South American Freshwater Fishes Research Articles

Accelerated body size evolution in upland environments is correlated with recent speciation in South American freshwater fishes

Speciation rates vary greatly among taxa and regions and are shaped by both biotic and abiotic factors. However, the relative importance and interactions of these factors are not well understood. Here we investigate the potential drivers of speciation rates in South American freshwater fishes, the most diverse continental vertebrate fauna, by examining the roles of multiple biotic and abiotic factors. We integrate a dataset on species geographic distribution, phylogenetic, morphological, climatic, and habitat data. We find that Late Neogene-Quaternary speciation events are strongly associated with body-size evolution, particularly in lineages with small body sizes that inhabit higher elevations near the continental periphery. Conversely, the effects of temperature, area, and diversity-dependence, often thought to facilitate speciation, are negligible. By evaluating multiple factors simultaneously, we demonstrate that habitat characteristics associated with elevation, as well as body size evolution, correlate with rapid speciation in South American freshwater fishes. Our study emphasizes the importance of integrative approaches that consider the interplay of biotic and abiotic factors in generating macroecological patterns of species diversity.

Hidden diversity and evolutionary diversification in Phalloceros harpagos Lucinda (Cyprinodontiformes: Poeciliidae)

AbstractThe distribution of Phalloceros species is generally allopatric and restricted; however, Phalloceros harpagos has a wide distribution and exhibits morphological variation among populations from different Brazilian river basins. Different species delimitation tests using cytochrome C oxidase subunit I gene sequences indicated that P. harpagos is a species complex that comprises between 7 and 9 phylogroups. The separation of the Paraná and Paraíba do Sul River basins most likely caused the diversification of these phylogroups in the Miocene period. The majority of phylogroups have a narrow distribution; however, one phylogroup has a broad distribution, being found primarily in the Paraná, Ribeira de Iguape, and Iguaçu River basins. This distribution can be attributed to possible headwater capture events caused by the reactivation of faults and erosion in the quaternary. Estimates about the times of divergence based on molecular data could be used to infer the biogeographic processes related to the diversification of South American freshwater fish.

Myxozoan Ceratomyxids Infecting the Gallbladder of Amazonian Ornamental Cichlid Fish: Description of Ellipsomyxa santarenensis n. sp. and Report of Ceratomyxa amazonensis in a New Host

Although most Myxozoa species of the genera Ceratomyxa and Ellipsomyxa have been described in marine hosts worldwide, an increasing diversity has been reported infecting South American freshwater fish, mainly in Amazonian waters. The present study deals with two species of myxozoan ceratomyxids parasitizing the gallbladder of Amazonian ornamental cichlids fish: Ceratomyxa amazonensis is identified from a new host—Geophagus altifrons; while Ellipsomyxa santarenensis n. sp. is described infecting Satanoperca jurupari. Morphological (light microscopy and transmission electron microscopy), molecular (small ribosomal subunit DNA—SSU-rDNA sequencing) and phylogenetic analyses were used to characterize both species. Ceratomyxa amazonensis showed a prevalence of 64.2%, with plasmodia showing a vermiform shape and motility. For E. santarenensis n. sp., the prevalence was 33.3%. Ultrastructural analysis revealed that the vermiform C. amazonensis plasmodia were composed of an outer cytoplasmic region and a large vacuole occupying the inner area. In E. santarenensis n. sp., cytoplasmic expansions were observed in pseudoplasmodia originating pseudopodia. SSU rDNA sequencing-based genetic distance analysis revealed a very small difference between C. amazonensis, parasite of G. altifrons, and C. amazonensis, parasite of S. discus—host of the original description, thus showing that they are the same species occurring in a new host. For Ellipsomyxa santarenensis n. sp., molecular data revealed a difference of 1.6% for Ellipsomyxa amazonensis and Ellipsomyxa paraensis. The phylogenetic analysis revealed the grouping of E. santarenensis n. sp. together with the other freshwater Ellipsomyxa species of the Amazonian region, and associated with the morphological data, it was possible to identify it as a new taxon within the genus Ellipsomyxa.

Landscape dynamics and diversification of the megadiverse South American freshwater fish fauna

Landscape dynamics are widely thought to govern the tempo and mode of continental radiations, yet the effects of river network rearrangements on dispersal and lineage diversification remain poorly understood. We integrated an unprecedented occurrence dataset of 4,967 species with a newly compiled, time-calibrated phylogeny of South American freshwater fishes-the most species-rich continental vertebrate fauna on Earth-to track the evolutionary processes associated with hydrogeographic events over 100 Ma. Net lineage diversification was heterogeneous through time, across space, and among clades. Five abrupt shifts in net diversification rates occurred during the Paleogene and Miocene (between 30 and 7 Ma) in association with major landscape evolution events. Net diversification accelerated from the Miocene to the Recent (c. 20 to 0 Ma), with Western Amazonia having the highest rates of insitu diversification, which led to it being an important source of species dispersing to other regions. All regional biotic interchanges were associated with documented hydrogeographic events and the formation of biogeographic corridors, including the Early Miocene (c. 23 to 16 Ma) uplift of the Serra do Mar and Serra da Mantiqueira and the Late Miocene (c. 10 Ma) uplift of the Northern Andes and associated formation of the modern transcontinental Amazon River. The combination of high diversification rates and extensive biotic interchange associated with Western Amazonia yielded its extraordinary contemporary richness and phylogenetic endemism. Our results support the hypothesis that landscape dynamics, which shaped the history of drainage basin connections, strongly affected the assembly and diversification of basin-wide fish faunas.

Integrative analysis of new Clinostomum metacercariae (Digenea, Clinostomidae) using COI mtDNA and morphology rises the number of lineages found in South American freshwater fishes.

Clinostomidae is a diverse family of digenean parasitizing fish-eating birds as adults and fishes as metacercariae. The species composition, within the genus Clinostomum has been steadily increasing in recent years. In Argentina, four named species of Clinostomum have been documented, accompanied by four metacercariae representing distinct genetic lineages whose adults have not been identified. This study focused on examining clinostomids in three fish species - Australoheros scitulus (ASI), Cichlasoma dimerus (CDIM), and Pimelodella laticeps (PLA) - at various localities in Argentina. We conducted both morphological and molecular characterizations of the Clinostomum metacercariae collected from these fish species. Molecular phylogenetic analyses using COI mtDNA were performed to determine the placement of these metacercariae within the clinostomid phylogenetic tree. Clinostomum ASC represents a distinct lineage, morphologically distinguishable from other sequenced metacercariae due to its body shape (widest anteriorly and becoming slender towards the posterior end); this lineage was found to be closely related to C. caffarae. While Clinostomum CDIM and Clinostomum PLA exhibited morphological differences, they clustered together genetically with metacercariae reported in previous studies as Clinostomum L3 and Clinostomum CVI. This outcome, coupled with a low genetic distance (0 to 3%), suggests that they are conspecific with metacercariae found in fish across Mexico, Costa Rica, and Argentina. In light of the extensive diversity of fish species in Argentine freshwater ecosystems (over 500 species), and considering the relatively constrained extent of prior investigations, the anticipation of unearthing additional Clinostomum species or lineages is plausible.

Occurrence of the host-parasite system Rhaphiodon vulpinus and Ceratomyxa barbata n. sp. in the two largest watersheds in South America.

While around world, species of the genus Ceratomyxa parasite majority marine hosts, growing diversity has been reported in South American freshwater fish. The present study reports Ceratomyxa barbata n. sp. parasitizing the gallbladder of the Rhaphiodon vulpinus fish from the Amazon and La Plata basins. Morphological (light and transmission electron microscopy), molecular (sequencing of small subunit ribosomal DNA - SSU rDNA), and phylogenetic analyses were used to characterize the new species. Worm-like plasmodia endowed with motility were found swimming freely in the bile. The myxospores were elongated, lightly arcuate, with rounded ends and had polar tubules with 3 coils in the polar capsules. Ultrastructural analysis revealed plasmodia composed of an outer cytoplasmic region, where elongated tubular mitochondria, a rough endoplasmic reticulum, sporogonic stages, and a large vacuole occupying the internal area were observed. Phylogenetic analysis, based on SSU rDNA, found that among all South America freshwater Ceratomyxa species, C. barbata n. sp. arises as an earlier divergent species. The present study reveals the occurrence of this host-parasite system (R. vulpinus/C. barbata n. sp.) in the two largest watersheds on the continent.

Fish otoliths from the middle Miocene Pebas Formation of the Peruvian Amazon

A small assemblage of 22 otoliths was identified from the historical collection of Bluntschli and Peyer gathered in 1912 on the Itaya riverbank at Iquitos, Peru (Amazonia), from the Pebas Formation. The Pebas Mega-Wetland System in western Amazonia during the Miocene represented a unique, albeit short-lived, biotope characterized by a pronounced endemic evolution with gigantism in some vertebrate groups (e.g., turtles, crocodylians). Thus far, fishes have mainly been recorded based on isolated skeletal remains and teeth. Here, we describe the first well-preserved otolith assemblage from the Pebas Formation. This otolith assemblage adds a new facet to the fauna by complementing the skeletal bony fish data, primarily with species of the Sciaenidae and, to a lesser extent, Ariidae and Cichlidae. The sciaenids and ariids indicate that migration must have occurred between the marginal marine environments to the north and the Pebas Wetland System. The otoliths also indicate the likelihood of endemic developments of adapted marine immigrants to the Pebas Wetland System, some of which have become extinct (Pogonias, Umbrina), while others now represent typical South American freshwater fish groups (Plagioscion). Six new species are described based on otoliths, one in the Cichlidae—Cichlasoma bluntschlii n. sp., one in the Ariidae—Cantarius ohei n. sp., and four in the Sciaenidae—Pebasciaena amazoniensis n. gen. et n. sp., Plagioscion peyeri n. sp., Pogonias tetragonus n. sp. and Umbrina pachaula n. sp.

Phylogenetic relationships of philometrid nematodes (Philometridae Baylis & Daubney, 1926) inferred from 18S rRNA, with molecular characterisation of recently described species.

Nematodes of the family Philometridae Baylis & Daubney, 1926 (Dracunculoidea Stiles, 1907) are generally poorly known, and there are many taxonomic issues within the family. Philometrids are parasites of fish and are found in various locations throughout the host, including within the subcutaneous tissues and musculature, the abdominal cavity and gonads; vast sexual dimorphism often means the males are not collected, leading to many species being described solely on female characteristics. Although there have been a number of studies utilising molecular data, the vast majority of species are yet to be sequenced. This study undertook genetic sequencing of 15 recently described species of philometrids across 4 genera, many of which were from specimens collected from waters off Australia. All of the sequences obtained were closely related with representatives of the family Philometridae. Species were found to be distributed in the phylogenetic trees within 4 clades based on a combination of site of infection within the host and host habitat. Family of host and geographical location was not as important for position within the trees. Clade A contained philometrids collected from the abdominal cavities and head tissues of South American freshwater fish. Clade B contained philometrids primarily from the abdominal cavities of freshwater European cyprinids. Clade C contained philometrids primarily from the ovaries of marine fish. Clade D contained philometrids from the body tissues of marine and freshwater fish. The potential co-evolutionary patterns between philometrids and their fish hosts are highlighted as an area of future research. This research also highlighted the importance of correct identification of any sequenced specimen.

The farming and husbandry ofColossoma macropomum: From Amazonian waters to sustainable production

AbstractWithin the rich diversity of South American freshwater fish,Colossoma macropomum(Characiformes: Serrasalmidae), known as tambaqui, cachama or blackfin pacu, can reach 30 kg, is a traditional product in regional fish markets and has drawn the attention of fish farmers since the 1930s. Considerable progress achieved in different fields of aquaculture science has contributed to the growth of tambaqui production. Tambaqui has proven suitable for both monoculture and polyculture systems, and for both extensive and intensive production systems aimed at achieving sustainable, higher productivity with minimal environmental impact. Studies of the reproductive anatomy and physiology of the species proved fundamental to development of techniques to boost commercial production, contributing to development of protocols for hormonally induced spawning and artificial propagation in the 1970s. Newly hatched larvae must be fed with live foods until they can be weaned to artificial feeds at about 100 mg weight. Despite its importance for aquaculture, only a few studies have reported components of quantitative genetic variance and parameters for weight at age, morphometric traits and disease resistance. Genomic tools currently available can be applied to detect variation relevant to performance and to accelerate the process of genetic improvement. While the species’ feeding habit allows the use of diets containing 75%–85% plant protein, much more work needs to be done to optimize aquafeeds. Refinement of tambaqui production methods has the potential to significantly boost South American aquaculture. We recommend research on diets, genetic improvement and system optimization to spur further productivity and achieve sustainable tambaqui culture.

How to build single-celled cnidarians with worm-like motility: Lessons from Myxozoa.

Metazoans with worm-like morphologies across diverse and disparate groups typically demonstrate motility generated by hydrostatic skeletons involving tissue layers (muscles and epithelia). Here we present representative morphological, behavioural and molecular data for parasitic cnidarians (myxozoans) that demonstrate unprecedented variation in form and function, developing as cellular hydrostats. Motile elongate plasmodia characterise a remarkable radiation of species in the genus Ceratomyxa. The vermiform plasmodia inhabit gall bladders of a range of South American freshwater fish and exhibit undulatory motility reminiscent of nematodes but achieved at the cellular level. Collective insights from ultrastructure, confocal and light microscopy along with videos depicting movements highlight key features that we propose explain the unique motility of the plasmodia. These features include cytoskeletal elements (net forming microfilaments and microtubules), a large internal vacuole, a relatively rigid outer glycocalyx and peripherally arranged mitochondria. These constituents provide collective evidence for repurposing of the cnidarian epitheliomuscular cell to support worm-like motility at the cellular level. The apparent restriction of vermiform ceratomyxids to South American freshwaters suggests an origination via Cretaceous or Miocene marine transgressions and subsequent radiation.

Candidate Species for Florida Aquaculture: Arapaima Arapaima gigas

Arapaima Arapaima gigas, also known as paiche and pirarucu, is a large South American freshwater fish with unique biology, favorable aquaculture characteristics, and potential for production in south Florida. This publication provides Arapaima gigas biology, specific culture methods known for each stage of development, and information about diseases and parasites, markets, and the laws associated with this species to potential Florida producers.

EVOLUÇÃO DA ICTIOFAUNA DE RIACHOS SUL-AMERICANOS (CASTRO, 1999) REVISITADO APÓS MAIS DE DUAS DÉCADAS

A little more than 20 years after the first publication of Castro (1999), the knowledge of the South American freshwater ichthyofauna, especially that of Brazil, has increased dramatically. This increase occurred both in terms of knowledge of its taxonomic diversity - in 1999, based on various sources in the scientific literature, a total of 2,800 species of South American freshwater fish was estimated, and a total of near 3,500 species it is currently known from Brazil alone - as to the knowledge of its evolution and also ecology. Consequently, all the hypotheses originally proposed in that paper are reexamined and critically discussed here considering this new knowledge accumulated in the past nearly two decades. Although the original 1999 hypothesis that the small adult size of their respective species is a general evolutionary pattern shared by the South American stream ichthyofauna has been firmly corroborated by several studies carried out in many regions of Brazil, the different patterns associated with most of the species of stream fish originally proposed are discussed in this chapter in the light of the new accumulated knowledge about their biology and ecology. Likewise, the possible role of heterochrony in their respective evolution is critically analyzed in the context of the great advance that has occurred in understanding the phylogenetic relationships of component taxa of freshwater neotropical ichthyofauna, using mainly the family Characidae as a possible model. Finally, a recent intriguing idea of the role of respiratory physiology in determining both the maximum size of teleost fish and their patterns of environmental occupation is examined in relation to its possible effect in the evolution of South American stream ichthyofauna.

Climate vulnerability of South American freshwater fish: Thermal tolerance and acclimation.

Freshwater fish are restricted by their physiology to rivers and lakes, and are generally limited in their capacity to disperse across basins. As a result, there is often a close match between the evolutionary history of river basins and their natural history. Thus, the regional landscape and ecological features, such as temperature, have shaped the evolution and adaptation of local fish assemblages. Climate change is expected to affect fish diversity and increase extinction, especially in low latitudes, and it has been suggested that species that inhabit low latitude species are more susceptible since they live close to their maximum thermal limits and have low capacity for acclimation. To understand the mechanisms of variation in thermal tolerance across a broad-scale of South American fishes is fundamental to be able to assess the vulnerability of species and habitat to global warming. Herein, we present the first attempt to analyze the vulnerability of South American freshwater fish species, based on the review of upper thermal limits of 106 species from a broad range of latitudinal habitats. Our findings show that upper thermal limits decrease with latitude, while the thermal safety margin (TSM) increase. Furthermore, the latitude has little effects on the acclimation response ratio, and the TSM decreased with rising temperatures. These data suggest that thermal phenotypic acclimation has low potential for mitigating global warming. These results indicate that South American fish species living in tropical areas are more susceptible to global warming since they are already living close to their maximum habitat temperature.

The Amazonian fish Colossoma macropomum harbors high myxosporean diversity: A description of three novel species from a fish farm

The farming of Colossoma macropomum has intensified in recent years, leading to an increased need for research into the health of the fish. We therefore investigated the diversity of myxosporeans (Cnidaria: Myxozoa) infecting C. macropomum in a breeding system in the municipality of Rio Branco, in the state of Acre, Brazil. Twenty-four fish specimens were examined from June to August 2018. Our results revealed a high prevalence of infection, with 23 specimens (95.8%) exhibiting myxosporean plasmodia. Morphological analysis, based on light and electron microscopies, and molecular analysis (small subunit ribosomal DNA [SSU rDNA] sequencing) revealed the occurrence of three novel species of the genus Myxobolus. Plasmodia of Myxobolus guttae n. sp. were found in the fins of 75% of the specimens, and the myxospores were pear-shaped, measuring 12.3 ± 0.6 (10.3–13.5) μm in length, 8.1 ± 0.3 (7.1–8.6) μm in width, and 5.1 ± 0.6 (4.5–6.5) μm in thickness. The polar capsules were elongated and equal in size, measuring 6.8 ± 0.5 (5.8–7.6) μm in length and 2.5 ± 0.3 (1.8–3.1) μm in width, exhibiting polar tubules with 9–10 coils. The plasmodia of Myxobolus longus n. sp. were found in the gills of 45.8% of the C. macropomum specimens, and the myxospores were fusiform, measuring 16.4 ± 0.6 (14.7–17.3) μm in length, 7.1 ± 0.2 (6.8–7.7) μm in width and 5.5 ± 0.6 (4.4–6.6) μm in thickness. The polar capsules occupied more than half of the myxospore, exhibiting different sizes, the largest measuring 9.2 ± 0.5 (7.3–10.1) μm in length and 2.5 ± 0.3 (2.0–3.1) μm in width, while the smallest measured 8.5 ± 0.4 (7.1–9.1) μm in length and 2.4 ± 0.2 (1.9–3.0) μm in width. Both polar capsules contained polar tubules with 10–12 coils. For Myxobolus tambaquiensis n. sp., plasmodia were found in the opercular cavity of 41.7% of the fish specimens, and the myxospores had an oval shape, measuring 10.0 ± 0.4 (9.5–11.3) μm in length, 6.5 ± 0.2 (6.1–7.1) μm in width, and 4.6 ± 0.4 (3.9–5.6) μm in thickness. The polar capsules were elongated and equal in size, measuring 4.9 ± 0.2 (4.4–5.3) μm in length and 1.9 ± 0.2 (1.5–2.2) μm in width, closing with 8–9 coils of the polar tubule. The morphological and sequencing data of the SSU rDNA showed that the three species studied herein remain unknown to science, increasing the diversity of myxosporeans infecting C. macropomum, an iconic fish in South American freshwater fish farming. The SSU rDNA based phylogenetic analysis revealed that Myxobolus spp. parasites of C. macropomum did not have a monophyletic origin, identifying different times and pathways of the acquisition of parasites by this host species.

Morphological abnormalities in cryopreserved semen from South American neotropical fish: A meta‐analysis

A systematic review (SR) and meta-analysis (MA) were performed to summarize the scientific evidence of the effects of cryopreservation of sperm from South American freshwater fish species on sperm morphology indicators. The search strategy was applied to four electronic databases, and specialists were contacted electronically. The main inclusion criteria involved studies on neotropical fish whose male gametes were submitted for cryopreservation. MA for random effects was performed for each indicator separately with the averages of the control and treated groups. A total of five publications, which comprise five studies and 15 trials, were included in the MA involving three different species. Heterogeneity was observed among the studies for all variables. In general, the percentage of normal cells did not change significantly after cryopreservation. Primary morphological abnormalities increased significantly at a level of 1.91% (95% confidence interval [CI]: −3.727–0.097; p = 0.021) after cryopreservation when compared with fresh semen. The percentage of secondary abnormalities did not show a significant change (p ≥ 0.05) between fresh semen and cryopreserved semen. Our results suggest a new classification for sperm abnormalities in South American neotropical fish based on the increase in primary abnormalities after cryopreservation.

Overlooked biodiversity of mitochondrial lineages in Hemiodus (Ostariophysi, Characiformes)

AbstractHemiodus is the largest genus of the South American freshwater fish family Hemiodontidae, with 23 valid species. However, the species‐level diversity of Hemiodus remains uncertain since morphological studies have indicated additional species. Also, the phylogenetic relationships among species and species boundaries within this genus are still unresolved. Herein, we sequenced the barcode (COI) gene of 19 species of Hemiodus on which undertook molecular species delimitation methods (automatic barcode gap discovery [ABGD], generalized mixed Yule coalescent [GMYC] and Poisson tree process [PTP]) and the standard DNA barcoding procedure to establish species boundaries and cast light on putative new species in the genus. We also provide a COI‐based phylogenetic hypothesis for Hemiodus by maximum‐likelihood (ML) analysis, the first at species level. The results of ABGD, GMYC and PTP indicate that around 30 MOTUs (range: 28–32) exist for Hemiodus within our data set of 19 valid species with concordance among strategies (i.e. equal results) in delimiting 24 MOTUs. Using classical DNA barcoding procedures, we settled on 28 MOTUs for Hemiodus. Undescribed morphospecies (i.e. feasible for description or taxonomic validation), as well as cryptic species (i.e. lineages with high genetic distance but indistinguishable in external morphology), are proposed. Even with the limitations of a single‐locus phylogeny, the ML tree recovered many species and species groups as clades, corroborating, thus, their taxonomic separation. Overall, our results highlight the existence of an overlooked diversity of mitochondrial lineages in Hemiodus that can represent cryptic or poorly scrutinized species. Moreover, this study provides a molecular backbone for future studies on the systematics and evolution of Hemiodontidae taxa.

Leucismo en el pez Lebiasina bimaculata (Characiformes: Lebiasinidae) en Guayas, Ecuador

Leucism is a rare chromatic aberration, manifested by the total or partial absence of melanin in the skin. This study reports a case of leucism in the fish Lebiasina bimaculata (Characiformes: Lebiasinidae), in the Chongon river, Ecuador. The individual with leucism presented depigmentation all over the body, except in the iris and on the base of the third scale of the fourth longitudinal scale-series. With this case, there are 19 records of chromatic aberrations in South American freshwater fish.

New genera and species of paramphistomes (Digenea: Paramphistomoidea: Cladorchiidae) parasitic in fishes from the Amazon basin in Peru.

Two new genera and three new species of paramphistomoid digeneans are described in the family Cladorchiidae Fischoeder, 1901 from doradid, heptapterid, pimelodid and pseudopimelodid fishes in the Amazon River in Peru. Goeldamphistomum amazonum n. g., n. sp. (type-species) from Goeldiella eques (Müller & Troschel) (type-host) and Tenellus trimaculatus (Boulenger), and Goeldamphistomum peruanum n. g., n. sp. from Duopalatinus peruanus Eigenmann & Allen (type-host), Calophysus macropterus (Lichtenstein) and Microglanis sp. are placed in the Dadayiinae Fukui, 1929. Both species have an accessory acetabular sucker, which distinguishes the genus from all taxa previously reported from South American freshwater fishes. They differ from each other primarily in that G. amazonum has a prebifurcal genital pore and oblique, separated testes, the levels of which rarely overlap longitudinally, whereas G. peruanum has a postbifurcal genital pore and testes directly to obliquely tandem. Iquitostrema papillatum n. g., n. sp. (Kalitrematinae Travassos, 1933) from the intestine of Hassar orestis (Steindachner) differs from other members of the subfamily in the combination of a massive acetabulum with a papillate luminal surface and symmetrical testes which overlie the caeca close to the caecal arch. These are the first records of paramphistomes from the five host species studied here.

Proteomic characterization of vitellogenins from three species of South American fresh water fish

Vitellogenins (Vtg) are glycolipophosphoproteins synthesized by oviparous vertebrates as yolk proteins precursors. These proteins have been studied for their role in reproduction and in endocrine disruption. In this work, we report the first proteomic study towards the characterization of Vtg from Pseudoplatystoma fasciatum , Piaractus brachypomus and Colossoma macropomum . Male specimens of each of the three fish species were estradiol-induced (experimental) and non-induced (control). The initial Vtg characterization was made by 2D protein gel electrophoresis of both groups The identification of the high molecular weight spots, presumed to be Vtgs, was assessed by MALDI-TOF mass spectrometry analysis. A post-translational modification study was performed by differential staining of 2D gels in order to visualize phosphoproteins and glycoproteins. Plasma samples from the three species, induced with estrogen, showed three high molecular weight spots with variable isoelectric points. Post-translational modifications showed that Vtgs from P. brachypomus and C. macropomum presented a phosphorylated and glycosylated subunit, while the same subunit in P. fasciatum was only glycosylated. This characterization will help in the development of reliable immunoassays, which could be used for studies of endocrine disruption or for the improvement of artificial spawning, by uncovering the time of fish maturation or sex determination.

First DNA Barcode Reference Library for the Identification of South American Freshwater Fish from the Lower Paraná River.

Valid fish species identification is essential for biodiversity conservation and fisheries management. Here, we provide a sequence reference library based on mitochondrial cytochrome c oxidase subunit I for a valid identification of 79 freshwater fish species from the Lower Paraná River. Neighbour-joining analysis based on K2P genetic distances formed non-overlapping clusters for almost all species with a ≥99% bootstrap support each. Identification was successful for 97.8% of species as the minimum genetic distance to the nearest neighbour exceeded the maximum intraspecific distance in all these cases. A barcoding gap of 2.5% was apparent for the whole data set with the exception of four cases. Within-species distances ranged from 0.00% to 7.59%, while interspecific distances varied between 4.06% and 19.98%, without considering Odontesthes species with a minimum genetic distance of 0%. Sequence library validation was performed by applying BOLDs BIN analysis tool, Poisson Tree Processes model and Automatic Barcode Gap Discovery, along with a reliable taxonomic assignment by experts. Exhaustive revision of vouchers was performed when a conflicting assignment was detected after sequence analysis and BIN discordance evaluation. Thus, the sequence library presented here can be confidently used as a benchmark for identification of half of the fish species recorded for the Lower Paraná River.