Phylogenomics, evolution of trophic traits and divergence times of hemiodontid fishes (Ostariophysi: Characiformes).

The South American characiform family Hemiodontidae comprises five genera and 34 species. The family lacks comprehensive phylogenetic hypotheses resolving its species relationships. The studies that addressed these questions exhibited a narrow taxon sampling or used single-locus markers. Herein we surveyed hundreds of ultraconserved elements (UCEs) loci to provide the first molecular phylogenetic hypothesis and divergence time estimates for hemiodontids encompassing all its genera and most species (27 of the 34 valid species). We also tracked the history of the protractile upper jaw in the genera Argonectes and Bivibranchia across the recovered phylogenies through ancestral state reconstruction. Our results corroborate the monophyly of Hemiodontidae and the genera Argonectes and Bivibranchia in all phylogenetic methods with maximum clade support. The genera Anodus and Hemiodus were not monophyletic because Anodus elongatus was sister to the monotypic Micromischodus instead of A. orinocensis, and H. immaculatus did not form a clade with its other congeners, but instead was sister to the clade including Anodus and Micromischodus. All remaining species of Hemiodus were placed together into a monophyletic group, where they were arranged into four major subclades. The relationship in the family is summarised as: (Bivibranchia, (Argonectes, ((H. immaculatus, (Anodus, Micromischodus)), Hemiodus clade))), in discordance with the morphological phylogeny that placed all genera monophyletic and resolved the family as: ((Anodus, Micromischodus), (Hemiodus, (Argonectes, Bivibranchia))). The origin of Hemiodontidae was estimated from the Late Cretaceous to the Middle Paleogene, with the mean age in the Paleocene, while the origin of most hemiodontid genera except Bivibranchia occurred in the Miocene. Unordered parsimony and likelihood reconstruction indicates that Argonectes and Bivibranchia developed their protractile upper jaw independently.

Early ontogeny of the commercially valuable fish red-bellied pacu Piaractus brachypomus (Characiformes, Serrasalmidae) from the Amazon, Brazil.

The initial development of the red-bellied pacu Piaractus brachypomus is described using morphological, meristic and morphometric characteristics. A total of 127 individuals were analysed (47 in the yolk-sac, 35 in pre-flexion, five in flexion, 20 in post-flexion and 20 in juvenile) with standard length varying between 2.92 and 48.61 mm. The larvae are born poorly developed and have a discoidal yolk at ~6.33 mm standard length. During early ontogeny, the mouth passes from terminal to subterminal and the anal opening reaches the vertical line over the midline region of the body. There are changes in body shape from long and moderate to deep, head length from small to large, and eye diameter from moderate to large. Dendritic chromatophores were present in the ventral, dorsal and upper part of the swim bladder in the early larval stages. Rounded spots are evident all over the body in juveniles. The total number of myomeres ranges from 39 to 41 (20-23 pre-anal, 17-20 post-anal). Through the morphometric relationships, it was evidenced that the greatest changes during the initial ontogeny of P. brachypomus occur in the transition from the post-flexion stage to the juvenile period, indicating changes in behaviour, foraging and physiology.

Knodus borari, a new species from the lower Rio Tapajós basin (Characiformes: Characidae).

A new species of Knodus from the Rio Cupari, a right-bank tributary of the lower Rio Tapajós, is described. Among its congeners, the new species shares exclusively with K. figueiredoi the presence of only uni-tricuspid teeth in the premaxilla, maxilla and dentary. Knodus borari differs from K. figueiredoi by the colour pattern and morphological characteristics. The new species is the fifth species of the genus described from the Rio Tapajós basin, and the second restricted to the lower portion of the basin. This is additional evidence that the lower Rio Tapajós basin is a region of ichthyofaunal endemism.

Phylogenomics of Piranhas and Pacus (Serrasalmidae) Uncovers How Dietary Convergence and Parallelism Obfuscate Traditional Morphological Taxonomy.

The Amazon and neighboring South American river basins harbor the world's most diverse assemblages of freshwater fishes. One of the most prominent South American fish families is the Serrasalmidae (pacus and piranhas), found in nearly every continental basin. Serrasalmids are keystone ecological taxa, being some of the top riverine predators as well as the primary seed dispersers in the flooded forest. Despite their widespread occurrence and notable ecologies, serrasalmid evolutionary history and systematics are controversial. For example, the sister taxon to serrasalmids is contentious, the relationships of major clades within the family are inconsistent across different methodologies, and half of the extant serrasalmid genera are suggested to be non-monophyletic. We analyzed exon capture to reexamine the evolutionary relationships among 63 (of 99) species across all 16 serrasalmid genera and their nearest outgroups, including multiple individuals per species to account for cryptic lineages. To reconstruct the timeline of serrasalmid diversification, we time-calibrated this phylogeny using two different fossil-calibration schemes to account for uncertainty in taxonomy with respect to fossil teeth. Finally, we analyzed diet evolution across the family and comment on associated changes in dentition, highlighting the ecomorphological diversity within serrasalmids. We document widespread non-monophyly of genera within Myleinae, as well as between Serrasalmus and Pristobrycon, and propose that reliance on traits like teeth to distinguish among genera is confounded by ecological homoplasy, especially among herbivorous and omnivorous taxa. We clarify the relationships among all serrasalmid genera, propose new subfamily affiliations, and support hemiodontids as the sister taxon to Serrasalmidae. [Characiformes; exon capture; ichthyochory; molecular time-calibration; piscivory.].

New species and records of Anacanthorus (Monogenoidea: Dactylogyridae) from the gills of Brycon amazonicus (Characiformes: Bryconidae) in the Peruvian Amazon.

Five species belonging to Anacanthorus Mizelle & Price, 1965 were found parasitizing the gills of Brycon amazonicus (Agassiz) collected in the Peruvian Amazon: A. spiralocirrus Kritsky, Thatcher & Kayton, 1979, A. femoris n. sp., A. kukamensis n. sp., A. rarus n. sp. and A. sabaloi n. sp. Anacanthorus femoris n. sp. presents a membranous accessory piece with flared tip, hooks with proximal bulbous base containing 1 translucent region; A. kukamensis n. sp. presents an accessory piece with two branches, one with can-opener-shaped termination and the other with denticles in the lateral margin and at both ends of the distal portion, hooks with expanded proximal bulbous base with central and elliptical translucent region; A. rarus n. sp. presents a MCO as a long sclerotized tube, slightly sinuous, with folds in its middle region, with proximal region with circular opening and distal region with rough termination. The accessory piece is small and curved, hooks with expanded proximal bulbous base with central, elliptical translucent region, elongate shank, truncate thumb, slightly curved shaft and inconspicuous point; in A. sabaloi n. sp. the MCO is a long sclerotized reflexive tube, with membranous appearance from its middle part towards the end, with distal and proximal end with an inwardly directed fold, the accessory piece is a membranous sheath that envelops the MCO just before the reflection zone. The four new species found in this study are the second records of species of Anacanthorus described from a fish host from the Peruvian Amazon.

Microstructural and compositional variation in pacu and piranha teeth related to diet specialization (Teleostei: Serrasalmidae).

In any vertebrate group, tooth shape is known to fit with a biological function related to diet. However, little is known about the relationships between diet and tooth microstructure and composition in teleost fishes. In this work, we describe the external morphology, internal microstructure and elemental composition of the oral teeth of three representative species of the family Serrasalmidae having different feeding habits (herbivorous vs. omnivorous vs. carnivorous). We used backscattered-electron imaging and low vacuum environmental scanning electron microscope to compare the organization and mineralization of tooth layers as well as energy dispersive X-ray microanalysis and Raman microspectrometry to investigate the elemental composition, Ca/P ratio and mineralogy of the most superficial layers. Oral teeth of each serrasalmid species have the same internal organization based on five distinctive layers (i.e. pulp, dentine, inner enameloid, outer enameloid and cuticle) but the general tooth morphology is different according to diet. Microstructural and compositional variation of the cuticle and iron-enrichment of superficial layers were highlighted between herbivorous and carnivorous species. Iron is more concentrated in teeth of the herbivorous species where it is associated with a thicker cuticle explaining the more intense red-pigmentation of the cutting edges of oral teeth. The iron-enrichment is interpreted as a substitution of Ca by Fe in the hydroxyapatite. These traits are discussed in the light of the evolutionary history of the family. Further considerations and hypotheses about the formation and origin of the mineralized tooth layers and especially the iron-rich superficial layers in teleost fishes are suggested.

An integrative approach assesses the intraspecific variations of Procamallanus (Spirocamallanus) inopinatus, a common parasite in Neotropical freshwater fishes, and the phylogenetic patterns of Camallanidae.

Integrative taxonomy was used to evaluate two component populations of Procamallanus (Spirocamallanus) inopinatus in Brazil and the phylogeny Camallanidae. Parasite populations were collected in the characiform Anostomoides passionis from River Xingu (Amazon basin) and Megaleporinus elongatus from River Miranda (Paraguay basin). Morphology was analysed using light and scanning electron microscopy (SEM). Genetic characterization was based on partial sequences of the 18S and 28S rDNA, and COI mtDNA. Phylogenies were based on 18S and COI due to data availability. Generalized Mixed Yule Coalescent (GMYC), Poisson Tree Process (PTP) and *BEAST were used for species delimitation and validation. SEM revealed for the first time the presence of minute denticles and pore-like structures surrounding the oral opening, phasmids in females and confirmed other important morphological aspects. Statistical comparison between the two-component populations indicated morphometric variations, especially among males. The different component population of P. (S.) inopinatus showed variable morphometry, but uniform morphology and were validated as conspecific by the GMYC, PTP and *BEAST. Some camallanid sequences in GenBank have incorrect taxonomic labelling. Host, environment and geographic aspects seem to be related to some lineages within Camallanidae; however, their real phylogenetic meanings are still unclear.

Microplastics and copper effects on the neotropical teleost Prochilodus lineatus: Is there any interaction?

Microplastics (MP) are emerging contaminants widely found in aquatic ecosystems. In addition to MP toxicity itself, there is increasing concern about the MP adsorption capacity and the interactive effects with other contaminants, such as copper. The objective of this research was to investigate the effects of polyethylene microplastic and its association with copper (Cu) in genotoxic, biochemical, and physiological biomarkers of the neotropical teleost Prochilodus lineatus. Fish were exposed for 24 and 96 h to MP (20 µg L-1) and Cu (10 µg L-1) and MP + Cu. The results showed that MP and Cu, both isolated and in combination, promoted DNA damage in erythrocytes (96 h) and liver cells (24 and 96 h) indicating that MP and Cu are genotoxic. Fish exposed only to Cu (96 h) showed a decrease in lipid peroxidation in the liver despite of the decrease in glutathione content, indicating the efficiency of other antioxidant defenses. Brain acetylcholinesterase was inhibited in the animals from all the treatments. Although MP did not influence on Cu accumulation in tissues, decreased plasma Na+ and Ca2+ (24 h) occurred after the exposure to MP and Cu, isolated and combined. Exposure to MP and MP + Cu resulted in decreased activity of Ca2+-ATPase (24 h). Taken altogether, these results showed that MP and Cu depicted genotoxic, neurotoxic, and physiological effects on P. lineatus, both alone and combined. An interaction between Cu and MP was observed in plasma Ca2+, where the combination of both contaminants caused a greater effect than the contaminants alone.

First record of Spirocamallanus krameri (Nematoda: Camallanidae) in Brazil: Morphological review.

This study aimed to describe the occurrence, morphology and prevalence of Spirocamallanus krameri, a parasite of Hoplerythrinus unitaeniatus collected in state of Pará, Brazil. The morphological and morphometric characteristics are consistent with the species S. krameri, by having a hexagonal mouth opening, 16 cephalic papillae and two lateral papillae on the amphids. The buccal capsule is internally different in both sexes, with (11-13) spiral ridges in males and (13-17) spiral ridges in females. The basal ring is well developed, and three teeth are present in males and absent in females. Ten sessile caudal papillae and a pair of phasmidial pores occur in males. The spicules are small, subequal, 81-101 µm in length and the tail is tapered. The vulva in females is postequatorial, the tail is conical with lateral phasmidial pores. Among the species of Spirocamallanus that have short spicules, the general morphology of S. krameri most resembles that of S. inopinatus, but differs from that species, which have two cephalic teeth visible (absent in S. krameri) and specimens of S. inopinatus do not have sexual dimorphism in the buccal capsule of present in S. krameri.

A new endangered species of Megaleporinus (Characiformes: Anostomidae) from the Rio de Contas basin, eastern Brazil.

A new species of Megaleporinus is described from the Rio de Contas, a coastal drainage of eastern Brazil, and its phylogenetic relationships are studied using molecular data. The new species is unique among Anostomidae by possessing two exclusive features: an irregular dark longitudinal stripe from supracleithrum to second midlateral blotch and anterior cranial fontanel partially closed. In addition, the new species is diagnosed by having three premaxillary teeth, three dentary teeth, 37 or 38 scales in lateral line, 16 scale rows around caudal peduncle, three dark midlateral blotches on body, and red fins in life. The new species is closely related to M. obtusidens from the São Francisco basin, corroborating previous studies that indicated that the latter represents a species complex as currently defined. The new species exhibits the first rib enlarged in mature males, a feature described for some congeners. The new species is herein considered to be Endangered under the IUCN criteria.

Comparative larval ontogeny of two fish species (Characiformes and Siluriformes) endemic to the São Francisco River in Brazil.

The aim of the present study was to perform comparative histological analyses of the ontogenetic development of two fish species endemic to the São Francisco River in Brazil: Prochilodus argenteus and Lophiosilurus alexandri. Histological analyses were performed every 24 h from the moment of hatching until 14 days post-hatching (dph) for the observation of larval development and until 39 dph for the observation of gonadal development. Whole larvae were fixed in Bouin's solution and the histological slides were stained with haematoxylin-eosin. Lophiosilurus alexandri larvae had a larger body size compared with P. argenteus larvae since hatching. Lophiosilurus alexandri larvae had mouth opening and pigmentation of the eyes upon hatching, whereas these events were observed at 1 dph in P. argenteus larvae. The visualisation and the inflation of the swim bladder occurred at 1 and 3 dph, respectively, in the P. argenteus, whereas these events occurred at 2 and 8 dph, respectively, in L. alexandri. Yolk granules were absorbed at 4 dph in P. argenteus and the 10 dph in L. alexandri. At 7 dph, the digestive tube was more differentiated in L. alexandri than P. argenteus and at 14 dph, the digestive system of both species had features of their eating habits: broad stomach and short intestine in L. alexandri, typical of carnivorous habits; stomach with a mechanical function and long intestine in P. argenteus, typical of detritivorous habits. The epithelial lining tissue, formed by a single layer of cells in the newly hatched larvae (0 dph), differentiated throughout the study, exhibiting scales in P. argenteus and numerous club cells in the middle epithelial region of L. alexandri at 39 dph. Undifferentiated gonads with somatic cells and primordial germ cells were observed at 39 dph, with caudal-cranial migration since 1 dph in both species. The anatomic changes during the ontogeny of P. argenteus and L. alexandri larvae are directly associated with the evolutionary history of each species, which explains their feeding habits, behaviour and distribution in the environment: Prochilodus argenteus is detritivorous and actively swims in the water column, whereas L. alexandri is carnivorous and inhabits bottom regions. At 39 dph neither species exhibited sexual differentiation.

Maternal genetic effects in Astyanax cavefish development.

The role of maternal factors in the evolution of development is poorly understood. Here we describe the use of reciprocal hybridization between the surface dwelling (surface fish, SF) and cave dwelling (cavefish, CF) morphs of the teleost Astyanax mexicanus to investigate the roles of maternal genetic effects in cavefish development. Reciprocal hybridization, a procedure in which F1 hybrids are generated by fertilizing SF eggs with CF sperm (SF × CF hybrids) and CF eggs with SF sperm (CF × SF hybrids), revealed that the CF degenerative eye phenotype showed maternal genetic effects. The eyes of CF × SF hybrids resembled the degenerate eyes of CF in showing ventral reduction of the retina and corresponding displacement of the lens within the optic cup, a smaller lens and eyeball, more lens apoptosis, a smaller cartilaginous sclera, and lens-specific gene expression characteristics compared to SF × CF hybrids, which showed eye and lens gene expression phenotypes resembling SF. In contrast, reciprocal hybridization failed to support roles for maternal genetic effects in the CF regressive pigmentation phenotype or in CF constructive changes related to enhanced jaw development. Maternal transcripts encoded by the pou2f1b, runx2b, and axin1 genes, which are involved in determining ventral embryonic fates, were increased in unfertilized CF eggs. In contrast, maternal mRNAs encoded by the ß-catenin and syntabulin genes, which control dorsal embryonic fates, showed similar expression levels in unfertilized SF and CF eggs. Furthermore, maternal transcripts of a sonic hedgehog gene were detected in SF and CF eggs and early cleaving embryos. This study reveals that CF eye degeneration is controlled by changes in maternal factors produced during oogenesis and introduces A. mexicanus as a model system for studying the role of maternal changes in the evolution of development.

Genetic linkage between altered tooth and eye development in lens-ablated Astyanax mexicanus.

The phenotype of lens-ablated Mexican tetra (Astyanax mexicanus) compared to wild-type surface fish has been described and includes, among other effects, eye degeneration, changes in tooth number and cranial bone changes. Here, we investigate the spatiotemporal expression patterns of several key genes involved in the development of these structures. Specifically, we show that the expression of pitx2, bmp4 and shh is altered in the eye, oral jaw, nasal pit and forebrain in these lens-ablated fish. Furthermore, for the first time, we show altered pitx2 expression in the cavefish, which also has altered eye and tooth phenotypes. We thus provide evidence for a genetic linkage between the eye and tooth modules in this fish species. Furthermore, the altered pitx2 expression pattern, together with the described morphological features of the lens-ablated fish suggests that Astyanax mexicanus could be considered as an alternative teleost model organism in which to study Axenfeld-Rieger syndrome (ARS), a rare autosomal dominant developmental disorder that is associated with PITX2 and which has both ocular and non-ocular abnormalities.

Tooth and consequences: Heterodonty and dental replacement in piranhas and pacus (Serrasalmidae).

Tooth replacement in piranhas is unusual: all teeth on one side of the head are lost as a unit, then replaced simultaneously. We used histology and microCT to examine tooth-replacement modes across carnivorous piranhas and their herbivorous pacu cousins (Serrasalmidae) and then mapped replacement patterns onto a molecular phylogeny. Pacu teeth develop and are replaced in a manner like piranhas. For serrasalmids, unilateral tooth replacement is not an "all or nothing" phenomenon; we demonstrate that both sides of the jaws have developing tooth rows within them, albeit with one side more mineralized than the other. All serrasalmids (except one) share unilateral tooth replacement, so this is not an adaptation for carnivory. All serrasalmids have interlocking teeth; piranhas interdigitate lateral tooth cusps with adjacent teeth, forming a singular saw-like blade, whereas lateral cusps in pacus clasp together. For serrasalmids to have an interlocking dentition, their teeth need to develop and erupt at the same time. We propose that interlocking mechanisms prevent tooth loss and ensure continued functionality of the feeding apparatus. Serrasalmid dentitions are ubiquitously heterodont, having incisiform and molariform dentitions reminiscent of mammals. Finally, we propose that simultaneous tooth replacement be considered as a synapomorphy for the family.

Different developmental environments reveal multitrait plastic responses in South American Anostomidae fish.

Complex phenotypes result from developmental processes integrating genetic, epigenetic, and environmental information. Although changing environments combine several signals that may induce multitrait plastic responses, literature often decodes developmental plasticity into single trait variation as a function of isolated environmental signals. To address the multivariate nature of developmental plasticity, we evaluated how different combinations of environmental signals influence the development of morphological and behavioral traits. We raised Megaleporinus macrocephalus (Anostomidae) in four different developmental environments, and found that foraging position and structural complexity during development induced different morphotypes, which overlapped with behavioral patterns. Foraging position induced distinct patterns of mouth and fin positioning and overall body shape, which were accentuated by structural complexity. Moreover, fish most often chose conditions similar to their developmental environments. Combined signals during development, therefore, revealed environment-specific phenotypic patterns associating morphology and behavior. Such results endorse the ability of developmental processes to influence the variation present in natural populations. Implications of addressing the multivariate essence of developmental plasticity transcend the evolutionary theory and inspire applications in several fields.

A new species of Piaractus (Characiformes: Serrasalmidae) from the Orinoco Basin with a redescription of Piaractus brachypomus.

Piaractus orinoquensis, a new species of serrasalmid fish, is described from the Orinoco River basin. The new species differs from congeners by having a slenderer body, relatively smaller head and snout, more compressed mid-body, fewer scales above and below the lateral line and diagnostic molecular characters in the coI mitochondrial gene region. We also provide a re-description of Piaractus brachypomus, restricting its geographic distribution to the Amazon River basin. Both species are economically important in their respective basins and need to be independently managed as distinct species.

A new species of Curimatopsis Steindachner (Characiformes: Curimatidae) from the Rio Nhamundá, Amazon basin.

A new species of Curimatopsis is described from the Rio Nhamundá, Amazon basin in northern Brazil. The new species is distinguished from congeners by the presence of a distinctive concentration of dark pigmentation over the entire lower lobe of the caudal fin, reticulate pattern of body pigmentation, lower jaw longer than and overlapping the anterior portion of the upper jaw, crescent-shaped posterior nostril and by morphometric and meristic data. Comments on the phylogenetic position of the new species within Curimatopsis are also provided.

A new Myleus species (Characiformes: Serrasalmidae) from the Rio Tapajós basin, Brazil.

Myleus pachyodus, a new serrasalmid species, is described from the Rio Teles Pires and Rio Jamanxim, both tributaries of the Rio Tapajós in Mato Grosso and Pará states, Brazil. The new species differs from all congeners by having a remarkable ontogenetic thickening of the teeth shifting from slender incisiform in juveniles to thick bulky teeth at standard length ≥ 100 mm, whereas congeners present slender, antero-posteriorly flat teeth throughout all life stages. The new species further differs from congeners by having 18-20 branched dorsal-fin rays, by the lack of a diastema between contralateral outer series of premaxillary teeth and by the presence of 16-25 prepelvic spines, with the anteriormost spine never reaching the vertical through the pectoral-fin origin.

Larval development of Brycon amazonicus (Teleostei, Bryconidae) with a focus on locomotory, respiratory and feeding structures.

In the present work we describe the larval development of Brycon amazonicus, with emphasis on structures linked to swimming, respiration and feeding. We monitored from larval hatching (13 h post-fertilization) to juvenile stage. Formation of the oral cavity and differentiation of gill arches began within 4 h post-hatching (hph) and within 11 hph the primordium of the pectoral fin appeared. At 25 hph, the head was in a rectilinear position with a terminal mouth, when the onset of tooth formation appeared. At 35 hph, gill arches were covered by the operculum and branchiostegal membrane and cusps of teeth pierced the epithelium of the premaxilla and anterior dentary region. Sharp teeth were observed in the maxilla at 171 hph and the pelvic fin primordium appeared at 243 hph, with taste buds on the tongue and the roof of the buccopharyngeal cavity. At 579 hph, all structures related to swimming and food capture were formed. Exogenous feeding of the larvae started at 29 hph, before the endogenous reserves were used up and the development of efficient swimming ability, reflecting the species' need to increasingly specialize on prey capture.

A new species of Hemibrycon (Characiformes, Characidae, Stevardiinae) from the upper Magdalena River basin in Colombia.

Hemibrycon iqueima sp. nov., is described from small streams in the Magdalena drainage at the foothills of the western slope of the Eastern Cordillera of the Colombian Andes, Suarez municipality, Tolima Department, Colombia. The new species is distinguished from its congeners in the Magdalena-Cauca River basin by a combination of characters related to snout-anal-fin origin length, head length, dorsal-pectoral fin distance, dorsal-fin-hypural distance, postorbital distance, orbital diameter, snout length, number of total vertebrae, pre-dorsal scales, scale rows between anal-fin origin and lateral line, number of branched rays of the anal fin, maxillary teeth number and number and arrangement of hooks on the branched rays of the pectoral and dorsal fins. In addition, the validity of this species is supported by previous molecular analyses that included specimens of the new species that had been erroneously identified. Phylogenetic relationships between the new species and congeners from Pacific coast basins are discussed.