 17) long, 39–68 (55 ± 8, n = 17) wide, 76–115 (97 ± 11, n = 17) in number, 9–19 (13 ± 3, n = 17) preporal, 24–38 (32 ± 4, n = 17) postporal, 41–64 (51 ± 7, n = 17) aporal, arranged anteroposteriorly in 4 regular columns, 2 layers deep in cross-section; each column extending from anterior margin of proglottid reaching anterior margin of ovary (Figs 4A, B, 5A–C). Cirrus sac pyriform in anterior third of proglottid, bent posteriorly, 103–200 (152 ± 32, n = 17) long, 43–135 (93 ± 26, n = 17) wide, containing coiled cirrus; cirrus covered with spinitriches (Figs 4C, 5C, F). Vas deferens coiled, extending posteriorly at level of cirrus sac to near anterior margin of ovary, entering cirrus sac at anteromedial margin (Figs 4A–C, 5C, F).

Figure 5. 

Light micrographs of cross-sections of mature proglottids of Anindobothrium danielae sp. nov. from Myliobatis goodei. A. Testes anterior to the cirrus sac (paratype MACN-Pa No. 799/3); B. At the level of the vagina (paratype MACN-Pa No. 799/4); C. At the level of the cirrus sac; arrowhead indicates the entrance of the vas deferens to the cirrus sac (paratype MACN-Pa No. 799/3); D. At the level of the ovarian isthmus (paratype MACN-Pa No. 799/3); E. Detail of the vagina showing vaginal sphincter and pigmented cells (paratype MACN-Pa No. 799/3); F. Detail of the cirrus sac showing armed cirrus; arrowhead indicates the entrance of the vas deferens to the cirrus sac (paratype MACN-Pa No. 799/3). Abbreviations: ci -cirrus; cs - cirrus sac; do - dorsal osmoregulatory duct; lm - longitudinal muscle; ov - ovary; pc - pigmented cells; sp - sphincter; ts - testes; ut - uterus; vd - vas deferens; vf - vitelline follicle; vg - vagina; vo - ventral osmoregulatory duct.

Vagina thick-walled, extending anteriorly from ootype, then running along midline of proglottid to anterior margin of cirrus sac, and laterally opening into genital atrium anterior to cirrus, vaginal sphincter present (Figs 4C, 5B, E). Ovary H-shaped in frontal view, poral lobe 185–450 (314 ± 66, n = 17) long, aporal lobe 218–440 (310 ± 61, n = 17) long, 170–380 (266 ± 57, n = 17) wide at isthmus, tetra-lobed in cross-section (Figs 4A, B, 5D). Mehlis’ gland posterior to ovarian isthmus, 65–115 (97 ± 16, n = 16) long, 70–120 (90 ± 17, n = 17) wide. Vitellarium follicular; vitelline follicles oval, 10–22 (15 ± 4, n = 16) long, 12–42 (27 ± 7, n = 16) wide, in 2 dorsal and 2 ventral columns on each lateral margin of proglottid, lateral to testes, extending from near anterior margin of proglottid to posterior margin of proglottid, partly interrupted by terminal genitalia and uninterrupted by ovary (Figs 4A–C, 5A–F). Uterus saccate, medial, and ventral, extending from the level of the genital atrium to ovarian isthmus (Fig. 4A–C). Osmoregulatory ducts 4, 1 dorsal, and 1 ventral pair on each lateral margin of proglottid, dorsal pair larger than ventral pair (Figs 4C, 5A–C).

Host

Myliobatis goodei Garman, 1855, Southern eagle ray (Myliobatiformes: Myliobatidae) (type host). Site of infection: spiral intestine. Prevalence of infection: 58% (7 hosts infected out of 12 examined).

Sequence data

GenBank accession numbers PQ346665, PQ346666; 2 hologenophores MACN-Pa No. 801/1-2.

Etymology

This species is named after Daniela Barbieri, the first author’s dear friend, in appreciation for her continued support and enthusiasm for science.

Distribution

Anindobothrium danielae sp. nov. occurs mainly along coastal waters off Buenos Aires Province, at depths of <100 m in the Warm Temperate SWA Marine Province.

Remarks

Anindobothrium danielae sp. nov. can easily be distinguished from A. anacolum, A. carrioni, and A. inexpectatum by the morphology of the bothridia. The new species has orbicular-shaped bothridia without longitudinal and transverse septa, whereas the three congeners exhibit ellipsoid-shaped bothridia with longitudinal and transverse septa (Figs 2C, 3A, 6). Anindobothrium danielae sp. nov. is different from A. anacolum, A. carrioni, A. inexpectatum, and A. lisae by having more testes per proglottid (76–115 vs. 24–50, 21–31, 23–44, and 30–72, respectively) along with the presence of two rows of testes arranged dorsoventrally instead of one. Furthermore, A. danielae sp. nov. possesses similar orbicular-shaped bothridia as A. lisae but can be distinguished from the freshwater species by the number of marginal loculi (101–121 vs. 40–58) and ovary length (59–159 vs. 185–450).

Figure 6. 

Schematic representation of the bothridia shape in the genus Anindobothrium. A. Ellipsoid-shaped bothridia, species subset 1: A. anacolum, A. carrioni, and A. inexpectatum; B. Orbicular-shaped bothridia, species subset 2: A. danielae sp. nov., A. lisae, and A. myliobatidis comb. nov.

Molecular and phylogenetic analysis

The phylogeny obtained in this study placed the two specimens recovered from M. goodei as members of the genus Anindobothrium (Fig. 7). These specimens were conspecific (no genetic variation) and corresponded to a new species of Anindobothrium (Suppl. material 1, Fig. 7).

Figure 7. 

Phylogram resulting from Bayesian inference of the 28S rDNA from the species of cestodes indicated in Table 1. Branch support values indicate posterior probabilities. Taxon labels include cestode species followed by GenBank accession numbers. New sequences are indicated in bold.

The genetic distance among 28S sequences varied between 0.00 and 0.31 (Suppl. material 1). Within Anindobothrium, there was a large clade, with support of 1.00, constituted by most of the species that comprise the genus (i.e., A. anacolum, A. carrioni, A. lisae, and A. inexpectatum), and that parasitize batoids belonging to the family Potamotrygonidae (Fig. 7). The sister taxon of this clade was A. danielae sp. nov., with a genetic distance of 0.08 (Suppl. material 1), associated with a new host family for Anindobothrium (Myliobatidae) (Fig. 7).

Anindobothrium myliobatidis (Brooks, Mayes & Thorson, 1981), comb. nov.

Fig. 8

Phyllobothrium myliobatidis Brooks, Mayes & Thorson, 1981 (Syn.).

Type material

Holotype • an entire, mature worm, Río de la Plata estuary near Montevideo, Uruguay, July 1979, T.B. Thorson leg., USNM No. 1371266.

Paratypes • three entire, mature worms, same data as preceding, USNM No. 1371267.

Amended diagnosis

Based on type material (holotype USNM No. 1371266 and 3 paratypes USNM No. 1371267). Worms euapolytic, 10.56–18.48 (14.04 ± 3.67, n = 4) mm long (Fig. 8A), proglottids acraspedote. Scolex 1,012–1,960 (1,385 ± 505, n = 3) long, 1,228–2,015 (1,517 ± 433, n = 3) wide, composed of four stalked bothridia (Fig. 8A, C, E). Bothridia orbicular-shaped, with marginal loculi and anterior apical sucker. Transverse and longitudinal septa absent (Fig. 8C, E). Cephalic peduncle present. Immature proglottid, initially wider than long, becoming longer than wide with maturity. Most terminal proglottids and some subterminal proglottids with sperm-filled vas deferens (Fig. 8A, B). Terminal mature proglottids 1,597–2,670 (2,333 ± 564, n = 3) long, 345–412 (387 ± 36, n = 3) wide, length to width ratio 4.6–6.6 (5.7 ± 1.0, n = 3):1. Testes round to oval, arranged anteroposteriorly in several columns, 2 layers deep; each column extending from anterior margin of proglottid reaching anterior margin of ovary (Fig. 8D). Vitellarium follicular; vitelline follicles oval, in 2 dorsal and 2 ventral columns on each lateral margin of proglottid, lateral to testes, extending from near anterior margin of proglottid to posterior margin of proglottid, partly interrupted at level of terminal genitalia, uninterrupted by ovary (Fig. 8A, B, D).

Figure 8. 

Light micrographs of Anindobothrium myliobatidis comb. nov. from Myliobatis goodei. A. Entire worm (holotype USNM No. 1371266); B. Terminal proglottid (holotype USNM No. 1371266); C. Scolex (holotype USNM No. 1371266); arrowhead indicates apical sucker; D. Mature proglottid (paratype USNM No. 1371267) showing testes arranged in two layers deep; arrowheads indicate testes in the top layer and circles indicate those in the deeper layer; E. Scolex (paratype USNM No. 1371267); arrowhead indicates apical sucker.

Host

Myliobatis goodei Garman, 1855, Southern eagle ray (Myliobatiformes: Myliobatidae). Site of infection: spiral intestine.

Distribution

Anindobothrium myliobatidis comb. nov. is known from off the estuary of Río de La Plata near Montevideo, Uruguay, in the Warm Temperate SWA Marine Province.

Remarks

Phyllobothrium myliobatidis of Brooks et al. (1981) is herein transferred to Anindobothrium on the basis of the type material examined in this study. The amended diagnosis includes modifications to accommodate the new observations of the morphological features, such as a scolex consisting of four orbicular-shaped bothridia with marginal loculi and an apical sucker, a terminal proglottid with a sperm-filled vas deferens, a reduced number of testes, and testes distributed in two layers deep (Fig. 8). Additionally, some morphological measurements (i.e., total length of tapeworms, size of the scolex, and terminal proglottids) were provided for the first time. Therefore, A. myliobatidis comb. nov. can be distinguished from its congeners as follows: A. anacolum, A. carrioni, and A. inexpectatum belong to subset 1 by having ellipsoid-shaped bothridia with longitudinal and transverse septa, whereas A. myliobatidis comb. nov. belongs to subset 2 by possessing orbicular-shaped bothridia with marginal loculi (Fig. 6A, B). Anindobothrium myliobatidis comb. nov. is also different from those three species along with A. danielae sp. nov. and A. lisae by possessing more proglottids per worm (50–75 vs. 8–33, 20–33, 23–49, 15–39, and 7–24, respectively) and more testes per proglottid (122–150 vs. 24–50, 21–31, 23–44, 76–115, and 30–72, respectively). Anindobothrium myliobatidis comb. nov. further differs from A. danielae sp. nov. in the number of marginal loculi (83–90 vs. 101–121).

Update of generic diagnosis, valid species, distribution, and hosts of Anindobothrium

Worms euapolytic. Ellipsoid-shaped bothridia with longitudinal and transverse septa (species subset 1) (Fig. 6A) or orbicular-shaped bothridia only with marginal loculi (species subset 2) (Fig. 6B). Microtriches on scolex surfaces include filitriches and gladiate or coniform spinitriches. Testes dorsoventrally distributed in 1–2 layers. Terminal proglottid typically with sperm-filled vas deferens. Vaginal sphincter present. Vitelline follicles partly or fully interrupted by terminal genitalia, interrupted or uninterrupted by ovary.

Type species. Anindobothrium anacolum (Brooks, 1977).

Additional species. Anindobothrium carrioni Trevisan, Primon & Marques, 2017, Anindobothrium danielae sp. nov., Anindobothrium inexpectatum Trevisan, Primon & Marques, 2017, Anindobothrium lisae Marques, Brooks & Lasso, 2001, and Anindobothrium myliobatidis (Brooks, Mayes & Thorson, 1981), comb. nov.

Geographic distribution. Marine realms including Tropical Eastern Pacific, Tropical Atlantic, and Temperate South America; also covering freshwater rivers in South America.

Hosts. Stingrays of the families Myliobatidae and Potamotrygonidae.

Discussion

The study of the rhinebothriidean specimens from Myliobatis caught along the coast of Argentina has led to the identification of a new species. Anindobothrium danielae sp. nov. parasitizes M. goodei in waters off Bahía San Blas and other localities off Buenos Aires Province in the SWA. This species is unique in a combination of morphological features, including the bothridial shape and loculi configuration; the number of marginal loculi, proglottids, and testes; and the distribution of testes. In addition, molecular support is also provided to recognize A. danielae sp. nov. as a new member of the genus Anindobothrium.

The examination of the type material of A. myliobatidis comb. nov. allowed us to verify the presence of four stalked orbicular-shaped bothridia, with marginal loculi and apical sucker, and proglottids with sperm-filled vas deferens, among other characters, providing an appropriate generic and ordinal placement for this species from the Southern eagle ray. Previously, Ruhnke (2011) treated this species as incertae sedis under the name P. myliobatidis, noting that its morphological features were not consistent with the generic diagnosis of Phyllobothrium (e.g., bothridia not foliose and posteriorly bifid), and, in fact, he suggested it was potentially a member of Anthocephalum Linton, 1890, a genus of the order Rhinebothriidea. The presence of bothridial stalks, marginal loculi, and a posteriorly recurved cirrus sac indicated a potential relationship between P. myliobatidis and Anthocephalum. However, the genital pore position in the anterior third of the proglottid and the apparent lack of apical sucker raised doubts about the generic placement at the time (Ruhnke 2011).

The presence of A. danielae sp. nov. and A. myliobatidis comb. nov. in M. goodei in the studied area not only allowed us to redefine the diagnosis of Anindobothrium but also to extend the geographical distribution of the genus to include coastal waters off Argentina in the SWA.

Morphology

Two subsets of members of Anindobothrium can be considered based on the morphology of the bothridia. Anindobothrium anacolum, A. carrioni, and A. inexpectatum have bothridia ellipsoid-shaped (longer than wide) characterized by a distal surface with longitudinal and transverse septa (subset 1), whereas A. danielae sp. nov., A. lisae, and A. myliobatidis comb. nov. are characterized by orbicular-shaped bothridia (wider than long) with only marginal loculi lacking transverse and longitudinal septa (subset 2) (Fig. 6). Additionally, the presence of an apical sucker is only evident in the species belonging to subset 2, in which it is typically rounded in shape and highly muscular, being easily distinguished from the remaining surface of the bothridia. (Figs 2B, C, 3B, 8C, E in this study; see fig. 10A, B in Trevisan et al. 2017).

In contrast, the species of Anindobothrium from subset 1, despite being described as having an apical sucker by Trevisan et al. (2017), appear to have an anteriormost loculus rather than an apical sucker sensu Caira et al. (1999), who essentially defined these structures based on the shape of their posterior margin, being straight for the first and rounded for the latter. Also, Bueno (2018) considered the apical suckers typically highly muscular and round in shape and the loculi weakly muscular and generally oblong. Thus, A. carrioni is, in fact, characterized by an anteriormost loculus as it is weakly muscular and oblong (see fig. 13A in Trevisan et al. 2017), while the remaining species of subset 1 seem to exhibit a weak musculature delimiting an anteriormost loculus (see figs 7A, 11A in Trevisan et al. 2017). Therefore, these two bothridial structures should be defined following objective criteria such as the musculature arrangement or the thickness of the muscular wall, as suggested by Franzese and Ivanov (2020) and Franzese et al. (2022), given that the shape of the posterior margin in both structures could be altered by fixation and processing methods. It would be important to study the muscular configuration of the anterior portion of the bothridia of the species of Anindobothrium to verify if the presence of the anteriormost loculus is only restricted to the species of subset 1 and the apical sucker is only present in species of subset 2.

The detailed study of the muscular morphology of the marginal septa of A. danielae sp. nov. is herein presented and is the first to be carried out within the genus. The disposition of the musculature in this type of septa has previously been studied in two species of Rhinebothriidea: Anthocephalum duszynskii Ruhnke, 1994, and Echeneibothrium williamsi Carvajal & Dailey, 1975 (see Healy 2006a; Franzese et al. 2022). Anindobothrium danielae sp. nov. shows the same muscular morphology of the marginal septa and a similar configuration of the marginal loculi than An. duszynskii (distal portion of septa with marginal muscle bundles and underlying radial muscles with proximal fibers ending adjacent to each other and small and numerous marginal loculi), differing from E. williamsi (distal portion of septa with marginal muscle bundles and underlying parallel radial muscles, and larger and less numerous marginal loculi), making it difficult to know if these loculi are homologous structures among the three species. It would be interesting to study the muscular morphology of the marginal septa among the species of Anindobothrium of subset 2, as well as in other rhinebothriideans with similar bothridial configuration, as in the case of members of Alveobothrium Boudaya, Neifar & Euzet, 2018, Anthocephalum, Biotobothrium Tan, Zhou & Yang, 2009, Cairaeanthus Kornyushin & Polyakova, 2012, and Clydonobothrium Euzet, 1959 (see Franzese and Ivanov 2020).

The tegumental study revealed that A. danielae sp. nov. has filitriches covering all the studied surfaces and coniform spinitriches only restricted to the distal bothridial surface. In contrast, in the four species previously described (i.e., A. anacolum, A. carrioni, A. inexpectatum, and A. lisae), the proximal surface is covered with both acicular filitriches and gladiate spinitriches. Also, the distal bothridial surface of A. anacolum, A. inexpectatum, and A. carrioni was described by having gladiate spinitriches and filitriches (see Trevisan et al. 2017). However, these species seem to have coniform spinitriches as they are like a cone with round bases (see figs 8F, G, 12F, G, 14F in Trevisan et al. 2017), such as those exhibited by A. danielae sp. nov. (see Fig. 3E). Therefore, the scolex surface of Anindobothrium is essentially characterized by having filitriches on the entire surface, with gladiate spinitriches covering the proximal bothridial surface and gladiate or coniform spinitriches in the distal bothridial surface.

The surface of the scolex of A. danielae sp. nov. also exhibits cilia (Fig. 3F). These structures with sensory functions have been detected in the scolex of several genera of rhinebothriideans including Anthocephalum, Crassuseptum Eyring, Healy & Reyda, 2012; Echeneibothrium van Beneden, 1850; Rhinebothrium Linton, 1890; and Stillabothrium Healy & Reyda, 2016 (Healy 2006b; Eyring et al. 2012; Reyda et al. 2016; Herzog and Jensen 2018; Franzese et al. 2022). Although Trevisan et al. (2017) did not mention the presence of cilia in any of the species of Anindobothrium, the distal bothridial surface of A. lisae is covered with spinitriches and cilia (see fig. 10F in Trevisan et al. 2017). Therefore, it would be interesting to verify if the third species of subset 2, A. myliobatidis comb. nov., also exhibits the microthrix pattern known so far, with spinitriches, coniform spinitriches, and cilia.

The tapeworms of Anindobothrium were considered apolytic by Trevisan et al. (2017), but this term has been used with subtle differences in different works (Franzese and Ivanov 2018). According to Caira et al. (1999), cestodes that retain mature proglottids in the strobila but never gravid proglottids are euapolytic. Since all known species of Anindobothrium have strobila with mature terminal proglottids, we consider that the genus is represented by euapolytic tapeworms.

In most species of Anindobothrium, the terminal proglottid has a voluminous vas deferens filled with sperm, testes restricted to the anterior portion of the proglottid with most of them already degenerated, and the vitelline follicles increasing in size, thickening the lateral bands, being quite different from the subterminal proglottid (Figs 4A, B). The exception to these observations is A. lisae, in which the terminal proglottid exhibits a vas deferens less voluminous and vitelline follicles smaller, delimiting narrower lateral bands, resembling the subterminal proglottid of their congeners.

The testes are arranged dorsoventrally in one layer in most species of Anindobothrium, while in A. danielae sp. nov. and A. myliobatidis comb. nov. they are distributed into two layers (Figs 4A, B, 5A–C, and Fig. 8D, respectively). The presence of a distinct seminal vesicle was included in the diagnosis of the Anindobothrium by Marques et al. (2001), but this feature was not mentioned in the amended diagnosis of the genus by Trevisan et al. (2017), not even in any of the species these authors discussed. We could not verify the presence of a seminal vesicle in the type material of A. myliobatidis comb. nov. due to the poor magnification of the images, nor was this character mentioned in the original description. So far, A. danielae sp. nov. is the only species in which the absence of this structure was effectively verified, based on whole mounts and cross-sections of several proglottids. Therefore, we include the testes arranged in 1–2 layers as a feature of the genus, but we do not consider the presence of a distinct seminal vesicle a diagnostic character of Anindobothrium.

Phylogeny

The molecular analysis based on 28S rDNA data unequivocally placed the two specimens recovered from M. goodei as members of the genus Anindobothrium (Fig. 7). The genetic distances observed concerning the rest of the species of the genus (Suppl. material 1) have allowed us to corroborate our morphological studies in which we considered these specimens as representatives of a new species of the genus.

Additionally, our phylogenetic hypothesis for Anindobothrium mirrors the phylogeny of their hosts (see Naylor et al. 2012), similar to the results of previous studies (Trevisan et al. 2017). However, our results suggest that there was no single codivergence event in the split between marine and freshwater lineages of Anindobothrium as proposed by Trevisan et al. (2017). Instead, there probably could have been a previous codivergence event that gave rise to a clade of marine species of Anindobothrium present in Myliobatidae and a sister group including marine and freshwater species parasitizing batoids of the family Potamotrygonidae (Fig. 7). Within the latter clade, the same codivergence referred by Trevisan et al. (2017) was observed in the present study, with the only freshwater species (A. lisae) as sister to a clade composed of the remaining species of Anindobothrium from marine potamotrygonids (i.e., A. anacolum, A. carrioni, and A. inexpectatum) (Fig. 7). However, we could not corroborate the grouping of species by oceans observed by Trevisan et al. (2017) (see fig. 16 in Trevisan et al. 2017), since in our study species distributed in different oceans (i.e., A. carrioni, A. inexpectatum) appeared as part of the same clade (Fig. 7).

The inclusion of molecular sequences of A. myliobatidis comb. nov. in future phylogenetic analyses is needed to verify the codivergence hypothesis proposed in the present study but also to validate the new combination herein considered, which was based on morphological characters only.

Host-parasite association and distribution

Prior to this study, the marine species of Anindobothrium were restricted to stingrays of the genus Styracura. Styracura schmardae (Werner, 1941), registered at the Caribbean Sea in the Tropical Atlantic, was found parasitized by A. anacolum and A. inexpectatum, whereas S. pacifica (Beebe & Tee-Van, 1941) off the Tropical Eastern Pacific was found infected with A. carrioni. Therefore, the presence of A. danielae sp. nov. and A. myliobatidis comb. nov. in the Southern eagle ray from off the Argentinean coast in Temperate South America represents the first report of batoids of the genus Myliobatis found parasitized by tapeworms of Anindobothrium.

To date, seven species of eagle stingrays of the genus Myliobatis have been reported from coastal waters of South America: M. californica Gill, 1865; M. chilensis Philippi, 1892; M. longirostris Apple & Fitch, 1964; and M. peruvianus Garman, 1913, from the Pacific; and M. freminvillei Lesueur, 1824; M. goodei, and M. ridens from the Atlantic (Bustamante et al. 2014; Cornejo et al. 2015; Calle-Morán and Béarez 2020; Froese and Pauly 2024). Among them, M. goodei hosts the greatest cestode species richness, including diphyllideans, lecanicephalideans, “tetraphyllideans,” trypanorhynchs, and the rhinebothriideans herein treated. Although M. californica, M. longirostris, and M. freminvillei have been reported as hosts of cestodes in North America (Mexico and USA) (see Caira et al. 2022), it appears that most species of Myliobatis in surrounding South American waters have not been surveyed for tapeworms. Particularly, we have not had the opportunity to study specimens of M. freminvillei so far, and only two individuals of M. ridens were examined prior to this study (Menoret et al. 2017). Given the great diversity of cestodes found in the Southern eagle ray in the studied area, we estimate that other species of Myliobatis can also be suitable hosts for new species of cestodes of the coasts of South America.

Finally, the results obtained in the present study allowed us to increase the number of species of Anindobothrium from eight to ten globally and to expand our knowledge of the rhinebothriideans and batoids of the family Myliobatidae association in the Southern Hemisphere.

Acknowledgments

We thank Gustavo Chiaramonte, who made laboratory facilities at the Estación Hidrobiológica Quequén, Museo Argentino de Ciencias Naturales-CONICET, available to us, and Sebastián Polimeni for fishing the Southern eagle rays at Puerto Quequén, Buenos Aires Province, Argentina. We also thank the Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET) for giving us the chance to work on board the research vessel “Puerto Deseado.” Special thanks are due to Anna Phillips from the Smithsonian National Museum of Natural History—Department of Invertebrate Zoology, Washington, D.C., USA, for providing us with digital micrographs of type material. This work was supported by the Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET) [grant numbers PIP 11220200101713CO, PIP 11220210100134CO] and Fondo para la Investigación Científica y Tecnológica (FONCyT) [grant numbers PICT-2021-I-INVI-00341, PICT-2020-SERIE A-01531, PICT-2020-SERIE A-00660, PICT-2016-3672]. This study was conducted under collecting permits No. 39 and No. 260 from the Dirección Provincial de Pesca-Ministerio de Asuntos Agrarios de la Provincia de Buenos Aires, Argentina.

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Supplementary material

Supplementary material 1 

Genetic divergence estimated through uncorrected p-distance of the 28S rDNA

Guillermina García Facal, Sebastián Franzese, Martín Miguel Montes, Adriana Menoret

Data type: xlsx

This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited.
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