An invasive bivalve with the potential to reconstruct chronologies of geomarkers in a large South American basin.

The Sr/Ca and 87Sr/86Sr ratios are used as natural indicators (geomarkers) in fish to reveal migratory patterns, due to significant relationships between these ratios measured in the water and in the fish calcified structures (otoliths, bones). The aim of this study was to assess the potential use of the Limnoperna fortunei shell as a proxy for monitoring the spatial and temporal variability of Sr/Ca and 87Sr/86Sr in water. These ratios were compared in water samples and bivalve shells proceeding from eight sites of four hydrogeological regions of the La Plata Basin (Argentina and Uruguay), collected in two hydrological periods (winter and summer) in order to depict the pattern of geographical and temporal variability and to evaluate the relationship between both matrices. Significant positive linear relationships were found between shell edge and water for 87Sr/86Sr (R2 = 0.96-0.97, p < 0.005) and Sr/Ca (R2 = 0.83-0.86, p < 0.05) in summer and winter. Slopes did not differ significantly from 1 (p > 0.05) for 87Sr/86Sr, while they differed from 1 for Sr/Ca in both seasons (p < 0.05), indicating that the elemental ratio would be influenced by endogenous/exogenous factors. No significant seasonal differences were found for both natural tags (p > 0.05), except for the lower Uruguay River (87Sr/86Sr) and outer estuary (Sr/Ca). These results indicate that the L. fortunei shell could be a valuable tool for monitoring spatio-temporal variations in water 87Sr/86Sr. Shell Sr/Ca shows high potential utility as a geomarker in freshwater, but additional assessments are needed to understand the impact of temperature and growth on this ratio.

Can species guilds act as hubs for energy transfer in macrophyte meadows of Amazonian floodplain lakes?

Aquatic macrophytes are the main autochthonous component of primary production in the Amazon Basin. Floating meadows of these plants support habitats with highly diverse animal communities. Fishes inhabiting these habitats have been assumed to use a broad range of food items and compose a particular food web. We employed carbon (δ13C) and nitrogen (δ15N) stable isotope analysis to draw the trophic structure of these habitats and to trace the energy flow by its trophic levels. Fishes and other animals from 18 independent macrophyte meadows of a floodplain lake of the Solimões River (Amazonia, Brazil) were analyzed. The food web of macrophyte meadows consists of four trophic levels above autotrophic sources. In general, primary consumers exhibited a broader range of food sources than the upper trophic levels. Some fish species depended on a large number of food sources and at the same time are consumed by several predators. The energy transfer from one trophic level to the next was then mainly accomplished by these species concentrating a high-energy flux and acting as hubs in the food web. The broad range of δ13C values observed indicates that the organisms living in the macrophyte meadows utilize a great diversity of autotrophic sources.

Ontogenetic change in the diet of Semaprochilodus insignis (Characiformes: Prochilodontidae) during migration between two limnologically distinct environments in the Amazon Basin

Semaprochilodus insignis is an Amazonian migratory fish species, moving in large shoals between white- and black-water rivers. It has long been classified as a detritivorous fish. However, it is possible that the trophic plasticity of S. insignis could be higher than previously assumed. The objective of this study was to investigate the relative contributions of autotrophic energy sources to the diet of S. insignis in the Negro and Solimões rivers and to determine if the species undergoes an ontogenetic change in the diet. We found variations between the δ13C and δ15N values of S. insignis between the rivers. In the Negro River, periphyton (84%) was the principal energy source for adults, while juveniles in the Solimões River foraged predominantly on terrestrial plants/C3 macrophytes (50%) and phytoplankton (42%). These variations in isotopic signatures are likely associated with migratory movements of S. insignis at different life stages and hydrological periods. Instead of the previously assumed dietary classification, we suggest that S. insignis varies its diet ontogenetically, with adults acting as illiophagous in black-water while the young are detritivorous in white-water rivers. The results show that this species creates complex links between food chains, thus emphasizing the importance of conserving flooded areas.(AU)

Semaprochilodus insignis é uma espécie de peixe migratório da Amazônia, movendo-se em cardumes entre rios de água branca e preta. Tem sido classificado como um peixe detritívoro-iliófago. Porém, é possível que a plasticidade trófica de S. insignis seja maior do que se supunha. O objetivo deste estudo foi investigar as contribuições relativas de fontes de energia autotróficas para a dieta do S. insignis nos rios Negro e Solimões e se a espécie muda ontogenticamente a sua dieta. Encontramos variações entre os valores de δ13C e δ15N de S. insignis entre os rios: Negro, o perifíton (84%) foi a principal fonte de energia para adultos, enquanto para os juvenis do rio Solimões, foram predominantemente as plantas terrestres/macrófitas C3 (50%) e fitoplâncton (42%). Essas variações nas assinaturas isotópicas provavelmente estão associadas a movimentos migratórios do S. insignis em diferentes estágios da vida e períodos hidrológicos. Ao invés da classificação alimentar previamente assumida, sugerimos que o S. insignis varie sua dieta ontogeneticamente, os adultos são iliófagos em águas negras, enquanto os jovens são detritívoros em rios de águas brancas. Os resultados demonstram que essa espécie promove ligações complexas entre as cadeias alimentares enfatizando, dessa forma, a importância da conservação das áreas inundadas amazônicas.(AU)

Discrimination of species and populations of the genus Cichla (Cichliformes: Cichlidae) in rivers of the Amazon basin using otolithic morphometry

ABSTRACT The genus Cichla is a highly diverse group, with 16 species already described. Externally, some species are very similar and discriminating between them may be very difficult. Nevertheless, discrimination of fish stocks is essential for management purposes. Morphometric analyses of otoliths have been successfully used to distinguish species and fish stocks, especially in marine environments. This study evaluated whether sagittal otolith shape can be used to discriminate among the species Cichla temensis, C. monoculus, and C. orinocensis, as well as within populations of C. temensis in rivers of the Amazon. Shape indices and Fourier coefficients were used to describe the shape of the otoliths. Among the groups of species, the morphology of the sagittal otolith of C. temensis was totally distinct from the species C. monoculus and C. orinocensis. While among populations of C. temensis, individuals from the Negro and Jatapú Rivers were different, regardless of the methods used. These results confirm the ability to differentiate species and populations by using the morphology of otoliths. However, more research is needed to verify the role of genetic versus environmental and biotic effects, and thus be able to explain the discrimination observed in otoliths.

RESUMO O gênero Cichla é bastante diverso, com 16 espécies descritas. Algumas espécies são externamente muito similares e sua discriminação pode ser bastante difícil. Ao mesmo tempo, a discriminação de estoques pesqueiros é essencial para propostas de manejo. Análises morfométricas em otólitos têm sido utilizadas com sucesso para a distinção de espécies e estoques pesqueiros, principalmente em ambientes marinhos. Este estudo avaliou se o formato do otólito sagittal pode ser utilizado para discriminar entre espécies Cichla temensis, C. monoculus e C. orinocensis, bem como dentro de populações de C. temensis em diferentes rios amazônicos. Índices de forma e coeficientes de Fourier foram utilizados para descrever a forma dos otólitos. Dentre as espécies, a morfologia do otólito sagittal do C. temensis mostrou ser totalmente distinta das espécies C. monoculus e C. orinocensis. Enquanto no grupo das populações de C. temensis, os indivíduos dos rios Negro e Jatapú mostraram-se diferentes independente dos métodos utilizados. Esses resultados confirmam a capacidade de diferenciação de espécies e populações através da morfologia dos otólitos. No entanto, são necessárias mais pesquisas para verificar o papel dos efeitos genéticos em comparação aos efeitos ambientais e bióticos para explicar a discriminação observada nos otólitos.

A database of freshwater fish species of the Amazon Basin.

The Amazon Basin is an unquestionable biodiversity hotspot, containing the highest freshwater biodiversity on earth and facing off a recent increase in anthropogenic threats. The current knowledge on the spatial distribution of the freshwater fish species is greatly deficient in this basin, preventing a comprehensive understanding of this hyper-diverse ecosystem as a whole. Filling this gap was the priority of a transnational collaborative project, i.e. the AmazonFish project - https://www.amazon-fish.com/. Relying on the outputs of this project, we provide the most complete fish species distribution records covering the whole Amazon drainage. The database, including 2,406 validated freshwater native fish species, 232,936 georeferenced records, results from an extensive survey of species distribution including 590 different sources (e.g. published articles, grey literature, online biodiversity databases and scientific collections from museums and universities worldwide) and field expeditions conducted during the project. This database, delivered at both georeferenced localities (21,500 localities) and sub-drainages grains (144 units), represents a highly valuable source of information for further studies on freshwater fish biodiversity, biogeography and conservation.

Seasonal oxygen isotope variations in freshwater bivalve shells as recorders of Amazonian rivers hydrogeochemistry.

Freshwater bivalve shell oxygen isotope values (δ18OS) may act as a recorder of river δ18O variations that can then be interpreted in terms of hydrology (e.g. precipitation-evaporation balance, precipitation and river discharge patterns). We investigated the potential of this proxy measured across the hinge of South American unionid shells: Anodontites elongatus collected in Peru and A. trapesialis in Brazil. The isotopic signatures were reproducible between individuals of the same species. A. trapesialis clearly showed a strong δ18OS cyclicity in accordance with its growth patterns while A. elongatus presented less clear δ18OS with lower amplitude. We confirm that the deposition of successive growth lines and increments is annual, with growth line corresponding to the wet season. Also, we suggest that low amplitude of δ18OS in the A. elongatus shells indicates a habitat close to the river while large amplitude of δ18OS cycles observed in A. trapesialis shells would reflect a floodplain lake habitat, seasonally disconnected from the river and thus subjected to higher seasonal fluctuations in water δ18O. Considering these promising first results, future studies could be directed towards the use of fossil shells to reconstruct the past and present hydrological and geochemical conditions of the Amazon.

The unique functioning of a pre-Columbian Amazonian floodplain fishery.

Archaeology provides few examples of large-scale fisheries at the frontier between catching and farming of fish. We analysed the spatial organization of earthen embankments to infer the functioning of a landscape-level pre-Columbian Amazonian fishery that was based on capture of out-migrating fish after reproduction in seasonal floodplains. Long earthen weirs cross floodplains. We showed that weirs bear successive V-shaped features (termed 'Vs' for the sake of brevity) pointing downstream for outflowing water and that ponds are associated with Vs, the V often forming the pond's downstream wall. How Vs channelled fish into ponds cannot be explained simply by hydraulics, because Vs surprisingly lack fishways, where, in other weirs, traps capture fish borne by current flowing through these gaps. We suggest that when water was still high enough to flow over the weir, out-migrating bottom-hugging fish followed current downstream into Vs. Finding deeper, slower-moving water, they remained. Receding water further concentrated fish in ponds. The pond served as the trap, and this function shaped pond design. Weir-fishing and pond-fishing are both practiced in African floodplains today. In combining the two, this pre-Columbian system appears unique in the world.

Present-day African analogue of a pre-European Amazonian floodplain fishery shows convergence in cultural niche construction.

Erickson [Erickson CL (2000) Nature 408 (6809):190-193] interpreted features in seasonal floodplains in Bolivia's Beni savannas as vestiges of pre-European earthen fish weirs, postulating that they supported a productive, sustainable fishery that warranted cooperation in the construction and maintenance of perennial structures. His inferences were bold, because no close ethnographic analogues were known. A similar present-day Zambian fishery, documented here, appears strikingly convergent. The Zambian fishery supports Erickson's key inferences about the pre-European fishery: It allows sustained high harvest levels; weir construction and operation require cooperation; and weirs are inherited across generations. However, our comparison suggests that the pre-European system may not have entailed intensive management, as Erickson postulated. The Zambian fishery's sustainability is based on exploiting an assemblage dominated by species with life histories combining high fecundity, multiple reproductive cycles, and seasonal use of floodplains. As water rises, adults migrate from permanent watercourses into floodplains, through gaps in weirs, to feed and spawn. Juveniles grow and then migrate back to dry-season refuges as water falls. At that moment fishermen set traps in the gaps, harvesting large numbers of fish, mostly juveniles. In nature, most juveniles die during the first dry season, so that their harvest just before migration has limited impact on future populations, facilitating sustainability and the adoption of a fishery based on inherited perennial structures. South American floodplain fishes with similar life histories were the likely targets of the pre-European fishery. Convergence in floodplain fish strategies in these two regions in turn drove convergence in cultural niche construction.

¿Qué factores determinan la distribución altitudinal de los peces de ríos tropicales andinos? / Which factors determine the altitudinal distribution of tropical Andean riverine fishes?

ResumenLos gradientes altitudinales representan un sistema apropiado para evaluar si existe una relación general entre los patrones de riqueza, variables ambientales, y los procesos ecológicos que determinan cuantas especies co-ocurren en un área dada.En cuanto a los peces de agua dulce en arroyos de montaña la relación prevalente es un decrecimiento monotónico en riqueza de especies con la altura. El objetivo de este estudio es evaluar cuatro hipótesis que pueden explicar la relación negativa entre la riqueza local de especies de peces y la altura, 1) hipótesis de la disminución de la energía disponible 2) hipótesis del aumento del rigor climático 3) hipótesis de la diversidad de hábitats e 4) hipótesis de la severidad de las condiciones físicas del hábitat que se presentan río arriba, relacionada a las dificultades para colonizar las partes altas de los ríos. Se recolectaron peces y macro-invertebrados en 83 sitios entre 200 a 4 000 m en dos cuencas en la Amazonía boliviana. La primera hipótesis fue evaluada mediante regresiones entre la densidad de macro-invertebrados, los peces invertívoros y la altura. Para evaluar la segunda y tercera hipótesis se realizó un análisis de regresión múltiple (GLM), entre la riqueza y varios factores locales y regionales. Se analizó también la disimilitud de los ensamblajes de peces sobre el gradiente altitudinal utilizando los índices βsim y βnes. Se encontró que la riqueza de peces disminuye de manera lineal con el aumento de la altura. La densidad de macroinvertebrados aumenta con la altura, contrariamente a la riqueza de peces invertívoros, sugiriendo que la energía no constituye un factor limitante para la colonización de las especies de peces en zonas altas. El GLM explicó un 86 % de la variación total de la riqueza, con un aporte significativo de la temperatura del agua, las pendientes máximas en el río principal y el ancho de río. El βsim presentó valores altos en niveles de altura bajos, mostrando mayor recambio de especies entre los sitios cercanos a la fuente de colonización. Inversamente, el βnes mostró valores mayores en las partes altas, correspondientes a cambios por pérdida de especies. Estos resultados sugieren que el rigor climático junto con la severidad física del hábitat crean barreras a la colonización y explican los cambios de riqueza en el gradiente altitudinal.

AbstractAltitudinal gradients represent an appropriate system to assess whether there is a relationship between richness patterns, environmental variables, and the ecological processes that determine the species type and number inhabiting a given area. In mountain streams freshwater fishes, the most prevalent relationship is a monotonic decrease in species richness with elevation. The objective of this study was to evaluate four hypotheses that can explain the negative relationship between local fish species richness and altitude, 1) the hypothesis of decreasing energy availability, 2) the hypothesis of increasing climate severity, 3) the hypothesis of habitat diversity, and 4) the hypothesis of isolation by physical severity of the environment. Fish and macro-invertebrates were collected following standard methods from 83 sites (between 200-4 000 meters) of two river basins in the Bolivian Amazon. The first hypothesis was tested by analyzing relationships between the density of macro-invertebrates, the richness of invertivorous fish species and altitude; while the second and third hypotheses were assessed by a multiple regression analysis (GLM) between fish species richness and several local and regional factors. Besides, assemblage dissimilarity between sites along the altitudinal gradient was analyzed using βsim and βness indices. Fish richness decreases linearly with increasing altitude. The density of macro-invertebrates tends to increase at higher altitudes, contrary to invertivorous fish species richness, suggesting that energy availability is not a limiting factor for fish species colonization. The GLM explained 86 % of the variation in fish species richness, with a significant contribution of water temperature, maximum slope in the river mainstem, and stream width. There is a higher species turnover (βsim) between sites at low elevation. Inversely, βness shows higher values in the upper parts, corresponding to change in assemblages mainly due to species loss. Taken together, these results suggest that climatic and physical severities create strong barriers to colonization, further explaining the decrease in fish richness along the altitudinal gradient.

[Which factors determine the altitudinal distribution of tropical Andean riverine fishes]? / Qué factores determinan la distribución altitudinal de los peces de ríos tropicales andinos?

Altitudinal gradients represent an appropriate system to assess whether there is a relationship between richness patterns, environmental variables, and the ecological processes that determine the species type and number inhabiting a given area. In mountain streams freshwater fishes, the most prevalent relationship is a monotonic decrease in species richness with elevation. The objective of this study was to evaluate four hypotheses that can explain the negative relationship between local fish species richness and altitude, 1) the hypothesis of decreasing energy availability, 2) the hypothesis of increasing climate severity, 3) the hypothesis of habitat diversity, and 4) the hypothesis of isolation by physical severity of the environment. Fish and macro-invertebrates were collected following standard methods from 83 sites (between 200-4 000 meters) of two river basins in the Bolivian Amazon. The first hypothesis was tested by analyzing relationships between the density of macro-invertebrates, the richness of invertivorous fish species and altitude; while the second and third hypotheses were assessed by a multiple regression analysis (GLM) between fish species richness and several local and regional factors. Besides, assemblage dissimilarity between sites along the altitudinal gradient was analyzed using ßsim and ßness indices. Fish richness decreases linearly with increasing altitude. The density of macro-invertebrates tends to increase at higher altitudes, contrary to invertivorous fish species richness, suggesting that energy availability is not a limiting factor for fish species colonization. The GLM explained 86 % of the variation in fish species richness, with a significant contribution of water temperature, maximum slope in the river mainstem, and stream width. There is a higher species turnover (ßsim) between sites at low elevation. Inversely, ßness shows higher values in the upper parts, corresponding to change in assemblages mainly due to species loss. Taken together, these results suggest that climatic and physical severities create strong barriers to colonization, further explaining the decrease in fish richness along the altitudinal gradient.

Geographical origin of Amazonian freshwater fishes fingerprinted by 87Sr/86Sr ratios on fish otoliths and scales.

Calcified structures such as otoliths and scales grow continuously throughout the lifetime of fishes. The geochemical variations present in these biogenic structures are particularly relevant for studying fish migration and origin. In order to investigate the potential of the (87)Sr/(86)Sr ratio as a precise biogeochemical tag in Amazonian fishes, we compared this ratio between the water and fish otoliths and scales of two commercial fish species, Hoplias malabaricus and Schizodon fasciatus, from three major drainage basins of the Amazon: the Madeira, Solimões, and Tapajós rivers, displaying contrasted (87)Sr/(86)Sr ratios. A comparison of the (87)Sr/(86)Sr ratios between the otoliths and scales of the same individuals revealed similar values and were very close to the Sr isotopic composition of the local river where they were captured. This indicates, first, the absence of Sr isotopic fractionation during biological uptake and incorporation into calcified structures and, second, that scales may represent an interesting nonlethal alternative for (87)Sr/(86)Sr ratio measurements in comparison to otoliths. Considering the wide range of (87)Sr/(86)Sr variations that exist across Amazonian rivers, we used variations of (87)Sr/(86)Sr to discriminate fish origin at the basin level, as well as at the sub-basin level between the river and savannah lakes of the Beni River (Madeira basin).

Trophic structure and mercury biomagnification in tropical fish assemblages, Iténez River, Bolivia.

We examined mercury concentrations in three fish assemblages to estimate biomagnification rates in the Iténez main river, affected by anthropogenic activities, and two unperturbed rivers from the Iténez basin, Bolivian Amazon. Rivers presented low to moderate water mercury concentrations (from 1.25 ng L(-1) to 2.96 ng L(-1)) and natural differences in terms of sediment load. Mercury biomagnification rates were confronted to trophic structure depicted by carbon and nitrogen stable isotopes composition (δ(15)N; δ(13)C) of primary trophic sources, invertebrates and fishes. Results showed a slight fish contamination in the Iténez River compared to the unperturbed rivers, with higher mercury concentrations in piscivore species (0.15 µg g(-1) vs. 0.11 µg g(-1) in the unperturbed rivers) and a higher biomagnification rate. Trophic structure analysis showed that the higher biomagnification rate in the Iténez River could not be attributed to a longer food chain. Nevertheless, it revealed for the Iténez River a higher contribution of periphyton to the diet of the primary consumers fish species; and more negative δ(13)C values for primary trophic sources, invertebrates and fishes that could indicate a higher contribution of methanotrophic bacteria. These two factors may enhance methylation and methyl mercury transfer in the food web and thus, alternatively or complementarily to the impact of the anthropogenic activities, may explain mercury differences observed in fishes from the Iténez River in comparison to the two other rivers.

Mercury bioaccumulation patterns in fish from the Iténez river basin, Bolivian Amazon.

The bioaccumulation mechanism expresses an increment of mercury concentration along the lifetime of each individual. It is generally investigated along the age or size range of organisms from a same population. Water chemistry and trophic position are important factors that may influence the emergence of bioaccumulation patterns. In order to detect the influence of these parameters on fish mercury bioaccumulation patterns, we explored the relations between mercury concentration, size and isotopic trophic position of fish populations of six species (three non piscivorous and three piscivorous) in three rivers of the Iténez basin (Bolivia) with different sediment load in water and anthropogenic impact. Fishes of the Iténez basin showed fairly lower mercury contamination in relation to the regional context. They presented lower total mercury concentrations in unperturbed clear water river (average of 0.051 µg g(-1) for non piscivores; 0.088 µg g(-1) for piscivores), intermediate values (average of 0.05 and 0.104 µg g(-1)) in unperturbed white water river, whereas the highest values (average of 0.062 and 0.194 µg g(-1)) were found in the perturbed clear water river. Piscivore and invertivore species showed significant positive bioaccumulation patterns in the perturbed river and in the unperturbed white water river. No positive pattern was detected in the unperturbed clear water river. Positive patterns could not be attributed to differences in trophic condition and mean fish mercury concentration between populations. Bioaccumulation seems not to be the main factor to explain increased mercury concentrations in fish from the perturbed river.

Anomalous mercury isotopic compositions of fish and human hair in the Bolivian Amazon.

We report mercury (Hg) mass-dependent isotope fractionation (MDF) and mass-independent isotope fractionation (MIF) in hair samples of the Bolivian Esse Ejjas native people and in several tropical fish species that constitute their daily diet. MDF with delta(202)Hg ranging from -0.40 to -0.92 per thousand for fish and +1.04 to +1.42 per thousand for hair was observed. Hair samples of native people with a fish-dominated diet are enriched by +2.0 +/- 0.2 per thousand in delta(202)Hg relative to the fish consumed. Both odd Hg isotopes, (199)Hg and (201)Hg, display MIF in fish (from -0.14 to +0.38 per thousand for Delta(201)Hg and from -0.09 to +0.55 per thousand for Delta(199)Hg) and in hair (from +0.12 to +0.66 per thousand for Delta(201)Hg and from +0.14 to +0.81 per thousand for Delta(199)Hg). No significant difference in MIF anomalies is observed between Hg in fish and in human hair, suggesting that the anomalies act as conservative source tracers between upper trophic levels of the tropical food chain. Fish Hg MIF anomalies are 10-fold lower than those published for fish species from midlatitude lakes. Grouping all Amazonian fish species per location shows that Delta(199)Hg:Delta(201)Hg regression slopes for the clear water Itenez River basin (0.95 +/- 0.08) are significantly lower than those for the white water Beni River basin (1.28 +/- 0.12). Assuming that the observed MIF originates from aquatic photoreactions, we calculated limited photodemethylation of monomethylmercury (MMHg) in the Beni River floodplains and insignificant photodemethylation in the Itenez River floodplains. This is possibly related to lower residence times of MMHg in the Itenez compared to the Beni River floodplains. Finally, a significantly negative Delta(201)Hg of -0.14 per thousand in Beni River fish suggests that the inorganic Hg precursor to the MMHg that bioaccumulates up the food chain defines an ecosystem specific non-zero Delta(201)Hg baseline. Calculation of photodemethylation intensities from Hg or MMHg MIF, therefore, requires a baseline correction.

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.