Comparative Characterization of Mitogenomes From Five Orders of Cestodes (Eucestoda: Tapeworms).

The recognized potential of using mitogenomics in phylogenetics and the more accessible use of high-throughput sequencing (HTS) offer an opportunity to investigate groups of neglected organisms. Here, we leveraged HTS to execute the most comprehensive documentation of mitogenomes for cestodes based on the number of terminals sequenced. We adopted modern approaches to obtain the complete mitogenome sequences of 86 specimens representing five orders of cestodes (three reported for the first time: Phyllobothriidea, "Tetraphyllidea" and Trypanorhyncha). These complete mitogenomes represent an increase of 41% of the mitogenomes available for cestodes (61-147) and an addition of 33% in the representativeness of the cestode orders. The complete mitochondrial genomes are conserved, circular, encoded in the same strand, and transcribed in the same direction, following the pattern observed previously for tapeworms. Their length varies from 13,369 to 13,795 bp, containing 36 genes in total. Except for the Trypanorhyncha specimen, the gene order of the other four cestode orders sequenced here suggests that it could be a synapomorphy for the acetabulate group (with a reversion for taenids). Our results also suggest that no single gene can tell all the evolutionary history contained in the mitogenome. Therefore, cestodes phylogenies based on a single mitochondrial marker may fail to capture their evolutionary history. We predict that such phylogenies would be improved if conducted under a total evidence framework. The characterization of the new mitochondrial genomes is the first step to provide a valuable resource for future studies on the evolutionary relationships of these groups of parasites.

The First Report and Description of a New Species of Rhinebothrium from a Dasyatid Stingray from the Brazilian Northeastern Coast with a Review of the Distribution of the Genus throughout Endemic Marine Ecoregions.

Here we describe a new species of RhinebothriumLinton, 1890, from Hypanus guttatus (Bloch and Schneider). Rhinebothrium ramosi n. sp. can be differentiated from all 51 valid species of the genus by having 4-5 testes and uterus that extends throughout the entire length of the proglottid. Only 8 of the above species closely resemble R. ramosi in total length (Rhinebothrium bunburyense, Rhinebothrium chollaense, Rhinebothrium corbatai, Rhinebothrium dasyatidis, Rhinebothrium kruppi, Rhinebothrium lintoni, Rhinebothrium margaritense, and Rhinebothrium reydai). Despite the resemblance, R. bunburyense, R. corbatai, R. dasyatidis, R. lintoni, and R. margaritense can be distinguished from the new species by possessing a larger number of proglottids. The remaining 3 species (R. chollaense, R. kruppi, and R. reydai) overlap in total length and number of proglottids with R. ramosi. However, they can be distinguished from the new species by possessing a single posterior-most bothridial loculus instead of arranged as a pair, as found in the new species. This is the first report of the genus from the coastal waters of Brazil and brings to 52 the number of valid species for this genus. Additionally, we use the patterns of infection and distribution for species of Rhinebothrium to make predictions of expected diversity within the genus, especially for unsurveyed hosts in endemic marine ecoregions of the world.

Genome skimming is a low-cost and robust strategy to assemble complete mitochondrial genomes from ethanol preserved specimens in biodiversity studies.

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.

Systematics and diversification of Anindobothrium Marques, Brooks & Lasso, 2001 (Eucestoda: Rhinebothriidea).

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.

Three new genera of rhinebothriidean cestodes from stingrays in Southeast Asia.

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 Müller 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 (Müller et Henle). The familial diagnosis of the Anthocephaliidae Ruhnke, Caira et Cox, 2015 is emended. The family now houses five genera.

Pararhinebothroides-Neither the Sister-Taxon of Rhinebothroides Nor a Valid Genus.

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.

Rhinebothrium jaimei sp. n. (Eucestoda: Rhinebothriidea: Rhinebothriidae): a new species from Neotropical freshwater stingrays (Potamotrygonidae).

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 Bahía de Marajó 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 Bahía de Marajó. The discovery of this new species of Rhinebothrium in Bahía de Marajó, 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.

Phylogeny of harvestmen family Gonyleptidae inferred from a multilocus approach (Arachnida: Opiliones).

Gonyleptidae is the second most diverse harvestmen family and the most studied in terms of morphology, behaviour, and ecology. Despite this, few phylogenetic studies have focused on gonyleptids, and those are based on a very limited number of taxa. We addressed this gap by constructing a phylogenetic hypothesis of the family using 101 taxa from all 16 gonyleptid subfamilies and four mitochondrial and nuclear loci (COI, 28S rRNA, 12S rRNA, and 16S rRNA). These were analysed under parsimony and likelihood optimality criteria (and using direct optimization for the former). Relationships among Gonyleptoidea and within each subfamily of Gonyleptidae were largely congruent between parsimony and maximum-likelihood approaches. Taxonomic actions from our phylogeny include the following: Tricommatidae, new status, is restored as a family; Metasarcidae, new status, is recognized as a family and considered sister to the Cosmetidae; and Cranainae and Manaosbiinae are suggested as members of Gonyleptidae, restoring Roewer's concept of the family. Within Gonyleptidae, the "K92" group-composed of Sodreaninae, Caelopyginae, Hernandariinae, Progonyleptoidellinae, and Gonyleptinae-forms a clade, although the latter two subfamilies are not monophyletic. The genus Parampheres is here transferred to Caelopyginae, and "Multumbo" dimorphicus to Gonyleptinae. Gonyleptidae is characterized by the presence of a ventral process on the penis glans and a bifid apophysis on the male coxa IV. The long-legged Mitobatinae can be considered monophyletic only if some short-legged pachylines are included, or if we assume that elongate legs arose twice independently (in the true mitobatine genera and in Longiperna). Pachylinae, the most diverse gonyleptid subfamily, represents several distinct lineages. We further conclude that the traditional use of a small set of morphological characters in the systematics of Gonyleptidae is unable to explain the complex evolution of the family.

Phylogenetic analysis and reconfiguration of genera in the cestode order Diphyllidea.

The generic boundaries of the Diphyllidea are reassessed based on parsimony and likelihood phylogenetic analyses of 28S rDNA (ribonucleic acid large subunit), 18S rDNA (ribonucleic acid small subunit), and COI (cytochrome oxidase subunit I) sequence data for 31 species representing morphological variation across the order. Trees resulting from these analyses yielded a number of well-supported clades that are congruent with unique morphological features mandating generic revision of the order and erection of at least two new genera. Species originally assigned to Echinobothriumvan Beneden, 1849 but bearing a corona of spines on the region of the scolex anterior to the bothria and posterior to the apical organ armature are transferred to Coronocestus n. gen.; members of this genus typically parasitize triakid sharks, although one report from a hemiscylliid shark exists. Species with lateral hooklets arranged in continuous bands, rather than in two distinct clusters, are transferred to Halysioncum n. gen.; all species parasitize batoids, mostly myliobatids and rhinopterids, but a few records also exist from arhynchobatids, rhinobatids, platyrhinids and urotrygonids. Our analyses support transfer of the five species originally assigned to MacrobothridiumKhalil and Abdul-Salam, 1989 owing to their lack of cephalic peduncle spines to Echinobothrium. As a consequence, Echinobothrium sensu stricto includes species both with and without spines on the cephalic peduncle, but all members of the genus possess lateral hooklets arranged in clusters on either side of the dorsal and ventral apical hooks. With respect to diphyllideans parasitizing catsharks, AhamulinaMarques, Jensen and Caira, 2012 is unique in possessing apical hooks but lacking lateral hooklets and DitrachybothridiumRees, 1959 is unique in entirely lacking scolex armature. By far the majority of species of Echinobothrium sensu stricto parasitize skates of the family Rajidae, guitarfish of the family Rhinobatidae, and stingrays of the dasyatid genera Taeniura Müller and Henle, Dasyatis Rafinesque, and Himantura Müller and Henle, although a single species each has been reported from Anacanthobatidae, Rhynchobatidae, Platyrhinidae and Myliobatidae. It now seems clear that while by far the majority of diphyllideans parasitize batoids, the diphyllideans parasitizing sharks, and catsharks in particular, remain problematic. Additional collections from these carcharhiniform hosts are likely to be particularly illuminating.

Diversification and species boundaries of Rhinebothrium (Cestoda; Rhinebothriidea) in South American freshwater stingrays (Batoidea; Potamotrygonidae).

BACKGROUND: Neotropical freshwater stingrays (Batoidea: Potamotrygonidae) host a diverse parasite fauna, including cestodes. Both cestodes and their stingray hosts are marine-derived, but the taxonomy of this host/parasite system is poorly understood. METHODOLOGY: Morphological and molecular (Cytochrome oxidase I) data were used to investigate diversity in freshwater lineages of the cestode genus Rhinebothrium Linton, 1890. Results were based on a phylogenetic hypothesis for 74 COI sequences and morphological analysis of over 400 specimens. Cestodes studied were obtained from 888 individual potamotrygonids, representing 14 recognized and 18 potentially undescribed species from most river systems of South America. RESULTS: Morphological species boundaries were based mainly on microthrix characters observed with scanning electron microscopy, and were supported by COI data. Four species were recognized, including two redescribed (Rhinebothrium copianullum and R. paratrygoni), and two newly described (R. brooksi n. sp. and R. fulbrighti n. sp.). Rhinebothrium paranaensis Menoret & Ivanov, 2009 is considered a junior synonym of R. paratrygoni because the morphological features of the two species overlap substantially. The diagnosis of Rhinebothrium Linton, 1890 is emended to accommodate the presence of marginal longitudinal septa observed in R. copianullum and R. brooksi n. sp. Patterns of host specificity and distribution ranged from use of few host species in few river basins, to use of as many as eight host species in multiple river basins. SIGNIFICANCE: The level of intra-specific morphological variation observed in features such as total length and number of proglottids is unparalleled among other elasmobranch cestodes. This is attributed to the large representation of host and biogeographical samples. It is unclear whether the intra-specific morphological variation observed is unique to this freshwater system. Nonetheless, caution is urged when using morphological discontinuities to delimit elasmobranch cestode species because the amount of variation encountered is highly dependent on sample size and/or biogeographical representation.

Phylogeny, biogeography, and electric signal evolution of Neotropical knifefishes of the genus Gymnotus (Osteichthyes: Gymnotidae).

The Neotropical knifefish genus Gymnotus is the most broadly distributed and the most diverse (34+species) gymnotiform genus. Its wide range includes both Central and South American drainages, including the Amazon, Orinoco, and La Plata Basins. Like all gymnotiforms, Gymnotus species produce weak electric fields for both navigation and communication, and these fields exhibit interspecific variation in electric waveform characteristics. Both biogeography and electric signal evolution can profitably be analyzed in a phylogenetic context. Here, we present a total evidence phylogeny for 19 Gymnotus species based on data from the mitochondrial cytochrome b and 16S genes (1558 bp), the nuclear RAG2 gene (1223 bp), and 113 morphological characters. Our phylogenetic hypothesis resolves five distinct Gymnotus lineages. In a previous morphology-based analysis, the Central American Gymnotus cylindricus lineage was hypothesized as the sister group to all other Gymnotus species. In our analysis, the G. cylindricus lineage is nested within South American species, and molecular age estimates support a relatively recent origin for the clade in Central America. Phylogenetic optimization of electric signal waveforms indicate that the ancestral state in Gymnotus is a multiphasic (4+phases of alternating polarity) condition, and independent phase loss has occurred in multiple lineages. Gymnotus is a model group for understanding Neotropical diversification and the evolution of communication at a continental scale.

Revision of Potamotrygonocotyle Mayes, Brooks & Thorson, 1981 (Platyhelminthes: Monogenoidea: Monocotylidae), with descriptions of four new species from the gills of the freshwater stingrays Potamotrygon spp. (Rajiformes: Potamotrygonidae) from the La Plata river basin.

The only known monocotylid genus to parasitise Neotropical freshwater stingrays (Potamotrygonidae) is Potamotrygonocotyle Mayes, Brooks & Thorson, 1981, a monotypic genus erected to accommodate P. tsalickisi Mayes, Brooks & Thorson, 1981. For more than 20 years, no other species has been recognised in this genus, but new efforts to survey the diversity of parasites inhabiting potamotrygonids have revealed the existence of new species and the need to redefine the genus. Here, the generic diagnosis of Potamotrygonocotyle is amended, P. tsalickisi is redescribed and four new species are recognised and described based on samples collected from the gills of freshwater potamotrygonids from the La Plata river basin: Potamotrygonocotyle chisholmae n. sp. and P. dromedarius n. sp. from Potamotrygon motoro; Potamotrygonocotyle eurypotamoxenus n. sp. from Potamotrygon cf. motoro (type-host), P. castexi, P. falkneri and P. histrix; and Potamotrygonocotyle uruguayensis n. sp. from Potamotrygon brachyura. Potamotrygonocotyle is characterised by species possessing: (1) slightly sinuous sclerotised ridges on all septa; (2) two pairs of the dorsal haptoral accessory structures associated with the four posterior peripheral loculi and with anterior dorsal haptoral accessory structure bilobate or semicircular; and (3) male copulatory organ without an accessory piece.

Monogenoidean parasites of freshwater stingrays (Rajiformes: Potamotrygonidae) from the Negro River, Amazon, Brazil: species of Potamotrygonocotyle (Monocotylidae) and Paraheteronchocotyle (Hexabothriidae).

Five new species of Potamotrygonocotyle (Monocotylidae) are described and Paraheteronchocotyle amazonense Mayes, Brooks et Thorson, 1981 (Hexabothriidae) is redescribed from monogenoideans collected on the gills of species of Potamotrygonidae from the Negro River, Amazon, Brazil. Potamotrygonocotyle quadracotyle sp. n. and P. umbella sp. n. were found parasitizing an undescribed species of Potamotrygon; Potamotrygonocotyle rarum sp. n. is described from Potamotrygon schroederi; Potamotrygonocotyle rionegrense inhabits Potamotrygon cf. motoro; Potamotrygonocotyle aramasae sp. n. is a parasite of Paratrygon aiereba; and Paraheteronchocotyle amazonense is reported from Potamotrygon orbignyi. The diagnosis of Paraheteronchocotyle is emended, and P. amazonense is redescribed.

Taxonomic revision of Rhinebothroides (Eucestoda: Tetraphyllidea: Phyllobothriidae), parasites of neotropical freshwater stingrays (Rajiformes: Myliobatoidei: Potamotrygonidae).

Extensive new collections of specimens of Rhinebothroides spp. from a wide range of Neotropical freshwater potamotrygonid stingrays in numerous localities permitted critical evaluation of the validity of the 7 nominal species in the genus. Some qualitative characters previously used in the taxonomy of this group are highly variable within and among populations, and most morphometric and meristic characters thought to have taxonomic value for this group overlapped among several previously defined nominal species. Characters that were consistent throughout populations and that clearly define morphotypes supported recognition of 4 species: R. freitasi (syn. R. circularisi and R. venezuelensis), R. glandularis (syn. R. mclennanae), R. moralaurai, and R. scorzai. We provide a taxonomic review of the genus, 8 additional host records, 7 new localities, and an identification key.