Fernando P. L. Marques

Among enduringly associated parasitic taxa, tapeworms (Platyhelminthes: Cestoda), particularly the family Anthocephaliidae Ruhnke, Caira & Cox, 2015, pose challenges to systematic history of the group. Within this family, the genus Anthocephalum Linton, 1890, remains insufficiently explored despite recent advancements. This study addresses the intricate taxonomy, phylogenetic relationships, diversity, and historical biogeography of tapeworms within the Anthocephaliidae, focusing on its type-genus Anthocephalum. To accomplish this objective, 15 specimens across various geographical regions including Alagoas and Pará in Brazil, Panama, and Senegal were selected for DNA extraction. For species of Anthocephalum, partial 18S and 28S rDNA sequences were amplified via PCR, purified, and sequenced using the ABI Big-Dye method. Three specimens of Alveobothrium grabatum Boudaya, Neifar & Euzet, 2018 were sequenced through Illumina technology. Phylogenetic analyses were conducted using IQTREE2 under two optimality criteria: Maximum Parsimony (MP) and Maximum Likelihood (ML). Biogeographic ancestral range estimations were performed using the R package BioGeoBEARS, incorporating multiple biogeographical models. Our phylogenetic analyses reaffirmed Anthocephaliidae’s monophyly. However, both the relationships with or within Anthocephaliidae require further investigation. One example within the family is the positioning of Alveobothrium Boudaya, Neifar & Euzet, 2018, which challenged Anthocephalum’s monophyly requiring taxonomic actions. Furthermore, when exploring new localities and/or new hosts for Anthocephalum, four independent lineages were identified, suggesting that Anthocephalum’s diversity remains underestimated especially in unexplored regions and hosts. About the biogeographic ancestral range estimation, the analysis suggested a Central Indo-Pacific origin for early Anthocephalum lineages, with subsequent colonization events shaping the current diversity. The preliminary biogeographical framework presented here underscores the importance of refining phylogenetic hypotheses and enhancing taxonomic understanding. As taxonomic actions taken, the four new lineages were formally described and the genus Alveobothrium was synonymized with Anthocephalum, for which we proposed an amended diagnosis. This revision brings the total number of valid species of Anthocephalum to 30. Therefore, we suggest that future research initiatives should prioritize time-calibrated analyses, multiple genetic loci, and broader taxonomic representation for a detailed exploration of systematic history of anthocephaliid’s.

Tachinidae is the second most species‐rich family of Diptera. It comprises four subfamilies, and all of its members have parasitoid habits. We present the first phylogenomic analysis of Tachinidae using transcriptomic data, based on 30 species. We constructed four datasets: three using translated data at the amino acid level (100% coverage, with 106 single‐copy protein‐coding genes; 75% coverage, with 1359 genes; and 50% coverage, with 1942 genes). The trees were estimated by analysing four matrices using maximum likelihood and maximum parsimony inferences, and only minor differences were found among them. Overall, our topologies are well resolved, with high node support. Polleniidae is corroborated as a sister group to Tachinidae. Within Tachinidae, our results confirm the hypothesis (Phasiinae + Dexiinae) + (Tachininae + Exoristinae). Phasiinae, Dexiinae and Exoristinae are recovered as monophyletic, and Tachininae as polyphyletic. Once again, the tribe Myiophasiini (Tachininae) composes a fifth lineage, clade sister to all the remaining Tachinidae. The Neotropical tribe Iceliini, formerly in Tachininae, is recovered within Exoristinae, sister to Winthemiini. In general, our results are congruent with recent phylogenetic studies that include tachinids, with the important confirmation of the subfamilial relationships and the existence of a fifth lineage of Tachinidae.

In maximum likelihood (ML), the support for a clade can be calculated directly as the likelihood ratio (LR) or log‐likelihood difference (S, LLD) of the best trees with and without the clade of interest. However, bootstrap (BS) clade frequencies are more pervasive in ML phylogenetics and are almost universally interpreted as measuring support. In addition to theoretical arguments against that interpretation, BS has several undesirable attributes for a support measure. For example, it does not vary in proportion to optimality or identify clades that are rejected by the evidence and can be overestimated due to missing data. Nevertheless, if BS is a reliable predictor of S, then it might be an efficient indirect method of measuring support—an attractive possibility, given the speed of many BS implementations. To assess the relationship between S and BS, we analyzed 106 empirical datasets retrieved from TreeBASE. Also, to evaluate the degree to which S and BS are affected by the number of replicates during suboptimal tree searches for S and pseudoreplicates during BS estimation, we randomly selected 5 of the 106 datasets and analyzed them using variable numbers of replicates and pseudoreplicates, respectively. The correlation between S and BS was extremely weak in the datasets we analyzed. Increasing the number of replicates during tree search decreased the estimated values of S for most clades, but the magnitude of change was small. In contrast, although increasing pseudoreplicates affected BS values for only approximately 40% of clades, values both increased and decreased, and they did so at much greater magnitudes. Increasing replicates/pseudoreplicates affected the rank order of clades in each tree for both S and BS. Our findings show decisively that BS is not an efficient indirect method of measuring support and suggest that even quite superficial searches to calculate S provide better estimates of support.

Global loss of biodiversity is an ongoing process that concerns both local and global authorities. Studies of biodiversity mainly involve traditional methods using morphological characters and molecular protocols. However, conventional methods are a time consuming and resource demanding task. The development of high-throughput sequencing (HTS) techniques has reshaped the way we explore biodiversity and opened a path to new questions and novel empirical approaches. With the emergence of HTS, sequencing the complete mitochondrial genome became more accessible, and the number of genome sequences published has increased exponentially during the last decades. Despite the current state of knowledge about the potential of mitogenomics in phylogenetics, this is still a relatively under-explored area for a multitude of taxonomic groups, especially for those without commercial relevance, non-models organisms and with preserved DNA. Here we take the first step to assemble and annotate the genomes from HTS data using a new protocol of genome skimming which will offer an opportunity to extend the field of mitogenomics to under-studied organisms. We extracted genomic DNA from specimens preserved in ethanol. We used Nextera XT DNA to prepare indexed paired-end libraries since it is a powerful tool for working with diverse samples, requiring a low amount of input DNA. We sequenced the samples in two different Illumina platform (MiSeq or NextSeq 550). We trimmed raw reads, filtered and had their quality tested accordingly. We performed the assembly using a baiting and iterative mapping strategy, and the annotated the putative mitochondrion through a semi-automatic procedure. We applied the contiguity index to access the completeness of each new mitogenome. Our results reveal the efficiency of the proposed method to recover the whole mitogenomes of preserved DNA from non-model organisms even if there are gene rearrangement in the specimens. Our findings suggest the potential of combining the adequate platform and library to the genome skimming as an innovative approach, which opens a new range of possibilities of its use to obtain molecular data from organisms with different levels of preservation.

The uplift of the Panamanian isthmus in the Pliocene caused the termination of gene flow among members of many ancestral populations of marine lineages leading the diversification of geminate species confined to opposite sides of Central America. This palaeogeographical event has been evoked to explain the diversification of few lineages of batoids with trans-isthmian distribution. As such, there is the expectation that this vicariance event also affected lineages of parasites associated with them. Our study suggests that this event can explain the diversification of Parachristianella Dollfus, 1946 (Trypanorhyncha:Eutetrarhynchidae) in the Caribbean Sea and tropical eastern Pacific Ocean. We provide molecular, morphological and biogeographical evidence to recognise five lineages within this genus inhabiting the coastal waters of Panama, including P. parva Campbell & Beveridge, 2007 and four new species: P. mendozai, sp. nov., P. kuchtai, sp. nov., P. campbelli, sp. nov. and P. soldanovae, sp. nov. These species can be diagnosed by unambiguous sets of molecular characters. The morphological cohesiveness of sister species, which most likely diverged from around 3million years ago through the uplift of the Panamanian isthmus, suggests that the traditional emphasis on the tentacular armature to circumscribe species within trypanorhynchs might underestimate the diversity of recently diverged lineages.

Tapeworms of the genus Anindobothrium Marques, Brooks & Lasso, 2001 are found in both marine and Neotropical freshwater stingrays of the family Potamotrygonidae. The patterns of host association within the genus support the most recent hypothesis about the history of diversification of potamotrygonids, which suggests that the ancestor of freshwater lineages of the Potamotrygonidae colonized South American river systems through marine incursion events. Despite the relevance of the genus Anindobothrium to understand the history of colonization and diversification of potamotrygonids, no additional efforts were done to better investigate the phylogenetic relationship of this taxon with other lineages of cestodes since its erection. This study is a result of recent collecting efforts to sample members of the genus in marine and freshwater potamotrygonids that enabled the most extensive documentation of the fauna of Anindobothrium parasitizing species of Styracura de Carvalho, Loboda & da Silva, Potamotrygon schroederi Fernández-Yépez, P. orbignyi (Castelnau) and P. yepezi Castex & Castello from six different countries, representing the eastern Pacific Ocean, Caribbean Sea, and river basins in South America (Rio Negro, Orinoco, and Maracaibo). The newly collected material provided additional specimens for morphological studies and molecular samples for subsequent phylogenetic analyses that allowed us to address the phylogenetic position of Anindobothrium and provide molecular and morphological evidence to recognize two additional species for the genus. The taxonomic actions that followed our analyses included the proposition of a new family, Anindobothriidae fam. n., to accommodate the genus Anindobothrium in the order Rhinebothriidea Healy, Caira, Jensen, Webster & Littlewood, 2009 and the description of two new species—one from the eastern Pacific Ocean, A. carrioni sp. n., and the other from the Caribbean Sea, A. inexpectatum sp. n. In addition, we also present a redescription of the type species of the genus, A. anacolum (Brooks, 1977) Marques, Brooks & Lasso, 2001, and of A. lisae Marques, Brooks & Lasso, 2001. Finally, we discuss the paleogeographical events mostly linked with the diversification of the genus and the protocols adopted to uncover cryptic diversity in Anindobothrium.

The present study contributes to the knowledge of the cestode fauna of species of Styracura de Carvalho, Loboda & da Silva, which is the putative sister taxon of freshwater potamotrygonids—a unique group of batoids restricted to Neotropical freshwater systems. We document species of Rhinebothrium Linton, 1890 as a result of the examination of newly collected specimens of Styracura from five different localities representing the eastern Pacific Ocean and the Caribbean Sea. Overall, we examined 33 spiral intestines, 11 from the eastern Pacific species Styracura pacifica (Beebe & Tee-Van) and 22 from the Caribbean species S. schmardae (Werner). However, only samples from the Caribbean were infected with members of Rhinebothrium. Rhinebothrium tetralobatum Brooks, 1977, originally described from S. schmardae—as Himantura schmardae (Werner)—off the Caribbean coast of Colombia based on six specimens is redescribed. This redescription provides the first data on the microthriches pattern, more details of internal anatomy (i.e., inclusion of histological sections) and expands the ranges for the counts and measurements of several features. We describe a new species of Rhinebothrium from S. schmardae collected off the Caribbean coast of Panama. Rhinebothrium reydai n. sp. is diagnosed by possessing four testes per proglottid, acraspedote proglottids, single anterior-most and porterior-most loculi, and bothridia divided into 34–44 loculi. Collectively, these features distinguish the new species from all 41 species of Rhinebothrium currently recognized as valid, with the exception of R. chollaensis Friggens & Duszynski, 2005. The latter species, a parasite of Urobatis halleri (Cooper) from the eastern Pacific Ocean, has a similar morphology in comparison to R. reydai n. sp., but can be distinguished by being apolytic instead of euapolytic and by the morphology of the aporal lobe of the ovary, which reaches the mid-lateral margin of the cirrus sac, whereas in R. reydai n. sp. the aporal lobe only reaches the posterior margin, since the cirrus sac takes approximately ¾ of the proglottid in width. Also, in R. reydai n. sp., the first square proglottid occurs within the anterior third of the strobila (13–30%), whereas in R. chollaensis it occurs near the middle of the strobila (42–62%). Further, we discuss the patterns of infection and biogeographical distribution for species of Rhinebothrium in species of Styracura. The apparent disjunctive distribution of R. tetralobatum and R. reydai n. sp. in the Caribbean Sea throughout their host distribution, S. schmardae, and the absence of species of Rhinebothrium in the eastern Pacific sister-host, S. pacifica, reveal the importance of sample size and biogeographical representation for documenting the parasite fauna of host lineages.

Three genera of rhinebothriideans, previously referred to as New genus 1, New genus 2 and New genus 4, are erected in the the Anthocephaliidae. New genus 1 is established as Divaricobothrium gen. n., with Divaricobothrium tribelum sp. n. as its type species; Echeneibothrium trifidum Shipley et Hornell, 1906 is transferred to the genus as Divaricobothrium trifidum (Shipley et Hornell, 1906) comb. n. This genus is unique among rhinebothriidean genera in bearing bothridia that are posteriorly deeply divided into two lobes with facial loculi but no apical sucker, and a vagina that extends to near the anterior margin of the proglottid. Its species parasitise Indo-Pacific members of the genera Brevitrygon Last, Naylor et Manjaji-Matsumoto, Maculabatis Last, Naylor et Manjaji-Matsumoto and Pateobatis Last, Naylor et Manjaji-Matsumoto. New genus 2 is established as Barbeaucestus gen. n., with Barbeaucestus jockuschae sp. n. as its type species; Barbeaucestus ralickiae sp. n. is also described. Anthobothrium sexorchidum Williams, 1964 and Rhinebothrium shipleyi Southwell, 1912 are transferred to the genus as Barbeaucestus sexorchidus (Williams, 1964) comb. n. and Barbeaucestus shipleyi (Southwell, 1912) comb. n., respectively. This genus is unique among rhinebothriidean genera in that its bothridia are substantially wider than long, bear an apical sucker and at least one row of two or more facial loculi in their anterior half. Its species parasitise the genera Neotrygon Castelnau and Taeniura M?ller et Henle. New genus 4 is established as Sungaicestus gen. n. with transfer of Rhinebothrium kinabatanganensis Healy, 2006, as Sungaicestus kinabatanganensis (Healy, 2006) comb. n., as its type species. Among the genera of its order, this genus most closely resembles Rhinebothrium Linton, 1890, however, despite the original description, the bothridia were found to bear, rather than lack, apical suckers. This monotypic genus is known only from the freshwater stingray Urogymnus polylepis (M?ller et Henle). The familial diagnosis of the Anthocephaliidae Ruhnke, Caira et Cox, 2015 is emended. The family now houses five genera.

The genus Pararhinebothroides was established for a species of cestode (Parahinebothroides hobergi) found parasitizing the Tumbes round stingray, Urobatis tumbesensis (McEachran & Chirichigno), in the inshore Pacific waters in the Gulf of Guayaquil, Ecuador. Its apparent affinities with the freshwater endemic genus Rhinebothroides were considered evidence to support the long-standing, yet controversial, biogeographical hypothesis that freshwater stingrays of the family Potamotrygonidae derived from a Pacific marine ancestor during the Cretaceous Period before the uplifting of the Andes. Here, we re-evaluate the phylogenetic and taxonomic status of P. hobergi based on examination of the available type material and newly collected material from the type host near the type locality. The new material allowed the description of tegumental structures using scanning electron microscopy and the generation of a hypothesis for the phylogenetic position of the species based on molecular data for the first time. Morphological investigations revealed that P. hobergi shares all the diagnostic features of the most recent concept of Anthocephalum, including the previously overlooked presence of bothridial apical suckers. Phylogenetic analyses based on partial 28S rDNA (D1–D3) and complete 18S rDNA sequence data for 4 specimens of P. hobergi, 45 species of other rhinebothriideans, and 5 non-rhinebothriidean outgroup species provided unequivocal support for the transfer of P. hobergi to Anthocephalum. Since this is the type and only species of the genus, Pararhinebothroides is considered a junior synonym of Anthocephalum, and Anthocephalum hobergi n. comb. is redescribed. Furthermore, our results reveal Rhinebothroides and Anthocephalum to be only distantly related among the Rhinebothriidea. Not only do our results confirm reservations expressed earlier about the affinities of P. hobergi, but they also substantially challenge inferences drawn previously about the biogeographical history of potamotrygonid stingrays based on parasitological data.

Neotropical freshwater stingrays (Batoidea: Potamotrygonidae) host a diversity of parasites, including some, like their hosts, that are marine-derived. Among the parasites of potamotrygonids, the cestode fauna is the most diverse, with multiple genera having been reported, including genera endemic to the freshwaters of the Neotropics and genera that have cosmopolitan distributions. Recent efforts have been made to document the diversity of cestodes of this host-parasite system and to refine the taxonomy of parasite lineages. The present study contributes to our knowledge of Rhinebothrium Linton, 1890, a diverse cosmopolitan genus of rhinebothriidean cestode, with 37 species reported from marine batoids, one species from a freshwater stingray in Borneo and six species from potamotrygonids. Rhinebothrium jaimei sp. n. is described from two species of potamotrygonids, Potamotrygon orbignyi (Castelnau) (type host) and Potamotrygon scobina Garman, from Bah?a de Maraj? of the lower Amazon region. It can be distinguished from most of its marine congeners via multiple attributes, including its possession of two, rather than one, posteriormost loculi on its bothridia and the lomeniform shape of its bothridium that is wider anteriorly. In addition, R. jaimei sp. n. can be distinguished from the six Rhinebothrium species described previously from potamotrygonids based on a unique combination of morphological features. Despite extensive stingray cestode sampling efforts throughout all major Neotropical river systems, we found that unlike most species of potamotrygonid Rhinebothrium species, which are widespread, R. jaimei sp. n. is restricted to the Bah?a de Maraj?. The discovery of this new species of Rhinebothrium in Bah?a de Maraj?, an area in which potamotrygonids occur sympatrically with some species of euryhaline batoids (e.g. Dasyatis spp.) and share some trophic resources, suggest that modern ecological processes may be contributing to the distribution patterns of cestodes infecting potamotrygonids.