Clinostominae Lühe, 1901

Simoes ˜, Mariana B., Alves, Philippe V., Lopez-Hern, Danimar, andez, Couto, Elimayke A., Moreira, Narcisa I. B. & Pinto, Hudson A., 2022, Size does not matter: molecular phylogeny reveals one of the largest trematodes from vertebrates, the enigmatic Ithyoclinostomum dimorphum, as a species of Clinostomum (Trematoda: Clinostomidae), International Journal for Parasitology: Parasites and Wildlife 19, pp. 84-88 : 87-88

Subfamily Clinostominae Lühe, 1901 .

(= Ithyoclinostominae Yamaguti, 1958 – new synonym).

Genus Clinostomum Leidy, 1856 .

Diagnosis: As in Kanev et al. (2002), updated (broader concept) by Caffara et al. (2019), but with the following change: Body middle-sized to very large (5–100 mm long), slender to stout, may be attenuated anteriorly. Space between ventral sucker and anteriormost vitelline follicles may be void of organs.

Clinostomum dimorphum ( Diesing, 1850) Braun,1901 .

(new synonym: Ithyoclinostomum dimorphum ).

Hosts: Hoplias malabaricus (Bloch, 1794) and Hoplias intermedius (Günther, 1864) .

Prevalence of infection: 1/1 (100%) for H. malabaricus , and 1/11 (9%) for H. intermedius .

Intensity of infection: 5 ( H. malabaricus ) and 1 ( H. intermedius ).

Site of infection: encysted at gill, heart and pericardial cavity ( H. malabaricus ), and liver ( H. intermedius ).

Locality: Doce River, municipalities of Colatina (19 ◦ 31 ′ 57.9"S; 40 ◦ 38 ′ 09.0"W) ( H. malabaricus ) and Baixo Guandu (19 ◦ 30 ′ 17.8"S; 41 ◦ 01 ′ 26.6"W ( H. intermedius ), State of Espirito Santo, Brazil.

Representative DNA sequences: Two nearly complete sequences of the ribosomal gene cluster ITS1-5.8S-ITS2 (length 970 bp ex H. malabaricus and 979 bp ex H. intermedius ; identical in their overlapping range); a partial sequence of the 28S gene (length 1253 bp ex H. malabaricus ); and two partial cox 1 sequences (length 666 bp ex both Hoplias species; 0.9% of nucleotide divergence).

Voucher material: Four whole-mounted metacercariae (2 hologenophores and 2 paragenophores) deposited at the Collection of Trematodes of the Universidade Federal de Minas Gerais (UFMG-TRE 125–126).

Data presented in this study reflects the complexity and problematic nature of the morphologically based classification involving clinostomids, complementing the recent study of Caffara et al. (2019) that molecularly confirmed the placement of the large-sized Clinostomoides brieni Dollfus, 1950 within Clinostomum ; besides the large size, C. brieni further differs from the ’typical’ Clinostomum species by having the genital pore post-testicular and the whole genital complex extremely close to the posterior end. These findings depict an opposing scenario to recent molecular phylogenetic studies ( Woodyard et al., 2017; Rosser et al., 2020) that have endorsed previous morphological arrangement for the family ( Kanev et al., 2002). Moreover, our data raise doubts concerning which morphological features are informative at higher-level classification schemes. Size, for instance, has been considered a major taxonomic criterion in most helminth classifications and was the main differential trait for the proposition of Ithyoclinostomum and Ithyoclinostominae ( Yamaguti, 1958; Kanev et al., 2002). However, despite the giant and unbelievable difference in body length between type species of the genus, Clinostomum complanatum (Rudolphi, 1814) and C. dimorphum (~ 5 mm vs up to 100 mm; more than 2000% larger), the phylogenetic analyses herein presented revealed there is no support for these species be considered as belonging to distinct clinostomid genera. To the best of our knowledge, such an enormous difference among congeneric trematodes, has never been reported for any other genus of trematodes, making the quote "looks can deceiving" well appropriated to describe our findings.

In our phylogenetic analyses, the recently described Ithyoclinostomum yamagutii presented as an early diverging and isolated lineage from other clinostomids, as previously shown by Briosio-Aguilar et al. (2019) and Rosser et al. (2020). On one hand, these authors assigned their specimens to Ithyoclinostomum primarily based on: the large body size, the position of the cirrus-sac (pre-testicular), the testes shape (deeply lobed), the position of gonads in the posterior fourth of the body, and the large free area (void of any internal organ) between the ventral sucker and anterior testis; these features are found in the type species of the genus, Ithyoclinostomum dimorphum , now C. dimorphum . On the other hand, they assertively comment that obtaining molecular data from the type species of Ithyoclinostomum is crucial either to confirm or reject their hypothesis as well as to assess the interrelationships among other clinostomid genera, which was done in this study. The phylogenies and the high molecular divergences verified between C. dimorphum and I. yamagutii (7.6% in 28S, 12.6% in ITS, and 18.6% in cox 1) reveal unequivocally these species are not congeneric. Based on the synonym between Clinostomum and Ithyoclinostomum required after our results, the taxon studied by Briosio-Aguilar et al. (2019) and Rosser et al. (2020) must be transferred to another genus, but for the sake of nomenclatural stability in the group, we provisionally retained the species in the non-natural Ithyoclinostomum [using quotation marks (’ Ithyoclinostomum ’ or ’ I.’) to refer to the genus] until key taxa (see below) are sequenced and compared with available data (see Table S1).

It is possible that ’ I.’ yamagutii may deserve a new clinostomid genus to accommodate it. However, given the complex scenario raised in the morphology-based taxonomy of Clinostomidae (traditionally used features such as body size and arrangement of the genital system were found unreliable for higher-level classification purposes), we chose not to erect a new genus for ’ I.’ yamagutii until molecular data on other clinostomid genera are available, especially for Clinostomatopsis sorbens (Braun, 1899) , the type and only species of the genus Clinostomatopsis Dollfus, 1932 ( Kanev et al., 2002). This clinostomid genus is the only one known from birds without molecular data available. Our conservative approach is also justified due to the morphological similarities among ’ I. ’ yamagutii and C. sorbens that include: gonads located toward the posterior extremity of the body and the presence of deeply lobed or irregular-shaped testes. Given the likely plesiomorphic/homoplastic nature of morphological traits otherwise used to differentiate subfamilies and genera (e.g., genital pore position and anterior extension of vitellaria — see Caffara et al., 2019; present results), we also opt by not to transfer ’ I.’ yamagutii to the genus Clinostomatopsis .

Despite the contribution of molecular phylogeny for proposing a more natural classification that reflects the evolution of the members of the family Clinostomidae , some key taxa (type genera and species) still need to be sequenced to test the traditional morphology-based classification system. For instance, in reptile clinostomids, O. incommodum was sequenced based on worms found in alligators from USA ( Woodyard et al., 2017). However, no molecular data is available for Odhneriotrema microcephala (Travassos, 1922) , the type species of the genus Odhneriotrema Travassos, 1928 , described from Brazil. Thus, despite morphological similarities between these species, the possibility they correspond to distinct genera cannot be ruled out. Such delay in the generation of molecular data is also verified for the genus Nephrocephalus Odhner, 1902 , found in African reptiles, which is the type genus of Nephrocephalinae Travassos, 1928 . Therefore, the phylogenetic position of its members in relation to the other clinostomids is unknown. A similar scenario can be found even at the species level, which can be evidenced by the case of Clinostomum marginatum (Rudolphi, 1819) . Despite the fact that this species has been sequenced from isolates obtained from birds and fish in North America ( Caffara et al., 2011; Rosser et al., 2017), its specific assignment should be confirmed once isolates from the type locality in Brazil, are sequenced ( Pinto et al., 2015; Montes et al., 2021).

Overall, the taxonomic issues raised in this study highlight the importance of the availability of molecular data for the type species of trematode genera described from South America. Since the XIX century, dozens of trematode genera were proposed from this continent, initially from material collected in Brazil by the naturalist Johann Natterer and described by European helminthologists. Later in the XX century, renowned trematode taxonomists such as Szidat, Travassos, Thatcher, and their disciples described several other trematode genera, especially in Brazil and Argentina ( Cribb and Bray, 2011). Unfortunately, most of the type species of these genera have not been sequenced so far. The lack of sequences for these species may be a reflection of limited access to

87

sequencing methodologies compared with developed countries. This bias is verified in different groups of trematodes ( Poulin and Jorge 2019), and despite advances verified in the last few years, most type-species of trematodes described from South America have not yet been sequenced. Such information is essential for more robust and natural classification and specially to support an inter- or transcontinental distribution of species assigned to the same genus.