Unveiling biogeographical patterns of the ichthyofauna in the Tuichi basin, a biodiversity hotspot in the Bolivian Amazon, using environmental DNA.

To date, more than 2400 valid fish species have been recorded in the Amazon basin. However, some regions remain poorly documented. This is the case in the Beni basin and in particular in one of its main sub-basins, the Tuichi, an Andean foothills rivers flowing through the Madidi National Park in the Bolivian Amazonia. The knowledge of its ichthyological diversity is, however, essential for the management and protection of aquatic ecosystems, which are threatened by the development of infrastructures (dams, factories and cities), mining and deforestation. Environmental DNA (eDNA) has been relatively little used so far in the Amazon basin. We sampled eDNA from water in 34 sites in lakes and rivers in the Beni basin including 22 sites in the Tuichi sub-basin, during the dry season. To assess the biogeographical patterns of the amazonian ichthyofauna, we implemented a metabarcoding approach using two pairs of specific primers designed and developed in our laboratory to amplify two partially overlapping CO1 fragments, one of 185bp and another of 285bp. We detected 252 fish taxa (207 at species level) among which 57 are newly identified for the Beni watershed. Species compositions are significantly different between lakes and rivers but also between rivers according to their hydrographic rank and altitude. Furthermore, the diversity patterns are related to the different hydro-ecoregions through which the Tuichi flows. The eDNA approach makes it possible to identify and complete the inventory of the ichthyofauna in this still poorly documented Amazon basin. However, taxonomic identification remains constrained by the lack of reference barcodes in public databases and does not allow the assignment of all OTUs. Our results can be taken into account in conservation and management strategies and could serve as a baseline for future studies, including on other Andean tributaries.

Species-level ichthyoplankton dynamics for 97 fishes in two major river basins of the Amazon using quantitative metabarcoding.

The Amazon basin holds the world's largest freshwater fish diversity. Information on the intensity and timing of reproductive ecology of Amazonian fish is scant. We use a metabarcoding method by capture using a single probe to quantify species-level ichthyoplankton dynamics. We sampled the Marañón and the Ucayali rivers in Peru monthly for 2 years. We identified 97 species that spawned mainly during the flood start, the flood end or the receding periods, although some species had spawning activity in more than one period. This information was new for 40 of the species in the Amazon basin and 80 species in Peru. Most species ceased spawning for a month during a strong hydrological anomaly in January 2016, demonstrating the rapidity with which they react to environmental modifications during the breeding season. We also document another unreported event in the Amazon basin, the inverse phenology of species belonging to one genus (Triportheus). Overall larval flow in the Marañón was more than twice that of the Ucayali, including for most commercial species (between two and 20 times higher), whereas the Ucayali accounts for ~80% of the fisheries landings in the region. Our results are discussed in the light of the main anthropogenic threats to fishes, hydropower dam construction and the Hidrovía Amazónica, and should serve as a pre-impact baseline.

Age validation and contrasted growth performances of Pseudoplatystoma punctifer (Siluriformes: Pimelodidae) in two river systems of the Western Amazon

The doncella Pseudoplatystoma punctifer is an economically and ecologically important catfish in the Amazon basin. However, little is known about its age, growth and population dynamics parameters. This study aims to validate the formation of growth marks in vertebrae of individuals collected from two rivers systems of the Peruvian Amazon (Amazon-Marañón-Ucayali and Putumayo) and compare growth parameters using the von Bertalanffy growth function between sexes and systems. A total of 372 individuals from the Amazon-Marañón-Ucayali (AMU) system and 93 from the Putumayo River were analyzed. The formation of one growth ring per year was validated and the individual ages ranged from zero to nine years old. Females grew significantly larger than males in both systems. Both females and males grew significantly better in the AMU system than in the Putumayo River. Maximum observed length and size at maturity in the AMU system were lower than those reported in previous studies in the area, and together with an important proportion of juveniles in the catches, suggest that the species is heavily exploited. Further studies on the reproductive biology and population dynamics of the doncella are needed in order to implement management measures more in line with the current situation.(AU)

La doncella Pseudoplatystoma punctifer es un bagre de importancia económica y ecológica en la cuenca Amazonica. Pese a ello, poco se conoce acerca de parámetros de edad, crecimiento y dinámica poblacional. Este estudio tuvo como objetivo validar la formación de marcas de crecimiento en vértebras de individuos colectados en dos sistemas fluviales de la Amazonia Peruana (Amazonas-Marañón-Ucayali y Putumayo) y comparar los parámetros de crecimiento usando la función de von Bertalanffy entre sexos y sistemas. Fueron analizados 372 individuos del sistema Amazonas-Marañón-Ucayali (AMU) y 93 del Putumayo. Se validó la formación de un anillo de crecimiento por año, la edad osciló entre cero y nueve años. Las hembras fueron significativamente más grandes que los machos en ambos sistemas. Para ambos sexos el crecimiento fue significativamente mayor en el sistema AMU que en el Putumayo. Las máximas tallas y edades de primera madurez observadas en el sistema AMU fueron menores a las reportadas en estudios previos en el área, y junto con un considerable porcentaje de juveniles presentes en las capturas, se sugiere que la especie está siendo fuertemente explotada. Son necesarios estudios de biología reproductiva y dinámica poblacional de doncella para implementar medidas de manejo acordes a la situación actual.(AU)

Molecular evidence for three genetic species of Dipteryx in the Peruvian Amazon.

There is a high international demand for timber from the genus Dipteryx, or "shihuahuaco" as it is known in Peru. Developing tools that allow the identification and discrimination of Dipteryx species is therefore important for supporting management of natural populations and to underpin legal trade of its timber. The objective of this study was the molecular characterization of Dipteryx species in the Peruvian Amazonia. Two plastid regions (cpDNA: trnH-psbA and matK) were sequenced and 11 microsatellite markers (nDNA) were genotyped for 32 individuals identified as Dipteryx charapilla, D. micrantha morphotype 1 and D. micrantha morphotype 2. Using the concatenated sequences of the plastid genes, we identified ten haplotypes that were not shared between the species or between the D. micrantha morphotypes. Haplotypic diversity was greater in D. micrantha morphotype 2 and D. charapilla than in D. micrantha morphotype 1, which presented only one haplotype with a wide distribution in Peru. The microsatellites allowed the discrimination of the same three clades and identified diagnostic alleles for each clade. These results allowed us to demonstrate that the two morphotypes of D. micrantha are different at both the plastid and nuclear markers, which supports the existence of three genetically distinct species in Peru. This study provides information for the genetic discrimination of Dipteryx species and emphasises the importance of conserving the genetic variability of this genus in the Peruvian Amazonia.

Age and growth of the Amazonian migratory catfish Brachyplatystoma rousseauxii in the Madeira River basin before the construction of dams

The goliath catfish Brachyplatystoma rousseauxii has crucial economical and ecological functions in the Amazon basin. Although its life history characteristics have been studied in the Amazon, there is little information in the Madeira River basin, which holds genetically distinct populations and where dams were recently built. Using fish collected in Bolivia, Brazil and Peru, this study provides a validation of growth rings deposition and details the growth patterns of B. rousseauxii in the Madeira before the dams' construction. Age structure and growth parameters were determined from 497 otolith readings. The species exhibits two growth rings per year and sampled fish were between 0 and 16 years old. In the Brazilian portion of the basin, mainly young individuals below 5 years old were found, whereas older fish (> 5 years) were caught only in the Bolivian and Peruvian stretches, indicating that after migrating upstream to reproduce, adults remain in the headwaters of the Madeira River. Comparing with previous publications, B. rousseauxii had a slower growth and 20 cm lower maximum standard length in the Madeira River than in the Amazon River. This study provides a baseline for future evaluation of changes in population dynamics of the species following dams closure.(AU)

Brachyplatystoma rousseauxii é um bagre de importante papel econômico e ecológico na bacia amazônica. Embora existam estudos acerca de sua história de vida na Amazônia, há pouca informação para a bacia do rio Madeira, onde existem populações geneticamente distintas e recentemente foram construídas duas usinas hidrelétricas. Este estudo validou a deposição das marcas de crescimento e detalhou os padrões de desenvolvimento dessa espécie no rio Madeira, antes da construção das barragens. As coletas abrangeram os territórios brasileiro, boliviano e peruano, com estrutura etária e parâmetros de crescimento determinados a partir de 497 otólitos. Foram observadas duas marcas de crescimento por ano e indivíduos entre 0 e 16 anos. Na porção brasileira foram encontrados principalmente jovens menores de 5 anos, enquanto que os peixes mais velhos (> 5 anos) foram capturados apenas na Bolívia e Peru, indicando que após a migração reprodutiva, os adultos permanecem nas cabeceiras do rio Madeira. Comparando com estudos prévios realizados na calha principal do rio Amazonas, B. rousseauxii apresentou crescimento mais lento e comprimento padrão máximo inferior de 20 cm no rio Madeira. Este estudo fornece uma base para a avaliação futura das mudanças na dinâmica populacional desse espécie após o implementação das barragens.(AU)

Tempo and rates of diversification in the South American cichlid genus Apistogramma (Teleostei: Perciformes: Cichlidae).

Evaluating biodiversity and understanding the processes involved in diversification are noticeable conservation issues in fishes subject to large, sometimes illegal, ornamental trade purposes. Here, the diversity and evolutionary history of the Neotropical dwarf cichlid genus Apistogramma from several South American countries are investigated. Mitochondrial and nuclear markers are used to infer phylogenetic relationships between 31 genetically identified species. The monophyly of Apistogramma is suggested, and Apistogramma species are distributed into four clades, corresponding to three morphological lineages. Divergence times estimated with the Yule process and an uncorrelated lognormal clock dated the Apistogramma origin to the beginning of the Eocene (≈ 50 Myr) suggesting that diversification might be related to marine incursions. Our molecular dating also suggests that the Quaternary glacial cycles coincide with the phases leading to Apistogramma speciation. These past events did not influence diversification rates in the speciose genus Apistogramma, since diversification appeared low and constant through time. Further characterization of processes involved in recent Apistogramma diversity will be necessary.

Molecular identification of a cryptic species in the Amazonian predatory catfish genus Pseudoplatystoma (Bleeker, 1962) from Peru.

Pseudoplatystoma species are highly prized South American Pimelodid migratory catfishes. Until recently, their taxonomy was not clearly established, with discrepancies between morphological and molecular analyses. Here, Pseudoplatystoma species from the Peruvian Amazon were characterized at the molecular level from a sample representing the observed range of their color pattern variations in the study area. Analyses were performed using seven microsatellite loci for 103 specimens and, for part of them (52), using sequences of two regions of their mitochondrial genome [Cytochrome Oxidase subunit I (COI) and Control Region (CR)]. Factorial correspondence analysis and assignment tests based on microsatellite polymorphism showed that the specimens originally identified as P. punctifer belonged to two different gene pools highly differentiated from P. tigrinum. Morphological examination identified two different morphotypes (with and without black stripes), suggesting the existence of two distinct taxa within P. punctifer. This result was corroborated by the ML tree based on CR sequences, where all individuals but four clustered in a similar way as in the FCA and Bayesian assignment tests. For these four individuals, mitochondrial introgression or retention of ancestral polymorphism was likely. In contrast, the ML tree based on COI sequences showed that reciprocal monophyly was not yet achieved for this marker for the two P. punctifer taxa. The existence of three sympatric species of Pseudoplatystoma in the Peruvian Amazon is discussed in relation to their molecular characteristics, color patterns and ecology. Evolutionary scenarios regarding their divergence are hypothesized.

Diferenciación morfológica y por ISSR (inter simple sequence repeats) de especies del género plukenetia (euphorbiaceae) de la Amazonía peruana: propuesta de una nueva especie / Morphological and differentiation by ISSR (inter simple sequence repeats) of species of the genus plukenetia (euphorbiaceae) in the Peruvian Amazon: proposal of a new species

En el presente trabajo se estudian cinco especies del género Plukenetia de la Amazonía peruana: P. brachybotrya, P. loretensis, P. polyadenia, P. huayllabambana, P. volubilis (procedencia San Martín); y de un supuesto morfotipo (P. volubilis, procedencia Cusco). Los 126 especímenes estudiados fueron identificados mediante claves de caracteres morfológicos (formas de hojas, tallos y semilla; posición de glándulas basilaminares) y posteriormente mediante marcadores moleculares ISSR (CAA, CAG, GACA). Los análisis morfológicos permitieron separar las cinco especies descritas: P. brachybotrya, P. loretensis, P. polyadenia, P. volubilis y P. huayllabambana. Los dos supuestos morfotipos de P. volubilis fueron discriminados por la posición de las glándulas, tamaño de semillas y forma del tallo. Los resultados proporcionados por el Análisis Factorial de Correspondencia (AFC) y corroborados por el Índice de fijación (FST), distancia genética y el dendograma estimado por el método UPGMA, evidencian una fuerte diferenciación entre los seis taxa, corroborando la identidad taxonómica molecular de las cinco especies ya descritas morfológicamente. Además, los resultados (Fst y la distancia genética) indicarian que P. volubilis (del Cusco) podría ser una nueva especie de Sacha Inchi, aún no descrita para la ciencia.

Are rapids a barrier for floodplain fishes of the Amazon basin? A demographic study of the keystone floodplain species Colossomamacropomum (Teleostei: Characiformes).

We investigated demographic history and population structuring of Colossoma macropomum sampled from 14 localities in the Amazon basin and the Bolivian sub-basin; the two basins are separated by a series of 16 rapids. Although genetically differentiated, IMa analyses suggest non-zero bi-directional migration rates, and inter-basin divergence of approximately 17 thousand years ago. Analyses in BEAST indicated that Bolivian C. macropomum has been demographically stable except for a moderate population increase in the last 12 thousand years, while Amazonian C. macropomum has been experiencing demographic growth over the last 350 thousand years, resulting in approximately one order of magnitude increase in coalescent N(e).

Delimiting species by reproductive isolation: the genetic structure of epigean and hypogean Trichomycterus spp. (Teleostei, Siluriformes) in the restricted area of Torotoro (Upper Amazon, Bolivia).

Genetic variability of Trichomycterus from the region of Torotoro (Bolivia, Upper Amazon), distributed in the same watershed where the habitat is structured by waterfalls, canyons and a cave, was studied by allozyme (twelve putative loci) and RFLP-mtDNA (DLoop and cytochrome b) analyses. Alloenzymatic variation studied by Correspondence Analysis and Maximum Likelihood Analysis revealed a four-group structure, which was largely congruent with the distribution of the 14 mtDNA haplotypes. Two of these four clusters (I and II) were differentiated by two diagnostic loci (IDH and G3PDH), two semi-diagnostic loci (PGM and 6PGDH) and consequently a very high F(st )value (estimator theta = 0.77). Therefore, clusters I and II are reproductively isolated. The distribution limit of these two (sibling) species does not correspond to those of the morphological species of Trichomycterus identified in this region: the epigean T. cf. barbouri and the hypogean T. chaberti. However, hypogean fish exhibited two mtDNA haplotypes, a private one and another shared with the epigean Trichomycterus from upstream reaches.

Isolation by distance and Pleistocene expansion of the lowland populations of the white piranha Serrasalmus rhombeus.

The genetic variability and distribution of Amazonian fish species have likely been influenced by major disturbance events in recent geological times. Alternatively, the great diversity of aquatic habitat in the Amazon is likely to shape ongoing gene flow and genetic diversity. In this context, complex patterns of genetic structure originating from a joint influence of historical and contemporary gene flow are to be expected. We explored the relative influence of Pleistocene climatic fluctuations and current water chemistry on the genetic structure of a piranha, Serrasalmus rhombeus, in the Upper Amazon by the simultaneous analysis of intron length polymorphism and mitochondrial DNA sequences. The Madeira river is well suited for that purpose as it is characterized by a great diversity of water types, the presence of one of the largest floodplain of the Amazon and the potential occurrence of two Pleistocene refuges. We found evidence of genetic structure even at a small geographical scale (less than 10 km), indicating that the floodplain is not a homogenizing factor promoting interdrainage dispersal in S. rhombeus. Likewise, the hierarchical genetic structure inferred was correlated to geographical distance instead of habitat characteristic. Our results also support the hypothesis that the area underwent population expansion during the last 800,000 years. In addition, a higher level of genetic diversity was found in the samples from the putative Aripuanã refuge. The present findings suggest that Pleistocene refuges contributed significantly to the colonization of the lowlands in the Upper Amazon valley during the Pleistocene.

Phylogeography of the piranha genera Serrasalmus and Pygocentrus: implications for the diversification of the Neotropical ichthyofauna.

The phylogenetic relationships within the piranhas were assessed using mitochondrial sequences with the aim of testing several hypotheses proposed to explain the origin of Neotropical diversity (palaeogeography, hydrogeology and museum hypotheses). Sequences of the ribosomal 16S gene (510 bp) and control region (980 bp) were obtained from 15 localities throughout the main South American rivers for 21 of the 28 extant piranha species. The results indicate that the genus Serrasalmus is monophyletic and comprises three major clades. The phylogeographical analyses of these clades allowed the identification of five vicariant events, extensive dispersal and four lineage duplications suggesting the occurrence of sympatric speciation. Biogeographical patterns are consistent with the prediction made by the museum hypothesis that lineages from the Precambrian shields are older than those from the lowlands of the Amazon. The vicariant events inferred here match the distribution of the palaeoarches and several postdispersal speciation events are identified, thereby matching the predictions of the palaeogeography and hydrogeology hypotheses, respectively. Molecular clock calibration of the control region sequences indicates that the main lineages differentiated from their most recent common ancestor at 9 million years ago in the proto Amazon-Orinoco and the present rate of diversification is the highest reported to date for large carnivorous Characiformes. The present results emphasize that an interaction among geology, sea-level changes, and hydrography created opportunities for cladogenesis in the piranhas at different temporal and geographical scales.